The problem investigated Four studies investigated the phenomenon of cancer-related fatigue (CRF) in women who had received adjuvant treatment for early-stage breast cancer, with a view to reducing the diagnostic uncertainty surrounding the syndrome and thus facilitating progress in both clinical management and aetiological research. Procedures and results A cross-sectional study of 109 women compared a ???cancer-specific??? self-report questionnaire (FACT-F) (canvassing fatigue symptoms) and a more generic questionnaire (SPHERE) (identifying depression and fatigue). Thirty-seven percent of women reported fatigue. Overall in 20%, fatigue was associated with psychological distress. Seventeen percent of women had fatigue but no depression. A qualitative study utilised focus groups to identify and compare the distinctive features of CRF with those of women with chronic fatigue syndrome (CFS). A similar set of symptoms was found in both groups, including overwhelming fatigue, un-refreshing sleep and subjective concentration problems. However, women with CFS also reported myalgia and arthralgia. Using the Structured Clinical Interview for Neurasthenia- SCIN, the third study compared the symptoms of three groups of women with fatigue: those with CRF, CFS or major depression. The detailed ???interviewer guide??? provided explicit directions for evaluating and classifying symptoms. This study confirmed the core symptom of ???profound fatigue unrelieved by rest???, and additional features that distinguished between the clinical diagnoses. The fourth study compared features of the evolution of clinically-identified fatigue syndromes in women from two prospective cohort studies; women with post-cancer fatigue (PCF) and women with post-infective fatigue syndrome (PIFS). Major conclusions A syndrome of PCF, present at least six months following adjuvant treatment and unexplained by medical or psychiatric disorder was investigated. The characteristics of PCF and those of CFS are very similar, with the fatigue state having indistinguishable descriptors. Longitudinal evaluation of the symptom complexes of PCF and PIFS suggests divergent pathways may be relevant. Co-morbid features like sleep disturbance; physical deconditioning and mood disturbance may be implicated as factors in the evolution and prolongation of PCF. These studies provide a basis for a more uniform and rigorous classification system - a necessary first step towards advancing the field both in investigating aetiology and new intervention strategies.

Immunotherapy as a means for cancer treatment has been investigated for over a century. While studies have been completed using different immunological strategies, development of a clinical therapeutic cancer vaccine has proven elusive. Recently, success has been seen with prophylactic vaccines for cancers with known viral origins (Gardasil® and Cervarix for Human Papiloma Virus). However, such strategies do not address the challenge in generating effective immune response against other tumor antigens, most of which are weakly immunogenic self-antigens. Tolerance to these self-antigens could ultimately limit the patient’s ability to mount an effective anti-tumor immune response. The US Food and Drug Administration recently approved the first DC cell-based cancer vaccine, Provenge®, for use in prostate cancer. This vaccine requires cell isolations from the patient as well as in vitro DC modifications, which ultimately leads to high cost as well as multiple procedures. However, results indicate that, on average, patients live only four months longer than those receiving a placebo. While this work remains important, and offers proof that priming DCs can improve the lifespan of a patient, it ultimately does not offer a long-term cure. Direct and highly efficient in vivo delivery of antigens to DCs could overcome the challenges associated with ex vivo DC manipulation and may offer a more scalable method for generating anti-tumor immunity. This research focuses on the development of novel formulations that allow simultaneous delivery of protein/peptide-based tumor antigens and immune-modulatory nucleic acids (siRNA and immune stimulatory CpG) to the same dendritic cells (DCs) in-vivo. Such formulations allow a synthetic immune-priming center to be created at the site of immunization and simultaneously deliver the tumor antigen to DCs and modulate their immune response through IL-10 silencing. Our hypothesis is that using such a DC-targeted dual delivery system we will be able to illicit strong T helper 1 (TH1) and Cytotxic T Lymphocyte (CTL) response in vivo against a wide array of tumor antigens. This can become a platform technology where the biomolecules (antigen and immunomodulatory agents) can be easily varied based on particular cancers. / text

Sammanfattning   Bakgrund: Bröstcancer är en av de vanligaste cancerformerna i vårt land. I huvudsak behandlas bröstcancer med kirurgi men tilläggsbehandling med cytostatika-, strål- och hormonell behandling är vanligt. Under behandlingen kommer kvinnorna att drabbas av bieffekter som kan orsaka lidande. Syfte: Studiens syfte var att beskriva kvinnors erfarenheter under tiden de får adjuvant cytostatika- och strålbehandling för bröstcancer; Hur upplever de det och hur hanterar de upplevelserna? Metod: En systematisk litteraturstudie gjordes. Efter genomförd sökning i databaserna, PubMed och Cinahl, kvalitetsgranskades och inkluderades tio artiklar. En kvalitativ innehållsanalys genomfördes. Resultat: Analysprocessen resulterade i två teman; Att vara drabbad av adjuvant behandling och Att finna ett sätt att hantera upplevelserna av adjuvant behandling. Resultatet visade att det upplevdes som ett stort lidande att få cytostatika- och strålbehandling men att det även fanns positiva aspekter. Lidandet låg i alla behandlingsrelaterade biverkningar, att de ofta hade en känsla av ensamhet och nedstämdhet, och att de upplevde brist på kontroll. Lusten fanns i att ha fått ett nytt perspektiv på livet och en ny syn på vad som var viktigt. Slutsats: Att kämpa för att upprätthålla ett meningsfullt liv och att inte ses som annorlunda var ett genomgående tema. Individanpassad vård behövs för att bemöta kvinnornas upplevelser och stödja hanteringen. / Maria Lidbäck

The problem investigated Four studies investigated the phenomenon of cancer-related fatigue (CRF) in women who had received adjuvant treatment for early-stage breast cancer, with a view to reducing the diagnostic uncertainty surrounding the syndrome and thus facilitating progress in both clinical management and aetiological research. Procedures and results A cross-sectional study of 109 women compared a ???cancer-specific??? self-report questionnaire (FACT-F) (canvassing fatigue symptoms) and a more generic questionnaire (SPHERE) (identifying depression and fatigue). Thirty-seven percent of women reported fatigue. Overall in 20%, fatigue was associated with psychological distress. Seventeen percent of women had fatigue but no depression. A qualitative study utilised focus groups to identify and compare the distinctive features of CRF with those of women with chronic fatigue syndrome (CFS). A similar set of symptoms was found in both groups, including overwhelming fatigue, un-refreshing sleep and subjective concentration problems. However, women with CFS also reported myalgia and arthralgia. Using the Structured Clinical Interview for Neurasthenia- SCIN, the third study compared the symptoms of three groups of women with fatigue: those with CRF, CFS or major depression. The detailed ???interviewer guide??? provided explicit directions for evaluating and classifying symptoms. This study confirmed the core symptom of ???profound fatigue unrelieved by rest???, and additional features that distinguished between the clinical diagnoses. The fourth study compared features of the evolution of clinically-identified fatigue syndromes in women from two prospective cohort studies; women with post-cancer fatigue (PCF) and women with post-infective fatigue syndrome (PIFS). Major conclusions A syndrome of PCF, present at least six months following adjuvant treatment and unexplained by medical or psychiatric disorder was investigated. The characteristics of PCF and those of CFS are very similar, with the fatigue state having indistinguishable descriptors. Longitudinal evaluation of the symptom complexes of PCF and PIFS suggests divergent pathways may be relevant. Co-morbid features like sleep disturbance; physical deconditioning and mood disturbance may be implicated as factors in the evolution and prolongation of PCF. These studies provide a basis for a more uniform and rigorous classification system - a necessary first step towards advancing the field both in investigating aetiology and new intervention strategies.

The mechanism of resistance of tumor cells to chemotherapeutic agents is explored using probabilistic methods where it is assumed that resistant cells arise spontaneously with a defined frequency. The resistance process is embedded in a discrete time Markov branching process which models the growth of the tumor and contains three seperate cell types: stem, transitional and end cells. Using the asymptotic properties of such models it is shown that the proportion of each type of cell converge to constants almost surely. It is shown that the parameters relating to stem cell behaviour determine the asymptotic behaviour of the system. It is argued that for biologically likely parameter values, cure of the tumor will occur if, and only if, all stem cells are eliminated. A model is developed for the acquisition of resistance by stem cells to a single drug. Probability generating functions are derived which describe the behaviour of the process after an arbitrary sequence of drug treatments. The probability of cure, defined as the probability of ultimate extinction of the stem cell compartment, is characterised as the central quantity reflecting the success of therapeutic intervention. Expressions for this function are derived for a number of experimental situations. The effects of variation in the parameter values are examined. The model is extended to the case where two anticancer drugs are available and formulae for the probability of cure are developed. The problem of therapeutic scheduling is examined and under situations where drugs are of "equal" effectiveness, but may not be given together, it is shown that the mean number of tumor cells is minimised by sequential alternation of the drugs. The models are applied to data collected on the L1210 leukemia treated by the drugs Cyclophosphamide and Arabinosylcytosine. In both cases the analysis of the data provide evidence that resistant cells arise spontaneously with a frequency of approximately 10⁻⁷ per division. When applied to human breast cancer, the model indicates that neoadjuvant therapy is unlikely to greatly influence the likelihood that the patient will die from the growth of drug-resistant cells. / Science, Faculty of / Statistics, Department of / Graduate

Mycolic acids, the characteristic, abundant waxes of the cell wall of Mycobacteria were purified by Counter Current Distribution (CCD) from alkaline methanolytic crude extracts of bacteria, aiming at investigating their role in eliciting immune responses. Crude mycobacterial cell-wall extracts were first made by saponification in potassium hydroxide methanol solution. Purification was then performed with CCD using a bi-phasic tricomponent system, consisting of double distilled deionized water (dddH2O), chloroform and methanol. Emulsions were formed in this system which in turn extended the purification time. The addition of a preliminary funnel extraction step, to reduce the saponified fatty acids in the crude extract, before CCD and the addition of NaCI as an emulsions breaker in the CCD solvent system, produced a high yield of pure mycolic acids. The purity of these mycolic acids were assessed using reversed-phase HPLC-analysis. This method proved not only to be applicable to purify mycolic acids from M. tuberculosis but was also applicable in purifying mycolic acids from other sources, such as M. vaccae. The immunogenic properties of the purified mycolic acids were confirmed in experiments in which they induced the formation of antibodies in Sprague-Dawley rats when immunized in Marcol 52 oil. The antibody response was monitored by ELISA after 3 months of repeated immunization every second week. A dose-related response was observed for the induction of antibodies specific for mycolic acids, immobilized on the ELISA plates. Mycolic acids also appeared to influence adjuvant arthritis. Pure mycolic acids, suspended in mineral oil were administered intradermally into Lewis rats one week before the intradermal administration of an arthritis-inducing dose of lyophilized M. tuberculosis H37Ra. Animals receiving Mycobacteria, but no mycolic acids treatment, developed severe symptoms of arthritis within two weeks after bacterial challenge. No arthritis symptoms were apparent in mycolic acids treated rats. Mycolic acids treatment alone, did not produce arthritis. Mycolic acids pre-treatment of M. tuberculosis H37Rv-infected mice, rendered tuberculosis susceptible Balb/c mice more resistant. This resistance was equivalent to that observed in tuberculosis resistant C57Bl/6 mice. Post-infection treatment of M. tuberculosis H37Rv-infected mice with MA had no effect. Resistance of C57Bl/6 mice is commonly associated with the expression of IL-12 and IFN-ã. The effect of mycolic acids in the spleens of M. tuberculosis-infected Balb/c mice was investigated. It was observed that there was no significant change on the THI and TH2 cytokines. The absence of mycolic acids-induced THI/TH2 cytokine bias implied that protection was not provided by the expression of IL-12 and IFN-ã in the spleen. These results support the hypothesis that mycolic acids are immunogenic in respect of being able to induce specific antibodies, to provide resistance against tuberculosis and to prevent the development of adjuvant arthritis. The mechanism by which mycolic acids perform these tasks is unknown, particularly in these rodent models, which differ from humans, in that they do not have the CD1b that presents mycolic acids in humans. Unravelling this mechanism, can possibly aid the development of a pharmaceutical formulation that introduces MA into the body to enhance resistance to TB and prevent arthritis as an associated side-reaction. / Dissertation (MSc)--University of Pretoria, 2010. / Biochemistry / unrestricted

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Doctor of Philosophy / Department of Anatomy and Physiology / Jishu Shi / Porcine reproductive and respiratory syndrome (PRRS) is one of the most economically important swine diseases worldwide that leads to severe reproductive failure in sows and high mortality in young pigs. Vaccination is currently the most effective way to control this disease. The protection ability provided by vaccines however is limited due to the large diversity of field PRRSV strains. In chapter 2, we compared immune responses induced by vaccination and/or PRRSV infection by using IngelVac® Modified Live PRRSV vaccine (MLV), its parental strain VR-2332, and the heterologous KS-06 strain. Our results showed that MLV provide complete protection to homologous virus and partial protection to heterologous challenge. The protection was associated with the levels of PRRSV neutralizing antibodies at the time of challenge. Besides developing new vaccines to combat PRRSV, adjuvants have been applied to PRRSV MLV vaccines to induce vaccination-mediated cross-protection against genetically dissimilar PRRSV strains. In chapter 3, we demonstrated that a commercial MontanideTM Gel01ST adjuvant provides enhanced protection to homologous PRRSV infection by regulating the production of PRRSV-specific antibodies. In chapter 4, we tested a novel peptide nanofiber hydrogel acting as a potent adjuvant for PRRSV MLV vaccines. We found that the hydrogel adjuvant enhanced vaccine efficacy by developing of higher titers of neutralizing antibodies and stronger IFN-γ cellular immune responses. Chinese highly pathogenic PRRSV (HP-PRRSV) variants were isolated in 2006 and they belong to genotype 2 of PRRSV. Compared with classic PRRSV, HP-PRRSV is characterized by robust proliferation ability and high morbidity/mortality with all ages of pigs. In chapter 5, we compared the difference of immune responses elicited by HV-PRRSV, a Chinese HP-PRRSV, and a US virulent strain of PRRSV NADC-20. Traditional PRRSV MLV vaccines developed in US offer no protection to HP-PRRSV. Vaccines specific to HP-PRRSV strains available in China provide protection to HP-PRRSV. In chapter 6, we demonstrated that pigs challenged with US NADC-20 strain were protected by vaccination with Chinese MLV HP-PRRSV vaccines. The availability of Chinese HP-PRRSV vaccines in North America may act to increase the preparedness of possible transmission of HP-PRRSV to North America.

The efficacy of some vaccines is enhanced by the presence of adjuvants added to their formulations or, in the case of live attenuated or killed whole cell vaccines, because of their endogenous adjuvant activity. The immune system responds robustly to these endogenous adjuvants, which includes Pathogen Associated Molecular Patterns, which stimulate innate immune responses through Pattern Recognition Receptors, such as TOLL-like receptors (TLRs). The development of most vaccine adjuvants has occurred despite little understanding of their overall mechanisms of immune enhancement. We hypothesized that TLR-dependent adjuvant activities are mediated through TLR stimulation of antigen presenting cells (APCs), and each APC type may play a unique role in the immune-stimulating ability of these adjuvants, including effects on downstream T cell stimulation. We used a mouse model where TLR/MyD88 signaling is prevented in specific APC types, in vivo, using loxP/cre recombinase transgenic mice (B cells, dendritic cells and macrophages) to investigate its role in vaccine adjuvant activity. We found that intact MyD88 signaling is essential, separately, in all three APC types for optimal TLR-ligand based adjuvant (PorB, CpG, MPLA), but not for TLR-independent (Alum, MF59) adjuvant activity. However, the immune responses were reduced to the greatest extent in mice with macrophage specific MyD88 deletion (Mac-MyD88-/-). We demonstrated that TLR-dependent adjuvants are potent inducers of germinal center (GCs) formation needed for an effective and robust immune response. Interestingly, GCs are nearly absent in Mac-MyD88-/- mice upon immunization with TLR-dependent adjuvants, but not with TLR-independent adjuvants. Further investigations revealed a significant impairment in T cell cytokines important for GC formation in Mac-MyD88-/- mice when immunized with TLR-dependent adjuvants. Through these studies we discovered that vaccine formulated with PorB/OVA induced a robust and diverse T cell response including highly functional OVA-CD4 and CD8 T cells. These CD8 T cells are protective and significantly reduced the bacterial burden and increased survival in a Listeria mouse infection model. Our findings reveal that PorB has broad adjuvant activity, signaling through all three APC types, inducing strong and diverse humoral and cellular responses. These insights will allow for a more intelligent use of adjuvants in future vaccine development.