Experimental murine and primate models for dissection of the immunosuppressive potential of photochemotherapy in autoimmune disease and transplantation

This paper reviews the results achieved in murine and primate models of autoimmune disease and transplantation. These studies have attempted to clarify the nature and specificity of the response induced by reinfusion of phototreated immunoactive lymphocytes. Results obtained in murine lupus have demonstrated that some of the disease features related to the abnormal proliferation of inducer T cells can be inhibited both prophylactically and therapeutically by exposure to photoinactivated autoimmune splenocytes. Radiolabeling studies performed in normal syngeneic mice have shown that, if immunoactive cells are phototreated and injected, their recirculation pattern is altered, and increased sequestration in the spleen, bone marrow, and kidney is noted. These studies suggest that reinfused, phototreated, antigen-activated lymphocytes may localize in sites where they are available for induction of immune responses. Primate cardiac xenotransplantation models have demonstrated that reinfusion of phototreated autologous leukocytes, administered with cyclosporine A and steroids, mediates enhanced specific suppression of both the cellular and humoral host response to foreign tissue. Taken as a whole, the experimental models suggest that photopheresis may provide a means of inducing specific suppression of immunoactive T cells. This form of therapy may have a role as an immunosuppressive agent in both autoimmune disease and transplantation.     

Experimental analysis and modelling of in vitro HUVECs proliferation in the presence of various types of drugs

Objectives: This study focuses on experimental analysis and corresponding mathematical simulation of in vitro HUVECs (human umbilical vein endothelial cells) proliferation in the presence of various types of drugs. Materials and methods: HUVECs, once seeded in Petri dishes, were expanded to confluence. Temporal profiles of total count obtained by classic haemocytometry and cell size distribution measured using an electronic Coulter counter, are quantitatively simulated by a suitable model based on the population balance approach. Influence of drugs on cell proliferation is also properly simulated by accounting for suitable kinetic equations. Results and discussion: The models’ parameters have been determined by comparison with experimental data related to cell population expansion and cell size distribution in the absence of drugs. Inhibition constant for each type of drug has been estimated by comparing the experimental data with model results concerning temporal profiles of total cell count. The reliability of the model and its predictive capability have been tested by simulating cell size distribution for experiments performed in the presence of drugs. The proposed model will be useful in interpreting effects of selected drugs on expansion of readily available human cells.     

The use of automated parameter searches to improve ion channel kinetics for neural modeling

The voltage and time dependence of ion channels can be regulated, notably by phosphorylation, interaction with phospholipids, and binding to auxiliary subunits. Many parameter variation studies have set conductance densities free while leaving kinetic channel properties fixed as the experimental constraints on the latter are usually better than on the former. Because individual cells can tightly regulate their ion channel properties, we suggest that kinetic parameters may be profitably set free during model optimization in order to both improve matches to data and refine kinetic parameters. To this end, we analyzed the parameter optimization of reduced models of three electrophysiologically characterized and morphologically reconstructed globus pallidus neurons. We performed two automated searches with different types of free parameters. First, conductance density parameters were set free. Even the best resulting models exhibited unavoidable problems which were due to limitations in our channel kinetics. We next set channel kinetics free for the optimized density matches and obtained significantly improved model performance. Some kinetic parameters consistently shifted to similar new values in multiple runs across three models, suggesting the possibility for tailored improvements to channel models. These results suggest that optimized channel kinetics can improve model matches to experimental voltage traces, particularly for channels characterized under different experimental conditions than recorded data to be matched by a model. The resulting shifts in channel kinetics from the original template provide valuable guidance for future experimental efforts to determine the detailed kinetics of channel isoforms and possible modulated states in particular types of neurons.     

Phospholipase C-delta extends intercellular signalling range and responses to injury-released growth factors in non-excitable cells

Abstract.   Objectives : Intercellular communication in non-excitable cells is restricted to a limited range close to the signal source. Here, we have examined whether modification of the intracellular microenvironment could prolong the spatial proposition of signal generation and could increase cell proliferation . Material and methods : Mathematical models and experimental studies of endothelial repair after controlled mechanical injury were used. The models predict the diffusion range of injury-released growth factors and identify important parameters involved in a signalling regenerative mode. Transfected human umbilical vein endothelial cells (HUVECs) were used to validate model results, by examining intercellular calcium signalling range, cell proliferation and wound healing rate. Results : The models predict that growth factors have a limited capacity of extracellular diffusion and that intercellular signals are specially sensitive to cell phospholipase C-delta (PLCδ) levels. As basal PLCδ levels are increased by transfection, a significantly increased intercellular calcium range, enhanced cell proliferation, and faster wound healing rate were observed. Conclusion : Our in silico and in vitro studies demonstrated that non-excitable endothelial cells respond to stimuli in a complex manner, in which intercellular communication is controlled by physicochemical properties of the stimulus and by the cell microenvironment. Such findings may have profound implications for our understanding of the tight nature of autocrine cell growth control, compensation to stress states and response to altered microenvironment, under pathological conditions.     

Comparison of analytical and inverse finite element approaches to estimate cell viscoelastic properties by micropipette aspiration

The viscoelastic properties of cells are important in predicting cell deformation under mechanical loading and may reflect cell phenotype or pathological transition. Previous studies have demonstrated that viscoelastic parameters estimated by finite element (FE) analyses of micropipette aspiration (MA) data differ from those estimated by the analytical half-space model. However, it is unclear whether these differences are statistically significant, as previous studies have been based on average cell properties or parametric analyses that do not reflect the inherent experimental and biological variability of real experimental data. To determine whether cell material parameters estimated by the half-space model are significantly different from those predicted by the FE method, we implemented an inverse FE method to estimate the viscoelastic parameters of a population of primary porcine aortic valve interstitial cells tested by MA. We found that inherent differences between the analytical and inverse FE estimation methods resulted in statistically significant differences in individual cell properties. However, in cases with small pipette to cell radius ratios and short loading periods, model-dependent differences were masked by experimental and cell-to-cell variability. Analytical models that account for finite cell-size and loading rate further relaxed the experimental conditions for which accurate cell material parameter estimates could be obtained. These data provide practical guidelines for analysis of MA data that account for the wide range of conditions encountered in typical experiments.     

Behaviour of telomere and telomerase during aging and regeneration in zebrafish

Telomere length and telomerase activity are important factors in the pathobiology of human diseases. Age-related diseases and premature aging syndromes are characterized by short telomeres, which can compromise cell viability, whereas tumour cells can prevent telomere loss by aberrantly upregulating telomerase. The zebrafish (Danio rerio) offers multiple experimental manipulation advantages over other vertebrate models and, therefore, it has been recently considered as a potential model for aging, cancer, and regeneration studies. However, it has only partially been exploited to shed light on these fundamental biological processes. The aim of this study was, therefore, to investigate telomere length and telomerase expression and activity in different strains of zebrafish obtained from different stock centres to determine whether they undergo any changes during aging and regeneration. We found that although both telomerase expression and telomere length increased from embryo to adulthood stages, they drastically declined in aged fish despite telomerase activity was detected in different tissues of old fish. In addition, we observed a weaker upregulation of telomerase expression in regenerating fins of old fish, which well correlates with their impaired regeneration capacity. Strikingly, telomeres were elongated or maintained during the fin regeneration process at all ages and after repeated amputations, likely to support high cell proliferation rates. We conclude that the expression of telomerase and telomere length are closely related during the entire life cycle of the fish and that these two parameters can be used as biomarkers of aging in zebrafish. Our results also reveal a direct relationship between the expression of telomerase, telomere length and the efficiency of tissue regeneration.     

Experimental analysis and modelling of in vitro proliferation of mesenchymal stem cells

Objectives:  Stem cell therapies based on differentiation of adult or embryonic stem cells into specialized ones appear to be effective for treating several human diseases. This work addresses the mathematical simulation of proliferation kinetics of stem cells.
Materials and methods:  Sheep bone marrow mesenchymal stem cells (phenotype characterized by flow cytometry analysis) seeded at different initial concentrations in Petri dishes were expanded to confluence. Sigmoid temporal profiles of total counts obtained through classic haemocytometry were quantitatively interpreted by both a phenomenological logistic equation and a novel model based on a one-dimensional, single-staged population balance approach capable of taking into account contact inhibition at confluence. The models' parameters were determined by comparison with experimental data on population expansion starting from single seeding concentration. Reliability of the models was tested by predicting cell proliferation carried out starting from different seeding concentrations.
Results and discussion:  It was found that the proposed population balance modelling approach was successful in predicting the experimental data over the whole range of initial cell numbers investigated, while prediction capability of phenomenological logistic equation was more limited.     

Fitted hyperelastic parameters for Human brain tissue from reported tension,compression, and shear tests

The mechanical properties of human brain tissue are the subject of interest because of their use in understanding brain trauma and in developing therapeutic treatments and procedures. To represent the behavior of the tissue, we have developed hyperelastic mechanical models whose parameters are fitted in accordance with experimental test results. However, most studies available in the literature have fitted parameters with data of a single type of loading, such as tension, compression, or shear. Recently, Jin et al. (Journal of Biomechanics 46:2795−2801, 2013) reported data from ex vivo tests of human brain tissue under tension, compression, and shear loading using four strain rates and four different brain regions. However, they do not report parameters of energy functions that can be readily used in finite element simulations. To represent the tissue behavior for the quasi-static loading conditions, we aimed to determine the best fit of the hyperelastic parameters of the hyperfoam, Ogden, and polynomial strain energy functions available in ABAQUS for the low strain rate data, while simultaneously considering all three loading modes. We used an optimization process conducted in MATLAB, calling iteratively three finite element models developed in ABAQUS that represent the three loadings. Results showed a relatively good fit to experimental data in all loading modes using two terms in the energy functions. Values for the shear modulus obtained in this analysis (897−1653 Pa) are in the range of those presented in other studies. These energy-function parameters can be used in brain tissue simulations using finite element models.     

Deformability of red blood cells from different species studied by resistive pulse shape analysis technique

The Cell Transit Analyzer (CTA) is now being used widely in clinical hemorheology. Most of the data obtained by CTA are limited to human blood, although the CTA has an important potential to be used in experimental studies on animal models. However, behavior of red blood cells (RBC) from various species might be different in CTA. Eight parameters reflecting different aspects of cell passage through pores with 5 μm diameter and 15 μm length were determined or human, guinea pig, dog, rabbit, rat, mouse and sheep RBC, together with instrument precision and biological variation. These parameters have a wide range when measured in different species and correlate with cell volume. Sensitivity of these parameters to the glutaraldehyde-induced alterations in RBC deformability was not same for different laboratory mammals. The main reason for this difference seems to be related to the cell size and thus sensitivity might be significantly limited if 5 μm pore-size filters are used to test the smaller RBC. The results of this study may help in designing experimental studies on laboratory mammals using the CTA.     

Immunoregulation of experimental allergic encephalomyelitis: conditions for induction of suppressor cells and analysis of mechanism.

We determined requirements for the induction of immunoregulatory suppressor cells in experimental allergic encephalomyelitis (EAE) in Lewis rats. Pretreatment of rats with myelin basic protein (BP) in incomplete Freund's adjuvant (IFA) stimulates the proliferation of suppressor cells that localize in lymph nodes and spleen (but not thymus) and exert control over the development of clinical EAE. Dosage studies revealed that 3 X 10(7) suppressor cells can adoptively transfer suppression to syngeneic recipients. Transferred unresponsiveness wanes within 3 weeks, indicating that the suppressor cells are short-lived lymphocytes, although actively induced unresponsiveness persists for at least 8 weeks, probably as a result of continual proliferation under the influence of antigen. No evidence was obtained to suggest that antigen carry-over or blocking antibody production accounts for adoptive transfer of unresponsiveness. Suppressor cells apparently act at the inductive phase of the immune response since they had no inhibitory effect on adoptive transfer of disease by effector lymph node cells. Other mechanisms also may play a role in unresponsiveness to EAE, since rats pretreated i.v. with high dosages of soluble BP were temporarily rendered unresponsive, although suppressor cells could not be detected in these animals.     

Global analysis of endothelial cell line proliferation patterns based on nutrient-depletion models: implications for a standardization of cell proliferation assays

It is known that cell populations growing in different environmental conditions may exhibit different proliferation patterns. However, it is not clear if, despite the diversity of the so-observed patterns, inherent cellular growth characteristics of the population can nevertheless be determined. This study quantifies the proliferative behaviour of the permanent endothelial human cell line, Eahy926, and establishes to which extent the estimation of the cell proliferation rate depends on variations of the experimental protocols. Cell proliferation curves were obtained for cells cultured over 16 days and the influences of cell seeding densities, foetal bovine serum content and frequency of culture medium changes were investigated. Quantitative dynamic modelling was conducted to evaluate the kinetic characteristics of this cell population. We proposed successive models and retained a nutrient-depletion toxicity dependant model, which takes into account the progressive depletion of nutrients, as well as the increase of toxicity in the cell culture medium. This model is shown to provide a very good and robust prediction of the experimental proliferation curves, whatever are the considered frequency of culture medium changes and serum concentrations. Thus, the model enables an intrinsic quantification of the parameters driving in vitro EAhy926 proliferation, including proliferation, nutrient consumption and toxicity increase rates, rather independently of the experiments design. We therefore propose that such models could provide a basis for a standardized quantification of intrinsic cell proliferation kinetics.     

Single Unpurified Breast Tumor-Initiating Cells from Multiple Mouse Models Efficiently Elicit Tumors in Immune-Competent Hosts

The tumor-initiating cell (TIC) frequency of bulk tumor cell populations is one of the criteria used to distinguish malignancies that follow the cancer stem cell model from those that do not. However, tumor-initiating cell frequencies may be influenced by experimental conditions and the extent to which tumors have progressed, parameters that are not always addressed in studies of these cells. We employed limiting dilution cell transplantation of minimally manipulated tumor cells from mammary tumors of several transgenic mouse models to determine their tumor-initiating cell frequency. We determined whether the tumors that formed following tumor cell transplantation phenocopied the primary tumors from which they were isolated and whether they could be serially transplanted. Finally we investigated whether propagating primary tumor cells in different tissue culture conditions affected their resident tumor-initiating cell frequency. We found that tumor-initiating cells comprised between 15% and 50% of the bulk tumor cell population in multiple independent mammary tumors from three different transgenic mouse models of breast cancer. Culture of primary mammary tumor cells in chemically-defined, serum-free medium as non-adherent tumorspheres preserved TIC frequency to levels similar to that of the primary tumors from which they were established. By contrast, propagating the primary tumor cells in serum-containing medium as adherent populations resulted in a several thousand-fold reduction in their tumor-initiating cell fraction. Our findings suggest that experimental conditions, including the sensitivity of the transplantation assay, can dramatically affect estimates of tumor initiating cell frequency. Moreover, conditional on cell culture conditions, the tumor-initiating cell fraction of bulk mouse mammary tumor cell preparations can either be maintained at high or low frequency in vitro thus permitting comparative studies of tumorigenic and non-tumorigenic cancer cells.     

Studies of mitochondrial respiration in muscle cells in situ: Use and misuse of experimental evidence in mathematical modelling

Applications of permeabilized cell and skinned fiber techniques in combination with methods of mathematical modelling for studies of mitochondrial function in the cell are critically evaluated. Mathematical models may be useful tools for explaining biological phenomena, but only if they are selected by fitting the computing results with real experimental data. Confocal microscopy has been used in experiments with permeabilized cardiomyocytes and myocardial fibers to determine the maximal diffusion distance from medium to the core of cells, which is shown not to exceed 8-10 microm. This is a principal index for correctly explaining high apparent Km for exogenous ADP (200-300 microM) in regulation of mitochondrial respiration in oxidative muscle cells in situ. The best fitting of the results of in silico studies may be achieved by using of the compartmentalized energy transfer model. From these results, it may be concluded that in cardiac muscle cells the mitochondria and ATPases are organized into intracellular energetic units (ICEUs) separated from the bulk phase of cytoplasm by some barriers which limit the diffusion of adenine nucleotides. In contrast, alternative models based on the concept of the cell as homogenous system do not explain the observed experimental phenomena and have led to misleading conclusions. The various sources of experimental and conceptual errors are analyzed.     

Stem cell characteristics of transplanted rat mammary clonogens

Rat mammary glands contain a subpopulation of clonogenic epithelial cells with large proliferation and differentiation potentials. When transplanted, the clonogens in monodispersed rat mammary epithelial cell suspensions give rise to either alveolar units (AUs) or ductal units (DUs) depending on the nature of the hormonal milieu in the graft recipient. Clonogens are also the primary cells of origin of mammary cancer following exposure to ionizing radiation or chemical carcinogens. Given the other stem cell characteristics of mammary clonogens, it would be expected that the primary AUs and DUs to which they give rise when grafted and hormonally stimulated (a) would be derived from the same clonogenic cell subpopulation, (b) would contain all of the functionally differentiated cell types of homologous parts of comparably stimulated mammary glands in situ, and (c) would also contain clonogen subpopulations capable when subtransplanted of giving rise to secondary AUs and DUs of similar cell composition. The current experiments were designed to test these expectations. The data are discussed in the context of results of previous studies with this and other experimental models. The results further support the conclusion that rat mammary clonogens are multipotent mammary stem cells.     

Coupled mathematical model of tumorigenesis and angiogenesis in vascular tumours

Objectives: Mathematical models are useful for studying vascular and avascular tumours, because these allow for more logical experimental design and provide valuable insights into the underlying mechanisms of their growth and development. The processes of avascular tumour growth and the development of capillary networks through tumour‐induced angiogenesis have already been extensively investigated, albeit separately. Despite the clinical significance of vascular tumours, few studies have combined these approaches to develop a single comprehensive growth and development model. Materials and methods: We develop a continuum‐based mathematical model of vascular tumour growth. In the model, angiogenesis is initiated through the release of angiogenic growth factors (AGFs) by cells in the hypoxic regions of the tumour. The nutrient concentration within the tumour reflects the influence of capillary growth and invasion induced by AGF. Results and conclusions: Parametric and sensitivity studies were performed to evaluate the influence of different model parameters on tumour growth and to identify the parameters with the most influence, which include the rates of proliferation, apoptosis and necrosis, as well as the diffusion of sprout tips and the size of the region affected by angiogenesis. An optimization was performed for values of the model parameters that resulted in the best agreement with published experimental data. The resulting model solution matched the experimental data with a high degree of correlation (r = 0.85).     

Size control models of Saccharomyces cerevisiae cell proliferation.

By using time-lapse photomicroscopy, the individual cycle times and sizes at bud emergence were measured for a population of saccharomyces cerevisiae cells growing exponentially under balanced growth conditions in a specially constructed filming slide. There was extensive variability in both parameters for daughter and parent cells. The data on 162 pairs of siblings were analyzed for agreement with the predictions of the transition probability hypothesis and the critical-size hypothesis of yeast cell proliferation and also with a model incorporating both of these hypotheses in tandem. None of the models accounted for all of the experimental data, but two models did give good agreement to all of the data. The wobbly tandem model proposes that cells need to attain a critical size, which is very variable, enabling them to enter a start state from which they exit with first order kinetics. The sloppy size control model suggests that cells have an increasing probability per unit time of traversing start as they increase in size, reaching a high plateau value which is less than one. Both models predict that the kinetics of entry into the cell division sequence will strongly depend on variability in birth size and thus will be quite different for daughters and parents of the asymmetrically dividing yeast cells. Mechanisms underlying these models are discussed.     

Testing of geroprotectors in experiments on cell cultures: Choosing the correct model system

We believe that cytogerontological models, such as the Hayflick model, though very useful for experimental gerontology, are based only on certain correlations and do not directly apply to the gist of the aging process. Thus, the Hayflick limit concept cannot explain why we age, whereas our “stationary phase aging” model appears to be a “gist model,” since it is based on the hypothesis that the main cause of both various “age-related” changes in stationary cell cultures and similar changes in the cells of aging multicellular organism is the restriction of cell proliferation. The model is applicable to experiments on a wide variety of cultured cells, including normal and transformed animal and human cells, plant cells, bacteria, yeasts, mycoplasmas, etc. The results of relevant studies show that cells in this model die out in accordance with the Gompertz law, which describes exponential increase of the death probability with time. Therefore, the “stationary phase aging” model may prove effective in testing of various geroprotectors (anti-aging factors) and geropromoters (pro-aging factors) in cytogerontological experiments. It should be emphasized, however, that even the results of such experiments do not always agree with the data obtained in vivo and therefore cannot be regarded as final but should be verified in studies on laboratory animals and in clinical trials (provided this complies with ethical principles of human subject research).     

Stochastic Models of Lymphocyte Proliferation and Death

Quantitative understanding of the kinetics of lymphocyte proliferation and death upon activation with an antigen is crucial for elucidating factors determining the magnitude, duration and efficiency of the immune response. Recent advances in quantitative experimental techniques, in particular intracellular labeling and multi-channel flow cytometry, allow one to measure the population structure of proliferating and dying lymphocytes for several generations with high precision. These new experimental techniques require novel quantitative methods of analysis. We review several recent mathematical approaches used to describe and analyze cell proliferation data. Using a rigorous mathematical framework, we show that two commonly used models that are based on the theories of age-structured cell populations and of branching processes, are mathematically identical. We provide several simple analytical solutions for a model in which the distribution of inter-division times follows a gamma distribution and show that this model can fit both simulated and experimental data. We also show that the estimates of some critical kinetic parameters, such as the average inter-division time, obtained by fitting models to data may depend on the assumed distribution of inter-division times, highlighting the challenges in quantitative understanding of cell kinetics.     

Mathematical modelling of human mesenchymal stem cell proliferation and differentiation inside artificial porous scaffolds

We present a mathematical model for the proliferation and differentiation of human mesenchymal stem cells grown inside artificial porous scaffolds under different oxygen concentrations. The values of parameters in the model are determined by comparison of the model solutions to published experimental data, complemented with a sensitivity analysis of the fitted parameters. It is shown that a simple hypothesis whereby the secretion of extra-cellular matrix (ECM) is oxygen dependent and that ECM itself stimulates cell proliferation is sufficient to explain the experimental data, which under conditions of low oxygen reveals increased total cell proliferation, upregulation of the numbers of undifferentiated cells, and extended lag phase. These results may help further to understand how cells proliferate inside artificial materials and are of importance to the field of tissue engineering.