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At least 649 records · Page 36Linked to original sources
PubMed2004

Low-dose total body irradiation causes clonal fluctuation of primate hematopoietic stem and progenitor cells.

Due to high frequency of side effects caused by high-dose total body irradiation (TBI) the nonmyeloablative regimen together with cytotoxic agents is currently used especially for elderly patients. However, immediate and long-term effects of low-dose irradiation used in allogeneic transplantation on stem cells is less well known. We have studied the effect of low-dose 3 Gy TBI on the number of hematopoietic stem cell (HSC) clones contributing simultaneously to granulocyte production in rhesus macaque. The number of clones after 3 Gy TBI decreased markedly by 2 to 3 weeks after 3 Gy TBI, followed by a period of clonal instability, and recovery to almost pre-3 Gy TBI clonal diversity. The clones accounting for this recovery contributed before 3 Gy TBI, suggesting the profound initial impact of TBI was on a pool of progenitor cells, whereas most of the more primitive HSCs remained unaffected and were able to again contribute to hematopoiesis after recovery. Clonal fluctuation may indirectly suggest the presence of short-term/long-term HSC populations in rhesus macaque bone marrow as reported in a mouse model. The results indicate that even low-dose irradiation affects hematopoietic clonal dynamics and have implications for design of conditioning regimens for transplantation purposes.

Animals↗
PubMed1999

Differential immediate early gene expression in conditional hepatitis B virus pX-transforming versus nontransforming hepatocyte cell lines.

We report construction and characterization of tetracycline-controlled hepatitis B virus pX-expressing hepatocyte (AML12) cell lines. These cell lines were constructed in AML12 clonal isolates (clones 3 and 4), which express constitutively the tetracycline-controlled transactivator. Since pX is implicated in HCC, this immortalized hepatocyte model system was used to investigate the mechanism of pX in transformation. Clonal isolates of 3pX and 4pX lineages display conditional synthesis of pX mRNA and protein and a 2-fold increase in growth saturation density following tetracycline removal, implicating pX in monolayer overgrowth. Interestingly, only 3pX clones display pX-dependent anchorage independence. Clone 3 lineages express hepatocyte nuclear factor-1alpha and hepatocyte-specific marker genes; clone 4 lineages express hepatocyte nuclear factor-1beta and reduced levels of hepatocyte-specific marker genes, suggesting the importance of the differentiated hepatocyte in pX-mediated oncogenic transformation. Importantly, 3pX and 4pX lineages display differential expression of immediate early genes c-fos and ATF3. The pX-transforming 3pX lineage displays early, pX-dependent induction of ATF3 and prolonged induction of c-fos. The nontransforming 4pX cells display an absence of pX-dependent ATF3 induction and transient induction of c-fos. Our results support the direct link of pX expression to oncogenic transformation in 3pX lineage clones and underscore the advantage of this conditional cellular model system for studying mechanisms of pX-mediated oncogenesis.

Activating Transcription Factor 3↗
PubMed1996

Single human T cells stimulated in the absence of feeder cells transcribe interleukin-2 and undergo long-term clonal growth in response to defined monoclonal antibodies and cytokine stimulation.

The two-signal model of T-cell activation postulates that T lymphocytes require at least two distinct signals for activation. This model has been established with bulk cultures of T cells in which T-cell-T-cell interaction can occur, possibly delivering further unrecognized costimulatory signals. The signal requirements of single T cells for the induction of clonal cell growth or the transcription of cytokines would best be studied in a cell cloning system in the absence of feeder cells; however, such an experimental system has not been reported so far. In this study, we report the long-term cloning of human resting peripheral blood CD4+CD45RO- T cells under feeder cell-free conditions in response to CD3 and CD28 stimulation in the presence of exogenous interleukin-2 (IL-2). Cloning efficiency ranged from 40% to 60% depending on the presence of additional cytokines IL-1 and IL-6. Single-call polymerase chain reaction showed that transcription of IL-2 occurred in cells stimulated through CD3 plus CD28 alone. T cells grown in response to CD3 plus CD28 plus IL-2 stimulation produced both IL-4 and interferon-gamma (IFN-gamma) on restimulation (Th0 cells) and could be functionally differentiated into Th1- or Th2-type cells by the addition of IFN-gamma or IL-4, respectively, during cell cloning. These data show on the single-cell level a two-signal model of T- cell activation for the transcription of IL-2. In addition, these experiments show that IFN-gamma and IL-4 exert their T-cell-differentiating effects directly on the T cell without any further need for antigen-presenting cells. Together, our experiments show the feasability of a defined long-term clonal cell culture system to study the growth and differentiation of human T lymphocytes.

Antibodies, Monoclonal↗
PubMed2006

Lung cancer risk in mice: analysis of fractionation effects and neutron RBE with a biologically motivated model.

Data from Argonne National Laboratory on lung cancer in 15,975 mice with acute and fractionated exposures to gamma rays and neutrons are analyzed with a biologically motivated model with two rate-limiting steps and clonal expansion. Fractionation effects and effects of radiation quality can be explained well by the estimated kinetic parameters. Both an initiating and a promoting action of neutrons and gamma rays are suggested. While for gamma rays the initiating event is described well with a linear dose-rate dependence, for neutrons a nonlinear term is needed, with less effectiveness at higher dose rates. For the initiating event, the neutron RBE compared to gamma rays is about 10 when the dose rate during each fraction is low. For higher dose rates this RBE decreases strongly. The estimated lifetime relative risk for radiation-induced lung cancers from 1 Gy of acute gamma-ray exposure at an age of 110 days is 1.27 for male mice and 1.53 for female mice. For doses less than 1 Gy, the effectiveness of fractionated exposure to gamma rays compared to acute exposure is between 0.4 and 0.7 in both sexes. For lifetime relative risk, the RBE from acute neutrons at low doses is estimated at about 10 relative to acute gamma-ray exposure. It decreases strongly with dose. For fractionated neutrons, it is lower, down to about 4 for male mice.

Animals↗
PubMed1989

Clonal rat parathyroid cell line expresses a parathyroid hormone-related peptide but not parathyroid hormone itself.

A novel parathyroid hormone-related peptide has been identified in tumors associated with the syndrome of humoral hypercalcemia of malignancy. Subsequently, mRNAs encoding this peptide have been found to be expressed in a number of normal tissues, including the parathyroids. Using Northern blotting, RNase protection, and immunochemical techniques, we examined a clonal rat parathyroid cell line originally developed as a model system for studying parathyroid cell physiology. We found that this line expresses the parathyroid hormone-related peptide but not parathyroid hormone itself. Secretion of the parathyroid hormone-related peptide varied inversely with extracellular calcium concentration, but neither calcium nor 1,25-dihydroxyvitamin D3 appeared to influence steady-state parathyroid hormone-related peptide mRNA levels. This clonal line may prove to be an interesting system for studying the factors responsible for tissue-specific parathyroid hormone and parathyroid hormone-related peptide gene expression.

Animals↗
PubMed2002

Murine models of paroxysmal nocturnal hemoglobinuria.

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal disorder characterized by chronic intravascular hemolysis, cytopenia, and an increased tendency to thrombosis. All patients with PNH studied so far have a somatic mutation of phosphatidyl inositol glycan complementation group A (PIG-A), an X-linked gene involved initially in the biosynthesis of the glycosyl phosphatidylinositol (GPI) molecule, which serves as an anchor for many cell surface proteins. The mutation occurs in a hematopoietic stem cell, and consequently, all cells derived from the mutated stem cell are devoid of GPI-linked proteins. The absence of GPI-linked proteins explains some clinical symptoms of the disease but not the mechanism that allows the expansion of the mutated clone. By using targeted disruption of the PIG-A gene in mouse embryonic stem cells, some mice models of PNH have been generated. These animals have a discrete proportion of blood cells devoid of GPI-linked proteins, and although not anemic, they have evidence of hemolysis. The clinical course of these animals is benign, and there are no signs of a substantial expansion of the PNH clone, as observed in human patients. The fact that these animals do not develop the disease strongly supports the notion that a mutation of PIG-A is not sufficient per se to cause PNH and that another factor, namely, bone marrow failure, is necessary to allow proliferation and expansion of the PNH clone.

Animals↗
PubMed1999

[Mechanisms and applications of oral tolerance].

The term oral tolerance (OT) describes the antigen-specific suppression of immune responses following the feeding of the antigen. While some common features with other forms of induction of systemic tolerance have been disclosed, OT can be distinguished by certain immunologic characteristics. Thus, work in experimental animal models revealed the importance of intestinal antigen processing, especially antigen processing in the Peyer's patches, in inducing OT. It has become clear that suppressive T cell cytokines derived from mucosal sites play a major role in mediating OT. The variation of the dose of fed antigen and the modulation of the cytokine milieu both have influences on the underlying immunologic mechanisms active suppression, clonal anergy and clonal deletion following oral antigen uptake. In several animal models of autoimmunity the disease activity can be suppressed by feeding oral autoantigen. Based on these results, recent clinical studies have begun to explore OT as a means to treat autoimmune disorders such as multiple sclerosis, rheumatoid arthritis and diabetes.

Administration, Oral↗
PubMed1995

[Analyses of T cell clonality in mice with autoimmune sialoadenitis].

T cells are believed to be deeply involved in the pathogenesis of organ-specific autoimmune diseases. However, it remains unknown whether antigen-specific immune responses occur at the inflammatory sites in vivo. As a model of human Sjögren's syndrome, we investigated the T cell clonality of mice with autoimmune sialoadenitis, i.e. IQI/Jcl-(Saegusa et al), thymus grafted (TG) nude-(Taguchi et al), and MRL/lpr-SCID (Hayashi et al) mice; using RT-PCR-SSCP method. As a result, accumulations of distinct T cell clones were demonstrated in the salivary and lacrimal glands in these mice, without V beta restriction. Moreover, a part of the accumulating clones were commonly detected among multiple glands, suggesting the existence of a common and specific immune response probably toward autoantigen(s) expressed on the affected glands.

Animals↗
PubMed1993

Fluoride-stimulated [3H]thymidine uptake in a human osteoblastic osteosarcoma cell line is dependent on transforming growth factor beta.

Controversy exists regarding the effect of fluoride on human osteoblast proliferation. To learn more of the cellular action of fluoride, we chose the clonal osteoblast cell line HOS TE85 as a model system. In these phenotypically osteoblast-like cells, sodium fluoride stimulated [3H]thymidine incorporation in a dose-dependent manner over the concentration range 1 x 10(-5)-2 x 10(-4) M. The fluoride-induced stimulation of [3H]thymidine uptake was dependent on cell density, being optimal at subconfluent cell numbers. Stimulation of [3H]thymidine uptake was inhibited by anti-transforming growth factor beta but not by antibody to insulin-like growth factor I or beta 2-microglobulin. Transforming growth factor beta was shown to be a biphasic stimulator of [3H]thymidine uptake in HOS TE85, with maximal stimulation occurring at 0.5 nM transforming growth factor beta. In the presence of fluoride the cells were more sensitive to stimulation by this growth factor, with maximum effect occurring at 0.1 nM. Fluoride did not increase mRNA for transforming growth factor beta following either 8 or 24 h of exposure. We conclude that fluoride activates osteoblast proliferation by modulating the cellular sensitivity to transforming growth factor beta, a known stimulator of bone growth.

Antibodies↗
PubMed1991

Control of self-reactivity in the intestine.

In the intestine maintenance of self-tolerance may involve tissue-specific self-Ags, APCs, 'second signals', and extrathymic pathways of T cell maturation. These factors combine to create a unique environment where autoimmune tissue destruction is prevented despite local inflammatory influences. In this review we summarize our findings using a TCR-gamma delta transgenic model where self-tolerance was maintained by clonal deletion for cells localizing to peripheral lymphoid tissue and by clonal anergy for cells localizing to the intraepithelial compartments. Several possible explanations exist for these results but in general, these findings have implications for the maintenance of self-tolerance of normal TCR-alpha beta and TCR-gamma delta IELs in epithelial tissues such as the intestine.

Animals↗
PubMed2006

Lymph node cells from BALB/c mice with chronic visceral leishmaniasis exhibiting cellular anergy and apoptosis: involvement of Ser/Thr phosphatase.

Visceral leishmaniasis (VL) produced in BALB/c mice through intracardial administration of Leishmania donovani amastigotes was accompanied by hepatosplenomegaly with high organ parasite load and lymphadenopathy when followed up to 4-months or so. To elucidate the mechanism of immunosuppression associated with VL, we report here progressive impairment of the proliferative response of lymph node cells (lymphocytes) from infected animals (I-LNC) to in vitro stimulation with the combination of phorbol 12-myristate 13-acetate (PMA) and ionomycin (Io) that could be related to the downregulation of PKC and MAP kinase (ERK 1/2) activation process. Further, pretreatment of I-LNC with the protein phosphatase inhibitor okadaic acid (OA), but not with calyculin A or sodium orthovanadate, significantly restored their proliferative response as well as PMA-induced activation of PKC. A population of LNC (primarily T-lymphocytes) from chronically infected animals was shown to undergo apoptosis, the number of which increased considerably following PMA+ Io stimulation. The apoptotic pathway, which was followed through binding of cells to Annexin V, activation of caspase-3 and fragmentation of DNA, involved destabilization of mitochondria, probably as a result of downregulation of PKC and Bcl-2. Interestingly, prior incubation of I-LNC with OA reversed the state of cell cycle arrest (anergy) and apoptosis through progression of cells from G0/G1 to S and G2/M phases with transcriptional activation of IL-2 and IL-2R genes. Our results suggest that the cellular (immune) dysfunction in VL could be attributed to dephosphorylation of key molecules in the T-lymphocyte signaling pathway by Ser/Thr phosphatase leading to their inactivation.

Animals↗
PubMed1989

Identification and characterization of D1 and D2 dopamine receptors in cultured neuroblastoma and retinoblastoma clonal cell lines.

The recent availability of high specific activity radiolabeled dopaminergic antagonists with specificity for dopamine receptor subtypes has allowed us to screen a wide variety of cultured mammalian cell lines for the presence of D1 and D2 dopamine receptors. Specific receptor binding of the D1 selective antagonists [3H]SCH 23390 and [125I]SCH 23982 was detected in membranes prepared from NS20Y cells, a clonal cell line derived from the C1300 murine neuroblastoma. Saturation analysis of [3H]SCH 23390 binding revealed the presence of saturable, high affinity binding sites with a dissociation constant (Kd) of 575 pM and a receptor density of 138 fmol/mg protein (approximately 9000 receptors/cell). Inhibition of [3H]SCH 23390 binding by a series of dopaminergic agonists and antagonists exhibited appropriate stereoselectivity and pharmacological specificity, verifying the D1 nature of this site. Dopamine inhibition of [3H]SCH 23390 binding revealed the presence of high and low affinity agonist binding sites which were converted to a homogeneous low affinity state by the addition of GppNHp. In membranes prepared from the WERI 27 human retinoblastoma cell line, specific receptor binding of the D2 antagonists [3H]methylspiperone and [125I]NAPS was observed. Saturation analysis of [3H]methylspiperone binding revealed the presence of a single class of high affinity, saturable binding sites with a Kd of 140 pM and a Bmax of 223 fmol/mg protein (approximately 2500 receptor sites/cell). Inhibition of [3H]methylspiperone binding by dopaminergic antagonists exhibited a rank order of potency consistent with the identification of a D2 dopamine receptor subtype. In addition, dopamine inhibition of [3H]methylspiperone binding exhibited both high and low affinity agonist binding sites which were converted to low affinity by the addition of GppNHp. These results represent the first direct demonstration of D1 and D2 dopamine receptors in cultured mammalian clonal cell lines. These cells should provide powerful model systems for investigating the molecular mechanisms involved in dopamine receptor/effector coupling and regulation.

Animals↗
PubMed1992

Antigen recognition and IL-2 receptor gene expression as evidence against clonal deletion in mice with neonatally induced transplantation tolerance.

Neonatal transplantation tolerance was induced in B10.A mice by the injection of spleen and bone marrow cells from semiallogeneic [C57BL/10(B10) x B10.A] F1 donors. The neonatally treated mice accepted skin grafts from B10 donors. Spleen cells from tolerant animals did not respond by proliferation to tolerated B10 antigens in vitro. However, spleen cells from tolerant mice recognized specific (B10) antigens and synthesized mRNA for the inducible 55-kDa interleukin-2 receptor (IL-2R) as did cells from normal animals. Maintenance of this early phase of cell activation upon contact with tolerated antigens is direct evidence against clonal deletion as a mechanism, in this particular model of neonatally induced transplantation tolerance.

Animals↗
PubMed1997

Isolation, cloning and characterization of a putative type-1 astrocyte cell line.

We have established a permanent cell line (1H91) of putative type-1 astrocyte precursor cells that were clonally derived from a single cell isolated from E16 mouse cerebellum. Epidermal growth factor (EGF) and transforming growth factor (TGF alpha) are strong mitogens for 1H91 cells (ED50 of 9.02 + 1.74 ng/ml and 15.98 +/- 2.34 ng/ml, respectively), while basic fibroblast growth factor (bFGF) is only weakly mitogenic and platelet derived growth factor (PDGF) has no mitogenic activity. In the proliferative state, the 1H91 cells are immunohistochemically positive for nestin and vimentin, and negative for A2B5, CNPase, neurofilament (NF), and neuron specific enolase (NSE). The majority of EGF-treated 1H91 cells are not immunoreactive for glial acid fibrillary protein (GFAP). In the presence of 5 ng/ml bFGF, 1H91 cells become non-mitotic and develop a morphology consistent with a fibrous astrocyte. In contrast to the proliferating cultures, the bFGF treated cultures were strongly immunoreactive for GFAP, only mildly immunoreactive for nestin and vimentin, and negative for A2B5, CNPase, NF, and NSE. Type-1 astrocytes are known to proliferate in response to EGF, and are immunohistochemically GFAP positive, A2B5 negative, and CNPase negative [38]. However, type-1 astrocytes only develop a fibrous morphology during the process of reactive gliosis [31]. Since EGF is a strong mitogen for 1H91 cells, and these cells may be differentiated into GFAP positive, A2B5 negative, CNPase negative astrocytes, we conclude that 1H91 cells conform to a type-1 astrocyte precursor phenotype. In addition, the fibrous morphology of the bFGF treated 1H91 cells suggests that these cells follow the process of reactive gliosis. Therefore, the 1H91 clonal cell line may provide an in vitro model for studying the underlying cellular mechanisms of the type-1 astrocyte in reactive gliosis.

Animals↗
PubMed1998

High-frequency ultrasound properties of multicellular spheroids during heating.

High-frequency ultrasound monitoring is a possible method for real-time imaging of thermal therapy in tissues at microscopic resolution. The objective of this work was to measure changes in the ultrasound properties of V79 spheroids (grown from Chinese Hamster lung fibroblasts) exposed to heating. Spheroids are clonal aggregates of cells that provide a useful model for investigating the ultrasound properties of cells in the absence of connective tissue. Relative echo signal power and attenuation coefficients were measured over the frequency range 30 MHz to 70 MHz, from spheroids heated from 37 degrees C to 50 degrees C or 60 degrees C. Echo signal power from the viable rim decreased during the first 5 min by a factor of 1.08 before the spheroid reached 50 degrees C. For the next 25 min, echo signal power rose to a factor of 1.27 above the initial level, after which it remained relatively constant over the remainder of the 50 degrees C heating period. At 60 degrees C, echo signal from the viable rim remained relatively constant, although it appeared to have possibly decreased slightly over the duration of the heating period. Echo signal power from the necrotic core fell to a factor of 1.4 and 1.54 below the initial level at 50 degrees C and 60 degrees C, respectively. First-order chemical rate analysis applied to the echo signal power results in the viable rim at 50 degrees C revealed a rate constant for the 5-15-min heating interval. Interpretation of the echo signal power results in terms of histological stains indicates that the rise in echo signal power at 50 degrees C was due to a loss of cell cohesion, and the possible drop in echo signal power at 60 degrees C was due to spheroid coagulation. Attenuation coefficients decreased by up to 1.54 dB mm-1 over a 30-min period at 60 degrees C. The appearance of a real-time ultrasound image of lesion formation in cells is discussed.

Animals↗
PubMed1989

Fetal calf serum and retinoic acid affect proliferation and terminal differentiation of a rat rhabdomyosarcoma cell line (BA-HAN-1C).

We report on the establishment of a model for differentiation induction in sarcomas, using the clonal rhabdomyosarcoma cell line BA-HAN-1C. This rhabdomyosarcoma cell line is composed of morphologically undifferentiated mononuclear stem cells, some of which spontaneously fuse to form terminally differentiated multinuclear myotube-like giant cells. The deprivation of fetal calf serum (FCS) or the exposure to retinoic acid, respectively, resulted in a significant inhibition of proliferation (P less than 0.001) and a marked increase in cellular differentiation as shown by a significant increase in the number of myotube-like giant cells (P less than 0.001) and in the creatine kinase activity (P less than 0.05) used as a biochemical marker of differentiation. Furthermore, after exposure to retinoic acid about 30% of the mononuclear tumour cells exhibited morphological features of rhabdomyogenic differentiation, such as bundles of thick and thin myofilaments, which had never been observed in the mononuclear cells of untreated cultures. These results confirm that the inverse linkage between proliferation and differentiation known from embryonic myogenesis is preserved in our rhabdomyosarcoma cell line. The failure to induce terminal differentiation by exposure to retinoic acid in all the cells of our clonal cell line indicates that some tumour cells might epigenetically be blocked from responding to retinoic acid. The temporary growth retardation observed after FCS-deprivation suggests that autocrine stimulation of proliferation may be operating in our cell line, too.

Animals↗
PubMed2006

The emergence of geometric order in proliferating metazoan epithelia.

The predominantly hexagonal cell pattern of simple epithelia was noted in the earliest microscopic analyses of animal tissues, a topology commonly thought to reflect cell sorting into optimally packed honeycomb arrays. Here we use a discrete Markov model validated by time-lapse microscopy and clonal analysis to demonstrate that the distribution of polygonal cell types in epithelia is not a result of cell packing, but rather a direct mathematical consequence of cell proliferation. On the basis of in vivo analysis of mitotic cell junction dynamics in Drosophila imaginal discs, we mathematically predict the convergence of epithelial topology to a fixed equilibrium distribution of cellular polygons. This distribution is empirically confirmed in tissue samples from vertebrate, arthropod and cnidarian organisms, suggesting that a similar proliferation-dependent cell pattern underlies pattern formation and morphogenesis throughout the metazoa.

Animals↗