Diss. (sammanfattning) Umeå : Univ., 2005. / Härtill 4 uppsatser.

Thesis (Ph. D.)--Case Western Reserve University, 2007. / [School of Medicine] Department of Molecular Biology and Microbiology. Includes bibliographical references. Available online via OhioLINK's ETD Center.

Early postnatal period is characterised by generally immature phenotype of the newborn's immune system. The maturation of the immune system including setting appropriate regulatory responses is occurring during this period and encountering pioneering bacteria colonizing neonate plays an important role. In the early days after birth, the immune system of a newborn is very limited, and the adaptive part is mostly represented by antibodies transferred from the mother by cord blood (CB) in the womb and then by colostrum and mother's milk after labour. Therefore, innate immunity plays a key role in defence (against pathogens) in neonates and is largely represented by neutrophils. This study aims to better understand neutrophil biology and phenotype in umbilical CB, compared to neutrophils from peripheral blood (PB) of mothers and healthy non pregnant women (referred to as HC). The assessment of neutrophil phenotype based on surface markers was performed using flow cytometry. Expression of genes linked to antimicrobial function was measured using quantitative PCR. Functional properties of neutrophils, metabolic activity during activation and phagocytosis, and suppressive properties were assessed using the SeaHorse machine and flow cytometry, respectively. Here we confirm the presence of immature CD16low...

Host defense peptides (HDPs) are a large group of small positively charged peptides that play an important role in innate immunity. Their role is more critical at early ages when other components of the immune system have not fully developed. There are three classes of avian HDPs: avian beta defensins (AvBDs), cathelicidins (Cath) and liver-expressed antimicrobial peptide 2 (LEAP-2). The objective was to compare expression of HDPs in male turkey poults at day of hatch (D0), D7, D14, D21 and D28 from the thymus, spleen, bursa, duodenum, jejunum and ileum. The expression of AvBD1, AvBD2, AvBD8, AvBD9, AvBD10, AvBD13, Cath2, Cath3 and LEAP-2 was measured using qPCR (n=6 birds/tissue/age). Data were analyzed by one-way ANOVA and Tukey's test, and significance considered at P ≤ 0.05. AvBDs and Caths exhibited greater expression in immune organs than intestinal tissues, with the greatest expression of AvBDs observed in the spleen. The intestinal tissues showed very low expression of AvBDs except for AvBD10 at D0. Similar to AvBDs, Caths expression in the immune organs was greater than the intestinal tissues with the spleen having the greatest expression among immune organs. Conversely, LEAP-2 showed greater expression in the intestinal tissues than in the immune tissues, which showed very low LEAP-2 expression unlike other HDPs. Understanding the differential expression of HDPs could reveal the innate immune status of poults, and may subsequently allow improvement of their health through appropriate mitigation strategies. / Master of Science

Plant Defensins type 1 (PDF1) sont principalement décrites pour leur rôle dans l'immunité innée en réponse à des attaques pathogènes via l'activation de la voie de signalisation de l'éthylène (Et) et de l'acide jasmonique (JA). Les défensines PDF1 du genre Arabidopsis sont également impliquées dans la tolérance cellulaire au zinc chez la levure. In planta, de nombreux résultats mettent en évidence une corrélation entre la forte accumulation des transcrits AhPDF1 et leur contribution dans la tolérance à un excès de zinc. Dans cette étude, l'analyse du transcriptome (qRT-PCR) révèle que les paralogues PDF1s, aussi bien chez A. thaliana que chez A. halleri sont très peu voire pas du tout sensibles au zinc. Toutefois, il y a une spécialisation des PDF1s en réponse à l'activation de la voie de l'acide jasmonique dans le genre Arabidopsis. De plus, la contribution fonctionnelle des membres de la famille PDF1s dans la tolérance au zinc a été caractérisée chez A. thaliana à l'aide d'une approche génétique combinant des mutants KO après insertion d'un ADN-T et la technologie de miRNA artificiel. L'étude de ces mutants souligne par ailleurs la diversité fonctionnelle au sein de la famille des défensines AtPDF1s qui ne confèrent pas toutes la tolérance au zinc. En effet, une diversité de déterminants moléculaires des PDF1s a été mise en évidence lors de cette étude. La forte accumulation des PDF1s n'est pas l'unique paramètre requis pour la tolérance au zinc. Il faut également considérer la spécificité de tissu où s'expriment ces PDF1s. A ces considérations s'ajoutent aussi des régulations post-transcriptionnelles et post-traductionnelles. L'étude de ces modifications est envisagée afin de comprendre la contribution des différentes défensines PDF1s dans la tolérance au zinc. / Plant Defensin type 1 (PDF1s) are mainly recognized for their response to pathogen attack via ethylene (Et)/jasmonate (JA) signaling activation pathway. However, PDF1s originating from Arabidopsis genus also showed their capacity to induce cellular zinc tolerance up on expression in yeast. In planta, a group of observation highlighted the correlation of AhPDF1 high transcript accumulation for their contribution to zinc tolerance. Here, transcriptomic analysis (qRT-PCR) revealed that in both A. thaliana and A. halleri species, PDF1 paralogues were barely or not at all responsive to zinc. Nevertheless, there is a species specialization of PDF1s in response to activation of JA-signaling in Arabidopsis genus. In addition, in A. thaliana, the functional contribution of PDF1 members in zinc tolerance was investigated through genetic approach. Examining combination of T-DNA insertion knockout mutant and artificial miRNA, these studies were first direct demonstration of the functional involvement of AtPDF1s in zinc tolerance. These also highlighted the functional diversity among AtPDF1s because not all of them could play a role in zinc tolerance. Indeed, a diversity of PDF1 molecular determinants for zinc tolerance in plants was underlined. Remarkably, PDF1 high transcript is not the only important parameter for zinc tolerance and PDF1 tissue specificity could be an important factor to consider. Moreover, post-transcriptional and post-translational regulation might occur. Studies on these modifications are now the further questions in order to understand the contribution of the different PDF1s to zinc tolerance.

Natural antimicrobials are peptides that are essential components of the innate immune system, providing broad-spectrum protection against bacteria, yeasts and some viruses. In addition to their innate immune activity, they exhibit properties suggesting they interact with the adaptive immune system. These functions imply they may be of particular importance in pregnancy. Intrauterine infection is responsible for approximately one third of cases of preterm labour, and normal labour is considered an inflammatory process, associated with leukocyte invasion of the uterine tissues and increased cytokine production. Little is known, however, about natural antimicrobial expression in pregnant reproductive tract. The aim of this thesis was thus to characterize natural antimicrobial production in pregnancy. The study focused on two main areas - the lower genital tract, comprised of the vagina and cervix; and the innermost fetal membrane, the amnion. In the lower genital tract, levels of natural antimicrobials were determined in samples of cervicovaginal secretions collected from pregnant women, using enzyme linked immunosorbance assay (ELISA). In addition Taqman quantitative PCR and ELISAs were used to investigate natural antimicrobial production by cell lines derived from endocervical, ectocervical and vaginal epithelium. It was found that elafin and secretory leucocyte protease inhibitor (SLPI) were found at high concentrations in cervicovaginal secretions, but levels were diminished in women with the common vaginal infection bacterial vaginosis (p<0.05). Cells derived from the vaginal epithelium expressed greater amounts of elafin than cervically derived cells. However, elafin and SLPI production could be stimulated in endocervical cells by the bacterial product lipopolysaccharide, a response that was not seen in the vaginal cell line. Natural antimicrobial production in the amnion was examined in tissue explants and primary cultured amnion cells, using a combination of Taqman PCR and ELISAs. In addition, cDNA microarray was carried out to investigate factors controlling amniotic antimicrobial production, and the involvement of signalling pathways was studied using specific pathway inhibitors. It was shown that the amnion expressed five antimicrobials: human beta defensins (HBD) 1, 2 and 3, SLPI and elafin. Expression of HBD2 was significantly upregulated following normal labour (p<0.05), with production in primary amnion epithelial cells dramatically increased by IL-1ß. The pattern of HBD2 expression in response to IL-1ß was biphasic, which suggested involvement of a secondary gene product. Several putative influential factors were identified by cDNA micorarray, including the NF-kappaB cofactor NFkappaBinhibitorζ. Its relationship to HBD2 was explored. The involvement of both NF-kappaB and mitogen activated protein (MAP) kinase p38 signalling appeared crucial in the response. This work has shown that natural antimicrobials are expressed by both the lower genital tract, where infections that are associated with preterm labour originate, and in the amnion, which is the fetus last line of defence to infection. They may have an important role in the prevention of infection associated preterm labour. Further characterization of these responses may increase understanding of the physiology, and pathophysiology of labour, and lead to strategies for the prevention of premature delivery.

The process of immune "self-nonself discrimination" is of utmost importance for the survival of all species as the biodestructive force of immune system can be directed towards the host as much as to pathogens. Thus, to shift this balance towards the latter, T cells bearing self- recognizing receptors are removed in the thymus (central tolerance) or their reactivity is harnessed through various additional mechanisms in periphery (peripheral tolerance). If the selfreactive T cells are not deleted and persist in the body, the regulation of self-tolerance can be breached, leading to the onset of autoimmunity. Presented thesis revolved around α-defensins, very effective bactericidal peptides that represent an important part of humoral innate immunity. There are two types of α-defensins: myeloid, expressed predominantly in neutrophils, and enteric, synthesized by intestinal Paneth cells. Data presented inhere are first to characterized the involvement of α-defensin- expressing cells in two types of autoimmune diseases, insulin-dependent diabetes mellitus (T1D) and autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED). The former relates to the identification of transcriptionally activated myeloid α-defensin- expressing eosinophils present in the thymus of diabetes prone rat. In...

Os peptídeos antimicrobianos são importantes ferramentas do sistema imune inato para controle das infecções e recentemente tem sido investigado seu potencial como estratégia terapêutica nas infecções e sepse. Estes peptídeos apresentam diversas atividades decorrentes de mecanismos de ação antimicrobianas e imuno estimuladoras. A indução de tolerância com a administração de pequenas doses de LPS reduz a mortalidade em modelos animais de sepse, modulando diversos aspectos do sistema imune. Nossa hipótese neste estudo foi que o efeito protetor da tolerância ao LPS está relacionado com a modulação da produção dos peptídeos antimicrobianos na sepse. A tolerância ao LPS foi induzida em camundongos C57bl/6 por injeção de lipopolissacarídeo de E. coli na dose de 1mg/kg por cinco dias. No oitavo dia os animais foram eutanasiados por overdose anestésica ou submetidos ao modelo de ligadura e punção cecal. Após seis horas os animais foram eutanasiados por overdose anestésica e o sangue, baço, intestino e pulmões foram coletados para determinação das concentrações de citocinas e peptídeos antimicrobianos. Nossos resultados mostram que o efeito da tolerância ao LPS sobre a produção de peptídeos antimicrobianos é tecido específica. Sistemicamente (plasma) a tolerância aumenta a concentração plasmática de beta defensina-3 e CRAMP somente em animais submetidos à CLP. No baço ocorre redução de beta defensinas 1 e 7 pela CLP e também pela tolerância. No pulmão há elevação de beta defensinas 1 e 3 pela CLP e esta é revertida pela tolerância. No intestino ocorre redução de defensinas pela tolerância tanto em animais controle quanto em animais submetidos à CLP. Observamos em nosso estudo um padrão invertido entre as concentrações de peptídeos antimicrobianos e citocinas no intestino e baço dos animais, principalmente nos submetidos à CLP. Quanto maior a concentração da citocina no tecido, menor a concentração da defensina em questão. No intestino podemos observar que a tolerância e a CLP aumentam a concentração de IL-6 e IL-10 e diminuem as concentrações de beta defensinas 1 e 7. No baço observamos esse padrão entre beta defensina-7 e IL-6 e entre beta defensina-1 e TNF-alfa. Não foi possível encontrar esse tipo de correlação no pulmão. Concluímos que os efeitos protetores da tolerância ao LPS estão relacionados com uma menor produção de defensinas no baço, pulmão e intestino de animais submetidos à sepse, e com maior concentração de peptídeos antimicrobianos no plasma / Antimicrobial peptides are important tools of the innate immune system to control infections and its potential as a therapeutic strategy in infections and sepsis has recently been investigated. These peptides present both antimicrobial and immuno-stimulatory activities. Lipopolysaccharide (LPS) tolerance is a defense mechanism against invading microorganisms also widely distributed in nature. Induction of tolerance with the administration of small doses of LPS reduces mortality in animal models of sepsis, modulating several immune system aspects. Our hypothesis was that the protective effect of LPS tolerance is related to the modulation of antimicrobial peptide production in sepsis. LPS tolerance was induced in C57bl / 6 mice by injection of E. coli LPS at 1mg / kg for five days. On the eighth day the animals were submitted to the sepsis model of cecal ligation and puncture. After six hours the animals were euthanized by anesthetic overdose and blood, spleen, intestine and lungs were collected for the determination of cytokines and antimicrobial peptides concentrations. Our results show that the effect of LPS tolerance on the production of antimicrobial peptides is tissue specific. LPS tolerance increases the plasma concentration of beta-defensin-3 and CRAMP only in animals undergoing CLP. In the spleen, there is reduction of ? defensins 1 and 7 by CLP and also by tolerance. In the lung, there is elevation of beta defensins 1 and 3 by PLC and this is reversed by tolerance. In the intestine, there is reduction of defensins by tolerance in both control and CLP animals. In our study, we observed an inverted pattern between the concentrations of antimicrobial peptides and cytokines in the intestine and spleen of animals, especially those submitted to CLP. The higher the cytokine concentration in the tissue, the lower the concentration of defensin in question. In the gut we can observe that tolerance and CLP increases IL-6 and IL-10 concentration and decreases ? defensins 1 and 7 concentrations. In the spleen, we observed this pattern between ?-defensin-7 and IL-6 and between alpha-defensin-1 and TNF-alpha. It was not possible to find this type of correlation in the lung. We conclude that the protective effects of LPS tolerance are related to a lower production of defensins in the spleen, lung and intestine of animals submitted to sepsis, and with a higher concentration of antimicrobial peptides in plasma

Orientador: Daniela Leal Zandim Barcelos / Resumo: O propósito deste estudo foi determinar a influência do tabagismo nos níveis de beta-defensina 3 (hBD 3) no fluido crevicular gengival (FCG) de indivíduos com periodontite crônica e avaliar sua relação com saúde e doença periodontal. Além disso, foi investigada a correlação deste peptídeo antimicrobiano com metaloproteinase da matriz (MMP-8) e citocinas pró (IL-10) e anti-inflamatórias (IL-1β, IFN-γ e TNF-α). Um total de 40 indivíduos com periodontite crônica, sendo 20 fumantes (PF) e 20 não fumantes (PNF), e 20 indivíduos sem doença periodontal (S) foram incluídos no estudo. Amostras de FCG de sítios sadios e doentes dos indivíduos com periodontite, e apenas de sítios sadios dos indivíduos periodontalmente saudáveis, foram coletadas com tiras de papel absorvente. A quantificação da hBD 3 foi feita pela técnica ELISA sanduíche e dos marcadores biológicos por ensaio Multiplex. Níveis significativamente menores de hBD 3 foram identificados nos sítios doentes de PF em comparação aos sítios doentes de PNF (p=0,02). Por outro lado, os níveis de hBD 3 nos sítios sadios de PF foram significativamente maiores que nos sítios sadios de PNF e S (p=0,006). Na comparação dos sítios dentro do grupo PF, foi verificado que os sítios doentes apresentavam níveis reduzidos de hBD 3 em comparação com sítios sadios (p=0,04). Já no grupo PNF, os níveis de hBD 3 foram mais elevados nos sítios doentes que nos sítios sadios (p=0,02). Uma correlação negativa foi observada entre os níveis de hBD 3 e MM... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The purpose of this study was to determine the influence of smoking on gingival crevicular fluid (GCF) levels of beta-defensin 3 (hBD 3) in individuals with chronic periodontitis and to evaluate its relationship with health and periodontal disease. In addition, the correlation of this antimicrobial peptide with matrix metalloproteinase (MMP-8) and pro (IL-10) and anti-inflammatory cytokines (IL-1β, IFN-γ and TNF-α) was investigated. A total of 40 individuals with chronic periodontitis, including 20 smokers (PS) and 20 non-smokers (PNS), and 20 subjects without periodontal disease (H) were included in the study. GCF samples from healthy and diseased sites of individuals with periodontitis, and only from healthy sites of periodontally healthy individuals, were collected with absorbent paper strips. Quantification of hBD 3 was performed by a sandwich ELISA assay and the biological markers levels were analyzed by a multiplex assay. Significantly lower levels of hBD 3 were identified in diseased sites of PS compared to diseased sites of PNS (p = 0.02). On the other hand, the levels of hBD 3 in healthy sites of PS were significantly higher than in healthy sites of PNS and H (p = 0.006). The comparison between the sites within the groups with periodontitis showed reduced levels of hBD 3 in diseased sites of PS compared to healthy sites (p = 0.04), while higher levels of this peptide was detected in diseased sites of PNS compared to healthy sites (p = 0.02). A negative correlation wa... (Complete abstract click electronic access below) / Mestre

Defensinas são peptídeos antimicrobianos produzidos na mucosa intestinal e fazem parte da imunidade inata, agindo sobre vários microrganismos luminais. Deficiência na expressão de defensinas tem sido relatada em doenças inflamatórias intestinais (DII), no entanto a contribuição de cada tipo de defensina, num cenário de polimorfismo genético, mantém alguma controversa. Βeta-defensinas humanas (HBDs) têm atividade antimicrobiana contra uma ampla variedade de fungos, bactérias e vírus e têm também, um papel na ligação entre a imunidade inata e adaptativa atuando como quimiotáticos. O gene DEFB1 (8p23), codificando a beta-defensina humana 1 (HBD-1), é expresso normalmente por células epiteliais de uma série de tecidos, mas sua expressão pode variar entre indivíduos e pode ser modificada durante processo inflamatório. Produção deficiente de defensinas parece contribuir para a patogênese de DII, e uma diminuição na expressão de HBD-1 tem sido relatada na mucosa de pacientes com doença de Crohn (DC) e retocolite ulcerativa (RCU). Nós avaliamos a possível associação de três polimorfismos do gene DEFB1 com a suscetibilidade a DII, RCU e DC, em 149 pacientes, 79 com DC e 70 com RCU; e 200 controles saudáveis do sul do Brasil. No nosso estudo não se observou diferença estatisticamente significativa entre a distribuição das frequências alélicas para DEFB1 SNPs -52G>A. -44C>G e -20G>A entre o total de pacientes com DII e controles. Porém, quando pacientes com DC foram estratificados de acordo com a localização anatômica, o alelo -20G>A foi mais frequente em pacientes com DC colônica do que em controles (65 % VS 44 %, p=0,048). De forma similar, o genótipo A/A foi mais frequente em pacientes com DC colônica do que em controles (36 % VS 16 %), mas neste caso, a diferença não foi estatisticamente significativa (p=0,07). Embora não se achou uma clara e forte associação entre os SNPs 5’-UTR DEFB1 e suscetibilidade/proteção à doença inflamatória intestinal, nossos resultados sugerem possível envolvimento do gene DEFB1 nestas enfermidades, especialmente com a localização colônica da doença de Crohn. Estudos com amostras maiores e populações diversas serão úteis para avaliar a tendência observada no nosso grupo. / Defensins are antimicrobial peptides produced by the intestinal mucosa and are part of the innate immune system, playing a protective role against various intestinal microorganisms. Deficiency in the expression of defensins has been reported in inflammatory bowel diseases (IBD), however there is some controversy over the contribution of each type of defensine, in a setting of great genetic polymorphism. Beta-defensins (HBDs) have an antimicrobial activity against a great variety of fungi, bacteria and viruses, and also have a role in connecting the innate and the adaptive immunity, acting as a chemostatic agent. Deficient production of defensins appears to contribute to the pathogenesis of IBD, and the lower expression of HBD-1 has been reported on the mucosa of Ulcerative colitis (UC) and Crohn’s disease (CD) patients. We evaluated a possible association of three polymorphisms of gene DEFB1 with susceptibility to develop IBD, UC and CD in 149 patients, 79 with CD and 70 with UC; and 200 healthy controls from the south of Brazil. The gene DEFB1 (8p23), which codifies human beta-defensin 1 (HBD-1), is constitutivelly expressed by epithelial cells of several tissues, but its expression may vary among different individuals and may be modified by inflammation. In our study we did not find a statistically significant difference between the distribution of the allelic frequencies for DEFB1 SNPs -52G>A, -44C.G and -20G>A between the total number of patients and controls. However, when patients were stratified according to the anatomic location, the allele -20G>A was more frequent in patients with colonic CD than in contros (65% VS 44%, p=0,048). Similarly, the genotype A/A was more frequent in patients with colonic CD than in controls (36% vs 16%), however, in this case, the difference wasn’t statistically significant (p=0,07). Although we did not find a clear and strong association between the 5’-UTR DEFB1 SNP and susceptibility to IBD, our results suggest a possible involvement of the DEFB1 gene and these diseases, particularlly colonic CD. Further studies with larger samples and diverse populations will be usefull to evaluate the trend observed by our group.