Résumé : Durant les dernières années, l’encapsulation d’îlots pancréatiques endocriniens a reçu une grande attention parce qu’elle pourrait constituer une solution pour diminuer les taux d'échecs des transplantations. Dans le contexte de la perte de la matrice extracellulaire (MEC) native des îlots lors de leur isolation et le rejet de greffes par le système immunitaire du receveur, cette thèse vise à améliorer la compréhension des interactions entre la MEC et les cellules des îlots pancréatiques endocriniens ainsi qu’à étudier les effets de stress apoptotiques associés à des éléments du système immunitaire sur la survie et les fonctions des îlots. Ces études pourraient permettre de raffiner notre compréhension des mécanismes associés au rejet des greffes d'îlots de Langerhans. Dans cette thèse, le premier chapitre constitue une revue de la littérature permettant de mettre en lumière les rôles réciproques de la MEC dans l'action des cellules immunitaires et l'influence de ces rôles sur le diabète de type 1 (DT1) et sur la transplantation d'îlots. Ce premier chapitre a été publié dans la revue Pathologie Biologie. Le premier travail expérimental comprend la culture de cellules d'insulinomes de rat (INS-1) sur des surfaces composées de carboxyméthyl dextrane (CMD) recouvertes de fibronectine, RGD ou YIGSR, un peptide synthétique de la laminine. Dans cette étude, l'effet bénéfique d’éléments de la MEC sur ces cellules productrices d'insuline a été démontré. Les cellules INS-1 ont davantage proliféré sur ces surfaces et sécrétaient plus d’insuline que les cellules INS-1 cultivées sur les surfaces contrôle de CMD, CMD+RGE et dans les plaques à multi-puits de polystyrène vendues pour la culture tissulaire (TCPS). Cette première étude a été publiée dans Acta Biomaterialia. La deuxième étude expérimentale avait pour objectif d’étudier l’effet protecteur de gels de fibrine pour enrober des îlots pancréatiques endocriniens isolés de jeunes porcs et exposés à deux concentrations de peroxyde d'hydrogène (H[indice inférieur 2]O[indice inférieur 2]). L’enrobage dans la fibrine a permis de réduire l'apoptose chez les cellules des îlots et d’améliorer la sécrétion d'insuline par ceux-ci lorsque les résultats étaient comparés à ceux des îlots non-enrobés. Ce travail a été publié dans la revue Islets. Dans la troisième étude expérimentale, des îlots porcins étaient enrobés dans des gels de fibrine et d'alginate et exposés à des monocytes humains pour comparer l’effet de l’enrobage par ces deux matériaux sur la survie et les fonctions des îlots. Les monocytes sécrétaient des concentrations importantes de cytokines TNFα, IL-6, IL-1β en réponse à la fibrine seule et aux îlots. Les cellules des îlots enrobés dans les gels de fibrine et d'alginate étaient moins apoptotiques et sécrétaient plus d'insuline que leurs contrôles respectifs non-enrobés. Cette étude a été acceptée dans la revue Pathologie Biologie. // Abstract : In recent years, the encapsulation of endocrine pancreatic islets has received enhanced attention as it might constitute a solution for islet transplantation failure. In the context of the loss of the native islet extracellular matrix (ECM) and graft rejection by the recipient’s immune system, this thesis aims to improve the understanding of ECM-islet cell interactions and immune system-related implications in islet survival and function in the context of type 1 diabetes mellitus (T1DM) and islet graft rejection. In the first chapter, a literature review introduces the reciprocal roles of the ECM in immune cell action and the influence of these interactions on T1DM and islet transplantation. The most important ECM components are discussed followed by an overview of immune cells and their possible implication in diabetes. Immune cell integrins and cytokines and their communication with and influence on ECM are highlighted, concluding in a brief discussion of the significance of these interactions for islet transplantation and encapsulation. This review has been accepted for publication by Pathologie Biologie. The first experimental work comprises the culture of rat insulinoma cells (INS-1) on welldefined low-fouling carboxymethyl-dextran (CMD) surfaces covalently grafted with fibronectin, RGD and YIGSR, a synthetic laminin peptide, resulting in higher cell proliferation and insulin secretion of INS-1 cells when compared to the controls CMD, CMD+RGE and tissue culture polystyrene (TCPS) plates. With this work, the beneficial effect of ECM cues on insulin-producing cells was proven. This study has been published in Acta Biomaterialia. The second experimental work aimed to study the effect of fibrin gels when used to embed endocrine pancreatic islets isolated from young pigs and exposed to hydrogen peroxide (H[subscript 2]O[subscript 2]). Fibrin-embedded islets showed less apoptosis and higher relative insulin secretion than islets on TCPS, verifying the protective effect of fibrin towards islets. This study has been published in Islets. In the third experimental study, porcine islets were encapsulated in fibrin and alginate gels and exposed to human monocytes to compare the two materials and to further investigate the immune protective properties of fibrin and alginate. Monocytes secreted high concentrations of TNFα, IL-6, and IL-1β in response to fibrin, but at the same time islets in both fibrin and alginate gels were less apoptotic and secreted more insulin then their TCPS controls. This study has been submitted to Pathologie Biologie.

Clustered regularly interspaced short palindromic repeats (CRISPRs) and associated (Cas) proteins form the basis of a prokaryotic adaptive immune system. Acquired sections of viral DNA are stored within the host genome as ‘spacers' flanked by ‘repeat' sequences. The CRISPR arrays are transcribed and processed to release mature CRISPR RNAs (crRNAs) – containing a single, intact spacer sequence – that are used by effector complexes to base-pair with matching hostile genetic elements and silence future infections. crRNA-biogenesis is thus an essential step within the defence pathway. Within Type I and III systems, the primary processing of the CRISPR transcript at repeat sites is performed almost exclusively by the CRISPR-specific riboendonuclease, Cas6. This thesis seeks to probe the catalytic mechanism of a Cas6 enzyme from the crenarchaeon Sulfolobus solfataricus (sso). Despite analogous generation of crRNA, ssoCas6 paralogues differ from previously characterised Cas6 examples in their lack of a canonical active site histidine residue. The work here builds on recent crystallographic evidence that the ssoCas6-1 paralogue unexpectedly adopts a dimeric conformation (PDB 3ZFV, 4ILR), to show that not only is the ssoCas6-1 dimer stable in solution but that this atypical arrangement is important to the activity of this particular enzyme. Furthermore, the ssoCas6-1 paralogue is shown to be the first in this family of endonucleases to employ multiple-turnover kinetics. The widespread diversity in Cas6 catalytic mechanisms reflects the plastic nature of the Cas6 active site and rapid co-evolution with substrate repeat sequences. The CRISPR/Cas environment within S. solfataricus is highly complex, containing three co-existing system types (Type I-A, III-A, III-B), five Cas6 paralogues and two families of CRISPR loci (AB and CD) that differ by repeat sequence. By probing the activity of an additional ssoCas6 paralogue (ssoCas6-3), which reveals different substrate specificities to those of ssoCas6-1, evidence emerges for functional coupling between ssoCas6 paralogues and downstream effector complexes, sufficient to regulate crRNA uptake and possibly even complex assembly.

Thesis (MScMedSc)--University of Stellenbosch, 2011. / ENGLISH SUMMARY: Background: Primary immunodeficiency disorders (PID) disrupt the proper functioning of the immune system. The prototypic PID is X-linked Agammaglobulinemia (XLA). This disorder is caused by mutations in the Bruton tyrosine kinase (Btk) gene and results in an arrest in B cell development which leads to a profound reduction of all classes of serum immunoglobulins (i.e antibodies). Patients with a lack of antibodies experience recurring bacterial infections during early childhood that can be fatal if not treated. Intravenous gammaglobulin replacement therapy (IVIg) is the standard treatment for XLA. It provides passive immunity thereby reducing the number and severity of infections as well as limiting many of the infectious complications. Early detection and treatment of XLA allows affected individuals to live a relatively normal life. Objective: The purpose of this study was to determine the molecular basis of XLA in South Africa using a direct sequence-based method to detect abnormalities in the Btk gene to aid clinical diagnosis of the disease. Methods : Male patients with a clinical diagnosis of XLA were included in this study. Genetic analysis was used to explore the exonic region of the Btk gene of 5 unrelated male patients and compared to 10 healthy controls. Family members were followed up to determine carrier status, where possible. Results: Mutational analysis revealed Btk abnormalities in 4 of the 5 patients leading to a definitive diagnosis of XLA. Two of the three mutations found in this study have been previously described while one mutation appears to be novel. The novel mutation is a one base pair deletion in exon 16 which leads to the truncation of the Btk protein. Despite the clinical findings suggestive of XLA, no mutation was identified in the exonic region of the Btk gene of the remaining patient, indicating that this patient might have a different form of PID. Maternal follow-up confirmed the maternal inheritance pattern as all mothers screened were carriers of the Btk mutation present in the affected individual. Discussion : Using a direct sequence-based method abnormalities were identified in the Btk gene of three patients. Molecular diagnosis coupled to clinical history of the patient provides a definitive XLA diagnosis. This study supports the use of molecular techniques in the diagnosis of PID and underlines the synergy that could be possible in a clinical setting. / AFRIKAANSE OPSOMMING: Agtergrond: Primêre immuungebrek siektes (PIGS) word gekenmerk aan ‘n gebrek aan teenliggame in die immuunsisteem wat lei tot herhaalde infeksies in jong kinders wat fataal kan wees indien dit nie vroegtydig behandel word nie. Die prototype van die bekende PIGS is X-gekoppelde Agammaglobulinemia (XGA). Die siekte word veroorsaak deur mutasies in die Bruton Tirosien kinase (Btk) geen en lei tot ʼn stilstand in B sel ontwikkeling en gevolglik ʼn vermindering van alle klasse van serum immuunoglobulins (teenliggaam). Intraveneuse gammaglobulien vervangingsterapie(IVIg) is die standaard behandeling vir XGA. Dit voorsien passiewe immunitiet en gevolglik verminder dit die getal en erns van infeksies en beperk baie van die aansteeklike komplikasies. Vroeë diagnose en behandeling van XGA laat toe dat geaffekteerde individue ʼn relatiewe normale lewe ly. Doel: Die doel van hierdie studie is om die molekulêre basis van XGA in Suid Afrika te ondersoek, deur gebruik te maak van direkte volgorde bepaling van die Btk geen in die hoop om die kliniese diagnose van die siekte aan te help. Metode : Manlike pasiente met ‘n kliniese diagnose wan XGA was by die studie ingesluit. Genetiese analise was gebruik om die “exonic” omgewing van die Btk geen te ondersoek van 5 onverwante manlike pasiente en vergelyk teenoor 10 gesonde kontrole. Waar moontlik was familie lede ogevolg om draers te bepaal. Resultaat: Mutasies in die Btk geen is geidentifiseer in 3 van die 4 pasiente, klinies gediagnoseer meet XGA. Die mutasies sluit 2 reeds beskryfde variante in en een nuwe mutasie, ‘n een basis paar delesie in ekson 16 van die Btk geen, Ten spyte van die kliniese profiel suggestief van XGA in die 5de pasient, was geen mutasies geidentifiseer in die “exconic” omgewing van die Btk geen nie, dit kan moontlik toegeskryf word aan die teenwoordigheid van ‘n ander vorm van PIGS in hierdie pasient. Opvolg analise op die DNA van die moeders van die pasiente het die moederlike oorerwings patroon van die siekte bevestig aangesien al die moeders draers van die geidentifiseerde mutasie in die Btk geen van die gaffekteerde individu was. Gevolgtrekking: Genetiese analise van die Btk geen blyk ʼn sensitiewe en spesefieke metode te wees om individue met XGA te diagnoseer. Hierdie studie ondersteun die gebruik van molekulêre metodes in die diagnose van PIGS en beklemtoon die moontlike sinergie wat kan bestaan tussen hierdie tipe benadering in die kliniese omgewing. / National Research Foundation / National Health Laboratory Services : Pathology Research Development Grant of NHLS Research Trust Grants

Thesis (MScEng (Process Engineering))--University of Stellenbosch, 2006. / The natural immune system is an exceptional pattern recognition system based on memory and learning that is capable of detecting both known and unknown pathogens. Artificial immune systems (AIS) employ some of the functionalities of the natural immune system in detecting change in dynamic process systems. The emerging field of artificial immune systems has enormous potential in the application of fault detection systems in process engineering. This thesis aims to firstly familiarise the reader with the various current methods in the field of fault detection and identification. Secondly, the notion of artificial immune systems is to be introduced and explained. Finally, this thesis aims to investigate the performance of AIS on data gathered from simulated case studies both with and without noise. Three different methods of generating detectors are used to monitor various different processes for anomalous events. These are: (1) Random Generation of detectors, (2) Convex Hulls, (3) The Hypercube Vertex Approach. It is found that random generation provides a reasonable rate of detection, while convex hulls fail to achieve the required objectives. The hypercube vertex method achieved the highest detection rate and lowest false alarm rate in all case studies. The hypercube vertex method originates from this project and is the recommended method for use with all real valued systems, with a small number of variables at least. It is found that, in some cases AIS are capable of perfect classification, where 100% of anomalous events are identified and no false alarms are generated. Noise has, expectedly so, some effect on the detection capability on all case studies. The computational cost of the various methods is compared, which concluded that the hypercube vertex method had a higher cost than other methods researched. This increased computational cost is however not exceeding reasonable confines therefore the hypercube vertex method nonetheless remains the chosen method. The thesis concludes with considering AIS’s performance in the comparative criteria for diagnostic methods. It is found that AIS compare well to current methods and that some of their limitations are indeed solved and their abilities surpassed in certain cases. Recommendations are made to future study in the field of AIS. Further the use of the Hypercube Vertex method is highly recommended in real valued scenarios such as Process Engineering.

Flavobacterium psychrophilum est l’agent pathogène des flavobactérioses d’eau froide touchant essentiellement les salmonidés dont la truite arc-en-ciel Oncorhynchus mykiss d’élevage. Cette bactérie Gram négative a un très fort impact économique en aquaculture car elle peut causer jusqu'à 70 % de mortalité dans les bassins d’élevage. La flavobactériose se décline sous deux formes pathologiques : la maladie de l’eau froide touchant les poissons adultes et le syndrome de l’alevin de truite arc-en-ciel touchant les juvéniles.Au cours de se travail, une méthode de PCR quantitative a été proposée. Elle permet en moins de trois heures de détecter et de quantifier un nombre de copies du gène codant l’ARNr 16S de la bactérie dans les tissus du poisson. Cette méthode a été testée sur différentes suspensions bactériennes (F. psychrophilum, autres flavobactéries, autres pathogènes) afin d’en valider la spécificité. La sensibilité de la méthode de détection a été évaluée à 1 et 2 bactéries par PCR en fonction de la matrice biologique utilisée.Une étude écotoxicologique a été menée et montre d’une part que F. psychrophilum est une bactérie hyper-sensible au cadmium comparée aux autres bactéries Gram négatives. Sa croissance est diminuée d’un facteur 2 en présence d’une contamination au Cd à 0,4 µM. D’autre part, nous avons constaté qu’une contamination de truites juvéniles par 1 µg CdCl2/L (2 mois) et une injection de 5 × 107 flavobactéries par individu (1 mois) ne provoque aucune mortalité. L’expression génique mesurée sur ses poissons démontre que le cadmium peut avoir des effets contradictoires sur le système immunitaire du poisson, pouvant soit exacerber ou diminuer la réponse immunitaire selon l’organe considéré. Un travail comparatif de la prévalence de la flavobactérie dans 7 sites aquacoles d’Aquitaine a démontré que la flavobactérie est omniprésente et que sa pathogénicité est contrôlée par le système immunitaire des poissons en bonne santé apparente. L’expression génique mesurée sur les poissons malades et apparemment sains nous apporte deux informations importantes : 1/ les gènes codant pour la métallothionéine A et l’interleukine 1-β sont de bons bio-marqueurs de la maladie et 2/ la répression des gènes codant pour le complexe majeur d’histocompatibilité 2-β, le facteur de croissance transformant β, le cluster de différentiation 8-α et l’immunoglobuline T dans la rate des poissons malades montre un effondrement du système immunitaire acquis nous permettant d’émettre l’hypothèse que ce phénomène déclenche l’apparition de la maladie. Ainsi, F. psychrophilum aurait un comportement de pathogène à virulence latente.Afin d’imaginer de nouvelles mesures prophylactiques et pour mieux comprendre la pathogénicité de la bactérie, une analyse du protéome de la membrane externe couplée à l’annotation du génome séquencé a été effectuée. Il a été identifié entre autres 1/ des protéines d’adhésion et d’invasion des tissus et 2/ des protéines d’acquisition de métabolites de l’hôte. De plus, un nombre important de protéines immunogènes chez la truite potentiellement utilisable dans un cocktail vaccinant a été détecté. Afin de chercher un vecteur pour ce cocktail, des nanoparticules d’acide γ-glutamique et phénylalanine d’environ 100 à 200 nm de diamètre ont été synthétisées. Ces dernières constituent une approche séduisante pour vacciner les poissons avec des antigènes de F. psychrophilum encapsulés puis incorporés dans la nourriture. / Flavobacterium psychrophilum is the causative agent of cold water flavobacteriosis, a condition affecting mostly salmonid fish, including the farmed rainbow trout Oncorhynchus mykiss. This Gram negative bacterium can cause up to 70% mortality in breeding tanks and has a very strong economic impact on the fish farming industry. Flavobacteriosis can take two pathological forms: the cold water disease affecting adult fish and the rainbow trout fry syndrome affecting juveniles.In the present study, a method of quantitative PCR was devised that allowed for the detection and the quantification, within three hours, of the 16S rRNA copy number in fish tissues. This method’s specificity was confirmed through the use of various bacterial suspensions (F.psychrophilum, others flavobacteria and others pathogens) and its detection limit was estimated to be 1 and 2 bacteria in broth and in biological matrices, respectively.An ecotoxicological study was then performed that showed that, on the one hand, F. psychrophilum is cadmium hypersensitive compared to others Gram negative bacteria because its growth rate, compared to a control, is decreased by a factor 2 at a cadmium concentration of 0,4 µM. On the other hand, we observed that subjecting rainbow trout juveniles to a concentration of 1 µg CdCl2/L for2 months prior to an injection of 5 × 107 F. psychrophilum by fish didn’t lead to any mortality. The gene expression which was measured on these fish demonstrated that cadmium can have contradictory effects on the immune system of fish, which could enhance or decrease the immune response depending of the organ. A comparative work of the prevalence of flavobacteria in 7 fish farms within the Aquitaine region (France) demonstrated that the bacterium was endemic and present in asymptomatic fish. Gene expression levels were measured on diseased and asymptomatic fish and demonstrated that the genes metallothionein A and interleukine 1-β were good biomarkers of the disease and that repression of the genes major histocompatibility complex 2-β, transforming growth factor -β, cluster of differentiation α and immunoglobulin T in the spleen of diseased fish was indicative of a collapse of the acquired immune system. We therefore hypothesized that this event marked the beginning of the disease and that F. psychrophilum is mostly an opportunistic pathogen.To prepare the development of new prophylactic techniques and to understand better the bacterium pathogenicity, an analysis of the outer membrane proteome coupled with sequencing of the bacterial genome was also performed. Furthermore, a significant number of immunogenic proteins were identified as good candidates for the preparation of a vaccine. Finally, γ-glutamic acid and phenylalanine nanoparticles of about 100 - 200 nm in diameter were synthesized to serve as potential vector for this vaccine. These nanoparticles should be tested to administrate F. psychrophilum antigens to fish through the digestive route.

Le fer, un métal de transition, est requis pour la survie de presque tout les organismes vivant à cause de son habilité à accepter ou donner un électron et donc à catalyser plusieurs réactions biochimique fondamentales. Cependant, la même propriété permet aussi au fer ionique d’accélérer la formation de radicaux libres et donc le fer peut potentiellement avoir des effets néfastes. Conséquemment, l’homéostasie du fer doit être étroitement régulé, tant au niveau cellulaire que systémique. Notre étude met l’emphase sur deux molécules importante pour régulation du métabolisme du fer : la lipocaline 2 (Lcn2) et l’hepcidine. Lcn2, une protéine de phase aiguë, est impliquée dans le transport du fer par les sidérophores. Lcn2 est un candidat potentiel comme transporteur du fer qui pourrait être responsable de l’accumulation excessive du fer non lié à la transferrine dans le foie des patients atteints d’hémochromatose héréditaire (HH). Nous avons généré des souris double-déficiente HfeLcn2 pour évaluer l’importance de Lcn2 dans la pathogenèse de surcharge en fer hépatique dans les souris knock-out Hfe (Hfe -/-). Notre étude révèle que la délétion de Lcn2 dans les souris Hfe-/- n’influence pas leur accumulation de fer hépatique ou leur réponse à une surcharge en fer. Le phénotype des souries HfeLcn2-/- demeure indiscernable de celui des souris Hfe-/-. Nos données impliquent que Lcn2 n’est pas essentiel pour la livraison du fer aux hépatocytes dans l’HH. L’hepcidine, un régulateur clé du métabolisme du fer, est un petit peptide antimicrobien produit par le foie et qui régule l’absorption intestinale du fer et son recyclage par les macrophages. L’expression de l’hepcidine est induite par la surcharge en fer et l’inflammation, tandis que, à l'inverse, elle est inhibée par l'anémie et l'hypoxie. Dans certaine situations pathologique, l’hepcidine est régulée dans des directions opposées par plus d’un régulateur. Nous avons, en outre, analysé comment les différents facteurs influencent l’expression de l’hepcidine in vivo en utilisant un modèle de souris avec un métabolisme du fer altéré. Nous avons examiné la régulation de l’hepcidine en présence de stimuli opposés, ainsi que la contribution des médiateurs et des voix de signalisation en aval de l’expression de l’hepcidine. Nous avons démontré que l'érythropoïèse, lorsque stimulé par l’érythropoïétine, mais pas par l’hypoxie, diminue l’expression de l’hepcidine d’une façon dépendante de la dose, même en présence de lipopolysaccharides ou de surcharge de fer alimentaire, qui peuvent agir de manière additive. De plus, l’entraînement érythropoïétique inhibe tant la voix inflammatoire que celle de détection du fer, du moins en partie, par la suppression du signal IL-6/STAT3 et BMP/SMAD4 in vivo. Au total, nos données suggèrent que le niveau d’expression de l’hepcidine en présence de signaux opposés est déterminé par la force du stimulus individuel plutôt que par une hiérarchie absolue. Ces découvertes sont pertinentes pour le traitement de l’anémie des maladies chronique et les désordres de surcharge en fer. / Iron, a transition metal, is required for survival by almost all living organisms due to its ability to accept or donate electrons and thus to catalyze many fundamental biochemical reactions. However, the same properties also allow ionic iron to accelerate the formation of free radicals and as such iron has the potential for deleterious effects. Consequently, iron homeostasis must be tightly regulated at both cellular and systemic levels. Our studies focused on two important molecules in the regulation of iron metabolism, namely, lipocalin 2 (Lcn2) and hepcidin. Lcn2, an acute phase protein, is involved in iron trafficking via siderophores. Lcn2 has emerged as a candidate iron-transporter that may be responsible for excessive non-transferrin-bound iron (NTBI) accumulation in the liver of hereditary hemochromatosis (HH) patients. We generated HfeLcn2 double-deficient mice to evaluate the importance of Lcn2 in the pathogenesis of hepatic iron loading in Hfe knockout mice. Our studies revealed that deletion of Lcn2 in Hfe-knockout mice does not influence hepatic iron accumulation in Hfe-/- mice, or their response to iron loading, as the phenotype of HfeLcn2-/- mice remained indistinguishable from that of Hfe-/- mice. Our data imply that Lcn2 is not essential for iron delivery to hepatocytes in HH. Hepcidin, a key regulator of iron metabolism, is a small antimicrobial peptide produced by the liver that regulates intestinal iron absorption and iron recycling by macrophages. Hepcidin expression is induced by iron-loading and inflammation while, conversely, being inhibited by anemia and hypoxia. Under certain pathologic situations, hepcidin is regulated in opposite directions by more than one regulator. We further investigated how different factors influence hepcidin expression in vivo using mouse models of altered iron metabolism. We examined hepcidin regulation in the presence of opposing stimuli as well as the contributions of mediators and downstream signaling pathways of hepcidin expression. We show that erythropoiesis drive, when stimulated by erythropoietin but not by hypoxia, down-regulates hepcidin in a dose-dependent manner, even in the presence of lipopolysaccharide or dietary iron-loading, which may act additively. Moreover, erythropoietic drive inhibited both the inflammatory and iron-sensing pathways, at least in part, via the suppression of IL-6/STAT3 and BMP/SMAD4 signaling in vivo. Altogether, our data suggest that hepcidin expression levels in the presence of opposing signaling are determined by the strength of the individual stimuli rather than by an absolute hierarchy. These findings are pertinent for the treatment of the anemia of chronic disease and iron-loading disorders.

Equine protozoal myeloencephalitis (EPM), caused by the protozoan parasite Sarcocystis neurona, is one of the most important neurological diseases of horses in the Americas. While seroprevalence of S. neurona in horses is high, clinical manifestation of EPM occurs in less than 1% of infected horses. Factors governing the occurrence and severity of EPM are largely unknown, although horse immunity might play an important role in clinical outcome. We hypothesize that EPM occurs due to an aberrant immune response, which will be discernable in the equine IgG subisotypes a, b, and (T) that recognize S. neurona in infected diseased horses versus infected but clinically healthy horses. Based on previously-established serum antibody concentrations for IgG subisotypes in healthy horses, standard curves were generated and served to establish the concentration of antigen-specific IgG subisotypes in equine serum and CSF in infected diseased and infected normal horses. The subisotype concentrations and ratios between subisotypes were analyzed to assess whether neurological disease is associated with detectable differences in the antibody response elicited by infection. Results indicate a type I biased immune response in infected diseased horses, implicating the role of immunity in the development of EPM.

O carrapato bovino Rhipicephalus (Boophilus) microplus é o principal vetor da riquétsia Anaplasma marginale, o agente etiológico da anaplasmose, uma doença que acomete os rebanhos e causa sérios prejuízos econômicos à pecuária no Brasil. Estabelecemos em nosso laboratório o cultivo da linhagem de células BME26 que são originárias do R. (B.) microplus e também a infecção dessas células por A. marginale, um patógeno que é naturalmente transmitido pelo carrapato. Detectamos que a expressão gênica da defensina e da ixodidina nas células é aumentada frente à infecção por A. marginale, embora nenhuma alteração da expressão gênica da microplusina foi constatada. As células foram expostas a microorganismos inativados por calor e LPS, sendo que a expressão gênica da microplusina é aumentada frente a todos os estímulos. Na exposição das células BME26 com a bactéria Microccocus luteus, a expressão gênica da defensina e da ixodidina não foi alterada e no estimulo com leveduras a expressão gênica da ixodidina foi reprimida. Frente à infecção por A. marginale detectamos, aumento expressão da defensina e ixodidina. Os genes da microplusina e defensina foram silenciadas por RNAi em células infectadas por A. marginale, mas não houve alteração no número de riquétsias / The cattle tick Rhipicephalus (Boophilus) microplus is the main vector of the rickettsia Anaplasma marginale, the etiological agent of anaplasmosis, a disease that affects cattle and causes serious economic losses to the Brazilian cattle industry. We established in our laboratory the embryonic cell culture line BME26 from R. (B.) microplus and infection by A. marginale, a pathogen naturally transmitted by R. (B.) microplus. We verified that defensin and ixodidin gene expression increased in these cells after an infection by A. marginale and no alteration in microplusin gene expression was detected. The BME26 cells were exposed to heat-inactivated microorganims or to LPS, microplusin gene expression increased after all stimuli. After exposure of BME26 cells to Micrococcus luteus, expression levels of defensin and ixodidin did not change and ixodidin gene expression reduced after exposure of these cells to yeast. In the infection by A. marginale we detected defensin and ixodidin gene expression. Also, microplusin and defensin genes were silenced by RNA interference (RNAi) in A. marginale-infected BME26 cells, but we did not observe alteration in the number of MSP4 rickettsias

Idealizou-se o presente ensaio com o objetivo de padronizar o perfil hematológico, bioquímico, eletroforético (proteinograma sérico) e quantificação das células linfocitárias CD4, CD5, CD8 por citometria de fluxo de cães da raça Golden Retrivier normais (grupos GR) e de cães distróficos (grupos GRMD). Para tanto, considerou-se a divisão dos grupos de acordo com a idade dos animais, desde o nascimento até a idade adulta, compondo assim seis grupos, sendo eles GR I, II, III e GRMD I, II, III. No presente projeto realizamos os estudos eletroforéticos de cães pertencentes a todos os grupos, estudos hematológicos e bioquímicos dos cães pertencentes aos grupos GR II, III, GRMD II e III, imunofenotipagem linfocitária dos grupos GR III e GRMD III. Os resultados eritroleucométricos e trombométricos obtidos para os cães pertencentes aos grupos GR II e GR III apresentaram valores médios dentro normalidade. Com relação aos cães pertencentes aos grupos GRMD III, observamos que o eritrograma se encontra dentro dos valores de referência. Contudo, considerando o leucograma, os valores médios (3,79/ mm3) referentes à mensuração de basófilos apresentaram-se acima dos valores de normalidade, variando de 0 a 159/mm3. Ademais, considerando os valores máximos, alguns animais pertencentes ao grupo em questão, apresentaram leucocitose com neutrofilia sem desvio à esquerda, além de trombocitose, monocitose e linfopenia, no momento das coletas. As dosagens bioquímicas séricas de todos os cães afetados apresentaram valores médios acima dos valores de normalidade para a dosagem de AST, ALT e CK. Para o estudo das proteínas séricas, a técnica SDS-PAGE permitiu o fracionamento de vinte e três proteínas, cujos pesos moleculares variaram de 16.000 a 260.000 daltons em todos os grupos estudados. Destas, foi possível identificar nominalmente doze frações protéicas: IgA (PM 142.000 Da), proteína C-reativa (PM122.000 Da), ceruloplasmina (PM 110.000 Da), fosforilase (PM 95.000 Da), transferrina (PM 82.000 Da), hemopexina (PM 78.000 Da), albumina (PM 66.000 Da), alfa1 antitripsina (PM 62.000 Da), IgG de cadeia pesada (PM 55.000 Da), haptoglobina (PM 45.000 Da), glicoproteína ácida (PM 40.000 Da) e IgG de cadeia leve (PM 23.000 Da). As demais proteínas foram identificadas pelos respectivos pesos moleculares. Não foi possível identificar nominalmente as frações protéicas de pesos moleculares 260.000 Da, 230.000 Da, 180.000 Da, 165.000 Da, 158.000 Da, 91.000 Da, 35.000 Da, 30.000 Da, 28.000 Da e 16.000 Da. Dentre essas proteínas foi possível observar que a proteína de peso molecular 91.000 Da foi encontrada apenas nos grupos GR I e GRMD I não sendo identificada nos outros grupos estudados. Considerando as alterações encontradas com relação às proteínas de fase aguda, evidenciamos as respostas de fase aguda frente às lesões musculares que ocorrem progressivamente em cães distróficos, principalmente em animais distróficos jovens, podendo inferir que os mesmos apresentaram alterações protéicas perante a injúria tecidual como é relatado em vários processos inflamatórios relacionados com outras patologias. Da mesma forma, os animais afetados pela distrofia, possuem alterações imunoglobulínicas quando comparados a animais normais. Com relação a imunofenotipagem linfocitária foi confeccionado um histograma das populações de linfócitos CD4+, CD5+ e CD8+ a partir da região do gate de linfócitos. Todos os animais afetados pela distrofia apresentaram porcentagem significativamente maior com relação à população linfocitária CD4+ e CD5+ quando comparados aos cães normais. Desta forma, podemos inferir que o processo progressivo da distrofia, esta relacionado com alterações nas populações celulares que compõe o sistema imune dos pacientes, pois devido a ausência de distrofina o músculo fica mais susceptível á lesões, sendo que na sua ocorrência, há liberação de citocinas que estimulam as células hepáticas a secretarem as proteínas de fase aguda e a promoverem uma co-estimulação de linfócitos T. Assim, podemos sugerir que os resultados encontrados em nosso trabalho são conseqüências da resposta inflamatória gerada pela lesão muscular. Esperamos com esse estudo, fornecer mais informações sobre a fisiopatogenia da doença, bem como promover um maior entendimento sobre a avaliação imunológica e resposta inflamatória neste modelo de estudo. Ademais, que os valores de base encontrados possam auxiliar na avaliação de possíveis reações nos testes pré-clínicos, facilitando assim, o entendimento das reações benéficas ou adversas para validar e explicar o mecanismo de ação de uma terapia celular, gênica ou mesmo medicamentosa / Devised to test this in order to standardize the hematological, biochemical, electrophoretic (Serum protein) and quantification of CD4 lymphocyte cells, CD5, CD8 by flow cytometry of Golden Retriever dogs normal (group GR) and dogs dystrophic (GRMD groups). To this end, we considered the division of groups according to age of animals, from birth to adulthood, thus composing six groups, as Gr I, II, III and GRMD I, II, III. In this project we performed electrophoretic studies of dogs belonging to all groups, haematological and biochemical studies of dogs belonging to the groups GR II, III, II and III GRMD, lymphocyte immunophenotyping in groups III and GR GRMD III. The results obtained for trombometric, erythometric and dogs belonging to the groups and GR II GR III showed mean values within normal limits. With respect to dogs belonging to groups III GRMD, we observed that the erythrocyte is within the reference values. However, considering the WBC, the mean (3.79 / mm3) relating to the measurement of basophils were above normal values, ranging from 0 to 159/mm3. Moreover, considering the maximum, some animals belonging to the group in question had leukocytosis with neutrophilia without left shift, and thrombocytosis, monocytosis and lymphopenia at the time of collection. The biochemical serum of all affected dogs had mean values above the normal range for the determination of AST, ALT and CK. For the study of proteins, SDS-PAGE technique allowed the fractionation of twenty-three proteins whose molecular weights ranged from 16,000 to 260,000 daltons in all groups. These could be identified by name twelve fractions: IgA (PM 142,000 Da), C-reactive protein (PM122.000 Da), ceruloplasmin (PM 110,000 Da), phosphorylase (95,000 Da PM), transferrin (82,000 Da PM), hemopexin (PM 78 000 Da), albumin (66,000 Da PM), alpha1 antitrypsin (PM 62,000 Da), IgG heavy chain (55,000 Da PM), haptoglobin (PM 45,000 Da), glycoprotein (PM 40,000 Da) and IgG light chain (PM 23 000 Da). The other proteins were identified by their molecular weights. We could not identify by name the protein fractions of molecular weight 260,000 Da, 230,000 Da, 180,000 Da, 165,000 Da, 158,000 Da, 91,000 Da, 35,000 Da, 30,000 Da, 28,000 Da and 16,000 Da. Among these proteins was observed that the protein molecular weight of 91,000 Da was found only in groups GR I and GRMD I was not identified in other groups. Considering the changes found with relation to acute phase proteins, we noted the acute phase response in the face of muscle injuries that occur gradually in dystrophic dogs, especially in young dystrophic animals, which may infer that they had abnormal protein before tissue injury as reported in many inflammatory-related pathologies. Likewise, animals affected by muscular dystrophy, alterations immunoglobulin when compared to normal animals. With respect to lymphocyte immunophenotyping was made a histogram of the populations of CD4, CD5 and CD8 from the region of the gate of lymphocytes. All animals affected by muscular dystrophy showed significantly higher percentage with respect to CD4 and CD5 lymphocyte population compared to normal dogs. Thus, we can infer that the process of progressive muscular dystrophy, is associated with changes in cell populations that make up the immune system of patients, because due to the absence of dystrophin, the muscle is more susceptible to damage, and its occurrence , a release of cytokines that stimulate liver cells to secrete acute phase proteins and promote a co-stimulation of T lymphocytes Thus, we suggest that the findings in our study are consequences of the inflammatory response generated by muscle injury.We hope that this study, provide more information about the pathophysiolgy of the disease, and promote a greater understanding of the immune and inflammatory responde assessment in this study model. Moreover, the base values found could help in assessing possible responses in preclinical testing, aiding the understanding of the beneficial or adverse reactions to validate and explain the mechanism of action of a cell therapy, gene or drug treatments

Estudo observacional em uma coorte 148 indivíduos infectados pelo HIV-1, em uso de terapia anti-retroviral, avaliando padrões de resposta imunológica, clínica e virológica em um período de 251 semanas, estratificados em três grupos de resposta virológica: Avirêmicos, Virêmicos e Virêmicos Atenuados. Este estudo observou melhor progressão clínica e imunológica foi observada no grupo Avirêmico, porém um significante ganho de linfócitos TCD4 (p < 0.013) e menor número de casos com evolução para Aids (p < 0.001) foi observado grupo Virêmico Atenuado comparado ao grupo Virêmico. / Observacional study in cohort of 148 HIV-1 infected patients under highly active antiretroviral therapy, analysed by different patterns of immunological reconstitution, clinical outcome and viral suppression in 251 weeks of follow up, estratified in Aviremic, Viremic and Virec Attenuated groups. This study observed better clinical and immunological response in Aviremic group, but Viremic Attenuated group shows a significant TCD4+ cells gain (p < 0.013) and less number of cases progressing to AIDS (p < 0.001) compared to Viremic group.