Macrophage transactivation for chemokine production identified as a negative regulator of granulomatous inflammation using agent-based modeling

Cellular activation in trans by interferons, cytokines and chemokines is a commonly recognized mechanism to amplify immune effector function and limit pathogen spread. However, an optimal host response also requires that collateral damage associated with inflammation is limited. This may be particularly so in the case of granulomatous inflammation, where an excessive number and / or excessively florid granulomas can have significant pathological consequences. Here, we have combined transcriptomics, agent-based modeling and in vivo experimental approaches to study constraints on hepatic granuloma formation in a murine model of experimental leishmaniasis. We demonstrate that chemokine production by non-infected Kupffer cells in the Leishmania donovani-infected liver promotes competition with infected KCs for available iNKT cells, ultimately inhibiting the extent of granulomatous inflammation. We propose trans-activation for chemokine production as a novel broadly applicable mechanism that may operate early in infection to limit excessive focal inflammation

Building Confidence in Quantitative Systems Pharmacology Models : An Engineer's Guide to Exploring the Rationale in Model Design and Development

This tutorial promotes good practice for exploring the rationale of systems pharmacology models. A safety systems engineering inspired notation approach provides much needed rigor and transparency in development and application of models for therapeutic discovery and design of intervention strategies. Structured arguments over a model's development, underpinning biological knowledge, and analyses of model behaviors are constructed to determine the confidence that a model is fit for the purpose for which it will be applied

Modelling fungal colonies and communities:challenges and opportunities

This contribution, based on a Special Interest Group session held during IMC9, focuses on physiological based models of filamentous fungal colony growth and interactions. Fungi are known to be an important component of ecosystems, in terms of colony dynamics and interactions within and between trophic levels. We outline some of the essential components necessary to develop a fungal ecology: a mechanistic model of fungal colony growth and interactions, where observed behaviour can be linked to underlying function; a model of how fungi can cooperate at larger scales; and novel techniques for both exploring quantitatively the scales at which fungi operate; and addressing the computational challenges arising from this highly detailed quantification. We also propose a novel application area for fungi which may provide alternate routes for supporting scientific study of colony behaviour. This synthesis offers new potential to explore fungal community dynamics and the impact on ecosystem functioning

Investigating The Dynamics of Hepatic Inflammation Through Simulation

Inflammation is a fundamental mechanism for the body to induce repair and healing in tissues, and exacerbated inflammatory responses are associated with a wide variety of diseases and disorders. Categorising the various cells, proteins, and precise mechanisms involved in initiating and driving inflammation poses significant challenges, due to the complex interplay that occurs between them. In this thesis, I will introduce a deadly parasitic disease called Visceral Leishmaniasis (VL) as a case study in using computational modelling techniques to elucidate the mechanisms underpinning inflammation. During VL infection, inflammatory aggregations of immune system cells form, these are called granulomas. Granulomas function to contain and subsequently remove infection. Whilst immunological studies have provided insights into the structure and function of granulomas, there remains a breadth of questions which laboratory techniques are currently incapable of answering. As such, the challenges facing biologists from a scientific perspective will be addressed, I will then argue after a thorough review of the relevant literature, that agent-based computational modelling is a logical choice for research into granuloma formation, and that such models can help answer some outstanding questions in the field. The thesis presents the process of designing and developing the first spatially resolved model of liver localised granuloma formation during VL. The development and use of modelling and simulation to study granulomas has involved close collaboration with immunologists at all stages through conceptualisation, modelling, implementation, and also results interpretation. I describe the use of established statistical techniques to instill confidence in both the model, and the results it can produce through simulation. Through iterative hypothesis generation and testing, the research undertaken has allowed for several predictions to be made, some of which have biological significance and which were later validated experimentally. Specifically, transcriptomic data analysis revealed that both infected and uninfected Kupffer cells are equally capable of responding to infection in a similar manner, something which wasn't previously evident in the literature. Using this transcriptomic data, I investigated through simulation, several experimental scenarios and elucidated a novel mechanism of immune system regulation in the liver microenvironment. Using an experimental model of Leishmania donovani infection, I demonstrated that such an immune regulatory mechanism can be overcome with the expansion of early promoter cells called Natural Killer T cells

Leishmania and other intracellular pathogens: selectivity, drug distribution and PK-PD.

New drugs and treatments for diseases caused by intracellular pathogens, such as leishmaniasis and the Leishmania species, have proved to be some of the most difficult to discover and develop. The focus of discovery research has been on the identification of potent and selective compounds that inhibit target enzymes (or other essential molecules) or are active against the causative pathogen in phenotypic in vitro assays. Although these discovery paradigms remain an essential part of the early stages of the drug R & D pathway, over the past two decades additional emphasis has been given to the challenges needed to ensure that the potential anti-infective drugs distribute to infected tissues, reach the target pathogen within the host cell and exert the appropriate pharmacodynamic effect at these sites. This review will focus on how these challenges are being met in relation to Leishmania and the leishmaniases with lessons learned from drug R & D for other intracellular pathogens

A PETRI NET MODEL OF LIVER RESPONSE TO VISCERAL LEISHMANIASIS: SELF-REGULATION AND COMPLEX INTERPLAY IN THE VERTEBRATE IMMUNE SYSTEM

Visceral leishmaniasis (also called "Kala-azar") is a widespread disease, which is usually fatal in the absence of treatment. Characteristic of the liver immune response to leishmaniasis is a type of inflammation (granulomatous inflammation) that leads to the formation of "granulomas". A granuloma provides a very interesting micro-environment, which is maintained by the coordination of many cells of the immune system. Due to the complexity of the immune response, only a limited amount of modeling work exists in the context of granulomatous infection, and most of the current models focus only on the formation stage of granulomas. The primary goal of this thesis is to gain insights into the process of formation and development of a granuloma. To this end, we built a model of the granuloma formation and resolution in the liver using stochastic Petri nets, and performed several in silico experiments to study the nature of the immune response to leishmaniasis, possible therapeutic options, and the role of the cells involved. Additionally, the building of the model is extensively documented, and the most important qualitative and quantitative assumptions are referenced and discussed, with the aim of presenting a \u201cconceptual framework\u201d to be used when facing similar problems. The model is validated against available biological data, and its robustness is assessed using sensitivity analysis

Development of a gamma glutamylcysteine synthetase vaccine to protect against Leishmania infection

Leishmaniasis is a public health problem and development of a vaccine to prevent infection is required. The overall aim of this study was to develop a vaccine to protect against Leishmania infection using L. donovani, L. mexicana and L. major gamma glutamylcysteine synthetase (γGCS) recombinant proteins. Studies to optimise the expression of L. donovani, L. mexicana and L. major γGCS recombinant proteins showed that induction with 0.1 mM isopropyl β-D-1-thiogalactopyranoside produced the highest amounts of recombinant protein, and incubation of bacteria at 18°C after induction increased the amount of soluble protein produced. Recombinant L. mexicana γGCS had significantly higher specific enzyme activity (p <0.001) compared to L. donovani and L. major γGCS. L. mexicana γGCS was the most resistant to L-buthionine sulphoximine (BSO) inhibition, the specific irreversible inhibitor of γGCS, at the maximum concentration tested (1.5 mM) and L. mexicana promastigotes were the most resistant to the cytotoxic effects of BSO compared to L. donovani and L. major promastigotes (p < 0.001). Vaccination with the recombinant γGCS proteins from L. donovani, L. major and L. mexicana (triple vaccine) induced significant parasite-specific Th1 and Th2 immune responses based on antibody titres and cytokine production by in vitro stimulated splenocytes from immunised mice. Vaccination by inhalation or subcutaneous injection with the triple vaccine was similar mean percentage reduction in parasite burdens compared to controls ± SE, was 98% ± 0.02 in L. mexicana infected mice. In L. major infected mice was 70% ± 0.1 by subcutaneous immunisation and 65%± 0.01 for inhalation vaccination. Treatment with the triple vaccine by inhalation failed to protect mice against L. donovani infection but was effective in hamsters, where a significant reduction in liver and bone marrow parasite burdens compared to control values (p < 0.05; mean percentage reduction compared to controls ± SE: spleen 89 ± 1; liver 83 ± 0.3; bone marrow 77 ± 1). In conclusion, the results of this study indicate that vaccination against leishmaniasis is feasible by the pulmonary route and that the triple vaccine is a potential vaccine candidate.Leishmaniasis is a public health problem and development of a vaccine to prevent infection is required. The overall aim of this study was to develop a vaccine to protect against Leishmania infection using L. donovani, L. mexicana and L. major gamma glutamylcysteine synthetase (γGCS) recombinant proteins. Studies to optimise the expression of L. donovani, L. mexicana and L. major γGCS recombinant proteins showed that induction with 0.1 mM isopropyl β-D-1-thiogalactopyranoside produced the highest amounts of recombinant protein, and incubation of bacteria at 18°C after induction increased the amount of soluble protein produced. Recombinant L. mexicana γGCS had significantly higher specific enzyme activity (p <0.001) compared to L. donovani and L. major γGCS. L. mexicana γGCS was the most resistant to L-buthionine sulphoximine (BSO) inhibition, the specific irreversible inhibitor of γGCS, at the maximum concentration tested (1.5 mM) and L. mexicana promastigotes were the most resistant to the cytotoxic effects of BSO compared to L. donovani and L. major promastigotes (p < 0.001). Vaccination with the recombinant γGCS proteins from L. donovani, L. major and L. mexicana (triple vaccine) induced significant parasite-specific Th1 and Th2 immune responses based on antibody titres and cytokine production by in vitro stimulated splenocytes from immunised mice. Vaccination by inhalation or subcutaneous injection with the triple vaccine was similar mean percentage reduction in parasite burdens compared to controls ± SE, was 98% ± 0.02 in L. mexicana infected mice. In L. major infected mice was 70% ± 0.1 by subcutaneous immunisation and 65%± 0.01 for inhalation vaccination. Treatment with the triple vaccine by inhalation failed to protect mice against L. donovani infection but was effective in hamsters, where a significant reduction in liver and bone marrow parasite burdens compared to control values (p < 0.05; mean percentage reduction compared to controls ± SE: spleen 89 ± 1; liver 83 ± 0.3; bone marrow 77 ± 1). In conclusion, the results of this study indicate that vaccination against leishmaniasis is feasible by the pulmonary route and that the triple vaccine is a potential vaccine candidate

New pharmacokinetic and PK/PD drug development methodologies for cutaneous leishmaniasis

Cutaneous leishmaniasis (CL) is a vector-borne neglected tropical disease caused by over 15 species of the intracellular Leishmania parasite. This skin infection is the most common and widely distributed form of leishmaniasis, with an estimated 0.7-1.2 million new cases annually, mainly in the Middle East and Latin America. CL is rarely fatal, but the ulcerative skin lesions often leave lifelong scars, disfigurement, and social stigma. Current treatment of CL is far from optimal. The pentavalent antimonials remain the standard of care since their discovery in the 1940s, but typically require three weeks of painful, toxic injections into the lesions. An alternative therapeutic option is liposomal amphotericin B, but cure rates are variable, access is problematic and invasive drug administration is required. Basic research tools that could accelerate the successful discovery and development of much needed new drugs are currently limited. This thesis aims to provide a coherent set of R&D methodologies to evaluate (i) the penetration of drugs into the infected skin lesions (pharmacokinetics, PK), (ii) the ability to kill the causative Leishmania parasites (pharmacodynamics, PD) and (iii) the interaction between PK and PD (PK/PD) parameters. In chapter 3.1, the limitations of the current in vitro models to predict in vivo drug activity were confirmed. Additionally, combination therapy of standard antileishmanial drugs with chloroquine was found unlikely to be a successful new treatment strategy for CL. In chapter 3.2, we demonstrated a clear relationship between PK and PD parameters for the standard liposomal amphotericin B formulation AmBisome in a mouse model of CL, providing a PK/PD basis for the rational design of better clinical dose regimens. In chapter 3.3, we compared the in vivo effects of AmBisome with Fungisome, an alternative liposomal amphotericin B formulation on the market in India. Fungisome was less efficacious than AmBisome due to lower drug accumulation in skin lesions and had a narrower therapeutic index in the treatment of murine CL. In chapter 3.4, we showed that the CL infection and associated local skin inflammation has a profound effect on the PK of AmBisome and can contribute to its variable therapeutic efficacy against Old and New World Leishmania species. The increased vascular permeability of the dermal capillaries might be exploited for enhanced delivery of small (< 500 Da) oral drugs to the infected skin. Finally, in chapter 3.5, we evaluated a new drug candidate from the Drugs for Neglected Diseases Initiative (DNDI) for visceral leishmaniasis. Our PK and PD data, based on innovative skin microdialysis and qPCR techniques, support the further preclinical development of the nitroimidazole DNDI-0690 as a promising new oral treatment for CL. Overall, the strategies, lessons and research tools provided in this PhD thesis could contribute to the development of safe, effective, affordable and patient-friendly new drugs for the treatment of CL

Engineering simulations for cancer systems biology

Computer simulation can be used to inform in vivo and in vitro experimentation, enabling rapid, low-cost hypothesis generation and directing experimental design in order to test those hypotheses. In this way, in silico models become a scientific instrument for investigation, and so should be developed to high standards, be carefully calibrated and their findings presented in such that they may be reproduced. Here, we outline a framework that supports developing simulations as scientific instruments, and we select cancer systems biology as an exemplar domain, with a particular focus on cellular signalling models. We consider the challenges of lack of data, incomplete knowledge and modelling in the context of a rapidly changing knowledge base. Our framework comprises a process to clearly separate scientific and engineering concerns in model and simulation development, and an argumentation approach to documenting models for rigorous way of recording assumptions and knowledge gaps. We propose interactive, dynamic visualisation tools to enable the biological community to interact with cellular signalling models directly for experimental design. There is a mismatch in scale between these cellular models and tissue structures that are affected by tumours, and bridging this gap requires substantial computational resource. We present concurrent programming as a technology to link scales without losing important details through model simplification. We discuss the value of combining this technology, interactive visualisation, argumentation and model separation to support development of multi-scale models that represent biologically plausible cells arranged in biologically plausible structures that model cell behaviour, interactions and response to therapeutic interventions

Development of Novel Predictive 2D and 3D in Vitro Models For Anti-Leishmanial Drug Testing

The failure rates of drugs once they reach clinical trials are high, with estimates up to nearly 90%. One reason is the lack of biologically relevant models in which potential drug candidates are screened, evaluated and selected during discovery and development phases. Leishmaniasis is a disease that suffers from this problem. Current therapies are weak, toxic and there is a growing problem of drug resistance. The aim of this thesis is to investigate different ways in which the current in vitro model systems could be made more predictive. Three methods of potentially improving reliability and predictability of in vitro models are investigated. Firstly, the differences between the media perfusion and static cell culture systems were studied. Using macrophages and L. major parasites, infections within the media perfusion system were optimised. The activities of standard drugs used for the treatment of leishmaniasis, were determined. A decrease in infection rates and in the activity of standard drugs was seen when using the media perfusion. Secondly, a 3D in vitro infection model was developed and used to determine the activity of standard drugs, compared with 2D cell culture. The model shows that 3D and 2D provide similar results for the activity of the standard drugs tested. Thirdly, a variety of macrophage cell types have been used as Leishmania host cells for intracellular amastigote assays. The use of macrophages differentiated from induced pluripotent stem cells was investigated as a viable and more predictive alternative. The option to use a cell type that is more biologically similar to the human in vivo situation, but can be maintained like cell lines, is a clear benefit to in in vitro assays. It was concluded that macrophages differentiated from induced pluripotent stem cells would be a suitable alternative to currently used cell types