Equine influenza virus causes a mild, self-limiting upper respiratory disease in its natural host. In stark contrast, equine influenza viruses of the H7N7 subtype produce lethal infection in BALB/c mice. This dissertation explored the mechanism underlying the differential pathogenicity of the equine H7N7 influenza virus observed in horses and BALB/c mice. Initially, a comparative study of the pathogenesis was conducted in BALB/c mice inoculated intranasally with a representative isolate of either H7N7 or H3N8 subtype equine influenza virus. All H3N8 virus-infected mice survived the infection whereas 100% mortality was documented for the mice receiving the H7N7 virus by day 8 post infection. Both viruses replicated to a similar degree in the lungs at the early stages of infection. However, after day 2 post infection until the death of the mice, the pulmonary viral loads of the H7N7 group were significantly higher than those of the control, whereas the H3N8 virus was eventually eradicated from the mice at day 7 p.i. Correspondingly, a vigorous pro-inflammatory cytokine response in the lung was induced by the H7N7 virus but not the H3N8 virus, which reflected a desperate attempt by the host immune responses to restrain the overwhelming infection. The H7N7 virus was poorly sensitive to the innate immune containment, resulting in a significant higher cumulative mortality rate than that of the control virus in chicken embryos aged 9 days and older. On the contrary, in horses, replication of the paired viruses was completely cleared by the host immune responses at day 7 p.i. and the infections produced an acute yet non-lethal illness, albeit the H3N8 virus induced generally more pronounced clinical manifestations than the H7N7 virus. The clinical severity correlated to the difference in cytokine-inducing capacity between the two viruses in horses, as evidenced by the finding that the H3N8 virus triggered significantly higher levels of gene transcription of multiple key inflammatory cytokines in the circulation than those seen for the H7N7 virus. In addition, equine peripheral monocyte-derived macrophages were found to be a target of equine influenza virus and can support the productive replication of the virus in vitro.

The mammalian immune system is equipped to both eliminate pathogenic microorganisms and tumors, while remaining in homeostasis with commensal species at mucosal surfaces and tolerant towards self. Suppressor regulatory T cells (Tregs) are a major sentinel of this immunological tolerance. Induced Tregs (iTregs) arise in the periphery following the integration of cues from the metabolites, cytokines, etc. which make up its milieu. Dysregulation of iTreg development, function or homing underlies the etiology of many autoimmune diseases and immunopathologies. The amelioration or prevention of multiple murine disease models by boosting Treg cell numbers foreshadows clinical efficacy of iTreg therapy, but an incomplete understanding of Treg development has thus far prevented successful translation. Therefore, we considered the basic biology of T cell fate decision making from two unique, but integrated angles. First, we show that the stimulation of PPARγ in human T cells upregulates RDH10, a molecule which catalyzes the rate limiting step in the oxidation of retinol to transcriptionally active all-trans retinoic acid (ATRA), a positive regulator of iTreg development. This functionally intact pathway endows T cells the ability to autonomously sense and respond to retinoid signals present during Treg development and at tissue sites. Next, we asked questions about how T cells sense nutrient and oxygen availability as they differentiate. Tregs lacking the serine/threonine kinase PINK1 have limited activation-induced phosphorylation of Akt and oxidative phosphorylation rates, and reduced suppressor function. Notably, the uncoupling of iTreg function from normal FoxP3 expression reinforces the recent hypothesis that the PI3K/Akt/mTORC1 axis and metabolic checkpoints are decisive players in the acquisition of suppressor activity. Ultimately, the studies described herein converge on Akt and metabolism, and contribute to our understanding of how T cells integrate diverse signals present during fate determinism, provoking future Treg based therapeutics.

This thesis presents the development and optimization of a high-throughput fluorescence microbead based approach for multiplexed, large scale medical diagnostics of biological fluids. Specifically, different sizes of semiconductor nanocrystals, called quantum dots, are infused into polystyrene microspheres, yielding a set of spectrally unique optical barcodes. The surface of these barcodes are then used for sandwich assays with target molecules and fluorophore-conjugated detection antibodies, changing the optical spectra of beads that have associated with (or captured) biomolecular targets. These assayed microbeads are analyzed at a single bead level in a high-throughput manner using an electrokinetic microfluidic system and laser induced fluorescence. Optical signals collected by solid state photodetectors are then processed using novel signal processing algorithms. This document will discuss developments made in each area of the platform as well as optimization of the platform for improved future performance.

Herpetic stromal keratitis (HSK) is an immunopathological and tissue destructive corneal lesion caused by herpes simplex virus (HSV) infection, which induces an intense inflammatory response and finally leads to blindness. Accumulating evidence using the murine model has shown that Th-1 phenotype CD4+ T cells orchestrating the inflammation mainly contribute to the immunopathological reaction in HSV-1 infected cornea. Initially various innate immune cells recruit and produce numerous inflammatory and angiogenic molecules into the corneal stroma those in turn drive the corneal immunopathology. While the basic principles of immunity to the influenza A viruses (IAV) are probably similar for all vertebrates, detailed understanding is based largely on experiments in laboratory mice. Virus clearance is normally mediated via CD8+ effector T cells but, in their absence, the class-switched antibody response can ultimately achieve the same goal. Influenza virus-specific plasma cells and CD8+ T cells persist in the long term and the recall of the CD8+ T cell response can lead to earlier virus clearance. The first part (Part I) of this dissertation focuses on the understanding of HSV-1 induced immunoinflammatory processes in the cornea and the secondary lymphoid tissues and the involvement of immuno-modulatory mechanisms following acute viral infections such as HSV and IAV. The next three parts (Part II-IV) focus on different inflammatory and counter-inflammatory mechanisms that are activated following acute viral infections. Results in Part II evaluate the role of small molecule inhibitors of VEGFR2/src kinase inhibitors in controlling the progression of the inflammatory lesions after ocular HSV infection. Results of the third section show that the host counter inflammatory mechanisms inhibit tissue damage but these may also act to constrain the effectiveness of immunity to acute infections. The fourth section describes the functional significance of HVEM expression on regulatory T cell in their expansion following HSV-1 infection. In this study, experiments were designed to understand the mechanisms involved in the regulation of immunity and resultant immunopathology using HSV-1 and IAV as the model systems and that modulation of these processes can enhance immune response and diminish immunopathology following acute infections.

No description available.

BACKGROUND: Infective endocarditis (IE) is an infectious disease, most often bacterial in etiology, which affects the endocardial tissue layer of the heart. Despite advances in diagnostic technology, surgical technique, and antimicrobial therapy, IE remains a high-mortality disease. OBJECTIVE: This is a proposed quality improvement initiative for the Boston Medical Center (BMC) inpatient medicine service. The initiative aims to identify predictors of mortality in patients with IE, and then use the predictors to create a mortality risk-assessment checklist. The checklist will serve as a clinical tool for medicine service providers to help determine if upgrade to ICU level of care is warranted. With early upgrade to an ICU setting, patients with a high risk of mortality will receive more individualized care and expedited medical intervention. The goal of this quality improvement initiative is to decrease mortality rate in patients with IE at BMC. METHODS: This quality improvement initiative will implement the PDSA (plan, do, study, act) model for quality improvement. The checklist will be integrated into the electronic health record system at BMC and will be implemented over a two-year time period. Each PDSA cycle will last one year, and between PDSA cycles the checklist will be modified according to medical provider feedback. The data will be gathered through chart reviews to determine pre and post-checklist differences in number of transfers to the ICU and overall mortality rates of IE patients at BMC. RESULTS: The literature review of this proposed quality improvement initiative has identified nine independent risk factors for mortality in patients with IE: Staphylococcus aureus as infective organism, New York Heart Association class IV heart failure, left ventricular ejection fraction < 40%, vegetation size ≥ 15 mm, age > 50 years, diabetes mellitus, peripheral dermatologic findings on physical examination, serum neutrophil-to-lymphocyte ratio > 5.45, and serum D-dimer level > 4.0 mg/L. CONCLUSION: If medical providers had access to a risk assessment tool to help identify IE patients with a high risk of mortality, they could more accurately determine appropriate level of care, expedite medical intervention, and possibly reduce rates of in-hospital death in patients with IE.

Medical cartography can provide valuable insights into the epidemiology and ecology of infectious diseases, providing a quantitative representation of the distribution of these pathogens. Such methods therefore provide a key step in informing public health policy decisions ranging from prioritising sites for further investigation to identifying targets for interventions. By increasing the resolution at which risk is defined, policymakers are provided with an increasingly informed approach for considering next steps as well as evaluating past progress. In spite of their benefits however, global maps of infectious disease are lacking in both quality and comprehensiveness. This thesis sets out to investigate the next steps for medical cartography and details the use of species distribution models in evaluating global distributions of a variety of zoonotic diseases of public health importance. Chapter 2 defines a methodology by which global targets for infectious disease mapping can be quantitatively assessed by comparing the global burden of each disease with the demand from national policymakers, non-governmental organisations and academic communities for global assessments of disease distribution. Chapter 3 introduces the use of boosted regression trees for mapping the distribution of a group of vector-borne diseases identified as being a high priority target, the leishmaniases. Chapter 4 adapts these approaches to consider Ebola virus disease. This technique shows that the West African outbreak was ecologically consistent with past infections and suggests a much wider area of risk than previously considered. Chapter 5 investigates Marburg virus disease and considers the variety of different factors relating to all aspects of the transmission cycle that must be considered in these analyses. Chapters 6 and 7 complete the mapping of the suite of viral haemorrhagic fevers by assessing the distribution of Crimean-Congo haemorrhagic fever and Lassa fever. Finally, Chapter 8 considers the risk that these viral haemorrhagic fevers present to the wider African continent, quantifying potential risk of spillover infections, local outbreaks and more widespread infection. This thesis addresses important information gaps in global knowledge of a number of pathogens of public health importance. In doing so, this work provides a template for considering the global distribution of a number of other zoonotic diseases.

In regions endemic for malaria, natural exposure results in an acquired immunity which protects individuals from severe disease. However, no vaccine against the blood-stage of malaria, against which naturally-acquired immunity is targeted, currently exists that is capable of emulating, or out-performing, natural protection. To rationally direct the next generation of blood-stage malaria vaccine development, a greater understanding of the immunological mechanisms involved in clinical protection is required. To date, the assessment of naturally-acquired and vaccine-induced immunity to the blood-stage of malaria has suffered from a paucity of in vitro immunological assays that are both robust and reproducible, whilst allowing for assessment of anti-parasitic activity induced by antibodies, either alone, or in conjunction with immune cells. Thus this Thesis describes the development of the antibody-dependent respiratory burst (ADRB) assay, for assessment of blood-stage immunity against Plasmodium falciparum, as well as the Duffy antigen receptor for chemokines (DARC) – Duffy binding protein (DBP) binding inhibition assay, for assessing antibody mediated immunity to P. vivax. A reproducible and standardised assay of ADRB activity was developed here and applied to studies of immunity in both mice and humans. ADRB activity, which assesses antibodies' ability to activate oxidative burst in neutrophils via Fc receptor (FcR)-dependent pathways, was shown to associate with clinical protection in a cohort from Mali where FcR-independent immunological assays, such as the assay of growth inhibition activity, did not. This work thus elucidates the importance of FcR-dependent immunity to P. falciparum malaria and establishes the ADRB assay as a useful tool for future vaccine development. In addition, the DARC-DBP binding inhibition assay was established and utilised to assess inhibitory activity of antibodies induced in the first Phase I clinical trial of this antigen. Results identify the need for significant improvements in vaccine design, and show the utility of the assay as a tool for assessing future blood-stage vaccine development efforts against this neglected parasite.

Made available in DSpace on 2014-06-11T19:27:16Z (GMT). No. of bitstreams: 0 Previous issue date: 2012-06-22Bitstream added on 2014-06-13T19:14:32Z : No. of bitstreams: 1 massa_r_me_jabo.pdf: 283971 bytes, checksum: 0b4319b9b239dc5b274d6820f39d809b (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / O presente estudo teve como objetivo avaliar a influência da brucelose, leptospirose, rinotraqueíte infecciosa bovina (IBR), diarreia viral bovina (BVD) e neosporose no indicador de reprodução mais comumente utilizado por pecuaristas brasileiros, a taxa de concepção (ou taxa de prenhez) após a estação de monta. Dois estudos epidemiológicos analíticos foram feitos: um estudo caso-controle, utilizando uma amostra das matrizes do rebanho que foram submetidas à estação de monta durante o período chuvoso, e um estudo de coortes retrospectivo, utilizando todas as novilhas submetidas à estação de monta durante a estação de “inverno”. A determinação do estado gestacional foi realizada por palpação retal entre 60 e 90 dias após o término da estação de monta. A pesquisa de anticorpos foi realizada pelos seguintes métodos: antígeno acidificado tamponado (AAT) e reação de fixação de complemento (RFC) para brucelose; soroaglutinação microscópica (SAM) para leptospirose; virusneutralização (VN) para IBR e BVD; e reação de imunofluorescência indireta (RIFI) para neosporose. A significância estatística da associação entre a doença e o fracasso na concepção foi avaliada pelos métodos de qui-quadrado ou teste exato de Fisher (p < 0,05) e pelo intervalo de confiança a 95% para o risco relativo (RR) ou a odds ratio (OR). Associações com relação causal possível foram encontradas entre falha na concepção e: infecção considerando qualquer sorovariedade de Leptospira spp. (Título ≥ 400) em primíparas (OR = 7,3636, IC 95%: 1,3373-40,5479; significativo), todas as quais foram infecções causadas por L. Wollfi; e infecção por L. Autmumnalis (título de ≥ 200), considerando todos os animais da estação de monta de “verão” (OR = 8,4058; IC95%: 1,0377 - 68,0882; significativo). Brucelose, neosporose IBR, BVD... / The present study aims to evaluate the influence of brucellosis, leptospirosis, infectious bovine rhinotracheitis (IBR), bovine viral diarrhea (BVD) and neosporosis in the reproductive indicator most commonly used among Brazilian ranchers, the conception rate (or pregnancy rate) after breeding season. Two analytical epidemiological studies were made: a case control study using a sample of matrices of the herd that were submitted to the breeding season during the rainy season, and a retrospective cohort study using all heifers submitted to the breeding season during the dry season. The determination of pregnancy status was performed by rectal palpation between 60 and 90 days after the end of the breeding season. The antibody search was performed by the following methods: rose bengal test and complement fixation test for brucellosis; microscopic agglutination test for leptospirosis, virus neutralization for IBR and BVD, and indirect immunofluorescence assay for neosporosis. The statistical significance of association between the disease and failure in the conception was held by the methods of chi-square or Fisher's exact test (p < 0,05) and by the 95% confidence interval for the relative risk (RR) or odds ratio (OR). Associations with possible causal relationship were found between conception failure and: Infection considering any serovar of Leptospira spp. (titer ≥ 400) in primiparous (OR = 7.3636, 95% CI: 1.3373 to 40.5479; significant), all of which were infections caused by L. Wollfi; and infection by L. Autmumnalis (titer of ≥ 200) considering all the animals of the rainy breeding season (OR = 8.4058; IC95%: 1.0377 – 68.0882; significative). Brucellosis, IBR, BVD, neosporosis and leptospirosis caused by other serovars did not influence the rate of conception in the breeding season... (Complete abstract click electronic access below)

Orientadores: José Dirceu Ribeiro, Renato Tetelbom Stein / Tese (doutorado) ¿ Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-24T01:47:34Z (GMT). No. of bitstreams: 1 Silva_EmersonRodriguesda_D.pdf: 2935794 bytes, checksum: a3a21e8b226c670f1ac8d027e5ed10a1 (MD5) Previous issue date: 2013 / Resumo: Introdução: A infecção do trato respiratório inferior (ITRI) é uma das principais causas de morbimortalidade em crianças, principalmente em países em desenvolvimento e em crianças menores de 3 anos de idade. Os vírus estão entre os principais agentes etiológicos das ITRI em crianças. No entanto, poucos estudos até o momento investigaram o impacto dos vírus respiratórios em populações de crianças de países em desenvolvimento e de que maneira as codetecções de dois ou mais vírus modificam a gravidade das infecções. Objetivo: identificar quais vírus respiratórios causam ITRI em lactentes e crianças até 3 anos de idade hospitalizadas e qual o impacto das codetecções virais sobre a gravidade destes episódios. Métodos: crianças de até 3 anos de idade internados em um hospital terciário no Brasil durante os meses de alta prevalência de vírus respiratórios tiveram amostras coletadas de aspiração nasofaríngea. Estas amostras foram testadas para 13 diferentes vírus respiratórios através de PCR em tempo real (RT-PCR). Os pacientes foram acompanhados durante a internação, e dados clínicos e das características da população foram registradas durante esse período e na alta para avaliar marcadores de gravidade, como tempo de internação e uso de oxigênio. Foi usada análise univariada para identificar potenciais fatores de risco e a regressão logística multivariada para determinar o impacto de detecções virais específicas sobre os desfechos, bem como para avaliar o efeito das codetecções sobre estes mesmos desfechos. Resultados: Foram analisados 260 episódios de ITRI com uma taxa de detecção viral de 85% (n = 222). Codetecção foi observada em 65% de todos os episódios de vírus-positivos. O vírus foi mais prevalente Vírus Sincicial Respiratório (RSV) (54%), seguido por Metapneumovirus humano (hMPV) (32%) e Rinovírus humano (HRV) (21%). Nos modelos multivariados, lactentes com codetecção de HRV + RSV permaneceram 4,5 dias a mais no hospital (p = 0,004), quando comparados ao grupo sem a codetecção. A mesma tendência foi observada para o número de dias de uso de oxigênio suplementar. Conclusões: Embora RSV permaneça como a principal causa da ITRI em crianças, mostrou-se um aumento no tempo de internação e uso de oxigênio em crianças com RSV e HRV codetectados por RT-PCR em comparação com aqueles com RSV mas sem HRV em codetecção. Além disso, nosso estudo identificou um número significativo de crianças infectadas por vírus recentemente identificados, tais como hMPV e bocavirus Humano (HBoV), e este é um achado relevante para comunidades pobres de países em desenvolvimento / Abstract: Introduction: lower respiratory tract infection (LRTI) is a major cause of morbidity and mortality in infants and children, especially in developing countries and in children under 3 years old. Viruses are among the major etiologic agents of LRTI in children. However, few studies to date have investigated the impact of respiratory viruses in populations of children in developing countries and how the co-detections of two or more viruses modify the severity of infections. Objective: To identify respiratory viruses in infants and children up to 3 years of age hospitalized due to LRTI and verify the impact of viral co-detections on the severity of these episodes. Methods: Children less than 3 years of age admitted to a tertiary hospital in Brazil during the months of high prevalence of respiratory viruses had collected samples of nasopharyngeal aspirate. These samples were tested for 13 different respiratory viruses by real-time PCR (RT-PCR). Patients were followed during hospitalization, and clinical and population characteristics were recorded during this period and at discharge to assess markers of severity, such as length of stay and use of oxygen. Univariate analysis was used to identify potential risk factors and multivariate logistic regression was used to determine the impact of specific viral detections on outcomes and to evaluate the effect of co-detections on these same outcomes. ix Results: We analyzed 260 episodes of LRTI with an overall viral detection rate of 85% (n = 222). Co-detection was observed in 65% of all virus-positive episodes. The most prevalent virus was Respiratory Syncytial Virus (RSV) (54%), followed by human metapneumovirus (hMPV) (32%) and human rhinovirus (HRV) (21%). In multivariate models, infants with co-detection of HRV + RSV stayed 4.5 days longer in the hospital (p = 0.004), when compared to the group without this co-detection. The same trend was observed for the number of days using supplemental oxygen. Conclusions: Although RSV remains the leading cause of LRTI in children, our study has shown an increase in the length of stay and use of oxygen in children with RSV and HRV co-detected by RT-PCR in comparison with those with RSV alone. Furthermore, our study identified a significant number of children infected by viruses recently identified, such as hMPV and Human bocavirus (HBoV), and this is an important finding for poor communities in developing countries / Doutorado / Pediatria / Doutor em Saude da Criança e do Adolescente