Em artrópodes o sistema imune inato contribui para a adaptação de animais como os escorpiões à diferentes ambientes. Esse sistema é composto por mecanismos capazes de agir contra injúrias e a ação de microrganismos e entre esses mecanismos estão os peptídeos antimicrobianos (PAMs). O objetivo deste trabalho foi identificar PAMs presentes na hemolinfa de Tityus serrulatus. Para isso sua hemolinfa foi extraída e separados os hemócitos e plasma, em seguida fracionamos em 3 concentrações de acetonitrila em TFA 0,05% (05, 40 e 80%). Estas frações foram submetidas a uma cromatografia liquida de alta eficiência (CLAE) e os picos foram avaliados quanto a sua ação antimicrobiana e hemolítica. Foram identificadas 16 frações que apresentam atividade antimicrobiana. Uma das frações com atividade antimicrobiana, presente nos hemócitos apresentou similaridade com defensina descrita em carrapatos da espécie Ixodes scapularis. Essa fração possui aproximadamente 3486 Da, não apresenta atividade hemolítica e foi denominada como Serrulina. / In arthropods, its innate immune system contributes to the adaptation of animals like scorpions to different environments. This system consists of mechanisms that act avoiding injuries and against the action of microorganisms, among these mechanisms are antimicrobial peptides (AMPs). The aim of this study was to identify AMPs, present in the hemolymph of Tityus serrulatus. The hemolymph was extracted and then we separated hemocyte and plasma. The samples were fractionated in different concentrations of acetonitrile in TFA 0.05% (05, 40 and 80%). These fractions were subjected to high-performance liquid chromatography (HPLC) and the peaks obtained were evaluated for its antimicrobial and hemolytic action. We found sixteen fractions with antimicrobial activity. One of the fractions with antimicrobial activity, present in hemocytes, is similar with a defensin described in ticks, Ixodes scapularis. This fraction has about 3486 Da, has no hemolytic activity and was named as Serrulina.

Irritable Bowel Syndrome (IBS) is the most common gastrointestinal disorder which affects approximately 4% of the population worldwide, according to the Rome IV criteria. It is characterized by abdominal pain and altered bowel movements in the absence of relevant structural abnormalities. The diagnosis of IBS is based on symptom profiles as no biomarkers exist to guide diagnosis or treatment stratification. Accumulating data suggests that altered gut microbiota and chronic low-grade inflammation play important roles in genesis of IBS. However, the mechanisms are unclear. My thesis first addresses the hypothesis that changes in fecal β-defensin secretion reflect compositional changes in the intestinal microbiota. This was driven by the understanding that compositional changes in the microbiota (“dysbiosis”) may play a role in the expression of IBS, and that a marker of these will identify those patients who might benefit from microbiota-directed therapies. I used a murine model in which I disrupted the microbiota using interventions relevant to the natural history of IBS i.e., antibiotics, stress, or dietary changes. I showed that experimentally induced compositional changes in the microbiota, with the exception of stress, altered the secretion of fecal β-defensin. My study indicates that monitoring fecal β-defensin over time identifies compositional changes in microbiota. I next investigated mechanisms and treatment options for a recently recognized variant of post-infectious IBS (PI-IBS) developed following antibiotic treatment in patients recovering from Clostridioides difficile infection (CDI). I refer to this variant as post-CDI gut dysfunction. I used a humanized mouse model in which germ-free mice were colonized with fecal microbiota from patients with post-CDI gut dysfunction, or age and sex matched healthy controls. I found that mice colonized with microbiota from a patient with severe slow transit constipation post-CDI reproduced the donor phenotype. Mice developed slow colonic transit due to macrophage mediated damage to the interstitial cells of Cajal (ICC) that maintain normal motility. These changes were reversed after fecal microbiota transplantation (FMT) from healthy donor mice thus confirming that the post-CDI gut dysfunction is microbiota driven. Similar results were obtained in a patient with slow transit constipation without a history of infection. My findings prompted me to next evaluate the therapeutic potential of microbiota-directed dietary therapies. I chose psyllium, the flavonoid quercetin, and pectin based on their demonstrated ability to alter microbiota composition. Psyllium and pectin each normalized colonic transit, and this was accompanied by an alteration in macrophages morphology, restoration of the disrupted ICC network and an increase in short chain fatty acids production. My results demonstrate the importance of a dysbiotic microbiota in this post infectious- IBS (PI-IBS) variant and, identify two potentially useful dietary based therapeutic approaches to improve gut dysfunction in these and similar patients. If findings from my thesis are confirmed in humans, it could offer novel approaches for identifying those IBS patients who might benefit from microbiota-directed therapeutics. / Thesis / Candidate in Philosophy

Thesis (MSc)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: Plant defensin peptides have become promising and attractive candidates to be used as antifungal agents in agricultural biotechnology. These peptides have a broad spectrum antifungal activity and play a vital role in the innate immune system of plants. Plant diseases caused by fungi are a major contributor to the decrease in the quality and safety of agricultural products. Due to the dangerous effects and negative environmental impact of pesticides, an effective, safe, natural and durable method to control crop pathogens has therefore become one of the major concerns in modern agriculture. Although these peptides are promising and attractive candidates, their precise mechanism of action is to date still unknown. Several common observations have been made. These include the antagonistic effect of cations on the activity of plant defensins. It is of vital importance to understand the underlying mechanism of the cation-antagonistic effect on the antifungal potency of defensin peptides in order to evaluate the possible contribution to defence reactions against microorganisms in planta. To this end we set out to characterize the effect of cations in the form of biological salts, NaCl, KCl, MgCl2 and CaCl2 on the structural stability and activity in terms of growth inhibition, morphological effects and permeabilization. In order to perform these characterization experiments, a production method resulting in a greater yield and involving simple and rapid purification methods was required. Heliophila coronopifolia peptides have previously been produced in a bacterial system, however the purification methods were tedious resulting in poor yields. Pichia pastoris was selected as production system as several other plant defensins have been successfully produced in this eukaryotic system. Hc-AFP1 and Hc-AFP3 was successfully produced using the Pichia production system and rendered active peptides. Hc-AFP2 and Hc- AFP4 was, however, not produced correctly, due to a post-translational modification event leading to the cyclization of the N-terminal glutamine to generate pyroglutamic acid. This modification negatively influenced the activity of these peptides. An active Hc-AFP2 could be produced by replacing the production buffer with a reduced ionic buffer. The effect of divalent and monovalent cations on the secondary structure of Hc-AFP1 was evaluated by circular dichroism spectroscopy. These cations induced a conformational change in the secondary structure of Hc-AFP1, with NaCl and MgCl2 inducing a more defined secondary structure and KCl and CaCl2 inducing a less defined secondary structure. Monovalent cations caused a slight reduction in the growth inhibition activity of Hc-AFP1 on Botrytis cinerea, however, characteristic hyperbranching and other morphogentic effects were still visible. Divalent cations had a greater antagonistic effect on the activity of Hc-AFP1, completely abolishing the growth inhibitory activity of the peptide, but the induced morphological effects on hyphae remained present. The activity of Hc-AFP1 to permeabilize B. cinerea hyphae was not influenced by the addition of cations, however it was in fact increased to up to 10-fold. However, since the growth inhibition activity of Hc-AFP1 was reduced in the presence of the biological salts indicates that permeabilization is not the sole activity responsible for growth inhibition caused by Hc-AFP1. This peptide probably has an alternative/primary target and more complex MOA. This is the first known report of the investigation of the influence of cations on the structure of plant defensin peptides. It is clear that cations induce a secondary structural conformational change in Hc-AFP1. This may be linked to the antagonism on the activity of this peptide. This study provides significant progress towards the structure-function analysis of plant defensins. / AFRIKAANSE OPSOMMING: Plantdefensinpeptiede word beskou as belowende en aantreklike kandidate vir gebruik as swammiddles in agribiotegnologie. Hierdie peptiede beskik oor breë spektrum antifungiese aktiwiteit en speel ‘n essensiële rol in die ingebore immuunsisteem van plante. Plant siektes wat deur swamme veroorsaak word dra betekenisvol by tot die afname in die kwaliteit en veiligheid van landbouprodukte. As gevolg van die skadelike effekte en negatiewe omgewingsimpak van plaagdoders, het effektiewe, veilige, natuurlike en duursame metodes om gewaspatogene te beheer, van die belangrikste vraagstukke van moderne landbou geword. Alhoewel hierdie peptiede belowende en aantreklike kandidate is vir die toepassing, is hulle presiese meganisme van aksie tot vandag toe steeds onbekend. Verskeie algemene waarnemings is egter al gemaak. Dit sluit die antagonistiese effek van katione op die aktiwiteit van plantdefensinpeptiede in. Dit is kernbelangrik om die onderliggende meganisme van die katioon-antagonistiese effek op die antifungiese effektiwiteit te verstaan om die moontlike bydrae van die peptiede tot die verdedigingsreaksies teen mikro-organismes in planta te evalueer. Met die doel voor oë het ons gemik om die effek van katione, spesifiek in die vorm van die biologiese soute NaCl, KCl, MgCl2 en CaCl2, op die strukturele stabiliteit en aktiwiteit in terme van groei inhibisie, morfologiese effekte en permeabilisasie te karakteriseer. Om uiteindelik hierdie karakterisasie eksperimente uit te voer was dit nodig om ‘n metode met ‘n groter opbrengs en wat vinnige suiwering van die peptied ondersteun, te optimiseer. Heliophila coronopifolia peptiede was voorheen in ‘n bakteriese sisteem geproduseer, maar die suiweringsmetodes was tydsaam en het gelei tot ‘n swak opbrengs. Pichia pastoris is dus geselekteer as die produksie sisteem aangesien verskeie ander plantdefensinpeptiede al suksesvol geproduseer is in hierdie eukariotiese sisteem. Hc-AFP1 and Hc-AFP3 is suksesvol vervaardig in die Pichia sisteem en het aktiewiteit getoon. Hc-AFP2 and Hc-AFP4 kon egter nie korrek vervaardig word nie as gevolg van ‘n na-vertalingsverandering wat gelei het tot die siklisering van die N-terminale glutamien, om piroglutamiensuur te lewer. Hierdie verandering het die aktiwiteit van die peptied negatief beinvloed. ‘n Aktiewe Hc-AFP2 kon wel vervaardig word deur die produksiebuffer te vervang met ‘n lae-ionise buffer. Die effek van divalente en monovalente katione op die sekondêre struktuur van Hc-AFP1 is ge-evalueer deur van sirkulêre dikroisme spektroskopie gebruik te maak. Hierdie katione het ‘n vouingsverandering in die sekondêre struktuur van Hc-AFP1 geïnduseer, NaCl and MgCl2 het ‘n meer gedefinieërde sekondêre struktuur induseer, terwyl KCl and CaCl2 ‘n minder gedefinieërde sekondêre struktuur geinduseer het. Monovalente katione het ‘n effense vermindering in die groei-inhibisie aktiwiteit van Hc-AFP1 op Botrytis cinerea veroorsaak, alhoewel kenmerkende hife-oorvertakking en ander morfologiese effekte nogsteeds sigbaar was. Divalente katione het ‘n sterker antagonistiese effek gehad op die aktiwiteit van Hc-AFP1, waar dit totaal en al die groei-inhibisie aktiwiteit van die peptied vernietig het, alhoewel die geïnduseerde morfologiese effekte op die hiffes steeds sigbaar was . Die aktiwiteit van Hc-AFP1 om B. cinerea hyphae te permeabiliseer is nie negatief beinvloed deur die byvoeging van katione nie, tewens dit het die aktiwiteit tot 10-voudig verhoog. Aangesien die groei-inhibisie aktiwiteit van Hc-AFP1 nie verminder is in die teenwoordigheid van die biologiese soute nie, dui dit aan dat permeabilisasie nie die enigste aktiwiteit is wat die groei inhibisie veroorsaak het nie. Die peptied het dus moontlik ‘n alternatiewe of primêre teiken en ‘n meer komplekse meganisme van aksie. Dit is die eerste verslag wat die invloed van katione op die struktuur van plantdefensinpeptiede ondersoek het. Dit is duidelik dat katione ‘n sekondêre strukturele vouingsverandering in Hc-AFP1 induseer. Hierdie verandering mag dalk bydra tot die antagonistiese uitwerking op die aktiwiteit van hierdie peptied. Hierdie studie het betekensisvolle vordering gemaak met die analise van die struktuur-funksie interaksie van plantdefensinpeptiede. / The National Research Foundation (NRF), Institute of Wine Biotechnology (IWBT), THRIP and Winetech for financial assistance.

The epithelial cells lining the intestinal mucosa separate the underlying tissue from components of the intestinal lumen. Innate immunity mediated by intestinal epithelial cells (IECs) provides rapid protective functions against microorganisms. Innate immunity also participates in orchestrating adaptive immunity. Key components in innate defence are defensins. To study the production of defensins and how it is affected by intestinal inflammation IECs were isolated from the small and large intestines of patients suffering from ulcerative colitis (UC), Crohn´s disease (MbC), celiac disease (CD), and from controls, and analyzed by quantitative RT-PCR (qRT-PCR) and immunoflow cytometry. Defensin expressing cells were also studied by in situ hybridization and immunohistochemistry. Normally, only small intestinal Paneth cells express human α-defensin 5 (HD-5) and HD-6. In UC colon IECs, HD-5, HD-6, and lysozyme mRNAs were expressed at high levels. In Crohn´s colitis colon the levels of HD-5 and lysozyme mRNAs were also increased although not to the same extent as in UC. No increase was detected in MbC with ileal localization. Metaplastic Paneth cell differentiation in UC colon was primarily responsible for the expression of the antimicrobial components. Human β-defensin 1 (hBD-1) mRNA was more abundant in large than in small intestine of controls, and remained unchanged in UC and MbC. hBD-2 mRNA was barely detectable in normal intestine and was induced in UC IECs but not in MbC IECs. mRNAs for the recently discovered hBD-3 and hBD-4, were detected in IECs from both small and large intestine. Both hBD-3 and hBD-4 mRNA were significantly increased in IECs of UC patients but not of MbC patients. Bacteria and IL-1β induced hBD-2 but not hBD-1 mRNA in colon carcinoma cell lines. IFN-γ, but not TNF-α or IL-1β, augmented hBD-3 expression in these cells, while none of the agents induced hBD-4. High antimicrobial activity of IECs in UC may be a consequence of changes in the epithelial lining, which permit the adherence of microorganisms. Unexpectedly, in situ hybridization revealed expression of hBD-3 and hBD-4 mRNAs by numerous lamina propria cells in colonic tissue from UC patients. These cells were identified as plasma cells (CD138+). hBD-3 and hBD-4 mRNAs were also demonstrated in the plasmacytoma cell line U266. This is the first demonstration of defensins in plasma cells. The four prominent constituents of the intestinal glycocalyx, carcinoembryonic antigen (CEA), CEA cell adhesion molecule 1 (CEACAM1), CEACAM6 and CEACAM7 all seem to play a critical role in innate defence of the intestinal mucosa by trapping and expelling microorganisms at the epithelial surface. The inducibility of these molecules in colonic epithelial cell lines was analyzed by qRT-PCR, immunoflow cytometry, and immunoelectron microscopy. IFN-g but not bacteria, LPS, TNF-α, or IL-1β modified the expression of CEA, CEACAM1 and CEACAM6. None of these agents modified CEACAM7 expression. IFN-γ was shown to have two effects: a direct effect on CEACAM1 transcription, and promotion of cell differentiation resulting in increased CEA and CEACAM6 and decreased CEACAM7 expression. Scanning electron microscopy of jejunal biopsies from children with CD revealed the presence of rod shaped bacteria in ~40% of patients with active CD, but only in 2% of controls. 19% of treated CD patients still had adhering bacteria. Presence of bacteria is not due to lack of antimicrobial factors. In fact, HD-5, HD-6, and lysozyme mRNA levels were significantly increased in IECs of patients with active CD. hBD-1 and hBD-2 were unchanged. Lack of induction of hBD-2 may reflect disturbed signalling in IECs of CD patients. Analysis of CEA and CEACAM1 mRNA/protein expression showed no differences between CD patients and controls. Analysis of the mucins MUC2 and MUC3 revealed significantly increased MUC2 levels in active disease and unchanged MUC3. Immunohistochemistry demonstrated goblet cell metaplasia as well as staining of the apical portion of absorptive cells. Glycosylation status of proteins was studied by lectin histochemistry. Goblet cells in the mucosa of CD patients were stained by the lectin UEAI. This was not seen in controls. The lectin PNA stained the glycocalyx of controls but not that of CD patients. Thus, unique carbohydrate structures of the glycocalyx/mucous layer are likely discriminating features of CD patients and may allow bacterial binding. We conclude that the intestinal epithelium is heavily involved in the innate defence of the mucosa and that its reactive pattern is affected by intestinal inflammation. Keywords: human intestinal mucosa; epithelial cells; innate immunity; defensin; ulcerative colitis; Crohn´s disease; celiac disease; glycoαcalyx; mucin

Diss. (sammanfattning) Umeå : Univ., 2007. / Härtill 4 uppsatser.

Made available in DSpace on 2015-03-26T13:47:13Z (GMT). No. of bitstreams: 1 texto completo.pdf: 1442239 bytes, checksum: 5f0041bc266aa2b219384708dc4dd876 (MD5) Previous issue date: 2012-11-20 / In response to an infection, blood cells such as neutrophils produce defensins within minutes to two hours to aid pathogens lysis and stimulate chemotaxis, reaching peak levels in the body in 24 hours. These peptides can also be produced without any stimulus by epithelial cells of the integumentary system. The aim of this work was to study the expression of β-defensins (cBD102 and cBD103) of canine skin diagnosed with canine visceral leishmaniasis (CVL). There were selected 43 skin samples from dogs positive to CVL and healthy, they were included in paraffin blocks and divided into three groups: I desquamative dermatitis (DD) with 16 blocks, II ulcerative dermatitis (UD) 21 blocks and the control group (6 blocks without canine skin problems). It was used the indirect immunoperoxidase technique (IIP) to confirm the LVC diagnosis and the identification of β-defensins, using human primary antibodies (hBD-2 and hBD-3, SIGMA®). The experimental delineation was completely casualized, with a confidence level of 0.95. Note that there was difference (p = 0.03) in the immunostaining intensity between groups, showing higher marcation intensity with cBD102 and cBD103 in the epidermis of the control group compared with DD and DU groups. In the dermis there was difference (p = 0.001) of the two β-defensins studied, being the cBD103 expression higher than the cBD102. This study is the first one relating to the immunological interaction of the extension and intensity expression between the β-defensin cBD102 and cBD103 and canine visceral leishmaniasis in the DD and DU dermatological standards, assessed by immunohistochemistry. / Em resposta a uma infeção as defensinas são produzidas em questão de minutos ou até 2 horas por células sanguíneas como neutrófilos, para auxiliar a lise de patógenos e estimular a quimiotaxia, alcançando níveis máximos no organismo em 24 horas. Também estes peptídeos podem ser produzidos sem estimulo algum pelas células epiteliais do sistema tegumentário. Com base no exposto anteriormente, o objetivo deste trabalho foi estudar a expressão de β-defensinas (cBD102 e cBD103) de pele de cães diagnosticados com leishmaniose visceral canina (LVC). Para tal, foram selecionados 43 amostras de pele entre cães acometidos por LVC e sadios incluídas em blocos de parafina, distribuídos em 3 grupos: I com dermatite descamativa (DD) por 16 blocos, II dermatite ulcerativa (DU) 21 blocos e o grupo controle (6 blocos de cães sem problemas dermatológicos). Foi empregada a técnica de imunoperoxidase indireta (IPI) para a confirmação do diagnóstico de LVC e identificação de β-defensinas, usando os anticorpos primários humanos (hBD102 e hBD103, SIGMA®). O delineamento foi inteiramente casualizado, adotando 0,05% de probabilidade para o erro Tipo I. Nota-se que houve diferença (p=0,003) na intensidade da imunomarcação entre os grupos, observando-se maior intensidade de marcação da cBD102 e cBD103 no grupo controle quando comparados com os grupos DD e DU. Na derme houve diferença (p=0,001) entre a expressão das duas β-defensinas estudadas, sendo a expressão de cBD103 superior a de cBD102. O presente estudo é o primeiro referente à interação imunológica da expressão de extensão e intensidade entre a β-defensina cBD102 e cBD103 e a leishmaniose visceral canina, nos padrões dermatológicos DD e DU, avaliados pela técnica de imunoistoquímica.

Objetivo: o presente estudo prospectivo avaliou a presença de microrganismos periodontopatogênicos, os níveis salivares de arginase e de HBD-2 em indivíduos com gengivite e periodontite tendo como controle indivíduos periodontalmente saudáveis, correlacionando-os aos respectivos parâmetros clínicos. Também foi avaliada expressão gênica do PAR2 crevicular em indivíduos saudáveis e com periodontite. Método: Inicialmente, foram avaliados 89 indivíduos sem doenças sistêmicas, nunca fumantes, sendo 31 saudáveis (média de idade 25,06 5,97), 27 com gengivite (média de idade 33,22 12,09) e 31 com periodontite (média de idade 52,16 11,54), todos submetidos à terapia periodontal não cirúrgica. Coleta salivar para avaliação dos níveis de arginase (quantificada por meio de espectrofotometria) foi realizada no início do tratamento em todos os indivíduos, e naqueles com gengivite e periodontite respectivamente em 30 e 50 dias pós-tratamento. Avaliação clínica de profundidade de sondagem, perda de inserção clínica e índices de placa e gengival e microbiana (Campylobacter rectus, Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Tanerella forsythia, Treponema denticola e Prevotella intermedia) foram também avaliados nos mesmos tempos. Dentre os 89 indivíduos, amostras do fluido gengival foram coletadas em 10 indivíduos saudáveis e 10 com periodontite para mensuração da atividade do PAR2 crevicular por meio de RT-PCR. Como esta relação foi positiva, foi verificada a quantidade de HBD-2 salivar por meio de ELISA de todos os 89 indivíduos e sua relação com os parâmetros clínicos e microbiológicos. A significância de todas as relações e quantificações foi analisada por meio de testes estatísticos apropriados. Resultados: foi observada uma melhora estatisticamente significativa dos parâmetros clínicos e microbianos após o tratamento periodontal. A arginase salivar estava significativamente mais elevada nos indivíduos com periodontite em relação àqueles com gengivite, e nestes em relação aos saudáveis. O tratamento periodontal promoveu melhora dos indivíduos doentes, cujos parâmetros avaliados tornaram-se estatisticamente semelhantes aos dos saudáveis. Houve maior atividade do PAR2 nos 10 indivíduos com periodontite em relação aos saudáveis e, após o tratamento, houve uma redução estatisticamente significativa deste parâmetro. Por fim, foram observados níveis estatisticamente mais elevados de HBD-2 salivar nos indivíduos com periodontite comparados àqueles com gengivite e aos saudáveis. Não foi possível observar uma correlação entre HBD-2 salivar e os microrganismos analisados. Conclusões: com base nos resultados observados, podemos concluir que: a arginase salivar está significativamente aumentada nos indivíduos periodontalmente comprometidos em relação aos saudáveis; o tratamento periodontal promoveu melhora dos indivíduos doentes em relação aos parâmetros avaliados; indivíduos com periodontite têm maior expressão gênica do PAR2 do que aqueles saudáveis e o tratamento tornou esta expressão semelhante nos dois grupos; indivíduos com periodontite têm níveis estatisticamente mais significativos de HBD-2 salivar do que aqueles saudáveis e aqueles com gengivite; a saliva parece ser uma ferramenta útil para o diagnóstico periodontal e para o monitoramento da eficácia do tratamento periodontal. / Objectives: This prospective study evaluated the presence of periodontopathogenics microorganisms, as it also examined the salivary levels of arginase and HBD-2 from subjects with gingivitis and periodontitis and periodontally healthy subjects as controls, correlating them to relevant clinical parameters. The gene expression of PAR2 crevicular in healthy subjects and periodontitis was also assessed. Methods: Initially, 89 individuals without systemic diseases, who were never smokers, were evaluated. Out of the 89, 31 were healthy subjects (average age 25.06 5.97), 27 have gingivitis (average age 33.22 12.09) and 31 were with periodontitis (average age 52.16 11.54), all underwent nonsurgical periodontal therapy. Saliva was collected for assessing levels of arginase at baseline in all subjects (quantified by spectrophotometry), and repeated on those with gingivitis and periodontitis respectively at 30 and 50 days post treatment. Clinical evaluation of probing depth, clinical attachment loss, both plaque and gingival index as well as microbial evaluation (Campylobacter rectus, Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Tanerella forsythia, Prevotella intermedia and Treponema denticola) were also assessed at the same time. Among the 89 individuals, gingival fluid samples were collected from 10 healthy ones and from 10 with periodontitis to measure crevicular PAR2 activity by RT-PCR. As the results came out positive, the amount of HBD-2 salivary was tested by ELISA for all 89 subjects assessing its relationship with clinical and microbiological parameters. The significance of all relationships and quantifications were analyzed using appropriate statistical tests. Results: we observed a statistically significant improvement of clinical and microbial parameters after periodontal treatment. Salivary arginase was significantly higher in subjects with periodontitis than in those with gingivitis, and those with gingivitis had higher results than the healthy ones. Periodontal treatment promoted improvement of the non-healthy individuals whose parameters became statistically similar to the healthy ones. There was a greater PAR2 activity in 10 individuals with periodontal disease compared to healthy ones, and after treatment the results showed a statistically significant reduction in this parameter. Finally, we observed statistically higher levels of salivary HBD-2 in individuals with periodontitis compared to both those with gingivitis and those individuals in the healthy group. It has not been possible to observe a correlation between HBD-2 and salivary microorganisms analyzed. Conclusion: Based on the observed results, we can conclude that salivary arginase is significantly increased in periodontally compromised individuals relative to the healthy ones; periodontal treatment promoted improvement of individuals in relation to the assessed parameters; individuals with periodontitis have higher gene expression of PAR2 than those healthy and the periodontal treatment brought similar results to both groups; individuals with periodontitis have statistically more significant levels of salivary HBD-2 than those with healthy gums and gingivitis; and, finally, saliva, besides being useful for periodontal diagnosis, appears to be also helpful for monitoring efficacy of periodontal treatment.

The global increasing problem of antibiotic resistance necessarily drives the pursuit and discovery of new antimicrobial agents. Antimicrobial peptides (AMPs) initially seemed like promising new drug candidates. Already members of the innate immune system, it was assumed that they would be bioactive and non-toxic. Their common trait for fundamental, non-specific mode of action also seemed likely to reduce resistance development. In this thesis, we demonstrate the ease with which two species of pathogenic bacteria, the gram-negative Salmonella typhimurium (S. typhimurium), and the gram-positive Staphylococcus aureus (S. aureus), can gain increased tolerance and stable resistance to various AMPs. By serially passaging each bacterial species separately under increasing AMP selection pressure we observed increasing AMP tolerance. Resulting in independent bacterial lineages exposed to four different AMPs (including a two-AMP combination) that exhibited 2 to 16-fold increases in MIC. Substantial cross-resistance between the AMPs was observed. Additionally, the S. aureus mutants were found to be cross-resistant to human beta-defensins 1, 2, 3, and 4. The LPS molecule, with mutations in the waaY, pmrB and phoP genes, was the principal target for S. typhimurium resistance development. The main target for S. aureus remained elusive. Reduced membrane potential was a common change for two of the mutants, but not for the others. All sequenced mutants had one or more mutations in various stress response pathways. Fitness of the resistant mutants was assayed by growth rate analysis and in vitro virulence factor testing (e.g. survival response to bile, superoxide, acidic pH). Furthermore an in vivo survival/virulence test involving a mouse competition experiment (S. typhimurium) and sepsis model (S. aureus) was performed. In the absence of AMPs there was often little or no fitness reduction in the mutants. Our results suggest that AMP resistance mechanisms do not irrevocably weaken either species with regard to virulence characteristics or survival within the host. In light of these findings, we suggest that the progression of therapeutic use of AMPs should proceed with great caution since otherwise we might select for AMP resistant mutants that are more resistant to our innate host defenses and thereby potentially more virulent.

La prévention du risque infectieux représente un enjeu de sécurité majeur pour l'Etablissement Français du Sang. A l'heure actuelle, le risque bactérien constitue le risque infectieux majoritaire dans les pays développés. Le risque bactérien ne se limite pas au domaine de la transfusion. La résistance aux antibiotiques constitue un problème majeur de santé publique. Les peptides antimicrobiens sont des armes importantes de l'immunité innée qui représentent une alternative intéressante aux antibiotiques classiques. Chez l'Homme, les α-défensines 1, 2 et 3 (HNPs 1-3) sont présentes dans les granules azurophiles des polynucléaires neutrophiles. Nous avons développé un procédé innovant de purification des HNPs 1-3 à partir des filtres de déleucocytation utilisés lors de la préparation des produits sanguins labiles. Ce procédé permet la production d'un cocktail pur d'HNPs 1-3 dont l'activité antibactérienne a été démontrée dans ce travail. Les défensines HNPs 1-3 ont par ailleurs été utilisées comme élément biorécepteur dans une approche innovante de détection bactérienne. Dans un premier temps, un immunocapteur électrochimique a été développé exploitant des microparticules magnétiques fonctionnalisées par des anticorps commerciaux. Dans un second temps, nous avons fonctionnalisé des microparticules magnétiques par les défensines HNPs 1-3 purifiées par notre protocole. Nous avons obtenu une première preuve de concept attestant de la capture des bactéries par cette approche innovante. La stabilité des peptides, associée aux performances des biocapteurs électrochimiques permettrait d'élaborer un test générique de détection de bactéries dans les produits sanguins labiles. / The prevention of the infectious risk is a major issue for the Etablissement Français du Sang. Currently, bacterial contamination is the most infectious risk in developed countries. The bacterial risk is not limited to blood transfusion safety. The antimicrobial resistance is a major public health problem. Antimicrobial peptides are important arm of the innate immune system which represents an interesting alternative to antibiotics. Human neutrophil peptides 1, 2 and 3 (HNPs 1-3) are found in the azurophilic granules of neutrophils. We have developed an original approach of HNPs 1-3 purification from leucodepletion filters used in blood processing. This process allows the production of a pure cocktail of HNPs 1-3 displaying high antibacterial activity as demonstrated by this work. HNPs 1-3 have also been used as bioreceptor in an innovative approach for bacterial detection. Initially, an electrochemical immunosensor was designed, exploiting magnetic microparticles coated with commercially available antibodies. In a second step, magnetic microparticles have been coated efficently with the HNPs 1-3 purified according our protocol. We have obtained a first proof of concept showing the bacterial capture by this innovative approach. The peptides stability combined with the electrochemical biosensors performances would allow the development of a generic bacteria detection assay in labile blood products.

Doctor of Philosophy / Department of Human Nutrition / Tonatiuh Melgarejo / Background and Objective: Crohn’s disease (CD) patients express low levels of host defense peptides (HDPs) especially β-defensins, which may compromise intestinal barrier function. Antibiotic treatment for bacterial infections in CD is limited and rarely curative, making it necessary to find alternative therapeutic approaches. We therefore investigated to what extent an essential amino acid; L-isoleucine (L-ILE) might induce the expression of human β-defensins (HBDs) in colonic epithelial cells as an alternative approach to help patients with CD. Antimicrobial activity of HBD2 was also assessed against four bacterial isolates which can cause secondary infections in CD. Methods: HTB-37 Caco-2 cells were stimulated with L-ILE at a concentration of 0 - 500µg/ml for 6 hours. Total RNA was extracted using RNeasy Micro Kit (QIAGEN). Reverse transcription was carried out with Superscript ®III First-Strand Synthesis System. The cDNA was amplified using specific primers for HBD1-3. Antimicrobial activity of HBD2 was determined using the broth dilution assay. Results: HBD1 was constitutively expressed under all conditions. HBD2 was expressed in HTB-37 cells after stimulation with L-ILE. Below 25µg/ml L- ILE stimulation, no expression of HBD2 was observed. HBD2 exhibited antimicrobial activity against bacterial isolates tested, with a MIC of 32, 64 and 128 µg/ml for both strains of E. coli, S. aureus and P. aeruginosa respectively. Conclusions: Our results indicate that L-ILE stimulation of HTB-37 Caco-2 cells can induce HBD2 expression. Data collected from our in vitro studies might have major implications for modifying the intestinal microbiota towards a healthier state in CD patients. Promoting the expression of HBD2 by colonic cells may lead to a lower rate of infection in these patients. Future in vivo studies are warranted to determine the potential clinical use of intra colonic administration of L-ILE in CD patients. The observed antimicrobial activity of HBD2 against bacterial isolates provides evidence that it is a crucial component of mucosal epithelial defense against infections which can complicate disease symptoms in CD.