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Protein-protein interactions play an important role in nearly all processes of the living cells and the function of many proteins is dependent on their specific interactions with other biomolecules. A reliable tool to modulate these interactions would be invaluable for the development of molecules suitable for diagnostics, medicine, and biotechnology. In this work, we aimed to study the specificity of interactions in the model system of Interferon gamma receptor 1 (IFNgR1) and its natural ligand Interferon gamma (IFNg), important in innate immunity. We searched for mutations within the interferon receptor molecule IFNgR1 to modulate (increase as well as decrease) its affinity to IFNg by in silico analysis of the existing crystal structures of the complex between IFNgR1 and IFNg. We modeled amino acid substitutions and gauged how they influenced the interaction using empirical force field implemented in software FoldX. All selected promising IFNgR1 variants were expressed in Escherichia coli, purified to homogeneity, characterized, and kinetics of their interactions with IFNg was measured by Surface Plasmon Resonance (SPR). The first set of IFNgR1 variants included mutations on the interface of the IFNg/IFNgR1 complex. According to our SPR measurements, the affinity of most of these receptor...

Orientador: Nicola Amanda Conran Zorzetto / Dissertação (mestrado) - Universidade Estadual de Campinas. Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-15T08:53:01Z (GMT). No. of bitstreams: 1 Miguel_LedianaIagalo_M.pdf: 1386194 bytes, checksum: b81f5aa45e96b255588df48747c1397c (MD5) Previous issue date: 2010 / Resumo: A adesão anormal das células brancas e vermelhas ao endotélio, que desencadeia numa diminuição do fluxo de sangue na microcirculação, é um dos principais fatores envolvidos na iniciação da vaso-oclusão em pacientes falciformes (AF). O estado inflamatório crônico, característico nos pacientes com AF, eleva a circulação de citocinas, as quais podem contribuir significativamente para a ativação e adesão das células vermelhas e brancas ao endotélio. O óxido nítrico (NO) e a via de sinalização dependente em NO têm importante efeito inibidor nas propriedades adesivas de leucócitos. Drogas que aumentem a biodisponibilidade de NO ou que atuem na via de sinalização NO-GMPc podem ser benéficas no tratamento de alguns aspectos da AF. Já é de conhecimento que pacientes com AF apresentam níveis elevados de algumas citocinas presentes no plasma, assim sendo, este estudo teve como objetivo avaliar os efeitos in vitro das citocinas nas propriedades adesivas de neutrófilos e células vermelhas de indivíduos controles e pacientes com AF. Adicionalmente, foram determinados os efeitos de BAY 73-6691, um inibidor da enzima hidrolizante de GMPc, fosfodiesterase 9A (PDE9A) e BAY 41-2272, um ativador de guanilato ciclase, na ausência ou presença da estimulação pelas citocinas na adesão dessas células. Os neutrófilos e as células vermelhas de indivíduos controles e pacientes com AF foram isolados de sangue periférico. A adesão das células à fibronectina foi determinada utilizando o ensaio de adesão estático na presença ou ausência das citocinas IL-8 (10-500ng/ml), TNF-alpha (10-100ng/ml) e GM-CSF (0,1-10ng/ml) e/ou na presença/ausência de BAY 73-6691 (60µM), BAY 41-2272 (60nM) ou DMSO como veículo (0.2%v/v). Como previamente demonstrado, os neutrófilos de pacientes com AF (neutrófilos AF) possuem uma maior capacidade de aderir à FN do que os neutrófilos de indivíduos controle (neutrófilos CON). A estimulação das células in vitro com as três citocinas aumentaram significativamente as adesões à FN dos neutrófilos CON e aumentou ainda mais a adesão dos neutrófilos AF. A incubação de ambos os neutrófilos, CON e AF, com BAY 73-6691, mas não BAY 41-2272, reduziu significativamente as propriedades adesivas à FN; esse evento foi acompanhado por uma diminuição da expressão das moléculas de adesão, L-selectina e CD11b (subunidade Mac-1) na superfície de neutrófilos AF. Além do mais, nas concentrações utilizadas, BAY 73-6691, mas não o BAY 41-2272, diminui significativamente a adesão de neutrófilos CON e AF após estimulação com IL-8, TNF-a e GM-CSF. No entanto, esse evento não foi acompanhado por alterações na expressão da moléculas de adesão na superfície de neutrófilos AF quando estimulados com IL-8. As células vermelhas de indivíduos AF também apresentaram uma maior capacidade de se aderir à FN quando comparadas às células de indivíduos controles. No entanto, ao contrário dos neutrófilos, na presença de IL-8 (10-500ng/ml) e TNF-a (0.1-1µg/ml), não houve alteração das propriedades adesivas dessas células tanto de indivíduos controles quanto das células de pacientes com AF. Além disso, BAY 73-6691 e BAY 41-2272, não alteraram a adesão basal tanto das células vermelhas de controles quanto pacientes com AF. Os principais mediadores inflamatórios, utilizados em concentrações fisiologicamente relevantes, foram capazes de aumentar as propriedades adesivas de neutrófilos, mas não das células vermelhas, de indivíduos controles e AF. Portanto, sugerimos que as citocinas inflamatórias circulantes podem desempenhar um papel na indução das propriedades adesivas dos neutrófilos em pacientes falciformes; em contrapartida, outros fatores além do estímulo inflamatório, podem ser mais importante para induzir a adesão das células vermelhas de pacientes AF. Dados sugerem que agentes que aumentam os níveis de GMPc intracelular podem ser úteis para reduzir as propriedades adesivas de neutrófilos AF, mesmo na presença de um estado inflamatório. PDE9A é altamente expressa pelas células hematopoiéticas e a inibição desta enzima, com conseqüente elevação de GMPc, pode representar um alvo terapêutico para drogas que são tecido/célula específicas, necessitando de mais estudos in vivo e in vitro para a terapêutica de AF / Abstract: The adhesion of both red and white cells to the vessel walls of the microcirculation initiates vaso-occlusion in sickle cell disease (SCD). The chronic inflammatory nature of SCD leads to elevation of circulating cytokines in patients, which may contribute significantly to the activation of red and white cells and their consequent adhesion. Nitric oxide (NO) and the NO-dependent signaling pathway have important inhibitory effects on cellular adhesive properties. Drugs that enhance NO bioavailability or NO-cGMP-dependent signaling may hold potential for treatment of various aspects of SCD. It is known that levels of certain cytokines are augmented in the plasma of SCD individuals; therefore, this study aimed to observe the effect of cytokines, on the in vitro adhesive properties of neutrophils (neu) and red blood cells (RBC) from healthy control (CON) and steady-state SCD (SCD) individuals. Furthermore, the effects of BAY 73-6691, an inhibitor of the cGMP-hydrolyzing enzyme, phosphodiesterase 9A (PDE9A) and BAY 41-2272, a guanylate cylase activator, on non-stimulated and cytokine-stimulated cell adhesion were determined. Neutrophils and red blood cells (RBC) were isolated from the peripheral blood of CON and SCD individuals. Cell adhesion to immobilized fibronectin was assessed using static adhesion assays in the presence or absence of the cytokines, IL-8 (10-500ng/ml), TNF-alpha (10-100ng/ml) and GM-CSF (0,1-10ng/ml) and/or in the presence/absence of BAY 73-6691 (10-60µM), BAY 41-2272 (60nM) or DMSO vehicle (0.2%v/v). As previously demonstrated, SCDneu have a greater capacity to adhere to FN than CONneu. Stimulation of cells in vitro with all three cytokines significantly augmented both CONneu adhesion to FN and further increased SCDneu adhesion. The incubation of both CONneu and SCDneu with BAY 73-6691, but not BAY 41-2272, significantly reduced their adhesions to FN; this was accompanied by a decrease in the expressions of the L-selectin and CD11b (Mac-1-subunit) adhesion molecules on the SCAneu surface. Furthermore, BAY 73-6691, but essentially not BAY 41-2272, significantly inhibited CONneu and SCDneu adhesion stimulated by IL-8, TNF-alpha and GM-CSF. However, this was not accompanied by alterations in adhesion molecule presentation on IL-8-stimulated SCAneu. As previously reported, SCD RBC have a greater capacity to adhere to FN, in vitro, compared to CON RBC. However, in contrast to neutrophils, cytokines IL-8 (10-500ng/ml) and TNF-alpha (0.1-1µg/ml) did not alter the capacities of neither CON RBC nor SCD RBC to adhere to FN. Furthermore, BAY 73-6691 and BAY 41-2272 did not affect either basal CON RBC or SCD RBC adhesion. Key SCD inflammatory mediators were found, at physiologically relevant concentrations, to augment the adhesive properties of neutrophils from control and SCD individuals. Circulating inflammatory cytokines may play a role in the induction of leukocyte adhesive properties in SCD; in contrast factors other than inflammatory stimuli may be more important for induction of SCD RBC adhesion. Data suggest that elevation of intracellular cGMP may be an important approach for reducing SCD leukocyte adhesive properties, even in an inflammatory environment. PDE9A is highly expressed in hematopoietic cells and inhibition of this enzyme, with consequent augmentation of cGMP, may represent a tissue/cell-specific therapeutic drug target worthy of further in vitro and in vivo studies as a therapy for SCD / Mestrado / Ciencias Biomedicas / Mestre em Ciências Médicas

Interaction between mouse dendritic cells (DCs) and Borrelia afzelii spichochetes was studied on three different levels: phagocytosis of borrelia by DCs, production of cytokines by borrelia-activated DCs and the ablilty of DCs to activate CD4+ T cells. The effect of Ixodes ricinus saliva on each of these levels was examined. Tick saliva was shown to decrease the number of phagocosing DCs. The ability of borrelia-activated DCs to induce both proliferation and IL-2 production in specific CD4+ T cells was significantly reduced by tick saliva. And surprisingly, we have shown an inhibitory effect of I. ricinus saliva on the production of both Th1 (IL-6 and TNF-{$\alpha$}) and Th2 (IL-10) cytokines. Our data reveal a complex inhibitory effect of tick saliva on DC function.

Dysregulation between pro- and anti-inflammatory cytokines activity in rheumatoid arthritis (RA) contributes to immune dysregulation, chronic inflammation and subsequent joint destruction. Interleukin-37 (IL-37) has been described as an anti-inflammatory cytokine in several autoimmune diseases. The main aim of this work was to determine the levels of IL-37 in serum and synovial fluid (SF) of RA patients and to compare them with the levels in patients with osteoarthritis (OA) and further explore the association of IL-37 with disease activity and other clinical parameters. Subsequent goal was to study its anti-inflammatory function on RA synovial fibroblasts and describe other cells types of synovial tissue contributing to its production. IL-37 levels were detected using enzyme-linked immunosorbent assay (ELISA). Synovial fibroblasts were stimulated by lipopolysaccharide (LPS) and recombinant IL-37 (rIL-37). The levels of studied genes were detected by PCR. Synovial tissues and immune cells were visualized by immunohistochemical and by immunofluorescence staining. We found increased levels of IL-37 in SF of patients with RA in comparison to OA patients. There was a significant correlation between serum and SF levels of IL-37. RA as well as OA patients showed increased levels of IL-37 in serum than in...

Cutaneous melanoma is a very aggressive cancer with increasing incidence. It originates from transformed pigmented skin cells (melanocytes). The main risk factor for melanoma development is exposure to UV light and repeated sunburns. In approximately 10 % of cases, melanoma occurs on hereditary basis. Patients with cutaneous melanoma diagnosed in early stages have very good prognosis, with surgical resection of the primary tumour being mostly sufficient for treatment. In contrast, the advanced melanoma stages with metastases are often progressive and refractory to conventional therapies. Cutaneous melanoma is referred to as an immunogenic tumour that is frequently infiltrated by cells of the immune system. Tumours with immune cell infiltration show better prognosis. Spontaneous regression may occur. Over the last few years, progress has been made in the treatment of melanoma using checkpoints molecules (anti-CTLA-4 and anti-PD-1) to activate patients own immune system to recognize tumour lesions. In the tumour microenvironment, cytokines play an important role, enabling communication between cells and regulation of cell proliferation and migration and thus the tumour development. Cytokines (IL-2, IFNα) can be used in adjuvant therapy of melanoma. This work analysed levels of expressed cytokines in...

Background: Interactions between the neuroendocrine and immune system play an importatnt role in maintaining homeostasis. This communication is mediated by cytokines, neurotransmiters and hormones through endocrine, paracrine and autocrine signaling. Prolactin (PRL), hormone of anterior pituitary, is produced by a number of other tissues and cells of immune system. On periphery, PRL is cytokine. Sepsis is an inflamatory response of the organism to severe infection, Th1 immune response is activated and PRL could participate in it. Toll-like receptors (TLR) play a key role in a recognition of bacteial components and mediate a systemic response (with PRL secretion) during infection. It is supposed that activated immune system leads to increasing of PRL, TLR2 and TLR4 gene expression. We detected PRL, TLR2 a TLR4 mRNA levels in monocytes from patiens with system inflammation. We studied influence of single nucleotide polymorphism (SNP -1149 G/T) in PRL gene promotor, it supposed that G allele increases PRL expression. Materials and Methods: For the pilot study 30 patients diagnose with severe infectious event. Collectoin of patiens blood samples was performed consequently three times. Control group comprised 40 healthy individuals. One blood sample was taken from each healthy subject. For testing of...

Teblad innehåller en mängd substanser som på olika sätt kan påverka biologiska processer i människan. I föreliggande arbete har en sammanställning gjorts utifrån vetenskapliga artiklar över teets antagna effekter på människans immunsystem .Främst har den mest studerade komponenten i te, epigallocatechin gallate, EGCG, undersökts. De variabler som studerats i immunförsvaret är neutrofiler, tumörnekrosfaktor-α, interferon-γ, interleukin-1β, interleukin-2, interleukin-5, interleukin-6, interleukin-8, interleukin-10 ,interleukin-12, interleukin-13, eikosanoidmetabolismen, lymfocyter, makrofager, dendritiskaceller och T-cellsdifferentiering. Sammafattningsvis föreföll te hämma följande: Neutrofilaktiviteten, TNF-α-bildningen, IFN-γ-aktiviteten, stimuli-inducerad IL-β-bildning, IL-8-aktivering och IL-10-bildning. EGCG visades öka dendritiska cellers apoptos och ändra dedenderitiska cellernas morfologi och minska deras förmüga att stimulera T-cellproliferationen. EGCG-behandling av celler ledde till ökad makrofagaktivitet, ökad produktion av T-regceller kontra andra T-cellslinjer. Tebehandling ökade bildningen av IL-12 ochIL-13. EGCGs effekt på IL-6 var tvetydig; enligt en del artiklar påverkade EGCG ej IL-6, medan det enligt andra artiklar antingen ökade eller minskade IL-6-bildningen. Trots att resultaten ej var entydiga kunde slutsatsen att EGCG (och därmed te) utövade immunsupprimerande effekt dras men att det sannolikt krävs betydligt högre koncentrationer än de som är fysiologiskt uppnåbara (1 μM i plasma och 3 mM i tarmen) genom reguljärt tedrickande för att få en avgörande effekt på människans immunsystem. EGCGs hämmnade effekt på immunsystemet kan dock vara av intresse i högre – farmakologiska – koncentrationer genom att EGCG skulle kunna nyttjas som komplement eller alternativ till för närvarande nyttjade immunförtryckande läkemedel som ciklosporin och glukokortikoider.

Hematopoiesis is a precisely regulated process, dependent on the activity of hematopoietic cytokines and their receptors. Due to an extra round of whole genome duplication in teleost fish, two paralogs of many important genes, including some hematopoietic cytokines and their receptors, are present in the zebrafish (Danio rerio) genome. In this project, we have been investigating the role of zebrafish Kit ligands in hematopoiesis. Kit ligand is a pleiotropic cytokine, which is essential for vertebrate erythropoiesis; however, in zebrafish, no such role has been reported so far. To determine the function of zebrafish paralogs of Kit ligand (Kitlga and Kitlgb) in hematopoiesis, we performed in vivo and ex vivo gain- and loss-of-function experiments. Strikingly, we were the first to report the synergistic cooperation of zebrafish Kitlga with erythropoietin and dexamethasone, enabling the growth of kidney marrow-derived suspension cells and providing optimal conditions for the expansion of adult erythroid progenitors. We assume that by using different cytokine combinations, optimal conditions for the growth of other hematopoietic cell types can be established, and therefore, this new approach now available for the...