Rozprawy doktorskie na temat „Adjuvant”

La grande maggioranza dei patogeni invade l’ospite o causa malattie attraverso le mucose. Lo sviluppo di nuove strategie di immunizzazione per indurre risposte immunitarie a livello delle mucose è ampiamente richiesto. Attualmente, sono disponibili pochi vaccini mucosali, nessuno dei quali è costituito da proteine ricombinanti o subunità di patogeni, questo principalmente a causa della mancanza di adiuvanti mucosali potenti e sicuri. Dato il ruolo cruciale dei metaboliti della vitamina A, come l’acido retinoico (RA), nel conferire alle cellule del sistema immunitario la capacità di migrare nei distretti mucosali, e di promuovere il differenziamento di cellule che producono IgA, i metaboliti della vitamina A sono potenziali molecole candidate per migliorare le risposte immunitarie a livello delle mucose. In questo studio, sono state investigate nuove strategie di immunizzazione per amplificare le risposte immunitarie sia sistemiche che mucosali tramite la somministrazione di RA. In una prima fase, abbiamo osservato che il trattamento con RA sinergizza con la tossina colerica (CT), nell’aumentare le risposte antigene (Ag)-specifiche mucosali e sistemiche in seguito ad un’immunizzazione intranasale. Nel corso di questi esperimenti, abbiamo osservato che la somministrazione dell’Ag intranasale in assenza di adjuvante era in grado di generare una risposta a livello mucosale in topi che erano stati trattati con RA. Ci siamo quindi chiesti se tale risposta potesse essere amplificata in seguito ad un boost sistemico con l’Ag in combinazione con un adjuvante sistemico. Abbiamo osservato che la combinazione di un priming mucosale con Ag da solo, seguito da un boost sistemico con Ag e Alum, induce risposte mucosali e sistemiche durevoli nel tempo. In una seconda fase, abbiamo valutato gli effetti della somministrazione di RA in concomitanza con una vaccinazionsistemica utilizzando un vaccino a subunità contro la tubercolosi. Tale vaccino è composto da un adjuvante sistemico CAF01 e da un Ag di fusione derivato da tre proteine antigeniche di Mycobacterium tuberculosis (Mtb), H56. Abbiamo osservato che il trattamento con RA promuove la risposta anticorpale Agspecifica sia sistemica (IgG) che mucosale (IgA) e determina la colonizzazione dei distretti mucosali da parte dei linfociti T CD4+ Ag-specifici. Infine, abbiamo valutato gli effetti di RA sulla protezione verso un challenge con il ceppo virulento di Mtb (H37Rv) di topi immunizzati con il vaccino a subunità CAF01/H56. Abbiamo osservato che l’immunizzazione sistemica con CAF01 e H56 in presenza di RA ha un impatto sul contenimento della crescita di Mtb a livello polmonare 14 giorni dopo l’infezione. Inoltre, è stata rilevata una più alta produzione di citochine pro-infiammatorie nei polmoni di topi immunizzati con CAF01 e H56 24 ore dopo l’infezione. In conclusione, questo approccio che ha lo scopo di generare le risposte a livello sistemico ed indirizzarle nei distretti mucosali utilizando modulatori della migrazione cellulare come l’RA, potrebbe contribuire a far progredirela ricerca degli adiuvanti. Risposte mucosali potrebbero essere generate utilizzando gli adiuvanti sistemici disponibili in assenza di adiuvanti mucosali.
The vast majority of pathogens invade the host through or cause disease at mucosal surfaces. Development of novel immunization strategies suitable for mucosal vaccines is widely desired to protect against infectious diseases. However, very few mucosal vaccines are available for human use, none of which are recombinant proteins or subunits of pathogens, owing to the lack of potent and safe mucosal adjuvants. Given the crucial role of Vitamin A metabolites, such as retinoic acid (RA) in imprinting a mucosal homing capacity on T and B cells, as well as its potential to promote the differentiation of IgA-producing plasma cells, RA holds potential as a candidate molecule to improve mucosal vaccinations. In this study, we investigated new strategies of immunization to amplify both systemic and mucosal immune responses by administering RA. First, we observed that treatment with RA synergises with the adjuvant capacity of cholera toxin (CT) to enhance both systemic and mucosal Ag-specific immune responses. The combination of mucosal priming with Ag alone, followed by a boost with systemic adjuvant was also evaluated. Mice treated with RA showed a higher titer of mucosal IgA compared to untreated mice, after intranasal priming with Ag followed by a systemic boost with Ag plus Alum. After eight months, higher IgG Ag-specific antibodies in the serum and a higher frequency of Ag-specific IgG and IgA secreting cells were detected in the bone marrow of mice treated with RA as compared to untreated mice. Higher percentages of proliferating CD4 and CD8 T cells upon Ag stimulation were found in the spleens, in the mesenteric lymph nodes and in the colonic lamina propria of mice treated with RA. Next, we evaluated the effects of RA on systemic vaccination with a subunit vaccine against tuberculosis. This vaccine includes CAF01 as adjuvant and the mycobacterial derived fusion protein H56. We found that mice treated with RA as compared with untreated ones, showed enhanced mucosal (IgA) H56 mycobacterial fusion proteinspecific antibody responses and enhanced Ag-specific CD4+ T lymphocytes harbouring the lung after systemic immunization with the TB subunit vaccine. Therefore, we evaluated the effects of RA on protection against challenge with virulent Mycobacterium tuberculosis (Mtb) strain after systemic vaccination with the TB subunits vaccine (CAF01 and H56). Vaccination with BCG was included in the experiment as control. We found that immunization with CAF01 and H56 in presence of RA leads to a lower bacterial colonization in the lungs 14 days after challenge as compared to control mice. Furthermore, higher pro-inflammatory cytokine production, such as IFNγ and IL-17 was found in the lung of mice immunized with CAF01 and H56 in presence of RA 24h after Mtb infection. Therefore, we hypothesize that the mucosal immune responses elicited during vaccination in presence of RA could have an impact on the containment of bacterial growth in the lungs. This approach can contribute to progress beyond the state of the art in adjuvant research by achieving mucosal immunity in the absence of mucosal adjuvants.

L’administration de vaccins par voie muqueuse (orale et nasale) est une alternative efficace aux injections classiques. En effet, au-delà d’une plus grande compliance pour les patients et les soignants, ces voies ont l’avantage de déclencher une immunité dite muqueuse, grâce à la présence d’un système immunitaire propre (aussi appelé MALT pour Mucosal Associated Lymphoid Tissue). Ce MALT est situé à la surface des épithélia de revêtement. Il est différencié en îlots distinguables, les Follicular Associated Epithelium (FAE), et est constitué de cellules épithéliales spécialisées dans la surveillance et le prélèvement de pathogènes, les cellules M. Celles-ci surplombent une zone riche en cellules présentatrices d’antigènes (CPA) et lymphocytes, les Interfollicular Regions (IFR). Ainsi lors d’une infection, les cellules M sont capables de prélever et transloquer des fragments antigéniques vers les CPA, qui initieront la réponse immunitaire auprès des lymphocytes. Cette réponse se traduira par une immunité humorale et cellulaire au niveau de l’épithélium infecté, au niveau des muqueuses plus distantes, mais aussi au niveau systémique. Comme la majorité des infections se produisent au niveau des muqueuses, cette stratégie d’immunisation est de plus en plus étudiée.Si les antigènes sous-unitaires sont moins toxiques que les vaccins vivants, ils sont aussi moins immunogènes, et leur administration nécessite la présence d’un adjuvant pour stimuler efficacement les CPA et initier une réponse immunitaire forte. Or, par la voie des muqueuses, les molécules immunomostimulantes classiquement utilisées pour les voies injectables (par exemple les toxines bactériennes, le LPS ou encore certaines émulsions) sont souvent reportées comme étant toxiques. L’utilisation de systèmes de délivrance pour vectoriser les antigènes jusque dans les CPA semble donc être une alternative séduisante.On distingue deux types de vecteurs d’antigènes : les particules immunomodulatrices, et les systèmes de délivrance purs. Les premiers délivrent les antigènes dans les CPA, et en parallèle, stimulent fortement les voies pro-inflammatoires, pour orienter la balance immunitaire Th1/Th2. Parmi ces vecteurs, les plus utilisés en tests cliniques sont les virus-like particles (VLP), les émulsions à base de saponines (ISCOMs) ou les liposomes contenant des éléments bactériens (MPL, CpG…). En revanche, leur utilisation par voie muqueuse, et notamment nasale, est confrontée aux mêmes risques de toxicité que les adjuvants classiques. Les systèmes de délivrance purs, quant à eux, n’améliorent l’immunogénicité des antigènes qu’en les délivrant dans les cellules, mimant ainsi une infection naturelle. Et bien qu’ils soient mieux tolérés par les muqueuses, leur efficacité doit s’orienter vers une pénétration du mucus, ainsi qu’une association, protection et délivrance des antigènes dans les CPA bien plus performantes.Durant cette thèse, nous avons ainsi étudié les mécanismes permettant à des nanoparticules (NPs) de maltodextrine cationiques et lipidées (NPL) de délivrer des antigènes par voie nasale.Nous avons tout d’abord évalué la capacité des NPL à franchir le mucus des voies respiratoires, en comparaison avec des nanoparticules mucopénétrantes (des PLGA recouvertes de PEG, ou PLGA-PEG) et des nanoparticules mucoadhérentes (des PLGA recouvertes de chitosan, ou PLGA-CS). En mesurant le déplacement des différentes NPs dans du mucus respiratoire reconstitué, nous avons observé que, grâce à la présence du coeur de phospholipides anioniques, la NPL était capable de se déplacer dans le mucus, contrairement aux PLGA-CS qui restaient immobiles [...]
The mucosal routes of immunization present several advantages compared to classical injection routes. Indeed, besides a better compliance towards patients, these routes possess their own immune system, also known as the Mucosal Associated Lymphoid Tissue (MALT), able to trigger a local mucosal response after immunization. This tissue is mainly located in the nasal and intestinal mucosa, where it is spread in small extents called Follicular Associated Epithelium (FAE). On their apical surface, the FAE contain specialized epithelial microfold cells (or M cells), whose role is to survey potential infections by sampling pathogenic fragments, and which overlay a lymphocyte and antigen presenting cells (APC) zone. Then, when an infection occurs, M cells sample and translocate antigenic fragments to CPA, which could therefore trigger lymphocyte maturation and the initiation of the subsequent immune response. This activation will lead to both humoral and cellular immunity in the infected epithelium and could also spread to distant mucosa. As many pathogens infect the body through mucosa, this way of immunization is often considered.Adjuvants are frequently added to subunit vaccines to enhance their immunogenicity toward APC. Indeed, despite their lower toxicity, they are also less immunogenic than live-attenuated vaccines. However, the administration of classical adjuvanting molecules, such as toxins or immunostimulating emulsions, via mucosal routes, has often led to serious adverse effects. Therefore, the alternative use of delivery systems to deliver antigen in APC after mucosal administration is more and more studied.Antigen delivery systems include immunomodulating particles, and inert delivery systems. The first ones can enhance the mucosal antigen bioavailability by vectorizing antigens to APC, and at the same time trigger intracellular pro-inflammatory pathways, to drive the Th1/Th2 immune balance. Among them, virus-like particles (VLP), saponin-based emulsions (ISCOMs) or MPL-containing liposomes are the most represented in clinical trials. However, their mucosal administration can lead to the same adverse effects than classical immunostimulating molecules. In parallel, true delivery systems can enhance the antigens immunogenicity by increasing their intracellular delivery, thus mimicking a natural infection. They are therefore far less toxic for the mucosa than immunomodulating particles but need to be more efficient in the mucus penetration, in the antigen association and in the APC intracellular delivery.During this thesis, we deciphered the mechanisms allowing cationic and lipidated maltodextrine nanoparticles (NPL) to deliver antigens after nasal administration.We first evaluated the ability of NPL to cross the airway mucus barrier, compared to mucopenetrant particles (PEG-coated PLGA or PLGA-PEG) and mucoadherent particles (chitosan-coated PLGA or PLGA-CS), by measuring their displacement in reconstituted mucus. We observed that in presence of the phospholipid core, the NPL were able to move in the mucus, while PLGA-CS NPs remained stuck in the gel. Moreover, we observed that the NPL uptake and the protein delivery in airway epithelial cells were not impaired by the presence of mucins, contrary to PLGA-CS that were hindered by the mucins, and to PLGA-PEG which were not taken up by the cells, due to their neutral surface charge. We finally demonstrated that the NPL mucopenetration was allowed thanks to steric and repulsive electrostatic forces between the anionic phospholipid core and the mucins.In parallel, we studied the mechanisms allowing the NPL to enhance the immunogenicity of subunit antigens after nasal administration, with a highlight on the importance of the NP’s density [...]

Breast cancer is commonly associated with bone metastases, with approximately 70% of patients dying from breast cancer having radiological evidence of skeletal involvement. Median survival after diagnosis on bone metastases can be 2-3 years and therefore patients are at a high risk for the development of skeletal-related events. Consequently, research in both the laboratory and the clinic has addressed the potential for bone targeted agents to reduce the risk of developing skeletal metastases. The AZURE clinical trial is an international randomised phase III clinical trial that recruited 3360 early breast cancer patients in which participants received either 19 doses of zoledronic acid (ZOL) in 5 years or observation. No other clinical trial has undertaken such an intensive schedule of adjuvant bisphosphonates and therefore the safety and longer term sequelae were imperative to investigate should the drug become a standard of care. This thesis describes sub-studies undertaken in AZURE participants to investigate i) the incidence of osteonecrosis of the jaw (a recognised complication of bisphosphonates) and oral health-related quality of life and ii) a quantitative bone scanning technique to describe the effects the intensive schedule of zoledronic acid on bone remodelling and how this changes with time. Finally, the use of bone-related biomarkers (1,25-OH vitamin D, P1NP, CTX and 1CTP) measured in serum collected at baseline (before commencing zoledronic acid) have been investigated for their prognostic and predictive potential. The principal findings described in this thesis are: i) relatively low rate of osteonecrosis of the jaw (2.1%) with no significant impact or oral health r-related quality of life; ii) patients with elevated bone turnover markers at baseline are at increased risk of bone metastasis but these markers cannot be used to identify patients who will benefit from zoledronic acid; iii) bone turnover continues to be significantly suppressed in the axial skeleton 2 years after the cessation of zoledronic acid. The quality of the safety data presented in this thesis has contributed to the introduction of bisphosphonates into standard practice in the UK and across the globe.

The aim of the thesis is to optimize adjuvant treatment for colorectal cancer (CRC) patients. CRC treatment has improved over the decades resulting in improved overall survival of patients. The 5-year overall survival for CRC in the US between 1975 – 1979 was 50.6% while by 2004; it had improved to 65.9%. This is largely due to improvements in surgical and radiation techniques, screening initiatives and more effective chemotherapy drugs. However, when compared to the 5-year overall survival for breast cancer in 2004 of 89.9% (SEER data), it is clear that further improvements in CRC treatment are needed. This thesis evaluated different approaches to further improve overall survival rates and to reduce the acute and late toxicities associated with adjuvant treatment. One of the approaches was to attempt to personalize treatment for colorectal cancer patients using prognostic and predictive biomarkers. The group of patients selected for review were Stage C colon and rectal patients as the risk of recurrence is very high and the 5 year overall survival remains poor at 28% for colon cancer and 33% for rectal cancer. In this era of personalized medicine, the hope is to be able to tailor treatment regimens according to the patient and disease stage to reduce toxicity and improve efficacy. A retrospective analysis of the survival of Stage C rectal cancer patients in a public teaching hospital who received adjuvant chemotherapy after a curative resection was conducted to evaluate the role of adjuvant chemotherapy alone without radiotherapy. An original research study evaluating the role of a candidate marker, s100A4 in the treatment of Stage C colon cancer was also performed to evaluate the possible role of a new candidate biomarker s100A4 in the prognostication of Stage C colon cancer.

Influenza, também conhecida como gripe, é uma doença infecciosa viral que acomete um grande número de indivíduos anualmente, sendo responsável por um elevado número de internações e óbitos. O agente etiológico é o Myxovirus influenzae, vírus envelopado, de RNA de fita simples e polaridade negativa. A vacinação é a forma mais eficaz de se prevenir a infecção pelo vírus, no entanto, a capacidade produtiva dessa vacina não é suficiente para a vacinação da totalidade da população mundial, principalmente em casos de pandemia. Esse projeto teve por objetivo desenvolver uma vacina influenza (fragmentada e inativada) adjuvada, visando aumentar a capacidade produtiva dessa vacina no Instituto Butantan, que hoje é estimada em aproximadamente 40 milhões de doses por campanha. A utilização de adjuvantes na formulação da vacina influenza é capaz de produzir a mesma resposta imunológica protetora contra esse vírus, utilizando uma quantidade menor dos antígenos vacinais, aumentando a capacidade de produção da vacina em até quatro vezes. Foram estudadas 23 formulações adjuvantes utilizando o esqualeno como referência (formulação similar ao MF59®, adjuvante desenvolvido pela Novartis), vitaminas lipossolúveis (vitaminas A, D e E), vitamina B2 (vitamina hidrossolúvel), MPLA (monofosforil lipídio A, produzido pelo Instituto Butantan como subproduto da vacina pertussis low) e gel de hidróxido de alumínio. Para tanto, foram avaliadas a resposta imune conferida a camundongos BALB/c após imunização com diferentes formulações de vacina influenza (fragmentada e inativada) adjuvada e a existência, ou não, de toxicidade induzida pelas formulações vacinais estudadas. As formulações vacinais mais promissoras farão parte das formulações candidatas para realizações de ensaios clínicos. Os animais foram imunizados por via intraperitoneal com as formulações vacinais e foram colhidas amostras de sangue para ensaios sorológicos (inibição de hemaglutinação e ELISA) e células esplênicas para avaliação celular (dosagem de citocinas por citometria de fluxo: IL-2, IL-4, IL-6, IL-10, IL-17 TNF-α e INF-γ). Além disso, em um dos experimentos avaliou-se a formação de memória imunológica contra influenza, parâmetro importante em se pensando em uma vacina. Os três primeiros experimentos foram uma triagem a partir da qual selecionaram-se as melhores formulações que foram testadas no último experimento. Nele foram avaliados além da indução de resposta imune a toxicidade e a memória imunológica. Todas as 23 formulações estudadas induziram resposta minimamente protetora nos animais, com exceção da formulação contendo apenas MPLA como adjuvante. As formulações que se mostraram mais promissoras continham além do gel de AI(OH)3 MPLA de B. pertussis ou vitamina B2. Isso sem considerar o tocoferol (vitamina E), que embora tenha apresentado bons resultados acabou preterido em decorrência de sua potencial relação com casos de narcolepsia descritos na literatura. O teste de memória foi capaz de demonstrar que essas formulações produzem resposta de memória imunológica duradoura. Assim, tem-se resultados promissores para novos estudos pré-clínicos e clínicos com a vacina influenza (fragmentada e inativada) sazonal (trivalente).
Influenza, also known as flu, is a viral infectious disease that infects a large number of people annually, being responsible by large morbidity and mortality rates. The etiologic agent is the Myxovirus influenzae, an enveloped virus with single-stranded RNA and negative polarity. Vaccination is the best way to prevent the virus infection; however, the production capacity of this vaccine is not sufficient to vaccinate the entire world population, especially in cases of pandemics. This project aimed to develop an adjuvanted influenza vaccine (split and inactivated), increasing the productive capacity of this vaccine in Instituto Butantan, which is estimated in approximately 40 million of doses by campaign. Influenza vaccines formulated with adjuvants can produce the same protective immunological response against the virus using less amount of antigen increasing the production capacity of this vaccine up to four times. Twenty-three adjuvants containing fat-soluble vitamins (vitamins A, D and E), vitamin B2 (water-soluble vitamin), MPLA (monophosphoryl lipid A, produced by Instituto Butantan as a byproduct of pertussis low vaccine production) and aluminum hydroxide gel were studied. An adjuvant similar to MF59® (Novartis adjuvant) containing squalene was used as control. The immune response elicited in BALB/c mice after immunization with the different formulations of the influenza vaccine and the existence or not of toxicity induced by the vaccines formulations were studied. The most promising formulation will be part of the candidate formulations of clinicai trials. The animais received the vaccine formulations intraperitoneally and at specific days blood samples were taken to serological tests (hemagglutination inhibition and ELISA). At the end, they were euthanized to collect the spleens and splenic cells were cultivated to evaluate cytokines by flow cytometry: IL-2, IL-4, IL-6, IL-10, IL-17 TNF-α and INF-γ. Furthermore, in one experiment the immunological memory against influenza was evaluated, an important parameter to vaccines. The most promising formulations contained besides to alum either B. pertussis MPLA or B2 vitamin. Tocopherol (vitamin E) presented good results too, however it has a potential relationship with reported cases of narcolepsy. The memory test was able to demonstrate that these formulations induced long lasting immune memory response. Thus, these are promising results for new pre-clinical and clinical trials with seasonal trivalent influenza vaccine (split and inactivated).

Os óleos vegetais são matérias primas de fontes renováveis. São metabolizáveis, biodegradáveis, de fácil disponibilidade e baixo custo. A necessidade de adjuvantes vacinais seguros e que possam modular a resposta imunológica Th1/Th2, favorece a busca por novas substâncias que assim se comportem. Este trabalho tem o objetivo de avaliar e identificar óleo vegetal capaz de mimetizar ação adjuvante dos óleos minerais comercialmente usados e modular a resposta imune. Foram realizados testes de toxicidade aguda in vivo, testes de formação de emulsões, de estabilidade, de qualidade e, por fim, de imunogenicidade com formulações com o vírus rábico. Algumas formulações derivadas dos óleos de girassol, canola e buriti se mostraram não tóxicas, estáveis e de boa qualidade. Os grupos de camundongos inoculados com estas formulações obtiveram respostas imunológicas que apoiam sua capacidade adjuvante, não diferenciando significativamente (p<0,05) dos resultados do óleo mineral comercial. Concluiu-se que é possível elaborar emulsões estáveis não tóxicas a partir de óleos vegetais para sua utilização como veículos e adjuvantes vacinais. Formulações vacinais em forma de emulsões de óleos vegetais, compostas na sua maior parte pelos óleos de girassol e de canola possuem potência e atividade adjuvante semelhantes e tão eficientes quanto aos do óleo mineral. Os óleos vegetais devem estar em seu estado bruto ou semirrefinado, sem a adição de antioxidantes e conservantes. Por último, parece haver uma tendência de equilíbrio de resposta Th1/Th2 para as formulações com óleos vegetais.
Vegetable oils are renewable raw materials. These substances are metabolizable, biodegradable, of easy availability and low cost. The need for safe vaccine adjuvants that can modulate the Th1/Th2 immune response drives the search for new substances with similar behavior. This study aims to evaluate and identify a vegetable oil able to mimic the adjuvant action of the mineral oils used commercially, and modulate the immune response. There were performed tests of in vivo acute toxicity, emulsion formation, stability, quality and immunogenicity with formulations with rabies virus. Some formulations derived from the sunflower, canola and buriti oils proved to be non-toxic, stable and of good quality. Groups of mice inoculated with these formulations had immune responses supporting their adjuvant capacity, not differing significantly (p <0.05) from the results of the commercial mineral oil. It was concluded that it is possible to prepare stable non-toxic emulsions from vegetable oils to be used as vaccine adjuvants and vehicles. Vaccine formulations as emulsions from vegetable oil, composed mostly by the oils of sunflower and canola had adjuvant activity and potency similar to and as effective as the mineral oil. The vegetable oils should be in its raw state or semi refined, without the addition of antioxidants and preservatives. Finally, there seems to be a tendency to balance Th1/Th2 response by formulations with vegetable oils.

Orientador: Maria Cecília Rui Luvizotto
Resumo: Nas últimas décadas biomarcadores de angiogênese e linfangiogênese tumoral são alvos de estudos e, em humanos, estes aspectos da vasculatura tumoral já são utilizados como fatores prognósticos em neoplasias mamárias. Em cadelas não há estudos suficientes para demonstrar o significado clínico-patológico da linfangiogênese em neoplasias mamárias. De maneira similar, não há estudos que demonstrem efeitos antilinfangiogênicos de terapias como a quimioterapia metronômica (QM), classicamente caracterizada como uma terapia antiangiogênica. Este estudo teve como objetivos estabelecer os efeitos da QM na angiogênese e linfangiogênese tumoral destas neoplasias, assim como obter o significado clínico-patológico da densidade de vasos linfáticos em neoplasias mamárias caninas. Foram utilizadas 40 cadelas portadoras de carcinomas mamários, divididas em dois grupos, sendo um tratado somente com mastectomia e outro tratado com quimioterapia metronômica (QM) seguida de mastectomia. A biópsia do linfonodo sentinela foi preconizada para estabelecimento do “status” do tecido linfoide sentinela. Após, foi realizada a classificação e graduação histopatológica das neoplasias, e procedeu-se a imunomarcação para determinar a densidade de vasos linfáticos (DVL), a densidade microvascular (DMV), índice de proliferação celular (IP), índice apoptótico (IA), escore de COX-2, grau do fator de crescimento endotelial vascular (VEGF) e metástase nodal. As imunomarcações foram comparadas entre os grupos e a DV... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: In recent decades biomarkers of tumor angiogenesis and lymphangiogenesis have been studied and, in humans, these aspects of tumor vasculature are already used as prognostic factors in breast cancer. In bitches there are no studies that prove the clinicopathological significance of lymphangiogenesis in mammary neoplasms. Similarly, there are no studies demonstrating anti-lymphangiogenic effects of therapies such as metronomic chemotherapy (MC), classically characterized as anti-angiogenic therapy. This study aimed to establish the effects of MC on tumor angiogenesis and lymphangiogenesis of these neoplasms, as well as to obtain the clinical-pathological significance of lymphatic vessel density in canine mammary tumors. Forty bitches with mammary carcinomas were used, divided into two groups, one treated with mastectomy and the other treated with MC followed by mastectomy. Sentinel lymph node biopsy was recommended for establishing the status of the sentinel lymph node. Afterwards, classification and histopathological graduation of the neoplasms was performed, followed by immunostaining and determination of lymphatic vessel density (LVD), microvascular density (MVD), cell proliferation index (PI), apoptotic index (AI), COX-2 score, and vascular endothelial growth factor (VEGF) grade. Immunostain were compared between the groups and the LVD compared to clinicopathological aspects. Antiangiogenic and pro-apoptotic effects were observed in the group treated with MC, but without an... (Complete abstract click electronic access below)
Doutor

Vaccination has important clinical potential in the immunotherapy of both infectious disease and cancer. The central aim of the studies reported in this thesis has been the development of vaccination strategies that will be effective for therapeutic applications in cancer. Using ovalbumin antigen in a mouse model, we have examined a combination of recently developed adjuvants referred to as CASAC (combined adjuvants for synergistic stimulation of cellular immunity) to optimise the efficacy of vaccination induced T cell mediated immunity. These studies have examined the effect of repeated rounds of vaccination with one, or alternating cycles of two different helper peptides. The hypothesis was that repeated stimulation of the same clonal population of CD4+ T helper cells with a single MHC class II peptide could induce anergy, exhaustion, clonal deletion and/or stimulation of regulatory T-cells, resulting in reduced and shorter lived immunity. These studies have also examined the effect of inclusion of other immune regulatory components, and in particular a cocktail of cytokines generated by phytohemagglutinin stimulation of human peripheral blood mononuclear cells (referred to as IRX-2). Another important issue for vaccination mediated immune therapy that was addressed is the age-associated loss of immunological competence (immunesenescence). A comparative analysis is reported of the magnitude and duration of responses to vaccination with a single MHC class-I presented peptide in combination with the same or alternating MHC class-II presented peptides. In addition, we have quantified the number of antigen specific CDS T cells, their effector and memory subsets and in vivo antigen specific cytolytic activity to examine the effect of CASAC vaccination alone or in combination with IRX-2. The induction of immunological responses in young and aged immune backgrounds was also examined. Vaccinations with ovalbumin peptides in the CASAC adjuvant have shown higher percentages and numbers of antigen specific CDS T cells with improved cytolytic activity when helper functions are stimulated by two different class-II peptides used in alternating cycles of vaccination, rather than repeated stimulation by the same class-II peptide. This conclusion can be confirmed with a repeated experiment. Analysis of T cells with a regulatory (Treg) phenotype (CD4+CD25+Foxp3+) showed that their expansion was reduced with the alternating T-helper peptide vaccination regimen. The addition of IRX-2 to the CASAC vaccination regimen was found to enhance the in vivo antigen specific cytolytic activity. This was particularly significant in the aged mice which were found to have increased levels of Tregs.

Lee, T. et al (1981) proposed the encapsulation of hormones such as progesterone into serum albumin beads, such that their in vivo proteolysis would allow a gradual release of hormone at low levels, for extended hormone action. It was proposed, in the Department of Microbiology, Rhodes University, to replace the hormone component of the above bead formulation, with virus as antigen, in the development of a vaccine. Beads optimally crosslinked at 1% final glutaraldehyde concentration, containing Nodamura virus, were shown to promote an adjuvant effect in vivo, analogous to the release of antigen from Freund's Complete Adjuvant (FCA), so that extended immunostimulation resulted. It was shown that soluble antigen promoted a short-lived primary immune response, peaking around day 25 following inoculation. Antigen presented in beads, on the other hand, initially elicited a low humoral response, but this response gradually increased up to a peak around day 110 post inoculation, before decreasing. No apparent adverse side-effects were noted following inoculation of antigen-containing serum albumin beads, compared to necrosis following antigen in FCA inoculation, supporting the proposal of using albumin homotypic for the test inoculee animal, so that the beads would themselves be non-immunogenic and would merely act as a vehicle in the vaccine formulation. The indirect enzyme-linked immunosorbent assay (ELISA) was used to monitor the humoral response to antigen following inoculation. Results showed that covalent crosslinking of albumin in the formation of the beads did not promote immunogenicity on the part of the chemically altered albumin. The ELISA test was used to indicate the kinetics of the IgG response to Nodamura virus when presented in formulations such as: Freely soluble virus or its subunit; soluble intact virus inactivated by treatment with glutaraldehyde; intact virus entrapped in serum albumin beads cross; linked at different percentage final glutaraldehyde concentrations and also virus subunit prepared in albumin beads. The presence of virus-neutral ising antibodies was noted in serum obtained from rabbits inoculated with virus entrapped in albumin beads. Virus infectivity, titrated in mice, showed protection against virus challenge after incubation of virus with serum obtained above.

En 2014 la Tuberculose (TB) à dépassé le VIH comme la principale cause de décès par maladie infectieuse dans le monde soulignant le besoin urgent de développer un vaccin plus efficace contre cette maladie. Le candidat vaccin contre la TB, ID93/GLA-SE, dévéloppé à l’Infectious Disease Research Institute (IDRI), est aujourd’hui en essai clinique de phase IIa et a montré des résultats pré-cliniques et cliniques promettants. Dans de modèle murin de TB, ce vaccin induit une forte réponse TH1, considérée comme centrale dans la protection contre la TB, et la production d’IgG2 par les lymphocytes B. Néanmoins, les mécanismes d’action de GLA-SE sont encore peu connus.L’objectif principal de cette thèse est donc d’élucider les méchanismes clés qui relient les réponses innées et adaptatives induites par cet adjuvant dans le modèle murin. Un objectif secondaire est d’établir un modèle murin de rechute de TB après traitement et d’évaluer l’utilisation d’ID93/GLA-SE en tant que vaccin immuno-thérapeutique et sa capacité à réduire les taux de rechute dans ce modèle. L‘ensemble de ce travail nous a permis de mieux comprendre les mécanismes impliqués dans la réponse immunitaire adaptative induite par GLA- SE et de montrer la capacité de ID93/GLA- SE a être utilisé comme un vaccin thérapeutique contre la tuberculose dans le but de réduire les taux de rechute post-thérapeutiques
In 2014 tuberculosis (TB) surpassed HIV as the leading cause of death by an infectious disease worldwide emphasizing the urgent need to develop a more effective vaccine against this airborne disease. The Infectious Disease Research Institute (IDRI) TB candidate vaccine ID93/GLA-SE is currently undergoing a Phase IIa clinical trial and has shown promising preclinical and clinical results. In murine models of TB this vaccine drives a strong CD4 TH1 response, which is thought to be important for protection against TB, and an IgG2c skewed B cell response. However, little is known about the cellular and molecular events that drive GLA-SE adjuvanticity.To that end, the main objective of my thesis was to elucidate the key mechanisms that connect innate and adaptive immune responses elicited by this adjuvant in the murine model. A secondary objective was to evaluate the possibility of using ID93/GLA-SE as adjunct therapy to existing antibiotic treatments to reduce relapse rates after TB treatment.Collectively the results obtained during this research project and thesis broaden our knowledge and our current understanding of the mechanisms involved in the adaptive immune response induced by GLA-SE and show the capacity of ID93/GLA-SE to be used as a therapeutic vaccine against TB to reduce post-therapeutic relapse rates

Made available in DSpace on 2014-06-11T19:28:32Z (GMT). No. of bitstreams: 0 Previous issue date: 2008-08-05Bitstream added on 2014-06-13T20:37:34Z : No. of bitstreams: 1 bachega_tf_me_jabo.pdf: 1220477 bytes, checksum: 87704e890386522830320809624f7570 (MD5)
Funep
O presente trabalho objetivou avaliar a lixiviação dos herbicidas sulfentrazone e amicarbazone aplicados a campo, com e sem óleo mineral, e correlacioná-la com o controle de corda-de-viola (Ipomoea nil e I. hederifolia), oriundas de sementes depositadas em diferentes profundidades e sob diferentes camadas de palha de cana-de-açúcar. Em área de plantio de canade- açúcar, após acumuladas precipitações de 35, 67 e 106 mm, tubos de PVC de 10 cm de diâmetro, seccionados longitudinalmente, foram enterrados até a profundidade de 35 cm. Os tubos foram retirados e, depois da última amostragem (106 mm), foram semeadas as plantas testes sorgo e Ipomoea nil em toda a seção dos tubos. Foram realizadas avaliações visuais de intoxicação aos 7, 10 e 15 dias após a semeadura (DAS) e aos 20 DAS procedeu-se a determinação da matéria seca das plântulas. O delineamento experimental utilizado foi o de blocos ao acaso, em esquema de parcelas subdivididas com quatro repetições. As parcelas consistiram da aplicação de dois herbicidas (amicarbazone e sulfentrazone), adicionados ou não de óleo, e uma testemunha sem herbicida. Nas subparcelas estudou-se as profundidades de lixiviação (0,0-2,5; 2,5-5,0; 5,0-10; 10-15; 15-20; 20-25; 25-30; 30-35 cm). Pelo bioensaio, a presença do sulfentrazone foi estimada na camada superior até os 10 cm de profundidade, mesmo com 106 mm de precipitação e independentemente da adição do óleo.
This study aimed to evaluate the lixiviation of sulfentrazone and amicarbazone applied to the field, with and without mineral oil, and its relationship with the control of morning glory (Ipomoea nil and I. hederifolia), from seed deposited at different depths and under different layers of sugar cane straw. In sugar cane field, after accumulated rainfall of 35, 67 and 106 mm, PVC pipes of 10 cm in diameter, sliced lengthwise, were buried by the depth of 35 cm. The tubes were removed and, after the last sampling (106 mm), the tests plants sorghum and Ipomoea nil were sown across the section of pipe. Visual assessments of intoxication were made at 7, 10 and 15 days after sowing (DAS) and 20 DAS the dry weight of the seedlings was determined. The experimental design was a randomized block in split plots scheme with four replications. The plot consisted of application of two herbicides (amicarbazone and sulfentrazone), with or without mineral oil, and a control without herbicide. In subplots was studied the depths of leaching (0,0-2,5; 2,5-5,0; 5,0-10, 10-15, 15-20, 20-25; 25-30; 30 - 35 cm). For the bioassay, the presence of sulfentrazone was estimated in the upper layer to the 10 cm deep, even with 106 mm of rainfall and whether adding the oil.

Made available in DSpace on 2016-12-08T16:24:21Z (GMT). No. of bitstreams: 1 PGCA15MA170.pdf: 1407687 bytes, checksum: 6d77197fef831a6cbdc6fcf9e39c8629 (MD5) Previous issue date: 2015-07-23
For a long time, Pasterella multocida has been considered only as a secondary agent in pneumonias in swine. However, studies developed in Brazil identified different isolates of P. multocida able to operate as primary disease agent in swine. The objective of his project was to study protection aspects of a vaccine with pathogenic samples of P. multocida in experimental model with high sanity status pigs. In addition, it aims to describe the expression of immune response in swine induced by P. multocida infection, and determinate the prevalence of P. multocida in tonsils of vaccinated and not vaccinated pigs through immunohistochemistry. The study was divided into three experiments: Immunization of mice with vaccines prepared with different adjuvants , Test of two adjuvants used in vaccine with a P. multocida homologous strain in swine and Experimental challenge in swine with a P. multocida heterologous strain to the vaccine . Four adjuvants were tested in mice: Al(OH)3, ISA 206, Montanide GEL 01 and ISA 760. Two of them were selected for testing in swine: Al(OH)3 which showed better results in mice-, and ISA 760 for being an oily adjuvant. At the second experiment, although two vaccines have been effective in the disease protection, the one with the adjuvant Al(OH)3 was selected, because showed lower vaccine reaction. Eighteen pigs were distributed into three groups for the specific test of the vaccine for pathogenic samples of P. multocida. Group 1 (G1) vaccine with Al(OH)3; group 2 (G2) infection control; and group 3 (G3) negative control. After the experiment, the animals were euthanized, and samples were collected in ice - for microbiological cultivation and relative quantification of cytokines gene expression (IL1 β, IL2, TNFα) and TLR4 , and in formaldehyde, for routine histopathology and immunohistochemistry. Results showed that the vaccine prototype with adjuvant Al(OH)3 was efficient in controlling pulmonary pasteurellosis. The immune response was mediated by TLR4, with increased expression of TNFα. IL-1β and IL2 were also expressed, however, there was no significant difference between the groups. The immunostaining of P. multocida in tonsil was positive in all pigs challenged with the bacteria
Por muito tempo, Pasteurella multocida foi considerada apenas como agente secundário nas pneumonias em suínos, todavia, estudos realizados no Brasil identificaram diferentes isolados de P. multocida capazes de atuar como agente primário de doença em suínos. Esse projeto teve como objetivo estudar aspectos de proteção de uma vacina com amostras patogênicas da P. multocida em modelo experimental com suínos de alto status sanitário . E de forma paralela descrever expressão da resposta imune dos suínos induzida pela infecção da P. multocida e determinar a prevalência dessa bactéria em tonsilas de suínos vacinados e não vacinados através de imunohistoquímica. O trabalho foi dividido em três experimentos: Imunização de camundongos com vacinas preparadas com diferentes adjuvantes ; Teste de dois adjuvantes utilizados na vacinação de suínos frente ao desafio com uma cepa homóloga de P. multocida ; e Desafio experimental em suínos com uma cepa de P. multocida heteróloga à da vacina . Em camundongo foram testados quatro adjuvantes: Al(OH)3, ISA 206, Montanide GEL 01 e ISA 760, dos quais, foram selecionados dois para teste em suínos: Al(OH)3, que apresentou melhor resultado nos camundongos e ISA 760, por ser um adjuvante oleoso. Embora as duas vacinas tenham sido efetivas na proteção da doença, no segundo experimento, selecionou-se àquela com adjuvante Al(OH)3, que apresentou menor reação vacinal. No terceiro experimento realizado em suínos, foram utilizados 18 suínos em três grupos: grupo 1 (G1) - vacina com Al(OH)3, e grupo 2 (G2) controle de infeção e grupo 3 (G3) controle negativo. Após o experimento os animais foram eutanasiados para avaliação patológica, coleta de amostras em gelo para cultivo microbiológico e quantificação relativa da expressão gênica das citocinas (IL1 β, IL2, TNFα) e TLR4, para histopatologia de rotina e imunohistoquimica. Os resultados demostraram que o protótipo de vacina com adjuvante Al(OH)3 foi eficiente no controle de pasteurelose pulmonar. O perfil da expressão da resposta imune foi mediado por TLR4, com aumento da expressão de TNFα. Também foram expressos IL-1β e IL2, porém sem diferença significativa entre os grupos. A imunomarcação de P. multocida na tonsila foi positiva em todos os suínos desafiados com a bactéria, independente de serem ou não vacinados

Forskning har visat att fysisk aktivitet ger en bättre prognos för kvinnor som drabbats av bröstcancer. Den beskrivs också ha betydelse för symtomlindring vid cytostatikarelaterade biverkningar, vilket i sin tur leder till ökad livskvalité hos dessa kvinnor. Det har även framkommit i studier att det råder brister inom sjukvården vad gäller att förmedla sådan information. Som ett första steg behövs en kartläggning av den fysiska aktiviteten hos kvinnor med bröstcancer, deras aktivitetsvanor och vilken betydelse fysisk aktivitet har för dem under pågående cytostatikabehandling. Denna studie genomfördes som en tvärsnittstudie med syftet att beskriva fysisk aktivitet hos kvinnor med bröstcancer som genomgår adjuvant cytostatikabehandling. 30 kvinnor som genomgick adjuvant cytostatikabehandling vid den onkologiska behandlingsenheten vid Universitetssjukhuset i Örebro (USÖ) besvarade en enkät med frågor om fysisk aktivitet. Enkäten bestod av både öppna och slutna frågor. Datamaterialet har analyserats med deskriptiv statistik. Den avslutande öppna frågan analyserades med modifierad manifest innehållsanalys. Resultatet visade att kvinnorna i lika hög grad var fysiskt aktiva före som under cytostatikabehandlingarna. Skälen som kvinnorna uppgav till att vara fysiskt aktiva var att de kände sig mindre trötta och att den kunde skingra tankarna. Många kvinnor upplevde att de mådde psykiskt bättre av fysisk aktivitet och att det gick lättare att återhämta sig mellan behandlingarna. Den aktivitetsform som främst utfördes var promenader. Ett flertal kvinnor hade fått information av vårdpersonal gällande fysisk aktivitet i samband med cytostatikabehandling men mindre än hälften av dessa kvinnor följdes upp under behandlingsperioden. Informationen gavs främst av sjuksköterskor och läkare.
Research has shown that physical activity improves survival for women diagnosed with breast cancer. Studies have also described that physical activity is a matter of importance for alleviating common side effects from chemotherapy which thereby increases the quality of life for these women. It has also emerged that there is insufficient information on this topic. As a first step, it is necessary to do a survey of the physical activity in women with breast cancer, their activity experiences and what importance physical activity play for them during adjuvant chemotherapy. This work was a cross-sectional study based on questionnaires. The purpose of this study was to describe physical activity in women with breast cancer receiving adjuvant chemotherapy. 30 women who received adjuvant chemotherapy at the medical treatment unit for oncology at Örebro University Hospital (USÖ) answered the questionnaire. The questionnaire contained both closed and open-ended questions. The data was analysed with descriptive statistics. The final open-ended questions were analysed with a modified manifest content analysis. The result showed that the women had the same activity level before as during the treatment period. The reasons for the women to be physically active were to decrease fatigue and to divert their minds. The majority of the women experienced feeling psychologically better and experienced better recovery between chemotherapy treatments. Walking was the most frequently used activity. The majority of the women received information about physical activity, but less than half of the women were followed-up. This information was primarily given by nurses and doctors.

L'artritis reumatoide (AR) és una malaltia crònica localitzada principalment en les articulacions, on es produeix inflamació de la membrana sinovial, degeneració del cartílag i l'os, que finalment originen la seva deformació i incapacitació. A més a més, l'AR és una malaltia autoimmunitària, on es desenvolupen diferents tipus d'autoanticossos, com el factor reumatoide dirigit contra la propia IgG i anticossos contra el col.làgen articular. Avui encara es desconeix l'etiologia i la patogènesi del procés artrític i per això resulta essencial disposar de models animals que presentin mecanismes patogènics semblants als de la malaltia humana per tal de dissenyar millors estratègies terapèutiques.

L'artritis adjuvant (AA) és una patologia induïda en rata que es considera model d'AR i que cursa amb inflor, eritema, dolor i impotència funcional de les articulacions dels animals afectats. Avui s'accepta que aquesta patologia és mitjançada fonamentaJment per limfòcits T.

En el present treball es va pretendre: identificar les poblacions cel.lulars presents en el teixit sinovial inflamat: esbrinar el possible paper dels limfòcits T gamma/delta, T CD4 i T CD8 en el desenvolupament i perpetuació de l'AA; i conèixer si la teràpia amb un anticòs monoclonal (AcMo) dirigit contra la molècula CD4 és capaç de modificar el curs de l'AA. Per assolir els objectius esmentats, és a dir, per delimitar quines subpoblacions de limfòcits T estan implicades en l'AA, es van realitzar diferents protocols d'immunoteràpia amb AcMo dirigits contra determinades molècules de superfície presents en els limfòcits T alfa/beta, T gamma/delta, T CD4 i T CD8.

Yoshino i col.laboradors, al 1990, ja van demostrar la implicació dels limfòcits T alfa/beta en el desenvolupament i en la perpetuació de l'AA, i en el present estudi el mateix AcMo R73 dirigit contra el TCR-alfa/beta de rata ha estat utilitzat com a control positiu de tractament. Fins a l'actualitat no es coneixia el paper dels limfòcits T gamma/delta a l'AA a causa de que no existia un AcMo anti-TCR-gamma/delta de rata. En el present treball, i gràcies al recent descobriment per part del grup del Prof. Hünig de Würzburg de l'AcMo V65 s'ha pogut avaluar la implicació d'aquestes cèl.lules en aquest model experimental. D'altra banda, també s'han realitzat estudis d'immunoteràpia emprant un AcMo anti-CD4. el W3/25, que no havia estat utilitzat anteriorment en l'AA, i que per tant, no es coneixia el seu potencial terapèutic en aquest model experimental. A més a més, s'ha assajat la teràpia combinada anti-CD4 + anti-CD8, que tampoc no ha estat descrita fins a l'actualitat, i que ha permès determinar els efectes de la població CD8+ sobre els Iimfbcits T CD4+-. Totes aquestes teràpies anaven encaminades a inhibir el desenvolupament de l'AA.

La inducció de l'AA en rates de la soca Wistar o Lewis es va realitzar mitjançant l'administració d'una suspensió de Mycobaeterium butyricum o de Mycobaeterium tuberculosis al 0,5% en vaselina liquida, per via intradèrmica a la base de la cua. Durant els primers dies després de la inducció no s'observa cap signe extern i a partir dels 10-12 dies es manifesta una.inflamació sistèmica que afecta a les extremitats posteriors i sovint també a les anteriors de l'animal. En els diferents experiments s'ha realitzat un seguiment de la inflamació dels animals mitjançant paràmetres clínics, com l'índex d'artritis, que comprèn la valoració clínica del grau d'eritema i d'edema de les quatre articulacions; i l'increment de volum articular de les extremitats posteriors, mesurat per pletismometria.

Per a l'anàlisi de les poblacions cel·lulars presents en teixit sinovial s'ha utilitzat una tècnica immunohistoquímica. Com a substrat s'han utilitzat talls criostàtics de membrana sinovial i la identificació de determinats antígens de membrana s'ha realitzat utilitzant AcMo dirigits contra aquests antígens de superfície cel·lular. La reacció s'ha revelat amb el mètode peroxidasa-anti-peroxidasa.

A partir de mostres de plasma dels animals artrítics. s'ha detenninat la concentració d'anticossos dirigits contra el micobacteri, la concentració dels AcMo administrats i de la resposta dirigida contra aquests, mitjançant tècniques d'ELISA.

Per a determinar l'efecte a nivell cel.lular dels AcMo administrats s'ha utilitzat la citometria de flux. Aquesta tècnica permet la identificació de poblacions cel.lulars mitjançant la detecció d'antígens de la seva superfície, prèviament marcats amb anticossos conjugats a un fluorocrom. Un pas previ per a l'anàlisi citomètrica de les poblacions de sang perifèrica és l'aïllament dels limfòcits. Amb el fi de conèixer quin mètode és el més adequat per analitzar els limtècits de sang perifèrica dels animals dels estudis d'immunoteràpia, es van comparar els efectes de quatre mètodes d'aïllament de limfòcits sobre sang de rata: gradient de Ficoll-Isopaque, lisi amb clorur amònic, lisi amb reactiu de Becton & Dickinson i lisi mitjançant el sistema Coulter Q-prep.

Els limfòcits es van marcar amb AcMo dirigits contra diferents subpoblacions limfocítiques. En considerar tots els marcatges realitzats no es van trobar diferències entre els mètodes, la qual cosa dona validesa als 4 mètodes per a sang de rata. Però l'anàlisi estadística que va considerar els resultats obtinguts per cada AcMo per separat va mostrar algunes diferències significatives. El gradient Ficoll-Isopaque, és el que va proporcionar uns percentatges més baixos de limfòcits CDS+, CD4+ i CD25+, suggerim una pèrdua selectiva d'alguns limfòcits T. Per altra banda, amb el clorur amònic, es van obtenir els percentatges més baixos de limfòcits B, i aquest fet es pot atribuir a que una part dels limfòcits B són sensibles a les condicions d'aquest mètode. En els dos mètodes comercials, els percentatges corresponents a la majoria de les poblacions estudiades van presentar valors intermitjos. En el nostre estudi, es va triar el clorur amònic com a mètode de lisi per els estudis d'immunoteràpia. Aquest mètode no afecta selectivament cap població de limfòcits T i, a més a més permet realitzar el marcatge sobre cèl.lules aïllades i presenta un baix cost.

Per conèixer quins tipus cel.lulars estan involucrats a nivell de l'articulació inflamada en l'AA, es va realitzar l'estudi histològic de la membrana sinovial de genoll. La membrana sinovial està formada per teixit conjuntiu i revesteix tota la cavitat articular, a excepció dels cartílags. El teixit sinovial artrític augmenta de mida i en general incrementa unes tres vegades el seu pes.

L'estudi histològic de la membrana sinovial mostra en el teixit control una capa íntima formada per 1 ó 2 fileres de sinoviòcits i una capa subíntima formada per teixit adipós amb alguns vasos sanguinis. Als 14 dies de la inducció, amb inflamació articular ja establerta, a nivell sinovial s'observa un augment en el nombre de fileres de cèl.lules de la capa íntima. També s'observen plegaments cap a la cavitat articular. Existeix un gran nombre de cèl.lules mononuclears i gran quantitat de fibres de col.làgen en la capa subíntima.

L'estudi immunohistoquímic de la membrana sinovial es va realitzar amb el mètode peroxidasa-antiperoxidasa. En alguns teixits sinovials inflamats, es van identificar algunes cèl.lules CD5+ amb l'AcMo OX19. Aquestes cèl.lules CD5+ no van aparèixer en cap teixit control sa. El nombre de cèl.lules CD8+ de la capa subíntima va incrementar significativament del dia 14 al 28 postinducció respecte als teixits control. L'AcMo W3/25, que s'uneix a l'antigen CD4 de limfòcits T col.laboradors i de macròfags, ja detecta cèl.lules positives en tots els teixits sinovials control i el seu nombre augmenta en els teixits artrítics. A causa de les poques cèl.lules CD5+ trobades i de la morfologia i la tinció citoplasmàtica d'aquestes cèl.lules es poden considerar macròfags. L'AcMo OX6 que detecta la molècula lA present en diferents tipus cel.lulars marca un gran nombre de cè1.lules a nivell de l'enzima sinovial dels animals control, i en els animals artrítics aquest nombre és molt elevat.

Com a primer estudi d'immunoteràpia i per tal d'analitzar la implicació dels limfòcits T gamma/detla en l'AA, van portar a terme diferents protocols terapèutics amb un AcMo dirigit contra el TCR-gamma/delta de rata, l'AcMo V65. Es van desenvolupar diferents protocols d'administració d'aquest anticòs: un tractament preventiu, realitzat a animals nounats des del moment del naixement i a dosis creixents durant 8 setmanes, és a dir durant tot el període previ a la inducció de l'AA; i 2 tractaments curatius. Un dels protocols va consistir en administrar els anticossos els dies 12, 15 i l8, és a dir, començant abans del màxim d'inflamació articular i l'altre protocol, en el qual es van assajar tres dosis diferents de l'anticòs, es va iniciar durant el màxim d'inflamació, és a dir el dia 15. En els dos tractaments curatius. es va realitzar un tractament paral.lel amb l'AcMo R73, dirigit contra el TCR-alfa/beta, com a control positiu del tractament.

En tractar amb l'AcMo V65 només es van observar algunes cèl.lules gamma/delta amb una expressió normal del seu TCR a nivell de sang perifèrica, ganglis limfàtics poplitis i mesentèrics. El que apareix és una població de limfòcits amb una expressió antigènica disminuïda de les molècules de TCR gamma/delta i de CD3 en la seva superfície. Aquest fet indica que l'AcMo V65 va produir la down-regulation del receptor i també la comodulació de la molecula CD3. Ara bé, el percentatge d'aquesta subpoblació amb el TCR modulat era inferior al percentatge de Iimfòcits T gamma/delta detectats abans del tractament, indicant que alguns limfòcits T van perdre totalment l'expressió del TCR o que es van depleccionar. El pretractament amb V65 des del naixement, que va provocar que aquests animals no tinguessin en cap moment Iimfòcits T gamma/delta amb expressió normal del seu TCR, no va aconseguir prevenir el desenvolupament de la patologia i els animals van seguir el mateix curs inflamatori que els animals artrítics control. El tractament curatiu amb l'AcMo V65, iniciat abans del màxim d'inflamació no va aconseguir modificar el volum articular respecte al grup artrític control d'isotip i el mateix va succeir amb la VSG. En canvi, l'AcMo R73, anti-TCR-alfa/beta, sí que mostra un efecte beneficiós, disminuint tant el volum articular com la VSG durant el tractament. El tractament curatiu iniciat durant el màxim d'inflamació tampoc va aconseguir modificar els paràmetres inflamatoris ni la VSG propis de les rates artrítiques, en cap de les dosis assajades.

Tot i la manca d'efecte curatiu de l'AcMo V6S observada en les variables estudiades quan el tractament es va iniciar abans del màxim d'ínflamació, es va observar un grau de destrucció articular significativament superior a l'observat en les rates artrítiques control en fases tardanes de la patologia. Aquest fet indica un paper protector fase-depenent dels limfòcits T gamma/delta a l'AA.

Després de descartar la intervenció directa dels limfòcits T gamma/delta en la patogènesi del procés artrític experimental, es va centrar l'atenció cap als limfòcits T alfa/beta, concretament cap a la població limfocítica CD4+ i la CD8+. Per estudiar la implicació d'aquestes dues poblacions limfocítiques en el desenvolupament de l'artritis, es va realitzar una immunoteràpia durant el període de latència, emprant l'AcMo anti-CD4 W3/25, a dosi de 3 mg, l'AcMo anti-CD8 OX8 a dosi d'1 mg i ambdós AcMo conjuntament.

Durant el tractament amb els AcMo W3/25 i OX8 es van estudiar també els seus efectes a nivell cel.lular per citometria de flux. L'administració de l'AcMo anti-CD4 W3/25 va produir la down-regulation dels limfòcits T CD4+, mentre que l'administració de l'AcMo OX8 va produir la deplecció dels limfòcits T CD8+ de sang perifèrica. L'administració de l'AcMo anti-CD4 W3/25 durant el període de latència administrat sol o conjuntament amb l'AcMo OX8, va inhibir el desenvolupament del procés inflamatori de l'AA, fins i tot després d'una segona inducció de la patologia, fet que confirma el paper essencial dels limfòcits T CD4+ en la patogènesi de l'AA. En canvi, l'administració de l'AcMo anti-CD8 no va modificar el curs de l'artritis, seguint els animals tractats amb aquest AcMo un curs similar al grup artrític control, la qual cosa indica que els limfòcits T CD8+ no intervenen directament ni exerceixen un paper regulador sobre els limfòcits T CD4+ en la patogènesi de l'AA.

Com a últim protocol terapèutic i amb el fi de conèixer la implicació dels limfòcits T CD4+ i els CD8+ en la perpetuació de l'AA, es va realitzar una immunoteràpia amb els mateixos AcMo que en l'estudi anterior però iniciant la teràpia el dia 14 postinducció. un cop l'artritis ja està establerta. En aquest estudi es va disposar també d'un grup tractat amb W3/25, un grup tractat amb OX8 i un grup tractat amb ambdós AcMo conjuntament.

El tractament amb l'AcMo OX8 no va aconseguir modificar el procés inflamatori ja existent. D'altra banda, el tractament amb W3/25 sol o conjuntament amb l'anticòs OX8 va ser capaç de revertir el procés inflamatori, fet que indica una implicació directa dels limfòcits T CD4+ en la perpetuació de l'AA. Aquest efecte, però, va ser transitori, ja que va desaparèixer després de 6 dosis. Aquest fet es pot atribuir al desenvolupament d'anticossos contra les 1g de ratolí administrades. que es van detectar en els animals tractats amb W3/25. A l'avaluar els nivells d'anticossos anti micobacteri desenvolupats pels diferents grups de l'estudi, cal destacar que ¡'administració de l'AcMo OX8 administrat sol va produir nivells 4 vegades superiors al dels altres grups i que van ser elevats durant tot l'estudi, el que suggereix que els limfòcits T CDS5+ tenen un efecte regulador sobre les cèl.lules responsables de la resposta humoral dirigida contra el micobacteri.

En resum, en aquest treball s'ha demostrat la implicació dels limfòcits T CD4+ o col.laboradors no només en la fase de desenvolupament del procés artrític. sinó també en el procés de cronificació, fet que permet considerar els limfòcits T CD4+ com a possibles dianes d'un tractament en l'AR humana. Cal tenir en compte, però, que aquest tractament s'ha de realitzar de forma contínua i amb molècules que no estimulin una resposta immunitària. A més a més, s'ha demostrat que els limfòcits T gamma/delta juguen un paper secundari en el desenvolupament de l'AA i que aquest paper és més aviat protector, modulant l'acció exercida per altres tipus cel.lulars en una determinada fase de la patologia. D'altra banda, els limtòcits T CD8+ no estan implicats en el desenvolupament de l'artritis, ni tan sols com a moduladors de l'acció dels limtòcits T CD4+ artritogènics. Per últim, malgrat la rellevància dels limfòcits T col.laboradors en el procés artrític, la presència d'aquestes cèl.lules a nivell inflamatori local és mínima, suggerint un efecte regulat majoritàriament per citocines des dels òrgans limfoides.
Rheumatoid arthritis (RA) is an autoimmune disease characterized by joint chronic inflammation, associated with synovitis and erosion of cartilage and bone. Adjuvant arthritis (AA) is an experimental autoimmune disease in rats which shares certain clinical and immunological features with RA. Although the pathogenesis of AA is unknown, a strong T lymphocyte dependence has been described.

The objectives of the present work were to identify cell populations present on inflamed tissue from arthritic rats and to determine the role of gamma/delta, CD4 and CDS T lymphocytes on the development and the perpetuation of adjuvant arthritis. For these purposes, we performed different immunotherapy protocols using monoclonal antibodies (MoAb) directed against alpha/beta, gamma/delta, CD4 and CD8 T cells.

The immunohistochemical analysis of synovia revealed that only a few CD5+ cells appeared on some arthritic tissues. No CD5+ cell appeared on healthy lissues. Arthritic tissues showed a higher number of CD5+, CD4+ and Ia+ cells than healthy tissues.

Immunotherapy with an anti-gamma/delta-TCR MoAb was applied according to a preventive and to a pre-peak and a late therapeutic schedule. Although all protocols down-regulated gamma/delta-TCR expression in peripheral blood and lymph nodes cells, none influenced clinical parameters of AA. If rats were treated before the clinical peak of AA, joint destruction was more severe than in vehicle-treated rats, suggesting a stage-dependent protective role of gamma/delta T cells in AA.

To study the pathogenic role of CD4 and CD8 T cells on arthritis, an immunotherapy using an anti-CD4 MoAb, W3/25, an anti-CDS MoAb, OX8 or both together was performed during the latency period of AA. W3/25 (which was found to be non-depleting) alone or in combination with OX8 prevented the inflammatory process of AA. To ascertain if any of the last MoAb could reverse AA, an immunotherapy beginning on day 14 postinduction was applied. During treatment with W3/25, a strong amelioration of inflammatory signals occurred. This study indicates that CD4+ cells play an important role both on the initiation and the perpetuation of AA, while CD8+ cells do not appear relevant for the development of the disease.

This simple quantitative research investigated whether patients with cancer developed depression whilst receiving adjuvant chemotherapy, and whether there was a particular time in the treatment cycle that it was likely to develop. A longitudinal survey method was chosen and the instrument of use was the Beck Depression Inventory II (BDI-II). The content of this survey include factors that reflect negative attitudes towards self, performance impairment and somatic disturbances as well as general factors of depression. Twenty-six participants were given the BDI-II, and asked to report on side effects that they had experienced every two weeks whilst they were receiving chemotherapy. The study took place in two regional oncology clinics. This report demonstrates that, patients can develop depression whilst receiving adjuvant chemotherapy. The study revealed that the 5th fortnight into a patient’s chemotherapy treatment was a more vulnerable point in their treatment to develop depression. Descriptive analysis illustrated that more females than males suffered depression and that those receiving treatment for breast cancer were more likely to develop depression. Correlation statistics demonstrated a relationship between fatigue and depressive symptoms. There was no statistically significant correlation between the number of side effects experienced by participants and depression. In summary, the findings suggest that patients were more vulnerable to developing depression around the 5th fortnight of their chemotherapy cycle. This research has demonstrated throughout that depression does occur in patients receiving adjuvant chemotherapy and that female patients are more likely to develop depression than males. Due to the small sample size though, the results were not statistically significant. The findings from this research could provide direction for more thorough studies in the future. The style of reporting used throughout this thesis has been the Harvard referencing style to ensure compliance with the university requirements and to maintain consistency.

BACKGROUND Treatment with an aromatase inhibitor for 5 years as up-front monotherapy or after tamoxifen therapy is the treatment of choice for hormone-receptor-positive early breast cancer in postmenopausal women. Extending treatment with an aromatase inhibitor to 10 years may further reduce the risk of breast-cancer recurrence. METHODS We conducted a double-blind, placebo-controlled trial to assess the effect of the extended use of letrozole for an additional 5 years. Our primary end point was disease-free survival. RESULTS We enrolled 1918 women. After a median follow-up of 6.3 years, there were 165 events involving disease recurrence or the occurrence of contralateral breast cancer (67 with letrozole and 98 with placebo) and 200 deaths (100 in each group). The 5-year disease-free survival rate was 95% (95% confidence interval [CI], 93 to 96) with letrozole and 91% (95% CI; 89 to 93) with placebo (hazard ratio for disease recurrence or the occurrence of contralateral breast cancer, 0.66; P = 0.01 by a two-sided log-rank test stratified according to nodal status, prior adjuvant chemotherapy, the interval from the last dose of aromatase-inhibitor therapy, and the duration of treatment with tamoxifen). The rate of 5-year overall survival was 93% (95% CI, 92 to 95) with letrozole and 94% (95% CI, 92 to 95) with placebo (hazard ratio, 0.97; P = 0.83). The annual incidence rate of contralateral breast cancer in the letrozole group was 0.21% (95% CI, 0.10 to 0.32), and the rate in the placebo group was 0.49% (95% CI, 0.32 to 0.67) (hazard ratio, 0.42; P = 0.007). Bone-related toxic effects occurred more frequently among patients receiving letrozole than among those receiving placebo, including a higher incidence of bone pain, bone fractures, and new-onset osteoporosis. No significant differences between letrozole and placebo were observed in scores on most subscales measuring quality of life. CONCLUSIONS The extension of treatment with an adjuvant aromatase inhibitor to 10 years resulted in significantly higher rates of disease-free survival and a lower incidence of contralateral breast cancer than those with placebo, but the rate of overall survival was not higher with the aromatase inhibitor than with placebo. ( Funded by the Canadian Cancer Society and others;)

Streptococcus pneumoniae is a major human pathogen and causes a significant burden of disease in both developed and developing countries. Currently, two pneumococcal vaccines are available, a polysaccharide conjugate vaccine for children <2 years of age and an adult polysaccharide vaccine for ‘at risk’ groups such as the elderly and immunocompromised. Unfortunately, due to the vast variation and highly recombinant nature of the pneumococcus vaccine escape through serotype replacement is significantly decreasing the efficacy of pneumococcal vaccines globally. New cost-effective and protective pneumococcal vaccines are urgently required. Pneumolysin (PLY) is a 53Kd cholesterol-dependent cytolysin that is largely conserved in all strains of Streptococcus pneumoniae, making it an ideal candidate for inclusion in a broad spectrum vaccine. It has been shown that PLY is not only a protective immunogen but also has potent adjuvant properties and stimulates both IgG and IgA antibody responses to antigens genetically coupled to the toxin (Douce et al., 2010). Both systemic and mucosal responses are induced when PLY is used as an adjuvant which may prevent colonization and therefore provide non-serotype specific herd immunity to Streptococcus pneumoniae. The cytolytic activity of PLY prevents its inclusion in a human vaccine; a non-lytic deletion mutant 76PLY was created for this purpose which retains adjuvanticity, albeit slightly reduced. The aim of this study was to elucidate the mechanism(s) of PLY/Δ6PLY adjuvanticity, it will be essential to have a basic model of adjuvant activity before PLY-based vaccines can be advanced to human clinical trials. This project used a combination of high-throughput methods such as protein pulldowns and gene expression profiling to examine the abilities of PLY, 76PLY and the truncation mutants D123PLY and D4PLY to bind to and be internalized by host cells and to differentially regulate gene expression. These studies highlighted specific and direct interactions between PLY variants and the host cytoskeleton that could mediate antigen/PLY uptake; they also revealed a pattern of gene expression that is similar to those of other adjuvants and could provide the basis for a model of adjuvanticity. Finally, through the use of reporter cell lines and transgenic TLR4-/- BMDM, the relationship between PLY and TLR4 has been further defined. A novel method for preparing vehicle controls provided evidence that the ligation of TLR4 in this system is PLY-dependent and is not an artefact caused by contaminating TLR ligands such as LPS. Once this was established it was possible to further investigate the role of TLR4 in the adjuvant activity of PLY, in particular the PLYdependent production of IL-1@. Through these studies a surprising role for TLR4 in in vitro PLY-dependent cytokine production was discovered. Additionally, it was found that complement has an essential role in the PLY-dependent production of IL-1@. The role(s) of complement and IL-1@ in the adjuvant activity were further investigated using an in vivo immunization model and the biological basis for the difference in adjuvant activity of PLY and 76PLY was defined.

Adjuvant chemotherapy treatment for breast cancer with the widely used anthracycline/taxane combination has potential myotoxic effects on skeletal muscle. More generally morbidity linked to breast cancer treatment may well be associated with physical deconditioning. Together these factors could lead to reduced muscle strength, increased fatigue and consequent decreases in quality of life. Resistance exercise offers the potential to promote improvements in muscle mass and strength, with related benefits in functional capacity and life quality in this population. The primary aims of this thesis were to investigate the effects of anthracycline/taxane adjuvant chemotherapy treatment and resistance exercise on body composition, knee muscle strength, quality of life (QoL) and perceived fatigue in women diagnosed with breast cancer. The study was designed: 1. To assess the effects of anthracycline/taxane chemotherapy on the following outcome measures in a group of volunteers following surgery for breast cancer: • whole body muscle and fat composition, • knee extension/flexion muscle strength • quality of life • perceived overall fatigue 2. To compare these outcome measures in women post chemotherapy with a group of age-matched sedentary healthy females. 3. To evaluate the effect of an individualised semi home-based 12-week resistance exercise intervention on the above variables in women with breast cancer compared to a non-exercising breast cancer control group. 4. To determine whether the changes in the above variables following the exercise intervention differs between women treated with chemotherapy compared with a healthy control group
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Allied Health
Griffith Health
Full Text

Este trabalho consistiu numa avaliação, sob um ponto de vista físico, da aplicabilidade da sílica mesoporosa ordenada tipo SBA-15 como adjuvante imunológico. Inicialmente foi estudado o método de preparação e reprodutibilidade das propriedades do material, condição necessária para a síntese de grandes quantidades (N 100g). Mostrou-se que a calcinação em vácuo, comparada com o processo em N2 e ar, resulta em material com estrutura mesoporosa mais bem ordenada. Para aplicações biológicas foi analisado o potencial de encapsulação de antígerios no material, através de estudos de incorporação de Albumina Bovina (BSA) e vacina contra Hepatite A. Foi observada uma incorporação bem-sucedida de BSA na sílica, com essa proteína alojando-se dentro da estrutura de poros. Resultado semelhante foi observado para a vacina contra hepatite A. O processo mais eficiente de incorporação foi determinado para uma mistura em repouso e seca através de evaporação. A aplicabilidade da sílica como adjuvante para uso animal foi avaliada através de análises, pelo método PIXE, da acumulação do material no organismo de camundongos. A sílica foi administrada a camundongos Swiss por via oral e intra-muscular, e o teor de silício em diferentes órgáos foi comparado aos teores em um grupo controle. Foi detectada a presença de sílica em determinados órgãos dos camundongos, cuja eliminação total se processa num período de 70 dias. Além disso, em testes toxicológicos realizados no Instituto Butantan, a sílica mostrou-se eficaz na indução de resposta humoral e atóxica.
This work consisted of an evaluation, from a physical standpoint, of the applicability of SB.4-15 type ordered rnesoporous silica as an inmunological adjuvant. The method of preparation and reproducibility of the material properties were initially studied. Those conditions are necessary to synthesize large quantities of the material (N 100g). Vacuum calcination, whem compared to the process executed in A5 and air, results in a better ordered mesoporous structure. For biological applications, the potential to encapsulate antigens in the material was analyzed through studies of incorporation of Bovine Serum Albumin (BSA) and vaccine for Hepatitis A. A successful incorporation of BSA in the silica was observed, with that protein being lodged inside the porous structure. A similar result was obtained for the vaccine for Hepatitis A. The most efficient incorporation process was determined by keeping the solution at rest and drying it through evaporation. The applicability of silica as an immunological adjuvant for animal use was evaluated through PIXE analyses of the silicon accumulation in mice\'s organs. Silica was administrated to Swiss mice through oral and intramuscular ways and the silicon content of different organs was compared to the figures of the control group. The silica´s presence was detected on certain organs, and it was completely eliminated after 70 days. Besides that, toxicological studies accomplished at the Butantan Institute showed that the silica is efficient for inductíon of humoral response and it is non-toxic.

Neste trabalho estudou-se a encapsulação de proteínas com diferentes pesos moleculares na sílica mesoporosa ordenada SBA-15 para avaliar sua aplicação como adjuvante imunológico. Para tanto, as proteínas IgG (150 kDa) e BSA (66,5 kDa) foram incorporadas à sílica mesoporosa com poros expandidos. Primeiramente estudou-se a dilatação dos poros utilizando-se um agente dilatador de estrutura no processo de síntese, através da preparação de amostras com diferentes quantidades de triisopropilbenzeno (TIPB). Resultados de isoterma de adsorção de nitrogênio (NAI) e espalhamento de raios X a baixo ângulo (SAXS) revelaram um aumento no diâmetro médio de poros da ordem de 23% e uma rede de poros mais desordenada. Para se ter uma estimativa das dimensões das proteínas, medidas de SAXS foram feitas e indicaram que ambas têm dimensões que permitiriam sua incorporação nos poros da SBA-15. As amostras com poros dilatados foram então utilizadas para a incorporação das proteínas IgG e BSA em solução tampão fosfato salina (PBS). Os resultados indicaram o preenchimento dos microporos pela solução de PBS com valor superior a 95%. Quanto ao preenchimento de mesoporos, observou-se maior variação no volume de poros e área superficial, para o material com maior diâmetro médio de poros, atingindo valores em torno de 80 % e 90%, respectivamente. Não houve diferença significativa nestas porcentagens entre a incorporação das duas proteínas, indicando que as proteínas devem estar encapsuladas na macroporosidade da sílica, obstruindo a entrada dos mesoporos. Um modelo teórico foi utilizado para analisar as medidas de SAXS e ele confirmou o preenchimento dos microporos e a presença da proteína nos mesoporos da sílica. Utilizando a técnica analítica de Particle Induced X-ray Emission (PIXE), o conteúdo de silício em fezes de camundongos isogênicos foi avaliado em função do tempo, com o intuito de analisar a liberação de sílica pelo organismo dos camundongos. Observou-se que após a primeira administração de sílica os camundongos inoculados por via oral liberaram uma quantidade de silício similar à liberada pelos camundongos controle, que receberam apenas a solução de PBS. Após a segunda administração os camundongos inoculados com sílica por via oral liberaram uma quantidade de silício superior à dos camundongos controle, devido à morte dos macrófagos e outras células que atuam na resposta imunológica, evidenciando a eliminação de silício pelo organismo. Um estudo paralelo de controle de qualidade foi realizado em amostras de SBA-15 preparadas pela Cristália Produtos Químicos Farmacêuticos Ltda. a fim de garantir a reprodutibilidade deste material, que será comercialmente utilizado para a formulação de vacina oral contra Hepatite B, produzida pelo Instituto Butantan. Os resultados mostraram propriedades texturais reprodutíveis das amostras produzidas pela Cristália indústria farmacêutica.
In this work we studied the encapsulation of proteins with different molecular weights into SBA-15 ordered mesoporous silica to evaluate its application as immunological adjuvant. Hence, the IgG protein (150 kDa) and BSA (66.5 kDa) were incorporated into the mesoporous silica with expanded pores. First we studied the expansion of the pores using a structure swelling agent in the synthesis process, by preparing samples with different amounts of triisopropylbenzene (TiPB). Results of Nitrogen Adsorption Isotherm (NAI) and Small Angle X-Ray Scattering (SAXS) showed an increase in average pore diameter of the order of 23% and a more disordered pore network. To provide an estimate of the size of proteins, SAXS measurements were performed and indicated that both have dimensions that allow its incorporation in the pores of SBA-15. These samples were used for the incorporation of IgG and BSA proteins in phosphate buffered saline (PBS) solution. The results indicated a filling of the micropores by PBS solution larger than 95%. Concerning the filling of mesopores, we observed a larger variation in pore volume and surface area for the material with highest average pore diameter, reaching values around 80% and 90%, respectively. There was no significant difference in these percentages between the incorporation of the two proteins, indicating that the proteins could be encapsulated in the silica macroporosity, blocking the entry of mesopores. A theoretical model was used to analyze the SAXS measurements and it confirmed the filling of micropores and the presence of protein in the silica mesopores. Using the analytical technique \"Particle-Induced X-ray Emission\"(PIXE), the silicon content in mice feces was evaluated as a function of time, in order to analyze the release of silica from the organism of the mice. We observed that after the first silica administration the isogenic mice inoculated by oral administration released a similar amount of silicon compared with the amount released by the isogenic control mice, which received only PBS solution. After the second oral administration, the mice inoculated with silica released a higher amount of silicon than the control mice, due to the death of macrophages and other cells that act on the immune response, indicating the elimination of silicon by the organism. A parallel study about quality control was performed on SBA-15 samples prepared by Cristália Produtos Químicos Farmacêuticos Ltda., to ensure the reproducibility of this material which will be used commercially for the formulation of an oral vaccine against hepatitis B, produced by the Butantan Institute. The results showed reproducible textural properties of the samples produced by the Cristália pharmaceutical industry.

Background: Southern Africa has a high incidence of bacterial meningitis in adults, often associated with HN co-infection. Mortality exceeds 50%, even with appropriate antibiotic therapy, and is not improved with corticosteroids. Glycerol adjuvant therapy reduces long-term morbidity in bacterial meningitis in children, and its use is being promoted. We aimed to assess the effectiveness of glycerol as an adjuvant therapy for adults with bacterial meningitis in Africa In addition, we aimed to measure the effect of glycerol on reducing intracranial pressure and deafness. Methods: The study was done in two phases. First, in an open-label dose-finding study, 45 adult patients with symptoms, signs, and cerebrospinal fluid findings consistent with bacterial meningitis received 50, 75, or 100 ml of glycerol four times a day for 4 days. We then did a randomised, double-blind, placebo-controlled trial of oral glycerol in adults with bacterial meningitis. Patients with clinical and cerebrospinal fluid findings suggestive of bacterial meningitis were randomly assigned in blocks of 12 by use of a random number list produced by an independent statistician to receive either glycerol or an equivalent volume of sugar solution. Glycerol and placebo were indistinguishable by colour or taste. The primary outcome was mortality at 40 days, with secondary outcomes including disability and mortality restricted to pneumococcal disease; reduction in cerebrospinal fluid opening pressure and optic nerve sheath diameter measurement at Day 2 and hearing loss at Day 40. All patients were analysed for the primary outcome excluding those who were lost to follow-up. This trial is registered at controlled-trials. com, number ISRClN70121840. Findings: 75 mL glycerol four times a day was the highest tolerated dose, and was used for the main study. 265 patients were assigned treatment: 137 glycerol and 128 placebo. The trial wa.s stopped early on the advice of the data and safety monitoring board after a planned interim analysis. By Day 40,61 (49%) of 125 patients in the placebo group and 86 (63%) of 136 in the glycerol group had died (adjusted odds ratio 2-4,95% CI 1·3-4·2, p=0·003). There was no benefit from glycerol for death and disability by Day 40, and glycerol did not improve death and disability by Day 40 or death at Day 40 in patients with proven bacterial disease or pneumococcal disease. Two serious adverse events occurred that were possibly due to the study drug. Optic nerve sheath diameter and CSF opening pressure were not reduced by glycerol. There was a trend towards less hearing loss at Day 40 in the glycerol arm five (5.6%) compared with placebo 16 (21.3%) (p=0.026). Interpretation: Oral glycerol therapy cannot be recommended as an adjuvant therapy in adults with bacterial meningitis in resource-poor settings with a high HIV prevalence.

Bakgrund: Adjuvant cytostatikabehandling används vid bröstcancer efter att tumören tagits bort. Biverkningar kan uppstå då cytostatika påverkar alla kroppens celler. För att sjuksköterskan ska kunna ge en god omvårdnad krävs det att hon/han får ta del av patienters upplevelser vid adjuvant cytostatikabehandling. Syfte: Syftet var att beskriva kvinnors upplevelser av adjuvant cytostatikabehandling vid bröstcancer. Metod: En litteraturöversikt som baserades på 13 vetenskapliga artiklar med kvalitativ ansats utfördes. Resultat: Resultatet utgjordes av tre huvudkategorier: uppleva en förändrad kropp, uppleva ett förändrat sinne och upplevd påverkan på det sociala livet. Det framkom att kvinnorna upplevde både fysiska och psykiska biverkningar och dessa hade en stor påverkan på deras sociala liv. Slutsats: I slutsatsen betonas vikten av sjuksköterskans stöd i form av information och omvårdnad.
Background: Adjuvant chemotherapy is used to treat breast cancer after the removal of the tumour. Side effects may occur since chemotherapy affects all of the body cells. The nurse should be able to provide good care, to do so it requires that she/he takes part of patients’ experiences of adjuvant chemotherapy. Aim: The aim was to describe women’s experiences of adjuvant chemotherapy for breast cancer Method: A literature review based on 13 scientific articles with qualitative approach was executed. Results: The result consisted of three main categories: experiencing a changed body, experiencing a changed mind and perceived impact on social life. It was revealed that women experienced both physical and psychological side effects and they had a great impact on their social life. Conclusion: The conclusion shows the importance of the nurse’s support in form of information and care.

SAMMANFATTNING Bakgrund: Enligt Regionala Cancercentrums nationella vårdplan för cancerrehabilitering rekommenderas alla patienter med cancerdiagnos att försöka röra på sig lika mycket som en frisk människa rekommenderas. Trots detta så händer det att personer med cancerdiagnos minskar sin träning. Därför är det viktigt att få en fördjupad förståelse av hur dessa patienter upplever träning under sin behandling. Syfte: Beskriva hur cancerpatienter upplever träning under pågående behandling samt vilka faktorer som påverkar patienternas träning (egenvård) under pågående behandling. Metod: Kvalitativ intervjustudie. Elva intervjuer från en pilotstudie i Phys-Can projektet analyserades med hjälp av en kvalitativ innehållsanalys. Resultat: Stöd från professionella, personer i en likartad situation, anhöriga samt stöd från sig själv upplever deltagarna varit viktiga för att kunna utföra träningen. Praktiska faktorer som tid till träning, möjligheten att ta sig till träningen samt lokalen upplever deltagarna påverka hur träningen uppfattas. Deltagarna har upplevt att träningen påverkar psykiska och fysiska faktorer hos dem mestadels till det positiva. Träningen har även påverkat några av deltagarnas upplevelse av deras sjukdom. Slutsats: Deltagarna i denna studie har upplevt att stöd i olika former har varit viktigt för att kunna utföra träningen som egenvård. De praktiska förhållandena runt träningen har upplevts kunna både försvåra eller förenkla träningen. Deltagarna har även upplevt att träningen har påverkat deras psykiska och fysiska hälsa oftast till det positiva. Det är av vikt att kunna se varje individs förutsättningar att kunna utföra egenvården och sedan anpassa stödet så att träningsförutsättningarna blir så bra som möjligt.
ABSTRACT Background: According to the Regional Cancer Centre's national treatment plan for cancer rehabilitation all patients with a cancer diagnosis are recommended to try to move as much as a healthy person. Even so it happens that people with cancer reduces their physical activity. Therefore, it is important to get a deeper understanding of how these patients experience training during their treatment. Aim: Describe how cancer patients experience exercise during treatment and the factors that affect patients’ exercise (self-care) during treatment. Method: A qualitative interview study. Eleven interviews from a pilot study in the Phys-Can project were analyzed using qualitative content analysis. Result: Support from professionals, people in a similar situation, relatives and support from her/himself participants experienced as important to carry out their training. Practical factors as time to exercise, the ability to get to the training venue and how they experienced the venue the participants affected how their training was perceived. The participants have experienced that exercise affects mental and physical factors in them mostly for the better. The traning has also affectes som of the participants’ experince of their disease. Conclusion: The participants in this study have experienced various forms of support that has been experienced as important to be able to perform the exercise as self-care. The practical conditions around the training could both complicate or simplify training. Participants also felt that the training has affected their mental and physical health, usually for the better. It is important to be able to see each individual's ability to perform self-care, and then align support so that exercise becomes as good as possible.

Includes abstract.
Includes bibliographical references.
South African children currently receive vaccines against Diphtheria, Tetanus, Pertussis (administered as DTP), Hepatitis B (HBV) and Haemophilus influenzae type b (Hib) at 6, 10 and 14 weeks. The use of combination vaccines provides a means of avoiding the logistical problems and costs associated with multiple injections of these different vaccines. A local vaccine manufacturer is in the process of developing a combined tetravalent DTP-HBV as well as a liquid pentavalent DTP-HBV-Hib vaccine. The Hib vaccine is a glycoconjugate in which Haemophilus influenzae type b capsular polysaccharide (polyribosylribitolphosphate or PRP) is conjugated to a carrier protein. The conjugate is immunogenic in infants who have a higher risk of infection while the polysaccharide vaccine is not. The compatibility of the Hib antigenic component in the presence of the other antigens and adjuvant presents a challenge. Aluminium containing adjuvants have been the most widely used adjuvants in human vaccines. Aluminium hydroxide has been found to catalyse the hydrolysis of PRP when added to Hib conjugate vaccines. Although this can be circumvented by the use of aluminium phosphate, there is a need for new adjuvants that elicit broader immune responses. This thesis presents a study of the size, structure and composition of a locally developed experimental adjuvant called Pheroid™ by use of NMR spectroscopy and Coulter Counter. NMR analysis of Pheroid™ formulations provided a structural fingerprint for the formulations and indicated the relative proportions of the major components present, whereas their particle size distribution was profiled using a Coulter Counter. The average size distribution was similar in the formulations tested including those that had been activated using nitrous oxide. The primary focus of this thesis was the application of appropriate physicochemical procedures for evaluation of the locally manufactured Hib vaccine alone and when in combination with DTP-HBV-Hib. Investigating the stability and integrity of Hib conjugate vaccines requires determination of the total saccharide and unbound or free saccharide which is expressed as the percentage of free saccharide present. In the absence of a suitable Hib conjugate, model compounds such as human serum albumin (HSA) , meningococcal group A polysaccharide (PsA) and the derived conjugate (Mn A-IT) were used to investigate three methods of free saccharide separation: solid phase extraction (SPE), acid precipitation using deoxycholate (DOC/HCI) and ultrafiltration (UF). At physiological pH, the binding capacity was low and so the SPE method was not investigated further. For Mn A-IT the DOC/HCI method generally gave lower free saccharide values than the UF method; this was attributed to entrapment and coprecipitation of free saccharide by DOC/HCI. In contrast, washing steps in the UF method ensured good free saccharide recovery. A colorimetric assay for phosphorus and high performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) were investigated for saccharide quantification in Mn A-TT vaccines. The HPAEC-PAD method for the monomer (mannosamine-6-phosphate from acid hydrolysis) permitted higher specificity and sensitivity for the free saccharide analysis compared to the phosphate assay. The DOC/HCI and UF methods were compared by their application to Mn A-TT samples subjected to an accelerated stability study.

Cyclin D1, a proto-oncogene, is required for progression from the G1 phase into the S phase of the cell cycle. Over-expression of cyclin D1 causes an increase in cell cycle progression and cell proliferation, implicating it in a variety of cancers including renal cell carcinoma (RCC). The rodent RCC cell model, QTRRE, and human RCC cell models, ACHN, 786-O and Caki-2, exhibit elevated levels of cyclin D1. Pentoxifylline (PTX), a non-specific phosphodiesterase inhibitor, is an FDA-approved hemorheologic agent used to treat intermittent claudication, stemming from peripheral vascular diseases, as well as other diseases involving defective locoregional blood flow. Treatment of QTRRE, ACHN, 786-O and Caki-2 with PTX caused a time- (0-24 hrs) and dose- (0-1.0 mg/mL) dependent decrease of cyclin D1 protein and p-Rb levels in whole cell lysate as well as cytosolic and nuclear fractions, albeit, to different extents within the models. Concomitant with cyclin D1 and p-Rb decrease, enhanced G1 phase cell cycle arrest was observed in the RCC models. Mechanistic studies in these RCC cell models were carried out to determine PTXs mechanism of action with regard to cyclin D1 protein level decrease. RT-PCR analysis showed no significant changes in cyclin D1 mRNA copy number in time- (0-24 hrs) and dose- (0-1.0 mg/mL) dependent PTX treatments. However, such treatments caused decrease in p-4EBP1 (Ser65), p-4EBP1 (Thr70), and p-4EBP1 (Thr37/46). Because PTX's ability to decrease cyclin D1 protein was prevented in the presence of the proteasome inhibitor, MG-132, studies were performed to determine whether cyclin D1 stability was decreased during PTX treatment. Cyclin D1 degradation is initiated by phosphorylation of residue Thr286 by GSK-3β. Inhibition of GSK-3β with LiCl or knockdown via siRNA in the presence of PTX failed to block cyclin D1 decrease. Moreover, PTX treatment in the presence of MG-132 revealed no significant increase in cyclin D1 p-Thr286 compared to control. Finally, using the protein synthesis inhibitor, CHX, PTX caused no significant decrease in cyclin D1 t₁/₂ (wt-HA and T286A-HA) compared to control. Sorafenib, a broad-spectrum (cRAF, bRAF, KIT, FLT-3, VEGFR-2, VEGFR-3, and PDGFR-β) kinase inhibitor, is FDA-approved for the treatment of RCC. Studies with sorafenib and PTX in the ACHN cell model were carried out to determine PTXs possible adjuvant role in inhibiting cell growth via cyclin D1 decrease and G1 phase arrest. MTS data showed PTX potentiates the anti-proliferative effects of sorafenib. PTX pre-treatment for 24 hrs was also lowered the effective dose of sorafenib from 50 μM to 5 μM. Further, ACHN xenograft tumor volumes from mice treated with PTX and sorafenib displayed significantly higher tumor growth inhibition compared to either drug treatment alone or vehicle. Finally, drug treated ACHN xenograft tissue displayed significantly lower cyclin D1, p-RB and p-4EBP1 levels. These results demonstrate a novel anti-cancer property of PTX and suggest its use as a possible adjuvant therapy in RCC treatment should be further explored.

Breast cancer is a leading cause of cancer related mortality globally, and epidemiological studies suggest a link between healthy nutrition and cancer prevention. Members of the Brassicaceae family, including watercress, have been extensively studied for their anti-cancer and anti-genotoxic potential. Watercress has a complex phytonutrient profile characterised by high levels of carotenoids, flavonols and glucosinolates. Extracts of watercress exhibit strong antioxidant capacity in vitro. Watercress and its components have been associated with the inhibition of the three stages of carcinogenesis: initiation, proliferation and metastasis in in vitro cancer cell models. Phenethyl isothiocyanate (PEITC) is a glucosinolate break-down product and watercress is the richest dietary source of it. It has received considerable attention for its anti-cancer properties and has been tested in a number of clinical trials. In this thesis, the effects of crude watercress extract and PEITC on the metabolic and phenotypic responses in breast cancer and healthy breast tissue cell lines were examined. Radiotherapy is the most common treatment modality for breast cancer patients; it functions by killing cancer cells but it simultaneously damages healthy tissues. We set out to examine synergistic responses to irradiation and watercress or PEITC exposures in breast cancer cells and we further investigated whether watercress or PEITC can be protective against radiation induced collateral damage. Watercress and PEITC effectively modulated important cancer cell metabolic pathways associated with anti-cancer endpoints such as cell cycle arrest and DNA damage. In this thesis, PEITC has been shown to enhance the sensitivity of cancer cells to irradiation making the cancer killing process more effective, whereas watercress can protect healthy breast cells from radiation induced damage. These observations appear to be mediated by the ability of PEITC and other phytochemicals in watercress to interact with the antioxidant glutathione. The results obtained from this work remain to be validated in a clinical setting.