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Sample records for murine peritoneal cells

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

    International Nuclear Information System (INIS)

    Tokita, N.; Raju, M.R.

    1982-01-01

    Methods have been developed to distinguish subpopulations of murine peritoneal cells, and these were applied to the measurement of early changes in peritoneal cells after irradiation. The ratio of the two major subpopulations in the peritoneal fluid, lymphocytes and macrophages, was measured rapidly by means of cell volume distribution analysis as well as by hypotonic propidium iodide (PI) staining. After irradiation, dose and time dependent changes were noted in the cell volume distributions: a rapid loss of peritoneal lymphocytes, and an increase in the mean cell volume of macrophages. The hypotonic PI staining characteristics of the peritoneal cells showed two or three distinctive G 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

  2. Rediscovering peritoneal macrophages in a murine endometriosis model.

    Science.gov (United States)

    Yuan, Ming; Li, Dong; An, Min; Li, Qiuju; Zhang, Lu; Wang, Guoyun

    2017-01-01

    What are the features of peritoneal macrophage subgroups and T helper cells in the development of murine endometriosis? During the development of endometriosis in a murine model, large peritoneal macrophages (LPMs) and small peritoneal macrophages (SPMs) are polarized into M1 and M2 cells, respectively, and the proportions of T helper (Th) 1, Th17 and T regulatory (T reg ) cells are increased. Numerous studies investigating the etiology and pathogenesis of endometriosis have focused on the polarization states of peritoneal macrophages in endometriosis models and patients, but the results are inconclusive. Further studies indicate that peritoneal macrophages are composed of two distinct subsets: LPMs and SPMs, although their roles in endometriosis are unknown. This study involves a prospective and randomized experiment. Fifty C57BL/6 female mice were randomly allocated to five control and five experimental groups (n = 5/group) according to the presence or absence of transplantation. The transplant periods are 0.25, 3, 14, 28 and 42 days. C57BL/6 mice were utilized to establish an endometriosis model by i.p. injection of allogeneic endometrial segments. Dynamic changes of peritoneal macrophage subsets and polarization profiles were evaluated by flow cytometry (FCM). Macrophage morphology and density were assessed by cell counting under a microscope. Dynamic changes of Th1, Th2, Th17 and T reg cells were estimated by FCM. Peritoneal macrophages are composed of two distinct subsets: LPMs and SPMs. The proportion of SPMs increased immediately after peritoneal injection of endometrial tissues, whereas LPMs showed an opposite trend. Peritoneal macrophages differentiated into both M1 and M2 macrophages. The bidirectional polarization of macrophages was caused by the inverse trends of polarization of LPMs and SPMs. Consistently, the proportions of Th1, Th17 and T reg cells were all increased in mice with endometriosis. N/A. In this study, detection was only performed in a

  3. Resident Peritoneal NK cells

    Science.gov (United States)

    Gonzaga, Rosemary; Matzinger, Polly; Perez-Diez, Ainhoa

    2011-01-01

    Here we describe a new population of NK cells that reside in the normal, un-inflamed peritoneal cavity. Phenotypically, they share some similarities with the small population of CD49b negative, CD27 positive immature splenic NK cells, and liver NK cells but differ in their expression of CD62L, TRAIL and EOMES. Functionally, the peritoneal NK cells resemble the immature splenic NK cells in their production of IFN-γ, GM-CSF and TNF-α and in the killing of YAC-1 target cells. We also found that the peritoneum induces different behavior in mature and immature splenic NK cells. When transferred intravenously into RAGγcKO mice, both populations undergo homeostatic proliferation in the spleen, but only the immature splenic NK cells, are able to reach the peritoneum. When transferred directly into the peritoneum, the mature NK cells survive but do not divide, while the immature NK cells proliferate profusely. These data suggest that the peritoneum is not only home to a new subset of tissue resident NK cells but that it differentially regulates the migration and homeostatic proliferation of immature versus mature NK cells. PMID:22079985

  4. The influences of a novel anti-adhesion device, thermally cross-linked gelatin film on peritoneal dissemination of tumor cells: The in vitro and in vivo experiments using murine carcinomatous peritonitis models.

    Science.gov (United States)

    Miyamoto, Hiroe; Tsujimoto, Hiroyuki; Horii, Tsunehito; Ozamoto, Yuki; Ueda, Joe; Takagi, Toshitaka; Saitoh, Naoto; Hagiwara, Akeo

    2017-10-10

    To create anti-adhesive materials to be more effective and safer, we developed a thermally cross-linked gelatin film that showed superior anti-adhesive effects with excellent peritoneal regeneration. However, it may act as a convenient scaffold for tumor cell growth, thereby accelerating peritoneal dissemination when used in surgery for abdominal tumors. In this study, we tried to clarify this issue using mouse carcinomatous peritonitis models. First, we examined the in vitro tumor cell growth of mouse B16 melanoma or Colon26 cells on the gelatin film or the conventional hyarulonate/carboxymethylcellulose film. Tumor cell growth on each film was significantly lower than that of the control (no film). Next, we conducted the following in vivo experiments: After the parietal peritoneum was partially removed and covered with each film or without any film, mice were inoculated intraperitoneally with B16 melanoma or Colon26/Nluc cells expressing NanoLuc luciferase gene. At 7 days after the operation, we measured the weight of B16 melanoma tumors or the NanoLuc activity of Colon26/Nluc cells using in vivo imaging at the injured sites. There were no significant differences in the weight of the tumors and the NanoLuc activity among the three groups. We also observed the survival time of mice receiving the same operation and treatments. There was no significant difference in the survival time among the three groups. These results suggest that the gelatin film will likely not accelerate peritoneal dissemination as a convenient scaffold for tumor cell growth when used in surgery for abdominal tumors. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2017. © 2017 Wiley Periodicals, Inc.

  5. Effects of protein-energy malnutrition on NF-kappaB signalling in murine peritoneal macrophages.

    Science.gov (United States)

    Fock, Ricardo Ambrósio; Rogero, Marcelo Macedo; Vinolo, Marco Aurélio Ramirez; Curi, Rui; Borges, Maria Carolina; Borelli, Primavera

    2010-04-01

    Protein-energy malnutrition (PEM) is an important public health problem affecting millions of people worldwide. PEM decreases resistance to infection, impairing a number of physiological processes. In unstimulated cells, NF-kappaB is kept from binding to its consensus sequence by the inhibitor I kappaB alpha, which retains NF-kappaB in the cytoplasm. Upon various signals, such as lipopolysaccharide (LPS), I kappaB alpha is rapidly degraded and NF-kappaB is induced to translocate into the nucleus, where it activates expression of various genes that participate in the inflammatory response, including those involved in the synthesis of TNF-alpha. TRAF-6 is a cytoplasmic adapter protein that links the stimulatory signal from Toll like receptor-4 to NF-kappaB. The aim of this study was to evaluate the effect of malnutrition on induction of TNF-alpha by LPS in murine peritoneal macrophages. We evaluated peritoneal cellularity, the expression of MyD88, TRAF-6, IKK, I kappaB alpha and NF-kappaB, NF-kappaB activation and TNF-alpha mRNA and protein synthesis in macrophages. Two-month-old male BALB/C mice were submitted to PEM with a low-protein diet that contained 2% protein, compared to 12% protein in the control diet. When the experimental group had lost about 20% of the original body weight, it was used in the subsequent experiments. Malnourished animals presented anemia, leucopenia and severe reduction in peritoneal cavity cellularity. TNF-alpha mRNA and protein levels of macrophages stimulated with LPS were significantly lower in malnourished animals. PEM also decreased TRAF-6 expression and NF-kappaB activation after LPS stimulation. These results led us to conclude that PEM changes NF-kB signalling pathway in macrophages to LPS stimulus.

  6. Pathophysiological Changes to the Peritoneal Membrane during PD-Related Peritonitis: The Role of Mesothelial Cells

    Science.gov (United States)

    Yung, Susan; Chan, Tak Mao

    2012-01-01

    The success of peritoneal dialysis (PD) is dependent on the structural and functional integrity of the peritoneal membrane. The mesothelium lines the peritoneal membrane and is the first line of defense against chemical and/or bacterial insult. Peritonitis remains a major complication of PD and is a predominant cause of technique failure, morbidity and mortality amongst PD patients. With appropriate antibiotic treatment, peritonitis resolves without further complications, but in some PD patients excessive peritoneal inflammatory responses lead to mesothelial cell exfoliation and thickening of the submesothelium, resulting in peritoneal fibrosis and sclerosis. The detrimental changes in the peritoneal membrane structure and function correlate with the number and severity of peritonitis episodes and the need for catheter removal. There is evidence that despite clinical resolution of peritonitis, increased levels of inflammatory and fibrotic mediators may persist in the peritoneal cavity, signifying persistent injury to the mesothelial cells. This review will describe the structural and functional changes that occur in the peritoneal membrane during peritonitis and how mesothelial cells contribute to these changes and respond to infection. The latter part of the review discusses the potential of mesothelial cell transplantation and genetic manipulation in the preservation of the peritoneal membrane. PMID:22577250

  7. Assessment of Antibody-based Drugs Effects on Murine Bone Marrow and Peritoneal Macrophage Activation.

    Science.gov (United States)

    Kozicky, Lisa; Sly, Laura M

    2017-12-26

    Macrophages are phagocytic innate immune cells, which initiate immune responses to pathogens and contribute to healing and tissue restitution. Macrophages are equally important in turning off inflammatory responses. We have shown that macrophages stimulated with intravenous immunoglobulin (IVIg) can produce high amounts of the anti-inflammatory cytokine, interleukin 10 (IL-10), and low levels of pro-inflammatory cytokines in response to bacterial lipopolysaccharides (LPS). IVIg is a polyvalent antibody, primarily immunoglobulin Gs (IgGs), pooled from the plasma of more than 1,000 blood donors. It is used to supplement antibodies in patients with immune deficiencies or to suppress immune responses in patients with autoimmune or inflammatory conditions. Infliximab, a therapeutic anti-tumor necrosis factor alpha (TNFα) antibody, has also been shown to activate macrophages to produce IL-10 in response to inflammatory stimuli. IVIg and other antibody-based biologics can be tested to determine their effects on macrophage activation. This paper describes methods for derivation, stimulation, and assessment of murine bone marrow macrophages activated by antibodies in vitro and murine peritoneal macrophages activated with antibodies in vivo. Finally, we demonstrate the use of western blotting to determine the contribution of specific cell signaling pathways to anti-inflammatory macrophage activity. These protocols can be used with genetically modified mice, to determine the effect of a specific protein(s) on anti-inflammatory macrophage activation. These techniques can also be used to assess whether specific biologics may act by changing macrophages to an IL-10-producing anti-inflammatory activation state that reduces inflammatory responses in vivo. This can provide information on the role of macrophage activation in the efficacy of biologics during disease models in mice, and provide insight into a potential new mechanism of action in people. Conversely, this may caution

  8. Modified pectin from Theobroma cacao induces potent pro-inflammatory activity in murine peritoneal macrophage.

    Science.gov (United States)

    Amorim, Juliana C; Vriesmann, Lucia Cristina; Petkowicz, Carmen L O; Martinez, Glaucia Regina; Noleto, Guilhermina R

    2016-11-01

    In vitro effects of acetylated pectin (OP) isolated from cacao pod husks (Theobroma cacao L.), its partially deacetylated and de-esterified form (MOP), and a commercial homogalacturonan (PG) were investigated on murine peritoneal macrophages. MOP stood out among the studied pectins. After 48h of incubation, compared with the control group, it was able to promote significant macrophage morphological differentiation from resident to activated stage and also stimulated nitric oxide production, which reached a level of 85% of that of LPS stimulus. In the presence of the highest tested concentration of MOP (200μg·mL -1 ), the levels of the cytokines TNF-α (6h) and IL-12 and IL-10 (48h) increased substantially in relation to untreated cells. Our results show that the partial deacetylation and de-esterification of pectin extracted from cacao pod husks (T. cacao L.) produced a polymer with greater ability than its native form to activate macrophages to a cytotoxic phenotype. Like this, they provide the possibility of a therapeutic application to MOP, which could lead to a decreased susceptibility to microbial infection besides antitumor activity. Additionally, the present results also corroborate with the proposition of that the chemical modifications of the biopolymers can result in an improved molecule with new possibilities of application. Copyright © 2016 Elsevier B.V. All rights reserved.

  9. Enterocyte-specific epidermal growth factor prevents barrier dysfunction and improves mortality in murine peritonitis.

    Science.gov (United States)

    Clark, Jessica A; Gan, Heng; Samocha, Alexandr J; Fox, Amy C; Buchman, Timothy G; Coopersmith, Craig M

    2009-09-01

    Systemic administration of epidermal growth factor (EGF) decreases mortality in a murine model of septic peritonitis. Although EGF can have direct healing effects on the intestinal mucosa, it is unknown whether the benefits of systemic EGF in peritonitis are mediated through the intestine. Here, we demonstrate that enterocyte-specific overexpression of EGF is sufficient to prevent intestinal barrier dysfunction and improve survival in peritonitis. Transgenic FVB/N mice that overexpress EGF exclusively in enterocytes (IFABP-EGF) and wild-type (WT) mice were subjected to either sham laparotomy or cecal ligation and puncture (CLP). Intestinal permeability, expression of the tight junction proteins claudins-1, -2, -3, -4, -5, -7, and -8, occludin, and zonula occludens-1; villus length; intestinal epithelial proliferation; and epithelial apoptosis were evaluated. A separate cohort of mice was followed for survival. Peritonitis induced a threefold increase in intestinal permeability in WT mice. This was associated with increased claudin-2 expression and a change in subcellular localization. Permeability decreased to basal levels in IFABP-EGF septic mice, and claudin-2 expression and localization were similar to those of sham animals. Claudin-4 expression was decreased following CLP but was not different between WT septic mice and IFABP-EGF septic mice. Peritonitis-induced decreases in villus length and proliferation and increases in apoptosis seen in WT septic mice did not occur in IFABP-EGF septic mice. IFABP-EGF mice had improved 7-day mortality compared with WT septic mice (6% vs. 64%). Since enterocyte-specific overexpression of EGF is sufficient to prevent peritonitis-induced intestinal barrier dysfunction and confers a survival advantage, the protective effects of systemic EGF in septic peritonitis appear to be mediated in an intestine-specific fashion.

  10. Peritoneal Cell-free DNA: an innovative method for determining acute cell damage in peritoneal membrane and for monitoring the recovery process after peritonitis.

    Science.gov (United States)

    Virzì, Grazia Maria; Milan Manani, Sabrina; Brocca, Alessandra; Cantaluppi, Vincenzo; de Cal, Massimo; Pastori, Silvia; Tantillo, Ilaria; Zambon, Roberto; Crepaldi, Carlo; Ronco, Claudio

    2016-02-01

    Cell-free DNA (cfDNA) is present in the peritoneal effluent of stable peritoneal dialysis (PD) patients, but there are no data on cfDNA in PD patients with peritonitis. We investigated the variation of peritoneal cfDNA levels subsequent to peritonitis in PD patients. We enrolled 53 PD patients: 30 without any history of systemic inflammation or peritonitis in the last 3 months (group A) and 23 with acute peritonitis (group B). CfDNA was quantified in the peritoneal effluent. Peritoneal samples on days 1, 3, 10, 30 and until day 120 from the start of peritonitis were collected for white blood cells (WBC) count and cfDNA evaluation in group B. Quantitative analysis of cfDNA showed significantly higher levels in group B on day 1, 3, 10 and 30 compared with group A (p peritoneal cfDNA levels tended to progressively decline during follow-up of peritonitis. From this decreasing curve, we estimated that 49 days are necessary to reach the value of 51 genome equivalents (GE)/ml (75th percentile in controls) and 63 days to reach 31 GE/ml (median). Our results demonstrate that cfDNA increases in peritoneal effluent of PD patients with peritonitis and tends to progressively decline in step with peritonitis resolution and membrane repair process. Peritoneal cfDNA quantification could be an innovative method to determine acute damage and an inverse index of the repair process.

  11. Efficacy of a synthetic antimicrobial peptidomimetic versus vancomycin in a Staphylococcus epidermidis device-related murine peritonitis model

    DEFF Research Database (Denmark)

    Cavanagh, Jorunn Pauline; Granslo, Hildegunn Norbakken; Fredheim, Elizabeth Aarag

    2013-01-01

    Objectives Biofilm-forming Staphylococcus epidermidis is a prevalent cause of peritonitis during peritoneal dialysis. We compared the efficacy of a synthetic antimicrobial peptidomimetic (Ltx21) versus vancomycin in a murine model mimicking a device-related peritonitis. Methods Silicone implants......, pre-colonized with an S. epidermidis biofilm, were inserted into the peritoneal cavity of BALB/c mice. Three groups (36 mice in each) with pre-colonized implants received intraperitoneal treatment with Ltx21, vancomycin or placebo. Mice were euthanized on day 3 (n = 12), day 6 (n = 12) or day 8 (n...... = 12) post-implantation. Controls were mice with sterile implants (n = 18) and mice without surgery (n = 6). Bacterial reductions in cfu were analysed from implants and peritoneal fluid (PF). Inflammatory responses in serum and PF were measured. Results Vancomycin resulted in a stronger reduction...

  12. Peritonitis

    Science.gov (United States)

    ... complication of colonoscopy or endoscopy. A ruptured appendix, stomach ulcer or perforated colon. Any of these conditions can ... risk of developing peritonitis: cirrhosis, appendicitis, Crohn's disease, stomach ulcers, diverticulitis and pancreatitis. History of peritonitis. Once you' ...

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

    International Nuclear Information System (INIS)

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

    1986-01-01

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

  14. Regional CAR-T cell infusions for peritoneal carcinomatosis are superior to systemic delivery.

    Science.gov (United States)

    Katz, S C; Point, G R; Cunetta, M; Thorn, M; Guha, P; Espat, N J; Boutros, C; Hanna, N; Junghans, R P

    2016-05-01

    Metastatic spread of colorectal cancer (CRC) to the peritoneal cavity is common and difficult to treat, with many patients dying from malignant bowel obstruction. Chimeric antigen receptor T cell (CAR-T) immunotherapy has shown great promise, and we previously reported murine and phase I clinical studies on regional intrahepatic CAR-T infusion for CRC liver metastases. We are now studying intraperitoneal (IP) delivery of CAR-Ts for peritoneal carcinomatosis. Regional IP infusion of CAR-T resulted in superior protection against carcinoembryonic antigen (CEA+) peritoneal tumors, when compared with systemically infused CAR-Ts. IP CAR-Ts also provided prolonged protection against IP tumor re-challenges and demonstrated an increase in effector memory phenotype over time. IP CAR-Ts provided protection against tumor growth at distant subcutaneous (SC) sites in association with increases in serum IFNγ levels. Given the challenges posed by immunoinhibitory pathways in solid tumors, we combined IP CAR-T treatment with suppressor cell targeting. High frequencies of myeloid-derived suppressor cells (MDSC) and regulatory T cells (Treg) were found within the IP tumors, with MDSC expressing high levels of immunosuppressive PD-L1. Combinatorial IP CAR-T treatment with depleting antibodies against MDSC and Treg further improved efficacy against peritoneal metastases. Our data support further development of combinatorial IP CAR-T immunotherapy for peritoneal malignancies.

  15. Differential regulation of tissue thiol-disulfide redox status in a murine model of peritonitis

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    Benton Shana M

    2012-10-01

    Full Text Available Abstract Background Glutathione (GSH/glutathione disulfide (GSSG and cysteine (Cys/cystine (CySS are major redox pools with important roles in cytoprotection. We determined the impact of septic peritonitis on thiol-disulfide redox status in mice. Methods FVB/N mice (6–12 week old; 8/group underwent laparotomy with cecal ligation and puncture (CLP or laparotomy alone (control. Sections of ileum, colon, lung and liver were obtained and GSH, GSSG, Cys and CySS concentrations determined by HPLC 24 h after laparotomy. Redox potential [Eh in millivolts (mV] of the GSH/GSSG and Cys/CySS pools was calculated using the Nernst equation. Data were analyzed by ANOVA (mean ± SE. Results GSH/GSSG Eh in ileum, colon, and liver was significantly oxidized in septic mice versus control mice (ileum: septic −202±4 versus control −228±2 mV; colon: -195±8 versus −214±1 mV; and liver: -194±3 vs. -210±1 mV, all Ph was unchanged with CLP, while liver and lung Cys/CySS Eh became significantly more reducing (liver: septic = −103±3 versus control −90±2 mV; lung: -101±5 versus −81±1 mV, each P Conclusions Septic peritonitis induced by CLP oxidizes ileal and colonic GSH/GSSG redox but Cys/CySS Eh remains unchanged in these intestinal tissues. In liver, CLP oxidizes the GSH/GSSG redox pool and CyS/CySS Eh becomes more reducing; in lung, CLP does not alter GSH/GSSG Eh, and Cys/CySS Eh is less oxidized. CLP-induced infection/inflammation differentially regulates major thiol-disulfide redox pools in this murine model.

  16. Apoptosis and T cell depletion during feline infectious peritonitis

    OpenAIRE

    Horzinek, M.C.; Haagmans, B.L.; Egberink, H.F.

    1996-01-01

    Cats that have succumbed to feline infectious peritonitis, an immune- mediated disease caused by variants of feline coronaviruses, show apoptosis and T-cell depletion in their lymphoid organs. The ascitic fluid that develops in the course of the condition causes apoptosis in vitro but only in activated T cells. Since feline infectious peritonitis virus does not infect T cells, and viral proteins did not inhibit T-cell proliferation, we postulate that soluble mediators released during the infe...

  17. Analysis of early mesothelial cell responses to Staphylococcus epidermidis isolated from patients with peritoneal dialysis-associated peritonitis.

    Science.gov (United States)

    McGuire, Amanda L; Mulroney, Kieran T; Carson, Christine F; Ram, Ramesh; Morahan, Grant; Chakera, Aron

    2017-01-01

    The major complication of peritoneal dialysis (PD) is the development of peritonitis, an infection within the abdominal cavity, primarily caused by bacteria. PD peritonitis is associated with significant morbidity, mortality and health care costs. Staphylococcus epidermidis is the most frequently isolated cause of PD-associated peritonitis. Mesothelial cells are integral to the host response to peritonitis, and subsequent clinical outcomes, yet the effects of infection on mesothelial cells are not well characterised. We systematically investigated the early mesothelial cell response to clinical and reference isolates of S. epidermidis using primary mesothelial cells and the mesothelial cell line Met-5A. Using an unbiased whole genome microarray, followed by a targeted panel of genes known to be involved in the human antibacterial response, we identified 38 differentially regulated genes (adj. p-value peritonitis. This study provides new insights into early mesothelial cell responses to infection with S. epidermidis, and confirms the importance of validating findings in primary mesothelial cells.

  18. Angiopoietin-like protein 2 induces proinflammatory responses in peritoneal cells

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    Umikawa, Masato, E-mail: umikawa@med.u-ryukyu.ac.jp [Department of Medical Biochemistry, Graduate School of Medicine, University of the Ryukyus, Okinawa (Japan); Umikawa, Asako; Asato, Tsuyoshi; Takei, Kimiko [Department of Medical Biochemistry, Graduate School of Medicine, University of the Ryukyus, Okinawa (Japan); Matsuzaki, Goro [Department of Tropical Infectious Diseases, COMB, Tropical Biosphere Research Center, University of the Ryukyus, Okinawa (Japan); Kariya, Ken-ichi [Department of Medical Biochemistry, Graduate School of Medicine, University of the Ryukyus, Okinawa (Japan); Zhang, Cheng Cheng, E-mail: alec.zhang@utsouthwestern.edu [Department of Physiology, University of Texas Southwestern Medical Center, Dallas, TX (United States)

    2015-11-13

    Monocytes and macrophages are important effectors and regulators of inflammation, and both their differentiation and activation are regulated strictly in response to environmental cues. Angiopoietin-like protein 2 (Angptl2) is a multifaceted protein, displaying many physiological and pathological functions in inflammation, angiogenesis, hematopoiesis, and tumor development. Although recent studies implicate Angptl2 in chronic inflammation, the mechanisms of inflammation caused by Angptl2 remain unclear. The purpose of the present study was to elucidate the role of Angptl2 in inflammation by understanding the effects of Angptl2 on monocytes/macrophages. We showed that Angptl2 directly activates resident murine peritoneal monocytes and macrophages and induces a drastic upregulation of the transcription of several inflammatory genes including nitric oxide synthase 2 and prostaglandin-endoperoxide synthase 2, and several proinflammatory cytokine genes such as interleukin (IL)-1β, IL-6, TNFα, and CSF2, along with activation of ERK, JNK, p38, and nuclear factor kappa B signaling pathways. Concordantly, proinflammatory cytokines IL-1β, IL-6, TNFα, and GM-CSF, were rapidly elevated from murine peritoneal monocytes and macrophages. These results demonstrate a novel role for Angptl2 in inflammation via the direct activation of peritoneal monocytes and macrophages. - Highlights: • Angptl2 directly activates resident murine peritoneal monocytes and macrophages. • Angptl2 induces a drastic upregulation of expression of inflammatory genes. • Angptl2 induces activation of ERK, JNK, p38, and nuclear factor kappa B signaling pathways. • Angptl2 does not activate bone marrow derived macrophages or macrophage cell lines.

  19. Effect of 60Co radiation on peritoneal cells

    International Nuclear Information System (INIS)

    Villavicencio, A.L.C.H.; Mastro, N.L. del.

    1988-07-01

    This work deals with the effect of 60 Co gamma irradiation on the levels and quality of peritoneal cells of albino mice. The cells were obtained from peritoneal exudate, fixed and stained in 30% glacial acetic acid containing 0,5% cristal violet. Os exudates from irradiated and control animals the qualitative analysis and the counting of different cell populations were performed one hour, three days and six days after irradiation with 9 Gy. All the cell populations from the peritoneal exudate shown a decrease 3 days after the irradiation with 9.0. Gy but the different cellular populations dimished in unlike proportions. The data reaffirm the discrepance in radiosensivity of the diverse peritoneal cell populations. (author) [pt

  20. Radiation effects of 60C in peritoneal cells

    International Nuclear Information System (INIS)

    Villavicencio, A.L.C.H.; Mastro, N.L. del

    1988-01-01

    This work deals with the effect of 60 Co gamma irradiation on the levels and quality of peritoneal cells of albino mice. The cells were obtained from peritoneal exudate, fixed and stained in 30% glacial acetic acid containing 0.5% cristal violet. On exudates from irradiated and control animals the qualitative analysis and the counting of different cell populations were performed one hour, three days and six days after irradiation with 9 Gy. All the cell populations from the peritoneal exudate shown a decrease 3 days after the irradiation with 9.0 Gy but the different cellular populations diminished in unlike proportions. The data reaffirm the discrepance in radiosensivity of the diverse peritoneal cell populations. (author) [pt

  1. Interaction of immunocompetent cells in peritonitis in the irradiated body

    International Nuclear Information System (INIS)

    Rasulev, B.K.

    1988-01-01

    The process of T- and B-lymphocyte cooperation under combined effect of ionizing radiation and wound peritonitis was investigated using CBA male rats. The animals were subjected to single total X irradiation with 5.5 Gy(10 17/30 ) dose. Then 1% fecal suspension was injected introperitoneally in 130 mg/kg dose and thus peritonitis of conventionally hard degree (HDP) was induced. It is shown that 5.5 Gy dose ionizing radiation suppresses the immune response at the expense of T- and B-lymphocyte function inhibition; peritonitis induction reduces immuene response, inhibiting the T-lymphocyte helper function, and does not effect the B-cell function; under combined irradiation and peritonitis effect the immune response is sufficiently suppressed at the expense of higher T-lymphocyte helper function inhibition while B-cell function is not so extremely violated

  2. Baicalein induces cell death in murine T cell lymphoma via inhibition of thioredoxin system.

    Science.gov (United States)

    Patwardhan, Raghavendra S; Pal, Debojyoti; Checker, Rahul; Sharma, Deepak; Sandur, Santosh K

    2017-10-01

    We have earlier demonstrated the radioprotective potential of baicalein using murine splenic lymphocytes. Here, we have studied the effect of baicalein on murine T cell lymphoma EL4 cells and investigated the underlying mechanism of action. We observed that baicalein induced a dose dependent cell death in EL4 cells in vitro and significantly reduced the frequency of cancer stem cells. Previously, we have reported that murine and human T cell lymphoma cells have increased oxidative stress tolerance capacity due to active thioredoxin system. Hence, we monitored the effect of baicalein on thioredoxin system in EL4 cells. Docking studies revealed that baicalein could bind to the active site of thioredoxin reductase. Baicalein treatment led to significant reduction in the activity of thioredoxin reductase and nuclear levels of thioredoxin-1 thereby increasing ASK1 levels and caspase-3 activity. Interestingly, CRISPR-Cas9 based knock-out of ASK1 or over-expression of thioredoxin-1 abolished anti-tumor effects of baicalein in EL4 cells. Further, baicalein administration significantly reduced intra-peritoneal tumor burden of EL4 cells in C57BL/6 mice. Thus, our study describes anti-tumor effects of baicalein in EL4 cells via inhibition of thioredoxin system. Copyright © 2017 Elsevier Ltd. All rights reserved.

  3. Rapid white blood cell detection for peritonitis diagnosis

    Science.gov (United States)

    Wu, Tsung-Feng; Mei, Zhe; Chiu, Yu-Jui; Cho, Sung Hwan; Lo, Yu-Hwa

    2013-03-01

    A point-of-care and home-care lab-on-a-chip (LoC) system that integrates a microfluidic spiral device as a concentrator with an optical-coding device as a cell enumerator is demonstrated. The LoC system enumerates white blood cells from dialysis effluent of patients receiving peritoneal dialysis. The preliminary results show that the white blood cell counts from our system agree well with the results from commercial flow cytometers. The LoC system can potentially bring significant benefits to end stage renal disease (ESRD) patients that are on peritoneal dialysis (PD).

  4. Genistein modulates the estrogen receptor and suppresses angiogenesis and inflammation in the murine model of peritoneal endometriosis

    Directory of Open Access Journals (Sweden)

    Sutrisno Sutrisno

    2018-04-01

    Full Text Available The purpose of this study was to investigate the effect of genistein administration on the modulation of the estrogen receptor, inhibition of inflammation and angiogenesis in the murine model of peritoneal endometriosis. A total of thirty-six mice (Mus musculus were divided into six groups (n = 6, including the control group, endometriosis group, endometriosis group treated with various doses of genistein (0.78; 1.04; 1.3 mg/day, and endometriosis group treated with leuprolide acetate (0.00975 mg/day every 5 days for 15 days. Analysis of estrogen receptor-α, estrogen receptor-β, TNF-α, IL-6, VEGF, and HIF-1α were performed immunohistochemically. Expression of estrogen receptor-α, estrogen receptor-β, TNF-α, IL-6, VEGF and HIF-1α increased significantly compared with the control group (p  0.05. Genistein also decreased the expression of TNF-α and IL-6 (1.04 and 1.3 mg/day compared with the endometriosis group, reaching level comparable to that of the control group (p > 0.05. It was concluded that genistein is able to modulate estrogen receptor-α and estrogen receptor-β and inhibit the development of inflammation and angiogenesis in the murine model of peritoneal endometriosis. Thus, genistein can be a candidate in the treatment of endometriosis. Keywords: Estrogen receptor, Growth factor, Inflammation, Angiogenesis, Peritoneum

  5. Effect of Shark Liver Oil on Peritoneal Murine Macrophages in Responses to Killed-Candida albicans

    Directory of Open Access Journals (Sweden)

    Monire Hajimoradi

    2009-09-01

    Full Text Available Objective(sShark Liver Oil (SLO is an immunomodulator. Macrophages play a key role in host defense against pathogens like fungi. Candida albicans have mechanisms to escape immune system. We determined the effect of killed-Candida on the in vitro viability of macrophages and the effect of SLO on augmentation of this potency.Materials and MethodsPeritoneal macrophages were separated and cultured (3×105/well. At first, the effect of killed-Candida (200 cells/well on macrophage viability was evaluated, using MTT test. Then, MTT was performed on macrophages stimulated with killed-Candida in the presence of SLO. ResultsKilled-Candida suppressed the ability of MTT reduction and hence macrophages viability (P=0.026, but addition of SLO (100 mg/ml significantly enhanced cell viability (P=0.00. So, SLO could neutralize the inhibitory effect of Candida.ConclusionSimultaneous with cytotoxic effect of killed-Candida cells on macrophages viability, SLO augment macrophages viability. So, it can be applied in candidiasis as a complement.

  6. Expression of Nocardia brasiliensis superoxide dismutase during the early infection of murine peritoneal macrophages.

    Science.gov (United States)

    Revol, Agnès; Espinoza-Ruiz, Marisol; Medina-Villanueva, Igor; Salinas-Carmona, Mario Cesar

    2006-12-01

    Nocardia brasiliensis is the main agent of actinomycetoma in Mexico, but little is known about its virulence and molecular pathogenic pathways. These facultative intracellular bacteria are able to survive and divide within the host phagocytic cells, in part by neutralizing the reactive oxygen intermediates. Superoxide dismutase (SOD) participates in the intracellular survival of several bacterial species and, in particular, constitutes one of Nocardia asteroides virulence factors. To clarify SOD participation in the N. brasiliensis early infective process, we report its isolation and the consequent comparison of its transcript level. A 630 bp polymerase chain reaction fragment that included most of the coding sequence of N. brasiliensis sodA was cloned. A competitive assay was developed, allowing comparison of bacterial sod expression in exponential culture and 1 h after infecting peritoneal macrophages from BALB/c mice. At that time, there were viable bacteria in the macrophages. The intracellular bacteria presented a clear decrease in their sod transcript amount, although their 16S rRNA (used as an internal control) and hsp levels were maintained or slightly increased, respectively. These results indicate that sodA transcription is not maintained within the SOS bacterial response induced by phagosomal conditions. Further kinetics will be necessary to precisely define sod transcriptional regulation during N. brasiliensis intra-macrophage growth.

  7. Thrombopoietin inhibits murine mast cell differentiation

    Science.gov (United States)

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

    2009-01-01

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

  8. Apoptosis and T cell depletion during feline infectious peritonitis

    NARCIS (Netherlands)

    Horzinek, M.C.; Haagmans, B.L.; Egberink, H.F.

    1996-01-01

    Cats that have succumbed to feline infectious peritonitis, an immune- mediated disease caused by variants of feline coronaviruses, show apoptosis and T-cell depletion in their lymphoid organs. The ascitic fluid that develops in the course of the condition causes apoptosis in vitro but only in

  9. Chronic alcohol ingestion increases mortality and organ injury in a murine model of septic peritonitis.

    Science.gov (United States)

    Yoseph, Benyam P; Breed, Elise; Overgaard, Christian E; Ward, Christina J; Liang, Zhe; Wagener, Maylene E; Lexcen, Daniel R; Lusczek, Elizabeth R; Beilman, Greg J; Burd, Eileen M; Farris, Alton B; Guidot, David M; Koval, Michael; Ford, Mandy L; Coopersmith, Craig M

    2013-01-01

    Patients admitted to the intensive care unit with alcohol use disorders have increased morbidity and mortality. The purpose of this study was to determine how chronic alcohol ingestion alters the host response to sepsis in mice. Mice were randomized to receive either alcohol or water for 12 weeks and then subjected to cecal ligation and puncture. Mice were sacrificed 24 hours post-operatively or followed seven days for survival. Septic alcohol-fed mice had a significantly higher mortality than septic water-fed mice (74% vs. 41%, p = 0.01). This was associated with worsened gut integrity in alcohol-fed mice with elevated intestinal epithelial apoptosis, decreased crypt proliferation and shortened villus length. Further, alcohol-fed mice had higher intestinal permeability with decreased ZO-1 and occludin protein expression in the intestinal tight junction. The frequency of splenic and bone marrow CD4+ T cells was similar between groups; however, splenic CD4+ T cells in septic alcohol-fed mice had a marked increase in both TNF and IFN-γ production following ex vivo stimulation. Neither the frequency nor function of CD8+ T cells differed between alcohol-fed and water-fed septic mice. NK cells were decreased in both the spleen and bone marrow of alcohol-fed septic mice. Pulmonary myeloperoxidase levels and BAL levels of G-CSF and TFG-β were higher in alcohol-fed mice. Pancreatic metabolomics demonstrated increased acetate, adenosine, xanthine, acetoacetate, 3-hydroxybutyrate and betaine in alcohol-fed mice and decreased cytidine, uracil, fumarate, creatine phosphate, creatine, and choline. Serum and peritoneal cytokines were generally similar between alcohol-fed and water-fed mice, and there were no differences in bacteremia, lung wet to dry weight, or pulmonary, liver or splenic histology. When subjected to the same septic insult, mice with chronic alcohol ingestion have increased mortality. Alterations in intestinal integrity, the host immune response, and

  10. Chronic alcohol ingestion increases mortality and organ injury in a murine model of septic peritonitis.

    Directory of Open Access Journals (Sweden)

    Benyam P Yoseph

    Full Text Available Patients admitted to the intensive care unit with alcohol use disorders have increased morbidity and mortality. The purpose of this study was to determine how chronic alcohol ingestion alters the host response to sepsis in mice.Mice were randomized to receive either alcohol or water for 12 weeks and then subjected to cecal ligation and puncture. Mice were sacrificed 24 hours post-operatively or followed seven days for survival.Septic alcohol-fed mice had a significantly higher mortality than septic water-fed mice (74% vs. 41%, p = 0.01. This was associated with worsened gut integrity in alcohol-fed mice with elevated intestinal epithelial apoptosis, decreased crypt proliferation and shortened villus length. Further, alcohol-fed mice had higher intestinal permeability with decreased ZO-1 and occludin protein expression in the intestinal tight junction. The frequency of splenic and bone marrow CD4+ T cells was similar between groups; however, splenic CD4+ T cells in septic alcohol-fed mice had a marked increase in both TNF and IFN-γ production following ex vivo stimulation. Neither the frequency nor function of CD8+ T cells differed between alcohol-fed and water-fed septic mice. NK cells were decreased in both the spleen and bone marrow of alcohol-fed septic mice. Pulmonary myeloperoxidase levels and BAL levels of G-CSF and TFG-β were higher in alcohol-fed mice. Pancreatic metabolomics demonstrated increased acetate, adenosine, xanthine, acetoacetate, 3-hydroxybutyrate and betaine in alcohol-fed mice and decreased cytidine, uracil, fumarate, creatine phosphate, creatine, and choline. Serum and peritoneal cytokines were generally similar between alcohol-fed and water-fed mice, and there were no differences in bacteremia, lung wet to dry weight, or pulmonary, liver or splenic histology.When subjected to the same septic insult, mice with chronic alcohol ingestion have increased mortality. Alterations in intestinal integrity, the host immune

  11. Proliferative capacity of murine hematopoietic stem cells

    International Nuclear Information System (INIS)

    Hellman, S.; Botnick, L.E.; Hannon, E.C.; Vigneulle, R.M.

    1978-01-01

    The present study demonstrates a decrease in self-renewal capacity with serial transfer of murine hematopoietic stem cells. Production of differentiated cell progeny is maintained longer than stem cell self-renewal. In normal animals the capacity for self-renewal is not decreased with increasing donor age. The stem cell compartment in normal animals, both young and old, appears to be proliferatively quiescent. After apparent recovery from the alkylating agent busulfan, the probability of stem cell self-renewal is decreased, there is a permanent defect in the capacity of the bone marrow for serial transplantation, and the stem cells are proliferatively active. These findings support a model of the hematopoietic stem cell compartment as a continuum of cells with decreasing capacities for self-renewal, increasing likelihood for differentiation, and increasing proliferative activity. Cells progress in the continuum in one direction and such progression is not reversible

  12. Murine macrophage response from peritoneal cavity requires signals mediated by chemokine receptor CCR-2 during Staphylococcus aureus infection.

    Science.gov (United States)

    Nandi, Ajeya; Bishayi, Biswadev

    2016-02-01

    C-C chemokine receptor-2 (CCR-2) is a cognate receptor for monocyte chemotactic protein-1 (MCP-1), and recent studies revealed that MCP-1-CCR-2 signaling is involved in several inflammatory diseases characterized by macrophage infiltration. Currently, there is no study on the involvement of CCR-2 in the killing of S. aureus by macrophages of Swiss albino mice, and its substantial role in host defense against S. aureus infection in murine macrophages is still unclear. Therefore, the present study was aimed to investigate the functional and interactive role of CCR-2 and MCP-1 in regulating peritoneal macrophage responses with respect to acute S. aureus infection. We found that phagocytosis of S. aureus can serve as an important stimulus for MCP-1 production by peritoneal macrophages, which is dependent directly or indirectly on cytokines, reactive oxygen species and nitric oxide. Neutralization of CCR-2 in macrophages leads to increased production of IL-10 and decreased production of IFN-γ and IL-6. In CCR-2 blocked macrophages, pretreatment with specific blocker of NF-κB or p38-MAPK causes elevation in MCP-1 level and subsequent downregulation of CCR-2 itself. We speculate that CCR-2 is involved in S. aureus-induced MCP-1 production via NF-κB or p38-MAPK signaling. We also hypothesized that unnaturally high level of MCP-1 that build up upon CCR-2 neutralization might allow promiscuous binding to one or more other chemokine receptors, a situation that would not occur in CCR-2 non-neutralized condition. This may be the plausible explanation for such observed Th-2 response in CCR-2 blocked macrophages infected with S. aureus in the present study.

  13. Interferon synthesis in mouse peritoneal cells damaged by x irradiation

    Energy Technology Data Exchange (ETDEWEB)

    Szolgay, E; T' alas, M

    1976-01-01

    NDV-induced interferon of peritoneal cells of irradiated (x-rays, 400 R) and control mice was investigated in vitro. Irradiation or treatment with hydroxyurea (10(-5) M) and mitomycin C (25 microng/ml) did not change interferon synthesis in spite of an 80 to 90% inhibition of 3H-thymidine incorporation. Increased doses of mitomycin C and treatment with actinomycin D and puromycin blocked interferon production. De novo interferon synthesis occurred in cells with damaged replicative activity of DNA caused by irradiation or by treatment with antimetabolites.

  14. Study of Nitric Oxide production by murine peritoneal macrophages induced by Brucella Lipopolysaccharide

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    Kavoosi G

    2001-07-01

    Full Text Available Brueclla is a gram negative bacteria that causes Brucellosis. Lipopolysaccharide (LPS ", the pathogenic agent of Brucella is composed of O-chain, core oligosaccharide and lipid A. in addition, the structural and biological properties of different LPS extracted from different strains are not identical. The first defense system against LPS is nonspecific immunity that causes macrophage activation. Activated macrophages produce oxygen and nitrogen radicals that enhance the protection against intracellular pathogens.In this experiment LPS was extracted by hot phenol- water procedure and the effect of various LPSs on nitric oxide prodution by peritoneal mouse macrophages was examined.Our results demonstrated that the effect of LPS on nitric oxide production is concentration-dependent we observed the maximum response in concentration of 10-20 microgram per milliliter. Also our results demonstrate that LPS extracted from vaccine Brucella abortus (S 19 had a highe effect on nitric oxide production than the LPS from other strains

  15. Dialysate White Blood Cell Change after Initial Antibiotic Treatment Represented the Patterns of Response in Peritoneal Dialysis-Related Peritonitis.

    Science.gov (United States)

    Tantiyavarong, Pichaya; Traitanon, Opas; Chuengsaman, Piyatida; Patumanond, Jayanton; Tasanarong, Adis

    2016-01-01

    Background. Patients with peritoneal dialysis-related peritonitis usually have different responses to initial antibiotic treatment. This study aimed to explore the patterns of response by using the changes of dialysate white blood cell count on the first five days of the initial antibiotic treatment. Materials and Methods. A retrospective cohort study was conducted. All peritoneal dialysis-related peritonitis episodes from January 2014 to December 2015 were reviewed. We categorized the patterns of antibiotic response into 3 groups: early response, delayed response, and failure group. The changes of dialysate white blood cell count for each pattern were determined by multilevel regression analysis. Results. There were 644 episodes in 455 patients: 378 (58.7%) of early response, 122 (18.9%) of delayed response, and 144 (22.3%) of failure episodes. The patterns of early, delayed, and failure groups were represented by the average rate reduction per day of dialysate WBC of 68.4%, 34.0%, and 14.2%, respectively (p value patterns, which were categorized by types of responses, have variable rates of WBC declining. Clinicians should focus on the delayed response and failure patterns in order to make a decision whether to continue medical therapies or to aggressively remove the peritoneal catheter.

  16. Immunomodulatory Role of Ocimum gratissimum and Ascorbic Acid against Nicotine-Induced Murine Peritoneal Macrophages In Vitro

    Directory of Open Access Journals (Sweden)

    Santanu Kar Mahapatra

    2011-01-01

    Full Text Available The aim of this present study was to evaluate the immune functions and immune responses in nicotine-induced (10 mM macrophages and concurrently establish the immunomodulatory role of aqueous extract of Ocimum gratissimum (Ae-Og and ascorbic acid. In this study, nitrite generations and some phenotype functions by macrophages were studied. Beside that, release of Th1 cytokines (TNF-α, IL-12 and Th2 cytokines (IL-10, TGF-β was measured by ELISA, and the expression of these cytokines at mRNA level was analyzed by real-time PCR. Ae-Og, at a dose of 10 μg/mL, significantly reduced the nicotine-induced NO generation and iNOSII expression. Similar kinds of response were observed with supplementation of ascorbic acid (0.01 mM. The administration of Ae-Og and ascorbic acid increased the decreased adherence, chemotaxis, phagocytosis, and intracellular killing of bacteria in nicotine-treated macrophages. Ae-Og and ascorbic acid were found to protect the murine peritoneal macrophages through downregulation of Th1 cytokines in nicotine-treated macrophages with concurrent activation of Th2 responses. These findings strongly enhanced our understanding of the molecular mechanism leading to nicotine-induced suppression of immune functions and provide additional rationale for application of anti-inflammatory therapeutic approaches by O. gratissimum and ascorbic acid for different inflammatory disease prevention and treatment during nicotine toxicity.

  17. Mast cell repopulation of the peritoneal cavity: contribution of mast cell progenitors versus bone marrow derived committed mast cell precursors

    Directory of Open Access Journals (Sweden)

    Pastor Maria

    2010-06-01

    Full Text Available Abstract Background Mast cells have recently gained new importance as immunoregulatory cells that are involved in numerous pathological processes. One result of these processes is an increase in mast cell numbers at peripheral sites. This study was undertaken to determine the mast cell response in the peritoneal cavity and bone marrow during repopulation of the peritoneal cavity in rats. Results Two mast cell specific antibodies, mAb AA4 and mAb BGD6, were used to distinguish the committed mast cell precursor from more mature mast cells. The peritoneal cavity was depleted of mast cells using distilled water. Twelve hours after distilled water injection, very immature mast cells could be isolated from the blood and by 48 hours were present in the peritoneal cavity. At this same time the percentage of mast cells in mitosis increased fourfold. Mast cell depletion of the peritoneal cavity also reduced the total number of mast cells in the bone marrow, but increased the number of mast cell committed precursors. Conclusions In response to mast cell depletion of the peritoneal cavity, a mast cell progenitor is released into the circulation and participates in repopulation of the peritoneal cavity, while the committed mast cell precursor is retained in the bone marrow.

  18. Cultivation of murine bone marrow macrophages in sponges: a method that permits recovery of viable cultured cells

    Energy Technology Data Exchange (ETDEWEB)

    Akporiaye, E T; Stewart, S; Stewart, C C

    1984-01-01

    Various investigators have cultured murine bone marrow or peritoneal cells in vitro on glass or plastic surfaces with the ultimate aim of retrieving adherent macrophages for morphologic and functional evaluation. The removal of these adherent macrophages by conventional techniques has been consistently accompanied by low yield and significant cell damage. The authors report here a simple technique for culturing murine bone marrow cells in gelatin sponges (Spongostan and Gelfoam) in growth medium containing 10% fetal bovine serum and 10% L-cell conditioned medium. Viable cells were retrieved from the sponges in 10 min by digestion with collagenase. The in situ growth kinetics were similar to those found for cells cultured on plastic dishes. The recovered cells were adherent, phagocytic, positive for Fc ..gamma.. receptors, and had esterase activity. 23 references, 1 figure, 1 table.

  19. Clear cell sarcoma of the abdominal wall with peritoneal sarcomatosis: CT features

    International Nuclear Information System (INIS)

    Sabate, J.M.; Fernandez, A.; Torrubia, S.; Villanueva, A.; Monill, J.M.

    1999-01-01

    Clear cell sarcoma, also called malignant melanoma of soft parts, is an uncommon neoplasm that involves tendons or aponeuroses of the lower extremity. The CT features of a clear cell sarcoma arising from the abdominal wall with later peritoneal dissemination are described. Peritoneal sarcomatosis from soft tissue sarcomas is a very rare condition previously unreported in the radiologic literature. Metastases to peritoneal surfaces must therefore be considered a possible site for systemic dissemination of soft tissue sarcomas. (orig.)

  20. Eradication of colon cancer cells before tumour formation in the peritoneal cavity of mice treated with intraperitoneal Re-186 radioimmunotherapy

    International Nuclear Information System (INIS)

    Kinuya, S.; Hiramatsu, T.; Michigishi, T.

    2006-01-01

    A treatment adjuvant to surgical resection of the primary lesion has been proven to be beneficial in improving the prognosis of patients with high risks of peritoneal dissemination of colon cancer. This study was performed to determine the comparative efficacy of intraperitoneal radioimmunotherapy (RIT) using Re-186 or I-131 labeled murine antibodies in the extermination of cancer cells. A murine anti-colorectal IgG1, A7 monoclonal antibody, was radio-labeled either with I-131 (by the chloramine-T method) or Re-186 (by the MAG3 pre-chelated method). A total number of 16 mice were subjected to RIT with Re-186 A7 (N=8) or I-131 A7 (N=8) at equitoxic doses in Balb/c bu/nu mice 10 min after intraperitoneal injection of LS180 human colon cancer cells. A third group of mice were subjected to chemotherapy with 5-fluorouracil at 30 mg/kg for 4 consecutive days following the intraperitoneal injection of the same LS180 human colon cancer cells. There were 19 mice in the control group who were not subjected to any form of therapy. The results revealed that the mean survival of mice in the control (N-19), I-131 A7 RIT (N=8) and Chemotherapy (N=6) groups were 33.8 ± 1.0, 80.1 ± 2.5 and 49.3 ± 5.3 days respectively. The eight mice who were subjected to Re-186 A7 RIT showed much better survival compared to the other groups. Two of the eight mice from this group died at 105 and 111 days following Re-186 A7 RIT. Other six mice were sacrificed at 172 days, and autopsy revealed no macroscopic peritoneal tumor growth. Based on this pilot study we concluded that individual tumor cells in the peritoneal cavity would be effectively exterminated by intraperitoneal RIT with Re-186 A7. (author)

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

    Science.gov (United States)

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

    2011-05-25

    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.

  2. Quercetin uptake and metabolism by murine peritoneal macrophages in vitro

    Directory of Open Access Journals (Sweden)

    Chieh-Jung Liu

    2015-12-01

    Full Text Available Quercetin (Q, a bioflavonoid ubiquitously distributed in vegetables, fruits, leaves, and grains, can be absorbed, transported, and excreted after oral intake. However, little is known about Q uptake and metabolism by macrophages. To clarify the puzzle, Q at its noncytotoxic concentration (44μM was incubated without or with mouse peritoneal macrophages for different time periods. Medium alone, extracellular, and intracellular fluids of macrophages were collected to detect changes in Q and its possible metabolites using high-performance liquid chromatography. The results showed that Q was unstable and easily oxidized in either the absence or the presence of macrophages. The remaining Q and its metabolites, including isorhamnetin and an unknown Q metabolite [possibly Q– (O-semiquinone], might be absorbed by macrophages. The percentage of maximal Q uptake by macrophages was found to be 2.28% immediately after incubation; however, Q uptake might persist for about 24 hours. Q uptake by macrophages was greater than the uptake of its methylated derivative isorhamnetin. As Q or its metabolites entered macrophages, those compounds were metabolized primarily into isorhamnetin, kaempferol, or unknown endogenous Q metabolites. The present study, which aimed to clarify cellular uptake and metabolism of Q by macrophages, may have great potential for future practical applications for human health and immunopharmacology.

  3. Gastric cancer cell supernatant causes apoptosis and fibrosis in the peritoneal tissues and results in an environment favorable to peritoneal metastases, in vitro and in vivo

    Directory of Open Access Journals (Sweden)

    Na Di

    2012-04-01

    Full Text Available Abstract Background In this study, we examined effects of soluble factors released by gastric cancer cells on peritoneal mesothelial cells in vitro and in vivo. Methods HMrSV5, a human peritoneal mesothelial cell line, was incubated with supernatants from gastric cancer cells. Morphological changes of HMrSV5 cells were observed. Apoptosis of HMrSV5 cells was observed under a transmission electron microscope and quantitatively determined by MTT assay and flow cytometry. Expressions of apoptosis-related proteins (caspase-3, caspase-8, Bax, bcl-2 were immunochemically evaluated. Results Conspicuous morphological changes indicating apoptosis were observed in HMrSV5 cells 24 h after treatment with the supernatants of gastric cancer cells. In vivo, peritoneal tissues treated with gastric cancer cell supernatant were substantially thickened and contained extensive fibrosis. Conclusions These findings demonstrate that supernatants of gastric cancer cells can induce apoptosis and fibrosis in HMrSV5 human peritoneal mesothelial cells through supernatants in the early peritoneal metastasis, in a time-dependent manner, and indicate that soluble factors in the peritoneal cavity affect the morphology and function of mesothelial cells so that the resulting environment can become favorable to peritoneal metastases.

  4. [Comparison of the immunomodulatory effects of spore polysaccharides and broken spore polysaccharides isolated from Ganoderma lucidum on murine splenic lymphocytes and peritoneal macrophages in vitro].

    Science.gov (United States)

    Wang, Peng-yun; Wang, Sai-zhen; Lin, Shu-qian; Lin, Zhi-bin

    2005-12-18

    To compare the immunomodulatory effects of spore polysaccharides (Gl-SP) and broken spore polysaccharides (Gl-BSP) isolated from Ganoderma lucidum(Leyss et Fr.) Karst. on murine splenic lymphocytes and peritoneal macrophages in vitro. Mixed lymphocyte culture reaction (MLR), lymphocyte proliferation in the presence or absence of mitogen, and the cytotoxic activity of splenic natural killer (NK) cells were detected with MTT assay in vitro. The percentage of phagocytosis of neutral red (NR) by mouse peritoneal macrophages was detected by colorimetric assay. Splenic T-lymphocyte subpopulations were measured with flow cytometry(FCM). IL-2, IFN-gamma and TNF-alpha in the culture supernatants were detected by ELISA and biological assay. Nitric oxide (NO) production was examined by Griess reaction. At the concentration range of 0.2-12.8 mg/L, Gl-SP and Gl-BSP were shown to increase lymphocyte proliferation in the presence or absence of mitogen, enhance NK cytotoxic activity, augment the production of TNF-alpha and NO in Gl-SP- or Gl-BSP-activated macrophages, as well the percentage of phagocytosis of NR by macrophages in vitro. Both Gl-SP and Gl-BSP could promote MLR, however, at the dose of 12.8 mg/L, Gl-BSP showed higher activity than Gl-SP in the proliferation of lymphocytes. These two kinds of polysaccharide could significantly increase the secretion of IL-2 and IFN-gamma in doublejway MLR at the concentrations of 0.2-12.8 mg/L, but Gl-BSP had stronger effects than Gl-SP at the same concentrations. Both Gl-SP and Gl-BSP could increase the ratio of T-lymphocyte subpopulations in double-way MLR. At the concentrations of 0.2-12.8 mg/L or 3.2-12.8 mg/L, Gl-BSP demonstrated more significant activity in increasing the percentage of the CD4(+) or CD8(+) subset than Gl-SP. At the concentrations of 0.2-0.8 mg/L, the ratio of the CD4(+) and CD8(+) subset in the Gl-BSP treated group was higher than that of the Gl-SP treated group. Gl-SP and Gl-BSP have similar immunomodulatory

  5. B-1 cells modulate the murine macrophage response to Leishmania major infection.

    Science.gov (United States)

    Arcanjo, Angelica F; Nunes, Marise P; Silva-Junior, Elias B; Leandro, Monique; da Rocha, Juliana Dutra Barbosa; Morrot, Alexandre; Decote-Ricardo, Debora; Freire-de-Lima, Celio Geraldo

    2017-05-26

    To investigate the modulatory effect of B-1 cells on murine peritoneal macrophages infected with Leishmania major ( L. major ) in vitro . Peritoneal macrophages obtained from BALB/c and BALB/c XID mice were infected with L. major and cultured in the presence or absence of B-1 cells obtained from wild-type BALB/c mice. Intracellular amastigotes were counted, and interleukin-10 (IL-10) production was quantified in the cellular supernatants using an enzyme-linked immunosorbent assay. The levels of the lipid mediator prostaglandin E2 (PGE 2 ) were determined using a PGE 2 enzyme immunoassay kit (Cayman Chemical, Ann Arbor, MI), and the number of lipid bodies was quantified in the cytoplasm of infected macrophages in the presence and absence of B-1 cells. Culturing the cells with selective PGE 2 -neutralizing drugs inhibited PGE 2 production and confirmed the role of this lipid mediator in IL-10 production. In contrast, we demonstrated that B-1 cells derived from IL-10 KO mice did not favor the intracellular growth of L. major . We report that B-1 cells promote the growth of L. major amastigotes inside peritoneal murine macrophages. We demonstrated that the modulatory effect was independent of physical contact between the cells, suggesting that soluble factor(s) were released into the cultures. We demonstrated in our co-culture system that B-1 cells trigger IL-10 production by L. major -infected macrophages. Furthermore, the increased secretion of IL-10 was attributed to the presence of the lipid mediator PGE 2 in supernatants of L. major -infected macrophages. The presence of B-1 cells also favors the production of lipid bodies by infected macrophages. In contrast, we failed to obtain the same effect on parasite replication inside L. major -infected macrophages when the B-1 cells were isolated from IL-10 knockout mice. Our results show that elevated levels of PGE 2 and IL-10 produced by B-1 cells increase L. major growth, as indicated by the number of parasites in cell

  6. Peritoneal macrophages mediated delivery of chitosan/siRNA nanoparticle to the lesion site in a murine radiation-induced fibrosis model

    DEFF Research Database (Denmark)

    Nawroth, Isabel; Alsner, Jan; Deleuran, B.W.

    2013-01-01

    of chitosan/siRNA nanoparticles directed towards silencing TNF alpha in local macrophage populations, but the mechanism for the therapeutic effect at the lesion site remains unclear. Methods. Using the same murine RIF model we utilized an optical imaging technique and fluorescence microscopy to investigate...... the uptake of chitosan/fluorescently labeled siRNA nanoparticles by peritoneal macrophages and their subsequent migration to the inflamed tissue in the RIF model. Results. We observed strong accumulation of the fluorescent signal in the lesion site of the irradiated leg up to 24 hours using the optical...... imaging system. We further confirm by immunohistochemical staining that Cy3 labeled siRNA resides in macrophages of the irradiated leg. Conclusion. We provide a proof-of-concept for host macrophage trafficking towards the inflamed region in a murine RIF model, which thereby suggests that the chitosan...

  7. Apoptosis transcriptional mechanism of feline infectious peritonitis virus infected cells.

    Science.gov (United States)

    Shuid, Ahmad Naqib; Safi, Nikoo; Haghani, Amin; Mehrbod, Parvaneh; Haron, Mohd Syamsul Reza; Tan, Sheau Wei; Omar, Abdul Rahman

    2015-11-01

    Apoptosis has been postulated to play an important role during feline infectious peritonitis virus (FIPV) infection; however, its mechanism is not well characterized. This study is focused on apoptosis and transcriptional profiling of FIPV-infected cells following in vitro infection of CRFK cells with FIPV 79-1146 WSU. Flow cytometry was used to determine mode of cell death in first 42 h post infection (hpi). FIPV infected cells underwent early apoptosis at 9 hpi (p apoptosis at 12 hpi (p apoptosis cluster (80 down-regulated and 51 up-regulated) along with increase of apoptosis, p53, p38 MAPK, VEGF and chemokines/cytokines signaling pathways were probably involved in apoptosis process. Six of the de-regulated genes expression (RASSF1, BATF2, MAGEB16, PDCD5, TNFα and TRAF2) and TNFα protein concentration were analyzed by RT-qPCR and ELISA, respectively, at different time-points. Up-regulations of both pro-apoptotic (i.e. PDCD5) and anti-apoptotic (i.e. TRAF2) were detected from first hpi and continuing to deregulate during apoptosis process in the infected cells.

  8. Methylglyoxal Induced Basophilic Spindle Cells with Podoplanin at the Surface of Peritoneum in Rat Peritoneal Dialysis Model

    Directory of Open Access Journals (Sweden)

    Ichiro Hirahara

    2015-01-01

    Full Text Available Peritoneal dialysis (PD is a common treatment for patients with reduced or absent renal function. Long-term PD leads to peritoneal injury with structural changes and functional decline. At worst, peritoneal injury leads to encapsulating peritoneal sclerosis (EPS, which is a serious complication of PD. In order to carry out PD safely, it is important to define the mechanism of progression of peritoneal injury and EPS. We prepared rat models of peritoneal injury by intraperitoneal administration of glucose degradation products, such as methylglyoxal (MGO or formaldehyde (FA, chlorhexidine gluconate (CG, and talc. In rats treated with MGO, peritoneal fibrous thickening with the appearance of basophilic spindle cells with podoplanin, cytokeratin, and α-smooth muscle actin at the surface of the peritoneum was observed. These cells may have been derived from mesothelial cells by epithelial-to-mesenchymal transition. In FA- or CG-treated rats, the peritoneum was thickened, and mesothelial cells were absent at the surface of the peritoneum. The CG- or MGO-treated rats presented with a so-called abdominal cocoon. In the talc-treated rats, extensive peritoneal adhesion and peritoneal thickening were observed. MGO-induced peritoneal injury model may reflect human histopathology and be suitable to analyze the mechanism of progression of peritoneal injury and EPS.

  9. Plasma Cell Alloantigen 1 and IL-10 Secretion Define Two Distinct Peritoneal B1a B Cell Subsets With Opposite Functions, PC1high Cells Being Protective and PC1low Cells Harmful for the Growing Fetus

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    Anne Schumacher

    2018-05-01

    Full Text Available B cells possess various immuno regulatory functions. However, research about their participation in tolerance induction toward the fetus is just emerging. Accumulating evidence supports the idea that B cells can play seemingly conflicting roles during pregnancy, either protecting or harming the fetus. Previous findings indicated the presence of two different peritoneal B cell subsets, defined by the expression of the plasma cell alloantigen 1 (PC1 and with distinct immune modulatory functions. Here, we aimed to study the participation of these two B cell subsets, on pregnancy outcome in a murine model of disturbed fetal tolerance. The frequencies and cell numbers of peritoneal and splenic CD19+IL-10+ and CD19+CD5+IL-10+PC1+ cells were assessed in virgin as well as normal pregnant (NP and abortion-prone (AP females during the course of gestation. Peritoneal PC1low or PC1high B1a B cells were sorted, analyzed for their ability to secrete IL-10 and adoptively transferred into NP or AP females. On gestation day (gd 12, the abortion rate as well as the frequencies and cell numbers of regulatory T cells, TH1 and TH17 cells were determined in spleens and decidua. In addition, mRNA expression of IL-10, TGF-β, IFN-γ, and TNF-α was analyzed in decidual tissue. Peritoneal CD19+IL-10+ and CD19+CD5+IL-10+PC1+ frequencies fluctuated during the progression of normal pregnancies while no significant changes were observed in spleen. AP females showed significantly reduced frequencies of both B cell populations and exhibited an altered peritoneal PC1high/PC1low ratio at gd10. Adoptive transfers of PC1low B1a B cells into NP females increased the abortion rate in association with a reduced splenic regulatory T/TH17 ratio. By contrast, the transfer of PC1high B1a B cells into AP females significantly diminished the fetal rejection rate and significantly reduced the numbers of splenic TH17 cells. Our results suggest that the peritoneum harbors two distinct B1a B

  10. Parasitic infection improves survival from septic peritonitis by enhancing mast cell responses to bacteria in mice.

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    Rachel E Sutherland

    Full Text Available Mammals are serially infected with a variety of microorganisms, including bacteria and parasites. Each infection reprograms the immune system's responses to re-exposure and potentially alters responses to first-time infection by different microorganisms. To examine whether infection with a metazoan parasite modulates host responses to subsequent bacterial infection, mice were infected with the hookworm-like intestinal nematode Nippostrongylus brasiliensis, followed in 2-4 weeks by peritoneal injection of the pathogenic bacterium Klebsiella pneumoniae. Survival from Klebsiella peritonitis two weeks after parasite infection was better in Nippostrongylus-infected animals than in unparasitized mice, with Nippostrongylus-infected mice having fewer peritoneal bacteria, more neutrophils, and higher levels of protective interleukin 6. The improved survival of Nippostrongylus-infected mice depends on IL-4 because the survival benefit is lost in mice lacking IL-4. Because mast cells protect mice from Klebsiella peritonitis, we examined responses in mast cell-deficient Kit(W-sh/Kit(W-sh mice, in which parasitosis failed to improve survival from Klebsiella peritonitis. However, adoptive transfer of cultured mast cells to Kit(W-sh/Kit(W-sh mice restored survival benefits of parasitosis. These results show that recent infection with Nippostrongylus brasiliensis protects mice from Klebsiella peritonitis by modulating mast cell contributions to host defense, and suggest more generally that parasitosis can yield survival advantages to a bacterially infected host.

  11. Induction of gastric cancer cell adhesion through transforming growth factor-beta1-mediated peritoneal fibrosis

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    Ma Xiao-Yang

    2010-10-01

    Full Text Available Abstract Background Peritoneal dissemination is one of the main causes of death in gastric cancer patients. Transforming growth factor-beta1 (TGF-β1, one of the most potent fibrotic stimuli for mesothelial cells, may play a key role in this processing. The purpose of this study is to elucidate the effects of TGF-β1 on regulation of gastric cancer adhesion to mesothelial cells. Methods Peritoneal tissues and peritoneal wash fluid were obtained for hematoxylin and eosin staining or ELISA to measure fibrosis and TGF-β1 levels, respectively. The peritoneal mesothelial cell line, HMrSV5, was used to determine the role of TGF-β1 in regulation of gastric cancer cell adhesion to mesothelial cells and expression of collagen, fibronectin, and Smad 2/3 by using adhesion assay, western blot, and RT-PCR. Results The data showed that TGF-β1 treatment was able to induce collagen III and fibronectin expression in the mesothelial cells, which was associated with an increased adhesion ability of gastric cancer cells, but knockdown of minimal sites of cell binding domain of extracellular matrix can partially inhibit these effects. Conclusion Peritoneal fibrosis induced by TGF-β1 may provide a favorable environment for the dissemination of gastric cancer.

  12. Peracetylated hydroxytyrosol, a new hydroxytyrosol derivate, attenuates LPS-induced inflammatory response in murine peritoneal macrophages via regulation of non-canonical inflammasome, Nrf2/HO1 and JAK/STAT signaling pathways.

    Science.gov (United States)

    Montoya, Tatiana; Aparicio-Soto, Marina; Castejón, María Luisa; Rosillo, María Ángeles; Sánchez-Hidalgo, Marina; Begines, Paloma; Fernández-Bolaños, José G; Alarcón-de-la-Lastra, Catalina

    2018-03-18

    The present study was designed to investigate the anti-inflammatory effects of a new derivative of hydroxytyrosol (HTy), peracetylated hydroxytyrosol (Per-HTy), compared with its parent, HTy, on lipopolysaccharide (LPS)-stimulated murine macrophages as well as potential signaling pathways involved. In particular, we attempted to characterize the role of the inflammasome underlying Per-HTy possible anti-inflammatory effects. Isolated murine peritoneal macrophages were treated with HTy or its derivative in the presence or absence of LPS (5 μg/ml) for 18 h. Cell viability was determined using sulforhodamine B (SRB) assay. Nitric oxide (NO) production was analyzed by Griess method. Production of pro-inflammatory cytokines was evaluated by enzyme-linked immunosorbent assay (ELISA) and inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2, janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway (STAT3), haem oxigenase 1 (HO1), nuclear factor (erythroid-derived 2)-like 2 (Nrf2) expression and mitogen-activated protein kinases (MAPKs) activation was determined by Western blot. Per-HTy significantly reduced the levels of NO and pro-inflammatory cytokines as well as both COX-2 and iNOS expressions. Furthermore, Per-HTy treatment inhibited STAT3 and increased Nrf2 and HO1 protein levels in murine macrophages exposed to LPS. In addition, Per-HTy anti-inflammatory activity was related with an inhibition of non-canonical nucleotide binding domain (NOD)-like receptor (NLRP3) inflammasome pathways by decreasing pro-inflammatory interleukin (IL)-1β and IL-18 cytokine levels as consequence of regulation of cleaved caspase-11 enzyme. These results support that this new HTy derivative may offer a new promising nutraceutical therapeutic strategy in the management of inflammatory-related pathologies. Copyright © 2018. Published by Elsevier Inc.

  13. Pure Δ9-tetrahydrocannabivarin and a Cannabis sativa extract with high content in Δ9-tetrahydrocannabivarin inhibit nitrite production in murine peritoneal macrophages.

    Science.gov (United States)

    Romano, Barbara; Pagano, Ester; Orlando, Pierangelo; Capasso, Raffaele; Cascio, Maria Grazia; Pertwee, Roger; Marzo, Vincenzo Di; Izzo, Angelo A; Borrelli, Francesca

    2016-11-01

    Historical and scientific evidence suggests that Cannabis use has immunomodulatory and anti-inflammatory effects. We have here investigated the effect of the non-psychotropic phytocannabinoid Δ 9 -tetrahydrocannabivarin (THCV) and of a Cannabis sativa extract with high (64.8%) content in THCV (THCV-BDS) on nitric oxide (NO) production, and on cannabinoid and transient receptor potential (TRP) channel expression in lipopolysaccharide (LPS)-stimulated murine peritoneal macrophages. THCV-BDS and THCV exhibited similar affinity in radioligand binding assays for CB 1 and CB 2 receptors, and inhibited, via CB 2 but not CB 1 cannabinoid receptors, nitrite production evoked by LPS in peritoneal macrophages. THCV down-regulated the over-expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2) and interleukin 1β (IL-1β) proteins induced by LPS. Furthermore, THCV counteracted LPS-induced up-regulation of CB 1 receptors, without affecting the changes in CB 2 , TRPV2 or TRPV4 mRNA expression caused by LPS. Other TRP channels, namely, TRPA1, TRPV1, TRPV3 and TRPM8 were poorly expressed or undetectable in both unstimulated and LPS-challenged macrophages. It is concluded that THCV - via CB 2 receptor activation - inhibits nitrite production in macrophages. The effect of this phytocannabinoid was associated with a down-regulation of CB 1 , but not CB 2 or TRP channel mRNA expression. Copyright © 2016 Elsevier Ltd. All rights reserved.

  14. Induction of histamine release in vitro from rat peritoneal mast cells by extracts of grain dust.

    Science.gov (United States)

    Warren, C P; Holford-Strevens, V

    1986-01-01

    The ability of extracts of grain dust and wheat to induce histamine release from rat peritoneal cells was investigated. Some grain dusts, with a high endotoxin content, were found to produce cytotoxic histamine release. Extract of wheat dust, with a low endotoxin release, produced noncytotoxic histamine release from peritoneal cells but not from purified mast cells. This reaction was dependent on the presence of phosphatidyl serine. The agent did not appear to be a lectin because histamine release was not enhanced by passive sensitization of mast cells with IgE. The activity occurred only over a narrow range of concentrations of the extract of wheat. The cause was unclear. PMID:2423321

  15. Vaccine-induced modulation of gene expression in turbot peritoneal cells. A microarray approach.

    Science.gov (United States)

    Fontenla, Francisco; Blanco-Abad, Verónica; Pardo, Belén G; Folgueira, Iria; Noia, Manuel; Gómez-Tato, Antonio; Martínez, Paulino; Leiro, José M; Lamas, Jesús

    2016-07-01

    We used a microarray approach to examine changes in gene expression in turbot peritoneal cells after injection of the fish with vaccines containing the ciliate parasite Philasterides dicentrarchi as antigen and one of the following adjuvants: chitosan-PVMMA microspheres, Freund́s complete adjuvant, aluminium hydroxide gel or Matrix-Q (Isconova, Sweden). We identified 374 genes that were differentially expressed in all groups of fish. Forty-two genes related to tight junctions and focal adhesions and/or actin cytoskeleton were differentially expressed in free peritoneal cells. The profound changes in gene expression related to cell adherence and cytoskeleton may be associated with cell migration and also with the formation of cell-vaccine masses and their attachment to the peritoneal wall. Thirty-five genes related to apoptosis were differentially expressed. Although most of the proteins coded by these genes have a proapoptotic effect, others are antiapoptotic, indicating that both types of signals occur in peritoneal leukocytes of vaccinated fish. Interestingly, many of the genes related to lymphocytes and lymphocyte activity were downregulated in the groups injected with vaccine. We also observed decreased expression of genes related to antigen presentation, suggesting that macrophages (which were abundant in the peritoneal cavity after vaccination) did not express these during the early inflammatory response in the peritoneal cavity. Finally, several genes that participate in the inflammatory response were differentially expressed, and most participated in resolution of inflammation, indicating that an M2 macrophage response is generated in the peritoneal cavity of fish one day post vaccination. Copyright © 2016 Elsevier Ltd. All rights reserved.

  16. Extra virgin olive oil polyphenolic extracts downregulate inflammatory responses in LPS-activated murine peritoneal macrophages suppressing NFκB and MAPK signalling pathways.

    Science.gov (United States)

    Cárdeno, A; Sánchez-Hidalgo, M; Aparicio-Soto, M; Sánchez-Fidalgo, S; Alarcón-de-la-Lastra, C

    2014-06-01

    Extra virgin olive oil (EVOO) is obtained from the fruit of the olive tree Olea europaea L. Phenolic compounds present in EVOO have recognized anti-oxidant and anti-inflammatory properties. However, the activity of the total phenolic fraction extracted from EVOO and the action mechanisms involved are not well defined. The present study was designed to evaluate the potential anti-inflammatory mechanisms of the polyphenolic extract (PE) from EVOO on LPS-stimulated peritoneal murine macrophages. Nitric oxide (NO) production was analyzed by the Griess method and intracellular reactive oxygen species (ROS) by fluorescence analysis. Moreover, changes in the protein expression of the pro-inflammatory enzymes, inducible nitric oxide synthase (iNOS), cyclooxygenase (COX)-2 and microsomal prostaglandin E synthase-1 (mPGES-1), as well as the role of nuclear transcription factor kappa B (NFκB) and mitogen-activated protein kinase (MAPK) signalling pathways, were analyzed by Western blot. PE from EVOO reduced LPS-induced oxidative stress and inflammatory responses through decreasing NO and ROS generation. In addition, PE induced a significant down-regulation of iNOS, COX-2 and mPGES-1 protein expressions, reduced MAPK phosphorylation and prevented the nuclear NFκB translocation. This study establishes that PE from EVOO possesses anti-inflammatory activities on LPS-stimulated murine macrophages.

  17. Primary peritoneal clear cell carcinoma versus ovarian carcinoma versus malignant transformation of endometriosis: a vexing issue.

    Science.gov (United States)

    Insabato, Luigi; Natella, Valentina; Somma, Anna; Persico, Marcello; Camera, Luigi; Losito, Nunzia Simona; Masone, Stefania

    2015-05-01

    Peritoneum is a site for both primary and secondary tumors. Primary peritoneal tumors are fairly rare. The most common primary tumors of the peritoneum are malignant mesothelioma and serous papillary adenocarcinoma. Clear cell carcinoma of the peritoneum is extremely rare and often misdiagnosed as mesothelioma, serous carcinoma, or metastatic adenocarcinoma, so it represents a diagnostic challenge for both clinicians and pathologists. Up to date, to the best of our knowledge, only 11 cases of primary peritoneal clear cell carcinoma have been reported in the English literature. Distinguishing this tumor of the peritoneum versus ovarian carcinoma can be problematic. Herein, we report a rare case of primary peritoneal clear cell carcinoma occurring in a 49-year-old woman, along with a review of the literature. © The Author(s) 2015.

  18. Exosomes Promote Ovarian Cancer Cell Invasion through Transfer of CD44 to Peritoneal Mesothelial Cells.

    Science.gov (United States)

    Nakamura, Koji; Sawada, Kenjiro; Kinose, Yasuto; Yoshimura, Akihiko; Toda, Aska; Nakatsuka, Erika; Hashimoto, Kae; Mabuchi, Seiji; Morishige, Ken-Ichirou; Kurachi, Hirohisa; Lengyel, Ernst; Kimura, Tadashi

    2017-01-01

    Epithelial ovarian cancer (EOC) cells metastasize within the peritoneal cavity and directly encounter human peritoneal mesothelial cells (HPMC) as the initial step of metastasis. The contact between ovarian cancer cells and the single layer of mesothelial cells involves direct communications that modulate cancer progression but the mechanisms are unclear. One candidate mediating cell-cell communications is exosomes, 30-100 nm membrane vesicles of endocytic origin, through the cell-cell transfer of proteins, mRNAs, or microRNAs. Therefore, the goal was to mechanistically characterize how EOC-derived exosomes modulate metastasis. Exosomes from ovarian cancer cells were fluorescently labeled and cocultured with HPMCs which internalized the exosomes. Upon exosome uptake, HPMCs underwent a change in cellular morphology to a mesenchymal, spindle phenotype. CD44, a cell surface glycoprotein, was found to be enriched in the cancer cell-derived exosomes, transferred, and internalized to HPMCs, leading to high levels of CD44 in HPMCs. This increased CD44 expression in HPMCs promoted cancer invasion by inducing the HPMCs to secrete MMP9 and by cleaning the mesothelial barrier for improved cancer cell invasion. When CD44 expression was knocked down in cancer cells, exosomes had fewer effects on HPMCs. The inhibition of exosome release from cancer cells blocked CD44 internalization in HPMCs and suppressed ovarian cancer invasion. In ovarian cancer omental metastasis, positive CD44 expression was observed in those mesothelial cells that directly interacted with cancer cells, whereas CD44 expression was negative in the mesothelial cells remote from the invading edge. This study indicates that ovarian cancer-derived exosomes transfer CD44 to HPMCs, facilitating cancer invasion. Mechanistic insight from the current study suggests that therapeutic targeting of exosomes may be beneficial in treating ovarian cancer. Mol Cancer Res; 15(1); 78-92. ©2016 AACR. ©2016 American

  19. Inhibition of PRL-3 gene expression in gastric cancer cell line SGC7901 via microRNA suppressed reduces peritoneal metastasis

    International Nuclear Information System (INIS)

    Li Zhengrong; Zhan Wenhua; Wang Zhao; Zhu Baohe; He Yulong; Peng Junsheng; Cai Shirong; Ma Jinping

    2006-01-01

    High expression of PRL-3, a protein tyrosine phosphatase, is proved to be associated with lymph node metastasis in gastric carcinoma from previous studies. In this paper, we examined the relationship between PRL-3 expression and peritoneal metastasis in gastric carcinoma. We applied the artificial miRNA (pCMV-PRL3miRNA), which is based on the murine miR-155 sequence, to efficiently silence the target gene expression of PRL-3 in SGC7901 gastric cancer cells at both mRNA and protein levels. Then we observed that, in vitro, pCMV-PRL3miRNA significantly depressed the SGC7901 cell invasion and migration independent of cellular proliferation. In vivo, PRL-3 knockdown effectively suppressed the growth of peritoneal metastases and improved the prognosis in nude mice. Therefore, we concluded that artificial miRNA can depress the expression of PRL-3, and that PRL-3 might be a potential therapeutic target for gastric cancer peritoneal metastasis

  20. Murine cell glycolipids customization by modular expression of glycosyltransferases.

    Science.gov (United States)

    Cid, Emili; Yamamoto, Miyako; Buschbeck, Marcus; Yamamoto, Fumiichiro

    2013-01-01

    Functional analysis of glycolipids has been hampered by their complex nature and combinatorial expression in cells and tissues. We report an efficient and easy method to generate cells with specific glycolipids. In our proof of principle experiments we have demonstrated the customized expression of two relevant glycosphingolipids on murine fibroblasts, stage-specific embryonic antigen 3 (SSEA-3), a marker for stem cells, and Forssman glycolipid, a xenoantigen. Sets of genes encoding glycosyltansferases were transduced by viral infection followed by multi-color cell sorting based on coupled expression of fluorescent proteins.

  1. Entry of Francisella tularensis into Murine B Cells: The Role of B Cell Receptors and Complement Receptors.

    Directory of Open Access Journals (Sweden)

    Lenka Plzakova

    Full Text Available Francisella tularensis, the etiological agent of tularemia, is an intracellular pathogen that dominantly infects and proliferates inside phagocytic cells but can be seen also in non-phagocytic cells, including B cells. Although protective immunity is known to be almost exclusively associated with the type 1 pathway of cellular immunity, a significant role of B cells in immune responses already has been demonstrated. Whether their role is associated with antibody-dependent or antibody-independent B cell functions is not yet fully understood. The character of early events during B cell-pathogen interaction may determine the type of B cell response regulating the induction of adaptive immunity. We used fluorescence microscopy and flow cytometry to identify the basic requirements for the entry of F. tularensis into B cells within in vivo and in vitro infection models. Here, we present data showing that Francisella tularensis subsp. holarctica strain LVS significantly infects individual subsets of murine peritoneal B cells early after infection. Depending on a given B cell subset, uptake of Francisella into B cells is mediated by B cell receptors (BCRs with or without complement receptor CR1/2. However, F. tularensis strain FSC200 ΔiglC and ΔftdsbA deletion mutants are defective in the ability to enter B cells. Once internalized into B cells, F. tularensis LVS intracellular trafficking occurs along the endosomal pathway, albeit without significant multiplication. The results strongly suggest that BCRs alone within the B-1a subset can ensure the internalization process while the BCRs on B-1b and B-2 cells need co-signaling from the co receptor containing CR1/2 to initiate F. tularensis engulfment. In this case, fluidity of the surface cell membrane is a prerequisite for the bacteria's internalization. The results substantially underline the functional heterogeneity of B cell subsets in relation to F. tularensis.

  2. Regulation of Murine Natural Killer Cell Commitment

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    Nicholas D Huntington

    2013-01-01

    Full Text Available NK cells can derive from the same precursors as B and T cells, however to achieve lineage specificity, several transcription factors need to be activated or annulled. While a few important transcription factors have identified for NK genesis the mechanisms of how this is achieved is far from resolved. Adding to the complexity of this, NK cells are found and potentially develop in diverse locations in vivo and it remains to be addressed if a common NK cell precursor seeds diverse niches and how transcription factors may differentially regulate NK cell commitment in distinct microenvironments. Here we will summarise some recent findings in NK cell commitment and discuss how a NK cell transcriptional network might be organised, while addressing some misconceptions and anomalies along the way.

  3. Haemopedia: An Expression Atlas of Murine Hematopoietic Cells

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    Carolyn A. de Graaf

    2016-09-01

    Full Text Available Hematopoiesis is a multistage process involving the differentiation of stem and progenitor cells into distinct mature cell lineages. Here we present Haemopedia, an atlas of murine gene-expression data containing 54 hematopoietic cell types, covering all the mature lineages in hematopoiesis. We include rare cell populations such as eosinophils, mast cells, basophils, and megakaryocytes, and a broad collection of progenitor and stem cells. We show that lineage branching and maturation during hematopoiesis can be reconstructed using the expression patterns of small sets of genes. We also have identified genes with enriched expression in each of the mature blood cell lineages, many of which show conserved lineage-enriched expression in human hematopoiesis. We have created an online web portal called Haemosphere to make analyses of Haemopedia and other blood cell transcriptional datasets easier. This resource provides simple tools to interrogate gene-expression-based relationships between hematopoietic cell types and genes of interest.

  4. Nanoelectroablation therapy for murine basal cell carcinoma

    Energy Technology Data Exchange (ETDEWEB)

    Nuccitelli, Richard, E-mail: rich@bioelectromed.com [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); Chang, Kris S.; Epstein, Ervin H. [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); Stanford University, Stanford, CA 94305 (United States)

    2012-08-03

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

  5. Immunohistochemical characterization of endometriosis-associated smooth muscle cells in human peritoneal endometriotic lesions.

    Science.gov (United States)

    Barcena de Arellano, Maria L; Gericke, Jessica; Reichelt, Uta; Okuducu, Ali Fuat; Ebert, Andreas D; Chiantera, Vito; Schneider, Achim; Mechsner, Sylvia

    2011-10-01

    Smooth muscle cells (SMC) are common components of endometriotic lesions. SMC have been characterized previously in peritoneal, ovarian and deep infiltrating endometriotic lesions and adenomyosis. The aim of this retrospective study was to investigate the extent of differentiation in endometriosis-associated SMC (EMaSMC) in peritoneal endometriotic lesions. We obtained biopsies from peritoneal endometriotic lesions (n = 60) and peritoneal sites distant from the endometriotic lesion (n = 60), as well as healthy peritoneum from patients without endometriosis (control tissue, n = 10). These controls were hysterectomy specimens from patients without endometriosis or adenomyosis. Histopathological examination of peritoneal specimens using antibodies against oxytocin receptor (OTR), vasopressin receptor (VPR), smooth muscle myosin heavy chain (SM-MHC), estrogen receptor (ER) or progesterone receptor (PR) was performed. To identify SMC and their level of differentiation, antibodies for smooth muscle actin desmin and caldesmon were used. SMC were detected in all endometriotic lesions. SMC were more abundant in unaffected peritoneum of women with endometriosis (38%) compared with women without endometriosis (6%; P endometriosis.

  6. Membrane Nanotubes between peritoneal mesothelial cells: functional connectivity and crucial participation during inflammatory reactions

    Directory of Open Access Journals (Sweden)

    Julia eRanzinger

    2014-10-01

    Full Text Available Peritoneal dialysis (PD has attained increased relevance as continuous renal replacement therapy over the past years. During this treatment, the peritoneum functions as dialysis membrane to eliminate diffusible waste products from the blood-stream. Success and efficacy of this treatment is dependent on the integrity of the peritoneal membrane. Chronic inflammatory conditions within the peritoneal cavity coincide with elevated levels of proinflammatory cytokines leading to the impairment of tissue integrity. High glucose concentrations and glucose metabolites in PD solutions contribute to structural and functional reorganization processes of the peritoneal membrane during long-term PD. The subsequent loss of ultrafiltration is causal for the treatment failure over time. It was shown that peritoneal mesothelial cells are functionally connected via Nanotubes (NTs and that a correlation of NT-occurrence and defined pathophysiological conditions exists. Additionally, an important participation of NTs during inflammatory reactions was shown. Here, we will summarize recent developments of NT-related research and provide new insights into NT-mediated cellular interactions under physiological as well as pathophysiological conditions.

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

    Energy Technology Data Exchange (ETDEWEB)

    Pissuwan, Dakrong [University of Technology Sydney, Institute for Nanoscale Technology (Australia); Valenzuela, Stella M. [University of Technology Sydney, Department of Medical and Molecular Biosciences (Australia)], E-mail: stella.valenzuela@uts.edu.au; Killingsworth, Murray C. [Sydney South West Pathology Service (Australia)], E-mail: murray.killingsworth@swsahs.nsw.gov.au; Xu, Xiaoda; Cortie, Michael B. [University of Technology Sydney, Institute for Nanoscale Technology (Australia)], E-mail: michael.cortie@uts.edu.au

    2007-12-15

    Gold nanorods manifest a readily tunable longitudinal plasmon resonance with light and consequently have potential for use in photothermal therapeutics. Recent work by others has shown how gold nanoshells and rods can be used to target cancer cells, which can then be destroyed using relatively high power laser radiation ({approx}1x10{sup 5} to 1x10{sup 10} W/m{sup 2}). Here we extend this concept to demonstrate how gold nanorods can be modified to bind to target macrophage cells, and show that high intensity laser radiation is not necessary, with even 5x10{sup 2} W/m{sup 2} being sufficient, provided that a total fluence of {approx}30 J/cm{sup 2} is delivered. We used the murine cell line RAW 264.7 and the monoclonal antibody CD11b, raised against murine macrophages, as our model system and a 5 mW solid state diode laser as our energy source. Exposure of the cells labeled with gold nanorods to a laser fluence of 30 J/cm{sup 2} resulted in 81% cell death compared to only 0.9% in the control, non-labeled cells.

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

    Science.gov (United States)

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

    2007-12-01

    Gold nanorods manifest a readily tunable longitudinal plasmon resonance with light and consequently have potential for use in photothermal therapeutics. Recent work by others has shown how gold nanoshells and rods can be used to target cancer cells, 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 gold nanorods can be modified to bind to target macrophage cells, and show that high intensity laser radiation is not necessary, with even 5×102 W/m2 being sufficient, provided that a total fluence of ˜30 J/cm2 is delivered. We used the murine cell line RAW 264.7 and the monoclonal antibody CD11b, raised against murine macrophages, as our model system and a 5 mW solid state diode laser as our energy source. Exposure of the cells labeled with gold nanorods to a laser fluence of 30 J/cm2 resulted in 81% cell death compared to only 0.9% in the control, non-labeled cells.

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

    International Nuclear Information System (INIS)

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

    2007-01-01

    Gold nanorods manifest a readily tunable longitudinal plasmon resonance with light and consequently have potential for use in photothermal therapeutics. Recent work by others has shown how gold nanoshells and rods can be used to target cancer cells, which can then be destroyed using relatively high power laser radiation (∼1x10 5 to 1x10 10 W/m 2 ). Here we extend this concept to demonstrate how gold nanorods can be modified to bind to target macrophage cells, and show that high intensity laser radiation is not necessary, with even 5x10 2 W/m 2 being sufficient, provided that a total fluence of ∼30 J/cm 2 is delivered. We used the murine cell line RAW 264.7 and the monoclonal antibody CD11b, raised against murine macrophages, as our model system and a 5 mW solid state diode laser as our energy source. Exposure of the cells labeled with gold nanorods to a laser fluence of 30 J/cm 2 resulted in 81% cell death compared to only 0.9% in the control, non-labeled cells

  10. Histamine and TNF-α release by rat peritoneal mast cells stimulated with Trichomonas vaginalis

    Directory of Open Access Journals (Sweden)

    Im S.J.

    2011-02-01

    Full Text Available Mast cells have been reported to be predominant in the vaginal smears of patients infected with T. vaginalis. In this study, we investigated whether T. vaginalis could induce mast cells to migrate and to produce TNF-α and histamine. Rat peritoneal mast cells (RPMC, a primary mast cell, were used for the study. T. vaginalis induced an increase in chemotactic migration of the mast cells toward excretory and secretory product (ESP of T. vaginalis, and the mast cells activated with T. vaginalis showed an increased release of histamine and TNF-α. Therefore, mast cells may be involved in the inflammatory response caused by T. vaginalis.

  11. Peritoneal milky spots serve as a hypoxic niche and favor gastric cancer stem/progenitor cell peritoneal dissemination through hypoxia-inducible factor 1α.

    Science.gov (United States)

    Miao, Zhi-Feng; Wang, Zhen-Ning; Zhao, Ting-Ting; Xu, Ying-Ying; Gao, Jian; Miao, Feng; Xu, Hui-Mian

    2014-12-01

    Peritoneal dissemination is the most common cause of death in gastric cancer patients. The hypoxic microenvironment plays a major role in controlling the tumor stem cell phenotype and is associated with patients' prognosis through hypoxia-inducible factor-1α (HIF-1α), a key transcriptional factor that responds to hypoxic stimuli. During the peritoneal dissemination process, gastric cancer stem/progenitor cells (GCSPCs) are thought to enter into and maintained in peritoneal milky spots (PMSs), which have hypoxic microenvironments. However, the mechanism through which the hypoxic environment of PMSs regulated GCSPC maintenance is still poorly understood. Here, we investigated whether hypoxic PMSs were an ideal cancer stem cell niche suitable for GCSPC engraftment. We also evaluated the mechanisms through which the HIF-1α-mediated hypoxic microenvironment regulated GCSPC fate. We observed a positive correlation between HIF-1α expression and gastric cancer peritoneal dissemination (GCPD) in gastric cancer patients. Furthermore, the GCSPC population expanded in primary gastric cancer cells under hypoxic condition in vitro, and hypoxic GCSPCs showed enhanced self-renewal ability, but reduced differentiation capacity, mediated by HIF-1α. In an animal model, GCSPCs preferentially resided in the hypoxic zone of PMSs; moreover, when the hypoxic microenvironment in PMSs was destroyed, GCPD was significantly alleviated. In conclusion, our results demonstrated that PMSs served as a hypoxic niche and favored GCSPCs peritoneal dissemination through HIF-1α both in vitro and in vivo. These results provided new insights into the GCPD process and may lead to advancements in the clinical treatment of gastric cancer. © 2014 The Authors. STEM CELLS Published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.

  12. Decreased Cytotoxicity of Peripheral and Peritoneal Natural Killer Cell in Endometriosis.

    Science.gov (United States)

    Jeung, InCheul; Cheon, Keunyoung; Kim, Mee-Ran

    2016-01-01

    Endometriosis causes significant chronic pelvic pain, dysmenorrhea, and infertility and affects 10% of all women. In endometriosis, ectopic endometrium surviving after retrograde menstruation exhibits an abnormal immune response characterized by increased levels of activated macrophages and inflammatory cytokines. Particularly, dysfunctional natural killer (NK) cells play an important role in the pathogenesis of the disease by either facilitating or inhibiting the survival, implantation, and proliferation of endometrial cells. NK cells in the peritoneum and peritoneal fluid exhibit reduced levels of cytotoxicity in women with endometriosis. Several cytokines and inhibitory factors in the serum and peritoneal fluid also dysregulate NK cell cytotoxicity. Additionally, increased numbers of immature peripheral NK cells and induction of NK cell apoptosis are evident in the peritoneal fluid of women with endometriosis. The high rate of endometriosis recurrence after pharmaceutical or surgical treatment, which is associated with dysfunctional NK cells, indicates that new immunomodulatory management strategies are required. A good understanding of immune dysfunction would enable improvement of current treatments for endometriosis.

  13. Decreased Cytotoxicity of Peripheral and Peritoneal Natural Killer Cell in Endometriosis

    Directory of Open Access Journals (Sweden)

    InCheul Jeung

    2016-01-01

    Full Text Available Endometriosis causes significant chronic pelvic pain, dysmenorrhea, and infertility and affects 10% of all women. In endometriosis, ectopic endometrium surviving after retrograde menstruation exhibits an abnormal immune response characterized by increased levels of activated macrophages and inflammatory cytokines. Particularly, dysfunctional natural killer (NK cells play an important role in the pathogenesis of the disease by either facilitating or inhibiting the survival, implantation, and proliferation of endometrial cells. NK cells in the peritoneum and peritoneal fluid exhibit reduced levels of cytotoxicity in women with endometriosis. Several cytokines and inhibitory factors in the serum and peritoneal fluid also dysregulate NK cell cytotoxicity. Additionally, increased numbers of immature peripheral NK cells and induction of NK cell apoptosis are evident in the peritoneal fluid of women with endometriosis. The high rate of endometriosis recurrence after pharmaceutical or surgical treatment, which is associated with dysfunctional NK cells, indicates that new immunomodulatory management strategies are required. A good understanding of immune dysfunction would enable improvement of current treatments for endometriosis.

  14. Fungal infection control by garlic extracts (Allium sativum L.) and modulation of peritoneal macrophages activity in murine model of sporotrichosis.

    Science.gov (United States)

    Burian, J P; Sacramento, L V S; Carlos, I Z

    2017-11-01

    Garlic (Allium sativum L.) is grown all over the world as seasoning and medicinal vegetable since 3,000 BC. Allicin is the main component of garlic, being attributed to it the most of its biological activities, such as bactericidal, antifungal and antiviral actions. However, other compounds of garlic present antioxidant, hypocholesterolemic, vasodilator activities, protective action against different types of cancer, and immunomodulatory. Fungal infections are important causes of morbidity and mortality in people mainly in immunosuppressed ones. Sporothrix schenckii, the causing agent of Sporotrichosis (most common subcutaneous mycosis in Latin America), is dimorphic fungus, of saprophytic life in soil or plants, infecting people and animals mainly through skin injuries and bruises. The main of this work was to evaluate the influence of garlic consuming on immune modulation of healthy and infected Swiss mice in induced way by S. schenckii, since these animals functioning of peritoneal macrophages as well as the nitric oxide and cytokines' production (IL-1β, IL-10 and IL-12) and to evaluate the antifungal potential of garlic with S. schenckii through minimum inhibitory concentration test and colony-forming units. The results showed that garlic offers antifungal potential with S. schenckii. The oral taking of garlic extracts influences the releasing of cytokines by macrophages, regular consuming shows anti-inflammatory effect, and its acute use may take to an inflammatory response. Mice that consumed garlic responded more effectively to fight against the infection.

  15. Fungal infection control by garlic extracts (Allium sativum L. and modulation of peritoneal macrophages activity in murine model of sporotrichosis

    Directory of Open Access Journals (Sweden)

    J. P. Burian

    2017-05-01

    Full Text Available Abstract Garlic (Allium sativum L. is grown all over the world as seasoning and medicinal vegetable since 3,000 BC. Allicin is the main component of garlic, being attributed to it the most of its biological activities, such as bactericidal, antifungal and antiviral actions. However, other compounds of garlic present antioxidant, hypocholesterolemic, vasodilator activities, protective action against different types of cancer, and immunomodulatory. Fungal infections are important causes of morbidity and mortality in people mainly in immunosuppressed ones. Sporothrix schenckii, the causing agent of Sporotrichosis (most common subcutaneous mycosis in Latin America, is dimorphic fungus, of saprophytic life in soil or plants, infecting people and animals mainly through skin injuries and bruises. The main of this work was to evaluate the influence of garlic consuming on immune modulation of healthy and infected Swiss mice in induced way by S. schenckii, since these animals functioning of peritoneal macrophages as well as the nitric oxide and cytokines’ production (IL-1β, IL-10 and IL-12 and to evaluate the antifungal potential of garlic with S. schenckii through minimum inhibitory concentration test and colony-forming units. The results showed that garlic offers antifungal potential with S. schenckii. The oral taking of garlic extracts influences the releasing of cytokines by macrophages, regular consuming shows anti-inflammatory effect, and its acute use may take to an inflammatory response. Mice that consumed garlic responded more effectively to fight against the infection.

  16. Mannose receptor is highly expressed by peritoneal dendritic cells in endometriosis.

    Science.gov (United States)

    Izumi, Gentaro; Koga, Kaori; Takamura, Masashi; Makabe, Tomoko; Nagai, Miwako; Urata, Yoko; Harada, Miyuki; Hirata, Tetsuya; Hirota, Yasushi; Fujii, Tomoyuki; Osuga, Yutaka

    2017-01-01

    To characterize peritoneal dendritic cells (DCs) in endometriosis and to clarify their role in its etiology. Experimental. University hospital. Sixty-three women (35 patients with endometriosis and 28 control women) who had undergone laparoscopic surgery. Peritoneal DCs from endometriosis and control samples were analyzed for the expression of cell surface markers. Monocyte-derived dendritic cells (Mo-DCs) were cultured with dead endometrial stromal cells (dESCs) to investigate changes in phagocytic activity and cytokine expression. Cell surface markers and cytokine expression and identification with the use of flow cytometry or reverse-transcription polymerase chain reaction (RT-PCR). Changes in cytokine expression and phagocytic activity of Mo-DCs cultured with dESCs and d-mannan were measured with the use of flow cytometry and RT-PCR. The proportion of mannose receptor (MR)-positive myeloid DC type 1 was higher in endometriosis samples than in control samples. The blocking of MR reduced phagocytosis of dESCs by Mo-DCs. Mo-DCs cultured with dESCs expressed higher levels of interleukin (IL) 1β and IL-6 than control samples. Peritoneal DCs in endometriosis tissue express high levels of MR, which promotes phagocytosis of dead endometrial cells and thereby contributes to the etiology of endometriosis. Copyright © 2016 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

  17. Correction of anemia in uremic mice by genetically modified peritoneal mesothelial cells.

    Science.gov (United States)

    Einbinder, Tom; Sufaro, Yuval; Yusim, Igor; Byk, Gerardo; Passlick-Deetjen, Jutta; Chaimovitz, Cidio; Douvdevani, Amos

    2003-06-01

    During peritoneal dialysis, mesothelial cells become detached from the peritoneum and accumulate in the dialysate. Our aim was to evaluate the potential of peritoneal effluent (PF)-derived human peritoneal mesothelial cells (HPMC) as target for gene therapy. We used erythropoietin (EPO) as our target gene. Various extracellular matrixes (ECM) were tested for optimal adhesion and growth of HPMC. The EPO gene was introduced to mouse peritoneal mesothelial cells (MPMC) and HPMC by transfection or retroviral transduction. EPO secretion from PMC was measured by enzyme-linked immunosorbent assay (ELISA) and by the TF-1 cell proliferation assay. We performed intraperitoneal or intramuscular transplantations of the genetically modified cells into regular or 5/6 nephrectomized Balb/c mice and nude mice. Finally, we measured serum EPO and hematocrit levels. ECM-coated plates provided up to sixfold increase in the efficiency of PMC isolation from PF. Gelatin coated dishes (20 microg/cm2) were found optimal for isolation of PF-HPMC. RPR-120535 liposome was found to be best for PMC transduction. In vitro studies showed EPO secretion from modified HPMC over 6 months. Intraperitoneal transplantation aided with collagen matrix was the most effective. EPO, in MPMC transplanted mice, was detected up to 3 weeks (peak at 13 +/- 1 mIU/mL), and anemia of uremic mice was corrected (35.3 +/- 0.9 mIU/mL to 41.9 +/- 1.1 mIU/mL). PF-HPMC can be considered as an appropriate target for gene therapy since these cells can be efficiently isolated, modified, and transplanted. Nevertheless, implantation techniques in the peritoneum should be directed at obtaining longer duration of transgene expression in vivo, and means should be developed for enabling regulated expression of the gene.

  18. P2X7 receptor activation induces cell death and microparticle release in murine erythroleukemia cells.

    NARCIS (Netherlands)

    Constantinescu, P.; Wang, B.; Kovacevic, K.; Jalilian, I.; Bosman, G.J.C.G.M.; Wiley, J.S.; Sluyter, R.

    2010-01-01

    Extracellular ATP induces cation fluxes in and impairs the growth of murine erythroleukemia (MEL) cells in a manner characteristic of the purinergic P2X7 receptor, however the presence of P2X7 in these cells is unknown. This study investigated whether MEL cells express functional P2X7. RT-PCR,

  19. Alterations of Intercellular Junctions in Peritoneal Mesothelial Cells from Patients Undergoing Dialysis: Effect of Retinoic Acid

    Science.gov (United States)

    Retana, Carmen; Sanchez, Elsa; Perez-Lopez, Alejandro; Cruz, Armando; Lagunas, Jesus; Cruz, Carmen; Vital, Socorro; Reyes, Jose L.

    2015-01-01

    ♦ Background: Dialysis patients are classified according to their peritoneal permeability as low transporter (LT, low solute permeability) or high transporter (HT, high solute permeability). Tight junction (TJ) proteins are critical to maintain ions, molecules and water paracellular transport through peritoneum. Exposure to peritoneal dialysis solutions causes damage to TJ in human peritoneal mesothelial cells (HPMCs). We analyzed the quantity, distribution and function of TJ proteins: claudin-1, -2 and -8, ZO-1 and occludin, in HPMC cultures from LT and HT patients. Since all-trans retinoic acid (ATRA) might modify the expression of TJ proteins, we studied its effect on HPMCs. ♦ Methods: Control HPMCs were isolated from human omentum, while HT or LT cells were obtained from dialysis effluents. Cells were cultured in presence of ATRA 0, 50 or 100 nM. Transepithelial electrical resistance (TER) measurement, immunostaining and Western blot analyses were performed. ♦ Results: HT exhibited lower TER than control and LT monolayers. Immunofluorescence for TJ was weak and discontinuous along the cell contour, in LT and HT. Furthermore, claudin-1, occludin and ZO-1 expressions were decreased. In all groups, claudin-2 was localized at nuclei. We observed that ATRA improved TJ distribution and increased TJ expression in HT. This retinoid did not modify claudin-2 and -8 expressions. All-trans retinoic acid decreased TER in HT, but had no effect in LT. ♦ Conclusions: Tight junctions were altered in HPMCs from dialyzed patients. The HT monolayer has lower TER than LT, which might be associated with the peritoneal permeability in these patients. ATRA might be a therapeutic alternative to maintain mesothelial integrity, since it improved TJ localization and expression. PMID:24584604

  20. Multifocal peritoneal splenosis in Tc-99m-labeled heat-denatured red blood cell scintigraphy

    Energy Technology Data Exchange (ETDEWEB)

    Yoon, Min Ki; Hwang, Kyung Hoon; Choe, Won Sick [Gachon University Gil Medical Center, Incheon (Korea, Republic of)

    2006-06-15

    A 44-year-old man with a past medical history of splenectomy came to hospital because of epigastric pain abdominopelvic computed tomography(CT) showed a soft tissue mass and multifocal variable-sized nodules as well as finding suggestive of cholecystitis. Subsequently, he underwent Tc-99m-labeled heat- denatured red blood cell(RBC) scintigraphy to evaluate the mass and nodules. The scintigraphy confirmed multifocal peritoneal splenosis in the abdominopelvic cavity.

  1. Effects of trichostatins on differentiation of murine erythroleukemia cells

    International Nuclear Information System (INIS)

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

    1987-01-01

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

  2. Sulfotanshinone IIA Sodium Ameliorates Glucose Peritoneal Dialysis Solution-Induced Human Peritoneal Mesothelial Cell Injury via Suppression of ASK1-P38-mediated Oxidative Stress

    Directory of Open Access Journals (Sweden)

    Yao Zhou

    2018-05-01

    Full Text Available Background/Aims: Long-term use of high-glucose peritoneal dialysis solution (PDS induces peritoneal mesothelial cell (PMC injury, peritoneal dysfunction, and peritoneal dialysis (PD failure in patients with end-stage renal disease. How to preserve PMCs in PD is a major challenge for nephrologists worldwide. In this study, we aimed to elucidate the efficacy and mechanisms of sulfotanshinone IIA sodium (Tan IIa in ameliorating high-glucose PDS-induced human PMC injury. Methods: The human PMC line HMrSV5 was incubated with 4.25% PDS in vitro to mimic the high-glucose conditions in PD. Cellular viability was measured by Cell Counting Kit 8. Generation of superoxide and reactive oxygen species (ROS was assessed using a Total ROS/Superoxide Detection Kit. Oxidative modification of protein was evaluated by OxyBlot Protein Oxidation Detection Kit. TUNEL (dT-mediated dUTP nick end labeling assay and DAPI (4,6-diamidino-2-phenylindole staining were used to evaluate apoptosis. Western blot analysis was performed to evaluate the efficacy and mechanisms of Tan IIa. Results: Tan IIa protected PMCs against PDS-induced injury as evidenced by alleviating changes in morphology and loss of cell viability. Consistent with their antioxidant properties, N-acetyl-L-cysteine (NAC and Tan IIa suppressed superoxide and ROS production, protein oxidation, and apoptosis elicited by PDS. Apoptosis signal-regulating kinase 1 (ASK1-p38 signaling was activated by PDS. Both Tan IIa and NAC suppressed ASK1 and p38 phosphorylation elicited by PDS. Moreover, genetic downregulation of ASK1 ameliorated cell injury and inhibited the phosphorylation of p38 and activation of caspase 3. Conclusion: Tan IIa protects PMCs against PDS-induced oxidative injury through suppression of ASK1-p38 signaling.

  3. Olopatadine Inhibits Exocytosis in Rat Peritoneal Mast Cells by Counteracting Membrane Surface Deformation

    Directory of Open Access Journals (Sweden)

    Asuka Baba

    2015-01-01

    Full Text Available Backgroud/Aims: Besides its anti-allergic properties as a histamine receptor antagonist, olopatadine stabilizes mast cells by inhibiting the release of chemokines. Since olopatadine bears amphiphilic features and is preferentially partitioned into the lipid bilayers of the plasma membrane, it would induce some morphological changes in mast cells and thus affect the process of exocytosis. Methods: Employing the standard patch-clamp whole-cell recording technique, we examined the effects of olopatadine and other anti-allergic drugs on the membrane capacitance (Cm in rat peritoneal mast cells during exocytosis. Using confocal imaging of a water-soluble fluorescent dye, lucifer yellow, we also examined their effects on the deformation of the plasma membrane. Results: Low concentrations of olopatadine (1 or 10 µM did not significantly affect the GTP-γ-S-induced increase in the Cm. However, 100 µM and 1 mM olopatadine almost totally suppressed the increase in the Cm. Additionally, these doses completely washed out the trapping of the dye on the cell surface, indicating that olopatadine counteracted the membrane surface deformation induced by exocytosis. As shown by electron microscopy, olopatadine generated inward membrane bending in mast cells. Conclusion: This study provides electrophysiological evidence for the first time that olopatadine dose-dependently inhibits the process of exocytosis in rat peritoneal mast cells. Such mast cell stabilizing properties of olopatadine may be attributed to its counteracting effects on the plasma membrane deformation in degranulating mast cells.

  4. The Role of the MHV Receptor and Related Glycoproteins in Murine Hepatitis Virus Infection of Murine Cell Lines

    Science.gov (United States)

    1995-04-13

    vaccinia virus-T7 RNA polymerase s y stem for e xpression of target genes . Mol . Cell . BioI . 7 : 2538-2544 . Gagneten , S ., Gout , 0 ., Dubois-Dalcq...glycoprotein. These results showed f or the first time that two murine CEA- related genes can be co-expressed in some cell lines from inbred mice...49 Southern Hybridization ................ . ............ 50 Subcloning of PCR Products and Gene Cloning ........ 51 Growth

  5. Responses of the Murine Myeloid Colony-Forming Cell to Ansamycin Antibiotics

    Science.gov (United States)

    Horoszewicz, Julius S.; Carter, William A.

    1974-01-01

    The in vitro susceptibility of murine myeloid colony-forming cells to the antiproliferative activities of three ansamycin antibiotics was determined. These cells were found to be 10- to 40-fold more susceptible than the corresponding human ones. PMID:4151701

  6. Peritoneal Dialysis

    Science.gov (United States)

    ... include: Infections. An infection of the abdominal lining (peritonitis) is a common complication of peritoneal dialysis. An ... day. You might have a lower risk of peritonitis because you connect and disconnect to the dialysis ...

  7. The kin17 Protein in Murine Melanoma Cells

    Directory of Open Access Journals (Sweden)

    Anelise C. Ramos

    2015-11-01

    Full Text Available kin17 has been described as a protein involved in the processes of DNA replication initiation, DNA recombination, and DNA repair. kin17 has been studied as a potential molecular marker of breast cancer. This work reports the detection and localization of this protein in the murine melanoma cell line B16F10-Nex2 and in two derived subclones with different metastatic potential, B16-8HR and B16-10CR. Nuclear and chromatin-associated protein fractions were analyzed, and kin17 was detected in all fractions, with an elevated concentration observed in the chromatin-associated fraction of the clone with low metastatic potential, suggesting that the kin17 expression level could be a marker of melanoma.

  8. The Presence of Endometrial Cells in Peritoneal Fluid of Women With and Without Endometriosis.

    Science.gov (United States)

    O, Dorien F; Roskams, Tania; Van den Eynde, Kathleen; Vanhie, Arne; Peterse, Daniëlle P; Meuleman, Christel; Tomassetti, Carla; Peeraer, Karen; D'Hooghe, Thomas M; Fassbender, Amelie

    2017-02-01

    To reinforce Sampson's theory of retrograde menstruation in the pathogenesis of endometriosis, proof should be provided that during menstruation endometrial cells are present in peritoneal fluid (PF). We hypothesize that the prevalence of PF samples containing endometrial cells is higher in patients with endometriosis than in controls without endometriosis during menstruation. We selected from our biobank PF samples of 17 reproductive-age women with (n = 9) or without (n = 8) endometriosis who had received a diagnostic laparoscopy for investigation of pain/infertility. Peritoneal fluid had been collected during laparoscopy in the menstrual phase of the cycle, centrifuged, and the resulting pellet was stored at -80°C. About 5-μm sections of frozen PF pellets were stained using the Dako Envision Flex system with primary antibodies against epithelial cell adhesion molecule (Ep-CAM; endometrial epithelial cells), CD10 (endometrial stromal cells), prekeratin (epithelial/mesothelial cells), vimentin (endometrial/mesothelial/immune cells), calretinin (mesothelial cells), and CD68 (macrophages). The PF cells positive for Ep-CAM were detected in 5 of 9 patients with endometriosis and 6 of 8 controls ( P = .62). CD10 stained positively in 6 of the 9 patients with endometriosis and 3 of the 8 controls ( P = .35). Calretinin and prekeratin staining showed the presence of mesothelial cells in all pellets. Vimentin stained approximately 100% of the PF cells. CD68+ macrophages represented >50% of cells in all pellets. The prevalence of PF samples containing endometrial epithelial and stromal cells was not higher in patients with endometriosis than in controls without endometriosis during menstruation. Our findings question the relevance of endometrial cells in PF for the pathogenesis of endometriosis and support the importance of other mechanisms such as immune dysfunction and/or endometrial stem cells.

  9. Increased storage and secretion of phosphatidylcholines by senescent human peritoneal mesothelial cells.

    Science.gov (United States)

    Bartosova, Maria; Rudolf, Andras; Pichl, Sebastian; Schmidt, Kathrin; Okun, Jürgen G; Straub, Beate K; Rutkowski, Rafael; Witowski, Janusz; Schmitt, Claus P

    2016-08-01

    Human peritoneal mesothelial cells (HPMC) secrete phosphatidylcholines (PC) which form a lipid bilayer lining the peritoneum. They prevent frictions and adhesions and act as a barrier to the transport of water-soluble solutes while permitting water flux. PC may play an essential role in peritoneal integrity and function, the role of PD induced HPMC senescence on PC homeostasis, however, is unknown. HPMC cell lines were isolated from four non-uremic patients. Expression of the three PC synthesis genes (rt-PCR), and cellular storage and secretion of PC (ESI-mass-spectrometry) were analyzed in young and senescent HPMC (>Hayflick-limit). Senescent cells displayed significantly altered morphology; flow cytometry demonstrated extensive staining for senescence-associated beta galactosidase. Nine different PC were detected in HPMC with palmitoyl-myristoyl phosphatidylcholine (PMPC) being most abundant. In senescent HPMC mRNA expression of the three key PC synthesis genes was 1.5-, 2.4- and 6-fold increased as compared to young HPMC, with the latter, phosphatidylcholine cytidylyltransferase, being rate limiting. Intracellular storage of the nine PC was 75-450 % higher in senescent vs. young HPMC, PC secretion rates were 100-300 % higher. Intracellular PC concentrations were not correlated with the PC secretion rates. Electron microscopy demonstrated lamellar bodies, the primary storage site of PC, in senescent but not in young cells. Senescent HPMC store and secrete substantially more PC than young cells. Our findings indicate a novel protective mechanism, which should counteract peritoneal damage induced by chronic exposure to PD fluids.

  10. Cellular renewal and improvement of local cell effector activity in peritoneal cavity in response to infectious stimuli.

    Directory of Open Access Journals (Sweden)

    Alexandra dos Anjos Cassado

    Full Text Available The peritoneal cavity (PerC is a singular compartment where many cell populations reside and interact. Despite the widely adopted experimental approach of intraperitoneal (i.p. inoculation, little is known about the behavior of the different cell populations within the PerC. To evaluate the dynamics of peritoneal macrophage (MØ subsets, namely small peritoneal MØ (SPM and large peritoneal MØ (LPM, in response to infectious stimuli, C57BL/6 mice were injected i.p. with zymosan or Trypanosoma cruzi. These conditions resulted in the marked modification of the PerC myelo-monocytic compartment characterized by the disappearance of LPM and the accumulation of SPM and monocytes. In parallel, adherent cells isolated from stimulated PerC displayed reduced staining for β-galactosidase, a biomarker for senescence. Further, the adherent cells showed increased nitric oxide (NO and higher frequency of IL-12-producing cells in response to subsequent LPS and IFN-γ stimulation. Among myelo-monocytic cells, SPM rather than LPM or monocytes, appear to be the central effectors of the activated PerC; they display higher phagocytic activity and are the main source of IL-12. Thus, our data provide a first demonstration of the consequences of the dynamics between peritoneal MØ subpopulations by showing that substitution of LPM by a robust SPM and monocytes in response to infectious stimuli greatly improves PerC effector activity.

  11. Na+ -K+ pump activity in rat peritoneal mast cells: inhibition by extracellular calcium

    DEFF Research Database (Denmark)

    Knudsen, Torben; Johansen, Torben

    1989-01-01

    1. Pure populations of rat peritoneal mast cells were used to study cellular potassium uptake. The radioactive potassium analogue, 86rubidium, was used as a tracer for potassium for measurements of the activity of the cellular potassium uptake process. 2. The ouabain-sensitive and the ouabain......-resistant potassium (86rubidium) uptake of mast cells incubated in the presence of calcium, 1 mmol l-1, were very low, 52 and 147 pmol per 10(6) cells min-1. 3. Calcium-deprivation of the cells uncovered a large capacity ouabain-sensitive potassium (86rubidium) uptake mechanism. The activity of the uptake mechanism...... was decreased by reintroduction of calcium into the cell suspension, and it was dependent on cellular energy metabolism, temperature and pH. 4. The potassium (86rubidium) uptake of mast cells incubated in a calcium-free medium occurs through an active and ouabain-sensitive mechanism that has the nature...

  12. The effect of iron-deficiency anemia on cytolytic activity of mice spleen and peritoneal cells against allogenic tumor cells

    International Nuclear Information System (INIS)

    Kuvibidila, S.R.; Baliga, B.S.; Suskind, R.M.

    1983-01-01

    The capacity of spleen and peritoneal cells from iron deficient mice, ad libitum fed control mice, and pair-fed mice to kill allogenic tumor cells (mastocytoma tumor P815) has been investigated. In the first study, mice were sensitized in vivo with 10(7) viable tumor cells 51 and 56 days after weaning. The capacity of splenic cells and peritoneal cells from sensitized and nonsensitized mice to kill tumor cells was evaluated 5 days after the second dose of tumor cells. At ratios of 2.5:1 to 100:1 of attacker to target cells, the percentage 51 Cr release after 4 h of incubation was significantly less in iron-deficient mice than control and/or pair-fed mice (p less than 0.05). Protein-energy undernutrition in pair-fed mice had no significant effect. In the second study, spleen cells and enriched T cell fractions were incubated in vitro for 5 days with uv irradiated Balb/C spleen cells in a 2:1 ratio. The cytotoxic capacity against the same allogenic tumor cells was again evaluated. The percentage chromium release at different attacker to target cells was less than 30% in the iron-deficient group compared to either control or pair-fed supporting the results of in vivo sensitized cells. The possible mode of impairment of the cytotoxic capacity is discussed

  13. In vitro assessment of curcumin against murine neuroblastoma cells.

    Science.gov (United States)

    Vanisree, Arambakkam Janardhanam; Ramanan, Ramya

    2007-04-01

    Neuroblastoma (NB) is a well-known malignant disease in infants, which comprises 10% of childhood malignancies. Despite recent advances in understanding the neuro-oncology, NB still accounts for more death in childhood than any other cancer. Research in childhood tumors should not only be focused on the malignant signatures of cancer cells but also novel drug prototypes using phytochemicals. The present study was aimed to determine the role of curcumin against murine neuroblastoma cell line (N2a). The in vitro assessment of curcumin against was made in N2a cell line in a dose-dependent manner (group I (control) and group II - IX (10 microM-80 microM). The efficacy of the drug was evaluated by estimating the levels of protein bound carbohydrates, glycoprotein, genomic DNA, total RNA levels, and inhibition of MMP-9 were studied. The gap junctional communication in the cells was also assessed. The levels of protein bound carbohydrates, DNA, RNA levels, glycoprotein were found to be altered on drug supplementation in NB cells. Inhibition of MMP-9 in curcumin-supplemented N2a cells was revealed by zymographic analysis. Assessment of Lucifer yellow dye uptake in curcumin-supplemented N2a cells showed the up-regulation of GJIC. These observations suggest that the curcumin, the active principle of curcuma longa, could be developed into an effective chemo preventive and chemotherapeutic agent. This selected concentration range needs further studies at molecular level, for conforming its role and its action against uncontrolled proliferation of NB.

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

    International Nuclear Information System (INIS)

    Tichy, Elisia D.; Stambrook, Peter J.

    2008-01-01

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

  15. Peritonitis - spontaneous bacterial

    Science.gov (United States)

    Spontaneous bacterial peritonitis (SBP); Ascites - peritonitis; Cirrhosis - peritonitis ... who are on peritoneal dialysis for kidney failure. Peritonitis may have other causes . These include infection from ...

  16. Murine macrophage heparanase: inhibition and comparison with metastatic tumor cells

    International Nuclear Information System (INIS)

    Savion, N.; Disatnik, M.H.; Nevo, Z.

    1987-01-01

    Circulating macrophages and metastatic tumor cells can penetrate the vascular endothelium and migrate from the circulatory system to extravascular compartments. Both activated murine macrophages and different metastatic tumor cells attach, invade, and penetrate confluent vascular endothelial cell monolayer in vitro, by degrading heparan sulfate proteoglycans in the subendothelial extracellular matrix. The sensitivity of the enzymes from the various sources degrading the heparan sulfate proteoglycan was challenged and compared by a series of inhibitors. Activated macrophages demonstrate a heparanase with an endoglycosidase activity that cleaves from the [ 35 S]O 4 - -labeled heparan sulfate proteoglycans of the extracellular matrix 10 kDa glycosaminoglycan fragments. The degradation of [ 35 S]O 4 - -labeled extracellular matrix proteoglycans by the macrophages' heparanase is significantly inhibited in the presence of heparan sulfate (10μg/ml), arteparon (10μg/ml), and heparin at a concentration of 3 μg/ml. Degradation of this heparan sulfate proteoglycan is a two-step sequential process involving protease activity followed by heparanase activity. B16-BL6 metastatic melanoma cell heparanase, which is also a cell-associated enzyme, was inhibited by heparin to the same extent as the macrophage haparanase. On the other hand, heparanase of the highly metastatic variant (ESb) of a methylcholanthrene-induced T lymphoma, which is an extracellular enzyme released by the cells to the incubation medium, was more sensitive to heparin and arteparon than the macrophages' heparanase. These results may indicate the potential use of heparin or other glycosaminoglycans as specific and differential inhibitors for the formation in certain cases of blood-borne tumor metastasis

  17. The Secretory Response of Rat Peritoneal Mast Cells on Exposure to Mineral Fibers.

    Science.gov (United States)

    Borelli, Violetta; Trevisan, Elisa; Francesca, Vita; Zabucchi, Giuliano

    2018-01-10

    Exposure to mineral fibers is of substantial relevance to human health. A key event in exposure is the interaction with inflammatory cells and the subsequent generation of pro-inflammatory factors. Mast cells (MCs) have been shown to interact with titanium oxide (TiO₂) and asbestos fibers. In this study, we compared the response of rat peritoneal MCs challenged with the asbestos crocidolite and nanowires of TiO₂ to that induced by wollastonite employed as a control fiber. Rat peritoneal MCs (RPMCs), isolated from peritoneal lavage, were incubated in the presence of mineral fibers. The quantities of secreted enzymes were evaluated together with the activity of fiber-associated enzymes. The ultrastructural morphology of fiber-interacting RPMCs was analyzed with electron microscopy. Asbestos and TiO₂ stimulate MC secretion. Secreted enzymes bind to fibers and exhibit higher activity. TiO₂ and wollastonite bind and improve enzyme activity, but to a lesser degree than crocidolite. (1) Mineral fibers are able to stimulate the mast cell secretory process by both active (during membrane interaction) and/or passive (during membrane penetration) interaction; (2) fibers can be found to be associated with secreted enzymes-this process appears to create long-lasting pro-inflammatory environments and may represent the active contribution of MCs in maintaining the inflammatory process; (3) MCs and their enzymes should be considered as a therapeutic target in the pathogenesis of asbestos-induced lung inflammation; and (4) MCs can contribute to the inflammatory effect associated with selected engineered nanomaterials, such as TiO₂ nanoparticles.

  18. Modulation of the nuclear factor-kappa B (NF-κB) signalling pathway by glutamine in peritoneal macrophages of a murine model of protein malnutrition.

    Science.gov (United States)

    da Silva Lima, Fabiana; Rogero, Marcelo Macedo; Ramos, Mayara Caldas; Borelli, Primavera; Fock, Ricardo Ambrósio

    2013-06-01

    Protein malnutrition affects resistance to infection by impairing the inflammatory response, modifying the function of effector cells, such as macrophages. Recent studies have revealed that glutamine-a non-essential amino acid, which could become conditionally essential in some situations like trauma, infection, post-surgery and sepsis-is able to modulate the synthesis of cytokines. The aim of this study was to evaluate the effect of glutamine on the expression of proteins involved in the nuclear factor-kappa B (NF-κB) signalling pathway of peritoneal macrophages from malnourished mice. Two-month-old male Balb/c mice were submitted to protein-energy malnutrition (n = 10) with a low-protein diet containing 2 % protein, whereas control mice (n = 10) were fed a 12 % protein-containing diet. The haemogram and analysis of plasma glutamine and corticosterone were evaluated. Peritoneal macrophages were pre-treated in vitro with glutamine (0, 0.6, 2 and 10 mmol/L) for 24 h and then stimulated with 1.25 μg LPS for 30 min, and the synthesis of TNF-α and IL-1α and the expression of proteins related to the NF-κB pathway were evaluated. Malnourished animals had anaemia, leucopoenia, lower plasma glutamine and increased corticosterone levels. TNF-α production of macrophages stimulated with LPS was significantly lower in cells from malnourished animals when cultivated in supraphysiological (2 and 10 mmol/L) concentrations of glutamine. Further, glutamine has a dose-dependent effect on the activation of macrophages, in both groups, when stimulated with LPS, inducing a decrease in TNF-α and IL-1α production and negatively modulating the NF-κB signalling pathway. These data lead us to infer that the protein malnutrition state interferes with the activation of macrophages and that higher glutamine concentrations, in vitro, have the capacity to act negatively in the NF-κB signalling pathway.

  19. Safety of peritoneal and pleural drain placement in pediatric stem cell transplant recipients with severe veno-occlusive disease.

    Science.gov (United States)

    Madenci, Arin L; Stetson, Alyssa; Weldon, Christopher B; Lehmann, Leslie E

    2016-08-01

    Hepatic VOD (veno-occlusive disease) is a serious complication of HSCT (hematopoietic stem cell transplantation) and has historically been associated with high mortality. This obstruction to hepatic flow often results in fluid collections in the peritoneal and pleural cavities. Catheter placement to drain ascites or pleural fluid may reduce intra-abdominal hypertension and/or improve respiratory parameters. The safety of these interventions among critically ill, immunocompromised children is unknown. Among 32 HSCT recipients (2000-2012) with severe VOD, we assessed the primary outcome of procedural complication from peritoneal drain placement. Twenty-four (75%) patients underwent peritoneal drain placement. No patient sustained visceral perforation or hemorrhage with drain placement. Overall mortality was 47% (n = 15). The procedure was not associated with increased overall mortality (p > 0.99). Eight (25%) peritoneal drains required replacement for malfunction. Of 24 patients with peritoneal drains, one (4%) patient had a positive culture from ascitic fluid. Eight (25%) patients underwent pleural drain placement. No pleural drain-related procedural complication or infection occurred. Four (50%) of the eight patients with pleural drains had de-escalation in oxygen requirement at drain removal, compared to time of placement. In this study, peritoneal and pleural drains were safe interventions for children with severe VOD. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  20. Snake venoms components with antitumor activity in murine melanoma cells

    International Nuclear Information System (INIS)

    Queiroz, Rodrigo Guimaraes

    2012-01-01

    Despite the constant advances in the treatment of cancer, this disease remains one of the main causes of mortality worldwide. So, the development of new treatment modalities is imperative. Snake venom causes a variety of biological effects because they constitute a complex mixture of substances as disintegrins, proteases (serine and metalo), phospholipases A2, L-amino acid oxidases and others. The goal of the present work is to evaluate a anti-tumor activity of some snake venoms fractions. There are several studies of components derived from snake venoms with this kind of activity. After fractionation of snake venoms of the families Viperidae and Elapidae, the fractions were assayed towards murine melanoma cell line B16-F10 and fibroblasts L929. The results showed that the fractions of venom of the snake Notechis ater niger had higher specificity and potential antitumor activity on B16-F10 cell line than the other studied venoms. Since the components of this venom are not explored yet coupled with the potential activity showed in this work, we decided to choose this venom to develop further studies. The cytotoxic fractions were evaluated to identify and characterize the components that showed antitumoral activity. Western blot assays and zymography suggests that these proteins do not belong to the class of metallo and serine proteinases. (author)

  1. Dietary n-3 PUFA affect TcR-mediated activation of purified murine T cells and accessory cell function in co-cultures

    Science.gov (United States)

    CHAPKIN, R S; ARRINGTON, J L; APANASOVICH, T V; CARROLL, R J; MCMURRAY, D N

    2002-01-01

    Diets enriched in n-3 polyunsaturated fatty acids (PUFA) suppress several functions of murine splenic T cells by acting directly on the T cells and/or indirectly on accessory cells. In this study, the relative contribution of highly purified populations of the two cell types to the dietary suppression of T cell function was examined. Mice were fed diets containing different levels of n-3 PUFA; safflower oil (SAF; control containing no n-3 PUFA), fish oil (FO) at 2% and 4%, or 1% purified docosahexaenoic acid (DHA) for 2 weeks. Purified (>90%) T cells were obtained from the spleen, and accessory cells (>95% adherent, esterase-positive) were obtained by peritoneal lavage. Purified T cells or accessory cells from each diet group were co-cultured with the alternative cell type from every other diet group, yielding a total of 16 different co-culture combinations. The T cells were stimulated with either concanavalin A (ConA) or antibodies to the T cell receptor (TcR)/CD3 complex and the costimulatory molecule CD28 (αCD3/αCD28), and proliferation was measured after four days. Suppression of T cell proliferation in the co-cultures was dependent upon the dose of dietary n-3 PUFA fed to mice from which the T cells were derived, irrespective of the dietary treatment of accessory cell donors. The greatest dietary effect was seen in mice consuming the DHA diet (P = 0·034 in the anova; P = 0·0053 in the Trend Test), and was observed with direct stimulation of the T cell receptor and CD28 costimulatory ligand, but not with ConA. A significant dietary effect was also contributed accessory cells (P = 0·033 in the Trend Test). We conclude that dietary n-3 PUFA affect TcR-mediated by T cell activation by both direct and indirect (accessory cell) mechanisms. PMID:12296847

  2. S1P Signalling Differentially Affects Migration of Peritoneal B Cell Populations In Vitro and Influences the Production of Intestinal IgA In Vivo.

    Science.gov (United States)

    Kleinwort, Annabel; Lührs, Felix; Heidecke, Claus-Dieter; Lipp, Martin; Schulze, Tobias

    2018-01-29

    Introduction: Sphingosine-1-phosphate (S1P) regulates the migration of follicular B cells (B2 cells) and directs the positioning of Marginal zone B cells (MZ B cells) within the spleen. The function of S1P signalling in the third B cell lineage, B1 B cells, mainly present in the pleural and peritoneal cavity, has not yet been determined. Methods: S1P receptor expression was analysed in peritoneal B cells by real-time polymerase chain reaction (qPCR). The chemotactic response to S1P was studied in vitro. The role of S1P signalling was further explored in a s1p₄ -/- mouse strain. Results: Peritoneal B cells expressed considerable amounts of the S1P receptors 1 and 4 (S1P₁ and S1P₄, respectively). S1P₁ showed differential expression between the distinct peritoneal B cell lineages. While B2 cells showed no chemotactic response to S1P, B1 B cells showed a migration response to S1P. s1p₄ -/- mice displayed significant alterations in the composition of peritoneal B cell populations, as well as a significant reduction of mucosal immunoglobulin A (IgA) in the gut. Discussion: S1P signalling influences peritoneal B1 B cell migration. S1P₄ deficiency alters the composition of peritoneal B cell populations and reduces secretory IgA levels. These findings suggest that S1P signalling may be a target to modulate B cell function in inflammatory intestinal pathologies.

  3. Peritoneal Dialysis in Children*

    African Journals Online (AJOL)

    1971-06-02

    Jun 2, 1971 ... cellular fluid' into the peritoneal cavity allows both the removal of excess fluid ... occur. In adults the maximum peritoneal urea clearance is ob- tained with a dialysis ..... and Records. Pulse and respiration rates, temperature and blood .... diffusion of urea out of the brain cells, with consequent movement of ...

  4. Role of the Na+/K+-ATPase in regulating the membrane potential in rat peritoneal mast cells

    DEFF Research Database (Denmark)

    Friis, U G; Praetorius, Birger Hans; Knudsen, T

    1997-01-01

    1. The aim of this study was to investigate the effect of the Na+/K+-ATPase on the membrane potential of peritoneal mast cells isolated from male Sprague-Dawley SPF-rats. 2. Experiments were performed at 22-26 degrees C in the tight-seal whole-cell configuration of the patch-clamp technique by use...

  5. Helicobacter pylori impairs murine dendritic cell responses to infection.

    Directory of Open Access Journals (Sweden)

    Ya-Hui Wang

    Full Text Available BACKGROUND: Helicobacter pylori, a human pathogen associated with chronic gastritis, peptic ulcer and gastric malignancies, is generally viewed as an extracellular microorganism. Here, we show that H. pylori replicates in murine bone marrow derived-dendritic cells (BMDCs within autophagosomes. METHODOLOGY/PRINCIPAL FINDINGS: A 10-fold increase of CFU is found between 2 h and 6 h p.i. in H. pylori-infected BMDCs. Autophagy is induced around the bacterium and participates at late time points of infection for the clearance of intracellular H. pylori. As a consequence of infection, LC3, LAMP1 and MHC class II molecules are retained within the H. pylori-containing vacuoles and export of MHC class II molecules to cell surface is blocked. However, formalin-fixed H. pylori still maintain this inhibitory activity in BMDC derived from wild type mice, but not in from either TLR4 or TLR2-deficient mice, suggesting the involvement of H. pylori-LPS in this process. TNF-alpha, IL-6 and IL-10 expression was also modulated upon infection showing a TLR2-specific dependent IL-10 secretion. No IL-12 was detected favoring the hypothesis of a down modulation of DC functions during H. pylori infection. Furthermore, antigen-specific T cells proliferation was also impaired upon infection. CONCLUSIONS/SIGNIFICANCE: H. pylori can infect and replicate in BMDCs and thereby affects DC-mediated immune responses. The implication of this new finding is discussed for the biological life cycle of H. pylori in the host.

  6. The effect of tartrazine on histamine release from rat peritoneal mast cells.

    Science.gov (United States)

    Safford, R J; Goodwin, B F

    1984-01-01

    The release of histamine from purified rat peritoneal mast cells induced by specific antigen (egg albumin), compound 48/80 and calcium ionophore A23187 was modified by tartrazine. Histamine release induced by 48/80 and antigen was inhibited by the presence of 10(-5) to 10(-2)M tartrazine. The inhibitory effect on egg albumin induced histamine release was maximal when the tartrazine was added simultaneously with egg albumin, and was reduced by increased preincubation of the cells with tartrazine. Tartrazine had a small inhibitory effect on ionophore induced release at high concentrations, but augmented histamine release at tartrazine concentrations of 10(-3) and 10(-4)M. Augmentation of ionophore induced release was maximal at between 0-5 min preincubation of the cells with tartrazine.

  7. A murine ESC-like state facilitates transgenesis and homologous recombination in human pluripotent stem cells

    NARCIS (Netherlands)

    C. Buecker (Christa); H.H. Chen; J.M. Polo (Jose); L. Daheron (Laurence); L. Bu (Lei); T.S. Barakat (Tahsin Stefan); P. Okwieka (Patricia); A. Porter (Andrew); J.H. Gribnau (Joost); K. Hochedlinger (Konrad); N. Geijsen (Niels)

    2010-01-01

    textabstractMurine pluripotent stem cells can exist in two functionally distinct states, LIF-dependent embryonic stem cells (ESCs) and bFGF-dependent epiblast stem cells (EpiSCs). However, human pluripotent cells so far seemed to assume only an epiblast-like state. Here we demonstrate that human

  8. Heterogeneity of functional properties of Clone 66 murine breast cancer cells expressing various stem cell phenotypes.

    Science.gov (United States)

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

    2013-01-01

    Breast cancer grows, metastasizes and relapses from rare, therapy resistant cells with a stem cell phenotype (cancer stem cells/CSCs). However, there is a lack of studies comparing the functions of CSCs isolated using different phenotypes in order to determine if CSCs are homogeneous or heterogeneous. Cells with various stem cell phenotypes were isolated by sorting from Clone 66 murine breast cancer cells that grow orthotopically in immune intact syngeneic mice. These populations were compared by in vitro functional assays for proliferation, growth, sphere and colony formation; and in vivo limiting dilution analysis of tumorigenesis. The proportion of cells expressing CD44(high)CD24(low/neg), side population (SP) cells, ALDH1(+), CD49f(high), CD133(high), and CD34(high) differed, suggesting heterogeneity. Differences in frequency and size of tumor spheres from these populations were observed. Higher rates of proliferation of non-SP, ALDH1(+), CD34(low), and CD49f(high) suggested properties of transit amplifying cells. Colony formation was higher from ALDH1(-) and non-SP cells than ALDH1(+) and SP cells suggesting a progenitor phenotype. The frequency of clonal colonies that grew in agar varied and was differentially altered by the presence of Matrigel™. In vivo, fewer cells with a stem cell phenotype were needed for tumor formation than "non-stem" cells. Fewer SP cells were needed to form tumors than ALDH1(+) cells suggesting further heterogeneities of cells with stem phenotypes. Different levels of cytokines/chemokines were produced by Clone 66 with RANTES being the highest. Whether the heterogeneity reflects soluble factor production remains to be determined. These data demonstrate that Clone 66 murine breast cancer cells that express stem cell phenotypes are heterogeneous and exhibit different functional properties, and this may also be the case for human breast cancer stem cells.

  9. Effect of peritoneal dialysis fluid containing osmo-metabolic agents on human endothelial cells

    Directory of Open Access Journals (Sweden)

    Bonomini M

    2016-11-01

    Full Text Available Mario Bonomini,1,2 Sara Di Silvestre,3,4 Pamela Di Tomo,3,4 Natalia Di Pietro,2,4 Domitilla Mandatori,3,4 Lorenzo Di Liberato,1 Vittorio Sirolli,1,2 Francesco Chiarelli,2,4 Cesare Indiveri,5 Assunta Pandolfi,3,4 Arduino Arduini6 1Unit of Nephrology and Dialysis, 2Department of Medicine and Aging Sciences, 3Department of Medical, Oral and Biotechnological Sciences, 4Aging Research Center and Translational Medicine, CeSI-MeT, University “G. d’Annunzio”, Chieti-Pescara, 5Department DiBEST (Biologia, Ecologia, Scienze della Terra, Unit of Biochemistry and Molecular Biotechnology, University of Calabria, Arcavacata di Rende CS, Italy; 6CoreQuest, Manno, Switzerland Background: The use of glucose as the only osmotic agent in peritoneal dialysis (PD solutions (PDSs is believed to exert local (peritoneal and systemic detrimental actions, particularly in diabetic PD patients. To improve peritoneal biocompatibility, we have developed more biocompatible PDSs containing xylitol and carnitine along with significantly less amounts of glucose and have tested them in cultured Human Vein Endothelial Cells (HUVECs obtained from the umbilical cords of healthy (C and gestational diabetic (GD mothers. Methods: Primary C- and GD-HUVECs were treated for 72 hours with our PDSs (xylitol 0.7% and 1.5%, whereas carnitine and glucose were fixed at 0.02% and 0.5%, respectively and two glucose-based PDSs (glucose 1.36% or 2.27%. We examined their effects on endothelial cell proliferation (cell count, viability (3-(4,5-dimethylthiazolyl-2-2,5-diphenyltetrazolium bromide assay, intracellular nitro-oxidative stress (peroxynitrite levels, Vascular Cell Adhesion Molecule-1 and Intercellular Adhesion Molecule-1 membrane exposure (flow cytometry, and HUVEC-monocyte interactions (U937 adhesion assay. Results: Compared to glucose-based PDSs, our in vitro studies demonstrated that the tested PDSs did not change the proliferative potential both in C- and GD-HUVECs. Moreover, our

  10. Peritoneal mesothelioma.

    OpenAIRE

    Anderson, J. H.; Stewart, C. J.; Hansell, D. T.; Anderson, J. R.

    1990-01-01

    We report two patients who presented with small bowel obstruction secondary to peritoneal mesothelioma. The difficulties in establishing this diagnosis at an early stage are illustrated. Recent advances in the management of peritoneal mesothelioma are reviewed.

  11. Shorter daily dwelling time in peritoneal dialysis attenuates the epithelial-to-mesenchymal transition of mesothelial cells

    Science.gov (United States)

    2014-01-01

    Background Peritoneal dialysis (PD) therapy is known to induce morphological and functional changes in the peritoneal membrane. Long-term exposure to conventional bio-incompatible dialysate and peritonitis is the main etiology of inflammation. Consequently, the peritoneal membrane undergoes structural changes, including angiogenesis, fibrosis, and hyalinizing vasculopathy, which ultimately results in technique failure. The epithelial-to-mesenchymal transition (EMT) of mesothelial cells (MCs) plays an important role during the above process; however, the clinical parameters associated with the EMT process of MCs remain to be explored. Methods To investigate the parameters impacting EMT during PD therapy, 53 clinical stable PD patients were enrolled. EMT assessments were conducted through human peritoneal MCs cultured from dialysate effluent with one consistent standard criterion (MC morphology and the expression of an epithelial marker, cytokeratin 18). The factors potentially associated with EMT were analyzed using logistic regression analysis. Primary MCs derived from the omentum were isolated for the in vitro study. Results Forty-seven percent of the patients presented with EMT, 28% with non-EMT, and 15% with a mixed presentation. Logistic regression analysis showed that patients who received persistent PD therapy (dwelling time of 24 h/day) had significantly higher EMT tendency. These results were consistent in vitro. Conclusions Dwelling time had a significant effect on the occurrence of EMT on MCs. PMID:24555732

  12. Amphipaths Differentially Modulate Membrane Surface Deformation in Rat Peritoneal Mast Cells During Exocytosis

    Directory of Open Access Journals (Sweden)

    Itsuro Kazama

    2013-04-01

    Full Text Available Background/Aims: Salicylate and chlorpromazine exert differential effects on the chemokine release from mast cells. Since these drugs are amphiphilic and preferentially partitioned into the lipid bilayers of the plasma membranes, they would induce some morphological changes in mast cells and thus affect the process of exocytosis. Methods: Employing the standard patch-clamp whole-cell recording technique, we examined the effects of salicylate and chlorpromazine on the membrane capacitance (Cm during exocytosis in rat peritoneal mast cells. Using confocal imaging of a water-soluble fluorescent dye, lucifer yellow, we also examined their effects on plasma membrane deformation of the cells. Results: Salicylate dramatically accelerated the GTP-γ-S-induced increase in the Cm immediately after its application, whereas chlorpromazine significantly suppressed the increase. Treatment with salicylate increased the trapping of the dye on the cell surface, while treatment with chlorpromazine completely washed it out, indicating that both drugs induced membrane surface deformation in mast cells. Conclusion: This study demonstrated for the first time that membrane amphipaths, such as salicylate and chlorpromazine, may oppositely modulate the process of exocytosis in mast cells, as detected by the changes in the Cm. The plasma membrane deformation induced by the drugs was thought to be responsible for their differential effects.

  13. Enhanced detection and study of murine norovirus-1 using a more efficient microglial cell line

    Directory of Open Access Journals (Sweden)

    Lu Yuanan

    2009-11-01

    Full Text Available Abstract Background Human Noroviruses are the predominant cause of non-bacterial gastroenteritis worldwide. To facilitate prevention and control, a norovirus isolated from mice can provide a model to understand human noroviruses. To establish optimal viral infectivity conditions for murine noroviruses, several cell lines of hematopoietic lineage, including murine BV-2, RAW 264.7, and TIB, as well as human CHME-5, were tested comparatively for their sensitivity to murine norovirus-1. Results Except for CHME-5, all three murine-derived cell lines were susceptible to MNV infection. Viral infection of these cells was confirmed by RT-PCR. Using both viral plaque and replication assays, BV-2 and RAW 264.7 cells were determined to have comparable sensitivities to MNV-1 infection. Comparisons of cell growth characteristics, general laboratory handling and potential in-field applications suggest the use of BV-2 to be more advantageous. Conclusion Results obtained from these studies demonstrate that an immortalized microglial cell line can support MNV-1 replication and provides a more efficient method to detect and study murine noroviruses, facilitating future investigations using MNV-1 as a model to study, detect, and control Human Norovirus.

  14. Increased percentage of Th17 cells in peritoneal fluid is associated with severity of endometriosis.

    Science.gov (United States)

    Gogacz, Marek; Winkler, Izabela; Bojarska-Junak, Agnieszka; Tabarkiewicz, Jacek; Semczuk, Andrzej; Rechberger, Tomasz; Adamiak, Aneta

    2016-09-01

    Th17 cells are a newly discovered T helper lymphocyte subpopulation, producing interleukin IL-17. Th17 cells are present in blood and peritoneal fluid (PF) at different stages of endometriosis. We aim to establish their potential importance in the pathogenesis and clinical features of the disease. The percentage of Th17 cells among T helper lymphocytes was determined in the PF and peripheral blood (PB) of patients with endometriosis and in the control group by flow cytometry using monoclonal antibodies: anti-CD-4-FITC, anti-CD-3-PE/Cy5, and anti-IL-17A-PE. Th17 percentage is increased in PF in comparison with PB in both endometriotic patients and in the control group. In severe endometriosis, the percentage of Th17 cells in PF was higher than with early (I/II stage) endometriosis. A positive correlation between the percentage of Th17 cells in PF and the white blood cell count in PB was found in patients with endometriosis. Targeting the activity of PF Th17 cells may have an influence on the proliferation of ectopic tissue and clinical manifestations of the disease. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  15. Regulation of the Na(+)-K+ pump activity and estimation of the reserve capacity in intact rat peritoneal mast cells

    DEFF Research Database (Denmark)

    Knudsen, Torben; Johansen, Torben

    1990-01-01

    Evidence is provided that regulation of the Na(+)-K+ pump activity in rat peritoneal mast cells occurs mainly through stimulation of the pump from inside the plasma membrane by sodium. It is demonstrated that there is a large reserve capacity for the exchange of intracellular sodium...... with extracellular potassium in these cells. The maximal pump activity was estimated to be 3230 pmol/10(6) cells per min and Km for extracellular potassium was 1.5 mM....

  16. Downregulation of connective tissue growth factor inhibits the growth and invasion of gastric cancer cells and attenuates peritoneal dissemination.

    Science.gov (United States)

    Jiang, Cheng-Gang; Lv, Ling; Liu, Fu-Rong; Wang, Zhen-Ning; Liu, Fu-Nan; Li, Yan-Shu; Wang, Chun-Yu; Zhang, Hong-Yan; Sun, Zhe; Xu, Hui-Mian

    2011-09-28

    Connective tissue growth factor (CTGF) has been shown to be implicated in tumor development and progression. However, the role of CTGF in gastric cancer remains largely unknown. In this study, we showed that CTGF was highly expressed in gastric cancer tissues compared with matched normal gastric tissues. The CTGF expression in tumor tissue was associated with histologic grade, lymph node metastasis and peritoneal dissemination (P cancer cells and decreased cyclin D1 expression. Moreover, knockdown of CTGF expression also markedly reduced the migration and invasion of gastric cancer cells and decreased the expression of matrix metalloproteinase (MMP)-2 and MMP-9. Animal studies revealed that nude mice injected with the CTGF knockdown stable cell lines featured a smaller number of peritoneal seeding nodules than the control cell lines. These data suggest that CTGF plays an important role in cell growth and invasion in human gastric cancer and it appears to be a potential prognostic marker for patients with gastric cancer.

  17. Turnover of T cells in murine gammaherpesvirus 68-infected mice

    DEFF Research Database (Denmark)

    Hamilton-Easton, A M; Christensen, Jan Pravsgaard; Doherty, P C

    1999-01-01

    Respiratory challenge of C57BL/6 mice with murine gammaherpesvirus 68 induces proliferation of T lymphocytes early after infection, as evidenced by incorporation of the DNA precursor bromodeoxyuridine. Using pulse-chase analysis, splenic and peripheral blood activated T lymphocytes were found...

  18. Type I collagen gel protects murine fibrosarcoma L929 cells from TNFα-induced cell death

    International Nuclear Information System (INIS)

    Wang, Hong-Ju; He, Wen-Qi; Chen, Ling; Liu, Wei-Wei; Xu, Qian; Xia, Ming-Yu; Hayashi, Toshihiko; Fujisaki, Hitomi; Hattori, Shunji; Tashiro, Shin-ichi; Onodera, Satoshi; Ikejima, Takashi

    2015-01-01

    Murine fibrosarcoma L929 cells have been used to test efficacy of proinflammatory cytokine TNFα. In the present study, we reported on protective effect of type I collagen gel used as L929 cell culture. L929 cell grew and proliferated well on collagen gel. However, the L929 cells exhibited cobblestone-like morphology which was much different from the spread fusiform shape when cultured on conventional cell dishes as well as the cells tended to aggregate. On conventional cell culture dishes, the cells treated with TNFα became round in shape and eventually died in a necroptotic manner. The cells cultured on collagen gel, however, were completely unaffected. TNFα treatment was reported to induce autophagy in L929 cells on the plastic dish, and therefore we investigated the effect of collagen gel on induction of autophagy. The results indicated that autophagy induced by TNFα treatment was much reduced when the cells were cultured on collagen gel. In conclusion, type I collagen gel protected L929 cell from TNFα-induced cell death. - Highlights: • Collagen gel culture changed the morphology of L929 cells. • L929 cell cultured on collagen gel were resistant to TNFα-induced cell death. • Collagen gel culture inhibited TNFα-induced autophagy in L929 cells

  19. Type I collagen gel protects murine fibrosarcoma L929 cells from TNFα-induced cell death

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Hong-Ju; He, Wen-Qi; Chen, Ling; Liu, Wei-Wei; Xu, Qian; Xia, Ming-Yu; Hayashi, Toshihiko [China-Japan Research Institute of Medical and Pharmaceutical Sciences, Shenyang Pharmaceutical University, Shenyang 110016 (China); Fujisaki, Hitomi; Hattori, Shunji [Nippi Research Institute of Biomatrix, Toride, Ibaraki 302-0017 (Japan); Tashiro, Shin-ichi [Institute for Clinical and Biomedical Sciences, Kyoto 603-8072 (Japan); Onodera, Satoshi [Department of Clinical and Pharmaceutical Sciences, Showa Pharmaceutical University, Tokyo 194-8543 (Japan); Ikejima, Takashi, E-mail: ikejimat@vip.sina.com [China-Japan Research Institute of Medical and Pharmaceutical Sciences, Shenyang Pharmaceutical University, Shenyang 110016 (China)

    2015-02-20

    Murine fibrosarcoma L929 cells have been used to test efficacy of proinflammatory cytokine TNFα. In the present study, we reported on protective effect of type I collagen gel used as L929 cell culture. L929 cell grew and proliferated well on collagen gel. However, the L929 cells exhibited cobblestone-like morphology which was much different from the spread fusiform shape when cultured on conventional cell dishes as well as the cells tended to aggregate. On conventional cell culture dishes, the cells treated with TNFα became round in shape and eventually died in a necroptotic manner. The cells cultured on collagen gel, however, were completely unaffected. TNFα treatment was reported to induce autophagy in L929 cells on the plastic dish, and therefore we investigated the effect of collagen gel on induction of autophagy. The results indicated that autophagy induced by TNFα treatment was much reduced when the cells were cultured on collagen gel. In conclusion, type I collagen gel protected L929 cell from TNFα-induced cell death. - Highlights: • Collagen gel culture changed the morphology of L929 cells. • L929 cell cultured on collagen gel were resistant to TNFα-induced cell death. • Collagen gel culture inhibited TNFα-induced autophagy in L929 cells.

  20. Neuroendocrine circuitry and endometriosis: progesterone derivative dampens corticotropin-releasing hormone-induced inflammation by peritoneal cells in vitro.

    Science.gov (United States)

    Tariverdian, Nadja; Rücke, Mirjam; Szekeres-Bartho, Julia; Blois, Sandra M; Karpf, Eva F; Sedlmayr, Peter; Klapp, Burghard F; Kentenich, Heribert; Siedentopf, Friederike; Arck, Petra C

    2010-03-01

    Clinical symptoms of endometriosis, such as pain and infertility, can be described as persistent stressors. Such continuous exposure to stress may severely affect the equilibrium and bidirectional communication of the endocrine and immune system, hereby further aggravating the progression of endometriosis. In the present study, we aimed to tease apart mediators that are involved in the stress response as well as in the progression of endometriosis. Women undergoing diagnostic laparoscopy due to infertility were recruited (n = 69). Within this cohort, early stage of endometriosis were diagnosed in n = 30 and advanced stage of endometriosis in n = 8. Levels of progesterone in serum were determined. Frequency of progesterone receptor (PR) expression on CD56(+) and CD8(+) peritoneal lymphocytes was analysed by flow cytometry. The production of tumour necrosis factor (TNF) and interleukin (IL)-10 by peritoneal leukocytes upon stimulation with the potent stress mediator corticotropin-releasing hormone (CRH) and the progesterone derivative dydrogesterone, or both, were evaluated. Furthermore, the production of progesterone-induced blocking factor (PIBF) by peritoneal leukocytes and the expression of PR in endometriotic tissue were investigated. Levels of progesterone in serum were decreased in women with endometriosis and inversely correlated to pain scores. Furthermore, an increased frequency of CD56(+)PR(+) and CD8(+)PR(+) peritoneal lymphocytes was present in advanced endometriosis. The TNF/IL-10 ratio, reflecting cytokine secretion by peritoneal cells, was higher in cells derived from endometriosis patients and could be further heightened by CRH stimulation, whereas stimulation with dydrogesterone abrogated the CRH-mediated inflammation. Finally, the expression of PIBF by peritoneal leukocytes was increased in endometriosis. Low levels of progesterone in the follicular phase could be responsible for the progression of endometriosis and related pain. Peripheral CRH

  1. Effect of fucoidan on B16 murine melanoma cell melanin formation ...

    African Journals Online (AJOL)

    Background:Fucoidan is a complex sulfated polysaccharide extracted from brown seaweed and has a wide variety of biological activities. It not only inhibits cancer cell growth but also inhibits tyrosinase in vitro. Therefore, it is of interest to investigate the effect of fucoidan on B16 murine melanoma cells as the findings may ...

  2. Downregulation of connective tissue growth factor inhibits the growth and invasion of gastric cancer cells and attenuates peritoneal dissemination

    Directory of Open Access Journals (Sweden)

    Zhang Hong-Yan

    2011-09-01

    Full Text Available Abstract Background Connective tissue growth factor (CTGF has been shown to be implicated in tumor development and progression. However, the role of CTGF in gastric cancer remains largely unknown. Results In this study, we showed that CTGF was highly expressed in gastric cancer tissues compared with matched normal gastric tissues. The CTGF expression in tumor tissue was associated with histologic grade, lymph node metastasis and peritoneal dissemination (P 1 expression. Moreover, knockdown of CTGF expression also markedly reduced the migration and invasion of gastric cancer cells and decreased the expression of matrix metalloproteinase (MMP-2 and MMP-9. Animal studies revealed that nude mice injected with the CTGF knockdown stable cell lines featured a smaller number of peritoneal seeding nodules than the control cell lines. Conclusions These data suggest that CTGF plays an important role in cell growth and invasion in human gastric cancer and it appears to be a potential prognostic marker for patients with gastric cancer.

  3. Murine Th9 cells promote the survival of myeloid dendritic cells in cancer immunotherapy.

    Science.gov (United States)

    Park, Jungsun; Li, Haiyan; Zhang, Mingjun; Lu, Yong; Hong, Bangxing; Zheng, Yuhuan; He, Jin; Yang, Jing; Qian, Jianfei; Yi, Qing

    2014-08-01

    Dendritic cells (DCs) are professional antigen-presenting cells to initiate immune responses, and DC survival time is important for affecting the strength of T-cell responses. Interleukin (IL)-9-producing T-helper (Th)-9 cells play an important role in anti-tumor immunity. However, it is unclear how Th9 cells communicate with DCs. In this study, we investigated whether murine Th9 cells affected the survival of myeloid DCs. DCs derived from bone marrow of C57BL/6 mice were cocultured with Th9 cells from OT-II mice using transwell, and the survival of DCs was examined. DCs cocultured with Th9 cells had longer survival and fewer apoptotic cells than DCs cultured alone in vitro. In melanoma B16-OVA tumor-bearing mice, DCs conditioned by Th9 cells lived longer and induced stronger anti-tumor response than control DCs did in vivo. Mechanistic studies revealed that IL-3 but not IL-9 secreted by Th9 cells was responsible for the prolonged survival of DCs. IL-3 upregulated the expression of anti-apoptotic protein Bcl-xL and activated p38, ERK and STAT5 signaling pathways in DCs. Taken together, our data provide the first evidence that Th9 cells can promote the survival of DCs through IL-3, and will be helpful for designing Th9 cell immunotherapy and more effective DC vaccine for human cancers.

  4. Senescence-Associated Changes in Proteome and O-GlcNAcylation Pattern in Human Peritoneal Mesothelial Cells

    Directory of Open Access Journals (Sweden)

    Rebecca Herzog

    2015-01-01

    Full Text Available Introduction. Senescence of peritoneal mesothelial cells represents a biological program defined by arrested cell growth and altered cell secretory phenotype with potential impact in peritoneal dialysis. This study aims to characterize cellular senescence at the level of global protein expression profiles and modification of proteins with O-linked N-acetylglucosamine (O-GlcNAcylation. Methods. A comparative proteomics analysis between young and senescent human peritoneal mesothelial cells (HPMC was performed using two-dimensional gel electrophoresis. O-GlcNAc status was assessed by Western blot under normal conditions and after modulation with 6-diazo-5-oxo-L-norleucine (DON to decrease O-GlcNAcylation or O-(2-acetamido-2-deoxy-D-glucopyranosylidene amino N-phenyl carbamate (PUGNAc to increase O-GlcNAcylation. Results. Comparison of protein pattern of senescent and young HPMC revealed 29 differentially abundant protein spots, 11 of which were identified to be actin (cytoplasmic 1 and 2, cytokeratin-7, cofilin-2, transgelin-2, Hsp60, Hsc70, proteasome β-subunits (type-2 and type-3, nucleoside diphosphate kinase A, and cytosolic 5′(3′-deoxyribonucleotidase. Although the global level of O-GlcNAcylation was comparable, senescent cells were not sensitive to modulation by PUGNAc. Discussion. This study identified changes of the proteome and altered dynamics of O-GlcNAc regulation in senescent mesothelial cells. Whereas changes in cytoskeleton-associated proteins likely reflect altered cell morphology, changes in chaperoning and housekeeping proteins may have functional impact on cellular stress response in peritoneal dialysis.

  5. Murine epidermal Langerhans cells and langerin-expressing dermal dendritic cells are unrelated and exhibit distinct functions

    NARCIS (Netherlands)

    Nagao, Keisuke; Ginhoux, Florent; Leitner, Wolfgang W.; Motegi, Sei-Ichiro; Bennett, Clare L.; Clausen, Björn E.; Merad, Miriam; Udey, Mark C.

    2009-01-01

    A new langerin(+) DC subset has recently been identified in murine dermis (langerin(+) dDC), but the lineage and functional relationships between these cells and langerin(+) epidermal Langerhans cells (LC) are incompletely characterized. Selective expression of the cell adhesion molecule EpCAM by LC

  6. Perforated small intestine in a patient with T-cell lymphoma; a rare cause of peritonitis

    Directory of Open Access Journals (Sweden)

    Petrişor Banu

    2016-04-01

    Full Text Available The nontraumatic perforations of the small intestine are pathological entities with particular aspects in respect to diagnosis and treatment. These peculiarities derive from the nonspecific clinical expression of the peritonitis syndrome, and from the multitude of causes that might be the primary sources of the perforation: foreign bodies, inflammatory diseases, tumors, infectious diseases, etc. Accordingly, in most cases intestinal perforation is discovered only by laparotomy and the definitive diagnosis is available only after histopathologic examination. Small bowel malignancies are rare; among them, lymphomas rank third in frequency, being mostly B-cell non Hodgkin lymphomas. Only 10% of non-Hodgkin lymphomas are with T-cell. We report the case of a 57 years’ old woman with intestinal T-cell lymphoma, whose first clinical symptomatology was related to a complication represented by perforation of the small intestine. Laparotomy performed in emergency identified an ulcerative lesion with perforation in the jejunum, which required segmental enterectomy with anastomosis. The nonspecific clinical manifestations of intestinal lymphomas make from diagnosis a difficult procedure. Due to the fact that surgery does not have a definite place in the treatment of the small intestinal lymphomas (for cases complicated with perforation, and beyond the morbidity associated with the surgery performed in emergency conditions, prognosis of these patients is finally given by the possibility to control the systemic disease through adjuvant therapy.

  7. S1P Signalling Differentially Affects Migration of Peritoneal B Cell Populations In Vitro and Influences the Production of Intestinal IgA In Vivo

    Directory of Open Access Journals (Sweden)

    Annabel Kleinwort

    2018-01-01

    Full Text Available Introduction: Sphingosine-1-phosphate (S1P regulates the migration of follicular B cells (B2 cells and directs the positioning of Marginal zone B cells (MZ B cells within the spleen. The function of S1P signalling in the third B cell lineage, B1 B cells, mainly present in the pleural and peritoneal cavity, has not yet been determined. Methods: S1P receptor expression was analysed in peritoneal B cells by real-time polymerase chain reaction (qPCR. The chemotactic response to S1P was studied in vitro. The role of S1P signalling was further explored in a s1p4−/− mouse strain. Results: Peritoneal B cells expressed considerable amounts of the S1P receptors 1 and 4 (S1P1 and S1P4, respectively. S1P1 showed differential expression between the distinct peritoneal B cell lineages. While B2 cells showed no chemotactic response to S1P, B1 B cells showed a migration response to S1P. s1p4−/− mice displayed significant alterations in the composition of peritoneal B cell populations, as well as a significant reduction of mucosal immunoglobulin A (IgA in the gut. Discussion: S1P signalling influences peritoneal B1 B cell migration. S1P4 deficiency alters the composition of peritoneal B cell populations and reduces secretory IgA levels. These findings suggest that S1P signalling may be a target to modulate B cell function in inflammatory intestinal pathologies.

  8. Therapeutic Cell-Cycle-Decoy Efficacy of a Telomerase-Dependent Adenovirus in an Orthotopic Model of Chemotherapy-Resistant Human Stomach Carcinomatosis Peritonitis Visualized With FUCCI Imaging.

    Science.gov (United States)

    Yano, Shuya; Takehara, Kiyoto; Tazawa, Hiroshi; Kishimoto, Hiroyuki; Urata, Yasuo; Kagawa, Shunsuke; Fujiwara, Toshiyoshi; Hoffman, Robert M

    2017-11-01

    We have established an orthotopic nude-mouse model of gastric cancer carcinomatosis peritonitis, a recalcitrant disease in human patients. Human MKN45 poorly-differentiated human gastric cancer cells developed carcinomatosis peritonitis upon orthotopic transplantation in nude mice. The MKN45 cells expressed the fluorescent ubiquitination-based cell cycle indicator (FUCCI) that color codes the phases of the cell cycle. The intra-peritoneal tumors and ascites contained mostly quiescent G 1 /G o cancer cells visualized as red by FUCCI imaging. Cisplatinum (CDDP) treatment did not reduce bloody ascites, and larger tumors formed in the peritoneal cavity after CDDP treatment in an early-stage carcinomatosis peritonitis orthotopic mouse model. Paclitaxel-treated mice had reduced ascites, but also had large tumor masses in the peritonium after treatment with cancer cells mostly in G 0 /G 1 , visualized by FUCCI red. In contrast, OBP-301 telomerase-dependent adenovirus-treated mice had no ascites and only small tumor nodules consisting of cancer cells mostly in S/G 2 phases in the early-stage carcinomatosis peritonitis model, visualized by FUCCI green. Furthermore, OBP-301 significantly reduced the size of tumors (P < 0.01) and ascites even in a late-stage carcinomatosis peritonitis model. These results suggest that quiescent peritoneally-disseminated gastric cancer cells are resistant to conventional chemotherapy, but OBP-301 significantly reduced the weight of the tumors and increased survival, suggesting clinical potential. J. Cell. Biochem. 118: 3635-3642, 2017. © 2016 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  9. Mast cells facilitate local VEGF release as an early event in the pathogenesis of postoperative peritoneal adhesions.

    LENUS (Irish Health Repository)

    Cahill, Ronan A

    2012-02-03

    BACKGROUND: Peritoneal injury sustained at laparotomy may evoke local inflammatory responses that result in adhesion formation. Peritoneal mast cells are likely to initiate this process, whereas vascular permeability\\/endothelial growth factor (VEGF) may facilitate the degree to which subsequent adhesion formation occurs. METHODS: Mast cell deficient mice (WBB6F1-\\/-), along with their mast cell sufficient counterparts (WBB6F1+\\/+), underwent a standardized adhesion-inducing operation (AIS) with subsequent sacrifice and adhesion assessment 14 days later in a blinded fashion. Additional CD-1 and WBB6F1+\\/+, and WBB6F1-\\/- mice were killed 2, 6, 12, and 24 hours after operation for measurement of VEGF by ELISA in systemic serum and peritoneal lavage fluid. Two further groups of CD-1 mice underwent AIS and received either a single perioperative dose of anti-VEGF monoclonal antibody (10 mug\\/mouse) or a similar volume of IgG isotypic antibody and adhesion formation 2 weeks later was evaluated. RESULTS: WBB6F1-\\/- mice had less adhesions then did their WBB6F1+\\/+ counterparts (median [interquartile range] adhesion score 3[3-3] vs 1.5[1-2] respectively; P < .003). Local VEGF release peaked 6 hours after AIS in both WBB6F1+\\/+ and CD-1 mice whereas levels remained at baseline in WBB6F1-\\/- mice. CD-1 mice treated with a single dose of anti-VEGF therapy during operation had less adhesions than controls (2[1.25-2] vs 3[2.25-3], P = .0002). CONCLUSIONS: Mast cells and VEGF are central to the formation of postoperative intra-abdominal adhesions with mast cells being responsible, either directly or indirectly, for VEGF release into the peritoneal cavity after operation. In tandem with the recent clinical success of anti-VEGF monoclonal antibodies in oncologic practice, our observations suggest an intriguing avenue for research and development of anti-adhesion strategy.

  10. Contrast-enhanced ultrasound and computed tomography findings of recurrent ovarian steroid cell tumor presenting with peritoneal seeding: A case report

    International Nuclear Information System (INIS)

    Im, A Lan; Lee, Young Hwan; Kim, Hye Won; Lee, Han Ah; Choi, Keum Ha

    2013-01-01

    We present ultrasonography and computed tomography (CT) findings of a case of recurrent ovarian steroid cell tumor presenting with peritoneal seeding in a 45-year-old woman. On abdominal ultrasonography, there were multiple hypoechoic round masses in the peritoneal cavity including the perihepatic area. Contrast-enhanced ultrasonography showed intense homogenous enhancement on the arterial phase and delayed prolonged enhancement of the masses. CT revealed multiple peritoneal solid masses with strong enhancement. Five years ago, the patient had been diagnosed with a steroid cell tumor of the left ovary. At that time, the CT showed a well-enhancing, lobulating, large solid mass at the left adnexa. Imaging findings of the peritoneal masses suggested peritoneal seeding from the preexisting ovarian steroid cell tumor. For treatment of the metastatic lesions in the perihepatic area, ultrasonography-guided radiofrequency ablation (RFA) was performed, and debulking surgery for the peritoneal masses was done. Six months later, complete ablation of the perihepatic metastases by RFA and a marked decrease in the peritoneal metastases by surgery were found on the follow-up CT.

  11. GPBAR1/TGR5 mediates bile acid-induced cytokine expression in murine Kupffer cells.

    Directory of Open Access Journals (Sweden)

    Guiyu Lou

    Full Text Available GPBAR1/TGR5 is a novel plasma membrane-bound G protein-coupled bile acid (BA receptor. BAs are known to induce the expression of inflammatory cytokines in the liver with unknown mechanism. Here we show that without other external stimuli, TGR5 activation alone induced the expression of interleukin 1β (IL-1β and tumor necrosis factor-α (TNF-α in murine macrophage cell line RAW264.7 or murine Kupffer cells. The TGR5-mediated increase of pro-inflammatory cytokine expression was suppressed by JNK inhibition. Moreover, the induced pro-inflammatory cytokine expression in mouse liver by 1% cholic acid (CA diet was blunted in JNK-/- mice. TGR5 activation by its ligands enhanced the phosphorylation levels, DNA-binding and trans-activities of c-Jun and ATF2 transcription factors. Finally, the induced pro-inflammatory cytokine expression in Kupffer cells by TGR5 activation correlated with the suppression of Cholesterol 7α-hydroxylase (Cyp7a1 expression in murine hepatocytes. These results suggest that TGR5 mediates the BA-induced pro-inflammatory cytokine production in murine Kupffer cells through JNK-dependent pathway. This novel role of TGR5 may correlate to the suppression of Cyp7a1 expression in hepatocytes and contribute to the delicate BA feedback regulation.

  12. In vitro activation of murine DRG neurons by CGRP-mediated mucosal mast cell degranulation

    NARCIS (Netherlands)

    De Jonge, F; De Laet, A; Van Nassauw, L; Miller, HRP; van Bogaert, PP; Timmermans, JP; Kroese, ABA

    Upregulation of CGRP-immunoreactive (IR) primary afferent nerve fibers accompanied by mastocytosis is characteristic for the Schistosoma mansoni-infected murine ileum. These mucosal mast cells (MMC) and CGRP-IR fibers, which originate from dorsal root (DRG) and nodose ganglia, are found in close

  13. Effect of ouabain, digoxin and digitoxigenin on potassium uptake and histamine release from rat peritoneal mast cells

    DEFF Research Database (Denmark)

    Knudsen, T; Ferjan, I; Johansen, Torben

    1993-01-01

    Rat peritoneal mast cells were used to study the effects of digitalis glycosides on potassium uptake and histamine release induced by compound 48/80, substance P and egg-albumin (immunological release). In the absence of calcium all glycosides inhibited potassium uptake. Ouabain and digoxin....... Hydrophilic digitalis glycosides seem to enhance histamine release secondary to an increase in intracellular sodium. Lipophilic glycosides have no effect on the release....

  14. Reconstitution of mammary epithelial morphogenesis by murine embryonic stem cells undergoing hematopoietic stem cell differentiation.

    Directory of Open Access Journals (Sweden)

    Shuxian Jiang

    2010-03-01

    Full Text Available Mammary stem cells are maintained within specific microenvironments and recruited throughout lifetime to reconstitute de novo the mammary gland. Mammary stem cells have been isolated through the identification of specific cell surface markers and in vivo transplantation into cleared mammary fat pads. Accumulating evidence showed that during the reformation of mammary stem cell niches by dispersed epithelial cells in the context of the intact epithelium-free mammary stroma, non-mammary epithelial cells may be sequestered and reprogrammed to perform mammary epithelial cell functions and to adopt mammary epithelial characteristics during reconstruction of mammary epithelium in regenerating mammary tissue in vivo.To examine whether other types of progenitor cells are able to contribute to mammary branching morphogenesis, we examined the potential of murine embryonic stem (mES cells, undergoing hematopoietic differentiation, to support mammary reconstitution in vivo. We observed that cells from day 14 embryoid bodies (EBs under hematopoietic differentiation condition, but not supernatants derived from these cells, when transplanted into denuded mammary fat pads, were able to contribute to both the luminal and myoepithelial lineages in branching ductal structures resembling the ductal-alveolar architecture of the mammary tree. No teratomas were observed when these cells were transplanted in vivo.Our data provide evidence for the dominance of the tissue-specific mammary stem cell niche and its role in directing mES cells, undergoing hematopoietic differentiation, to reprogram into mammary epithelial cells and to promote mammary epithelial morphogenesis. These studies should also provide insights into regeneration of damaged mammary gland and the role of the mammary microenvironment in reprogramming cell fate.

  15. Hepatocyte growth factor secreted by ovarian cancer cells stimulates peritoneal implantation via the mesothelial-mesenchymal transition of the peritoneum.

    Science.gov (United States)

    Nakamura, Michihiko; Ono, Yoshihiro J; Kanemura, Masanori; Tanaka, Tomohito; Hayashi, Masami; Terai, Yoshito; Ohmichi, Masahide

    2015-11-01

    A current working model for the metastatic process of ovarian carcinoma suggests that cancer cells are shed from the ovarian tumor into the peritoneal cavity and attach to the layer of mesothelial cells that line the inner surface of the peritoneum, and several studies suggest that hepatocyte growth factor (HGF) plays an important role in the dissemination of ovarian cancer. Our objectives were to evaluate the HGF expression of ovarian cancer using clinical data and assess the effect of HGF secreted from human ovarian cancer cells to human mesothelial cells. HGF expression was immunohistochemically evaluated in 165 epithelial ovarian cancer patients arranged as tissue microarrays. HGF expression in four ovarian cancer cell lines was evaluated by using semi-quantitative polymerase chain reaction, Western blotting and enzyme-linked immunosorbent assay. The effect of ovarian cancer cell derived HGF to the human mesothelial cells was assessed by using morphologic analysis, Western blotting and cell invasion assay. The effect of HGF on ovarian cancer metastasis was assessed by using in vivo experimental model. The clinical data showed a significantly high correlation between the HGF expression and the cancer stage. The in vivo and in vitro experimental models revealed that HGF secreted by ovarian cancer cells induces the mesothelial-to-mesenchymal transition and stimulates the invasion of mesothelial cells. Furthermore, manipulating the HGF activity affected the degree of dissemination and ascite formation. We demonstrated that HGF secreted by ovarian cancer cells plays an important role in cancer peritoneal implantation. Copyright © 2015 Elsevier Inc. All rights reserved.

  16. PERITONEAL CARCINOMATOSIS

    Directory of Open Access Journals (Sweden)

    I. V. Stepanov

    2014-01-01

    Full Text Available The literature review examines the main pathological and morphogenetic aspects of the development of peritoneal carcinomatosis and its molecular and biological features. In addition, the basic principles of peritoneal carcinomatosis classification have been presented and groups of tumors, which may be complicated by dissemination to the peritoneum, have been designated. Particular attention is paid to the problems of diagnosis and treatment.

  17. Herpes simplex virus type 2 induces rapid cell death and functional impairment of murine dendritic cells in vitro

    NARCIS (Netherlands)

    Jones, CA; Fernandez, M; Herc, K; Bosnjak, L; Miranda-Saksena, M; Boadle, RA; Cunningham, A

    2003-01-01

    Dendritic cells (DC) are critical for stimulation of naive T cells. Little is known about the effect of herpes simplex virus type 2 (HSV-2) infection on DC structure or function or if the observed effects of HSV-1 on human DC are reproduced in murine DC. Here, we demonstrate that by 12 h

  18. Activation of peritoneal macrophages to cytoxicity against B16 melanoma cells by Serratia marcescens polyribosome fractions

    International Nuclear Information System (INIS)

    Hoover, S.K.

    1985-01-01

    Serratia marcescens polyribosomes (SMPR) have been shown to elicit an anti-tumor response in vivo. The in-vitro effects of SMPR on macrophages as the nonspecific mediators of the anti-tumor response have not previously been examined. The first objective of this research project is to corroborate and analyze the in-vivo results by the development and application of an in-vitro cytotoxicity assay. The second objective is to examine the effect of SMPR upon previously unstimulated peritoneal macrophages as representing the mechanism of cytotoxicity. The third objective is to identify the minimal effective component of SMPR responsible for an effect on macrophages. Results revealed that SMPR preparations exert a number of effects upon macrophages. Morphologic changes included increased spreading and increased perinuclear vacuolization. Macrophages were shown to be metabolically activate by two lines of evidence. SMPR-treated macrophages exhibited increased cellular metabolism by the increased uptake of 3 H-thymidine and by the increased levels of secreted leucine aminopeptidase as compared to control macrophages. Results also showed that SMPR activates macrophages to cytotoxicity against syngeneic tumor target cells. Buoyant-density fractions were isolated and assayed for macrophage activating ability. Results showed 50S ribosomal subunits to be the smallest fraction effective for macrophage activation. Both the RNA and protein were necessary for complete effectiveness

  19. Transduction of hematopoietic stem cells to stimulate RNA interference against feline infectious peritonitis.

    Science.gov (United States)

    Anis, Eman A; Dhar, Madhu; Legendre, Alfred M; Wilkes, Rebecca P

    2017-06-01

    Objectives The goals of the study were: (1) to develop and evaluate non-replicating lentivirus vectors coding for feline coronavirus (FCoV)-specific micro (mi)RNA as a potential antiviral therapy for feline infectious peritonitis (FIP); (2) to assess the feasibility of transducing hematopoietic stem cells (HSCs) with ex vivo introduction of the miRNA-expressing lentivirus vector; and (3) to assess the ability of the expressed miRNA to inhibit FCoV replication in HSCs in vitro. Methods HSCs were obtained from feline bone marrow and replicated in vitro. Three lentiviruses were constructed, each expressing a different anti-FCoV miRNA. HSCs were stably transduced with the miRNA-expressing lentivirus vector that produced the most effective viral inhibition in a feline cell line. The effectiveness of the transduction and the expression of anti-FCoV miRNA were tested by infecting the HSCs with two different strains of FCoV. The inhibition of coronavirus replication was determined by relative quantification of the inhibition of intracellular viral genomic RNA synthesis using real-time, reverse-transcription PCR. The assessment of virus replication inhibition was determined via titration of extracellular virus using the TCID 50 assay. Results Inhibition of FCoV was most significant in feline cells expressing miRNA-L2 that targeted the viral leader sequence, 48 h postinfection. miRNA-L2 expression in stably transduced HSCs resulted in 90% and 92% reductions in FIPV WSU 79-1146 genomic RNA synthesis and extracellular virus production, respectively, as well as 74% and 80% reduction in FECV WSU 79-1683 genomic RNA synthesis and extracellular virus production, respectively, as compared with an infected negative control sample producing non-targeting miRNA. Conclusions and relevance These preliminary results show that genetic modification of HSCs for constitutive production of anti-coronavirus miRNA will reduce FCoV replication.

  20. Proliferation and Differentiation of Murine Myeloid Precursor 32D/G-CSF-R Cells

    Czech Academy of Sciences Publication Activity Database

    Zjablovskaja, Polina; Daněk, Petr; Kardošová, Miroslava; Alberich-Jorda, Meritxell

    č. 132 (2018), č. článku e57033. ISSN 1940-087X R&D Projects: GA ČR GA15-03796S Institutional support: RVO:68378050 Keywords : 32D/G-CSF-R cells * murine myeloid precursor cells * liquid culture * differentiation * neutrophils * proliferation * cytokines * IL-3 * G-CSF Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 1.232, year: 2016

  1. Peritoneal carcinomatosis

    International Nuclear Information System (INIS)

    Raptopoulos, V.; Gourtsoyiannis, N.

    2001-01-01

    The peritoneum is a mesothelial lining of the abdominal cavity (parietal) and intraperitoneal viscera (visceral). The cavity contains a small amount of fluid, which circulates from cephalad to caudal to cephalad, influenced by negative pressure under the diaphragm during exhalation, gravity, and bowel peristalsis. Peritoneal reflections and mesenteries divide the cavity into various compartments (supramesocolic, inframesocolic, and pelvis). These reflections support the peritoneal organs and provide vascular and nervous connections while within the cavity they influence pathway of intraperitoneal fluid circulation. Capillary force over convex surfaces influence stasis of fluid and promotes peritoneal seeding; thus, there are numerous areas in which peritoneal masses are seen more commonly. These areas include the undersurface of the diaphragm (negative pressure and capillary force), the omentum (bathed in fluid), the right lower quadrant (oblique course from left superior to right inferior of the small bowel mesentery), the left lower quadrant (transverse course of the sigmoid), and the pelvis (gravity). Peritoneal carcinomatosis may be either primary (mesothelioma) or metastatic. The mode of spread is by direct invasion, lymphatic permeation, peritoneal seeding or hematogenous. The imaging patterns include fibronodular stranding, nodules, plaques, and masses. Mesenteric thickening may produce pleated or stellate patterns. Spiral CT is the most useful modality in diagnosis and follow-up of peritoneal tumors. (orig.)

  2. Selective host range restriction of goat cells for recombinant murine leukemia virus and feline leukemia virus type A.

    OpenAIRE

    Fischinger, P J; Thiel, H J; Blevins, C S; Dunlop, N M

    1981-01-01

    We isolated a strain of normal goat fibroblasts which was uniquely selective in that it allowed the replication of xenotropic murine leukemia virus but not polytropic recombinant murine leukemia virus. In addition, feline leukemia virus type A replication was severely diminished in these goat cells, whereas feline leukemia virus type B and feline endogenous RD114-CCC viruses replicated efficiently. No other known cells exhibit this pattern of virus growth restriction. These goat cells allow t...

  3. Intestinal and peritoneal mast cells differ in kinetics of quantal release

    Energy Technology Data Exchange (ETDEWEB)

    Balseiro-Gomez, Santiago, E-mail: sanbalgom@alum.us.es; Ramirez-Ponce, M. Pilar, E-mail: pponce@us.es; Acosta, Jorge, E-mail: jorgealo@us.es; Ales, Eva, E-mail: eales@us.es; Flores, Juan A., E-mail: jaflores@us.es

    2016-01-15

    5-hydroxytriptamine (5-HT, serotonin) storage and release in mast cell (MC) secretory granules (SG) are dependent on serglycin proteoglycans (PG). This notion is based on the studies of MC of the connective tissue subtype that predominantly contain PG of the heparin type, whereas intestinal mucosal MC, which contain predominantly chondroitin sulfate, have been poorly explored. In the present study, we addressed the possibility that PG contents may differently affect the storage and release of preformed mediators in these two MC subclasses and explain in part their different functional properties. Rat peritoneal (PMC) and intestinal mast cells (IMC) were isolated and purified using a percoll gradient, and the efflux of 5-HT from each SG was measured by amperometric detection. IMC exhibited a ∼34% reduction in the release of 5-HT compared with PMC because of a lower number of exocytotic events, rather than a lower secretion per single exocytotic event. Amperometric spikes from IMC exhibited a slower decay phase and increased half-width but a similar ascending phase and foot parameters, indicating that the fusion pore kinetics are comparable in both MC subclasses. We conclude that both PG subtypes are equally efficient systems, directly involved in serotonin accumulation, and play a crucial role in regulating the kinetics of exocytosis from SG, providing specific secretory properties for the two cellular subtypes. - Highlights: • We improved a method for isolating and purifying IMC. • There was a reduction in total serotonin release in IMC with respect to PMC. • This decrease was not due to less secretion per quantum but a lower number of exocytotic events. • There was also a deceleration of exocytosis in IMC with respect to PMC.

  4. Connective tissue growth factor is a positive regulator of epithelial-mesenchymal transition and promotes the adhesion with gastric cancer cells in human peritoneal mesothelial cells.

    Science.gov (United States)

    Jiang, Cheng-Gang; Lv, Ling; Liu, Fu-Rong; Wang, Zhen-Ning; Na, Di; Li, Feng; Li, Jia-Bin; Sun, Zhe; Xu, Hui-Mian

    2013-01-01

    Connective tissue growth factor (CTGF) is involved in human cancer development and progression. Epithelial to mesenchymal transition (EMT) plays an important role in many biological processes. In this study, we wished to investigate the role of CTGF in EMT of peritoneal mesothelial cells and the effects of CTGF on adhesion of gastric cancer cells to mesothelial cells. Human peritoneal mesothelial cells (HPMCs) were cultured with TGF-β1 or various concentrations of CTGF for different time. The EMT process was monitored by morphology. Real-time RT-PCR and Western blot were used to evaluate the expression of vimentin, α-SMA , E-cadherin and β-catenin. RNA interference was used to achieve selective and specific knockdown of CTGF. We demonstrated that CTGF induced EMT of mesothelial cells in a dose- and time-dependent manner. HPMCs were exposed to TGF-β1 also underwent EMT which was associated with the induction of CTGF expression. Transfection with CTGF siRNA was able to reverse the EMT partially after treatment of TGF-β1. Moreover, the induced EMT of HPMCs was associated with an increased adhesion of gastric cancer cells to mesothelial cells. These findings suggest that CTGF is not only an important mediator but a potent activator of EMT in peritoneal mesothelial cells, which in turn promotes gastric cancer cell adhesion to peritoneum. Copyright © 2012 Elsevier Ltd. All rights reserved.

  5. Alanyl-glutamine dipeptide restores the cytoprotective stress proteome of mesothelial cells exposed to peritoneal dialysis fluids.

    Science.gov (United States)

    Kratochwill, Klaus; Boehm, Michael; Herzog, Rebecca; Lichtenauer, Anton Michael; Salzer, Elisabeth; Lechner, Michael; Kuster, Lilian; Bergmeister, Konstantin; Rizzi, Andreas; Mayer, Bernd; Aufricht, Christoph

    2012-03-01

    Exposure of mesothelial cells to peritoneal dialysis fluids (PDF) results in cytoprotective cellular stress responses (CSR) that counteract PDF-induced damage. In this study, we tested the hypothesis that the CSR may be inadequate in relevant models of peritoneal dialysis (PD) due to insufficient levels of glutamine, resulting in increased vulnerability against PDF cytotoxicity. We particularly investigated the role of alanyl-glutamine (Ala-Gln) dipeptide on the cytoprotective PDF stress proteome. Adequacy of CSR was investigated in two human in vitro models (immortalized cell line MeT-5A and mesothelial cells derived from peritoneal effluent of uraemic patients) following exposure to heat-sterilized glucose-based PDF (PD4-Dianeal, Baxter) diluted with medium and, in a comparative proteomics approach, at different levels of glutamine ranging from depletion (0 mM) via physiological (0.7 mM) to pharmacological levels (8 mM administered as Ala-Gln). Despite severe cellular injury, expression of cytoprotective proteins was dampened upon PDF exposure at physiological glutamine levels, indicating an inadequate CSR. Depletion of glutamine aggravated cell injury and further reduced the CSR, whereas addition of Ala-Gln at pharmacological level restored an adequate CSR, decreasing cellular damage in both PDF exposure systems. Ala-Gln specifically stimulated chaperoning activity, and cytoprotective processes were markedly enhanced in the PDF stress proteome. Taken together, this study demonstrates an inadequate CSR of mesothelial cells following PDF exposure associated with low and physiological levels of glutamine, indicating a new and potentially relevant pathomechanism. Supplementation of PDF with pharmacological doses of Ala-Gln restored the cytoprotective stress proteome, resulting in improved resistance of mesothelial cells to exposure to PDF. Future work will study the clinical relevance of CSR-mediated cytoprotection.

  6. Benfotiamine protects against peritoneal and kidney damage in peritoneal dialysis.

    Science.gov (United States)

    Kihm, Lars P; Müller-Krebs, Sandra; Klein, Julia; Ehrlich, Gregory; Mertes, Laura; Gross, Marie-Luise; Adaikalakoteswari, Antonysunil; Thornalley, Paul J; Hammes, Hans-Peter; Nawroth, Peter P; Zeier, Martin; Schwenger, Vedat

    2011-05-01

    Residual renal function and the integrity of the peritoneal membrane contribute to morbidity and mortality among patients treated with peritoneal dialysis. Glucose and its degradation products likely contribute to the deterioration of the remnant kidney and damage to the peritoneum. Benfotiamine decreases glucose-induced tissue damage, suggesting the potential for benefit in peritoneal dialysis. Here, in a model of peritoneal dialysis in uremic rats, treatment with benfotiamine decreased peritoneal fibrosis, markers of inflammation, and neovascularization, resulting in improved characteristics of peritoneal transport. Furthermore, rats treated with benfotiamine exhibited lower expression of advanced glycation endproducts and their receptor in the peritoneum and the kidney, reduced glomerular and tubulointerstitial damage, and less albuminuria. Increased activity of transketolase in tissue and blood contributed to the protective effects of benfotiamine. In primary human peritoneal mesothelial cells, the addition of benfotiamine led to enhanced transketolase activity and decreased expression of advanced glycation endproducts and their receptor. Taken together, these data suggest that benfotiamine protects the peritoneal membrane and remnant kidney in a rat model of peritoneal dialysis and uremia. Copyright © 2011 by the American Society of Nephrology

  7. Differential gene expression in the murine gastric fundus lacking interstitial cells of Cajal

    Directory of Open Access Journals (Sweden)

    Ward Sean M

    2003-06-01

    Full Text Available Abstract Background The muscle layers of murine gastric fundus have no interstitial cells of Cajal at the level of the myenteric plexus and only possess intramuscular interstitial cells and this tissue does not generate electric slow waves. The absence of intramuscular interstitial cells in W/WV mutants provides a unique opportunity to study the molecular changes that are associated with the loss of these intercalating cells. Method The gene expression profile of the gastric fundus of wild type and W/WV mice was assayed by murine microarray analysis displaying a total of 8734 elements. Queried genes from the microarray analysis were confirmed by semi-quantitative reverse transcription-polymerase chain reaction. Results Twenty-one genes were differentially expressed in wild type and W/WV mice. Eleven transcripts had 2.0–2.5 fold higher mRNA expression in W/WV gastric fundus when compared to wild type tissues. Ten transcripts had 2.1–3.9 fold lower expression in W/WV mutants in comparison with wild type animals. None of these genes have ever been implicated in any bowel motility function. Conclusions These data provides evidence that several important genes have significantly changed in the murine fundus of W/WV mutants that lack intramuscular interstitial cells of Cajal and have reduced enteric motor neurotransmission.

  8. Ouabain enhancement of compound 48/80 induced histamine secretion from rat peritoneal mast cells: dependence on extracellular sodium

    DEFF Research Database (Denmark)

    Knudsen, T; Bertelsen, Niels Haldor; Johansen, Torben

    1992-01-01

    Purified populations of rat peritoneal mast cells were used to study the effect of ouabain on compound 48/80-induced histamine secretion and on 86Rb+ uptake. 86Rb+ was used as a tracer for extracellular K+. The calculated value of the ouabain-sensitive uptake of K+ and 86Rb+ was considered...... on the secretion occurs in the presence of sodium but not when sodium was replaced by lithium. Preservation by ouabain of a high intracellular sodium content in sodium-loaded cells was associated with preservation of the secretory response in a calcium-free medium. In the presence of lanthanum in a calcium...

  9. ArtinM Mediates Murine T Cell Activation and Induces Cell Death in Jurkat Human Leukemic T Cells

    Science.gov (United States)

    Oliveira-Brito, Patrícia Kellen Martins; Gonçalves, Thiago Eleutério; Vendruscolo, Patrícia Edivânia; Roque-Barreira, Maria Cristina

    2017-01-01

    The recognition of cell surface glycans by lectins may be critical for the innate and adaptive immune responses. ArtinM, a d-mannose-binding lectin from Artocarpus heterophyllus, activates antigen-presenting cells by recognizing TLR2 N-glycans and induces Th1 immunity. We recently demonstrated that ArtinM stimulated CD4+ T cells to produce proinflammatory cytokines. Here, we further studied the effects of ArtinM on adaptive immune cells. We showed that ArtinM activates murine CD4+ and CD8+ T cells, augmenting their positivity for CD25, CD69, and CD95 and showed higher interleukin (IL)-2 and interferon (IFN)-γ production. The CD4+ T cells exhibited increased T-bet expression in response to ArtinM, and IL-2 production by CD4+ and CD8+ T cells depended on the recognition of CD3εγ-chain glycans by ArtinM. The ArtinM effect on aberrantly-glycosylated neoplastic lymphocytes was studied in Jurkat T cells, in which ArtinM induced IL-2, IFN-γ, and IL-1β production, but decreased cell viability and growth. A higher frequency of AnnexinV- and propidium iodide-stained cells demonstrated the induction of Jurkat T cells apoptosis by ArtinM, and this apoptotic response was reduced by caspases and protein tyrosine kinase inhibitors. The ArtinM effects on murine T cells corroborated with the immunomodulatory property of lectin, whereas the promotion of Jurkat T cells apoptosis may reflect a potential applicability of ArtinM in novel strategies for treating lymphocytic leukemia. PMID:28665310

  10. 4T1 Murine Mammary Carcinoma Cells Enhance Macrophage-Mediated Innate Inflammatory Responses.

    Directory of Open Access Journals (Sweden)

    Laurence Madera

    Full Text Available Tumor progression and the immune response are intricately linked. While it is known that cancers alter macrophage inflammatory responses to promote tumor progression, little is known regarding how cancers affect macrophage-dependent innate host defense. In this study, murine bone-marrow-derived macrophages (BMDM were exposed to murine carcinoma-conditioned media prior to assessment of the macrophage inflammatory response. BMDMs exposed to 4T1 mammary carcinoma-conditioned medium demonstrated enhanced production of pro-inflammatory cytokines tumor necrosis factor α, interleukin-6, and CCL2 in response to lipopolysaccharide (LPS while production of interleukin-10 remained unchanged. The increased LPS-induced production of pro-inflammatory cytokines was transient and correlated with enhanced cytokine production in response to other Toll-like receptor agonists, including peptidoglycan and flagellin. In addition, 4T1-conditioned BMDMs exhibited strengthened LPS-induced nitric oxide production and enhanced phagocytosis of Escherichia coli. 4T1-mediated augmentation of macrophage responses to LPS was partially dependent on the NFκB pathway, macrophage-colony stimulating factor, and actin polymerization, as well as the presence of 4T1-secreted extracellular vesicles. Furthermore, peritoneal macrophages obtained from 4T1 tumor-bearing mice displayed enhanced pro-inflammatory cytokine production in response to LPS. These results suggest that uptake of 4T1-secreted factors and actin-mediated ingestion of 4T1-secreted exosomes by macrophages cause a transient enhancement of innate inflammatory responses. Mammary carcinoma-mediated regulation of innate immunity may have significant implications for our understanding of host defense and cancer progression.

  11. Peritoneal mesothelioma

    International Nuclear Information System (INIS)

    Ros, P.R.; Yuschok, T.J.; Buck, J.L.; Shekitka, K.M.; Kaude, J.V.; Armed Forces Inst. of Pathology, Washington, DC

    1991-01-01

    Previous imaging reports of peritoneal mesothelioma have described a variety of radiologic appearances, but have not included its pathologic classification. We retrospectively reviewed 10 cases of peritoneal mesothelioma representing the following histologic categories: 7 epithelial, 2 sarcomatoid, and one biphasic. By imaging, epithelial mesotheliomas demonstrated diffuse thickening of the peritoneum and mesentery and/or multiple small nodules. The sarcomatoid-type appeared as a mass and the biphasic-type had radiologic and gross pathologic features of both sarcomatoid and epithelial types. We conclude that peritoneal mesothelioma presents with a wide spectrum of radiographic appearances and should therefore be included in the differential diagnoses of diffuse as well as localized peritoneal processes. (orig.)

  12. Quantitative analysis of thymus lymphoid cells during murine radioleukemogenesis

    International Nuclear Information System (INIS)

    Boniver, J.; Simar, L.J.; Courtoy, R.; Betz, E.H.

    1978-01-01

    Fractionated irradiation by four doses of 150 R leads to the development of lymphoma in the thymus of C57BL mice, after a long latent period (4 to 12 months) during which thymic subcapsular blast cells undergo neoplastic transformation. Electron microscope studies on this blast cell population have revealed several types that are distinguishable on the basis of nuclear ultrastructure and activity in the cell cycle. These blast cells are lymphoblasts, ring-shaped nucleolus cells, and x-cells. A stereological analysis demonstrates that the result of the irradiations is not an increase of the thymic blast cell number during the preleukemic period, as generally thought before, but important modifications in the relative proportion of the three cell types forming the blastic population. X-cells, which essentially appear in S phase, are more numerous after the third and fourth doses of radiation. They might be more ''sensitive'' to the action of the leukemogenic virus and facilitate the leukemogenic process. In contrast, in the preleukemic atrophic thymus just before the appearance of the lymphoma, x-cells are scarce, and ring-shaped nucleolus cells are more frequent than in the controls, suggesting important changes in thymus cell kinetics. The only modification detected in the blast cells at any time after irradiation concerns the surface of Golgi membranes, which decreases in x-cells and ring-shaped nucleolus cells of the atrophic thymus. Because the same change is observed later on in the lymphomatous cells, it is suggested that the change might be related to the transformation of the thymic blast cells into cancerous cells

  13. Heterogenous populations of cytotoxic cells in the peritoneal cavity of BALB/c mice immunized with allogeneic EL4 leukemia cells

    International Nuclear Information System (INIS)

    Zighelboim, J.; Bonavida, B.; Fahey, J.L.

    1974-01-01

    Adherent cells, presumably macrophages, obtained from the peritoneal cavity shortly after rejection of the allogeneic leukemia EL4, produced effective cell-mediated cytotoxicity (CMC) in vitro. These cytotoxic cells were sensitive to anti-macrophage serum and resistant to anti-thymocyte serum and 10,000 roentgen irradiation. In contrast, a second population of specifically cytotoxic cells were nonadherent, sensitive to x-rays and anti-thymocyte serum, but not to anti-macrophage serum. The two cell populations had a cooperative cytotoxic effect in vitro against allogeneic tumor cells

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

    NARCIS (Netherlands)

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

    2013-01-01

    Multiple myeloma and B cell lymphoma are leading causes of death in Gaucher's disease but the nature of the stimulus driving the often noted clonal expansion of immunoglobulin-secreting B cells and cognate lymphoid malignancy is unknown. We investigated the long-term development of B cell

  15. 17-AAG mediated targeting of Hsp90 limits tert activity in peritoneal sarcoma related malignant ascites by downregulating cyclin D1 during cell cycle entry.

    Science.gov (United States)

    Chaklader, M; Das, P; Pereira, J A; Law, A; Chattopadhyay, S; Chatterjee, R; Mondal, A; Law, S

    2012-07-01

    Peritoneal or retro-peritoneal sarcomatosis related malignant ascites formation is a rare but serious consequence of the locoregional metastatic event. The present work aimed to study the effect of the Hsp90 inhibitor (17-AAG), an ansamycin analog, on cell cycle and DNA replication specific chaperone-clients interaction in the event of peritoneal sarcoma related malignant ascites formation in mouse model at the late stage of malignant growth. We administered 17-AAG, an Hsp90 inhibitor, divided doses (330 μg/kg b.w./day for first five days then next ten days with166 μg/kg b.w./day) through intra-peritoneal route of inbred Swiss albino mice bearing full grown peritoneal malignant ascites of sarcoma-180. Our study was evaluated by peripheral blood hemogram analysis, malignant ascitic cytology, cell viability test, survival time and mitotic indexing. Furthermore, flowcytometric HSP90, TERT, CyclinD1, PCNA and GM-CSF expression analysis has been considered for special objective of the study. Our experimental efforts reduced the aggressive proliferation of malignant ascites by drastic downregulation of TERT and cyclin D1 on the verge of cell cycle entry along with DNA replication processivity factor PCNA by directly modulating their folding machinery - heat shock protein 90. Consequently, we observed that malignant ascitic cells became error prone during the event of karyokinesis and produced micronucleus containing malignant cells with low viability. Peripheral neutrophilia due to over-expression of GM-CSF by the peritoneal malignant ascites were also controlled by the treatment with 17-AAG and overall, the treatment modality improved the median survival time. Finally we can conclude that 17AAG administration might serve as a prospective pharmacological agent for the management of peritoneal sarcoma related malignant ascites and throws light towards prolonged survival of the patients concerned.

  16. Role of P2X7 on steroid synthesis in murine luteal cells

    Directory of Open Access Journals (Sweden)

    Chunping Zhang

    2016-03-01

    Full Text Available The extracellular adenosine triphosphate (ATP regulates different cellular functions through activating purinergic receptors as a signalling molecule or neurotransmitter. P2X7 is highly expressed in murine small luteal cells. In this study, murine luteal cells were cultured in vitro and treated with P2X7 agonists – ATP and 2′(3′-O-(4-benzoyl-benzoyl-adenosine 50-triphosphate (BzATP and with P2X7 antagonist – brilliant blue G (BBG. We found that ATP and BzATP increased the production of progesterone and had no influence on the production of estradiol. BBG reversed the effect of BzATP and ATP. Further studies demonstrated that ATP and BzATP promoted the expression of CYP11A. These results revealed that P2X7 receptor activation is involved in the steroid synthesis in corpus luteum.

  17. Clearance and binding of radiolabeled glycoproteins by cells of the murine mononuclear phagocyte system

    International Nuclear Information System (INIS)

    Imber, M.J.

    1982-01-01

    The clearance and binding of radiolabeled lactoferrin and fast α 2 -macroglobulin were studied. Both glycoproteins cleared rapidly following intravenous injection in mice, and both bound specifically to discrete receptors on murine peritoneal macrophages. The simultaneous presence of excess, unlabeled ligands specific for receptors recognizing terminal fucose, mannose, N-acetylglucosamine or galactose residues did not inhibit the clearance or binding of either lactoferrin or fast-α 2 M. The clearance and binding of enzymatically defucosylated lactoferrin was indistinguishable from native lactoferrin, indicating that terminal α(1-3)-linked fucose on lactoferrin is not necessary for receptor recognition. The clearance and binding of two fast -α 2 M forms, α 2 M-trypsin and α 2 M-MeNH 2 cross compete with each other. Saturation binding studies indicated that the total binding of mannosyl -BSA, fusocyl-BSA, and N-acetylglucosaminyl-BSA to macrophages activated by BCG was approximately 15% of the levels observed with inflammatory macrophages elicited by thioglycollate broth. Cross-competition binding studies demonstrated a common surface receptor mediated binding of all three neoglycoprotein ligands and was identical to the receptor on mononuclear phagocytes that binds mannosyl- and N-acetylglucosaminyl-terminated glycoproteins. These results suggest that difference between discrete states of macrophage function may be correlated with selective changes in levels of the surface receptor for mannose-containing glycoproteins

  18. Nature of leukemic stem cells in murine myelogenous leukemia

    International Nuclear Information System (INIS)

    Yoshida, K.; Nemoto, K.; Nishimura, M.; Hayata, I.; Inoue, T.; Seki, M.

    1986-01-01

    We investigated the nature of myelogenous leukemic stem cells in mice. L-8057, a megakaryoblastic leukemia cell line used in this study, produces in vivo and in vitro colonies. By means of typical chromosomal aberrations in L-8057, one can conveniently detect the origin of the cells in each colony derived from a leukemic stem cell. Direct evidence of whether cells from each colony had leukemogenicity in recipient mice was successfully obtained by the colony transplantation assay. Both leukemic colony-forming unit-spleen (L-CFU-s) and leukemic colony-forming unit-culture (L-CFU-c) in L-8057 may have belonged to the same differentiating stage in the stem cells because of their similar radiosensitivity, although some parts of the L-CFU of L-8057 seemed to have lost their capability to regenerate L-CFU-s when the cells were plated in dishes. This leukemic stem cell preserves high self-renewal ability in vitro after 10 passages. In addition, in vitro colony formation by this leukemic cell during the above course of serial passages did not require any additional exogenous stimulators. The same sort of trials have been made on other types of leukemias. Leukemic stem cells showed remarkable variety in their response to stimulating factors and in their self-renewal activity, which suggests that they may have consisted of heterogeneous populations

  19. The role of adrenergic activation on murine luteal cell viability and progesterone production.

    Science.gov (United States)

    Wang, Jing; Tang, Min; Jiang, Huaide; Wu, Bing; Cai, Wei; Hu, Chuan; Bao, Riqiang; Dong, Qiming; Xiao, Li; Li, Gang; Zhang, Chunping

    2016-09-15

    Sympathetic innervations exist in mammalian CL. The action of catecholaminergic system on luteal cells has been the focus of a variety of studies. Norepinephrine (NE) increased progesterone secretion of cattle luteal cells by activating β-adrenoceptors. In this study, murine luteal cells were treated with NE and isoprenaline (ISO). We found that NE increased the viability of murine luteal cells and ISO decreased the viability of luteal cells. Both NE and ISO promoted the progesterone production. Nonselective β-adrenergic antagonist, propranolol reversed the effect of ISO on cell viability but did not reverse the effect of NE on cell viability. Propranolol blocked the influence of NE and ISO on progesterone production. These results reveal that the increase of luteal cell viability induced by NE is not dependent on β-adrenergic activation. α-Adrenergic activation possibly contributes to it. Both NE and ISO increased progesterone production through activating β-adrenergic receptor. Further study showed that CyclinD2 is involved in the increase of luteal cell induced by NE. 3β-Hydroxysteroid dehydrogenase, LHR, steroidogenic acute regulatory protein (StAR), and PGF2α contribute to the progesterone production induced by NE and ISO. Copyright © 2016 Elsevier Inc. All rights reserved.

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

    International Nuclear Information System (INIS)

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

    1986-01-01

    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 - ) for murine Cu-Zn-SOD was determined to be 6.8 x 10 -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

  1. Selection of viable cell subpopulations from murine tumours using FACS

    International Nuclear Information System (INIS)

    Chaplin, D.J.; Durand, R.E.; Olive, P.L.

    1985-01-01

    The authors developed a technique which enables isolation of viable tumour cells subpopulation as a function of their distance from the blood supply. The basis for this separation procedure is that the fluorochrome, Hoechst 33342, as a result of its high avidity for cellular DNA, exhibits a marked diffusion/consumption gradient when it has to pass through several cell layers. As a result intravenous injection of Hoechst 33342 into tumour bearing animals, results in a heterogeneous straining pattern within the tumour with cells close to blood vessels being brightly fluorescent while those more distant are less intensely stained. Since these differences in staining intensity persist after tumour disaggregation, cells can be sorted into subpopulations on the basis of their fluorescence intensity using a fluorescence activated cell sorter. This technique offers the unique possibility of identifying the location of those cell subpopulations resistant to treatment with either radiation or chemotherapeutic drugs

  2. Ontogeny and function of murine epidermal Langerhans cells.

    Science.gov (United States)

    Kaplan, Daniel H

    2017-09-19

    Langerhans cells (LCs) are epidermis-resident antigen-presenting cells that share a common ontogeny with macrophages but function as dendritic cells (DCs). Their development, recruitment and retention in the epidermis is orchestrated by interactions with keratinocytes through multiple mechanisms. LC and dermal DC subsets often show functional redundancy, but LCs are required for specific types of adaptive immune responses when antigen is concentrated in the epidermis. This Review will focus on those developmental and functional properties that are unique to LCs.

  3. Free cancer cell detection in peritoneal cavity in gastric cancer patients by RT-PCR for CEA

    International Nuclear Information System (INIS)

    Lee, Jong Inn; Moon, Nan Mo; Paik, Nam Sun; Choi, Dong Wook; Bang, Ho Yun; Hong, Seok Il

    1997-12-01

    Authors applied RT-PCR assay to detecting CEA expressing free cancer cells in peritoneal cavity of 114 gastric cancer patients to find an indication for prophylactic treatment to prevent peritoneal recurrence. Sixty-three of 114 cases were positive for RT-PCR, of which 16 cases were positive for cytologic examination and 47 cases were negative. Forty-nine of 51 cases who were negative for RT-PCR were negative for cytologic examination. Positivity for RT-PCR according to the depth of invasion were as follows : two (28.6 %) of seven cases whose cancer invaded mucosal or submucosal layer were positive. Ten (45.5 %) of 22 cases whose cancer invaded muscular or subserosal layer were positive. Forty-one (57.7 %) of 71 serosa involved cases were positive. Eleven (78.6 %) of cases who had grossly perioneal seedings were positive (p=0.026). However, all of 7 EGC cases, 19 of 22 cases whose cancer invaded to muscle layer or to subserosa were negative for cytologic examination, and eight of 13 cases who had had peritoneal seedings were positive. Positivity for RT-PCR according to cell differentiation were as follows: forty-two (61.8 %) of 68 cases who cancer were poorly differentiated type were positive. (p=0.163) Serum level of CEA of RT-PCR positive group and that of negative group were not statistically different. It was revealed that RT-PCR was more sensitive than cytologic examination in detecting free tumor cells, especially in pm, ss and serosa positive cancers, so if further study with more cases and longer follow-up is performed, its role as prognostic factor and an indication of prophylactic therapy will be clarified. (author). 22 refs., 5 tabs

  4. Free cancer cell detection in peritoneal cavity in gastric cancer patients by RT-PCR for CEA

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Jong Inn; Moon, Nan Mo; Paik, Nam Sun; Choi, Dong Wook; Bang, Ho Yun; Hong, Seok Il [Korea Cancer Center Hospital, Seoul (Korea, Republic of)

    1997-12-01

    Authors applied RT-PCR assay to detecting CEA expressing free cancer cells in peritoneal cavity of 114 gastric cancer patients to find an indication for prophylactic treatment to prevent peritoneal recurrence. Sixty-three of 114 cases were positive for RT-PCR, of which 16 cases were positive for cytologic examination and 47 cases were negative. Forty-nine of 51 cases who were negative for RT-PCR were negative for cytologic examination. Positivity for RT-PCR according to the depth of invasion were as follows : two (28.6 %) of seven cases whose cancer invaded mucosal or submucosal layer were positive. Ten (45.5 %) of 22 cases whose cancer invaded muscular or subserosal layer were positive. Forty-one (57.7 %) of 71 serosa involved cases were positive. Eleven (78.6 %) of cases who had grossly perioneal seedings were positive (p=0.026). However, all of 7 EGC cases, 19 of 22 cases whose cancer invaded to muscle layer or to subserosa were negative for cytologic examination, and eight of 13 cases who had had peritoneal seedings were positive. Positivity for RT-PCR according to cell differentiation were as follows: forty-two (61.8 %) of 68 cases who cancer were poorly differentiated type were positive. (p=0.163) Serum level of CEA of RT-PCR positive group and that of negative group were not statistically different. It was revealed that RT-PCR was more sensitive than cytologic examination in detecting free tumor cells, especially in pm, ss and serosa positive cancers, so if further study with more cases and longer follow-up is performed, its role as prognostic factor and an indication of prophylactic therapy will be clarified. (author). 22 refs., 5 tabs.

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

    Science.gov (United States)

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

    2004-04-01

    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 Suillus granulatus, S. luteus). The last one had never been investigated for its cytotoxic compounds before.

  6. A novel CCR-2/TLR-2 triggered signaling in murine peritoneal macrophages intensifies bacterial (Staphylococcus aureus) killing by reactive oxygen species through TNF-R1.

    Science.gov (United States)

    Nandi, Ajeya; Bishayi, Biswadev

    2017-10-01

    Macrophages are remarkably versatile in their ability to recognize and respond to a wide range of stimuli by expressing a variety of surface and intracellular receptors and triggering multiple signal transduction pathways. The onset of microbial infection is primarily determined by the initial contacts made by the microbes with the host macrophages. Although there prevail a relationship between the chemokine receptor and Toll like receptors during disease, particularly TLR-2 and CCR-2 signaling interdependence on each other has not been yet investigated during acute staphylococcal infection. Thus, the present study was aimed to trace possible interaction between CCR-2 and TLR-2 in peritoneal macrophages during acute Staphylococcus aureus infection. We found that neutralization of CCR-2 attenuates TLR-2 expression and restricts S. aureus burden but TLR-2 neutralization augments CCR-2 expression in macrophages, along with compromised host-derived reactive oxygen species production. S. aureus infection to CCR-2 intact but TLR-2 neutralized macrophages triggered production of IL-1β, TNF-α, IL-6, IFN-γ, MCP-1 and expression of iNOS, TNFR-1 and GPx with concomitant decrease in IL-10 production. Further, study with NG-monomethyl-l-arginine (L-NMMA) [iNOS blocker] and buthionine sulfoximine (BSO) [GPx blocker] revealed that S. aureus infection enhanced TLR-2 expression in CCR-2 intact and TLR-2 neutralized macrophages possibly via iNOS and TNFR-1 up regulation and GPx down regulation. Overall, our data indicate that targeting CCR-2 with neutralizing antibody in the early phase of S. aureus infection could restrict excessive inflammation with less compromised bacterial killing. It certainly would be a therapeutic strategy in S. aureus induced inflammatory and infective diseases. Copyright © 2017 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.

  7. Peritoneal mesothelioma

    International Nuclear Information System (INIS)

    Raptopoulos, V.

    1985-01-01

    The definitive diagnosis of peritoneal mesothelioma and its differentiation from metastatic peritoneal carcinomatosis may be difficult because of the clinical, macroscopic, and microscopic variability of the tumor. To this purpose, a combination of criteria, including the clinical picture, the gross pathologic findings, the exclusion of other primary neoplasms, and the microscopic findings, must be taken into consideration. Conventionally, these criteria may be established only after surgical exploration and extensive sampling. Experience with patients with peritoneal mesothelioma and metastatic peritoneal carcinomatosis, as well as a review of the recent imaging literature, shows excellent correlation between computed tomography or ultrasound and the operative or autopsy findings. These imaging modalities showed soft-tissue masses or nodules; thickened omentum (omental cake), peritoneum, mesentery, and bowel wall; pleural plaques; and usually disproportionally small, if any, ascites. The latter two observations may be useful in differentiating mesothelioma from carcinomatosis macroscopically. Furthermore, fine-needle aspiration biopsy, after performing wide sampling of the tumors in different locations under ultrasonic or computed tomographic guidance, produced diagnostic cytologic specimens. Thus, the need for exploratory surgery may be alleviated, and the diagnosis of peritoneal mesothelioma may be made prospectively and relatively noninvasively with the use of computed tomography or ultrasound and fine-needle aspiration biopsy. Since epidemiologic studies predict increasing incidence of this neoplasm, especially among asbestos workers, it is suggested that these techniques be seriously considered as screening methods for high-risk populations.67 references

  8. Characterization of the binding of radioiodinated hybrid recombinant IFN-alpha A/D to murine and human lymphoid cell lines

    International Nuclear Information System (INIS)

    Faltynek, C.R.; Princler, G.L.; Schwabe, M.; Shata, M.T.; Lewis, G.K.; Kamin-Lewis, R.M.

    1990-01-01

    The hybrid recombinant human interferon (IFN) rIFN-alpha A/D was radioiodinated. Specific binding of [125I]rIFN-alpha A/D was observed with both human and murine cell lines. The binding of [125I]rIFN-alpha A/D to human Daudi cells had similar characteristics to the previously described binding of [125I]rIFN-alpha A or -alpha 2. The following lines of evidence demonstrated that [125I]rIFN-alpha A/D bound with high affinity to the same receptor on murine cells as murine IFN-alpha and -beta: (i) the binding of [125I]rIFN-alpha A/D to murine LBRM cells was inhibited to a similar extent by natural murine IFN-alpha, natural murine IFN-beta, and rIFN-A/D; (ii) the Kd (approximately 2 X 10(-10) M) obtained from both competition experiments and saturation binding experiments with [125I]rIFN-alpha A/D was comparable to the previously reported Kd for the binding of natural murine IFN-alpha and -beta to other murine cell lines; (iii) the size of the cross-linked [125I]rIFN-alpha A/D receptor complex formed on murine LBRM cells was similar to the previously reported cross-linked complex formed after binding radioiodinated natural murine IFN-beta to other murine cell lines. Due to the current lack of readily available recombinant murine IFN-alpha or -beta for radiolabeling and the previously demonstrated biological activity of rIFN-alpha A/D on murine cells, [125I]rIFN-alpha A/D should prove to be a useful reagent for further studies of murine IFN receptors

  9. H4 histamine receptors mediate cell cycle arrest in growth factor-induced murine and human hematopoietic progenitor cells.

    Directory of Open Access Journals (Sweden)

    Anne-France Petit-Bertron

    Full Text Available The most recently characterized H4 histamine receptor (H4R is expressed preferentially in the bone marrow, raising the question of its role during hematopoiesis. Here we show that both murine and human progenitor cell populations express this receptor subtype on transcriptional and protein levels and respond to its agonists by reduced growth factor-induced cell cycle progression that leads to decreased myeloid, erythroid and lymphoid colony formation. H4R activation prevents the induction of cell cycle genes through a cAMP/PKA-dependent pathway that is not associated with apoptosis. It is mediated specifically through H4R signaling since gene silencing or treatment with selective antagonists restores normal cell cycle progression. The arrest of growth factor-induced G1/S transition protects murine and human progenitor cells from the toxicity of the cell cycle-dependent anticancer drug Ara-C in vitro and reduces aplasia in a murine model of chemotherapy. This first evidence for functional H4R expression in hematopoietic progenitors opens new therapeutic perspectives for alleviating hematotoxic side effects of antineoplastic drugs.

  10. Potent and reversible lentiviral vector restriction in murine induced pluripotent stem cells.

    Science.gov (United States)

    Geis, Franziska K; Galla, Melanie; Hoffmann, Dirk; Kuehle, Johannes; Zychlinski, Daniela; Maetzig, Tobias; Schott, Juliane W; Schwarzer, Adrian; Goffinet, Christine; Goff, Stephen P; Schambach, Axel

    2017-05-31

    Retroviral vectors are derived from wild-type retroviruses, can be used to study retrovirus-host interactions and are effective tools in gene and cell therapy. However, numerous cell types are resistant or less permissive to retrovirus infection due to the presence of active defense mechanisms, or the absence of important cellular host co-factors. In contrast to multipotent stem cells, pluripotent stem cells (PSC) have potential to differentiate into all three germ layers. Much remains to be elucidated in the field of anti-viral immunity in stem cells, especially in PSC. In this study, we report that transduction with HIV-1-based, lentiviral vectors (LV) is impaired in murine PSC. Analyses of early retroviral events in induced pluripotent stem cells (iPSC) revealed that the restriction is independent of envelope choice and does not affect reverse transcription, but perturbs nuclear entry and proviral integration. Proteasomal inhibition by MG132 could not circumvent the restriction. However, prevention of cyclophilin A (CypA) binding to the HIV-1 capsid via use of either a CypA inhibitor (cyclosporine A) or CypA-independent capsid mutants improved transduction. In addition, application of higher vector doses also increased transduction. Our data revealed a CypA mediated restriction in iPSC, which was acquired during reprogramming, associated with pluripotency and relieved upon subsequent differentiation. We showed that murine PSC and iPSC are less susceptible to LV. The block observed in iPSC was CypA-dependent and resulted in reduced nuclear entry of viral DNA and proviral integration. Our study helps to improve transduction of murine pluripotent cells with HIV-1-based vectors and contributes to our understanding of retrovirus-host interactions in PSC.

  11. The acquisition of cytokine responsiveness by murine B cells

    DEFF Research Database (Denmark)

    Poudrier, J; Owens, T

    1994-01-01

    chains and mRNA to levels comparable to those seen in activated T cells. Anti-mu-stimulated B cells responded to IL-2 by incorporation of [3H]thymidine and high rate immunoglobulin (Ig) secretion. Both IL-5 (at optimal concentration) and suboptimal lipopolysaccharide (LPS; 20 ng/ml) induced surface...... expression of IL-2R alpha. The level of expression induced by IL-5 was equivalent to that on anti-Ig-activated B cells. Neither stimulus induced detectable expression of IL-2R beta, and neither induced B cells to respond to IL-2. IL-2R alpha expression was strongly enhanced, and low levels of IL-2R beta...

  12. Collateral methotrexate resistance in cisplatin-selected murine leukemia cells

    Directory of Open Access Journals (Sweden)

    Bhushan A.

    1999-01-01

    Full Text Available Resistance to anticancer drugs is a major cause of failure of many therapeutic protocols. A variety of mechanisms have been proposed to explain this phenomenon. The exact mechanism depends upon the drug of interest as well as the tumor type treated. While studying a cell line selected for its resistance to cisplatin we noted that the cells expressed a >25,000-fold collateral resistance to methotrexate. Given the magnitude of this resistance we elected to investigate this intriguing collateral resistance. From a series of investigations we have identified an alteration in a membrane protein of the resistant cell as compared to the sensitive cells that could be the primary mechanism of resistance. Our studies reviewed here indicate decreased tyrosine phosphorylation of a protein (molecular mass = 66 in the resistant cells, which results in little or no transfer of methotrexate from the medium into the cell. Since this is a relatively novel function for tyrosine phosphorylation, this information may provide insight into possible pharmacological approaches to modify therapeutic regimens by analyzing the status of this protein in tumor samples for a better survival of the cancer patients.

  13. Puerarin Facilitates T-Tubule Development of Murine Embryonic Stem Cell-Derived Cardiomyocytes

    Directory of Open Access Journals (Sweden)

    Lu Wang

    2014-07-01

    Full Text Available Aims: The embryonic stem cell-derived cardiomyocytes (ES-CM is one of the promising cell sources for repopulation of damaged myocardium. However, ES-CMs present immature structure, which impairs their integration with host tissue and functional regeneration. This study used murine ES-CMs as an in vitro model of cardiomyogenesis to elucidate the effect of puerarin, the main compound found in the traditional Chinese medicine the herb Radix puerariae, on t-tubule development of murine ES-CMs. Methods: Electron microscope was employed to examine the ultrastructure. The investigation of transverse-tubules (t-tubules was performed by Di-8-ANEPPS staining. Quantitative real-time PCR was utilized to study the transcript level of genes related to t-tubule development. Results: We found that long-term application of puerarin throughout cardiac differentiation improved myofibril array and sarcomeres formation, and significantly facilitated t-tubules development of ES-CMs. The transcript levels of caveolin-3, amphiphysin-2 and junctophinlin-2, which are crucial for the formation and development of t-tubules, were significantly upregulated by puerarin treatment. Furthermore, puerarin repressed the expression of miR-22, which targets to caveolin-3. Conclusion: Our data showed that puerarin facilitates t-tubule development of murine ES-CMs. This might be related to the repression of miR-22 by puerarin and upregulation of Cav3, Bin1 and JP2 transcripts.

  14. Trp53 activity is repressed in radio-adapted cultured murine limb bud cells

    International Nuclear Information System (INIS)

    Vares, Guillaume; Wang, Bing; Tanaka, Kaoru; Shang, Yi; Fujita, Kazuko; Hayata, Isamu; Nenoi, Mitsuru

    2011-01-01

    Understanding the effects of ionizing radiation (IR) at low dose in fetal models is of great importance, because the fetus is considered to be at the most radiosensitive stage of the development and prenatal radiation might influence subsequent development. We previously demonstrated the existence of an adaptive response (AR) in murine fetuses after pre-exposure to low doses of X-rays. Trp53-dependent apoptosis was suggested to be responsible for the teratogenic effects of IR; decreased apoptosis was observed in adapted animals. In this study, in order to investigate the role of Trp53 in AR, we developed a new model of irradiated micromass culture of fetal limb bud cells, which replicated proliferation, differentiation and response to IR in murine embryos. Murine fetuses were exposed to whole-body priming irradiation of 0.3 Gy or 0.5 Gy at embryonic day 11 (E11). Limb bud cells (collected from digital ray areas exhibiting radiation-induced apoptosis) were cultured and exposed to a challenging dose of 4 Gy at E12 equivalent. The levels of Trp53 protein and its phosphorylated form at Ser18 were investigated. Our results suggested that the induction of AR in mouse embryos was correlated with a repression of Trp53 activity. (author)

  15. Proliferation differentiation and therapeutic effect of short-term cultured murine bone marrow cells

    International Nuclear Information System (INIS)

    Zhao Zekun; Cong Jianbo

    1986-01-01

    Murine bone marrow cells were cultured in conditioned medium of muscle. After 24 hours of culture, both adherent and suspended cells appeared in the culture. The adherent cells mainly consisted of macrophages and the suspended cells were predominantly granulocytes. After 6 days, the total number of nucleated cells and CFU-C in the culture increased about 400% and 600% respectively, but CFU-S reduced to 21% approximately. Lymphocytes persisted only for 4 days. The stem cells (CFU-S) from 6-day culture were injected into the lethally irradiated syngenic mice. The 30 day survival rate of the treated mice was 89% whereas that of the controls was only 7%. The bone marrow cells in 2/8 of recipients sacrificed at 30 or 60 days were of donor type and 6/8 of the recipients were chimeras

  16. Gut-homing CD4+ T cell receptor alpha beta+ T cells in the pathogenesis of murine inflammatory bowel disease

    DEFF Research Database (Denmark)

    Rudolphi, A; Boll, G; Poulsen, S S

    1994-01-01

    reconstituted a CD3+ T cell receptor alpha beta+ CD4+ T cell subset. CD4+ cells of this subset expressed the surface phenotype of mucosa-seeking, memory T cells. In the immunodeficient scid host, this gut-derived CD4+ T cell subset was found in spleen, peritoneal cavity, mesenteric lymph nodes (LN), epithelial...... compartments with CD4+ T cells from normal GALT plays an essential role in the pathogenesis of IBD in an immunodeficient host.......We studied which T cell subsets from the gut-associated lymphoid tissue (GALT) can migrate out of the gut mucosa and repopulate GALT compartments of an immunodeficient (semi)syngeneic host. Many distinct lymphocyte subsets were found in GALT of immunocompetent H-2d (BALB/c, BALB/cdm2, C.B-17...

  17. 5-FU-hydrogel inhibits colorectal peritoneal carcinomatosis and tumor growth in mice

    International Nuclear Information System (INIS)

    Wang, Yongsheng; Gong, Changyang; Yang, Li; Wu, Qinjie; Shi, Shuai; Shi, Huashan; Qian, Zhiyong; Wei, Yuquan

    2010-01-01

    Colorectal peritoneal carcinomatosis (CRPC) is a common form of systemic metastasis of intra-abdominal cancers. Intraperitoneal chemotherapy is a preferable option for colorectal cancer. Here we reported that a new system, 5-FU-loaded hydrogel system, can improve the therapeutic effects of intraperitoneal chemotherapy. A biodegradable PEG-PCL-PEG (PECE) triblock copolymer was successfully synthesized. The biodegradable and temperature sensitive hydrogel was developed to load 5-FU. Methylene blue-loaded hydrogel were also developed for visible observation of the drug release. The effects and toxicity of the 5-FU-hydrogel system were evaluated in a murine CRPC model. The hydrogel system is an injectable flowing solution at ambient temperature and forms a non-flowing gel depot at physiological temperature. 5-FU-hydrogel was subsequently injected into abdominal cavity in mice with CT26 cancer cells peritoneal dissemination. The results showed that the hydrogel delivery system prolonged the release of methylene blue; the 5-FU-hydrogel significantly inhibited the peritoneal dissemination and growth of CT26 cells. Furthermore, intraperitoneal administration of the 5-FU-hydrogel was well tolerated and showed less hematologic toxicity. Our data indicate that the 5-FU-hydrogel system can be considered as a new strategy for peritoneal carcinomatosis, and the hydrogel may provide a potential delivery system to load different chemotherapeutic drugs for peritoneal carcinomatosis of cancers

  18. The regulation of CD5 expression in murine T cells

    Directory of Open Access Journals (Sweden)

    Herzenberg Leonard A

    2001-05-01

    Full Text Available Abstract Background CD5 is a pan-T cell surface marker that is also present on a subset of B cells, B-1a cells.Functional and developmental subsets of T cells express characteristic CD5 levels that vary over roughly a 30-fold range. Previous investigators have cloned a 1.7 Kb fragment containing the CD5 promoter and showed that it can confer similar lymphocyte-specific expression pattern as observed for endogenous CD5 expression. Results We further characterize the CD5 promoter and identify minimal and regulatory regions on the CD5 promoter. Using a luciferase reporter system, we show that a 43 bp region on the CD5 promoter regulates CD5 expression in resting mouse thymoma EL4 T cells and that an Ets binding site within the 43 bp region mediates the CD5 expression. In addition, we show that Ets-1, a member of the Ets family of transcription factors, recognizes the Ets binding site in the electrophoretic mobility shift assay (EMSA. This Ets binding site is directly responsible for the increase in reporter activity when co-transfected with increasing amounts of Ets-1 expression plasmid. We also identify two additional evolutionarily-conserved regions in the CD5 promoter (CD5X and CD5Y and demonstrate the respective roles of the each region in the regulation of CD5 transcription. Conclusion Our studies define a minimal and regulatory promoter for CD5 and show that the CD5 expression level in T cells is at least partially dependent on the level of Ets-1 protein. Based on the findings in this report, we propose a model of CD5 transcriptional regulation in T cells.

  19. T-Cell lymphoproliferative disorder of hand-mirror cell morphology presenting in an eosinophilic loculated peritoneal effusion, with omental "caking"

    Directory of Open Access Journals (Sweden)

    Tufankjian Dearon

    2006-01-01

    Full Text Available Abstract Background Cells with "hand mirror" morphology have not, to the best of our knowledge, been described in a primary effusion sample. This paper describes a case of T-cell lymphoma with eosinophilia in a patient with suspected peritoneal carcinomatosis. Rarely, a T-cell lymphoproliferative process may mimic primary peritoneal carcinomatosis, clinically suggested by a presentation in CT imaging of omental caking with bilateral massive loculated effusions in a patient without lymphadenopathy or splenomegaly. Methods A 60 year old caucasian male presented with vague abdominal discomfort and increasing abdominal girth. Computed tomography showed a two centimeter thick omental cake and a small loculated effusion. The clinical presentation and imaging findings were most consistent with peritoneal carcinomatosis. Cytologic evaluation of the effusion was undertaken for diagnostic study. Results Rapid intraprocedural interpretation of the effusion sample showed a monomorphic population of cells with "hand-mirror" cell morphology exhibiting cytoplasmic extensions (uropodia with 3–5 course dark cytoplasmic granules and a rim of vacuolated cytoplasm capping the opposing "mirror head" side. These cells were seen within a background of mature eosinophils. Flow cytometric evaluation of the ascites fluid demonstrated an atypical T-cell population with the following immunophenotype: CD2-, CD3+, CD4-, CD5-, CD7-, CD8+, CD56+. T-cell receptor (TCR gene rearrangement was positive for clonal TCR-gamma gene rearrangement, supporting the diagnosis of a T-lymphoprolifereative disorder. Conclusion A T-cell lymphoproliferative process may present with "hand mirror" morphology in an effusion sample. These cells may show polar cytoplasmic vacuolization and 3–5 course granules within the "handle" of these unique cells. Cytoplasm shows peripheral constriction around the nucleus.

  20. A method for high efficiency YAC lipofection into murine embryonic stem cells.

    Science.gov (United States)

    Lee, J T; Jaenisch, R

    1996-01-01

    We describe a modified protocol for introducing yeast artificial chromosomes (YACs) into murine embryonic stem (ES) cells by lipofection. With a decreased DNA:cell ratio, increased concentration of condensing agents and altered culture conditions, this protocol reduces the requirement for YAC DNA to a few micrograms, improves the recovery of neomycin-resistant ES colonies and increases the yield of clones containing both flanking vector markers and insert. These modifications enable generation of sufficient 'intact' transgenic clones for biological analysis with a single experiment. PMID:9016681

  1. Disruption of canonical TGFβ-signaling in murine coronary progenitor cells by low level arsenic

    Energy Technology Data Exchange (ETDEWEB)

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

    2013-10-01

    Exposure to arsenic results in several types of cancers as well as heart disease. A major contributor to ischemic heart pathologies is coronary artery disease, however the influences by environmental arsenic in this disease process are not known. Similarly, the impact of toxicants on blood vessel formation and function during development has not been studied. During embryogenesis, the epicardium undergoes proliferation, migration, and differentiation into several cardiac cell types including smooth muscle cells which contribute to the coronary vessels. The TGFβ family of ligands and receptors is essential for developmental cardiac epithelial to mesenchymal transition (EMT) and differentiation into coronary smooth muscle cells. In this in vitro study, 18 hour exposure to 1.34 μM arsenite disrupted developmental EMT programming in murine epicardial cells causing a deficit in cardiac mesenchyme. The expression of EMT genes including TGFβ2, TGFβ receptor-3, Snail, and Has-2 are decreased in a dose-dependent manner following exposure to arsenite. TGFβ2 cell signaling is abrogated as detected by decreases in phosphorylated Smad2/3 when cells are exposed to 1.34 μM arsenite. There is also loss of nuclear accumulation pSmad due to arsenite exposure. These observations coincide with a decrease in vimentin positive mesenchymal cells invading three-dimensional collagen gels. However, arsenite does not block TGFβ2 mediated smooth muscle cell differentiation by epicardial cells. Overall these results show that arsenic exposure blocks developmental EMT gene programming in murine coronary progenitor cells by disrupting TGFβ2 signals and Smad activation, and that smooth muscle cell differentiation is refractory to this arsenic toxicity. - Highlights: • Arsenic blocks TGFβ2 induced expression of EMT genes. • Arsenic blocks TGFβ2 triggered Smad2/3 phosphorylation and nuclear translocation. • Arsenic blocks epicardial cell differentiation into cardiac mesenchyme.

  2. Adaptive changes in NAD+ metabolism in ultraviolet light-irradiated murine lymphoma cells

    International Nuclear Information System (INIS)

    Kleczkowska, H.E.; Szumiel, I.; Althaus, F.R.

    1990-01-01

    We have determined the ability of UV254nm-irradiated murine lymphoma cells to adapt their NAD+ metabolism to the increased NAD+ consumption for the poly ADP-ribosylation of chromatin proteins. Two murine lymphoma sublines with differential UV-sensitivity and poly(ADP-ribose) turnover were used as a model system. The first subline, designated LY-R is UV254nm-sensitive and tumorigenic in DBA/2 mice. The second subline, LY-S is UV254nm-resistant and nontumorigenic. Following treatment of these cells with 2 mM benzamide, an inhibitor of the NAD(+)-utilizing enzyme poly(ADP-ribose) polymerase, NAD+ levels slowly increased up to about 160% of control levels after 3 hours. When benzamide was added to these cultures 20 min after UV254nm irradiation, a dramatic transient increase of NAD+ levels was observed within 4 min in LY-R cells and more moderately in LY-S cells. At later times after UV254nm irradiation, the NAD+ levels increased in both sublines reaching up to 200% of the concentrations prior to benzamide treatment. These results demonstrate an adaptative response of NAD+ metabolism to UV254nm irradiation. In parallel, we observed a differential repartitioning of ADP-ribosyl residues between the NAD+ and poly(ADP-ribose) pools of LY-R and LY-S cells that correlates with the differential UV sensitivity of these cells

  3. Single-dose-response curves of murine gastrointestinal crypt stem cells

    International Nuclear Information System (INIS)

    Masuda, K.; Withers, H.R.; Mason, K.A.; Chen, K.Y.

    1977-01-01

    Dose-response curves for the reproductive capacity of crypt stem cells of murine colonic, jejunal, and gastric mucosae exposed in situ to multifractionated gamma ray exposures were analyzed and single-dose-survival curves of these cells were constructed. The following conclusions were drawn: (1) The single-dose-response curves bend downward over a dose range of approximately 200 to 1500 rad; (2) cell death seems to be due to nonrepairable damage at doses less than 250 rad for colon, and 220 rad for jejunum; (3) there are 21, 110, and 140 stem cells per crypt of gastric, colonic, and jejunal mucosa, respectively; and (4) jejunal stem cells are the most radiosensitive and gastric mucosal stem cells are the most resistant

  4. The Natural Time Course of Membrane Alterations During Peritoneal Dialysis Is Partly Altered by Peritonitis.

    Science.gov (United States)

    van Esch, Sadie; Struijk, Dirk G; Krediet, Raymond T

    2016-01-01

    patients who experienced peritonitis (p peritonitis. The two groups showed a similar decrease in the mesothelial cell mass marker cancer antigen 125 during follow-up. ♦ On top of the natural course of peritoneal function, peritonitis episodes to some extent influence the time-course of small-solute and fluid transport-especially the transport of solute-free water. Those modifications increase the risk for overhydration. Copyright © 2016 International Society for Peritoneal Dialysis.

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

    Science.gov (United States)

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

    2008-01-01

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

  6. Murine scid cells complement ataxia-telangiectasia cells and show a normal port-irradiation response of DNA synthesis

    International Nuclear Information System (INIS)

    Komatsu, K.; Yoshida, M.; Okumura, Y.

    1993-01-01

    The murine severe combined immunodeficient mutation (scid) is characterized by a lack of both B and T cells, due to a deficit in lymphoid variable-(diversity)-joining (V(D)J) rearrangement. Scid cells are highly sensitive to both radiation-induced killing and chromosomal aberrations. Significantly reduced D 0 and n values were demonstrated in scid cells and were similar to ataxia-telangiectasia (AT) cells (a unique human disease conferring whole body radiosensitivity). However, the kinetics of DNA synthesis after irradiation were different between the two cell types. In contrast with the radioresistant DNA synthesis of AT cells, DNA synthesis of scid cells was markedly inhibited after irradiation. The existence of different mutations was also supported by evidence of complementation in somatic cell hybrids between scid cells and AT cells. Results indicate that the radiobiological character of scid is similar to AT but is presumably caused by different mechanisms. (author)

  7. Slow and steady cell shrinkage reduces osmotic stress in bovine and murine oocyte and zygote vitrification.

    Science.gov (United States)

    Lai, D; Ding, J; Smith, G W; Smith, G D; Takayama, S

    2015-01-01

    Does the use of a new cryoprotectant agent (CPA) exchange protocol designed to minimize osmotic stress improve oocyte or zygote vitrification by reducing sublethal cryodamage? The use of a new CPA exchange protocol made possible by automated microfluidics improved oocyte and zygote vitrification with superior morphology as indicated by a smoother cell surface, higher sphericity, higher cytoplasmic lipid retention, less cytoplasmic leakage and higher developmental competence compared with conventional methods. The use of more 'steps' of CPA exposure during the vitrification protocol increases cryosurvival and development in the bovine model. However, such an attempt to eliminate osmotic stress is limited by the practicality of performing numerous precise pipetting steps in a short amount of time. Murine meiotically competent germinal vesicle intact oocytes and zygotes were harvested from the antral follicles in ovaries and ampulla, respectively. Bovine ovaries were obtained from a local abattoir at random stages of the estrous cycle. A total of 110 murine oocytes, 802 murine zygotes and 52 bovine oocytes were used in this study. Microfluidic devices were fabricated using conventional photo- and soft-lithography. CPAs used were 7.5% ethylene glycol (EG) and 7.5% dimethyl sulfoxide (DMSO) for equilibration solution and 15% EG, 15% DMSO and 0.5 M sucrose for vitrification solution. End-point analyses include mathematical modeling using Kedem-Katchalsky equations, morphometrics assessed by conventional and confocal microscopy, cytoplasmic lipid quantification by nile red staining, cytoplasmic leakage quantification by fluorescent dextran intercalation and developmental competence analysis by 96 h embryo culture and blastomere quantification. The automated microfluidics protocol decreased the shrinkage rate of the oocyte and zygote by 13.8 times over its manual pipetting alternative. Oocytes and zygotes with a lower shrinkage rate during CPA exposure experienced less

  8. Induction of differentiation of murine embryonal carcinoma cells by ouabain

    International Nuclear Information System (INIS)

    Zimmerman, B.T.

    1986-01-01

    Embryonal carcinoma (EC) cells can be induced to differentiate by ouabain at concentrations which inhibit Na + , K + -ATPase activity as measured by inhibition of 86 Rb + uptake. Since the pharmacologic action of ouabain is thought to be specific, the authors investigated the role of Na + , K + -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 + , K + -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 + , K + -ATPase is a major consumer of ATP. They therefore examined the effect of Na + , K + -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

  9. Highly efficient gene transfer using a retroviral vector into murine T cells for preclinical chimeric antigen receptor-expressing T cell therapy

    International Nuclear Information System (INIS)

    Kusabuka, Hotaka; Fujiwara, Kento; Tokunaga, Yusuke; Hirobe, Sachiko; Nakagawa, Shinsaku; Okada, Naoki

    2016-01-01

    Adoptive immunotherapy using chimeric antigen receptor-expressing T (CAR-T) cells has attracted attention as an efficacious strategy for cancer treatment. To prove the efficacy and safety of CAR-T cell therapy, the elucidation of immunological mechanisms underlying it in mice is required. Although a retroviral vector (Rv) is mainly used for the introduction of CAR to murine T cells, gene transduction efficiency is generally less than 50%. The low transduction efficiency causes poor precision in the functional analysis of CAR-T cells. We attempted to improve the Rv gene transduction protocol to more efficiently generate functional CAR-T cells by optimizing the period of pre-cultivation and antibody stimulation. In the improved protocol, gene transduction efficiency to murine T cells was more than 90%. In addition, almost all of the prepared murine T cells expressed CAR after puromycin selection. These CAR-T cells had antigen-specific cytotoxic activity and secreted multiple cytokines by antigen stimulation. We believe that our optimized gene transduction protocol for murine T cells contributes to the advancement of T cell biology and development of immunotherapy using genetically engineered T cells. - Highlights: • We established highly efficient gene transduction protocols for murine T cells. • CD8"+ CAR-T cells had antigen-specific cytotoxic activity. • CD4"+ CAR-T cells secreted multiple cytokines by antigen stimulation. • This finding can contribute to the development of T-cell biology and immunotherapy.

  10. Highly efficient gene transfer using a retroviral vector into murine T cells for preclinical chimeric antigen receptor-expressing T cell therapy

    Energy Technology Data Exchange (ETDEWEB)

    Kusabuka, Hotaka; Fujiwara, Kento; Tokunaga, Yusuke; Hirobe, Sachiko; Nakagawa, Shinsaku, E-mail: nakagawa@phs.osaka-u.ac.jp; Okada, Naoki, E-mail: okada@phs.osaka-u.ac.jp

    2016-04-22

    Adoptive immunotherapy using chimeric antigen receptor-expressing T (CAR-T) cells has attracted attention as an efficacious strategy for cancer treatment. To prove the efficacy and safety of CAR-T cell therapy, the elucidation of immunological mechanisms underlying it in mice is required. Although a retroviral vector (Rv) is mainly used for the introduction of CAR to murine T cells, gene transduction efficiency is generally less than 50%. The low transduction efficiency causes poor precision in the functional analysis of CAR-T cells. We attempted to improve the Rv gene transduction protocol to more efficiently generate functional CAR-T cells by optimizing the period of pre-cultivation and antibody stimulation. In the improved protocol, gene transduction efficiency to murine T cells was more than 90%. In addition, almost all of the prepared murine T cells expressed CAR after puromycin selection. These CAR-T cells had antigen-specific cytotoxic activity and secreted multiple cytokines by antigen stimulation. We believe that our optimized gene transduction protocol for murine T cells contributes to the advancement of T cell biology and development of immunotherapy using genetically engineered T cells. - Highlights: • We established highly efficient gene transduction protocols for murine T cells. • CD8{sup +} CAR-T cells had antigen-specific cytotoxic activity. • CD4{sup +} CAR-T cells secreted multiple cytokines by antigen stimulation. • This finding can contribute to the development of T-cell biology and immunotherapy.

  11. [Combined effect of cisplatin and caffeine on murine B16-BL6 melanoma cells].

    Science.gov (United States)

    Yasutake, H; Tsuchiya, H; Sugihara, M; Tomita, K; Ueda, Y; Tanaka, M; Sasaki, T

    1989-05-01

    Combined effect of cisplatin and caffeine on murine B16-BL6 melanoma cells was studied. Synergistic inhibition of the cell growth was observed when caffeine (2 mM) was added continuously after one hour exposure of cisplatin. On the other hand, when caffeine was added before one hour exposure of cisplatin or one hour simultaneous exposure with cisplatin, synergistic effect was not shown. In the analysis of DNA histogram obtained from flow cytometry, S and G2/M accumulation was observed by the treatment of cisplatin and that accumulation was reduced by the combination of cisplatin and caffeine. From this findings, it was suggested that caffeine would inhibit DNA repair process. Furthermore, according to morphological studies with hematoxylin-eosin stain and Fontana-Masson stain, the addition of caffeine alone resulted in mild swelling of melanoma cells and the decrease of nuclear-cytoplasmic ratio. The combination of cisplatin and caffeine caused marked swelling of melanoma cells and remarkable increase of dendrite-like processes. Melanogenesis was also enhanced by the addition of these two drugs. Many matured melanosomes, increases of mitochondria, Golgi's apparatus and endoplasmic reticula were observed by the use of electron microscope. These findings implied that the combination of cisplatin and caffeine induced a differentiation of murine melanoma cells.

  12. Hyphal growth of phagocytosed Fusarium oxysporum causes cell lysis and death of murine macrophages.

    Directory of Open Access Journals (Sweden)

    Katja Schäfer

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

  13. Hyphal growth of phagocytosed Fusarium oxysporum causes cell lysis and death of murine macrophages.

    Science.gov (United States)

    Schäfer, Katja; Bain, Judith M; Di Pietro, Antonio; Gow, Neil A R; Erwig, Lars P

    2014-01-01

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

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

    Science.gov (United States)

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

    2007-07-15

    (-)-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 24-72 h after addition of cubebin to B16 cells, and then intracellular melanin amount was increased at 48-96 h after the treatment. The expression levels of tyrosinase were time-dependently enhanced after the treatment with cubebin. At the same time, the expression levels of tyrosinase mRNA were also increased after addition of cubebin. Furthermore Western blot analysis revealed that cubebin elevated the level of phosphorylation of p38 mitogen-activated protein kinase (MAPK). SB203580, a selective inhibitor of p38 MAPK, completely blocked cubebin-induced expression of tyrosinase mRNA in B16 cells. These results suggested that cubebin increased melanogenesis in B16 cells through the enhancement of tyrosinase expression mediated by activation of p38 MAPK.

  15. Study of homing patterns of x-irradiated murine lymphoid cells

    International Nuclear Information System (INIS)

    Crouse, D.A.

    1974-01-01

    Effects of in vitro x-ray exposure of murine lymphoid cells on their subsequent in vivo homing patterns were studied. The homing of lymphoid cells to various tissues and organs was followed by using radio-labeled cell preparations or by following the distribution of cells with a specific immunological memory. X irradiation of 51 Cr-labeled spleen, lymph node, bone marrow, or thymus cells was found to significantly alter their subsequent in vivo distribution. Irradiated cells demonstrated an increased distribution to the liver and a significantly lower retention in the lungs. Cells going to the lymph nodes of Peyer's patches showed a significant exposure dependent decrease in homing following irradiation. Irradiated lymph node cells homed in greater numbers to the spleen and bone marrow, while irradiated cells from other sources showed a decrease or no change indistribution to the same tissues. Lymph node cell suspensions from dinitrophenyl-bovine gamma globulin (DNP-BGG) immune LBN rats were prepared, irradiated (0 and 200 R) and injected into intermediate (LBN) hosts and controls. Irradiated memory cells provided a secondary antibody response, which was delayed but not suppressed when compared to unirradiated cells. Alteration in homing of lymphocytes caused by various physical and chemical agents was a result of effects on cell membrane characteristics which controlled some aspects of the phenomenon. Radiation (100 to 200 R) may have had a similar effect or it may have resulted in the selective elimination of a population of cells. (U.S.)

  16. Electrospun poly(ε-caprolactone)/Ca-deficient hydroxyapatite nanohybrids: Microstructure, mechanical properties and cell response by murine embryonic stem cells

    International Nuclear Information System (INIS)

    Bianco, Alessandra; Di Federico, Erica; Moscatelli, Ilana; Camaioni, Antonella; Armentano, Ilaria; Campagnolo, Luisa; Dottori, Mariaserena; Kenny, Jose Maria; Siracusa, Gregorio; Gusmano, Gualtiero

    2009-01-01

    Nanohybrid scaffolds mimicking extracellular matrix are promising experimental models to study stem cell behaviour, in terms of adhesion and proliferation. In the present study, the structural characterization of a novel electrospun nanohybrid and the analysis of cell response by a highly sensitive cell type, embryonic stem (ES) cells, are investigated. Ca-deficient hydroxyapatite nanocrystals (d-HAp) were synthesized by precipitation. Fibrous PCL/d-HAp nanohybrids were obtained by electrospinning, d-HAp content ranging between 2 and 55 wt.%. Electrospun mats showed a non-woven architecture, average fiber size was 1.5 ±0.5 μm, porosity 80-90%, and specific surface area 16 m 2 g -1 . Up to 6.4 wt.% d-HAp content, the nanohybrids displayed comparable microstructural, mechanical and dynamo-mechanical properties. Murine ES cell response to neat PCL and to nanohybrid PCL/d-HAp (6.4 wt.%) mats was evaluated by analyzing morphological, metabolic and functional markers. Cells growing on either scaffold proliferated and maintained pluripotency markers at essentially the same rate as cells growing on standard tissue culture plates with no detectable signs of cytotoxicity, despite a lower cell adhesion at the beginning of culture. These results indicate that electrospun PCL scaffolds may provide adequate supports for murine ES cell proliferation in a pluripotent state, and that the presence of d-HAp within the mat does not interfere with their growth.

  17. Peritoneal fluid reduces angiogenesis-related microRNA expression in cell cultures of endometrial and endometriotic tissues from women with endometriosis.

    Directory of Open Access Journals (Sweden)

    Aitana Braza-Boïls

    Full Text Available UNLABELLED: Endometriosis, defined as the presence of endometrium outside the uterus, is one of the most frequent gynecological diseases. It has been suggested that modifications of both endometrial and peritoneal factors could be implicated in this disease. Endometriosis is a multifactorial disease in which angiogenesis and proteolysis are dysregulated. MicroRNAs (miRNAs are small non-coding RNAs that regulate the protein expression and may be the main regulators of angiogenesis. Our hypothesis is that peritoneal fluid from women with endometriosis could modify the expression of several miRNAs that regulate angiogenesis and proteolysis in the endometriosis development. The objective of this study has been to evaluate the influence of endometriotic peritoneal fluid on the expression of six miRNAs related to angiogenesis, as well as several angiogenic and proteolytic factors in endometrial and endometriotic cell cultures from women with endometriosis compared with women without endometriosis. METHODS: Endometrial and endometriotic cells were cultured and treated with endometriotic and control peritoneal fluid pools. We have studied the expression of six miRNAs (miR-16, -17-5p, -20a, -125a, -221, and -222 by RT-PCR and protein and mRNA levels of vascular endothelial growth factor-A, thrombospondin-1, urokinase plasminogen activator and plasminogen activator inhibitor-1 by ELISA and qRT-PCR respectively. RESULTS: Control and endometriotic peritoneal fluid pools induced a significant reduction of all miRNAs levels in endometrial and endometriotic cell cultures. Moreover, both peritoneal fluids induced a significant increase in VEGF-A, uPA and PAI-1 protein levels in all cell cultures without significant increase in mRNA levels. Endometrial cell cultures from patients treated with endometriotic peritoneal fluid showed lower expression of miRNAs and higher expression of VEGF-A protein levels than cultures from controls. In conclusion , this "in vitro

  18. Effects of Wnt-10b on proliferation and differentiation of murine melanoma cells

    International Nuclear Information System (INIS)

    Misu, Masayasu; Ouji, Yukiteru; Kawai, Norikazu; Nishimura, Fumihiko; Nakamura-Uchiyama, Fukumi; Yoshikawa, Masahide

    2015-01-01

    In spite of the strong expression of Wnt-10b in melanomas, its role in melanoma cells has not been elucidated. In the present study, the biological effects of Wnt-10b on murine B16F10 (B16) melanoma cells were investigated using conditioned medium from Wnt-10b-producing COS cells (Wnt-CM). After 2 days of culture in the presence of Wnt-CM, proliferation of B16 melanoma cells was inhibited, whereas tyrosinase activity was increased. An in vitro wound healing assay demonstrated that migration of melanoma cells to the wound area was inhibited with the addition of Wnt-CM. Furthermore, evaluation of cellular senescence revealed prominent induction of SA-β-gal-positive senescent cells in cultures with Wnt-CM. Finally, the growth of B16 melanoma cell aggregates in collagen 3D-gel cultures was markedly suppressed in the presence of Wnt-CM. These results suggest that Wnt-10b represses tumor cell properties, such as proliferation and migration of B16 melanoma cells, driving them toward a more differentiated state along a melanocyte lineage. - Highlights: • Wnt-10b inhibited proliferation and migration of melanoma cells. • Wnt-10b induced tyrosinase activity and senescence of melanoma cells. • Wnt-10b suppressed growth of cell aggregates in collagen 3D-gel cultures. • Wnt-10b represses tumor cell properties, driving them toward a more differentiated state along a melanocyte lineage

  19. Effects of Wnt-10b on proliferation and differentiation of murine melanoma cells

    Energy Technology Data Exchange (ETDEWEB)

    Misu, Masayasu [Department of Pathogen, Infection and Immunity, Nara Medical University, 840 Shijo-cho, Kashihara, Nara 634-8521 (Japan); Ouji, Yukiteru, E-mail: oujix@naramed-u.ac.jp [Department of Pathogen, Infection and Immunity, Nara Medical University, 840 Shijo-cho, Kashihara, Nara 634-8521 (Japan); Kawai, Norikazu [Department of Pathogen, Infection and Immunity, Nara Medical University, 840 Shijo-cho, Kashihara, Nara 634-8521 (Japan); Nishimura, Fumihiko [Department of Neurosurgery, Nara Medical University, 840 Shijo-cho, Kashihara, Nara 634-8521 (Japan); Nakamura-Uchiyama, Fukumi [Department of Pathogen, Infection and Immunity, Nara Medical University, 840 Shijo-cho, Kashihara, Nara 634-8521 (Japan); Yoshikawa, Masahide, E-mail: myoshika@naramed-u.ac.jp [Department of Pathogen, Infection and Immunity, Nara Medical University, 840 Shijo-cho, Kashihara, Nara 634-8521 (Japan)

    2015-08-07

    In spite of the strong expression of Wnt-10b in melanomas, its role in melanoma cells has not been elucidated. In the present study, the biological effects of Wnt-10b on murine B16F10 (B16) melanoma cells were investigated using conditioned medium from Wnt-10b-producing COS cells (Wnt-CM). After 2 days of culture in the presence of Wnt-CM, proliferation of B16 melanoma cells was inhibited, whereas tyrosinase activity was increased. An in vitro wound healing assay demonstrated that migration of melanoma cells to the wound area was inhibited with the addition of Wnt-CM. Furthermore, evaluation of cellular senescence revealed prominent induction of SA-β-gal-positive senescent cells in cultures with Wnt-CM. Finally, the growth of B16 melanoma cell aggregates in collagen 3D-gel cultures was markedly suppressed in the presence of Wnt-CM. These results suggest that Wnt-10b represses tumor cell properties, such as proliferation and migration of B16 melanoma cells, driving them toward a more differentiated state along a melanocyte lineage. - Highlights: • Wnt-10b inhibited proliferation and migration of melanoma cells. • Wnt-10b induced tyrosinase activity and senescence of melanoma cells. • Wnt-10b suppressed growth of cell aggregates in collagen 3D-gel cultures. • Wnt-10b represses tumor cell properties, driving them toward a more differentiated state along a melanocyte lineage.

  20. Evaluation of expansile nanoparticle tumor localization and efficacy in a cancer stem cell-derived model of pancreatic peritoneal carcinomatosis

    Science.gov (United States)

    Herrera, Victoria LM; Colby, Aaron H; Tan, Glaiza AL; Moran, Ann M; O’Brien, Michael J; Colson, Yolonda L; Ruiz-Opazo, Nelson; Grinstaff, Mark W

    2016-01-01

    Aim: To evaluate the tumor localization and efficacy pH-responsive expansile nanoparticles (eNPs) as a drug delivery system for pancreatic peritoneal carcinomatosis (PPC) modeled in nude rats. Methods & materials: A Panc-1-cancer stem cell xeno1graft model of PPC was validated in vitro and in vivo. Tumor localization was tracked via in situ imaging of fluorescent eNPs. Survival of animals treated with paclitaxel-loaded eNPs (PTX-eNPs) was evaluated in vivo. Results: The Panc-1-cancer stem cell xenograft model recapitulates significant features of PPC. Rhodamine-labeled eNPs demonstrate tumor-specific, dose- and time-dependent localization to macro- and microscopic tumors following intraperitoneal injection. PTX-eNPs are as effective as free PTX in treating established PPC; but, PTX-eNPs result in fewer side effects. Conclusion: eNPs are a promising tool for the detection and treatment of PPC. PMID:27078118

  1. Reemergence of apoptotic cells between fractionated doses in irradiated murine tumors

    International Nuclear Information System (INIS)

    Meyn, R.E.; Hunter, N.R.; Milas, L.

    1994-01-01

    The purpose of this investigation was to follow up our previous studies on the development of apoptosis in irradiated murine tumors by testing whether an apoptotic subpopulation of cells reemerges between fractionated exposures. Mice bearing a murine ovarian carcinoma, OCa-I, were treated in vivo with two fractionation protocols: two doses of 12.5 Gy separated by various times out to 5 days and multiple daily fractions of 2.5 Gy. Animals were killed 4 h after the last dose in each protocol, and the percent apoptosis was scored from stained histological sections made from the irradiated tumors according to the specific features characteristic of this mode of cell death. The 12.5+12.5 Gy protocol yielded a net total percent apoptosis of about 45% when the two doses were separated by 5 days (total dose = 25 Gy), whereas the 2.5 Gy per day protocol yielded about 50% net apoptotic cells when given for 5 days (total dose = 12.5 Gy). These values are to be compared to the value of 36% apoptotic cells that is yielded by large single doses (> 25 Gy). Thus, these results indicate that an apoptotic subpopulation of cells reemerged between the fractions in both protocols, but the kinetics appeared to be delayed in the 12.5+12.5 Gy vs. the multiple 2.5 Gy protocol. This reemergence of cells with the propensity for radiation-induced apoptosis between fractionated exposures is consistent with a role for this mode of cell death in the response of tumors to radiotherapy and may represent the priming of a new subpopulation of tumor cells for apoptosis as part of normal tumor homeostasis to counterbalance cell division. 25 refs., 3 figs., 1 tab

  2. Murine but not human basophil undergoes cell-specific proteolysis of a major endoplasmic reticulum chaperone.

    Directory of Open Access Journals (Sweden)

    Bei Liu

    Full Text Available Basophil has been implicated in anti-parasite defense, allergy and in polarizing T(H2 response. Mouse model has been commonly used to study basophil function although the difference between human and mouse basophils is underappreciated. As an essential chaperone for multiple Toll-like receptors and integrins in the endoplasmic reticulum, gp96 also participates in general protein homeostasis and in the ER unfolded protein response to ensure cell survival during stress. The roles of gp96 in basophil development are unknown.We genetically delete gp96 in mice and examined the expression of gp96 in basophils by Western blot and flow cytometry. We compared the expression pattern of gp96 between human and mouse basophils.We found that gp96 was dispensable for murine basophil development. Moreover, gp96 was cleaved by serine protease(s in murine but not human basophils leading to accumulation of a nun-functional N-terminal ∼50 kDa fragment and striking induction of the unfolded protein response. The alteration of gp96 was unique to basophils and was not observed in any other cell types including mast cells. We also demonstrated that the ectopic expression of a mouse-specific tryptase mMCP11 does not lead to gp96 cleavage in human basophils.Our study revealed a remarkable biochemical event of gp96 silencing in murine but not human basophils, highlighting the need for caution in using mouse models to infer the function of basophils in human immune response. Our study also reveals a novel mechanism of shutting down gp96 post-translationally in regulating its function.

  3. Murine but not human basophil undergoes cell-specific proteolysis of a major endoplasmic reticulum chaperone.

    Science.gov (United States)

    Liu, Bei; Staron, Matthew; Li, Zihai

    2012-01-01

    Basophil has been implicated in anti-parasite defense, allergy and in polarizing T(H)2 response. Mouse model has been commonly used to study basophil function although the difference between human and mouse basophils is underappreciated. As an essential chaperone for multiple Toll-like receptors and integrins in the endoplasmic reticulum, gp96 also participates in general protein homeostasis and in the ER unfolded protein response to ensure cell survival during stress. The roles of gp96 in basophil development are unknown. We genetically delete gp96 in mice and examined the expression of gp96 in basophils by Western blot and flow cytometry. We compared the expression pattern of gp96 between human and mouse basophils. We found that gp96 was dispensable for murine basophil development. Moreover, gp96 was cleaved by serine protease(s) in murine but not human basophils leading to accumulation of a nun-functional N-terminal ∼50 kDa fragment and striking induction of the unfolded protein response. The alteration of gp96 was unique to basophils and was not observed in any other cell types including mast cells. We also demonstrated that the ectopic expression of a mouse-specific tryptase mMCP11 does not lead to gp96 cleavage in human basophils. Our study revealed a remarkable biochemical event of gp96 silencing in murine but not human basophils, highlighting the need for caution in using mouse models to infer the function of basophils in human immune response. Our study also reveals a novel mechanism of shutting down gp96 post-translationally in regulating its function.

  4. Angiogenesis gene expression in murine endothelial cells during post-pneumonectomy lung growth

    Directory of Open Access Journals (Sweden)

    Konerding Moritz A

    2011-07-01

    Full Text Available Abstract Although blood vessel growth occurs readily in the systemic bronchial circulation, angiogenesis in the pulmonary circulation is rare. Compensatory lung growth after pneumonectomy is an experimental model with presumed alveolar capillary angiogenesis. To investigate the genes participating in murine neoalveolarization, we studied the expression of angiogenesis genes in lung endothelial cells. After left pneumonectomy, the remaining right lung was examined on days 3, 6, 14 and 21days after surgery and compared to both no surgery and sham thoracotomy controls. The lungs were enzymatically digested and CD31+ endothelial cells were isolated using flow cytometry cell sorting. The transcriptional profile of the CD31+ endothelial cells was assessed using quantitative real-time polymerase chain reaction (PCR arrays. Focusing on 84 angiogenesis-associated genes, we identified 22 genes with greater than 4-fold regulation and significantly enhanced transcription (p

  5. Proteomic analysis of erythroid differentiation induced by hexamethylene bisacetamide in murine erythroleukemia cells

    Czech Academy of Sciences Publication Activity Database

    Petrák, J.; Myslivcová, D.; Man, Petr; Čmejlová, J.; Čmejla, R.; Vyoral, D.

    2007-01-01

    Roč. 35, - (2007), s. 193-202 ISSN 0301-472X R&D Projects: GA MŠk LC545 Grant - others:CZ(CZ) 023736; GA ČR(CZ) GA303/04/0003; GA MŠk(CZ) LC06044 Institutional research plan: CEZ:AV0Z50200510 Source of funding: V - iné verejné zdroje ; V - iné verejné zdroje ; V - iné verejné zdroje Keywords : murine erythroleukemia cells * erythroid differentiation * hexamethylene bisacetamide Subject RIV: EE - Microbiology, Virology Impact factor: 3.147, year: 2007

  6. Bifidobacterium bifidum Actively Changes the Gene Expression Profile Induced by Lactobacillus acidophilus in Murine Dendritic Cells

    DEFF Research Database (Denmark)

    Weiss, Gudrun Margarethe; Rasmussen, Simon; Fink, Lisbeth Nielsen

    2010-01-01

    Dendritic cells (DC) play a pivotal regulatory role in activation of both the innate as well as the adaptive immune system by responding to environmental microorganisms. We have previously shown that Lactobacillus acidophilus induces a strong production of the pro-inflammatory and Th1 polarizing...... cytokine IL-12 in DC, whereas bifidobacteria do not induce IL-12 but inhibit the IL-12 production induced by lactobacilli. In the present study, genome-wide microarrays were used to investigate the gene expression pattern of murine DC stimulated with Lactobacillus acidophilus NCFM and Bifidobacterium...

  7. Contemplating the murine test tube: lessons from natural killer cells and Cryptococcus neoformans.

    Science.gov (United States)

    Marr, Kaleb J; Jones, Gareth J; Mody, Christopher H

    2006-06-01

    Murine experimentation has provided many useful tools, including the ability to knockout or over-express genes and to perform experiments that are limited by ethical considerations. Over the past century, mice have imparted valuable insights into the biology of many systems, including human immunity. However, although there are many similarities between the immune response of humans and mice, there are also many differences; none is more prominent than when examining natural killer cell biology. These differences include tissue distribution, effector molecules, receptor repertoire, and cytokine responses, all of which have important implications when extrapolating the studies to the human immune responses to Cryptococcus neoformans.

  8. γ-ray dose rate effect in DNA double-strand break repair deficient murine cells

    International Nuclear Information System (INIS)

    Li Liya; Li Peiwen

    2002-01-01

    Objective: To analyze the dose rate effect and potentially lethal damage repair in DNA double-strand break repair deficient murine cells (SCID) irradiated by γ-ray. Methods: The wild type (CB.17+/+) and SCID cells were exposed to γ-ray at high and low dose rates. The high dose rate exposure was fractionated into two equal doses at 24 h intervals. The survival rates of irradiated cells were calculated by clone-forming analysis. Results: When γ-ray was given to wild type (CB.17+/+) cells in two fractions at 24 h intervals, the survival rate was significantly higher than that when the same total dose was given singly. In contrast, there was no difference in the survival rates between the single and fractionated exposure in SCID cells. SCID cells were more sensitive than CB.17+/+ cells to both low and high dose rates γ-ray exposure for cell killing. The survival rate by low dose rate exposure was significantly higher than that by high dose rate exposure, not only in CB.17+/+ cells but also in SCID cells. Conclusions: SCID cells are deficient in repairing γ-ray induced double-strand breaks. There is dose rate effect in both SCID and CB.17+/+ cells

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

    Science.gov (United States)

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

    2015-01-01

    Stem cell (SC) division, deployment, and differentiation are processes that contribute to corneal epithelial renewal. Until now studying the destiny of these cells in a living mammal has not been possible. However, the advent of inducible multicolor genetic tagging and powerful imaging technologies has rendered this achievable in the translucent and readily accessible murine cornea. K14CreER(T2)-Confetti mice that harbor two copies of the Brainbow 2.1 cassette, yielding up to 10 colors from the stochastic recombination of fluorescent proteins, were used to monitor K-14(+) progenitor cell dynamics within the corneal epithelium in live animals. Multicolored columns of cells emerged from the basal limbal epithelium as they expanded and migrated linearly at a rate of 10.8 µm/day toward the central cornea. Moreover, the permanent expression of fluorophores, passed on from progenitor to progeny, assisted in discriminating individual clones as spectrally distinct streaks containing more than 1,000 cells within the illuminated area. The centripetal clonal expansion is suggestive that a single progenitor cell is responsible for maintaining a narrow corridor of corneal epithelial cells. Our data are in agreement with the limbus as the repository for SC as opposed to SC being distributed throughout the central cornea. This is the first report describing stem/progenitor cell fate determination in the murine cornea using multicolor genetic tracing. This model represents a powerful new resource to monitor SC kinetics and fate choice under homeostatic conditions, and may assist in assessing clonal evolution during corneal development, aging, wound-healing, disease, and following transplantation. © 2014 AlphaMed Press.

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

    Energy Technology Data Exchange (ETDEWEB)

    Morizane, Ryuji [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); Itoh, Hiroshi [Department of Internal Medicine, Keio University School of Medicine, Tokyo (Japan)

    2009-12-25

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

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

    International Nuclear Information System (INIS)

    Lepore, D.A.; Roeszler, K.; Wagner, J.; Ross, S.A.; Bauer, K.; Thomas, P.Q.

    2005-01-01

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

  12. Phenotypic correction of Fanconi anemia cells in the murine bone marrow after carrier cell mediated delivery of lentiviral vector.

    Science.gov (United States)

    Chakkaramakkil Verghese, Santhosh; Goloviznina, Natalya A; Kurre, Peter

    2016-11-19

    Fanconi anemia (FA) is an autosomal-recessive disorder associated with hematopoietic failure and it is a candidate for hematopoietic stem cell (HSC)-directed gene therapy. However, the characteristically reduced HSC numbers found in FA patients, their ineffective mobilization from the marrow, and re-oxygenation damage during ex vivo manipulation have precluded clinical success using conventional in vitro approaches. We previously demonstrated that lentiviral vector (LV) particles reversibly attach to the cell surface where they gain protection from serum complement neutralization. We reasoned that cellular delivery of LV to the bone marrow niche could avoid detrimental losses during FA HSC mobilization and in vitro modification. Here, we demonstrate that a VSV-G pseudotyped lentivector, carrying the FANCC transgene, can be transmitted from carrier to bystander cells. In cell culture and transplantation models of FA, we further demonstrate that LV carrier cells migrate along SDF-1α gradients and transfer vector particles that stably integrate and phenotypically correct the characteristic DNA alkylator sensitivity in murine and human FA-deficient target bystander cells. Altogether, we demonstrate that cellular homing mechanisms can be harnessed for the functional phenotype correction in murine FA hematopoietic cells.

  13. Phenotypic correction of Fanconi anemia cells in the murine bone marrow after carrier cell mediated delivery of lentiviral vector

    Directory of Open Access Journals (Sweden)

    Santhosh Chakkaramakkil Verghese

    2016-11-01

    Full Text Available Abstract Fanconi anemia (FA is an autosomal-recessive disorder associated with hematopoietic failure and it is a candidate for hematopoietic stem cell (HSC-directed gene therapy. However, the characteristically reduced HSC numbers found in FA patients, their ineffective mobilization from the marrow, and re-oxygenation damage during ex vivo manipulation have precluded clinical success using conventional in vitro approaches. We previously demonstrated that lentiviral vector (LV particles reversibly attach to the cell surface where they gain protection from serum complement neutralization. We reasoned that cellular delivery of LV to the bone marrow niche could avoid detrimental losses during FA HSC mobilization and in vitro modification. Here, we demonstrate that a VSV-G pseudotyped lentivector, carrying the FANCC transgene, can be transmitted from carrier to bystander cells. In cell culture and transplantation models of FA, we further demonstrate that LV carrier cells migrate along SDF-1α gradients and transfer vector particles that stably integrate and phenotypically correct the characteristic DNA alkylator sensitivity in murine and human FA-deficient target bystander cells. Altogether, we demonstrate that cellular homing mechanisms can be harnessed for the functional phenotype correction in murine FA hematopoietic cells.

  14. Interplay between Misplaced Müllerian-Derived Stem Cells and Peritoneal Immune Dysregulation in the Pathogenesis of Endometriosis

    Directory of Open Access Journals (Sweden)

    Antonio Simone Laganà

    2013-01-01

    Full Text Available In the genetic regulation of Müllerian structures development, a key role is played by Hoxa and Wnt clusters, because they lead the transcription of different genes according to the different phases of the organogenesis, addressing correctly cell-to-cell interactions, allowing, finally, the physiologic morphogenesis. Accumulating evidence is suggesting that dysregulation of Wnt and/or Hox genes may affect cell migration during organogenesis and differentiation of Müllerian structures of the female reproductive tract, with possible dislocation and dissemination of primordial endometrial stem cells in ectopic regions, which have high plasticity to differentiation. We hypothesize that during postpubertal age, under the influence of different stimuli, these misplaced and quiescent ectopic endometrial cells could acquire new phenotype, biological functions, and immunogenicity. So, these kinds of cells may differentiate, specializing in epithelium, glands, and stroma to form a functional ectopic endometrial tissue. This may provoke a breakdown in the peritoneal cavity homeostasis, with the consequent processes of immune alteration, documented by peripheral mononuclear cells recruitment and secretion of inflammatory cytokines in early phases and of angiogenic and fibrogenic cytokines in the late stages of the disease.

  15. Interplay between Misplaced Müllerian-Derived Stem Cells and Peritoneal Immune Dysregulation in the Pathogenesis of Endometriosis

    Science.gov (United States)

    Sturlese, Emanuele; Retto, Giovanni; Sofo, Vincenza; Triolo, Onofrio

    2013-01-01

    In the genetic regulation of Müllerian structures development, a key role is played by Hoxa and Wnt clusters, because they lead the transcription of different genes according to the different phases of the organogenesis, addressing correctly cell-to-cell interactions, allowing, finally, the physiologic morphogenesis. Accumulating evidence is suggesting that dysregulation of Wnt and/or Hox genes may affect cell migration during organogenesis and differentiation of Müllerian structures of the female reproductive tract, with possible dislocation and dissemination of primordial endometrial stem cells in ectopic regions, which have high plasticity to differentiation. We hypothesize that during postpubertal age, under the influence of different stimuli, these misplaced and quiescent ectopic endometrial cells could acquire new phenotype, biological functions, and immunogenicity. So, these kinds of cells may differentiate, specializing in epithelium, glands, and stroma to form a functional ectopic endometrial tissue. This may provoke a breakdown in the peritoneal cavity homeostasis, with the consequent processes of immune alteration, documented by peripheral mononuclear cells recruitment and secretion of inflammatory cytokines in early phases and of angiogenic and fibrogenic cytokines in the late stages of the disease. PMID:23843796

  16. Suppressed histamine release from rat peritoneal mast cells by ultraviolet B irradiation: decreased diacylglycerol formation as a possible mechanism

    International Nuclear Information System (INIS)

    Danno, K.; Fujii, K.; Tachibana, T.; Toda, K.; Horio, T.

    1988-01-01

    This study was designed to investigate the effect of ultraviolet B (UVB) irradiation on mast cell functions. Purified mast cells obtained from rat peritoneal cavity were irradiated with UVB and subsequently exposed to a degranulator, compound 48/80, or the calcium ionophore A-23187. The amount of histamine released from mast cells measured by the enzyme isotopic assay was significantly decreased by UVB irradiation (100-400 mJ/cm2). Within this dose range, UVB alone was not cytotoxic to the cells because it did not induce histamine release. The suppression was observed when mast cells were subjected to degranulation without intervals after UVB irradiation, and even after 5 h postirradiation. The wavelength of 300 nm from a monochromatic light source showed the maximum effect. When mast cells prelabeled with [ 3 H]arachidonate were irradiated and challenged by compound 48/80, label accumulation in diacylglycerol produced by the phosphatidylinositol cycle was considerably decreased by UVB irradiation. From these results, we hypothesize that, within an adequate irradiation dose, UVB irradiation suppresses histamine release from mast cells, probably by causing noncytotoxic damage to the membrane phospholipid metabolism, which is tied to the degranulation mechanisms

  17. Meconium peritonitis

    International Nuclear Information System (INIS)

    Kuroda, Tatsuo; Honna, Toshiro; Morikawa, Nobuyuki

    2008-01-01

    Recent advances in imaging the fetus in uterine have brought about the knowledge of prenatal process of the meconium peritonitis (mp), a chemical peritonitis by intestinal perforation, which was unknown hitherto. This paper describes the retrospective examination of perinatal diagnosis, treatment and management of mp essentially based on authors' experiences. Subjects are 18 fetuses (6.9%) with mp or suspicious mp/260 with possible diseases necessary for surgery during the period Mar. 2002-Dec. 2007. All subjects received ultrasonography (US) and MR imaging and were diagnosed as mp in 12 cases (66.7%). Seven cases were found to be in the fibroadhesive type (FA), 4 in cystic type (C) and 1 in generalized type (G), and neonates with the second and third types tended to be resulted in premature birth. The causes were found by laparotomy to be due to intestinal atresia (10 cases, 83.3%), atresia complicated with aproctia (1) and midgut axial volvulus (1). FA underwent intestinal anastomosis, and C and G, peritoneal drainage followed by anastomosis. For more appropriate perinatal management and possible prenatal surgery, needed is further elucidation of clinical features and pathophysiology of the disease. (R.T.)

  18. Suppression of NK cells and regulatory T lymphocytes in cats naturally infected with feline infectious peritonitis virus.

    Science.gov (United States)

    Vermeulen, Ben L; Devriendt, Bert; Olyslaegers, Dominique A; Dedeurwaerder, Annelike; Desmarets, Lowiese M; Favoreel, Herman W; Dewerchin, Hannah L; Nauwynck, Hans J

    2013-05-31

    A strong cell-mediated immunity (CMI) is thought to be indispensable for protection against infection with feline infectious peritonitis virus (FIPV) in cats. In this study, the role of natural killer (NK) cells and regulatory T cells (Tregs), central players in the innate and adaptive CMI respectively, was examined during natural FIPV infection. When quantified, both NK cells and Tregs were drastically depleted from the peripheral blood, mesenteric lymph node (LN) and spleen in FIP cats. In contrast, mesentery and kidney from FIP cats did not show any difference when compared to healthy non-infected control animals. In addition, other regulatory lymphocytes (CD4+CD25-Foxp3+ and CD3+CD8+Foxp3+) were found to be depleted from blood and LN as well. Phenotypic analysis of blood-derived NK cells in FIP cats revealed an upregulation of activation markers (CD16 and CD25) and migration markers (CD11b and CD62L) while LN-derived NK cells showed upregulation of only CD16 and CD62L. LN-derived NK cells from FIPV-infected cats were also significantly less cytotoxic when compared with healthy cats. This study reveals for the first time that FIPV infection is associated with severe suppression of NK cells and Tregs, which is reflected by cell depletion and lowered cell functionality (only NK cells). This will un-doubtfully lead to a reduced capacity of the innate immune system (NK cells) to battle FIPV infection and a decreased capacity (Tregs) to suppress the immunopathology typical for FIP. However, these results will also open possibilities for new therapies targeting specifically NK cells and Tregs to enhance their numbers and/or functionality during FIPV infection. Copyright © 2013 Elsevier B.V. All rights reserved.

  19. Identification of murine T-cell epitopes in Ebola virus nucleoprotein

    International Nuclear Information System (INIS)

    Simmons, Graham; Lee, Anee; Rennekamp, Andrew J.; Fan Xin; Bates, Paul; Shen Hao

    2004-01-01

    CD8 T cells play an important role in controlling Ebola infection and in mediating vaccine-induced protective immunity, yet little is known about antigenic targets in Ebola that are recognized by CD8 T cells. Overlapping peptides were used to identify major histocompatibility complex class I-restricted epitopes in mice immunized with vectors encoding Ebola nucleoprotein (NP). CD8 T-cell responses were mapped to a H-2 d -restricted epitope (NP279-288) and two H-2 b -restricted epitopes (NP44-52 and NP288-296). The identification of these epitopes will facilitate studies of immune correlates of protection and the evaluation of vaccine strategies in murine models of Ebola infection

  20. Antigen-specific murine T cell clones produce soluble interleukin 2 receptor on stimulation with specific antigens

    International Nuclear Information System (INIS)

    Wagner, D.K.; York-Jolley, J.; Malek, T.R.; Berzofsky, J.A.; Nelson, D.L.

    1986-01-01

    In this study, monoclonal antibodies were used to the murine IL 2 receptor (IL 2R) termed 3C7 and 7D4, which bind to different epitopes on the murine IL 2R, to develop an ELISA to measure soluble murine IL 2R. Surprisingly, stimulated murine spleen cells not only expressed cell-associated IL 2R, but also produced a considerable level of cellfree IL 2R in the culture supernatant fluid. To assess the fine specificity of this response, myoglobin-immune murine T cell clones were stimulated with appropriate or inappropriate antigen and syngeneic or allogeneic presenting cells. Proliferation, measured by [ 3 H] thymidine incorporation, and levels of soluble IL 2R were determined at day 4. The production of soluble IL2R displayed the same epitope fine specificity, genetic restriction, and antigen dose-response as the proliferative response. Indeed, in some cases there was sharper discrimination of epitope specificity and genetic restriction with the soluble IL 2R levels. There was also reproducible clone-to-clone variation in the amount of soluble receptor produced in response to antigen among 12 T cell clones and lines tested. In time course experiments, proliferation was greatest at day 3, whereas soluble IL 2R levels continued to rise in subsequent days. To the authors' knowledge, this is the first demonstration of release of secretion of soluble IL 2R by murine T cells, and the first demonstration of the fine specificity and genetic restriction of the induction of soluble IL 2R by specific antigen

  1. Acute and delayed effect of (-) deprenyl and (-) 1-phenyl-2-propylaminopentane (PPAP) on the serotonin content of peritoneal cells (white blood cells and mast cells).

    Science.gov (United States)

    Csaba, G; Kovács, P; Pállinger, Eva

    2006-01-01

    Acute and delayed (hormonal imprinting) effect of (-) deprenyl and its derivative without MAO-B inhibitory activity (-) PPAP, were studied on cells of the peritoneal fluid (lymphocytes, monocytes, granulocytes and mast cells) by flow cytometric and confocal microscopic analysis. Thirty minutes after treatment of 6-week-old female animals, deprenyl was ineffective while PPAP significantly increased the serotonin level of these cells. Three weeks after treatment at weaning, deprenyl drastically decreased the serotonin level of each cell type, while PPAP moderately but significantly increased the serotonin level of monocytes, granulocytes and mast cells. This means that the two related molecules have different effects on the immune cells, which seem to be independent of MAO-B inhibition. The experiments emphasize the necessity of studying the prolonged effects of biologically active molecules, even if they are without acute effects. As serotonin is a modulator of the immune system, the influence on immune cells of the molecules studied can contribute to their enhancing effect. Copyright 2004 John Wiley & Sons, Ltd.

  2. Peritoneal Fluid Analysis

    Science.gov (United States)

    ... Get Tested? To help diagnose the cause of peritonitis, an inflammation of the membrane lining the abdomen, ... fever and your healthcare practitioner suspects you have peritonitis or ascites Sample Required? A peritoneal fluid sample ...

  3. Dual effect of LPS on murine myeloid leukemia cells: Pro-proliferation and anti-proliferation

    Energy Technology Data Exchange (ETDEWEB)

    Yu, Lingling [Department of Pediatrics, Jingjiang People' s Hospital, Yangzhou University, Jingjiang 214500 (China); Noncoding RNA Center, Yangzhou University, Yangzhou 225001 (China); Zhao, Yingmin [Department of Pediatrics, Jingjiang People' s Hospital, Yangzhou University, Jingjiang 214500 (China); Gu, Xin; Wang, Jijun; Pang, Lei; Zhang, Yanqing; Li, Yaoyao; Jia, Xiaoqin; Wang, Xin [Noncoding RNA Center, Yangzhou University, Yangzhou 225001 (China); Gu, Jian [Department of Hematology, Yangzhou University School of Clinical Medicine, Yangzhou 225001 (China); Yu, Duonan, E-mail: duonan@yahoo.com [Department of Pediatrics, Jingjiang People' s Hospital, Yangzhou University, Jingjiang 214500 (China); Noncoding RNA Center, Yangzhou University, Yangzhou 225001 (China); Jiangsu Key Laboratory of Integrated Traditional Chinese and Western Medicine for Prevention and Treatment of Senile Disease, Yangzhou 225001 (China); Institute of Comparative Medicine, Yangzhou University, Yangzhou 225001 (China); Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Disease and Zoonosis, Yangzhou 225001 (China)

    2016-06-10

    Modification of the bone marrow microenvironment is considered as a promising strategy to control leukemic cell proliferation, diseases progression and relapse after treatment. However, due to the diversity and complexity of the cellular and molecular compartments in the leukemic microenvironment, it is extremely difficult to dissect the role of each individual molecule or cell type in vivo. Here we established an in vitro system to dissect the role of lipopolysaccharide (LPS), stromal cells and endothelial cells in the growth of mouse myeloid tumor cells and B-lymphoma cells. We found that either LPS or bone marrow stromal cells as a feeder layer in culture is required for the proliferation of myeloid tumor cells. Surprisingly, the growth of myeloid leukemic cells on stromal cells is strongly inhibited when coupled with LPS in culture. This opposing effect of LPS, a complete switch from pro-proliferation to antitumor growth is due, at least in part, to the rapidly increased production of interleukin 12, Fas ligand and tissue inhibitor of metalloproteinases-2 from stromal cells stimulated by LPS. These results demonstrate that LPS can either facilitate or attenuate tumor cell proliferation, thus changing the disease course of myeloid leukemias through its direct effect or modulation of the tumor microenvironment. - Highlights: • LPS alone in culture is required for the proliferation of murine myeloid tumor cells. • Bone marrow stromal cells as a feeder layer is also required for the proliferation of myeloid tumor cells. • However, the growth of myeloid tumor cells is inhibited when LPS and stromal cells are both available in culture. • Thus LPS can either facilitate or attenuate tumor growth through its direct effect or modulation of tumor microenvironment.

  4. Dual effect of LPS on murine myeloid leukemia cells: Pro-proliferation and anti-proliferation

    International Nuclear Information System (INIS)

    Yu, Lingling; Zhao, Yingmin; Gu, Xin; Wang, Jijun; Pang, Lei; Zhang, Yanqing; Li, Yaoyao; Jia, Xiaoqin; Wang, Xin; Gu, Jian; Yu, Duonan

    2016-01-01

    Modification of the bone marrow microenvironment is considered as a promising strategy to control leukemic cell proliferation, diseases progression and relapse after treatment. However, due to the diversity and complexity of the cellular and molecular compartments in the leukemic microenvironment, it is extremely difficult to dissect the role of each individual molecule or cell type in vivo. Here we established an in vitro system to dissect the role of lipopolysaccharide (LPS), stromal cells and endothelial cells in the growth of mouse myeloid tumor cells and B-lymphoma cells. We found that either LPS or bone marrow stromal cells as a feeder layer in culture is required for the proliferation of myeloid tumor cells. Surprisingly, the growth of myeloid leukemic cells on stromal cells is strongly inhibited when coupled with LPS in culture. This opposing effect of LPS, a complete switch from pro-proliferation to antitumor growth is due, at least in part, to the rapidly increased production of interleukin 12, Fas ligand and tissue inhibitor of metalloproteinases-2 from stromal cells stimulated by LPS. These results demonstrate that LPS can either facilitate or attenuate tumor cell proliferation, thus changing the disease course of myeloid leukemias through its direct effect or modulation of the tumor microenvironment. - Highlights: • LPS alone in culture is required for the proliferation of murine myeloid tumor cells. • Bone marrow stromal cells as a feeder layer is also required for the proliferation of myeloid tumor cells. • However, the growth of myeloid tumor cells is inhibited when LPS and stromal cells are both available in culture. • Thus LPS can either facilitate or attenuate tumor growth through its direct effect or modulation of tumor microenvironment.

  5. Functional Heterogeneity in the CD4+ T Cell Response to Murine γ-Herpesvirus 68

    Science.gov (United States)

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

    2015-01-01

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

  6. Bone marrow-derived cultured mast cells and peritoneal mast cells as targets of a growth activity secreted by BALB/3T3 fibroblasts

    International Nuclear Information System (INIS)

    Jozaki, K.; Kuriu, A.; Hirota, S.; Onoue, H.; Ebi, Y.; Adachi, S.; Ma, J.Y.; Tarui, S.; Kitamura, Y.

    1991-01-01

    When fibroblast cell lines were cultured in contact with bone marrow-derived cultured mast cells (CMC), both NIH/3T3 and BALB/3T3 cell lines supported the proliferation of CMC. In contrast, when contact between fibroblasts and CMC was prohibited by Biopore membranes or soft agar, only BALB/3T3 fibroblasts supported CMC proliferation, suggesting that BALB/3T3 but not NIH/3T3 cells secreted a significant amount of a mast cell growth activity. Moreover, the BALB/3T3-derived growth activity induced the incorporation of [3H]thymidine by CMC and the clonal growth of peritoneal mast cells in methylcellulose. The mast cell growth activity appeared to be different from interleukin 3 (IL-3) and interleukin 4 (IL-4), because mRNAs for these interleukins were not detectable in BALB/3T3 fibroblasts. Although mast cells are genetically deficient in tissues of W/Wv mice, CMC did develop when bone marrow cells of W/Wv mice were cultured with pokeweed mitogen-stimulated spleen cell-conditioned medium. Because BALB/3T3 fibroblast-conditioned medium (BALB-FCM) did not induce the incorporation of [3H]thymidine by W/Wv CMC, the growth activity in BALB-FCM appeared to be a ligand for the receptor encoded by the W (c-kit) locus. Because CMC and peritoneal mast cells are obtained as homogeneous suspensions rather easily, these cells may be potentially useful as targets for the fibroblast-derived mast cell growth activity

  7. A novel in vivo adjuvant activity of kaempferol: enhanced Tbx-21, GATA-3 expression and peritoneal CD11c+MHCII+ dendritic cell infiltration.

    Science.gov (United States)

    Singh, Divya; Tanwar, Himanshi; Das, Sudeshna; Ganju, Lilly; Singh, Shashi Bala

    2018-02-28

    Kaempferol, a natural flavonol present in various traditional medicinal plants, is known to possess potent anti-inflammatory properties. This study was designed to study the adjuvant effect of kaempferol administration along with ovalbumin antigen (K + O) in balb/c mice. Mice were immunized with kaempferol (100 and 50 mg/kg body weight) without or with ovalbumin (20 µg/mouse). After priming, booster was administered on day 21. Antigen specific IgG titers and its subtypes, on day 28, were estimated by indirect ELISA. Effect of kaempferol administration on CD11c + MHCII + peritoneal dendritic cells was studied by flow cytometry. Expression levels of proteins Tbx21, GATA-3, BLIMP-1, Caspase-1 and Oct-2 were studied by western blotting. LPS activated IL-1β production by peritoneal cells of immunized mice was estimated by sandwich ELISA. Ovalbumin specific IgG, IgG1 and IgG2a antibody titers in sera samples of K + O immunized mice increased significantly (p Kaempferol increased the infiltration of peritoneal CD11c + MHCII + dendritic cells but failed to enhance LPS activated IL-1β by peritoneal macrophages and suppressed caspase-1 protein expression as compared to that in ovalbumin immunized mice. Present study strongly demonstrates the novel adjuvant activity of kaempferol in vivo and its potential as an immunostimulatory agent.

  8. Peritoneal Dialysis and Retroperitoneal Laparoscopic Radical Nephrectomy: A Favorable Experience With a Patient Complicated by Renal Cell Carcinoma

    Directory of Open Access Journals (Sweden)

    Reika Imai

    2017-12-01

    Full Text Available Peritoneal dialysis (PD is an accepted modality for managing end-stage kidney disease. We herein report a 75-year-old female patient on chronic PD who was complicated by renal cell carcinoma. She was successfully treated with retroperitoneal laparoscopic radical nephrectomy followed by a prompt resumption of the procedure. Various surgeries disturbing the abdominal wall integrity often disrupt the regular PD schedule, and using minimally invasive approaches is therefore an attractive therapeutic option. Our experience emphasizes the feasibility and safety of a retroperitoneal approach–based laparoscopic technique based on several empirical examples. However, systemic studies on this topic are obviously lacking, so we strongly recommend the accumulation of more cases similar to our own. Several surgical concerns that need to be dealt with among PD patients are also discussed.

  9. Dendritic cell-based vaccination in cancer: therapeutic implications emerging from murine models

    Directory of Open Access Journals (Sweden)

    Soledad eMac Keon

    2015-05-01

    Full Text Available Dendritic cells (DCs play a pivotal role in the orchestration of immune responses, and are thus key targets in cancer vaccine design. Since the 2010 FDA approval of the first cancer DC-based vaccine (Sipuleucel T there has been a surge of interest in exploiting these cells as a therapeutic option for the treatment of tumors of diverse origin. In spite of the encouraging results obtained in the clinic, many elements of DC-based vaccination strategies need to be optimized. In this context, the use of experimental cancer models can help direct efforts towards an effective vaccine design. This paper reviews recent findings in murine models regarding the antitumoral mechanisms of DC-based vaccination, covering issues related to antigen sources, the use of adjuvants and maturing agents, and the role of DC subsets and their interaction in the initiation of antitumoral immune responses. The summary of such diverse aspects will highlight advantages and drawbacks in the use of murine models, and contribute to the design of successful DC-based translational approaches for cancer treatment.

  10. Mesenchymal stem cells ameliorate the histopathological changes in a murine model of chronic asthma.

    Science.gov (United States)

    Firinci, Fatih; Karaman, Meral; Baran, Yusuf; Bagriyanik, Alper; Ayyildiz, Zeynep Arikan; Kiray, Muge; Kozanoglu, Ilknur; Yilmaz, Osman; Uzuner, Nevin; Karaman, Ozkan

    2011-08-01

    Asthma therapies are effective in reducing inflammation but airway remodeling is poorly responsive to these agents. New therapeutic options that have fewer side effects and reverse chronic changes in the lungs are essential. Mesenchymal stem cells (MSCs) are promising for the development of novel therapies in regenerative medicine. This study aimed to examine the efficacy of MSCs on lung histopathology in a murine model of chronic asthma. BALB/c mice were divided into four groups: Group 1 (control group, n=6), Group 2 (ovalbumin induced asthma only, n=10), Group 3 (ovalbumin induced asthma + MSCs, n=10), and Group 4 (MSCs only, n=10). Histological findings (basement membrane, epithelium, subepithelial smooth muscle thickness, numbers of goblet and mast cells) of the airways and MSC migration were evaluated by light, electron, and confocal microscopes. In Group 3, all early histopathological changes except epithelial thickness and all of the chronic changes were significantly ameliorated when compared with Group 2. Evaluation with confocal microscopy showed that no noteworthy amount of MSCs were present in the lung tissues of Group 4 while significant amount of MSCs was detected in Group 3. Serum NO levels in Group 3, were significantly lower than Group 2. The results of this study revealed that MSCs migrated to lung tissue and ameliorated bronchial asthma in murine model. Further studies are needed to evaluate the efficacy of MSCs for the treatment of asthma. Copyright © 2011 Elsevier B.V. All rights reserved.

  11. Effects of murine and human bone marrow-derived mesenchymal stem cells on cuprizone induced demyelination.

    Directory of Open Access Journals (Sweden)

    Jasmin Nessler

    Full Text Available For the treatment of patients with multiple sclerosis there are no regenerative approaches to enhance remyelination. Mesenchymal stem cells (MSC have been proposed to exert such regenerative functions. Intravenous administration of human MSC reduced the clinical severity of experimental autoimmune encephalomyelitis (EAE, an animal model mimicking some aspects of multiple sclerosis. However, it is not clear if this effect was achieved by systemic immunomodulation or if there is an active neuroregeneration in the central nervous system (CNS. In order to investigate remyelination and regeneration in the CNS we analysed the effects of intravenously and intranasally applied murine and human bone marrow-derived MSC on cuprizone induced demyelination, a toxic animal model which allows analysis of remyelination without the influence of the peripheral immune system. In contrast to EAE no effects of MSC on de- and remyelination and glial cell reactions were found. In addition, neither murine nor human MSC entered the lesions in the CNS in this toxic model. In conclusion, MSC are not directed into CNS lesions in the cuprizone model where the blood-brain-barrier is intact and thus cannot provide support for regenerative processes.

  12. A Murine Herpesvirus Closely Related to Ubiquitous Human Herpesviruses Causes T-Cell Depletion.

    Science.gov (United States)

    Patel, Swapneel J; Zhao, Guoyan; Penna, Vinay R; Park, Eugene; Lauron, Elvin J; Harvey, Ian B; Beatty, Wandy L; Plougastel-Douglas, Beatrice; Poursine-Laurent, Jennifer; Fremont, Daved H; Wang, David; Yokoyama, Wayne M

    2017-05-01

    The human roseoloviruses human herpesvirus 6A (HHV-6A), HHV-6B, and HHV-7 comprise the Roseolovirus genus of the human Betaherpesvirinae subfamily. Infections with these viruses have been implicated in many diseases; however, it has been challenging to establish infections with roseoloviruses as direct drivers of pathology, because they are nearly ubiquitous and display species-specific tropism. Furthermore, controlled study of infection has been hampered by the lack of experimental models, and until now, a mouse roseolovirus has not been identified. Herein we describe a virus that causes severe thymic necrosis in neonatal mice, characterized by a loss of CD4 + T cells. These phenotypes resemble those caused by the previously described mouse thymic virus (MTV), a putative herpesvirus that has not been molecularly characterized. By next-generation sequencing of infected tissue homogenates, we assembled a contiguous 174-kb genome sequence containing 128 unique predicted open reading frames (ORFs), many of which were most closely related to herpesvirus genes. Moreover, the structure of the virus genome and phylogenetic analysis of multiple genes strongly suggested that this virus is a betaherpesvirus more closely related to the roseoloviruses, HHV-6A, HHV-6B, and HHV-7, than to another murine betaherpesvirus, mouse cytomegalovirus (MCMV). As such, we have named this virus murine roseolovirus (MRV) because these data strongly suggest that MRV is a mouse homolog of HHV-6A, HHV-6B, and HHV-7. IMPORTANCE Herein we describe the complete genome sequence of a novel murine herpesvirus. By sequence and phylogenetic analyses, we show that it is a betaherpesvirus most closely related to the roseoloviruses, human herpesviruses 6A, 6B, and 7. These data combined with physiological similarities with human roseoloviruses collectively suggest that this virus is a murine roseolovirus (MRV), the first definitively described rodent roseolovirus, to our knowledge. Many biological and

  13. Adult Murine Skeletal Muscle Contains Cells That Can Differentiate into Beating Cardiomyocytes In Vitro

    Directory of Open Access Journals (Sweden)

    Winitsky Steve O

    2005-01-01

    Full Text Available It has long been held as scientific fact that soon after birth, cardiomyocytes cease dividing, thus explaining the limited restoration of cardiac function after a heart attack. Recent demonstrations of cardiac myocyte differentiation observed in vitro or after in vivo transplantation of adult stem cells from blood, fat, skeletal muscle, or heart have challenged this view. Analysis of these studies has been complicated by the large disparity in the magnitude of effects seen by different groups and obscured by the recently appreciated process of in vivo stem-cell fusion. We now show a novel population of nonsatellite cells in adult murine skeletal muscle that progress under standard primary cell-culture conditions to autonomously beating cardiomyocytes. Their differentiation into beating cardiomyocytes is characterized here by video microscopy, confocal-detected calcium transients, electron microscopy, immunofluorescent cardiac-specific markers, and single-cell patch recordings of cardiac action potentials. Within 2 d after tail-vein injection of these marked cells into a mouse model of acute infarction, the marked cells are visible in the heart. By 6 d they begin to differentiate without fusing to recipient cardiac cells. Three months later, the tagged cells are visible as striated heart muscle restricted to the region of the cardiac infarct.

  14. Adult murine skeletal muscle contains cells that can differentiate into beating cardiomyocytes in vitro.

    Directory of Open Access Journals (Sweden)

    Steve O Winitsky

    2005-04-01

    Full Text Available It has long been held as scientific fact that soon after birth, cardiomyocytes cease dividing, thus explaining the limited restoration of cardiac function after a heart attack. Recent demonstrations of cardiac myocyte differentiation observed in vitro or after in vivo transplantation of adult stem cells from blood, fat, skeletal muscle, or heart have challenged this view. Analysis of these studies has been complicated by the large disparity in the magnitude of effects seen by different groups and obscured by the recently appreciated process of in vivo stem-cell fusion. We now show a novel population of nonsatellite cells in adult murine skeletal muscle that progress under standard primary cell-culture conditions to autonomously beating cardiomyocytes. Their differentiation into beating cardiomyocytes is characterized here by video microscopy, confocal-detected calcium transients, electron microscopy, immunofluorescent cardiac-specific markers, and single-cell patch recordings of cardiac action potentials. Within 2 d after tail-vein injection of these marked cells into a mouse model of acute infarction, the marked cells are visible in the heart. By 6 d they begin to differentiate without fusing to recipient cardiac cells. Three months later, the tagged cells are visible as striated heart muscle restricted to the region of the cardiac infarct.

  15. Evaluation of PLGA containing anti-CTLA4 inhibited endometriosis progression by regulating CD4+CD25+Treg cells in peritoneal fluid of mouse endometriosis model.

    Science.gov (United States)

    Liu, Qi; Ma, Pingchuan; Liu, Lanxia; Ma, Guilei; Ma, Jingjing; Liu, Xiaoxuan; Liu, Yijin; Lin, Wanjun; Zhu, Yingjun

    2017-01-01

    Our study investigated poly(lactic-co-glycolic acid) (PLGA) as protein delivery vehicles encapsulate CTLA-4-antibody (anti-CTLA-4) which is essential for CD4+CD25+Treg cells suppressive function exposing superior potential for inhibiting endometriosis progress in mouse model than single anti-CTLA-4. Anti-CTLA-4 loaded PLGA combined to ligands CTLA-4 in surface of CD4+CD25+Treg cells which distributed in peritoneal fluid of mouse endometriosis model. The particle size, zeta potential of the anti-CTLA-4 loaded nanoparticles was detected by dynamic light scattering. Morphology of nanoparticles was evaluated by transmission electron microscopy (TEM). Confocal laser scanning microscopy (CLSM) indicated distribution of anti-CTLA-4 with PLGA or without in peritoneal fluid. Cumulative anti-CTLA-4 release from nanoparticles was evaluated by Micro BCA assay. The percentage of CD4+CD25+Treg cells in peritoneal fluid was demonstrated by flow cytometer. In vitro experiment we co-culture ectopic endometrial cells (EEC) with isolated CD4+CD25+Treg cells in peritoneal fluid (PF), proliferation and invasion of ectopic endometrial cells (EEC) was measured by BrdU ELISA assay and Matrigel invasion assay. In comparison with anti-CTLA-4 without nanoparticles, the bioconjugates PLGA/anti-CTLA-4 were tolerated in peritoneal fluid with a controlled release of anti-CTLA-4 in 3, 7, 14days. Moreover, PLGA/anti-CTLA-4 had superior protective regulation ability to reduce level of CD4+CD25+Treg cells in peritoneal fluid. Most strikingly, in vitro experiment, PLGA/anti-CTLA-4 exhibited better ability in inhibiting proliferation and invasion of ectopic endometrial cells in co-culture system compared with anti-CTLA-4. Progressively, PLGA/anti-CTLA-4 had better suppressive activity to inhibited IL-10 and TGF-beta secreted by CD4+CD25+Treg cells which indicating that PLGA/anti-CTLA-4 suppressed cells proliferation and invasion through reduced IL-10 and TGF-beta production. Thus, PLGA/anti-CTLA-4 may

  16. 1.8 Å structure of murine GITR ligand dimer expressed in Drosophila melanogaster S2 cells

    Energy Technology Data Exchange (ETDEWEB)

    Chattopadhyay, Kausik [Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, New York 10461 (United States); Ramagopal, Udupi A. [Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461 (United States); Nathenson, Stanley G., E-mail: nathenso@aecom.yu.edu [Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, New York 10461 (United States); Department of Cell Biology, Albert Einstein College of Medicine, Bronx, New York 10461 (United States); Almo, Steven C., E-mail: nathenso@aecom.yu.edu [Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461 (United States); Department of Physiology and Biophysics, Albert Einstein College of Medicine, Bronx, New York 10461 (United States); Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, New York 10461 (United States)

    2009-05-01

    1.8 Å X-ray crystal structure of mouse GITRL expressed in D. melanogaster S2 cells shows an identical ‘strand-exchanged’ dimeric assembly similar to that observed previously for the E. coli-expressed protein. 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 Å 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.

  17. 1.8 Å structure of murine GITR ligand dimer expressed in Drosophila melanogaster S2 cells

    International Nuclear Information System (INIS)

    Chattopadhyay, Kausik; Ramagopal, Udupi A.; Nathenson, Stanley G.; Almo, Steven C.

    2009-01-01

    1.8 Å X-ray crystal structure of mouse GITRL expressed in D. melanogaster S2 cells shows an identical ‘strand-exchanged’ dimeric assembly similar to that observed previously for the E. coli-expressed protein. 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 Å 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

  18. Vitamin D Can Ameliorate Chlorhexidine Gluconate-Induced Peritoneal Fibrosis and Functional Deterioration through the Inhibition of Epithelial-to-Mesenchymal Transition of Mesothelial Cells

    Directory of Open Access Journals (Sweden)

    Yi-Che Lee

    2015-01-01

    Full Text Available Background. Peritoneal dialysis (PD can induce fibrosis and functional alterations in PD patients’ peritoneal membranes, due to long-term unphysiological dialysate exposure, partially occurring via triggering of epithelial-to-mesenchymal transition (EMT in peritoneal mesothelial cells (MCs. Vitamin D can ameliorate these negative effects; however, the mechanism remains unexplored. Therefore, we investigated its possible links to MCs EMT inhibition. Methods. Peritoneal fibrosis was established in Sprague-Dawley rats by chlorhexidine gluconate (CG intraperitoneal injection for 21 days, with and without 1α,25(OH2D3 treatment. Morphological and functional evaluation and western blot analysis of EMT marker were performed upon peritoneum tissue. In vitro study was also performed in a primary human peritoneal MC culture system; MCs were incubated with transforming growth factor-β1 (TGF-β1 in the absence or presence of 1α,25(OH2D3. EMT marker expression, migration activities, and cytoskeleton redistribution of MCs were determined. Results. 1α,25(OH2D3 ameliorated CG-induced morphological and functional deterioration in animal model, along with CG-induced upregulation of α-SMA and downregulation of E-cadherin expression. Meanwhile, 1α,25(OH2D3 also ameliorated TGF-β1-induced decrease in E-cadherin expression, increase in Snai1 and α-SMA expression, intracellular F-actin redistribution, and migration activity in vitro. Conclusion. 1α,25(OH2D3 can ameliorate CG-induced peritoneal fibrosis and attenuate functional deterioration through inhibiting MC EMT.

  19. ZFP521 regulates murine hematopoietic stem cell function and facilitates MLL-AF9 leukemogenesis in mouse and human cells.

    Science.gov (United States)

    Garrison, Brian S; Rybak, Adrian P; Beerman, Isabel; Heesters, Balthasar; Mercier, Francois E; Scadden, David T; Bryder, David; Baron, Roland; Rossi, Derrick J

    2017-08-03

    The concept that tumor-initiating cells can co-opt the self-renewal program of endogenous stem cells as a means of enforcing their unlimited proliferative potential is widely accepted, yet identification of specific factors that regulate self-renewal of normal and cancer stem cells remains limited. Using a comparative transcriptomic approach, we identify ZNF521 / Zfp521 as a conserved hematopoietic stem cell (HSC)-enriched transcription factor in human and murine hematopoiesis whose function in HSC biology remains elusive. Competitive serial transplantation assays using Zfp521 -deficient mice revealed that ZFP521 regulates HSC self-renewal and differentiation. In contrast, ectopic expression of ZFP521 in HSCs led to a robust maintenance of progenitor activity in vitro. Transcriptional analysis of human acute myeloid leukemia (AML) patient samples revealed that ZNF521 is highly and specifically upregulated in AMLs with MLL translocations. Using an MLL-AF9 murine leukemia model and serial transplantation studies, we show that ZFP521 is not required for leukemogenesis, although its absence leads to a significant delay in leukemia onset. Furthermore, knockdown of ZNF521 reduced proliferation in human leukemia cell lines possessing MLL-AF9 translocations. Taken together, these results identify ZNF521/ZFP521 as a critical regulator of HSC function, which facilitates MLL-AF9-mediated leukemic disease in mice.

  20. Inhibition of Na(+) -K+ pump activity by divalent cations in intact peritoneal mast cells of the rat

    DEFF Research Database (Denmark)

    Knudsen, T; Berthelsen, Carsten; Johansen, Torben

    1990-01-01

    1. The inhibition by the divalent cations magnesium, barium and strontium and the trivalent ion lanthanum of the Na(+) -K+ pump in the plasma membrane of rat peritoneal mast cells was studied in pure mast cell populations by measurement of the ouabain-sensitive uptake of the radioactive potassium...... or more, but no decrease was observed after 2 min incubation when the cells are supposed to be loaded with sodium due to the cell isolation procedure. 3. Barium and strontium caused concentration-dependent decreases in the ouabain-sensitive K(+) -(86Rb+) -uptake of the cells but the ouabain......-resistant uptake was not changed. Half maximum decrease in the ouabain-sensitive K+(86Rb+)-uptake was observed with 1.8 mM magnesium, 1.2mM barium and 0.7 mM strontium. 4. The trivalent ion lanthanum blocked almost completely the ouabain-sensitive K+(86Rb+)-uptake at a concentration of 1 microM as does 1 m...

  1. Characterization of immortalized MARCO and SR-AI/II-deficient murine alveolar macrophage cell lines

    Directory of Open Access Journals (Sweden)

    Imrich Amy

    2008-05-01

    Full Text Available Abstract Background Alveolar macrophages (AM avidly bind and ingest unopsonized inhaled particles and bacteria through class A scavenger receptors (SRAs MARCO and SR-AI/II. Studies to characterize the function of these SRAs have used AMs from MARCO or SR-AI/II null mice, but this approach is limited by the relatively low yield of AMs. Moreover, studies using both MARCO and SR-AI/II-deficient (MS-/- mice have not been reported yet. Hence, we sought to develop continuous cell lines from primary alveolar macrophages from MS-/- mice. Results We used in vitro infection of the primary AMs with the J2 retrovirus carrying the v-raf and v-myc oncogenes. Following initial isolation in media supplemented with murine macrophage colony-stimulating factor (M-CSF, we subcloned three AM cell lines, designated ZK-1, ZK-2 and ZK-6. These cell lines grow well in RPMI-1640-10% FBS in the absence of M-CSF. These adherent but trypsin-sensitive cell lines have a doubling time of approximately 14 hours, exhibit typical macrophage morphology, and express macrophage-associated cell surface Mac-1 (CD11b and F4/80 antigens. The cell lines show robust Fc-receptor dependent phagocytosis of opsonized red blood cells. Similar to freshly isolated AMs from MS-/- mice, the cell lines exhibit decreased phagocytosis of unopsonized titanium dioxide (TiO2, fluorescent latex beads and bacteria (Staphylococcus aureus compared with the primary AMs from wild type (WT C57BL/6 mice. Conclusion Our results indicated that three contiguous murine alveolar macrophage cell lines with MS-/- (ZK1, ZK2 and ZK6 were established successfully. These cell lines demonstrated macrophage morphology and functional activity. Interestingly, similar to freshly isolated AMs from MS-/- mice, the cell lines have a reduced, but not absent, ability to bind and ingest particles, with an altered pattern of blockade by scavenger receptor inhibitors. These cell lines will facilitate in vitro studies to further define

  2. Mitochondrial biogenesis and energy production in differentiating murine stem cells: a functional metabolic study.

    Science.gov (United States)

    Han, Sungwon; Auger, Christopher; Thomas, Sean C; Beites, Crestina L; Appanna, Vasu D

    2014-02-01

    The significance of metabolic networks in guiding the fate of the stem cell differentiation is only beginning to emerge. Oxidative metabolism has been suggested to play a major role during this process. Therefore, it is critical to understand the underlying mechanisms of metabolic alterations occurring in stem cells to manipulate the ultimate outcome of these pluripotent cells. Here, using P19 murine embryonal carcinoma cells as a model system, the role of mitochondrial biogenesis and the modulation of metabolic networks during dimethyl sulfoxide (DMSO)-induced differentiation are revealed. Blue native polyacrylamide gel electrophoresis (BN-PAGE) technology aided in profiling key enzymes, such as hexokinase (HK) [EC 2.7.1.1], glucose-6-phosphate isomerase (GPI) [EC 5.3.1.9], pyruvate kinase (PK) [EC 2.7.1.40], Complex I [EC 1.6.5.3], and Complex IV [EC 1.9.3.1], that are involved in the energy budget of the differentiated cells. Mitochondrial adenosine triphosphate (ATP) production was shown to be increased in DMSO-treated cells upon exposure to the tricarboxylic acid (TCA) cycle substrates, such as succinate and malate. The increased mitochondrial activity and biogenesis were further confirmed by immunofluorescence microscopy. Collectively, the results indicate that oxidative energy metabolism and mitochondrial biogenesis were sharply upregulated in DMSO-differentiated P19 cells. This functional metabolic and proteomic study provides further evidence that modulation of mitochondrial energy metabolism is a pivotal component of the cellular differentiation process and may dictate the final destiny of stem cells.

  3. Radioautographic analysis of changes in different phases of cell kinetics in murine oral mucosa

    International Nuclear Information System (INIS)

    Park, Chang Suck; You, Dong Soo

    1983-01-01

    The age related changes in the life cycle of the progenitor cell population of murine oral epithelia was studied. Using radioautographic methods which have been adopted in previous cell cycle studies, the age-related changes of different phases in renewing cells of the palatal, buccal and lingual mucosae were determined. The results confirm published findings on cell cycle changes of epithelia with aging and illustrated further that mitotic phases which has hither to been considered stationary, also changes with aging. The major parts revealed by this study are as follows: 1. The basal progenitor cells in different regions of oral mucosa have different generation times. 2. The basal cell cycle time increases as a function of aging and the region most affected by aging appears to be the epithelium of the cheek. 3. The phases of the cell cycle affected by the process of aging are in increasing order of magnitude: M-, S- and G1-phase. 4. The age related change in the number of DNA synthesizing basal progenitor cells occurs at two age periods. Between 1 and 12 months of life it decreases, while from 12 to 20 months it increases.

  4. The NLRP3 Inflammasome Has a Critical Role in Peritoneal Dialysis-Related Peritonitis.

    Science.gov (United States)

    Hautem, Nicolas; Morelle, Johann; Sow, Amadou; Corbet, Cyril; Feron, Olivier; Goffin, Eric; Huaux, François; Devuyst, Olivier

    2017-07-01

    Bacterial peritonitis remains the main cause of technique failure in peritoneal dialysis (PD). During peritonitis, the peritoneal membrane undergoes structural and functional alterations that are mediated by IL-1 β The NLRP3 inflammasome is a caspase-1-activating multiprotein complex that links sensing of microbial and stress products to activation of proinflammatory cytokines, including IL-1 β The potential roles of the NLRP3 inflammasome and IL-1 β in the peritoneal membrane during acute peritonitis have not been investigated. Here, we show that the NLRP3 inflammasome is activated during acute bacterial peritonitis in patients on PD, and this activation associates with the release of IL-1 β in the dialysate. In mice, lipopolysaccharide- or Escherichia coli -induced peritonitis led to IL-1 β release in the peritoneal membrane. The genetic deletion of Nalp3 , which encodes NLRP3, abrogated defects in solute transport during acute peritonitis and restored ultrafiltration. In human umbilical vein endothelial cells, IL-1 β treatment directly enhanced endothelial cell proliferation and increased microvascular permeability. These in vitro effects require endothelial IL-1 receptors, shown by immunofluorescence to be expressed in peritoneal capillaries in mice. Furthermore, administration of the IL-1 β receptor antagonist, anakinra, efficiently decreased nitric oxide production and vascular proliferation and restored peritoneal function in mouse models of peritonitis, even in mice treated with standard-of-care antibiotherapy. These data demonstrate that NLRP3 activation and IL-1 β release have a critical role in solute transport defects and tissue remodeling during PD-related peritonitis. Blockade of the NLRP3/IL-1 β axis offers a novel method for rescuing morphologic alterations and transport defects during acute peritonitis. Copyright © 2017 by the American Society of Nephrology.

  5. Activation of the Na+/K(+)-pump in rat peritoneal mast cells following histamine release: a possible role in cell recovery

    DEFF Research Database (Denmark)

    Knudsen, T; Ferjan, I; Johansen, Torben

    1993-01-01

    1. The activity of the Na+/K(+)-pump in rat peritoneal mast cells was measured at various time intervals after induction of cellular histamine release by compound 48/80 or by the antigen-antibody reaction. The Na+/K(+)-pump activity was assessed as the ouabain-sensitive potassium uptake....... On the basis of the present results, we suggest a role for the Na+/K(+)-pump in the recovery process of the mast cell following histamine release....... of the cells using 86Rb+ as a tracer for potassium (K+(86Rb+)-uptake). 2. Stimulation of the cells with compound 48/80 induced a time and concentration dependent increase of the Na+/K(+)-pump activity. The pump activity was maximal 2 min after stimulation of the cells. Then, the activity gradually decreased...

  6. Decreased Progesterone Receptor B/A Ratio in Endometrial Cells by Tumor Necrosis Factor-Alpha and Peritoneal Fluid from Patients with Endometriosis.

    Science.gov (United States)

    Chae, Uisoo; Min, Jin Young; Kim, Sung Hoon; Ihm, Hyo Jin; Oh, Young Sang; Park, So Yun; Chae, Hee Dong; Kim, Chung Hoon; Kang, Byung Moon

    2016-11-01

    Progesterone resistance is thought to be a major factor that contributes to progression of endometriosis. However, it is not clear what causes progesterone resistance in endometriosis. This study aimed to assess whether cytokines or peritoneal fluid can affect progesterone receptor (PR) expression in endometrial cells and to verify whether PR expression is reduced in endometriosis. The PR-B/A ratio was measured via real-time polymerase chain reaction after in vitro culture, in which endometrial cells were treated with either tumor necrosis factor-alpha (TNF-α), interleukin-1 beta, or peritoneal fluid obtained from women with advanced-stage endometriosis. Immunohistochemistry was performed to compare PR-B expression between eutopic and ectopic endometrial tissues from women with and without advanced-stage endometriosis. The PR-B/A ratio was significantly decreased by treatment with either TNF-α (p=0.011) or peritoneal fluid from women with advanced-stage endometriosis (p=0.027). Immunoreactivity of PR-B expression was significantly lower during the secretory phase than during the proliferative phase in endometrial tissues from control subjects (pendometriosis compared with eutopic endometrium tissues from control subjects. Progesterone resistance in endometriosis may be caused by proinflammatory conditions in the pelvic peritoneal microenvironment.

  7. Fibulin-2 is present in murine vascular lesions and is important for smooth muscle cell migration

    DEFF Research Database (Denmark)

    Ström, A.; Olin, A. I.; Aspberg, A.

    2006-01-01

    /hyaluronan complexes, an ECM network that has been suggested to be important during tissue repair. In this study we have analysed the presence of fibulin-2 in two different models of murine vascular lesions. We have also examined how the fibulin-2/versican network influences SMC migration. Methods: Presence of fibulin......Objective: The vascular extracellular matrix (ECM) can affect smooth muscle cell (SMC) adhesion, migration and proliferation-events that are important during the atherosclerotic process. Fibulin-2 is a member of the ECM protein family of fibulins and has been found to cross-link versican...... and is upregulated during SMC phenotypic modulation in cell culture. Moreover, treatments with peptides that block the interaction between versican and fibulin-2 inhibit SMC migration in vitro. Conclusions: Fibulin-2 can be produced by SMC as a response to injury and may participate in the ECM organisation...

  8. Circulating endothelial progenitor cells do not contribute to regeneration of endothelium after murine arterial injury

    DEFF Research Database (Denmark)

    Hagensen, Mette; Raarup, Merete Krog; Mortensen, Martin Bødtker

    2012-01-01

    into endothelial cells (ECs). We tested this theory in a murine arterial injury model using carotid artery transplants and fluorescent reporter mice. METHODS AND RESULTS: Wire-injured carotid artery segments from wild-type mice were transplanted into TIE2-GFP transgenic mice expressing green fluorescent protein...... (GFP) in ECs. We found that the endothelium regenerated with GFP(+) ECs as a function of time, evolving from the anastomosis sites towards the centre of the transplant. A migration front of ECs at Day 7 was verified by scanning electron microscopy and by bright-field microscopy using recipient TIE2-lac......Z mice with endothelial β-galactosidase expression. These experiments indicated migration of flanking ECs rather than homing of circulating cells as the underlying mechanism. To confirm this, we interposed non-injured wild-type carotid artery segments between the denuded transplant and the TIE2-GFP...

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

    Science.gov (United States)

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

    2006-04-01

    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.

  10. The response of hypoxic cells in SCCVII murine tumors to treatment with cisplatin and x rays

    International Nuclear Information System (INIS)

    Yan, R.D.; Durand, R.E.

    1991-01-01

    Possible mechanisms of enhancement of radiation effects by cisplatin, including radiosensitization of hypoxic cells, drug-induced tumor reoxygenation, and inhibition of repair of sublethal radiation damage, were examined in the murine SCCVII model. Combination radiation/drug treatments were most effective when drug exposure preceded irradiation of animals breathing a reduced oxygen atmosphere, indicating that the primary interaction between the modalities was a cisplatin-induced increase in the oxygenation status of the acutely hypoxic cells in those tumors. Delivering cisplatin prior to or immediately after the first of two 5 Gy fractions was more effective than combinations with a single x-ray exposure, suggesting that proper sequences of the combined modalities may augment natural reoxygenation processes

  11. The effect of medium composition on interleukin-2 production by murine EL-4 thymoma cells

    Directory of Open Access Journals (Sweden)

    Galesi A. L. L.

    2004-01-01

    Full Text Available Due to the role of interleukin-2 (IL-2 in the mediation of immune response, this cytokine has been used in the treatment of some types of cancer and infectious diseases. However, relatively high levels of this cytokine are required to achieve significant activity. The aim of this work was to study a culture medium composition designed to increase the production of IL-2 by suspended murine EL-4 cells. The cultivations were carried out aiming at producing IL-2 in stirred bioreactors. The effects of concentration of glutamine, phorbol-12-myristate-13-acetate (PMA, concanavalin A (Con A, Pluronic F68, and fetal calf serum (FCS on cell viability and IL-2 production were evaluated. PMA alone was more efficient in IL-2 production than it was in association with Con A. The maximum IL-2 production was around 162 ng/mL with 856 ng/mL PMA and 1.45% (v/v FCS.

  12. Effects of interferon gamma and specific polyclonal antibody on the infection of murine peritoneal macrophages and murine macrophage cell line PMJ2-R with Encephalitozoon cuniculi

    Czech Academy of Sciences Publication Activity Database

    Jelínek, Jiří; Salát, Jiří; Sak, Bohumil; Kopecký, Jan

    2007-01-01

    Roč. 54, č. 3 (2007), s. 172-176 ISSN 0015-5683 R&D Projects: GA ČR GP524/03/D167 Institutional research plan: CEZ:AV0Z60220518 Keywords : microsporidia * Encephalitozoon cuniculi * antibody * macrophage s * interferon gamma (IFN-gamma) Subject RIV: EC - Immunology Impact factor: 1.000, year: 2007

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

    International Nuclear Information System (INIS)

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

    2006-01-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

    Reynertson, Kurt A. [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); Department of Medicine, Weill Cornell Medical College, 1300 York Avenue, New York, NY 10065 (United States); Gudas, Lorraine J., E-mail: ljgudas@med.cornell.edu [Center for Complementary and Integrative Medicine, Weill Cornell Medical College, 1300 York Avenue, New York, NY 10065 (United States); Department of Pharmacology, Weill Cornell Medical College, 1300 York Avenue, New York, NY 10065 (United States); Department of Medicine, Weill Cornell Medical College, 1300 York Avenue, New York, NY 10065 (United States)

    2011-01-01

    Epidemiological evidence indicates that diets high in fruits and vegetables provide a measure of cancer chemoprevention due to phytochemical constituents. Natural products are a rich source of cancer chemotherapy drugs, and primarily target rapidly cycling tumor cells. Increasing evidence indicates that many cancers contain small populations of resistant, stem-like cells that have the capacity to regenerate tumors following chemotherapy and radiation, and have been linked to the initiation of metastases. Our goal is to discover natural product-based clinical or dietary interventions that selectively target cancer stem cells, inducing differentiation. We adapted an alkaline phosphatase (AP) stain to assay plant extracts for the capacity to induce differentiation in embryonic stem (ES) cells. AP is a characteristic marker of undifferentiated ES cells, and this represents a novel approach to screening medicinal plant extracts. Following a survey of approximately 100 fractions obtained from 12 species of ethnomedically utilized plants, we found fractions from 3 species that induced differentiation, decreasing AP and transcript levels of pluripotency markers (Nanog, Oct-4, Rex-1). These fractions affected proliferation of murine ES, and human embryonal, prostate, and breast carcinoma cells in a dose-dependent manner. Several phytochemical constituents were isolated; the antioxidant phytochemicals ellagic acid and gallic acid were shown to affect viability of cultured breast carcinoma cells.

  15. In vitro nanotoxicity of single-walled carbon nanotube-dendrimer nanocomplexes against murine myoblast cells.

    Science.gov (United States)

    Cancino, J; Paino, I M M; Micocci, K C; Selistre-de-Araujo, H S; Zucolotto, V

    2013-05-10

    Single-wall carbon nanotubes (SWCNTs) and polyamidoamine dendrimers (PAMAM) have been proposed for a variety of biomedical applications. The combination of both molecules makes this new composite nanomaterial highly functionalizable and versatile to theranostic and drug-delivery systems. However, recent toxicological studies have shown that nanomaterials such as SWCNTs and PAMAM may have high toxicity in biological environments. Aiming to elucidate such behavior, in vitro studies with different cultured cells have been conducted in the past few years. This study focuses on the effects of SWCNT-PAMAM nanomaterials and their individual components on the C2C12 murine cell line, which is a mixed population of stem and progenitor cells. The interactions between the cells and the nanomaterials were studied with different techniques usually employed in toxicological analyses. The results showed that SWCNT-PAMAM and PAMAM inhibited the proliferation and caused DNA damage of C2C12 cells. Data from flow cytometry revealed a less toxicity in C2C12 cells exposed to SWCNT compared to the other nanomaterials. The results indicated that the toxicity of SWCNT, SWCNT-PAMAM and PAMAM in C2C12 cells can be strongly correlated with the charge of the nanomaterials. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  16. Effects of atmospheric pressure plasma jet with floating electrode on murine melanoma and fibroblast cells

    Science.gov (United States)

    Xu, G.; Liu, J.; Yao, C.; Chen, S.; Lin, F.; Li, P.; Shi, X.; Zhang, Guan-Jun

    2017-08-01

    Atmospheric pressure cold plasma jets have been recently shown as a highly promising tool in certain cancer therapies. In this paper, an atmospheric pressure plasma jet (APPJ) with a one inner floating and two outer electrode configuration using helium gas for medical applications is developed. Subjected to a range of applied voltages with a frequency of 19.8 kHz at a fixed rate of gas flow (i.e., 3 l/min), electrical and optical characteristics of the APPJ are investigated. Compared with the device only with two outer electrodes, higher discharge current, longer jet, and more active species in the plasma plume at the same applied voltage together with the lower gas breakdown voltage can be achieved through embedding a floating inner electrode. Employing the APPJ with a floating electrode, the effects of identical plasma treatment time durations on murine melanoma cancer and normal fibroblast cells cultured in vitro are evaluated. The results of cell viability, cell apoptosis, and DNA damage detection show that the plasma can inactivate melanoma cells in a time-dependent manner from 10 s to 60 s compared with the control group (p cells compared with their control group, the plasma with treatment time from 30 s to 60 s can induce significant changes (p cells at the same treatment time. The different basal reactive oxygen species level and antioxidant superoxide dismutase level of two kinds of cells may account for their different responses towards the identical plasma exposure.

  17. CrxOS maintains the self-renewal capacity of murine embryonic stem cells

    International Nuclear Information System (INIS)

    Saito, Ryota; Yamasaki, Tokiwa; Nagai, Yoko; Wu, Jinzhan; Kajiho, Hiroaki; Yokoi, Tadashi; Noda, Eiichiro; Nishina, Sachiko; Niwa, Hitoshi; Azuma, Noriyuki; Katada, Toshiaki; Nishina, Hiroshi

    2009-01-01

    Embryonic stem (ES) cells maintain pluripotency by self-renewal. Several homeoproteins, including Oct3/4 and Nanog, are known to be key factors in maintaining the self-renewal capacity of ES cells. However, other genes required for the mechanisms underlying this process are still unclear. Here we report the identification by in silico analysis of a homeobox-containing gene, CrxOS, that is specifically expressed in murine ES cells and is essential for their self-renewal. ES cells mainly express the short isoform of endogenous CrxOS. Using a polyoma-based episomal expression system, we demonstrate that overexpression of the CrxOS short isoform is sufficient for maintaining the undifferentiated morphology of ES cells and stimulating their proliferation. Finally, using RNA interference, we show that CrxOS is essential for the self-renewal of ES cells, and provisionally identify foxD3 as a downstream target gene of CrxOS. To our knowledge, ours is the first delineation of the physiological role of CrxOS in ES cells.

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

    Science.gov (United States)

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

    2011-01-01

    Epidemiological evidence indicates that diets high in fruits and vegetables provide a measure of cancer chemoprevention due to phytochemical constituents. Natural products are a rich source of cancer chemotherapy drugs, and primarily target rapidly cycling tumor cells. Increasing evidence indicates that many cancers contain small populations of resistant, stem-like cells that have the capacity to regenerate tumors following chemotherapy and radiation, and have been linked to the initiation of metastases. Our goal is to discover natural product-based clinical or dietary interventions that selectively target cancer stem cells, inducing differentiation. We adapted an alkaline phosphatase (AP) stain to assay plant extracts for the capacity to induce differentiation in embryonic stem (ES) cells. AP is a characteristic marker of undifferentiated ES cells, and this represents a novel approach to screening medicinal plant extracts. Following a survey of approximately 100 fractions obtained from 12 species of ethnomedically utilized plants, we found fractions from 3 species that induced differentiation, decreasing AP and transcript levels of pluripotency markers (Nanog, Oct-4, Rex-1). These fractions affected proliferation of murine ES, and human embryonal, prostate, and breast carcinoma cells in a dose-dependent manner. Several phytochemical constituents were isolated; the antioxidant phytochemicals ellagic acid and gallic acid were shown to affect viability of cultured breast carcinoma cells. Copyright © 2010 Elsevier Inc. All rights reserved.

  19. A bioengineered murine model using CD24+CD44+ pancreatic cancer stem cells for chemotherapy study

    International Nuclear Information System (INIS)

    Qin, Shengqi; Li, Jianshe; Zhang, Zhongtao; Deng, Yiming

    2015-01-01

    In this work we first developed a murine pancreatic tumor model using CD24 + CD44 + pancreatic cancer stem cells (CSC) supported by an electrospun scaffold. Unlike conventional models, the use of CSC and the scaffold, which were biologically and chemically defined, afforded scientists a reliable platform to evaluate novel chemotherapy regimens. CD24 + CD44 + CSC successfully initiated tumorigenesis in vitro on the scaffold without suffering apoptosis, evidencing the lack of cytotoxicity of scaffolding materials. Also, the scaffold contributed to the acceleration of in vivo tumorigenesis and increased the likelihood of tumor formation. Using this model, we set out to explore the effectiveness of irinotecan/gemcitabine (IRIN-GEM), a chemotherapy regimen, for pancreatic cancer. Our study showed that IRIN-GEM induced a tumor regression whereas gemcitabine alone could only arrest the tumor growth. Further study suggested that the superior performance of IRIN-GEM could be attributed to its capacity to demolish the CD24 + CD44 + CSC sub-population by inducing a large-scale apoptosis. The use of highly proliferative yet homogenous CD24 + CD44 + CSC along with a chemically defined scaffold accelerated the tumor formation and significantly reduced the variability associated with conventional murine models. Armed with this new model, we discovered that IRIN-GEM would be a promising chemotherapy candidate for patients with advanced pancreatic cancer. (paper)

  20. Inhibition of Murine Pulmonary Microvascular Endothelial Cell Apoptosis Promotes Recovery of Barrier Function under Septic Conditions

    Directory of Open Access Journals (Sweden)

    Lefeng Wang

    2017-01-01

    Full Text Available Sepsis is characterized by injury of the pulmonary microvasculature and the pulmonary microvascular endothelial cells (PMVEC, leading to barrier dysfunction and acute respiratory distress syndrome (ARDS. Our recent work identified a strong correlation between PMVEC apoptosis and microvascular leak in septic mice in vivo, but the specific role of apoptosis in septic PMVEC barrier dysfunction remains unclear. Thus, we hypothesize that PMVEC apoptosis is likely required for PMVEC barrier dysfunction under septic conditions in vitro. Septic stimulation (mixture of tumour necrosis factor α, interleukin 1β, and interferon γ [cytomix] of isolated murine PMVEC resulted in a significant loss of barrier function as early as 4 h after stimulation, which persisted until 24 h. PMVEC apoptosis, as reflected by caspase activation, DNA fragmentation, and loss of membrane polarity, was first apparent at 8 h after cytomix. Pretreatment of PMVEC with the pan-caspase inhibitor Q-VD significantly decreased septic PMVEC apoptosis and was associated with reestablishment of PMVEC barrier function at 16 and 24 h after stimulation but had no effect on septic PMVEC barrier dysfunction over the first 8 h. Collectively, our data suggest that early septic murine PMVEC barrier dysfunction driven by proinflammatory cytokines is not mediated through apoptosis, but PMVEC apoptosis contributes to late septic PMVEC barrier dysfunction.

  1. Identification of distinct topographical surface microstructures favoring either undifferentiated expansion or differentiation of murine embryonic stem cells.

    Science.gov (United States)

    Markert, Lotte D'Andrea; Lovmand, Jette; Foss, Morten; Lauridsen, Rune Hoff; Lovmand, Michael; Füchtbauer, Ernst-Martin; Füchtbauer, Annette; Wertz, Karin; Besenbacher, Flemming; Pedersen, Finn Skou; Duch, Mogens

    2009-11-01

    The potential of embryonic stem (ES) cells for both self-renewal and differentiation into cells of all three germ layers has generated immense interest in utilizing these cells for tissue engineering or cell-based therapies. However, the ability to culture undifferentiated ES cells without the use of feeder cells as well as means to obtain homogeneous, differentiated cell populations devoid of residual pluripotent ES cells still remain major challenges. Here we have applied murine ES cells to topographically microstructured surface libraries, BioSurface Structure Arrays (BSSA), and investigated whether these could be used to (i) identify topographically microstructured growth supports alleviating the need for feeder cells for expansion of undifferentiated ES cells and (ii) identify specific types of microstructures enforcing differentiation of ES cells. The BSSA surfaces arrays consisted of 504 different topographical microstructures each located in a tester field of 3 x 3 mm. The murine ES cell lines CJ7 and KH2 were seeded upon the BSSA libraries and specific topographical structures facilitating either undifferentiated ES cell growth or enhancing spreading indicative of differentiation of the ES cells were identified. Secondly serial passage of undifferentiated CJ7 ES cells on selected microstructures, identified in the screening of these BSSA libraries, showed that these cells had retained germ-line potential. These results indicate that one specific type of topographical surface microstructures, identified by the BSSA technology, can substitute for feeder cells and that another subset may be used to eliminate undifferentiated ES cells from a population of differentiated ES cells.

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

    International Nuclear Information System (INIS)

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

    1984-01-01

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

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

    International Nuclear Information System (INIS)

    McQuillan, D.J.; Yanagishita, M.; Hascall, V.C.; Bickel, M.

    1989-01-01

    Murine monocytic leukemic (M1) cells were cultured in the presence of [ 3 H]glucosamine and [ 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

  4. Upregulation of cellular glutathione levels in human ABCB5- and murine Abcb5-transfected cells.

    Science.gov (United States)

    Kondo, Shingo; Hongama, Keita; Hanaya, Kengo; Yoshida, Ryota; Kawanobe, Takaaki; Katayama, Kazuhiro; Noguchi, Kohji; Sugimoto, Yoshikazu

    2015-12-15

    Previously, we have demonstrated that human ABCB5 is a full-sized ATP-binding cassette transporter that shares strong homology with ABCB1/P-glycoprotein. ABCB5-transfected cells showed resistance to taxanes and anthracyclines. Herein, we further screened ABCB5 substrates, and explored the mechanism of resistance. Sensitivity of the cells to test compounds was evaluated using cell growth inhibition assay. Cellular levels of buthionine sulfoximine (BSO), glutathione and amino acids were measured using HPLC and an enzyme-based assay. Cellular and vesicular transport of glutathione was evaluated by a radiolabeled substrate. Expression levels of glutathione-metabolizing enzymes were assessed by RT-PCR. Human ABCB5-transfected 293/B5-11 cells and murine Abcb5-transfected 293/mb5-8 cells showed 6.5- and 14-fold higher resistance to BSO than the mock-transfected 293/mock cells, respectively. BSO is an inhibitor of gamma-glutamylcysteine ligase (GCL), which is a key enzyme of glutathione synthesis. 293/B5-11 and 293/mb5-8 cells also showed resistance to methionine sulfoximine, another GCL inhibitor. A cellular uptake experiment revealed that BSO accumulation in 293/B5-11 and 293/mb5-8 cells was similar to that in 293/mock cells, suggesting that BSO is not an ABCB5 substrate. The cellular glutathione content in 293/B5-11 and 293/mb5-8 cells was significantly higher than that in 293/mock cells. Evaluation of the BSO effect on the cellular glutathione content showed that compared with 293/mock cells the BSO concentration required for a 50 % reduction in glutathione content in 293/B5-11 and 293/mb5-8 cells was approximately 2- to 3-fold higher. This result suggests that the BSO resistance of the ABCB5- and Abcb5-transfected cells can be attributed to the reduced effect of BSO on the transfectants. Cellular and vesicular transport assays showed that the transport of radiolabeled glutathione in 293/B5-11 cells was similar to that in 293/mock cells. The mRNA expression of genes

  5. Upregulation of endothelial cell adhesion molecules characterizes veins close to granulomatous infiltrates in the renal cortex of cats with feline infectious peritonitis and is indirectly triggered by feline infectious peritonitis virus-infected monocytes in vitro.

    Science.gov (United States)

    Acar, Delphine D; Olyslaegers, Dominique A J; Dedeurwaerder, Annelike; Roukaerts, Inge D M; Baetens, Wendy; Van Bockstael, Sebastiaan; De Gryse, Gaëtan M A; Desmarets, Lowiese M B; Nauwynck, Hans J

    2016-10-01

    One of the most characteristic pathological changes in cats that have succumbed to feline infectious peritonitis (FIP) is a multifocal granulomatous phlebitis. Although it is now well established that leukocyte extravasation elicits the inflammation typically associated with FIP lesions, relatively few studies have aimed at elucidating this key pathogenic event. The upregulation of adhesion molecules on the endothelium is a prerequisite for stable leukocyte-endothelial cell (EC) adhesion that necessarily precedes leukocyte diapedesis. Therefore, the present work focused on the expression of the EC adhesion molecules and possible triggers of EC activation during the development of FIP. Immunofluorescence analysis revealed that the endothelial expression of P-selectin, E-selectin, intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) was elevated in veins close to granulomatous infiltrates in the renal cortex of FIP patients compared to non-infiltrated regions and specimens from healthy cats. Next, we showed that feline venous ECs become activated when exposed to supernatant from feline infectious peritonitis virus (FIPV)-infected monocytes, as indicated by increased adhesion molecule expression. Active viral replication seemed to be required to induce the EC-stimulating activity in monocytes. Finally, adhesion assays revealed an increased adhesion of naive monocytes to ECs treated with supernatant from FIPV-infected monocytes. Taken together, our results strongly indicate that FIPV activates ECs to increase monocyte adhesion by an indirect route, in which proinflammatory factors released from virus-infected monocytes act as key intermediates.

  6. Cyanotoxins at low doses induce apoptosis and inflammatory effects in murine brain cells: Potential implications for neurodegenerative diseases

    OpenAIRE

    Takser, Larissa; Benachour, Nora; Husk, Barry; Cabana, Hubert; Gris, Denis

    2016-01-01

    Cyanotoxins have been shown to be highly toxic for mammalian cells, including brain cells. However, little is known about their effect on inflammatory pathways. This study investigated whether mammalian brain and immune cells can be a target of certain cyanotoxins, at doses approximating those in the guideline levels for drinking water, either alone or in mixtures. We examined the effects on cellular viability, apoptosis and inflammation signalling of several toxins on murine macrophage-like ...

  7. Expression of human cationic trypsinogen (PRSS1) in murine acinar cells promotes pancreatitis and apoptotic cell death

    Science.gov (United States)

    Athwal, T; Huang, W; Mukherjee, R; Latawiec, D; Chvanov, M; Clarke, R; Smith, K; Campbell, F; Merriman, C; Criddle, D; Sutton, R; Neoptolemos, J; Vlatković, N

    2014-01-01

    Hereditary pancreatitis (HP) is an autosomal dominant disease that displays the features of both acute and chronic pancreatitis. Mutations in human cationic trypsinogen (PRSS1) are associated with HP and have provided some insight into the pathogenesis of pancreatitis, but mechanisms responsible for the initiation of pancreatitis have not been elucidated and the role of apoptosis and necrosis has been much debated. However, it has been generally accepted that trypsinogen, prematurely activated within the pancreatic acinar cell, has a major role in the initiation process. Functional studies of HP have been limited by the absence of an experimental system that authentically mimics disease development. We therefore developed a novel transgenic murine model system using wild-type (WT) human PRSS1 or two HP-associated mutants (R122H and N29I) to determine whether expression of human cationic trypsinogen in murine acinar cells promotes pancreatitis. The rat elastase promoter was used to target transgene expression to pancreatic acinar cells in three transgenic strains that were generated: Tg(Ela-PRSS1)NV, Tg(Ela-PRSS1*R122H)NV and Tg(Ela-PRSS1*N29I)NV. Mice were analysed histologically, immunohistochemically and biochemically. We found that transgene expression is restricted to pancreatic acinar cells and transgenic PRSS1 proteins are targeted to the pancreatic secretory pathway. Animals from all transgenic strains developed pancreatitis characterised by acinar cell vacuolisation, inflammatory infiltrates and fibrosis. Transgenic animals also developed more severe pancreatitis upon treatment with low-dose cerulein than controls, displaying significantly higher scores for oedema, inflammation and overall histopathology. Expression of PRSS1, WT or mutant, in acinar cells increased apoptosis in pancreatic tissues and isolated acinar cells. Moreover, studies of isolated acinar cells demonstrated that transgene expression promotes apoptosis rather than necrosis. We therefore

  8. Airway delivery of soluble factors from plastic-adherent bone marrow cells prevents murine asthma.

    Science.gov (United States)

    Ionescu, Lavinia I; Alphonse, Rajesh S; Arizmendi, Narcy; Morgan, Beverly; Abel, Melanie; Eaton, Farah; Duszyk, Marek; Vliagoftis, Harissios; Aprahamian, Tamar R; Walsh, Kenneth; Thébaud, Bernard

    2012-02-01

    Asthma affects an estimated 300 million people worldwide and accounts for 1 of 250 deaths and 15 million disability-adjusted life years lost annually. Plastic-adherent bone marrow-derived cell (BMC) administration holds therapeutic promise in regenerative medicine. However, given the low cell engraftment in target organs, including the lung, cell replacement cannot solely account for the reported therapeutic benefits. This suggests that BMCs may act by secreting soluble factors. BMCs also possess antiinflammatory and immunomodulatory properties and may therefore be beneficial for asthma. Our objective was to investigate the therapeutic potential of BMC-secreted factors in murine asthma. In a model of acute and chronic asthma, intranasal instillation of BMC conditioned medium (CdM) prevented airway hyperresponsiveness (AHR) and inflammation. In the chronic asthma model, CdM prevented airway smooth muscle thickening and peribronchial inflammation while restoring blunted salbutamol-induced bronchodilation. CdM reduced lung levels of the T(H)2 inflammatory cytokines IL-4 and IL-13 and increased levels of IL-10. CdM up-regulated an IL-10-induced and IL-10-secreting subset of T regulatory lymphocytes and promoted IL-10 expression by lung macrophages. Adiponectin (APN), an antiinflammatory adipokine found in CdM, prevented AHR, airway smooth muscle thickening, and peribronchial inflammation, whereas the effect of CdM in which APN was neutralized or from APN knock-out mice was attenuated compared with wild-type CdM. Our study provides evidence that BMC-derived soluble factors prevent murine asthma and suggests APN as one of the protective factors. Further identification of BMC-derived factors may hold promise for novel approaches in the treatment of asthma.

  9. DNA supercoiling in proliferating and quiescent 67 murine mammary tumor cells

    International Nuclear Information System (INIS)

    Cochran-Sandhu, L.; Warters, R.L.; Dethlefsen, L.A.

    1985-01-01

    The nucleoid sedimentation assay, which is a measure of DNA ''compactness'' or supercoiling, was used to evaluate the supercoiling state of proliferating (P) and quiescent (Q) murine mammary tumor cells. Two day old cultures are referred to as P cells, whereas 7 day old cultures maintained without media replenishment are referred to as Q cells (>95% arrested in G/sub 1/). Q nucleoids sedimented significantly less far into neutral sucrose gradients than P nucleoids, suggesting a less compact DNA structure. This was further confirmed by the utilization of two other probes of DNA supercoiling: ionizing radiation and sedimentation through gradients containing the intercalator ethidium bromide (EtBr). Whereas nucleoids from P cells showed a decrease in sedimentation following ionizing radiation and an initial decrease, followed by an increase, in sedimentation through gradients containing increasing concentrations of EtBr, the sedimentation of nucleoids from Q cells did not change following either treatment. These data indicate that the DNA of nucleoids isolated from Q cells is in a ''relaxed'' state. The potential significance of these results is discussed

  10. Different concentrations of kaempferol distinctly modulate murine embryonic stem cell function.

    Science.gov (United States)

    Correia, Marcelo; Rodrigues, Ana S; Perestrelo, Tânia; Pereira, Sandro L; Ribeiro, Marcelo F; Sousa, Maria I; Ramalho-Santos, João

    2016-01-01

    Kaempferol (3,4',5,7-tetrahydroxyflavone) is a natural flavonoid with several beneficial and protective effects. It has been demonstrated that kaempferol has anticancer properties, particularly due to its effects on proliferation, apoptosis and the cell cycle. However, possible effects on pluripotent embryonic stem cell function have not yet been addressed. Embryonic stem cells have the ability to self-renew and to differentiate into all three germ layers with potential applications in regenerative medicine and in vitro toxicology. We show that exposure of murine embryonic stem cells (mESC) to high concentrations of kaempferol (200 μM) leads to decreased cell numbers, although the resulting smaller cell colonies remain pluripotent. However, lower concentrations of this compound (20 μM) increase the expression of pluripotency markers in mESCs. Mitochondrial membrane potential and mitochondrial mass are not affected, but a dose-dependent increase in apoptosis takes place. Moreover, mESC differentiation is impaired by kaempferol, which was not related to apoptosis induction. Our results show that low concentrations of kaempferol can be beneficial for pluripotency, but inhibit proper differentiation of mESCs. Additionally, high concentrations induce apoptosis and increase mitochondrial reactive oxygen species (ROS). Copyright © 2015 Elsevier Ltd. All rights reserved.

  11. Mechanistic investigation of toxicity of chromium oxide nanoparticles in murine fibrosarcoma cells

    Directory of Open Access Journals (Sweden)

    Alarifi S

    2016-03-01

    Full Text Available Saud Alarifi, Daoud Ali, Saad Alkahtani Department of Zoology, College of Science, King Saud University, Riyadh, Saudi Arabia Abstract: Chromium oxide nanoparticles (Cr2O3NPs are widely used in polymers and paints. In the present study, we aimed to determine the toxicity of Cr2O3NPs in murine fibrosarcoma (L929 cells. The cytotoxicity of Cr2O3NPs was measured by MTT and neutral red uptake assays; Cr2O3NPs had significant cytotoxic effects on L929 cells. Enhancement of intracellular reactive oxygen species was observed in L929 cells after exposure to Cr2O3NPs. Cr2O3NPs produced caspase-3, indicating that exposure to Cr2O3NPs induced apoptosis. After exposure to Cr2O3NPs, the cellular glutathione level decreased and lipid peroxidation, superoxide dismutase, and catalase increased in a dose- and time-dependent manner. By using single-cell gel tests, we also observed increased DNA damage in a Cr2O3NP exposure-duration- and dose-dependent fashion. Cell toxicity and DNA damage may be useful biomarkers for determining the safety of Cr2O3NPs in human and animal health. Keywords: Cr2O3NPs, L929 cells, MTT assay, oxidative stress 

  12. The human and murine hematopoietic stem cell niches: are they comparable?

    Science.gov (United States)

    van Pel, Melissa; Fibbe, Willem E; Schepers, Koen

    2016-04-01

    Hematopoietic stem cells (HSCs) reside in specific niches that provide various instructive cues that regulate HSC self-renewal and their development into all mature cells of the peripheral blood. Progress in this research field has largely been guided by mouse studies. However, parallel studies with human subjects, tissues, and cells, in combination with xenotransplantation experiments in immunodeficient mice, have contributed to our increased understanding of the human HSC niche. Here, we summarize our current knowledge of the various specialized subsets of both stromal and hematopoietic cells that support HSCs through cell-cell interactions and secreted factors, and the many parallels between the murine and human HSC niches. Furthermore, we discuss recent technological advances that are likely to improve our understanding of the human HSC niche, a better understanding of which may allow further identification of unique molecular and cellular pathways in the HSC niche. This information may help to further improve the outcome of HSC transplantation and refine the treatment of hematopoietic diseases. © 2015 New York Academy of Sciences.

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

    Science.gov (United States)

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

  14. Assessment of DNA synthesis in Islet-1{sup +} cells in the adult murine heart

    Energy Technology Data Exchange (ETDEWEB)

    Weinberger, Florian, E-mail: f.weinberger@uke.de; Mehrkens, Dennis, E-mail: dennis.mehrkens@uk-koeln.de; Starbatty, Jutta, E-mail: starbatty@uke.uni-hamburg.de; Nicol, Philipp, E-mail: Philipp.Nicol@gmx.de; Eschenhagen, Thomas, E-mail: t.eschenhagen@uke.de

    2015-01-02

    Highlights: • Islet-1 was expressed in the adult heart. • Islet-1-positive cells did not proliferate in the adult heart. • Sinoatrial node cells did not proliferate in the adult heart. - Abstract: Rationale: Islet-1 positive (Islet-1{sup +}) cardiac progenitor cells give rise to the right ventricle, atria and outflow tract during murine cardiac development. In the adult heart Islet-1 expression is limited to parasympathetic neurons, few cardiomyocytes, smooth muscle cells, within the proximal aorta and pulmonary artery and sinoatrial node cells. Its role in these cells is unknown. Here we tested the hypothesis that Islet-1{sup +} cells retain proliferative activity and may therefore play a role in regenerating specialized regions in the heart. Methods and results: DNA synthesis was analyzed by the incorporation of tritiated thymidine ({sup 3}H-thymidine) in Isl-1-nLacZ mice, a transgenic model with an insertion of a nuclear beta-galactosidase in the Islet-1 locus. Mice received daily injections of {sup 3}H-thymidine for 5 days. DNA synthesis was visualized throughout the heart by dipping autoradiography of cryosections. Colocalization of an nLacZ-signal and silver grains would indicate DNA synthesis in Islet-1{sup +} cells. Whereas Islet{sup −} non-myocyte nuclei were regularly marked by accumulation of silver grains, colocalization with nLacZ-signals was not detected in >25,000 cells analyzed. Conclusions: Islet-1{sup +} cells are quiescent in the adult heart, suggesting that, under normal conditions, even pacemaking cells do not proliferate at higher rates than normal cardiac myocytes.

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

    Directory of Open Access Journals (Sweden)

    Christopher M Collins

    2014-05-01

    Full Text Available 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.

  16. Assessment of DNA synthesis in Islet-1+ cells in the adult murine heart

    International Nuclear Information System (INIS)

    Weinberger, Florian; Mehrkens, Dennis; Starbatty, Jutta; Nicol, Philipp; Eschenhagen, Thomas

    2015-01-01

    Highlights: • Islet-1 was expressed in the adult heart. • Islet-1-positive cells did not proliferate in the adult heart. • Sinoatrial node cells did not proliferate in the adult heart. - Abstract: Rationale: Islet-1 positive (Islet-1 + ) cardiac progenitor cells give rise to the right ventricle, atria and outflow tract during murine cardiac development. In the adult heart Islet-1 expression is limited to parasympathetic neurons, few cardiomyocytes, smooth muscle cells, within the proximal aorta and pulmonary artery and sinoatrial node cells. Its role in these cells is unknown. Here we tested the hypothesis that Islet-1 + cells retain proliferative activity and may therefore play a role in regenerating specialized regions in the heart. Methods and results: DNA synthesis was analyzed by the incorporation of tritiated thymidine ( 3 H-thymidine) in Isl-1-nLacZ mice, a transgenic model with an insertion of a nuclear beta-galactosidase in the Islet-1 locus. Mice received daily injections of 3 H-thymidine for 5 days. DNA synthesis was visualized throughout the heart by dipping autoradiography of cryosections. Colocalization of an nLacZ-signal and silver grains would indicate DNA synthesis in Islet-1 + cells. Whereas Islet − non-myocyte nuclei were regularly marked by accumulation of silver grains, colocalization with nLacZ-signals was not detected in >25,000 cells analyzed. Conclusions: Islet-1 + cells are quiescent in the adult heart, suggesting that, under normal conditions, even pacemaking cells do not proliferate at higher rates than normal cardiac myocytes

  17. Bactericidal Activity of Ceragenin CSA-13 in Cell Culture and in an Animal Model of Peritoneal Infection.

    Science.gov (United States)

    Bucki, Robert; Niemirowicz, Katarzyna; Wnorowska, Urszula; Byfield, Fitzroy J; Piktel, Ewelina; Wątek, Marzena; Janmey, Paul A; Savage, Paul B

    2015-10-01

    Ceragenins constitute a novel family of cationic antibiotics characterized by a broad spectrum of antimicrobial activities, which have mostly been assessed in vitro. Using a polarized human lung epithelial cell culture system, we evaluated the antibacterial activities of the ceragenin CSA-13 against two strains of Pseudomonas aeruginosa (PAO1 and Xen5). Additionally, the biodistribution and bactericidal activity of a CSA-13-IRDye 800CW derivate were assessed using an animal model of peritoneal infection after PAO1 challenge. In cell culture, CSA-13 bactericidal activities against PAO1 and Xen5 were higher than the activities of the human cathelicidin peptide LL-37. Increased CSA-13 activity was observed in polarized human lung epithelial cell cultures subjected to butyric acid treatment, which is known to increase endogenous LL-37 production. Eight hours after intravenous or intraperitoneal injection, the greatest CSA-13-IRDye 800CW accumulation was observed in mouse liver and kidneys. CSA-13-IRDye 800CW administration resulted in decreased bacterial outgrowth from abdominal fluid collected from animals subjected to intraperitoneal PAO1 infection. These observations indicate that CSA-13 may synergistically interact with antibacterial factors that are naturally present at mucosal surfaces and it maintains its antibacterial activity in the infected abdominal cavity. Cationic lipids such as CSA-13 represent excellent candidates for the development of new antibacterial compounds. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  18. Transcriptional profiling of feline infectious peritonitis virus infection in CRFK cells and in PBMCs from FIP diagnosed cats.

    Science.gov (United States)

    Harun, Mohammad Syamsul Reza; Kuan, Choong Oi; Selvarajah, Gayathri Thevi; Wei, Tan Sheau; Arshad, Siti Suri; Hair Bejo, Mohd; Omar, Abdul Rahman

    2013-11-09

    Feline Infectious Peritonitis (FIP) is a lethal systemic disease, caused by the FIP Virus (FIPV); a virulent mutant of Feline Enteric Coronavirus (FECV). Currently, the viruses virulence determinants and host gene expressions during FIPV infection are not fully understood. RNA sequencing of Crandell Rees Feline Kidney (CRFK) cells, infected with FIPV strain 79-1146 at 3 hours post infection (h.p.i), were sequenced using the Illumina next generation sequencing approach. Bioinformatic's analysis, based on Felis catus 2X annotated shotgun reference genome, using CLC bio Genome Workbench mapped both control and infected cell reads to 18899 genes out of 19046 annotated genes. Kal's Z test statistical analysis was used to analyse the differentially expressed genes from the infected CRFK cells. Real time RT-qPCR was developed for further transcriptional profiling of three genes (PD-1, PD-L1 and A3H) in infected CRFK cells and Peripheral Blood Mononuclear Cells (PBMCs) from healthy and FIP-diseased cats. Based on Kal's Z-test, with False Discovery Rate (FDR) 1.99 fold change on gene expressions, a total of 61 genes were differentially expressed by both samples, where 44 genes were up-regulated and the remainder were down-regulated. Most genes were closely clustered together, suggesting a homogeneous expression. The majority of the genes that were significantly regulated, were those associated with monocytes-macrophage and Th1 cell functions, and the regulation of apoptosis. Real time RT-qPCR developed focusing on 2 up-regulated genes (PD-L1 and A3H) together with an apoptosis associated gene PD-1 expressions in FIPV infected CRFK cells and in PBMCs from healthy and FIP diagnosed cats produced concordant results with transcriptome data. The possible roles of these genes, and their importance in feline coronaviruses infection, are discussed.

  19. Establishment of a murine graft-versus-myeloma model using allogeneic stem cell transplantation.

    Directory of Open Access Journals (Sweden)

    Marilène Binsfeld

    Full Text Available 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.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 Vβ spectratyping analysis identified Vβ families within CD4 and CD8 T cells, which were associated with both GvM effects and GvHD, whereas other Vβ families within CD4 T cells were associated exclusively with either GvM or GvHD responses.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 Vβ spectratyping, we also demonstrated the presence of GvM unique responses

  20. The M1 muscarinic receptor and its second messenger coupling in human neuroblastoma cells and transfected murine fibroblast cells

    International Nuclear Information System (INIS)

    Mei, Lin.

    1989-01-01

    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 [ 3 H]IP 1 accumulation in the SH-SY5Y cells was decreased in the presence of 1μg/ml pertussis toxin, suggesting that a pertussis toxin sensitive G-protein may be involved in the coupling. Several cell clones which express only M 1 mAChR were generated by transfecting the murine fibroblast B82 cells with the cloned rat genomic m 1 gene. The transfected B82 cells (cTB10) showed specific [ 3 H](-)QNB binding activity. The mAChRs in these cells are of the M 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 1 mAChR density and the receptor-mediated hydrolysis of inositol lipids was studied in 7 clones. The M 1 mAChR densities in these cells characterized by [ 3 H](-)MQNB binding ranged from 12 fmol/10 6 cells in LK3-1 cells to 260 fmol/10 6 cells in the LK3-8 cells

  1. Growth inhibition and differentiation of murine melanoma B16-BL6 cells caused by the combination of cisplatin and caffeine.

    Science.gov (United States)

    Tsuchiya, H; Tomita, K; Yasutake, H; Ueda, Y; Tanaka, M; Sasaki, T

    1989-12-01

    We preliminarily investigated the combined effects of cisplatin and caffeine on murine melanoma B16-BL6 cells in vitro. When caffeine was added before or simultaneously with cisplatin, there was little growth inhibition. The addition of 2.0 mM caffeine after 1 h of exposure to cisplatin inhibited growth and induced cell differentiation. This treatment resulted in fewer cells, and the numbers of melanosomes and mitochondria and the amount of Golgi's complex and endoplasmic reticulum were increased. DNA histograms obtained by flow cytometry showed that cells treated with cisplatin alone accumulated in the G2/M phase, with a partial G2 block. The addition of 2.0 mM caffeine after 1 h of treatment with cisplatin reduced this block. Caffeine caused murine melanoma B16-BL6 cells treated with cisplatin to differentiate, and this inhibited growth.

  2. Notch Signaling Is Associated With ALDH Activity And An Aggressive Metastatic Phenotype In Murine Osteosarcoma Cells

    Directory of Open Access Journals (Sweden)

    Xiaodong eMu

    2013-06-01

    Full Text Available Osteosarcoma (OS is the most common primary malignancy of bone, and pulmonary metastatic disease accounts for nearly all mortality. However, little is known about the biochemical signaling alterations that drive the progression of metastatic disease. Two murine OS cell populations, K7M2 and K12, are clonally related but differ significantly in their metastatic phenotypes and therefore represent excellent tools for studying metastatic OS molecular biology. K7M2 cells are highly metastatic, whereas K12 cells display limited metastatic potential. Here we report that the expression of Notch genes (Notch1, 2, 4 are up-regulated, including downstream targets Hes1 and Stat3, in the highly metastatic K7M2 cells compared to the less metastatic K12 cells, indicating that the Notch signaling pathway is more active in K7M2 cells. We have previously described that K7M2 cells exhibit higher levels of aldehyde dehydrogenase (ALDH activity. Here we report that K7M2 cell ALDH activity is reduced with Notch inhibition, suggesting that ALDH activity may be regulated in part by the Notch pathway. Notch signaling is also associated with increased resistance to oxidative stress, migration, invasion, and VEGF expression in vitro. However, Notch inhibition did not significantly alter K7M2 cell proliferation. In conclusion, we provide evidence that Notch signaling is associated with ALDH activity and increased metastatic behavior in OS cells. Both Notch and ALDH are putative molecular targets for the treatment and prevention of OS metastasis.

  3. A novel murine T-cell receptor targeting NY-ESO-1.

    Science.gov (United States)

    Rosati, Shannon F; Parkhurst, Maria R; Hong, Young; Zheng, Zhili; Feldman, Steven A; Rao, Mahadev; Abate-Daga, Daniel; Beard, Rachel E; Xu, Hui; Black, Mary A; Robbins, Paul F; Schrump, David A; Rosenberg, Steven A; Morgan, Richard A

    2014-04-01

    Cancer testis antigens, such as NY-ESO-1, are expressed in a variety of prevalent tumors and represent potential targets for T-cell receptor (TCR) gene therapy. DNA encoding a murine anti-NY-ESO-1 TCR gene (mTCR) was isolated from immunized HLA-A*0201 transgenic mice and inserted into a γ-retroviral vector. Two mTCR vectors were produced and used to transduce human PBL. Transduced cells were cocultured with tumor target cell lines and T2 cells pulsed with the NY-ESO-1 peptide, and assayed for cytokine release and cell lysis activity. The most active TCR construct was selected for production of a master cell bank for clinical use. mTCR-transduced PBL maintained TCR expression in short-term and long-term culture, ranging from 50% to 90% efficiency 7-11 days after stimulation and 46%-82% 10-20 days after restimulation. High levels of interferon-γ secretion were observed (1000-12000 pg/mL), in tumor coculture assays and recognition of peptide-pulsed cells was observed at 0.1 ng/mL, suggesting that the new mTCR had high avidity for antigen recognition. mTCR-transduced T cells also specifically lysed human tumor targets. In all assays, the mTCR was equivalent or better than the comparable human TCR. As the functional activity of TCR-transduced cells may be affected by the formation of mixed dimers, mTCRs, which are less likely to form mixed dimers with endogenous hTCRs, may be more effective in vivo. This new mTCR targeted to NY-ESO-1 represents a novel potential therapeutic option for adoptive cell-transfer therapy for a variety of malignancies.

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

    Science.gov (United States)

    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

    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.

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

    Directory of Open Access Journals (Sweden)

    Bin Zheng

    Full Text Available 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.

  6. Anti-Melanogenic Property of Geoditin A in Murine B16 Melanoma Cells

    Directory of Open Access Journals (Sweden)

    Chun-Tao Che

    2012-02-01

    Full Text Available 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.

  7. Antitumour Effects of Isocurcumenol Isolated from Curcuma zedoaria Rhizomes on Human and Murine Cancer Cells

    Science.gov (United States)

    Lakshmi, S.; Padmaja, G.; Remani, P.

    2011-01-01

    Curcuma zedoaria belonging to the family Zingiberaceae has been used in the traditional system of medicine in India and Southwest Asia in treating many human ailments and is found to possess many biological activities. The rationale of the present study was to isolate, identify, and characterize antitumour principles from the rhizomes of Curcuma zedoaria, to assess its cytotoxic effects on human and murine cancer cells, to determine its apoptosis inducing capacity in cancer cells, and to evaluate its tumour reducing properties in in vivo mice models. Isocurcumenol was characterized as the active compound by spectroscopy and was found to inhibit the proliferation of cancer cells without inducing significant toxicity to the normal cells. Fluorescent staining exhibited the morphological features of apoptosis in the compound-treated cancer cells. In vivo tumour reduction studies revealed that a dose of 35.7 mg/kg body weight significantly reduced the ascitic tumour in DLA-challenged mice and increased the lifespan with respect to untreated control mice. PMID:27429805

  8. X-ray induced alterations in the differentiation and mineralization potential of murine preosteoblastic cells

    Science.gov (United States)

    Hu, Yueyuan; Lau, Patrick; Baumstark-Khan, Christa; Hellweg, Christine E.; Reitz, Günther

    2012-05-01

    To evaluate the effects of ionizing radiation (IR) on murine preosteoblastic cell differentiation, we directed OCT-1 cells to the osteoblastic lineage by treatment with a combination of β-glycerophosphate (β-GP), ascorbic acid (AA), and dexamethasone (Dex). In vitro mineralization was evaluated based on histochemical staining and quantification of the hydroxyapatite content of the extracellular bone matrix. Expression of mRNA encoding Runx2, transforming growth factor β1 (TGF-β1), osteocalcin (OCN), and p21CDKN1A was analyzed. Exposure to IR reduced the growth rate and diminished cell survival of OCT-1 cells under standard conditions. Notably, calcium content analysis revealed that deposition of mineralized matrix increased significantly under osteogenic conditions after X-ray exposure in a time-dependent manner. In this study, higher radiation doses exert significant overall effects on TGF-β1, OCN, and p21CDKN1A gene expression, suggesting that gene expression following X-ray treatment is affected in a dose-dependent manner. Additionally, we verified that Runx2 was suppressed within 24 h after irradiation at 2 and 4 Gy. Although further studies are required to verify the molecular mechanism, our observations strongly suggest that treatment with IR markedly alters the differentiation and mineralization process of preosteoblastic cells.

  9. Prospective Isolation of Murine and Human Bone Marrow Mesenchymal Stem Cells Based on Surface Markers

    Directory of Open Access Journals (Sweden)

    Yo Mabuchi

    2013-01-01

    Full Text Available Mesenchymal stem cells (MSCs are currently defined as multipotent stromal cells that undergo sustained in vitro growth and can give rise to cells of multiple mesenchymal lineages, such as adipocytes, chondrocytes, and osteoblasts. The regenerative and immunosuppressive properties of MSCs have led to numerous clinical trials exploring their utility for the treatment of a variety of diseases (e.g., acute graft-versus-host disease, Crohn’s disease, multiple sclerosis, osteoarthritis, and cardiovascular diseases including heart failure and myocardial infarction. On the other hand, conventionally cultured MSCs reflect heterogeneous populations that often contain contaminating cells due to the significant variability in isolation methods and the lack of specific MSC markers. This review article focuses on recent developments in the MSC research field, with a special emphasis on the identification of novel surface markers for the in vivo localization and prospective isolation of murine and human MSCs. Furthermore, we discuss the physiological importance of MSC subtypes in vivo with specific reference to data supporting their contribution to HSC niche homeostasis. The isolation of MSCs using selective markers (combination of PDGFRα and Sca-1 is crucial to address the many unanswered questions pertaining to these cells and has the potential to enhance their therapeutic potential enormously.

  10. Effects of taurolidine on radiosensitivity of murine melanoma cells and its mechanism

    International Nuclear Information System (INIS)

    Sun Baosheng; Liu Shixin; Wang Tiejun; Liu Linlin; Huang Guomin; Gong Shouliang

    2008-01-01

    Objective: To observe the effects of taurolidine on radiosensitivity of B16-F10 cells of murine melanoma via the enhancement of Bax and Bad proteins and induction of Bcl-2 protein. Methods: The apoptosis of B16-F10 cells was assessed after treated with 0, 10, 25, 50, 100 and 150 μmol·L -1 taurolidine, clone survival assay was used to detect the radiosensitivity of B16-F10 cells, and protein expressions were determined by Western blotting. Results: The apoptosis of 5% cells was induced in a dose-and time-dependent manner after B16-F10 cells were treated with 50 μmol·L -1 taurolidine. The survival rate decreased after treated with tautolidine in combination with 2 Gy X-irradiation with the increase of taurolidine concentration and doses of irradiation (P 0 and SER D q ) also increased with the increase of its concentration, there was significant difference between 50 μmol·L -1 taurolidine group and 10 μmol·L -1 taurolidine group (P<0.05); meantime, the level of proapototic protein Bax and Bad increased and the level of antiapoptotic protein Bcl-2 reduced. Conclusion: Taurolidine in combination with irradiation can enhance the radiosensitivity by the mediation of Bcl-2 family protein. (authors)

  11. Cytotoxic and toxicological effects of phthalimide derivatives on tumor and normal murine cells

    Directory of Open Access Journals (Sweden)

    PAULO MICHEL PINHEIRO FERREIRA

    2015-03-01

    Full Text Available Eleven phthalimide derivatives were evaluated with regards to their antiproliferative activity on tumor and normal cells and possible toxic effects. Cytotoxic analyses were performed against murine tumors (Sarcoma 180 and B-16/F-10 cells and peripheral blood mononuclear cells (PBMC using MTT and Alamar Blue assays. Following, the investigation of cytotoxicity was executed by flow cytometry analysis and antitumoral and toxicological potential by in vivo techniques. The molecules 3b, 3c, 4 and 5 revealed in vitro cytotoxicity against Sarcoma 180, B-16/F-10 and PBMC. Since compound 4 was the most effective derivative, it was chosen to detail the mechanism of action after 24, 48 and 72 h exposure (22.5 and 45 µM. Sarcoma 180 cells treated with compound 4 showed membrane disruption, DNA fragmentation and mitochondrial depolarization in a time- and dose-dependent way. Compounds 3c, 4 and 5 (50 mg/kg/day did not inhibit in vivotumor growth. Compound 4-treated animals exhibited an increase in total leukocytes, lymphocytes and spleen relative weight, a decreasing in neutrophils and hyperplasia of spleen white pulp. Treated animals presented reversible histological changes. Molecule 4 had in vitro antiproliferative action possibly triggered by apoptosis, reversible toxic effects on kidneys, spleen and livers and exhibited immunostimulant properties that can be explored to attack neoplasic cells.

  12. Influence and timing of arrival of murine neural crest on pancreatic beta cell development and maturation.

    Science.gov (United States)

    Plank, Jennifer L; Mundell, Nathan A; Frist, Audrey Y; LeGrone, Alison W; Kim, Thomas; Musser, Melissa A; Walter, Teagan J; Labosky, Patricia A

    2011-01-15

    Interactions between cells from the ectoderm and mesoderm influence development of the endodermally-derived pancreas. While much is known about how mesoderm regulates pancreatic development, relatively little is understood about how and when the ectodermally-derived neural crest regulates pancreatic development and specifically, beta cell maturation. A previous study demonstrated that signals from the neural crest regulate beta cell proliferation and ultimately, beta cell mass. Here, we expand on that work to describe timing of neural crest arrival at the developing pancreatic bud and extend our knowledge of the non-cell autonomous role for neural crest derivatives in the process of beta cell maturation. We demonstrated that murine neural crest entered the pancreatic mesenchyme between the 26 and 27 somite stages (approximately 10.0 dpc) and became intermingled with pancreatic progenitors as the epithelium branched into the surrounding mesenchyme. Using a neural crest-specific deletion of the Forkhead transcription factor Foxd3, we ablated neural crest cells that migrate to the pancreatic primordium. Consistent with previous data, in the absence of Foxd3, and therefore the absence of neural crest cells, proliferation of insulin-expressing cells and insulin-positive area are increased. Analysis of endocrine cell gene expression in the absence of neural crest demonstrated that, although the number of insulin-expressing cells was increased, beta cell maturation was significantly impaired. Decreased MafA and Pdx1 expression illustrated the defect in beta cell maturation; we discovered that without neural crest, there was a reduction in the percentage of insulin-positive cells that co-expressed Glut2 and Pdx1 compared to controls. In addition, transmission electron microscopy analyses revealed decreased numbers of characteristic insulin granules and the presence of abnormal granules in insulin-expressing cells from mutant embryos. Together, these data demonstrate that

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

    Energy Technology Data Exchange (ETDEWEB)

    Dianhar, Hanhan, E-mail: liadewi@chem.itb.ac.id; Syah, Yana Maolana, E-mail: liadewi@chem.itb.ac.id; Mujahidin, Didin, E-mail: liadewi@chem.itb.ac.id; Hakim, Euis Holisotan, E-mail: liadewi@chem.itb.ac.id; Juliawaty, Lia Dewi, E-mail: liadewi@chem.itb.ac.id [Natural Product Chemistry Research Group, Organic Chemistry Division, Program Study of Chemistry, Faculty of Mathematics and Natural Sciences, Institut Teknologi Bandung, Jalan Ganeca 10, Bandung 40132 (Indonesia)

    2014-03-24

    Sesbania sesban, locally named as Jayanti, is one of Indonesia plants belonging to Fabaceae family. This species is traditionally used by Indonesian people to cure digestive disorders, fever, or headache. Jayanti can grow well in tropical to subtropical region, such as in Asia and Africa. Based on literature, qualitative analysis of the methanol extract of leaves of S. sesban showed that it contained flavonoids, alkaloids, saponins and glycosides. In addition, the activity assay of extracts of different tissues of this species showed antitumor, antimalarial, and antidiabetic activityies (leaves and seed extracts), antioxidants (flower extract), and analgesic (wood extract). Though the extracts of S. sesban parts showed interesting activities, chemical study of those extracts have not been widely reported. Therefore, the objective of this research was to isolate the secondary metabolites from methanol extract of leaves of S. sesban and to determine their cytotoxicity against murine leukemia P-388 cells. One compound has been obtained and identified as 3-hydroxy-4',7-dimethoxyflavone (1), a new isolated compound from nature. This compound was obtained through separation of methanol extract using various chromatographic techniques, such as vacuum liquid chromatography and radial chromatography. The structure elucidation of isolated compound was based on 1D NMR ({sup 1}H-NMR and {sup 13}C-NMR) and 2D NMR (HMBC). The cytotoxicity of methanol extract and compound 1 against murine leukemia P-388 cells examined through MTT assay showed IC{sub 50} value of 60.04 μg/mL and 5.40 μg/mL, respectively.

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

    Energy Technology Data Exchange (ETDEWEB)

    Dianhar, Hanhan; Syah, Yana Maolana, E-mail: liadewi@chem.itb.ac.id; Mujahidin, Didin; Hakim, Euis Holisotan, E-mail: liadewi@chem.itb.ac.id; Juliawaty, Lia Dewi, E-mail: liadewi@chem.itb.ac.id [Natural Product Chemistry Research Group, Organic Chemistry Division, Program Study of Chemistry, Faculty of Mathematics and Natural Sciences, Institut Teknologi Bandung, Jalan Ganeca 10, Bandung 40132 (Indonesia)

    2014-03-24

    Sesbania sesban, locally named as Jayanti, is one of Indonesia plants belonging to Fabaceae family. This species is traditionally used by Indonesian people to cure digestive disorders, fever, or headache. Jayanti can grow well in tropical to subtropical region, such as in Asia and Africa. Based on literature, qualitative analysis of the methanol extract of leaves of S. sesban showed that it contained flavonoids, alkaloids, saponins and glycosides. In addition, the activity assay of extracts of different tissues of this species showed antitumor, antimalarial, and antidiabetic activityies (leaves and seed extracts), antioxidants (flower extract), and analgesic (wood extract). Though the extracts of S. sesban parts showed interesting activities, chemical study of those extracts have not been widely reported. Therefore, the objective of this research was to isolate the secondary metabolites from methanol extract of leaves of S. sesban and to determine their cytotoxicity against murine leukemia P-388 cells. One compound has been obtained and identified as 3-hydroxy-4',7-dimethoxyflavone (1), a new isolated compound from nature. This compound was obtained through separation of methanol extract using various chromatographic techniques, such as vacuum liquid chromatography and radial chromatography. The structure elucidation of isolated compound was based on 1D NMR ({sup 1}H-NMR and {sup 13}C-NMR) and 2D NMR (HMBC). The cytotoxicity of methanol extract and compound 1 against murine leukemia P-388 cells examined through MTT assay showed IC{sub 50} value of 60.04 μg/mL and 5.40 μg/mL, respectively.

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

    Science.gov (United States)

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

    2014-03-01

    Sesbania sesban, locally named as Jayanti, is one of Indonesia plants belonging to Fabaceae family. This species is traditionally used by Indonesian people to cure digestive disorders, fever, or headache. Jayanti can grow well in tropical to subtropical region, such as in Asia and Africa. Based on literature, qualitative analysis of the methanol extract of leaves of S. sesban showed that it contained flavonoids, alkaloids, saponins and glycosides. In addition, the activity assay of extracts of different tissues of this species showed antitumor, antimalarial, and antidiabetic activityies (leaves and seed extracts), antioxidants (flower extract), and analgesic (wood extract). Though the extracts of S. sesban parts showed interesting activities, chemical study of those extracts have not been widely reported. Therefore, the objective of this research was to isolate the secondary metabolites from methanol extract of leaves of S. sesban and to determine their cytotoxicity against murine leukemia P-388 cells. One compound has been obtained and identified as 3-hydroxy-4',7-dimethoxyflavone (1), a new isolated compound from nature. This compound was obtained through separation of methanol extract using various chromatographic techniques, such as vacuum liquid chromatography and radial chromatography. The structure elucidation of isolated compound was based on 1D NMR (1H-NMR and 13C-NMR) and 2D NMR (HMBC). The cytotoxicity of methanol extract and compound 1 against murine leukemia P-388 cells examined through MTT assay showed IC50 value of 60.04 μg/mL and 5.40 μg/mL, respectively.

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

    International Nuclear Information System (INIS)

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

    2014-01-01

    Sesbania sesban, locally named as Jayanti, is one of Indonesia plants belonging to Fabaceae family. This species is traditionally used by Indonesian people to cure digestive disorders, fever, or headache. Jayanti can grow well in tropical to subtropical region, such as in Asia and Africa. Based on literature, qualitative analysis of the methanol extract of leaves of S. sesban showed that it contained flavonoids, alkaloids, saponins and glycosides. In addition, the activity assay of extracts of different tissues of this species showed antitumor, antimalarial, and antidiabetic activityies (leaves and seed extracts), antioxidants (flower extract), and analgesic (wood extract). Though the extracts of S. sesban parts showed interesting activities, chemical study of those extracts have not been widely reported. Therefore, the objective of this research was to isolate the secondary metabolites from methanol extract of leaves of S. sesban and to determine their cytotoxicity against murine leukemia P-388 cells. One compound has been obtained and identified as 3-hydroxy-4',7-dimethoxyflavone (1), a new isolated compound from nature. This compound was obtained through separation of methanol extract using various chromatographic techniques, such as vacuum liquid chromatography and radial chromatography. The structure elucidation of isolated compound was based on 1D NMR ( 1 H-NMR and 13 C-NMR) and 2D NMR (HMBC). The cytotoxicity of methanol extract and compound 1 against murine leukemia P-388 cells examined through MTT assay showed IC 50 value of 60.04 μg/mL and 5.40 μg/mL, respectively

  17. Changes in numbers and types of mast cell colony-forming cells in the peritoneal cavity of mice after injection of distilled water: evidence that mast cells suppress differentiation of bone marrow-derived precursors

    International Nuclear Information System (INIS)

    Kanakura, Y.; Kuriu, A.; Waki, N.; Nakano, T.; Asai, H.; Yonezawa, T.; Kitamura, Y.

    1988-01-01

    Two different types of cells in the peritoneal cavity of mice produce mast cell colonies in methylcellulose. Large mast cell colonies are produced by bone marrow-derived precursors resembling lymphoid cells by light microscopy (L-CFU-Mast), whereas medium and small mast cell colonies are produced by morphologically identifiable mast cells (M-CFU-Mast and S-CFU-Mast, respectively). In the present study we eradicated peritoneal mast cells by intraperitoneal (IP) injection of distilled water. The regeneration process was investigated to clarify the relationship between L-CFU-Mast, M-CFU-Mast, and S-CFU-Mast. After injection of distilled water, M-CFU-Mast and S-CFU-Mast disappeared, but L-CFU-Mast increased, and then M-CFU-Mast and S-CFU-Mast appeared, suggesting the presence of a hierarchic relationship. When purified peritoneal mast cells were injected two days after the water injection, the L-CFU-Mast did not increase. In the peritoneal cavity of WBB6F1-+/+ mice that had been lethally irradiated and rescued by bone marrow cells of C57BL/6-bgJ/bgJ (beige, Chediak-Higashi syndrome) mice, L-CFU-Mast were of bgJ/bgJ type, but M-CFU-Mast and S-CFU-Mast were of +/+ type. The injection of distilled water to the radiation chimeras resulted in the development of bgJ/bgJ-type M-CFU-Mast and then S-CFU-Mast. The presence of mast cells appeared to suppress the recruitment of L-CFU-Mast from the bloodstream and to inhibit the differentiation of L-CFU-Mast to M-CFU-Mast

  18. Inhibition of erythroid cell growth by allogeneic murine lymphocytes. Evidence for a synergism between lymph node cells and thymocytes

    International Nuclear Information System (INIS)

    Blomgren, H.; Jacobsson, H.

    1974-01-01

    Murine lymphoid cells from thymus and lymph nodes were tested for synergistic response in a graft-vs-host test. The test is based on the principle that allogeneic lymphocytes inhibit erythroid cell proliferation in the spleens of irradiated mice infused with syngeneic bone marrow cells. Mixtures of thymocytes and lymph node cells from the same parental strain yielded graft-vs-host responses in irradiated F 1 -hybrids higher than expected by summing the responses of the two cell populations tested separately. A similar synergistic response was obtained using mixtures of thymocytes and lymph node cells obtained from the two parental strains of the hybrid, whereas such an effect was not detected using mixtures of lymph node cells or mixtures of thymocytes from the two parental strains. Nor could synergy be demonstrated between parental strain lymph node cells and thymocytes syngeneic with the bone marrow target cells. Thymocytes obtained from one parental strain which was injected into its irradiated F 1 -hybrid transformed into a population of sensitized cells in the spleens of the recipients. This transformation was suppressed by the simultaneous injection of lymph node cells from the second parental strain. Since there is a synergistic immune response by such cell mixtures it is concluded that thymocytes may enhance the graft-vs-host response of lymph node cells. Parental strain thymocytes and lymph node cells, the latter being specifically immunologically tolerant to the bone marrow target cells, failed to give a synergistic response indicating that thymocytes do not transform unresponsive lymphocytes into responsive, but rather enhance the reactivity of existing, specifically responsive cells. The results thus show that thymocytes may enhance the response of lymph node cells in this specific graft-vs-host assay

  19. Increased endothelial cell-leukocyte interaction in murine schistosomiasis: possible priming of endothelial cells by the disease.

    Directory of Open Access Journals (Sweden)

    Suellen D S Oliveira

    Full Text Available BACKGROUND AND AIMS: Schistosomiasis is an intravascular parasitic disease associated with inflammation. Endothelial cells control leukocyte transmigration and vascular permeability being modulated by pro-inflammatory mediators. Recent data have shown that endothelial cells primed in vivo in the course of a disease keep the information in culture. Herein, we evaluated the impact of schistosomiasis on endothelial cell-regulated events in vivo and in vitro. METHODOLOGY AND PRINCIPAL FINDINGS: The experimental groups consisted of Schistosoma mansoni-infected and age-matched control mice. In vivo infection caused a marked influx of leukocytes and an increased protein leakage in the peritoneal cavity, characterizing an inflamed vascular and cellular profile. In vitro leukocyte-mesenteric endothelial cell adhesion was higher in cultured cells from infected mice as compared to controls, either in the basal condition or after treatment with the pro-inflammatory cytokine tumor necrosis factor (TNF. Nitric oxide (NO donation reduced leukocyte adhesion to endothelial cells from control and infected groups; however, in the later group the effect was more pronounced, probably due to a reduced NO production. Inhibition of control endothelial NO synthase (eNOS increased leukocyte adhesion to a level similar to the one observed in the infected group. Besides, the adhesion of control leukocytes to endothelial cells from infected animals is similar to the result of infected animals, confirming that schistosomiasis alters endothelial cells function. Furthermore, NO production as well as the expression of eNOS were reduced in cultured endothelial cells from infected animals. On the other hand, the expression of its repressor protein, namely caveolin-1, was similar in both control and infected groups. CONCLUSION/SIGNIFICANCE: Schistosomiasis increases vascular permeability and endothelial cell-leukocyte interaction in vivo and in vitro. These effects are partially

  20. [Inhibitory effect of murine cytomegalovirus infection on neural stem cells' differentiation and its mechanisms].

    Science.gov (United States)

    Zhou, Yu-feng; Fang, Feng; Dong, Yong-sui; Zhou, Hua; Zhen, Hong; Liu, Jin; Li, Ge

    2006-07-01

    Cytomegalovirus (CMV) is the leading infectious cause of congenital anomalies of the central nervous system caused by intrauterine infection. However, the exact pathogenesis of these brain abnormalities has not been fully elucidated. It has been reported that periependymitis, periventricular necrosis and calcification are the most frequent findings in the brains of congenital CMV infection. Because a number of multipotential neural stem cells (NSCs) have been identified from ventricular zone, it is possible that NSCs in this area are primary targets for viral infection, which seems to be primarily responsible for the generation of the brain abnormalities. Therefore, the objective of the present study was to investigate the effect and mechanism of murine cytomegalovirus (MCMV) infection on neural stem cells' differentiation in vitro and its role in the mechanisms of brain abnormalities caused by congenital cytomegalovirus infection. NSCs were prepared from fetal BALB/c mouse and were infected with recombinant MCMV RM461 inserted with a report gene LacZ at 1 multiplicity of infection (MOI = 1). The effect of MCMV infection on neural stem cells' differentiation was observed by detecting the ratio of nestin, GFAP and NSE positive cells with immunohistochemistry and flow cytometry on day 2 postinfection. The effects of MCMV infection on gene expression of Wnt-1 and neurogenin 1 (Ngn1) related to neural differentiation were detected by RT-PCR. NSCs isolated from embryonic mouse brains strongly expressed nestin, a specific marker of NSCs and had the capacity to differentiate into NF-200 and NSE positive neurons or GFAP positive astrocytes. At MOI = 1, the results of flow cytometry assay showed that nestin positive cells' proportion in the infection group [(62.2 +/- 1.8)%] was higher than that in the normal group [(37.2 +/- 2.4)%] (t = 4.62, P differentiation, which may be primary causes of disorders of brain development in congenital CMV infection. The decreased

  1. The potential role of ribosomal protein S5 on cell cycle arrest and initiation of murine erythroleukemia cell differentiation.

    Science.gov (United States)

    Matragkou, Christina N; Papachristou, Eleni T; Tezias, Sotirios S; Tsiftsoglou, Asterios S; Choli-Papadopoulou, Theodora; Vizirianakis, Ioannis S

    2008-07-01

    Evidence now exists to indicate that some ribosomal proteins besides being structural components of the ribosomal subunits are involved in the regulation of cell differentiation and apoptosis. As we have shown earlier, initiation of erythroid differentiation of murine erythroleukemia (MEL) cells is associated with transcriptional inactivation of genes encoding ribosomal RNAs and ribosomal proteins S5 (RPS5) and L35a. In this study, we extended these observations and investigated whether transfection of MEL cells with RPS5 cDNA affects the onset of initiation of erythroid maturation and their entrance in cell cycle arrest. Stably transfected MEL cloned cells (MEL-C14 and MEL-C56) were established and assessed for their capacity to produce RPS5 RNA transcript and its translated product. The impact of RPS5 cDNA transfection on the RPS5 gene expression patterns and the accumulation of RPS5 protein in inducible transfected MEL cells were correlated with their ability to: (a) initiate differentiation, (b) enter cell cycle arrest at G(1)/G(0) phase, and (c) modulate the level of cyclin-dependent kinases CDK2, CDK4, and CDK6. The data presented indicate that deregulation of RPS5 gene expression (constitutive expression) affects RPS5 protein level and delays both the onset of initiation of erythroid maturation and entrance in cell cycle arrest in inducer-treated MEL cells. 2008 Wiley-Liss, Inc.

  2. Particle Size-Dependent Antibacterial Activity and Murine Cell Cytotoxicity Induced by Graphene Oxide Nanomaterials

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    Lin Zhao

    2016-01-01

    Full Text Available Recent studies have indicated that graphene and its derivative graphene oxide (GO engage in a wide range of antibacterial activities with limited toxicity to human cells. Here, we systematically evaluate the dependence of GO toxicity on the size of the nanoparticles used in treatments: we compare the cytotoxic effects of graphene quantum dots (GQDs, <15 nm, small GOs (SGOs, 50–200 nm, and large GOs (LGOs, 0.5–3 μm. We synthesize the results of bacterial colony count assays and SEM-based observations of morphological changes to assess the antibacterial properties that these GOs bring into effect against E. coli. We also use Live/Dead assays and morphological analysis to investigate changes to mammalian (Murine macrophage-like Raw 264.7 cells induced by the presence of the various GO particle types. Our results demonstrate that LGOs, SGOs, and GQDs possess antibacterial activities and cause mammalian cell cytotoxicity at descending levels of potency. Placing our observations in the context of previous simulation results, we suggest that both the lateral size and surface area of GO particles contribute to cytotoxic effects. We hope that the size dependence elucidated here provides a useful schematic for tuning GO-cell interactions in biomedical applications.

  3. Immunological response induced by cryoablation against murine H22 hepatoma cell line in vivo.

    Science.gov (United States)

    Yang, Xueling; Li, Xiaoli; Guo, Zhi; Si, Tongguo; Yu, Haipeng; Xing, Wenge

    2018-02-01

    To describe immunological consequences induced by cryoablation against H22 cells in vivo. Adult BALB/c mice underwent subcutaneous implantation of H22 cells. All of them were assigned into three groups randomly: group A (false surgery), group B (cryoablation) and group C (cryoablation plus Freund's adjuvant). Animals were sacrificed 1, 2 and 3 weeks after treatment. Serum IFN-γ and IL-4, Th1/Th2 in spleens and cytotoxicity were detected. Compared with that of group A, (1) INF-γ of group B was higher, but IL-4 was lower; cryoablation plus Freund's adjuvant enhanced these effects. (2) Th1/Th2 rose significantly in both group B and group C. (3) Strong cytolytic activity against H22 cells of group B and group C was found on day 7, 14 and 21. Our study showed a marked shift toward Th1 and IFN-γ expression after cryoablation, with an immuno-stimulatory effect against murine H22 hepatoma Cell. Copyright © 2017. Published by Elsevier Inc.

  4. Mediator Med23 deficiency enhances neural differentiation of murine embryonic stem cells through modulating BMP signaling.

    Science.gov (United States)

    Zhu, Wanqu; Yao, Xiao; Liang, Yan; Liang, Dan; Song, Lu; Jing, Naihe; Li, Jinsong; Wang, Gang

    2015-02-01

    Unraveling the mechanisms underlying early neural differentiation of embryonic stem cells (ESCs) is crucial to developing cell-based therapies of neurodegenerative diseases. Neural fate acquisition is proposed to be controlled by a 'default' mechanism, for which the molecular regulation is not well understood. In this study, we investigated the functional roles of Mediator Med23 in pluripotency and lineage commitment of murine ESCs. Unexpectedly, we found that, despite the largely unchanged pluripotency and self-renewal of ESCs, Med23 depletion rendered the cells prone to neural differentiation in different differentiation assays. Knockdown of two other Mediator subunits, Med1 and Med15, did not alter the neural differentiation of ESCs. Med15 knockdown selectively inhibited endoderm differentiation, suggesting the specificity of cell fate control by distinctive Mediator subunits. Gene profiling revealed that Med23 depletion attenuated BMP signaling in ESCs. Mechanistically, MED23 modulated Bmp4 expression by controlling the activity of ETS1, which is involved in Bmp4 promoter-enhancer communication. Interestingly, med23 knockdown in zebrafish embryos also enhanced neural development at early embryogenesis, which could be reversed by co-injection of bmp4 mRNA. Taken together, our study reveals an intrinsic, restrictive role of MED23 in early neural development, thus providing new molecular insights for neural fate determination. © 2015. Published by The Company of Biologists Ltd.

  5. Effects of ultraviolet irradiation and postirradiation incubation on heterogeneous nuclear RNA size in murine cells

    International Nuclear Information System (INIS)

    Ali, R.; Sauerbier, W.

    1978-01-01

    We have analyzed the decrease in synthesis of individual size classes of heterogeneous nuclear RNA (hnRNA) in ultraviolet (uv)-irradiated Merwin plasmacytoma (MPC-11) cells at various times of postirradiation incubation. HnRNA from nonirradiated control cells is distributed over a wide range from approximately 60S to 5S, with 42S RNA carrying more label than any other size class. HnRNA from uv-irradiated cells shows a dose-dependent shift in size distribution toward lower molecular weight. The size distribution of hnRNA synthesized after prolonged times of postirradiation incubation is restored toward normal, i.e., synthesis of long RNA molecules increases relative to the synthesis of short ones. Analysis of the total number of hnRNA chains synthesized during a 20-min [ 3 H]uridine pulse shows a considerable eduction in their number with increasing uv dose. Murine cell lines are excision-repair-deficient but capable of post replication repair inhibited by caffeine. HnRNA transcripts of cells incubated in its presence were studied. The caffeine, which has no effect on hnRNA size in control cells, inhibits to a considerable extent the restoration of full-length transcripts during postirradiation incubation. The lack of excision repair in MPC-11 was confirmed by the analysis of pyrimidine dimers in trichloracetic acid-insoluble and soluble fractions within 8 h of postirradiation incubation. The size of parental and daughter strand DNA in uv-irradiated cells was correlated with RNA transcript size. The parental DNA in these experiments does not change its size as a consequence of uv exposure and postirradiation incubation. In contrast, daughter DNA strands are short in uv-irradiated cells and they increase in size during postirradiation incubation to reach the size of parental strands after 8 h

  6. Agmatine promotes the migration of murine brain endothelial cells via multiple signaling pathways.

    Science.gov (United States)

    Jung, Hyun-Joo; Jeon, Yong-Heui; Bokara, Kiran Kumar; Koo, Bon-Nyeo; Lee, Won Taek; Park, Kyung Ah; Lee, Jong-Eun

    2013-01-17

    The combination of adhesion and migration of endothelial cells (ECs) is an integral process for evolution, organization, repair and vessel formation in living organisms. Agmatine, a polycationic amine existing in brain, has been investigated to exert neuroprotective effects. Up to date, there are no studies reporting that agmatine modulates murine brain endothelial (bEnd.3) cells migration. In the present study, we intend to investigate the role of agmatine in bEnd.3 cells migration and the molecular mechanism mediating this action. The effect of agmatine on the bEnd.3 cells migration was examined by migration assay, and the mechanism involved for this effect was investigated by western blot analysis and NO contents measurements. Agmatine treatment (50, 100 and 200 μM) significantly accelerated bEnd.3 cells migration in a concentration-dependent manner. Western blotting revealed that agmatine treatment significantly induced vascular endothelial growth factor (VEGF), VEGF receptor 2 (Flk-1/KDR or VEGFR2), phosphatidylinositol 3-kinase (PI3K), Akt/protein kinase B (also known as PKB, PI3K downstream effector protein), endothelial nitric oxide synthase (eNOS) nitric oxide (NO; product by eNOS) and intercellular adhesion molecule 1 (ICAM-1) expressions during bEnd.3 cells migration. The expression of ICAM-1 and migration of bEnd.3 cells, induced by agmatine, were significantly attenuated by treatment of wortmannin, a specific PI3K inhibitor. Taken together, we provide the first evidence that activation of VEGF/VEGFR2 and the consequential PI3K/Akt/eNOS/NO/ICAM-1 signaling pathways are serial events, through which the treatment of agmatine could lead to bEnd.3 cells migration. Copyright © 2012 Elsevier Inc. All rights reserved.

  7. Human cardiac-derived adherent proliferating cells reduce murine acute Coxsackievirus B3-induced myocarditis.

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    Kapka Miteva

    Full Text Available BACKGROUND: Under conventional heart failure therapy, inflammatory cardiomyopathy typically has a progressive course, indicating a need for alternative therapeutic strategies to improve long-term outcomes. We recently isolated and identified novel cardiac-derived cells from human cardiac biopsies: cardiac-derived adherent proliferating cells (CAPs. They have similarities with mesenchymal stromal cells, which are known for their anti-apoptotic and immunomodulatory properties. We explored whether CAPs application could be a novel strategy to improve acute Coxsackievirus B3 (CVB3-induced myocarditis. METHODOLOGY/PRINCIPAL FINDINGS: To evaluate the safety of our approach, we first analyzed the expression of the coxsackie- and adenovirus receptor (CAR and the co-receptor CD55 on CAPs, which are both required for effective CVB3 infectivity. We could demonstrate that CAPs only minimally express both receptors, which translates to minimal CVB3 copy numbers, and without viral particle release after CVB3 infection. Co-culture of CAPs with CVB3-infected HL-1 cardiomyocytes resulted in a reduction of CVB3-induced HL-1 apoptosis and viral progeny release. In addition, CAPs reduced CD4 and CD8 T cell proliferation. All CAPs-mediated protective effects were nitric oxide- and interleukin-10-dependent and required interferon-γ. In an acute murine model of CVB3-induced myocarditis, application of CAPs led to a decrease of cardiac apoptosis, cardiac CVB3 viral load and improved left ventricular contractility parameters. This was associated with a decline in cardiac mononuclear cell activity, an increase in T regulatory cells and T cell apoptosis, and an increase in left ventricular interleukin-10 and interferon-γ mRNA expression. CONCLUSIONS: We conclude that CAPs are a unique type of cardiac-derived cells and promising tools to improve acute CVB3-induced myocarditis.

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

    Science.gov (United States)

    Meng, Qing-Yuan; Akaike, Toshihiro

    2013-03-01

    Induced embryonic stem (ES) cells are expected to be promising cell resources for the observation of the cell behaviors in developmental biology as well as the implantation in cell treatments in human diseases. A recombinant E-cadherin substratum was developed as a cell recognizable substratum to maintain the ES cells' self-renewal and pluripotency at single cell level. Furthermore, the generation of various cell lineages in different germ layers, including hepatic or neural cells, was achieved on the chimeric protein layer precisely and effectively. The induction and isolation of specific cell population was carried out with the enhancing effect of other artificial extracellular matrices (ECMs) in enzyme-free process. The murine ES cell-derived cells showed highly morphological similarities and functional expressions to matured hepatocytes or neural progenitor cells.

  9. WNT Signaling Is Required for Peritoneal Membrane Angiogenesis.

    Science.gov (United States)

    Padwal, Manreet Kaur; Cheng, Genyang; Liu, Limin; Boivin, Felix J; Gangji, Azim; Brimble, Kenneth Scott; Bridgewater, Darren; Margetts, Peter J

    2018-01-24

    The WNT signaling pathway is involved in wound healing and fibrosis. We evaluated the WNT signaling pathway in peritoneal membrane injury. We assessed WNT1 protein expression in the peritoneal effluents of 54 stable peritoneal dialysis (PD) patients and WNT-related gene expression in ex vivo mesothelial cell cultures from 21 PD patients. In a transforming growth factor beta (TGFB) mediated animal model of peritoneal fibrosis, we evaluated regulation of the WNT pathway and the effect of WNT inhibition on peritoneal fibrosis and angiogenesis. WNT1 and WNT2 gene expression were positively correlated with peritoneal membrane solute transport in PD patients. In the mouse peritoneum, TGFΒ-induced peritoneal fibrosis was associated with increased expression of WNT2 and WNT4. Peritoneal b-catenin protein was significantly upregulated after infection with AdTGFB along with elements of the WNT signaling pathway. Treatment with a b-catenin inhibitor (ICG-001) in mice with AdTGFB-induced peritoneal fibrosis resulted in attenuation of peritoneal angiogenesis and reduced vascular endothelial growth factor. Similar results were also observed with the WNT antagonist Dickkopf related protein (DKK) 1. In addition to this, DKK-1 blocked epithelial to mesenchymal transition and increased levels of the cell adhesion protein E-cadherin. We provide evidence that WNT signaling is active in the setting of experimental peritoneal fibrosis and WNT1 correlates with patient peritoneal membrane solute transport in PD patients. Intervention in this pathway is a possible therapy for peritoneal membrane injury.

  10. HIV-1 Myristoylated Nef Treatment of Murine Microglial Cells Activates Inducible Nitric Oxide Synthase, NO2 Production and Neurotoxic Activity.

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    Giorgio Mangino

    Full Text Available The potential role of the human immunodeficiency virus-1 (HIV-1 accessory protein Nef in the pathogenesis of neuroAIDS is still poorly understood. Nef is a molecular adapter that influences several cellular signal transduction events and membrane trafficking. In human macrophages, Nef expression induces the production of extracellular factors (e.g. pro-inflammatory chemokines and cytokines and the recruitment of T cells, thus favoring their infection and its own transfer to uninfected cells via exosomes, cellular protrusions or cell-to-cell contacts. Murine cells are normally not permissive for HIV-1 but, in transgenic mice, Nef is a major disease determinant. Both in human and murine macrophages, myristoylated Nef (myr+Nef treatment has been shown to activate NF-κB, MAP kinases and interferon responsive factor 3 (IRF-3, thereby inducing tyrosine phosphorylation of signal transducers and activator of transcription (STAT-1, STAT-2 and STAT-3 through the production of proinflammatory factors.We report that treatment of BV-2 murine microglial cells with myr+Nef leads to STAT-1, -2 and -3 tyrosine phosphorylation and upregulates the expression of inducible nitric oxide synthase (iNOS with production of nitric oxide. We provide evidence that extracellular Nef regulates iNOS expression through NF-κB activation and, at least in part, interferon-β (IFNβ release that acts in concert with Nef. All of these effects require both myristoylation and a highly conserved acidic cluster in the viral protein. Finally, we report that Nef induces the release of neurotoxic factors in the supernatants of microglial cells.These results suggest a potential role of extracellular Nef in promoting neuronal injury in the murine model. They also indicate a possible interplay between Nef and host factors in the pathogenesis of neuroAIDS through the production of reactive nitrogen species in microglial cells.

  11. Melanogenesis stimulation in murine B16 melanoma cells by Piper nigrum leaf extract and its lignan constituents.

    Science.gov (United States)

    Matsuda, Hideaki; Kawaguchi, Yoshiko; Yamazaki, Miho; Hirata, Noriko; Naruto, Shunsuke; Asanuma, Yusuke; Kaihatsu, Takayuki; Kubo, Michinori

    2004-10-01

    A methanolic extract from the leaves of Piper nigrum L. showed a significant stimulatory effect on melanogenesis in cultured murine B16 melanoma cells. Activity-guided fractionation of the methanolic extract led to the isolation of two known lignans, (-)-cubebin (1) and (-)-3,4-dimethoxy-3,4-desmethylenedioxycubebin (2), together with a new lignan, (-)-3-desmethoxycubebinin (3). Among these lignans, 1 and 2 showed a significant stimulatory activity of melanogenesis without any significant effects on cell proliferation.

  12. [Peritonitis in pediatric patients receiving peritoneal dialysis].

    Science.gov (United States)

    Jellouli, Manel; Ferjani, Meriem; Abidi, Kamel; Hammi, Yosra; Boutiba, Ilhem; Naija, Ouns; Zarrouk, Chokri; Ben Abdallah, Taieb; Gargah, Tahar

    2015-12-01

    Peritonitis on catheter of dialysis represents the most frequent complication of the peritoneal dialysis (PD) in the pediatric population. It remains a significant cause of morbidity and mortality. In this study, we investigated the risk factors for peritonitis in children. In this study, we retrospectively collected the records of 85 patients who were treated with PD within the past ten years in the service of pediatrics of the University Hospital Charles-Nicolle of Tunis. Peritonitis rate was 0.75 episode per patient-year. Notably, peritonitis caused by Gram-positive organisms were more common. Analysis of infection risk revealed three significant independent factors: the poor weight (P=0.0045), the non-automated PD (P=0.02) and the short delay from catheter insertion to starting PD (P=0.02). The early onset peritonitis was significantly associated with frequent peritonitis episodes (P=0.0008). The mean duration between the first and second episode of peritonitis was significantly shorter than between PD commencement and the first episode of peritonitis. We revealed a significant association between Gram-negative peritonitis and the presence of ureterostomy (0.018) and between Gram-positive peritonitis and the presence of exit-site and tunnel infections (0.02). Transition to permanent hemodialysis was needed in many children but no death occurred in patients with peritonitis. Considering the important incidence of peritonitis in our patients, it is imperative to establish a targeted primary prevention. Nutritional care must be provided to children to avoid poor weight. The automated dialysis has to be the modality of choice. Copyright © 2015 Association Société de néphrologie. Published by Elsevier SAS. All rights reserved.

  13. Prolonged exposure of resveratrol induces reactive superoxide species-independent apoptosis in murine prostate cells.

    Science.gov (United States)

    Kumar, Sanjay; Stokes, James; Singh, Udai P; Scissum-Gunn, Karyn; Singh, Rajesh; Manne, Upender; Mishra, Manoj K

    2017-10-01

    Nitric oxide, a signaling molecule, inhibits mitochondrial respiration by binding with cytochrome c oxidase, resulting in elevated production of reactive superoxide species (reactive oxygen and nitrogen) in the mitochondria and increased susceptibility to cell death. Generation of mitochondrial superoxide species can be suppressed by natural compounds such as resveratrol, a dietary polyphenol found in the skin of red fruits. In various cancer cells, resveratrol shows anti-oxidant and cancer preventive properties. Since, the effect of resveratrol on reactive superoxide species-independent apoptosis in prostate cancer cells is not well illustrated; therefore, we investigated this phenomenon in TRAMP murine prostate cancer cells. To accomplish this, TRAMP cells were incubated with resveratrol, resveratrol + DETA-NONOate, DETA-NONOate (nitric oxide donor), resveratrol + L-NMMA, or L-NMMA (nitric oxide inhibitor) for 48 h, and reactive superoxide species in the mitochondria and culture supernatant were measured. In addition, the mitochondrial membrane potential, cell viability, expression of apoptotic markers (Bax and Bcl2), γ-H2A.x, p53, and caspase-3 was determined. We found that resveratrol suppressed reactive superoxide species such as reactive oxygen species in the mitochondria and nitric oxide in culture supernatant when compared to the DETA-NONOate treatment and disrupted the mitochondrial membrane potential. Resveratrol also reduced cell viability, altered the expression of apoptotic markers (Bax and Bcl2), and increased expression of γ-H2A.x (indicative marker of DNA fragmentation) and p53 (a critical DNA damage response protein). However, there was no appreciable modulation of the caspase-3. Therefore, our data suggest that resveratrol induces superoxide species-independent apoptosis and may act as a therapeutic agent against prostate cancer.

  14. Irradiated murine fibroblasts as feeder layer used in human cell culture

    International Nuclear Information System (INIS)

    Almeida, Tiago L.; Klingbeil, Fatima G.; Yoshito, Daniele; Caproni, Priscila; Mathor, Monica B.; Herson, Marisa R.

    2007-01-01

    In 1975, Rheinwald and Green published an in vitro model for keratinocyte cell cultures in which the use of murine fibroblasts, as a feeder layer was introduced. These cells are modified fibroblasts, which presence render keratinocyte cells to remain proliferative for longer periods of time. This optimization of culture outputs has allowed for several clinical applications of confluent keratinocyte cultures as skin substitutes or wound dressings in situations such as post burn extensive skin loss, loss of oral mucosa, and other skin disorders. Nevertheless, proliferation of fibroblast in co-culture with keratinocytes must be controlled by anti-proliferative measures such as irradiation; at the same time, keratinocytes require specific nutrients in the culture medium, which may interfere with the fibroblast feeder layer viability. Therefore, the thorough understanding of the impact of different issues such as culture media composition, irradiation dose and pre-plating storage conditions of irradiated fibroblast to be used as feeder layer in these co-culture systems is important. In this work, changes as far as viability and proliferative rates of irradiated fibroblasts in culture were evaluated in relation to the type of culture medium used, dose of gamma radiation exposure, storage and timing of cell plating post irradiation. Results indicate that the type of culture medium used and time-lag between irradiation, refrigeration and plating of irradiated cells do not have significant impact in culture outcomes. However, the dose of gamma radiation administered to the cells may influence the final quality of these cells if to be used as a feeder layer. (author)

  15. Human Peritoneal Mesothelial Cell Death Induced by High-Glucose Hypertonic Solution Involves Ca2+ and Na+ Ions and Oxidative Stress with the Participation of PKC/NOX2 and PI3K/Akt Pathways

    Directory of Open Access Journals (Sweden)

    Felipe Simon

    2017-06-01

    Full Text Available Chronic peritoneal dialysis (PD therapy is equally efficient as hemodialysis while providing greater patient comfort and mobility. Therefore, PD is the treatment of choice for several types of renal patients. During PD, a high-glucose hyperosmotic (HGH solution is administered into the peritoneal cavity to generate an osmotic gradient that promotes water and solutes transport from peritoneal blood to the dialysis solution. Unfortunately, PD has been associated with a loss of peritoneal viability and function through the generation of a severe inflammatory state that induces human peritoneal mesothelial cell (HPMC death. Despite this deleterious effect, the precise molecular mechanism of HPMC death as induced by HGH solutions is far from being understood. Therefore, the aim of this study was to explore the pathways involved in HGH solution-induced HPMC death. HGH-induced HPMC death included influxes of intracellular Ca2+ and Na+. Furthermore, HGH-induced HPMC death was inhibited by antioxidant and reducing agents. In line with this, HPMC death was induced solely by increased oxidative stress. In addition to this, the cPKC/NOX2 and PI3K/Akt intracellular signaling pathways also participated in HGH-induced HPMC death. The participation of PI3K/Akt intracellular is in agreement with previously shown in rat PMC apoptosis. These findings contribute toward fully elucidating the underlying molecular mechanism mediating peritoneal mesothelial cell death induced by high-glucose solutions during peritoneal dialysis.

  16. Pathway aberrations of murine melanoma cells observed in Paired-End diTag transcriptomes

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    Liu Edison

    2007-06-01

    Full Text Available Abstract Background Melanoma is the major cause of skin cancer deaths and melanoma incidence doubles every 10 to 20 years. However, little is known about melanoma pathway aberrations. Here we applied the robust Gene Identification Signature Paired End diTag (GIS-PET approach to investigate the melanoma transcriptome and characterize the global pathway aberrations. Methods GIS-PET technology directly links 5' mRNA signatures with their corresponding 3' signatures to generate, and then concatenate, PETs for efficient sequencing. We annotated PETs to pathways of KEGG database and compared the murine B16F1 melanoma transcriptome with three non-melanoma murine transcriptomes (Melan-a2 melanocytes, E14 embryonic stem cells, and E17.5 embryo. Gene expression levels as represented by PET counts were compared across melanoma and melanocyte libraries to identify the most significantly altered pathways and investigate the expression levels of crucial cancer genes. Results Melanin biosynthesis genes were solely expressed in the cells of melanocytic origin, indicating the feasibility of using the PET approach for transcriptome comparison. The most significantly altered pathways were metabolic pathways, including upregulated pathways: purine metabolism, aminophosphonate metabolism, tyrosine metabolism, selenoamino acid metabolism, galactose utilization, nitrobenzene degradation, and bisphenol A degradation; and downregulated pathways: oxidative phosphorylation, ATPase synthesis, TCA cycle, pyruvate metabolism, and glutathione metabolism. The downregulated pathways concurrently indicated a slowdown of mitochondrial activities. Mitochondrial permeability was also significantly altered, as indicated by transcriptional activation of ATP/ADP, citrate/malate, Mg++, fatty acid and amino acid transporters, and transcriptional repression of zinc and metal ion transporters. Upregulation of cell cycle progression, MAPK, and PI3K/Akt pathways were more limited to certain

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

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    Eric R. Secor

    2013-01-01

    Full Text Available 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.

  18. The acute exposure effects of inhaled nickel nanoparticles on murine endothelial progenitor cells.

    Science.gov (United States)

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

    2014-08-01

    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, generation of reactive oxygen species, altered vasomotor tone and potentiated atherosclerosis in murine species. Following an acute whole body inhalation exposure to 500 µg/m(3) of nickel nanoparticles for 5 h, 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. 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. These data provide new insight into how an acute nickel nanoparticle exposure to half of the current Occupational Safety & Health Administration (OSHA) permissible exposure limit may adversely affect EPCs and exacerbate cardiovascular disease states.

  19. Interactions of Francisella tularensis with Alveolar Type II Epithelial Cells and the Murine Respiratory Epithelium.

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    Matthew Faron

    data extend our understanding of early Francisella infection by demonstrating that Francisella enter significant numbers of AT-II cells within the lung and that the capsule and LPS of wild type Schu S4 helps prevent murine lung damage during infection. Furthermore, our data identified that human AT-II cells allow growth of Schu S4, but these same cells supported poor growth of the attenuated LVS strain in vitro. Collectively, these data further our understanding of the role of AT-II cells in Francisella infections.

  20. Murine Lung Cancer Increases CD4+ T Cell Apoptosis and Decreases Gut Proliferative Capacity in Sepsis.

    Science.gov (United States)

    Lyons, John D; Mittal, Rohit; Fay, Katherine T; Chen, Ching-Wen; Liang, Zhe; Margoles, Lindsay M; Burd, Eileen M; Farris, Alton B; Ford, Mandy L; Coopersmith, Craig M

    2016-01-01

    Mortality is significantly higher in septic patients with cancer than in septic patients without a history of cancer. We have previously described a model of pancreatic cancer followed by sepsis from Pseudomonas aeruginosa pneumonia in which cancer septic mice have higher mortality than previously healthy septic mice, associated with increased gut epithelial apoptosis and decreased T cell apoptosis. The purpose of this study was to determine whether this represents a common host response by creating a new model in which both the type of cancer and the model of sepsis are altered. C57Bl/6 mice received an injection of 250,000 cells of the lung cancer line LLC-1 into their right thigh and were followed three weeks for development of palpable tumors. Mice with cancer and mice without cancer were then subjected to cecal ligation and puncture and sacrificed 24 hours after the onset of sepsis or followed 7 days for survival. Cancer septic mice had a higher mortality than previously healthy septic mice (60% vs. 18%, p = 0.003). Cancer septic mice had decreased number and frequency of splenic CD4+ lymphocytes secondary to increased apoptosis without changes in splenic CD8+ numbers. Intestinal proliferation was also decreased in cancer septic mice. Cancer septic mice had a higher bacterial burden in the peritoneal cavity, but this was not associated with alterations in local cytokine, neutrophil or dendritic cell responses. Cancer septic mice had biochemical evidence of worsened renal function, but there was no histologic evidence of renal injury. Animals with cancer have a significantly higher mortality than previously healthy animals following sepsis. The potential mechanisms associated with this elevated mortality differ significantly based upon the model of cancer and sepsis utilized. While lymphocyte apoptosis and intestinal integrity are both altered by the combination of cancer and sepsis, the patterns of these alterations vary greatly depending on the models used.

  1. Murine Lung Cancer Increases CD4+ T Cell Apoptosis and Decreases Gut Proliferative Capacity in Sepsis.

    Directory of Open Access Journals (Sweden)

    John D Lyons

    Full Text Available Mortality is significantly higher in septic patients with cancer than in septic patients without a history of cancer. We have previously described a model of pancreatic cancer followed by sepsis from Pseudomonas aeruginosa pneumonia in which cancer septic mice have higher mortality than previously healthy septic mice, associated with increased gut epithelial apoptosis and decreased T cell apoptosis. The purpose of this study was to determine whether this represents a common host response by creating a new model in which both the type of cancer and the model of sepsis are altered.C57Bl/6 mice received an injection of 250,000 cells of the lung cancer line LLC-1 into their right thigh and were followed three weeks for development of palpable tumors. Mice with cancer and mice without cancer were then subjected to cecal ligation and puncture and sacrificed 24 hours after the onset of sepsis or followed 7 days for survival.Cancer septic mice had a higher mortality than previously healthy septic mice (60% vs. 18%, p = 0.003. Cancer septic mice had decreased number and frequency of splenic CD4+ lymphocytes secondary to increased apoptosis without changes in splenic CD8+ numbers. Intestinal proliferation was also decreased in cancer septic mice. Cancer septic mice had a higher bacterial burden in the peritoneal cavity, but this was not associated with alterations in local cytokine, neutrophil or dendritic cell responses. Cancer septic mice had biochemical evidence of worsened renal function, but there was no histologic evidence of renal injury.Animals with cancer have a significantly higher mortality than previously healthy animals following sepsis. The potential mechanisms associated with this elevated mortality differ significantly based upon the model of cancer and sepsis utilized. While lymphocyte apoptosis and intestinal integrity are both altered by the combination of cancer and sepsis, the patterns of these alterations vary greatly depending on

  2. The role of Hibiscus sabdariffa L. (Roselle) in maintenance of ex vivo murine bone marrow-derived hematopoietic stem cells.

    Science.gov (United States)

    Abdul Hamid, Zariyantey; Lin Lin, Winnie Hii; Abdalla, Basma Jibril; Bee Yuen, Ong; Latif, Elda Surhaida; Mohamed, Jamaludin; Rajab, Nor Fadilah; Paik Wah, Chow; Wak Harto, Muhd Khairul Akmal; Budin, Siti Balkis

    2014-01-01

    Hematopoietic stem cells- (HSCs-) based therapy requires ex vivo expansion of HSCs prior to therapeutic use. However, ex vivo culture was reported to promote excessive production of reactive oxygen species (ROS), exposing HSCs to oxidative damage. Efforts to overcome this limitation include the use of antioxidants. In this study, the role of Hibiscus sabdariffa L. (Roselle) in maintenance of cultured murine bone marrow-derived HSCs was investigated. Aqueous extract of Roselle was added at varying concentrations (0-1000 ng/mL) for 24 hours to the freshly isolated murine bone marrow cells (BMCs) cultures. Effects of Roselle on cell viability, reactive oxygen species (ROS) production, glutathione (GSH) level, superoxide dismutase (SOD) activity, and DNA damage were investigated. Roselle enhanced the survival (P Roselle increased (P Roselle showed significant cellular genoprotective potency against H2O2-induced DNA damage. Conclusively, Roselle shows novel property as potential supplement and genoprotectant against oxidative damage to cultured HSCs.

  3. Phenolic compounds from Viscum album tinctures enhanced antitumor activity in melanoma murine cancer cells.

    Science.gov (United States)

    Melo, Michelle Nonato de Oliveira; Oliveira, Adriana Passos; Wiecikowski, Adalgisa Felippe; Carvalho, Renato Sampaio; Castro, Juliana de Lima; de Oliveira, Felipe Alves Gomes; Pereira, Henrique Marcelo Gualberto; da Veiga, Venicio Feo; Capella, Marcia Marques Alves; Rocha, Leandro; Holandino, Carla

    2018-03-01

    Cancer is one of the biggest problems in public health worldwide. Plants have been shown important role in anticancer research. Viscum album L. (Santalaceae), commonly known as mistletoe, is a semi-parasitic plant that grows on different host trees. In complementary medicine, extracts from European mistletoe ( Viscum album L.) have been used in the treatment of cancer. The study was conducted to identify chemical composition and antitumor potential of Viscum album tinctures. Chemical analysis performed by high resolution chromatography equipped with high resolution mass spectrometer identified caffeic acid, chlorogenic acid, sakuranetin, isosakuranetin, syringenin 4-O-glucoside, syringenin 4-O-apiosyl-glucoside, alangilignoside C and ligalbumoside A compounds. Some of these compounds are probably responsible for the reduction of tumoral cellular growth in a dose-dependent manner. It was observed that melanoma murine cells (B16F10) were more sensitive to V. album tinctures than human leukaemic cells (K562), besides non-tumoral cells (MA-104) had a much lower cytotoxicity to them. Apoptotic-like cells were observed under light microscopy and were confirmed by a typical DNA fragmentation pattern. Additionally, flow cytometry results using Annexin-V/FITC permitted to quantify increased expression of early and late apoptotic markers on tumoral cells, confirming augmented Sub G0 population, which was probably associated with a consistent decrease in G1, and an increase in S or G2/M populations. Results indicate the chemical composition of V. album tinctures influences the mechanisms of in vitro tumoral cell death, suggesting a potential use in cancer pharmacotherapy research.

  4. Local transplantation is an effective method for cell delivery in the osteogenesis imperfecta murine model.

    Science.gov (United States)

    Pauley, Penelope; Matthews, Brya G; Wang, Liping; Dyment, Nathaniel A; Matic, Igor; Rowe, David W; Kalajzic, Ivo

    2014-09-01

    Osteogenesis imperfecta is a serious genetic disorder that results from improper type I collagen production. We aimed to evaluate whether bone marrow stromal cells (BMSC) delivered locally into femurs were able to engraft, differentiate into osteoblasts, and contribute to formation of normal bone matrix in the osteogenesis imperfect murine (oim) model. Donor BMSCs from bone-specific reporter mice (Col2.3GFP) were expanded in vitro and transplanted into the femoral intramedullary cavity of oim mice. Engraftment was evaluated after four weeks. We detected differentiation of donor BMSCs into Col2.3GFP+ osteoblasts and osteocytes in cortical and trabecular bone of transplanted oim femurs. New bone formation was detected by deposition of dynamic label in the proximity to the Col2.3GFP+ osteoblasts, and new bone showed more organized collagen structure and expression of type I α2 collagen. Col2.3GFP cells were not found in the contralateral femur indicating that transplanted osteogenic cells did not disseminate by circulation. No osteogenic engraftment was observed following intravenous transplantation of BMSCs. BMSC cultures derived from transplanted femurs showed numerous Col2.3GFP+ colonies, indicating the presence of donor progenitor cells. Secondary transplantation of cells recovered from recipient femurs and expanded in vitro also showed Col2.3GFP+ osteoblasts and osteocytes confirming the persistence of donor stem/progenitor cells. We show that BMSCs delivered locally in oim femurs are able to engraft, differentiate into osteoblasts and osteocytes and maintain their progenitor potential in vivo. This suggests that local delivery is a promising approach for introduction of autologous MSC in which mutations have been corrected.

  5. Mesenchymal stem cells from different murine tissues have differential capacity to metabolize extracellular nucleotides.

    Science.gov (United States)

    Iser, Isabele C; Bracco, Paula A; Gonçalves, Carlos E I; Zanin, Rafael F; Nardi, Nance B; Lenz, Guido; Battastini, Ana Maria O; Wink, Márcia R

    2014-10-01

    Mesenchymal stem cells (MSCs) have shown a great potential for cell-based therapy and many different therapeutic purposes. Despite the recent advances in the knowledge of MSCs biology, their biochemical and molecular properties are still poorly defined. Ecto-nucleoside triphosphate diphosphohydrolases (E-NTPDases) and ecto-5'-nucleotidase (eNT/CD73) are widely expressed enzymes that hydrolyze extracellular nucleotides, generating an important cellular signaling cascade. Currently, studies have evidenced the relationship between the purinergic system and the development, maintenance, and differentiation of stem cells. The objective of this study is to identify the NTPDases and eNT/CD73 and compare the levels of nucleotide hydrolysis on MSCs isolated from different murine tissues (bone marrow, lung, vena cava, kidney, pancreas, spleen, skin, and adipose tissue). MSCs from all tissues investigated expressed the ectoenzymes at different levels. In MSCs from pancreas and adipose tissue, the hydrolysis of triphosphonucleosides was significantly higher when compared to the other cells. The diphosphonucleosides were hydrolyzed at a higher rate by MSC from pancreas when compared to MSC from other tissues. The differential nucleotide hydrolysis activity and enzyme expression in these cells suggests that MSCs play different roles in regulating the purinergic system in these tissues. Overall MSCs are an attractive adult-derived cell population for therapies, however, the fact that ecto-nucleotide metabolism can affect the microenvironment, modulating important events, such as immune response, makes the assessment of this metabolism an important part of the characterization of MSCs to be applied therapeutically. © 2014 Wiley Periodicals, Inc.

  6. Abnormally banded chromosomal regions in doxorubicin-resistant B16-BL6 murine melanoma cells.

    Science.gov (United States)

    Slovak, M L; Hoeltge, G A; Ganapathi, R

    1986-08-01

    B16-BL6 murine melanoma cells were selected for cytogenetic evaluation during the stepwise development of increasing resistance in vitro to the antitumor antibiotic, doxorubicin (DOX). Karyotypic studies demonstrated extensive heteroploidy with both numerical and structural abnormalities which were not present in the parental DOX-sensitive B16-BL6 cells. Trypsin-Giemsa banding revealed the presence of several marker chromosomes containing abnormally banding regions (ABRs) in the 44-fold B16-BL6 DOX-resistant subline. These ABRs appeared to be more homogeneously staining at the higher DOX concentrations. Length measurements (ABR index) in seven banded metaphases indicated a direct correlation with increasing DOX concentration. When the DOX-resistant cells were grown in drug-free medium for 1 yr, the drug-resistant phenotype gradually declined in parallel with the level of resistance and the ABR index. DOX-induced cytogenetic damage examined by sister chromatid exchange methodology in parental B16-BL6 cells indicated a linear sister chromatid exchange:DOX dose-response relationship. However, after continuous treatment of parental B16-BL6 cells with DOX (0.01 microgram/ml) for 30 days, sister chromatid exchange scores were found to return to base-line values. The B16-BL6 resistant cells demonstrated a cross-resistant phenotype with N-trifluoroacetyladriamycin-14-valerate, actinomycin D, and the Vinca alkaloids but not with 1-beta-D-arabinofuranosylcytosine. The results suggest that ABR-containing chromosomes in DOX-resistant sublines may represent cytogenetic alterations of specific amplified genes involved in the expression of DOX resistance. Further studies are required to identify and define the possible gene products and to correlate their relationship to the cytotoxic action of doxorubicin.

  7. Enniatin B-induced cell death and inflammatory responses in RAW 267.4 murine macrophages

    Energy Technology Data Exchange (ETDEWEB)

    Gammelsrud, A. [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); Dendelé, B. [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); Ivanova, L. [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); Lagadic-Gossmann, D. [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); Eriksen, G. [Norwegian Veterinary Institute, P.O. Box 750, Centrum, N-0106 Oslo (Norway); Holme, J.A., E-mail: jorn.holme@fhi.no [Department of Air Pollution and Noise, Division of Environmental Medicine, Norwegian Institute of Public Health, N-0403 Oslo (Norway)

    2012-05-15

    The mycotoxin enniatin B (EnnB) is predominantly produced by species of the Fusarium genera, and often found in grain. The cytotoxic effect of EnnB has been suggested to be related to its ability to form ionophores in cell membranes. The present study examines the effects of EnnB on cell death, differentiation, proliferation and pro-inflammatory responses in the murine monocyte–macrophage cell line RAW 264.7. Exposure to EnnB for 24 h caused an accumulation of cells in the G0/G1-phase with a corresponding decrease in cyclin D1. This cell cycle-arrest was possibly also linked to the reduced cellular ability to capture and internalize receptors as illustrated by the lipid marker ganglioside GM1. EnnB also increased the number of apoptotic, early apoptotic and necrotic cells, as well as cells with elongated spindle-like morphology. The Neutral Red assay indicated that EnnB induced lysosomal damage; supported by transmission electron microscopy (TEM) showing accumulation of lipids inside the lysosomes forming lamellar structures/myelin bodies. Enhanced levels of activated caspase-1 were observed after EnnB exposure and the caspase-1 specific inhibitor ZYVAD-FMK reduced EnnB-induced apoptosis. Moreover, EnnB increased the release of interleukin-1beta (IL-1β) 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-1β 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

  8. Enniatin B-induced cell death and inflammatory responses in RAW 267.4 murine macrophages

    International Nuclear Information System (INIS)

    Gammelsrud, A.; Solhaug, A.; Dendelé, B.; Sandberg, W.J.; Ivanova, L.; Kocbach Bølling, A.; Lagadic-Gossmann, D.; Refsnes, M.; Becher, R.; Eriksen, G.; Holme, J.A.

    2012-01-01

    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-1β) 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-1β 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

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

    International Nuclear Information System (INIS)

    Krieg, A.M.; Gourley, M.F.; Steinberg, A.D.

    1991-01-01

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

  10. CD8+ T Cells Contribute to the Development of Coronary Arteritis in the Lactobacillus casei Cell Wall Extract-Induced Murine Model of Kawasaki Disease.

    Science.gov (United States)

    Noval Rivas, Magali; Lee, Youngho; Wakita, Daiko; Chiba, Norika; Dagvadorj, Jargalsaikhan; Shimada, Kenichi; Chen, Shuang; Fishbein, Michael C; Lehman, Thomas J A; Crother, Timothy R; Arditi, Moshe

    2017-02-01

    Kawasaki disease (KD) is the leading cause of acquired heart disease among children in developed countries. Coronary lesions in KD in humans are characterized by an increased presence of infiltrating CD3+ T cells; however, the specific contributions of the different T cell subpopulations in coronary arteritis development remain unknown. Therefore, we sought to investigate the function of CD4+ and CD8+ T cells, Treg cells, and natural killer (NK) T cells in the pathogenesis of KD. We addressed the function of T cell subsets in KD development by using a well-established murine model of Lactobacillus casei cell wall extract (LCWE)-induced KD vasculitis. We determined which T cell subsets were required for development of KD vasculitis by using several knockout murine strains and depleting monoclonal antibodies. LCWE-injected mice developed coronary lesions characterized by the presence of inflammatory cell infiltrates. Frequently, this chronic inflammation resulted in complete occlusion of the coronary arteries due to luminal myofibroblast proliferation (LMP) as well as the development of coronary arteritis and aortitis. We found that CD8+ T cells, but not CD4+ T cells, NK T cells, or Treg cells, were required for development of KD vasculitis. The LCWE-induced murine model of KD vasculitis mimics many histologic features of the disease in humans, such as the presence of CD8+ T cells and LMP in coronary artery lesions as well as epicardial coronary arteritis. Moreover, CD8+ T cells functionally contribute to the development of KD vasculitis in this murine model. Therapeutic strategies targeting infiltrating CD8+ T cells might be useful in the management of KD in humans. © 2016, American College of Rheumatology.

  11. Osteogenic gene expression of murine osteoblastic (MC3T3-E1) cells under cyclic tension

    International Nuclear Information System (INIS)

    Kao, C T; Chen, C C; Cheong, U-I; Liu, S L; Huang, T H

    2014-01-01

    Low-level laser therapy (LLLT) can promote cell proliferation. The remodeling ability of the tension side of orthodontic teeth affects post-orthodontic stability. The purpose of the present study was to investigate the osteogenic effects of LLLT on osteoblast-like cells treated with a simulated tension system that provides a mechanical tension regimen. Murine osteoblastic (MC3T3-E1) cells were cultured in a Flexcell strain unit with programmed loads of 12% elongation at a frequency of 0.5 Hz for 24 and 48 h. The cultured cells were treated with a low-level diode laser using powers of 5 J and 10 J. The proliferation of MC3T3-E1 cells was determined using the Alamar Blue assay. The expression of osteogenic genes (type I collagen (Col-1), osteopontin (OPN), osteocalcin (OC), osteoprotegerin (OPG), receptor activator of nuclear factor kappa B ligand (RANKL), bone morphologic protein (BMP-2), and bone morphologic protein (BMP-4)) in MC3T3-E1 cells was analyzed using reverse transcription polymerase chain reaction (RT-PCR). The data were analyzed using one-way analysis of variance. The proliferation rate of tension-cultured MC3T3-E1 cells under 5 J and 10 J LLLT increased compared with that of the control group (p < 0.05). Prominent mineralization of the MC3T3-E1 cells was visible using a von Kossa stain in the 5 J LLLT group. Osteogenic genes (Col-1, OC, OPG and BMP-2) were significantly expressed in the MC3T3-E1 cells treated with 5 J and 10 J LLLT (p < 0.05). LLLT in tension-cultured MC3T3-E1 cells showed synergistic osteogenic effects, including increases in cell proliferation and Col-1, OPN, OC, OPG and BMP-2 gene expression. LLLT might be beneficial for bone remodeling on the tension side of orthodontics. (paper)

  12. G2M arrest and apoptosis in murine T lymphoma cells following exposure to 212Bi alpha particle irradiation

    International Nuclear Information System (INIS)

    Palayoor, S.T.; Humm, J.L.; Macklis, R.M.

    1993-01-01

    Asynchronous exponentially growing EL4 murine T lymphoma cells were exposed either to high LET α-radiation from 212 Bi-DTPA or to γ-radiation from a 137 Cs source. Radiation-induced cell cycle perturbation was studied by flow cytometry. Alpha irradiation, like γ, transiently arrested cells in the G2M phase in a dose-dependent manner. The maximum percentages of cells accumulated in G2M 18 h after α- and γ-irradiation were comparable, though the dose-response relationships differed. The ''RBE'' value for G2M block for α- versus γ-radiation was approx. 4. (author)

  13. Androgen-independent proliferation of LNCaP prostate cancer cells infected by xenotropic murine leukemia virus-related virus

    International Nuclear Information System (INIS)

    Kakoki, Katsura; Kamiyama, Haruka; Izumida, Mai; Yashima, Yuka; Hayashi, Hideki; Yamamoto, Naoki; Matsuyama, Toshifumi; Igawa, Tsukasa; Sakai, Hideki; Kubo, Yoshinao

    2014-01-01

    Highlights: • XMRV infection induces androgen-independent growth in LNCaP cells. • XMRV infection reduces expression of androgen receptor. • XMRV promotes appearance of androgen blocker-resistant prostate cancer cells. - Abstract: Xenotropic murine leukemia virus-related virus (XMRV) is a novel gammaretrovirus that was originally isolated from human prostate cancer. It is now believed that XMRV is not the etiologic agent of prostate cancer. An analysis of murine leukemia virus (MLV) infection in various human cell lines revealed that prostate cancer cell lines are preferentially infected by XMRV, and this suggested that XMRV infection may confer some sort of growth advantage to prostate cancer cell lines. To examine this hypothesis, androgen-dependent LNCaP cells were infected with XMRV and tested for changes in certain cell growth properties. We found that XMRV-infected LNCaP cells can proliferate in the absence of the androgen dihydrotestosterone. Moreover, androgen receptor expression is significantly reduced in XMRV-infected LNCaP cells. Such alterations were not observed in uninfected and amphotropic MLV-infected LNCaP cells. This finding explains why prostate cancer cell lines are preferentially infected with XMRV

  14. Melanoma cells treated with GGTI and IFN-gamma allow murine vaccination and enhance cytotoxic response against human melanoma cells.

    Directory of Open Access Journals (Sweden)

    Guillaume Sarrabayrouse

    Full Text Available BACKGROUND: Suboptimal activation of T lymphocytes by melanoma cells is often due to the defective expression of class I major histocompatibility antigens (MHC-I and costimulatory molecules. We have previously shown that geranylgeranyl transferase inhibition (done with GGTI-298 stimulates anti-melanoma immune response through MHC-I and costimulatory molecule expression in the B16F10 murine model [1]. METHODOLOGY/PRINCIPAL FINDINGS: In this study, it is shown that vaccination with mIFN-gand GGTI-298 pretreated B16F10 cells induces a protection against untreated tumor growth and pulmonary metastases implantation. Furthermore, using a human melanoma model (LB1319-MEL, we demonstrated that in vitro treatment with hIFN-gamma and GGTI-298 led to the up regulation of MHC-I and a costimulatory molecule CD86 and down regulation of an inhibitory molecule PD-1L. Co-culture experiments with peripheral blood mononuclear cells (PBMC revealed that modifications induced by hIFN-gamma and GGTI-298 on the selected melanoma cells, enables the stimulation of lymphocytes from HLA compatible healthy donors. Indeed, as compared with untreated melanoma cells, pretreatment with hIFN-gamma and GGTI-298 together rendered the melanoma cells more efficient at inducing the: i activation of CD8 T lymphocytes (CD8+/CD69+; ii proliferation of tumor-specific CD8 T cells (MelanA-MART1/TCR+; iii secretion of hIFN-gamma; and iv anti-melanoma specific cytotoxic cells. CONCLUSIONS/SIGNIFICANCE: These data indicate that pharmacological treatment of melanoma cell lines with IFN-gamma and GGTI-298 stimulates their immunogenicity and could be a novel approach to produce tumor cells suitable for vaccination and for stimulation of anti-melanoma effector cells.

  15. Signaling profiling at the single-cell level identifies a distinct signaling signature in murine hematopoietic stem cells.

    Science.gov (United States)

    Du, Juan; Wang, Jinyong; Kong, Guangyao; Jiang, Jing; Zhang, Jingfang; Liu, Yangang; Tong, Wei; Zhang, Jing

    2012-07-01

    Hematopoietic stem cell (HSC) function is tightly regulated by cytokine signaling. Although phospho-flow cytometry allows us to study signaling in defined populations of cells, there has been tremendous hurdle to carry out this study in rare HSCs due to unrecoverable critical HSC markers, low HSC number, and poor cell recovery rate. Here, we overcame these difficulties and developed a "HSC phospho-flow" method to analyze cytokine signaling in murine HSCs at the single-cell level and compare HSC signaling profile to that of multipotent progenitors (MPPs), a cell type immediately downstream of HSCs, and commonly used Lin(-) cKit(+) cells (LK cells, enriched for myeloid progenitors). We chose to study signaling evoked from three representative cytokines, stem cell factor (SCF) and thrombopoietin (TPO) that are essential for HSC function and granulocyte macrophage-colony-stimulating factor (GM-CSF) that is dispensable for HSCs. HSCs display a distinct TPO and GM-CSF signaling signature from MPPs and LK cells, which highly correlates with receptor surface expression. In contrast, although majority of LK cells express lower levels of cKit than HSCs and MPPs, SCF-evoked ERK1/2 activation in LK cells shows a significantly increased magnitude for a prolonged period. These results suggest that specific cellular context plays a more important role than receptor surface expression in SCF signaling. Our study of HSC signaling at the homeostasis stage paves the way to investigate signaling changes in HSCs under conditions of stress, aging, and hematopoietic diseases. Copyright © 2012 AlphaMed Press.

  16. Melanogenesis stimulation in murine b16 melanoma cells by umberiferae plant extracts and their coumarin constituents.

    Science.gov (United States)

    Matsuda, Hideaki; Hirata, Noriko; Kawaguchi, Yoshiko; Yamazaki, Miho; Naruto, Shunsuke; Shibano, Makio; Taniguchi, Masahiko; Baba, Kimiye; Kubo, Michinori

    2005-07-01

    Melanogenesis stimulation activities of seven ethanolic extracts obtained from Umbelliferae plants used as Chinese crude drugs, namely the roots of Angelica dahurica BENTH. et HOOK., A. biserrata SHEN et YUAN, Notopterygium incisum TING, Heracleum lanatum MICHX., and H. candicans WALL., and the fruits of Cinidium monnieri (L.) CUSSON and C. formosanum YABE, were examined by using cultured murine B16 melanoma cells. Among them, the extract (5, 25 microg/ml) of H. lanatum showed a potent stimulatory effect on melanogenesis with significant enhancement of cell proliferation in a dose-dependent manner. The melanogenesis stimulatory effects of sixteen coumarins (1-16) isolated from the seven Umbelliferae crude drugs were also examined. Among them, linear-furocoumarins [psoralen (1), xanthotoxin (2), bergapten (3), and isopimpinellin (4)] and angular-furocoumarin [sphondin (13)] exhibited potent melanogenesis stimulation activity. From the view point of structure-activity relationships, it may be assumed that a linear-furocoumarin ring having a hydrogen and/or methoxyl group at 5 and 8 positions such as 1, 2, 3 and 4 was preferable for the melanogenesis stimulation activity. The introduction of a prenyl group into the furocoumarin ring was disadvantageous. Coumarin derivatives having a simple coumarin ring were inactive.

  17. Effects of melanin-induced free radicals on the isolated rat peritoneal mast cells

    International Nuclear Information System (INIS)

    Ranadive, N.S.; Shirwadkar, S.; Persad, S.; Menon, I.A.

    1986-01-01

    Pheomelanin from human red hair (RHM) produces considerably more cellular damage in Ehrlich ascites carcinoma cells when subjected to radiations of wavelength 320-700 nm than eumelanin from black hair (BHM). Irradiation of RHM generated large amounts of superoxide while BHM did not produce detectable amounts of superoxide. The present investigations describe the effects of irradiation of mast cells in the presence of various natural and synthetic melanins. Irradiation of mast cells in the presence of RHM and red hair melanoprotein released large amounts of histamine while BHM and synthetic melanins prepared from dopa, cysteinyldopa, or a mixture of dopa and cysteinyldopa did not release histamine. The release of histamine at lower concentrations of RHM was not accompanied by the release of 51 Cr from chromium-loaded cells, suggesting that this release was of noncytotoxic nature. On the other hand, the release of histamine at higher concentrations of RHM was due to cell lysis since both histamine and cytoplasmic marker 51 Cr were released to the same extent. The release evoked by large concentration RHM was not inhibited by superoxide dismutase or catalase. This suggests that the cell lysis under these conditions was not due to H 2 O 2 or O-2. The finding that mast cells release histamine when irradiated in the presence of RHM suggests that the immediate and late-phase reactions seen in sunburn may in part be due to the release of mediators from these cells

  18. Effects of AC magnetic field and carboxymethyldextran-coated magnetite nanoparticles on mice peritoneal cells

    International Nuclear Information System (INIS)

    Araujo Guedes, Maria Helena; Sadeghiani, Neda; Lima Guedes Peixoto, Danielle; Poubel Coelho, Julia; Santos Barbosa, Luzirlane; Bentes Azevedo, Ricardo; Kueckelhaus, Selma; Silva, Maria de Fatima da; Morais, Paulo Cesar; Guerrero Marques Lacava, Zulmira

    2005-01-01

    A portable apparatus was developed to perform magnetohyperthermia (MHT) assays. In order to investigate its efficiency on cell lysis, biological effects of the AC magnetic field exposure after carboxymethyldextran-coated magnetite-nanoparticles (CMDC) treatment were investigated. Phagocyte capacity, cell viability, and morphology data evidenced that the CMDC sample and the apparatus are useful to further investigate MHT in cancer therapy

  19. Effects of melanin-induced free radicals on the isolated rat peritoneal mast cells

    Energy Technology Data Exchange (ETDEWEB)

    Ranadive, N.S.; Shirwadkar, S.; Persad, S.; Menon, I.A.

    1986-03-01

    Pheomelanin from human red hair (RHM) produces considerably more cellular damage in Ehrlich ascites carcinoma cells when subjected to radiations of wavelength 320-700 nm than eumelanin from black hair (BHM). Irradiation of RHM generated large amounts of superoxide while BHM did not produce detectable amounts of superoxide. The present investigations describe the effects of irradiation of mast cells in the presence of various natural and synthetic melanins. Irradiation of mast cells in the presence of RHM and red hair melanoprotein released large amounts of histamine while BHM and synthetic melanins prepared from dopa, cysteinyldopa, or a mixture of dopa and cysteinyldopa did not release histamine. The release of histamine at lower concentrations of RHM was not accompanied by the release of /sup 51/Cr from chromium-loaded cells, suggesting that this release was of noncytotoxic nature. On the other hand, the release of histamine at higher concentrations of RHM was due to cell lysis since both histamine and cytoplasmic marker /sup 51/Cr were released to the same extent. The release evoked by large concentration RHM was not inhibited by superoxide dismutase or catalase. This suggests that the cell lysis under these conditions was not due to H/sub 2/O/sub 2/ or O-2. The finding that mast cells release histamine when irradiated in the presence of RHM suggests that the immediate and late-phase reactions seen in sunburn may in part be due to the release of mediators from these cells.

  20. Appendiceal Goblet Cell Carcinoids: Management Considerations from a Reference Peritoneal Tumour Service Centre and ENETS Centre of Excellence.

    Science.gov (United States)

    Lamarca, Angela; Nonaka, Daisuke; Lopez Escola, Cristina; Hubner, Richard A; O'Dwyer, Sarah; Chakrabarty, Bipasha; Fulford, Paul; Valle, Juan W

    2016-01-01

    Appendix goblet cell carcinoids are known to share histological features of adenocarcinoma and neuroendocrine tumours. Due to their low incidence, quality evidence is lacking for the management of these patients. We performed a single-centre retrospective study of patients with a confirmed diagnosis of appendiceal goblet cell carcinoid (GCC; 1996-2014). Patients were divided into curative intent (CI) and palliative intent (PI) cohorts. Our primary end point was overall survival (OS). Seventy-four patients were eligible; 76% were treated with CI [surgery only (36%), cytoreductive surgery (CRS) and hyperthermic intra-peritoneal chemotherapy (HIPEC; 36%), adjuvant chemotherapy (20%) and a combination of CRS and HIPEC followed by adjuvant chemotherapy (9%)], and 23% had advanced-stage disease amenable to palliative treatment (chemotherapy or supportive care) only. Completion right hemicolectomy, performed in 64% of the CI cohort, did not impact on the relapse rate or disease-free survival. FOLFOX chemotherapy was used in both the adjuvant and palliative settings; safety was as expected, and we observed a high rate (60%) of disease control in the palliative cohort. The estimated median OS (all patients), disease-free survival (CI patients) and progression-free survival (PI patients) were 52.1 (95% CI 29.4-90.3), 75.9 (26.6-not reached) and 5.3 (0.6-5.7) months, respectively. Age and stage were independent factors associated with OS in the multivariable analysis. Tang classification showed a trend for impact on OS. No benefit from specific adjuvant approach was identified; however, selection bias for treatment approach was observed. Prospective trials are needed to define optimal approaches in GCC. All GCC patients should be managed by specialized centres due to their esoteric behaviour; we provide management considerations based on our experience and conclusions. © 2015 S. Karger AG, Basel.

  1. Differential expression of the Slc4 bicarbonate transporter family in murine corneal endothelium and cell culture.

    Science.gov (United States)

    Shei, William; Liu, Jun; Htoon, Hla M; Aung, Tin; Vithana, Eranga N

    2013-01-01

    To characterize the relative expression levels of all the solute carrier 4 (Slc4) transporter family members (Slc4a1-Slc4a11) in murine corneal endothelium using real-time quantitative (qPCR), to identify further important members besides Slc4a11 and Slc4a4, and to explore how close to the baseline levels the gene expressions remain after cells have been subjected to expansion and culture. Descemet's membrane-endothelial layers of 8-10-week-old C57BL6 mice were stripped from corneas and used for both primary cell culture and direct RNA extraction. Total RNA (from uncultured cells as well as cultured cells at passages 2 and 7) was reverse transcribed, and the cDNA was used for real time qPCR using specific primers for all the Slc4 family members. The geNorm method was applied to determine the most stable housekeeping genes and normalization factor, which was calculated from multiple housekeeping genes for more accurate and robust quantification. qPCR analyses revealed that all Slc4 bicarbonate transporter family members were expressed in mouse corneal endothelium. Slc4a11 showed the highest expression, which was approximately three times higher than that of Slc4a4 (3.4±0.3; p=0.004). All Slc4 genes were also expressed in cultured cells, and interestingly, the expression of Slc4a11 in cultured cells was significantly reduced by approximately 20-fold (0.05±0.001; p=0.000001) in early passage and by approximately sevenfold (0.14±0.002; p=0.000002) in late passage cells. Given the known involvement of SLC4A4 and SLC4A11 in corneal dystrophies, we speculate that the other two highly expressed genes in the uncultured corneal endothelium, SLC4A2 and SLC4A7, are worthy of being considered as potential candidate genes for corneal endothelial diseases. Moreover, as cell culture can affect expression levels of Slc4 genes, caution and careful design of experiments are necessary when undertaking studies of Slc4-mediated ion transport in cultured cells.

  2. Hematopoietic Stem and Progenitor Cell Migration After Hypofractionated Radiation Therapy in a Murine Model

    Energy Technology Data Exchange (ETDEWEB)

    Kane, Jonathan [Department of Biological Sciences, Oakland University, Rochester, Michigan (United States); Radiation Oncology, William Beaumont Health System, Royal Oak, Michigan (United States); Krueger, Sarah A.; Dilworth, Joshua T.; Torma, John T.; Wilson, George D.; Marples, Brian [Radiation Oncology, William Beaumont Health System, Royal Oak, Michigan (United States); Madlambayan, Gerard J., E-mail: madlamba@oakland.edu [Department of Biological Sciences, Oakland University, Rochester, Michigan (United States)

    2013-12-01

    Purpose: To characterize the recruitment of bone marrow (BM)-derived hematopoietic stem and progenitor cells (HSPCs) within tumor microenvironment after radiation therapy (RT) in a murine, heterotopic tumor model. Methods and Materials: Lewis lung carcinoma tumors were established in C57BL/6 mice and irradiated with 30 Gy given as 2 fractions over 2 days. Tumors were imaged with positron emission tomography/computed tomography (PET/CT) and measured daily with digital calipers. The HSPC and myelomonocytic cell content was assessed via immunofluorescent staining and flow cytometry. Functionality of tumor-associated HSPCs was verified in vitro using colony-forming cell assays and in vivo by rescuing lethally irradiated C57BL/6 recipients. Results: Irradiation significantly reduced tumor volumes and tumor regrowth rates compared with nonirradiated controls. The number of CD133{sup +} HSPCs present in irradiated tumors was higher than in nonirradiated tumors during all stages of regrowth. CD11b{sup +} counts were similar. PET/CT imaging and growth rate analysis based on standardized uptake value indicated that HSPC recruitment directly correlated to the extent of regrowth and intratumor cell activity after irradiation. The BM-derived tumor-associated HSPCs successfully formed hematopoietic colonies and engrafted irradiated mice. Finally, targeted treatment with a small animal radiation research platform demonstrated localized HSPC recruitment to defined tumor subsites exposed to radiation. Conclusions: Hypofractionated irradiation resulted in a pronounced and targeted recruitment of BM-derived HSPCs, possibly as a mechanism to promote tumor regrowth. These data indicate for the first time that radiation therapy regulates HSPC content within regrowing tumors.

  3. Hematopoietic Stem and Progenitor Cell Migration After Hypofractionated Radiation Therapy in a Murine Model

    International Nuclear Information System (INIS)

    Kane, Jonathan; Krueger, Sarah A.; Dilworth, Joshua T.; Torma, John T.; Wilson, George D.; Marples, Brian; Madlambayan, Gerard J.

    2013-01-01

    Purpose: To characterize the recruitment of bone marrow (BM)-derived hematopoietic stem and progenitor cells (HSPCs) within tumor microenvironment after radiation therapy (RT) in a murine, heterotopic tumor model. Methods and Materials: Lewis lung carcinoma tumors were established in C57BL/6 mice and irradiated with 30 Gy given as 2 fractions over 2 days. Tumors were imaged with positron emission tomography/computed tomography (PET/CT) and measured daily with digital calipers. The HSPC and myelomonocytic cell content was assessed via immunofluorescent staining and flow cytometry. Functionality of tumor-associated HSPCs was verified in vitro using colony-forming cell assays and in vivo by rescuing lethally irradiated C57BL/6 recipients. Results: Irradiation significantly reduced tumor volumes and tumor regrowth rates compared with nonirradiated controls. The number of CD133 + HSPCs present in irradiated tumors was higher than in nonirradiated tumors during all stages of regrowth. CD11b + counts were similar. PET/CT imaging and growth rate analysis based on standardized uptake value indicated that HSPC recruitment directly correlated to the extent of regrowth and intratumor cell activity after irradiation. The BM-derived tumor-associated HSPCs successfully formed hematopoietic colonies and engrafted irradiated mice. Finally, targeted treatment with a small animal radiation research platform demonstrated localized HSPC recruitment to defined tumor subsites exposed to radiation. Conclusions: Hypofractionated irradiation resulted in a pronounced and targeted recruitment of BM-derived HSPCs, possibly as a mechanism to promote tumor regrowth. These data indicate for the first time that radiation therapy regulates HSPC content within regrowing tumors

  4. BCG vaccination drives accumulation and effector function of innate lymphoid cells in murine lungs.

    Science.gov (United States)

    Steigler, Pia; Daniels, Naomi J; McCulloch, Tim R; Ryder, Brin M; Sandford, Sarah K; Kirman, Joanna R

    2018-04-01

    The tuberculosis (TB) vaccine bacille Calmette-Guérin (BCG) prevents disseminated childhood TB; however, it fails to protect against the more prevalent pulmonary TB. Limited understanding of the immune response to Mycobacterium tuberculosis, the causative agent of TB, has hindered development of improved vaccines. Although memory CD4 T cells are considered the main mediators of protection against TB, recent studies suggest there are other key subsets that contribute to antimycobacterial immunity. To that end, innate cells may be involved in the protective response. In this study, we investigated the primary response of innate lymphoid cells (ILCs) to BCG exposure. Using a murine model, we showed that ILCs increased in number in the lungs and lymph nodes in response to BCG vaccination. Additionally, there was significant production of the antimycobacterial cytokine IFN-γ by ILCs. As ILCs are located at mucosal sites, it was investigated whether mucosal vaccination (intranasal) stimulated an enhanced response compared to the traditional vaccination approach (intradermal or subcutaneous). Indeed, in response to intranasal vaccination, the number of ILCs, and IFN-γ production in NK cells and ILC1s in the lungs and lymph nodes, were higher than that provoked through intradermal or subcutaneous vaccination. This work provides the first evidence that BCG vaccination activates ILCs, paving the way for future research to elucidate the protective potential of ILCs against mycobacterial infection. Additionally, the finding that lung ILCs respond rigorously to mucosal vaccination may have implications for the delivery of novel TB vaccines. © 2018 Australasian Society for Immunology Inc.

  5. Novel allelic mutations in murine Serca2 induce differential development of squamous cell tumors

    Energy Technology Data Exchange (ETDEWEB)

    Toki, Hideaki; Minowa, Osamu; Inoue, Maki; Motegi, Hiromi; Karashima, Yuko; Ikeda, Ami [Team for Advanced Development and Evaluation of Human Disease Models, Riken BioResource Center (BRC), Tsukuba, Ibaraki (Japan); Kaneda, Hideki [Technology and Development Team for Mouse Phenotype Analysis, Riken BRC, Tsukuba, Ibaraki (Japan); Sakuraba, Yoshiyuki [Mutagenesis and Genomics Team, Riken BRC, Tsukuba, Ibaraki (Japan); Saiki, Yuriko [Department of Molecular Pathology, Tohoku University Graduate School of Medicine, Sendai, Miyagi (Japan); Wakana, Shigeharu [Technology and Development Team for Mouse Phenotype Analysis, Riken BRC, Tsukuba, Ibaraki (Japan); Suzuki, Hiroshi [Department of Biochemistry, Asahikawa Medical University, Asahikawa, Hokkaido (Japan); Gondo, Yoichi [Mutagenesis and Genomics Team, Riken BRC, Tsukuba, Ibaraki (Japan); Shiroishi, Toshihiko [Mammalian Genetics Laboratory, National Institute of Genetics, Mishima, Shizuoka (Japan); Noda, Tetsuo, E-mail: tnoda@jfcr.or.jp [Team for Advanced Development and Evaluation of Human Disease Models, Riken BioResource Center (BRC), Tsukuba, Ibaraki (Japan); Department of Cell Biology, Cancer Institute, The Japanese Foundation for Cancer Research, Tokyo (Japan)

    2016-08-05

    Dominant mutations in the Serca2 gene, which encodes sarco(endo)plasmic reticulum calcium-ATPase, predispose mice to gastrointestinal epithelial carcinoma [1–4] and humans to Darier disease (DD) [14–17]. In this study, we generated mice harboring N-ethyl-N-nitrosourea (ENU)-induced allelic mutations in Serca2: three missense mutations and one nonsense mutation. Mice harboring these Serca2 mutations developed tumors that were categorized as either early onset squamous cell tumors (SCT), with development similar to null-type knockout mice [2,4] (aggressive form; M682, M814), or late onset tumors (mild form; M1049, M1162). Molecular analysis showed no aberration in Serca2 mRNA or protein expression levels in normal esophageal cells of any of the four mutant heterozygotes. There was no loss of heterozygosity at the Serca2 locus in the squamous cell carcinomas in any of the four lines. The effect of each mutation on Ca{sup 2+}-ATPase activity was predicted using atomic-structure models and accumulated mutated protein studies, suggesting that putative complete loss of Serca2 enzymatic activity may lead to early tumor onset, whereas mutations in which Serca2 retains residual enzymatic activity result in late onset. We propose that impaired Serca2 gene product activity has a long-term effect on squamous cell carcinogenesis from onset to the final carcinoma stage through an as-yet unrecognized but common regulatory pathway. -- Highlights: •Novel mutations in murine Serca2 caused early onset or late onset of tumorigenesis. •They also caused higher or lower incidence of Darier Disease phenotype. •3D structure model suggested the former mutations led to severer defect on ATPase. •Driver gene mutations via long-range effect on Ca2+ distributions are suggested.

  6. Effects of ionizing radiation on differentiation of murine bone marrow cells into mast cells

    International Nuclear Information System (INIS)

    Murakami, Sho; Yoshino, Hironori; Ishikawa, Junya; Yamaguchi, Masaru; Tsujiguchi, Takakiyo; Nishiyama, Ayaka; Yokoyama, Kouki; Kashiwakura, Ikuo

    2015-01-01

    Mast cells, immune effector cells produced from bone marrow cells, play a major role in immunoglobulin E–mediated allergic responses. Ionizing radiation affects the functions of mast cells, which are involved in radiation-induced tissue damage. However, whether ionizing radiation affects the differential induction of mast cells is unknown. Here we investigated whether bone marrow cells of X-irradiated mice differentiated into mast cells. To induce mast cells, bone marrow cells from X-irradiated and unirradiated mice were cultured in the presence of cytokines required for mast cell induction. Although irradiation at 0.5 Gy and 2 Gy decreased the number of bone marrow cells 1 day post-irradiation, the cultured bone marrow cells of X-irradiated and unirradiated mice both expressed mast cell–related cell-surface antigens. However, the percentage of mast cells in the irradiated group was lower than in the unirradiated group. Similar decreases in the percentage of mast cells induced in the presence of X-irradiation were observed 10 days post irradiation, although the number of bone marrow cells in irradiated mice had recovered by this time. Analysis of mast cell function showed that degranulation of mast cells after immunoglobulin E–mediated allergen recognition was significantly higher in the X-irradiated group compared with in the unirradiated group. In conclusion, bone marrow cells of X-irradiated mice differentiated into mast cells, but ionizing radiation affected the differentiation efficiency and function of mast cells. (author)

  7. Newly Identified TLR9 Stimulant, M6-395 Is a Potent Polyclonal Activator for Murine B Cells.

    Science.gov (United States)

    Park, Mi-Hee; Jung, Yu-Jin; Kim, Pyeung-Hyeun

    2012-02-01

    Toll-like receptors (TLRs) have been extensively studied in recent years. However, functions of these molecules in murine B cell biology are largely unknown. A TLR4 stimulant, LPS is well known as a powerful polyclonal activator for murine B cells. In this study, we explored the effect of a murine TLR9 stimulant, M6-395 (a synthetic CpG ODNs) on B cell proliferation and Ig production. First, M6-395 was much more potent than LPS in augmenting B cell proliferation. As for Ig expression, M6-395 facilitated the expression of both TGF-β1-induced germ line transcript α (GLTα) and IL-4-induced GLTγ1 as levels as those by LPS and Pam3CSK4 (TLR1/2 agonist) : a certain Ig GLT expression is regarded as an indicative of the corresponding isotype switching recombination. However, IgA and IgG1 secretion patterns were quite different--these Ig isotype secretions by M6-395 were much less than those by LPS and Pam3CSK4. Moreover, the increase of IgA and IgG1 production by LPS and Pam3CSK4 was virtually abrogated by M6-395. The same was true for the secretion of IgG3. We found that this unexpected phenomena provoked by M6-395 is attributed, at least in part, to its excessive mitogenic nature. Taken together, these results suggest that M6-395 can act as a murine polyclonal activator but its strong mitogenic activity is unfavorable to Ig isotype switching.

  8. Poly-thymidine oligonucleotides mediate activation of murine glial cells primarily through TLR7, not TLR8.

    Directory of Open Access Journals (Sweden)

    Min Du

    Full Text Available The functional role of murine TLR8 in the inflammatory response of the central nervous system (CNS remains unclear. Murine TLR8 does not appear to respond to human TLR7/8 agonists, due to a five amino acid deletion in the ectodomain. However, recent studies have suggested that murine TLR8 may be stimulated by alternate ligands, which include vaccinia virus DNA, phosphothioate oligodeoxynucleotides (ODNs or the combination of phosphothioate poly-thymidine oligonucleotides (pT-ODNs with TLR7/8 agonists. In the current study, we analyzed the ability of pT-ODNs to induce activation of murine glial cells in the presence or absence of TLR7/8 agonists. We found that TLR7/8 agonists induced the expression of glial cell activation markers and induced the production of multiple proinflammatory cytokines and chemokines in mixed glial cultures. In contrast, pT-ODNs alone induced only low level expression of two cytokines, CCL2 and CXCL10. The combination of pT-ODNs along with TLR7/8 agonists induced a synergistic response with substantially higher levels of proinflammatory cytokines and chemokines compared to CL075. This enhancement was not due to cellular uptake of the agonist, indicating that the pT-ODN enhancement of cytokine responses was due to effects on an intracellular process. Interestingly, this response was also not due to synergistic stimulation of both TLR7 and TLR8, as the loss of TLR7 abolished the activation of glial cells and cytokine production. Thus, pT-ODNs act in synergy with TLR7/8 agonists to induce strong TLR7-dependent cytokine production in glial cells, suggesting that the combination of pT-ODNs with TLR7 agonists may be a useful mechanism to induce pronounced glial activation in the CNS.

  9. microRNA Regulation of Peritoneal Cavity Homeostasis in Peritoneal Dialysis

    Directory of Open Access Journals (Sweden)

    Melisa Lopez-Anton

    2015-01-01

    Full Text Available Preservation of peritoneal cavity homeostasis and peritoneal membrane function is critical for long-term peritoneal dialysis (PD treatment. Several microRNAs (miRNAs have been implicated in the regulation of key molecular pathways driving peritoneal membrane alterations leading to PD failure. miRNAs regulate the expression of the majority of protein coding genes in the human genome, thereby affecting most biochemical pathways implicated in cellular homeostasis. In this review, we report published findings on miRNAs and PD therapy, with emphasis on evidence for changes in peritoneal miRNA expression during long-term PD treatment. Recent work indicates that PD effluent- (PDE- derived cells change their miRNA expression throughout the course of PD therapy, contributing to the loss of peritoneal cavity homeostasis and peritoneal membrane function. Changes in miRNA expression profiles will alter regulation of key molecular pathways, with the potential to cause profound effects on peritoneal cavity homeostasis during PD treatment. However, research to date has mainly adopted a literature-based miRNA-candidate methodology drawing conclusions from modest numbers of patient-derived samples. Therefore, the study of miRNA expression during PD therapy remains a promising field of research to understand the mechanisms involved in basic peritoneal cell homeostasis and PD failure.

  10. Expression of bovine herpesvirus 1 glycoproteins gI and gIII in transfected murine cells

    International Nuclear Information System (INIS)

    Fitzpatrick, D.R.; Zamb, T.; Parker, M.D.; van Drunen Littel-van den Hurk, S.; Babiuk, L.A.; Lawman, M.J.P.

    1988-01-01

    Genes encoding two of the major glycoproteins of bovine herpesvirus 1 (BHV-1), gI and gIII, were cloned into the eucaryotic expression vectors pRSVcat and pSV2neo and transfected into murine LMTK - cells, and cloned cell lines were established. The relative amounts of gI or gIII expressed from the two vectors were similar. Expression of gI was cell associated and localized predominantly in the perinuclear region, but nuclear and plasma membrane staining was also observed. Expression of gI was additionally associated with cell fusion and the formation of polykaryons and giant cells. Expression of gIII was localized predominantly in the nuclear and plasma membranes. Radioimmunoprecipitation in the presence or absence of tunicamycin revealed that the recombinant glycoproteins were proteolytically processed and glycosylated and had molecular weights similar to those of the forms of gI and gIII expressed in BHV-1 infected bovine cells. However, both recombinant glycoproteins were glycosylated to a lesser extent than were the forms found in BHV-1 infected bovine cells. For gI, a deficiency in N-linked glycosylated of the amino-terminal half of the protein was identified; for gIII, a deficiency in O-linked glycosylation was implicated. The reactivity pattern of a panel of gI- and gIII-specific monoclonal antibodies, including six which recognize conformation-dependent epitopes, was found to be unaffected by the glycosylation differences and was identical for transfected of BHV-1-infected murine cells. Use of the transfected cells as targets in immune-mediated cytotoxicity assays demonstrated the functional recognition of recombinant gI and gIII by murine antibody and cytotoxic T lymphocytes

  11. Stem Cell Heterogeneity of Mononucleated Cells from Murine Peripheral Blood: Molecular Analysis

    Directory of Open Access Journals (Sweden)

    Muhammad Dain Yazid

    2011-01-01

    Full Text Available The main purpose of this paper was to determine the heterogeneity of primary isolated mononucleated cells that originated from the peripheral blood system by observing molecular markers. The isolated cells were cultured in complete medium for 4 to 7 days prior to the separation of different cell types, that is, adherent and suspension. Following a total culture time of 14 days, adherent cells activated the Cd105 gene while suspension cells activated the Sca-1 gene. Both progenitor markers, Cbfa-1 and Ostf-1, were inactivated in both suspension and adherent cells after 14-day culture compared to cells cultured 3 days in designated differentiation medium. In conclusion, molecular analyses showed that primary mononucleated cells are heterogeneous, consisting of hematopoietic stem cells (suspension and mesenchymal stem cells (adherent while both cells contained no progenitor cells.

  12. Fungal peritonitis in children on peritoneal dialysis.

    NARCIS (Netherlands)

    Raaijmakers, R.; Schroder, C.; Monnens, L.A.H.; Cornelissen, E.A.M.; Warris, A.

    2007-01-01

    Fungal peritonitis is a rare but serious complication in children on peritoneal dialysis (PD). In this study, risk factors were evaluated, and therapeutic measures were reviewed. A retrospective, multi-centre study was performed in 159 Dutch paediatric PD patients, between 1980 and 2005 (3,573

  13. Peritoneal fluid culture

    Science.gov (United States)

    Culture - peritoneal fluid ... sent to the laboratory for Gram stain and culture. The sample is checked to see if bacteria ... The peritoneal fluid culture may be negative, even if you have ... diagnosis of peritonitis is based on other factors, in addition ...

  14. Myths in peritoneal dialysis.

    Science.gov (United States)

    Lee, Martin B; Bargman, Joanne M

    2016-11-01

    To clarify misconceptions about the feasibility and risks of peritoneal dialysis that unnecessarily limit peritoneal dialysis uptake or continuation in patients for whom peritoneal dialysis is the preferred dialysis modality. The inappropriate choice of haemodialysis as a result of these misconceptions contributes to low peritoneal dialysis penetrance, increases transfer from peritoneal dialysis to haemodialysis, increases expenditure on haemodialysis and compromises quality of life for these patients. Peritoneal dialysis is an excellent renal replacement modality that is simple, cost-effective and provides comparable clinical outcomes to conventional in-centre haemodialysis. Unfortunately, many patients are deemed unsuitable to start or continue peritoneal dialysis because of false or inaccurate beliefs about peritoneal dialysis. Here, we examine some of these 'myths' and critically review the evidence for and against each of them. We review the feasibility and risk of peritoneal dialysis in patients with prior surgery, ostomies, obesity and mesh hernia repairs. We examine the fear of mediastinitis with peritoneal dialysis after coronary artery bypass graft surgery and the belief that the use of hypertonic glucose dialysate causes peritoneal membrane failure. By clarifying common myths about peritoneal dialysis, we hope to reduce overly cautious practices surrounding this therapy.

  15. Tuberculous peritonitis: CT appearance

    International Nuclear Information System (INIS)

    Hanson, R.D.; Hunter, T.B.

    1985-01-01

    Rare, sporadic cases of tuberculous peritonitis do occur in the United States and other advanced countries. Because there are few descriptions of the CT appearance of the peritoneal forms of tuberculous (TB), this report illustrates a case of tuberculous peritonitis with prominent CT findings and discusses the differentiation of this entity from other, more common diseases

  16. Regulation of hemopoiesis: inhibitors and stimulators produced by a murine bone marrow stromal cell line (H-1)

    International Nuclear Information System (INIS)

    Cronkite, E.P.; Miller, M.E.; Garnett, H.; Harigaya, K.

    1982-01-01

    A murine cell line (H-1) probably derived from the adventitial reticular cell has been isolated and preserved. This line produces: (1) CSF; (2) a labile inhibitor of CFU-c proliferation with preferential action on granulopoiesis; (3) PGE; (4) proliferation inhibitors of BFU-E and GEMM; and (5) suppression of entry of CFU-S into DNA synthesis in vitro. It is postulated that the adventitial reticular cell (ARC) may play a major regulatory role in hemopoiesis through intramedullary production of the factors described. The nature of the signals that activate the genes in the ARC which are coded for the factors described is not known

  17. Comparative microscopic and biochemical study of the uptake of fluorescent and 125I-labeled lipoproteins by skin fibroblasts, smooth muscle cells, and peritoneal macrophages in culture

    International Nuclear Information System (INIS)

    Reynolds, G.D.; St Clair, R.W.

    1985-01-01

    Uptake of low density lipoprotein (LDL) and of acetyl LDL was compared in skin fibroblasts, smooth muscle cells, and peritoneal macrophages with the use of lipoproteins labeled with either 125 I or the fluorescent probe 3,3'-dioctadecylindocarbocyanine (DiI). The uptake of DiI-labeled lipoproteins was assessed by quantitative spectrofluorometry and by fluorescence microscopy. The DiI was quantitatively retained by the cells, while the 125 I-LDL was degraded and 125 I-labeled degradation products were excreted from the cells. In smooth muscle cells and fibroblasts the uptake of LDL was virtually the same whether measured with the use of the DiI or 125 I-label. The labeling of acetyl LDL with DiI enhanced its uptake in peritoneal macrophages by an average of 18%. With the DiI label, lipoprotein uptake could be determined after as little as 10 minutes of incubation at 37 C. The pattern of uptake of the DiI-labeled lipoproteins was consistent with binding to specific receptors, because no DiI could be detected in mutant cells without LDL receptors, and uptake was competitively inhibited by addition of excess unlabeled lipoprotein. When the DiI-labeled lipoproteins were removed from the medium, there was a 5-15% loss of DiI from all cell types studied over the first 24 hours

  18. Generation of murine tumor cell lines deficient in MHC molecule surface expression using the CRISPR/Cas9 system.

    Directory of Open Access Journals (Sweden)

    Krishna Das

    Full Text Available In this study, the CRISPR/Cas9 technology was used to establish murine tumor cell lines, devoid of MHC I or MHC II surface expression, respectively. The melanoma cell line B16F10 and the murine breast cancer cell line EO-771, the latter stably expressing the tumor antigen NY-BR-1 (EO-NY, were transfected with an expression plasmid encoding a β2m-specific single guide (sgRNA and Cas9. The resulting MHC I negative cells were sorted by flow cytometry to obtain single cell clones, and loss of susceptibility of peptide pulsed MHC I negative clones to peptide-specific CTL recognition was determined by IFNγ ELISpot assay. The β2m knockout (KO clones did not give rise to tumors in syngeneic mice (C57BL/6N, unless NK cells were depleted, suggesting that outgrowth of the β2m KO cell lines was controlled by NK cells. Using sgRNAs targeting the β-chain encoding locus of the IAb molecule we also generated several B16F10 MHC II KO clones. Peptide loaded B16F10 MHC II KO cells were insusceptible to recognition by OT-II cells and tumor growth was unaltered compared to parental B16F10 cells. Thus, in our hands the CRISPR/Cas9 system has proven to be an efficient straight forward strategy for the generation of MHC knockout cell lines. Such cell lines could serve as parental cells for co-transfection of compatible HLA alleles together with human tumor antigens of interest, thereby facilitating the generation of HLA matched transplantable tumor models, e.g. in HLAtg mouse strains of the newer generation, lacking cell surface expression of endogenous H2 molecules. In addition, our tumor cell lines established might offer a useful tool to investigate tumor reactive T cell responses that function independently from MHC molecule surface expression by the tumor.

  19. Rosiglitazone inhibits metastasis development of a murine mammary tumor cell line LMM3

    International Nuclear Information System (INIS)

    Magenta, Gabriela; Borenstein, Ximena; Rolando, Romina; Jasnis, María Adela

    2008-01-01

    Activation of peroxisome proliferator-activated receptors γ (PPARγ) induces diverse effects on cancer cells. The thiazolidinediones (TZDs), such as troglitazone and ciglitazone, are PPARγ agonists exhibiting antitumor activities; however, the underlying mechanism remains inconclusive. Rosiglitazone (RGZ), a synthetic ligand of PPARγ used in the treatment of Type 2 diabetes, inhibits growth of some tumor cells and is involved in other processes related to cancer progression. Opposing results have also been reported with different ligands on tumor cells. The purpose of this study was to determine if RGZ and 15d-PGJ 2 induce antitumor effects in vivo and in vitro on the murine mammary tumor cell line LMM3. The effect on LMM3 cell viability and nitric oxide (NO) production of different doses of RGZ, 15-dPGJ 2 , BADGE and GW9662 were determined using the MTS colorimetric assay and the Griess reaction respectively. In vivo effect of orally administration of RGZ on tumor progression was evaluated either on s.c. primary tumors as well as on experimental metastasis. Cell adhesion, migration (wound assay) and invasion in Transwells were performed. Metalloproteinase activity (MMP) was determined by zymography in conditioned media from RGZ treated tumor cells. PPARγ expression was detected by inmunohistochemistry in formalin fixed tumors and by western blot in tumor cell lysates. RGZ orally administered to tumor-bearing mice decreased the number of experimental lung metastases without affecting primary s.c. tumor growth. Tumor cell adhesion and migration, as well as metalloproteinase MMP-9 activity, decreased in the presence of 1 μM RGZ (non-cytotoxic dose). RGZ induced PPARγ protein expression in LMM3 tumors. Although metabolic activity -measured by MTS assay- diminished with 1–100 μM RGZ, 1 μM-treated cells recovered their proliferating capacity while 100 μM treated cells died. The PPARγ antagonist Biphenol A diglicydyl ether (BADGE) did not affect RGZ activity

  20. A multicenter study to standardize reporting and analyses of fluorescence-activated cell-sorted murine intestinal epithelial cells

    Science.gov (United States)

    Magness, Scott T.; Puthoff, Brent J.; Crissey, Mary Ann; Dunn, James; Henning, Susan J.; Houchen, Courtney; Kaddis, John S.; Kuo, Calvin J.; Li, Linheng; Lynch, John; Martin, Martin G.; May, Randal; Niland, Joyce C.; Olack, Barbara; Qian, Dajun; Stelzner, Matthias; Swain, John R.; Wang, Fengchao; Wang, Jiafang; Wang, Xinwei; Yan, Kelley; Yu, Jian

    2013-01-01

    Fluorescence-activated cell sorting (FACS) is an essential tool for studies requiring isolation of distinct intestinal epithelial cell populations. Inconsistent or lack of reporting of the critical parameters associated with FACS methodologies has complicated interpretation, comparison, and reproduction of important findings. To address this problem a comprehensive multicenter study was designed to develop guidelines that limit experimental and data reporting variability and provide a foundation for accurate comparison of data between studies. Common methodologies and data reporting protocols for tissue dissociation, cell yield, cell viability, FACS, and postsort purity were established. Seven centers tested the standardized methods by FACS-isolating a specific crypt-based epithelial population (EpCAM+/CD44+) from murine small intestine. Genetic biomarkers for stem/progenitor (Lgr5 and Atoh 1) and differentiated cell lineages (lysozyme, mucin2, chromogranin A, and sucrase isomaltase) were interrogated in target and control populations to assess intra- and intercenter variability. Wilcoxon's rank sum test on gene expression levels showed limited intracenter variability between biological replicates. Principal component analysis demonstrated significant intercenter reproducibility among four centers. Analysis of data collected by standardized cell isolation methods and data reporting requirements readily identified methodological problems, indicating that standard reporting parameters facilitate post hoc error identification. These results indicate that the complexity of FACS isolation of target intestinal epithelial populations can be highly reproducible between biological replicates and different institutions by adherence to common cell isolation methods and FACS gating strategies. This study can be considered a foundation for continued method development and a starting point for investigators that are developing cell isolation expertise to study physiology and

  1. Pacific ciguatoxin 1B-induced modulation of inflammatory mediators in a murine macrophage cell line.

    Science.gov (United States)

    Matsui, Mariko; Kumar-Roine, Shilpa; Darius, H Taiana; Chinain, Mireille; Laurent, Dominique; Pauillac, Serge

    2010-10-01

    Ciguatoxins, potent marine neurotoxins responsible for ciguatera, exert their numerous damaging effects through primary binding to the voltage-sensitive sodium channels of excitable cells. Using RAW 264.7 murine macrophages, we report the first experimental study presenting evidence that P-CTX-1B (the most potent congener from the Pacific) could modulate mRNA expression of pro- and anti-inflammatory cytokines as well as of inducible nitric oxide synthase (iNOS). P-CTX-1B, unlike other less potent marine polyether toxins, P-CTX-3C and PbTx-3, induced the overexpression of interleukin (IL)-1beta, IL-6, IL-10, tumor necrosis factor-alpha and iNOS with different magnitude and kinetic profiles, as compared to bacterial lipopolysaccharide (LPS). Unlike LPS, P-CTX-1B did not modulate IL-11 expression. In this report, we provide new evidence of the P-CTX-1B iNOS- and cytokines-inducing ability and shed new light on host response to potent neurotoxins. Copyright 2009 Elsevier Ltd. All rights reserved.

  2. Expression of mink cell focus-forming murine leukemia virus-related transcripts in AKR mice

    International Nuclear Information System (INIS)

    Khan, A.S.; Laigret, F.; Rodi, C.P.

    1987-01-01

    The authors used a synthetic 16-base-pair mink cell focus-forming (MCF) env-specific oligomer as radiolabeled probe to study MCF murine leukemia virus (MuLV)-related transcripts in brain, kidney, liver, spleen, and thymus tissues of AKR mice ranging from 5 weeks to 6 months (mo) of age. Tissue-specific expression of poly(A) + RNAs was seen. In addition, all the tissues tested contained 3.0-kb messages. The transcription of these MCF-related mRNAs was independent of the presence of ecotropic and xenotropic MuLVs. In general, expression of the MCF env-related transcripts appeared to peak at 2 mo of age; these messages were barely detectable in brain, kidney, liver, and spleen tissues after 2 mo and in thymus tissue after 4 mo of age. All of the subgenomic MCF env-related mRNAs appeared to contain the 190-base-pair cellular DNA insert, characteristic of the long terminal repeats associated with endogenous MCF env-related proviruses. No genomic-size (8.4-kb) transcripts corresponding to endogenous MCF-related proviruses were detected. An 8.4-kb MCF env-related mRNA was first seen at 3 mo of age, exclusively in thymus tissue. This species most likely represents the first appearance of a recombinant MCF-related MuLV genome. The transcripts which were detected in thymus tissue might be involved in the generation of leukemogenic MCF viruses

  3. Essential role of the TFIID subunit TAF4 in murine embryogenesis and embryonic stem cell differentiation.

    Science.gov (United States)

    Langer, Diana; Martianov, Igor; Alpern, Daniel; Rhinn, Muriel; Keime, Céline; Dollé, Pascal; Mengus, Gabrielle; Davidson, Irwin

    2016-03-30

    TAF4 (TATA-binding protein-associated factor 4) and its paralogue TAF4b are components of the TFIID core module. We inactivated the murine Taf4a gene to address Taf4 function during embryogenesis. Here we show that Taf4a(-/-) embryos survive until E9.5 where primary germ layers and many embryonic structures are identified showing Taf4 is dispensable for their specification. In contrast, Taf4 is required for correct patterning of the trunk and anterior structures, ventral morphogenesis and proper heart positioning. Overlapping expression of Taf4a and Taf4b during embryogenesis suggests their redundancy at early stages. In agreement with this, Taf4a(-/-) embryonic stem cells (ESCs) are viable and comprise Taf4b-containing TFIID. Nevertheless, Taf4a(-/-) ESCs do not complete differentiation into glutamatergic neurons and cardiomyocytes in vitro due to impaired preinitiation complex formation at the promoters of critical differentiation genes. We define an essential role of a core TFIID TAF in differentiation events during mammalian embryogenesis.

  4. Acyl Chain Preference in Foam Cell Formation from Mouse Peritoneal Macrophages.

    Science.gov (United States)

    Fujiwara, Yuko; Hama, Kotaro; Tsukahara, Makoto; Izumi-Tsuzuki, Ryosuke; Nagai, Toru; Ohe-Yamada, Mihoko; Inoue, Keizo; Yokoyama, Kazuaki

    2018-01-01

    Macrophage foam cells play critical roles in the initiation and development of atherosclerosis by synthesizing and accumulating cholesteryl ester (CE) in lipid droplets. However, in analyzing lipid metabolism in foam cell formation, studies have focused on the sterol group, and little research has been done on the acyl chains. Therefore, we adapted a model system using liposomes containing particular acyl chains and examined the effect of various acyl chains on foam cell formation. Of the phosphatidylserine (PS) liposomes tested containing PS, phosphatidylcholine, and cholesterol, we found that unsaturated (C18:1), but not saturated (C16:0 and C18:0), PS liposomes induced lipid droplet formation, indicating that foam cell formation depends on the nature of the acyl chain of the PS liposomes. Experiments on the uptake and accumulation of cholesterol from liposomes by adding [ 14 C]cholesterol suggested that foam cell formation could be induced only when cholesterol was converted to CE in the case of C18:1 PS liposomes. Both microscopic observations and metabolic analysis suggest that cholesterol incorporated into either C16:0 or C18:0 PS liposomes may stay intact after being taken in by endosomes. The [ 14 C]C18:1 fatty acyl chain in the C18:1 PS liposome was used to synthesize CE and triacylglycerol (TG). Interestingly, the [ 14 C]C16:0 in the C18:1 PS liposome was metabolized to sphingomyelin rather than being incorporated into either CE or TG, which could be because of enzymatic acyl chain selectivity. In conclusion, our results indicate that the acyl chain preference of macrophages could have some impact on their progression to foam cells.

  5. Radiobiological studies on target cell populations in murine bone marrow transplantation recipients.

    NARCIS (Netherlands)

    van Os, Ronald Peter

    1994-01-01

    The experiments presented in this thesis were designed to investigate the role of total body irradiation (TBI) in conditioning murine recipients of syngeneic and allogeneic bone marrow transplantation (BMT). ... Zie: Summary

  6. Kinetic of magnetic nanoparticles uptake evaluated by morphometry of mice peritoneal cells

    International Nuclear Information System (INIS)

    Silva, L.P.; Kuckelhaus, S.; Guedes, M.H.A.; Lacava, Z.G.M.; Tedesco, A.C.; Morais, P.C.; Azevedo, R.B.

    2005-01-01

    The development of magnetic fluids (MFs) has led to a wide range of new biomedical applications. Nevertheless, few studies have examined the kinetics of the magnetic nanoparticles (MNPs) internalization by phagocytes. In this study, we present morphometry as a method to quantify the cell surface covered by MNPs. The maximum cell surface covered by MNPs aggregates was 32.5% (8.5 min), 18.3% (24.1 min), and 18.0% (20.2 min) in DMSA, citric acid and dextran-coated MNPs, respectively. We concluded that the phagocytosis process of MNPs is strongly dependent upon the coating species

  7. B cell depletion reduces T cell activation in pancreatic islets in a murine autoimmune diabetes model.

    Science.gov (United States)

    Da Rosa, Larissa C; Boldison, Joanne; De Leenheer, Evy; Davies, Joanne; Wen, Li; Wong, F Susan

    2018-06-01

    Type 1 diabetes is a T cell-mediated autoimmune disease characterised by the destruction of beta cells in the islets of Langerhans, resulting in deficient insulin production. B cell depletion therapy has proved successful in preventing diabetes and restoring euglycaemia in animal models of diabetes, as well as in preserving beta cell function in clinical trials in the short term. We aimed to report a full characterisation of B cell kinetics post B cell depletion, with a focus on pancreatic islets. Transgenic NOD mice with a human CD20 transgene expressed on B cells were injected with an anti-CD20 depleting antibody. B cells were analysed using multivariable flow cytometry. There was a 10 week delay in the onset of diabetes when comparing control and experimental groups, although the final difference in the diabetes incidence, following prolonged observation, was not statistically significant (p = 0.07). The co-stimulatory molecules CD80 and CD86 were reduced on stimulation of B cells during B cell depletion and repopulation. IL-10-producing regulatory B cells were not induced in repopulated B cells in the periphery, post anti-CD20 depletion. However, the early depletion of B cells had a marked effect on T cells in the local islet infiltrate. We demonstrated a lack of T cell activation, specifically with reduced CD44 expression and effector function, including IFN-γ production from both CD4 + and CD8 + T cells. These CD8 + T cells remained altered in the pancreatic islets long after B cell depletion and repopulation. Our findings suggest that B cell depletion can have an impact on T cell regulation, inducing a durable effect that is present long after repopulation. We suggest that this local effect of reducing autoimmune T cell activity contributes to delay in the onset of autoimmune diabetes.

  8. Ingestion of Staphylococcus aureus, Staphylococcus epidermidis, and Escherichia coli by human peritoneal mesothelial cells

    NARCIS (Netherlands)

    Visser, C. E.; Brouwer-Steenbergen, J. J.; Schadee-Eestermans, I. L.; Meijer, S.; Krediet, R. T.; Beelen, R. H.

    1996-01-01

    In the present study we examined whether mesothelial cells can ingest and digest bacteria. The results showed that all strains were ingested. Ingested staphylococci proliferated abundantly, and only a few were digested. Escherichia coli, however, was digested during the first 8 h, whereafter the

  9. Suppression of annexin A2 by prostaglandin E₂ impairs phagocytic ability of peritoneal macrophages in women with endometriosis.

    Science.gov (United States)

    Wu, Meng-Hsing; Chuang, Pei-Chin; Lin, Yiu-Juian; Tsai, Shaw-Jenq

    2013-04-01

    Is annexin A2 involved in the reduced phagocytic ability of macrophages in endometriosis? Data from women with endometriosis and a murine model of the disease show that expression of annexin A2 in peritoneal macrophages is inhibited by prostaglandin E2 (PGE2) and this impairs the phagocytic ability of macrophages. Endometriosis is a chronic inflammatory disease that recruits many immune cells, especially macrophages, to the peritoneal cavity. The phagocytic ability of peritoneal macrophages isolated from women with endometriosis is reduced. A laboratory study. Thirty-five patients (20 with and 15 without endometriosis) of reproductive age with normal menstrual cycles were recruited. Peritoneal macrophages isolated from women with or without endometriosis were cultured and treated with vehicle, PGE2 and different EP receptor agonists, and the expression of annexin A2 was quantified by RT-PCR and western blotting. Annexin A2 was knocked down (by small interfering RNA) in normal macrophages or overexpressed (by treatment with recombinant protein) in endometriotic macrophages and their phagocytic ability was measured by flow cytometry. Peritoneal macrophages were isolated from a mouse model of endometriosis and treated with PGE2 or cyclo-oxygenase (COX) inhibitors, and annexin A2 mRNA was quantified. Levels of annexin A2 were markedly reduced in peritoneal macrophages from women with endometriosis versus controls (mRNA: P endometriosis versus control) via the EP2/EP4 receptor-dependent signaling pathway. Treatment with PGE2 or knockdown of annexin A2 inhibited the phagocytic ability of macrophages (P peritoneal macrophages were markedly reduced in mice treated with PGE2 (P peritoneal macrophages (P peritoneal cells from patients with endometriosis or that their endometriotic fluid contains increased amounts of PGE2 when compared with control subjects. Inhibiting PGE2 signaling, in order to restore or enhance the phagocytic capability of macrophages, may represent a new

  10. Differential Activation of Peritoneal Cells by Subcutaneous Treatment of Rats with Cryptococcal Antigens▿

    OpenAIRE

    Baronetti, José L.; Chiapello, Laura S.; Garro, Ana P.; Masih, Diana T.

    2009-01-01

    Previous studies in our laboratory have shown that the subcutaneous pretreatment of rats with heat-killed cells (HKC) of Cryptococcus neoformans emulsified in complete Freund adjuvant (CFA) promotes protective immunity against an intraperitoneal challenge with C. neoformans. In contrast, subcutaneous treatment with the capsular polysaccharide (PSC) emulsified in CFA exacerbates the cryptococcal infection. The purpose of this study was to analyze the mechanisms involved in these phenomena. Adh...

  11. Hydrocortisone selectively inhibits IgE-dependent arachidonic acid release from rat peritoneal mast cells

    International Nuclear Information System (INIS)

    Heiman, A.S.; Crews, F.T.

    1984-01-01

    Purified rat mst cells were used to study the effects of antiinflammatory steroids on the release of [1-14C]-arachidonic acid ([1-14C]AA) and metabolites. Mast cell were incubated overnight with glucocorticoids, [1-14C]AA incorporated into cellular phospholipids and the release of [1-14C]AA, and metabolites determined using a variety of secretagogues. Release of [1-14C]AA and metabolites by concanavalin A, the antigen ovalbumin and anti-immunoglobulin E antibody was markedly reduced by glucocorticoid treatment. Neither the total incorporation of [1-14C]AA nor the distribution into phospholipids was altered by hydrocortisone pretreatment. Glucocorticoid pretreatment did not alter [1-14C]AA release stimulated by somatostatin, compound 48/80, or the calcium ionophore, A23187. These data indicate that antiinflammatory steroids selectively inhibit immunoglobulin dependent release of arachidonic acid from rat mast cells. These findings question the role of lipomodulin and macrocortin as general phospholipase inhibitors and suggest that they may be restricted to immunoglobulin stimuli

  12. Metabolism of murine TH 17 cells: Impact on cell fate and function.

    Science.gov (United States)

    Wang, Ran; Solt, Laura A

    2016-04-01

    An effective adaptive immune response relies on the ability of lymphocytes to rapidly act upon a variety of insults. In T lymphocytes, this response includes cell growth, clonal expansion, differentiation, and cytokine production, all of which place a significant energy burden on the cell. Recent evidence shows that T-cell metabolic reprogramming is an essential component of the adaptive immune response and specific metabolic pathways dictate T-cell fate decisions, including the development of TH 17 versus T regulatory (Treg) cells. TH 17 cells have garnered significant attention due to their roles in the pathology of immune-mediated inflammatory diseases. Attempts to characterize TH 17 cells have demonstrated that they are highly dynamic, adjusting their function to environmental cues, which dictate their metabolic program. In this review, we highlight recent data demonstrating the impact of cellular metabolism on the TH 17/Treg balance and present factors that mediate TH 17-cell metabolism. Some examples of these include the differential impact of the mTOR signaling complexes on T-helper-cell differentiation, hypoxia inducible factor 1 alpha (HIF1α) promotion of glycolysis to favor TH 17-cell development, and ACC1-dependent de novo fatty acid synthesis favoring TH 17-cell development over Treg cells. Finally, we discuss the potential therapeutic options and the implications of modulating TH 17-cell metabolism for the treatment of TH 17-mediated diseases. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  13. Caffeine inhibits nonselective cationic currents in interstitial cells of Cajal from the murine jejunum.

    Science.gov (United States)

    Jin, Nan Ge; Koh, Sang Don; Sanders, Kenton M

    2009-10-01

    Interstitial cells of Cajal (ICC) discharge unitary potentials in gastrointestinal muscles that constitute the basis for pacemaker activity. Caffeine has been used to block unitary potentials, but the ionic conductance responsible for unitary potentials is controversial. We investigated currents in cultured ICC from murine jejunum that may underlie unitary potentials and studied the effects of caffeine. Networks of ICC generated slow wave events under current clamp, and these events were blocked by caffeine in a concentration-dependent manner. Single ICC generated spontaneous transient inward currents (STICs) under voltage clamp at -60 mV and noisy voltage fluctuations in current clamp. STICs were unaffected when the equilibrium potential for Cl- (ECl) was set to -60 mV (excluding Cl- currents) and reversed at 0 mV, demonstrating that a nonselective cationic conductance, and not a Cl- conductance, is responsible for STICs in ICC. Caffeine inhibited STICs in a concentration-dependent manner. Reduced intracellular Ca2+ and calmidazolium (CMZ; 1 microM) activated persistent inward, nonselective cation currents in ICC. Currents activated by CMZ and by dialysis of cells with 10 mM BAPTA were also inhibited by caffeine. Excised inside-out patches contained channels that exhibited spontaneous openings, and resulting currents reversed at 0 mV. Channel openings were increased by reducing Ca2+ concentration from 10(-6) M to 10(-8) M. CMZ (1 microM) also increased openings of nonselective cation channels. Spontaneous currents and channels activated by CMZ were inhibited by caffeine (5 mM). The findings demonstrate that the Ca2+-inhibited nonselective cation channels that generate STICs in ICC are blocked directly by caffeine. STICs are responsible for unitary potentials in intact muscles, and the block of these events by caffeine is consistent with the idea that a nonselective cation conductance underlies unitary potentials in ICC.

  14. The third helix of the murine Hoxc8 homeodomain facilitates protein transduction in mammalian cells

    International Nuclear Information System (INIS)

    Kong, Kyoung-Ah; Gadi, Jogeswar; Park, Hyoung Woo; Bok, Jinwoong; Kim, Myoung Hee

    2008-01-01

    Previously, we have demonstrated that purified Hoxc8 homeoprotein has the ability to penetrate the cellular membrane and can be transduced efficiently into COS-7 cells. Moreover, the Hoxc8 protein is able to form a complex with DNA molecules in vitro and helps the DNA be delivered intracellularly, serving as a gene delivery vehicle. Here, we further analyzed the membrane transduction activity of Hoxc8 protein and provide the evidence that the 16 amino acid (a.a.191-206, 2.23 kDa) third helix of murine Hoxc8 protein is an efficient protein transduction domain (PTD). When the 16 amino acid peptide was fused at the carboxyl terminal of enhanced green fluorescence protein (EGFP), the fusion proteins were transduced efficiently into the primary pig fetal fibroblast cells. The transduction efficiency increased in a concentration-dependent manner up to 1 μM, and appeared to plateau above a concentration of 1 μM. When tandem multimers of PTD, EGFP-PTD(2), EGFP-PTD(3), EGFP-PTD(4), and EGFP-PTD(5), were analyzed at 500 nM of concentration, the penetrating efficiency increased in a dose-dependent manner. As the number of PTDs increased, the EGFP signal also increased, although the signal maintained plateau after EGFP-PTD(3). These results indicate that the 16 amino acid third helix is the key element responsible for the membrane transduction activity of Hoxc8 proteins, and further suggest that the small peptide could serve as a therapeutic delivery vehicle for large cargo proteins

  15. Effect of selenium compounds on murine B16 melanoma cells and pigmented cloned pB16 cells

    International Nuclear Information System (INIS)

    Siwek, B.; Bahbouth, E.; Serra, M.A.; Sabbioni, E.; Pauw-Gillet, M.C. de; Bassleer, R.

    1994-01-01

    The effects of selenium compounds such as sodium selenite, sodium selenate, seleno-DL-cystine and seleno-DL-methionine (100 μM and 10 μM) on B16 and pigmented cloned pB 16 murine melanoma cells were investigated in vitro. At the tested concentrations, B16 cells showed a greater sensitivity to the toxic effects of sodium selenite and seleno-DL-cystine than pB 16 cells, whereas no decrease of B 16 and pB 16 cell number was observed after incubation with sodium selenate or seleno-DL-methionine. Glutathione (GSH) percentages were strongly decreased only by selenite and seleno-DL-cystine; it was marked more in B 16 than in pB 16 cells. The pretreatment of B 16 cells with a GSH depleting agent (10 μM buthionine-[S,R]-sulfoximine) did not significantly influence the cytotoxic effects of selenite and seleno-DL-cystine. On both cell populations. GSH preincubation (50 μM) enhanced the cytotoxicity of selenite whereas the survival of seleno-DL-cystine treated cells was increased. Glutathione peroxidase (GSH-Px) activity in B 16 cells was more sensitive than in pB 16 cells to the activating effect of selenite, and particularly of seleno-DL-cystine; however, cell-free controls indicated that activation was mainly due to glutathione reductase. The rate of 75 Se (as sodium selenite) uptake in both cell populations was maximal within the first hour of incubation, with a preferential accumulation in the cytosol; after 24 h of incubation, the amount of 75 Se in cytosol and pellet was approximately the same. Gel filtration chromatography of lysed cells after incubation for 6 h with 10 μM 75 Se-selenite showed that the radioactivity was eluted as two peaks corresponding to low (4-9 kDa) and high (280-320 kDa) molecular weights. Possible toxicological mechanisms are discussed at molecular level. (orig./MG)

  16. Cell kinetics of hypoxic cells in a murine tumour in vivo: flow cytometric determination of the radiation-induced blockage of cell cycle progression

    International Nuclear Information System (INIS)

    Rutgers, D.H.; Niessen, D.P.P.; Linden, P.M. van der

    1987-01-01

    Cells from the small cell population of viable cells in the large necrotic centre of murine M8013 tumours were investigated with respect to their cell kinetics. Flow cytometry (FCM) of this part of subcutaneously transplanted tumours revealed the presence of tumour cells with G1,S and G2 + M phase DNA-contents. These severely hypoxic cells could have stopped cell cycle progression due to the nutritional deprivation, irrespective of their position within the cell cycle. Labelling methods, used to disclose the cell kinetics of this cell population, are hampered by the absence of a transport system in these large necrotic areas. Therefore FCM was used to monitor radiation induced changes in the cell cycle distribution. From this investigation it was concluded that hypoxic cells in the necrotic centre of the M8013 tumour progress through the cell cycle. As well as a cell population with a cell cycle time (Tsub(c)) of approximately 84 hr, a subpopulation with a Tsub(c) of approximately 21 hr occurred. (author)

  17. FDG small animal PET permits early detection of malignant cells in a xenograft murine model

    International Nuclear Information System (INIS)

    Nanni, Cristina; Spinelli, Antonello; Trespidi, Silvia; Ambrosini, Valentina; Castellucci, Paolo; Farsad, Mohsen; Franchi, Roberto; Fanti, Stefano; Leo, Korinne di; Tonelli, Roberto; Pession, Andrea; Pettinato, Cinzia; Rubello, Domenico

    2007-01-01

    The administration of new anticancer drugs in animal models is the first step from in vitro to in vivo pre-clinical protocols. At this stage it is crucial to ensure that cells are in the logarithmic phase of growth and to avoid vascular impairment, which can cause inhomogeneous distribution of the drug within the tumour and thus lead to bias in the final analysis of efficacy. In subcutaneous xenograft murine models, positivity for cancer is visually recognisable 2-3 weeks after inoculation, when a certain amount of necrosis is usually already present. The aim of this study was to evaluate the accuracy of FDG small animal PET for the early detection of malignant masses in a xenograft murine model of human rhabdomyosarcoma. A second goal was to analyse the metabolic behaviour of this xenograft tumour over time. We studied 23 nude mice, in which 7 x 10 6 rhabdomyosarcoma cells (RH-30 cell line) were injected in the dorsal subcutaneous tissues. Each animal underwent four FDG PET scans (GE, eXplore Vista DR) under gas anaesthesia. The animals were studied 2, 5, 14 and 20 days after inoculation. We administered 20 MBq of FDG via the tail vein. Uptake time was 60 min, and acquisition time, 20 min. Images were reconstructed with OSEM 2D iterative reconstruction and the target to background ratio (TBR) was calculated for each tumour. Normal subcutaneous tissue had a TBR of 0.3. Necrosis was diagnosed when one or more cold areas were present within the mass. All the animals were sacrificed and histology was available to verify PET results. PET results were concordant with the findings of necropsy and histology in all cases. The incidence of the tumour was 69.6% (16/23 animals); seven animals did not develop a malignant mass. Ten of the 23 animals had a positive PET scan 2 days after inoculation. Nine of these ten animals developed a tumour; the remaining animal became negative, at the third scan. The positive predictive value of the early PET scan was 90% (9/10 animals

  18. The clinical role of imaging in peritoneal carcinomatosis

    International Nuclear Information System (INIS)

    Prassopoulos, P.

    2015-01-01

    Full text: Intraperitoneal seeding, the so-called peritoneal carcinomatosis (PC) is a common mechanism of spread in advanced intra-abdominal malignancies including ovarian (71%), gastric (17%) and colorectal (10%) cancers. When cancer cells from a growing primary neoplasm reach the peritoneal surface, they are carried out by the peritoneal fluid and disseminated throughout the peritoneal cavity.the location of implants development is governed mostly by peritoneal fluid circulation and by anatomic pathways formed by peritoneal reflections, namely ligaments, mesenteries and omenta. The most common sites where the peritoneal fluid may temporarily arrested facilitating implantation of cancer cells include cul-de-sac, distal small bowel mesentery, right paracolic gutter, posterior sub-hepatic space, greater omentum and subphrenic spaces. MDCT with thin collimation and i.v contrast material supplemented by multiplanar reconstructions is the primary imaging modality for the investigation of peritoneal carcinomatosis. Ascitis, contrast enhanced smooth, nodular, or plaque-like peritoneal thickening, peritoneal nodules, plaques or masses, rounded, ill-defined soft-tissue or cystic mesenteric masses, mesenteric fixation with increased attenuation values and thickening, irregular soft-tissue permeation of omental fat or confluent solid omental masses are the most frequent CT findings of peritoneal carcinomatosis. CT has a sensitivity and specificity between 85-95%, depending on the size, location and applied examination protocol. MR imaging using a post-gadolinium enhanced 3dFlASH sequence with fat saturation may alternatively

  19. Activation of p38 MAPK by feline infectious peritonitis virus regulates pro-inflammatory cytokine production in primary blood-derived feline mononuclear cells.

    Science.gov (United States)

    Regan, Andrew D; Cohen, Rebecca D; Whittaker, Gary R

    2009-02-05

    Feline infectious peritonitis (FIP) is an invariably fatal disease of cats caused by systemic infection with a feline coronavirus (FCoV) termed feline infectious peritonitis virus (FIPV). The lethal pathology associated with FIP (granulomatous inflammation and T-cell lymphopenia) is thought to be mediated by aberrant modulation of the immune system due to infection of cells such as monocytes and macrophages. Overproduction of pro-inflammatory cytokines occurs in cats with FIP, and has been suggested to play a significant role in the disease process. However, the mechanism underlying this process remains unknown. Here we show that infection of primary blood-derived feline mononuclear cells by FIPV WSU 79-1146 and FIPV-DF2 leads to rapid activation of the p38 MAPK pathway and that this activation regulates production of the pro-inflammatory cytokine tumor necrosis factor alpha (TNF-alpha) and interleukin-1 beta (IL-1 beta). FIPV-induced p38 MAPK activation and pro-inflammatory cytokine production was inhibited by the pyridinyl imidazole inhibitors SB 203580 and SC 409 in a dose-dependent manner. FIPV-induced p38 MAPK activation was observed in primary feline blood-derived mononuclear cells individually purified from multiple SPF cats, as was the inhibition of TNF-alpha production by pyridinyl imidazole inhibitors.

  20. Production and Functional Characterization of Murine Osteoclasts Differentiated from ER-Hoxb8-Immortalized Myeloid Progenitor Cells.

    Directory of Open Access Journals (Sweden)

    Frank Zach

    Full Text Available In vitro differentiation into functional osteoclasts is routinely achieved by incubation of embryonic stem cells, induced pluripotent stem cells, or primary as well as cryopreserved spleen and bone marrow-derived cells with soluble receptor activator of nuclear factor kappa-B ligand and macrophage colony-stimulating factor. Additionally, osteoclasts can be derived from co-cultures with osteoblasts or by direct administration of soluble receptor activator of nuclear factor kappa-B ligand to RAW 264.7 macrophage lineage cells. However, despite their benefits for osteoclast-associated research, these different methods have several drawbacks with respect to differentiation yields, time and animal consumption, storage life of progenitor cells or the limited potential for genetic manipulation of osteoclast precursors. In the present study, we therefore established a novel protocol for the differentiation of osteoclasts from murine ER-Hoxb8-immortalized myeloid stem cells. We isolated and immortalized bone marrow cells from wild type and genetically manipulated mouse lines, optimized protocols for osteoclast differentiation and compared these cells to osteoclasts derived from conventional sources. In vitro generated ER-Hoxb8 osteoclasts displayed typical osteoclast characteristics such as multi-nucleation, tartrate-resistant acid phosphatase staining of supernatants and cells, F-actin ring formation and bone resorption activity. Furthermore, the osteoclast differentiation time course was traced on a gene expression level. Increased expression of osteoclast-specific genes and decreased expression of stem cell marker genes during differentiation of osteoclasts from ER-Hoxb8-immortalized myeloid progenitor cells were detected by gene array and confirmed by semi-quantitative and quantitative RT-PCR approaches. In summary, we established a novel method for the quantitative production of murine bona fide osteoclasts from ER-Hoxb8 stem cells generated from

  1. Currently available murine Leydig cell lines can be applied to study early steps of steroidogenesis but not testosterone synthesis

    Directory of Open Access Journals (Sweden)

    Roger T. Engeli

    2018-02-01

    Full Text Available Androgen biosynthesis in males occurs to a large extent in testicular Leydig cells. This study focused on the evaluation of three murine Leydig cell lines as potential screening tool to test xenobiotics interfering with gonadal androgen synthesis. The final step of testosterone (T production in Leydig cells is catalyzed by the enzyme 17β-hydroxysteroid dehydrogenase 3 (17β-hsd3. The endogenous 17β-hsd3 mRNA expression and Δ4-androstene-3,17-dione (AD to T conversion were determined in the murine cell lines MA-10, BLTK1 and TM3. Additionally, effects of 8-Br-cAMP and forskolin stimulation on steroidogenesis and T production were analyzed. Steroids were quantified in supernatants of cells using liquid chromatography–tandem mass spectrometry. Unstimulated cells incubated with AD produced only very low T but substantial amounts of the inactive androsterone. Stimulated cells produced low amounts of T, moderate amounts of AD, but high amounts of progesterone. Gene expression analyses revealed barely detectable 17β-hsd3 levels, absence of 17β-hsd5 (Akr1c6, but substantial 17β-hsd1 expression in all three cell lines. Thus, MA-10, BLTK1 and TM3 cells are not suitable to study the expression and activity of the gonadal T synthesizing enzyme 17β-hsd3. The low T production reported in stimulated MA-10 cells are likely a result of the expression of 17β-hsd1. This study substantiates that the investigated Leydig cell lines MA-10, BLTK1, and TM3 are not suitable to study gonadal androgen biosynthesis due to altered steroidogenic pathways. Furthermore, this study emphasizes the necessity of mass spectrometry-based steroid quantification in experiments using steroidogenic cells such as Leydig cells.

  2. Exposure to low level GSM 935 MHz radiofrequency fields does not induce apoptosis in proliferating or differentiated murine neuroblastoma cells

    International Nuclear Information System (INIS)

    Moquet, J.; Ainsbury, E.; Bouffler, S.; Lloyd, D.

    2008-01-01

    The aim of this study was to investigate whether radiofrequency (RF) fields characteristic of mobile phones at non-thermal levels can induce apoptosis in murine neuroblastoma (N2a) cells in both proliferating and differentiated states. Cells were exposed continuously for 24 h to one of the three 935-MHz RF signals: global system for mobile communication (GSM) basic, GSM talk and a continuous wave, unmodulated signal; all at a specific energy absorption rate of 2 W kg -1 . The measured increase in temperature of the cells due to the RF fields was around 0.06 deg. C. At a number of time points between 0 and 48 h post-exposure, the cells were assessed for apoptosis under a fluorescence microscope using three independent assays: Annexin V, caspase activation and in situ end-labelling. No statistically significant differences in apoptosis levels were observed between the exposed and sham-exposed cells using the three assays at any time point post-exposure. These data suggest that RF exposures, characteristic of GSM mobile phones, do not significantly affect the apoptosis levels in proliferating and differentiated murine neuroblastoma cell line N2a. (authors)

  3. Possible Immune Regulation of Natural Killer T Cells in a Murine Model of Metal Ion-Induced Allergic Contact Dermatitis

    Directory of Open Access Journals (Sweden)

    Kenichi Kumagai

    2016-01-01

    Full Text Available Metal often causes delayed-type hypersensitivity reactions, which are possibly mediated by accumulating T cells in the inflamed skin, called irritant or allergic contact dermatitis. However, accumulating T cells during development of a metal allergy are poorly characterized because a suitable animal model is unavailable. We have previously established novel murine models of metal allergy and found accumulation of both metal-specific T cells and natural killer (NK T cells in the inflamed skin. In our novel models of metal allergy, skin hypersensitivity responses were induced through repeated sensitizations by administration of metal chloride and lipopolysaccharide into the mouse groin followed by metal chloride challenge in the footpad. These models enabled us to investigate the precise mechanisms of the immune responses of metal allergy in the inflamed skin. In this review, we summarize the immune responses in several murine models of metal allergy and describe which antigen-specific responses occur in the inflamed skin during allergic contact dermatitis in terms of the T cell receptor. In addition, we consider the immune regulation of accumulated NK T cells in metal ion–induced allergic contact dermatitis.

  4. Daphnetin inhibits invasion and migration of LM8 murine osteosarcoma cells by decreasing RhoA and Cdc42 expression

    Energy Technology Data Exchange (ETDEWEB)

    Fukuda, Hiroki [Department of Clinical and Translational Physiology, Kyoto Pharmaceutical University, Kyoto (Japan); Nakamura, Seikou [Department of Pharmacognosy, Kyoto Pharmaceutical University, Kyoto (Japan); Chisaki, Yugo [Education and Research Center for Clinical Pharmacy, Kyoto Pharmaceutical University, Kyoto (Japan); Takada, Tetsuya [Department of Clinical and Translational Physiology, Kyoto Pharmaceutical University, Kyoto (Japan); Toda, Yuki [Department of Medicinal Chemistry, Kyoto Pharmaceutical University, Kyoto (Japan); Murata, Hiroaki [Department of Orthopedics, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kyoto (Japan); Department of Orthopedic Surgery, Matsushita Memorial Hospital, Osaka (Japan); Itoh, Kazuyuki [Department of Biology, Osaka Medical Center of Cancer and Cardiovascular Diseases, Osaka (Japan); Yano, Yoshitaka [Education and Research Center for Clinical Pharmacy, Kyoto Pharmaceutical University, Kyoto (Japan); Takata, Kazuyuki [Department of Clinical and Translational Physiology, Kyoto Pharmaceutical University, Kyoto (Japan); Ashihara, Eishi, E-mail: ash@mb.kyoto-phu.ac.jp [Department of Clinical and Translational Physiology, Kyoto Pharmaceutical University, Kyoto (Japan)

    2016-02-26

    Daphnetin, 7,8-dihydroxycoumarin, present in main constituents of Daphne odora var. marginatai, has multiple pharmacological activities including anti-proliferative effects in cancer cells. In this study, using a Transwell system, we showed that daphnetin inhibited invasion and migration of highly metastatic murine osteosarcoma LM8 cells in a dose-dependent manner. Following treatment by daphnetin, cells that penetrated the Transwell membrane were rounder than non-treated cells. Immunofluorescence analysis revealed that daphnetin decreased the numbers of intracellular stress fibers and filopodia. Moreover, daphnetin treatment dramatically decreased the expression levels of RhoA and Cdc42. In summary, the dihydroxycoumarin derivative daphnetin inhibits the invasion and migration of LM8 cells, and therefore represents a promising agent for use against metastatic cancer. - Highlights: • Daphnetin, a coumarin-derivative, inhibited invasion and migration of LM8 cells. • Stress fibers and filopodia were decreased by daphnetin treatment. • Daphnetin decreased RhoA and Cdc42 protein expression.

  5. Daphnetin inhibits invasion and migration of LM8 murine osteosarcoma cells by decreasing RhoA and Cdc42 expression

    International Nuclear Information System (INIS)

    Fukuda, Hiroki; Nakamura, Seikou; Chisaki, Yugo; Takada, Tetsuya; Toda, Yuki; Murata, Hiroaki; Itoh, Kazuyuki; Yano, Yoshitaka; Takata, Kazuyuki; Ashihara, Eishi

    2016-01-01

    Daphnetin, 7,8-dihydroxycoumarin, present in main constituents of Daphne odora var. marginatai, has multiple pharmacological activities including anti-proliferative effects in cancer cells. In this study, using a Transwell system, we showed that daphnetin inhibited invasion and migration of highly metastatic murine osteosarcoma LM8 cells in a dose-dependent manner. Following treatment by daphnetin, cells that penetrated the Transwell membrane were rounder than non-treated cells. Immunofluorescence analysis revealed that daphnetin decreased the numbers of intracellular stress fibers and filopodia. Moreover, daphnetin treatment dramatically decreased the expression levels of RhoA and Cdc42. In summary, the dihydroxycoumarin derivative daphnetin inhibits the invasion and migration of LM8 cells, and therefore represents a promising agent for use against metastatic cancer. - Highlights: • Daphnetin, a coumarin-derivative, inhibited invasion and migration of LM8 cells. • Stress fibers and filopodia were decreased by daphnetin treatment. • Daphnetin decreased RhoA and Cdc42 protein expression.

  6. The Na{sup +}/K{sup +} -pump in rat peritoneal mast cells: Some aspects of regulatio of activity and cellular fusion

    Energy Technology Data Exchange (ETDEWEB)

    Knudsen, T. [Odense Univ., Dept. of Pharmacology, Inst. of Medical Biology, The Faculty of Health Scineces (Denmark)

    1995-12-31

    The mast cell contains potent mediators of inflammation which are released after IgE-directed and non-IgE-directed stimulation of the cell. This highly specialized cell is therefore ascribed a role in the pathogenesis of disease states in which the inflammatory response plays a role for the development of the clinical symptoms. Thus, besides being of interest in basic research, studies of the cellular processes leading to release of inflammatory mediators from the mast cell also also have important clinical implications. The aim of the present work has been to document the existence of the Na{sup +}/K{sup +}-pump in rat peritoneal mast cells, to investigate the regulation of the pump activity and to explore whether modulation of the pump activity interferes with the cellular stimulus/secretion coupling mechanism. The Na{sup +}/K{sup +}-pump activity following stimulation of the mast cell was also investigated. The pump activity was assessed as the ouabain-sensitive cellular potassium uptake with {sup 86}Rb{sup +} as a tracer for potassium. The histamine release from the mast cell following IgE-directed and non-IgE-directed stimulation of the cell was used as a parameter of cellular degranulation. Histamine was measured by spectrofluorometry. Besides describing aspects of the function and regulation of the Na{sup +}/K{sup +}-pump in the rat peritoneal mast cell, this thesis points to the potential role of sodium transport mechanisms in mast cell physiology. Pharmacological manipulations of such transport mechanisms might in the future add to the treatment of allergic diseases. (au) 253 refs.

  7. Dek overexpression in murine epithelia increases overt esophageal squamous cell carcinoma incidence

    Science.gov (United States)

    Cimperman, Katherine A.; Haas, Sarah R.; Guasch, Geraldine; Waclaw, Ronald R.; Komurov, Kakajan; Lane, Adam; Wikenheiser-Brokamp, Kathryn A.

    2018-01-01

    Esophageal cancer occurs as either squamous cell carcinoma (ESCC) or adenocarcinoma. ESCCs comprise almost 90% of cases worldwide, and recur with a less than 15% five-year survival rate despite available treatments. The identification of new ESCC drivers and therapeutic targets is critical for improving outcomes. Here we report that expression of the human DEK oncogene is strongly upregulated in esophageal SCC based on data in the cancer genome atlas (TCGA). DEK is a chromatin-associated protein with important roles in several nuclear processes including gene transcription, epigenetics, and DNA repair. Our previous data have utilized a murine knockout model to demonstrate that Dek expression is required for oral and esophageal SCC growth. Also, DEK overexpression in human keratinocytes, the cell of origin for SCC, was sufficient to cause hyperplasia in 3D organotypic raft cultures that mimic human skin, thus linking high DEK expression in keratinocytes to oncogenic phenotypes. However, the role of DEK over-expression in ESCC development remains unknown in human cells or genetic mouse models. To define the consequences of Dek overexpression in vivo, we generated and validated a tetracycline responsive Dek transgenic mouse model referred to as Bi-L-Dek. Dek overexpression was induced in the basal keratinocytes of stratified squamous epithelium by crossing Bi-L-Dek mice to keratin 5 tetracycline transactivator (K5-tTA) mice. Conditional transgene expression was validated in the resulting Bi-L-Dek_K5-tTA mice and was suppressed with doxycycline treatment in the tetracycline-off system. The mice were subjected to an established HNSCC and esophageal carcinogenesis protocol using the chemical carcinogen 4-nitroquinoline 1-oxide (4NQO). Dek overexpression stimulated gross esophageal tumor development, when compared to doxycycline treated control mice. Furthermore, high Dek expression caused a trend toward esophageal hyperplasia in 4NQO treated mice. Taken together, these

  8. The Effects of Inhaled Nickel Nanoparticles on Murine Endothelial Progenitor Cells

    Science.gov (United States)

    Liberda, Eric N.

    Introduction. Particulate air pollution, specifically nickel found on or in particulate matter, has been associated with an increased risk of mortality in human population studies and can cause increases in vascular inflammation, generate reactive oxygen species, alter vasomotor tone, and potentiate atherosclerosis in murine exposures. With the discovery of endothelial progenitor cells (EPCs), a door has been opened which may explain these observed cardiovascular effects associated with inhaled air particles and nickel exposure. In order to further quantify the effects of inhaled nickel nanoparticles and attempt to elucidate how the observed findings from other studies may occur, several whole body inhalation exposure experiments to nickel nanoparticles were performed. Methods. Following whole body exposure to approximately 500mug/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, circulating endothelial cells (CECs), and endothelial microparticles (EMPs) were quantified on a BD FACSCalibur to examine endothelial damage and repair associated with the inhalation exposure. Plasma proteins were assessed using the 2D DIGE proteomic approach and commercially available ELISAs. Results and Conclusions. Exposure to inhaled nickel nanoparticles significantly increased both bone marrow EPCs as well as their levels in circulation. CECs were significantly upregulated suggesting 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. This decrease in EPC function

  9. Selected microRNAs define cell fate determination of murine central memory CD8 T cells.

    Directory of Open Access Journals (Sweden)

    Gonzalo Almanza

    2010-06-01

    Full Text Available During an immune response T cells enter memory fate determination, a program that divides them into two main populations: effector memory and central memory T cells. Since in many systems protection appears to be preferentially mediated by T cells of the central memory it is important to understand when and how fate determination takes place. To date, cell intrinsic molecular events that determine their differentiation remains unclear. MicroRNAs are a class of small, evolutionarily conserved RNA molecules that negatively regulate gene expression, causing translational repression and/or messenger RNA degradation. Here, using an in vitro system where activated CD8 T cells driven by IL-2 or IL-15 become either effector memory or central memory cells, we assessed the role of microRNAs in memory T cell fate determination. We found that fate determination to central memory T cells is under the balancing effects of a discrete number of microRNAs including miR-150, miR-155 and the let-7 family. Based on miR-150 a new target, KChIP.1 (K (+ channel interacting protein 1, was uncovered, which is specifically upregulated in developing central memory CD8 T cells. Our studies indicate that cell fate determination such as surface phenotype and self-renewal may be decided at the pre-effector stage on the basis of the balancing effects of a discrete number of microRNAs. These results may have implications for the development of T cell vaccines and T cell-based adoptive therapies.

  10. Cell division requirement for activation of murine leukemia virus in cell culture by irradiation

    International Nuclear Information System (INIS)

    Otten, J.A.; Quarles, J.M.; Tennant, R.W.

    1976-01-01

    Actively dividing cultures of AKR mouse cells were exposed to relatively low dose-rates of γ radiation and tested for activation of endogenous leukemia viruses. Efficient and reproducible induction of virus was obtained with actively dividing cells, but cultures deprived of serum to inhibit cell division before and during γ irradiation were not activated, even when medium with serum was added immediately after irradiation. These results show that cell division was required for virus induction but that a stable intermediate similar to the state induced by halogenated pyrimidines was not formed. In actively dividing AKR cell cultures, virus activation appeared to be proportional to the dose of γ radiation; the estimated frequency of activation was 1-8 x 10 - 5 per exposed cell and the efficiency of activation was approximately 0.012 inductions per cell per rad. Other normal primary and established mouse cell cultures tested were not activated by γ radiation. The requirement of cell division for radiation and chemical activation may reflect some common mechanism for initiation of virus expression

  11. Maturational steps of bone marrow-derived dendritic murine epidermal cells. Phenotypic and functional studies on Langerhans cells and Thy-1+ dendritic epidermal cells in the perinatal period.

    Science.gov (United States)

    Elbe, A; Tschachler, E; Steiner, G; Binder, A; Wolff, K; Stingl, G

    1989-10-15

    The adult murine epidermis harbors two separate CD45+ bone marrow (BM)-derived dendritic cell systems, i.e., Ia+, ADPase+, Thy-1-, CD3- Langerhans cells (LC) and Ia-, ADPase-, Thy-1+, CD3+ dendritic epidermal T cells (DETC). To clarify whether the maturation of these cells from their ill-defined precursors is already accomplished before their entry into the epidermis or, alternatively, whether a specific epidermal milieu is required for the expression of their antigenic determinants, we studied the ontogeny of CD45+ epidermal cells (EC). In the fetal life, there exists a considerable number of CD45+, Ia-, ADPase+ dendritic epidermal cells. When cultured, these cells become Ia+ and, in parallel, acquire the potential of stimulating allogeneic T cell proliferation. These results imply that CD45+, Ia-, ADPase+ fetal dendritic epidermal cells are immature LC precursors and suggest that the epidermis plays a decisive role in LC maturation. The day 17 fetal epidermis also contains a small population of CD45+, Thy-1+, ADPase-, CD3- round cells. Over the course of 2 to 3 wk, they are slowly replaced by an ever increasing number of round and, finally, dendritic CD45+, Thy-1+, CD3+ EC. Thus, CD45+, Thy-1+, ADPase-, CD3- fetal EC may either be DETC precursors or, alternatively, may represent a distinctive cell system of unknown maturation potential. According to this latter theory, these cells would be eventually outnumbered by newly immigrating CD45+, Thy-1+, CD3+ T cells--the actual DETC.

  12. Memory Th1 Cells Are Protective in Invasive Staphylococcus aureus Infection

    NARCIS (Netherlands)

    A.F. Brown (Aisling F.); A.G. Murphy (Alison G.); S.J. Lalor (Stephen J.); J.M. Leech (John M.); K.M. O’Keeffe (Kate M.); M. Mac Aogáin (Micheál); D.P. O’Halloran (Dara P.); K.A. Lacey (Keenan A.); M. Tavakol (Mehri); C.H. Hearnden (Claire H.); D. Fitzgerald-Hughes (Deirdre); H. Humphreys (Hilary); J.P. Fennell (Jérôme P.); W.J.B. van Wamel (Willem); T.J. Foster (Timothy J.); J.A. Geoghegan (Joan A.); E.C. Lavelle (Ed C.); T.R. Rogers (Thomas R.); R.M. McLoughlin (Rachel M.)

    2015-01-01

    textabstractMechanisms of protective immunity to Staphylococcus aureus infection in humans remain elusive. While the importance of cellular immunity has been shown in mice, T cell responses in humans have not been characterised. Using a murine model of recurrent S. aureus peritonitis, we

  13. Dynamic O-linked N-acetylglucosamine modification of proteins affects stress responses and survival of mesothelial cells exposed to peritoneal dialysis fluids.

    Science.gov (United States)

    Herzog, Rebecca; Bender, Thorsten O; Vychytil, Andreas; Bialas, Katarzyna; Aufricht, Christoph; Kratochwill, Klaus

    2014-12-01

    The ability of cells to respond and survive stressful conditions is determined, in part, by the attachment of O-linked N-acetylglucosamine (O-GlcNAc) to proteins (O-GlcNAcylation), a post-translational modification dependent on glucose and glutamine. This study investigates the role of dynamic O-GlcNAcylation of mesothelial cell proteins in cell survival during exposure to glucose-based peritoneal dialysis fluid (PDF). Immortalized human mesothelial cells and primary mesothelial cells, cultured from human omentum or clinical effluent of PD patients, were assessed for O-GlcNAcylation under normal conditions or after exposure to PDF. The dynamic status of O-GlcNAcylation and effects on cellular survival were investigated by chemical modulation with 6-diazo-5-oxo-L-norleucine (DON) to decrease or O-(2-acetamido-2-deoxy-D-glucopyranosylidene)amino N-phenyl carbamate (PUGNAc) to increase O-GlcNAc levels. Viability was decreased by reducing O-GlcNAc levels by DON, which also led to suppressed expression of the cytoprotective heat shock protein 72. In contrast, increasing O-GlcNAc levels by PUGNAc or alanyl-glutamine led to significantly improved cell survival paralleled by higher heat shock protein 72 levels during PDF treatment. Addition of alanyl-glutamine increased O-GlcNAcylation and partly counteracted its inhibition by DON, also leading to improved cell survival. Immunofluorescent analysis of clinical samples showed that the O-GlcNAc signal primarily originates from mesothelial cells. In conclusion, this study identified O-GlcNAcylation in mesothelial cells as a potentially important molecular mechanism after exposure to PDF. Modulating O-GlcNAc levels by clinically feasible interventions might evolve as a novel therapeutic target for the preservation of peritoneal membrane integrity in PD. Copyright © 2014 by the American Society of Nephrology.

  14. Viable Cancer Cells in the Remnant Stomach are a Potential Source of Peritoneal Metastasis after Curative Distal Gastrectomy for Gastric Cancer.

    Science.gov (United States)

    Murata, Satoshi; Yamamoto, Hiroshi; Yamaguchi, Tsuyoshi; Kaida, Sachiko; Ishida, Mitsuaki; Kodama, Hirokazu; Takebayashi, Katsushi; Shimizu, Tomoharu; Miyake, Toru; Tani, Tohru; Kushima, Ryoji; Tani, Masaji

    2016-09-01

    The mechanisms underlying peritoneal metastasis (PM) after curative gastrectomy for gastric cancer (GC) are not well elucidated. This study assessed whether viable cancer cells, including cancer stemlike cells (CSCs), were present in the remnant stomach immediately before gastrointestinal (GI) tract reconstruction because these could be a source of PM after gastrectomy. Saline fluid used for remnant stomach lumen irrigation before GI reconstruction was prospectively collected from 142 consecutive patients undergoing distal gastrectomy for GC and cytologically examined. Proliferative activity (Ki67 staining) and stemness (expression of the CSC surface markers CD44s or CD44v6) were evaluated in detected cancer cells. Viable cancer cells were detected in 33 (23.2 %) of the 142 remnant stomachs. These cells formed clusters and stained positively for Ki67, indicating proliferation. Cancer cells in remnant stomachs and surface cancer cells in primary GCs from 10 (30.3 %) of these 33 cases also stained positively for CD44s or CD44v6. In a multiple logistic regression analysis, advanced cancer (odds ratio [OR], 4.65; 95 % confidence interval [CI], 1.32-16.4; P = 0.017), tumor size of 40 mm or larger (OR, 3.78; 95 % CI, 1.12-12.8; P = 0.033), and histologic differentiation (OR, 3.10; 95 % CI, 1.30-7.40; P = 0.011) were associated independently with the presence of cancer cells in the remnant stomach. Viable, proliferative, and clustered cancer cells, including CSCs, were found in remnant gastric lumens immediately before GI reconstruction, indicating a possible cellular source of PM after curative gastrectomy for GC. Dissemination of gastric contents into the peritoneal cavity should be avoided during GI reconstruction.

  15. Phenethyl ester and amide of Ferulic Acids: Synthesis and bioactivity against P388 Leukemia Murine Cells

    Science.gov (United States)

    Firdaus; Soekamto, N. H.; Seniwati; Islam, M. F.; Sultan

    2018-03-01

    Bioactivity of a compound is closely related to the molecular structure of the compound concerned, its strength being the quantitative relation of the strength of the activity of the group it possesses. The combining of moieties of the active compounds will produce more active compounds. Most phenolic compounds as well as compounds containing moiety phenethyl groups have potential activity as anticancer. Combining phenolic groups and phenethyl groups in a compound will result in compounds having strong anticancer bioactivity. This study aims to combine the feruloyl and phenethyl groups to form esters and amides by synthesize of phenethyl trans-3-(4-hydroxy-3-methoxyphenyl)acrylate (5) and trans-3-(4- hydroxy-3-methoxyphenyl)-N-phenethylacrylamide (6) from ferulic acid with phenethyl alcohol and phenethylamine, and to study their bioactivity as anticancer. The synthesis of both compounds was conducted via indirect reaction, including acetylation, chlorination, esterfication/amidation, and deacetylation. Structures of products were characterized by FTIR and NMR data, and their bioactivity assay of the compounds against P388 Leukemia Murine Cells was conducted by an MTT method. Results showed that the compound 5 was obtained as a yellow gel with the IC50 of 10.79 μg/mL (36.21 μΜ), and the compound 6 was a yellowish solid with a melting point of 118-120°C and the IC50 of 29.14 μg/mL (97.79 μΜ). These compounds were more active than the analog compounds.

  16. Stable integration of recombinant adeno-associated virus vector genomes after transduction of murine hematopoietic stem cells.

    Science.gov (United States)

    Han, Zongchao; Zhong, Li; Maina, Njeri; Hu, Zhongbo; Li, Xiaomiao; Chouthai, Nitin S; Bischof, Daniela; Weigel-Van Aken, Kirsten A; Slayton, William B; Yoder, Mervin C; Srivastava, Arun

    2008-03-01

    We previously reported that among single-stranded adeno-associated virus (ssAAV) vectors, serotypes 1 through 5, ssAAV1 is the most efficient in transducing murine hematopoietic stem cells (HSCs), but viral second-strand DNA synthesis remains a rate-limiting step. Subsequently, using double-stranded, self-complementary AAV (scAAV) vectors, serotypes 7 through 10, we observed that scAAV7 vectors also transduce murine HSCs efficiently. In the present study, we used scAAV1 and scAAV7 shuttle vectors to transduce HSCs in a murine bone marrow serial transplant model in vivo, which allowed examination of the AAV proviral integration pattern in the mouse genome, as well as recovery and nucleotide sequence analyses of AAV-HSC DNA junction fragments. The proviral genomes were stably integrated, and integration sites were localized to different mouse chromosomes. None of the integration sites was found to be in a transcribed gene, or near a cellular oncogene. None of the animals, monitored for up to 1 year, exhibited pathological abnormalities. Thus, AAV proviral integration-induced risk of oncogenesis was not found in our study, which provides functional confirmation of stable transduction of self-renewing multipotential HSCs by scAAV vectors as well as promise for the use of these vectors in the potential treatment of disorders of the hematopoietic system.

  17. Prognostic Factors for Peritonitis Outcome

    NARCIS (Netherlands)

    van Esch, Sadie; Krediet, Raymond T.; Struijk, Dirk G.

    2012-01-01

    Despite advances in treatment and prevention, peritonitis remains a major problem in peritoneal dialysis (PD) patients with often technique failure as a consequence. The last decades the focus of PD peritonitis has changed from lowering peritonitis incidence to improvement of peritonitis outcome.

  18. Peritoneal dialysis-related peritonitis: challenges and solutions

    Science.gov (United States)

    Salzer, William L

    2018-01-01

    Peritoneal dialysis is an effective treatment modality for patients with end-stage renal disease. The relative use of peritoneal dialysis versus hemodialysis varies widely by country. Data from a 2004 survey reports the percentage of patients with end-stage renal disease treated with peritoneal dialysis to be 5%–10% in economically developed regions like the US and Western Europe to as much as 75% in Mexico. This disparity is probably related to the availability and access to hemodialysis, or in some cases patient preference for peritoneal over hemodialysis. Peritoneal dialysis-related peritonitis remains the major complication and primary challenge to the long-term success of peritoneal dialysis. Fifty years ago, with the advent of the Tenckhoff catheter, patients averaged six episodes of peritonitis per year on peritoneal dialysis. In 2016, the International Society for Peritoneal Dialysis proposed a benchmark of 0.5 episodes of peritonitis per year or one episode every 2 years. Despite the marked reduction in peritonitis over time, peritonitis for the individual patient is problematic. The mortality for an episode of peritonitis is 5% and is a cofactor for mortality in another 16% of affected patients. Prevention of peritonitis and prompt and appropriate management of peritonitis is essential for the long-term success of peritoneal dialysis in all patients. In this review, challenges and solutions are addressed regarding the pathogenesis, clinical features, diagnosis, treatment, and prevention of peritoneal dialysis-related peritonitis from the viewpoint of an infectious disease physician.

  19. Enhanced therapeutic effect on murine melanoma and angiosarcoma cells by boron neutron capture therapy using a boronated metalloporphyrin

    International Nuclear Information System (INIS)

    Yamada, Yoshihiko; Ichihashi, Masamitsu; Kahl, S.B.; Toda, Ken-ichi.

    1994-01-01

    We have already achieved successful treatment of several human patients with malignant melanoma by boron neutron capture therapy (BNCT) using 10 B 1 -paraboronophenylalanine ( 10 B 1 -BPA·HCl). In this study we used a new compound, a manganese boronated protoporphyrin (Mn- 10 BOPP), and compared it to 10 B 1 -BPA·HCl with respect to uptake in murine melanoma and angiosarcoma cells as well as to their cell killing effect. 10 B uptake was measured in a new method, and the new compound was much more incorporated into both cells than 10 B 1 -BPA·HCl. Furthermore, melanoma and angiosarcoma cells preincubated with the new compound were 15 to 20 times more efficiently killed by BNCT than cells preincubated with 10 B 1 -BPA·HCl. (author)

  20. Murine T cell activation is regulated by surfen (bis-2-methyl-4-amino-quinolyl-6-carbamide)

    Energy Technology Data Exchange (ETDEWEB)

    Warford, Jordan, E-mail: jordan.warford@dal.ca [Department of Pathology, Dalhousie University, Tupper Building, 5850 College Street, Halifax, Nova Scotia B3H 4R2 (Canada); Doucette, Carolyn D., E-mail: carolyn.doucette@dal.ca [Department of Microbiology and Immunology, Dalhousie University, Tupper Building, 5850 College Street, Halifax, Nova Scotia B3H 4R2 (Canada); Hoskin, David W., E-mail: d.w.hoskin@dal.ca [Department of Pathology, Dalhousie University, Tupper Building, 5850 College Street, Halifax, Nova Scotia B3H 4R2 (Canada); Department of Microbiology and Immunology, Dalhousie University, Tupper Building, 5850 College Street, Halifax, Nova Scotia B3H 4R2 (Canada); Easton, Alexander S., E-mail: alexander.easton@dal.ca [Department of Pathology, Dalhousie University, Tupper Building, 5850 College Street, Halifax, Nova Scotia B3H 4R2 (Canada); Department of Microbiology and Immunology, Dalhousie University, Tupper Building, 5850 College Street, Halifax, Nova Scotia B3H 4R2 (Canada); Department of Surgery (Neurosurgery), Dalhousie University, Tupper Building, 5850 College Street, Halifax, Nova Scotia B3H 4R2 (Canada)

    2014-01-10

    Highlights: •Surfen is the first inhibitor of glycosaminoglycan function to be studied in murine T cells. •Surfen reduces T cell proliferation stimulated in vitro and in vivo. •Surfen reduces CD25 expression in T cells activated in vivo but not in vitro. •Surfen increases T cell proliferation when T cell receptor activation is bypassed. •Surfen’s effects are blocked by co-administration of heparin sulfate. -- Abstract: Surfen (bis-2-methyl-4-amino-quinolyl-6-carbamide) binds to glycosaminoglycans (GAGs) and has been shown to influence their function, and the function of proteoglycans (complexes of GAGs linked to a core protein). T cells synthesize, secrete and express GAGs and proteoglycans which are involved in several aspects of T cell function. However, there are as yet no studies on the effect of GAG-binding agents such as surfen on T cell function. In this study, surfen was found to influence murine T cell activation. Doses between 2.5 and 20 μM produced a graduated reduction in the proliferation of T cells activated with anti-CD3/CD28 antibody-coated T cell expander beads. Surfen (20 mg/kg) was also administered to mice treated with anti-CD3 antibody to activate T cells in vivo. Lymphocytes from surfen-treated mice also showed reduced proliferation and lymph node cell counts were reduced. Surfen reduced labeling with a cell viability marker (7-ADD) but to a much lower extent than its effect on proliferation. Surfen also reduced CD25 (the α-subunit of the interleukin (IL)-2 receptor) expression with no effect on CD69 expression in T cells treated in vivo but not in vitro. When receptor activation was bypassed by treating T cells in vitro with phorbyl myristate acetate (10 ng/ml) and ionomycin (100 ng/ml), surfen treatment either increased proliferation (10 μM) or had no effect (2.5, 5 and 20 μM). In vitro treatment of T cells with surfen had no effect on IL-2 or interferon-γ synthesis and did not alter proliferation of the IL-2 dependent cell

  1. Menstrual endometrial cells from women with endometriosis demonstrate increased adherence to peritoneal cells and increased expression of CD44 splice variants.

    Science.gov (United States)

    Griffith, Jason S; Liu, Ya-Guang; Tekmal, Rajeshwar R; Binkley, Peter A; Holden, Alan E C; Schenken, Robert S

    2010-04-01

    We previously demonstrated that adherence of endometrial epithelial (EECs) and stromal cells (ESCs) to peritoneal mesothelial cells (PMCs) is partly regulated by ESC/EEC CD44 interactions with PMC associated hyaluronan. CD44, a transmembrane glycoprotein and major ligand for hyaluronan, has numerous splice variants which may impact hyaluronan binding. Here, we assessed whether ESCs and EECs from women with endometriosis demonstrate increased adherence to PMCs and examined CD44 splice variants' potential role in this process. In vitro study. Academic medical center. Fertility patients with and without endometriosis. Menstrual endometrium was collected from women with and without endometriosis confirmed surgically. The adherence of ESC/EECs to PMCs was measured. The ESC/EEC CD44 splice variants were assessed using dot-blot analysis. The ESCs and EECs from women with endometriosis demonstrated increased adherence to PMCs. The predominant CD44 splice variants expressed by ESCs and EECs from women with and without endometriosis were v3, v6, v7, v8, v9, and v10. The ESCs and EECs from women with endometriosis were more likely to express v6, v7, v8, and v9. Increased eutopic endometrial-PMC adherence and CD44 splice variant expression may contribute to the histogenesis of endometriotic lesions. Elucidation of factors controlling this expression may lead to novel endometriosis therapies. Copyright 2010 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

  2. Human and murine very small embryonic-like cells represent multipotent tissue progenitors, in vitro and in vivo.

    Science.gov (United States)

    Havens, Aaron M; Sun, Hongli; Shiozawa, Yusuke; Jung, Younghun; Wang, Jingcheng; Mishra, Anjali; Jiang, Yajuan; O'Neill, David W; Krebsbach, Paul H; Rodgerson, Denis O; Taichman, Russell S

    2014-04-01

    The purpose of this study was to determine the lineage progression of human and murine very small embryonic-like (HuVSEL or MuVSEL) cells in vitro and in vivo. In vitro, HuVSEL and MuVSEL cells differentiated into cells of all three embryonic germ layers. HuVSEL cells produced robust mineralized tissue of human origin compared with controls in calvarial defects. Immunohistochemistry demonstrated that the HuVSEL cells gave rise to neurons, adipocytes, chondrocytes, and osteoblasts within the calvarial defects. MuVSEL cells were also able to differentiate into similar lineages. First round serial transplants of MuVSEL cells into irradiated osseous sites demonstrated that ∼60% of the cells maintained their VSEL cell phenotype while other cells differentiated into multiple tissues at 3 months. Secondary transplants did not identify donor VSEL cells, suggesting limited self renewal but did demonstrate VSEL cell derivatives in situ for up to 1 year. At no point were teratomas identified. These studies show that VSEL cells produce multiple cellular structures in vivo and in vitro and lay the foundation for future cell-based regenerative therapies for osseous, neural, and connective tissue disorders.

  3. Effects of Electromagnetic Stimulation on Cell Density and Neural Markers in Murine Enteric Cell Cultures

    International Nuclear Information System (INIS)

    Carreon-Rodriguez, A.; Belkind-Gerson, J.; Serrano-Luna, G.; Canedo-Dorantes, L.

    2008-01-01

    Availability of adult stem cells from several organs like bone marrow, umbilical cord blood or peripheral blood has become a powerful therapeutic tool for many chronic diseases. Potential of adult stem cells for regeneration extents to other tissues among them the nervous system. However two obstacles should be resolved before such cells could be currently applied in clinical practice: a) slow growth rate and b) ability to form enough dense colonies in order to populate a specific injury or cellular deficiency. Many approaches have been explored as genetic differentiation programs, growth factors, and supplemented culture media, among others. Electromagnetic field stimulation of differentiation, proliferation, migration, and particularly on neurogenesis is little known. Since the biological effects of ELF-EMF are well documented, we hypothesize ELF-EMF could affect growth and maturation of stem cells derived of enteric tissue

  4. The Role of Hibiscus sabdariffa L. (Roselle) in Maintenance of Ex Vivo Murine Bone Marrow-Derived Hematopoietic Stem Cells

    Science.gov (United States)

    Abdul Hamid, Zariyantey; Lin Lin, Winnie Hii; Abdalla, Basma Jibril; Bee Yuen, Ong; Latif, Elda Surhaida; Mohamed, Jamaludin; Rajab, Nor Fadilah; Paik Wah, Chow; Budin, Siti Balkis

    2014-01-01

    Hematopoietic stem cells- (HSCs-) based therapy requires ex vivo expansion of HSCs prior to therapeutic use. However, ex vivo culture was reported to promote excessive production of reactive oxygen species (ROS), exposing HSCs to oxidative damage. Efforts to overcome this limitation include the use of antioxidants. In this study, the role of Hibiscus sabdariffa L. (Roselle) in maintenance of cultured murine bone marrow-derived HSCs was investigated. Aqueous extract of Roselle was added at varying concentrations (0–1000 ng/mL) for 24 hours to the freshly isolated murine bone marrow cells (BMCs) cultures. Effects of Roselle on cell viability, reactive oxygen species (ROS) production, glutathione (GSH) level, superoxide dismutase (SOD) activity, and DNA damage were investigated. Roselle enhanced the survival (P < 0.05) of BMCs at 500 and 1000 ng/mL, increased survival of Sca-1+ cells (HSCs) at 500 ng/mL, and maintained HSCs phenotype as shown from nonremarkable changes of surface marker antigen (Sca-1) expression in all experimental groups. Roselle increased (P < 0.05) the GSH level and SOD activity but the level of reactive oxygen species (ROS) was unaffected. Moreover, Roselle showed significant cellular genoprotective potency against H2O2-induced DNA damage. Conclusively, Roselle shows novel property as potential supplement and genoprotectant against oxidative damage to cultured HSCs. PMID:25405216

  5. The Role of Hibiscus sabdariffa L. (Roselle in Maintenance of Ex Vivo Murine Bone Marrow-Derived Hematopoietic Stem Cells

    Directory of Open Access Journals (Sweden)

    Zariyantey Abdul Hamid

    2014-01-01

    Full Text Available Hematopoietic stem cells- (HSCs- based therapy requires ex vivo expansion of HSCs prior to therapeutic use. However, ex vivo culture was reported to promote excessive production of reactive oxygen species (ROS, exposing HSCs to oxidative damage. Efforts to overcome this limitation include the use of antioxidants. In this study, the role of Hibiscus sabdariffa L. (Roselle in maintenance of cultured murine bone marrow-derived HSCs was investigated. Aqueous extract of Roselle was added at varying concentrations (0–1000 ng/mL for 24 hours to the freshly isolated murine bone marrow cells (BMCs cultures. Effects of Roselle on cell viability, reactive oxygen species (ROS production, glutathione (GSH level, superoxide dismutase (SOD activity, and DNA damage were investigated. Roselle enhanced the survival (P<0.05 of BMCs at 500 and 1000 ng/mL, increased survival of Sca-1+ cells (HSCs at 500 ng/mL, and maintained HSCs phenotype as shown from nonremarkable changes of surface marker antigen (Sca-1 expression in all experimental groups. Roselle increased (P<0.05 the GSH level and SOD activity but the level of reactive oxygen species (ROS was unaffected. Moreover, Roselle showed significant cellular genoprotective potency against H2O2-induced DNA damage. Conclusively, Roselle shows novel property as potential supplement and genoprotectant against oxidative damage to cultured HSCs.

  6. Murine bone marrow-derived mesenchymal stem cells as vehicles for interleukin-12 gene delivery into Ewing sarcoma tumors.

    Science.gov (United States)

    Duan, Xiaoping; Guan, Hui; Cao, Ying; Kleinerman, Eugenie S

    2009-01-01

    This study evaluated the therapeutic efficacy of interleukin 12 (IL-12) gene therapy in Ewing sarcoma and whether murine mesenchymal stem cells (MSCs) could serve as vehicles for IL-12 gene delivery. MSCs were isolated from murine bone marrow cells. Cells were phenotyped using flow cytometry. Cultured MSCs differentiated into osteocytes and adipocytes using the appropriate media. Freshly isolated MSCs were transfected with adenoviral vectors containing either the beta-galactosidase (Ad:beta-gal) or the IL-12 (Ad:IL-12) gene. Expression of IL-12 was confirmed using reverse transcription polymerase chain reaction. Mice with TC71 Ewing sarcoma tumors were then treated intravenously with MSCs transfected with Ad:beta-gal or Ad:IL-12. Tumors were measured and analyzed by immunohistochemical analysis for expression of IL-12 protein. Expression of both p35 and p40 IL-12 subunits was demonstrated in MSCs transfected in vitro with Ad:IL-12. IL-12 expression was seen in tumors from mice treated with MSCs transfected with Ad:IL-12. Tumor growth was also significantly inhibited compared with that in mice treated with MSCs transfected with Ad:beta-gal. MSCs can be transfected with the IL-12 gene. These transfected cells localize to tumors after intravenous injection and induce local IL-12 protein production and the regression of established tumors. Copyright (c) 2008 American Cancer Society.

  7. Inhibition of Angiogenic Factor Production from Murine Mast Cells by an Antiallergic Agent (Epinastine Hydrochloride In Vitro

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    K. Asano

    2008-01-01

    Full Text Available Angiogenesis is an important event both in the development of allergic inflammatory responses and in the pathophysiology of tissue remodeling in allergic diseases. In the present study, therefore, we examined the influence of antihistamines on angiogenesis through the choice of epinastine hydrochloride (EP and murine mast cells in vitro. Mast cells (5×105 cells/mL presensitized with murine IgE specific for ovalbumin (OVA were stimulated with 10 ng/mL OVA in the presence of various concentrations of EP for 4 hours. The levels of angiogenesis factors, keratinocyte-derived chemokine (KC, tumor necrosis factor-α (TNF, and vascular endothelial growth factor (VEGF in culture supernatants, were examined by ELISA. We also examined mRNA expression for the angiogenesis factors by RT-PCR. EP significantly inhibited the production of KC, TNF, and VEGF induced by IgE-dependent mechanism at more than 25 ng/mL. Semiquantitative analysis using RT-PCR showed that EP also significantly reduced mRNA expressions for KC, TNF, and VEGF. These results strongly suggest that EP suppresses angiogenesis factor production through the inhibition of mRNA expression in mast cells and results in favorable modification of clinical conditions of allergic diseases.

  8. Stem cell factor enhances the survival of murine intestinal stem cells after photon irradiation

    International Nuclear Information System (INIS)

    Leigh, B.R.; Khan, W.; Hancock, S.L.

    1995-01-01

    Recombinant rat stem cell factor (SCF) has been shown to decrease lethality in mice exposed to total-body irradiation (TBI) in the lower range of lethality through radioprotection of hematopoietic stem cells and acceleration of bone marrow repopulation. This study evaluates the effect of SCF on the survival of the intestinal mucosal stem cell after TBI. This non-hematopoietic cell is clinically relevant. Gastrointestinal toxicity is common during and after abdominal and pelvic radiation therapy and limits the radiation dose in these regions. As observed with bone marrow, the administration of SCF to mice prior to TBI enhanced the survival of mouse duodenal crypt stem cells. The maximum enhancement of survival was seen when 100 μ/kg of SCF was given intraperitoneally 8 h before irradiation. This regimen increased the survival of duodenal crypt stem cells after 12.0 Gy TBI from 22.5 ± 0.7 per duodenal cross section for controls to 30.0 ± 1.7 after treatment with SCF (P=0.03). The TBI dose producing 50% mortality of 6 days (LD 50/6 ) was increased from 14.9 Gy for control mice to 19.0 Gy for mice treated with SCF (dose modification factor = 1.28). These findings demonstrate that SCF (dose modification factor = 1.28). These findings demonstrate that SCF has radioprotective effects on a non-hematopoietic stem cell population and suggest that SCF may be of clinical value in preventing radiation injury to the intestine. 29 refs., 4 figs

  9. Protein tyrosine phosphatase 1B deficiency ameliorates murine experimental colitis via the expansion of myeloid-derived suppressor cells.

    Directory of Open Access Journals (Sweden)

    Jing Zhang

    Full Text Available Protein tyrosine phosphatase 1B (PTP1B is a key molecule in modulating low-degree inflammatory conditions such as diabetes. The role of PTP1B in other chronic inflammations, however, remains unknown. Here, we report that PTP1B deficiency ameliorates Dextran Sulfate Sodium (DSS-induced murine experimental colitis via expanding CD11b(+Gr-1(+ myeloid-derived suppressor cells (MDSCs. Employing DSS-induced murine experimental colitis as inflammatory animal model, we found that, compared with wild-type littermates, PTP1B-null mice demonstrated greater resistance to DSS-induced colitis, as reflected by slower weight-loss, greater survival rates and decreased PMN and macrophage infiltration into the colon. The evidence collectively also demonstrated that the resistance of PTP1B-null mice to DSS-induced colitis is based on the expansion of MDSCs. First, PTP1B-null mice exhibited a greater frequency of MDSCs in the bone marrow (BM, peripheral blood and spleen when compared with wild-type littermates. Second, PTP1B levels in BM leukocytes were significantly decreased after cells were induced into MDSCs by IL-6 and GM-CSF, and the MDSC induction occurred more rapidly in PTP1B-null mice than in wild-type littermates, suggesting PTP1B as a negative regulator of MDSCs. Third, the adoptive transfer of MDSCs into mice with DSS-colitis significantly attenuated colitis, which accompanies with a decreased serum IL-17 level. Finally, PTP1B deficiency increased the frequency of MDSCs from BM cells likely through enhancing the activities of signal transducer and activator of transcription 3 (STAT3 and Janus kinase 2 (JAK2. In conclusion, our study provides the first evidences that PTP1B deficiency ameliorates murine experimental colitis via expanding MDSCs.

  10. Enhanced granulocyte growth on peritoneal cell-coated membranes following irradiation: a dual effect of humoral stimulation and repair of x ray-induced damage to the microenvironment

    International Nuclear Information System (INIS)

    Turner, A.R.; Pfrimmer, W.J.; Boggs, D.R.; Carpe, A.I.

    1978-01-01

    An experimental model of the hematopoietic microenvironment was created by allowing a peritoneal cell coating to form on a disk of cellulose acetate placed in the peritoneal cavity of mice. An effective microenvironment capable of supporting colony growth, primarily granulocytic, was established if the cellulose acetate disk was in the peritoneum for 3 to 5 days. Its effectiveness was hampered by transferring it to another mouse or by exposure to toxic agents such as a propylene glycol-ethanol mixture or irradiation. An exponential dose-related decrease in colony formation was seen with increasing doses or irradiation of the microenvironment before colonization. After a low dose of irradiation, recovery of colony support capacity occurred over a 6-day period. Enhancement of colony growth was seen when cell injection was delayed for 2 to 3 days after irradiation. The effects of irradiation on the cellular stroma were separated from the systemic changes in the host by transferring an established hematopoietic microenvironment to a secondary host. It was shown that there are two distinct effects of irradiation on granulocytic colony growth; one was a short-lived period, 2 to 3 days of stimulation, presumably humoral, and the other was dose-dependent reversible microenvironment damage

  11. [Characteristics of postoperative peritonitis].

    Science.gov (United States)

    Lock, J F; Eckmann, C; Germer, C-T

    2016-01-01

    Postoperative peritonitis is still a life-threatening complication after abdominal surgery and approximately 10,000 patients annually develop postoperative peritonitis in Germany. Early recognition and diagnosis before the onset of sepsis has remained a clinical challenge as no single specific screening test is available. The aim of therapy is a rapid and effective control of the source of infection and antimicrobial therapy. After diagnosis of diffuse postoperative peritonitis surgical revision is usually inevitable after intestinal interventions. Peritonitis after liver, biliary or pancreatic surgery is managed as a rule by means of differentiated therapy approaches depending on the severity.

  12. Applied peritoneal anatomy

    International Nuclear Information System (INIS)

    Patel, R.R.; Planche, K.

    2013-01-01

    The peritoneal cavity is a complex anatomical structure with multiple attachments and connections. These are better understood with reference to the embryological development of this region. Armed with this knowledge, the diagnosis and assessment of a wide range of common intra-abdominal diseases becomes straightforward. This article will review and simplify the terminology, complex embryological development, and anatomy of the peritoneum, peritoneal attachments, and the reflections forming the peritoneal boundaries. Normal anatomy will be described using schematic diagrams with corresponding computed tomography (CT) and magnetic resonance imaging (MRI) images, including CT peritoneograms. The relevance of intra- and extra-peritoneal anatomy to common pathological processes will be demonstrated

  13. Production of antibodies against glycolipids from the Mycobacterium tuberculosis cell wall in aerosol murine models of tuberculosis.

    Science.gov (United States)

    Cardona, P J; Julián, E; Vallès, X; Gordillo, S; Muñoz, M; Luquin, M; Ausina, V

    2002-06-01

    Evolution of antibodies against glycolipids from the Mycobacterium tuberculosis cell wall has been studied for the first time in experimental murine models of tuberculosis induced by aerosol, in which infection, reinfection, reactivation, prophylaxis and treatment with antibiotics have been assayed. Results show a significant humoral response against these antigens, where diacyltrehaloses (DAT) and sulpholipid I (SL-I) elicited higher antibody levels than protein antigens like antigen 85 protein complex (Ag85), culture filtrate proteins (CFP) and purified protein derivative (PPD). Only immunoglobulin M (IgM) antibodies have been detected against DAT and SL-I. Their evolution has a positive correlation with bacillary concentration in tissues.

  14. Growth-related variations in the glycosaminoglycan synthesis of ultraviolet light-induced murine cutaneous fibrosarcoma cells

    International Nuclear Information System (INIS)

    Piepkorn, M.; Carney, H.; Linker, A.

    1985-01-01

    Glycosaminoglycan synthesis was studied in cell populations of ultraviolet light-induced murine cutaneous fibrosarcoma cells under conditions of varying growth rates in vitro. After labeling with the precursors, 3 H-glucosamine and 35 SO 4 , sulfated glycosaminoglycans recoverable by direct proteolysis of the culture monolayers increased approximately 5-fold on a per cell basis from sparsely populated, exponential cell cultures (greater than 85% of cells in S, G2, or M phases) to stationary cultures inhibited by high cell density (greater than 50% of cells in G1). Within this cell surface-associated material, the relative ratio of heparan sulfate to the chondroitin sulfates was approximately 60/40% under conditions of exponential growth; in the growth-arrested cultures, the reverse ratio was found. The substratum attached material, obtained from the flask surface after ethyl glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid (EGTA)-mediated detachment of the monolayers, contained relatively more hyaluronic acid, heparan sulfate, and chondroitin sulfates in the most actively proliferating cultures compared with the growth-inhibited cell populations. Furthermore, heparan sulfate and the chondroitin sulfates, which were enriched in the substratum material and in the cell pellet of exponential cultures, showed a relative shift to the cell surface-associated compartment (releasable by mild trypsinization after EGTA-mediated cell detachment) and to the compartment loosely associated with the pericellular matrix (i.e., released into the supernatant during detachment of the monolayers in the presence of EGTA)

  15. CT findings of lymphoma with peritoneal, omental and mesenteric involvement: Peritoneal lymphomatosis

    International Nuclear Information System (INIS)

    Karaosmanoglu, Devrim; Karcaaltincaba, Musturay; Oguz, Berna; Akata, Deniz; Ozmen, Mustafa; Akhan, Okan

    2009-01-01

    Purpose: We aimed to describe computed tomography (CT) findings in patients with peritoneal, omental and mesenteric lymphoma involvement. Materials and methods: We searched our archive retrospectively to find out patients with peritoneal, omental and mesenteric lymphoma involvement. We found 16 patients with non-hodgkin lymphoma meeting these criteria. CT studies of these patients were reevaluated for the presence of peritoneal involvement, ascites, omental mass, organomegaly, retroperitoneal lymphadenopathy, bowel wall thickening and other associated findings. Results: There were 14 males and 2 females with peritoneal and/or mesenteric and omental lymphoma involvement. Mean age was 39 (range 4-76). Subgroups of non-hodgkin lymphoma were diffuse large B-cell lymphoma (n = 11), small cell lymphocytic lymphoma (n = 2), small cleaved cell lymphoma (n = 1), T-cell lymphoma (n = 1) and Burkitt's lymphoma (n = 1). Peritoneal involvement was seen in 15 patients (93.8%) in the form of linear (n = 12) and nodular (n = 3) thickening. Ascites was seen in 12 (75%) patients. Omental and mesenteric masses were present in 10 (66.6%) and 10 (66.6%) patients, respectively. Bowel wall thickening, retroperitoneal lymphadenopathy and hepatosplenomegaly were also common and observed in 10, 10 and 11 patients, respectively. Solid organ involvement in the form of liver and splenic lesions was seen in 9 (56%) patients. Conclusion: Peritoneal involvement can be seen in many subtypes of lymphoma and most frequently in diffuse large B-cell lymphoma. Peritoneal lymphomatosis can mimic peritoneal carcinomatosis and should be included in the differential diagnosis list in patients with ascites, hepatosplenic lesions and unidentified cause of peritoneal thickening on CT in a male patient.

  16. Vitrification by Ultra-fast Cooling at a Low Concentration of Cryoprotectants in a Quartz Microcapillary: A Study Using Murine Embryonic Stem Cells

    Science.gov (United States)

    He, Xiaoming; Park, Eric Y.H.; Fowler, Alex; Yarmush, Martin L.; Toner, Mehmet

    2009-01-01

    Conventional cryopreservation protocols for slow-freezing or vitrification involve cell injury due to ice formation/cell dehydration or toxicity of high cryoprotectant (CPA) concentrations, respectively. In this study, we developed a novel cryopreservation technique to achieve ultra-fast cooling rates using a quartz microcapillary (QMC). The QMC enabled vitrification of murine embryonic stem (ES) cells using an intracellular cryoprotectant concentration in the range used for slowing freezing (1–2 M). The cryoprotectants used included 2 M 1,2-propanediol (PROH, cell membrane permeable) and 0.5 M extracellular trehalose (cell membrane impermeable). More than 70% of the murine ES cells post-vitrification attached with respect to non-frozen control cells, and the proliferation rates of the two groups were similar. Preservation of undifferentiated properties of the pluripotent murine ES cells post vitrification cryopreservation was verified using three different types of assays: the expression of transcription factor Oct-4, the presentation of the membrane surface glycoprotein SSEA-1, and the elevated expression of the intracellular enzyme alkaline phosphatase. These results indicate that vitrification at a low concentration (2 M) of intracellular cryoprotectants is a viable and effective approach for the cryopreservation of murine embryonic stem cells. PMID:18462712

  17. Lack of spontaneous and radiation-induced chromosome breakage at interstitial telomeric sites in murine scid cells.

    Science.gov (United States)

    Wong, H-P; Mozdarani, H; Finnegan, C; McIlrath, J; Bryant, P E; Slijepcevic, P

    2004-01-01

    Interstitial telomeric sites (ITSs) in chromosomes from DNA repair-proficient mammalian cells are sensitive to both spontaneous and radiation-induced chromosome breakage. Exact mechanisms of this chromosome breakage sensitivity are not known. To investigate factors that predispose ITSs to chromosome breakage we used murine scid cells. These cells lack functional DNA-PKcs, an enzyme involved in the repair of DNA double-strand breaks. Interestingly, our results revealed lack of both spontaneous and radiation-induced chromosome breakage at ITSs found in scid chromosomes. Therefore, it is possible that increased sensitivity of ITSs to chromosome breakage is associated with the functional DNA double-strand break repair machinery. To investigate if this is the case we used scid cells in which DNA-PKcs deficiency was corrected. Our results revealed complete disappearance of ITSs in scid cells with functional DNA-PKcs, presumably through chromosome breakage at ITSs, but their unchanged frequency in positive and negative control cells. Therefore, our results indicate that the functional DNA double-strand break machinery is required for elevated sensitivity of ITSs to chromosome breakage. Interestingly, we observed significant differences in mitotic chromosome condensation between scid cells and their counterparts with restored DNA-PKcs activity suggesting that lack of functional DNA-PKcs may cause a defect in chromatin organization. Increased condensation of mitotic chromosomes in the scid background was also confirmed in vivo. Therefore, our results indicate a previously unanticipated role of DNA-PKcs in chromatin organisation, which could contribute to the lack of ITS sensitivity to chromosome breakage in murine scid cells. Copyright 2003 S. Karger AG, Basel

  18. Protective effect of kombucha tea against tertiary butyl hydroperoxide induced cytotoxicity and cell death in murine hepatocytes.

    Science.gov (United States)

    Bhattacharya, Semantee; Manna, Prasenjit; Gachhui, Ratan; Sil, Parames C

    2011-07-01

    Kombucha (KT), a fermented black tea (BT), is known to have many beneficial properties. In the present study, antioxidant property of KT has been investigated against tertiary butyl hydroperoxide (TBHP) induced cytotoxicity using murine hepatocytes. TBHP, a reactive oxygen species inducer, causes oxidative stress resulting in organ pathophysiology. Exposure to TBHP caused a reduction in cell viability, increased membrane leakage and disturbed the intra-cellular antioxidant machineries in hepatocytes. TBHP exposure disrupted mitochondrial membrane potential and induced apoptosis as evidenced by flow cytometric analyses. KT treatment, however, counteracted the changes in mitochondrial membrane potential and prevented apoptotic cell death of the hepatocytes. BT treatment also reverted TBHP induced hepatotoxicity, however KT was found to be more efficient. This may be due to the formation of antioxidant molecules like D-saccharic acid-1,4-lactone (DSL) during fermentation process and are absent in BT. Moreover, the radical scavenging activities of KT were found to be higher than BT. Results of the study showed that KT has the potential to ameliorate TBHP induced oxidative insult and cell death in murine hepatocytes more effectively than BT.

  19. The inhibition effect of 211At-Te colloid and Na211 at injections on murine Ehrlich ascites tumor cells

    International Nuclear Information System (INIS)

    Wang Juan; Wang Xizhong; Zhang Jiazao

    1992-01-01

    Na 211 At and 211 At-Te colloid injections are prepared. It has been demonstrated that the 211 At-Te colloid is stable in vivo and in vitro, and can be applied in the study of biology and medicine. In the report, the model of Murine Ehrlich Ascites Cells cultured in vivo and in vitro is elected for a series of experiments. It has been proved that Na 211 At and 211 At-Te colloid injections possess an inhibition effect on tumor cells. The inhibition effect was expressed in surviving of the mice and inhibiting growth of tumor as well as the changes of enzyme activity. Meanwhile, it was also noticed that Na 211 At and 211 At-Te colloid injections of various dose inhibited the absorb of pyrimidine nucleosides in Murine Ehrlich Ascites Cells. And the effect is not reversible. It is closely related to the dose administrated and 50% inhibition rate needs about 1.48 x 10 5 Bq/ml culture

  20. The potential role of HMGB1 release in peritoneal dialysis-related peritonitis.

    Directory of Open Access Journals (Sweden)

    Shirong Cao

    Full Text Available High mobility group box 1 (HMGB1, a DNA-binding nuclear protein, has been implicated as an endogenous danger signal in the pathogenesis of infection diseases. However, the potential role and source of HMGB1 in the peritoneal dialysis (PD effluence of patients with peritonitis are unknown. First, to evaluate HMDB1 levels in peritoneal dialysis effluence (PDE, a total of 61 PD patients were enrolled in this study, including 42 patients with peritonitis and 19 without peritonitis. Demographic characteristics, symptoms, physical examination findings and laboratory parameters were recorded. HMGB1 levels in PDE were determined by Western blot and ELISA. The concentrations of TNF-α and IL-6 in PDE were quantified by ELISA. By animal model, inhibition of HMGB1 with glycyrrhizin was performed to determine the effects of HMGB1 in LPS-induced mice peritonitis. In vitro, a human peritoneal mesothelial cell line (HMrSV5 was stimulated with lipopolysaccharide (LPS, HMGB1 extracellular content in the culture media and intracellular distribution in various cellular fractions were analyzed by Western blot or immunofluorescence. The results showed that the levels of HMGB1 in PDE were higher in patients with peritonitis than those in controls, and gradually declined during the period of effective antibiotic treatments. Furthermore, the levels of HMGB1 in PDE were positively correlated with white blood cells (WBCs count, TNF-α and IL-6 levels. However, pretreatment with glycyrrhizin attenuated LPS-induced acute peritoneal inflammation and dysfunction in mice. In cultured HMrSV5 cells, LPS actively induced HMGB1 nuclear-cytoplasmic translocation and release in a time and dose-dependent fashion. Moreover, cytosolic HMGB1 was located in lysosomes and secreted via a lysosome-mediated secretory pathway following LPS stimulation. Our study demonstrates that elevated HMGB1 levels in PDE during PD-related peritonitis, at least partially, from peritoneal mesothelial cells

  1. Spatially Fractionated Radiation Induces Cytotoxicity and Changes in Gene Expression in Bystander and Radiation Adjacent Murine Carcinoma Cells

    Science.gov (United States)

    Asur, Rajalakshmi S.; Sharma, Sunil; Chang, Ching-Wei; Penagaricano, Jose; Kommuru, Indira M.; Moros, Eduardo G.; Corry, Peter M.; Griffin, Robert J.

    2012-01-01

    Radiation-induced bystander effects have been extensively studied at low doses, since evidence of bystander induced cell killing and other effects on unirradiated cells were found to be predominant at doses up to 0.5 Gy. Therefore, few studies have examined bystander effects induced by exposure to higher doses of radiation, such as spatially fractionated radiation (GRID) treatment. In the present study, we evaluate the ability of GRID treatment to induce changes in GRID adjacent (bystander) regions, in two different murine carcinoma cell lines following exposure to a single irradiation dose of 10 Gy. Murine SCK mammary carcinoma cells and SCCVII squamous carcinoma cells were irradiated using a brass collimator to create a GRID pattern of nine circular fields 12 mm in diameter with a center-to-center distance of 18 mm. Similar to the typical clinical implementation of GRID, this is approximately a 50:50 ratio of direct and bystander exposure. We also performed experiments by irradiating separate cultures and transferring the medium to unirradiated bystander cultures. Clonogenic survival was evaluated in both cell lines to determine the occurrence of radiation-induced bystander effects. For the purpose of our study, we have defined bystander cells as GRID adjacent cells that received approximately 1 Gy scatter dose or unirradiated cells receiving conditioned medium from irradiated cells. We observed significant bystander killing of cells adjacent to the GRID irradiated regions compared to sham treated controls. We also observed bystander killing of SCK and SCCVII cells cultured in conditioned medium obtained from cells irradiated with 10 Gy. Therefore, our results confirm the occurrence of bystander effects following exposure to a high-dose of radiation and suggest that cell-to-cell contact is not required for these effects. In addition, the gene expression profile for DNA damage and cellular stress response signaling in SCCVII cells after GRID exposure was studied

  2. The Murine Natural Cytotoxic Receptor NKp46/NCR1 Controls TRAIL Protein Expression in NK Cells and ILC1s

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    Sam Sheppard

    2018-03-01

    Full Text Available Summary: TRAIL is an apoptosis-inducing ligand constitutively expressed on liver-resident type 1 innate lymphoid cells (ILC1s and a subset of natural killer (NK cells, where it contributes to NK cell anti-tumor, anti-viral, and immunoregulatory functions. However, the intrinsic pathways involved in TRAIL expression in ILCs remain unclear. Here, we demonstrate that the murine natural cytotoxic receptor mNKp46/NCR1, expressed on ILC1s and NK cells, controls TRAIL protein expression. Using NKp46-deficient mice, we show that ILC1s lack constitutive expression of TRAIL protein and that NK cells activated in vitro and in vivo fail to upregulate cell surface TRAIL in the absence of NKp46. We show that NKp46 regulates TRAIL expression in a dose-dependent manner and that the reintroduction of NKp46 in mature NK cells deficient for NKp46 is sufficient to restore TRAIL surface expression. These studies uncover a link between NKp46 and TRAIL expression in ILCs with potential implications in pathologies involving NKp46-expressing cells. : Sheppard et al. find that mice deficient in the activating receptor NCR1/NKp46 (Ncr1−/− fail to express the apoptosis-inducing ligand TRAIL at the surface of group 1 innate lymphoid cells (ILC1s. Keywords: NK cell, natural killer cell, NKp46, ILC1, TRAIL, IL-15, IL-2

  3. Inactivation of the small GTP binding protein Rho induces multinucleate cell formation and apoptosis in murine T lymphoma EL4.

    Science.gov (United States)

    Moorman, J P; Bobak, D A; Hahn, C S

    1996-06-01

    The small G-protein Rho regulates the actin microfilament-dependent cytoskeleton. Exoenzyme C3 of Clostridium botulinum ADP-ribosylates Rho at Asn41, a modification that functionally inactivates Rho. Using a Sindbis virus-based transient gene expression system, we studied the role of Rho in murine EL4 T lymphoma cells. We generated a double subgenomic infectious Sindbis virus (dsSIN:C3) recombinant which expressed C3 in >95% of EL4 cells. This intracellular C3 resulted in modification and inactivation of virtually all endogenous Rho. dsSIN:C3 infection led to the formation of multinucleate cells, likely by inhibiting the actin microfilament-dependent step of cytokinesis. Intriguingly, in spite of the inhibition of cytokinesis, karyokinesis continued, with the result that cells containing a nuclear DNA content as high as 16N (eight nuclei) were observed. In addition, dsSIN:C3-mediated inactivation of Rho was a potent activator of apoptosis in EL4 cells. To discern whether the formation of multinucleate cells was responsible for the activation of apoptosis, 5-fluorouracil (5-FUra) was used to induce cell cycle arrest. As expected, EL4 cells treated with 5-FUra were prevented from forming multinucleate cells upon infection with dsSIN:C3. dsSIN:C3 infection, however, still caused marked apoptosis in 5-FUra-treated cells, indicating that this activation of apoptosis was independent of multinucleate cell formation.

  4. CRISPR/Cas9 Editing of Murine Induced Pluripotent Stem Cells for Engineering Inflammation-Resistant Tissues.

    Science.gov (United States)

    Brunger, Jonathan M; Zutshi, Ananya; Willard, Vincent P; Gersbach, Charles A; Guilak, Farshid

    2017-05-01

    Proinflammatory cytokines such as interleukin-1 (IL-1) are found in elevated levels in diseased or injured tissues and promote rapid tissue degradation while preventing stem cell differentiation. This study was undertaken to engineer inflammation-resistant murine induced pluripotent stem cells (iPSCs) through deletion of the IL-1 signaling pathway and to demonstrate the utility of these cells for engineering replacements for diseased or damaged tissues. Targeted deletion of the IL-1 receptor type I (IL-1RI) gene in murine iPSCs was achieved using the RNA-guided, site-specific clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 genome engineering system. Clonal cell populations with homozygous and heterozygous deletions were isolated, and loss of receptor expression and cytokine signaling was confirmed by flow cytometry and transcriptional reporter assays, respectively. Cartilage was engineered from edited iPSCs and tested for its ability to resist IL-1-mediated degradation in gene expression, histologic, and biomechanical assays after a 3-day treatment with 1 ng/ml of IL-1α. Three of 41 clones isolated possessed the IL-1RI +/- genotype. Four clones possessed the IL-1RI -/- genotype, and flow cytometry confirmed loss of IL-1RI on the surface of these cells, which led to an absence of NF-κB transcription activation after IL-1α treatment. Cartilage engineered from homozygous null clones was resistant to cytokine-mediated tissue degradation. In contrast, cartilage derived from wild-type and heterozygous clones exhibited significant degradative responses, highlighting the need for complete IL-1 blockade. This work demonstrates proof-of-concept of the ability to engineer custom-designed stem cells that are immune to proinflammatory cytokines (i.e., IL-1) as a potential cell source for cartilage tissue engineering. © 2016, American College of Rheumatology.

  5. Virulence-associated genome mutations of murine rotavirus identified by alternating serial passages in mice and cell cultures.

    Science.gov (United States)

    Tsugawa, Takeshi; Tatsumi, Masatoshi; Tsutsumi, Hiroyuki

    2014-05-01

    Although significant clinical efficacy and safety of rotavirus vaccines were recently revealed in many countries, the mechanism of their attenuation is not well understood. We passaged serially a cell culture-adapted murine rotavirus EB strain in mouse pups or in cell cultures alternately and repeatedly and fully sequenced all 11 genes of 21 virus samples passaged in mice or in cell cultures. Sequence analysis revealed that mouse-passaged viruses that regained virulence almost consistently acquired four kinds of amino acid (aa) substitutions in VP4 and substitution in aa 37 (Val to Ala) in NSP4. In addition, they gained and invariably conserved the 3' consensus sequence in NSP1. The molecular changes occurred along with the acquisition of virulence during passages in mice and then disappeared following passages in cell cultures. Intraperitoneal injection of recombinant NSP4 proteins confirmed the aa 37 site as important for its diarrheagenic activity in mice. These genome changes are likely to be correlated with rotavirus virulence. Serial passage of a virulent wild-type virus in vitro often results in loss of virulence of the virus in an original animal host, while serial passage of a cell culture-adapted avirulent virus in vivo often gains virulence in an animal host. Actually, live attenuated virus vaccines were originally produced by serial passage in cell cultures. Although clinical efficacy and safety of rotavirus vaccines were recently revealed, the mechanism of their attenuation is not well understood. We passaged serially a murine rotavirus by alternating switch of host (mice or cell cultures) repeatedly and sequenced the eleven genes of the passaged viruses to identify mutations associated with the emergence or disappearance of virulence. Sequence analysis revealed that changes in three genes (VP4, NSP1, and NSP4) were associated with virulence in mice. Intraperitoneal injection of recombinant NSP4 proteins confirmed its diarrheagenic activity in mice

  6. Identification of BC005512 as a DNA damage responsive murine endogenous retrovirus of GLN family involved in cell growth regulation.

    Directory of Open Access Journals (Sweden)

    Yuanfeng Wu

    Full Text Available Genotoxicity assessment is of great significance in drug safety evaluation, and microarray is a useful tool widely used to identify genotoxic stress responsive genes. In the present work, by using oligonucleotide microarray in an in vivo model, we identified an unknown gene BC005512 (abbreviated as BC, official full name: cDNA sequence BC005512, whose expression in mouse liver was specifically induced by seven well-known genotoxins (GTXs, but not by non-genotoxins (NGTXs. Bioinformatics revealed that BC was a member of the GLN family of murine endogenous retrovirus (ERV. However, the relationship to genotoxicity and the cellular function of GLN are largely unknown. Using NIH/3T3 cells as an in vitro model system and quantitative real-time PCR, BC expression was specifically induced by another seven GTXs, covering diverse genotoxicity mechanisms. Additionally, dose-response and linear regression analysis showed that expression level of BC in NIH/3T3 cells strongly correlated with DNA damage, measured using the alkaline comet assay,. While in p53 deficient L5178Y cells, GTXs could not induce BC expression. Further functional studies using RNA interference revealed that down-regulation of BC expression induced G1/S phase arrest, inhibited cell proliferation and thus suppressed cell growth in NIH/3T3 cells. Together, our results provide the first evidence that BC005512, a member from GLN family of murine ERV, was responsive to DNA damage and involved in cell growth regulation. These findings could be of great value in genotoxicity predictions and contribute to a deeper understanding of GLN biological functions.

  7. Virulence-Associated Genome Mutations of Murine Rotavirus Identified by Alternating Serial Passages in Mice and Cell Cultures

    Science.gov (United States)

    Tatsumi, Masatoshi; Tsutsumi, Hiroyuki

    2014-01-01

    ABSTRACT Although significant clinical efficacy and safety of rotavirus vaccines were recently revealed in many countries, the mechanism of their attenuation is not well understood. We passaged serially a cell culture-adapted murine rotavirus EB strain in mouse pups or in cell cultures alternately and repeatedly and fully sequenced all 11 genes of 21 virus samples passaged in mice or in cell cultures. Sequence analysis revealed that mouse-passaged viruses that regained virulence almost consistently acquired four kinds of amino acid (aa) substitutions in VP4 and substitution in aa 37 (Val to Ala) in NSP4. In addition, they gained and invariably conserved the 3′ consensus sequence in NSP1. The molecular changes occurred along with the acquisition of virulence during passages in mice and then disappeared following passages in cell cultures. Intraperitoneal injection of recombinant NSP4 proteins confirmed the aa 37 site as important for its diarrheagenic activity in mice. These genome changes are likely to be correlated with rotavirus virulence. IMPORTANCE Serial passage of a virulent wild-type virus in vitro often results in loss of virulence of the virus in an original animal host, while serial passage of a cell culture-adapted avirulent virus in vivo often gains virulence in an animal host. Actually, live attenuated virus vaccines were originally produced by serial passage in cell cultures. Although clinical efficacy and safety of rotavirus vaccines were recently revealed, the mechanism of their attenuation is not well understood. We passaged serially a murine rotavirus by alternating switch of host (mice or cell cultures) repeatedly and sequenced the eleven genes of the passaged viruses to identify mutations associated with the emergence or disappearance of virulence. Sequence analysis revealed that changes in three genes (VP4, NSP1, and NSP4) were associated with virulence in mice. Intraperitoneal injection of recombinant NSP4 proteins confirmed its

  8. In vitro phenotypic, genomic and proteomic characterization of a cytokine-resistant murine β-TC3 cell line.

    Directory of Open Access Journals (Sweden)

    Antonina Coppola

    Full Text Available Type 1 diabetes mellitus (T1DM is caused by the selective destruction of insulin-producing β-cells. This process is mediated by cells of the immune system through release of nitric oxide, free radicals and pro-inflammatory cytokines, which induce a complex network of intracellular signalling cascades, eventually affecting the expression of genes involved in β-cell survival.The aim of our study was to investigate possible mechanisms of resistance to cytokine-induced β-cell death. To this purpose, we created a cytokine-resistant β-cell line (β-TC3R by chronically treating the β-TC3 murine insulinoma cell line with IL-1β + IFN-γ. β-TC3R cells exhibited higher proliferation rate and resistance to cytokine-mediated cell death in comparison to the parental line. Interestingly, they maintained expression of β-cell specific markers, such as PDX1, NKX6.1, GLUT2 and insulin. The analysis of the secretory function showed that β-TC3R cells have impaired glucose-induced c-peptide release, which however was only moderately reduced after incubation with KCl and tolbutamide. Gene expression analysis showed that β-TC3R cells were characterized by downregulation of IL-1β and IFN-γ receptors and upregulation of SOCS3, the classical negative regulator of cytokines signaling. Comparative proteomic analysis showed specific upregulation of 35 proteins, mainly involved in cell death, stress response and folding. Among them, SUMO4, a negative feedback regulator in NF-kB and JAK/STAT signaling pathways, resulted hyper-expressed. Silencing of SUMO4 was able to restore sensitivity to cytokine-induced cell death in β-TC3R cells, suggesting it may play a key role in acquired cytokine resistance by blocking JAK/STAT and NF-kB lethal signaling.In conclusion, our study represents the first extensive proteomic characterization of a murine cytokine-resistant β-cell line, which might represent a useful tool for studying the mechanisms involved in resistance to

  9. Boron neutron capture therapy induces cell cycle arrest and DNA fragmentation in murine melanoma cells

    Energy Technology Data Exchange (ETDEWEB)

    Faiao-Flores, F. [Biochemical and Biophysical Laboratory, Butantan Institute, 1500 Vital Brasil Avenue, Sao Paulo (Brazil)] [Faculty of Medicine, University of Sao Paulo, 455 Doutor Arnaldo Avenue, Sao Paulo (Brazil); Coelho, P.R.P. [Institute for Nuclear and Energy Research, 2242 Lineu Prestes Avenue, Sao Paulo (Brazil); Arruda-Neto, J. [Physics Institute, University of Sao Paulo, 187 Matao Street, Sao Paulo (Brazil)] [FESP, Sao Paulo Engineering School, 5520 Nove de Julho Avenue, Sao Paulo (Brazil); Maria, Durvanei A., E-mail: durvaneiaugusto@yahoo.br [Biochemical and Biophysical Laboratory, Butantan Institute, 1500 Vital Brasil Avenue, Sao Paulo (Brazil)

    2011-12-15

    The melanoma is a highly lethal skin tumor, with a high incidence. Boron Neutron Capture Therapy (BNCT) is a radiotherapy which combines Boron with thermal neutrons, constituting a binary system. B16F10 melanoma and L929 fibroblasts were treated with Boronophenylalanine and irradiated with thermal neutron flux. The electric potential of mitochondrial membrane, cyclin D1 and caspase-3 markers were analyzed. BNCT induced a cell death increase and cyclin D1 amount decreased only in B16F10 melanoma. Besides, there was not caspase-3 phosphorylation.

  10. Vernix caseosa peritonitis: report of two cases.

    Science.gov (United States)

    Val-Bernal, José-Fernando; Mayorga, Marta; García-Arranz, Pilar; Salcedo, Waleska; León, Alicia; Fernández, Fidel A

    2015-01-01

    Vernix caseosa peritonitis is a rare complication caused by inflammatory response to amniotic fluid spilled into the maternal peritoneal cavity. Most cases occur after cesarean section. We discuss herein two patients, aged 33 and 29 years, who presented with vernix caseosa peritonitis seven to nine days after a cesarean delivery. Laparotomy was performed and it revealed neither uterine rupture nor other surgical emergencies, but cheesy exudates on the serosal surface of all viscera. Appendicectomy was performed. Histopathologic study revealed acute fibrinous serositis and a mixed cellular infiltrate, rich in neutrophils, around fetal desquamated anucleate squamous cells. Patients´ recovery was complete. Clinical diagnosis of vernix caseosa peritonitis should be suspected in patients presenting post-cesarean section with an acute abdomen. Distinctive histopathologic findings allow making the correct diagnosis. Vigilant monitoring after diagnosis is essential as delayed morbidities may appear.

  11. Characterization of a spontaneously generated murine retinal pigmented epithelium cell line; a model for in vitro experiments

    International Nuclear Information System (INIS)

    Ranaei Pirmardan, Ehsan; Soheili, Zahra-Soheila; Samiei, Shahram; Ahmadieh, Hamid; Mowla, Seyed Javad; Ezzati, Razie; Naseri, Marzieh

    2016-01-01

    Retinal pigmented epithelium (RPE), the outermost layer of the retina, has a key role in maintaining retinal cells’ functions. Severity of the culture of RPE cells has exerted many limitations to both in vitro and in vivo studies and its therapeutic applications. Therefore, establishment of RPE cell lines with high proliferative potential can considerably improve study of RPE cell biology. Here we report generation of a spontaneously immortalized murine RPE cell line in primary mouse RPE cell culture. Founded colonized cells were picked up and expression of RPE and retinal progenitor cells’ (RPC) markers were studied using immunocytochemistry (ICC). Emerged cells cultured over 35 passages and population doubling times in different serum concentrations were calculated. We also investigated the ability of cells for becoming transfected by calcium-phosphate method and for becoming infected by adeno-associated virus serotype 2 (AAV2) using flow cytometry. Data showed that the cobblestone constituent cells expressed RPE65, cytokeratin and ZO1 and moreover several progenitor markers such as Pax6, Sox2, Nestin and Chx10. It revealed that, despite primary RPE cells, the newly emerged cells were easily transfectable and were highly infectable when compared with HEK293T cells. Our data indicated that the emerged mouse RPE cell line pretended RPC-like phenotype and also simultaneously expressed RPE markers. It would be a promising model for leading studies on RPE and RPC cells and substantially confirmed the great RPE plasticity and its invaluable potential in research studies. - Highlights: • Isolation of a spontaneously generated retinal pigmented epithelium cell line is reported. • The cells express some of the retinal progenitor cell markers in addition to the RPE markers. • The aforesaid cell line is highly transfecable and considerably infectable by AAV2. • These results confirm the great RPE plasticity and its invaluable potential in research studies.

  12. Characterization of a spontaneously generated murine retinal pigmented epithelium cell line; a model for in vitro experiments

    Energy Technology Data Exchange (ETDEWEB)

    Ranaei Pirmardan, Ehsan [Department of Molecular Genetics, Faculty of Biological Sciences, Tarbiat Modares University, Tehran (Iran, Islamic Republic of); Soheili, Zahra-Soheila [Department of Molecular Medicine, National Institute of Genetic Engineering and Biotechnology, Tehran (Iran, Islamic Republic of); Samiei, Shahram [Blood Transfusion Research Center, High Institute for Research and Education in Transfusion Medicine, Tehran (Iran, Islamic Republic of); Ahmadieh, Hamid [Ophthalmic Research Center, Shahid Beheshti University of Medical Sciences, Tehran (Iran, Islamic Republic of); Mowla, Seyed Javad [Department of Molecular Genetics, Faculty of Biological Sciences, Tarbiat Modares University, Tehran (Iran, Islamic Republic of); Ezzati, Razie [Department of Molecular Medicine, National Institute of Genetic Engineering and Biotechnology, Tehran (Iran, Islamic Republic of); Naseri, Marzieh [Department of Molecular Medicine, Faculty of Advanced Technology, Iran University of Medical Sciences, Tehran (Iran, Islamic Republic of)

    2016-10-01

    Retinal pigmented epithelium (RPE), the outermost layer of the retina, has a key role in maintaining retinal cells’ functions. Severity of the culture of RPE cells has exerted many limitations to both in vitro and in vivo studies and its therapeutic applications. Therefore, establishment of RPE cell lines with high proliferative potential can considerably improve study of RPE cell biology. Here we report generation of a spontaneously immortalized murine RPE cell line in primary mouse RPE cell culture. Founded colonized cells were picked up and expression of RPE and retinal progenitor cells’ (RPC) markers were studied using immunocytochemistry (ICC). Emerged cells cultured over 35 passages and population doubling times in different serum concentrations were calculated. We also investigated the ability of cells for becoming transfected by calcium-phosphate method and for becoming infected by adeno-associated virus serotype 2 (AAV2) using flow cytometry. Data showed that the cobblestone constituent cells expressed RPE65, cytokeratin and ZO1 and moreover several progenitor markers such as Pax6, Sox2, Nestin and Chx10. It revealed that, despite primary RPE cells, the newly emerged cells were easily transfectable and were highly infectable when compared with HEK293T cells. Our data indicated that the emerged mouse RPE cell line pretended RPC-like phenotype and also simultaneously expressed RPE markers. It would be a promising model for leading studies on RPE and RPC cells and substantially confirmed the great RPE plasticity and its invaluable potential in research studies. - Highlights: • Isolation of a spontaneously generated retinal pigmented epithelium cell line is reported. • The cells express some of the retinal progenitor cell markers in addition to the RPE markers. • The aforesaid cell line is highly transfecable and considerably infectable by AAV2. • These results confirm the great RPE plasticity and its invaluable potential in research studies.

  13. Triggering receptor expressed on myeloid cells-2 fine-tunes inflammatory responses in murine Gram-negative sepsis

    DEFF Research Database (Denmark)

    Schmidt Thøgersen, Mariane; Gawish, Riem; Martins, Rui

    2015-01-01

    During infections, TLR-mediated responses require tight regulation to allow for pathogen removal, while preventing overwhelming inflammation and immunopathology. The triggering receptor expressed on myeloid cells (TREM)-2 negatively regulates inflammation by macrophages and impacts on phagocytosis...... was followed by an accelerated resolution and ultimately improved survival, associated with the induction of the negative regulator A20. Upon infection with Escherichia coli, the otherwise beneficial effect of an exaggerated early immune response in TREM-2(-/-) animals was counteracted by a 50% reduction...... in bacterial phagocytosis. In line with this, TREM-2(-/-) peritoneal macrophages (PMs) exhibited augmented inflammation following TLR4 stimulation, demonstrating the presence and negative regulatory functionality of TREM-2 on primary PMs. Significant