Mathematical modelling of tumour growth and interaction with host tissue and the immune system

In this thesis, three mathematical models describing the growth of solid tumour incorporating the host tissue and the immune system response are developed and investigated. The initial model describes the dynamics of the growing tumour and immune response before being extended in the second model by introducing a time-varying dendritic cell-based treatment strategy. Finally, in the third model, we present a mathematical model of a growing tumour using a hybrid cellular automata. These models can provide information to pre-experimental work to assist in designing more effective and efficient laboratory experiments related to tumour growth and interactions with the immune system and immunotherapy.

The development of an effective vaccine against Chlamydia : utilisation of a non-toxic mucosal adjuvant to generate a protective mucosal response

Chlamydia is responsible for a wide range of diseases with enormous global economic and health burden. As the majority of chlamydial infections are asymptomatic, a vaccine has greatest potential to reduce infection and disease prevalence. Protective immunity against Chlamydia requires the induction of a mucosal immune response, ideally, at the multiple sites in the body where an infection can be established. Mucosal immunity is most effectively stimulated by targeting vaccination to the epithelium, which is best accomplished by direct vaccine application to mucosal surfaces rather than by injection. The efficacy of needle-free vaccines however is reliant on a powerful adjuvant to overcome mucosal tolerance. As very few adjuvants have proven able to elicit mucosal immunity without harmful side effects, there is a need to develop non-toxic adjuvants or safer ways to administered pre-existing toxic adjuvants. In the present study we investigated the novel non-toxic mucosal adjuvant CTA1-DD. The immunogenicity of CTA1-DD was compared to our "gold-standard" mucosal adjuvant combination of cholera toxin (CT) and cytosine-phosphate-guanosine oligodeoxynucleotide (CpG-ODN). We also utilised different needle-free immunisation routes, intranasal (IN), sublingual (SL) and transcutaneous (TC), to stimulate the induction of immunity at multiple mucosal surfaces in the body where Chlamydia are known to infect. Moreover, administering each adjuvant by different routes may also limit the toxicity of the CT/CpG adjuvant, currently restricted from use in humans. Mice were immunised with either adjuvant together with the chlamydial major outer membrane protein (MOMP) to evaluate vaccine safety and quantify the induction of antigen-specific mucosal immune responses. The level of protection against infection and disease was also assessed in vaccinated animals following a live genital or respiratory tract infectious challenge. The non-toxic CTA1-DD was found to be safe and immunogenic when delivered via the IN route in mice, inducing a comparable mucosal response and level of protective immunity against chlamydial challenge to its toxic CT/CpG counterpart administered by the same route. The utilisation of different routes of immunisation strongly influenced the distribution of antigen-specific responses to distant mucosal surfaces and also abrogated the toxicity of CT/CpG. The CT/CpG-adjuvanted vaccine was safe when administered by the SL and TC routes and conferred partial immunity against infection and pathology in both challenge models. This protection was attributed to the induction of antigen-specific pro-inflammatory cellular responses in the lymph nodes regional to the site of infection and rather than in the spleen. Development of non-toxic adjuvants and effective ways to reduce the side effects of toxic adjuvants has profound implications for vaccine development, particularly against mucosal pathogens like Chlamydia. Interestingly, we also identified two contrasting vaccines in both infection models capable of preventing infection or pathology exclusively. This indicated that the development of pathology following an infection of vaccinated animals was independent of bacterial load and was instead the result of immunopathology, potentially driven by the adaptive immune response generated following immunisation. While both vaccines expressed high levels of interleukin (IL)-17 cytokines, the pathology protected group displayed significantly reduced expression of corresponding IL-17 receptors and hence an inhibition of signalling. This indicated that the balance of IL-17-mediated responses defines the degree of protection against infection and tissue damage generated following vaccination. This study has enabled us to better understand the immune basis of pathology and protection, necessary to design more effective vaccines.

Molecular characterisation and expression analysis of interferon gamma in response to natural chlamydia infection in the koala, phascolarctos cinereus

Interferon gamma (IFNγ) is a key Th1 cytokine, with a principal role in the immune response against intracellular organisms such as Chlamydia. Along with being responsible for significant morbidity in human populations, Chlamydia is also responsible for wide spread infection and disease in many animal hosts, with reports that many Australian koala subpopulations are endemically infected. An understanding of the role played by IFNγ in koala chlamydial diseases is important for the establishment of better prophylactic and therapeutic approaches against chlamydial infection in this host. A limited number of IFNγ sequences have been published from marsupials and no immune reagents to measure expression have been developed. Through preliminary analysis of the koala transcriptome, we have identified the full coding sequence of the koala IFNγ gene. Transcripts were identified in spleen and lymph node tissue samples. Phylogenetic analysis demonstrated that koala IFNγ is closely related to other marsupial IFNγ sequences and more distantly related to eutherian mammals. To begin to characterise the role of this important cytokine in the koala's response to chlamydial infection, we developed a quantitative real time PCR assay and applied it to a small cohort of koalas with and without active chlamydial disease, revealing significant differences in expression patterns between the groups. Description of the IFNγ sequence from the koala will not only assist in understanding this species' response to its most important pathogen but will also provide further insight into the evolution of the marsupial immune system

Progesterone activates multiple innate immune pathways inChlamydia trachomatis-Infected endocervical cells

Problem Susceptibility to Chlamydia trachomatis infection is increased by oral con- traceptives and modulated by sex hormones. We therefore sought to determine the effects of female sex hormones on the innate immune response to C. trachomatis infection. Method of study ECC-1 endometrial cells, pre-treated with oestradiol or progesterone, were infected with C. trachomatis and the host transcriptome analysed by Illumina Sentrix HumanRef-8 microarray. Primary endocervical epithe- lial cells, prepared at either the proliferative or secretory phase of the menstrual cycle, were infected with C. trachomatis and cytokine gene expression determined by quantitative RT-PCR analysis. Results Chlamydia trachomatis yield from progesterone-primed ECC-1 cells was significantly reduced compared with oestradiol-treated cells. Genes upregulated in progesterone-treated and Chlamydia-infected cells only included multiple CC and CXC chemokines, IL-17C, IL-29, IL-32, TNF-a, DEFB4B, LCN2, S100A7-9, ITGAM, NOD2, JAK1, IL-6ST, type I and II interferon receptors, numerous interferon-stimulated genes and STAT6. CXCL10, CXCL11, CX3CL1 and IL-17C, which were also upregu- lated in infected secretory-stage primary cells, and there was a trend towards higher levels of immune mediators in infected secretory-phase compared with proliferative-phase cells. Conclusion Progesterone treatment primes multiple innate immune pathways in hormone-responsive epithelial cells that could potentially increase resis- tance to chlamydial infection.

Innate transcriptional networks activated in bladder in response to uropathogenic Escherichia coli drive diverse biological pathways and rapid synthesis of IL-10 for defense against bacterial urinary tract infection

Early transcriptional activation events that occur in bladder immediately following bacterial urinary tract infection (UTI) are not well defined. In this study, we describe the whole bladder transcriptome of uropathogenic Escherichia coli (UPEC) cystitis in mice using genome-wide expression profiling to define the transcriptome of innate immune activation stemming from UPEC colonization of the bladder. Bladder RNA from female C57BL/6 mice, analyzed using 1.0 ST-Affymetrix microarrays, revealed extensive activation of diverse sets of innate immune response genes, including those that encode multiple IL-family members, receptors, metabolic regulators, MAPK activators, and lymphocyte signaling molecules. These were among 1564 genes differentially regulated at 2 h postinfection, highlighting a rapid and broad innate immune response to bladder colonization. Integrative systems-level analyses using InnateDB (http://www.innatedb.com) bioinformatics and ingenuity pathway analysis identified multiple distinct biological pathways in the bladder transcriptome with extensive involvement of lymphocyte signaling, cell cycle alterations, cytoskeletal, and metabolic changes. A key regulator of IL activity identified in the transcriptome was IL-10, which was analyzed functionally to reveal marked exacerbation of cystitis in IL-10–deficient mice. Studies of clinical UTI revealed significantly elevated urinary IL-10 in patients with UPEC cystitis, indicating a role for IL-10 in the innate response to human UTI. The whole bladder transcriptome presented in this work provides new insight into the diversity of innate factors that determine UTI on a genome-wide scale and will be valuable for further data mining. Identification of protective roles for other elements in the transcriptome will provide critical new insight into the complex cascade of events that underpin UTI.

The Escherichia coli O157:H7 EhaB autotransporter protein binds to laminin and collagen I and induces a serum IgA response in O157:H7 challenged cattle

Enterohaemorrhagic Escherichia coli (EHEC) are a subgroup of Shiga toxin-producing E. coli that cause gastrointestinal disease with the potential for life-threatening sequelae. Cattle serve as the natural reservoir for EHEC and outbreaks occur sporadically as a result of contaminated beef and other farming products. While certain EHEC virulence mechanisms have been extensively studied, the factors that mediate host colonization are poorly defined. Previously, we identified four proteins (EhaA,B,C,D) from the prototypic EHEC strain EDL933 that belong to the autotransporter (AT) family. Here we characterize the EhaB AT protein. EhaB was shown to be located at the cell surface and overexpression in E. coli K-12 resulted in significant biofilm formation under continuous flow conditions. Overexpression of EhaB in E. coli K12 and EDL933 backgrounds also promoted adhesion to the extracellular matrix proteins collagen I and laminin. An EhaB-specific antibody revealed that EhaB is expressed in E. coli EDL933 following in vitro growth. EhaB also cross-reacted with serum IgA from cattle challenged with E. coli O157:H7, indicating that EhaB is expressed in vivo and elicits a host IgA immune response.

Resistance to amoebic gill disease (AGD) is characterised by the transcriptional dysregulation of immune and cell cycle pathways

Amoebic gill disease (AGD) is a parasite-mediated proliferative gill disease capable of affecting a range of teleost hosts. While a moderate heritability for AGD resistance in Atlantic salmon has been reported previously, the mechanisms by which individuals resist the proliferative effects remain poorly understood. To gain more knowledge of this commercially important trait, we compared gill transcriptomes of two groups of Atlantic salmon, one designated putatively resistant, and one designated putatively susceptible to AGD. Utilising a 17k Atlantic salmon cDNA microarray we identified 196 transcripts that were differentially expressed between the two groups. Expression of 11 transcripts were further examined with real-time quantitative RT-PCR (qPCR) in the AGD-resistant and AGD-susceptible animals, as well as non-infected naïve fish. Gene expression determined by qPCR was in strong agreement with the microarray analysis. A large number of differentially expressed genes were involved in immune and cell cycle responses. Resistant individuals displayed significantly higher expression of genes involved in adaptive immunity and negative regulation of the cell cycle. In contrast, AGD-susceptible individuals showed higher expression of acute phase proteins and positive regulators of the cell cycle. Combined with the gill histopathology, our results suggest AGD resistance is acquired rather than innately present, and that this resistance is for the most part associated with the dysregulation of immune and cell cycle pathways. © 2008 Elsevier Ltd. All rights reserved.

An agent-based approach to immune modelling

This study focuses on trying to understand why the range of experience with respect to HIV infection is so diverse, especially as regards to the latency period. The challenge is to determine what assumptions can be made about the nature of the experience of antigenic invasion and diversity that can be modelled, tested and argued plausibly. To investigate this, an agent-based approach is used to extract high-level behaviour which cannot be described analytically from the set of interaction rules at the cellular level. A prototype model encompasses local variation in baseline properties contributing to the individual disease experience and is included in a network which mimics the chain of lymphatic nodes. Dealing with massively multi-agent systems requires major computational efforts. However, parallelisation methods are a natural consequence and advantage of the multi-agent approach. These are implemented using the MPI library.

Evaluation of immune responses to influenza vaccination in chronic obstructive pulmonary disease

Background: Given that viral infections are common triggers for exacerbations of Chronic Obstructive Pulmonary Disease (COPD), current clinical guidelines recommend that all patients receive annual influenza vaccinations. A detailed examination of the immune response to vaccination in COPD has not previously been undertaken, so this study aimed to compare immune responses to influenza vaccination between COPD patients and healthy subjects. Methods: Twenty one COPD patients and fourteen healthy subjects were recruited and cellular immune function was assessed pre- and post- vaccination with trivalent inactivated influenza vaccine. Results: One month after vaccination, H1N1 specific antibody titres were significantly lower in COPD patients than in healthy controls (p=0.02). Multivariate analysis demonstrated that post vaccination antibody titres were independently associated with COPD, but not with age or smoking status. Innate immune responses to the vaccine preparation did not differ between the two populations. Serum concentrations of IL-21, a cytokine that is important for B cell development and antibody synthesis, were also lower in COPD patients than in healthy subjects (p<0.01). In vitro functional differences were also observed, with fewer proliferating B cells expressing CD27 (p=0.04) and reduced T-cell IFN-γ synthesis (p<0.01) in COPD patients, relative to healthy subjects. Conclusions: In conclusion, COPD was associated with altered immune responses to influenza vaccination compared to healthy controls with reductions in both T-cell and B-cell function. These findings provide a foundation for future research aimed at optimising the effectiveness of influenza vaccination in COPD.

Interleukin 17A is an immune marker for chlamydial disease severity and pathogenesis in the koala (Phascolarctos cinereus)

The koala (Phascolarctos cinereus) is an iconic Australian marsupial species that is facing many threats to its survival. Chlamydia pecorum infections are a significant contributor to this ongoing decline. A major limiting factor in our ability to manage and control chlamydial disease in koalas is a limited understanding of the koala’s cell-mediated immune response to infections by this bacterial pathogen. To identify immunological markers associated with chlamydial infection and disease in koalas, we used koala-specific Quantitative Real Time PCR (qrtPCR) assays to profile the cytokine responses of Peripheral Blood Mononuclear Cells (PBMCs) collected from 41 koalas with different stages of chlamydial disease. Target cytokines included the principal Th1 (Interferon gamma; IFNγ), Th2 (Interleukin 10; IL10), and pro-inflammatory cytokines (Tumor Necrosis Factor alpha; TNFα). A novel koala-specific IL17A qrtPCR assay was also developed as part of this study to quantitate the gene expression of this Th17 cytokine in koalas. A statistically significant higher IL17A gene expression was observed in animals with current chlamydial disease compared to animals with asymptomatic chlamydial infection. A modest up-regulation of pro-inflammatory cytokines, such as TNFα and IFNγ, was also observed in these animals with signs of current chlamydial disease. IL10 gene expression was not evident in the majority of animals from both groups. Future longitudinal studies are now required to confirm the role played by cytokines in pathology and/or protection against C. pecorum infection in the koala.

Stimulating immune responses to fight cancer: Basic biology and mechanisms

Chronic inflammation is now recognized as a major cause of malignant disease. In concert with various mechanisms (including DNA instability), hypoxia and activation of inflammatory bioactive lipid pathways and pro-inflammatory cytokines open the doorway to malignant transformation and proliferation, angiogenesis, and metastasis in many cancers. A balance between stimulatory and inhibitory signals regulates the immune response to cancer. These include inhibitory checkpoints that modulate the extent and duration of the immune response and may be activated by tumor cells. This contributes to immune resistance, especially against tumor antigen-specific T-cells. Targeting these checkpoints is an evolving approach to cancer immunotherapy, designed to foster an immune response. The current focus of these trials is on the programmed cell death protein 1 (PD-1) receptor and its ligands (PD-L1, PD-L2) and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4). Researchers have developed anti-PD-1 and anti-PDL-1 antibodies that interfere with the ligands and receptor and allow the tumor cell to be recognized and attacked by tumor-infiltrating T-cells. These are currently being studied in lung cancer. Likewise, CTLA-4 inhibitors, which have had success treating advanced melanoma, are being studied in lung cancer with encouraging results.

Chlamydial infection and spatial ascension of the female genital tract : a novel hybrid cellular automata and continuum mathematical model

Sexually transmitted chlamydial infection initially establishes in the endocervix in females, but if the infection ascends the genital tract, significant disease, including infertility, can result. Many of the mechanisms associated with chlamydial infection kinetics and disease ascension are unknown. We attempt to elucidate some of these processes by developing a novel mathematical model, using a cellular automata–partial differential equation model. We matched our model outputs to experimental data of chlamydial infection of the guinea-pig cervix and carried out sensitivity analyses to determine the relative influence of model parameters. We found that the rate of recruitment and action of innate immune cells to clear extracellular chlamydial particles and the rate of passive movement of chlamydial particles are the dominant factors in determining the early course of infection, magnitude of the peak chlamydial time course and the time of the peak. The rate of passive movement was found to be the most important factor in determining whether infection would ascend to the upper genital tract. This study highlights the importance of early innate immunity in the control of chlamydial infection and the significance of motility-diffusive properties and the adaptive immune response in the magnitude of infection and in its ascension.

Characterisation of mesenchymal cells from osteophytes in osteoarthritis

Osteophytes form through the process of chondroid metamorphosis of fibrous tissue followed by endochondral ossification. Osteophytes have been found to consist of three different mesenchymal tissue regions including endochondral bone formation within cartilage residues, intra-membranous bone formation within fibrous tissue and bone formation within bone marrow spaces. All these features provide evidence of mesenchymal stem cells (MSC) involvement in osteophyte formation; nevertheless, it remains to be characterised. MSC from numerous mesenchymal tissues have been isolated but bone marrow remains the “ideal” due to the ease of ex vivo expansion and multilineage potential. However, the bone marrow stroma has a relatively low number of MSC, something that necessitates the need for long-term culture and extensive population doublings in order to obtain a sufficient number of cells for therapeutic applications. MSC in vitro have limited proliferative capacity and extensive passaging compromises differentiation potential. To overcome this barrier, tissue derived MSC are of strong interest for extensive study and characterisation, with a focus on their potential application in therapeutic tissue regeneration. To date, no MSC type cell has been isolated from osteophyte tissue, despite this tissue exhibiting all the hallmark features of a regenerative tissue. Therefore, this study aimed to isolate and characterise cells from osteophyte tissues in relation to their phenotype, differentiation potential, immuno-modulatory properties, proliferation, cellular ageing, longevity and chondrogenesis in in vitro defect model in comparison to patient matched bone marrow stromal cells (bMSC). Osteophyte derived cells were isolated from osteophyte tissue samples collected during knee replacement surgery. These cells were characterised by the expression of cell surface antigens, differentiation potential into mesenchymal lineages, growth kinetics and modulation of allo-immune responses. Multipotential stem cells were identified from all osteophyte samples namely osteophyte derived mesenchymal stem cells (oMSC). Extensively expanded cell cultures (passage 4 and 9 respectively) were used to confirm cytogenetic stability and study signs of cellular aging, telomere length and telomerase activity. Cultured cells at passage 4 were used to determine 84 pathway focused stem cell related gene expression profile. Micro mass pellets were cultured in chondrogenic differentiation media for 21 days for phenotypic and chondrogenic related gene expression. Secondly, cell pellets differentiated overnight were placed into articular cartilage defects and cultured for further 21 days in control medium and chondrogenic medium to study chondrogenesis and cell behaviour. The surface antigen expression of oMSC was consistent with that of mesenchymal stem cells, such as lacking the haematopoietic and common leukocyte markers (CD34, CD45) while expressing those related to adhesion (CD29, CD166, CD44) and stem cells (CD90, CD105, CD73). The proliferation capacity of oMSC in culture was superior to that of bMSC, and they readily differentiated into tissues of the mesenchymal lineages. oMSC also demonstrated the ability to suppress allogeneic T-cell proliferation, which was associated with the expression of tryptophan degrading enzyme indoleamine 2,3 dioxygenase (IDO). Cellular aging was more prominent in late passage bMSC than in oMSC. oMSC had longer telomere length in late passages compared with bMSC, although there was no significant difference in telomere lengths in the early passages in either cell type. Telomerase activity was detectable only in early passage oMSC and not in bMSC. In osteophyte tissues telomerase positive cells were found to be located peri vascularly and were Stro-1 positive. Eighty-four pathway-focused genes were investigated and only five genes (APC, CCND2, GJB2, NCAM and BMP2) were differentially expressed between bMSC and oMSC. Chondrogenically induced micro mass pellets of oMSC showed higher staining intensity for proteoglycans, aggrecan and collagen II. Differential expression of chondrogenic related genes showed up regulation of Aggrecan and Sox 9 in oMSC and collagen II in bMSC. The in vitro defect models of oMSC in control medium showed rounded and aggregated cells staining positively for proteoglycan and presence of some extracellular matrix. In contrast, defects with bMSC showed fragmentation and loss of cells, fibroblast-like cell morphology staining positively for proteoglycans. For defects maintained in chondrogenic medium, rounded, aggregated and proteoglycan positive cells were found in both oMSC and bMSC cultures. Extracellular matrix and cellular integration into newly formed matrix was evident only in oMSC defects. For analysis of chondrocyte hypertrophy, strong expression of type X collagen could be noticed in the pellet cultures and transplanted bMSC. In summary, this study demonstrated that osteophyte derived cells had similar properties to mesenchymal stem cells in the expression of antigen phenotype, differential potential and suppression of allo-immune response. Furthermore, when compared to bMSC, oMSC maintained a higher proliferative capacity due to a retained level of telomerase activity in vitro, which may account for the relatively longer telomeres delaying growth arrest by replicative senescence compared with bMSC. oMSC behaviour in defects supported chondrogenesis which implies that cells derived from regenerative tissue can be an alternative source of stem cells and have a potential clinical application for therapeutic stem cell based tissue regeneration.