Avaliação do tratamento com um inibidor para serinoprotease em modelo experimental de enfisema induzido por exposição à fumaça de cigarro

Introdução: Demonstramos previamente que em modelo experimental de enfisema pulmonar induzido por instilação de elastase, o inibidor de serinoprotease rBmTI-A promoveu a melhora da destruição tecidual em camundongos. Considerando que o tabagismo é o principal fator de risco para o desenvolvimento da Doença Pulmonar Obstrutiva Crônica (DPOC) e que o modelo de exposição à fumaça de cigarro é considerado o que melhor mimetiza esta doença em humanos, este estudo teve por objetivo verificar a ação do inibidor para serinoproteases rBmTI-A sobre os processos fisiopatológicos envolvidos no desenvolvimento do enfisema pulmonar, em modelo de exposição ao tabaco. Métodos: Para a indução do enfisema pulmonar, os animais foram expostos à fumaça de cigarro (duas vezes ao dia/ 30 minutos/ 5 dias por semana/ durante 12 semanas), e os animais controle permaneceram expostos ao ar ambiente. Dois protocolos de tratamento com o inibidor rBmTI-A foram realizados. No primeiro, os animais receberam duas administrações do inibidor rBmTI-A ou de seu veículo (Solução Salina 0,9%) por via intranasal, sendo a primeira após 24h do término das exposições ao cigarro e outra, 7 dias após à primeira instilação do inibidor. No segundo protocolo, os animais receberam 3 administrações do inibidor rBmTI-A, durante o tempo de exposição (1ª dose: 24h antes do início da exposição à fumaça de cigarro; 2ª dose: um mês após o início da exposição; 3ª dose: dois meses após o início). Após o término dos protocolos de exposição e tratamento, os animais foram submetidos aos procedimentos para coleta dos dados de mecânica respiratória e avaliação do Intercepto Linear Médio (Lm). Para o segundo protocolo, realizamos também as medidas para quantificação de fibras de colágeno e elástica, da densidade de células positivas para MAC-2, MMP-12 e 9, TIMP-1, Gp91phox e TNFalfa; no parênquima através de imunohistoquímica, contagem de células polimorfonucleares além da expressão gênica de MMP-12 e 9 no pulmão através de RT-qPCR. Resultados e Discussão: O tratamento com o inibidor para serinoprotease rBmTI-A atenuou o desenvolvimento do enfisema pulmonar apenas no segundo protocolo, quando foi administrado durante a exposição à fumaça de cigarro. Embora os grupos Fumo-rBmTIA e Fumo-VE apresentem aumento de Lm comparados aos grupos controles, houve uma redução deste índice no grupo Fumo-rBmTIA comparado ao grupo Fumo-VE. O mesmo comportamento foi observado para as análises de proporção em volume de fibras de elástica e colágeno no parênquima. Além disto, observamos aumento de macrófagos, MMP-12, MMP-9 e TNFalfa; nos grupos expostos à fumaça de cigarro, mas o tratamento com o inibidor rBmTI-A diminuiu apenas a quantidade de células positivas para MMP-12. Na avaliação da expressão gênica para MMP-12 e 9, não observamos diferença entre os grupos experimentais e o mesmo comportamento foi observado para a quantidade de células polimorfonucleares no parênquima. Além disso, observamos aumento de GP91phox e TIMP-1 nos grupos tratados com rBmTIA. Conclusões: Tais resultados sugerem que o inibidor rBmTI-A não foi efetivo como tratamento da lesão após a doença instalada. Entretanto, atenuou o desenvolvimento da doença quando administrado durante a indução do enfisema, possivelmente através do aumento de GP91phox e TIMP-1, acompanhados pela diminuição de MMP-12.

Novel particulate BCG-loaded delivery system for mucosal immunization against tuberculosis

Tese de doutoramento, Farmácia (Tecnologia Farmacêutica), Universidade de Lisboa, Faculdade de Farmácia, 2016

Regulatory T Cells Accumulate in the Lung Allergic Inflammation and Efficiently Suppress T-Cell Proliferation but Not Th2 Cytokine Production

Foxp3(+)CD25(+)CD4(+) regulatory T cells are vital for peripheral tolerance and control of tissue inflammation. In this study, we characterized the phenotype and monitored the migration and activity of regulatory T cells present in the airways of allergic or tolerant mice after allergen challenge. To induce lung allergic inflammation, mice were sensitized twice with ovalbumin/aluminum hydroxide gel and challenged twice with intranasal ovalbumin. Tolerance was induced by oral administration of ovalbumin for 5 consecutive days prior to OVA sensitization and challenge. We detected regulatory T cells (Foxp3(+)CD25(+)CD4(+) T cells) in the airways of allergic and tolerant mice; however, the number of regulatory T cells was more than 40-fold higher in allergic mice than in tolerant mice. Lung regulatory T cells expressed an effector/memory phenotype (CCR4(high)CD62L(low)CD44(high)CD54(high)CD69(+)) that distinguished them from naive regulatory T cells (CCR4(int)CD62L(high)CD44(int)CD54(int)CD69(-)). These regulatory T cells efficiently suppressed pulmonary T-cell proliferation but not Th2 cytokine production.

Glutathione in Parkinson's disease

Thesis (Ph.D.)--University of Washington, 2016-03

Lipid and carbohydrate based adjuvant/carriers in immunology

This review discusses various issues regarding vaccines:what are they and how they work, safety aspects, the role of adjuvants and carriers in vaccination, synthetic peptides as immunogens, and new technologies for vaccine development and delivery including the identification of novel adjuvants for mucosal vaccine delivery. There has been a recent increase of interest, in the use of lipids and carbohydrates as adjuvants, and so a particular emphasis is placed on adjuvants derived from lipids or carbohydrates, or from both. Copyright (C) 2003 European Peptide Society and John Wiley Sons, Ltd.

Downregulation of IgE antibody and allergic responses in the lung by epidermal biolistic microparticle delivery

Background: Biolistic injections provide a needle-free delivery of antigen-laden microparticles to the epithelium. The precision of the injection preferentially targets the Langerhans cell network, which, although ideal for vaccination, might not be suitable for the downregulation of immune responses in immunotherapy. Objective: We sought to determine the ability of biolistic injection of antigen into the epithelium of sensitized mice to inhibit IgE antibody and lung inflammatory responses produced by further exposure to antigen. Methods: Mice were sensitized by means of a needle injection of ovalbumin (OVA) in alum and given a series of biolistic injections of OVA or vehicle control, followed by a boost of OVA in alum. Serum IgE and IgG antibodies were measured before and after the boost. The mice were then challenged intranasally, and the infiltration of inflammatory cells was measured by means of bronchoalveolar lavage. Airway reactivity of the challenged mice was measured by examining responses to methacholine with forced oscillatory techniques. Results: Biolistic injection of OVA into the dorsal skin of sensitized mice markedly inhibited IgE and IgG1 antibody responses induced by boosting. IgG2a antibody responses were reduced rather than stimulated. The eosinophilic inflammation in the bronchoalveolar lavage fluid induced by intranasal challenge was also markedly inhibited. Lung hyperreactivity showed an initial increase and then a decrease of responsiveness to methacholine, with elastance returning to the level of unsensitized mice. Biolistic injection into the buccal epithelium was also inhibitory. Conclusions: Biolistic injection of allergen inhibited the boosting of IgE antibody and eosinophilic lung inflammatory responses without inducing TO immunity.

Novel bioadhesive formulations for mucosal and parenteral delivery of vaccines

In this work we have established the efficient mucosal delivery of vaccines using absorption enhancers and chitosan. In addition, the use of chitosan was shown to enhance the action of other known adjuvants, such as CTB or Quil-A. Collectively, the results presented herein indicate that chitosan has excellent potential as a mucosal adjuvant. We have evaluated a number of absorption enhancers for their adjuvant activity in vivo. Polyornithine was shown to engender high scrum immune reasons to nasally delivered antigens, with higher molecular weight polyornithine facilitating the best results. We have demonstrated for the first time that vitamin E TPGS can act as mucosal adjuvant. Deoxycholic acid, cyclodextrins and acylcarnitines were also identified as effective mucosal adjuvants and showed enhanced immune responses to nasally delivered TT, DT and Yersinia pestis V and F1 antigens. Previously, none of these agents, common in their action as absorption enhancing agents, have been shown to have immunopotentiating activity for mucosal immunisation. We have successfully developed novel surface modified microspheres using chitosan as an emulsion stabiliser during the preparation of PLA microspheres. It was found that immune responses could be substantially increased, effectively exploiting the immunopenetrating characteristics of both chitosan and PLA microspheres in the same delivery vehicle. In the same study, comparison of intranasal and intramuscular routes of administration showed that with these formulations, the nasal route could be as effective as intramuscular delivery, highlighting the potential of mucosal administration for these particulate delivery systems. Chitosan was co-administered with polymer microspheres. It was demonstrated that this strategy facilitates markedly enhanced immune responses in both magnitude and duration following intramuscular administration. We conclude that this combination shows potential for single dose administration of vaccines. In another study, we have shown that the addition of chitosan to alum adsorbed TT was able to enhance immune responses. PLA micro/nanospheres were prepared and characterised with discreet particle size ranges. A smaller particle size was shown to facilitate higher scrum IgG responses following nasal administration. A lower antigen loading was additionally identified as being preferential for the induction of immune responses in combination with the smaller particle size. This may be due to the fact that the number of particles will be increased when antigen loading is low, which may in turn facilitate a more widespread uptake of particles. PLA lamellar particles were prepared and characterised. Adsorbed TT was evaluated for the potential to engender immune responses in vivo. These formulations were shown to generate effective immune responses following intramuscular administration. Positively charged polyethylcyanoacrylate and PLA nanoparticies were designed and characterised and their potential as delivery vehicles for DNA vaccines was investigated. Successful preparation of particles with narrow size distribution and positive surface charge (imparted by the inclusion of chitosan) was achieved. In the evaluation of antibody responses to DNA encoded antigen in the presence of alum administered intranasally, discrimination between the groups was only seen following intramuscular boosting with the corresponding protein. Our study showed that DNA vaccines in the presence of either alum or Quil-A may advantageously influence priming of the immune system by a mucosal route. The potential for the combination of adjuvants, Quil-A and chitosan, to enhance antibody responses to plasmid encoded antigen co-administered with the corresponding protein antigen was shown and this is worthy of further investigation. The findings here have identified novel adjuvants and approaches to vaccine delivery. In particular, chitosan or vitamin E TPGS are shown here to have considerable promise as non-toxic, safe mucosal adjuvants. In addition, biodegradable mucoadhesive delivery systems, surface modified with chitosan in a single step process, may have application for other uses such as drug and gene delivery.

Improving brain drug targeting through exploitation of the nose-to-brain route:a physiological and pharmacokinetic perspective

With an ageing population and increasing prevalence of central-nervous system (CNS) disorders new approaches are required to sustain the development and successful delivery of therapeutics into the brain and CNS. CNS drug delivery is challenging due to the impermeable nature of the brain microvascular endothelial cells that form the blood-brain barrier (BBB) and which prevent the entry of a wide range of therapeutics into the brain. This review examines the role intranasal delivery may play in achieving direct brain delivery, for small molecular weight drugs, macromolecular therapeutics and cell-based therapeutics, by exploitation of the olfactory and trigeminal nerve pathways. This approach is thought to deliver drugs into the brain and CNS through bypassing the BBB. Details of the mechanism of transfer of administrated therapeutics, the pathways that lead to brain deposition, with a specific focus on therapeutic pharmacokinetics, and examples of successful CNS delivery will be explored. © 2014 Bentham Science Publishers.

Imunoexpressão de receptores de calcitonina e corticosteroides em lesões centrais de células gigantes dos ossos maxilares

The aim of this study was to analyze the immunoexpression of calcitonin (CTR) and glucorticoid (GCR) receptors in aggressive and non-aggressive central giant cell lesions (CGCL). This is an immunohistochemistry study (immunoperoxidase technique) of 52 cases of CGCL of the jaws, in which 12 patients were treated with intralesional triamcinolone injections and one with calcitonin nasal spray. The mean of immunostaining was compared between the cell types and clinical subtype of the lesion. The correlations among means were analyzed by Mann-Whitney test. Of the 52 cases studied, 53.8% were females, with a mean of 25.69 years. Most lesions were located in the mandible. Thirty patients (57.7%) had aggressive lesions and 22 (42.3%) of the cases consisted of non-aggressive lesions. Surgery was the treatment of choice in 75% of the cases. In 56.7% of the aggressive CGCL surgery was performed, while 43.4% of patients were submitted to conservative treatment. Among cases submitted to conservative treatment, the majority (n = 8; 61.5%) responded well to treatment. CTR expression was observed in 67.3% and GCR in 96.15% of cases. There was no significant statistical difference between the expression of CTRs and GCRs in mononuclear and multinucleated CGCLscells, regarding aggressiveness, treatment performed for aggressive lesions and the response to conservative treatment (p>0.05). The results of our research suggest that the immunoreactivity of CTRs and GCRs did not influence the response to clinical treatment with calcitonin or triamcinolone in the sample studied and it exhibited a varied expression regardless of the aggressiveness of the lesion.

Imunoexpressão de receptores de calcitonina e corticosteroides em lesões centrais de células gigantes dos ossos maxilares

The aim of this study was to analyze the immunoexpression of calcitonin (CTR) and glucorticoid (GCR) receptors in aggressive and non-aggressive central giant cell lesions (CGCL). This is an immunohistochemistry study (immunoperoxidase technique) of 52 cases of CGCL of the jaws, in which 12 patients were treated with intralesional triamcinolone injections and one with calcitonin nasal spray. The mean of immunostaining was compared between the cell types and clinical subtype of the lesion. The correlations among means were analyzed by Mann-Whitney test. Of the 52 cases studied, 53.8% were females, with a mean of 25.69 years. Most lesions were located in the mandible. Thirty patients (57.7%) had aggressive lesions and 22 (42.3%) of the cases consisted of non-aggressive lesions. Surgery was the treatment of choice in 75% of the cases. In 56.7% of the aggressive CGCL surgery was performed, while 43.4% of patients were submitted to conservative treatment. Among cases submitted to conservative treatment, the majority (n = 8; 61.5%) responded well to treatment. CTR expression was observed in 67.3% and GCR in 96.15% of cases. There was no significant statistical difference between the expression of CTRs and GCRs in mononuclear and multinucleated CGCLscells, regarding aggressiveness, treatment performed for aggressive lesions and the response to conservative treatment (p>0.05). The results of our research suggest that the immunoreactivity of CTRs and GCRs did not influence the response to clinical treatment with calcitonin or triamcinolone in the sample studied and it exhibited a varied expression regardless of the aggressiveness of the lesion.

Insights into Nonpilus Adhesin Functionality and the Molecular Determinants of Nontypeable Haemophilus influenzae Colonization

Bacterial colonization of the upper respiratory tract is the first step in the pathogenesis of nontypeable Haemophilus influenzae (NTHi) disease. Examination of the determinants of NTHi colonization process has been hampered by the lack of an appropriate animal model. To address this, we have developed a model of NTHi colonization in adult rhesus macaques that involves intranasal inoculation of 1x105 CFU and results in persistent colonization of the upper respiratory tract for at least three weeks with no signs of disease, mimicking asymptomatic colonization of humans. Using this model, we assessed the contributions to colonization of the HMW1 and HMW2 adhesive proteins. In competition experiments, the parent strain expressing both HMW1 and HMW2 was able to efficiently out-compete an isogenic mutant strain expressing neither HMW1 nor HMW2. In experiments involving inoculation of single isogenic derivatives of NTHi strain 12, the strains expressing HMW1 or HMW2 or both were able to colonize efficiently, while the strain expressing neither HMW1 nor HMW2 colonized inefficiently. Furthermore, colonization resulted in antibody production against HMW1 and HMW2 in one-third of the animals, demonstrating that colonization can be an immunizing event. In conclusion, we have established that NTHi is capable of colonizing the upper respiratory tract of rhesus macaques, in some cases associated with stimulation of an immune response. The HMW1 and HMW2 adhesive proteins play a major role in the process of colonization.

After establishing that the HMW1 and HMW2 proteins are colonization factors we further investigated the determinants of HMW1 function. HMW1 is encoded in the same genetic locus as two other proteins, HMW1B and HMW1C, with which HMW1 must interact in order to be functional. Interaction with HMW1C in the cytoplasm results in the glycosylation of HMW1. By employing homologues of HMW1C that glycosylate HMW1 in slightly different patterns we show that the pattern of modification is critical to HMW1 function. Structural analysis showed a change in protein structure when the pattern of HMW1 modification differed. We also identified two specific sites which must be glycosylated for HMW1 to function properly. These point mutations did not have a significant effect on protein structure, suggesting that glycosylation at those specific sites is instead necessary for interaction of HMW1 with its receptor. HMW1B is an outer membrane pore through which HMW1 is transported to reach the bacterial cell surface. We observed that HMW1 isolated from the cytoplasm has a different structure than HMW1 isolated from the bacterial cell surface. By forcing HMW1 to be secreted in a non-HMW1B dependent manner, we show that secretion alone is not sufficient for HMW1 to obtain a functional structure. This leads us to hypothesize that there is something specific in the interaction between HMW1 and HMW1B that aids in proper HMW1 folding.

The NTHi HMW1C glycosyltransferase mediates unconventional N-linked glycosylation of HMW1. In this system, HMW1 is modified in the cytoplasm by sequential transfer of hexose residues. To determine if this mechanism of N-linked glycosylation is employed by species other than NTHi, we examined Kingella kingae and Aggregatibacter aphrophilus homologues of HMW1C. We found both homologues to be functional glycosyltransferases and identified their substrates as the K. kingae Knh and the A. aphrophilus EmaA trimeric autotransporter proteins. LC-MS/MS analysis revealed multiple sites of N-linked glycosylation on Knh and EmaA. Without glycosylation, Knh and EmaA failed to facilitate wild type levels of bacterial autoaggregation or adherence to human epithelial cells, establishing that glycosylation is essential for proper protein function.

Effect of experimental parameters on alginate/chitosan microparticles for BCG encapsulation

The aim of the present study was to develop novel Mycobacterium bovis bacille Calmette-Guérin (BCG)-loaded polymeric microparticles with optimized particle surface characteristics and biocompatibility, so that whole live attenuated bacteria could be further used for pre-exposure vaccination against Mycobacterium tuberculosis by the intranasal route. BCG was encapsulated in chitosan and alginate microparticles through three different polyionic complexation methods by high speed stirring. For comparison purposes, similar formulations were prepared with high shear homogenization and sonication. Additional optimization studies were conducted with polymers of different quality specifications in a wide range of pH values, and with three different cryoprotectors. Particle morphology, size distribution, encapsulation efficiency, surface charge, physicochemical properties and biocompatibility were assessed. Particles exhibited a micrometer size and a spherical morphology. Chitosan addition to BCG shifted the bacilli surface charge from negative zeta potential values to strongly positive ones. Chitosan of low molecular weight produced particle suspensions of lower size distribution and higher stability, allowing efficient BCG encapsulation and biocompatibility. Particle formulation consistency was improved when the availability of functional groups from alginate and chitosan was close to stoichiometric proportion. Thus, the herein described microparticulate system constitutes a promising strategy to deliver BCG vaccine by the intranasal route.

Sinusite maxilar odontogénica

O seio maxilar é o seio paranasal mais susceptível a invasões bacterianas, tanto pelo óstio nasal, como pela cavidade oral. As sinusites maxilares têm como causas mais frequentes, as infecções víricas, rinites alérgicas ou não alérgicas, variações anatómicas, diabetes mellitus, fumar, nadar, mergulhar, escalar a altas atitudes, e as infecções e tratamentos dentários. A pesquisa bibliográfica, foi realizada sem quaisquer limitações temporais, com restrição linguística a Português, Espanhol e Inglês, sendo excluídos os artigos de outros idiomas; em vários livros e revistas, assim como artigos científicos obtidos, entre Maio e Julho de 2015, nos motores de busca Pubmed, ScienceDirect, Scielo, Elsevier e B-on. A sinusite maxilar odontogénica é uma doença infecto-inflamatória, habitualmente associada à ruptura da membrana de Schneider e a processos infecciosos dentários crónicos. Causa hiperplasia e hipertrofia da mucosa, o que origina sinais e sintomas próprios, assim como mudanças radiográficas perceptíveis. Existem diferentes etiologias de causa odontogénica: cárie, doença periodontal, quistos odontogénicos e iatrogenia – tratamento endodôntico não cirúrgico, cirurgia endodôntica, comunicações oro-antrais, implantes dentários, elevação do seio maxilar, cirurgia pré-protética e cirurgia ortognática – sendo que a iatrogenia é a mais comum (cerca de 56%). Esta patologia afecta com mais frequência indivíduos dos 42,7 aos 51, 7 anos, e preferencialmente a região molar, seguida dos pré-molares e em alguns casos, caninos. Os organismos que dominam na fase aguda e crónica, são sensivelmente os mesmos, mas em número diferente, e existe uma conexão entre a flora comensal periapical e a flora patogénica em caso de sinusite maxilar odontogénica. O diagnóstico é essencialmente clínico, no entanto existem diferentes exames complementares para confirmarem ou formarem o diagnóstico. Pela grande acessibilidade ao método radiográfico, torna-se fundamental que o médico dentista saiba diferencial as diversas patologias que afectam o seio maxilar. O tratamento abrange a eliminação da causa dentária e o tratamento farmacológico, da infecção, essencialmente à base de antibióticos, e da dor se esta existir. E o tratamento cirúrgico, que contempla a punção-lavagem sinusal, antrostomia intranasal, técnica de Caldwell-Luc e cirurgia sinusal endoscópica. Concluindo, o médico dentista deve ter um amplo conhecimento sobre esta patologia para que a possa reconhecer, tratar ou preveni-la.

Investigating the Diabetic Brain: The Effects of Pioglitazone and Insulin on the Cellular Processes and Pathology of Alzheimer's Disease

Alzheimer’s disease (AD) is the sixth leading cause of death in the US. Some researchers refer to AD as “Type III Diabetes” because of reported glucose metabolism dysfunction. Preclinical studies suggest increasing insulin decreases AD pathology, although the mechanism remains unclear. To sensitize insulin signaling, this study activated Peroxisome Proliferator-Activated Receptor Gamma using intranasal co-administration of pioglitazone (PGZ) and insulin. This method targeted the site of action to reduce peripheral effects and to maximize impact in transgenic mice expressing AD pathology. Data from GC-MS fluxomics analysis suggested that PGZ+Insulin increased glucose metabolism in the brain. Immunohistochemistry with relevant antibodies was used to identify AD pathological markers in the subiculum, indicating that PGZ+Insulin decreased pathology compared to Insulin and Saline. This suggests that increasing glucose uptake in the brain alleviated AD pathology, further clarifying the role of insulin signaling in AD pathology.Gemstone