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PubMed2000

Estimation problems associated with stochastic modeling of proliferation and differentiation of O-2A progenitor cells in vitro.

Our previous research effort has resulted in a stochastic model that provides an excellent fit to our experimental data on proliferation and differentiation of oligodendrocyte type-2 astrocyte progenitor cells at the clonal level. However, methods for estimation of model parameters and their statistical properties still remain far away from complete exploration. The main technical difficulty is that no explicit analytic expression for the joint distribution of the number of progenitor cells and oligodendrocytes, and consequently for the corresponding likelihood function, is available. In the present paper, we overcome this difficulty by using computer-intensive simulation techniques for estimation of the likelihood function. Since the output of our simulation model is essentially random, stochastic optimization methods are necessary to maximize the estimated likelihood function. We use the Kiefer-Wolfowitz procedure for this purpose. Given sufficient computing resources, the proposed estimation techniques significantly extend the spectrum of problems that become approachable. In particular, these techniques can be applied to more complex branching models of multi-type cell systems with dependent evolutions of different types of cells.

Animals↗
PubMed1991

Major histocompatibility complex controls clonal proliferation of CD5+ B cells in H-2-congenic New Zealand mice: a model for B cell chronic lymphocytic leukemia and autoimmune disease.

By employing H-2-congenic NZB, NZW and (NZB x NZW)F1 mice with either the homozygous H-2d/H-2d, H-2z/H-2z or heterozygous H-2d/H-2z haplotype, we found that in the spleen of all the congenic strains homozygous for H-2z, there were extremely high frequencies of CD5+ B cells. These cells eventually proliferated in an oligoclonal or even monoclonal fashion, and B cell-chronic lymphocytic leukemia (B-CLL) developed in some cases. Because this feature was not observed in H-2d/H-2d homozygotes or H-2d/H-2z heterozygotes, the high CD5+ B cell frequencies are apparently controlled by the homozygosity of a locus or cluster of loci closely linked to H-2z complex of NZW strain. As the CD5+ B cell frequencies in the peritoneal cavity did not differ among the H-2-congenic strains, the frequencies of these cells in the peritoneal cavity and in the spleen appear to be at least in part under separate control. Flow cytometry and Southern blot analyses using an immunoglobulin gene JH probe revealed that the H-2z/H-2z homozygotes, there was a propagation of distinct clonal populations between the spleen and the peritoneal cavity, a finding which suggested that in the major histocompatibility complex (MHC)-related microenvironments for CD5+ B cell propagation differ between the two compartments. All our findings taken together imply that certain different but related MHC haplotypes may predispose either to B-CLL or to autoimmune disease, in close relatives.

Animals↗
PubMed2003

Generation of a transgenic animal model of hyperthyroid Graves' disease.

Graves' disease (GD) is an organ-specific autoimmune disease characterized by hyperthyroidism. Agonistic anti-thyrotropin receptor antibodies (thyroid-stimulating antibodies, TSAb), which mimic the thyrotropin (TSH) action, are thought to cause GD. The precise immunological mechanism of TSAb production, however, remains elusive. Previous immunization approaches using TSH receptor led to transient hyperthyroidism, but did not seem sufficient for comprehensive understanding of the development of autoimmune responses. To create GD-related autoimmunity in mice, we here generated TSAb-transgenic mice in which a patient-derived TSAb is expressed in B cells. Expression of the human TSAb in mice resulted in various manifestations of hyperthyroidism including increased free thyroxine levels with concomitantly decreased TSH levels, increased thyroid uptake of technetium pertechnetate, hyperthermia and thyroid hyperplasia. We found a correlation between the serum levels of human TSAb immunoglobulin and free thyroxine. In addition, conventional B cells expressing the TSAb were partially deleted in the periphery while B1 cells expressing the TSAb persisted and accumulated in the peritoneal cavity, a finding consistent with previous demonstrations that the maintenance of B1 cells plays an important role in the development of autoimmune diseases. Thus, our transgenic mouse may provide a novel and useful animal model for elucidating the pathogenesis and pathophysiology of GD.

Animals↗
PubMed1992

Clonal hybrids of the common laboratory fish Fundulus heteroclitus.

All-female hybrids of the killifishes Fundulus heteroclitus and Fundulus diaphanus, known from two sites in Nova Scotia, Canada, are shown to reproduce clonally. Isozyme analysis of crosses between female hybrids and male F. heteroclitus reveals that their progeny are genetically identical and show no evidence of recombination or paternal inheritance. Flow cytometric measurement of DNA content shows the hybrids to be diploid, with DNA values intermediate to those of the parental species. Because they are related to F. heteroclitus, a fish used widely as a model organism in experimental biology, the clonal hybrids are potentially valuable for experimental studies requiring subjects with a constant genetic background. In addition, the discovery of unisexuality and cloning in a fish whose reproductive physiology and development are so well characterized provides a unique opportunity to examine the underlying causes of clonal reproduction in vertebrates.

Alleles↗
PubMed1997

Establishment and characterization of a human stroma-dependent myeloma cell line (MM5.1) and its stroma-independent variant (MM5.2).

Although IL-6 has been identified as a major growth factor in multiple myeloma (MM), it is believed that maintenance of tumor growth in vivo depends on one or more additional stroma-derived factors. We describe a new human myeloma cell line (MM5.1) that can be maintained in the presence of bone marrow-derived stromal cell layers, and not only when cultured with exogeneous IL-6. This cell line expresses the same immunoglobulin kappa light chain RNA sequence as the patient's original tumor cells, has a plasma cell morphology and expresses plasma cell antigens (cytoplasmic kappa light chain, CD38, BB4). Without the presence of stromal factors, MM5.1 cells become apoptotic. A low proliferative effect was observed in the presence of oncostatin M (OSM) but other cytokines (IL-10, IL-11, stem cell factor (SCF) and leukemia inhibitory factor (LIF)) had no effect at all. We observed that MM5.1 cells also grow when physically separated from stromal cell layers by a 0.45 microm microporous membrane or when cultured in conditioned medium from stromal marrow cells. Unexpectedly, the growth in stromal supernatants was markedly inhibited by an anti-IL-6 antiserum and an anti-IL-6 receptor transducer chain (gp130) mAb in a dose-dependent manner. This implies that MM5.1 cells are IL-6 responsive only when exposed to one or more additional soluble factor(s) derived from bone marrow stroma. Coculturing MM5.1 cells with IL-6 and cytokines that were described to increase the IL-6 responsiveness of myeloma cells (G-CSF, GM-CSF and IL-3) had no effect on the growth or survival. A strong proliferative effect was observed when MM5.1 cells were cultured with IL-6 and soluble IL-6 receptor (sgp80). However no sgp80 could be detected in stromal supernatants using a sensitive immunoassay. This indicates that sustained proliferation of the MM5.1 cell line depends on a combination of IL6 and at least one, thus far unidentified, stroma-derived factor. After more than 1 year in continuous culture, we could obtain a variant of the line (MM5.2) that shows an improved growth rate and grows stroma independently. Molecular analysis revealed clonal identity with the early passage form and Epstein-Barr virus antigen expression was negative. The two variants of this cell line offer a useful model to identify molecular mechanisms involved in clonal evolution towards stroma-independent growth of myeloma cells.

Adipose Tissue↗
PubMed2002

Immunobiology and long-term graft function in a transplant heterotopic renal rat model.

BACKGROUND: The Th1-Th2 paradigm proposes clonal expansion of Th2 lymphocytes as the basis of allograft tolerance. The Th2 cells have been found to be present in recipients with long-term allograft survival. However, the presence of Th2 cells and tolerance is not a uniform finding. Previously we have shown that pre-engraftment single dose rapamycin and a 7-d course of cyclosporin induce transplantation tolerance to 120 d. In the present study, we investigated the immunobiology of grafts in a long-term follow-up (>350 d). METHODS: Kidney allografts (n = 7), isografts (n = 5) and single nephrectomy (n = 3) groups were followed for 350 +/- 87 d. Heterotopic kidney transplant was performed by the same surgeon in the allograft group (ACI-Lewis) and the isograft group (Lewis-Lewis). The left kidney was removed in the single nephrectomy group. The allograft group was treated with pre-engraftment single dose rapamycin and a 7-d course of cyclosporin. A kidney biopsy was performed at midpoint time for histological study and tissue was frozen for measuring intragraft cytokine expression (IL-4, IL-10) in all animals. Prior to biopsy, serum blood urea nitrogen (BUN) and creatinine (Cr) levels were studied. Serum BUN, Cr levels, plus 24-h urinary protein (PRO) were measured prior to sacrifice. Randomly, four allograft rats received skin grafts (ACI, Lewis and Buffalo skin donors) after kidney biopsy. Skin grafts were studied for a mean of 6 weeks for signs of acceptance or rejection. Analysis of variance (ANOVA) with Tukey's test was used; p < 0.05 was considered statistically significant. RESULTS: The mean follow up was 352 +/- 87 d. BUN and Cr levels at biopsy time (mean 214 d) were not statistically different between the three groups (p = 0.19 and p = 0.66). At sacrifice (mean 352 d), BUN, Cr and PRO were statistically different between allograft and isograft groups (p = 0.013), and between allograft and single nephrectomy groups (p = 0.027). Functional and histological signs of graft loss occurred in three of seven (42.8%) of the allografts at 352 d. Using BANFF criteria, three allografts at biopsy time and seven allografts (100%) and four isografts (80%) at sacrifice time developed chronic histologic changes. Intragraft overexpression of IL-4 and IL-10 was seen at biopsy and sacrifice time in six of seven allografts and one of five isografts. All donor specific skin grafts (ACI-Lewis) on allografts were accepted and third party (Buffalo) donor skin grafts were rejected in all animals (>95% skin necrosis). CONCLUSIONS: This highly stringent, functional, renal transplant model yields 100% normal renal function as compared with isografts at 120 d follow-up. With the follow-up extended to 350 d, 43% of the allografts loose function and develop a chronic allograft histology despite a demonstrated intragraft Th2 cytokine dominance and donor specific skin graft acceptance.

Animals↗
PubMedDate not supplied

Immunophenotyping of lymphocytes in the lung interstitium and expression of osteopontin and interleukin-2 mRNAs in two different murine models of pulmonary fibrosis.

In the surfactant protein C/tumor necrosis factor (SP-C/TNF) transgenic mouse, the TNF-alpha transgene is overexpressed in type II pneumocytes. Pulmonary lymphocytic infiltration develops which is followed by fibrotic changes including accumulation of fibroblasts and deposition of extracellular matrix. We hypothesized that lymphocytes played a role in the development of pulmonary fibrosis in this model. Lymphocytes were recovered from the interstitium of the lung and analyzed by flow cytometry. The absolute number of lymphocytes recovered from transgenic mice were approximately four times of that in littermates. Flow cytometric analysis showed the presence of gamma delta T cells and B1 cells in the former group but these cells were almost absent in the lung of non-transgenic littermates. We also studied lymphocytes accumulating in the lung during bleomycin (BLM)-induced pneumopathy. Serial analyses showed a progressive increase of CD4/CD8 ratio after injection of BLM, reaching a peak at day 14, then decreased to the normal level by day 48. Northern blot analysis of the lung showed an enhanced expression of interleukin (IL)-2 and osteopontin (OPN) mRNA in those two models of pulmonary fibrosis. Expansion of clonal alpha beta T cells as detected by reverse transcriptase-polymerase chain reaction/single strand conformation polymorphism (RT-PCR/SSCP) suggests involvement of antigen-driven mechanisms in the development of pulmonary fibrosis.

Animals↗
PubMed1998

Regulation of the effector stages of experimental autoimmune encephalomyelitis via neuroantigen-specific tolerance induction. III. A role for anergy/deletion.

Our previous work has shown that specific peripheral immune tolerance induced by the intravenous administration of ECDI-fixed, antigen-coupled syngeneic splenocytes is an extremely efficient method for prevention and treatment of chronic relapsing experimental autoimmune encephalomyelitis (R-EAE) in susceptible SJL/J mice. The current study examined the mechanisms by which unresponsiveness is induced in primed encephalitogenic T cells. The results indicate that the inhibition of MBP-specific T cells by the i.v. injection of MBP-coupled splenocytes is not due to the induction of antigen-specific regulatory T cells, but rather to the induction of anergy/deletion of the effector cells. This conclusion is supported by the findings that spleen or lymph node cells isolated from MBP-tolerant mice fail to inhibit the adoptive transfer of R-EAE in cotransfer assays, and that tolerance is not inhibited by prior thymectomy or prior treatment with cyclophosphamide or anti-CD8 monoclonal antibody. In contrast, we demonstrate that splenocytes from MBP-tolerized, asymptomatic mice have a significantly reduced ability to serially transfer R-EAE to naive secondary recipients following antigen re-activation in vitro, in the first several weeks following tolerization, but that the ability to serially transfer R-EAE returns to sham tolerant control levels within 1-2 months. We also demonstrate a significantly reduced precursor frequency of MBP-specific, IL-2-producing T cells in the MBP-tolerant within three days of treatment. Collectively, the data most closely support a model wherein inhibition of MBP-specific encephalitogenic CD4+ effector T cells by i.v. injected MBP-coupled splenocytes is due to the direct induction of anergy/deletion from which they can recover over time.

Adoptive Transfer↗
PubMed2005

Prediction of penicillin resistance in Staphylococcus aureus isolates from dairy cows with mastitis, based on prior test results.

AIM: To gauge how well prior laboratory test results predict in vitro penicillin resistance of Staphylococcus aureus isolates from dairy cows with mastitis. METHODS: Population-based data on the farm of origin (n=79), genotype based on pulsed-field gel electrophoresis (PFGE) results, and the penicillin-resistance status of Staph. aureus isolates (n=115) from milk samples collected from dairy cows with mastitis submitted to two diagnostic laboratories over a 6-month period were used. Data were mined stochastically using the all-possible-pairs method, binomial modelling and bootstrap simulation, to test whether prior test results enhance the accuracy of prediction of penicillin resistance on farms. RESULTS: Of all Staph. aureus isolates tested, 38% were penicillin resistant. A significant aggregation of penicillin-resistance status was evident within farms. The probability of random pairs of isolates from the same farm having the same penicillin-resistance status was 76%, compared with 53% for random pairings of samples across all farms. Thus, the resistance status of randomly selected isolates was 1.43 times more likely to correctly predict the status of other isolates from the same farm than the random population pairwise concordance probability (p=0.011). This effect was likely due to the clonal relationship of isolates within farms, as the predictive fraction attributable to prior test results was close to nil when the effect of within-farm clonal infections was withdrawn from the model. CONCLUSIONS: Knowledge of the penicillin-resistance status of a prior Staph. aureus isolate significantly enhanced the predictive capability of other isolates from the same farm. In the time and space frame of this study, clinicians using previous information from a farm would have more accurately predicted the penicillin-resistance status of an isolate than they would by chance alone on farms infected with clonal Staph. aureus isolates, but not on farms infected with highly genetically heterogeneous bacterial strains.

Animals↗
PubMed2005

Control of genotypic allelic inclusion through TCR surface expression.

To gain insight into the molecular causes and functional consequences of allelic inclusion of TCR alpha-chains, we develop a computational model for thymocyte selection in which the signal that determines cell fate depends on surface expression. Analysis of receptor pairs on selected dual TCR cells reveals that allelic inclusion permits both autoreactive TCR and receptors not in the single TCR cell repertoire to be selected. However, in comparison with earlier theoretical studies, relatively few dual TCR cells display receptors with high avidity for thymic ligands because their alpha-chains compete aggressively for the beta-chain, which hinders rescue from clonal deletion. This feature of the model makes clear that allelic inclusion does not in itself compromise central tolerance. A specific experiment based on modulation of TCR surface expression levels is proposed to test the model.

Alleles↗
PubMed1996

Critical determinants of melanoma metastasis.

The process of metastasis consists of a series of linked, sequential steps. Although some of the steps in this process contain stochastic elements, metastasis as a whole favors the survival and growth of a few subpopulations of cells that preexist within the parent neoplasm. Metastases can have a clonal origin, and different metastases can originate from the proliferation of single cells. The outcome of metastasis depends on the interaction of metastatic cells with various host factors. Organ-specific metastases have been demonstrated in a variety of tumors and may even be specific to a particular site within one organ. Studies from our laboratory and from others have shown that the implantation of human cancer cells derived from surgical specimens into correct anatomic sites of nude mice provides a suitable model of metastasis of human tumors. Clonal analysis of human melanomas revealed that these tumors are heterogeneous for metastatic properties and that growth in the environment of specific organs is selective. These data suggest that systemic physiologic signals can be recognized by neoplastic cells, presumably by mechanisms similar to those shared by their normal cell counterparts. Elucidation of the mechanisms that regulate metastasis should lead to better therapeutic interventions.

Animals↗
PubMed2000

Immune selection in neoplasia: towards a microevolutionary model of cancer development.

The dual properties of genetic instability and clonal expansion allow the development of a tumour to occur in a microevolutionary fashion. A broad range of pressures are exerted upon a tumour during neoplastic development. Such pressures are responsible for the selection of adaptations which provide a growth or survival advantage to the tumour. The nature of such selective pressures is implied in the phenotype of tumours that have undergone selection. We have reviewed a range of immunologically relevant adaptations that are frequently exhibited by common tumours. Many of these have the potential to function as mechanisms of immune response evasion by the tumour. Thus, such adaptations provide evidence for both the existence of immune surveillance, and the concept of immune selection in neoplastic development. This line of reasoning is supported by experimental evidence from murine models of immune involvement in neoplastic development. The process of immune selection has serious implications for the development of clinical immunotherapeutic strategies and our understanding of current in vivo models of tumour immunotherapy.

Adaptation, Physiological↗
PubMed1994

Clonal expansion of T lymphocytes causes arthritis and mortality in mice infected with toxic shock syndrome toxin-1-producing staphylococci.

Erosive arthritis is a common and feared complication of staphylococcal infection. The reason(s) for the progressive course of the arthritis is unknown. It has been recently established that enterotoxins produced by Staphylococcus aureus display superantigen properties leading to stimulation of T cells carrying distinct T cell receptor V beta elements. This finding provides a potential connection between staphylococcal exoproteins and endogenous immune mechanisms participating in the infectious process. We have recently describe successful induction of infections arthritis in mice after intravenous inoculation of a toxic shock syndrome toxin-1 (TSST-1)-producing S. aureus LS-1 strain. Using this model we have now found a clonal expansion of T cells expressing V beta 11+ T cell receptor in the synovial tissue of arthritic mice. The role of TSST-1 as a superantigen inducing oligoclonal expansion was confirmed in an in vitro culture system. The expansion of V beta 11+ T cells proved to be of arthritogenic significance since mice genomically deleted of the V beta 11+ T cells did not develop arthritis and since pretreatment of healthy mice with anti-CD4 or anti-V beta 11 monoclonal antibodies inhibited arthritis. In addition, CD4+ and V beta 11+ T cells showed themselves to be of pathogenic significance in staphylococcal-induced mortality, since mice depleted of such populations showed increased survival. We propose that in hematogenously spread S. aureus-induced arthritis the TSST-1-dependent clonal expansion of CD4+ V beta 11+ T cells is a driving pathogenic force.

Animals↗
PubMed1994

In favor of the selective model of positive selection.

The mechanisms of thymocyte commitment towards the CD4+ and CD8+ lineage remain unresolved. Two models--one based on instruction, the other on selection--have previously been proposed. The instructional model has been popularly received based on results of earlier studies. However, our data from MHC class II, class I, and double-deficient mice suggest otherwise. There exists a significant population of CD4+ cells that is intermediate in maturity between CD4+ CD8+ and fully mature CD4+ CD8- thymocytes in class II-deficient animals; an analogous population of CD4-CD8+ cells exists in class I-negative mice. We suggest that a selective model in which two TCR-MHC molecule engagements are required: the first induces a random down-modulation of either CD4 or CD8 and some differentiation; the second, involving the participation of the appropriate coreceptor, permits end-stage differentiation.

Animals↗
PubMed1995

Use of tissue culture models to study environmental-genetic interactions relevant to neurodegenerative diseases.

1. Clonal cell lines, primary cultured neurones and transgenic animals expressing mutant genes linked to familial forms of neurodegenerative diseases provide models in which to examine the interaction between expression of a predisposing gene and exposure to neurotoxic chemicals. Methods of establishing these models are reviewed. 2. Mutations in the gene encoding Cu/Zn-superoxide dismutase (SOD-1) have been identified in cases of familial amyotrophic lateral sclerosis linked to chromosome 21. We report that in clonal lines of PC12 cells, the cytotoxicity of a glutathione-depleting epoxide, styrene oxide, varied with SOD activity in a manner similar to that previously demonstrated for redox cycling chemicals. These preliminary data suggest that either low or high SOD-1 activities may be associated with greater toxicity of a variety of neurotoxic chemicals and their metabolites.

Amyotrophic Lateral Sclerosis↗
PubMed1990

Critical factors in the biology of human cancer metastasis: twenty-eighth G.H.A. Clowes memorial award lecture.

The process of metastasis is not random. Rather, it consists of a series of linked, sequential steps that must be completed by tumor cells if a metastasis is to develop. Although some of the steps in this process contain stochastic elements, as a whole, metastasis favors the survival and growth of a few subpopulations of cells that preexist within the parent neoplasm. Moreover, metastases can have a clonal origin, and different metastases can originate from the proliferation of single cells. The outcome of metastasis depends on the interaction of metastatic cells with different organ environments. Organ-specific metastases have been demonstrated in a variety of experimental tumor systems. Moreover, we have found tumor growth that is specific to a particular site within one organ. Whether the same conclusions can be reached for human cancers remained unanswered until very recently. Studies from our laboratory and from others have shown that the implantation of human cancer cells derived from surgical specimens into correct anatomical sites of nude mice can provide a suitable model of metastasis of human tumors. Clonal analysis of a human renal carcinoma, colon carcinomas, and melanomas has revealed that these tumors are indeed heterogeneous for metastatic properties, an observation made only after orthotopic implantation. Thus, growth in the environment of specific organs can be selective and the environment per se influences this process. While it is clear that vascularity and local immunity can facilitate or retard tumor growth, we have concentrated on understanding how damage to an organ and the subsequent repair process can facilitate tumor cell proliferation. Accelerated growth of human colon cancer cells was found in hepatectomized nude mice, whereas accelerated growth of human renal cancer cells was found in nephrectomized nude mice. These data suggest that systemic physiological signals can be recognized by neoplastic cells presumably by mechanisms similar to those shared by their normal cell counterparts. In summary, the critical factors that regulate metastasis are the intrinsic properties of metastatic cells and host factors involved in homeostasis. The recent increase in our understanding of metastasis should provide important leads for developing more effective approaches to the treatment of disseminated cancer.

Animals↗
PubMed2006

Dual isotype expressing B cells [kappa(+)/lambda(+)] arise during the ontogeny of B cells in the bone marrow of normal nontransgenic mice.

Central to the clonal selection theory is the tenet that a single B cell expresses a single receptor with a single specificity. Previously, based on our work in anti-phosphocholine transgenic mouse models, we suggested that B cells escaped clonal deletion by coexpression of more than one receptor on their cell surface. We argued that "receptor dilution" was necessary when: (i) the expressed immunoglobulin receptor is essential for immune protection against pathogens and (ii) this protective receptor is autoreactive and would be clonally deleted, leaving a hole in the B cell repertoire. Here, we demonstrate that dual isotype expressing B cells arise during the normal ontogeny of B cells in the bone marrow and populate both the spleen and peritoneal cavity of nontransgenic mice. Furthermore, single cell analysis of the expressed immunoglobulin light chains suggests that receptor editing may play a role in the generation of a significant fraction of dual isotype expressing B cells.

Animals↗