Intensive study of interleukin-4 for more than three decades has revealed multiple functions of this cytokine in diverse processes. Nevertheless, the wide distribution of Interleukin-4 suggests the possibility of unexplored roles. Indeed, in here we present a novel role of IL-4 for the maintenance of different populations of stromal cells in peripheral lymph nodes at homeostasis and describe a role of IL-4 in the expansion of these stromal populations following antigen challenge. In consequence, IL-4 is fundamental for mounting an appropriate humoral response to a primary immunization, and absence of this cytokine is detrimental for the development of a Type 2 response. Furthermore, we describe the role of IL-4 in the immune responses of offspring antenatally exposed to <i>Schistosoma mansoni</i>antigens. Diminished IL-4 production is linked to reduced cellular T and B cells responses in offspring derived from infected mothers, which is of critical relevance to understand vaccination failure. Finally, we describe the protective role of Schistosomiasis infection in atherosclerosis and propose possible mechanism that helps explain the athero-protection. This will contribute to the discovery of novel pathways inducing protection from cardiovascular disease and help to identify possible targets for novel treatments

京都大学 / 新制・課程博士 / 博士(医科学) / 甲第23108号 / 医科博第119号 / 新制||医科||8(附属図書館) / 京都大学大学院医学研究科医科学専攻 / (主査)教授 藤渕 航, 教授 松田 道行, 教授 黒田 知宏 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

<div>Pulsed electric fields (PEFs) can induce numerous biophysical phenomena, especially perturbation of the outer and inner membranes, that may be used for applications that include nonthermal pasteurization, enhanced permeabilization of tumors to improve the transport of chemotherapeutics for cancer therapy, and enhanced membrane permeabilization of individual cells to enhance RNA and DNA delivery for gene therapy. The applied electric field and pulse duration determine the density, size, and reversibility of the created membrane pores. PEFs with durations longer than the outer membrane’s charging time will induce pore formation with the potential for application in irreversible electroporation for cancer therapy and microorganism inactivation. Shorter duration PEFs, particularly on the nanosecond timescale (nsPEFs), induce a larger density of smaller membrane pores with the potential to permeabilize intracellular membranes, such as the mitochondria, to induce programmed cell death. Thus, the PEFs can effectively kill multiple types of cells, dependent upon the cells. This thesis assesses the ability of nsPEFs to kill different cell types, specifically microorganisms with and without antibiotics as well as varying the parameters to affect populations of immortalized leukemia cells (Jurkats).</div><div>Antibiotic resistance has been an acknowledged challenge since the initial development of penicillin; however, recent discoveries by the CDC and the WHO of microorganisms resistant to last line of defense drugs combined with predictions of potential infection cases reaching 50 million a year globally and the absence new drugs in the discovery pipeline highlight the need to develop novel ways to combat and overcome these resistance mechanisms. Repurposing drugs, exploring nature for new drugs, and developing enzymes to counter the resistance mechanisms may provide potential alternatives for addressing the scarcity of antibiotics effective against gram-negative infections. One may also leverage the abundance of drugs effective against gram-positive infections by using nsPEFs to make them effective against gram-negative infections, including bacterial species with multiple natural and acquired resistance mechanisms. Numerous drug and microbial combinations for different doses and pulse treatments were tested and presented here.</div><div>Low intensity PEFs may selectively target cell populations at different stages of the cell cycle (quiescence and mitosis) to modify cancer cell population dynamics. Experimental studies of cancer cell growth when exposed to a low number of nsPEFs, while varying pulse duration, field intensity and number of pulses reveals a threshold beyond which cell recovery is not possible, but also a point of diminishing returns if cell death is the intention. A theory comprised of coupled differential equations representing the proliferating and quiescent cells showed how changing PEF parameters altered the behavior of these cell populations after treatment. These results may provide important information on the impact of PEFs with sub-threshold intensities and durations on cell population growth and potential recurrence.</div>

This thesis extends an existing cell population modelling framework to investigate two different hypotheses for what drives the phenomenon of cell sorting, which is the spontaneous self-reorganization of cell populations. This behaviour cause cells to find their way back into their original configuration after they have been scrambled. Original tissue function may also be regained. The modelling framework is called discrete Laplacian cell mechanics (DLCM), and models cell movement on a lattice as a result of pressure differences. The first hypothesis suggests that cells exhibit type-specific adhesion properties which cause cells of the same type to adhere more to each other than to cells of a different kind. The other, more recent, hypothesis explains cell sorting behaviour as a consequence of interfacial tension, where cells of different types exhibit larger tension between them compared to cells of the same type. Adhesion is implemented as a passive force between cells of the same type, which counteract the pressure-driven events, while interfacial tension is implemented as pressure sources arising due to contact with cells of a different type. This thesis investigates whether these additions on the scale of individual cells can be sufficient to induce sorting behaviour on the cell population scale. Subsequently the suitability of implementing these effects in the DLCM framework can be evaluated. Starting from a scrambled cell configuration of two types, the results show that differential adhesion can result in the cell population sorting into smaller clusters, with the addition of Brownian motion improving the sorting ability significantly. Differential interfacial tension as it is implemented here demonstrates the effect of dissociation between cells of different type, but this is not sufficient to achieve sorting. The behaviour can be likened to a form of localized Brownian motion where more unsorted areas are prone to more movement events. Therefore, differential tension is not deemed suitable within the DLCM framework on its own. The cohesive effect of differential adhesion together with the dissociative effect of differential interfacial tension proved to work well together, acheiving a high degree of sorting both overall and compared to the case of only differential adhesion with some Brownian motion. Full separation into one distinct cell mass for each cell type present could not be achieved.

Computational modelling of populations of cells has been applied to further understanding in a range of biological fields, from cell sorting to tumour development. The ability to analyse the emergent population-level effects of variation at the cellular and subcellular level makes it a powerful approach. As more detailed models have been proposed, the demand for computational power has increased. While developments in microchip technology continue to increase the power of individual compute units available to the research community, the use of parallel computing offers an immediate increase in available computing power. To make full use of parallel computing technology it is necessary to develop specialised algorithms. To that end, this thesis is concerned with the development, implementation and application of a novel parallel algorithm for the simulation of an off-lattice individual-based model of a population of cells. We first use the Message Passing Interface to develop a parallel algorithm for the overlapping spheres model which we implement in the Chaste software library. We draw on approaches for parallelising molecular dynamics simulations to develop a spatial decomposition approach to dividing data between processors. By using functions designed for saving and loading the state of simulations, our implementation allows for the parallel simulation of all subcellular models implemented in Chaste, as well as cell-cell interactions that depend on any of the cell state variables. Our implementation allows for faithful replication of model cells that migrate between processors during a simulation. We validate our parallel implementation by comparing results with the extensively tested serial implementation in Chaste. While the use of the Message Passing Interface means that our algorithm may be used on shared- and distributed-memory systems, we find that parallel performance is limited due to high communication costs. To address this we apply a series of optimisations that improve the scaling of our algorithm both in terms of compute time and memory consumption for given benchmark problems. To demonstrate an example application of our work to a biological problem, we extend our algorithm to enable parallel simulation of the Subcellular Element Model (S.A. Sandersius and T.J. Newman. Phys. Biol., 5:015002, 2008). By considering subcellular biomechanical heterogeneity we study the impact of a stiffer nuclear region within cells on the initiation of buckling of a compressed epithelial layer. The optimised parallel algorithm decreases computation time for a single simulation in this study by an order of magnitude, reducing computation time from over a week to a single day.

Mathematical models, that depict the dynamics of a cancer cell population growing out of the human body (in vitro) in unconstrained microenvironment conditions, are considered in this thesis. Cancer cells in vitro grow and divide much faster than cancer cells in the human body, therefore, the effects of various cancer treatments applied to them can be identified much faster. These cell populations, when not exposed to any cancer treatment, exhibit exponential growth that we refer to as the balanced exponential growth (BEG) state. This observation has led to several effective methods of estimating parameters that thereafter are not required to be determined experimentally. We present derivation of the age-structured model and its theoretical analysis of the existence of the solution. Furthermore, we have obtained the condition for BEG existence using the Perron-Frobenius theorem. A mathematical description of the cell-cycle control is shown for one-compartment and two-compartment populations, where a compartment refers to a cell population consisting of cells that exhibit similar kinetic properties. We have incorporated into our mathematical model the required growing/aging times in each phase of the cell cycle for the biological viability. Moreover, we have derived analytical formulae for vital parameters in cancer research, such as population doubling time, the average cell-cycle age, and the average removal age from all phases, which we argue is the average cell-cycle time of the population. An estimate of the average cell-cycle time is of a particular interest for biologists and clinicians, and for patient survival prognoses as it is considered that short cell-cycle times correlate with poor survival prognoses for patients. Applications of our mathematical model to experimental data have been shown. First, we have derived algebraic expressions to determine the population doubling time from single experimental observation as an alternative to empirically constructed growth curve. This result is applicable to various types of cancer cell lines. One option to extend this model would be to derive the cell cycle time from a single experimental measurement. Second, we have applied our mathematical model to interpret and derive dynamic-depicting parameters of five melanoma cell lines exposed to radiotherapy. The mathematical result suggests there are shortcomings in the experimental methods and provides an insight into the cancer cell population dynamics during post radiotherapy. Finally, a mathematical model depicting a theoretical cancer cell population that comprises two sub-populations with different kinetic properties is presented to describe the transition of a primary culture to a cell line cell population.

B-lymphocytes are a subset of immune cells that can be distinguished mainly by carrying clonally diversified membrane-bound immunoglobulin specialized to specific antigen recognition. Together with other immunocytes B-lymphocytes play a central role in adaptive immune system which takes part in defense of the host against wide variety of pathogens. Recently the evidence has supported the emerging concept of different B-cell subpopulations to play a direct or indirect role in a pathogenesis of spectrum of disorders. However, so far the knowledge has been limited mainly in the way of how the specific differentiation stages of B-lymphocytes are involved in pathogenesis of diseases and how course of disease, stage, and eventually different treatment can affect B-cell homeostasis. That is the reason for the thesis to be focused on an analysis of B-cell population profile changes in disease, identification of any association present among specific B-cell subpopulations, as well as association between these subpopulations and clinical parameters. Using polychromatic flow cytometry we analyzed frequencies of 11 B-cell subpopulations including stages of transient B-lymphocytes through effector antibody-producing plasma cells. We examined 81 individuals including 22 healthy controls and 59 patients with...

Plasmacytoid dendritic cells (pDC) constitute a very rare blood cell population and play a significant role in immune response and immune-mediated disorders. Investigations on primary pDCs are hindered not only due to their rarity but also because they represent a heterogeneous cell population which is difficult to culture ex vivo. We generated a conditionally immortalized pDC line (Dox-pDC) from mice with Doxycycline-inducible SV40 Large T Antigen with a comparable immune profile to primary pDCs. The Dox-pDC secrete pro- and anti-inflammatory cytokines upon Toll-like receptor 9 stimulation and upregulate their MHCI, MHCII and costimulatory molecules. Further, the Dox-pDC activate and polarize naïve T cells in vivo and in vitro in response to the model antigen Ovalbumin. Due to their long-term culture stability and their robust proliferation Dox-pDC represent a reliable alternative to primary mouse pDC.

Plasmacytoid dendritic cells (pDC) constitute a very rare blood cell population and play a significant role in immune response and immune-mediated disorders. Investigations on primary pDCs are hindered not only due to their rarity but also because they represent a heterogeneous cell population which is difficult to culture ex vivo. We generated a conditionally immortalized pDC line (Dox-pDC) from mice with Doxycycline-inducible SV40 Large T Antigen with a comparable immune profile to primary pDCs. The Dox-pDC secrete pro- and anti-inflammatory cytokines upon Toll-like receptor 9 stimulation and upregulate their MHCI, MHCII and costimulatory molecules. Further, the Dox-pDC activate and polarize naïve T cells in vivo and in vitro in response to the model antigen Ovalbumin. Due to their long-term culture stability and their robust proliferation Dox-pDC represent a reliable alternative to primary mouse pDC.

While adenovirus (Ad) vectors have been shown to elicit potent antigen-specific T cell responses, the kinetics and nature of antigen-specific mucosa! and systemic T-cell responses has rarely been examined, especially following mucosal administration of Ad-based vectors. In the present studies, the phenotypic and functional characterization of antigen-specific CD8+ T cell responses following intrarectal (i.r.) vaccination with an Ad vector expressing Gallus gallus ovalbumin (OVA) was conducted. The frequencies of OVA-specific CD8+ T cells was maximal at 2 weeks post-vaccination in all tissues examined and then declined, demonstrating normal expansion and contraction kinetics. CD8+ T cells induced in the course of immunization exhibited phenotypic characteristics of effector memory T cells including up-regulation of the cell surface molecules CD43, CD44 and a low level of expression of CD127 at both local and systemic sites. While the discordance between the number of tetramer-reactive and cytokine-producing OVA-specific CD8+ T cells was observed, CD8+ T cells appeared to be fully functional in vivo. Upon secondary antigen exposure, the CD8+ T cell population expanded dramatically, particularly at the mucosa! surfaces. In addition, the CD8+ T cell response generated in the course of i.r. priming protected mice from intravaginal (i. vag.) vaccinia virus one month after immunization, thus underscoring the importance of inducing a tissue-resident effector memory T cell subset for protection against pathogens at mucosal surfaces. In developing future vaccines for mucosal diseases, the induction of a tissue-resident effector memory T cell subset should be one of the immunization objectives. Lentiviral vectors represent an attractive mode of genetic vaccination. Most commonly used, vesicular stomatitis virus glycoprotein (VSVG)-pseudotyped lentiviral vectors do not efficiently infect epithelial cells from the apical side, and, therefore, are not suitable as mucosa! vaccines. In the present studies, Ebola Zaïre strain glycoprotein (EboZ)-pseudotyped lentiviral vectors, which have been previously used to deliver transgene to the lung epithelium, were delivered i.r. and evaluated as a mucosal booster vaccine. Rectal delivery of EboZ-pseudotyped lentiviral vectors expressing β-galactosidase (β-gal) had resulted in low, but detectable levels of β-gal expression 2 weeks after administration. When delivered on its own, EboZ-pseudotyped lentivirus did not prime detectable antigen-specific immune response. However, when delivered i.r. 30 days after i.r. Adβ-gal immunization, a significant enlargement (boost) of β-gal-specific CD8+ T cell responses, especially in the colonic lamina propria (LP), was observed as compared to the delivery of EboZ-pseudotyped vector encoding different transgenes or VSVG-pseudotyped lentivirus expressing β-gal. When these animals were i. vag. challenged with vaccinia virus expressing β-gal, a dramatic expansion of β-gal-specific CD8+ T cells, especially in the vaginal tract, was observed. In addition, this prime and boost strategy protected the mice from i. vag. vaccinia virus challenge. Therefore, i.r. Ad-based priming followed by i.r. EboZ-pseudotyped lentiviral boosting was an effective strategy for eliciting protective mucosal CD8+ T cell responses. / Thesis / Doctor of Philosophy (PhD)