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Results for “clonal modeling”

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PubMed2000

Growth hormone-regulated intracellular signaling in UMR 106 osteosarcoma cells.

Growth hormone (GH) and insulin-like growth factor 1 (IGF-1) are important growth factors for postnatal longitudinal bone growth. Although many effects of GH on bone growth are mediated by IGF-1, GH can directly influence bone cells. Limited knowledge exists regarding specific intracellular signaling pathways and genes activated by GH in bone cells. GH is known to activate several intracellular signaling pathways, among them the Janus kinase (JAK)/signal transducers and activators of transcription (STAT) pathway. GH mainly activates JAK2 and both isoforms of STAT5, A and B. STAT5 gene deletion experiments have shown the importance of these transcription factors for growth. To understand the molecular mechanism(s) behind this, different experimental models are needed. The UMR 106 cell line is a rat clonal osteosarcoma cell line with osteoblast-like phenotypic properties, one is the endogenous expression of GH receptor (GHR). The present study focused on whether these cells express a functional GH-responsive JAK2/STAT5 pathway. Analysis of cell extracts by immunoprecipitation and Western blot showed that physiological concentrations of GH activated JAK2. Western blot analysis of nuclear extracts from GH-stimulated UMR 106 cells showed that physiological concentrations of GH induced nuclear translocation of both STAT5 isoforms, but with STAT5A being predominant. Both isoforms displayed similar nuclear turnover after GH stimulation of cells. Gel electrophoretic mobility shift assay (GEMSA) of nuclear extract revealed that both STAT5A and STAT5B obtained DNA-binding capacity after GH stimulation. Thus, we have shown, for the first time, the expression and GH-induced activation of JAK2 and STAT5A/B in UMR 106 osteoblast-like cells. This study also shows that this cell line is a suitable experimental model to study unique GH effects in osteoblasts mediated by STAT5.

Animals↗

PubMed2006

Generation of human cortical neurons from a new immortal fetal neural stem cell line.

Isolation and expansion of neural stem cells (NSCs) of human origin are crucial for successful development of cell therapy approaches in neurodegenerative diseases. Different epigenetic and genetic immortalization strategies have been established for long-term maintenance and expansion of these cells in vitro. Here we report the generation of a new, clonal NSC (hc-NSC) line, derived from human fetal cortical tissue, based on v-myc immortalization. Using immunocytochemistry, we show that these cells retain the characteristics of NSCs after more than 50 passages. Under proliferation conditions, when supplemented with epidermal and basic fibroblast growth factors, the hc-NSCs expressed neural stem/progenitor cell markers like nestin, vimentin and Sox2. When growth factors were withdrawn, proliferation and expression of v-myc and telomerase were dramatically reduced, and the hc-NSCs differentiated into glia and neurons (mostly glutamatergic and GABAergic, as well as tyrosine hydroxylase-positive, presumably dopaminergic neurons). RT-PCR analysis showed that the hc-NSCs retained expression of Pax6, Emx2 and Neurogenin2, which are genes associated with regionalization and cell commitment in cortical precursors during brain development. Our data indicate that this hc-NSC line could be useful for exploring the potential of human NSCs to replace dead or damaged cortical cells in animal models of acute and chronic neurodegenerative diseases. Taking advantage of its clonality and homogeneity, this cell line will also be a valuable experimental tool to study the regulatory role of intrinsic and extrinsic factors in human NSC biology.

Cell Differentiation↗

PubMed1998

Liver disease and compensatory growth: unexpected lessons from genetically altered mice.

Over the last decade, several animal models have been established that permit exploration of liver biology and disease. Although these models have been developed using diverse strategies, including transgene targeting, homozygous gene disruption and administration of hepatotoxic chemicals, each approach creates an animal with hepatocyte damage, resulting in an hepatic microenvironment that supports proliferation of healthy hepatocytes. These models have been used to demonstrate: (1) the remarkable ability of adult hepatocytes to clonally proliferate in response to liver growth signals, (2) the effectiveness of transplanted donor hepatocytes in repopulating damaged liver parenchyma, and (3) the feasibility of reconstituting liver with xenogeneic hepatocytes. This paper reviews the development and use of these models, and outlines their potential future application to the study of hepatic stem cells, therapy of liver disease and hepatic toxicology.

Albumins↗

PubMed2005

Polyclonality of familial murine adenomas: analyses of mouse chimeras with low tumor multiplicity suggest short-range interactions.

In previous studies demonstrating the polyclonal structure of familial intestinal adenomas, high tumor multiplicity made it difficult to eliminate the possibility that polyclonality arose by the random collision of distinct initiated clones as opposed to some form of clonal interaction. We sought to test further the random collision hypothesis. Chimeric mice carrying the multiple intestinal neoplasia (Min) mutation of the adenomatous polyposis coli gene (Apc) and homozygous for the tumor resistance allele of the Mom1 locus were established. These chimeras also display a strong propensity for tumors of polyclonal structure, despite their markedly reduced tumor multiplicity. Considering tumor sizes and multiplicities, the observed fraction of overtly polyclonal heterotypic adenomas was significantly higher than predicted by the random collision hypothesis. This finding supports models of polyclonality involving interaction among multiple initiated clones. The extent of clonal interaction was assessed by statistical analyses that relate the observed frequency of overtly polyclonal heterotypic tumors to the geometry of the chimeric patches and the pattern of underlying crypts. These statistical calculations indicate that the familial adenomas of the Apc(Min/+) mouse may commonly form through interactions between clones as close as 1-2 crypt diameters apart.

Adenoma↗

PubMed1990

Mechanisms of autoimmunity in the context of T-cell tolerance: insights from natural and transgenic animal model systems.

There are a number of mechanisms which cooperate to produce and maintain T-cell tolerance. First, and perhaps most important, is the clonal deletion in the thymus of T cells with high affinity for self antigens. However, to ensure that a wide repertoire of T cells is available in the periphery to combat foreign antigens, the threshold of clonal deletion may be set low enough so that T cells whose TCR's have sub-threshold affinity for self antigens mature and migrate to the periphery. T cells which recognize self antigen-derived peptides not expressed or presented in the thymus will also fail to be deleted. For those self-reactive T cells which are not deleted in the thymus, other mechanisms may produce tolerance, including an undefined alteration of signalling pathways which produces clonal anergy, and lowering the avidity of the TCR for its ligand by downregulating coreceptor and accessory molecules. Active suppression of T-cell responses in another well-described phenomenon whose mechanism is undefined. From our observations with the model systems discussed here, we have observed three distinct mechanisms by which T-cell tolerance can be circumvented, allowing autoimmune phenomena to occur. These mechanisms may have relevance for different types of autoimmune diseases seen in humans. In gld mice, the autoimmune disease seems to be related to a global defect in T-cell differentiation and function, which allows for the expansion of autoimmune B cells. While we showed that clonal deletion of V beta-bearing T cells is appropriate in certain cases, aberrant lymphokine secretion by the abnormal T cells or disruption of immune system regulation are most probably responsible for allowing autoantibody production. While human lupus erythematosis shares much of the pathology of lpr and gld mice, there is no expansion of T cells with a similar phenotype in human lupus. There are environmental factors which must play a role in the development of human lupus, since the incidence of the disease does not follow an absolute genetic pattern. The escape from clonal deletion and subsequent reactivation of autoimmune T cells which we observed in V beta 8.1 TCR-transgenic mice can be a model for human autoimmune diseases such as multiple sclerosis and type I diabetes, in which T cells are directed against a specific autoantigen. According to this model, susceptibility loci for autoimmune disease such as the MHC would function by producing different repertoires of T cells which in some cases could gain autoreactivity following activation.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗

PubMed2001

Evidence of genetic instability in 3 Gy X-ray-induced mouse leukaemias and 3 Gy X-irradiated haemopoietic stem cells.

PURPOSE: If radiation-induced genetic instability is causal in mouse radiation leukaemogenesis, then genetic instability should be detectable in the irradiated target untransformed haemopoietic stem cell, and evidence of genetic instability detected in the clonal radiation-induced leukaemia. We have tested this hypothesis using the CBA/H mouse model of radiation-induced acute myeloid leukaemia (r-AML). MATERIALS AND METHODS: Fluorescence in situ hybridisation (FISH) was employed to screen for chromosomal aberrations in mouse 3 Gy X-ray-induced r-AMLs and in the clonal descendents of control and 3 Gy X-irradiated bone marrow haemopoietic stem cells using the in vitro clonogenic CFU-A colony assay. RESULTS: High levels of clonal non-specific chromosomal aberrations were detected in the r-AML (approximately 4-5 aberrations/r-AML), and ongoing chromosomal instability as defined by subclonal variants detected in 5/10 r-AML. A similar analysis of CFU-A colonies revealed chromosomal aberrations in 25% of colonies derived from irradiated bone marrow (2% in controls). However, 66% of the aberrant colonies (2% in controls) exhibited ongoing genetic instability as defined by non-clonal chromosomal aberrations. Overall, 6% (121/1884) of the CFU-A cells derived from irradiated bone marrow were aberrant (0.05% in controls) of which 12% (15/121) were subclonal. No one CFU-A cell exhibited aberrations on more than one of the three chromosomes painted. CONCLUSIONS: The high levels of non-specific genetic damage observed in the r-AMLs is therefore attributed to the accumulation of genetic lesions in the target haemopoietic stem cell over a longer time-scale after exposure than assessed in the in vitro CFU-A clonogenic assay. This is consistent with the long latency of the multi-stage radiation leukaemogenic process, and a role for radiation-induced genetic instability is inferred.

Animals↗

PubMed1993

Clonal predominance of T cell receptors within the CD8+ CD45RO+ subset in normal human subjects.

Structural models for the TCR alpha/beta predict that the CDR1, CDR2, and CDR3 loops of both the alpha- and beta-chains contribute to specific interactions with the Ag/MHC complex. The CDR3 loops are constructed by joining events involving the V-(D)-J segments, and thus may vary in both sequence and length. We have developed a polymerase chain reaction assay to assess the length variation of the CDR3 loop in TCR derived from seven V beta segment families (V beta 2, V beta 3, V beta 4, V beta 9, V beta 14, V beta 16, and V beta 17). Peripheral blood T cells from 10 normal adults as well as five cord blood samples were studied. CD4+ and CD8+ T cells were analyzed separately. We observed extreme predominance of particular CDR3 lengths in half of the normal adults. These TCR were shown to be clonal by direct sequence analysis. This clonal dominance was found in the CD8+, CD45RO+ T cell population, and was observed in various V segment families. These patterns of TCR clonality were persistent over many months of observation in some individuals. In one subject, the new appearance of a predominant clone was associated with a booster vaccination for hepatitis B. These studies reveal a surprising degree of oligoclonality in the CD8+ cells of normal subjects which may be due to both environmental and genetic factors; the functional significance of persistent clonal dominance in the CD8 compartment remains to be determined.

Adult↗

PubMed2001

Phage spread dynamics in clonal bacterial populations is depending on features of the founder cell.

Plate-cultured bacterial colonies are intriguing models to study host-parasite interactions in senescent populations. During the growth of bacteriophage-infected colonies there is a synchronous prophage induction episode among lysogenic cells that allows a dramatic but time-restricted amplification of viral particles. We report here that the dynamics of phage spread depends on the history of the lysogenic cell that establishes the clonal population, the duration of the pre-burst period being shorter when the founder, infected cell derives from older colonies. These results offer a physiologic explanation for the self-contained progression of the viral spread in closed environments, that ensures both viral dissemination but also survival of most of the host cells.

Age Factors↗

PubMed1993

Immune reactions in Drosophila and other insects: a model for innate immunity.

All insects defend themselves against bacteria and parasites, using cellular and humoral systems that are rapidly activated in infected animals. Among the induced effector molecules are antibacterial proteins and peptides such as cecropins, attacins, lysozymes and insect defensins, the genes for many of which have now been cloned. The induction of this system, which lacks clonally selected receptors like those of vertebrate B and T cells, may provide a model for innate immune reactions in other animals, including vertebrates.

Amino Acid Sequence↗

PubMed1997

Mathematical models of parasite responses to host immune defences.

We examine the evolution of microparasites in response to the immune system of vertebrate hosts. We first describe a simple model for an acute infection. This model suggests that the within-host dynamics of the microparasite will be a 'race' between parasite multiplication and a clonally expanding response by the host immune system, resulting either in immune-mediated clearance or host death. In this very simple model, in which there is only a single parasite and host genotype, maximum transmission is obtained by parasites with intermediate rates of growth (and virulence). We examine how these predictions depend on key assumptions about the parasite and the host, and consider how this model may be expanded to incorporate the effect of additional complexities such as host-parasite co-evolution, host polymorphism, and multiple infections.

Animals↗

PubMed1983

Comparative study of Cyclomunine and Cyclosporin A on human lymphocyte proliferation in vitro. The lack of an immunosuppressive effect by specific clonal deletion.

The effect of Cyclomunine (Cym) and Cyclosporin A (CyA) on in vitro human lymphocyte proliferation was assessed and it was noted that Cym and CyA suppressed lymphocyte response to mitogens, allogeneic cells or antigen in a dose--and time--dependent fashion. Furthermore, using a secondary mixed lymphocyte reaction model, we showed that neither CyA nor Cym exerted their immunosuppressive activity by producing specific clonal deletion and that their constant presence was necessary to obtain an antilymphocytic activity. These data suggest that treatment with these drugs in allotransplantation should be on a continuous basis.

Clone Cells↗

PubMed1989

A proposal for the development of clonal hemopoietic leukemias.

We elaborate on the pathogenesis of (human) hemopoietic leukemias according to a model whereby malignant growth follows the anachronistic expression of gene P in a rare cell at a certain stage of differentiation (1,2). P is a stimulus needed for renewal of hemopoietic cells. When illegally autocatalytic for P any mitotically competent cell (not necessarily pluripotent, at least originally) acquires the marginal (seldom complete) autonomous growth of malignant cells. Rather than confrontational, these views complement advantageously the mutation model and account for certain peculiarities of (human) leukemic cells, such as the propensity to differentiate terminally when isolated from their in vivo microenvironments. In addition, we discuss the rare occurrence of certain human leukemias (e.g., erythroleukemias and polycythemia veras) comparatively to the surprisingly more frequent chronic and acute myelogenous leukemias. This occurs in spite of the similarity between erythroid and myeloid populations in terms of size (mass of cells) and number of targets at mutational risk. Thus the classic mutation model predicts that random hits, after radiation exposure for example, should induce erythroid and myeloid leukemias with similar frequencies in contrast to clinical findings (3) and the predictions from our model.

Cell Division↗

PubMed1984

Is a bi-clonal interaction, analogous to that underlying embryogenesis the origin of the earliest malignant phenotype?

We propose a theoretical model of carcinogenesis which parallels embryonic induction. It is based on the interaction between two clones of cells. One of them is of germ cell ancestry and is the source of transforming effector cells. The second clone is the potential target of such travelling "lymphocytoid" cells and emerges in tissues exhibiting the precancerous picture of dysplasia. In atypical fields each endopolyploid cell, arrested in G2 and involved in genomic changes, is the stem of a clonal growth. This emerging new phenotype is subjected to policing control by wandering effector cells. This situation establishes the probability of a random collision between two cells pertaining to different lineages, with two possible antithetical results, cytolysis or cell fusion comparable to fertilization. The latter event is suggested to determine malignancy.

Animals↗

PubMed1994

Further studies of veto activity in rhesus monkey bone marrow in relation to allograft tolerance and chimerism.

Infusing the DR-/dim fraction of bone marrow cells (BMC) from an allogeneic kidney donor into rabbit antithymocyte globulin-treated transplant recipients delivers a tolerogenic signal, leading to functional allograft tolerance in rhesus monkeys without additional drug therapy. Our updated results in an expanded series show a median 131-day graft survival of recipients given DR-/dim donor BMC with a 23% 1-year survival (P < 0.00001 vs. rabbit antithymocyte globulin controls). Removing DRbright cells from donor BMC appeared to have a significant effect (P < 0.05). We have further investigated the tolerogenic mechanism within the experimental framework of the veto hypothesis in this preclinical model. In limiting dilution assays, we demonstrated the donor specificity of clonal inactivation of CTL precursors (CTLp) after in vitro or in vivo exposure to DR-/dim donor BMC, confirming specific tolerance. Additionally, in vitro studies confirmed the allogeneic specificity of CTLp inactivation in 3-cell MLR assays; minimal bystander effects were seen on normal CTLp responses to third party stimulator cells, while CTLp responses to the BMC donor's cells were abrogated in the same cultures. BMC mediating the veto effect were found to be resistant to L-leucyl-L-leucine methyl ester (Leu-leu-OMe), which excluded BMC-mediated cytotoxicity by NK or lymphokine-activated killer cells, CTL, or activated macrophages. In contrast, veto activity was abolished if the BMC were pretreated with either high dose UV-B light irradiation, mitomycin, or gamma-irradiation, indicating that BMC contained a UV-B-sensitive precursor of the veto effector, and that a proliferative step separated the two. Irradiation of DR-/dim donor BMC or administration of cyclophosphamide after infusion of nonirradiated BMC prevented the tolerogenic effect. Only recipients given nonirradiated DR-/dim donor BMC demonstrated PBL chimerism, which associated with functional deletion of antidonor CTLp and duration of graft survival. The Leu-leu-OMe resistance and the other properties of the allogeneic monkey CD3- CD2+ CD8+ BMC subpopulation that exhibits tolerance-promoting activity in vitro and in vivo lead us to postulate that a donor BMC-derived precursor population, possibly a dendritic cell population, may induce allogeneic unresponsiveness in this model.

Animals↗

PubMed1993

Requirement for CD8+ cells in T cell receptor peptide-induced clonal unresponsiveness.

T cell receptor (TCR) vaccination in rats prevents the development of experimental allergic encephalomyelitis (EAE), an animal model of multiple sclerosis. The mechanism of this potential immunotherapy was examined by vaccinating mice with an immunogenic peptide fragment of the variable region of the TCR V beta 8.2 gene. Another immunogen that usually induces an immune response mediated by V beta 8.2+ T cells was subsequently inhibited because specific clonal unresponsiveness (anergy) had been induced. Depletion of CD8+ cells before TCR peptide vaccination blocked such inhibition. Thus, the clonal anergy was dependent on CD8+ T cells, and such immunoregulatory T cells may participate in the normal course of EAE.

Animals↗

PubMed1996

Kinetic discrimination in T-cell activation.

We propose a quantitative model for T-cell activation in which the rate of dissociation of ligand from T-cell receptors determines the agonist and antagonist properties of the ligand. The ligands are molecular complexes between antigenic peptides and proteins of the major histocompatibility complex on the surfaces of antigen-presenting cells. Binding of ligand to receptor triggers a series of biochemical reactions in the T cell. If the ligand dissociates after these reactions are complete, the T cell receives a positive activation signal. However, dissociation of ligand after completion of the first reaction but prior to generation of the final products results in partial T-cell activation, which acts to suppress a positive response. Such a negative signal is brought about by T-cell ligands containing the variants of antigenic peptides referred to as T-cell receptor antagonists. Results of recent experiments with altered peptide ligands compare favorably with T-cell responses predicted by this model.

Animals↗

PubMed2025

SURROGATE SELECTION OVERSAMPLES EXPANDED T CELL CLONOTYPES.

Surrogate selection is an experimental design that without sequencing any DNA can restrict a sample of cells to those carrying certain genomic mutations. In immunological disease studies, this design may provide a relatively easy approach to enrich a lymphocyte sample with cells relevant to the disease response because the emergence of neutral mutations associates with the proliferation history of clonal subpopulations. A statistical analysis of clonotype sizes provides a structured, quantitative perspective on this useful property of surrogate selection. Our model specification couples within-clonotype birth-death processes with an exchangeable model across clonotypes. Beyond enrichment questions about the surrogate selection design, our framework enables a study of sampling properties of elementary sample diversity statistics; it also points to new statistics that may usefully measure the burden of somatic genomic alterations associated with clonal expansion. We examine statistical properties of immunological samples governed by the coupled model specification, and we illustrate calculations in surrogate selection studies of melanoma and in single-cell genomic studies of T cell repertoires.

Bayes’s rule↗

PubMed1985

LLC-PK1 cysts: a model for the study of epithelial polarity.

In the present work, we have taken advantage of the properties of two recently isolated clonal subpopulations of the pig kidney-derived LLC-PK1 cell line to study aspects of the establishment of epithelial polarity. When grown in suspension, LLC-PK1/D + Sc cells reaggregated within a few hours and, during the following days of culture, formed free-floating, hollow spheres or cysts, lined by a monolayer of polarized cells. In contrast, LLC-PK1/D- cells were unable to develop such polarized structures even upon prolonged culture in suspension. The polarity of the LLC-PK1/D + Sc cells lining the cysts was inverted compared to that in intact renal tubules, the microvilli-rich "apical" pole being oriented toward the external medium. However, upon embedding these preformed cysts in collagen gels, a reversal of polarity was observed within hours, the microvilli-rich pole now facing the cyst cavity. Thus, in the same clonally derived cell population, cell-to-cell contact and interaction with the extracellular matrix differentially affect the orientation of cellular polarity. The LLC-PK1/D + Sc cysts provide a suitable in vitro model system for further study of the sequential events by which extracellular matrix components induce an appropriately oriented polarization. In addition, the comparison between LLC-PK1/D + Sc and D- cells, which differ in their ability to polarize in response to cell-to-cell contact, should help define some of the cellular determinants involved in epithelial organization.

Animals↗