No description available.

No description available.

No description available.

Much of the research that has been done on the fish immune system has focussed on innate immunity. Very little is known about the adaptive immune system of fish and how it is regulated. This study has identified and characterised key cytokines that had not been found in any teleost species to date involved in T helper 2 (Th2) and T helper 17 (Th17) responses. By exploiting the synteny that exists between the human and zebrafish genomes two IL-4 homologues were identified, and their full length cDNA cloned and sequenced, which were termed IL-4 like (IL-4L) and IL-4 related (IL-4rel). This work describes the finding of what we consider to be the actual IL-4 homologue in fish and establishes reasons why the already published ‘IL-4’ gene in Tetraodon (Li, Shao et al. 2007b) may not in fact be the actual IL-4 homologue of mammals but an IL-4 related (IL-4rel) gene which we have also been able to locate within the zebrafish genome. To help with determining the identity of these genes within fish, recombinant proteins of the IL-4L and IL-4rel genes were produced in zebrafish and the IL-4rel molecule in trout. Preliminary expression studies have displayed some evidence of bioactivity in the zebrafish IL-4L and trout IL-4rel proteins, through their impact on the regulation of various immune related genes. These include IL-1β, IFN-γ, the IFN-γR1 and the IL-4 receptor-α chain. This work has also identified and characterised for the first time in any lower vertebrate, the zebrafish IL-23 p19 subunit, which is very important in Th17 responses. In addition the p19 sequence has been predicted from other available fish genomes (stickleback, Fugu, Tetraodon) for comparison. Expression analysis of this subunit along with the p40 subunit within a zebrafish disease model for tuberculosis shows up regulation of this gene after 1 day.

Abstract Mortality of patients requiring intensive care treatment for greater than 5 days has been shown to be about 20% worldwide. Hyperglycaemia is common in critically ill patients. Strict glucose control with insulin in critically ill patients was shown to reduce mortality and morbidity significantly. Several interrelated mechanisms are involved in the development of “stress hyperglycaemia” in critically ill patients. These include dextrose containing intravenous infusions and total parenteral nutrition; the counter regulatory hormones (catecholamines, cortisol, glucagon and growth hormone) which oppose the effects of insulin; nervous system signaling; increased insulin clearance; and excess production of cytokines that interfere with intracellular insulin signaling pathways. Aim of study: To determine if the cytokines TNFα, IL-6 and adiponectin are significant determinants of insulin resistance in critically ill patients. Methods: The study was a prospective observational study conducted in the intensive care unit (ICU) at the Chris Hani Baragwanath hospital. Forty sequential adult ICU admissions that met with the inclusion criteria were enrolled. Blood specimens were drawn for adiponectin, TNF, and IL-6 at the time of ICU admission, on day 3, day 7 and on discharge from the ICU. Demographic data and clinical data were recorded, and body mass index (BMI) and APACHE II scores were calculated on admission. Blood glucose was measured every 2 to 4 hours, recorded and a mean value was calculated over the 24 hour period. Insulin infusions were started when the blood glucose values exceeded 6.0mmol/l. Administration of insulin was according to a fixed sliding scale. The total amount of insulin administered intravenously over that 24 hour period was recorded. Other factors known to be related to insulin sensitivity, such as inflammation (as indicated by C-reactive protein), vii triglycerides, insulin, C-peptide and cortisol levels were also drawn in addition to the blood drawn for routine investigations. Results: Duration of stay in ICU correlated with severity of illness as assessed by the APACHE II score (r = 0.44, p = 0.004). There was no significant difference in the mean 24 hour plasma glucose concentration throughout the duration of stay in ICU, there were however significant differences in the amount of insulin administered to maintain normoglycaemia. The amount of administered insulin required was found to peak on day 3 and decline thereafter. The main determinant of insulin administered was mean glucose (r = 0.79, p < 0.00001). The measured insulin concentrations on admission correlated with mean plasma glucose (r = 0.41, p = 0.009) and C-peptide (r = 0.45, p = 0.004) levels. The main determinants of mean plasma glucose levels on admission were BMI (r = 0.38, p = 0.013) and serum cortisol (r = 0.41, p = 0.008) levels. Serum triglycerides levels showed a significant difference from admission to discharge, with values increasing from admission levels. Adiponectin levels showed a significant increase from admission to discharge. IL-6 levels showed a significant decrease. TNFα levels did not show statistically significant changes. No statistically significant correlations were found between the levels of TNFα or IL-6 and administered insulin. Adiponectin concentrations showed a negative correlation with amount of administered insulin on discharge (r = -0.457, p = 0.0049). There were significant gender differences in BMI, administered insulin on admission, serum cortisol and C-peptide concentrations, with females having higher values than males. BMI was shown to account for the gender differences in administered insulin and C-peptide levels. viii There were significant differences in IL-6 and TNFα concentrations between the survivors and nonsurvivors, with higher levels being seen in non-survivors. Adiponectin levels were lower in nonsurvivors, but this did not reach statistical significance. Conclusion: Although there was a demonstrable change in insulin sensitivity during the stay in ICU, there was no statistically significant association between the cytokines TNFα or IL-6 and insulin administration. There was a negative correlation between adiponectin concentrations and administered insulin on discharge. This data also demonstrates that mortality is associated with increased levels of proinflammatory cytokines.

Univerzita Karlova v Praze Přírodovědecká fakulta Studijní program: Doktorský studijní program v biomedicíně Studijní obor: Imunologie Mgr. Libor Kolesár Klinický význam polymorfismu cytokinových genů Clinical significance of cytokine gene polymorphism Disertační práce Vedoucí závěrečné práce/Školitel: Prof. MUDr. Ilja Stříž, CSc Praha 2012 Abstract The human genome is full of different sequence variants. They are different mainly in size but also in their influence on phenotype. The smallest unit of genetic polymorphism is single nucleotide polymorphism (SNP). SNPs represent a single nucleotide change between two alleles and might affect the gene expression. We have studied SNPs in three distinct fields as: (1) marker of risky patients after the organ transplantation, (2) diagnostic marker of patients with interstitial lung diseases (ILD) or (3) with uterine fibroid (UF). We have come to the following results. Ethnicity or even nationality plays a role in the distribution of genetic polymorphism. This must be absolutely taken into account when one would like to transfer findings of a clinical study from a certain nation or ethnic and applied them to his studied group for the comparative purposes. Our first clinical gene-association study has found that even gene polymorphism of the IL-18 gene may...

Cytokine based therapies can be targeted to the sites of active inflammation by modifying a given cytokine as a LAP-cytokine. IL-17A has been shown to directly contribute to pathogenesis of rheumatoid arthritis (RA). IL-17F, another member of the IL-17 cytokines family shares structural homology, receptor binding and biological properties with IL-17A but is 30-100 times less potent than IL-17A. (H161R) IL-17F mutant, a natural variant of IL-17F was shown to be protective against asthma in Japanese population. In vitro, IL-17F mutant competitively inhibited wild-type IL-17F and lacked the ability to activate downstream signaling pathways. I hypothesized that (H161R) IL-17F mutant is an additional inhibitor of IL-17A and if modified as LAP-IL-17F mutant, would be an effective targeted therapy for RA. (H161R) IL-17F mutant was created by substituting nucleotide A at position 485 in the wild type IL-17F by G. In vitro assays showed that the IL-17F mutant could bind to IL-17RC but lacked the ability to stimulate IL-6 secretion in HFFF2, 3T3 and HeLa cells and phosphorylate ERK1/2 in HeLa cells. IL-17F mutant also inhibited IL-17A induced secretion of IL-6 in all these cell lines. In order to assess in vivo therapeutic efficacy of LAP-IL-17F mutant in collagen induced arthritis mice, three mouse analogues of human IL-17F mutant were developed. Of these, (Q158R) IL-17F mutant displayed IL-17 agonistic properties, (H157R) IL-17F mutant could not be expressed in vitro and the truncated IL-17F mutant could not bind to mouse IL-17RC. Investigation of in vivo expression and pharmacokinetics of intravenous hydrodynamically delivered human full-length and LAP-IL-17 plasmid DNAs in naïve SCID and C57BL/6 mice showed that human IL-17 transgene expression was detectable in mouse serum at 48 hours post-delivery. The transgene expression however declined rapidly over the next two weeks. The local expression of transgene in C57BL/6 airpouch lavage fluid was less than 5% of its systemic levels. Taken together, the findings of the study warrant an investigation of in vivo therapeutic efficacy of human (H161F) IL-17F mutant in a suitable preclinical RA model, such as RA synovium/SCID mice.

No description available.

Orientador: Ângela Maria Victoriano de Campos Soares / Coorientador: Silvio Luis de Oliveira / Banca: José Maurício Sforcin / Banca: Sueli Aparecida Calvi / Banca: Carlos Artério Sorgi / Banca: Wafa Hanna Koury Cabrera / Resumo: O Paracoccidioides brasiliensis é o agente etiológico da paracoccidioidomicose a micose sistêmica de maior prevalência na América Latina. Dentre os vários mecanismos da resposta inata contra esse fungo, os envolvendo as células fagocitárias desempenham papel central na contenção do processo infeccioso, com destaque para a participação na atividade fungicida e modulação da resposta inflamatória. As funções das células fagocitárias contra os diversos microrganismos, incluindo os fungos, são iniciadas a partir de mecanismos de ativação dessas células, gerados após o reconhecimento dos mesmos por receptores de reconhecimento de estruturas moleculares padrões (PRRs) que reconhecem estruturas moleculares compartilhadas por determinados grupos de microrganismos, os chamados padrões moleculares associados à patógenos (PAMPS). Nosso estudo objetivou estudar a participação dos receptores TLR2, TLR4, receptor de manose (MR) e dectina-1, na produção de citocinas pró e anti-inflamatórias e dos eicosanóides PGE2 e LTB4, por monócitos e neutrófilos humanos em resposta a cepas de P. brasiliensis de alta (Pb18) e baixa virulência (Pb265). Verificamos que as duas cepas do fungo, mas principalmente a Pb265 são capazes de induzir a produção de TNF- por monócitos. TLR2 e TLR4 foram os receptores que se mostraram mais envolvidos nesse processo. Adicionalmente, mostramos a capacidade de essas células liberarem IL-12 em resposta ao fungo, sendo os maiores níveis detectados para a cepa Pb265 e ocorrendo um importante papel do TLR4 e MR. Altos níveis de IL-10 também foram detectados, principalmente em resposta à cepa Pb18 para a qual houve um envolvimento do TLR2 e do MR. Para a cepa 265, o receptor de maior envolvimento foi o TLR2. As duas cepas, de forma semelhante, foram capazes de induzir uma maior produção de PGE2 e LTB4 pelos monócitos. No entanto, nenhum dos PRRs mostrou-se envolvido na produção dos ... / Abstract: Paracoccidioides brasiliensis is the etiological agent of paracoccidiodomycosis, the most prevalent deep mycosis in Latin America. During the innate immune response of the host against this fungus, phagocytic cells play an essential role, highlighting their participation in fungicidal activity and modulation of the inflammatory response. The functions of phagocytic cells against various microorganisms, including fungi, are initiated by the recognition of patterns molecular structures, shared by a group of microorganisms, called pathogen-associated molecular patterns (PAMPs), by pattern recognizing receptors (PRRs) expressed by these cells. In this study, we assessed the involvement of the PRRs : TLR2, TLR4, mannose receptor (MR) and dectin-1, in the production of pró and anti-inflammatory cytokines and the eicosanoid PGE2 and LTB4, by human monocytes and neutrophils in response to high- (Pb18) and a low- (Pb265) virulence strain of P. brasiliensis. We showed that monocytes produce TNF-alpha and IL-12 in response to both strains, but with higher levels to Pb 265. TLR2 and TLR4 were the main receptors involved in TNF-alpha production, while for IL-12, the participation of TLR4 and MR was detected. High levels of IL-10 were also observed, mainly in response to Pb18, with involvement of TLR2 and MR. For Pb265, the receptor responsible was TLR2. Boths strains were able to induce PGE2 and LTB4 by monocytes. However, none of the studied receptors was shown to be involved in this production. Neutrophils did not produce TNF-alpha in response to both strains. On the other hand, these cells produce IL-12, mainly in response to Pb265, with participation of TLR2, dectin-1 and TLR4. These cells also produce L-10, whose levels were higher for Pb18 with involvement of TLR2 and MR and only TLR2 for Pb265. The production of PGE2 and LTB4 was also detected for these cells, similarly for the two strains. For PGE2, it ... / Doutor

Graduation date: 1998