Examination of the clonal deletion theory in the guinea pig to Lewis rat cardiac xenograft model.
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Animals↗
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Results for “clonal modeling”
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Clonal variations in complement activation and deposition of C3b and C4b on model immune complexes.
This study examined the relationship between complement activation and the deposition of C3b and C4b on a panel of model immune complexes (IC). IC were constructed by combining murine monoclonal IgM, IgA, IgG1, IgG2a or IgG3 anti-dinitrophenyl (DNP) antibodies with DNP-bovine serum albumin (DNP-BSA). The IC were incubated with human plasma as a complement source and the formation of C4a and C3a, as well as the deposition of C4b and C3b on the IC, measured by radioimmunoassay. The results indicate that there were isotype-independent variations in the capacity of different types of IC to activate the classical pathway, especially for isotype-matched pairs of IC containing IgG1, IgG2a and IgG3 antibodies. In most cases, there was a direct relationship between classical pathway activation and the cleavage of C3. There was, for most of the IC, a direct correlation between cleavage of C4 and C3 and the subsequent deposition of C4b and C3b on the IC. However, a pair of IC constructed with independently derived IgG1 antibodies was virtually identical with respect to C3 cleavage and yet differed in the number of C3b molecules deposited on the IC. Collectively, these data suggest that the immunoglobulin variable region can play a significant role in both complement activation and the deposition of C3b and C4b on IC.
Antigen-Antibody Complex↗
Maintenance of clonal diversity in Dipsa bifurcata (Fallén, 1810) (Diptera: Lonchopteridae). I. Fluctuating seasonal selection moulds long-term coexistence.
The deterministic maintenance of clonal diversity in thelytokous taxa can be seen as a model for understanding how environmental heterogeneity both can stabilize genetic diversity and can allow coexistence of competing species. We here analyze the temporal fluctuations in clonal diversity in the thelytokous Lonchopterid fly, Dipsa bifurcata (Fallén, 1810), at four localities in Sweden over an 8-year period. Estimated fitness values for clones are cyclical, synchronous among populations and correlated with seasonal changes in the environment. Differential winter viability and emergence from overwintering along with differential reproductive rate during the summer appear to be the selective mechanisms by which long-term clonal diversity is maintained. In a companion paper (Tomiuk et al, 2004), we present a model for the maintenance of clonal diversity through the mechanism of differential diapause among clones, utilizing fitness values estimated from the data presented here. In general, our results imply that fluctuating seasonal fitnesses can maintain stable genetic polymorphism within populations, as well as coexistence between closely related competitors, when coupled with differences in diapause phenology.
Analysis of Variance↗
Immortalized dopamine neurons: A model to study neurotoxicity and neuroprotection.
6-Hydroxydopamine (6-OHDA) causes selective degeneration of dopaminergic neurons in the rat brain and has been used to produce an animal model of Parkinsonism. Recently, a clonal line of immortalized dopamine (DA) neurons (1RB3AN27), which expresses varying levels of tyrosine hydroxylase, dopamine transporter, neuron specific enolase, and nestin, was established. These DA neurons reduce behavioral deficits in 6-OHDA-lesioned rats. The relative sensitivity of fetal and adult neurons to potential neurotoxins is not well defined. The availability of immortalized DA neurons provides a unique opportunity to compare the relative neurotoxicity of 6-OHDA in differentiated and undifferentiated DA neurons in vitro and identify neuroprotective agents. Our results showed that 6-OHDA treatment for 24 hr decreased the viability of undifferentiated and differentiated immortalized DA neurons in vitro, as determined by the MTT assay, and increased the rate of apoptosis in differentiated DA neurons. The differentiated DA neurons (IC50 = 33 microM) were about 2-fold more sensitive to 6-OHDA than undifferentiated DA neurons (IC50 = 75 microM) in cell culture. Similarly, the differentiated DA neurons were more sensitive to another neurotoxin, 1-methyl-4-phenylpyridinium (MPP+), which is commonly used to induce Parkinsonism in animal models, than were the undifferentiated DA neurons in culture. Among growth factors tested, only glial cell line-derived neurotrophic factor (GDNF) partially protected differentiated DA neurons against 6-OHDA-induced toxicity. These results suggest that undifferentiated and differentiated immortalized DA neurons can be a useful experimental model to study relative sensitivity to neurotoxins and neuroprotective agents that could have relevance to fetal and adult neurons.
1-Methyl-4-phenylpyridinium↗
Yersinia pseudotuberculosis disseminates directly from a replicating bacterial pool in the intestine.
Dissemination of Yersinia pseudotuberculosis within mice after oral inoculation was analyzed. Y. pseudotuberculosis translocated to organs such as the liver and spleen shortly after oral inoculation, but was quickly cleared. In contrast, a second temporally distinct bacterial translocation event resulted in successful hepatosplenic replication of the bacteria. Replicating pools of bacteria could be established in these organs in mouse mutants that lacked Peyer's patches. These animals frequently had sterile mesenteric lymph nodes, a finding consistent with translocation taking place independently of regional lymph node colonization. In further contradiction to accepted models for dissemination of enteropathogens, clonal analysis revealed that bacteria causing disease in the spleen and liver of C57BL/6J mice were derived from populations located outside the intestinal lymph nodes. Replication of bacteria in the intestine before translocation appeared critical for dissemination, as transient selective suppression by streptomycin of bacterial growth in the intestine delayed dissemination of Y. pseudotuberculosis. These results collectively indicate that hepatosplenic colonization appears intimately connected with the ability of Y. pseudotuberculosis to successfully establish replication in the intestinal lumen and does not result from ordered spread leading from the intestine to regional lymph nodes before dissemination.
Animals↗
Natural selection in neoplastic progression of Barrett's esophagus.
Neoplasms progress to cancer through a process of natural selection. The rate of evolution, and thus progression is determined by three parameters: mutation rate, population size of the evolving neoplastic cells, and intensity of selection or rate of clonal expansion. All three parameters are reviewed in the context of Barrett's esophagus, a pre-malignant neoplasm. Although Barrett's esophagus is an ideal model for the study of neoplastic clonal evolution, similar studies may be carried out in a wide variety of human neoplasms. Evolutionary analyses provide insights for clinical management, including rates of progression to cancer and emergence of resistance to interventions.
Barrett Esophagus↗
Erythroid progenitor cells from pig bone marrow and peripheral blood.
With the intention of using the pig as a large animal model in haematopoietic research, a clonal assay in methylcellulose was developed and the optimal conditions for raising erythroid progenitors from adult pig bone marrow (BM) and peripheral blood (PB) have been established. Progenitor cells were stimulated to proliferate and differentiate in vitro by growth factors containing leucocyte condition medium (LCM), and with recombinant human erythropoietin (rhEpo). The number of PB BFU-E (burst forming units - erythroid) directly depended on the concentration of LCM, but BM BFU-E were not dependent on LCM. Both CFU-E (colony forming units - erythroid) and BFU-E were rhEpo dependent. Despite relatively high but expected individual variations, the mean number of colonies, as well as the functional characteristics of progenitor cells investigated, were similar to those of miniature pigs and some other mammals.
Animals↗
Phenytoin stimulates chondrogenic differentiation in mouse clonal chondrogenic EC cells, ATDC5.
Phenytoin (DPH) is known to affect bone formation. However, the mechanism of this effect has not been well understood. In this study, we evaluated the effects of DPH on cartilage formation in a model system using ATDC5 cells, a clonal murine chondrogenic cell line. Alcian blue staining for cartilage nodules and real-time reverse-transcription polymerase chain reaction for the expression of genes encoding type II collagen, aggrecan, transforming growth factor (TGF)-beta1, bone morphogenetic protein (BMP)-4, parathyroid hormone-related peptide (PTHrP), indian hedgehog (Ihh), and patched (Ptc) were performed in ATDC5 cells cultured with DPH. The ATDC5 cells demonstrated enhanced cartilage formation in cultures with DPH. During promoted chondrogenic differentiation, it was observed that DPH increased the mRNA expression of TGF-beta1, BMP-4, Ihh, and Ptc, in a dose-dependent manner on Days 5 to 15. In contrast, other antiepileptic drugs, phenobarbital and valproic acid had no effects on chondrogenesis in ATDC5 cells and osteogenesis in MC3T3-E1 cells. Our results provide fundamental evidence that DPH has a direct stimulatory effect on cartilage formation by regulating TGF-beta and hedgehog signaling molecules, and that DPH effect on bone formation, including chondrogenesis and osteogenesis, is distinct from other antiepileptic drugs as suggested in clinical settings.
3T3 Cells↗
Inference of bacterial microevolution using multilocus sequence data.
We describe a model-based method for using multilocus sequence data to infer the clonal relationships of bacteria and the chromosomal position of homologous recombination events that disrupt a clonal pattern of inheritance. The key assumption of our model is that recombination events introduce a constant rate of substitutions to a contiguous region of sequence. The method is applicable both to multilocus sequence typing (MLST) data from a few loci and to alignments of multiple bacterial genomes. It can be used to decide whether a subset of isolates share common ancestry, to estimate the age of the common ancestor, and hence to address a variety of epidemiological and ecological questions that hinge on the pattern of bacterial spread. It should also be useful in associating particular genetic events with the changes in phenotype that they cause. We show that the model outperforms existing methods of subdividing recombinogenic bacteria using MLST data and provide examples from Salmonella and Bacillus. The software used in this article, ClonalFrame, is available from http://bacteria.stats.ox.ac.uk/.
Bacteria↗
The heat shock proteins in cellular aging: is zinc the missing link?
T-cell functions are critical for the efficiency of the adaptive immune response. It is now clear that aging is associated with changes in the T-cell response to antigenic stimulation, one of the many changes collectively resulting in immune senescence. Several hypotheses have been proposed to explain such changes. We believe that chronic stimulation of T-cells enhances the appearance of apoptosis-resistant anergic dysfunctional cells; in humans in vivo these are predominantly specific for antigens of persistent viruses, especially CMV. Concomitantly, age-associated zinc deficiency is common and one hypothesis is that lack of zinc bioavailability contributes to impaired T-cell function. This could further compromise the integrity of T-cells under chronic antigenic stress, which can be modelled in long-term clonal cultures in vitro. Newly synthesized heat-shock proteins (HSPs) protect the cellular proteins from degradation under such conditions. In this short review we will briefly outline the role of heat-shock proteins and zinc deficiency in aging in order to finally discuss our own results in the context of a link between HSPs, aging and zinc.
Aging↗
Demonstration of hematopoietic stem cells in ginbuna carp (Carassius auratus langsdorfii) kidney.
A model system of ginbuna carp (clonal triploid fish, Carassius auratus langsdorfii) and ginbuna-goldfish hybrids (tetraploid fish, having three sets of chromosomes from a triploid clone and a haploid set of chromosomes from goldfish) was employed to demonstrate the presence of hematopoietic stem cells (HSCs) in kidney hematopoietic tissues. Kidney hematopoietic cells were obtained from ginbuna carp (S3n, donor) and injected into a ginbuna-gold fish hybrid (S4n, recipient) via the caudal sinus. The percentage of S3n cells in the S4n blood was measured by flow cytometry by the difference of DNA content from 3 to 18 months after the injection. Donor-derived major types of blood cells including erythrocytes, thrombocytes, neutrophils, basophils, monocytes, T and B lymphocytes were detected in the recipient blood over a long period (9-18 month posttransplantation). Moreover, recipient lympho-hematopoietic organs such as head kidney, body kidney, spleen, and thymus were reconstituted by donor cells. These results indicate that long-term multipotent repopulating HSCs reside in the donor kidney.
Acridine Orange↗
An immunohistochemical study of p16(INK4a) expression in multistep thyroid tumourigenesis.
Normal human thyroid follicular epithelial cells exhibit a very low proliferative rate which in vitro is dramatically increased by RAS oncogene activation, resulting in clones displaying a phenotype consistent with that of a ras-induced follicular adenoma in vivo. Eventual spontaneous cessation of growth of these clones is closely correlated with increasing expression of the tumour suppressor gene p16(INK4a), suggesting that p16 may limit clonal expansion in this tumour model. We therefore hypothesised that p16 expression would also increase in vivo in follicular adenomas, and further that escape from growth control in follicular cancers would be accompanied by loss of p16 expression. This was tested using tissue microarrays, representing multiple stages of thyroid tumourigenesis. Whereas the majority of normal thyroids showed no immunostaining, p16 protein was readily detectable in follicular adenomas. Unexpectedly, however, p16 expression was also observed in follicular and papillary carcinomas. Poorly differentiated (insular) carcinomas showed either very intense staining, or a complete loss of staining. We conclude that loss of p16 is not necessary for malignant transformation in thyroid follicular cells, but that it may form one of two or more events needed for progression to more aggressive forms of thyroid cancer.
Adenoma↗
A computational method for inferring growth parameters and shape changes during development based on clonal analysis.
We describe a method for estimating growth parameters in various regions of a developing organ undergoing cell divisions, along with the corresponding changes in organ shape. Growth parameters are computed by coupling clonal analysis with a growth model, allowing a wide range of developmental stages to be covered. The method was applied to the development of dorsal petal lobes of Antirrhinum majus. The resulting description of growth patterns and shape changes is consistent with direct observations using scanning electron microscopy. This method can potentially be applied to other organs, and opens the way to comparative studies of growth and gene expression patterns.
Antirrhinum↗
Ionotropic glutamate receptors expressed in human retinoblastoma Y79 cells.
Mammalian retinal photoreceptors and pinealocytes have common characteristic that they secrete melatonin and L-glutamate as chemical transmitters. Although pinealocytes express glutamate receptors and receive glutamate signals, whether or not photoreceptors express glutamate receptors is unknown. Here, we investigated the expression of the glutamate receptors in cultured Y79 clonal human retinoblastoma cells, as model systems of photoreceptors. Reverse transcription-polymerase chain reaction (RT-PCR) analysis indicated that GluR1, GluR5, GluR7, EAA2, NR1, NR2A and NR2D mRNAs were present in the cultured cells. Northern analysis confirmed the presence of GluR7, EAA2, NR1, NR2A and NR2D mRNAs, while other mRNAs were under the detection limit. Addition of (RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid or kainate increases intracellular (Ca(2+)) in Fura-2 loaded cells, which is blocked by 6-cyano-7-nitroquinoxaline-2,3-dione. N-methyl-D-aspartate also increases intracellular (Ca(2+)). These results demonstrated the presence of functional ionotropic receptors in Y79 cells.
6-Cyano-7-nitroquinoxaline-2,3-dione↗
In vitro and in vivo inhibition of SK-N-MC neuroblastoma growth using cyclic nucleotide phosphodiesterase inhibitors.
The effect of cyclic nucleotide phosphodiesterase (PDE) inhibitors Zaprinast and DC-TA-46 has been tested on SK-N-MC neuroblastoma growth. Antiproliferative activity of the tested drugs was assayed both in vitro and in the xenograft model of nude mice. In clonal density experiments, the IC50 value was 3.3 microM for Zaprinast and 1.9 microM for DC-TA-46, while 7.5 microM BCNU alkylating agent was required to obtain the same effect. SK-N-MC cells xenografted in the nude mouse showed that the administration of Zaprinast and DC-TA-46 caused a significant 50% decrease of the tumour weight. These data demonstrate that PDE inhibitors may be useful for at least reducing tumour growth; they may be of interest for further evaluation as alternative molecules in the design of multiple agent protocols for neuroblastoma treatment.
3',5'-Cyclic-AMP Phosphodiesterases↗
Cross-linking of surface IgM in the Burkitt's lymphoma cell line ST486 provides protection against arsenite- and stress-induced apoptosis that is mediated by ERK and phosphoinositide 3-kinase signaling pathways.
The ST486 cell line, derived from a human Burkitt's lymphoma, is a model for antigen-induced clonal deletion in germinal center B-lymphocytes, with apoptosis induced upon cross-linking of surface IgM. Moreover, this cell line is highly sensitive to the induction of apoptosis by many chemicals, including sodium arsenite, a significant environmental contaminant with immunotoxic activity. In contrast to arsenite and other chemicals, surface IgM cross-linking induces apoptosis in ST486 cells with delayed kinetics. Moreover, the initial signaling events following IgM stimulation are associated with cell survival and proliferation and include activation of the extracellular-signal regulated kinase (ERK) and the phosphoinositide 3-kinase (PI3K) pathways. We examined the question of whether IgM-mediated activation of the ERK and PI3K pathways can influence the apoptotic response of ST486 cells following exposure to arsenite and selected drugs with different molecular targets, including cycloheximide, etoposide, and camptothecin, and a physical stress, hyperthermia. Our findings show that IgM-stimulated cells are significantly protected against arsenite and drug-induced apoptosis during a window of several hours after surface IgM cross-linking, as evidenced by an inhibition of cleavage of poly(ADP-ribose) polymerase and lack of morphological changes indicative of apoptosis. Significantly, surface IgM cross-linking also protects against arsenite-induced mitochondrial depolarization as well as caspase-9 cleavage. Furthermore, we demonstrate that this IgM-mediated protection requires the activation of the ERK and PI3K pathways, because inhibition of either pathway blocks the ability of antigen receptor activation to protect against apoptosis. Our study also provides evidence for p90(S6) ribosomal kinase as a point of convergence between the two signaling pathways resulting in the phosphorylation of the pro-apoptotic Bcl-2 family member Bad at serine 112. This investigation demonstrates, for the first time, that specific signals transduced by activation of the B-cell receptor protect cells at a common point of regulation in the apoptotic pathways for diverse stresses.
Apoptosis↗
Heterogeneous radiation and heat sensitivity in vitro of human melanoma xenograft lines established from different lesions in the same patient. Comparisons with the radiation and heat sensitivity of cells isolated from the donor patient's surgical specimens.
Human melanoma xenograft lines were established in athymic nude mice (BALB/c-nu/nu/BOM) from the primary tumour (OKL-PRI), a s.c. metastasis (OKL-SCM) and a lymph node metastasis (OKL-LNM) in the same patient. The three lines differed in growth rate, melanin content, and radiation and heat sensitivity in vitro. The OKL-PRI line grew more slowly than the OKL-SCM and OKL-LNM lines and was the only line that synthesized significant amounts of melanin. The D0 values were 0.96 +/- 0.07 Gy, 0.87 +/- 0.07 Gy and 1.52 +/- 0.09 Gy (X-rays); 143 +/- 21 min, 109 +/- 12 min and 195 +/- 40 min (heat, 42.5 degrees C); and 21.3 +/- 2.7 min, 15.3 +/- 1.7 min and 26.7 +/- 3.0 min (heat, 44.5 degrees C) for the OKL-PRI, OKL-SCM and OKL-LNM line, respectively. The ranking of the lines in treatment sensitivity was equal for radiation and heat. The radiation and heat sensitivities were similar to those for cells isolated directly from the surgical specimens of the donor patient. The lines were thus established from a single neoplastic disease without artificial cloning in vitro or in vivo, and the cellular radiation and heat sensitivity did not change during the establishment procedure, suggesting that they constitute a relevant experimental model system for studies of clonal tumour heterogeneity in response to radiation and hyperthermia treatments.
Acclimatization↗
Receptors for monoamines on cultured cells.
Clonal cell lines are ideal model systems for the study of monoamine receptors. Cell lines that express dopamine and serotonin receptors have been isolated from pituitary tumor 7315a. The SUP1 cell line contains mRNA for the dopamine D-2 receptor and expresses D-2 receptors coupled to the inhibition of cAMP production and to the inhibition of prolactin secretion. The P11 cell line expresses 5-HT2 receptors coupled to the stimulation of phosphoinositide hydrolysis. These cell lines will be used to investigate manipulations that affect the expression and function of dopamine and serotonin receptors.
Adenylyl Cyclase Inhibitors↗