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Results for “clonal modeling”

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PubMed2001

A model for the detection of clonality in marked hematopoietic stem cells.

The semirandom location of retroviral integration in the target cell genome introduces a marker in the form of a fusion sequence composed of a genomic and a proviral part that is unique for each transduced cell and its clonal progeny. High-sensitivity detection of these fusion sequences would allow the tracking of clonal contributions of individual, marked hematopoietic progenitor, and stem cells in vivo. Clone detection by Southern blot has helped to analyze models of oligoclonal repopulation but is limited in sensitivity and specificity. Inverse PCR (Nolta et al., Proc. Natl. Acad. Sci. USA 93: 2414-2419) can demonstrate the clonal identity by sequencing but does not permit simultaneous detection of multiple clones. In an efficiently transduced rhesus macaque model (Tisdale et al., Blood 92: 2681-2687; Wu et al., Mol. Ther. 1: 285-293) Kim et al. (Blood 96: 1-8) have identified more than 40 insertion sequences from marrow CFU by inverse PCR. However, no previous study has been able to directly analyze the number of clones active in vivo. Here we demonstrate that the application of a recently developed PCR technology allows the simultaneous visualization of multiple integration sites from small clonal contributions to hematopoietic cells. By combining solid-phase primer extension with ligation-mediated PCR, direct genomic sequencing of retroviral integration sites was obtained in murine bone marrow samples. Further development of this technology will allow analysis of the clonal composition of marked hematopoiesis in small and large animals as well as in human gene transfer.

Animals↗

PubMed2000

An orthotopic model of murine osteosarcoma with clonally related variants differing in pulmonary metastatic potential.

To provide an investigative tool for the study of osteosarcoma (OSA) biology we have developed a syngeneic (balb/c) murine model of OSA, using cell lines derived from a spontaneously occurring murine OSA (Schmidt et al. Differentiation 1988; 39: 151-60). This model is characterized by orthotopic primary tumor growth, a period of minimal residual disease, spontaneous pulmonary metastasis, and clonally related variants (K7M2 and K12) that differ in pulmonary metastatic potential. Primary tumor and pulmonary metastasis histology was consistent with OSA in human patients. Expression of bone sialoprotein, biglyan, decorrin, and osteopontin was suggestive of bone lineage cells. The development and use of a more aggressive OSA cell line (K7M2) resulted in spontaneous metastasis to the lungs in over 90% of mice, whereas metastases were seen in only 33% of mice when a less aggressive OSA cell line (K12; Schmidt et al. Differentiation 1988; 39: 151-60) was used. Death from metastasis occurred at a median of 76 days using K7M2 whereas no median was achieved after 140 days using K12. Angiogenic potential, characterized by CD31 and factor VIII staining of primary tumors and pulmonary metastases, was greater in the K7M2 model compared to the K12 model. No significant differences in the in vitro or in vivo expression of angiogenesis associated genes (flt1, flt4, TIE1, TIE2, and VEGF) was found between K7M2 and K12. This well characterized and relevant model of OSA will be a valuable resource to improve our understanding of the biology and treatment of metastasis in OSA.

Animals↗

PubMedDate not supplied

Time sequence of cancer occurrence. Implications in low level radiation risk assessment.

A total of 8,229 C57 Black mice of both sexes were randomly assigned to various groups. In some groups, mice aged 33 +/- 3 days were submitted to either sham, neutron or cobalt external radiation at 32, 45, 63, 88 and 123 mGy or at 18, 25, 36 and 51 cGy dose levels, respectively. In other groups, mice either at birth or weaning, were injected with tritiated thymidine or tritiated water, or were given tritiated water as drinking water for the entire lifespan. The main purpose of the experiment was to investigate the low dose-response relationship of cancer induction, especially leukemogenesis and to evaluate the excess risk, using actuarial age-specific rates. Following neutron or cobalt exposure, the phenotypic occurrence of lymphocytic lymphomas was earlier in appearance and higher in yield during the first decades of lifespan in irradiated groups versus matching controls, whereas such occurrence was markedly lower in yield at a later age. Under parallel experimental conditions, induction of reticulum cell lymphomas, however, was uniformly enhanced throughout the entire lifespan. Induction rates of all tumors (reticular and solid) pooled were significantly increased, and more so following cobalt than neutron irradiation. In mice injected with tritiated thymidine, the overall tumor incidence was increased monotonically throughout the lifespan. In mice exposed to tritiated water, the incidence of lymphocytic lymphomas was markedly increased throughout the lifespan, whereas no such effect was observed for reticulum cell tumors. In the light of tumor data analysis, it appears that selection of a particular type of tumor as a dependent variable for dose-response assessment cannot disregard the primary modulation of the whole tumor spectrum, In C57 Black murine leukemogenesis, the shape and structure of the dose-response regression curve over the entire lifespan dose not fit the linear-quadratic model. It is, however, theorized that our data are consistent with the two-mutation clonal expansion model, assuming creation of initiated cells which are added to the pool of spontaneously occurring initiated cells and implying that the excess risk is initially high at early age and then decreases with increasing age following exposure. It is concluded that murine radiocarcinogenesis investigation may contribute to improving the assessment as well as understanding the underlying mechanisms of low level radiation hazards.

Animals↗

PubMed2000

Glucose-mediated Ca(2+) signalling in single clonal insulin-secreting cells: evidence for a mixed model of cellular activation.

Using clonal insulin-secreting BRIN-BD11 cells, we have assessed whether the graded response of the whole cell population to glucose can be accounted for by a dose-dependent recruitment of individual cells, an amplification of the response of the recruited cells or both. Cytosolic free Ca(2+) concentration ([Ca(2+)](i)) is an established index of beta-cell function. We used fura-2 microfluorescence techniques to assess the [Ca(2+)](i) responsiveness of single BRIN-BD11 cells to glucose and other secretagogues. Glucose (1-16.7 mM) evoked oscillatory [Ca(2+)](i) rises in these cells resembling those found in parental rat pancreatic beta-cells. The percentage of glucose-responsive cells was 11% at 1 mM and increased to 40-70% at 3-16.7 mM glucose, as assessed by a single-stimulation protocol. This profile was unrelated to possible differences in the cell cycle, as inferred from experiments where the cultured cells were synchronized by a double thymidine block protocol. Individual cells exhibited variable sensitivities to glucose (threshold range: 1-5 mM) and a variable dose-dependent amplification of the [Ca(2+)](i) responses (EC(50) range: 2-10 mM), as assessed by a multiple-stimulation protocol. Glyceraldehyde and alpha-ketoisocaproic acid had glucose-like effects on [Ca(2+)](i). The data support a mixed model for the activation of insulin-secreting cells. Specifically, the graded secretory response of the whole cell population is likely to reflect both a recruitment of individual cells with different sensitivities to glucose and a dose-dependent amplification of the response of the recruited cells.

Animals↗

PubMed1999

Insulin-like growth factor (IGF-I) induces myotube hypertrophy associated with an increase in anaerobic glycolysis in a clonal skeletal-muscle cell model.

Insulin-like growth factor-I (IGF-I) is an important autocrine/paracrine mediator of skeletal-muscle growth and development. To develop a definitive cultured cell model of skeletal-muscle hypertrophy, C2C12 cells were stably transfected with IGF-I and clonal lines developed and evaluated. Quantitative morphometric analysis showed that IGF-I-transfected myotubes had a larger area (2381+/-60 micrometer2 versus 1429+/-39 micrometer2; P<0.0001) and a greater maximum width (21.4+/-0.6 micrometer versus 13.9+/-0.3 micrometer; P<0.0001) than control C2C12 myotubes, independent of the number of cell nuclei per myotube. IGF-I-transfected myotubes had higher levels of protein synthesis but no difference in DNA synthesis when compared with control myotubes, indicating the development of hypertrophy rather than hyperplasia. Both lactate dehydrogenase and alanine aminotransferase activities were increased (3- and 5-fold respectively), and total lactate levels were higher (2.3-fold) in IGF-I-transfected compared with control myotubes, indicating an increase in anaerobic glycolysis in the hypertrophied myotubes. However, expression of genes involved in skeletal-muscle growth or hypertrophy in vivo, e.g. myocyte nuclear factor and myostatin, was not altered in the IGF-I myotubes. Finally, myotube hypertrophy could also be induced by treatment of C2C12 cells with recombinant IGF-I or by growing C2C12 cells in conditioned media from IGF-I-transfected cells. This quantitative model should be uniquely useful for elucidating the molecular mechanisms of skeletal-muscle hypertrophy.

Animals↗

PubMed2026

Ex vivo long-term expansion of human hematopoietic stem and progenitor cells as a tool for modeling vector integration sites and clonality.

BACKGROUND: Gene therapy (GT) using retroviral vectors (RVs) is efficacious in treating monogenic diseases. However, there is an inherent risk for severe adverse effects due to insertional mutagenesis. Preclinical safety assessment and patient monitoring are inevitable in GT. To assess the genotoxic risk of novel RV vectors, mainly murine hematopoietic stem and progenitor cells (HPSCs) are routinely used, because human HSPCs cannot be immortalized in vitro using mutagenic vectors. In this study, we aim to identify early signs of clonal outgrowth by performing integration site analyses (ISA). METHODS: The small molecules A83-01, pomalidomide, and UM171 (APU) were used for the ex vivo expansion, lentiviral transduction, and long-term cultivation of umbilical cord blood-derived HSPCs. We determined the influence of APU on the stemness of HSPCs and their differentiation capacity via single-cell RNA sequencing (scRNA seq) and in xenotransplantation studies. To track vector insertion site dynamics, we transduced 7-day expanded HSPCs with a mutagenic or a safer RV. ISA was conducted in human HSPCs over a 5-week cultivation in vitro and compared to the bone marrow of xenotransplanted mice to assess clonal skewings. RESULTS: APU supported the expansion of CD34+CD38-CD45RA-CD90+EPCR+ HSPCs. scRNA seq confirmed the enrichment of HSC signature genes in APU-expanded HSPCs compared to the clinically used medium SFT3 (SCF, FLT3-L, TPO, IL-3). After RV transduction, APU still maintained around 30% of CD34+ cells for 5 more weeks. Without the compounds, already 2 weeks post-transduction, less than 10% of cells were CD34+. The long-term culture allowed the detection of high-risk integrations of the mutagenic SIN-LV.SF in MEIS1 or SUSD6 due to their increasing abundance over time. Bone marrow of xenotransplanted mice was less clonal but did not support the outgrowth of insertional mutants. Overall, APU increased clonal diversity. CONCLUSIONS: Our findings propose that long-term cultivation of transduced HSPC in APU allows for outgrowth of clonal integration sites. The decrease of clonality has been observed in gene therapy patient's years after treatment. Thus, the in vitro model could be used to develop novel human HSPC-based genotoxicity assays that predict insertional mutagenesis, in addition to existing preclinical biosafety assays.

Humans↗

PubMed2000

Analysis of lung tumour risk in radon-exposed rats: an intercomparison of multi-step modelling.

Three carcinogenesis modelling groups have both jointly and separately applied a multi-step carcinogenesis model with clonal expansion to one data set of lung tumours in rats exposed to radon (CEA, France). This study was designed to investigate the differences in modelling approach and fitting procedures used by the three groups in detail, and to explore possible discrepancies in the results. Using the same model assumptions and a (linear) radiation dependence on the first model step only, the three groups arrived at identical best fits, proving that the mathematical formalisms and fitting procedures do not lead to different results. However, when each group was allowed to find its own preferred fit for this data set, all three found a significantly better, but different fit to the data. All solutions indicated radiation to be an initiating agent and found additional radiation action necessary. The character of this additional radiation dependence, however, could not be unambiguously pinpointed. Tumour incidence data were described equally well when radiation dependence was taken into account in clonal expansion ("promotion") or in the second mutational step ("transformation"); extension to three model stages also resulted in an adequate description. The study showed that, although the three groups used one carcinogenesis model in principle, different model assumptions and/or different methods of finding the "best fit" could result in different descriptions of experimental data. This implies that on statistical grounds, different interpretations can be given for the action that radiation had in this data set. Different data, i.e. other data sets with age-dependent tumour data and/or information from cellular radiobiology experiments, are needed to specifically pin down the radiation dependence in the multi-step carcinogenesis process.

Age Factors↗

PubMed1986

Limiting dilution analysis of proliferating and helper T cells in the in vivo immune response to KLH: derepression of helper T cells at moderately increased frequencies.

While it is clear that some T cells have the capacity for almost indefinite proliferation in vitro, it is a controversial issue how much of this proliferative capacity is utilized by T cells in a response to antigen in vivo. In the framework of a strict clonal selection model the functional activities of normal and immune lymphocyte populations are essentially determined by the frequencies of antigen-specific cells which are clonally expanded after recognition of antigen. In contrast, our group has proposed a network model in which the more prominent effect of immunization is a release of antigen-specific T cells from a state of suppression (= derepression) which exists in the non-immune situation and which is generated through interactions between T cells involving their antigen-specific receptors. In this model, derepression is achieved by competition of antigen with the interactions among T cells through which suppression is exerted. To test our model, we analyze in this paper how much of the immune response to keyhole limpet hemocyanin (KLH) in draining lymph nodes is accounted for by an increase in the numbers of KLH-reactive T cells or by their derepression. To this end, we immunize mice subcutaneously with KLH in CFA. For a period of 14 days after immunization draining lymph nodes are removed, and the frequencies of KLH-reactive proliferating T cells and of KLH-reactive helper T cells determined. We find that proliferating T cells increase 5 to 8 fold in frequency from day 1 to 4 after immunization (approximately 1/30,000 to approximately 1/5000) and no evidence for suppression of these T cells in the non-immune situation can be obtained. In contrast, T helper cells are strongly suppressed in non-immune lymph nodes and become derepressed suddenly between days 3 and 4 following immunization. From day 0 to day 3 T helper cell frequencies are in the order of 1/14,000-1/38,000, then increase suddenly approximately 3-6 fold within 1 day from 1/16,000-1/8,000 to 1/3,000-1/5,000 with no further change until day 14. Thus, helper T cell immunity in draining lymph nodes appears to be generated by a combination of increased frequencies of specific T cells with their release from suppression. In addition, we have reasons to suspect that we overestimate the increase in T cell frequencies. We therefore think that derepression is a major factor in the response of T helper cells to antigen.

Animals↗

PubMed2000

An analysis of bone and head sinus cancers in radium dial painters using a two-mutation carcinogenesis model.

Bone and head sinus cancer incidence after ingestion of 226Ra and 228Ra by radium dial painters is analysed using a two-mutation clonal expansion model for radiation carcinogenesis, taking into account the retention and radiation patterns of these nuclides in the body. The best fit is obtained for compact bone retention and efficient diffusion of 222Rn to the bone cavities and radiation action on both mutation rates of the cancer model, as found in a similar analysis of bone sarcomas after 226Ra injection in beagles. The model parameters of the best fit are consistent with cellular radiobiological data and a previous analysis of lung cancer in uranium miners. Due to the low background incidence of bone and head sinus cancer, the resulting dose-effect relationships for these cancers are linear-quadratic with radium ingestion and alpha radiation dose. These results do not support a threshold dose concept, but the risks at low doses calculated by the model come out to about a factor 10 lower than using a linear extrapolation of the data to low doses, a procedure currently applied by ICRP and EPA. Furthermore, the model results indicate radiation risks at low doses to be related with background cancer incidence between relative and absolute radiation risk projections. The results, which are dependent on the model assumptions, might be more generally applicable for bone seekers and will therefore need further study to arrive at better radiation risk estimations.

Adult↗

PubMed1999

Two-step model for the risk of fatal and incidental lung tumors in rats exposed to radon.

Data from 4276 rats with radon exposures up to 10,000 WLM at rates up to 1000 WL are analyzed with a two-step clonal expansion model. The age dependences of the hazard for the risks for fatal and for incidental tumors are very different. Therefore, two different parameterizations of the model are used in the two cases. In both cases radiation acts only on the initiating mutation and the clonal expansion, but not on the second mutation. Average exposure rates of 5 WL for fatal tumors and 0.5 WL for incidental tumors double the rate of spontaneous mutations. While the fatal tumors show a linear increase in the effective clonal expansion rate up to about 100 WL average exposure rate and a saturation at higher exposure rates, the incidental tumors follow a step-like behavior of this parameter. It is proposed that only the fatal lung tumors among the rats be used for generalizations to models for lung cancer in humans. The fitted model for fatal tumors shows an inverse dose-rate effect at average exposure rates above 20 WL. However, below 10 WL the lung cancer risk per unit exposure decreases with increasing duration of exposure. Between 10 and 20 WL, the difference in ERR/WLM between acute and protracted exposure is small.

Age Factors↗

PubMed2005

Interaction of smoking and radon in rats: a biologically based mechanistic model.

Data on rats exposed to cigarette smoke before or after exposure to radon are used to estimate smoke-dependent parameters of the biologically based two-stage clonal expansion model. The baseline parameters and the action of radon acting on initiation and promotion were fixed based on earlier work. Cigarette smoke acting on transformation and inducing a reduction of the radon dose to the target cells after a smoking period gives an acceptable description of the data.

Animals↗

PubMed2000

An unsolved problem of the clonal selection theory and the model of an oligomeric B-cell antigen receptor.

The B cell antigen receptor (BCR) plays a central role in the development, survival and activation of B lymphocytes. As the pre-BCR, it controls allelic exclusion of heavy chains and the expansion of pre-B cells. As the BCR, it controls the positive and negative selection of immature B cells as well as the survival and activation of mature B cells. Recent studies of receptors have shown that it is the ligand that brings them into the conformation necessary for signaling. How the multiple and structurally diverse antigens could fulfill this task for the BCR is unknown, and we regard this as an unsolved problem of Burnet's clonal selection theory This question and our recent biochemical studies lead us to propose a new model for the BCR, according to which the BCR exists as a precise oligomeric complex on the B cell surface. In this form, it can signal positive selection and survival of B cells. Binding to self- or foreign antigen results in a distortion of the oligomeric complex that gives the signal for negative selection of immature and activation of mature B cells.

Animals↗

PubMed1999

A random walk model of oligodendrocyte generation in vitro and associated estimation problems.

A branching stochastic process proposed earlier to model oligodendrocyte generation by O-2A progenitor cells under in vitro conditions does not allow invoking the maximum likelihood techniques for estimation purposes. To overcome this difficulty, we propose a partial likelihood function based on an embedded random walk model of clonal growth and differentiation of O-2A progenitor cells. Under certain conditions, the partial likelihood function yields consistent estimates of model parameters. The usefulness of this approach is illustrated with computer simulations and data analyses.

Algorithms↗

PubMed1997

Multistage carcinogenesis modeling and the initiation event.

Carcinogenesis is generally considered to be a multistage process classified under "initiation," "promotion," and "progression," and it is therefore of interest to know how radiation might affect them. Models which are currently being used with some success to analyze a variety of data are based on the two-mutation with clonal expansion model developed by Moolgavkar and Knudson [J Natl Cancer Inst 66:1037-1052, 1981]. These models imply that the "initiation" event would be a mutation but also imply that "progression" also involves mutation and that both these events could be influenced by radiation. The models offer the possibility of calculating the incidence of cancer over lifetime and can simultaneously provide the age and dose dependence of cancer. The models have implications for radiation protection and imply that radiation risk is related to spontaneous cancer incidence and that spontaneously induced "initiation" may also be associated with radiation-induced cancer.

Age Factors↗

PubMed2026

Integrating genomic additive relationship matrices improves the efficiency in diploid banana breeding.

Partitioning of genetic variance into additive and non-additive components using the pedigree-based best linear unbiased prediction (P-BLUP) model is possible because of the family structure and replicated clones in clonally propagated crops, but this model may overestimate these components. However, the genomic best linear unbiased prediction (G-BLUP) method, which integrates the genetic relationship through molecular marker information reduces the overestimation. Alternatively, a combination of the P-BLUP and G-BLUP, sourcing to create a hybrid matrix that estimates hybrid best linear unbiased prediction (H-BLUP), is proposed. We investigated if integrating molecular information into the clonal model could improve the partitioning of the variance components leading to more accurate estimates of genetic parameters and prediction accuracy of breeding values of 14 key traits in diploid banana. In this study, we used clones of 14 full-sib families from a factorial mating design of four female and five diploid male banana (Musa acuminata) parents, generated at the International Institute of Tropical Agriculture in Arusha. The genomic-based relationship matrices were constructed using a set of 2792 filtered single-nucleotide polymorphism markers. Additive variance and heritability derived from G-BLUP and H-BLUP models reduced bias compared to the P-BLUP model. The H-BLUP estimated the highest prediction accuracies for yield-related and cycling traits, while the P-BLUP model had the highest prediction accuracy estimates for agronomic traits. The use of marker-based models enhances the accuracy of predicting breeding values, contributing to accurate estimates of genetic gain while paving a way for further genomic exploration in diploid banana breeding programs.

Journal Article↗

PubMed2005

Central tolerance: learning self-control in the thymus.

In the past few years, there has been a flurry of discoveries and advancements in our understanding of how the thymus prepares T cells to exist at peace in normal healthy tissue: that is, to be self-tolerant. In the thymus, one of the main mechanisms of T-cell central tolerance is clonal deletion, although the selection of regulatory T cells is also important and is gaining enormous interest. In this Review, we discuss the emerging consensus about which models of clonal deletion are most physiological, and we review recent data that define the molecular mechanisms of central tolerance.

Animals↗

PubMed2005

Restricted IgA repertoire in both B-1 and B-2 cell-derived gut plasmablasts.

Mucosal IgA is the most abundantly produced Ig upon colonization of the intestinal tract with commensal organisms in the majority of mammals. The repertoire of these IgA molecules is still largely unknown; a large amount of the mucosal IgA cannot be shown to react with the inducing microorganisms. Analysis of the repertoire of used H chain Ig (V(H)) genes by H-CDR3 spectrotyping, cloning, and sequencing of V(H) genes from murine intestinal IgA-producing plasma cells reveals a very restricted usage of V(H) genes and multiple clonally related sequences. The restricted usage of V(H) genes is a very consistent observation, and is observed for IgA plasma cells derived from B-1 or conventional B-2 cells from different mouse strains. Clonal patterns from all analyzed V(H) gene sequences show mainly independently acquired somatic mutations in contrast to the clonal evolution patterns often observed as a consequence of affinity maturation in germinal center reactions in peripheral lymphoid organs and Peyer's patches. Our data suggest a model of clonal expansion in which many mucosal IgA-producing B cells develop in the absence of affinity maturation. The affinity of most produced IgA might not be the most critical factor for its possible function to control the commensal organisms, but simply the abundance of large amounts of IgA that can bind with relatively unselected affinity to redundant epitopes on such organisms.

Animals↗

PubMed2002

Chromosomal size variation in Trypanosoma cruzi is mainly progressive and is evolutionarily informative.

The evolutionary significance of chromosome size polymorphism was explored in a representative panel of 26 Trypanosoma cruzi stocks. We tested a progressive model (aCSDI) assuming that the larger the size difference between homologous chromosomes, the more divergent the parasites are. This was contrasted with a non-progressive model (Jaccard's distance), in which any chromosome size difference has the same weight. ACSDI-based dendrograms were very similar to those built-up from multilocus enzyme electrophoresis (MLEE) and random amplified polymorphic DNA (RAPD) data: structuring in 2 major lineages (T. cruzi I and T. cruz II) and 5 small subdivisions within T. cruzi II was identical, and branching was very similar. Furthermore, a significant correlation (P < 0.001) was observed between aCSDI and phenetic distances calculated from MLEE and RAPD data. In contrast, analysis of chromosome size polymorphism with Jaccard's distance generated dendrograms with relatively long branches, causing most branching points to cluster close together, which generates statistically uncertain branching points. Our results thus support a model of progressive chromosome size-variation and show that despite an extensive polymorphism, chromosomal sizes constitute valuable characters for evolutionary analyses. Furthermore, our data are consistent with the clonal evolution model previously proposed for T. cruzi.

Animals↗