Rozprawy doktorskie na temat „Respiratory agents”

Dissertação de Mestrado Integrado em Medicina Veterinária
Several fungi are described to cause invasive infections in dolphins, the respiratory system being a common site of involvement. Mucormycosis is considered one of the most devastating fungal infections in dolphins, associated with an elevated mortality rate, where hyphae are capable of invading blood vessels, producing tissue infarction and necrosis. A one-year-old male bottlenose dolphin (Tursiops truncatus) calf presented with a history of recurrent episodes of leukocytosis and occasional respiratory signs. During a routine faecal examination, a myriad of hyphae were found. Fungal culture revealed a mucormycete isolation, the aetiologic agent of mucormycosis. Molecular studies allowed to identify Cunninghamella bertholletiae. Thoracic radiographs showed the presence of a bronchoalveolar pattern on both the right and left lung apexes. A bronchoscopy was performed, which revealed multiple whitish lesions, diffusely distributed on the tracheal and bronchial submucosa. The antifungal therapy prescribed was a combination of posaconazole and aerosolized liposomal amphotericin B. Adjunctive therapies included bromhexine, vitamin C, vitamin B complex, probiotics, silymarin, Imuno-2865™ and ozone therapy. Follow-ups were conducted with haematology and blood biochemistry, faecal and sputum culture and direct microscopy, and bronchoscopies. There was a good overall response to treatment and antifungal therapy was discontinued. However, the infection relapsed and posaconazole therapy was restarted. Serum concentrations of posaconazole were retrospectively evaluated and the set of results did not appear to show subtherapeutic concentrations as a plausible explanation for the relapse. Although complete clinical resolution was not obtained during the timeframe considered, this case corroborates the idea that medical management of mucormycosis is possible, especially with a prompt diagnosis and treatment as well as a tight follow-up protocol. As described in the literature, mucormycosis treatment may take several years and relapses are common.
RESUMO - Maneio médico de mucormicose respiratória numa cria de golfinho-roaz (Tursiops truncatus) em contexto zoológico - Várias espécies de fungos estão descritas como agentes etiológicos de infeções invasivas em golfinhos, sendo o sistema respiratório um dos locais comuns de infeção. A mucormicose é uma das infeções fúngicas invasivas com efeitos mais devastadores, associada a uma elevada taxa de mortalidade em cetáceos. Nesta dissertação é apresentado um caso clínico referente a uma cria de golfinho-roaz (Tursiops truncatus) com um ano de idade, com uma história clínica que incluía episódios recorrentes de leucocitose e ocasionais sinais clínicos de etiologia respiratória. Como parte do programa de medicina preventiva instituído, uma análise microscópica de fezes permitiu a visualização de estruturas fúngicas. A cultura fúngica permitiu o isolamento de um mucormicete, o agente etiológico da mucormicose, e através de PCR e sequenciação foi possível identificar Cunninghamella bertholletiae. Estudos imagiológicos demonstraram a presença de um ligeiro padrão broncoalveolar nos ápices pulmonares e o exame endoscópico permitiu visualizar múltiplas lesões esbranquiçadas, difusamente distribuídas pelas mucosas traqueal e brônquica. O tratamento antifúngico consistiu na administração de comprimidos gastrorresistentes de posaconazol e nebulizações com anfotericina B lipossómica. Tratamentos adjuvantes incluíram bromexina, silimarina, suplementação vitamínica, probióticos, Imuno-2865TM e ozonoterapia. O acompanhamento do caso foi feito com base em dados hematológicos e bioquímicos, análises microscópicas/cultura de fezes e exsudado respiratório e broncoscopias. O tratamento com posaconazol foi descontinuado após 95 dias de terapia, tendo em conta os resultados constantemente negativos na cultura e observação microscópica de amostras fecais e exsudado respiratório. No entanto, verificou-se a recidiva da infeção e o tratamento antifúngico foi recomeçado. As concentrações séricas do fármaco ao longo do caso clínico foram retrospetivamente analisadas e esta monitorização permitiu descartar a hipótese de não terem sido atingidas concentrações séricas terapêuticas como causa da recidiva da infeção. Apesar de não ter existido uma completa resolução clínica no período considerado, este caso corrobora o facto do maneio médico da mucormicose em cetáceos ser possível, através de um diagnóstico e tratamento precoces, além de um plano apertado de seguimento clínico. Como descrito na bibliografia, o tratamento da mucormicose pode demorar vários anos e recidivas são comuns.
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BACKGROUND: Dampness and mould exposure have been repeatedly associated with respiratory health. However, less is known about the specific agents provoking or arresting health effects in adult populations. We aimed to assess predictors of microbial agents in mattress dust throughout Europe and to investigate associations between microbial exposures, home characteristics and respiratory health. METHODS: Seven different fungal and bacterial parameters were assessed in mattress dust from 956 adult ECRHS II participants in addition to interview based home characteristics. Associations between microbial parameters and the asthma score and lung function were examined using mixed negative binomial regression and linear mixed models, respectively. RESULTS: Indoor dampness and pet keeping were significant predictors for higher microbial agent concentrations in mattress dust. Current mould and condensation in the bedroom were significantly associated with lung function decline and current mould at home was positively associated with the asthma score. Higher concentrations of muramic acid were associated with higher mean ratios of the asthma score (aMR 1.37, 95%CI 1.17-1.61). There was no evidence for any association between fungal and bacterial components and lung function. CONCLUSION: Indoor dampness was associated with microbial levels in mattress dust which in turn was positively associated with asthma symptoms.

CoordenaÃÃo de AperfeÃoamento de Pessoal de NÃvel Superior
Nosocomial infections (NI) are a serious public health problem. Knowledge about the etiology of NI is important for the development of control measures, prevention and treatment. Viruses are important etiologic agent of NI has been studied in populations considered at risk as premature, heart disease, lung disease, and immunosuppressed. Respiratory hospital infection (RHI) generate discomfort to patients, postponing medical interventions, postoperative complications, use more drugs and, in some cases, intensive care, may progress to cure or to death. Viruses are responsible for outbreaks of RHI in wards and intensive care units, with the virus as detected respiratory syncytial virus. In our country there are few data on the impact of RHI caused by respiratory viruses in children. Aims of the study were: identify and describe RHI cases in children and submit nasopharyngeal aspirates collected from January to December 2013 to search for molecular diagnosis 13 respiratory viruses [respiratory syncytial virus (RSV), adenovirus, influenza A and B, parainfluenza virus -1 ,-2 , -3 and -4 , metapneumovirus and human coronavirus OC43 , NL63 , 229E and HKU - 1]. During the study period, 120 samples were included in the study and 65 % were positive for at least one virus. A total of 114 viruses were detected (33 RSV, 32 adenovirus, 14 Parainfluenza -3, 14 influenza A , 12 Parainfluenza -4 , 5 parainfluenza -1 , 3 metapneumovirus and 1 coronavirus OC43). Co-detections occurred in 26 cases: 16 with two viruses and 10 with three viruses. No clinical differences between positive and negative RHI for viruses investigated were observed. Respiratory virus were detected in four of five deaths (5/120 4.16%) associated RHI. The knowledge about the occurrence of viral RHI in association with the period of viruses circulation in the community, as described in the study, allows to develop specific actions steps to prevent and control hospital outbreaks caused by viruses.
As infecÃÃes relacionadas à assistÃncia à saÃde (IRAS) sÃo um sÃrio problema de saÃde pÃblica. O conhecimento sobre a etiologia das IRAS à importante para o desenvolvimento de medidas de controle, prevenÃÃo e tratamento. A importÃncia dos vÃrus na etiologia das IRAS tem sido estudada em populaÃÃes consideradas de risco, como prematuros, cardiopatas, pneumopatas e imunodeprimidos. As infecÃÃes respiratÃrias hospitalares (IRH) geram aos pacientes desconforto, adiamento de intervenÃÃes mÃdicas, complicaÃÃes pÃs-cirÃrgicas, uso adicional de medicamentos e, em alguns casos, cuidados intensivos, podendo cursar para a cura ou para o Ãbito. Os vÃrus sÃo responsÃveis por surtos de IRH em enfermarias e unidades de terapia intensiva, sendo o vÃrus sincicial respiratÃrio o mais detectado. Em nosso paÃs sÃo escassos os dados sobre o impacto das IRH causadas por vÃrus respiratÃrios em pediatria. Os objetivos deste estudo foram: identificar e descrever os casos de IRH em crianÃas e submeter aspirados de nasofaringe coletados no perÃodo de janeiro a dezembro de 2013 ao diagnÃstico molecular para pesquisa de 13 vÃrus respiratÃrios [vÃrus sincicial respiratÃrio (VSR), adenovÃrus, influenza A e B, parainfluenza 1, 2, 3 e 4, metapneumovÃrus e coronavÃrus humanos OC43, NL63, 229E e HKU-1]. Para tanto, as amostras foram extraÃdas para obtenÃÃo do material genÃtico viral e, posteriormente, para os vÃrus de RNA, parte deste produto foi transformada em DNA complementar e depois a amplificado. Para detecÃÃo do adenovÃrus, foram realizados PCR e Nested PCR. No perÃodo de estudo, 120 amostras foram incluÃdas e 65% delas foram positivas para pelo menos um vÃrus. Um total de 114 vÃrus foram detectados (33 VSR; 32 adenovÃrus; 14 parainfluenza-3; 14 influenza A; 12 parainfluenza-4; 5 parainfluenza-1; 3 metapneumovÃrus e 1 coronavÃrus OC43). Co-detecÃÃes ocorreram em 26 casos: 16 com dois vÃrus e 10 com trÃs vÃrus. NÃo foram observadas diferenÃas clÃnicas entre as IRH positivas e negativas para os vÃrus pesquisados. VÃrus respiratÃrios foram identificados em quatro dos cinco casos de Ãbito (4,16%; 5/120) associados à IRH. O conhecimento sobre a ocorrÃncia da IRH virais em associaÃÃo com o perÃodo de circulaÃÃo dos vÃrus na comunidade, como descrito no estudo, permite desenvolver aÃÃes especÃficas de medidas para prevenir e controlar surtos hospitalares causados pelos vÃrus.

The human respiratory syncytial virus (hRSV) infection is a global public health burden in children aged under 2 years and immunocompromised or elderly adults. The choice for prophylaxis and therapy of hRSV infection was constrained to Palivizumab and Ribavirin. Therefore, this study aimed to develop effective anti-hRSV infection agents, such as short interfering RNA (siRNA) and β-defensin-4 (β-D-4). Since there still is no compatible animal model to evaluate antiviral effect of anti-hRSV agents, we first attempted to establish suitable animal model. A clinical isolate of hRSV (KI-RSV-W) was adapted in BALB/c mice by serial passages. Old male mice (age > 8 months) were used for establishment of hRSV infection model, because the more efficient viral infection in lungs of the old male mice than that old female mice and young mice (age < 3 weeks). After the virus was propagated in old male mice for 20 passages, a virus variant KI-RSV-P70-4 exhibited more efficient infection/replication in the mice. Its viral load was about 100-fold higher than that of wild type strain KI-RSV-W. The infection of KI-RSV-P70-4 also caused more severe histopathological changes in lung tissues. Although KI-RSV-P70-4 could not result in death of the infected mice, both viral load and pathological change in lungs may be good indicators for evaluating antiviral effect. The mouse model and adapted hRSV strain solidly laid the foundation for evaluation of anti-hRSV agents. We then designed and evaluated anti-hRSV effect of siRNAs. A total of 25 siRNAs targeting 4 viral genes (M2-1, NS2, N and F) were designed and their anti-hRSV effect was assessed in vitro. The results showed that 6 siRNAs respectively targeting M2-1, N and F genes exhibited higher anti-hRSV effect than that of the positive control, whereas those targeting NS2 gene did not show significant antiviral effect. The 50% inhibitory concentrations (IC50) of three most potent siRNAs (M2-1-361, N889 and F-1143) were 0.51, 2.14 and 0.64 nM, respectively. Antiviral activity of β-D-4 against hRSV infection was evaluated in vitro and in vivo. In vitro experiments showed supreme antiviral effect with IC50 around 3.4 μg/ml when the virus was pretreated with β-D-4, but no significant inhibitory effect was observed when the cells were pretreated with β-D-4 or β-D-4 was maintained in the culture medium after viral infection. These results indicated that the inhibitory effect of β-D-4 was associated with direct interaction with the virus itself and blocked virus entry of the cells. Furthermore, a single dose (13.6 μg) of β-D-4 intratracheal (i.t.) administration in old male mice after the viral infection resulted in about 0.7 log reduce of viral load in lung tissues, while inoculation of premixed β-D-4 and the virus caused about 3 logs decrease of viral load in lungs. These results have demonstrated that β-D-4 may be an effective anti-hRSV agent. Taken together, old male BALB/c mice might be used to establish hRSV infection model. Three siRNAs and the β-D-4 were validated as the potent anti-hRSV agents, respectively.
published_or_final_version
Microbiology
Doctoral
Doctor of Philosophy

P. aeruginosa is one of the major opportunistic pathogen colonizing the respiratory tract of cystic fibrosis (CF) patients and causing chronic airways infection. Once P.aeruginosa established chronically in the CF lung, bacterial density increases and the microorganism switches to a mucoid form and to a stable biofilm mode of growth in which susceptibility to antimicrobials decreases. The high resistance of P.aeruginosa to multiple antimicrobials led to scenarios in which almost no treatment options are available. In this regard, the research on the introduction of less toxic antimicrobials as well as the use of pharmaceutical forms enabling dose reductions, longer administration intervals, and reduced systemic toxicity has been stimulated. Therefore, the aim of this thesis was to develop nanoencapsulated colistin and tobramycin in lipid nanoparticles (SLN: Solid Lipid Nanoparticles and NLC: Nanostrucutured Lipid Carriers) and explore their antimicrobial activity versus free drug against P.aeruginosa clinical isolates from CF patients and to investigate the efficacy of these novel formulations in the eradication of biofilms, one of the most relevant mechanisms involved in persistence and in chronic infections. ELABORATION AND CHARACTERIZATION The main objective of the first part of this thesis was to elaborate and characterize lipid nanoparticles (SLN and NLC) as colistin and tobramycin carriers to treat P.aeruginosa lung infection. The nanoparticles obtained displayed a 200–400 nm size, high drug encapsulation (79–94%) and a sustained drug release profile. The integrity of the nanoparticles was not affected by nebulization through a mesh vibrating nebulizer. Next, tobramycin-NLCs were able to overcome an artificial mucus barrier in the presence of mucolytic agents. Moreover, lipid nanoparticles loaded with both antimicrobials appeared to be less toxic than free drug in cell culture. Finally, an in vivo distribution experiment showed that nanoparticles spread homogenously through the lung and there was no migration of lipid nanoparticles to other organs, such as liver, spleen or kidneys. STABILITY The second essential point of this work concerns the stability of both types of lipid nanoparticles after freeze-drying. The results showed that colistin-SLNs lost their antimicrobial activity at the third month; on the contrary, the antibacterial activity of colistin-NLCs was maintained throughout the study within an adequate range. In addition, colistin-NLCs exhibited suitable physic-chemical properties at 5 °C and 25 °C/60% relative humidity over one year. Altogether, colistin-NLCs proved to have better stability than colistin- SLNs. The last part focuses on the study of the antimicrobial activity of SLN and NLC loaded with colistin and tobramycin against P.aeruginosa isolates from Sant Joan de Déu and Vall d’Hebrón hospitals CF patients. Regarding the data documenting planktonic experiments, colistin nanoparticles had the same antimicrobial activity as free drug. The activity of tobramycin-loaded SLN was less than that of either tobramycin-loaded NLC or free tobramycin. However, in the relation to biofilms, nanoencapsulated antimicrobials were much more efficient than their free form. Moreover, the results showed the more rapid killing of P. aeruginosa bacterial biofilms by NLC-colistin than by free colistin. Nevertheless, the two formulations did not differ in terms of the final percentages of living and dead cells, which were higher in the inner than in the outer layers of the treated biofilms. Since it seems clear than biofilms play a key role in respiratory infections in CF patients by P. aeruginosa, these formulations seem to us encouraging alternative to the currently available CF therapies.
P.aeruginosa és un dels principals patògens oportunistes colonitzadors del tracte respiratori dels pacients amb fibrosi quística (FQ) causant una infecció crònica. Una vegada aquest microorganisme ja està establert de manera crònica al pulmó, la densitat bacteriana augmenta i P.aeruginosa canvia de morfologia no mucosa a mucosa afavorint la formació de biofilm en el qual la susceptibilitat als antibiòtics es veu enormement disminuïda. L’elevada resistència de P.aeruginosa a múltiples antimicrobians ens condueix a un escenari on gairebé no hi ha opcions de tractament disponibles. En aquest sentit, la recerca en la introducció d’antimicrobians menys tòxics així com l’ús de noves formes farmacèutiques amb la capacitat de reduir la dosi, allargar els intervals d’administració així com reduir la toxicitat adquireix molta rellevància. Per tant, l’objectiu d’aquesta tesi va ser desenvolupar nanopartícules lipídiques (Solid Lipid Nanoparticles: SLN y Nanostructured Lipid Carriers: NLC) carregades amb colistina I també les partícules amb tobramicina, explorar la seva activitat antimicrobiana comparant-la amb la seva forma lliure contra soques clíniques de P.aeruginosa aïllades de pacients amb FQ, i investigar l’eficàcia d’aquestes noves nanoformulacions en l’eradicació dels biofilms ja que és un dels mecanismes més rellevants associat a les infeccions cròniques.

Human respiratory syncytial viruses (hRSV) are a major cause of lower respiratory tract disease on primary infection of infants and children. There is no effective vaccine against the virus, but high risk infants can be protected by administration of palivizumab (PZ), a humanised anti-fusion glycoprotein. In previous studies, hRSV isolated from the naso-pharyngeal secretions of infected infants were found to be dominated by slow growing variants which were largely refractory to neutralization by PZ. On further passage in tissue culture, the slow growing variants were replaced by fast growing, neutralization susceptible variants. The aim of this study was to investigate the mechanism of neutralization phenotype shift between slow-growing neutralization resistant and fast-growing neutralization susceptible clones. Neutralization resistance was found to be cell-line dependent with cell lines varying in their permissiveness to antibody treated virus. Antibody resistant and susceptible viruses showed no differences in the amount of the membrane expressed F glycoprotein or post-translational processing of the F polypeptide. They were also equally susceptible to inhibition by clathrin endocytosis inhibitors (monodansylcadaverine and chlorpromazine) suggesting that endocytosis was required for entry of both. They were further tested for susceptibility to anti-fusion inhibitors. These included a peptide derived from the F glycoprotein heptad repeat 2 and two small molecular weight compouds BMS-433771 and BTA9881. It was found that all three compounds failed to inhibit slow growing PZ resistant virus clones in parallel with PZ, but efficiently blocked fast growing PZ susceptible virus clones. These studies suggest that differences in antibody susceptibility stem from differences in the mechanism of fusion for the two virus clones. The full genomes of virus clones with resistant and susceptible phenotypes were sequenced to identify mutations which correlate with the difference in antibody susceptibility. The two clones differed at four sites in the SH, G, F and L genes. These mutations were sought in a number of virus clones expressing a range of susceptibility to antibody neutralization. No single mutation was associated with the shift from neutralization resistant to susceptible phenotype in all clones. However, a mutation at nucleotide 6162 in the L gene was associated with the shift from resistance to susceptibility in one virus lineage.

AIMS: To evaluate the use of Manual Muscle Strength Tests (MMST), Timed Functional Tests (TFT) and Respiratory Function Tests (RFT) as measures of muscle strength in young boys with Duchenne Muscular Dystrophy (DMD) and specifically to evaluate the use of Peak Expiratory Flow (PEF). BACKGROUND: There is a need to measure the effect of treatments that potentially increase muscle strength in DMD. PEF may have advantages over Vital Capacity (VC) as a measure of respiratory function in young boys with DMD. METHODS: 17 boys with DMD (aged 5-10 years) were assessed regularly over one year. Assessment involved Respiratory Function Testing (PEF, VC, Forced Expiratory Volume in one second [FEV1]), Timed Functional Testing (walking 9 metres, climbing four stairs, arising from supine) and MMST. A single investigator performed MMST and TFTs. A separate investigator performed RFTs. For RFTs a percentage of predicted was calculated [PEF(%), FEV1(%), VC(%)].11/17 boys were treated with prednisolone which increases strength in DMD. RESULTS: At baseline, all boys had significant weakness. Mean (+/- SD) PEF(%) 69 +/- 13% and VC(%) 77 +/- 18% were abnormal. Baseline PEF(%) predicted correlated with MMST (P=0.003) and time to walk 9 metres (P=0.022). Baseline VC(%) correlated with MMST (P=0.049). There was a consistent statistically significant correlation between MMST and all TFTs. PEF was performed well on 80% of occasions, spirometry on 65%. Changes in PEF(%) showed statistically significant correlation with changes in all TFTs. The correlation was not statistically significant for VC(%) or FEV1(%). Prednisolone treated boys did better than those not treated. PEF, time to walk 9 metres and time to climb 4 stairs showed statistically significant improvement. The mean improvement from baseline in PEF(%) was 19 +/-14% in treated and 2 +/- 7% in untreated boys (P=0.012). CONCLUSIONS: MMST, TFTs and RFTs are valid measures of muscle strength in young boys with DMD. PEF is abnormal in young boys with DMD; correlates with other measures of strength and is sensitive to changes in strength. PEF is more easily performed than spirometry and has a role in monitoring muscle strength in young boys with DMD.

Aspirin exacerbated respiratory disease (AERD) is a clinical syndrome characterized by the combination of nasal polyps (NPs), chronic hypertrophic eosinophilic sinusitis, asthma and severity to any medication that inhibits cyclooxygenase (COX)-1 enzymes, namely aspirin and other non-steroidal anti-inflammatory drugs. AERD is characterized by intense inflammation of the respiratory mucosa, with large number of activated tissue eosinophils, mast cells, monocytes, and neutrophils. The persistent inflammation likely contributes to the recurrent development of severe nasal polyposis. The pathogenesis of AERD is not fully understood and many questions remain unsolved. However, several studies have reported that the pathogenic mechanisms of this condition may be due, at least in part, to a marked imbalance in eicosanoid metabolism, possibly increasing and perpetuating the process of inflammation. A central feature of AERD is its association with a profound overproduction and overresponsiveness to cysteinyl leukotrienes (cys-LTs) occurring concomitantly with a marked underproduction and underresponsiveness to prostaglandin (PG) E2, which result in a disproportion between pro-inflammatory and anti-inflammatory mediators. The downregulation of PG pathways impedes its mediators to participate in the attenuation of pro-inflammatory responses. Recent studies have demonstrated that the pattern of cytokine expression could differentiate patients with AERD and patients with asthma or eosinophilic sinusitis who tolerate aspirin. We hypothesized that the characteristics of the inflammatory airway milieu (cytokine profile) in AERD is responsible for the deficient expression of EP2 , modifying its capacity to respond to PGE2, resulting in an altered regulation of the autocrine positive loop of the COX pathway that contributes to the exacerbated inflammation and remodeling processes usually present in AERD. In summary, this study provides evidence supporting the hypothesis that the inflammatory environment in the airways of patients with AERD induces alterations in the expression of EP2. This anomaly alters the induction of interleukin-1 receptor type I (IL-1RI), the main factor receptor responsible for the activation of COX-2 by interleukin (IL)-1β, which, in turn, results in a low production of PGE2 which, associated with the low expression of the EP2 receptor, alters the autocrine feedback loop that regulates the COX pathway. The EP2 receptor plays a central role in the alteration of PGE2 synthesis, since its reestablishment can recover the normal function of the autocrine loop and thereby normalizing the expression and function of all the components that constitute it (IL-1RI, COX-2, microsomal PGE synthase-1 (mPGES-1) and PGE2). The decrease in the synthesis of PGE2 may contribute to the perpetuation of the eosinophilic inflammation and remodeling processes in the airways of patients who suffer from AERD.
La enfermedad respiratoria exacerbada por antiinflamatorios no esteroides (EREA) se caracteriza por la presencia de asma bronquial, rinosinusitis crónica con pólipos nasales e hipersensibilidad a la aspirina y otros antiinflamatorios no esteroides. La inflamación de las vías aéreas en los pacientes con EREA se ha relacionada con diversas alteraciones en el metabolismo del ácido araquidónico (AA) las cuales podrían contribuir al incremento y perpetuación de los procesos inflamatorios. Además, estudios recientes también han demostrado que el perfil de citocinas (sobreexpresión de interferón (IFN)-γ e interleucina (IL)-4) en las vías aéreas, podría tener un papel relevante en la regulación del metabolismo del AA en la EREA. La hipótesis general del estudio establece que las características del entorno inflamatorio de las vías aéreas (perfil de citocinas) en la EREA es responsable de la expresión deficiente del receptor EP2, modificando su capacidad de responder a la PGE2, lo que provoca una alteración en la regulación del bucle de retroalimentación positivo autocrina de la vía de la COX, lo cual contribuye a la inflamación descontrolada y a los procesos de remodelado de las vías aéreas normalmente presentes en la EREA. En suma, este estudio aporta evidencias que soportan la hipótesis de que el entorno inflamatorio en las vías aéreas de los pacientes con enfermedad respiratoria exacerbada por antiinflamatorios no esteroides induce la alteración en la expresión del receptor EP2 de la PGE2. Esta anomalía altera la inducción del receptor tipo I de la interleucina-1 (IL-1RI), principal responsable de la activación de la COX-2 por la citocina IL-1β, lo que, a su vez, da como resultado una baja producción de PGE2 que asociada a la baja expresión de su receptor EP2 altera el bucle de retroalimentación autocrina que regula la vía de la COX. El receptor EP2 juega un papel central en la alteración de la síntesis de la prostaglandina E2 ya que su reparación permite recuperar el funcionamiento normal del bucle autocrino y con ello la normalización en la expresión y funcionamiento de todos los componentes del mismo (IL-1RI, COX-2, PGE sintasa microsomal-1 (mPGES-1) y PGE2). La disminución en la síntesis de PGE2 contribuye a perpetuar la inflamación eosinofílica y el proceso de remodelado de las vías aéreas de los pacientes con enfermedad respiratoria exacerbada por antiinflamatorios no esteroides.

Background: Acute respiratory infections (ARIs) represent an important cause of morbidity and mortality in children, remaining a major public health concern, especially affecting children under 5 years old from low-income countries. Unfortunately, information regarding their epidemiology is still limited in Peru. Methods: A secondary data analysis was performed from a previous cross-sectional study conducted in children with a probable diagnosis of Pertussis from January 2010 to July 2012. All samples were analyzed via Polymerase Chain Reaction (PCR) for the following etiologies: Influenza-A, Influenza-B, RSV-A, RSV-B, Adenovirus, Parainfluenza 1 virus, Parainfluenza 2 virus, Parainfluenza 3 virus, Mycoplasma pneumoniae and Chlamydia pneumoniae. Results: A total of 288 patients were included. The most common pathogen isolated was Adenovirus (49%), followed by Bordetella pertussis (41%) from our previous investigation, the most prevelant microorganisms were Mycoplasma pneumonia (26%) and Influenza-B (19.8%). Coinfections were reported in 58% of samples and the most common association was found between B. pertussis and Adenovirus (12.2%). Conclusions: There was a high prevalence of Adenovirus, Mycoplasma pneumoniae and other etiologies in patients with a probable diagnosis of pertussis. Despite the presence of persistent cough lasting at least two weeks and other clinical characteristics highly suspicious of pertussis, secondary etiologies should be considered in children under 5 years-old in order to give a proper treatment.
Revisión por pares

Orientador: Antonio Fernando Ribeiro
Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas
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Resumo: Considerando que a fibrose cística é a mais importante doença hereditária, potencialmente letal, incidente na raça branca, que a infecção pulmonar é reconhecida por ter o maior papel na morbimortalidade levando à morte prematura em 90% dos pacientes, e que a principal causa das exacerbações é as infecções recorrentes ou crônicas; torna-se fundamental para um centro de referência, o conhecimento do perfil microbiológico de seus pacientes. A correlação entre a exacerbação dos sintomas pulmonares e a contagem de colônias de bactérias na cultura rotina diagnóstica (CRD) serve para orientar o controle das infecções. Norteados por este fundamento, para o perfil microbiológico utilizaram-se resultados CRD do: Registro eletrônico do Paciente (REP), Arquivo do Laboratório de Microbiologia (ALM) e Prontuário do paciente (PP). As três bases dados pertencem à mesma casuística, sendo que os dois últimos foram utilizados para verificar a coerência entre os perfis e estudo da susceptibilidade. Estatística: x2, Fisher, Pearson, regressão linear, e, nível significância p<0.05 e IC 95%. Foram resgatados 38.480 registros do REP, referentes a 975 CRD's, 402 nos ALM's e 371 dos PP's. Foram isoladas: Pseudomonas aeruginosa em 80,9% REP (43% pertenciam ao morfotipo não mucóide), nos ALM em 70,8% (43% não mucóide) e em 100% dos PP (60,6% não mucóide) e; Staphylococcus aureus em (50,1% REP, 48% ALM, e em 55% PP). Microrganismos emergentes como Burkholderia cepacia em (3,69% REP, 1% ALM, e em 3,5% dos PP's), Strenotrophomonas maltophilia em (3% REP, 2,8% ALM e 1,6% PP). Observamos uma elevada prevalência dos P. aeruginosa, durante o 1° ano de vida (57%), diferença significante se comparadas aos resultados da Cystic Fibrosis Fundation e Consenso Europeu que relatam uma prevalência de 20% entre 0-5 anos. Em relação à prevalência da B. cepacia, foi utilizado o meio seletivo, elevando a prevalência para 13% no ano de 2003, e, se observados ao longo dos anos, o valor se assemelha aos demais estudos (3,6%). Em relação ao estudo de susceptibilidade antimicrobiana, foram encontradas 13 cepas de P.aeruginosa multidroga-resistente aos 5 antibióticos incluídos no estudo (3,23% CRD's em 3 pacientes). Das P.aeruginosa 13,4% foram sensíveis a todos os antimicrobianos (ALM). Foi possível observar resistência das cepas P. aeruginosa aos antibióticos freqüentemente utilizados como a Gentamicina (50%) e Amicacina (31%). As mesmas cepas foram sensíveis à Ceftazidima (45%), Ciprofloxacina (48,7%). Foram isoladas em 389 dos 402 CRD's (ALM), ambos morfotipos (mucóide e não mucóide) apresentando elevada sensibilidade a Ceftazidima, Imipenem e Amicacina (66,9, 56,3% e 60% respectivamente) e, dentre eles, o que apresentou maior eficácia e menor resistência foi a Ceftazidima (5,4%). As cepas de S. aureus apresentaram elevada sensibilidade (72,7% ALM e 100% PP) sendo que dos 183 antibiogramas somente 11 cepas (6% em 5 pacientes ALM) apresentaram-se Oxa-Resistente, apesar de uma prevalência elevada e persistente. O perfil microbiológico, utilizando REP, foi coerente com ALM e PP, sugerindo que dados eletrônicos podem se amoldar às perspectivas de futuras pesquisas, levando-se em consideração a necessidade de novos estudos e maior interação entre as equipes, ajustadas as correções de possíveis vieses
Abstract: Considering that the Cystic Fibrosis is the most important hereditary illness potentially lethal incident in Caucasoid, and the pulmonary infections has been recognized as having the greatest role in the morbid mortality, being cause of death in 90% of the patients, and the main exacerbations cause are the recurrent or chronic infection/ the knowledge in the microbiological profile from patients becomes basic for all reference centers. When infected, the treatment will depends of microorganisms characteristics (antimicrobial resistance and ambient conditions) and, the prognostic depends of the nutritionals and immunological conditions; The correlation between the exacerbation from pulmonary symptoms and the counting of bacteria colonies by the culture routine diagnosis (CRD), serves to guide and control the infections. Guided by this bedding, our objective was to delineate this profile, using the CRD results from: Electronic Patient Register (REP), Archives from Microbiology Laboratory (ALM) and Handbooks of Patient (PP). All the databases belong to the same casuistry, being ALM data and PP collected to verify if the electronic registers correspond to the findings and notations in these archives. For the statistical calculations: descriptive analysis, x2, fisher, correlation Pearson and linear regression and the significance were p <0, 05 and IC 95%. Were analyzed 975 CRD's results between 38.480 registers from REP (100 patients), 402 from ALM (100 patients) and 371(9 patients) from PP's. Were identified: Pseudomonas aeruginosa in 80,9% from REP (43.1% belonged to morphotype mucoid), from ALM in 70,8% (27.8% mucoid) and PP (51.4% mucoid); Staphylococcus aureus in (50.1% REP, 48% ALM, and in 55% PP). Emergent microorganisms as Burkholderia cepacia in (3.69% REP, 1% ALM, and in 3,5% of the PP's), Strenotrophomonas maltophilia in (3% REP, 2.8% ALM and 1.6% PP). In relation of microorganisms prevalence, we observed a high significant prevalence of the Pseudomonas aeruginosa during the first year life (57%), greater than the results from Cystic Fibrosis Foundation and European Consensus (20% of 0-5 yrs). In relation of Burkholderia cepacia prevalence, the selective media was used (year 2003), increasing the prevalence to 13%, if observed during a long period, it would be equivalent to found (3,6%). About antimicrobial susceptibility, 13 of P. aeruginosa multi-drugs resistant were found to all usual antibiotics (3,3% CRD of 3 patients). The P. aeruginosa, 13,4% were sensitive to all antimycrobiane. We observed a great resistance between P. aeruginosa against to usual antibiotics as Gentamicine (50%) and Amicacine (31%). The same cepas were sensitive to Ceftazidima (45%), Ciprofloxacin (48,7%). Of 389 CRD's (ALM), were simultaneously found the both morphotypes (mucoid and nonmucoid) that showed a high sensitive to Ceftazidima, Imipenem e Amicacin (66,9, 56,3% e 60%) and the more effective and less resistant was Ceftazidima (5,4%). The Staphylococcus aureus were high sensitive (72,7% ALM and 100% PP) and just 11 cepas in 183 (5,9%) were Oxa-Resistant in 5 patients, even thought the high and persistent prevalence. The microbiological profile of cystic fibrosis patient from REP corresponds to the results from ALM and PP, suggesting that the electronic register can be molder to the perspectives of future researches, with news studies on this subject, and to plan more interaction between teams to correct any possible vises to appropriate research
Mestrado
Saude da Criança e do Adolescente
Mestre em Saude da Criança e do Adolescente

In title 12, 22, 11 are in subscript.
Thesis (PhD)--Stellenbosch University, 2002.
ENGLISH ABSTRACT: The aim of this study was to test a synthetic protein-free surfactant preparation, LPM-l, with the same chemical composition as commercially available Exosurf (Glaxo Wellcome), but containing in addition, a sugar, trehalose (TRE). Towards this end, a study was designed to firstly test the hypothesis that the true difference in acute physiological effects between a mixture of oppe, tyloxapol, hexadecanol and trehalose (LPM-l), and Exosurf, (Oppe, tyloxapol and hexadecanol) is zero, in a surfactantdeficient animal model. A second study addressed the physiological effects of oppe, hexadecanol, tyloxapol and trehalose (LPM-l) compared to treatment with trehalose (TRE) or saline, in order to determine (1) the contribution of TRE to the mixture of oppe, hexadecanol and tyloxapol, and (2) to assess the effect of the LPM-l surfactant replacement on the epithelial lining fluid composition by means of analysing bronchoalveolar lavage fluid. Thirdly, the effects of TRE and / or calcium were studied on the surface properties of oppe suspensions, by in vitro analysis using the ring detachment method of Du Nouy The in vivo research comprised of two studies, performed in randomised controlled fashion. In the first study, 24 New Zealand White adult rabbits were randomised into 4 groups, while in the second study, 15 animals were randomised into 3 groups. In the first in vivo study, three synthetic surfactants, LPM-l, Exosurf and LPM-2, and a saline group were tested. LPM-l is a new formulation that consists ofa mixture of Df'PC, TRE, hexadecanol and tyloxapol. LPM-2 is a formulation with a composition equivalent to that of commercially available Exosurf, prepared on site. In both studies animals were subjected to repeated lavage with large volumes of warm saline (25 ml/kg) in order to establish surfactant deficiency and acute lung injury. Five minutes after the last lavage, vehicle, i.e. surfactants LPM-l, Exosurf, or LPM-2, or saline, in the first in vivo study, and LPM-l, TRE or saline in the second in vivo study, was instilled, and the course of the animals followed over the next 3 hours. Ventilator settings were standardized before and after lavage. The effects of surfactant treatment on gas exchange (arterial Pa02, oxygenation index (Ol), arterial-alveolar oxygen (a/A) ratio), percentage calculated shunt, and total dynamic respiratory compliance (CRSdyn), and histopathological changes were compared with changes in saline treated controls. Arterial blood gases in 100% oxygen and CRSdynwere measured before and after lavage, at 15 minute intervals for the first 30 min, then at 60, 90, 120, and 180 min after vehicle instillation. Oxygenation improved to a similar extent after LPM-l and Exosurf instillation, surpassing that of LPM-2 or saline. Overall, intratracheal instillation of both Exosurf and LPM-l, rapidly improved the gas exchange and reduced the intrapulmonary shunt, but did not restore the lung to its pre-lavage condition. From the 2nd in vivo study it was evident that trehalose-only, was inefficient as a lung surfactant, failing to improve oxygenation indices or the calculated percentage shunt, or influencing respiratory compliance. The addition of the sugar, trehalose (TRE), to the on-site 'Exosurf mixture (LPM-2) brought the activity of the resultant LPM-l to the same level as that of commercial Exosurf, but failed to raise the activity above that of Exosurf. These physiological improvements were sustained for up to 3 hours. Saline-treated animals had no improvement in gas exchange despite management with variable PIP (to maintain a tidal volume of -1 0 ml / kg) and constant PEEP of 5 cm H20. In-vitro results, obtained by the Ou Nouy tensiometer, showed higher mean ordinate surface tension values for the OPPC-only and DPPC + TRE mixtures, and the slopes of their respective graphs smaller in magnitude than those of the other formulations, suggesting that these formulations had less surface tension-lowering capability than the other surfactants. At 20°C (20 mg / ml DPPC-surfactants) the mean ordinate values of OPPC and OPPC + TRE, 70.13 and 69.47 dyne / cm, respectively, were not significantly different from each other. The mean ordinate values of LPM-l and the formulation containing OPPC + TRE + tyloxapol + CaCh were lower, but similar, as were the values of LPM-2 (on-site Exosurf) and LPM-2 + CaCho Thus, three internally homogeneous subgroups could be identified which differed significantly, namely: DPPC and DPPC + TRE, LPM-2 and LPM-2 + CaCh, and DPPC + TRE + tyloxapol + CaCh and LPM-l. Similar conclusions apply to the ordinate values of the surfactants at 37°C, and to the mean slope values at 20°C, with the exception that the subgroups, LPM-2 and LPM-2 + CaCh, and LPM-l and OPPC + TRE + tyloxapol + CaCh are not so clearly separated. A similar analysis of mean slope values was performed. Here too a significant difference between substances was found, OPPC alone or in combination with TRE, again being significantly different from the other surfactants. The most prominent light microscopy findings of the lungs of animals included general lymphatic dilatation, congestion and lung polymorphonuclear infiltration, with no difference between study groups. Hyaline membranes were present in all surfactant groups, but significantly more so in the saline treated group. In the first in vivo study, the presence of neutrophils in the lung interstitiwn as well as alveoli, was a common finding in all of the study groups towards the end of the study protocol. A significant increase in the BAL-fluid neutrophil count occurred in all animals, concurrent with a significant decrease in the BAL macrophage count. No significant change occurred in the peripheral neutrophil count during the 3-hour study, suggesting recruitment of neutrophils from storage pools. Treatment with synthetic surfactant (LPM -1) did not have a significant effect on modifying the inflammatory response, since there was no significant difference in the BAL-derived cell counts between the LPM-1 and -saline groups. Epithelial damage was a consistent finding in all groups. The damage was more evident by electron microscopy examination and included hydropic changes, most readily observed in the mitochondria. The airspaces of study subjects showed the presence of oedema fluid. This luminal oedema appeared to be more prominent in the control group and LPM-2 (on site 'Exosurf') group. Organellar debris, probably originating from lysis of epithelial cells, was present, despite treatment with synthetic surfactant. The electron microscopical appearance of the epithelial-lined substance ("hyaline membranes") in the present study showed a marked variability within groups as well as within the same case. The majority of cases showed a mix of membrane types with both granular and fibrillar materials present within the same membrane. In some cases there were layering of the membranes into distinct bands. The instillation of LPM-l resulted in the formation of a slightly different type of epithelial lining fluid after lavage, when compared to the prelavage composition. The most pronounced changes occurred within the fatty acids, whilst the phosphatidylcholine values remained unchanged. Palmitic acid concentrations (C16:0) increased significantly, suggesting enrichment of the epithelial lining fluid after instillation of LPM-l. This increase in C16:0 was concurrent with significant decreases in the percentage C16:1, C18:0, and C18:2. In contrast to previous studies, we describe higher levels for phosphatidyldimethylethanolarnine (PEA). An explanation may be that the lipid identified as PEA, was in fact partly phosphatidylglycerol (PG)-a lipid whose accurate identification was precluded for technical reasons. After surfactant instillation, the PC/SM ratio, a reflection of the lecithin / sphingomyelin (LIS), decreased significantly in the TRE-group between the first and final lavage, but remained statistically unchanged in the animals treated with LPM-l or saline. The change in ratio was mainly accounted for by a decrease in BAL-fluid PC content together with a rise in SM content. A poor correlation existed between the BAL-derived PC/SM ratio and indices reflecting oxygenation status (a/A ratio, Ol), as well as the CRSdynat the time of the final lavage. In conclusion, the primary hypothesis was accepted, LPM-l performed similarly to Exosurf in vivo, improving oxygenation, but not CRSdyn.None was clearly superior to the other. Some questions remain. The reason why LPM-l (LPM-2 + TRE) did not behave in a superior manner, in vivo, to Exosurf, is partly unclear. This finding was somewhat surprising since the chemical composition of Exosurf and LPM-2 did not differ, and the addition of TRE to LPM-2 (on-site Exosurf), did improve the in vivo activity of the resultant LPM-l, above that of LPM-2. A possible explanation for observed differences in performance include methodological issues, i.e. the preparation of the on-site formulations, especially that of LPM-2 (on-site Exosurf), may differ from the way in which true commercial Exosurf is prepared.
AFRIKAANSE OPSOMMING: Die doel van die studie was om 'n sintetiese proteïn vrye surfaktant te ontwikkel en die produk te vergelyk met 'n kunsmatige surfaktant reeds in kliniese gebruik. Die bekende uit die literatuur en die onbekende van die produk wat evalueer sou word, lei op tot die samestelling van die nul hipotese van die PhD naamlik dat geen verskil in longfunksie sou gewys word tussen die toetsproduk en reeds gebruikte kommersiële surfaktant nie. Die hipotese was dat 'n suiker (trehalose), in kombinasie met Dipalmitoiel fosfatidielcholine (DPPC), gaswisseling en longfunksies sal verbeter vir 'n long met 'n lae surfaktant konsentrasie. Vir die studie is jong volwasse wit New Zealand konyne gebruik en is hulle met 'n gestandaardiseerde en menslike manier gebruik in eksperimentele werk. Die diere is onder intraveneuse narkose geplaas en verskillende kardiovaskulêre en pulmonologiese aspekte is gemeet. Die long surfaktant is uitgewas deur middel van fisiologiese soutoplossing wat tot liggaam temperatuur verhit is en daarna is die diere prospektief gerandomiseer tot eksperimentele groepe. Met vooraf bepaalde tydsintervalle is die fisiologiese metings herhaal en was die metings toegespits daarop om longmeganiese funksie en gasoordrag vermoë te evalueer. Lig mikroskopiese en elektron mikroskopiese studies is ook op die longe gedoen en verder is brongoalveolêre vloeistof ook ontleed. Die groepe met ondersoek was: I. oppe, heksadekanol, tyloxapol en trehalose (LPM-I). 2. oppe, heksadekanol, tyloxapol (LPM-2 :. LPM-I sonder trehalose). Hierdie is 'n proteïnvrye surfaktant plaaslik berei ( dieselfde samestelling as Exosurf). 3. Exosurf®. (Kommersiële preperaat reeds in gebruik). Hierdie is 'n proteïnvrye sintetiese surfaktant. 4. Trehalose, 'n non-reduserende disakklaried van glukose. Addisioneel is daar ook in vitro studies gedoen waann die oppervlakte spanmngs aktiwiteite van die verskillende surfaktant oplossings vergelyk is. Die statistiese analise is gedoen in samewerking met Prof. J. Maritz wat 'n unieke metode ontwikkel en gepubliseer het om herhalende veranderlikes op 'n statisties verantwoordbare manier te ontleed. In die eerste van die studies, is LPM-I, Exosurf®, fisiologiese soutoplossing en 'n plaaslik bereide "Exosurf" (LPM-2), met 'n chemiese samestelling identies aan dié van kommersiële Exosurf®, evalueer. In 'n tweede studie is die fisologiese effekte van LPM-I vergelyk met trehalose of fisiologiese soutoplossing om die volgende te ondersoek: 1) Die bydrae van trehalose tot 'n mengsel van oppe, heksadekanol en tyloxapol (LPM-2). 2) Die gevolg van LPM-l surfaktant toediening op die konyn se brongo-alveolêre vloeistof samestelling. 'n Derde, in vitro studie, het die oppervlaktespannings-effekte van trehalose en of kalsiumbyvoegings tot DPPC-oplossings gemeet deur middel van die ring metode van Du Nouy, In die eerste in vivo studie verbeter oksigenasie en persentasie longaftakking tot dieselfde mate na LPM-l en Exosurf® toediening en word die hipotese van die proefskrif bevestig. In die breë gesien, is die tydsprofiele van LPM-l en Exosurf® ten opsigte van oksigenasie en persentasie longaftakking statisties betekenisvol beter en van 'n sneller aard, as die tydsprofiele van dieselfde indekse na die toediening van fisiologiese soutoplossing of LPM-2. Die tydsprofiel van dinamiese longvervormbaarheid, na die toediening van LPM-I of Exosurf®, is dieselfde, maar betekenisvol beter as die vervormbaarheid na toediening van LPM-2 of fisiologiese soutoplossing. Alhoewel die oksigenasie indekse in die geval van LPM-l en Exosurf® betekenisvol verbeter oor die studietydperk, vind volkome herstel tot die basislynwaardes (voor spoeling) nie plaas nie. Bykomend, geen van die surfaktante het na toediening enige noemenswaardige verbetering in longvervormbaarheid tot gevolg gehad nie. Die rede vir die swakker vertoning van LPM-2 en Exosurf is onbekend en sal in opvolg studie ondersoek word. In die tweede in vivo studie is dit duidelik dat trehalose op sy eie, 'n oneffektiewe surfaktant is aangesien die preperaat na toediening geen verbetering teweegbring ten opsigte van oksigenasie indekse, persentasie longaftakking, of long-dinamiese vervormbaarheid nie. Die toevoeging van trehalose tot LPM-2, om LPM-l te lewer, neem wel die aktiwiteit van LPM-l tot dieselfde in vivo vlak as dié van kommersiële Exosurf®, maar slaag nie daarim om 'n hoër fisiologiese in vivo aktiwiteit as dié produk te bereik nie. Die diere wat met fisiologiese soutoplossing behandel is toon geen verbetering in enige fisiologiese parameter nie. Die in vitro resultate wat verkry is deur die Du Nouy tensiometer toon hoër gemiddelde ordinaat oppervlaktespannings waardes vir 'n formule wat slegs uit DPPC bestaan, asook vir 'n mengsel van DPPC + trehalose. Die helling van die grafieke van hierdie oplossings is ook kleiner as die van die ander formulas wat daarop dui dat DPPC op sigself, en DPPC + trehalose, weinig vermoë het om oppervlaktespanning te verminder. Daarteenoor verlaag die volgende oplossings die oppervlaktespanning ten opsigte van gedistilleerde water betekenisvol en wel in In konsentrasie afhanklike manier by beide 21°C en 3rc: LMP-I-, LPM-2-, DPPC + trehalose + tyloxapol + CaCf2-, en LPM-2 + CaCf2. Die prominentste ligmikroskopiese bevindinge van die longe van die diere sluit in: Algemene limfvat dilatasie, stuwing, en long neutrofiel infiltrasie. Betreffende hierdie histologiese bevindinge is daar geen verskille aangetoon tussen die groepe nie. Hialienmembrane was teenwoordig in al die groepe, maar betekenisvol meer in die groep wat fisiologiese soutoplossing ontvang as vervangingsterapie. In die tweede in vivo studie is daar 'n betekenisvolle styging in die neutrofiel- en daling in makrofaagtelling, van die brongoalveolêre vloeistof spoeling in al drie die groep aangetoon. Terselfdertyd vind geen noemenswaardige daling in die perifêre (sistematiese) neutrofieltelling plaas nie. Hierdie bevindinge dui daarop dat die brongoalveolêre selveranderinge toegeskryf kan word aan verwerwing van neutrofiele vanuit 'n longstoringspoel eerder as rekrutering vanuit die sistemiese sirkulatoriese poel. Surfaktant (LPM-l), behandeling het geen betekenisvolle vermindering in long inflammasie teweeggebring nie. Epiteelskade was 'n algemene ligmikroskopiese bevinding in al die groepe. Die samestelling van die brongoalveolêre vloeistof verander na installering van LPM-I. Die prominentste verandering word waargeneem in die vetsuur samestelling terwyl die DPPC waardes onveranderd bly. Die vetsuur, palmitiensuur (palmitic acid), (CI6:0), verhoog betekenisvol na toediening van LPM-l. Daarteenoor verminder die konsentrasie van C16:1, C18:0 en C18:2. In kontras met vorige studies, beskryf die huidige studie hoër konsentrasies van fosfatidieletanolamien, moontlik as gevolg van tegniese verskille in die metingsmetodes. 'n Betekenisvolle verlaging in die fosfatidielcholine:sfingomiëlien (PC/SM) verhouding word waargeneem tussen die eerste en die finale longspoeling van die trehalose-groep, terwyl dit onveranderd bly in die diere wat LPM-1 of fisiologiese soutoplossing ontvang.

Les infections aiguës des voies respiratoire inférieures (ALRI) sont un problème persistant et dominant de santé publique. De nombreux virus peuvent causer une ALRI, y compris le virus respiratoire syncitial, le virus de la grippe, le virus parainfluenza, le rhinovirus, l'enterovirus, l'adénovirus et le coronavirus. Depuis l'épidémie de SRAS en 2004 et les épidémies récentes de grippe aviaire H5N1 hautement pathogène, la transmission des virus zoonotiques à l'homme pose un problème majeur de santé publique, car les contacts rapprochés entre l'animal et l'homme ou entre les hommes faciliteraient le réassortiment et la recombinaison entre les virus pour générer des nouveaux virus qui pourraient passer la barrière d'espèce. L'introduction de ces nouveaux virus dans la population immunologiquement naïve pourrait être à l'origine d'épidémies ou de pandémies. . Par ailleurs, de nouveaux virus comme le métapneumovirus humain, les coronavirus humains NL63 et HKU1, et le bocavirus humain, ont été identifiés grâce au développement des nouvelles techniques moléculaires. Toutes ces approches ont changé le profil étiologique des ALRI. Pour mieux identifier les causes d'épidémies, il est nécessaire de surveiller la distribution et l'évolution génétique des virus respiratoires. L'objectif de ce travail a été dicté en priorité par la nécessité de développer des méthodes rapides, spécifiques et sensibles de diagnostic pouvant détecter non seulement des pathogènes viraux importants mais également de nombreuses co-infections virales. Les objectifs de ce travail étaient de développer différentes techniques moléculaires multiplexées et de mettre en place une plateforme de diagnostic des virus respiratoires. La stratégie a été de développer de nouvelles techniques en rapport avec les types de virus à détecter: (1) virus prioritaires comme la grippe aviaire H5N1 ou le virus pandémique SOI-H1N1v, détectés par RT-PCR en temps réel et multiplexée; (2) virus à haute prévalence de circulation dans la population possédant une diversité génétique élevée, détectés par RT-PCR multiplexée ; virus rares et émergents, détectés et étudiés par micropuce ADN et séquençage à haut-débit
Acute respiratory infections (ARIs) are one persistent and Worldwide problem to public health and the leading cause of morbidity and mortality in developing countries. In China, nearly 21 million cases occur every year [1]. Numerous viruses can cause ALRI, including respiratory syncitial virus, influenza virus, parainfluenza virus, rhinovirus, adenovirus and coronavirus [2-12]. Since the outbreak of SARS in 2004 and the recent epidemics of highly pathogenic avian influenza H5N1 virus in China [13-15] as well as in other countries of Southeast Asia, the transmission of zoonotic viruses from animals to human has become a big concern to public health because the increasing close contacts of animal-human and human-human would largely facilitate the reassortment and recombination of viruses to generate new viruses which could cross the species barrier. The introduction of new viruses to immune naïve population would cause epidemics or pandemics. Meanwhile, new viruses like human metapneumovirus, human coronaviruses NL63 and HKU1, and human bocavirus, were identified as the result of development of new molecular techniques. All these approaches have largely changed the etiological profile in ARI. To better react in case of epidemics, it is necessary to monitor the distribution and the genetic evolution of respiratory viruses. Sustained global surveillance project was required to improve the capacity in many developing countries to detect endemic, epidemic and newly emerging respiratory pathogens [16]. To set up such project, reliable and standardized diagnostic methods were requested. With sequencing and phylogenetic analysis, the project could identify a wide variety of agents, to differentiate highly pathogenic viruses from less virulent seasonal respiratory viruses and to identify new emerging viruses. Meanwhile, the epidemiological and etiological profile of ARI should be thoroughly studied to describe the background and set up a baseline for epidemic alert. In 2006, the project "Surveillance and Investigation of Endemic Situations in South-East Asia (SISEA)" was implemented (http://www. Hku. Hk/respari/research_07. Htm), which supported my PhD work. Shanghai, as the biggest metropolis of China, is an important center for population migration and with distinct four seasons including very cold winter

Le complexe bc₁ de la chaîne respiratoire mitochondriale est une bonne cible thérapeutique pour traiter le paludisme car cette enzyme est essentielle au parasite. Ses deux sites actifs, Qo et Qi, formés par le cytochrome b, ne sont pas totalement conservés entre les espèces, facilitant la découverte d’inhibiteurs à affinité différentielle, ce qui est important dans le développement de médicaments. L’atovaquone est le seul antipaludique ciblant le complexe bc₁ utilisé en médecine. L’émergence de résistance rend urgente l’étude de nouveaux inhibiteurs. Les ELQs (Endochin-like Quinolones) sont une classe d’antipaludiques particulièrement prometteuse.Pour étudier la liaison des inhibiteurs dans les sites actifs et l’effet de mutations de résistance, nous utilisons la levure et des méthodes biochimiques et bio-informatiques. Dans ce travail, nous avons étudié la relation entre mutations de résistance à l’atovaquone dans le site Qo et perte de fonction. Nous avons aussi modifié le site Qo de la levure pour qu’il mime mieux le site de l’enzyme du parasite. Les résidus «Plasmodium» altèrent le fonctionnement du site, résultant en une surproduction d’ions superoxides et une perte de croissance respiratoire, qui est restaurée par la modification d’une autre sous-unité du complexe, ISP, partenaire du site Qo, suggérant que les deux sous-unités doivent s’ajuster pour un fonctionnement correct. Nous avons analysé des polymorphismes de la région Qo observés chez l’Homme et trouvé qu’ils peuvent modifier la sensibilité du complexe à l’atovaquone, ce qui pourrait avoir un impact sur les effets secondaires du traitement. Nous avons ensuite étudié le mode d’action d’ELQ-400 et montré que ce nouvel antipaludique cible les deux sites Qo et Qi, ce qui rend l’apparition de résistance peu probable. Enfin, nous avons commencé la reconstruction du site Qi de la levure pour mimer le site du parasite.Les mutants de levure avec un complexe bc₁ «Plasmodium» semblent être de bons outils pour l’étude des inhibiteurs. Leur étude a aussi permis de comprendre mieux la structure et le fonctionnement du complexe bc₁
The bc₁ complex of the mitochondrial respiratory chain is a good therapeutic target for the treatment of malaria as the enzyme is essential for pathogen proliferation. The two catalytic sites, Qo and Qi, formed by cytochrome b, are not fully conserved between species, facilitating the development of inhibitors with differential saffinity, which is important for the development of new drugs. At present, Atovaquone is the only antimalarial drug targeting the bc₁ complex used in medicine. The emergence of resistance makes it important to find new inhibitors, and the ELQs (Endochin-like Quinolones) are promising antimalarial candidates.In order to study the inhibitor binding to the active sites and the effect of resistance mutations, we have used yeast and a combination of biochemical and bioinformatic methods. We have studied the relationship between atovaquone resistance mutations in the Qo site and loss of function. We have also modified the yeast Qo site to make it more like the parasite site. The “Plasmodium” residues in the yeast Qo site altered its activity, which resulted in the overproduction of superoxide and the loss of respiratory growth. This could be restored by the modification of another bc₁ complex subunit interacting with the Qo site, ISP, suggesting that both these subunits need to be readjusted for correct activity. We then analyzed polymorphisms of the Qo region reported in Humans and found that they could alter the enzyme sensitivity to atovaquone, which could impact the side-effects linked to atovaquone treatment. We have also studied the mode of action of ELQ-400 and showed that this new antimalarial drug targets both the Qo and Qi sites, which would make the emergence of resistance less likely. Finally, we have started the reconstruction of yeast Qi site to make it resemble the parasite site.The yeast mutants with a “Plasmodium-like” bc₁ complex could be useful tools for the study of antimalarial drugs. These analyses have also resulted in a better understanding of the structure and function of the bc₁ complex

INTRODUÇÃO: A dosagem ideal de salbutamol através de inaladores dosimetrados para o tratamento da asma aguda na infância não está bem estabelecida. Este estudo visou comparar dois regimes de dosagem de salbutamol via inalador dosimetrado com espaçador em crianças com crises moderadas ou graves de asma. As hipóteses deste estudo foram: I. Pacientes em vigência de crises de sibilância moderadas ou graves necessitam doses de salbutamol maiores que as até recentemente recomendadas pelo Global Initiative for Asthma (GINA). II. Doses maiores de salbutamol são seguras para crianças com idade igual ou superior a dois anos de idade. OBJETIVOS: I. Avaliar a eficácia (principalmente tempo de permanência na sala de emergência e taxas de internação hospitalar) de um regime de doses maiores de salbutamol em comparação com as até recentemente recomendadas pelo GINA. II. Verificar a segurança destas dosagens de salbutamol através do monitoramento de possíveis efeitos colaterais e dos níveis plasmáticos desta droga. MÉTODOS: Este foi um estudo prospectivo, randomizado, duplo-cego e controlado realizado em salas de emergência de três centros na cidade de São Paulo. Foram incluídos pacientes com 2-17 anos de idade com asma aguda moderada a grave (escore PRAM, Pediatric Respiratory Assessment Measure, >= 5). As dosagens de salbutamol via inalador dosimetrado com espaçador administradas durante a primeira hora foram: 6 (até 25 kg) ou 12 jatos ( > 25 kg) no grupo controle e 9 (até 15 kg), 12 ( > 15 a 20 kg), 15 ( > 20 a 25 kg) ou 18 jatos ( > 25 kg) no grupo estudo. Cada jato continha 100 mcg de salbutamol. Os pacientes dos dois grupos receberam corticosteroides e brometo de ipratrópio. Os desfechos principais do estudo foram o tempo de permanência na sala de emergência para os pacientes não internados e a necessidade ou não de internação hospitalar. Os desfechos secundários foram: mudança no volume expiratório forçado no primeiro segundo (VEF1) após uma hora, mudanças no escore PRAM, na oximetria de pulso e na frequência respiratória após uma hora e no tempo final (momento da alta ou internação hospitalar, máximo de quatro horas) e a necessidade de tratamentos adicionais após a primeira hora. Os desfechos de segurança incluíram mudanças nos níveis séricos de potássio, glicose, bicarbonato e pH no tempo final em relação ao tempo inicial, bem como possíveis anormalidades no eletrocardiograma, níveis plasmáticos de salbutamol, mudanças na frequência cardíaca e presença ou ausência de tremores, os dois últimos após uma hora e no tempo final. RESULTADOS: Foram incluídos 119 pacientes com condições basais semelhantes e não foram observadas diferenças significativas entre os grupos no tempo de permanência na sala de emergência (p=0.55) ou nas taxas de internação hospitalar (p=0.48). Não foram observadas diferenças significativas entre os grupos nas mudanças de VEF1 após uma hora, nas mudanças de escore PRAM, na oximetria de pulso e na frequência respiratória após uma hora e no tempo final. Não houve diferenças significativas entre os grupos na necessidade de tratamentos adicionais administrados após a primeira hora. Não foram observadas diferenças significativas nos desfechos de segurança entre os grupos. CONCLUSÕES: O uso de doses maiores de salbutamol administradas através de inalador dosimetrado com espaçador em crianças com asma aguda moderada ou grave não resultou em menor taxa de internação, menor tempo de permanência na sala de emergência ou melhora em outros desfechos de eficácia em comparação com o regime de dosagens até recentemente proposto pelo GINA. Os dois regimes de dosagens mostraram perfis de segurança semelhantes
INTRODUCTION: The ideal dosing of albuterol via metered-dose inhalers for acute childhood asthma is not well established. This study aimed to compare two dosing regimens of albuterol via metered-dose inhaler with spacer in children with moderate to severe asthma attacks. The hypothesis of this study were: I. Patients with moderate to severe asthma attacks would benefit from higher doses of albuterol than those recommended until recently by the Global Initiative for Asthma (GINA). II. Higher doses of albuterol are safe for children two years of age and older. OBJECTIVES: I. To compare the efficacy (mainly length of stay in the emergency room and admission rates) of higher doses of albuterol with those recommended until recently by the GINA. II. To assess the safety of different doses of albuterol by monitoring for possible side effects and measuring drug plasma levels. METHODS: This was a prospective, randomized, controlled, double-blind study conducted in emergency rooms of the three participating centers in the city of São Paulo. We included patients with 2-17 years old with moderate to severe acute asthma (Pediatric Respiratory Assessment Measure, PRAM, score >= 5). Dosages of albuterol via metered-dose inhaler with spacer administered during the first hour included: 6 (up to 25 kg) or 12 puffs ( > 25 kg) in the control group and 9 (up to 15 kg), 12 ( > 15 to 20 kg), 15 ( > 20 to 25 kg) or 18 puffs ( > 25 kg) in the study group. Each puff contained 100 mcg of albuterol. All patients received corticosteroids and ipratropium bromide. Primary outcomes were the length of stay in the emergency room for non-admitted patients, and rate of admission. Secondary outcomes included forced expiratory volume in one second (FEV1) changes following one hour, PRAM score, pulse oximetry and respiratory rate changes following one hour and at the final time (discharge or admission, maximum four hours) and the need for additional therapies after the first hour. Safety outcomes included changes in serum potassium, glucose, bicarbonate and pH at the final time in comparison with the initial time, as well as electrocardiogram abnormalities, plasma albuterol levels, heart rate, and tremors (the last two after one hour and at the final time). RESULTS: We included 119 patients with similar baseline conditions, and no significant differences were observed between groups in the length of stay in the emergency room (p=0.55) or admission rates (p=0.48). No significant differences were observed between groups in FEV1 changes after one hour, and PRAM score, pulse oximetry and respiratory rate changes after one hour and at the final time. There were no significant differences between groups in additional therapies administered after the first hour. No significant differences were observed in safety outcomes between groups. CONCLUSIONS: Higher dosage regimens of albuterol via metered-dose inhaler with spacer for children with moderate to severe acute asthma did not result in lower admission rate, shorter length of stay in the emergency room or improvement in other efficacy outcomes in comparison with those recommended until recently by the GINA. Both dosage regimens showed similar safety profile

Orientadores: Clarice Weis Arns, Luciana Konecny Kohn
Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia
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Resumo: Para avaliar a atividade antiviral dos extratos de plantas brasileiras foram eleitos o Metapneumovirus aviário (aMPV) e o vírus Respiratório sincicial bovino (BRSV) pertences à família Paramyxoviridae, subfamília Pneumovirinae, gêneros Metapneumovirus e Pneumovirus respectivamente. Tanto o aMPV quanto o BRSV são vírus semelhantes aos que causam doenças em humanos como o vírus respiratório sincicial humano (HRSV) e metapneumovírus humano (hMPV). O objetivo do presente trabalho foi avaliar a atividade antiviral de 12 diferentes espécies de plantas: Pterodon emarginatus Vogel.; Kielmeyera coriacea Mart. & Zucc.; Pfaffia glomerata (Spreng.) Pedersen; Virola sebifera Aubl.; Gaylussacia brasiliensis (Spreng.) Meisn.; Maytenus ilicifolia (Schrad.) Planch.; Bursera aloexylon (Schiede ex Schltdl.) Engl.; Aspidosperma tomentosum Mart.; Copaifera langsdorffii Desf; Baccharis dracunculifolia DC.; Arrabideae chica (Humb. & Bonpl.) B.Verl.; Aniba rosaeodora Ducke (Lin 3). Para realizar os estudos antivirais foi utilizada concentrações máximas não tóxicas para as diferentes linhagens celulares utilizadas frente aos dois vírus. Para os ensaios antivirais foram utilizadas a linhagens CER (Chicken embryo related cells) e CRIB (bovine viral diarrhea virus-resistant clone of MDBK cells) para o aMPV e BRSV respectivamente. Os extratos brutos com atividade antiviral foram submetidos a uma curva concentração resposta com diferentes concentrações de extrato na presença de 100 DICC/mL de cada amostra viral através do ensaio colorimétrico MTT [3-(4,5- dimetiltiazol-z-yl)-2,5 difeniltertrazolim brometo] determinando assim a concentração antiviral 50% (EC50). Para determinar o mecanismo de ação dos extratos e os vírus nas células foram utilizados três diferentes tratamentos: (i) Pré-tratamento (célula tratada com extrato e posterior inoculação com a amostra viral); (ii) Pós-tratamento (célula inoculada com a amostra viral e depois tratada com o extrato); (iii) Virucida (extrato e vírus mantidos juntos e posterior inoculação na cultura celular). O extrato foi considerado ativo quando houve diminuição do título viral em 1,5 log em relação ao titulo viral controle. Os resultados mostraram que todos os extratos testados obtiveram concentrações não tóxicas para as diferentes linhagens celulares. Em relação à atividade antiviral o extrato da espécie Aspidosperma tomentosum apresentou atividade frente ao BRSV e aMPV, ambos no pré-tratamento. Os extratos das espécies Virola sebifera; Arrabidaea chica; Gaylussacia brasiliensis e Anniba rosaeodora apresentaram atividade antiviral para o aMPV no pós-tratamento. Nenhum extrato bruto apresentou atividade de "vírus-inativação" em relação aos vírus avaliados. Os demais extratos não apresentaram atividade antiviral significativa para nenhum destas espécies virais. O ensaio antiviral deste estudo poderá continuar sendo utilizado como triagem para outras espécies em busca de substâncias com potencial medicinal. As diferentes atividades de ação dos extratos sugerem a ocorrência de mecanismo de ação distinto entre os vírus avaliados
Abstract: To evaluate the antiviral activity of extracts from Brazilian plants two different viruses were elected Avian metapneumovirus (aMPV) and bovine respiratory syncytial virus (BRSV) members of the family Paramyxoviridae, subfamily Pneumovirinae, Pneumovirus and Metapneumovirus genera respectively. Both viruses are similar to those that cause humans diseases such as respiratory syncytial virus (HRSV) and human metapneumovirus (hMPV). The purpose of this study was to assess the antiviral activity of 12 different plant species: Pterodon emarginatus Vogel; Kielmeyera coriácea Mart. & Zucc.; Pfaffia glomerata (Spreng.) Pedersen; Virola sebifera Aubl.; Gaylussacia brasiliensis (Spreng.) Meisn.; Maytenus ilicifolia (Schrad.) Planch. ex. Reiss; Bursera aloexylon (Schiede ex Schltdl.) Engl.; Aspidosperma tomentosum Mart.; Copaífera langsdorffii Desf.; Baccharis dracunculifolia DC.; Arrabideae chica (Humb. & Bonpl.) B.Verl.; Aniba rosaeodora Ducke. (Lin 3). To perform the antiviral assay we used non toxic maximum concentrations of the plants extracts with different cell lines used against the two viruses. The cell lines used were CER (Chicken embryo related cells) and CRIB (bovine viral diarrhea resistant clone of MDBK cells) for AMPV and BRSV, respectively. The extracts with antiviral activity were subjected to a concentration response curve with varying concentrations of extract in the presence of 100 TCID / mL of each viral sample by colorimetric MTT assay [3 - (4,5- dimethylthiazol-z-yl) -2 , 5-diphenyltetrazolium bromide] to identify the antiviral 50% concentration (EC50). To define the mechanism of action of extracts and viruses in cells three different treatments were used: (i) pre-treatment (cells treated with plants extracts and subsequent inoculation with a viral sample), (ii) Post-treatment (cells inoculated with the viral sample and afterwards treated with the extract), (iii) Virucidal (viruses and plants extracts were kept together and after that inoculated into cell cultures). The extract was considered active when there was a decrease of virus titers by 1.5 log in contrast to the control viral titer. The results revealed that all tested extracts had no toxic concentrations for the different cell lines and detected the antiviral activity of the extract of the species Aspidosperma tomentosum against the BRSV and aMPV, both in the pretreatment conditions. The extracts of the species Virola sebifera; Arrabidaea chica; Gaylussacia brasiliensis and Anniba rosaeodora (Lin 3) exhibited antiviral activity for aMPV after treatment. The other extracts showed no significant antiviral activity for any of these viral species. The antiviral test of this study may still be used as screening for other species in search for substances with medicinal potential. The varied action activities of the extracts suggest that the occurrence of distinct mechanism of action between the viruses evaluated
Mestrado
Microbiologia
Mestre em Genética e Biologia Molecular

Introdução: A bronquiolite aguda é a principal causa de internação de lactentes menores de um ano de idade e tem como principal agente etiológico o vírus sincicial respiratório. As principais diretrizes baseadas em evidências recomendam o tratamento de suporte com hidratação e oxigenoterapia, quando necessário e não indicam o uso rotineiro de corticosteroides, broncodilatadores e antibióticos. No entanto, estudos anteriores mostraram que o uso inadvertido dessas medicações é frequente na prática clínica. Objetivo: Analisar o tratamento aplicado a lactentes com bronquiolite viral aguda em um hospital público e compará-lo a diretrizes nacionais e internacionais. Casuística e métodos: Foi realizado um estudo observacional, transversal, descritivo e analítico que incluiu crianças menores de 2 anos, internadas no Hospital do Servidor Público Estadual durante dois anos (2012 - 2014), com primeiro episódio de sibilância e que tiveram coletado aspirado de nasofaringe, para pesquisa de vírus sincicial respiratório na admissão. Foram excluídos os lactentes com fatores de risco conhecidos para desenvolver doença grave. As informações foram coletadas dos prontuários e por contato telefônico com os responsáveis. Resultados: Dentre os 129 pacientes com resultado positivo para a pesquisa do vírus sincicial respiratório, 7 foram excluídos por apresentarem alguma comorbidade ou fator de risco prognóstico e 2 não tiveram o seus prontuários encontrados. A idade média, dos 120 estudados, foi de 6,8 meses e 51,6% foram do sexo feminino. Broncodilatadores, corticosteroides, antibióticos e inalação com solução salina hipertônica foram prescritos, durante a internação, a 90%, 72,5%, 40% e 66,7% dos casos, respectivamente. O uso dessas medicações foi excessivo e não compatível com as diretrizes, exceto o uso de solução salina hipertônica inalatória que esteve em acordo com a recomendação da Sociedade Brasileira de Pediatria. Após ajuste para confundidores, a chance de prescrição de antibióticos foi menor em menores de 3 meses (OR=0,21, p= 0,007) e também associada à presença de febre na admissão (OR=3, p = 0,013) e maior tempo de internação (OR=2,53, p= 0,003). A introdução de oxigênio suplementar apresentou associação com maior tempo de internação (OR=12,9, p < 0,001). Os lactentes com idade abaixo de 3 meses tiveram menor chance de prescrição de corticosteroides (OR=0,67, p < 0,001) que também foi associada à saturação menor que 95% no momento da admissão (OR=3,17, p= 0,037) e ao maior tempo de internação. O uso de solução salina hipertônica também foi associado ao maior tempo de internação (OR=3,07, p < 0,001). Conclusão: Em lactentes hospitalizados em um hospital público, o uso de antibióticos, corticosteroides e broncodilatadores para tratamento da bronquiolite aguda foi elevado. Essas condutas não seguiram as recomendações das principais diretrizes nacionais e internacionais. O uso de solução salina hipertônica inalatória seguiu a diretriz da Sociedade Brasileira de Pediatria
Background: Acute bronchiolitis is the main cause of hospitalization of infants under one year of age and has respiratory syncytial virus as the main etiological agent. The main evidence-based guidelines recommend hydration and oxygen therapy support when necessary and do not indicate the routine use of corticosteroids, bronchodilators and antibiotics. However, previous studies have shown that inadvertent use of these medications is common in clinical practice. Objective: To analyze the treatment of infants with acute viral bronchiolitis in a public hospital and to compare it with national and international guidelines. Patients and methods: An observational, transversal, descriptive and analytical study was carried out, including children under 2 years of age, hospitalized between two years (2012 - 2014), with first episode of wheezing and nasopharyngeal test for respiratory syncytial virus on admission. Infants with known risk factors for developing severe disease were excluded. The information was collected from the medical records and by telephone contact with those parents. Result: Of the 129 patients with a positive test for respiratory syncytial virus, 7 were excluded because they presented some comorbidity or a prognostic risk factor, and 2 did not have their charts found. The mean age of the 120 studied was 6.8 months and 51.6% were female. Bronchodilators, corticosteroids, antibiotics and nebulised hypertonic saline were prescribed, during hospitalization, at 90%, 72.5%, 40% and 66.7% of the cases, respectively. The use of these medications was excessive and not compatible with the guidelines, except the use of nebulised hypertonic saline that was in agreement with the recommendation of the Brazilian Society of Pediatrics. After adjusting for confounders, the chance of antibiotic prescription was lower in children younger than 3 months (OR = 0.21, p = 0.007) and also associated with the presence of fever at admission (OR = 3, p = 0.013) and a longer stay (OR = 2.53, p = 0.003). The introduction of supplemental oxygen showed an association with longer hospitalization (OR = 12.9, p < 0.001). Infants less than 3 months of age had a lower chance of prescribing corticosteroids (OR = 0.67, p < 0.001), which was also associated with a saturation of less than 95% on admission (OR = 3.17, p = 0.037 ) and longer hospitalization time. The use of nebulised hypertonic saline was also associated with longer length of stay (OR = 3.07, p < 0.001). Conclusion: In infants hospitalized in a public hospital, the use of antibiotics, corticosteroids and bronchodilators to treat acute bronchiolitis was high. These conducts did not follow the recommendations of the main national and international guidelines. The use of nebulised hypertonic saline followed the guideline of the Brazilian Society of Pediatrics

Uvod: Interleukin 33 (IL-33) ima značajnu ulogu u inflamatornim i autoimunskim oboljenjima, ali se sve više proučava njegov značaj u imunopatogenezi različitih alergijskih oboljenja, uključujući i alergijsku astmu (AA). Cilj: Ispitivanje vrednosti IL-33 u serumu pacijenata sa AA pre i posle šestomesečne inhalatorne kortikosteroidne terapije (ICS Th) i povezanosti dobijenih vrednosti IL-33 sa određenim kliničkim i laboratorijskim karakteristikama ovih pacijenata. Metode: Vrednost IL-33 u serumu određena je kod 61 pacijenta sa AA pre započinjanja i posle sprovedne šestomesečne ICS Th i kod 30 zdrave dece. U obradi podataka primenjene su standardne metode deskriptivne i analitičke statistike. Rezultati: Kod pacijenata sa nelečenom AA, serumske vrednosti IL-33 su signifikantno veće u odnosu na pacijente kod kojih je sprovedena šestomesečna ICS Th (p<0,05), kao i u odnosu na zdravu decu (p<0,01). Pacijenti sa AA koji su tokom 6 meseci lečeni sa ICS Th i zdrava deca imaju slične vrednosti IL-33 u serumu (p>0,05). Kod pacijenata sa AA pre započinjanja i 6 meseci posle primene ICS Th ne postoji signifikantna korelacija između vrednosti IL-33 u serumu i eozinofilnih granulocita periferne krvi (p>0,05), eozinofilnih granulocita u nazalnom sekretu (p>0,05) i ukupnog IgE u serumu (p>0,05). Kod pacijenata sa nelečenom AA postoji signifikantna negativna korelacija između vrednosti serumskog nivoa IL-33 i sledećih parametara plućne fukcije: FEV1 (p<0,05), FEV1/FVC (p<0,05), PEF(p<0,05) i MEF 25/75 (p<0,05). Posle šestomesečne ICS Th poboljšava se plućna funkcija, odnosno dolazi do porasta brzine protoka vazduha u disajnim putevima kao i promena u plućnim volumenima u zavisnosti od stepena opstrukcije u odnosu na vrednosti pre uključenja antiinflamatorne terapije (FEV1, FVC, FEV1/FVC, PEF, MEF 25/75, za sve vrednosti p<0,01). Dok je signifikantna negativna korelacija dokazana između IL-33 i vrednosti FEV1 (p<0,01), FVC (p<0,01) i PEF (p<0,05). Zaključak: Serumski nivo IL-33 je značajno povišen kod dece sa nelečenom, odnosno nekontrolisanom AA. Šestomesečna primena ICS dovodi do značajne redukcije IL-33 u serumu čije su vrednosti u negativnoj korelaciji sa vrednostima FEV1, FVC i PEF, odnosno pozitivnoj korelaciji sa težinom i kontrolom AA. Rezultati naše studije ističu da IL-33 ima značajnu ulogu u imunopatogenezi AA. Određivanje serumske vrednosti IL-33 može biti koristan indikator težine AA.
Introduction: Interleukin 33 (IL-33) plays a significant role in inflammatory and autoimmune diseases, but its significance in the immunopathogenesis of various allergic diseases including allergic asthma (AA) has gained increasing attention in research over recent years. Objective: Testing serum levels of IL-33 in patients with AA before and after a six-month inhaled corticosteroid therapy (ICS Th) and correlation of IL-33 values with specific clinical and laboratory characteristics of these patients. Methods: Serum levels of IL-33 were determined in 61 patients with AA prior to the initiation of ICS Th and following the six-month ICS Th as well as in 30 healthy children. Data processing was performed applying standard methods of descriptive and analytical statistics. Results: In patients with untreated AA, serum levels of IL-33 were significantly higher as compared to the patients who have received a six month ICS Th (p <0.05) as well as to healthy children (p <0.01). Patients with AA, who were treated with ICS Th for six months, and healthy children have similar serum IL-33 (p> 0.05). In patients with AA, significant correlation between serum IL-33 levels and eosinophilic peripheral blood granulocytes (p>0.05), eosinophilic granulocytes in nasal secretion (p>0.05) and the total IgE in serum has not been observed for the period prior to initiation and 6 months after the administration of ICS Th. In patients with untreated AA, there is significant negative correlation between serum IL-33 and the following pulmonary functions test results: FEV1 (p<0.05), FEV1/FVC (p<0.05), PEF (p<0.05) and MEF 25/75 (p<0.05). After a six-month ICS Th, significant improvement of pulmonary functions was evident, that is, increase in airflow speed and lung volume change as compared to the values determined before the initiation of the anti-inflammatory therapy (FEV1, FVC, FEV1/FVC, PEF, MEF 25/75, for all values p<0.01). Significant negative correlation between IL-33 and the values of FEV1 (p<0.01), FVC (p<0.01)and PEF (p<0.05) has been established. Conclusion: Serum level of IL-33 is significantly elevated in children with untreated, i.e., uncontrolled AA. A six-month ICS Th asthma treatment results in significant reduction of serum levels of IL-33. This level is negatively correlated with FEV1, FVC and PEF values while positively correlated with the severity of the disease and control of AA. The results of our study point out that IL-33 plays an important role in the immunopathogenesis of AA. Quantification of serum IL-33 levels can be a useful indicator of the severity of AA.

O surfactante pulmonar é uma substância fundamental na mecânica pulmonar, com atividade biofísica e de proteção alveolar por reduzir a tensão superficial e impedir o seu colabamento.Na síndrome do desconforto respiratório tipo agudo(SDRA) ocorre uma diminuição quantitativa e disfunção qualitativa do surfactante com agravamento do quadro clínico.Estudamos, em um modelo experimental de SDRA em coelho adulto, os efeitos da adição de polietilenoglicol ao surfactante pulmonar exógeno quanto à melhora da complacência pulmonar,pressão ventilatória,índice de oxigenação,diferença alvéolo-arterial de oxigênio,gradiente alvéolo-arterial de oxigênio pressão arteial parcial de CO2, pelo índice de eficiência ventilatória,diâmetro alveolar médio e índice de distorção.A utilização do surfactante melhorou a oxigenação, e a mecânica pulmonar, sem no entanto, haver diferença entre os grupos surfactante e surfactante mais polietilenoglicol
Lung surfactant is a fundamental substance in lung mechanics, with biophysical activity to reduce alveolar surface tension and to avoid pulmonary collapse. In the acute respiratory distress syndrome (ARDS) occurs a quantitative and qualitative surfactant dysfunction with worsening of clinical status. We study, in an experimental model of ARDS in adult rabbit, the effects of polyethyeneglycol addition to the exogenous surfactant to improve the pulmonary compliance, ventilatory pressure, oxygenation index, arterial-alveolar oxygen ratio , alveolar-arterial oxygen gradient, carbon dioxide partial arterial pressure, ventilatory efficiency index , alveolar medium diameter and ditorsion index. Surfactant treatment improved arterial oxygenation and the lung mechanics, with no differences between the study groups

La enfermedad pulmonar obstructiva crónica (EPOC) es una patología prevenible caracterizada por una limitación al flujo aéreo progresiva y no completamente reversible, disnea, producción de esputo y tos crónica. La obstrucción al flujo aéreo se asocia a un proceso inflamatorio crónico en la vía aérea y el parénquima pulmonar en respuesta a partículas nocivas o gases inhalados, en particular al humo de cigarrillo (HC) (Figura. Esta respuesta inflamatoria crónica puede inducir la destrucción del parénquima pulmonar o enfisema y alteraciones estructurales de la vía aérea pequeña al alterar los mecanismos de reparación y defensa. De esta manera, se produce limitación progresiva al flujo aéreo debido a la menor retracción elástica y aumento de la distensibilidad pulmonar. La inflamación y el estrechamiento de la vía aérea periférica también contribuyen a la limitación del flujo aéreo produciendo una disminución del volumen de aire espirado en el primer segundo de la espiración forzada (FEV1) en la espirometría forzada. Además, alteraciones en las relaciones ventilación-perfusión (VA/Q) pueden dar lugar a hipoxemia e hipercapnia en estos pacientes. Por otro lado, la inflamación inducida por el HC lleva a la hipersecreción mucosa, que también se asocia con el declive del FEV1, debida al mayor número de células caliciformes en la epitelio bronquial y al aumento de las glándulas submucosas, resultando en tos productiva que es característica de la bronquitis crónica, entidad clínica independiente. En el curso evolutivo de la EPOC puede desarrollarse hipertensión pulmonar (HP) debida al remodelado vascular caracterizado por la hiperplasia de la capa íntima y la muscularización de arteriolas (8). Estos cambios se han atribuido a la acción directa del HC sobre el endotelio vascular, a la cual puede añadirse la vasoconstricción pulmonar hipóxica (VPH). Por otro lado, se postula que la perdida de lecho capilar pulmonar debido al enfisema podría contribuir al desarrollo de la HP asociada a la EPOC. También se ha demostrado infiltración por células inflamatorias y disfunción endotelial en las arterias pulmonares de pacientes con EPOC. Por su parte, la HP sostenida puede promover la hipertrofia del ventrículo derecho (VD) y llevar al desarrollo de “cor pulmonale” e insuficiencia cardiaca derecha. Los pacientes con EPOC también presentan comorbilidades a nivel sistémico, incluyendo enfermedades cardiovasculares, disfunción del musculo esquelético, y cáncer de pulmón. Vistos los antecedentes, la presente tesis doctoral se plantea en base a las siguientes hipótesis: 1. El modelo experimental de EPOC por exposición al HC en cobayos reproduce la destrucción del parénquima y las alteraciones en la vía aérea y vasos pulmonares características de la EPOC, en los que diferentes mediadores químicos y células inflamatorias tendrían un papel fisiopatológico destacable. Por este motivo, hipotetizamos que la exposición crónica al HC producirá en cobayos un proceso inflamatorio asociado con cambios morfológicos y funciónales en las estructuras pulmonares similares a lo que se observa en los pacientes con EPOC. 2. La adecuada caracterización del modelo experimental de EPOC en cobayos permitiría su uso en la evaluación de los efectos de nuevos fármacos y dianas terapéuticas en el tratamiento de la EPOC. En este sentido, se evalúan los efectos de bromuro de aclidinio, un antagonista muscarínico de acción prolongada, y sildenafilo, un inhibidor selectivo de la PDE5. Teniendo en cuenta todo ello, los objetivos que se plantearon en los tres artículos que conforman esta tesis doctoral fueron: 1.- Primer artículo. “Pulmonary inflammatory reaction and structural changes induced by cigarette smoke exposure in the Guinea pig.” A) Objetivo general: Evaluar la naturaleza y características de la reacción inflamatoria en el pulmón, y su implicación en los cambios estructurales que tienen lugar a nivel pulmonar en el modelo experimental de EPOC en cobayos expuestos crónicamente al HC. B) Objetivos concretos: I. Caracterizar el tipo de células inflamatorias y su distribución en las estructuras pulmonares (vía aérea, vasos y parénquima). II. Analizar el remodelado de la vía aérea y de los vasos pulmonares. III. Evaluar la presencia de fibrosis y la aparición de enfisema en el parénquima. IV. Explorar los mecanismos que interconectan la infiltración de células inflamatorias con las alteraciones estructurales características de la EPOC. V. Establecer la importancia, el orden secuencial y la dinámica de estas alteraciones en el desarrollo de la EPOC. 2.- Segundo artículo. “Effects of Aclidinium Bromide in a Cigarette Smoke-Exposed Guinea Pig Model of Chronic Obstructive Pulmonary Disease”. A) Objetivo general. Investigar los efectos del broncodilatador bromuro de aclidinio sobre los cambios histopatológicos y el infiltrado de células inflamatorias en los pulmones del modelo de EPOC en cobayos crónicamente expuestos al HC. B) Objetivos concretos. I. Evaluar el efecto de aclidinio sobre las alteraciones morfológicas, como potencial agente antirremodelado de la vía aérea. II. Evaluar el efecto de aclidinio sobre la infiltración de células inflamatorias en las estructuras pulmonares, como potencial agente antiinflamatorio. III. Evaluar el efecto de aclidinio sobre la función pulmonar y los signos respiratorios característicos de la EPOC, particularmente su efecto broncodilatador. IV. Evaluar el efecto de aclidinio sobre otras alteraciones típicas de la EPOC como la metaplasia de células caliciformes y el desarrollo de enfisema pulmonar. V. Explorar posibles mecanismos que interconectan los diferentes cambios observados con la administración de aclidinio. 3.- Tercer artículo. “Sildenafil in a cigarette smoke-induced model of COPD in the guinea pig”. A) Objetivo general. Evaluar los efectos del vasodilatador sildenáfilo sobre la hemodinámica pulmonar, función endotelial, y el remodelado vascular y del parénquima, en el modelo de EPOC en cobayos crónicamente expuestos al HC. B) Objetivos concretos. I. Evaluar el efecto de sildenafilo sobre la hemodinámica pulmonar, como agente vasodilatador de la circulación pulmonar. II. Evaluar el efecto de sildenafilo sobre la hipertrofia del VD y la función endotelial de arterias pulmonares. III. Evaluar el efecto de sildenafilo sobre las alteraciones morfológicas y el remodelado vascular pulmonar, como potencial agente antiproliferativo. IV. Evaluar el efecto de sildenafilo sobre el funcionalismo respiratorio. V. Integrar posibles mecanismos que expliquen los cambios producidos con la administración de sildenafilo.

INTRODUÇÃO: O transplante de pulmão é parte fundamental no tratamento das doenças terminais do pulmão, constituindo uma modalidade terapêutica eficaz para pacientes com doença pulmonar incapacitante, progressiva e em estágio final. No entanto, as drogas imunossupressoras usadas para evitar a rejeição do enxerto podem causar efeitos colaterais em diversos tecidos. O sistema mucociliar, presente nas vias aéreas, é um dos principais mecanismos de defesa do trato respiratório e pode ser alterado por ação das drogas imunossupressoras. Desta forma, o objetivo deste estudo foi avaliar o sistema mucociliar traqueobrônquico de ratos submetidos a dois esquemas de terapia tríplice imunossupressora. MÉTODOS: Foram utilizados 90 ratos machos Wistar distribuídos em 3 grupos conforme o tratamento: controle (C) = solução salina; terapia 1 (TI) = tacrolimus + micofenolato de mofetil + prednisona; terapia 2 (TII) = ciclosporina + azatioprina + prednisona. Após o período de tratamento (7, 15 ou 30 dias), os animais foram sacrificados e realizadas as seguintes medidas: transportabilidade do muco (TM), frequência de batimento ciliar (FBC), quantificação de muco neutro e ácido, velocidade de transporte mucociliar (VTMC), e contagem total e diferencial de células no lavado broncoalveolar (LBA). RESULTADOS: A TM não foi afetada pelas terapias em nenhum dos tempos estudados. Ambas as terapias causaram significativa redução da FBC dos animais tratados por 7 e 15 dias. A produção de muco neutro foi menor nos animais tratados com a TI por 7, 15 e 30 dias. Porém, com a TII, essa redução ocorreu apenas aos 7 dias. Por outro lado, a quantidade de muco ácido foi significativamente maior em todos os animais tratados com as duas terapias. Todos os animais tratados com as terapias imunossupressoras apresentaram redução da VTMC nos três tempos. Houve aumento do número total de células e de macrófagos e neutrófilos no grupo TI em 7 dias. CONCLUSÕES: Ambas as terapias imunossupressoras foram prejudiciais ao transporte mucociliar das vias aéreas de ratos, tanto pela redução da FBC e da VTMC, quanto pela maior produção de muco ácido e menor produção de muco neutro. A TI foi mais prejudicial ao sistema mucociliar em comparação à TII
INTRODUCTION: Lung transplantation is an essential part in the treatment of terminal lung diseases, providing an effective therapeutic modality for patients with disabling, progressive and final stage lung disease. However, the immunosuppressant drugs used to prevent graft rejection may cause side effects in several tissues. The mucociliary system, present in the airways, is a major defense mechanism of the respiratory tract and can be changed by action of immunosuppressive drugs. Thus, the aim of this study was to evaluate the tracheobronchial mucociliary system of rats submitted to two triple immunosuppressive therapy regimens. METHODS: We used 90 male Wistar rats divided into 3 groups according to treatment: control (C) = saline solution; therapy 1 (TI) = tacrolimus + mycophenolate mofetil + prednisone therapy; therapy 2 (TII) = cyclosporine + azathioprine + prednisone. After the period of treatment (7, 15, or 30 days), the animals were sacrificed and the following measures taken: mucus transportability (MT), ciliary beating frequency (CBF), quantification of neutral and acid mucus, mucociliary transport velocity (MCTV), and total and differential counting of cells in bronchoalveolar lavage (BAL). RESULTS: MT was not affected by treatments in any of the periods studied. Both therapies have caused significant reduction of CBF of animals treated for 7 and 15 days. The neutral mucus production was lower in animals treated with TI for 7, 15 and 30 days. But with TII, this reduction occurred only at 7 days. Moreover, the amount of acid mucus was significantly higher in all animals treated with both therapies. All animals treated with immunosuppressive therapies had reduced MCTV at the three times. There was an increase of total cells and macrophages and neutrophils in the TI group in 7 days. CONCLUSIONS: Both immunosuppressive therapies were harmful to the mucociliary clearance of the airways of rats, either by reducing the CBF and MCTV, as by the increased production of acid mucus and decreased production of neutral mucus. TI was more harmful to the mucociliary system in comparison to TII

Orientador: Sandra Elisa Contir Pitton
Banca: Ari Menardi Junior
Banca: Lucy Marion Calderini Philadelpho Machado
Resumo: O trabalho científico em questão buscou de um modo claro, unir todas as nuances relevantes ao tema proposto, mesclando Geografia, Medicina e Direito, numa redação coesa e enriquecida com dados estatísticos e muita discussão teórica. Inicialmente, discutiu-se questões preliminares da Geografia da Saúde, pontuando observações terminológicas e metodológicas, consagrando assim uma ruptura institucional com antigas formas de produção científica. Num segundo plano, destacou-se a qualidade de vida como de difícil constatação qualitativa ou quantitativa, verificando-se a saúde como um de seus indicadores de grande importância. Neste contexto, procurou-se nos dados coletados, sublimar as doenças mais corriqueiras, com influência internacional, que tivessem vínculos com atitudes omissivas ou comissivas de Estados independentes e de notoriedade indispensável à boa qualidade de vida, ou com a própria sobrevivência humana. Emergiu a questão do ar como objeto de estudo, levantando cientificamente a circulação das massas de ar e, por conseguinte, o caminhar das impurezas aéreas, causadoras e catalisadoras das doenças respiratórias. Isto posto, verificou-se que o tema possuía grande inserção na questão da soberania de Estados independentes, tendo sido de suma importância discorrer sobre a gênese do Estado moderno e assim, discutir nas considerações finais, sobre as necessidades institucionais para que se garanta a soberania, respeitando o planeta como um grande Sistema.
Abstract: This scientific work aimed at clearly putting together all the relevant nuances considering the proposed issue, mixing up Geography, Medicine and Laws, in a cohesive writing enriched by statistical data and lots of oral discussion. At the beginning the preliminary questions like the Geography of Health were discussed, pointing out terminological and methodological observations, therefore obtaining an institutional breakup with the old forms of scientific production. In a second phase, the quality of life was shown to be of difficult qualitative and quantitative verification, health being one of its most important indicators. In such context, we tried to point out from the collected data, the most common deceases with international influences, which had to do with negligent or wrong attitudes by independent states and of high significance to the quality of life or to human survival. Then the issue of the air as an object of study arose, scientifically studying the circulation of masses of air and therefore the trips of air impurities, which cause respiratory deceases. Afterwards we could realize both that the issue was of great value towards the question of the sovereignty of independent states and that the discussion on the genesis of the modern state was highly important, which led us to the final considerations about the institutional needs for the sovereignty to be guaranteed, respecting the planet as a great System.
Mestre

Les élevages avicoles familiaux tels que les basses-cours rurales, les poulaillers urbains et les élevages de volailles de loisir sont un secteur important de la production avicole française. Estimés à 2,5 millions de propriétaires à ce jour, ces élevages sont suspectés de pouvoir transmettre des agents pathogènes aux élevages avicoles commerciaux. Dans cette étude, nous avons étudié le rôle des élevages avicoles familiaux dans la circulation et la transmission d'agents pathogènes aux élevages avicoles commerciaux. Afin de mieux caractériser les acteurs de la filière avicole familiale, une enquête participative nationale a été réalisée afin d'analyser les pratiques d'élevage et d'identifier différentes sous-populations. En parallèle, les prévalences de 14 agents infectieux à tropisme majoritairement respiratoire ayant un intérêt en santé publique et/ou animale ont été estimées. L'identification d'agents pathogènes comme marqueurs d'infection et de transmission a été effectuée en comparant les prévalences entre secteurs familial et commercial. Dans le secteur familial, cinq sous-populations ont été mises en évidence : les poulaillers urbains, les basses-cours traditionnelles, les poulaillers d'étudiants, les élevages familiaux "de compagnie", et les élevages de poules de race et de loisir. Des agents pathogènes comme Mycoplasma synoviae et Avibacterium paragallinarum, présentent une prévalence élevée dans les basses-cours, mais sont rarement identifiés dans les élevages commerciaux, ce qui pourrait en faire des marqueurs pertinents de transmission du secteur familial au secteur commercial. D'une manière générale, les résultats suggèrent un rôle limité des basses-cours dans la contamination des élevages commerciaux. A l'inverse, dans un contexte épizootique d'influenza aviaire, il a été montré que les liens humains entre secteur familial et secteur commercial représentaient un risque de contamination des basses-cours, conduisant ainsi à privilégier l'hypothèse inverse d'une possible contamination des élevages familiaux par les élevages commerciaux
Backyard poultry farming involving for example traditional family flocks and hobby poultry flocks is an important sector of the French poultry production. Today, it is estimated that they represent 2.5 million owners, and they are suspected of transmitting pathogens to commercial poultry farms (and/or humans). In this study, we investigated the role of backyard poultry flocks in the circulation and transmission of pathogens to commercial poultry farms. In order to better characterize the stakeholders in the backyard poultry sector, a national participatory survey was conducted to analyze owners' practices and identify different sub-populations. Simultaneously, the prevalence of 14 pathogens of interest in human and/or animal health and predominantly presenting a respiratory tropism were studied. The identification of pathogens as markers of infection and transmission was carried out by comparing their prevalence levels in the familial and commercial poultry sectors. In the familial poultry sector, five sub-populations were identified: urban poultry, traditional poultry, student poultry, "pet" family poultry, and hobby poultry. Pathogens such as Mycoplasma synoviae and Avibacterium paragallinarum are highly prevalent in French family poultry flocks but are rarely identified in commercial farms. Consequently, they could be considered as relevant markers of transmission from backyard to commercial flocks. In general, the results indicate the limited role of backyard poultry flocks in the contamination of commercial poultry farms. On the contrary, human links existing between backyard and commercial premises were identified as a risk factor for backyard flocks in an epizootic avian influenza context. These results lead us to consider the reverse hypothesis of a possible pathogens transmission from commercial to backyard poultry flocks during epidemics

Surveillance of acute respiratory infections (ARI): innovative approaches for the identification and characterization of viral agents Introduction. Acute respiratory infections (ARI) are ubiquitous, air-borne transmitted and highly contagious infections, characterized by typical epidemic pattern. Though ARI causative agents may be bacterial or viral, viruses are by far the most common causes of ARI. In recent years, the global epidemiological scenario has been enlivened by the identification and emergence of many airborne pathogens (such as influenza viruses A/H5N1, A/H7N7, A/H7N9, coronaviruses SARS and MERS), which have been co-circulating along with the already known viral strains (i.e. seasonal influenza viruses, parainfluenza viruses, respiratory syncytial viruses, etc.), thus highlighting the public health concern about emerging infectious disease. Objectives. The project aimed at applying new molecular biology techniques, "virus discovery" methodology and bioinformatics analyses to investigate the etiology of ARI. The specific objectives were: 1) Identification of viral pathogens responsible of severe acute respiratory infections (SARI) and acute respiratory distress syndrome (ARDS) during the pandemic and post-pandemic (2009-2011). On purpose, the proportion of SARI/ARDS cases and deaths due to A(H1N1)pdm09 infection and the impact of other respiratory pathogens were evaluated during the pandemic and post-pandemic period in Lombardy. Additionally, unknown viruses were investigated in those cases for which diagnosis remained negative by using VIDISCA-454 methodology, a “virus discovery” technique. This analysis was performed at the Laboratory of experimental virology, Academic Medical Center (AMC), University of Amsterdam, where I completed an internship under the supervision of Prof. Lia van der Hoek. 2) Evaluating the genetic variability and molecular evolution of respiratory syncytial virus (RSV). In order to reconstruct the origin and phylodynamic history of RSV, the genetic diversity and evolutionary dynamics of RSV A and RSV B identified in respiratory specimens collected from children aged ≤ 3 years hospitalized in Lombardy for ARI over six epidemic seasons (2006 to 2012) were analyzed. Materials and methods 1) From October 2009 to December 2011, 206 respiratory samples were collected from patients (61.2% males, median age: 44.3 years) hospitalized for SARI/ARDS. Nucleic acids were purified by NucliSENS® easyMAG® (bioMérieux, France), and analyzed by real-time RT-PCR assay to identify influenza virus. The clinical specimens that resulted negative to influenza virus detection were then screened by real-time RT-PCR/PCR for a panel of respiratory pathogens (Respiratory MWS r-gene™ Real-time PCR, bioMérieux, France) to detect: RSV A and B; human metapneumovirus (hMPV) A and B; human rhinovirus (hRV) and enterovirus (hEV); adenovirus (AdV); human bocavirus (hBoV) 1-4; human coronavirus (hCoV) 229E, NL63, OC43, HKU1; human parainfluenza virus (hPIV) 1-4; Chlamydophila pneumoniae; Mycoplasma pneumoniae. Cases resulted negative to all diagnostic assays were further investigated by VIDISCA-454 (virus discovery cDNA-AFLP) technique. This is a virus discovery method based on recognition of restriction enzyme cleavage sites, ligation of adaptors and subsequent amplification by PCR combined with high-throughput sequencing 454 FLX/Titanium system of Roche. 2) RSV A (n=23) and RSV B (n=12) sequences obtained from oro-pharyngeal swabs of RSV-infected children aged ≤3 years hospitalized for ARI from 2006 to 2012 were considered for molecular characterization. Sequences were obtained by multiplex-PCR to amplify a fragment of RSV G gene and several bioinformatic programs were used for the phylogenetic and phylodynamic analysis. Phylogenetic trees of RSV A and RSV B sequences were constructed by MEGA 5 program using the Neighbor-Joining method to identify RSV genotypes circulating and a bootstrap re-sampling analysis was performed to test tree robustness. To clarify RSV variability, amino acid mutations analysis was performed, and potential N-glycosylation and O-glycosylation sites were predicted by NetNGlyc 1.0 and NetOGlyc 3.1 programs, respectively. To evaluate site-specific selection pressure, different Maximum Likelihood approaches were applied (SLAC, FEL/IFEL, and MEME) by DATAMONKEY. To assess the evolutionary dynamics of RSV A and B, dated trees and evolutionary rates were estimated by BEAST with a Bayesian Markov Chain Monte Carlo (MCMC) approach. This analysis allowed identifying the time of the most recent common ancestor (tMRCA). Results and discussion 1) During the pandemic and post-pandemic different pathogens have co-circulated and were associated with clinical cases in the study, but influenza virus A(H1N1)pdm09 showed the greatest impact. Influenza A(H1N1)pdm09 virus was detected in 58.3% (120/206) of SARI/ARDS cases (61.7% males; 13.3% aged ≤5 years, 67.5% aged 6-64 years). A(H1N1)pdm09 was identified in 77.8% of fatal ARDS cases. The impact of respiratory pathogens other than A(H1N1)pdm09 was 19.4% (40/206) (65% males; 30% aged ≤5 years, 47.5% aged 6-64 years). The influence of other respiratory viruses was significantly lower (19.4% vs. 58.3%, p<0.0000001): hRV/hEV were the most commonly detected viruses, but also A(H3N2) influenza virus has played a significant role. Forty-six (46/206: 22.3%) SARI/ARDS cases (including two fatalities) resulted negative to all diagnostic assays and were further investigated by VIDISCA-454 that revealed no sequence reads that could belong to a novel virus or viral variant; however it enabled the identification of one case of undiagnosed measles. VIDISCA-454 methodology proved to be a sensitive and specific method, successfully applied to the monitoring of viral respiratory infections. Anyway, nearly 22% of SARI/ARDS cases did not obtain a definite diagnosis. In clinical practice, great efforts should be devoted to improve diagnosis of severe respiratory infections and to reduce such “diagnostic gap”. The advantage from relying upon more accurate diagnosis could benefit the patient - in term of receiving the more appropriate antiviral drugs -, and could provide more detailed information on viruses circulating in the community, thus making public health authorities aware so as to adjust their policies accordingly. 2) From phylogenetic analysis resulted that 3 RSV A sequences clustered in genotype GA2 and all the other isolates clustered with NA1 genotype. Compared to the reference strain, 31 amino acid substitutions were identified. Phylogenetic analysis of RSV B sequences showed that study sequences were included in BA genotype tended to cluster within a clade including also reference sequence BA4 and 8 amino acid substitutions were identified among all of them. Similar mean evolutionary rates for RSV A and RSV B were estimated, 2.1x10-3 subs/site/year (95% highest density probability (HPD): 1.7-2.5x10-3) and 3.03x10-3 subs/site/year (95%HPD: 2.1-3.9x10-3), respectively. The tMRCA for the RSV A tree root was 71 years (95%HPD: 60-85), suggesting an origin of the currently circulating strains back to 1940s. The study strains within clade NA1 shared a single significant internal node with an estimated mean tMRCA of 7 years ago (2005). The three GA2 strains had a significant tMRCA dating back to 16 years ago (95%HPD = 14-18). The dated tree obtained with RSV B strains showed a temporal-structure similar to RSV A. In particular, the tree root had an estimated mean tMRCA of 55 years ago (1957). All the studying strains clustered within a significant subclade, having a tMRCA estimate of 9 years ago (2003), including also a single BA4 strain. The RSV A Bayesian skyline plots (BSP) showed a first pick of the number of effective infections in the second half of 1980s, followed by a decrease of transmission events ending in about 2005, when a sharp growth restored the original viral population size. The RSV B BSP showed a similar trend, with a decrease in the effective number of infections occurring between the mid-1980s and 1990s, followed by a rapid growth in early 2000s. The RSV A site-specific selection analysis identified 10 codons under positive selection and a total of 39 sites were found to be under negative selection. The RSV B codon-specific selection analysis identified only one positively selected site and 27 negatively selected. Different patterns of O- and N-glycosilation sites were found by analyzing RSV A and RSV B studied sequences. Phylogenetic and phylodynamic analysis permitted to better understand the natural history of the virus, even if RSV remains difficult to control due to its antigenic diversity. G protein variability may play a significant role in RSV pathogenesis by allowing immune evasion. It is important monitoring changes in sequences coding this protein to permit the identification of future epidemic strains and to implement both vaccine and therapy strategies. This study thus contributed to have a better knowledge on the molecular epidemiology of RSV in Lombardy and provided data for a comparative analysis with other strains circulating in other regions of the world. Conclusions. This project showed that respiratory virology needs a constant update of diagnostic procedures and surveillance scheme, essential support in the study and monitoring of viral infections of both classic and, above all, emerging or newly identified viruses. The application of cutting-edge technologies can be a successful tool for public health to face such emerging threats.

The overall objectives of this thesis are: one, characterize the expression of mucin at baseline human nasal mucosa in healthy and inflamed (nasal polyps), and two, to analyze the expression of mucin and the regulation exerted by glucocorticoids on it in patients with nasal polyposis ( "in vivo") and a respiratory cell line (in vitro).
D'entre totes les estructures que composen el tracte respiratori, el nas, a través de la mucosa nasal, és l'òrgan encarregat de la preparació del moc inhalat mitjançant la filtració, l'escalfament i la humidificació d'aquest abans que arribi als pulmons, exercint d'aquesta manera una acció protectora sobre les vies aèries vers agents irritants i patògens. Per tal de desenvolupar aquesta tasca, la mucosa nasal, concretament l'epiteli i les glàndules submucoses d'aquesta, secreta moc i alhora promou l'aclariment mucociliar a través del seu epiteli ciliat, el qual es troba submergit en les secrecions nasals.

El moc respiratori està constituït per aigua, ions, secrecions pulmonars, trasudats de proteïnes del sèrum, proteïnes antimicrobianes i glicoproteïnes mucoses o mucines, les quals són responsables de la viscoelasticitat i adhesivitat del moc. Fins ara, han estat descrits 20 gens que codifiquen per mucines, subdividits en dos grups principals: mucines secretades i de membrana.

Tot i que ambdós tipus de mucines (de membrana i secretades) comparteixen una característica comuna en la seva estructura proteica, que consisteix en la presència de diverses repeticions en tandem de regions riques en residus de serina i treonina altament glicosilats, aquestes presenten diferències estructurals que determinaran en cert grau la seva funcionalitat.

En general, en patologies respiratòries s'ha trobat un increment en la expressió de mucines respecte teixits sans. Aquestes malalties, a més de compartir, entre d'altres símptomes l'obstrucció nasal i la hipersecreció de moc, semblen presentar una composició mucínica anormal del moc en referència a la quantitat, tipus i mida de les mucines (8-10). Aquests canvis podrien contribuir a les propietats reològiques del moc del tracte respiratori, produint un moc hiperviscós en el cas de la fibrosi quística i l'asma, i un moc aquós en el de la rinitis al·lèrgica i la poliposis nasal. No obstant això, les conseqüències funcionals del moc amb composició mucínica diferent han estat poc estudiades.

Un dels objectius actuals en l'estudi de la secreció mucosa i de la regulació dels gens MUC és investigar la relació potencial entre els seus patrons d'expressió, les seves propietats fisiològiques i les seves manifestacions clíniques en patologies respiratòries com ara la poliposi nasal i l'asma. Amb els estudis que composen aquesta tesi, es pretén determinar el paper dels gens MUC en l'etiologia de malalties com la poliposi nasal i l'asma, veure si existeixen patrons de diagnòstic definits, i alhora investigar els mecanismes de regulació d'aquests gens. Els resultats d'aquests estudis contribuiran a augmentar el coneixement de l'etiologia de la poliposi nasosinusal i obrirà noves perspectives per a la millora del tractament actual, donant la possibilitat de dissenyar nous fàrmacs i noves estratègies de tractament per a la rinosinusitis crònica i la poliposi nasosinusal, especialment en relació a la hipersecreció mucosa que acompanya en aquestes malalties.

En concret, els objectius generals d'aquesta tesi sòn: un, caracteritzar l'expressió de mucines a nivell basal en mucosa nasal humana sana i inflamada (pòlips nasals), i dos, analitzar l'expressió de mucines i la regulació que exerceixen els glucocorticoides sobre aquesta en pacients amb poliposi nasal (in vivo) i en una línia cel·lular respiratòria (in vitro).

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O trabalho científico em questão buscou de um modo claro, unir todas as nuances relevantes ao tema proposto, mesclando Geografia, Medicina e Direito, numa redação coesa e enriquecida com dados estatísticos e muita discussão teórica. Inicialmente, discutiu-se questões preliminares da Geografia da Saúde, pontuando observações terminológicas e metodológicas, consagrando assim uma ruptura institucional com antigas formas de produção científica. Num segundo plano, destacou-se a qualidade de vida como de difícil constatação qualitativa ou quantitativa, verificando-se a saúde como um de seus indicadores de grande importância. Neste contexto, procurou-se nos dados coletados, sublimar as doenças mais corriqueiras, com influência internacional, que tivessem vínculos com atitudes omissivas ou comissivas de Estados independentes e de notoriedade indispensável à boa qualidade de vida, ou com a própria sobrevivência humana. Emergiu a questão do ar como objeto de estudo, levantando cientificamente a circulação das massas de ar e, por conseguinte, o caminhar das impurezas aéreas, causadoras e catalisadoras das doenças respiratórias. Isto posto, verificou-se que o tema possuía grande inserção na questão da soberania de Estados independentes, tendo sido de suma importância discorrer sobre a gênese do Estado moderno e assim, discutir nas considerações finais, sobre as necessidades institucionais para que se garanta a soberania, respeitando o planeta como um grande Sistema.
This scientific work aimed at clearly putting together all the relevant nuances considering the proposed issue, mixing up Geography, Medicine and Laws, in a cohesive writing enriched by statistical data and lots of oral discussion. At the beginning the preliminary questions like the Geography of Health were discussed, pointing out terminological and methodological observations, therefore obtaining an institutional breakup with the old forms of scientific production. In a second phase, the quality of life was shown to be of difficult qualitative and quantitative verification, health being one of its most important indicators. In such context, we tried to point out from the collected data, the most common deceases with international influences, which had to do with negligent or wrong attitudes by independent states and of high significance to the quality of life or to human survival. Then the issue of the air as an object of study arose, scientifically studying the circulation of masses of air and therefore the trips of air impurities, which cause respiratory deceases. Afterwards we could realize both that the issue was of great value towards the question of the sovereignty of independent states and that the discussion on the genesis of the modern state was highly important, which led us to the final considerations about the institutional needs for the sovereignty to be guaranteed, respecting the planet as a great System.

Les mouvements de larges sous-unités protéiques sont très importants dans la liaison des substrats et les propriétés catalytiques des enzymes. Le but de notre étude a été de combiner la modélisation des réactions d'oxydo-réduction et les changements de conformations des complexes enzymatiques redox afin de décrire la dynamique réactionnelle de ces complexes. Nous avons réutilisé des algorythmes précédemment décrits dans la littérature afin de déterminer les changements conformationnels au sein des complexes redox. Nous avons ensuite développé un Système Multi-Agent permettant la simulation des activités redox de ces complexes. Nous avons appliqué notre approche à l'étude du complexe III de la Chaine Respiratoire Mitochondriale. Notre modèle nous permet de retrouver le onctionnement normal du complexe III lié à la dynamique de ces réactions redox et de ces mouvements internes
Because movements of large protein structures are key components in ligand docking and enzymatic catalysis, our aim was to combine modeling of the redox reactions and modeling of the conformational changes of enzymatic oxydoreduction complex in order to describe their dynamical functioning. We decided to reused previously described algorithms in order to uncover the conformational changes of redox complexes. We have then developed a Multi-Agent System to simulate the redox activiities of these complexes. We have applied our method to the complex III of the Mitochondrial Respiratory Chain. With this modeling we are able to find the normal functioning of the complex III as a consequence of the reaction mechanisms taking into account the tridimensional structure of the complex and its conformational changes

EN EL PRESENTE TRABAJO SE DESCRIBEN LOS EXPERIMENTOS REALIZADOS CON EL OBJETO DE DEMOSTRAR LA EXISTENCIA O NO DE UNA ASOCIACION ENTRE EL PRRSV Y H. PARASUIS. PARA ELLO SE HAN REALIZADO ESTUDIOS TANTO IN VIVO COMO IN VITRO, HACIENDO HINCAPIE EN LOS ESTUDIOS PATOLOGICOS Y DE PATOGENIA EN LOS PRIMEROS, Y ULTRAESTRUCTURALES Y FUNCIONALES EN LOS SEGUNDOS. SE REALIZARON 3 EXPERIMENTOS CON ANIMALES CON EL OBJETO DE DILUCIDAR SI LA PRESENCIA PEVIA DE PRRSV PROVOCABA UN AUMENTO EN LA PREVALENCIA DE POLISEROSITIS FIBRINOSA EN LOS ANIMALES INOCULADOS POSTERIORMENTE CON H. PARASUIS (EXPERIMENTOS 1 Y 3), Y OBTENER UN MODELO ADECUADO DE REPRODUCCION DE ENFERMEDAD DE GLASSER (EXPERIMENTO 2). EN LOS 2 PRIMEROS EXPERIMENTOS SE OBSERVO QUE LA UTILIZACION DE ANIMALES LIBRES DE PRRSV DIFICILMENTE PERMITE LA REPRODUCCION DE LA ENFERMEDAD DE GLASSER, LO CUAL SUGIRIO QUE EL COMPONENTE DE INMUNIDAD SISTEMICA PUEDE SER DETERMINANTE DE CARA A DESARROLLAR O NO LA INFECCION POR H. PARASUIS. EN EL TERCER EXPERIMENTO, REALIZADO CON ANIMALES LIBRES DE LOS DOS AGENTES PATOGENOS, SE LOGRO REPRODUCIR POLISEROSITIS FIBRINOSA EN 7 DE 10 CERDOS INOCULADOS EXCLUSIVAMENTE CON LA BACTERIA, PERO LA INOCULACION DE PRRSV PRIMERO Y H. PARASUIS (SEROVAR 5) 5 DIAS DESPUES NO RESULTO EN UN AUMENTO DE ENFERMEDAD DE GLASSER SINO EN UNA DISMINUCION DE ESTA PERO UN AUMENTO EN LOS NIVELES DE MORTALIDAD SUBITA RESPECTO AL GRUPO INOCULADO SOLO CON LA BACTERIA. ESTOS RESULTADOS SUGIEREN QUE EL EFECTO INMUNOMODULADOR DEL PRRSV, EN LOS CUALES SE PRODUCE UNA DISMINUCION DE LAS DEFENSAS A NIVEL PULMONAR PERO UNA MEJORA DE LOS MECANISMOS DE DEFENSA SISTEMICOS, PODRIA SER LA RAZON DE LOS RESULTADOS OBTENIDOS. EN LOS EXPERIMENTOS ULTRAESTRUCTURALES IN VITRO SE PRETENDIO ESTUDIAR LOS CAMBIOS SUFRIDOS POR LOS MAP INOCULADOS CON PRRSV Y DUALMENTE CON PRRSV Y H. PARASUIS, UTILIZANDO UNA DOSIS VIRICA DE 1 MOI. LA INFECCION DE MAP POR PRRSV DENOTO CAMBIOS MINIMOS EN LA ULTRAESTRUCTURA DE ESTOS, PRODUCIENDOSE BASICAMENTE UN AUMENTO DEL NUMERO DE LISOSOMAS. LA PRESENCIA DE LA BACTERIA PRODUJO UN CAMBIO DE ULTRAESTRUCTURA MUY EVIDENTE EN MAP, CON UNA MASIVA FORMACION DE FAGOSOMAS Y FAGOLISOSOMAS. LOS MAP SE MOSTRARON, ADEMAS, COMO CELULAS DE EFICACIA RELATIVA EN FAGOCITOSIS DE H. PARASUIS OPSONIZADA. EN LOS EXPERIMENTOS IN VITRO DE FUNCIONALIDAD SE PRETENDIO VALORAR LAS CAPACIDADES DE FAGOCITOSIS Y MUERTE INTRACELULAR DE H. PARASUIS POR PARTE DE MAP INFECTADOS O NO CON PRRSV. LOS RESULTADOS DE ESTAS PRUEBAS REVELARON QUE LA BACTERIA OPSONIZADA ERA FAGOCITADA POR MAP A UNOS NIVELES RELATIVAMENTE BAJOS (PORCENTAJES DE FAGOCITOSIS INFERIORES AL 25%), PERO QUE LA BACTERIA NO OPSONIZADA NO ES PRACTICAMENTE FAGOCITADA. POR OTRO LADO, SE PRESENTO UNA TENDENCIA DE MENOR CAPACIDAD DE MUERTE INTRACELULAR A MEDIDA QUE AUMENTAVA EL TIEMPO PI VIRICO.

The respiratory syncytial virus is the main causative agent of bronchiolitis during infancy. During my thesis, I have evaluated in mice a novel subunit vaccine based on the nasal delivery of the viral nucleoprotein (N SRS). Adult responses primed by N SRS immunization were protective against an experimental viral challenge. However, when administered to neonates, this vaccine caused an immunopathologic Th2 immune response characterized by lung eosinophilia. In order to decipher the mechanisms leading to lung immunity in neonates, I have compared the activation and polarization of neonatal versus adult CD4 T cells. Phenotypical and functional characterisation of pulmonary dendritic cells showed no differences related to the age of the cells. On the other hand, I have observed an intrinsic bias of neonatal T lymphocytes towards Th2 polarization that correlated with the strong expression of the transcription factor GATA-3
Le virus respiratoire syncytial (VRS) est l’agent étiologique majeur des bronchiolites du nourisson. Au cours de mon travail de thèse, une stratégie de vaccination sous-unitaire par voie nasale basée sur la nucléoprotéine virale (N SRS) a été évaluée. Chez la souris adulte, N SRS induit une réponse protectrice contre une épreuve virale. Appliquée au souriceau, cette vaccination aboutit à un recrutement d’éosinophiles dans le poumon, signe d’une réponse immunopathologique de type Th2. Afin de comprendre les mécanismes gouvernant l’immunité du tractus respiratoire du nouveau-né, j’ai comparé l’activation et l’orientation des lymphocytes T CD4 du nouveau né et de l’adulte. La caractérisation phénotypique et fonctionnelle des cellules dendritiques pulmonaires n’a pas mis en évidence de différence liée à l’âge. En revanche j’ai confirmé chez le nouveau-né une orientation des lymphocytes T dans la voie Th2, qui corrèle avec la surexpression du facteur de transcription GATA-3

Muito se tem pesquisado sobre os efeitos adversos dos antidepressivos tricíclicos (p.e. imipramina) sobre o sistema respiratório, embora pouco ou quase nada se encontre com relação a tal aspecto na literatura médica sobre a fluoxetina (Prozac®)– um inibidor seletivo da recaptação de serotonina, até porque esta droga começou a ser utilizada somente há cerca de quinze anos. Ambas substâncias (fluoxetina e imipramina, ou seus congêneres) agem basicamente sobre quadros depressivos, fóbicos e obsessivo-compulsivos, obesidade, anorexia, e outras indicações de várias naturezas, indo desde a ansiedade até a sindrome do pânico. Nota-se entretanto, que na maioria dos pacientes com tais quadros (sob tratamento com a fluoxetina ou não), são muito frequentes as queixas de natureza respiratória, como tosse, falta de ar, “angústia" no peito etc. Alguns efeitos adversos da fluoxetina e seus derivados são descritos na literatura, embora raros. Entre eles, destaca-se o comprometimento do aparelho respiratório na forma de doença pulmonar intersticial, pneumonia de hipersensibilidade e fosfolipidose. Apresentamos a seguir um projeto de trabalho experimental em cobaias com intuito de verificar a ação da fluoxetina sobre o aparelho respiratório, com relação à mecânica pulmonar, influência sobre o óxido nítrico exalado e resposta histopatológica pulmonar frente a possíveis injúrias, comparados a animais controles sem efeito da droga. Nosso modelo descreve um protocolo em que os animais foram submetidos ao cloridrato de fluoxetina por via oral durante 30 dias consecutivos, sendo ainda submetidos a uma reação de estresse tipo pânico– a natação forçada.
Although high-affinity imipramine binding sites have been reported in both rat and human lung, the role of the lungs in the pharmacokinetics of antidepressants like fluoxetine has not received much attention. Imipramine and fluoxetine have their action in depression, obesity, panic and anxiety among other indications. However, accumulation of selective serotonin-reuptake inhibitors- like fluoxetine, in the human lungs has been reported. Besides, it is very frequent to most patients (under fluoxetine treatment or not) present respiratory symptoms and/or signs, just like dyspnea, cough, chest anguish and so on. There also have been some adverse effects of fluoxetine described in the literature, although being few and isolated ones, related to hypersensitivity pneumonitis, phospholipidosis and interstitial lung disease. The purpose of this project is to investigate the fluoxetine action in respiratory tract, more specifically over the pulmonary interstitium, using an experimental model in guinea-pigs. Our project comprehends an experimental model with the animals under treatment of fluoxetine so that we could evaluate the substance effects on the respiratory system, concerning the mechanics and the lung histopathological aspects, compared to the control animals. We created then a protocol where the animals were treated with fluoxetine for 30 consecutive days and submmited to the forced swimmig test, a kind of panic-like reaction.

Objective: To study the presence of 8 respiratory viruses (Influenza-A, Influenza-B, RSV-A, RSV-B, Adenovirus, Parainfluenza-1, Parainfluenza-2 and Parainfluenza-3) and atypical bacteria (Mycoplasma pneumonia and Chlamydia pneumonia) in samples from Peruvian children under 5 years-old previously analyzed for Bordetella. Pertussis. Methods: A secondary data analysis was performed from a previous cross-sectional study conducted in children with a probable diagnosis of Pertussis from January 2010 to July 2012. All samples were analyzed via Polymerase Chain Reaction (PCR) for the following etiologies: Influenza-A, Influenza-B, RSV-A, RSV-B, Adenovirus, Parainfluenza 1 virus, Parainfluenza 2 virus, Parainfluenza 3 virus, Mycoplasma pneumoniae and Chlamydia pneumoniae. Results: A total of 288 patients were included. The most common pathogen isolated was Adenovirus (49%), followed by Bordetella pertussis (41%) from our previous investigation; the most prevalent microorganisms were Mycoplasma pneumonia (26%) and Influenza-B (19.8%). Coinfections were reported in 58% of samples and the most common association was found between B. pertussis and Adenovirus (12.2%). Conclusions: There was a high prevalence of Adenovirus, Mycoplasma pneumoniae and other etiologies in patients with a probable diagnosis of pertussis. Despite the presence of persistent cough lasting at least two weeks and other clinical characteristics highly suspicious of pertussis, secondary etiologies should be considered in children under 5 years-old in order to give a proper treatment.
Objetivo: Estudiar la presencia de 8 virus respiratorios (Influenza-A, Influenza-B, RSV-A, RSV-B, Adenovirus, Parainfluenza-1, Parainfluenza-2 y Parainfluenza-3) y bacterias atípicas (Mycoplasma pneumoniae y Chlamydia pneumonia) en muestras de niños peruanos menores de 5 años analizados previamente para Bordetella pertussis Métodos: se realizó un análisis de datos secundarios de un estudio transversal previo realizado en niños con un diagnóstico probable de Pertussis desde enero del 2010 hasta julio del 2012. Todas las muestras se analizaron mediante reacción en cadena de la polimerasa (PCR) para las siguientes etiologías: Influenza-A, Influenza-B, RSV-A, RSV-B, Adenovirus, virus Parainfluenza 1, virus Parainfluenza 2, virus Parainfluenza 3, Mycoplasma pneumoniae y Chlamydia pneumoniae. Resultados: un total de 288 pacientes fueron incluidos en el estudio. El patógeno aislado más común fue el Adenovirus (49%), seguido de Bordetella pertussis (41%) de nuestra investigación previa; los microorganismos más prevelantes fueron Mycoplasma pnemoniae (26%) e Influenza-B (19.8%). Las coinfecciones fueron reportadas en el 58% de las muestras y la asociación más común se encontró entre B. pertussis y Adenovirus (12.2%). Conclusiones: hubo una alta prevalencia de Adenovirus, Mycoplasma pneumoniae y otras etiologías en pacientes con un diagnóstico probable de pertussis. A pesar de la presencia de tos persistente que dura por lo menos dos semanas y otras características clínicas altamente sospechosas de pertussis, se deben considerar las etiologías secundarias en niños menores de 5 años para poder administrar un tratamiento adecuado.
Tesis

Des inhibiteurs du complexe bc1 de la chaîne respiratoire mitochondriale ont été développés comme agents antimicrobiens pour lutter contre des pathogènes de l'Homme et de plantes. Ces drogues ciblent les poches catalytiques Qo et Qi formées par le cytochrome b. La comparaison de séquences de cette protéine montre que les sites Qo et Qi sont bien conservés entre les organismes mais qu'il existe toutefois des variations qui pourraient expliquer leur différence de sensibilité aux drogues. A l'aide du modèle levure S. cerevisiae, nous avons étudié les déterminants de la résistance/sensibilité naturelle à deux antipaludiques se liant au site Qo de Plasmodium: l'atovaquone et RCQ06. Nous avons notamment montré que le résidu 275 joue un rôle clé dans ce phénomène. Une approche similaire est actuellement utilisée pour identifier les facteurs de la sensibilité différentielle à deux drogues ciblant le site Qi des oomycètes. Malheureusement, des cas de résistance acquise à ces antimicrobiens ont été rapportés et ont pour origine des mutations dans le cytochrome b. De ce fait, de nouvelles molécules sont requises pour court-circuiter ces résistances. Au cours de ma thèse, nous avons mis au point un test qui permet de cribler des molécules capables d'inhiber la fonction respiratoire. Nous avons ainsi pu identifier un nouvel inhibiteur du complexe bc1 : D12. Nous avons ensuite déterminé le mode de liaison de cette molécule ainsi que celui d'un composé capable d'inhiber la prolifération de Plasmodium, HDQ, grâce à une collection de mutants des poches catalytiques. HDQ s'est avéré être un inhibiteur du site Qi. Il pourrait être utilisé avec un inhibiteur du site Qo afin de limiter l'apparition de mutations de résistance. D12 est un inhibiteur du site Qo qui est capable notamment de court-circuiter la mutation de résistance à des fongicides du site Qo G143A. Cette dernière a été trouvée chez de nombreux phytopathogènes, mais n'est cependant pas apparue chez des champignons possédant un intron immédiatement après le codon codant pour la glycine 143. En utilisant la levure, nous avons montré que la mutation empêche l'épissage de l'intron en altérant la structure exon/intron. Nous avons également identifié des mécanismes de " by-pass " qui permettent de restaurer la fonction respiratoire du mutant et qui pourraient apparaître chez les pathogènes. Les mutants créés au cours de ma thèse pourront aider à identifier, concevoir et caractériser de nouveaux antimicrobiens et à étudier l'apparition de mutations de résistance.

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Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico
The objective of this work - which is characterized analyze the search for symptomatic tuberculosis in practice and perspective of the Community Health Agent (ACS) in the districts of Natal. Methods: This is a cross-sectional study. The study population was 646 professionals, and conducted a probabilistic random sampling, stratified by districts. The data were collected from one instrument to collect data based on Primary Care Assesment Toll (PCAT) and analyzed by descriptive statistics. The sample consisted of ACS was 87% female. Among the study participants 58% completed high school and 120 months of exercise training (95% CI 111.9 to 129.5) on average. 90% were USF. The average follow-up of cases found were 2 cases of TB since the beginning of the career of the ACS and the last three years the average is presented in a case accompanied. The ACS received satisfactory ratings on the bond of trust with the user, so as access to homes in the community. The ACS reported for denying the fear of being positive result was the biggest reason for not performing the sputum. All units have a professional that responds to the Tuberculosis Control Program. Regarding the structural capacity of primary care settings for the diagnosis of TB, we observed satisfactory levels in different districts of pots for sputum collection, however, a point that deserves attention from managers is lack of materials for packaging sputum. Fear of positive result was one of the reasons for the refusal of sputum collection, followed by alcoholism. With regard to TB suspects, all responded that ACS always suspect when the user has TB coughs, but in all districts were noticed at low delivery of requests for applications for smear. BSR in TB control, is characterized in practice as a complex action goes beyond technical expertise and contact with the family that breaks with the Cartesian. The BSR is part of the ACS can perform them from the daily visits. We conclude that the ACS is difficult to achieve. This practice should not be the privilege of this actor, but the entire team of primary care. We must rethink the practices of TB care, seeing the health surveillance while aegis of the working process of primary care teams for early diagnosis and thereby reduce TB in communities
Uma das prioridades nas pol?ticas de controle da TB refere-se ao diagn?stico precoce da doen?a, nas visitas domiciliares dos Agentes Comunit?rios de Sa?de (ACS), em que se espera oportunamente a identifica??o dos Sintom?ticos Respirat?rios na comunidade. Assim, prop?s-se analisar a busca de sintom?ticos respirat?rios de tuberculose na pr?tica e perspectiva do Agente Comunit?rio de Sa?de nos distritos de Natal. Trata-se de um estudo de corte transversal, sendo realizado inqu?rito com os ACS do munic?pio de Natal/RN. A popula??o do estudo foi constitu?da por ACS inseridos nas Unidades B?sicas de Sa?de e Unidades Sa?de da Fam?lia totalizando 646 profissionais, sendo realizado amostragem de forma probabil?stica aleat?ria, estratificada por distritos, Utilizou-se instrumento de coleta de dados baseado no Primary care Assesment Toll, (PCAT), adaptado para aten??o ? TB por Ruffino-Netto e Villa (2009). O question?rio tem como marco te?rico os atributos da aten??o prim?ria, uma vez que os sistemas de sa?de que se baseiam nesses atributos apresentam-se com melhor desempenho. RESULTADOS: a amostra de ACS foi composta de 87% do sexo feminino. Quanto ao grau de forma??o, 58% conclu?ram o ensino m?dio, em que os ACS apresentaram em m?dia de 120 meses de exerc?cio profissional (IC 95% 111,9 129,5. 90% eram USF. A m?dia encontrada de acompanhamento de casos foi de 2 casos de TB desde o in?cio da carreira profissional do ACS e nos ?ltimos tr?s anos essa m?dia se apresenta de 1 caso acompanhado.Os ACS obtiveram ?ndices satisfat?rios quanto ao elo de confian?a com o usu?rio, assim como o acesso aos domic?lios na comunidade. Os ACS relataram para a recusa o medo do resultado ser positivo foi o maior motivo da n?o realiza??o da baciloscopia de escarro. A maioria dos ACS referiu que se sente preparado para orientar quanto ? coleta de escarro na comunidade. No que se refere ao acesso do usu?rio aos servi?os de sa?de, verificou-se proximidade dos domic?lios aos servi?os de sa?de, entretanto o tempo de espera para o atendimento ? longo. Todas as unidades possuem um profissional que responde pelo Programa de Controle da Tuberculose. Em rela??o a capacidade estrutural das unidades de aten??o prim?ria para o diagn?stico de TB, observou-se nos diferentes distritos n?veis satisfat?rios de potes para a coleta de escarro, no entanto, um ponto que merece aten??o por parte dos gestores ? falta de insumos para o acondicionamento do escarro. O medo do resultado positivo foi um dos motivos para a recusa da coleta do escarro, seguido do Alcoolismo. No que tange ? suspeita de TB, todos os ACS responderam que sempre suspeitam de TB quando o usu?rio apresenta tosse, por?m em todos os distritos foram verificados a baixa entrega de solicita??es de pedidos de baciloscopia. A BSR no controle da TB, caracteriza-se na pr?tica como uma a??o complexa vai al?m das habilidades t?cnicas e o contato com a fam?lia que rompe com o cartesianismo. A BSR ? papel do ACS, podendo realiz?-las a partir das visitas di?rias. Conclui-se que o ACS encontra dificuldades para a consecu??o. Essa pr?tica n?o deve ser privil?gio desse ator, mas de toda a equipe da aten??o b?sica. ? preciso repensar as pr?ticas de aten??o ? TB, vislumbrando a vigil?ncia em sa?de enquanto ?gide do processo de trabalho das equipes da aten??o b?sica, para o diagn?stico precoce e a redu??o da TB nas comunidades

Orientador: José Corrêa de Lacerda Neto
Banca: Raquel Micarelli Albernaz
Banca: Gustavo Garkalns de Souza Oliveira
Banca: Kamila Gravena
Banca: Daniela Gomes da Silva
Resumo: O grande desafio do sistema de defesa respiratório é a manutenção dos animais em baias. Pois, devido a diversos fatores, acaba por aumentar as chances do desenvolvimento de afecções respiratórias e a qualidade do material utilizado como cama é fator agravante. Diante disto, três grupos com 5 equinos cada foram submetidos a 45 dias sob o mesmo manejo, sendo dois grupos estabulados com diferentes tipos de cama, um com maravalha esterilizada (ME) e o outro com maravalha não esterilizada (MNE), e o terceiro grupo a pasto (Pasto). Foram realizadas análises do lavado broncoalveolar (LBA) (celularidade e marcadores de estresse oxidativo) e hemograma antes do início do manejo (Basal) e posteriormente a cada 15 dias (M15, M30 e M45). Também foi avaliada a presença de gêneros fúngicos nas amostras de cama, e do feno utilizado na alimentação dos animais. Os hemogramas permaneceram dentro dos valores da normalidade. Os valores obtidos de malondialdeído (MDA) e ácido úrico no LBA não apresentaram diferença entre os momentos e grupos avaliados. A vitamina C no LBA apresentou queda em seus valores em M30. Os três grupos apresentaram queda nas concentrações de glutationa reduzida em M30, tendo havido diferença significativas entre os grupos Pasto e MNE. Os grupos ME e MNE apresentaram queda nos valores de glutationa oxidada em M45, já o grupo Pasto apresentou queda constante a partir de M15, com diferença significativa em relação ao grupo ME em M30. A superóxido dismutase a... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: The great challenge of the respiratory defense system is the maintenance of animals in stalls, which due to several factors ends up increasing the chances of development of respiratory diseases. The quality of the material used as bed is an aggravating factor. Three groups of 5 horses were submitted to 45 days under the same management, and two groups were housed with different types of beds, one with sterilized wood shaving (ME) and the other with unsterilized wood shaving (MNE), and the third group was maintened in the pasture. Bronchoalveolar lavage fluid (BALF) and hemoglobin analyzes were performed before baseline and then every 15 days (M15, M30 and M45). It was also evaluated the possible presence of fungal genera in bed samples, and in hay used in animal feeding. The hemograms remained within normal values. The values of malondialdehyde (MDA) and uric acid in BALF showed no difference between the moments and groups evaluated. Vitamin C in BALF showed a decrease in M30 values. The three groups showed a decrease in the concentrations of glutathione reduced in M30, and there were significant differences between the groups pasture and MNE. The ME and MNE groups showed a decrease in the values of oxidized glutathione in M45, whereas the pasture group presented a constant drop from M15, with a significant difference in relation to the ME group in M30. Superoxide dismutase increased in M30 in the MNE group, leading to a significant difference in relation to... (Complete abstract click electronic access below)
Doutor

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
O grande desafio do sistema de defesa respiratório é a manutenção dos animais em baias. Pois, devido a diversos fatores, acaba por aumentar as chances do desenvolvimento de afecções respiratórias e a qualidade do material utilizado como cama é fator agravante. Diante disto, três grupos com 5 equinos cada foram submetidos a 45 dias sob o mesmo manejo, sendo dois grupos estabulados com diferentes tipos de cama, um com maravalha esterilizada (ME) e o outro com maravalha não esterilizada (MNE), e o terceiro grupo a pasto (Pasto). Foram realizadas análises do lavado broncoalveolar (LBA) (celularidade e marcadores de estresse oxidativo) e hemograma antes do início do manejo (Basal) e posteriormente a cada 15 dias (M15, M30 e M45). Também foi avaliada a presença de gêneros fúngicos nas amostras de cama, e do feno utilizado na alimentação dos animais. Os hemogramas permaneceram dentro dos valores da normalidade. Os valores obtidos de malondialdeído (MDA) e ácido úrico no LBA não apresentaram diferença entre os momentos e grupos avaliados. A vitamina C no LBA apresentou queda em seus valores em M30. Os três grupos apresentaram queda nas concentrações de glutationa reduzida em M30, tendo havido diferença significativas entre os grupos Pasto e MNE. Os grupos ME e MNE apresentaram queda nos valores de glutationa oxidada em M45, já o grupo Pasto apresentou queda constante a partir de M15, com diferença significativa em relação ao grupo ME em M30. A superóxido dismutase apresentou aumento em M30 no grupo MNE, levando a diferença significativa em relação aos grupos Pasto e ME. A glutationa peroxidase no LBA apresentou queda significativa no grupo ME em M45. Quanto a celularidade do LBA a contagem de células nucleadas totais e eosinófilos não apresentaram diferenças significativas. O grupo ME apresentou elevação nos valores de neutrófilos em M30, levando a diferença significativa em relação aos grupos MNE e Pasto, já o grupo MNE apresentou queda significativa em M45. Os grupos ME e MNE apresentaram queda nos valores de linfócitos no LBA em M30, já o grupo Pasto apresentou queda em M15. O grupo Pasto apresentou aumento dos macrófagos no LBA em M15 e M30. As análises das amostras de cama e feno apresentaram baixas porcentagens de gêneros fúngicos. Grande parte das alterações apresentadas ocorreram em M30, momento em que foram registradas as menores temperaturas e maiores velocidades dos ventos durante todo período experimental, o que possivelmente levou a um desequilíbrio oxidativo pontual, com pequenas variações na celularidade do LBA. Acredita-se que o manejo, as boas condições de higiene e ventilação das baias tenham contribuído para que não houvesse o desenvolvimento de alterações inflamatórias no sistema respiratório dos animais avaliados. Deste modo, podemos concluir que não houve diferenças significativas na manutenção dos animais nos diferentes tipos de cama em relação a resposta inflamatória, estresse oxidativo e desenvolvimento fúngico.
The great challenge of the respiratory defense system is the maintenance of animals in stalls, which due to several factors ends up increasing the chances of development of respiratory diseases. The quality of the material used as bed is an aggravating factor. Three groups of 5 horses were submitted to 45 days under the same management, and two groups were housed with different types of beds, one with sterilized wood shaving (ME) and the other with unsterilized wood shaving (MNE), and the third group was maintened in the pasture. Bronchoalveolar lavage fluid (BALF) and hemoglobin analyzes were performed before baseline and then every 15 days (M15, M30 and M45). It was also evaluated the possible presence of fungal genera in bed samples, and in hay used in animal feeding. The hemograms remained within normal values. The values of malondialdehyde (MDA) and uric acid in BALF showed no difference between the moments and groups evaluated. Vitamin C in BALF showed a decrease in M30 values. The three groups showed a decrease in the concentrations of glutathione reduced in M30, and there were significant differences between the groups pasture and MNE. The ME and MNE groups showed a decrease in the values of oxidized glutathione in M45, whereas the pasture group presented a constant drop from M15, with a significant difference in relation to the ME group in M30. Superoxide dismutase increased in M30 in the MNE group, leading to a significant difference in relation to the pasture and ME groups. Glutathione peroxidase in BALF showed a significant decrease in the ME group in M45. As for the cellularity of BALF, total nucleated and eosinophil counts did not showed significant differences. The ME group presented elevation in neutrophil values in M30, leading to a significant difference in relation to the MNE and pasture groups, whereas the MNE group presented a significant decrease in M45. The ME and MNE groups presented a decrease in lymphocyte values in the BALF in M30, whereas the pasture group presented a decrease in M15. The pasture group presented increase of the macrophages in the BALF in M15 and M30. Bed and hay samples showed low percentages of fungal genera. Most of the alterations presented occurred in M30, at which time the lowest temperatures were recorded throughout the experimental period, possibly leading to a punctual oxidative imbalance, with small variations in BALF cellularity. It is believed that management, good conditions of hygiene and ventilation of the boxes contributed to the no development of inflammatory changes in the respiratory system of the animals evaluated. Thus, we can conclude that there were no significant differences in the maintenance of the animals in the different types of bed in relation to the inflammatory response, oxidative stress and fungal development.

L'apparition de souches muqueuses de pseudomonas aeruginosa, par production d'exopolysaccharides, est responsable du passage a la chronicite des infections a p. Aeruginosa et de l'echec therapeutique. La composition de ces exopolysaccharides, riches en alginate, est heterogene et pourrait expliquer les differences de comportement des souches productrices vis-a-vis des reponses de l'hote. Pour aborder ce probleme, nous avons choisi d'etudier le role de la composition chimique de ces exopolysaccharides a deux niveaux : (1) l'adhesion de p. Aeruginosa aux cellules de l'epithelium respiratoire et (2) la reponse anti-infectieuse des macrophages. Sur les macrophages, nous avons examine la phagocytose de p. Aeruginosa et la production de radicaux libres oxygenes. Nous montrons que les alginates et les polysaccharides neutres, associes dans la composition des exopolysaccharides, ont une activite inhibitrice sur la phagocytose de p. Aeruginosa par les macrophages peritoneaux de souris et l'adhesion aux cellules epitheliales respiratoires. L'adhesion a ces cellules est plus importante pour les cellules de mucoviscidose (mutation