These are representative sample records from Science.gov related to your search topic.
For comprehensive and current results, perform a real-time search at Science.gov.
1

Sertoli cells as biochambers  

NASA Technical Reports Server (NTRS)

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.

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

2004-01-01

2

Tumor de células de Sertoli esclerosante Sclerosing Sertoli Cell Tumor  

Microsoft Academic Search

SUMMARY Background: Sertoli cell tumor is an unfrequent neo- plasm of the testis and the recently reported sclerosing variant is even rarer, and seems to have a different progno- sis. Patients and Methods: A 28 year-old man consulted the urologist because of a slight increase in right testicular volume. Echographic examination revealed an intratesticu- lar solid tumor, 1 cm in

Leire Etxegarai; Leire Andrés; Cosme Ereño; Francisco José Bilbao; José Ignacio López

3

Pure cultures and characterization of yak Sertoli cells.  

PubMed

The culture of primary Sertoli cells has become an important resource in the study of their function. However, their use is limited because of contamination of isolated cells with other testicular cells, mainly germ cells. The aim was to establish technique to obtain pure yak Sertoli cells as well as to study the growth kinetics and biological characteristics of Sertoli cells in vitro. Two-step enzyme digestion was used to separate and culture yak Sertoli cells. Cultured using starvation method and the hypotonic treatment were also invented to get pure yak Sertoli cells. Furthermore, the purification of Yak Sertoli cells were identified according to their characteristics, such as bipolar corpuscular around the nucleus and expression of Fasl, in addition to their morphology. The average viability of the Sertoli cells was 97% before freezing and 94.5% after thawing, indicating that cryopreservation in liquid nitrogen had little influence on the viability of Sertoli cells. The growth tendency of yak Sertoli cells was similar to an S-shaped growth curve. Purified yak Sertoli cells frequently exhibited bipolar corpuscula in nucleus after Feulgen staining, and did have a positive reaction of Fasl by the immunocytochemical identification. After recovery chromosomal analysis of Sertoli cells had a normal chromosomal number of 60, comprising 29 pairs of autosomes and one pair of sex chromosomes. Assays for bacteria, fungi and mycoplasmas were negative. In conclusion, yak Sertoli cells have been successfully purified and cultured in vitro, and maintain stable biological characteristics after thawing. Therefore, it will not only preserve the genetic resources of yaks at the cellular level, but also provide valuable materials for transgenic research and feeder layer and nuclear donor cells in yak somatic cell cloning technology. PMID:23938058

Zhang, Hua; Liu, Ben; Qiu, Yuan; Fan, Jiang feng; Yu, Si jiu

2013-12-01

4

Purinergic signalling mobilizes mitochondrial Ca2+ in mouse Sertoli cells  

PubMed Central

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

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

5

Sertoli-Leydig cell tumor  

MedlinePLUS

... the female ovaries, usually in younger women. The cancer cells release a male sex hormone. As a result, the woman may develop symptoms such as: A deep voice Enlarged clitoris Facial hair Loss in breast size Stopping of menstrual periods Pain in the ...

6

Fine structure of Sertoli cells in three marine snails with a discussion on the functional morphology of Sertoli cells in general  

Microsoft Academic Search

The fine structure of Sertoli cells in three marine prosobranch molluscs has been studied with light- and electron microscopy. Sertoli cells of prosobranchs are modified columnar epithelial cells that maintain continuous contact with the basal lamina and extend from it to the lumen of a testicular tubule. Spermatogenesis takes place between adjacent Sertoli cells, but a continuous layer of cytoplasm

John Buckland-Nicks; Fu-Shiang Chia

1986-01-01

7

[Large cell calcifying Sertoli cell tumour: A case report].  

PubMed

A 19-year-old male Caucasian, without prior medical history, noticed a painless right testicular mass. Physical examination revealed neither gynecomastia nor abnormal skin pigmentation. Serum alpha-fetoprotein, ?-HCG and testosterone levels were normal. Sonography depicted an intratesticular diffusely hyperechoic lesion with acoustic shadowing. The patient underwent right orchiectomy. Histology revealed a benign large cell calcifying Sertoli cell tumour. This tumour is rare and may be associated with genetic abnormalities. PMID:23954122

Coulibaly, Béma; Mesturoux, Laura; Lanoe, Matthieu; Delage-Corre, Manuela; Labrousse, François

2013-08-01

8

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

E-print Network

). In typical A cells, some organelles (endoplasmic reticulum, microfilaments, microtubules) were modified or of isolated Sertoli cells has confirmed all the results obtained so far (Hansson et al., 1978). The Sertoli

Boyer, Edmond

9

NOTCH signaling in Sertoli cells regulates gonocyte fate  

PubMed Central

Gonocytes (or prospermatogonia) are the precursors to spermatogonial stem cells (SSCs), which provide the foundation for spermatogenesis through their ability to both self-renew and generate daughter cells. Despite their relative importance, the regulatory mechanisms that govern gonocyte maintenance and transition to SSCs are poorly understood. Recently, we reported that constitutive activation of NOTCH1 signaling in Sertoli cells causes gonocyte exit from quiescence—the first suggestion of the potential role of this signaling pathway in the testis. This Extra View will review what is known about NOTCH signaling, particularly in Sertoli cells and germ cells in the testes, by providing a background on germ cell biology and a summary of our recently published data on NOTCH1 signaling in Sertoli cells. We also describe additional data showing that aberrant proliferation and differentiation of gonocytes in response to constitutive activation of NOTCH1 signaling in Sertoli cells involves de novo expression of cell cycle proteins and a marked upregulation of the KIT receptor. These data further suggest that NOTCH signaling orchestrates a dynamic balance between maintenance and differentiation of gonocytes in the perinatal testis. PMID:23907117

Garcia, Thomas Xavier; Hofmann, Marie-Claude

2013-01-01

10

Biochemical aspects of the interaction of androgens with Sertoli cells  

E-print Network

and exhibiting a Kd of 6.3 nM for 3H-testosterone has been found in Sertoli cell cytosol. Effectiveness of unlabeled steroids in competing for 3H-testosterone binding sites was found to be testosterone > estradiol. After labeling the cells in culture with 3H-testosterone, macromolecular bound steroid could

Paris-Sud XI, Université de

11

Sertoli cells maintain Leydig cell number and peritubular myoid cell activity in the adult mouse testis.  

PubMed

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

Rebourcet, Diane; O'Shaughnessy, Peter J; Monteiro, Ana; Milne, Laura; Cruickshanks, Lyndsey; Jeffrey, Nathan; Guillou, Florian; Freeman, Tom C; Mitchell, Rod T; Smith, Lee B

2014-01-01

12

Pattern of Sertoli cell degeneration in cryptorchid prepubertal testes.  

PubMed

Seventy-three testicular biopsies from 54 children (aged 2 months-14 years) with undescended testes were examined by light and electron microscopy. The biopsies included abdominal, inguinally fixed, inguinally moveable, and retractile testes. Alterations in Sertoli cell morphology were found in all biopsies. The alterations included dilated elements of rough endoplasmic reticulum, vacuolization of the cytoplasm, mitochondria with poorly preserved cristae, increase in electron density of the matrix, elongation of the nuclei, and irregularities of the nuclear membrane. According to the numerical appearance of these cells and to the extent of lesions in single Sertoli cells, seven phases in the continuous process of tubular alteration were distinguished. The most severe tubular damaged (phase VII) occurred when the seminiferous epithelium consisted exclusively of necrotic cells. All phases of tubular alterations were seen regularly in each of the biopsies investigated. Germ cells occurred only in phases I-IV and were never observed in tubules in phases V-VII. Significant differences became evident between inguinal and retractile testes by morphometric evaluation. It was demonstrated that the number of germ cells per cross-sectioned tubule (S/T value) correlated negatively with the percentage of tubules in phases V-VII. In contrast to inguinal testes, a complete absence of Sertoli cells and an S/T value less than 0.1 were never found in retractile testes and the percentage of tubules in phases V-VII was reduced significantly compared with inguinal testes. Our findings indicate that (i) maldescended testis in patients between 1 and 15 years-of-age is associated with a special pattern of Sertoli cell degeneration; (ii) Sertoli cell degeneration is a continuous process, which can lead eventually to complete dissolution of the seminiferous epithelium; (iii) total degeneration is not related to age but is dependent on testicular position; (iv) a defined phase of degeneration excludes germ cell development, and therefore enhanced Sertoli cell degeneration in cryptorchid testes must also account for the reduction in germ cell number. PMID:1347512

Rune, G M; Mayr, J; Neugebauer, H; Anders, C; Sauer, H

1992-02-01

13

Heregulins or Neu Differentiation Factors and the Interactions between Peritubular Myoid Cells and Sertoli Cells  

Microsoft Academic Search

Interactions between (mesenchymal) peritubular myoid cells and (epithelial) Sertoli cells play an important role in the control of Sertoli cell function and spermatogenesis. The factors involved, however, have only partially been identified. Heregulins or neu differentiation factors (NDFs) mediate mesenchymal-epithelial interactions in a va- riety of tissues, but their role in the testis has not been investigated. Here we demonstrate

EEF HOEBEN; JOHANNES V. SWINNEN; WALTER HEYNS; GUIDO VERHOEVEN

1999-01-01

14

Gene silencing by RNAi in mouse Sertoli cells  

PubMed Central

Background RNA interference (RNAi) is a valuable tool in the investigation of gene function. The purpose of this study was to examine the availability, target cell types and efficiency of RNAi in the mouse seminiferous epithelium. Methods The experimental model was based on transgenic mice expressing EGFP (enhanced green fluorescent protein). RNAi was induced by in vivo transfection of plasmid vectors encoding for short hairpin RNAs (shRNAs) targeting EGFP. shRNAs were transfected in vivo by microinjection into the seminiferous tubules via the rete testis followed by square wave electroporation. As a transfection reporter, expression of red fluorescent protein (HcRed 1) was used. Cell types, the efficiency of both transfections and RNAi were all evaluated. Results Sertoli cells were the main transfected cells. A reduction of about 40% in the level of EGFP protein was detected in cells successfully transfected both in vivo and in vitro. However, the efficiency of in vivo transfection was low. Conclusion In adult seminiferous epithelial cells, in vivo post-transcriptional gene silencing mediated by RNAi via shRNA is efficient in Sertoli cells. Similar levels of RNAi were detected both in vivo and in vitro. This also indicates that Sertoli cells have the necessary silencing machinery to repress the expression of endogenous genes via RNAi. PMID:18620581

Gonzalez-Gonzalez, Emilio; Lopez-Casas, Pedro P; del Mazo, Jesus

2008-01-01

15

Granular transformation of Sertoli cells in testicular disorders.  

PubMed

In order to study the granular transformation of Sertoli cells the following testicular specimens were reviewed: 58 postmortem biopsies from 21 children and 37 young adult males with normal histologic pattern; 165 biopsies from prepubertal cryptorchid testes; 38 biopsies and 18 surgical specimens from postpubertal-cryptorchid testes; bilateral biopsies from eight men with Del Castillo's syndrome, 14 men with retractile testes, and five men with obstructive azospermia; 17 bilateral and seven unilateral biopsies from 24 men with varicocele; seven unilateral biopsies plus five surgical specimens from 12 men with male pseudohermaphroditism; one biopsy and one surgical specimen from two men with macroorchidism; and the autopsy specimens from 28 adult men with acquired immunodeficiency syndrome (AIDS). Sertoli cells with eosinophilic granular cytoplasm were found in the testes of one prepubertal and four postpubertal cryptorchid males, two males with Del Castillo's syndrome, two males with retractile testes, four males with varicocele, two male pseudohermaphrodites, two males with macroorchidism, and one male with AIDS and interstitial orchitis. Histochemical and ultrastructural examination of granular Sertoli cells revealed that these cells accumulate secondary lysosomes and show scant cytoplasmic organelles. In the males with varicocele or retractile testes, these lysosomes were probably heterolysosomes that had degraded the germ cells and testicular fluid accumulated in the lumen of the ectatic seminiferous tubules of these testes. A similar mechanism is also probable in the male with interstitial orchitis that had caused germ cell destruction. In the other cases, in which the tubules showed reduced lumen and severe germ cell depletion, the abundant lysosomes are probably cytolysosomes. The development of these cytolysosomes might be related to the Sertoli cell dysgenesis present in these testes. PMID:1672120

Nistal, M; Garcia-Rodeja, E; Paniagua, R

1991-02-01

16

Clinicopathologic features of ovarian Sertoli-Leydig cell tumors  

PubMed Central

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.

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

2014-01-01

17

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

PubMed Central

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

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

2010-01-01

18

DHT and E2 as modulators of apoptotic signaling in rat Sertoli cells.  

E-print Network

??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… (more)

Simões, Vera Lúcia Silveira

2012-01-01

19

Polycyclic Aromatic Hydrocarbon-Induced Cytotoxicity in Cultured Rat Sertoli Cells Involves Differential Apoptotic Response  

Microsoft Academic Search

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous and persistent environmental contami- nants. Some PAHs are carcinogens and may affect the male reproductive system. Therefore, we exposed cultured rat Sertoli cells to a variety of PAHs to determine possible direct toxic effects on the cells of the seminiferous epithelium. Sertoli cells were chosen because they support germ cell development and maintain spermatogenesis.

Samir S. Raychoudhury; Dana Kubinski

2002-01-01

20

Stimulation of rat Sertoli cell secretory activity in vitro by germ cells and residual bodies  

Microsoft Academic Search

Summary. The direct influence of germ cells and residual bodies on Sertoli cell basal and FSH-stimulated secretion of androgen-binding protein (ABP) was studied using Sertoli cells, recovered from 20-day-old rats, cultured alone or cocultured with a crude germ cell preparation from adult rats or with pachytene spermatocytes, round sperma- tids or populations of residual bodies enriched by centrifugal elutriation. The

B. Le Magueresse; F. Le Gac; M. Loir; B. Jegou

1986-01-01

21

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

E-print Network

Effects of FSH and testosterone on Sertoli cells and spermatocytes from rat testis F. F. G was stimulated in the presence of testosterone, FSH or dibutyryl cAMP. In addition Sertoli cells appeared observations support the view that the effects of follitropin (FSH) and testosterone on germinal cells might

Paris-Sud XI, Université de

22

Sertoli cell index and spermatic reserves in adult captive African lions ( Panthera leo, Linnaeus, 1758)  

Microsoft Academic Search

The intrinsic yield of spermatogenesis and the supporting indexes of the Sertoli cells are the best indicators for the spermatic production capacity in a species. The aim of the present study was to quantify the intrinsic yield of the spermatogenetic process, as well as the Sertoli cell index and spermatic reserves. Testicular fragments of five adult African lions was fixed

João Bosco Gonçalves de Barros; Tarcízio Antônio Rego de Paula; Sérgio Luis Pinto da Matta; Cláudio César Fonseca; Flaviana Lima Guião Leite; João Luiz Rossi Jr; Priscila Carvalho de Oliveira; Eduardo Paulino da Costa

2007-01-01

23

Sertoli cell tumor in a prepubertal boy mimicking testicular torsion.  

PubMed

A 9-year-old boy presented with left, intermittent testicular pain that was present for 3 days. On physical examination, left testis was grossly enlarged and firm but mildly tender. Serum alpha-fetoprotein and beta-human chorionic gonadotropin levels were within normal range. Color doppler ultrasonography which was performed to rule out testicular torsion revealed an intratesticular mass located at the upper pole of left testis and left radical orchiectomy was performed. The histopathological diagnosis was Sertoli cell tumor. PMID:11112872

Tan, M; Uygur, C; Ozer, E; Erol, D

2000-01-01

24

Vectorial Production of Interleukin 1 and Interleukin 6 by Rat Sertoli Cells Cultured in a Dual Culture Compartment System  

Microsoft Academic Search

The bidirectional production of interleukin-1 (IL-1) and IL-6 by Sertoli cells and its regulation by inflammatory and physiological stimuli has been studied using a dual compartment culture system allowing the study of Sertoli cell apical and basal secretory activities. Another Sertoli cell activity, the vectorial transferrin production was also studied in all culture conditions. A low constitutive IL-1 produc- tion

CORINNE CUDICINI; HENRI KERCRET; ANNE-MARIE TOUZALIN; FRANCOIS BALLET; BERNARD JEGOU

1997-01-01

25

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

PubMed

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

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

2014-12-01

26

Immunoprotective Properties of Primary Sertoli Cells in Mice: Potential Functional Pathways that Confer Immune Privilege1  

PubMed Central

ABSTRACT Primary Sertoli cells isolated from mouse testes survive when transplanted across immunological barriers and protect cotransplanted allogeneic and xenogeneic cells from rejection in rodent models. In contrast, the mouse Sertoli cell line (MSC-1) lacks immunoprotective properties associated with primary Sertoli cells. In this study, enriched primary Sertoli cells or MSC-1 cells were transplanted as allografts into the renal subcapsular area of naive BALB/c mice, and their survival in graft sites was compared. While Sertoli cells were detected within the grafts with 100% graft survival throughout the 20-day study, MSC-1 cells were rejected between 11 and 14 days, with 0% graft survival at 20 days posttransplantation. Nonetheless, the mechanism for primary Sertoli cell survival and immunoprotection remains unresolved. To identify immune factors or functional pathways potentially responsible for immune privilege, gene expression profiles of enriched primary Sertoli cells were compared with those of MSC-1 cells. Microarray analysis identified 2369 genes in enriched primary Sertoli cells that were differentially expressed at ±4-fold or higher levels than in MSC-1 cells. Ontological analyses identified multiple immune pathways, which were used to generate a list of 340 immune-related genes. Three functions were identified in primary Sertoli cells as potentially important for establishing immune privilege: suppression of inflammation by specific cytokines and prostanoid molecules, slowing of leukocyte migration by controlled cell junctions and actin polymerization, and inhibition of complement activation and membrane-associated cell lysis. These results increase our understanding of testicular immune privilege and, in the long-term, could lead to improvements in transplantation success. PMID:21900683

Doyle, Timothy J.; Kaur, Gurvinder; Putrevu, Saroja M.; Dyson, Emily L.; Dyson, Mathew; McCunniff, William T.; Pasham, Mithun R.; Kim, Kwan Hee; Dufour, Jannette M.

2011-01-01

27

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

PubMed

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

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

2014-11-01

28

Effect of hypotonic treatment on sertoli cell purity and function in culture  

Microsoft Academic Search

Summary  Commonly used enzymic methods for the isolation of rat Seroli cells yield populations containing ?15% germ cells. Although\\u000a the germ cells become eliminated after several media changes, they could interferen with the use of Sertoli cells for critical\\u000a studies during the first several days of culture. A brief treatment of Sertoli cell monolayer cultures with 20 mM Tris-HCl (pH 7.4)

J. R. Wagle; J. J. Heindel; A. Steinberger; B. M. Sanborn

1986-01-01

29

Involvement of intratesticular IL1 system in the regulation of Sertoli cell functions  

Microsoft Academic Search

The Interleukin-1 (IL-1) system has been suggested to be involved in the cell–cell cross talk within the testis. To investigate the testicular autocrine, paracrine and endocrine factors involved in the regulation of Sertoli cell functions, we have examined the capacity of Sertoli cell cultures, from immature mice, to produce IL-1?, IL-1? and IL-1 receptor antagonist (IL-1ra) under in vitro cultures

Mahmoud Huleihel; Eitan Lunenfeld

2002-01-01

30

ETV5 regulates sertoli cell chemokines involved in mouse stem/progenitor spermatogonia maintenance.  

PubMed

Spermatogonial stem cells are the only stem cells in the body that transmit genetic information to offspring. Although growth factors responsible for self-renewal of these cells are known, the factors and mechanisms that attract and physically maintain these cells within their microenvironment are poorly understood. Mice with targeted disruption of Ets variant gene 5 (Etv5) show total loss of stem/progenitor spermatogonia following the first wave of spermatogenesis, resulting in a Sertoli cell-only phenotype and aspermia. Microarray analysis of primary Sertoli cells from Etv5 knockout (Etv5(-/-)) versus wild-type (WT) mice revealed significant decreases in expression of several chemokines. Chemotaxis assays demonstrated that migration of stem/progenitor spermatogonia toward Etv5(-/-) Sertoli cells was significantly decreased compared to migration toward WT Sertoli cells. Interestingly, differentiating spermatogonia, spermatocytes, and round spermatids were not chemoattracted by WT Sertoli cells, whereas stem/progenitor spermatogonia showed a high and significant chemotactic index. Rescue assays using recombinant chemokines indicated that C-C-motif ligand 9 (CCL9) facilitates Sertoli cell chemoattraction of stem/progenitor spermatogonia, which express C-C-receptor type 1 (CCR1). In addition, there is protein-DNA interaction between ETV5 and Ccl9, suggesting that ETV5 might be a direct regulator of Ccl9 expression. Taken together, our data show for the first time that Sertoli cells are chemoattractive for stem/progenitor spermatogonia, and that production of specific chemokines is regulated by ETV5. Therefore, changes in chemokine production and consequent decreases in chemoattraction by Etv5(-/-) Sertoli cells helps to explain stem/progenitor spermatogonia loss in Etv5(-/-) mice. PMID:20799334

Simon, Liz; Ekman, Gail C; Garcia, Thomas; Carnes, Kay; Zhang, Zhen; Murphy, Theresa; Murphy, Kenneth M; Hess, Rex A; Cooke, Paul S; Hofmann, Marie-Claude

2010-10-01

31

Sertoli Cell-Specific Deletion of the Androgen Receptor Compromises Testicular Immune Privilege in Mice1  

PubMed Central

In the mammalian testis, meiotic and postmeiotic germ cell antigens are granted immune privilege. Both local immune suppression and specialized intercellular junctions between somatic Sertoli cells have been proposed to contribute to a highly restricted and effective blood-testis barrier (BTB) that helps maintain tolerance to germ cell antigens. Several studies have suggested that androgens play a role in immune suppression, although direct evidence for this is lacking. We previously reported that Sertoli cell-specific ablation of the androgen receptor (Ar) decreases expression of Cldn3, an androgen-regulated gene and component of Sertoli cell tight junctions, and increases the permeability of the BTB to biotin, a small-molecular-weight tracer. The physiological consequences of Sertoli cell-specific Ar (S-Ar) ablation on immune privilege are unknown. Here we show that in the testes of S-Ar mutant mice, the ultrastructure of Sertoli cell tight junctions is defective and testicular IgG levels are elevated. The interstitium of S-Ar mutant testes becomes populated with macrophages, neutrophils, plasma cells, and eosinophils, and serum samples of mutant mice contain antibodies against germ cell antigens. Together, these results suggest that Sertoli cell-specific deletion of the androgen receptor results in loss of testicular immune privilege. Suppressed levels of androgen signaling may be a contributing factor in idiopathic male infertility. PMID:21543771

Meng, Jing; Greenlee, Anne R.; Taub, Chloe J.; Braun, Robert E.

2011-01-01

32

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

Microsoft Academic Search

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,

Yoshiro Toyama; Mamiko Maekawa; Shigeki Yuasa

2003-01-01

33

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

PubMed Central

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

Fumel, Betty; Roy, Stephanie; Fouchecourt, Sophie; Livera, Gabriel; Parent, Anne-Simone; Casas, Francois; Guillou, Florian

2013-01-01

34

Genomic-independent action of thyroid hormones on NTPDase activities in Sertoli cell cultures from congenital hypothyroid rats  

Microsoft Academic Search

The Sertoli cells play an essential role in the maintenance and control of spermatogenesis. The ecto-nucleoside triphosphate diphosphohydrolase (E-NTPDase) and 5?-nucleotidase activities can modulate the extracellular adenine nucleotide levels, controlling nucleotide-mediated signaling events in Sertoli cells. Since thyroid hormones (TH) and adenine nucleotides and nucleosides play important modulatory roles in Sertoli cell proliferation and differentiation, the aim of our study

Ariane Zamoner; Alessandra Nejar Bruno; Emerson André Casali; Patrícia Frasson Corbelini; Gabriela Placoná Diniz; Maria Luiza Morais Barreto-Chaves; Fátima Regina Mena Barreto Silva; João José Freitas Sarkis; Regina Pessoa-Pureur

2006-01-01

35

Altered Lipid Homeostasis in Sertoli Cells Stressed by Mild Hyperthermia  

PubMed Central

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

Valles, Ana S.; Aveldano, Marta I.; Furland, Natalia E.

2014-01-01

36

Direct reprogramming of fibroblasts into embryonic Sertoli-like cells by defined factors  

PubMed Central

SUMMARY 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 with their immunosuppressive and neurotrophic properties make them an attractive cell type for therapeutic applications. Here we demonstrate the generation of embryonic sertoli-like cells (ieSCs) by ectopic expression of five transcription factors. We characterize the role of specific transcription factor combinations in the transition from fibroblasts to ieSCs and identify key steps in the process. Initially, transduced fibroblasts underwent a mesenchymal to epithelial transition and then, acquired the ability to aggregate, formed tubular-like structures, and expressed embryonic Sertoli specific markers. These Sertoli-like cells facilitated neuronal differentiation and self-renewal of NPC, supported the survival of germ cells in culture and cooperated with endogenous embryonic Sertoli and primordial germ cells in the generation of testicular cords in the fetal gonad. PMID:22958931

Buganim, Yosef; Itskovich, Elena; Hu, Yueh-Chiang; Cheng, Albert W.; Ganz, Kibibi; Sarkar, Sovan; Fu, Dongdong; Welstead, Grant; Page, David C.; Jaenisch, Rudolf

2012-01-01

37

Somatostatin Inhibits Stem Cell Factor Messenger RNA Expression by Sertoli Cells and Stem Cell Factor-Induced DNA Synthesis in Isolated Seminiferous Tubules1  

Microsoft Academic Search

Immature porcine Sertoli cells have been reported to be tar- gets for the regulatory peptide somatostatin (SRIF), which inhibits the basal and FSH-induced proliferation of Sertoli cells through a decrease of cAMP production. In the present study, we show that SRIF inhibits both basal and FSH-stimulated expression of the stem cell factor (SCF), a Sertoli cell-specific gene. The SRIF-me- diated

Isabelle Goddard; Sylvian Bauer; Alain Gougeon; Frederic Lopez; Nathalie Giannetti; Christiane Susini; Mohamed Benahmed; Slavica Krantic

38

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

Microsoft Academic Search

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

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

1998-01-01

39

Sertoli-cell prostaglandin synthesis. Effects of (follitropin) differentiation and dietary vitamin E.  

PubMed Central

The synthesis of prostanoids by the Sertoli cell was assessed as part of a study on the role of vitamin E in maintaining spermatogenesis. Analyses of eicosanoid synthesis from endogenous substrate were carried out using freshly isolated Sertoli-cell-enriched preparations from both pre-pubertal and adult rats fed purified diets with and without vitamin E, as well as cells carried in primary culture. Freshly isolated cells from both the immature and fully differentiated adult testes produced PGI2 (prostaglandin I2) and PGE2, but PGF2 alpha was produced only by cells of the adult vitamin E-deficient rat. Cells from adult controls synthesized PGF2 alpha after primary culture. In contrast with other hormone responses of this cell, which are refractory in the adult, FSH (follitropin) potentiated prostaglandin production by freshly isolated cells of both immature and adult rats. The FSH response of Sertoli cells from immature animals did not change after primary culture. Adult cells were refractory to the hormone after culture, but the total amounts of prostaglandins produced by these cells were 10-fold higher than by either freshly isolated or cells of the immature in culture. Analogues of cyclic AMP did not potentiate prostaglandin synthesis. However, mepacrine, a phospholipase inhibitor, blocked the FSH effect. The finding that Sertoli cells synthesize prostaglandins and FSH enhances prostaglandin production implicates a potential role for eicosanoids in spermatogenesis and suggests that vitamin E may affect intratesticular regulators. PMID:3109380

Cooper, D R; Carpenter, M P

1987-01-01

40

Testicular neoplasms (interstitial and Sertoli cell tumours) in a domestic ferret (Mustela putorius furo).  

PubMed

Unilateral testicular enlargement was detected in a 5-years-old domestic ferret during a routine sterilization. The right testicle showed two different types of proliferative lesions: (i) round nodules, well demarcated, showing a soft yellow tissue; (ii) white nodules, firm, with irregular-shaped invaginations. Microscopically, the neoplastic proliferations were identified as an interstitial neoplasm and Sertoli cell tumour, respectively. The left testicle was small and showed intense testicular atrophy. Clinical evaluation of the ferret did not show any other apparent pathological processes. This study is the first case reporting the concomitant occurrence of a Sertoli cells tumour and an interstitial cell tumour in a domestic ferret. PMID:20088849

Batista-Arteaga, M; Suárez-Bonnet, A; Santana, M; Niño, T; Reyes, R; Alamo, D

2011-02-01

41

Nonylphenol induces apoptosis in rat testicular Sertoli cells via endoplasmic reticulum stress  

Microsoft Academic Search

Nonylphenol (NP) is a widely distributed environment contaminant and has been documented to disrupt testicular development and decrease male fertility. Amongst possible targets of this compound are testicular Sertoli cells, which play a crucial role in supporting and nourishing sperm cells. In the present study, we found that NP treatment could cause dramatic morphological changes as well as decreased cell

Yi Gong; Jiang Wu; Yufeng Huang; Sunan Shen; Xiaodong Han

2009-01-01

42

Reduced Intratesticular Testosterone Concentration Alters the Polymerization State of the Sertoli Cell Intermediate Filament Cytoskeleton by Degradation of Vimentin  

Microsoft Academic Search

The Sertoli cell intermediate filament cytoskeleton is com- posed of the type III family member vimentin. The distribu- tion of Sertoli cell vimentin varies with the stage of spermat- ogenesis, with shortening of the filaments at stages VII-VIII, the stages of spermiation. Experimental reduction in intra- testicular testosterone (T) concentration also results in the sloughing of advanced spermatids from the

MATTHEW D. SHOW; MATTHEW D. ANWAY; JANET S. FOLMER; BARRY R. ZIRKIN

2003-01-01

43

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

PubMed

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

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

2014-06-01

44

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

PubMed Central

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

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

45

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

SciTech Connect

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

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

1989-12-12

46

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

PubMed

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

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

47

Regulation of Sertoli-Germ Cell Adhesion and Sperm Release by FSH and Nonclassical Testosterone Signaling  

PubMed Central

Testosterone and FSH act in synergy to produce the factors required to maximize the production of spermatozoa and male fertility. However, the molecular mechanisms by which these hormones support spermatogenesis are not well established. Recently, we identified a nonclassical mechanism of testosterone signaling in cultured rat Sertoli cells. We found that testosterone binding to the androgen receptor recruits and activates Src tyrosine kinase. Src then causes the activation of the epidermal growth factor receptor, which results in the phosphorylation and activation of the ERK MAPK and the cAMP response element-binding protein transcription factor. In this report, we find that FSH inhibits testosterone-mediated activation of ERK and the MAPK pathway in Sertoli cells via the protein kinase A-mediated inhibition of Raf kinase. In addition, FSH, as well as inhibitors of Src and ERK kinase activity, reduced germ cell attachment to Sertoli cells in culture. Using pathway-specific androgen receptor mutants we found that the nonclassical pathway is required for testosterone-mediated increases in germ cell attachment to Sertoli cells. Studies of seminiferous tubule explants determined that Src kinase, but not ERK kinase, activity is required for the release of sperm from seminiferous tubule explants. These findings suggest the nonclassical testosterone-signaling pathway acts via Src and ERK kinases to facilitate the adhesion of immature germ cells to Sertoli cells and through Src to permit the release of mature spermatozoa. In contrast, FSH acts to limit testosterone-mediated ERK kinase activity and germ cell attachment. PMID:21177760

Shupe, John; Cheng, Jing; Puri, Pawan; Kostereva, Nataliya

2011-01-01

48

Loss of Dicer in Sertoli cells has a major impact on the testicular proteome of mice.  

PubMed

Sertoli cells (SCs) are the central, essential coordinators of spermatogenesis, without which germ cell development cannot occur. We previously showed that Dicer, an RNaseIII endonuclease required for microRNA (miRNA) biogenesis, is absolutely essential for Sertoli cells to mature, survive, and ultimately sustain germ cell development. Here, using isotope-coded protein labeling, a technique for protein relative quantification by mass spectrometry, we investigated the impact of Sertoli cell-Dicer and subsequent miRNA loss on the testicular proteome. We found that, a large proportion of proteins (50 out of 130) are up-regulated by more that 1.3-fold in testes lacking Sertoli cell-Dicer, yet that this protein up-regulation is mild, never exceeding a 2-fold change, and is not preceeded by alterations of the corresponding mRNAs. Of note, the expression levels of six proteins of interest were further validated using the Absolute Quantification (AQUA) peptide technology. Furthermore, through 3'UTR luciferase assays we identified one up-regulated protein, SOD-1, a Cu/Zn superoxide dismutase whose overexpression has been linked to enhanced cell death through apoptosis, as a likely direct target of three Sertoli cell-expressed miRNAs, miR-125a-3p, miR-872 and miR-24. Altogether, our study, which is one of the few in vivo analyses of miRNA effects on protein output, suggests that, at least in our system, miRNAs play a significant role in translation control. PMID:20467044

Papaioannou, Marilena D; Lagarrigue, Mélanie; Vejnar, Charles E; Rolland, Antoine D; Kühne, Françoise; Aubry, Florence; Schaad, Olivier; Fort, Alexandre; Descombes, Patrick; Neerman-Arbez, Marguerite; Guillou, Florian; Zdobnov, Evgeny M; Pineau, Charles; Nef, Serge

2011-04-01

49

Sertoli Cell Tumor Causing Precocious Puberty in a Girl with Peutz-Jeghers Syndrome  

Microsoft Academic Search

Distinctive ovarian and cervical tumors are associated with Peutz-Jeghers syndrome (PJS). The most common gynecological tumors in this syndrome are adenoma malignum of the uterine cervix and ovarian sex cord tumor, particularly sex cord tumor with annular tubules (SCTAT). Other kinds of ovarian tumors have been rarely reported in association of PJS, including Sertoli cell tumors. We report a case

Amnon Zung; Zeev Shoham; Magda Open; Yehudit Altman; Ram Dgani; Zvi Zadik

1998-01-01

50

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

E-print Network

Variations in the plasma levels of gonadotrophin and testosterone and in Leydig and Sertoli cell Petit-Bourg, Guadeloupe. Summary. Plasma gonadotrophin and testosterone levels during the prepuberal of testosterone pulses per hour was higher in spring than in autumn animals. In the prepuberal period, the ratio

Boyer, Edmond

51

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

SciTech Connect

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.

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

52

Tumor necrosis factor- inhibits glutathione S-transferase- expression in cultured porcine Sertoli cells  

Microsoft Academic Search

Glutathione S-transferases (GSTs) are a family of soluble enzymes of detoxification that use reduced glutathione in conjugation and reduction reactions. Toxic electrophiles are substrates for the GSTs. GST is expressed at high levels in different tissues such as the testis. Among the different GSTs present in the testis, GST is specifically expressed in Leydig and Sertoli cells known to be

L Benbrahim-Tallaa; F Boussouar; C Rey; M Benahmed

2002-01-01

53

Basolateral Uptake of Nucleosides by Sertoli Cells Is Mediated Primarily by Equilibrative Nucleoside Transporter 1  

PubMed Central

The blood-testis barrier (BTB) prevents the entry of many xenobiotic compounds into seminiferous tubules thereby protecting developing germ cells. Understanding drug transport across the BTB may improve drug delivery into the testis. Members of one class of drug, nucleoside reverse transcriptase inhibitors (NRTIs), do penetrate the BTB, presumably through interaction with physiologic nucleoside transporters. By investigating the mechanism of nucleoside transport, it may be possible to design other drugs to bypass the BTB in a similar manner. We present a novel ex vivo technique to study transport at the BTB that employs isolated, intact seminiferous tubules. Using this system, we found that over 80% of total uptake by seminiferous tubules of the model nucleoside uridine could be inhibited by 100 nM nitrobenzylmercaptopurine riboside (NBMPR, 6-S-[(4-nitrophenyl)methyl]-6-thioinosine), a concentration that selectively inhibits equilibrative nucleoside transporter 1 (ENT1) activity. In primary cultured rat Sertoli cells, 100 nM NBMPR inhibited all transepithelial transport and basolateral uptake of uridine. Immunohistochemical staining showed ENT1 to be located on the basolateral membrane of human and rat Sertoli cells, whereas ENT2 was located on the apical membrane of Sertoli cells. Transepithelial transport of uridine by rat Sertoli cells was partially inhibited by the NRTIs zidovudine, didanosine, and tenofovir disoproxil fumarate, consistent with an interaction between these drugs and ENT transporters. These data indicate that ENT1 is the primary route for basolateral nucleoside uptake into Sertoli cells and a possible mechanism for nucleosides and nucleoside-based drugs to undergo transepithelial transport. PMID:23639800

Klein, David M.; Evans, Kristen K.; Hardwick, Rhiannon N.; Dantzler, William H.; Wright, Stephen H.

2013-01-01

54

Antigens on Murine Erythroid Cells  

Microsoft Academic Search

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

ANDREAS HELIAS SARRIS; GEORGE E. PALADE

55

Environmentally induced epigenetic transgenerational inheritance of altered Sertoli cell transcriptome and epigenome: molecular etiology of male infertility.  

PubMed

Environmental toxicants have been shown to induce the epigenetic transgenerational inheritance of adult onset disease, including testis disease and male infertility. The current study was designed to determine the impact of an altered sperm epigenome on the subsequent development of an adult somatic cell (Sertoli cell) that influences the onset of a specific disease (male infertility). A gestating female rat (F0 generation) was exposed to the agriculture fungicide vinclozolin during gonadal sex determination and then the subsequent F3 generation progeny used for the isolation of Sertoli cells and assessment of testis disease. As previously observed, enhanced spermatogenic cell apoptosis was observed. The Sertoli cells provide the physical and nutritional support for the spermatogenic cells. Over 400 genes were differentially expressed in the F3 generation control versus vinclozolin lineage Sertoli cells. A number of specific cellular pathways were identified to be transgenerationally altered. One of the key metabolic processes affected was pyruvate/lactate production that is directly linked to spermatogenic cell viability. The Sertoli cell epigenome was also altered with over 100 promoter differential DNA methylation regions (DMR) modified. The genomic features and overlap with the sperm DMR were investigated. Observations demonstrate that the transgenerational sperm epigenetic alterations subsequently alters the development of a specific somatic cell (Sertoli cell) epigenome and transcriptome that correlates with adult onset disease (male infertility). The environmentally induced epigenetic transgenerational inheritance of testis disease appears to be a component of the molecular etiology of male infertility. PMID:23555832

Guerrero-Bosagna, Carlos; Savenkova, Marina; Haque, Md Muksitul; Nilsson, Eric; Skinner, Michael K

2013-01-01

56

RiboTag Analysis of Actively Translated mRNAs in Sertoli and Leydig Cells In Vivo  

PubMed Central

Male spermatogenesis is a complex biological process that is regulated by hormonal signals from the hypothalamus (GnRH), the pituitary gonadotropins (LH and FSH) and the testis (androgens, inhibin). The two key somatic cell types of the testis, Leydig and Sertoli cells, respond to gonadotropins and androgens and regulate the development and maturation of fertilization competent spermatozoa. Although progress has been made in the identification of specific transcripts that are translated in Sertoli and Leydig cells and their response to hormones, efforts to expand these studies have been restricted by technical hurdles. In order to address this problem we have applied an in vivo ribosome tagging strategy (RiboTag) that allows a detailed and physiologically relevant characterization of the “translatome” (polysome-associated mRNAs) of Leydig or Sertoli cells in vivo. Our analysis identified all previously characterized Leydig and Sertoli cell-specific markers and identified in a comprehensive manner novel markers of Leydig and Sertoli cells; the translational response of these two cell types to gonadotropins or testosterone was also investigated. Modulation of a small subset of Sertoli cell genes occurred after FSH and testosterone stimulation. However, Leydig cells responded robustly to gonadotropin deprivation and LH restoration with acute changes in polysome-associated mRNAs. These studies identified the transcription factors that are induced by LH stimulation, uncovered novel potential regulators of LH signaling and steroidogenesis, and demonstrate the effects of LH on the translational machinery in vivo in the Leydig cell. PMID:23776628

Sanz, Elisenda; Evanoff, Ryan; Quintana, Albert; Evans, Elizabeth; Miller, Jeremy A.; Ko, Chemyong; Amieux, Paul S.; Griswold, Michael D.; McKnight, G. Stanley

2013-01-01

57

Sertoli cell index and spermatic reserves in adult captive African lions (Panthera leo, Linnaeus, 1758).  

PubMed

The intrinsic yield of spermatogenesis and the supporting indexes of the Sertoli cells are the best indicators for the spermatic production capacity in a species. The aim of the present study was to quantify the intrinsic yield of the spermatogenetic process, as well as the Sertoli cell index and spermatic reserves. Testicular fragments of five adult African lions was fixed in 4% glutaric aldehyde, dehydrated at increasing alcohol concentrations, included into hydroxyethyl methacrylate, and were cut into 4 microm thickness. In the seminiferous epithelium of the African lions, 10.3 primary spermatocytes at pre-leptotene phase are produced by the type-A spermatogonia. During meiotic divisions, only 2.7 spermatids were produced from the primary spermatocytes. The general spermatogenesis production in the African lions was approximately 22.1 cells, and each Sertoli cell was able to sustain and maintain approximately 14.9 cells of the germinative line, from which 7.9 are round spermatids. A total of 103x10(6) spermatozoa are produced by each testis gram at each cycle of the seminiferous epithelium. The spermatic reserve of lion is below the amplitude observed in mammals. PMID:17499460

de Barros, João Bosco Gonçalves; de Paula, Tarcízio Antônio Rego; da Matta, Sérgio Luis Pinto; Fonseca, Cláudio César; Leite, Flaviana Lima Guião; Rossi Jr, João Luiz; de Oliveira, Priscila Carvalho; da Costa, Eduardo Paulino

2007-12-01

58

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

Microsoft Academic Search

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

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

59

Bilateral Sertoli and Interstitial Cell Tumours in Abdominal Testes of a Goat with Polled Intersex Syndrome (PIS).  

PubMed

An 8-year-old, mixed breed, polled goat was presented for evaluation of male-like behaviour. Clinical findings included clitoromegaly, a heavily muscled neck, pronounced beard, and erect dorsal guard hairs, which are phenotypic characteristics commonly observed in intersex animals. Transrectal ultrasonography revealed the presence of two abdominal masses caudolateral to the uterine horns. Serum concentration of estradiol was elevated. Genetic evaluation was compatible with polled intersex syndrome defined by an XX karyotype without a Y chromosome or SRY gene. Based on gross and histologic evaluation, the abdominal masses were determined to be intra-abdominal testes, each of which was effaced by Sertoli cell and interstitial (Leydig) cell tumours. The Sertoli cell tumours (SCTs) represented two unique histologic patterns. Regardless of pattern, neoplastic Sertoli cells were consistently lipid laden and positive for vimentin. Interstitial cell tumours (ICTs) were negative for vimentin. Clinical and histopathologic findings suggest that prolonged exposure to steroids secreted by neoplastic Sertoli cells contributed to virilization. In addition, results from immunohistochemistry indicated that vimentin may be a valuable immunodiagnostic tool for differentiation between interstitial and Sertoli cell tumours in goats. PMID:25219569

Canisso, If; Coffee, Ll; Ortved, K; Fubini, Sl; Monteagudo, Lv; Schlafer, Dh; Gilbert, Ro

2014-12-01

60

Gene expression profiling of rat spermatogonia and Sertoli cells reveals signaling pathways from stem cells to niche and testicular cancer cells to surrounding stroma  

PubMed Central

Background Stem cells and their niches are studied in many systems, but mammalian germ stem cells (GSC) and their niches are still poorly understood. In rat testis, spermatogonia and undifferentiated Sertoli cells proliferate before puberty, but at puberty most spermatogonia enter spermatogenesis, and Sertoli cells differentiate to support this program. Thus, pre-pubertal spermatogonia might possess GSC potential and pre-pubertal Sertoli cells niche functions. We hypothesized that the different stem cell pools at pre-puberty and maturity provide a model for the identification of stem cell and niche-specific genes. We compared the transcript profiles of spermatogonia and Sertoli cells from pre-pubertal and pubertal rats and examined how these related to genes expressed in testicular cancers, which might originate from inappropriate communication between GSCs and Sertoli cells. Results The pre-pubertal spermatogonia-specific gene set comprised known stem cell and spermatogonial stem cell (SSC) markers. Similarly, the pre-pubertal Sertoli cell-specific gene set comprised known niche gene transcripts. A large fraction of these specifically enriched transcripts encoded trans-membrane, extra-cellular, and secreted proteins highlighting stem cell to niche communication. Comparing selective gene sets established in this study with published gene expression data of testicular cancers and their stroma, we identified sets expressed genes shared between testicular tumors and pre-pubertal spermatogonia, and tumor stroma and pre-pubertal Sertoli cells with statistic significance. Conclusions Our data suggest that SSC and their niche specifically express complementary factors for cell communication and that the same factors might be implicated in the communication between tumor cells and their micro-enviroment in testicular cancer. PMID:21232125

2011-01-01

61

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

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.

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

62

Follicle-stimulating hormone stimulates estradiol-17beta synthesis in cultured Sertoli cells.  

PubMed Central

Sertoli cells isolated from testes of 20-day-old rats and maintained in primary culture synthesized estradiol-17beta [1,3,5(10)-estratriene-3,17beta-diol] (measured by specific radioimmunoassay) when testosterone (17beta-hydroxy-4-androsten-3-one) 0.5 muM, was added to the culture medium. No detectable estradiol synthesis occurred when cells were incubated in medium containing pregnenolone (3beta-hydroxypregn-5-en-20-one), 0.5 muM, or containing no added steroid substrate. Follicle-stimulating hormone (FSH) (NIH-FSH-S10, 5 mug/ml) stimulated estradiol synthesis 12- to 80-fold when added to medium containing testosterone, but not when added to medium containing pregnenolone or no exogenous steroid substrate. A highly purified FSH preparation, with FSH potency 50 times that of the NIH-FSH, caused a similar stimulation at a concentration of 0.25 mug/ml of medium, whereas luteinizing hormone (NIH-LH-S18, 5 MUG/ML) Caused only marginal stimulation. Dibutyryl-adenosine 3':5' cyclic monophosphate, 0.1 mM, caused a 30-fold increase in estradiol synthesis by Sertoli cells cultured in medium containing testosterone. These studies provide direct demonstration of estradiol-17beta production by Seroli cells from normal animals, and offer evidence that the synthesis of this steroid is regulated at the level of the aromatizing enzyme system by FSH and adenosine 3':5' cyclic monophosphate. PMID:170613

Dorrington, J H; Armstrong, D T

1975-01-01

63

Malignant Large Cell Calcifying Sertoli Cell Tumor of Testis with Skip Metastasis to Lung Presented With Peutz-Jeghers Syndrome  

PubMed Central

Large cell calcifying Sertoli cell tumor of the testis (LCCSCT) is a rare tumor that is usually benign and multifocal. It may be associated with hereditary endocrine anomalies such as Carney's and Peutz-Jeghers syndromes. It is a rare histological variant of sex cord stromal tumors. It is exceptional in elderly men and the outcome is rarely fatal. We report a case of LCCSCT in a 44 year-old man with fatal outcome. The tumor involved the right testis and several areas of the tunica albuginea were grossly invaded. It composed of cords and trabeculae of large polygonal cells embedded in a myxoid and fibrous stroma with areas of calcification along with nuclear atypia, necrosis, and abundant mitoses. The Peutz-Jeghers syndrome (PJS) is known to be commonly associated with ovarian tumors. However, its association with testicular tumors is uncommon. To the best of our knowledge, this is the eight such case being reported in the literature. Our case, to our knowledge, is the only other reported case of malignant large cell calcifying Sertoli cell tumor with clinical and histopathological features related to aggressiveness, such as large tumor size, cellular pleomorphism, high mitotic rate, necrosis and aneuploid deoxyribonucleic acid. Such characteristics are not found in benign large cell calcifying Sertoli cell tumors.

Sarkar, Debasis; Ray, Arindam; Thakur, Indranil; Mukherjee, Sekhar; Chatterjee, Sanjoy Kumar

2012-01-01

64

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

PubMed

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

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

2014-10-28

65

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

E-print Network

Hormonal regulation of Sertoli cell function in the rat V. HANSSON, K. PURVIS E. M. RITZÃ?N 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

Boyer, Edmond

66

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

PubMed Central

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

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

67

Sertoli-Leydig cell tumor with heterologous element: a case report and a review of the literature  

PubMed Central

The patient was a 19-year-old female who presented with a chief complaint of progressive pelvic pain. Preoperative ultrasound of the right ovary revealed an ovarian torsion as the cause of the patient’s progressive pain. Laparoscopy confirmed the torsion and revealed a right ovary measuring 10 cm in greatest diameter. Intraoperative incision into the ovary revealed a simple ovarian cystic mass measuring 3.0 x 1.5 x 0.8 cm. A solid component within the cyst was identified. Histological sections of the cystic mass demonstrated mononuclear and hyperchromatic Sertoli cells with a trabecular growth pattern. Clusters of medium-sized epithelioid cells with abundant eosinophilic cytoplasm consistent with Leydig cells were also identified between the trabeculae of Sertoli cells. In addition, focal areas of intestinal type mucinous epithelium were identified embedded within the trabeculae of Sertoli cells. Immunohistochemical studies revealed that the Sertoli cells were positive for calretinin (bright) while the Leydig cells were positive for calretinin (dim), inhibin, CAM5.2 and AE1&3. CEA showed positivity mainly of the intraluminal contents of the mucinous type intestinal epithelium. The patient had an uneventful post-operative course and was disease-free for 3 years. PMID:24696734

Chen, Longwen; Tunnell, Cairo Dana; Petris, Giovanni De

2014-01-01

68

Inflammatory mediators increase surface expression of integrin ligands, adhesion to lymphocytes, and secretion of interleukin 6 in mouse Sertoli cells.  

PubMed Central

The expression of the cell adhesion molecules ICAM-1, ICAM-2, and VCAM-1 and the secretion of the cytokine interleukin 6 have been measured in mouse Sertoli cells cultured in vitro. Cytometric analysis revealed that, in basal conditions, low levels of ICAM-1 and VCAM-1 were present on the surface of the cells, whereas treatment with interleukin 1, tumor necrosis factor alpha, lipopolysaccharide, or interferon gamma induced, with different kinetics, increases in their expression. ICAM-2 was not detectable in basal conditions, nor was it inducible. Electron microscopic analysis and binding experiments using 51Cr-labeled lymphocytes demonstrated that increased expression of ICAM-1 and VCAM-1 on the surface of Sertoli cells, induced by inflammatory mediators, determines an augmented adhesion between the two cell types. The same stimuli, with the exception of interferon gamma, produced a rapid and remarkable increment of interleukin 6 production by Sertoli cells. These results suggest the presence of both direct and paracrine mechanisms of interaction between Sertoli and immune-competent cells, possibly involved in the control of immune reactions in the testis. Such mechanisms are of interest for the understanding of autoimmune pathologies of the testis and, if confirmed in humans, they could be involved in the sexual transmission of human immunodeficiency virus infection. Images Fig. 3 PMID:7541137

Riccioli, A; Filippini, A; De Cesaris, P; Barbacci, E; Stefanini, M; Starace, G; Ziparo, E

1995-01-01

69

Isolation of endothelial cells from murine tissue  

Microsoft Academic Search

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

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

2000-01-01

70

The role of androgens in sertoli cell proliferation and functional maturation: studies in mice with total or Sertoli cell-selective ablation of the androgen receptor.  

PubMed

The role of androgens in the proliferation and maturation of Sertoli cells (SC) and the development of their capacity to support spermatogenesis remains poorly understood. We evaluated these functions in complete androgen receptor knockout (ARKO) and SC-selective androgen receptor knockout (SCARKO) mice. Compared with controls, ARKO mice exhibited a progressive reduction in SC number/testis, whereas SCARKOs showed minor changes, suggesting that androgen effects on SC number are not mediated via direct action on SCs. Immunoexpression of anti-Mullerian hormone (AMH), p27(kip1), GATA-1, and sulfated glycoprotein-2, which changes according to SC maturational status, occurred normally in ARKOs and SCARKOs. Functional capacity of SCs to support spermatogonia was similar in SCARKOs and controls, whereas ARKOs showed reduced capacity with age. SC capacity to support total germ cells revealed major deficits in ARKO and SCARKO adults, particularly with respect to postmeiotic germ cells. Using quantitative RT-PCR, the expression of SC markers was compared in d 50 testes. In ARKOs, expression of Pem, fatty acid binding protein, platelet-derived growth factor-A, and transferrin were all significantly reduced, whereas FSH receptor and AMH were increased. In SCARKOs, there were modest reductions in expression of cystatin-related gene highly expressed in testis and epididymis (cystatin-TE) and claudin-11, whereas expression of Pem, fatty acid binding protein, and platelet-derived growth factor-A was markedly reduced, highlighting these as potentially androgen-regulated SC genes that merit further study. In conclusion, androgen action is not required for maturation-dependent changes in immunoexpression of the SC markers AMH, p27(kip1), GATA-1, and sulfated glycoprotein-2 but is essential for expression of other SC genes, the attainment of normal SC number, and the support of meiotic and postmeiotic germ cell development. PMID:15761038

Tan, Karen A L; De Gendt, Karel; Atanassova, Nina; Walker, Marion; Sharpe, Richard M; Saunders, Philippa T K; Denolet, Evi; Verhoeven, Guido

2005-06-01

71

Cytoskeleton Vimentin Disruption of Mouse Sertoli Cells Injured by Nitrogen Mustard In Vitro  

Microsoft Academic Search

Reproductive toxicity is one of the potential side effects of anticancer alkylating agents, with potential effects on vimentin intermediate filaments, one of the main components of the Sertoli cytoskeleton. Research suggests (Aumuller et al, 1988; Aumuller et al, 1992) that the highly organized and active Sertoli cytoskeleton is important in spermatogenesis. The aim of the current study was to investigate

DAWEI HE; DEYING ZHANG; GUANGHUI WEI; TAO LIN; XULIANG LI

2006-01-01

72

Glycan composition of follicle (Sertoli) cells of the amphibian Pleurodeles waltl. A lectin histochemical study  

PubMed Central

The glycan composition of the N- and O-linked oligosaccharides of the follicle (Sertoli) cells of the urodele amphibian Pleurodeles waltl testis were identified by lectin histochemistry, performed alone or in combination with enzymatic and chemical deglycosylation methods. The follicle cells were shown to contain: (1) Fuc, Gal?(1,4)GlcNAc, GalNAc and Neu5Ac?(2,3)Gal?(1,4)GlcNAc in both N- and O-linked oligosaccharides; (2) Man in N-linked glycans; and (3) Gal?(1,3)GalNAc in O-linked sugar chains. The follicle cells at the pre- and postmeiotic stages showed some differences in the UEA-I-positive Fuc characterisation, suggesting differences in the glycan composition. In addition, the sequence Neu5Ac?(2,6)Gal/GalNAc was shown in the follicle cells only after spermiation, in the sperm-empty lobules of the developing glandular tissue. These results suggest that the follicle cells modify their glycoprotein content, probably for the performance of new roles, as the spermatogenetic cells develop. Thus the follicle cells surrounding male germ cells at different spermatogenetic stages would contain different glycoproteins involved in specific roles during male germ cell proliferation and maturation. PMID:11465860

SAEZ, FRANCISCO JOSE; MADRID, JUAN FRANCISCO; ALONSO, EDURNE; HERNANDEZ, FRANCISCO

2001-01-01

73

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

PubMed Central

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

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

2013-01-01

74

Phorbol myristate acetate induces changes on F-actin and vinculin content in immature rat Sertoli cells  

Microsoft Academic Search

Actin and vinculin are two of the most abundant cytoskeletal proteins, widely expressed in nearly all types of eukaryotic cells. It has been well established that long-term exposure to the tumor promoter phorbol myristate acetate (PMA) affects Sertoli cell morphology, as well as F-actin and vinculin organization in vitro. To analyze in a quantitative manner the F-actin and vinculin content

M Kouloukoussa; V Aleporou-Marinou; B Angelopoulou; I. P Trougakos; E Panagopoulou; Chr Kittas; Evangelos Marinos

2004-01-01

75

A chimerical phagocytosis model reveals the recruitment by Sertoli cells of autophagy for the degradation of ingested illegitimate substrates  

PubMed Central

Phagocytosis and autophagy are typically dedicated to degradation of substrates of extrinsic and intrinsic origins respectively. Although overlaps between phagocytosis and autophagy were reported, the use of autophagy for ingested substrate degradation by nonprofessional phagocytes has not been described. Blood-separated tissues use their tissue-specific nonprofessional phagocytes for homeostatic phagocytosis. In the testis, Sertoli cells phagocytose spermatid residual bodies produced during germ cell differentiation. In the retina, pigmented epithelium phagocytoses shed photoreceptor tips produced during photoreceptor renewal. Spermatid residual bodies and shed photoreceptor tips are phosphatidylserine-exposing substrates. Activation of the tyrosine kinase receptor MERTK, which is implicated in phagocytosis of phosphatidylserine-exposing substrates, is a common feature of Sertoli and retinal pigmented epithelial cell phagocytosis. The major aim of our study was to investigate to what extent phagocytosis by Sertoli cells may be tissue specific. We analyzed in Sertoli cell cultures that were exposed to either spermatid residual bodies (legitimate substrates) or retina photoreceptor outer segments (illegitimate substrates) the course of the main phagocytosis stages. We show that whereas substrate binding and ingestion stages occur similarly for legitimate or illegitimate substrates, the degradation of illegitimate but not of legitimate substrates triggers autophagy as evidenced by the formation of double-membrane wrapping, MAP1LC3A-II/LC3-II clustering, SQSTM1/p62 degradation, and by marked changes in ATG5, ATG9 and BECN1/Beclin 1 protein expression profiles. The recruitment by nonprofessional phagocytes of autophagy for the degradation of ingested cell-derived substrates is a novel feature that may be of major importance for fundamentals of both apoptotic substrate clearance and tissue homeostasis. PMID:23439251

Yefimova, Marina G.; Messaddeq, Nadia; Harnois, Thomas; Meunier, Annie-Claire; Clarhaut, Jonathan; Noblanc, Anaïs; Weickert, Jean-Luc; Cantereau, Anne; Philippe, Michel; Bourmeyster, Nicolas; Benzakour, Omar

2013-01-01

76

Expression of IL-1?, IL-6, TGF-?, FasL and ZNF265 During Sertoli Cell Infection by Ureaplasma Urealyticum  

PubMed Central

To investigate immunoregulatory mechanisms of Sertoli cells in the testis in vitro and in vivo, we utilized our well-characterized Ureaplasma Urealyticum (UU)-induced model. We investigated the expressions of IL-1?, IL-6, TGF-?, FasL and ZNF265 at the first, second and third weeks post-infection. During recovery from inflammation and with the help of negative regulators TGF-? and FasL, the high levels of IL-1? and IL-6 expressions were observed in the early stages of the infection, and decreased gradually in the later weeks both in vitro and in vivo. The trend of varied expression of ZNF265 was similar to those of TGF-? and FasL in vitro and in vivo for Sertoli cells infected with UU. PMID:19567205

Li, Rongping; Xi, Yebin; Liu, Xiuzhi; Chen, Guangjie; Wang, Baoguo; Jiang, Lihua; Li, Weiyi

2009-01-01

77

An integrative omics strategy to assess the germ cell secretome and to decipher sertoli-germ cell crosstalk in the Mammalian testis.  

PubMed

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

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

2014-01-01

78

Maternal Exposure to Octylphenol Suppresses Ovine Fetal Follicle-Stimulating Hormone Secretion, Testis Size, and Sertoli Cell Number  

Microsoft Academic Search

We have tested the hypothesis that maternal exposure to oc- tylphenol, a putative endocrine disrupting chemical, will suppress gonadotropin secretion with a concomitant decrease in testis size and Sertoli cell number during fetal life in the lamb. In Exp 1, pregnant ewes received a continuous iv infusion of diethylstilbes- trol (DES; 50 mg\\/kgzday), octylphenol (1000 mg\\/kgzday), or vehicle (1:4, alcohol-saline)

T. Sweeney; L. NICOL; J. F. ROCHE; A. N. BROOKS

2000-01-01

79

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

Microsoft Academic Search

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

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

2010-01-01

80

Modulation of m-dinitrobenzene and m-nitrosonitrobenzene toxicity in rat Sertoli--germ cell cocultures  

Microsoft Academic Search

Previous work has shown that m-dinitrobenzene is a testicular toxicant in rats in vivo, and in vitro produces comparable morphological changes in rat testicular Sertoli-germ cell cocultures. m-Dinitrobenzene is metabolized both in vivo and in the in vitro system to m-nitroaniline m-nitroaniline and m-nitroacetanilide. These metabolites do not provoke testicular toxicity in vivo or in vitro. We have therefore proposed

D. A. Cave; P. M. Foster

1990-01-01

81

Transcriptional Suppression of Sertoli Cell Timp2 in Rodents Following Mono-(2-ethylhexyl) Phthalate Exposure Is Regulated by CEBPA and MYC1  

PubMed Central

ABSTRACT Our previous studies showed that the prototypical testicular toxic phthalate monoester, mono-(2-ethylhexyl) phthalate (MEHP), suppresses Sertoli cell TIMP2 levels and allows for the activation of MMP2 in seminiferous epithelium. Activation of MMP2 is important for triggering germ cell apoptosis and instigating germ cell detachment from Sertoli cells. These novel findings led us to examine the transcriptional regulation of the Timp2 gene that accounts for the decrease in Sertoli cell TIMP2 levels following MEHP exposure. Sequential deletion of the Timp2 5?-upstream activating sequence (1200 bp) was used to survey transcriptional activation in the Timp2 promoter region in response to MEHP. Results indicate that under control conditions in rat Sertoli cells, CCAAT enhancer-binding protein alpha (CEBPA) acts as a transactivator to initiate Timp2 gene transcription, and its action is deactivated by exposure to MEHP. By contrast, MYC protein acts as an inhibitor of Timp2 gene transcription, and its activity is increased after MEHP treatment. Addition of follicle-stimulating hormone (FSH) to cells causes translocation of CEBPA into the Sertoli cell nucleus and rescues MEHP-suppressed TIMP2 levels. Down-regulation of TIMP2 expression by MEHP exposure is blocked by forskolin (a cAMP-elevating agent), suggesting that the decrease in Sertoli cell TIMP2 expression following MEHP exposure is cAMP-dependent. Taken together, these data indicate that MEHP both disrupts the FSH-stimulated cAMP signaling pathway and activates the inhibitory signaling mediated by MYC protein, to ultimately account for the cellular mechanism underlying the decreased expression of TIMP2 in Sertoli cells. PMID:21832167

Yao, Pei-Li; Lin, Yi-Chen; Richburg, John H.

2011-01-01

82

Identification of IL6 as one of the important cytokines responsible for the ability of mononuclear cells to stimulate Sertoli cell functions  

Microsoft Academic Search

There is increasing evidence that locally produced cytokines may play an important role in the control of testicular function. In a previous report we demonstrated that medium conditioned by activated human peripheral blood mononuclear cells (PBMC-CM), which is a rich source of cytokines, has extremely potent effects on Sertoli cell transferrin and cGMP secretion. Part of this activity could be

Eef Hoeben; Anja Wuyts; Paul Proost; Jo Van Damme; Guido Verhoeven

1997-01-01

83

Hybrid GPCR\\/Cadherin (Celsr) Proteins in Rat Testis Are Expressed With Cell Type Specificity and Exhibit Differential Sertoli Cell-Germ Cell Adhesion Activity  

Microsoft Academic Search

Spermatogenesis requires Sertoli cell-germ cell ad- hesion for germ cell survival and maturation. Cadherins are a diverse superfamily of adhesion proteins; structurally unique members of this superfamily (celsr cadherins) are hybrid molecules containing extra- cellular cadherin repeats connected to a G protein-coupled receptor transmembrane motif. Here we demonstrate postnatal testicular mRNA expression of the 3 celsr paralogs (celsr1, celsr2, and

STEPHANIE A. BEALL; KIM BOEKELHEIDE; KAMIN J. JOHNSON

2005-01-01

84

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

PubMed

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

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

2014-05-01

85

Melatonin alters the glycolytic profile of Sertoli cells: implications for male fertility.  

PubMed

Melatonin co-operates with insulin in the regulation of glucose homeostasis. Within the testis, glucose metabolism in the somatic Sertoli cells (SCs) is pivotal for spermatogenesis. Since the effects of melatonin on male reproductive physiology remain largely unknown, we hypothesized that melatonin may affect spermatogenesis by modulating SC metabolism, interacting with insulin. To test our hypothesis, rat SCs were maintained in culture for 24 h in the presence of insulin, melatonin or both and metabolite production/consumption was determined by proton nuclear magnetic resonance ((1)H-NMR). Protein levels of glucose transporters (GLUT1 and GLUT3), phosphofructokinase 1, lactate dehydrogenase (LDH) and monocarboxylate transporter 4 were determined by western blot. LDH activity was also assessed. SCs treated with melatonin showed an increase in glucose consumption via modulation of GLUT1 levels, but decreased LDH protein expression and activity, which resulted in lower lactate production. Moreover, SCs exposed to melatonin produced and accumulated less acetate than insulin-exposed cells. The combined treatment (insulin plus melatonin) increased acetate production by SCs, but intracellular acetate content remained lower than in insulin exposed cells. Finally, the intracellular redox state, as reflected by intracellular lactate/alanine ratio, was maintained at control levels in SCs by melatonin exposure (i.e. melatonin, alone or with insulin, increased the lactate/alanine ratio versus cells treated with insulin). Furthermore, SCs exposed to insulin plus melatonin produced more lactate and maintained the protein levels of some glycolysis-related enzymes and transporters at control levels. These findings illustrate that melatonin regulates SCs metabolism, and thus may affect spermatogenesis. Since lactate produced by SCs provides nutritional support and has an anti-apoptotic effect in developing germ cells, melatonin supplementation may be an effective therapy for diabetic male individuals facing subfertility/infertility. PMID:25205674

Rocha, Cátia S; Martins, Ana D; Rato, Luís; Silva, Branca M; Oliveira, Pedro F; Alves, Marco G

2014-11-01

86

Copy Number Variants in Patients with Severe Oligozoospermia and Sertoli-Cell-Only Syndrome  

PubMed Central

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

Tuttelmann, Frank; Simoni, Manuela; Kliesch, Sabine; Ledig, Susanne; Dworniczak, Bernd; Wieacker, Peter; Ropke, Albrecht

2011-01-01

87

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

PubMed

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

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

2013-01-01

88

Starvation is more efficient than the washing technique for purification of rat Sertoli cells.  

PubMed

Sertoli cells (SCs), one of the most important components of seminiferous tubules, are vital for normal spermatogenesis and male fertility. In recent years, numerous in vitro studies have shown the potential and actual activities of SCs. However, pure SCs are necessary for various in vitro studies. In this study, we have evaluated the efficiency of the starvation method for SC purification as compared with the washing method. Seminiferous tubule-derived cells (STDCs) of rats' testes underwent two different techniques for SC purification. In the first group (washing group), the medium was changed every 3-4 d, and cells were washed twice with phosphate-buffered saline that lacked CaC12 and MgSO4 (PBS(-)) before the addition of fresh medium. In the second group (starvation), the medium was changed every 7-8 d. Primary culture (P0), passage 1 (P1), and passage 2 (P2) cells were analyzed for the expression of SC-specific genes, vimentin, Wilm's tumor 1 (WT1), germ cell gene (vasa), Leydig cell marker, 17beta-hydroxysteroid dehydrogenase type 3 (Hsd17b3), and a marker of peritubular myoid cells, alpha smooth muscle actin (?Sma), by reverse transcriptase polymerase chain reaction (RT-PCR) and real-time RT-PCR. Gene expression analysis showed that P0 cells expressed all tested genes except Hsd17b3. The starvation method caused significant downregulation of vasa and ?Sma expression in P0, P1, and P2 cells, whereas vimentin and WT1 were upregulated. In contrast, the washing method was less effective than the starvation method for the removal of germ and pretubular myoid cells (p?cells in SC cultures, starvation has a stronger effect and is a suitable, affordable technique for SC purification. We propose that starvation is an efficient, inexpensive method that can be used for purification of SCs in animal species. PMID:24789729

Ghasemzadeh-Hasankolaei, Mohammad; Eslaminejad, Mohamadreza Baghaban; Sedighi-Gilani, Mohammadali; Mokarizadeh, Aram

2014-09-01

89

Expression characterization of testicular DMRT1 in both Sertoli cells and spermatogenic cells of polyploid gibel carp.  

PubMed

Dmrt1 has been suggested to play significant roles in sex determination and differentiation, but various expression patterns and cell types have been observed in the testis of vertebrates. Polyploid gibel carp, because of the multiple modes of unisexual gynogenesis and sexual reproduction, has become a unique case to explore the evolution of sex determination and differentiation. However, the sex-determination related genes in gibel carp have remained unknown. In this study, we identified and characterized 4 cDNAs of Dmrt1 genes. Subsequently, a polyclonal antibody specific to CagDMRT1 was prepared to examine its expression and distribution patterns at protein level. Significantly, both relative real-time PCR and Western blot detection confirmed predominant expression of CagDmrt1 in the adult testis of gibel carp. Moreover, the intensive expression of CagDMRT1 around spermatogenic cysts was revealed during spermatogenesis. And, following immunofluorescence co-localization of CagDMRT1 and CagVASA, a prominent CagDMRT1 expression in Sertoli cells and a mild CagDMRT1 expression in spermatogenic cells including spermatogonia and primary spermatocytes were clearly characterized. The CagDMRT1 signal in Sertoli cells is extensively distributed in both nuclei and cytoplasm, while the CagDMRT1 in spermatogonia and primary spermatocytes is mainly expressed in nuclei, and there is only the remained CagDMRT1 signal in the cytoplasm of secondary spermatocytes. These findings suggest that DMRT1 should be related to testis differentiation and spermatogenesis in gibel carp. PMID:25020260

Li, Xi-Yin; Li, Zhi; Zhang, Xiao-Juan; Zhou, Li; Gui, Jian-Fang

2014-09-10

90

Prenatal testosterone excess alters Sertoli and germ cell number and testicular FSH receptor expression in rams.  

PubMed

Exposure to excess testosterone (T) during fetal life has a profound impact on the metabolic and reproductive functions in the female's postnatal life. However, less is known about the effects of excess testosterone in males. The aim of the present study was to evaluate the impact (consequences) of an excess of T during fetal development on mature male testis. The testicular evaluation was by histological analysis and by determination of mRNA expression of the FSH receptor (FSH-R), transforming growth factor-? type I receptor (T?R-I), and two members of the TGF-? superfamily, transforming growth factor-?3 (TGF?3) and anti-Müllerian hormone (AMH) in males born to mothers receiving an excess of T during pregnancy. At 42 wk of age, postpubertal males born to mothers treated with 30 mg of T propionate twice weekly from day 30 to 90, followed by 40 mg of T propionate from day 90 to 120 of pregnancy (T males), showed higher concentrations of FSH in response to a GnRH analog, a higher number of Sertoli cells/seminiferous tubule cross-section, and a lower number of germ cells/tubules (P < 0.05) than control males (C males) born to mothers treated with the vehicle. The mRNA expression of FSH-R and of T?R-I was higher in T males compared with C males (P < 0.05). Moreover, in T males, AMH expression level correlated negatively with the expression level of TGF?3. In C males, this latter correlation was not observed. These results suggest that prenatal exposure to an excess of T can negatively modify some histological and molecular characteristics of the mature testis. PMID:20858754

Rojas-García, Pedro P; Recabarren, Mónica P; Sarabia, Luis; Schön, Jennifer; Gabler, Christoph; Einspanier, Ralf; Maliqueo, Manuel; Sir-Petermann, Teresa; Rey, Rodolfo; Recabarren, Sergio E

2010-12-01

91

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

PubMed Central

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

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

2014-01-01

92

Transfection with steroid-responsive reporter constructs shows glucocorticoid rather than androgen responsiveness in cultured Sertoli cells.  

PubMed

It remains unclear why it has proven so difficult to identify androgen target genes in cultured Sertoli cells. Given the lack of useful endogenous reporter genes, we studied the androgen and glucocorticoid responsiveness of these cells by transfection with three different steroid-responsive reporter constructs. The constructs were driven by the tyrosine aminotransferase steroid-responsive region (TAT-GRE4x-Luc), the mouse mammary tumor virus promoter (MMTV-Luc) and the Pem homeobox gene proximal promoter respectively (Pem-Luc). These constructs can be activated either by both the glucocorticoid receptor (GR) and the androgen receptor (AR) (TAT-GRE4x-Luc and MMTV-Luc) or selectively by the AR (Pem-Luc). Despite high transfection efficiency (30-40%) none of the constructs could be activated by treatment of the Sertoli cells with testosterone, 5alpha-dihydrotestosterone or synthetic androgens. Even pretreatment with follicle-stimulating hormone to raise AR levels (from 31 up to 82fmol/mg protein) did not result in androgen responsiveness. In contrast, treatment with dexamethasone markedly stimulated TAT-GRE4x-Luc and MMTV-Luc activity. GR levels reached a value of 172fmol/mg protein in the cultured cells and both AR and GR displayed homogeneous distribution by immunocytochemical evaluation. Androgen responsiveness was restored and glucocorticoid responsiveness was increased by cotransfection with AR or GR expression constructs. Under cotransfection conditions, 1nM of testosterone (a concentration that is some 100 times lower than that estimated to be present in the testis) was sufficient to stimulate the TAT-GRE4x-Luc maximally. Our data indicate that cultured Sertoli cells respond better to glucocorticoids than to androgens and that one of the factors limiting androgen responsiveness is the availability of AR. Other factors limiting the transactivation capacity of the (endogenous) AR, however, cannot be excluded. PMID:16388947

Denolet, Evi; Gendt, Karel De; Swinnen, Johannes V; Verrijdt, Guy; Deboel, Ludo; Roskams, Tania; Verhoeven, Guido

2006-02-01

93

Organic Anion Transporter 6 (Slc22a20) Specificity and Sertoli Cell-Specific Expression Provide New Insight on Potential Endogenous Roles  

PubMed Central

Organic anion transporter 6 (Oat6; Slc22a20), a member of the OAT family, was demonstrated previously to mediate the transport of organic anions (Am J Physiol Renal Physiol 291:F314–F321, 2006). In the present study, we sought to further delineate the function of murine Oat6 (mOat6) by analyzing the effect of select organic anions on mOat6-mediated transport by using a Chinese hamster ovary (CHO) cell line stably expressing mOat6 (CHO-mOat6). When examined, kinetic analysis demonstrated that the mechanism of inhibition of mOat6 and mOat3 was competitive. Homovanillic acid, 5-hydroxyindole acetic acid, 2,4-dihydroxyphenylacetate, hippurate, and dehydroepiandrosterone sulfate (DHEAS) each significantly reduced mOat6 activity with inhibitory constant (Ki) values of 3.0 ± 0.5, 48.9 ± 10.3, 61.4 ± 7.1, 59.9 ± 4.9, and 38.8 ± 3.1 ?M, respectively. Comparison to Ki values determined for mOat3 (67.8 ± 7.2, 134.5 ± 27.0, 346.7 ± 97.9, 79.3 ± 4.0, and 3.8 ± 1.1 ?M, respectively) revealed that there are significant differences in compound affinity between each transporter. Fluoroquinolone antimicrobials and reduced folates were without effect on mOat6-mediated uptake. Investigation of testicular cell type-specific expression of mOat6 by laser capture microdissection and quantitative polymerase chain reaction revealed significant mRNA expression in Sertoli cells, but not in Leydig cells or spermatids. Overall, these data should aid further refinements to the interpretation and modeling of the in vivo disposition of OAT substrates. Specifically, expression in Sertoli cells suggests Oat6 may be an important determinant of blood-testis barrier function, with Oat6-mediated transport of estrone sulfate and DHEAS possibly representing a critical step in the maintenance of testicular steroidogenesis. PMID:20519554

Schnabolk, Gloriane W.; Gupta, Bhawna; Mulgaonkar, Aditi; Kulkarni, Mrugaya

2010-01-01

94

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

SciTech Connect

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

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

1988-07-01

95

Sertoli cell function in diabetic, insulin-treated diabetic, and semi-starved rats.  

PubMed

It is well documented that long-term diabetes mellitus results in numerous deleterious consequences. However, considerable controversy exists concerning male reproductive function in diabetes. The purpose of this investigation was to study several endocrine parameters in diabetic male rats with emphasis on Sertoli cell function. Male Wistar rats were injected with streptozotocin and then either left untreated for 30 days or injected with insulin so as to prevent spillover of glucose into the urine. These two groups were compared with control animals that had only been injected with the vehicle for streptozotocin. Semi-starved control animals were included to determine if any of the potential endocrine alterations were related to body weight changes which occur in streptozotocin-injected rats. It was found that FSH, LH, PRL, and GH serum levels were reduced in diabetic animals. Only FSH was restored to normal by insulin injections. The testis, seminal vesicle, and epididymis weights were all reduced in diabetic animals. Insulin injections raised all organ weights; however, only testis weights were fully restored. Levels of epididymal ABP activity were found to be higher in diabetic animals when expressed per mg protein. Similar patterns of organ weight loss and hormonal alterations were observed in semi-starved rats. However, epididymal levels of ABP activity were unaffected by the semi-starved condition. While weight loss should be taken into consideration when interpreting cause and effect relationships in streptozotocin-treated animals, epididymal ABP levels appear to be well correlated with the altered metabolic state characteristic of diabetes. PMID:6219025

Hutson, J C; Stocco, D M; Campbell, G T; Wagoner, J

1983-02-01

96

Perfluorooctanesulfonate (PFOS) perturbs male rat Sertoli cell blood-testis barrier function by affecting F-actin organization via p-FAK-Tyr(407): an in vitro study.  

PubMed

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)-Tyr(407) 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-Tyr(407) was found to worsen PFOS-mediated Sertoli cell tight junction disruption. In summary, PFOS-induced BTB disruption is mediated by down-regulating phosphorylated FAK-Tyr(407) 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

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

2014-01-01

97

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

PubMed Central

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

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

2013-01-01

98

p,p?-DDE Induces Apoptosis of Rat Sertoli Cells via a FasL-Dependent Pathway  

PubMed Central

One,1-dichloro-2,2 bis(p-chlorophenyl) ethylene (p,p?-DDE), the major metabolite of 2,2-bis(4-Chlorophenyl)-1,1,1-trichloroethane (DDT), is a known persistent organic pollutant and male reproductive toxicant. It has antiandrogenic effect. However, the mechanism by which p,p?-DDE exposure causes male reproductive toxicity remains unknown. In the present study, rat Sertoli cells were used to investigate the molecular mechanism involved in p,p?-DDE-induced toxicity in male reproductive system. The results indicated that p,p?-DDE exposure at over 30 ?M showed the induction of apoptotic cell death. p,p?-DDE could induce increases in FasL mRNA and protein, which could be blocked by an antioxidant agent, N-acetyl-l-cysteine (NAC). In addition, caspase-3 and -8 were activated by p,p?-DDE treatment in these cells. The activation of NF-?B was enhanced with the increase of p,p?-DDE dose. Taken together, these results suggested that exposure to p,p?-DDE might induce apoptosis of rat Sertoli cells through a FasL-dependent pathway. PMID:19644561

Shi, Yuqin; Song, Yang; Wang, Yinan; Liang, Xianmin; Hu, Yafei; Guan, Xia; Cheng, Jin; Yang, Kedi

2009-01-01

99

Poorly Differentiated Ovarian Sertoli-Leydig Cell Tumor in a 16-Year-Old Single Woman: A Case Report and Literature Review  

PubMed Central

Sertoli-Leydig cell tumor (SLCT) of ovary is an exceedingly unusual neoplasm that belongs to a group of sex cord-stromal tumors of ovary and accounts for less than 0.5% of all primary ovarian neoplasms. Very few case reports have been documented in the literature so far. Herein, we report a case of primary poorly differentiated ovarian Sertoli-Leydig cell tumor (SLCT) involving the left ovary in a 16-year-old single woman who presented with a 3-month history of a pelviabdominal mass, acne, hirsutism, and menstrual irregularities. In addition, a literature review on ovarian SLCTs is provided. PMID:23878752

Abu-Zaid, Ahmed; Azzam, Ayman; Alghuneim, Lama Abdulhamid; Metawee, Mona Tarek; Amin, Tarek; Al-Hussain, Turki Omar

2013-01-01

100

Modulation of m-dinitrobenzene and m-nitrosonitrobenzene toxicity in rat Sertoli--germ cell cocultures  

SciTech Connect

Previous work has shown that m-dinitrobenzene is a testicular toxicant in rats in vivo, and in vitro produces comparable morphological changes in rat testicular Sertoli-germ cell cocultures. m-Dinitrobenzene is metabolized both in vivo and in the in vitro system to m-nitroaniline m-nitroaniline and m-nitroacetanilide. These metabolites do not provoke testicular toxicity in vivo or in vitro. We have therefore proposed a pathway for the metabolism of m-dinitrobenzene to m-nitroaniline and m-nitroacetanilide, which involved the intermediate m-nitrosonitrobenzene (1-nitroso-3-nitrobenzene, NNB). When tested, m-nitrosonitrobenzene, at equimolar doses to m-dinitrobenzene, produced similar morphological changes in the culture system to those exhibited by m-dinitrobenzene. However, m-nitrosonitrobenzene produced a greater toxicity than did m-dinitrobenzene (as measured by germ cell detachment). When the intracellular thiol levels were reduced in the cocultures pretreated with diethyl maleate, the toxicity of both m-dinitrobenzene and m-nitrosonitrobenzene was enhanced. In contrast, pretreatment of cocultures with agents known to increase cellular thiol (cysteamine) or scavenge reactive intermediates (cysteamine or ascorbate) reduced the toxicity of m-dinitrobenzene and m-nitrosonitrobenzene. We propose that m-dinitrobenzene requires metabolic activation before it can exert its toxicity to Sertoli cells, and it appears that the toxic species is m-nitrosonitrobenzene or a further metabolite of m-nitrosonitrobenzene.

Cave, D.A.; Foster, P.M. (Central Toxicology Laboratory, Macclesfield, Cheshire (England))

1990-01-01

101

Modulation of m-dinitrobenzene and m-nitrosonitrobenzene toxicity in rat Sertoli--germ cell cocultures.  

PubMed

Previous work has shown that m-dinitrobenzene is a testicular toxicant in rats in vivo, and in vitro produces comparable morphological changes in rat testicular Sertoli-germ cell cocultures. m-Dinitrobenzene is metabolized both in vivo and in the in vitro system to m-nitroaniline m-nitroaniline and m-nitroacetanilide. These metabolites do not provoke testicular toxicity in vivo or in vitro. We have therefore proposed a pathway for the metabolism of m-dinitrobenzene to m-nitroaniline and m-nitroacetanilide, which involved the intermediate m-nitrosonitrobenzene (1-nitroso-3-nitrobenzene, NNB). When tested, m-nitrosonitrobenzene, at equimolar doses to m-dinitrobenzene, produced similar morphological changes in the culture system to those exhibited by m-dinitrobenzene. However, m-nitrosonitrobenzene produced a greater toxicity than did m-dinitrobenzene (as measured by germ cell detachment). When the intracellular thiol levels were reduced in the cocultures pretreated with diethyl maleate, the toxicity of both m-dinitrobenzene and m-nitrosonitrobenzene was enhanced. In contrast, pretreatment of cocultures with agents known to increase cellular thiol (cysteamine) or scavenge reactive intermediates (cysteamine or ascorbate) reduced the toxicity of m-dinitrobenzene and m-nitrosonitrobenzene. We propose that m-dinitrobenzene requires metabolic activation before it can exert its toxicity to Sertoli cells, and it appears that the toxic species is m-nitrosonitrobenzene or a further metabolite of m-nitrosonitrobenzene. PMID:2307317

Cave, D A; Foster, P M

1990-01-01

102

Large Cell Calcifying Sertoli Cell Tumor of the Testis: A Case Study and Review of the Literature  

PubMed Central

A 24-year-old man was admitted due to an incidentally detected mass in his left testis, which showed radiopaque calcification on plain X-ray film. Left orchiectomy was performed, and the resected testis contained a well-demarcated, hard mass measuring 1.1 cm. Histological analysis revealed that the tumor was composed of neoplastic cells, fibrotic stroma, and laminated or irregularly shaped calcific bodies. The individual cells had abundant eosinophilic or clear cytoplasm with round nuclei, each of which contained one or two conspicuous nucleoli. They were arranged in cords, trabeculae, clusters, and diffuse sheets. There were several foci of intra-tubular growth patterns, with thickening of the basal lamina. Immunohistochemically, the neoplastic cells were positive for S-100 protein and vimentin, focally positive for inhibin alpha, and negative for cytokeratin, CD10, and Melan-A. In addition to reporting this rare case, we also review the relevant literature regarding large cell calcifying Sertoli cell tumors. PMID:24627695

Song, Dae Hyun; Jeong, Seong Muk; Park, Jong Tak; Yun, Gak Won; Kim, Byoung Kwon

2014-01-01

103

B Cells Regulate Murine Gammaherpesvirus 68 Latency  

Microsoft Academic Search

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

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

1999-01-01

104

Sertoli cells in culture secrete paracrine factor(s) that inhibit peritubular myoid cell proliferation: identification of heparinoids as likely candidates  

SciTech Connect

Conditioned medium from Sertoli cells, prepared from testes of 20-day-old rats, contains component(s) that inhibit the incorporation of (3H)-thymidine into DNA of peritubular myoid cells (PMC) and inhibit the proliferation of PMC. These components are trypsin-resistant, heat-stable compounds having a molecular weight less than 30,000. The active inhibitory components in Sertoli cell conditioned medium are inactivated by treatment with heparinase, but not by treatment with hyaluronidase or chondroitin sulfate lyases. Addition of heparin or heparan sulfate results in inhibition of DNA synthesis by PMC in a dose-dependent manner, whereas other glycosaminoglycans (GAGs) examined (hyaluronic acid, keratan sulfate, and chondroitin sulfate) have no detectable effects. Heparin and heparan sulfate are unique among GAGs tested in inhibiting the characteristic multilayer growth pattern of PMC following the attainment of confluence in serum-rich medium. On the basis of these and other data presented, it is concluded that heparin and other heparin-like GAGs synthesized by Sertoli cells are implicated in the modulation of growth of PMC in vitro during co-culture. It is postulated that heparin may play a similar role in maintaining the quiescent peritubular myoid cell phenotype in vivo.

Tung, P.S.; Fritz, I.B. (Banting and Best Department of Medical Research, University of Toronto, Ontario (Canada))

1991-06-01

105

The dynamic of the apical ectoplasmic specialization between spermatids and Sertoli cells: the case of the small GTPase Rap1.  

PubMed

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

Berruti, Giovanna; Paiardi, Chiara

2014-01-01

106

Differential Gene-Expression of Metallothionein 1M and 1G in Response to Zinc in Sertoli TM4 Cells  

PubMed Central

Background: Zinc (Zn) as an important trace element is essential for testicular development and spermatogenesis. Molecular mechanism of Zn action in the reproductive system may be related to metal binding low-molecular weight proteins, metallothioneins (MT). Our objective was to determine the effect of Zn on two important isoforms of MT, MT1M and MT1G genes expression on testicular sertoli cells. Methods: Cultured sertoli TM4 cells were exposed to different concentrations of Zn at different time points. Cellular uptake of Zn was tested using flame atomic absorption spectrometry. The cellular viability and gene expression were assessed by MTT and real-time PCR methods, respectively. Results: The treated cells resulted in higher Zn concentration and cellular viability. The expression of MT1M and MT1G genes in the treated cells were greater than those of the untreated cells (P<0.05). In the high dosage treated group (100 and 500 ?M), Zn concentration and expression of MT1M and MT1G genes increased three h after treatment; MT1G gene expression increased more at sixth h. At 18th h of treatment, the expression of both genes especially MT1G, increased dramatically while Zn concentration decreased. Conclusion: Since the increase of MT1G mRNA was coincident with cellular Zn level, it seems that MT1G has a more prominent role than MT1M in the homeostasis of Zn. In addition, Zn at dosage of 50 ?M (pharmacologic concentration) may protect cells by increasing the expression of MT genes at longer periods. PMID:20683493

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

2010-01-01

107

Varicocele-caused progressive damage in bilateral testis and sertoli cell-only syndrome in homolateral testis in rats.  

PubMed

Background We aimed to investigate whether varicocele (VC) in rats can cause Sertoli cell-only syndrome (SCOS). Material and 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

Liu, Jianjun; Ding, Degang; Liu, Jie

2014-01-01

108

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

PubMed Central

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

Liu, Jianjun; Ding, Degang; Liu, Jie

2014-01-01

109

1,3Dinitrobenzene induces apoptosis in TM4 mouse Sertoli cells: Involvement of the c-Jun N-terminal kinase (JNK) MAPK pathway  

Microsoft Academic Search

Several studies have shown that 1,3-dinitrobenzene (1,3-DNB) causes injury to Sertoli cells and induces apoptosis in the surrounding germinal cells in male laboratory rats; however, the mechanism by which 1,3-DNB functions is not well understood. In this study, we investigated whether 1,3-DNB induces apoptosis and which pathways are undertaken in TM4 cells. When cells were treated with 1,3-DNB, a dose-dependent

Young Sook Lee; Hea-Jin Yoon; Jung-Hwa Oh; Han-Jin Park; Eun-Hee Lee; Chang-Woo Song; Seokjoo Yoon

2009-01-01

110

TNF Alpha-Mediated Disruption of Spermatogenesis in Response to Sertoli Cell Injury in Rodents Is Partially Regulated by MMP21  

PubMed Central

Mono-(2-ethylhexyl) phthalate (MEHP)-induced Sertoli cell injury in peripubertal rodents results in the stimulation of germ cell apoptosis through an interaction of FAS/FASL between these two cell types. During this peripubertal period, an early spike in the incidence of germ cell apoptosis occurs during the first wave of spermatogenesis and is essential for the development of functional spermatogenesis in adults. Our previous observations revealed that soluble tumor necrosis factor alpha (sTNFA) released by germ cells after MEHP exposure consequently resulted in a robust induction of FASL by Sertoli cells. Metalloproteinases (MPs) are essential for processing the TNFA precursor to its soluble form and its ability to bind to TNFRSF1A. The activity of MPs is regulated by the tissue inhibitors of MPs (TIMPs) family. Herein we report that TIMP2 is predominately expressed in Sertoli cells and that protein levels decrease in a time-dependent manner after MEHP exposure. The secretion of matrix MP 2 (MMP2) in primary rat Sertoli cell-germ cell cocultures is induced after MEHP exposure, and its activity increases in a time-dependent manner. The addition of SB-3CT, a specific gelatinase inhibitor, decreases the activity of MMP2 and significantly reduces MEHP-enhanced sTNFA production in primary cocultures. In vivo challenges with SB-3CT decrease sTNFA and reduce MEHP-induced testicular germ cell apoptosis. In primary cocultures, MEHP exposure causes a 9.46-fold increase in sTNFA, while the addition of recombinant MMP2 protein results in a 5.4-fold increase in sTNFA, suggesting that MEHP-induced MMP2 is in part responsible for the activation of TNFA in the testis. Taken together, these observations indicate the distinct role of specific MPs in response to toxicant-induced Sertoli cell injury, providing further insights into the mechanism by which Sertoli cells control the sensitivity of germ cells to undergo apoptosis. PMID:19038859

Yao, Pei-Li; Lin, Yi-Chen; Richburg, John H.

2008-01-01

111

Gelatinase A secretion and its control in peritubular and Sertoli cell cultures: effects of hormones, second messengers and inducers of cytokine production  

Microsoft Academic Search

Extracellular matrix components as well as enzymes and enzyme-inhibitors controlling the turn-over of these components play an important role in the local control of testicular function. Zymographic analysis was used to study the secretion and the control of the secretion of gelatinase A (MMP-2) and B (MMP-9) by primary cultures of rat Sertoli cells and by subcultures of peritubular cells.

Eef Hoeben; Ilse Van Aelst; Johannes V. Swinnen; Ghislain Opdenakker; Guido Verhoeven

1996-01-01

112

Sertoli-secreted FGF-2 induces PFKFB4 isozyme expression in mouse spermatogenic cells by activation of the MEK/ERK/CREB pathway.  

PubMed

Sertoli cells play a central role in the control and maintenance of spermatogenesis by secreting growth factors, in response to hormonal stimulation, that participate in the paracrine regulation of this process. In this study, we investigated how the hormonal regulation of spermatogenesis modulates 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB) isozyme expression in two mouse spermatogenic cell lines, GC-1 spg and GC-2 spd (ts). For this purpose, TM4 Sertoli cells were used to obtain conditioned medium that was treated or not with dihydrotestosterone for 2 days [dihydrotestosterone conditioned medium (TCM) and basal conditioned medium (BCM), respectively]. We observed an increase in the expression of PFKFB4 along with a decrease in PFKFB3 in spermatogenic cell lines treated with TCM. These effects were inhibited by the antiandrogen drug flutamide and by heat-inactivated TCM, indicating the protein nature of the TCM mediator and its dependence on Sertoli cell stimulation by dihydrotestosterone. In addition, adult rat testes treated with the GnRH antagonist Degarelix exhibited a reduction in the expression of PFKFB4 in germ cells. Addition of exogenous FGF-2 mimicked the changes in the Pfkfb gene expression, whereas neutralizing antibodies against FGF-2 abolished them. Interestingly, similar effects on Pfkfb gene expression were observed using different MAPK inhibitors (U-0126, PD-98059, and H-89). Luciferase analysis of Pfkfb4 promoter constructs demonstrated that a putative CRE-binding sequence located at -1,463 relative to the transcription start site is required to control Pfkfb4 gene expression after TCM treatment. Pulldown assays showed the binding of the CREB transcription factor to this site. Altogether, these results show how the paracrine regulation orchestrated by Sertoli cells in response to testosterone controls glycolysis in germ cells. PMID:22811469

Gómez, Marta; Manzano, Anna; Figueras, Agnes; Viñals, Francesc; Ventura, Francesc; Rosa, Jose Luis; Bartrons, Ramon; Navarro-Sabaté, Àurea

2012-09-15

113

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

Microsoft Academic Search

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

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

2003-01-01

114

Cytokines, Polarity Proteins, and Endosomal Protein Trafficking and Signaling--The Sertoli Cell Blood-Testis Barrier System In Vitro as a Study Model  

PubMed Central

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

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

2014-01-01

115

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

PubMed

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

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

2014-01-01

116

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

Microsoft Academic Search

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

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

2000-01-01

117

Isolation of murine valve endothelial cells.  

PubMed

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

Miller, Lindsey J; Lincoln, Joy

2014-01-01

118

Control of Sertoli cell metabolism by sex steroid hormones is mediated through modulation in glycolysis-related transporters and enzymes.  

PubMed

Sertoli cells (SCs) glucose metabolism is crucial for spermatogenesis since developing germ cells consume lactate produced by SCs as their main energy source. Recently, androgens and estrogens have been implicated in SCs energy metabolism modulation, although the molecular mechanisms remained undisclosed. Here, we report the effect of sex steroid hormones on key points of cultured rat SCs glycolytic pathway. We used primary cultures of immature rat SCs treated with 17?-estradiol (E2) or 5?-dihydrotestosterone (DHT). The transcript levels of glucose transporters (GLUTs), phosphofructokinase 1 (PFK-1) and lactate dehydrogenase C (LDH C) were analyzed after 25 and 50 h of culture by qPCR. Protein levels of GLUTs, PFK-1, LDH and monocarboxylate transporter 4 (MCT4) after 25 and 50 h were determined by western blot and LDH activity was also assessed. Our results show that both E2 and DHT downregulated the transcript levels of PFK-1, GLUT1 and GLUT3 after 50 h. However, only DHT-treated cells presented a downregulation of LDH C transcript levels. Interestingly, the protein levels of these enzymes and transporters remained unaltered except in DHT-treated cells that presented a significant decrease on GLUT1 protein levels evidencing a possible site for the regulation of SCs glucose metabolism by androgens. Taken together, our results provide evidence that sex steroid hormones action in SCs energy metabolism is mediated through modulation in glycolysis-related transporters and enzymes, particularly at the transcriptional level. DHT decreased GLUT1 protein levels and increased LDH activity after 25 h, evidencing key points for this hormone action in the regulation of SCs metabolism. PMID:24057877

Martins, Ana D; Alves, Marco G; Simões, Vera L; Dias, Tânia R; Rato, Luís; Moreira, Paula I; Socorro, Sílvia; Cavaco, José E; Oliveira, Pedro F

2013-12-01

119

Lectin binding sites in normal rat ovary and ENU-induced Sertoli cell tumors of the ovaries.  

PubMed

A panel of seven fluorescein isothiocyanate (FITC) labeled lectins were used to study the distribution of specific binding sites in histologic sections of rat ovaries and ENU-induced Sertoli cell tumors (SCT) of the ovaries. Ten SCT and 5 normal ovaries derived from Berlin Druckey IV (BD-IV) rats were examined by FITC lectins. The tissues examined were fixed in 10% buffered formalin and embedded in paraffin blocks. In normal ovaries, lectin binding sites were more uniform, ordered and consistent than in ovarian SCT where some lectin staining appeared disorderly inconsistent and varied with the degree of tumor differentiation. Two lectins, (from Triticum vulgaris [WGA] and Arachis hypogaea [PNA], uniformly stained the apices of the ovarian surface epithelium and subadjacent tunica vaginalis. The ovarian stroma, oocyte nucleus, follicular and granulosa-theca cells, stained uniformly strong with succinated Con A (from Con-canavalia ensiformis). Three lectins (from Triticum vulgaris, Ulex europeaus [UEA-1] and Arachis hypogeae) accentuated the basal lamina in the SCT and normal ovarian follicles. The zona pellucida was strongly labeled with lectin derived from Triticum vulgaris, Ricinus communis (RCA) and moderately with lectin derived from Arachis hypogeae. The oviduct ampulla exhibited an intracytoplasmic strong vesicular labeling with lectins derived from Triticum vulgare, Dolichos biflorus (DBA), Glycin max (Soybean-SBA) and Arachis hypogeae. The SCT cells showed an inconsistent, irregular labeling pattern with lectins derived from Ulex europaeus, Dolichos biflorus and Soybean mostly as a coarse granular cytoplasmic labeling. Neuraminidase digestion enhanced lectin staining with PNA in normal ovary and in SCT. This data provided at list of lectin markers for distinct components of the BD-IV rat ovary and ovarian SCT. PMID:2764514

Stoica, G; O'Leary, M

1989-01-01

120

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

PubMed

The effects of dinoseb (DNBP), a known testicular toxicant in the rat, on germ cells were investigated in Sertoli-germ cell co-cultures. Two DNBP-related dinitrophenolic compounds, 4,6-dinitro-o-cresol (DNOC) and 2,4-dinitrophenol (DNP), were also examined, as testicular toxicity of these compounds had not been elucidated. Cultures were exposed to each compound (10(-7)-10(-4)M) for 24h and examined for the number and viability of detached cells and morphologic alterations under a light microscope. DNBP significantly increased the number of detached cells (10(-5) and 10(-4)M) and suppressed their viability (10(-6)-10(-4)M). Morphologic observations revealed degenerative alterations in the germ cells and Sertoli cells. Similar effects as observed after DNBP exposure were evident at 10(-4)M DNOC and 10(-4)M DNP. These results demonstrate that DNBP, DNOC, and DNP have in vitro toxicity to these cell populations at high concentration, and suggest the possibility that DNOC and DNP also cause testicular damage in experimental animals and humans. PMID:12642158

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

2003-01-01

121

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

Microsoft Academic Search

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

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

2010-01-01

122

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

PubMed

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

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

2013-03-01

123

Telomere sister chromatid exchange in telomerase deficient murine cells  

SciTech Connect

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.

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

2005-01-01

124

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

Microsoft Academic Search

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

Manuel Grez; Ercan Akgun; Frank Hilberg; Wolfram Ostertag

1990-01-01

125

Assessment of testicular function after acute and chronic irradiation: Further evidence for an influence of late spermatids on Sertoli cell function in the adult rat  

SciTech Connect

To study cell to cell communications within the testis of adult Sprague-Dawley rats, we used acute whole body neutron plus gamma-irradiation over 7-121 days postirradiation and chronic whole body gamma-irradiation over 14-84 days of irradiation and 7-86 days postirradiation. Neither irradiation protocol had an effect on the body weight of the animals. Neutron plus gamma-rays induced dramatic damages to spermatogonia, preleptotene spermatocytes, spermatozoa, and, to a lesser extent, pachytene spermatocytes. In contrast, gamma-rays induced a selective destruction of spermatogonia. Subsequently, in both experiments a maturation-depletion process led to a marked decrease in all germ cell types. A complete or near complete recovery of the different germ cell types and spermatozoa took place during the two postirradiation periods. Under both irradiation protocols Sertoli cells number was unchanged. Androgen-binding protein and FSH levels were normal in spite of the disappearance of most germ cells from spermatogonia to early spermatids. However, the decline of androgen-binding protein as well as the rise of FSH and their subsequent recovery were highly correlated to the number of late spermatids and spermatozoa. Moreover, it appeared that spermatocytes may also interfere with the production of inhibin (Exp B). With neither irradiation was Leydig cell function altered, except in Exp B in which elevated LH levels were temporarily observed. Correlation analysis suggested a relationship between preleptotene spermatocytes and Leydig cell function. In conclusion, this study establishes that chronic gamma-irradiation is particularly useful in the study of intratesticular paracrine regulation in vivo and provides further support to the concept that late spermatids play a major role in controlling some aspects of Sertoli cell function in the adult rat.

Pineau, C.; Velez de la Calle, J.F.; Pinon-Lataillade, G.; Jegou, B.

1989-06-01

126

Testicular Leukemia Inhibitory Factor (LIF) and LIF Receptor Mediate Phosphorylation of Signal Transducers and Activators of Transcription (STAT)-3 and STAT1 and Induce c-fos Transcription and Activator Protein1 Activation in Rat Sertoli But Not Germ Cells  

Microsoft Academic Search

Increasing amounts of evidence suggest noninflammatory roles for growth factor and cytokines in development and differentiation. Leu- kemia inhibitory factor (LIF) belongs to a gp130 pleiotropic family of growth factors that has recently been shown to enhance the survival of rat testicular gonocytes and Sertoli cells. In this study, we show the expression of gp130 and LIF messenger RNAs (mRNAs)

SHIRZAD JENAB; PATRICIA L. MORRIS

1998-01-01

127

A novel DICER1 mutation identified in a female with ovarian Sertoli-Leydig cell tumor and multinodular goiter: a case report  

PubMed Central

Introduction Germ-line mutations in the micro-ribonucleic acid processing gene DICER1 have been shown to predispose to a subset of benign tumors susceptible to malignant transformation, including ovarian Sertoli-Leydig cell tumor, nontoxic multinodular goiter, multilocular cystic nephroma and pleuropulmonary blastoma, which can occur in children and young adults. This may be due to reduced Dcr-1 homolog expression in carriers of germline mutations, which causes impairment of micro-ribonucleic acid processing and deregulates the growth and differentiation of target cells, leading to an increased risk of tumorigenesis. Many carriers of germ-line DICER1 mutations remain unaffected, but development of tumors within carriers is associated with varying prognoses. Case presentation Despite the Dcr-1 homolog syndrome phenotype being incompletely defined, a DICER1 mutation was suspected when a girl (case 1 patient) of Danish ethnicity presented with both an ovarian Sertoli-Leydig cell tumor and a multinodular goiter at the age of 13 years. In addition, family history included a male sibling (case 2 patient) who also had a multinodular goiter and had undergone a hemithyroidectomy at the age of 14 years. Subsequent DICER1 screening of the girl identified two novel mutations in exon 21 - a nonsense (c.3647C>A, p.Ser1216*) and a missense (c.3649T>A, p.Tyr1217Asn) mutation. The siblings had inherited the mutations from their father and paternal grandfather, which both currently were asymptomatic, indicating reduced penetrance of the nonsense mutation. Analysis of the parents revealed that the mutations were present in cis, making the contribution of the missense mutation less significant. Conclusion We report a novel pathogenic DICER1 mutation (p.Ser1216*) in a Danish family associated with ovarian Sertoli-Leydig cell tumor and a multinodular goiter. A multinodular goiter was diagnosed in the siblings during childhood. Clinicians should be aware of a potential germ-line DICER1 mutation when evaluating multinodular goiter in young patients with or without a family history of thyroid diseases. PMID:24708902

2014-01-01

128

SYNCHRONIZATION OF RAPID GLOBIN EXPRESSION IN MURINE ERYTHROLEUKEMIC CELLS  

EPA Science Inventory

The addition of butyric acid (BA) to murine erythroleukemia cells (MELC) produces the expression of primarily A and E2 hemoglobins while DMSO incubation produces the expression of primarily A hemoglobin. Preincubation of MELC with DMSO followed by BA induction accelerates the exp...

129

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

PubMed

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

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

2014-01-01

130

Treatment of High Risk Sertoli-Leydig Cell Tumors of the Ovary Using a Gonadotropin Releasing Hormone (GnRH) Analog  

PubMed Central

Sertoli–Leydig cell tumors are rare ovarian neoplasms. We report two unusual cases with bilateral SLCTs suggesting evidence of genetic predisposition and at high risk of recurrence. To reduce this risk, we exploited the use of GnRH analog to lower gondadotropin and potentially directly inhibit the tumors through expressed GnRH receptors. We used it as maintenance antitumor therapy for 2 years after completion of chemotherapy, to cover the period of risk for recurrence. Both patients remain in complete remission at >2 years after completing leuprorelin therapy. Of note, both patients carry DICER1 mutations, frequently found in pleuropulmonary blastoma syndrome. Pediatr Blood Cancer 2013; 60: E16–E18. © 2012 Wiley Periodicals, Inc. PMID:23193086

Lashkari, Harsha Prasada; Nash, Ruth; Albanese, Assunta; Okoye, Bruce; Millar, Robert; Pritchard-Jones, Kathy

2013-01-01

131

Defining suitable reference genes for RT-qPCR analysis on human sertoli cells after 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure.  

PubMed

Quantitative real-time RT-PCR (qPCR) has proven to be a valuable molecular technique to quantify gene expression. There are few studies in the literature that describe suitable reference genes to normalize gene expression data. Studies of transcriptionally disruptive toxins, like tetrachlorodibenzo-p-dioxin (TCDD), require careful consideration of reference genes. The present study was designed to validate potential reference genes in human Sertoli cells after exposure to TCDD. 32 candidate reference genes were analyzed to determine their applicability. geNorm and NormFinder softwares were used to obtain an estimation of the expression stability of the 32 genes and to identify the most suitable genes for qPCR data normalization. PMID:25078986

Ribeiro, Mariana Antunes; Dos Reis, Mariana Bisarro; de Moraes, Leonardo Nazário; Briton-Jones, Christine; Rainho, Cláudia Aparecida; Scarano, Wellerson Rodrigo

2014-11-01

132

Immunosuppression in murine renal cell carcinoma  

Microsoft Academic Search

Summary In our companion paper we have reported that cell-mediated immunity of mice bearing renal cell carcinoma is profoundly suppressed. The non-responsiveness of such animals was found to be attributable to Renca cells themselves and to splenic lymphoid cells that down-regulate other fully capable lymphoid cells. In this communication the lymphoid cell source of suppression within Rencabearing mice has been

Shahik K. Gregorian; Jack R. Battisto

1990-01-01

133

Protective effects of ginsenosides against Bisphenol A-induced cytotoxicity in 15P-1 Sertoli cells via extracellular signal-regulated kinase 1/2 signalling and antioxidant mechanisms.  

PubMed

Numerous studies have demonstrated that Bisphenol A (BPA) can cause reproductive toxicity. Ginseng has wide range of pharmacological actions and, more importantly, has proven its worth with respect to reproductive function in several reports. We have suggested that ginsenosides, the main active components of ginseng, may protect against BPA-induced cell damage. Therefore, an in vitro culture model of 15P-1 Sertoli cells was employed to investigate whether ginsenosides have protective effects on BPA-stimulated 15P-1 Sertoli cells. The results revealed that ginsenosides (75 ?g/ml) significantly inhibited BPA-induced decreases in cell viability and increases in apoptosis. Immunofluorescence staining showed that BPA exposure-induced collapse of vimentin intermediate filaments was prevented by the application of ginsenosides. Ginsenosides also inhibited extracellular signal-regulated kinase (ERK1/2) phosphorylation and BPA-induced alterations of Bcl-2 and Bax protein expression in 15P-1 Sertoli cells. Furthermore, the alterations of T-AOC, superoxide dismutase, glutathione peroxidase, glutathione reductase, glutathione and malondialdehyde levels in BPA-stimulated cells were partially prevented with pre-treatment with ginsenosides. Taken together, these results suggest that ginsenosides have protective effects against BPA-induced cell damage and that these effects are mediated by preventing ERK1/2 phosphorylation and through the enhancement of cellular antioxidant capacity. Ginsenosides may therefore be beneficial in the prevention of environmental BPA-induced, reproduction-related toxicity. PMID:22269103

Wang, Limin; Hao, Jie; Hu, Jiangang; Pu, Jun; Lü, Zilan; Zhao, Lina; Wang, Qi; Yu, Qiubo; Wang, Yingxiong; Li, Gang

2012-07-01

134

Effect of temperature on cell population balance in Dexter's culture of murine bone marrow hematopoietic cells with stromal cells  

Microsoft Academic Search

The effect of temperature in the range from 25 to 37°C on the population balance of stromal and multiple-lineage hematopoietic cells from murine bone marrow at various stages of differentiation in Dexter's culture was investigated. The length of time required for stromal cells to reach confluence after inoculation of both cell types harvested from murine bone marrow was shorter at

Mutsumi Takagi; Daiki Kubomura; Toshiomi Yoshida

1999-01-01

135

Generation and establishment of murine adherent cell lines.  

PubMed

We describe a method to derive cell lines and clones from cells of the murine midgestation aorta-gonads-mesonephros (AGM) microenvironment. We start from subdissected AGM regions in "explant" or "single cell suspension" type cultures from embryos transgenic for tsA58, a temperature-sensitive mutant of the SV40 T antigen gene. The number of cells in such cultures initially expand, but in most cases, this expansion phase is followed by a stable or even decline in cell number. After this so-called crisis phase, cell proliferation is noticeable in more than 90% of the cultures. Stromal cell clones can be isolated from these cultures, some of which have been cultured for more than 50 population doublings, and functionally characterized using various methods These stromal cell clones are valuable tools for the study of the regulation of hematopoietic stem and progenitor cells in the midgestation mouse embryo. PMID:23179840

Istvanffy, Rouzanna; Oostendorp, Robert A J

2013-01-01

136

Nanoelectroablation therapy for murine basal cell carcinoma  

SciTech Connect

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.

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

137

Light scattering properties of murine hemopoietic cells.  

PubMed

The light scatter of cells contains information about cell structure and size. Much of this information can be obtained by measuring the light scattered in a flow cytometer in two directions: forward (1.5 degrees-13 degrees) and perpendicular (65 degrees-115 degrees) with respect to the direction of the laser light. For different mouse bone marrow cell types and Sephadex G-25 beads of 10-50 micron diameter, the forward light scatter intensity can be shown to be linearly proportional to the cross-sectional area. The perpendicular light scatter intensity can be shown to depend both on size and degree of structuredness. Therefore, light scatter measurements may be used to obtain overall morphological descriptions of rare cells. By sorting on the basis of light scatter measurements and by subsequent in vivo and in vitro culture assays, it can be shown that the pluripotent hemopoietic stem cell and three committed progenitor cells which represent consecutive stages in the granulocyte/monocyte differentiation series have diameters of 7.1-7.5 micron, and show a complexity of structuredness which increases with differentiation. Since these cells have a low incidence and are only described by their function, such morphological information cannot be obtained by direct microscopic examination of bone marrow. Furthermore, most measurements by flow cytometers can be improved by simultaneous light scatter measurements. Examples are presented which illustrate this in studies of immunofluorescence, leukemic bone marrow, and stem cell purification. PMID:7406983

Visser, J W; van den Engh, G J; van Bekkum, D W

1980-01-01

138

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

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

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

139

Bioengineering murine mastocytoma cells to produce anticoagulant heparin.  

PubMed

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

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

2014-03-01

140

Flow cytometric quantification of radiation responses of murine peritoneal cells  

SciTech Connect

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.

Tokita, N.; Raju, M.R.

1982-01-01

141

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

SciTech Connect

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

Johnson, G.P.

1987-01-01

142

Sertoli cell dedifferentiation in human cryptorchidism and gender reassignment shows similarities between fetal environmental and adult medical treatment estrogen and antiandrogen exposure.  

PubMed

Studies over the last years show an increase in testicular cancer, hypospadias and cryptorchidism in industrial countries, leading to the concept of testicular dysgenesis syndrome (TDS). It is hypothesized that TDS is caused by estrogen and antiandrogen exposure during fetal life, accompanied by incomplete maturation of testicular Sertoli cells (SC). However, it is not known if SC disruption is a primary cause or a response to fetal Leydig cell testosterone production changes. To determine if SC differentiation is directly affected by estrogens, we compared SC maturation between adult gender reassignment cases exposed to estrogen and antiandrogen therapy, and those of typical TDS in adult cryptorchidism. We found similar expression of immature SC markers M2A antigen, inhibin bodies and Anti Mullerian Hormone, and the absence of maturation marker androgen receptor in SC of both types of patients. These data supports the occurrence of true SC dedifferentiation caused by estrogen exposure in adult humans. Our data also suggests that SC maturation is directly disrupted in TDS. PMID:24012888

Nistal, Manuel; Gonzalez-Peramato, Pilar; De Miguel, Maria P

2013-12-01

143

Surface Markers for the Murine Oval Cell Response  

PubMed Central

The biology of progenitor activation in the liver is of considerable medical and scientific interest. The powerful genetic tools available for the mouse make it an ideal model system to study this complex process involving many different cell types. However, reagents for the isolation and study of distinct hepatic subpopulations have been quite limited compared to those available for hematopoietic cells. To produce cell surface reactive reagents more specific for the oval cell response, we generated a new collection of monoclonal antibodies by immunization of Fischer rats with enzymatically dispersed nonparenchymal cells from the livers of adult mice treated with 3,5-diethoxycarbonyl-1,4-dihydrocollidine. Each of the resulting antibodies recognized a surface antigen present on a liver cell subset and permitted the viable isolation of the associated subpopulation by fluorescence-activated cell sorting. Differential activity was observed on normal liver cells and at different stages of oval cell activation, indicating potential utility for progenitor cell identification. The subdivision of liver cells using these tools should facilitate the study of the biology of ductal and periductal hepatic cell types, including progenitors. Conclusion A new panel of surface reactive monoclonal antibodies to support investigation of the murine oval cell response has been developed. PMID:18726953

Dorrell, Craig; Erker, Laura; Lanxon-Cookson, Kelsea M.; Abraham, Stephanie L.; Victoroff, Tristan; Ro, Simon; Canaday, Pamela S.; Streeter, Philip R.; Grompe, Markus

2011-01-01

144

Development of Phase II Xenobiotic Metabolizing Enzymes in Differentiating Murine Clara Cells  

E-print Network

disease is the leading cause of death in infants under 1 year of age (American Lung Association, 1999Development of Phase II Xenobiotic Metabolizing Enzymes in Differentiating Murine Clara Cells Xenobiotic Metabolizing Enzymes in Differentiating Murine Clara Cells. Fanucchi, M. V., Buckpitt, A. R

Hammock, Bruce D.

145

Murine Norovirus Transcytosis across an In Vitro Polarized Murine Intestinal Epithelial Monolayer Is Mediated by M-Like Cells  

PubMed Central

Noroviruses (NoVs) are the causative agent of the vast majority of nonbacterial gastroenteritis worldwide. Due to the inability to culture human NoVs and the inability to orally infect a small animal model, little is known about the initial steps of viral entry. One particular step that is not understood is how NoVs breach the intestinal epithelial barrier. Murine NoV (MNV) is the only NoV that can be propagated in vitro by infecting murine macrophages and dendritic cells, making this virus an attractive model for studies of different aspects of NoV biology. Polarized murine intestinal epithelial mICcl2 cells were used to investigate how MNV interacts with and crosses the intestinal epithelium. In this in vitro model of the follicle-associated epithelium (FAE), MNV is transported across the polarized cell monolayer in the absence of viral replication or disruption of tight junctions by a distinct epithelial cell with microfold (M) cell properties. In addition to transporting MNV, these M-like cells also transcytose microbeads and express an IgA receptor. Interestingly, B myeloma cells cultured in the basolateral compartment underlying the epithelial monolayer did not alter the number of M-like cells but increased their transcytotic activity. Our data demonstrate that MNV can cross an intact intestinal epithelial monolayer in vitro by hijacking the M-like cells' intrinsic transcytotic pathway and suggest a potential mechanism for MNV entry into the host. PMID:24049163

Gonzalez-Hernandez, Mariam B.; Liu, Thomas; Blanco, Luz P.; Auble, Heather; Payne, Hilary C.

2013-01-01

146

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

Microsoft Academic Search

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

Rudolf P Wuthrich; Tracey L Snyder

1992-01-01

147

Effects of trichostatins on differentiation of murine erythroleukemia cells  

SciTech Connect

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.

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

1987-07-15

148

On the heterogeneity of murine natural killer cells  

PubMed Central

The heterogeneity of cells capable exerting spontaneous cytotoxicity in vitro was explored using antisera to several genetically determined surface markers on mouse lymphocytes. Four phenotypes of cells derived either from fresh or cultured murine lymphoid tissue were found to exert natural killer (NK) activity in vitro. One affector cell subset, termed NKI cells, had the serological phenotype of Thy-1-, Lyt-2-, Qa5+, and lysed measles virus persistently infected target cells (HeLa- Ms) but not P815 mastocytoma cells. It corresponds with the NK cells described in most systems in which lymphoma targets are commonly used. A second subset, with the same target cell specificity, termed NKT is a thymus-independent cell with the phenotype Thy-1+, Lyt-2-, Qa-5+, Ly- 5+. A third subset of NK cells, termed T killer (TK) cells deriving from cultures of conventional but not nude mouse spleens, mediated spontaneous cytotoxicity of P815 mastocytoma cells, but not of virus- infected targets. It has a phenotype of Thy-1+, Lyt-2+, Qa-5-, Ly-5+, apparently identical with that of conventional, antigen-specific cytotoxic T lymphocytes. The fourth phenotype of NK cells, termed NKM, derived primarily from cultures of bone marrow, is cytotoxic for HeLa- measles but not P815, and expresses only Ly-5+ among the various markers tested. Beige mice possess normal TK and NKM activities, but had normal NKI, NKT as well as NKM activity. All NK cell subsets express the Ly-5 surface marker. The existence of four phenotypically distinct NK effector cells was strengthened by studies on selective regulation of their activity by two different biological factors. Interferon (IFN) augmented NK activity of primarily one of the subsets examined, the NKI cell; the activity of IFN on NKT cells could not be directly tested, but IFN was without positive effect on TK or NKM cells. In contrast, partially purified IFN-free interleuken 2 (IL-2) augmented the activities of both the TK and NKT subsets, but not of NKI or NKM cell. IL-2 was active in augmenting NK activity in spleen cells obtained from both conventional and nu/nu mice, but was without effect on spleens of nu/nu mice depleted of Thy-1+ cells. These and other data suggest that IL-2 acts primarily, if not exclusively, on THy-1+ cells. These results strengthen the view that natural cytotoxicity in vitro can be mediated by several distinct cell populations under different genetic and regulatory control and indicate the importance of defining and delineating the cell lineages of each and the role of the independent subsets in resistance to virus infections and tumors in vivo. PMID:6168724

1981-01-01

149

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

PubMed Central

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

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

2013-01-01

150

Differential effects of c-Src and c-Yes on the endocytic vesicle-mediated trafficking events at the Sertoli cell blood-testis barrier: an in vitro study.  

PubMed

The blood-testis barrier (BTB) is one of the tightest blood-tissue barriers in the mammalian body. However, it undergoes cyclic restructuring during the epithelial cycle of spermatogenesis in which the "old" BTB located above the preleptotene spermatocytes being transported across the immunological barrier is "disassembled," whereas the "new" BTB found behind these germ cells is rapidly "reassembled," i.e., mediated by endocytic vesicle-mediated protein trafficking events. Thus, the immunological barrier is maintained when preleptotene spermatocytes connected in clones via intercellular bridges are transported across the BTB. Yet the underlying mechanism(s) in particular the involving regulatory molecules that coordinate these events remains unknown. We hypothesized that c-Src and c-Yes might work in contrasting roles in endocytic vesicle-mediated trafficking, serving as molecular switches, to effectively disassemble and reassemble the old and the new BTB, respectively, to facilitate preleptotene spermatocyte transport across the BTB. Following siRNA-mediated specific knockdown of c-Src or c-Yes in Sertoli cells, we utilized biochemical assays to assess the changes in protein endocytosis, recycling, degradation and phagocytosis. c-Yes was found to promote endocytosed integral membrane BTB proteins to the pathway of transcytosis and recycling so that internalized proteins could be effectively used to assemble new BTB from the disassembling old BTB, whereas c-Src promotes endocytosed Sertoli cell BTB proteins to endosome-mediated protein degradation for the degeneration of the old BTB. By using fluorescence beads mimicking apoptotic germ cells, Sertoli cells were found to engulf beads via c-Src-mediated phagocytosis. A hypothetical model that serves as the framework for future investigation is thus proposed. PMID:25117412

Xiao, Xiang; Mruk, Dolores D; Wong, Elissa W P; Lee, Will M; Han, Daishu; Wong, Chris K C; Cheng, C Yan

2014-10-01

151

Complementation analysis of the murine scid cell line  

SciTech Connect

It has been shown that several X-ray-sensitive Chinese hamster cell mutants defective in repair of DNA double-strand breaks (DSBs) are also impaired in the process of V(D)J recombination. The hamster mutants with this phenotype represent three distinct complementation groups, represented by the xrs series, XR-1 and V-3. The murine scid cell line also shows the same phenotype, and therefore we examined whether the scid mutant represents a new complementation group or belongs to one of the existing groups. Scid cells were fused with hamster cell mutants representing the three complementation groups. Hybrids between V-3 and scid cells were only partially complemented for X-ray sensitivity, whereas hybrids derived from fusions with the other mutants were resistant to X rays. These results suggest that V-3 and scid cells are defective in the same gene. To confirm this finding, a single human chromosome 8, which is known to carry the scid gene, was introduced into V-3 cells by microcell-mediated chromosome transfer. Nine hybrid clones derived from V-3 and carrying human chromosome 8 were obtained, and seven were found to be partially complemented for X-ray sensitivity. When human chromosome 8 was introduced into scid cells, seven of eight hybrid clones became resistant to X rays. The results indicate that the defective genes in V-3 and scid are both localized on human chromosome 8. This supports the results from the fusion analysis that V-3 and scid cells are defective in the same gene. 53 refs., 4 figs., 1 tab.

Zdzienicka, M.Z. [Univ. of Leiden, Wassenaarseweg (Netherlands)]|[J.A. Cohen Institute, Leiden (Netherlands); Priestly, A.; Jeggo, P.A. [Sussex Univ., Brighton (United Kingdom)] [and others

1995-09-01

152

High-dimensional analysis of the murine myeloid cell system.  

PubMed

Advances in cell-fate mapping have revealed the complexity in phenotype, ontogeny and tissue distribution of the mammalian myeloid system. To capture this phenotypic diversity, we developed a 38-antibody panel for mass cytometry and used dimensionality reduction with machine learning-aided cluster analysis to build a composite of murine (mouse) myeloid cells in the steady state across lymphoid and nonlymphoid tissues. In addition to identifying all previously described myeloid populations, higher-order analysis allowed objective delineation of otherwise ambiguous subsets, including monocyte-macrophage intermediates and an array of granulocyte variants. Using mice that cannot sense granulocyte macrophage-colony stimulating factor GM-CSF (Csf2rb(-/-)), which have discrete alterations in myeloid development, we confirmed differences in barrier tissue dendritic cells, lung macrophages and eosinophils. The methodology further identified variations in the monocyte and innate lymphoid cell compartment that were unexpected, which confirmed that this approach is a powerful tool for unambiguous and unbiased characterization of the myeloid system. PMID:25306126

Becher, Burkhard; Schlitzer, Andreas; Chen, Jinmiao; Mair, Florian; Sumatoh, Hermi R; Teng, Karen Wei Weng; Low, Donovan; Ruedl, Christiane; Riccardi-Castagnoli, Paola; Poidinger, Michael; Greter, Melanie; Ginhoux, Florent; Newell, Evan W

2014-12-01

153

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

SciTech Connect

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

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

1990-08-01

154

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

Microsoft Academic Search

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

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

2007-01-01

155

DNA repair in murine embryonic stem cells and differentiated cells  

SciTech Connect

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.

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

156

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

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

Xiang, Zou; Qian, Weiping; Han, Xiaodong; Li, Dongmei

2014-01-01

157

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

PubMed Central

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

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

2013-01-01

158

Murine Bladder Carcinoma Cells Present Antigen to BCG-specific CD4+ T-Cells1  

Microsoft Academic Search

Intravesical administration of Bacillus Calmette-Guérin (BCG) is the most effective therapy for superficial transitional cell carcinoma of the bladder although its mechanism of action is not known. To determine if bladder tumors are capable of antigen presentation and thus might interact directly with BCG-specific I -cells, we studied the murine blad der tumor MB49. M»49 (MlK Class II negative) (IA

Edmund C. Lattime; Leonard G. Cornelia; Peter A. McCue

159

Antisense oligodeoxynucleotide inhibition of delta protein kinase C expression accelerates induced differentiation of murine erythroleukaemia cells.  

PubMed Central

The potential regulatory role of delta protein kinase C (delta PKC) in murine erythroleukaemia cell differentiation was studied by using antisense oligodeoxynucleotides targeting the translation initiation region of mouse delta PKC mRNA. Cell treatment with antisense oligonucleotides, at a concentration of 20 microM, followed by hexamethylenebisacetamide induction, produced a specific 2-fold increase in the differentiation rate of both slowly and rapidly differentiating murine erythroleukaemia cell clones. Cell permeabilization by a cationic lipid resulted in a decrease of one order of magnitude in the amounts of antisense oligonucleotides necessary to elicit the maximal response, and accelerated the kinetics of the stimulatory effect. These changes in murine erythroleukaemia cell differentiation rates, observed in both cell clones, were associated with 60% and 50% decreases, respectively, in delta PKC immunoreactive protein in slowly and rapidly differentiating cells. The present results indicate strongly that basal levels of delta PKC in murine erythroleukaemia cells are essential in regulating the initial differentiation rate of these cells in response to chemical induction, and provide further evidence that this PKC isoform plays a fundamental role in maintaining the undifferentiated phenotype of murine erythroleukaemia cells. Images Figure 4 Figure 5 Figure 6 PMID:8526869

Pessino, A; Passalacqua, M; Sparatore, B; Patrone, M; Melloni, E; Pontremoli, S

1995-01-01

160

Localization of Label-Retaining Cells in Murine Vocal Fold Epithelium  

ERIC Educational Resources Information Center

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

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

2011-01-01

161

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

PubMed Central

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

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

162

Transformation of murine LMTK- cells with purified HLA class I genes. I. Modification of conformation of murine beta 2-microglobulin upon its association with HLA heavy chains.  

PubMed

Murine LMTK- cells were unexpectedly found to cross-react with a murine anti-human beta 2-microglobulin (beta 2-m)monoclonal antibody (m.Ab) after transformation with cosmid clones containing different purified HLA class I genes. The same cross-reactivity was observed with CTP 34 B4 (murine x human) somatic hybrid cells, which express class I molecules constituted of human HLA heavy chains and murine beta 2-m. Inhibition studies of the complement-dependent cytolysis mediated by the cross-reacting m.Ab indicated that isolated murine beta 2-m does not express the cross-reacting determinant, suggesting that its expression by the transformed cells reflects conformational modification of murine beta 2-m upon its association with HLA heavy chains. These results illustrate one of the possible post-translational mechanisms through which the antigenicity of a polypeptide chain can be modified. They might provide a serologic marker of the third domain of HLA class I heavy chains. Finally, because quantitative differences of reactivity with the anti-human beta 2-m m.Ab were observed, depending on the HLA class I genes used for transformation, these results individualize two families of HLA class I heavy chains responsible for different conformational modifications of murine beta 2-m. PMID:6185587

Lemonnier, F A; Le Bouteiller, P P; Malissen, B; Golstein, P; Malissen, M; Mishal, Z; Caillol, D H; Jordan, B R; Kourilsky, F M

1983-03-01

163

Mycoplasma contamination of murine embryonic stem cells affects cell parameters, germline transmission and chimeric progeny  

Microsoft Academic Search

Murine embryonic stem cells (mESCs) inoculated at passage P13 with the mycoplasma species M. hominis, M. fermentans and M. orale and cultured over 20 passages showed reduced growth rate and viability (P < 0.0001) compared to control mESCs. Spectral karyotypic analysis of mycoplasma-infected mESCs showed a number of non-clonal\\u000a chromosomal aberrations which increased with the duration of infection. The differentiation status of

Kyriaki Markoullis; Diana Bulian; Gabriele Hölzlwimmer; Leticia Quintanilla-Martinez; Katrin-Janine Heiliger; Horst Zitzelsberger; Hagen Scherb; Josef Mysliwietz; Cord C. Uphoff; Hans G. Drexler; Thure Adler; Dirk H. Busch; Jörg Schmidt; Esther Mahabir

2009-01-01

164

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

PubMed Central

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

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

2014-01-01

165

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

E-print Network

elucidate key sets of intracellular signaling protein activities that combine to govern cell phenotypic that regulate ES cell processes operate through multiple intracellular signaling pathways, which are generallyMultivariate proteomic analysis of murine embryonic stem cell self-renewal versus differentiation

Zandstra, Peter W.

166

Hemopoietic stem cells in murine embryonic yolk sac and peripheral blood  

Microsoft Academic Search

Disaggregated embryonic yolk sac cells and circulating peripheral blood cells were obtained from normal murine day 9 embryos, prior to the formation of the fetal liver. These cells were microinjected transplacentally into days 11-15 W mutant anemic fetuses, when the fetal liver was the major hemopoietic organ. In a small proportion of the recipient animals examined after birth, long-term repopulation

J. F. Toles; D H Chui; L W Belbeck; E Starr; J E Barker

1989-01-01

167

Neurogenic differentiation of murine and human adipose-derived stromal cells  

Microsoft Academic Search

The identification of cells capable of neuronal differentiation has great potential for cellular therapies. We examined whether murine and human adipose-derived adult stem (ADAS) cells can be induced to undergo neuronal differentiation. We isolated ADAS cells from the adipose tissue of adult BalbC mice or from human liposuction tissue and induced neuronal differentiation with valproic acid, butylated hydroxyanisole, insulin, and

Kristine M Safford; Kevin C Hicok; Shawn D Safford; Yuan-Di C Halvorsen; William O Wilkison; Jeffrey M Gimble; Henry E Rice

2002-01-01

168

Natural Species-Restricted Attachment of Human and Murine T Lymphocytes to Various Cells  

Microsoft Academic Search

Murine and human T lymphocytes bear on their surface a receptor that confers on them the ability to attach to a variety of target cells from the same species, derived in vivo and in vitro. Thymocytes and activated T cells attached readily to target cells, while blood T lymphocytes were able to do so only after the removal of sialic

Uri Galili; Naomi Galili; Farkas Vanky; Eva Klein

1978-01-01

169

Apigenin protects HT22 murine hippocampal neuronal cells against endoplasmic reticulum stress-induced apoptosis  

Microsoft Academic Search

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

A Young Choi; Ji Hyun Choi; Jung Yeon Lee; Kyung-Sik Yoon; Wonchae Choe; Joohun Ha; Eui-Ju Yeo; Insug Kang

2010-01-01

170

A stem cell for stem cells in murine haematopoiesis  

Microsoft Academic Search

Mature erythrocytes and granulocytes have limited lifespans, do not replicate and must therefore be replenished constantly. They are derived from pluripotent stem cells (PSCs) which are capable of self-renewal1. The numbers and properties of PSCs can be inferred in part from studies of their progeny. Such studies have depended largely on highly artificial experimental systems, involving such procedures as X-ray

D. I. Burton; J. D. Ansell; R. A. Gray; H. S. Micklem

1982-01-01

171

Specific lysis of murine cells expressing HLA molecules by allospecific human and murine H-2-restricted anti-HLA T killer lymphocytes.  

PubMed

The lysis by human and murine anti-HLA cytolytic T lymphocytes (CTL) of murine cells expressing class I HLA molecule after gene transfection has been studied using two different murine cells: LMTK- and P815-HTR-TK-. Weak but significant HLA-A11-specific lysis was found occasionally with human CTL on the HLA-A11+ L cells. On the contrary, P815-A11 or P815-A2 cells were lysed strongly and specifically by HLA-A11 or HLA-A2-specific human CTL. The T8+T4- phenotype of the effector cells was confirmed and the reaction was inhibited by anti-HLA class I monoclonal antibodies. Despite their higher sensitivity to human CTL, the P815-HLA+ cells did not express higher levels of HLA antigens than L cells, and the presence or the absence of human beta 2 microglobulin was irrelevant. Anti-human LFA-1 antibodies abrogated the lysis of P815-A11+ cells showing that the LFA-1 receptor which is apparently lacking on the L cell surface was on the contrary expressed on P815 cells. On the other hand, murine anti-HLA CTL have been prepared by immunizing mice against syngeneic HLA-A11+ L cells. They lysed very efficiently and specifically these cells, but appeared completely devoid of activity against human HLA-A11 target cells. This barrier was apparently due to the H-2 restriction of these H-2k anti-HLA murine CTL, as shown by their inability to lyse allogeneic H-2d cells expressing HLA-A11, and by the blocking of their activity by anti H-2k antibodies. By contrast, xenogeneic anti-HLA CTL obtained by immunizing murine lymphocytes against human cells lysed both human and murine HLA+ cells but they reacted with a monomorphic epitope of the HLA molecule in a nonrestricted way. These results show that human cells lyse very efficiently P815 murine cells expressing HLA class I antigens; the higher sensitivity of P815 cells compared to L cells is probably due to the presence of a LFA-1 receptor on these cells; a class I molecule of human origin can be seen as an H-2-restricted minor histocompatibility antigen in another species. PMID:3522244

Achour, A; Begue, B; Gomard, E; Paul, P; Sayagh, B; Van Pel, A; Levy, J P

1986-06-01

172

Haemopoietic cell proliferation in murine bone marrow cells exposed to extreme low frequency (ELF) electromagnetic fields.  

PubMed

As leukemia is one of the health hazards that is sometimes associated with exposure to extreme low frequency fields, we studied the in vitro effects of ELF fields on haemopoietic cell proliferation. First, the cytotoxic effect of 80 microT, 50 Hz magnetic fields on 3T3 cell proliferation was investigated using the neutral red test. Many chemicals are believed to cause damage because they interfere with basal or "housekeeping" cell functions. The basal cell functions are present in every cell. Non-specialized, actively dividing cells are suitable for measuring cytotoxic effects. Cytotoxic doses can be identified by exposing actively dividing cells in vitro and measuring growth inhibition caused by interference with these basal cell functions. 80 microT, 50 Hz magnetic fields caused no cytotoxicity: we were not able to demonstrate any interference with essential cell functions in the non-differentiated 3T3 cell line. Furthermore, the in vitro effects of ELF fields on murine haemopoietic and stromal stem cell proliferation were studied. Haemopoiesis is a continuous process, where mature blood cells are replaced by the proliferation and differentiation of more primitive progenitor and stem cells. Blood formation is tightly regulated by the stromal micro-environment. Exposure of murine bone marrow cells, from male and female mice, to 80 microT (50 Hz) magnetic fields showed a reduction in the proliferation and differentiation of the granulocyte-macrophage progenitor (CFU-GM) compared to non-exposed bone marrow cells. The results on the effect of the ELF-field on stromal stem cell proliferation (CFU-f) are somewhat equivocal at the moment. CFU-f from female mice showed a reduction while CFU-f from male mice were not decreased. PMID:11566562

Van Den Heuvel, R; Leppens, H; Nêmethova, G; Verschaeve, L

2001-01-01

173

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

PubMed Central

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

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

2013-01-01

174

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 Research and INSERM U899 - ANRS Center for Human Vaccines, Dallas, TX 75204, USA 4Centre d'Immunologie de: dankaplan@umn.edu DOI 10.1016/j.immuni.2011.06.005 SUMMARY Skin-resident dendritic cells (DCs) are well

175

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

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.

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

1989-12-01

176

Cytotoxic effects of glutathione synthesis inhibition by L-buthionine-(SR)-sulfoximine on human and murine tumor cells  

Microsoft Academic Search

The glutathione (GSH) synthesis inhibitor, buthionine sulfoximine (BSO) was tested for cytotoxicity and thiol depletion in murine and human tumor cells in vitro, and for its antitumor activity and toxicity in vivo. The cell lines used in these studies included murine L-1210 leukemia, human RPMI 8226 myeloma, MCF-7 breast cancer and WiDr colon carcinoma. Soft agar colony forming assays showed

Robert T. Dorr; James D. Liddil; Michelle J. Soble

1986-01-01

177

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

Microsoft Academic Search

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

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

1997-01-01

178

Tumor Growth Inhibitory and Natural Suppressor Activities of Murine Bone Marrow Cells: A Comparative Study  

Microsoft Academic Search

The murine bone marrow (BM) cells having a certain phenotypic similarity to null natural suppressor (NS) cells have been previously established to be able to inhibitin vitroleukemic cell growth in a genetically unrestricted manner. In this study we found that the treatment of normal (C57BL\\/6 × DBA)F1 BM cells with a lysosomotropic agent,l-leucine methyl ester (LME), largely abrogated their ability

Victor I. Seledtsov; Vadim Y. Taraban; Galina V. Seledtsova; Denis M. Samarin; Ilias V. Avdeev; Vladimir V. Senyukov; Vladimir A. Kozlov

1997-01-01

179

Host Cell Cathepsins Potentiate Moloney Murine Leukemia Virus Infection  

Microsoft Academic Search

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

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

2007-01-01

180

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

PubMed Central

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

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

1983-01-01

181

Cryoprotective effects of low-density lipoproteins, trehalose and soybean lecithin on murine spermatogonial stem cells.  

PubMed

Summary Spermatogonial stem cells (SSCs) have the ability to self-renew and offer a pathway for genetic engineering of the male germ line. Cryopreservation of SSCs has potential value for the treatment of male infertility, spermatogonial transplantation, and so on. In order to investigate the cryopreservation effects of different cryoprotectants on murine SSCs, 0.2 M of low-density lipoproteins (LDL), trehalose and soybean lecithin were added to the cryoprotective medium, respectively, and the murine SSCs were frozen at -80°C or -196°C. The results indicated that the optimal recovery rates of murine SSCs in the cryoprotective medium supplemented with LDL, trehalose and soybean lecithin were 92.53, 76.35 and 75.48% at -80°C, respectively. Compared with freezing at -196°C, the optimum temperature for improvement of recovery rates of frozen murine SSCs, cryopreservation in three different cryoprotectants at -80°C, were 17.11, 6.68 and 10.44% respectively. The recovery rates of murine SSCs in the cryoprotective medium supplemented with 0.2 M LDL were significantly higher than that of other cryoprotectants (P < 0.05). Moreover, the recovery rates were demonstrated to be greater at -80°C compared with at -196°C (P < 0.05). In conclusion, 0.2 M of LDL could significantly protect murine SSCs at -80°C. In the freezing-thawing process, LDL is responsible for the cryopreservation of murine SSCs because it can form a protective film at the surface of membranes. However, more research is needed to evaluate and understand the precise role of LDL during the freezing-thawing of SSCs. PMID:22974447

Wang, Peng; Li, Ying; Hu, Xiao-Chen; Cai, Xiao-Li; Hou, Li-Peng; Wang, Yan-Feng; Hu, Jian-Hong; Li, Qing-Wang; Suo, Li-Juan; Fan, Zhi-Guo; Zhang, Bo

2014-05-01

182

Dendritic Cells Are the Major Antigen Presenting Cells in Inflammatory Lesions of Murine Mycoplasma Respiratory Disease  

PubMed Central

Mycoplasmas cause chronic respiratory diseases in animals and humans, and to date, development of vaccines have been problematic. Using a murine model of mycoplasma pneumonia, lymphocyte responses, specifically T cells, were shown to confer protection as well as promote immunopathology in mycoplasma disease. Because T cells play such a critical role, it is important to define the role of antigen presenting cells (APC) as these cells may influence either exacerbation of mycoplasma disease pathogenesis or enhancement of protective immunity. The roles of APC, such as dendritic cells and/or macrophages, and their ability to modulate adaptive immunity in mycoplasma disease are currently unknown. Therefore, the purpose of this study was to identify individual pulmonary APC populations that may contribute to the activation of T cell responses during mycoplasma disease pathogenesis. The present study indeed demonstrates increasing numbers of CD11c? F4/80+ cells, which contain macrophages, and more mature/activated CD11c+ F4/80? cells, containing DC, in the lungs after infection. CD11c? F4/80+ macrophage-enriched cells and CD11c+ F4/80? dendritic cell-enriched populations showed different patterns of cytokine mRNA expression, supporting the idea that these cells have different impacts on immunity in response to infection. In fact, DC containing CD11c+ F4/80? cell populations from the lungs of infected mice were most capable of stimulating mycoplasma-specific CD4+ Th cell responses in vitro. In vivo, these CD11c+F4/80? cells were co-localized with CD4+ Th cells in inflammatory infiltrates in the lungs of mycoplasma-infected mice. Thus, CD11c+F4/80? dendritic cells appear to be the major APC population responsible for pulmonary T cell stimulation in mycoplasma-infected mice, and these dendritic cells likely contribute to responses impacting disease pathogenesis. PMID:23390557

Sun, Xiangle; Jones, Harlan P.; Dobbs, Nicole; Bodhankar, Sheetal; Simecka, Jerry W.

2013-01-01

183

Effect of Tunisian Capparis spinosa L. extract on melanogenesis in B16 murine melanoma cells  

Microsoft Academic Search

The effect of Tunisian Capparis spinosa L. aromatic plant extract on melanogenesis regulation in B16 murine melanoma cells was investigated. B16 cells were treated\\u000a with 0.0005, 0.005, and 0.05% (w\\/v) C. spinosa extract after which the melanin content and cell viability were measured. To clarify the mechanism behind melanogenesis regulation,\\u000a the expression of tyrosinase was determined. Results showed that the

Kyoko Matsuyama; Myra O. Villareal; Abdelfatteh El Omri; Junkyu Han; Mohamed Elyes Kchouk; Hiroko Isoda

2009-01-01

184

Mechanism of the melanogenesis stimulation activity of (?)-cubebin in murine B16 melanoma cells  

Microsoft Academic Search

(?)-Cubebin showed a melanogenesis stimulation activity in a concentration-dependent manner in murine B16 melanoma cells without any significant effects on cell proliferation. Tyrosinase activity was increased at 24–72h after addition of cubebin to B16 cells, and then intracellular melanin amount was increased at 48–96h after the treatment. The expression levels of tyrosinase were time-dependently enhanced after the treatment with cubebin.

Noriko Hirata; Shunsuke Naruto; Kenji Ohguchi; Yukihiro Akao; Yoshinori Nozawa; Munekazu Iinuma; Hideaki Matsuda

2007-01-01

185

Two functionally distinct pools of glycosaminoglycan in the substrate adhesion site of murine cells  

Microsoft Academic Search

Footpad adhesion sites pinch off from the rest of the cell surface during EGTA- mediated detachment of normal or virus-transformed murine cells from their tissue culture substrates. In these studies, highly purified trypsin and testicular hyaluronidase were used to investigate the selective destruction or solubilization of proteins and polysaccharides in this substrate-attached material (SAM). Tryp- sin-mediated detachment of cells or

LLOYD A. CULP; BARRETT J. ROLLINS; JOSEFINA BUNIEL

1978-01-01

186

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 ...

187

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

E-print Network

Anthrax Lethal Toxin-Mediated Killing of Human and Murine Dendritic Cells Impairs the Adaptive Immune Response Abdelkrim Alileche¤ , Evan R. Serfass, Stefan M. Muehlbauer, Steven A. Porcelli, Ju, United States of America Many pathogens have acquired strategies to combat the immune response. Bacillus

Brojatsch, Jürgen

188

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

E-print Network

IL-10 from Regulatory T Cells Determines Vaccine Efficacy in Murine Leishmania major Infection1 in balance from IL-4 to IFN- was the key to vaccine success. Recently, a role for IL-10 and regulatory of the receptor for activated C kinase (LACK) or tryparedoxin peroxidase (TRYP). Both induced low IL-4 and high

Arnold, Jonathan

189

In vitro uptake of gelatin nanoparticles by murine dendritic cells and their intracellular localisation  

Microsoft Academic Search

The long term goal of this study is to develop an efficient nanoscopic vaccine delivery system, based on the biodegradable and natural polymer gelatin, to deliver therapeutic protein antigens along with adjuvants into dendritic cells (DCs). In this study, gelatin nanoparticles were tested for qualitative and quantitative uptake in murine DCs in vitro. A second aim of this study was

Conrad Coester; Paras Nayyar; John Samuel

2006-01-01

190

Immobilization of leukemia inhibitory factor (LIF) to culture murine embryonic stem cells  

Microsoft Academic Search

Murine embryonic stem (ES) cells were cultured on a material containing immobilized leukemia inhibitory factor (LIF). To immobilize LIF, we synthesized photoreactive gelatin mixed with LIF and cast the mixture on a polystyrene plate, which was then dried. LIF was immobilized by photoirradiation in the presence or absence of a photo mask. The plate was washed until LIF was no

Hiroshi Makino; Hirokazu Hasuda; Yoshihiro Ito

2004-01-01

191

Cytotoxic activity of methanol extracts from Basidiomycete mushrooms on murine cancer cell lines.  

PubMed

Crude methanol extracts of 58 mushroom species were screened for their cytotoxic activities against two murine cancer cell lines, L1210 and 3LL, using the tetrazolium assay. A majority of extracts (74%) exhibited IC50 > 100 microg/ml against both cell lines. A most marked activity against one of the cell lines was noted for nine species (14% of the tested species). While Amanitales and Russulales tested were not found active, Polyporales and Boletales gave better results. Four species exhibited a significant cytotoxic activity (IC50 < or = 20 microg/ml) against at least one of the two murine cancer cell lines (Ganoderma lucidum, Meripilus giganteus, Suillus granulatus, S. luteus). The last one had never been investigated for its cytotoxic compounds before. PMID:15125575

Tomasi, S; Lohézic-Le Dévéhat, F; Sauleau, P; Bézivin, C; Boustie, J

2004-04-01

192

Effects of lymphokines and immune complexes on murine placental cell growth in vitro  

SciTech Connect

Isolated murine placental cells obtained at Day 16 of allogeneic gestation (C3H x DBA/2J) were cultured for 3 days alone or in coculture with irradiated mouse splenocytes at the end of which 3H-thymidine was added for an additional 18-h culture to assess cell proliferation. Placental cell proliferation was significantly enhanced at spleen cell:placental cell ratios of 10:1 and 25:1 above that observed in the absence of added spleen cells. The stimulatory effect of irradiated allogeneic (C3H plus Balb/cJ) spleen cell cultures was significantly greater (approximately 2-fold) than that of isogeneic spleen cells (C3H alone). Conditioned medium from murine spleen cells cultured with concanavalin A (ConA) to induce lymphokine production had dose-dependent inhibitory effects on proliferation when added to placental cell cultures over a range of concentrations from 10 to 40% (vol:vol). Addition of pseudo immune complexes in the form of heat-aggregated human gamma globulin (AHGG) to culture medium failed to alter placental cell proliferation over a range of concentrations from 2 to 200 micrograms/ml either in the absence or presence of ConA-conditioned medium. In contrast to late-gestational stage placental cells, cell suspensions obtained from Days 8-9 murine ectoplacental cone (EPC) outgrowths, or from earlier stage placentas (Days 12-14) responded to low concentrations of conditioned medium from ConA-stimulated splenocytes with increased proliferation. The effect was less impressive on placental cells at gestational ages later than 12 days than on earlier stage preparations. On all placental cell suspensions tested, as well as EPC cells, a clear-cut inhibition of growth was observed at high doses of conditioned medium.

Armstrong, D.T.; Chaouat, G. (Clinique Universitaire Baudelocque, Paris (France))

1989-03-01

193

Cell density-dependent regulation of mdr-1 gene expression in murine colon cancer cells.  

PubMed

We studied the regulation of mdr-1 and P-glycoprotein in sparse and confluent cultures of murine CT-26 colon carcinoma cells. The expression level of mdr-1 mRNA transcripts (analyzed by Northern blot and in situ hybridization) and P-glycoprotein (analyzed by flow cytometry) inversely correlated with cell density. The modulation of mdr gene expression in sparse and confluent cells was not related to cell division, nutrient depletion, inhibition of protein synthesis, gap junction status, extracellular ATP, or the presence of various extracellular matrixes, but may be related to cell-density and cell-contact mediated changes in phosphatase activity. The confluence-mediated downmodulation of mdr-1 increased the chemosensitivity of the cells to several anticancer drugs commonly associated with an in vitro MDR phenotype by increasing the intracellular accumulation of the drugs. These data may explain some of the discrepancies in results obtained when analyzing mdr gene expression in tumors growing in vivo or in vitro, and why mdi expression in tumors is localized to the periphery of the lesions. PMID:21541589

Fan, D; Beltran, P; Wang, Y; Bucana, C; Yoon, S; Deguzman, A; Fidler, I

1996-11-01

194

DNA Methylation Levels in Human and Murine Melanoma Cell Lines of Varying Metastatic Potential  

Microsoft Academic Search

DNA methylation levels were measured in a seríesof murine and human melanoma cell lines consisting of matched variants of low and high experimental metastatic capacity. The percentage of cytosine resi dues modified to 5-methylcytosine ranged between 2.13-3.92% in these lines. Ten cell lines were established in culture from individual lung tumor nodules produced in nude mice by i.v. injection of

E. Jane Ormerod; Christine A. Everett; Mavis Finch; Ian R. Hart

1986-01-01

195

Effect of propolis extract on malignant cell transformation by moloney murine sarcoma virus  

Microsoft Academic Search

Summary.  ?An aqueous extract of propolis was found to significantly inhibit NIH\\/3T3 cell malignant transformation by Moloney murine\\u000a sarcoma virus (MuSV-124). The inhibitory effect of propolis extract was most effective when it was added 2?h before infection\\u000a or at the time of infection. The continuous presence of propolis extract in the culture medium was essential for full prevention\\u000a of malignant cell

M. Huleihel; V. Ishano

2001-01-01

196

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

NASA Technical Reports Server (NTRS)

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.

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

1986-01-01

197

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

PubMed

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

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

198

Stimulation of cell proliferation in the subventricular zone by synthetic murine pheromones  

PubMed Central

Adult neurogenesis in female mice is known to be enhanced by exposure to soiled bedding from males, although the identity of the relevant chemosignals has remained unknown. Here we show that the previously recognized male murine pheromones, the farnesenes and 2-sec-butyl-4,5-dihydrothiazole (SBT), strongly increase cell proliferation in the subventricular zone (SVZ) of adult female mice, but not younger female mice. In addition, we found that a unique female murine pheromone, 2,5-dimethylpyrazine, facilitates similar changes in males. SBT stimulated cell proliferation in the SVZ of only adult females and not in young adult or pre- and post-puberty females. Our study suggests that pheromonal communication between males and females is enhancing reproductive success by controlling the estrous cycle and by promoting cell proliferation in a reciprocal manner. PMID:23964214

Koyama, Sachiko; Soini, Helena A.; Foley, John; Novotny, Milos V.; Lai, Cary

2013-01-01

199

In vivo murine and in vitro M-like cell models of gastrointestinal anthrax.  

PubMed

Bacillus anthracis is the causative agent of anthrax and is acquired by three routes of infection: inhalational, gastrointestinal and cutaneous. Gastrointestinal (GI) anthrax is rare, but can rapidly result in severe, systemic disease that is fatal in 25%-60% of cases. Disease mechanisms of GI anthrax remain unclear due to limited numbers of clinical cases and the lack of experimental animal models. Here, we developed an in vivo murine model of GI anthrax where spore survival was maximized through the neutralization of stomach acid followed by an intragastric administration of a thiabendazole paste spore formulation. Infected mice showed a dose-dependent mortality rate and pathological features closely mimicking human GI anthrax. Since Peyer's patches in the murine intestine are the primary sites of B. anthracis growth, we developed a human M (microfold)-like-cell model using a Caco-2/Raji B-cell co-culturing system to study invasive mechanisms of GI anthrax across the intestinal epithelium. Translocation of B. anthracis spores was higher in M-like cells than Caco-2 monolayers, suggesting that M-like cells may serve as an initial entry site for spores. Here, we developed an in vivo murine model of GI anthrax and an in vitro M-like cell model that could be used to further our knowledge of GI anthrax pathogenesis. PMID:23108317

Tonry, Jessica H; Popov, Serguei G; Narayanan, Aarthi; Kashanchi, Fatah; Hakami, Ramin M; Carpenter, Calvin; Bailey, Charles; Chung, Myung-Chul

2013-01-01

200

Analysis of apoptosis in murine embryonic stem cells  

E-print Network

Currently, efforts to generate transgenic swine using embryonic germ (EG) cells are hampered by the loss of the cells to apostasis in vitro. Yet, due to complex culture conditions, it is difficult to study apostasis directly in these cells. A...

Weaks, Regina Lanell

2012-06-07

201

FimH can directly activate human and murine natural killer cells via TLR4.  

PubMed

Although the importance of natural killer (NK) cells in innate immune responses against tumors or viral infections are well documented, their ability to directly recognize pathogens is less well defined. We have recently reported FimH, a bacterial fimbrial protein, as a novel Toll-like receptor (TLR)4 ligand that potently induces antiviral responses. Here, we investigated whether FimH either directly or indirectly can activate human and murine NK cells. We demonstrate that FimH potently activates both human and murine NK cells in vitro to induce cytokines [interferon (IFN)-gamma and tumor necrosis factor (TNF)-alpha] and cytotoxicity. Importantly, NK cells directly recognize FimH-expressing pathogens as FimH(+), but not FimH(-), bacteria were able to activate human NK cells. FimH activation of NK cells required TLR4 and MyD88 signaling, as NK cells from both TLR4(-/-) and MyD88(-/-) mice as well as human NK-92 cells, which lack TLR4, were all unresponsive to FimH. In addition, TLR4 neutralization significantly abrogated the response of human NK cells to FimH. Activation of purified NK cells by FimH was independent of lipopolysaccharide (LPS) or other bacterial contaminations. These data demonstrate for the first time that highly purified NK cells directly recognize and respond to FimH via TLR4-MyD88 pathways to aid innate protection against cancer or microbial infections. PMID:20442710

Mian, M Firoz; Lauzon, Nicole M; Andrews, David W; Lichty, Brian D; Ashkar, Ali A

2010-07-01

202

Stem-cell-based Tissue Engineering of Murine Teeth  

Microsoft Academic Search

Teeth develop from reciprocal interactions between mesenchyme cells and epithelium, where the epithelium provides the instructive information for initiation. Based on these initial tissue interactions, we have replaced the mesenchyme cells with mesenchyme created by aggregation of cultured non-dental stem cells in mice. Recombinations between non-dental cell-derived mesenchyme and embryonic oral epithelium stimulate an odontogenic response in the stem cells.

A. Ohazama; S. A. C. Modino; I. Miletich; P. T. Sharpe

2004-01-01

203

Doxorubicin treatment induces tumor cell death followed by immunomodulation in a murine neuroblastoma model  

PubMed Central

Chemotherapy of malignant tumors induces tumor cell death. Numerous antitumor agents induce apoptosis of tumor cells, which are subsequently engulfed by phagocytes, initiating an immune reaction. The induction of immunogenic cell death by antitumor agents may be advantageous for antitumor immunity. The purpose of this study was to determine whether doxorubicin is capable of inducing an immunogenic reaction in murine neuroblastoma cells. The murine neuroblastoma cell line (neuro-2a cells) was cultured in a medium containing doxorubicin or cisplatin (CDDP), and induction of cell death was confirmed by cell viability assays. Cluster of differentiation (CD)8?+ lymphocytes were co-cultured with neuro-2a cells that had died following treatment with either doxorubicin or CDDP, and CD11b+ spleen cells or bone marrow-derived dendritic cells (BM-DCs) were added to the culture. Proliferation of CD8?+ lymphocytes and interferon (IFN)-? production were evaluated. When CD8?+ cells were co-cultured with doxorubicin-treated neuro-2a cells and BM-DCs, CD8?+ cells reacted to anti-CD3/CD28 antibody stimulation, proliferated and increased IFN-? production. IFN-? production was more effectively promoted by co-culture with doxorubicin-treated neuro-2a cells than by co-culture with CDDP-treated neuro-2a cells. These findings suggest that doxorubicin is capable of inducing immunogenic cell death in neuroblastoma cells, and thus has an immunological advantage for chemotherapy of neuroblastoma compared with CDDP. BM-DCs are considered to be the key antigen-presenting cells in the immune reaction following the induction of immunogenic neuroblastoma cell death and phagocytosis. PMID:24520271

INOUE, SEIICHIRO; SETOYAMA, YUMIKO; ODAKA, AKIO

2014-01-01

204

Conserved signaling through vascular endothelial growth (VEGF) receptor family members in murine lymphatic endothelial cells.  

PubMed

Lymphatic vessels guide interstitial fluid, modulate immune responses by regulating leukocyte and antigen trafficking to lymph nodes, and in a cancer setting enable tumor cells to track to regional lymph nodes. The aim of the study was to determine whether primary murine lymphatic endothelial cells (mLECs) show conserved vascular endothelial growth factor (VEGF) signaling pathways with human LECs (hLECs). LECs were successfully isolated from murine dermis and prostate. Similar to hLECs, vascular endothelial growth factor (VEGF) family ligands activated MAPK and pAkt intracellular signaling pathways in mLECs. We describe a robust protocol for isolation of mLECs which, by harnessing the power of transgenic and knockout mouse models, will be a useful tool to study how LEC phenotype contributes to alterations in lymphatic vessel formation and function. PMID:21839079

Coso, Sanja; Zeng, Yiping; Sooraj, Dhanya; Williams, Elizabeth D

2011-10-15

205

Sorting and Expansion of Murine Embryonic Stem Cell Colonies using Micropallet Arrays  

PubMed Central

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

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

2008-01-01

206

Purification and immunophenotypic characterization of murine B10 B cells.  

PubMed

Regulatory B10 cells represent a rare CD1d(hi)CD5(+) IL-10-secreting B cell subset in mice which is induced to produce IL-10 after 5 h of in vitro stimulation with a combination of B cell mitogen and chemical stimulants. Although B10 cells only constitute 1-2 % of splenic CD19(+) B cells, they play important roles in controlling T cell-mediated immune responses in an antigen-specific and IL-10-dependent manner. The regulatory effects of B10 cells have been demonstrated in multiple mouse models of inflammatory and autoimmune diseases. Herein, we described current methods for identification and purification of CD1d(hi)CD5(+) B10 cells. PMID:25015271

Hong, Chao; Gao, Xiao-Ming

2014-01-01

207

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

Microsoft Academic Search

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

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

2002-01-01

208

Influence of a stationary magnetic field on acetylcholinesterase in murine bone marrow cells  

Microsoft Academic Search

A thirty-minute exposure of mice to a homogeneous stationary magnetic field (SMF) of 1.4 Tesla at either 27° C or 37° C body temperature causes an inhibition of about 20 percent of acetylcholinesterase (AChE, E.C. 3.11.7) in murine bone marrow cells (BMC) after 3.5 and 2 h, respectively, at the two aforementioned body temperatures. The extent of enzyme inhibition is

S. Stegemann; K. I. Altman; H. Mühlensiepen; L. E. Feinendegent

1993-01-01

209

Enhancement of Porcine Natural Killer Cell Activity by Recombinant Human and Murine IL12  

Microsoft Academic Search

These studies describe the effects of recombinant human (rHu) and recombinant murine (rMu) interleukin 12 (IL-12) in the porcine system. To examine the effects of rHu and rMu IL-12, porcine PBL were cultured with rHu and rMu IL-12, and then used for a NK assay. Significant enhancement of porcine NK cell cytotoxicity was observed, and maximal enhancement was reached after

Daeho Cho; Wang J. Lee; Patrick J. Halloran; Giorgio Trinchieri; Yoon B. Kim

1996-01-01

210

Leukotriene C: A Slow-Reacting Substance from Murine Mastocytoma Cells  

Microsoft Academic Search

Murine mastocytoma cells treated with calcium ionophore A23187 produced a slow-reacting substance (SRS) that caused guinea pig ileum to contract. The response was reversed by the SRS antagonist FPL 55712. On the basis of isotope incorporation experiments, spectroscopy, and chemical degradations, the SRS was identified as a cysteine-containing derivative of 5-hydroxy-7,9,11,14-icosatetraenoic acid. This amino acid was attached in thioether linkage

Robert C. Murphy; Sven Hammarstrom; Bengt Samuelsson

1979-01-01

211

Purification of murine endothelial cell cultures by flow cytometry using fluorescein-labeled griffonia simplicifolia agglutinin.  

PubMed Central

Griffonia simplicifolia agglutinin (GSA) is a valuable histochemical tool in the identification of endothelium. In this study GSA labeled with fluorescein isothiocyanate (GSA-FITC) was used to purify cultures of murine cerebral microvascular endothelium. Cultures were stained with GSA-FITC, then sorted using a fluorescence-activated cell sorter (FACS). GSA-positive endothelial cells were collected, re-cultured, and subsequently re-analyzed by FACS using GSA-FITC. Cultures that initially contained 80 +/- 3 to 89 +/- 3% (X +/- SE) GSA-positive cells were purified to 98 +/- 1% positivity. Immunohistochemistry with an anti-muscle-action antibody confirmed that FACS sorting of GSA-FITC-stained cells effectively removed contaminating smooth muscle cells from endothelial cell cultures. Viability, proliferation, and prostaglandin production of the cells was unaltered by lectin staining and FACS sorting. Thus, GSA-FITC can be used in conjunction with flow cytometry to enhance the purity of murine endothelial cell cultures without adversely affecting cell viability, growth, or metabolism. Images Figure 2 PMID:2757116

Sahagun, G.; Moore, S. A.; Fabry, Z.; Schelper, R. L.; Hart, M. N.

1989-01-01

212

Cytoplasmic superoxide dismutase and catalase activity and resistance to radiation lethality in murine tumor cells  

SciTech Connect

Reduced species of molecular oxygen are produced by the interaction of ionizing radiation with aqueous solutions containing molecular oxygen. The enzymes catalase and superoxide dismutase (SOD) are thought to function in vivo as scavengers of metabolically produced peroxide and superoxide respectively. SOD has been shown to protect against the lethal effects of ionizing radiation in vitro and in vivo. The authors have investigated the relationship between the cytosolic SOD catalase content and the sensitivity to radiation lethality of a number of murine cell lines (402AX, EL-4, MB-2T3, MB-4, MEL, P-815, SAI, SP-2, and SV-3T3). K/sub i/(CN/sup -/) for murine Cu-Zn-SOD was determined to be 6.8 x 10/sup -6/ M. No cytosolic Mn-SOD activity was found in any of the cell lines studied. No correlation was found between the cytosolic Cu-Zn-SOD or cytosolic catalase activity and the resistance to radiation lethality or the murine cell lines studied.

Davy, C.A.; Tesfay, Z.; Jones, J.; Rosenberg, R.C.; McCarthy, C.; Rosenberg, S.O.

1986-05-01

213

Inducible expression of endomorphins in murine dendritic cells?  

PubMed Central

Bone marrow precursor cells were extracted from C57BL/6J mice aged 7–8 weeks, and dendritic cells were purified using anti-CD11c (a specific marker for dendritic cells) antibody-coated magnetic beads. Immunofluorescence staining revealed that the expression levels of endomorphin-1 and endomorphin-2 were upregulated in dendritic cells activated by lipopolysaccharide. An enzyme immunoassay showed that lipopolysaccharide and other Toll-like receptor ligands promoted the secretion of endomorphin-1 and endomorphin-2 from activated dendritic cells. [3H]-thymidine incorporation demonstrated that endomorphin-1 and endomorphin-2 both inhibited the proliferation of T lymphocyte induced by activated dendritic cells. Furthermore, this immunosuppressive effect was blocked by CTOP, a specific antagonist of µ-opioid receptors. Our experimental findings indicate that activated dendritic cells can induce the expression and secretion of endomorphins, and that endomorphins suppress T lymphocyte proliferation through activation of µ-opioid receptors.

Yang, Xiaohuai; Xia, Hui; Chen, Yong; Liu, Xiaofen; Zhou, Cheng; Gao, Qin; Li, Zhenghong

2012-01-01

214

Testicular Somatic Cells, not Gonocytes, Are the Major Source of Functional Activin A during Testis Morphogenesis  

PubMed Central

Proper development of the seminiferous tubules (or testis cords in embryos) is critical for male fertility. Sertoli cells, somatic components of the seminiferous tubules, serve as nurse cells to the male germline, and thus their numbers decide the quantity of sperm output in adulthood. We previously identified activin A, the protein product of the activin ?A (Inhba) gene, as a key regulator of murine Sertoli cell proliferation and testis cord expansion during embryogenesis. Although our genetic studies implicated fetal Leydig cells as the primary producers of testicular activin A, gonocytes are another potential source. To investigate the relative contribution of gonocyte-derived activin A to testis morphogenesis, we compared testis development in the Inhba global knockout mouse, which lacks activin A production in all cells (including the gonocytes), and a steroidogenic factor 1 (Sf1)-specific conditional knockout model in which activin A expression in testicular somatic cells is disrupted but gonocyte expression of activin A remains intact. Surprisingly, testis development was comparable in these two models of activin A insufficiency, with similar reductions in Sertoli cell proliferation and minor differences in testis histology. Thus, our findings suggest activin A from male gonocytes is insufficient to promote Sertoli cell proliferation and testis cord expansion in the absence of somatic cell-derived activin A. Evaluation of adult male mice with fetal disruption of activin A revealed reduced testis size, lowered sperm production, altered testicular histology, and elevated plasma FSH levels, defects reminiscent of human cases of androgen-sufficient idiopathic oligozoospermia. PMID:21952240

Archambeault, Denise R.; Tomaszewski, Jessica; Childs, Andrew J.; Anderson, Richard A.

2011-01-01

215

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

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.

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

216

Aryl hydrocarbon receptor controls murine mast cell homeostasis  

PubMed Central

We propose that the aryl hydrocarbon receptor (AhR), a unique chemical sensor, is critical in controlling mast cell differentiation, growth, and function in vitro and in vivo. In antigen-stimulated mast cells, exposure to AhR ligands resulted in a calcium- and reactive oxygen species (ROS)-dependent increase of reversible oxidation in and reduced activity of SHP-2 phosphatase, leading to enhanced mast cell signaling, degranulation, and mediator and cytokine release, as well as the in vivo anaphylactic response. Surprisingly, significant mast cell deficiency was noted in AhR-null mice due to defective calcium signaling and mitochondrial function, concomitant with reduced expression of c-kit and cytosolic STAT proteins, as well as enhanced intracellular ROS and apoptosis. Consequently, AhR-null mast cells responded poorly to stimulation, demonstrating a critical role of AhR signaling in maintaining mast cell homeostasis. PMID:23462117

Zhou, Yufeng; Tung, Hui-Ying; Tsai, Ying-Ming; Hsu, Shih-Chang; Chang, Hui-Wen; Kawasaki, Hirokazu; Tseng, Hsiao-Chun; Plunkett, Beverly; Gao, Peisong; Hung, Chih-Hsing; Vonakis, Becky M.

2013-01-01

217

Aryl hydrocarbon receptor controls murine mast cell homeostasis.  

PubMed

We propose that the aryl hydrocarbon receptor (AhR), a unique chemical sensor, is critical in controlling mast cell differentiation, growth, and function in vitro and in vivo. In antigen-stimulated mast cells, exposure to AhR ligands resulted in a calcium- and reactive oxygen species (ROS)-dependent increase of reversible oxidation in and reduced activity of SHP-2 phosphatase, leading to enhanced mast cell signaling, degranulation, and mediator and cytokine release, as well as the in vivo anaphylactic response. Surprisingly, significant mast cell deficiency was noted in AhR-null mice due to defective calcium signaling and mitochondrial function, concomitant with reduced expression of c-kit and cytosolic STAT proteins, as well as enhanced intracellular ROS and apoptosis. Consequently, AhR-null mast cells responded poorly to stimulation, demonstrating a critical role of AhR signaling in maintaining mast cell homeostasis. PMID:23462117

Zhou, Yufeng; Tung, Hui-Ying; Tsai, Ying-Ming; Hsu, Shih-Chang; Chang, Hui-Wen; Kawasaki, Hirokazu; Tseng, Hsiao-Chun; Plunkett, Beverly; Gao, Peisong; Hung, Chih-Hsing; Vonakis, Becky M; Huang, Shau-Ku

2013-04-18

218

Cholinergic epithelial cell with chemosensory traits in murine thymic medulla.  

PubMed

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

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

219

Regulation of Glycan Structures in Murine Embryonic Stem Cells  

PubMed Central

The abundance and structural diversity of glycans on glycoproteins and glycolipids are highly regulated and play important roles during vertebrate development. Because of the challenges associated with studying glycan regulation in vertebrate embryos, we have chosen to study mouse embryonic stem (ES) cells as they differentiate into embryoid bodies (EBs) or into extraembryonic endodermal (ExE) cells as a model for cellular differentiation. We profiled N- and O-glycan structures isolated from these cell populations and examined transcripts encoding the corresponding enzymatic machinery for glycan biosynthesis in an effort to probe the mechanisms that drive the regulation of glycan diversity. During differentiation from mouse ES cells to either EBs or ExE cells, general trends were detected. The predominance of high mannose N-glycans in ES cells shifted to an equal abundance of complex and high mannose structures, increased sialylation, and increased ?-Gal termination in the differentiated cell populations. Whereas core 1 O-glycan structures predominated in all three cell populations, increased sialylation and increased core diversity characterized the O-glycans of both differentiated cell types. Increased polysialylation was also found in both differentiated cell types. Differences between the two differentiated cell types included greater sialylation of N-glycans in EBs, whereas ?-Gal-capped structures were more prevalent in ExE cells. Changes in glycan structures generally, but not uniformly, correlated with alterations in transcript abundance for the corresponding biosynthetic enzymes, suggesting that transcriptional regulation contributes significantly to the regulation of glycan expression. Knowledge of glycan structural diversity and transcript regulation should provide greater understanding of the roles of protein glycosylation in vertebrate development. PMID:22988249

Nairn, Alison V.; Aoki, Kazuhiro; dela Rosa, Mitche; Porterfield, Mindy; Lim, Jae-Min; Kulik, Michael; Pierce, J. Michael; Wells, Lance; Dalton, Stephen; Tiemeyer, Michael; Moremen, Kelley W.

2012-01-01

220

Isolation and Characterization of Murine Mandibular Condylar Cartilage Cell Populations  

PubMed Central

Objectives The mandibular condylar cartilage is a heterogeneous tissue containing cells at various stages of chondrocyte maturation organized into 4 zones: superficial, polymorphic, flattened, and hypertrophic. The goal of this study was to use transgenic mice containing chondrocyte maturation markers fused to fluorescent protein transgenes to isolate and characterize homogenous cell populations of the mandibular condylar cartilage. Methods Fluorescent reporter expression in the mandibular condylar cartilage of transgenic mice containing the 3.6-kb fragment of the rat collagen type 1 promoter fused to a topaz-fluorescent protein (Col3.6-tpz), collagen type 2 promoter fused to a cyan-fluorescent protein (Col2-cyan), and/or collagen type 10 promoter fused to cherry-fluorescent protein (Col10-cherry) was examined. Mandibular condylar cartilage cells were analyzed by fluorescence-activated cell sorting (FACS) and either used for gene expression analysis or plated in cell cultures and exposed to adipogenic, osteogenic, or chondrogenic conditions. To determine cell fate, transgenic mice containing the Col3.6-cre recombinase were bred with cre reporter mice. Results Localization and analysis of gene expression revealed that Col3.6-tpz-positive cells corresponded to the polymorphic/flattened zones and Col2-cyan-positive cells corresponded to the flattened/hypertrophic zones of the mandibular condylar cartilage. Mandibular condylar cartilage FACS-sorted Col3.6-tpz-positive cells have the potential to differentiate into bone, cartilage, and fat. Cell fate mapping revealed that Col3.6 cells are precursors of some of the hypertrophic chondrocytes in the mandibular condylar cartilage. Conclusion Col3.6-tpz cells represent an earlier stage of the mandibular condylar cartilage maturation pathway. PMID:21646777

Chen, J.; Utreja, A.; Kalajzic, Z.; Sobue, T.; Rowe, D.; Wadhwa, S.

2012-01-01

221

Identification and in vivo analysis of murine hematopoietic stem cells.  

PubMed

Hematopoietic stem cells (HSCs) can self-renew and give rise to all the cells of the blood and the immune system. As they differentiate, HSCs progressively lose their self-renewal capacity and generate lineage-restricted multipotential progenitor cells that in turn give rise to mature cells. The development of rigorous quantitative in vivo assays for HSC activity combined with multicolor flow cytometry and high-speed sorting have resulted in the phenotypic definition of HSCs to virtual purity. Here, we describe the isolation and identification of HSCs by flow cytometry and the use of competitive repopulation to assess HSC number and function. PMID:20691879

Avagyan, Serine; Amrani, Yacine M; Snoeck, Hans-Willem

2010-01-01

222

Regulation of murine hematopoietic stem cell quiescence by Dmtf1  

PubMed Central

The cell-cycle status of hematopoietic stem cells (HSCs) is tightly regulated, most likely to balance maintenance of stem-cell status through quiescence and expansion/differentiation of the hematopoietic system. Tumor-suppressor genes (TSGs), with their cell cycle–regulatory functions, play important roles in HSC regulation. The cyclin-D binding myb-like transcription factor 1 (Dmtf1) was recently recognized as a TSG involved in human cancers by repressing oncogenic Ras/Raf signaling. However, the role of Dmtf1 in the hematopoietic system is entirely unknown. In the present study, we demonstrate that Dmtf1 regulates HSC function under both steady-state and stress conditions. Dmtf1?/? mice showed increased blood cell counts in multiple parameters, and their progenitor cells had increased proliferation and accelerated cell-cycle progression. In addition, long-term HSCs from Dmtf1?/? mice had a higher self-renewal capacity that was clearly demonstrated in secondary recipients in serial transplantation studies. Dmtf1?/? BM cells showed hyper proliferation after 5-fluorouracil–induced myeloablation. Steady-state expression and Induction of CDKN1a (p21) and Arf were impaired in HSCs from Dmtf1?/? mice. The function of Dmtf1 was mediated by both Arf-dependent and Arf-independent pathways. Our results implicate Dmtf1 in the regulation of HSC function through novel cell cycle–regulatory mechanisms. PMID:22039255

Kobayashi, Michihiro

2011-01-01

223

The effects of simulated hypogravity on murine bone marrow cells  

NASA Technical Reports Server (NTRS)

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.

Lawless, Desales

1989-01-01

224

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...

225

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...

226

Glycomics of Proteoglycan Biosynthesis in Murine Embryonic Stem Cell Differentiation  

Microsoft Academic Search

Glycosaminoglycans (GAGs) play a critical role in binding and activation of growth factors involved in cell signaling critical for developmental biology. The biosynthetic pathways for GAGs have been elucidated over the past decade and now analytical methodology makes it possible to determine GAG composition in as few as 10 million cells. A glycomics approach was used to examine GAG content,

Alison V. Nairn; Akiko Kinoshita-Toyoda; Hidenao Toyoda; Jin Xie; Kyle Harris; Stephen Dalton; Michael Kulik; J. Michael Pierce; Toshihiko Toida; Kelley W. Moremen; Robert J. Linhardt

2007-01-01

227

Abscisic acid does not evoke calcium influx in murine primary microglia and immortalised murine microglial BV-2 and N9 cells.  

PubMed

Brain microglia are resident macrophage-like cells representing the first and main form of active immune response during brain injury. Microglia-mediated inflammatory events in the brain are known to be associated with chronic degenerative diseases such as Multiple Sclerosis, Parkinson's, or Alzheimer's disease. Therefore, identification of mechanisms activating microglia is not only important in the understanding of microglia-mediated brain pathologies, but may also lead to the development of new anti-inflammatory drugs for the treatment of chronic neurodegenerative diseases. Recently, abscisic acid (ABA), a phytohormone regulating important physiological functions in higher plants, has been proposed to activate murine microglial cell line N9 through increased intracellular calcium. In the present study, we determined the response to ABA and its analogues from murine primary microglia and immortalized murine microglial cell line BV-2 and N9 cells. A Fura-2-acetoxymethyl ester (Fura-2AM)-based ratiometric calcium imaging and measurement technique was used to determine the intracellular calcium changes in these cells when treated with (-)-ABA, (+)-ABA, (-)-trans-ABA and (+)-trans-ABA. Both primary microglia and microglial cell lines (BV-2 and N9 cells) showed significant increase in intracellular calcium ([Ca(2+)]i) in response to treatment with ATP and ionomycine. However, ABAs failed to evoke dose- and time-dependent [Ca(2+)]i changes in mouse primary microglia, BV-2 and N9 cells. Together, these surprising findings demonstrate that, contrary to that reported in N9 cells [3], ABAs do not evoke intracellular calcium changes in primary microglia and microglial cell lines. The broad conclusion that ABA evokes [Ca(2+)]i in microglia requires more evidence and further careful examination. PMID:20869945

Jiang, Susan X; Benson, Chantel L; Zaharia, L Irina; Abrams, Suzanne R; Hou, Sheng T

2010-10-22

228

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

PubMed Central

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

1981-01-01

229

Effect of verapamil on cell cycle transit and c-myc gene expression in normal and malignant murine cells.  

PubMed Central

Verapamil, the prototype calcium channel blocker, reversibly inhibits cell proliferation in many normal and tumour cell lines (Schmidt et al., Cancer Res., 48, 3617, 1988). We have found that two closely related cell lines - B16 murine melanoma cells and B10.BR normal murine melanocytes growing in culture - behave differently in the presence of verapamil, and we are now utilising these two related cell lines to help elucidate the molecular basis of verapamil's antiproliferative effect. In this study, we studied cell cycle phase distribution and c-myc gene expression in both cell lines in the absence of verapamil, during incubation with verapamil and after the cells were washed free of verapamil. Our studies show that 100 microM verapamil rapidly blocks DNA synthesis in melanocytes but not in B16 cells. Similarly, incubation with verapamil for 6-24 h results in a decreased c-myc signal in melanocytes, but a transient increase in c-myc expression in B16 cells. After verapamil is washed from the cells following a 24-h incubation with drug, c-myc expression increases in melanocytes as they begin again to proliferate, but decreases in B16 cells as they begin to die. Our disparate results with these cell lines suggest that c-myc gene expression, regardless of its known involvement in growth control, is not the immediate target for verapamil's inhibitory action. Images Figure 1 Figure 2 Figure 4 PMID:2736205

Huber, K. R.; Schmidt, W. F.; Thompson, E. A.; Forsthoefel, A. M.; Neuberg, R. W.; Ettinger, R. S.

1989-01-01

230

Murine mast cells secrete and respond to interleukin-33.  

PubMed

Interleukin-33 (IL-33) appears to play a crucial role in the expression of allergic diseases, but its cellular source and regulatory mechanisms remain to be fully elucidated. Mast cells, one of the major effecter cell populations in mediating allergy, express high levels of IL-33 receptor, ST2, and have been shown to express IL-33 transcripts. In this study, we aimed to examine the secretion of IL-33 in mast cells and their response to IL-33. We have successfully detected secreted IL-33 from cell supernatants through a modified enzyme-linked immunosorbent assay (ELISA) technique-cell-based ELISA. Activation of bone marrow-derived cultured mast cells (BMMCs) by crosslinkage of an antigen [ovalbumin (OVA)] and OVA-specific IgE mAbs significantly induced the expression of IL-33 transcripts, cytosolic and secreted proteins. In addition, the Toll-like receptor (TLR) 2 and TLR-9 ligands could trigger IL-33 mRNA expression. Exposure of BMMCs to IL-33 significantly increased the levels of IL-13 and IL-6 expression, concomitant with enhanced activation of mitogen-activated protein kinase (MAPKs) (ERK, p38, and JNK) and nuclear factor-kappa B. These results suggest that mouse BMMCs are capable of producing and serving as endogenous sources of IL-33, and that IL-33 plays an important role in regulating mast cell functions. PMID:24028396

Tung, Hui-Ying; Plunkett, Beverly; Huang, Shau-Ku; Zhou, Yufeng

2014-03-01

231

Investigation of xenotropic murine leukemia virus-related virus (XMRV) in human and other cell lines.  

PubMed

Xenotropic murine leukemia virus-related virus (XMRV) was discovered in human prostate tumors and later in some chronic fatigue syndrome (CFS) patients. However, subsequent studies have identified various sources of potential contamination with XMRV and other murine leukemia virus (MLV)-related sequences in test samples. Biological and nucleotide sequence analysis indicates that XMRV is distinct from known xenotropic MLVs and has a broad host range and cell tropism including human cells. Therefore, it is prudent to minimize the risk of human exposure to infection by evaluating XMRV contamination in cell lines handled in laboratory research and particularly those used in the manufacture of biological products. Nested DNA PCR assays were optimized for investigating XMRV gag and env sequences in various cell lines, which included MRC-5, Vero, HEK-293, MDCK, HeLa, and A549, that may be used in the development of some vaccines and other cell lines broadly used in research. The sensitivity of the DNA PCR assays was <10 copies in approximately 1.8 x 10(5) cells equivalent of human DNA. The results indicated the absence of XMRV in the cell lines tested; although in some cases DNA fragments identified as cellular sequences were seen following the first round of PCR amplification with the env primer pair. PMID:21996050

Williams, Dhanya K; Galvin, Teresa A; Ma, Hailun; Khan, Arifa S

2011-11-01

232

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

SciTech Connect

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.

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

2009-01-01

233

Polycomb complexes repress developmental regulators in murine embryonic stem cells  

Microsoft Academic Search

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

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

234

Identification of Multipotent Stem/Progenitor Cells in Murine Sclera  

PubMed Central

Purpose. The sclera forms the fibrous outer coat of the eyeball and acts as a supportive framework. The purpose of this study was to examine whether the sclera contains mesenchymal stem/progenitor cells. Method. Scleral tissue from C57BL6/J mice was separated from the retina and choroid and subsequently enzyme digested to release single cells. Proliferation capacity, self-renewal capacity, and ability for multipotent differentiation were analyzed by BrdU labeling, flow cytometry, reverse transcriptase–polymerase chain reaction, immunocytochemistry, and in vivo transplantation. Results. The scleral stem/progenitor cells (SSPCs) possessed clonogenic and high doubling capacities. These cells were positive for the mesenchymal markers Sca-1, CD90.2, CD44, CD105, and CD73 and negative for the hematopoietic markers CD45, CD11b, Flk1, CD34, and CD117. In addition to expressing stem cell genes ABCG2, Six2, Notch1, and Pax6, SSPCs were able to differentiate to adipogenic, chondrogenic, and neurogenic lineages. Conclusions. This study indicates that the sclera contains multipotent mesenchymal stem cells. Further study of SSPCs may help elucidate the cellular and molecular mechanism of scleral diseases such as scleritis and myopia. PMID:21788434

Tsai, Chia-Ling; Wu, Pei-Chang; Fini, M. Elizabeth; Shi, Songtao

2011-01-01

235

Neurogenic potential of dental pulp stem cells isolated from murine incisors  

PubMed Central

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

2014-01-01

236

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

PubMed

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

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

237

Antigen processing and presentation by a murine myoblast cell line.  

PubMed Central

The ability of non-professional antigen-presenting cells (APC) to process and present antigen to the immune system has been the subject of debate in autoimmunity and tumour immunology. The role of muscle cells in the processing and presentation of antigen to T cells via class I and class II MHC pathways is of increasing interest. Muscle cells are the targets of autoimmune attack in the inflammatory muscle diseases, and direct intramuscular injection of antigen-expressing DNA constructs is under scrutiny as a means of vaccination. Furthermore, the immunological properties of muscle cells are of relevance in attempts to transfer myoblasts as replacement cells in dystrophic diseases or as depot cells for the secretion of certain molecules in deficiency states. Using class I and class II MHC transfectant clones of the C2C12 myoblast cell line, myoblasts have been shown to be capable of presenting antigen to, and stimulating secretion of IL-2 by, T cell hybridomas via both of these pathways. The epitopes which are dominantly presented by professional APC after processing of native antigens were also presented by the myoblast cell line after processing of either ovalbumin (class I) or hen egg lysozyme (class II). Further, antigen processing and presentation via the class II pathway were enhanced by pretreatment of the myoblasts with interferon-gamma (IFN-gamma). Up-regulation of invariant chain expression by this treatment may have contributed to this enhanced presentation, but an effect of IFN-gamma on the expression of other molecules such as H-2 DM may have also played a role. The demonstration of the antigen-presenting properties of these myoblasts is of relevance to all three areas mentioned above. In each situation myoblasts comprise a significant population within muscle. In the case of inflammatory muscle diseases the process of muscle degeneration and regeneration is on-going, while in the vaccination procedure some muscle damage occurs, and vaccination is more effective when muscle damage has preceded inoculation. Images Fig. 5 PMID:8536381

Garlepp, M J; Chen, W; Tabarias, H; Baines, M; Brooks, A; McCluskey, J

1995-01-01

238

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

SciTech Connect

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.

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

239

Characterization of tumor cell lines derived from murine gammaherpesvirus-68-infected mice.  

PubMed Central

Cell lines were derived from mice with murine gammaherpesvirus-68 (MHV-68)-associated lymphoproliferative disease. Four were of an ambiguous phenotype and were MHV-68 negative. One, S11, was a B lymphocyte that contained MHV-68 genomes in both linear and episomal forms and released virus. The line was clonable and grew into tumors in nude mice. This is the first naturally occurring MHV-68-positive B-cell line to be generated, and it will be an invaluable tool for the study of MHV-68 latency. PMID:8709292

Usherwood, E J; Stewart, J P; Nash, A A

1996-01-01

240

Understanding the murine cutaneous dendritic cell network to improve intradermal vaccination strategies.  

PubMed

Dendritic cells (DCs) form a heterogeneous group of antigen presenting cells that play different roles in tissue immunity. Recent studies have revealed the presence of distinct DC populations in murine skin, highlighting the complexity of the cutaneous DC network. In this review, we will define the major DC subsets that populate the different layers of the skin, focusing on their origin and the mechanisms controlling their homeostasis. We will also review recent evidence underlining the functional specialization of dermal DC subsets and its relevance in the design of novel vaccine approaches. PMID:21058006

Ginhoux, F; Ng, L G; Merad, M

2012-01-01

241

Toll-like receptor 3 agonist enhances IFN-? and TNF-? production by murine uterine NK cells  

Microsoft Academic Search

To understand the response of murine uterine natural killer (uNK) cells to Toll-like receptor (TLR) 3 agonist at the early gestation stage, CBA×DBA\\/2 mice were intraperitoneally (i.p.) injected with polyinosinic–polycytidylic acid (poly I:C), the specific TLR3 agonist, at a dose of 10 ?g\\/g BW or PBS at gestation day (gd) 6.5. The CD69 expression of uNK (DX5+CD3?) cells was highly up-regulated

Jianhong Zhang; Rui Sun; Haiming Wei; Dongmei Wu; Zhigang Tian

2007-01-01

242

Immortalization of human endothelial cells by murine sarcoma viruses, without morphologic transformation.  

PubMed

Amphotropic murine leukemia virus pseudotypes of murine sarcoma viruses containing the ras or mos oncogenes were constructed to permit efficient introduction of the sarcoma virus genome into early-passage human umbilical vein endothelial cells. The resulting cell lines were morphologically and phenotypically unchanged, retaining properties characteristic of differentiated endothelial cells. For example, the cells in a Kirsten sarcoma virus-modified line were found to biosynthesize and secrete von Willebrand factor in both a constitutive and regulated manner, and they contained ultrastructurally identifiable Weibel-Palade bodies, an endothelial cell-specific organelle. In contrast to the parent cultures, sarcoma virus-modified cells were able to proliferate indefinitely in culture. Examination of both Kirsten sarcoma and Moloney leukemia virus-modified lines indicated that the immortalized cells retained a diploid female karyotype after over 18 months in culture. In addition, the sarcoma virus-modified cells were able to grow independently of added endothelial cell growth factor. This growth factor autonomy does not appear to be due to autocrine production of a biologically cross-reactive growth factor. These immortal, virus-modified endothelial cells express large amounts of sarcoma virus-specific mRNA but no detectable helper virus or transforming virus activity. This technique for immortalization of primary human cells without alteration of the differentiated characteristics of the cell type is readily applied to a variety of human cell types. Moreover, the ability to separate the immortalizing and transforming activities of viral oncogenes should provide further understanding as to mechanisms of oncogene action. PMID:2826502

Faller, D V; Kourembanas, S; Ginsberg, D; Hannan, R; Collins, T; Ewenstein, B M; Pober, J S; Tantravahi, R

1988-01-01

243

Antigen-Presenting Cell Population Dynamics during Murine Silicosis  

PubMed Central

Silicosis is an occupational lung disease resulting from the inhalation of silica particles over prolonged periods of time, which causes chronic inflammation and progressive pulmonary fibrosis. Alveolar macrophages (AM) are critical effector cells, while less is known about the role and function of pulmonary dendritic cells (DC) in silicosis. We hypothesize that a balance exists between the suppressive nature of AM and the stimulatory capacity of DC to regulate lung immunity, and that this equilibrium may be overcome by silica exposure in vivo. Our results demonstrate that in response to silica exposure, both the percent and absolute number of AM significantly decreased over time, with a concomitant significant increase in DC. Both AM and DC exhibited cellular activation in response to silica, indicated by increased expression of cell surface markers. In the absence of silica-induced AM apoptosis (TNFR 1/2–null and Gld mice), no change was observed in the percent or absolute number of either cell type. Furthermore, bone marrow–derived DC, but not bone marrow–derived macrophages, migrated from the alveoli into the lung parenchyma in response to silica, resulting in significantly increased numbers of activated T lymphocytes. Collectively, the results demonstrate that AM and DC are distinct antigen-presenting cells within the respiratory tract that respond to silica exposure in vivo in unique ways, with significant implications for immune reactivity of the lung in response to environmental pathogens. PMID:17641296

Beamer, Celine A; Holian, Andrij

2007-01-01

244

Dendritic cells therapy confers a protective microenvironment in murine pregnancy.  

PubMed

The fetal-placental unit is a semi-allograft and immunological recognition of pregnancy, together with the subsequent response of the maternal immune system, is necessary for a successful pregnancy. Dendritic cells (DC) show a biological plasticity that confers them special characteristics regulating both immunity and tolerance. Therapy employing DC proved to diminish the abortion in the DBA/2J-mated CBA/J females; however, the underlying mechanisms remain unknown. Here, we evaluated whether DC therapy influences the presence of immunoregulatory populations of cells at the fetal-maternal interface. To address this hypothesis, we analysed the pregnancy-protective CD8, gammadelta cell populations as well as transforming growth factor (TGF)-beta1 and progesterone-induced blocking factor (PIBF) expression at the fetal-maternal interface from abortion-prone female mice that had previously received adoptive transfer of syngeneic DC. Syngeneic DC therapy induced an increase in the number of CD8 and gammadelta cells. Additionally, an upregulation of TGF-beta1 and PIBF expression could be detected after DC transfer. We suggest that DC therapy differentially upregulates a regulatory/protective population of cells at the fetal-maternal interface. It is reasonable to assure that this mechanism would be responsible for the lower abortion rate. PMID:17032241

Miranda, S; Litwin, S; Barrientos, G; Szereday, L; Chuluyan, E; Bartho, J S; Arck, P C; Blois, S M

2006-11-01

245

A dense network of dendritic cells populates the murine epididymis  

PubMed Central

One of the most intriguing aspects of male reproductive physiology is the ability to generate spermatogenic cells - which are “foreign” to the host - without triggering immune activation. After leaving the testis, spermatozoa enter the epididymis where they mature and are stored. We report here a previously unrecognized dense network of dendritic cells located at the base of the epididymal epithelium. This network was detected in transgenic mice expressing CD11c-EYFP and CX3CR1-GFP reporters. Epididymal dendritic cells (eDCs) establish intimate interactions with the epithelium and project long dendrites between epithelial cells toward the lumen. We show that isolated eDCs express numerous leukocyte markers described previously in other organs that are in contact with the external environment, and present and cross-present ovalbumin to T cells in vitro. eDCs are, therefore, strategically positioned to regulate the complex interplay between immune tolerance and activation, a balance that is fundamental to male fertility. PMID:21310816

Da Silva, Nicolas; Cortez-Retamozo, Virna; Reinecker, Hans-Christian; Wildgruber, Moritz; Hill, Eric; Brown, Dennis; Swirsk, Filip K.; Pittet, Mikael J.; Breton, Sylvie

2013-01-01

246

Cytotoxic activity of some lichen extracts on murine and human cancer cell lines.  

PubMed

Eight lichens were extracted successively with n-hexane, diethyl ether and methanol using a Soxhlet process. The cytotoxic activity of the 24 lichen extracts was evaluated in vitro using two murine (the L1210: lymphocytic leukaemia, and the 3LL: Lewis lung carcinoma) and four human (the K-562: chronic myelogenous leukaemia, the U251: glioblastoma, the DU145: prostate carcinoma, and the MCF7: breast adenocarcinoma) cancer cell lines and non-cancerous cells, the Vero cell line (African green monkey kidney cell line). The MTT assay revealed significant cytotoxicity (IC50 < or = 20 microg/ml) on one of the tested cancer cell lines for at least one extract of each lichen species. Some extracts of Cladonia convoluta, Cladonia rangiformis, Parmelia caperata, Platismatia glauca and Ramalina cuspidata demonstrated interesting activities particularly on human cancer cell lines as good selectivity indices were recorded (SI > 3). PMID:13678234

Bézivin, C; Tomasi, S; Lohézic-Le Dévéhat, F; Boustie, J

2003-01-01

247

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

SciTech Connect

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.

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

248

Expression and Function of the Murine B7 Antigen, the Major Costimulatory Molecule Expressed by Peritoneal Exudate Cells  

Microsoft Academic Search

The murine B7 (mB7) protein is a potent costimulatory molecule for the T-cell receptor (TCR)-mediated activation of murine CD4^+ T cells. We have previously shown that stable mB7-transfected Chinese hamster ovary (CHO) cells but not vector-transfected controls synergize with either anti-CD3 monoclonal antibody-induced or concanavalin A-induced T-cell activation, resulting ultimately in lymphokine production and proliferation. We now have generated a

Ziba Razi-Wolf; Gordon J. Freeman; Frances Galvin; Baruj Benacerraf; Lee Nadler; Hans Reiser

1992-01-01

249

Retrovirally transduced murine T lymphocytes expressing FasL mediate effective killing of prostate cancer cells  

PubMed Central

Adoptively transferred T cells possess anticancer activities partially mediated by T-cell FasL engagement of Fas tumor targets. However, antigen-induced T-cell activation and clonal expansion, which stimulates FasL activity, is often inefficient in tumors. As a gene therapy approach to overcome this obstacle, we have created oncoretroviral vectors to overexpress FasL or non-cleavable FasL (ncFasL) on murine T cells of a diverse T-cell receptor repertoire. Expression of c-FLIP was also engineered to prevent apoptosis of transduced cells. Retroviral transduction of murine T lymphocytes has historically been problematic, and we describe optimized T-cell transduction protocols involving CD3/CD28 co-stimulation of T cells, transduction on ice using concentrated oncoretrovirus, and culture with IL-15. Genetically modified T cells home to established prostate cancer tumors in vivo. Co-stimulated T cells expressing FasL, ncFasL and ncFasL/c-FLIP each mediated cytotoxicity in vitro against RM-1 and LNCaP prostate cancer cells. To evaluate the compatibility of this approach with current prostate cancer therapies, we exposed RM-1, LNCaP, and TRAMP-C1 cells to radiation, mitoxantrone, or docetaxel. Fas and H-2b expression were upregulated by these methods. We have developed a novel FasL-based immuno-gene therapy for prostate cancer that warrants further investigation given the apparent constitutive and inducible Fas pathway expression in this malignancy. PMID:19096446

Symes, JC; Siatskas, C; Fowler, DH; Medin, JA

2010-01-01

250

Murine erythroleukemia cell line GM979 contains factors that can activate silent chromosomal human. gamma. -globin genes  

SciTech Connect

The authors introduced a normal chromosome 11 into GM979 murine erythroleukemia cells by fusing them with Epstein-Barr virus-transformed lymphocytes from a normal individual. In contrast to precious data obtained with other murine erythroleukemia cells, they detected activation of human chromosomal {gamma}-globin genes in GM979 cells. GM979, unlike previously used murine erythroleukemia cell lines, expresses murine embryonic globin in addition to adult globin. While all the hybrids expressed {gamma}- and {beta}-globin, they displayed a wide range of {gamma}-globin expression in relation to that of {beta}-globin. No correlation, however, was found in quantitative expression between murine embryonic globin and human {gamma}-globin in these hybrids, suggesting that the two globins are regulated independently, at least in this cell line. These data indicate that {gamma}-globin genes from normal, nonerythroid chromosomes are not irreversibly silenced, and they can be activated by a positive trans factor(s) present in GM979 cells.

Zitnik, G.; Hines, P.; Stamatoyannopoulos, G.; Papayannopoulou, T. (Univ. of Washington, Seattle (United States))

1991-03-15

251

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

PubMed

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

Kim, Myun Soo; Kim, Tae Sung

2013-05-01

252

Complex 2B4 Regulation of Mast Cells and Eosinophils in Murine Allergic Inflammation.  

PubMed

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

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

2014-12-01

253

Epifluorescence Intravital Microscopy of Murine Corneal Dendritic Cells  

PubMed Central

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

Rosenbaum, James T.; Planck, Stephen R.

2010-01-01

254

Targeted destruction of murine macrophage cells with bioconjugated gold nanorods  

Microsoft Academic Search

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

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

2007-01-01

255

Differential effects of protoporphyrin and uroporphyrin on murine mast cells  

SciTech Connect

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

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

1987-03-01

256

Induction of differentiation of murine embryonal carcinoma cells by ouabain  

SciTech Connect

Embryonal carcinoma (EC) cells can be induced to differentiate by ouabain at concentrations which inhibit Na/sup +/, K/sup +/-ATPase activity as measured by inhibition of /sup 86/Rb/sup +/ uptake. Since the pharmacologic action of ouabain is thought to be specific, the authors investigated the role of Na/sup +/, K/sup +/-ATPase inhibition and specific metabolic consequences of this inhibition in the induction of EC differentiation, and explored whether this might be a common mode of action for a variety of structurally diverse inducers. The Na/sup +/, K/sup +/-ATPase maintains ionic gradients in cells. However, results of studies utilizing specific ionophores, channel blockers, and media deficient in specific components failed to demonstrate a consistent role for ion flux or concentration in the differentiation process. The Na/sup +/, K/sup +/-ATPase is a major consumer of ATP. They therefore examined the effect of Na/sup +/, K/sup +/-ATPase inhibition on the adenylate energy charge as measured by high performance liquid chromatography of adenylate nucleotides. Ouabain was found to significantly decrease the energy charge in sensitive cells suggesting a role for suppression of ATP turnover is triggering differentiation. However, direct inhibition of glycolysis also induced differentiation without decreasing the energy charge, suggesting that reduction of the energy charge is not a common mechanism for induction of differentiation of EC.

Zimmerman, B.T.

1986-01-01

257

Rat pachytene spermatocytes down-regulate a polo-like kinase and up-regulate a thiol-specific antioxidant protein, whereas sertoli cells down-regulate a phosphodiesterase and up-regulate an oxidative stress protein after exposure to methoxyethanol and methoxyacetic acid.  

PubMed

2-Methoxyethanol (ME) and its metabolite, methoxyacetic acid (MAA), produce testicular lesions characterized by pachytene spermatocyte degeneration. To understand the molecular basis of this action on meiotic prophase cells, mRNA differential display was used to identify gene expression changes in control and treated cells. When pachytene spermatocytes were cultured with 5 mM ME or 5 mM MAA for 24 h, two complementary DNAs (cDNAs), of 557 nucleotides (clone 5) and 388 nucleotides (clone 6), were up-regulated; and a cDNA of 648 nucleotides (clone 1) was down-regulated. The altered expression pattern shown by differential display was confirmed by Northern blotting. Sequence analyses indicate that clones 1 and 6 have 83% and 79% homology at the nucleotide level to a polo-like kinase and a thiol-specific antioxidant, respectively. Clone 5 shows no homology to any known gene in the database. Messenger RNAs (mRNAs) encoding the thiol-specific antioxidant and clone 5 are up-regulated within 30 min of the addition of MAA, whereas the polo-like kinase mRNA decreased to undetectable levels after 6 h. Changes in Sertoli cell gene expression were also detected when Sertoli cells were cultured with 5 mM ME or MAA for 24 h. Two cDNAs, of 367 nucleotides (clone 2) and 676 nucleotides (clone 3), were up-regulated; and a cDNA of 538 nucleotides (clone 4) was down-regulated. Homology searches revealed that clones 3 and 4 have 90 and 91% homology at the nucleotide level to an oxidative stress protein and a phosphodiesterase (PDE), respectively. Northern blotting confirmed the differential display expression pattern for the PDE and oxidative stress protein. mRNAs for the latter were induced within 30 min, and PDE mRNAs were down-regulated within one h, after the addition of MAA. To determine whether the changes in gene expression seen with cells in culture also occur in vivo, rats were given a single oral dose of 250 mg/kg ME or MAA. After 24 h, total testis RNAs from control and treated rats were purified and hybridized. The expression patterns seen in vivo for the differentially expressed cDNAs were identical to those seen in vitro. We conclude that, although pachytene spermatocytes seem to be selectively affected by ME and MAA, changes in gene expression are also detected in Sertoli cells, suggesting that the action(s) of ME or MAA on pachytene spermatocytes could be mediated through Sertoli cells. PMID:9681501

Syed, V; Hecht, N B

1998-08-01

258

Oral administration of kefiran induces changes in the balance of immune cells in a murine model.  

PubMed

The aim of the present study was to evaluate the effect of the oral administration of kefiran on the balance of immune cells in a murine model. Six week old BALB/c mice were treated with kefiran (300 mg/L) for 0, 2 and 7 days. Kefiran treatment increased the number of IgA+ cells in lamina propria after 2 and 7 days. Percentage of B220+/MHCII(high) cells in mesenteric lymph nodes (2 days) and Peyer's patches (7 days) was higher compared to untreated control mice. An increase of macrophages (F4/80+ cells) was observed in lamina propria and peritoneal cavity (2 and 7 days). In contrast, at day 7, macrophage population decreased in Peyer's patches. These results show the ability of kefiran to modify the balance of immune cells in intestinal mucosa. This property could be highly relevant for the comprehension of the probiotic effect attributed to kefir. PMID:21504180

Medrano, Micaela; Racedo, Silvia M; Rolny, Ivanna S; Abraham, Analía G; Pérez, Pablo F

2011-05-25

259

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

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

Paris-Sud XI, Université de

260

Marker Polypeptides Distinguishing between Cancer Cell Clones with High and Low Potential for Spontaneous Metastasis in Murine Fibrosarcoma Cells1  

Microsoft Academic Search

Consistent differences in expression of specific proteins were observed between numerous cancer cell clones with high and low potential for spontaneous metastasis. Seventeen clones from two unrelated murine fibrosareomas were examined concomitantly for spontaneous formation of lung métastasesand for occurrence of individual polypeptide differ ences by high resolution two-dimensional gel electrophoresis. One of the identified marker polypeptides, designated Hi:2, was

Ivar Amund Grimstad; Anne Karine Thorsrud; Egil Jellum

1988-01-01

261

Isolation, Expansion and Transplantation of Postnatal Murine Progenitor Cells of the Enteric Nervous System  

PubMed Central

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

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

262

Mismatch and Base Excision Repair Proficiency in Murine Embryonic Stem Cells  

PubMed Central

Accumulation of mutations in embryonic stem (ES) cells would be detrimental to an embryo derived from these cells, and would adversely affect multiple organ systems and tissue types. ES cells have evolved multiple mechanisms to preserve genomic integrity that extend beyond those found in differentiated cell types. The present study queried whether mismatch repair (MMR) and base-excision repair (BER) may play a role in the maintenance of murine ES cell genomes. The MMR proteins Msh2 and Msh6 are highly elevated in mouse ES cells compared with mouse embryo fibroblasts (MEFs), as are Pms2 and Mlh1, albeit to a lesser extent. Cells transfected with an MMR reporter plasmid showed that MMR repair capacity is low in MEFs, but highly active in wildtype ES cells. As expected, an ES cell line defective in MMR was severalfold less effective in repair level than wildtype ES cells. Like proteins that participate in MMR, the level of proteins involved in BER was elevated in ES cells compared with MEFs. When BER activity was examined biochemically using a uracil-containing oligonucleotide template, repair activity was higher in ES cells compared with MEFs. The data are consistent with the suggestion that ES cells have multiple mechanisms, including highly active MMR and BER that preserve genetic integrity and minimize the accumulation of mutations. PMID:21315663

Tichy, Elisia D.; Liang, Li; Deng, Li; Tischfield, Jay; Schwemberger, Sandy; Babcock, George; Stambrook, Peter J.

2011-01-01

263

Modulation of prostaglandin biosynthesis in murine mammary adenocarcinoma tumor cells  

SciTech Connect

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.

Shalinsky, D.R.

1988-01-01

264

Analyzing Murine Schwann Cell Development Along Growing Axons  

PubMed Central

The development of peripheral nerves is an intriguing process. Neurons send out axons to innervate specific targets, which in humans are often more than 100 cm away from the soma of the neuron. Neuronal survival during development depends on target-derived growth factors but also on the support of Schwann cells (SCs). To this end SC ensheath axons from the region of the neuronal soma (or the transition from central to peripheral nervous system) to the synapse or neuromuscular junction. Schwann cells are derivatives of the neural crest and migrate as precursors along emerging axons until the entire axon is covered with SCs. This shows the importance of SC migration for the development of the peripheral nervous system and underlines the necessity to investigate this process. In order to analyze SC development, a setup is needed which next to the SCs also includes their physiological substrate for migration, the axon. Due to intrauterine development in vivo time-lapse imaging, however, is not feasible in placental vertebrates like mouse (mus musculus). To circumvent this, we adapted the superior cervical ganglion (SCG) explant technique. Upon treatment with nerve growth factor (NGF) SCG explants extend axons, followed by SC precursors migrating along the axons from the ganglion to the periphery. The beauty of this system is that the SC are derived from a pool of endogenous SC and that they migrate along their own physiological axons which are growing at the same time. This system is especially intriguing, because the SC development along axons can be analyzed by time-lapse imaging, opening further possibilities to gain insights into SC migration. PMID:23208118

Heermann, Stephan; Krieglstein, Kerstin

2012-01-01

265

Combined Treatment of Murine Fibrosarcoma with Chemotherapy (Paclitaxel), Radiotherapy, and Intratumoral Injection of Dendritic Cells  

PubMed Central

Background New antitumor therapeutic strategies aim to combine different approaches that are able to induce tumor-specific effector and memory T cell responses that might control tumor growth. Dendritic cells (DCs) have the capacity to induce antigen-specific cytotoxic T lymphocytes. We have previously shown that the combined treatment of paclitaxel chemotherapy (Chemo) and injection of DCs led to complete tumor regression. Objective The goal of this study was to evaluate synergistic antitumor effect of a triple combination treatment comprising radiotherapy, paclitaxel Chemo and intratumoral injection of syngeneic bone marrow-derived DCs on murine fibrosarcoma, compared to other single or double combination treatments. Methods For the murine fibrosarcoma model, naïve C57BL/6 mice were inoculated intradermally with 2×103 MCA102 cells in the right upper flank. Mice were assigned to five groups (untreatedcontrol, RT alone, RT+Chemo, RT+DC, and RT+Chemo+DC), with eight mice in each group. In vitro cytotoxicity assays were performed to assess the immune activity. The persistence of tumor-specific immunity was determined by second tumor challenge in mice with complete tumor regression. Results The triple combination treatment showed a significantly enhanced therapeutic efficacy by decreasing tumor size and inducing complete tumor regression, resulting in a cure of 50% of mice. The results of in vitro cytotoxicity assays and the second tumor challenge experiment strongly indicated the induction of a tumor-specific cytotoxic T lymphocyte response and acquisition of prolonged tumor immunity. Conclusion These findings suggest that the triple combination treatment can be a promising strategy for the treatment of murine fibrosarcoma. PMID:24648686

Byun, Ji-Won; Lee, Hyeon-Sook; Song, Sun-Uk; Lee, Si-Won; Kim, Soon-Ki; Kim, Woo-Chul; Lee, Moon-Hee

2014-01-01

266

Accessing the Genomic Effects of Naked Nanoceria in Murine Neuronal Cells  

PubMed Central

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

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

2011-01-01

267

Mitotic recombination is responsible for the loss of heterozygosity in cultured murine cell lines.  

PubMed Central

Heterozygous mammalian cell lines normally express both parental alleles at most autosomal loci. However, mutants can be isolated that fail to express one of the alleles. Using a murine pre-B cell line that is heterozygous for several loci on chromosome 12, including one encoding the cell surface antigen Ly-18, we found that one of the two Ly-18 antigenic forms was lost at a rate of 1.5 x 10(-5) per cell per generation. Molecular analysis revealed that a genetic marker distal to Ly-18 became homozygous. Analysis of the genotype of the mutants at the rDNA cluster, located close to the centromere, strongly suggests that the mutants arose by mitotic recombination within this multicopy locus. Images PMID:2725499

Nelson, F K; Frankel, W; Rajan, T V

1989-01-01

268

Induction of the anti-carcinogenic enzyme quinone reductase by food extracts using murine hepatoma cells.  

PubMed

Over 145 extracts of vegetables, fruits, herbs, spices and beverages which are consumed regularly in the European diet have been surveyed for potential anti-carcinogenic activity using an assay which measures the induction of NAD(P)H: (quinone acceptor) menadione oxidoreductase (quinone reductase, QR) activity in murine cells challenged with solutions of potential inducers. When appropriate the study has included extracts prepared from cooked and autolysed material. The results indicate that extracts of some brassicas, legumes (peas), lettuces, red pepper, grapefruit and some herbs including basil, tarragon and rosemary are inducers of QR activity. Inducing activity is strongly dependent on processing and on variety. PMID:8061594

Tawfiq, N; Wanigatunga, S; Heaney, R K; Musk, S R; Williamson, G; Fenwick, G R

1994-05-01

269

An autologous leukemia cell vaccine prevents murine acute leukemia relapse after cytarabine treatment.  

PubMed

Acute leukemias with adverse prognostic features carry a high relapse rate without allogeneic stem cell transplantation (allo-SCT). Allo-SCT has a high morbidity and is precluded for many patients because of advanced age or comorbidities. Postremission therapies with reduced toxicities are urgently needed. The murine acute leukemia model C1498 was used to study the efficacy of an intravenously administered vaccine consisting of irradiated leukemia cells loaded with the natural killer T (NKT)-cell agonist ?-galactosylceramide (?-GalCer). Prophylactically, the vaccine was highly effective at preventing leukemia development through the downstream activities of activated NKT cells, which were dependent on splenic langerin(+)CD8?(+) dendritic cells and which led to stimulation of antileukemia CD4(+) and CD8(+) T cells. However, hosts with established leukemia received no protective benefit from the vaccine, despite inducing NKT-cell activation. Established leukemia was associated with increases in regulatory T cells and myeloid-derived suppressor cells, and the leukemic cells themselves were highly suppressive in vitro. Although this suppressive environment impaired both effector arms of the immune response, CD4(+) T-cell responses were more severely affected. When cytarabine chemotherapy was administered prior to vaccination, all animals in remission posttherapy were protected against rechallenge with viable leukemia cells. PMID:25237205

Gibbins, John D; Ancelet, Lindsay R; Weinkove, Robert; Compton, Benjamin J; Painter, Gavin F; Petersen, Troels R; Hermans, Ian F

2014-11-01

270

Genetic engineering of murine CD8+ and CD4+ T cells for pre-clinical adoptive immunotherapy studies  

PubMed Central

T-cell-receptor (TCR) gene therapy enables for the rapid creation of antigen-specific T cells from mice of any strain and represents a valuable tool for pre-clinical immunotherapy studies. Here, we describe the superiority of gamma-retroviral vectors compared to lentiviral vectors for transduction of murine T cells and surprisingly illustrate robust gene-transfer into phenotypically naïve/memory-stem cell (CD62Lhi/CD44low) and central memory (CD62Lhi/CD44hi) CD8+ T cells using murine-stem-cell-based gamma-retroviral vectors (MSGV1). We created MSGV1 vectors for a MHC-class I restricted T-cell receptor (TCR) specific for the melanocyte-differentiation antigen, gp100 (MSGV1-pmel-1), and a MHC-class II restricted TCR specific for tyrosinase-related-protein-1 (MSGV1-TRP-1), and found that robust gene expression required codon optimization of TCR sequences for the pmel-1 TCR. To test for functionality, we adoptively transferred TCR-engineered T cells into mice bearing B16 melanomas and observed delayed growth of established tumors with pmel-1TCR engineered CD8+ T cells and significant tumor regression with TRP-1 TCR transduced CD4+ T cells. We simultaneously created lentiviral vectors encoding the pmel-1TCR, but found that these vectors mediated low TCR expression in murine T cells, but robust gene expression in other murine and human cell lines. These results indicate that preclinical murine models of adoptive immunotherapies are more practical using gamma-retroviral rather than lentiviral vectors. PMID:21499127

Kerkar, Sid P; Sanchez-Perez, Luis; Yang, Shicheng; Borman, Zachary; Muranski, Pawel; Ji, Yun; Chinnasamy, Dhanalakshmi; Kaiser, Andrew DM; Hinrichs, Christian; Klebanoff, Christopher A; Scott, Christopher; Gattinoni, Luca; Morgan, Richard A; Rosenberg, Steven A; Restifo, Nicholas P

2011-01-01

271

Cue, signal, response analysis of murine embryonic stem cell differentiation : multivariable analysis of cytokine and ECM effects on commitment to differentiation  

E-print Network

The highly complex nature of developmental cell fate decisions is exemplified by murine embryonic stem cell (ES cell) differentiation, for which almost two decades of study has identified a small set of putative individual ...

Prudhomme, Wendy A. (Wendy Adele), 1975-

2004-01-01

272

Diverse type III secretion phenotypes among Pseudomonas aeruginosa strains upon infection of murine macrophage-like and endothelial cell lines  

Microsoft Academic Search

The interaction of over 100 isolates of Pseudomonas aeruginosa representing different genotypes of type III secretion system (TTSS) with RAW 264.7 murine macrophage-like cells and pulmonary microvascular endothelial (PME) cells were studied. The strains were isolated from clinical materials and from stool specimens of healthy carriers and were analyzed by pulsed field gel electrophoresis (PFGE) to characterize their heterogeneity. In

Ma?gorzata St?pi?ska; El?bieta A. Trafny

2008-01-01

273

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

SciTech Connect

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.

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

274

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

PubMed

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 500bp 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

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

2014-11-01

275

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

SciTech Connect

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.

Grdina, D.J.; Hunter, N.

1982-10-01

276

Murine Epidermal Label-Retaining Cells Isolated by Flow Cytometry do not Express the Stem Cell Markers CD34, Sca1, or Flk-1  

Microsoft Academic Search

Keratinocyte stem cells are present in the murine epidermis, based on both in vitro and in vivo evidence, and better characterization of these cells remains an active goal. Because keratinocyte stem cells are believed to cycle slowly, a good method for identification is based on their ability to retain nucleoside analog, such as bromodeoxyuridine. Adult stem cells have been identified

Michael R. Albert; Ruth-Ann Foster; Jonathan C. Vogel

2001-01-01

277

Immunoelectron microscopic study on interactions of noninfectious sendai virus and murine cells.  

PubMed Central

The early interactions of LLC-MK2 cell-grown noninfectious Sendai virus and a murine cell line, P815 mastocytoma ascitic cells, were studied by electron microscopy, using the ferritin-conjugated antibody technique with anti-virus glycoprotein serum. For comparison, the interactions of egg-grown infectious Sendai virus with the same cells were also examined. When noninfectious virus was adsorbed to the cells in the cold, the cell membranes become partially invaginated at the site of contact of adsorbed virions, but ferritin-conjugated antibodies did not penetrate into the areas of envelope-cell membrane association. This pattern of virus attachment was similar to that of infectious virus attachment. Upon subsequent incubation at 37 degrees C, most of the adsorbed noninfectious virions were taken into cytoplasmic vesicles and then degraded, although a few virions remained attached to the cell membrane. No evidence of fusion of envelopes of noninfectious virions was obtained. On the other hand, envelopes of infectious virions fused with the cell membrane, and the transferred viral antigens diffused on the cell surfaces and then decreased in number. Images PMID:6268815

Yasuda, Y; Hosaka, Y; Fukami, Y; Fukai, K

1981-01-01

278

Induction of multiple independent T-cell lymphomas in rats inoculated with MOloney murine leukemia virus.  

PubMed Central

Tumor cell DNA derived from different lymphoid organs of 30 rats serially inoculated at birth with Moloney murine leukemia virus (MoMuLV) was examined by Southern blot analysis and hybridization to the following DNA probes: MoMuLV long terminal repeat (LTR), Moloney leukemia virus integration regions 1, 2, 3, and 4 (Mlvi-1, Mlvi-2, Mlvi-3, and Mlvi-4), T-cell receptor beta locus, and immunoglobulin heavy chain locus. This analysis revealed that the tumors segregating in different lymphoid organs in 10% of the animals were clonally unrelated. These findings are consistent with the hypothesis that the MoMuLV-induced rat thymic lymphomas are polyclonal in origin. At least two factors may be responsible for this phenomenon: (i) increase in the number of the available target cells in virus-infected animals, and (ii) genetic instability associated with provirus integration in the developing premalignant clones. Images PMID:2786211

Bellacosa, A; Lazo, P A; Bear, S E; Shinton, S; Tsichlis, P N

1989-01-01

279

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

PubMed

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

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

2014-01-01

280

Reprogramming murine telomerase rapidly inhibits the growth of mouse cancer cells in vitro and in vivo.  

PubMed

Telomerase plays a critical role in cancer, prompting the pursuit of various telomerase-based therapeutic strategies. One such strategy, telomerase interference, exploits the high telomerase activity in cancer cells and reprograms telomerase to encode "toxic" telomeres. To date, telomerase interference has been tested in human cancer cells xenografted into mice, an approach that does not recapitulate spontaneous malignancy and offers few insights about host toxicities, because human telomerase is targeted in a mouse host. To address these limitations, we designed and validated two new gene constructs specifically targeting mouse telomerase: mutant template mouse telomerase RNA (MT-mTer) and small interfering RNA against wild-type mouse telomerase RNA (?-mTer-siRNA). Using lentiviral delivery in mouse prostate cancer cells, we achieved ?-mTer-siRNA-mediated knockdown of wild-type mTer (80% depletion) and concurrent overexpression of MT-mTer (50-fold). We showed that the two constructs effectively synergize to reprogram murine telomerase to add mutant instead of wild-type telomeric repeats, resulting in rapid telomeric uncapping (5-fold increase in DNA damage foci). This, in turn, led to rapid and significant apoptosis (>90% of cells) and growth inhibition in vitro (90% reduction in viable cell mass) and in vivo (75% reduction in tumor allograft wet weight). In summary, we have shown that mouse cancer cells are vulnerable to direct telomerase interference using novel murine telomerase-targeting constructs; this approach can now be used to study the true therapeutic potential of telomerase interference in mouse spontaneous cancer models. PMID:20124445

Xu, Tong; Xu, Yucheng; Liao, Chun-Peng; Lau, Roy; Goldkorn, Amir

2010-02-01

281

A flavone derivative from Sesbania sesban leaves and its cytotoxicity against murine leukemia P-388 cells  

NASA Astrophysics Data System (ADS)

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.

Dianhar, Hanhan; Syah, Yana Maolana; Mujahidin, Didin; Hakim, Euis Holisotan; Juliawaty, Lia Dewi

2014-03-01

282

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

SciTech Connect

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.

Grdina, D.J.; Hunter, N.

1982-10-01

283

Indirect inhibition of generation of murine lymphokine-activated killer cell activity in splenocyte cultures by interferon-gamma.  

PubMed Central

The effect of mouse recombinant interferon-gamma (rIFN-gamma) on murine lymphokine-activated killer (LAK) cell activity was investigated using natural killer (NK)-resistant, spontaneously developed, weakly immunogenic and highly tumourigenic, syngeneic murine mammary adenocarcinoma, JC, mimicking that of human disease, as the target. Murine YAC-1 also was used as a target cell line. rIFN-gamma, when used in combination with recombinant interleukin-2 (rIL-2), was shown to exhibit a suppressed effect on LAK cell activity generated from BALB/c mouse splenocytes, compared to that with rIL-2 alone. The decrease in LAK cell activity was rIFN-gamma dose-dependent. Kinetic study revealed that this inhibitory effect was demonstrated only when rIFN-gamma was added to the medium at the early phase of rIL-2 culture. The inhibitory effect on LAK cell generation by rIFN-gamma was completely abrogated when the nylon-wool-treated non-adherent 'macrophage-free' splenocytes were incubated with rIL-2 and rIFN-gamma. These results indicated that the LAK cell activity generated from murine splenocytes cultured with rIL-2 could be depressed by rIFN-gamma, and that the macrophages may be involved as mediators. PMID:2113034

Chao, T Y; Ohnishi, H; Chu, T M

1990-01-01

284

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

PubMed Central

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

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

2011-01-01

285

Dynamic expression of BCL6 in murine conventional dendritic cells during in vivo development and activation.  

PubMed

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

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

2014-01-01

286

Effects of ionizing radiation on bone cell differentiation in an experimental murine bone cell model  

NASA Astrophysics Data System (ADS)

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

Baumstark-Khan, Christa; Lau, Patrick; Hellweg, Christine; Reitz, Guenther

287

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

Microsoft Academic Search

During spermatogenesis in the mamma- lian testis, preleptotene\\/leptotene spermatocytes differ- entiate from type B spermatogonia and traverse the blood-testis barrier (BTB) at stage VIII of the seminifer- ous epithelial cycle for further development. This timely movement of germ cells involves extensive junction re- structuring at the BTB. Previous studies have shown that these events are regulated by testosterone (T) and

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

2008-01-01

288

Changes in calcium influx affect the differentiation of murine erythroleukaemia cells.  

PubMed Central

As indicated by direct evidence, obtained by altering the cell-membrane permeability for Ca2+ in murine erythroleukaemia (MEL) cells, calpain is the triggering factor which connects fluctuations of the intracellular Ca2+ concentrations to the decay of protein kinase C (PKC), as well as to the kinetics of cell differentiation induced by hexamethylenebisacetamide. Cell exposure to verapamil caused a profound decrease in the rate of PKC down-regulation and a slower initial rate of accumulation of mature erythroid cells, whereas addition of the Ca2+ ionophore A23187 produced opposite effects. The high susceptibility of PKC-delta to calpain degradation, at concentrations of Ca2+ much lower than those required for degradation of the other PKC isoforms, may be explained by the finding that this kinase isoform is predominantly associated with the cell membrane. The different cellular localizations, as well as the different susceptibilities to calpain digestion, further support the hypothesis that in MEL cells the various PKC isoforms play distinct biological functions that are critical for the maintenance of the undifferentiated state of the cell and for its commitment to terminal erythroid differentiation. PMID:7826342

Sparatore, B; Pessino, A; Patrone, M; Passalacqua, M; Melloni, E; Pontremoli, S

1995-01-01

289

Stimulation of Mesothelial Cell Proliferation by Exudate Macrophages Enhances Serosal Wound Healing in a Murine Model  

PubMed Central

Examination of thermally induced serosal lesions in mice displayed collections of inflammatory cells, predominantly macrophages, on and surrounding the wound within 48 hours of injury. Furthermore, by 2 days a large number of uninjured mesothelial cells adjacent to the wound were synthesizing DNA. From these findings, it was hypothesized that macrophages play a major role in serosal repair by stimulating mesothelial cell proliferation. Again, using a murine model of mesothelial regeneration, depletion of circulating monocytes significantly delayed serosal healing whereas addition of peritoneal exudate cells to the wound site 36 hours before injury increased the healing rate. In vivo assessment of mesothelial cell proliferation using tritiated thymidine incorporation and autoradiography demonstrated that peritoneal exudate cells stimulated mesothelial cell proliferation (12.44 ± 1.63% labeling index, compared with controls in which medium only was used 4.48 ± 0.71%). The mesothelial proliferation was predominantly because of macrophage-secreted products with molecular weights of 36 to 53 kd or 67 to 100 kd. These data support the hypothesis that macrophages play an important role in serosal healing by stimulating mesothelial cell proliferation. PMID:11839589

Mutsaers, Steven E.; Whitaker, Darrel; Papadimitriou, John M.

2002-01-01

290

Murine Spleen Tissue Regeneration from Neonatal Spleen Capsule Requires Lymphotoxin Priming of Stromal Cells  

PubMed Central

Spleen is a tissue with regenerative capacity, which allows autotransplantation of human spleen fragments to counteract the effects of splenectomy. We now reveal in a murine model that transplant of neonatal spleen capsule alone leads to the regeneration of full spleen tissue. This finding indicates that graft-derived spleen stromal cells, but not lymphocytes, are essential components of tissue neogenesis, a finding verified by transplant and regeneration of Rag1KO spleen capsules. We further demonstrate that lymphotoxin and lymphoid tissue inducer cells participate in two key elements of spleen neogenesis, bulk tissue regeneration and white pulp organization, identifying a lymphotoxin-dependent pathway for neonatal spleen regeneration that contrasts with previously defined lymphotoxin-independent embryonic spleen organogenesis. PMID:24951816

Tan, Jonathan K. H.

2014-01-01

291

Identification and characterization of a beta-globin promoter-binding factor from murine erythroleukemia cells.  

PubMed Central

We have identified a DNA-binding activity with specificity for the beta DRE, an evolutionarily conserved transcriptional regulatory element in mammalian adult beta-globin promoters. This binding activity, which we term beta DRf, for beta-globin direct repeat factor, was detected in fractionated nuclear extracts from the murine erythroleukemia cell line and has been partially purified from undifferentiated cells. beta DRf makes symmetric contacts on the two copies of its recognition sequence on both strands and introduces a bend into the DNA helix upon binding. While the factor displays a low binding affinity for the beta DRE in isolation, it binds to the intact beta-globin promoter and DNA fragments containing multiple beta DRE-binding sites with high affinity. A correlation between beta DRf binding affinity and transcriptional activity of beta DRE mutant promoters suggests that this factor stimulates transcription of the beta-globin promoter in vivo. Images PMID:8321233

Stuve, L L; Myers, R M

1993-01-01

292

Melanogenesis stimulation in murine B16 melanoma cells by Kava (Piper methysticum) rhizome extract and kavalactones.  

PubMed

Melanogenesis stimulation activity of aqueous ethanolic extracts obtained from several different parts of five Piper species, namely Piper longum, P. kadsura, P. methysticum, P. betle, and P. cubeba, were examined by using cultured murine B16 melanoma cells. Among them, the extract of P. methysticum rhizome (Kava) showed potent stimulatory effect on melanogenesis as well as P. nigrum leaf extract. Activity-guided fractionation of Kava extract led to the isolation of two active kavalactones, yangonin (2) and 7,8-epoxyyangonin (5), along with three inactive kavalactones, 5,6-dehydrokawain (1), (+)-kawain (3) and (+)-methysticin (4), and a glucosylsterol, daucosterin (6). 7,8-Epoxyyangonin (5) showed a significant stimulatory effect on melanogenesis in B16 melanoma cells. Yangonin (2) exhibited a weak melanogenesis stimulation activity. PMID:16595931

Matsuda, Hideaki; Hirata, Noriko; Kawaguchi, Yoshiko; Naruto, Shunsuke; Takata, Takanobu; Oyama, Masayoshi; Iinuma, Munekazu; Kubo, Michinori

2006-04-01

293

The detailed analysis of the changes of murine dendritic cells (DCs) induced by thymic peptide  

PubMed Central

The aim of present research is to analyze the detailed changes of dendritic cells (DCs) induced by pidotimod(PTD). These impacts on DCs of both bone marrow derived DCs and established DC2.4 cell line were assessed with use of conventional scanning electron microscopy (SEM), flow cytometry (FCM), transmission electron microscopy (TEM), cytochemistry assay FITC-dextran, bio-assay and enzyme linked immunosorbent assay (ELISA). We demonstrated the ability of PTD to induce DC phynotypic and functional maturation as evidenced by higher expression of key surface molecules such as MHC II, CD80 and CD86. The functional tests proved the downregulation of ACP inside the DCs, occurred when phagocytosis of DCs decreased, with simultaneously antigen presentation increased toward maturation. Finally, PTD also stimulated production of more cytokine IL-12 and less TNF-?. Therefore it is concluded that PTD can markedly exert positive induction to murine DCs. PMID:22863756

Hu, Xiaofang; Zheng, Wei; Wang, Lu; Wan, Nan; Wang, Bing; Li, Weiwei; Hua, Hui; Hu, Xu; Shan, Fengping

2012-01-01

294

Mouse Embryonic Stem Cells Inhibit Murine Cytomegalovirus Infection through a Multi-Step Process  

PubMed Central

In humans, cytomegalovirus (CMV) is the most significant infectious cause of intrauterine infections that cause congenital anomalies of the central nervous system. Currently, it is not known how this process is affected by the timing of infection and the susceptibility of early-gestational-period cells. Embryonic stem (ES) cells are more resistant to CMV than most other cell types, although the mechanism responsible for this resistance is not well understood. Using a plaque assay and evaluation of immediate-early 1 mRNA and protein expression, we found that mouse ES cells were resistant to murine CMV (MCMV) at the point of transcription. In ES cells infected with MCMV, treatment with forskolin and trichostatin A did not confer full permissiveness to MCMV. In ES cultures infected with elongation factor-1? (EF-1?) promoter-green fluorescent protein (GFP) recombinant MCMV at a multiplicity of infection of 10, less than 5% of cells were GFP-positive, despite the fact that ES cells have relatively high EF-1? promoter activity. Quantitative PCR analysis of the MCMV genome showed that ES cells allow approximately 20-fold less MCMV DNA to enter the nucleus than mouse embryonic fibroblasts (MEFs) do, and that this inhibition occurs in a multi-step manner. In situ hybridization revealed that ES cell nuclei have significantly less MCMV DNA than MEF nuclei. This appears to be facilitated by the fact that ES cells express less heparan sulfate, ?1 integrin, and vimentin, and have fewer nuclear pores, than MEF. This may reduce the ability of MCMV to attach to and enter through the cellular membrane, translocate to the nucleus, and cross the nuclear membrane in pluripotent stem cells (ES/induced pluripotent stem cells). The results presented here provide perspective on the relationship between CMV susceptibility and cell differentiation. PMID:21407806

Kawasaki, Hideya; Kosugi, Isao; Arai, Yoshifumi; Iwashita, Toshihide; Tsutsui, Yoshihiro

2011-01-01

295

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

E-print Network

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

Paris-Sud XI, Université de

296

Antitumor Activity of T Cells Generated from Lymph Nodes Draining the SEA-expressing Murine B16 Melanoma and Secondarily Activated with Dendritic Cells  

Microsoft Academic Search

The successful use of tumor-draining lymph nodes (TDLN) as a source of effector cells for cancer immunotherapy depends largely on the immunogenicity of the tumor drained by the lymph nodes as well as the methods for secondary in vitro T cell activation and expansion. We transferred the bacterial superantigen staphylococcal enterotoxin A (SEA) gene into B16 murine melanoma tumor cells,

Jiyun Yu; Rong Tian; Bingshui Xiu; Jinqi Yan; Rui Jia; Liang Zhang; Alfred E. Chang; Hongbin Song; Qiao Li

2009-01-01

297

Identification of the earliest prethymic T-cell progenitors in murine fetal blood.  

PubMed

During murine fetal development, hemato-poietic progenitors start to colonize the thymic anlage at day 11 of gestation via blood stream. The present study aims at identifying the earliest prethymic progenitors in circulation. Here, we show that the interleukin-7 receptor-positive (IL-7R+) cells in Lin- c-kit+ population are circulating exclusively between days 11 and 14 of fetal age. Clonal analysis revealed that these IL-7R+ cells mostly contain T-cell lineage-restricted progenitors (p-Ts). The proportion of circulating p-Ts reaches 30% of the total p-Ts during these fetal ages, whereas virtually all B-cell lineage-restricted progenitors stay in the fetal liver, suggesting that the p-Ts are selectively released to the circulation. The circulating p-Ts retain the potential to generate natural killer cells and dendritic cells and exhibit extensive proliferation before the occurrence of T-cell receptor beta (TCRbeta) chain gene rearrangement. We propose that the wave of p-Ts in fetal blood disclosed by this study represents the ontogenically earliest thymic immigrants. PMID:14512296

Ikawa, Tomokatsu; Masuda, Kyoko; Lu, Min; Minato, Nagahiro; Katsura, Yoshimoto; Kawamoto, Hiroshi

2004-01-15

298

Two distinct types of murine blast colony-forming cells are multipotential hematopoietic precursors  

PubMed Central

Two types of blast colonies can be stimulated to develop in semisolid agar cultures of murine bone marrow cells. Typically, these are either multicentric colonies stimulated by stem cell factor (SCF) plus interleukin-6 (IL-6) or dispersed colonies stimulated by Flt3 ligand (FL) plus IL-6. Both types of blast colony-forming cells (BL-CFCs) can generate large numbers of lineage-committed granulocyte-macrophage progenitor cells and exhibit some capacity for self-generation and the formation of eosinophil and megakaryocyte progenitor cells. However, the two populations of BL-CFCs are largely distinct and partially separable by fluorescence-activated cell sorting and are distinguished by differing capacity to form granulocyte-committed progeny. Both types of BL-CFCs can generate dendritic cells and small numbers of lymphocytes but the FL-responsive BL-CFCs have a greater capacity to form both B and T lymphocytes. Both types of blast colonies offer remarkable opportunities to analyze multilineage commitment at a clonal level in vitro. PMID:19011094

Metcalf, D.; Greig, K. T.; de Graaf, C. A.; Loughran, S. J.; Alexander, W. S.; Kauppi, M.; Hyland, C. D.; Di Rago, L.; Mifsud, S.

2008-01-01

299

Induction of murine embryonic stem cell differentiation by medicinal plant extracts  

SciTech Connect

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.

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

300

Extracellular high-mobility group 1 protein is essential for murine erythroleukaemia cell differentiation.  

PubMed Central

A high-mobility group 1 (HMG1) protein type isolated from murine erythroleukaemia (MEL) cells promotes acceleration of the differentiation process when added to a MEL cell culture together with the inducer hexamethylene bisacetamide. We now provide direct evidence that the presence of HMG1 protein in the extracellular medium is essential for terminal erythroid differentiation. An extracellular function for HMG1 protein in MEL cell is further supported by a demonstration that this protein is released from MEL cells exposed to the chemical inducer and that the addition of an anti-(HMG1 protein) monoclonal antibody to the cell culture inhibits the differentiation process almost completely. The release of HMG1 protein from MEL cells is modulated by compounds affecting cell calcium homoeostasis, such as a calcium ionophore or verapamil. In fact, in the presence of the ionophore an increased rate of differentiation is accompanied by an enhanced extracellular release of HMG1 protein, whereas in the presence of verapamil both phenomena are significantly decreased. PMID:8947495

Sparatore, B; Passalacqua, M; Patrone, M; Melloni, E; Pontremoli, S

1996-01-01

301

Induction of murine embryonic stem cell differentiation by medicinal plant extracts  

PubMed Central

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

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

2010-01-01

302

Infection of human cells with murine amphotropic replication-competent retroviruses.  

PubMed

Replication of the murine wild-type 4070A amphotropic retrovirus and a recombinant amphotropic replication-competent retrovirus arising from the PA12 packaging cell line varied considerably among the primate cell types tested. Medium from infected primate fibroblasts and endothelial cells contained the highest viral titers [10(4)-10(5) focus-forming units (ffu)/ml], while most hematopoietic cell lines, such as K562 and MOLT4, were associated with viral titers in the range of 10(3)-10(4) ffu/ml. Interestingly, HTLV-1-transformed T cell lines (TJF-2 and HM) and primary tumor infiltrating lymphocytes (TIL) had very low viral titer (0-10(1) ffu/ml). The low production of virus was not due to low infectivity and, in contrast to the virus, retroviral vectors were expressed without difficulty. Because screening for replication-competent retrovirus (RCR) is an important component of human retroviral-mediated gene therapy clinical protocols, a variety of assays were tested for their ability to detect RCR in virus-exposed cell lines. A biologic assay (3T3 amplification) and polymerase chain reaction (PCR) for the 4070A viral envelope are effective screening methods for RCR, even in cell lines associated with low virus production. PMID:8280796

Cornetta, K; Nguyen, N; Morgan, R A; Muenchau, D D; Hartley, J W; Blaese, R M; Anderson, W F

1993-10-01

303

Of mice and men: Different functions of the murine and human 2B4 (CD244) receptor on NK cells  

Microsoft Academic Search

2B4 was initially discovered on murine NK cells and T cells displaying non-MHC dependent cytotoxicity. Human 2B4 was cloned based on sequence homology with mouse 2B4. Recent evidence suggests that the function of this receptor might be different in the two species. Human 2B4 activates NK cell cytotoxicity and interferon gamma production when engaged by CD48, its ligand, on target

Swapnil V. Vaidya; Porunelloor A. Mathew

2006-01-01

304

Visualization and Characterization of Migratory Langerhans Cells in Murine Skin and Lymph Nodes by Antibodies Against Langerin\\/CD207  

Microsoft Academic Search

Dendritic cells are professional antigen-presenting cells that initiate primary immunity. Migration from sites of antigen uptake to lymphoid organs is crucial for the generation of immune responses. We investigated the migratory pathways specifically of epidermal Langerhans cells by tracing them from the epidermis to the draining lymph nodes. This was possible with a new monoclonal antibody, directed against murine Langerin\\/CD207,

Patrizia Stoitzner; Sandra Holzmann; Alexander D. McLellan; Lennart Ivarsson; Hella Stössel; Michaela Kapp; Ulrike Kämmerer; Patrice Douillard; Eckhart Kämpgen; Franz Koch; Sem Saeland; Nikolaus Romani

2003-01-01

305

Proteoglycan biosynthesis in murine monocytic leukemic (M1) cells before and after differentiation  

SciTech Connect

Murine monocytic leukemic (M1) cells were cultured in the presence of ({sup 3}H)glucosamine and ({sup 35}S)sulfate. Labeled proteoglycans were purified by anion exchange chromatography and characterized by gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis in combination with chemical and enzymatic degradation. M1 cells synthesize a single predominant species of proteoglycan which distributes almost equally between the cell and medium after 17 h labeling. The cell-associated proteoglycan has an overall size of about 135 kDa and contains three to five chondroitin sulfate chains (28-31 kDa each) attached to a chondroitinase-generated core protein of 28 kDa. The synthesis and subsequent secretion of this proteoglycan was enhanced 4-5-fold in cells induced to differentiate into macrophages. This was not a phenomenon of arrest in the G0/G1 stage of the cell cycle, since density inhibited undifferentiated cells arrested at this stage did not increase proteoglycan synthesis. The chondroitin sulfate chains contained exclusively chondroitin 4- and 6-sulfate; however, the ratio of these two disaccharides differed between the medium- and cell-associated proteoglycans, and changed during progression of the cells into a fully differentiated phenotype. Pulse-chase kinetics indicate the presence of two distinct pools of proteoglycan; one that is secreted very rapidly from the cell after a approximately 1-h lag, and a second pool that is turned over in the cell with a half-time of approximately 3.5 h. Subtle differences in the glycosylation patterns of the medium- and cell-associated species are consistent with synthesis of two pools. Papain digestion suggests that the chondroitin sulfate chains are clustered on a small protease resistant peptide. The data suggest that this proteoglycan is similar to the serglycin proteoglycan family.

McQuillan, D.J.; Yanagishita, M.; Hascall, V.C.; Bickel, M. (National Institute of Dental Research, Bethesda, MD (USA))

1989-08-05

306

Impurity of Stem Cell Graft by Murine Embryonic Fibroblasts - Implications for Cell-Based Therapy of the Central Nervous System  

PubMed Central

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

Molcanyi, Marek; Mehrjardi, Narges Zare; Schafer, Ute; Haj-Yasein, Nadia Nabil; Brockmann, Michael; Penner, Marina; Riess, Peter; Reinshagen, Clemens; Rieger, Bernhard; Hannes, Tobias; Hescheler, Jurgen; Bosche, Bert

2014-01-01

307

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

Microsoft Academic Search

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,

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

2012-01-01

308

Role of the LTR Region between the Enhancer and Promoter in Mink Cell Focus-Forming Murine Leukemia Virus Pathogenesis  

Microsoft Academic Search

Long terminal repeat (LTR) sequences are important determinants of mink cell focus-forming (MCF) murine leukemia virus pathogenesis. These sequences include the enhancer and sequences between the enhancer and promoter (DEN). In a previous study we showed that a virus missing the DEN region in its LTR was severely attenuated in its ability to induce thymic lymphoma. In this study we

Fayth K. Yoshimura; Tao Wang

2001-01-01

309

Sage weed (Salvia plebeia) extract antagonizes foam cell formation and promotes cholesterol efflux in murine macrophages.  

PubMed

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

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

310

Minocycline inhibits apoptotic cell death in a murine model of partial flap loss.  

PubMed

For breast reconstruction, the deep inferior epigastric perforator (DIEP) flap has become standard therapy. A feared complication is partial or even total flap loss. In a novel murine model of partial DIEP flap loss, the contribution of apoptotis to flap loss was investigated. The clinically available apoptosis-inhibiting compound minocycline was tested for its ability to reduce cell death. The effect of minocycline on cell proliferation was studied in cell cultures of breast carcinoma. In 12 mice, pedicled DIEP flaps were raised, which were subjected to 15 minutes of ischemia and 4 days of reperfusion. Six mice were treated with minocycline 2 hours before surgery and every 24 hours for 4 days. Apoptosis was revealed by injecting annexin A5 30 minutes before sacrifice. Annexin A5 binds to phosphatidylserines, which are expressed on the cell membrane during apoptotis. Prior to sacrifice, necrosis was measured using planimetry. Minocycline reduced cell death after 4 days from 35.9% (standard deviation = 10.6) to 13.9% (standard deviation = 8.0; P < 0.05). Apoptosis, as shown by annexin A5 binding in nontreated animals, was abundant. Minocycline did not influence tumor growth in cell cultures of human breast cancer. Minocycline treatment leads to increased DIEP flap viability in mice. This study widens the perspective in the improvement of free flap survival in patients. PMID:20648419

Dumont, Ewald A W J; Lutgens, Suzanne P M; Reutelingsperger, Christopher P M; Bos, Gerard M J; Hofstra, Lenoard

2010-10-01

311

Effect of Tunisian Capparis spinosa L. extract on melanogenesis in B16 murine melanoma cells.  

PubMed

The effect of Tunisian Capparis spinosa L. aromatic plant extract on melanogenesis regulation in B16 murine melanoma cells was investigated. B16 cells were treated with 0.0005, 0.005, and 0.05% (w/v) C. spinosa extract after which the melanin content and cell viability were measured. To clarify the mechanism behind melanogenesis regulation, the expression of tyrosinase was determined. Results showed that the extract had a significant stimulative effect on melanogenesis in B16 cells in a dose-dependent manner without cytotoxicity. Western blot analysis showed that expression of tyrosinase in cells treated with 0.03% (w/v) C. spinosa extract increased by 12.5- and 20-fold after 24 and 48 h of incubation, respectively, compared with untreated cells. HPLC analysis of the extract revealed the presence of 1% quercetin, a known melanogenesis stimulator, indicating that our findings may be attributed to quercetin; however, other compounds present in the extract may also have an effect on the overall ability of the extract to stimulate melanogenesis. We report here that Tunisian C. spinosa leaf extract can stimulate melanogenesis in a dose-dependent manner without cytotoxicity by increasing tyrosinase protein expression and has the potential to be used as a possible tanning agent or as a treatment for hair depigmentation. PMID:19685105

Matsuyama, Kyoko; Villareal, Myra O; El Omri, Abdelfatteh; Han, Junkyu; Kchouk, Mohamed Elyes; Isoda, Hiroko

2009-10-01

312

Modification of Glycosylation Mediates the Invasive Properties of Murine Hepatocarcinoma Cell Lines to Lymph Nodes  

PubMed Central

Among the various posttranslational modification reactions, glycosylation is the most common, and nearly 50% of all known proteins are thought to be glycosylated. In fact, changes in glycosylation readily occur in carcinogenesis, invasion and metastasis. This report investigated the modification of glycosylation mediated the invasive properties of Hca-F and Hca-P murine hepatocarcinoma cell lines, which have high, low metastatic potential in the lymph nodes, respectively. Analysis revealed that the N-glycan composition profiling, expression of glycogenes and lectin binding profiling were different in Hca-F cells, as compared to those in Hca-P cells. Further analysis of the N-glycan regulation by tunicamycin (TM) application or PNGase F treatment in Hca-F cells showed partial inhibition of N-glycan glycosylation and decreased invasion both in vitro and in vivo. We targeted glycogene ST6GAL1, which was expressed differently in Hca-F and Hca-P cells, and regulated the expression of ST6GAL1. The altered levels of ST6GAL1 were also responsible for changed invasive properties of Hca-F and Hca-P cells both in vitro and in vivo. These findings indicate a role for glycosylation modification as a mediator of tumor lymphatic metastasis, with its altered expression causing an invasive ability differentially. PMID:23840320

Hao, Lihong; Liu, Chunqing; Ma, Hongye; Jia, Li

2013-01-01

313

Genetic complexity of glucocorticoid-induced lysis of murine T-lymphoma cells.  

PubMed

Several well characterized murine T-lymphoma cell lines were used in somatic cell hybridization experiments to study the genetic regulation of glucocorticoid-induced lysis. Cell fusions were carried out among the SL12-derived cloned lines and between the W7 and SAK8 lines all of which have functional hormone receptors. These cell lines differ in their sensitivity to glucocorticoid-induced lysis. The resultant hybrids were characterized by their growth response to 1 microM dexamethasone, their hormone receptor content, their chromosome number, and the expression of surface antigens. Fusion of the hormone-sensitive W7 parent to a number of glucocorticoid-resistant cell lines resulted in hybrids which were of the sensitive phenotype. In contrast the fusion of another hormone-sensitive clone, SL12.4, with glucocorticoid-resistant SL12 clones or with SAK8 always resulted in hybrids resistant to glucocorticoid lysis. These results reveal a complex genetic regulation of the hormone response or the requirement for multiple gene activity in the mechanism for glucocorticoid-induced cell lysis. PMID:3875400

Weinroth, S E; MacLeod, C L; Minning, L; Hays, E F

1985-10-01

314

Characterization of a tachykinin peptide NK sub 2 receptor transfected into murine fibroblast B82 cells  

SciTech Connect

Membranes isolated from a murine fibroblast B82 cell line (SKLKB82{number sign}3) transfected with the bovine stomach cDNA pSKR56S exhibited binding of (His({sup 125}I){sup 1})neurokinin A ({sup 125}I-NKA) to a single population of sites with a B{sub max} of 147 fmol/mg of protein and a K{sub d} of 0.59 nM. The ligand binding in SKLKB82{number sign}3 cells was reversible. Thus, SKLKB82{number sign}3 cells have been transfected with NK{sub 2} receptors that have become associated with an endogenous guanine nucleotide-binding protein. In comparison with membranes from the hamster urinary bladder, a tissue enriched in NK{sub 2} receptors, NK{sub 2} receptor antagonists displayed markedly different potencies, either more or less potent, in inhibiting specific binding in membranes of the transfected cells. Furthermore, inhibition of {sup 125}I-NKA binding by nucleotide analogues was markedly different in SKLKB82{number sign}3 cells compared with hamster bladder tissue. The different binding profile in the cells is not due to an artefact introduced during cDNA transfection because a similar profile was also observed in bovine stomach membranes. These results may indicate the existence of two distinct NK{sub 2} receptors.

Van Giersbergen, P.L.M. (Marion Merrell Dow Research Inst., Cincinnati, OH (United States) Univ. of Cincinnati, OH (United States)); Shatzer, S.A.; Buck, S.H. (Marion Merrell Dow Research Inst., Cincinnati, OH (United States)); Henderson, A.K.; Lai, J.; Yamamura, Henry, I. (Univ. of Arizona, Tucson (United States)); Nakanishi, Shigetada (Kyoto Univ. (Japan))

1991-03-01

315

Dendritic Cells Produce CXCL13 and Participate in the Development of Murine Small Intestine Lymphoid Tissues  

PubMed Central

In the adult intestine, luminal microbiota induce cryptopatches to transform into isolated lymphoid follicles (ILFs), which subsequently act as sites for the generation of IgA responses. The events leading to this conversion are incompletely understood. Dendritic cells (DCs) are components of cryptopatches (CPs) and ILFs and were therefore evaluated in this process. We observed that the adult murine intestine contains clusters of DCs restricted to the CP/ILF continuum. A numerical and cell associative hierarchy in the adult intestine and a chronologic hierarchy in the neonatal intestine demonstrated that these clusters form after the coalescence of CD90+ cells to form CPs and before the influx of B220+ B lymphocytes to form ILFs. Cluster formation was dependent on lymphotoxin and the lymphotoxin ? receptor and independent of lymphocytes. The ILF DC population was distinguished from that of the lamina propria by the absence of CD4+CD11c+ cells and an increased proportion of CD11c+B220+ cells. The formation of clusters was not limited by DC numbers but was induced by luminal microbiota. Moreover, in the absence of the chemokine CXCL13, CP transformation into ILF was arrested. Furthermore, ILF DCs express CXCL13, and depletion of DCs resulted in regression of ILFs and disorganization of CPs. These results reveal DC participation in ILF transformation and maintenance and suggest that in part this may be due to CXCL13 production by these cells. PMID:20304952

McDonald, Keely G.; McDonough, Jacquelyn S.; Dieckgraefe, Brian K.; Newberry, Rodney D.

2010-01-01

316

Gene expression profiling of murine T-cell lymphoblastic lymphoma identifies deregulation of S-phase initiating genes.  

PubMed

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

Dabrowska, Magdalena Julia; Ejegod, Ditte; Lassen, Louise Berkhoudt; Johnsen, Hans Erik; Wabl, Matthias; Pedersen, Finn Skou; Dybkær, Karen

2013-10-01

317

Adenoviral Gene Transfer in Bovine Adrenomedullary and Murine Pheochromocytoma Cells: Potential Clinical and Therapeutic Relevance  

PubMed Central

Recombinant adenoviruses (rAd) have been widely used as gene transfer vectors both in the laboratory and in human clinical trials. In the present study, we investigated the effects of adenoviral-mediated gene transfer in primary bovine adrenal chromaffin cells (BACC) and a murine pheochromocytoma cell line (MPC). Cells were infected with one of three nonreplicating E1/E3-deleted (E1-/E3-) rAd vectors: Ad.GFP, expressing a green fluorescent protein (GFP); Ad.null, expressing no transgene; or Ad.C2.TK, expressing the herpes simplex virus-1 thymidine kinase gene (TK). Forty-eight hours after exposure to Ad.GFP, the percentage of GFP-expressing BACC ranged from 23.5-97% in a dose-dependent manner and similarly from 1.06 - 84.4% in the MPC, indicating that adrenomedullary cells are a potentially valuable target for adenoviral-mediated gene transfer. Ultrastructural analysis, however, revealed profound changes in the nucleus and mitochondria of cells infected with rAd. Furthermore, infection of BACC with Ad.null was accompanied by a time- and dose-dependent decrease in cell survival due to the vector alone. Specific whole-cell norepinephrine uptake was also decreased in a time- and dose-dependent fashion in BACC. Infection of MPC cells with the Ad.C2.TK vector sensitized them to the cytotoxic effect of the antiviral drug ganciclovir, in direct proportion to the fraction of cells infected with the virus. We conclude that rAd may alter the structural and functional integrity of adrenomedullary cells, potentially interfering with the normal stress response. At the same time, in light of their ability to effectively deliver and express genes in pheochromocytoma cells, they may be applicable to the gene therapy of adrenomedullary tumors. PMID:17525127

Alesci, Salvatore; Perera, Shiromi M.; Lai, Edwin W.; Kukura, Christina; Abu-Asab, Mones; Tsokos, Maria; Morris, John C.; Pacak, Karel

2008-01-01

318

Differentiation and selection of hepatocyte precursors in suspension spheroid culture of transgenic murine embryonic stem cells.  

PubMed

Embryonic stem cell-derived hepatocyte precursor cells represent a promising model for clinical transplantations to diseased livers, as well as for establishment of in vitro systems for drug metabolism and toxicology investigations. This study aimed to establish an in vitro culture system for scalable generation of hepatic progenitor cells. We used stable transgenic clones of murine embryonic stem cells possessing a reporter/selection vector, in which the enhanced green fluorescent protein- and puromycin N-acetyltransferase-coding genes are driven by a common alpha-fetoprotein gene promoter. This allowed for "live" monitoring and puromycin selection of the desired differentiating cell type possessing the activated alpha-fetoprotein gene. A rotary culture system was established, sequentially yielding initially partially selected hepatocyte lineage-committed cells, and finally, a highly purified cell population maintained as a dynamic suspension spheroid culture, which progressively developed the hepatic gene expression phenotype. The latter was confirmed by quantitative RT-PCR analysis, which showed a progressive up-regulation of hepatic genes during spheroid culture, indicating development of a mixed hepatocyte precursor-/fetal hepatocyte-like cell population. Adherent spheroids gave rise to advanced differentiated hepatocyte-like cells expressing hepatic proteins such as albumin, alpha-1-antitrypsin, cytokeratin 18, E-cadherin, and liver-specific organic anion transporter 1, as demonstrated by fluorescent immunostaining. A fraction of adherent cells was capable of glycogen storage and of reversible up-take of indocyanine green, demonstrating their hepatocyte-like functionality. Moreover, after transplantation of spheroids into the mouse liver, the spheroid-derived cells integrated into recipient. These results demonstrate that large-scale hepatocyte precursor-/hepatocyte-like cultures can be established for use in clinical trials, as well as in in vitro screening assays. PMID:23028675

Gabriel, Elke; Schievenbusch, Stephanie; Kolossov, Eugen; Hengstler, Jan G; Rotshteyn, Tamara; Bohlen, Heribert; Nierhoff, Dirk; Hescheler, Jürgen; Drobinskaya, Irina

2012-01-01

319

Expansion of murine gammaherpesvirus latently infected B cells requires T follicular help.  

PubMed

X linked lymphoproliferative disease (XLP) is an inherited immunodeficiency resulting from mutations in the gene encoding the slam associated protein (SAP). One of the defining characteristics of XLP is extreme susceptibility to infection with Epstein-Barr virus (EBV), a gammaherpesvirus belonging to the genus Lymphocryptovirus, often resulting in fatal infectious mononucleosis (FIM). However, infection of SAP deficient mice with the related Murine gammaherpesvirus 68 (MHV68), a gammaherpesvirus in the genus Rhadinovirus, does not recapitulate XLP. Here we show that MHV68 inefficiently establishes latency in B cells in SAP deficient mice due to insufficient CD4 T cell help during the germinal center response. Although MHV68 infected B cells can be found in SAP-deficient mice, significantly fewer of these cells had a germinal center phenotype compared to SAP-sufficient mice. Furthermore, we show that infected germinal center B cells in SAP-deficient mice fail to proliferate. This failure to proliferate resulted in significantly lower viral loads, and likely accounts for the inability of MHV68 to induce a FIM-like syndrome. Finally, inhibiting differentiation of T follicular helper (TFH) cells in SAP-sufficient C57Bl/6 mice resulted in decreased B cell latency, and the magnitude of the TFH response directly correlated with the level of infection in B cells. This requirement for CD4 T cell help during the germinal center reaction by MHV68 is in contrast with EBV, which is thought to be capable of bypassing this requirement by expressing viral proteins that mimic signals provided by TFH cells. In conclusion, the outcome of MHV68 infection in mice in the setting of loss of SAP function is distinct from that observed in SAP-deficient patients infected with EBV, and may identify a fundamental difference between the strategies employed by the rhadinoviruses and lymphocryptoviruses to expand B cell latency during the early phase of infection. PMID:24789087

Collins, Christopher M; Speck, Samuel H

2014-05-01

320

Establishment of a Murine Graft-versus-Myeloma Model Using Allogeneic Stem Cell Transplantation  

PubMed Central

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

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

321

Characterization of the high-affinity cell-surface receptor for murine B-cell-stimulating factor 1  

SciTech Connect

Radiolabeled recombinant murine B-cell-stimulatory factory 1 (BSF-1) was used to characterize receptors specific for this lymphokine on the surface of primary B and T cells and in vitro cell lines representing the B-cell, T-cell, mast cell, macrophage, and myelomonocytic lineages. BSF-1 binding was rapid and saturable at 4/sup 0/C and 37/sup 0/C with a slow dissociation rate. On all cell types examined, BSF-1 bound to a single class of high-affinity receptor (<20000 receptors per cell) with a K/sub a/ of 10/sup 10/-10/sup 11/ M/sup -1/. Receptor expression on resting primary B and T cells was low (<100 receptors per cell), whereas activation with lipopolysaccharide or Con A produced a 5- to 10-fold increase in receptor numbers. Among a panel of lymphokines and growth hormones, only unlabeled BSF-1 was able to compete for the binding of /sup 125/I-labeled BSF-1. Affinity crosslinking experiments resulted in the identification on all cells tested on a receptor protein with an average M/sub r/ of 75,000.

Park, L.S.; Friend, D.; Grabstein, K.; Urdal, D.L.

1987-03-01

322

Protease inhibitors selectively block T cell receptor-triggered programmed cell death in a murine T cell hybridoma and activated peripheral T cells  

PubMed Central

The hypothesis that cytoplasmic proteases play a functional role in programmed cell death was tested by examining the effect of protease inhibitors on the T cell receptor-mediated death of the 2B4 murine T cell hybridoma and activated T cells. The cysteine protease inhibitors trans-epoxysuccininyl-L-leucylamido-(4-guanidino) butane (E-64) and leupeptin, the calpain selective inhibitor acetyl-leucyl-leucyl- normethional, and the serine protease inhibitors diisopropyl fluorophosphate and phenylmethylsulfonyl fluoride, all showed dose- dependent blocking of the 2B4 death response triggered by the T cell receptor complex and by anti-Thy-1. These protease inhibitors enhanced rather than inhibited IL-2 secretion triggered by T cell receptor cross- linking, showing that they did not act by preventing signal transduction. Growth inhibition induced by cross-linking the 2B4 T cell receptor, measured by inhibition of thymidine incorporation, was not generally blocked by these protease inhibitors. All five of these protease inhibitors enhanced rather than blocked 2B4 cell death triggered by dexamethasone, an agent previously shown to have a death pathway antagonistic with that of the TCR. 2B4 cytolysis by the cytotoxic agents staphylococcal alpha-toxin and dodecyl imidazole, and that caused by hypotonic conditions, was not significantly affected by the five protease inhibitors tested. The selected protease inhibitors blocked both the apoptotic nuclear morphology changes and DNA fragmentation induced by T cell receptor cross-linking, and enhanced both these properties induced by dexamethasone in 2B4 cells. The T cell receptor-induced death of activated murine lymph node T cells and human peripheral blood CD4+ T cells was blocked by both cysteine and serine protease inhibitors, showing that the protease-dependent death pathway also operates in these systems. PMID:8228816

1993-01-01

323

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

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 University of New York, Stony Brook, NY 11794, USA 4 National Synchrotron Light Source, Brookhaven National

Terasaki, Mark

324

Bifidobacterium breve Attenuates Murine Dextran Sodium Sulfate-Induced Colitis and Increases Regulatory T Cell Responses  

PubMed Central

While some probiotics have shown beneficial effects on preventing or treating colitis development, others have shown no effects. In this study, we have assessed the immunomodulating effects of two probiotic strains, Lactobacillus rhamnosus (L. rhamnosus) and Bifidobacterium breve (B. breve) on T cell polarization in vitro, using human peripheral blood mononuclear cells (PBMC), and in vivo, using murine dextran sodium sulfate (DSS) colitis model. With respect to the latter, the mRNA expression of T cell subset-associated transcription factors and cytokines in the colon was measured and the T helper type (Th) 17 and regulatory T cell (Treg) subsets were determined in the Peyer's patches. Both L. rhamnosus and B. breve incubations in vitro reduced Th17 and increased Th2 cell subsets in human PBMCs. In addition, B. breve incubation was also able to reduce Th1 and increase Treg cell subsets in contrast to L. rhamnosus. In vivo intervention with B. breve, but not L. rhamnosus, significantly attenuated the severity of DSS-induced colitis. In DSS-treated C57BL/6 mice, intervention with B. breve increased the expression of mRNA encoding for Th2- and Treg-associated cytokines in the distal colon. In addition, intervention with B. breve led to increases of Treg and decreases of Th17 cell subsets in Peyer's patches of DSS-treated mice. B. breve modulates T cell polarization towards Th2 and Treg cell-associated responses in vitro and in vivo. In vivo B. breve intervention ameliorates DSS-induced colitis symptoms and this protective effect may mediated by its effects on the T-cell composition. PMID:24787575

Zheng, Bin; van Bergenhenegouwen, Jeroen; Overbeek, Saskia; van de Kant, Hendrik J. G.; Garssen, Johan; Folkerts, Gert; Vos, Paul; Morgan, Mary E.; Kraneveld, Aletta D.

2014-01-01

325

Effects of brevetoxins on murine myeloma SP2/O cells: Aberrant cellular division  

USGS Publications Warehouse

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.

Han, T. K.; Derby, M.; Martin, D. F.; Wright, S. D.; Dao, M. L.

2003-01-01

326

Characterization of peripheral benzodiazepine type sites in a cultured murine BV-2 microglial cell line.  

PubMed

It is known that the density of peripheral benzodiazepine receptors (PBR) increases after brain damage. Astrocytes are among the cell types where PBR ligand binding has been detected and may be involved in the response to neuronal injury and regeneration. Consistent with the hypothesis, the apparent density of PBR sites in astrocytes is increased by both cytokines and neurotoxins. However, microglia, the resident macrophages which represent 5-15% of glial cell populations have not been evaluated for the presence of the PBR. In the present study, we report the presence of [3H]Ro5-4864 binding in microglial cells. In particular, we used BV-2 cells, an immortalized cell line of murine microglial cells. High affinity binding of [3H]Ro5-4864 to a single site was detected in membranes prepared from BV-2 cells (KD = 4.4 nM, Bmax = 3,800 fmoles/mg protein). Various ligands for the PBR displaced [3H]Ro5-4864 binding with the following rank order of potencies: PK11195 = Ro5-4864 > FGIN-1-27 > triazolam = diazepam > beta-pro-pyl-beta-carboline-3-carboxylate = clonazepam > lorazepam = flurazepam > chlordiazepoxide = clorazepate. Subcellular fractionationstudies indicate that the majority of the Ro5-4864 binding sites is in the mitochondrial fraction. The remainder is found in nonmitochondrial cell fractions. The [3H]Ro5-4864 binding observed on intact cells had characteristics similar to those found on membranes. The presence of a high density of PBRs in these cells establish the basis for additional investigations into their possible functional role, if any, in the microglial response to neuronal injury. PMID:8787774

Park, C H; Carboni, E; Wood, P L; Gee, K W

1996-01-01

327

An increase in telomere sister chromatid exchange in murine embryonic stem cells possessing critically shortened telomeres  

SciTech Connect

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.

Wang, Yisong [ORNL; Giannone, Richard J [ORNL; Wu, Jun [ORNL; Gomez, Marla V [ORNL; Liu, Yie [ORNL

2005-01-01

328

Bifidobacterium breve attenuates murine dextran sodium sulfate-induced colitis and increases regulatory T cell responses.  

PubMed

While some probiotics have shown beneficial effects on preventing or treating colitis development, others have shown no effects. In this study, we have assessed the immunomodulating effects of two probiotic strains, Lactobacillus rhamnosus (L. rhamnosus) and Bifidobacterium breve (B. breve) on T cell polarization in vitro, using human peripheral blood mononuclear cells (PBMC), and in vivo, using murine dextran sodium sulfate (DSS) colitis model. With respect to the latter, the mRNA expression of T cell subset-associated transcription factors and cytokines in the colon was measured and the T helper type (Th) 17 and regulatory T cell (Treg) subsets were determined in the Peyer's patches. Both L. rhamnosus and B. breve incubations in vitro reduced Th17 and increased Th2 cell subsets in human PBMCs. In addition, B. breve incubation was also able to reduce Th1 and increase Treg cell subsets in contrast to L. rhamnosus. In vivo intervention with B. breve, but not L. rhamnosus, significantly attenuated the severity of DSS-induced colitis. In DSS-treated C57BL/6 mice, intervention with B. breve increased the expression of mRNA encoding for Th2- and Treg-associated cytokines in the distal colon. In addition, intervention with B. breve led to increases of Treg and decreases of Th17 cell subsets in Peyer's patches of DSS-treated mice. B. breve modulates T cell polarization towards Th2 and Treg cell-associated responses in vitro and in vivo. In vivo B. breve intervention ameliorates DSS-induced colitis symptoms and this protective effect may mediated by its effects on the T-cell composition. PMID:24787575

Zheng, Bin; van Bergenhenegouwen, Jeroen; Overbeek, Saskia; van de Kant, Hendrik J G; Garssen, Johan; Folkerts, Gert; Vos, Paul; Morgan, Mary E; Kraneveld, Aletta D

2014-01-01

329

Infection of Xenotransplanted Human Cell Lines by Murine Retroviruses: A Lesson Brought Back to Light by XMRV.  

PubMed

Infection of xenotransplanted human cells by xenotropic retroviruses is a known phenomenon in the scientific literature, with examples cited since the early 1970s. However, arguably, until recently, the importance of this phenomenon had not been largely recognized. The emergence and subsequent debunking of Xenotropic Murine leukemia virus-Related Virus (XMRV) as a cell culture contaminant as opposed to a potential pathogen in several human diseases, notably prostate cancer and Chronic Fatigue Syndrome, highlighted a potential problem of murine endogenous gammaretroviruses infecting commonly used human cell lines. Subsequent to the discovery of XMRV, many additional cell lines that underwent xenotransplantation in mice have been shown to harbor murine gammaretroviruses. Such retroviral infection poses the threat of not only confounding experiments performed in these cell lines via virus-induced changes in cellular behavior but also the potential infection of other cell lines cultured in the same laboratory. Thus, the possibility of xenotropic retroviral infection of cell lines may warrant additional precautions, such as periodic testing for retroviral sequences in cell lines cultured in the laboratory. PMID:23785669

Hempel, Heidi A; Burns, Kathleen H; De Marzo, Angelo M; Sfanos, Karen S

2013-01-01

330

Brain Ingress of Regulatory T Cells in a Murine Model of HIV-1 Encephalitis  

PubMed Central

CD4+CD25+ regulatory T cells (Treg) transform the HIV-1 infected macrophage from a neurotoxic to a neuroprotective phenotype. This was demonstrated previously in a murine model of HIV-1 encephalitis induced by intracranial injection of HIV-1/vesicular stomatitis virus-infected bone marrow macrophages. Relationships between Treg ingress of end organ tissues, notably the brain, and neuroprotection were investigated. Treg from EGFP-transgenic donor mice were expanded, labeled with indium-111, and adoptively transferred. Treg distribution was assayed by computed tomography/single photon emission computed tomography and immunohistochemistry. Treg readily migrated across the blood brain barrier and were retained within virus-induced neuroinflammatory sites. In non-inflamed peripheral tissues (liver and spleen) Treg were depleted. These observations demonstrate that Treg migrate to sites of inflammation and modulate immune responses at disease sites. PMID:20846730

Gong, Nan; Liu, Jianuo; Reynolds, Ashley D.; Gorantla, Santhi; Mosley, R. Lee; Gendelman, Howard E.

2010-01-01

331

Photothermal Response of Human and Murine Cancer Cells to Multiwalled Carbon Nanotubes after Laser Irradiation  

PubMed Central

This study demonstrates the capability of multiwalled carbon nanotubes (MWNTs) coupled with laser irradiation to enhance treatment of cancer cells through enhanced and more controlled thermal deposition, increased tumor injury, and diminished heat shock protein (HSP) expression. We also explored the potential promise of MWNTs as drug delivery agents by observing the degree of intracellular uptake of these nanoparticles. To determine the heat generation capability of MWNTs, the absorption spectra and temperature rise during heating were measured. Higher optical absorption was observed for MWNTs in water compared with water alone. For identical laser parameters, MWNT-containing samples produced a significantly greater temperature elevation compared to samples treated with laser alone. Human prostate cancer (PC3) and murine renal carcinoma (RENCA) cells were irradiated with a 1,064-nm laser with an irradiance of 15.3 W/cm2 for 2 heating durations (1.5 and 5 minutes) alone or in combination with MWNT inclusion. Cytotoxicity and HSP expression following laser heating was used to determine the efficacy of laser treatment alone or in combination with MWNTs. No toxicity was observed for MWNTs alone. Inclusion of MWNTs dramatically decreased cell viability and HSP expression when combined with laser irradiation. MWNT cell internalization was measured using fluorescence and transmission electron microscopy following incubation of MWNTs with cells. With increasing incubation duration, a greater number of MWNTs were observed in cellular vacuoles and nuclei. These findings offer an initial proof of concept for the application of MWNTs in cancer therapy. PMID:21098701

Fisher, Jessica W.; Sarkar, Saugata; Buchanan, Cara F.; Szot, Christopher S.; Whitney, Jon; Hatcher, Heather C.; Torti, Suzy V.; Rylander, Christopher G.; Rylander, Marissa Nichole

2013-01-01

332

Anti-melanogenic property of geoditin A in murine B16 melanoma cells.  

PubMed

Geoditin A, an isomalabaricane triterpene isolated from marine sponge Geodia japonica, has been demonstrated to induce apoptosis in leukemia HL60 cells and human colon HT29 cancer cells through an oxidative stress, a process also interfering with normal melanogenesis in pigment cells. Treatment of murine melanoma B16 cells with geoditin A decreased expression of melanogenic proteins and cell melanogenesis which was aggravated with adenylate cyclase inhibitor SQ22536, indicating melanogenic inhibition was mediated through a cAMP-dependent signaling pathway. Immunofluorescence microscopy and glycosylation studies revealed abnormal glycosylation patterns of melanogenic proteins (tyrosinase and tyrosinase-related protein 1), and a co-localization of tyrosinase with calnexin (CNX) and lysosome-associated membrane protein 1 (LAMP-1), implicating a post-translational modification in the ER and a degradation of tyrosinase in the lysosome. Taken together, potent anti-melanogenic property and the relatively low cytotoxicity of geoditin A have demonstrated its therapeutic potential as a skin lightening agent. PMID:22412813

Cheung, Florence W K; Guo, Jia; Ling, Yick-Hin; Che, Chun-Tao; Liu, Wing-Keung

2012-02-01

333

Identification of Immunomodulatory Signatures Induced by American Ginseng in Murine Immune Cells  

PubMed Central

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

Yan, Jian; Ma, Yonghui; Zhao, Fusheng; Gu, Weikuan; Jiao, Yan

2013-01-01

334

Heparin effect on DNA synthesis in a murine fibrosarcoma cell line: influence of anionic density  

SciTech Connect

The effects of heparin subfractions on DNA synthesis in a murine cutaneous fibrosarcoma cell line were examined. Porcine mucosal heparin was preparatively fractionated for anionic charge density by DEAE-Sephadex chromatography and for molecular weight by Sephadex G-100 filtration. The cell line was plated from confluent monolayer cultures and grown in medium and fetal bovine serum, with or without a heparin fraction at a final concentration of 10 micrograms/ml. At intervals thereafter, the cells were pulsed with (/sup 3/H)thymidine. A low-charge density heparin fraction stimulated (/sup 3/H)thymidine incorporation (cpm/mg protein and cpm/cell) during the first 3 days of growth compared to control values without added heparin, whereas a high-charge density heparin fraction had little of this effect (186 +/- 35% of control vs. 101 +/- 14%, respectively; P less than .05). The augmentation of DNA synthesis observed with the low-charge density fraction correlated with increased proportions of cells in S and G2 phases compared with those of the controls, as determined by flow cytofluorometry. Low- and high-molecular-weight heparin fractions did not significantly alter DNA synthesis. Heparin subfractions are thus heterogeneous with respect to their effect on cellular DNA synthesis in this tumor line.

Piepkorn, M.W.; Daynes, R.A.

1983-09-01

335

Silencing of GM3 synthase suppresses lung metastasis of murine breast cancer cells  

PubMed Central

Background Gangliosides are sialic acid containing glycosphingolipids that are ubiquitously distributed on vertebrate plasma membranes. GM3, a precursor for most of the more complex ganglioside species, is synthesized by GM3 synthase. Although total ganglioside levels are significantly higher in breast tumor tissue than in normal mammary tissue, the roles played by gangliosides in breast cancer formation and metastasis are not clear. Methods To investigate the roles of gangliosides in breast tumor development, GM3 synthase was silenced in the highly metastatic 4T1 cells and over-expressed in the non-metastatic 67NR cells. The behavior of breast cancer cells was examined in vitro using migration assay, invasion assay, and soft agar assay. Tumor formation and metastasis in vivo were examined using a well established mouse mammary tumor model. Results GM3 synthase silencing in 4T1 cells significantly inhibited cell migration, invasion and anchorage-independent growth in vitro, and lung metastasis in vivo. In addition, over-expression of GM3 synthase in nonmetastatic 67NR cells significantly induced cell migration and anchorage-independent growth. Further studies indicated that activation of the phosphoinositide-3 kinase/Akt pathway, and consequently inhibition of nuclear factor of activated T cell (NFAT)1 expression, could be the mechanism underlying the suppression of breast cancer migration/invasion induced by GM3 synthase silencing. Conclusion Our findings indicate that GM3 synthase silencing suppressed lung metastasis in murine breast cancer cells. The molecular mechanism that underlies GM3 synthase mediated migration and invasion was inhibition of the phosphoinositide-3 kinase/Akt pathway. The findings suggest that GM3 synthase may be of value as a therapeutic target in breast cancer. PMID:18171481

Gu, Yuchao; Zhang, Junhua; Mi, Wenyi; Yang, Jing; Han, Feng; Lu, Xinzhi; Yu, Wengong

2008-01-01

336

Murine Splenic CD4+ T Cells, Induced by Innate Immune Cell Interactions and Secreted Factors, Develop Antileukemia Cytotoxicity.  

PubMed

Inciting the cellular arm of adaptive immunity has been the fundamental goal of cancer immunotherapy strategies, specifically focusing on inducing tumor antigen-specific responses by CD8(+) cytotoxic T lymphocytes (CTL). However, there is an emerging appreciation that the cytotoxic function of CD4(+) T cells can be effective in a clinical setting. Harnessing this potential will require an understanding of how such cells arise. In this study, we use an IL12-transduced variant of the 70Z/3 leukemia cell line in a B6D2F1 (BDF1) murine model system to reveal a novel cascade of cells and soluble factors that activate anticancer CD4(+) killer cells. We show that natural killer T cells play a pivotal role by activating dendritic cells in a contact-dependent manner; soluble products of this interaction, including MCP-1, propagate the activation signal, culminating in the development of CD4(+) CTLs that directly mediate an antileukemia response while also orchestrating a multipronged attack by other effector cells. A more complete picture of the conditions that induce such a robust response will allow us to capitalize on CD4(+) T-cell plasticity for maximum therapeutic effect. Cancer Immunol Res; 2(11); 1113-24. ©2014 AACR. PMID:25154710

Nelles, Megan E; Moreau, Joshua M; Furlonger, Caren L; Berger, Alexandra; Medin, Jeffrey A; Paige, Christopher J

2014-11-01

337

Maintenance and induction of murine embryonic stem cell differentiation using E-cadherin-Fc substrata without colony formation  

NASA Astrophysics Data System (ADS)

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.

Meng, Qing-Yuan; Akaike, Toshihiro

2013-03-01

338

Tsc2 Null Murine Neuroepithelial Cells Are a Model for Human Tuber Giant Cells, and Show Activation of an mTOR Pathway  

Microsoft Academic Search

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

Hiroaki Onda; Peter B. Crino; Hongbing Zhang; Ryan D. Murphey; Luca Rastelli; Bonnie E. Gould Rothberg; David J. Kwiatkowski

2002-01-01

339

CpG-matured Murine Plasmacytoid Dendritic Cells Are Capable of In Vivo Priming of Functional CD8 T Cell Responses to Endogenous but Not Exogenous Antigens  

Microsoft Academic Search

Plasmacytoid dendritic cells (PDCs) are a unique leukocyte population capable of secreting high levels of type I interferon (IFN) in response to viruses and bacterial stimuli. In vitro exper- iments have shown that upon maturation, human and murine PDCs develop into potent immunostimulatory cells; however, their ability to prime an immune response in vivo remains to be addressed. We report

Mariolina Salio; Michael J. Palmowski; Ann Atzberger; Ian F. Hermans; Vincenzo Cerundolo

340

Functional differentiation of stem cell-derived neurons from different murine backgrounds  

PubMed Central

Murine stem cell-derived neurons have been used to study a wide variety of neuropsychiatric diseases with a hereditary component, ranging from autism to Alzheimer’s. While a significant amount of data on their molecular biology has been generated, there is little data on the physiology of these cultures. Different mouse strains show clear differences in behavioral and other neurobiologically relevant readouts. We have studied the physiology of early differentiation and network formation in neuronal cultures derived from three different mouse embryonic stem cell lines. We have found largely overlapping patterns with some significant differences in the timing of the functional milestones. Neurons from R1 showed the fastest development of intrinsic excitability, while E14Tg2a and J1 were slower. This was also reflected in an earlier appearance of synaptic activity in R1 cultures, while E14Tg2a and J1 were delayed by up to 2 days. In conclusion, stem cells from all backgrounds could be successfully differentiated into functioning neural networks with similar developmental patterns. Differences in the timing of specific milestones, suggest that control cell lines and time-points should be carefully chosen when investigating genetic alterations that lead to subtle deficits in neuronal function. PMID:24600351

Barth, Lydia; Sutterlin, Rosmarie; Nenniger, Markus; Vogt, Kaspar E.

2014-01-01

341

OCILRP2 signaling synergizes with LPS to induce the maturation and differentiation of murine dendritic cells.  

PubMed

Osteoclast Inhibitory Lectin-related Protein 2 (OCILRP2) is a typical type II transmembrane protein and belongs to C-type lectin-related protein family. It is preferentially expressed in dendritic cells (DC), B lymphocytes, and activated T lymphocytes. Upon binding to its ligand, OCILRP2 can promote CD28-mediated co-stimulation and enhance T cell activation. However, the role of OCILRP2 in DC development and activation is unclear. In this report, we present evidence that recombinant protein OCILRP2-Fc inhibits the generation and LPS-induced maturation of murine bone marrow-derived dendritic cells (BMDCs) by downregulating the expression of CD11c, MHC-II, and co-stimulators CD80 and CD86. OCILRP2-Fc also reduces the capacity of BMDCs to take up antigens, activates T cells, and secret inflammatory cytokines such as IL-6, IL-12, and TNF-?. Additionally, we show that OCILRP2-Fc may cause the aforementioned effects through inhibiting NF-?B activation. Therefore, OCILRP2 is a new regulator of DC maturation and differentiation following TLR4 activation. PMID:24631687

Chai, Lihui; Wu, Suxia; Liu, Guangchao; Wang, Zhanzheng; Tian, Wenzhi; Ma, Yuanfang

2014-04-18

342

Radiosensitizing and toxic effects of RSU-1069 on hypoxic cells in a murine tumor  

SciTech Connect

RSU-1069 is one of a group of compounds of particular interest in radiobiology, since it combines the nitroimidazole ring with a side chain bearing a monofunctional alkylating agent. This compound has been shown to be a potent radiosensitizer both in vitro and in vivo. Furthermore, it has recently been shown to be an effective hypoxic cell cytotoxin in vitro. Our studies have been carried out using the SCCVII squamous carcinoma implanted subcutaneously in C/sub 3/H mice, using a technique we recently developed which facilitates isolation of tumor cell subpopulations from known locations relative to the tumor blood supply. The response of the separated tumor subpopulations was assessed using a soft agar clonogenic assay. For radiosensitization studies, RSU-1069 was administered i.p. at 0.5 mumol/g 20 min before irradiation and the tumors excised 20 min after irradiation. For toxicity studies, tumors were excised 16-18 hr after RSU-1069 administration. The results obtained to date clearly demonstrate that RSU-1069 is an efficient hypoxic cell radiosensitizer and cytotoxin in this murine tumor and has little effect on well perfused (i.e., oxic) cells.

Chaplin, D.J.; Durand, R.E.; Stratford, I.J.; Jenkins, T.C.

1986-07-01

343

Neddylation plays an important role in the regulation of murine and human dendritic cell function  

PubMed Central

Posttranslational protein modifications (PTMs) are necessary for cells to function properly. The role of PTMs in regulating immune responses, specifically those mediated by dendritic cells (DCs), which are critical for both innate and adaptive immunity, is not well understood. Utilizing multiple but complementary approaches, we determined the role of an important but less understood type of PTM, namely, neddylation, in regulating DC functions. Inhibition of neddylation suppressed the release of proinflammatory cytokines by DCs in response to Toll-like receptor, nucleotide oligomerization domain–like receptor, and noninfectious CD40L stimulation. These effects were more profound than those mediated by the proteasome inhibitor bortezomib or a commonly used antiinflammatory agent, dexamethasone. Targeting neddylation also suppressed the ability of DCs to stimulate murine allogeneic T cells in vitro and in vivo and human allogeneic T-cell responses in vitro. Mechanistic studies demonstrated that inhibition of neddylation reduced both canonical and noncanonical nuclear factor-?B (NF-?B) activity. Neddylation inhibition prevented the degradation of inhibitor-?B and thus reduced the translocation and activation of NF-?B, but without perturbation of the mitogen-activated protein kinase/extracellular signal-regulated kinase pathway. Thus, blocking neddylation could be a novel strategy for mitigating immune-mediated disease processes. PMID:23863900

Mathewson, Nathan; Toubai, Tomomi; Kapeles, Steven; Sun, Yaping; Oravecz-Wilson, Katherine; Tamaki, Hiroya; Wang, Ying; Hou, Guoqing; Sun, Yi

2013-01-01

344

NF-?B-Dependent Induction of Cathelicidin-Related Antimicrobial Peptide in Murine Mast Cells by Lipopolysaccharide  

Microsoft Academic Search

Background: An important aspect of the innate immune response to pathogens is the production of anti-microbial peptides such as cathelicidin-related antimicrobial peptide (CRAMP), the murine homologue of human cathelicidin LL-37. In this study, mechanisms regulating LPS-induction of CRAMP gene expression in mast cells were investigated. NF-?B and MAPK pathways were the focus of investigation. Methods: Mouse bone marrow-derived mast cells

Guiming Li; Joanne Domenico; Yi Jia; Joseph J. Lucas; Erwin W. Gelfand

2009-01-01

345

HPV 16 E2 Protein Induces Apoptosis in Human and Murine HPV 16 Transformed Epithelial Cells and Has Antitumoral Effects in vivo  

Microsoft Academic Search

Objective: Our aims were to examine the ability of the human papillomaviruse (HPV) 16 E2 protein to induce apoptosis in a murine HPV-transformed cell line, and to evaluate its antitumor properties on HPV-associated tumors in vivo in immunocompetent mice. Methods: HPV-transformed murine BMK-16\\/myc cells and human SiHa cells were transfected with the HPV 16 E2 gene to examine the effects

V. H. Bermúdez-Morales; O. Peralta-Zaragoza; E. Guzmán-Olea; A. García-Carrancá; M. Bahena-Román; J. M. Alcocer-González; V. Madrid-Marina

2009-01-01

346

Generation of Ugt1-Deficient Murine Liver Cell Lines Using TALEN Technology  

PubMed Central

The Crigler-Najjar Syndrome Type I (CNSI) is a rare genetic disorder caused by mutations in the Ugt1a1 gene. It is characterized by unconjugated hyperbilirubinemia that may result in severe neurologic damage and death if untreated. To date, liver transplantation is the only curative treatment. With the aim of generating mutant cell lines of the Ugt1 gene, we utilized the TALEN technology to introduce site-specific mutations in Ugt1 exon 4. We report a fast and efficient method to perform gene knockout in tissue culture cells, based on the use of TALEN pairs targeting restriction enzyme (RE) sites in the region of interest. This strategy overcame the presence of allele-specific single nucleotide polymorphisms (SNPs) and pseudogenes, conditions that limit INDELs' detection by Surveyor. We obtained liver-derived murine N-Muli cell clones having INDELs with efficiency close to 40%, depending on the TALEN pair and RE target site. Sequencing of the target locus and WB analysis of the isolated cell clones showed a high proportion of biallelic mutations in cells treated with the most efficient TALEN pair. Ugt glucuronidation activity was reduced basal levels in the biallelic mutant clones. These mutant liver-derived cell lines could be a very useful tool to study biochemical aspects of Ugt1 enzyme activity in a more natural context, such as substrate specificity, requirement of specific co-factors, the study of inhibitors and other pharmacological aspects, and to correlate enzyme activity to the presence of specific mutations in the gene, by adding back to the mutant cell clones specific variants of the Ugt1 gene. In addition, since genome editing has recently emerged as a potential therapeutic approach to cure genetic diseases, the definition of the most efficient TALEN pair could be an important step towards setting up a platform to perform genome editing in CNSI. PMID:25118822

Porro, Fabiola; Bockor, Luka; De Caneva, Alessia; Bortolussi, Giulia; Muro, Andres F.

2014-01-01

347

Sustained CTL activation by murine pulmonary epithelial cells promotes the development of COPD-like disease  

PubMed Central

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

Borchers, Michael T.; Wesselkamper, Scott C.; Curull, Victor; Ramirez-Sarmiento, Alba; Sanchez-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

348

Bromelain Inhibits Allergic Sensitization and Murine Asthma via Modulation of Dendritic Cells  

PubMed Central

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

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

349

Stimulatory Effects of Polysaccharide Fraction from Solanum nigrum on RAW 264.7 Murine Macrophage Cells  

PubMed Central

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

Razali, Faizan Naeem; Ismail, Amirah; Abidin, Nurhayati Zainal; Shuib, Adawiyah Suriza

2014-01-01

350

The Acute Exposure Effects of Inhaled Nickel Nanoparticles on Murine Endothelial Progenitor Cells  

PubMed Central

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

Liberda, Eric N; Cuevas, Azita K; Qu, Qingshan; Chen, Lung Chi

2014-01-01

351

Stimulatory Effects of Polysaccharide Fraction from Solanum nigrum on RAW 264.7 Murine Macrophage Cells.  

PubMed

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

Razali, Faizan Naeem; Ismail, Amirah; Abidin, Nurhayati Zainal; Shuib, Adawiyah Suriza

2014-01-01

352

Two conserved essential motifs of the murine immunoglobulin lambda enhancers bind B-cell-specific factors.  

PubMed Central

Two highly homologous enhancers associated with the two murine immunoglobulin lambda constant-region clusters were recently identified. In order to better understand the molecular basis for the developmental stage- and cell-type-restricted expression of lambda genes, we have undertaken an analysis of the putative regulatory domains of these enhancers. By using a combination of DNase I footprinting, electrophoretic mobility shift assay, and site-specific mutations, four candidate protein binding sites have been identified at analogous positions in both enhancers. A mutation of any of these sites decreases enhancer activity. Two of the sites, lambda A and lambda B, are essential for enhancer function, and both of these sites appear to bind both B-cell-specific and general factors. Nevertheless, isolated lambda A and lambda B sites show no evidence of inherent transactivating potential, alone or together, even when present in up to three copies. We suggest that the generation of transactivating signals from these enhancers may require the complex interaction of multiple B-cell-specific and nonspecific DNA-binding factors. Images PMID:1729607

Rudin, C M; Storb, U

1992-01-01

353

Menstrual Blood-derived Cells Confer Human Dystrophin Expression in the Murine Model of Duchenne Muscular Dystrophy via Cell Fusion and Myogenic Transdifferentiation  

PubMed Central

Duchenne muscular dystrophy (DMD), the most common lethal genetic disorder in children, is an X-linked recessive muscle disease characterized by the absence of dystrophin at the sarcolemma of muscle fibers. We examined a putative endometrial progenitor obtained from endometrial tissue samples to determine whether these cells repair muscular degeneration in a murine mdx model of DMD. Implanted cells conferred human dystrophin in degenerated muscle of immunodeficient mdx mice. We then examined menstrual blood–derived cells to determine whether primarily cultured nontransformed cells also repair dystrophied muscle. In vivo transfer of menstrual blood–derived cells into dystrophic muscles of immunodeficient mdx mice restored sarcolemmal expression of dystrophin. Labeling of implanted cells with enhanced green fluorescent protein and differential staining of human and murine nuclei suggest that human dystrophin expression is due to cell fusion between host myocytes and implanted cells. In vitro analysis revealed that endometrial progenitor cells and menstrual blood–derived cells can efficiently transdifferentiate into myoblasts/myocytes, fuse to C2C12 murine myoblasts by in vitro coculturing, and start to express dystrophin after fusion. These results demonstrate that the endometrial progenitor cells and menstrual blood–derived cells can transfer dystrophin into dystrophied myocytes through cell fusion and transdifferentiation in vitro and in vivo. PMID:17314403

Cui, Chang-Hao; Uyama, Taro; Miyado, Kenji; Terai, Masanori; Kyo, Satoru; Kiyono, Tohru

2007-01-01

354

Association of murine lupus and thymic full-length endogenous retroviral expression maps to a bone marrow stem cell  

SciTech Connect

Recent studies of thymic gene expression in murine lupus have demonstrated 8.4-kb (full-length size) modified polytropic (Mpmv) endogenous retroviral RNA. In contrast, normal control mouse strains do not produce detectable amounts of such RNA in their thymuses. Prior studies have attributed a defect in experimental tolerance in murine lupus to a bone marrow stem cell rather than to the thymic epithelium; in contrast, infectious retroviral expression has been associated with the thymic epithelium, rather than with the bone marrow stem cell. The present study was designed to determine whether the abnormal Mpmv expression associated with murine lupus mapped to thymic epithelium or to a marrow precursor. Lethally irradiated control and lupus-prone mice were reconstituted with T cell depleted bone marrow; one month later their thymuses were studied for endogenous retroviral RNA and protein expression. Recipients of bone marrow from nonautoimmune donors expressed neither 8.4-kb Mpmv RNA nor surface MCF gp70 in their thymuses. In contrast, recipients of bone marrow from autoimmune NZB or BXSB donors expressed thymic 8.4-kb Mpmv RNA and mink cell focus-forming gp70. These studies demonstrate that lupus-associated 8.4-kb Mpmv endogenous retroviral expression is determined by bone marrow stem cells.

Krieg, A.M.; Gourley, M.F.; Steinberg, A.D. (National Institutes of Health, Bethesda, MD (USA))

1991-05-01

355

Non-genomic actions of estradiol and 4-OH-tamoxifen on murine breast cancer cells.  

PubMed

Estrogens and tamoxifen do not only exert their effects at the genomic level, but also play a role at the cell membrane activating downstream signaling pathways. We recently characterized an estrogen receptor-positive epithelial murine breast cancer cell line, LM05-E. Utilizing this cell line and MCF-7 cells, we compared the non-genomic effects of estradiol and 4-OH-tamoxifen. We showed that, similar to estradiol, tamoxifen activated the MAPK/ERK 1/2 pathway; however, we did not find activation of PI3K/AKT by either estradiol or tamoxifen. Short-term treatments with estradiol stimulated, whereas tamoxifen inhibited cell proliferation. Using pharmacological inhibitors we showed that the effect of estradiol was mediated by the MAPK/ERK 1/2 pathway, but that inhibition of this pathway did not affect tamoxifen. Surprisingly, however, blocking of PI3K/AKT signaling interfered with the inhibitory effect of tamoxifen. Analysis of the involvement of the EGFR support previous findings that designate this receptor as a mediator of the non-genomic effects of estradiol; blocking EGFR also reverses the inhibitory effect of tamoxifen. Finally, matrix metalloproteinases (MMPs) were confirmed to be involved in the proliferative effect of estradiol. These results demonstrated the novel non-genomic effects of tamoxifen and revealed that pathways downstream of EGFR and PI3K/AKT are involved in the inhibition of cell proliferation. Caution should be exercised when analyzing strategies that aim at combining endocrine therapy with specific signaling inhibitors. PMID:25338647

Raffo, Diego; Pontiggia, Osvaldo; Bal de Kier Joffé, Elisa; Simian, Marina

2015-01-01

356

Distribution and development of the postnatal murine V?1 T-cell receptor repertoire  

PubMed Central

Murine ?/? T cells express canonical V?5V?1 chains in the epidermis and V?6V?1 chains at reproductive sites. Both subsets carry an identical V?1-D?2-J?2 chain which completely lacks junctional diversity. These cells are thought to monitor tissue integrity via recognition of stress-induced self antigens. In this study, we showed by reverse transcription–polymerase chain reaction (RT-PCR), complementarity determining region 3 (CDR3) spectratyping and sequencing of the junctional regions of V?1 chains from C57BL/6 mice (aged 1 day to 14 months) that the canonical V?1-D?2-J?2 chain is also consistently present at other sites such as the thymus, gut, lung, liver, spleen and peripheral blood. In addition, we found multiple V?1 chains with fetal type rearrangements which were also shared among organs and among animals. These V?1 chains were typically characterized by a conserved amino acid motif, ‘GGIRA’. Furthermore, by analysing the early postnatal period at days 10 and 16, we demonstrated that the diversification of the thymic V?1 repertoire is not paralleled by a diversification of extrathymic V?1+?/? T cells. This indicates that only fetal type rearrangements survive at extrathymic sites. In conclusion, ?/? T cells expressing the canonical V?1-D?2-J?2 chain are not unique to the skin and reproductive sites. Furthermore, we found other ?/? T cells expressing fetal type V?1 chains which were shared among different organs and animals. Thus, ?/? T cells expressing conserved V?1 chains are likely to have important functions. We suggest a model in which this subset continuously recirculates throughout the organism and rapidly responds to stress-induced self antigens. PMID:20465568

Holtmeier, Wolfgang; Gille, Jens; Zeuzem, Stefan; Sinkora, Marek

2010-01-01

357

Investigation of xenotropic murine leukemia virus-related virus (XMRV) in human and other cell lines  

Microsoft Academic Search

Xenotropic murine leukemia virus-related virus (XMRV) was discovered in human prostate tumors and later in some chronic fatigue syndrome (CFS) patients. However, subsequent studies have identified various sources of potential contamination with XMRV and other murine leukemia virus (MLV)-related sequences in test samples. Biological and nucleotide sequence analysis indicates that XMRV is distinct from known xenotropic MLVs and has a

Dhanya K. Williams; Teresa A. Galvin; Hailun Ma; Arifa S. Khan

2011-01-01

358

Enniatin B-induced cell death and inflammatory responses in RAW 267.4 murine macrophages  

SciTech Connect

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.

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

359

Distinct populations of dendritic cells are present in the subepithelial dome and T cell regions of the murine Peyer's patch  

PubMed Central

Despite the fact that the Peyer's patch (PP) is the primary site for antigen uptake in the intestine, the cellular basis of antigen handling after transport into the PP is poorly understood. We performed immunohistology of murine PPs using the dendritic cell (DC)-reactive monoclonal antibodies N418, NLDC-145, M342, and 2A1, as well as antibodies to other T cell, B cell, and macrophage markers. N418+, 2A1+, NLDC-145-, M342- cells form a dense layer of cells in the subepithelial dome (SED), just beneath the follicle epithelium, and are scattered throughout the follicle, sparing the germinal center. In contrast, N418+, 2A1+, NLDC-145+, and M342+ DCs are present in the interfollicular T cell regions (IFR). CD3+ and CD4+, but no CD8+ T cells were present in the SED and the follicle, including the germinal center, while CD3+, CD4+, and CD8+ T cells were present in the IFR. B cells and macrophages were poorly represented in the SED as no B220+ cells, only few Mac-1lo cells, and no F4/80+ cells were present at this site. In contrast, Mac-1hi cells were found in the IFR and lamina propria of intestinal villi, while F4/80+ cells were found only in the latter. In further phenotypic studies, we analyzed surface molecules of PP and spleen DCs by flow cytometry and found that these cells had similar fluorescence profiles when stained with N418, NLDC-145, and 33D1 DC-reactive antibodies, and antibodies to the costimulatory molecules B7-1 (1G10) and B7-2 (GL1). In contrast, PP DCs expressed 5- 10-fold higher levels of major histocompatibility complex class II antigens (IEk) than spleen DCs. Finally, in functional studies, we demonstrated that both PP and spleen DCs process soluble protein antigens during overnight culture and induce similar levels of proliferation in CD3+ T cells, and CD4+/Mel 14hi T cells from T cell receptor transgenic mice. The in vivo relevance of such presentation was shown by the fact that PP DCs isolated from Balb/c mice after being fed ovalbumin stimulated proliferation in ovalbumin T cell receptor T cells. Taken together, our data suggest that DCs in the SED of the PP are uniquely positioned for the processing of antigens passed into the PP from the overlying M cell, and that PP DCs are effective at processing and presenting oral antigens to naive T cells. PMID:8551227

1996-01-01

360

Phorbol 12-myristate 13-acetate-mediated signalling in murine bone marrow cells.  

PubMed Central

Phorbol 12-myristate 13-acetate (PMA)-mediated signalling was investigated in relation to the ability of murine (CBA) bone marrow cells to form colonies in vitro. Treatment of marrow cells with PMA did not influence the 1,2-diacylglycerol or cyclic AMP concentrations, the intracellular Ca2+ concentration or phospholipase D activity. PMA increased particulate phospholipase A2 (PLA2) activity, lysophosphatidylcholine formation and arachidonic acid release from bone marrow cells; these effects were abolished when cells were pretreated with the putative PLA2 inhibitors heparin and mepacrine. While indomethacin and nordihydroguaiaretic acid inhibited either the cyclo-oxygenase or lipoxygenase pathway of arachidonic acid metabolism, as measured by their products prostaglandin E2 and leukotriene B4, they did not influence PMA-mediated PLA2 activation or translocation of protein kinase C (PKC) from the soluble to the particulate fraction. Treatment of cells with PMA increased the amounts of membrane-bound alpha, beta, delta, epsilon and zeta isoforms of PKC in bone marrow cells. Pretreatment of cells with PLA2 inhibitors reduced the amount of membrane-bound PKC-zeta in unstimulated cells and diminished PMA-induced translocation of PKC-zeta to membranes without affecting other PKC isoforms. This effect could be overcome by exogenous addition of arachidonic acid, suggesting that PKC-zeta may operate downstream of the activated PLA2. On the other hand, wortmannin, an inhibitor of phosphatidylinositol 3-kinase, did not influence the amount of PKC-zeta associated with particulate fractions in control cells and could not abolish the PMA-mediated translocation of this isoform. Short-term exposure (45 min) of bone marrow cells to PMA, phorbol 12,13-dibutyrate or arachidonic acid increased the number of colonies formed over 7 days in a methylcellulose-based culture in vitro. The effects of PMA, but not those of arachidonic acid, could be prevented by putative PLA2 inhibitors. This suggests that PMA-mediated activation of conventional PKCs and novel PKCs leads to PLA2 activation which, by releasing arachidonic acid from phospholipids, activates PKC-zeta. This signalling pathway appears to be mitogenic for bone marrow cells. Images Figure 4 PMID:7646440

Visnji?, D; Batini?, D; Lasi?, Z; Knotek, M; Marusi?, M; Banfi?, H

1995-01-01

361

Interplay of autophagy and apoptosis during murine cytomegalovirus infection of RPE cells  

PubMed Central

Purpose Previous studies have demonstrated that autophagy is involved in the pathogenesis of human cytomegalovirus (HCMV) infection. However, whether autophagy is regulated by murine cytomegalovirus (MCMV) infection has not yet been investigated. The purpose of these studies was to determine how autophagy is affected by MCMV infection of the retinal pigment epithelial (RPE) cells and whether there is a functional relationship between autophagy and apoptosis; and if so, how regulation of autophagy impacts apoptosis. Methods RPE cells were isolated from C57BL/6 mice and infected with MCMV K181. The cells were cultured in medium containing rapamycin, chloroquine, or ammonium chloride. Green fluorescent protein–light chain 3 (GFP-LC3) plasmid was transfected to RPE cells, and the GFP-LC3 positive puncta were counted. Electron microscopic (EM) images were taken to visualize the structure of the autophagic vacuoles. Western blot was performed to detect the expression of related proteins. Trypan blue exclusion assay was used to measure the percentage of viable cells. Results Although the LC3B-II levels consistently increased during MCMV infection of RPE cells, administration of chloroquine or ammonium chloride increased LC3B-II expression only at the early stage of infection (6 h post-inoculation [p.i.] and 12 h p.i.), not at or after 24 h p.i. The punctate autophagic vacuoles in the GFP-LC3 transfected RPE cells were counted using light microscopy or by EM examination, the number of autophagic vacuoles was significantly increased in the MCMV-infected RPE cells compared to the uninfected controls. Compared to untreated MCMV-infected control cells, rapamycin treatment resulted in a significant decrease in the cleaved caspase 3 levels as well as a significant decrease in the ratio of phosphorylated mammalian target of rapamycin (mTOR) to total mTOR and in the ratio of phosphorylated P70S6K to total P70S6K. In contrast, chloroquine treatment resulted in a significant increase in the cleaved caspase 3 levels in the MCMV-infected RPE cells. Conclusions Autophagic vacuole accumulation was detected during MCMV infection of RPE cells. In contrast, autophagic flux was greatly decreased at or after 24 h p.i. The results suggest that MCMV might have a strategy for inhibiting or blocking autophagy activity by targeting a later autophagy process, such as the formation of autolysosomes or degradation of their content. Our data also suggest that there is a functional relationship between autophagy and apoptosis, which plays an important role during MCMV infection of the RPE. PMID:25324684

Mo, Juan; Zhang, Ming; Marshall, Brendan; Smith, Sylvia; Covar, Jason

2014-01-01

362

Genotoxicity of amorphous silica particles with different structure and dimension in human and murine cell lines.  

PubMed

Although amorphous silica is used in food products, cosmetics and paints and as vector for drug delivery, data on its potential health hazard are limited. The aim of this study was to investigate the cytotoxic and genotoxic potential of silica particles of different sizes (250 and 500nm) and structures (dense and mesoporous). Dense silica (DS) spheres were prepared by sol-gel synthesis, mesoporous silica particles (MCM-41) were prepared using hexadecyltrimethyl ammonium bromide as a structure-directing agent and tetraethylorthosilicate as silica source. Particles were accurately characterised by dynamic light scattering, nitrogen adsorption, X-ray diffraction and field emission scanning electron microscopy. Murine macrophages (RAW264.7) and human epithelial lung (A549) cell lines were selected for investigation. Genotoxicity was evaluated by Comet assay and micronucleus test. Cytotoxicity was tested by the trypan blue method. Cells were treated with 0, 5, 10, 20, 40 and 80 µg/cm(2) of different silica powders for 4 and 24 h. The intracellular localisation of silica was investigated by transmission electron microscopy. Amorphous particles penetrated into the cells, being compartmentalised within endocytic vacuoles. DS and MCM-41 particles induced cytotoxic and genotoxic effects in A549 and RAW264.7 although to different extent in the two cell lines. A549 were resistant in terms of cell viability, but showed a generalised induction of DNA strand breaks. RAW264.7 were susceptible to amorphous silica exposure, exhibiting both cytotoxic and genotoxic responses as DNA strand breaks and chromosomal alterations. The cytotoxic response of RAW264.7 was particularly relevant after MCM-41 exposure. The genotoxicity of amorphous silica highlights the need for a proper assessment of its potential hazard for human health. PMID:23325795

Guidi, Patrizia; Nigro, Marco; Bernardeschi, Margherita; Scarcelli, Vittoria; Lucchesi, Paolo; Onida, Barbara; Mortera, Renato; Frenzilli, Giada

2013-03-01

363

Urea and hypertonicity increase expression of heme oxygenase-1 in murine renal medullary cells.  

PubMed

Epithelial cells derived from the mammalian kidney medulla are responsive to urea at the levels of signal transduction and gene regulation. Hybridization of RNA harvested from control- and urea-treated murine inner medullary collecting duct (mIMCD3) cells with a cDNA expression array encoding stress-responsive genes suggested that heme oxygenase (HO)-1 mRNA was upregulated by urea. RNase protection assay confirmed this upregulation; hypertonicity also increased HO-1 mRNA expression but neither hypertonic NaCl nor urea were effective in the nonrenal 3T3 cell line. The effect on HO-1 expression appeared to be transcriptionally mediated on the basis of mRNA half-life studies and reporter gene analyses using the promoters of both human and chicken HO-1. Although urea signaling resembles that of heavy metal signaling in other contexts, the effect of urea on HO-1 transcription was independent of the cadmium response element in this promoter. Urea-inducible HO-1 expression was sensitive to antioxidants but not to scavengers of nitric oxide. Urea also upregulated HO-1 protein expression and pharmacological inhibition of HO-1 action with zinc protoporphyrin-sensitized mIMCD3 cells to the adverse effects of hypertonicity but not to urea. Coupled with the prior observation of others that HO-1 expression increases along the renal corticomedullary gradient, these data suggest that HO-1 expression may comprise an element of the adaptive response to hypertonicity and/or urea in renal epithelial cells. PMID:11592956

Tian, W; Bonkovsky, H L; Shibahara, S; Cohen, D M

2001-11-01

364

Leucine and calcium regulate fat metabolism and energy partitioning in murine adipocytes and muscle cells.  

PubMed

Dietary calcium modulation of adiposity is mediated, in part, by suppression of calcitriol, while the additional effect of dairy products is mediated by additional components; these include the high concentration of leucine, a key factor in the regulation of muscle protein turnover. We investigated the effect of leucine, calcitriol and calcium on energy metabolism in murine adipocytes and muscle cells and on energy partitioning between adipocytes and skeletal muscle. Leucine induced a marked increase in fatty acid oxidation in C2C12 muscle cells (P<0.001) and decreased FAS expression by 66% (P<0.001) in 3T3-L1 adipocytes. Calcitriol decreased muscle cell fatty acid oxidation by 37% (P<0.001) and increased adipocyte FAS gene expression by threefold (P<0.05); these effects were partially reversed by either leucine or calcium channel antagonism with nifedipine. Co-culture of muscle cells with adipocytes or incubation with 48-h adipocyte conditioned medium decreased muscle fatty acid oxidation by 62% (P<0.001), but treating adipocytes with leucine and/or nifedipine attenuated this effect. Leucine, nifedipine and calcitriol also modulated adiponectin production and thereby exerted additional indirect effects on fatty acid oxidation in C2C12 myotubes. Adiponectin increased IL-15 and IL-6 release by myotubes and partially reversed the inhibitory effects of calcitriol. Comparable effects of leucine, calcitriol and adiponectin were found in myotubes treated with conditioned medium derived from adipocytes or co-cultured with adipocytes. These data suggest that leucine and nifedipine promote energy partitioning from adipocytes to muscle cells, resulting in decreased energy storage in adipocytes and increasing fatty acid utilization in muscle. PMID:17406924

Sun, Xiaocun; Zemel, Michael B

2007-04-01

365

The murine AIDS defective provirus acts as an insertional mutagen in its infected target B cells.  

PubMed Central

In susceptible mice, the murine AIDS (MAIDS) defective virus can induce marked expansion of its target cells, the majority of which belong to the B-cell lineage. This expansion, which appears to be critical for the development of the immunodeficiency syndrome, is initially polyclonal but becomes oligoclonal late in the disease, suggesting the involvement of a secondary genetic event(s) during this proliferation. To determine whether integration of the MAIDS defective provirus into particular regions of the cellular genome contributes to this oligoclonal expansion, we searched for common provirus integration sites in enlarged lymphoid organs of MAIDS mice. We identified two common proviral integration sites, Dis-1 and Dis-2, which were occupied by a defective provirus at frequencies of 20 and 13%, respectively. Our analysis revealed that the Dis-1 region corresponds to the Sfpil1 (Spi-1, PU.1) locus, which maps on chromosome 2, and encodes a transcription factor. Insertion of the MAIDS defective provirus into this region led to a two- to threefold increase in the expression of Sfpi1 RNA. The Dis-2 locus was found to map to mouse chromosome 11, between Hox2 and Scya. It appears to be a novel locus probably harboring a gene involved in B-cell proliferation. The present study indicates that the MAIDS defective provirus can act as an insertional mutagen, thus contributing to the oligoclonal expansion of infected cells. The detection of two common proviral integration sites, each of which targetted at a low frequency in diseased organs, suggests that the deregulation of a unique gene through provirus insertion is essential for neither proliferation of infected B cells nor development of the immunodeficiency syndrome. PMID:7769664

Huang, M; Takac, M; Kozak, C A; Jolicoeur, P

1995-01-01

366

Functionalization density dependent toxicity of oxidized multiwalled carbon nanotubes in a murine macrophage cell line.  

PubMed

The present study investigates the effect of functionalization density on the toxicity and cellular uptake of oxidized multiwalled carbon nanotubes (f-MWCNTs) in vitro. The toxicity of f-MWCNTs at varying degrees of carboxylation was assessed in a murine macrophage RAW 264.7 cell line, a model for liver Kupffer cells. In vitro cytotoxicity of oxidized MWCNTs was directly proportional to their functionalization density. The increased cytotoxicity was associated with a concurrent increase in the number of apoptotic cells and production of reactive nitrogen species (RNS). In contrast, reactive oxygen species (ROS) generation was the highest in the case of pristine MWCNTs and decreased with increased functionalization density. Quantitative cellular uptake studies indicated that endogenous ROS production was independent of the concentration of CNTs internalized by a specific cell population and was directly proportional to their surface hydrophobicity. Mechanistic studies suggested that cellular uptake of CNTs was critically charge-dependent and mediated through scavenger receptors, albeit the involvement of nonscavenger receptor mechanisms at low CNT concentrations and their saturation at the experimental concentration cannot be ruled out. A mathematical model was established to correlate between the cellular uptake of CNTs with their length and zeta potential. In an attempt to correlate the results of in vitro toxicity experiments with those of the in vivo toxicity in the mouse model, we found that the toxicity trends in vitro and in vivo are rather opposing. The apparent anomaly was explained on the basis of different experimental conditions and doses associated with cells under in vivo and in vitro culture conditions. PMID:22994501

Singh, Raman Preet; Das, Manasmita; Thakare, Vivek; Jain, Sanyog

2012-10-15

367

Regulated Intramembrane Proteolysis and Degradation of Murine Epithelial Cell Adhesion Molecule mEpCAM  

PubMed Central

Epithelial cell adhesion molecule EpCAM is a transmembrane glycoprotein, which is highly and frequently expressed in carcinomas and (cancer-)stem cells, and which plays an important role in the regulation of stem cell pluripotency. We show here that murine EpCAM (mEpCAM) is subject to regulated intramembrane proteolysis in various cells including embryonic stem cells and teratocarcinomas. As shown with ectopically expressed EpCAM variants, cleavages occur at ?-, ?-, ?-, and ?-sites to generate soluble ectodomains, soluble A?-like-, and intracellular fragments termed mEpEX, mEp-?, and mEpICD, respectively. Proteolytic sites in the extracellular part of mEpCAM were mapped using mass spectrometry and represent cleavages at the ?- and ?-sites by metalloproteases and the b-secretase BACE1, respectively. Resulting C-terminal fragments (CTF) are further processed to soluble A?-like fragments mEp-? and cytoplasmic mEpICD variants by the g-secretase complex. Noteworthy, cytoplasmic mEpICD fragments were subject to efficient degradation in a proteasome-dependent manner. In addition the ?-secretase complex dependent cleavage of EpCAM CTF liberates different EpICDs with different stabilities towards proteasomal degradation. Generation of CTF and EpICD fragments and the degradation of hEpICD via the proteasome were similarly demonstrated for the human EpCAM ortholog. Additional EpCAM orthologs have been unequivocally identified in silico in 52 species. Sequence comparisons across species disclosed highest homology of BACE1 cleavage sites and in presenilin-dependent ?-cleavage sites, whereas strongest heterogeneity was observed in metalloprotease cleavage sites. In summary, EpCAM is a highly conserved protein present in fishes, amphibians, reptiles, birds, marsupials, and placental mammals, and is subject to shedding, ?-secretase-dependent regulated intramembrane proteolysis, and proteasome-mediated degradation. PMID:24009667

Hachmeister, Matthias; Bobowski, Karolina D.; Hogl, Sebastian; Dislich, Bastian; Fukumori, Akio; Eggert, Carola; Mack, Brigitte; Kremling, Heidi; Sarrach, Sannia; Coscia, Fabian; Zimmermann, Wolfgang; Steiner, Harald; Lichtenthaler, Stefan F.; Gires, Olivier

2013-01-01

368

The M sub 1 muscarinic receptor and its second messenger coupling in human neuroblastoma cells and transfected murine fibroblast cells  

SciTech Connect

The data of this study indicate that pirenzepine (PZ)-high affinity muscarinic receptors (mAChRs) are coupled to the hydrolysis of inositol lipids and not to the adenylate cyclase system in human neuroblastoma SH-SY5Y cells. The maximal carbachol(CCh)-stimulated ({sup 3}H)IP{sub 1} accumulation in the SH-SY5Y cells was decreased in the presence of 1{mu}g/ml pertussis toxin, suggesting that a pertussis toxin sensitive G-protein may be involved in the coupling. Several cell clones which express only M{sub 1} mAChR were generated by transfecting the murine fibroblast B82 cells with the cloned rat genomic m{sub 1} gene. The transfected B82 cells (cTB10) showed specific ({sup 3}H)(-)QNB binding activity. The mAChRs in these cells are of the M{sub 1} type defined by their high affinity for PZ and low affinity for AF-DX 116 and coupled to hydrolysis of inositol lipids, possibly via a pertussis toxin sensitive G protein. The relationship between the M{sub 1} mAChR density and the receptor-mediated hydrolysis of inositol lipids was studied in 7 clones. The M{sub 1} mAChR densities in these cells characterized by ({sup 3}H)(-)MQNB binding ranged from 12 fmol/10{sup 6} cells in LK3-1 cells to 260 fmol/10{sup 6} cells in the LK3-8 cells.

Mei, Lin.

1989-01-01

369

B cell dependence on and response to accessory signals in murine lupus strains  

PubMed Central

B cell hyperactivity, a feature common to all lupus-prone murine strains, may be caused by hyperresponsiveness to, overproduction of, or bypassing of certain signals required for B cell activation, proliferation, and differentiation. In this study, we have compared the responses of B cells from three lupus-prone strains of mice (BXSB males, MRL and NZB/W females) and normal strains in a number of assays for which two or more signals are required to obtain a response. In medium to low density cultures of B cells from BXSB and NZB/W but not MRL/l lupus mice, the cells' proliferation induced by bacterial lipopolysaccharide (LPS) or anti-mu antibody was much higher than that of B cells from normal controls. At low B cell density, polyclonal activation by these substances and subsequent Ig secretion were dependent on accessory signals present in supernatants of concanavalin A-treated normal lymphocytes (CAS) or on the MRL/l proliferating T cell- derived B cell differentiation factor (L-BCDF) in both lupus-prone and immunologically normal mice. However, the responses of B cells from BXSB and NZB/W, but not MRL/l, mice to these accessory signals were higher than those of normal mice. Ig synthesis by fresh B cells of BXSB and NZB/W mice cultured in the absence of mitogens but in the presence of CAS or L-BCDF was higher than by similar cells from other strains, suggesting an increased frequency of B cells activated in vivo in these two autoimmune strains of mice. The patterns of IgG subclass secretion in response to LPS (without added CAS or L-BCDF) were abnormal in all lupus strains, with a predominance of IgG2b and/or IgG2a and low levels of IgG3, contrary to normal B cells for which IgG3 synthesis predominated. However, IgG1 synthesis in vitro by autoimmune and normal B cells alike was highly dependent on T cell-derived soluble mediators. Antigen-specific responses to SRBC in vitro of B cells from all lupus strains, like those of B cells from normal strains, required a minimum of three signals (antigen, LPS, T cell-derived antigen nonspecific helper factors). Yet, once triggered, B cells of BXSB and NZB/W mice gave higher responses than those of the other strains. We conclude that B cells of lupus mice have signal requirements similar to those of normal mice. Nevertheless, B cells of BXSB and NZB/W, but not MRL/l, lupus mice hyperrespond or process some accessory signals abnormally. PMID:6406639

1983-01-01

370

Cyclosporin-binding sites of murine sensitive and resistant lymphoid cell lines.  

PubMed

Cyclosporin (Cs)-binding sites on murine spleen lymphocytes and on Cs-sensitive (CsS) and Cs-resistant (CsR) cloned lymphoma lines were compared using a ditritiated derivative of cyclosporin C (d3H-CsC). All three types of lymphocytes displayed similar d3H-CsC-binding characteristics. There were no major differences in the d3H-CsC-binding sites in terms of their cell surface density (number per surface area), their affinity and their specificity (capacity to discrimi