Sample records for murine sertoli cells

  1. A tumorigenic murine Sertoli cell line that is temperature-sensitive for differentiation.

    PubMed

    Boekelheide, K; Lee, J W; Hall, S J; Rhind, N R; Zaret, K S

    1993-10-01

    The Sertoli cell is the epithelial cell within the seminiferous tubule responsible for supporting germ cells. Most current in vitro studies of Sertoli cell function use primary cultures because of the limited number of available Sertoli cell lines. In addition, few in vivo models of Sertoli cell malignancy have been described. In this study, a tumorigenic Sertoli cell line was developed by infection of isolated murine Sertoli cells by simian virus 40 tsA255; the ts mutation causes the inactivation of the large T antigen at elevated temperatures. A cloned Sertoli cell line, called S14-1, demonstrated temperature-dependent growth in soft agar and formed tumors in nude mice. Electron microscopy of the S14-1-derived tumor revealed extensive basal intercellular junctions and tubulobulbarlike processes supporting its Sertoli cell origin. Cytogenetic analysis showed that S14-1 cells were aneuploid with an average of 70 chromosomes per cell. At the nonpermissive (40 C) temperature, S14-1 cells in vitro demonstrated a reduced growth rate, enhanced secretion of transferrin, and increased expression of sulfated glycoprotein-2 messenger RNA, indicating the cells manifested increased differentiation following large T antigen inactivation. The murine S14-1 Sertoli cell line should be useful for both in vitro studies of Sertoli cell function and in vivo studies of Sertoli cell malignancy. PMID:8214009

  2. Computer-Assisted Annotation of Murine Sertoli Cell Small RNA Transcriptome1

    PubMed Central

    Ortogero, Nicole; Hennig, Grant W.; Langille, Chad; Ro, Seungil; McCarrey, John R.; Yan, Wei

    2012-01-01

    ABSTRACT Mammalian genomes encode a large number of small noncoding RNAs (sncRNAs) that play regulatory roles during development and adulthood by affecting gene expression. Several sncRNA species, including microRNAs (miRNAs), piwi-interacting RNAs (piRNAs), endogenous small interfering RNAs (endo-siRNAs), and small nucleolar RNAs (snoRNAs), are abundantly expressed in the testis and required for normal testicular development and spermatogenesis. To evaluate global changes in sncRNA expression, the next-generation sequencing (NGS)-based sncRNA transcriptomic analysis has become routine, because it allows rapid determination of the small RNA transcriptome of a particular testicular cell type. However, annotation of small RNA NGS reads can be challenging due to the volume of reads obtained, which is usually in the millions. Therefore, we developed a computer-assisted sncRNA annotation protocol that could identify not only known sncRNAs but also previously uncharacterized ones. Using this protocol, we annotated NGS reads of a Sertoli cell sncRNA library, and we report to our knowledge the first comprehensive annotation of the sncRNA transcriptome of immature murine Sertoli cells. Moreover, the computer-assisted sncRNA annotation pipeline that we report is applicable for annotating NGS reads derived from other cell types and/or sequencing platforms. PMID:23136297

  3. Sertoli cells as biochambers

    NASA Technical Reports Server (NTRS)

    Cameron, Don F. (Inventor); Sanberg, Paul R. (Inventor); Saporta, Samuel (Inventor); Hushen, Joelle J. (Inventor)

    2004-01-01

    According to the present invention, there is provided a biological chamber system having a biochamber defined by outer walls of Sertoli cells. Also provided is a transplantation facilitator including a biochamber. A method of making biochambers by co-culturing facilitator cells and therapeutic cells and then aggregating the facilitator celes is also provided. Also provided is a method of transplanting cells by incorporating transplant cells into a biochamber and transplanting the biochamber containing the transplant cells.

  4. Expression of the Pem Homeobox Gene in Sertoli Cells Increases the Frequency of Adjacent Germ Cells with

    E-print Network

    Wilkinson, Miles F.

    Expression of the Pem Homeobox Gene in Sertoli Cells Increases the Frequency of Adjacent Germ Cells, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030 Pem is a member of the homeobox. In the murine testis, Pem is specifically ex- pressed in Sertoli cells, where it is dramatically induced

  5. Sertoli Cell Differentiation in Pubertal Boars

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Meishan boars experience puberty at a younger age than crossbred (BX) boars in association with earlier cessation of Sertoli cell proliferation and smaller post pubertal testicular size. The current study defined changes in expression, assessed by immunohistochemistry, of anti-Mullerian hormone (AMH...

  6. Defined pattern of Sertoli cell differentiation in pubertal porcine testes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Number of Sertoli cells is a primary determinant of mature testicular size and sperm production. In boars, formation of the blood/testis barrier, which occurs by 4 mo of age in commercial breeds, signals the end of Sertoli cell proliferation. Previous studies established that expression of p27Kip1, ...

  7. Aromatase expression in prepuberal Sertoli cells: effect of thyroid hormone

    Microsoft Academic Search

    S. Andò; R. Sirianni; P. Forastieri; I. Casaburi; M. Lanzino; V. Rago; F. Giordano; C. Giordano; A. Carpino; V. Pezzi

    2001-01-01

    Aromatase activity has recently been assumed as a Sertoli cell functional maturation marker since it is maximally expressed in prepuberal age then it dramatically decreases at puberty and is virtually absent in adult age. Neonatal hypothyroidism is associated with a prolonged proliferation of Sertoli cells. This immature stage persists concomitantly with a dramatic enhancement of aromatase activity reversed by triiodothyronine

  8. Testicular Sertoli cell function in ankylosing spondylitis.

    PubMed

    Almeida, Breno Pires; Saad, Carla Gonçalves Schahin; Souza, Fernando Henrique Carlos; Moraes, Julio Cesar Bertacini; Nukumizu, Lucia Akemi; Viana, Vilma Santos Trindade; Bonfá, Eloísa; Silva, Clovis Artur

    2013-07-01

    To assess the testicular Sertoli cell function according to inhibin B levels in ankylosing spondylitis (AS) patients and the possible effect of anti-TNF therapy on this hormone production, 20 consecutive AS patients and 24 healthy controls were evaluated. At study entry, AS patients were not receiving sulfasalazine/methotrexate and never have used biological/cytotoxic agents. They were assessed by serum inhibin B levels, hormone profile, urological examination, testicular ultrasound, seminal parameters, and clinical features. Ten of these patients received anti-TNF treatment and they were reevaluated for Sertoli function and disease parameters at 6 months. Four of them agreed to repeat sperm analysis. At study entry, the median of inhibin B (68 vs. 112.9 pg/mL, p = 0.111), follicle-stimulating hormone levels (3.45 vs. 3.65 IU/L, p = 0.795), and the other hormones was comparable in AS patients and controls (p > 0.05). Sperm analysis was similar in AS patients and controls (p > 0.05) with one AS patient presenting borderline low inhibin B levels. Further analysis at 6 months of the 10 patients referred for anti-TNF therapy, including one with borderline inhibin B, revealed that median inhibin B levels remained stable (116.5 vs. 126.5 pg/mL, p = 0.431) with a significant improvement in C-reactive protein (27.8 vs. 2.27 mg/L, p = 0.039). Sperm motility and concentration were preserved in the four patients who repeated this analysis after TNF blockage. In conclusion, this was the first study to report, using a specific marker, a normal testicular Sertoli cell function in AS patients with mild to moderate disease activity. PMID:23417428

  9. Sertoli cells- Immunological sentinels of spermatogenesis

    PubMed Central

    Kaur, Gurvinder; Thompson, Lea Ann; Dufour, Jannette M.

    2014-01-01

    Testicular germ cells, which appear after the establishment of central tolerance, express novel cell surface and intracellular proteins that can be recognized as ‘foreign antigens’ by the host’s immune system. However, normally these germ cells do not evoke an auto-reactive immune response. The focus of this manuscript is to review the evidence that the Blood-Testis-Barrier (BTB)/Sertoli cell (SC) barrier along with the SCs ability to modulate the immune response is vital for protecting auto-antigenic germ cells. In normal testis, the BTB/SC barrier protects the majority of the auto-antigenic germ cells by limiting access by the immune system and sequestering these ‘new antigens’. SCs also modulate testis immune cells (induce regulatory immune cells) by expressing several immunoregulatory factors, thereby creating a local tolerogenic environment optimal for survival of nonsequesetred auto-antigenic germ cells. Collectively, the fortress created by the BTB/SC barrier along with modulation of the immune response is pivotal for completion of spermatogenesis and species survival. PMID:24603046

  10. Sertoli cells secrete both testis-specific and serum proteins.

    PubMed Central

    Wright, W W; Musto, N A; Mather, J P; Bardin, C W

    1981-01-01

    The secretions of the Sertoli cell were examined with two polyvalent antisera--one prepared against proteins in rat serum and the other against testis-specific proteins in rete testis fluid. These antisera detected 12 serum and 9 testis-specific proteins in rete testis fluid. To determine the origin of these proteins, primary cultures enriched in Sertoli cells were incubated with [35S]methionine, and the radiolabeled proteins in the medium were immunoprecipitated. Gel electrophoresis of the two immunoprecipitates resolved eight serum and nine testis-specific proteins. These two sets of proteins were specifically bound to their respective antiserum and were immunologically distinct. Medium from Sertoli cell cultures contained 10 times more of the testis-specific proteins than did cultures enriched for testicular myoid or interstitial cells. The concentration of the serum proteins in Sertoli cell medium was 5 and 10 times greater, respectively, than in myoid or interstitial cell preparations. The proteins from Sertoli cells were next characterized on two-dimensional gels. Seven of the proteins recognized by antiserum against serum proteins had identical molecular weights and isoelectric points as serum proteins. Three of these proteins were ceruloplasmin, transferrin, and glycoprotein 2. In addition to the proteins immunoprecipitated by the two antisera, more than 60 other proteins were detected on two-dimensional gels of the total secretory proteins. We conclude that the Sertoli cell secretes many proteins, some of which are specific to the testis and others of which are similar to serum proteins. Images PMID:6950398

  11. Effects of FSH and testosterone on Sertoli cells and spermatocytes from rat testis

    E-print Network

    Paris-Sud XI, Université de

    Effects of FSH and testosterone on Sertoli cells and spermatocytes from rat testis F. F. G biochemical parameters have been studied in isolated Sertoli cells and germinal cells from rat testis the same testis tissue. Isolated Sertoli cells when incubated with 3H-leucine secreted in addition

  12. Sertoli Cells Maintain Leydig Cell Number and Peritubular Myoid Cell Activity in the Adult Mouse Testis

    PubMed Central

    Monteiro, Ana; Milne, Laura; Cruickshanks, Lyndsey; Jeffrey, Nathan; Guillou, Florian; Freeman, Tom C.; Mitchell, Rod T.; Smith, Lee B.

    2014-01-01

    The Sertoli cells are critical regulators of testis differentiation and development. In the adult, however, their known function is restricted largely to maintenance of spermatogenesis. To determine whether the Sertoli cells regulate other aspects of adult testis biology we have used a novel transgenic mouse model in which Amh-Cre induces expression of the receptor for Diphtheria toxin (iDTR) specifically within Sertoli cells. This causes controlled, cell-specific and acute ablation of the Sertoli cell population in the adult animal following Diphtheria toxin injection. Results show that Sertoli cell ablation leads to rapid loss of all germ cell populations. In addition, adult Leydig cell numbers decline by 75% with the remaining cells concentrated around the rete and in the sub-capsular region. In the absence of Sertoli cells, peritubular myoid cell activity is reduced but the cells retain an ability to exclude immune cells from the seminiferous tubules. These data demonstrate that, in addition to support of spermatogenesis, Sertoli cells are required in the adult testis both for retention of the normal adult Leydig cell population and for support of normal peritubular myoid cell function. This has implications for our understanding of male reproductive disorders and wider androgen-related conditions affecting male health. PMID:25144714

  13. Numerical Relation of Spermatozoa to Sertoli Cells

    E-print Network

    Hague, Florence S.

    1914-01-01

    ization of light and even says that he has never been able to identify with certainty these crystals of Charcot. Besides the rod or crystal hoth authors describe rodlets or accessory strips (Batonnets), which Montgomery says persist longer, while... Sntw. Wirbeltiere, 1 Jena. Walker Sc Embleton 1906 On the origin of the Sertoli or Foot-oells of the Testis. University of Liverpool and Royal Infirmary Cancer Reseach Laboratories. First Report on the Cytological Investigation of Cancer. Watase...

  14. 4-Methylcatechol-induced cell damage in TM4 Sertoli cells.

    PubMed

    Li, Chunjin; Li, Wanhong; Chen, Shuxiong; Kang, Zhichen; Sun, Lina; Li, Hongjiao; Chen, Lu; Rao, Jiahui; Zhao, Yun; Yu, Jiaxin; Zhou, Xu

    2015-06-01

    4-Methylcatechol (4-MC) is one of the metabolites of quercetin, which is a potential drug for neuroprotection and tumorigenesis inhibition. This study was performed to investigate the cytotoxic effect of 4-MC in mouse TM4 Sertoli cells. TM4 Sertoli cell viability was significantly inhibited by 4-MC in a time- and dose-dependent manner. The number of apoptotic and dead cells was significantly increased after 4-MC treatment. Caspase 3 activity increased by prolonged exposure of TM4 Sertoli cells to 200??M 4-MC. The 4-MC significantly upregulated the mRNA level of Bax gene and considerably downregulated the Bcl-2 gene expression in a concentration-dependent manner. Results showed that 4-MC could induce TM4 Sertoli cell apoptosis, and the cytotoxic effect of 4-MC on TM4 Sertoli cells may be associated with upregulated Bax gene expression, which induced caspase cascade activation. PMID:25639863

  15. Canine hypertrophic osteoarthropathy associated with a malignant Sertoli cell tumour.

    PubMed

    Barrand, K R; Scudamore, C L

    2001-03-01

    A 15-year-old crossbred dog was presented with a severe cough of acute onset and an enlarged right testis. Symptomatic treatment for presumed 'kennel cough' failed to produce any improvement and at re-examination the dog had developed a swollen right forelimb. Radiographic examination suggested a diagnosis of hypertrophic pulmonary osteoarthropathy (Marie's disease) associated with pulmonary metastases from a testicular tumour. The dog was re-presented five days later with acute-onset severe vomiting and the owner elected for euthanasia. Necropsy was performed and histopathological assessment confirmed the presence of a Sertoli cell tumour in the right testis with multiple pulmonary and renal metastases. Hypertrophic pulmonary osteoarthropathy is a rare complication of metastatic canine Sertoli cell tumour. The authors know of no previously reported cases. PMID:11303857

  16. Thyroid hormone effects on androgen receptor messenger RNA expression in rat Sertoli and peritubular cells

    Microsoft Academic Search

    N K Arambepola; D Bunick; P S Cooke

    1998-01-01

    Postnatal Sertoli cell maturation is characterized by a pronounced rise in androgen receptor (AR) expression, which increases several fold between birth and adulthood. Since both 3,3*,5-triiodothyronine (T3) and FSH regulate Sertoli cell proliferation and diVerentiation, we have determined the eVects of T3 and FSH on AR mRNA expression in cultured Sertoli cells from 5-day-old rats. These cultures contain 5-9% peritubular

  17. Adjudin-mediated Sertoli-germ cell junction disassembly affects Sertoli cell barrier function in vitro and in vivo

    PubMed Central

    Su, Linlin; Cheng, C. Yan; Mruk, Dolores D.

    2010-01-01

    Adjudin, an analogue of lonidamine, affects adhesion between Sertoli and most germ cells, resulting in reversible infertility in rats, rabbits and dogs. Previous studies have described the apical ectoplasmic specialization, a hybrid-type of Sertoli cell–elongating/elongated spermatid adhesive junction, as a key target of adjudin. In this study, we ask if the function of the blood-testis barrier which is constituted by co-existing tight junctions, desmosome-gap junctions and basal ectoplasmic specializations can be maintained when the seminiferous epithelium is under assault by adjudin. We report herein that administration of a single oral dose of adjudin to adult rats increased the levels of several tight junction and basal ectoplasmic specialization proteins during germ cell loss from the seminiferous epithelium. These findings were corroborated by a functional in vitro experiment when Sertoli cells were cultured on Matrigel™-coated bicameral units in the presence of adjudin and transepithelial electrical resistance was quantified across the epithelium. Indeed, the Sertoli cell permeability barrier was shown to become tighter after adjudin treatment as evidenced by an increase in transepithelial electrical resistance. Equally important, the blood-testis barrier in adjudin-treated rats was shown to be intact 2 weeks post-treatment when its integrity was monitored following vascular administration of inulinfluorescein isothiocyanate which failed to permeate past the barrier and enter into the adluminal compartment. These results illustrate that a unique mechanism exists to maintain blood-testis barrier integrity at all costs, irrespective of the presence of germ cells in the seminiferous epithelium of the testis. PMID:20713173

  18. Aromatase expression in prepuberal Sertoli cells: effect of thyroid hormone.

    PubMed

    Andò, S; Sirianni, R; Forastieri, P; Casaburi, I; Lanzino, M; Rago, V; Giordano, F; Giordano, C; Carpino, A; Pezzi, V

    2001-06-10

    Aromatase activity has recently been assumed as a Sertoli cell functional maturation marker since it is maximally expressed in prepuberal age then it dramatically decreases at puberty and is virtually absent in adult age. Neonatal hypothyroidism is associated with a prolonged proliferation of Sertoli cells. This immature stage persists concomitantly with a dramatic enhancement of aromatase activity reversed by triiodothyronine (T3) either in vivo or in vitro administration. Therefore, in the present study, after immunolocalisation of aromatase in the cytoplasm of cultured Sertoli cells as well as in testis section, we investigate the regulatory effects of T3 in the same cells just at the age when aromatase activity is reported to be maximally expressed. In this aim, the effects of thyroid hormone have been evaluated in 2-weeks-old rats, in basal condition and upon stimulation with dibutyryl cyclic AMP [(Bu)(2)cAMP] by simultaneously analysing three functional levels of aromatase, mRNA expression; protein content; enzymatic activity. Western-blot analysis of Sertoli cell extracts revealed a protein, which co-migrated with a 55 kDa protein detected in human placenta used a positive control. The presence of a functional P450 aromatase protein in purified Sertoli cells was confirmed by the measurement [3H]H(2)O released after incubation with [1beta-(3)H]androst-4-3,17-dione. At the dose used, T3 down-regulates basal aromatase activity, while aromatase mRNA expression was apparently not inhibited. It is noteworthy that aromatase content pattern evaluated by Western blot analysis did not tightly parallel the aromatase activity pattern which clearly displays the inhibitory effects of T3, in basal condition ad upon (Bu)(2)cAMP stimulation, simulating FSH stimulation. The detection of mRNA altered transcript coding for putative protein lacking both aromatic and heme-binding regions upon T3 treatment and unable to convert androgens into estrogens, provides a reasonable explanation for the observed discrepancies between aromatase protein pattern, P450arom mRNA levels and aromatase activity. The authors conclude that although the altered transcript induced by prolonged exposure to T3 is a mechanism by which T3 may down regulate aromatase activity, it cannot be ruled out a direct effect of this hormone at the transcription levels since a recognisable emisite for potential TR(s) binding is located in the promoter region of aromatase gene. Thus a further investigation on T3 modulator role on aromatase gene promoter should be pursued even utilising higher doses of T3. PMID:11403889

  19. Effects of simulated microgravity on mouse Sertoli cells in culture

    NASA Astrophysics Data System (ADS)

    Angela, Masini Maria; Prato, Paola; Linda, Scarabelli; Lanza, Cristina; Palmero, Silvio; Pointis, Georges; Ricci, Franco; Strollo, Felice

    With the advent of space flights questions concerning the effects of microgravity (0xG) on hu-man reproduction physiology have got priority Spermatogenesis is a complex, highly ordered process of cell division and differentiation by which spermatogonial cells give rise to mature spermatozoa. Sertoli cells play a crucial role in the development of germ cells and the regulation of spermatogenesis. In this study the influence of 0xG on Sertoli cells was evaluated. A Sertoli cell line from mouse testis (42GPA9) was analyzed for cytoskeletal (using the 3D reconstruction generated from a stack of confocal images) and SHBG changes by immunohistochemistry, for antioxidant agents by RT-PCR and for culture medium lactate concentrations by wet chemistry. Cells were cultured for 6, 24 and 48 hrs on a three-dimensional Random Positioning Machine (3D-RPM); static controls (1xG) were positioned on the supporting frame. At the end of each experiment, cultured cells were either fixed in paraformaldehyde or RNA-extracted or used for culture medium lactate measurements as needed. At 0xG Sertoli cytoskeleton got disorganized, microtubules fragmented and SHBG undetectable already after 24 hrs, with alterations wors-ening further until 48 hrs; various antioxidant systems (SOD, GST, PARP, MTs) appreciably increased during the first 24 hrs but significantly decreased at 48 hrs. No changes occurred in 1xG samples. At least initially, 0xG seems to perturb antioxidant protection strategies allowing the testes to support sperm production, thus generating an aging-like state of oxidative stress. Lactate production at 0xG slightly decreased only after 24 hrs. Further experiments need to be carried out in space to investigate upon steroidogenesis and germ cell differentiation within the testis, to rule out eventually pending male infertility consequences, which would be a problem nowadays, when life expectancy increases and male fertility might become a social issue often extending into 60 years and over. (experiment funded by ASI, through a grant within the OSMA project).

  20. Proliferation and functional maturation of Sertoli cells, and their relevance to disorders of testis function in adulthood

    Microsoft Academic Search

    Richard M. Sharpe; Chris McKinnell; Catrina Kivlin; Jane S. Fisher

    2003-01-01

    Disorders of testicular function may have their origins in fetal or early life as a result of abnormal development or proliferation of Sertoli cells. Failure of Sertoli cells to mature, with consequent inability to express functions capable of supporting spermatogenesis, is ap rime example. In a similar way, failure of Sertoli cells to proliferate normally at the appropriate period in

  1. Maturational and hormonal influences on Sertoli cell function.

    PubMed

    Sanborn, B M; Wagle, J R; Steinberger, A; Greer-Emmert, D

    1986-04-01

    The maturation of Sertoli cell function was studied by observing properties of cells obtained from 15-, 25-, and 35-day-old rats after 3 days in culture gamma-Glutamyl transpeptidase activity, expressed per mg DNA or per mg protein, and total and soluble protein to DNA ratios increased with age. In contrast, lactate dehydrogenase activity was unchanged between 15 and 35 days of age. Secreted protein to DNA ratios and the secretion of lactate, androgen-binding protein (ABP), and transferrin per mg DNA also increased with age. Two-dimensional electrophoresis maps of [35S]methionine-labeled secretory proteins indicated the appearance of two specific bands [mol wt, 66,000; pI 6.0-6.8 (band 1); mol wt, 56,000; pI 5.3-6.0 (band 2)] between 15 and 35 days of age. The hormone dependence of these parameters was studied in Sertoli cells isolated from rats hypophysectomized at 20 days of age and subsequently treated with oil, testosterone propionate, or testosterone propionate plus FSH for 15-21 days. Hypophysectomy decreased total, soluble, and secreted protein to DNA ratios; hormone treatment in vivo increased these ratios compared to oil treatment. gamma-Glutamyl transpeptidase activity was significantly decreased by hypophysectomy and increased, compared to oil treatment, by hormone treatment. In contrast, lactate dehydrogenase activity per mg DNA was also decreased by hypophysectomy, but was unaffected by hormone treatment. [35S]Methionine incorporation into secreted protein and secretion of ABP and lactate per mg DNA were all decreased by hypophysectomy, whereas transferrin secretion per mg DNA was unaffected. While the hormone treatments increased ABP secretion, they had no effect on lactate or transferrin secretion, expressed per mg DNA. The [35S]methionine-labeled secretory proteins (bands 1 and 2) were not visible in two-dimensional electrophoresis maps after hypophysectomy of the donor animals. Treatment with testosterone propionate or testosterone propionate plus FSH in vivo resulted in the appearance of the acidic components of band 2. These data demonstrate that changes reflecting in vivo maturational patterns and hormonal influences on Sertoli cell function persist, at least in a relative sense, after 3 days in culture. Although the majority of [35S]methionine-labeled Sertoli cell cellular and secretory proteins were present under all conditions, maturation- and hormone-dependent changes in a number of specific functions were demonstrated. PMID:2868884

  2. Co-culture with Sertoli cells promotes proliferation and migration of umbilical cord mesenchymal stem cells

    SciTech Connect

    Zhang, Fenxi, E-mail: fxzhang0824@gmail.com [Department of Anatomy, Sanquan College, Xinxiang Medical University, Henan 453003, People's Republic of China (China)] [Department of Anatomy, Sanquan College, Xinxiang Medical University, Henan 453003, People's Republic of China (China); Hong, Yan; Liang, Wenmei [Department of Histology and Embryology, Guiyang Medical University, Guizhou 550004, People's Republic of China (China)] [Department of Histology and Embryology, Guiyang Medical University, Guizhou 550004, People's Republic of China (China); Ren, Tongming [Department of Anatomy, Sanquan College, Xinxiang Medical University, Henan 453003, People's Republic of China (China)] [Department of Anatomy, Sanquan College, Xinxiang Medical University, Henan 453003, People's Republic of China (China); Jing, Suhua [ICU Center, The Third Hospital of Xinxiang Medical University, Henan 453003, People's Republic of China (China)] [ICU Center, The Third Hospital of Xinxiang Medical University, Henan 453003, People's Republic of China (China); Lin, Juntang [Stem Cell Center, Xinxiang Medical University, Henan 453003, People's Republic of China (China)] [Stem Cell Center, Xinxiang Medical University, Henan 453003, People's Republic of China (China)

    2012-10-12

    Highlights: Black-Right-Pointing-Pointer Co-culture of Sertoli cells (SCs) with human umbilical cord mesenchymal stem cells (UCMSCs). Black-Right-Pointing-Pointer Presence of SCs dramatically increased proliferation and migration of UCMSCs. Black-Right-Pointing-Pointer Presence of SCs stimulated expression of Mdm2, Akt, CDC2, Cyclin D, CXCR4, MAPKs. -- Abstract: Human umbilical cord mesenchymal stem cells (hUCMSCs) have been recently used in transplant therapy. The proliferation and migration of MSCs are the determinants of the efficiency of MSC transplant therapy. Sertoli cells are a kind of 'nurse' cells that support the development of sperm cells. Recent studies show that Sertoli cells promote proliferation of endothelial cells and neural stem cells in co-culture. We hypothesized that co-culture of UCMSCs with Sertoli cells may also promote proliferation and migration of UCMSCs. To examine this hypothesis, we isolated UCMSCs from human cords and Sertoli cells from mouse testes, and co-cultured them using a Transwell system. We found that UCMSCs exhibited strong proliferation ability and potential to differentiate to other cell lineages such as osteocytes and adipocytes. The presence of Sertoli cells in co-culture significantly enhanced the proliferation and migration potential of UCMSCs (P < 0.01). Moreover, these phenotypic changes were accompanied with upregulation of multiple genes involved in cell proliferation and migration including phospho-Akt, Mdm2, phospho-CDC2, Cyclin D1, Cyclin D3 as well as CXCR4, phospho-p44 MAPK and phospho-p38 MAPK. These findings indicate that Sertoli cells boost UCMSC proliferation and migration potential.

  3. Formation of insulin-secreting, Sertoli-enriched tissue constructs by microgravity coculture of isolated pig islets and rat Sertoli cells

    Microsoft Academic Search

    Don F. Cameron; Joelle J. Hushen

    2001-01-01

    Summary  Pancreatic islets, isolated from neonatal pigs, and Sertoli cells, isolated from prepubertal rats, were cocultured in simulated\\u000a microgravity utilizing the NASA-developed highly accelerating, rotating vessel (HARV) biochamber. Following 5 d of incubation,\\u000a three-dimensional Sertoli-islet cell aggregates (SICA) retained the ability to secrete insulin when exposed to elevated glucose.\\u000a SICA contained FasL-positive Sertoli cells and insulin-positive ?-cells randomly organized within the

  4. Intracellular pH regulation in human Sertoli cells: role of membrane transporters

    Microsoft Academic Search

    P F Oliveira; M Sousa; A Barros; T Moura; A Rebelo da Costa; Abel Salazar

    2009-01-01

    Sertoli cells are responsible for regulating a wide range of processes that lead to the differentiation of male germ cells into spermatozoa. Intracellular pH (pHi) is an important parameter in cell physiology regulating namely cell metabolism and differentiation. However, pHi regulation mechanisms in Sertoli cells have not yet been systematically elucidated. In this work, pHi was determined in primary cultures

  5. RBPJ in mouse Sertoli cells is required for proper regulation of the testis stem cell niche.

    PubMed

    Garcia, Thomas Xavier; Farmaha, Jaspreet Kaur; Kow, Sean; Hofmann, Marie-Claude

    2014-12-01

    Stem cells are influenced by their surrounding microenvironment, or niche. In the testis, Sertoli cells are the key niche cells directing the population size and differentiation fate of spermatogonial stem cells (SSCs). Failure to properly regulate SSCs leads to infertility or germ cell hyperplasia. Several Sertoli cell-expressed genes, such as Gdnf and Cyp26b1, have been identified as being indispensable for the proper maintenance of SSCs in their niche, but the pathways that modulate their expression have not been identified. Although we have recently found that constitutively activating NOTCH signaling in Sertoli cells leads to premature differentiation of all prospermatogonia and sterility, suggesting that there is a crucial role for this pathway in the testis stem cell niche, a true physiological function of NOTCH signaling in Sertoli cells has not been demonstrated. To this end, we conditionally ablated recombination signal binding protein for immunoglobulin kappa J region (Rbpj), a crucial mediator of NOTCH signaling, in Sertoli cells using Amh-cre. Rbpj knockout mice had: significantly increased testis sizes; increased expression of niche factors, such as Gdnf and Cyp26b1; significant increases in the number of pre- and post-meiotic germ cells, including SSCs; and, in a significant proportion of mice, testicular failure and atrophy with tubule lithiasis, possibly due to these unsustainable increases in the number of germ cells. We also identified germ cells as the NOTCH ligand-expressing cells. We conclude that NOTCH signaling in Sertoli cells is required for proper regulation of the testis stem cell niche and is a potential feedback mechanism, based on germ cell input, that governs the expression of factors that control SSC proliferation and differentiation. PMID:25406395

  6. Regulation of spermatogonial stem cell self-renewal and spermatocyte meiosis by Sertoli cell signaling.

    PubMed

    Chen, Su-Ren; Liu, Yi-Xun

    2015-04-01

    Spermatogenesis is a continuous and productive process supported by the self-renewal and differentiation of spermatogonial stem cells (SSCs), which arise from undifferentiated precursors known as gonocytes and are strictly controlled in a special 'niche' microenvironment in the seminiferous tubules. Sertoli cells, the only somatic cell type in the tubules, directly interact with SSCs to control their proliferation and differentiation through the secretion of specific factors. Spermatocyte meiosis is another key step of spermatogenesis, which is regulated by Sertoli cells on the luminal side of the blood-testis barrier through paracrine signaling. In this review, we mainly focus on the role of Sertoli cells in the regulation of SSC self-renewal and spermatocyte meiosis, with particular emphasis on paracrine and endocrine-mediated signaling pathways. Sertoli cell growth factors, such as glial cell line-derived neurotrophic factor (GDNF) and fibroblast growth factor 2 (FGF2), as well as Sertoli cell transcription factors, such as ETS variant 5 (ERM; also known as ETV5), nociceptin, neuregulin 1 (NRG1), and androgen receptor (AR), have been identified as the most important upstream factors that regulate SSC self-renewal and spermatocyte meiosis. Other transcription factors and signaling pathways (GDNF-RET-GFRA1 signaling, FGF2-MAP2K1 signaling, CXCL12-CXCR4 signaling, CCL9-CCR1 signaling, FSH-nociceptin/OPRL1, retinoic acid/FSH-NRG/ERBB4, and AR/RB-ARID4A/ARID4B) are also addressed. PMID:25504872

  7. Structural characterization of proteoglycans produced by testicular peritubular cells and Sertoli cells

    SciTech Connect

    Skinner, M.K.; Fritz, I.B.

    1985-09-25

    The structural characteristics of proteoglycans produced by seminiferous peritubular cells and by Sertoli cells are defined. Peritubular cells secrete two proteoglycans designated PC I and PC II. PC I is a high molecular mass protein containing chondroitin glycosaminoglycan (GAG) chains (maximum 70 kDa). PC II has a protein core of 45 kDa and also contains chondroitin GAG chains (maximum 70 kDa). Preliminary results imply that PC II may be a degraded or processed form of PC I. Sertoli cells secrete two different proteoglycans, designated SC I and SC II. SC I is a large protein containing both chondroitin (maximum 62 kDa) and heparin (maximum 15 kDa) GAG chains. Results obtained suggest that this novel proteoglycan contains both chondroitin and heparin GAG chains bound to the same core protein. SC II has a 50-kDa protein core and contains chondroitin (maximum 25 kDa) GAG chains. A proteoglycan obtained from extracts of Sertoli cells is described which contains heparin (maximum 48 kDa) GAG chains. In addition, Sertoli cells secrete a sulfoprotein, SC III, which is not a proteoglycan. The stimulation by follicle-stimulating hormone of the incorporation of (TVS)SO2) U) into moieties secreted by Sertoli cells is shown to represent an increased production or sulfation of SC III, and not an increased production or sulfation of proteoglycans. Results are discussed in relation to the possible functions of proteoglycans in the seminiferous tubule.

  8. Xenotransplantation of neonatal porcine islets and Sertoli cells into nonimmunosuppressed streptozotocin-induced diabetic rats.

    PubMed

    Wang, D Z; Skinner, S; Elliot, R; Escobar, L; Salto-Tellez, M; Garkavenko, O; Khoo, A; Lee, K O; Calne, R; Isaac, J R

    2005-01-01

    The testis has been shown to be a privileged site for transplantation of allogenic islets in rodents, and the testicular cell aggregates are thought to confer this immunologic privilege. Recently, a group in Mexico reported transplantation of cocultured neonatal porcine islets and Sertoli cells resulting in insulin independence in nonimmunosuppressed type 1 diabetes patients. We have transplanted similar islets alone (naked islets) or cocultured islets with Sertoli cells (islet/Sertoli cells) into an omental site and other locations of nonimmunosuppressed, streptozotocin-induced diabetic male Sprague Dawley (SD) rats. Histologic examination showed viable neonatal porcine islets survived in xenografted rodents for at least 2 days, and some glucagon and inhibin stained cells appear to have survived for 4 days posttransplantation. However, histological examination did not demonstrate any difference in xenograft survival in the islets/Sertoli cells mixture compared to naked islets when transplanted into these nonimmunosuppressed diabetic rats. PMID:15808679

  9. The Warburg effect revisited--lesson from the Sertoli cell.

    PubMed

    Oliveira, Pedro F; Martins, Ana D; Moreira, Ana C; Cheng, C Yan; Alves, Marco G

    2015-01-01

    Otto Warburg observed that cancerous cells prefer fermentative instead of oxidative metabolism of glucose, although the former is in theory less efficient. Since Warburg's pioneering works, special attention has been given to this difference in cell metabolism. The Warburg effect has been implicated in cell transformation, immortalization, and proliferation during tumorigenesis. Cancer cells display enhanced glycolytic activity, which is correlated with high proliferation, and thus, glycolysis appears to be an excellent candidate to target cancer cells. Nevertheless, little attention has been given to noncancerous cells that exhibit a "Warburg-like" metabolism with slight, but perhaps crucial, alterations that may provide new directions to develop new and effective anticancer therapies. Within the testis, the somatic Sertoli cell (SC) presents several common metabolic features analogous to cancer cells, and a clear "Warburg-like" metabolism. Nevertheless, SCs actively proliferate only during a specific time period, ceasing to divide in most species after puberty, when they become terminally differentiated. The special metabolic features of SC, as well as progression from the immature but proliferative state, to the mature nonproliferative state, where a high glycolytic activity is maintained, make these cells unique and a good model to discuss new perspectives on the Warburg effect. Herein we provide new insight on how the somatic SC may be a source of new and exciting information concerning the Warburg effect and cell proliferation. PMID:25043918

  10. Malignant Sertoli cell tumor in the retained abdominal testis of a unilaterally cryptorchid horse.

    PubMed

    Pratt, Suzanne M; Stacy, Brian A; Whitcomb, Mary Beth; Vidal, Justin D; De Cock, Hilde E V; Wilson, W David

    2003-02-15

    A 13-year-old Morgan gelding was evaluated because of a mass in the caudal region of the abdomen. The horse had been presumed to be a gelding, but necropsy findings revealed a retained testis in the right retroperitoneal space. Histologically, the retained testis contained neoplastic cells; metastases were identified in the liver, spleen, lungs, and sublumbar lymph nodes. Immunohistochemical examination of the testis and metastatic tissues confirmed the diagnosis of malignant Sertoli cell tumor. Testicular neoplasms are infrequently reported in stallions. Seminomas are most commonly reported, whereas Sertoli cell tumors are considered to be rare. Typical biological behavior of Sertoli cell tumors in horses is unknown. To the authors' knowledge, there have been 2 reports of Sertoli cell tumors in horses; the tumors developed in descended testes, and 1 tumor was malignant. PMID:12597422

  11. Krüppel-like factor 4 is involved in functional differentiation of testicular Sertoli cells

    PubMed Central

    Godmann, Maren; Katz, Jonathan P.; Guillou, Florian; Simoni, Manuela; Kaestner, Klaus H.; Behr, Rüdiger

    2008-01-01

    Krüppel-like factor 4 (KLF4) is a pleiotropic zinc finger transcription factor that regulates genes being involved in differentiation and cell-cycle control. Knock out studies revealed a critical function for KLF4 in the terminal differentiation of many epithelial cells. In testicular Sertoli cells, Klf4 is strongly inducible by the glycoprotein Follicle stimulating hormone (FSH). Since KLF4 is essential for postnatal survival in mice, we deleted Klf4 specifically in Sertoli cells using the Cre/loxP system. Importantly, around postnatal day 18, a critical period of terminal Sertoli cell differentiation, mutant seminiferous tubules exhibited a disorganized germinal epithelium and delayed lumen formation. The ultrastructural finding of highly vacuolized Sertoli cell cytoplasm and the identification of differentially expressed genes, which are known to play roles during vesicle transport and fusion or for maintenance of the differentiated cell state, suggest impaired apical secretion of the Sertoli cell. Interestingly, a high proportion of all identified genes was localized in a small subregion of chromosome 7 suggesting coordinated regulation. Intriguingly, adult mutant mice are fertile and show normal testicular morphology, although the testosterone levels are decreased. In summary, KLF4 plays a significant role for proper and timely Sertoli cell differentiation in pubertal mice. PMID:18243172

  12. Ultrastructural study of crystalloids in Sertoli cells of the three-toed sloth ( Bradypus tridactylus )

    Microsoft Academic Search

    Yoshiro Toyama; Francisco Ureña Calderón; Rafael Quesada

    1990-01-01

    Summary Crystalloids were found in Sertoli cells of the testis of the three-toed sloth by examination at the lightand electron-microscopic levels. Needle-, or spindle-shaped crystalloids, varying in length, were located in the basal part of the Sertoli cells. They consisted of bundles of filaments each measuring ~ 11 nm in diameter. Several filaments were packed hexagonally to form a bundle.

  13. PERMEABILITY OF SERTOLI CELL TIGHT JUNCTIONS TO LANTHANUM AFTER LIGATION OF DUCTUS DEFERENS AND DUCTULI EFFERENTES

    PubMed Central

    Neaves, William B.

    1973-01-01

    The permeability of Sertoli cell tight junctions to lanthanum administered during fixation has been compared in rats after ligation of the ductus deferens and after ligation of the ductuli efferentes. In both control and vasoligated testes, lanthanum penetrated only short distances into the Sertoli cell tight junctions before stopping abruptly. The tight junction, consisting of numerous pentalaminar fusions of contiguous Sertoli cell membranes, prevented diffusion of lanthanum into the adluminal compartment of the seminiferous epithelium. In rats with ligated ductuli efferentes, lanthanum completely permeated many Sertoli cell tight junctions and occupied intercellular spaces of the adluminal compartment. In spite of their newly acquired permeability to lanthanum, tight junctions retained characteristic ultrastructural features, including numerous membrane fusions. When lanthanum-filled tight junctions were sectioned en face, membrane fusions appeared as pale lines in lakes of electron-opaque tracer. These linearly extensive fasciae occludentes occasionally ended blindly, suggesting that lanthanum may have traversed the junction by diffusing around such incomplete barriers. The increased permeability of Sertoli cell tight junctions after efferent ductule ligation, which caused rapid testicular weight gain followed by atrophy, indicates that tight junctions are sensitive to enforced retention of testicular secretions inside the seminiferous tubules. The apparent normalcy of Sertoli cell tight junctions after vasoligation, which had no effect on testis weight, supports the view that blockage of testicular secretions distal to the epididymis is relatively innocuous. PMID:4761331

  14. Depletion of the p43 Mitochondrial T3 Receptor Increases Sertoli Cell Proliferation in Mice

    PubMed Central

    Fumel, Betty; Roy, Stéphanie; Fouchécourt, Sophie; Livera, Gabriel; Parent, Anne-Simone; Casas, François; Guillou, Florian

    2013-01-01

    Among T3 receptors, TR?1 is ubiquitous and its deletion or a specific expression of a dominant-negative TR?1 isoform in Sertoli cell leads to an increase in testis weight and sperm production. The identification of a 43-kDa truncated form of the nuclear receptor TR?1 (p43) in the mitochondrial matrix led us to test the hypothesis that this mitochondrial transcription factor could regulate Sertoli cell proliferation. Here we report that p43 depletion in mice increases testis weight and sperm reserve. In addition, we found that p43 deletion increases Sertoli cell proliferation in postnatal testis at 3 days of development. Electron microscopy studies evidence an alteration of mitochondrial morphology observed specifically in Sertoli cells of p43?/? mice. Moreover, gene expression studies indicate that the lack of p43 in testis induced an alteration of the mitochondrial-nuclear cross-talk. In particular, the up-regulation of Cdk4 and c-myc pathway in p43?/? probably explain the extended proliferation recorded in Sertoli cells of these mice. Our finding suggests that T3 limits post-natal Sertoli cell proliferation mainly through its mitochondrial T3 receptor p43. PMID:24040148

  15. Age-dependent changes in the in-vitro response of a pig Sertoli cell-enriched population to FSH

    Microsoft Academic Search

    C. Monet-Kuntz; I. Fontaine

    1989-01-01

    Summary. The response of pig Sertoli cell-enriched cultures to FSH was investigated by measuring plasminogen activator (PA) secretion in culture, throughout the non- pubertal and prepubertal periods. Sertoli cell-enriched populations could be isolated from birth until a testicular weight of 56 g. FSH elicited a dose-dependent increase in PA secretion by pig Sertoli cell-enriched cultures. The ED50 was minimal for

  16. Reduced endogenous estrogen and hemicastration interact synergistically to increase porcine sertoli cell proliferation.

    PubMed

    Berger, Trish; Conley, Alan

    2014-05-01

    Both reduced endogenous estrogen and hemicastration stimulate proliferation of porcine Sertoli cells. The objective of these experiments was to compare the temporal patterns of response to each stimulus with the response to the combined stimuli as indications of shared or separate mechanisms. Within a replicate, one littermate was treated weekly with canola oil vehicle and remained intact; a second littermate was treated weekly with vehicle, and one testis was removed at Day 8; a third littermate was treated weekly with the aromatase inhibitor letrozole to reduce endogenous estrogens and remained intact; and the fourth littermate was treated weekly with letrozole, and one testis was removed at Day 8. Four replicates were evaluated at 2 wk of age, five replicates evaluated at 6.5 wk of age, and five replicates were evaluated at 11 wk of age, with treatment ceasing at 6 wk of age. Numbers of Sertoli cells were determined following GATA4 labeling using the optical dissector method. Levels of estradiol, estrogen conjugates, follicle-stimulating hormone (FSH), luteinizing hormone (LH), and inhibin were determined by radioimmunoassay. Hemicastration appeared to have a rapid effect on Sertoli cell proliferation, but letrozole treatment had no apparent effect on Sertoli cell numbers at 2 wk of age. Both letrozole treatment and hemicastration had stimulated Sertoli cell proliferation by 6.5 wk of age, although the magnitude of the hemicastration response was much greater. Letrozole appeared to have minimal interaction with hemicastration at this age. Letrozole and hemicastration together increased Sertoli cell numbers at 11 wk of age compared with either treatment alone. Estradiol and estrogen conjugates were dramatically reduced by aromatase inhibition as anticipated; treatment-induced changes in inhibin, LH, or FSH were minimal. Differences in timing of response and positive interaction at 11 wk of age suggest that hemicastration and letrozole stimulate proliferation of Sertoli cells by two initially different pathways. PMID:24740600

  17. Receptors and signaling pathways involved in proliferation and differentiation of Sertoli cells

    PubMed Central

    Lucas, Thaís FG; Nascimento, Aline R; Pisolato, Raisa; Pimenta, Maristela T; Lazari, Maria Fatima M; Porto, Catarina S

    2014-01-01

    The identification of the hormones and other factors regulating Sertoli cell survival, proliferation, and maturation in neonatal, peripubertal, and pubertal life remains one of the most critical questions in testicular biology. The regulation of Sertoli cell proliferation and differentiation is thought to be controlled by cell–cell junctions and a set of circulating and local hormones and growth factors. In this review, we will focus on receptors and intracellular signaling pathways activated by androgen, follicle-stimulating hormone, thyroid hormone, activin, retinoids, insulin, insulin-like growth factor, relaxin, and estrogen, with special emphasis on estrogen receptors. Estrogen receptors activate intracellular signaling pathways that converge on cell cycle and transcription factors and play a role in the regulation of Sertoli cell proliferation and differentiation. PMID:25225624

  18. Disruption of Sertoli cell vimentin filaments in prepubertal rats: an acute effect of butylparaben in vivo and in vitro.

    PubMed

    Alam, Mohammad Shah; Kurohmaru, Masamichi

    2014-06-01

    Parabens are p-hydroxybenzoic acid ester compounds widely used as preservatives in foods, cosmetics, toiletries and pharmaceuticals. We have recently shown that butylparaben induces spermatogenic cell apoptosis in prepubertal rats. We have conducted the present study for further information. Three-week-old male Sprague-Dawley rats (n=8) were given a single oral dose of 1,000 mg/kg butylparaben. The rats were sacrificed under anesthesia at 3, 6 and 24h after administration and their testes were collected for histopathological and immunohistochemical examination. Results showed a gradual collapse of Sertoli cell vimentin filaments and decreased actin staining intensity without accompanying changes in the pattern of tubulin expression, while spermatogenic cells became separated from the basement membrane and sloughed into the lumen in the butylparaben-treated rats, compared to the controls. To determine the direct effects of butylparaben on Sertoli cells, primary Sertoli cell cultures with and without butylparaben treatment were examined. Toluidine blue staining in butylparaben treated-cultured Sertoli cells showed an increased number and size of vacuoles in their cytoplasm. In agreement with the in vivo experiment, the in vitro study also clearly demonstrated disruption of vimentin filaments in Sertoli cells after butylparaben treatment. Considering both our present and previous reports, we can speculate that butylparaben-induced disruption of Sertoli cell vimentin filaments may lead to precocious release of spermatogenic cells from underlying Sertoli cells, and the released cells may undergo apoptosis owing to loss of support provided by the Sertoli cells. PMID:24444665

  19. Roles of miRNAs in microcystin-LR-induced Sertoli cell toxicity.

    PubMed

    Zhou, Yuan; Wang, Hui; Wang, Cong; Qiu, Xuefeng; Benson, Mikael; Yin, Xiaoqin; Xiang, Zou; Li, Dongmei; Han, Xiaodong

    2015-08-15

    Microcystin (MC)-LR, a cyclic heptapeptide, is a potent reproductive system toxin. To understand the molecular mechanisms of MC-induced reproductive system cytotoxicity, we evaluated global changes of miRNA and mRNA expression in mouse Sertoli cells following MC-LR treatment. Our results revealed that the exposure to MC-LR resulted in an altered miRNA expression profile that might be responsible for the modulation of mRNA expression. Bio-functional analysis indicated that the altered genes were involved in specific cellular processes, including cell death and proliferation. Target gene analysis suggested that junction injury in Sertoli cells exposed to MC-LR might be mediated by miRNAs through the regulation of the Sertoli cell-Sertoli cell pathway. Collectively, these findings may enhance our understanding on the modes of action of MC-LR on mouse Sertoli cells as well as the molecular mechanisms underlying the toxicity of MC-LR on the male reproductive system. PMID:25986756

  20. Altered protein prenylation in Sertoli cells is associated with adult infertility resulting from childhood mumps infection

    PubMed Central

    Wang, Xiu-Xing; Ying, Pu; Diao, Fan; Wang, Qiang; Ye, Dan; Jiang, Chen; Shen, Ning; Xu, Na; Chen, Wei-Bo; Lai, Shan-Shan; Jiang, Shan; Miao, Xiao-Li; Feng, Jin; Tao, Wei-Wei; Zhao, Ning-Wei; Yao, Bing; Xu, Zhi-Peng; Sun, Hai-Xiang; Sha, Jia-Hao; Huang, Xing-Xu; Shi, Qing-Hua; Tang, Hong

    2013-01-01

    Mumps commonly affects children 5–9 yr of age, and can lead to permanent adult sterility in certain cases. However, the etiology of this long-term effect remains unclear. Mumps infection results in progressive degeneration of the seminiferous epithelium and, occasionally, Sertoli cell–only syndrome. Thus, the remaining Sertoli cells may be critical to spermatogenesis recovery after orchitis healing. Here, we report that the protein farnesylation/geranylgeranylation balance is critical for patients’ fertility. The expression of geranylgeranyl diphosphate synthase 1 (GGPPS) was decreased due to elevated promoter methylation in the testes of infertile patients with mumps infection history. When we deleted GGPPS in mouse Sertoli cells, these cells remained intact, whereas the adjacent spermatogonia significantly decreased after the fifth postnatal day. The proinflammatory MAPK and NF-?B signaling pathways were constitutively activated in GGPPS?/? Sertoli cells due to the enhanced farnesylation of H-Ras. GGPPS?/? Sertoli cells secreted an array of cytokines to stimulate spermatogonia apoptosis, and chemokines to induce macrophage invasion into the seminiferous tubules. Invaded macrophages further blocked spermatogonia development, resulting in a long-term effect through to adulthood. Notably, this defect could be rescued by GGPP administration in EMCV-challenged mice. Our results suggest a novel mechanism by which mumps infection during childhood results in adult sterility. PMID:23825187

  1. E-Box and Cyclic Adenosine Monophosphate Response Elements Are Both Required for Follicle-Stimulating Hormone-Induced Transferrin Promoter Activation in Sertoli Cells

    Microsoft Academic Search

    JAIDEEP CHAUDHARY; MICHAEL K. SKINNER

    1999-01-01

    Sertoli cells are the epithelial cells responsible for the onset of pubertal development and maintenance of spermatogenesis in the adult. Transferrin is one of the major secretory products expressed by differentiated Sertoli cells. Investigation of the transcriptional control of transferrin gene expression provides insight into the regulation of Sertoli cell differentiation. Analysis of the mouse transferrin (mTf) promoter reveals the

  2. Thyroid hormone, retinoic acid, and testosterone suppress proliferation and induce markers of differentiation in cultured rat sertoli cells.

    PubMed

    Buzzard, Jeremy J; Wreford, Nigel G; Morrison, John R

    2003-09-01

    This study uses a high purity cell culture system to extend previous observations of factors controlling the end of the Sertoli cell proliferative phase. Thyroid hormone, retinoic acid, and testosterone were assessed for their ability to halt the proliferative phase and regulate the expression of markers associated with maturation of the Sertoli cell. We show that these hormones share similar suppressive effects on the rate of Sertoli cell division without any apparent additive effects. We demonstrate that these hormones induce the progressive accumulation of cell cycle inhibitors p27Kip1 and p21Cip1 in Sertoli cells, a likely regulatory mechanism controlling the suppression of proliferation. We used real-time RT-PCR to examine the effects of these factors on the expression of mRNA encoding the Id proteins, demonstrating an increase in Id2 and Id3 expression in Sertoli cells treated with thyroid hormone, retinoic acid, or testosterone. Finally, we examined the expression of a number of genes that have been implicated in the Sertoli cell differentiation process. Our results suggest that these hormones can induce aspects of Sertoli cell differentiation in vitro, providing a valuable in vitro model for studying Sertoli cell function. PMID:12933640

  3. Selective Ablation of the Androgen Receptor in Mouse Sertoli Cells Affects Sertoli Cell Maturation, Barrier Formation and Cytoskeletal Development

    PubMed Central

    Willems, Ariane; Batlouni, Sergio R.; Esnal, Arantza; Swinnen, Johannes V.; Saunders, Philippa T. K.; Sharpe, Richard M.; França, Luiz R.; De Gendt, Karel; Verhoeven, Guido

    2010-01-01

    The observation that mice with a selective ablation of the androgen receptor (AR) in Sertoli cells (SC) (SCARKO mice) display a complete block in meiosis supports the contention that SC play a pivotal role in the control of germ cell development by androgens. To delineate the physiological and molecular mechanism responsible for this control, we compared tubular development in pubertal SCARKO mice and littermate controls. Particular attention was paid to differences in SC maturation, SC barrier formation and cytoskeletal organization and to the molecular mediators potentially involved. Functional analysis of SC barrier development by hypertonic perfusion and lanthanum permeation techniques and immunohistochemical analysis of junction formation showed that SCARKO mice still attempt to produce a barrier separating basal and adluminal compartment but that barrier formation is delayed and defective. Defective barrier formation was accompanied by disturbances in SC nuclear maturation (immature shape, absence of prominent, tripartite nucleoli) and SC polarization (aberrant positioning of SC nuclei and cytoskeletal elements such as vimentin). Quantitative RT-PCR was used to study the transcript levels of genes potentially related to the described phenomena between day 8 and 35. Differences in the expression of SC genes known to play a role in junction formation could be shown from day 8 for Cldn11, from day 15 for Cldn3 and Espn, from day 20 for Cdh2 and Jam3 and from day 35 for ZO-1. Marked differences were also noted in the transcript levels of several genes that are also related to cell adhesion and cytoskeletal dynamics but that have not yet been studied in SC (Actn3, Ank3, Anxa9, Scin, Emb, Mpzl2). It is concluded that absence of a functional AR in SC impedes the remodeling of testicular tubules expected at the onset of spermatogenesis and interferes with the creation of the specific environment needed for germ cell development. PMID:21152390

  4. SOX9 is up-regulated by the transient expression of SRY specifically in Sertoli cell precursors

    Microsoft Academic Search

    Ryohei Sekido; Isabelle Bar; Véronica Narváez; Graeme Penny; Robin Lovell-Badge

    2004-01-01

    The Y chromosome gene Sry encodes a transcription factor required to initiate testis development. The related autosomal gene Sox9 is up-regulated shortly after the onset of Sry transcription and is thought essential for the differentiation of Sertoli cells. The lineage that gives rise to Sertoli cells has its origins within the coelomic epithelium (CE) of the genital ridge, but from

  5. Lipopolysaccharide inhibits the self-renewal of spermatogonial stem cells in vitro via downregulation of GDNF expression in Sertoli cells.

    PubMed

    Zhang, Xiaoli; Shi, Kun; Li, Yi; Zhang, Haiyu; Hao, Jing

    2014-06-01

    Lipopolysaccharide (LPS) can reduce sperm count and sperm quality. The molecular mechanisms underlying this process are not fully understood. In this report, we investigated the effects of LPS-treated Sertoli cells on self-renewal and differentiation of spermatogoinial stem cells (SSCs). Sertoli cell cultures were established and incubated with LPS (10?g/ml) for 1, 2 or 3 days, respectively. The culture media were collected and used as conditioned media (CM) to culture SSCs. The expression of glial cell-derived neurotrophic factor (GDNF), stem cell factor (SCF) and bone morphogenetic protein 4 (BMP4) in Sertoli cells treated with LPS was analyzed by RT-PCR and Western blotting. The results showed that the expression of SSC differentiation markers, c-kit and Sohlh2, was increased, while the expression of SSC self-renewal markers, plzf, oct4, and PCNA, was repressed when cultured in CM from LPS-treated Sertoli cells. GDNF levels in Sertoli cells and CM reduced dramatically after LPS treatments, while SCF and BMP4 levels did not show any significant changes. Moreover, correlated with the GDNF levels in CM, GDNF target genes, Bcl6b and Etv5, were reduced markedly in SSCs. Our results suggest that LPS inhibits the expression of GDNF in Sertoli cells, and might prevent the SSC self-renewal via down-regulation of GDNF target genes. PMID:24503145

  6. Specific deficiency of Plzf paralog, Zbtb20, in Sertoli cells does not affect spermatogenesis and fertility in mice.

    PubMed

    Jiang, Xiaohua; Zhang, Huan; Yin, Shi; Zhang, Yuanwei; Yang, Weimei; Zheng, Wei; Wang, Liu; Wang, Zheng; Bukhari, Ihtisham; Cooke, Howard J; Iqbal, Furhan; Shi, Qinghua

    2014-01-01

    Ztbt20 is a POK family transcription factor and primarily functions through its conserved C2H2 Krüppel type zinc finger and BTB/POZ domains. The present study was designed to define the function of the Zbtb20, in vivo, during mouse spermatogenesis. Immunohistochemical studies revealed that ZBTB20 protein was localized specifically in the nuclei of Sertoli cells in seminiferous tubules. To investigate its role during spermatogenesis, we crossed Amh-Cre transgenic mice with Zbtb20 floxp mice to generate conditionally knockout mice (cKO) in which Zbtb20 was specifically deleted in Sertoli cells. The cKO mice were fertile and did not show any detectable abnormalities in spermatogenesis. Taken together, though specific deletion of transcription factor Zbtb20 in Sertoli cells has no apparent influence on spermatogenesis, its specific localization in Sertoli cells makes Zbtb20 a useful marker for the identification of Sertoli cells in seminiferous tubules. PMID:25395169

  7. Specific deficiency of Plzf paralog, Zbtb20, in Sertoli cells does not affect spermatogenesis and fertility in mice

    PubMed Central

    Jiang, Xiaohua; Zhang, Huan; Yin, Shi; Zhang, Yuanwei; Yang, Weimei; Zheng, Wei; Wang, Liu; Wang, Zheng; Bukhari, Ihtisham; Cooke, Howard J.; Iqbal, Furhan; Shi, Qinghua

    2014-01-01

    Ztbt20 is a POK family transcription factor and primarily functions through its conserved C2H2 Krüppel type zinc finger and BTB/POZ domains. The present study was designed to define the function of the Zbtb20, in vivo, during mouse spermatogenesis. Immunohistochemical studies revealed that ZBTB20 protein was localized specifically in the nuclei of Sertoli cells in seminiferous tubules. To investigate its role during spermatogenesis, we crossed Amh-Cre transgenic mice with Zbtb20 floxp mice to generate conditionally knockout mice (cKO) in which Zbtb20 was specifically deleted in Sertoli cells. The cKO mice were fertile and did not show any detectable abnormalities in spermatogenesis. Taken together, though specific deletion of transcription factor Zbtb20 in Sertoli cells has no apparent influence on spermatogenesis, its specific localization in Sertoli cells makes Zbtb20 a useful marker for the identification of Sertoli cells in seminiferous tubules. PMID:25395169

  8. Characteristics of retinol accumulation from serum retinol-binding protein by cultured sertoli cells

    SciTech Connect

    Shingleton, J.L.; Skinner, M.K.; Ong, D.E. (Vanderbilt Univ. School of Medicine, Nashville, TN (USA))

    1989-12-12

    The uptake of retinol was examined in cultured Sertoli cells when retinol was provided as a complex with the transport protein retinol-binding protein (RBP). Sertoil cells accumulated ({sup 3}H)retinol in a time- and temperature-dependent manner. The change in rate of retinol accumulation occurred when the cells had accumulated approximately 0.53 pmol of retinol/{mu}g of cellular DNA. Extraction and HPLC analysis of the cell-associated radioactivity yielded retinol and retinyl esters, indicating that a significant proportion of the accumulated retinol was esterified. Excess unlabeled retinol-RBP competed with ({sup 3}H)retinol-RBP for ({sup 3}H)retinol delivery to the cells, indicating that RBP delivery of retinol was a saturable and competable process. However, free ({sup 3}H)retinol associated with Sertoli cells in a noncompetable manner. The transport constant for specific retinol accumulation from RBP was 3.0 {mu}M. Neither iodinated nor reductively methylated RBP was accumulated by or tightly bound to Sertoli cells. Competition studies indicated, however, that protein recognition is important in the retinol uptake process. RBP, CRBP, and CRBP(II) competed with ({sup 3}H)retinol-RBP for ({sup 3}H)retinol accumulation, but free retinol, retinol-bovine serum albumin, and retinol-{beta}-lactoglobulin did not. These studies indicated that Sertoli cell uptake of retinol involved recognition of the retinol-RBP complex at the cell surface with subsequent internalization of retinol, but not RBP.

  9. Nesprin-3 connects plectin and vimentin to the nuclear envelope of Sertoli cells but is not required for Sertoli cell function in spermatogenesis

    PubMed Central

    Ketema, Mirjam; Kreft, Maaike; Secades, Pablo; Janssen, Hans; Sonnenberg, Arnoud

    2013-01-01

    Nesprin-3 is a nuclear envelope protein that connects the nucleus to intermediate filaments by interacting with plectin. To investigate the role of nesprin-3 in the perinuclear localization of plectin, we generated nesprin-3–knockout mice and examined the effects of nesprin-3 deficiency in different cell types and tissues. Nesprin-3 and plectin are coexpressed in a variety of tissues, including peripheral nerve and muscle. The expression level of nesprin-3 in skeletal muscle is very low and decreases during myoblast differentiation in vitro. Of interest, plectin was concentrated at the nuclear envelope in only a few cell types. This was most prominent in Sertoli cells of the testis, in which nesprin-3 is required for the localization of both plectin and vimentin at the nuclear perimeter. Testicular morphology and the position of the nucleus in Sertoli cells were normal, however, in the nesprin-3–knockout mice and the mice were fertile. Furthermore, nesprin-3 was not required for the polarization and migration of mouse embryonic fibroblasts. Thus, although nesprin-3 is critical for the localization of plectin to the nuclear perimeter of Sertoli cells, the resulting link between the nuclear envelope and the intermediate filament system seems to be dispensable for normal testicular morphology and spermatogenesis. PMID:23761073

  10. Development of Sertoli cell populations in organ culture of immature pig testis

    E-print Network

    Boyer, Edmond

    Development of Sertoli cell populations in organ culture of immature pig testis Michelle CHEVALIER 45, 63170 Aubière, France. Summary. Small samples of porcine testis were maintained in organ culture in the testis is the FSH target cell (Steinberger and Steinberger, 1977 ; Means, 1977 ; Hansson et al., 1978

  11. Androgen-Dependent Sertoli Cell Tight Junction Remodeling Is Mediated by Multiple Tight Junction Components

    PubMed Central

    Chakraborty, Papia; William Buaas, F.; Sharma, Manju; Smith, Benjamin E.; Greenlee, Anne R.; Eacker, Stephen M.

    2014-01-01

    Sertoli cell tight junctions (SCTJs) of the seminiferous epithelium create a specialized microenvironment in the testis to aid differentiation of spermatocytes and spermatids from spermatogonial stem cells. SCTJs must be chronically broken and rebuilt with high fidelity to allow the transmigration of preleptotene spermatocytes from the basal to adluminal epithelial compartment. Impairment of androgen signaling in Sertoli cells perturbs SCTJ remodeling. Claudin (CLDN) 3, a tight junction component under androgen regulation, localizes to newly forming SCTJs and is absent in Sertoli cell androgen receptor knockout (SCARKO) mice. We show here that Cldn3-null mice do not phenocopy SCARKO mice: Cldn3?/? mice are fertile, show uninterrupted spermatogenesis, and exhibit fully functional SCTJs based on imaging and small molecule tracer analyses, suggesting that other androgen-regulated genes must contribute to the SCARKO phenotype. To further investigate the SCTJ phenotype observed in SCARKO mutants, we generated a new SCARKO model and extensively analyzed the expression of other tight junction components. In addition to Cldn3, we identified altered expression of several other SCTJ molecules, including down-regulation of Cldn13 and a noncanonical tight junction protein 2 isoform (Tjp2iso3). Chromatin immunoprecipitation was used to demonstrate direct androgen receptor binding to regions of these target genes. Furthermore, we demonstrated that CLDN13 is a constituent of SCTJs and that TJP2iso3 colocalizes with tricellulin, a constituent of tricellular junctions, underscoring the importance of androgen signaling in the regulation of both bicellular and tricellular Sertoli cell tight junctions. PMID:24825397

  12. Thyroid hormone modulates androgen and oestrogen receptor content in the Sertoli cells of peripubertal rats.

    PubMed

    Panno, M L; Sisci, D; Salerno, M; Lanzino, M; Pezzi, V; Morrone, E G; Mauro, L; Palmero, S; Fugassa, E; Andò, S

    1996-01-01

    A possible role of tri-iodothyronine (T3) on the interplay between testicular steroids and Sertoli cells has been investigated on the basis of previous findings demonstrating a direct inhibitory influence of T3 on aromatase activity and oestradiol production in peripuberal Sertoli cells. In this context, the present study was focused on the effects of T3 on oestrogen receptor (ER) and androgen receptor (AR) contents in the cytosol and nucleus of Sertoli cells isolated from 2-, 3- and 4-week-old euthyroid, hypothyroid and hypothyroid treated rats. Hypothyroidism was induced by the oral administration of 0.025% methimazole (MMI) from birth until the rats were killed at 2, 3 and 4 weeks of age. Half of the MMI-treated animals were injected i.p. with L-tri-iodothyronine (T3; 3 micrograms/100 g body weight) during the last week before death. Sertoli cells from all groups were initially cultured under basal conditions for the first 24 h and subsequently in the presence of testosterone with or without T3 for an additional 24 h. Hypothyroidism was associated with severe impairment of body as well as testicular growth. Euthyroid ERs showed an elevated Kd (0.76 nM) which was similar in the different age groups investigated. The in vitro addition of T3 or testosterone induced a decrease in ER content and this decrease was greater after exposure to both hormones. In 2- and 3-week-old hypothyroid rats, ER content was markedly increased and was reversed in euthyroid rats when T3 was given in vivo. When ERs were assayed in the Sertoli cell nucleus and cytoplasm of 2- and 3-week-old animals, a strong relationship in ER content in the two cellular compartments was observed. Neither of the hormones tested seemed to affect the AR content in the nucleus significantly, while the in vitro addition of testosterone or T3 or both hormones together augmented the ARs in the cytosol to a greater extent, resulting in an increase in their total (cytosolic and nuclear) content in the cells. The present data suggest that T3 down-regulates ERs and up-regulates ARs in peripuberal Sertoli cells. The additive effect of testosterone and T3 in up-regulating ARs could possibly involve a role for T3 in influencing the androgen responsiveness of the Sertoli cells during spermatogenesis. PMID:8568470

  13. Variations in the plasma levels of gonadotrophin and testosterone and in Leydig and Sertoli cell populations

    E-print Network

    Boyer, Edmond

    . Despite these endocrinological differences, testis weight, total Sertoli and Leydig cell numbers per testis, total number of gonocytes per testis at birth and daily production of round spermatids per testis spring and autumn-born Romanov lambs. The cellular composition of the testis at birth and in adult

  14. Pem Homeobox Gene Promoter Sequences that Direct Transcription in a Sertoli Cell-Specific, Stage-

    E-print Network

    Wilkinson, Miles F.

    - Specific, and Androgen-Dependent Manner in the Testis in Vivo MANJEET K. RAO, CHAD M. WAYNE, MARVIN L Sertoli cell-specific expression in the postnatal and adult testis in vivo have not been identified in the testis and the epididymis but not in any other tissues tested. In the adult testis, this promoter frag

  15. Control of Sertoli and germ cell populations in the cock and sheep testes

    E-print Network

    Paris-Sud XI, Université de

    Control of Sertoli and germ cell populations in the cock and sheep testes M. de REVIERS, Marie in the cock and ram testes to determine their controlling factors : initial number, endocrine environment to the natural environment and hemicastrated as impuberal when 6 weeks old. They were compared to normal animals

  16. Bilateral retiform variant of sertoli leydig cell tumour of ovary: An uncommon tumor with review of literature.

    PubMed

    Rathi, Monika; Budania, Satish Kumar; Khalid, Mohammad; Mittal, Ankur

    2015-01-01

    Sertoli-leydig cell tumors are the uncommon sex-cord stromal tumors of the ovary. We report a case of 42-year-old female with retiform variant of sertoli-leydig cell tumour. She presented with the complaint of mass in abdomen for 7 years. Ultrasound revealed bilateral ovarian mass suggestive of malignancy. Bilateral salpingo-oopherectomy with surgical staging was done. The tumor was diagnosed as stage I retiform variant of sertoli-leydig cell tumor on histopathology and immunohistochemistry. PMID:25861207

  17. Bilateral retiform variant of sertoli leydig cell tumour of ovary: An uncommon tumor with review of literature

    PubMed Central

    Rathi, Monika; Budania, Satish Kumar; Khalid, Mohammad; Mittal, Ankur

    2015-01-01

    Sertoli-leydig cell tumors are the uncommon sex-cord stromal tumors of the ovary. We report a case of 42-year-old female with retiform variant of sertoli-leydig cell tumour. She presented with the complaint of mass in abdomen for 7 years. Ultrasound revealed bilateral ovarian mass suggestive of malignancy. Bilateral salpingo-oopherectomy with surgical staging was done. The tumor was diagnosed as stage I retiform variant of sertoli-leydig cell tumor on histopathology and immunohistochemistry. PMID:25861207

  18. Condensation behavior of the human x chromosome in male germ cells and Sertoli cells examined by flourescence in situ hybridisation

    SciTech Connect

    Kofman-Alfaro, S.; Cervantes, A. [Servicio de Genetica (Mexico); Speed, R.M. [WGH, Edinburgh (United Kingdom)] [and others

    1994-09-01

    The chromatin condensation behavior of the human x chromosome has been studied by FISH analysis in germ cells and Sertoli cells of the adult testes. Comparisons are made with previous findings for the human Y chromosome and for chromosome 7. In meiotic prophase, the X chromosome can be seen to extend greatly at zygotene and to contract through pachytene into the sex vesicle. Such extension, which has also been noted for the human Y chromosome at this state of meiosis, could be a prerequisite for XY pairing crossing-over. In patients with {open_quotes}Sertoli-cell-only{close_quotes} syndrome, the sex chromosomes, by in situ hybridization analysis, appear extremely contracted compared with their normal extended state seen in adult Sertoli cells of fertile men. By contrast, the state of expansion of chromosome 7 in Sertoli cells appears identical for sterile and fertile testes. This could suggest an association between gene-controlled germ cell losses and failure of expansion of the sex chromosome axes. The variable patterns of extension and contraction for the X and Y chromosome axes in germ cells and Sertoli cells might provide underlying clues to pattern of expression noted for sex-linked genes in the human testis.

  19. Specific deletion of Cdh2 in Sertoli cells leads to altered meiotic progression and subfertility of mice.

    PubMed

    Jiang, Xiaohua; Ma, Tieliang; Zhang, Yuanwei; Zhang, Huan; Yin, Shi; Zheng, Wei; Wang, Liu; Wang, Zheng; Khan, Manan; Sheikh, Salma W; Bukhari, Ihtisham; Iqbal, Furhan; Cooke, Howard J; Shi, Qinghua

    2015-03-01

    CDH2 (cadherin 2, Neural-cadherin, or N-cadherin) is the predominant protein of testicular basal ectoplasmic specializations (basal ES; a testis-specific type of adhesion junction), one of the major cell junctions composing the blood-testis barrier (BTB). The BTB is found between adjacent Sertoli cells in seminiferous tubules, which divides the tubules into basal and adluminal compartments and prevents the deleterious exchange of macromolecules between blood and seminiferous tubules. However, the exact roles of basal ES protein CDH2 in BTB function and spermatogenesis is still unknown. We thus generated mice with Cdh2 specifically knocked out in Sertoli cells by crossing Cdh2 loxP mice with Amh-Cre mice. Cdh2 deletion in Sertoli cells did not affect Sertoli cell counts, but led to compromised BTB function, delayed meiotic progression from prophase to metaphase I in testes, increased germ cell apoptosis, sloughing of meiotic cells, and, subsequently, reduced sperm counts in epididymides and subfertility of mice. However, the testes with Cdh2-specific deletion in germ cells did not show any difference from the normal control testes, and phenotypes observed in Sertoli cell and germ cell Cdh2 double-knockout mice were indistinguishable from those in mice with Cdh2 specifically knocked out only in Sertoli cells. Taken together, our data demonstrate that the adhesion junction component, Cdh2, functions just in Sertoli cells, but not in germ cells during spermatogenesis, and is essential for the integrity of BTB function, its deletion in Sertoli cells would lead to the BTB damage and subsequently meiosis and spermatogenesis failure. PMID:25631347

  20. Sertoli cell death by apoptosis in the immature rat testis following x-irradiation

    SciTech Connect

    Allan, D.J.; Gobe, G.C.; Harmon, B.V.

    1988-03-01

    The importance of the morphological study of cell death has recently been emphasized by the recognition that the ultrastructural features of dying cells allow categorization of the death as either apoptosis or necrosis. This classification enables inferences to be drawn about the mechanism and biological significance of the death occurring in a particular set of circumstances. In this study, Sertoli cell death induced in the immature testis of three and four day old rats by 5 Gy (500 rads) x-irradiation was described by light and transmission electron microscopy with the objective of categorizing the death as apoptosis or necrosis. The testes were examined 1, 2, 3, 4, 8, and 24 h after irradiation. Following irradiation, there was a wave of apoptosis of the Sertoli cells starting in three to four hours and reaching a peak between four and eight hours. At 24 hours, only 61% of the expected number of Sertoli cells remained. These findings are in accord with recent ultrastructural reports that ionizing radiation induces cell death by apoptosis in rapidly proliferating cell populations. New insights into the pathogenesis of radiation-induced cell death might thus be expected to stem from future elucidation of the general molecular events involved in triggering apoptosis.

  1. Bilateral Sertoli-Leydig Cell Tumor in a Primigravida: A Rare Case

    PubMed Central

    Tyagi, Ruchita; Agrawal, Parimal; Nijhawan, Raje; Prasad, GRV

    2014-01-01

    We present a unique case of incidentally discovered bilateral Sertoli Leydig cell tumour in a primigravida who displayed no features of virilization. The apha fetoprotein levels were elevated. Magnetic resonance imaging was suggestive of ovarian tumors, possibly germ cell tumor. Bilateral salpingo-oophorectomy was performed and histopathology showed features of Sertoli Leydig cell tumor with intermediate to poor differentiation. Immunohistochemistry was positive for calretinin and inhibin, while cytokeratin was negative. Four courses of bleomycin-, etoposide- and cisplatin-based chemotherapy regimen was started, but the patient aborted while receiving the second cycle of chemotherapy. She received the remaining two cycles of chemotherapy and is now on close follow up with monitoring of serum inhibin levels to detect any tumor recurrence. Bilateral Sertloli Leydig cell tumor has not been reported previously in a pregnant female. The aim of this article is to describe the clinical, radiological and pathological features and management of this rare entity. PMID:25002956

  2. Lycopene supplementation prevents reactive oxygen species mediated apoptosis in Sertoli cells of adult albino rats exposed to polychlorinated biphenyls

    PubMed Central

    Krishnamoorthy, Gunasekaran; Selvakumar, Kandaswamy; Venkataraman, Prabhu; Elumalai, Perumal

    2013-01-01

    Sertoli cell proliferation is attenuated before attaining puberty and the number is fixed in adult testes. Sertoli cells determine both testis size and daily sperm production by providing physical and metabolic support to spermatogenic cells. Polychlorinated biphenyls (PCBs) exposure disrupts functions of Sertoli cells causing infertility with decreased sperm count. On the other hand, lycopene is improving sperm count and motility by reducing oxidative stress in humans and animals. Hence we hypothesized that PCBs-induced infertility might be due to Sertoli cell apoptosis mediated by oxidative stress and lycopene might prevent PCBs-induced apoptosis by acting against oxidative stress. To test this hypothesis, animals were treated with vehicle control, lycopene, PCBs and PCBs + lycopene for 30 days. After the experimental period, the testes and cauda epididymidis were removed for isolation of Sertoli cells and sperm, respectively. We observed increased levels of oxidative stress markers (H2O2 and LPO) levels, increased expression of apoptotic molecules (caspase-8, Bad, Bid, Bax, cytochrome C and caspase-3), decreased anti-apoptotic (Bcl2) molecule and elevated apoptotic marker activity (caspase-3) in Sertoli cells of PCBs-exposed animals. These results were associated with decreased sperm count and motility in PCBs exposed animals. On the other hand, lycopene prevented the elevation of Sertoli cellular apoptotic parameters and prevented the reduction of sperm parameters (count and motility). The data confirmed that lycopene as an antioxidant scavenged reactive oxygen substances, prevented apoptosis, maintained normal function in Sertoli cells and helped to provide physical and metabolic support for sperm production, thereby treating infertility in men. PMID:24179434

  3. Ultrastructural study of crystalloids in Sertoli cells of the three-toed sloth (Bradypus tridactylus).

    PubMed

    Toyama, Y; Calderón, F U; Quesada, R

    1990-03-01

    Crystalloids were found in Sertoli cells of the testis of the three-toed sloth by examination at the light- and electron-microscopic levels. Needle-, or spindle-shaped crystalloids, varying in length, were located in the basal part of the Sertoli cells. They consisted of bundles of filaments each measuring approximately 11 nm in diameter. Several filaments were packed hexagonally to form a bundle. The center-to-center distance between individual filaments of a bundle was approximately 17 nm. Periodical lateral projections emanated from the filaments. Cross sections of crystalloids showed that the projections radiated from each filament in three directions, forming an equilateral triangle with a side length of approximately 15 nm. Scattered polyribosomes were found between and around the bundles. PMID:2317847

  4. Antigens on Murine Erythroid Cells

    Microsoft Academic Search

    ANDREAS HELIAS SARRIS; GEORGE E. PALADE

    A sialoglycoprotein fraction isolated from murine (DBA\\/2) erythrocytic ghosts (see companion article, Sarris and Palade, 1982, J. Cell . Biol . 93:583-590) was used to raise antibodies in rabbits . By immune-IgG (serum)-(\\

  5. Environmental toxicants perturb human Sertoli cell adhesive function via changes in F-actin organization mediated by actin regulatory proteins

    PubMed Central

    Xiao, Xiang; Mruk, Dolores D.; Tang, Elizabeth I.; Wong, Chris K.C.; Lee, Will M.; John, Constance M.; Turek, Paul J.; Silvestrini, Bruno; Cheng, C. Yan

    2014-01-01

    STUDY QUESTION Can human Sertoli cells cultured in vitro and that have formed an epithelium be used as a model to monitor toxicant-induced junction disruption and to better understand the mechanism(s) by which toxicants disrupt cell adhesion at the Sertoli cell blood–testis barrier (BTB)? SUMMARY ANSWER Our findings illustrate that human Sertoli cells cultured in vitro serve as a reliable system to monitor the impact of environmental toxicants on the BTB function. WHAT IS KNOWN ALREADY Suspicions of a declining trend in semen quality and a concomitant increase in exposures to environmental toxicants over the past decades reveal the need of an in vitro system that efficiently and reliably monitors the impact of toxicants on male reproductive function. Furthermore, studies in rodents have confirmed that environmental toxicants impede Sertoli cell BTB function in vitro and in vivo. STUDY DESIGN, SIZE AND DURATION We examined the effects of two environmental toxicants: cadmium chloride (0.5–20 µM) and bisphenol A (0.4–200 µM) on human Sertoli cell function. Cultured Sertoli cells from three men were used in this study, which spanned an 18-month period. PARTICIPANTS/MATERIALS, SETTING, METHODS Human Sertoli cells from three subjects were cultured in F12/DMEM containing 5% fetal bovine serum. Changes in protein expression were monitored by immunoblotting using specific antibodies. Immunofluorescence analyses were used to assess changes in the distribution of adhesion proteins, F-actin and actin regulatory proteins following exposure to two toxicants: cadmium chloride and bisphenol A (BPA). MAIN RESULTS AND THE ROLE OF CHANCE Human Sertoli cells were sensitive to cadmium and BPA toxicity. Changes in the localization of cell adhesion proteins were mediated by an alteration of the actin-based cytoskeleton. This alteration of F-actin network in Sertoli cells as manifested by truncation and depolymerization of actin microfilaments at the Sertoli cell BTB was caused by mislocalization of actin filament barbed end capping and bundling protein Eps8, and branched actin polymerization protein Arp3. Besides impeding actin dynamics, endocytic vesicle-mediated trafficking and the proper localization of actin regulatory proteins c-Src and annexin II in Sertoli cells were also affected. Results of statistical analysis demonstrate that these findings were not obtained by chance. LIMITATIONS, REASONS FOR CAUTION (i) This study was done in vitro and might not extrapolate to the in vivo state, (ii) conclusions are based on the use of Sertoli cell samples from three men and (iii) it is uncertain if the concentrations of toxicants used in the experiments are reached in vivo. WIDER IMPLICATIONS OF THE FINDINGS Human Sertoli cells cultured in vitro provide a robust model to monitor environmental toxicant-mediated disruption of Sertoli cell BTB function and to study the mechanism(s) of toxicant-induced testicular dysfunction. PMID:24532171

  6. Effect of argemone oil and argemone alkaloid, sanguinarine on Sertoli-germ cell coculture.

    PubMed

    Mishra, Vivek; Saxena, Daya Krishna; Das, Mukul

    2009-04-25

    Several incidences of reduction in the fertility (sperm count) have been reported in India and worldwide as well. Adulteration of food and consumption of adulterated mustard oil with argemone oil (AO) are presumed to be the factors for reduction in sperm count. In the present study we have studied the exfoliation of germ cells from Sertoli cell, its viability after detachment, cytotoxicity and execution of apoptosis via mitochondrial pathway for different concentration of AO, argemone alkaloid (AA) and its major constituent sanguinarine (SA). A dose dependent increase in germ cell detachment and decrease in viability of detached germ cells were observed (P<0.05). A significant inhibition was observed via 3-(4,5-dimethylthiazol-2-yl)-2,5-dipehyl tetrazolium bromide (MTT) assay in the proliferative activity of germ cell and leakage of cytosolic enzyme was observed via Lactate dehydrogenase(LDH) assay (P<0.05). A time and dose dependent inhibition of mitochondrial membrane potential was observed (P<0.05). Treatment of Sertoli-germ cells with the lowest concentration of AO/AA and SA for 24h resulted in 5.2- , 4.4- and 3.6-fold increase in the percentage of early apoptotic cells, respectively. This increase was enhanced to 8.3, 4.75 and 5.81-fold, respectively at 48 h in detached germ cells undergoing early apoptosis. These results suggest that alterations in germ cell apoptosis by a disruption in contact mediated communication between the Sertoli cells and germ cells, may subsequently lead to testicular impairment. PMID:19429230

  7. The death of sertoli cells and the capacity to phagocytize elongated spermatids during testicular regression due to short photoperiod in Syrian hamster (Mesocricetus auratus).

    PubMed

    Seco-Rovira, Vicente; Beltrán-Frutos, Esther; Ferrer, Concepción; Sáez, Francisco José; Madrid, Juan Francisco; Pastor, Luis Miguel

    2014-05-01

    In the Syrian hamster (Mesocricetus auratus), an animal that displays testicular regression due to short photoperiod, germ cells are removed by apoptosis during this process and the apoptotic remains are phagocytized by Sertoli cells. The aim of this work was to investigate morphologically whether the testicular regression process due to short photoperiod leads to the apoptosis of Sertoli cells, and whether, during testicular regression, the elongated spermatids are eliminated through phagocytosis by Sertoli cells. To this end, we studied testis sections during testicular regression in Syrian hamster subjected to short photoperiod by means of several morphological techniques using conventional light microscopy (hematoxylin and eosin [H&E], semi-thin section vimentin, immunohistochemistry, SBA lectin, and TUNEL staining), fluorescence microscopy, and transmission electron microscopy (TEM). H&E and semi-thin sections identified Sertoli cells with a degenerated morphology. Greater portion of Sertoli cells that were positive for TUNEL staining were observed especially during the mild regression (MR) and strong regression (SR) phases. In addition, TEM identified the characteristic apoptotic changes in the nucleus and cytoplasm of Sertoli cells. Moreover, during testicular regression and using light microscopy, some elongated spermatids were seen in basal position next to the Sertoli cell nucleus. This Sertoli phagocytic activity was higher in MR and SR phases. TEM confirmed this to be the result of the phagocytic activity of Sertoli cells. In conclusion, during testicular regression in Syrian hamster due to short photoperiod, when germ cells are known to be lost through apoptosis, there is morphological evidences that Sertoli cells are also lost through apoptosis, while some elongated spermatids are phagocytized and eliminated by the Sertoli cells. PMID:24719257

  8. 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced cytotoxicity accompanied by oxidative stress in rat Sertoli cells: Possible role of mitochondrial fractions of Sertoli cells

    SciTech Connect

    Aly, Hamdy A.A., E-mail: hamdyaali@yahoo.com [Department of Pharmacology and Toxicology, Faculty of Pharmacy, King Abdulaziz University, Jeddah (Saudi Arabia); Khafagy, Rasha M. [Physics Department, Girls College for Arts, Science and Education, Ain Shams University, Cairo (Egypt)

    2011-05-01

    TCDD, as an endocrine disruptor, is known to impair testicular functions and fertility. To elucidate the mechanism(s) underlying the testicular effects of TCDD, the potential toxicity of TCDD on Sertoli cells was investigated. Furthermore, the study aims to delineate whether mitochondrial fractions of Sertoli cells are involved in mediating the testicular effects of TCDD. Adult rat Sertoli cells were incubated with (5, 10 or 15 nM) of TCDD for 6, 12 or 24 h. Cell viability, lactate and LDH leakage into media along with lipid peroxidation, ROS generation, SOD, CAT, GPx, GR, {gamma}-GT and {beta}-glucuronidase activities, GSH content and {Delta}{psi}{sub m} were measured. Superoxide anion production, COX and cardiolipin content were measured in mitochondrial fractions. Cell viability was significantly decreased while lactate and LDH leakage into media were increased. ROS generation along with lipid peroxidation was also increased. SOD, CAT, GPx, GR activities and GSH content were significantly decreased. {gamma}-GT and {beta}-glucuronidase activities were also decreased. Superoxide anion production was increased while COX activity and cardiolipin content were decreased in mitochondrial fractions. Moreover, the {Delta}{psi}{sub m} was significantly decreased as measured in Sertoli cells. In conclusion, TCDD impairs Sertoli cell functions and this effect is, at least in part, attributed to oxidative stress. We have also found that TCDD increases mitochondrial superoxide anion production and decreases {Delta}{psi}{sub m}, COX activity and mitochondrial cardiolipin content. Our findings suggest that mitochondria may play an important role in ROS production, leading to the TCDD-induced oxidative stress response and resulting toxicological consequences in rat Sertoli cells.

  9. Effects of tri-iodothyronine on alternative splicing events in the coding region of cytochrome P450 aromatase in immature rat Sertoli cells

    Microsoft Academic Search

    V Pezzi; M L Panno; R Sirianni; P Forastieri; I Casaburi; M Lanzino; V Rago; F Giordano; C Giordano; A Carpino

    2001-01-01

    Transient postnatal hypothyroidism in male rats induces a prolonged proliferation of immature Sertoli cells. This change in Sertoli cell replication at young ages is coinci- dent with enhanced and prolonged aromatase activity that leads to a marked increase in the conversion of androgens into estrogens. Both events are drastically inhibited by tri-iodothyronine (T3) replacement either in vivo or in vitro.

  10. Action of hormones on Sertoli cells during maturation I. B. FRITZ B. G. LOUIS P. S. TUNG, Jennifer DORRINGTON

    E-print Network

    Paris-Sud XI, Université de

    antigenic determinant on the plasma membrane of Sertoli cells at a time cor- responding approximately., 1975). Greatest effects are obtained in cells from testes of immature animals, or from regressed testes nucleus of responsive cells. In tfm mutants, this androgen receptor is non-functional, and such animals

  11. Large-Cell Calcifying Sertoli Cell Tumour of the Testis Detected at Screening of a Family with Carney Syndrome

    Microsoft Academic Search

    S. N. Jayasena; J. T. N. Ariyasinghe; D. M. R. Gunawardena; S. A. S. Gunawardena; M. V. C. de Silva

    2005-01-01

    We report the detection of a large-cell calcifying Sertoli cell tumour (LCCSCT) in a 34-year-old male during screening of a family with Carney syndrome. The patient had ignored the testicular swelling for 7 years. He also had a cardiac myxoma. The LCCSCT in this patient had prognostically unfavourable features such as large size (>6 cm) and a high mitotic rate.

  12. Effects of glial cell line-derived neurotrophic factor on isolated developing mouse Sertoli cells in vitro.

    PubMed

    Wu, Zhenyu; Templeman, Jenny L; Smith, Robert A; Mackay, Sarah

    2005-02-01

    Cell proliferation is a key factor in sex determination where a size increase relative to the XX gonad is one of the first signs of testis differentiation. Moreover, proliferation of Sertoli cells during development is important in building up the stock of supporting cells necessary for subsequent successful fertility. Because proliferation is such an essential part of testis development, the hypothesis under long-term investigation is that it is under fail-safe control by multiple alternative growth factors. This study was undertaken to investigate the role of glial cell-derived neurotrophic factor (GDNF) on developing mouse Sertoli cells in vitro. Sertoli cells, isolated from mouse embryos at three stages of testis development, were maintained for 2-7 days in vitro (div) in the presence or absence of GDNF at 1, 10 and 100 ng mL(-1). Overall the presence of extracellular matrix gel had little effect on proliferative activity, but encouraged expression of the epithelial phenotype. A statistically significant difference in proliferation, assessed by immunocytochemical staining for proliferating cell nuclear antigen, was seen with GDNF at embryonic day (E)12.5 after 2 div (at both 10 and 100 ng mL(-1), P < 0.001) and 7 div (at both 10 and 100 ng mL(-1), P < 0.05); at E13.5 after 3 div (at both 10 and 100 ng mL(-1), P < 0.05) and at E14.5 after 7 div (100 ng mL(-1), P < 0.01), compared with controls cultured without growth factor. In conclusion, GDNF stimulates mitosis throughout this critical developmental window. The in vitro approach used here is a useful adjunct to the knockout mouse model and has been applied to show that GDNF exerts a proliferative effect on developing mouse Sertoli cells. PMID:15730482

  13. Differential proteomic profile of spermatogenic and Sertoli cells from peri-pubertal testes of three different bovine breeds

    PubMed Central

    Tripathi, Utkarsh K.; Aslam, Muhammad K. M.; Pandey, Shashank; Nayak, Samiksha; Chhillar, Shivani; Srinivasan, A.; Mohanty, T. K.; Kadam, Prashant H.; Chauhan, M. S.; Yadav, Savita; Kumaresan, Arumugam

    2014-01-01

    Sub-fertility is one of the most common problems observed in crossbred males, but the etiology remains unknown in most of the cases. Although proteomic differences in the spermatozoa and seminal plasma between breeds have been investigated, the possible differences at the sperm precursor cells and supporting/nourishing cells have not been studied. The present study reports the differential proteomic profile of spermatogenic and Sertoli cells in crossbred and purebred bulls. Testis was removed by unilateral castration of 12 peri-pubertal bulls (10 months age), four each from crossbred (Holstein Friesian × Tharparkar), exotic purebred [Holstein Friesian (HF)] and indigenous purebred [Tharparkar (TP)] bulls. Spermatogenic and Sertoli cells were isolated and subjected to proteomic analysis. Protein extracts from the Sertoli and spermatogenic cells of each breed were analyzed with 2-dimensional difference gel electrophoresis (2D-DIGE) and analyzed with Decyder™ software. Compared to HF, 26 protein spots were over expressed and 14 protein spots were under expressed in spermatogenic cells of crossbred bulls. Similarly, 7 protein spots were over expressed and 15 protein spots were under expressed in the spermatogenic cells of TP bulls compared to that of crossbred bulls. Out of 12 selected protein spots identified through mass spectrometry, Phosphatidyl ethanolamine binding protein was found to be over expressed in the spermatogenic cells of crossbred bulls compared to TP bulls. The protein, gamma actin was found to be over expressed in the Sertoli cells of HF bulls, whereas Speedy Protein-A was found to be over expressed in Sertoli cells of crossbred bulls. It may be concluded that certain proteomic level differences exist in sperm precursor cells and nourishing cells between breeds, which might be associated with differences in the fertility among these breeds. PMID:25364731

  14. Microparticle-loaded neonatal porcine Sertoli cells for cell-based therapeutic and drug delivery system.

    PubMed

    Giovagnoli, S; Mancuso, F; Vannini, S; Calvitti, M; Piroddi, M; Pietrella, D; Arato, I; Falabella, G; Galli, F; Moretti, M; Neri, L M; Bodo, M; Capitani, S; Cameron, D F; Ricci, M; Luca, G; Calafiore, R

    2014-10-28

    Neonatal porcine Sertoli cells (NPSC) are immune privileged cells showing innate phagocytic and antibacterial activities. NPSC have been shown capable of immunoaltering the body's response and possess lung homing capacity. These properties encourage investigation of NPSC as functional components of cell-based therapeutic protocols to treat lung infections and related complications. In this work, for the first time, NPSC were tailored to carry an antibiotic drug loaded into poly(d,l lactic) acid microparticles (MP). A loading protocol was developed, which afforded 30% drug uptake and high stability over time, with little or no effects on NPSC viability, morphology, reactive oxygen species production and DNA integrity. FSH receptor integrity, and TGF? (transforming growth factor ?) and AMH (anti-Müllerian hormone) expressions were unchanged after 1month of cryopreservation. Protein tyrosine kinase activation due to phagocytosis may have had resulted in changes in inhibin B expression. The activity of MP-loaded or NPSC alone against Pseudomonas aeruginosa was maintained throughout 1month of storage. NPSC couple an innate antibacterial activity with the capacity to embody drug loaded MP. We showed for the first time that engineered NPSC can be cryopreserved with no loss of their basic properties, thereby possibly representing a novel approach for cell-based therapeutic and drug delivery system. PMID:25111130

  15. Sertoli cell synthesizes and secretes a protease inhibitor,. alpha. sub 2 -macroglobulin

    SciTech Connect

    Cheng, C.Y. (Population Council, New York, NY (USA) Rockefeller Univ., New York, NY (USA)); Grima, J.; Stahler, M.S.; Guglielmotti, A.; Bardin, C.W. (Population Council, New York, NY (USA)); Silvestrini, B. (Univ. of Rome (Italy))

    1990-01-30

    The mechanism by which the seminiferous epithelium limits the damaging effects of proteases that are released from degenerating late spermatids does not depend upon protease inhibitors in the systemic circulation since these proteins are excluded from the seminiferous tubule by the blood-testis barrier. The purpose of this study was to identify the major protease inhibitor of the testis and determine its cellular origin. Sertoli cells, the major epithelial components of the seminiferous epithelium, release a protease inhibitor, testicular {alpha}{sub 2}-macroglobulin, in vitro. Immunoprecipitation using ({sup 35}S)methionine and a monospecific polyclonal antibody prepared against purified testicular {alpha}{sub 2}-macroglobulin establishes that this protein is actively synthesized and secreted by Sertoli cells. Measurements of immunoreactive protease inhibitors in tubular and rete testis fluids collected by micropuncture suggest that {alpha}{sub 2}-macroglubulin rather than {alpha}{sub 1}-antitrypsin is the major protease inhibitor in the seminiferous tubules in vivo. The ability of {alpha}{sub 2}-macroglobulin to inactivate proteases and growth factors such as TGF-{beta} by a common mechanism suggests that this protein may have a dual function in the testis.

  16. SRY Induced TCF21 Genome-Wide Targets and Cascade of bHLH Factors During Sertoli Cell Differentiation and Male Sex Determination in Rats1

    PubMed Central

    Bhandari, Ramji K.; Schinke, Ellyn N.; Haque, Md. M.; Sadler-Riggleman, Ingrid; Skinner, Michael K.

    2012-01-01

    ABSTRACT Male sex determination is initiated through the testis-determining factor SRY that promotes Sertoli cell differentiation and subsequent gonadal development. The basic helix-loop-helix (bHLH) gene Tcf21 was identified as one of the direct downstream targets of SRY. The current study was designed to identify the downstream targets of TCF21 and the potential cascade of bHLH genes that promote Sertoli cell differentiation. A modified ChIP-Chip comparative hybridization analysis identified 121 direct downstream binding targets for TCF21. The gene networks and cellular pathways potentially regulated by these TCF21 targets were identified. One of the main bHLH targets for TCF21 was the bHLH gene scleraxis (Scx). An embryonic ovarian gonadal cell culture was used to examine the functional role of Sry, Tcf21, and Scx to promote an in vitro sex reversal and induction of Sertoli cell differentiation. SRY and TCF21 were found to induce the initial stages of Sertoli cell differentiation, whereas SCX was found to induce the later stages of Sertoli cell differentiation associated with pubertal development using transferrin gene expression as a marker. Therefore, a cascade of SRY followed by TCF21 followed by SCX appears to promote, in part, Sertoli cell fate determination and subsequent differentiation. The current observations help elucidate the initial molecular events involved in the induction of Sertoli cell differentiation and testis development. PMID:23034159

  17. Sclerosing Sertoli cell tumor of the testis: a clinicopathologic study of 20 cases.

    PubMed

    Kao, Chia-Sui; Kum, Jennifer B; Idrees, Muhammad T; Ulbright, Thomas M

    2014-04-01

    Sclerosing Sertoli cell tumor (SSCT) of the testis is rare, with only 22 previously reported cases. Most have been small, circumscribed masses, and none has had a malignant clinical course; however, follow-up is limited. We have examined 20 new SSCTs to better characterize their features and report long-term follow-up. At least focal tubule formation by sex cord cells in a dense, hypocellular fibrous stroma occupying at least 50% of the lesion was required. The patient age ranged from 23 to 52 years (mean, 37; median, 39). All SSCTs were unilateral with 11 left sided and 9 right sided. The average size was 1.7 cm (range, 0.5 to 6 cm). In most cases, the stroma represented 50% to 70% of the mass but was at least 80% in 2. The Sertoli cells formed cords, trabeculae, small nests, focal tubules (sometimes with a vague pseudovascular or retiform appearance), and rarely single cells. Most tumor cells had small, round, oval to polygonal nuclei with finely granular chromatin, small nucleoli, and modest amounts of pale, eosinophilic cytoplasm. No mitotic figures, significant atypia, or necrosis was seen. All tumors but 1 were circumscribed and lacked lymphovascular invasion. Follow-up in 15 patients (3 mo to 16 y; mean, 6.1 y) showed 9 alive and free of disease, 5 alive with unknown disease status, and 1 patient, who presented with bone metastases, dead of disease at 27 months. The only features in the malignant case that differed from all others in our study were lymphovascular invasion and lack of circumscription. Combining our cases with previously reported ones shows that SSCTs are unilateral, usually small (80% <2 cm) tumors that occur in a wide age range (18 to 80 y old; mean, 35 y) and lack necrosis. Only 1 of 31 with follow-up (mean, 4.4 y) metastasized; this tumor was 3.8 cm and had lymphovascular invasion and invasive growth. We conclude that SSCTs<2 cm with the typical features and lacking those associated with malignancy in Sertoli cell tumors, not otherwise specified, have a negligible risk of metastasis and are adequately managed by orchiectomy alone. PMID:24552667

  18. Structural and Functional Modifications of Sertoli Cells in the Testis of Adult Follicle-Stimulating Hormone Receptor Knockout Mice1

    Microsoft Academic Search

    Amit Grover; M. Ram Sairam; Charles E. Smith; Louis Hermo

    Follicle stimulating hormone (FSH) plays important roles dur- ing testicular development and in the maintenance of spermato- genesis in the adult. However, the cellular events or pathways that FSH regulates to achieve these effects in Sertoli cells, where the FSH receptors (FSH-R) are located, is still not fully eluci- dated. The development of FSH-R knockout (FORKO) mice pro- vides a

  19. Reversal of experimental Laron Syndrome by xenotransplantation of microencapsulated porcine Sertoli cells.

    PubMed

    Luca, Giovanni; Calvitti, Mario; Mancuso, Francesca; Falabella, Giulia; Arato, Iva; Bellucci, Catia; List, Edward O; Bellezza, Enrico; Angeli, Giovanni; Lilli, Cinzia; Bodo, Maria; Becchetti, Ennio; Kopchick, John J; Cameron, Don F; Baroni, Tiziano; Calafiore, Riccardo

    2013-01-10

    Recombinant human IGF-1 currently represents the only available treatment option for the Laron Syndrome, a rare human disorder caused by defects in the gene encoding growth hormone receptor, resulting in irreversibly retarded growth. Unfortunately, this treatment therapy, poorly impacts longitudinal growth (13% in females and 19% in males), while burdening the patients with severe side effects, including hypoglycemia, in association with the unfair chore of taking multiple daily injections that cause local intense pain. In this study, we have demonstrated that a single intraperitoneal graft of microencapsulated pig Sertoli cells, producing pig insulin-like growth factor-1, successfully promoted significant proportional growth in the Laron mouse, a unique animal model of the human Laron Syndrome. These findings indicate a novel, simply, safe and successful method for the cell therapy-based cure of the Laron Syndrome, potentially applicable to humans. PMID:22964394

  20. Isolation of endothelial cells from murine tissue

    Microsoft Academic Search

    Federica M Marelli-Berg; Emma Peek; Elaine A Lidington; Hans J Stauss; Robert I Lechler

    2000-01-01

    The isolation and long-term culture of murine endothelial cells (ECs) has often proven a difficult task. In this paper we describe a quick, efficient protocol for the isolation of microvascular endothelial cells from murine tissues. Murine lung or heart are mechanically minced and enzymatically digested with collagenase and trypsin. The single cell suspension obtained is then incubated with an anti-CD31

  1. Rhox8 Ablation in the Sertoli Cells Using a Tissue-Specific RNAi Approach Results in Impaired Male Fertility in Mice.

    PubMed

    Welborn, Joshua P; Davis, Matthew G; Ebers, Steven D; Stodden, Genna R; Hayashi, Kanako; Cheatwood, Joseph L; Rao, Manjeet K; MacLean, James A

    2015-07-01

    The reproductive homeobox X-linked, Rhox, genes encode transcription factors that are selectively expressed in reproductive tissues. While there are 33 Rhox genes in mice, only Rhox and Rhox8 are expressed in Sertoli cells, suggesting that they may regulate the expression of somatic-cell gene products crucial for germ cell development. We previously characterized Rhox5-null mice, which are subfertile, exhibiting excessive germ cell apoptosis and compromised sperm motility. To assess the role of Rhox8 in Sertoli cells, we used a tissue-specific RNAi approach to knockdown RHOX8 in vivo, in which the Rhox5 promoter was used to drive Rhox8-siRNA transgene expression in the postnatal Sertoli cells. Western and immunohistochemical analysis confirmed Sertoli-specific knockdown of RHOX8. However, other Sertoli markers, Gata1 and Rhox5, maintained normal expression patterns, suggesting that the knockdown was specific. Interestingly, male RHOX8-knockdown animals showed significantly reduced spermatogenic output, increased germ cell apoptosis, and compromised sperm motility, leading to impaired fertility. Importantly, our results revealed that while some RHOX5-dependent factors were also misregulated in Sertoli cells of RHOX8-knockdown animals, the majority were not, and novel putative RHOX8-regulated genes were identified. This suggests that while reduction in levels of RHOX5 and RHOX8 in Sertoli cells elicits similar phenotypes, these genes are not entirely redundant. Taken together, our study underscores the importance of Rhox genes in male fertility and suggests that Sertoli cell-specific expression of Rhox5 and Rhox8 is critical for complete male fertility. PMID:25972016

  2. Loss of Sertoli-germ cell adhesion determines the rapid germ cell elimination during the seasonal regression of the seminiferous epithelium of the large hairy armadillo Chaetophractus villosus.

    PubMed

    Luaces, Juan Pablo; Rossi, Luis Francisco; Sciurano, Roberta Beatriz; Rebuzzini, Paola; Merico, Valeria; Zuccotti, Maurizio; Merani, Maria Susana; Garagna, Silvia

    2014-03-01

    The armadillo Chaetophractus villosus is a seasonal breeder whose seminiferous epithelium undergoes rapid regression with massive germ cell loss, leaving the tubules with only Sertoli cells and spermatogonia. Here, we addressed the question of whether this regression entails 1) the disassembly of cell junctions (immunolocalization of nectin-3, Cadm1, N-cadherin, and beta-catenin, and transmission electron microscopy [TEM]); 2) apoptosis (immunolocalization of cytochrome c and caspase 3; TUNEL assay); and 3) the involvement of Sertoli cells in germ cell phagocytosis (TEM). We showed a dramatic reduction in the extension of vimentin filaments associated with desmosomelike junctions at the interface between Sertoli and germ cells, and an increased diffusion of the immunosignals of nectin-3, Cadm1, N-cadherin, and beta-catenin. Together, these results suggest loss of Sertoli-germ cell adhesion, which in turn might determine postmeiotic cell sloughing at the beginning of epithelium regression. Then, loss of Sertoli-germ cell adhesion triggers cell death. Cytochrome c is released from mitochondria, but although postmeiotic cells were negative for late apoptotic markers, at advanced regression spermatocytes were positive for all apoptotic markers. Transmission electron microscopy analysis showed cytoplasmic engulfment of cell debris and lipid droplets within Sertoli cells, a sign of their phagocytic activity, which contributes to the elimination of the residual meiocytes still present in the latest regression phases. These findings are novel and add new players to the mechanisms of seminiferous epithelium regression occurring in seasonal breeders, and they introduce the armadillo as an interesting model for studying seasonal spermatogenesis. PMID:24451984

  3. Hemicastration causes and testosterone prevents enhanced uptake of (/sup 3/H)thymidine by Sertoli cells in testes of immature rats

    SciTech Connect

    Orth, J.M.; Higginbotham, C.A.; Salisbury, R.L.

    1984-02-01

    Rat pups were hemicastrated and uptake of (/sup 3/H)thymidine by Sertoli cells in the remaining testis was compared to that in testes of sham-operated pups at intervals of from 8 h to 21 days after surgery. Labeled thymidine was administered subcutaneously 2 h before sacrifice. Testes were processed for light microscope autoradiography and the percent of Sertoli cell nuclei that had incorporated (/sup 3/H)thymidine was determined by scoring nuclei in tissue sections as labeled or unlabeled. The percentage of cells labeled was increased in hemicastrates over intact controls by 8 h after surgery and testicular hypertrophy became apparent in hemicastrates by the following day. Labeling of Sertoli cells in hemicastrates remained elevated for 4 days and then returned to normal. When plasma levels of gonadotropins were measured in both groups 4 days after surgery, follicle-stimulating hormone (FSH) was found to be more than twice normal in hemicastrates while luteinizing hormone (LH) was unchanged. The effect of testosterone on the response of Sertoli cells to hemicastration was also examined. In hemicastrates, 2 days of androgen therapy depressed, and an additional 2 days abolished, the proliferative response of the Sertoli cells. Our findings suggest that increased proliferation of Sertoli cells within the remaining testis is involved in the enlargement of the testis that follows hemicastration. They also imply that prevention of compensatory hypertrophy by testosterone involves interference with this response of Sertoli cells in some way. Finally, our data implicate FSH in control of Sertoli cell proliferation in vivo in immature rats.

  4. Retinol-induced elevation of ornithine decarboxylase activity in cultured rat Sertoli cells is attenuated by free radical scavenger and by iron chelator

    Microsoft Academic Search

    Fábio Klamt; Felipe Dal-Pizzol; Nede Carlos Ribeiro; Elena Aida Barnard; Mara Silveira Benfato; José Cláudio Fonseca Moreira

    2000-01-01

    We investigated retinol effects in ornithine decarboxylase activity in Sertoli cells. We also tested the hypothesis that free radical scavengers and iron chelators may attenuate the effect of retinol. Sertoli cells isolated from 15-day-old Wistar rats were previously cultured for 48 h and then treated with retinol by 24 h with or without mannitol (1 mM) or 1,10 phenanthroline (100

  5. Cell Death during Development of Testis and Cerebellum in the Mutant Mouse Weaver

    Microsoft Academic Search

    S. M. W. Harrison; S. K. Roffler-Tarlov

    1998-01-01

    The murine mutationweaverconfers early death during development on cells in testes, cerebellum, and midbrain. The results reported here support the hypothesis that the action ofweaveris intrinsic to testes and independent of Sertoli cells: germ cells are the only testicular cell type seen to die in weaver homozygotes, while Sertoli cell-dependent development of the blood testis barrier is normal. This report

  6. An Integrative Omics Strategy to Assess the Germ Cell Secretome and to Decipher Sertoli-Germ Cell Crosstalk in the Mammalian Testis

    PubMed Central

    Lavigne, Régis; Hernio, Nolwen; Teixeira-Gomes, Ana-Paula; Dacheux, Jean-Louis; Pineau, Charles

    2014-01-01

    Mammalian spermatogenesis, which takes place in complex testicular structures called seminiferous tubules, is a highly specialized process controlled by the integration of juxtacrine, paracrine and endocrine information. Within the seminiferous tubules, the germ cells and Sertoli cells are surrounded by testicular fluid (TF), which probably contains most of the secreted proteins involved in crosstalk between these cells. It has already been established that germ cells can modulate somatic Sertoli cell function through the secretion of diffusible factors. We studied the germ cell secretome, which was previously considered inaccessible, by analyzing the TF collected by microsurgery in an “integrative omics” strategy combining proteomics, transcriptomics, genomics and interactomics data. This approach identified a set of proteins preferentially secreted by Sertoli cells or germ cells. An interaction network analysis revealed complex, interlaced cell-cell dialog between the secretome and membranome of seminiferous cells, mediated via the TF. We then focused on germ cell-secreted candidate proteins, and we identified several potential interacting partners located on the surface of Sertoli cells. Two interactions, APOH/CDC42 and APP/NGFR, were validated in situ, in a proximity ligation assay (PLA). Our results provide new insight into the crosstalk between germ cells and Sertoli cells occurring during spermatogenesis. Our findings also demonstrate that this “integrative omics” strategy is powerful enough for data mining and highlighting meaningful cell-cell communication events between different types of cells in a complex tissue, via a biological fluid. This integrative strategy could be applied more widely, to gain access to secretomes that have proved difficult to study whilst avoiding the limitations of in vitro culture. PMID:25111155

  7. Weight reduction and pioglitazone ameliorate polycystic ovary syndrome after removal of a Sertoli-stromal cell tumor

    PubMed Central

    Baba, Tsuyoshi; Endo, Toshiaki; Ikeda, Keiko; Shimizu, Ayumi; Morishita, Miyuki; Kuno, Yoshika; Honnma, Hiroyuki; Kiya, Tamotsu; Ishioka, Shin-ichi; Saito, Tsuyoshi

    2012-01-01

    This report presents an unusual case of Sertoli-stromal cell tumor and polycystic ovary syndrome successfully treated with weight reduction and an insulin-sensitizing agent. A 22-year-old woman, gravida 0, para 0, visited our hospital for the first time with a 12-year history of secondary amenorrhea and hypertrichosis. Transvaginal ultrasonography revealed a solid tumor in the right ovary. Right salpingo-oophorectomy was performed and pathological examination confirmed a Sertoli-stromal cell tumor. The patient’s serum androgen levels declined postoperatively, but remained above normal. Pioglitazone treatment for 6 months also significantly reduced serum androgen levels, but they still remained above normal. However, after losing 12 kg of body weight, the patient’s serum androgen levels declined to normal, and spontaneous menstruation became regular. Weight reduction with pioglitazone is an effective means of treating hyperandrogenism. PMID:23226075

  8. Profiling spermatogenic failure in adult testes bearing Sox9-deficient Sertoli cells identifies genes involved in feminization, inflammation and stress

    Microsoft Academic Search

    Aurélie Lardenois; Frédéric Chalmel; Francisco Barrionuevo; Philippe Demougin; Gerd Scherer; Michael Primig

    2010-01-01

    BACKGROUND: Sox9 (Sry box containing gene 9) is a DNA-binding transcription factor involved in chondrocyte development and sex determination. The protein's absence in testicular Sertoli nurse cells has been shown to disrupt testicular function in adults but little is known at the genome-wide level about molecular events concomitant with testicular break-down. METHODS: To determine the genome-wide effect on mRNA concentrations

  9. Sertoli cell as a model in male reproductive toxicology: Advantages and disadvantages.

    PubMed

    Reis, Mariana M S; Moreira, Ana C; Sousa, Mário; Mathur, Premendu P; Oliveira, Pedro F; Alves, Marco G

    2015-08-01

    Pressure towards population aging in the demographic pyramid is not only due to sociological/personal choices but also due to subfertility or infertility. There are several chemicals and mixtures that impair male fertility. While experimental animal models are crucial to identify compounds that affect male fertility, it is essential to use reliable in vitro models to determine cellular targets and intracellular pathways that mediate chemical toxicity in the male reproductive system. In this review, we focused on the somatic Sertoli cell (SC) that, within the testis, is a major target for hormonal signaling and provides physical and nutritional support to developing germ cells. The different outcomes possible in each type of study: in vivo versus in vitro (either in primary or immortalized cell cultures) are analyzed. Herein, we intend to clarify the unique features that render SCs as excellent candidates for a robust in vitro model to study the deleterious effects of chemicals on male reproductive health. The sensitivity of SCs to toxicants/pharmaceuticals is discussed and, based on the literature reviewed we propose the in vitro study of SC physiology as a model to disclose deleterious effects of substances to male fertility. Copyright © 2015 John Wiley & Sons, Ltd. PMID:25693974

  10. Insulinoma Presenting with Long-Standing Depression, Primary Hypogonadism, and Sertoli Cell Only Syndrome

    PubMed Central

    Malabu, Usman H.; Gowda, Durgesh; Tan, Yong Mong

    2013-01-01

    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. PMID:24455334

  11. Sertoli cells control peritubular myoid cell fate and support adult Leydig cell development in the prepubertal testis.

    PubMed

    Rebourcet, Diane; O'Shaughnessy, Peter J; Pitetti, Jean-Luc; Monteiro, Ana; O'Hara, Laura; Milne, Laura; Tsai, Yi Ting; Cruickshanks, Lyndsey; Riethmacher, Dieter; Guillou, Florian; Mitchell, Rod T; van't Hof, Rob; Freeman, Tom C; Nef, Serge; Smith, Lee B

    2014-05-01

    Sertoli cells (SCs) regulate testicular fate in the differentiating gonad and are the main regulators of spermatogenesis in the adult testis; however, their role during the intervening period of testis development, in particular during adult Leydig cell (ALC) differentiation and function, remains largely unknown. To examine SC function during fetal and prepubertal development we generated two transgenic mouse models that permit controlled, cell-specific ablation of SCs in pre- and postnatal life. Results show that SCs are required: (1) to maintain the differentiated phenotype of peritubular myoid cells (PTMCs) in prepubertal life; (2) to maintain the ALC progenitor population in the postnatal testis; and (3) for development of normal ALC numbers. Furthermore, our data show that fetal LCs function independently from SC, germ cell or PTMC support in the prepubertal testis. Together, these findings reveal that SCs remain essential regulators of testis development long after the period of sex determination. These findings have significant implications for our understanding of male reproductive disorders and wider androgen-related conditions affecting male health. PMID:24803659

  12. Sertoli cells control peritubular myoid cell fate and support adult Leydig cell development in the prepubertal testis

    PubMed Central

    Rebourcet, Diane; O'Shaughnessy, Peter J.; Pitetti, Jean-Luc; Monteiro, Ana; O'Hara, Laura; Milne, Laura; Tsai, Yi Ting; Cruickshanks, Lyndsey; Riethmacher, Dieter; Guillou, Florian; Mitchell, Rod T.; van ’t Hof, Rob; Freeman, Tom C.; Nef, Serge; Smith, Lee B.

    2014-01-01

    Sertoli cells (SCs) regulate testicular fate in the differentiating gonad and are the main regulators of spermatogenesis in the adult testis; however, their role during the intervening period of testis development, in particular during adult Leydig cell (ALC) differentiation and function, remains largely unknown. To examine SC function during fetal and prepubertal development we generated two transgenic mouse models that permit controlled, cell-specific ablation of SCs in pre- and postnatal life. Results show that SCs are required: (1) to maintain the differentiated phenotype of peritubular myoid cells (PTMCs) in prepubertal life; (2) to maintain the ALC progenitor population in the postnatal testis; and (3) for development of normal ALC numbers. Furthermore, our data show that fetal LCs function independently from SC, germ cell or PTMC support in the prepubertal testis. Together, these findings reveal that SCs remain essential regulators of testis development long after the period of sex determination. These findings have significant implications for our understanding of male reproductive disorders and wider androgen-related conditions affecting male health. PMID:24803659

  13. Ovarian Sertoli-Leydig cell tumor with heterologous elements of gastrointestinal type associated with elevated serum alpha-fetoprotein level: an unusual case and literature review.

    PubMed

    Horta, Mariana; Cunha, Teresa Margarida; Marques, Rita Canas; Félix, Ana

    2014-11-01

    Here we describe the case of a 19-year-old woman with a poorly differentiated ovarian Sertoli-Leydig cell tumor and an elevated serum alpha-fetoprotein level. The patient presented with diffuse abdominal pain and bloating. Physical examination, ultrasound, and magnetic resonance imaging revealed a right ovarian tumor that was histopathologically diagnosed as a poorly differentiated Sertoli-Leydig cell tumor with heterologous elements. Her alpha-fetoprotein serum level was undetectable after tumor resection. Sertoli-Leydig cell tumors are rare sex cord-stromal tumors that account for 0.5% of all ovarian neoplasms. Sertoli-Leydig cell tumors tend to be unilateral and occur in women under 30 years of age. Although they are the most common virilizing tumor of the ovary, about 60% are endocrine-inactive tumors. Elevated serum levels of alpha-fetoprotein are rarely associated with Sertoli-Leydig cell tumors, with only approximately 30 such cases previously reported in the literature. The differential diagnosis should include common alpha-fetoprotein-producing ovarian entities such as germ cell tumors, as well as other non-germ cell tumors that have been rarely reported to produce this tumor marker. PMID:25926909

  14. Combined Leydig cell and Sertoli cell dysfunction in 46,XX males lacking the sex determining region Y gene

    SciTech Connect

    Turner, B.; Vordermark, J.S. [Texas Tech Univ. Health Sciences Center, Lubbock, TX (United States); Fechner, P.Y. [Johns Hopkins Univ. School of Medicine, Baltimore, MD (United States)] [and others

    1995-07-03

    We have evaluated 3 individuals with a rare form of 46,XX sex reversal. All of them had ambiguous external genitalia and mixed wolffian and muellerian structures, indicating both Leydig cell and Sertoli cell dysfunction, similar to that of patients with true hermaphroditism. However, gonadal tissue was not ovotesticular but testicular with varying degrees of dysgenesis. SRY sequences were absent in genomic DNA from peripheral leukocytes in all 3 subjects. Y centromere sequences were also absent, indicating that testis development did not occur because of a low level mosaicism of Y-bearing cells. The subjects in this report demonstrate that there is a continuum in the extent of the testis determination in SRY-negative 46,XX sex reversal, ranging from nearly normal to minimal testicular development. 20 refs.

  15. Sertoli-leydig cell tumour of ovary with menorrhagia: a rare case report.

    PubMed

    Kanade, Umesh Sidheshwar; Dantkale, Sunita Sanjay; Narkhede, Rahul Ravindra; Kurawar, Rupali Ramrao; Bansode, Shubhada Yadavrao

    2014-10-01

    Sertoli-Leydig cell tumours (SLCTs) are rare sex cord stromal neoplasms of ovary accounting for less than 0.5% of all ovarian tumours. These are found in women of all age groups (2-75 y), but are most common in reproductive age group with an average age of 25 y. Mostly these are unilateral, confined to ovaries and usually stage I at the time of clinical diagnosis. The common presenting complaints in these patients are due to either mass occupying lesion (mostly pelviabdominal mass and/or pain) or hormonal production (mostly androgen and more rarely oestrogen). Androgenic manifestations, seen in 80% of patients with SLCT, are virilism, hirsutism, receding hairline, breast atrophy, clitoromegaly, acne, hoarseness of voice, etc. Estrogenic manifestations are precocious puberty, abnormal uterine bleeding, abnormal vaginal bleeding, menstrual irregularities, generalised oedema, weight gain, breast hypertrophy, endometrial hyperplasia, endometrial polyps and endometrial carcinoma. Histologically these are classified (WHO) as well-differentiated, intermediately differentiated, poorly differentiated, with heterologous components and retiform type. Prognosis depends upon degree of tumour differentiation (grading) and tumour extent (staging). We herein report an unusual case of SLCT of ovary with oestrogenic manifestation of menorrhagia. PMID:25478358

  16. Acceleration of Functional Maturation and Differentiation of Neonatal Porcine Islet Cell Monolayers Shortly In Vitro Cocultured with Microencapsulated Sertoli Cells

    PubMed Central

    Mancuso, Francesca; Calvitti, Mario; Luca, Giovanni; Nastruzzi, Claudio; Baroni, Tiziano; Mazzitelli, Stefania; Becchetti, Ennio; Arato, Iva; Boselli, Carlo; Ngo Nselel, Monique D.; Calafiore, Riccardo

    2010-01-01

    The limited availability of cadaveric human donor pancreata as well as the incomplete success of the Edmonton protocol for human islet allografts fasten search for new sources of insulin the producing cells for substitution cell therapy of insulin-dependent diabetes mellitus (T1DM). Starting from isolated neonatal porcine pancreatic islets (NPIs), we have obtained cell monolayers that were exposed to microencapsulated monolayered Sertoli cells (ESCs) for different time periods (7, 14, 21 days). To assess the development of the cocultured cell monolayers, we have studied either endocrine cell phenotype differentiation markers or c-kit, a hematopoietic stem cell marker, has recently been involved with growth and differentiation of ?-cell subpopulations in human as well as rodent animal models. ESC which were found to either accelerate maturation and differentiation of the NPIs ?-cell phenotype or identify an islet cell subpopulation that was marked positively for c-kit. The insulin/c-kit positive cells might represent a new, still unknown functionally immature ?-cell like element in the porcine pancreas. Acceleration of maturation and differentiation of our NPI cell monolayers might generate a potential new opportunity to develop insulin-producing cells that may suite experimental trials for cell therapy of T1DM. PMID:21048849

  17. Toxicity of cadmium on Sertoli cell functional competence: an in vitro study.

    PubMed

    Luca, G; Lilli, C; Bellucci, C; Mancuso, F; Calvitti, M; Arato, I; Falabella, G; Giovagnoli, S; Aglietti, M C; Lumare, A; Muzi, G; Calafiore, R; Bodo, M

    2013-01-01

    Cadmium (Cd), an ubiquitous environmental metal, mainly used for industrial purposes, may be toxic at level of the reproductive system. Testis tubular-based Sertoli cells (SC), play a major role in constituting the blood-testis barrier and provide a unique microenvironment for the genesis and differentiation of germ cells. Hence SC strictly control sperm qualitative and quantitative parameters. We aimed to assess whether exposure to Cd would adversely affect superior mammal SC viability and function. We isolated and purified SC from pre-pubertal pig testes according to our method and incubated the retrieved cells with three different Cadmium chloride concentrations (5-10-15 microM). Parameters of SC function such as inhibin B and anti-Mullerian hormone (AMH) were depressed by Cd exposure, contrary to what observed in untreated controls. No impairment of the FSH receptor integrity on the SC, as assessed by 17-beta-estradiol production, upon stimulation with FSH, was observed in either 5 microM Cd-treated or untreated controls. Differences, on the contrary, were observed for higher Cd concentrations (10 and 15 mM), in terms of FSH receptor integrity, that was altered, as compared to untreated controls, in terms of lower production of 17-beta-estradiol. In addition, the apoptotic test showed a significant increase of early (ANNEXIN V-/Propidium Iodide+) (AV-/PI+) and late apoptotic cells (AV+/ PI+) in all Cd -treated SC conditions as compared to controls. In conclusion, the Cd -related toxicity on SC, clearly demonstrated by our study, even at low concentrations, is expected to damage spermatogenesis that directly is dependent upon retention of SC viability and function. PMID:24152845

  18. Mixture effects of nonylphenol and di-n-butyl phthalate (monobutyl phthalate) on the tight junctions between Sertoli cells in male rats in vitro and in vivo.

    PubMed

    Hu, Yang; Wang, Ruoyu; Xiang, Zou; Qian, Weiping; Han, Xiaodong; Li, Dongmei

    2014-12-01

    The estrogenic chemical nonylphenol (NP) and the antiandrogenic agent di-n-butyl phthalate (DBP) are regarded as widespread environmental endocrine disruptors (EEDs) which at high doses in some species of laboratory animals have adverse effects on male reproduction and development. Given the ubiquitous coexistence of various classes of EEDs in the environment, their combined effects warrant investigation. In this study, we attempted to clarify the interactions of NP and DBP on tight junctions (TJs) between rat Sertoli cells. In the in vitro experiment, monobutyl phthalate (MBP), the active metabolite of DBP, was used instead of DBP. Sertoli cells were isolated from Sprague-Dawley rats, and treated with NP and MBP, singly or combined. The morphology of Sertoli cells, and structure and functionality of TJs were measured. In the in vivo experiment, rats were gavaged on postnatal day 23-35 with a single or combined NP and DBP treatment. Testicular weight and morphology of TJs were recorded. These data indicated that NP and DBP/MBP, either in single or in combination, induced the structural and function changes of Sertoli cell tight junctions, both in vivo and in vitro. The combined effect on the regulation of TJ proteins at both the protein and gene levels was correlated to the effect exerted by NP, suggesting that the structure and function of Sertoli cells were more sensitive to exposure to NP than MBP. PMID:25200483

  19. An ultrastructural study of developing spermatids and associated Sertoli cells during spermiogenesis in the kowari (crest-tailed marsupial rat), Dasyuroides byrnei.

    PubMed

    Kurohmaru, M; Nishida, T; Hayashi, Y; Yamashiro, S; Matsuzaki, T

    1990-03-01

    Ultrastructural changes of developing spermatids and associated Sertoli cells during spermiogenesis in the kowari, Dasyuroides byrnei, were observed by transmission electron microscopy. The early round spermatid in the kowari showed an acrosomal vacuole containing sparsely distributed material. The acrosomal vacuole grew to some degree and then collapsed upon itself and decreased in size. After the diminution of the vacuole, flattening and condensation of the nucleus began. At this period, the manchette, nuclear ring and caudal plaque appeared and the ectoplasmic specialization of the Sertoli cell developed in association with the acrosomal region of the spermatid head. Microtubules of the manchette were arranged obliquely or perpendicular to the long axis of the spermatid. As the spermatid developed further, the nucleus displayed a horseshoe shape in cross section and was flattened in longitudinal section. The ectoplasmic specialization which was the most developed at this period appeared like horns protruding from the spermatid nucleus. Immediately before spermiation, the flattened aspect of the spermatid head contacted an apical process of the Sertoli cell. The Sertoli cell apical process extended the spermatid head into the lumen. Long tubulobulbar complexes appeared in the Sertoli cell, after the ectoplasmic specialization had dissociated. PMID:2336246

  20. Characterization of rat transferrin receptor cDNA: the regulation of transferrin receptor mRNA in testes and in Sertoli cells in culture.

    PubMed

    Roberts, K P; Griswold, M D

    1990-04-01

    A 3.4 kilobase cDNA complementary to rat transferrin receptor mRNA has been isolated from an adult rat testis cDNA library. The rat transferrin receptor nucleotide sequence was shown to be 82% similar to the human transferrin receptor sequence over the amino acid coding region and over 90% similar in the sequences known to be responsible for iron regulation in the human mRNA. The mRNA was shown by Northern blot analysis to be regulated by iron levels in Sertoli cells in culture. Iron depletion resulted in at least a 5-fold increase in receptor message in Sertoli cells, as well as in an actively growing testicular cell line (S10-7). The level of transferrin receptor mRNA in cultured Sertoli cells was not influenced by hormones; however, chronic administration of testosterone or FSH to hypophysectomized rats resulted in increased transferrin receptor mRNA levels in the testis. Northern blot analysis of mRNAs from testes of rats synchronized at various stages of the cycle of the seminiferous epithelium showed that transferrin receptor mRNA was differentially regulated throughout the cycle. Northern blots of mRNA from germinal cell populations derived from synchronized tests showed that the message was regulated in the nongerminal cell components of the tubule, most likely the Sertoli cell. The comparison of transferrin receptor mRNA levels in normal testes and testes from hypophysectomized rats, as well as in isolated germinal cells and cultured Sertoli cells, suggested that transferrin receptor mRNA levels were considerably higher in Sertoli cells than in other cell types of the seminiferous tubules. PMID:2126342

  1. Activation of innate immune system in response to lipopolysaccharide in chicken Sertoli cells.

    PubMed

    Michailidis, Georgios; Anastasiadou, Maria; Guibert, Edith; Froment, Pascal

    2014-09-01

    Sertoli cells (SCs) play an important physiological role in the testis, as they support, nourish, and protect the germ cells. As protection of the developing spermatozoa is an emerging aspect of reproductive physiology, this study examined the expression pattern of innate immune-related genes, including avian ?-defensins (AvBDs), Toll-like receptors (TLRs), and cytokines, and investigated the time course of an inflammatory response in rooster SCs triggered by exposure to the bacterial endotoxin lipopolysaccharide (LPS). SCs were isolated from 6-week-old chicken, cultured in vitro, and stimulated with 1 ?g/ml LPS at different time courses (0, 6, 12, 24, and 48? h). Data on expression analysis revealed that all ten members of the chicken TLR family, nine members of the AvBD family, as well as eight cytokine genes were expressed in SCs. Quantitative real-time PCR analysis revealed that LPS treatment resulted in significant induction of the expression levels of six TLRs, six AvBDs, and four cytokine genes, while two cytokine genes were downregulated and two other genes were unchanged. The increasing interleukin 1? (IL1?) production was confirmed in the conditioned medium. Furthermore, the phagocytosis of SCs was increased after LPS treatment. In conclusion, these findings provide evidence that SCs express innate immune-related genes and respond directly to bacterial ligands. These genes represent an important component of the immune system, which could be integrated into semen, and present a distinctive constituent of the protective repertoire of the testis against ascending infections. PMID:24920664

  2. Perfluorooctanesulfonate (PFOS) Perturbs Male Rat Sertoli Cell Blood-Testis Barrier Function by Affecting F-Actin Organization via p-FAK-Tyr407: An in Vitro Study

    PubMed Central

    Wan, Hin-Ting; Mruk, Dolores D.; Wong, Chris K. C.

    2014-01-01

    Environmental toxicants such as perfluorooctanesulfonate (PFOS) have been implicated in male reproductive dysfunction, including reduced sperm count and semen quality, in humans. However, the underlying mechanism(s) remains unknown. Herein PFOS at 10–20 ?M (?5–10 ?g/mL) was found to be more potent than bisphenol A (100 ?M) in perturbing the blood-testis barrier (BTB) function by disrupting the Sertoli cell tight junction-permeability barrier without detectable cytotoxicity. We also delineated the underlying molecular mechanism by which PFOS perturbed Sertoli cell BTB function using an in vitro model that mimics the BTB in vivo. First, PFOS perturbed F-actin organization in Sertoli cells, causing truncation of actin filaments at the BTB. Thus, the actin-based cytoskeleton was no longer capable of supporting the distribution and/or localization of actin-regulatory and adhesion proteins at the cell-cell interface necessary to maintain BTB integrity. Second, PFOS was found to perturb inter-Sertoli cell gap junction (GJ) communication based on a dye-transfer assay by down-regulating the expression of connexin-43, a GJ integral membrane protein. Third, phosphorylated focal adhesion kinase (FAK)-Tyr407 was found to protect the BTB from the destructive effects of PFOS as shown in a study via an overexpression of an FAK Y407E phosphomimetic mutant. Also, transfection of Sertoli cells with an FAK-specific microRNA, miR-135b, to knock down the expression of phosphorylated FAK-Tyr407 was found to worsen PFOS-mediated Sertoli cell tight junction disruption. In summary, PFOS-induced BTB disruption is mediated by down-regulating phosphorylated FAK-Tyr407 and connexin-43, which in turn perturbed F-actin organization and GJ-based intercellular communication, leading to mislocalization of actin-regulatory and adhesion proteins at the BTB. PMID:24169556

  3. Regulation of the phosphoinositide pathway in cultured Sertoli cells from immature rats: effects of follicle-stimulating hormone and fluoride

    SciTech Connect

    Quirk, S.M.; Reichert, L.E. Jr.

    1988-07-01

    Many hormones elicit effects on target cells by stimulating the enzyme phospholipase-C, which catalyzes the hydrolysis of phosphoinositides to the intracellular second messengers diacylglycerol and inositol phosphates. The present study examined the roles of FSH and guanine nucleotide-binding proteins (G-proteins) in regulating the hydrolysis of phosphoinositides in Sertoli cells. Sertoli cell cultures prepared from 16- to 18-day-old rats were incubated for 24 h with myo-(2-3H) inositol to label endogenous phospholipids. Treatment of cells from 0.5-20 min with preparations of ovine FSH ranging in potency from 1-60 times that of NIH FSH S1 did not affect accumulation of inositol phosphates. Levels of total (3H)inositol phosphates ((3H)inositol mono-, di-, and triphosphates (IP, IP2, and IP3)) in FSH-treated cultures was 75-120% the levels in control cultures over the various time intervals studied. Addition of testosterone and the combination of testosterone plus retinoic acid, agents that have been shown to potentiate effects of FSH in other systems, did not affect accumulation of inositol phosphates in response to FSH. In contrast to the lack of effect on accumulation of inositol phosphates, FSH stimulated 4- to 11-fold increases in estradiol secretion over 24 h of culture, indicating that Sertoli cells were viable and responsive to FSH. AIF4- has been shown to activate G-proteins involved in regulation of adenylate cyclase activity. In the present study, AIF4- induced 4- to 5-fold increases in IP, IP2, and IP3 in experiments wherein FSH had no effect. Pretreatment of Sertoli cells with pertussis toxin (100 and 1000 ng/ml) for 24 h inhibited fluoride-induced generation of IP, IP2, and IP3 by 24-51%. Similar treatment with cholera toxin had no effect on basal or fluoride-induced generation of IP2 or IP3, but increased fluoride-induced generation of IP by 20-34%.

  4. In vitro effect of nanosilver on gene expression of superoxide dismutases and nitric oxide synthases in chicken Sertoli cells.

    PubMed

    Hassanpour, H; Mirshokraei, P; Sadrabad, E Khalili; Dehkordi, A Esmailian; Layeghi, S; Afzali, A; Mohebbi, A

    2015-02-01

    To evaluate effects of different concentrations of nanosilver colloid on the cell culture of Sertoli cells, the proportion of lipid peroxidation, antioxidant capacity, nitric oxide (NO) production and genes expression of superoxide dismutases (SOD1 and SOD2) and nitric oxide synthases (eNOS and iNOS) were measured. Sertoli cells were incubated at concentrations of 25, 75 and 125 ppm nanosilver for 48 h. There was progressive lipid peroxidation in treatments according to increasing of nanosilver. Lipid peroxidation, as indicated by malondialdehyde levels, was significantly elevated by the highest concentration of silver colloid (125 ppm), although antioxidant capacity, as measured by ferric ion reduction, was unaffected. Nitrite, as an index of NO production was reduced only in 125 ppm of nanosilver. Expression of SOD1 gene was reduced in nanosilver-treated cells at all concentrations, whereas expression of SOD2 gene was reduced only in cells treated with 125 ppm nanosilver. Expression of iNOS gene was progressively increased with higher concentrations of nanosilver. Expression of eNOS gene was also increased in 125 ppm of nanosilver. In conclusion, toxic effects of nanosilver could be due to high lipid peroxidation and suppression of antioxidant mechanisms via reduced expression of SOD genes and increased expression of NOS genes. PMID:25229128

  5. Prolongation of skin allograft survival in rats by the transplantation of microencapsulated xenogeneic neonatal porcine Sertoli cells.

    PubMed

    Bistoni, Giovanni; Calvitti, Mario; Mancuso, Francesca; Arato, Iva; Falabella, Giulia; Cucchia, Rosa; Fallarino, Francesca; Becchetti, Alessio; Baroni, Tiziano; Mazzitelli, Stefania; Nastruzzi, Claudio; Bodo, Maria; Becchetti, Ennio; Cameron, Don F; Luca, Giovanni; Calafiore, Riccardo

    2012-07-01

    Skin rejection remains a major hurdle in skin reconstructive transplantation surgery. In fact, 85% of the grafted patients experience at least one episode of acute skin rejection in the first year. It has been observed that Sertoli cells (SC), when co-transplanted with allo- or xenogeneic cell/tissues, can induce graft acceptance in the absence of systemic immunosuppression. A method aimed at significantly prolonging skin allografts in rats transplanted with barium alginate-based microencapsulated xenogeneic porcine SC (SC-MCs) is described. Results demonstrated that intraperitoneal (IP) transplantation of SC-MCs with high cellular viability and function can significantly prolong allogeneic skin grafts when compared to transplantation controls receiving only empty alginate capsules (E-MCs). Lymphocytic infiltration at the skin graft site was not observed in 80% of the SC-MCs transplanted rats and these recipient animals showed a significant increased expression of T regulatory (Tregs) cells when compared to E-MCs transplantation controls. The findings of this report further substantiate the positive therapeutic effects of SC on transplantation technology mediated by Sertoli cell-induced alterations of the host's immune system and indicate new perspectives and new strategies for successful skin tissue allografts. PMID:22560198

  6. Vectorial secretion of inhibin by immature rat Sertoli cells in vitro: reexamination of previous results.

    PubMed

    Janecki, A; Jakubowiak, A; Steinberger, A

    1990-10-01

    The vectorial secretion of immunoactive and bioactive inhibin by immature rat Sertoli cells (Sc) cultured in a two-compartment system was investigated using various culture supports. When Sc were cultured on Millipore-HA filters (used in all previous studies on vectorial secretion of inhibin), both immuno- and bioactive inhibin were found almost exclusively in the apical compartment, suggesting predominantly apical secretion of the glycoprotein. However, the cell-free Millipore-HA filters completely blocked the passage of Sc-conditioned medium (SCCM) inhibin, even after pretreatment with BSA and SCCM to saturate the protein-binding sites. On the other hand, polycarbonate Nucleopore filters or Millicell-CM membranes, both exhibiting extremely low protein-binding capacity, did not significantly block the passage of SCCM inhibin. When Sc were cultured on Nucleopore filters, the immunoactive inhibin was detected in both culture compartments; the basal compartment/apical compartment (BC/AC) ratio was about 1.5 (range, 1.2-1.9). The maximal effective dose of FSH or (Bu)2cAMP caused a 6- to 9-fold increase in the total (BC plus AC) secretion of immunoactive inhibin, but only a 60% increase in the secretion of bioactive inhibin, as evaluated by RIA and pituitary cell bioassay, respectively. The latter phenomenon was not accompanied by any significant change in the basal/apical distribution of either bioactive nor immunoactive inhibin. The presence of testosterone alone (10(-6) M) did not affect either total immunoactive inhibin secretion or its BC/AC ratio. The effects of the concomitant presence of FSH and testosterone did not differ significantly from those of FSH alone. Similarly to testosterone, the lack of any significant effect was observed for 17 beta-estradiol, dihydrotestosterone, androstenediol, and androstenedione regardless of the presence or absence of FSH. The striking dissimilarity of BC/AC ratios of inhibin noted in cultures maintained on Millipore-HA and Nucleopore filters was not due to differences in permeability barrier or Sc functional polarity. When cultured on either support, Sc monolayers developed comparable permeability barriers, as evaluated by measuring the passage of [3H]inulin and development of electrical resistance. The maximal electrical resistance (130-150 omega cm2) developed after 6-8 days of culture on either support. Also, total transferrin secretion and transferrin BC/AC ratio were similar on both supports, suggesting comparable cell numbers and functional polarities. These findings demonstrate that immature Sc in vitro secrete inhibin bidirectionally (BC/AC ratio, approximately 1.5); the polarity of secretion is unaffected by either FSH or various naturally occurring steroids, including testosterone.(ABSTRACT TRUNCATED AT 400 WORDS) PMID:2169406

  7. Follicle-Stimulating Hormone-Independent Functions of Primate Sertoli Cells: Potential Implications in the Diagnosis and Management of Male Infertility

    Microsoft Academic Search

    Yendrembam Sangeeta Devi; Kanchan Sarda; Benjamin Stephen; P. Nagarajan; Subeer S. Majumdar

    Context: FSH is known to augment the production of essential germ cell (Gc) survival factors, lactate and estradiol, by Sertoli cells (Sc) of 18-d-old pubertal rats. However, the failure of gonadotropin and an- drogen treatment to initiate spermatogenesis in testis of some infer- tile men bearing Sc and Gc is intriguing. The role of FSH in regulation of lactate and

  8. Insulin-like growth factor binding protein-3 secretion from cultured rat sertoli cells: dual regulation by follicle stimulating hormone and insulin-like growth factor-I.

    PubMed

    Smith, E P; Dickson, B A; Chernausek, S D

    1990-12-01

    The insulin-like growth factors (IGFs) are found in extracellular fluids bound to carrier proteins which influence the biological activity of the IGFs. Three structurally different binding proteins (BPs) have been isolated and cloned; each has distinct tissue specific expression and unique properties. We report here that testicular cells synthesize a specific subset of these binding proteins. Ligand blot analysis and RNA blot hybridization indicates that cultured peritubular cells synthesize primarily IGFBP-2. In contrast, as determined by ligand blot, RNA blot hybridization and N-linked deglycosylated studies, IGFBP-3 is predominantly synthesized by the Sertoli cell. In a dose dependent fashion, FSH markedly reduces the levels of IGFBP-3 in Sertoli cell conditioned medium. Similarly, isoproterenol, (Bu)2cAMP and cholera toxin also markedly reduce the abundance of IGFBP-3 in conditioned media. In contrast, IGF-I increases the concentrations of IGFBP-3 with the concentration required for half-maximal stimulation, approximately 20 ng/ml. Consistent with a peritubular cell origin, IGFBP-2 may be the predominant species found in interstitial fluid. In summary, our data reveal that the IGFBPs are expressed in a cell type specific manner in the testis. The opposing effects of FSH and IGF-I on Sertoli cell IGFBP-3 expression suggests a mechanism by which the IGF-I biological activity on Sertoli cell might be influenced. PMID:1701126

  9. The Sertoli Cell Only Syndrome and Glaucoma in a Sex – Determining Region Y (SRY) Positive XX Infertile Male

    PubMed Central

    Jain, Manish; V, Veeramohan; Chaudhary, Isha; Halder, Ashutosh

    2013-01-01

    The XX male syndrome is a rare genetic disorder. The phenotype is variable; it ranges from a severe impairment of the external genitalia to a normal male phenotype with infertility. It generally results from an unequal crossing over between the short arms of the sex chromosomes (X and Y). We are reporting a case of a 38-year-old man who presented with infertility and the features of hypogonadism and glaucoma. The examinations revealed normal external male genitalia, soft small testes, gynaecomastia and glaucoma. The semen analysis showed azoospermia. The serum gonadotropins were high, with low Anti Mullerian Hormone (AMH) and Inhibin B levels. The chromosomal analysis demonstrated a 46, XX karyotype. Fluorescent In-Situ Hybridization (FISH) and Polymerase Chain Reaction (PCR) revealed the presence of a Sex-determining Region Y (SRY). Testicular Fine Needle Aspiration Cytology (FNAC) revealed the Sertoli Cell Only Syndrome (SCOS). The presence of only Sertoli Cells in the testes, with glaucoma in the XX male syndrome, to our knowledge, has not been reported in the literature. PMID:23998093

  10. Role of type I receptors for anti-Müllerian hormone in the SMAT-1 Sertoli cell line.

    PubMed

    Belville, Corinne; Jamin, Soazik P; Picard, Jean-Yves; Josso, Nathalie; di Clemente, Nathalie

    2005-07-21

    Anti-Müllerian hormone (AMH) is a member of the transforming growth factor-beta family responsible for regression of Müllerian ducts during male sexual differentiation and for regulation of gonadal steroidogenesis. AMH is also a gonadal tumor suppressor which mediates its effects through a specific type II receptor and the bone morphogenetic protein (BMP)-specific Smad proteins, suggesting that AMH and BMPs could also share type I receptors, namely activin-like kinases (ALKs)2, 3 or 6. However, attempts to identify a unique AMH type I receptor among them were unsuccessful. Here, using kinase-deficient type I receptors and small interfering RNA technology, we demonstrate that, in an AMH Sertoli target cell line, ALK3 mediates AMH effects on both Smad1 activation and P450 side-chain cleavage enzyme. In addition, transfecting a combination of normal and kinase-deficient receptors, we show that ALK2 can compensate for the absence of ALK3 and probably acts in synergy with ALK3 at high concentrations of AMH to activate Smad1, whereas ALK6 has a competitive inhibitory effect. These results are a first step in understanding how AMH transduces its effects in immature Sertoli cells. PMID:15897891

  11. Postnatal testis development, Sertoli cell proliferation and number of different spermatogonial types in C57BL/6J mice made transiently hypo- and hyperthyroidic during the neonatal period

    PubMed Central

    Auharek, Sarah Alves; de França, Luiz Renato

    2010-01-01

    The role of thyroid hormones in testis structure and function has been fairly well studied in laboratory rodents. However, there are no comprehensive data in the literature for mice regarding the effects of transiently induced neonatal hypo- and hyperthyroidism on testis and spermatogonial cell development from birth to adulthood. Our goals were to evaluate the effects of propylthiouracil (PTU) and triidothyronine (T3) on Sertoli cell proliferation/differentiation and to correlate these events with the evolution of the spermatogenic process, tubular lumen formation, blood vessel volume density, and size and number of different spermatogonial types. Although Sertoli cell maturation was accelerated or delayed, respectively, in T3- and PTU-treated mice, the pace of the germ cell maturation was only slightly altered before puberty and the period of Sertoli cell proliferation was apparently not affected by the treatments. However, compared with controls, the total number of Sertoli cells per testis from 10 days of age to adulthood was significantly increased and decreased in PTU- and T3-treated mice, respectively. In comparison to all other spermatogonia, type A2 was the largest cell in all ages and groups investigated. The PTU-treated mice had a significantly increased total number of undifferentiated spermatogonia as well as volume and percentage of vessels/capillaries, probably due to the higher number of Sertoli cells, particularly at 10 days of age. Taken together, our results suggest that neonatal hypothyroidism may be a valuable tool for studying spermatogonial biology as well as a means for providing more spermatogonial stem cells that could potentially be used for spermatogonial transplantation, thereby optimizing the efficiency of this technique when young mice are used as donors. PMID:20525087

  12. High TGFbeta1, estrogen receptor, and aromatase gene expression in a large cell calcifying sertoli cell tumor (LCCSCT): implications for the mechanism of oncogenesis.

    PubMed

    Saraco, Nora; Berensztein, Esperanza; Sciara, Mariela; de Davila, Maria T G; Ciaccio, Marta; Ferrari, Patricia; Belgorosky, Alicia; Rivarola, Marco A

    2006-01-01

    Large cell calcifying Sertoli cell tumors (LCCSCT) are associated with Carney complex and Peutz-Jeghers syndrome. The mechanisms linking these 2 genetic defects to the genesis of this tumor are obscure. Studies of CYP19 (aromatase) and transforming growth factor (TGF)-beta1 messenger RNA (mRNA) abundance, estrogen receptor (ER), TGFbeta1, and TGFbeta type II receptor (R) immunochemistry were carried out in the testis of a patient with this tumor to gain information on possible mechanisms of cell tumor development. Testicular tissue of a prepubertal patient, collected at gonadectomy, was separated into 2 macroscopically distinct fractions: tumoral nodules (Tu) and extratumoral, normal-looking testicular tissue (ExTu). The patient was a 9.5-year-old boy with a 5-year history of bilateral gynecomastia (Tanner stage 4), no pubic hair, incipient genital development, and bilateral testicular nodules. Multiple pigmented lesions of the skin were present. Bilateral mammectomy and gonadectomy was performed. RNA was extracted from Tu and ExTu for semiquantitative reverse transcriptase-polymerase chain reaction of CYP19 and TGFbeta1. Protein expression of ER, TGFbeta1, and TGFbeta type II R in Tu and ExTu was detected by immunohistochemistry. Cell proliferation was estimated by Ki-67 antigen immunochemistry and apoptosis using a modified TUNEL assay. Mean expression of aromatase and TGFbeta1 mRNAs in Tu was 6- and 2.3-fold higher than in ExTu, respectively (P<0.05). Tumoral cells exhibited ER staining with a predominant extranuclear localization. Positive staining of Sertoli cells in Tu was higher than in ExTu. TGFbeta1 immunostaining of the interstitial cells in Tu was higher than in ExTu. TGFbeta type II R immunostaining was detected in most Sertoli and interstitial cells, but intensity in ExTu was lower than in Tu. No significant difference was detected in the proliferation index, but in Tu, the percentage of Sertoli cells in apoptosis (1.4%) was significantly lower (P<0.01) than in ExTu (14.0%). The following hypothesis is proposed. The congenital gene defects of Carney complex or of Peutz-Jeghers syndrome might trigger a cascade of intracellular events that leads to overexpression of aromatase in Sertoli cells, favoring the development of a LCCSCT. At some point in the evolution of the disease, a mutational event might induce a higher expression of the ER. Also, TGFbeta1 protein expression is increased in neighboring cells. In this environment, TGFbeta1 might switch from tumor suppressor to oncogenic factor and, along with estrogen-ER complexes, might favor tumor progression by inhibiting apoptosis. PMID:16944977

  13. The Dynamic of the Apical Ectoplasmic Specialization between Spermatids and Sertoli Cells: The Case of the Small GTPase Rap1

    PubMed Central

    2014-01-01

    Despite advances in assisted reproductive technologies, infertility remains a consistent health problem worldwide. Spermiation is the process through which mature spermatids detach from the supporting Sertoli cells and are released into the tubule lumen. Spermiation failure leads to lack of mature spermatozoa and, if not occasional, could result into azoospermia, major cause of male infertility in human population. Spermatids are led through their differentiation into spermatozoa by the apical ectoplasmic specialization (aES), a testis-specific, actin-based anchoring junction restricted to the Sertoli-spermatid interface. The aES helps spermatid movement across the seminiferous epithelium, promotes spermatid positioning, and prevents the release of immature spermatozoa. To accomplish its functions, aES needs to undergo tightly and timely regulated restructuring. Even if components of aES are partly known, the mechanism/s through which aES is regulated remains still elusive. In this review, we propose a model by which the small GTPase Rap1 could regulate aES assembly/remodelling. The characterization of key players in the dynamic of aES, such as Rap1, could open new possibility to develop prognostic, diagnostic, and therapeutic approaches for male patients under treatment for infertility as well as it could lead to the identification of new target for male contraception. PMID:24719879

  14. Varicocele-Caused Progressive Damage in Bilateral Testis and Sertoli Cell-Only Syndrome in Homolateral Testis in Rats

    PubMed Central

    Liu, Jianjun; Ding, Degang; Liu, Jie

    2014-01-01

    Background We aimed to investigate whether varicocele (VC) in rats can cause Sertoli cell-only syndrome (SCOS). Material/Methods Forty adolescent SD rats were randomly divided into 4 groups: 4-weeks control group, 4-weeks experimental group, 12-weeks control group, and 12-weeks experimental group. Left varicocele models were introduced by partially ligating left kidney veins for the experimental groups, and the sham surgery groups as controls were executed with exactly the same surgery as in the experimental groups except for the ligation. Rats in control and experimental groups for 4 and 12 weeks were killed after laparotomy at 4 and 12 weeks, respectively, the testes were taken out and fixed in fixative containing 4% polyformaldehyde, then were stained by hematoxylin and eosin (HE). The density and viability of sperm were analyzed by computer-aided sperm analysis. Results Compared with rats in 4-weeks and 12-weeks control group, histological structures of bilateral testes in both experimental groups were impaired, most of them showing as focal focuses. The pathological changes of testes in rats of the 12-weeks experimental group were bilateral, and included atrophy of seminiferous tubules, turbulence of spermatogenic cells in seminiferous tubules, defluvium of most spermatogenic cells, abortion of spermatogenesis, and degradation of spermatogenic epithelia. One rat in the 12-weeks experimental group was shown having SCOS, with the spermatogenic cells in seminiferous tubules completely flaked, degraded, or absent, and only Sertoli cells lined the seminiferous tubules. Conclusions Laboratory VC caused progressive impairment of homolateral testes, and SCOS could be induced when the damage was severe. Our results indicate that asthenozoospermia, azoospermia, and SCOS can be prevented by the earlier treatment of VC. PMID:25313556

  15. Extratesticular interstitial and Sertoli cell tumors in previously neutered dogs and cats: A report of 17 cases

    PubMed Central

    Doxsee, Angela L.; Yager, Julie A.; Best, Susan J.; Foster, Robert A.

    2006-01-01

    Primary neoplasms derived from testicular tissue and in an extratesticular location are extremely rare. Clinical and surgical information was collected and verified from 15 different submitting practices for 12 dogs and 5 cats that spontaneously developed neoplasms of testicular origin after castration. Eleven dogs had Sertoli cell tumors in an extratesticular location. One dog and all 5 cats had an extratesticular interstitial cell tumor. Six animals (1 dog, 5 cats) had developed secondary sexual characteristics that reversed after removal of the tumor. All had a palpable mass in the scrotum or at the site of the original prescrotal incision. No animals died of neoplasia-related disease and no metastases were identified. Several possibilities, including the presence of embryological ectopic tissue or the presence of testicular tissue transplanted during castration, are considered as causal. PMID:16933553

  16. Thrombopoietin inhibits murine mast cell differentiation

    PubMed Central

    Martelli, Fabrizio; Ghinassi, Barbara; Lorenzini, Rodolfo; Vannucchi, Alessandro M; Rana, Rosa Alba; Nishikawa, Mitsuo; Partamian, Sandra; Migliaccio, Giovanni; Migliaccio, Anna Rita

    2009-01-01

    We have recently shown that Mpl, the thrombopoietin receptor, is expressed on murine mast cells and on their precursors and that targeted deletion of the Mpl gene increases mast cell differentiation in mice. Here we report that treatment of mice with thrombopoietin, or addition of this growth factor to bone marrow-derived mast cell cultures, severely hampers the generation of mature cells from their precursors by inducing apoptosis. Analysis of the expression profiling of mast cells obtained in the presence of thrombopoietin suggests that thrombopoietin induces apoptosis of mast cells by reducing expression of the transcription factor Mitf and its target anti-apoptotic gene Bcl2. PMID:18276801

  17. Commonly dysregulated genes in murine APL cells

    PubMed Central

    Yuan, Wenlin; Payton, Jacqueline E.; Holt, Matthew S.; Link, Daniel C.; Watson, Mark A.; DiPersio, John F.; Ley, Timothy J.

    2007-01-01

    To identify genes that are commonly dysregulated in a murine model of acute promyelocytic leukemia (APL), we first defined gene expression patterns during normal murine myeloid development; serial gene expression profiling studies were performed with primary murine hematopoietic progenitors that were induced to undergo myeloid maturation in vitro with G-CSF. Many genes were reproducibly expressed in restricted developmental “windows,” suggesting a structured hierarchy of expression that is relevant for the induction of developmental fates and/or differentiated cell functions. We compared the normal myeloid developmental transcriptome with that of APL cells derived from mice expressing PML-RAR? under control of the murine cathepsin G locus. While many promyelocyte-specific genes were highly expressed in all APL samples, 116 genes were reproducibly dysregulated in many independent APL samples, including Fos, Jun, Egr1, Tnf, and Vcam1. However, this set of commonly dysregulated genes was expressed normally in preleukemic, early myeloid cells from the same mouse model, suggesting that dysregulation occurs as a “downstream” event during disease progression. These studies suggest that the genetic events that lead to APL progression may converge on common pathways that are important for leukemia pathogenesis. PMID:17008535

  18. The PGD2 pathway, independently of FGF9, amplifies SOX9 activity in Sertoli cells during male sexual differentiation.

    PubMed

    Moniot, Brigitte; Declosmenil, Faustine; Barrionuevo, Francisco; Scherer, Gerd; Aritake, Kosuke; Malki, Safia; Marzi, Laetitia; Cohen-Solal, Anne; Georg, Ina; Klattig, Jürgen; Englert, Christoph; Kim, Yuna; Capel, Blanche; Eguchi, Naomi; Urade, Yoshihiro; Boizet-Bonhoure, Brigitte; Poulat, Francis

    2009-06-01

    Activation by the Y-encoded testis determining factor SRY and maintenance of expression of the Sox9 gene encoding the central transcription factor of Sertoli cell differentiation are key events in the mammalian sexual differentiation program. In the mouse XY gonad, SOX9 upregulates Fgf9, which initiates a Sox9/Fgf9 feedforward loop, and Sox9 expression is stimulated by the prostaglandin D2 (PGD2) producing lipocalin prostaglandin D synthase (L-PGDS, or PTDGS) enzyme, which accelerates commitment to the male pathway. In an attempt to decipher the genetic relationships between Sox9 and the L-Pgds/PGD2 pathway during mouse testicular organogenesis, we found that ablation of Sox9 at the onset or during the time window of expression in embryonic Sertoli cells abolished L-Pgds transcription. By contrast, L-Pgds(-/-) XY embryonic gonads displayed a reduced level of Sox9 transcript and aberrant SOX9 protein subcellular localization. In this study, we demonstrated genetically that the L-Pgds/PGD2 pathway acts as a second amplification loop of Sox9 expression. Moreover, examination of Fgf9(-/-) and L-Pgds(-/-) XY embryonic gonads demonstrated that the two Sox9 gene activity amplifying pathways work independently. These data suggest that, once activated and maintained by SOX9, production of testicular L-PGDS leads to the accumulation of PGD2, which in turn activates Sox9 transcription and nuclear translocation of SOX9. This mechanism participates together with FGF9 as an amplification system of Sox9 gene expression and activity during mammalian testicular organogenesis. PMID:19429785

  19. Characterization of ferritin in murine erythroleukaemia cells.

    PubMed

    Peto, T E; Thompson, J L

    1986-03-19

    Murine erythroleukaemic cells were studied to determine whether different isoferritins have different functions. The cells were labelled with radioactive iron and the pattern of isoferritins was analysed by chromatofocussing. No change was found after iron-loading the cells but after inducing erythroid differentiation with dimethyl sulphoxide (DMSO), iron was incorporated into both more basic and more acidic isoferritins. This was compared to ferritin subunit synthesis; DMSO induced the synthesis of a third, minor subunit whereas iron-loading had no effect. The fate of murine erythroleukaemic cell ferritin iron was followed after incubations in iron-deficient medium containing DMSO; some, but not all, of the ferritin iron was mobilized and used for haem synthesis, and the remaining iron was found amongst the more basic isoferritins. Finally, sequential radioactive iron labels were used to demonstrate that the movement of iron from ferritin to haem was compatible with the 'last-in-first-out' principle, but this could not be related to different isoferritins. These results show firstly that DMSO changes the pattern of isoferritins and ferritin subunits in murine erythroleukaemic cells. Secondly, iron associated with more basic isoferritins seems to be less easily mobilized for haem synthesis. These results support the concept that different isoferritins have different functions. PMID:3456243

  20. Early Postnatal Exposure to a Low Dose of Decabromodiphenyl Ether Affects Expression of Androgen and Thyroid Hormone Receptor-Alpha and Its Splicing Variants in Mouse Sertoli Cells

    PubMed Central

    Miyaso, Hidenobu; Nakamura, Noriko; Naito, Munekazu; Hirai, Shuichi; Matsuno, Yoshiharu; Itoh, Masahiro; Mori, Chisato

    2014-01-01

    Decabromodiphenyl ether (decaBDE) adversely affects reproduction and development. Our previous study showed that postnatal exposure to a low dose of decaBDE (0.025 mg/kg body weight/day) by subcutaneous injection on postnatal days (PNDs) 1 through 5 leads to reductions in testicular size and number of Sertoli cells and sperm, while higher dose of decaBDE (2.5 mg/kg body weight/day) had no significant differences about these. In the present study, we examined the molecular mechanism of these effects on mouse testes following postnatal exposure to a low decaBDE dose. We hypothesized that postnatal exposure to decaBDE may alter levels of serum thyroid hormones (THs) and testosterone, or the level of TH receptor alpha (Thra) transcripts and its splicing variants and androgen receptor (Ar) in Sertoli cells, adversely affecting spermatogenesis. To test this hypothesis, we examined serum TH and testosterone levels and the levels of transcripts of the Ar, Thra and its splicing variants, and Thra splicing factors (Hnrnpa1, Srsf1, and Hnrnph1) with qPCR in isolated mouse Sertoli cells exposed postnatally to decaBDE (0.025, 0.25, and 2.5 mg/kg). Levels of serum testosterone and transcripts encoding Ar, Thra, and its variant, Thra1, declined significantly in Sertoli cells of mice exposed to 0.025 mg decaBDE/kg. No significant differences in serum TH level or Thra2, Hnrnph1, or Srsf1 transcript levels were observed between control and decaBDE-exposed mice. However, the Thra1:Thra2 and Hnrnpa1:Srsf1 ratios were altered in Sertoli cells of mice exposed to 0.025 mg decaBDE/kg but not in cells exposed to 0.25 or 2.5 mg decaBDE/kg. These results indicate that postnatal exposure to a low dose of decaBDE on PNDs 1 through 5 lowers the testosterone level and the levels of Ar and Thra transcripts in Sertoli cells, accompanied by an imbalance in the ratios of Thra splicing variants, resulting in smaller testicular size and impaired spermatogenesis. PMID:25479311

  1. Effects of tri-iodothyronine on alternative splicing events in the coding region of cytochrome P450 aromatase in immature rat Sertoli cells.

    PubMed

    Pezzi, V; Panno, M L; Sirianni, R; Forastieri, P; Casaburi, I; Lanzino, M; Rago, V; Giordano, F; Giordano, C; Carpino, A; Andò, S

    2001-08-01

    Transient postnatal hypothyroidism in male rats induces a prolonged proliferation of immature Sertoli cells. This change in Sertoli cell replication at young ages is coincident with enhanced and prolonged aromatase activity that leads to a marked increase in the conversion of androgens into estrogens. Both events are drastically inhibited by tri-iodothyronine (T(3)) replacement either in vivo or in vitro. This study, after the immunolocalization of aromatase in cultured rat Sertoli cells, examined the effects elicited by T(3) on this enzyme, by simultaneously investigating three functional levels of aromatase: mRNA expression, protein content, and enzymatic activity. The immunolocalization of cytochrome P450 aromatase (P450 arom) was shown in the cytoplasm of cultured Sertoli cells from 15- and 21-day-old rats. Western blot analysis revealed an enhancement of aromatase protein content upon stimulation with N(6),2'-O-dibutyryladenosine-3':5'-cyclic monophosphate ((Bu)(2)cAMP) that was clearly down-regulated by T(3). The presence of a functional P450 arom protein in purified Sertoli cells was confirmed by the measurement of [(3)H]H(2)O released after incubation with [1 beta-(3)H]androst-4-ene-3,17-dione. With 100 nM T3, a decrease in both P450 arom mRNA levels and aromatase activity was observed. The aromatase enzymatic activity was strongly stimulated by (Bu)(2)cAMP and markedly down-regulated by T(3). In contrast, the strong increase in aromatase mRNA upon (Bu)(2)cAMP stimulation was apparently unaffected by T(3) administration. This paper shows how the identification of an altered transcript induced by T(3) coding for putative truncated and inactive aromatase protein might explain such a decrease in aromatase activity in T(3)-treated cells. On the basis of these results, it is concluded that at least two mechanisms could be involved in the down-regulatory effect of T(3) on aromatase activity in prepuberal Sertoli cells. The first mechanism is linked to a possible direct modulatory role for T(3) in the regulation of the aromatase promoter, whilst the second one is represented by the induction of altered transcripts coding for truncated and inactive aromatase proteins. PMID:11479134

  2. Blood-testis barrier dynamics are regulated by testosterone and cytokines via their differential effects on the kinetics of protein endocytosis and recycling in Sertoli cells

    PubMed Central

    Yan, Helen H. N.; Mruk, Dolores D.; Lee, Will M.; Cheng, C. Yan

    2009-01-01

    During spermatogenesis in the mammalian testis, preleptotene/leptotene spermatocytes differentiate from type B spermatogonia and traverse the blood-testis barrier (BTB) at stage VIII of the seminiferous epithelial cycle for further development. This timely movement of germ cells involves extensive junction restructuring at the BTB. Previous studies have shown that these events are regulated by testosterone (T) and cytokines [e.g., the transforming growth factor (TGF) -?s], which promote and disrupt the BTB assembly, respectively. However, the mechanisms underlying the “opening” of the BTB above a migrating preleptotene/leptotene spermatocyte and the “resealing” of the barrier underneath this cell remain obscure. We now report findings on a novel mechanism utilized by the testes to regulate these events. Using cell surface protein biotinylation coupled with immunoblotting and immunofluorescent microscopy, we assessed the kinetics of endocytosis and recycling of BTB-associated integral membrane proteins: occludin, JAM-A, and N-cadherin. It was shown that these proteins were continuously endocytosed and recycled back to the Sertoli cell surface via the clathrin-mediated but not the caveolin-mediated pathway. When T or TGF-?2 was added to Sertoli cell cultures with established functional BTB, both factors accelerated the kinetics of internalization of BTB proteins from the cell surface, perhaps above the migrating preleptotene spermatocyte, thereby opening the BTB. Likewise, T also enhanced the kinetics of recycling of internalized biotinylated proteins back to the cell surface, plausibly relocating these proteins beneath the migrating spermatocyte to reassemble the BTB. In contrast, TGF-?2 targeted internalized biotinylated proteins to late endosomes for degradation, destabilizing the BTB. In summary, the transient opening of the BTB that facilitates germ cell movement is mediated via the differential effects of T and cytokines on the kinetics of endocytosis and recycling of integral membrane proteins at the BTB. The net result of these interactions, in turn, determines the steady-state protein levels at the Sertoli-Sertoli cell interface at the BTB. PMID:18192323

  3. The role of connexins in the differentiation of NT2 cells in Sertoli-NT2 cell tissue constructs grown in the rotating wall bioreactor.

    PubMed

    Shamekh, R; Cameron, D F; Willing, A E; Saporta, S

    2006-04-01

    Neural transplantation is developing as a successful treatment for neurodegenerative diseases such as Parkinson's disease. The human Ntera-2/D1 (NT2) cell line is an attractive alternative to the use of human fetal neurons as a cell source for transplantation. We have explored combining NT2 cells, as a neuronal source, and Sertoli cells, which may act as a graft facilitator to enhance neuronal survival and differentiation, and ameliorate the host immune response, into a tissue construct for use in cell replacement therapy for neurodegenerative disease. This Sertoli-NT2-aggregated cell (SNAC) tissue construct is formed in the high aspect ratio vessel (HARV) bioreactor. NT2 cells differentiate to dopaminergic NT2N neurons within the SNAC tissue construct without retinoic acid. We report here that the gap junction protein connexin 43 is decreased among differentiated NT2N neurons. Inhibition of connexin 43 with 18beta glycyrrhetinic acid and carbenoxolone, a glycyrrhetinic acid derivative, during formation of the SNAC tissue constructs disrupts the differentiation of NT2 cells. Therefore, connexin 43 is important in the differentiation of NT2 cells in the SNAC tissue construct. PMID:16328273

  4. Effects of dinoseb, 4,6-dinitro- o-cresol, and 2,4-dinitrophenol on rat Sertoli-germ cell co-cultures

    Microsoft Academic Search

    Ken L Takahashi; Hiroaki Aoyama; Kunio Kawashima; Shoji Teramoto

    2003-01-01

    The effects of dinoseb (DNBP), a known testicular toxicant in the rat, on germ cells were investigated in Sertoli-germ cell co-cultures. Two DNBP-related dinitrophenolic compounds, 4,6-dinitro-o-cresol (DNOC) and 2,4-dinitrophenol (DNP), were also examined, as testicular toxicity of these compounds had not been elucidated. Cultures were exposed to each compound (10?7–10?4M) for 24h and examined for the number and viability of

  5. Cytokines, polarity proteins, and endosomal protein trafficking and signaling-the sertoli cell blood-testis barrier system in vitro as a study model.

    PubMed

    Xiao, Xiang; Wong, Elissa W P; Lie, Pearl P Y; Mruk, Dolores D; Wong, Chris K C; Cheng, C Yan

    2014-01-01

    Endosomal signaling is emerging as one of the most important cellular events that regulate signaling function in mammalian cells or an epithelium in response to changes in environment such as the presence of stimuli mediated by cytokines, toxicants, heat, ions during growth and development, and other cellular processes such as cytokinesis and spermatogenesis. Recent studies have shown that protein endocytosis-the initial step of endosomal signaling-involves the participation of polarity proteins, such as partitioning defective protein 6 (Par6), Cdc42 and 14-3-3 (also known as Par5), which in turn is regulated by cytokines (e.g., TGF-?2, TGF-?3) and testosterone at the Sertoli cell blood-testis barrier (BTB) in the mammalian testis. In this short method paper, we provide a detailed protocol of assessing protein endocytosis, the initial and also the most critical step of endosomal signaling at the Sertoli cell BTB. This biochemical endocytosis assay summarizes our experience for the last decade, which should likely be performed in conjunction with the dual-labeled immunofluorescence analysis to assess protein endocytosis. While we are using a Sertoli cell in vitro system that mimics the BTB in vivo, this approach should be applicable to virtually all mammalian cells. PMID:24359954

  6. Implication of actin microfilaments in maintenance of intercellular bridges and the Sertoli cell barrier in the rat seminiferous epithelium

    SciTech Connect

    Weber, J.E.

    1987-01-01

    Within the seminiferous epithelium, germ cells are connected to one another by intercellular bridges. Additionally, young germ cells are separated from more advanced germ cells by the Sertoli cell barrier, the occluding junctions of which are associated with actin microfilaments. To examine how microfilaments influence these structures in the rat the actin-disrupting agent cytochalasin D (CD) was injected intratesticularly (i.t.). In preliminary studies the optical injection volume was found to be 50 {mu}l and, by using the dye trypan blue, the injected solution was shown to enter the lymphatic system and rapidly spread throughout the testis. A 50% clearance of {sup 3}H-insulin from the testis was achieved at 3 hr and 95% by 24 hr. Vehicles with varying solubility properties did not cause testicular damage. Intercellular bridges were found to be dynamic structures. As spermatogenesis progressed, the bridge diameter gradually increased. The formation and degradation of bridge partitioning complexes within pre-existing bridges of dividing cells were described.

  7. Localization of rDNA transcription sites in nucleoli of human Sertoli cells: an EM quantitative autoradiographic study using 3H-uridine.

    PubMed

    Brechard, M P; Hartung, M; de Lanversin, A; Cau, P; Stahl, A

    1994-01-01

    The sites of rDNA transcription within human Sertoli cell nucleoli have been localized using EM autoradiography after a 45-min pulse of 3H-uridine and an exposure time of 6 months. Two successive quantitative image analyses, one derived from the 50% probability circle method and the other from the cross-fire method, allowed us to estimate the radioactivity incorporated within each nucleolus compartment. This study demonstrated that rDNA transcription occurred mainly at the border between fibrillar centers and dense fibrillar components and to a lesser extent within the dense fibrillar component. The other Sertoli cell nucleoli compartments did not incorporate 3H-uridine and therefore were not involved in rDNA transcription. PMID:7696977

  8. A Single Dose of Di(2-ethylhexyl) Phthalate in Neonatal Rats Alters Gonocytes, Reduces Sertoli Cell Proliferation, and Decreases Cyclin D2 Expression

    Microsoft Academic Search

    Ling-Hong Li; William F. Jester; Andrew L. Laslett; Joanne M. Orth

    2000-01-01

    In this study, we explored the impact on both Sertoli cells and gonocytes of a single, relatively low dose of di-(2-ethylhexyl) phthalate (DEHP; 20–500 mg\\/kg) administered in vivo to 3-day-old rat pups. In parallel, we assessed the potential for two immediate metabolites of DEHP to produce similar testicular changes and began to explore the possible mechanisms involved. Morphological examination revealed

  9. Nutritional management during fetal and postnatal life, and the influence on testicular stereology and Sertoli cell numbers in Corriedale ram lambs

    Microsoft Academic Search

    Alejandro Bielli; Helena Katz; Graciela Pedrana; Mar??a Teresa Gastel; Antonio Moraña; Alejandro Castrillejo; Nils Lundeheim; Mats Forsberg; Heriberto Rodriguez-Martinez

    2001-01-01

    The aim of the experiment was to determine whether supplementation of the lamb-ewe unit during intra-uterine and postnatal life affects testicular stereology, particularly Sertoli cell numbers, in 120 pregnant Corriedale ewes grazed either native pastures (control group) or improved pastures+grain supplement (treated group). Ewes bearing single ram lambs were maintained under the same feeding regime until lambs were castrated (99

  10. Genes involved in nonpermissive temperature-induced cell differentiation in Sertoli TTE3 cells bearing temperature-sensitive simian virus 40 large T-antigen

    SciTech Connect

    Tabuchi, Yoshiaki [Division of Molecular Genetics, Life Scientific Research Center, Toyama Medical and Pharmaceutical University, Toyama 930-0194 (Japan)]. E-mail: ytabu@ms.toyama-mpu.ac.jp; Kondo, Takashi [Department of Radiological Sciences, Faculty of Medicine, Toyama Medical and Pharmaceutical University, Toyama 930-0194 (Japan); Suzuki, Yoshihisa [Department of Cell Biology, Institute of Development, Aging and Cancer, Tohoku University, Sendai 980-0872 (Japan); Obinata, Masuo [Department of Cell Biology, Institute of Development, Aging and Cancer, Tohoku University, Sendai 980-0872 (Japan)

    2005-04-15

    Sertoli TTE3 cells, derived from transgenic mice bearing temperature-sensitive simian virus 40 large T (tsSV40LT)-antigen, proliferated continuously at a permissive temperature (33 deg C) whereas inactivation of the large T-antigen by a nonpermissive temperature (39 deg C) led to differentiation as judged by elevation of transferrin. To clarify the detailed mechanisms of differentiation, we investigated the time course of changes in gene expression using cDNA microarrays. Of the 865 genes analyzed, 14 genes showed increased levels of expression. Real-time quantitative PCR revealed that the mRNA levels of p21{sup waf1}, milk fat globule membrane protein E8, heat-responsive protein 12, and selenoprotein P were markedly elevated. Moreover, the differentiated condition induced by the nonpermissive temperature significantly increased mRNA levels of these four genes in several cell lines from the transgenic mice bearing the oncogene. The present results regarding changes in gene expression will provide a basis for a further understanding of molecular mechanisms of differentiation in both Sertoli cells and cell lines transformed by tsSV40LT-antigen.

  11. Predictive Factors of Successful Microdissection Testicular Sperm Extraction in Patients with Presumed Sertoli Cell-Only Syndrome

    PubMed Central

    Modarresi, Tahereh; Hosseinifar, Hani; Daliri Hampa, Ali; Chehrazi, Mohammad; Hosseini, Jalil; Farrahi, Faramarz; Dadkhah, Farid; Sabbaghian, Marjan; Sadighi Gilani, Mohammad Ali

    2015-01-01

    Background To evaluate predictive factors of successful microdissection-testicular sperm extraction (MD-TESE) in patients with presumed Sertoli cell-only syndrome (SCOS). Materials and Methods In this retrospective analysis, 874 men with non-obstructive azoospermia (NOA), among whom 148 individuals with diagnosis of SCOS in prior biopsy, underwent MD-TESE at Department of Andrology, Royan Institute, Tehran, Iran. The predictive values of follicle stimulating hormone (FSH), luteinizing hormone (LH), and testosterone (T) levels, testicular volume, as well as male age for retrieving testicular sperm by MD-TESE were analyzed by multiple logistic regression analysis. Results Testicular sperm were successfully retrieved in 23.6% men with presumed SCOS. Using receiver operating characteristic (ROC) curve analysis, it was shown that sperm retrieval rate in the group of men with FSH values >15.25% was 28.9%. This was higher than the group of men with FSH ?15.25 (11.8%). Conclusion Sperm retrieval rate (SRR) was 23.6% in men with presumed SCOS and FSH level can be a fair predictor for SPR at MD-TESE. MD-TESE appears to be recommendable in such cases (SCOS with high FSH concentration) with reasonable results. PMID:25918598

  12. Novel Role for p110? PI 3-Kinase in Male Fertility through Regulation of Androgen Receptor Activity in Sertoli Cells.

    PubMed

    Guillermet-Guibert, Julie; Smith, Lee B; Halet, Guillaume; Whitehead, Maria A; Pearce, Wayne; Rebourcet, Diane; León, Kelly; Crépieux, Pascale; Nock, Gemma; Strömstedt, Maria; Enerback, Malin; Chelala, Claude; Graupera, Mariona; Carroll, John; Cosulich, Sabina; Saunders, Philippa T K; Huhtaniemi, Ilpo; Vanhaesebroeck, Bart

    2015-07-01

    The organismal roles of the ubiquitously expressed class I PI3K isoform p110? remain largely unknown. Using a new kinase-dead knockin mouse model that mimics constitutive pharmacological inactivation of p110?, we document that full inactivation of p110? leads to embryonic lethality in a substantial fraction of mice. Interestingly, the homozygous p110? kinase-dead mice that survive into adulthood (maximum ~26% on a mixed genetic background) have no apparent phenotypes, other than subfertility in females and complete infertility in males. Systemic inhibition of p110? results in a highly specific blockade in the maturation of spermatogonia to spermatocytes. p110? was previously suggested to signal downstream of the c-kit tyrosine kinase receptor in germ cells to regulate their proliferation and survival. We now report that p110? also plays a germ cell-extrinsic role in the Sertoli cells (SCs) that support the developing sperm, with p110? inactivation dampening expression of the SC-specific Androgen Receptor (AR) target gene Rhox5, a homeobox gene critical for spermatogenesis. All extragonadal androgen-dependent functions remain unaffected by global p110? inactivation. In line with a crucial role for p110? in SCs, selective inactivation of p110? in these cells results in male infertility. Our study is the first documentation of the involvement of a signalling enzyme, PI3K, in the regulation of AR activity during spermatogenesis. This developmental pathway may become active in prostate cancer where p110? and AR have previously been reported to functionally interact. PMID:26132308

  13. Novel Role for p110? PI 3-Kinase in Male Fertility through Regulation of Androgen Receptor Activity in Sertoli Cells

    PubMed Central

    Guillermet-Guibert, Julie; Smith, Lee B.; Halet, Guillaume; Whitehead, Maria A.; Pearce, Wayne; Rebourcet, Diane; León, Kelly; Crépieux, Pascale; Nock, Gemma; Strömstedt, Maria; Enerback, Malin; Chelala, Claude; Graupera, Mariona; Carroll, John; Cosulich, Sabina; Saunders, Philippa T. K.; Huhtaniemi, Ilpo; Vanhaesebroeck, Bart

    2015-01-01

    The organismal roles of the ubiquitously expressed class I PI3K isoform p110? remain largely unknown. Using a new kinase-dead knockin mouse model that mimics constitutive pharmacological inactivation of p110?, we document that full inactivation of p110? leads to embryonic lethality in a substantial fraction of mice. Interestingly, the homozygous p110? kinase-dead mice that survive into adulthood (maximum ~26% on a mixed genetic background) have no apparent phenotypes, other than subfertility in females and complete infertility in males. Systemic inhibition of p110? results in a highly specific blockade in the maturation of spermatogonia to spermatocytes. p110? was previously suggested to signal downstream of the c-kit tyrosine kinase receptor in germ cells to regulate their proliferation and survival. We now report that p110? also plays a germ cell-extrinsic role in the Sertoli cells (SCs) that support the developing sperm, with p110? inactivation dampening expression of the SC-specific Androgen Receptor (AR) target gene Rhox5, a homeobox gene critical for spermatogenesis. All extragonadal androgen-dependent functions remain unaffected by global p110? inactivation. In line with a crucial role for p110? in SCs, selective inactivation of p110? in these cells results in male infertility. Our study is the first documentation of the involvement of a signalling enzyme, PI3K, in the regulation of AR activity during spermatogenesis. This developmental pathway may become active in prostate cancer where p110? and AR have previously been reported to functionally interact. PMID:26132308

  14. Effects of 4-nonylphenol isomers on cell receptors and mitogen-activated protein kinase pathway in mouse Sertoli TM4 cells.

    PubMed

    Liu, Xiaozhen; Nie, Shaoping; Chen, Yangjie; Huang, Danfei; Xie, Mingyong

    2014-12-01

    In the present study, experiments were performed to investigate the effects of nonylphenol (NP) isomers (4-[1,2, 4-trimethylhexyl]-phenol (NP41), 4-[1,2, 5-trimethylhexyl]-phenol (NP42)) on Sertoli TM4 cells. NP41 decreased mRNA expression levels of androgen receptor and toll-like receptor (TLR)-4 in 20-40?M (P<0.05), and increased mRNA levels of estrogen receptor (ER)-? and progesterone receptor in 1-40?M (P<0.05). NP42 treatment only evoked significant decrease in mRNA expression levels of ER-? in 20-40?M (P<0.05). Similarly, NP41 (1-40?M) drastically increased the protein expression of ER-?, which was significantly decreased in 20-40?M NP42 groups (P<0.01). Both NP41 and NP42 showed no effect on the expression of ER-?. Protein levels of follicle stimulating hormone receptor were increased significantly in high concentrations of NP41 (40?M) and NP42 (10-40?M) challenged cells. Furthermore, NP41 and NP42 showed various effects on the expression of junction-associated molecules and inhibin B secretion in TM4 cells. Additionally, activation of JNK1/2 pathway was induced by NP41 and NP42. However, ERK1/2 and p38 pathways were inhibited in TM4 cells exposed to low concentrations of NP41 (0.1-20?M) and NP42 (0.1-1?M), and high concentrations of NP41 (40?M) and NP42 (10-40?M) resulted in a return of p-ERK1/2 and p-p38 to control levels. We proposed that molecular mechanism of reproductive damage in Sertoli cells induced by NPs may be mediated by cell receptors and/or cell signaling pathways, and the effects may be related to the structure of NP isomer. PMID:25242005

  15. Sertoli-cell-specific knockout of connexin 43 leads to multiple alterations in testicular gene expression in prepubertal mice

    PubMed Central

    Giese, Sarah; Hossain, Hamid; Markmann, Melanie; Chakraborty, Trinad; Tchatalbachev, Svetlin; Guillou, Florian; Bergmann, Martin; Failing, Klaus; Weider, Karola; Brehm, Ralph

    2012-01-01

    SUMMARY A significant decline in human male reproductive function has been reported for the past 20 years but the molecular mechanisms remain poorly understood. However, recent studies showed that the gap junction protein connexin-43 (CX43; also known as GJA1) might be involved. CX43 is the predominant testicular connexin (CX) in most species, including in humans. Alterations of its expression are associated with different forms of spermatogenic disorders and infertility. Men with impaired spermatogenesis often exhibit a reduction or loss of CX43 expression in germ cells (GCs) and Sertoli cells (SCs). Adult male transgenic mice with a conditional knockout (KO) of the Gja1 gene [referred to here as connexin-43 (Cx43)] in SCs (SCCx43KO) show a comparable testicular phenotype to humans and are infertile. To detect possible signaling pathways and molecular mechanisms leading to the testicular phenotype in adult SCCx43KO mice and to their failure to initiate spermatogenesis, the testicular gene expression of 8-day-old SCCx43KO and wild-type (WT) mice was compared. Microarray analysis revealed that 658 genes were significantly regulated in testes of SCCx43KO mice. Of these genes, 135 were upregulated, whereas 523 genes were downregulated. For selected genes the results of the microarray analysis were confirmed using quantitative real-time PCR and immunostaining. The majority of the downregulated genes are GC-specific and are essential for mitotic and meiotic progression of spermatogenesis, including Stra8, Dazl and members of the DM (dsx and map-3) gene family. Other altered genes can be associated with transcription, metabolism, cell migration and cytoskeleton organization. Our data show that deletion of Cx43 in SCs leads to multiple alterations of gene expression in prepubertal mice and primarily affects GCs. The candidate genes could represent helpful markers for investigators exploring human testicular biopsies from patients showing corresponding spermatogenic deficiencies and for studying the molecular mechanisms of human male sterility. PMID:22699423

  16. Cardiotonic steroid ouabain stimulates expression of blood-testis barrier proteins claudin-1 and -11 and formation of tight junctions in Sertoli cells.

    PubMed

    Dietze, Raimund; Shihan, Mazen; Stammler, Angelika; Konrad, Lutz; Scheiner-Bobis, Georgios

    2015-04-15

    The interaction of ouabain with the sodium pump induces signalling cascades resembling those triggered by hormone/receptor interactions. In the rat Sertoli cell line 93RS2, ouabain at low concentrations stimulates the c-Src/c-Raf/Erk1/2 signalling cascade via its interaction with the ?4 isoform of the sodium pump expressed in these cells, leading to the activation of the transcription factor CREB. As a result of this signalling sequence, ouabain stimulates expression of claudin-1 and claudin-11, which are also controlled by a CRE promoter. Both of these proteins are known to be essential constituents of tight junctions (TJ) between Sertoli cells, and as a result of the ouabain-induced signalling TJ formation between neighbouring Sertoli cells is significantly enhanced by the steroid. Thus, ouabain-treated cell monolayers display higher transepithelial resistance and reduced free diffusion of FITC-coupled dextran in tracer diffusion assays. Taking into consideration that the formation of TJ is indispensable for the maintenance of the blood-testis barrier (BTB) and therefore for male fertility, the actions of ouabain described here and the fact that this and other related cardiotonic steroids (CTS) are produced endogenously suggest a direct influence of ouabain/sodium pump interactions on the maintenance of the BTB and thereby an effect on male fertility. Since claudin-1 and claudin-11 are also present in other blood-tissue barriers, one can speculate that ouabain and perhaps other CTS influence the dynamics of these barriers as well. PMID:25666991

  17. Trafficking of sulfated glycoprotein-1 (prosaposin) to lysosomes or to the extracellular space in rat Sertoli cells.

    PubMed

    Igdoura, S A; Rasky, A; Morales, C R

    1996-03-01

    Sulfated glycoprotein-1 (prosaposin) exists in 2 forms: a 65kDa form targeted to lysosomes and a 70kDa form secreted extracellularly. In order to understand the sorting and targeting mechanisms of the two forms of SGP-1, we have compared their maturation, processing, and secretion in rat Sertoli cells in vivo. Metabolic labeling experiments in vivo demonstrated that the 65kDa form is synthesized first, then post-translationally modified to the 70kDa form of SGP-1. Subcellular fractionation of testicular homogenate was used to obtain Golgi fractions containing up to 50-fold enrichment in galactosyltransferase. Permeabilization of enriched Golgi fractions with saponin released the 70kDa form, but did not affect the 65kDa protein. While excess free mannose 6-phosphate did not release lysosomal SGP-1, it released the 35kDa cathepsin L from Golgi membranes. Using quantitative electron-microscopic immunocytochemistry, the lysosomal contents of SGP-1 were shown to increase significantly after the administration of tunicamycin in vivo. Therefore, the trafficking of the 65kDa form of SGP-1 to the lysosomes appears to be independent of the M6P-receptor pathway. The 70kDa form of SGP-1 was found to aggregate within perforated Golgi fractions in a process which depends on low pH and calcium ions. We conclude that the targeting of the 65kDa form of SGP-1 to the lysosomes involves an early association with Golgi membrane that is independent of mannose 6-phosphate receptors. PMID:8593668

  18. Cardiac glycoside ouabain induces activation of ATF-1 and StAR expression by interacting with the ?4 isoform of the sodium pump in Sertoli cells.

    PubMed

    Dietze, Raimund; Konrad, Lutz; Shihan, Mazen; Kirch, Ulrike; Scheiner-Bobis, Georgios

    2013-03-01

    Sertoli cells express ?1 and ?4 isoforms of the catalytic subunit of Na(+),K(+)-ATPase (sodium pump). Our recent findings demonstrated that interactions of the ?4 isoform with cardiotonic steroids (CTS) like ouabain induce signaling cascades that resemble the so-called non-classical testosterone pathway characterized by activation of the c-Src/c-Raf/Erk1/2/CREB signaling cascade. Here we investigate a possible physiological significance of the activated cascade. The results obtained in the current investigation show that the ouabain-induced signaling cascade also leads to the activation of the CREB-related activating transcription factor 1 (ATF-1) in the Sertoli cell line 93RS2 in a concentration- and time-dependent manner, as demonstrated by detection of ATF-1 phosphorylated on Ser63 in western blots. The ouabain-activated ATF-1 protein was found to localize to the cell nuclei. The sodium pump ?4 isoform mediates this activation, as it is ablated when cells are incubated with siRNA to the ?4 isoform. Ouabain also leads to increased expression of steroidogenic acute regulator (StAR) protein, which has been shown to be a downstream consequence of CREB/ATF-1 activation. Taking into consideration that CTS are most likely produced endogenously, the demonstrated induction of StAR expression by ouabain establishes a link between CTS, the ?4 isoform of the sodium pump, and steroidogenesis crucial for male fertility and reproduction. PMID:23220124

  19. Follicular dendritic cell function and murine AIDS.

    PubMed Central

    Masuda, A; Burton, G F; Fuchs, B A; Bhogal, B S; Rupper, R; Szakal, A K; Tew, J G

    1994-01-01

    Infection of mice with LP-BM5 elicits an immunodeficiency state referred to as murine acquired immune deficiency syndrome (MAIDS). Shortly after infection, retrovirus particles become associated with follicular dendritic cells (FDC) and this study was undertaken to determine whether retroviruses alter FDC functions. The FDC functions examined included the ability to: (1) retain antigen (Ag) trapped prior to infection; (2) trap new Ag after infection; (3) maintain specific IgG responses; and (4) provide co-stimulatory signals to B cells. Mice were infected with LP-BM5 and the ability of their FDC to trap and retain 125I-Ag (HSA) was assessed. Serum anti-HSA levels were monitored and FDC co-stimulatory activity was indicated by increased B-cell proliferation. HSA trapped on FDC prior to infection began to disappear by 3 weeks and was practically gone by 6 weeks. Serum anti-HSA titres were maintained normally for about 3 weeks after infection and then declined precipitously. The ability of FDC to trap new Ag began to disappear around the second and third week of infection and was markedly depressed by the fourth week. However, FDC recovered from infected mice retained their ability to co-stimulate anti-mu- and interleukin-4 (IL-4)-activated B cells throughout a 5-week period. In short, the ability of FDC to trap and retain specific Ag and maintain specific antibody levels was markedly depressed after retrovirus infection. However, FDC from infected mice continued to provide co-stimulatory signals and these signals may contribute to the lymphadenopathy and splenomegaly characteristic of MAIDS. Images Figure 4 PMID:8132218

  20. Short survival of phosphatidylserine-exposing red blood cells in murine sickle cell anemia

    Microsoft Academic Search

    Kitty de Jong; Renee K. Emerson; James Butler; Jacob Bastacky; Narla Mohandas; Frans A. Kuypers

    2010-01-01

    Several transgenic murine models for sickle cell anemia have been developed that closely reproduce the biochemical and physiological disorders in the human disease. A comprehensive characteriza- tion is described of hematologic parame- ters of mature red blood cells, reticulo- cytes, and red cell precursors in the bone marrow and spleen of a murine sickle cell model in which erythroid cells

  1. Azoospermia and Sertoli-cell-only syndrome: hypoxia in the sperm production site due to impairment in venous drainage of male reproductive system.

    PubMed

    Gat, Yigal; Gornish, M; Perlow, A; Chakraborty, J; Levinger, U; Ben-Shlomo, I; Pasqualotto, F

    2010-10-01

    Sertoli-cell-only (SCO) syndrome, or germ cell aplasia, is diagnosed on testicular biopsy when germ cells are seen to be absent without histological impairment of Sertoli or Leydig cells. It is considered a situation of irreversible infertility. Recent studies have shown that varicocele, a bilateral disease, causes hypoxia in the testicular microcirculation. Destruction of one-way valves in the internal spermatic veins (ISV) elevates hydrostatic pressure in the testicular venules, exceeding the pressure in the arteriolar system. The positive pressure gradient between arterial and venous system is reversed, causing hypoxia in the sperm production site. Sperm production deteriorates gradually, progressing to azoospermia. Our prediction was that, if genetic problems are excluded, SCO may be the final stage of longstanding hypoxia which deteriorates sperm production in a progressive process over time. This would indicate that SCO is not always an independent disease entity, but may represent deterioration of the testicular parenchyma beyond azoospermia. Our prediction is confirmed by histology of the seminiferous tubules demonstrating that SCO is associated with extensive degenerative ischaemic changes and destruction of the normal architecture of the sperm production site. Adequate treatment of bilateral varicocele by microsurgery or by selective sclerotherapy of the ISV resumes, at least partially, the flow of oxygenated blood to the sperm production site and restored sperm production in 4 out of 10 patients. Based on our findings the following statements can be made: (i) SCO may be related in part of the cases to persistent, longstanding testicular parenchymal hypoxia; (ii) germ cells may still exist in other areas of the testicular parenchyma; and (iii) if genetic problems are excluded, adequate correction of the hypoxia may restore very limited sperm production in some patients. PMID:20860630

  2. Xenograft of microencapsulated Sertoli cells for the cell therapy of type 2 diabetes mellitus in spontaneously diabetic nonhuman primates: preliminary data.

    PubMed

    Luca, G; Cameron, D F; Arato, I; Mancuso, F; Linden, E H; Calvitti, M; Falabella, G; Szekeres, K; Bodo, M; Ricci, G; Hansen, B C; Calafiore, R

    2014-01-01

    Insulin resistance in type 2 diabetes mellitus (T2DM) may be due to a chronic inflammation of the visceral adipose tissue (VAT) leading to local and systemic increases in proinflammatory cytokines. Microencapsulated porcine Sertoli cells (MC-pSC), by provision of immunomodulatory and trophic factors, have been successfully used to reduce such inflammation in rodent animal models of type 1 diabetes with no complications or deleterious side effects. Herein, we have begun to investigate this novel and safe therapeutic approach in the spontaneously obese nonhuman primate with spontaneous, insulin-dependent T2DM. After MC-pSC intraperitoneal injection we have evaluated, throughout a 6-month follow-up period, daily ad libitum fed glucose levels, daily exogenous insulin supplementation, biweekly body weight measurements, periodic fasting blood glucose concentrations, glycated hemoglobin (HbA1c) levels, glucose tolerance tests (GTT), and fluorescence-activated cell sorting cytometry (FACS) assessment of peripheral blood mononuclear cells. Very preliminarily, we have observed a slight reduction in fasting (FPG) and mean nonfasting (NF) plasma glucose levels. We found minimal changes, only in 1 animal, in daily exogenous insulin requirements and HbA1c levels. Flow cytometric analysis was associated with decrease in CD8(+) cells only in 1 recipient with a reduction in mean regulatory T Cells (Treg), whereas interestingly, decrease of B lymphocytes was observed in both animals. These results may suggest that this novel MC-SC-based transplantation protocol might possibly impact the metabolic status of T2DM in higher mammals that are close to humans. PMID:25131093

  3. Mitogen-activated protein kinase and Akt pathways are involved in 4-n-nonyphenol induced apoptosis in mouse Sertoli TM4 cells.

    PubMed

    Liu, Xiaozhen; Nie, Shaoping; Huang, Danfei; Xie, Mingyong

    2015-03-01

    Nonylphenol (NP) is considered an important environmental toxicant, which may disrupt male reproductive system. The aim of this study was to investigate 4-n-nonylphenol (4-n-NP) induced apoptosis and its related mechanism in mouse Sertoli cell line, TM4 cells. Our results showed that NP treatment (0.1, 1, 10, 20 and 30 ?M) decreased cell viability and induced apoptosis in the cells, accompanied by alteration of Bcl-2 family mRNA expression, activation of caspases-3, release of Ca(2+), and increase of reactive oxygen species (ROS) generation. Subsequently, it was found that the levels of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-PX) in the cells were markedly decreased, and maleic dialdehyde (MDA) content was increased by NP treatment. Then activation of the mitogen-activated protein kinases (MAPKs) pathways and inhibition of Akt pathway were simultaneously detected in NP challenged TM4 cells. Taken together, it was concluded that NP induced cytotoxicity and apoptosis in TM4 cells, and the apoptosis may be mediated via MAPKs and Akt pathways in addition to Ca(2+) release and ROS generation. PMID:25748095

  4. Poliovirus type 1 infection of murine PRNP -knockout neuronal cells

    Microsoft Academic Search

    Andreina Baj; Alessia Bettaccini; Takuya Nishimura; Takashi Onodera; Antonio Toniolo

    2005-01-01

    Transfection of the prion protein gene (Prnp) into prion-deficient mouse cells was shown to reduce the replication of coxsackievirus B3, an enterovirus. Because mice\\u000a can be susceptible to poliovirus infection by parenteral routes, the authors tested the susceptibility to poliovirus-1 (PV-1)\\u000a of a panel of murine neuronal cell lines differing in their ability to express Prnp. The investigated cell lines

  5. Hexavalent chromium at low concentration alters Sertoli cell barrier and connexin 43 gap junction but not claudin-11 and N-cadherin in the rat seminiferous tubule culture model

    SciTech Connect

    Carette, Diane [INSERM U 1065, Team 5 “Physiopathology of Germ Cell Control: Genomic and Non Genomic Mechanisms” C3M, University of Nice Sophia Antipolis, Nice (France); UMR S775, University Paris Descartes, 45 rue des Saints Pères, 75006, Paris (France); Perrard, Marie-Hélène, E-mail: marie-helene.durand@ens-lyon.fr [Institut de Génomique Fonctionnelle de Lyon, Université de Lyon, Université Lyon I, CNRS, INRA, Ecole Normale Supérieure de Lyon, Lyon (France); Prisant, Nadia [University of Versailles/St Quentin-en-Yvelines (France); UMR S775, University Paris Descartes, 45 rue des Saints Pères, 75006, Paris (France); Gilleron, Jérome; Pointis, Georges [INSERM U 1065, Team 5 “Physiopathology of Germ Cell Control: Genomic and Non Genomic Mechanisms” C3M, University of Nice Sophia Antipolis, Nice (France); Segretain, Dominique [University of Versailles/St Quentin-en-Yvelines (France); UMR S775, University Paris Descartes, 45 rue des Saints Pères, 75006, Paris (France); Durand, Philippe [Institut de Génomique Fonctionnelle de Lyon, Université de Lyon, Université Lyon I, CNRS, INRA, Ecole Normale Supérieure de Lyon, Lyon (France); Kallistem SAS Ecole Normale Supérieure de Lyon, Lyon (France)

    2013-04-01

    Exposure to toxic metals, specifically those belonging to the nonessential group leads to human health defects and among them reprotoxic effects. The mechanisms by which these metals produce their negative effects on spermatogenesis have not been fully elucidated. By using the Durand's validated seminiferous tubule culture model, which mimics the in vivo situation, we recently reported that concentrations of hexavalent chromium, reported in the literature to be closed to that found in the blood circulation of men, increase the number of germ cell cytogenetic abnormalities. Since this metal is also known to affect cellular junctions, we investigated, in the present study, its potential influence on the Sertoli cell barrier and on junctional proteins present at this level such as connexin 43, claudin-11 and N-cadherin. Cultured seminiferous tubules in bicameral chambers expressed the three junctional proteins and ZO-1 for at least 12 days. Exposure to low concentrations of chromium (10 ?g/l) increased the trans-epithelial resistance without major changes of claudin-11 and N-cadherin expressions but strongly delocalized the gap junction protein connexin 43 from the membrane to the cytoplasm of Sertoli cells. The possibility that the hexavalent chromium-induced alteration of connexin 43 indirectly mediates the effect of the toxic metal on the blood–testis barrier dynamic is postulated. - Highlights: ? Influence of Cr(VI) on the Sertoli cell barrier and on junctional proteins ? Use of cultured seminiferous tubules in bicameral chambers ? Low concentrations of Cr(VI) (10 ?g/l) altered the trans-epithelial resistance. ? Cr(VI) did not alter claudin-11 and N-cadherin. ? Cr(VI) delocalized connexin 43 from the membrane to the cytoplasm of Sertoli cells.

  6. Human Pontine Glioma Cells Can Induce Murine Tumors

    PubMed Central

    Caretti, Viola; Sewing, A. Charlotte P.; Lagerweij, Tonny; Schellen, Pepijn; Bugiani, Marianna; Jansen, Marc H. A.; van Vuurden, Dannis G.; Navis, Anna C.; Horsman, Ilona; Vandertop, W. Peter; Noske, David P.; Wesseling, Pieter; Kaspers, Gertjan J.L.; Nazarian, Javad; Vogel, Hannes; Hulleman, Esther; Monje, Michelle; Wurdinger, Thomas

    2014-01-01

    Diffuse intrinsic pontine glioma (DIPG), with a median survival of only nine months, is the leading cause of pediatric brain cancer mortality. Dearth of tumor tissue for research has limited progress in this disease until recently. New experimental models for DIPG research are now emerging. To develop preclinical models of DIPG, two different methods were adopted: cells obtained at autopsy 1) were directly xenografted orthotopically into the pons of immunodeficient mice without an intervening cell culture step or 2) were first cultured in vitro and, upon successful expansion, injected in vivo. Both strategies resulted in pontine tumors histopathologically similar to the original human DIPG tumors. However, following the direct transplantation method all tumors proved to be composed of murine and not of human cells. This is in contrast to the indirect method that included initial in vitro culture and resulted in xenografts comprised of human cells. Of note, direct injection of cells obtained post mortem from the pons and frontal lobe of human brains not affected by cancer did not give rise to neoplasms. The murine pontine tumors exhibited an immunophenotype similar to human DIPG, but were also positive for microglia/macrophage markers, such as CD45, CD68 and CD11b. Serial orthotopic injection of these murine cells results in lethal tumors in recipient mice. Direct injection of human DIPG cells in vivo can give rise to malignant murine tumors. This represents an important caveat for xenotransplantation models of DIPG. In contrast, an initial in vitro culture step can allow establishment of human orthotopic xenografts. The mechanism underlying this phenomenon observed with direct xenotransplantation remains an open question. PMID:24777482

  7. Regulation of follitropin-sensitive adenylate cyclase by stimulatory and inhibitory forms of the guanine nucleotide regulatory protein in immature rat Sertoli cells

    SciTech Connect

    Johnson, G.P.

    1987-01-01

    Studies have been designed to examine the role of guanine nucleotides in mediating FSH-sensitive adenylate cyclase activity in Sertoli cell plasma membranes. Analysis of ({sup 3}H)GDP binding to plasma membranes suggested a single high affinity site with a K{sub d} = 0.24 uM. Competition studies indicated that GTP{sub {gamma}}S was 7-fold more potent than GDP{sub {beta}}S. Bound GDP could be released by FSH in the presence of GTP{sub {gamma}}S, but not by FSH alone. Adenylate cyclase activity was enhanced 5-fold by FSH in the presence of GTP. Addition of GDP{sub {beta}}S to the activated enzyme (FSH plus GTP) resulted in a time-dependent decay to basal activity within 20 sec. GDP{sub {beta}}S competitively inhibited GTP{sub {gamma}}S-stimulated adenylate cyclase activity with a K{sub i} = 0.18 uM. Adenylate cyclase activity was also demonstrated to be sensitive to the nucleotide bound state. In the presence of FSH, only the GTP{sub {gamma}}S-bound form persisted even if GDP{sub {beta}}S previously occupied all available binding sites. Two membrane proteins, M{sub r} = 43,000 and 48,000, were ADP{centered dot}ribosylated using cholera toxin and labeling was enhanced 2 to 4-fold by GTP{sub {gamma}}S but not by GDP{sub {beta}}S. The M{sub r} = 43,000 and 48,000 proteins represented variant forms of G{sub S}. A single protein of M{sub r} = 40,000 (G{sub i}) was ADP-ribosylated by pertussis toxin in vitro. GTP inhibited forskolin-stimulated adenylate cyclase activity with an IC{sub 50} = 0.1 uM. The adenosine analog, N{sup 6}{centered dot}phenylisopropyl adenosine enhanced GTP inhibition of forskolin-stimulated adenylate cyclase activity by an additional 15%. GTP-dependent inhibition of forskolin-sensitive adenylate cyclase activity was abolished in membranes prepared from Sertoli cells treated in culture with pertussis toxin.

  8. Cell fusion induced by the murine leukemia virus envelope glycoprotein.

    PubMed Central

    Jones, J S; Risser, R

    1993-01-01

    To determine whether ecotropic murine leukemia virus (MuLV) envelope glycoproteins are sufficient to cause cell-to-cell fusion when expressed in the absence of virus production, we used an ecotropic MuLV, AKV, to construct env expression vectors that lack the gag and pol genes. The rat cell line XC, which undergoes cell-to-cell fusion upon infection with ecotropic MuLV, was transfected with wild-type env expression vectors, and high levels of syncytium formation resulted. Transfection of the murine cell line NIH 3T3 with expression vectors containing the wild-type or mutated env region did not result in syncytium formation. Immunoprecipitation analysis of the envelope glycoproteins expressed in NIH 3T3 and XC cells showed that the mature surface glycoprotein expressed in XC cells was of a much lower apparent molecular weight than that expressed in NIH 3T3 cells. Further characterization showed that most if not all of this difference was the result of differences in glycosylation. Finally, site-directed mutagenesis was used to introduce several conservative and nonconservative changes into the amino-terminal region of the transmembrane protein. Analysis of the effect of these mutations confirmed that this region is a fusion domain. Images PMID:8416389

  9. Murine Oligodendroglial Cells Express Nerve Growth Factor

    Microsoft Academic Search

    Sujatha Byravan; Lyndon M. Foster; Tommy Phan; A. Neil Verity; Anthony T. Campagnoni

    1994-01-01

    The studies reported here present evidence for the expression of nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) by an oligodendroglial cell line and of NGF by oligodendrocytes in mouse primary culture. An immortalized oligodendroglial cell line (N19) expressing markers for immature oligodendrocytes stimulated PC12 cells to elaborate processes. Polymerase chain reaction analysis with degenerate primers indicated that the

  10. Isolation of murine pluripotent hemopoietic stem cells

    PubMed Central

    1984-01-01

    A method described to purify pluripotent hemopoietic stem cells ( PHSC ) from adult mouse bone marrow. The method consists of three separation steps. First, bone marrow cells are centrifuged in a discontinuous metrizamide gradient and simultaneously labeled with wheat germ agglutinin-fluorescein isothiocyanate (WGA-FITC). Second, the low density cells are analyzed by a fluorescence-activated cell sorter (FACS) and the WGA-positive cells with medium forward and low perpendicular light scatter intensities are sorted. The WGA-FITC is removed from the cells by incubation with N-acetyl-D-glucosamine. Finally, the sorted cells are incubated with anti-H-2K-biotin and avidin-FITC and sorted a second time to enrich cells with high H-2K density. The sorted cells gave rise to 2 spleen colonies per 100 injected cells at 8 d and 6.6 colonies per 100 cells at 12 d after transplantation into lethally irradiated syngeneic recipients. The average enrichment factor for day 12 CFU-S (colony-forming unit/spleen) was 135 (range, 90--230; n = 15) and was similar to that for the cell type that provides radioprotection (180 +/- 70), indicating that these functional properties were copurified. Indirect evidence suggests that the spleen-seeding efficiency (f factor) of these cells is 0.10 and, therefore, the average purity of the sorted PHSC was 65% (range in 15 experiments, 35--110%). The sorted cells were all in the G1 or G0 phase of the cell cycle. They appeared to be undifferentiated blasts by morphological criteria. Electron microscopy revealed that the sorted cells consisted primarily of two cell types, possibly representing G0 and G1 cells. The FACS was used to deposit single selected cells into individual microwells of Terasaki trays. 32% of the sorted cells could be induced to form myeloid progeny in vitro. This procedure should be useful for direct studies on the regulation of hemopoietic cell differentiation. PMID:6427383

  11. Isolation of murine natural killer cells.

    PubMed

    Pak-Wittel, Melissa A; Piersma, Sytse J; Plougastel, Beatrice F; Poursine-Laurent, Jennifer; Yokoyama, Wayne M

    2014-01-01

    This unit describes the isolation of natural killer (NK) cells from mouse spleen. The basic protocol describes a method for preparing a highly purified NK cell population from mouse spleen by depletion of contaminating cells with selected monoclonal antibodies (MAbs) and magnetic separation. There are several advantages to this negative selection process. One of these is that the NK cells are not coated with antibody and, therefore, are not at risk of functional perturbation by antibody cross-linking. Additionally, negative selection provides a way to isolate diverse subpopulations of NK cells without selectively purifying a specific subpopulation. Following enrichment, NK cell purity can be assessed by cell surface phenotype using flow cytometry. Curr. Protoc. Immunol.. 105:3.22.1-3.22.9. © 2014 by John Wiley & Sons, Inc. PMID:24700324

  12. Research Resource: Genome-Wide Identification of AR-Regulated Genes Translated in Sertoli Cells In Vivo Using the RiboTag Approach

    PubMed Central

    De Gendt, Karel; Verhoeven, Guido; Amieux, Paul S.

    2014-01-01

    An understanding of the molecular mechanisms by which androgens drive spermatogenesis has been thwarted by the fact that few consistent androgen receptor (AR) target genes have been identified. Here, we addressed this issue using next-generation sequencing coupled with the RiboTag approach, which purifies translated mRNAs expressed in cells that express cyclic recombinase (CRE). Using RiboTag mice expressing CRE in Sertoli cells (SCs), we identified genes expressed specifically in SCs in both prepubertal and adult mice. Unexpectedly, this analysis revealed that the SC-specific gene program is already largely defined at the initiation of spermatogenesis despite the subsequent dramatic maturational changes known to occur in SCs. To identify AR-regulated genes, we generated triple-mutant mice in which the SCs express the RiboTag but lack ARs. RNA sequencing analysis revealed hundreds of SC-expressed AR-regulated genes that had previously gone unnoticed, including suppressed genes involved in ovarian development. Comparison of the SC-enriched dataset with that from the whole testes allowed us to classify genes in terms of their degree of expression in SCs. This revealed that a greater fraction of AR–up-regulated genes than AR–down-regulated genes were expressed predominantly in SCs. Our results also revealed that AR signaling in SCs causes a large number of genes not detectably expressed in SCs to undergo altered expression, thereby providing genome-wide evidence for wide-scale communication between SCs and other cells. Taken together, our results identified novel classes of genes expressed in a hormone-dependent manner in different testicular cell subsets and highlight a new approach to analyze cell type–specific gene regulation. PMID:24606126

  13. Genome-wide identification of AR-regulated genes translated in Sertoli cells in vivo using the RiboTag approach.

    PubMed

    De Gendt, Karel; Verhoeven, Guido; Amieux, Paul S; Wilkinson, Miles F

    2014-04-01

    An understanding of the molecular mechanisms by which androgens drive spermatogenesis has been thwarted by the fact that few consistent androgen receptor (AR) target genes have been identified. Here, we addressed this issue using next-generation sequencing coupled with the RiboTag approach, which purifies translated mRNAs expressed in cells that express cyclic recombinase (CRE). Using RiboTag mice expressing CRE in Sertoli cells (SCs), we identified genes expressed specifically in SCs in both prepubertal and adult mice. Unexpectedly, this analysis revealed that the SC-specific gene program is already largely defined at the initiation of spermatogenesis despite the subsequent dramatic maturational changes known to occur in SCs. To identify AR-regulated genes, we generated triple-mutant mice in which the SCs express the RiboTag but lack ARs. RNA sequencing analysis revealed hundreds of SC-expressed AR-regulated genes that had previously gone unnoticed, including suppressed genes involved in ovarian development. Comparison of the SC-enriched dataset with that from the whole testes allowed us to classify genes in terms of their degree of expression in SCs. This revealed that a greater fraction of AR-up-regulated genes than AR-down-regulated genes were expressed predominantly in SCs. Our results also revealed that AR signaling in SCs causes a large number of genes not detectably expressed in SCs to undergo altered expression, thereby providing genome-wide evidence for wide-scale communication between SCs and other cells. Taken together, our results identified novel classes of genes expressed in a hormone-dependent manner in different testicular cell subsets and highlight a new approach to analyze cell type-specific gene regulation. PMID:24606126

  14. Directed Ig class switch recombination in activated murine B cells.

    PubMed Central

    Winter, E; Krawinkel, U; Radbruch, A

    1987-01-01

    Immunoglobulin class switch recombination occurs at frequencies of up to 10%/cell/generation in activated murine B-lymphocytes. We analysed cH gene rearrangements and switch recombinations from active and inactive IgH loci of B-cells activated in various ways and immortalized by cell fusion. Although about half of the IgM+ cells show rearrangement of c mu genes, the deletion of c mu is a rare event. Half of the IgG3+ and IgG1+ cells show rearrangement of c mu genes on the inactive IgH locus and the other half of the IgG+ cells have deleted c mu from both IgH loci by switch recombination. This recombination is directed to the same switch regions on both IgH loci in 60-80% of all cases. Interleukin 4 may play a critical role in programming murine B-lymphocytes for specific switch recombination. Images Fig. 1. Fig. 2. Fig. 6. PMID:3038529

  15. Flow cytometric quantification of radiation responses of murine peritoneal cells

    SciTech Connect

    Tokita, N.; Raju, M.R.

    1982-01-01

    Methods have been developed to distinguish subpopulations of murine peritoneal cells, and these were applied to the measurement of early changes in peritoneal cells after irradiation. The ratio of the two major subpopulations in the peritoneal fluid, lymphocytes and macrophages, was measured rapidly by means of cell volume distribution analysis as well as by hypotonic propidium iodide (PI) staining. After irradiation, dose and time dependent changes were noted in the cell volume distributions: a rapid loss of peritoneal lymphocytes, and an increase in the mean cell volume of macrophages. The hypotonic PI staining characteristics of the peritoneal cells showed two or three distinctive G/sub 1/ peaks. The ratio of the areas of these peaks was also found to be dependent of the radiation dose and the time after irradiation. These results demonstrate that these two parameters may be used to monitor changes induced by irradiation (biological dosimetry), and to sort different peritoneal subpopulations.

  16. A murine model of NK cell mediated resorption.

    PubMed

    Kinsky, R; Delage, G; Rosin, N; Thang, M N; Hoffmann, M; Chaouat, G

    1990-07-01

    There is increasing evidence that some models of immunologically mediated murine embryo demise involve nonspecific lytic effector cells. In this paper, we use two double stranded synthetic RNAs, known as potent interferon inducers and NK cell activators, the Poly (I). Poly (C) and the less toxic Poly (I). Poly (C12U). These polynucleotides enhance fetal resorption rates in both resorption prone and none-resorption prone strains of mice. We have studied the kinetics of the phenomenon, and observed an anti-implantation-like effect of early injection during early pregnancy. The abortifacient effects can be adoptively transferred to naive recipients by spleen cells from Ds RNA injected donors. Such effects are abrogated if the cells are pretreated with anti-NK cell antiserum. The relevance of these findings to the survival of the conceptus is suggested. PMID:2257054

  17. Generation of skeletal muscle stem/progenitor cells from murine induced pluripotent stem cells.

    PubMed

    Mizuno, Yuta; Chang, Hsi; Umeda, Katsutsugu; Niwa, Akira; Iwasa, Toru; Awaya, Tomonari; Fukada, So-ichiro; Yamamoto, Hiroshi; Yamanaka, Shinya; Nakahata, Tatsutoshi; Heike, Toshio

    2010-07-01

    Induced pluripotent stem (iPS) cells, which are a type of pluripotent stem cell generated from reprogrammed somatic cells, are expected to have potential for patient-oriented disease investigation, drug screening, toxicity tests, and transplantation therapies. Here, we demonstrated that murine iPS cells have the potential to develop in vitro into skeletal muscle stem/progenitor cells, which are almost equivalent to murine embryonic stem cells. Cells with strong in vitro myogenic potential effectively were enriched by fluorescence-activated cell sorting using the anti-satellite cell antibody SM/C-2.6. Furthermore, on transplantation into mdx mice, SM/C-2.6(+) cells exerted sustained myogenic lineage differentiation in injured muscles, while providing long-lived muscle stem cell support. Our data suggest that iPS cells have the potential to be used in clinical treatment of muscular dystrophies. PMID:20181939

  18. Cytomegalovirus infection of murine testicular interstitial Leydig cells.

    PubMed Central

    Baskar, J F; Stanat, S C; Huang, E S

    1983-01-01

    We studied the susceptibility of mouse testicular interstitial Leydig cells to cytomegalovirus both in vivo and in vitro. The in vivo studies included intratesticular and intraperitoneal infection of 6-week-old mice with murine cytomegalovirus (MCMV); the in vitro studies involved an MCMV-Leydig cell interaction using a Leydig tumor cell line (I-10). MCMV-specific antigens were detected in interstitial Leydig cells in sections of MCMV-inoculated testes by an indirect immunofluorescence test. MCMV DNA was also localized in the same testes cells derived from mice, which received intratesticular and intraperitoneal MCMV inoculations, respectively, by in situ DNA-RNA hybridization. Cytopathic effects were seen in MCMV-infected I-10 cell cultures 2 or more days after exposure to MCMV. The infected cells showed intranuclear inclusions characteristic of cytomegalovirus when stained with May-Grunwald-Giemsa stain. The indirect immunofluorescence test was also positive with MCMV-infected I-10 cells. MCMV DNA was detected in these cells by in situ DNA-RNA cytohybridization, and the presence of viral particles in MCMV-infected I-10 cells was confirmed by electron microscopy. Thus, we conclude that the interstitial Leydig cell is susceptible to MCMV infection both in vivo and in vitro. Images PMID:6302003

  19. Quantitative image analysis of cell colocalization in murine bone marrow.

    PubMed

    Mokhtari, Zeinab; Mech, Franziska; Zehentmeier, Sandra; Hauser, Anja E; Figge, Marc Thilo

    2015-06-01

    Long-term antibody production is a key property of humoral immunity and is accomplished by long-lived plasma cells. They mainly reside in the bone marrow, whose importance as an organ hosting immunological memory is becoming increasingly evident. Signals provided by stromal cells and eosinophils may play an important role for plasma cell maintenance, constituting a survival microenvironment. In this joint study of experiment and theory, we investigated the spatial colocalization of plasma cells, eosinophils and B cells by applying an image-based systems biology approach. To this end, we generated confocal fluorescence microscopy images of histological sections from murine bone marrow that were subsequently analyzed in an automated fashion. This quantitative analysis was combined with computer simulations of the experimental system for hypothesis testing. In particular, we tested the observed spatial colocalization of cells in the bone marrow against the hypothesis that cells are found within available areas at positions that were drawn from a uniform random number distribution. We find that B cells and plasma cells highly colocalize with stromal cells, to an extent larger than in the simulated random situation. While B cells are preferentially in contact with each other, i.e., form clusters among themselves, plasma cells seem to be solitary or organized in aggregates, i.e., loosely defined groups of cells that are not necessarily in direct contact. Our data suggest that the plasma cell bone marrow survival niche facilitates colocalization of plasma cells with stromal cells and eosinophils, respectively, promoting plasma cell longevity. © 2015 International Society for Advancement of Cytometry. PMID:25652548

  20. Follicle-stimulating hormone receptor-mediated uptake of sup 45 Ca sup 2+ by cultured rat Sertoli cells does not require activation of cholera toxin- or pertussis toxin-sensitive guanine nucleotide binding proteins or adenylate cyclase

    SciTech Connect

    Grasso, P.; Reichert, L.E. Jr. (Albany Medical College, NY (USA))

    1990-08-01

    We have previously reported that FSH stimulates flux of 45Ca2+ into cultured Sertoli cells from immature rats via voltage-sensitive and voltage-independent calcium channels. In the present study, we show that this effect of FSH does not require cholera toxin (CT)- or pertussis toxin (PT)-sensitive guanine nucleotide binding (G) protein or activation of adenylate cyclase (AC). Significant stimulation of 45Ca2+ influx was observed within 1 min, and maximal response (3.2-fold over basal levels) was achieved within 2 min after exposure to FSH. FSH-stimulated elevations in cellular cAMP paralleled increases in 45Ca2+ uptake, suggesting a possible coupling of AC activation to 45Ca2+ influx. (Bu)2cAMP, however, was not able to enhance 45Ca2+ uptake over basal levels at a final concentration of 1000 microM, although a concentration-related increase in androstenedione conversion to estradiol was evident. Exposure of Sertoli cells to CT (10 ng/ml) consistently stimulated basal levels of androstenedione conversion to estradiol but had no effect on basal levels of 45Ca2+ uptake. Similarly, CT had no effect on FSH-induced 45Ca2+ uptake, but potentiated FSH-stimulated estradiol synthesis. PT (10 ng/ml) augmented basal and FSH-stimulated estradiol secretion without affecting 45Ca2+ influx. The adenosine analog N6-phenylisopropyladenosine, which binds to Gi-coupled adenosine receptors on Sertoli cells, inhibited FSH-stimulated androgen conversion to estradiol in a dose-related (1-1000 nM) manner, but FSH-stimulated 45Ca2+ influx remained unchanged. Our results show that in contrast to FSH-stimulated estradiol synthesis, the flux of 45Ca2+ into Sertoli cells in response to FSH is not mediated either directly or indirectly by CT- or PT-sensitive G protein, nor does it require activation of AC. Our data further suggest that the FSH receptor itself may function as a calcium channel.

  1. Vascular cell adhesion molecule-1 (VCAM-1) expression in murine lupus nephritis

    Microsoft Academic Search

    Rudolf P Wuthrich; Tracey L Snyder

    1992-01-01

    Vascular cell adhesion molecule-1 (VCAM-1) expression in murine lupus nephritis. Vascular cell adhesion molecule-1 (VCAM-1) is a cell surface protein which mediates adherence of inflammatory cells to target cells by binding with the ?1-integrin ligand Very Late Antigen-4 (VLA-4) on leukocytes. The expression of VCAM-1 was investigated in a murine model of lupus nephritis, the autoimmune MRL\\/lpr mouse. Compared with

  2. A Murine ESC-like State Facilitates Transgenesis and Homologous Recombination in Human Pluripotent Stem Cells

    Microsoft Academic Search

    Christa Buecker; Hsu-Hsin Chen; Jose Maria Polo; Laurence Daheron; Lei Bu; Tahsin Stefan Barakat; Patricia Okwieka; Andrew Porter; Joost Gribnau; Konrad Hochedlinger; Niels Geijsen

    2010-01-01

    Murine pluripotent stem cells can exist in two functionally distinct states, LIF-dependent embryonic stem cells (ESCs) and bFGF-dependent epiblast stem cells (EpiSCs). However, human pluripotent cells so far seemed to assume only an epiblast-like state. Here we demonstrate that human iPSC reprogramming in the presence of LIF yields human stem cells that display morphological, molecular, and functional properties of murine

  3. Non-Apoptotic Toxicity of Pseudomonas aeruginosa toward Murine Cells

    PubMed Central

    Roy, Sanhita; Bonfield, Tracey; Tartakoff, Alan M.

    2013-01-01

    Although P. aeruginosa is especially dangerous in cystic fibrosis (CF), there is no consensus as to how it kills representative cell types that are of key importance in the lung. This study concerns the acute toxicity of the sequenced strain, PAO1, toward a murine macrophage cell line (RAW 264.7). Toxicity requires brief contact with the target cell, but is then delayed for more than 12 h. None of the classical toxic effectors of this organism is required and cell death occurs without phagocytosis or acute perturbation of the actin cytoskeleton. Apoptosis is not required for toxicity toward either RAW 264.7 cells or for alveolar macrophages. Transcriptional profiling shows that encounter between PAO1 and RAW 264.7 cells elicits an early inflammatory response, followed by growth arrest. As an independent strategy to understand the mechanism of toxicity, we selected variant RAW 264.7 cells that resist PAO1. Upon exposure to P. aeruginosa, they are hyper-responsive with regard to classical inflammatory cytokine production and show transient downregulation of transcripts that are required for cell growth. They do not show obvious morphologic changes. Although they do not increase interferon transcripts, when exposed to PAO1 they dramatically upregulate a subset of the responses that are characteristic of exposure to g-interferon, including several guanylate-binding proteins. The present observations provide a novel foundation for learning how to equip cells with resistance to a complex challenge. PMID:23358229

  4. FLOW CYTOMETRIC COMPARISON OF THE EFFECTS OF TRIALKYTING ON THE MURINE ERYTHROLEUKEMIC CELL

    EPA Science Inventory

    Cellular effects of exposure to tributyltin (TBT), triethyltin (TET), or trimethyltin (TMT) were investigated by flow cytometry employing the murine erythroleukemic cell (MELC) as a model cellular system. Cell viability was investigated by the carboxyfluorescein diacetate (CFDA) ...

  5. Antagonistic effects of a mixture of low-dose nonylphenol and di-n-butyl phthalate (monobutyl phthalate) on the Sertoli cells and serum reproductive hormones in prepubertal male rats in vitro and in vivo.

    PubMed

    Hu, Yang; Wang, Ruoyu; Xiang, Zou; Qian, Weiping; Han, Xiaodong; Li, Dongmei

    2014-01-01

    The estrogenic chemical nonylphenol (NP) and the antiandrogenic agent di-n-butyl phthalate (DBP) are regarded as widespread environmental endocrine disruptors (EDCs) which at high doses in some species of laboratory animals, such as mice and rats, have adverse effects on male reproduction and development. Given the ubiquitous coexistence of various classes of EDCs in the environment, their combined effects warrant clarification. In this study, we attempted to determine the mixture effects of NP and DBP on the testicular Sertoli cells and reproductive endocrine hormones in serum in male rats based on quantitative data analysis by a mathematical model. In the in vitro experiment, monobutyl phthalate (MBP), the active metabolite of DBP, was used instead of DBP. Sertoli cells were isolated from 9-day-old Sprague-Dawley rats followed by treatment with NP and MBP, singly or combined. Cell viability, apoptosis, necrosis, membrane integrity and inhibin-B concentration were tested. In the in vivo experiment, rats were gavaged on postnatal days 23-35 with a single or combined NP and DBP treatment. Serum reproductive hormone levels were recorded. Next, Bliss Independence model was employed to analyze the quantitative data obtained from the in vitro and in vivo investigation. Antagonism was identified as the mixture effects of NP and DBP (MBP). In this study, we demonstrate the potential of Bliss Independence model for the prediction of interactions between estrogenic and antiandrogenic agents. PMID:24676355

  6. Adrenocortical Cell Transplantation Reverses a Murine Model of Adrenal Failure

    PubMed Central

    Zupekan, Tatiana; Dunn, James C.Y.

    2011-01-01

    Purpose Although adrenal insufficiency can be managed with steroid replacement, transplantation of adrenocortical cells may represent a more definitive therapy. Methods An adrenal failure model was created by adding stress to mice that underwent staged bilateral adrenalectomy. Murine adrenocortical cells were seeded onto collagen sponges. The grafts were implanted under the renal capsule during the first adrenalectomy. Some mice had an additional graft placed next to the kidney. A contralateral adrenalectomy and a laparotomy were performed one week after the first adrenalectomy. Two weeks later, blood was collected for costicosterone measurement, and implants were retrieved for adrenal-specific mRNA analysis and histology. Mice that underwent the same procedures but received a graft without cells served as controls. Results Control group mortality was 100%. Mice that had only one cell-seeded implant had 42% survival, whereas mice that had two cell-seeded implants had 100% survival. Retrieved implants demonstrated viable cells and expression of adrenocortical genes. The plasma corticosterone concentration of survived animals was similar to that in normal mice. Conclusion Cells transplantation restored the adrenocortical function in these mice. Further optimization of this technique could bring a curative therapy to patients with adrenal insufficiency. PMID:21683224

  7. Control of Murine Cytomegalovirus Infection by ?? T Cells

    PubMed Central

    Sell, Sabrina; Dietz, Monika; Schneider, Andrea; Holtappels, Rafaela; Mach, Michael; Winkler, Thomas H.

    2015-01-01

    Infections with cytomegalovirus (CMV) can cause severe disease in immunosuppressed patients and infected newborns. Innate as well as cellular and humoral adaptive immune effector functions contribute to the control of CMV in immunocompetent individuals. None of the innate or adaptive immune functions are essential for virus control, however. Expansion of ?? T cells has been observed during human CMV (HCMV) infection in the fetus and in transplant patients with HCMV reactivation but the protective function of ?? T cells under these conditions remains unclear. Here we show for murine CMV (MCMV) infections that mice that lack CD8 and CD4 ??-T cells as well as B lymphocytes can control a MCMV infection that is lethal in RAG-1-/- mice lacking any T- and B-cells. ?? T cells, isolated from infected mice can kill MCMV infected target cells in vitro and, importantly, provide long-term protection in infected RAG-1-/- mice after adoptive transfer. ?? T cells in MCMV infected hosts undergo a prominent and long-lasting phenotypic change most compatible with the view that the majority of the ?? T cell population persists in an effector/memory state even after resolution of the acute phase of the infection. A clonotypically focused V?1 and V?2 repertoire was observed at later stages of the infection in the organs where MCMV persists. These findings add ?? T cells as yet another protective component to the anti-CMV immune response. Our data provide clear evidence that ?? T cells can provide an effective control mechanism of acute CMV infections, particularly when conventional adaptive immune mechanisms are insufficient or absent, like in transplant patient or in the developing immune system in utero. The findings have implications in the stem cell transplant setting, as antigen recognition by ?? T cells is not MHC-restricted and dual reactivity against CMV and tumors has been described. PMID:25658831

  8. The murine Fhit locus: isolation, characterization, and expression in normal and tumor cells.

    PubMed

    Pekarsky, Y; Druck, T; Cotticelli, M G; Ohta, M; Shou, J; Mendrola, J; Montgomery, J C; Buchberg, A M; Siracusa, L D; Manenti, G; Fong, L Y; Dragani, T A; Croce, C M; Huebner, K

    1998-08-01

    The murine Fhit locus maps near the centromere nu proximal Ptprg locus on mouse chromosome 14. The cDNA sequence and structure are similar to those of the human gene, with exons 5-9 encoding the protein. The predominant mRNA in the tissues and cell lines tested was an alternatively spliced form missing exon 3. Most murine cell lines tested, including lines established from normal mouse embryos and tumors, expressed very low or undetectable levels of Fhit mRNA. Most normal mouse tissues expressed wild-type Fhit mRNA, whereas approximately 40% of murine lung carcinomas expressed wild-type and aberrant Fhit RT-PCR products that lacked various exons. Several tumorigenic mouse cell lines exhibited homozygous deletions of Fhit exons. We conclude that the murine Fhit gene, like its human counterpart, is a target of alterations involved in murine carcinogenesis. PMID:9699672

  9. Toxicity of Calcium Hydroxide Nanoparticles on Murine Fibroblast Cell Line

    PubMed Central

    Dianat, Omid; Azadnia, Sina; Mozayeni, Mohammad Ali

    2015-01-01

    Introduction: One of the major contributing factors, which may cause failure of endodontic treatment, is the presence of residual microorganisms in the root canal system. For years, most dentists have been using calcium hydroxide (CH) as the intracanal medicament between treatment sessions to eliminate remnant microorganisms. Reducing the size of CH particles into nanoparticles enhances the penetration of this medicament into dentinal tubules and increases their antimicrobial efficacy. This in vitro study aimed to compare the cytotoxicity of CH nanoparticles and conventional CH on fibroblast cell line using the Mosmann’s Tetrazolium Toxicity (MTT) assay. Methods and Materials: This study was conducted on L929 murine fibroblast cell line by cell culture and evaluation of the direct effect of materials on the cultured cells. Materials were evaluated in two groups of 10 samples each at 24, 48 and 72 h. At each time point, 10 samples along with 5 positive and 5 negative controls were evaluated. The samples were transferred into tubes and exposed to fibroblast cells. The viability of cells was then evaluated. The Two-way ANOVA was used for statistical analysis and the level of significance was set at 0.05. Results: Cytotoxicity of both materials decreased over time and for conventional CH was lower than that of nanoparticles. However, this difference was not statistically significant (P>0.05). Conclusion: The cytotoxicity of CH nanoparticles was similar to that of conventional CH. PMID:25598810

  10. Molecular Profiling of Single Sca-1+/CD34+,? Cells—The Putative Murine Lung Stem Cells

    PubMed Central

    Huesemann, Yves; Maneck, Matthias; Botteron, Catherine; Kaeufl, Stephanie; Klein, Christoph A.; Polzer, Bernhard

    2013-01-01

    Murine bronchioalveolar stem cells play a key role in pulmonary epithelial maintenance and repair but their molecular profile is poorly described so far. In this study, we used antibodies directed against Sca-1 and CD34, two markers originally ascribed to pulmonary cells harboring regenerative potential, to isolate single putative stem cells from murine lung tissue. The mean detection rate of positive cells was 8 per 106 lung cells. We then isolated and globally amplified the mRNA of positive cells to analyze gene expression in single cells. The resulting amplicons were then used for molecular profiling by transcript specific polymerase chain reaction (PCR) and global gene expression analysis using microarrays. Single marker-positive cells displayed a striking heterogeneity for the expression of epithelial and mesenchymal transcripts on the single cell level. Nevertheless, they could be subdivided into two cell populations: Sca-1+/CD34? and Sca-1+/CD34+ cells. In these subpopulations, transcripts of the epithelial marker Epcam (CD326) were exclusively detected in Sca-1+/CD34? cells (p?=?0.03), whereas mRNA of the mesenchymal marker Pdgfr? (CD140a) was detected in both subpopulations and more frequently in Sca-1+/CD34+ cells (p?=?0.04). FACS analysis confirmed the existence of a Pdgfr? positive subpopulation within Epcam+/Sca-1+/CD34? epithelial cells. Gene expression analysis by microarray hybridization identified transcripts differentially expressed between the two cell types as well as between epithelial reference cells and Sca-1+/CD34+ single cells, and selected transcripts were validated by quantitative PCR. Our results suggest a more mesenchymal commitment of Sca-1+/CD34+ cells and a more epithelial commitment of Sca-1+/CD34? cells. In summary, the study shows that single cell analysis enables the identification of novel molecular markers in yet poorly characterized populations of rare cells. Our results could further improve our understanding of Sca-1+/CD34+,? cells in the biology of the murine lung. PMID:24391845

  11. Lycium barbarum polysaccharides regulate phenotypic and functional maturation of murine dendritic cells

    Microsoft Academic Search

    Jie Zhu; Lu-Hang Zhao; Xiao-Ping Zhao; Zhi Chen

    2007-01-01

    Lycium barbarum polysaccharides (LBPs) have been known to have a variety of immunomodulatory functions including activation of T cells, B cells and NK cells. Dendritic cells (DC) are potent antigen-presenting cells that play pivotal roles in the initiation of the primary immune response. However, little is known about the immunomodulatory effects of LBPs on murine bone marrow derived dendritic cells

  12. Induction of regulatory T cells by a murine ?-defensin.

    PubMed

    Navid, Fatemeh; Boniotto, Michele; Walker, Catherine; Ahrens, Kerstin; Proksch, Ehrhardt; Sparwasser, Tim; Müller, Werner; Schwarz, Thomas; Schwarz, Agatha

    2012-01-15

    ?-Defensins are antimicrobial peptides of the innate immune system produced in the skin by various stimuli, including proinflammatory cytokines, bacterial infection, and exposure to UV radiation (UVR). In this study we demonstrate that the UVR-inducible antimicrobial peptide murine ?-defensin-14 (mBD-14) switches CD4(+)CD25(-) T cells into a regulatory phenotype by inducing the expression of specific markers like Foxp3 and CTLA-4. This is functionally relevant because mBD-14-treated T cells inhibit sensitization upon adoptive transfer into naive C57BL/6 mice. Accordingly, injection of mBD-14, comparable to UVR, suppresses the induction of contact hypersensitivity and induces Ag-specific regulatory T cells (Tregs). Further evidence for the ability of mBD-14 to induce Foxp3(+) T cells is provided using DEREG (depletion of Tregs) mice in which Foxp3-expressing cells can be depleted by injecting diphtheria toxin. mBD-14 does not suppress sensitization in IL-10 knockout mice, suggesting involvement of IL-10 in mBD-14-mediated immunosuppression. However, unlike UVR, mBD-14 does not appear to mediate its immunosuppressive effects by affecting dendritic cells. Accordingly, UVR-induced immunosuppression is not abrogated in mBD-14 knockout mice. Together, these data suggest that mBD-14, like UVR, has the capacity to induce Tregs but does not appear to play a major role in UVR-induced immunosuppression. Through this capacity, mBD-14 may protect the host from microbial attacks on the one hand, but tame T cell-driven reactions on the other hand, thereby enabling an antimicrobial defense without collateral damage by the adaptive immune system. PMID:22174455

  13. Malignant Potential of Murine Stromal Cells after Transplantation of Human Tumors into Nude Mice

    NASA Astrophysics Data System (ADS)

    Goldenberg, David M.; Pavia, Rose A.

    1981-04-01

    Human malignant cancer tumors grafted into nude mice produce tumors containing both human cancer cells and the host's stromal cells. After short-term propagation of these tumors in vitro, the murine mesenchymal cells appear transformed and are tumorigenic in nude mice. However, established human cancer cell lines fail to similarly alter adjacent murine stromal cells when used to produce tumors in nude mice. These experiments suggest that cancer cells may recruit normal cells to become malignant, qualifying the view of the clonal (unicellular) origin of cancer.

  14. DNA repair in murine embryonic stem cells and differentiated cells

    SciTech Connect

    Tichy, Elisia D. [Department of Cell and Cancer Biology, University of Cincinnati, Cincinnati, OH 45267 (United States)], E-mail: tichyed@email.uc.edu; Stambrook, Peter J. [Department of Cell and Cancer Biology, University of Cincinnati, Cincinnati, OH 45267 (United States)

    2008-06-10

    Embryonic stem (ES) cells are rapidly proliferating, self-renewing cells that have the capacity to differentiate into all three germ layers to form the embryo proper. Since these cells are critical for embryo formation, they must have robust prophylactic mechanisms to ensure that their genomic integrity is preserved. Indeed, several studies have suggested that ES cells are hypersensitive to DNA damaging agents and readily undergo apoptosis to eliminate damaged cells from the population. Other evidence suggests that DNA damage can cause premature differentiation in these cells. Several laboratories have also begun to investigate the role of DNA repair in the maintenance of ES cell genomic integrity. It does appear that ES cells differ in their capacity to repair damaged DNA compared to differentiated cells. This minireview focuses on repair mechanisms ES cells may use to help preserve genomic integrity and compares available data regarding these mechanisms with those utilized by differentiated cells.

  15. Generation of Murine Sympathoadrenergic Progenitor-Like Cells from Embryonic Stem Cells and Postnatal Adrenal Glands

    PubMed Central

    Saxena, Shobhit; Wahl, Joachim; Huber-Lang, Markus S.; Stadel, Dominic; Braubach, Peter; Debatin, Klaus-Michael; Beltinger, Christian

    2013-01-01

    Sympathoadrenergic progenitor cells (SAPs) of the peripheral nervous system (PNS) are important for normal development of the sympathetic PNS and for the genesis of neuroblastoma, the most common and often lethal extracranial solid tumor in childhood. However, it remains difficult to isolate sufficient numbers of SAPs for investigations. We therefore set out to improve generation of SAPs by using two complementary approaches, differentiation from murine embryonic stem cells (ESCs) and isolation from postnatal murine adrenal glands. We provide evidence that selecting for GD2 expression enriches for ESC-derived SAP-like cells and that proliferating SAP-like cells can be isolated from postnatal adrenal glands of mice. These advances may facilitate investigations about the development and malignant transformation of the sympathetic PNS. PMID:23675538

  16. B cell lymphoma and myeloma in murine Gaucher's disease.

    PubMed

    Pavlova, E V; Wang, S Z; Archer, J; Dekker, N; Aerts, J M F G; Karlsson, S; Cox, T M

    2013-09-01

    Multiple myeloma and B cell lymphoma are leading causes of death in Gaucher's disease but the nature of the stimulus driving the often noted clonal expansion of immunoglobulin-secreting B cells and cognate lymphoid malignancy is unknown. We investigated the long-term development of B cell malignancies in an authentic model of non-neuronopathic Gaucher's disease in mice: selective deficiency of ?-glucocerebrosidase in haematopoietic cells [Gba(tm1Karl/tm1Karl)Tg(Mx1-cre)1Cgn/0, with excision of exons 9-11 of the murine GBA1 gene, is induced by poly[I:C]. Mice with Gaucher's disease showed visceral storage of ?-glucosylceramide and greatly elevated plasma ?-glucosylsphingosine [median 57.9 (range 19.8-159) nm; n = 39] compared with control mice from the same strain [median 0.56 (range 0.04-1.38) nm; n = 29] (p < 0.0001). Sporadic fatal B cell lymphomas developed in 11 of 21 GD mice (6-24 months) but only two of eight control animals developed tumours by age 24 months. Unexpectedly, most mice with overt lymphoma had absent or few Gaucher cells but local inflammatory macrophages were present. Eleven of 39 of Gaucher mice developed monoclonal gammopathy, but in the control group only one animal of 25 had clonal immunoglobulin abnormalities. Seven of 10 of the B cell lymphomas were found to secrete a monoclonal paraprotein and the lymphomas stained intensely for pan-B cell markers; reactive T lymphocytes were also present in tumour tissue. In the Gaucher mouse strain, it was notable that, as in patients with this disease, CD138(+) plasma cells frequently surrounded splenic macrophages engorged with glycosphingolipid. Our strain of mice, with inducible deficiency of ?-glucocerebrosidase in haematopoietic cells and a high frequency of sporadic lethal B cell malignancies, faithfully recapitulates human Gaucher's disease: it serves as a tractable model to investigate the putative role of bioactive sphingolipids in the control of B cell proliferation and the pathogenesis of myelomatosis-the most prevalent human cancer associated with this disorder. PMID:23775597

  17. Growth and differentiation potential of main- and side-population cells derived from murine skeletal muscle

    Microsoft Academic Search

    Tetsuro Tamaki; Akira Akatsuka; Yoshinori Okada; Yumi Matsuzaki; Hideyuki Okano; Minoru Kimura

    2003-01-01

    Skeletal muscle-derived CD34+\\/45? (Sk-34) cells were identified as a new candidate for stem cells. However, the relationship between Sk-34 cells and side-population (SP) cells is unknown. Here, we demonstrate that Sk-34 cells prepared from murine skeletal muscles consist wholly of main-population (MP) cells. The Sk-34 cells included only a few SP cells (1:1000, SP:MP). Colony-forming units of Sk-34 cells of

  18. Molecular cloning and adhesive properties of murine platelet/endothelial cell adhesion molecule 1.

    PubMed Central

    Xie, Y; Muller, W A

    1993-01-01

    We describe the isolation and characterization of a functional murine platelet/endothelial cell adhesion molecule (PECAM) 1 cDNA clone from a mouse lung library. At the nucleotide level, the coding sequence of murine PECAM-1 is 73% identical to human PECAM-1, and at the amino acid level, the sequence is 79% homologous to its human counterpart. Southern hybridization reveals that one copy of the gene exists in the mouse genome; Northern hybridization reveals a single mRNA species in mouse lung tissue. COS-7 and mouse L cells transfected with murine PECAM-1 expressed a 130-kDa glycoprotein on their surfaces that reacted with anti-murine PECAM-1 monoclonal antibody and comigrated on SDS/PAGE with human PECAM-1. Stable L-cell transfectants aggregate with each other in a PECAM-dependent, homophilic manner. Images Fig. 3 Fig. 6 PMID:8516303

  19. Reprogramming of murine and human somatic cells using a single polycistronic vector

    E-print Network

    Saha, Krishanu

    Reprogramming of murine and human somatic cells using a single polycistronic vector Bryce W. Careya concerns associated with somatic cell nuclear transfer (SCNT) and human embryonic stem (hES) cells and human, a major impediment to the use of iPS cells for therapeutic purposes has been the viral

  20. Expansion of Intestinal Epithelial Stem Cells during Murine Development

    PubMed Central

    Dehmer, Jeffrey J.; Garrison, Aaron P.; Speck, Karen E.; Dekaney, Christopher M.; Van Landeghem, Laurianne; Sun, Xiaofei; Henning, Susan J.; Helmrath, Michael A.

    2011-01-01

    Murine small intestinal crypt development is initiated during the first postnatal week. Soon after formation, overall increases in the number of crypts occurs through a bifurcating process called crypt fission, which is believed to be driven by developmental increases in the number of intestinal stem cells (ISCs). Recent evidence suggests that a heterogeneous population of ISCs exists within the adult intestine. Actively cycling ISCs are labeled by Lgr5, Ascl2 and Olfm4; whereas slowly cycling or quiescent ISC are marked by Bmi1 and mTert. The goal of this study was to correlate the expression of these markers with indirect measures of ISC expansion during development, including quantification of crypt fission and side population (SP) sorting. Significant changes were observed in the percent of crypt fission and SP cells consistent with ISC expansion between postnatal day 14 and 21. Quantitative real-time polymerase chain reaction (RT-PCR) for the various ISC marker mRNAs demonstrated divergent patterns of expression. mTert surged earliest, during the first week of life as crypts are initially being formed, whereas Lgr5 and Bmi1 peaked on day 14. Olfm4 and Ascl2 had variable expression patterns. To assess the number and location of Lgr5-expressing cells during this period, histologic sections from intestines of Lgr5-EGFP mice were subjected to quantitative analysis. There was attenuated Lgr5-EGFP expression at birth and through the first week of life. Once crypts were formed, the overall number and percent of Lgr5-EGFP positive cells per crypt remain stable throughout development and into adulthood. These data were supported by Lgr5 in situ hybridization in wild-type mice. We conclude that heterogeneous populations of ISCs are expanding as measured by SP sorting and mRNA expression at distinct developmental time points. PMID:22102874

  1. Liver sinusoidal endothelial cells promote lymphohematopoietic differentiation from murine embryonic stem cells: role of soluble factors.

    PubMed

    Silva-Cote, Ingrid; Cardier, Jose E

    2014-09-01

    Liver sinusoid endothelial cells (LSEC) constitute an in vitro and in vivo microenvironment for the proliferation and differentiation of hematopoietic stem cells (HSC). Previously, we have shown that LSEC support the survival and growth of murine embryonic stem cells (ESC). In this study, we investigated the capacity of LSEC to promote hematopoietic differentiation from the murine ESC cell line, CGR8. Undifferentiated ESC were cultured on LSEC monolayers in the absence of exogenous cytokines. After 10 and 20 days, cells were harvested and examined by their morphology, phenotype and capacity of hematopoietic colony formation. Microscopic observation of LSEC/ESC cocultures showed the presence of cobblestone areas formation, which indicates active hematopoiesis. Morphological analysis of cell from these foci showed the presence of hematopoietic cells at different stages of differentiation. Cells expressing B lymphoid markers (B220 and CD19) were detected by flow cytometry, and clonogenic assays showed the formation of CFU-pre B colonies. Similar results were observed when ESC were cultured with LSEC conditioned media. Myeloid precursors were also detected by the presence of CFU-GM colonies and cells expressing myeloid markers. These results indicate that LSEC provided an in vitro microenvironment mainly for B cell development, but also myeloid differentiation from ESC. Coculture of ESC with LSEC may constitute a very powerful tool to study the mechanisms involved in B cell generation from ESC. PMID:24892988

  2. Trop2 identifies a subpopulation of murine and human prostate basal cells with stem cell characteristics.

    PubMed

    Goldstein, Andrew S; Lawson, Devon A; Cheng, Donghui; Sun, Wenyi; Garraway, Isla P; Witte, Owen N

    2008-12-30

    The epithelium of the adult prostate contains 3 distinct cell types: basal, luminal, and neuroendocrine. Tissue-regenerative activity has been identified predominantly from the basal cells, isolated by expression of CD49f and stem cell antigen-1 (Sca-1). An important question for the field is whether all basal cells have stem cell characteristics. Prostate-specific microarray databases were interrogated to find candidate surface antigens that could subfractionate the basal cell population. Tumor-associated calcium signal transducer 2 (TACSTD2/Trop2/M1S1/GA733-1) was identified because it was enriched after castration, in prostate sphere cells and in the basal fraction. In the murine prostate, Trop2 shows progenitor characteristics such as localization to the region of the gland proximal to the urethra and enrichment for sphere-forming and colony-forming cells. Trop2 subfractionates the basal cells into 2 populations, both of which express characteristic basal cell markers by quantitative PCR. However, only the basal cells expressing high levels of Trop2 were able to efficiently form spheres in vitro. In the human prostate, where Sca-1 is not expressed, sphere-forming progenitor cells were also isolated based on high expression of Trop2 and CD49f. Trop2-expressing murine basal cells could regenerate prostatic tubules in vivo, whereas the remaining basal cells had minimal activity. Evidence was found for basal, luminal, and neuroendocrine cells in prostatic tubules regenerated from Trop2(hi) basal cells. In summary, functionally distinct populations of cells exist within the prostate basal compartment and an epithelial progenitor can give rise to neuroendocrine cells in vivo. PMID:19088204

  3. Melanoma Cells Treated with GGTI and IFN-c Allow Murine Vaccination and Enhance Cytotoxic Response

    E-print Network

    Paris-Sud XI, Université de

    Melanoma Cells Treated with GGTI and IFN-c Allow Murine Vaccination and Enhance Cytotoxic Response against Human Melanoma Cells Guillaume Sarrabayrouse1,2,3 , Christine Pich1,2,3 , Raphae¨l Moriez4 activation of T lymphocytes by melanoma cells is often due to the defective expression of class I major

  4. Trafficking of Murine Hematopoietic Stem and Progenitor Cells in Health and Vascular Disease

    E-print Network

    von Andrian, Ulrich H.

    Trafficking of Murine Hematopoietic Stem and Progenitor Cells in Health and Vascular Disease Medical School, Boston, Massachusetts, USA ABSTRACT Hematopoietic stem cells (HSCs) possess the unique capacity for self-renewal and differentiation into various hematopoietic cell lineages. Here we summarize

  5. In vitro differentiation of murine embryonic stem cells into keratinocyte-like cells.

    PubMed

    Haase, Ingo; Knaup, Renate; Wartenberg, Maria; Sauer, Heinrich; Hescheler, Jürgen; Mahrle, Gustav

    2007-12-01

    Embryonic stem (ES) cells are omnipotent; they can differentiate into every cell type of the body. The development of culture conditions that allow their differentiation has made it conceivable to produce large numbers of cells with lineage-specific characteristics in vitro. Here, we describe a method by which murine ES cells can be differentiated into cells with characteristics of epidermal keratinocytes. Keratinocyte-like cells were isolated from embryoid bodies and grown in culture. Potential applications of this method are the in vitro differentiation of cells of interest from ES cells of mice with lethal phenotypes during embryonic development and the production of genetically modified epidermal keratinocytes that could be used as temporary wound dressing or as carriers of genes of interest in gene therapeutic treatments. PMID:17716780

  6. Immunohistochemical localization of murine stage-specific embryonic antigens in human testicular germ cell tumors

    Microsoft Academic Search

    I Damjanov; N Fox; B B Knowles; D Solter; P H Lange; E E Fraley

    1982-01-01

    Monoclonal antibodies raised against and\\/or recognizing stage-specific antigens on preimplantation mouse embryos and stem cells of murine teratocarcinoma were used to localize these antigens immunohistochemically on human testicular germ cell tumors. SSEA-1, the antigen found on mouse embryonal carcinoma (EC) cells and embryonic cells from the 8-cell stage embryo onward, including the fetal primordial germ cells, was detected on yolk

  7. Regulation of Sertoli-germ cell adherens junction dynamics in the testis via the nitric oxide synthase (NOS)/cGMP/protein kinase G (PRKG)/beta-catenin (CATNB) signaling pathway: an in vitro and in vivo study.

    PubMed

    Lee, Nikki P Y; Mruk, Dolores D; Wong, Ching-Hang; Cheng, C Yan

    2005-09-01

    During spermatogenesis, extensive restructuring of cell junctions takes place in the seminiferous epithelium to facilitate germ cell movement. However, the mechanism that regulates this event remains largely unknown. Recent studies have shown that nitric oxide (NO) likely regulates tight junction (TJ) dynamics in the testis via the cGMP/protein kinase G (cGMP-dependent protein kinase, PRKG) signaling pathway. Due to the proximity of TJ and adherens junctions (AJ) in the testis, in particular at the blood-testis barrier, it is of interest to investigate if NO can affect AJ dynamics. Studies using Sertoli-germ cell cocultures in vitro have shown that the levels of NOS (nitric oxide synthase), cGMP, and PRKG were induced when anchoring junctions were being established. Using an in vivo model in which adult rats were treated with adjudin [a molecule that induces adherens junction disruption, formerly called AF-2364, 1-(2,4-dichlorobenzyl)-IH-indazole-3-carbohydrazide], the event of AJ disruption was also associated with a transient iNOS (inducible nitric oxide synthase, NOS2) induction. Immunohistochemistry has illustrated that NOS2 was intensely accumulated in Sertoli and germ cells in the epithelium during adjudin-induced germ cell loss, with a concomitant accumulation of intracellular cGMP and an induction of PRKG but not cAMP or protein kinase A (cAMP-dependent protein kinase, PRKA). To identify the NOS-mediated downstream signaling partners, coimmunoprecipitation was used to demonstrate that NOS2 and eNOS (endothelial nitric oxide synthase, NOS3) were structurally associated with the N-cadherin (CDH2)/beta-catenin (CATNB)/actin complex but not the nectin-3 (poliovirus receptor-related 3, PVRL 3)/afadin (myeloid/lymphoid or mixed lineage-leukemia tranlocation to 4 homolog, MLLT4) nor the integrin beta1 (ITB1)-mediated protein complexes, illustrating the spatial vicinity of NOS with selected AJ-protein complexes. Interestingly, CDH2 and CATNB were shown to dissociate from NOS during the adjudin-mediated AJ disruption, implicating the CDH2/CATNB protein complex is the likely downstream target of the NO signaling. Furthermore, PRKG, the downstream signaling protein of NOS, was shown to interact with CATNB in the rat testis. Perhaps the most important of all, pretreatment of testes with KT5823, a specific PRKG inhibitor, can indeed delay the adjudin-induced germ cell loss, further validating NOS/NO regulates Sertoli-germ cell AJ dynamics via the cGMP/PRKG pathway. These results illustrate that the CDH2/CATNB-mediated adhesion function in the testis is regulated, at least in part, via the NOS/cGMP/PRKG/CATNB pathway. PMID:15858215

  8. The NS2 Polypeptide of Parvovirus MVM Is Required for Capsid Assembly in Murine Cells

    Microsoft Academic Search

    Susan F Cotmore; Anthony M D'abramo; Luis F Carbonell; Jessica Bratton; Peter Tattersall

    1997-01-01

    Mutants of minute virus of mice (MVM) which express truncated forms of the NS2 polypeptide are known to exhibit a host range defect, replicating productively in transformed human cells but not in cells from their normal murine host. To explore this deficiency we generated viruses with translation termination codons at various positions in the second exon of NS2. In human

  9. Enhanced chondrogenic differentiation of murine embryonic stem cells in hydrogels with glucosamine

    Microsoft Academic Search

    Nathaniel S. Hwang; Shyni Varghese; Parnduangjai Theprungsirikul; Adam Canver; Jennifer Elisseeff

    2006-01-01

    Differentiation of embryonic stem (ES) cells generally occurs after formation of three-dimensional cell aggregates, known as embryoid bodies (EBs). We have previously reported that hydrogels provide EBs a supportive environment for in vitro chondrogenic differentiation and three dimensional tissue formation [Hwang NS, et al. The Effects of three dimensional culture and growth factors on the chondrogenic differentiation of murine ES

  10. Butyric acid-induced apoptosis of murine thymocytes, splenic T cells, and human Jurkat T cells.

    PubMed Central

    Kurita-Ochiai, T; Fukushima, K; Ochiai, K

    1997-01-01

    The purpose of this study was to examine the effect of butyric acid, an extracellular metabolite from periodontopathic bacteria, on apoptosis induction in murine thymocytes, splenic T cells, and human Jurkat T cells. Butyric acid significantly suppressed T-cell viability in both a concentration- and time-dependent fashion. The results of DNA fragmentation assay indicated that butyric acid rapidly induced apoptosis in thymocytes (with 1.25 mM butyric acid and 6 h after treatment) and in splenic T cells and Jurkat cells (with 2.5 mM butyric acid and 16 h after treatment). Incubation of thymocytes or Jurkat cells with 5 mM butyric acid for 21 h resulted in the typical ladder pattern of DNA fragmentation. Furthermore, Jurkat cells treated with 5 mM butyric acid showed the characteristic pattern of apoptotic cells such as chromatin condensation and hypodiploid nuclei. Experiments with fractionated subpopulations of splenic T cells revealed that DNA fragmentation was predominantly observed in CD4+ T cells. Butyric acid-induced apoptosis of thymocytes was decreased by the protein kinase inhibitors H7 and staurosporine. These inhibitors were less effective with similarly treated splenic T cells and Jurkat cells. These data suggest that butyric acid, one of the volatile fatty acids produced by periodontopathic bacteria and one that easily penetrates the oral mucosa, can modulate the immunoregulatory cell population in periodontal tissue by inducing T-cell death through apoptosis. PMID:8975889

  11. Intrapulmonary Administration of CCL21 Gene-Modified Dendritic Cells Reduces Tumor Burden in Spontaneous Murine Bronchoalveolar Cell Carcinoma

    Microsoft Academic Search

    Seok-Chul Yang; Raj K. Batra; Sven Hillinger; Karen L. Reckamp; Robert M. Strieter; Steven M. Dubinett; Sherven Sharma

    The antitumor efficiency of dendritic cells transduced with an adenovirus vector expressing secondary lymphoid chemokine (CCL21) was evaluated in a murine model of spontaneous bronchoalveolar cell carcinoma. The transgenic mice (CC-10 TAg) express the SV40 large T antigen (TAg) under the Clara cell promoter, develop bilateral, multifocal, and pulmonary adenocarcinomas, and die at 4 months as a result of progressive

  12. Characterization of Definitive Lymphohematopoietic Stem Cells in the Day 9 Murine Yolk Sac

    Microsoft Academic Search

    Mervin C Yoder; Kelly Hiatt; Parmesh Dutt; Pinku Mukherjee; David M Bodine; Donald Orlic

    1997-01-01

    The site of origin of lymphohematopoietic stem cells (HSC) that initiate definitive blood cell production in the murine fetal liver is controversial. Contrary to reports that the preliver yolk sac does not contain definitive HSC, we observed that CD34+ day 9 yolk sac cells repopulated multiple blood cell lineages in newborn hosts for at least 1 year. Furthermore, 100 CD34+c-Kit+

  13. Prevalence and Characterization of Murine Leukemia Virus Contamination in Human Cell Lines

    PubMed Central

    Uphoff, Cord C.; Lange, Sandra; Denkmann, Sabine A.; Garritsen, Henk S. P.; Drexler, Hans G.

    2015-01-01

    Contaminations of cell cultures with microbiological organisms are well documented and can be managed in cell culture laboratories applying reliable detection, elimination and prevention strategies. However, the presence of viral contaminations in cell cultures is still a matter of debate and cannot be determined with general detection methods. In the present study we screened 577 human cell lines for the presence of murine leukemia viruses (MLV). Nineteen cell lines were found to be contaminated with MLV, including 22RV1 which is contaminated with the xenotropic murine leukemia virus-related virus variant of MLV. Of these, 17 cell lines were shown to produce active retroviruses determined by product enhanced reverse transcriptase PCR assay for reverse transcriptase activity. The contaminated cell lines derive from various solid tumor types as well as from leukemia and lymphoma types. A contamination of primary human cells from healthy volunteers could not be substantiated. Sequence analyses of 17 MLV PCR products and five complete MLV genomes of different infected cell lines revealed at least three groups of related MLV genotypes. The viruses harvested from the supernatants of infected cell cultures were infectious to uninfected cell cultures. In the course of the study we found that contamination of human genomic DNA preparations with murine DNA can lead to false-positive results. Presumably, xenotransplantations of the human tumor cells into immune-deficient mice to determine the tumorigenicity of the cells are mainly responsible for the MLV contaminations. Furthermore, the use of murine feeder layer cells during the establishment of human cell lines and a cross-contamination with MLV from infected cultures might be sources of infection. A screening of cell cultures for MLV contamination is recommended given a contamination rate of 3.3%. PMID:25927683

  14. Identification of murine T-cell epitopes on the S4 subunit of pertussis toxin.

    PubMed Central

    Petersen, J W; Holm, A; Ibsen, P H; Hasløv, K; Heron, I

    1993-01-01

    The aim of the present study was to identify murine T-cell epitopes on pertussis toxin subunit S4. Six mouse strains with five different haplotypes at the H-2 locus were immunized with the pertussis toxin B oligomer. Lymph node lymphocytes were isolated and stimulated in an in vitro proliferation assay with pertussis toxin components and 11 overlapping synthetic peptides synthesized on the basis of the primary sequence of S4. In vitro proliferative responses to the synthetic peptides revealed the presence of four distinct murine T-cell epitopes on subunit S4. The recognition of the peptides was major histocompatibility complex restricted. Immunizing four of the six mouse strains with the synthetic peptides showed that the peptides which were demonstrated to contain T-cell epitopes following immunization with the B oligomer were able to induce proliferative responses to detoxified pertussis toxin and pertussis toxin components containing subunit S4. One of the identified murine T-cell epitopes corresponded to one of the major human T-cell epitopes previously identified on subunit S4. It is hoped that this murine model system will facilitate the development of a synthetic immunogen mimicking the protective properties of pertussis toxin. PMID:7678102

  15. Transcriptional and post-transcriptional regulation of globin gene accumulation in murine erythroleukemia cells.

    PubMed Central

    Profous-Juchelka, H R; Reuben, R C; Marks, P A; Rifkind, R A

    1983-01-01

    The mechanism responsible for the accumulation of newly synthesized alpha- and beta-globin mRNA in the cytoplasm of induced murine erythroleukemia cells was examined by nuclear mRNA nascent chain elongation (run-off transcription). Hexamethylenebisacetimide, a potent inducer of murine erythroleukemia cell differention, induced high levels of both alpha- and beta-globin gene transcription within 48 to 72 h in culture. Butyric acid, a modest inducer of murine erythroleukemia cells, induced a somewhat lower level of globin gene transcription. With both inducers, alpha-globin transcriptional rates exceeded those of beta-globin. Hemin, on the other hand, showed no detectable increase over the basal rate observed in uninduced cells, even at a time (48 h) when newly synthesized globin mRNA was accumulating in the cytoplasm. These results suggest that there are at least two mechanisms responsible for regulating alpha- and beta-globin structural gene expression in induced murine erythroleukemia cells and that the mechanisms involved are inducer dependent. Hexamethylenebisacetimide and butyric acid increase the rate at which globin genes are transcribed, but hemin appears to allow constitutive levels of transcripts to accumulate. PMID:6572784

  16. Isolation of murine hair-inducing cells using the cell surface marker prominin-1/CD133.

    PubMed

    Ito, Yuriko; Hamazaki, Tatsuo S; Ohnuma, Kiyoshi; Tamaki, Kunihiko; Asashima, Makoto; Okochi, Hitoshi

    2007-05-01

    Hair is a mini-organ in which dermal papilla (DP) cells play important roles in hair follicle morphogenesis and formation via interactions with epithelial cells. DP cells have previously been difficult to analyze because of the lack of a specific surface marker. We have demonstrated that prominin-1/CD133 (CD133) is a useful marker for murine DP cells. DP cells express CD133 during the early anagen stage (active growth phase) not only during hair morphogenesis, but also during the growth phase of hairs after birth. Gene expression and flow cytometric analysis revealed that CD133-positive (+) cells in the skin possess the characteristics of DP cells. The CD133(+) cells isolated from embryonic or adult skin-induced new hair follicles in vivo when they were transplanted into nude mice mixed with embryonic epithelial cells, but CD133-negative (-) cells could not. We propose that the CD133 is a novel surface marker useful for collecting DP cells in the anagen stage and for analyzing the function of DP. PMID:17185982

  17. Cellular Pharmacology in Murine and Human Leukemic Cell Lines of Diaziquone (NSC 182986)1

    Microsoft Academic Search

    Merrill J. Egorin; Bonnie M. Fox; James F. Spiegel; Peter L. Gutierrez; Rosalind D. Friedman; Nicholas R. Bachur

    We investigated the in vitro interaction with and antitumor effect on several murine and human leukemic cell lines of diazi- quone (AZQ). L1210 cells accumulated AZQ from Roswell Park Memorial Institute Medium 1640 with or without newborn calf serum by a temperature-dependent and sodium azide-resistant process. AZQ inhibited, in a dose-dependent fashion, (3H)thy- midine incoporation into L1210 cells, but this

  18. Identification of tissue-specific vasculogenic cells originating from murine uterus

    Microsoft Academic Search

    Narumi Onodera; Tetsuro Tamaki; Yoshinori Okada; Akira Akatsuka; Daisuke Aoki

    2006-01-01

    Endometrium is a highly regenerative adult tissue that undergoes repeated degeneration and regeneration following menarche.\\u000a Therefore, it is believed that endometrium contains stem and\\/or progenitor cells in order to compensate for the regeneration\\u000a of tissue components. We report here that stem-like cells having vasculogenic potential are present in the uterus. Enzymatically\\u000a extracted cells from murine uteri were characterized and fractionated

  19. METHODOLOGICAL CONSIDERATIONS FOR THE MURINE HEPATOMA CELL BIOASSAY-DIRECTED ISOLATION OF CANCER CHEMOPREVENTIVE AGENTS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The development of a murine hepatoma (Hepa 1c1c7) cell line over a decade ago for the isolation of phase II enzyme inducing agents by a group from Johns Hopkins University led to the identification of sulforaphane as a major cancer chemopreventive agent in broccoli. We have found that choices among...

  20. Involvement of Retinal Neurons and Pigment Epithelial Cells in a Murine Model of Sandhoff Disease

    Microsoft Academic Search

    Kazunori Sango; Shoji Yamanaka; Kyoko Ajiki; Nobutaka Arai; Masahiko Takano

    2008-01-01

    Background\\/Aims: To investigate the effects of deficient degradation of glycolipids on the morphological appearance of all retinal cells in a murine model of GM2 gangliosidosis (Sandhoff disease). Methods: The morphological appearance of the retina in Sandhoff mice at symptomatic stages (3 and 4 months of age) was examined by immunohistochemistry, lectin histochemistry and electron microscopy. Results: Under a light microscope,

  1. Anthrax Lethal Toxin-Mediated Killing of Human and Murine Dendritic Cells Impairs

    E-print Network

    Brojatsch, Jürgen

    Anthrax Lethal Toxin-Mediated Killing of Human and Murine Dendritic Cells Impairs the Adaptive anthracis interferes with host defenses by releasing anthrax lethal toxin (LT), which inactivates mitogen that anthrax LT impairs adaptive immunity by specifically targeting DCs. This may represent an immune- evasion

  2. Isolation of a slowly adhering cell fraction containing stem cells from murine skeletal muscle by the preplate technique

    Microsoft Academic Search

    Burhan Gharaibeh; Aiping Lu; Jessica Tebbets; Bo Zheng; Joe Feduska; Mihaela Crisan; Bruno Péault; James Cummins; Johnny Huard

    2008-01-01

    This protocol details a procedure, known as the modified preplate technique, which is currently used in our laboratory to isolate muscle cells on the basis of selective adhesion to collagen-coated tissue culture plates. By employing this technique to murine skeletal muscle, we have been able to isolate a rapidly adhering cell (RAC) fraction within the earlier stages of the process,

  3. SPECIFIC MURINE B-CELL ACTIVATION BY SYNTHETIC SINGLE- AND DOUBLE-STRANDED POLYNUCLEOTIDES

    PubMed Central

    Scher, Irwin; Strong, Douglas M.; Ahmed, Aftab; Knudsen, Richard C.; Sell, Kenneth W.

    1973-01-01

    The synthetic single- and double-stranded polynucleotides, poly I, poly C, and poly I·C, were shown to induce thymidine incorporation in six inbred strains of murine spleen cells. This stimulation was shown to be secondary to B-cell activation and not due to contamination of the polynucleotides with bacterial lipopolysaccharide (LPS). The ability of poly I·C to act as a B-cell mitogen, in addition to its behavior as a thymic-independent antigen, suggested that these two phenomena may be related. The similarity of the molecular structure of poly I·C to LPS, a material which also acts as a thymic-independent antigen and a B-cell mitogen, supports the hypothesis that the polyvalent nature of these materials accounts for their functional interaction with murine B cells. PMID:4543458

  4. A model system for testing gene vectors using murine tumor cells on the chorioallantoic membrane of the chick embryo

    Microsoft Academic Search

    Sergio U. Dani; Rachel Espindola

    2002-01-01

    We developed a model system for testing gene vectors, based on the growth of murine tumors on the chorioallantoic membrane (CAM) of embryonic chickens. The ability of selected murine cells to grow on the CAM was rated according to the following criteria: i) for- mation of tumor masses; ii) metastasis formation; iii) reproducibility; iv) yield, indicated as the number of

  5. Genetic and Clonal Dissection of Murine Small Cell Lung Carcinoma Progression by Genome Sequencing

    PubMed Central

    McFadden, David G.; Papagiannakopoulos, Thales; Taylor-Weiner, Amaro; Stewart, Chip; Carter, Scott L.; Cibulskis, Kristian; Bhutkar, Arjun; McKenna, Aaron; Dooley, Alison; Vernon, Amanda; Sougnez, Carrie; Malstrom, Scott; Heimann, Megan; Park, Jennifer; Chen, Frances; Farago, Anna F.; Dayton, Talya; Shefler, Erica; Gabriel, Stacey; Getz, Gad; Jacks, Tyler

    2014-01-01

    Summary Small cell lung carcinoma (SCLC) is a highly lethal, smoking-associated cancer with few known targetable genetic alterations. Using genome sequencing, we characterized the somatic evolution of a genetically engineered mouse model (GEMM) of SCLC initiated by loss of Trp53 and Rb1. We identified alterations in DNA copy number and complex genomic rearrangements and demonstrated a low somatic point mutation frequency in the absence of tobacco mutagens. Alterations targeting the tumor suppressor Pten occurred in the majority of murine SCLC studied, and engineered Pten deletion accelerated murine SCLC and abrogated loss of Chr19 in Trp53; Rb1; Pten compound mutant tumors. Finally, we found evidence for polyclonal and sequential metastatic spread of murine SCLC by comparative sequencing of families of related primary tumors and metastases. We propose a temporal model of SCLC tumorigenesis with implications for human SCLC therapeutics and the nature of cancer-genome evolution in GEMMs. PMID:24630729

  6. Neonatal testicular cell transplantation restores murine spermatogenesis damaged in the course of herpes simplex virus-induced orchitis.

    PubMed

    Malolina, Ekaterina A; Kulibin, Andrey Yu; Kushch, Alla A

    2014-11-17

    Genital tract infection and inflammation may affect male fertility, causing germ and Sertoli cell loss. We determined if testicular cell transplantation is effective at repairing testicular injury induced by herpes simplex virus (HSV) orchitis. ROSA26 mice were used as donors and the recipients were C57BL/6 mice after HSV testicular inoculation; some of the recipients were treated with the antiviral drug acyclovir (ACV). ACV reduced the amount of HSV antigen in testes on Day 3 after transplantation and enhanced the efficacy of transplantation at Day 30. In recipient testes, donor Sertoli cells formed new seminiferous tubules; significantly more new tubules were observed in the testes of ACV-treated mice compared with mice not treated with ACV (17.8% vs 3.6%). Over half (50.4%) of new tubules in ACV-treated testes contained germ cells and round spermatids were detected in 14.2% of new tubules compared with 15.9% and 5.3% in testes not treated with ACV, respectively. At Day 150 the seminiferous epithelium was completely recovered in some donor tubules and elongated spermatids were observed inside it. Thus, our findings reveal the effectiveness of the combination of antiviral therapy with neonatal testis-cell transplantation for the restoration of spermatogenesis damaged by viral infection. PMID:25399480

  7. Murine interleukin 7 (IL-7) receptor. Characterization on an IL-7- dependent cell line

    PubMed Central

    1990-01-01

    A murine cell line (IxN/2b) absolutely dependent upon exogenous IL-7 for continued growth has been obtained that expresses lymphoid precursor and class I MHC antigens and also contains a rearranged mu heavy chain. This cell line has been used to define the binding and structural characteristics of the murine IL-7 receptor using 125I- labeled recombinant murine IL-7. 125I-IL-7 binding to IxN/2b cell was rapid and saturable at both 4 degrees and 37 degrees C. Equilibrium binding studies produced curvilinear Scatchard plots at both temperatures with high and low affinity Ka values of approximately 1 x 10(10) M-1 and 4 x 10(8) M-1, respectively, and a total of 2,000-2,500 IL-7 binding sites expressed per cell. Experiments measuring inhibition of binding of 125I-IL-7 by unlabeled IL-7 also produced data consistent with the existence of two classes of IL-7 receptors. Evidence concerning the possible molecular nature of two classes of IL-7 receptors was provided by dissociation kinetics and affinity crosslinking experiments. The dissociation rate of 125I-IL-7 was markedly increased when measured in the presence of unlabeled IL-7 at both 37 degrees and 4 degrees C, which is diagnostic of a receptor population displaying negative cooperativity. Crosslinking studies showed that under both reducing and nonreducing conditions, the major crosslinked species observed corresponded to a receptor size of 75-79 kD while a less intense higher molecular mass crosslinked species was also seen which corresponded to a receptor size approximately twice as large (159-162 kD). Both types of experiments suggest that the IL-7 receptor may form noncovalently associated dimers in the membrane. The IL-7 receptor was expressed on pre-B cells, but not detected on several murine B cell lines or primary mature B cells. It was also expressed on murine thymocytes, some T lineage cell lines, and on bone marrow- derived macrophage. All cells binding 125I-IL-7 exhibited curvilinear Scatchard plots. No cytokines or growth factors tested were able to inhibit binding of 125I-IL-7 to its receptor. These results define the initial binding and structural characteristics, and the cellular distribution, of the murine IL-7 receptor. PMID:2324686

  8. Dermal dendritic cells, but not Langerhans cells, are critical in murine single epicutaneous sensitization.

    PubMed

    Lee, Chih-Hung; Chen, Jau-Shiuh; Chiu, Hsien-Ching; Hong, Chien-Hui; Liu, Ching-Yi; Ta, Yng-Cun; Clausen, Bj?rn E; Ho, Ji-Chen; Wang, Li-Fang

    2015-01-01

    A murine repeated protein-patch model has been established to study epicutaneous sensitization in atopic dermatitis. This model has shown a predominant Th2 and a weak Th1 response in both BALB/c and C57BL/6 mice. However, Th responses induced in the repeated model are not consistent with the generally accepted theory that BALB/c and C57BL/6 mice are Th2 and Th1 prone and are representatives of human atopy and non-atopy, respectively. In this study, a single protein-patch model was established, which showed in addition to the Th2 response, a remarkable Th1 response in C57BL/6 mice, but not in BALB/c mice. Moreover, using muLangerin-DTR mice, we demonstrated that dermal dendritic cells, but not Langerhans cells, are critical in single epicutaneous sensitization in both strains of mice. PMID:25363677

  9. Investigations on the mechanisms of methotrexate resistance in a cisplatin-resistant L1210 murine leukemia cell subline

    Microsoft Academic Search

    Diana H. Wroblewski; Alok Bhushan; Yongzhi Xuan; Bradford T. Brinton; Thomas R. Tritton; Miles P. Hacker

    1996-01-01

    We report a murine leukemia cell variant (L1210\\/DDP), selected for cisplatin (DDP) resistance, to be cross-resistant to methotrexate\\u000a (MTX). Cross-resistance of L1210 cells to DDP and MTX has been observed by others, and has also been recorded in P388 murine\\u000a leukemia and SSC-25 human squamous carcinoma cells. We demonstrated that MTX resistance is not due to dihydrofolate reductase\\u000a (DHFR) gene

  10. Lactational exposure of phthalate causes long-term disruption in testicular architecture by altering tight junctional and apoptotic protein expression in Sertoli cells of first filial generation pubertal Wistar rats.

    PubMed

    Sekaran, S; Balaganapathy, P; Parsanathan, R; Elangovan, S; Gunashekar, J; Bhat, F A; Jagadeesan, A

    2015-06-01

    Di(2-ethylhexyl) phthalate (DEHP) is a ubiquitous environmental contaminant and a well-known endocrine disruptor (ED) that interferes with the reproductive function in both humans and animals. This study aimed to find out the impact of lactational exposure of DEHP in testes of first filial generation (F1) progeny male rat postnatal day (PND)-60. Lactating dams were orally treated with DEHP (0, 1, 10 and 100 mg/kg body weight/day, respectively) from the PND-1 to PND-21. Rats were killed at PND 60. Testes were removed and used for histological analysis and for isolation of Sertoli cells (SCs). The histoarchitecture of DEHP-treated rats showed disturbed testicular structure. DEHP-treated rats also showed increased oxidative stress by decreasing antioxidant levels in the SCs; it disrupted SC tight junctional proteins occludin, claudin, junctional adhesion molecule, zona occludens protein-1 (ZO-1), zona occludens protein-2 (ZO-2), and afadin-6 (AF-6), increased apoptosis by altering the apoptotic genes Bax, cytochrome c, caspase-8, -9, -3 and antiapoptotic gene Bcl-2. It is concluded that early postnatal exposure to DEHP disturbs histoarchitecture of testis and SC function in pubertal Wistar rats. PMID:25352649

  11. Cleavage Map and Proteolytic Processing of the Murine Norovirus Nonstructural Polyprotein in Infected Cells

    Microsoft Academic Search

    Stanislav V. Sosnovtsev; G. Belliot; K.-O. Chang; V. G. Prikhodko; L. B. Thackray; C. E. Wobus; S. M. Karst; H. W. Virgin; K. Y. Green

    2006-01-01

    Murine norovirus (MNV) is presently the only member of the genus Norovirus in the Caliciviridae that can be propagated in cell culture. The goal of this study was to elucidate the proteolytic processing strategy of MNV during an authentic replication cycle in cells. A proteolytic cleavage map of the ORF1 polyprotein was generated, and the virus-encoded 3C-like (3CL) proteinase (Pro)

  12. Binding Kinetics of Ecotropic (Moloney) Murine Leukemia Retrovirus with NIH 3T3 Cells

    Microsoft Academic Search

    HONG YU; FRENCH ANDERSON

    1995-01-01

    A quantitative analysis of the binding kinetics of intact Moloney murine leukemia retrovirus (MoMuLV) particles with NIH 3T3 cells was performed with an immunofluorescenceflow cytometry assay. The virus-cell binding equilibrium dissociation constant (KD), expressed in terms of virus particle concentration, was measured to be 8.5 (66.4) 310 212 Mat4 &C and was three- to sixfold lower at temperatures above 15&C.

  13. Transfusion of fresh murine red blood cells reverses adverse effects of older stored red blood cells

    PubMed Central

    Hendrickson, Jeanne E; Hod, Eldad A.; Hudson, Krystalyn E.; Spitalnik, Steven L.; Zimring, James C.

    2011-01-01

    Background Although a subset of recent studies have suggested red blood cell (RBC) storage length is associated with adverse patient outcomes, others have shown no such relationship. Adults may be transfused with RBC units of different storage lengths, and existing studies do not take into consideration that fresh RBCs may alter responses to concurrently transfused stored RBCs. To test this possibility, we utilized a murine model and investigated transfusion outcomes of fresh, stored, or fresh plus stored RBCs. Study Design and Methods Fresh, 14-day stored, or fresh plus 14-day stored leukoreduced RBCs from HOD transgenic donors (with RBC specific expression of hen egg lysozyme, ovalbumin, and human Duffyb) were transfused into naïve C57BL/6 recipients. Serum cytokines and anti-HOD alloimmunization were evaluated following transfusion. Results In 6 of 6 experiments (n=90 mice total), a pro-inflammatory serum cytokine storm of interleukin-6, keratinocyte-derived chemokine/CXCL1, and monocyte chemoattractant protein-1 was observed in transfusion recipients of stored but not fresh RBCs, along with high degrees of anti-HOD alloimmunization. However, concurrent transfusion of fresh HOD RBCs along with stored HOD RBCs significantly decreased these adverse outcomes (p<0.05). Conclusions These results are consistent with fresh murine HOD RBCs losing protective properties during storage, and introduce a previously unrecognized variable in RBC storage studies. If translatable to humans, uniform “old blood” groups may be needed in future clinical studies to most accurately investigate the biological effects of older RBC units. PMID:21645005

  14. Monoamine oxidase A regulates neural differentiation of murine embryonic stem cells

    PubMed Central

    Wang, Zhi-qiang; Chen, Kevin; Ying, Qi-long; Li, Ping

    2012-01-01

    Monoamine oxidase (MAO) A is the major metabolizing enzyme of serotonin (5-hydroxytryptamine, 5-HT) which regulates early brain development. In this study, wild-type (WT) and MAO Aneo embryonic stem (ES) cell lines were established from the inner cell mass of murine blastocysts and their characteristics during ES and differentiating stages were studied. Our results show that the differentiation to neural cells in MAO Aneo ES cells was reduced compared to WT, suggesting MAO A played a regulatory role in stem cells neural differentiation. PMID:21607742

  15. Development and characterization of Histoplasma capsulatum-reactive murine T-cell lines and clones

    NASA Technical Reports Server (NTRS)

    Deepe, George S., Jr.; Smith, James G.; Denman, David; Bullock, Ward E.; Sonnenfeld, Gerald

    1986-01-01

    Several Histoplasma capsulatum-reactive murine cloned T-cell lines (TCLs) were isolated from spleens of C57BL/6 mice immunized with viable H. capsulatum yeast cells, using the methodology of Kimoto and Fathman (1980). These T-cells were characterized phenotypically as Thy-1.2(+) Lyt-1(+) L3T4(+) Lyt-2(-), that is, as the helper/inducer phenotype. The cloned T cells proliferate in response to histoplasmin and, in some cases, to heterologous fungal anigens. Upon injection of mice with the antigen, the T-cells mediate local delayed-type hypersensitivity responses and, after stimulation, release regulatory lymphokines.

  16. RANKL-mediated osteoclast formation from murine RAW 264.7 cells.

    PubMed

    Collin-Osdoby, Patricia; Osdoby, Philip

    2012-01-01

    Extensive research efforts over the years have provided us with great insights into how bone-resorbing osteoclasts (OCs) develop and function and, based on such work, valuable antiresorptive therapies have been developed to help combat the excessive bone loss that occurs in numerous skeletal disorders. The RAW 264.7 murine cell line has proven to be an important tool for in vitro studies of OC formation and function, having particular advantages over the use of OCs generated from primary bone marrow cell populations or directly isolated from murine bones. These include their ready access and availability, simple culture for this pure macrophage/pre-OC population, sensitive and rapid development into highly bone-resorptive OCs expressing hallmark OC characteristics following their RANKL stimulation, abundance of RAW cell-derived OCs that can be generated to provide large amounts of study material, relative ease of transfection for genetic and regulatory manipulation, and close correlation in characteristics, gene expression, signaling, and developmental or functional processes between RAW cell-derived OCs and OCs either directly isolated from murine bones or formed in vitro from primary bone marrow precursor cells. Here, we describe methods for the culture and RANKL-mediated differentiation of RAW cells into bone-resorptive OCs as well as procedures for their enrichment, characterization, and general use in diverse analytical assays. PMID:22130930

  17. Murine dendritic cell development: Difficulties associated with subset analysis

    Microsoft Academic Search

    Heather L Wilson; Helen C O'Neill

    2003-01-01

    Dendritic cells are bone marrow-derived professional antigen presenting cells that play major roles in both the induction of primary immune responses and tolerance. It has become clear that dendritic cells are a heterogenous group of cells that vary in cell surface marker expression and function. Multiple dendritic cell subsets have now been defined in mouse lymphoid organs and peripheral tissues.

  18. Behaviour of four different B16 murine melanoma cell sublines: C57BL/6J skin.

    PubMed

    Danciu, Corina; Oprean, Camelia; Coricovac, Dorina E; Andreea, Cioca; Cimpean, Anca; Radeke, Heinfried; Soica, Codruta; Dehelean, Cristina

    2015-04-01

    Transplantable murine melanomas are well-established models for the study of experimental cancer therapies. The aim of this study was to explore the behaviour of four different B16 murine melanoma cell sublines after inoculation into C57BL/6J mice; and, more specifically to analyse skin changes, with respect to two specific parameters: clinical (tumour volume, melanin amount, erythema) and histological (H & E, S100, VEGF expression). Both non-invasive and invasive analysis showed that B164A5 is the most aggressive melanoma cell line for C57BL/6J's skin, followed by B16F10 and then by diminished aggressive growth pattern by the B16GMCSF and B16FLT3 cell lines. PMID:25664478

  19. Expression Profiling of Murine Acute Promyelocytic Leukemia Cells Reveals Multiple Model-Dependent Progression Signatures†

    PubMed Central

    Walter, Matthew J.; Park, John S.; Lau, Steven K. M.; Li, Xia; Lane, Andrew A.; Nagarajan, Rakesh; Shannon, William D.; Ley, Timothy J.

    2004-01-01

    Leukemia results from the expansion of self-renewing hematopoietic cells that are thought to contain mutations that contribute to disease initiation and progression. Studies of the gene expression profiles of human acute myeloid leukemia samples has allowed their classification based on the presence of translocations and French-American-British subtypes, but it is not yet clear whether their molecular signatures reflect the initiating mutations or mutations acquired during progression. To begin to address this question, we examined the expression profiles of normal murine promyelocyte-enriched samples, nontransformed murine promyelocytes expressing human promyelocytic leukemia-retinoic acid receptor alpha (PML-RAR?) fusion gene, and primary acute promyelocytic leukemia cells. The expression profile of nontransformed cells expressing PML-RAR? was remarkably similar to that of wild-type promyelocytes. In contrast, the expression profiles of fully transformed cells from three acute promyelocytic leukemia model systems were all different, suggesting that the expression signature of acute promyelocytic leukemia cells reflects the genetic changes that contributed to progression. To further evaluate these progression events, we compared two high-penetrance acute promyelocytic leukemia models that both commonly acquire an interstitial deletion of chromosome 2 during progression. The two models exhibited distinct gene expression profiles, suggesting that the dominant molecular signatures of murine acute promyelocytic leukemia can be influenced by several independent progression events. PMID:15572690

  20. Reduced serum concentration is permissive for increased in vitro endocrine differentiation from murine embryonic stem cells.

    PubMed

    Vincent, Robert K; Odorico, Jon S

    2009-07-01

    Embryonic stem cells (ESCs) have been shown to be capable of differentiating into pancreatic progenitors and insulin-producing cells in vitro. However, before ESC derivatives can be used in clinical settings, efficient selective differentiation needs to be achieved. Essential to improving ESC differentiation to islet endocrine cells is an understanding of the influences of extrinsic signals and transcription factors on cell specification. Herein, we investigate the influence of serum-supplemented growth conditions on the differentiation of murine ESCs to endocrine lineages in the context of over-expression of two pancreatic transcription factors, Pdx1 and Ngn3. To study the effect of different serum formulations and concentrations on the ability of murine ESCs to differentiate into endocrine cells in vitro, cells were grown into embryoid bodies and then differentiated in various serum replacement (SR), fetal calf serum (FCS) and serum-free conditions. Using immunohistochemistry and quantitative real-time RT-PCR (QPCR), we found that, of the conditions tested, 1% SR differentiation medium resulted in the highest levels of insulin-1 mRNA and significantly increased the total number of insulin-expressing cells. Applying this knowledge to cell lines in which Pdx1 or Ngn3 transgene expression could be induced by exposure to doxycycline we differentiated TetPDX1 and TetNgn3 ESCs under conditions of either 10% FCS or 1% SR medium. In the presence of 10% serum, induced expression of either Pdx1 or Ngn3 in differentiating ESCs resulted in modest increases in hormone transcripts and cell counts. However, changing the serum formulation from 10% FCS to 1% SR significantly enhanced the number of insulin+/C-peptide+ cells in parallel with increased insulin-1 transcript levels in both inducible cell lines. In summary, these data demonstrate that induced expression of key pancreatic transcription factors in combination with low serum/SR concentrations increases endocrine cell differentiation from murine ESCs. PMID:19446949

  1. Icariin promotes expression of PGC1?, PPAR?, and NRF-1 during cardiomyocyte differentiation of murine embryonic stem cells in vitro

    Microsoft Academic Search

    Ling Ding; Xing-guang Liang; Dan-yan Zhu; Yi-jia Lou

    2007-01-01

    Aim:To investigate the effect of icariin on the expression of peroxisome proliferator-activated receptor ? coactivator-1 alpha (PGC-1?), peroxisome proliferator-activated receptor alpha (PPAR?), and nuclear respiratory factor 1 (NRF-1) on cardiomyocyte differentiation of murine embryonic stem (ES) cells in vitro.Methods:The cardiomyocytes derived from murine ES cells were verified by immunocytochemistry using confocal laser scanning microscopy. Cardiac-specific sarcomeric proteins (ie ?-actinin, troponin

  2. Induction of stress proteins in murine and human melanoma cell cultures.

    PubMed

    Mattei, E; Delpino, A; Mileo, A M; Ferrini, U

    1986-04-30

    The induction of stress proteins was studied in two human and two murine melanoma cell lines. Exposure for 1 h to heat (42 degrees C), to ethanol (6%), to arsenate (100 microM) and to disulfiram (50 microM) induced the expression of SPs with apparent molecular weights of 100, 86, 70-72 and 24-26 Kd. Quantitation of the single SPs indicated that the basal level as well as the enhanced synthesis following the various stressors were different in each cell line. The induction of the 100 Kd species occurred in only one murine melanoma and not in the others. The 86 and in particular the 70-72 Kd species were the most prominent groups, whereas the 24-26 SPs were induced only following arsenate and disulfiram exposure in the three melanoma cell lines. In one of the murine melanomas, the expression of SPs was markedly reduced compared to the other cell lines. No definite specific patterns of SP expression could be identified in tumors of the same histologic type. PMID:3705184

  3. Densely Granulated Murine NK Cells Eradicate Large Solid Tumors

    PubMed Central

    Liu, Rebecca B.; Engels, Boris; Arina, Ainhoa; Schreiber, Karin; Hyjek, Elizabeth; Schietinger, Andrea; Binder, David C.; Butz, Eric; Krausz, Thomas; Rowley, Donald A.; Jabri, Bana; Schreiber, Hans

    2013-01-01

    NK cells inhibit early stages of tumor formation, recurrence and metastasis. Here we show that NK cells can also eradicate large solid tumors. Eradication depended on the massive infiltration of proliferating NK cells due to IL15 released and presented by the cancer cells in the tumor microenvironment. Infiltrating NK cells had the striking morphological feature of being densely loaded with PAS-positive, diastase-resistant granules, resembling uterine NK cells. Perforin-mediated killing by these densely granulated NK cells was essential for tumor eradication. Expression of the IL15 receptor ? on cancer cells was needed to efficiently induce granulated NK cells and expression on host stromal cells was essential to prevent tumor relapse after near complete destruction. These results indicate that IL15 released at the cancer site induces highly activated NK cells that lead to eradication of large solid tumors. PMID:22374983

  4. Sorting and expansion of murine embryonic stem cell colonies using micropallet arrays.

    PubMed

    Shadpour, Hamed; Sims, Christopher E; Thresher, Randy J; Allbritton, Nancy L

    2009-02-01

    Isolation of cell colonies is an essential task in most stem cell studies. Conventional techniques for colony selection and isolation require significant time, labor, and consumption of expensive reagents. New microengineered technologies hold the promise for improving colony manipulation by reducing the required manpower and reagent consumption. Murine embryonic stem cells were cultured on arrays composed of releasable elements termed micropallets created from a biocompatible photoresist. Micropallets containing undifferentiated colonies were released using a laser-based technique followed by cell collection and expansion in culture. The micropallet arrays provided a biocompatible substrate for maintaining undifferentiated murine stem cells in culture. A surface coating of 0.025% gelatin was shown to be optimal for cell culture and collection. Arrays composed of surface-roughened micropallets provided further improvements in culture and isolation. Colonies of viable stem cells were efficiently isolated and collected. Colonies sorted in this manner were shown to remain undifferentiated even after collection and further expansion in culture. Qualitative and quantitative analyses of sorting, collection efficiency, and cell viability after release and expansion of stem cell colonies demonstrated that the micropallet array technology is a promising alternative to conventional sorting methods for stem cell applications. PMID:19012319

  5. TH1 and TH2 T-cell subsets are differentially activated by macrophages and B cells in murine leishmaniasis.

    PubMed Central

    Rossi-Bergmann, B; Müller, I; Godinho, E B

    1993-01-01

    The role of antigen-presenting cells in the differential expansion of TH1 and TH2 T cells in murine leishmaniasis was investigated. In general, macrophages preferentially induced gamma interferon and interleukin-2 secretion by syngeneic Leishmania-specific T cells, whereas B cells were more efficient in activating interleukin 4 production. B cells from susceptible BALB/c mice were better in inducing TH2 responses than B cells from resistant C57BL/6 mice, whereas macrophages from C57BL/6 mice were superior to BALB/c macrophages in inducing TH1 responses. PMID:8478122

  6. Human Mesenchymal Stem Cells Differentiate to a Cardiomyocyte Phenotype in the Adult Murine Heart

    Microsoft Academic Search

    Catalin Toma; Mark F. Pittenger; Kevin S. Cahill; Barry J. Byrne; Paul D. Kessler

    2002-01-01

    Background—Cellular cardiomyoplasty has been proposed as an alternative strategy for augmenting the function of diseased myocardium. We investigated the potential of human mesenchymal stem cells (hMSCs) from adult bone marrow to undergo myogenic differentiation once transplanted into the adult murine myocardium. Methods and Results—A small bone marrow aspirate was taken from the iliac crest of healthy human volunteers, and hMSCs

  7. Modification of the fatty acid composition of L1210 murine leukemia cells

    Microsoft Academic Search

    C. Patrick Burns; DIANA G. LUTTENEGGERo; Shiao-Ping L. Wei; Arthur A. Spector

    1977-01-01

    We have compared the effect of diets containing 16% sunflower seed oil (polyunsaturated fat-rich) or 16% coconut oil (saturated\\u000a fat-rich) fed for 3–7 weeks on the composition of L1210 murine leukemia cells which were transplanted into the peritoneal\\u000a cavity during the final week of feeding. The L1210 phospholipids of mice fed the sunflower oil diet contained 43% polyenoic\\u000a fatty acids

  8. Optimization of Gene Transfection in Murine Myeloma Cell Lines using Different Transfection Reagents.

    PubMed

    Shabani, Mahdi; Hemmati, Sheyda; Hadavi, Reza; Amirghofran, Zahra; Jeddi-Tehrani, Mahmood; Rabbani, Hodjatallah; Shokri, Fazel

    2010-07-01

    Purification and isolation of cellular target proteins for monoclonal antibody (MAb) production is a difficult and time-consuming process. Immunization of mice with murine cell lines stably transfected with genes coding for xenogenic target molecules is an alternative method for mouse immunization and MAb production. Here we present data on transfection efficiency of some commercial reagents used for transfection of murine myeloma cell lines. Little is known about transfectability of murine myeloma cell lines by different transfection reagents. Mouse myeloma cell lines (SP2/0, NS0, NS1, Ag8, and P3U1) were transfected with pEGFP-N1 vector using Lipofectamine 2000, jetPEI and LyoVec commercial transfection reagents in different combinations. The transfection permissible HEK293-FT cell line was used as a control in transfection procedure. Transfected cells, expressing the Enhanced Green Fluorescent Protein (EGFP), were analyzed by flow cytometry 48 hrs post transfection. Our results showed transfection efficiency of 71%, 57% and 22% for HEK293-FT, 5.5%, 3.4% and 1% for SP2/0, 55.7%, 21.1% and 9.3% for NS0, 8.2%, 6% and 5.5% for NS1, 22%, 49.2% and 5.5% for Ag8 and 6.3%, 21.5% and 4.6% for P3U1 cell lines after transfection with Lipofectamine 2000, jetPEI and LyoVec reagents, respectively. Our data indicate that NS0 and Ag8 are efficiently transfected by Lipofectamine 2000 and jetPEI reagents. Finally, we propose Ag8 and NS0 cell lines as suitable host cells for efficient expression of target genes which can be used for mouse immunization and MAb production. PMID:23408356

  9. Targeting Syk-activated B cells in murine and human chronic graft-versus-host disease.

    PubMed

    Flynn, Ryan; Allen, Jessica L; Luznik, Leo; MacDonald, Kelli P; Paz, Katelyn; Alexander, Kylie A; Vulic, Ante; Du, Jing; Panoskaltsis-Mortari, Angela; Taylor, Patricia A; Poe, Jonathan C; Serody, Jonathan S; Murphy, William J; Hill, Geoffrey R; Maillard, Ivan; Koreth, John; Cutler, Corey S; Soiffer, Robert J; Antin, Joseph H; Ritz, Jerome; Chao, Nelson J; Clynes, Raphael A; Sarantopoulos, Stefanie; Blazar, Bruce R

    2015-06-25

    Novel therapies for chronic graft-versus-host disease (cGVHD) are needed. Aberrant B-cell activation has been demonstrated in mice and humans with cGVHD. Having previously found that human cGVHD B cells are activated and primed for survival, we sought to further evaluate the role of the spleen tyrosine kinase (Syk) in cGVHD in multiple murine models and human peripheral blood cells. In a murine model of multiorgan system, nonsclerodermatous disease with bronchiolitis obliterans where cGVHD is dependent on antibody and germinal center (GC) B cells, we found that activation of Syk was necessary in donor B cells, but not T cells, for disease progression. Bone marrow-specific Syk deletion in vivo was effective in treating established cGVHD, as was a small-molecule inhibitor of Syk, fostamatinib, which normalized GC formation and decreased activated CD80/86(+) dendritic cells. In multiple distinct models of sclerodermatous cGVHD, clinical and pathological disease manifestations were not eliminated when mice were therapeutically treated with fostamatinib, though both clinical and immunologic effects could be observed in one of these scleroderma models. We further demonstrated that Syk inhibition was effective at inducing apoptosis of human cGVHD B cells. Together, these data demonstrate a therapeutic potential of targeting B-cell Syk signaling in cGVHD. PMID:25852057

  10. Isolation, cultivation, and characterization of adult murine prostate stem cells

    PubMed Central

    Lukacs, Rita U.; Goldstein, Andrew S.; Lawson, Devon A.; Cheng, Donghui; Witte, Owen N.

    2010-01-01

    ABSTRACT/SUMMARY The successful isolation and cultivation of prostate stem cells will allow us to study their unique biological properties and their application in therapeutic approaches. Here we provide step-by-step procedures on the basis of previous work in our laboratory for: the harvesting of primary prostate cells from adolescent male mice by a modified enzymatic procedure; the isolation of an enriched population of prostate stem cells through cell sorting; the cultivation of prostate stem cells in vitro; and characterization of these cells and their stem-like activity, including in vivo tubule regeneration. Normally it will take approximately 8 hours to harvest prostate cells, isolate the stem cell enriched population, and set up the in vitro sphere assay. It will take up to 8 weeks to analyze the unique properties of the stem cells, including their regenerative capacity in vivo. PMID:20360765

  11. ?? T Cells Confer Protection against Murine Cytomegalovirus (MCMV)

    PubMed Central

    Villacreces, Arnaud; Juzan, Marina; Rousseau, Benoît; Dulanto, Sara; Giese, Alban; Costet, Pierre; Praloran, Vincent; Moreau, Jean-François; Dubus, Pierre; Vermijlen, David

    2015-01-01

    Cytomegalovirus (CMV) is a leading infectious cause of morbidity in immune-compromised patients. ?? T cells have been involved in the response to CMV but their role in protection has not been firmly established and their dependency on other lymphocytes has not been addressed. Using C57BL/6 ?? and/or ?? T cell-deficient mice, we here show that ?? T cells are as competent as ?? T cells to protect mice from CMV-induced death. ?? T cell-mediated protection involved control of viral load and prevented organ damage. ?? T cell recovery by bone marrow transplant or adoptive transfer experiments rescued CD3??/? mice from CMV-induced death confirming the protective antiviral role of ?? T cells. As observed in humans, different ?? T cell subsets were induced upon CMV challenge, which differentiated into effector memory cells. This response was observed in the liver and lungs and implicated both CD27+ and CD27? ?? T cells. NK cells were the largely preponderant producers of IFN? and cytotoxic granules throughout the infection, suggesting that the protective role of ?? T cells did not principally rely on either of these two functions. Finally, ?? T cells were strikingly sufficient to fully protect Rag?/??c?/? mice from death, demonstrating that they can act in the absence of B and NK cells. Altogether our results uncover an autonomous protective antiviral function of ?? T cells, and open new perspectives for the characterization of a non classical mode of action which should foster the design of new ?? T cell based therapies, especially useful in ?? T cell compromised patients. PMID:25747674

  12. Analysis of apoptosis in murine embryonic stem cells 

    E-print Network

    Weaks, Regina Lanell

    1999-01-01

    of this research were to induce apostasis in both differentiated (-LIF) and undifferentiated (+LIF) mouse ES cells, and to study the effects of the apostasis inhibitors N-acetyl-L-cysteine (NAC), ?-macroglobulin (MAC), and glutathione (GSH) on cell numbers...

  13. Analysis of apoptosis in murine embryonic stem cells

    E-print Network

    Weaks, Regina Lanell

    1999-01-01

    of this research were to induce apostasis in both differentiated (-LIF) and undifferentiated (+LIF) mouse ES cells, and to study the effects of the apostasis inhibitors N-acetyl-L-cysteine (NAC), ?-macroglobulin (MAC), and glutathione (GSH) on cell numbers...

  14. Interleukin 3 mediates interleukin 6 production in murine interleukin 3-dependent hemopoietic cells.

    PubMed

    Hültner, L; Szöts, H; Van Snick, J; Moeller, J; Welle, M; Dörmer, P

    1989-01-01

    A series of murine interleukin 3 (IL-3)-dependent hemopoietic cell lines was studied for the capacity to produce interleukin 6 (IL-6) in vitro. These included a bone marrow-derived mast cell line (L138.8A) and several early myeloid cell lines described in the literature (DA-1, DA-3, NFS-60, NFS-78, FDC-P1, FDC-P2, FDC-PmixA4, and 32Dcl.23). All of these cell lines produced growth factor activity for IL-6-dependent hybridoma cells (7TD1), which was completely neutralized by the monoclonal anti-IL-6-antibody 6B4. IL-6 expression was also evident at the mRNA level using a murine IL-6-specific cDNA probe. In 32Dcl.23 cells (2 x 10(5)/ml) stimulated for 24 hr with serial dilutions of purified murine IL-3, a positive correlation was found between the IL-3 dose and the amount of IL-6 measured in the conditioned media. At 24 hr this correlation was not evident at the mRNA level. However, prolonged exposure of 32Dcl.23 cells (up to 72 hr) to either a high (60 U/ml) or a low IL-3 concentration (1 U/ml) revealed a time-dependent increase and decrease, respectively, of IL-6 mRNA levels. At both IL-3 concentrations 32Dcl.23 cells remained in a fully viable and proliferative state. The influence of IL-3 on IL-6 release could be specifically counteracted by anti-IL-3-antiserum. IL-6 added alone or in concert with IL-3 did not stimulate 32Dcl.23 proliferation.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:2635055

  15. Mechanisms of murine dendritic cell antitumor dysfunction in aging

    Microsoft Academic Search

    Annabelle Grolleau-Julius; Lisa Abernathy; Erin Harning; Raymond L. Yung

    2009-01-01

    Effective cancer immunotherapy depends on the body’s ability to generate tumor antigen-presenting cells and tumor-reactive\\u000a effector lymphocytes. As the most potent antigen presenting cells (APCs), dendritic cells (DCs) are capable of sensitizing\\u000a T cells to new and recall antigens. Clinical trials of antigen-pulsed autologous DCs have been conducted in patients with\\u000a a number of hematological and solid cancers, including malignant

  16. Eicosapentaenoic and docosahexaenoic acid ethyl esters differentially enhance B-cell activity in murine obesity.

    PubMed

    Teague, Heather; Harris, Mitchel; Fenton, Jenifer; Lallemand, Perrine; Shewchuk, Brian M; Shaikh, Saame Raza

    2014-05-17

    EPA and DHA are not biologically equivalent; however, their individual activity on B cells is unknown. We previously reported fish oil enhanced murine B-cell activity in obesity. To distinguish between the effects of EPA and DHA, we studied the ethyl esters of EPA and DHA on murine B-cell function as a function of time. We first demonstrate that EPA and DHA maintained the obese phenotype, with no improvements in fat mass, adipose inflammatory cytokines, fasting insulin, or glucose clearance. We then tested the hypothesis that EPA and DHA would increase the frequency of splenic B cells. EPA and DHA differentially enhanced the frequency and/or percentage of select B-cell subsets, correlating with increased natural serum IgM and cecal IgA. We next determined the activities of EPA and DHA on ex vivo production of cytokines upon lipopolysaccharide stimulation of B cells. EPA and DHA, in a time-dependent manner, enhanced B-cell cytokines with DHA notably increasing IL-10. At the molecular level, EPA and DHA differentially enhanced the formation of ordered microdomains but had no effect on Toll-like receptor 4 mobility. Overall, the results establish differential effects of EPA and DHA in a time-dependent manner on B-cell activity in obesity, which has implications for future clinical studies. PMID:24837990

  17. Rapamycin Inhibits ALDH Activity, Resistance to Oxidative Stress, and Metastatic Potential in Murine Osteosarcoma Cells.

    PubMed

    Mu, Xiaodong; Isaac, Christian; Schott, Trevor; Huard, Johnny; Weiss, Kurt

    2013-01-01

    Osteosarcoma (OS) is the most common primary malignancy of bone. Mortality is determined by the presence of metastatic disease, but little is known regarding the biochemical events that drive metastases. Two murine OS cell lines, K7M2 and K12, are related but differ significantly in their metastatic potentials: K7M2 is highly metastatic whereas K12 displays much less metastatic potential. Using this experimental system, the mammalian target of rapamycin (mTOR) pathway has been implicated in OS metastasis. We also discovered that aldehyde dehydrogenase (ALDH, a stem cell marker) activity is higher in K7M2 cells than K12 cells. Rapamycin treatment reduces the expression and enzymatic activity of ALDH in K7M2 cells. ALDH inhibition renders these cells more susceptible to apoptotic death when exposed to oxidative stress. Furthermore, rapamycin treatment reduces bone morphogenetic protein-2 (BMP2) and vascular endothelial growth factor (VEGF) gene expression and inhibits K7M2 proliferation, migration, and invasion in vitro. Inhibition of ALDH with disulfiram correlated with decreased mTOR expression and activity. In conclusion, we provide evidence for interaction between mTOR activity, ALDH activity, and metastatic potential in murine OS cells. Our work suggests that mTOR and ALDH are therapeutic targets for the treatment and prevention of OS metastasis. PMID:23476113

  18. CRKL overexpression suppresses in vitro proliferation, invasion and migration of murine hepatocarcinoma Hca-P cells.

    PubMed

    Lin, Qiuyue; Sun, Ming-Zhong; Guo, Chunmei; Shi, Ji; Chen, Xin; Liu, Shuqing

    2015-02-01

    The signal adaptor CRK family protein play important roles in cancer cell progression, proliferation, migration and invasion. Previously, we showed that CRK was involved in lymphatic metastatic potential of murine hepatocarcinoma cells. In current work, as a member of CRK family, chicken tumour virus number 10 regulator of kinase-like protein (CRKL) was revealed to be associated with malignant behaviors of Hca-P, a murine HCC cell with lymph node metastatic (LNM) rate of ?25%. CRKL overexpression in Hca-P by a constructed eukaryotic expression vector of pcDNA3.1/V5-HisB-CRKL significantly ameliorated its malignant biological properties. CCK-8 and soft agar colony formation assays indicated CRKL overexpression significantly inhibits the cell proliferation and colony formation abilities of Hca-P. Additionally, transwell assays indicated that the Hca-P cell migration and invasion capacities were apparently reduced following CRKL overexpression. As Hca-P is an ideal hepatocarcinoma cell model with low (initial) LNM potential, CRKL is shown to act as a potential suppressor and to provide new insight for both the malignant behaviors of hepatocarcinoma cells and lymphatic metastasis mechanism of hepatocarcinoma. PMID:25661331

  19. T cells in murine lupus: propagation and regulation of disease

    Microsoft Academic Search

    Stanford L. Peng; Joe Craft

    1996-01-01

    MRL\\/Mp-lpr\\/lpr mice develop a spontaneous lupus syndrome, including hypergammaglobulinemia, autoantibodies, glomerulonephritis, and lymphadenopathy. To investigate the role of lymphocyte subsets in the pathogenesis of disease, lupus-prone MRL mice deficient in aß T cells, ?d T cells, or both were generated. Mice deficient in aß T cells developed a partially penetrant lupus syndrome, characterized by lymphadenopathy, elevated levels of class-switched immunoglobulins,

  20. In vitro tolerance induction of neonatal murine B cells

    PubMed Central

    1976-01-01

    The susceptibility of neonatal and adult B lymphocytes to tolerance induction was analyzed by a modification of the in vitro splenic focus technique. This technique permits stimulation of individual hapten- specific clonal precursor cells from both neonatal and adult donors. Neonatal or adult BALB/c spleen cells were adoptively transferred into irradiated, syngeneic, adult recipients which had been carrier-primed to hemocyanin (Hy), thus maximizing stimulation to the hapten 2,4- dinitrophenyl coupled by Hy (DNP-Hy). Cultures were initially treated with DNP on several heterologous (non-Hy) carriers and subsequently stimulated with DNP-Hy. Whereas the responsiveness of adult B cells was not diminished by pretreatment with any DNP conjugate, the majority of the neonatal B-cell response was abolished by in vitro culture with all of the DNP-protein conjugates. During the 1st wk of life, the ability to tolerize neonatal splenic B cells progressively decreased. Thus, tolerance in this system is: (a) restricted to B cells early in development; (b) established by both tolerogens and immunogens; (c) achieved at low (10(-9) M determinant) antigen concentrations; and (d) highly specific, discriminating between DNP- and TNP-specific B cells. We conclude that: (a) B lymphocytes, during their development, mature through a stage in which they are extremely susceptible to tolerogenesis; (b) the specific interaction of B-cell antigen receptors with multivalent antigens, while irrelevant to mature B cells, is tolerogenic to neonatal (immature) B cells unless antigen is concomitantly recognized by primed T cells; and (c) differences in the susceptibility of immature and mature B lymphocytes to tolerance induction suggest intrinsic differences between neonatal and adult B cells and may provide a physiologically relevant model for the study of tolerance to self-antigens. PMID:58052

  1. Extracortical origin of some murine subplate cell populations

    PubMed Central

    Pedraza, María; Hoerder-Suabedissen, Anna; Albert-Maestro, María Amparo; Molnár, Zoltán; De Carlos, Juan A.

    2014-01-01

    The subplate layer, the deepest cortical layer in mammals, has important roles in cerebral cortical development. The subplate contains heterogeneous cell populations that are morphologically diverse, with several projection targets. It is currently assumed that these cells are generated in the germinative zone of the earliest cortical neuroepithelium. Here we identify a pallial but extracortical area located in the rostromedial telencephalic wall (RMTW) that gives rise to several cell populations. Postmitotic neurons migrate tangentially from the RMTW toward the cerebral cortex. Most RMTW-derived cells are incorporated into the subplate layer throughout its rostrocaudal extension, with others contributing to the GABAergic interneuron pool of cortical layers V and VI. PMID:24778253

  2. Engineering skeletal muscle tissues from murine myoblast progenitor cells and application of electrical stimulation.

    PubMed

    van der Schaft, Daisy W J; van Spreeuwel, Ariane C C; Boonen, Kristel J M; Langelaan, Marloes L P; Bouten, Carlijn V C; Baaijens, Frank P T

    2013-01-01

    Engineered muscle tissues can be used for several different purposes, which include the production of tissues for use as a disease model in vitro, e.g. to study pressure ulcers, for regenerative medicine and as a meat alternative (1). The first reported 3D muscle constructs have been made many years ago and pioneers in the field are Vandenburgh and colleagues (2,3). Advances made in muscle tissue engineering are not only the result from the vast gain in knowledge of biochemical factors, stem cells and progenitor cells, but are in particular based on insights gained by researchers that physical factors play essential roles in the control of cell behavior and tissue development. State-of-the-art engineered muscle constructs currently consist of cell-populated hydrogel constructs. In our lab these generally consist of murine myoblast progenitor cells, isolated from murine hind limb muscles or a murine myoblast cell line C2C12, mixed with a mixture of collagen/Matrigel and plated between two anchoring points, mimicking the muscle ligaments. Other cells may be considered as well, e.g. alternative cell lines such as L6 rat myoblasts (4), neonatal muscle derived progenitor cells (5), cells derived from adult muscle tissues from other species such as human (6) or even induced pluripotent stem cells (iPS cells) (7). Cell contractility causes alignment of the cells along the long axis of the construct (8,9) and differentiation of the muscle progenitor cells after approximately one week of culture. Moreover, the application of electrical stimulation can enhance the process of differentiation to some extent (8). Because of its limited size (8 x 2 x 0.5 mm) the complete tissue can be analyzed using confocal microscopy to monitor e.g. viability, differentiation and cell alignment. Depending on the specific application the requirements for the engineered muscle tissue will vary; e.g. use for regenerative medicine requires the up scaling of tissue size and vascularization, while to serve as a meat alternative translation to other species is necessary. PMID:23542531

  3. Engineering Skeletal Muscle Tissues from Murine Myoblast Progenitor Cells and Application of Electrical Stimulation

    PubMed Central

    van der Schaft, Daisy W. J.; van Spreeuwel, Ariane C. C.; Boonen, Kristel J. M.; Langelaan, Marloes L. P.; Bouten, Carlijn V. C.; Baaijens, Frank P. T.

    2013-01-01

    Engineered muscle tissues can be used for several different purposes, which include the production of tissues for use as a disease model in vitro, e.g. to study pressure ulcers, for regenerative medicine and as a meat alternative 1. The first reported 3D muscle constructs have been made many years ago and pioneers in the field are Vandenburgh and colleagues 2,3. Advances made in muscle tissue engineering are not only the result from the vast gain in knowledge of biochemical factors, stem cells and progenitor cells, but are in particular based on insights gained by researchers that physical factors play essential roles in the control of cell behavior and tissue development. State-of-the-art engineered muscle constructs currently consist of cell-populated hydrogel constructs. In our lab these generally consist of murine myoblast progenitor cells, isolated from murine hind limb muscles or a murine myoblast cell line C2C12, mixed with a mixture of collagen/Matrigel and plated between two anchoring points, mimicking the muscle ligaments. Other cells may be considered as well, e.g. alternative cell lines such as L6 rat myoblasts 4, neonatal muscle derived progenitor cells 5, cells derived from adult muscle tissues from other species such as human 6 or even induced pluripotent stem cells (iPS cells) 7. Cell contractility causes alignment of the cells along the long axis of the construct 8,9 and differentiation of the muscle progenitor cells after approximately one week of culture. Moreover, the application of electrical stimulation can enhance the process of differentiation to some extent 8. Because of its limited size (8 x 2 x 0.5 mm) the complete tissue can be analyzed using confocal microscopy to monitor e.g. viability, differentiation and cell alignment. Depending on the specific application the requirements for the engineered muscle tissue will vary; e.g. use for regenerative medicine requires the up scaling of tissue size and vascularization, while to serve as a meat alternative translation to other species is necessary. PMID:23542531

  4. Lack of Expression from a Retroviral Vector After Transduction of Murine Hematopoietic Stem Cells is Associated with Methylation in vivo

    Microsoft Academic Search

    Pia-Maria Challita; Donald B. Kohn

    1994-01-01

    We describe studies of gene transfer and expression of the human glucocerebrosidase cDNA by a Moloney murine leukemia virus (MoMuLV)-based retroviral vector in a murine gene transfer\\/bone marrow transplant (BMT) model. Pluripotent hematopoietic stem cells (HSCs) were assayed as the colony-forming units, spleen (CFU-S) generated after serial transplantation. Transcriptional expression from the MoMuLV long-terminal repeat (LTR) was detected at a

  5. Generation of bone marrow derived murine dendritic cells for use in 2-photon imaging.

    PubMed

    Matheu, Melanie P; Sen, Debasish; Cahalan, Michael D; Parker, Ian

    2008-01-01

    Several methods for the preparation of murine dendritic cells can be found in the literature. Here, we present a method that produces greater than 85% CD11c high dendritic cells in culture that home to the draining lymph node after subcutaneous injection and present antigen to antigen specific T cells (see video). Additionally, we use Essen Instruments Incucyte to track dendritic cell maturation, where, at day 10, the morphology of the cultured cells is typical of a mature dendritic cell and <85% of cells are CD11chigh. The study of antigen presentation in peripheral lymph nodes by 2-photon imaging revealed that there are three distinct phases of dendritic cell and T cell interaction. Phase I consists of brief serial contacts between highly motile antigen specific T cells and antigen carrying dendritic cells. Phase two is marked by prolonged contacts between antigen-specific T cell and antigen bearing dendritic cells. Finally, phase III is characterized by T cells detaching from dendritic cells, regaining motility and beginning to divide. This is one example of the type of antigen-specific interactions that can be analyzed by two-photon imaging of antigen-loaded cell tracker dye-labeled dendritic cells. PMID:19066518

  6. Disruption of canonical TGF?-signaling in murine coronary progenitor cells by low level arsenic

    SciTech Connect

    Allison, Patrick; Huang, Tianfang; Broka, Derrick; Parker, Patti [Department of Pharmacology and Toxicology College of Pharmacy, Southwest Environmental Health Sciences Center, Steele Children's Research Center and Bio5 Institute, University of Arizona, Tucson, AZ 85721 (United States); Barnett, Joey V. [Department of Pharmacology, Vanderbilt Medical University, Nashville, TN (United States); Camenisch, Todd D., E-mail: camenisch@pharmacy.arizona.edu [Department of Pharmacology and Toxicology College of Pharmacy, Southwest Environmental Health Sciences Center, Steele Children's Research Center and Bio5 Institute, University of Arizona, Tucson, AZ 85721 (United States)

    2013-10-01

    Exposure to arsenic results in several types of cancers as well as heart disease. A major contributor to ischemic heart pathologies is coronary artery disease, however the influences by environmental arsenic in this disease process are not known. Similarly, the impact of toxicants on blood vessel formation and function during development has not been studied. During embryogenesis, the epicardium undergoes proliferation, migration, and differentiation into several cardiac cell types including smooth muscle cells which contribute to the coronary vessels. The TGF? family of ligands and receptors is essential for developmental cardiac epithelial to mesenchymal transition (EMT) and differentiation into coronary smooth muscle cells. In this in vitro study, 18 hour exposure to 1.34 ?M arsenite disrupted developmental EMT programming in murine epicardial cells causing a deficit in cardiac mesenchyme. The expression of EMT genes including TGF?2, TGF? receptor-3, Snail, and Has-2 are decreased in a dose-dependent manner following exposure to arsenite. TGF?2 cell signaling is abrogated as detected by decreases in phosphorylated Smad2/3 when cells are exposed to 1.34 ?M arsenite. There is also loss of nuclear accumulation pSmad due to arsenite exposure. These observations coincide with a decrease in vimentin positive mesenchymal cells invading three-dimensional collagen gels. However, arsenite does not block TGF?2 mediated smooth muscle cell differentiation by epicardial cells. Overall these results show that arsenic exposure blocks developmental EMT gene programming in murine coronary progenitor cells by disrupting TGF?2 signals and Smad activation, and that smooth muscle cell differentiation is refractory to this arsenic toxicity. - Highlights: • Arsenic blocks TGF?2 induced expression of EMT genes. • Arsenic blocks TGF?2 triggered Smad2/3 phosphorylation and nuclear translocation. • Arsenic blocks epicardial cell differentiation into cardiac mesenchyme. • Arsenic does not block TGF?2 induced smooth muscle cell differentiation.

  7. Selective induction of apoptosis in murine skin carcinoma cells (CH72) by an ethanol extract of Lentinula edodes

    Microsoft Academic Search

    Yu-Huan Gu; Martha A. Belury

    2005-01-01

    The effects of ethanol extracts from four species of mushroom fruiting bodies, mushroom spores and mushroom cultured broth, were assessed for modulation of cell proliferation and apoptosis in murine skin carcinoma cells (CH72) and non-tumorigenic epidermal cells (C50). While extracts from mycelia of Grifola frondosa, Ganoderma lucidum, Hericium erinaceus, or from spores of G. lucidum exerted little, if any, effect

  8. Murine Tumor Cell Lysis by Antibody-dependent Macrophage-mediated Cytotoxicity Using Syngeneic Monoclonal Antibodies1

    Microsoft Academic Search

    Ichiro Kawase; Kiyoshi Komuta; Takeshi Ogura; Hiromi Fujiwara; Toshiyuki Hamaoka; Susumu Kishimoto

    Four monoclonal antibodies to MH134 murine syngeneic hep- atoma cells, 3H1, 7C2, 11G2, and 12A2, were produced by hybridomas constructed by fusing P3-X63-Ag8-U1 murine mye loma cells with spleen cells of a C3H\\/HeN mouse immunized with the syngeneic tumor cells. Immunodiffusion analysis with rabbit anti-mouse immunoglobulin antisera showed that 3H1, 7C2,11G2, and 12A2 are lgG2a, IgM, lgG1, and lgG2a, respec

  9. Dexamethasone facilitates erythropoiesis in murine embryonic stem cells differentiating into hematopoietic cells in vitro

    SciTech Connect

    Srivastava, Anand S. [Departments of Medicine, Moores UCSD Cancer Center, University of California San Diego, San Diego, CA 92093-0820 (United States); Kaushal, Sharmeela [Departments of Medicine, Moores UCSD Cancer Center, University of California San Diego, San Diego, CA 92093-0820 (United States); Mishra, Rangnath [Division of Nephrology, Department of Medicine, Case Western Reserve University, Cleveland, OH 44106 (United States); Lane, Thomas A. [Departments of Medicine, Moores UCSD Cancer Center, University of California San Diego, San Diego, CA 92093-0820 (United States); Carrier, Ewa [Departments of Medicine, Moores UCSD Cancer Center, University of California San Diego, San Diego, CA 92093-0820 (United States)]. E-mail: assrivastava@ucsd.edu

    2006-07-28

    Differentiating embryonic stem (ES) cells are increasingly emerging as an important source of hematopoietic progenitors with a potential to be useful for both basic and clinical research applications. It has been suggested that dexamethasone facilitates differentiation of ES cells towards erythrocytes but the mechanism responsible for sequential expression of genes regulating this process are not well-understood. Therefore, we in vitro induced differentiation of murine ES cells towards erythropoiesis and studied the sequential expression of a set of genes during the process. We hypothesized that dexamethasone-activates its cognate nuclear receptors inducing up-regulation of erythropoietic genes such as GATA-1, Flk-1, Epo-R, and direct ES cells towards erythropoietic differentiation. ES cells were cultured in primary hematopoietic differentiation media containing methyl-cellulose, IMDM, IL-3, IL-6, and SCF to promote embryoid body (EB) formation. Total RNA of day 3, 5, and 9-old EBs was isolated for gene expression studies using RT-PCR. Cells from day 9 EBs were subjected to secondary differentiation using three different cytokines and growth factors combinations: (1) SCF, EPO, dexamethasone, and IGF; (2) SCF, IL-3, IL-6, and TPO; and (3) SCF IL-3, IL-6, TPO, and EPO. Total RNA from day 12 of secondary differentiated ES cells was isolated to study the gene expression pattern during this process. Our results demonstrate an up-regulation of GATA-1, Flk-1, HoxB-4, Epo-R, and globin genes ({alpha}-globin, {beta}H-1 globin, {beta}-major globin, {epsilon} -globin, and {zeta}-globin) in the 9-day-old EBs, whereas, RNA from 5-day-old EBs showed expression of HoxB-4, {epsilon}-globin, {gamma}-globin, {beta}H1-globin, and Flk-1. Three-day-old EBs showed only HoxB-4 and Flk-1 gene expression and lacked expression of all globin genes. These findings indicate that erythropoiesis-specific genes are activated later in the course of differentiation. Gene expression studies on the ES cells of secondary EB origin cultured in media containing dexamethasone showed a down-regulation of GATA-3 and an up-regulation of GATA-1, Flk-1, and Epo-R in comparison to the two other cytokines and growth factor combinations containing media. The secondary differentiation also showed an enhanced production of erythrocytic precursors in dexamethasone containing media in comparison to that in the control media. Our results indicate that dexamethasone can prove to be an effective agent which can be employed to enhance differentiation towards erythrocytic progenitors from ES cells.

  10. Recognition of thyroglobulin autoantigenic epitopes by murine T and B cells.

    PubMed Central

    Champion, B R; Page, K; Rayner, D C; Quartey-Papafio, R; Byfield, P G; Henderson, G

    1987-01-01

    We have used a large panel of thyroglobulins (Tg) prepared from a wide range of mammalian species to study the Tg autoantigenic epitopes recognized by populations of monoclonal and polyclonal murine T and B cells. This approach showed the existence of at least six different epitopes; three recognized by T cells (in association with I-Ak on antigen-presenting cells) and three by B cells (monoclonal antibodies). The majority of serum and monoclonal autoantibodies were found to be highly specific for mouse Tg, with some cross-reactive binding to rat Tg. In contrast, T-cell lines/clones and hybridomas recognized cross-reactive epitopes on Tg that were highly conserved throughout most of the mammalian orders. Moreover, two hybrid clones, which showed similar patterns of cross-reactivity, differed in their responsiveness to tryptic digests of human Tg. Thus, autoreactive T and B cells recognize distinct areas of the Tg molecule. PMID:2445667

  11. Measuring Intracellular Calcium Signaling in Murine NK Cells by Flow Cytometry

    PubMed Central

    MacFarlane, Alexander W.; Oesterling, James F.; Campbell, Kerry S.

    2009-01-01

    Summary This chapter describes a method by which activating receptor-mediated calcium signaling can be measured in individual murine NK cells using a flow cytometer fitted with a UV laser. One major advantage of this method is that the calcium response of the minority NK cell population and even smaller NK cell sub-populations can be measured simultaneously from a mixture of freshly prepared total splenocytes without resorting to prior cell sorting or expansion in culture. Briefly, cells are harvested and stained to mark the populations of interest, then loaded with indo-1 AM dye and analyzed on the flow cytometer. After an appropriate baseline is established, the cells are treated with a biotinylated antibody to activating receptors, which are subsequently cross-linked by addition of streptavidin. The increase in intracellular calcium is quantified by measuring a shift in the indo-1 emission spectrum that takes place when the dye becomes bound to calcium. PMID:20033639

  12. Cell-type-resolved quantitative proteomics of murine liver.

    PubMed

    Azimifar, S Babak; Nagaraj, Nagarjuna; Cox, Juergen; Mann, Matthias

    2014-12-01

    Mass spectrometry (MS)-based proteomics provides a powerful approach to globally investigate the biological function of individual cell types in mammalian organs. Here, we applied this technology to the in-depth analysis of purified hepatic cell types from mouse. We quantified 11,520 proteins, making this the most comprehensive proteomic resource of any organ to date. Global protein copy number determination demonstrated that a large proportion of the hepatocyte proteome is dedicated to fatty acid and xenobiotic metabolism. We identified as-yet-unknown components of the TGF-? signaling pathway and extracellular matrix in hepatic stellate cells, uncovering their regulative role in liver physiology. Moreover, our high-resolution proteomic data set enabled us to compare the distinct functional roles of hepatic cell types in cholesterol flux, cellular trafficking, and growth factor receptor signaling. This study provides a comprehensive resource for liver biology and biomedicine. PMID:25470552

  13. Distinct transcriptional signatures of aneuploidy in murine pluripotent cell populations 

    E-print Network

    Skylaki, Stavroula

    2012-11-30

    Genomic integrity in mouse embryonic and induced pluripotent stem cells can be compromised by factors such as extended time in culture and cellular reprogramming. Surprising, only a few studies have thus far examined the ...

  14. Guardians of the Gut – Murine Intestinal Macrophages and Dendritic Cells

    PubMed Central

    Gross, Mor; Salame, Tomer-Meir; Jung, Steffen

    2015-01-01

    Intestinal mononuclear phagocytes find themselves in a unique environment, most prominently characterized by its constant exposure to commensal microbiota and food antigens. This anatomic setting has resulted in a number of specializations of the intestinal mononuclear phagocyte compartment that collectively contribute the unique steady state immune landscape of the healthy gut, including homeostatic innate lymphoid cells, B, and T cell compartments. As in other organs, macrophages and dendritic cells (DCs) orchestrate in addition the immune defense against pathogens, both in lymph nodes and mucosa-associated lymphoid tissue. Here, we will discuss origins and functions of intestinal DCs and macrophages and their respective subsets, focusing largely on the mouse and cells residing in the lamina propria.

  15. Peroxisomal and Mitochondrial Status of Two Murine Oligodendrocytic Cell Lines (158N, 158JP): Potential Models for the Study of Peroxisomal Disorders Associated with

    E-print Network

    Paris-Sud XI, Université de

    1 Peroxisomal and Mitochondrial Status of Two Murine Oligodendrocytic Cell Lines (158N, 158JP title: Peroxisomal and Mitochondrial Status of Two Differentiated Murine Oligodendrocytic Cell Lines, the defect of peroxisomal functions on myelin-producing cells (oligodendrocytes) are poorly understood

  16. Diabetes, insulin-mediated glucose metabolism and Sertoli/blood-testis barrier function

    PubMed Central

    Alves, Marco G.; Martins, Ana D.; Cavaco, José E.; Socorro, Sílvia; Oliveira, Pedro F.

    2013-01-01

    Blood testis barrier (BTB) is one of the tightest blood-barriers controlling the entry of substances into the intratubular fluid. Diabetes Mellitus (DM) is an epidemic metabolic disease concurrent with falling fertility rates, which provokes severe detrimental BTB alterations. It induces testicular alterations, disrupting the metabolic cooperation between the cellular constituents of BTB, with dramatic consequences on sperm quality and fertility. As Sertoli cells are involved in the regulation of spermatogenesis, providing nutritional support for germ cells, any metabolic alteration in these cells derived from DM may be responsible for spermatogenesis disruption, playing a crucial role in fertility/subfertility associated with this pathology. These cells have a glucose sensing machinery that reacts to hormonal fluctuations and several mechanisms to counteract hyper/hypoglycemic events. The role of DM on Sertoli/BTB glucose metabolism dynamics and the metabolic molecular mechanisms through which DM and insulin deregulation alter its functioning, affecting male reproductive potential will be discussed. PMID:24665384

  17. Distinct functions of antigen-specific CD4 T cells during murine Mycobacterium tuberculosis infection

    PubMed Central

    Reiley, William W.; Shafiani, Shahin; Wittmer, Susan T.; Tucker-Heard, Glady's; Moon, James J.; Jenkins, Marc K.; Urdahl, Kevin B.; Winslow, Gary M.; Woodland, David L.

    2010-01-01

    The immune response elicited after Mycobacterium tuberculosis (Mtb) infection is critically dependent on CD4 T cells during both acute and chronic infection. How CD4 T-cell responses are maintained throughout infection is not well understood, and evidence from other infection models has suggested that, under conditions of chronic antigen stimulation, T cells can undergo replicative exhaustion. These findings led us to determine whether subpopulations of CD4 T cells existed that displayed markers of terminal differentiation or exhaustion during murine Mtb infection. Analysis of antigen-specific effector CD4 T cells revealed that programmed death-1 (PD-1) and the killer cell lectin-like receptor G1 (KLRG1) delineated subpopulations of T cells. PD-1–expressing CD4 T cells were highly proliferative, whereas KLRG1 cells exhibited a short lifespan and secreted the cytokines IFN? and TNF?. Adoptive transfer studies demonstrated that proliferating PD-1–positive CD4 T cells differentiated into cytokine-secreting KLRG1-positive T cells, but not vice versa. Thus, proliferating PD-1–positive cells are not exhausted, but appear to be central to maintaining antigen-specific effector T cells during chronic Mtb infection. Our findings suggest that antigen-specific T-cell responses are maintained during chronic mycobacterial infection through the continual production of terminal effector cells from a proliferating precursor population. PMID:20962277

  18. Specific uptake of serotonin by murine lymphoid cells

    SciTech Connect

    Jackson, J.C.; Walker, R.F.; Brooks, W.H.; Roszman, T.L.

    1986-03-01

    Recently the authors confirmed and extended earlier observations that serotonin (5-hydroxytryptamine, 5HT) can influence immune function. Both 5HT and its precursor, 5-hydroxytryptophan inhibit the primary, in vivo antibody response to sheep red blood cells, in mice. Here, the authors report specific in vitro association of this amine with mouse splenocytes. Spleen cells from 6-8 week old CBA/J mice incorporated /sup 3/H-5HT(10/sup -8/ to 2.5 x 10/sup -6/M) in a saturable manner, at 37/sup 0/C. Specificity of uptake was indicated by competition with excess (10/sup -5/M) unlabelled 5HT and with 10/sup -5/M fluoxetine, a selective inhibitor of active 5HT reuptake in rat brain. The 5HT receptor antagonists, methysergide and cyproheptadine, also blocked 5HT uptake. Cell lysis and displacement studies revealed largely intracellular accumulation of /sup 3/H-5HT with little membrane association, in splenocytes. Hofstee analysis of uptake kinetics yielded an apparent Km of 0.82 +/- 0.22 x 10/sup -7/M and Vmax of 501 +/- 108 pM/3 x 10/sup 6/ cells/10 min. Spleen cells fractionated on Sephadex G10 showed virtually no specific 5HT uptake while peritoneal exudate cells from thioglycollate treated mice displayed 5HT uptake kinetics similar to those of splenocytes. The site of specific /sup 3/H-5HT incorporation within a population of spleen cells and the functional significance of this phenomenon to immunomodulation by 5HT remain to be elucidated.

  19. R-phycoerythrin-conjugated antibodies are inappropriate for intracellular staining of murine plasma cells.

    PubMed

    Kim, Myun Soo; Kim, Tae Sung

    2013-05-01

    Phycoerythrin (PE) is a type of phycobiliproteins found in cyanobacteria and red algae. PE-conjugated antibodies are broadly used for flow cytometry and immunofluorescence microscopy. Because nonspecific binding of antibodies results in decreased analytic accuracy, numerous efforts have been made to unveil cases and mechanisms of nonspecific bindings. However, nonspecific binding of specific cell types by a fluorescent dye-conjugated form of antibody has been rarely reported. In the present study, we discovered that PE-conjugated antibodies, but not FITC- or APC-antibodies, selectively stained lamina propria plasma cells (LP-PCs) from the murine small intestine after membrane permeabilization. We demonstrated that LP-PC-selective staining with PE-antibodies was not due to interactions of antibody-epitope or antibody-Fc receptor. This unexpected staining by PE-antibody was not dependent on the mouse strain of LP-PCs, experimental methods, or origin species of the antibody, but dependent on PE itself. This phenomenon was also observed in plasma cells isolated from bone marrow, spleen, and mesenteric lymph nodes. Furthermore, in vitro activated B cells and in vivo generated LP-PCs were also selectively stained by PE-conjugated antibodies. Taken together, these results show that PE-conjugated antibodies are inappropriate for intracellular staining of murine plasma cells. PMID:23463627

  20. An ES-Like Pluripotent State in FGF-Dependent Murine iPS cells

    PubMed Central

    Ungaro, Federica; Prigione, Alessandro; Chen, Hsu-Hsin; Welling, Maaike; Eijpe, Maureen; Mostoslavsky, Gustavo; Tesar, Paul; Adjaye, James; Geijsen, Niels; Broccoli, Vania

    2010-01-01

    Recent data demonstrates that stem cells can exist in two morphologically, molecularly and functionally distinct pluripotent states; a naïve LIF-dependent pluripotent state which is represented by murine embryonic stem cells (mESCs) and an FGF-dependent primed pluripotent state represented by murine and rat epiblast stem cells (EpiSCs). We find that derivation of induced pluripotent stem cells (iPSCs) under EpiSC culture conditions yields FGF-dependent iPSCs from hereon called FGF-iPSCs) which, unexpectedly, display naïve ES-like/ICM properties. FGF-iPSCs display X-chromosome activation, multi-lineage differentiation, teratoma competence and chimera contribution in vivo. Our findings suggest that in 129 and Bl6 mouse strains, iPSCs can dominantly adopt a naive pluripotent state regardless of culture growth factor conditions. Characterization of the key molecular signalling pathways revealed FGF-iPSCs to depend on the Activin/Nodal and FGF pathways, while signalling through the JAK-STAT pathway is not required for FGF-iPS cell maintenance. Our findings suggest that in 129 and Bl6 mouse strains, iPSCs can dominantly adopt a naive pluripotent state regardless of culture growth factor conditions. PMID:21209851

  1. Dielectrophoresis and electrorotation of neurospora slime and murine myeloma cells.

    PubMed Central

    Gimsa, J; Marszalek, P; Loewe, U; Tsong, T Y

    1991-01-01

    Dielectrophoresis and electrorotation are commonly used to measure dielectric properties and membrane electrical parameters of biological cells. We have derived quantitative relationships for several critical points, defined in Fig. A 1, which characterize the dielectrophoretic spectrum and the electrorotational spectrum of a cell, based on the single-shell model (Pauly, H., and H.P. Schwan, 1959. Z. Naturforsch. 14b:125-131; Sauer, F.A. 1985. Interactions between Electromagnetic Field and Cells. A. Chiabrera, C. Nicolini, and H.P. Schwan, editors. Plenum Publishing Corp., New York. 181-202). To test these equations and to obtain membrane electrical parameters, a technique which allowed simultaneous measurements of the dielectrophoresis and the electrorotation of single cells in the same chamber, was developed and applied to the study of Neurospora slime and the Myeloma Tib9 cell line. Membrane electrical parameters were determined by the dependence of the first critical frequency of dielectrophoresis (fct1) and the first characteristic frequency of electrorotation (fc1) on the conductivity of the suspending medium. Membrane conductances of Neurospora slime and Myeloma also were found to be 500 and 380 S m-2, respectively. Several observations indicate that these cells are more complex than that described by the single-shell model. First, the membrane capacities from fct1 (0.81 x 10(-2) and 1.55 x 10(-2) F m-2 for neurospora slime and Myeloma, respectively) were at least twice those derived from fc1. Second, the electrorotation spectrum of Myeloma cells deviated from the single-shell like behavior. These discrepancies could be eliminated by adapting a three-shell model (Furhr, G., J. Gimsa, and R. Glaser. 1985. Stud. Biophys. 108:149-164). Apparently, there was more than one membrane relaxation process which could influence the lower frequency region of the beta-dispersion. fct1 of Myeloma in a medium of given external conductivity were found to be similar for most cells, but for some a dramatically increased fct1 was recorded. Model analysis suggested that a decrease in the cytoplasmatic conductivity due to a drastic ion loss in a cell could cause this increase in fct1. Model analysis also suggested that the electrorotation spectrum in the counter-field rotation range and fc1 would be more sensitive to conductivity changes of the cytoplasmic fluid and to the influence of internal membranes than would fct1, although the latter would be sensitive to changes in capacitance of the cytoplasmic membranes. PMID:1835890

  2. Disruption of canonical TGF?-signaling in murine coronary progenitor cells by low level arsenic.

    PubMed

    Allison, Patrick; Huang, Tianfang; Broka, Derrick; Parker, Patti; Barnett, Joey V; Camenisch, Todd D

    2013-10-01

    Exposure to arsenic results in several types of cancers as well as heart disease. A major contributor to ischemic heart pathologies is coronary artery disease, however the influences by environmental arsenic in this disease process are not known. Similarly, the impact of toxicants on blood vessel formation and function during development has not been studied. During embryogenesis, the epicardium undergoes proliferation, migration, and differentiation into several cardiac cell types including smooth muscle cells which contribute to the coronary vessels. The TGF? family of ligands and receptors is essential for developmental cardiac epithelial to mesenchymal transition (EMT) and differentiation into coronary smooth muscle cells. In this in vitro study, 18hour exposure to 1.34?M arsenite disrupted developmental EMT programming in murine epicardial cells causing a deficit in cardiac mesenchyme. The expression of EMT genes including TGF?2, TGF? receptor-3, Snail, and Has-2 are decreased in a dose-dependent manner following exposure to arsenite. TGF?2 cell signaling is abrogated as detected by decreases in phosphorylated Smad2/3 when cells are exposed to 1.34?M arsenite. There is also loss of nuclear accumulation pSmad due to arsenite exposure. These observations coincide with a decrease in vimentin positive mesenchymal cells invading three-dimensional collagen gels. However, arsenite does not block TGF?2 mediated smooth muscle cell differentiation by epicardial cells. Overall these results show that arsenic exposure blocks developmental EMT gene programming in murine coronary progenitor cells by disrupting TGF?2 signals and Smad activation, and that smooth muscle cell differentiation is refractory to this arsenic toxicity. PMID:23732083

  3. Magnetite- and maghemite-induced different toxicity in murine alveolar macrophage cells.

    PubMed

    Park, Eun-Jung; Umh, Ha Nee; Choi, Dong-Hyuk; Cho, Myung Haing; Choi, Wookhee; Kim, Sang-Wook; Kim, Younghun; Kim, Jae-Ho

    2014-08-01

    The unique properties of nanoparticles and biological systems are important factors affecting the biological response following nanoparticle exposure. Iron oxide nanoparticles are classified mainly as magnetite (M-FeNPs) and maghemite (NM-FeNPs). In our previous study, NM-FeNPs induced autophagic cell death in RAW264.7, a murine peritoneal macrophage cell line, which has excellent lysosomal activity. In this study, we compared the toxicity of M-FeNPs and NM-FeNPs in MH-S, a murine alveolar macrophage cell line, which has relatively low lysosomal activity. At 24 h post-exposure, M-FeNPs decreased cell viability and ATP production, and elevated the levels of reactive oxygen species, nitric oxide, and pro-inflammatory cytokines to a higher extent than NM-FeNPs. Damage of mitochondria and the endoplasmic reticulum and the down-regulation of mitochondrial function and transcription-related genes were also higher in cells exposed to M-FeNPs than in cells exposed to NM-FeNPs (50 ?g/ml). In addition, cells exposed to M-FeNPs (50 ?g/ml) showed an increase in the number of autophagosome-like vacuoles, whereas cells exposed to NM-FeNPs formed large vacuoles in the cytosol. However, an autophagy-related molecular response was not induced by exposure to either FeNPs, unlike the results seen in our previous study with RAW264.7 cells. We suggest that M-FeNPs induced higher toxicity compared to NM-FeNPs in MH-S cells, and lysosomal activity plays an important role in determining cell death pathway. PMID:24525745

  4. Therapeutic liver reconstitution with murine cells isolated long after death

    PubMed Central

    Erker, Laura; Azuma, Hisaya; Lee, Andrew Y.; Guo, Changsheng; Orloff, Susan; Eaton, Laura; Benedetti, Eric; Jensen, Bryan; Finegold, Milton; Willenbring, Holger; Grompe, Markus

    2013-01-01

    Background and Aims Due to the shortage of donor organs many patients needing liver transplantation cannot receive one. For some liver diseases hepatocyte transplantation could be a viable alternative, but donor cells are currently procured from the same sources as whole organs and thus the supply is severely limited. Methods Here, we investigated the possibility of isolating viable hepatocytes for liver cell therapy from the plentiful source of morgue cadavers. In order to determine the utility of this approach, cells were isolated from the livers of non-heart-beating cadaveric mice long after death and transplanted into fumarylacetoacetate hydrolase (Fah) deficient mice, a model for the human metabolic liver disease hereditary tyrosinemia type I and a stringent in vivo model for hepatic cell transplantation. Results Surprisingly, complete and therapeutic liver repopulation could be achieved with hepatocytes derived up to 27 hours post-mortem. Conclusions Competitive repopulation experiments demonstrated that cadaveric liver cells had a repopulation capacity similar to freshly isolated hepatocytes. Importantly, viable hepatocytes could also be efficiently isolated from cadaveric primate liver (monkey and human). These data provide evidence that non-heart-beating donors could be a suitable source of hepatocytes for much longer time periods than previously thought possible. PMID:20621682

  5. Hyphal Growth of Phagocytosed Fusarium oxysporum Causes Cell Lysis and Death of Murine Macrophages

    PubMed Central

    Schäfer, Katja; Bain, Judith M.

    2014-01-01

    Fusarium oxysporum is an important plant pathogen and an opportunistic pathogen of humans. Here we investigated phagocytosis of F. oxysporum by J774.1 murine cell line macrophages using live cell video microscopy. Macrophages avidly migrated towards F. oxysporum germlings and were rapidly engulfed after cell-cell contact was established. F. oxysporum germlings continued hyphal growth after engulfment by macrophages, leading to associated macrophage lysis and escape. Macrophage killing depended on the multiplicity of infection. After engulfment, F. oxysporum inhibited macrophages from completing mitosis, resulting in large daughter cells fused together by means of a F. oxysporum hypha. These results shed new light on the initial stages of Fusarium infection and the innate immune response of the mammalian host. PMID:25025395

  6. Tetrahydro-6-biopterin is associated with tetrahydro-7-biopterin in primary murine mast cells.

    PubMed

    Ziegler, I; Hültner, L

    1992-07-28

    Murine bone marrow-derived mast cells proliferate in response to interleukin 3. In addition to 6-biopterin, 7-biopterin was identified in these cells by HPLC analysis of iodine oxidized extracts and by alkaline permanganate oxidation to the 6- and 7-carboxylic acids. 7-Biopterin comprised 31.9 (+/- 7.7)% of the total biopterin. It was absent in cells which were grown with of L-p-chlorophenylalanine, an inhibitor of tryptophan 5-mono-oxygenase. Both 6- and 7-biopterin were present in the cell as their tetrahydro forms. From these data we conclude that 7-biopterin, in contrast to e.g. brain tissue, regularly occurs as a normal metabolite in primary mast cells and that it is generated during hydroxylation of tryptophan. PMID:1644167

  7. Involvement of tumor necrosis factor (TNF-?) in arsenic trioxide induced apoptotic cell death of murine myeloid leukemia cells

    Microsoft Academic Search

    N. K Mak; R. N. S Wong; K. N Leung; M. C Fung

    2002-01-01

    Arsenic trioxide (As2O3) has recently been shown to be effective to inhibit the growth and to induce apoptosis in acute promyelocytic leukemia (APL) but not in acute myeloid leukemia (AML) cells. Recently, we have isolated an As2O3 sensitive subclone JCS-16 from the murine myeloid leukemia WEHI 3B (JCS). At the concentrations of 0.3–3 ?M, As2O3 induces a dose-dependent cytotoxicity and

  8. Estrogen and progesterone together expand murine endometrial epithelial progenitor cells

    PubMed Central

    Janzen, DM; Cheng, D; Schafenacker, AM; Paik, DY; Goldstein, AS; Witte, ON; Jaroszewicz, A; Pellegrini, M; Memarzadeh, S

    2013-01-01

    Synchronous with massive shifts in reproductive hormones, the uterus and its lining the endometrium expand to accommodate a growing fetus during pregnancy. In the absence of an embryo the endometrium, composed of epithelium and stroma, undergoes numerous hormonally regulated cycles of breakdown and regeneration. The hormonally mediated regenerative capacity of the endometrium suggests that signals that govern the growth of endometrial progenitors must be regulated by estrogen and progesterone. Here we report an antigenic profile for isolation of mouse endometrial epithelial progenitors. These cells are EpCAM+CD44+ITGA6hiThy1?PECAM1?PTPRC?Ter119?, comprise a minor subpopulation of total endometrial epithelia and possess a gene expression profile that is unique and different from other cells of the endometrium. The epithelial progenitors of the endometrium could regenerate in vivo, undergo multi-lineage differentiation and proliferate. We show that the number of endometrial epithelial progenitors is regulated by reproductive hormones. Co-administration of estrogen and progesterone dramatically expanded the endometrial epithelial progenitor cell pool. This effect was not observed when estrogen or progesterone was administered alone. Despite the remarkable sensitivity to hormonal signals, endometrial epithelial progenitors do not express estrogen or progesterone receptors. Therefore their hormonal regulation must be mediated through paracrine signals resulting from binding of steroid hormones to the progenitor cell niche. Discovery of signaling defects in endometrial epithelial progenitors or their niche can lead to development of better therapies in diseases of the endometrium. PMID:23341289

  9. Estrogen and progesterone together expand murine endometrial epithelial progenitor cells.

    PubMed

    Janzen, Deanna M; Cheng, Donghui; Schafenacker, Amanda M; Paik, Daniel Y; Goldstein, Andrew S; Witte, Owen N; Jaroszewicz, Artur; Pellegrini, Matteo; Memarzadeh, Sanaz

    2013-04-01

    Synchronous with massive shifts in reproductive hormones, the uterus and its lining the endometrium expand to accommodate a growing fetus during pregnancy. In the absence of an embryo the endometrium, composed of epithelium and stroma, undergoes numerous hormonally regulated cycles of breakdown and regeneration. The hormonally mediated regenerative capacity of the endometrium suggests that signals that govern the growth of endometrial progenitors must be regulated by estrogen and progesterone. Here, we report an antigenic profile for isolation of mouse endometrial epithelial progenitors. These cells are EpCAM(+) CD44(+) ITGA6(hi) Thy1(-) PECAM1(-) PTPRC(-) Ter119(-), comprise a minor subpopulation of total endometrial epithelia and possess a gene expression profile that is unique and different from other cells of the endometrium. The epithelial progenitors of the endometrium could regenerate in vivo, undergo multilineage differentiation and proliferate. We show that the number of endometrial epithelial progenitors is regulated by reproductive hormones. Coadministration of estrogen and progesterone dramatically expanded the endometrial epithelial progenitor cell pool. This effect was not observed when estrogen or progesterone was administered alone. Despite the remarkable sensitivity to hormonal signals, endometrial epithelial progenitors do not express estrogen or progesterone receptors. Therefore, their hormonal regulation must be mediated through paracrine signals resulting from binding of steroid hormones to the progenitor cell niche. Discovery of signaling defects in endometrial epithelial progenitors or their niche can lead to development of better therapies in diseases of the endometrium. PMID:23341289

  10. Cell Death in the Midbrain of the Murine Mutation weaver

    Microsoft Academic Search

    Suzanne Roffler-Tarlov; Baby Martin; Ann M. Graybiel; John S. Kauer

    1996-01-01

    The midbrain of the adult homozygous weaver (WV\\/WV) mouse is notable for a reduction in the numbers of dopamine- containing cells in the substantia nigra (A9) and the retrorubral nucleus (A8). We have determined that the reduction in tyrosine hydroxylase (TH)-positive neurons in the ventral midbrain of the weaver is attributable to the loss of neurons after postnatal day 7

  11. Differential effects of protoporphyrin and uroporphyrin on murine mast cells

    SciTech Connect

    Lim, H.W.; Gigli, I.; Wasserman, S.I.

    1987-03-01

    To investigate the mechanisms responsible for the distinct cutaneous manifestations of erythropoietic protoporphyria and porphyria cutanea tarda, the effects of protoporphyrin (PP) and uroporphyrin (URO), the predominant porphyrins in the respective disease, on mast cells were examined. Release of preformed and generated mediators was assessed by the release of radioactivity from cells labeled with (/sup 3/H)serotonin and (/sup 14/C)arachidonic acid, respectively. Clinically relevant doses of PP (25-500 ng/ml) and 396-407 nm irradiation (3-16 X 10(2)J/m2) induced maximal net release of preformed mediators ,f 44.52 +/- 6.6 to 58.01 +/- 4.0% (mean +/- SE). In contrast, irradiation in the presence of URO (50-5000 ng/ml) resulted in less than 5% net release. (3H)Serotonin release induced by PP and irradiation was calcium-independent, and was not enhanced by phorbol 12-myristate 13-acetate, a known activator of protein kinase C. This release was suppressed by catalase, a scavenger of hydrogen peroxide. Furthermore, irradiation in the presence of PP, but not in the presence of URO, resulted in perturbation of cell membrane. Irradiation in the presence of PP also resulted in a maximal net release of generated mediators of 9.98 +/- 3.5% (mean +/- SE), whereas similar treatment in the presence of URO induced less than 0.5% net release. These results suggested that the burning, stinging, erythema, and edema experienced by patients with erythropoietic protoporphyria following sun exposure, and the lack of such findings in patients with porphyria cutanea tarda, may be explained, at least in part, by the differential effects of PP and URO on mast cells.

  12. Toll-like receptor ligand activation of murine bone marrow-derived dendritic cells.

    PubMed

    Dearman, Rebecca J; Cumberbatch, Marie; Maxwell, Gavin; Basketter, David A; Kimber, Ian

    2009-04-01

    Dendritic cells (DCs) are required for the initiation of primary immune responses. The pattern of Toll-like receptor (TLR) expression on various subsets of these cells has been shown to differ, suggestive of distinct roles in influencing immune responses. We have examined here the responses of immature DCs derived from murine bone marrow (BMDCs) to a range of TLR ligands. BMDCs cultured for 6 days in the presence of granulocyte-macrophage colony-stimulating factor were stimulated for 24 hr with ligands to TLR1-2 [Pam(3)Cys-Ser-(Lys)(4) (PAM)], TLR2-6 (macrophage-activating lipopeptide-2 (MALP-2); zymosan or peptidoglycan (PG)], TLR3 (polyinosinic-polycytidylic acid), TLR4 [lipopolysaccharide R515 (LPS)], TLR5 (flagellin), TLR7 (polyuridylic acid) and TLR9 [CpG ODN2395 (CpG)]. DC activation was monitored using membrane marker expression and analysis of culture supernatants for cytokine/chemokine release. Ligands to TLR3 and TLR7 failed to activate BMDCs. All other TLR ligands caused elevated expression of membrane markers. PAM, MALP-2 and LPS induced high-level expression of proinflammatory cytokines and chemokines. Treatment with CpG was associated with a preferential type 1 cytokine and chemokine profile. Zymosan and PG were proinflammatory but also skewed towards a type 2 pattern of cytokines and chemokines. In contrast, flagellin did not cause marked secretion by BMDCs of cytokines or chemokines. These data for BMDCs are largely consistent with the reported TLR repertoire of freshly isolated murine Langerhans cells. In addition, murine BMDCs show selective responses to TLR ligands with respect to general activation, with differentiated cytokine patterns suggestive of potential priming for divergent immune responses. PMID:18778283

  13. Toll-like receptor ligand activation of murine bone marrow-derived dendritic cells

    PubMed Central

    Dearman, Rebecca J; Cumberbatch, Marie; Maxwell, Gavin; Basketter, David A; Kimber, Ian

    2009-01-01

    Dendritic cells (DCs) are required for the initiation of primary immune responses. The pattern of Toll-like receptor (TLR) expression on various subsets of these cells has been shown to differ, suggestive of distinct roles in influencing immune responses. We have examined here the responses of immature DCs derived from murine bone marrow (BMDCs) to a range of TLR ligands. BMDCs cultured for 6 days in the presence of granulocyte–macrophage colony-stimulating factor were stimulated for 24 hr with ligands to TLR1-2 [Pam3Cys-Ser-(Lys)4 (PAM)], TLR2-6 (macrophage-activating lipopeptide-2 (MALP-2); zymosan or peptidoglycan (PG)], TLR3 (polyinosinic-polycytidylic acid), TLR4 [lipopolysaccharide R515 (LPS)], TLR5 (flagellin), TLR7 (polyuridylic acid) and TLR9 [CpG ODN2395 (CpG)]. DC activation was monitored using membrane marker expression and analysis of culture supernatants for cytokine/chemokine release. Ligands to TLR3 and TLR7 failed to activate BMDCs. All other TLR ligands caused elevated expression of membrane markers. PAM, MALP-2 and LPS induced high-level expression of proinflammatory cytokines and chemokines. Treatment with CpG was associated with a preferential type 1 cytokine and chemokine profile. Zymosan and PG were proinflammatory but also skewed towards a type 2 pattern of cytokines and chemokines. In contrast, flagellin did not cause marked secretion by BMDCs of cytokines or chemokines. These data for BMDCs are largely consistent with the reported TLR repertoire of freshly isolated murine Langerhans cells. In addition, murine BMDCs show selective responses to TLR ligands with respect to general activation, with differentiated cytokine patterns suggestive of potential priming for divergent immune responses. PMID:18778283

  14. Spatio-temporal organization of DNA replication in murine embryonic stem, primary, and immortalized cells.

    PubMed

    Panning, Margaret M; Gilbert, David M

    2005-05-01

    The extent to which chromosomal domains are reorganized within the nucleus during differentiation is central to our understanding of how cells become committed to specific developmental lineages. Spatio-temporal patterns of DNA replication are a reflection of this organization. Here, we demonstrate that the temporal order and relative duration of these replication patterns during S-phase are similar in murine pluripotent embryonic stem (ES) cells, primary adult myoblasts, and an immortalized fibroblast line. The observed patterns were independent of fixation and denaturation techniques. Importantly, the same patterns were detected when fluorescent nucleotides were introduced into living cells, demonstrating their physiological relevance. These data suggest that heritable gene silencing during commitment to specific cell lineages is not mediated by global changes in the sub-nuclear organization and replication timing of chromosome domains. PMID:15723284

  15. Accessing the Genomic Effects of Naked Nanoceria in Murine Neuronal Cells

    PubMed Central

    Lee, Tin-Lap; Raitano, Joan M.; Rennert, Owen M; Chan, Siu-Wai; Chan, Wai-Yee

    2011-01-01

    Cerium oxide nanoparticles (nanoceria) are versatile engineered nanoparticles (ENPs) due to their unique redox properties. We and others have previously demonstrated naked nanoceria could act as antioxidants to protect cells against oxidative damage. While the redox properties may be beneficial, the genome-wide effects of nanoceria on gene transcription and associated biological processes remain elusive. Here we applied functional genomic approach to examine the genome-wide effects of nanoceria on global gene transcription and cellular functions in mouse neuronal cells. Importantly, we demonstrated nanoceria induced chemical- and size-specific changes in the murine neuronal cell transcriptome. The nanoceria specially contributed more than 83% of uniquely altered gene population and associate with a unique spectrum of genes related to neurological disease, cell cycle control and growth. These observations suggest an in-depth assessment of potential health effects of naked nanoceria and other naked nanoparticles is both necessary and imminent. PMID:21889474

  16. Selective cytotoxicity of ricin A chain immunotoxins towards murine cytomegalovirus-infected cells.

    PubMed Central

    Barnett, B B; Smee, D F; Malek, S M; Sidwell, R W

    1996-01-01

    Immunotoxins were constructed by linking immunoglobulins specific for murine cytomegalovirus (MCMV) to deglycosylated ricin A chain. Toxicities toward MCMV-infected and uninfected cells were determined by measuring the inhibition of protein synthesis following a 48-h exposure to immunotoxins commencing 24 h after infection. The 50% inhibitory concentrations ranged from 0.4 to 4 micrograms/ml for infected cells and from 22 to 120 micrograms/ml for uninfected cells. Selectivity indices ranged from 30 to 157. Control immunotoxins, which were constructed identically except that the immunoglobulin moiety had no specificity toward MCMV antigens, had 50% inhibitory concentrations of 50 and 100 micrograms/ml toward infected and uninfected cells, respectively. PMID:8834901

  17. Modification of acute murine cytomegalovirus adrenal gland infection by adoptive spleen cell transfer.

    PubMed Central

    Shanley, J D

    1987-01-01

    Homozygous (nu/nu), athymic nude mice, infected with murine cytomegalovirus (MCMV), develop unremitting and ablative virus infection involving both the adrenal cortex and medulla. During acute infection, adoptive transfer of MCMV-immune, but not naive, spleen cells suppressed virus replication in the adrenal glands, but not the lungs or salivary gland. T lymphocytes, not macrophages or B cells, were responsible for limiting viral replication. The effect by donor cells was restricted by compatibility at the major histocompatibility locus. Restriction of MCMV replication in the adrenal gland was associated with T lymphocytes of the L3T4 phenotype. Thus, T-cell immunity is critical in regulating MCMV replication in the adrenal glands, and T lymphocytes restricted by class II major histocompatibility antigens mediate this effect. PMID:3023703

  18. Radiation enhances tumor necrosis factor alpha production by murine brain cells.

    PubMed

    Chiang, C S; McBride, W H

    1991-12-01

    Astrocytes and microglial cells cultured from murine brain were stimulated to produce tumor necrosis factor alpha (TNF) by exposure to lipopolysaccharide (LPS). TNF alpha production began within 2 h with maximum production between 4 and 8 h after stimulation. Clinically relevant low (2 Gy), but not high (8 Gy), doses of radiation significantly increased TNF production by astrocytes and microglial cells in response to LPS. The radiation effect was even more marked with multiple 2 Gy doses. TNF is cytotoxic for oligodendrocytes and for certain tumor cells. It increases vascular permeability and enhances immune responses as well as having other biological effects. It is conceivable that production of TNF by astrocytes and microglial cells during clinical radiation therapy might influence the responses of tumor and/or normal CNS tissues. PMID:1814542

  19. Index-sorting resolves heterogeneous murine hematopoietic stem cell populations

    E-print Network

    Schulte, Reiner; Wilson, Nicola K.; Prick, Janine C. M.; Cossetti, Chiara; Maj, Michael K.; Gottgens, Berthold; Kent, David G.

    2015-01-01

    to hold individual 1.4mL polypropylene round bottom tubes (See Methods and Figure 1A). Index sorting used as a tool for determining transcriptional differences in single stem cells. To develop a tool to understand the molecular heterogeneity within... . To create a fitting holder a 96 well polycarbonate rack typically used to hold individual 1.4mL polypropylene round bottom tubes was used. By removing the legs of the rack and shaving the bottom surface to be flat, we were able to create a rigid fit...

  20. The role of the e3 ligase cbl-B in murine dendritic cells.

    PubMed

    Wallner, Stephanie; Lutz-Nicoladoni, Christina; Tripp, Christoph H; Gastl, Günther; Baier, Gottfried; Penninger, Josef M; Stoitzner, Patrizia; Wolf, Dominik

    2013-01-01

    Dendritic cells (DCs) are potent antigen-presenting cells with a promising potential in cancer immunotherapy. Cbl proteins are E3 ubiquitin ligases and have been implicated in regulating the functional activity of various immune cells. As an example, c-Cbl negatively affects DC activation. We here describe that another member of the Cbl-protein family (i.e. Cbl-b) is highly expressed in murine bone-marrow-derived DCs (BMDCs). Differentiation of cblb-/- bone marrow mononuclear cells into classical BMDCs is unaltered, except enhanced induction of DEC-205 (CD205) expression. When tested in mixed-lymphocyte reaction (MLR), cblb-/- BMDCs exhibit increased allo-stimulatory capacity in vitro. BMDCs were next in vitro stimulated by various toll like receptor (TLR)-agonists (LPS, Poly(I:C), CpG) and exposed to FITC-labeled dextran. Upon TLR-stimulation, cblb-/- BMDCs produce higher levels of proinflammatory cytokines (IL-1?, IL-6 and TNF-?) and exhibit a slightly higher level of FITC-dextran uptake. To further characterize the functional significance of cblb-/- BMDCs we tested them in antigen-specific T cell responses against ovalbumin (OVA) protein and peptides, activating either CD8(+) OT-I or CD4(+) OT-II transgenic T cells. However, cblb-/- BMDCs are equally effective in inducing antigen-specific T cell responses when compared to wildtype BMDCs both in vitro and in vivo. The migratory capacity into lymph nodes during inflammation was similarly not affected by the absence of Cbl-b. In line with these observations, cblb-/- peptide-pulsed BMDCs are equally effective vaccines against OVA-expressing B16 tumors in vivo when compared to wildtype BMDCs. We conclude that in contrast to c-Cbl, Cbl-b plays only a limited role in the induction of Ag-specific T cell responses by murine BMDCs in vitro and in vivo. PMID:23762309

  1. The Role of the E3 Ligase Cbl-B in Murine Dendritic Cells

    PubMed Central

    Wallner, Stephanie; Lutz-Nicoladoni, Christina; Tripp, Christoph H.; Gastl, Günther; Baier, Gottfried; Penninger, Josef M.; Stoitzner, Patrizia; Wolf, Dominik

    2013-01-01

    Dendritic cells (DCs) are potent antigen-presenting cells with a promising potential in cancer immunotherapy. Cbl proteins are E3 ubiquitin ligases and have been implicated in regulating the functional activity of various immune cells. As an example, c-Cbl negatively affects DC activation. We here describe that another member of the Cbl-protein family (i.e. Cbl-b) is highly expressed in murine bone-marrow-derived DCs (BMDCs). Differentiation of cblb?/? bone marrow mononuclear cells into classical BMDCs is unaltered, except enhanced induction of DEC-205 (CD205) expression. When tested in mixed-lymphocyte reaction (MLR), cblb?/? BMDCs exhibit increased allo-stimulatory capacity in vitro. BMDCs were next in vitro stimulated by various toll like receptor (TLR)-agonists (LPS, Poly(I:C), CpG) and exposed to FITC-labeled dextran. Upon TLR-stimulation, cblb?/? BMDCs produce higher levels of proinflammatory cytokines (IL-1?, IL-6 and TNF-?) and exhibit a slightly higher level of FITC-dextran uptake. To further characterize the functional significance of cblb?/? BMDCs we tested them in antigen-specific T cell responses against ovalbumin (OVA) protein and peptides, activating either CD8+ OT-I or CD4+ OT-II transgenic T cells. However, cblb?/? BMDCs are equally effective in inducing antigen-specific T cell responses when compared to wildtype BMDCs both in vitro and in vivo. The migratory capacity into lymph nodes during inflammation was similarly not affected by the absence of Cbl-b. In line with these observations, cblb?/? peptide-pulsed BMDCs are equally effective vaccines against OVA-expressing B16 tumors in vivo when compared to wildtype BMDCs. We conclude that in contrast to c-Cbl, Cbl-b plays only a limited role in the induction of Ag-specific T cell responses by murine BMDCs in vitro and in vivo. PMID:23762309

  2. Tracing the fate of limbal epithelial progenitor cells in the murine cornea.

    PubMed

    Di Girolamo, N; Bobba, S; Raviraj, V; Delic, N C; Slapetova, I; Nicovich, P R; Halliday, G M; Wakefield, D; Whan, R; Lyons, J G

    2015-01-01

    Stem cell (SC) division, deployment, and differentiation are processes that contribute to corneal epithelial renewal. Until now studying the destiny of these cells in a living mammal has not been possible. However, the advent of inducible multicolor genetic tagging and powerful imaging technologies has rendered this achievable in the translucent and readily accessible murine cornea. K14CreER(T2)-Confetti mice that harbor two copies of the Brainbow 2.1 cassette, yielding up to 10 colors from the stochastic recombination of fluorescent proteins, were used to monitor K-14(+) progenitor cell dynamics within the corneal epithelium in live animals. Multicolored columns of cells emerged from the basal limbal epithelium as they expanded and migrated linearly at a rate of 10.8 µm/day toward the central cornea. Moreover, the permanent expression of fluorophores, passed on from progenitor to progeny, assisted in discriminating individual clones as spectrally distinct streaks containing more than 1,000 cells within the illuminated area. The centripetal clonal expansion is suggestive that a single progenitor cell is responsible for maintaining a narrow corridor of corneal epithelial cells. Our data are in agreement with the limbus as the repository for SC as opposed to SC being distributed throughout the central cornea. This is the first report describing stem/progenitor cell fate determination in the murine cornea using multicolor genetic tracing. This model represents a powerful new resource to monitor SC kinetics and fate choice under homeostatic conditions, and may assist in assessing clonal evolution during corneal development, aging, wound-healing, disease, and following transplantation. PMID:24966117

  3. Histamine synthesis is required for granule maturation in murine mast cells.

    PubMed

    Nakazawa, Shunsuke; Sakanaka, Mariko; Furuta, Kazuyuki; Natsuhara, Mayuko; Takano, Hirotsugu; Tsuchiya, Soken; Okuno, Yasushi; Ohtsu, Hiroshi; Nishibori, Masahiro; Thurmond, Robin L; Hirasawa, Noriyasu; Nakayama, Kazuhisa; Ichikawa, Atsushi; Sugimoto, Yukihiko; Tanaka, Satoshi

    2014-01-01

    Mast cells are the major sources of histamine, which is released in response to immunological stimulations. The synthesis of histamine is catalyzed by histidine decarboxylase (HDC). Previous studies have shown that Hdc(-/-) mast cells exhibit aberrant granule morphology with severely decreased granule content. Here, we investigated whether the histamine synthesized in mast cells regulates the granule maturation of murine mast cells. Several genes, including those encoding granule proteases and enzymes involved in heparin biosynthesis, were downregulated in Hdc(-/-) peritoneal mast cells. Impaired granule maturation was also found in Hdc(-/-) BM-derived cultured mast cells when they were cocultured with fibroblasts in the presence of c-kit ligand. Exogenous application of histamine and several H4 receptor agonists restored the granule maturation of Hdc(-/-) cultured mast cells. However, the maturation of granules was largely normal in Hrh4(-/-) peritoneal mast cells. Depletion of cellular histamine with tetrabenazine, an inhibitor of vesicular monoamine transporter-2, did not affect granule maturation. In vivo experiments with mast cell deficient Kit(W) /Kit(W-v) mice indicated that the expression of the Hdc gene in mast cells is required for granule maturation. These results suggest that histamine promotes granule maturation in mast cells and acts as an proinflammatory mediator. PMID:24002822

  4. Local origin of mesenchymal cells in a murine orthotopic lung transplantation model of bronchiolitis obliterans.

    PubMed

    Mimura, Takeshi; Walker, Natalie; Aoki, Yoshiro; Manning, Casey M; Murdock, Benjamin J; Myers, Jeffery L; Lagstein, Amir; Osterholzer, John J; Lama, Vibha N

    2015-06-01

    Bronchiolitis obliterans is the leading cause of chronic graft failure and long-term mortality in lung transplant recipients. Here, we used a novel murine model to characterize allograft fibrogenesis within a whole-lung microenvironment. Unilateral left lung transplantation was performed in mice across varying degrees of major histocompatibility complex mismatch combinations. B6D2F1/J (a cross between C57BL/6J and DBA/2J) (Haplotype H2b/d) lungs transplanted into DBA/2J (H2d) recipients were identified to show histopathology for bronchiolitis obliterans in all allogeneic grafts. Time course analysis showed an evolution from immune cell infiltration of the bronchioles and vessels at day 14, consistent with acute rejection and lymphocytic bronchitis, to subepithelial and intraluminal fibrotic lesions of bronchiolitis obliterans by day 28. Allografts at day 28 showed a significantly higher hydroxyproline content than the isografts (33.21 ± 1.89 versus 22.36 ± 2.33 ?g/mL). At day 40 the hydroxyproline content had increased further (48.91 ± 7.09 ?g/mL). Flow cytometric analysis was used to investigate the origin of mesenchymal cells in fibrotic allografts. Collagen I-positive cells (89.43% ± 6.53%) in day 28 allografts were H2Db positive, showing their donor origin. This novel murine model shows consistent and reproducible allograft fibrogenesis in the context of single-lung transplantation and represents a major step forward in investigating mechanisms of chronic graft failure. PMID:25848843

  5. Conditional Expression of Murine Flt3 Ligand Leads to Expansion of Multiple Dendritic Cell Subsets in Peripheral Blood and Tissues of Transgenic Mice1

    Microsoft Academic Search

    Denise J. Manfra; Shu-Cheng Chen; Kristian K. Jensen; Jay S. Fine; Maria T. Wiekowski; Sergio A. Lira

    The analysis of the development and function of distinct subsets of murine dendritic cells (DC) has been hampered by the limited number of these cells in vivo. To circumvent this limitation we have developed a conditional transgenic mouse model for producing large numbers of DC. We used the tetracycline-inducible system to conditionally express murine Flt3 ligand (FL), a potent hemopoietic

  6. Injury of neoplastic cells by murine macrophages leads to inhibition of mitochondrial respiration.

    PubMed Central

    Granger, D L; Taintor, R R; Cook, J L; Hibbs, J B

    1980-01-01

    Cytotoxic activated macrophages (CM) inhibited the growth of neoplastic L1210 cells in vitro but L1210 cell death was minimal to nonexistent. L1210 cells injured by CM were separated from macrophages and studied in an isolated system. CM-injured L1210 cells had an absolute requirement for glucose or another glycolyzable hexose (mannose or fructose) for at least 40 h after removal from macrophages. If the culture medium lacked sufficient concentration of one of these sugars, CM-injured L1210 cells died within 4 h. Uninjured L1210 cells cultured alone or with peptone-stimulated macrophages had no such requirement and maintained complete viability in hexoseless medium. The hexose requirement of CM-injured L1210 cells could not be fulfilled by other naturally occurring monosaccharides, glucose or mannose derivatives, or substrates that can be oxidized by mitochondria. The concentration requirements for glucose, mannose, and fructose by CM-injured L1210 cells correlated with the concentrations required to support maximal glycolysis of these sugars by other murine ascites cells. A concentration of 2-deoxy-D-glucose which completely inhibited L1210 cell glycolysis also complete prevented the ability of glucose or mannose to maintain viability of CM-injured L1210 cells. Interaction with CM led to inhibition of L1210 cell mitochondrial oxidative phosphorylation. This was supported by the findings that: (a) CM-injured L1210 cells had no Pasteur effect; their rate of aerobic glycolysis was the same as the rate of anaerobic glycolysis of uninjured L1210 cells, (b) Endogenous respiration of CM-injured L1210 cells was 15% of normal. Maximal inhibition of uninjured L1210 cell respiration by a specific mitochondrial poison (oligomycin) was nearly the same (13% of normal). It followed that CM-injured L1210 cells required hexose for chemical energy production via the glycolytic pathway. CM-induced mitochondrial injury occurred in five other neoplastic cell lines tested. Images PMID:7356685

  7. Modulation of murine bone marrow-derived dendritic cells and B-cells by MCS-18 a natural product isolated from Helleborus purpurascens.

    PubMed

    Littmann, Leonie; Rössner, Susanne; Kerek, Franz; Steinkasserer, Alexander; Zinser, Elisabeth

    2008-01-01

    MCS-18, a natural product isolated from Helleborus purpurascens has been shown to have several beneficial effects in inflammatory and autoimmune disorders. However, very little is known regarding the immuno-modulatory capacity of MCS-18 in respect to murine bone marrow-derived dendritic cells (BM-DC) and B-cells. Thus, in the present study we examined the effect of MCS-18 on murine BM-DC and B-cells. Interestingly MCS-18 inhibited the expression of important DC-specific molecules and lead to an impaired T-cell stimulation capacity. In addition, MCS-18 also reduced B-cell proliferation and immunoglobulin production. PMID:18926301

  8. Human and Murine Clonal CD8+ T Cell Expansions Arise during Tuberculosis Because of TCR Selection

    PubMed Central

    Nunes-Alves, Cláudio; Booty, Matthew G.; Carpenter, Stephen M.; Rothchild, Alissa C.; Martin, Constance J.; Desjardins, Danielle; Steblenko, Katherine; Kløverpris, Henrik N.; Madansein, Rajhmun; Ramsuran, Duran; Leslie, Alasdair; Correia-Neves, Margarida; Behar, Samuel M.

    2015-01-01

    The immune system can recognize virtually any antigen, yet T cell responses against several pathogens, including Mycobacterium tuberculosis, are restricted to a limited number of immunodominant epitopes. The host factors that affect immunodominance are incompletely understood. Whether immunodominant epitopes elicit protective CD8+ T cell responses or instead act as decoys to subvert immunity and allow pathogens to establish chronic infection is unknown. Here we show that anatomically distinct human granulomas contain clonally expanded CD8+ T cells with overlapping T cell receptor (TCR) repertoires. Similarly, the murine CD8+ T cell response against M. tuberculosis is dominated by TB10.44-11-specific T cells with extreme TCR? bias. Using a retrogenic model of TB10.44-11-specific CD8+ T cells, we show that TCR dominance can arise because of competition between clonotypes driven by differences in affinity. Finally, we demonstrate that TB10.4-specific CD8+ T cells mediate protection against tuberculosis, which requires interferon-? production and TAP1-dependent antigen presentation in vivo. Our study of how immunodominance, biased TCR repertoires, and protection are inter-related, provides a new way to measure the quality of T cell immunity, which if applied to vaccine evaluation, could enhance our understanding of how to elicit protective T cell immunity. PMID:25945999

  9. Development and functional analysis of eosinophils from murine embryonic stem cells.

    PubMed

    Hamaguchi-Tsuru, Emi; Nobumoto, Atsuya; Hirose, Noriyuki; Kataoka, Sayo; Fujikawa-Adachi, Kiyomi; Furuya, Masato; Tominaga, Akira

    2004-03-01

    We have established a culture system for the development of eosinophils from murine embryonic stem (ES) cells. After transferring ES cells from embryonic fibroblast cells onto macrophage colony-stimulating factor-deficient stromal cells, OP9, ES cells were cultured in the presence of interleukin (IL)-5 with either IL-3 or granulocyte-macrophage colony stimulating factor (GM-CSF) for 20 d to obtain approximately 50% eosinophils. Electron microscopy confirmed the presence of crystallized major basic protein (MBP) in the granules of some of these cells. Neither IL-5, IL-3, GM-CSF nor eotaxin alone could induce eosinophils as efficiently as the conditions described above. Eotaxin induced eosinophil development in combination with either IL-3 or IL-5. Levels of GATA-1, Friend of GATA (FOG)-1, PU.1, CCAAT/enhancer binding protein (C/EBP)alpha, C/EBPbeta, IL-3 receptor alpha (IL-3Ralpha), GM-CSF receptor alpha (GM-CSFRalpha), and MBP mRNAs were increased in ES cells 10 d after transfer onto OP9 cells. In contrast, C/EBPepsilon, IL-5Ralpha, and eosinophil peroxidase mRNAs were induced in response to IL-3 and IL-5 after transfer onto OP9 cells. Eosinophils that developed in this system expressed Gr-1, F4/80, B220, CCR3, IL-3Ralpha, IL-5Ralpha, and DX5. Finally, eosinophils developed from ES cells produced reactive oxygen species in response to Leishmania as do peripheral blood eosinophils. PMID:15009071

  10. 2,4-Dinitrophenol induces neural differentiation of murine embryonic stem cells.

    PubMed

    Freitas-Correa, Léo; Lourenco, Mychael V; Acquarone, Mariana; da Costa, Rodrigo F Madeiro; Galina, Antonio; Rehen, Stevens K; Ferreira, Sergio T

    2013-11-01

    2,4-Dinitrophenol (DNP) is a neuroprotective compound previously shown to promote neuronal differentiation in a neuroblastoma cell line and neurite outgrowth in primary neurons. Here, we tested the hypothesis that DNP could induce neurogenesis in embryonic stem cells (ESCs). Murine ESCs, grown as embryoid bodies (EBs), were exposed to 20 ?M DNP (or vehicle) for 4 days. Significant increases in the proportion of nestin- and ?-tubulin III-positive cells were detected after EB exposure to DNP, accompanied by enhanced glial fibrillary acidic protein (GFAP), phosphorylated extracellular signal-regulated kinase (p-ERK) and ATP-linked oxygen consumption, thought to mediate DNP-induced neural differentiation. DNP further protected ESCs from cell death, as indicated by reduced caspase-3 positive cells, and increased proliferation. Cell migration from EBs was significantly higher in DNP-treated EBs, and migrating cells were positive for nestin, ß-tubulin III and MAP2, similar to that observed with retinoic acid (RA)-treated EBs. Compared to RA, however, DNP exerted a marked neuritogenic effect on differentiating ESCs, increasing the average length and number of neurites per cell. Results establish that DNP induces neural differentiation of ESCs, accompanied by cell proliferation, migration and neuritogenesis, suggesting that DNP may be a novel tool to induce neurogenesis in embryonic stem cells. PMID:24148244

  11. Measurement of Gross cell-surface antigen and p30 level in murine retrovirus-infected cell lines.

    PubMed Central

    Gerlier, D.; Gisselbrecht, S.; Guillemain, B.; Doré, J. F.

    1981-01-01

    The level of Gross cell-surface antigen (GCSAa) expression at the surface of murine retrovirus-infected fibroblasts was determined by quantitative absorption of the anti-GCSAa activity of a serum produced in syngeneic W/Fu rats immunized against (C58NT)D lymphoma, and tested in a cytotoxicity assay against E male G2 lymphoma cells. While GCSAa was specifically expressed on Gross-type virus (G-MuLV)-induced lymphoma cells, and while G-MuLV and G-related MuLV induced a high level of GCSAa expression on murine fibroblasts, the Friend-Moloney-Rauscher (FMR) group viruses (FMR MuLV) and xenotropic isolates were also able to induce a high or intermediate level of GCSAa. Since GCSAa has been shown to be borne by glycosylated precursors of the viral nucleocapside (gp95gag and gp85gag), the amount of GCSAa expressed on these cells was compared to the level of cytoplasmic p30. In G- and G-related MuLV-infected cell lines, a significant relationship was found between the amount of GCSAa and the level of p30, whereas in FMR-MuLV or xenotropic virus-infected cells the amount of GCSAa varied independently of the p30 level. These results could explain the discrepancy in the specificity of expression of GCSAa in vivo and in vitro. PMID:7248150

  12. Characterization of the Murine Myeloid Precursor Cell Line MuMac-E8

    PubMed Central

    Fricke, Stephan; Riemschneider, Sina; Kohlschmidt, Janine; Hilger, Nadja; Fueldner, Christiane; Knauer, Jens; Sack, Ulrich; Emmrich, Frank; Lehmann, Jörg

    2014-01-01

    Starting point for the present work was the assumption that the cell line MuMac-E8 represents a murine cell population with stem cell properties. Preliminary studies already pointed to the expression of stem-cell associated markers and a self-regenerative potential of the cells. The cell line MuMac-E8 should be examined for their differential stage within stem cell hierarchy. MuMac-E8 cells were derived from a chimeric mouse model of arthritis. It could be shown that MuMac-E8 cells express mRNA of some genes associated with pluripotent stem cells (Nanog, Nucleostemin), of genes for hematopoietic markers (EPCR, Sca-1, CD11b, CD45), for the mesenchymal marker CD105 and of genes for the neural markers Pax-6 and Ezrin. In methylcellulose and May-Grünwald-Giemsa staining, hematopoietic colonies were obtained but the hematopoietic system of lethally irradiated mice could not be rescued. Osteogenic differentiation was not detectable. Thus, it became evident that MuMac-E8 represents not a stem cell line. However, MuMac-E8 cells expressed several myeloid surface markers (i.e. CD11b, F4/80, CD14, CD64), showed phagocytosis and is capable of producing nitric oxide. Thus, this cell line seems to be arrested an advanced stage of myeloid differentiation. Adherence data measured by impedance-based real-time cell analysis together with cell morphology data suggested that MuMac-E8 represents a new macrophage precursor cell line exhibiting weak adherence. This cell line is suitable as an in-vitro model for testing of macrophage functions. Moreover, it might be also useful for differentiation or reprogramming studies. PMID:25546418

  13. Interleukin 21 Signaling in B Cells Is Required for Efficient Establishment of Murine Gammaherpesvirus Latency

    PubMed Central

    Collins, Christopher M.; Speck, Samuel H.

    2015-01-01

    The human gammaherpesviruses take advantage of normal B cell differentiation pathways to establish life-long infection in memory B cells. Murine gammaherpesvirus 68 (MHV68) infection of laboratory strains of mice also leads to life-long infection in memory B cells. To gain access to the memory B cell population, MHV68 infected B cells pass through the germinal center reaction during the onset of latency and require signals from T follicular helper (TFH) cells for proliferation. Interleukin 21 (IL-21), one of the secreted factors produced by TFH cells, plays an important role in both the maintenance of the germinal center response as well as in the generation of long-lived plasma cells. Using IL-21R deficient mice, we show that IL-21 signaling is required for efficient establishment of MHV68 infection. In the absence of IL-21 signaling, fewer infected splenocytes are able to gain access to either the germinal center B cell population or the plasma cell population – the latter being a major site of MHV68 reactivation. Furthermore, the germinal center B cell population in IL-21R-/- mice is skewed towards the non-proliferating centrocyte phenotype, resulting in reduced expansion of infected B cells. Additionally, the reduced frequency of infected plasma cells results in a significant reduction in the frequency of splenocytes capable of reactivating virus. This defect in establishment of MHV68 infection is intrinsic to B cells, as MHV68 preferentially establishes infection in IL-21R sufficient B cells in mixed bone marrow chimeric mice. Taken together, these data indicate that IL-21 signaling plays multiple roles during establishment of MHV68 infection, and identify IL-21 as a critical TFH cell-derived factor for efficient establishment of gammaherpesvirus B cell latency. PMID:25875847

  14. Simvastatin-enhanced expression of promyogenic nuclear factors and cardiomyogenesis of murine embryonic stem cells.

    PubMed

    Yang, ChenMin; Madonna, Rosalinda; Li, Yangxin; Zhang, Qi; Shen, Wei-Feng; McNamara, Katharine; Yang, Yue-Jin; Geng, Yong-Jian

    2014-01-01

    A combination of statin and stem cell therapies has been shown to benefit in experimental models of myocardial infarction. This study tests whether treatment with simvastatin has a direct impact on the cardiomyogenic development of murine embryonic stem cells (ESCs) in embryoid bodies. In a concentration-dependent manner, simvastatin treatment enhanced expression of several promyogenic nuclear transcription factors, including GATA4, Nkx2.5, DTEF-1 and myocardin A. The statin-treated cells also displayed higher levels of cardiac proteins, including myosin, ?-actinin, Ryanodine receptor-2, and atrial natriuretic peptide, and they developed synchronized contraction. The statin's promyogenic effect was partially diminished by the addition of the two isoprenoids FPP and GGPP, which are intermediates of cholesterol synthesis. Thus, simvastatin treatment enhances ESC myogenesis during early development perhaps via a mechanism inhibiting the mevalonate-FPP/GGPP pathway. PMID:24200505

  15. Chemically linked phage idiotype vaccination in the murine B cell lymphoma 1 model

    PubMed Central

    2013-01-01

    Background B cell malignancies are characterized by clonal expansion of B cells expressing tumor-specific idiotypes on their surface. These idiotypes are ideal target antigens for an individualized immunotherapy. However, previous idiotype vaccines mostly lacked efficiency due to a low immunogenicity of the idiotype. The objective of the present study was the determination of the feasibility, safety and immunogenicity of a novel chemically linked phage idiotype vaccine. Methods In the murine B cell lymphoma 1 model, tumor idiotypes were chemically linked to phage particles used as immunological carriers. For comparison, the idiotype was genetically expressed on the major phage coat protein g8 or linked to keyhole limpet hemocynanin. After intradermal immunizations with idiotype vaccines, tolerability and humoral immune responses were assessed. Results Feasibility and tolerability of the chemically linked phage idiotype vaccine was demonstrated. Vaccination with B cell lymphoma 1 idiotype expressing phage resulted in a significant survival benefit in the murine B cell lymphoma 1 protection model (60.2?±?23.8 days vs. 41.8?±?1.6 days and 39.8?±?3.8 days after vaccination with wild type phage or phosphate buffered saline, respectively). Superior immunogenicity of the chemically linked phage idiotype vaccine compared to the genetically engineered phage idiotype and keyhole limpet hemocynanin-coupled idiotype vaccine was demonstrated by significantly higher B cell lymphoma 1 idiotype-specific IgG levels after vaccination with chemically linked phage idiotype. Conclusion We present a novel, simple, time- and cost-efficient phage idiotype vaccination strategy, which represents a safe and feasible therapy and may produce a superior immune response compared to previously employed idiotype vaccination strategies. PMID:24152874

  16. Use of laser microdissection in the analysis of renal-infiltrating T cells in murine lupus

    PubMed Central

    Guo, Zhentao; Wang, Yingge; Li, Rongqian; Huang, Haifeng

    2014-01-01

    Objective To clarify the role of T cells in kidney pathology of three widely used murine lupus models. Material and methods Cells infiltrating the glomeruli and perivascular areas in MRL/lpr (n = 10 female), NZB× NZW F1 (B/W F1) (n = 9 female), and BXSB (n = 10 male) mice were captured by laser microdissection (LMD). Samples were subjected to nested reverse transcription polymerase chain reaction (RT-PCR) with primers specific to ?-actin, T-cell receptor ? chain (TCR-C?), interleukin (IL)-10, IL-13, IL-17, and interferon-g (IFN-?). Frozen sections of lesions were also stained immunohistochemically for tissue and cellular characterization. Results T cells infiltrating the glomeruli and perivascular areas predominantly produced IFN-?, IL-13, and IL-17 in MRL/lpr, B/W F1, and BXSB mice, with IL-17 expression in glomeruli of BXSB mice being significantly lower than that of MRL/lpr and B/W F1 mice. IL-10 was detected only in the perivascular areas of MRL/lpr and B/W F1 mice and not in glomeruli isolates. Immunohistochemical staining revealed positive for the expression of Thy-1, CD4, CD8, and B220 in glomeruli and perivascular areas from all three strains of mice. Conclusions Cytokine balance in murine SLE is complex and cannot be attributed simply to the balance between Th1 and Th2 cells. Th17 cells may play a critical role in disease pathology, possibly with greater contribution toward disease progression in MRL/lpr and B/W F1 mice than in BXSB mice. Furthermore, these findings lend support to the concept that different molecular mechanisms underlie glomerulonephritis as compared to vasculitis.

  17. Cloning of murine interferon gamma receptor cDNA: expression in human cells mediates high-affinity binding but is not sufficient to confer sensitivity to murine interferon gamma.

    PubMed Central

    Hemmi, S; Peghini, P; Metzler, M; Merlin, G; Dembic, Z; Aguet, M

    1989-01-01

    A full-length cDNA encoding the murine interferon gamma (IFN-gamma) receptor was isolated from a lambda gt11 library using a human IFN-gamma receptor cDNA probe. The deduced amino acid sequence of the murine IFN-gamma receptor shows approximately 53% homology to its human counterpart but no homology to other known proteins. Murine IFN-gamma receptor cDNA was expressed in human HEp-2 cells, which do not bind murine IFN-gamma and are insensitive to its action. Transfectants displayed the same binding properties as mouse cells. The biological responsiveness of such transfectants to various biological effects of both human and murine IFN-gamma was investigated, including modulation of major histocompatibility complex class I and class II antigen expression, inhibition of cell growth, and antiviral activity. Like parental HEp-2 cells, these transfectants responded only to human, but not to murine, IFN-gamma. Inversely, mouse L929 cells transfected with human IFN-gamma receptor cDNA were insensitive to human IFN-gamma. These results confirm and extend previous findings, suggesting that species-specific cofactors are needed for IFN-gamma-mediated signal transduction. Images PMID:2532365

  18. A comparative study on cannabidiol-induced apoptosis in murine thymocytes and EL-4 thymoma cells.

    PubMed

    Lee, Chi-Ya; Wey, Shiaw-Pyng; Liao, Mei-Hsiu; Hsu, Wei-Lun; Wu, Hsin-Ying; Jan, Tong-Rong

    2008-05-01

    It has been shown that leukemia and glioma cells are sensitive to cannabidiol (CBD)-induced apoptosis, whereas primary monocytes and glia cells are relatively insensitive. In the current study, the cellular events and sensitivity to CBD-induced apoptosis between murine thymocytes and EL-4 thymoma cells were compared. Cannabidiol markedly induced apoptosis in a time- and concentration-related manner in both cells. The efficacy of CBD to induce apoptosis was comparable between the 2 types of T cells, whereas CBD induced apoptosis in thymocytes with a slightly greater potency than in EL4 cells. Time-course analyses revealed CBD-mediated apoptosis occurred earlier in EL-4 cells than that in thymocytes. An increased level of cellular reactive oxygen species (ROS) was detected in both cells with the peak response at 2 h post CBD treatment. Concordantly, CBD triggered a gradual diminishment in the cellular thiols. The presence of N-acetyl-L-cysteine (NAC), a precursor of glutathione, markedly attenuated the induction of apoptosis, and restored the diminished levels of cellular thiols. The results demonstrated that both thymocytes and EL-4 thymoma cells were susceptible to CBD-induced apoptosis and that ROS played a critical role in the apoptosis induction. PMID:18387516

  19. Effects of Wnt-10b on proliferation and differentiation of murine melanoma cells.

    PubMed

    Misu, Masayasu; Ouji, Yukiteru; Kawai, Norikazu; Nishimura, Fumihiko; Nakamura-Uchiyama, Fukumi; Yoshikawa, Masahide

    2015-08-01

    In spite of the strong expression of Wnt-10b in melanomas, its role in melanoma cells has not been elucidated. In the present study, the biological effects of Wnt-10b on murine B16F10 (B16) melanoma cells were investigated using conditioned medium from Wnt-10b-producing COS cells (Wnt-CM). After 2 days of culture in the presence of Wnt-CM, proliferation of B16 melanoma cells was inhibited, whereas tyrosinase activity was increased. An in vitro wound healing assay demonstrated that migration of melanoma cells to the wound area was inhibited with the addition of Wnt-CM. Furthermore, evaluation of cellular senescence revealed prominent induction of SA-?-gal-positive senescent cells in cultures with Wnt-CM. Finally, the growth of B16 melanoma cell aggregates in collagen 3D-gel cultures was markedly suppressed in the presence of Wnt-CM. These results suggest that Wnt-10b represses tumor cell properties, such as proliferation and migration of B16 melanoma cells, driving them toward a more differentiated state along a melanocyte lineage. PMID:26056007

  20. A flavone derivative from Sesbania sesban leaves and its cytotoxicity against murine leukemia P-388 cells

    NASA Astrophysics Data System (ADS)

    Dianhar, Hanhan; Syah, Yana Maolana; Mujahidin, Didin; Hakim, Euis Holisotan; Juliawaty, Lia Dewi

    2014-03-01

    Sesbania sesban, locally named as Jayanti, is one of Indonesia plants belonging to Fabaceae family. This species is traditionally used by Indonesian people to cure digestive disorders, fever, or headache. Jayanti can grow well in tropical to subtropical region, such as in Asia and Africa. Based on literature, qualitative analysis of the methanol extract of leaves of S. sesban showed that it contained flavonoids, alkaloids, saponins and glycosides. In addition, the activity assay of extracts of different tissues of this species showed antitumor, antimalarial, and antidiabetic activityies (leaves and seed extracts), antioxidants (flower extract), and analgesic (wood extract). Though the extracts of S. sesban parts showed interesting activities, chemical study of those extracts have not been widely reported. Therefore, the objective of this research was to isolate the secondary metabolites from methanol extract of leaves of S. sesban and to determine their cytotoxicity against murine leukemia P-388 cells. One compound has been obtained and identified as 3-hydroxy-4',7-dimethoxyflavone (1), a new isolated compound from nature. This compound was obtained through separation of methanol extract using various chromatographic techniques, such as vacuum liquid chromatography and radial chromatography. The structure elucidation of isolated compound was based on 1D NMR (1H-NMR and 13C-NMR) and 2D NMR (HMBC). The cytotoxicity of methanol extract and compound 1 against murine leukemia P-388 cells examined through MTT assay showed IC50 value of 60.04 ?g/mL and 5.40 ?g/mL, respectively.

  1. Dendritic Cell-Based Vaccination in Cancer: Therapeutic Implications Emerging from Murine Models

    PubMed Central

    Mac Keon, Soledad; Ruiz, María Sol; Gazzaniga, Silvina; Wainstok, Rosa

    2015-01-01

    Dendritic cells (DCs) play a pivotal role in the orchestration of immune responses, and are thus key targets in cancer vaccine design. Since the 2010 FDA approval of the first cancer DC-based vaccine (Sipuleucel-T), there has been a surge of interest in exploiting these cells as a therapeutic option for the treatment of tumors of diverse origin. In spite of the encouraging results obtained in the clinic, many elements of DC-based vaccination strategies need to be optimized. In this context, the use of experimental cancer models can help direct efforts toward an effective vaccine design. This paper reviews recent findings in murine models regarding the antitumoral mechanisms of DC-based vaccination, covering issues related to antigen sources, the use of adjuvants and maturing agents, and the role of DC subsets and their interaction in the initiation of antitumoral immune responses. The summary of such diverse aspects will highlight advantages and drawbacks in the use of murine models, and contribute to the design of successful DC-based translational approaches for cancer treatment. PMID:26042126

  2. Optical imaging of Ca2+-evoked fluid secretion by murine nasal submucosal gland serous acinar cells.

    PubMed

    Lee, Robert J; Limberis, Maria P; Hennessy, Michael F; Wilson, James M; Foskett, J Kevin

    2007-08-01

    Airway submucosal glands are sites of high expression of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel and contribute to fluid homeostasis in the lung. However, the molecular mechanisms of gland ion and fluid transport are poorly defined. Here, submucosal gland serous acinar cells were isolated from murine airway, identified by immunofluorescence and gene expression profiling, and used in physiological studies. Stimulation of isolated acinar cells with carbachol (CCh), histamine or ATP was associated with marked decreases in cell volume (20 +/- 2% within 62 +/- 5 s) that were tightly correlated with increases in cytoplasmic Ca(2+) concentration ([Ca(2+)](i)) as revealed by simultaneous DIC and fluorescent indicator dye microscopy. Simultaneous imaging of cell volume and the Cl(-)-sensitive fluorophore SPQ indicated that the 20% shrinkage was associated with a fall of [Cl(-)](i) from 65 mm to 28 mm, reflecting loss of 67% of cell Cl(-) content, accompanied by parallel efflux of K(+). Upon agonist removal, [Ca(2+)](i) relaxed and the cells swelled back to resting volume via a bumetanide-sensitive Cl(-) influx pathway, likely to be NKCC1. Accordingly, agonist-induced serous acinar cell shrinkage and swelling are caused by activation of solute efflux and influx pathways, respectively, and cell volume reflects the secretory state of these cells. In contrast, elevation of cAMP failed to elicit detectible volume responses, or enhance those induced by submaximal [CCh], because the magnitude of the changes were likely to be below the threshold of detection using optical imaging. Finally, when stimulated with cholinergic or cAMP agonists, cells from mice that lacked CFTR, as well as wild-type cells treated with a CFTR inhibitor, exhibited identical rates and magnitudes of shrinkage and Cl(-) efflux compared with control cells. These results provide insights into the molecular mechanisms of salt and water secretion by lung submucosal glands, and they suggest that while murine submucosal gland fluid secretion in response to cholinergic stimulation can originate from CFTR-expressing serous acinar cells, it is not dependent upon CFTR function. PMID:17525116

  3. Galectin-8 Ameliorates Murine Autoimmune Ocular Pathology and Promotes a Regulatory T Cell Response

    PubMed Central

    Sampson, James F.; Hasegawa, Eiichi; Mulki, Lama; Suryawanshi, Amol; Jiang, Shuhong; Chen, Wei-Sheng; Rabinovich, Gabriel A.; Connor, Kip M.; Panjwani, Noorjahan

    2015-01-01

    Galectins have emerged as potent immunoregulatory agents that control chronic inflammation through distinct mechanisms. Here, we report that treatment with Galectin-8 (Gal-8), a tandem-repeat member of the galectin family, reduces retinal pathology and prevents photoreceptor cell damage in a murine model of experimental autoimmune uveitis. Gal-8 treatment increased the number of regulatory T cells (Treg) in both the draining lymph node (dLN) and the inflamed retina. Moreover, a greater percentage of Treg cells in the dLN and retina of Gal-8 treated animals expressed the inhibitory coreceptor cytotoxic T lymphocyte antigen (CTLA)-4, the immunosuppressive cytokine IL-10, and the tissue-homing integrin CD103. Treg cells in the retina of Gal-8-treated mice were primarily inducible Treg cells that lack the expression of neuropilin-1. In addition, Gal-8 treatment blunted production of inflammatory cytokines by retinal T helper type (TH) 1 and TH17 cells. The effect of Gal-8 on T cell differentiation and/or function was specific for tissues undergoing an active immune response, as Gal-8 treatment had no effect on T cell populations in the spleen. Given the need for rational therapies for managing human uveitis, Gal-8 emerges as an attractive therapeutic candidate not only for treating retinal autoimmune diseases, but also for other TH1- and TH17-mediated inflammatory disorders. PMID:26126176

  4. Murine hematopoietic reconstitution after tagging and selection of retrovirally transduced bone marrow cells.

    PubMed

    García-Hernández, B; Castellanos, A; López, A; Orfao, A; Sánchez-García, I

    1997-11-25

    A major problem facing the effective treatment of patients with cancer is how to get the specific antitumor agent into every tumor cell. In this report we describe the use of a strategy that, by using retroviral vectors encoding a truncated human CD5 cDNA, allows the selection of only the infected cells, and we show the ability to obtain, before bone marrow transplantation, a population of 5-fluouracil-treated murine bone marrow cells that are 100% marked. This marked population of bone marrow cells is able to reconstitute the hematopoietic system in lethally irradiated mice, indicating that the surface marker lacks deleterious effects on the functionality of bone marrow cells. No gross abnormalities in hematopoiesis were detected in mice repopulated with CD5-expressing cells. Nevertheless, a significant proportion of the hematopoietic cells no longer expresses the surface marker CD5 in the 9-month-old recipient mice. This transcriptional inactivity of the proviral long terminal repeat (LTR) was accompanied by de novo methylation of the proviral sequences. Our results show that the use of the CD5 as a retrovirally encoded marker enables the rapid, efficient, and nontoxic selection in vitro of infected primary cells, which can entirely reconstitute the hematopoietic system in mice. These results should now greatly enhance the power of studies aimed at addressing questions such as generation of cancer-negative hematopoiesis. PMID:9371830

  5. Murine hematopoietic reconstitution after tagging and selection of retrovirally transduced bone marrow?cells

    PubMed Central

    García-Hernández, B.; Castellanos, A.; López, A.; Orfao, A.; Sánchez-García, I.

    1997-01-01

    A major problem facing the effective treatment of patients with cancer is how to get the specific antitumor agent into every tumor cell. In this report we describe the use of a strategy that, by using retroviral vectors encoding a truncated human CD5 cDNA, allows the selection of only the infected cells, and we show the ability to obtain, before bone marrow transplantation, a population of 5-fluouraci-treated murine bone marrow cells that are 100% marked. This marked population of bone marrow cells is able to reconstitute the hematopoietic system in lethally irradiated mice, indicating that the surface marker lacks deleterious effects on the functionality of bone marrow cells. No gross abnormalities in hematopoiesis were detected in mice repopulated with CD5-expressing cells. Nevertheless, a significant proportion of the hematopoietic cells no longer expresses the surface marker CD5 in the 9-month-old recipient mice. This transcriptional inactivity of the proviral long terminal repeat (LTR) was accompanied by de novo methylation of the proviral sequences. Our results show that the use of the CD5 as a retrovirally encoded marker enables the rapid, efficient, and nontoxic selection in vitro of infected primary cells, which can entirely reconstitute the hematopoietic system in mice. These results should now greatly enhance the power of studies aimed at addressing questions such as generation of cancer-negative hematopoiesis. PMID:9371830

  6. Effects of ionizing radiation on bone cell differentiation in an experimental murine bone cell model

    NASA Astrophysics Data System (ADS)

    Baumstark-Khan, Christa; Lau, Patrick; Hellweg, Christine; Reitz, Guenther

    During long-term space travel astronauts are exposed to a complex mixture of different radiation types under conditions of dramatically reduced weight-bearing activity. It has been validated that astronauts loose a considerable amount of bone mass at a rate up to one to two percent each month in space. Therapeutic doses of ionizing radiation cause bone damage and increase fracture risks after treatment for head-and-neck cancer and in pelvic irradiation. For low radiation doses, the possibility of a disturbed healing potential of bone was described. Radiation induced damage has been discussed to inflict mainly on immature and healing bone. Little is known about radiation effects on bone remodelling and even less on the combined action of microgravity and radiation. Bone remodelling is a life-long process performed by balanced action of cells from the osteoblast and osteoclast lineages. While osteoblasts differentiate either into bone-lining cells or into osteocytes and play a crucial role in bone matrix synthesis, osteoclasts are responsible for bone resorption. We hypothesize that the balance between bone matrix assembly by osteocytes and bone degradation by osteoclasts is modulated by microgravity as well as by ionizing radiation. To address this, a cell model consisting of murine cell lines with the potential to differentiate into bone-forming osteoblasts (OCT-1, MC3T3-E1 S24, and MC3T3-E1 S4) was used for studying radiation response after exposure to simulated components of cosmic radiation. Cells were exposed to graded doses of 150 kV X-rays, ? particles (0.525 MeV/u, 160 keV/µm; PTB, Braunschweig, Germany) and accelerated heavy ions (75 MeV/u carbon, 29 keV/µm; 95 MeV/u argon, 230 keV/µm; GANIL, Caen, France). Cell survival was measured as colony forming ability; cell cycle progression was analyzed via fluorescence-activated cell scanning (FACS) by measurement of the content of propidium iodide-stained DNA, DNA damage was visualized by ?H2AX-immunostaining. Osteoblastogenesis was estimated by measurement of alkaline phosphatase (ALP) activity and production of mineralized matrix (von-Kossa staining, Alizarin Red staining). During the process of osteoblastic cell differentiation, the expression of the bone specific marker genes osteocalcin (OCN) and osteopontin (OPN) were recorded by quantitative real time reverse transcription PCR (qRT-PCR). Compared with standard culture conditions, the osteogenic marker genes OCN and OPN were highly expressed during the differentiation process induced either by osteo-inductive media additives (50 µg/ml ascorbic acid, 10 mmol/l ?-glycero phosphate) or by sparsely ionizing radiation (X-rays). After 21 days of postirradiation incubation sparsely ionizing radiation could be shown to induce the formation of bone-like nodules (von-Kossa staining) for OCT-1 and MC3T3-E1 S4 cells but nor for MC3T3- E1 S24 cells. Ionizing radiation leads to a cell cycle arrest which is resolved in a dose and time dependent way. This was accompanied by a dose dependent regulation of the cyclin kinase inhibitor CDKN1A (p21/WAF) and transforming growth factor beta 1 (TGF-?1). TGF-?1 is known to affect osteoblast differentiation, matrix formation and mineralization. Modulation of its expression could influence the expression of main osteogenic transcription factors. For exposure with high LET radiation a pronounced cell cycle block was evident. The expression of the osteogenic marker genes OCN and Osterix (OSX) was increased in the OCT-1 cells with differentiation potential for exposure to ? particles and accelerated carbon and argon ions. The results on the expression of differentiation markers during radiation-induced premature differentiation of bone cells of the osteoblast lineage show that densely ionizing radiation results in expression of proteins essential for bone formation and consequently in an increase in bone volume. Such an effect has been observed in in-vivo carbon ion irradiated rats. As radiation dependent permanent cell cycle blocks lead to a depletion of proliferation-competent cel

  7. A model system for testing gene vectors using murine tumor cells on the chorioallantoic membrane of the chick embryo.

    PubMed

    Dani, Sergio U; Espindola, Rachel

    2002-01-01

    We developed a model system for testing gene vectors, based on the growth of murine tumors on the chorioallantoic membrane (CAM) of embryonic chickens. The ability of selected murine cells to grow on the CAM was rated according to the following criteria: i) formation of tumor masses; ii) metastasis formation; iii) reproducibility; iv) yield, indicated as the number of embryos surviving to assessment time with visible tumors on the CAM; v) maintainability of the cell, both in the original host and the embryonic chick, or 'shuttle maintainability'; vi) detection by the naked eye, and vii) cost/benefit relation. The murine melanoma cell lineage, B16F10, which efficiently forms distinct, pigmented tumor masses and metastases on the CAM, performed better in this model than the murine B61 cell line. In vitro transduction of B16F10 cells with a recombinant adenovirus carrying a construct of the E. coli LacZ gene followed by inoculation onto the CAM resulted in beta-galactosidase expression in the tumor mass growing on the CAM. This model is potentially applicable to preclinical evaluation of gene vectors, especially for gene therapy of cancer. PMID:14963844

  8. Murine Models of B-Cell Lymphomas: Promising Tools for Designing Cancer Therapies

    PubMed Central

    Donnou, Sabrina; Galand, Claire; Touitou, Valérie; Sautès-Fridman, Catherine; Fabry, Zsuzsanna; Fisson, Sylvain

    2012-01-01

    Human B-cell lymphomas, the fourth most common hematologic malignancy, are currently the subject of extensive research. The limited accessibility of biopsies, the heterogeneity among patients, and the subtypes of lymphomas have necessitated the development of animal models to decipher immune escape mechanisms and design new therapies. Here, we summarize the cell lines and murine models used to study lymphomagenesis, the lymphoma microenvironment, and the efficacy of new therapies. These data allow us to understand the role of the immune system in the fight against tumors. Exploring the advantages and limitations of immunocompetent versus immunodeficient models improves our understanding of the molecular and cellular mechanisms of tumor genesis and development as well as the fundamental processes governing the interaction of tumors and their host tissues. We posit that these basic preclinical investigations will open up new and promising approaches to designing better therapies. PMID:22400032

  9. Release of prostaglandin D2 by murine mast cells: importance of metabolite formation for antiproliferative activity.

    PubMed Central

    Haberl, C; Hültner, L; Flügel, A; Falk, M; Geuenich, S; Wilmanns, W; Denzlinger, C

    1998-01-01

    Prostaglandin (PG) D2, PGJ2 and delta12-PGJ2 are antiproliferative eicosanoids. We investigated the production of PGD2 by murine bone marrow-derived mast cells (BMMC) taking into consideration metabolism of PGD2 to PGD2 and delta12-PGJ2. PG-metabolites were quantified by high performance liquid chromatography (HPLC) combined with radioimmunoassay (RIA). Stimulated with calcium ionophore A23187 BMMC released eight-fold more PGJ2 and delta12-PGJ2 than PGD2. Conversion of endogenously produced PGD2 to PGJ2 and delta12-PGJ2 proceeded rapidly in contrast to metabolism of exogenously added PGD2. The antiproliferative potency of these prostaglandins is demonstrated in vitro. We conclude that determination of PGD2 production by mast cells must take into consideration rapid conversion to active derivatives, which may play a significant role in growth regulation. PMID:9836493

  10. Distribution and Ca2+ signalling of fibroblast-like (PDGFR?+) cells in the murine gastric fundus

    PubMed Central

    Baker, Salah A; Hennig, Grant W; Salter, Anna K; Kurahashi, Masaki; Ward, Sean M; Sanders, Kenton M

    2013-01-01

    Platelet-derived growth factor receptor ? positive (PDGFR?+) cells are suggested to mediate purinergic inputs in GI muscles, but the responsiveness of these cells to purines in situ has not been evaluated. We developed techniques to label and visualize PDGFR?+ cells in murine gastric fundus, load cells with Ca2+ indicators, and follow their activity via digital imaging. Immunolabelling demonstrated a high density of PDGFR?+ cells in the fundus. Cells were isolated and purified by fluorescence-activated cell sorting (FACS) using endogenous expression of enhanced green fluorescent protein (eGFP) driven off the Pdgfra promoter. Quantitative PCR showed high levels of expression of purinergic P2Y1 receptors and SK3 K+ channels in PDGFR?+ cells. Ca2+ imaging was used to characterize spontaneous Ca2+ transients and responses to purines in PDGFR?+ cells in situ. ATP, ADP, UTP and ?-NAD elicited robust Ca2+ transients in PDGFR?+ cells. Ca2+ transients were also elicited by the P2Y1-specific agonist (N)-methanocarba-2MeSADP (MRS-2365), and inhibited by MRS-2500, a P2Y1-specific antagonist. Responses to ADP, MRS-2365 and ?-NAD were absent in PDGFR?+ cells from P2ry1(?/?) mice, but responses to ATP were retained. Purine-evoked Ca2+ transients were mediated through Ca2+ release mechanisms. Inhibitors of phospholipase C (U-73122), IP3 (2-APB), ryanodine receptors (Ryanodine) and SERCA pump (cyclopiazonic acid and thapsigargin) abolished Ca2+ transients elicited by purines. This study provides a link between purine binding to P2Y1 receptors and activation of SK3 channels in PDGFR?+ cells. Activation of Ca2+ release is likely to be the signalling mechanism in PDGFR?+ cells responsible for the transduction of purinergic enteric inhibitory input in gastric fundus muscles. PMID:24144881

  11. CrxOS maintains the self-renewal capacity of murine embryonic stem cells

    SciTech Connect

    Saito, Ryota; Yamasaki, Tokiwa; Nagai, Yoko; Wu, Jinzhan [Department of Developmental and Regenerative Biology, Medical Research Institute, Tokyo Medical and Dental University, Tokyo 113-8510 (Japan) [Department of Developmental and Regenerative Biology, Medical Research Institute, Tokyo Medical and Dental University, Tokyo 113-8510 (Japan); Department of Physiological Chemistry, Graduate School of Pharmaceutical Sciences, University of Tokyo, Tokyo 113-0033 (Japan); Kajiho, Hiroaki [Department of Physiological Chemistry, Graduate School of Pharmaceutical Sciences, University of Tokyo, Tokyo 113-0033 (Japan)] [Department of Physiological Chemistry, Graduate School of Pharmaceutical Sciences, University of Tokyo, Tokyo 113-0033 (Japan); Yokoi, Tadashi; Noda, Eiichiro [Department of Developmental and Regenerative Biology, Medical Research Institute, Tokyo Medical and Dental University, Tokyo 113-8510 (Japan) [Department of Developmental and Regenerative Biology, Medical Research Institute, Tokyo Medical and Dental University, Tokyo 113-8510 (Japan); Department of Ophthalmology, National Center for Child Health and Development, Tokyo 157-8535 (Japan); Nishina, Sachiko [Department of Ophthalmology, National Center for Child Health and Development, Tokyo 157-8535 (Japan)] [Department of Ophthalmology, National Center for Child Health and Development, Tokyo 157-8535 (Japan); Niwa, Hitoshi [Laboratory for Pluripotent Cell Studies, RIKEN Center for Developmental Biology, Hyogo 650-0047 (Japan)] [Laboratory for Pluripotent Cell Studies, RIKEN Center for Developmental Biology, Hyogo 650-0047 (Japan); Azuma, Noriyuki [Department of Ophthalmology, National Center for Child Health and Development, Tokyo 157-8535 (Japan)] [Department of Ophthalmology, National Center for Child Health and Development, Tokyo 157-8535 (Japan); Katada, Toshiaki [Department of Physiological Chemistry, Graduate School of Pharmaceutical Sciences, University of Tokyo, Tokyo 113-0033 (Japan)] [Department of Physiological Chemistry, Graduate School of Pharmaceutical Sciences, University of Tokyo, Tokyo 113-0033 (Japan); Nishina, Hiroshi, E-mail: nishina.dbio@mri.tmd.ac.jp [Department of Developmental and Regenerative Biology, Medical Research Institute, Tokyo Medical and Dental University, Tokyo 113-8510 (Japan)] [Department of Developmental and Regenerative Biology, Medical Research Institute, Tokyo Medical and Dental University, Tokyo 113-8510 (Japan)

    2009-12-25

    Embryonic stem (ES) cells maintain pluripotency by self-renewal. Several homeoproteins, including Oct3/4 and Nanog, are known to be key factors in maintaining the self-renewal capacity of ES cells. However, other genes required for the mechanisms underlying this process are still unclear. Here we report the identification by in silico analysis of a homeobox-containing gene, CrxOS, that is specifically expressed in murine ES cells and is essential for their self-renewal. ES cells mainly express the short isoform of endogenous CrxOS. Using a polyoma-based episomal expression system, we demonstrate that overexpression of the CrxOS short isoform is sufficient for maintaining the undifferentiated morphology of ES cells and stimulating their proliferation. Finally, using RNA interference, we show that CrxOS is essential for the self-renewal of ES cells, and provisionally identify foxD3 as a downstream target gene of CrxOS. To our knowledge, ours is the first delineation of the physiological role of CrxOS in ES cells.

  12. Induction of murine embryonic stem cell differentiation by medicinal plant extracts

    SciTech Connect

    Reynertson, Kurt A. [Center for Complementary and Integrative Medicine, Weill Cornell Medical College, 1300 York Avenue, New York, NY 10065 (United States) [Center for Complementary and Integrative Medicine, Weill Cornell Medical College, 1300 York Avenue, New York, NY 10065 (United States); Department of Pharmacology, Weill Cornell Medical College, 1300 York Avenue, New York, NY 10065 (United States); Charlson, Mary E. [Center for Complementary and Integrative Medicine, Weill Cornell Medical College, 1300 York Avenue, New York, NY 10065 (United States) [Center for Complementary and Integrative Medicine, Weill Cornell Medical College, 1300 York Avenue, New York, NY 10065 (United States); Department of Medicine, Weill Cornell Medical College, 1300 York Avenue, New York, NY 10065 (United States); Gudas, Lorraine J., E-mail: ljgudas@med.cornell.edu [Center for Complementary and Integrative Medicine, Weill Cornell Medical College, 1300 York Avenue, New York, NY 10065 (United States); Department of Pharmacology, Weill Cornell Medical College, 1300 York Avenue, New York, NY 10065 (United States); Department of Medicine, Weill Cornell Medical College, 1300 York Avenue, New York, NY 10065 (United States)

    2011-01-01

    Epidemiological evidence indicates that diets high in fruits and vegetables provide a measure of cancer chemoprevention due to phytochemical constituents. Natural products are a rich source of cancer chemotherapy drugs, and primarily target rapidly cycling tumor cells. Increasing evidence indicates that many cancers contain small populations of resistant, stem-like cells that have the capacity to regenerate tumors following chemotherapy and radiation, and have been linked to the initiation of metastases. Our goal is to discover natural product-based clinical or dietary interventions that selectively target cancer stem cells, inducing differentiation. We adapted an alkaline phosphatase (AP) stain to assay plant extracts for the capacity to induce differentiation in embryonic stem (ES) cells. AP is a characteristic marker of undifferentiated ES cells, and this represents a novel approach to screening medicinal plant extracts. Following a survey of approximately 100 fractions obtained from 12 species of ethnomedically utilized plants, we found fractions from 3 species that induced differentiation, decreasing AP and transcript levels of pluripotency markers (Nanog, Oct-4, Rex-1). These fractions affected proliferation of murine ES, and human embryonal, prostate, and breast carcinoma cells in a dose-dependent manner. Several phytochemical constituents were isolated; the antioxidant phytochemicals ellagic acid and gallic acid were shown to affect viability of cultured breast carcinoma cells.

  13. The MHP36 line of murine neural stem cells expresses functional CXCR1 chemokine receptors that initiate chemotaxis in vitro

    Microsoft Academic Search

    John Samuel Beech; Daniel Wren Wheeler; Jill Reckless; Andrew James Grant; Jack Price; Pietro Mastroeni; David John Grainger; David Krishna Menon

    2007-01-01

    Chemokines help to establish cerebral inflammation after ischemia, which comprises a major component of secondary brain injury. The CXCR4 chemokine receptor system induces neural stem cell migration, and hence has been implicated in brain repair. We show that CXCR1 and interleukin-8 also stimulate chemotaxis in murine neural stem cells from the MHP36 cell line. The presence of CXCR1 was confirmed

  14. Effects of nicotine on cellular proliferation, macromolecular synthesis and cell cycle phase distribution in human and murine cells

    SciTech Connect

    Konno, S.; Chiao, J.; Rossi, J.; Wang, C.H.; Wu, J.M.

    1986-05-01

    Addition of nicotine causes a dose- and time-dependent inhibition of cell growth in established human and murine cells. In the human promyelocytic HL-60 leukemic cells, 3 mM nicotine results in a 50% inhibition of cellular proliferation after 80 h. Nicotine was also found to affect the cell cycle distribution of HL-60 cells. Treatment with 4 mM nicotine for 20 h causes an increase in proportion of Gl-phase cells (from 49% to 57%) and a significant decrease in the proportion of S-phase cells (from 41% to 32%). These results suggest that nicotine causes cell arrest in the Gl-phase which may in part account for its effects on cell growth. To determine whether nicotine has a primary effect on the uptake/transport of macromolecular precursors into cells, HL-60 cells were treated with 2-6 mM nicotine for 30 h/sub 3/ at the end of which time cells were labeled with (/sup 3/H)thymidine, (/sup 3/H)uridine, (/sup 14/C)lysine and (/sup 35/S)methionine, the trichloroacetic acid (TCA) soluble and insoluble radioactivities from each of the labeling conditions were determined. These studies show that nicotine primarily affect the synthesis of proteins.

  15. Impurity of stem cell graft by murine embryonic fibroblasts - implications for cell-based therapy of the central nervous system.

    PubMed

    Molcanyi, Marek; Mehrjardi, Narges Zare; Schäfer, Ute; Haj-Yasein, Nadia Nabil; Brockmann, Michael; Penner, Marina; Riess, Peter; Reinshagen, Clemens; Rieger, Bernhard; Hannes, Tobias; Hescheler, Jürgen; Bosche, Bert

    2014-01-01

    Stem cells have been demonstrated to possess a therapeutic potential in experimental models of various central nervous system disorders, including stroke. The types of implanted cells appear to play a crucial role. Previously, groups of the stem cell network NRW implemented a feeder-based cell line within the scope of their projects, examining the implantation of stem cells after ischemic stroke and traumatic brain injury. Retrospective evaluation indicated the presence of spindle-shaped cells in several grafts implanted in injured animals, which indicated potential contamination by co-cultured feeder cells (murine embryonic fibroblasts - MEFs). Because feeder-based cell lines have been previously exposed to a justified criticism with regard to contamination by animal glycans, we aimed to evaluate the effects of stem cell/MEF co-transplantation. MEFs accounted for 5.3?±?2.8% of all cells in the primary FACS-evaluated co-culture. Depending on the culture conditions and subsequent purification procedure, the MEF-fraction ranged from 0.9 to 9.9% of the cell suspensions in vitro. MEF survival and related formation of extracellular substances in vivo were observed after implantation into the uninjured rat brain. Impurity of the stem cell graft by MEFs interferes with translational strategies, which represents a threat to the potential recipient and may affect the graft microenvironment. The implications of these findings are critically discussed. PMID:25249934

  16. Impurity of Stem Cell Graft by Murine Embryonic Fibroblasts – Implications for Cell-Based Therapy of the Central Nervous System

    PubMed Central

    Molcanyi, Marek; Mehrjardi, Narges Zare; Schäfer, Ute; Haj-Yasein, Nadia Nabil; Brockmann, Michael; Penner, Marina; Riess, Peter; Reinshagen, Clemens; Rieger, Bernhard; Hannes, Tobias; Hescheler, Jürgen; Bosche, Bert

    2014-01-01

    Stem cells have been demonstrated to possess a therapeutic potential in experimental models of various central nervous system disorders, including stroke. The types of implanted cells appear to play a crucial role. Previously, groups of the stem cell network NRW implemented a feeder-based cell line within the scope of their projects, examining the implantation of stem cells after ischemic stroke and traumatic brain injury. Retrospective evaluation indicated the presence of spindle-shaped cells in several grafts implanted in injured animals, which indicated potential contamination by co-cultured feeder cells (murine embryonic fibroblasts – MEFs). Because feeder-based cell lines have been previously exposed to a justified criticism with regard to contamination by animal glycans, we aimed to evaluate the effects of stem cell/MEF co-transplantation. MEFs accounted for 5.3?±?2.8% of all cells in the primary FACS-evaluated co-culture. Depending on the culture conditions and subsequent purification procedure, the MEF-fraction ranged from 0.9 to 9.9% of the cell suspensions in vitro. MEF survival and related formation of extracellular substances in vivo were observed after implantation into the uninjured rat brain. Impurity of the stem cell graft by MEFs interferes with translational strategies, which represents a threat to the potential recipient and may affect the graft microenvironment. The implications of these findings are critically discussed. PMID:25249934

  17. Fetal Calf Serum-Free Generation of Functionally Active Murine Dendritic Cells Suitable for In Vivo Therapeutic Approaches

    Microsoft Academic Search

    Gabriele Müller; Anke Müller; Helmut Jonuleit; Kerstin Steinbrink; Claudia Szalma; Lydia Paragnik; Karen Lingnau; Edgar Schmidt; Jürgen Knop; Alexander H. Enk

    2000-01-01

    Standard protocols to generate mouse dendritic cells (DC) generally use culture medium supplemented with fetal calf serum; however, reinjection in vivo of DC cultured in fetal calf serum results in priming to xenogeneic proteins that clearly limits the use of such DC. We therefore established a fetal calf serum-free culture system for the generation of murine DC from bone marrow

  18. Induced Trefoil Factor Family 1 Expression by Trans-Differentiating Clara Cells in a Murine Asthma Model

    Microsoft Academic Search

    Irina Kouznetsova; Caroline E. Chwieralski; Ralf Balder; Margitta Hinz; Armin Braun; Norbert Krug; Werner Hoffmann

    Asthma is a chronic inflammatory disease of the airways that is accompanied by goblet cell metaplasia and mucus hypersecretion. Trefoil factor family (TFF) peptides represent major secretory prod- ucts of the respiratory tract and are synthesized together with mucins. In the murine lung, TFF2 is mainly expressed, whereas TFF1 transcripts represent only a minor species. TFF peptides are well known

  19. Establishment and characterization of an immortalized Z310 choroidal epithelial cell line from murine choroid plexus

    PubMed Central

    Zheng, Wei; Zhao, Qiuqu

    2014-01-01

    The choroid plexus plays a wide range of roles in brain development, maturation, aging process, endocrine regulation, and pathogenesis of certain neurodegenerative diseases. To facilitate in vitro study, we have used a gene transfection technique to immortalize murine choroidal epithelial cells. A viral plasmid (pSV3neo) was inserted into the host genome of primary choroidal epithelia by calcium phosphate precipitation. The transfected epithelial cells, i.e., Z310 cells, that survived from cytotoxic selection expressed SV40 large-T antigen throughout the life span, suggesting a successful gene transfection. The cells displayed the same polygonal epithelial morphology as the starting cells by light microscopy. Immunocytochemical studies demonstrate the presence of transthyretin (TTR), a thyroxine transport protein known to be exclusively produced by the choroidal epithelia in the CNS, in both transfected and starting cells. Western blot analyses further confirm the production and secretion of TTR by these cells. The mRNAs encoding transferrin receptor (TfR) were identified by Northern blot analyses. The cells grow at a steady rate, currently in the 110th passage with a population doubling time of 20–22 h in the established culture. When Z310 cells were cultured onto a Trans-well apparatus, the cells formed an epithelial monolayer similar to primary choroidal cells, possessing features such as an uneven fluid level between inner and outer chambers and an electrical resistance approximately 150–200 ?-cm2. These results indicate that immortalized Z310 cells possess the characteristics of choroidal epithelia and may have the potential for application in blood-CSF barrier (BCB) research. PMID:12470873

  20. Proteoglycan biosynthesis in murine monocytic leukemic (M1) cells before and after differentiation

    SciTech Connect

    McQuillan, D.J.; Yanagishita, M.; Hascall, V.C.; Bickel, M. (National Institute of Dental Research, Bethesda, MD (USA))

    1989-08-05

    Murine monocytic leukemic (M1) cells were cultured in the presence of ({sup 3}H)glucosamine and ({sup 35}S)sulfate. Labeled proteoglycans were purified by anion exchange chromatography and characterized by gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis in combination with chemical and enzymatic degradation. M1 cells synthesize a single predominant species of proteoglycan which distributes almost equally between the cell and medium after 17 h labeling. The cell-associated proteoglycan has an overall size of about 135 kDa and contains three to five chondroitin sulfate chains (28-31 kDa each) attached to a chondroitinase-generated core protein of 28 kDa. The synthesis and subsequent secretion of this proteoglycan was enhanced 4-5-fold in cells induced to differentiate into macrophages. This was not a phenomenon of arrest in the G0/G1 stage of the cell cycle, since density inhibited undifferentiated cells arrested at this stage did not increase proteoglycan synthesis. The chondroitin sulfate chains contained exclusively chondroitin 4- and 6-sulfate; however, the ratio of these two disaccharides differed between the medium- and cell-associated proteoglycans, and changed during progression of the cells into a fully differentiated phenotype. Pulse-chase kinetics indicate the presence of two distinct pools of proteoglycan; one that is secreted very rapidly from the cell after a approximately 1-h lag, and a second pool that is turned over in the cell with a half-time of approximately 3.5 h. Subtle differences in the glycosylation patterns of the medium- and cell-associated species are consistent with synthesis of two pools. Papain digestion suggests that the chondroitin sulfate chains are clustered on a small protease resistant peptide. The data suggest that this proteoglycan is similar to the serglycin proteoglycan family.

  1. Pertussis Toxin Nullifies the Depolarization of the Membrane Potential and the Stimulation of the Rapid Phase of 45Ca2+ Entry Through L-type Calcium Channels that are Produced by Follicle Stimulating Hormone in 10- to 12-Day-Old Rat Sertoli Cells

    PubMed Central

    Jacobus, Ana Paula; Loss, Eloísa Silveira; Wassermann, Guillermo Federico

    2010-01-01

    The aim of this study was to evaluate the effect of pertussis toxin (PTX) on the depolarizing component of the action of follicle stimulating hormone (FSH) on the membrane potential (MP) of Sertoli cells, which is linked to the rapid entry of Ca2+ into cells and to the Ca2+-dependent transport of neutral amino acids by the A system. This model allowed us to analyze the involvement of Gi proteins in the action of FSH in these phenomena. In parallel, using an inactive analog of insulin-like growth factor type I (IGF-1), JB1, and an anti-IGF-I antibody we investigated the possible mediating role of IGF-I on these effects of FSH because IGF-I is produced and released by testicular cells in response to stimulation by FSH and shows depolarization effects on MP similar to those from FSH. Our results have the following implications: (a) the rapid membrane actions of FSH, which occur in a time-frame of seconds to minutes and include the depolarization of the MP, and stimulation of 45Ca2+ uptake and [14C]-methyl aminoisobutyric acid ([14C]-MeAIB) transport, are nullified by the action of PTX and, therefore, are probably mediated by GiPCR activation; (b) the effects of FSH were also nullified by verapamil, an L-type voltage-dependent Ca2+ channel blocker; (c) wortmannin, an inhibitor of phosphoinositide 3-kinase (PI3K), prevented FSH stimulation of 45Ca2+ entry and [14C]-MeAIB transport; and (d) these FSH actions are independent of the IGF-I effects. In conclusion, these results strongly suggest that the rapid action of FSH on L-type Ca2+ channel activity in Sertoli cells from 10- to 12-day-old rats is mediated by the Gi/??/PI3K? pathway, independent of the effects of IGF-I. PMID:21423378

  2. Interleukin-17A: a T-cell-derived growth factor for murine and human mesenchymal stem cells.

    PubMed

    Huang, Weitao; La Russa, Vincent; Alzoubi, Azam; Schwarzenberger, Paul

    2006-06-01

    Interleukin-17A (IL-17A) is a proinflammatory cytokine expressed in activated T-cells. It is required for microbial host defense and is a potent stimulator of granulopoiesis. In a dose-dependent fashion, IL-17A expanded human mesenchymal stem cells (MSCs) and induced the proliferation of mature stroma cells in bone marrow-derived stroma cultures. Recombinant human interleukin-17A (rhIL-17A) nearly doubled colony-forming unit-fibroblast (CFU-f) frequency and almost tripled the surface area covered by stroma. In a murine transplant model, in vivo murine (m)IL-17A expression enhanced CFU-f by 2.5-fold. Enrichment of the graft with CD4(+) T-cell resulted in a 7.5-fold increase in CFU-f in normal C57BL/6, but only threefold in IL-17Ra(-/-) mice on day 14 post-transplant. In this transplant model, in vivo blockade of IL-17A in C57BL/6 mice resembled the phenotype of IL-17Ra(-/-) mice. Approximately half of the T-cell-mediated effect on MSC recovery following radiation-conditioned transplantation was attributed to the IL-17A/IL-17Ra pathway. Pluripotent MSCs have the potential of regenerating various tissues, and mature stroma cells are critical elements of the hematopoietic microenvironment (HME). The HME is pivotal for formation and maintenance of functional blood cells. As a newly identified stroma cell growth factor, IL-17A might have potential applications for novel treatment approaches involving MSCs, such as tissue graft engineering. PMID:16513762

  3. Interaction of interleukin-5 with its receptors on murine leukemic BCL1 cells and its implication in biological activity.

    PubMed

    Tsuruoka, N; Funakoshi, K; Kodama, S; Tsujimoto, M

    1990-02-01

    Interaction of interleukin (IL)-5 with its receptors on murine leukemic cell line, BCL1 cells was examined. 125I-labeled recombinant murine IL-5(rmIL-5) bound specifically to high-affinity receptors on BCL1 cells. rmIL-5, which was about 2500-fold more active than recombinant human IL-5(rhIL-5) in IgM-inducing activity on BCL1 cells, also showed about 5000-fold higher affinity to receptors. These results suggest that the bioactivity of IL-5 correlates with its receptor-binding activity. When disulfide bond formation was blocked, rmIL-5 dissociated into a monomer and lost its biological activity. This monomeric form of rmIL-5 also lost its ability to bind to cells, suggesting that dimer formation is essential for the biological activity of IL-5. PMID:2297794

  4. Interaction of interleukin-5 with its receptors on murine leukemic BCL1 cells and its implication in biological activity

    SciTech Connect

    Tsuruoka, N.; Funakoshi, K.; Kodama, S.; Tsujimoto, M. (Suntory Institute for Biomedical Research, Mishima, Osaka (Japan))

    1990-02-01

    Interaction of interleukin (IL)-5 with its receptors on murine leukemic cell line, BCL1 cells was examined. {sup 125}I-labeled recombinant murine IL-5(rmIL-5) bound specifically to high-affinity receptors on BCL1 cells. rmIL-5, which was about 2500-fold more active than recombinant human IL-5(rhIL-5) in IgM-inducing activity on BCL1 cells, also showed about 5000-fold higher affinity to receptors. These results suggest that the bioactivity of IL-5 correlates with its receptor-binding activity. When disulfide bond formation was blocked, rmIL-5 dissociated into a monomer and lost its biological activity. This monomeric form of rmIL-5 also lost its ability to bind to cells, suggesting that dimer formation is essential for the biological activity of IL-5.

  5. Antiviral activity of biological response modifiers in a murine model of aids. requirement for augmentation of natural killer cell activity and synergy with oral AZT

    Microsoft Academic Search

    Paul L. Black; Katherine M. McKinnon; Sharon L. Wooden; Michael A. Ussery

    1996-01-01

    We employed the Rauscher murine leukemia virus (RMuLV) as a murine retrovirus model of AIDS, to test biological response modifiers (BRM) and antiviral agents for potential therapeutic activity against the human immunodeficiency virus (HIV). We examined the relationship between the augmentation of natural killer (NK) cell activity and antiviral efficacy of a series of BRM, most of which are known

  6. Gold nanoparticles enhance the radiation therapy of a murine squamous cell carcinoma This article has been downloaded from IOPscience. Please scroll down to see the full text article.

    E-print Network

    Terasaki, Mark

    Gold nanoparticles enhance the radiation therapy of a murine squamous cell carcinoma This article) 3045­3059 doi:10.1088/0031-9155/55/11/004 Gold nanoparticles enhance the radiation therapy of a murine The purpose of this study is to test the hypothesis that gold nanoparticle (AuNP, nanogold)-enhanced radiation

  7. Growth Regulated Oncogene-? expression by murine squamous cell carcinoma promotes tumor growth, metastasis, leukocyte infiltration and angiogenesis by a host CXC Receptor2 dependent mechanism

    Microsoft Academic Search

    Elena Loukinova; Gang Dong; Ileana Enamorado-Ayalya; Giovana R Thomas; Zhong Chen; Hans Schreiber; Carter Van Waes

    2000-01-01

    Growth Regulated Oncogene-? (GRO-?) is an autocrine growth factor in melanoma and is a member of the C-X-C family of chemokines which promote chemotaxis of granulocytes and endothelia through binding to CXC Receptor 2. We found previously that variants of murine squamous cell carcinoma PAM 212 which grow and metastasize more rapidly in vivo constitutively express increased levels of murine

  8. Morphologic and Functional Characteristics of Long-Term Cultures of Murine Myeloma Cells

    PubMed Central

    Sorenson, George D.; Pettengill, Olive S.

    1972-01-01

    Murine myeloma cells proliferate well in cell culture even after up to 5 years in vitro and the globulin synthesized, as well as the ultrastructure, continues to be comparable to that of original tumor cells. Globulin synthesis by cultured cells from the C3H mouse tumor (×5563) ranged from 6 to 12.2 mg/g/48 hr and by cultured cells from Balb/c mouse plasmacytomas MOPC 21, 31C and 315 up to 40 mg/g/48 hr. This production is greater and occurs over a more prolonged period than those reported in other known published observations on globulin synthesis by normal or neoplastic cells from mice or other sources. However, both rate and quantity of globulin synthesis tend to decrease with time in vitro and this is inversely related to growth rate, which tends to increase with time in vitro. No evidence was found for increased globulin breakdown either intra- or extracellularly in these cultures. ImagesFig 2Fig 6Fig 7Fig 3Fig 4Fig 5Fig 1 PMID:4336548

  9. Evidence for multiple mechanisms of polyclonal T cell activation in murine lupus.

    PubMed Central

    Singh, R R; Hahn, B H; Tsao, B P; Ebling, F M

    1998-01-01

    Individuals with systemic autoantibody-mediated diseases such as lupus have polyclonal T and B cell activation. Yet, autoantibody production is restricted to certain autoantigens. The mechanisms underlying this phenomenon remain unclear. We propose three potential mechanisms by which autoreactive helper T cell responses diversify to become polyclonal, yet are restricted to certain antigens. First, using a model where self-Ig peptides spontaneously activate T cells and modulate disease in lupus mice, we demonstrate that the numbers of autoantibody-augmenting T helper peptides increased across the Ig molecule as mice aged ("intramolecular determinant spreading"). Secondly, a single T cell hybridoma established from a (NZB x NZW)F1 mouse immunized with one self-Ig peptide recognized several Ig-derived determinants, which had little sequence homology with the immunizing peptide. Such determinant degeneracy can lead to polyclonality. To explore a mechanism for restriction to certain autoantigens, a protein database search was done for homologies with sequences of selected stimulatory Ig peptides. Identical sequences of such determinants were not found in murine proteins other than Ig. These occurred infrequently in nonautoantibody Ig, but quite commonly in lupus-related autoantibodies such as antibodies to DNA, cardiolipin, and erythrocytes. Thus, determinant spreading and degenerate recognition in T cells coupled with recurring use of T cell determinant sequences among autoantibodies result in polyclonality that is restricted to certain autoantigens. PMID:9819370

  10. The in vitro migration of murine fetal liver cells to thymic rudiments.

    PubMed

    Pyke, K W; Bach, J F

    1979-04-01

    The migration of murine fetal liver cells to thymus rudiments was studied in vitro using a migration under agar technique. There appeared to be a minor population that migrated specifically to the thymus from the age of 10 to 14 days of gestation. The specificity of migration was demonstrated in 12-day fetal liver cells by a series of competition studies. The ability of these cells to colonize a thymus rudiment was shown by further incubation after invasion of the epithelial thymus rudiments: small colonies of lymphoid cells were present in invaded tissue but absent from uninvaded control tissue. At 13 to 14 days of gestation, there appeared an additional population that migrated specifically to the spleen, as demonstrated, again, with a competition protocol. Studies with avian and human tissue as attractants in the same system showed that migration was specific to the thymus and did cross species barriers. This observation was used to demonstrate a similar attractant activity in cell-free conditioned medium from human thymus epithelial cultures, and to demonstrate the absence of such a cell-attractant factor in the conditioned medium from the thymus of a child with previously documented severe combined immunodeficiency disease. PMID:313881

  11. Activation of murine dendritic cells and macrophages induced by Salmonella enterica serovar Typhimurium

    PubMed Central

    Kalupahana, Ruwani Sagarika; Mastroeni, Pietro; Maskell, Duncan; Blacklaws, Barbara Ann

    2005-01-01

    Macrophages and dendritic cells (DCs) are antigen-presenting cells (APCs), and the direct involvement of both cell types in the immune response to Salmonella has been identified. In this study we analysed the phenotypic and functional changes that take place in murine macrophages and DCs in response to live and heat-killed Salmonella enterica serovar Typhimurium. Both types of cell secreted proinflammatory cytokines and nitric oxide (NO) in response to live and heat-killed salmonellae. Bacterial stimulation also resulted in up-regulation of costimulatory molecules on macrophages and DCs. The expression of major histocompatibility complex (MHC) class II molecules by macrophages and DCs was differentially regulated by interferon (IFN)-? and salmonellae. Live and heat-killed salmonellae as well as lipopolysaccharide (LPS) inhibited the up-regulation of MHC class II expression induced by IFN-? on macrophages but not on DCs. Macrophages as well as DCs presented Salmonella-derived antigen to CD4 T cells, although DCs were much more efficient than macrophages at stimulating CD4 T-cell cytokine release. Macrophages are effective in the uptake and killing of bacteria whilst DCs specialize in antigen presentation. This study showed that the viability of salmonellae was not essential for activation of APCs but, unlike live bacteria, prolonged contact with heat-killed bacteria was necessary to obtain maximal expression of the activation markers studied. PMID:16011515

  12. Modeling murine yolk sac hematopoiesis with embryonic stem cell culture systems

    PubMed Central

    COOK, Brandoch D.

    2014-01-01

    The onset of hematopoiesis in mammals is defined by generation of primitive erythrocytes and macrophage progenitors in embryonic yolk sac. Laboratories have met the challenge of transient and swiftly changing specification events from ventral mesoderm through multipotent progenitors and maturing lineage-restricted hematopoietic subtypes, by developing powerful in vitro experimental models to interrogate hematopoietic ontogeny. Most importantly, studies of differentiating embryonic stem cell derivatives in embryoid body and stromal coculture systems have identified crucial roles for transcription factor networks (e.g. Gata1, Runx1, Scl) and signaling pathways (e.g. BMP, VEGF, WNT) in controlling stem and progenitor cell output. These and other relevant pathways have pleiotropic biological effects, and are often associated with early embryonic lethality in knockout mice. Further refinement in subsequent studies has allowed conditional expression of key regulatory genes, and isolation of progenitors via cell surface markers (e.g. FLK1) and reporter-tagged constructs, with the purpose of measuring their primitive and definitive hematopoietic potential. These observations continue to inform attempts to direct the differentiation, and augment the expansion, of progenitors in human cell culture systems that may prove useful in cell replacement therapies for hematopoietic deficiencies. The purpose of this review is to survey the extant literature on the use of differentiating murine embryonic stem cells in culture to model the developmental process of yolk sac hematopoiesis. PMID:25558247

  13. Chronic liver disease in murine hereditary tyrosinemia type 1 induces resistance to cell death.

    PubMed

    Vogel, Arndt; van Den Berg, Inge E T; Al-Dhalimy, Muhsen; Groopman, John; Ou, Ching-Nan; Ryabinina, Olga; Iordanov, Mihail S; Finegold, Milton; Grompe, Markus

    2004-02-01

    The murine model of hereditary tyrosinemia type 1 (HT1) was used to analyze the relationship between chronic liver disease and programmed cell death in vivo. In healthy fumarylacetoacetate hydrolase deficient mice (Fah(-/-)), protected from liver injury by the drug 2-(2- nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione (NTBC), the tyrosine metabolite homogentisic acid (HGA) caused rapid hepatocyte death. In contrast, all mice survived the same otherwise lethal dose of HGA if they had preexisting liver damage induced by NTBC withdrawal. Similarly, Fah(-/-) animals with liver injury were also resistant to apoptosis induced by the Fas ligand Jo-2 and to necrosis-like cell death induced by acetaminophen (APAP). Molecular studies revealed a marked up-regulation of the antiapoptotic heat shock proteins (Hsp) 27, 32, and 70 and of c-Jun in hepatocytes of stressed mice. In addition, the p38 and Jun N-terminal kinase (JNK) stress-activated kinase pathways were markedly impaired in the cell-death resistant liver. In conclusion, these results provide evidence that chronic liver disease can paradoxically result in cell death resistance in vivo. Stress-induced failure of cell death programs may lead to an accumulation of damaged cells and therefore enhance the risk for cancer as observed in HT1 and other chronic liver diseases. PMID:14767996

  14. Characterization of a tachykinin peptide NK sub 2 receptor transfected into murine fibroblast B82 cells

    SciTech Connect

    Van Giersbergen, P.L.M. (Marion Merrell Dow Research Inst., Cincinnati, OH (United States) Univ. of Cincinnati, OH (United States)); Shatzer, S.A.; Buck, S.H. (Marion Merrell Dow Research Inst., Cincinnati, OH (United States)); Henderson, A.K.; Lai, J.; Yamamura, Henry, I. (Univ. of Arizona, Tucson (United States)); Nakanishi, Shigetada (Kyoto Univ. (Japan))

    1991-03-01

    Membranes isolated from a murine fibroblast B82 cell line (SKLKB82{number sign}3) transfected with the bovine stomach cDNA pSKR56S exhibited binding of (His({sup 125}I){sup 1})neurokinin A ({sup 125}I-NKA) to a single population of sites with a B{sub max} of 147 fmol/mg of protein and a K{sub d} of 0.59 nM. The ligand binding in SKLKB82{number sign}3 cells was reversible. Thus, SKLKB82{number sign}3 cells have been transfected with NK{sub 2} receptors that have become associated with an endogenous guanine nucleotide-binding protein. In comparison with membranes from the hamster urinary bladder, a tissue enriched in NK{sub 2} receptors, NK{sub 2} receptor antagonists displayed markedly different potencies, either more or less potent, in inhibiting specific binding in membranes of the transfected cells. Furthermore, inhibition of {sup 125}I-NKA binding by nucleotide analogues was markedly different in SKLKB82{number sign}3 cells compared with hamster bladder tissue. The different binding profile in the cells is not due to an artefact introduced during cDNA transfection because a similar profile was also observed in bovine stomach membranes. These results may indicate the existence of two distinct NK{sub 2} receptors.

  15. [Decrease in tumorigenic activity of murine hepatoma cells after treatment with antioxidants and melatonin].

    PubMed

    Filatova, N A; Kirpichnikova, K M; Aksenov, N D; Vakhromova, E A; Gamale?, I A

    2011-01-01

    We studied the effect of antioxidants such as N-acetylcysteine (NAC, 10 mM) and alpha-lipoic acid (ALA, 1.25 mM) and of the hormone melatonin (1 microM) on the ability of murine hepatoma cells MH22a to develop tumors in syngenic mice (C3HA) after subsutaneous injection. Tumor formation and development slowed down and mouse mortality decreased when the injected cells were pretreated by NAC, ALA or melatonin during 20 h. Melatonin had the most marked effect. Tumors appeared in 100 % cases after 10 days in control mice when untreated cells had been injected; injection of cells pretreated by NAC or ALA resulted in tumor formation only in 40 and 53 % of mice, respectively. When cells were pretreated with melatonin the tumors appeared only in 18-20 days after injection. Until the end of the observation (36 days) 67 % of control mice died, but when the cells were pretreated by NAC or ALA mouse death-rate was 20 and 53 %, respectively. In the case of melatonin we did not observed any dead mice at all. We showed that treatment by antioxidants delayed (NAC) or completely inhibited (ALA) cell cycle of hepatoma cells. Cell cycle was restored after removal of the antioxidants. Melatonin did not change cell cycle phase distribution. We conclude that there is no direct correlation between loss of tumorigenic properties and changing of proliferative activity of hepatoma cells. Different mechanisms of antioxidants and melatonin action resulting in transient tumor phenotype normalization are discussed. PMID:21786683

  16. Membrane permeability coefficients of murine primary neural brain cells in the presence of cryoprotectant.

    PubMed

    Paynter, S J; Andrews, K J; Vinh, N N; Kelly, C M; Rosser, A E; Amso, N N; Dunnett, S B

    2009-06-01

    Neural cells isolated from the brain have a number of research and clinical applications, including transplantation to patients with neurodegenerative conditions. Tissue supply is one of the major limiting factors to clinical transplantation. Cryopreservation of primary neural cells would improve supply, aid in organisation of transplantation surgery and facilitate research. To date, cryopreservation using standard methods has resulted in reduced yield and/or viability of primary neural tissue. In order to optimise freezing protocols specifically for such cells, the non-osmotic volume (V(b)), water permeability (L(p)) and permeability to cryoprotectant (P(cpa)) were determined. Murine foetal brain tissue from the ganglionic eminence (GE), ventral mesencephalon (VM), or neocortical mantle (Ctx) was trypsinised to a single cell suspension. To determine V(b,) cell volume was measured after exposure to anisotonic solutions of sucrose (150-1500 mOsmol/kg). L(p) (mum/min.atm) and P(cpa) (mum/s) were determined for GE cells by measuring cell volume during exposure to 1.5 mol/l cryoprotectant. Cell volume was determined using an electronic particle counting method. V(b) was 27% for Ctx and GE, and 30% for VM. The osmotic response of GE cells was similar in the presence of propane-1,2-diol and dimethyl sulphoxide. In the presence of ethylene glycol, cell volume decrease was greater on initial exposure to cryoprotectant and recovery slower. Differences in L(p,) but not P(cpa), were found between cryoprotectants. The present results provide key parameters for optimisation of freezing protocols for cryopreservation of primary foetal brain tissues for application in neural cell transplantation. PMID:19285056

  17. Unique roles of infiltrating myeloid cells in the murine uterus during early to midpregnancy.

    PubMed

    Zhao, Hui; Kalish, Flora; Schulz, Stephanie; Yang, Yang; Wong, Ronald J; Stevenson, David K

    2015-04-15

    Leukocyte infiltration into the uterus is a characteristic feature in early to midpregnancy, but the composition and function of these leukocytes are not well understood. Using a pregnant murine model, we showed that myeloid cells and uterine NK (uNK) cells were the predominant populations in uteri during early to midgestation, whereas T and B cells were constrained. Uterine myeloid populations included cells that infiltrated from the circulation (myeloid-derived suppressor cells [MDSCs], monocyte-derived macrophages [M?s], and dendritic cells [DCs]) or proliferated from resident precursors (resident M?s [Re-M?s] and DCs). CD11b(hi)Ly6-G(hi) cells, representing neutrophils in both blood and uterine MDSCs, significantly increased from embryonic days 8.5 to 9.5. To understand their putative functions, we used anti-Gr-1 Ab to deplete circulating neutrophils and uterine MDSCs. In the absence of MDSC suppression, uterine DCs, T cells, and regulatory T cells expanded. Conversely, uterine MDSCs responded to LPS-induced inflammation and transformed into CD14(+)-activated neutrophils, resulting in an upregulation of tolerogenic DCs. A high dose of LPS (2.5 ?g/mouse) significantly increased the influx of neutrophils and production of proinflammatory cytokines, such as IL-1? and TNF-?, resulting in the reduction of Re-M?s and uNK cells, and led to placental hemorrhages and fetal deaths. In summary, uterine MDSCs are important in early to midpregnancy by responding to the maternal immunologic milieu and protecting uNK cells and Re-M?s via MDSC's suppressive and anti-inflammatory functions. Upsetting this delicate immune balance by factors leading to either insufficient MDSCs or excessive neutrophil infiltration in the fetomaternal interface may contribute to pregnancy failure. PMID:25780045

  18. Infection of Xenotransplanted Human Cell Lines by Murine Retroviruses: A Lesson Brought Back to Light by XMRV.

    PubMed

    Hempel, Heidi A; Burns, Kathleen H; De Marzo, Angelo M; Sfanos, Karen S

    2013-01-01

    Infection of xenotransplanted human cells by xenotropic retroviruses is a known phenomenon in the scientific literature, with examples cited since the early 1970s. However, arguably, until recently, the importance of this phenomenon had not been largely recognized. The emergence and subsequent debunking of Xenotropic Murine leukemia virus-Related Virus (XMRV) as a cell culture contaminant as opposed to a potential pathogen in several human diseases, notably prostate cancer and Chronic Fatigue Syndrome, highlighted a potential problem of murine endogenous gammaretroviruses infecting commonly used human cell lines. Subsequent to the discovery of XMRV, many additional cell lines that underwent xenotransplantation in mice have been shown to harbor murine gammaretroviruses. Such retroviral infection poses the threat of not only confounding experiments performed in these cell lines via virus-induced changes in cellular behavior but also the potential infection of other cell lines cultured in the same laboratory. Thus, the possibility of xenotropic retroviral infection of cell lines may warrant additional precautions, such as periodic testing for retroviral sequences in cell lines cultured in the laboratory. PMID:23785669

  19. Specific subsets of murine dendritic cells acquire potent T cell regulatory functions following CTLA4-mediated induction of indoleamine 2,3 dioxygenase

    Microsoft Academic Search

    Andrew L. Mellor; Phillip Chandler; Babak Baban; Anna M. Hansen; Brendan Marshall; Jeanene Pihkala; Herman Waldmann; Stephen Cobbold; Elizabeth Adams; David H. Munn

    2004-01-01

    Murine dendritic cells (DCs) expressing indoleamine 2,3 dioxygenase (IDO) catabolize tryptophan and can suppress T cell responses elicited in vivo. Here, we identify specific subsets of splenic (CD11c1) dendritic cells competent to mediate IDO-dependent T cell suppression following CTLA4-mediated ligation of B7 molecules. IDO-competent DC subsets acquired potent and dominant T cell suppressive properties as a consequence of IDO up-regulation,

  20. Global microRNA expression is essential for murine mast cell development in vivo.

    PubMed

    Oh, Sun Young; Brandal, Stephanie; Kapur, Reuben; Zhu, Zhou; Takemoto, Clifford M

    2014-10-01

    MicroRNAs (miRNAs) are small, noncoding RNAs that have been shown to play a critical role in normal physiology and disease, such as hematopoietic development and cancer. However, their role in mast-cell function and development is poorly understood. The major objective of this study was to determine how global miRNA expression affects mast-cell physiology. The RNase III endonuclease, Dicer, is required for the processing of pre-miRNAs into mature miRNAs. To investigate the effect of global miRNA depletion on mast cells in vivo, we generated a mast-cell-specific knock out of Dicer in mice. Transgenic mice (Mcpt5-Cre) that express Cre selectively in connective tissue mast cells were crossed with mice carrying the floxed conditional Dicer allele (Dicer fl/fl). Mcpt5-Cre × Dicer fl/fl mice with homozygous Dicer gene deletion in mast cells were found to have a profound mast-cell deficiency with near complete loss of peritoneal, gastrointestinal, and skin mast cells. We examined the in vivo functional consequence of mast-cell-specific Dicer deletion using an immunoglobulin-E-dependent passive systemic anaphylaxis murine model. Immunoglobulin-E-sensitized wild type Mcpt5-Cre × Dicer +/+ and heterozygous Mcpt5-Cre × Dicer fl/+ mice show marked hypothermia with antigen; however, homozygous Mcpt5-Cre × Dicer fl/fl mice were completely unresponsive to antigen challenge. These studies suggest a critical role for Dicer and miRNA expression for establishment of tissue compartments of functional mast cells in vivo. PMID:25201754

  1. Engineering vascularized bone: osteogenic and proangiogenic potential of murine periosteal cells.

    PubMed

    van Gastel, Nick; Torrekens, Sophie; Roberts, Scott J; Moermans, Karen; Schrooten, Jan; Carmeliet, Peter; Luttun, Aernout; Luyten, Frank P; Carmeliet, Geert

    2012-11-01

    One of the key challenges in bone tissue engineering is the timely formation of blood vessels that promote the survival of the implanted cells in the construct. Fracture healing largely depends on the presence of an intact periosteum but it is still unknown whether periosteum-derived cells (PDC) are critical for bone repair only by promoting bone formation or also by inducing neovascularization. We first established a protocol to specifically isolate murine PDC (mPDC) from long bones of adult mice. Mesenchymal stem cells were abundantly present in this cell population as more than 50% of the mPDC expressed mesenchymal markers (CD73, CD90, CD105, and stem cell antigen-1) and the cells exhibited trilineage differentiation potential (chondrogenic, osteogenic, and adipogenic). When transplanted on a collagen-calcium phosphate scaffold in vivo, mPDC attracted numerous blood vessels and formed mature bone which comprises a hematopoiesis-supportive stroma. We explored the proangiogenic properties of mPDC using in vitro culture systems and showed that mPDC promote the survival and proliferation of endothelial cells through the production of vascular endothelial growth factor. Coimplantation with endothelial cells demonstrated that mPDC can enhance vasculogenesis by adapting a pericyte-like phenotype, in addition to their ability to stimulate blood vessel ingrowth from the host. In conclusion, these findings demonstrate that periosteal cells contribute to fracture repair, not only through their strong osteogenic potential but also through their proangiogenic features and thus provide an ideal cell source for bone regeneration therapies. PMID:22911908

  2. Entry of Francisella tularensis into Murine B Cells: The Role of B Cell Receptors and Complement Receptors

    PubMed Central

    Plzakova, Lenka; Krocova, Zuzana; Kubelkova, Klara; Macela, Ales

    2015-01-01

    Francisella tularensis, the etiological agent of tularemia, is an intracellular pathogen that dominantly infects and proliferates inside phagocytic cells but can be seen also in non-phagocytic cells, including B cells. Although protective immunity is known to be almost exclusively associated with the type 1 pathway of cellular immunity, a significant role of B cells in immune responses already has been demonstrated. Whether their role is associated with antibody-dependent or antibody-independent B cell functions is not yet fully understood. The character of early events during B cell–pathogen interaction may determine the type of B cell response regulating the induction of adaptive immunity. We used fluorescence microscopy and flow cytometry to identify the basic requirements for the entry of F. tularensis into B cells within in vivo and in vitro infection models. Here, we present data showing that Francisella tularensis subsp. holarctica strain LVS significantly infects individual subsets of murine peritoneal B cells early after infection. Depending on a given B cell subset, uptake of Francisella into B cells is mediated by B cell receptors (BCRs) with or without complement receptor CR1/2. However, F. tularensis strain FSC200 ?iglC and ?ftdsbA deletion mutants are defective in the ability to enter B cells. Once internalized into B cells, F. tularensis LVS intracellular trafficking occurs along the endosomal pathway, albeit without significant multiplication. The results strongly suggest that BCRs alone within the B-1a subset can ensure the internalization process while the BCRs on B-1b and B-2 cells need co-signaling from the co receptor containing CR1/2 to initiate F. tularensis engulfment. In this case, fluidity of the surface cell membrane is a prerequisite for the bacteria’s internalization. The results substantially underline the functional heterogeneity of B cell subsets in relation to F. tularensis. PMID:26161475

  3. Mutational Analysis of the Murine Coronavirus Spike Protein: Effect on Cell-to-Cell Fusion

    Microsoft Academic Search

    EVELYNE C. W. BOS; LEO HEIJNEN; WILLEM LUYTJES; WILLY J. M. SPAAN

    1995-01-01

    The spike (S) protein of murine coronavirus strain A59 (MHV-A59) is a type I membrane protein that induces membrane fusion. In this study we have analyzed the role of two domains in the S protein on fusion. The 180-kDa mature S protein is partially cleaved into two 90-kDa subunits during transport to the plasma membrane. We have identified several amino

  4. Dynamic DNA methylation and histone modifications contribute to lentiviral transgene silencing in murine embryonic carcinoma cells.

    PubMed

    He, Jin; Yang, Qing; Chang, Lung-Ji

    2005-11-01

    Embryonic stem cells are subjected to a dynamic genome regulation during development. Here we report that the ectopic lentiviral transgenes are quickly silenced in murine embryonic carcinoma P19 cells. The silencing was correlated with CpG hypermethylation in the transgene promoter. Using high-resolution sodium bisulfite genome sequencing, we detected distinct DNA methylation kinetics in different proviral regions. DNase I sensitivity and chromatin immunoprecipitation assays revealed condensed chromatin structure and histone code switch during silencing. Longitudinal analysis of nonsilenced and silenced identical single-cell clones revealed that the silencing was coupled with CpG methylation in the promoter, as well as a global histone H3 deacetylation. Interestingly, the primer binding site and the packaging signal region appeared to serve as a DNA methylation initiation center which was rapidly hypermethylated regardless of transgene silencing and chromatin modifications. Analysis of cellular genes 45 to 50 kbp upstream and downstream of the integration site indicated that transcriptional activities of the flanking host genes were not affected. Genetic modifications of stem cells have great therapeutic potentials and our results picture a dynamic embryonic genome response to ectopic transgene integration that may have important implications in the future safety and efficacy modifications of stem cells. PMID:16227270

  5. Nuclear receptor expression patterns in murine plasmacytoid and conventional dendritic cells.

    PubMed

    Karthaus, Nina; Hontelez, Saartje; Looman, Maaike W G; van Spriel, Annemiek B; Ansems, Marleen; Adema, Gosse J

    2013-10-01

    Dendritic cells (DC) play a central role in the immune system. They can either induce immunity or promote tolerance. The DC family is generally comprised of two functionally distinct DC subsets. Conventional dendritic cells (cDC) are the classical antigen presenting cells; plasmacytoid dendritic cells (pDC) are the main producers of type I interferons thereby serving innate immunity. Upon activation DCs are able to present antigen and stimulate T cells. The immune modulatory functions of DCs largely depend on the recognition of soluble cues. Besides pathogen derived cues, recent data indicate that the tissue micro-environment, i.e. of the gut and skin affects cDC function. Many of these micro-environmental factors are ligands for the nuclear receptor (NR) family of transcription regulators known to affect immunity and tolerance. Whether pDC function is also influenced by tissue derived cues, like hormones, vitamins and metabolic products, is largely unknown. Here, we investigated the NR expression profile of murine pDCs and cDCs. We assessed the mRNA levels of 19 NRs of in vitro derived as well as ex vivo isolated DCs from four different lymphoid tissues. We observed that cDCs and pDCs expressed the same repertoire of NRs. Expression levels, however, differed between the two subsets, especially upon maturation of DCs. These data imply that NR ligands do impact pDC function and that their activity might be regulated in a DC-specific manner. PMID:23597769

  6. Hepatic Differentiation of Murine Disease-Specific Induced Pluripotent Stem Cells Allows Disease Modelling In Vitro

    PubMed Central

    Eggenschwiler, Reto; Loya, Komal; Sgodda, Malte; André, Francoise; Cantz, Tobias

    2011-01-01

    Direct reprogramming of somatic cells into pluripotent cells by retrovirus-mediated expression of OCT4, SOX2, KLF4, and C-MYC is a promising approach to derive disease-specific induced pluripotent stem cells (iPSCs). In this study, we focused on three murine models for metabolic liver disorders: the copper storage disorder Wilson's disease (toxic-milk mice), tyrosinemia type 1 (fumarylacetoacetate-hydrolase deficiency, FAH?/? mice), and alpha1-antitrypsin deficiency (PiZ mice). Colonies of iPSCs emerged 2-3 weeks after transduction of fibroblasts, prepared from each mouse strain, and were maintained as individual iPSC lines. RT-PCR and immunofluorescence analyses demonstrated the expression of endogenous pluripotency markers. Hepatic precursor cells could be derived from these disease-specific iPSCs applying an in vitro differentiation protocol and could be visualized after transduction of a lentiviral albumin-GFP reporter construct. Functional characterization of these cells allowed the recapitulation of the disease phenotype for further studies of underlying molecular mechanisms of the respective disease. PMID:21977043

  7. Identification of immunomodulatory signatures induced by american ginseng in murine immune cells.

    PubMed

    Yan, Jian; Ma, Yonghui; Zhao, Fusheng; Gu, Weikuan; Jiao, Yan

    2013-01-01

    Background. American ginseng (Panax quinquefolius, AG) has been used for more than 300 years. Some of its claimed benefits can be attributed to the immunomodulatory activities, whose molecular mechanisms are largely unknown. Methods. Murine splenic cells from adult male C57BL/6 (B6) mice were isolated and divided into 4 groups to mimic 4 basic pathophysiological states: (1) normal naïve; (2) normal activated; (3) deficient naïve; (4) deficient activated. Then, different AG extracts were added to all groups for 24?h incubation. MTT proliferation assays were performed to evaluate the phenotypic features of cells. Finally, microarray assays were carried out to identify differentially expressed genes associated with AG exposure. Real-time PCR was performed to validate the expression of selected genes. Results. Microarray data showed that most of gene expression changes were identified in the deficient naïve group, suggesting that the pathophysiological state has major impacts on transcriptomic changes associated with AG exposure. Specifically, this study revealed downregulation of interferon- ? signaling pathway in the deficient group of cells. Conclusion. Our study demonstrated that only specific groups of immune cells responded to AG intervention and immunocompromised cells were more likely regulated by AG treatment. PMID:24319490

  8. Efficacy of Cell Wall-Deficient Spheroplasts Against Experimental Murine Listeriosis.

    PubMed

    Ansari, M A; Zia, Q; Kazmi, S; Ahmad, E; Azhar, A; Johnson, K E; Zubair, S; Owais, M

    2015-07-01

    Various strategies adapted to develop an efficient vaccine against foodborne pathogen, Listeria monocytogenes, have met with little success. Spheroplasts (bacterial cell devoid of cell wall) are likely to undergo membrane-membrane fusion, leading to the delivery of their content to the cytosol of antigen-presenting cells, thus facilitating MHC class I antigen processing and presentation. In this study, we evaluated the prophylactic potential of Listeria spheroplast-based vaccine against experimental murine listeriosis in comparison with heat-killed Listeria (HKL) and archaeosome-entrapped Listeria whole-cell protein (LWCP). Compared with HKL, the spheroplast-based vaccine was found to evoke better Th1 response as exhibited by the presence of type 1 cytokines in the host (interferon-? and IL-12) and a high IgG2a /IgG1 ratio. Robust lympho-proliferative efficacy was apparent in both spheroplast-immunized and archaeosome-entrapped LWCP-immunized groups. The upregulation of costimulatory and effector memory markers upon immunization with spheroplasts was found to be at par with that evoked by archaeosome-entrapped LWCP-immunized group. Central memory response in gated CD8(+) T cell was much higher in spheroplast-immunized animals when compared with archaeosome-entrapped LWCP group. The data presented here clearly demonstrate that spheroplasts evoked a robust immune response and offer better prophylactic potential against L. monocytogenes. PMID:25833403

  9. Anti-Melanogenic Property of Geoditin A in Murine B16 Melanoma Cells

    PubMed Central

    Cheung, Florence W. K.; Guo, Jia; Ling, Yick-Hin; Che, Chun-Tao; Liu, Wing-Keung

    2012-01-01

    Geoditin A, an isomalabaricane triterpene isolated from marine sponge Geodia japonica, has been demonstrated to induce apoptosis in leukemia HL60 cells and human colon HT29 cancer cells through an oxidative stress, a process also interfering with normal melanogenesis in pigment cells. Treatment of murine melanoma B16 cells with geoditin A decreased expression of melanogenic proteins and cell melanogenesis which was aggravated with adenylate cyclase inhibitor SQ22536, indicating melanogenic inhibition was mediated through a cAMP-dependent signaling pathway. Immunofluorescence microscopy and glycosylation studies revealed abnormal glycosylation patterns of melanogenic proteins (tyrosinase and tyrosinase-related protein 1), and a co-localization of tyrosinase with calnexin (CNX) and lysosome-associated membrane protein 1 (LAMP-1), implicating a post-translational modification in the ER and a degradation of tyrosinase in the lysosome. Taken together, potent anti-melanogenic property and the relatively low cytotoxicity of geoditin A have demonstrated its therapeutic potential as a skin lightening agent. PMID:22412813

  10. Elevated natural killer cell responses in mice infected with recombinant vaccinia virus encoding murine IL-2.

    PubMed

    Karupiah, G; Coupar, B E; Andrew, M E; Boyle, D B; Phillips, S M; Müllbacher, A; Blanden, R V; Ramshaw, I A

    1990-01-01

    The role of cytotoxic T cells and NK cells in the recovery of immunodeficient, athymic, nude mice infected with a recombinant vaccinia virus (VV) encoding murine IL-2 was investigated. Kinetic studies with the IL-2-encoding recombinant (VV-HA-IL2) and control (VV-HA-TK) viruses excluded a role for cytotoxic T cells but suggested the possible involvement of NK cells. In athymic nude mice given VV-HA-IL2, NK activity was at least threefold higher than mice infected with VV-HA-TK and this activity persisted for at least 6 days after infection. The effectors mediating the NK-like activity were asialo-GM1+ (as-GM1+), Thy1.2+/-, CD4- and CD8-, the phenotype of conventional NK cells. Elevated NK activity coincided with the rapid clearance of VV-HA-IL2 from ovaries of infected normal CBA/H mice but not from ovaries of CBA beige mice which had no detectable NK activity in spleens or ovaries. The expression of IL-2 in recombinant VV infection probably induces a cascade of immunologic effects of which elevated NK activity is one. We speculate that the chemoattractant and NK activity augmenting effects of IL-2 may contribute to recovery from VV-infection. PMID:2295796

  11. Maintenance and induction of murine embryonic stem cell differentiation using E-cadherin-Fc substrata without colony formation

    NASA Astrophysics Data System (ADS)

    Meng, Qing-Yuan; Akaike, Toshihiro

    2013-03-01

    Induced embryonic stem (ES) cells are expected to be promising cell resources for the observation of the cell behaviors in developmental biology as well as the implantation in cell treatments in human diseases. A recombinant E-cadherin substratum was developed as a cell recognizable substratum to maintain the ES cells' self-renewal and pluripotency at single cell level. Furthermore, the generation of various cell lineages in different germ layers, including hepatic or neural cells, was achieved on the chimeric protein layer precisely and effectively. The induction and isolation of specific cell population was carried out with the enhancing effect of other artificial extracellular matrices (ECMs) in enzyme-free process. The murine ES cell-derived cells showed highly morphological similarities and functional expressions to matured hepatocytes or neural progenitor cells.

  12. Complementation of the beige mutation in cultured cells by episomally replicating murine yeast artificial chromosomes

    SciTech Connect

    Perou, C.M.; Pryor, R.J.; Kaplan, J. [Univ. of Utah, Salt Lake City, UT (United States)] [Univ. of Utah, Salt Lake City, UT (United States); Justice, M.J. [Oak Ridge National Labortory, TN (United States)] [Oak Ridge National Labortory, TN (United States)

    1996-06-11

    Chediak-Higashi syndrome in man and the beige mutation of mice are phenotypically similar disorders that have profound effects upon lysosome and melansosome morphology and function. We isolated two murine yeast artificial chromosomes (YACs) that, when introduced into beige mouse fibroblasts, complement the beige mutation. The complementing YACs exist as extrachromosomal elements that are amplified in high concentrations of G418. When YAC-complemented beige cells were fused to human Chediak-Higashi syndrome or Aleutian mink fibroblasts, complementation of the mutant phenotype also occurred. These results localize the beige gene to a 500-kb interval and demonstrate that the same or homologous genes are defective in mice, minks, and humans. 16 refs., 5 figs.

  13. Mulberry leaf polysaccharides modulate murine bone-marrow-derived dendritic cell maturation.

    PubMed

    Xue, Ming; Sun, Haiyan; Cao, Yan; Wang, Guangchuan; Meng, Yiming; Wang, Dongmei; Hong, Yansong

    2015-04-01

    Various components of mulberry leaves, such as iminosugars, flavonoids and polysaccharides, have been reported to exert anti-diabetic activity. The purpose of our present study was to examine the modulating effect of mulberry leaf polysaccharides (MLPs) on murine bone-marrow-derived dendritic cells (BMDCs). The ultrastructure, phenotype and functional maturation of BMDCs were studied using transmission electron microscopy (TEM), ?ow cytometry (FCM), and tested for phagocytosis, acid phosphatase (ACP) activity using an enzyme linked immunosorbent assay (ELISA). Our results demonstrated that MLPs could markedly induce BMDC maturation by up-regulating the expression of membrane phenotypic markers, such as CD80, CD86, CD83,CD40, and MHC II, down-regulating phagocytosis and ACP activity, and by enhancing the production of interleukin 12 (IL-12) and tumor necrosis factor ? (TNF-?) secreted by BMDCs. We therefore concluded that MLPs can positively modulate BMDCs. PMID:25830302

  14. Suppression of tumorigenicity and metastasis in murine UV2237 fibrosarcoma cells by infection with a retroviral vector harboring the interferon-beta gene

    Microsoft Academic Search

    Zhongyun Dong; Shin-Hun Juang; Rakesh Kumar; Ines Eue; Keping Xie; Diane Bielenberg; Weixin Lu; Corazon Bucana; Xiulan Yang; Isaiah J. Fidler

    1998-01-01

    In this study, we endeavored to determine the effectiveness of interferon ? (IFN?) gene therapy against highly metastatic\\u000a murine UV-2237m fibrosarcoma cells. UV-2237m cells were engineered to produce murine IFN? constitutively following infection\\u000a by a retroviral vector harboring the murine IFN? gene. Parental (UV-2237m-P), control-vector-transduced (UV-2237m-Neo), and\\u000a IFN?-transduced (UV-2237m-IFN?) cells were injected subcutaneously (s.c.) or intravenously (i.v.) into syngeneic mice.

  15. The Development of Murine Plasmacytoid Dendritic Cell Precursors Is Differentially Regulated by FLT3-ligand and Granulocyte\\/Macrophage Colony-Stimulating Factor

    Microsoft Academic Search

    Michel Gilliet; Andre Boonstra; Carine Paturel; Svetlana Antonenko; Xiu-Ling Xu; Giorgio Trinchieri; Anne O'Garra; Yong-Jun Liu

    2002-01-01

    Plasmacytoid predendritic cells or type 1 interferon (IFN)-producing cells (IPCs) have recently been identified in mice. Although culture systems giving rise to different murine dendritic cell subsets have been established, the developmental regulation of murine plasmacytoid IPCs and the culture conditions leading to their generation remain unknown. Here we show that large numbers of over 40% pure CD11cCD11bB220 ? Gr-1

  16. Vaccine effect of granulocyte–macrophage colony-stimulating factor or CD80 gene-transduced murine hematopoietic tumor cells and their cooperative enhancement of antitumor immunity

    Microsoft Academic Search

    Y Nakazaki; K Tani; Z-T Lin; H Sumimoto; H Hibino; T Tanabe; M-S Wu; K Izawa; H Hase; S Takahashi; A Tojo; M Azuma; H Hamada; S Mori; S Asano

    1998-01-01

    To develop immunogene therapy targeting minimal residual hematopoietic tumor cells in patients, we transduced murine GM-CSF or CD80 gene into murine WEHI 3B myelomonocytic leukemia and EL-4 thymic lymphoma cells using retroviral vectors and evaluated their effects on inducing antitumor responses in syngeneic host mice. Subcutaneously injected GM-CSF- and CD80 gene-transduced WEHI 3B (GMCSF\\/WEHI\\/3.2 or CD80\\/WEHI\\/1.8, respectively) cells lost their

  17. Different processing of LH/hCG receptors in cultured rat luteal cells and murine Leydig tumor cells (MLTC-1)

    SciTech Connect

    Kellokumpu, S.

    1987-02-01

    The metabolic fate of LH/hCG receptors after exposure to human chorionic gonadotropin (hCG) was examined in cultured rat luteal cells and murine Leydig tumor cells (MLTC-1). Kinetic studies performed after pulse-labelling of the cells with (/sup 125/I)hCG indicated that the bound hormone was lost much more rapidly from the tumor cells than from the luteal cells. The tumor cells were also found to internalize and degrade the hormone more effectively than the luteal cells. Chemical cross-linking and analyses by SDS-PAGE of this material revealed that both cell types also released, in addition to intact hCG, two previously characterized receptor fragment-(/sup 125/I)hCG complexes (M/sub r/ 96,000 and 74,000) into the medium, although their amount was negligible in MLTC-1 cells. Possibly due to rapid discharge of the ligand from its receptor, no similar complexes could be detected inside the MLTC-1 cells, suggesting that they were released directly from the cell surface. However, the M/sub r/ 74,000 complex was observed inside MLTC-1 cells if chloroquine, a lysosomotropic agent, was present during the incubations. This suggests that the internalized receptor also becomes degraded, at least when complexed to hCG. The results thus provide evidence that there exist two different mechanisms for proteolytic processing of LH/hCG receptors in these target cells. In tumor cells, the degradation seems to occur almost exclusively intracellularly, whereas in luteal cells a substantial portion of the receptors is also degraded at the cell surface.

  18. Signed weighted gene co-expression network analysis of transcriptional regulation in murine embryonic stem cells

    PubMed Central

    Mason, Mike J; Fan, Guoping; Plath, Kathrin; Zhou, Qing; Horvath, Steve

    2009-01-01

    Background Recent work has revealed that a core group of transcription factors (TFs) regulates the key characteristics of embryonic stem (ES) cells: pluripotency and self-renewal. Current efforts focus on identifying genes that play important roles in maintaining pluripotency and self-renewal in ES cells and aim to understand the interactions among these genes. To that end, we investigated the use of unsigned and signed network analysis to identify pluripotency and differentiation related genes. Results We show that signed networks provide a better systems level understanding of the regulatory mechanisms of ES cells than unsigned networks, using two independent murine ES cell expression data sets. Specifically, using signed weighted gene co-expression network analysis (WGCNA), we found a pluripotency module and a differentiation module, which are not identified in unsigned networks. We confirmed the importance of these modules by incorporating genome-wide TF binding data for key ES cell regulators. Interestingly, we find that the pluripotency module is enriched with genes related to DNA damage repair and mitochondrial function in addition to transcriptional regulation. Using a connectivity measure of module membership, we not only identify known regulators of ES cells but also show that Mrpl15, Msh6, Nrf1, Nup133, Ppif, Rbpj, Sh3gl2, and Zfp39, among other genes, have important roles in maintaining ES cell pluripotency and self-renewal. We also report highly significant relationships between module membership and epigenetic modifications (histone modifications and promoter CpG methylation status), which are known to play a role in controlling gene expression during ES cell self-renewal and differentiation. Conclusion Our systems biologic re-analysis of gene expression, transcription factor binding, epigenetic and gene ontology data provides a novel integrative view of ES cell biology. PMID:19619308

  19. Cryptococcus neoformans fails to induce nitric oxide synthase in primed murine macrophage-like cells.

    PubMed Central

    Naslund, P K; Miller, W C; Granger, D L

    1995-01-01

    Nitric oxide (NO) is a microbiostatic gas generated by activated murine macrophages. Cytokine signals, gamma interferon (IFN-gamma) and tumor necrosis factor alpha (TNF-alpha) act synergistically to induce production of a macrophage nitric oxide synthase (NOS). A variety of intracellular pathogens, when recognized by macrophages primed with IFN-gamma, induce NOS by eliciting TNF-alpha secretion, which then functions as a positive autocrine signal. In cell culture assays, a murine macrophage cell line (J774), primed with IFN-gamma, was tested for NOS induction upon challenge with virulent Cryptococcus neoformans. C. neoformans failed to induce macrophage NOS as measured by nitrite production. This was true irrespective of the C. neoformans-to-J774 ratio. Other nonpathogenic Cryptococcus species likewise failed to induce NOS, yet Saccharomyces cerevisiae, Histoplasma capsulatum, and Candida albicans were efficient inducers of NOS. Conditions which promoted attachment and/or phagocytosis of C. neoformans did not lead to NOS induction (including opsonization with specific antibodies against C. neoformans). Assays for transcriptional repressors of NOS were negative. Tests for consumption of nitrite by measurement of additional products of NOS induction were negative. No TNF-alpha was detected by enzyme-linked immunosorbent assay in supernatants from C. neoformans-J774 cocultures. A mutant C. neoformans strain with a minimal, but visible, polysaccharide capsule also failed to induce NOS; however, several nonencapsulated mutants of C. neoformans did induce NOS. Failure of C. neoformans to act as an inducer of NOS may be related to the virulence of this pathogen in mice; C. neoformans is a unique example of a facultative intracellular pathogen which fails to induce NOS in primed macrophages. The mechanism appears to involve the failure of TNF-alpha secretion once the macrophage comes in contact with the fungus. The presence of the polysaccharide capsule appears to mask the signal necessary for TNF-alpha secretion and, ultimately, NOS induction. PMID:7534274

  20. A flavone derivative from Sesbania sesban leaves and its cytotoxicity against murine leukemia P-388 cells

    SciTech Connect

    Dianhar, Hanhan, E-mail: liadewi@chem.itb.ac.id; Syah, Yana Maolana, E-mail: liadewi@chem.itb.ac.id; Mujahidin, Didin, E-mail: liadewi@chem.itb.ac.id; Hakim, Euis Holisotan, E-mail: liadewi@chem.itb.ac.id; Juliawaty, Lia Dewi, E-mail: liadewi@chem.itb.ac.id [Natural Product Chemistry Research Group, Organic Chemistry Division, Program Study of Chemistry, Faculty of Mathematics and Natural Sciences, Institut Teknologi Bandung, Jalan Ganeca 10, Bandung 40132 (Indonesia)

    2014-03-24

    Sesbania sesban, locally named as Jayanti, is one of Indonesia plants belonging to Fabaceae family. This species is traditionally used by Indonesian people to cure digestive disorders, fever, or headache. Jayanti can grow well in tropical to subtropical region, such as in Asia and Africa. Based on literature, qualitative analysis of the methanol extract of leaves of S. sesban showed that it contained flavonoids, alkaloids, saponins and glycosides. In addition, the activity assay of extracts of different tissues of this species showed antitumor, antimalarial, and antidiabetic activityies (leaves and seed extracts), antioxidants (flower extract), and analgesic (wood extract). Though the extracts of S. sesban parts showed interesting activities, chemical study of those extracts have not been widely reported. Therefore, the objective of this research was to isolate the secondary metabolites from methanol extract of leaves of S. sesban and to determine their cytotoxicity against murine leukemia P-388 cells. One compound has been obtained and identified as 3-hydroxy-4',7-dimethoxyflavone (1), a new isolated compound from nature. This compound was obtained through separation of methanol extract using various chromatographic techniques, such as vacuum liquid chromatography and radial chromatography. The structure elucidation of isolated compound was based on 1D NMR ({sup 1}H-NMR and {sup 13}C-NMR) and 2D NMR (HMBC). The cytotoxicity of methanol extract and compound 1 against murine leukemia P-388 cells examined through MTT assay showed IC{sub 50} value of 60.04 ?g/mL and 5.40 ?g/mL, respectively.

  1. A characterization of four B16 murine melanoma cell sublines molecular fingerprint and proliferation behavior

    PubMed Central

    2013-01-01

    Background One of the most popular and versatile model of murine melanoma is by inoculating B16 cells in the syngeneic C57BL6J mouse strain. A characterization of different B16 modified cell sub-lines will be of real practical interest. For this aim, modern analytical tools like surface enhanced Raman spectroscopy/scattering (SERS) and MTT were employed to characterize both chemical composition and proliferation behavior of the selected cells. Methods High quality SERS signal was recorded from each of the four types of B16 cell sub-lines: B164A5, B16GMCSF, B16FLT3, B16F10, in order to observe the differences between a parent cell line (B164A5) and other derived B16 cell sub-lines. Cells were incubated with silver nanoparticles of 50–100 nm diameter and the nanoparticles uptake inside the cells cytoplasm was proved by transmission electron microscopy (TEM) investigations. In order to characterize proliferation, growth curves of the four B16 cell lines, using different cell numbers and FCS concentration were obtained employing the MTT proliferation assay. For correlations doubling time were calculated. Results SERS bands allowed the identification inside the cells of the main bio-molecular components such as: proteins, nucleic acids, and lipids. An "on and off" SERS effect was constantly present, which may be explained in terms of the employed laser power, as well as the possible different orientations of the adsorbed species in the cells in respect to the Ag nanoparticles. MTT results showed that among the four tested cell sub-lines B16 F10 is the most proliferative and B164A5 has the lower growth capacity. Regarding B16FLT3 cells and B16GMCSF cells, they present proliferation ability in between with slight slower potency for B16GMCSF cells. Conclusion Molecular fingerprint and proliferation behavior of four B16 melanoma cell sub-lines were elucidated by associating SERS investigations with MTT proliferation assay. PMID:23890195

  2. DNA-transformed murine teratocarcinoma cells: regulation of expression of simian virus 40 tumor antigen in stem versus differentiated cells.

    PubMed Central

    Linnenbach, A; Huebner, K; Croce, C M

    1980-01-01

    Thymidine kinase-deficient (TK-; ATP:thymidine 5'-phosphotransferase, EC 2.7.1.21)F9 teratocarcinoma stem cells have been transformed with a recombinant plasmid genome consisting of the pBR322 genome linked to a herpes simplex virus type 1 thymidine kinase gene (HSV-1 tk) and a simian virus 40 (SV40) genome. A clonal line of stem cells was obtained that contains only one copy of plasmid DNA, which is integrated into murine chromosomal DNA through a site on the pBRR322 genome. The HSV-1 tk gene, which is adjacent to the SV40 genome, is expressed in stem cells, whereas SV40 gene expression is not detectable. If differentiation of these stem cells is induced, the differentiated cells express SV40 early gene products. Thus, we have constructed a stem cell which contains a set of genes (SV40), the expression of which is regulated differently in stem and differentiated cells. This cell line could be used to determine the mechanism of suppression of expression of these genes in stem cells. Images PMID:6254045

  3. Basal medium composition and serum or serum replacement concentration influences on the maintenance of murine embryonic stem cells

    Microsoft Academic Search

    Muhammad A. Chaudhry; Timothy Z. Vitalis; Bruce D. Bowen; James M. Piret

    2008-01-01

    The expansion of stem cell numbers while retaining their developmental properties is a bioprocess challenge. We compared the\\u000a growth rates and embryoid body (EB) formation yields of R1 and EFC murine embryonic stem cells (mESC) cultured in two basal\\u000a media (DMEM or DMEM:F12) with additions of 1.7–15% fetal bovine serum (FBS) or serum replacer (KOSR). Whereas the basal medium\\u000a or

  4. HIV-1 Myristoylated Nef Treatment of Murine Microglial Cells Activates Inducible Nitric Oxide Synthase, NO2 Production and Neurotoxic Activity

    PubMed Central

    Capone, Caterina; Veroni, Caterina; Percario, Zulema Antonia; Leone, Stefano; Fiorucci, Gianna; Lülf, Sebastian; Romeo, Giovanna; Agresti, Cristina; Persichini, Tiziana; Geyer, Matthias; Affabris, Elisabetta

    2015-01-01

    Background The potential role of the human immunodeficiency virus-1 (HIV-1) accessory protein Nef in the pathogenesis of neuroAIDS is still poorly understood. Nef is a molecular adapter that influences several cellular signal transduction events and membrane trafficking. In human macrophages, Nef expression induces the production of extracellular factors (e.g. pro-inflammatory chemokines and cytokines) and the recruitment of T cells, thus favoring their infection and its own transfer to uninfected cells via exosomes, cellular protrusions or cell-to-cell contacts. Murine cells are normally not permissive for HIV-1 but, in transgenic mice, Nef is a major disease determinant. Both in human and murine macrophages, myristoylated Nef (myr+Nef) treatment has been shown to activate NF-?B, MAP kinases and interferon responsive factor 3 (IRF-3), thereby inducing tyrosine phosphorylation of signal transducers and activator of transcription (STAT)-1, STAT-2 and STAT-3 through the production of proinflammatory factors. Methodology/Principal Findings We report that treatment of BV-2 murine microglial cells with myr+Nef leads to STAT-1, -2 and -3 tyrosine phosphorylation and upregulates the expression of inducible nitric oxide synthase (iNOS) with production of nitric oxide. We provide evidence that extracellular Nef regulates iNOS expression through NF-?B activation and, at least in part, interferon-? (IFN?) release that acts in concert with Nef. All of these effects require both myristoylation and a highly conserved acidic cluster in the viral protein. Finally, we report that Nef induces the release of neurotoxic factors in the supernatants of microglial cells. Conclusions These results suggest a potential role of extracellular Nef in promoting neuronal injury in the murine model. They also indicate a possible interplay between Nef and host factors in the pathogenesis of neuroAIDS through the production of reactive nitrogen species in microglial cells. PMID:26066624

  5. Muscle-derived stem\\/progenitor cell dysfunction limits healthspan and lifespan in a murine progeria model

    Microsoft Academic Search

    Mitra Lavasani; Andria R. Robinson; Aiping Lu; Minjung Song; Joseph M. Feduska; Bahar Ahani; Jeremy S. Tilstra; Chelsea H. Feldman; Paul D. Robbins; Laura J. Niedernhofer; Johnny Huard

    2012-01-01

    With ageing, there is a loss of adult stem cell function. However, there is no direct evidence that this has a causal role in ageing-related decline. We tested this using muscle-derived stem\\/progenitor cells (MDSPCs) in a murine progeria model. Here we show that MDSPCs from old and progeroid mice are defective in proliferation and multilineage differentiation. Intraperitoneal administration of MDSPCs,

  6. Role of virulence factors, cell components and adhesion in Helicobacter pylori-mediated iNOS induction in murine macrophages

    Microsoft Academic Search

    Ilka A. Assmann; Georg A. Enders; Jürgen Püls; Gabriele Rieder; Rainer Haas; Rudolf A. Hatz

    2001-01-01

    To investigate the mechanisms involved in Helicobacter pylori-mediated inducible nitric oxide synthase (iNOS) upregulation in mononuclear cells we cocultivated human THP-1 acute monocytic leukemia cells and murine J774A.1 professional macrophages with different H. pylori wild-type strains and mutants. We have shown that H. pylori-mediated iNOS induction in J774A.1 is independent of established virulence factors but dependent on direct interaction between

  7. Neddylation plays an important role in the regulation of murine and human dendritic cell function

    PubMed Central

    Mathewson, Nathan; Toubai, Tomomi; Kapeles, Steven; Sun, Yaping; Oravecz-Wilson, Katherine; Tamaki, Hiroya; Wang, Ying; Hou, Guoqing; Sun, Yi

    2013-01-01

    Posttranslational protein modifications (PTMs) are necessary for cells to function properly. The role of PTMs in regulating immune responses, specifically those mediated by dendritic cells (DCs), which are critical for both innate and adaptive immunity, is not well understood. Utilizing multiple but complementary approaches, we determined the role of an important but less understood type of PTM, namely, neddylation, in regulating DC functions. Inhibition of neddylation suppressed the release of proinflammatory cytokines by DCs in response to Toll-like receptor, nucleotide oligomerization domain–like receptor, and noninfectious CD40L stimulation. These effects were more profound than those mediated by the proteasome inhibitor bortezomib or a commonly used antiinflammatory agent, dexamethasone. Targeting neddylation also suppressed the ability of DCs to stimulate murine allogeneic T cells in vitro and in vivo and human allogeneic T-cell responses in vitro. Mechanistic studies demonstrated that inhibition of neddylation reduced both canonical and noncanonical nuclear factor-?B (NF-?B) activity. Neddylation inhibition prevented the degradation of inhibitor-?B and thus reduced the translocation and activation of NF-?B, but without perturbation of the mitogen-activated protein kinase/extracellular signal-regulated kinase pathway. Thus, blocking neddylation could be a novel strategy for mitigating immune-mediated disease processes. PMID:23863900

  8. Radiosensitizing and toxic effects of RSU-1069 on hypoxic cells in a murine tumor

    SciTech Connect

    Chaplin, D.J.; Durand, R.E.; Stratford, I.J.; Jenkins, T.C.

    1986-07-01

    RSU-1069 is one of a group of compounds of particular interest in radiobiology, since it combines the nitroimidazole ring with a side chain bearing a monofunctional alkylating agent. This compound has been shown to be a potent radiosensitizer both in vitro and in vivo. Furthermore, it has recently been shown to be an effective hypoxic cell cytotoxin in vitro. Our studies have been carried out using the SCCVII squamous carcinoma implanted subcutaneously in C/sub 3/H mice, using a technique we recently developed which facilitates isolation of tumor cell subpopulations from known locations relative to the tumor blood supply. The response of the separated tumor subpopulations was assessed using a soft agar clonogenic assay. For radiosensitization studies, RSU-1069 was administered i.p. at 0.5 mumol/g 20 min before irradiation and the tumors excised 20 min after irradiation. For toxicity studies, tumors were excised 16-18 hr after RSU-1069 administration. The results obtained to date clearly demonstrate that RSU-1069 is an efficient hypoxic cell radiosensitizer and cytotoxin in this murine tumor and has little effect on well perfused (i.e., oxic) cells.

  9. Involvement of PIKE in icariin induced cardiomyocyte differentiation from murine embryonic stem cells.

    PubMed

    Zhou, Limin; Zheng, Bei; Tang, Leilei; Huang, Yujie; Zhu, Danyan

    2014-03-01

    Icariin (ICA) has demonstrated to induce cardiomyocyte differentiation from murine embryonic stem (ES) cells in vitro, however, the mechanisms have not been fully elucidated. In the present study, we investigated whether phosphatidylinositol 3-kinase enhancer (PIKE) was involved in ICA induced cardiomyocyte differentiation of ES cells. Small interfering RNA (siRNA) of PIKE was applied to investigate the role of PIKE in ICA induced cardiomyocyte differentiation. The cardiomyocytes derived from ES cells were verified using immunofluorescence. The expressions of Troponin T, PIKE, phosphatidylinositol 3-kinase (PI3K), and nuclear factor-kappaB (NF-kappaB) were detected by western blot. The change of reactive oxygen species (ROS) generation was estimated using the fluorescent dye 2', 7' - dichlorodihydrofluorescein diacetate. The results showed that PIKE expression increased during cardiomyocyte differentiation. ICA markedly enhanced PIKE and PI3K expression in a time-dependent manner. Knockdown of PIKE by siRNAs blocked the differentiation of ES cells into cardiomyocytes expressing alpha-actinin for cardiac sarcomeric structures. Moreover, reduced ROS generation and NF-kappaB nuclear translocation were responsible for the inhibitory effect of si-PIKE. In conclusion, PIKE was involved in ICA induced cardiomyocyte differentiation, and ROS generation and NF-kappaB nuclear translocation were associated with PIKE activation. PMID:24716409

  10. Mechanism of leukemogenesis induced by mink cell focus-forming murine leukemia viruses.

    PubMed Central

    Li, J P; Baltimore, D

    1991-01-01

    The Friend or Moloney mink cell focus-forming (MCF) virus encodes a recombinant-type envelope glycoprotein, gp70, that is closely related to the membrane glycoprotein, gp55, of Friend spleen focus-forming virus (SFFV). We have shown previously that gp55 has the ability to activate cell growth by binding to the cellular receptor for erythropoietin. Here we show that gp70 encoded by either the Friend or Moloney MCF virus also binds to the erythropoietin receptor and that coexpression of the receptor and gp70 in an interleukin-3 (IL-3)-dependent cell line can activate IL-3-independent growth. Furthermore, when the cDNA for the human IL-2 receptor beta chain, which is related by sequence to the erythropoietin receptor, was introduced into this cell line, it became growth factor independent after infection either with SFFV or with one of the two MCF viruses but not with an ecotropic virus. Based on these observations, we propose a mechanism for the early stage of leukemogenesis induced by the MCF-type murine leukemia viruses. Images PMID:1850020

  11. Treadmill exercise induces murine cardiac allograft survival and generates regulatory T cell

    PubMed Central

    Uchiyama, Masateru; Jin, Xiangyuan; Yin, Enzhi; Shimokawa, Tomoki; Niimi, Masanori

    2015-01-01

    Exercise therapy has been associated with improvement in functional capacity and quality of life. The role of exercise therapy in heart transplant recipients is of great interest for the transplant society, although concerning the effect of exercise therapy, there is little knowledge at present. We analyzed the effects of exercise on alloimmune responses in murine cardiac allograft transplantation. CBA mice (H2k) underwent transplantation of C57Bl/6 (H2b) hearts and exercised on a treadmill. Untreated CBA recipients rejected C57Bl/6 cardiac grafts acutely (median survival time [MST], 7 days). CBA recipients treated with treadmill for 1 week after transplantation, and for 1 week both before and after transplantation prolonged allograft survivals (MSTs, 35 and 18 days, respectively). However, treadmill exercise recipients for 1 week before transplantation were not effective to allograft survival (MST, 8 days). Adoptive transfer of whole splenocytes and CD4+ cells from treadmill exercise recipients significantly prolonged allograft survival in naive secondary recipients (MSTs, 30 and 52 days, respectively), suggesting that regulatory cells was generated after treadmill exercise. Moreover, flow cytometry studies showed that CD4+CD25+Foxp3+ cell population increased in treadmill exercise recipients. Therefore, postoperative but not pre-operative exercise could induce prolongation of survival of fully allogeneic cardiac allografts and generate CD4+CD25+Foxp3+ regulatory T cells. PMID:25406375

  12. Induction of murine T-helper-cell responses to the filarial nematode Brugia malayi.

    PubMed Central

    Pearlman, E; Hazlett, F E; Boom, W H; Kazura, J W

    1993-01-01

    The purpose of this study was to examine the murine T-helper-cell (Th) cytokine response to the human filarial parasite Brugia malayi. In the first 14 days following intraperitoneal inoculation of live microfilariae into BALB/c mice, filarial antigen-driven splenic lymphoid cells produced gamma interferon (IFN-gamma) and little or no interleukin-5 (IL-5). After this time, IL-5 production increased (to 10 to 12 ng per 5 x 10(6) cells) coincident with a marked diminution in IFN-gamma generation. A single subcutaneous immunization with soluble microfilarial antigens also induced an IFN-gamma but no IL-5 response, whereas immunization three times elicited a predominant Th2-like reaction characterized by IL-4 and IL-5 production by CD4+ lymph node lymphocytes and a 10-fold increase in serum immunoglobulin E. The importance of IL-10 in establishing the balance between parasite-specific Th1 and Th2 responses was demonstrated by the ability of neutralizing monoclonal antibody to this cytokine to increase IFN-gamma production by splenic and lymph node cells from mice chronically exposed to live microfilariae or immunized multiple times with soluble filarial antigens. PMID:8094378

  13. Club cell secretory protein improves survival in a murine obliterative bronchiolitis model.

    PubMed

    Wendt, Christine; Tram, Kevin; Price, Andrew; England, Kristen; Stiehm, Andrew; Panoskaltsis-Mortari, Angela

    2013-11-01

    Club cell secretory protein (CCSP) is an indirect phospholipase A2 inhibitor with some immunosuppressive and antiproliferative properties that is expressed in bronchiolar Club cells. In our murine bone marrow transplant (BMT) model of obliterative bronchiolitis (OB), CCSP is diminished; however, its role is unknown. To determine the role of CCSP, B6 wild-type (WT) or CCSP-deficient (CCSP(-/-)) mice were lethally conditioned and given allogeneic bone marrow with a sublethal dose of allogeneic splenic T cells to induce OB. We found that CCSP(-/-) mice demonstrated a higher mortality following BMT-induced OB compared with WT mice. Mice were analyzed 60 days post-BMT for protein expression, pulmonary function, and histology. CCSP levels were reduced in WT mice with BMT-induced OB, and lower levels correlated to decreased lung compliance. CCSP(-/-) had a higher degree of injury and fibrosis as measured by hydroxy proline, along with an increased lung resistance and the inflammatory markers, leukotriene B4 and CXCL1. Replacement with recombinant intravenous CCSP partially reversed the weight loss and improved survival in the CCSP(-/-) mice. In addition, CCSP replacement improved histology and decreased inflammatory cells and markers. These findings indicate that CCSP has a regulatory role in OB and may have potential as a preventive therapy. PMID:23997179

  14. Functional Th1 Cells Are Required for Surgical Adhesion Formation in a Murine Model1

    PubMed Central

    Zheng, Xin-Xiao; Stucchi, Arthur F.; Kuchroo, Vijay K.; Strom, Terry B.; Glimcher, Laurie H.; Cruikshank, William W.

    2013-01-01

    Tissue trauma in the peritoneal and pelvic cavities following surgery or bacterial infection results in adhesions that are a debilitating cause of intestinal obstruction, chronic pelvic pain, and infertility in women. We recently demonstrated that CD4+ ?? T cells are essential for development of this process. Using a murine model of experimental adhesion formation, we now demonstrate that adhesion formation is characterized by the selective recruitment of Tim-3+, CCR5+, CXCR3+, IFN-?+ cells, indicating the presence of a Th1 phenotype. We further demonstrate that adhesion formation is critically dependent on the function of Th1 cells because mice genetically deficient for IFN-?, T-bet, or treated with Abs to the Th1-selective chemoattractant IL-16 show significantly less adhesion formation than wild-type mice. In addition, disrupting the interaction of the Th1-specific regulatory molecule Tim-3, with its ligand, significantly exacerbates adhesion formation. This enhanced response is associated with increases in the level of neutrophil-attracting chemokines KC and MIP-2, known to play a role in adhesiogenesis. These data demonstrate that the CD4+ T cells orchestrating adhesion formation are of the Th1 phenotype and delineate the central role of T-bet, Tim-3, IFN-?, and IL-16 in mediating this pathogenic tissue response. PMID:18453619

  15. Inhibition of NF-kappaB/Rel induces apoptosis of murine B cells.

    PubMed Central

    Wu, M; Lee, H; Bellas, R E; Schauer, S L; Arsura, M; Katz, D; FitzGerald, M J; Rothstein, T L; Sherr, D H; Sonenshein, G E

    1996-01-01

    Apoptosis of the WEHI 231 immature B cell lymphoma line following membrane interaction with an antibody against the surface IgM chains (anti-IgM) is preceded by dramatic changes in Nuclear Factor-kappaB (NF-kappaB)/ Rel binding activities. An early transient increase in NF-kappaB/Rel binding is followed by a significant decrease in intensity below basal levels. Here we have explored the role of these changes in Rel-related factors in B cell apoptosis. Treatment of WEH1 231 cells with N-tosyl-L-phenylalanine chloromethyl ketone (TPCK), a protease inhibitor which prevents degradation of the inhibitor of NF-kappaB (IkappaB)-alpha, or with low doses of pyrrolidinedithiocarbamate (PDTC) selectively inhibited NF-kappaB/Rel factor binding and induced apoptosis. Bcl-XL expression protected WEHI 231 cells from apoptosis induced by these agents. Microinjection of WEHI 231 cells with either IkappaB-alpha-GST protein or a c-Rel affinity-purified antibody induced apoptosis. Ectopic c-Rel expression ablated apoptosis induced by TPCK or anti-IgM. Treatment of BALENLM 17 and A20 B lymphoma cells or normal murine splenic B lymphocytes with either TPCK or PDTC also resulted in apoptosis. These findings indicate that the drop in NF-kappaB/Rel binding following anti-IgM treatment activates apoptosis of WEHI 231 cells; furthermore, they implicate the NF-kappaB/Rel family in control of apoptosis of normal and transformed B cells. Images PMID:8887559

  16. Mitochondrial biogenesis and energy production in differentiating murine stem cells: a functional metabolic study.

    PubMed

    Han, Sungwon; Auger, Christopher; Thomas, Sean C; Beites, Crestina L; Appanna, Vasu D

    2014-02-01

    The significance of metabolic networks in guiding the fate of the stem cell differentiation is only beginning to emerge. Oxidative metabolism has been suggested to play a major role during this process. Therefore, it is critical to understand the underlying mechanisms of metabolic alterations occurring in stem cells to manipulate the ultimate outcome of these pluripotent cells. Here, using P19 murine embryonal carcinoma cells as a model system, the role of mitochondrial biogenesis and the modulation of metabolic networks during dimethyl sulfoxide (DMSO)-induced differentiation are revealed. Blue native polyacrylamide gel electrophoresis (BN-PAGE) technology aided in profiling key enzymes, such as hexokinase (HK) [EC 2.7.1.1], glucose-6-phosphate isomerase (GPI) [EC 5.3.1.9], pyruvate kinase (PK) [EC 2.7.1.40], Complex I [EC 1.6.5.3], and Complex IV [EC 1.9.3.1], that are involved in the energy budget of the differentiated cells. Mitochondrial adenosine triphosphate (ATP) production was shown to be increased in DMSO-treated cells upon exposure to the tricarboxylic acid (TCA) cycle substrates, such as succinate and malate. The increased mitochondrial activity and biogenesis were further confirmed by immunofluorescence microscopy. Collectively, the results indicate that oxidative energy metabolism and mitochondrial biogenesis were sharply upregulated in DMSO-differentiated P19 cells. This functional metabolic and proteomic study provides further evidence that modulation of mitochondrial energy metabolism is a pivotal component of the cellular differentiation process and may dictate the final destiny of stem cells. PMID:24350892

  17. Curcumin Suppresses the Production of Pro-inflammatory Cytokine Interleukin-18 in Lipopolysaccharide Stimulated Murine Macrophage-Like Cells.

    PubMed

    Yadav, Renu; Jee, Babban; Awasthi, Sudhir Kumar

    2015-01-01

    Curcumin is a major bioactive compound of turmeric that exerts its anti-inflammatory effects by suppressing the many pro-inflammatory cytokines and chemokines in a number of cell types and pathologic conditions. Interleukin-18 (IL-18) is a novel pro-inflammatory cytokine which plays an important role not only in generating Th1 responses but also in inducing severe inflammatory reactions. As curcumin induced inhibition of IL-18 production in keratinocytes and mice is well known, effect of curcumin on IL-18 release in macrophages remains unknown. Hence, this present study has been designed to evaluate the effect of curcumin on IL-18 production and necrotic cell death in murine macrophages-like cells treated with or without lipopolysaccharide (LPS). The IL-18 secretion in cell culture supernatants was assayed by enzyme-linked immunosorbent assay and cytotoxicity was determined by lactate dehydrogenase release assay. Our results demonstrate that curcumin significantly inhibited the production of pro-inflammatory cytokine IL-18 in E.coli LPS stimulated murine macrophage-like cells RAW264.7 in a concentration-dependent manner. Interestingly, curcumin had no cytotoxic effect on murine macrophage-like cells. Our findings suggest that curcumin may be used as a potential therapeutic agent for the treatment of inflammatory diseases. PMID:25646051

  18. Menstrual Blood-derived Cells Confer Human Dystrophin Expression in the Murine Model of Duchenne Muscular Dystrophy via Cell Fusion and Myogenic Transdifferentiation

    PubMed Central

    Cui, Chang-Hao; Uyama, Taro; Miyado, Kenji; Terai, Masanori; Kyo, Satoru; Kiyono, Tohru

    2007-01-01

    Duchenne muscular dystrophy (DMD), the most common lethal genetic disorder in children, is an X-linked recessive muscle disease characterized by the absence of dystrophin at the sarcolemma of muscle fibers. We examined a putative endometrial progenitor obtained from endometrial tissue samples to determine whether these cells repair muscular degeneration in a murine mdx model of DMD. Implanted cells conferred human dystrophin in degenerated muscle of immunodeficient mdx mice. We then examined menstrual blood–derived cells to determine whether primarily cultured nontransformed cells also repair dystrophied muscle. In vivo transfer of menstrual blood–derived cells into dystrophic muscles of immunodeficient mdx mice restored sarcolemmal expression of dystrophin. Labeling of implanted cells with enhanced green fluorescent protein and differential staining of human and murine nuclei suggest that human dystrophin expression is due to cell fusion between host myocytes and implanted cells. In vitro analysis revealed that endometrial progenitor cells and menstrual blood–derived cells can efficiently transdifferentiate into myoblasts/myocytes, fuse to C2C12 murine myoblasts by in vitro coculturing, and start to express dystrophin after fusion. These results demonstrate that the endometrial progenitor cells and menstrual blood–derived cells can transfer dystrophin into dystrophied myocytes through cell fusion and transdifferentiation in vitro and in vivo. PMID:17314403

  19. Reproducible generation of autonomous malignant sublines from non-tumorogenic murine interleukin 3-dependent mast cell lines.

    PubMed

    Hültner, L; Moeller, J; Dörmer, P

    1986-12-01

    Murine interleukin 3 (IL-3)-dependent permanent mast cell lines derived from normal mouse bone marrow were established using pokeweed mitogen-stimulated spleen cell conditioned medium (SCM) as a source of IL-3. When propagated continuously in media containing a high concentration of IL-3 (20% SCM or 20 U/ml murine recombinant IL-3 (rIL-3], all the cell lines remained strictly factor-dependent in vitro and non-tumorogenic in vivo. However, we were able to reproducibly generate autonomous sublines from cultures supplemented with low amounts of IL-3 (1% SCM or 2 U/ml rIL-3). Abrogation of exogeneous growth factor dependency was always associated with neoplastic transformation. In newly generated autonomous sublines an autocrine mechanism of growth regulation was evident in vitro. PMID:3492230

  20. Characterization of thyroid cancer cell lines in murine orthotopic and intracardiac metastasis models.

    PubMed

    Morrison, Jennifer A; Pike, Laura A; Lund, Greg; Zhou, Qiong; Kessler, Brittelle E; Bauerle, Kevin T; Sams, Sharon B; Haugen, Bryan R; Schweppe, Rebecca E

    2015-06-01

    Thyroid cancer incidence has been increasing over time, and it is estimated that ?1950 advanced thyroid cancer patients will die of their disease in 2015. To combat this disease, an enhanced understanding of thyroid cancer development and progression as well as the development of efficacious, targeted therapies are needed. In vitro and in vivo studies utilizing thyroid cancer cell lines and animal models are critically important to these research efforts. In this report, we detail our studies with a panel of authenticated human anaplastic and papillary thyroid cancer (ATC and PTC) cell lines engineered to express firefly luciferase in two in vivo murine cancer models-an orthotopic thyroid cancer model as well as an intracardiac injection metastasis model. In these models, primary tumor growth in the orthotopic model and the establishment and growth of metastases in the intracardiac injection model are followed in vivo using an IVIS imaging system. In the orthotopic model, the ATC cell lines 8505C and T238 and the PTC cell lines K1/GLAG-66 and BCPAP had take rates >90 % with final tumor volumes ranging 84-214 mm(3) over 4-5 weeks. In the intracardiac model, metastasis establishment was successful in the ATC cell lines HTh74, HTh7, 8505C, THJ-16T, and Cal62 with take rates ?70 %. Only one of the PTC cell lines tested (BCPAP) was successful in the intracardiac model with a take rate of 30 %. These data will be beneficial to inform the choice of cell line and model system for the design of future thyroid cancer studies. PMID:25800363

  1. Inhibition of proliferation, migration and invasiveness of endothelial murine cells culture induced by resveratrol

    PubMed Central

    Zdanowski, Robert; Lewicki, S?awomir; Le?niak, Monika; Suska, Milena; Wojdat, El?bieta; Skopi?ska-Ró?ewska, Ewa; Skopi?ski, Piotr

    2014-01-01

    Angiogenesis is a multi-stage process of new vessel development which involves migration, proliferation and differentiation of endothelial cells. Pathological angiogenesis plays a crucial role in the pathomechanism of various ischemic, malignant and inflammatory disorders. Among eye diseases, macular degeneration (AMD) and proliferative diabetic retinopathy are a major public health issue as the most common causes of blindness. Since angiogenesis plays a crucial role in these conditions, there has been an increased interest in evaluating anti-angiogenic agents in their treatment. The polyphenol resveratrol found in the skin of red grapes, red wine, peanuts and other natural sources, controls proliferation of the cells, induces differentiation and induces apoptosis in various malignant cell lines. Modulation of angiogenesis by this compound has been considered as a very exciting topic and subject of further investigations. The aim of our study was in vitro assessment of resveratrol's influence on proliferation, migration and invasion of an immortalized murine endothelial cell line from peripheral lymph node HEC clone a10. Resveratrol was shown to inhibit the proliferation of the endothelial cells in MTT (at 1, 10 and 50 µM) and AlamarBlue (at 50 µM) assays, and at a concentration of 50 µM significantly inhibited migration of endothelial cells. A concentration-dependent decrease in invasion potential of endothelial cells incubated with resveratrol 10 µM and 50 µM was detected. These promising in vitro results might encourage investigators to test efficacy and safety of resveratrol more extensively in the clinical practice, as a natural and safe anti-angiogenic agent.

  2. Generation of Ugt1-Deficient Murine Liver Cell Lines Using TALEN Technology

    PubMed Central

    Porro, Fabiola; Bockor, Luka; De Caneva, Alessia; Bortolussi, Giulia; Muro, Andrés F.

    2014-01-01

    The Crigler-Najjar Syndrome Type I (CNSI) is a rare genetic disorder caused by mutations in the Ugt1a1 gene. It is characterized by unconjugated hyperbilirubinemia that may result in severe neurologic damage and death if untreated. To date, liver transplantation is the only curative treatment. With the aim of generating mutant cell lines of the Ugt1 gene, we utilized the TALEN technology to introduce site-specific mutations in Ugt1 exon 4. We report a fast and efficient method to perform gene knockout in tissue culture cells, based on the use of TALEN pairs targeting restriction enzyme (RE) sites in the region of interest. This strategy overcame the presence of allele-specific single nucleotide polymorphisms (SNPs) and pseudogenes, conditions that limit INDELs' detection by Surveyor. We obtained liver-derived murine N-Muli cell clones having INDELs with efficiency close to 40%, depending on the TALEN pair and RE target site. Sequencing of the target locus and WB analysis of the isolated cell clones showed a high proportion of biallelic mutations in cells treated with the most efficient TALEN pair. Ugt glucuronidation activity was reduced basal levels in the biallelic mutant clones. These mutant liver-derived cell lines could be a very useful tool to study biochemical aspects of Ugt1 enzyme activity in a more natural context, such as substrate specificity, requirement of specific co-factors, the study of inhibitors and other pharmacological aspects, and to correlate enzyme activity to the presence of specific mutations in the gene, by adding back to the mutant cell clones specific variants of the Ugt1 gene. In addition, since genome editing has recently emerged as a potential therapeutic approach to cure genetic diseases, the definition of the most efficient TALEN pair could be an important step towards setting up a platform to perform genome editing in CNSI. PMID:25118822

  3. Characterization of a tachykinin peptide NK2 receptor transfected into murine fibroblast B82 cells.

    PubMed Central

    van Giersbergen, P L; Shatzer, S A; Henderson, A K; Lai, J; Nakanishi, S; Yamamura, H I; Buck, S H

    1991-01-01

    Membranes isolated from a murine fibroblast B82 cell line (SKLKB82#3) transfected with the bovine stomach cDNA pSKR56S exhibited binding of [His(125I)1]neurokinin A (125I-NKA) to a single population of sites with a Bmax of 147 fmol/mg of protein and a Kd of 0.59 nM. Control cell lines had little or no specific binding. The ligand binding in SKLKB82#3 cells was reversible and was inhibited by peptides in the potency rank of neuropeptide gamma greater than neuropeptide K greater than neurokinin A greater than [10-norleucine]neurokinin A-(4-10) greater than substance P much greater than senktide (succinyl-Asp-Phe-MePhe-Gly-Leu-Met-NH2). Specific binding was enhanced by Mn2+, Mg2+, and Ca2+ and was inhibited by guanine nucleotide analogues. Thus, SKLKB82#3 cells have been transfected with NK2 receptors that have become associated with an endogenous guanine nucleotide-binding protein. In comparison with membranes from the hamster urinary bladder, a tissue enriched in NK2 receptors, NK2 receptor antagonists displayed markedly different potencies, either more or less potent, in inhibiting specific binding in membranes of the transfected cells. Furthermore, inhibition of 125I-NKA binding by nucleotide analogues was markedly different in SKLKB82#3 cells compared with hamster bladder tissue. The different binding profile in the cells is not due to an artefact introduced during cDNA transfection because a similar profile was also observed in bovine stomach membranes. These results may indicate the existence of two distinct NK2 receptors. PMID:1848006

  4. Assessment of immunosuppressive activity of human mesenchymal stem cells using murine antigen specific CD4 and CD8 T cells in vitro

    PubMed Central

    2013-01-01

    Introduction Mesenchymal stem cells (MSCs) have immunosuppressive activity. They do not induce allospecific T cell responses, making them promising tools for reducing the severity of graft versus host disease (GVHD) as well as treating various immune diseases. Currently, there is a need in the MSC field to develop a robust in vitro bioassay which can characterize the immunosuppressive function of MSCs. Methods Murine clonal CD4 and CD8 T cells were stimulated with cognate peptide antigen and antigen presenting cells (APCs) in the absence or presence of human MSCs, different aspects of T cell activation were monitored and analyzed using flow cytometery, real time RT-PCR and cytokine measurement. Results Human MSCs (hMSCs) can alter multiple aspects of murine T cell activation induced by stimulation with specific antigen, including: reduced proliferation, inhibited or stimulated cell surface marker expression (CD25, CD69, CD44 and CD62L), inhibited mRNA expression of transcription factors (T-bet and GATA-3) and decreased cytokine expression (interferon-gamma, interleukin-10). Disappearance of activation-induced cluster formation and decreased apoptosis of CD8 T cells were also observed. Moreover, the effects are specific to MSCs; incubating the T cells with non-MSC control cell lines had no effect on T cell proliferation and activation. Conclusions Clonal murine T cells can be used to measure, characterize, and quantify the in vitro immunosuppressive activity of human MSCs, representing a promising approach to improve bioassays for immunosuppression. PMID:24406271

  5. Storage of murine red blood cells enhances alloantibody responses to an erythroid-specific model antigen

    PubMed Central

    Hendrickson, Jeanne E.; Hod, Eldad A.; Spitalnik, Steven L.; Hillyer, Christopher D.; Zimring, James C.

    2013-01-01

    BACKGROUND Red blood cell (RBC) alloimmunization can be a serious complication of blood transfusion, but factors influencing the development of alloantibodies are only partially understood. Within FDA-approved time limits, RBCs are generally transfused without regard to length of storage. However, recent studies have raised concerns that RBCs stored for more than 14 days have altered biologic properties that may affect medical outcomes. To test the hypothesis that storage time alters RBC immunogenicity, we utilized a murine model of RBC storage and alloimmunization. STUDY DESIGN AND METHODS Blood from transgenic HOD donor mice, which express a model antigen (hen egg lysozyme [HEL]) specifically on RBCs, was filter leukoreduced and stored for 14 days under conditions similar to those used for human RBCs. Fresh or 14-day-stored RBCs were transfused into wild-type recipients. The stability of the HOD antigen and post-transfusion RBC survival were analyzed by flow cytometry. RBC alloimmunization was monitored by measuring circulating anti-HEL immunoglobulin levels. RESULTS Transfusion of 14-day-stored, leukoreduced HOD RBCs resulted in 10- to 100-fold higher levels of anti-HEL alloantibodies as detected by enzyme-linked immunosorbent assay than transfusion of freshly collected, leukoreduced RBCs. RBC expression of the HOD antigen was stable during storage. CONCLUSIONS These findings demonstrate that HOD murine RBCs become more immunogenic with storage and generate the rationale for clinical trials to test if the same phenomenon is observed in humans. Length of storage of RBCs may represent a previously unappreciated variable in whether or not a transfusion recipient becomes alloimmunized. PMID:19906034

  6. TGF-?-Dependent Dendritic Cell Chemokinesis in Murine Models of Airway Disease.

    PubMed

    Hashimoto, Mitsuo; Yanagisawa, Haruhiko; Minagawa, Shunsuke; Sen, Debasish; Ma, Royce; Murray, Lynne A; Tsui, Ping; Lou, Jianlong; Marks, James D; Baron, Jody L; Krummel, Matthew F; Nishimura, Stephen L

    2015-08-01

    Small airway chronic inflammation is a major pathologic feature of chronic obstructive pulmonary disease (COPD) and is refractory to current treatments. Dendritic cells (DCs) accumulate around small airways in COPD. DCs are critical mediators of Ag surveillance and Ag presentation and amplify adaptive immune responses. How DCs accumulate around airways remains largely unknown. We use 2-photon DC imaging of living murine lung sections to directly visualize the dynamic movement of living DCs around airways in response to either soluble mediators (IL-1?) or environmental stimuli (cigarette smoke or TLR3 ligands) implicated in COPD pathogenesis. We find that DCs accumulate around murine airways primarily by increasing velocity (chemokinesis) rather than directional migration (chemotaxis) in response to all three stimuli. DC accumulation maximally occurs in a specific zone located 26-50 ?m from small airways, which overlaps with zones of maximal DC velocity. Our data suggest that increased accumulation of DCs around airways results from increased numbers of highly chemokinetic DCs entering the lung from the circulation with balanced rates of immigration and emigration. Increases in DC accumulation and chemokinesis are partially dependent on ccr6, a crucial DC chemokine receptor, and fibroblast expression of the integrin ?v?8, a critical activator of TGF-?. ?v?8-Mediated TGF-? activation is known to enhance IL-1?-dependent fibroblast expression of the only known endogenous ccr6 chemokine ligand, ccl20. Taken together, these data suggest a mechanism by which ?v?8, ccl20, and ccr6 interact to lead to DC accumulation around airways in response to COPD-relevant stimuli. PMID:26109638

  7. Association of murine lupus and thymic full-length endogenous retroviral expression maps to a bone marrow stem cell

    SciTech Connect

    Krieg, A.M.; Gourley, M.F.; Steinberg, A.D. (National Institutes of Health, Bethesda, MD (USA))

    1991-05-01

    Recent studies of thymic gene expression in murine lupus have demonstrated 8.4-kb (full-length size) modified polytropic (Mpmv) endogenous retroviral RNA. In contrast, normal control mouse strains do not produce detectable amounts of such RNA in their thymuses. Prior studies have attributed a defect in experimental tolerance in murine lupus to a bone marrow stem cell rather than to the thymic epithelium; in contrast, infectious retroviral expression has been associated with the thymic epithelium, rather than with the bone marrow stem cell. The present study was designed to determine whether the abnormal Mpmv expression associated with murine lupus mapped to thymic epithelium or to a marrow precursor. Lethally irradiated control and lupus-prone mice were reconstituted with T cell depleted bone marrow; one month later their thymuses were studied for endogenous retroviral RNA and protein expression. Recipients of bone marrow from nonautoimmune donors expressed neither 8.4-kb Mpmv RNA nor surface MCF gp70 in their thymuses. In contrast, recipients of bone marrow from autoimmune NZB or BXSB donors expressed thymic 8.4-kb Mpmv RNA and mink cell focus-forming gp70. These studies demonstrate that lupus-associated 8.4-kb Mpmv endogenous retroviral expression is determined by bone marrow stem cells.

  8. Interactions of Francisella tularensis with Alveolar Type II Epithelial Cells and the Murine Respiratory Epithelium

    PubMed Central

    Faron, Matthew; Fletcher, Joshua R.; Rasmussen, Jed A.; Apicella, Michael A.; Jones, Bradley D.

    2015-01-01

    Francisella tularensis is classified as a Tier 1 select agent by the CDC due to its low infectious dose and the possibility that the organism can be used as a bioweapon. The low dose of infection suggests that Francisella is unusually efficient at evading host defenses. Although ~50 cfu are necessary to cause human respiratory infection, the early interactions of virulent Francisella with the lung environment are not well understood. To provide additional insights into these interactions during early Francisella infection of mice, we performed TEM analysis on mouse lungs infected with F. tularensis strains Schu S4, LVS and the O-antigen mutant Schu S4 waaY::TrgTn. For all three strains, the majority of the bacteria that we could detect were observed within alveolar type II epithelial cells at 16 hours post infection. Although there were no detectable differences in the amount of bacteria within an infected cell between the three strains, there was a significant increase in the amount of cellular debris observed in the air spaces of the lungs in the Schu S4 waaY::TrgTn mutant compared to either the Schu S4 or LVS strain. We also studied the interactions of Francisella strains with human AT-II cells in vitro by characterizing the ability of these three strains to invade and replicate within these cells. Gentamicin assay and confocal microscopy both confirmed that F. tularensis Schu S4 replicated robustly within these cells while F. tularensis LVS displayed significantly lower levels of growth over 24 hours, although the strain was able to enter these cells at about the same level as Schu S4 (1 organism per cell), as determined by confocal imaging. The Schu S4 waaY::TrgTn mutant that we have previously described as attenuated for growth in macrophages and mouse virulence displayed interesting properties as well. This mutant induced significant airway inflammation (cell debris) and had an attenuated growth phenotype in the human AT-II cells. These data extend our understanding of early Francisella infection by demonstrating that Francisella enter significant numbers of AT-II cells within the lung and that the capsule and LPS of wild type Schu S4 helps prevent murine lung damage during infection. Furthermore, our data identified that human AT-II cells allow growth of Schu S4, but these same cells supported poor growth of the attenuated LVS strain in vitro. Collectively, these data further our understanding of the role of AT-II cells in Francisella infections. PMID:26010977

  9. Human muscle–derived stem/progenitor cells promote functional murine peripheral nerve regeneration

    PubMed Central

    Lavasani, Mitra; Thompson, Seth D.; Pollett, Jonathan B.; Usas, Arvydas; Lu, Aiping; Stolz, Donna B.; Clark, Katherine A.; Sun, Bin; Péault, Bruno; Huard, Johnny

    2014-01-01

    Peripheral nerve injuries and neuropathies lead to profound functional deficits. Here, we have demonstrated that muscle-derived stem/progenitor cells (MDSPCs) isolated from adult human skeletal muscle (hMDSPCs) can adopt neuronal and glial phenotypes in vitro and ameliorate a critical-sized sciatic nerve injury and its associated defects in a murine model. Transplanted hMDSPCs surrounded the axonal growth cone, while hMDSPCs infiltrating the regenerating nerve differentiated into myelinating Schwann cells. Engraftment of hMDSPCs into the area of the damaged nerve promoted axonal regeneration, which led to functional recovery as measured by sustained gait improvement. Furthermore, no adverse effects were observed in these animals up to 18 months after transplantation. Following hMDSPC therapy, gastrocnemius muscles from mice exhibited substantially less muscle atrophy, an increase in muscle mass after denervation, and reorganization of motor endplates at the postsynaptic sites compared with those from PBS-treated mice. Evaluation of nerve defects in animals transplanted with vehicle-only or myoblast-like cells did not reveal histological or functional recovery. These data demonstrate the efficacy of hMDSPC-based therapy for peripheral nerve injury and suggest that hMDSPC transplantation has potential to be translated for use in human neuropathies. PMID:24642464

  10. Bromelain Inhibits Allergic Sensitization and Murine Asthma via Modulation of Dendritic Cells

    PubMed Central

    Secor, Eric R.; Szczepanek, Steven M.; Castater, Christine A.; Adami, Alexander J.; Matson, Adam P.; Rafti, Ektor T.; McNamara, Jeffrey T.; Schramm, Craig M.; Thrall, Roger S.; Silbart, Lawrence K.

    2013-01-01

    The incidence of atopic conditions has increased in industrialized countries. Persisting symptoms and concern for drug side-effects lead patients toward adjunctive treatments such as phytotherapy. Previously, we have shown that Bromelain (sBr), a mixture of cysteine proteases from pineapple, Ananas comosus, inhibits ovalbumin (OVA)-induced murine model of allergic airway disease (AAD). However, sBr's effect on development of AAD when treatment is administered throughout OVA-alum sensitization was unknown and is the aim of the present study. C57BL/6J mice were sensitized with OVA/alum and challenged with 7 days OVA aerosol. sBr 6?mg/kg/0.5?ml or PBS vehicle were administered throughout sensitization. Lung, bronchoalveolar lavage (BAL), spleen, and lymph nodes were processed for flow cytometry and OVA-specific IgE was determined via ELISA. sBr treatment throughout OVA-alum sensitization significantly reduced the development of AAD (BAL eosinophils and lymphocytes). OVA-specific IgE and OVA TET+ cells were decreased. sBr reduced CD11c+ dendritic cell subsets, and in vitro treatment of DCs significantly reduced CD44, a key receptor in both cell trafficking and activation. sBr was shown to reduce allergic sensitization and the generation of AAD upon antigen challenge. These results provide additional insight into sBr's anti-inflammatory and antiallergic properties and rationale for translation into the clinical arena. PMID:24381635

  11. Differential Nucleosome Occupancies across Oct4-Sox2 Binding Sites in Murine Embryonic Stem Cells

    PubMed Central

    Sebeson, Amy; Xi, Liqun; Zhang, Quanwei; Sigmund, Audrey; Wang, Ji-Ping; Wang, Xiaozhong

    2015-01-01

    The binding sequence for any transcription factor can be found millions of times within a genome, yet only a small fraction of these sequences encode functional transcription factor binding sites. One of the reasons for this dichotomy is that many other factors, such as nucleosomes, compete for binding. To study how the competition between nucleosomes and transcription factors helps determine a functional transcription factor site from a predicted transcription factor site, we compared experimentally-generated in vitro nucleosome occupancy with in vivo nucleosome occupancy and transcription factor binding in murine embryonic stem cells. Using a solution hybridization enrichment technique, we generated a high-resolution nucleosome map from targeted regions of the genome containing predicted sites and functional sites of Oct4/Sox2 regulation. We found that at Pax6 and Nes, which are bivalently poised in stem cells, functional Oct4 and Sox2 sites show high amounts of in vivo nucleosome displacement compared to in vitro. Oct4 and Sox2, which are active, show no significant displacement of in vivo nucleosomes at functional sites, similar to nonfunctional Oct4/Sox2 binding. This study highlights a complex interplay between Oct4 and Sox2 transcription factors and nucleosomes among different target genes, which may result in distinct patterns of stem cell gene regulation. PMID:25992972

  12. Murine Double Minute-2 Prevents p53-Overactivation-Related Cell Death (Podoptosis) of Podocytes.

    PubMed

    Thomasova, Dana; Bruns, Hauke A; Kretschmer, Victoria; Ebrahim, Martrez; Romoli, Simone; Liapis, Helen; Kotb, Ahmed M; Endlich, Nicole; Anders, Hans-Joachim

    2015-07-01

    Murine double minute-2 (MDM2), an E3 ligase that regulates the cell cycle and inflammation, is highly expressed in podocytes. In podocyte injury, MDM2 drives podocyte loss by mitotic catastrophe, but the function of MDM2 in resting podocytes has not been explored. Here, we investigated the effects of podocyte MDM2 deletion in vitro and in vivo. In vitro, MDM2 knockdown by siRNA caused increased expression of p53 and podocyte death, which was completely rescued by coknockdown of p53. Apoptosis, pyroptosis, pyronecrosis, necroptosis, ferroptosis, and parthanatos were excluded as modes of occurrence for this p53-overactivation-related cell death (here referred to as podoptosis). Podoptosis was associated with cytoplasmic vacuolization, endoplasmic reticulum stress, and dysregulated autophagy (previously described as paraptosis). MDM2 knockdown caused podocyte loss and proteinuria in a zebrafish model, which was consistent with the phenotype of podocyte-specific MDM2-knockout mice that also showed the aforementioned ultrastructual podocyte abnormalities before and during progressive glomerulosclerosis. The phenotype of both animal models was entirely rescued by codeletion of p53. We conclude that MDM2 maintains homeostasis and long-term survival in podocytes by preventing podoptosis, a p53-regulated form of cell death with unspecific features previously classified as paraptosis. PMID:25349197

  13. Cannabidiol-induced apoptosis in murine microglial cells through lipid raft.

    PubMed

    Wu, Hsin-Ying; Goble, Kathleen; Mecha, Miriam; Wang, Chia-Chi; Huang, Chung-Hsiung; Guaza, Carmen; Jan, Tong-Rong

    2012-07-01

    Cannabidiol (CBD), the major nonpsychotropic phytocannabinoid, induces apoptosis in both immortalized and primary lymphocytes and monocytes. However, contrasting effects of CBD on the apoptosis between normal and immortalized glial cells have been reported. This study investigated the proapoptotic effect of CBD on primary microglial cells. Treatment of murine primary microglial cultures with CBD resulted in a time- and concentration-dependent induction of apoptosis, as shown by increase in hypodiploid cells and DNA strand breaks, and marked activation of both caspase-8 and -9. Mechanistic studies revealed that antioxidants, including N-acetyl-L-cysteine and glutathione, the G protein-coupled receptor 55 agonist abnormal-CBD and specific antagonists for vanilloid, and CB1 and CB2 cannabinoid receptors did not counteract the apoptosis induced by CBD. In contrast, methyl-?-cyclodextrin (MCD), a lipid raft disruptor, potently attenuated CBD-induced microglial apoptosis and caspase activation. Furthermore, CBD induced lipid raft coalescence and augmented the expression of GM1 ganglioside and caveolin-1, all of which were attenuated by MCD. Taken together, these results suggest that CBD induces a marked proapoptotic effect in primary microglia through lipid raft coalescence and elevated expression of GM1 ganglioside and caveolin-1. PMID:22535572

  14. Phosphatidylinositol turnover and transformation of cells by Abelson murine leukaemia virus.

    PubMed Central

    Fry, M J; Gebhardt, A; Parker, P J; Foulkes, J G

    1985-01-01

    The transforming protein of the Abelson murine leukaemia virus encodes a protein-tyrosine kinase. Previously, we have shown that in Abelson-transformed cells, the Abelson kinase regulates the phosphoserine content of ribosomal protein S6. Phorbol 12-myristate 13-acetate (TPA), which activates protein kinase C, induces the phosphorylation of S6 at the same five phosphopeptides as found in S6 isolated from Abelson-transformed cells. We have investigated three models whereby the Abelson kinase might regulate S6 phosphorylation via the activation of protein kinase C. First, the Abelson kinase could phosphorylate protein kinase C on tyrosine. However, we do not detect significant amounts of phosphotyrosine in protein kinase C in vivo. Second, it has been suggested that protein-tyrosine kinases might phosphorylate phosphatidylinositol. This could increase the intracellular levels of diacylglycerol and thereby activate protein kinase C. Our data strongly suggest that direct phosphorylation of phosphatidylinositol by the Abelson protein-tyrosine kinase has no physiological role. Third, an indirect activation of protein kinase C may occur via an increase in the rate of phosphoinositide breakdown. We have found that phosphoinositide breakdown appears to be constitutively activated in Abelson-transformed cells. The implications of these observations are discussed with regard to S6 phosphorylation and the mechanism of Abelson-induced transformation. Images Fig. 1. Fig. 2. PMID:3004937

  15. Transcriptional analysis of glial cell differentiation in the postnatal murine spinal cord.

    PubMed

    Raddatz, Barbara B; Lehmbecker, Annika; Kalkuhl, Arno; Deschl, Ulrich; Baumgärtner, Wolfgang; Ulrich, Reiner

    2015-05-01

    Postnatal murine spinal cord represents a good model system to study mammalian central nervous system myelination in vivo as a basis for further studies in demyelinating diseases. Transcriptional changes were analyzed in SJL/J mice on postnatal day 0, 14, 49 and 231 (P0, P14, P49, P231) employing Affymetrix GeneChip Mouse Genome 430 2.0 Arrays. Additionally, marker gene signatures for astrocyte and oligodendrocyte lineage-stages were defined to study their gene expression in more detail. In addition, immunohistochemistry was used to quantify the abundance of commonly used glial cell markers. 6092 differentially regulated genes (DEGs) were identified. The up-regulated DEGs at P14, P49 and P231 compared to P0 exhibited significantly enriched associations to gene ontology terms such as myelination and lipid metabolic transport and down-regulated DEGs to neurogenesis and axonogenesis. Expression values of marker gene signatures for neural stem cells, oligodendrocyte precursor cells, and developing astrocytes were constantly decreasing, whereas myelinating oligodendrocyte and mature astrocyte markers showed a steady increase. Molecular findings were substantiated by immunohistochemical observations. The transcriptional changes observed are an important reference for future analysis of degenerative and inflammatory conditions in the spinal cord. PMID:25702526

  16. Low-density lipoprotein-mediated delivery of docosahexaenoic acid selectively kills murine liver cancer cells

    PubMed Central

    Reynolds, Lacy; Mulik, Rohit S.; Wen, Xiaodong; Dilip, Archana; Corbin, Ian R.

    2014-01-01

    Aim The natural omega-3 polyunsaturated fatty acid, docosahexaenoic acid (DHA), has recently been credited for possessing anticancer properties. Herein, we investigate the cytotoxic actions of DHA-loaded low-density lipoprotein (LDL) nanoparticles in normal and liver cancer cells. Materials & methods LDL-DHA nanoparticles were prepared and subjected to extensive biophysical characterization. The therapeutic utility of LDL-DHA nanoparticles was evaluated in normal and malignant murine hepatocyte cell lines, TIB-73 and TIB-75, respectively. Results & discussion The engineered LDL-DHA nanoparticles possessed enhanced physical and oxidative stabilities over native LDL and free DHA. Dose–response studies showed that therapeutic doses of LDL-DHA nanoparticles that completely killed TIB-75 were innocuous to TIB-73. The selective induction of lipid peroxidation and reactive oxygen species in the cancer cells was shown to play a central role in LDL-DHA nanoparticle-mediated cytotoxicity. Conclusion In summary, these findings indicate that LDL-DHA nanoparticles show great promise as a selective anticancer agent against hepatocellular carcinoma. PMID:24397600

  17. Interactome analysis of myeloid-derived suppressor cells in murine models of colon and breast cancer

    PubMed Central

    Aliper, Alexander M.; Frieden-Korovkina, Victoria P.; Buzdin, Anton; Roumiantsev, Sergey A.; Zhavoronkov, Alex

    2014-01-01

    In solid cancers, myeloid derived suppressor cells (MDSC) infiltrate (peri)tumoral tissues to induce immune tolerance and hence to establish a microenvironment permissive to tumor growth. Importantly, the mechanisms that facilitate such infiltration or a subsequent immune suppression are not fully understood. Hence, in this study, we aimed to delineate disparate molecular pathways which MDSC utilize in murine models of colon or breast cancer. Using pathways enrichment analysis, we completed interactome maps of multiple signaling pathways in CD11b+/Gr1(high/low) MDSC from spleens and tumor infiltrates of mice with c26GM colon cancer and tumor infiltrates of MDSC in 4T1 breast cancer. In both cancer models, infiltrating MDSC, but not CD11b+ splenic cells, have been found to be enriched in multiple signaling molecules suggestive of their enhanced proliferative and invasive phenotypes. The interactome data has been subsequently used to reconstruct a previously unexplored regulation of MDSC cell cycle by the c-myc transcription factor which was predicted by the analysis. Thus, this study represents a first interactome mapping of distinct multiple molecular pathways whereby MDSC sustain cancer progression. PMID:25294811

  18. Mechanism-based combination treatment dramatically increases therapeutic efficacy in murine globoid cell leukodystrophy.

    PubMed

    Hawkins-Salsbury, Jacqueline A; Shea, Lauren; Jiang, Xuntian; Hunter, Daniel A; Guzman, A Miguel; Reddy, Adarsh S; Qin, Elizabeth Y; Li, Yedda; Gray, Steven J; Ory, Daniel S; Sands, Mark S

    2015-04-22

    Globoid cell leukodystrophy (GLD, Krabbe disease) is a lysosomal storage disease (LSD) caused by a deficiency in galactocerebrosidase (GALC) activity. In the absence of GALC activity, the cytotoxic lipid, galactosylsphingosine (psychosine), accumulates in the CNS and peripheral nervous system. Oligodendrocytes and Schwann cells are particularly sensitive to psychosine, thus leading to a demyelinating phenotype. Although hematopoietic stem-cell transplantation provides modest benefit in both presymptomatic children and the murine model (Twitcher), there is no cure for GLD. In addition, GLD has been relatively refractory to virtually every experimental therapy attempted. Here, Twitcher mice were simultaneously treated with CNS-directed gene therapy, substrate reduction therapy, and bone marrow transplantation to target the primary pathogenic mechanism (GALC deficiency) and two secondary consequences of GALC deficiency (psychosine accumulation and neuroinflammation). Simultaneously treating multiple pathogenic targets resulted in an unprecedented increase in life span with improved motor function, persistent GALC expression, nearly normal psychosine levels, and decreased neuroinflammation. Treating the primary pathogenic mechanism and secondary targets will likely improve therapeutic efficacy for other LSDs with complex pathological and clinical presentations. PMID:25904800

  19. Murine leukemia P388 vinorelbine-resistant cell lines are sensitive to vinflunine.

    PubMed

    Aggarwal, Ashish; Kruczynski, Anna; Frankfurter, Anthony; Correia, John J; Lobert, Sharon

    2008-08-01

    The work presented here was initiated to explore the mechanisms underlying vinorelbine resistance in two previously established murine leukemia P388 cell lines (N.63 and N2.5). IC(50) measurements demonstrated that the vinorelbine-resistant cell line N.63 was sensitive to both vinblastine and vinflunine. In addition, vinorelbine-resistant cell line N2.5 retained sensitivity to vinflunine. We used flow cytometry with propidium iodide to measure G2/M arrest in response to drug treatment. Annexin V labeling was used as a marker of apoptosis and JC-1 dye labeling as a marker of mitochondrial membrane depolarization to explore differential responses that might help explain the absence of cross resistance to vinflunine. At equipotent (10X IC(50)) doses, after 8 h of drug treatment, vinflunine induced G2/M arrest in a significantly larger fraction of vinorelbine- resistant cells compared to vinorelbine. At the same drug doses, at 16 h after initiation of drug treatment, vinflunine induced a statistically significant greater apoptotic response and mitochondrial depolarization. The mitochondrial depolarization at 16 h was confirmed by Western blotting that showed release of cytochrome c. Comparison of apoptotic and mitochondrial depolarization responses in vinorelbine-resistant cells upon exposure to vinorelbine, vinblastine and vinflunine demonstrated the following pattern of drug activity: vinflunine > vinblastine > vinorelbine, confirming the importance of a antimitotic-induced mitochondria-mediated pathways in these P388 cell lines. We conclude that vinflunine may be preferred for treatment of specific cancers compared to other vinca alkaloids due to its enhanced effects on apoptotic pathways that follow G2/M arrest. PMID:18071633

  20. Thymoquinone Inhibits Murine Leukemia WEHI-3 Cells In Vivo and In Vitro

    PubMed Central

    Ali Salim, Landa Zeenelabdin; Othman, Rozana; Abdulla, Mahmood Ameen; Al-Jashamy, Karim; Mohd Ali, Hapipah; Hassandarvish, Pouya; Dehghan, Firouzeh; Ibrahim, Mohamed Yousif; Omer, Fatima Abd Elmutaal Ahmed; Mohan, Syam

    2014-01-01

    Background Thymoquinone is an active ingredient isolated from Nigella sativa (Black Seed). This study aimed to evaluate the in vitro and in vivo anti-leukemic effects of thymoquinone on WEHI-3 cells. Methodology/Principal Findings The cytotoxic effect of thymoquinone was assessed using an MTT assay, while the inhibitory effect of thymoquinone on murine WEHI-3 cell growth was due to the induction of apoptosis, as evidenced by chromatin condensation dye, Hoechst 33342 and acridine orange/propidium iodide fluorescent staining. In addition, Annexin V staining for early apoptosis was performed using flowcytometric analysis. Apoptosis was found to be associated with the cell cycle arrest at the S phase. Expression of Bax, Bcl2 and HSP 70 proteins were observed by western blotting. The effects of thymoquinone on BALB/c mice injected with WEHI-3 cells were indicated by the decrease in the body, spleen and liver weights of the animal, as compared to the control. Conclusion Thymoquinone promoted natural killer cell activities. This compound showed high toxicity against WEHI-3 cell line which was confirmed by an increase of the early apoptosis, followed by up-regulation of the anti-apoptotic protein, Bcl2, and down-regulation of the apoptotic protein, Bax. On the other hand, high reduction of the spleen and liver weight, and significant histopathology study of spleen and liver confirmed that thymoquinone inhibited WEHI-3 growth in the BALB/c mice. Results from this study highlight the potential of thymoquinone to be developed as an anti-leukemic agent. PMID:25531768

  1. DNA methylation analysis of murine hematopoietic side population cells during aging.

    PubMed

    Taiwo, Oluwatosin; Wilson, Gareth A; Emmett, Warren; Morris, Tiffany; Bonnet, Dominique; Schuster, Eugene; Adejumo, Tomas; Beck, Stephan; Pearce, Daniel J

    2013-10-01

    Stem cells have been found in most tissues/organs. These somatic stem cells produce replacements for lost and damaged cells, and it is not completely understood how this regenerative capacity becomes diminished during aging. To study the possible involvement of epigenetic changes in somatic stem cell aging, we used murine hematopoiesis as a model system. Hematopoietic stem cells (HSCs) were enriched for via Hoechst exclusion activity (SP-HSC) from young, medium-aged and old mice and subjected to comprehensive, global methylome (MeDIP-seq) analysis. With age, we observed a global loss of DNA methylation of approximately 5%, but an increase in methylation at some CpG islands. Just over 100 significant (FDR<0.2) aging-specific differentially methylated regions (aDMRs) were identified, which are surprisingly few considering the profound age-based changes that occur in HSC biology. Interestingly, the polycomb repressive complex -2 (PCRC2) target genes Kiss1r, Nav2 and Hsf4 were hypermethylated with age. The promoter for the Sdpr gene was determined to be progressively hypomethylated with age. This occurred concurrently with an increase in gene expression with age. To explore this relationship further, we cultured isolated SP-HSC in the presence of 5-aza-deoxycytdine and demonstrated a negative correlation between Sdpr promoter methylation and gene expression. We report that DNA methylation patterns are well preserved during hematopoietic stem cell aging, confirm that PCRC2 targets are increasingly methylated with age, and suggest that SDPR expression changes with age in HSCs may be regulated via age-based alterations in DNA methylation. PMID:23949429

  2. Enhancement of radiation cytotoxicity in murine cancer cells by electroporation: in vitro and in vivo studies.

    PubMed

    Shil, Pratip; Sanghvi, Surendra H; Vidyasagar, Pandit B; Mishra, Kaushala P

    2005-01-01

    Increasing evidence has accumulated in recent years to suggest that the cell membrane forms the vital common target for action by ionizing radiation and electroporation. The present work describes the use of electric pulses for enhancement of radiation-induced cytotoxicity of cancer cells both in vitro and in vivo. In vitro: low dose rate (0.37 Gy/min) Co60 gamma-rays (2 Gy) in combination with electric pulses of square wave (2 kV/cm, 200 micros duration, 8 pulses/burst, 10 times) significantly enhanced the cytotoxicity in Ehrlich ascites carcinoma cells (EAC), probably through enhanced production of intracellular reactive oxygen species (ROS). The intracellular generation of ROS and changes in oxidative damage-mediated membrane fluidity were determined by fluorescence using DCH-FDA and DPH, respectively, as probes. Both radiation and electroporation, separately, have been observed to produce ROS in a dose-dependent fashion. We show that the combined treatment of cells with radiation and electroporation significantly increased intracellular ROS and changed membrane fluidity of EAC cells as compared to the effects by each individual treatment. In vivo studies have been carried out with murine fibrosarcoma as a model system. The localized treatment of a fibrosarcoma tumor, grown in the right hind leg of Swiss mice, had been carried out using radiation (Co60 gamma-rays, 2 Gy, dose rate: 0.37 Gy/min) and electric pulses (1 kV/cm, 200 micros, 8 pulses/burst, 10 times). Studies on tumor growth kinetics have shown a significant growth delay (by 50% to that of control) 7 days after treatment of tumor with radiation and electroporation. The results suggest that radiocytotoxicity of tumor cells in vitro as well as in vivo were enhanced significantly by electric pulses, which may offer a potentially improved treatment of cancer. PMID:16393122

  3. Estrogen receptor ? activation impairs prostatic regeneration by inducing apoptosis in murine and human stem/progenitor enriched cell populations.

    PubMed

    Hussain, Shirin; Lawrence, Mitchell G; Taylor, Renea A; Lo, Camden Yeung-Wah; Frydenberg, Mark; Ellem, Stuart J; Furic, Luc; Risbridger, Gail P

    2012-01-01

    Androgen depletion is the primary treatment for prostate disease; however, it fails to target residual castrate-resistant cells that are regenerative and cells of origin of prostate cancer. Estrogens, like androgens, regulate survival in prostatic cells, and the goal of this study was to determine the advantages of selective activation of estrogen receptor ? (ER?) to induce cell death in stem cells that are castrate-resistant. Here we show two cycles of short-term ER? agonist (8?-VE2) administration this treatment impairs regeneration, causing cystic atrophy that correlates with sustained depletion of p63+ basal cells. Furthermore, agonist treatment attenuates clonogenicity and self-renewal of murine prostatic stem/progenitor cells and depletes both murine (Lin(-)Sca1(+)CD49f(hi)) and human (CD49f(hi)Trop2(hi)) prostatic basal cells. Finally, we demonstrate the combined added benefits of selective stimulation of ER?, including the induction of cell death in quiescent post-castration tissues. Subsequent to castration ER?-induces further apoptosis in basal, luminal and intermediate cells. Our results reveal a novel benefit of ER? activation for prostate disease and suggest that combining selective activation of ER? with androgen-deprivation may be a feasible strategy to target stem cells implicated in the origin of prostatic disease. PMID:22808245

  4. Human Mesenchymal Stromal Cells Transplantation May Enhance or Inhibit 4T1 Murine Breast Adenocarcinoma through Different Approaches

    PubMed Central

    Jazedje, T.; Ribeiro, A. L.; Pellati, M.; de Siqueira Bueno, H. M.; Nagata, G.; Trierveiler, M.; Rodrigues, E. G.; Zatz, M.

    2015-01-01

    The use of Mesenchymal Stromal Cells (MSCs) aiming to treat cancer has shown very contradictory results. In an attempt to clarify the contradictory results reported in the literature and the possible role of human fallopian tube Mesenchymal Stromal Cells (htMSCs) against breast cancer, the aim of this study was to evaluate the clinical effect of htMSCs in murine mammary adenocarcinoma using two different approaches: (1) coinjections of htMSCs and 4T1 murine tumor cell lineage and (2) injections of htMSCs in mice at the initial stage of mammary adenocarcinoma development. Coinjected animals had a more severe course of the disease and a reduced survival, while tumor-bearing animals treated with 2 intraperitoneal injections of 106 htMSCs showed significantly reduced tumor growth and increased lifespan as compared with control animals. Coculture of htMSCs and 4T1 tumor cells revealed an increase in IL-8 and MCP-1 and decreased VEGF production. For the first time, we show that MSCs isolated from a single source and donor when injected in the same animal model and tumor can lead to opposite results depending on the experimental protocol. Also, our results demonstrated that htMSCs can have an inhibitory effect on the development of murine mammary adenocarcinoma. PMID:26000020

  5. Regulated Intramembrane Proteolysis and Degradation of Murine Epithelial Cell Adhesion Molecule mEpCAM

    PubMed Central

    Hachmeister, Matthias; Bobowski, Karolina D.; Hogl, Sebastian; Dislich, Bastian; Fukumori, Akio; Eggert, Carola; Mack, Brigitte; Kremling, Heidi; Sarrach, Sannia; Coscia, Fabian; Zimmermann, Wolfgang; Steiner, Harald; Lichtenthaler, Stefan F.; Gires, Olivier

    2013-01-01

    Epithelial cell adhesion molecule EpCAM is a transmembrane glycoprotein, which is highly and frequently expressed in carcinomas and (cancer-)stem cells, and which plays an important role in the regulation of stem cell pluripotency. We show here that murine EpCAM (mEpCAM) is subject to regulated intramembrane proteolysis in various cells including embryonic stem cells and teratocarcinomas. As shown with ectopically expressed EpCAM variants, cleavages occur at ?-, ?-, ?-, and ?-sites to generate soluble ectodomains, soluble A?-like-, and intracellular fragments termed mEpEX, mEp-?, and mEpICD, respectively. Proteolytic sites in the extracellular part of mEpCAM were mapped using mass spectrometry and represent cleavages at the ?- and ?-sites by metalloproteases and the b-secretase BACE1, respectively. Resulting C-terminal fragments (CTF) are further processed to soluble A?-like fragments mEp-? and cytoplasmic mEpICD variants by the g-secretase complex. Noteworthy, cytoplasmic mEpICD fragments were subject to efficient degradation in a proteasome-dependent manner. In addition the ?-secretase complex dependent cleavage of EpCAM CTF liberates different EpICDs with different stabilities towards proteasomal degradation. Generation of CTF and EpICD fragments and the degradation of hEpICD via the proteasome were similarly demonstrated for the human EpCAM ortholog. Additional EpCAM orthologs have been unequivocally identified in silico in 52 species. Sequence comparisons across species disclosed highest homology of BACE1 cleavage sites and in presenilin-dependent ?-cleavage sites, whereas strongest heterogeneity was observed in metalloprotease cleavage sites. In summary, EpCAM is a highly conserved protein present in fishes, amphibians, reptiles, birds, marsupials, and placental mammals, and is subject to shedding, ?-secretase-dependent regulated intramembrane proteolysis, and proteasome-mediated degradation. PMID:24009667

  6. Genotoxicity of amorphous silica particles with different structure and dimension in human and murine cell lines.

    PubMed

    Guidi, Patrizia; Nigro, Marco; Bernardeschi, Margherita; Scarcelli, Vittoria; Lucchesi, Paolo; Onida, Barbara; Mortera, Renato; Frenzilli, Giada

    2013-03-01

    Although amorphous silica is used in food products, cosmetics and paints and as vector for drug delivery, data on its potential health hazard are limited. The aim of this study was to investigate the cytotoxic and genotoxic potential of silica particles of different sizes (250 and 500nm) and structures (dense and mesoporous). Dense silica (DS) spheres were prepared by sol-gel synthesis, mesoporous silica particles (MCM-41) were prepared using hexadecyltrimethyl ammonium bromide as a structure-directing agent and tetraethylorthosilicate as silica source. Particles were accurately characterised by dynamic light scattering, nitrogen adsorption, X-ray diffraction and field emission scanning electron microscopy. Murine macrophages (RAW264.7) and human epithelial lung (A549) cell lines were selected for investigation. Genotoxicity was evaluated by Comet assay and micronucleus test. Cytotoxicity was tested by the trypan blue method. Cells were treated with 0, 5, 10, 20, 40 and 80 µg/cm(2) of different silica powders for 4 and 24 h. The intracellular localisation of silica was investigated by transmission electron microscopy. Amorphous particles penetrated into the cells, being compartmentalised within endocytic vacuoles. DS and MCM-41 particles induced cytotoxic and genotoxic effects in A549 and RAW264.7 although to different extent in the two cell lines. A549 were resistant in terms of cell viability, but showed a generalised induction of DNA strand breaks. RAW264.7 were susceptible to amorphous silica exposure, exhibiting both cytotoxic and genotoxic responses as DNA strand breaks and chromosomal alterations. The cytotoxic response of RAW264.7 was particularly relevant after MCM-41 exposure. The genotoxicity of amorphous silica highlights the need for a proper assessment of its potential hazard for human health. PMID:23325795

  7. Trientine, a copper-chelating agent, induced apoptosis in murine fibrosarcoma cells by activation of the p38 MAPK pathway.

    PubMed

    KADOWAKI, Shingo; ENDOH, Daiji; OKUI, Toyo; HAYASHI, Masanobu

    2009-11-01

    We have reported that treatment with trientine, Cu-chelating agent, inhibits tumor growth in a murine transplantation model using fibrosarcoma and induces apoptosis in tumor cells in vivo and in vitro. When fibrosarcoma cells were treated with 10 mM trientine, the activities of p38 MAPK in treated cells were approximately 3-4 times higher than those in untreated cells. Proportions of cells in which apoptosis was induced by trientine increased in an incubation time-dependent manner from days 2 to 6. The proportions of apoptotic cells in the cells treated with trientine and SB203580, an inhibitor of p38 MAPK, were approximately 50% in those of cells treated with trientine alone. The present results showed that the p38 MAPK pathway may play an important role in induction of apoptosis in fibrosarcoma cells by trientine. PMID:19959910

  8. Expression of glycophosphatidylinositol-anchored and -non-anchored isoforms of vascular cell adhesion molecule 1 in murine stromal and endothelial cells

    Microsoft Academic Search

    Tatsuo Kinashi; Timothy A. Springer

    1995-01-01

    Monoclonal antibodies to murine vascular cell adhesion molecule-i (VCAM-1, CD1O6) revealed not only the expected VCAM-1 molecule with an apparent molecular weight of 100 kDa, but also a molecule with a smaller size of 46 kDa in stromal cells and stimulated endothelial cells. Peptide mapping suggested the 46 kDa and 100 kDa proteins were closely related. The 46 kDa, but

  9. MURINE TUMOR CELLS TRANSDUCED WITH THE GENE FOR TUMOR NECROSIS FACTOR-?

    PubMed Central

    ASHER, A. L.; MULÉ, J . J.; KASID, A; RESTIFO, N. P.; SALO, J . C.; REICHERT, C. M.; JAFFE, G.; FENDLY, B; KRIEGLER, M.; ROSENBERG, S. A.

    2007-01-01

    Studies of the anti-tumor activity of TNF-? in vivo have been hampered by the need to administer systemically toxic doses of the cytokine to obtain a curative response. To facilitate studies of the effect of high local concentrations of TNF-? on tumor growth and host immunity, a newly induced murine sarcoma was transduced with the gene for human TNF-? and the biologic characteristics of these cells were examined. We identified high and low TNF-producing tumor clones which exhibited stable TNF secretion over time. Significant amounts of membrane associated TNF were found in a high-TNF producing clone as well. No difference in the in vitro growth rates between TNF-producing and nonproducing cell lines was observed. In contrast, in vivo studies demonstrate that although unmodified parental tumor cells grew progressively when implanted s.c. in animals, tumor cells transduced with the TNF gene were found to regress in a significant number of animals after an initial phase of growth. This effect correlated with the amount of TNF produced and could be blocked with a specific anti-TNF antibody. Regressions of TNF-producing cells occurred in the absence of any demonstrable toxicity in the animals bearing these tumors. TNF-producing tumor cells could function in a paracrine fashion by inhibiting the growth of unmodified, parental tumor cells implanted at the same site. The ability of tumor cells to regress was abrogated by in vivo depletion of CD4+ or CD8+ T cell subsets and animals that had experienced regression of TNF-producing tumors rejected subsequent challenges of parental tumor. Our studies thus show that tumor cells elaborating high local concentrations of TNF regress in the absence of toxicity in the host and that this process requires the existence of intact host immunity. Studies of the lymphocytes infiltrating the gene modified tumors and attempts to use TNF gene modified tumor infiltrating lymphocytes to deliver high local concentrations of TNF to the tumor site without inducing systemic toxicity are underway. PMID:2016545

  10. Allium sativum potentiates suicide gene therapy for murine transitional cell carcinoma.

    PubMed

    Moon, D G; Cheon, J; Yoon, D H; Park, H S; Kim, H K; Kim, J J; Koh, S K

    2000-01-01

    This study evaluated the synergistic effect of Allium sativum (AS) with suicide gene therapy for transitional cell carcinoma (TCC) of the bladder. Subcutaneous TCCs were established in syngeneic C3H/He mice with 1 x 10(5) MBT-2 cells. AS liquid extract was injected at the site of tumor transplantation on Day 1 for three weeks (Experiment I) and into the established tumors weekly for five weeks (Experiment II) in combination with or without gene therapy using a replication-defective adenoviral vector containing a herpes simplex virus thymidine kinase (HSV-TK) gene under the transcriptional control of Rous sarcoma virus (RSV) promoter (Ad-RSV-TK, 5 x 10(8) plaque-forming units) plus ganciclovir (20 mg/kg/day i.p.). AS demonstrated a statistically significant reduction in incidence of TCC (cumulative dose 25 mg of AS). Combination AS-suicide gene therapy significantly inhibited the tumor growth compared with the controls, which was evidenced by apoptosis on histomorphological and immunohistochemical studies. These results suggest that AS had a definite antitumor effect in inhibiting tumorigenesis and growth of TCC in a murine model. AS treatment combined with suicide gene therapy had significant additive antitumor effects on TCC and may provide a novel and effective treatment modality for TCC of the bladder. PMID:11341051

  11. Melanogenesis stimulation in murine b16 melanoma cells by umberiferae plant extracts and their coumarin constituents.

    PubMed

    Matsuda, Hideaki; Hirata, Noriko; Kawaguchi, Yoshiko; Yamazaki, Miho; Naruto, Shunsuke; Shibano, Makio; Taniguchi, Masahiko; Baba, Kimiye; Kubo, Michinori

    2005-07-01

    Melanogenesis stimulation activities of seven ethanolic extracts obtained from Umbelliferae plants used as Chinese crude drugs, namely the roots of Angelica dahurica BENTH. et HOOK., A. biserrata SHEN et YUAN, Notopterygium incisum TING, Heracleum lanatum MICHX., and H. candicans WALL., and the fruits of Cinidium monnieri (L.) CUSSON and C. formosanum YABE, were examined by using cultured murine B16 melanoma cells. Among them, the extract (5, 25 microg/ml) of H. lanatum showed a potent stimulatory effect on melanogenesis with significant enhancement of cell proliferation in a dose-dependent manner. The melanogenesis stimulatory effects of sixteen coumarins (1-16) isolated from the seven Umbelliferae crude drugs were also examined. Among them, linear-furocoumarins [psoralen (1), xanthotoxin (2), bergapten (3), and isopimpinellin (4)] and angular-furocoumarin [sphondin (13)] exhibited potent melanogenesis stimulation activity. From the view point of structure-activity relationships, it may be assumed that a linear-furocoumarin ring having a hydrogen and/or methoxyl group at 5 and 8 positions such as 1, 2, 3 and 4 was preferable for the melanogenesis stimulation activity. The introduction of a prenyl group into the furocoumarin ring was disadvantageous. Coumarin derivatives having a simple coumarin ring were inactive. PMID:15997104

  12. A mouse gene on chromosome 5 that restricts infectivity of mink cell focus-forming recombinant murine leukemia viruses

    PubMed Central

    1983-01-01

    DBA/1, DBA/2, CBA/N, and CBA/Ca mice carry a gene which specifically restricts infectivity of mink cell focus-forming (MCF) murine leukemia viruses. The gene, designated Rmcfr, is dominant or semidominant and maps to chromosome 5; it is closely linked to the morphologic marker gene Hm. The Rmcf gene may be of much use as a means of determining the role of MCF viruses in various forms of leukemogenesis. PMID:6306133

  13. Effects of inhibitors of ion-motive ATPases on the plasma membrane potential of murine erythroleukemia cells

    Microsoft Academic Search

    A. Arcangeli; M. R. Del Bene; A. Becchetti; E. Wanke; M. Olivotto

    1992-01-01

    Summary  The membrane electric effects of N,N-dicyclohexylcarbodiimide (DCCD) and vanadate were studied in murine erythroleukemia cells (MELC), comparing the patch-clamp technique and the accumulation ratio (AR\\u000aexp) of [3H]-tetraphenylphosphonium (TPP+). Electrophysiological measurements showed that both these inhibitors produce, at micromolar concentrations, a 20–30 mV hyperpolarization of resting potential (\\u000ap\\u000a) of MELC, which is abolished when the electrochemical equilibrium potential

  14. Characterization, isolation, and differentiation of murine skin cells expressing hematopoietic stem cell markers

    Microsoft Academic Search

    Simone Meindl; Uwe Schmidt; Christine Vaculik; Adelheid Elbe-Burger

    2006-01-01

    As the phenotype of adult dermal stem cells is still elusive, and the hematopoietic stem cell is one of the best-characterized stem cells in the body, we tested dermal cell suspensions, sections, and wholemounts in newborn and adult mice for hematopoietic stem cell marker expression. Phe- notypic analysis revealed that a small population of CD45 cells and a large population

  15. Vaccination with Irradiated Tumor Cells Engineered to Secrete Murine Granulocyte-Macrophage Colony-Stimulating Factor Stimulates Potent, Specific, and Long-Lasting AntiTumor Immunity

    Microsoft Academic Search

    Glenn Dranoff; Elizabeth Jaffee; Audrey Lazenby; Paul Golumbek; Hyam Levitsky; Katja Brose; Valerie Jackson; Hirofumi Hamada; Drew Pardoll; Richard C. Mulligan

    1993-01-01

    To compare the ability of different cytokines and other molecules to enhance the immunogenicity of tumor cells, we generated 10 retroviruses encoding potential immunomodulators and studied the vaccination properties of murine tumor cells transduced by the viruses. Using a B16 melanoma model, in which irradiated tumor cells alone do not stimulate significant anti-tumor immunity, we found that irradiated tumor cells

  16. Hematopoietic Stem and Progenitor Cell Migration After Hypofractionated Radiation Therapy in a Murine Model

    SciTech Connect

    Kane, Jonathan [Department of Biological Sciences, Oakland University, Rochester, Michigan (United States); Radiation Oncology, William Beaumont Health System, Royal Oak, Michigan (United States); Krueger, Sarah A.; Dilworth, Joshua T.; Torma, John T.; Wilson, George D.; Marples, Brian [Radiation Oncology, William Beaumont Health System, Royal Oak, Michigan (United States); Madlambayan, Gerard J., E-mail: madlamba@oakland.edu [Department of Biological Sciences, Oakland University, Rochester, Michigan (United States)

    2013-12-01

    Purpose: To characterize the recruitment of bone marrow (BM)-derived hematopoietic stem and progenitor cells (HSPCs) within tumor microenvironment after radiation therapy (RT) in a murine, heterotopic tumor model. Methods and Materials: Lewis lung carcinoma tumors were established in C57BL/6 mice and irradiated with 30 Gy given as 2 fractions over 2 days. Tumors were imaged with positron emission tomography/computed tomography (PET/CT) and measured daily with digital calipers. The HSPC and myelomonocytic cell content was assessed via immunofluorescent staining and flow cytometry. Functionality of tumor-associated HSPCs was verified in vitro using colony-forming cell assays and in vivo by rescuing lethally irradiated C57BL/6 recipients. Results: Irradiation significantly reduced tumor volumes and tumor regrowth rates compared with nonirradiated controls. The number of CD133{sup +} HSPCs present in irradiated tumors was higher than in nonirradiated tumors during all stages of regrowth. CD11b{sup +} counts were similar. PET/CT imaging and growth rate analysis based on standardized uptake value indicated that HSPC recruitment directly correlated to the extent of regrowth and intratumor cell activity after irradiation. The BM-derived tumor-associated HSPCs successfully formed hematopoietic colonies and engrafted irradiated mice. Finally, targeted treatment with a small animal radiation research platform demonstrated localized HSPC recruitment to defined tumor subsites exposed to radiation. Conclusions: Hypofractionated irradiation resulted in a pronounced and targeted recruitment of BM-derived HSPCs, possibly as a mechanism to promote tumor regrowth. These data indicate for the first time that radiation therapy regulates HSPC content within regrowing tumors.

  17. Comparative cytotoxicity studies of carbon-encapsulated iron nanoparticles in murine glioma cells.

    PubMed

    Grudzinski, Ireneusz P; Bystrzejewski, Michal; Cywinska, Monika A; Kosmider, Anita; Poplawska, Magdalena; Cieszanowski, Andrzej; Fijalek, Zbigniew; Ostrowska, Agnieszka

    2014-05-01

    Carbon-encapsulated iron nanoparticles (CEINs) have recently emerged as a new class of magnetic nanomaterials with a great potential for an increasing number of biomedical applications. To address the current deficient knowledge of cellular responses due to CEIN exposures, we focused on the investigation of internalization profile and resulting cytotoxic effects of CEINs (0.0001-100 ?g/ml) in murine glioma cells (GL261) in vitro. The studied CEIN samples were characterized (TEM, FT-IR, Zeta potential, Boehm titration) and examined as raw and purified nanomaterials with various surface chemistry composition. Of the four type CEINs (the mean diameter 47-56 nm) studied here, the as-synthesized raw nanoparticles (Fe@C/Fe) exhibited high cytotoxic effects on the plasma cell membrane (LDH, Calcein AM/PI) and mitochondria (MTT, JC-1) causing some pro-apoptotic evens (Annexin V/PI) in glioma cells. The effects of the purified (Fe@C) and surface-modified (Fe@C-COOH and Fe@C-(CH2)2COOH) CEINs were found in quite similar patterns; however, most of these cytotoxic events were slightly diminished compared to those induced by Fe@C/Fe. The study showed that the surface-functionalized CEINs affected the cell cycle progression in both S and G2/M phases to a greater extent compared to that of the rest of nanoparticles studied to data. Taken all together, the present results highlight the importance of the rational design of CEINs as their physicochemical features such as morphology, hydrodynamic size, impurity profiles, and especially surface characteristics are critical determinants of different cytotoxic responses. PMID:24632386

  18. Glycosylation of the envelope glycoprotein from a polytropic murine retrovirus in two different host cells.

    PubMed

    Geyer, H; Kempf, R; Schott, H H; Geyer, R

    1990-11-13

    A polytropic recombinant retrovirus containing the envelope gene of Friend mink cell focus-inducing virus plus the remainder of the genome of an amphoropic murine leukemia virus was propagated on mouse embryo fibroblasts and mink lung cells. Virus particles, metabolically labeled with [2-3H]mannose, were harvested from the culture supernatants and lysed with detergents. The viral envelope glycoprotein was isolated from the lysates by immunoaffinity chromatography and purified by preparative SDS/PAGE. Oligosaccharides were liberated by sequential treatment of tryptic glycopeptides with endo-beta-N-acetylglucosaminidase H and peptide-N4-(N-acetyl-beta-glucosaminyl) asparagine amidase F and fractionated by high-performance liquid chromatography. Individual glycans were characterized chromatographically, by methylation analyses and in part, by enzymic microsequencing. The results demonstrated that viral glycoproteins, synthesized in mouse embryo fibroblasts, carried as major constituents partially fucosylated diantennary, 2,4- and 2,6-branched triantennary and tetraantennary complex type N-glycans with 0-4 sialic acid residues and only small amounts of high-mannose type species with 5-9 mannose residues. As a characteristic feature, part of the complex type glycans contained additional Gal(alpha 1-3) substituents. Glycoprotein obtained from virions propagated on mink lung cells, contained partially fucosylated diantennary and 2,4-branched triantennary oligosaccharides with 1-3 sialic acid residues, in addition to trace amounts of high-mannose type species with 8 or 9 mannose residues. Thus, the results reveal that predominantly, the complex type N-glycans of the retroviral envelope glycoprotein display cell-specific variations including differences in oligosaccharide branching, sialylation and substitution by additional Gal(alpha 1-3) residues. PMID:2174368

  19. The Antiviral Effect of High-Molecular Weight Poly-Gamma-Glutamate against Newcastle Disease Virus on Murine Macrophage Cells

    PubMed Central

    Talactac, Melbourne; Lee, Jong-Soo; Moon, Hojin; Chowdhury, Mohammed Y. E.; Kim, Chul Joong

    2014-01-01

    This study demonstrates the capacity of HM-?-PGA treatment to significantly protect murine macrophage cells (RAW 264.7 cells) against NDV infection. Such protection can be explained by the induction of antiviral state of HM-?-PGA in RAW 264.7 cells via TLR4-mediated IRF-3, IRF-7, IFN-?, and IFN-related gene induction as shown in time-dependent changes in mRNA expression confirmed by polymerase chain reaction (PCR). Moreover, the present research also showed that HM-?-PGA can induce proinflammatory cytokine secretion in RAW 264.7 as measured by enzyme-linked immunosorbent assay (ELISA). Therefore, our findings suggest that HM-?-PGA can be a potential antiviral substance that can inhibit NDV infection through its stimulation of antiviral state on RAW 264.7 cells. These results have been consistent with the previous studies showing that HM-?-PGA can protect RAW 264.7 cells and mice against influenza infection. However, it should be noted that although murine macrophage cells are susceptible to NDV, they are not the natural host cells of the virus; thus further in vivo and in vitro studies involving chicken and chicken immune cells are needed to fully assess the efficacy and applicability of HM-?-PGA in the poultry industry. PMID:25610463

  20. Eicosapentaenoic and docosahexaenoic acid ethyl esters differentially enhance B-cell activity in murine obesity[S

    PubMed Central

    Teague, Heather; Harris, Mitchel; Fenton, Jenifer; Lallemand, Perrine; Shewchuk, Brian M.; Shaikh, Saame Raza

    2014-01-01

    EPA and DHA are not biologically equivalent; however, their individual activity on B cells is unknown. We previously reported fish oil enhanced murine B-cell activity in obesity. To distinguish between the effects of EPA and DHA, we studied the ethyl esters of EPA and DHA on murine B-cell function as a function of time. We first demonstrate that EPA and DHA maintained the obese phenotype, with no improvements in fat mass, adipose inflammatory cytokines, fasting insulin, or glucose clearance. We then tested the hypothesis that EPA and DHA would increase the frequency of splenic B cells. EPA and DHA differentially enhanced the frequency and/or percentage of select B-cell subsets, correlating with increased natural serum IgM and cecal IgA. We next determined the activities of EPA and DHA on ex vivo production of cytokines upon lipopolysaccharide stimulation of B cells. EPA and DHA, in a time-dependent manner, enhanced B-cell cytokines with DHA notably increasing IL-10. At the molecular level, EPA and DHA differentially enhanced the formation of ordered microdomains but had no effect on Toll-like receptor 4 mobility. Overall, the results establish differential effects of EPA and DHA in a time-dependent manner on B-cell activity in obesity, which has implications for future clinical studies. PMID:24837990

  1. A multicenter study to standardize reporting and analyses of fluorescence-activated cell-sorted murine intestinal epithelial cells

    PubMed Central

    Magness, Scott T.; Puthoff, Brent J.; Crissey, Mary Ann; Dunn, James; Henning, Susan J.; Houchen, Courtney; Kaddis, John S.; Kuo, Calvin J.; Li, Linheng; Lynch, John; Martin, Martin G.; May, Randal; Niland, Joyce C.; Olack, Barbara; Qian, Dajun; Stelzner, Matthias; Swain, John R.; Wang, Fengchao; Wang, Jiafang; Wang, Xinwei; Yan, Kelley; Yu, Jian

    2013-01-01

    Fluorescence-activated cell sorting (FACS) is an essential tool for studies requiring isolation of distinct intestinal epithelial cell populations. Inconsistent or lack of reporting of the critical parameters associated with FACS methodologies has complicated interpretation, comparison, and reproduction of important findings. To address this problem a comprehensive multicenter study was designed to develop guidelines that limit experimental and data reporting variability and provide a foundation for accurate comparison of data between studies. Common methodologies and data reporting protocols for tissue dissociation, cell yield, cell viability, FACS, and postsort purity were established. Seven centers tested the standardized methods by FACS-isolating a specific crypt-based epithelial population (EpCAM+/CD44+) from murine small intestine. Genetic biomarkers for stem/progenitor (Lgr5 and Atoh 1) and differentiated cell lineages (lysozyme, mucin2, chromogranin A, and sucrase isomaltase) were interrogated in target and control populations to assess intra- and intercenter variability. Wilcoxon's rank sum test on gene expression levels showed limited intracenter variability between biological replicates. Principal component analysis demonstrated significant intercenter reproducibility among four centers. Analysis of data collected by standardized cell isolation methods and data reporting requirements readily identified methodological problems, indicating that standard reporting parameters facilitate post hoc error identification. These results indicate that the complexity of FACS isolation of target intestinal epithelial populations can be highly reproducible between biological replicates and different institutions by adherence to common cell isolation methods and FACS gating strategies. This study can be considered a foundation for continued method development and a starting point for investigators that are developing cell isolation expertise to study physiology and pathophysiology of the intestinal epithelium. PMID:23928185

  2. Murine AIDS Protects Mice Against Experimental Cerebral Malaria: Down-Regulation by Interleukin 10 a T-Helper Type 1 CD4^+ Cell-Mediated Pathology

    NASA Astrophysics Data System (ADS)

    Eckwalanga, Michel; Marussig, Myriam; Dias Tavares, Marisa; Bouanga, Jean Claude; Hulier, Elisabeth; Henriette Pavlovitch, Jana; Minoprio, Paola; Portnoi, Denis; Renia, Laurent; Mazier, Dominique

    1994-08-01

    The retrovirus LP-BM5 murine leukemia virus induces murine AIDS in C57BL/6 mice that has many similarities with human AIDS; Plasmodium berghei ANKA causes experimental cerebral malaria in the same strain of mice. The outcome of malaria infection was studied in mice concurrently infected with the two pathogens. The retrovirus significantly reduced the gravity of the neurological manifestations associated with Plasmodium berghei ANKA infection. The protection against experimental cerebral malaria induced by murine AIDS increased with duration of viral infection and, hence, with the severity of the immunodeficiency. Interleukin 10, principally from splenic T cells, was shown to play a crucial role in this protection.

  3. Chemokine Transfer by Liver Sinusoidal Endothelial Cells Contributes to the Recruitment of CD4+ T Cells into the Murine Liver

    PubMed Central

    Neumann, Katrin; Erben, Ulrike; Kruse, Nils; Wechsung, Katja; Schumann, Michael; Klugewitz, Katja

    2015-01-01

    Leukocyte adhesion and transmigration are central features governing immune surveillance and inflammatory reactions in body tissues. Within the liver sinusoids, chemokines initiate the first crucial step of T-cell migration into the hepatic tissue. We studied molecular mechanisms involved in endothelial chemokine supply during hepatic immune surveillance and liver inflammation and their impact on the recruitment of CD4+ T cells into the liver. In the murine model of Concanavalin A-induced T cell-mediated hepatitis, we showed that hepatic expression of the inflammatory CXC chemokine ligands (CXCL)9 and CXCL10 strongly increased whereas homeostatic CXCL12 significantly decreased. Consistently, CD4+ T cells expressing the CXC chemokine receptor (CXCR)3 accumulated within the inflamed liver tissue. In histology, CXCL9 was associated with liver sinusoidal endothelial cells (LSEC) which represent the first contact site for T-cell immigration into the liver. LSEC actively transferred basolaterally internalized CXCL12, CXCL9 and CXCL10 via clathrin-coated vesicles to CD4+ T cells leading to enhanced transmigration of CXCR4+ total CD4+ T cells and CXCR3+ effector/memory CD4+ T cells, respectively in vitro. LSEC-expressed CXCR4 mediated CXCL12 transport and blockage of endothelial CXCR4 inhibited CXCL12-dependent CD4+ T-cell transmigration. In contrast, CXCR3 was not involved in the endothelial transport of its ligands CXCL9 and CXCL10. The clathrin-specific inhibitor chlorpromazine blocked endothelial chemokine internalization and CD4+ T-cell transmigration in vitro as well as migration of CD4+ T cells into the inflamed liver in vivo. Moreover, hepatic accumulation of CXCR3+ CD4+ T cells during T cell-mediated hepatitis was strongly reduced after administration of chlorpromazine. These data demonstrate that LSEC actively provide perivascularly expressed homeostatic and inflammatory chemokines by CXCR4- and clathrin-dependent intracellular transport mechanisms thereby contributing to the hepatic recruitment of CD4+ T-cell populations during immune surveillance and liver inflammation. PMID:26052942

  4. Chemokine Transfer by Liver Sinusoidal Endothelial Cells Contributes to the Recruitment of CD4+ T Cells into the Murine Liver.

    PubMed

    Neumann, Katrin; Erben, Ulrike; Kruse, Nils; Wechsung, Katja; Schumann, Michael; Klugewitz, Katja; Scheffold, Alexander; Kühl, Anja A

    2015-01-01

    Leukocyte adhesion and transmigration are central features governing immune surveillance and inflammatory reactions in body tissues. Within the liver sinusoids, chemokines initiate the first crucial step of T-cell migration into the hepatic tissue. We studied molecular mechanisms involved in endothelial chemokine supply during hepatic immune surveillance and liver inflammation and their impact on the recruitment of CD4+ T cells into the liver. In the murine model of Concanavalin A-induced T cell-mediated hepatitis, we showed that hepatic expression of the inflammatory CXC chemokine ligands (CXCL)9 and CXCL10 strongly increased whereas homeostatic CXCL12 significantly decreased. Consistently, CD4+ T cells expressing the CXC chemokine receptor (CXCR)3 accumulated within the inflamed liver tissue. In histology, CXCL9 was associated with liver sinusoidal endothelial cells (LSEC) which represent the first contact site for T-cell immigration into the liver. LSEC actively transferred basolaterally internalized CXCL12, CXCL9 and CXCL10 via clathrin-coated vesicles to CD4+ T cells leading to enhanced transmigration of CXCR4+ total CD4+ T cells and CXCR3+ effector/memory CD4+ T cells, respectively in vitro. LSEC-expressed CXCR4 mediated CXCL12 transport and blockage of endothelial CXCR4 inhibited CXCL12-dependent CD4+ T-cell transmigration. In contrast, CXCR3 was not involved in the endothelial transport of its ligands CXCL9 and CXCL10. The clathrin-specific inhibitor chlorpromazine blocked endothelial chemokine internalization and CD4+ T-cell transmigration in vitro as well as migration of CD4+ T cells into the inflamed liver in vivo. Moreover, hepatic accumulation of CXCR3+ CD4+ T cells during T cell-mediated hepatitis was strongly reduced after administration of chlorpromazine. These data demonstrate that LSEC actively provide perivascularly expressed homeostatic and inflammatory chemokines by CXCR4- and clathrin-dependent intracellular transport mechanisms thereby contributing to the hepatic recruitment of CD4+ T-cell populations during immune surveillance and liver inflammation. PMID:26052942

  5. CD34+ Cells Represent Highly Functional Endothelial Progenitor Cells in Murine Bone Marrow

    Microsoft Academic Search

    Junjie Yang; Masaaki II; Naosuke Kamei; Cantas Alev; Sang-Mo Kwon; Atsuhiko Kawamoto; Hiroshi Akimaru; Haruchika Masuda; Yoshiki Sawa; Takayuki Asahara; Maurizio C. Capogrossi

    2011-01-01

    BackgroundEndothelial progenitor cells (EPCs) were shown to have angiogenic potential contributing to neovascularization. However, a clear definition of mouse EPCs by cell surface markers still remains elusive. We hypothesized that CD34 could be used for identification and isolation of functional EPCs from mouse bone marrow.Methodology\\/Principal FindingsCD34+ cells, c-Kit+\\/Sca-1+\\/Lin? (KSL) cells, c-Kit+\\/Lin? (KL) cells and Sca-1+\\/Lin? (SL) cells were isolated from

  6. Triazine herbicides and their chlorometabolites alter steroidogenesis in BLTK1 murine Leydig cells.

    PubMed

    Forgacs, Agnes L; D'Souza, Michelle L; Huhtaniemi, Ilpo T; Rahman, Nafis A; Zacharewski, Timothy R

    2013-07-01

    The triazine herbicides, atrazine (ATR), simazine (SIM), propazine (PRO), terbuthylazine (TBA), and their chlorinated metabolites have been implicated in the etiology of testicular dysgenesis by altering steroidogenesis. To further investigate their effects on testosterone biosynthesis, BLTK1 cells were used to evaluate steroid hormone levels and genome-wide gene expression. BLTK1 cells are a novel murine Leydig cell line possessing an intact steroidogenic pathway with constitutive low basal testosterone (T) levels that can be induced by recombinant human chorionic gonadotropin (rhCG). Triazines (ATR, SIM, PRO, and TBA) and their chlorometabolites (DEA, DIA, and DACT) induced concentration-dependent (1, 3, 10, 30, 100, 300, and 600 µM) increases in progesterone (P) and T levels relative to solvent control at 24h. Temporal analysis (300 µM at 1, 2, 4, 8, 12, 24, or 48h) elicited comparable P and T profiles by all compounds with varying efficacies (ATR > TBA > PRO > DEA > DIA > DACT > SIM) that were similar to rhCG. ATR and TBA elicited time- and concentration-dependent induction of Star, Hsd3b6, and Hsd17b3 mRNA levels, whereas Hsd3b1, Cyp17a1, and Srd5a1 mRNA expression was repressed. PRO elicited similar albeit weaker effects, whereas SIM had negligible effects consistent with their induction of P and T levels. Whole-genome microarrays identified 797 differentially regulated genes elicited by 300 µM ATR, occurring primarily at later time points (> 12h) with overrepresented functions associated with steroidogenesis and cholesterol metabolism. These results indicate that changes in P and T levels can be partially attributed to triazine-elicited alterations in steroidogenic gene expression. PMID:23591565

  7. Progesterone-induced activation of membrane-bound progesterone receptors in murine macrophage cells

    PubMed Central

    Lu, Jing; Reese, Joshua; Zhou, Ying; Hirsch, Emmet

    2014-01-01

    Parturition is an inflammatory process mediated to a significant extent by macrophages. Progesterone maintains uterine quiescence in pregnancy, and a proposed functional withdrawal of progesterone classically regulated by nuclear progesterone receptors (nPRs) leads to labor. Progesterone can impact the functions of macrophages despite the reported lack of expression of nPRs in these immune cells. Therefore, in this study we investigated the effects of the activation of the putative membrane-associated progesterone receptor on the function of macrophages (a key cell for parturition) and discuss the implications of these findings for pregnancy and parturition. In murine macrophage cells (RAW264.7), activation of mPRs by progesterone modified to be active only extracellularly by conjugation to BSA (P4BSA, 1.0×10?7 mol/l) caused a pro-inflammatory shift in mRNA expression profile, with significant up-regulation of the expression of cyclooxygenase 2 (Ptgs2), Il1B, and Tnf and down-regulation of membrane progesterone receptor alpha (Paqr7) and oxytocin receptor (Oxtr). Pretreatment with PD98059, a MEK 1/2 inhibitor, significantly reduced P4BSA-induced Il1B, Tnf and Ptgs2 mRNA. Inhibition of protein kinase A (PKA) by H89 blocked P4BSA-induced Il1B and Tnf mRNA levels. P4BSA induced rapid phosphorylation of MEK1/2 and cAMP responsive element binding protein (CREB, a downstream target of PKA). This phosphorylation was inhibited by pretreatment with PD98059 and H89, respectively, revealing that MEK1/2 and PKA are two of the components involved in mPR signaling. Taken together, these data demonstrate that changes in membrane progesterone receptor alpha expression and signaling in macrophages are associated with the inflammatory responses; and that these changes might contribute to the functional withdrawal of progesterone related to labor. PMID:25472814

  8. Modulation of cytochrome P4501A1 activity by ascorbigen in murine hepatoma cells.

    PubMed

    Stephenson, P U; Bonnesen, C; Bjeldanes, L F; Vang, O

    1999-10-01

    Modulation of cytochrome P4501A1 (CYP1A1) activity is a mechanism whereby indoles present in cruciferous vegetables could affect the metabolism of xenobiotics. Ascorbigen (ASG) is the predominant indole formed during the degradation of glucobrassicin, although the mechanism by which ASG modulates CYP1A1 activity is not known. The major focus of this study was to examine the mechanism of CYP induction by ASG using a murine hepatoma-derived cell line (Hepa 1c1c7). ASG was shown to induce the activity of 7-ethoxyresorufin O-deethylase, a marker for CYP1A1, in a concentration-responsive manner with a maximum induction at 700 microM. Maximum ASG induction after 24-hr treatment was 7% of maximal CYP1Al activity induced by the well-known potent CYP1A1 inducer, indolo[3,2-b]carbazole (ICZ) (1 microM), and the EC50 values differed by 2-fold. The CYP1A1 activity increased continuously up to 72 hr, where ASG showed an induction efficiency in the same range as for the positive control (1 microM ICZ) after 24 hr, whereas the CYP1A1 protein level, measured by Western blot analysis, was maximally induced after 24 hr. ASG significantly inhibited CYP1A1 activity in whole cells at concentrations above 1 microM. ASG increased the chloramphenicol acetyl transferase (CAT) activity via a CAT reporter construct containing a dioxin-responsive element in Hepa 1c1c7 cells, indicating involvement of the aryl hydrocarbon receptor. ASG was shown to be transformed into ICZ, or a compound with the same chromatographic mobility as ICZ, in the medium. Taken together, the results indicate that ASG inhibits CYP1A1 activity at low concentrations, but induces the same activity at higher concentrations. PMID:10484072

  9. Interleukin-1 production after treatment with non-ionic surfactants in a murine keratinocytes cell line.

    PubMed

    Corsini, E; Marinovich, M; Marabini, L; Chiesara, E; Galli, C L

    1994-06-01

    Detergents are well known irritating agents in human as well as in animal models. Using a murine keratinocyte cell line (HEL30) changes in the interleukin-1alpha profile were characterized in response to three non-ionic detergents, all widely used in the cosmetics industry. The compounds used in this study were the most active (dodoxynol-9, Delta-9), moderate (polyglyceryl-4-lauryl ether, PEL) and mild (PEG-20-glyceryl ricinoleate + ricinoleamide DEA, PEG) in inducing cytotoxicity, measured as lactate dehydrogenase leakage and de novo protein synthesis, on the same cell line after 2 hr of treatment. All of the surfactants tested were able to induce IL-1alpha production both at a secretory and cell-associated level. However, in order to achieve a similar IL-1 production different concentrations of surfactants were necessary. It was possible to calculate an EC(50) for IL-1alpha release of 52.9 mug/ml for Delta-9, of 293.7 mug/ml for PEL and of greater than 9000 mug/ml for PEG. At the concentration of 30 mug/ml no release could be detected even after 24 hr of treatment with PEL or PEG. A time-course experiment also showed significant amounts of IL-1alpha 20 min after treatment with Delta-9. These data confirmed Delta-9 as the most potent of the three non-ionic detergents tested in inducing IL-1alpha release. The surfactants were also tested in vivo using the modified Draize test. Once again Delta-9 was the most active, followed by PEL and PEG. Considering the key role of IL-1 in the inflammatory response, the release of this cytokine by keratinocytes in vitro could be used as a more specific (in comparison with classical cytotoxic markers) and early marker to determine the irritant potential of water-soluble chemicals. PMID:20692927

  10. Expression of mink cell focus-forming murine leukemia virus-related transcripts in AKR mice

    SciTech Connect

    Khan, A.S.; Laigret, F.; Rodi, C.P.

    1987-03-01

    The authors used a synthetic 16-base-pair mink cell focus-forming (MCF) env-specific oligomer as radiolabeled probe to study MCF murine leukemia virus (MuLV)-related transcripts in brain, kidney, liver, spleen, and thymus tissues of AKR mice ranging from 5 weeks to 6 months (mo) of age. Tissue-specific expression of poly(A)/sup +/ RNAs was seen. In addition, all the tissues tested contained 3.0-kb messages. The transcription of these MCF-related mRNAs was independent of the presence of ecotropic and xenotropic MuLVs. In general, expression of the MCF env-related transcripts appeared to peak at 2 mo of age; these messages were barely detectable in brain, kidney, liver, and spleen tissues after 2 mo and in thymus tissue after 4 mo of age. All of the subgenomic MCF env-related mRNAs appeared to contain the 190-base-pair cellular DNA insert, characteristic of the long terminal repeats associated with endogenous MCF env-related proviruses. No genomic-size (8.4-kb) transcripts corresponding to endogenous MCF-related proviruses were detected. An 8.4-kb MCF env-related mRNA was first seen at 3 mo of age, exclusively in thymus tissue. This species most likely represents the first appearance of a recombinant MCF-related MuLV genome. The transcripts which were detected in thymus tissue might be involved in the generation of leukemogenic MCF viruses.

  11. Type I Interferon Signaling Enhances CD8+ T Cell Effector Function and Differentiation during Murine Gammaherpesvirus 68 Infection

    PubMed Central

    Jennings, Ryan N.; Grayson, Jason M.

    2014-01-01

    ABSTRACT CD8+ T cell responses are critical to the control of replication and reactivation associated with gammaherpesvirus infection. Type I interferons (IFNs) have been shown to have direct and indirect roles in supporting CD8+ T cell development and function during viral infection; however, the role of type I interferons during latent viral infection has not been examined. Mice deficient in type I IFN signaling (IFNAR1?/? mice) have high levels of reactivation during infection with murine gammaherpesvirus 68 (MHV68), a murine gammaherpesvirus model for Epstein-Barr virus. We hypothesized that type I IFNs function to enhance the anti-gammaherpesvirus CD8+ T cell response. To test this, IFNAR1?/? mice were infected with MHV68 and the CD8+ T cell response was analyzed. In the absence of type I IFN signaling, there was a marked increase in short-lived effector CD8+ T cells, and MHV68-specific CD8+ T cells had upregulated expression of PD-1 and reduced tumor necrosis factor alpha (TNF-?), gamma IFN (IFN-?), and interleukin-2 (IL-2) production. Suppressing MHV68 replication early in infection using the antiviral cidofovir rescued CD8+ T cell cytokine production and reduced PD-1 expression. However, suppressing high levels of reactivation in IFNAR1?/? mice failed to improve CD8+ T cell cytokine production during latency. T cell-specific abrogation of type I IFN signaling showed that the effects of type I IFNs on the CD8+ T cell response during MHV68 infection are independent of direct type I IFN signaling on T cells. Our findings support a model in which type I IFNs likely suppress MHV68 replication, thus limiting viral antigen and facilitating an effective gammaherpesvirus-directed CD8+ T cell response. IMPORTANCE The murine gammaherpesvirus MHV68 has both genetic and biologic homology to the human gammaherpesvirus Epstein-Barr virus (EBV), which infects over 90% of humans. Latent EBV infection and reactivation are associated with various life-threatening diseases and malignancies. Host suppression of gammaherpesvirus latency and reactivation requires both CD8+ T cells as well as type I interferon signaling. Type I IFNs have been shown to critically support the antiviral CD8+ T cell response in other virus models. Here, we identify an indirect role for type I IFN signaling in enhancing gammaherpesvirus-specific CD8+ T cell cytokine production. Further, this function of type I IFN signaling can be partially rescued by suppressing viral replication during early MHV68 infection. Our data suggest that type I IFN signaling on non-T cells can enhance CD8+ T cell function during gammaherpesvirus infection, potentially through suppression of MHV68 replication. PMID:25253356

  12. Optimization of fibrin scaffolds for differentiation of murine embryonic stem cells into neural lineage cells

    Microsoft Academic Search

    Stephanie M. Willerth; Kelly J. Arendas; David I. Gottlieb; Shelly Elese Sakiyama-Elbert

    2006-01-01

    The objective of this research was to determine the appropriate cell culture conditions for embryonic stem (ES) cell proliferation and differentiation in fibrin scaffolds by examining cell seeding density, location, and the optimal concentrations of fibrinogen, thrombin, and aprotinin (protease inhibitor). Mouse ES cells were induced to become neural progenitors by adding retinoic acid for 4 days to embryoid body

  13. Molecular Characterization of c-Abl\\/c-Src Kinase Inhibitors Targeted against Murine Tumour Progenitor Cells that Express Stem Cell Markers

    Microsoft Academic Search

    Thomas Kruewel; Silvia Schenone; Marco Radi; Giovanni Maga; Astrid Rohrbeck; Maurizio Botta; Juergen Borlak; Maria A. Deli

    2010-01-01

    BackgroundThe non-receptor tyrosine kinases c-Abl and c-Src are overexpressed in various solid human tumours. Inhibition of their hyperactivity represents a molecular rationale in the combat of cancerous diseases. Here we examined the effects of a new family of pyrazolo [3,4-d] pyrimidines on a panel of 11 different murine lung tumour progenitor cell lines, that express stem cell markers, as well

  14. Generation and functional characterization of a clonal murine periportal Kupffer cell line from H-2Kb tsA58 mice

    Microsoft Academic Search

    Daniel Dory; Hakim Echchannaoui; Maryse Letiembre; Fabrizia Ferracin; Jean Pieters; Yoshiyuki Adachi; Sachiko Akashi; Werner Zimmerli; Regine Landmann

    2003-01-01

    Murine Kupffer cells (KCs) are heter- ogeneous and survive only for a short time in vitro. Here, a clonal, murine KC line was generated from transgenic mice, expressing the thermolabile mu- tant tsA58 of the Simian virus 40 large T antigen under the control of the H-2Kb promoter. Thirty- three degrees Celcius and 37°C but not 39°C have been permissive

  15. Highly malignant behavior of a murine oligodendrocyte precursor cell line following transplantation into the demyelinated and nondemyelinated central nervous system.

    PubMed

    Hansmann, Florian; Pringproa, Kidsadagon; Ulrich, Reiner; Sun, Yanyong; Herder, Vanessa; Kreutzer, Mihaela; Baumgärtner, Wolfgang; Wewetzer, Konstantin

    2012-01-01

    Understanding the basic mechanisms that control CNS remyelination is of direct clinical relevance. Suitable model systems include the analysis of naturally occurring and genetically generated mouse mutants and the transplantation of oligodendrocyte precursor cells (OPCs) following experimental demyelination. However, aforementioned studies were exclusively carried out in rats and little is known about the in vivo behavior of transplanted murine OPCs. Therefore in the present study, we (i) established a model of ethidium bromide-induced demyelination of the caudal cerebellar peduncle (CCP) in the adult mouse and (ii) studied the distribution and marker expression of the murine OPC line BO-1 expressing the enhanced green fluorescent protein (eGFP) 10 and 17 days after stereotaxic implantation. Injection of ethidium bromide (0.025%) in the CCP resulted in a severe loss of myelin, marked astrogliosis, and mild to moderate axonal alterations. Transplanted cells formed an invasive and liquorogenic metastasizing tumor, classified as murine giant cell glioblastoma. Transplanted BO-1 cells displayed substantially reduced CNPase expression as compared to their in vitro phenotype, low levels of MBP and GFAP, prominent upregulation of NG2, PDGFR?, nuclear p53, and an unaltered expression of signal transducer and activator of transcription (STAT)-3. Summarized environmental signaling in the brain stem was not sufficient to trigger oligodendrocytic differentiation of BO-1 cells and seemed to block CNPase expression. Moreover, the lack of the remyelinating capacity was associated with tumor formation indicating that BO-1 cells may serve as a versatile experimental model to study tumorigenesis of glial tumors. PMID:22420305

  16. T-cell Intracellular Antigen (TIA)-Proteins Deficiency in Murine Embryonic Fibroblasts Alters Cell Cycle Progression and Induces Autophagy

    PubMed Central

    Sánchez-Jiménez, Carmen; Izquierdo, José M.

    2013-01-01

    Mice lacking either T-cell intracellular antigen 1 (TIA1) or TIA1 related/like protein (TIAR/TIAL1) show high rates of embryonic lethality, suggesting a relevant role for these proteins during embryonic development. However, intrinsic molecular and cellular consequences of either TIA1 or TIAR deficiency remain poorly defined. By using genome-wide expression profiling approach, we demonstrate that either TIA1 or TIAR inactivation broadly alter normal development-associated signalling pathways in murine embryonic fibroblasts (MEF). Indeed, these analyses highlighted alterations of cytokine-cytokine and ECM-receptor interactions and Wnt, MAPK, TGF-beta dependent signalling pathways. Consistent with these results, TIA1 and TIAR knockout (KO) MEF show reduced rates of cell proliferation, cell cycle progression delay and increased cell size. Furthermore, TIA-proteins deficiency also caused metabolic deficiencies, increased ROS levels and DNA damage, promoting a gentle rise of cell death. Concomitantly, high rates of autophagy were detected in both TIA1 and TIAR KO MEF with induction of the formation of autophagosomes, as evidenced by the up-regulation of the LC3B protein, and autolysosomes, measured by colocalization of LC3B and LAMP1, as a survival mechanism attempt. Taken together, these observations support that TIA proteins orchestrate a transcriptome programme to activate specific developmental decisions. This program is likely to contribute to mouse physiology starting at early stages of the embryonic development. TIA1/TIAR might function as cell sensors to maintain homeostasis and promote adaptation/survival responses to developmental stress. PMID:24086455

  17. Exposure to low level GSM 935 MHZ radiofrequency fields does not induce apoptosis in proliferating or differentiated murine neuroblastoma cells.

    PubMed

    Moquet, J; Ainsbury, E; Bouffler, S; Lloyd, D

    2008-01-01

    The aim of this study was to investigate whether radiofrequency (RF) fields characteristic of mobile phones at non-thermal levels can induce apoptosis in murine neuroblastoma (N2a) cells in both proliferating and differentiated states. Cells were exposed continuously for 24 h to one of the three 935-MHz RF signals: global system for mobile communication (GSM) basic, GSM talk and a continuous wave, unmodulated signal; all at a specific energy absorption rate of 2 W kg(-1). The measured increase in temperature of the cells due to the RF fields was around 0.06 degrees C. At a number of time points between 0 and 48 h post-exposure, the cells were assessed for apoptosis under a fluorescence microscope using three independent assays: Annexin V, caspase activation and in situ end-labelling. No statistically significant differences in apoptosis levels were observed between the exposed and sham-exposed cells using the three assays at any time point post-exposure. These data suggest that RF exposures, characteristic of GSM mobile phones, do not significantly affect the apoptosis levels in proliferating and differentiated murine neuroblastoma cell line N2a. PMID:18550513

  18. Overexpression of VEGF183 promotes murine breast cancer cell proliferation in vitro and induces dilated intratumoral microvessels.

    PubMed

    Zhang, Huiyong; Chen, Ying; Fan, Binglin; Wang, Wenfeng; Zhu, Wuling

    2015-05-01

    Vascular endothelial growth factor (VEGF) was considered as a critical growth factor for tumor expansion. The roles of VEGF121, VEGF165, and VEGF189 in tumor growth have been intensely investigated; however, involvements of another extracellular matrix (ECM)-binding VEGF isoform, namely VEGF183 (six amino acids shorter than VEGF189 in exon 6a), in physiological or pathological processes are still unclear although the wide tissue distribution. To investigate the role of VEGF183 in carcinogenesis, we generated murine breast cancer cell (EMT-6) clones stably overexpressing VEGF183, VEGF121, VEGF165, and VEGF189 shortened as V183, V121, V165, and V189, respectively. Methylthiazolyldiphenyl-tetrazolium bromide (MTT) results showed that VEGF183, like all other VEGF-overexpressing isoforms except for VEGF121, could enhance the proliferation of mouse breast cancer EMT-6 cells. Immunochemistry results displayed that overexpressing VEGF183 and VEGF189 in EMT-6 cells induced larger proportional dilated microvessels. On the other hand, results from cell wound healing experiments demonstrated that all of the VEGF-overexpressing isoforms could increase the chemotaxis of EMT-6 cells in vitro. In conclusion, our results supported the idea that overexpression of VEGF183 promotes murine breast cancer cell proliferation in vitro and induces dilated intratumoral microvessels, and it plays a dissimilar role in comparison with that of VEGF189. PMID:25577246

  19. Influence of extremely-low-frequency magnetic field on antioxidative melatonin properties in AT478 murine squamous cell carcinoma culture

    Microsoft Academic Search

    K. ?wirska-Korczala; M. Adamczyk-Sowa; R. Polaniak; P. Sowa; E. Birkner; Z. Drzazga; T. Brzozowski; S. J. Konturek

    2004-01-01

    Effects of melatonin, extremely-low-frequency magnetic field (ELF-MF), and their combination on AT478 murine squamous cell\\u000a carcinoma line were studied. Manganese superoxide dismutase (MnSOD), copper-zinc superoxide dismutase (Cu\\/ZnSOD), and glutathione\\u000a peroxidase (GSH-Px) were used as markers of cells antioxidative status, and malondialdehyde (MDA) level was used as a marker\\u000a of lipid peroxidation. After melatonin treatment, antioxidative enzyme activities were increased and

  20. The third helix of the murine Hoxc8 homeodomain facilitates protein transduction in mammalian cells

    SciTech Connect

    Kong, Kyoung-Ah; Gadi, Jogeswar; Park, Hyoung Woo [Department of Anatomy, Embryology Laboratory, Brain Korea 21 Project for Medical Science, Yonsei University College of Medicine, 134 Shinchon-dong, Sodaemun-gu, Seoul 120-752 (Korea, Republic of); Bok, Jinwoong [Department of Anatomy, Embryology Laboratory, Brain Korea 21 Project for Medical Science, Yonsei University College of Medicine, 134 Shinchon-dong, Sodaemun-gu, Seoul 120-752 (Korea, Republic of)], E-mail: bokj@yuhs.ac; Kim, Myoung Hee [Department of Anatomy, Embryology Laboratory, Brain Korea 21 Project for Medical Science, Yonsei University College of Medicine, 134 Shinchon-dong, Sodaemun-gu, Seoul 120-752 (Korea, Republic of)], E-mail: mhkim1@yuhs.ac

    2008-12-05

    Previously, we have demonstrated that purified Hoxc8 homeoprotein has the ability to penetrate the cellular membrane and can be transduced efficiently into COS-7 cells. Moreover, the Hoxc8 protein is able to form a complex with DNA molecules in vitro and helps the DNA be delivered intracellularly, serving as a gene delivery vehicle. Here, we further analyzed the membrane transduction activity of Hoxc8 protein and provide the evidence that the 16 amino acid (a.a.191-206, 2.23 kDa) third helix of murine Hoxc8 protein is an efficient protein transduction domain (PTD). When the 16 amino acid peptide was fused at the carboxyl terminal of enhanced green fluorescence protein (EGFP), the fusion proteins were transduced efficiently into the primary pig fetal fibroblast cells. The transduction efficiency increased in a concentration-dependent manner up to 1 {mu}M, and appeared to plateau above a concentration of 1 {mu}M. When tandem multimers of PTD, EGFP-PTD(2), EGFP-PTD(3), EGFP-PTD(4), and EGFP-PTD(5), were analyzed at 500 nM of concentration, the penetrating efficiency increased in a dose-dependent manner. As the number of PTDs increased, the EGFP signal also increased, although the signal maintained plateau after EGFP-PTD(3). These results indicate that the 16 amino acid third helix is the key element responsible for the membrane transduction activity of Hoxc8 proteins, and further suggest that the small peptide could serve as a therapeutic delivery vehicle for large cargo proteins.

  1. DEFICIENT IN VITRO AND IN VIVO PHAGOCYTOSIS OF APOPTOTIC T CELLS BY RESIDENT MURINE ALVEOLAR MACROPHAGES

    PubMed Central

    Hu, Bin; Sonstein, Joanne; Christensen, Paul J.; Punturieri, Antonello; Curtis, Jeffrey L.

    2015-01-01

    Apoptotic lymphocytes are readily identified in murine lungs, both during the response to particulate antigen and in normal mice. Because apoptotic lymphocytes are seldom detected in other organs, we hypothesized that alveolar macrophages (AMø) clear apoptotic lymphocytes poorly. To test this hypothesis, we compared in vitro phagocytosis of apoptotic thymocytes by resident AMø and peritoneal Mø (PMø) from normal C57BL/6 mice. AMø were deficient relative to PMø both in percentage containing apoptotic thymocytes (19.1 ± 1.0% versus 96.0 ± 2.6% positive) and in phagocytic index (0.23 ± 0.02 versus 4.2 ± 0.67). This deficiency was not due to kinetic differences, was seen with six other inbred mouse strains, and was not observed using carboxylate-modified polystyrene microbeads. Annexin V blockade indicated that both Mø types cleared apoptotic T cells by a mechanism involving phosphatidylserine expression. By contrast, neither mAb blockade of a variety of receptors (CD11b, CD29, CD51, and CD61) known to be involved in clearance of apoptotic cells, nor the tetrapeptide RGDS, blocked ingestion by either type of Mø. To confirm these studies, apoptotic thymocytes were given intratracheally or intraperitoneally to normal mice and then AMø or PMø were recovered 30–240 min later. Ingestion of apoptotic thymocytes by AMø in vivo was significantly decreased at all times. Defective ingestion of apoptotic lymphocytes may preserve AMø capacity to produce proinflammatory cytokines in host defense, but could contribute to development of autoimmunity by failing to eliminate nucleosomes. PMID:10925298

  2. Murine Peyer's patch dendritic cells prime naïve CD4 + T cells to produce interferon-?

    Microsoft Academic Search

    Ayuko Sato; Masaaki Hashiguchi; Satoshi Hachimura; Shuichi Kaminogawa

    2002-01-01

    We investigated the role of Peyer's patch (PP) dendritic cells (DCs) in the production of interferon (IFN)-? from naïve CD4+ T cells of T cell receptor transgenic mice. PP DCs were found to prime naïve CD4+ T cells for the production of higher levels of IFN-?, when compared to spleen (SP) DCs. However, a similar level of interleukin-12\\u000a (IL-12) production

  3. Bilirubin and light induced cell death in a murine lymphoma cell line

    Microsoft Academic Search

    Terje Christensen; Ellen B. Roll; Alicja Jaworska; Gunnar Kinn

    2000-01-01

    Cells from the mouse lymphoma cell line L5178Y-R were exposed to blue light from phototherapy lamps in the presence of solutions of 160 ?M bilirubin supplemented with serum albumin. HPLC analysis showed that the bilirubin solution was photooxidised as a function of increasing light dose. The cells were stained with trypan blue to score necrosis, and apoptosis was assayed by

  4. Comparative neoplastic transformation responses of Balb\\/3T3 cells, Syrian hamster embryo cells, and Rauscher murine leukemia virus-infected Fischer 344 rat embryo cells to chemical carcinogens

    Microsoft Academic Search

    V. C. Dunkel; R. J. Pienta; A. Sivak; K. A. Traul

    1981-01-01

    This study provides a preliminary comparative evaluation of the responses to a series of 49 chemicals, in in vitro transformation assays, of Balb\\/3T3 cells, Syrian hamster embryo cells, and Fischer 344 rat embryo cells infected with Rauscher murine leukemia virus. The chemicals assayed included aromatic amines; polycylic aromatic hydrocarbons; alkylating agents; nitrosamines, hydrazines, and related compounds; heterocyclic compounds, amides, ureas,

  5. Murine T cell activation is regulated by surfen (bis-2-methyl-4-amino-quinolyl-6-carbamide)

    SciTech Connect

    Warford, Jordan, E-mail: jordan.warford@dal.ca [Department of Pathology, Dalhousie University, Tupper Building, 5850 College Street, Halifax, Nova Scotia B3H 4R2 (Canada)] [Department of Pathology, Dalhousie University, Tupper Building, 5850 College Street, Halifax, Nova Scotia B3H 4R2 (Canada); Doucette, Carolyn D., E-mail: carolyn.doucette@dal.ca [Department of Microbiology and Immunology, Dalhousie University, Tupper Building, 5850 College Street, Halifax, Nova Scotia B3H 4R2 (Canada); Hoskin, David W., E-mail: d.w.hoskin@dal.ca [Department of Pathology, Dalhousie University, Tupper Building, 5850 College Street, Halifax, Nova Scotia B3H 4R2 (Canada); Department of Microbiology and Immunology, Dalhousie University, Tupper Building, 5850 College Street, Halifax, Nova Scotia B3H 4R2 (Canada); Easton, Alexander S., E-mail: alexander.easton@dal.ca [Department of Pathology, Dalhousie University, Tupper Building, 5850 College Street, Halifax, Nova Scotia B3H 4R2 (Canada); Department of Microbiology and Immunology, Dalhousie University, Tupper Building, 5850 College Street, Halifax, Nova Scotia B3H 4R2 (Canada); Department of Surgery (Neurosurgery), Dalhousie University, Tupper Building, 5850 College Street, Halifax, Nova Scotia B3H 4R2 (Canada)

    2014-01-10

    Highlights: •Surfen is the first inhibitor of glycosaminoglycan function to be studied in murine T cells. •Surfen reduces T cell proliferation stimulated in vitro and in vivo. •Surfen reduces CD25 expression in T cells activated in vivo but not in vitro. •Surfen increases T cell proliferation when T cell receptor activation is bypassed. •Surfen’s effects are blocked by co-administration of heparin sulfate. -- Abstract: Surfen (bis-2-methyl-4-amino-quinolyl-6-carbamide) binds to glycosaminoglycans (GAGs) and has been shown to influence their function, and the function of proteoglycans (complexes of GAGs linked to a core protein). T cells synthesize, secrete and express GAGs and proteoglycans which are involved in several aspects of T cell function. However, there are as yet no studies on the effect of GAG-binding agents such as surfen on T cell function. In this study, surfen was found to influence murine T cell activation. Doses between 2.5 and 20 ?M produced a graduated reduction in the proliferation of T cells activated with anti-CD3/CD28 antibody-coated T cell expander beads. Surfen (20 mg/kg) was also administered to mice treated with anti-CD3 antibody to activate T cells in vivo. Lymphocytes from surfen-treated mice also showed reduced proliferation and lymph node cell counts were reduced. Surfen reduced labeling with a cell viability marker (7-ADD) but to a much lower extent than its effect on proliferation. Surfen also reduced CD25 (the ?-subunit of the interleukin (IL)-2 receptor) expression with no effect on CD69 expression in T cells treated in vivo but not in vitro. When receptor activation was bypassed by treating T cells in vitro with phorbyl myristate acetate (10 ng/ml) and ionomycin (100 ng/ml), surfen treatment either increased proliferation (10 ?M) or had no effect (2.5, 5 and 20 ?M). In vitro treatment of T cells with surfen had no effect on IL-2 or interferon-? synthesis and did not alter proliferation of the IL-2 dependent cell line CTLL-2. The effect of surfen was antagonized dose-dependently by co-treatment with heparin sulfate. We conclude that surfen inhibits T cell proliferation in vitro and in vivo. When T cell receptor-driven activation is bypassed surfen had a neutral or stimulatory effect on T cell proliferation. The results imply that endogenous GAGs and proteoglycans play a complex role in promoting or inhibiting different aspects of T cell activation.

  6. Bilirubin- and light induced cell death in a murine lymphoma cell line.

    PubMed

    Christensen, T; Roll, E B; Jaworska, A; Kinn, G

    2000-11-01

    Cells from the mouse lymphoma cell line L5178Y-R were exposed to blue light from phototherapy lamps in the presence of solutions of 160 microM bilirubin supplemented with serum albumin. HPLC analysis showed that the bilirubin solution was photooxidised as a function of increasing light dose. The cells were stained with trypan blue to score necrosis, and apoptosis was assayed by the terminal deoxynucleotide transferase assay (TdT) or by studying the nuclear structure in cells stained with propidium iodide. A rapidly developing apoptosis was observed after light doses killing 60-80% of the cells as judged from the trypan blue exclusion test. The fraction of apoptotic cells was smaller than the fraction of necrotic cells. Exposure of the cells to fractions of light at a high dose rate was compared to the effect of the same total dose at a lower dose rate given as a single fraction. No large differences were found, however, there was a tendency of a higher degree of necrosis as well as apoptosis in the cells receiving the light in fractions at a high dose rate. PMID:11233646

  7. Selective induction of apoptosis in murine skin carcinoma cells (CH72) by an ethanol extract of Lentinula edodes.

    PubMed

    Gu, Yu-Huan; Belury, Martha A

    2005-03-18

    The effects of ethanol extracts from four species of mushroom fruiting bodies, mushroom spores and mushroom cultured broth, were assessed for modulation of cell proliferation and apoptosis in murine skin carcinoma cells (CH72) and non-tumorigenic epidermal cells (C50). While extracts from mycelia of Grifola frondosa, Ganoderma lucidum, Hericium erinaceus, or from spores of G. lucidum exerted little, if any, effect on proliferation, the ethanol-soluble extract of Lentinula edodes (L. edodes) significantly decreased cell proliferation of CH72 cells. There were no changes in the proliferative response of the non-tumorigenic keratinocyte cell line, C50, to any of the mushroom extracts tested. To analyze cell proliferation and apoptosis, fluorescent DNA-microscopy with ethidium bromide and acridine orange staining of cells revealed L. edodes reduced cell proliferation and induced apoptosis in time- and dose-dependent manners in carcinoma cells but had no effect in non-tumorigenic cells (C50). Cell cycle analysis demonstrated that L. edodes extract induced a transient G(1) arrest, with no changes observed in the non-tumorigenic cells (C50). PMID:15737684

  8. Anti-CD20-interferon-? fusion protein therapy of murine B cell lymphomas

    PubMed Central

    Trinh, K. Ryan; Vasuthasawat, Alex; Steward, Kristopher K.; Yamada, Reiko E.; Timmerman, John M.; Morrison, Sherie L.

    2013-01-01

    Type I interferons (IFN?/?) are cytokines with a broad spectrum of anti-tumor activities including anti-proliferative, pro-apoptotic, and immunostimulatory effects, and are potentially useful in the treatment of B cell malignancies and other cancers. To improve anti-tumor potency and diminish the systemic side effects of IFN, we recently developed anti-CD20-IFN? fusion proteins with in vitro and in vivo efficacy against both mouse and human lymphomas expressing CD20. Since IFN? binds more tightly to the IFN?/? receptor (IFNAR) and has more potent anti-tumor activities, we have now constructed an anti-CD20 fusion protein with murine IFN? (mIFN?). Anti-CD20-mIFN? was more potent than recombinant mIFN? and anti-CD20-mIFN? in inhibiting the proliferation of a mouse B cell lymphoma expressing human CD20 (38C13-huCD20). Growth inhibition was accompanied by caspase-independent apoptosis and DNA fragmentation. The efficacy of anti-CD20-mIFN? required the physical linkage of mIFN? to anti-CD20 antibody (Ab). Importantly, anti-CD20-mIFN? was active against tumor cells expressing low levels of IFNAR (38C13-huCD20 IFNARlo). In vivo, established 38C13-huCD20 tumors were largely insensitive to rituximab or a non-targeted mIFN? fusion protein, yet treatment with anti-CD20-mIFN? eradicated 83% of tumors. Anti-CD20-mIFN? was also more potent in vivo against 38C13-huCD20 than anti-CD20-mIFN?, curing 75% versus 25% of tumors (p = 0.001). Importantly, while anti-CD20-mIFN? could not eradicate 38C13-huCD20 IFNARlo tumors, anti-CD20-mIFN? treatment prolonged survival (p = 0.0003), and some animals remained tumor-free. Thus, Ab fusion proteins targeting mIFN? to tumors show promise as therapeutic agents, especially for use against tumors resistant to the effects of mIFN?. PMID:23719241

  9. The role of Hibiscus sabdariffa L. (Roselle) in maintenance of ex vivo murine bone marrow-derived hematopoietic stem cells.

    PubMed

    Abdul Hamid, Zariyantey; Lin Lin, Winnie Hii; Abdalla, Basma Jibril; Bee Yuen, Ong; Latif, Elda Surhaida; Mohamed, Jamaludin; Rajab, Nor Fadilah; Paik Wah, Chow; Wak Harto, Muhd Khairul Akmal; Budin, Siti Balkis

    2014-01-01

    Hematopoietic stem cells- (HSCs-) based therapy requires ex vivo expansion of HSCs prior to therapeutic use. However, ex vivo culture was reported to promote excessive production of reactive oxygen species (ROS), exposing HSCs to oxidative damage. Efforts to overcome this limitation include the use of antioxidants. In this study, the role of Hibiscus sabdariffa L. (Roselle) in maintenance of cultured murine bone marrow-derived HSCs was investigated. Aqueous extract of Roselle was added at varying concentrations (0-1000 ng/mL) for 24 hours to the freshly isolated murine bone marrow cells (BMCs) cultures. Effects of Roselle on cell viability, reactive oxygen species (ROS) production, glutathione (GSH) level, superoxide dismutase (SOD) activity, and DNA damage were investigated. Roselle enhanced the survival (P < 0.05) of BMCs at 500 and 1000 ng/mL, increased survival of Sca-1(+) cells (HSCs) at 500 ng/mL, and maintained HSCs phenotype as shown from nonremarkable changes of surface marker antigen (Sca-1) expression in all experimental groups. Roselle increased (P < 0.05) the GSH level and SOD activity but the level of reactive oxygen species (ROS) was unaffected. Moreover, Roselle showed significant cellular genoprotective potency against H2O2-induced DNA damage. Conclusively, Roselle shows novel property as potential supplement and genoprotectant against oxidative damage to cultured HSCs. PMID:25405216

  10. PI3K-AKT signaling is a downstream effector of retinoid prevention of murine basal cell carcinogenesis.

    PubMed

    So, Po-Lin; Wang, Grace Y; Wang, Kevin; Chuang, Mindy; Chiueh, Venice Calinisan; Kenny, Paraic A; Epstein, Ervin H

    2014-04-01

    Basal cell carcinoma (BCC) is the most common human cancer. We have demonstrated previously that topical application of the retinoid prodrug tazarotene profoundly inhibits murine BCC carcinogenesis via retinoic acid receptor ?-mediated regulation of tumor cell transcription. Because topical retinoids can cause adverse cutaneous effects and because tumors can develop resistance to retinoids, we have investigated mechanisms downstream of tazarotene's antitumor effect in this model. Specifically we have used (i) global expression profiling to identify and (ii) functional cell-based assays to validate the phosphoinositide 3-kinase (PI3K)/AKT/mTOR pathway as a downstream target pathway of tazarotene's action. Crucially, we have demonstrated that pharmacologic inhibition of this downstream pathway profoundly reduces murine BCC cell proliferation and tumorigenesis both in vitro and in vivo. These data identify PI3K/AKT/mTOR signaling as a highly attractive target for BCC chemoprevention and indicate more generally that this pathway may be, in some contexts, an important mediator of retinoid anticancer effects. PMID:24449057

  11. PI3K-AKT signaling is a downstream effector of retinoid prevention of murine basal cell carcinogenesis

    PubMed Central

    So, Po-Lin; Wang, Grace Y.; Wang, Kevin; Chuang, Mindy; Calinisan Chiueh, Venice; Kenny, Paraic A.; Epstein, Ervin H.

    2014-01-01

    Basal cell carcinoma (BCC) is the most common human cancer. We have demonstrated previously that topical application of the retinoid prodrug tazarotene profoundly inhibits murine BCC carcinogenesis via RAR?-mediated regulation of tumor cell transcription. Since topical retinoids can cause adverse cutaneous effects and since tumors can develop resistance to retinoids, we have investigated mechanisms downstream of tazarotene’s anti-tumor effect in this model. Specifically we have used (i) global expression profiling to identify and (ii) functional cell-based assays to validate the PI3K/AKT/mTOR pathway as a downstream target pathway of tazarotene’s action. Crucially, we have demonstrated that pharmacologic inhibition of this downstream pathway profoundly reduces murine BCC cell proliferation and tumorigenesis both in vitro and in vivo. These data identify PI3K/AKT/mTOR signaling as a highly attractive target for BCC chemoprevention and indicate more generally that this pathway may be, in some contexts, an important mediator of retinoid anti-cancer effects. PMID:24449057

  12. Paracrine Action of Mesenchymal Stem Cells Revealed by Single Cell Gene Profiling in Infarcted Murine Hearts

    PubMed Central

    Geng, Yongjian; Qian, Haiyan; Wang, Fan; Liu, Xiaohui; Shang, Meisheng; Nie, Shaoping; Liu, Nian; Du, Xin; Dong, Jianzeng; Ma, Changsheng

    2015-01-01

    Background Mesenchymal stem cells (MSCs) have been recently demonstrated as a promising stem cell type to rescue damaged myocardium after acute infarction. One of the most important mechanisms underlying their therapeutic effects is the secretion of paracrine factors. However, the expression profile of paracrine factors of MSCs in infarcted hearts, especially at single cell level, is poorly defined. Methods and Results We aimed to depict the transcriptional profile of paracrine factors secreted by MSCs in vivo, with particular interest in the comparison between normal and infarcted hearts. Bone marrow mesenchymal stem cells were isolated and injected into mice hearts immediately after infarction surgery. Bioluminescence imaging (BLI) indicated a proportion of cells still alive even up to 10 days post surgery. Paralleled with survived cells, cardiac function was significantly improved after MSC injection compared to that in PBS-injected mice, indicated by MRI and histology. Despite increased number of vessels in MSC-injected hearts, endothelial cells and cardiomyocytes transdifferentiation were not observed in infarcted hearts 5 days after infarction. Furthermore, laser capture microdissection (LCM) followed by high through-put real time PCR was employed in our study, uncovering that the injected MSCs, compared to local cardiomyocytes, displayed elevated levels of secreted factors. To further investigate the regulation of those factors, we performed single cell analysis to dissect the gene expression profile of MSCs at single cell level in infarcted and normal hearts, respectively. Consistent with the in vivo observation, a similar regulation pattern of those factors was detected in cultured MSCs under hypoxia. Conclusions Our study, for the first time, elucidated gene expression profiles, as well as regulation of paracrine factors, of MSCs at single cell level in vivo, indicating that paracrine factors from MSCs account for the improvement of cardiac function after infarction. PMID:26043119

  13. P hosp hol i pid (d iacyl , al kylacy I, al kenylacyl) and fatty acyl chain composition in murine mastocytoma cells

    Microsoft Academic Search

    Shim Yoshioka; Shigeru Nakashima; Yukio Okano; Himyuki Hasegawa; Anta Ichiyama; Yoshinori Nozawa

    The phospholipids from murine mastocytoma FMA3 and P-815 clone cells were quantitatively analyzed, and the major glycerophospholipids were examined for their fatty acyl chain distribution. In these cells, the content of histamine was less than 1\\/100 of nor