Sample records for murine sertoli cells

  1. 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.

  2. Genetically engineered immune privileged Sertoli cells

    PubMed Central

    Kaur, Gurvinder; Long, Charles R.; Dufour, Jannette M.

    2012-01-01

    Sertoli cells are immune privileged cells, important for controlling the immune response to male germ cells as well as maintaining the tolerogenic environment in the testis. Additionally, ectopic Sertoli cells have been shown to survive and protect co-grafted cells when transplanted across immunological barriers. The survival of ectopic Sertoli cells has led to the idea that they could be used in cell based gene therapy. In this review, we provide a brief overview of testis immune privilege and Sertoli cell transplantation, factors contributing to Sertoli cell immune privilege, the challenges faced by viral vector gene therapy, the use of immune privileged cells in cell based gene therapy and describe several recent studies on the use of genetically engineered Sertoli cells to provide continuous delivery of therapeutic proteins. PMID:22553487

  3. Sertoli cell tumour in an Amur tiger.

    PubMed

    Scudamore, C L; Meredith, A L

    2001-01-01

    The histological and immunohistochemical characteristics of a malignant Sertoli cell tumour in a 17-year-old Amur tiger (Panthera tigris altaica) are described. Histological examination of the primary lesion in the right testis and metastatic lesions throughout the internal organs showed a variable cellular pattern with an admixture of tubular structures divided by fine stroma filled with fusiform to stellate cells, and sheets of polygonal cells with abundant vacuolated cytoplasm. Immunohistochemical techniques demonstrated strong positive staining for neuron-specific enolase and variable positive staining for vimentin in neoplastic cells, supporting a diagnosis of a tumour of Sertoli cell origin. PMID:11428192

  4. Sertoli cell only syndrome: Status of sertoli cell maturation and function

    PubMed Central

    Jain, Manish; Halder, Ashutosh

    2012-01-01

    Background of the study: Mature and functional Sertoli cells are essential for the survival of germ cells in testes. In Sertoli cell only syndrome (SCOS), there is no germ cells. Then, question arises whether absence of germ cells in SCOS secondary to Sertoli cells immaturity or mal function. Sertoli cells maturational and functional status is unclear in SCOS. This study investigated status of maturation and function of Sertoli cells in patients with SCOS. Materials and Methods: The present study was comprised of 37 cases of SCOS and 50 normal control males. Detailed clinical examination and investigation were carried out as per pre-determined proforma. Semen analysis, hormonal analysis (FSH, LH, testosterone, etc.), and fine needle aspiration cytology (FNAC) of testes (bilateral) were performed. Fluorescence in situ hybridization (FISH) with XY probes was carried out in addition to conventional chromosome analysis to find out chromosomal abnormalities, in particular sex chromosome aneuploidy, including mosaicism. Yq microdeletion status was also investigated. The anti-mullerian hormone (AMH), inhibin B, and seminal lactate were estimated by ELISA methods. Results: The study did not find any case of high AMH. About 78% cases had low inhibin B, and 60% had low AMH. FSH was high in about 78% cases. Low level of lactate was found in 49% cases. There was one case of high level of inhibin B. There were 6 (16.2%) cases of chromosomal abnormality (2 mosaic Klinefelter and 4 Klinefelter syndrome) and 4 (10.8%) cases of Yq microdeletion. Conclusion: We conclude that Sertoli cell immaturity does not play any role in SCOS (no case of high AMH). It seems, in majority cases, Sertoli cells are functionally- and/or numerically-deficient (low inhibin B, AMH and lactate). However, in about 22% cases, Sertoli cell function and/or number remains normal (normal inhibin B, AMH). Inhibin B and FSH seems best predictor/marker of Sertoli cell function. PMID:23565483

  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. Numerical Relation of Spermatozoa to Sertoli Cells

    E-print Network

    Hague, Florence S.

    1914-01-01

    of the Sertoli cell, often clustering close about the nucleus. iSven when passing out the nucleus is frequent- ly very close to the spermatozoa. ^ Montgomery in speaking of ultimate spermatogonia one quarter of which he claims contain rods and three quarters...

  7. Sertoli cell signaling by Desert hedgehog regulates the male germline

    Microsoft Academic Search

    Mark J Bitgood; Liya Shen; Andrew P McMahon

    1996-01-01

    Background: In mammals, testis development is initiated in the embryo in response to the expression of the sex determining gene, Sry, in Sertoli cell precursors. Subsequently, Sertoli cells are thought to play a central role in male-specific cell interactions, including those that occur during spermatogenesis. However, the molecular nature of these interactions is poorly understood. Desert hedgehog (Dhh) encodes a

  8. Purinergic signalling mobilizes mitochondrial Ca2+ in mouse Sertoli cells

    PubMed Central

    Veitinger, Sophie; Veitinger, Thomas; Cainarca, Silvia; Fluegge, Daniela; Engelhardt, Corinna H; Lohmer, Stefan; Hatt, Hanns; Corazza, Sabrina; Spehr, Jennifer; Neuhaus, Eva M; Spehr, Marc

    2011-01-01

    Abstract Intimate bidirectional communication between Sertoli cells and developing germ cells ensures the integrity and efficiency of spermatogenesis. Yet, a conceptual mechanistic understanding of the physiological principles that underlie Sertoli cell autocrine and paracrine signalling is lacking. Here, we characterize a purinergic Ca2+ signalling network in immature mouse Sertoli cells that consists of both P2X2 and P2Y2 purinoceptor subtypes, the endoplasmic reticulum and, notably, mitochondria. By combining a transgenic mouse model with a dedicated bioluminescence imaging device, we describe a novel method to monitor mitochondrial Ca2+ mobilization in Sertoli cells at subcellular spatial and millisecond temporal resolution. Our data identify mitochondria as essential components of the Sertoli cell signalling ‘toolkit’ that control the shape of purinergic Ca2+ responses, and probably several other paracrine Ca2+-dependent signals. PMID:21859825

  9. GATA4 regulates Sertoli cell function and fertility in adult male mice

    PubMed Central

    Kyrönlahti, Antti; Euler, Rosemarie; Bielinska, Malgorzata; Schoeller, Erica L.; Moley, Kelle H.; Toppari, Jorma; Heikinheimo, Markku; Wilson, David B.

    2011-01-01

    Transcription factor GATA4 is expressed in Sertoli and Leydig cells and is required for proper development of the murine fetal testis. The role of GATA4 in adult testicular function, however, has remained unclear due to prenatal lethality of mice harboring homozygous mutations in Gata4. To characterize the function of GATA4 in the adult testis, we generated mice in which Gata4 was conditionally deleted in Sertoli cells using Cre-LoxP recombination with Amhr2-Cre. Conditional knockout (cKO) mice developed age-dependent testicular atrophy and loss of fertility, which coincided with decreases in the quantity and motility of sperm. Histological analysis demonstrated Sertoli cell vacuolation, impaired spermatogenesis, and increased permeability of the blood-testis barrier. RT-PCR analysis of cKO testes showed decreased expression of germ cell markers and increased expression of testicular injury markers. Our findings support the premise that GATA4 is a key transcriptional regulator of Sertoli cell function in adult mice. PMID:21172404

  10. Ultrastructure of the nucleus of human sertoli cells in normal and pathological testes

    Microsoft Academic Search

    E. Bustos-Obregón; P. Esponda

    1974-01-01

    The ultrastructure of the nucleus of human Sertoli cells has been studied in testicular samples from normal adults and patients with hypospermatogenesis or absence of the germinal cell line (Sertoli cell only syndrome).

  11. Sertoli cells and development of seminiferous epithelium Marie-Thrse HOCHEREAU-de REVIERS M. COUROT

    E-print Network

    Paris-Sud XI, Université de

    Sertoli cells and development of seminiferous epithelium Marie-Thérèse HOCHEREAU-de REVIERS M., Nouzilly, 37380 Monnaie, France. Summary. Sertoli cells partially control the development of impuberal lambs results in a decrease in the total number of Sertoli cells/testis while that of stem cells

  12. Vitamin A Modifies the Glycopeptide Composition of Cultured Sertoli Cells

    Microsoft Academic Search

    M. GALDIERI; L. NISTICO

    1986-01-01

    Sertoli cells obtained from prepubertal rat testes were cultured in the presence or absence of retinol. Incorpora- tion of monosaccharides and glycopeptide composition of the cells were studied under two experimental condi- tions. The results indicate that retinol increases the amount of mannose and glucosamine incorporated into cellular glycoconjugates. The labeled glycopeptides obtained from control and retinol-treated cells were separated

  13. Establishment and Characterization of Neonatal Mouse Sertoli Cell Lines

    Microsoft Academic Search

    MARIE-CLAUDE HOFMANN; KATHERINE S. VAN DER WEE; JAMIE L. DARGART; GHENIMA DIRAMI; LUIS DETTIN; MARTIN DYM

    2003-01-01

    Sertoli cells isolated from 6-day postpartum mouse testes were conditionally immortalized with the simian virus 40 large tumor antigen gene (SV40-LTAg) under the control of a promoter inducible with ponasterone A, an analog of ecdysone. This strategy produced 2 cell lines, which exhibited mixed phenotypes. We first tested the conditional expression of the LTAg gene in the presence or absence

  14. 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

  15. Establishment and characterization of neonatal mouse sertoli cell lines.

    PubMed

    Hofmann, Marie-Claude; Van Der Wee, Katherine S; Dargart, Jamie L; Dirami, Ghenima; Dettin, Luis; Dym, Martin

    2003-01-01

    Sertoli cells isolated from 6-day postpartum mouse testes were conditionally immortalized with the simian virus 40 large tumor antigen gene (SV40-LTAg) under the control of a promoter inducible with ponasterone A, an analog of ecdysone. This strategy produced 2 cell lines, which exhibited mixed phenotypes. We first tested the conditional expression of the LTAg gene in the presence or absence of ponasterone A. The results showed that both cell lines expressed LTAg when the inducer was present in the culture media. When ponasterone A was removed, the majority of the cells died. After 60 generations, however, the continued expression of LTAg in the absence of the hormone indicated that unknown changes may have occurred in the genome of the cells. One of the cell lines was further subcloned, resulting in 7 new lines exhibiting a morphology resembling that of Sertoli cells in tissue culture. Reverse transcriptase-polymerase chain reaction (RT-PCR) was performed on RNA collected from each cell line in order to determine which cells were phenotypically similar to Sertoli cells in vivo. All cell lines expressed the products of the Sertoli cell-specific genes stem cell factor (SCF) and sulfated glycoprotein-2 (SGP-2), in addition to alpha-inhibin, GATA-1, and steroidogenic factor-1. Further, the lines express growth and differentiation factors known to act upon germ cells in vivo and in vitro such as leukemia inhibitory factor (LIF), transforming growth factor beta (TGF-beta), and basic fibroblast growth factor (bFGF). Moreover, when used as feeder layers in cocultures, at least 2 of these lines are able to maintain the viability of type A spermatogonia for at least 7 days and to support the first steps of spermatogonial differentiation. PMID:12514093

  16. Malignant sertoli cell tumor in a goose (Anser cygnoides domesticus).

    PubMed

    Uetsuka, Koji; Suzuki, Takayuki; Doi, Kunio; Nunoya, Tetsuo

    2012-12-01

    This paper describes the pathologic features of a malignant Sertoli cell tumor found in an adult goose (Anser cygnoides domesticus). At necropsy, in addition to one large tumor mass (15 cm in diameter), multiple small tumor masses were observed over the peritoneum and mesenterium in the coelomic cavity. The large tumor mass was composed of sheets, lobules, and small islands of tumor cells, and elongated tumor cells lying perpendicular to fibrous connective tissue were characteristic. Such histopathologic characteristics were common to all the tumors. The tumor cells were immunohistochemically positive for neuron-specific enolase and S-100, and some tumor cells contained fine intracytoplasmic pigments that stained red by oil red O staining. These findings, taken together with the fact that one testis was markedly atrophied and bore no tumor cells and the other testis was not discernible, the present case was diagnosed as unilateral malignant Sertoli cell tumor arising from the unilateral testis. To our knowledge, this is the first report of Sertoli cell tumor in the goose. PMID:23397858

  17. Clinicopathologic features of ovarian Sertoli-Leydig cell tumors

    PubMed Central

    Zhang, Hai-Yan; Zhu, Jia-Er; Huang, Wen; Zhu, Jin

    2014-01-01

    Background: Ovarian Stertoli-Ledig cell tumor (SLCT) is a rare type of sex cord-stromal tumor of the ovary. The present study was to evaluate clinicalopahologic features and prognosis of patients with Sertoli-Leydig cell tumor treated by surgery and adjuvant chemotherapy during short term follow-up. Methods: A total of sixteen patients with ovarian Sertoli-Leydig cell tumor treated at the Obstetrics and Gynecology Hospital, Shanghai, China, between Jan 2001 and Dec 2011 were reviewed. The clinical data, treatment and prognosis were obtained from medical records. Results: The median age of the patients with ovarian Sertoli-Leydig cell tumor was about 27.5 years old in non-menopausal women, while the median age of menopausal women was about 63 years old. The most common complaint was with hormonal-related symptoms in the form of secondary amenorrhea and infinity, features of virilization, abdominal mass or irregular vaginal bleeding. All of sixteen patients underwent surgical staging and all were found to have stage I disease at the time of diagnosis. Eleven patients with intermediate and two patients with poorly differentiated tumors received adjuvant chemotherapy. There were differences found in operative time, blood loss and postoperative recovery time between laparotomy and laparoscopy. There were no disease-related deaths and all patients were under complete remission at the last follow-up. Conclusions: Ovarian Sertoli-Leydig cell tumors could happen in any period age of women. However, the tumors typically occur in the single side while still at the early stage, a favorable outcome could be achieved by surgery and adjuvant chemotherapy. Laparoscopy has similar surgical effects as laparotomy, but has a number of advantages. PMID:25400781

  18. Sertoli cell tumor with benign peritoneal implants associated with gonadoblastoma.

    PubMed

    Onida, Giovanna A; Bosincu, Luisanna; Dessole, Salvatore; Nicolae, Alina; Preda, Ovidiu; Cossu-Rocca, Paolo; Aneiros-Fernandez, Jose; Nogales, Francisco F

    2010-09-01

    We present a unique case of bilateral gonadoblastoma in a 23-year-old patient with Swyer syndrome. The gonadoblastoma on both sides underwent synchronous neoplastic transformation, into a stage I germinoma in the right streak gonad and a highly differentiated Sertoli cell tumor in the left one. The latter was associated with a myriad of microscopic, Sertoli cell implants on the peritoneal surface, which were considered benign as they had a high grade of differentiation, minimal proliferative activity, and an absence of invasion. Most probably, the pathogenesis of this abdominal dissemination was iatrogenic, with implantation occurring mechanically as a result of the multiple laparoscopic biopsies performed on both of the streak gonads 2 months before the abdominal surgery. The pathogenesis of other benign abdominal implants is discussed. PMID:20736766

  19. In vitro effects of simulated microgravity on Sertoli cell function

    NASA Astrophysics Data System (ADS)

    Masini, M. A.; Prato, P.; Scarabelli, L.; Lanza, C.; Palmero, S.; Pointis, G.; Ricci, F.; Strollo, F.

    2011-02-01

    With the advent of space flights questions concerning the effects of microgravity (0×G) on human reproductive physiology have received great attention. The aim of this study was to evaluate the influence of 0×G on Sertoli cells. A Sertoli cell line from mouse testis (42GPA9) was analyzed for cytoskeletal and Sex Hormone Binding Globilin (SHBG) changes by immunohistochemistry, for antioxidant content by RT-PCR and for culture medium lactate concentrations by protein chemistry. Cells were cultured for 6, 24 and 48 h on a three-dimensional Random Positioning Machine (3D-RPM); static controls (1×G) were positioned on the supporting frame. At the end of each experiment, cultured cells were either fixed in paraformaldehyde or lysed and RNA-extracted or used for culture medium lactate measurements as needed. At 0×G, Sertoli cytoskeleton became disorganized, microtubules fragmented and SHBG undetectable already after 24 h, with alterations worsening by 48 h. It was evident that various antioxidant systems appreciably increased during the first 24 h but significantly decreased at 48 h. No changes occurred in the 1×G samples. Initially, 0×G seemed to disturb antioxidant protection strategies allowing the testes to support sperm production, thus generating an aging-like state of oxidative stress. Lactate production at 0×G slightly decreased after 24 h. Further experiments are needed in space to investigate upon steroidogenesis and germ cell differentiation within the testis, to rule out male infertility as a possible consequence, which could be a problem, as life expectancy increases.

  20. Intrauterine growth retardation associated with precocious puberty and sertoli cell hyperplasia.

    PubMed

    Lodish, M B; Gartner, L A; Albini, P; Sabnis, G; Brodie, A; Meck, J M; Meloni-Ehrig, A M; Hill, S; Tsilou, E; Valera, V A; Walter, B A; Merino, M J; Stratakis, C A

    2010-08-01

    The original description of patients with Russell-Silver syndrome included precocious puberty, the mechanism of which was unclear. We describe a child with a Russell-Silver syndrome-like phenotype who presented with precocious puberty that was associated with hyperplasia of the Sertoli cells. The patient was found to have an immature cryptorchid testicle; hyperplastic Sertoli cells were also aneuploid carrying trisomy 8. This chromosomal abnormality was present in Sertoli cells only and could not be detected in peripheral lymphocytes, tunica vaginalis, or other, normal, testicular tissue. Sertoli cells in culture showed excess aromatization providing an explanation for the rapid advancement of the patient's bone age. We conclude that in a patient with a Russell-Silver syndrome-like phenotype, Sertoli cell hyperplasia was associated with somatic trisomy 8, increased aromatization, and gonadotropin-independent precocious puberty. PMID:20411478

  1. Testicular Sertoli cells influence the proliferation and immunogenicity of co-cultured endothelial cells

    SciTech Connect

    Fan, Ping, E-mail: fanpinggoodluck@163.com [Department of Rheumatism and Immunity, The First Affiliated Hospital Xi'an Jiaotong University School of Medicine, Xi'an, Shaanxi 710061 (China)] [Department of Rheumatism and Immunity, The First Affiliated Hospital Xi'an Jiaotong University School of Medicine, Xi'an, Shaanxi 710061 (China); He, Lan; Pu, Dan; Lv, Xiaohong; Zhou, Wenxu; Sun, Yining; Hu, Nan [Department of Rheumatism and Immunity, The First Affiliated Hospital Xi'an Jiaotong University School of Medicine, Xi'an, Shaanxi 710061 (China)] [Department of Rheumatism and Immunity, The First Affiliated Hospital Xi'an Jiaotong University School of Medicine, Xi'an, Shaanxi 710061 (China)

    2011-01-21

    Research highlights: {yields} The proliferation of dramatic increased by co-cultured with Sertoli cells. {yields} VEGF receptor-2 expression of ECs was up-regulated by co-cultured with Sertoli cells. {yields} The MHC expression of ECs induced by INF-{gamma} and IL-6, IL-8 and sICAM induced by TNF-{alpha} decreased respectively after co-cultured with Sertoli cells. {yields} ECs co-cultured with Sertoli cells also didn't increase the stimulation index of spleen lymphocytes. -- Abstract: The major problem of the application of endothelial cells (ECs) in transplantation is the lack of proliferation and their immunogenicity. In this study, we co-cultured ECs with Sertoli cells to monitor whether Sertoli cells can influence the proliferation and immunogenicity of co-cultured ECs. Sertoli cells were isolated from adult testicular tissue. ECs were divided into the control group and the experimental group, which included three sub-groups co-cultured with 1 x 10{sup 3}, 1 x 10{sup 4} or 1 x 10{sup 5} cell/ml of Sertoli cells. The growth and proliferation of ECs were observed microscopically, and the expression of vascular endothelial growth factor (VEGF) receptor-2 (KDR) was examined by Western blotting. In another experiment, ECs were divided into the control group, the single culture group and the co-culture group with the optimal concentration of Sertoli cells. After INF-{gamma} and TNF-{alpha} were added to the culture medium, MHC II antigen expression was detected by immunofluorescence staining and western blotting; interleukin (IL)-6, IL-8 and soluble intercellular adhesion molecule (sICAM) were measured in the culture medium by ELISA. We demonstrated that 1 x 10{sup 4} cell/ml Sertoli cells promoted the proliferation of co-cultured ECs more dramatically than that in other groups (P < 0.05). Western blotting showed that 1 x 10{sup 4} cell/ml of the Sertoli cells was most effective in the up-regulation of KDR expression in the co-cultured ECs (P < 0.05). Sertoli cells can effectively suppress INF-{gamma}-induced MHC II antigen expression in co-cultured ECs compared with single culture group (P < 0.05). TNF-{alpha} induced the expression of IL-6, IL-8 and sICAM in ECs. When co-cultured with Sertoli cells, their expressions were significantly lower than in the EC single culture group (P < 0.05). ECs co-cultured with Sertoli cells also did not significantly increase the stimulation index of spleen lymphocytes compared to the single culture group (P < 0.05). Our results suggested that co-culturing with Sertoli cells can significantly promote the proliferation of ECs, accelerate post-transplant angiogenesis, while reduce EC immunogenicity and stimulus to lymphocytes.

  2. Long-term culture and analysis of cashmere goat Sertoli cells.

    PubMed

    Su, Huimin; Luo, Fenhua; Bao, Jiajing; Wu, Sachula; Zhang, Xueming; Zhang, Yan; Duo, Shuguang; Wu, Yingji

    2014-12-01

    Sertoli cells have important functions in the testis for spermatogenesis. Thus, Sertoli cell culture systems have been established in many animals, such as rat, mouse, human, dog, cow, and pig, but a goat culture has not been reported. This study describes the isolation and culture of Sertoli cells from 3- to 4-month-old cashmere goat (Capra hircus) testes. These proliferative cells were expanded for 20 passages and repeatedly cryopreserved in vitro, in contrast to previous study in human, of which maintain steady growth for up to seven passages and only passages 1 to 5 could be refrozen. The microstructure and ultrastructure of the culture were typical of Sertoli cells, bearing irregular nuclei and a cytoplasm that was rich in smooth and rough endoplasmic reticulum, mitochondria, Golgi, lysosomes, lipid drops, and glycogenosomes. By immunofluorescence analysis, the all cells expressed SRY-related HMG box gene 9 (Sox9). Growth curves and 5-bromo-2'-deoxyuridine (BrdU) incorporation were used to analyze the proliferation of the cultured cells. With increasing passage times, the proliferation of the Sertoli cells declined, but the transcription of glial cell-derived neurotrophic factor (GDNF), stem cell factor (SCF), and ?1-integrin was constant. By flow cytometry, the cells retained the ability to proliferate after 5 yr of cryopreservation. Thus, cashmere goat Sertoli cells have significant proliferative potential in vitro, expressing germ cell regulatory factors and have important applications in studying Sertoli cell-germ cell interactions, spermatogenesis, reproductive toxicology, and male infertility. PMID:25164184

  3. 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.

  4. Expression of Dominant-Negative Thyroid Hormone Receptor Alpha1 in Leydig and Sertoli Cells Demonstrates No Additional Defect Compared with Expression in Sertoli Cells Only

    PubMed Central

    Fumel, Betty; Froment, Pascal; Holzenberger, Martin; Livera, Gabriel; Monget, Philippe; Fouchécourt, Sophie

    2015-01-01

    Background In the testis, thyroid hormone (T3) regulates the number of gametes produced through its action on Sertoli cell proliferation. However, the role of T3 in the regulation of steroidogenesis is still controversial. Methods The TR?AMI knock-in allele allows the generation of transgenic mice expressing a dominant-negative TR?1 (thyroid receptor ?1) isoform restricted to specific target cells after Cre-loxP recombination. Here, we introduced this mutant allele in both Sertoli and Leydig cells using a novel aromatase-iCre (ARO-iCre) line that expresses Cre recombinase under control of the human Cyp19(IIa)/aromatase promoter. Findings We showed that loxP recombination induced by this ARO-iCre is restricted to male and female gonads, and is effective in Sertoli and Leydig cells, but not in germ cells. We compared this model with the previous introduction of TR?AMI specifically in Sertoli cells in order to investigate T3 regulation of steroidogenesis. We demonstrated that TR?AMI-ARO males exhibited increased testis weight, increased sperm reserve in adulthood correlated to an increased proliferative index at P3 in vivo, and a loss of T3-response in vitro. Nevertheless, TR?AMI-ARO males showed normal fertility. This phenotype is similar to TR?AMI-SC males. Importantly, plasma testosterone and luteinizing hormone levels, as well as mRNA levels of steroidogenesis enzymes StAR, Cyp11a1 and Cyp17a1 were not affected in TR?AMI-ARO. Conclusions/Significance We concluded that the presence of a mutant TR?AMI allele in both Leydig and Sertoli cells does not accentuate the phenotype in comparison with its presence in Sertoli cells only. This suggests that direct T3 regulation of steroidogenesis through TR?1 is moderate in Leydig cells, and that Sertoli cells are the main target of T3 action in the testis. PMID:25793522

  5. Effects of cadmium on survival and morphology of cultured rat Sertoli cells.

    PubMed

    Espevik, T; Lamvik, M K; Sunde, A; Eik-Nes, K B

    1982-07-01

    The toxicity of different metals on isolated Sertoli cells grown in culture has been investigated. Methyl mercury (CH3HgCl) and mercury chloride (HgCl2) were more toxic than cadmium (CdCl2) which was slightly more toxic than arsenic (As2O3). Isolated peritubular cells and Sertoli cells were equally sensitive to cadmium. Cadmium reduced the Sertoli cell survival over the concentration range of 1--10 microM. Freeze-etch electron microscopy of cadmium-exposed Sertoli cells revealed circular areas of average diameter 500 nm devoid of intramembrane particles in the nuclear membrane, and general signs of degeneration such as vesiculation of the plasma membrane and intramembrane particle aggregation. However, cadmium did not dissolve junctional complexes between Sertoli cells. Isolated Sertoli cells were protected against cadmium-induced damage when the cells were preincubated for 48 h with selenium, zinc or low doses of cadmium. Preincubation with cobalt, FSH, testosterone or oestradiol did not protect against cadmium-induced damage. Cadmium bound to metallothionein had no toxic effects on isolated Sertoli cells. PMID:7097653

  6. Extracellular ATP activates different signalling pathways in rat Sertoli cells.

    PubMed Central

    Foresta, C; Rossato, M; Bordon, P; Di Virgilio, F

    1995-01-01

    1. The present study describes effects of extracellular ATP (ATPe) on plasma membrane potential and cytoplasmic Ca2+ concentrations ([Ca2+]i) in rat Sertoli cells. Sertoli cells in suspension were stimulated with ATPe and other nucleotides and ionic changes were monitored utilizing the fluorescent dyes bis-oxonol and fura-2/AM. ATPe induced a prompt plasma membrane depolarization which was dependent on Na+ influx from the extracellular medium, since it was abolished by omission of extracellular Na+. Depolarization was independent of [Ca2+]i rise as it also occurred in the absence of extracellular Ca2+ and after intracellular Ca2+ stores were discharged with thapsigargin. ATPe also stimulated a rapid and biphasic increase in [Ca2+]i: a prompt spike was followed by a prolonged sustained plateau. The initial spike was dependent on Ca2+ release from intracellular stores since it was also present when cells were incubated in EGTA-supplemented Ca(2+)-free medium and was abolished by pretreatment with ionomycin and thapsigargin, agents that discharge intracellular Ca2+ stores. The sustained phase was dependent on Ca2+ influx from the extracellular medium as it was abolished when cells were incubated in EGTA-supplemented Ca(2+)-free medium. Ca2+ influx was due to activation of voltage-operated calcium channels (VOCCs) since it was abolished by the VOCC inhibitors verapamil and nifedipine or incubation in sucrose medium, an experimental condition which precludes plasma membrane depolarization by ATPe. 2. ATPe-induced rises in intracellular Ca2+ concentration and plasma membrane depolarization were reduced by pretreatment with pertussis toxin, suggesting that ATPe-activated transduction mechanisms are in part under the control of pertussis toxin-sensitive G-proteins. These data show that Sertoli cells possess P2-purinergic receptor subtypes coupled to influx of Na+ and release of Ca2+ from intracellular stores and provide evidence for an activation of different pathways by extracellular ATPe. Activation of these receptors induces Na+ influx that causes a rapid plasma membrane depolarization. Furthermore, ATPe also triggers Ca2+ release from intracellular stores and Ca2+ influx from extracellular space via dihydropyridine-sensitive VOCCs. PMID:7575464

  7. 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

  8. Expression of genomic functional estrogen receptor 1 in mouse sertoli cells.

    PubMed

    Lin, Jing; Zhu, Jia; Li, Xian; Li, Shengqiang; Lan, Zijian; Ko, Jay; Lei, Zhenmin

    2014-11-01

    There is no consensus whether Sertoli cells express estrogen receptor 1 (Esr1). Reverse transcription-polymerase chain reaction, Western blot, and immunofluorescence demonstrated that mouse Sertoli cell lines, TM4, MSC-1, and 15P-1, and purified primary mouse Sertoli cells (PSCs) contained Esr1 messenger RNA and proteins. Incubation of Sertoli cells with 17?-estradiol (E2) or ESR1 agonist stimulated the expression of an estrogen responsive gene Greb1, which was prevented by ESR inhibitor or ESR1 antagonist. Overexpression of Esr1 in MSC-1 enhanced E2-induced Greb1 expression, while knockdown of Esr1 by small interfering RNA in TM4 attenuated the response. Furthermore, E2-induced Greb1 expression was abolished in the PSCs isolated from Amh-Cre/Esr1-floxed mice in which Esr1 in Sertoli cells were selectively deleted. Chromatin immunoprecipitation assays indicated that E2-induced Greb1 expression in Sertoli cells was mediated by binding of ESR1 to estrogen responsive elements. In summary, ligand-dependent nuclear ESR1 was present in mouse Sertoli cells and mediates a classical genomic action of estrogens. PMID:24615934

  9. 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

  10. Direct Reprogramming of Fibroblasts into Embryonic Sertoli-like Cells by Defined Factors

    E-print Network

    Buganim, Yosef

    Sertoli cells are considered the “supporting cells” of the testis that play an essential role in sex determination during embryogenesis and in spermatogenesis during adulthood. Their essential roles in male fertility along ...

  11. Male Differentiation of Germ Cells Induced by Embryonic Age-Specific Sertoli Cells in Mice1

    PubMed Central

    Ohta, Kohei; Yamamoto, Miyuki; Lin, Yanling; Hogg, Nathanael; Akiyama, Haruhiko; Behringer, Richard R.; Yamazaki, Yukiko

    2012-01-01

    ABSTRACT Retinoic acid (RA) is a meiosis-inducing factor. Primordial germ cells (PGCs) in the developing ovary are exposed to RA, resulting in entry into meiosis. In contrast, PGCs in the developing testis enter mitotic arrest to differentiate into prospermatogonia. Sertoli cells express CYP26B1, an RA-metabolizing enzyme, providing a simple explanation for why XY PGCs do not initiate meios/is. However, regulation of entry into mitotic arrest is likely more complex. To investigate the mechanisms that regulate male germ cell differentiation, we cultured XX and XY germ cells at 11.5 and 12.5 days postcoitus (dpc) with an RA receptor inhibitor. Expression of Stra8, a meiosis initiation gene, was suppressed in all groups. However, expression of Dnmt3l, a male-specific gene, during embryogenesis was elevated but only in 12.5-dpc XY germ cells. This suggests that inhibiting RA signaling is not sufficient for male germ cell differentiation but that the male gonadal environment also contributes to this pathway. To define the influence of Sertoli cells on male germ cell differentiation, Sertoli cells at 12.5, 15.5, and 18.5 dpc were aggregated with 11.5 dpc PGCs, respectively. After culture, PGCs aggregated with 12.5 dpc Sertoli cells increased Nanos2 and Dnmt3l expression. Furthermore, these PGCs established male-specific methylation imprints of the H19 differentially methylated domains. In contrast, PGCs aggregated with Sertoli cells at late embryonic ages did not commit to the male pathway. These findings suggest that male germ cell differentiation is induced both by inhibition of RA signaling and by molecule(s) production by embryonic age-specific Sertoli cells. PMID:22262692

  12. Comparative immunohistochemical characterization of canine seminomas and Sertoli cell tumors

    PubMed Central

    Yu, Chi-Ho; Hwang, Du-Na; Yhee, Ji-Young; Kim, Jong-Hyuk; Im, Keum-Soon; Nho, Whan-Gook; Lyoo, Young-Soo

    2009-01-01

    Primary testicular tumors are the most common causes of cancer in male dogs. Overall, the majority of canine patients should be cured by testicular surgery. However, tumor markers are not well-known in veterinary medicine. We sought to determine using immunohistochemistry whether the combined human testicular tumor markers (placental alkaline phosphatase, OCT3/4, CD30, alpha-fetoprotein, inhibin-alpha, vimentin, c-KIT, and desmin) are expressed in canine seminomas and Sertoli cell tumors (SCTs). We examined 35 canine testicular tumors, 20 seminomas and 15 SCTs. c-KIT was expressed markedly in canine seminomas. Both inhibin-alpha and vimentin were expressed significantly in canine SCTs. The results of this study demonstrate differences and similarities between tumor marker expression of testicular tumors in dogs and humans. All the main markers in current routine use are discussed as well as potential useful markers for benign and malignant tumors, and tumor progression. PMID:19255517

  13. Testicular Sclerosing Sertoli Cell Tumor: A Case Report and Review of the Literature

    PubMed Central

    Coleman, Sarah; Barazani, Yagil; Roma, Andres A; Sabanegh, Edmund

    2014-01-01

    Sertoli cell tumors are very rare testicular tumors, representing 0.4% to 1.5% of all testicular malignancies. They are subclassified as classic, large-cell calcifying, and sclerosing Sertoli cell tumors (SSCT) based on distinct clinical features. Only 42 cases of SSCTs have been reported in the literature. We present a case of a 23-year-old man diagnosed with SSCT. PMID:25548547

  14. Late morfofunctional alterations of the Sertoli cell caused by doxorubicin administered to prepubertal rats

    PubMed Central

    2012-01-01

    Background Doxorubicin is a potent chemotherapeutic drug used against a variety of cancers. It acts through interaction with polymerases and topoisomerase II and free radical production. Doxorubicin activity is not specific to cancer cells and can also damage healthy cells, especially those undergoing rapid proliferation, such as spermatogonia. In previous studies our group showed that etoposide, another topoisomarese II poison, causes irreversible damage to Sertoli cells. Thus, the aim of this study was to address the effects of doxorubicin on Sertoli cell morphology and function and on the seminiferous epithelium cycle when administered to prepubertal rats. Methods Prepubertal rats received the dose of 5?mg/Kg of doxorubicin, which was fractioned in two doses: 3?mg/Kg at 15dpp and 2?mg/Kg at 22dpp. The testes were collected at 40, 64 and 127dpp, fixed in Bouin’s liquid and submitted to transferrin immunolabeling for Sertoli cell function analysis. Sertoli cell morphology and the frequency of the stages of the seminiferous epithelium cycle were analyzed in PAS?+?H-stained sections. Results The rats treated with doxorubicin showed reduction of transferrin labeling in the seminiferous epithelium at 40 and 64dpp, suggesting that Sertoli cell function is altered in these rats. All doxorubicin-treated rats showed sloughing and morphological alterations of Sertoli cells. The frequency of the stages of the seminiferous epithelium cycle was also affected in all doxorubicin-treated rats. Conclusions and discussion These data show that doxorubicin administration during prepuberty causes functional and morphological late damage to Sertoli cells; such damage is secondary to the germ cell primary injury and contributed to enhance the spermatogenic harm caused by this drug. However, additional studies are required to clarify if there is also a direct effect of doxorubicin on Sertoli cells producing a primary damage on these cells. PMID:22967030

  15. Constitutive activation of NOTCH1 signaling in Sertoli cells causes gonocyte exit from quiescence

    PubMed Central

    Garcia, Thomas Xavier; DeFalco, Tony; Capel, Blanche; Hofmann, Marie-Claude

    2013-01-01

    Notch signaling components have long been detected in Sertoli and germ cells in the developing and mature testis. However, the role of this pathway in testis development and spermatogenesis remains unknown. Using reporter mice expressing green fluorescent protein following Notch receptor activation, we found that Notch signaling was active in Sertoli cells at various fetal, neonatal, and adult stages. Since Notch signaling specifies stem cell fate in many developing and mature organ systems, we hypothesized that maintenance and differentiation of gonocytes and/or spermatogonial stem cells would be modulated through this pathway in Sertoli cells. To this end, we generated mutant mice constitutively expressing the active, intracellular domain of NOTCH1 (NICD1) in Sertoli cells. We found that mutant Sertoli cells were morphologically normal before and after birth, but presented a number of functional changes that drastically affected gonocyte numbers and physiology. We observed aberrant exit of gonocytes from mitotic arrest, migration toward cord periphery, and premature differentiation before birth. These events, presumably unsupported by the cellular microenvironment, were followed by gonocyte apoptosis and near complete disappearance of the gonocytes by day 2 after birth. Molecular analysis demonstrated that these effects are correlated with a dysregulation of Sertoli-expressed genes that are required for germ cell maintenance, such as Cyp26b1 and Gdnf. Taken together, our results demonstrate that Notch signaling is active in Sertoli cells throughout development and that proper regulation of Notch signaling in Sertoli cells is required for the maintenance of gonocytes in an undifferentiated state during fetal development. PMID:23391689

  16. Reduced Immunogenicity of Induced Pluripotent Stem Cells Derived from Sertoli Cells

    PubMed Central

    Wang, Xiaoying; Qin, Jie; Zhao, Robert Chunhua; Zenke, Martin

    2014-01-01

    Sertoli cells constitute the structural framework in testis and provide an immune-privileged environment for germ cells. Induced pluripotent stem cells (iPS cells) resemble embryonic stem cells (ES cells) and are generated from somatic cells by expression of specific reprogramming transcription factors. Here, we used C57BL/6 (B6) Sertoli cells to generate iPS cells (Ser-iPS cells) and compared the immunogenicity of Ser-iPS cells with iPS cells derived from mouse embryonic fibroblast (MEF-iPS cells). Ser-iPS cells were injected into syngeneic mice to test for their in vivo immunogenicity in teratoma assay. Teratoma assay allows assessing in vivo immunogenicity of iPS cells and of their differentiated progeny simultaneously. We observed that early-passage Ser-iPS cells formed more teratomas with less immune cell infiltration and tissue damage and necrosis than MEF-iPS cells. Differentiating Ser-iPS cells in embryoid bodies (EBs) showed reduced T cell activation potential compared to MEF-iPS cells, which was similar to syngeneic ES cells. However, Ser-iPS cells lost their reduced immunogenicity in vivo after extended passaging in vitro and late-passage Ser-iPS cells exhibited an immunogenicity similar to MEF-iPS cells. These findings indicate that early-passage Ser-iPS cells retain some somatic memory of Sertoli cells that impacts on immunogenicity of iPS cells and iPS cell-derived cells in vivo and in vitro. Our data suggest that immune-privileged Sertoli cells might represent a preferred source for iPS cell generation, if it comes to the use of iPS cell-derived cells for transplantation. PMID:25166861

  17. Sertoli cells in the testis of caecilians, Ichthyophis tricolor and Uraeotyphlus cf. narayani (Amphibia: Gymnophiona): light and electron microscopic perspective.

    PubMed

    Smita, Mathew; Oommen, Oommen V; George, Jancy M; Akbarsha, M A

    2003-12-01

    The caecilians have evolved a unique pattern of cystic spermatogenesis in which cysts representing different stages in spermatogenesis coexist in a testis lobule. We examined unsettled issues relating to the organization of the caecilian testis lobules, including the occurrence of a fatty matrix, the possibility of both peripheral and central Sertoli cells, the origin of Sertoli cells from follicular cells, and the disengagement of older Sertoli cells to become loose central Sertoli cells. We subjected the testis of Ichthyophis tricolor (Ichthyophiidae) and Uraeotyphlus cf. narayani (Uraeotyphliidae) from the Western Ghats of Kerala, India, to light and transmission electron microscopic studies. Irrespective of the functional state of the testis, whether active or regressed, Sertoli cells constitute a permanent feature of the lobules. The tall Sertoli cells adherent to the basal lamina with basally located pleomorphic nuclei extend deeper into the lobule to meet at the core. There they provide for association of germ cells at different stages of differentiation, an aspect that has earlier been misconceived as the fatty matrix. Germ cells up to the 4-cell stage remain in the intercalating region of the Sertoli cells and they are located at the apices of the Sertoli cells from the 8-cell stage onwards. The developing germ cells are intimately associated with the Sertoli cell adherent to the basal lamina until spermiation. There are ameboid cells in the core of the lobules that appear to interact with the germ cells at the face opposite to their attachment with the Sertoli cells. Adherence of the Sertoli cells to the basal lamina is a permanent feature of the caecilian testicular lobules. The ameboid cells in the core are neither Sertoli cells nor their degeneration products. PMID:14584033

  18. Ectoplasmic specializations in the Sertoli cell: new vistas based on genetic defects and testicular toxicology

    Microsoft Academic Search

    Yoshiro Toyama; Mamiko Maekawa; Shigeki Yuasa

    2003-01-01

    The ectoplasmic specialization is a unique junctional complex formed in two cortical areas of the Sertoli cell in the mammalian\\u000a testis: one near the base of the seminiferous epithelium forming the blood—testis barrier, and the other near the lumen of\\u000a the seminiferous tubule embracing the acrosome region of the developing spermatids. The specialization consists of the Sertoli\\u000a cell plasma membrane,

  19. 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.

  20. Depletion of the p43 mitochondrial T3 receptor increases Sertoli cell proliferation in mice.

    PubMed

    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

  1. 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

  2. Effect of TGF-beta/Smad signaling on sertoli cell and possible mechanism related to complete sertoli cell-only syndrome.

    PubMed

    Sun, Tao; Xin, Zhongcheng; Jin, Zhe; Wu, Yiguang; Gong, Yanqing

    2008-12-01

    The roles of TGF-beta and the interaction between TGF-beta and EGFR signaling are critical in Sertoli cell, though the knowledge about them is limited. RT-PCR was used to characterize the status of TGF-beta signaling in clinical testicular specimens with complete Sertoli cell-only syndrome (SCOS). The mouse Sertoli cell TM4 was used to investigate the interaction between TGF-beta and EGFR signaling by using mitogenic assay, luciferase assay, and western blot, while TM3 (mouse leydig cell), 3T3 (mouse embryo fibroblasts), and B82 (mouse lung fibroblasts) were selected as control. The RT-PCR assay indicated that the expression levels of TbetaRII and Smad2 in SCOS testes were upregulated compared to that in the normal controls. In the in vitro experiment, the TGF-beta1 downregulated cellular proliferation of TM3 and B82 cell (P < 0.05), but it did not changed the proliferation of TM4 and 3T3 cells (P > 0.05). On contrast, TGF-beta1 only increased the TGF response elements p3TP-lux activity significantly (P < 0.05) in Sertoli cell TM4. Also, the Western blot assay shows an obvious increase of Smad2 in TM4, 3T3, and TM3 cells after TGF-beta1 treatment while the EGFR expression level was significantly increased in TM4 cells only. In conclusion, the TGF-beta pathway and the cross-link between TGF-beta and EGFR signaling may play an important role on the dysfunction of Sertoli cells which induce germ stem cells' disappearance in SCOS. PMID:18648910

  3. CEACAM2-L on spermatids interacts with poliovirus receptor on Sertoli cells in mouse seminiferous epithelium.

    PubMed

    Salaheldeen, Elsaid; Howida, Ali; Wakayama, Tomohiko; Iida, Hiroshi

    2014-09-01

    The removal of excess cytoplasm from elongated spermatids by Sertoli cells is the last essential step in spermatogenesis. It requires cell-to-cell recognition between a Sertoli cell and an elongating spermatid through protein-protein interactions. CEACAM2-L, an adhesion molecule of the immunoglobulin superfamily (IgSF), is present at the plasma membrane covering the excess cytoplasm of elongated spermatids, and is possibly involved in the cell-to-cell recognition. In this study, we investigated the interaction between CEACAM2-L and Poliovirus receptor (PVR), which is also from the IgSF and is expressed by Sertoli cells. Immunohistochemical analysis showed that CEACAM2-L expressed on elongated spermatids was in close contact with PVR-positive cell processes of Sertoli cells. Immunoprecipitation experiments both in vivo and in vitro demonstrated a direct heterophilic interaction between CEACAM2-L and PVR. We show that the N-terminal Ig domain of CEACAM2-L was critical for its interaction with PVR. In addition, we found that CEACAM2-L formed heterophilic trans-tetramers with PVR in transfected COS-7 cells. From these data, we propose that Sertoli cells recognize the excess cytoplasm of elongated spermatids through the PVR-CEACAM2-L interaction in mouse testis. PMID:24948196

  4. 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.

  5. Altered Lipid Homeostasis in Sertoli Cells Stressed by Mild Hyperthermia

    PubMed Central

    Vallés, Ana S.; Aveldaño, Marta I.; Furland, Natalia E.

    2014-01-01

    Spermatogenesis is known to be vulnerable to temperature. Exposures of rat testis to moderate hyperthermia result in loss of germ cells with survival of Sertoli cells (SC). Because SC provide structural and metabolic support to germ cells, our aim was to test the hypothesis that these exposures affect SC functions, thus contributing to germ cell damage. In vivo, regularly repeated exposures (one of 15 min per day, once a day during 5 days) of rat testes to 43°C led to accumulation of neutral lipids. This SC-specific lipid function took 1–2 weeks after the last of these exposures to be maximal. In cultured SC, similar daily exposures for 15 min to 43°C resulted in significant increase in triacylglycerol levels and accumulation of lipid droplets. After incubations with [3H]arachidonate, the labeling of cardiolipin decreased more than that of other lipid classes. Another specifically mitochondrial lipid metabolic function, fatty acid oxidation, also declined. These lipid changes suggested that temperature affects SC mitochondrial physiology, which was confirmed by significantly increased degrees of membrane depolarization and ROS production. This concurred with reduced expression of two SC-specific proteins, transferrin, and Wilms' Tumor 1 protein, markers of SC secretion and differentiation functions, respectively, and with an intense SC cytoskeletal perturbation, evident by loss of microtubule network (?-tubulin) and microfilament (f-actin) organization. Albeit temporary and potentially reversible, hyperthermia-induced SC structural and metabolic alterations may be long-lasting and/or extensive enough to respond for the decreased survival of the germ cells they normally foster. PMID:24690895

  6. Metformin and male reproduction: effects on Sertoli cell metabolism

    PubMed Central

    Alves, M G; Martins, A D; Vaz, C V; Correia, S; Moreira, P I; Oliveira, P F; Socorro, S

    2014-01-01

    Background and Purpose Metformin is commonly used to treat type 2 diabetes (T2D). While new clinical applications have been ascribed to metformin, including treatment of anovulatory infertility, its effects on male reproduction have not been investigated. The Sertoli cell (SC) is crucial for germ cell development, exerting metabolic control of spermatogenesis, therefore, we investigated the effects of metformin on SC metabolism. Experimental Approach Rat SCs were cultured in the absence and presence of metformin (5, 50 and 500??M). mRNA and protein levels of glucose transporters (GLUT1 and GLUT3), phosphofructokinase 1 (PFK 1), lactate dehydrogenase (LDH) and monocarboxylate transporter 4 (MCT4) were determined by quantitative PCR and Western blot respectively. LDH activity was assessed and metabolite production/consumption determined by 1H-NMR. Key Results Metformin (50??M) decreased mRNA and protein levels of GLUT1, GLUT3, MCT4 and PFK 1 but did not affect LDH mRNA or protein levels. However, although glucose consumption was maintained in metformin-treated cells, LDH activity, lactate and alanine production were increased, indicating an enhanced glycolytic flux. No metabolic cytotoxicity was detected in SCs exposed to supra-pharmacological concentration of metformin. Conclusions and Implications Our results indicate that metformin: (i) decreases mRNA and protein levels of glycolysis-related transporters in SCs but increases their activity; and (ii) stimulates alanine production, which induces antioxidant activity and maintains the NADH/NAD+ equilibrium. The increased lactate in metformin-treated SCs provides nutritional support and has an anti-apoptotic effect in developing germ cells. Thus, metformin can be considered as a suitable antidiabetic drug for male patients of reproductive age with T2D. PMID:24261663

  7. 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

  8. Unilateral cryptorchidism induces morphological changes of testes and hyperplasia of Sertoli cells in a dog.

    PubMed

    Moon, Joon Ho; Yoo, Dae Young; Jo, Young Kwang; Kim, Geon A; Jung, Hyo Young; Choi, Jung Hoon; Hwang, In Koo; Jang, Goo

    2014-12-01

    Cryptorchidism is one of the most common genital defects in dogs. This study investigated the effects of abdominal cryptorchidism on morphology, cell proliferation, and Sertoli cell condition in a dog with spontaneous unilateral cryptorchidism. Elective orchidectomy was performed on the abdominal right testis and the scrotal left testis. Significant reductions in numbers of spermatogonia, spermatocytes, and spermatids were observed in hematoxylin and eosin stained sections of the cryptorchid testis. The size of the epididymal duct was smaller than that of the control testis. Based on Ki67 immunohistochemistry, the proliferative activity of spermatogonia and spermatocytes was significantly decreased in the cryptorchid testis. However, proliferative activity was increased in the epididymal duct. Based on GATA-4 immunohistochemistry, Sertoli cells were relatively resistant to cryptorchidism, and the proliferative activity of Sertoli cells was markedly increased in the cryptorchid testis than in the control testis. These results suggest that spontaneous unilateral cryptorchidism causes morphological defects in spermatogonia and spermatocytes in the testis and changes the size of the efferent ductule of the epididymis. In addition, spontaneous unilateral cryptorchidism increases proliferative activity of Sertoli cells, which may be a predisposing factor for Sertoli cell cancer in cryptorchid testes. PMID:25628730

  9. Unilateral cryptorchidism induces morphological changes of testes and hyperplasia of Sertoli cells in a dog

    PubMed Central

    Moon, Joon Ho; Yoo, Dae Young; Jo, Young Kwang; Kim, Geon A; Jung, Hyo Young; Choi, Jung Hoon

    2014-01-01

    Cryptorchidism is one of the most common genital defects in dogs. This study investigated the effects of abdominal cryptorchidism on morphology, cell proliferation, and Sertoli cell condition in a dog with spontaneous unilateral cryptorchidism. Elective orchidectomy was performed on the abdominal right testis and the scrotal left testis. Significant reductions in numbers of spermatogonia, spermatocytes, and spermatids were observed in hematoxylin and eosin stained sections of the cryptorchid testis. The size of the epididymal duct was smaller than that of the control testis. Based on Ki67 immunohistochemistry, the proliferative activity of spermatogonia and spermatocytes was significantly decreased in the cryptorchid testis. However, proliferative activity was increased in the epididymal duct. Based on GATA-4 immunohistochemistry, Sertoli cells were relatively resistant to cryptorchidism, and the proliferative activity of Sertoli cells was markedly increased in the cryptorchid testis than in the control testis. These results suggest that spontaneous unilateral cryptorchidism causes morphological defects in spermatogonia and spermatocytes in the testis and changes the size of the efferent ductule of the epididymis. In addition, spontaneous unilateral cryptorchidism increases proliferative activity of Sertoli cells, which may be a predisposing factor for Sertoli cell cancer in cryptorchid testes. PMID:25628730

  10. Sertoli cell-conditioned medium induces germ cell differentiation in human embryonic stem cells

    Microsoft Academic Search

    Mieke Geens; Karen Dora Sermon; Hilde Van de Velde; Herman Tournaye

    2011-01-01

    Purpose  To investigate the spontaneous germ cell differentiation capacity of VUB hESC lines, develop a protocol for the induction\\u000a of germ cell differentiation using conditioned medium from Sertoli cells (SCCM) and compare it to existing protocols.\\u000a \\u000a \\u000a \\u000a \\u000a Methods  hESC were allowed to differentiate spontaneously or after the addition of bone morphogenetic proteins (BMPs) and\\/or SCCM.\\u000a VASA transcripts were measured by relative quantification real-time

  11. Experimental Sertoli cell tumors in the mouse and their progression into a mixed germ cell-sex cord proliferation.

    PubMed Central

    Paquis-Flucklinger, V.; Rassoulzadegan, M.; Michiels, J. F.

    1994-01-01

    Males of transgenic families where the large T protein of polyoma virus is expressed in the seminiferous epithelium of the testis (Sertoli and germ cells) develop bilateral testicular tumors when they become old (15 to 18 months). The histological features of these tumors revealed a neoplastic proliferation of Sertoli cell origin. Occasional isolated germ cells arrested at premeiotic stages were seen in the tumor. They did not participate in tumoral proliferation and their malignant character could not at first be established. Tumor cells injected in athymic (nu/nu) mice generated secondary tumors. In this case, a proliferative component of non-Sertoli origin was clearly evident. Its ultrastructural characteristics and the expression of genes that are transcribed in vivo in male germ cells (c-kit, LDH-X, and Hox a-4) suggest the progression of an initial, apparently pure Sertoli cell tumor into a mixed proliferation. Images Figure 1 Figure 2 Figure 3 PMID:7510454

  12. Thyroid Hormone and Follicle-Stimulating Hormone Regulate Mullerian-Inhibiting Substance Messenger Ribonucleic Acid Expression in Cultured Neonatal Rat Sertoli Cells

    Microsoft Academic Search

    NIROMI K. ARAMBEPOLA; DAVID BUNICK; PAUL S. COOKE

    1998-01-01

    Thyroid hormone is a major regulator of Sertoli cell development, and the present study sought to determine the role of T3 in Müllerian- inhibiting substance (MIS) messenger RNA (mRNA) expression. MIS, a Sertoli cell secretory protein that induces Mullerian duct regression and also may be critical for germ and Leydig cell development, is maximal perinatally, then decreases as Sertoli cells

  13. A case of Sertoli-Leydig cell tumor in a postmenopausal woman.

    PubMed

    Caringella, A; Loizzi, V; Resta, L; Ferreri, R; Loverro, G

    2006-01-01

    Sertoli-Leydig cell tumor belongs to the group of sex cord-stromal tumors of the ovary. These neoplasms account for less than 0.5% of all ovarian tumors and are more often encountered in young women between the ages of 20 and 30 years who usually become virilized. We described an unusual case of Sertoli-Leydig cell tumor in a postmenopausal women who presented with a solid right pelvic mass, a large amount of ascites, and laboratory tests revealing an elevated CA125, all suggesting a pelvic malignancy. Although five similar cases of postmenopausal women with Sertoli-Leydig cell tumor of ovary have been reported in the literature, we believe that this is an useful addition to the literature. PMID:16445673

  14. Testosterone regulates the autophagic clearance of androgen binding protein in rat Sertoli cells

    PubMed Central

    Ma, Yi; Yang, Hao-Zheng; Xu, Long-Mei; Huang, Yi-Ran; Dai, Hui-Li; Kang, Xiao-Nan

    2015-01-01

    Dysregulation of androgen-binding protein (ABP) is associated with a number of endocrine and andrology diseases. However, the ABP metabolism in Sertoli cells is largely unknown. We report that autophagy degrades ABP in rat Sertoli cells, and the autophagic clearance of ABP is regulated by testosterone, which prolongs the ABP biological half-life by inhibiting autophagy. Further studies identified that the autophagic clearance of ABP might be selectively regulated by testosterone, independent of stress (hypoxia)-induced autophagic degradation. These data demonstrate that testosterone up-regulates ABP expression at least partially by suppressing the autophagic degradation. We report a novel finding with respect to the mechanisms by which ABP is cleared, and by which the process is regulated in Sertoli cells. PMID:25745956

  15. Testosterone regulates the autophagic clearance of androgen binding protein in rat Sertoli cells.

    PubMed

    Ma, Yi; Yang, Hao-Zheng; Xu, Long-Mei; Huang, Yi-Ran; Dai, Hui-Li; Kang, Xiao-Nan

    2015-01-01

    Dysregulation of androgen-binding protein (ABP) is associated with a number of endocrine and andrology diseases. However, the ABP metabolism in Sertoli cells is largely unknown. We report that autophagy degrades ABP in rat Sertoli cells, and the autophagic clearance of ABP is regulated by testosterone, which prolongs the ABP biological half-life by inhibiting autophagy. Further studies identified that the autophagic clearance of ABP might be selectively regulated by testosterone, independent of stress (hypoxia)-induced autophagic degradation. These data demonstrate that testosterone up-regulates ABP expression at least partially by suppressing the autophagic degradation. We report a novel finding with respect to the mechanisms by which ABP is cleared, and by which the process is regulated in Sertoli cells. PMID:25745956

  16. 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

  17. Stage-specific effects of the fungicide carbendazim on Sertoli cell microtubules in rat testis.

    PubMed

    Nakai, M; Miller, M G; Carnes, K; Hess, R A

    2002-04-01

    The aim of the present study is to provide a morphological explanation of carbendazim (CBZ)-induced sloughing of germ cells that occurs in a stage-specific manner. Therefore, very early alterations in the seminiferous tubule epithelium were examined histologically in the rat testis after oral administration of CBZ (400mg/kg). Gaps between the elongated and round spermatids, the first indication of germ cell sloughing (pre-sloughing), were observed in stage late VI-early VII seminiferous tubules at 90-min post-treatment. Tubulin immunoreaction in the Sertoli cells was reduced in intensity in tubules with pre-sloughing. However, electron microscopy demonstrated that there were some intact microtubules in these cells. At 120 min, sloughing was seen in stage late VI-early VII and XIII-XIV. Tubulin immunoreaction in the Sertoli cells was greatly decreased in intensity in tubules where cell sloughing was observed. Electron microscopy showed that there were few microtubules in the body region of these cells. Stages II-V and mid-VII-VIII were exempt from the sloughing effect at 180 min. These changes in microtubules were not observed in Sertoli cells that did not exhibit sloughing characteristics, regardless of the post-treatment intervals. The present results suggest that stage specificity of sloughing is due to the stage-specific susceptibility of Sertoli cell microtubules to CBZ. PMID:12165241

  18. 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

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

    PubMed

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

    2013-08-01

    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

  20. Identification of Hsf1 as a novel androgen receptor-regulated gene in mouse Sertoli cells.

    PubMed

    Yang, Lihua; Wang, Yadong; Zhang, Qiang; Lai, Yongqing; Li, Cailing; Zhang, Qiaoxia; Huang, Weiren; Duan, Yonggang; Jiang, Zhimao; Li, Xianxin; Cai, Zhiming; Mou, Lisha; Gui, Yaoting

    2014-06-01

    Androgen signaling plays a crucial role in spermatogenesis, yet few downstream targets for this signaling pathway have been identified. In the current study, we found that the expression of heat-shock transcription factor 1 (Hsf1) was increased in the testes of Sertoli cell-selective androgen receptor knockout (S-AR(-/y) ) mice compared with wild-type mice by quantitative real-time PCR, and the expression of HSF1 in the S-AR(-/y) Sertoli cells was significantly increased, based on immunofluorescence analysis. In vitro cell-culture studies showed that testosterone repressed the expression of Hsf1 in TM4 cells, a mouse Sertoli cell line. Moreover, a luciferase assay, electrophoretic mobility shift assay, and chromatin immunoprecipitation assay showed that testosterone repressed Hsf1 expression by facilitating the binding of androgen receptor to the Hsf1 promoter. Our experiments also demonstrated that testosterone-mediated inhibition of Hsf1 transcription down-regulated the expression of heat-shock proteins HSP105 and HSP60. Taken together, these results reveal that Hsf1 is a novel target of androgen receptor in mouse Sertoli cells, and testosterone and its receptor regulate the process of spermatogenesis partially by inhibiting Hsf1 expression. PMID:24599545

  1. Morphological changes in the rat Sertoli cell induced by the microtubule poison carbendazim.

    PubMed

    Nakai, M; Hess, R A

    1994-12-01

    Early morphological changes in the rat Sertoli cell induced by the fungicide carbendazim (methyl-2-benzimidazole carbamate; MBC), a metabolite of benomyl, were examined. Adult rats were treated with single doses of MBC (400 mg/kg) or vehicle and examined by light and electron microscopy at 3 hr post-treatment. Sloughing of elongating spermatid clusters was observed in all stages of spermatogenesis, except for Stages III-V. Cleavage occurred near the apical region of the seminiferous epithelium where cytoplasmic processes of the Sertoli cell surround the heads of elongating spermatids. The cleaved cytoplasm remained attached to the sloughed spermatids and ectoplasmic specializations remained undamaged. Intact microtubules were observed in the apical Sertoli cell cytoplasm (including sloughed tissues) but were decreased in the body region, where aggregates of mitochondria were found. Cytoplasm near the cleavage site exhibited rarefaction, which was associated with swollen cisternae of endoplasmic reticulum. It appears that the mechanism of germ cell sloughing induced by MBC treatment involves the disruption of microtubules in the body region of the Sertoli cell, the retraction of cytoplasmic organelles and the swelling of endoplasmic reticulum. PMID:7886678

  2. CTNNB1 Signaling in Sertoli Cells Downregulates Spermatogonial Stem Cell Activity via WNT4

    PubMed Central

    Boyer, Alexandre; Yeh, Jonathan R.; Zhang, Xiangfan; Paquet, Marilène; Gaudin, Aurore; Nagano, Makoto C.; Boerboom, Derek

    2012-01-01

    Constitutive activation of the WNT signaling effector CTNNB1 (?-catenin) in the Sertoli cells of the Ctnnb1tm1Mmt/+;Amhr2tm3(cre)Bhr/+ mouse model results in progressive germ cell loss and sterility. In this study, we sought to determine if this phenotype could be due to a loss of spermatogonial stem cell (SSC) activity. Reciprocal SSC transplants between Ctnnb1tm1Mmt/+;Amhr2tm3(cre)Bhr/+ and wild-type mice showed that SSC activity is lost in Ctnnb1tm1Mmt/+;Amhr2tm3(cre)Bhr/+ testes over time, whereas the mutant testes could not support colonization by wild-type SSCs. Microarray analyses performed on cultured Sertoli cells showed that CTNNB1 induces the expression of genes associated with the female sex determination pathway, which was also found to occur in Ctnnb1tm1Mmt/+;Amhr2tm3(cre)Bhr/+ testes. One CTNNB1 target gene encoded the secreted signaling molecule WNT4. We therefore tested the effects of WNT4 on SSC-enriched germ cell cultures, and found that WNT4 induced cell death and reduced SSC activity without affecting cell cycle. Conversely, conditional inactivation of Wnt4 in the Ctnnb1tm1Mmt/+;Amhr2tm3(cre)Bhr/+ model rescued spermatogenesis and male fertility, indicating that WNT4 is the major effector downstream of CTNNB1 responsible for germ cell loss. Furthermore, WNT4 was found to signal via the CTNNB1 pathway in Sertoli cells, suggesting a self-reinforcing positive feedback loop. Collectively, these data indicate for the first time that ectopic activation of a signaling cascade in the stem cell niche depletes SSC activity through a paracrine factor. These findings may provide insight into the pathogenesis of male infertility, as well as embryonic gonadal development. PMID:22253774

  3. The Sertoli cell of the water buffalo ( Bubalus bubalis ) during the spermatogenic cycle

    Microsoft Academic Search

    Harcharan Singh Pawar; Karl-Heinz Wrobel

    1991-01-01

    Ultrastructural features and morphometric evaluations of buffalo Sertoli cells are reported for the six phases of the spermatogenic cycle. The phases of the tubular seminiferous epithelium are identified according to characteristic cellular associations with completed spermiation as demarcation between two cycles. Average tubular diameter (245 µm) and epithelial height (61 µm) do not vary significantly during the cycle. The relative

  4. Reduced numbers of Sertoli, germ, and spermatogonial stem cells in impaired spermatogenesis

    Microsoft Academic Search

    Anna Hentrich; Martin Wolter; Carolin Szardening-Kirchner; Georg H Lüers; Martin Bergmann; Sabine Kliesch; Lutz Konrad

    2011-01-01

    A key step in the investigation of male infertility is the appropriate classification of impaired spermatogenesis. In this study, we precisely identified Sertoli and distinct germ-cell types in the rat, the mouse, and in the human testis. As a proof of principle, we studied testis biopsy samples from azoospermic patients with defined spermatogenic defects. Remarkably, we found that already the

  5. Effect of continuous low-dose ?-irradiation on rat Sertoli cell function (*)

    E-print Network

    Paris-Sud XI, Université de

    by the treatment from 70 days on, as attested by androgen binding protein (ABP) and transferrin secretions which, transferrin and Sertoli cell paracrine factors. Introduction. Numerous studies related to seminiferous tubule binding protein : ABP, transferrin, cyclic protein 2) vary markedly at different stages

  6. Rat Sertoli cell-specific regulation of the transferrin gene. F Guillou 1, EF Guillou E

    E-print Network

    Paris-Sud XI, Université de

    Rat Sertoli cell-specific regulation of the transferrin gene. F Guillou 1, EF Guillou E Schaeffer2 transferrin. It is an important marker of testis function since its synthesis and secretion are in- volved in the control of spermatogenesis. Our purpose is to elucidate the mechanisms control- ling the transferrin gene

  7. Dynamin 2 is required for actin assembly in phagocytosis in Sertoli cells

    SciTech Connect

    Otsuka, Atsushi; Abe, Tadashi [Department of Neuroscience, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Okayama 700-8558 (Japan); Watanabe, Masami [Department of Urology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Okayama 700-8558 (Japan); Yagisawa, Hitoshi [Laboratory of Biological Signaling, Graduate School of Life Science, University of Hyogo, Harima Science Garden City, Kouto 3-chome, Kamigori, Hyogo 678-1297 (Japan); Takei, Kohji [Department of Neuroscience, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Okayama 700-8558 (Japan); Yamada, Hiroshi [Department of Neuroscience, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Okayama 700-8558 (Japan)], E-mail: hiroyama@md.okayama-u.ac.jp

    2009-01-16

    Dynamin 2 has been reported to be implicated in phagocytosis. However, the mode of action of dynamin is poorly understood. In this study, we examined whether dynamin 2 participates in actin assembly during phagocytosis in Sertoli cells. In the presence of dynasore, a dynamin inhibitor, phagocytosis was reduced by 60-70% in Sertoli cells and macrophages. Scanning electron microscopy revealed that Sertoli cells treated with dynasore were unable to form phagocytic cups. In addition, dysfunction of dynamin 2 reduced both actin polymerization and recruitment of actin and dynamin 2 to phosphatidylinositol (4,5) bisphosphate [PI(4,5)P{sub 2}]-containing liposomes. The formation of dynamin 2-positive ruffles of Sertoli cells was decreased by 60-70% by sequestering PI(4,5)P{sub 2} either by expression of PH domain of PLC{delta} or treatment with neomycin. These results strongly suggest that dynamin 2 is involved in actin dynamics and the formation of dynamin 2-positive ruffles during phagocytosis.

  8. Primary Mesenteric Sertoli-Leydig Cell Tumor: A Case Report and Review of the Literature

    PubMed Central

    Trabelsi, Amel; Ben Abdelkarim, Soumaya; Hadfi, Mohamed; Fatnaci, Ridha; Stita, Wided; Sriha, Badreddine; Korbi, Sadok

    2008-01-01

    The occurrence of primary sex cord-stromal tumors at extraovarian sites is exceedingly rare. We report a new case of Sertoli-Leydig cell tumor in the mesentery of a 78-year-old woman who presented with occlusive syndrome and reviewed the previously reported cases of extraovarian sex cord-stromal tumors in the English literature. PMID:19266086

  9. 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.

  10. 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

  11. 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

  12. Short-term stimulatory effect of Sertoli cell conditioned medium on Leydig cell steroidogenesis is not mediated by inhibin.

    PubMed

    Grootenhuis, A J; Melsert, R; Timmerman, M A; Hoogerbrugge, J W; Rommerts, F F; de Jong, F H

    1990-08-14

    Addition of concentrated rat Sertoli cell conditioned medium (rSCCM) to isolated Leydig cells from immature rats stimulated steroid production more than 13-fold within 4 h. LH-stimulated steroidogenesis was not enhanced by addition of rSCCM. The biological activity of the concentrated rSCCM was higher after incubation of Sertoli cells with FSH, whereas FSH alone did not stimulate steroid production. This effect of rSCCM was not due to inhibin, since highly purified 32 kDa rat inhibin, in doses equivalent to those present in rSCCM, had no effect on steroidogenesis during the 4 h incubation period. Furthermore, inhibin could be separated from the Leydig cell stimulating factor by anion-exchange chromatography. These results indicate a short-term paracrine control of Leydig cell steroidogenesis by Sertoli cell derived factors, which differ from inhibin. PMID:2120516

  13. Metabolic modulation induced by oestradiol and DHT in immature rat Sertoli cells cultured in vitro.

    PubMed

    Rato, Luís; Alves, Marco G; Socorro, Sílvia; Carvalho, Rui A; Cavaco, José E; Oliveira, Pedro F

    2012-02-01

    Sertoli cells actively metabolize glucose that is converted into lactate, which is used by developing germ cells for their energy metabolism. Androgens and oestrogens have general metabolic roles that reach far beyond reproductive processes. Hence, the main purpose of this study was to examine the effect of sex hormones on metabolite secretion/consumption in primary cultures of rat Sertoli cells. Sertoli cell-enriched cultures were maintained in a defined medium for 50 h. Glucose and pyruvate consumption, and lactate and alanine secretion were determined, by 1H-NMR (proton NMR) spectra analysis, in the presence or absence of 100 nM E2 (17?-oestradiol) or 100 nM 5?-DHT (dihydrotestosterone). Cells cultured in the absence (control) or presence of E2 consumed the same amount of glucose (29±2 pmol/cell) at similar rates during the 50 h. After 25 h of treatment with DHT, glucose consumption and glucose consumption rate significantly increased. Control and E2-treated cells secreted similar amounts of lactate during the 50 h, while the amount of lactate secreted by DHT-treated cells was significantly lower. Such a decrease was concomitant with a significant decrease in LDH A [LDH (lactate dehydrogenase) chain A] and MCT4 [MCT (monocarboxylate transporter) isoform 4] mRNA levels after 50 h treatment in hormonally treated groups, being more pronounced in DHT-treated groups. Finally, alanine production was significantly increased in E2-treated cells after 25 h treatment, which indicated a lower redox/higher oxidative state for the cells in those conditions. Together, these results support the existence of a relation between sex hormones action and energy metabolism, providing an important assessment of androgens and oestrogens as metabolic modulators in rat Sertoli cells. PMID:21671886

  14. Notch Signaling in Sertoli Cells Regulates Cyclical Gene Expression of Hes1 but Is Dispensable for Mouse Spermatogenesis

    PubMed Central

    Hasegawa, Kazuteru; Okamura, Yoshiaki

    2012-01-01

    Mammalian spermatogenesis is a highly regulated system dedicated to the continuous production of spermatozoa from spermatogonial stem cells, and the process largely depends on microenvironments created by Sertoli cells, unique somatic cells that reside within a seminiferous tubule. Spermatogenesis progresses with a cyclical program known as the “seminiferous epithelial cycle,” which is accompanied with cyclical gene expression changes in Sertoli cells. However, it is unclear how the cyclicity in Sertoli cells is regulated. Here, we report that Notch signaling, which is known to play an important role for germ cell development in Drosophila and Caenorhabditis elegans, is cyclically activated in Sertoli cells and regulates stage-dependent gene expression of Hes1. To elucidate the regulatory mechanism of stage-dependent Hes1 expression and the role of Notch signaling in mouse spermatogenesis, we inactivated Notch signaling in Sertoli cells by deleting protein O-fucosyltransferase 1 (Pofut1), using the cre-loxP system, and found that stage-dependent Hes1 expression was dependent on the activation of Notch signaling. Unexpectedly, however, spermatogenesis proceeded normally. Our results thus indicate that Notch signaling regulates cyclical gene expression in Sertoli cells but is dispensable for mouse spermatogenesis. This highlights the evolutionary divergences in regulation of germ cell development. PMID:22037762

  15. Heat Treatment Induces Liver Receptor Homolog1 Expression in Monkey and Rat Sertoli Cells

    Microsoft Academic Search

    Jian Guo; Shi-Xin Tao; Min Chen; Yu-Qiang Shi; Zhu-Qiang Zhang; Yin-Chuan Li; Xue-Sen Zhang; Zhao-Yuan Hu; Yi-Xun Liu

    2006-01-01

    We demonstrated in this study that liver receptor homolog-1 (LRH-1) was expressed in the round spermatids in normal monkey testis, and no LRH-1 signal was observed in the Ser- toli cells. After local warming (43 C) the monkey testis, how- ever, LRH-1 expression was induced in the Sertoli cells in coincidence with activation of cytokeratin 18 (CK-18), a Ser- toli

  16. Spermatogenesis Associated 4 Promotes Sertoli Cell Proliferation Modulated Negatively by Regulatory Factor X1

    PubMed Central

    Jiang, Junjun; Zhang, Nannan; Shiba, Hiroshi; Li, Liyuan; Wang, Zhao

    2013-01-01

    Spermatogenesis associated 4 (Spata4), a testis-specific and CpG island associated gene, is involved in regulating cell proliferation, differentiation and apoptosis. To obtain insight into the role of Spata4 in cell cycling control, we characterized the promoter region of Spata4 and investigated its transcriptional regulation mechanism. The Spata4 promoter is unidirectional transcribed and possesses multiple transcription start sites. Moreover, we present evidence that regulatory factor X1 (RFX1) could bind the typical 14-bp cis-elements of Spata4 promoter, modulate transcriptional activity and endogenous expression of Spata4, and further regulate the proliferation of Sertoli cells. Overexpression of RFX1 was shown to down-regulate both the promoter activity and mRNA expression of Spata4, whereas knockdown of RFX1 demonstrated the opposite effects. Our studies provide insight into Spata4 gene regulation and imply the potential role of RFX1 in growth of Sertoli cells. RFX1 may have negative effect on cell proliferation of Sertoli cells via modulating Spata4 expression levels by binding the conserved 14-bp cis-elements of Spata4 promoter. PMID:24146794

  17. Spermatogenesis associated 4 promotes Sertoli cell proliferation modulated negatively by regulatory factor X1.

    PubMed

    Jiang, Junjun; Zhang, Nannan; Shiba, Hiroshi; Li, Liyuan; Wang, Zhao

    2013-01-01

    Spermatogenesis associated 4 (Spata4), a testis-specific and CpG island associated gene, is involved in regulating cell proliferation, differentiation and apoptosis. To obtain insight into the role of Spata4 in cell cycling control, we characterized the promoter region of Spata4 and investigated its transcriptional regulation mechanism. The Spata4 promoter is unidirectional transcribed and possesses multiple transcription start sites. Moreover, we present evidence that regulatory factor X1 (RFX1) could bind the typical 14-bp cis-elements of Spata4 promoter, modulate transcriptional activity and endogenous expression of Spata4, and further regulate the proliferation of Sertoli cells. Overexpression of RFX1 was shown to down-regulate both the promoter activity and mRNA expression of Spata4, whereas knockdown of RFX1 demonstrated the opposite effects. Our studies provide insight into Spata4 gene regulation and imply the potential role of RFX1 in growth of Sertoli cells. RFX1 may have negative effect on cell proliferation of Sertoli cells via modulating Spata4 expression levels by binding the conserved 14-bp cis-elements of Spata4 promoter. PMID:24146794

  18. Distribution of vimentin-type intermediate filaments in Sertoli cells of the human testis, normal and pathologic

    Microsoft Academic Search

    Gerhard Aumiiller; Manfred Steinbriick; Walter Krause; Hans-Joachim Wagner

    1988-01-01

    Summary  The presence, distribution and spatial arrangement of vimentin-type intermediate filaments in Sertoli cells from human testis\\u000a biopsies, were studied in semithin and ultrathin sections using a polyclonal rabbit antiserum. At the ultrastructural level,\\u000a vimentin immunoreactivity was seen concentrated around the nuclei, along fibrillary material within the cytoplasm and at the\\u000a ectoplasmic specializations of the Sertoli cell junctions, as well as

  19. 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

  20. Cytologic, Immunocytologic, Histopathologic and Immunohistologic Diagnosis of the Poorly Differentiated Sertoli-Leydig Cell Tumor

    Microsoft Academic Search

    H. Soleimanpour; S. Shirian; A. Oryan; K. Daneshbod; N. Bagheri; Y. Daneshbod

    2011-01-01

    Background: Sertoli-Leydig cell tumors (SLCTs), also known as arrhenoblastomas, are tumors of the sex cord-stromal group of ovary and testis cancers. They comprise <1% of all ovarian tumors. They are divided into 6 categories based on the degree of differentiation and the presence of heterologous elements. However, <15% of these tumors are poorly differentiated. Case: A 23-year-old unmarried female presented

  1. Can electrons travel through actin microfilaments and generate oxidative stress in retinol treated Sertoli cell?

    Microsoft Academic Search

    Ramatis Birnfeld de Oliveira; Matheus Augusto de Bittencourt Pasquali; Alfeu Zanotto Filho; Rodrigo Juliani Siqueira Dalmolin; Daniel Pens Gelain; Carmem Gottfried; José Luiz Rodrigues; Fábio Klamt; José Cláudio Fonseca Moreira

    2007-01-01

    In early reports our research group has demonstrated that 7 ?M retinol (vitamin A) treatment leads to many changes in Sertoli\\u000a cell metabolism, such as up-regulation of antioxidant enzyme activities, increase in damage to biomolecules, abnormal cellular\\u000a division, pre-neoplasic transformation, and cytoskeleton conformational changes. These effects were observed to be dependent\\u000a on the production of reactive oxygen species (ROS), suggesting extra-nuclear

  2. Multiple fucosyltransferases and their carbohydrate ligands are involved in spermatogenic cell-Sertoli cell adhesion in vitro in rats.

    PubMed

    Raychoudhury, S S; Millette, C F

    1997-05-01

    We have identified multiple fucosyltransferases (FTs) (alpha[1-2]-, alpha[1-3]-, alpha[1-4]-FTs) on cells of the rat seminiferous epithelium as demonstrated by fucose incorporation into phenyl-beta-D-galactoside (Ph-beta-D-Gal), 2'-fucosyllactose (2'-FL), and lacto-N-fucopentaose-l (LNF I), respectively. Now, using fluorescence laser scanning cytometry, we report that multiple FTs are implicated in germ cell-Sertoli cell adhesion in vitro. Sertoli cells were isolated from 19- to 21-day-old CD rats and cultured for 6-10 days. Mixed germ cells were obtained by enzymatic dispersion of adult rat testis and cultured overnight before labeling with 10 microM acetoxymethyl ester derivative of the fluorescent indicator, calcein. The adherent cell analysis and sorting 570 interactive laser cytometer was used to determine the number of labeled adherent germ cells on Sertoli cell monolayers in the presence or absence of a variety of low molecular weight acceptors for fucose. Coincubation of labeled germ cells with Sertoli cell monolayers in the presence of GDP-fucose, UDP-galactose, Ph-beta-D-Gal, 2'-FL, LNF I, and Lewis-X and 3'-sialyl-Lewis-X oligosaccharides resulted in significant reduction of germ cell binding when compared to that of the untreated controls or of control samples incubated with cellobiose, melibiose, and alpha-D-mannopyranose, which do not serve as fucose acceptors. Our results suggest that multiple FTs and their lectin/selectin ligands are involved in mediating germ cell-Sertoli cell adhesion to form a cohesive epithelium and thus aid germ cell adluminal translocation within the seminiferous epithelium. PMID:9160727

  3. Survey with follow-up of 67 dogs with testicular sertoli cell tumours.

    PubMed

    Weaver, A D

    1983-07-30

    Sixty-seven cases of canine sertoli cell tumour are reviewed. The mean age at diagnosis was 9.5 years (sd +/- 2.1, range 3 to 17). The most commonly affected breeds were boxer, cairn terrier, labrador retriever, border collie, German shepherd and rough collie. The left side was affected in 28 dogs (42 per cent), the right in 35 (52.5 per cent) and both sides in four (6 per cent). Twenty testicles were abdominal (left, seven; right, 13), seven were inguinal (left, four; right, three) and 44 were scrotal (left, 21; right, 23). Hormonal signs were evident in 38 (57 per cent) animals (feminisation with alopecia in 21, symmetrical alopecia alone in 17). Other types of testicular tumours were found in 12 dogs (18 per cent) of this series. Of 42 dogs which were castrated and discharged, 38 were available for follow-up study and survived for between one week and five years. Two of the 15 dogs which were subject to post mortem examination had metastases. Two others which died after failure of remission, or a recurrence of the hormonal changes probably also had metastases. Two of the 25 dogs which were presented initially for other reasons, but had sertoli cell tumours, were found to have metastases post mortem. The boxer, cairn terrier, border collie, Shetland collie and pekingese had a higher risk of sertoli cell tumour than other breeds. PMID:6137897

  4. Transplantation of sertoli-islet cell aggregates formed by microgravity: prolonged survival in diabetic rats.

    PubMed

    Han, Xiaoming; Qiu, Liyuan; Zhang, Ye; Kong, Qingxue; Wang, Hong; Wang, Haibin; Li, Hongbo; Duan, Cuimi; Wang, Yanmeng; Song, Ying; Wang, Changyong

    2009-05-01

    Transplantation of pancreatic islets is a potentially attractive treatment for type I diabetes. We generated the transplantable, tissue-like aggregates composed of Sertoli cells and islets in rotating wall vessel bioreactors, SICA (Sertoli-islet cell aggregates), to improve their biological function in vitro and in vivo. The isolated islet equivalent and Sertoli cells were purified from Wistar rats and cocultured for 5 days in bioreactor to generate SICA. The SICA, islets aggregates, and fresh isolated islets were transplanted under the kidney capsule of diabetic Sprague-Dawley (SD) rats, respectively. The functions of different grafts were ascertained by blood glucose level measurements and an in vivo glucose tolerance test. In response to elevated glucose, insulin secretion from SICA was 1.4-fold higher (P<0.05, n=5) than islet aggregates cultured alone. Of the rats that received SICA, 90% (9/10) remained normoglycemic at 60 days post-transplantation, and the survival significantly increased compared with recipients bearing homotypic islets aggregates or freshly isolated islets. The former responded similarly with healthy rats to the glucose tolerance test. Our results support the usefulness of SICA for the treatment of type 1 diabetes without any immunosuppressive agents. PMID:19244542

  5. 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.

  6. Rapid differentiation of NT2 cells in Sertoli–NT2 cell tissue constructs grown in the rotating wall bioreactor

    Microsoft Academic Search

    Samuel Saporta; Alison E. Willing; Rania Shamekh; Paula Bickford; Daniel Paredes; Don F. Cameron

    2004-01-01

    Cell replacement therapy is of great interest as a long-term treatment of neurodegenerative diseases such as Parkinson's disease (PD). We have previously shown that Sertoli cells (SC) provide neurotrophic support to transplants of dopaminergic fetal neurons and NT2N neurons, derived from the human clonal precursors cell line NTera2\\/D1 (NT2), which differentiate into dopaminergic NT2N neurons when exposed to retinoic acid.

  7. Bilateral large-cell calcifying Sertoli cell tumor of the testes with Peutz-Jeghers syndrome: a case report.

    PubMed

    Dreyer, L; Jacyk, W K; du Plessis, D J

    1994-12-01

    A 13-year-old boy with Peutz-Jeghers syndrome (PJs), gynecomastia, and bilateral multifocal testicular tumors is described. Histology of the testicular tissue disclosed large-cell calcifying Sertoli cell tumors. Females with PJs are known to be at increased risk of developing gonadal tumors. This case and a review of other reported cases suggest that males with PJs are also at risk for developing gonadal tumors. PMID:7899185

  8. The total number of Leydig and Sertoli cells in the testes of men across various age groups - a stereological study.

    PubMed

    Petersen, Peter M; Seierøe, Karina; Pakkenberg, Bente

    2015-02-01

    The aim of this study was to estimate the total number of Sertoli and Leydig cells in testes from male subjects across the human lifespan, using an optimized stereological method for cell-counting. In comparison with many other organs, estimation of the total cell numbers in the testes is particularly sensitive to methodological problems. Therefore, using the optical fractionator technique and a sampling design specifically optimized for human testes, we estimated the total number of Sertoli and Leydig cells in the testes from 26 post mortem male subjects ranging in age from 16 to 80?years. The mean unilateral total number of Sertoli cells was 407?×?10(6) [range: 86?×?10(6) to 665?×?10(6) , coefficient of variation (CV)?=?0.33], and the mean unilateral total number of Leydig cells was 99?×?10(6) (range: 47?×?10(6) to 245?×?10(6) , CV?=?0.48). There was a significant decline in the number of Sertoli cells with age; no such decline was found for Leydig cells. Quantitative stereological analysis of post mortem tissue may help understand the influence of age or disease on the number of human testicular cells. PMID:25545958

  9. Hormonal regulation of Sertoli cell function in the rat V. HANSSON, K. PURVIS E. M. RITZN F. S. FRENCH

    E-print Network

    Boyer, Edmond

    Hormonal regulation of Sertoli cell function in the rat V. HANSSON, K. PURVIS E. M. RITZÉN F. S. ' The hormonal regulation of spermatogenesis involves an interplay of sex steroids and pituitary gonadotrophic hormones acting on specific cells of the testis. Several reviews have brought together earlier work

  10. 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

  11. 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.

  12. Dmrt1 Expression Is Regulated by Follicle-Stimulating Hormone and Phorbol Esters in Postnatal Sertoli Cells*

    PubMed Central

    CHEN, JIANG KAI; HECKERT, LESLIE L.

    2006-01-01

    Dmrt1 is a recently described gene that is expressed exclusively in the testis and is required for postnatal testis differentiation. Here we describe the expression of Dmrt1 in postnatal rat testis and Sertoli cells. RNase protection analysis was used to examine Dmrt1 messenger RNA (mRNA) levels in intact testis during postnatal development and in primary cultures of Sertoli cells under various culture conditions. We show that Dmrt1 mRNA levels rise significantly beginning approximately 10 days after birth and remain elevated until after the third postnatal week. Thereafter, mRNA levels drop coincident with the proliferation of germ cells in the testis. In freshly isolated Sertoli cells, Dmrt1 mRNA levels were robust but decreased significantly when the cells were placed in culture for 24 h. Treatment of Sertoli cells with either FSH or 8-bromo-cAMP resulted in a significant rise in Dmrt1 mRNA levels. This cAMP response was sensitive to treatment with the transcriptional inhibitor actinomycin D but not to the translational inhibitor cycloheximide. The cAMP-dependent rise in Dmrt1 mRNA also required activation of protein kinase A, as mRNA induction was sensitive to the inhibitor H89. Studies also show that Dmrt1 expression was inhibited by phorbol esters (PMA) but only modestly effected by serum. PMID:11181532

  13. 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

  14. SERTOLI CELLS IN THE BOAR TESTIS: CHANGES DURING DEVELOPMENT AND COMPENSATORY HYPERTROPHY AFTER HEMICASTRATION AT DIFFERENT AGES

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Changes in Sertoli cell numbers and testicular structure during normal development and during compensatory hypertrophy were assessed in crossbred Meishan x White Composite males. Boars were assigned at birth to unilateral castration at 1, 10, 56 or 112 days, or remained as intact controls through 22...

  15. Dietary antioxidant, quercetin, protects sertoli-germ cell coculture from atrazine-induced oxidative damage.

    PubMed

    Abarikwu, Sunny O; Pant, Aditya B; Farombi, Ebenezer O

    2012-11-01

    Quercetin (QT), a dietary-derived flavonoid, is ubiquitous in fruits and vegetables and plays an important role in human health by virtue of its antioxidant function. The present study was designed to examine the effects of QT on oxidative damage that was induced by the herbicide, atrazine (ATZ), in mixed cultures of Sertoli-germ cells. Results showed that treatment with QT increased cell viability and decreased catalase activity, malondialdehyde, and reactive oxygen species (ROS) levels. QT treatment also increased the mRNA expression of glutathione peroxidase (GSH-Px), glutathione reductase (GR), glutathione-S-transferase, and superoxide dismutase-1 and could not reversed to the control levels ATZ-induced steady-state mRNA levels of these antioxidant genes as well as the level of glutathione and activities of GSH-Px and GR. QT has protective effect against ATZ-induced oxidative stress through a reduction in ROS levels and lipid peroxidation. PMID:23132811

  16. KATNAL1 Regulation of Sertoli Cell Microtubule Dynamics Is Essential for Spermiogenesis and Male Fertility

    PubMed Central

    Smith, Lee B.; Milne, Laura; Nelson, Nancy; Eddie, Sharon; Brown, Pamela; Atanassova, Nina; O'Bryan, Moira K.; O'Donnell, Liza; Rhodes, Danielle; Wells, Sara; Napper, Diane; Nolan, Patrick; Lalanne, Zuzanna; Cheeseman, Michael; Peters, Josephine

    2012-01-01

    Spermatogenesis is a complex process reliant upon interactions between germ cells (GC) and supporting somatic cells. Testicular Sertoli cells (SC) support GCs during maturation through physical attachment, the provision of nutrients, and protection from immunological attack. This role is facilitated by an active cytoskeleton of parallel microtubule arrays that permit transport of nutrients to GCs, as well as translocation of spermatids through the seminiferous epithelium during maturation. It is well established that chemical perturbation of SC microtubule remodelling leads to premature GC exfoliation demonstrating that microtubule remodelling is an essential component of male fertility, yet the genes responsible for this process remain unknown. Using a random ENU mutagenesis approach, we have identified a novel mouse line displaying male-specific infertility, due to a point mutation in the highly conserved ATPase domain of the novel KATANIN p60-related microtubule severing protein Katanin p60 subunit A-like1 (KATNAL1). We demonstrate that Katnal1 is expressed in testicular Sertoli cells (SC) from 15.5 days post-coitum (dpc) and that, consistent with chemical disruption models, loss of function of KATNAL1 leads to male-specific infertility through disruption of SC microtubule dynamics and premature exfoliation of spermatids from the seminiferous epithelium. The identification of KATNAL1 as an essential regulator of male fertility provides a significant novel entry point into advancing our understanding of how SC microtubule dynamics promotes male fertility. Such information will have resonance both for future treatment of male fertility and the development of non-hormonal male contraceptives. PMID:22654668

  17. mRNA-selective translation induced by FSH in primary Sertoli cells.

    PubMed

    Musnier, Astrid; León, Kelly; Morales, Julia; Reiter, Eric; Boulo, Thomas; Costache, Vlad; Vourc'h, Patrick; Heitzler, Domitille; Oulhen, Nathalie; Poupon, Anne; Boulben, Sandrine; Cormier, Patrick; Crépieux, Pascale

    2012-04-01

    FSH is a key hormonal regulator of Sertoli cell secretory activity, required to optimize sperm production. To fulfil its biological function, FSH binds a G protein-coupled receptor, the FSH-R. The FSH-R-transduced signaling network ultimately leads to the transcription or down-regulation of numerous genes. In addition, recent evidence has suggested that FSH might also regulate protein translation. However, this point has never been demonstrated conclusively yet. Here we have addressed this issue in primary rat Sertoli cells endogenously expressing physiological levels of FSH-R. We observed that, within 90 min of stimulation, FSH not only enhanced overall protein synthesis in a mammalian target of rapamycin-dependent manner but also increased the recruitment of mRNA to polysomes. m(7)GTP pull-down experiments revealed the functional recruitment of mammalian target of rapamycin and p70 S6 kinase to the 5'cap, further supported by the enhanced phosphorylation of one of p70 S6 kinase targets, the eukaryotic initiation factor 4B. Importantly, the scaffolding eukaryotic initiation factor 4G was also recruited, whereas eukaryotic initiation factor 4E-binding protein, the eukaryotic initiation factor 4E generic inhibitor, appeared to play a minor role in translational regulations induced by FSH, in contrast to what is generally observed in response to anabolic factors. This particular regulation of the translational machinery by FSH stimulation might support mRNA-selective translation, as shown here by quantitative RT-PCR amplification of the c-fos and vascular endothelial growth factor mRNA but not of all FSH target mRNA, in polysomal fractions. These findings add a new level of complexity to FSH biological roles in its natural target cells, which has been underappreciated so far. PMID:22383463

  18. The AMP-activated protein kinase activator, 5-aminoimidazole-4-carboxamide-1-b-D-ribonucleoside, regulates lactate production in rat Sertoli cells

    Microsoft Academic Search

    Maria Noel Galardo; Maria Fernanda Riera; Eliana Herminia Pellizzari; Selva Beatriz Cigorraga; Silvina Beatriz Meroni

    2007-01-01

    The aim of the present study was to investigate whether the AMP-activated protein kinase (AMPK), a key regulator of cellular energy homeostasis, is present in Sertoli cells and whether its activation by 5-aminoimidazole-4-carboxamide-1-b-D- ribonucleoside (AICAR) results in the regulation of cell metabolism to ensure lactate supply for germ cell development. Sertoli cell cultures from 20-day-old rats were used. Western blot

  19. Ecotropic murine leukemia virus-induced fusion of murine cells

    SciTech Connect

    Pinter, A.; Chen, T.; Lowy, A.; Cortez, N.G.; Silagi, S.

    1986-03-01

    Extensive fusion occurs upon cocultivation of murine fibroblasts producing ecotropic murine leukemia viruses (MuLVs) with a large variety of murine cell lines in the presence of the polyene antibiotic amphotericin B, the active component of the antifungal agent Fungizone. The resulting polykaryocytes contain nuclei from both infected and uninfected cells, as evidenced by autoradiographic labeling experiments in which one or the other parent cell type was separately labeled with (/sup 3/H)thymidine and fused with an unlabeled parent. This cell fusion specifically requires the presence of an ecotropic MuLV-producing parent and is not observed for cells producing xenotropic, amphotropic, or dualtropic viruses. Mouse cells infected with nonecotropic viruses retain their sensitivity toward fusion, whereas infection with ecotropic viruses abrogates the fusion of these cells upon cocultivation with other ecotropic MuLV-producing cells. Nonmurine cells lacking the ecotropic gp70 receptor are not fused under similar conditions. Fusion is effectively inhibited by monospecific antisera to gp70, but not by antisera to p15(E), and studies with monoclonal antibodies identify distinct amino- and carboxy-terminal gp70 regions which play a role in the fusion reaction. The enhanced fusion which occurs in the presence of amphotericin B provides a rapid and sensitive assay for the expression of ecotropic MuLVs and should facilitate further mechanistic studies of MuLV-induced fusion of murine cells.

  20. Electrochemical sensors, MTT and immunofluorescence assays for monitoring the proliferation effects of cissus populnea extracts on Sertoli cells

    PubMed Central

    2011-01-01

    Background We describe the development of an electrochemical sensor array for monitoring the proliferation effects of cissus populnea plant extracts on TM4 Sertoli cells. Methods The proliferation activities of the extracts on Sertoli cells were studied using a high-throughput electrochemical sensor array (DOX-96) and the analytical sensor characteristics were compared with conventional colorimetric MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay and fluorescence spectroscopy. Results This work shows that there is a definite positive trend in the proliferation effect of the extract of Cissus populnea on the TM4 Sertoli cells. All of the three techniques confirmed that the most effective concentration for the proliferation is 10 ppm. At this concentration, the proliferation effect was established around 120% for both DOX-96 and MTT techniques, whereas fluorescence assays showed a higher level (120-150%). DOX-96 showed a lower limit of detection (1.25 × 10(4) cells/ml); whereas the LOD recorded for both MTT and fluorescence techniques was 2.5 × 10(4) cells/ml. Visual examination of the cells by means of confocal fluorescence microscopy confirmed the proliferation of Sertoli cells as was determined using the MTT assay. This investigation provides a confident interpretation of the results and proved that the most effective concentration for the proliferation using Cissus populnea plant extract is 10 ppm. Conclusions Overall, the DOX results compared well with the conventional methods of checking proliferation of cells. The fascinating feature of the sensor array is the ability to provide continuous proliferation experiments with no additional reagents including 96 simultaneous electrochemical experiments. The use of the DOX-96 could reduce a typical bioassay time by 20-fold. Thus the DOX-96 can be used as both a research tool and for practical cell culture monitoring. PMID:21575213

  1. Up-Regulation of SOX9 in Sertoli Cells from Testiculopathic Patients Accounts for Increasing Anti-Mullerian Hormone Expression via Impaired Androgen Receptor Signaling

    PubMed Central

    Lan, Kuo-Chung; Chen, Yen-Ta; Chang, Chawnshang; Chang, Yung-Chiao; Lin, Hsin-Jung; Huang, Ko-En; Kang, Hong-Yo

    2013-01-01

    Background Testosterone provokes Sertoli cell maturation and represses AMH production. In adult patients with Sertoli-cells-only syndrome (SCOS) and androgen insensitivity syndrome (AIS), high level of AMH expression is detected in Sertoli cells due to defect of androgen/AR signaling. Objective We postulated that up-regulation of SOX9 due to impairment of androgen/AR signaling in Sertoli cells might explain why high level of anti-Mullerian hormone (AMH) expression occur in these testiculopathic patients. Methods Biological research of testicular specimens from men with azoospermia or mouse. The serum hormone levels were studied in 23 men with obstructive azoospermia, 33 men with SCOS azoospermia and 21 volunteers with normal seminograms during a period of 4 years. Immunohistochemical staining and reverse-transcription PCR were used to examine the relationships among AR, SOX9 and AMH expression in adult human and mouse testes. The ability of AR to repress the expression of SOX9 and AMH was evaluated in vitro in TM4 Sertoli cells and C3H10T1/2 cells. Results SCOS specimens showed up-regulation of SOX9 and AMH proteins but down-regulation of AR proteins in Sertoli cells. The mRNA levels of AR were significantly lower and the SOX9, AMH mRNA levels higher in all SCOS patients compared to controls (P< 0.05). The testosterone levels in the SCOS patients were within the normal range, but most were below the median of the controls. Furthermore, our in vitro cell line experiments demonstrated that androgen/AR signaling suppressed the gene and protein levels of AMH via repression of SOX9. Conclusions Our data show that the functional androgen/AR signaling to repress SOX9 and AMH expression is essential for Sertoli cell maturation. Impairment of androgen/AR signaling promotes SOX9-mediated AMH production, accounts for impairments of Sertoli cells in SCOS azoospermic patients. PMID:24098470

  2. Identification of meiotic and postmeiotic gene expression in testicular tissue of patients histologically classified as Sertoli cell only

    Microsoft Academic Search

    Pasquale Patrizio; Stacey M Ricci; John E Tomaszewski; Norman B Hecht

    2000-01-01

    Objective: To determine whether meiotic and postmeiotic germ cell gene products could be detected in biopsy specimen from patients with Sertoli cell only (SCO) and maturation arrest.Design: Prospective clinical study.Setting: University-based departments and laboratories.Patient(s): Nine patients, seven with nonobstructive azoospermia (12 biopsies) and two with obstructive azoospermia (controls) (2 biopsies).Intervention(s): Specimens were divided into three parts: IVF laboratory, histology, and

  3. Identification, characterization, and hormonal regulation of 3', 5'-cyclic adenosine monophosphate-dependent protein kinases in rat Sertoli cells.

    PubMed

    Landmark, B F; Fauske, B; Eskild, W; Skålhegg, B; Lohmann, S M; Hansson, V; Jahnsen, T; Beebe, S J

    1991-11-01

    Recent studies have disclosed multiple isoforms of regulatory (R) and catalytic (C) subunits of cAMP-dependent protein kinase (PKA) at the protein and messenger RNA (mRNA) levels. The purpose of the present study was to identify, characterize, and quantify individual R subunits in rat Sertoli cells both at the mRNA and protein levels. Unstimulated Sertoli cells contain high levels of R (approximately 9.2 +/- 0.8 pmol/mg protein) and C (approximately 7.3 +/- 0.7 pmol/mg protein). Stimulation with (Bt)2cAMP (0.1 mM) for 24 and 48 h revealed a time-dependent increase in [3H]cAMP-binding activity. During the same time period the catalytic activity remained relatively constant, resulting in an increase in the R/C ratio from approximately 1.3 to 3.0. Using diethylaminoethyl cellulose chromatography, 8-N3-[32P]cAMP photoaffinity labeling, autophosphorylation by gamma-[32P]ATP, and specific antibodies, we show that unstimulated Sertoli cells contain approximately 75% RI alpha, 25% RII alpha, and very low levels of RII beta. Stimulation of Sertoli cells with (Bt)2cAMP (0.1 mM, 48 h) was associated with a 2.1-fold increase in RI alpha (6.6-14 pmol/mg) and a 10- to 20-fold increase in RII beta (less than 0.1-1.1 pmol/mg), with little or no change in RII alpha (1.9-2.3 pmol/mg). Treatment with cAMP was associated with a slight increase in RI/RII ratio (3.3-4.1). mRNA levels for RII beta increased 30- to 50-fold after (Bt)2cAMP stimulation, whereas only minor changes in mRNA levels for RI alpha, RII alpha, and C alpha were observed (1.5- to 2.0-fold). mRNA levels for RI beta, C beta, and C gamma were not detected in either unstimulated or in cAMP-stimulated Sertoli cells. It is concluded that chronic treatment with cAMP changes the relative proportion of R subunits of PKA in a manner reflecting the changing levels in respective mRNAs. Furthermore, such treatment is associated with the appearance of a new PKA R subunit (RII beta), which is absent in untreated Sertoli cells. PMID:1657573

  4. 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.

  5. Research resource: the dynamic transcriptional profile of sertoli cells during the progression of spermatogenesis.

    PubMed

    Zimmermann, Céline; Stévant, Isabelle; Borel, Christelle; Conne, Béatrice; Pitetti, Jean-Luc; Calvel, Pierre; Kaessmann, Henrik; Jégou, Bernard; Chalmel, Frédéric; Nef, Serge

    2015-04-01

    Sertoli cells (SCs), the only somatic cells within seminiferous tubules, associate intimately with developing germ cells. They not only provide physical and nutritional support but also secrete factors essential to the complex developmental processes of germ cell proliferation and differentiation. The SC transcriptome must therefore adapt rapidly during the different stages of spermatogenesis. We report comprehensive genome-wide expression profiles of pure populations of SCs isolated at 5 distinct stages of the first wave of mouse spermatogenesis, using RNA sequencing technology. We were able to reconstruct about 13 901 high-confidence, nonredundant coding and noncoding transcripts, characterized by complex alternative splicing patterns with more than 45% comprising novel isoforms of known genes. Interestingly, roughly one-fifth (2939) of these genes exhibited a dynamic expression profile reflecting the evolving role of SCs during the progression of spermatogenesis, with stage-specific expression of genes involved in biological processes such as cell cycle regulation, metabolism and energy production, retinoic acid synthesis, and blood-testis barrier biogenesis. Finally, regulatory network analysis identified the transcription factors endothelial PAS domain-containing protein 1 (EPAS1/Hif2?), aryl hydrocarbon receptor nuclear translocator (ARNT/Hif1?), and signal transducer and activator of transcription 1 (STAT1) as potential master regulators driving the SC transcriptional program. Our results highlight the plastic transcriptional landscape of SCs during the progression of spermatogenesis and provide valuable resources to better understand SC function and spermatogenesis and its related disorders, such as male infertility. PMID:25710594

  6. Retinoblastoma protein (RB) interacts with E2F3 to control terminal differentiation of Sertoli cells.

    PubMed

    Rotgers, E; Rivero-Müller, A; Nurmio, M; Parvinen, M; Guillou, F; Huhtaniemi, I; Kotaja, N; Bourguiba-Hachemi, S; Toppari, J

    2014-01-01

    The retinoblastoma protein (RB) is essential for normal cell cycle control. RB function depends, at least in part, on interactions with the E2F family of DNA-binding transcription factors (E2Fs). To study the role of RB in the adult testis, a Sertoli cell (SC)-specific Rb knockout mouse line (SC-RbKO) was generated using the Cre/loxP recombination system. SC-RbKO mice exhibited an age-dependent testicular atrophy, impaired fertility, severe SC dysfunction, and spermatogenic defects. Removal of Rb in SC induced aberrant SC cycling, dedifferentiation, and apoptosis. Here we show that E2F3 is the only E2F expressed in mouse SCs and that RB interacts with E2F3 during mouse testicular development. In the absence of RB, the other retinoblastoma family members p107 and p130 began interacting with E2F3 in the adult testes. In vivo silencing of E2F3 partially restored the SC maturation and survival as well as spermatogenesis in the SC-RbKO mice. These results point to RB as a key regulator of SC function in adult mice and that the RB/E2F3 pathway directs SC maturation, cell cycle quiescence, and RB protects SC from apoptosis. PMID:24901045

  7. 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

  8. Insulinoma presenting with long-standing depression, primary hypogonadism, and sertoli cell only syndrome.

    PubMed

    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

  9. Involvement of CaM-CaMKII-ERK in bisphenol A-induced Sertoli cell apoptosis.

    PubMed

    Qian, Wenyi; Zhu, Jingying; Mao, Changfei; Liu, Jingli; Wang, Yixin; Wang, Qiang; Liu, Yanqing; Gao, Rong; Xiao, Hang; Wang, Jun

    2014-10-01

    Bisphenol A (BPA), one of the most prevalent chemicals for daily use, has been reported as a xenoestrogen to induce reproductive toxicity, but its mechanism is poorly understood. In the present study, we aimed to explore whether CaM-CaMKII-ERK1/2 signaling pathway was involved in BPA-induced Sertoli cells injury via the mitochondrial apoptotic pathway. TM4 cells were cultured with 0, 0.02, 0.2, 2.0, 20?M BPA, and cell viability, mitochondrial function and CaM-CaMKII-ERK1/2 signal pathway were examined. With the MTT assay, BPA was found to suppress cell viability in a dose- and time-dependent manner. Moreover, mitochondrial mass loss, membrane potential decrease, cytochrome c release, Bcl-2 family members down-regulation and caspases-3 up-regulation were obviously observed when the TM4 cells were exposed to BPA. Additionally, the expression of calmodulin (CaM) and phosphorylation of calcium/calmodulin dependent kinase II (CaMKII) significantly increased, and pretreatment with 10?M antagonist of CaM (W-7) or CaMKII (KN62) prevented cell damage through mitochondrial apoptotic pathway. In parallel, ERK1/2 pathway was proved to participate in BPA-induced cell damage, since W-7 and KN62 partially suppressed ERK1/2 activation, and PD98059, the ERK1/2 antagonist, significantly attenuated BPA-induced cell damage. These data, taken together, indicated that CaM-CaMKII-ERK axis might transmit apoptotic signals to the mitochondria during BPA-induced cell apoptosis. By exploring the mechanisms of the Ca(2+) homeostasis and the corresponding proteins, our study provides new insight into BPA-induced reproductive toxicity. PMID:24905940

  10. 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

  11. Modulation of endogenous GATA-4 activity reveals its dual contribution to Müllerian inhibiting substance gene transcription in Sertoli cells.

    PubMed

    Tremblay, J J; Robert, N M; Viger, R S

    2001-09-01

    Secretion of Müllerian inhibiting substance by fetal Sertoli cells is essential for normal male sex differentiation since it induces regression of the Müllerian ducts in the developing male embryo. Proper spatiotemporal expression of the MIS gene requires a specific combination of transcription factors, including the zinc finger factor GATA-4 and the nuclear receptor steroidogenic factor-1, which both colocalize with Müllerian inhibiting substance in Sertoli cells. To establish the molecular mechanisms through which GATA-4 contributes to MIS transcription, we have generated and characterized novel GATA-4 dominant negative competitors. The first one, which consisted solely of the GATA-4 zinc finger DNA-binding domain, was an efficient competitor of GATA transcription mediated both by direct GATA binding to DNA and protein-protein interactions involving GATA factors. The second type of competitor consisted of the same GATA-4 zinc finger DNA-binding domain but harboring mutations that prevented DNA binding. This second class of competitors repressed GATA-dependent transactivation by specifically competing for GATA protein-protein interactions without affecting the DNA-binding activity of endogenous GATA factors. These competitors, along with the GATA-4 cofactor FOG-2 (friend of GATA-2), were used to specifically modulate endogenous GATA-4 activity in Sertoli cells. Our results indicate that GATA-4 contributes to MIS promoter activity through two distinct mechanisms. Moreover, the GATA competitors described here should provide invaluable in vitro and in vivo tools for the study of GATA- dependent transcription and the identification of new target genes. PMID:11518812

  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. Genotyping of Tunisian azoospermic men with Sertoli cell-only and maturation arrest.

    PubMed

    Hadjkacem-Loukil, Lobna; Hadj-Kacem, Hassen; Hadj Salem, Ikhlass; Bahloul, Ali; Fakhfakh, Faiza; Ayadi, Hammadi

    2011-07-01

    Azoospermia factor (AZF) deletions were associated with severe oligospermia and azoospermia with testicular histologies varying from maturation arrest (MA) to Sertoli cell-only (SCO) phenotypes. Abnormal androgen receptor (AR) structure or function has also been implicated in male infertility. To assess the contribution of these genetic defects to azoospermic patients, 19 Tunisian men with SCO (n?=?13) or MA (n?=?6) were enrolled in this study. Using immunohistochemistry method, we evaluated the expression of AR in testicular biopsy for the two phenotypes. PCR with primers flanking the AR-(CAG)n region and direct sequencing were used to determine AR-(CAG)n length. And PCR amplification of 14 sequence-tagged sites (STSs) located at Yq was used to determine the rate and extent of Y microdeletions. We found a significant difference of the AR expression between SCO and MA cases. Hence, this expression in the testis depends on the status of spermatogenesis. However, we did not find any relationship between the (CAG) repeat and the testicular histology (neither for SCO nor MA). On the other hand, we found a high frequency of AZF deletions (46.2%) in SCOS and in MA (50%). The present results also suggest the contribution of Y chromosome microdeletions in SCO and MA pathogenesis. PMID:21729129

  14. Copy number variants in patients with severe oligozoospermia and Sertoli-cell-only syndrome.

    PubMed

    Tüttelmann, Frank; Simoni, Manuela; Kliesch, Sabine; Ledig, Susanne; Dworniczak, Bernd; Wieacker, Peter; Röpke, Albrecht

    2011-01-01

    A genetic origin is estimated in 30% of infertile men with the common phenotypes of oligo- or azoospermia, but the pathogenesis of spermatogenic failure remains frequently obscure. To determine the involvement of Copy Number Variants (CNVs) in the origin of male infertility, patients with idiopathic severe oligozoospermia (N?=?89), Sertoli-cell-only syndrome (SCOS, N?=?37)) and controls with normozoospermia (N?=?100) were analysed by array-CGH using the 244A/400K array sets (Agilent Technologies). The mean number of CNVs and the amount of DNA gain/loss were comparable between all groups. Ten recurring CNVs were only found in patients with severe oligozoospermia, three only in SCOS and one CNV in both groups with spermatogenic failure but not in normozoospermic men. Sex-chromosomal, mostly private CNVs were significantly overrepresented in patients with SCOS. CNVs found several times in all groups were analysed in a case-control design and four additional candidate genes and two regions without known genes were associated with SCOS (P<1×10(-3)). In conclusion, by applying array-CGH to study male infertility for the first time, we provide a number of candidate genes possibly causing or being risk factors for the men's spermatogenic failure. The recurring, patient-specific and private, sex-chromosomal CNVs as well as those associated with SCOS are candidates for further, larger case-control and re-sequencing studies. PMID:21559371

  15. Sertoli-Leydig Cell Tumour of Ovary with Menorrhagia: A Rare Case Report

    PubMed Central

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

    2014-01-01

    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. 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

  17. 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.

  18. Regulation of apoptotic signaling pathways by 5?-dihydrotestosterone and 17?-estradiol in immature rat Sertoli cells.

    PubMed

    Simões, V L; Alves, M G; Martins, A D; Dias, T R; Rato, L; Socorro, S; Oliveira, P F

    2013-05-01

    Apoptosis is an important regulatory event in testicular homeostasis and optimization of sperm production. Sertoli cells (SCs) form the blood-testis barrier creating a special microenvironment where germ cells develop and are under strict hormonal control. Estrogens and androgens are known to play critical roles in SCs functioning, improving their in vitro survival by preventing apoptotic progression. Herein, we studied the influence of 17?-estradiol (E2) and 5?-dihydrotestosterone (DHT) on the apoptotic signaling pathways of immature rat cultured SCs. For that we chose key points of the apoptotic pathway that interact with the mitochondria and evaluated the mRNA expression and/or protein levels of several apoptotic markers such as p53, the anti-apoptotic protein Bcl2, the pro-apoptotic Bcl2 family member Bax, the apoptosis-inducing factor (AIF) and caspase-3 and 9. Caspase-3 activity and DNA fragmentation were also evaluated as endpoint markers of apoptosis. E2 and DHT down-regulated the mRNA transcript levels of p53, Bax, caspase-9 and caspase-3. The protein levels of AIF were reduced after DHT treatment while E2-treated cells presented decreased levels of cleaved caspase-9 protein. Moreover, Bax/Bcl2 ratio was significantly decreased in E2-treated cells. The apoptotic endpoints caspase-3 activity and DNA fragmentation presented significant decreased levels after hormonal treatment. Taken together, these results show that E2 and DHT act as apoptotic signaling modulators in in vitro immature rat SCs suggesting that androgens and estrogens may be capable of modulating independent pathways of the apoptotic event by regulating different pro-apoptotic factors. PMID:23220551

  19. Roundup disrupts male reproductive functions by triggering calcium-mediated cell death in rat testis and Sertoli cells.

    PubMed

    de Liz Oliveira Cavalli, Vera Lúcia; Cattani, Daiane; Heinz Rieg, Carla Elise; Pierozan, Paula; Zanatta, Leila; Benedetti Parisotto, Eduardo; Wilhelm Filho, Danilo; Mena Barreto Silva, Fátima Regina; Pessoa-Pureur, Regina; Zamoner, Ariane

    2013-12-01

    Glyphosate is the primary active constituent of the commercial pesticide Roundup. The present results show that acute Roundup exposure at low doses (36 ppm, 0.036 g/L) for 30 min induces oxidative stress and activates multiple stress-response pathways leading to Sertoli cell death in prepubertal rat testis. The pesticide increased intracellular Ca(2+) concentration by opening L-type voltage-dependent Ca(2+) channels as well as endoplasmic reticulum IP3 and ryanodine receptors, leading to Ca(2+) overload within the cells, which set off oxidative stress and necrotic cell death. Similarly, 30 min incubation of testis with glyphosate alone (36 ppm) also increased (45)Ca(2+) uptake. These events were prevented by the antioxidants Trolox and ascorbic acid. Activated protein kinase C, phosphatidylinositol 3-kinase, and the mitogen-activated protein kinases such as ERK1/2 and p38MAPK play a role in eliciting Ca(2+) influx and cell death. Roundup decreased the levels of reduced glutathione (GSH) and increased the amounts of thiobarbituric acid-reactive species (TBARS) and protein carbonyls. Also, exposure to glyphosate-Roundup stimulated the activity of glutathione peroxidase, glutathione reductase, glutathione S-transferase, ?-glutamyltransferase, catalase, superoxide dismutase, and glucose-6-phosphate dehydrogenase, supporting downregulated GSH levels. Glyphosate has been described as an endocrine disruptor affecting the male reproductive system; however, the molecular basis of its toxicity remains to be clarified. We propose that Roundup toxicity, implicated in Ca(2+) overload, cell signaling misregulation, stress response of the endoplasmic reticulum, and/or depleted antioxidant defenses, could contribute to Sertoli cell disruption in spermatogenesis that could have an impact on male fertility. PMID:23820267

  20. Dose-dependent effects of sertoli cell toxicants 2,5-hexanedione, carbendazim, and mono-(2-ethylhexyl) phthalate in adult rat testis.

    PubMed

    Moffit, Jeffrey S; Bryant, Bronwyn H; Hall, Susan J; Boekelheide, Kim

    2007-08-01

    Sertoli cells are the primary cellular target for a number of pharmaceutical and environmental testicular toxicants, including 2,5-hexanedione, carbendazim, and mono-(2-ethylhexyl) phthalate. Exposure to these individual compounds can result in impaired Sertoli cell function and subsequent germ cell loss. The loss of testicular function is marked by histopathological changes in seminiferous tubule diameter, seminiferous epithelial sloughing, vacuolization, spermatid head retention, germ cell apoptosis, and altered microtubule assembly. The present study investigates dose-response relationships for these classic Sertoli cell toxicants using histopathology endpoints. Understanding the relationship between the Sertoli cell toxicant dose and its histopathologic manifestations will help establish the sensitivity of these endpoints as markers of testicular injury. The results indicate that no single histopathology endpoint was sensitive on its own in identifying altered testicular morphology resulting from toxicant exposure. However, when multiple endpoints were combined dose-response relationships could be associated with incremental alterations in histopathology. The data generated from these experiments will be useful in further investigating the effects of Sertoli cell toxicant exposure in animal toxicity studies. In addition, this work is fundamental to a planned investigation of the histopathologic and gene expression changes associated with testicular toxicant co-exposures, which may occur both occupationally and environmentally. PMID:17763286

  1. NC1 domain of collagen ?3(IV) derived from the basement membrane regulates Sertoli cell blood-testis barrier dynamics

    PubMed Central

    Wong, Elissa W.P.; Cheng, C. Yan

    2013-01-01

    The blood-testis barrier (BTB) is an important ultrastructure for spermatogenesis. Delay in BTB formation in neonatal rats or its irreversible damage in adult rats leads to meiotic arrest and failure of spermatogonial differentiation beyond type A. While hormones, such as testosterone and FSH, are crucial to BTB function, little is known if there is a local regulatory mechanism in the seminiferous epithelium that modulates BTB function. Herein, we report that collagen ?3(IV) chain, a component of the basement membrane in the rat testis, could generate a noncollagenous (NC1) domain peptide [Col?3(IV) NC1] via limited proteolysis by matrix metalloproteinase-9 (MMP-9), and that the expression of MMP-9 was upregulated by TNF?. While recombinant Col?3(IV) NC1 protein produced in E. coli failed to perturb Sertoli cell tight junction (TJ)-permeability barrier function, possibly due to the lack of glycosylation, Col?3(IV) NC1 recombinant protein produced in mammalian cells and purified to apparent homogeneity by affinity chromatography was found to reversibly perturb the Sertoli cell TJ-barrier function. Interestingly, Col?3(IV) NC1 recombinant protein did not perturb the steady-state levels of several TJ- (e.g., occludin, CAR, JAM-A, ZO-1) and basal ectoplasmic specialization- (e.g., N-cadherin, ?-catenin, ?-catenin) proteins at the BTB but induced changes in protein localization and/or distribution at the Sertoli cell-cell interface in which these proteins moved from the cell surface into the cell cytosol, thereby destabilizing the TJ function. These findings illustrate the presence of a local regulatory axis known as the BTB-basement membrane axis that regulates BTB restructuring during spermatogenesis. PMID:23885308

  2. 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

  3. Aquaporin-4 as a molecular partner of cystic fibrosis transmembrane conductance regulator in rat Sertoli cells.

    PubMed

    Jesus, Tito T; Bernardino, Raquel L; Martins, Ana D; Sá, Rosália; Sousa, Mário; Alves, Marco G; Oliveira, Pedro F

    2014-04-18

    Sertoli cells (SCs) form the blood-testis barrier (BTB) that controls the microenvironment where the germ cells develop. The cystic fibrosis transmembrane conductance regulator (CFTR) plays an essential role to male fertility and it was recently suggested that it may promote water transport. Interestingly, Aquaporin-4 (AQP4) is widely expressed in blood barriers, but was never identified in SCs. Herein we hypothesized that SCs express CFTR and AQP4 and that they can physically interact. Primary SCs cultures from 20-day-old rats were maintained and CFTR and AQP4 mRNA and protein expression was assessed by RT-PCR and Western blot, respectively. The possible physical interaction between CFTR and AQP4 was studied by co-immunoprecipitation. We were able to confirm the presence of CFTR at mRNA and protein level in cultured rat SCs. AQP4 mRNA analysis showed that cultured rat SCs express the transcript variant c of AQP4, which was followed by immunodetection of the correspondent protein. The co-immunoprecipitation experiments showed a direct interaction between AQP4 and CFTR in cultured rat SCs. Our results suggest that CFTR physically interacts with AQP4 in rat SCs evidencing a possible mechanism by which CFTR can control water transport through BTB. The full enlightenment of this particular relation between CFTR and AQP4 may point towards possible therapeutic targets to counteract male subfertility/infertility in men with Cystic Fibrosis and mutations in CFTR gene, which are known to impair spermatogenesis due to defective water transport. PMID:24657265

  4. Environmental Tobacco Smoke Exposure during Intrauterine Period, Promotes Caspase Dependent and Independent DNA Fragmentation in Sertoli-Germ Cells

    PubMed Central

    Yüksel, Beril; Kilic, Sevtap; Lortlar, Nese; Tasdemir, Nicel; Sertyel, Semra; Bardakci, Yesim; Aksu, Tarik; Batioglu, Sertaç

    2014-01-01

    Objectives. To investigate the effect of cigarette smoke exposure during intrauterine period on neonatal rat testis. Methods. Twenty-five rats were randomized to be exposed to cigarette smoke with the Walton Smoking Machine or to room air during their pregnancies. The newborn male rats (n = 21) were grouped as group 1 (n = 15) which were exposed to cigarette smoke during intrauterine life and group 2 (n = 6) which were exposed to room air during intrauterine life. The orchiectomy materials were analyzed with TUNEL immunofluorescent staining for detection of DNA damage. To detect apoptosis, immunohistochemical analyses with caspase-3 were performed. Primary outcomes were apoptotic index and immunohistochemical scores (HSCORES); secondary outcomes were Sertoli-cell count and birth-weight of rats. Results. Sertoli cell apoptosis was increased in group 1 (HSCORE = 210.6 ± 41.9) when compared to group 2 (HSCORE = 100.0 ± 17.8) (P = 0.001). Sertoli cell count was decreased in group 1 (P = 0.043). The HSCORE for the germ cells was calculated as 214.0 ± 46.2 in group 1 and 93.3 ± 10.3 in group 2 (P = 0.001) referring to an increased germ cell apoptosis in group 1. The apoptotic indexes for group 1 were 49.6 ± 9.57 and 29.98 ± 2.34 for group 2 (P = 0.001). The immunofluorescent technique demonstrated increased DNA damage in seminiferous epithelium in group 1. Conclusions. Intrauterine exposure to cigarette smoke adversely affects neonatal testicular structuring and diminishes testicular reserve. PMID:25045542

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

    Microsoft Academic Search

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

    2006-01-01

    Neural transplantation is developing as a successful treatment for neurodegenerative diseases such as Parkinson’s disease.\\u000a 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.\\u000a We have explored combining NT2 cells, as a neuronal source, and Sertoli cells, which may act as a graft facilitator to enhance

  6. Zinc and low-dose of cadmium protect sertoli cells against toxic-dose of cadmium: The role of metallothionein

    PubMed Central

    Kheradmand, Fatemeh; Nourmohammadi, Issa; Ahmadi-Faghih, Mohamad Amin; Firoozrai, Mohsen; Modarressi, Mohammad Hossein

    2013-01-01

    Background: The impact of cadmium (Cd) on male infertility may be related to the interaction with metal-binding proteins known as metallothioneins (Mts). Trace elements like zinc (Zn) have protective effects on testicular damage induced by Cd. Objective: We determined the effect of Zn and low-dose Cd pre-treatment on the expression of Mt1 and Mt2 genes on testicular Sertoli cells. Materials and Methods: The cultured TM4 mouse sertoli cells were treated with 50 ?M ZnSO4 (Zn pre-treated group; ZnPG), 2 ?M CdCl2 (Cd pre-treated group; CdPG), or distilled water (DW pre-treated group; DWPG). After 18 hour, all of these groups were exposed to 100 ?M CdCl2 for different periods of time (1, 2, 3, and 6 hours). There was also a control group for all three groups, which was treated only with distilled water (without Cd or Zn pre-treatment). Cellular viability, Zn and Cd concentrations and gene expression were assessed by MTT, atomic absorption spectrometry and real time PCR methods, respectively. Results: The expression of Mt1 and Mt2 genes in ZnPG, CdPG, and DWPG was greater than the control group (p=0.02 and p=0.01, respectively). Cd concentrations in CdPG and DWPG were greater than the control group (p=0.00). Expression of both genes in ZnPG and CdPG increased after 3 hours of treatment and Cd concentration decreased simultaneously, which was more obvious in ZnPG. Conclusion: Zn and short term low-dose Cd pre-treatment might reduce the adverse effects of Cd by increasing expression of Mts genes in Sertoli cells. The protective effect of Zn was stronger than Cd. PMID:24639783

  7. 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

  8. The AMP-activated protein kinase activator, 5-aminoimidazole-4-carboxamide-1-b-D-ribonucleoside, regulates lactate production in rat Sertoli cells.

    PubMed

    Galardo, María Noel; Riera, María Fernanda; Pellizzari, Eliana Herminia; Cigorraga, Selva Beatriz; Meroni, Silvina Beatriz

    2007-10-01

    The aim of the present study was to investigate whether the AMP-activated protein kinase (AMPK), a key regulator of cellular energy homeostasis, is present in Sertoli cells and whether its activation by 5-aminoimidazole-4-carboxamide-1-b-d-ribonucleoside (AICAR) results in the regulation of cell metabolism to ensure lactate supply for germ cell development. Sertoli cell cultures from 20-day-old rats were used. Western blot analysis for the alpha-subunit of AMPK showed that high levels of AMPK are present in Sertoli cells. Treatment of the cultures with AICAR resulted in a dose- and time-dependent increase of P-AMPK levels indicating activation of the enzyme. A possible effect of AICAR on Sertoli cell lactate production was then analyzed. A dose- and time-dependent increment in lactate secretion was observed. The participation of AMPK activation in different biochemical processes that may be implicated in the regulation of lactate production was also analyzed. AICAR stimulated glucose uptake in a dose- and time-dependent manner. Additionally, AICAR increased the glucose transporter 1 (GLUT1) and decreased the glucose transporter 3 (GLUT3) mRNA levels. As for the role of AMPK in the regulation of the monocarboxylate transporters 1 and 4 (MCT1 and MCT4), it has been observed that AICAR treatment decreased MCT1 and increased MCT4 mRNA levels. In summary, the results presented herein show that AMPK is present in Sertoli cells and that its activation by AICAR increases lactate production as a result, at least in part, of a) an increase in glucose uptake, b) an increase in GLUT1 expression, and c) a decrease in MCT1 and an increase in MCT4 levels. Altogether, these results suggest an important role of AMPK in modulating the nutritional function of Sertoli cells. PMID:17909267

  9. Chlamydia muridarum infection-induced destruction of male germ cells and sertoli cells is partially prevented by Chlamydia major outer membrane protein-specific immune CD4 cells.

    PubMed

    Sobinoff, Alexander P; Dando, Samantha J; Redgrove, Kate A; Sutherland, Jessie M; Stanger, Simone J; Armitage, Charles W; Timms, Peter; McLaughlin, Eileen A; Beagley, Kenneth W

    2015-01-01

    Chlamydia trachomatis infections are increasingly prevalent worldwide. Male chlamydial infections are associated with urethritis, epididymitis, and orchitis; however, the role of Chlamydia in prostatitis and male factor infertility remains controversial. Using a model of Chlamydia muridarum infection in male C57BL/6 mice, we investigated the effects of chlamydial infection on spermatogenesis and determined the potential of immune T cells to prevent infection-induced outcomes. Antigen-specific CD4 T cells significantly reduced the infectious burden in the penile urethra, epididymis, and vas deferens. Infection disrupted seminiferous tubules, causing loss of germ cells at 4 and 8 wk after infection, with the most severely affected tubules containing only Sertoli cells. Increased mitotic proliferation, DNA repair, and apoptosis in spermatogonial cells and damaged germ cells were evident in atrophic tubules. Activated caspase 3 (casp3) staining revealed increased (6-fold) numbers of Sertoli cells with abnormal morphology that were casp3 positive in tubules of infected mice, indicating increased levels of apoptosis. Sperm count and motility were both decreased in infected mice, and there was a significant decrease in morphologically normal spermatozoa. Assessment of the spermatogonial stem cell population revealed a decrease in promyelocytic leukemia zinc finger (PLZF)-positive cells in the seminiferous tubules. Interestingly, adoptive transfer of antigen-specific CD4 cells, particularly T-helper 2-like cells, prior to infection prevented these effects in spermatogenesis and Sertoli cells. These data suggest that chlamydial infection adversely affects spermatogenesis and male fertility, and that vaccination can potentially prevent the spread of infection and these adverse outcomes. PMID:25472923

  10. Arsenic trioxide-induced apoptosis in TM4 Sertoli cells: the potential involvement of p21 expression and p53 phosphorylation.

    PubMed

    Kim, Yoon-Jae; Chung, Jin-Yong; Lee, Seung Gee; Kim, Ji Young; Park, Ji-Eun; Kim, Won Rok; Joo, Bo Sun; Han, Seong Ho; Yoo, Ki Soo; Yoo, Young Hyun; Kim, Jong-Min

    2011-07-29

    Arsenic is a toxic metalloid that exists ubiquitously in the environment, and exhibits carcinogenicity. Conversely, arsenic trioxide (AsTO) has successfully been employed in the treatment of acute promyelocytic leukemia (APL). It has been shown that AsTO efficiently induces apoptosis in the malignant cells of APL in vitro. Although the mechanisms underlying AsTO-induced apoptosis in certain types of cancer cells, such as APL cells, have been delineated, the mechanism underlying AsTO-induced cell death in non-cancer cells remains unknown. In the present study, we examined AsTO-provoked cytotoxicity and cell death mechanism(s) in TM4 Sertoli cells. Exposure of these cells to AsTO generates reactive oxygen species and alters mitochondrial apoptosis, inducing cell death via both caspase-dependent and caspase-independent pathways. AsTO-induced apoptosis was concomitant with the downregulation of p53, phosphorylation of p53 at serine residues, and G2/M cell cycle arrest. Particularly, the interaction of p21 with caspase-3 proteins during AsTO treatment suggested an antiapoptotic role of p21 against genotoxic stresses in TM4 Sertoli cells. However, clinically relevant concentrations of AsTO failed to induce cell death in TM4 Sertoli cells, indicating that these cells could be resistant to cancer treatment. The results presented herein may not represent the actual effect of AsTO on Sertoli cells in vivo. Thus, further studies on the exposure effects of AsTO on the morphology and function of Sertoli cells in animal experiments will provide a more precise knowledge of AsTO cytotoxicity on male reproduction. PMID:21565247

  11. HnRNPL as a key factor in spermatogenesis: Lesson from functional proteomic studies of azoospermia patients with sertoli cell only syndrome.

    PubMed

    Li, Jingping; Guo, Wenbin; Li, Fei; He, Jincan; Yu, Qingfeng; Wu, Xiaoqiang; Li, Jianming; Mao, Xiangming

    2012-06-01

    Sertoli cell only syndrome (SCOS) is one of the main causes leading to the abnormal spermatogenesis. However, the mechanisms for abnormal spermatogenesis in SCOS are still unclear. Here, we analyzed the clinical testis samples of SCOS patients by two-dimensional gel electrophoresis (2-DE) and matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF/TOF MS) to find the key factors contributing to SCOS. Thirteen differential proteins were identified in clinical testis samples between normal spermatogenesis group and SCOS group. Interestingly, in these differential proteins, Heterogeneous nuclear ribonucleoprotein L(HnRNPL) was suggested as a key regulator involved in apoptosis, death and growth of spermatogenic cells by String and Pubgene bioinformatic programs. Down-regulated HnRNPL in testis samples of SCOS patients was further confirmed by immunohistochemical staining and western blotting. Moreover, in vitro and in vivo experiments demonstrated that knockdown of HnRNPL led to inhibited proliferation, increased apoptosis of spermatogenic cell but decreased apoptosis of sertoli cells. Expression of carcinoembryonic antigen-related cell adhesion molecule 1 in GC-1 cells or expression of inducible nitric oxide synthases in TM4 sertoli cells, was found to be regulated by HnRNPL. Our study first shows HnRNPL as a key factor involved in the spermatogenesis by functional proteomic studies of azoospermia patients with sertoli cell only syndrome. This article is part of a Special Issue entitled: Proteomics: The clinical link. PMID:22245417

  12. 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

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

    PubMed

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

    2013-07-01

    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

  14. Expression of ATP-binding cassette membrane transporters in rodent and human sertoli cells: relevance to the permeability of antiretroviral therapy at the blood-testis barrier.

    PubMed

    Robillard, Kevin R; Hoque, Tozammel; Bendayan, Reina

    2012-01-01

    The blood-testis barrier (BTB), composed primarily of Sertoli cells, is responsible for protecting developing germ cells from xenobiotic exposure. ATP-binding cassette (ABC) membrane-associated drug efflux transporters, P-glycoprotein (P-gp), breast cancer resistance protein (BCRP), and the multidrug resistance-associated proteins (Mrps), have been shown to restrict antiretroviral drug permeability at blood-tissue barriers such as the blood-brain barrier. However, it remains unclear whether these transporters are functional at the level of Sertoli cells and can regulate anti-HIV drug permeability at the BTB. This study investigated the functional expression of ABC transporters in a mouse Sertoli cell line system (TM4) and in primary cultures of human Sertoli cells (HSECs). Expression of multidrug resistance Mdr1a/1b/MDR1/P-gp, Mrp1/MRP1, and Mrp4/MRP4 is confirmed by quantitative polymerase chain reaction and immunoblotting analysis in TM4 cells and HSECs. Immunofluorescence studies revealed plasma membrane localization of P-gp, Mrp1/MRP1, and Mrp4/MRP4 in both cell systems. However, Bcrp expression and localization was only detected in rodent cells. Accumulation of 1) rhodamine-6G (R-6G), a fluorescent P-gp substrate, 2) [³H]atazanavir, a HIV protease inhibitor and known P-gp substrate, 3) 2'7'-bis-(2-carboxyethyl)-5-(and-6)carboxyfluorescein (BCECF), a fluorescent Mrp substrate, and 4) [³H]mitoxantrone, a BCRP substrate, by TM4 monolayer cells in the presence of established inhibitors demonstrates that these transporters are functional. In addition, several anti-HIV drugs significantly enhance the accumulation of R-6G, [³H]atazanavir, BCECF, and [³H]mitoxantrone by TM4 cells. This study provides the first evidence of ABC transporter expression and activity in Sertoli cells and suggests that these transporters could play an important role in restricting antiretroviral drug permeability at the BTB. PMID:21990609

  15. Testicular juvenile granulosa cell and Sertoli cell tumours: a clinicopathological study of 29 cases from the Kiel Paediatric Tumour Registry.

    PubMed

    Harms, D; Kock, L R

    1997-04-01

    Testicular Sertoli cell tumours (SCT) and juvenile granulosa cell tumours (JGCT) are rare in childhood. This study was designed to investigate the clinical picture, morphology and disease course in a comparatively large series of cases (total number = 29). Of 198 cases of childhood testicular tumour documented in the Kiel Paediatric Tumour Registry 18 were cases of infantile SCT (9.1%) and 11 of JGCT (5.6%). The average age at the time of diagnosis was 4.2 months for infantile SCT and 0.4 months for IGCT. SCT and JGCT often showed infiltrative growth into adjacent testicular tissue, dense cellularity and considerable proliferation activity. Immunohistochemically all cases expressed vimentin intermediate filaments in both tumour types. Next in frequency of expression were cytokeratins (SCT: 7/16; JGCT: 7/10) and smooth-muscle actin (SCT: 9/15; JGCT: 4/10). Follow-up studies (24/29) showed that in cases of tumour manifestation in infancy and after complete tumour removal (usually orchiectomy) no local recurrences and no metastases occurred. The most important conclusion for diagnosis and therapy is that despite infiltrative growth, incomplete differentiation, dense cellularity and considerable proliferation activity, after surgical excision infantile SCT and JGCT have a good prognosis. Adjuvant chemotherapy or more extensive operations with lymphadenectomy are thus not indicated. PMID:9134041

  16. Direct in-vivo detection of atypical hormonal expression of a Sertoli-Leydig cell tumour following stimulation with human chorionic gonadotrophin.

    PubMed

    Cohen, I; Shapira, M; Cuperman, S; Goldberger, S; Siegal, A; Altaras, M; Beyth, Y

    1993-10-01

    A 60-year-old woman presented with progressive hirsutism and elevated serum testosterone levels. Selective bilateral ovarian and adrenal vein catheterization demonstrated mild elevated testosterone and androstenedione levels in the right ovarian vein, which increased considerably 15 minutes following intravenous injection of 5000 IU human chorionic gonadotrophin. Androgen levels decreased remarkably after administration of gonadotrophin hormone releasing hormone-agonist (GnRH-a). On histological examination, diffuse stromal hyperplasia of both ovaries was noted, with a small Sertoli-Leydig cell tumour in the right ovary. This is the first report of preoperative, direct selective diagnosis of a small Sertoli-Leydig cell tumour with such a hormonal expression. Ovarian Sertoli-Leydig cell tumours are rare sex cord stromal tumours that exhibit testicular-like structure and differentiation. These tumours are potentially malignant, can cause progressive virilization (Young & Scully, 1985), and are often clinically manifested as palpable pelvic masses and virilization (Meldrum & Abraham, 1979; Friedman et al., 1985). We describe a patient with postmenopausal virilization due to Sertoli-Leydig cell tumour, in whom a remarkable increase in androgens was detected following intravenous human chorionic gonadotrophin injection, during adrenal and ovarian selective vein blood sampling. A remarkable decrease in the serum androgen level was noticed following an injection of gonadotrophin hormone releasing hormone-agonist (GnRH-a). PMID:8287578

  17. Sertoli cell processes have axoplasmic features: an ordered microtubule distribution and an abundant high molecular weight microtubule- associated protein (cytoplasmic dynein)

    PubMed Central

    1988-01-01

    Microtubules in the cytoplasm of rat Sertoli cell stage VI-VIII testicular seminiferous epithelium were studied morphometrically by electron microscopy. The Sertoli cell microtubules demonstrated axonal features, being largely parallel in orientation and predominantly spaced one to two microtubule diameters apart, suggesting the presence of microtubule-bound spacer molecules. Testis microtubule-associated proteins (MAPs) were isolated by a taxol, salt elution procedure. Testis MAPs promoted microtubule assembly, but to a lesser degree than brain MAPs. High molecular weight MAPs, similar in electrophoretic mobilities to brain MAP-1 and MAP-2, were prominent components of total testis MAPs, though no shared immunoreactivity was detected between testis and brain high molecular weight MAPs using both polyclonal and monoclonal antibodies. Unlike brain high molecular weight MAPs, testis high molecular weight MAPs were not heat stable. Testis MAP composition, studied on postnatal days 5, 10, 15, and 24 and in the adult, changed dramatically during ontogeny. However, the expression of the major testis high molecular weight MAP, called HMW-2, was constitutive and independent of the development of mature germ cells. The Sertoli cell origin of HMW-2 was confirmed by identifying this protein as the major MAP found in an enriched Sertoli cell preparation and in two rat models of testicular injury characterized by germ cell depletion. HMW-2 was selectively released from testis microtubules by ATP and co-purified by sucrose density gradient centrifugation with MAP- 1C, a neuronal cytoplasmic dynein. The inhibition of the microtubule- activated ATPase activity of HMW-2 by vanadate and erythro-(2-hydroxy-3- nonyl)adenine and its proteolytic breakdown by vanadate-dependent UV photocleavage confirmed the dynein-like nature of HMW-2. As demonstrated by this study, the neuronal and Sertoli cell cytoskeletons share morphological, structural and functional properties. PMID:2972729

  18. 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

  19. 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

  20. DICER1 mutations in Familial Multi-Nodular Goiter with and without Ovarian Sertoli-Leydig Cell Tumors

    PubMed Central

    Frio, Thomas Rio; Bahubeshi, Amin; Kanellopoulou, Chryssa; Hamel, Nancy; Niedziela, Marek; Sabbaghian, Nelly; Pouchet, Carly; Gilbert, Lucy; O’Brien, Paul K.; Serfas, Kim; Broderick, Peter; Houlston, Richard S.; Lesueur, Fabienne; Bonora, Elena; Muljo, Stefan; Schimke, R. Neil; Soglio, Dorothée Bouron-Dal; Arseneau, Jocelyne; Schultz, Kris Ann; Priest, John R.; Nguyen, Van-Hung; Harach, H. Ruben; Livingston, David M.; Foulkes, William D.; Tischkowitz, Marc

    2012-01-01

    Context Non-toxic multinodular goiter (MNG) is frequently observed in the general population, but little is known about the underlying genetic susceptibility to this disease. Familial cases of MNG have been reported and there are five such published families which also contain individuals with Sertoli-Leydig cell tumors of the ovary (SLCT). Germline mutations in DICER1, a gene that codes for an RNase III endoribonuclease, have recently been identified in families affected pleuropulmonary blastoma (PPB), some of whom include cases of MNG and gonadal tumors such as SLCT. Objective To determine whether familial MNG with or without SLCT in the absence of PPB was caused by mutations in DICER1. Design, Setting and Patients From September 2009 to September 2010, we studied two MNG families and three MNG/SLCT families. We screened affected probands for mutations in the DICER1 gene. We investigated blood lymphocytes, MNG and SLCT tissue from family members for loss of the wild-type allele (loss of heterozygosity), DICER1 expression and microRNA dysregulation. Main Outcome Measure(s) Detection of germline DICER1 gene mutations in familial MNG with and without SLCT. Results We identified and characterized germline DICER1 mutations in all five families. Molecular analysis of the three SLCTs showed no loss of heterozygosity at DICER1, and IHC analysis in two available samples showed strong expression of DICER1 in Sertoli cells, but weak staining of Leydig cells. MicroRNA profiling of RNA derived from lymphoblastoid cell lines from both affected and unaffected members of the familial MNG cases revealed miRNA perturbations in DICER1 mutation carriers. Conclusions DICER1 mutations predispose to both familial MNG and MNG with SLCT, independent of PPB and germline DICER1 mutations lead to dysregulation of miRNA. This could be investigated further as a possible novel mechanism of tumorigenesis. PMID:21205968

  1. Expression of inhibin-alpha is regulated synergistically by Wilms' tumor gene 1 (Wt1) and steroidogenic factor-1 (Sf1) in sertoli cells.

    PubMed

    Ji, Shao-Yang; Hao, Jian-Xiu; Li, Lei; Zhang, Jun; Zheng, Qiao-Song; Li, Xi-Xia; Wang, Xiao-Na; Han, Chun-Sheng; Gao, Fei; Liu, Yi-Xun

    2013-01-01

    Wt1 encodes a zinc finger nuclear transcriptional factor, which is specifically expressed in testicular Sertoli cells and knockdown of Wt1 in Sertoli cells causes male mice subfertility. However, the underlying mechanism is still unclear. In this study, we found that expression of inhibin-? is significantly reduced in Wt1-deficient Sertoli cells. Luciferase assays using the inhibin-? promoter indicated that the inhibin-? promoter is transactivated by the Wt1 A, and B isoforms (-KTS), but not the C, and D isoforms (+KTS). Analysis of the Wt1 responsive element of the inhibin-? promoter region using site-directed mutagenesis showed that the nucleotides between -58 and -49 are essential for Wt1-dependent transactivation of the inhibin-? promoter. ChIP assays indicated that Wt1 directly interacts with the inhibin-? promoter. In addition, the inhibin-? promoter is activated synergistically by Wt1 and Sf1. Mutation of the ligand binding domain (LBD) of Sf1 (residues 235-238) completely abolished the synergistic action between Wt1 and Sf1, but did not affect the physical interaction between these two proteins, suggesting that other factor(s) may also be involved in the regulation of inhibin-? in Sertoli cells. Further studies demonstrated that ?-catenin enhances the synergistic activation of Wt1 and Sf1 on the inhibin-? promoter. Given the fact that inhibin-?, a subunit of inhibin, is known to be involved in the regulation of spermatogenesis and testicular steroidogenesis, this study reveals a new regulatory mechanism of inhibin-? in Sertoli cells and also sheds light on the physiological functions of Wt1 in gonad development and spermatogenesis. PMID:23326390

  2. B Cells Regulate Murine Gammaherpesvirus 68 Latency

    Microsoft Academic Search

    KAREN E. WECK; SUSANNE S. KIM; HERBERT W. VIRGIN; SAMUEL H. SPECK

    1999-01-01

    The dynamics of the establishment of, and reactivation from, gammaherpesviruses latency has not been quantitatively analyzed in the natural host. Gammaherpesvirus 68 (gHV68) is a murine gammaherpesvirus genetically related to primate gammaherpesviruses that establishes a latent infection in infected mice. We used limiting dilution reactivation (frequency of cells reactivating gHV68 in vitro) and limiting dilution PCR (frequency of cells carrying

  3. Anti-Müllerian hormone and inhibin B levels reflect altered Sertoli cell function in men with metabolic syndrome.

    PubMed

    Robeva, R; Tomova, A; Kirilov, G; Kumanov, P

    2012-05-01

    The aim of the present study was to investigate the Sertoli cell markers inhibin B and anti-Müllerian hormone (AMH) in men with metabolic syndrome (MS). Twenty patients with MS according to the criteria of the International Diabetes Federation and 20 non obese age-matched men were investigated. The levels of testosterone, sex hormone binding globulin (SHBG), gonadotropins, inhibin B and AMH were measured in all of them. In obese patients with MS total testosterone (15.74 ± 6.95 versus 27.84 ± 12.80 nmol l(-1), P = 0.001), SHBG (21.71 ± 11.08 versus 38.80 ± 17.51 nmol l(-1), P = 0.001) and free testosterone (430.35 ± 237.40 versus 613.85 ± 303.65 pmol l(-1), P = 0.040) were significantly lower than in the controls. Interestingly, the inhibin B (103.64 ± 56.77 versus 149.88 ± 68.31 pg ml(-1), P = 0.025) and AMH levels (30.84 ± 13.14 versus 43.14 ± 9.66 pmol l(-1), P = 0.002) were also significantly lower in MS group in comparison to the other participants. The lowest levels of AMH were found in patients with MS and carbohydrate disturbances. The decreased concentrations of testosterone, inhibin B and AMH in patients with MS could reflect an impaired Leydig and Sertoli cell function. Further studies in men with obesity, insulin resistance and diabetes type 2 could reveal more information about the interrelations between the metabolic disturbances and reproductive function in men. PMID:21749433

  4. Regulation of Germ Cell and Sertoli Cell Development by Activin, Follistatin, and FSH

    Microsoft Academic Search

    Terri Meehan; Stefan Schlatt; Moira K. O'Bryan; David M. de Kretser; Kate Lakoski Loveland

    2000-01-01

    We have demonstrated a role for activin A, follistatin, and FSH in male germ cell differentiation at the time when spermatogonial stem cells and committed spermatogonia first appear in the developing testis. Testis fragments from 3-day-old rats were cultured for 1 or 3 days with various combinations of these factors, incubated with bromodeoxyuridine (BrdU) to label proliferating cells, and then

  5. 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

  6. EB1 regulates tubulin and actin cytoskeletal networks at the sertoli cell blood-testis barrier in male rats: an in vitro study.

    PubMed

    Tang, Elizabeth I; Mok, Ka-Wai; Lee, Will M; Cheng, C Yan

    2015-02-01

    During spermatogenesis, developing germ cells are transported across the seminiferous epithelium. Studies propose that because microtubules (MTs) serve as the tracks for transporting cell organelles, they may also serve a similar function in the transport of developing germ cells. Polarized MTs may provide the tracks along which polarized actin microfilaments, which act as vehicles to transport cargo, such as preleptotene spermatocytes through the blood-testis barrier (BTB) and spermatids across the epithelium. Yet the molecular mechanism(s) underlying these events remain unknown. Using an established in vitro Sertoli cell system to study BTB function, we demonstrated herein that a MT regulatory protein end-binding protein 1 (EB1) regulates the MT- and also the actin-based cytoskeleton of the Sertoli cell BTB in the rat. EB1 serves as a coordinator between the two cytoskeletons by regulating MT polymerization and actin filament bundling to modulate germ cell transport at the Sertoli cell BTB. A knockdown of EB1 by RNA interference was found to perturb the tight junction (TJ)-permeability barrier, as evidenced by mislocalization of junctional proteins critical for barrier function to facilitate spermatocyte transport, which was likely achieved by two coordinated events. First, EB1 knockdown resulted in changes in MT polymerization, thereby perturbing MT organization in Sertoli cells in which polarized MT no longer stretched properly across the cell cytosol to serve as the tracks. Second, EB1 knockdown perturbed actin organization via its effects on the branched actin polymerization-inducing protein called Arp3 (actin-related protein 3), perturbing microfilament bundling capability based on a biochemical assay, thereby causing microfilament truncation and misorganization, disrupting the function of the vehicle. This reduced actin microfilament bundling capability thus perturbed TJ-protein distribution and localization at the BTB, destabilizing the TJ barrier, leading to its remodeling to facilitate spermatocyte transport. In summary, EB1 provides a functional link between tubulin- and actin-based cytoskeletons to confer spermatocyte transport at the BTB. PMID:25456071

  7. 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

  8. Identification of genetic networks involved in the cell injury accompanying endoplasmic reticulum stress induced by bisphenol A in testicular Sertoli cells

    SciTech Connect

    Tabuchi, Yoshiaki [Division of Molecular Genetics Research, Life Science Research Center, University of Toyama, Toyama (Japan)]. E-mail: ytabu@cts.u-toyama.ac.jp; Takasaki, Ichiro [Division of Molecular Genetics Research, Life Science Research Center, University of Toyama, Toyama (Japan); Kondo, Takashi [Department of Radiological Sciences, Faculty of Medicine, University of Toyama, Toyama (Japan)

    2006-07-07

    To identify detailed mechanisms by which bisphenol A (BPA), an endocrine-disrupting chemical, induces cell injury in mouse testicular Sertoli TTE3 cells, we performed genome-wide microarray and computational gene network analyses. BPA (200 {mu}M) significantly decreased cell viability and simultaneously induced an increase in mRNA levels of HSPA5 and DDIT3, endoplasmic reticulum (ER) stress marker genes. Of the 22,690 probe sets analyzed, BPA down-regulated 661 probe sets and up-regulated 604 probe sets by >2.0-fold. Hierarchical cluster analysis demonstrated nine gene clusters. In decreased gene clusters, two significant genetic networks were associated with cell growth and proliferation and the cell cycle. In increased gene clusters, two significant genetic networks including many basic-region leucine zipper transcription factors were associated with cell death and DNA replication, recombination, and repair. The present results will provide additional novel insights into the detailed molecular mechanisms of cell injury accompanying ER stress induced by BPA in Sertoli cells.

  9. Altered LKB1/AMPK/TSC1/TSC2/mTOR signaling causes disruption of Sertoli cell polarity and spermatogenesis

    PubMed Central

    Tanwar, Pradeep S.; Kaneko-Tarui, Tomoko; Zhang, LiHua; Teixeira, Jose M.

    2012-01-01

    Male patients with Peutz–Jeghers syndrome (PJS) have defective spermatogenesis and are at increased risk of developing Sertoli cell tumors. Mutations in the Liver Kinase B1 (LKB1/STK11) gene are associated with the pathogenesis of PJS and have been identified in non-PJS patients with sporadic testicular cancers. The mechanisms controlled by LKB1 signaling in Sertoli cell functions and testicular biology have not been described. We have conditionally deleted the Lkb1 gene (Lkb1cko) in somatic testicular cells to define the molecular mechanisms involved in the development of the testicular phenotype observed in PJS patients. Focal vacuolization in some of the seminiferous tubules was observed in 4-week-old mutant testes but germ cell development appeared to be normal. However, similar to PJS patients, we observed progressive germ cell loss and Sertoli cell only tubules in Lkb1cko testes from mice older than 10 weeks, accompanied by defects in Sertoli cell polarity and testicular junctional complexes and decreased activation of the MAP/microtubule affinity regulating and focal adhesion kinases. Suppression of AMP kinase and activation of mammalian target of rapamycin (mTOR) signaling were also observed in Lkb1cko testes. Loss of Tsc1 or Tsc2 copies the progressive Lkb1cko phenotype, suggesting that dysregulated activation of mTOR contributes to the pathogenesis of the Lkb1cko testicular phenotype. Ptencko mice had a normal testicular phenotype, which could be explained by the comparative lack of mTOR activation detected. These studies describe the importance of LKB1 signaling in testicular biology and the possible molecular mechanisms driving the pathogenesis of the testicular defects observed in PJS patients. PMID:22791749

  10. Murine CD4+ T cell subsets defined

    PubMed Central

    1988-01-01

    We have used two monoclonal anti-murine T cell autoantibodies (SM3G11 and SM6C10) and multi-color immunofluorescence staining to resolve splenic CD4+ cells into four populations. Two of these populations (Fr. I and Fr. III, 35% and 10% of CD4+ cells) show mutually exclusive expression of these determinants and exhibit distinct functions. Fr. III secretes IL-4, but not IL-2 when activated by Con A, and includes memory T cells responsible for secondary antibody formation. In contrast, Fr. I secretes IL-2 but not IL-4 in response to Con A, and does not contribute to the secondary antibody response. Furthermore, these two fractions exhibit differential accessory cell dependence. Whereas Fr. III responds with B cells (and also non-B cells) as accessory cells in Con A-induced activation, Fr. I requires non-B cells. However, we found that many CD4+ cells (Fr. II, 40% of CD4+ cells) express both determinants and are not distinguishable with regard to lymphokine secretion, accessory cell effect, and memory T cell activity. Curiously, the fraction expressing neither determinant (Fr. IV, 10% of CD4+ cells) is unresponsive to experimental conditions used here. We discuss the possible relationships between these T cell subsets and the implications of differential expression of these determinants. PMID:2903214

  11. Pachytene spermatocyte protein(s) stimulate sertoli cells grown in bicameral chambers: Dose-dependent secretion of ceruloplasmin, sulfated glycoprotein-1, sulfated glycoprotein-2, and transferrin

    Microsoft Academic Search

    Makoto Onoda; Daniel Djakiew

    1991-01-01

    Summary  Interactions between pachytene spermatocytes and Sertoli cells were investigated using the bicameral culture chamber system.\\u000a Pachytene spermatocytes were isolated from adult rats with a purity in excess of 90% by centrifugal elutriation. The pachytene\\u000a spermatocytes were cultured in a defined media and pachytene spermatocyte protein prepared from the conditioned media by dialysis\\u000a and lyophilization. This pachytene spermatocyte protein was reconstituted

  12. 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.

  13. Ovarian Sertoli-Leydig cell tumor: a report of seven cases and a review of the literature.

    PubMed

    Xiao, Huiting; Li, Bin; Zuo, Jing; Feng, Xiaoli; Li, Xiaoguang; Zhang, Rong; Wu, Lingying

    2013-03-01

    The aim of this study was to investigate the clinicopathologic features, treatment and outcome of seven patients with an ovarian Sertoli-Leydig cell tumor (SLCT). Five patients presented with feminization, two with accompanying virilization. One presented with amenorrhea alone. Three of the five patients showing feminization symptoms had endocrine-related diseases. Histologically, five tumors were well differentiated, the other two were poorly differentiated. The latter two patients were misdiagnosed as having an ovarian epithelial carcinoma or granulosa cell tumor from frozen sections. Immunohistochemistry showed that the tumors were calretinin-positive in two patients and one was inhibin-positive. Four patients underwent total abdominal hysterectomy and bilateral salpingo-oophorectomy(TAH/BSO) and two were treated by unilateral salpingo-oophorectomy. Among them, two patients received adjuvant chemotherapy. Six patients were free of disease in a follow-up of 2-34 years and one achieved a pregnancy. The remaining patient recurred 4 years later. Feminization as well as virilization might provide important clues for a preoperative diagnosis. Histological misdiagnosis is probable in poorly differentiated tumors. Conservative surgery including retention of fertility can be considered. However, the tendency for recurrence in poorly differentiated tumors should be considered. PMID:23173550

  14. Sperm retrieval and live birth rates in presumed Sertoli-cell-only syndrome in testis biopsy: a single centre experience.

    PubMed

    Gul, U; Turunc, T; Haydardedeoglu, B; Yaycioglu, O; Kuzgunbay, B; Ozkardes, H

    2013-01-01

    We aimed to investigate sperm retrieval rates (SRR) by testicular sperm extraction (TESE), factors affecting SRR, and fertilization rate (FR), implantation rate (IR), clinical pregnancy rate (CPR) and live birth rate (LBR) in patients with presumed Sertoli-cell-only syndrome in testis biopsy (SCOS). We retrospectively evaluated files of 134 patients with SCOS who underwent TESE. Group I were patients in whom spermatozoa were retrieved and Group II were patients in whom no spermatozoa could be retrieved. SRR, Follicle stimulating hormone (FSH), Luteinizing hormone (LH), and testosterone levels, and the volume of testicles were compared between groups. In addition, FR, IR, CPR and LBR were determined. Sperm retrieval was achieved in 37 (27.6%) patients (Group I), and the remaining 97 (72.4%) patients made Group II. There were no significant differences in age, infertility time, testicular volume, serum FSH, LH and testosterone levels between Groups I and II (p > 0.05). Intracytoplasmic sperm injection (ICSI) was performed in 36 patients. FR, IR, and CPR were 60.86 ± 23.03, 36.53 ± 41.78 and 51.3% respectively. Cycle and patient based LBRs were 37.8 and 45.1% respectively. SRR in SCOS is lower than patients with non-obstructive azoospermia (NOA) in general. No parameters to predict spermatozoa retrieval were determined. In patients with SCOS, ICSI achieves similar live birth rate to other patients with NOA. PMID:23258629

  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. New Candidate Genes Identified for Controlling Mouse Gonadal Sex Determination and the Early Stages of Granulosa and Sertoli Cell Differentiation1

    PubMed Central

    Bouma, Gerrit J.; Hudson, Quanah J.; Washburn, Linda L.; Eicher, Eva M.

    2009-01-01

    Mammalian gonadal sex-determining (GSD) genes are expressed in a unique population of somatic cells that differentiate into granulosa cells in XX gonads or Sertoli cells in XY gonads. The ability to efficiently isolate these somatic support cells (SSCs) during the earliest stages of gonad development would facilitate identifying 1) new candidate GSD genes that may be involved in cases of unexplained abnormal gonad development and 2) genes involved in the earliest stages of granulosa and Sertoli cell differentiation. We report the development of a unique mouse carrying two transgenes that allow XX and XY mice to be distinguished as early as Embryonic Day 11.5 (E11.5) and allow SSCs to be isolated from undifferentiated (E11.5) and early differentiated (E12.5) fetal gonads. The Mouse Genome 430v2.0 GeneChip (Affymetrix) was used to identify transcripts exhibiting a sexual dimorphic expression pattern in XX and XY isolated SSCs. The analysis revealed previously unidentified sexually dimorphic transcripts, including low-level expressed genes such as Sry, a gene not identified in other microarray studies. Multigene real-time PCR analysis of 57 genes verified that 53 were expressed in fetal gonads in a sexually dimorphic pattern, and whole-mount in situ hybridization analysis verified 4930563E18Rik, Pld1, and Sprr2d are expressed in XX gonads, and Fbln2, Ppargc1a, and Scrn1 are expressed in XY gonads. Taken together, the data provide a comprehensive resource for the spatial-temporal expression pattern of genes that are part of the genetic network underlying the early stages of mammalian fetal gonadal development, including the development of granulosa and Sertoli cells. PMID:19864314

  17. Isolation of murine valve endothelial cells.

    PubMed

    Miller, Lindsey J; Lincoln, Joy

    2014-01-01

    Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and surrounded by a monolayer of valve endothelial cells (VECs). VECs play essential roles in establishing the valve structures during embryonic development, and are important for maintaining life-long valve integrity and function. In contrast to a continuous endothelium over the surface of healthy valve leaflets, VEC disruption is commonly observed in malfunctioning valves and is associated with pathological processes that promote valve disease and dysfunction. Despite the clinical relevance, focused studies determining the contribution of VECs to development and disease processes are limited. The isolation of VECs from animal models would allow for cell-specific experimentation. VECs have been isolated from large animal adult models but due to their small population size, fragileness, and lack of specific markers, no reports of VEC isolations in embryos or adult small animal models have been reported. Here we describe a novel method that allows for the direct isolation of VECs from mice at embryonic and adult stages. Utilizing the Tie2-GFP reporter model that labels all endothelial cells with Green Fluorescent Protein (GFP), we have been successful in isolating GFP-positive (and negative) cells from the semilunar and atrioventricular valve regions using fluorescence activated cell sorting (FACS). Isolated GFP-positive VECs are enriched for endothelial markers, including CD31 and von Willebrand Factor (vWF), and retain endothelial cell expression when cultured; while, GFP-negative cells exhibit molecular profiles and cell shapes consistent with VIC phenotypes. The ability to isolate embryonic and adult murine VECs allows for previously unattainable molecular and functional studies to be carried out on a specific valve cell population, which will greatly improve our understanding of valve development and disease mechanisms. PMID:25177896

  18. Chimeric Human Immunodeficiency Virus Type 1 Containing Murine Leukemia Virus Matrix Assembles in Murine Cells

    Microsoft Academic Search

    Margaret Reed; Roberto Mariani; Liana Sheppard; Katja Pekrun; Nathaniel R. Landau; Nay-Wei Soong

    2002-01-01

    Murine cells do not support efficient assembly and release of human immunodeficiency virus type 1 (HIV-1) virions. HIV-1-infected mouse cells that express transfected human cyclin T1 synthesize abundant Gag precursor polyprotein, but inefficiently assemble and release virions. This assembly defect may result from a failure of the Gag polyprotein precursor to target to the cell membrane. Plasma membrane targeting of

  19. rpS6 Regulates Blood-Testis Barrier Dynamics Through Arp3-Mediated Actin Microfilament Organization in Rat Sertoli Cells. An In Vitro Study.

    PubMed

    Mok, Ka-Wai; Chen, Haiqi; Lee, Will M; Cheng, C Yan

    2015-05-01

    In the seminiferous epithelium of rat testes, preleptotene spermatocytes residing in the basal compartment are transported across the blood-testis barrier (BTB) to enter the adluminal compartment at stage VIII of the epithelial cycle. This process involves redistribution of tight junction (TJ) proteins via reorganization of actin cytoskeleton in Sertoli cells that serves as attachment site for adhesion protein complexes. Ribosomal protein S6 (rpS6), a downstream molecule of mTORC1 (mammalian target of rapamycin complex 1), participates in this process via a yet-to-be defined mechanism. Here, we constructed an rpS6 quadruple phosphomimetic mutant by converting Ser residues at 235, 236, 240, and 244 to Glu via site-directed mutagenesis, making this mutant constitutively active. When this rpS6 mutant was overexpressed in Sertoli cells cultured in vitro with an established TJ barrier mimicking the BTB in vivo, it perturbed the TJ permeability by down-regulating and redistributing TJ proteins at the cell-cell interface. These changes are mediated by a reorganization of actin microfilaments, which was triggered by a redistribution of activated actin-related protein 3 (Arp3) as well as changes in Arp3-neuronal Wiskott-Aldrich Syndrome protein (N-WASP) interaction. This in turn induced reorganization of actin microfilaments, converting them from a "bundled" to an "unbundled/branched" configuration, concomitant with a reduced actin bundling activity, thereby destabilizing the TJ-barrier function. These changes were mediated by Akt (transforming oncogene of v-akt), because an Akt knockdown by RNA interference was able to mimic the phenotypes of rpS6 mutant overexpression at the Sertoli cell BTB. In summary, this study illustrates a mechanism by which mTORC1 signal complex regulates BTB function through rpS6 downstream by modulating actin organization via the Arp2/3 complex, which may be applicable to other tissue barriers. PMID:25714812

  20. 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

  1. Primary ovarian mucinous cystic tumor with prominent theca cell proliferation and focal granulosa cell tumor in its stroma: case report, literature review, and comparison with Sertoli-Leydig cell tumor with heterologous elements.

    PubMed

    Staats, Paul N; Coutts, Michael A; Young, Robert H

    2010-05-01

    A 73-year-old woman was found to have a 22 cm unilateral multilocular mucinous cystic tumor of the ovary. Microscopic examination showed a routine appearance of the epithelial component, which ranged from benign to borderline to low-grade carcinoma. The stromal component was unusual because of a striking cellular theca cell component in the stroma, which, in turn, merged with a component of adult granulosa cell tumor. The "parent" neoplasm in this case and 3 other similar cases in the literature appears to be the mucinous neoplasm, in contrast with the other example of mucinous neoplasia associated with sex cord neoplasia, the Sertoli-Leydig cell tumor with heterologous elements, in which the "parent" neoplasm is likely the Sertoli-Leydig cell tumor. PMID:20407320

  2. AZF and DAZ gene copy-specific deletion analysis in maturation arrest and Sertoli cell-only syndrome.

    PubMed

    Ferrás, C; Fernandes, S; Marques, C J; Carvalho, F; Alves, C; Silva, J; Sousa, M; Barros, A

    2004-10-01

    Deletions of the AZFc region in Yq11.2, which include the DAZ gene family, are responsible for most cases of male infertility and were associated with severe oligozoospermia and also with a variable testicular pathology. To uncover the functional contribution of DAZ to human spermatogenesis, a DAZ gene copy-specific deletion analysis was previously established and showed that DAZ1/DAZ2 deletions associate with oligozoospermia. In this study we applied the same screening method to 50 control fertile males and 91 non-obstructive azoospermic males, 39 with Sertoli cell-only syndrome (SCOS) and 52 with meiotic arrest (MA). Samples were also screened with 24 sequence-tagged sites to the different AZF regions, including 114 control fertile males. After biopsy (testicular sperm extraction, TESE), residual spermiogenesis was found in 57.7% MA and 30.8% SCOS cases (incomplete syndromes). DAZ1/DAZ2 deletions were associated with the testicular phenotype of residual spermiogenesis as they were only found in two patients (8%) with incomplete MA. Differences between incomplete (23.3%) and complete (4.5%) MA cases regarding AZFc and DAZ1/DAZ2 deletion frequencies, and between incomplete (58.3%) and complete (11.1%) SCOS cases for AZFc deletions, suggest that incomplete syndromes might represent an aggravation of the oligozoospermic phenotype. As successful TESE was achieved in 87.5% of MA cases with AZFc and DAZ1/DAZ2 deletions and in 58.3% of SCOS cases with AZFc deletions, the present results also suggest that these molecular markers might be used for the establishment of a prognosis before TESE. PMID:15347736

  3. Sertoli cell ultrastructure. II. Morphological effects of hypophysectomy in pubescent pigs.

    E-print Network

    Paris-Sud XI, Université de

    effect on the testicular structure of pigs (Dufaure et al., 1974 ; Morat, 1977), on pig Leydig cell ultrastructure (Dufaure et al., 1974 ; Chambon et al., 1975 ; Chambon, 1976 ; Morat, 1977) and on that of some accessory glands of the genital tract (Morat et al., in progress).

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

    E-print Network

    Boyer, Edmond

    ). Research on hypophysectomized animals (Weddington et al., 1975 ; Elkington et al., 1975, 1977) suggests cell has been studied in different physiological conditions, i.e. imma- ture, pubescent, adult in animals in which the sexual functions were disturbed or suppressed (cryptorchidism, hypophysectomy

  5. Murine Mueller cells are progenitor cells for neuronal cells and fibrous tissue cells

    SciTech Connect

    Florian, Christian; Langmann, Thomas; Weber, Bernhard H.F. [Institute of Human Genetics, University of Regensburg, Franz-Josef-Strauss-Allee 11, 93053 Regensburg (Germany); Morsczeck, Christian [Institute of Human Genetics, University of Regensburg, Franz-Josef-Strauss-Allee 11, 93053 Regensburg (Germany)], E-mail: Christian.morsczeck@klinik.uni-regensburg.de

    2008-09-19

    Mammalian Mueller cells have been reported to possess retinal progenitor cell properties and generate new neurons after injury. This study investigates murine Mueller cells under in vitro conditions for their capability of dedifferentiation into retinal progenitor cells. Mueller cells were isolated from mouse retina, and proliferating cells were expanded in serum-containing medium. For dedifferentiation, the cultured cells were transferred to serum-replacement medium (SRM) at different points in time after their isolation. Interestingly, early cell passages produced fibrous tissue in which extracellular matrix proteins and connective tissue markers were differentially expressed. In contrast, aged Mueller cell cultures formed neurospheres in SRM that are characteristic for neuronal progenitor cells. These neurospheres differentiated into neuron-like cells after cultivation on laminin/ornithine cell culture substrate. Here, we report for the first time that murine Mueller cells can be progenitors for both, fibrous tissue cells and neuronal cells, depending on the age of the cell culture.

  6. 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

  7. Erk-dependent cytosolic phospholipase A2 activity is induced by CD95 ligand cross-linking in the mouse derived Sertoli cell line TM4 and is required to trigger apoptosis in CD95 bearing cells

    Microsoft Academic Search

    S Ulisse; B Cinque; G Silvano; N Rucci; L Biordi; M G Cifone; M D'Armiento

    2000-01-01

    In the present study we demonstrated that CD95L cross-linking generated reverse signalling in the mouse derived Sertoli cell line TM4. Treatment of TM4 cells with mAb anti-CD95L induced activation of the cytosolic phospholipase A2 (cPLA2). Cytosolic PLA2 activation was controlled by the MAPK pathway as indicated by the ability of the specific MEK inhibitor, PD098059, to abolish cPLA2 activation. In

  8. Telomere sister chromatid exchange in telomerase deficient murine cells

    SciTech Connect

    Wang, Yisong [ORNL; Giannone, Richard J [ORNL; Liu, Yie [ORNL

    2005-01-01

    We have recently demonstrated that several types of genomic rearrangements (i.e., telomere sister chromatid exchange (T-SCE), genomic-SCE, or end-to-end fusions) were more often detected in long-term cultured murine telomerase deficient embryonic stem (ES) cells than in freshly prepared murine splenocytes, even through they possessed similar frequencies of critically short telomeres. The high rate of genomic rearrangements in telomerase deficient ES cells, when compared to murine splenocytes, may reflect the cultured cells' gained ability to protect chromosome ends with eroded telomeres allowing them to escape 'end crisis'. However, the possibility that ES cells were more permissive to genomic rearrangements than other cell types or that differences in the microenvironment or genetic background of the animals might consequentially determine the rate of T-SCEs or other genomic rearrangements at critically short telomeres could not be ruled out.

  9. Embryonic Stem Cell Virus, a Recombinant Murine Retrovirus with Expression in Embryonic Stem Cells

    Microsoft Academic Search

    Manuel Grez; Ercan Akgun; Frank Hilberg; Wolfram Ostertag

    1990-01-01

    The expression of Moloney murine leukemia virus and vectors derived from it is restricted in undifferentiated mouse embryonal carcinoma and embryonal stem (ES) cells. We have developed a retroviral vector, the murine embryonic stem cell virus (MESV), that is active in embryonal carcinoma and ES cells. MESV was derived from a retroviral mutant [PCC4-cell-passaged myeloproliferative sarcoma virus (PCMV)] expressed in

  10. Maternal undernutrition does not alter Sertoli cell numbers or the expression of key developmental markers in the mid-gestation ovine fetal testis

    PubMed Central

    2013-01-01

    Background The aim of this study was to determine the effects of maternal undernutrition on ovine fetal testis morphology and expression of relevant histological indicators. Maternal undernutrition, in sheep, has been reported, previously, to alter fetal ovary development, as indicated by delayed folliculogenesis and the altered expression of ovarian apoptosis-regulating gene products, at day 110 of gestation. It is not known whether or not maternal undernutrition alters the same gene products in the day 110 fetal testis. Design and methods Mature Scottish Blackface ewes were fed either 100% (Control; C) or 50% (low; L) of estimated metabolisable energy requirements of a pregnant ewe, from mating to day 110 of gestation. All pregnant ewes were euthanized at day 110 and a sub-set of male fetuses was randomly selected (6 C and 9?L) for histology studies designed to address the effect of nutritional state on several indices of testis development. Sertoli cell numbers were measured using a stereological method and Ki67 (cell proliferation index), Bax (pro-apoptosis), Mcl-1 (anti-apoptosis), SCF and c-kit ligand (development and apoptosis) gene expression was measured in Bouins-fixed fetal testis using immunohistochemistry. Results No significant differences were observed in numbers of Sertoli cells or testicular Ki67 positive cells. The latter were localised to the testicular cords and interstitium. Bax and Mcl-1 were localised specifically to the germ cells whereas c-kit was localised to both the cords and interstitium. SCF staining was very sparse. No treatment effects were observed for any of the markers examined. Conclusions These data suggest that, unlike in the fetal ovary, maternal undernutrition for the first 110?days of gestation affects neither the morphology of the fetal testis nor the expression of gene products which regulate apoptosis. It is postulated that the effects of fetal undernutrition on testis function may be expressed through hypothalamic-pituitary changes. PMID:23295129

  11. Murine model of CD40-activation of B cells.

    PubMed

    Liebig, Tanja M; Fiedler, Anne; Klein-Gonzalez, Nela; Shimabukuro-Vornhagen, Alexander; von Bergwelt-Baildon, Michael

    2010-01-01

    Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand (CD40L), human B cells can be expanded without difficulties from small amounts of peripheral blood within 14 days to very large amounts of highly-pure CD40-B cells (>10(9) cells per patient) from healthy donors as well as cancer patients. CD40-B cells express important lymph node homing molecules and can attract T cells in vitro. Furthermore they efficiently take up, process and present antigens to T cells. CD40-B cells were shown to not only prime naíve, but also expand memory T cells. Therefore CD40-activated B cells (CD40-B cells) have been studied as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cell-based immunotherapy1,5,10. In order to further study whether CD40-B cells induce effective T cell responses in vivo and to study the underlying mechanism we established a cell culture system for the generation of murine CD40-activated B cells. Using splenocytes or purified B cells from C57BL/6 mice for CD40-activation, optimal conditions were identified as follows: Starting from splenocytes of C57BL/6 mice (haplotype H-2b) lymphocytes are purified by density gradient centrifugation and co-cultured with HeLa cells expressing recombinant murine CD40 ligand (tmuCD40L HeLa). Cells are recultured every 3-4 days and key components such as CD40L, interleukin-4, -Mercaptoethanol and cyclosporin A are replenished. In this protocol we demonstrate how to obtain fully activated murine CD40-B cells (mCD40B) with similar APC-phenotype to human CD40-B cells (Fig 1a,b). CD40-stimulation leads to a rapid outgrowth and expansion of highly pure (>90%) CD19+ B cells within 14 days of cell culture (Fig 1c,d). To avoid contamination with non-transfected cells, expression of the murine CD40 ligand on the transfectants has to be controlled regularly (Fig 2). Murine CD40-activated B cells can be used to study B-cell activation and differentiation as well as to investigate their potential to function as APC in vitro and in vivo. Moreover, they represent a promising tool for establishing therapeutic or preventive vaccination against tumors and will help to answer questions regarding safety and immunogenicity of this approach. PMID:20208476

  12. Involvement of activating ERK1/2 through G protein coupled receptor 30 and estrogen receptor ?/? in low doses of bisphenol A promoting growth of Sertoli TM4 cells.

    PubMed

    Ge, Li-Chen; Chen, Zhuo-Jia; Liu, Han-Yan; Zhang, Kun-Shui; Liu, Hao; Huang, Hong-Bin; Zhang, Ge; Wong, Chris K C; Giesy, John P; Du, Jun; Wang, Hong-Sheng

    2014-04-01

    Sertoli cells play a pivotal role in supporting proliferation of germ cells and differentiation during spermatogenesis in mammals. Nanomolar concentrations of Bisphenol A (BPA) can significantly stimulate the proliferation of mouse immature Sertoli (TM4) cells. However, mechanisms by which BPA caused these effects were still unclear. In the present study, an inverse U-shaped curve was observed when treating TM4 cells with increasing doses of BPA: 1 to 10nM BPA significantly stimulated the proliferation of TM4 cells and increased the proportion of cells in S phase; >1 ?M BPA caused lesser proliferation of cells. Exposure of TM4 cells to G15 or ICI 182,780, which are specific antagonists of GPR30 and estrogen receptor ?/? (ER?/?), respectively, abolished BPA-induced proliferation of cells, which suggests that both GPR30 and ER?/? were involved in the observed effects of BPA. Furthermore, exposure to BPA caused rapid (5 min) activation of ERK1/2 via both GPR30 and ER?/?. Blocking the GPR30/EGFR signal transduction pathway by antagonists suppressed both phosphorylation of ERK and BPA-induced cell proliferation. BPA up-regulated mRNA and protein expression of GPR30 in a concentration-dependent manner. In summary, the results reported here indicated that activating ERK1/2 through GPR30 and ER?/? is involved in low doses of BPA that promoted growth of Sertoli TM4 cells. The GPR30/EGFR/ERK signal is the downstream transduction pathway in BPA-induced proliferation of TM4 Sertoli cells. PMID:24495410

  13. [Cardiomyocyte differentiation of individual clones murine induced pluripotent stem cells].

    PubMed

    Malysheva, S V; Budash, H V; Bil'ko, N M; Heschheller, J

    2013-01-01

    Cardiomyocyte differentiation of certain clones of murine induced pluripotent stem cells (iPS) was estimated. iPS were obtained due to reprogramming of murine embryonic fibroblasts with transposon-based Sleeping beauty plasmids as sene delivery systems. Differentiation was performed in suspension culture and in attached to tissue-culture plates embryoid bodies (EBs). Ascorbic acid was applied as inductor. According to the obtained results, the differentiation was tenfold more effective in attached EBs. Ascorbic acid stimulated the generation of cardiomiocytes. PMID:23957159

  14. The Wilms Tumor Gene, Wt1, Is Critical for Mouse Spermatogenesis via Regulation of Sertoli Cell Polarity and Is Associated with Non-Obstructive Azoospermia in Humans

    PubMed Central

    Wang, Ya Qing; Chen, Min; Zhang, Jun; Hao, Jian Xiu; Wang, Yan Bo; Sha, Ri Na; Huang, Yi; Liu, Xiao; Hu, Jing Chu; Sun, Guang Qing; Li, Hong Gang; Xiong, Cheng Liang; Xie, Jun; Jiang, Zhi Mao; Cai, Zhi Ming; Wang, Jun; Wang, Jian; Huff, Vicki; Gui, Yao Ting; Gao, Fei

    2013-01-01

    Azoospermia is one of the major reproductive disorders which cause male infertility in humans; however, the etiology of this disease is largely unknown. In the present study, six missense mutations of WT1 gene were detected in 529 human patients with non-obstructive azoospermia (NOA), indicating a strong association between WT1 mutation and NOA. The Wilms tumor gene, Wt1, is specifically expressed in Sertoli cells (SCs) which support spermatogenesis. To examine the functions of this gene in spermatogenesis, Wt1 was deleted in adult testis using Wt1flox and Cre-ERTM mice strains. We found that inactivation of Wt1 resulted in massive germ cell death and only SCs were present in most of the seminiferous tubules which was very similar to NOA in humans. In investigating the potential mechanism for this, histological studies revealed that the blood–testis barrier (BTB) was disrupted in Wt1 deficient testes. In vitro studies demonstrated that Wt1 was essential for cell polarity maintenance in SCs. Further studies found that the expression of cell polarity associated genes (Par6b and E-cadherin) and Wnt signaling genes (Wnt4, Wnt11) were downregulated in Wt1 deficient SCs, and that the expression of Par6b and E-cadherin was regulated by Wnt4. Our findings suggest that Wt1 is important in spermatogenesis by regulating the polarity of SCs via Wnt signaling pathway and that WT1 mutation is one of the genetic causes of NOA in humans. PMID:23935527

  15. 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.

  16. Improving in vitro Sertoli cell/gonocyte co-culture model for assessing male reproductive toxicity: Lessons learned from comparisons of cytotoxicity versus genomic responses to phthalates

    SciTech Connect

    Yu Xiaozhong; Hong, Sung Woo [Institute for Risk Analysis and Risk Communication, Department of Environmental and Occupational Health Sciences, University of Washington, 4225 Roosevelt Way NE, Suite 100, Seattle, WA 98105-6099 (United States); Moreira, Estefania G. [Institute for Risk Analysis and Risk Communication, Department of Environmental and Occupational Health Sciences, University of Washington, 4225 Roosevelt Way NE, Suite 100, Seattle, WA 98105-6099 (United States); Dept of Physiological Sciences, State University of Londrina (UEL), Londrina, PR (Brazil); Faustman, Elaine M. [Institute for Risk Analysis and Risk Communication, Department of Environmental and Occupational Health Sciences, University of Washington, 4225 Roosevelt Way NE, Suite 100, Seattle, WA 98105-6099 (United States)], E-mail: faustman@u.washington.edu

    2009-09-15

    Gonocytes exist in the neonatal testis and represent a transient population of male germ-line stem cells. It has been shown that stem cell self-renewal and progeny production is probably controlled by the neighboring differentiated cells and extracellular matrix (ECM) in vivo known as niches. Recently, we developed an in vitro three-dimensional (3D) Sertoli cell/gonocyte co-culture (SGC) model with ECM overlay, which creates an in vivo-like niche and supports germ-line stem cell functioning within a 3D environment. In this study, we applied morphological and cytotoxicity evaluations, as well as microarray-based gene expression to examine the effects of different phthalate esters (PE) on this model. Known in vivo male developmentally toxic PEs (DTPE) and developmentally non-toxic PEs (DNTPE) were evaluated. We observed that DTPE induced significantly greater dose-dependent morphological changes, a decrease in cell viability and an increase in cytotoxicity compared to those treated with DNTPE. Moreover, the gene expression was more greatly altered by DTPE than by DNTPE and non-supervised cluster analysis allowed the discrimination of DTPE from the DNTPE. Our systems-based GO-Quant analysis showed significant alterations in the gene pathways involved in cell cycle, phosphate transport and apoptosis regulation with DTPE but not with DNTPE treatment. Disruptions of steroidogenesis related-gene expression such as Star, Cyp19a1, Hsd17b8, and Nr4a3 were observed in the DTPE group, but not in the DNTPE group. In summary, our observation on cell viability, cytotoxicity, and microarray-based gene expression analysis induced by PEs demonstrate that our in vitro 3D-SGC system mimicked in vivo responses for PEs and suggests that the 3D-SGC system might be useful in identifying developmental reproductive toxicants.

  17. Discordant expression of selectin ligands and sialyl Lewis x-related epitopes on murine myeloid cells

    Microsoft Academic Search

    Marcin M. A. Kobzdej; Anne Leppanen; Vishwanath Ramachandran; Richard D. Cummings; Rodger P. McEver

    2002-01-01

    Murine leukocytes are thought to express 2-3-sialylated and 1-3-fucosylated se- lectin ligands such as sialyl Lewis x (sLex), although monoclonal antibodies (mAbs) to sLex or Lex reportedly do not bind to murine leukocytes. We observed that P- and E-selectin bound to pronase-sensi- tive ligands on murine monocytic WEHI-3 cells and murine neutrophils, indicating that the ligands for both selectins are

  18. Effects of trichostatins on differentiation of murine erythroleukemia cells

    Microsoft Academic Search

    Milioni Yoshida; Shintaro Nomura; Teruhiko Beppu

    1987-01-01

    The fungistatic antibiotics trichostatins (TS) A and C were isolated from culture broth of Streptomyces platensis No. 145 and were found to be potent inducers of differentiation in murine erythroleukemia (Friend and RV133) cells at concentrations of 1.5 X 10(-8) M for TSA and 5 X 10(-7) M for TSC. Differentiation induced by TS was cooperatively enhanced by UV irradiation

  19. Elderly men have low levels of anti-Müllerian hormone and inhibin B, but with high interpersonal variation: a cross-sectional study of the sertoli cell hormones in 615 community-dwelling men.

    PubMed

    Chong, Yih Harng; Dennis, Nicola A; Connolly, Martin J; Teh, Ruth; Jones, Gregory T; van Rij, Andre M; Farrand, Stephanie; Campbell, A John; McLennan, Ian S; Mlennan, Ian S

    2013-01-01

    The Sertoli cells of the testes secrete anti-Müllerian hormone (Müllerian inhibiting Substance, AMH) and inhibin B (InhB). AMH triggers the degeneration of the uterine precursor in male embryos, whereas InhB is part of the gonadal-pituitary axis for the regulation of sperm production in adults. However, both hormones are also putative regulators of homeostasis, and age-related changes in these hormones may therefore be important to the health status of elderly men. The levels of AMH in elderly men are unknown, with limited information being available about age-related changes in InhB. We have therefore used ELISAs to measure Sertoli cell hormone levels in 3 cohorts of community-dwelling men in New Zealand. In total, 615 men were examined, 493 of which were aged 65 or older. Serum AMH and InhB levels inversely correlated with age in men older than 50 years (p<0.001) but not in the younger men. A minority of elderly men had undetectable levels of AMH and InhB. The variation in hormone levels between similarly aged men increased with the age of men. AMH and InhB partially correlated with each other as expected (r = 0.48, p<0.001). However, the ratio of the two Sertoli hormones varied significantly between men, with this variation increasing with age. Elderly men selected for the absence of cardiovascular disease had AMH levels similar to those of young men whereas their InhB levels did not differ from aged-matched controls. These data suggests that Sertoli cell number and function changes with age, but with the extent and nature of the changes varying between men. PMID:23940675

  20. Single-nucleotide polymorphisms in the SEPTIN12 gene may be a genetic risk factor for Japanese patients with Sertoli cell-only syndrome.

    PubMed

    Miyakawa, Hiroe; Miyamoto, Toshinobu; Koh, Eitetsu; Tsujimura, Akira; Miyagawa, Yasushi; Saijo, Yasuaki; Namiki, Mikio; Sengoku, Kazuo

    2012-01-01

    Genetic mechanisms have been implicated as a cause of some cases of male infertility. Recently, 10 novel genes involved in human spermatogenesis, including human SEPTIN12, were identified by expression microarray analysis of human testicular tissue. Septin12 is a member of the septin family of conserved cytoskeletal GTPases that form heteropolymeric filamentous structures in interphase cells. It is expressed specifically in the testis. Therefore, we hypothesized that mutation or polymorphisms of SEPTIN12 participate in male infertility, especially Sertoli cell-only syndrome (SCOS). To investigate whether SEPTIN12 gene defects are associated with azoospermia caused by SCOS, mutational analysis was performed in 100 Japanese patients by direct sequencing of coding regions. Statistical analysis was performed in patients with SCOS and in 140 healthy control men. No mutations were found in SEPTIN12 ; however, 8 coding single-nucleotide polymorphisms (SNP1-SNP8) could be detected in the patients with SCOS. The genotype and allele frequencies in SNP3, SNP4, and SNP6 were notably higher in the SCOS group than in the control group (P < .001). These results suggest that SEPTIN12 might play a critical role in human spermatogenesis. PMID:21636737

  1. An essential role for insulin and IGF1 receptors in regulating sertoli cell proliferation, testis size, and FSH action in mice.

    PubMed

    Pitetti, Jean-Luc; Calvel, Pierre; Zimmermann, Céline; Conne, Béatrice; Papaioannou, Marilena D; Aubry, Florence; Cederroth, Christopher R; Urner, Françoise; Fumel, Betty; Crausaz, Michel; Docquier, Mylène; Herrera, Pedro Luis; Pralong, François; Germond, Marc; Guillou, Florian; Jégou, Bernard; Nef, Serge

    2013-05-01

    Testis size and sperm production are directly correlated to the total number of adult Sertoli cells (SCs). Although the establishment of an adequate number of SCs is crucial for future male fertility, the identification and characterization of the factors regulating SC survival, proliferation, and maturation remain incomplete. To investigate whether the IGF system is required for germ cell (GC) and SC development and function, we inactivated the insulin receptor (Insr), the IGF1 receptor (Igf1r), or both receptors specifically in the GC lineage or in SCs. Whereas ablation of insulin/IGF signaling appears dispensable for GCs and spermatogenesis, adult testes of mice lacking both Insr and Igf1r in SCs (SC-Insr;Igf1r) displayed a 75% reduction in testis size and daily sperm production as a result of a reduced proliferation rate of immature SCs during the late fetal and early neonatal testicular period. In addition, in vivo analyses revealed that FSH requires the insulin/IGF signaling pathway to mediate its proliferative effects on immature SCs. Collectively, these results emphasize the essential role played by growth factors of the insulin family in regulating the final number of SCs, testis size, and daily sperm output. They also indicate that the insulin/IGF signaling pathway is required for FSH-mediated SC proliferation. PMID:23518924

  2. Nanoelectroablation therapy for murine basal cell carcinoma

    SciTech Connect

    Nuccitelli, Richard, E-mail: rich@bioelectromed.com [BioElectroMed Corp., 849 Mitten Rd., Suite 104, Burlingame, CA 94010 (United States)] [BioElectroMed Corp., 849 Mitten Rd., Suite 104, Burlingame, CA 94010 (United States); Tran, Kevin; Athos, Brian; Kreis, Mark; Nuccitelli, Pamela [BioElectroMed Corp., 849 Mitten Rd., Suite 104, Burlingame, CA 94010 (United States)] [BioElectroMed Corp., 849 Mitten Rd., Suite 104, Burlingame, CA 94010 (United States); Chang, Kris S.; Epstein, Ervin H. [The Children's Hospital Oakland Research Institute, Oakland, CA 94609 (United States)] [The Children's Hospital Oakland Research Institute, Oakland, CA 94609 (United States); Tang, Jean Y. [The Children's Hospital Oakland Research Institute, Oakland, CA 94609 (United States) [The Children's Hospital Oakland Research Institute, Oakland, CA 94609 (United States); Stanford University, Stanford, CA 94305 (United States)

    2012-08-03

    Highlights: Black-Right-Pointing-Pointer Nanoelectroablation is a new, non-thermal therapy that triggers apoptosis in tumors. Black-Right-Pointing-Pointer Low energy, ultrashort, high voltage pulses ablate the tumor with little or no scar. Black-Right-Pointing-Pointer Nanoelectroablation eliminates 99.8% of the BCC but may leave a few remnants behind. Black-Right-Pointing-Pointer Pilot clinical trials on human BCCs are ongoing and leave no remnants in most cases. -- Abstract: When skin tumors are exposed to non-thermal, low energy, nanosecond pulsed electric fields (nsPEF), apoptosis is initiated both in vitro and in vivo. This nanoelectroablation therapy has already been proven effective in treating subdermal murine allograft tumors. We wanted to determine if this therapy would be equally effective in the treatment of autochthonous BCC tumors in Ptch1{sup +/-}K14-Cre-ER p53 fl/fl mice. These tumors are similar to human BCCs in histology and in response to drug therapy . We have treated 27 BCCs across 8 mice with either 300 pulses of 300 ns duration or 2700 pulses of 100 ns duration, all at 30 kV/cm and 5-7 pulses per second. Every nsPEF-treated BCC began to shrink within a day after treatment and their initial mean volume of 36 {+-} 5 (SEM) mm{sup 3} shrunk by 76 {+-} 3% over the ensuing two weeks. After four weeks, they were 99.8% ablated if the size of the treatment electrode matched the tumor size. If the tumor was larger than the 4 mm wide electrode, multiple treatments were needed for complete ablation. Treated tumors were harvested for histological analysis at various times after treatment and exhibited apoptosis markers. Specifically, pyknosis of nuclei was evident as soon as 2 days after nsPEF treatment, and DNA fragmentation as detected via TUNEL staining was also evident post treatment. Nanoelectroablation is effective in triggering apoptosis and remission of radiation-induced BCCs with a single 6 min-long treatment of 2700 pulses.

  3. 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

  4. Murine Virus-Induced Proteins Synthesized by Hamster Tumor Cells Transformed by, But Not Producing, Murine Sarcoma Virus

    PubMed Central

    Ikegami, Nobuko; Gomatos, Peter J.

    1974-01-01

    The 8303 hamster tumor cells transformed by Moloney strain of murine sarcoma virus (M-MSV), but which do not produce virus, do contain murine virus-induced proteins. The virus-induced proteins within the cell were identified either as free proteins or in association with membranous material, including the plasma membrane. In addition, some were excreted by the 8303 hamster tumor cells into the growth medium. Most virus-induced proteins were larger than 68,000 daltons, and they did not dissociate into components of smaller size in the presence of detergent and a reducing agent. A small amount of virus-induced protein with a molecular weight of less than 20,000 was also found in the hamster tumor cells. No virus-specific proteins with the identical antigenic specificity or size of the major internal group specific antigen (molecular weight about 30,000) of the murine leukemia viruses were present in these cells. There is a common cell surface antigen present in three other tumor cell lines, both virus-producing and non-virus-producing, identical in reactivity to that of the murine virus-induced antigen of the 8303 hamster tumor cell. This antigen is not present on the cell surface of normal mouse embryo cells. Images PMID:4359302

  5. Bioengineering murine mastocytoma cells to produce anticoagulant heparin

    PubMed Central

    Gasimli, Leyla; Glass, Charles A; Datta, Payel; Yang, Bo; Li, Guoyun; Gemmill, Trent R; Baik, Jong Youn; Sharfstein, Susan T; Esko, Jeffrey D; Linhardt, Robert J

    2014-01-01

    Heparin (HP), an important anticoagulant polysaccharide, is produced in a complex biosynthetic pathway in connective tissue-type mast cells. Both the structure and size of HP are critical factors determining the anticoagulation activity. A murine mastocytoma (MST) cell line was used as a model system to gain insight into this pathway. As reported, MST cells produce a highly sulfated HP-like polysaccharide that lacks anticoagulant activity (Montgomery RI, Lidholt K, Flay NW, Liang J, Vertel B, Lindahl U, Esko JD. 1992. Stable heparin-producing cell lines derived from the Furth murine mastocytoma. Proc Natl Acad Sci USA 89:11327–11331). Here, we show that transfection of MST cells with a retroviral vector containing heparan sulfate 3-O-sulfotransferase-1 (Hs3st1) restores anticoagulant activity. The MST lines express N-acetylglucosamine N-deacetylase/N-sulfotransferase-1, uronosyl 2-O-sulfotransferase and glucosaminyl 6-O-sulfotransferase-1, which are sufficient to make the highly sulfated HP. Overexpression of Hs3st1 in MST-10H cells resulted in a change in the composition of heparan sulfate (HS)/HP and CS/dermatan sulfate (DS) glycosaminoglycans. The cell-associated HS/HP closely resembles HP with 3-O-sulfo group-containing glucosamine residues and shows anticoagulant activity. This study contributes toward a better understanding of the HP biosynthetic pathway with the goal of providing tools to better control the biosynthesis of HP chains with different structures and activities. PMID:24326668

  6. Membrane heterogeneity in murine stem cells

    SciTech Connect

    Myers, C.P.; Kerk, D.K.; Norman, A.

    1980-01-01

    The cell membrane's role in hemopoietic differentiation and regulation is increasingly evident. Certain fluorescent molecules interact with membrane components at the molecular level. The organic dye merocyanine 540(MCN) is such a tool for studying hemopoietic stem cell membranes at the molecular level. This paper present work involving biologic interactions of MCN with CLL (chronic lymphocytic leukemia) and hemopoietic cell membranes. The authors work indicates that MNC staining reveals membrane differences that accompany cellular transformation and differentiation. Changes in MNC fluorescene indicate that hemopoietic cells are heterogeneous with respect to membrane properties, and sensitivity to MNC photoinactivation reveals that membrane properties may change as a function of differentiative state between pluripotent and committed granulocytic precursor cells.

  7. Sertoli-Leydig cell tumors: hormonal profile after dynamic test with GnRH analogue: triptorelin represents a useful tool to evaluate tumoral hyperandrogenism.

    PubMed

    Turra, J; Granzotto, M; Gallea, M; Faggian, D; Conte, L; Litta, P; Vettor, R; Mioni, R

    2015-01-01

    We report the case of a 15-year-old woman with signs of hyperandrogenism affected by a Sertoli-Leydig cell tumor (SLCT). In our patient, blood analysis showed a high testosterone (T) level (T: 8.53?nmol/L; nv?

  8. Stromal cells participate in the murine esophageal mucosal injury response

    PubMed Central

    Binkley, Jana; Darwech, Isra; Swietlicki, Elzbieta; McDonald, Keely; Newberry, Rodney; Rubin, Deborah C.

    2013-01-01

    We identified ?-smooth muscle actin (?-SMA)- and vimentin-expressing spindle-shaped esophageal mesenchymal cells in the adult and neonate murine esophageal lamina propria. We hypothesized that these esophageal mesenchymal cells express and secrete signaling and inflammatory mediators in response to injury. We established primary cultures of esophageal mesenchymal cells using mechanical and enzymatic digestion. We demonstrate that these primary cultures are nonhematopoietic, nonendothelial, stromal cells with myofibroblast-like features. These cells increase secretion of IL-6 in response to treatment with acidified media and IL-1?. They also increase bone morphogenetic protein (Bmp)-4 secretion in response to sonic hedgehog. The location of these cells and their biological functions demonstrate their potential role in regulating esophageal epithelial responses to injury and repair. PMID:23370675

  9. 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.

  10. Packaging of Endogenous Retroviral Sequences in Retroviral Vectors Produced by Murine and Human Packaging Cells

    Microsoft Academic Search

    CLIVE PATIENCE; YASUHIRO TAKEUCHI; FRANCOIS-LOIC COSSET; ROBIN A. WEISS

    1998-01-01

    Interaction of retrovirus vectors and endogenous retroviruses present in packaging cell lines and target cells may result in unwanted events, such as the formation of recombinant viruses and the mobilization of therapeu- tic vectors. Using sensitive reverse transcriptase PCR assays, we investigated human and murine gene therapy packaging cell lines for incorporation of endogenous retrovirus transcripts into murine leukemia virus

  11. Multivariate proteomic analysis of murine embryonic stem cell self-renewal versus differentiation signaling

    E-print Network

    Zandstra, Peter W.

    Multivariate proteomic analysis of murine embryonic stem cell self-renewal versus differentiation soluble cytokines and insoluble matrix factors, are known to influence murine embryonic stem cell self stimulation of mouse embryonic stem cells by fibronectin, laminin, leukemia-inhibitory factor, and fibro

  12. Generation of Eosinophils from Cryopreserved Murine Bone Marrow Cells

    PubMed Central

    Schollaert, Kaila L.; Stephens, Michael R.; Gray, Jerilyn K.; Fulkerson, Patricia C.

    2014-01-01

    Eosinophils are produced in the bone marrow from CD34+ eosinophil lineage–committed progenitors, whose levels in the bone marrow are elevated in a variety of human diseases. These findings suggest that increased eosinophil lineage–committed progenitor production is an important process in disease-associated eosinophilia. The pathways central to the biology of the eosinophil lineage–committed progenitor remain largely unknown. Thus, developing new methods to investigate the regulators of eosinophil lineage–committed progenitor differentiation is needed to identify potential therapeutic targets to specifically inhibit eosinophil production. We tested cytokine regimens to optimize liquid cultures for the study of eosinophil lineage–committed progenitor and eosinophil precursor differentiation into mature eosinophils. Stem cell factor (but not fms-related tyrosine kinase 3 ligand) was required for optimal yield of eosinophils. Furthermore, we evaluated the effects of cell preservation and scale on the culture, successfully culturing functional eosinophils from fresh and frozen murine bone marrow cells and in a standard-sized and 96-well culture format. In summary, we have developed an adaptable culture system that yields functionally competent eosinophils from murine low-density bone marrow cells and whose cytokine regime includes expansion of progenitors with stem cell factor alone with subsequent differentiation with interleukin 5. PMID:25551463

  13. 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

  14. Dickkopf Homolog 3 (DKK3) Plays a Crucial Role Upstream of WNT/?-CATENIN Signaling for Sertoli Cell Mediated Regulation of Spermatogenesis

    PubMed Central

    Kunj, Neetu; Sarda, Kanchan; Pradhan, Bhola Shankar; Majumdar, Subeer S.

    2013-01-01

    Testicular Sertoli cells (Sc) are main somatic component of seminiferous tubules that govern the differentiation of germ cells (Gc) and provide them physical support. Sc are the target of follicle stimulating hormone (FSH) and testosterone (T) which are known to regulate spermatogenesis. FSH and T levels in human and sub-human male primates remain high during infancy (4–6 months post birth), similar to those during puberty. Subsequently, juvenile phase is marked with low levels of these hormones. In spite of prolonged hormonal exposure, spermatogenesis is not discerned during infancy unlike that during puberty. Situation during infancy is similar to certain idiopathic male infertility, where prolonged hormone supplementation fails to initiate spermatogenesis. In our quest to determine non hormonal causes of idiopathic infertility which may reside within the Sc, we investigated the association between spermatogenesis and Sc specific gene(s) expressed differentially during puberty and infancy. Although products of several genes may be necessary for quantitatively normal spermatogenesis, one needs to investigate their roles one by one. Differential display and real time PCR analysis revealed higher expression of a known tumor suppressor, Dickkopf homolog 3 (DKK3), by pubertal monkey Sc as compared to infant Sc. To evaluate role of DKK3 in spermatogenesis, we generated DKK3 knock down mice (DKDM) using shRNA construct targeted to DKK3. In testis of adult DKDM, expression of DKK3 mRNA and protein were significantly (p<0.05) low and was associated with elevated WNT-4/?-CATENIN activity. Elevated ?-CATENIN activity is known to restrict Sc maturation. Abundant expression of infant Sc marker, Mullerian inhibiting substance (MIS), in the testes of adult DKDM confirmed lack of Sc maturation in DKDM. Gc differentiation and fertility was severely compromised in DKDM. This is the first report of role of DKK3 in the testis and DKK3 mediated regulation of spermatogenesis via WNT-4/?-CATENIN modulation. PMID:23667645

  15. Low Levels of G?s and Ric8b in Testicular Sertoli Cells May Underlie Restricted FSH Action During Infancy in Primates.

    PubMed

    Bhattacharya, Indrashis; Basu, Sayon; Sarda, Kanchan; Gautam, Mukkesh; Nagarajan, Perumal; Pradhan, Bhola Shankar; Sarkar, Hironmoy; Devi, Yendrembam Sangeeta; Majumdar, Subeer S

    2015-03-01

    FSH acts via testicular Sertoli cells (Sc) bearing FSH receptor (FSH-R) for regulating male fertility. Despite an adult-like FSH milieu in infant boys and monkeys, spermatogenesis is not initiated until the onset of puberty. We used infant and pubertal monkey Sc to reveal the molecular basis underlying developmental differences of FSH-R signaling in them. Unlike pubertal Sc, increasing doses of FSH failed to augment cAMP production by infant Sc. The expression of G?s subunit and Ric8b, which collectively activate adenylyl cyclase (AC) for augmenting cAMP production and gene transcription, were significantly low in infant Sc. However, forskolin, which acts directly on AC bypassing FSH-R, augmented cAMP production and gene transcription uniformly in both infant and pubertal Sc. FSH-induced G?s mRNA expression was higher in pubertal Sc. However, G?i-2 expression was down-regulated by FSH in pubertal Sc, unlike infant Sc. FSH failed, but forskolin or 8-Bromoadenosine 3',5'-cyclic monophosphate treatment to infant Sc significantly augmented the expression of transferrin, androgen binding protein, inhibin-?-B, stem cell factor, and glial-derived neurotropic factor, which are usually up-regulated by FSH in pubertal Sc during spermatogenic onset. This suggested that lack of FSH mediated down-regulation of G?i-2 expression and limited expression of G?s subunit as well as Ric8b may underlie limited FSH responsiveness of Sc during infancy. This study also divulged that intracellular signaling events downstream of FSH-R are in place and can be activated exogenously in infant Sc. Additionally, this information may help in the proper diagnosis and treatment of infertile individuals having abnormal G protein-coupled FSH-R. PMID:25549048

  16. Effects of a murine germ cell-specific knockout of Connexin 43 on Connexin expression in testis and fertility.

    PubMed

    Günther, Sabine; Fietz, Daniela; Weider, Karola; Bergmann, Martin; Brehm, Ralph

    2013-06-01

    Connexin 43 (Cx 43)--expressed by germ cells (GC), Sertoli cells (SC) and Leydig cells--is one of at least eleven Cx in the murine testis. A general knockout (KO) of Cx 43 in mice results in perinatal death and a SC-specific KO of Cx 43 (SCCx43KO) causes infertility of male mice by preventing the initiation of spermatogenesis. To further elucidate the role of Cx 43 in the testis, a new mouse model with a GC-specific KO of Cx 43 (GCCx43KO) was created by using the Cre/loxP recombination system. A transgenic mouse line expressing the Cre gene under the tissue non-specific alkaline phosphatase promoter and a transgenic floxed Cx 43-LacZ mouse line were mated. The resulting F1-generation was backcrossed with homozygous Cx 43 floxed mice, and offspring was genotyped. Immunohistochemical analysis of testes of different aged homozygous mice revealed normal spermatogenesis and reduced Cx 43 immunoreactions. RT-qPCR and Western blots showed a downregulation of Cx 43 mRNA and protein, and a nearly unchanged mRNA expression of Cx 26, Cx 33 and Cx 45 in pubertal and adult KO mice. Western blots revealed considerable immunoreactive bands for Cx 26 and Cx 45. Male and female homozygous GCCx43KO mice were viable and fertile. Our data suggest, in contrast to inter SC and inter SC-GC cross talk in SCCx43KO mice which depends selectively on Cx 43 expression, that Cx 43 in GC seems not to be essential in GC-SC communication, when other Cx persist to be expressed. PMID:23188169

  17. Synthetic peptides corresponding to residues 551 to 555 and 650 to 653 of the rat testicular follicle-stimulating hormone (FSH) receptor are sufficient for post-receptor modulation of Sertoli cell responsiveness to FSH stimulation.

    PubMed

    Grasso, P; Deziel, M R; Reichert, L E

    1995-12-14

    We have recently demonstrated that synthetic peptides corresponding to the third cytoplasmic (3i) loop (residues 533 to 555) and a region in the carboxy-terminal cytoplasmic tail (residues 645 to 653) of the rat testicular follicle-stimulating hormone receptor (FSHR) affected signal transduction in rat testis membranes and cultured rat Sertoli cells. In order to define more precisely the peptide domains involved, we synthesized truncated peptide amides corresponding to FSHR residues 551-555 (KIAKR) and 650-653 (RKSH), respectively. These two regions were chosen since they contained a minimal structural motif present in G protein activator regions of several other G protein-coupled receptors (i.e., B-X-X-B-B or B-B-X-B, B representing a basic amino acid). Neither peptide inhibited binding of FSH to testis membrane receptors. Each peptide significantly reduced FSH-stimulated estradiol biosynthesis by intact cultured rat Sertoli cells. The same results were obtained with streptolysin O-permeabilized Sertoli cells. No effect was noted on forskolin-induced steroidogenesis, indicating that the peptide effects were not due to interaction with adenylyl cyclase. Each peptide amide, however, induced concentration-dependent increases in guanine nucleotide exchange in rat testis membranes. Our results indicate that interaction of FSH receptor with its associated G protein may involve relatively restricted peptide sequences, and include residues 551-555 (KIAKR) in the third cytoplasmic loop, and residues 650-653 (RKSH) in the carboxy-terminal cytoplasmic tail of the FSH receptor. PMID:8746544

  18. Functional expression of SK channels in murine detrusor PDGFR?+ cells

    PubMed Central

    Lee, Haeyeong; Koh, Byoung H; Peri, Lauren E; Sanders, Kenton M; Koh, Sang Don

    2013-01-01

    We sought to characterize molecular expression and ionic conductances in a novel population of interstitial cells (PDGFR?+ cells) in murine bladder to determine how these cells might participate in regulation of detrusor excitability. PDGFR?+ cells and smooth muscle cells (SMCs) were isolated from detrusor muscles of PDGFR?+/eGFP and smMHC/Cre/eGFP mice and sorted by FACS. PDGFR?+ cells were highly enriched in Pdgfra (12 fold vs. unsorted cell) and minimally positive for Mhc (SMC marker), Kit (ICC marker) and Pgp9.5 (neuronal marker). SK3 was dominantly expressed in PDGFR?+ cells in comparison to SMCs. ?Slo (BK marker) was more highly expressed in SMCs. SK3 protein was observed in PDGFR?+ cells by immunohistochemistry but could not be resolved in SMCs. Depolarization evoked voltage-dependent Ca2+ currents in SMCs, but inward current conductances were not activated in PDGFR?+ cells under the same conditions. PDGFR?+ cells displayed spontaneous transient outward currents (STOCs) at potentials positive to ?60 mV that were inhibited by apamin. SK channel modulators, CyPPA and SKA-31, induced significant hyperpolarization of PDGFR?+ cells and activated SK currents under voltage clamp. Similar responses were not resolved in SMCs at physiological potentials. Single channel measurements confirmed the presence of functional SK3 channels (i.e. single channel conductance of 10 pS and sensitivity to intracellular Ca2+) in PDGFR?+ cells. The apamin-sensitive stabilizing factor regulating detrusor excitability is likely to be due to the expression of SK3 channels in PDGFR?+ cells because SK agonists failed to elicit resolvable currents and hyperpolarization in SMCs at physiological potentials. PMID:23148317

  19. Effects of trichostatins on differentiation of murine erythroleukemia cells

    SciTech Connect

    Yoshida, M.; Nomura, S.; Beppu, T.

    1987-07-15

    The fungistatic antibiotics trichostatins (TS) A and C were isolated from culture broth of Streptomyces platensis No. 145 and were found to be potent inducers of differentiation in murine erythroleukemia (Friend and RV133) cells at concentrations of 1.5 X 10(-8) M for TSA and 5 X 10(-7) M for TSC. Differentiation induced by TS was cooperatively enhanced by UV irradiation but not by treatment with dimethyl sulfoxide. This enhanced activity was completely inhibited by adding cycloheximide to the culture medium 2 h after exposure to TS, suggesting that TS are dimethyl sulfoxide-type inducers of erythroid differentiation. No inhibitory effect of TS was observed on macromolecular synthesis in cultured cells.

  20. Effects of trichostatins on differentiation of murine erythroleukemia cells.

    PubMed

    Yoshida, M; Nomura, S; Beppu, T

    1987-07-15

    The fungistatic antibiotics trichostatins (TS) A and C were isolated from culture broth of Streptomyces platensis No. 145 and were found to be potent inducers of differentiation in murine erythroleukemia (Friend and RV133) cells at concentrations of 1.5 X 10(-8) M for TSA and 5 X 10(-7) M for TSC. Differentiation induced by TS was cooperatively enhanced by UV irradiation but not by treatment with dimethyl sulfoxide. This enhanced activity was completely inhibited by adding cycloheximide to the culture medium 2 h after exposure to TS, suggesting that TS are dimethyl sulfoxide-type inducers of erythroid differentiation. No inhibitory effect of TS was observed on macromolecular synthesis in cultured cells. PMID:2439196

  1. Isolation of Primary Murine Brain Microvascular Endothelial Cells

    PubMed Central

    Ruck, Tobias; Bittner, Stefan; Epping, Lisa; Herrmann, Alexander M.; Meuth, Sven G.

    2014-01-01

    The blood-brain-barrier is ultrastructurally assembled by a monolayer of brain microvascular endothelial cells (BMEC) interconnected by a junctional complex of tight and adherens junctions. Together with other cell-types such as astrocytes or pericytes, they form the neurovascular unit (NVU), which specifically regulates the interchange of fluids, molecules and cells between the peripheral blood and the CNS. Through this complex and dynamic system BMECs are involved in various processes maintaining the homeostasis of the CNS. A dysfunction of the BBB is observed as an essential step in the pathogenesis of many severe CNS diseases. However, specific and targeted therapies are very limited, as the underlying mechanisms are still far from being understood. Animal and in vitro models have been extensively used to gain in-depth understanding of complex physiological and pathophysiological processes. By reduction and simplification it is possible to focus the investigation on the subject of interest and to exclude a variety of confounding factors. However, comparability and transferability are also reduced in model systems, which have to be taken into account for evaluation. The most common animal models are based on mice, among other reasons, mainly due to the constantly increasing possibilities of methodology. In vitro studies of isolated murine BMECs might enable an in-depth analysis of their properties and of the blood-brain-barrier under physiological and pathophysiological conditions. Further insights into the complex mechanisms at the BBB potentially provide the basis for new therapeutic strategies. This protocol describes a method to isolate primary murine microvascular endothelial cells by a sequence of physical and chemical purification steps. Special considerations for purity and cultivation of MBMECs as well as quality control, potential applications and limitations are discussed. PMID:25489873

  2. Genes and signals regulating murine trophoblast cell development

    PubMed Central

    El-Hashash, Ahmed H.K.; Warburton, David; Kimber, Susan J.

    2010-01-01

    A fundamental step in embryonic development is cell differentiation whereby highly specialised cell types are developed from a single undifferentiated, fertilised egg. One of the earliest lineages to form in the mammalian conceptus is the trophoblast, which contributes exclusively to the extraembryonic structures that form the placenta. Trophoblast giant cells (TGCs) in the rodent placenta form the outermost layer of the extraembryonic compartment, establish direct contact with maternal cells, and produce a number of pregnancy-specific cytokine hormones. Giant cells differentiate from proliferative trophoblasts as they exit the cell cycle and enter a genome-amplifying endocycle. Normal differentiation of secondary TGCs is a critical step toward the formation of the placenta and normal embryonic development. Trophoblast development is also of particular interest to the developmental biologist and immunobiologist, as these cells constitute the immediate cellular boundary between the embryonic and maternal tissues. Abnormalities in the development of secondary TGCs results in severe malfunction of the placenta. Herein we review new information that has been accumulated recently regarding the molecular and cellular regulation of trophoblast and placenta development. In particular, we discuss the molecular aspects of murine TGC differentiation. We also focus on the role of growth and transcription factors in TGC development. PMID:19755154

  3. 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 Central

    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

  4. Human p32 protein relieves a post-transcriptional block to HIV replication in murine cells

    Microsoft Academic Search

    Yong-Hui Zheng; Hai-Feng Yu; B. Matija Peterlin

    2003-01-01

    In the mouse, replication of human immunodeficiency virus type 1 (HIV) is blocked at the levels of entry, transcription and assembly. For the latter effect, the amounts of unspliced viral genomic RNA could have an important function. Indeed, in murine cells, HIV transcripts are spliced excessively, a process that is not inhibited by the murine splicing inhibitor p32 (mp32). In

  5. 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

  6. Chinese medicinal herbs inhibit growth of murine renal cell carcinoma.

    PubMed

    Lau, B H; Ruckle, H C; Botolazzo, T; Lui, P D

    1994-01-01

    Tumors are known to produce factors suppressing immune functions. We previously showed that a murine renal cell carcinoma (Renca) suppressed macrophage function in vitro and that this suppression was abolished by co-incubation with extracts of two Chinese medicinal herbs. We now report that these phytochemicals are capable of inhibiting growth of Renca in vivo. BALB/c mice were transplanted intraperitoneally (IP) with 1-2 x 10(5) Renca cells. One day after tumor transplant, mice were randomized into two groups. One group was treated IP, daily for 10 days, with 100 microliters of phytochemicals containing 500 micrograms each of Astragalus membranaceus and Ligustrum lucidum, while the other group received saline as controls. A cure rate of 57% was obtained with these phytochemicals when the initial tumor load was 2 x 10(5), and 100% when the initial tumor load was 1 x 10(5). Additional experiments were performed to investigate the mechanisms involved in this protection. Splenic macrophages from tumor-bearing mice were shown to have depressed chemiluminescent oxidative burst activity, and this depression was restored with phytochemical treatment. Splenocytes from mice transplanted with Renca responded less favorably to interleukin-2 (IL-2) in generating lymphokine-activated killer (LAK) cells; again this depression was restored with phytochemical treatment. Our data suggest that these phytochemicals may have exerted their antitumor effects via augmentation of phagocyte and LAK cell activities. PMID:7812364

  7. Simvastatin induces osteogenic differentiation of murine embryonic stem cells.

    PubMed

    Pagkalos, Joseph; Cha, Jae Min; Kang, Yunyi; Heliotis, Manolis; Tsiridis, Eleftherios; Mantalaris, Athanasios

    2010-11-01

    Statins are potent inhibitors of cholesterol synthesis. Several statins are available with different molecular and pharmacokinetic properties. Simvastatin is more lipophilic than pravastatin and has a higher affinity to phospholipid membranes than atorvastatin, allowing its passive diffusion through the cell membrane. In vitro studies on bone marrow stromal cells, osteoblast-like cells, and embryonic stem cells have shown statins to have cholesterol-independent anabolic effects on bone metabolism; alas, statins were supplemented in osteogenic medium, which does not facilitate elucidation of their potential osteoinductive properties. Embryonic stem cells (ESCs), derived from the inner cell mass of the blastocyst, are unique in that they enjoy perpetual self-proliferation, are pluripotent, and are able to differentiate toward all the cellular lineages composing the body, including the osteogenic lineage. Consequently, ESCs represent a potentially potent cell source for future clinical cellular therapies of various bone diseases, even though there are several hurdles that still need to be overcome. Herein we demonstrate, for the first time to our knowledge, that simvastatin induces murine ESC (mESC) differentiation toward the osteogenic lineage in the absence of osteoinductive supplements. Specifically, we found that a simvastatin concentration in the micromolar range and higher was toxic to the cells and that an effective concentration for osteoinduction is 0.1 nM, as shown by increased alizarin red staining as well as increased osteocalcin and osetrix gene expression. These results suggest that in the future, lipophilic simvastatin may provide a novel pharmacologic agent for bone tissue engineering applications. PMID:20564244

  8. L* Protein of Theiler's Murine Encephalomyelitis Virus Is Required for Virus Growth in a Murine Macrophage-Like Cell Line

    Microsoft Academic Search

    MASATSUGU OBUCHI; JIRO YAMAMOTO; TAKATO ODAGIRI; MOHAMMED NASIR UDDIN; HIDEAKI IIZUKA; YOSHIRO OHARA

    2000-01-01

    We sought to confirm the importance of L* protein for growth of Theiler's murine encephalomyelitis virus (TMEV) in a macrophage-like cell line, J774-1. The protein is out of frame with the polyprotein and synthesized in DA but not GDVII subgroup strains of TMEV. A recombinant virus, DANCL*\\/GD, which substitutes the DA 5* noncoding and L* coding regions for the corresponding

  9. 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.

  10. 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

  11. Sertoli-Leydig cell tumor

    MedlinePLUS

    Surgery is done to remove one or both ovaries. If the tumor is advanced stage, chemotherapy or radiation therapy may ... Ramirez PT, Gershenson DM. Neoplastic diseases of the ovary: ... stromal tumors. In: Lentz GM, Lobo RA, Gershenson DM, Katz ...

  12. Vitamin D controls murine and human plasmacytoid dendritic cell function.

    PubMed

    Karthaus, Nina; van Spriel, Annemiek B; Looman, Maaike W G; Chen, Shuo; Spilgies, Lisanne M; Lieben, Liesbet; Carmeliet, Geert; Ansems, Marleen; Adema, Gosse J

    2014-05-01

    Topical application of the vitamin D (VitD) analog calcipotriol is a highly effective standard treatment modality of psoriatic skin lesions. However, the immune modulatory effects of the treatment are incompletely understood. VitD is well known to induce tolerogenic responses in conventional dendritic cells (cDCs). Plasmacytoid DCs (pDCs) comprise a specialized, naturally occurring DC subset known to be important in autoimmune diseases including psoriasis. pDCs from the blood rapidly infiltrate psoriatic skin and are key to the initiation of the immune-mediated pathogenesis of the disease. We now demonstrate that pDCs express various proteins of the VitD receptor (VDR) pathway, including the VitD-metabolizing enzymes Cyp27B1 and Cyp24A1, and that VDR is transcriptionally active in pDCs. Moreover, VitD impairs the capacity of murine and human pDCs to induce T-cell proliferation and secretion of the T-helper 1 cytokine IFN?. The inhibitory effect of VitD is dependent on the expression of the VDR in the DCs. This study demonstrates that VitD signaling can act as a natural inhibitory mechanism on both cDCs and pDCs, which may instigate the development of VitD-based therapeutic applications for psoriasis and other inflammatory skin diseases. PMID:24352045

  13. Localization of Label-Retaining Cells in Murine Vocal Fold Epithelium

    ERIC Educational Resources Information Center

    Leydon, Ciara; Bartlett, Rebecca S.; Roenneburg, Drew A.; Thibeault, Susan L.

    2011-01-01

    Purpose: Epithelial homeostasis is critical for vocal fold health, yet little is known about the cells that support epithelial self-renewal. As a known characteristic of stem cells is that they are slow-cycling in vivo, the purpose of this prospective controlled study was to identify and quantify slow-cycling cells or putative stem cells in murine

  14. Interferon-Gamma-Induced Nitric Oxide Inhibits the Proliferation of Murine Renal Cell Carcinoma Cells

    PubMed Central

    Tate Jr., David J.; Patterson, John R.; Velasco-Gonzalez, Cruz; Carroll, Emily N.; Trinh, Janie; Edwards, Daniel; Aiyar, Ashok; Finkel-Jimenez, Beatriz; Zea, Arnold H.

    2012-01-01

    Renal cell carcinoma (RCC) remains one of the most resistant tumors to systemic chemotherapy, radiotherapy, and immunotherapy. Despite great progress in understanding the basic biology of RCC, the rate of responses in animal models and clinical trials using interferons (IFNs) has not improved significantly. It is likely that the lack of responses can be due to the tumor's ability to develop tumor escape strategies. Currently, the use of targeted therapies has improved the clinical outcomes of patients with RCC and is associated with an increase of Th1-cytokine responses (IFN?), indicating the importance of IFN? in inhibiting tumor proliferation. Thus, the present study was designed to investigate a new mechanism by which IFN? mediates direct anti-proliferative effects against murine renal cell carcinoma cell lines. When cultured RCC cell lines were exposed to murine recombinant IFN?, a dose dependent growth inhibition in CL-2 and CL-19 cells was observed; this effect was not observed in Renca cells. Growth inhibition in CL-2 and CL-19 cell lines was associated with the intracellular induction of nitric oxide synthase (iNOS) protein, resulting in a sustained elevation of nitric oxide (NO) and citrulline, and a decrease in arginase activity. The inhibition of cell proliferation appears to be due to an arrest in the cell cycle. The results indicate that in certain RCC cell lines, IFN? modulates L-arginine metabolism by shifting from arginase to iNOS activity, thereby developing a potent inhibitory mechanism to encumber tumor cell proliferation and survival. Elucidating the cellular events triggered by IFN? in murine RCC cell lines will permit anti-tumor effects to be exploited in the development of new combination therapies that interfere with L-arginine metabolism to effectively combat RCC in patients. PMID:22991499

  15. Derivation of hematopoietic stem cells from murine embryonic stem cells.

    PubMed

    McKinney-Freeman, Shannon; Daley, George

    2007-01-01

    A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny. Embryonic stem cells (ESC) are derived from the blastocyst of the early embryo and are pluripotent in differentiative ability. Their vast differentiative potential has made them the focus of much research centered on deducing how to coax them to generate clinically useful cell types. The successful derivation of hematopoietic stem cells (HSC) from mouse ESC has recently been accomplished and can be visualized in this video protocol. HSC, arguably the most clinically exploited cell population, are used to treat a myriad of hematopoietic malignancies and disorders. However, many patients that might benefit from HSC therapy lack access to suitable donors. ESC could provide an alternative source of HSC for these patients. The following protocol establishes a baseline from which ESC-HSC can be studied and inform efforts to isolate HSC from human ESC. In this protocol, ESC are differentiated as embryoid bodies (EBs) for 6 days in commercially available serum pre-screened for optimal hematopoietic differentiation. EBs are then dissociated and infected with retroviral HoxB4. Infected EB-derived cells are plated on OP9 stroma, a bone marrow stromal cell line derived from the calvaria of M-CSF-/- mice, and co-cultured in the presence of hematopoiesis promoting cytokines for ten days. During this co-culture, the infected cells expand greatly, resulting in the generation a heterogeneous pool of 100 s of millions of cells. These cells can then be used to rescue and reconstitute lethally irradiated mice. PMID:18830431

  16. Multiple effects of dendritic cell depletion on murine norovirus infection

    PubMed Central

    Elftman, Michael D.; Gonzalez-Hernandez, Mariam B.; Kamada, Nobuhiko; Perkins, Cheryl; Henderson, Kenneth S.; Núñez, Gabriel

    2013-01-01

    Dendritic cells (DCs) are permissive to murine norovirus (MNV) infection in vitro and in vivo. However, their roles during infection in vivo are not well defined. To determine the role of DCs during infection, conventional DCs were depleted from CD11c-DTR mice and infected with a persistent MNV strain. Viral titres in the intestine and secondary lymphoid organs were determined at early time points during infection, and anti-MNV antibody responses were analysed later during infection. Depletion of conventional DCs resulted in increased viral loads in intestinal tissues, impaired generation of antibody responses, and a failure of MNV to efficiently infect lymphoid tissues. These data suggest that DCs play multiple roles in MNV pathogenesis, in both innate immunity and the efficient generation of adaptive immune responses against MNV, as well as by promoting the dissemination of MNV to secondary lymphoid tissues. This is the first study to probe the roles of DCs in controlling and/or facilitating a norovirus infection in vivo and provides the basis for further studies aimed at defining mechanisms by which DCs control MNV replication and promote viral dissemination. PMID:23636823

  17. Molecular cloning of a novel murine cell-surface glycoprotein homologous to killer cell inhibitory receptors.

    PubMed

    Hayami, K; Fukuta, D; Nishikawa, Y; Yamashita, Y; Inui, M; Ohyama, Y; Hikida, M; Ohmori, H; Takai, T

    1997-03-14

    We have isolated a cDNA clone encoding a novel murine cell-surface glycoprotein. This polypeptide is predicted to be composed of a signal peptide of 23 amino acids, an extracellular region of 620 amino acids that contains six immunoglobulin-like domains with five potential N-glycosylation sites, a transmembrane sequence of 20 amino acids, and a cytoplasmic tail of 178 amino acids with four sets of sequences similar to the immunoreceptor tyrosine-based inhibition motif. The relative molecular mass of the mature polypeptide is calculated to be 90,520 Da. The polypeptide, designated as p91, shows striking homologies to human killer cell inhibitory receptors, a murine gp49B1 protein, a bovine Fcgamma2 receptor, and a human Fcalpha receptor. The mRNA of p91 was especially abundant in murine macrophages. Western blot analysis using p91-specific anti-peptide sera detected a 130-kDa polypeptide in macrophages. Surface biotinylation and immunoprecipitation analysis verified the surface expression of the translation products on COS-1 cells transfected with the p91 cDNA, but the cells failed to show any Fc binding activity. PMID:9054430

  18. Heterogeneity of Functional Properties of Clone 66 Murine Breast Cancer Cells Expressing Various Stem Cell Phenotypes

    PubMed Central

    Mukhopadhyay, Partha; Farrell, Tracy; Sharma, Gayatri; McGuire, Timothy R.; O’Kane, Barbara; Sharp, J. Graham

    2013-01-01

    Introduction Breast cancer grows, metastasizes and relapses from rare, therapy resistant cells with a stem cell phenotype (cancer stem cells/CSCs). However, there is a lack of studies comparing the functions of CSCs isolated using different phenotypes in order to determine if CSCs are homogeneous or heterogeneous. Methods Cells with various stem cell phenotypes were isolated by sorting from Clone 66 murine breast cancer cells that grow orthotopically in immune intact syngeneic mice. These populations were compared by in vitro functional assays for proliferation, growth, sphere and colony formation; and in vivo limiting dilution analysis of tumorigenesis. Results The proportion of cells expressing CD44highCD24low/neg, side population (SP) cells, ALDH1+, CD49fhigh, CD133high, and CD34high differed, suggesting heterogeneity. Differences in frequency and size of tumor spheres from these populations were observed. Higher rates of proliferation of non-SP, ALDH1+, CD34low, and CD49fhigh suggested properties of transit amplifying cells. Colony formation was higher from ALDH1? and non-SP cells than ALDH1+ and SP cells suggesting a progenitor phenotype. The frequency of clonal colonies that grew in agar varied and was differentially altered by the presence of Matrigel™. In vivo, fewer cells with a stem cell phenotype were needed for tumor formation than “non-stem” cells. Fewer SP cells were needed to form tumors than ALDH1+ cells suggesting further heterogeneities of cells with stem phenotypes. Different levels of cytokines/chemokines were produced by Clone 66 with RANTES being the highest. Whether the heterogeneity reflects soluble factor production remains to be determined. Conclusions These data demonstrate that Clone 66 murine breast cancer cells that express stem cell phenotypes are heterogeneous and exhibit different functional properties, and this may also be the case for human breast cancer stem cells. PMID:24265713

  19. Anti-leukemic properties of deferasirox via apoptosis in murine leukemia cell lines

    PubMed Central

    Jeon, Sol-Rim; Lee, Jae-Wook; Jang, Pil-Sang; Cho, Bin; Jeong, Dae-Chul

    2015-01-01

    Background Although deferasirox (DFX) is reported to have anti-tumor effects, its anti-leukemic activity remains unclear. We evaluated the effect of DFX treatment on two murine lymphoid leukemia cell lines, and clarified the mechanisms underlying its potential anti-leukemic activity. Methods L1210 and A20 murine lymphoid leukemia cell lines were treated with DFX. Cell viability and apoptosis were evaluated by the 3-(4,5-dimethylthaizol-2-yl)-5-(3-carboxymethylphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay and fluorescence-activated cell sorting (FACS) analysis, respectively. Immunoblotting was performed to detect the expression of key apoptotic proteins. Results In dose- and time-dependent manner, DFX decreased viability and increased apoptosis of murine leukemic cells. Fas expression was significantly higher in A20 cells than in L1210 cells at all DFX concentrations tested. Although both cell lines exhibited high caspase 3 and caspase 9 expression, a critical component of the intrinsic mitochondrial apoptotic pathway, expression was greater in L1210 cells. In contrast, caspase 8, a key factor in the extrinsic apoptotic pathway, showed greater expression in A20 cells. Cytochrome c expression was significantly higher in L1210 cells. In both cell lines, co-treatment with ferric chloride and DFX diminished the expression of these intracellular proteins, as compared to DFX treatment alone. Conclusion Treatment with DFX increased caspase-dependent apoptosis in two murine lymphoid leukemia cell lines, with differing apoptotic mechanisms in each cell line.

  20. Generation and characterization of regulatory dendritic cells derived from murine induced pluripotent stem cells

    PubMed Central

    Zhang, Qi; Fujino, Masayuki; Iwasaki, Shizue; Hirano, Hiroshi; Cai, Songjie; Kitajima, Yuya; Xu, Jinhua; Li, Xiao-Kang

    2014-01-01

    Regulatory dendritic cells (DCregs) represent a potential therapeutic tool for assessing a variety of immune overreaction conditions; however, current approaches for generating DCregs for therapeutic purposes are limited. We attempted to generate and characterize DCregs from murine induced pluripotent stem (iPS) cells. The iPS cells co-cultured with OP9 cells displayed mesodermally differentiated flat colonies. GM-CSF drove most of the colonies exhibiting a differentiated morphology. Thereafter, cells became morphologically heterologous under the effects of TGF-? and IL-10. Most of the floating cells developed an irregular shape with areas of protrusion. The generated iPS-DCregs demonstrated high CD11b/c and low CD40, CD80, CD86 and MHC-II expressions with a high antigen uptake ability and poor T-cell stimulatory function. Importantly, iPS-DCregs showed immune responsiveness regulation effects both in vitro and in vivo and the ability to generate regulatory T-cells in vitro. Our result illustrates a feasible approach for generating functional DCregs from murine iPS cells. PMID:24496181

  1. Alternariol induces abnormal nuclear morphology and cell cycle arrest in murine RAW 264.7 macrophages.

    E-print Network

    Boyer, Edmond

    1 Alternariol induces abnormal nuclear morphology and cell cycle arrest in murine RAW 264: AOH induces abnormal nuclear morphology and cell cycle arrest. inserm-00871659,version1-11Apr2014, is known to induce DNA damage with subsequent cell cycle arrest. Here we elucidated the effects of AOH

  2. Skin-Resident Murine Dendritic Cell Subsets Promote Distinct and Opposing Antigen-Specific

    E-print Network

    Immunity Article Skin-Resident Murine Dendritic Cell Subsets Promote Distinct and Opposing Antigen: dankaplan@umn.edu DOI 10.1016/j.immuni.2011.06.005 SUMMARY Skin-resident dendritic cells (DCs) are well. There are at least three subsets of skin DC-- Langer- hans cells (LC), Langerin+ dermal DCs (dDCs), and classic d

  3. Follicle-stimulating hormone receptor-mediated uptake of sup 45 Ca sup 2+ by proteoliposomes and cultured rat sertoli cells: Evidence for involvement of voltage-activated and voltage-independent calcium channels

    SciTech Connect

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

    1989-12-01

    We have previously reported incorporation into liposomes of Triton X-100-solubilized FSH receptor-G-protein complexes derived from purified bovine calf testis membranes. In the present study we have used this model system to show that FSH induces flux of 45Ca2+ into such proteoliposomes in a hormone-specific concentration-dependent manner. FSH, inactivated by boiling, had no stimulatory effect on 45Ca2+ flux, nor did isolated alpha- or beta-subunits of FSH. Addition of GTP (or its analogs 5'-guanylylimidodiphosphate and guanosine-5'-O-(3-thiotriphosphate)) or sodium fluoride (in the presence or absence of GTP or its analogs) failed to induce 45Ca2+ flux into proteoliposomes, suggesting that the uptake of 45Ca2+ was receptor, and not G-protein, related. Voltage-independent (ruthenium red and gadolinium chloride) and voltage-activated (methyoxyverapamil and nifedipine) calcium channel-blocking agents reduced FSH-stimulated 45Ca2+ flux into proteoliposomes to control levels. FSH also induced uptake of 45Ca2+ by cultured rat Sertoli cells. Ruthenium red and gadolinium chloride had no effect on basal levels of 45Ca2+ uptake or estradiol secretion by cultured rat Sertoli cells, nor did methoxyverapamil or nifedipine. All four calcium channel blockers, however, were able to reduce FSH-induced 45Ca2+ uptake to basal levels and FSH-stimulated conversion of androstenedione to estradiol by up to 50%, indicating an involvement of Ca2+ in FSH-stimulated steroidogenesis. Our results suggest that the well documented changes in intracellular calcium levels consequent to FSH binding may be due, at least in part, to an influx of calcium through FSH receptor-regulated calcium channels.

  4. 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

  5. Pseudolaric Acid B Induced Cell Cycle Arrest, Autophagy and Senescence in Murine Fibrosarcoma L929 Cell

    PubMed Central

    hua Yu, Jing; yu Liu, Chun; bin Zheng, Gui; Zhang, Li Ying; hui Yan, Ming; yan Zhang, Wen; ying Meng, Xian; fang Yu, Xiao

    2013-01-01

    Objective: PAB induced various cancer cell apoptosis, cell cycle arrest and senescence. But in cell line murine fibrosarcoma L929, PAB did not induce apoptosis, but autophagy, therefore it was thought by us as a good model to research the relationship of cell cycle arrest, autophagy and senescence bypass apoptosis. Methods: Inhibitory ratio was assessed by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) analysis. Phase contrast microscopy visualized cell morphology. Hoechst 33258 staining for nuclear change, propidium iodode (PI) staining for cell cycle, monodansylcadaverine (MDC) staining for autophagy, and rodanmine 123 staining for mitochondrial membrane potential (MMP) were measured by fluorescence microscopy or flowcytometry. Apoptosis was determined by DNA ladder test. Protein kinase C (PKC) activity was detected by PKC assay kit. SA-?-galactosidase assay was used to detect senescence. Protein expression was examined by western blot. Results: PAB inhibited L929 cell growth in time-and dose-dependent manner. At 12 h, 80 ?mol/L PAB induced obvious mitotic arrest; at 24 h, PAB began to induce autophagy; at 36 h, cell-treated with PAB slip into G1 cell cycle; and 3 d PAB induced senescence. In time sequence PAB induced firstly cell cycle arrest, then autophagy, then slippage into G1 phase, lastly senescence. Senescent cells had high level of autophagy, inhibiting autophagy led to apoptosis, and no senescence. PAB activated PKC activity to induce cell cycle arrest, autophagy and senescence, inhibiting PKC activity suppressed cell cycle arrest, autophagy and senescence. Conclusion: PAB induced cell cycle arrest, autophagy and senescence in murine fibrosarcoma L929 cell through PKC. PMID:23630435

  6. Plasmacytoid dendritic cells promote rotavirus-induced human and murine B cell responses

    PubMed Central

    Deal, Emily M.; Lahl, Katharina; Narváez, Carlos F.; Butcher, Eugene C.; Greenberg, Harry B.

    2013-01-01

    B cell–dependent immunity to rotavirus, an important intestinal pathogen, plays a significant role in viral clearance and protects against reinfection. Human in vitro and murine in vivo models of rotavirus infection were used to delineate the role of primary plasmacytoid DCs (pDCs) in initiating B cell responses. Human pDCs were necessary and sufficient for B cell activation induced by rotavirus. Type I IFN recognition by B cells was essential for rotavirus-mediated B cell activation in vitro and murine pDCs and IFN-?/?–mediated B cell activation after in vivo intestinal rotavirus infection. Furthermore, rotavirus-specific serum and mucosal antibody responses were defective in mice lacking functional pDCs at the time of infection. These data demonstrate that optimal B cell activation and virus-specific antibody secretion following mucosal infection were a direct result of pDC-derived type I IFN. Importantly, viral shedding significantly increased in pDC-deficient mice, suggesting that pDC-dependent antibody production influences viral clearance. Thus, mucosal pDCs critically influence the course of rotavirus infection through rotavirus recognition and subsequent IFN production and display powerful adjuvant properties to initiate and enhance humoral immunity. PMID:23635775

  7. Comparative Study of Cytotoxicity, Tumor Necrosis Factor, and Prostaglandin Release After Stimulation of Rat Kupffer Cells, Murine Kupffer Cells, and Murine Inflammatory Liver Macrophages

    Microsoft Academic Search

    Thomas Decker; Marie-Luise Lohmann-Matthes; UIrich Karck; Thomas Peters; Karl Decker

    Resident murine liver macrophages had no natural cytotoxicity for the TNF-resistant target cell line P815. Activation of these cells was only obtained by a combination of IFNy and LPS. Inflammatory murlne macrophages were in a primed stage and could be actIvated by LPS alone in the absence of lFNy. Rat resident macrophages resembled functionally the Inflammatory macrophages of the mouse

  8. Hepatic Differentiation from Murine and Human iPS Cells Using Nanofiber Scaffolds.

    PubMed

    Yamazoe, Taiji; Shiraki, Nobuaki; Kume, Shoen

    2014-11-20

    The induced pluripotent stem (iPS) cells of murine and human are capable to differentiate into any cell type of the body through recapitulating normal development, similarly as the embryonic stem (ES) cells. Lines of evidence support that both ES cells and iPS cells are induced to differentiate in vitro by sequential treatment of humoral cues such as growth factors and chemicals, combined with the use of certain microenvironments including extracellular matrices and scaffolds.Here, we describe the procedure to potentiate hepatic lineage cells differentiation from murine and human iPS cells, using growth factor cocktails and nanofiber scaffolds. Nanofiber scaffolds have a three-dimensional surface mimicking the fine structures of the basement membrane in vivo, allow the iPS cells to differentiate into the definitive endoderm and mature hepatocyte-like cells more efficiently than the two-dimensional conventional culture plates. PMID:25410288

  9. Lung Epithelial Cells Are a Major Site of Murine Gammaherpesvirus Persistence

    Microsoft Academic Search

    James P. Stewart; Edward J. Usherwood; Alan Ross; Heather Dyson; Tony Nash

    2010-01-01

    Summary It is currently believed that latently infected, resting B lymphocytes are central to gammaher- pesvirus persistence, whereas mucosal epithelial cells are considered nonessential. We have re- addressed the question of nonlymphoid persistence using murine gammaherpesvirus 68 (MHV- 68). To dissect lymphoid from nonlymphoid persistence, we used m MT transgenic mice that are defective in B cells. MHV-68 DNA persisted

  10. Expression of Adrenergic and Cholinergic Receptors in Murine Renal Intercalated Cells

    PubMed Central

    JUN, Jin-Gon; MAEDA, Seishi; KUWAHARA-OTANI, Sachi; TANAKA, Koichi; HAYAKAWA, Tetsu; SEKI, Makoto

    2014-01-01

    ABSTRACT Neurons influence renal function and help to regulate fluid homeostasis, blood pressure and ion excretion. Intercalated cells (ICCs) are distributed throughout the renal collecting ducts and help regulate acid/base equilibration. Because ICCs are located among principal cells, it has been difficult to determine the effects that efferent nerve fibers have on this cell population. In this study, we examined the expression of neurotransmitter receptors on the murine renal epithelial M-1 cell line. We found that M-1 cells express a2 and b2 adrenergic receptor mRNA and the b2 receptor protein. Further, b2 receptor-positive cells in the murine cortical collecting ducts also express AQP6, indicating that these cells are ICCs. M-1 cells were found to express m1, m4 and m5 muscarinic receptor mRNAs and the m1 receptor protein. Cells in the collecting ducts also express the m1 receptor protein, and some m1-positive cells express AQP6. Acetylcholinesterase was detected in cortical collecting duct cells. Interestingly, acetylcholinesterase-positive cells neighbored AQP6-positive cells, suggesting that principal cells may regulate the availability of acetylcholine. In conclusion, our data suggest that ICCs in murine renal collecting ducts may be regulated by the adrenergic and cholinergic systems. PMID:25069412

  11. Efficient marking of neural stem cell-derived neurons with a modified murine embryonic stem cell virus, MESV2

    Microsoft Academic Search

    GC Owens; S Mistry; GM Edelman; KL Crossin

    2002-01-01

    Treatments for nervous system disorders that involve transplanting genetically modified neural stem cells may ultimately be feasible. As a step towards this therapeutic approach, a novel murine embryonic stem cell gammaretroviral vector was developed with features designed to optimize transgene expression in neural stem cells and to increase vector safety. All potential start sites of translation in the 5’ leader

  12. Host Cell Cathepsins Potentiate Moloney Murine Leukemia Virus Infection

    Microsoft Academic Search

    Pankaj Kumar; Deepa Nachagari; Carolyn Fields; John Franks; Lorraine M. Albritton

    2007-01-01

    The roles of cellular proteases in Moloney murine leukemia virus (MLV) infection were investigated using MLV particles pseudotyped with vesicular stomatitis virus (VSV) G glycoprotein as a control for effects on core MLV particles versus effects specific to Moloney MLV envelope protein (Env). The broad-spectrum inhibitors cathepsin inhibitor III and E-64d gave comparable dose-dependent inhibition of Moloney MLV Env and

  13. Identification of a murine ICAM-1-specific peptide by subtractive phage library selection on cells

    Microsoft Academic Search

    Anna-Karine Bélizaire; Lioudmila Tchistiakova; Yves St-Pierre; Valery Alakhov

    2003-01-01

    The ICAM-1 adhesion molecule is expressed selectively at low levels on endothelial cells but is strongly upregulated in dysfunctional endothelial cells associated with inflammation, cancer, and atherogenesis. Using COS-7 cells transfected with murine ICAM-1 (mICAM-1) as a target receptor, a phage display library was screened. Clones were selected by elution with a mAb specific for a functional epitope of ICAM-1

  14. REGULATION OF GROWTH-RELATED GENES BY INTERLEUKIN6 IN MURINE MYELOMA CELLS

    Microsoft Academic Search

    Serge Côté; Carl Simard; Réal Lemieux

    2002-01-01

    Interleukin-6 (IL-6), a pleiotropic cytokine with effects on several hematopoietic and other normal cells, is also important for the growth and survival of tumor cells such as murine plasmacytomas and human myelomas. Exploiting the 11A3 hybridoma cell line for its IL-6 requirement to proliferate in vitro, we used subtractive suppression hybridization (SSH) to identify genes whose expression is stimulated and\\/or

  15. A transformed murine Leydig cell line expresses the ET A receptor subtype

    Microsoft Academic Search

    Adviye Ergul; Marilyn K Glassberg; Marc E Freeman; David Puett

    1994-01-01

    We recently demonstrated that transformed murine Leydig cells (MA-10) responded to endothelin-1 (ET-1) via increased steroidogenesis. This study addresses the endothelin receptor subtype present on this cell line and whether or not the cells produce ET-1. The expression of the preproendothelin-1 (PPET-1) gene was investigated by Northern blot analysis, and PPET-1 mRNA was found to be 125I]ET-1 and unlabeled ET-1,

  16. 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

  17. IL-10 from Regulatory T Cells Determines Vaccine Efficacy in Murine Leishmania major Infection1

    E-print Network

    Arnold, Jonathan

    IL-10 from Regulatory T Cells Determines Vaccine Efficacy in Murine Leishmania major Infection1 * Leishmaniasis affects 12 million people, but there are no vaccines. Immunological correlates of vaccine efficacy in balance from IL-4 to IFN- was the key to vaccine success. Recently, a role for IL-10 and regulatory

  18. COMPARATIVE TOXICITY OF DIFFERENT EMISSION PARTICLES IN MURINE PULMONARY EPITHELIAL CELLS AND MACROPHAGES

    EPA Science Inventory

    Comparative Toxicity of Different Emission Particles in Murine Pulmonary Epithelial Cells and Macrophages. T Stevens1, M Daniels2, P Singh2, M I Gilmour2. 1 UNC, Chapel Hill 27599 2Experimental Toxicology Division, NHEERL, RTP, NC 27711 Epidemiological studies have shown ...

  19. 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,

  20. TGF-? Induces Surface LAP Expression on Murine CD4 T Cells Independent of Foxp3 Induction

    PubMed Central

    Oida, Takatoku; Weiner, Howard L.

    2010-01-01

    Background It has been reported that human FOXP3+ CD4 Tregs express GARP-anchored surface latency-associated peptide (LAP) after activation, based on the use of an anti-human LAP mAb. Murine CD4 Foxp3+ Tregs have also been reported to express surface LAP, but these studies have been hampered by the lack of suitable anti-mouse LAP mAbs. Methodology/Principal Findings We generated anti-mouse LAP mAbs by immunizing TGF-??/? animals with a mouse Tgfb1-transduced P3U1 cell line. Using these antibodies, we demonstrated that murine Foxp3+ CD4 Tregs express LAP on their surface. In addition, retroviral transduction of Foxp3 into mouse CD4+CD25? T cells induced surface LAP expression. We then examined surface LAP expression after treating CD4+CD25? T cells with TGF-? and found that TGF-? induced surface LAP not only on T cells that became Foxp3+ but also on T cells that remained Foxp3? after TGF-? treatment. GARP expression correlated with the surface LAP expression, suggesting that surface LAP is GARP-anchored also in murine T cells. Conclusions/Significance Unlike human CD4 T cells, surface LAP expression on mouse CD4 T cells is controlled by Foxp3 and TGF-?. Our newly described anti-mouse LAP mAbs will provide a useful tool for the investigation and functional analysis of T cells that express LAP on their surface. PMID:21124798

  1. 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

  2. Cell differentiation mediated by co-culture of human umbilical cord blood stem cells with murine hepatic cells.

    PubMed

    Stecklum, Maria; Wulf-Goldenberg, Annika; Purfürst, Bettina; Siegert, Antje; Keil, Marlen; Eckert, Klaus; Fichtner, Iduna

    2015-02-01

    In the present study, purified human cord blood stem cells were co-cultivated with murine hepatic alpha mouse liver 12 (AML12) cells to compare the effect on endodermal stem cell differentiation by either direct cell-cell interaction or by soluble factors in conditioned hepatic cell medium. With that approach, we want to mimic in vitro the situation of preclinical transplantation experiments using human cells in mice. Cord blood stem cells, cultivated with hepatic conditioned medium, showed a low endodermal differentiation but an increased connexin 32 (Cx32) and Cx43, and cytokeratin 8 (CK8) and CK19 expression was monitored by reverse transcription polymerase chain reaction (RT-PCR). Microarray profiling indicated that in cultivated cord blood cells, 604 genes were upregulated 2-fold, with the highest expression for epithelial CK19 and epithelial cadherin (E-cadherin). On ultrastructural level, there were no major changes in the cellular morphology, except a higher presence of phago(ly)some-like structures observed. Direct co-culture of AML12 cells with cord blood cells led to less incisive differentiation with increased sex-determining region Y-box 17 (SOX17), Cx32 and Cx43, as well as epithelial CK8 and CK19 expressions. On ultrastructural level, tight cell contacts along the plasma membranes were revealed. FACS analysis in co-cultivated cells quantified dye exchange on low level, as also proved by time relapse video-imaging of labelled cells. Modulators of gap junction formation influenced dye transfer between the co-cultured cells, whereby retinoic acid increased and 3-heptanol reduced the dye transfer. The study indicated that the cell-co-cultured model of human umbilical cord blood cells and murine AML12 cells may be a suitable approach to study some aspects of endodermal/hepatic cell differentiation induction. PMID:25270685

  3. Membrane transport changes in an adriamycin-resistant murine leukemia cell line and in its sensitive parental cell line

    Microsoft Academic Search

    Ratna Bose; Hing-Yat Peter Lain

    1988-01-01

    Multidrug resistance in cancer chemotherapy occurs when cells develop resistance towards structurally and functionally unrelated drugs. It is speculated that alteration of some fundamental process(es) in the cells leads to the development of multidrug resistance. The sodium pump activity of murine leukemia cell lines P388\\/S (sensitive) and P388\\/ADR (resistant) was measured and found to be different in the two cell

  4. Qa-2–Dependent Selection of Cd8?/? T Cell Receptor ?/?+ Cells in Murine Intestinal Intraepithelial Lymphocytes

    PubMed Central

    Das, Gobardhan; Gould, Dina S.; Augustine, Mathew M.; Fragoso, Gladis; Scitto, Edda; Stroynowski, Iwona; Van Kaer, Luc; Schust, Danny J.; Ploegh, Hidde; Janeway, Charles A.

    2000-01-01

    Murine intestinal intraepithelial lymphocytes (iIELs) are made up of a heterogeneous mix of T cells with unique phenotypes. Whereas CD8+ T cells in peripheral lymphoid organs use CD8?/? and are selected on MHC class Ia molecules, a majority of iIELs use CD8?/?. Here, we report that the presence of CD8?/? TCR-?/? cells in iIELs is independent of classical MHC class I molecules Kb and Db, as illustrated by their presence in Kb/Db double-knockout mice and in mice lacking a nonclassical MHC class I molecule, CD1d. Most strikingly, their presence is decreased by ?70% in mice lacking transporter associated with antigen processing (TAP). The TAP-dependent nonclassical MHC class I molecule Qa-2 is strongly implicated in the presence of these cells, as inferred from the low numbers of CD8?/? TCR-?/? T cells in mice deficient in Qa-2 genes. Second, a Qa-2–transgenic mouse made in a Qa-2? strain showed an increase in the numbers of CD8?/? cells among its iIELs. Thus, the presence of CD8?/? TCR-?/? cells in iIELs is mainly dependent on the nonclassical MHC class I molecule Qa-2. PMID:11085754

  5. Murine and Human Myogenic Cells Identified by Elevated Aldehyde Dehydrogenase Activity: Implications for Muscle Regeneration and Repair

    Microsoft Academic Search

    Joseph B. Vella; Seth D. Thompson; Mark J. Bucsek; Minjung Song; Johnny Huard

    2011-01-01

    BackgroundDespite the initial promise of myoblast transfer therapy to restore dystrophin in Duchenne muscular dystrophy patients, clinical efficacy has been limited, primarily by poor cell survival post-transplantation. Murine muscle derived stem cells (MDSCs) isolated from slowly adhering cells (SACs) via the preplate technique, induce greater muscle regeneration than murine myoblasts, primarily due to improved post-transplantation survival, which is conferred by

  6. 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)

  7. Efficient genetic modification of murine dendritic cells by electroporation with mRNA

    Microsoft Academic Search

    Sonja Van Meirvenne; Lieven Straetman; Carlo Heirman; Melissa Dullaers; Catherine De Greef; Viggo Van Tendeloo; Kris Thielemans

    2002-01-01

    Recently, human dendritic cells (DCs) pulsed with mRNA encoding a broad range of tumor antigens have proven to be potent activators of a primary anti–tumor-specific T-cell response in vitro. The aim of this study was to improve the mRNA pulsing of murine DC. Compared to a standard lipofection protocol and passive pulsing, electroporation was, in our hands, the most efficient

  8. Is trisomy cause or consequence of murine T cell leukemia development? Studies on Robertsonian translocation mice.

    PubMed Central

    Spira, J; Wiener, F; Ohno, S; Klein, G

    1979-01-01

    Trypsin-Giemsa banding studies on T cell leukemias induced in Robertsonian translocation mice by dimethylbenz[a]anthracene and Moloney leukemia virus show a trisomy of chromosome 15 even in cases in which chromosome 15 has undergone centromeric fusion with chromosomes 1, 5, or 6. These results suggest that the duplication of gene(s) located on chromosome 15 is of critical importance for murine T cell leukemia development. Images PMID:316545

  9. Cytokine-Based Log-Scale Expansion of Functional Murine Dendritic Cells

    Microsoft Academic Search

    Yui Harada; Yasuji Ueda; Hiroaki Kinoh; Atsushi Komaru; Terumi Fuji-Ogawa; Aki Furuya; Akihiro Iida; Mamoru Hasegawa; Tomohiko Ichikawa; Yoshikazu Yonemitsu

    2009-01-01

    Background: Limitations of the clinical efficacy of dendritic cell (DC)-based immunotherapy, as well as difficulties in their industrial production, are largely related to the limited number of autologous DCs from each patient. We here established a possible breakthrough, a simple and cytokine-based culture method to realize a log-scale order of functional murine DCs (.1,000-fold), which cells were used as a

  10. Primate Gammaretroviruses Require an Ancillary Factor Not Required for Murine Gammaretroviruses To Infect BHK Cells?

    PubMed Central

    Xu, Wenqin; Eiden, Maribeth V.

    2011-01-01

    BHK cells remain resistant to xenotropic murine retrovirus-related virus (XMRV) or gibbon ape leukemia virus (GALV) infection, even when their respective receptors, Xpr1 or PiT1, are expressed. We set out to determine the stage at which viral infection is blocked and whether this block is mediated by a dominant-negative factor or the absence of a requisite ancillary factor. BHK cells bind neither XMRV nor GALV envelope proteins. BHK cells expressing the appropriate receptors bind XMRV or GALV envelope proteins. BHK cells can be infected by NZB-XMV(New Zealand Black mouse xenotropic murine virus)-enveloped vectors, expressing an envelope derived from a xenotropic retrovirus that, like XMRV, employs Xpr1 as a receptor, and also by vectors bearing the envelope of 10A1 murine leukemia virus (MLV), a murine retrovirus that can use PiT1 as a receptor. The retroviral vectors used in these analyses differ solely in their viral envelope proteins, suggesting that the block to XMRV and GALV infection is mediated at the level of envelope-receptor interactions. N-linked glycosylation of the receptors was not found to mediate resistance of receptor-expressing BHK cells to GALV or XMRV, as shown by tunicamycin treatment and mutation of the specific glycosylation site of the PiT1 receptor. Hybrid cells produced by fusing BHKXpr1 or BHKPiT1 to XMRV- or GALV-resistant cells, respectively, can mediate efficient XMRV or GALV infection. These findings indicate that BHK cells lack a factor that is required for infection by primate xenotropic viruses. This factor is not required for viruses that use the same receptors but were directly isolated from mice. PMID:21270153

  11. Development of Resting Membrane Potentials of Embryonic Murine Spinal Cord Cells Evaluated by Flow Cytometric Analysis

    Microsoft Academic Search

    C. Krieger; E. Puil; S. U. Kim

    1991-01-01

    The membrane electrical properties of embryonic murine spinal cord cells of embryonic day-10 (E10) to E14 were studied using a voltage-sensitive oxonol dye combined with fluorescence-activated cell sorting techniques. This period of development corresponds to the time when neurons differentiate from their neuroblast precursors. Bovine oligodendrocytes were also investigated in these studies as they have an exclusive K+ dependence on

  12. Differential gene expression in murine large cell B-cell lymphoma metastatic variants.

    PubMed

    Joshi, Shantaram S; Mittal, Amit K; Wang, Peng; Joshi, Avadhut D; Vu, Eileen; Wang, Xioujun

    2008-09-01

    Previous studies from this laboratory have characterized RAW117-P murine large cell B-cell lymphoma and its in vivo selected highly malignant and liver metastatic RAW117-H10 subline for their biological and biochemical properties. In this study, to understand the molecular basis of low and high metastatic behavior of these variant sublines, we have investigated the molecular phenotypes of these cells using differential display techniques and cDNA array analysis. Differential display analysis indicated a significant difference in expression of several genes between these two metastatic variant lymphoma cells. Further analyses of these cells using microarray showed an increased expression of several genes including uPAR1, CRE-BP1, Chop-10, IGF, insulin-like growth factor-IA, STAT6, Cyclin-D1, Cyclin-E, ERBB-3, Alpha NGF, Kruppel-like factor LKLF, (P)19INK4 in metastatic RAW117-H10 cells compared to parental RAW117-P cells. On the other hand, MIP1beta, CD14 antigen, Cathepsin B and MOD are expressed more in RAW117-P cells compared to RAW117-H10 cells. Differential expression of the selected genes was confirmed using semiquantitative RT-PCR techniques. The combination of plasminogen activator and its receptor and IGF-like growth factors, cell cycle regulatory molecules and transcription factors might provide an ideal environment for RAW117-H10 cells to metastasize to distant organs and colonize. Thus these results identify certain differentially expressed genes that are involved in the metastatic properties of these lymphoma cells and lay foundation for further in depth analyses to use this information to develop therapy for metastatic lymphoma. PMID:18602072

  13. 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.

  14. Involvement of a chromatin modifier in response to mono-(2-ethylhexyl) phthalate (MEHP)-induced Sertoli cell injury: Probably an indirect action via the regulation of NF?B/FasL circuitry

    SciTech Connect

    Chen, Shiwei [Department of Urology, 174th Hospital of PLA, Fujian 361001 (China)] [Department of Urology, 174th Hospital of PLA, Fujian 361001 (China); Dong, Yushu [Department of Neurosurgery, 463rd Hospital of PLA, Shenyang 110042 (China)] [Department of Neurosurgery, 463rd Hospital of PLA, Shenyang 110042 (China); Xu, Chun; Jiang, Liming; Chen, Yongjie; Jiang, Cheng [Department of Urology, 174th Hospital of PLA, Fujian 361001 (China)] [Department of Urology, 174th Hospital of PLA, Fujian 361001 (China); Hou, Wugang, E-mail: gangwuhou@163.com [Department of Anesthesiology, Xijing Hospital, Fourth Military Medical University, Xi’an 710032 (China)] [Department of Anesthesiology, Xijing Hospital, Fourth Military Medical University, Xi’an 710032 (China); Li, Wei, E-mail: liweipepeyato@163.com [Department of Human Anatomy, Histology and Embryology, Fourth Military Medical University, Xi’an 710032 (China)] [Department of Human Anatomy, Histology and Embryology, Fourth Military Medical University, Xi’an 710032 (China)

    2013-11-01

    Highlights: •MTA1 expression is upregulated in SCs upon MEHP treatment. •Knockdown of MTA1 in SCs impairs the MEHP-induced NF?B signaling activation. •Knockdown of MTA1 inhibits recruitment of NF?B onto FasL promoter in MEHP-treated SCs. -- Abstract: The Fas/FasL signaling pathway, controlled by nuclear factor-?B (NF?B) at the transcriptional level, is critical for triggering germ cell apoptosis in response to mono-(2-ethylhexyl) phthalate (MEHP)-induced Sertoli cell (SC) injury, but the exact regulation mechanism remain unknown. Here, we discovered that expression level of Metastasis associated protein 1 (MTA1), a component of the Mi-2/nucleosome remodeling and deacetylase complex, was upregulated in SCs during the early recovery after MEHP exposure. This expression change was in line with the dynamic changes in germ cell apoptosis in response to MEHP treatment. Furthermore, a knockdown of MTA1 by RNAi in SCs was found to impair the MEHP-induced early activation of NF?B pathway and abolish the recruitment of NF?B onto FasL promoter, which consequently diminished the MEHP-triggered FasL induction. Considering that Fas/FasL is a well characterized apoptosis initiating signaling during SCs injury, our results point to a potential “switch on” effect of MTA1, which may govern the activation of NF?B/FasL cascade in MEHP-insulted SCs. Overall, the MTA1/NF?B/FasL circuit may serve as an important defensive/repairing mechanism to help to control the germ cell quality after SCs injury.

  15. Peptide nucleic acids targeting ?-globin mRNAs selectively inhibit hemoglobin production in murine erythroleukemia cells

    PubMed Central

    MONTAGNER, GIULIA; GEMMO, CHIARA; FABBRI, ENRICA; MANICARDI, ALEX; ACCARDO, IGEA; BIANCHI, NICOLETTA; FINOTTI, ALESSIA; BREVEGLIERI, GIULIA; SALVATORI, FRANCESCA; BORGATTI, MONICA; LAMPRONTI, ILARIA; BRESCIANI, ALBERTO; ALTAMURA, SERGIO; CORRADINI, ROBERTO; GAMBARI, ROBERTO

    2015-01-01

    In the treatment of hemoglobinopathies, amending altered hemoglobins and/or globins produced in excess is an important part of therapeutic strategies and the selective inhibition of globin production may be clinically beneficial. Therefore the development of drug-based methods for the selective inhibition of globin accumulation is required. In this study, we employed peptide nucleic acids (PNAs) to alter globin gene expression. The main conclusion of the present study was that PNAs designed to target adult murine ?-globin mRNA inhibit hemoglobin accumulation and erythroid differentiation of murine erythroleukemia (MEL) cells with high efficiency and fair selectivity. No major effects were observed on cell proliferation. Our study supports the concept that PNAs may be used to target mRNAs that, similar to globin mRNAs, are expressed at very high levels in differentiating erythroid cells. Our data suggest that PNAs inhibit the excess production of globins involved in the pathophysiology of hemoglobinopathies. PMID:25405921

  16. Identification of replication competent murine gammaretroviruses in commonly used prostate cancer cell lines.

    PubMed

    Sfanos, Karen Sandell; Aloia, Amanda L; Hicks, Jessica L; Esopi, David M; Steranka, Jared P; Shao, Wei; Sanchez-Martinez, Silvia; Yegnasubramanian, Srinivasan; Burns, Kathleen H; Rein, Alan; De Marzo, Angelo M

    2011-01-01

    A newly discovered gammaretrovirus, termed XMRV, was recently reported to be present in the prostate cancer cell line CWR22Rv1. Using a combination of both immunohistochemistry with broadly-reactive murine leukemia virus (MLV) anti-sera and PCR, we determined if additional prostate cancer or other cell lines contain XMRV or MLV-related viruses. Our study included a total of 72 cell lines, which included 58 of the 60 human cancer cell lines used in anticancer drug screens and maintained at the NCI-Frederick (NCI-60). We have identified gammaretroviruses in two additional prostate cancer cell lines: LAPC4 and VCaP, and show that these viruses are replication competent. Viral genome sequencing identified the virus in LAPC4 and VCaP as nearly identical to another known xenotropic MLV, Bxv-1. We also identified a gammaretrovirus in the non-small-cell lung carcinoma cell line EKVX. Prostate cancer cell lines appear to have a propensity for infection with murine gammaretroviruses, and we propose that this may be in part due to cell line establishment by xenograft passage in immunocompromised mice. It is unclear if infection with these viruses is necessary for cell line establishment, or what confounding role they may play in experiments performed with these commonly used lines. Importantly, our results suggest a need for regular screening of cancer cell lines for retroviral "contamination", much like routine mycoplasma testing. PMID:21698104

  17. Peptidoglycan Induces Necrosis and Regulates Cytokine Production in Murine Trophoblast Stem Cells

    PubMed Central

    Rose, Jennifer A.; Rabenold, Jessica J.; Parast, Mana M.; Milstone, David S.; Abrahams, Vikki M.; Riley, Joan K.

    2011-01-01

    Problem Intrauterine bacterial infection during pregnancy may lead to adverse outcome. The objective of this study was to assess whether peptidoglycan (PGN) derived from Gram-positive bacteria induces trophoblast stem (TS) cell death or alters TS cell cytokine production. Method of study TLR transcript expression was assessed by RT-PCR. Protein expression was determined by confocal microscopy or flow cytometry. 7-Aminoactinomycin D (7-AAD) staining was used to assess TS cell death. Morphological features of cell death were evaluated by transmission electron microscopy. The presence of cleaved caspase-3 and HMGB1 protein was examined by western blot. Cytokine levels in cell supernatants were determined using a mouse cytokine 23-plex panel. Results TLR2 and TLR4 protein was expressed from the 1-cell through the blastocyst stage of murine embryo development. Murine TS cells expressed TLR2 and TLR6 but not TLR1 or TLR4 RNA. Only TLR2 protein was detected at the plasma membrane of TS cells. PGN induced TS cell death by a caspase-3 independent mechanism. The cell death pathway induced by PGN was morphologically consistent with necrosis. Finally, PGN induced HMGB1 release and increased MIP-1? secretion while inhibiting the constitutive release of RANTES. Conclusion PGN-induced TS cell necrosis and the subsequent release of HMGB1 and MIP-1? may regulate an infection-induced inflammatory response at the maternal-fetal interface and thus may play a role in the pathogenesis of infection-associated pregnancy complications. PMID:21385270

  18. 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

  19. Potent Inhibition of Junín Virus Infection by Interferon in Murine Cells

    PubMed Central

    Huang, Cheng; Walker, Aida G.; Grant, Ashley M.; Kolokoltsova, Olga A.; Yun, Nadezhda E.; Seregin, Alexey V.; Paessler, Slobodan

    2014-01-01

    The new world arenavirus Junín virus (JUNV) is the causative agent of Argentine hemorrhagic fever, a lethal human infectious disease. Adult laboratory mice are generally resistant to peripheral infection by JUNV. The mechanism underlying the mouse resistance to JUNV infection is largely unknown. We have reported that interferon receptor knockout mice succumb to JUNV infection, indicating the critical role of interferon in restricting JUNV infection in mice. Here we report that the pathogenic and vaccine strains of JUNV were highly sensitive to interferon in murine primary cells. Treatment with low concentrations of interferon abrogated viral NP protein expression in murine cells. The replication of both JUNVs was enhanced in IRF3/IRF7 deficient cells. In addition, the vaccine strain of JUNV displayed impaired growth in primary murine cells. Our data suggested a direct and potent role of host interferon response in restricting JUNV replication in mice. The defect in viral growth for vaccine JUNV might also partially explain its attenuation in mice. PMID:24901990

  20. Identification of ganglio-N-tetraosylceramide as a new cell surface marker for murine natural killer (NK) cells.

    PubMed

    Young, W W; Hakomori, S I; Durdik, J M; Henney, C S

    1980-01-01

    The BCG-induced NK cell activity of murine peritoneal exudate cells was abolished by preincubation of effector cells with anti-ganglio-N-tetraosylceramide (anti-asialo GM1) and C but not with other anti-glycolipid antibodies, anti-ganglioside GM1, anti-globoside, and anti-ganglio-N-triosylceramide (anti-asialo GM2). In contrast, the cytotoxic activity of alloimmune T cells was not affected by treatment with anti-asialo GM1 antisera. These findings suggest that asialo GM1 display may be characteristic of NK cell populations and aid in the isolation of this population of cytotoxic cells. PMID:6985637

  1. 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

  2. RHOF PROMOTES MURINE MARGINAL ZONE B CELL DEVELOPMENT

    PubMed Central

    KISHIMOTO, MAYUKO; MATSUDA, TAKENORI; YANASE, SHOUGO; KATSUMI, AKIRA; SUZUKI, NOBUAKI; IKEJIRI, MAKOTO; TAKAGI, AKIRA; IKAWA, MASAHITO; KOJIMA, TETSUHITO; KUNISHIMA, SHINJI; KIYOI, HITOSHI; NAOE, TOMOKI; MATSUSHITA, TADASHI; MARUYAMA, MITSUO

    2014-01-01

    ABSTRACT RhoF is a member of the Rho GTPase family that has been implicated in various cell functions including long filopodia formation, adhesion, and migration of cells. Although RhoF is expressed in lymphoid tissues, the roles of RhoF in B cell development remain largely unclear. On the other hand, other members of the Rho GTPase family, such as Cdc42, RhoA, and Rac, have been intensively studied and are known to be required for B cell development in the bone marrow and spleen. We hypothesized that RhoF is also involved in B cell development. To examine our hypothesis, we analyzed B cell development in RhoF knockout (KO) mice and found a significant reduction in marginal zone (MZ) B cells in the spleen, although T cell development in the thymus and spleen was not affected. Consistent with these results, the width of the MZ B cell region in the spleen was significantly reduced in the RhoF KO mice. However, the antigen-specific antibody titer of IgM and IgG3 after MZ B cell-specific antigen (T cell-independent antigen, type I) stimulation was not affected by RhoF deletion. Furthermore, we demonstrated that RhoF was dispensable for stromal cell-derived factor-1?- and B lymphocyte chemoattractant-induced B cell migration. These results suggest that RhoF promotes MZ B cell development in the spleen. PMID:25741038

  3. Isolation and Characterization of Novel Murine Epiphysis Derived Mesenchymal Stem Cells

    Microsoft Academic Search

    Chun-Chun Cheng; Wei-Shiung Lian; Felix Shih-Hsiang Hsiao; I-Hsuan Liu; Shau-Ping Lin; Yen-Hua Lee; Chia-Chun Chang; Guan-Yu Xiao; Hsin-Yi Huang; Ching-Feng Cheng; Winston Teng-Kuei Cheng; Shinn-Chih Wu

    2012-01-01

    BackgroundWhile bone marrow (BM) is a rich source of mesenchymal stem cells (MSCs), previous studies have shown that MSCs derived from mouse BM (BMMSCs) were difficult to manipulate as compared to MSCs derived from other species. The objective of this study was to find an alternative murine MSCs source that could provide sufficient MSCs.Methodology\\/Principal FindingsIn this study, we described a

  4. 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

  5. ?? 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

  6. Murine CLCA5 is uniquely expressed in distinct niches of airway epithelial cells.

    PubMed

    Dietert, Kristina; Mundhenk, Lars; Erickson, Nancy A; Reppe, Katrin; Hocke, Andreas C; Kummer, Wolfgang; Witzenrath, Martin; Gruber, Achim D

    2015-03-01

    The murine mCLCA5 protein is a member of the chloride channel regulators, calcium-activated (CLCA) family and is suspected to play a role in airway mucus cell differentiation. Although mCLCA5 mRNA was previously found in total lung extracts, the expressing cells and functions in the naive murine respiratory tract are unknown. Therefore, mCLCA5 protein expression was identified by immunohistochemistry and confocal laser scanning microscopy using entire lung sections of naive mice. Moreover, we determined mRNA levels of functionally related genes (mClca3, mClca5, Muc5ac and Muc5b) and quantified mCLCA5-, mCLCA3- and CC10-positive cells and periodic acid-Schiff-positive mucus cells in naive, PBS-treated or Staphylococcus aureus-infected mice. We also investigated mCLCA5 protein expression in Streptococcus pneumoniae and influenza virus lung infection models. Finally, we determined species-specific differences in the expression patterns of the murine mCLCA5 and its human and porcine orthologs, hCLCA2 and pCLCA2. The mCLCA5 protein is uniquely expressed in highly select bronchial epithelial cells and submucosal glands in naive mice, consistent with anatomical locations of progenitor cell niches. Under conditions of challenge (PBS, S. aureus, S. pneumoniae, influenza virus), mRNA and protein expression strongly declined with protein recovery only in models retaining intact epithelial cells. In contrast to mice, human and porcine bronchial epithelial cells do not express their respective mCLCA5 orthologs and submucosal glands had fewer expressing cells, indicative of fundamental differences in mice versus humans and pigs. PMID:25212661

  7. No attenuation of the ATM-dependent DNA damage response in murine telomerase-deficient cells

    PubMed Central

    Erdmann, Natalie; Harrington, Lea A.

    2009-01-01

    Inactivation of mammalian telomerase leads to telomere attrition, eventually culminating in uncapped telomeres, which elicit a DNA damage response and cell cycle arrest or death. In some instances, telomerase modulation evokes a response not obviously attributable to changes in telomere length. One such example is the suppression of the DNA damage response (DDR) and changes in histone modification that occur upon repression of the telomerase reverse transcriptase, TERT, in human primary cells [1]. Here, we evaluate the contribution of TERT to the DDR in murine Tert?/? cells without critically shortened telomeres. We treated mTert?/? embryonic stem (ES) cells and murine embryonic fibroblasts (MEFs) with etoposide and irradiation, and assessed the status of p53pS15, 53BP1, ATMpS1981, SMC1pS957, and ?H2AX by indirect immunofluorescence or western blotting. In four independently derived mTert?/? ES cell lines, there was no significant difference in the induction of ?H2AX, 53BP1 foci formation, or the phosphorylation of ATM targets (ATM, SMC1, p53) between wildtype and mTert?/? ES cells and MEFs. A slight difference in post-translational modification of histones H3 and H4 was observed in a subset of mTert?/? ES cells, however this difference was reflected in the cellular levels of H3 and H4. Thus, in contrast to previous studies in human cells, the absence of Tert does not overtly affect the ATM-dependent response to DNA damage in murine cells. PMID:19071232

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

    PubMed Central

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

    2010-01-01

    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. 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

  10. CD166 regulates human and murine hematopoietic stem cells and the hematopoietic niche.

    PubMed

    Chitteti, Brahmananda Reddy; Kobayashi, Michihiro; Cheng, Yinghua; Zhang, Huajia; Poteat, Bradley A; Broxmeyer, Hal E; Pelus, Louis M; Hanenberg, Helmut; Zollman, Amy; Kamocka, Malgorzata M; Carlesso, Nadia; Cardoso, Angelo A; Kacena, Melissa A; Srour, Edward F

    2014-07-24

    We previously showed that immature CD166(+) osteoblasts (OB) promote hematopoietic stem cell (HSC) function. Here, we demonstrate that CD166 is a functional HSC marker that identifies both murine and human long-term repopulating cells. Both murine LSKCD48(-)CD166(+)CD150(+) and LSKCD48(-)CD166(+)CD150(+)CD9(+) cells, as well as human Lin(-)CD34(+)CD38(-)CD49f(+)CD166(+) cells sustained significantly higher levels of chimerism in primary and secondary recipients than CD166(-) cells. CD166(-/-) knockout (KO) LSK cells engrafted poorly in wild-type (WT) recipients and KO bone marrow cells failed to radioprotect lethally irradiated WT recipients. CD166(-/-) hosts supported short-term, but not long-term WT HSC engraftment, confirming that loss of CD166 is detrimental to the competence of the hematopoietic niche. CD166(-/-) mice were significantly more sensitive to hematopoietic stress. Marrow-homed transplanted WT hematopoietic cells lodged closer to the recipient endosteum than CD166(-/-) cells, suggesting that HSC-OB homophilic CD166 interactions are critical for HSC engraftment. STAT3 has 3 binding sites on the CD166 promoter and STAT3 inhibition reduced CD166 expression, suggesting that both CD166 and STAT3 may be functionally coupled and involved in HSC competence. These studies illustrate the significance of CD166 in the identification and engraftment of HSC and in HSC-niche interactions, and suggest that CD166 expression can be modulated to enhance HSC function. PMID:24740813

  11. Ex vivo imaging of T cells in murine lymph node slices with widefield and confocal microscopes.

    PubMed

    Salmon, Hélène; Rivas-Caicedo, Ana; Asperti-Boursin, François; Lebugle, Camille; Bourdoncle, Pierre; Donnadieu, Emmanuel

    2011-01-01

    Naïve T cells continuously traffic to secondary lymphoid organs, including peripheral lymph nodes, to detect rare expressed antigens. The migration of T cells into lymph nodes is a complex process which involves both cellular and chemical factors including chemokines. Recently, the use of two-photon microscopy has permitted to track T cells in intact lymph nodes and to derive some quantitative information on their behavior and their interactions with other cells. While there are obvious advantages to an in vivo system, this approach requires a complex and expensive instrumentation and provides limited access to the tissue. To analyze the behavior of T cells within murine lymph nodes, we have developed a slice assay, originally set up by neurobiologists and transposed recently to murine thymus. In this technique, fluorescently labeled T cells are plated on top of an acutely prepared lymph node slice. In this video-article, the localization and migration of T cells into the tissue are analyzed in real-time with a widefield and a confocal microscope. The technique which complements in vivo two-photon microscopy offers an effective approach to image T cells in their natural environment and to elucidate mechanisms underlying T cell migration. PMID:21775968

  12. Murine fertilized ovum, blastomere and morula cells lacking SP phenotype

    Microsoft Academic Search

    YiXin Xu; ZhiYing He; HaiYing Zhu; XueSong Chen; JianXiu Li; HongXia Zhang; XingHua Pan; YiPing Hu

    2007-01-01

    In the field of stem cell research, SP (side population) phenotype is used to define the property that cells maintain a high\\u000a efflux capability for some fluorescent dye, such as Hoechst 33342. Recently, many researches proposed that SP phenotype is\\u000a a phenotype shared by some stem cells and some progenitor cells, and that SP phenotype is regarded as a candidate

  13. Therapy of a fatal murine cytomegalovirus infection with thymic humoral factor (THF-gamma 2) treated immune spleen cells.

    PubMed Central

    Rager-Zisman, B; Zuckerman, F; Benharroch, D; Pecht, M; Burstein, Y; Trainin, N

    1990-01-01

    Infection of mice with murine cytomegalovirus (CMV) presents a model for the study of the role of the immune system in the pathogenesis of human CMV. We performed adoptive transfer experiments to evaluate the prospects for enhancing the anti-viral potential of murine CMV immune spleen cells by THF-gamma 2. Adult BALB/c mice resistant to murine CMV become highly susceptible following immunosuppression by cyclophosphamide. Recipient mice were injected with murine CMV and cyclophosphamide concomitantly, and 24 h later adoptive transfers of syngeneic immune spleen cells were performed. We showed that passive transfers of murine CMV immune spleen cells prevented the development of a fatal disease in 38% of the recipient mice. Daily injections of murine CMV immune donor mice with THF-gamma 2 enhanced considerably (93%) the therapeutic potential of virus-specific immune cells. These experiments provide direct evidence for the antiviral capacity of THF-gamma 2 through its immunomodulatory effect on immune T cells. PMID:2155728

  14. Liver Sinusoidal Endothelial Cells Are a Site of Murine Cytomegalovirus Latency and Reactivation?

    PubMed Central

    Seckert, Christof K.; Renzaho, Angélique; Tervo, Hanna-Mari; Krause, Claudia; Deegen, Petra; Kühnapfel, Birgit; Reddehase, Matthias J.; Grzimek, Natascha K. A.

    2009-01-01

    Latent cytomegalovirus (CMV) is frequently transmitted by organ transplantation, and its reactivation under conditions of immunosuppressive prophylaxis against graft rejection by host-versus-graft disease bears a risk of graft failure due to viral pathogenesis. CMV is the most common cause of infection following liver transplantation. Although hematopoietic cells of the myeloid lineage are a recognized source of latent CMV, the cellular sites of latency in the liver are not comprehensively typed. Here we have used the BALB/c mouse model of murine CMV infection to identify latently infected hepatic cell types. We performed sex-mismatched bone marrow transplantation with male donors and female recipients to generate latently infected sex chromosome chimeras, allowing us to distinguish between Y-chromosome (gene sry or tdy)-positive donor-derived hematopoietic descendants and Y-chromosome-negative cells of recipients' tissues. The viral genome was found to localize primarily to sry-negative CD11b? CD11c? CD31+ CD146+ cells lacking major histocompatibility complex class II antigen (MHC-II) but expressing murine L-SIGN. This cell surface phenotype is typical of liver sinusoidal endothelial cells (LSECs). Notably, sry-positive CD146+ cells were distinguished by the expression of MHC-II and did not harbor latent viral DNA. In this model, the frequency of latently infected cells was found to be 1 to 2 per 104 LSECs, with an average copy number of 9 (range, 4 to 17) viral genomes. Ex vivo-isolated, latently infected LSECs expressed the viral genes m123/ie1 and M122/ie3 but not M112-M113/e1, M55/gB, or M86/MCP. Importantly, in an LSEC transfer model, infectious virus reactivated from recipients' tissue explants with an incidence of one reactivation per 1,000 viral-genome-carrying LSECs. These findings identified LSECs as the main cellular site of murine CMV latency and reactivation in the liver. PMID:19535440

  15. 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

  16. Extracellular matrix promotes the growth and differentiation of murine hematopoietic cells in vitro.

    PubMed Central

    Campbell, A; Wicha, M S; Long, M

    1985-01-01

    We have developed a long-term culture system in which murine marrow cells are cultured on a complex extracellular matrix (ECM) that is derived from marrow and extracted with guanidine hydrochloride and dithiothreitol. Marrow cultures were established with fresh murine marrow cells and recharged at 2 wk (week 0). Phase microscopy showed a dramatically increased adherent cell layer development on ECM compared with controls within a week after recharge. By electron microscopy, this adherent layer was composed of numerous reticular cells apparently attached to the ECM which extended cytoplasmic projections to the surrounding hematopoietic cells. Adherent cellularity on ECM-coated dishes increased to 30 times the control values by week 2. Cumulative suspension cells on ECM dishes were eight times controls. ECM influenced both hematopoietic progenitor cell proliferation and differentiation. Adherent colony-forming unit-granulocyte/macrophage and colony-forming unit-megakaryocyte were greater than 30 and 15 times the control values, respectively, by week 2 (P less than or equal to 0.05). There were more mature granulocytic and megakaryocytic cells in ECM-coated dishes than in controls at all time points. This new culture system directly demonstrates that ECM is an important component of the hematopoietic microenvironment. Images PMID:4008654

  17. Cholinergic epithelial cell with chemosensory traits in murine thymic medulla.

    PubMed

    Panneck, Alexandra Regina; Rafiq, Amir; Schütz, Burkhard; Soultanova, Aichurek; Deckmann, Klaus; Chubanov, Vladimir; Gudermann, Thomas; Weihe, Eberhard; Krasteva-Christ, Gabriela; Grau, Veronika; del Rey, Adriana; Kummer, Wolfgang

    2014-12-01

    Specialized epithelial cells with a tuft of apical microvilli ("brush cells") sense luminal content and initiate protective reflexes in response to potentially harmful substances. They utilize the canonical taste transduction cascade to detect "bitter" substances such as bacterial quorum-sensing molecules. In the respiratory tract, most of these cells are cholinergic and are approached by cholinoceptive sensory nerve fibers. Utilizing two different reporter mouse strains for the expression of choline acetyltransferase (ChAT), we observed intense labeling of a subset of thymic medullary cells. ChAT expression was confirmed by in situ hybridization. These cells showed expression of villin, a brush cell marker protein, and ultrastructurally exhibited lateral microvilli. They did not express neuroendocrine (chromogranin A, PGP9.5) or thymocyte (CD3) markers but rather thymic epithelial (CK8, CK18) markers and were immunoreactive for components of the taste transduction cascade such as G?-gustducin, transient receptor potential melastatin-like subtype 5 channel (TRPM5), and phospholipase C?2. Reverse transcription and polymerase chain reaction confirmed the expression of G?-gustducin, TRPM5, and phospholipase C?2. Thymic "cholinergic chemosensory cells" were often in direct contact with medullary epithelial cells expressing the nicotinic acetylcholine receptor subunit ?3. These cells have recently been identified as terminally differentiated epithelial cells (Hassall's corpuscle-like structures in mice). Contacts with nerve fibers (identified by PGP9.5 and CGRP antibodies), however, were not observed. Our data identify, in the thymus, a previously unrecognized presumptive chemosensitive cell that probably utilizes acetylcholine for paracrine signaling. This cell might participate in intrathymic infection-sensing mechanisms. PMID:25300645

  18. Sertoli Cell-specific Expression of Metastasis-associated Protein 2 (MTA2) Is Required for Transcriptional Regulation of the Follicle-stimulating Hormone Receptor (FSHR) Gene during Spermatogenesis*

    PubMed Central

    Zhang, Shun; Li, Wei; Zhu, Chuchao; Wang, Xiaohong; Li, Zhen; Zhang, Jinshan; Zhao, Jie; Hu, Jing; Li, Teng; Zhang, Yuanqiang

    2012-01-01

    The effect of follicle-stimulating hormone (FSH) on spermatogenesis is modulated at a fundamental level by controlling the number of competent receptors present at the surface of Sertoli cells (SCs). One underlying mechanism is the down-regulation of the expression levels of the FSH receptor (FSHR) gene after exposure to FSH. Here we report that metastasis-associated protein 2 (MTA2), a component of histone deacetylase and nucleosome-remodeling complexes, as a gene product induced directly by testosterone or indirectly by FSH, is exclusively expressed in SCs. Stimulation of SCs with FSH is accompanied by up-regulation of MTA2 expression and enhancement of deacetylase activity. This effect requires the integrity of functional androgen receptor. Furthermore, MTA2 is a potent corepressor of FSHR transcription, because it can recruit histone deacetylase-1 onto the FSHR promoter and participates in the down-regulation of FSHR expression upon FSH treatment. Abolishment of endogenous MTA2 by siRNA treatment disrupted the desensitization of the FSH response and thereafter impaired the FSH-dependent secretory function of SCs. From a clinical standpoint, deregulated expression of MTA2 in SCs of human pathological testes negatively correlates to the deregulated level of serum FSH. Overall, our present results provide the first evidence that the FSH/androgen receptor/MTA2 cascade may serve as an indispensable negative feedback mechanism to modulate the transduction events of SCs in response to FSH. These data also underscore an unexpected reproductive facet of MTA2, which may operate as a novel integrator linking synergistic actions of FSH and androgen signaling in SCs. PMID:23086931

  19. Inactivation of the Moloney murine leukemia virus long terminal repeat in murine fibroblast cell lines is associated with methylation and dependent on its chromosomal position.

    PubMed Central

    Hoeben, R C; Migchielsen, A A; van der Jagt, R C; van Ormondt, H; van der Eb, A J

    1991-01-01

    The expression of a retroviral vector with the Moloney murine leukemia virus (Mo-MuLV) long terminal repeat (LTR) promoter after integration into the genome of murine fibroblast cell lines was monitored with the Escherichia coli-derived beta-galactosidase (beta-gal) gene as the reporter. Monoclonal cell lines derived after retroviral infection exhibited a marked heterogeneity in their expression of the reporter gene. We studied two monoclonal cell lines with a single unrearranged copy of the vector provirus integrated into their genome. The first, BB10, expressed the marker enzyme in only 8% of its cell population, whereas in the second, BB16, beta-gal expression could be detected in over 98% of the cells. Treatment of BB10 with the DNA-demethylating agent 5-azacytidine raised the number of beta-gal-positive cells to over 60%. Transfection experiments showed that the Mo-MuLV LTR promoter-enhancer is potentially fully functional in both the BB10 and BB16 cell lines. The inactivated provirus from BB10 cells was cloned and subsequently used to generate retrovirus stocks. The promoter-enhancer activity of its LTR after infection with these BB10-derived viruses showed a variation similar to that of the original virus stocks. Our data showed that (1) inactivation of the Mo-MuLV LTR is a frequent event in murine fibroblast cell lines, (2) inactivation is associated with de novo methylation of cytidine residues, (3) the frequency of inactivation of the provirus must be determined by its chromosomal position, (4) the process of methylation of sequences within the LTR is not necessarily the same as the transcription-repression mechanism that is operating in undifferentiated embryonal carcinoma cells. Images PMID:1702844

  20. Calcium regulates the commitment of murine erythroleukemia cells to terminal erythroid differentiation

    PubMed Central

    1981-01-01

    An alteration in the rate of calcium transport appears to be the rate- limiting event for the commitment of murine erythroleukemia (MEL) cells to initiate a program of terminal erythroid differentiation. The dimethyl sulfoxide (DMSO)-induced commitment of MEL cells to erythroid differentiation can be inhibited by treatment of cells with the calcium- chelating agent EGTA. Upon removal of EGTA, cells initiate commitment without the 12-h lag normally observed after treatment with DMSO alone. Treatment of cells with DMSO in the presence of calcium ionophore A23187 causes cells to initiate commitment from time zero with no lag. These results suggest that the lag is the time required for DMSO to alter the calcium transport properties of the cell. PMID:6793600

  1. 1.8 Astroms Structure of Murine GITR Ligand Dimer Expressed in Drosophila Melanogaster S2 Cells

    SciTech Connect

    Chattopadhyay, K.; Ramagopal, U; Nathenson, S; Almo, S

    2009-01-01

    Glucocorticoid-induced TNF receptor ligand (GITRL), a prominent member of the TNF superfamily, activates its receptor on both effector and regulatory T cells to generate critical costimulatory signals that have been implicated in a wide range of T-cell immune functions. The crystal structures of murine and human orthologs of GITRL recombinantly expressed in Escherichia coli have previously been determined. In contrast to all classical TNF structures, including the human GITRL structure, murine GITRL demonstrated a unique 'strand-exchanged' dimeric organization. Such a novel assembly behavior indicated a dramatic impact on receptor activation as well as on the signaling mechanism associated with the murine GITRL costimulatory system. In this present work, the 1.8 {angstrom} resolution crystal structure of murine GITRL expressed in Drosophila melanogaster S2 cells is reported. The eukaryotic protein-expression system allows transport of the recombinant protein into the extracellular culture medium, thus maximizing the possibility of obtaining correctly folded material devoid of any folding/assembly artifacts that are often suspected with E. coli-expressed proteins. The S2 cell-expressed murine GITRL adopts an identical 'strand-exchanged' dimeric structure to that observed for the E. coli-expressed protein, thus conclusively demonstrating the novel quaternary structure assembly behavior of murine GITRL.

  2. Impaired NK-cell education diminishes resistance to murine CMV infection.

    PubMed

    Wei, Hairong; Nash, William T; Makrigiannis, Andrew P; Brown, Michael G

    2014-11-01

    Ly49G2 (G2+) NK cells mediate murine (M)CMV resistance in MHC D(k) -expressing mice. Bone marrow transplantation (BMT) studies revealed that G2+ NK cell-mediated MCMV resistance requires D(k) in both hematopoietic and nonhematopoietic cells. As a Ly49G2 ligand, D(k) in both cell lineages may contribute to lysis of virus-infected cells. Alternatively, cellular differences in self-MHC D(k) may have affected NK-cell education, and consequently NK cell-mediated viral clearance. We investigated the D(k) -licensing effect on BM-derived NK cells in BMT recipients by analyzing cytokines, cytotoxicity and MCMV resistance. In BMT recipients with lineage-restricted D(k) , G2+ NK-cell reactivity and cytotoxicity was diminished in comparison to BMT recipients with self-MHC in all cells. Reduced G2+ NK-mediated MCMV resistance in BMT recipients with lineage-restricted self-MHC indicates that licensing of G2+ NK cells is related to NK-cell reactivity and viral control. Titrating donor BM with self-MHC-bearing hematopoietic cells, as well as adoptive transfer of mature G2+ NK cells into BMT recipients with self-MHC in non-hematopoietic cells only, enhanced NK-cell licensing and rescued MCMV resistance. This disparate self-MHC NK-cell education model would suggest that inadequately licensed NK cells corresponded to inefficient viral sensing and clearance. PMID:25187217

  3. AUGMENTATION OF MURINE NATURAL KILLER CELL ACTIVITY BY MANGANESE CHLORIDE

    EPA Science Inventory

    Natural Killer (NK) cell activity of spleen cells from male CBA/J mice was augmented by a single parenteral injection of MnCl2 administered 1 day prior to testing by in vitro and in vivo isotope release assays. Increased cytotoxic activity was observed in vitro against both NK-se...

  4. AN IN VITRO MODEL FOR MURINE URETERIC EPITHELIAL CELLS

    EPA Science Inventory

    This report presents a model developed to study growth and differentiation of primary cultures of ureteric epithelial cells from embryonic C57BL/6N mouse urinary tracts. Single cells were resuspended in medium and plated onto transwells coated with collagen IV and laminin. Basa...

  5. The effects of simulated hypogravity on murine bone marrow cells

    NASA Technical Reports Server (NTRS)

    Lawless, Desales

    1989-01-01

    Mouse bone marrow cells grown in complete medium at unit gravity were compared with a similar population cultured in conditions that mimic some aspects of microgravity. After the cells adjusted to the conditions that simulated microgravity, they proliferated as fetal or oncogenic populations; their numbers doubled in twelve hour periods. Differentiated subpopulations were depleted from the heterogeneous mixture with time and the undifferentiated hematopoietic stem cells increased in numbers. The cells in the control groups in unit gravity and those in the bioreactors in conditions of microgravity were monitored under a number of parameters. Each were phenotyped as to cell surface antigens using a panel of monoclonal antibodies and flow cytometry. Other parameters compared included: pH, glucose uptake, oxygen consumption and carbon-dioxide production. Nuclear DNA was monitored by flow cytometry. Functional responses were studied by mitogenic stimulation by various lectins. The importance of these findings should have relevance to the space program. Cells should behave predictably in zero gravity; specific populations can be eliminated from diverse populations and other populations isolated. The availability of stem cell populations will enhance both bone marrow and gene transplant programs. Stem cells will permit developmental biologists study the paths of hematopoiesis.

  6. Pontin is essential for murine hematopoietic stem cell survival

    PubMed Central

    Bereshchenko, Oxana; Mancini, Elena; Luciani, Luisa; Gambardella, Adriana; Riccardi, Carlo; Nerlov, Claus

    2012-01-01

    Pontin is a highly conserved DNA helicase/ATPase which is a component of several macromolecular complexes with functions that include DNA repair, telomere maintenance and tumor suppression. While Pontin is known to be essential in yeast, fruit flies and frogs, its physiological role in mammalian organisms remains to be determined. We here find that Pontin is highly expressed in embryonic stem cells and hematopoietic tissues. Through germline inactivation of Ruvbl1, the gene encoding Pontin, we found it to be essential for early embryogenesis, as Ruvbl1 null embryos could not be recovered beyond the blastocyst stage where proliferation of the pluripotent inner cell mass was impaired. Conditional ablation of Ruvbl1 in hematopoietic tissues led to bone marrow failure. Competitive repopulation experiments showed that this included the loss of hematopoietic stem cells through apopotosis. Pontin is, therefore, essential for the function of both embryonic pluripotent cells and adult hematopoietic stem cells. PMID:22371176

  7. 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

  8. Neurogenic potential of dental pulp stem cells isolated from murine incisors

    PubMed Central

    2014-01-01

    Introduction Interest in the use of dental pulp stem cells (DPSC) to enhance neurological recovery following stroke and traumatic injury is increasing following successful pre-clinical studies. A murine model of autologous neural stem cell transplantation would be useful for further pre-clinical investigation of the underlying mechanisms. However, while human-derived DPSC have been well characterised, the neurogenic potential of murine DPSC (mDPSC) has been largely neglected. In this study we demonstrate neuronal differentiation of DPSC from murine incisors in vitro. Methods mDPSC were cultured under neuroinductive conditions and assessed for neuronal and glial markers and electrophysiological functional maturation. Results mDPSC developed a neuronal morphology and high expression of neural markers nestin, ßIII-tubulin and GFAP. Neurofilament M and S100 were found in lower abundance. Differentiated cells also expressed protein markers for cholinergic, GABAergic and glutaminergic neurons, indicating a mixture of central and peripheral nervous system cell types. Intracellular electrophysiological analysis revealed the presence of voltage-gated L-type Ca2+ channels in a majority of cells with neuronal morphology. No voltage-gated Na+ or K+ currents were found and the cultures did not support spontaneous action potentials. Neuronal-like networks expressed the gap junction protein, connexin 43 but this was not associated with dye coupling between adjacent cells after injection of the low-molecular weight tracers Lucifer yellow or Neurobiotin. This indicated that the connexin proteins were not forming traditional gap junction channels. Conclusions The data presented support the differentiation of mDPSC into immature neuronal-like networks. PMID:24572146

  9. Analysis of apoptosis in murine embryonic stem cells

    E-print Network

    Weaks, Regina Lanell

    1999-01-01

    in detail in the nematode Caenorhabdi tis eiegans, providing the basis for understanding this process in higher organisms. Three genes have been found to regulate programmed cell death in C. elegans, ced-3, ced-4, and ced- 9. CED-3 and CED-4 are death... promoting factors, while CED-9 promotes cell survival. CED-3 is a member of the caspase family and is homologous to the mammalian protease interleukin-i+converting enzyme (ICE) (Yuan et al. , 1993). CED-4 acts as an effector of cell death in C. elegans...

  10. 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.

  11. 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.

  12. Polycomb complexes repress developmental regulators in murine embryonic stem cells

    Microsoft Academic Search

    Laurie A. Boyer; Kathrin Plath; Julia Zeitlinger; Tobias Brambrink; Lea A. Medeiros; Tong Ihn Lee; Stuart S. Levine; Marius Wernig; Adriana Tajonar; Mridula K. Ray; George W. Bell; Arie P. Otte; Miguel Vidal; David K. Gifford; Richard A. Young; Rudolf Jaenisch

    2006-01-01

    The mechanisms by which embryonic stem (ES) cells self-renew while maintaining the ability to differentiate into virtually all adult cell types are not well understood. Polycomb group (PcG) proteins are transcriptional repressors that help to maintain cellular identity during metazoan development by epigenetic modification of chromatin structure. PcG proteins have essential roles in early embryonic development and have been implicated

  13. Estimation of heterokaryon formation and hybridoma growth in murine and human cell fusions.

    PubMed

    Alkan, S S; Mestel, F; Jiricka, J; Blaser, K

    1987-08-01

    Four mouse myelomas commonly used for cell fusions (X63.Ag8.653, SP2/0, NS1, P3U1), 3 human myeloma-like cell lines (ARH77, U-266, GM1500) and 3 human x mouse hybridomas (SPAZ4, SA2, SA3) were compared for their heterokaryon formation and successful hybridoma growth after cell fusion with polyethylene glycol. The cells were stained with different fluorescent dyes which do not alter hybridoma growth or antibody secretion. After fusion myeloma cells containing at least 1 nucleus from a lymphocyte (heterokaryons) were counted from fluorescence photomicrographs and the heterokaryon frequency was calculated. Mouse myelomas fused at a frequency of 1-7%, whereas human myeloma lines showed a higher heterokaryon frequency ranging from 3-25%. In mouse fusions almost every well contained growing hybridomas showing a minimum hybridoma frequency of 2/10(6) lymphocytes. In human fusions the SPAZ4 and SA2 lines showed a heterokaryon frequency nearly as good as mouse myelomas, whereas U-266 yielded no growing hybridomas despite 20% heterokaryon frequency. Furthermore, human cell lines showed a high tendency of multikaryon formation whereas this phenomenon was rarely observed with murine and murine x human heterohybrids. In individual fusion experiments no correlation was found between heterokaryon formation and the number of growing hybridomas. Thus, our study shows that defects in hybridoma growth may not always result from lack of a successful fusion and human hybridomas might be more sensitive to post-fusion conditions. PMID:3623581

  14. Isolation, Long-Term Expansion, and Differentiation of Murine Neural Stem Cells.

    PubMed

    Bizy, Alexandra; Ferrón, Sacri R

    2014-07-26

    Stem cells are capable of extensive self-renewal while preserving the ability to generate cell progeny that can differentiate into different cell types. Here, we describe some methods for the isolation of neural stem cells (NSCs) from the adult murine subependymal zone (SEZ), their extensive culturing and the assessment of their full developmental potential, particularly with respect to their differentiation capacity. The procedure includes chemically defined conditions such as absence of serum and addition of specific growth factors, in which differentiated cells die and are rapidly eliminated from the culture. In contrast, undifferentiated precursors become hypertrophic and proliferate, forming clonal spherical clusters called "neurospheres." Experimental manipulation of NSCs identifies populations of cells with differential restriction in their self-renewal potential and introduces a great interest in defining the conditions that guide their differentiation into a variety of neuronal and glial subtypes, aspects that have important implications for their use in future clinical purposes. PMID:25063500

  15. Murine CD83-positive T cells mediate suppressor functions in vitro and in vivo.

    PubMed

    Kreiser, Simon; Eckhardt, Jenny; Kuhnt, Christine; Stein, Marcello; Krzyzak, Lena; Seitz, Christine; Tucher, Christine; Knippertz, Ilka; Becker, Christoph; Günther, Claudia; Steinkasserer, Alexander; Lechmann, Matthias

    2015-02-01

    The CD83 molecule (CD83) is a well-known surface marker present on mature dendritic cells (mDC). In this study, we show that CD83 is also expressed on a subset of T cells which mediate regulatory T cell (Treg)-like suppressor functions in vitro and in vivo. Treg-associated molecules including CD25, cytotoxic T lymphocyte antigen-4 (CTLA-4), glucocorticoid-induced TNFR family-related gene (GITR), Helios and neuropilin-1 (NRP-1) as well as forkhead box protein 3 (FOXP3) were specifically expressed by these CD83(+) T cells. In contrast, CD83(-) T cells showed a naive T cell phenotype with effector T cell properties upon activation. Noteworthy, CD83(-) T cells were not able to upregulate CD83 despite activation. Furthermore, CD83(+) T cells suppressed the proliferation and inflammatory cytokine release of CD83(-) T cells in vitro. Strikingly, stimulated CD83(+) T cells released soluble CD83 (sCD83), which has been reported to possess immunosuppressive properties. In vivo, using the murine transfer colitis model we could show that CD83(+) T cells were able to suppress colitis symptoms while CD83(-) T cells possessed effector functions. In addition, this CD83 expression is also conserved on expanded human Treg. Thus, from these studies we conclude that CD83(+) T cells share important features with regulatory T cells, identifying CD83 as a novel lineage marker to discriminate between different T cell populations. PMID:25151500

  16. Murine lipid phosphate phosphohydrolase-3 acts as a cell-associated integrin ligand

    SciTech Connect

    Humtsoe, Joseph O. [Center for Extracellular Matrix Biology, Institute of Biosciences and Technology, Texas A and M University System Health Science Center, Texas Medical Center, 2121 W. Holcombe Blvd, Houston, TX 77030 (United States); Bowling, Rodney A. [Center for Extracellular Matrix Biology, Institute of Biosciences and Technology, Texas A and M University System Health Science Center, Texas Medical Center, 2121 W. Holcombe Blvd, Houston, TX 77030 (United States); Feng, Shu [Center for Extracellular Matrix Biology, Institute of Biosciences and Technology, Texas A and M University System Health Science Center, Texas Medical Center, 2121 W. Holcombe Blvd, Houston, TX 77030 (United States); Wary, Kishore K. [Center for Extracellular Matrix Biology, Institute of Biosciences and Technology, Texas A and M University System Health Science Center, Texas Medical Center, 2121 W. Holcombe Blvd, Houston, TX 77030 (United States)]. E-mail: kwary@ibt.tamhsc.edu

    2005-09-30

    Lipid phosphate phosphohydrolase-3 (LPP3) is a cell surface protein that exhibits ectoenzyme activity. Previously, we identified human LPP3 in a functional assay of angiogenesis and showed that the Arg-Gly-Asp (RGD) motif in the proposed second extracellular domain interacts with a subset of integrins to mediate cell-cell adhesion. In contrast to the RGD domain of human LPP3, murine Lpp3 contains a variant sequence, Arg-Gly-Glu (RGE). Whether the RGE motif of murine Lpp3 mediates cell-cell interaction has not been studied. In this report, we test the hypothesis that the cell adhesion function of the LPP3 protein is conserved across mouse and human. A glutathione S-transferase (GST) fusion protein of the proposed second extracellular loop of the murine Lpp3 sequence (GST-mLpp3-RGE) promoted attachment of cells in a long-term cell adhesion assay. GST-mLpp3-RGE interacted with {alpha}{sub 5}{beta}{sub 1} and {alpha}{sub v}{beta}{sub 3} integrins in a solid-phase ELISA, while a mutant control, GST-hLPP3-RAD, did not. Long-term adhesion of endothelial cells to GST-mLpp3-RGE induced phosphorylation of FAK, SHC, and CAS, whereas adhesion to GST-hLPP3-RAD failed to do so. Upon long-term adhesion both the GST-hLPP3-RGD and GST-mLpp3-RGE substrates bound to the {alpha}{sub 5}{beta}{sub 1} integrin of FRT-{alpha}{sub 5}(+) cells, an interaction that was inhibited by an anti-{alpha}{sub 5} integrin antibody. In addition, a cell aggregation assay showed that the intact mLpp3-RGE protein interacts with {alpha}{sub 5}{beta}{sub 1} and {alpha}{sub v}{beta}{sub 3} integrins expressed by adjacent cells, an interaction that can be blocked by GRGDSP peptides and anti-LPP3-RGD antibodies. These data, together with the known importance of integrins in angiogenesis, provide a mechanism for the function of LPP3 in cell-cell interactions in both human and mouse.

  17. Oatp-associated uptake and toxicity of microcystins in primary murine whole brain cells

    SciTech Connect

    Feurstein, D.; Holst, K.; Fischer, A. [Human and Environmental Toxicology, University of Konstanz, Konstanz (Germany); Dietrich, D.R. [Human and Environmental Toxicology, University of Konstanz, Konstanz (Germany)], E-mail: daniel.dietrich@uni-konstanz.de

    2009-01-15

    Microcystins (MCs) are naturally occurring cyclic heptapeptides that exhibit hepato-, nephro- and possibly neurotoxic effects in mammals. Organic anion transporting polypeptides (rodent Oatp/human OATP) appear to be specifically required for active uptake of MCs into hepatocytes and kidney epithelial cells. Based on symptoms of neurotoxicity in MC-intoxicated patients and the presence of Oatp/OATP at the blood-brain-barrier (BBB) and blood-cerebrospinal-fluid-barrier (BCFB) it is hypothesized that MCs can be transported across the BBB/BCFB in an Oatp/OATP-dependent manner and can induce toxicity in brain cells via inhibition of protein phosphatase (PP). To test these hypotheses, the presence of murine Oatp (mOatp) in primary murine whole brain cells (mWBC) was investigated at the mRNA and protein level. MC transport was tested by exposing mWBCs to three different MC-congeners (MC-LR, -LW, -LF) with/without co-incubation with the OATP/Oatp-substrates taurocholate (TC) and bromosulfophthalein (BSP). Uptake of MCs and cytotoxicity was demonstrated via MC-Western blot analysis, immunocytochemistry, cell viability and PP inhibition assays. All MC congeners bound covalently and inhibited mWBC PP. MC-LF was the most cytotoxic congener followed by -LW and -LR. The lowest toxin concentration significantly reducing mWBC viability after 48 h exposure was 400 nM (MC-LF). Uptake of MCs into mWBCs was inhibited via co-incubation with excess TC (50 and 500 {mu}M) and BSP (50 {mu}M). MC-Western blot analysis demonstrated a concentration-dependent accumulation of MCs. In conclusion, the in vitro data support the assumed MC-congener-dependent uptake in a mOatp-associated manner and cytotoxicity of MCs in primary murine whole brain cells.

  18. 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

  19. Complex 2B4 regulation of mast cells and eosinophils in murine allergic inflammation.

    PubMed

    Elishmereni, Moran; Fyhrquist, Nanna; Singh Gangwar, Roopesh; Lehtimäki, Sari; Alenius, Harri; Levi-Schaffer, Francesca

    2014-12-01

    The cell surface molecule 2B4 (CD244) is an important regulator of lymphocyte activation, and its role in antiviral immunity and lymphoproliferative disorders is well established. Although it is also expressed on mast cells (MCs) and eosinophils (Eos), the functions of 2B4 on these allergy-orchestrating cells remain unclear. We therefore investigated the role of 2B4 on murine MCs and Eos, particularly how this molecule affects allergic and nonallergic inflammatory processes involving these effector cells. Experiments in bone marrow-derived cultures revealed an inhibitory effect for 2B4 in MC degranulation, but also an opposing stimulatory effect in eosinophil migration and delayed activation. Murine disease models supported the dual 2B4 function: In 2B4-/- mice with nonallergic peritonitis and mild atopic dermatitis (AD), modest infiltrates of Eos into the peritoneum and skin (respectively) confirmed that 2B4 boosts eosinophil trafficking. In a chronic AD model, 2B4-/- mice showed overdegranulated MCs, confirming the inhibiting 2B4 effect on MC activation. This multifunctional 2B4 profile unfolded in inflammation resembles a similar mixed effect of 2B4 in natural killer cells. Taken together, our findings provide evidence for physiological 2B4 stimulatory/inhibitory effects in MCs and Eos, pointing to a complex role for 2B4 in allergy. PMID:24999594

  20. Retroviral transduction of murine and human hematopoietic progenitors and stem cells.

    PubMed

    Ciuculescu, Marioara F; Brendel, Christian; Harris, Chad E; Williams, David A

    2014-01-01

    Genetic modification of cells using retroviral vectors is the method of choice when the cell population is difficult to transfect and/or requires persistent transgene expression in progeny cells. There are innumerable potential applications for these procedures in laboratory research and clinical therapeutic interventions. One paradigmatic example is the genetic modification of hematopoietic stem and progenitor cells (HSPCs). These are rare nucleated cells which reside in a specialized microenvironment within the bone marrow, and have the potential to self-renew and/or differentiate into all hematopoietic lineages. Due to their enormous regenerative capacity in steady state or under stress conditions these cells are routinely used in allogeneic bone marrow transplantation to reconstitute the hematopoietic system in patients with metabolic, inflammatory, malignant, and other hematologic disorders. For patients lacking a matched bone marrow donor, gene therapy of autologous hematopoietic stem cells has proven to be an alternative as highlighted recently by several successful gene therapy trials. Genetic modification of HSPCs using retrovirus vectors requires ex vivo manipulation to efficiently introduce the new genetic material into cells (transduction). Optimal culture conditions are essential to facilitate this process while preserving the stemness of the cells. The most frequently used retroviral vector systems for the genetic modifications of HSPCs are derived either from Moloney murine leukemia-virus (Mo-MLV) or the human immunodeficiency virus-1 (HIV-1) and are generally termed according to their genus gamma-retroviral (?-RV) or lentiviral vectors (LV), respectively. This chapter describes in a step-by-step fashion some techniques used to produce research grade vector supernatants and to obtain purified murine or human hematopoietic stem cells for transduction, as well as follow-up methods for analysis of transduced cell populations. PMID:25062637

  1. Effects of glial cell line-derived neurotrophic factor on cultured murine retinal progenitor cells

    PubMed Central

    Wang, Jinmei; Yang, Jing; Gu, Ping

    2010-01-01

    Purpose Glial cell line-derived neurotrophic factor (GDNF) is neuroprotective of retinal neurons, and transduced retinal progenitor cells (RPCs) can deliver this cytokine for the treatment of retinal diseases, yet the potential effects of GDNF on RPCs have received little attention. Methods Murine RPCs were assessed under multiple conditions in the presence or absence of epidermal growth factor (EGF, 20 ng/ml) and/or GDNF (10 ng/ml) using a variety of techniques, including live-cell imaging, caspase-3 activity assay, whole genome microarray, quantitative polymerase chain reaction (qPCR), and western blotting. Results Live monitoring revealed that formation of initial aggregates resulted largely from the collision and adherence of dissociated RPCs, as opposed to clonal proliferation. Spheres enlarged in size and number, with more reaching the threshold criteria for cross-sectional areas in the EGF+GDNF condition. Proliferation was measurably augmented in association with EGF+GDNF, and Ki-67 expression was modestly increased (1.07 fold), as were hairy and enhancer of split 5 (Hes5), mammalian achaete-scute homolog 1 (Mash1), and Vimentin. However, global gene expression did not reveal a notable treatment-related response, and the expression of the majority of progenitor and lineage markers examined remained stable. GDNF reduced RPC apoptosis, compared to complete growth-factor withdrawal, although it could not by itself sustain mitotic activity. Conclusions These data support the feasibility of developing GDNF-transduced RPCs as potential therapeutic agents for use in retinal diseases. PMID:21203407

  2. IMMUNE AND NATURAL ANTIBODIES TO SYNGENEIC MURINE PLASMA CELL TUMORS

    PubMed Central

    Herberman, Ronald B.; Aoki, Tadao

    1972-01-01

    Cytotoxic antibody to a plasma cell tumor antigen was produced in syngeneic BALB mice by immunization with viable or inactivated plasma cell tumors. Antibody with the same specificity was found in the sera of normal BALB and other strains of mice. This natural antibody reacted with an antigen with characteristics indistinguishable from the previously described alloantigen, PC.1, and with viral envelope antigen, ?VEA. The incidence of cytotoxic reactivity and the antibody titers reached a peak in normal BALB mice at 3–4 months of age, and were lower in 9–12-month old mice. The sera of germfree mice had lower reactivity; but when the mice were transferred to conventional conditions, their sera soon became as active as those of conventional mice. A virus common to all plasma cell tumors, which is present in latent form in some normal tissues of BALB and other PC.1 positive strains, is suggested as the cause for the PC.1 antigen and for the appearance of natural antibody to it. The considerable evidence for the close association of a virus with plasma cell tumors is presented. PMID:5033423

  3. Murine mammary tumor cells with a claudin-low genotype

    PubMed Central

    2011-01-01

    Background Molecular classification of human breast cancers has identified at least 5 distinct tumor subtypes; luminal A, luminal B, Her2-enriched, basal-like and claudin-low. The claudin-low subtype was identified in 2007 and is characterized by low expression of luminal differentiation markers and claudins 3, 4 and 7 and high levels of mesenchymal markers. Claudin-low tumors have a reported prevalence of 7-14% and these tumors have a poor prognosis. Results In this study we report the characterization of several cell lines established from mammary tumors that develop in MTB-IGFIR transgenic mice. Two lines, RM11A and RJ348 present with histological features and gene expression patterns that resemble claudin-low breast tumors. Specifically, RM11A and RJ348 cells express high levels of the mesenchymal genes Zeb1, Zeb2, Twist1 and Twist2 and very low levels of E-cadherin and claudins 3, 4 and 7. The RM11A and RJ348 cells are also highly tumorigenic when re-introduced into the mammary fat pad of mice. Conclusions Mammary tumor cells established from MTB-IGFIR transgenic mice can be used as in vitro and in vivo model systems to further our understanding of the poorly characterized, claudin-low, breast cancer subtype. PMID:21846397

  4. 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

  5. Label-Retaining Cells in the Adult Murine Salivary Glands Possess Characteristics of Adult Progenitor Cells

    PubMed Central

    Chibly, Alejandro M.; Querin, Lauren; Harris, Zoey; Limesand, Kirsten H.

    2014-01-01

    Radiotherapy is the primary treatment for patients with head and neck cancer, which account for roughly 500,000 annual cases worldwide. Dysfunction of the salivary glands and associated conditions like xerostomia and dysphagia are often developed by these patients, greatly diminishing their life quality. Current preventative and palliative care fail to deliver an improvement in the quality of life, thus accentuating the need for regenerative therapies. In this study, a model of label retaining cells (LRCs) in murine salivary glands was developed, in which LRCs demonstrated proliferative potential and possessed markers of putative salivary progenitors. Mice were labeled with 5-Ethynyl-2?-deoxyuridine (EdU) at postnatal day 10 and chased for 8 weeks. Tissue sections from salivary glands obtained at the end of chase demonstrated co-localization between LRCs and the salivary progenitor markers keratin 5 and keratin 14, as well as kit mRNA, indicating that LRCs encompass a heterogeneous population of salivary progenitors. Proliferative potential of LRCs was demonstrated by a sphere assay, in which LRCs were found in primary and secondary spheres and they co-localized with the proliferation marker Ki67 throughout sphere formation. Surprisingly, LRCs were shown to be radio-resistant and evade apoptosis following radiation treatment. The clinical significance of these findings lie in the potential of this model to study the mechanisms that prevent salivary progenitors from maintaining homeostasis upon exposure to radiation, which will in turn facilitate the development of regenerative therapies for salivary gland dysfunction. PMID:25238060

  6. Transformation of Murine Cells by Two “Slow Viruses,” Visna Virus and Progressive Pneumonia Virus

    PubMed Central

    Takemoto, Kenneth K.; Stone, Lawrence B.

    1971-01-01

    Visna and progressive pneumonia virus (PPV), two antigenically related, non-oncogenic “slow viruses” which have ribonucleic acid (RNA)-dependent deoxyribonucleic acid (DNA) polymerase activity, were examined for their ability to transform cells. Murine cells which had been exposed to either visna or PPV developed foci of altered, spindle-shaped cells 3 to 4 weeks after infection. Visna and PPV transformed lines were established from these cultures. There was no evidence that other oncogenic DNA or RNA viruses were involved in the observed transformation. Visna or PPV could be “rescued” from all transformed lines by co-cultivation with normal sheep testis cells. “Rescued” virus was identified as visna or PPV, and they retained the capacity to transform mouse cells. These experiments may have important implications in the understanding of both viral carcinogenesis and “slow” viral infections. Images PMID:4998321

  7. 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

  8. 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

  9. Isolation, expansion and transplantation of postnatal murine progenitor cells of the enteric nervous system.

    PubMed

    Dettmann, Heike Monika; Zhang, Ying; Wronna, Nadine; Kraushaar, Udo; Guenther, Elke; Mohr, Roland; Neckel, Peter Helmut; Mack, Andreas; Fuchs, Joerg; Just, Lothar; Obermayr, Florian

    2014-01-01

    Neural stem or progenitor cells have been proposed to restore gastrointestinal function in patients suffering from congenital or acquired defects of the enteric nervous system. Various, mainly embryonic cell sources have been identified for this purpose. However, immunological and ethical issues make a postnatal cell based therapy desirable. We therefore evaluated and quantified the potential of progenitor cells of the postnatal murine enteric nervous system to give rise to neurons and glial cells in vitro. Electrophysiological analysis and BrdU uptake studies provided direct evidence that generated neurons derive from expanded cells in vitro. Transplantation of isolated and expanded postnatal progenitor cells into the distal colon of adult mice demonstrated cell survival for 12 weeks (end of study). Implanted cells migrated within the gut wall and differentiated into neurons and glial cells, both of which were shown to derive from proliferated cells by BrdU uptake. This study indicates that progenitor cells isolated from the postnatal enteric nervous system might have the potential to serve as a source for a cell based therapy for neurogastrointestinal motility disorders. However, further studies are necessary to provide evidence that the generated cells are capable to positively influence the motility of the diseased gastrointestinal tract. PMID:24871092

  10. Entry of diphtheria toxin linked to concanavalin A into primate and murine cells.

    PubMed

    Guillemot, J C; Sundan, A; Olsnes, S; Sandvig, K

    1985-02-01

    Diphtheria toxin linked by a disulfide bridge to concanavalin A was highly toxic to HeLa S3 and Vero cells, as well as to murine L cells. The cells could be protected with alpha-methyl mannoside, indicating that the conjugate binds mainly through its concanavalin A moiety. Treatment of Vero cells with phospholipase C, TPA (12-O-tetradecanoylphorbol-13-acetate), and vanadate, which strongly reduce the ability of the cells to bind free diphtheria toxin, had little protective effect against the conjugate, whereas SITS (L-acetamido-4'-isothiocyano-stilbene-2,2'disulfonic acid), which inhibits diphtheria toxin binding, as well as the subsequent entry, protected Vero cells, but not L cells. Both types of cells are protected against the conjugate by NH4Cl and monensin, indicating that an acidified compartment is necessary for entry into the cytosol. Exposure of cells, bound with surface conjugate, to low pH induced entry of the toxin into Vero cells, but not into L Cells. Phospholipase C, TPA, and vanadate did not protect L cells against the conjugate. It is concluded that toxin in the conjugate enters L cells by a route which involves low pH, but which is not identical to that in Vero cells. PMID:3844014

  11. Epifluorescence Intravital Microscopy of Murine Corneal Dendritic Cells

    PubMed Central

    Rosenbaum, James T.; Planck, Stephen R.

    2010-01-01

    Purpose. Dendritic cells (DCs) are antigen-presenting cells vital for initiating immune responses. In this study the authors examined the in vivo migratory capability of resident corneal DCs to various stimuli. Methods. The authors used mice expressing enhanced yellow fluorescent protein (eYFP) under control of the CD11c promoter to visualize corneal DCs. To assess the distribution and mobility of DCs, normal corneas were imaged in vivo and ex vivo with fluorescence microscopy. Intravital microscopy was used to examine the responses of resident central and peripheral corneal DCs to silver nitrate injury, lipopolysaccharide, microspheres, and tumor necrosis factor (TNF-?). In some experiments, TNF-? injection was used to first induce centripetal migration of DCs to the central cornea, which was subsequently reinjected with microspheres. Results. In normal corneas, DCs were sparsely distributed centrally and were denser in the periphery, with epithelial-level DCs extending into the epithelium. Videomicroscopy showed that though cell processes were in continuous movement, cells generally did not migrate. Within the first 6 hours after stimulation, neither central nor peripheral corneal DCs exhibited significant lateral migration, but central corneal DCs assumed extreme morphologic changes. An increased number of DCs in the TNF-?–stimulated central cornea were responsive to subsequent microsphere injection by adopting a migratory behavior, but not with increased speed. Conclusions. In vivo imaging reveals minimal lateral migration of corneal DCs after various stimuli. In contrast, DCs within the central cornea after initial TNF-? injection are more likely to respond to a secondary insult with lateral migration. PMID:20007837

  12. T cells and post menopausal osteoporosis in murine models

    Microsoft Academic Search

    Roberto Pacifici

    2007-01-01

    Estrogen deficiency is one of the most frequent causes of osteoporosis in women and a possible cause of bone loss in men.\\u000a But the mechanism involved remains largely unknown. Estrogen deficiency leads to an increase in the immune function, which\\u000a culminates in an increased production of tumor necrosis factor (TNF) by activated T cells. TNF increases osteoclast formation\\u000a and bone

  13. Targeted destruction of murine macrophage cells with bioconjugated gold nanorods

    Microsoft Academic Search

    Dakrong Pissuwan; Stella M. Valenzuela; Murray C. Killingsworth; Xiaoda Xu; Michael B. Cortie

    2007-01-01

    Gold nanorods manifest a readily tunable longitudinal plasmon resonance with light and consequently have potential for use\\u000a in photothermal therapeutics. Recent work by others has shown how gold nanoshells and rods can be used to target cancer cells,\\u000a which can then be destroyed using relatively high power laser radiation (?1×105 to 1×1010 W\\/m2). Here we extend this concept to demonstrate how

  14. Generation of a murine hepatic angiosarcoma cell line and reproducible mouse tumor model.

    PubMed

    Rothweiler, Sonja; Dill, Michael T; Terracciano, Luigi; Makowska, Zuzanna; Quagliata, Luca; Hlushchuk, Ruslan; Djonov, Valentin; Heim, Markus H; Semela, David

    2015-03-01

    Hepatic angiosarcoma (AS) is a rare and highly aggressive tumor of endothelial origin with dismal prognosis. Studies of the molecular biology of AS and treatment options are limited as animal models are rare. We have previously shown that inducible knockout of Notch1 in mice leads to spontaneous formation of hepatic AS. The aims of this study were to: (1) establish and characterize a cell line derived from this murine AS, (2) identify molecular pathways involved in the pathogenesis and potential therapeutic targets, and (3) generate a tumor transplantation model. AS cells retained specific endothelial properties such as tube formation activity, as well as expression of CD31 and Von Willebrand factor. However, electron microscopy analysis revealed signs of dedifferentiation with loss of fenestrae and loss of contact inhibition. Microarray and pathway analysis showed substantial changes in gene expression and revealed activation of the Myc pathway. Exposing the AS cells to sorafenib reduced migration, filopodia dynamics, and cell proliferation but did not induce apoptosis. In addition, sorafenib suppressed ERK phosphorylation and expression of cyclin D2. Injection of AS cells into NOD/SCID mice resulted in formation of undifferentiated tumors, confirming the tumorigenic potential of these cells. In summary, we established and characterized a murine model of spontaneous AS formation and hepatic AS cell lines as a useful in vitro tool. Our data demonstrate antitumor activity of sorafenib in AS cells with potent inhibition of migration, filopodia formation, and cell proliferation, supporting further evaluation of sorafenib as a novel treatment strategy. In addition, AS cell transplantation provides a subcutaneous tumor model useful for in vivo preclinical drug testing. PMID:25418579

  15. Cloning of the human homologue of the murine flt3 ligand: a growth factor for early hematopoietic progenitor cells.

    PubMed

    Lyman, S D; James, L; Johnson, L; Brasel, K; de Vries, P; Escobar, S S; Downey, H; Splett, R R; Beckmann, M P; McKenna, H J

    1994-05-15

    Using a fragment of the murine flt3 ligand as a probe, we have succeeded in cloning a human flt3 ligand from a human T-cell lambda gt10 cDNA library. The human and murine ligands are 72% identical at the amino acid level. Analysis of multiple cDNA clones shows that alternative splicing of the human flt3 mRNA can occur at a number of positions. A recombinant soluble form of the human flt3 ligand stimulates the proliferation and colony formation of a subpopulation of human bone marrow cells that are CD34+ and are enriched for primitive hematopoietic cells. In addition, the human flt3 ligand also stimulates the proliferation of cells expressing murine flt3 receptors. Northern blot analysis shows widespread expression of flt3 ligand mRNA transcripts in human tissues. PMID:8180375

  16. A CD133-expressing murine liver oval cell population with bilineage potential.

    PubMed

    Rountree, C Bart; Barsky, Lora; Ge, Shundi; Zhu, Judy; Senadheera, Shantha; Crooks, Gay M

    2007-10-01

    Although oval cells are postulated to be adult liver stem cells, a well-defined phenotype of a bipotent liver stem cell remains elusive. The heterogeneity of cells within the oval cell fraction has hindered lineage potential studies. Our goal was to identify an enriched population of bipotent oval cells using a combination of flow cytometry and single cell gene expression in conjunction with lineage-specific liver injury models. Expression of cell surface markers on nonparenchymal, nonhematopoietic (CD45-) cells were characterized. Cell populations were isolated by flow cytometry for gene expression studies. 3,5-Diethoxycarbonyl-1,4-dihydrocollidine toxic injury induced cell cycling and expansion specifically in the subpopulation of oval cells in the periportal zone that express CD133. CD133+CD45- cells expressed hepatoblast and stem cell-associated genes, and single cells coexpressed both hepatocyte and cholangiocyte-associated genes, indicating bilineage potential. CD133+CD45- cells proliferated in response to liver injury. Following toxic hepatocyte damage, CD133+CD45- cells demonstrated upregulated expression of the hepatocyte gene Albumin. In contrast, toxic cholangiocyte injury resulted in upregulation of the cholangiocyte gene Ck19. After 21-28 days in culture, CD133+CD45- cells continued to generate cells of both hepatocyte and cholangiocyte lineages. Thus, CD133 expression identifies a population of oval cells in adult murine liver with the gene expression profile and function of primitive, bipotent liver stem cells. In response to lineage-specific injury, these cells demonstrate a lineage-appropriate genetic response. Disclosure of potential conflicts of interest is found at the end of this article. PMID:17585168

  17. Murine cytomegalovirus stimulates natural killer cell function but kills genetically resistant mice treated with radioactive strontium.

    PubMed Central

    Masuda, A; Bennett, M

    1981-01-01

    Treatment of C3H/St mice with 100 microCi of 89Sr weakened their genetic resistance to murine cytomegalovirus (MCMV) infection. The criteria utilized to detect increased susceptibility were: (i) survival of mice; (ii) numbers of MCMV-infected cells in the spleens and liver; and (iii) serum glutamic pyruvic transaminase levels. The natural killer (NK) cell activity of spleen cells from mice treated with 89Sr is very low. However, the NK activities of spleen cells of both normal and 89Sr-treated mice were greatly augmented 3 days after infection with MCMV. These NK cells lysed a variety of tumor cells and shared several features with conventional NK cells, but were not lysed by anti-Nk-1.2 serum (specific for NK cells) plus complement. Splenic adherent cells did not lyse tumor cells themselves but were necessary for the stimulation of NK cells by MCMV. The paradox of high NK cell function and poor survival in 89Sr-treated mice infected with MCMV was a surprise. We conclude that these augmented NK cells, of themselves, cannot account for the genetic resistance of C3H/St mice to infection with MCMV. Images PMID:6277794

  18. Loss of Cytotoxicity and Gain of Cytokine Production in Murine Tumor-Activated NK Cells

    PubMed Central

    Müller, Jürgen R.; Waldmann, Thomas A.; Dubois, Sigrid

    2014-01-01

    NK cells are able to form a functional memory suggesting that some NK cells are surviving the activation process. We hypothesized that NK cell activation causes the development of a distinct NK cell subset and studied the fate of murine post-activation NK cells. Activation was achieved by in vivo and in vitro exposures to the melanoma tumor cell line B16 that was followed by differentiation in IL-2. When compared with control NK cells, post-activation CD25+ NK cells expressed little granzyme B or perforin and had low lysis activity. Post-activation NK cells expressed CD27, CD90, CD127, and were low for CD11b suggesting that tumor-induced activation is restricted to an early NK cell subset. Activation of NK cells led to decreases of CD16, CD11c and increases of CD62L and the IL-18 receptor. In vivo activated but not control NK cells expressed a variety of cytokines that included IFN?, TNF?, GM-CSF and IL-10. These data suggest that the exposure of a subset of peripheral NK cells to the B16 tumor environment caused an exhaustion of their cytolytic capacity but also a gain in their ability to produce cytokines. PMID:25101668

  19. Changes in CD45 isoform expression accompany antigen-induced murine T-cell activation.

    PubMed Central

    Birkeland, M L; Johnson, P; Trowbridge, I S; Puré, E

    1989-01-01

    Leukocytes express a family of plasma membrane proteins called CD45 or the leukocyte common antigen. Isoforms of various molecular masses, 180-240 kDa, are produced by alternative splicing and usage of three exons, named A, B, and C, that encode the N-terminal portion of the external domain. By using monoclonal antibodies that precipitate B exon-dependent and B exon-independent isoforms we find that both murine CD4+ and murine CD8+ T cells selectively down-regulate the B exon-dependent forms of CD45 during an immune response. This change was monitored by using fluorescence-activated cell sorter (FACS) analysis and immunoprecipitation from surface radioiodinated and metabolically labeled cells. The loss of the 190-kDa B exon-dependent isoform during T-cell activation is accompanied by an increased production of a 180-kDa form, which does not contain the B exon-encoded sequence. This accounts for our observation that the overall expression of CD45, as assessed by FACS analysis, does not change. Images PMID:2528147

  20. Role of Germination in Murine Airway CD8+ T-Cell Responses to Aspergillus Conidia

    PubMed Central

    Templeton, Steven P.; Buskirk, Amanda D.; Law, Brandon; Green, Brett J.; Beezhold, Donald H.

    2011-01-01

    Pulmonary exposure to Aspergillus fumigatus has been associated with morbidity and mortality, particularly in immunocompromised individuals. A. fumigatus conidia produce ?-glucan, proteases, and other immunostimulatory factors upon germination. Murine models have shown that the ability of A. fumigatus to germinate at physiological temperature may be an important factor that facilitates invasive disease. We observed a significant increase in IFN-?-producing CD8+ T cells in bronchoalveolar lavage fluid (BALF) of immunocompetent mice that repeatedly aspirated A. fumigatus conidia in contrast to mice challenged with A. versicolor, a species that is not typically associated with invasive, disseminated disease. Analysis of tissue sections indicated the presence of germinating spores in the lungs of mice challenged with A. fumigatus, but not A. versicolor. Airway IFN-?+CD8+ T-cells were decreased and lung germination was eliminated in mice that aspirated A. fumigatus conidia that were formaldehyde-fixed or heat-inactivated. Furthermore, A. fumigatus particles exhibited greater persistence in the lungs of recipient mice when compared to non-viable A. fumigatus or A. versicolor, and this correlated with increased maintenance of airway memory-phenotype CD8+ T cells. Therefore, murine airway CD8+ T cell-responses to aspiration of Aspergillus conidia may be mediated in part by the ability of conidia to germinate in the host lung tissue. These results provide further evidence of induction of immune responses to fungi based on their ability to invade host tissue. PMID:21533200

  1. Diminished organelle motion in murine Kupffer cells during the erythrocytic stage of malaria

    PubMed Central

    Bellows, Charles F.; Molina, Ramon M.; Brain, Joseph D.

    2011-01-01

    Parasitized erythrocytes are ingested by murine hepatic macrophages during malaria infection. We non-invasively monitored how this altered the motion of intracellular phagosomes in Kupffer cells using magnetometry. Submicrometric ?Fe2O3 particles were injected prior to malaria infection. They were cleared from the blood, primarily by Kupffer cells, and retained within their phagosomes. The mice were periodically magnetized. After removing this external magnet, the aligned iron particles created a remnant magnetic field (RMF) which then decayed (relaxation), reflecting the motion of particle-containing phagosomes. After baseline measurements of relaxation, the mice were injected intravenously with Plasmodium chabaudi-parasitized or normal murine red blood cells (RBCs). During the next 15 days, relaxation measurements, parasitaemia and haematocrit values were monitored. At 6 days post injection with 3 × 107 parasitized RBCs, relaxation rates had decreased. At this time, all mice had parasitaemias greater than 58 per cent and haematocrits less than 20 per cent. At day 7, while the parasitaemias were declining, the rate of relaxation continued to decrease. Throughout the experiment, relaxation remained constant in animals injected with normal RBCs. Electron microscopy revealed Kupffer cells filled with damaged and parasitized erythrocytes, and haemoglobin degradation pigment. We conclude that ingestion and metabolism of parasitized erythrocytes by liver macrophages during malaria infection decreases their organelle motion with likely consequences of compromised host defences. PMID:21068031

  2. Replicative cis-advantage of polyomavirus regulatory region mutants in different murine cell lines.

    PubMed Central

    De Simone, V; Amati, P

    1987-01-01

    To determine the relative growth advantages of polyomavirus regulatory region mutants selected from Friend leukemic and neuroblastoma cells persistently infected with polyomavirus A2 wild type, different murine cell lines, some of which are capable of further differentiation in vitro, were used as hosts in mixed infection and transfection. The tests allowed the determination, through the measurement of cis-advantage in replication, of the most effective polyomavirus regulatory region constitutions for a given cell line and, in some cases, for specific stages of cell differentiation. Furthermore, different domains of the viral regulatory region were shown to have different effects--positive, neutral, or negative--depending on the host analyzed. Images PMID:3033287

  3. Modulation of prostaglandin biosynthesis in murine mammary adenocarcinoma tumor cells

    SciTech Connect

    Shalinsky, D.R.

    1988-01-01

    In efforts to exploit the differential oxygen levels within the subcompartments of solid neoplasms, this project has focused on modulating prostaglandin (PG) biosynthesis under aerobic and hypoxic conditions. Mammary adenocarcinoma tumor cells (Line 4526), either intact or sonicated, were incubated with either 2.0 uM {sup 14}C-arachidonic acid (AA) or 20.0 uM {sup 14}C-PGH{sub 2}, respectively. Following metabolism, products were extracted, separated by thin layer chromatography and analyzed by radiochromatographic scan. PGE{sub 2} was predominantly formed with minimal amounts of PGF{sub 2a} or PGD{sub 2}. Indomethacin and ibuprofen inhibited the PGE{sub 2} formation from AA with an IC{sub 50} value of 6.3 {times} 10{sup {minus}8} and 9.6 {times} 10{sup {minus}5}M, respectively. Suspended cells in glass vials were made hypoxic by flushing with N{sub 2} for varying time intervals to study AA metabolism. A time-dependent inhibition of PG biosynthesis was observed under hypoxia, and by 30 min, the PGE{sub 2} synthesis was reduced by 50% which was further inhibited by indomethacin. Misonidazole, a 2-nitroimidazole analogue, partially reversed the inhibition of PGE{sub 2} synthesis under hypoxia by 49% at 100 uM. However, misonidazole did not affect PG biosynthesis under aerobic conditions. The stimulation of PGE{sub 2} biosynthesis by misonidazole under hypoxia was blocked by indomethacin, suggesting that misonidazole can not act independently of the cyclooxygenase.

  4. 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

  5. Differentiation of murine embryonic stem and induced pluripotent stem cells to renal lineage in vitro

    SciTech Connect

    Morizane, Ryuji [Department of Internal Medicine, Keio University School of Medicine, Tokyo (Japan)] [Department of Internal Medicine, Keio University School of Medicine, Tokyo (Japan); Monkawa, Toshiaki, E-mail: monkawa@sc.itc.keio.ac.jp [Department of Internal Medicine, Keio University School of Medicine, Tokyo (Japan)] [Department of Internal Medicine, Keio University School of Medicine, Tokyo (Japan); Itoh, Hiroshi [Department of Internal Medicine, Keio University School of Medicine, Tokyo (Japan)] [Department of Internal Medicine, Keio University School of Medicine, Tokyo (Japan)

    2009-12-25

    Embryonic stem (ES) cells which have the unlimited proliferative capacity and extensive differentiation potency can be an attractive source for kidney regeneration therapies. Recent breakthroughs in the generation of induced pluripotent stem (iPS) cells have provided with another potential source for the artificially-generated kidney. The purpose of this study is to know how to differentiate mouse ES and iPS cells into renal lineage. We used iPS cells from mouse fibroblasts by transfection of four transcription factors, namely Oct4, Sox2, c-Myc and Klf4. Real-time PCR showed that renal lineage markers were expressed in both ES and iPS cells after the induction of differentiation. It also showed that a tubular specific marker, KSP progressively increased to day 18, although the differentiation of iPS cells was slower than ES cells. The results indicated that renal lineage cells can be differentiated from both murine ES and iPS cells. Several inducing factors were tested whether they influenced on cell differentiation. In ES cells, both of GDNF and BMP7 enhanced the differentiation to metanephric mesenchyme, and Activin enhanced the differentiation of ES cells to tubular cells. Activin also enhanced the differentiation of iPS cells to tubular cells, although the enhancement was lower than in ES cells. ES and iPS cells have a potential to differentiate to renal lineage cells, and they will be an attractive resource of kidney regeneration therapy. This differentiation is enhanced by Activin in both ES and iPS cells.

  6. Identification and enrichment of colony-forming cells from the adult murine pituitary

    SciTech Connect

    Lepore, D.A. [Pituitary Research Unit, Murdoch Childrens Research Institute, Royal Children's Hospital, Parkville, Victoria 3052 (Australia); Roeszler, K. [Pituitary Research Unit, Murdoch Childrens Research Institute, Royal Children's Hospital, Parkville, Victoria 3052 (Australia); Wagner, J. [Pituitary Research Unit, Murdoch Childrens Research Institute, Royal Children's Hospital, Parkville, Victoria 3052 (Australia); Department of Paediatrics, University of Melbourne, Melbourne, Victoria (Australia); Ross, S.A. [Pituitary Research Unit, Murdoch Childrens Research Institute, Royal Children's Hospital, Parkville, Victoria 3052 (Australia); Bauer, K. [Max-Planck-Institut fuer Experimentelle Endokrinologie, Hannover (Germany); Thomas, P.Q. [Pituitary Research Unit, Murdoch Childrens Research Institute, Royal Children's Hospital, Parkville, Victoria 3052 (Australia)], E-Mail: paul.thomas@mcri.edu.au

    2005-08-01

    Stem and progenitor cells have been identified in many adult tissues including bone marrow, the central nervous system, and skin. While there is direct evidence to indicate the activity of a progenitor cell population in the pituitary gland, this putative subpopulation has not yet been identified. Herein we describe the isolation and characterization of a novel clonogenic cell type in the adult murine pituitary, which we have termed Pituitary Colony-Forming Cells (PCFCs). PCFCs constitute 0.2% of pituitary cells, and generate heterogeneous colonies from single cells. PCFCs exhibit variable proliferative potential, and may exceed 11 population doublings in 14 days. Enrichment of PCFCs to 61.5-fold with 100% recovery can be obtained through the active uptake of the fluorescent dipeptide, {beta}-Ala-Lys-N{epsilon}-AMCA. PCFCs are mostly contained within the large, agranular subpopulation of AMCA{sup +} cells, and constitute 28% of this fraction, corresponding to 140.5-fold enrichment. Interestingly, the AMCA{sup +} population contains rare cells that are GH{sup +} or PRL{sup +}. GH{sup +} cells were also identified in PCFC single cell colonies, suggesting that PCFCs have the potential to differentiate into GH{sup +} cells. Together, these data show that the pituitary contains a rare clonogenic population which may correspond to the somatotrope/lactotrope progenitors suggested by previous experiments.

  7. Identification of two regulatory elements controlling Fucosyltransferase 7 transcription in murine CD4+ T cells.

    PubMed

    Pink, Matthias; Ratsch, Boris A; Mardahl, Maibritt; Schröter, Micha F; Engelbert, Dirk; Triebus, Julia; Hamann, Alf; Syrbe, Uta

    2014-11-01

    Fucosyltransferase VII encoded by the gene Fut7 is essential in CD4(+) T cells for the generation of E- and P-selectin ligands (E- and P-lig) which facilitate recruitment of lymphocytes into inflamed tissues and into the skin. This study aimed to identify regulatory elements controlling the inducible Fut7 expression in CD4(+) T cells that occurs upon activation and differentiation of naive T cells into effector cells. Comparative analysis of the histone modification pattern in non-hematopoetic cells and CD4(+) T cell subsets revealed a differential histone modification pattern within the Fut7 locus including a conserved non-coding sequence (CNS) identified by cross-species conservation comparison suggesting that regulatory elements are confined to this region. Cloning of the CNS located about 500 bp upstream of the Fut7 locus, into a luciferase reporter vector elicited reporter activity after transfection of the ??-WT T cell line, but not after transfection of primary murine CD4(+) Th1 cells. As quantification of different Fut7 transcripts revealed a predominance of transcripts lacking the first exons in primary Th1 cells we searched for an alternative promoter. Cloning of an intragenic region spanning a 1kb region upstream of exon 4 into an enhancer-containing vector indeed elicited promoter activity. Interestingly, also the CNS enhanced activity of this intragenic minimal promoter in reporter assays in primary Th1 cells suggesting that both elements interact in primary CD4(+) T cells to induce Fut7 transcription. PMID:24915132

  8. Liver sinusoidal endothelial cells are a site of murine cytomegalovirus latency and reactivation.

    PubMed

    Seckert, Christof K; Renzaho, Angélique; Tervo, Hanna-Mari; Krause, Claudia; Deegen, Petra; Kühnapfel, Birgit; Reddehase, Matthias J; Grzimek, Natascha K A

    2009-09-01

    Latent cytomegalovirus (CMV) is frequently transmitted by organ transplantation, and its reactivation under conditions of immunosuppressive prophylaxis against graft rejection by host-versus-graft disease bears a risk of graft failure due to viral pathogenesis. CMV is the most common cause of infection following liver transplantation. Although hematopoietic cells of the myeloid lineage are a recognized source of latent CMV, the cellular sites of latency in the liver are not comprehensively typed. Here we have used the BALB/c mouse model of murine CMV infection to identify latently infected hepatic cell types. We performed sex-mismatched bone marrow transplantation with male donors and female recipients to generate latently infected sex chromosome chimeras, allowing us to distinguish between Y-chromosome (gene sry or tdy)-positive donor-derived hematopoietic descendants and Y-chromosome-negative cells of recipients' tissues. The viral genome was found to localize primarily to sry-negative CD11b(-) CD11c(-) CD31(+) CD146(+) cells lacking major histocompatibility complex class II antigen (MHC-II) but expressing murine L-SIGN. This cell surface phenotype is typical of liver sinusoidal endothelial cells (LSECs). Notably, sry-positive CD146(+) cells were distinguished by the expression of MHC-II and did not harbor latent viral DNA. In this model, the frequency of latently infected cells was found to be 1 to 2 per 10(4) LSECs, with an average copy number of 9 (range, 4 to 17) viral genomes. Ex vivo-isolated, latently infected LSECs expressed the viral genes m123/ie1 and M122/ie3 but not M112-M113/e1, M55/gB, or M86/MCP. Importantly, in an LSEC transfer model, infectious virus reactivated from recipients' tissue explants with an incidence of one reactivation per 1,000 viral-genome-carrying LSECs. These findings identified LSECs as the main cellular site of murine CMV latency and reactivation in the liver. PMID:19535440

  9. Murine T-cell response to native and recombinant protein antigens of Rickettsia tsutsugamushi.

    PubMed Central

    Hickman, C J; Stover, C K; Joseph, S W; Oaks, E V

    1993-01-01

    A polyclonal T-cell line with TH1 characteristics was used to assess the murine cellular immune response to native and recombinant Rickettsia tsutsugamushi antigens. Proliferation of this T-cell line was observed in response to numerous native antigen fractions, which indicates that the murine T-helper-cell response is directed at multiple scrub typhus antigens with no apparent antigenic immunodominance. Subsequent analysis of recombinant R. tsutsugamushi antigens made it possible to identify a 47-kDa scrub typhus antigen (Sta47) that was stimulatory for the polyclonal T-cell line. Recombinant clones encoding 56-, 58-, and 110-kDa antigens (Sta56, Sta58, and Sta110, respectively) were unable to induce proliferation of this T-cell line. DNA sequence analysis of the cloned rickettsial insert encoding the Sta47 protein revealed the presence of four open reading frames potentially encoding proteins of 47, 30, 18, and 13 kDa. Analysis of sodium dodecyl sulfate-polyacrylamide gel electrophoresis-separated and eluted fractions of lysates from the recombinant HB101(pRTS47B4.3) demonstrated that the fractions containing the 47-kDa protein as well as those containing proteins less than 18 kDa were stimulatory. Selected synthetic amphipathic peptides derived from the Sta47 antigen sequence identified a 20-amino-acid peptide that gave a 10-fold increase in T-cell proliferation over a control malarial peptide of similar length. Recognition of the 47-kDa antigen by a T-cell line with TH1 characteristics implicates this protein as one of potential importance in protection studies and future vaccine development. Images PMID:8478055

  10. Cyclic-radiation response of murine fibrosarcoma cells grown as pulmonary nodules

    SciTech Connect

    Grdina, D.J.; Hunter, N.

    1982-10-01

    The radiation age response of murine fibrosarcoma (FSa) cells grown as pulmonary nodules in C/sub 3/Hf/Kam mice was determined. FSa cells were irradiated in vivo either with 10 Gy as 14 day-old lung tumors (i.e., artificial macrometastases) prior to cell separation or with 5 Gy as single cells trapped in the lungs of recipient mice (i.e., artificial micrometastases) following cell separation and synchronization by centrifugal elutriation. Flow microfluorometry (FMF) was used to determine cell-cycle parameters and the relative synchrony of the separated populations, as well as the percent contamination of normal diploid cells in each of the tumor cell populations. Tumor populations containing up to 90% G/sub 1/, 60% S-, and 75% G/sub 2/+M-phase tumor cells were obtained. Cell clonogenicity, determined using a lung colony assay, ranged from 0.7 to 6% for control FSa cells from the various elutriator fractions. The radiation sensitivity of these separated cell populations varied by a factor of 6, regardless of whether the cells were irradiated as artificial micro or macro-metastases. In each experiment, tumor populations most enriched in s-phase cells exhibited the greatest radiation sensitivity. To confirm that these populations were highly enriched in S-phase cells and to demonstrate that they were more radiosensitive than FSa cells in other parts of the cell cycle, the elutriated tumor populations were exposed to either suicide labeling by high specific activity tritiated thymidine or hydroxyurea. The resultant age response curves were qualitatively similar to those obtained following irradiation and reflected the S-phase sensitivity of FSa cells to these agents.

  11. Functional Heterogeneity in the CD4+ T Cell Response to Murine ?-Herpesvirus 68.

    PubMed

    Hu, Zhuting; Blackman, Marcia A; Kaye, Kenneth M; Usherwood, Edward J

    2015-03-15

    CD4(+) T cells are critical for the control of virus infections, T cell memory, and immune surveillance. We studied the differentiation and function of murine ?-herpesvirus 68 (MHV-68)-specific CD4(+) T cells using gp150-specific TCR-transgenic mice. This allowed a more detailed study of the characteristics of the CD4(+) T cell response than did previously available approaches for this virus. Most gp150-specific CD4(+) T cells expressed T-bet and produced IFN-?, indicating that MHV-68 infection triggered differentiation of CD4(+) T cells largely into the Th1 subset, whereas some became follicular Th cells and Foxp3(+) regulatory T cells. These CD4(+) T cells were protective against MHV-68 infection in the absence of CD8(+) T cells and B cells, and protection depended on IFN-? secretion. Marked heterogeneity was observed in the CD4(+) T cells, based on lymphocyte Ag 6C (Ly6C) expression. Ly6C expression positively correlated with IFN-?, TNF-?, and granzyme B production; T-bet and KLRG1 expression; proliferation; and CD4(+) T cell-mediated cytotoxicity. Ly6C expression inversely correlated with survival, CCR7 expression, and secondary expansion potential. Ly6C(+) and Ly6C(-) gp150-specific CD4(+) T cells were able to interconvert in a bidirectional manner upon secondary Ag exposure in vivo. These results indicate that Ly6C expression is closely associated with antiviral activity in effector CD4(+) T cells but is inversely correlated with memory potential. Interconversion between Ly6C(+) and Ly6C(-) cells may maintain a balance between the two Ag-specific CD4(+) T cell populations during MHV-68 infection. These findings have significant implications for Ly6C as a surface marker to distinguish functionally distinct CD4(+) T cells during persistent virus infection. PMID:25662997

  12. Mast Cells Modulate Acute Toxoplasmosis in Murine Models

    PubMed Central

    Chen, Ying; Zheng, Huanqin; Shen, Jilong; Lun, Zhao-Rong; Wang, Yong; Kasper, Lloyd H.; Lu, Fangli

    2013-01-01

    The role of mast cells (MCs) in Toxoplasma gondii infection is poorly known. Kunming outbred mice were infected intraperitoneally with RH strain T. gondii, either treated with compound 48/80 (C48/80, MC activator) or disodium cromoglycate (DSCG, MC inhibitor). Compared with infected controls, infected mice treated with C48/80 exhibited significantly increased inflammation in the liver (P < 0.01), spleen (P < 0.05), and mesentery (P < 0.05) tissues, higher parasite burden in the peritoneal lavage fluids (P < 0.01), and increased levels of mRNA transcripts of T. gondii tachyzoite surface antigen 1 (SAG1) gene in the spleen and liver tissues (P < 0.01), accompanied with significantly increased Th1 cytokine (IFN-?, IL-12p40, and TNF-?) (P < 0.01) and decreased IL-10 (P < 0.01) mRNA expressions in the liver, and increased IFN-? (P < 0.01) and IL-12p40 (P < 0.01) but decreased TNF-? (P < 0.01) and IL-4 (P < 0.01) in the spleens of infected mice treated with C48/80 at day 9-10 p.i. Whereas mice treated with DSCG had significantly decreased tissue lesions (P < 0.01), lower parasite burden in the peritoneal lavage fluids (P < 0.01) and decreased SAG1 expressions in the spleen and liver tissues (P < 0.01), accompanied with significantly increased IFN-? (P < 0.01) and IL-12p40 (P < 0.05) in the liver, and decreased IFN-? (P < 0.05) and TNF-? (P < 0.01) in the spleens; IL-4 and IL-10 expressions in both the spleen and liver were significantly increased (P < 0.01) in the infected mice treated with DSCG. These findings suggest that mediators associated with the MC activation may play an important role in modulating acute inflammatory pathogenesis and parasite clearance during T. gondii infection in this strain of mice. Thus, MC activation/inhibition mechanisms are potential novel targets for the prevention and control of T. gondii infection. PMID:24146978

  13. Tumor-initiating cells are enriched in CD44(hi) population in murine salivary gland tumor.

    PubMed

    Shen, Shukun; Yang, Wenjun; Wang, Zhugang; Lei, Xia; Xu, Liqun; Wang, Yang; Wang, Lizhen; Huang, Lei; Yu, Zhiwei; Zhang, Xinhong; Li, Jiang; Chen, Yan; Zhao, Xiaoping; Yin, Xuelai; Zhang, Chenping

    2011-01-01

    Tumor-initiating cells (T-ICs) discovered in various tumors have been widely reported. However, T-IC populations in salivary gland tumors have yet to be elucidated. Using the established Pleomorphic Adenoma Gene-1 (Plag1) transgenic mouse model of a salivary gland tumor, we identified CD44(high) (CD44(hi)) tumor cells, characterized by high levels of CD44 cell surface expression, as the T-ICs for pleomorphic adenomas. These CD44(hi) tumor cells incorporated 5-bromo-2-deoxyuridine (BrdU), at a lower rate than their CD44(negative) (CD44(neg)) counterparts, and also retained BrdU for a long period of time. Cell surface maker analysis revealed that 25% of the CD44(hi) tumor cells co-express other cancer stem cell markers such as CD133 and CD117. As few as 500 CD44(hi) tumor cells were sufficient to initiate pleomorphic adenomas in one third of the wildtype mice, whereas more than 1×10(4) CD44(neg) cells were needed for the same purpose. In NIH 3T3 cells, Plag1 was capable of activating the gene transcription of Egr1, a known upregulator for CD44. Furthermore, deletion of sequence 81-96 in the Egr1 promoter region abolished the effect of Plag1 on Egr1 upregulation. Our results establish the existence of T-ICs in murine salivary gland tumors, and suggest a potential molecular mechanism for CD44 upregulation. PMID:21858056

  14. Perforin Gene Transfer Into Hematopoietic Stem Cells Improves Immune Dysregulation in Murine Models of Perforin Deficiency

    PubMed Central

    Carmo, Marlene; Risma, Kimberly A; Arumugam, Paritha; Tiwari, Swati; Hontz, Adrianne E; Montiel-Equihua, Claudia A; Alonso-Ferrero, Maria E; Blundell, Michael P; Schambach, Axel; Baum, Christopher; Malik, Punam; Thrasher, Adrian J; Jordan, Michael B; Gaspar, H Bobby

    2015-01-01

    Defects in perforin lead to the failure of T and NK cell cytotoxicity, hypercytokinemia, and the immune dysregulatory condition known as familial hemophagocytic lymphohistiocytosis (FHL). The only curative treatment is allogeneic hematopoietic stem cell transplantation which carries substantial risks. We used lentiviral vectors (LV) expressing the human perforin gene, under the transcriptional control of the ubiquitous phosphoglycerate kinase promoter or a lineage-specific perforin promoter, to correct the defect in different murine models. Following LV-mediated gene transfer into progenitor cells from perforin-deficient mice, we observed perforin expression in mature T and NK cells, and there was no evidence of progenitor cell toxicity when transplanted into irradiated recipients. The resulting perforin-reconstituted NK cells showed partial recovery of cytotoxicity, and we observed full recovery of cytotoxicity in polyclonal CD8+ T cells. Furthermore, reconstituted T cells with defined antigen specificity displayed normal cytotoxic function against peptide-loaded targets. Reconstituted CD8+ lymphoblasts had reduced interferon-? secretion following stimulation in vitro, suggesting restoration of normal immune regulation. Finally, upon viral challenge, mice with >30% engraftment of gene-modified cells exhibited reduction of cytokine hypersecretion and cytopenias. This study demonstrates the potential of hematopoietic stem cell gene therapy as a curative treatment for perforin-deficient FHL. PMID:25523759

  15. Growth of Trypanosoma musculi in cultures of murine spleen cells and analysis of the requirement for supportive spleen cells.

    PubMed Central

    Albright, J W; Albright, J F

    1978-01-01

    Growth of Trypanosoma musculi in vitro has been achieved. The number of parasites increased by more than 1,500-fold in less than 8 days under the most suitable conditions. The rate and magnitude of growth was comparable to that which occurs in inoculated murine hosts. Maximum growth was displayed in cultures composed of RPMI 1640 medium supplemented with fetal calf serum, murine spleen cells, and foreign erythrocytes (sheep). No growth occurred in the absence of spleen cells. The adherent, macrophage-rich population supported parasite growth much better than did the nonadherent population. Parasite growth was excellent in the presence of irradiated spleen cells or of cells from thymectomized, irradiated, bone marrow-reconstituted mice. The important cells appeared to be macrophages. The beneficial effect of sheep erythrocytes probably resulted from preoccupation or stimulation of phagocytes. Soluble substances released by spleen cell cultures promote parasite growth, as was shown by experiments with double-compartment culture vessels. The utility of this culture system for analysis of host immune responses against the trypanosome was demonstrated. PMID:730358

  16. Single-nucleotide polymorphisms in the LRWD1 gene may be a genetic risk factor for Japanese patients with Sertoli cell-only syndrome.

    PubMed

    Miyamoto, T; Koh, E; Tsujimura, A; Miyagawa, Y; Saijo, Y; Namiki, M; Sengoku, K

    2014-04-01

    Genetic mechanisms have been implicated as a cause of some cases of male infertility. Recently, ten novel genes involved in human spermatogenesis, including human LRWD1, have been identified by expression microarray analysis of human testictissue. The human LRWD1 protein mediates the origin recognition complex in chromatin, which is critical for the initiation of pre-replication complex assembly in G1 and chromatin organization in post-G1 cells. The Lrwd1 gene expression is specific to the testis in mice. Therefore, we hypothesized that mutation or polymorphisms of LRWD1 participate in male infertility, especially azoospermia. To investigate whether LRWD1 gene defects are associated with azoospermia caused by SCOS and meiotic arrest (MA), mutational analysis was performed in 100 and 30 Japanese patients by direct sequencing of the coding regions, respectively. Statistical analysis was performed for patients with SCOS and MA and in 100 healthy control men. No mutations were found in LRWD1; however, three coding single-nucleotide polymorphisms (SNP1-SNP3) could be detected in the patients. The genotype and allele frequencies in SNP1 and SNP2 were notably higher in the SCOS group than in the control group (P < 0.05). These results suggest the critical role of LRWD1 in human spermatogenesis. PMID:23445371

  17. 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

  18. 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

  19. Interleukin 21 signaling in B cells is required for efficient establishment of murine gammaherpesvirus latency.

    PubMed

    Collins, Christopher M; Speck, Samuel H

    2015-04-01

    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

  20. 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

  1. Macrophage Matrix Metalloproteinase-9 Mediates Epithelial-Mesenchymal Transition in Vitro in Murine Renal Tubular Cells

    PubMed Central

    Tan, Thian Kui; Zheng, Guoping; Hsu, Tzu-Ting; Wang, Ying; Lee, Vincent W.S.; Tian, Xinrui; Wang, Yiping; Cao, Qi; Wang, Ya; Harris, David C.H.

    2010-01-01

    As a rich source of pro-fibrogenic growth factors and matrix metalloproteinases (MMPs), macrophages are well-placed to play an important role in renal fibrosis. However, the exact underlying mechanisms and the extent of macrophage involvement are unclear. Tubular cell epithelial?mesenchymal transition (EMT) is an important contributor to renal fibrosis and MMPs to induction of tubular cell EMT. The aim of this study was to investigate the contribution of macrophages and MMPs to induction of tubular cell EMT. The murine C1.1 tubular epithelial cell line and primary tubular epithelial cells were cultured in activated macrophage-conditioned medium (AMCM) derived from lipopolysaccharide-activated J774 macrophages. MMP-9, but not MMP-2 activity was detected in AMCM. AMCM-induced tubular cell EMT in C1.1 cells was inhibited by broad-spectrum MMP inhibitor (GM6001), MMP-2/9 inhibitor, and in AMCM after MMP-9 removal by monoclonal Ab against MMP-9. AMCM-induced EMT in primary tubular epithelial cells was inhibited by MMP-2/9 inhibitor. MMP-9 induced tubular cell EMT in both C1.1 cells and primary tubular epithelial cells. Furthermore, MMP-9 induced tubular cell EMT in C1.1 cells to an extent similar to transforming growth factor-?. Transforming growth factor-?-induced tubular cell EMT in C1.1 cells was inhibited by MMP-2/9 inhibitor. Our in vitro study provides evidence that MMPs, specifically MMP-9, secreted by effector macrophages can induce tubular cell EMT and thereby contribute to renal fibrosis. PMID:20075196

  2. Cyclic-radiation response of murine fibrosarcoma cells grown as pulmonary nodules

    SciTech Connect

    Grdina, D.J.; Hunter, N.

    1982-10-01

    The radiation age response of murine fibrosarcoma (FSa) cells grown as pulmonary nudules in C/sub 3/Hf/Kam mice was determined. FSa cells were irradiated in vivo either with 10 Gy as 14 day-old lung tumors (i.e., artifical micrometastases) following cell separation and synchronization by centrifugal elutriation. Flow microfluorometry (FMF) was used to determine cell-cycle parameters and the relative synchrony of the separated populations, as well as the percent contamination of normal diploid cells in each of the tumor cells populations. Tumor populations containing up to 90% G/sub 1/-, 60% S-, and 75% G/sub 2/+M-phase tumor cells were obtained. Cell clonogenicity, determined using a lung colony assay, ranged from 0.7 to 6% for control FSa cells from the various elutriator fractions. The radiation sensitivity of these separated cell populations varied by a factor of 6, regardless of whether the cells were irradiated as artifical micro or macro-metastases. In each experiment, tumor population most enriched in S-phase cells exhibited the greatest radiation sensitivity. To confirm that these populations were highly enriched in S-phase cells and to demonstrate that they were more radiosensitive than FSa cells in other parts of the cell cycle, the elutriated tumor population were exposed to either suicide labeling by high specific activity tritated thymidine or hydroxyurea. The resultant age response curves were qualitatively similar to those obtained following irradiation and reflected the S-phase sensitivity of FSa cells to these agents.

  3. 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.

  4. Histamine suppresses regulatory T cells mediated by TGF-? in murine chronic allergic contact dermatitis.

    PubMed

    Tamaka, Kyoko; Seike, Masahiro; Hagiwara, Tamio; Sato, Atsushi; Ohtsu, Hiroshi

    2015-04-01

    Regulatory T cells (Tregs) suppress effector T cells and ameliorate contact hypersensitivity (CH); however, the role of Tregs in chronic allergic contact dermatitis (CACD) has not been assessed. Repeated elicitation of CH has been used to produce CACD models in mice. We previously showed that the presence of histamine facilitates the creation of eczematous lesions in this model using histidine decarboxylase (HDC) (-/-) mice. Therefore, the effects of histamine on Tregs in the CACD model were investigated in this study. CACD was developed by repeated epicutaneous application of 2, 4, 6-trinitro-1-chlorobenzene (TNCB) on HDC (+/+) and HDC (-/-) murine skin to assess the effects of histamine in CACD. Histamine aggravated CACD in the murine model and suppressed the number of Tregs in the skin. Histamine also suppressed the level of TGF-?1 in this model. Recombinant TGF-?1 or anti-TGF-?1 antibody was injected into the dorsal dermis of HDC (+/+) mice daily just before TNCB challenge to determine the effects of histamine-regulated TGF-? on the Treg population in CACD. Recombinant TGF-?1 injection promoted the infiltration of Tregs in the skin and the production of IL-10; however, anti-TGF-?1 antibody injection suppressed the number of Tregs in the skin and the production of IL-10. Histamine suppresses the number of Tregs in CACD, and this effect is mediated by TGF-?. PMID:25651189

  5. Interferons modulate the expression of hormone receptors on the surface of murine melanoma cells.

    PubMed Central

    Kameyama, K; Tanaka, S; Ishida, Y; Hearing, V J

    1989-01-01

    The effects of IFN-alpha, IFN-beta, and IFN-gamma on the differentiation of murine melanoma cells has been studied, in the presence and absence of melanocyte-stimulating hormone (MSH); the cells were highly responsive to treatment with MSH, which increased the rate of melanin production 25-fold and tyrosinase activity 6-fold within 4 d. Treatment of melanoma cells with IFN-alpha, IFN-beta, or IFN-gamma alone had no stimulatory effect on melanin production, but when the cells were cultured with IFN in the presence of MSH, pigment production was significantly and synergistically increased relative to cells cultured with MSH only. Flow cytometric analysis revealed that levels of tyrosinase in the cells were not affected by MSH or by IFN, which suggests that stimulation of melanogenic activity occurred by activation of a preexisting cellular enzyme. Scatchard analyses showed that the number of MSH receptors on IFN-treated cells was significantly increased (approximately 2.5-fold) relative to untreated cells (approximately 61,000/cell). These findings demonstrate that IFN stimulate differentiation (that is, pigmentation) of melanocytes by increasing the expression of surface MSH receptors; this in turn suggests that such a mechanism may in part be responsible for postinflammatory skin pigmentation, and provides an additional basis for action in the clinical responses of melanoma to IFN treatment. PMID:2463267

  6. Erythropoietin acts as an anti-inflammatory signal on murine mast cells.

    PubMed

    Wiedenmann, Tanja; Ehrhardt, Stefanie; Cerny, Daniela; Hildebrand, Dagmar; Klein, Sabrina; Heeg, Klaus; Kubatzky, Katharina F

    2015-05-01

    Recently it was found that the erythropoietin receptor (EpoR) is expressed on innate immune cells, such as dendritic cells and macrophages. We found that murine bone marrow-derived mast cells express the EpoR and that its expression is increased under hypoxic conditions. Interestingly, Epo stimulation of the cells did not activate signal transducer and activator of transcription molecules, nor did we find differences in the expression of typical STAT-dependent genes, the proliferation rate, and the ability to differentiate or to protect the cells from apoptosis. Instead, we demonstrate that stimulation of mast cells with Epo leads to phosphorylation of the receptor tyrosine kinase c-kit. We hypothesize that this is due to the formation of a receptor complex between the EpoR and c-kit. The common beta chain of the IL-3 receptor family, which was described as part of the tissue protective receptor (TPR) on other non-erythroid cells, however is not activated. To investigate whether the EpoR/c-kit complex has tissue protective properties, cells were treated with the Toll-like receptor ligand LPS. Combined Epo and LPS treatment downregulated the inflammatory response of the cells as detected by a decrease in IL-6 and TNF-? secretion. PMID:25645506

  7. Production and Characterization of Monoclonal Antibodies to Fcy 2a-Binding Protein Isolated From the Detergent Lysate of a Murine Macrophagelike Cell Line, P388D1

    Microsoft Academic Search

    Masaru Kagami; Yoshiyuki Funatsu; Tsuneo Suzuki

    Hybridoma cell lines were produced by fusion of SP2\\/0 murine myeloma cell line with the spleen cells of Wister rats which were immunized with IgG?-bindlng protein isolated from the detergent lysate of a murine macrophagelike cell line, P388D1, by affinity chromatography on IgG-Sepharose 4B. A monoclonai clone (designated as 3A2) out of a total of 13 different antibody-secreting cell lines

  8. Knockdown of the L3/Lhx8 gene suppresses cholinergic differentiation of murine embryonic stem cell-derived spheres.

    PubMed

    Manabe, Takayuki; Tatsumi, Kouko; Inoue, Masahide; Matsuyoshi, Hiroko; Makinodan, Manabu; Yamauchi, Takahira; Makinodan, Eri; Yokoyama, Shohei; Sakumura, Ryoko; Okuda, Hiroaki; Wanaka, Akio

    2008-04-01

    L3/Lhx8, a member of the Lim-homeobox gene family, is selectively and specifically expressed in the murine embryonic medial ganglionic eminence (MGE). Our previous study demonstrated that L3/Lhx8-deficient mice specifically lack cholinergic neurons in the basal forebrain. In this manuscript, we report the in vitro effects of reduced L3/Lhx8 gene expression on cholinergic differentiation in murine embryonic stem (ES) cell-derived spheres without dissociation. The knockdown of L3/Lhx8 gene expression dramatically decreased the cholinergic phenotype of spheres without altering other known phenotypes (TuJ1, GABA and GFAP). These results strongly suggest that L3/Lhx8 is a key factor for cholinergic differentiation of murine ES cell-derived spheres and is involved in basal forebrain development. PMID:18248940

  9. Anti-inflammatory effects of natural product formulations on murine dendritic cells

    PubMed Central

    Miller, Andrea K.; Benson, Jenna M.; Muanza, Dave N.; Smith, Jerry R.; Shepherd, David M.

    2011-01-01

    The popularity and availability of herbal extracts has increased dramatically over the last decade, providing an inexpensive manner of self-medication. Although the efficacy of individual extracts is currently being intensively studied, research regarding complex mixtures is limited. Therefore, we evaluated the effects of three complex formulations including BRC-301, a polyherbal extract; BRC-304, a mixture of vitamins, minerals, antioxidant enzymes, botanical extracts and carotenoids; and BRC-306, a proprietary blend of Uncaria tomentosa (cat’s claw) and Phytolens® on murine dendritic cells (DCs). We hypothesized that these formulations would decrease the inflammatory responsiveness and innate function of DCs. To address this hypothesis, we evaluated the effects of BRC-301, 304, and 306 on DC2.4 cells and further assessed the effects of BRC-301 on bone marrow-derived DCs (bmDCs). LPS stimulation of DC2.4 cells and bmDCs induced production of NO, TNF-?, and IL-6, a response that was modulated by concomitant treatment with non-cytotoxic concentrations of BRC-301. In contrast, only the production of NO or IL-6 by LPS-activated DC2.4 cells was affected by BRC-304 or BRC-306, respectively. Flow cytometric evaluation following concurrent BRC-301 and LPS treatment revealed an increased relative expression of CD11c, CD86, and CD54 on bmDCs and an increased frequency of bmDCs expressing MHC II. Finally, BRC-301 enhanced the uptake of FITC-conjugated ovalbumin by bmDCs. Taken together, these results suggest that these commercially available formulations modulate the innate responsiveness of murine DCs and may enhance their ability to initiate T cell-mediated immunity. PMID:21399725

  10. Anti-inflammatory effects of natural product formulations on murine dendritic cells.

    PubMed

    Miller, Andrea K; Benson, Jenna M; Muanza, Dave N; Smith, Jerry R; Shepherd, David M

    2011-03-01

    The popularity and availability of herbal extracts has increased dramatically over the last decade, providing an inexpensive manner of self-medication. Although the efficacy of individual extracts is currently being studied intensively, research regarding complex mixtures is limited. Therefore, we evaluated the effects of three complex formulations, including BRC-301, a polyherbal extract; BRC-304, a mixture of vitamins, minerals, antioxidant enzymes, botanical extracts, and carotenoids; and BRC-306, a proprietary blend of Uncaria tomentosa (cat's claw) and Phytolens(®) on murine dendritic cells (DCs). We hypothesized that these formulations would decrease the inflammatory responsiveness and innate function of DCs. In order to address this hypothesis, we evaluated the effects of BRC-301, BRC-304, and BRC-306 on DC2.4 cells and assessed the effects of BRC-301 on bone marrow-derived DCs (bmDCs). Lipopolysaccharide (LPS) stimulation of DC2.4 cells and bmDCs induced production of nitric oxide (NO), TNF-?, and IL-6, a response that was modulated by concomitant treatment with non-cytotoxic concentrations of BRC-301. In contrast, only the production of NOor IL-6 by LPS-activated DC2.4 cells was affected by BRC-304 or BRC-306, respectively. Flow cytometric evaluation following concurrent BRC-301 and LPS treatment revealed an increased relative expression of CD11c, CD86, and CD54 on bmDCs and an increased frequency of bmDCs expressing MHC II. Finally, BRC-301 enhanced the uptake of fluorescein isothiocyanate-conjugated ovalbumin by bmDCs. Taken together, these results suggest that these commercially available formulations modulate the innate responsiveness of murine DCs and may enhance their ability to initiate T cell-mediated immunity. PMID:21399725

  11. Selective erythroid replacement in murine beta-thalassemia using fetal hematopoietic stem cells.

    PubMed Central

    Bethel, C. A.; Murugesh, D.; Harrison, M. R.; Mohandas, N.; Rubin, E. M.

    1993-01-01

    We have explored the application of fetal hematopoietic stem cell (HSC) transplants for cellular replacement in a murine model of beta-thalassemia. Liver-derived HSCs from nonthalassemic syngeneic murine fetal donors were transplanted into nonirradiated neonatal beta-thalassemic recipients. Significant erythrocyte chimerism (9-27%) was demonstrated in the majority of recipients at 1 month and remained stable or increased (up to 55%) during long-term follow-up in almost all cases. Chimeras had improved phenotypes, as evidenced by decreased reticulocyte counts, increased mean erythrocyte deformability, and decreased iron deposits in comparison to controls. To investigate whether the high degree of peripheral blood chimerism was predominantly a feature of erythroid elements or was a general feature of all hematopoietic elements, chimeras were created using donor HSCs "tagged" with a DNA transgene. Whereas donor hemoglobin comprised > 30% of total hemoglobin, nucleated tagged nonerythroid donor cells comprised < 1% of peripheral blood elements. Explanations for the observed selective increase in erythroid chimerism include longer survival of normal donor red cells compared to that of thalassemic red cells and the effective maturation of the donor erythroid elements in the bone marrow in chimeric animals. The latter explanation bears consideration because it is consistent with the process of ineffective erythropoiesis, well documented to occur in thalassemia, in which the majority of thalassemic erythroid cells are destroyed during erythropoiesis prior to release from the bone marrow. Overall, these data demonstrate the potential for significant erythroid chimerism and suggest that fetal HSC transplantation may play a significant role in future treatment. Images Fig. 1 Fig. 4 Fig. 7 PMID:7980734

  12. Dynamic Expression of BCL6 in Murine Conventional Dendritic Cells during In Vivo Development and Activation

    PubMed Central

    Zhang, Ting-ting; Liu, Dong; Calabro, Samuele; Eisenbarth, Stephanie C.; Cattoretti, Giorgio; Haberman, Ann M.

    2014-01-01

    The transcriptional repressor BCL6 plays an essential role in the development of germinal center B cells and follicular helper T cells. However, much less is known about the expression and function of BCL6 in other cell types. Here we report that during murine dendritic cell (DC) ontogeny in vivo, BCL6 is not expressed in bone marrow hematopoietic stem cells, common DC precursors and committed precursors of conventional DCs (pre-cDCs), but is elevated in peripheral pre-cDCs. BCL6 protein levels rise as pre-cDCs differentiate into cDCs in secondary lymphoid organs. Elevated protein levels of Bcl6 are observed in all cDC subsets, with CD8?+ cDCs displaying the greatest levels. Co-staining of Ki-67 revealed BCL6hi cDCs to be more proliferative than BCL6lo cDCs. After adjuvant inoculation, BCL6 levels are significantly reduced in the CD11cint MHC class IIhi CD86hi cDCs. Activation-induced BCL6 reduction correlated with reduced proliferation. A LPS injection study further confirmed that, in response to microbial stimuli, BCL6 levels are dynamically regulated during the maturation of CD11cint MHC class IIhi splenic cDCs. This reduction of BCL6 levels in cDCs does not occur after LPS injection in MyD88?/? TRIF?/? mice. Thus, regulation of Bcl6 protein levels is dynamic in murine cDCs during development, maturation and activation in vivo. PMID:24979752

  13. Distribution of cells bearing receptors for a colony-stimulating factor (CSF-1) in murine tissues

    PubMed Central

    1981-01-01

    CSF-1 is a subclass of the colony-stimulating factors that specifically stimulates the growth of mononuclear phagocytes. We used the binding of 125I-CSF-1 at 0 degrees C by single cell suspensions from various murine tissues, in conjunction with radioautography, to determine the frequency of binding cells, their identity, and the number of binding sites per binding cell. For all tissues examined, saturation of binding sites was achieved within 2 h at 2--3 x 10(-10) M 125I-CSF-1. The binding was irreversible and almost completely blocked by a 2 h preincubation with 5 x 10(-10) M CSF-1. 125I-CSF-1 binding was exhibited by 4.3% of bone marrow cells, 7.5% of blood mononuclear cells, 2.4% of spleen cells, 20.5% of peritoneal cells, 11.8% of pulmonary alveolar cells and 0.4% of lymph node cells. Four morphologically distinguishable cell types bound 125I-CSF-1: blast cells; mononuclear cells with a ratio of nuclear to cytoplasmic area (N/C) greater than 1; cells with indented nuclei; and mononuclear cells with N/C less than or equal to 1. No CSF-1 binding cells were detected among blood granulocytes or thymus cells. Bone marrow promyelocytes, myelocytes, neutrophilic granulocytes, eosinophilic granulocytes, nucleated erythroid cells, enucleated erythrocytes, and megakaryocytes also failed to bind. The frequency distribution of grain counts per cell for blood mononuclear cells was homogenous. In contrast, those for bone marrow, spleen, alveolar, and peritoneal cells were heterogeneous. The monocytes in blood or bone marrow (small cells, with either indented nuclei or with N/C greater than 1) were relatively uniformly labeled, possessing approximately 3,000 binding sites per cell. Larger binding cells (e.g., alveolar cells) may possess higher numbers of receptors. It is concluded that CSF-1 binding is restricted to mononuclear phagocytic cells and their precursors and that it can be used to identify both mature and immature cells of this series. PMID:6276411

  14. 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

  15. CYLD deletion triggers nuclear factor-?B-signaling and increases cell death resistance in murine hepatocytes

    PubMed Central

    Urbanik, Toni; Koehler, Bruno Christian; Wolpert, Laura; Elßner, Christin; Scherr, Anna-Lena; Longerich, Thomas; Kautz, Nicole; Welte, Stefan; Hövelmeyer, Nadine; Jäger, Dirk; Waisman, Ari; Schulze-Bergkamen, Henning

    2014-01-01

    AIM: To analyze the role of CYLD for receptor-mediated cell death of murine hepatocytes in acute liver injury models. METHODS: Hepatocyte cell death in CYLD knockout mice (CYLD-/-) was analyzed by application of liver injury models for CD95- (Jo2) and tumor necrosis factor (TNF)-?- [D-GalN/lipopolysaccharide (LPS)] induced apoptosis. Liver injury was assessed by measurement of serum transaminases and histological analysis. Apoptosis induction was quantified by cleaved PARP staining and Western blotting of activated caspases. Nuclear factor (NF)-?B, ERK, Akt and jun amino-terminal kinases signaling were assessed. Primary Hepatocytes were isolated by two step-collagenase perfusion and treated with recombinant TNF-? and with the CD95-ligand Jo2. Cell viability was analyzed by MTT-assay. RESULTS: Livers of CYLD-/- mice showed increased anti-apoptotic NF-?B signaling. In both applied liver injury models CYLD-/- mice showed a significantly reduced apoptosis sensitivity. After D-GalN/LPS treatment CYLD-/- mice exhibited significantly lower levels of alanine aminotransferase (ALT) (295 U/L vs 859 U/L, P < 0.05) and aspartate aminotransferase (AST) (560 U/L vs 1025 U/L, P < 0.01). After Jo injection CYLD-/- mice showed 2-fold lower ALT (50 U/L vs 110 U/L, P < 0.01) and lower AST (250 U/L vs 435 U/L, P < 0.01) serum-levels compared to WT mice. In addition, isolated CYLD-/- primary murine hepatocytes (PMH) were less sensitive towards death receptor-mediated apoptosis and showed increased levels of Bcl-2, XIAP, cIAP1/2, survivin and c-FLIP expression upon TNF- and CD95-receptor triggering, respectively. Inhibition of NF-?B activation by the inhibitor of NF-?B phosphorylation inhibitor BAY 11-7085 inhibited the expression of anti-apoptotic proteins and re-sensitized CYLD-/- PMH towards TNF- and CD95-receptor mediated cell death. CONCLUSION: CYLD is a central regulator of apoptotic cell death in murine hepatocytes by controlling NF-?B dependent anti-apoptotic signaling. PMID:25493017

  16. Hybrid liposomes inhibit tumor growth and lung metastasis of murine osteosarcoma cells.

    PubMed

    Kitajima, Hideki; Komizu, Yuji; Ichihara, Hideaki; Goto, Koichi; Ueoka, Ryuichi

    2013-06-01

    Antitumor effects of hybrid liposomes (HL) composed of l-?-dimyristoylphosphatidylcholine (DMPC) and polyoxyethylene(23) dodecyl ether (C??(EO)??) on the metastatic growth of murine osteosarcoma (LM8) cells were investigated in vitro and in vivo. Remarkable inhibitory effects of HL-23 on the growth of LM8 cells were obtained through the induction of apoptotic cell death in vitro. It was also indicated that HL-23 should dramatically suppress the invasion of LM8 cells and the formation of filopodia on the cell surface in vitro. Furthermore, significantly high therapeutic effects were observed in the homograft mouse models of LM8 cells with lung metastasis after the treatment with HL-23 in vivo. That is, the histological analysis demonstrated that the primary tumor growth of LM8 cells implanted subcutaneously into the mice was inhibited along with the induction of apoptosis. In addition, it was found that HL-23 significantly decreased the lung metastasis of LM8 cells in the mouse models through the inhibition of primary tumor invasion. These results suggest that HL-23 could be a novel agent for the chemotherapy of osteosarcoma. PMID:23930203

  17. Localized colonic stem cell transplantation enhances tissue regeneration in murine colitis

    PubMed Central

    Zhou, QiQi; Price, Donald D; Dreher, Kara L; Pronold, Barry; Callam, Christopher S; Sharma, Jay; Verne, G Nicholas

    2012-01-01

    Abstract Many patients suffer from chronic gastrointestinal diseases characterized by chronic inflammation, increased intestinal permeability and visceral pain in which there is no definitive treatment. Adult stem cells have recently been used in various disease states to contribute wound-healing processes. In the current study we investigated the ability of intra-colonic adult stem cells application to heal colonic inflammation in IL-10?/? mice with active colitis. The aims of this study were to determine whether intra-colonic infusion of adult colonic stem cells (CSCs) (local stem cell transplantation): (i) restores intestinal permeability; (ii) attenuates visceral hypersensitivity; (iii) heals murine colitis. IL-10?/? mice with active colitis were transplanted with adult stem cells. Mice received either a single intracolonic infusion of CSCs or colonic epithelial cells. Two weeks after transplantation, we measured visceral hypersensitivity and intestinal permeability and correlated these with histological improvement of colitis. IL-10?/? mice that received stem cell transplantation showed histopathologic evidence of recovery from colitis. Improvement in colitis as graded by pathology scores correlated with restoration of intestinal permeability and decreased visceral hypersensitivity. Intra-colonic administration of CSCs is a potential therapeutic method for treating refractory symptoms in patients with chronic gastrointestinal diseases associated with chronic inflammation and visceral hypersensitivity. This method may be safer and should have far fewer side effects than systemic stem cell administration. PMID:22050903

  18. Molecular Mechanisms of Differentiation of Murine Pro-Inflammatory ?? T Cell Subsets

    PubMed Central

    Serre, Karine; Silva-Santos, Bruno

    2013-01-01

    ?? T cells are unconventional innate-like lymphocytes that actively participate in protective immunity against tumors and infectious organisms including bacteria, viruses, and parasites. However, ?? T cells are also involved in the development of inflammatory and autoimmune diseases. ?? T cells are functionally characterized by very rapid production of pro-inflammatory cytokines, while also impacting on (slower but long-lasting) adaptive immune responses. This makes it crucial to understand the molecular mechanisms that regulate ?? T cell effector functions. Although they share many similarities with ?? T cells, our knowledge of the molecular pathways that control effector functions in ?? T cells still lags significantly behind. In this review, we focus on the segregation of interferon-? versus interleukin-17 production in murine thymic-derived ?? T cell subsets defined by CD27 and CCR6 expression levels. We summarize the most recent studies that disclose the specific epigenetic and transcriptional mechanisms that govern the stability or plasticity of discrete pro-inflammatory ?? T cell subsets, whose manipulation may be valuable for regulating (auto)immune responses. PMID:24367369

  19. Phenotypic correction of murine hemophilia A using an iPS cell-based therapy.

    PubMed

    Xu, Dan; Alipio, Zaida; Fink, Louis M; Adcock, Dorothy M; Yang, Jianchang; Ward, David C; Ma, Yupo

    2009-01-20

    Hemophilia A is caused by mutations within the Factor VIII (FVIII) gene that lead to depleted protein production and inefficient blood clotting. Several attempts at gene therapy have failed for various reasons-including immune rejection. The recent generation of induced pluripotent stem (iPS) cells from somatic cells by the ectopic expression of 3 transcription factors, Oct4, Sox2, and Klf4, provides a means of circumventing the immune rejection barrier. To date, iPS cells appear to be indistinguishable from ES cells and thus provide tremendous therapeutic potential. Here we prepared murine iPS cells from tail-tip fibroblasts and differentiated them to both endothelial cells and endothelial progenitor cells by using the embryoid body differentiation method. These iPS cells express major ES cell markers such as Oct4, Nanog, SSEA-1, alkaline phosphatase, and SALL4. Endothelial/endothelial progenitor cells derived from iPS cells expressed cell-specific markers such as CD31, CD34, and Flk1 and secreted FVIII protein. These iPS-derived cells were injected directly into the liver of irradiated hemophilia A mice. At various times after transplantation (7-90 days) hemophilia A mice and their control mice counterparts were challenged by a tail-clip bleeding assay. Nontransplanted hemophilia A mice died within a few hours, whereas transplanted mice survived for more than 3 months. Plasma FVIII levels increased in transplanted hemophilia A mice during this period to 8% to 12% of wild type and corrected the hemophilia A phenotype. Our studies provide additional evidence that iPS cell therapy may be able to treat human monogenetic disorders in the future. PMID:19139414

  20. Developing cell therapy techniques for respiratory disease: Intratracheal delivery of genetically engineered stem cells in a murine model of airway injury

    E-print Network

    Paris-Sud XI, Université de

    of genetically engineered stem cells in a murine model of airway injury Anne-Laure Leblond 1,2 , Patrice Naud 1: Stem cell therapy for respiratory disease. Page 1 of 48 Human-reviewedandacceptedforpublication,buthasyettoundergocopyeditingandproofcorrection.Thefinalpublishedversionmaydifferfromthisproof. #12;- 2 - Abstract Over the past decade, interest has increased in the use of exogenous stem cells

  1. Cytokine dysregulation induced by apoptotic cells is a shared characteristic of murine lupus.

    PubMed

    Koh, J S; Wang, Z; Levine, J S

    2000-10-15

    Of the multiple murine models of autoimmunity, the three most closely resembling human systemic lupus erythematosus (SLE) are the MRL/lpr, New Zealand Black/White F(1), and male BXSB. Although these strains share many disease characteristics, no common cellular defect has previously been found in prediseased mice from all these strains. We show in this study that macrophages from prediseased mice of all three SLE-prone strains, as well as macrophages from mice whose genomes contribute to the development of SLE (MRL/+, New Zealand White, New Zealand Black, female BXSB, and LG/J), have an identical and profound defect in cytokine expression that is triggered by apoptotic cells. Strikingly, none of 13 nonautoimmune strains tested exhibited this defect. Given that apoptotic Ags have been increasingly recognized as the target of autoantibodies, a defect in cytokine expression that is triggered by apoptotic cells has broad potential to upset the balance between tolerance and immunity. PMID:11035051

  2. Side population of a murine mantle cell lymphoma model contains tumour-initiating cells responsible for lymphoma maintenance and dissemination

    PubMed Central

    Vega, Francisco; Davuluri, Yogesh; Cho-Vega, Jeong Hee; Singh, Rajesh R; Ma, Shuguang; Wang, Rui-Yu; Multani, Asha S; Drakos, Elias; Pham, Lan V; Lee, Yen-Chiu Lin; Shen, Long; Ambrus Jr, Julian; Medeiros, L Jeffrey; Ford, Richard J

    2010-01-01

    Abstract ‘Cancer stem cells’ or ‘tumour initiating cells’ in B-cell non-Hodgkin lymphomas have not been demonstrated, although some studies focused on other cancer types suggest that such populations exist and represent tumour cells resistant to therapy and involved in relapse. These cells may also represent a putative neoplastic ‘cell of origin’ in lymphomas, but there is little substantive data to support this suggestion. Using cell lines derived from a recently established murine IL-14?× c-Myc double transgenic/mantle cell lymphoma-blastoid variant model, heretofore referred to as DTG cell lines, we identified a subset of cells within the side population (SP) with features of ‘tumour-initiating cells’. These features include higher expression of ABCG2 and BCL-2, longer telomere length, greater self-renewal ability and higher in vitro clonogenic and in vivo tumorigenic capacities compared with non-SP. In addition, in vitro viability studies demonstrated that the non-SP lymphoma subpopulation has a limited lifespan in comparison with the SP fraction. Syngenic transplant studies showed that non-SP derived tumours, in comparison to the SP-derived tumours, exhibit greater necrosis/apoptosis and less systemic dissemination capability. In conclusion, our data support the interpretation that the DTG SP fraction contains a cell population highly capable of tumour maintenance and systemic dissemination and lends support to the concept that ‘tumour-initiating cells’ occur in lymphomas. PMID:19656242

  3. 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.

  4. Induction of murine embryonic stem cell differentiation by medicinal plant extracts

    PubMed Central

    Reynertson, Kurt A.; Charlson, Mary E.; Gudas, Lorraine J.

    2010-01-01

    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 twelve species of ethnomedically utilized plants, we found fractions from three 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. PMID:20955699

  5. Regulation of growth-related genes by interleukin-6 in murine myeloma cells.

    PubMed

    Côté, Serge; Simard, Carl; Lemieux, Réal

    2002-11-01

    Interleukin-6 (IL-6), a pleiotropic cytokine with effects on several hematopoietic and other normal cells, is also important for the growth and survival of tumor cells such as murine plasmacytomas and human myelomas. Exploiting the 11A3 hybridoma cell line for its IL-6 requirement to proliferate in vitro, we used subtractive suppression hybridization (SSH) to identify genes whose expression is stimulated and/or repressed in response to IL-6. Northern blot analysis of 100 arbitrarily picked subtracted cDNA clones revealed that expression of 11 mRNAs were IL-6-modulated. Among these, eight were genes known to encode a variety of proteins such as enzymes (PCK, MTDNI), structural proteins (Tropoelastin), transcriptional regulators (BRG1) and proteins involved in cell division control (Cyclin A, OAZi) or cell signaling (PIX, TOPK/PBK). The recently identified MAPKK-like protein kinase TOPK/PBK gene represents a likely candidate IL-6 target gene as suggested by its significant up-regulated expression in hybridoma cells induced to grow by a brief IL-6 pulse. The diversity of growth-related genes identified in this study further emphasizes the central role of IL-6 in the regulation of myeloma cell expansion in addition to its previously demonstrated role in the inhibition of apoptosis. PMID:12453469

  6. Apoptosis induced by oxysterols in murine lymphoma cells and in normal thymocytes.

    PubMed Central

    Christ, M; Luu, B; Mejia, J E; Moosbrugger, I; Bischoff, P

    1993-01-01

    Oxygenated derivatives of cholesterol (oxysterols), a family of naturally occurring compounds, possess marked anti-proliferative and immunosuppressive activities, in particular they have been shown to inhibit T-cell responses to different stimuli. 25-Hydroxycholesterol (25-OHC) and 7 beta,25-dihydroxycholesterol (7.25-OHC) are able to kill not only RDM4 murine lymphoma in vitro, but also, surprisingly, mouse thymocytes after several hours of incubation. In this study, we report that the death of RDM4 and thymocytes induced by oxysterols exhibits the features of apoptosis. This phenomenon was identified by agarose gel electrophoresis of DNA fragments extracted from the cells and quantified by flow cytometric analysis of the DNA fluorescence of propidium iodide-stained cells. Cycloheximide and actinomycin D were found to decrease the number of apoptotic cells and to increase cell viability, indicating a requirement for the synthesis of macromolecules in oxysterol-induced programmed cell death. The pathway by which 25-OHC and 7.25-OHC are able to induce apoptosis in this type of cell and the possible contribution of these compounds to thymus involution during development are discussed. Images Figure 2 Figure 4 PMID:7682990

  7. 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.

  8. Isolating And Immunostaining Lymphocytes and Dendritic Cells from Murine Peyer's Patches

    PubMed Central

    De Jesus, Magdia; Ahlawat, Sarita; Mantis, Nicholas J.

    2013-01-01

    Peyer's patches (PPs) are integral components of the gut-associated lymphoid tissues (GALT) and play a central role in intestinal immunosurveillance and homeostasis. Particulate antigens and microbes in the intestinal lumen are continuously sampled by PP M cells in the follicle-associated epithelium (FAE) and transported to an underlying network of dendritic cells (DCs), macrophages, and lymphocytes. In this article, we describe protocols in which murine PPs are (i) dissociated into single cell suspensions and subjected to flow cytometry and (ii) prepared for cryosectioning and immunostaining. For flow cytometry, PPs are mechanically dissociated and then filtered through 70 ?m membranes to generate single cell suspensions free of epithelial cells and large debris. Starting with 20-25 PPs (from four mice), this quick and reproducible method yields a population of >2.5 x 106 cells with >90% cell viability. For cryosectioning, freshly isolated PPs are immersed in Optimal Cutting Temperature (OCT) medium, snap-frozen in liquid nitrogen, and then sectioned using a cryomicrotome. Tissue sections (5-12 ?m) are air-dried, fixed with acetone or methanol, and then subjected to immunolabeling. PMID:23525039

  9. Development of resting membrane potentials in differentiating murine neuroblastoma cells (N1E-115) evaluated by flow cytometry

    Microsoft Academic Search

    William S. Kisaalita; John M. Bowen

    1997-01-01

    With the aid of a voltage-sensitive oxonol dye, flow cytometry was used to measure relative changes in resting membrane potential\\u000a (Vm) and forward angle light scatter (FALS) profiles of a differentiating\\/differentiated murine neuroblastoma cell line (N1E-115).\\u000a Electrophysiological differentiation was characterized by Vm establishment. The (Vm)-time profile was found to be seed cell concentration-dependent for cell densities of less than 2

  10. 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

  11. Thy-1+ dendritic cells in murine epidermis are bone marrow-derived

    SciTech Connect

    Breathnach, S.M.; Katz, S.I.

    1984-07-01

    Thy-1+, Ly-5+ dendritic cells have recently been described as a resident cell population in murine epidermis, but their ontogeny and function are unknown. The origin and turnover of epidermal Thy-1+ cells utilizing chimeric mice were investigated. Lethally x-irradiated AKR/J (Thy-1.1+) and AKR/Cum (Thy-1.2+) mice were reconstituted with allogeneic bone marrow cells with or without thymocytes from congenic AKR/Cum or AKR/J mice, respectively. The density of residual indigenous Thy-1.1+ cells in AKR/J chimeras and Thy-1.2+ cells in AKR/Cum chimeras was substantially reduced following x-irradiation, as determined by immunofluorescence staining of epidermal sheets. Epidermal repopulation by allogeneic Thy-1+ dendritic epidermal cells was first observed at 5 weeks in AKR/J chimeras and at 7 weeks in AKR/Cum chimeras and progressed slowly. Repopulation was not enhanced by increasing the number of allogeneic bone marrow cells injected from 2 X 10(7) to 10(8) cells or by the addition of 8 X 10(7) allogeneic thymocytes to the donor inoculate. Epidermal repopulation by allogeneic Thy-1.2+ cells was not seen in AKR/J mice reconstituted with syngeneic bone marrow cells and allogeneic Thy-1.2+ AKR/Cum thymocytes. Taken together, these results indicate that Thy-1+ dendritic epidermal cells are derived from the bone marrow and suggest that they are not related to conventional peripheral T-lymphocytes.

  12. Kinetics of ocular and systemic antigen-specific T-cell responses elicited during murine cytomegalovirus retinitis

    Microsoft Academic Search

    Martin S Zinkernagel; Claire Petitjean; Matthew E Wikstrom; Mariapia A Degli-Esposti

    2012-01-01

    Cytomegalovirus (CMV) reactivation in the retina of immunocompromized patients is a cause of significant morbidity as it can lead to blindness. The adaptive immune response is critical in controlling murine CMV (MCMV) infection in MCMV-susceptible mouse strains. CD8+ T cells limit systemic viral replication in the acute phase of infection and are essential to contain latent virus. In this study,

  13. Effects of Mistletoe Extract on Murine Thymocytes in vivo and on Glucocorticoid-Induced Cell Count Reduction

    Microsoft Academic Search

    Tibor Hajtò; Timea Berki; László Pàlinkàs; Ferenc Boldizsàr; Péter Németh

    2006-01-01

    Background: Mistletoe extracts are widely used in cancer patients due to their cytostatic and immunomodulatory effects. Essential components include mistletoe lectins which act as biomodulators with proinflammatory and apoptosisinducing effects. This study investigates the acute and longterm effects of standardized mistletoe extract (Iscador® M spec 5 mg) on thymocyte subpopulations and peripheral T-cells using a murine (Balb\\/c) model. Materials and

  14. Markers of murine embryonic and neural stem cells, neurons and astrocytes: reference points for developmental neurotoxicity testing

    EPA Science Inventory

    Developmental neurotoxicity (DNT) is a significant concern for environmental chemicals, as well as for food and drug constituents. The sensitivity of animal-based DNT models is unclear, and they are expensive and time consuming. Murine embryonic stem cells (mESC) recapitulate sev...

  15. Differentiation of murine embryonic stem cells toward renal lineages by conditioned medium from ureteric bud cells in vitro.

    PubMed

    Ren, Xiaohui; Zhang, Jingya; Gong, Xiaowen; Niu, Xin; Zhang, Xuejin; Chen, Peng; Zhang, Xuejun

    2010-07-01

    The kidney is formed from two tissue populations derived from the intermediate mesoderm, the ureteric bud, and the metanephric mesenchyme. Metanephric mesenchyme is a pluripotent renal stem population, and conversion of renal mesenchyme into epithelia depends on the ureteric bud in vivo and in vitro. Embryonic stem (ES) cells have been induced to differentiate into a broad spectrum of specialized cell types in vitro, including hematopoietic, pancreatic, and neuronal cells. Such ES-derived cells can provide a valuable source of progenitor cells. However, whether ES cells can be stimulated by factors secreted from the fetal renal cells to differentiate into renal precursor cells in vitro has not been reported. In this study, we showed that murine ES cells can give rise to embryoid bodies in the absence of leukemia inhibitory factor. Culture conditions were optimized [6 days, 10 ng/ml activin and 10(-7) M retinoic acid (RA)] to generate maximal mesoderm populations specifically expressing Pax2 and brachyury. Results showed that 72% of the cells were brachyury positive by fluorescent activated cell sorter on Day 6 of EB cell differentiation. Conditioned medium collected from cultures of ureteric bud cells from renal cells of a 13-day-old fetus was added to the culture medium. Mesoderm cells were cultured for up to 10 days before showing expression of renal markers, initiation of nephrogenesis (WT-1 and Pax2), and terminally differentiated renal cell types (POD-1 and E-cadherin). This study suggests that ES cells pre-treated by RA and activin can interact with secreted molecules of the fetal renal cells to specifically differentiate into renal precursor cells. Our results provide an experimental basis for the development of in vitro assays to steer differentiation of ES cells toward renal lineages. PMID:20705585

  16. cDNA cloning and expression of murine macrophage colony-stimulating factor from L929 cells.

    PubMed Central

    Ladner, M B; Martin, G A; Noble, J A; Wittman, V P; Warren, M K; McGrogan, M; Stanley, E R

    1988-01-01

    A 4-kilobase and a 2-kilobase cDNA clone encoding a murine macrophage colony-stimulating factor have been isolated. Except for 2 amino acid residue differences, these two clones encode the same 520 amino acid residue protein, which is preceded by a 32-amino acid residue signal peptide. The two clones, whose molecular masses correspond to the two transcripts observed in murine L929 fibroblasts, contain 3' untranslated regions that are markedly different in sequence and length. Both clones can be expressed in COS cells and the recombinant protein is active in a mouse bone marrow colony assay. Images PMID:2457916

  17. Sage weed (Salvia plebeia) extract antagonizes foam cell formation and promotes cholesterol efflux in murine macrophages.

    PubMed

    Park, Sin-Hye; Kim, Jung-Lye; Kang, Min-Kyung; Gong, Ju-Hyun; Han, Seon-Young; Shim, Jae-Hoon; Lim, Soon Sung; Kang, Young-Hee

    2012-11-01

    Lipid-laden peripheral tissue cells release cholesterol to an extracellular acceptor such as high-density lipoprotein (HDL). Foam cells are formed at the first stage of atherosclerosis development. This study investigated whether sage weed (Salvia plebeia) extract (SWE) influences cholesterol handling of J774A1 murine macrophages. A murine macrophage cell line, J774A1, was used in this study. Oxidized low-density lipoproteins (LDL) treatment was used for foam cell formation, which was confirmed using Oil red O staining. The oxidized LDL uptake and cholesterol efflux from lipid-laden foam cell-associated proteins were detected by western blot analysis. Also, transcriptional levels of these associated genes were examined using reverse transcription-PCR. Also, cholesterol efflux was measured using NBD-cholesterol efflux assay. Non-toxic SWE at ?10 µg/ml attenuated scavenger receptor (SR)-B1 expression of macrophages induced by oxidized LDL for 6 h, which was achieved at its transcriptional levels. Consistently, SWE suppressed oxidized LDL-stimulated cellular lipid accumulation and foam cell formation due to downregulated SR-B1. SWE upregulated the protein expression and mRNA levels of ATP-binding cassette transporter A1 (ABCA1) and ATP-binding cassette transporter G1 (ABCG1) in lipid-laden foam cells, both responsible for cholesterol efflux. In addition, SWE promoted apolipoprotein E (apoE) secretion from oxidized LDL-induced foam cells. Cholesterol efflux was enhanced by ?10 µg/ml SWE most likely through the induction of ABCA1 and ABCG1 and the secretion of apoE. Although 10 µM homoplantaginin, a compound mainly present in sage weeds, did not influence cellular expression of ABCA1 and ABCG1, it suppressed oxidized LDL-enhanced SR-B1 induction and foam cell formation. These results demonstrate that SWE antagonized oxidized LDL uptake and promoted cholesterol efflux in lipid-laden macrophages. Therefore, SWE may serve as a protective therapeutic agent against the development of atherosclerosis. PMID:22922992

  18. 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

  19. Antivirus antibody-dependent cell-mediated cytotoxicity during murine cytomegalovirus infection.

    PubMed Central

    Manischewitz, J E; Quinnan, G V

    1980-01-01

    BALB/c mice infected with murine cytomegalovirus were studied to determine whether antibody-dependent cell-mediated cytotoxicity contributes to the immune control of this infection. Antibody-dependent killer cells from uninfected mice were used as effector cells to assay for antibody in sera of infected mice. Secondary immune sera were found to contain both cytomegalovirus-specific and autoreactive antibodies. After primary infection only cytomegalovirus-specific antibodies were found. These were detected by antibody-dependent cell-mediated cytotoxicity within 8 to 10 days after onset of infection, but usually not until day 21, by a neutralizing antibody assay. Antibody titers were about 10-fold higher by antibody-dependent cell-mediated cytotoxicity than by neutralization. The results indicate that cellular immunity to cytomegalovirus infection includes an antibody-dependent cell-mediated cytotoxicity response which is likely to be highly efficient and may contribute significantly to control of both acute and later stages of infection. PMID:6253395

  20. Glycine modulates membrane potential, cell volume, and phagocytosis in murine microglia.

    PubMed

    Komm, Barbara; Beyreis, Marlena; Kittl, Michael; Jakab, Martin; Ritter, Markus; Kerschbaum, Hubert H

    2014-08-01

    Phagocytes form engulfment pseudopodia at the contact area with their target particle by a process resembling cell volume (CV) regulatory mechanisms. We evaluated whether the osmoregulatory active neutral amino acid glycine, which contributes to CV regulation via activation of sodium-dependent neutral amino acid transporters (SNATs) improves phagocytosis in isotonic and hypertonic conditions in the murine microglial cell line BV-2 and primary microglial cells (pMG). In BV-2 cells and pMG, RT-PCR analysis revealed expression of SNATs (Slc38a1, Slc38a2), but not of GlyRs (Glra1-4). In BV-2 cells, glycine (5 mM) led to a rapid Na(+)-dependent depolarization of membrane potential (V mem). Furthermore, glycine increased CV by about 9%. Visualizing of phagocytosis of polystyrene microspheres by scanning electron microscopy revealed that glycine (1 mM) increased the number of BV-2 cells containing at least one microsphere by about 13%. Glycine-dependent increase in phagocytosis was suppressed by the SNAT inhibitor ?-(methylamino)isobutyric acid (MeAIB), by replacing extracellular Na(+) with choline, and under hypertonic conditions, but not by the GlyR antagonist strychnine or the GlyR agonist taurine. Interestingly, hypertonicity-induced suppression of phagocytosis was rescued by glycine. These findings demonstrate that glycine increases phagocytosis in iso- and hypertonic conditions by activation of SNATs. PMID:24760586

  1. Autologous apoptotic cells preceding transplantation enhance survival in lethal murine graft-versus-host models.

    PubMed

    Florek, Mareike; Sega, Emanuela I; Leveson-Gower, Dennis B; Baker, Jeanette; Müller, Antonia M S; Schneidawind, Dominik; Meyer, Everett; Negrin, Robert S

    2014-09-11

    Acute graft-versus-host disease (GVHD) is induced by alloreactivity of donor T cells toward host antigens presented on antigen-presenting cells (APCs). Apoptotic cells are capable of inducing tolerance by altering APC maturation. Apoptosis can be induced by extracorporeal photopheresis (ECP). We demonstrate that the use of ECP as a prophylaxis prior to conditioning significantly improves survival (P < .0001) after bone marrow transplantation (BMT) by inhibiting the initiation phase of acute GVHD in a murine BMT model. ECP-treated autologous splenocytes resulted in immune tolerance in the host, including reduced dendritic cell activation with decreased nuclear factor-?B engagement, increased regulatory T-cell (Treg) numbers with enhanced expression of cytolytic T lymphocyte-associated antigen 4, potentiating their suppressive function. The protective effect required host production of interleukin-10 and host Tregs. Conventional T cells that entered this tolerant environment experienced reduced proliferation, as well as a reduction of tissue homing and expression of activation markers. The induction of this tolerant state by ECP was obviated by cotreatment with lipopolysaccharide, suggesting that the inflammatory state of the recipient prior to treatment would play a role in potential clinical translation. The use of prophylactic ECP may provide an alternative and safe method for immunosuppression in the bone marrow transplant setting. PMID:25030062

  2. 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

  3. 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

  4. Osteogenic gene expression of murine osteoblastic (MC3T3-E1) cells under cyclic tension

    NASA Astrophysics Data System (ADS)

    Kao, C. T.; Chen, C. C.; Cheong, U.-I.; Liu, S. L.; Huang, T. H.

    2014-08-01

    Low-level laser therapy (LLLT) can promote cell proliferation. The remodeling ability of the tension side of orthodontic teeth affects post-orthodontic stability. The purpose of the present study was to investigate the osteogenic effects of LLLT on osteoblast-like cells treated with a simulated tension system that provides a mechanical tension regimen. Murine osteoblastic (MC3T3-E1) cells were cultured in a Flexcell strain unit with programmed loads of 12% elongation at a frequency of 0.5?Hz for 24 and 48?h. The cultured cells were treated with a low-level diode laser using powers of 5?J and 10?J. The proliferation of MC3T3-E1 cells was determined using the Alamar Blue assay. The expression of osteogenic genes (type I collagen (Col-1), osteopontin (OPN), osteocalcin (OC), osteoprotegerin (OPG), receptor activator of nuclear factor kappa B ligand (RANKL), bone morphologic protein (BMP-2), and bone morphologic protein (BMP-4)) in MC3T3-E1 cells was analyzed using reverse transcription polymerase chain reaction (RT-PCR). The data were analyzed using one-way analysis of variance. The proliferation rate of tension-cultured MC3T3-E1 cells under 5?J and 10?J LLLT increased compared with that of the control group (p < 0.05). Prominent mineralization of the MC3T3-E1 cells was visible using a von Kossa stain in the 5?J LLLT group. Osteogenic genes (Col-1, OC, OPG and BMP-2) were significantly expressed in the MC3T3-E1 cells treated with 5?J and 10?J LLLT (p < 0.05). LLLT in tension-cultured MC3T3-E1 cells showed synergistic osteogenic effects, including increases in cell proliferation and Col-1, OPN, OC, OPG and BMP-2 gene expression. LLLT might be beneficial for bone remodeling on the tension side of orthodontics.

  5. Gene expression profiling of murine T-cell lymphoblastic lymphoma identifies deregulation of S-phase initiating genes?

    PubMed Central

    Dabrowska, Magdalena Julia; Ejegod, Ditte; Lassen, Louise Berkhoudt; Johnsen, Hans Erik; Wabl, Matthias; Pedersen, Finn Skou; Dybkær, Karen

    2014-01-01

    In a search for genes and pathways implicated in T-cell lymphoblastic lymphoma (T-LBL) development, we used a murine lymphoma model, where mice of the NMRI-inbred strain were inoculated with murine leukemia virus mutants. The resulting tumors were analyzed by integration analysis and global gene expression profiling to determine the effect of the retroviral integrations on the nearby genes, and the deregulated pathways in the tumors. Gene expression profiling identified increased expression of genes involved in the minichromosome maintenance and origin of recognition pathway as well as downregulation in negative regulators of G1/S transition, indicating increased S-phase initiation in murine T-LBLs. PMID:23896059

  6. 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

  7. 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

  8. Establishment of a Murine Graft-versus-Myeloma Model Using Allogeneic Stem Cell Transplantation

    PubMed Central

    Binsfeld, Marilène; Beguin, Yves; Belle, Ludovic; Otjacques, Eléonore; Hannon, Muriel; Briquet, Alexandra; Heusschen, Roy; Drion, Pierre; Zilberberg, Jenny; Bogen, Bjarne; Baron, Frédéric; Caers, Jo

    2014-01-01

    Background Multiple myeloma (MM) is a malignant plasma cell disorder with poor long-term survival and high recurrence rates. Despite evidence of graft-versus-myeloma (GvM) effects, the use of allogeneic hematopoietic stem cell transplantation (allo-SCT) remains controversial in MM. In the current study, we investigated the anti-myeloma effects of allo-SCT from B10.D2 mice into MHC-matched myeloma-bearing Balb/cJ mice, with concomitant development of chronic graft-versus-host disease (GvHD). Methods and results Balb/cJ mice were injected intravenously with luciferase-transfected MOPC315.BM cells, and received an allogeneic (B10.D2 donor) or autologous (Balb/cJ donor) transplant 30 days later. We observed a GvM effect in 94% of the allogeneic transplanted mice, as the luciferase signal completely disappeared after transplantation, whereas all the autologous transplanted mice showed myeloma progression. Lower serum paraprotein levels and lower myeloma infiltration in bone marrow and spleen in the allogeneic setting confirmed the observed GvM effect. In addition, the treated mice also displayed chronic GvHD symptoms. In vivo and in vitro data suggested the involvement of effector memory CD4 and CD8 T cells associated with the GvM response. The essential role of CD8 T cells was demonstrated in vivo where CD8 T-cell depletion of the graft resulted in reduced GvM effects. Finally, TCR V? spectratyping analysis identified V? families within CD4 and CD8 T cells, which were associated with both GvM effects and GvHD, whereas other V? families within CD4 T cells were associated exclusively with either GvM or GvHD responses. Conclusions We successfully established an immunocompetent murine model of graft-versus-myeloma. This is the first murine GvM model using immunocompetent mice that develop MM which closely resembles human MM disease and that are treated after disease establishment with an allo-SCT. Importantly, using TCR V? spectratyping, we also demonstrated the presence of GvM unique responses potentially associated with the curative capacity of this immunotherapeutic approach. PMID:25415267

  9. An increase in telomere sister chromatid exchange in murine embryonic stem cells possessing critically shortened telomeres

    SciTech Connect

    Wang, Yisong [ORNL; Giannone, Richard J [ORNL; Wu, Jun [ORNL; Gomez, Marla V [ORNL; Liu, Yie [ORNL

    2005-01-01

    Telomerase deficiency leads to a progressive loss of telomeric DNA that eventually triggers cell apoptosis in human primary cells during prolonged growth in culture. Rare survivors can maintain telomere length through either activation of telomerase or recombination-based telomere lengthening, and thus proliferate indefinitely. We have explored the possibility that telomeres may be maintained through telomere sister chromatid exchange (T-SCE) in murine telomere reverse transcriptase-deficient (mTert -/-) splenocytes and ES cells. Because telomerase deficiency leads to gradual loss of telomeric DNA in mTert -/- splenocytes and ES cells and eventually to chromosomes with telomere signal-free ends (SFEs), we examined these cell types for evidence of sister chromatid exchange at telomeres, and observed an increase in T-SCEs only in a subset of mTert -/- splenocytes or ES cells that possessed multiple SFEs. Furthermore, T-SCEs were more often detected in ES cells than in splenocytes that harbored a similar frequency of SFEs. In mTert heterozygous (mTert +/-) ES cells or splenocytes, which are known to exhibit a decrease in average telomere length but no SFEs, no increase in T-SCE was observed. In addition to T-SCE, other genomic rearrangements (i.e., SCE) were also significantly increased in mTert -/- ES cells possessing critically short telomeres, but not in splenocytes. Our results suggest that animals and cell culture differ in their ability to carry out genomic rearrangements as a means of maintaining telomere integrity when telomeres become critically shortened.

  10. Effects of brevetoxins on murine myeloma SP2/O cells: Aberrant cellular division

    USGS Publications Warehouse

    Han, T.K.; Derby, M.; Martin, D.F.; Wright, S.D.; Dao, M.L.

    2003-01-01

    Massive deaths of manatees (Trichechus manatus latirostris) during the red tide seasons have been attributed to brevetoxins produced by the dinoflagellate Karenia brevis (formerly Ptychodiscus breve and Gymnodinium breve). Although these toxins have been found in macrophages and lymphocytes in the lung, liver, and secondary lymphoid tissues of these animals, the molecular mechanisms of brevetoxicosis have not yet been identified. To investigate the effects of brevetoxins on immune cells, a murine myeloma cell line (SP2/O) was used as a model for in vitro studies. By adding brevetoxins to cultures of the SP2/O cells at concentrations ranging from 20 to 600 ng/ml, an apparent increase in proliferation was observed at around 2 hours post challenge as compared to the unchallenged cell cultures. This was followed by a drop in cell number at around 3 hours, suggesting an aberrant effect of brevetoxins on cellular division, the cells generated at 2 hours being apparently short-lived. In situ immunochemical staining of the SP2/O cells at 1 and 2 hour post challenge showed an accumulation of the toxins in the nucleus. A 21-kDa protein was subsequently isolated from the SP2/O cells as having brevetoxin-binding properties, and immunologically identified as p21, a nuclear factor known to down-regulate cellular proliferation through inhibition of cyclin-dependent kinases. These data are the first on a possible effect of brevetoxins on the cell cycle via binding to p21, a phenomenon that needs to be further investigated and validated in normal immune cells.

  11. 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

  12. Notch Signaling is Associated with ALDH Activity and an Aggressive Metastatic Phenotype in Murine Osteosarcoma Cells

    PubMed Central

    Mu, Xiaodong; Isaac, Christian; Greco, Nicholas; Huard, Johnny; Weiss, Kurt

    2013-01-01

    Osteosarcoma (OS) is the most common primary malignancy of bone, and pulmonary metastatic disease accounts for nearly all mortality. However, little is known about the biochemical signaling alterations that drive the progression of metastatic disease. Two murine OS cell populations, K7M2 and K12, are clonally related but differ significantly in their metastatic phenotypes and therefore represent excellent tools for studying metastatic OS molecular biology. K7M2 cells are highly metastatic, whereas K12 cells display limited metastatic potential. Here we report that the expression of Notch genes (Notch1, 2, 4) are up-regulated, including downstream targets Hes1 and Stat3, in the highly metastatic K7M2 cells compared to the less metastatic K12 cells, indicating that the Notch signaling pathway is more active in K7M2 cells. We have previously described that K7M2 cells exhibit higher levels of aldehyde dehydrogenase (ALDH) activity. Here we report that K7M2 cell ALDH activity is reduced with Notch inhibition, suggesting that ALDH activity may be regulated in part by the Notch pathway. Notch signaling is also associated with increased resistance to oxidative stress, migration, invasion, and VEGF expression in vitro. However, Notch inhibition did not significantly alter K7M2 cell proliferation. In conclusion, we provide evidence that Notch signaling is associated with ALDH activity and increased metastatic behavior in OS cells. Both Notch and ALDH are putative molecular targets for the treatment and prevention of OS metastasis. PMID:23805413

  13. Effects of brevetoxins on murine myeloma SP2/O cells: aberrant cellular division.

    PubMed

    Han, Thomas K; Derby, Melissa; Martin, Dean F; Wright, Scott D; Dao, My Lien

    2003-01-01

    Massive deaths of manatees (Trichechus manatus latirostris) during the red tide seasons have been attributed to brevetoxins produced by the dinoflagellate Karenia brevis (formerly Ptychodiscus breve and Gymnodinium breve). Although these toxins have been found in macrophages and lymphocytes in the lung, liver, and secondary lymphoid tissues of these animals, the molecular mechanisms of brevetoxicosis have not yet been identified. To investigate the effects of brevetoxins on immune cells, a murine myeloma cell line (SP2/O) was used as a model for in vitro studies. By adding brevetoxins to cultures of the SP2/O cells at concentrations ranging from 20 to 600 ng/ml, an apparent increase in proliferation was observed at around 2 hours post challenge as compared to the unchallenged cell cultures. This was followed by a drop in cell number at around 3 hours, suggesting an aberrant effect of brevetoxins on cellular division, the cells generated at 2 hours being apparently short-lived. In situ immunochemical staining of the SP2/O cells at 1 and 2 hour post challenge showed an accumulation of the toxins in the nucleus. A 21-kDa protein was subsequently isolated from the SP2/O cells as having brevetoxin-binding properties, and immunologically identified as p21, a nuclear factor known to down-regulate cellular proliferation through inhibition of cyclin-dependent kinases. These data are the first on a possible effect of brevetoxins on the cell cycle via binding to p21, a phenomenon that needs to be further investigated and validated in normal immune cells. PMID:12745987

  14. Prospective Isolation of Murine and Human Bone Marrow Mesenchymal Stem Cells Based on Surface Markers

    PubMed Central

    Houlihan, Diarmaid D.; Akazawa, Chihiro; Okano, Hideyuki; Matsuzaki, Yumi

    2013-01-01

    Mesenchymal stem cells (MSCs) are currently defined as multipotent stromal cells that undergo sustained in vitro growth and can give rise to cells of multiple mesenchymal lineages, such as adipocytes, chondrocytes, and osteoblasts. The regenerative and immunosuppressive properties of MSCs have led to numerous clinical trials exploring their utility for the treatment of a variety of diseases (e.g., acute graft-versus-host disease, Crohn's disease, multiple sclerosis, osteoarthritis, and cardiovascular diseases including heart failure and myocardial infarction). On the other hand, conventionally cultured MSCs reflect heterogeneous populations that often contain contaminating cells due to the significant variability in isolation methods and the lack of specific MSC markers. This review article focuses on recent developments in the MSC research field, with a special emphasis on the identification of novel surface markers for the in vivo localization and prospective isolation of murine and human MSCs. Furthermore, we discuss the physiological importance of MSC subtypes in vivo with specific reference to data supporting their contribution to HSC niche homeostasis. The isolation of MSCs using selective markers (combination of PDGFR? and Sca-1) is crucial to address the many unanswered questions pertaining to these cells and has the potential to enhance their therapeutic potential enormously. PMID:23766770

  15. CD4+ T cells are required for development of a murine retrovirus- induced immunodeficiency syndrome (MAIDS)

    PubMed Central

    1988-01-01

    Mice depleted in vivo of CD4+ Th cells by treatment with mAb GK1.5 were found to be resistant to the lymphoproliferative/immunodeficiency disease (MAIDS) induced in intact mice by infection with the mixture of LP-BM5 murine leukemia viruses. Depleted mice did not develop lymphadenopathy or splenomegaly, had normal serum IgM levels, normal CTL responses to alloantigens, and were able to generate PFC responses to Th-independent antigens even though frequencies of virus-producing spleen cells were comparable in depleted and intact mice. Depletion of CD4+ Th cells after infection resulted in a reversal of many abnormalities exhibited by infected controls; spleen weights, serum IgM levels, and allogeneic CTL responses of treated mice were comparable to those of uninfected controls. These results demonstrate that dysfunction of CD4+ Th cells is central to the induction and progression of both T and B cell abnormalities in MAIDS. PMID:2842430

  16. A murine monoclonal antibody raised against human non-small cell carcinoma of the lung.

    PubMed

    Stein, R; Goldenberg, D M

    1988-12-01

    Murine monoclonal antibodies (MAbs) reactive with human non-small cell carcinoma of the lung (NSCCL) were produced following immunization with a membrane preparation of Calu-3, a human adenocarcinoma of the lung cell line grown in nude mice. Positive hybrids unreactive with normal liver membranes and human peripheral white blood cells were selected for further testing. One MAb (RS5-4H6) recognized an antigen expressed in a variety of lung and other carcinoma cell lines as detected by flow cytometry. Immunohistology showed a selectivity for normal and neoplastic lung epithelium, as well as other cancers. The antigen was detected by immunohistology in 87% of tumor specimens derived from the lung, breast, colon, kidney, and ovary. Most other normal human tissues were not stained but occasional cells of the stomach, salivary and other glands, as well as kidney tubules were reactive. This MAb is an IgG1. Western blot analysis indicated that the antibody reacts selectively with an antigen greater than 300 kD. The antigen is resistant to neuraminidase treatment and periodate oxidation, but sensitive to pronase treatment, suggesting that the epitope is peptide in nature. This antibody may be potentially useful as a targeting agent for radioimmunodetection and immunoconjugate therapy. PMID:2853133

  17. Characterization of Lgr5-positive epithelial cells in the murine thymus.

    PubMed

    Vroegindeweij, Eric; van Mourik, Irene; Cupedo, Tom; Cornelissen, Jan J

    2013-05-01

    Leucine-rich repeat-containing G protein-coupled receptor (Lgr)5 is a marker for epithelial stem cells in the adult intestine of mice. Lgr5 transcripts have also been detected in the developing murine thymus, leading to speculation that Lgr5 is a marker for the long-sought stem cell of the thymus. To address the nature of the Lgr5-expressing thymic epithelial cells (TECs), we used Lgr5-GFP reporter mice. We show that epithelial cells expressing Lgr5 protein are present in the fetal thymus during a specific developmental window yet are no longer detectable at birth. To analyze the function of the Lgr5 protein during thymus development, we generated Lgr5(-/-) mice. These experiments unequivocally show that thymus development is not perturbed in the absence of Lgr5, that all TEC subsets develop in Lgr5(-/-) mice and that T cells are produced in the expected ratios. Finally, by using an inducible lineage tracing system to track the progeny of Lgr5(+) fetal TECs in vivo, we demonstrated that Lgr5(+) fetal TECs have no detectable progeny in the later fetal thymus. In sum, we show that presence of the Lgr5 protein is not a prerequisite for proper thymus organogenesis. PMID:23423968

  18. X-ray induced alterations in the differentiation and mineralization potential of murine preosteoblastic cells

    NASA Astrophysics Data System (ADS)

    Hu, Yueyuan; Lau, Patrick; Baumstark-Khan, Christa; Hellweg, Christine E.; Reitz, Günther

    2012-05-01

    To evaluate the effects of ionizing radiation (IR) on murine preosteoblastic cell differentiation, we directed OCT-1 cells to the osteoblastic lineage by treatment with a combination of ?-glycerophosphate (?-GP), ascorbic acid (AA), and dexamethasone (Dex). In vitro mineralization was evaluated based on histochemical staining and quantification of the hydroxyapatite content of the extracellular bone matrix. Expression of mRNA encoding Runx2, transforming growth factor ?1 (TGF-?1), osteocalcin (OCN), and p21CDKN1A was analyzed. Exposure to IR reduced the growth rate and diminished cell survival of OCT-1 cells under standard conditions. Notably, calcium content analysis revealed that deposition of mineralized matrix increased significantly under osteogenic conditions after X-ray exposure in a time-dependent manner. In this study, higher radiation doses exert significant overall effects on TGF-?1, OCN, and p21CDKN1A gene expression, suggesting that gene expression following X-ray treatment is affected in a dose-dependent manner. Additionally, we verified that Runx2 was suppressed within 24 h after irradiation at 2 and 4 Gy. Although further studies are required to verify the molecular mechanism, our observations strongly suggest that treatment with IR markedly alters the differentiation and mineralization process of preosteoblastic cells.

  19. Expansion of undifferentiated murine embryonic stem cells as aggregates in suspension culture bioreactors.

    PubMed

    Cormier, Jaymi T; zur Nieden, Nicole I; Rancourt, Derrick E; Kallos, Michael S

    2006-11-01

    Pluripotent embryonic stem cells (ESCs) have recently been considered as a primary material for regenerating tissues lost to injuries and degenerative diseases. For clinical implementation of this technology, a quality controlled, reproducible culture system is necessary for the expansion and differentiation of the cells. Used in many bioprocess applications, suspension bioreactors have gained considerable attention for the regulated large-scale expansion of cells. The current study presents a bioreactor process for the large-scale expansion of undifferentiated murine ESCs as aggregates. In this system, the level of ESC aggregation and differentiation was effectively controlled by adjusting shear forces and inoculation density, achieving a 31-fold expansion in 5 days. Pluripotency markers Oct-4, Nanog, SSEA-1, ALP, and rex-1 were assessed using flow cytometry analysis and gene expression profiles and showed that the undifferentiated nature of the cells within the ESC aggregates was maintained. Colony-forming efficiencies and embryoid body formation tests of the expanded cultures demonstrated that characteristic functional attributes of undifferentiated cells were not lost. Overcoming a major impediment in the area of ESC expansion, this study describes a successful process for the controlled and reproducible largescale expansion of ESCs using suspension culture bioreactors. PMID:17518637

  20. Identification of Immunomodulatory Signatures Induced by American Ginseng in Murine Immune Cells

    PubMed Central

    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

  1. 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.

  2. Growth-promoting activity of desmopressin in murine leukemia cells treated in vitro.

    PubMed

    Beenken, S W; Batra, J K; Gerrard, J M; Goldenberg, G J

    1988-12-15

    The synthetic vasopressin analogue, desmopressin (dDAVP), has been shown to influence membrane transport of melphalan in murine L5178Y lymphoblasts. Accordingly, the effect of dDAVP on the cytocidal activity of melphalan in L5178Y cells was evaluated. dDAVP did not affect the cytocidal activity of melphalan in these cells, but significantly affected the cloning efficiency of stationary phase or slowly dividing L5178Y cells over a range of concentrations. In particular, stationary phase cells showed an increase in cloning efficiency from 4.3 +/- 0.5% in control cells to 7.0 +/- 0.3% in cells treated with 25 nM dDAVP (P less than 0.001), whereas cells doubling every 26 h showed an increase from 10.8 +/- 1.2% in control cells to 21.0 +/- 2.0% in cells treated with 150 nM dDAVP (P less than 0.001). This phenomenon was associated with significant elevations of 1,2[3H] diacylglycerol after incubation with dDAVP for 9 min (P less than 0.01) and of total [3H]diacylglycerols after incubation for both 3 min (P less than 0.05) and 9 min (P less than 0.02). Within 10 s of treatment with 100 nM dDAVP, there was a marked decrease in the levels of inositol 1,4,5-trisphosphate and inositol 1-phosphate, but subsequently no change was observed for up to 9 min after treatment. We postulate that the increase of diacylglycerol content produced by dDAVP might be primarily from a phosphatidylcholine source and that the growth-promoting activity of desmopressin may be a consequence of activation of protein kinase C. PMID:3191481

  3. 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

  4. 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.

  5. Generation of pancreatic hormone-expressing islet-like cell aggregates from murine adipose tissue-derived stem cells.

    PubMed

    Chandra, Vikash; G, Swetha; Phadnis, Smruti; Nair, Prabha D; Bhonde, Ramesh R

    2009-08-01

    The success of cell replacement therapy for diabetes depends on the availability and generation of an adequate number of islets, preferably from an autologous origin. Stem cells are now being probed for the generation of physiologically competent, insulin-producing cells. In this investigation, we explored the potential of adipose tissue-derived stem cells (ASCs) to differentiate into pancreatic hormone-expressing islet-like cell aggregates (ICAs). We initiated ASC culture from epididymal fat pads of Swiss albino mice to obtain mesenchymal cells, murine epididymal (mE)-ASCs. Subsequent single-cell cloning resulted in a homogeneous cell population with a CD29(+)CD44(+)Sca-1(+) surface antigen expression profile. We formulated a 10-day differentiation protocol to generate insulin-expressing ICAs from mE-ASCs by progressively changing the differentiation cocktail on day 1, day 3, and day 5. Our stage-specific approach successfully differentiated mesodermic mE-ASCs into definitive endoderm (cells expressing Sox17, Foxa2, GATA-4, and cytokeratin [CK]-19), then into pancreatic endoderm (cells expressing pancreatic and duodenal homeobox [PDX]-1, Ngn3, NeuroD, Pax4, and glucose transporter 2), and finally into cells expressing pancreatic hormones (insulin, glucagon, somatostatin). Fluorescence-activated cell sorting analysis showed that day 5 ICAs contained 64.84% +/- 7.03% PDX-1(+) cells, and in day 10 mature ICAs, 48.17% +/- 3% of cells expressed C-peptide. Day 10 ICAs released C-peptide in a glucose-dependent manner, exhibiting in vitro functionality. Electron microscopy of day 10 ICAs revealed the presence of numerous secretory granules within the cell cytoplasm. Calcium alginate-encapsulated day 10 ICAs (1,000-1,200), when transplanted i.p. into streptozotocin-induced diabetic mice, restored normoglycemia within 2 weeks. The data presented here demonstrate the feasibility of using ASCs as a source of autologous stem cells to differentiate into the pancreatic lineage. PMID:19544426

  6. An in vitro embryotoxicity assay based on the disturbance of the differentiation of murine embryonic stem cells into endothelial cells. I: Establishment of the differentiation protocol

    Microsoft Academic Search

    Matthias Festag; Claudia Sehner; Pablo Steinberg; Bruno Viertel

    2007-01-01

    The aim of the present study was to establish an experimental protocol to differentiate murine embryonic stem (ES) cells into endothelial cells in vitro. The spinner flask technique as well as the hanging drop method were used to generate so-called embryoid bodies (EBs). In order to find out the optimal differentiation environment, EBs were cultured under various experimental conditions for

  7. Tsc2 Null Murine Neuroepithelial Cells Are a Model for Human Tuber Giant Cells, and Show Activation of an mTOR Pathway

    Microsoft Academic Search

    Hiroaki Onda; Peter B. Crino; Hongbing Zhang; Ryan D. Murphey; Luca Rastelli; Bonnie E. Gould Rothberg; David J. Kwiatkowski

    2002-01-01

    Cortical tubers are developmental brain malformations in the tuberous sclerosis complex (TSC) that cause epilepsy and autism in TSC patients whose pathogenesis is uncertain. Tsc2 null murine neuroepithelial progenitor (NEP) cells display persistent growth when growth factors are withdrawn, express GFAP at high levels, and have reduced expression of a set of early neuronal lineage markers. Tsc2 null NEP cells

  8. Role of prostaglandin E2 receptors in migration of murine and human breast cancer cells.

    PubMed

    Timoshenko, Alexander V; Xu, Guoxiong; Chakrabarti, Sumontra; Lala, Peeyush K; Chakraborty, Chandan

    2003-10-01

    Aberrant upregulation of COX-2 enzyme resulting in accumulation of PGE2 in a cancer cell environment is a marker for progression of many cancers, including breast cancer. Four subtypes of cell surface receptors (EP1, EP2, EP3, and EP4), which are coupled with different G-proteins, mediate PGE2 actions. Since migration is an essential step in invasion and metastasis, in the present study we defined the expression of EP receptors and their roles in migratory function of breast cancer cells of murine (C3L5) and human (MDA-MB-231 and MCF-7) origin. Highly metastatic C3L5 and MDA-MB-231 cells, found to be highly migratory in a Transwell migration assay, were shown to accumulate much higher levels of PGE2 in culture media in comparison with nonmetastatic and poorly migrating MCF-7 cells; the levels of PGF2alpha and 6-keto-PGF1alpha were low in all cases. The elevated PGE2 production by metastatic cancer cells was due to COX-2 activity since dual COX-1/2 inhibitor indomethacin and selective COX-2 inhibitor NS-398 equally suppressed both basal and inducible (by IFN-gamma/LPS or Ca2+-ionophores) PGE2 accumulation. RT-PCR analysis revealed that murine C3L5 cells expressed mRNA of EP1, EP3, and EP4 but not EP2 receptors. On the other hand, human MDA-MB-231 and MCF-7 cells expressed all the above receptors. High levels of expression of functional EP4 receptors coupled with Gs-protein was confirmed in C3L5 cells by biochemical assay showing a dose-dependent increase of intracellular cAMP synthesis in response to PGE2. EP receptor antagonists SC-19220, AH-6809, and AH-23848B, having highest affinity for EP1, EP1/EP2/DP, and EP4 receptors, respectively, variably inhibited migration of metastatic breast cancer cells. An autocrine PGE2-mediated migratory activity of these cells appeared to be associated predominantly with EP4 receptor-mediated signaling pathway, which uses cAMP as a second messenger. This conclusion is based on several observations: (1) selective EP4 antagonist AH-23848B effectively inhibited migration of both C3L5 and MDA-MB-231 cells in a dose-dependent manner; (2) exogenous PGE2 and EP4 agonist PGE1 alcohol increased migration of C3L5 cells; (3) forskolin, a potent activator of adenylate cyclase, as well as membrane-permeable analogues of cAMP (8-bromo-cAMP, dibutyryl-cAMP) stimulated migration of C3L5 cells; and (4) Rp-cAMPS, a selective protein kinase A inhibitor, reduced migration of C3L5 cells. Migration of poorly migratory MCF-7 cells remained unaffected with either PGE2 or EP4 antagonist. These findings are relevant for designing therapeutic strategies against breast cancer metastasis. PMID:14499627

  9. Inhibition of Amino Acid Transport by c\\/s-Diamminedichloroplatinum(ll) Derivatives in L1210 Murine Leukemia Cells1

    Microsoft Academic Search

    K. J. Scanlon; R. L. Safirstein; H. Thies; R. B. Gross; S. Waxman; J. B. Guttenplan

    The uptake of c;s-diamminedichloroplatinum(ll) (cisplatin) has been studied in the L1210 murine lymphoid leukemia cell line. Labeled cisplatin and its aquated derivatives were resolved by high-performance liquid chromatography on a strong cationic exchange column. After 10 min of incubation of cisplatin with the cells, the major portion of the non-protein-bound platinum was in the form of cisplatin. However, a portion

  10. Genetic Modification of a Murine Fibrosarcoma to Produce Interleukin 7 Stimulates Host Cell Infiltration and Tumor Immunity1

    Microsoft Academic Search

    William H. McBride; J. Dean Thacker; Sian Coinora; James S. Economou; David Kelley; Donna Hogge; Steven M. Dubinett; Graeme J. Dougherty

    1992-01-01

    Retroviral-mediated gene transfer was used to introduce and express the gene for murine interleukin 7 (II,-7) in a fibrosarcoma tumor (FSA). The tumorigenicity of these genetically modified FSA cells was greatly decreased in immunologically intact syngeneic mice but was unaltered in T-cell-deprived mice. IL-7-infected tumors that did grow in intact ani mals from large size inocula did so slowly and

  11. Induction of erythroid differentiation by inhibition of Ras\\/ERK pathway in a Friend murine leukemia cell line

    Microsoft Academic Search

    Tomoko Matsuzaki; Ken-ichi Aisaki; Yasuko Yamamura; Makoto Noda; Yoji Ikawa

    2000-01-01

    The role of Ras and MAP kinases (MAPKs) in the regulation of erythroid differentiation was studied using a cell line (SKT6) derived from Friend virus (Anemic strain)-induced murine erythroleukemia. This cell line undergoes differentiation in vitro in response to erythropoietin (EPO) or other chemical inducers such as dimethylsulfoxide (DMSO). When a constitutively active ras mutant (ras12V) was expressed in SKT6

  12. A Novel Dendritic Cell-Based Cancer Vaccine Produces Promising Results in a Syngenic CC36 Murine Colon Adenocarcinoma Model

    Microsoft Academic Search

    Angela M. Jack; Nebil Aydin; Grace Montenegro; Khorshed Alam; Marc Wallack

    Background. This study was conducted to test the efficacy of a new cancer vaccine, composed of dendritic cells (DCs) pulsed with an interleukin-2 gene-encoded vaccinia virus tumor oncolysate (DC-IL-2VCO) in a CC-36 murine colon adenocarcinoma model. Materials and methods. CC-36 tumor cells were in- jected subcutaneously into the left flank of four- to six-week old male BALB\\/c mice. The mice

  13. Promoter analysis of the murine T-cell protein tyrosine phosphatase gene.

    PubMed

    Wee, C; Muise, E S; Coquelet, O; Ennis, M; Wagner, J; Lemieux, N; Branton, P E; Nepveu, A; Tremblay, M L

    1999-09-17

    The T-cell protein tyrosine phosphatase (TC PTP) is expressed ubiquitously at all stages of mammalian development. However, mRNA levels fluctuate in a cell-cycle-dependent manner, reaching peak levels in late G1, and rapidly decreasing in S phase. Furthermore, TC PTP being present in higher amounts in lymphoid tissues, we have recently shown that it is essential for proper maintenance of both the bone marrow micro-environment and B- and T-cell functions. In order to better understand the elements controlling the expression pattern of this gene, we have isolated and characterized approx. 4kb of the murine TC PTP promoter. DNA sequencing of the proximal 5' region revealed the absence of both TATAA and CAAT boxes. Primer extension analysis and S1 nuclease mapping techniques identified multiple transcription initiation sites. Functional promoter activity was determined using transfection experiments of promoter deletion constructs fused to a CAT reporter construct. Our results indicate that the minimal promoter sequence required for functional expression is contained within the first 147bp of the TC PTP promoter. In addition, consistent with the cell-cycle-dependent expression of TC PTP, we localized a domain between 492 and 1976bp from the transcription initiation site through which repression occurs. In conclusion, although initiator-driven transcription allows for ubiquitous expression of TC PTP, we define general transcription motifs present within the promoter that may mediate specific modulations of the TC PTP gene. PMID:10521659

  14. Mechanical Activation of ?-catenin Regulates Phenotype in Adult Murine Marrow-Derived Mesenchymal Stem Cells

    PubMed Central

    Case, Natasha; Xie, Zhihui; Sen, Buer; Styner, Maya; Zou, Minxu; O’Conor, Chris; Horowitz, Mark; Rubin, Janet

    2010-01-01

    Regulation of skeletal remodeling appears to influence the differentiation of multipotent mesenchymal stem cells (MSC) resident in the bone marrow. As murine marrow cultures are contaminated with hematopoietic cells, they are problematic for studying direct effects of mechanical input. Here we use a modified technique to isolate marrow-derived MSC (mdMSC) from adult mice, yielding a population able to differentiate into adipogenic and osteogenic phenotypes that is devoid of hematopoietic cells. In pure mdMSC populations, a daily strain regimen inhibited adipogenic differentiation, suppressing expression of PPAR? and adiponectin. Strain increased ?-catenin and inhibition of adipogenesis required this effect. Under osteogenic conditions, strain activated ?-catenin signaling and increased expression of WISP1 and COX2. mdMSC were also generated from mice lacking caveolin-1, a protein known to sequester ?-catenin: caveolin-1(?/?) mdMSC exhibited retarded differentiation along both adipogenic and osteogenic lineages, but retained mechanical responses that involved ?-catenin activation. Interestingly, caveolin-1(?/?) mdMSC failed to express bone sialoprotein and did not form mineralized nodules. In summary, mdMSC from adult mice respond to both soluble factors and mechanical input, with mechanical activation of ?-catenin influencing phenotype. As such, these cells offer a useful model for studies of direct mechanical regulation of MSC differentiation and function. PMID:20872592

  15. 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

  16. 3D culture of murine neural stem cells on decellularized mouse brain sections.

    PubMed

    De Waele, Jorrit; Reekmans, Kristien; Daans, Jasmijn; Goossens, Herman; Berneman, Zwi; Ponsaerts, Peter

    2015-02-01

    Transplantation of neural stem cells (NSC) in diseased or injured brain tissue is widely studied as a potential treatment for various neurological pathologies. However, effective cell replacement therapy relies on the intrinsic capacity of cellular grafts to overcome hypoxic and/or immunological barriers after transplantation. In this context, it is hypothesized that structural support for grafted NSC will be of utmost importance. With this study, we present a novel decellularization protocol for 1.5 mm thick mouse brain sections, resulting in the generation of acellular three-dimensional (3D) brain sections. Next, the obtained 3D brain sections were seeded with murine NSC expressing both the eGFP and luciferase reporter proteins (NSC-eGFP/Luc). Using real-time bioluminescence imaging, the survival and growth of seeded NSC-eGFP/Luc cells was longitudinally monitored for 1-7 weeks in culture, indicating the ability of the acellular brain sections to support sustained ex vivo growth of NSC. Next, the organization of a 3D maze-like cellular structure was examined using confocal microscopy. Moreover, under mitogenic stimuli (EGF and hFGF-2), most cells in this 3D culture retained their NSC phenotype. Concluding, we here present a novel protocol for decellularization of mouse brain sections, which subsequently support long-term 3D culture of undifferentiated NSC. PMID:25522971

  17. Fam40b is required for lineage commitment of murine embryonic stem cells

    PubMed Central

    Wagh, V; Doss, M X; Sabour, D; Niemann, R; Meganathan, K; Jagtap, S; Gaspar, J A; Ardestani, M A; Papadopoulos, S; Gajewski, M; Winkler, J; Hescheler, J; Sachinidis, A

    2014-01-01

    FAM40B (STRIP2) is a member of the striatin-interacting phosphatase and kinase (STRIPAK) complex that is involved in the regulation of various processes such as cell proliferation and differentiation. Its role for differentiation processes in embryonic stem cells (ESCs) is till now completely unknown. Short hairpin RNA (shRNA)-mediated silencing of Fam40b expression in ESCs and differentiating embryoid bodies (EBs) led to perturbed differentiation to embryonic germ layers and their derivatives including a complete abrogation of cardiomyogenesis. Pluripotency factors such as Nanog, Oct4 and Sox2 as well as epigenetic factors such as histone acetyltransferase type B (HAT1) and DNA (cytosine-5)-methyltransferase 3-? (Dnmt3b) were highly upregulated in Fam40b knockdown EBs as compared with control and scrambled EBs. To examine the relevance of Fam40b for development in vivo, Fam40b was knocked down in developing zebrafish. Morpholino-mediated knockdown of Fam40b led to severe abnormalities of the cardiovascular system, including an impaired expression of ventricular myosin heavy chain (vmhc) and of cardiac myosin light chain 2 (cmlc2) in the heart. We identified the gene product of Fam40b in ESCs as a perinuclear and nucleolar protein with a molecular weight of 96?kDa. We conclude that the expression of Fam40b is essential for the lineage commitment of murine embryonic stem cells (mESCs) into differentiated somatic cells via mechanisms involving pluripotency and epigenetic networks. PMID:25010986

  18. 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

  19. Myeloid-Derived Suppressor Cells Prevent Type 1 Diabetes in Murine Models

    PubMed Central

    Yin, Bingjiao; Ma, Ge; Yen, Chun-Yu; Zhou, Zuping; Wang, George X.; Divino, Celia M.; Casares, Sofia; Chen, Shu-Hsia; Yang, Wen-Chin; Pan, Ping-Ying

    2015-01-01

    Effective immunotherapy for type 1 diabetes (T1D) relies on active induction of peripheral tolerance. Myeloid-derived suppressor cells (MDSCs) play a critical role in suppressing immune responses in various pathologic settings via multiple mechanisms, including expansion of regulatory T cells (Tregs). In this study, we investigated whether MDSCs could act as APCs to induce expansion of Ag-specific Tregs, suppress T cell proliferation, and prevent autoimmune T1D development. We found that MDSC-mediated expansion of Tregs and T cell suppression required MHC-dependent Ag presentation. A murine T1D model was established in INS-HA/RAG?/? mice in which animals received CD4-HA-TCR transgenic T cells via adoptive transfer. We found a significant reduction in the incidence of diabetes in recipients receiving MDSC plus HA, but not OVA peptide, leading to 75% diabetes-free mice among the treated animals. To test further whether MDSCs could prevent diabetes onset in NOD mice, nondiabetic NOD/SCID mice were injected with inflammatory T cells from diabetic NOD mice. MDSCs significantly prevented diabetes onset, and 60% of MDSC-treated mice remained diabetes free. The pancreata of treated mice showed significantly lower levels of lymphocyte infiltration in islet and less insulitis compared with that of the control groups. The protective effects of MDSCs might be mediated by inducing anergy in autoreactive T cells and the development of CD4+CD25+Foxp3+ Tregs. Thist study demonstrates a remarkable capacity of transferred MDSCs to downregulate Ag-specific autoimmune responses and prevent diabetes onset, suggesting that MDSCs possess great potential as a novel cell-based tolerogenic therapy in the control of T1D and other autoimmune diseases. PMID:20956337

  20. The in vitro effects of Bordetella pertussis lymphocytosis-promoting factor on murine lymphocytes: II. Nature of the responding cells

    PubMed Central

    1977-01-01

    The mitogenic response of murine lymphocytes to the lymphocytosis- promoting factor of Bordetella pertussis has been shown to be due to activation of T cells. The selectivity of responsiveness to LPF with respect to the population of T cells which is stimllated, differs from that of PHA as well as Con A, and the surface receptors are different. A population of adherent cells, which does not appear to consist of macrophages or other phagocytic cells, is required for the T-cell response. PMID:187716

  1. Evidence that a slowly cycling subpopulation of adult murine epidermal cells retains carcinogen.

    PubMed

    Morris, R J; Fischer, S M; Slaga, T J

    1986-06-01

    The distribution and persistence of radioactively labeled benzo(a)pyrene [B(a)P] in the skin of adult female SENCAR mice were investigated by autoradiography of epidermal whole mounts and cross-sections at intervals following a single initiating application of 200 nmol of either [3H]B(a)P (2 mCi) or [14C]B(a)P (23 muCi). One day after treatment, the entire thickness of the skin was labeled; the grain density was greatest over hair follicles, sebaceous glands, and interfollicular epidermis. At 1 and 2 weeks, decreases in the nuclear grain density were consistent with the overall pattern of epidermal renewal. One month after treatment, carcinogen label-retaining cells made up approximately 2% of the interfollicular basal cells. They were also present in the hair follicles, approximately 4 and 5% of basal cells in the infundibulum and external root sheath, respectively. They were rare in the germ region and dermal papilla. Carcinogen label-retaining cells were compared with slowly cycling [3H]thymidine label-retaining cells and "maturing" basal cells, two distinct proliferative subsets of adult murine epidermis. Carcinogen label-retaining cells were found to have characteristics of the slowly cycling cells: (a) most of the carcinogen labeled nuclei were found in the central regions of the epidermal proliferative units; (b) treatment of the carcinogen label-retaining cells with 2 micrograms of 12-O-tetradecanoylphorbol-13-acetate elicited labeled mitoses within 1 day, and a general decrease in grain density over basal nuclei. In contrast, maturing basal cells 4 days after a single injection of [3H]thymidine were found at the periphery of the epidermal proliferative units. Within 1 day after treatment with 2 micrograms of 12-O-tetradecanoylphorbol-13-acetate, maturing basal cells were displaced to the suprabasal layers. Double isotope-double emulsion autoradiographs demonstrated doubly labeled cells 1 month after continuous labeling with [3H]thymidine and [14C]B(a)P and provide evidence that the radioactive carcinogen is retained by the slowly cycling [3H]thymidine label-retaining cells. These observations suggest that a slowly cycling population of epidermal cells may be relevant to the initiation phase of two-stage carcinogenesis. PMID:3698024

  2. Role of NK cell subsets in organ-specific murine melanoma metastasis.

    PubMed

    Ballas, Zuhair K; Buchta, Claire M; Rosean, Timothy R; Heusel, Jonathan W; Shey, Michael R

    2013-01-01

    Tumor metastasis plays a major role in the morbidity and mortality of cancer patients. Among solid tumors that undergo metastasis, there is often a predilection to metastasize to a particular organ with, for example, prostate cancer preferentially metastasizing to bones and colon cancer preferentially metastasizing to the liver. Although many factors are thought to be important in establishing permissiveness for metastasis, the reasons for organ-specific predilection of each tumor are not understood. Using a B16 murine melanoma model, we tested the hypothesis that organ-specific NK cell subsets play a critical role in organ-specific metastasis of this tumor. Melanoma cells, given intravenously, readily colonized the lungs but not the liver. NK cell depletion (either iatrogenically or by using genetically targeted mice) resulted in substantial hepatic metastasis. Analysis of NK cell subsets, defined by the differential expression of a combination of CD27 and CD11b, indicated a significant difference in the distribution of NK cell subsets in the lung and liver with the mature subset being dominant in the lung and the immature subset being dominant in the liver. Several experimental approaches, including adoptive transfer, clearly indicated that the immature hepatic NK cell subset, CD27+ CD11b-, was protective against liver metastasis; this subset mediated its protection by a perforin-dependent cytotoxic mechanism. In contrast, the more mature NK cell subsets were more efficient at reducing pulmonary tumor load. These data indicate that organ-specific immune responses may play a pivotal role in determining the permissiveness of a given organ for the establishment of a metastatic niche. PMID:23776508

  3. Role of NK Cell Subsets in Organ-Specific Murine Melanoma Metastasis

    PubMed Central

    Ballas, Zuhair K.; Buchta, Claire M.; Rosean, Timothy R.; Heusel, Jonathan W.; Shey, Michael R.

    2013-01-01

    Tumor metastasis plays a major role in the morbidity and mortality of cancer patients. Among solid tumors that undergo metastasis, there is often a predilection to metastasize to a particular organ with, for example, prostate cancer preferentially metastasizing to bones and colon cancer preferentially metastasizing to the liver. Although many factors are thought to be important in establishing permissiveness for metastasis, the reasons for organ-specific predilection of each tumor are not understood. Using a B16 murine melanoma model, we tested the hypothesis that organ-specific NK cell subsets play a critical role in organ-specific metastasis of this tumor. Melanoma cells, given intravenously, readily colonized the lungs but not the liver. NK cell depletion (either iatrogenically or by using genetically targeted mice) resulted in substantial hepatic metastasis. Analysis of NK cell subsets, defined by the differential expression of a combination of CD27 and CD11b, indicated a significant difference in the distribution of NK cell subsets in the lung and liver with the mature subset being dominant in the lung and the immature subset being dominant in the liver. Several experimental approaches, including adoptive transfer, clearly indicated that the immature hepatic NK cell subset, CD27+ CD11b–, was protective against liver metastasis; this subset mediated its protection by a perforin-dependent cytotoxic mechanism. In contrast, the more mature NK cell subsets were more efficient at reducing pulmonary tumor load. These data indicate that organ-specific immune responses may play a pivotal role in determining the permissiveness of a given organ for the establishment of a metastatic niche. PMID:23776508

  4. Hemolysin-Positive Enteroaggregative and Cell-Detaching Escherichia coli Strains Cause Oncosis of Human Monocyte-Derived Macrophages and Apoptosis of Murine J774 Cells

    Microsoft Academic Search

    CARMEN FERNANDEZ-PRADA; BEN D. TALL; SIMON E. ELLIOTT; DAVID L. HOOVER; JAMES P. NATARO; MALABI M. VENKATESAN

    Infection of human monocyte-derived macrophages (HMDM) and J774 cells (murine macrophage cell line) with several enteroaggregative and cytodetaching Escherichia coli (EAggEC and CDEC, respectively) strains demonstrated that some strains could induce macrophage cell death accompanied by release of lactate dehydrogenase activity and interleukin 1b (IL-1b) into culture supernatants. The mode of cell death differed in the two types of macrophages.

  5. Intracoronary administration of cardiac stem cells in mice: a new, improved technique for cell therapy in murine models

    PubMed Central

    Li, Qianhong; Guo, Yiru; Ou, Qinghui; Chen, Ning; Wu, Wen-Jian; Yuan, Fangping; O’Brien, Erin; Wang, Tao; Luo, Li; Hunt, Gregory N.; Zhu, Xiaoping

    2013-01-01

    A model of intracoronary stem cell delivery that enables transgenesis/gene targeting would be a powerful tool but is still lacking. To address this gap, we compared intracoronary and intramyocardial delivery of lin?/c-kit+/GFP+ cardiac stem cells (CSCs) in a murine model of reperfused myocardial infarction (MI). Lin?/c-kit+/GFP+ CSCs were successfully expanded from GFP transgenic hearts and cultured with no detectable phenotypic change for up to ten passages. Intracoronary delivery of CSCs 2 days post-MI resulted in significant alleviation of adverse LV remodeling and dysfunction, which was at least equivalent, if not superior, to that achieved with intramyocardial delivery. Compared with intramyocardial injection, intracoronary infusion was associated with a more homogeneous distribution of CSCs in the infarcted region and a greater increase in viable tissue in this region, suggesting greater formation of new cardiomyocytes. Intracoronary CSC delivery resulted in improved function in the infarcted region, as well as in improved global LV systolic and diastolic function, and in decreased LV dilation and LV expansion index; the magnitude of these effects was similar to that observed after intramyocardial injection. We conclude that, in the murine model of reperfused MI, intracoronary CSC infusion is at least as effective as intramyocardial injection in limiting LV remodeling and improving both regional and global LV function. The intracoronary route appears to be superior in terms of uniformity of cell distribution, myocyte regeneration, and amount of viable tissue in the risk region. To our knowledge, this is the first study to report that intracoronary infusion of stem cells in mice is feasible and effective. PMID:21516491

  6. 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

  7. 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

  8. 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

    2014-10-27

    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

  9. Sustained CTL activation by murine pulmonary epithelial cells promotes the development of COPD-like disease

    PubMed Central

    Borchers, Michael T.; Wesselkamper, Scott C.; Curull, Victor; Ramirez-Sarmiento, Alba; Sánchez-Font, Albert; Garcia-Aymerich, Judith; Coronell, Carlos; Lloreta, Josep; Agusti, Alvar G.; Gea, Joaquim; Howington, John A.; Reed, Michael F.; Starnes, Sandra L.; Harris, Nathaniel L.; Vitucci, Mark; Eppert, Bryan L.; Motz, Gregory T.; Fogel, Kevin; McGraw, Dennis W.; Tichelaar, Jay W.; Orozco-Levi, Mauricio

    2009-01-01

    Chronic obstructive pulmonary disease (COPD) is a lethal progressive lung disease culminating in permanent airway obstruction and alveolar enlargement. Previous studies suggest CTL involvement in COPD progression; however, their precise role remains unknown. Here, we investigated whether the CTL activation receptor NK cell group 2D (NKG2D) contributes to the development of COPD. Using primary murine lung epithelium isolated from mice chronically exposed to cigarette smoke and cultured epithelial cells exposed to cigarette smoke extract in vitro, we demonstrated induced expression of the NKG2D ligand retinoic acid early transcript 1 (RAET1) as well as NKG2D-mediated cytotoxicity. Furthermore, a genetic model of inducible RAET1 expression on mouse pulmonary epithelial cells yielded a severe emphysematous phenotype characterized by epithelial apoptosis and increased CTL activation, which was reversed by blocking NKG2D activation. We also assessed whether NKG2D ligand expression corresponded with pulmonary disease in human patients by staining airway and peripheral lung tissues from never smokers, smokers with normal lung function, and current and former smokers with COPD. NKG2D ligand expression was independent of NKG2D receptor expression in COPD patients, demonstrating that ligand expression is the limiting factor in CTL activation. These results demonstrate that aberrant, persistent NKG2D ligand expression in the pulmonary epithelium contributes to the development of COPD pathologies. PMID:19197141

  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. Stimulatory Effects of Polysaccharide Fraction from Solanum nigrum on RAW 264.7 Murine Macrophage Cells

    PubMed Central

    Razali, Faizan Naeem; Ismail, Amirah; Abidin, Nurhayati Zainal; Shuib, Adawiyah Suriza

    2014-01-01

    The polysaccharide fraction from Solanum nigrum Linne has been shown to have antitumor activity by enhancing the CD4+/CD8+ ratio of the T-lymphocyte subpopulation. In this study, we analyzed a polysaccharide extract of S. nigrum to determine its modulating effects on RAW 264.7 murine macrophage cells since macrophages play a key role in inducing both innate and adaptive immune responses. Crude polysaccharide was extracted from the stem of S. nigrum and subjected to ion-exchange chromatography to partially purify the extract. Five polysaccharide fractions were then subjected to a cytotoxicity assay and a nitric oxide production assay. To further analyze the ability of the fractionated polysaccharide extract to activate macrophages, the phagocytosis activity and cytokine production were also measured. The polysaccharide fractions were not cytotoxic, but all of the fractions induced nitric oxide in RAW 264.7 cells. Of the five fractions tested, SN-ppF3 was the least toxic and also induced the greatest amount of nitric oxide, which was comparable to the inducible nitric oxide synthase expression detected in the cell lysate. This fraction also significantly induced phagocytosis activity and stimulated the production of tumor necrosis factor-? and interleukin-6. Our study showed that fraction SN-ppF3 could classically activate macrophages. Macrophage induction may be the manner in which polysaccharides from S. nigrum are able to prevent tumor growth. PMID:25299340

  12. Interactome analysis of myeloid-derived suppressor cells in murine models of colon and breast cancer.

    PubMed

    Aliper, Alexander M; Frieden-Korovkina, Victoria P; Buzdin, Anton; Roumiantsev, Sergey A; Zhavoronkov, Alex

    2014-11-30

    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

  13. 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

  14. Efficient Production and Purification of Recombinant Murine Kindlin-3 from Insect Cells for Biophysical Studies

    PubMed Central

    Yates, Luke A.; Gilbert, Robert J. C.

    2014-01-01

    Kindlins are essential coactivators, with talin, of the cell surface receptors integrins and also participate in integrin outside-in signalling, and the control of gene transcription in the cell nucleus. The kindlins are ~75 kDa multidomain proteins and bind to an NPxY motif and upstream T/S cluster of the integrin ?-subunit cytoplasmic tail. The hematopoietically-important kindlin isoform, kindlin-3, is critical for platelet aggregation during thrombus formation, leukocyte rolling in response to infection and inflammation and osteoclast podocyte formation in bone resorption. Kindlin-3's role in these processes has resulted in extensive cellular and physiological studies. However, there is a need for an efficient method of acquiring high quality milligram quantities of the protein for further studies. We have developed a protocol, here described, for the efficient expression and purification of recombinant murine kindlin-3 by use of a baculovirus-driven expression system in Sf9 cells yielding sufficient amounts of high purity full-length protein to allow its biophysical characterization. The same approach could be taken in the study of the other mammalian kindlin isoforms. PMID:24686835

  15. The Acute Exposure Effects of Inhaled Nickel Nanoparticles on Murine Endothelial Progenitor Cells

    PubMed Central

    Liberda, Eric N; Cuevas, Azita K; Qu, Qingshan; Chen, Lung Chi

    2014-01-01

    Introduction The discovery of endothelial progenitor cells (EPCs) may help to explain observed cardiovascular effects associated with inhaled nickel nanoparticle exposures such as increases in vascular inflammation, generate reactive oxygen species, alter vasomotor tone, and potentiated atherosclerosis in murine species. Methods Following an acute whole body inhalation exposure to 500?g/m3 of nickel nanoparticles for 5 hrs, bone marrow EPCs from C57BL/6 mice were isolated. EPCs were harvested for their RNA or used in a variety of assays including chemotaxis, tube formation, and proliferation. Gene expression was assessed for important receptors involved in EPC mobilization and homing using RT-PCR methods. EPCs, circulating endothelial progenitor cells (CEPCs), circulating endothelial cells (CECs), and endothelial microparticles (EMPs) were quantified on a BD FACSCalibur to examine endothelial damage and repair associated with the exposure. Results and Conclusions Acute exposure to inhaled nickel nanoparticles significantly increased both bone marrow EPCs as well as their levels in circulation (CEPCs). CECs were significantly elevated indicating that endothelial damage occurred due to the exposure. There was no significant difference in EMPs between the two groups. Tube formation and chemotaxis, but not proliferation, of bone marrow EPCs was impaired in the nickel nanoparticle exposed group. These results coincided with a decrease in the mRNA of receptors involved in EPC mobilization and homing. This data provides new insight into how an acute nickel nanoparticle exposure to half of the current Occupational Safety & Health Administration permissible exposure limit may adversely affect EPCs. PMID:25144474

  16. Bromelain Inhibits Allergic Sensitization and Murine Asthma via Modulation of Dendritic Cells.

    PubMed

    Secor, Eric R; Szczepanek, Steven M; Castater, Christine A; Adami, Alexander J; Matson, Adam P; Rafti, Ektor T; Guernsey, Linda; Natarajan, Prabitha; 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

  17. Antileukemic effect of zerumbone-loaded nanostructured lipid carrier in WEHI-3B cell-induced murine leukemia model.

    PubMed

    Rahman, Heshu Sulaiman; Rasedee, Abdullah; How, Chee Wun; Zeenathul, Nazariah Allaudin; Chartrand, Max Stanley; Yeap, Swee Keong; Abdul, Ahmad Bustamam; Tan, Sheau Wei; Othman, Hemn Hassan; Ajdari, Zahra; Namvar, Farideh; Arulselvan, Palanisamy; Fakurazi, Sharida; Mehrbod, Parvaneh; Daneshvar, Nasibeh; Begum, Hasina

    2015-01-01

    Cancer nanotherapy is progressing rapidly with the introduction of many innovative drug delivery systems to replace conventional therapy. Although the antitumor activity of zerumbone (ZER) has been reported, there has been no information available on the effect of ZER-loaded nanostructured lipid carrier (NLC) (ZER-NLC) on murine leukemia cells. In this study, the in vitro and in vivo effects of ZER-NLC on murine leukemia induced with WEHI-3B cells were investigated. The results from 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide, Hoechst 33342, Annexin V, cell cycle, and caspase activity assays showed that the growth of leukemia cells in vitro was inhibited by ZER-NLC. In addition, outcomes of histopathology, transmission electron microscopy, and Tdt-mediated dUTP nick-end labeling analyses revealed that the number of leukemia cells in the spleen of BALB/c leukemia mice significantly decreased after 4 weeks of oral treatment with various doses of ZER-NLC. Western blotting and reverse-transcription quantitative polymerase chain reaction assays confirmed the antileukemia effects of ZER-NLC. In conclusion, ZER-NLC was shown to induce a mitochondrial-dependent apoptotic pathway in murine leukemia. Loading of ZER in NLC did not compromise the anticancer effect of the compound, suggesting ZER-NLC as a promising and effective delivery system for treatment of cancers. PMID:25767386

  18. Anti-tumor activity of Acinetobacter baumannii outer membrane protein A on dendritic cell-based immunotherapy against murine melanoma.

    PubMed

    Lee, Jun Sik; Kim, Jung Wook; Choi, Chul Hee; Lee, Won Kee; Chung, Hae Young; Lee, Je Chul

    2008-04-01

    Acinetobacter baumannii outer membrane protein A (AbOmpA) is a major surface protein that is an important pathogen-associated molecular pattern. Based on our previous findings that AbOmpA induced the phenotypic maturation of dendritic cells (DCs) and drove the Th1 immune response in vitro, we investigated the therapeutic efficacy of AbOmpA-pulsed DC vaccines in a murine melanoma model. The surface expression of co-stimulatory molecules (CD80 and CD86) and major histocompatibility complex class I and II molecules was higher in DCs pulsed with AbOmpA alone or with a combination of B16F10 cell lysates than that of DCs pulsed with B16F10 cell lysates. AbOmpA stimulated the maturation of murine splenic DCs in vivo. In a therapeutic model of murine melanoma, AbOmpA-pulsed DCs significantly retarded tumor growth and improved the survival of tumor-bearing mice. AbOmpA-pulsed DCs significantly enhanced CD8+, interleukin-2+ T cells and CD4+, interferon-gamma+ T cells in tumor-bearing mice. These results provide evidence that AbOmpA may be therapeutically useful in adjuvant DC immunotherapy against poorly immunogenic melanoma without tumor-specific antigens. PMID:18545973

  19. Antileukemic effect of zerumbone-loaded nanostructured lipid carrier in WEHI-3B cell-induced murine leukemia model

    PubMed Central

    Rahman, Heshu Sulaiman; Rasedee, Abdullah; How, Chee Wun; Zeenathul, Nazariah Allaudin; Chartrand, Max Stanley; Yeap, Swee Keong; Abdul, Ahmad Bustamam; Tan, Sheau Wei; Othman, Hemn Hassan; Ajdari, Zahra; Namvar, Farideh; Arulselvan, Palanisamy; Fakurazi, Sharida; Mehrbod, Parvaneh; Daneshvar, Nasibeh; Begum, Hasina

    2015-01-01

    Cancer nanotherapy is progressing rapidly with the introduction of many innovative drug delivery systems to replace conventional therapy. Although the antitumor activity of zerumbone (ZER) has been reported, there has been no information available on the effect of ZER-loaded nanostructured lipid carrier (NLC) (ZER-NLC) on murine leukemia cells. In this study, the in vitro and in vivo effects of ZER-NLC on murine leukemia induced with WEHI-3B cells were investigated. The results from 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide, Hoechst 33342, Annexin V, cell cycle, and caspase activity assays showed that the growth of leukemia cells in vitro was inhibited by ZER-NLC. In addition, outcomes of histopathology, transmission electron microscopy, and Tdt-mediated dUTP nick-end labeling analyses revealed that the number of leukemia cells in the spleen of BALB/c leukemia mice significantly decreased after 4 weeks of oral treatment with various doses of ZER-NLC. Western blotting and reverse-transcription quantitative polymerase chain reaction assays confirmed the antileukemia effects of ZER-NLC. In conclusion, ZER-NLC was shown to induce a mitochondrial-dependent apoptotic pathway in murine leukemia. Loading of ZER in NLC did not compromise the anticancer effect of the compound, suggesting ZER-NLC as a promising and effective delivery system for treatment of cancers.

  20. 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

  1. Murine Cardiosphere-Derived Cells Are Impaired by Age but Not by Cardiac Dystrophic Dysfunction

    PubMed Central

    Hsiao, Lien-Cheng; Perbellini, Filippo; Gomes, Renata S.M.; Tan, Jun Jie; Vieira, Silvia; Faggian, Giuseppe; Clarke, Kieran

    2014-01-01

    To be clinically relevant as a therapy for heart failure, endogenous progenitor cells must be isolated and expanded from aged and/or diseased tissue. Here, we investigated the effect of age and cardiac impairment resulting from lack of dystrophin on murine cardiosphere-derived cells (CDCs). CDCs were isolated and expanded from atrial biopsies from wild-type mice aged 1.5, 6, 18, and 24 months and from mdx mice aged 6 and 18 months. Cardiac function was measured in mdx mice and age-matched wild-type mice using high-resolution cine magnetic resonance imaging. CDCs could be isolated and expanded from all mice, however, the number of cells obtained, and their regenerative potential, decreased with age, as demonstrated by decreased expression of stem cell markers, c-kit and Sca-1, and decreased cell proliferation, migration, clonogenicity, and differentiation. Six-month-old mdx mice showed right ventricular (RV) dilation and reduced RV ejection fraction (EF) in comparison to wild-type mice. Older mdx mice displayed significant RV and left ventricular dilation and decreased EF in both ventricles, compared with age-matched wild-type mice. Mdx mouse hearts contained significantly more fibrotic tissue than age-matched wild-type mouse hearts. However, CDCs isolated from mice aged 6 and 18 months had the same number and regenerative potential from mdx mice and age-matched wild-type mice. Thus, the cardiac progenitor cell population is impaired by age but is not substantially altered by the progressive deterioration in function of the dystrophic heart. PMID:24351030

  2. Cytotoxic Effects of Biosynthesized Zinc Oxide Nanoparticles on Murine Cell Lines

    PubMed Central

    Namvar, Farideh; Rahman, Heshu Sulaiman; Mohamad, Rosfarizan; Azizi, Susan; Tahir, Paridah Mohd; Chartrand, Max Stanley

    2015-01-01

    The aim of this study is to evaluate the in vitro cytotoxic activity and cellular effects of previously prepared ZnO-NPs on murine cancer cell lines using brown seaweed (Sargassum muticum) aqueous extract. Treated cancer cells with ZnO-NPs for 72 hours demonstrated various levels of cytotoxicity based on calculated IC50 values using MTT assay as follows: 21.7?±?1.3??g/mL (4T1), 17.45?±?1.1??g/mL (CRL-1451), 11.75?±?0.8??g/mL (CT-26), and 5.6?±?0.55??g/mL (WEHI-3B), respectively. On the other hand, ZnO-NPs treatments for 72 hours showed no toxicity against normal mouse fibroblast (3T3) cell line. On the other hand, paclitaxel, which imposed an inhibitory effect on WEHI-3B cells with IC50 of 2.25?±?0.4, 1.17?±?0.5, and 1.6?±?0.09??g/mL after 24, 48, and 72 hours treatment, respectively, was used as positive control. Furthermore, distinct morphological changes were found by utilizing fluorescent dyes; apoptotic population was increased via flowcytometry, while a cell cycle block and stimulation of apoptotic proteins were also observed. Additionally, the present study showed that the caspase activations contributed to ZnO-NPs triggered apoptotic death in WEHI-3 cells. Thus, the nature of biosynthesis and the therapeutic potential of ZnO-NPs could prepare the way for further research on the design of green synthesis therapeutic agents, particularly in nanomedicine, for the treatment of cancer. PMID:25784947

  3. Cytotoxic effects of biosynthesized zinc oxide nanoparticles on murine cell lines.

    PubMed

    Namvar, Farideh; Rahman, Heshu Sulaiman; Mohamad, Rosfarizan; Azizi, Susan; Tahir, Paridah Mohd; Chartrand, Max Stanley; Yeap, Swee Keong

    2015-01-01

    The aim of this study is to evaluate the in vitro cytotoxic activity and cellular effects of previously prepared ZnO-NPs on murine cancer cell lines using brown seaweed (Sargassum muticum) aqueous extract. Treated cancer cells with ZnO-NPs for 72 hours demonstrated various levels of cytotoxicity based on calculated IC50 values using MTT assay as follows: 21.7?±?1.3??g/mL (4T1), 17.45?±?1.1??g/mL (CRL-1451), 11.75?±?0.8??g/mL (CT-26), and 5.6?±?0.55??g/mL (WEHI-3B), respectively. On the other hand, ZnO-NPs treatments for 72 hours showed no toxicity against normal mouse fibroblast (3T3) cell line. On the other hand, paclitaxel, which imposed an inhibitory effect on WEHI-3B cells with IC50 of 2.25?±?0.4, 1.17?±?0.5, and 1.6?±?0.09??g/mL after 24, 48, and 72 hours treatment, respectively, was used as positive control. Furthermore, distinct morphological changes were found by utilizing fluorescent dyes; apoptotic population was increased via flowcytometry, while a cell cycle block and stimulation of apoptotic proteins were also observed. Additionally, the present study showed that the caspase activations contributed to ZnO-NPs triggered apoptotic death in WEHI-3 cells. Thus, the nature of biosynthesis and the therapeutic potential of ZnO-NPs could prepare the way for further research on the design of green synthesis therapeutic agents, particularly in nanomedicine, for the treatment of cancer. PMID:25784947

  4. 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

  5. Apigenin protects HT22 murine hippocampal neuronal cells against endoplasmic reticulum stress-induced apoptosis.

    PubMed

    Choi, A Young; Choi, Ji Hyun; Lee, Jung Yeon; Yoon, Kyung-Sik; Choe, Wonchae; Ha, Joohun; Yeo, Eui-Ju; Kang, Insug

    2010-09-01

    Endoplasmic reticulum (ER) stress has been implicated in neurodegenerative diseases including Alzheimer's disease, Parkinson disease, and cerebral ischemia. In this study, we investigated the effects of apigenin on ER stress-induced apoptosis in murine HT22 hippocampal neuronal cells. Apigenin reduced apoptotic cell death of HT22 cells induced by thapsigargin (TG) and brefeldin A (BFA), two representative ER stress inducers. Consistent with these findings, apigenin blocked TG- and BFA-induced activation of caspase-12 and -3 and cleavage of poly (ADP-ribose) polymerase. Apigenin also reduced the TG- and BFA-induced expression of ER stress-associated proteins, including C/EBP homologous protein (CHOP), glucose-regulated protein (GRP) 78 and GRP94, the cleavage of activating transcription factor 6alpha, the phosphorylation of eukaryotic initiation factor 2alpha and inositol-requiring enzyme 1alpha, and the activation of mitogen-activated protein kinases, such as p38, c-Jun NH(2)-terminal kinase, and extracellular-regulated kinase. We also found that antioxidants such as N-acetylcysteine and glutathione blocked TG- and BFA-induced cell death and the expression of CHOP and GRP78. These results suggest that TG- and BFA-induced reactive oxygen species (ROS) accumulation plays an important role in ER stress-induced apoptosis. Apigenin also reduced TG- and BFA-induced ROS accumulation, suggesting that it exerts an antioxidant effect against ER stress inducers. Moreover, apigenin recovered TG- and BFA-induced reduction of the mitochondrial membrane potential in HT22 cells. Taken together, these results suggest that apigenin could protect HT22 neuronal cells against ER stress-induced apoptosis by reducing CHOP induction as well as ROS accumulation and mitochondrial damage. PMID:20493918

  6. Enniatin B-induced cell death and inflammatory responses in RAW 267.4 murine macrophages

    SciTech Connect

    Gammelsrud, A. [Norwegian Veterinary Institute, P.O. Box 750, Centrum, N-0106 Oslo (Norway) [Norwegian Veterinary Institute, P.O. Box 750, Centrum, N-0106 Oslo (Norway); Department of Air Pollution and Noise, Division of Environmental Medicine, Norwegian Institute of Public Health, N-0403 Oslo (Norway); Solhaug, A. [Norwegian Veterinary Institute, P.O. Box 750, Centrum, N-0106 Oslo (Norway)] [Norwegian Veterinary Institute, P.O. Box 750, Centrum, N-0106 Oslo (Norway); Dendelé, B. [EA 4427 SeRAIC, IRSET, Université de Rennes 1, IFR 140, Rennes (France)] [EA 4427 SeRAIC, IRSET, Université de Rennes 1, IFR 140, Rennes (France); Sandberg, W.J. [Department of Air Pollution and Noise, Division of Environmental Medicine, Norwegian Institute of Public Health, N-0403 Oslo (Norway)] [Department of Air Pollution and Noise, Division of Environmental Medicine, Norwegian Institute of Public Health, N-0403 Oslo (Norway); Ivanova, L. [Norwegian Veterinary Institute, P.O. Box 750, Centrum, N-0106 Oslo (Norway)] [Norwegian Veterinary Institute, P.O. Box 750, Centrum, N-0106 Oslo (Norway); Kocbach Bølling, A. [Department of Air Pollution and Noise, Division of Environmental Medicine, Norwegian Institute of Public Health, N-0403 Oslo (Norway)] [Department of Air Pollution and Noise, Division of Environmental Medicine, Norwegian Institute of Public Health, N-0403 Oslo (Norway); Lagadic-Gossmann, D. [EA 4427 SeRAIC, IRSET, Université de Rennes 1, IFR 140, Rennes (France)] [EA 4427 SeRAIC, IRSET, Université de Rennes 1, IFR 140, Rennes (France); Refsnes, M.; Becher, R. [Department of Air Pollution and Noise, Division of Environmental Medicine, Norwegian Institute of Public Health, N-0403 Oslo (Norway)] [Department of Air Pollution and Noise, Division of Environmental Medicine, Norwegian Institute of Public Health, N-0403 Oslo (Norway); Eriksen, G. [Norwegian Veterinary Institute, P.O. Box 750, Centrum, N-0106 Oslo (Norway)] [Norwegian Veterinary Institute, P.O. Box 750, Centrum, N-0106 Oslo (Norway); Holme, J.A., E-mail: jorn.holme@fhi.no [Department of Air Pollution and Noise, Division of Environmental Medicine, Norwegian Institute of Public Health, N-0403 Oslo (Norway)

    2012-05-15

    The mycotoxin enniatin B (EnnB) is predominantly produced by species of the Fusarium genera, and often found in grain. The cytotoxic effect of EnnB has been suggested to be related to its ability to form ionophores in cell membranes. The present study examines the effects of EnnB on cell death, differentiation, proliferation and pro-inflammatory responses in the murine monocyte–macrophage cell line RAW 264.7. Exposure to EnnB for 24 h caused an accumulation of cells in the G0/G1-phase with a corresponding decrease in cyclin D1. This cell cycle-arrest was possibly also linked to the reduced cellular ability to capture and internalize receptors as illustrated by the lipid marker ganglioside GM1. EnnB also increased the number of apoptotic, early apoptotic and necrotic cells, as well as cells with elongated spindle-like morphology. The Neutral Red assay indicated that EnnB induced lysosomal damage; supported by transmission electron microscopy (TEM) showing accumulation of lipids inside the lysosomes forming lamellar structures/myelin bodies. Enhanced levels of activated caspase-1 were observed after EnnB exposure and the caspase-1 specific inhibitor ZYVAD-FMK reduced EnnB-induced apoptosis. Moreover, EnnB increased the release of interleukin-1beta (IL-1?) in cells primed with lipopolysaccharide (LPS), and this response was reduced by both ZYVAD-FMK and the cathepsin B inhibitor CA-074Me. In conclusion, EnnB was found to induce cell cycle arrest, cell death and inflammation. Caspase-1 appeared to be involved in the apoptosis and release of IL-1? and possibly activation of the inflammasome through lysosomal damage and leakage of cathepsin B. -- Highlights: ? The mycotoxin EnnB induced cell cycle arrest, cell death and inflammation. ? The G0/G1-arrest was linked to a reduced ability to internalize receptors. ? EnnB caused lysosomal damage, leakage of cathepsin B and caspase-1 cleavage. ? Caspase-1 was partly involved in both apoptosis and release of IL-1?. ? There was a synergistic action between EnnB and bacterial LPS.

  7. Interleukin 2-induced proliferation of murine natural killer cells in vivo

    PubMed Central

    1990-01-01

    The growth factor, IL-2, was administered to mice to evaluate the in vivo responsiveness of NK cells to this factor. The immediate effects of this factor on NK cells were determined by examining cytotoxic activity at 18-24 h after a single treatment with rIL-2. Although moderate doses of rIL-2 (3 x 10(4) U) could be shown to activate existing cytotoxic cells on a per cell basis, higher doses (10(6) U) were required to elicit blast size killer cells. The elicited killer cells were characterized as NK cells by the following criteria: (a) they were readily induced in athymic mice; (b) they mediated killing of NK-sensitive YAC-1 target cells but not NK-resistant P815 target cells; and (c) they expressed the NK cell determinants asialo ganglio-n- tetraosylceramide and NK1.1, but not the T cell determinants CD3, L3T4, or Lyt-2. High-dose IL-2 treatment induced not only the appearance of blast size NK cells, but also the expansion of this population. After treatments, the number of large granular lymphocytes and the number of NK1.1+ cells were increased at least twofold. Analysis of DNA content within the NK1.1+ cell subset demonstrated that IL-2 preferentially drove NK1.1+ cells into S and G2/M phases of the cell cycle. The in vivo elicited blast lymphocytes were examined by Northern blot analysis and in situ hybridization for expression of the IL-2-R p55 alpha chain gene. As previous work from this laboratory has demonstrated that NK cells proliferate in response to IFNs and IFN induce