<p>The prevalence of allergic disease such as peanut (PN) allergy has increased within the last century. Environmental factors have been associated with an increased risk of developing allergic diseases. The severity of allergic diseases has also increased and clinical trials are investigating allergen-specific immunotherapy as a method to treat allergies. The purpose of this work was to identify a vaccine adjuvant that induced potent antigen-specific Th1 immune responses and determine its ability to reduce the development and severity of Th2- mediated allergic disease, using models of peanut hypersensitivity.</p><p>Three studies were performed. The first study compared a variety of vaccine adjuvants to identify a potent adjuvant with strong Th1-associated activity. This study verified that the Toll-like receptor (TLR) ligand CpG could induce potent Th1-associated immune responses. The second study tested the ability of environmental endotoxin levels and alum-adjuvanted vaccines to modulate the development of allergic disease using a mouse model of peanut allergy. Additionally, the TLR ligands, CpG and MPL, were combined with alum-adjuvanted vaccines to determine their ability to further impact allergic disease development. Results suggested that the addition of CpG to an alum-adjuvanted vaccine indirectly modified host immunity in a manner that decreased the development of PN-induced allergic disease. The last study evaluated the ability of CpG to reduce the severity of peanut allergy symptoms when combined with peanut in an immunotherapy formulation administered to peanut-hypersensitive mice. Nasal immunotherapy with PN + CpG but not PN alone or CpG alone reduced the severity of PN-induced anaphylaxis in hypersensitive mice. PN-hypersensitive mice treated with PN + CpG displayed an increased PN-specific IgG2c and IFN-γ responses. A reduction in allergic disease severity in PN-hypersensitive mice correlated with an increase in PN-specific IgG2c, IFN-γ and IL-10 responses and a reduction in PN-specific IL-13 responses, suggesting a shift from Th2 responses towards Th1 and/or T regulatory cell responses.</p><p>Taken together, the data obtained from these studies demonstrate the potent activity of CpG to induce antigen-specific Th1-associated immune responses and also reduce the severity of peanut-hypersensitivity in mice through direct and indirect association with peanut allergens.</p> / Dissertation

No description available.

A number of mathematical models of cancer growth and treatment are presented. The most significant model presented is of the interactions between a growing tumor and the immune system. The equations and parameters of the model are based on experimental and clinical results from published studies. The model includes the primary cell populations involved in effector-T-cell-mediated tumor killing: regulatory T cells, helper T cells, and dendritic cells. A key feature is the inclusion of multiple mechanisms of immunosuppression through the main cytokines and growth factors mediating the interactions between the cell populations. Decreased access of effector cells to the tumor interior with increasing tumor size is accounted for.The model is applied to tumors of different growth rates and antigenicities to gauge the relative importance of the various immunosuppressive mechanisms in a tumor. The results suggest that there is an optimum antigenicity for maximal immune system effect. The immunosuppressive effects of further increases in antigenicity outweigh the increase in tumor cell control due to larger populations of tumor-killing effector T cells. The model is applied to situations involving cytoreductive treatment, specifically chemotherapy and a number of immunotherapies. The results how that for some types of tumors, the immune system is able to remove any tumor cells remaining after the therapy is finished. In other cases, the immune system acts to prolong remission periods. A number of immunotherapies are found to be ineffective at removing a tumor burden alone, but offer significant improvement on therapeutic outcome when used in combination with chemotherapy.Two simplified classes of cancer models are also presented. A model of cellular metabolism is formulated. The goal of the model is to understand the differences between normal cell and tumor cell metabolism. Several theories explaining the Crabtree Effect, hereby tumor cells reduce their aerobic respiration in the presence of glucose, have been put forth in the literature; the models test some of these theories, and examine their plausibility.A model of elastic tissue mechanics for a cylindrical tumor growing within a ductal membrane is used to determine the buildup of residual stress due to growth. These results can have possible implications for tumor growth rates and morphology.

Infection of people with human immunodeficiency virus (HIV) as well as LPBM5 infection in mice results in progressive deterioration of the immune system in the majority of untreated hosts. Peptide immunotherapy has been shown to be effective in the stimulation or immunoregulation of T-helper 1 (TH1) and T-helper 2 (TH2) response subsets. In murine acquired immunodeficiency syndrome (AIDS), TH1 deficiency enables the host to be susceptible to coxsackievirus infection, inducing cardiopathology in a short period. T-cell receptor (TCR) Vβ8.1 peptide, a 16-mer peptide containing the entire CFR1 segment and part of the FR2 region of human Vβ8, showed both an immunoregulating and immunostimulating effect in murine AIDS. TCR Vβ8.1 peptide acts on T cells promoting interleukin-2 production and therefore enhancing a cellmediated immune response. It retarded development of cardiopathology due to coxsackievirus infection. Retrovirus infected mice treated with the peptide showed a longer life span than the nontreated retrovirus infected animals.

There are currently no ideal treatments or acceptable vaccines for cutaneous leishmaniasis, a worldwide health problem caused by infection with a number of species of the dimorphic protozoa Leishmania. Therefore, there is an urgent need to search for simple, safe, effective, and affordable treatments. Imiquimod is an immune-response modifying agent. Recently, 5% imiquimod cream (Aldara(TM)) received approval by the Food and Drug Administration in the United States and is currently available for the treatment of external genital and perianal warts caused by human papillomavirus infection. The antiviral activity of this drug is mediated through stimulation of cytokine release from many cell types including macrophages resulting in a local immune response at the site of application. Moreover, imiquimod has been shown to enhance cell-mediated immune responses (CMIR). Since imiquimod activates macrophages, the exclusive host cells of Leishmania, and stimulates CMIR which are required for host defence against Leishmania, we have investigated the potential of using imiquimod and its related compound, S-28463, as agents for treating leishmaniasis. It is demonstrated within that imiquimod and S-28463 effectively stimulated leishmanicidal activity both in vitro in macrophages and in vivo in a mouse model. These compounds also stimulated signal transduction associated with the induction of nitric oxide synthesis in macrophages. Imiquimod and S-28463 induced leishmanicidal activity in macrophages in the absence of any other cell types. We have demonstrated that S-28463 generated macrophage leishmanicidal activity by inducing genes involved in macrophage activation and inflammatory responses. Finally, we have also performed an analysis on the influence of L. donovani on macrophage gene expression using a cDNA array analysis, a similar methodology to study the effect of S-28463 on macrophage gene expression. Intramacrophage infection with L. donovani was shown to cause general

No description available.

Endotoxin (LPS, lipopolysaccharide) forms an integral part of the outer cellular membrane of gram negative bacteria (GNB). The canines' intestine always contains large amounts of GNB, and hence LPS. If these GNB with their LPS, remain within the intestinal lumen, they are not harmful to the host. When GNB do gain entry into a hosts' circulation a bacteraemia will occur with a concurrent endotoxaemia. In the past, it had been accepted that GNB were, themselves, primarily responsible for the mortality and morbidity of bacteraemic and septicaemic patients. Evidence has emerged to indicate that this is not altogether true as isolated LPS, without the presence of GNB, can also lead to fatalities. Circulating LPS is exceptionally chemically stable and highly toxic to host cells. Antimicrobial chemotherapy can destroy GNB, but this therapy does not reduce the toxicity of LPS, nor does it clear LPS from the circulation. Destruction of the GNB by certain antibiotics can, in fact, increase the concentration of circulating plasma LPS in a host. The functional integrity of the intestinal wall is highly dependent upon an adequate blood supply, and the mucosal cells acts as the primary defence against the potentially pathogenic, endogenous and exogenous GNB and LPS. Once these pathogens become intravascular then the liver is the next most important organ of defence. Shock, irrespective of its aetiology, without adequate therapy, leads to reduced micro-vascular circulation, and thus a state of either localised or generalised hypoxia occurs. Partial or complete intestinal vascular ischaemia will produce a state of regional hypoxia, and lead to damage of the intestinal wall allowing GNB, with their LPS, or LPS by itself, to enter into the hosts' blood circulation. Therefore, an aetiology that gives rise to any type of "classified shock," may eventually give rise to concurrent endotoxaemia. In clinical practice there are numerous different diseases, physical onslaughts, and either acquired or congenital anatomical defects, that can give rise to intestinal vascular ischaemia, and hence, endotoxaemia. Many treatment regimens to combat the effects of an endotoxaemia have been advocated over the years, but this problem still has an unacceptably high mortality and morbidity index, probably because almost all such therapeutic regimens fail to destroy the LPS molecule. Recent clinical studies have shown that immunotherapy is effective in combating gram negative bacteraemia and septicaemia in humans and animals. Research workers have been able to produce a "broad- spectrum" or "polyvalent" equine, hyperimmune, anti-endotoxir, antibody-enriched plasma (ANTI- LPS), with favourab"^ responses recorded when this plasma was used to treat a variety of experimentally-induced endotoxin-shocked subjects. ANTI-LPS significantly reduced the mortality in experimentally produced superior mesenteric arterial occlusion endotoxaemia in rabbits, presumably by neutralizing and opsonizing the circulating plasma LPS. Equine practitioners have reported successful results when ANTI-LPS was incorporated into the treatment of certain medical and surgical equine endotoxic related problems. A ^/ery recent, independent, Canadian study showed the effectivness of ANTI-LPS, where this preparation was tested against other anti-LPS products, to treat experimentally-induced sepsis in rats. The polyvalent equine ANTI- LPS was the most effective, in that its use resulted in the longest survival. In order to establish the generality of the use of equine ANTI-LPS plasma, I have extended these studies to the canine, since an abdominal vascular ischaemia carries a serious, high-risk, surgical emergency with unsatisfactorily high mortality rates, despite successful surgical intervention with concurrent supportive medical therapy. Twenty healthy dogs were divided into four groups; a control group (n=5) and three experimentally treated groups (n=5 in each group). All twenty dogs were subjected to the well-documented cranial (superior) mesenteric arterial occlusion (CMAO) shock model. The three experimental groups received the polyvalent equine, ANTI-LPS at different times and by two different routes, with no side effects being observed in any of these dogs. One group (n=5)received ANTI-LPS s.c. before CMAO was performed, a second group (n= 5) received their dosage of ANTI-LPS i.v. during the three-hour occlusion period, and a third group (n=5) received their dose s.c, within three minutes after the CMAO was released. Survival was recorded when any dog lived for a minimum of 14 days after the occluded vessel was released. All 5/5 (100%) controls died within 17 hours after the release of the occluded vessel, whereas only one of the 15 (6,5%) experimentally ANTI-LPS treated dogs died (P / Thesis (M. Med.Sc.)-University of Natal, Durban, 1986.

Replicating viruses for the treatment of cancer have a number of advantages over traditional therapeutic modalities. They are highly targeted, self-amplifying, and have the added potential to act as both gene-therapy delivery vehicles and oncolytic agents. ORFV, (Parapoxvirus ovis, or Orf virus) is the prototypic species of the Parapoxvirus genus, causing a benign disease in its natural ungulate host. ORFV possesses a number of unique properties that make it an ideal viral backbone for the development of a cancer therapeutic: it is safe in humans, has the ability to cause repeat infections even in the presence of antibody, and it induces a potent Th-1 dominated immune response. Here I show for the first time that live replicating ORFV induces an anti-tumour immune response in multiple syngeneic mouse models of cancer that is mediated largely by the potent activation of both cytokine-secreting, and tumouricidal natural killer (NK) cells. I have also highlighted the clinical potential of the virus by demonstration of human cancer cell oncolysis including efficacy in an A549 xenograft model of cancer. The mechanism of ORFV-mediated activation of NK cells has been explored, where I have demonstrated activation via direct ex vivo infection of NK cells. I have also highlighted ORFV-mediated activation of dendritic cells (DCs), both in vivo and by direct infection ex vivo. An in vivo DC depletion study demonstrated an indirect mechanism for ORFV NK cell activation, where in the absence of DCs, NK cell activation was diminished, as was the ability of ORFV to clear lung metastases. The ORFV innate immune stimulatory profile has been harnessed for therapeutic application in an experimental surgery model of cancer, where ORFV therapy at the time of surgery reduces the number of cancer metastases. These data highlight the clinical potential of a live, immune stimulating Parapoxvirus therapeutic.

For the treatment of cancer, immunotherapy has some inherent advantages over other treatment modalities: disseminated disease can be eradicated due to the systemic nature of immunity, the immune system is effective against a wide range of targets, long-term memory can offer added protection against disease relapse, immunotherapy should be relatively non-toxic, and it can be synergistically combined with other treatment platforms such as radiation and chemotherapy. Type 1 immune responses are thought to be superior for the treatment of cancer and, as the quintessential Th1 polarizing cytokine, interleukin-12 (IL-12) holds much promise; however, optimal therapeutic protocols have yet to be developed and clinical results have fallen short of this promise. The in vivo IL-12 experiments described here highlight a characteristic of cellular therapy that has not previously been appreciated. That is, the effect of cell-mediated cytokine delivery on the immediate microenvironment and how that affects the immune response initiated. This observation has implications for the clinical application of IL-12 therapy but may also prove to be an important consideration when studying other immunostimulants. I have herein developed a novel in vitro assay system that I have used to dissect the cellular responses to IL-12 and to identify the signals that are required for activation of a cluster of differentiation 4 (CD4)+ effector population that affects leukaemia cell clearance both in vitro and in vivo. This work, and the future studies proposed, will expand our understanding of the potential of IL-12 immunotherapy and enhance our ability to manipulate therapeutic conditions to favour the desired response. Moreover, the in vitro assay system offers a method for further characterization of CD4+ effector cells and the development of protocols to initiate their potent anticancer activity.

Prostate cancer (PC) is the most common visceral cancer amongst men in Australia and elsewhere in the world. The American Cancer Society estimated that in 2006, PC alone would account for about 33% of newly diagnosed cancer in men, and be responsible for 9% of cancer related deaths in men. In men with advanced, metastatic PC, hormone therapy is widely accepted as the treatment of choice and produces good initial responses in most patients. However, many patients will relapse and become resistant to further hormone manipulation. Currently used treatment modalities for these patients are intended to palliate symptoms and therefore improve quality of life; the long term survival benefit of currently used management strategies is marginal at best. The limited treatment modalities with survival benefit for patients with advanced PC results in the need for development of novel therapies. Enhancement of the natural anti-tumour defences of the immune system to recognise and destroy tumour cells appears as a favourable alternative to prior therapies. Use of autologous dendritic cells (DC) as stimulators of an anti-tumour response has shown promise in several phase I, II and III trials. The Mater Medical Research Institute has developed a novel system for the isolation of blood DC (BDC). This system utilizes an antibody selection system based on a human/mouse chimeric CMRF-56 (huCMRF-56) monoclonal antibody (mAb) which has been engineered from the prototype murine IgG1 CMRF-56 mAb. Pre-clinical studies have demonstrated that the huCMRF-56 mAb isolates a CMRF-56+ cell population comparable to that obtained with the murine IgG1 CMRF-56 mAb with a sufficient CMRF-56+ BDC purity and yield to warrant its use as a BDC based immunotherapy. The objective of this research project was to validate the use of the huCMRF-56 mAb isolated BDC preparations in PC immunotherapy. This was achieved by; i) determining the optimal concentration of the huCMRF-56 mAb at which to perform isolations in order to obtain a CMRF-56+ cellular preparation with purity, yield and cellular composition, in terms of DC subsets, B cells, monocytes and contaminating cells, comparable to that obtained with the murine mAb, which has shown efficacy in in vitro studies; ii) Demonstrating that CMRF-56+ preparations obtained from PC patients using the huCMRF-56 BDC mAb are comparable to those from healthy donors (HD) and iii) demonstrating functional capacity of both freshly isolated and cryopreserved CMRF-56+ BDC isolated preparations to induce anti-tumour cytotoxic T lymphocyte (CTL) responses in both HD and PC patients. These studies utilised the appropriate immunostaining techniques and flow cytometry to determine the CMRF-56+ cell yield, CMRF-56+ BDC purity and viability of the preparation. To determine functional capacity CMRF-56+ preparations were isolated from HD and PC patients, and loaded with the prostate tumour associated antigens (TAA) and control antigen peptides: prostate specific antigen-3 (PSA-3), prostatic acid phosphatase- 5 (PAP-5), prostatic specific membrane antigen-2 (PSMA-2), flu matrix protein (FMP) and/or melanoma antigen recognised by T cells (MART). Co-culture experiments were set up with autologous peripheral blood mononuclear cells (PBMC) and induction of CTL responses were assessed using pentamer staining and ELISPOT assay. The working concentration of the huCMRF-56 mAb of 1.28mg/ml was determined to immunoselect a cellular preparation with maximal BDC purity, BDC yield and total viable cell number. Phenotyping studies of this preparation demonstrated it to be predominately comprised of antigen presenting cells (APC) and enriched for BDC with several BDC subsets immunoselected. CMRF-56+ preparations immunoselected from HD and PC donors were similar and comparable to previously described preparations immunoselected using the original murine CMRF-56 mAb. From antigen loaded CMRF-56+ preparations, specific major histocompatibility complex (MHC) class 1 restricted CD8 lymphocyte responses were generated against prostate TAAs in 2 of 5 HD and 1of 3 PC donors and against FMP in 1 of 3 PC and 5 of 5 HD. Cryopreserved antigen loaded CMRF-56+ BDC preparations from HD generated antigen specific FMP or MART-1 CTL responses in 3 of 4 HD, however anti-prostate TAA CTL responses were not observed. The humanised CMRF-56 mAb immunoselects a cellular preparation enriched in BDC and capable of effective uptake and presentation of antigen. The CMRF-56+ BDC enriched preparation, loaded with PC TAA is capable of inducing specific anti tumour responses in vitro. While further preclinical studies are required, the preparation, loaded with PC TAA shows promise as a potential immunotherapy for PC.