A asma alérgica é uma doença pulmonar de inflamação crônica caracterizada por uma resposta imune do tipo Th2 e uma das principais abordagens terapêuticas para o seu tratamento no futuro, poderia ser a imunoterapia baseada na modulação da resposta imune Th2 para um perfil Th1 e anti-inflamatório. Nosso grupo já demonstrou a eficácia de uma imunoterapia, baseada em um plasmídeo de DNA contendo o gene hsp65 de M. Leprae (DNAHsp65) em modelo murino de asma alérgica. No entanto, apesar dos excelentes resultados, o grupo está procurando outras alternativas imunoterapêuticas, usando a Hsp65 micobacteriana, para futura utilização clinica na área de saúde humana e veterinária. Efeitos benéficos na prevenção e tratamento de doenças alérgicas vêm sendo obtidos com o uso de probióticos. Nossa hipótese é de que uma nova formulação terapêutica, como o probiótico Lactococcus lactis expressando Hsp65 micobacteriana (LLHsp65), apresentaria vantagens significativas no desenvolvimento de pesquisas translacionais. Diante disso, este estudo teve como objetivo avaliar se o probiótico LLHsp65 apresenta atividades imunoterapêuticas no modelo de asma experimental induzida por ovalbumina (OVA). Em diferentes grupos experimentais, 5x109 CFU de LLHsp65 ou de L. lactis selvagem (LLSELV), foram administrados por via oral durante 10 dias consecutivos aos camundongos BALB/c previamente sensibilizados e desafiados com OVA. Em seguida, investigamos os efeitos do tratamento na inflamação alérgica, modulação do padrão de citocinas, produção de anticorpos específicos, hiper-responsividade das vias aéreas e inflamação pulmonar. Nossos resultados demonstraram que o tratamento oral com LLhsp65 modula a resposta imune alérgica de padrão Th2 para o perfil Th1 com aumento dos níveis de IFN-?, IL-12, TNF-?, IL-10, IL-6 e IL- 17, e com a redução das citocinas IL-4, IL-5 e IL-13. Os níveis de IgE e IgG1 anti-OVA no soro também foram significativamente diminuídos. Como consequência desses resultados, também observamos diminuição significativa do infiltrado de eosinófilos no lavado broncoalveolar, na hiper-responsividade nas vias aéreas e a atenuação da inflamação e produção de muco no tecido pulmonar, quando comparados com o grupo controle (OVA/SAL). Por conseguinte, nossos resultados demonstraram que a administração oral de LLHsp65 proporciona uma melhora significativa do processo inflamatório alérgico induzido por OVA, sendo portanto, uma estratégia terapêutica promissora para o desenvolvimento de pesquisa translacional no tratamento de asma alérgica. / Allergic asthma is a chronic inflammatory lung disease characterized by a Th2-type immune response and one of the main therapeutic approaches to its treatment in the future could be immunotherapy based on the modulation of the Th2 immune response to a Th1 and anti-inflammatory profile. Our group has already demonstrated the efficacy of an immunotherapy based on a DNA plasmid carrying the mycobacterial hsp65 gene (DNAHsp65) in murine model of allergic asthma. However, despite the excellent results, our group is looking for other immunotherapeutic alternatives, using mycobacterial Hsp65, for future clinical use in the area of human and veterinary health. Beneficial effects in the prevention and treatment of allergic diseases have been obtained with the use of probiotics. Our hypothesis is that a new therapeutic formulation, such as the probiotic Lactococcus lactis expressing mycobacterial Hsp65 (LLHsp65), would present significant advantages in the development of translational research. The objective of this study was to evaluate whether the probiotic LLHsp65 has immunotherapeutic activities in the ovalbumin-induced asthma model. In different experimental groups, 5x109 CFU of LLHsp65 or control L. lactis (ctLL) were administered orally for 10 consecutive days to BALB/c mice previously sensitized and challenged with OVA. We then investigated the effects of treatment on allergic inflammation, modulation of the cytokine pattern, production allergen-specific antibodies, airway hyperresponsiveness and pulmonary inflammation. Our results demonstrated that oral treatment with LLhsp65 modulates the allergic Th2 immune response to Th1 profile with increased levels of IFN-?, IL-12, TNF-?, IL-10, IL-6 and IL-17 and with the reduction of IL-4, IL-5 and IL-13 cytokines. Serum anti-OVA IgE and IgG1 levels were also significantly decreased. Correspondingly with these results, we also observed a significant decrease in eosinophil infiltrate in bronchoalveolar lavage, airway hyper responsiveness and attenuation of inflammation and mucus production in lung tissue when compared to the control group (OVA/SAL). Therefore, our results demonstrated that oral administration of LLHsp65 provides a significant improvement of the OVA-induced allergic inflammatory process and is therefore a promising therapeutic strategy for the development of translational research in the treatment of allergic asthma.

The production of Heat Shock Protein 72, the inducible for~ of the highly conserved 70 kilodalton heat shock protein family, was investigated in MRL++ mouse intestine during the two weeks of a Trichinella spiralis infection. Within hours of an oral infection using the encysted Trichinella spiralis found in the diaphragm of an infected mouse, the larvae are released from the cyst in the stomach. They travel to the intestine and burrow into the epithelial layer of the intestine. The jejunum is the primary site of the intestinal phase of trichinosis (Despommier 1983). This stage of infection in the jejunum was the focus of this study. Heat shock protein (HSP) synthesis is precipitated by stressful stimuli: in vitro by chemicals such as sodium arsenite and in vivo by cytoskeletal disturbance and/or toxic 02 radicals (Linquist 1986). The latter in vivo studies lend support to the inflammatory response induction of HSPs. Heat shock protein 72 (HSP72) is rarely expressed constitutively especially in non-primates and is a good indicator of various stresses. This study hypothesized that HSP72 would be induced by cells in the jejunum of the MRL++ mouse during a Trichinella spiralis infection due to the stress of the parasitic infection. Different techniques were employed to investigate this hypothesis. Immunohistochemistry and immunoblots facilitated this study. Although immunoblots did not demonstrate HSP72 induction, immunohistochemical analysis suggested the presence of HSP72 in various cells in the lamina propria of the jejunal villi.

Upon forming a biofilm, bacteria undergo several changes that prevent them from being eradicated with antimicrobials alone. These biofilms manifest as persistent infections and biofouling in the medical and industrial world, respectively, constituting an ongoing medical crisis and creating a huge financial burden. Biofilms on implanted medical devices cause thousands of patients each year to undergo multiple surgeries to explant and replace the implant, driving billions of dollars in increased health care costs due to the lack of viable treatment options for in situ biofilm eradication. Heat has been used to reliably eliminate biofilms for many years, but the temperatures employed are infeasible for many applications, particularly in vivo medical treatment. Remotely activated localized heat can be applied through a superparamagnetic iron oxide nanoparticle polymer coating when paired with an alternating magnetic field. However, there is very little known about the temperatures required to kill the biofilms and the effects of the heat in conjunction with antibiotics. To better understand the required parameters to effectively kill off bacteria in biofilms a variety of heat treatments were investigated for a variety of Pseudomonas aeruginosa biofilms grown in different conditions. Additionally, these heat treatments were combined with antibiotics to better understand any combined effects of the two orthogonal treatment plans. It was found that heat is an effective method for killing the bacteria in biofilms. Temperatures ranging from body temperature, 37 °C, to 80 °C were used to heat shock the biofilms for 1 to 30 minutes. Higher temperatures for short exposure times yielded similar results to lower temperatures for longer exposure time. Biofilms grown in different conditions did vary in their susceptibility to the heat shocks; however, at the higher temperatures the differences became negligible. Therefore, the more effective treatments were the higher temperature heat shocks with shorter exposure times to maximize bacterial cell death and minimize the potential heat transfer to the surrounding tissue. Regrowth studies indicate a critical post-shock bacterial loading (~103 CFU/cm2) below which the biofilms were no longer viable, while films above that loading slowly regrew to their previous population density. Combined treatments with antibiotics had synergistic effects for all antibiotics across a window of heat shock conditions. Erythromycin in particular, which showed no effect on the biofilm alone, decreased biofilm population by six orders of magnitude at temperatures which had no effect in the absence of antibiotics. These studies will evolve the understanding of biofilms and how to efficiently eradicate them on implant surfaces. The introduction of such a novel coating in conjunction with antibiotics could obviate thousands of surgeries and save billions of dollars spent on explantation, recovery, and re-implantation.

Thesis (Ph. D.)--University of Hong Kong, 2006. / Title proper from title frame. Also available in printed format.

<p>New efficient vaccines against infectious diseases are in demand. Some important factors impeding the vaccine development are the poor immunogenicity and the MHC restriction of the immune responses to a number of antigens. The use of novel vaccine adjuvants or carrier proteins, which are known to enhance the immunogenicity of the subunit antigens and provide T-cell help, can circumvent these problems. The potential of heat shock proteins (HSPs) to function as adjuvants when fused to or co-delivered with protein antigens, make them attractive vaccine candidates. In this thesis we have evaluated the potency of heat shock protein 70 (HSP70) as a possible vaccine adjuvant and studied the mechanisms behind the adjuvanticity.</p><p>The first article aims to evaluate the carrier effect of glutathione-S-transferase (GST) on a malarial antigen EB200 that induces a MHC restricted response in mice. Immunization of CBA and C57BL/6 mice, high and low responders to EB200, respectively, with the GST-EB200 fusion protein elicited EB200 specific antibody responses in both strains of mice, which indicated that MHC restriction was broken in C57BL/6 mice. However, the antibody affinity and the magnitude of the response were lower in the C57BL/6 mice compared with that in CBA. To improve the response, the efficacy of various adjuvants like alum, HSP70 from <i>Trypanosoma cruzi</i>, and the adjuvant combination (HSP70 and cholera toxin) was evaluated. The results indicated that cholera toxin and HSP70 act synergistically and improve the immunogenicity of EB200 antigen by increasing the affinity and magnitude of the response.</p><p>HSP belongs to a family of conserved molecules and the maximum homology lies on the N-terminal region of the protein, therefore there is a risk that use of a complete molecule would give rise to autoimmunity. Thus, in our second study we first evaluated the adjuvant effect of the less conserved portion of HSP70 derived from <i>Plasmodium falciparum</i> (Pf70C). We found that the Pf70C exhibited similar adjuvant properties as the whole molecule. We further analyzed the adjuvant potential of Pf70C against EB200 formulated as a chimeric DNA vaccine construct. These constructs alone failed to generate substantial levels of EB200 specific antibodies in mice. However, the DNA immunization efficiently primed the immune system. This was evident as the subsequent boosting with the corresponding recombinant fusion proteins Pf70C-EB200 elicited strong EB200 specific Th-1 antibody responses. In contrast, no such priming effect was observed for <i>ex vivo</i> IFN-γ production, however stimulation with the Pf70C-EB200 fusion protein induced an enhanced secretion of IFN-γ <i>in vitro</i>.</p><p>During the infection process, the synthesis of bacterial HSP is up-regulated, which is known to sensitize T cells in the infected host. Since a high degree of homology exists within the phylogenetic families of HSPs, we postulated that exposure of mice to microorganisms could prime the immune system for evolutionary diverse HSPs and for any antigen coupled to them. We tested this hypothesis by priming mice with different microorganisms such as BCG, <i>Mycobacterium vaccae</i> or <i>Chlamydia pneumoniae</i> and boosted with a recombinant fusion protein Pf70C-EB200 or with a panel of HSPs. We found that BCG and <i>M. vaccae</i> but not <i>C. pneumoniae</i> could provide priming of the immune system to induce secondary IgG responses to Pf70C as well as to other HSPs tested. The priming effect was also observed when the EB200 antigen was coupled to Pf70C. Analysis of the IgG1 and IgG2a profiles and IFN-g production induced against the HSPs revealed a mixture of Th1/Th2 type of responses. We also observed that HSP70 specific sera cross-reacted some extent with certain autoreactive antigens. However, no deposits were observed in the kidneys of HSP treated animals.</p><p>Finally, we investigated the role of TLR2 and TLR4 on HSP70-mediated adjuvanticity. We found that HSPs displayed different degrees of adjuvanticity regarding both the strength and the profile of the induced immune response. Also, they possessed different requirements for signaling through TLRs. While HSP70 from <i>T. cruzi</i> induced antigen-specific humoral responses in wild type as well as in both the TLR2 and TLR4 knockout mice, the response was diminished in the TLR4 knockout mice when both the whole and C-terminal fragment of HSP70 from <i>Mycobacterium tuberculosis</i> was used. However, the C-terminal fragment of <i>P. falciparum</i> HSP70 elicited responses only in wild type mice but not in TLR2 or TLR4 knockout mice indicating that the adjuvant function differ for phylogenetically related HSPs. Taken together our data suggest that HSPs can be promising candidates in future vaccines.</p>

Graduation date: 1995

Neurons need to be maintained to persist throughout adulthood for proper brain function. However neuronal activity, injury and aging exert physical stress on the nervous system, which compromise nervous system function. Healthy neurons are able to maintain their integrity throughout the lifespan of the animal, suggesting the existence of a maintenance mechanism that allows neurons to sustain or even repair damage. A forward genetic screening in Drosophila identified mutations in a gene called nmnat that cause a rapid and severe neurodegeneration immediately post neuronal differentiation and development. NMNAT protein was required to maintain neuronal integrity in an activity-dependent manner. When probing for the exact role of NMNAT in neuronal maintenance, a novel stress responsive chaperone function was identified, in addition to its essential housekeeping NAD synthase role. In this work, the mechanism of NMNAT-mediated neuroprotection is investigated. First, the transcriptional regulation of Drosophila NMNAT during acute stress is analyzed. Here, both stress transcription factors heat shock factor (HSF) and hypoxia inducible factor alpha (HIF1-α) have been shown to upregulate NMNAT during stress through a heat shock element in the nmnat promoter. In addition, the role of NMNAT for stress tolerance in Drosophila is revealed. Second, to elucidate the neuroprotective capacity of NMNAT in neurodegenerative disease, mouse models of tauopathy have been used. In the P301L Tau-transgenic mouse model, the levels of endogenous NMNAT2 have been studied at various ages to link a reduction in NMNAT2 as a precursor for neurodegeneration. The underlying mechanism of NMNAT2 downregulation is further studied in this model. Third, using Drosophila model of Tauopathy, the protective capacity of both wild type and enzyme-inactive NMNAT in ameliorating the pathological and behavioral impairments from Tau-induced neurodegeneration were studied extensively. The possible protective mechanism of NMNAT is uncovered by identifying novel interactions of NMNAT with hyperphosphorylated and ubiquitinated Tau in regulating the levels of toxic Tau species. Finally, this study also identified endogenous proteins that NMNAT interacts with to provide insight into a neuroprotective chaperone role of NMNAT. Together, these studies improve our understanding of the mechanisms of neuronal maintenance, by providing a comprehensive investigation of the stress-responsive regulation of NMNAT in both Drosophila and mammalian models, and its role as a chaperone both in protein foldopathies and in healthy neurons.

Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (leaves 119-141).

Alzheimer’s disease (AD) is a neurodegenerative disorder that is one of such diseases associated with protein aggregation. Aβ is the main protein component of senile plaques in AD, and is neurotoxic when aggregated. In particular, soluble oligomeric forms of Aβ are closely related to neurotoxicity. In this dissertation, we examine the differences in Aβ aggregation intermediates, and final structures formed when only a simple modification in Aβ aggregation conditions is made, the presence or absence of mixing during aggregation. We show that intermediates in the aggregation pathway show significantly different structural rearrangements. The protein stabilities of Αβ species show that spherical aggregates corresponding to the most toxic Αβ species change their structure the most rapidly in denaturant, and that in general, increased toxicity correlated with decreased aggregate stability. In Alzheimer’s disease, even delaying Aβ aggregation onset or slowing its progression might be therapeutically useful, as disease onset is late in life. Small heat shock proteins (sHsps) may be useful for prevention of Αβ aggregation, since sHsps can interact with partly folded intermediate states of proteins to prevent incorrect folding and aggregation. In this research, several small heat shock proteins (sHsps) are tested to prevent Aβ aggregation and toxicity. sHsps used in this research are Hsp17.7, Hsp27, and Hsp20. All types of Hsp20, Hsp20-MBP, His-Hsp20 and His-Hsp20 without 11 residues in C-terminus, can prevent Aβ1-40 aggregation. Hsp20 also prevents Aβ toxicity in the same concentration ranges of it aggregation prevention activity. Hsp17.7 and Hsp27, however, can inhibit Αβ1-40 aggregation but not toxicity. A number of experiments to examine the mechanism of Hsp20 suggest that multivalent binding of sHsp to Aβ is necessary for the toxicity prevention activity. Conclusively, different Aβ incubation conditions in vitro can affect the rate of Aβ fibril formation, the morphology, the toxicity and the conformation of intermediates in the aggregation pathway. Hsp20 rather than other sHsps may be a useful molecular model for the drug design of the next generation of Aβ aggregation inhibitors to be used in the treatment of AD.

New efficient vaccines against infectious diseases are in demand. Some important factors impeding the vaccine development are the poor immunogenicity and the MHC restriction of the immune responses to a number of antigens. The use of novel vaccine adjuvants or carrier proteins, which are known to enhance the immunogenicity of the subunit antigens and provide T-cell help, can circumvent these problems. The potential of heat shock proteins (HSPs) to function as adjuvants when fused to or co-delivered with protein antigens, make them attractive vaccine candidates. In this thesis we have evaluated the potency of heat shock protein 70 (HSP70) as a possible vaccine adjuvant and studied the mechanisms behind the adjuvanticity. The first article aims to evaluate the carrier effect of glutathione-S-transferase (GST) on a malarial antigen EB200 that induces a MHC restricted response in mice. Immunization of CBA and C57BL/6 mice, high and low responders to EB200, respectively, with the GST-EB200 fusion protein elicited EB200 specific antibody responses in both strains of mice, which indicated that MHC restriction was broken in C57BL/6 mice. However, the antibody affinity and the magnitude of the response were lower in the C57BL/6 mice compared with that in CBA. To improve the response, the efficacy of various adjuvants like alum, HSP70 from Trypanosoma cruzi, and the adjuvant combination (HSP70 and cholera toxin) was evaluated. The results indicated that cholera toxin and HSP70 act synergistically and improve the immunogenicity of EB200 antigen by increasing the affinity and magnitude of the response. HSP belongs to a family of conserved molecules and the maximum homology lies on the N-terminal region of the protein, therefore there is a risk that use of a complete molecule would give rise to autoimmunity. Thus, in our second study we first evaluated the adjuvant effect of the less conserved portion of HSP70 derived from Plasmodium falciparum (Pf70C). We found that the Pf70C exhibited similar adjuvant properties as the whole molecule. We further analyzed the adjuvant potential of Pf70C against EB200 formulated as a chimeric DNA vaccine construct. These constructs alone failed to generate substantial levels of EB200 specific antibodies in mice. However, the DNA immunization efficiently primed the immune system. This was evident as the subsequent boosting with the corresponding recombinant fusion proteins Pf70C-EB200 elicited strong EB200 specific Th-1 antibody responses. In contrast, no such priming effect was observed for ex vivo IFN-γ production, however stimulation with the Pf70C-EB200 fusion protein induced an enhanced secretion of IFN-γ in vitro. During the infection process, the synthesis of bacterial HSP is up-regulated, which is known to sensitize T cells in the infected host. Since a high degree of homology exists within the phylogenetic families of HSPs, we postulated that exposure of mice to microorganisms could prime the immune system for evolutionary diverse HSPs and for any antigen coupled to them. We tested this hypothesis by priming mice with different microorganisms such as BCG, Mycobacterium vaccae or Chlamydia pneumoniae and boosted with a recombinant fusion protein Pf70C-EB200 or with a panel of HSPs. We found that BCG and M. vaccae but not C. pneumoniae could provide priming of the immune system to induce secondary IgG responses to Pf70C as well as to other HSPs tested. The priming effect was also observed when the EB200 antigen was coupled to Pf70C. Analysis of the IgG1 and IgG2a profiles and IFN-g production induced against the HSPs revealed a mixture of Th1/Th2 type of responses. We also observed that HSP70 specific sera cross-reacted some extent with certain autoreactive antigens. However, no deposits were observed in the kidneys of HSP treated animals. Finally, we investigated the role of TLR2 and TLR4 on HSP70-mediated adjuvanticity. We found that HSPs displayed different degrees of adjuvanticity regarding both the strength and the profile of the induced immune response. Also, they possessed different requirements for signaling through TLRs. While HSP70 from T. cruzi induced antigen-specific humoral responses in wild type as well as in both the TLR2 and TLR4 knockout mice, the response was diminished in the TLR4 knockout mice when both the whole and C-terminal fragment of HSP70 from Mycobacterium tuberculosis was used. However, the C-terminal fragment of P. falciparum HSP70 elicited responses only in wild type mice but not in TLR2 or TLR4 knockout mice indicating that the adjuvant function differ for phylogenetically related HSPs. Taken together our data suggest that HSPs can be promising candidates in future vaccines.