Orientadores: Simone Appenzeller, Lilian Tereza Lavras Costallat, Roberto Marini / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-22T13:05:21Z (GMT). No. of bitstreams: 1 Pelicari_KarinadeOliveira_M.pdf: 707715 bytes, checksum: 41d920f7671a64c2ce3b93fd4dbcdf3f (MD5) Previous issue date: 2013 / Resumo: Lúpus Eritematoso Sistêmico (LES) e uma doença autoimune, crônica e mutissistemica, caracterizada por períodos de atividade e remissão. Anormalidades hematológicas e imunológicas são comumente encontrados. A avaliação laboratorial, incluindo o perfil de citosinas, auxilia no diagnostico e na determinacao da atividade da doença. O presente estudo, de característica transversal, teve como objetivo avaliar os níveis de IL- 17 em pacientes com LESj (inicio da doença ?16 anos), familiares de primeiro grau e controles sadios e ainda elucidar sua associação com a atividade da doença, dados laboratoriais e de tratamento. Foram selecionados pacientes consecutivos com LESj acompanhados na Unidade de Reumatologia Pediátrica da UNICAMP entre 2009/2011. Manifestações clinicas, laboratoriais, atividade da doença [SLE Disease Activity Index (SLEDAI)], dano cumulativo [Lúpus International Collaborating Clinics / American College of Rheumatology Damage Index (SDI)] e medicação em uso foram avaliados. Os transtornos de humor foram determinados através dos inventários de Depressão (BDI) e Ansiedade (BAI) de Beck. A dosagem da citosina foi realizada por ELISA. Níveis séricos de IL-17 estavam aumentados no LESj (p?0,05) quando comparado controles sadios. Observamos uma associação entre os níveis séricos de IL-17 e migranea (p = 0,03) e nefrite (p = 0,01). Níveis de IL-17 não foram associados com atividade da doença (p = 0,32), dano cumulativo (p = 0,34), medicação (p = 0,63), ansiedade (p = 0,42) e depressão (p = 0,42). Estudos longitudinais sao necessários para determinar se a dosagem de IL-17 pode ser utilizado como um biomarcador em LESj / Abstract: Systemic lupus erythematosus (SLE) is a chronic, multisistemic autoimmune disease, , characterized by periods of activity and remission. Haematological and immunological abnormalities are commonly observed. Laboratory evaluation, including the cytokine profile, aids in the diagnosis and determination of disease activity. The present transverse study, we aimed to evaluate levels of IL-17 in patients with childhood-onset SLE (cSLE) (disease onset ? 16 years), first-degree relatives and healthy controls and to determine the association between IL-17 levels and disease activity, laboratory data and treatment. We selected consecutive patients with cSLE followed at the Pediatric Rheumatology Unit at UNICAMP between 2009/2011. Clinical, laboratory, disease activity [SLE Disease Activity Index (SLEDAI)], cumulative damage [Systemic Lupus International Collaborating Clinics / American College of Rheumatology Damage Index (SDI)] and medication use were assessed. Mood disorders were measured using the Beck's Depression Inventory (BDI) and Anxiety Inventory (BAI). Determination of cytokine levels was carried out by ELISA. Serum levels of IL-17 were increased in SLE (p ? 0.05) when compared to healthy controls. An association was observed between serum IL-17 levels and migraine (p = 0.03) and nephritis (p = 0.01). IL-17 levels were not associated with disease activity (p = 0.32), cumulative damage (p = 0.34), medication (p = 0.63), anxiety (p = 0.42) and depression (p = 0.42). Longitudinal studies are needed to determine if serum IL-17 levels can be used as a biomarker in SLE / Mestrado / Clinica Medica / Mestra em Ciências

No description available.

Antibodies to tropomyosin, cardiac troponin I, myosin, and the beta-adrenergic receptors have been implicated in myocarditis, dilated cardiomyopathy, and heart failure. However, in systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA), there are only a few studies on how autoantibodies play a role in autoimmune mediated heart disease, despite the prevalence of these conditions. Ro52 antibodies have been implicated in fetal heart block, but their role in adult myocarditis remains elusive. In this study, we look beyond Ro52 and characterized the relevant autoantibodies in adult patients with SLE and RA myocarditis. An optimized immunoprecipitation followed by liquid chromatography mass spectrometry methodology was performed to determine putative auto-antigens in the human heart. The quantity and specificity of auto-antibodies was correlated with clinical measures of myocardial cellular infiltration, as determined by fluorodeoxyglucose (FDG)-positron emission tomography (PET) in patients with SLE and RA. We created autoantibody profiles that are complimentary to SLE and RA patients' clinical profile. Autoantibodies that correlated with cellular infiltration included TPI1, TPM1, MYL2, XRCC6 and APOA4. We then explored methodologies for testing patient autoantibodies using engineered cardiac tissues derived from human induced pluripotent stem cells (iPSCs). These tissues are increasingly used for drug discovery, pharmacology and in models of development and disease. While there are numerous platforms with engineered cardiac tissues, they often require expensive and non-conventional equipment and utilize complex video processing algorithms. As a result, only specialized academic labs have been able to harness this technology. In addition, methodologies and tissue features have been challenging to reproduce between different groups and models. Here, we describe a facile technology (milliPillar) that covers the entire pipeline required for studies of engineered cardiac tissues: (i) platform fabrication, (ii) cardiac tissue generation, (iii) electrical stimulation, (iv) automated real-time data acquisition, and (v) advanced video analyses. We validate these methodologies and demonstrate the versatility of the platform by showcasing the fabrication of tissues in different hydrogel materials and by using cardiomyocytes derived from different iPSC lines in combination with different types of stromal cells. We also validate the long-term culture (100 days) of tissues within the platform and provide protocols for automated analysis of force generation and calcium flux using both brightfield and fluorescent imaging. Lastly, we demonstrate the compatibility of the milliPillar platform with electromechanical stimulation to enhance cardiac tissue function. milliPillar tissues were cultured in the presence of patient autoantibodies to recapitulate the phenotype of myocardial disease, and the calcium transients and force generation were measured. Our results indicated that milliPillar tissues exhibited a decrease in force generation after 6 days in culture with SLE autoantibodies. Separately, our results indicated a prolonged calcium transient after 7 days in culture with SLE and RA autoantibodies. Changes to the downstroke of the calcium transient correlated most with patients’ autoantibody profiles and cellular infiltration. We confirmed autoantibody binding to live tissues/cells in 25% of the patients with SLE and myocarditis. Finally, we used changes in cardiac tissue function in the presence of autoantibodies to classify patients with SLE myocarditis with an accuracy of 87.5%.

Immune system functionality and lymphocyte activity are gaining traction as a relevant therapeutic source for potentially addressing diseases such as cancer and autoimmune disorders. One such promising technique, adoptive cell therapy, revolves around successful ex vivo T cell activation and the ability to elicit a specific immune response. Key studies have recently suggested that mechanical forces play an important role in the ability of T cells to expand and proliferate and that T cell activation is sensitive to the mechanical properties of activating substrates. T cells initiate adaptive immune responses through interactions with antigen presenting cells (APCs). When T cells interact with APCs, they form the immune synapse, a multistep process that leads to downstream signaling and cellular function. Previous research has suggested that this process is both dynamic and mechanically sensitive. Gaining insight into the mechanisms through which T cells carry out mechanosensing and the associated effector functionalities will be advantageous in developing approaches for controlling T cell activation through mechanics and will allow for more accurate and efficient methods of promoting cell expansions for targeted therapies. This dissertation serves to generate a new mechanically dynamic 3D system to be utilized towards these understandings and contribute to the fields of immunology and mechanobiology. We first establish the development of a novel variable rigidity system actuated by magnetic field application. Validation experiments conclude that this device provides rapid, dynamic, and reversible control of substrate rigidity, without affecting the physical or biochemical properties of the system. The novel system is first used to explore mechanistic activity of T cells during activation in the face of a dynamic biomechanical environmental; we discover that T cells modulate the deflection and protrusive nature of their physical behaviors towards their targets in response to variable rigidity changes. We then utilize the magnetically driven system to characterize the biological mechanisms involved in these mechanosensitively associated behavior phenotypes. We demonstrate that activation patterns of T cells, defined by cytokine secretion profiles and TCR stimulation, correspond with varying cellular deformation directionality of activating substrates of variable increasing rigidity. In this process we discover a possible rigidity threshold upon which TCR triggering is sustained. Furthermore we reveal cytoskeleton components associated with identified mechanosensitive behaviors that cells produce in response to dynamic biomechanical cues. Together this work highlights the dynamic physicality and biomechanical mechanisms of T cell activation in response to a variable rigidity environment. These conclusions reveal insights into T cell mechanosensing activity within the natural mechanically complex atmosphere of the body. Encompassing those understandings, this thesis will help address current scientific gaps between mechanobiology and immunology and advance the biomechanical parameters of cell expansion driven adoptive immunotherapies.

No description available.

Thesis: M.S., Massachusetts Institute of Technology, Department of Nutrition and Food Science, 1980 / Bibliography: leaves 56-57. / by Julianne Elizabeth Radkowski Opperman. / M.S. / M.S. Massachusetts Institute of Technology, Department of Nutrition and Food Science

The autoimmune disease known as autoimmune thrombocytopenic purpura (ITP) is clinically defined by a low numbers of platelets in the circulation blood. Anti-platelet antibodies bind to glycoprotein molecules on the membranes of platelets and result in their dysfunction and destruction. Despite a growing body of information about ITP, it is difficult to isolate and characterise anti-platelet antibodies, because only limited monoclonal antibodies are available from ITP patients. This study used a phage display system to recognise Fab anti-platelet antibodies. Anti-platelet Fab-expressing phage was isolated by sequential panning of an ITP Fab library against normal non-ITP platelets. After isolation, the anti-platelet Fab-expressing phage was characterised by ELISA and Western blotting. The Fab-bearing phage pool obtained from five rounds of panning was analysed in order to determine its anti-platelet reactivity. Of the phage colonies obtained, 100 colonies of different sizes were randomly selected for reaction with whole platelets, using Ml3 phage as a negative control. 12 colonies of them had strong reactions against the whole platelet preparation, but only four colonies showed substantial reactivity against the lysed platelet preparation (lysate). Colony S7 showed highest the greatest degree of binding to both the lysate and the whole platelet preparation. The specificity of the four colonies (S2, S7, S8 and S9) that had strong positive reactions against platelet antigens was determined for the glycoprotein component GP Ilb/IIIa. Further characterisation of the proteins in the lysate preparation was carried out using blotting techniques. The protein content of the four Fab-bearing phage colonies was quantified under the non-reducing conditions of Western blotting to evaluate their ability to recognise platelet antigens. Three of the four colonies showed three bands representing proteins with different molecular weights. Each of these three colonies had one band that corresponded to a protein of molecular weight 92 kD. The fourth colony showed only a single band, but this band also corresponded to a 92-kD protein.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2005. / Härtill 6 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2006. / Härtill 4 uppsatser.