Introdução: Rosácea é uma doença inflamatória cutânea crônica relativamente comum, com incidência que varia de 2 a 10%. Caracteriza-se pelo surgimento de pápulas e pápulo-pustulas, eritema e telangiectasias precedidas por episódios de flushing. Apesar de não ser doença que comprometa o estado geral dos doentes, por ter acometimento preferencial da face, representa problema estético acentuado que interfere na socialização e qualidade de vida dos doentes. A etiologia da rosácea permanece incerta. A participação da imunidade inata tem sido implicada recentemente. Objetivo: Este estudo avaliou o envolvimento da imunidade inata na patogenia da rosácea através de pesquisa de células de Langerhans, células plasmocitóides (PDC), receptores \"toll-like\" (TLR) e expressão da forma induzida da enzima óxido nítrico sintase (iNOS) em biopsias de pele de pacientes com diagnóstico de rosácea. Métodos: Biopsias de 28 pacientes com diagnóstico clínico e histopatológico de Rosácea foram classificadas de acordo com características histopatológicas em Rosácea Granulomatosa (RG) (n = 10) e Rosácea Não Granulomatosa (RNG) (n = 18), e submetidas à técnica imunoistoquímica para demonstração de células de Langerhans (anticorpo anti-CD1a) (n = 26), PCD (anticorpo anti- CD123) (n = 24) e expressão dos receptores toll-like 2 e 4, bem como da forma induzida da óxido nítrico sintase (iNOS) (n = 28). Todos foram comparados com controles de pele normal (n = 15). Resultados: A população de células de Langerhans epidérmicas foi menor no grupo rosácea. Foram encontradas PDC dérmicas isoladas ou agrupadas no grupo rosácea, representando um novo dado no estudo da sua etiopatogenia. A expressão de TLR 2, TLR 4 e iNOS foi maior no grupo rosácea do que no grupo controle, estando distribuída com forte predominância na epiderme e anexos. Não houve diferença dos achados entre os grupos RG e RNG. Conclusão: Demonstrou-se, pela primeira vez, a presença de PDC nas lesões de rosácea. Juntamente com os outros marcadores estudados, os resultados apresentados confirmam a participação da imunidade inata na patogênese da rosácea através de mecanismos interdependentes e associados / Introduction: Rosacea is a common, chronic inflammatory condition with a reported prevalence between 2 and 10%. The disease has a variety of clinical manifestations that include flushing, persistent erythema, papules, pustules and telangiectasia. Because the facial skin is the predominant site of involvement, many patients sense that rosacea alters their social interactions affecting quality of life. The etiology of rosacea remains unknown. Recent studies have suggested that aberrant innate immunity is central to this disease. Objective: The aim of the present study was to examine the presence of Langerhans cells, plasmacytoid dentritic cells (PDC), and the expression of toll-like receptors (TLR) and inducible oxide nitric synthase (iNOS) in skin of patients with rosacea, in order to highlight the participation of innate immunity in the pathogenesis of this disease. Methods: 28 biopsy specimens were taken from patients with clinical and histopathological findings of rosacea. The samples were classified as Granulomatous rosacea (GR) (n= 10) or Non-Granulomatous rosacea (NGR) (n =18) according to histopathological features. Immunohistochemical demonstration of Langerhans cells (anti-CD1a antibody) (n = 24), PDC (anti-CD 123 antibody) (n = 26), TLR 2, TLR 4 and iNOS (n = 28) was performed in skin samples. The results were compared to normal skin control group (n = 15). Results: The number of Langerhans cells was lower in rosacea group than in control group. PDC were found in skin samples of rosacea as isolated cells and forming small clusters which represents a new contribution to the researches of its etiology. Expression of TLR2, TLR4 and iNOS was higher in rosacea samples than in normal skin controls, predominatly located in epidermal and adnexal structures. The comparison between GR and NGR groups did not show significant statistical difference. Conclusion: This research demonstrates, for the first time, the presence of PDC in lesions of rosacea, which together with the other results of this study, ratifies the existence of an altered innate immunity in pathogenesis of rosacea

Atualmente existem diversas opções terapêuticas para o tratamento do fotoenvelhecimento cutâneo.Uma das formas de tratamento muito utilizada é a fototerapia com laser. Com o desenvolvimento da tecnologia em lasers surgiu um novo conceito de tratamento da pele envelhecida, a fototermólise fracionada microablativa. Essa tecnologia fracionada visa combinar os efeitos visíveis de uma terapia ablativa com o conforto e segurança dos métodos não ablativos. Antes de atingir a pele o laser passa por uma lente óptica especial que o divide em microrraios. Tal procedimento produz colunas de lesões térmicas microscópicas que penetram na epiderme e derme, sem danificar o tecido circunvizinho. A grande vantagem de ser fracionado é que a pele íntegra ao redor de cada coluna funciona como um reservatório de células potentes para a rápida cicatrização e regeneração da pele, culminando com a produção de colágeno e resultando em uma pele mais jovial, sem o inconveniente de um longo período de recuperação pós-tratamento. 20 pacientes foram selecionadas para o tratamento das rítides periorais com uma sessão de laser Erbium-YAG de 2940nm, fracionado. Após 28 dias do procedimento observou-se melhora na textura da pele, clareamento de manchas e atenuação de rugas finas periorais. Foi realizado um estudo comparativo da quantificação das células de Langerhans e receptores toll-like ( TLRs ) 2, 3 e 9 na epiderme, antes e após 3, 7, 14 e 28 dias do tratamento e da quantificação das fibras de colágeno na derme superior, antes e após 28 dias do laser. Houve diferenças estatisticamente significativas entre as medianas dos valores de CD1a (p = 0,0085), TLR 2 (p = 0,0108), TLR 3 (p = 0,0011) e TLR 9 (p = 0,0012) na epiderme pré e pós-tratamento. Foi constatado que a significância se deu entre os valores: antes e após 14 dias para CD1a (diminuição), antes e após 7 dias para TLR 2 (diminuição), antes e após 14 dias para TLR 3 (aumento), antes e após 7 dias para TLR 9 (diminuição). Não se encontraram diferenças estatisticamente significativas entre os valores medianos das fibras colágenas do tipo I (p = 1,0000) e III (p = 0,3125) antes e após 28 dias do tratamento. O protocolo desse estudo mostrou-se bastante seguro, pois apresentou efeitos colaterais transitórios e nenhuma complicação permanente. / There are several treatment options for skin photoaging. One of them widely used nowadays is lasertherapy. The development of lasers technology introduced a new therapeutic concept for aging skin: ablative fractional photothermolysis. This technology combines the visible effects of an ablative therapy with the comfort and safety of nonablative methods. Before reaching the skin the laser passes through a special optical lens that splits it into tiny rays. This procedure produces columns of microscopic thermal injuries that penetrate the epidermis and dermis, without damaging the surrounding tissue. The great advantage of the fractional photothermolysis is that the intact skin around each column acts as a potent reservoir of cells for fast healing and skin regeneration, resulting in collagen production and a youthful skin without the inconvenience of a long period of recovery after treatment. 20 patients were selected for perioral wrinkles treatment with a session of fractional Erbium-YAG 2940nm. After 28 days of the procedure the perioral skin showed improvement in texture, bleaching of sunspots and attenuation of superficial wrinkles. A comparative study was conducted between the expression of Langerhans cells and toll-like receptors 2, 3 and 9 in the epidermis, before and after 3,7,14 and 28 days of treatment and among collagen fibers in the dermis, before and after 28 days of the laser. There were statistically significant differences between the median values of CD1a (p = 0.0085), TLR 2 (p = 0.0108), TLR 3 (p = 0.0011) and TLR 9 (p = 0.0012) in the epidermis pre and post-treatment. The significance occurred between the values: before and after 14 days for CD1a (decrease) before and after 7 days to TLR 2 (decrease) before and after 14 days for TLR 3 (increase) before and after 7 days for TLR 9 (decrease). There were not statistically significant differences between the median values of collagen type I (p = 1.0000) and III (p = 0.3125) before and after 28 days of treatment. The irradiation of skin with fractional Erbium-YAG was safe, as it showed transitory side effects and no permanent complication.

A administração de deidroepiandrosterona (DHEA) tem resultado em efeitos anti-diabetogênicos em animais de experimentação e no homem. Assim, o objetivo desse trabalho é avaliar o efeito do DHEA in vitro na expressão protéica do IR, do IRS-1, IRS-2, PI3-K, Akt, ERK-1/2; na expressão gênica do PDX-1, do PGC-1, da insulina, do GLUT-2 e da glicocinase; e avaliar a secreção estática de insulina de ilhotas pancreáticas de ratos. O cultivo das ilhotas por 24 horas com DHEA, não induziu nenhuma alteração tanto na expressão das proteínas quanto na secreção estática de insulina estimulada por glicose. Ocorreu aumento da fosforilação de ERK-1/2 e na expressão gênica do PGC-1. As células RINm5F, cultivadas por 72 horas com DHEA, apresentaram aumento da expressão total de IRS-1 e IRS-2. Concluímos, que 24 horas de cultura com ilhotas não é tempo suficiente para observar nenhuma alteração induzida pelo DHEA, na secreção de insulina, e na expressão das proteínas da via IRS/PI3-K/Akt. Células RINm5F podem ser um modelo alternativo para investigar os efeitos diretos do DHEA. / The dehydroepiandrosterone (DHEA) administration has resulted in reduction of abdominal fat and protection against insulin resistance from experimental animals and humans. So, the purpose of this project is measure the in vitro effects from DHEA: on protein expression of insulin receptor, the proteins IRS-1, IRS-2, PI3-K, Akt, and ERK-1/2; on gene expression of transcriptional factors PDX-1 and PGC-1, insulin, glucose transport GLUT-2 and glicocinase; and to measure the static insulin secretion, on cultured pancreatic islets of the rat. The culture of pancreatic islet for 24 hours with DHEA, did not induce nothing alteration on protein expression of the IR, IRS-1, IRS-2, PI3-K, Akt-1 and ERK-1/2, and static insulin secretion induced by glucose. However, happened increase ERK-1/2 phosphorylation and PGC-1 gene expression. The RINm5F cells, cultured by 72 hours, showed increase of the IRS-1 and IRS-2 expression. We conclude that 24 hours of the pancreatic islets culture are not sufficient time to look any alteration induced by DHEA, on insulin secretion, and on protein expression involved on IRS/PI3-K/Akt pathway. RINm5F cells can be an alternative model to research the direct effects from DHEA.

A secreção de insulina é estimulada pela glicose, porém os ácidos graxos (AG) podem influenciar o processo secretório. A oxidação de AG é importante para a estimulação da secreção de insulina por aumentar o ATP, porém, existem vias dependentes e independentes de ATP. Os AG <font face=\"Symbol\">w-3 interferem em processos fisiológicos e na composição e função da membrana plasmática, promovendo potente ação anti-inflamatória. Considerando a importante relação da NAD(P)H oxidase com a secreção de insulina, o estudo das alterações induzidas pela suplementação com AG <font face=\"Symbol\">w-3 sobre o conteúdo de superóxido (O2<font face=\"Symbol\">&#183;) e a expressão da NAD(P)H oxidase, é importante para a compreensão da fisiologia das células <font face=\"Symbol\">b-pancreáticas. Neste estudo, o grupo suplementado apresentou redução do conteúdo de O2<font face=\"Symbol\">&#183;, redução da expressão das subunidades da NAD(P)H oxidase e aumento na expressão da superóxido dismutase (SOD1 e 2), quando comparado ao grupo controle. Embora desconhecido o mecanismo, este dado é relevante, pois pressupõe melhor regulação do estado redox durante a secreção de insulina. / Insulin secretion is stimulated by glucose (GSIS), but fatty acid (FA) may influence the secretory process. The oxidation of FA is important for the stimulation of insulin secretion by increasing the ATP, although there are dependent and independent ATP pathways. The <font face=\"Symbol\">w-3 FA change physiological processes, and affect the composition and function of the plasma membrane, promote potent anti-inflammatory action. Considering the important relationship of NAD(P)H oxidase with insulin secretion, the study of changes induced by supplementation with <font face=\"Symbol\">w-3 FA on the superoxide (O2<font face=\"Symbol\">&#183;) content, and expression of NAD(P)H oxidase, becomes of great importance for understanding the pancreatic <font face=\"Symbol\">b cells physiology. In this study, the group supplemented with <font face=\"Symbol\">w-3 FA showed a reduction of the O2<font face=\"Symbol\">&#183; content, reduced expression of NAD(P)H oxidase subunits, and increased the expression of the enzyme superoxide dismutase (SOD 1 and 2), compared to control. Although unknown the mechanism, this data is relevant, because it represents better regulation of the redox state during GSIS .

L. (L.) amazonensis (La) e L. (V.) braziliensis (Lb) podem causar um espectro de manifestações clínicas e imunopatológicas no homem, sendo La responsável pela forma anérgica difusa e Lb pela forma mucocutânea da doença, formas polares e de elevada gravidade. Neste sentido, o objetivo do presente estudo foi avaliar os aspectos da resposta imune celular no ponto de inoculação e no linfonodo de drenagem de camundongos BALB/c inoculados no coxim plantar com 106 promastigotas de La e Lb. A evolução da lesão foi avaliada semanalmente sendo que na 4ª e 8ª semana PI biópsias do ponto de inoculação foram coletadas para determinação da densidade de células dendríticas (CD207+ e CD11c+), linfócitos T CD4+ e CD8+ e células iNOS+ por imunoistoquímica, e o linfonodo de drenagem para caracterização de subpopulações de células dendríticas e de linfócitos T CD4 e CD8 por citometria de fluxo. Células de linfonodo de drenagem foram cultivadas, com estímulo homólogo, para quantificação de citocinas (IL-4, IL- 10 e IFN-g) e nitrito nos sobrenadantes. A infecção por La levou à progressão da doença, com aumento do tamanho da lesão e da carga parasitária tanto na pele quanto no linfonodo de drenagem, enquanto que a infecção por Lb mostrou um discreto aumento da lesão entre a 6ª e 7ª semana PI com posterior regressão e redução da carga parasitária na pele e no linfonodo de drenagem. Aumento do número de células dendríticas dérmicas e de Langerhans foi observado na pele de camundongos inoculados com La na 4ª semana PI, juntamente com o aumento no número de células de Langerhans nos linfonodos de drenagem. Resposta imune celular preferencial de células T CD4+ foi observada tanto na pele quanto no linfonodo de camundongos inoculados com La, que mostrou ser predominantemente do tipo Th2 com a produção aumentada de IL-10 e IL-4. Já a infecção por Lb levou ao aumento na expressão de células dendríticas dérmicas e Langerhans na pele dos animais inoculados com Lb somente na 8ª semana PI, assim como aumento do número de células dendríticas dérmicas no linfonodo. A resposta imune celular foi caracterizada por células T CD4+ e CD8+ em pele e linfonodo dos animais infectados com Lb, vinculada a um perfil Th1 com a produção preferencial de IFN-g e altos níveis de NO. Aumento no número de células T regulatórias foi observado na infecção por Lb, que mostrou correlação direta com o número de linfócitos T CD4+ produtores de IL-10. Assim, a infecção por La foi relacionada à suscetibilidade, enquanto que a infecção por Lb foi relacionada à resistência do hospedeiro vertebrado. Estes resultados evidenciam não só o papel do parasita na modulação da resposta imune do hospedeiro como também das células dendríticas à infecção por Leishmania / L. (L.) amazonensis (La) and L. (V.) braziliensis (Lb) are responsible for a spectrum of clinical and immunopathological manifestations in humans, La is able to cause anergic diffuse leishmaniasis and Lb mucocutaneous leishmaniasis, polar forms with high severity. In this way, the aim of the present study was to evaluate aspects of the cellular immune response in the site of infection and in the draining lymph node of BALB/c mice inoculated in the hind footpad with 106 promastigotes of La and Lb. The evolution of the lesion size was evaluated weekly and in the 4th and 8th week PI biopsies from the site of infection were collected to determine the density of dendritic cells (CD207+ and CD11c+), CD4+ and CD8+ T cells and iNOS+ cells by immunohistochemistry, and the draining lymph node to characterize subsets of dendritic cells and CD4+ and CD8+ T cells by flow citometry. The draining lymph nodes cells were cultured with specific antigen to determine the cytokines (IL-4, IL-10 e IFN-g), and the nitric oxide in the supernatants. The infection caused by La led to the progression of disease with increase on lesion size and parasite load in the skin and draining lymph node, while Lb infection showed a discrete increase on the lesion size between 6th and 7th week PI with late regression and reduction in the skin as well as lymph node parasite load. An increase on the number of dermal dendritic and Langerhans cells were observed in the skin of BALB/c mice infected with La at 4th week PI together with an increase of Langerhans cells in the draining lymph node. The preferential CD4+ T cell immune response was observed in skin and lymph node of mice infected with La, which showed to be rather Th2 with an increase on the levels of IL-4 and IL-10. However, Lb infection led an increase of dermal dendritic cells and Langerhans cells in the skin only at 8th week PI, as well as an increase in the dermal dendritic cells in lymph node. The cellular immune response were characterized by CD4+ and CD8+ T cells in the skin and draining lymph node of mice infected with Lb, which was related to a Th1 immune response with production of high levels of IFN-g and nitric oxide. An increase of Regulatory T cells was observed in Lb infection, which showed the positive correlation with the IL-10 producing CD4+ T cells. So, the La infection was related to the susceptibility, while Lb infection was related to the resistance in the vertebrate host. These results emphasize the role of the parasite in the modulation of the host immune response to Leishmania infection

Ma thèse a permis de développer une nouvelle approche pour étudier, par imagerie in vivo, le comportement des cellules présentatrices d’antigènes (CPA) de la peau suite à la vaccination par voie intradermique chez le primate non-humain. Le ciblage des CPA a été réalisé par injection in vivo d’un anticorps monoclonal anti-HLA-DR fluorescent. L’effet sur les CPA d’un adjuvant (R-848, ligand du TLR7/8) et l’immuno-ciblage des cellules de Langerhans par une protéine de fusion vaccinale anti-VIH (anti-langérine-VIHGag) ont ainsi été évalués par imagerie in vivo, vidéomicroscopie confocale ex vivo et cytométrie en flux. Ce travail a contribué à améliorer nos connaissances immunologiques sur les effets locaux et précoces des vaccins et /ou adjuvants. / My pHD project allowed the development of in vivo imaging approaches to study the skin antigen presenting cell (APC) behavior post-intradermal vaccination in non-human primates. APC targeting was performed by in vivo injection of fluorescent anti-HLA-DR monoclonal antibody. The effect of an adjuvant (R-848, ligand of TLR7/8) on skin APC and the immunotargeting of Langerhans cells by anti-HIV vaccinal fusion protein (anti-langerin-HIVGag) were assessed by in vivo fluorescent imaging, ex vivo confocal videomicroscopy and flow cytometry. This work contributed to improve immunological knowledge on local and early events post-vaccination with or without adjuvant.

Les îlots de Langerhans, principalement composés de cellules bêta sécrétant l’insuline, jouent un rôle majeur dans l’homéostasie glucidique grâce à leur sécrétion hormonale finement régulée. Dans un contexte d’insulino-résistance associée à l’obésité, la masse fonctionnelle des cellules bêta pancréatiques augmente, en partie grâce à une prolifération accrue. Le diabète de type 2 survient lorsque les mécanismes de compensation échouent et que la sécrétion d’insuline devient insuffisante. Par conséquent, augmenter la prolifération des cellules bêta a été proposée comme approche thérapeutique afin de retarder l’apparition du diabète de type 2. Parmi les différents facteurs pouvant moduler la prolifération des cellules bêta, les nutriments, en particulier le glucose et les acides gras, jouent un rôle important et plusieurs études chez le rongeur montrent que les nutriments augmentent la prolifération et la masse des cellules bêta avant l’apparition de l’insulino-résistance. De plus, des travaux de notre laboratoire ont montré que l’infusion d’un mélange d’acide gras, le ClinOleic (65% oléate, 20% linoléate et 15% palmitate) et de glucose provoquait une augmentation marquée de la prolifération des cellules bêta chez le rat. L’objectif de cette thèse est donc d’évaluer les mécanismes par lesquels les acides gras stimulent la prolifération des cellules bêta. Dans un premier article, seul l’oléate, parmi plusieurs acides gras testé, a démonté un effet significatif sur l’augmentation de la prolifération des cellules bêta en présence de glucose ex vivo. La prolifération induite par l’oléate nécessite la formation de sphingolipides à très longue chaîne monoinsaturée, tandis que la perturbation de leur synthèse provoque une diminution de la réponse proliférative. Dans une seconde étude, l’analyse par séquençage d’ARN sur cellules uniques a mis en évidence le rôle important des espèces réactives de l’oxygène, des peroxyrédoxines et du proto-oncogène MYC dans le processus prolifératif des cellules bêta induit par l’oléate. Dans l’ensemble, les travaux présentés dans cette thèse apportent un éclairage nouveau sur le potentiel prolifératif encore énigmatique des cellules bêta pancréatiques et soulignent le rôle des sphingolipides et des espèces réactives de l’oxygène dans ce processus. / The islets of Langerhans, mainly composed of insulin-secreting beta cells, plays a major role in glucose homeostasis due to their finely regulated hormone secretion. In a context of insulin resistance associated with obesity, the functional mass of pancreatic beta cells increases, in part due to increased proliferation. Type 2 diabetes occurs when these compensatory mechanisms fail and insulin secretion becomes insufficient. Therefore, increasing beta cell proliferation has been proposed as a therapeutic approach to delay the onset of type 2 diabetes. Among the various factors that can modulate the proliferation of beta cells, nutrients, in particular glucose and fatty acids, play an important role, and several studies in rodents show that nutrients increase beta-cell proliferation before insulin resistance can be detected. Previous work from our laboratory has shown that infusion of the fatty-acid mixture ClinOleic (65% oleate, 20% linoleate and 15% palmitate) in the presence of glucose markedly increases beta-cell proliferation in rats. The objective of this thesis is to evaluate the underlying mechanisms by which fatty acids stimulate beta-cell proliferation. In a first study, among several fatty acids tested, only oleate increased beta cell proliferation in presence of glucose ex vivo. Oleate-induced beta-cell proliferation requires the formation of monounsaturated very long chain sphingolipids, while blockade of their biosynthesis dampens the proliferative response. In a second study, single-cell RNA sequencing analysis highlighted the role of reactive oxygen species, peroxiredoxins, and the proto-oncogene MYC in oleate-induced beta cell proliferation. Overall, the work presented in this thesis sheds new light on the enigmatic proliferative potential of pancreatic beta cells and identifies a role for sphingolipids and reactive oxygen species in this process.

A l'interface entre l'immunité innée et adaptative, les cellules dendritiques (DC) jouent un rôle clef dans l'induction de la réponse immunitaire. Dans la peau et les muqueuses, deux types de DC sont présentes : les cellules de Langerhans (CL) dans l'épiderme et les épithéliums et les DC dans le derme et le chorion (DDC). Dans une première partie, nous nous sommes intéressées aux DDC humaines, qui contrairement aux CL, restent méconnues. Les principales caractéristiques phénotypiques et fonctionnelles des DDC humaines isolées ex vivo à partir de derme humain ont été étudiées et notamment leur expression des récepteurs de l'immunité innée tels que les Toll Like Receptor (TLR) ou les lectines de type C. En parallèle, le profil d'expression des TLR par les CL humaines fraîchement isolées a également été analysé. Ainsi, par l'expression différentielle des récepteurs de l'immunité innée, les CL et les DDC joueraient chacune un rôle spécifique dans l'immunité de la peau et des muqueuses. Au sein des muqueuses génitales, plus particulièrement, elles interviennent dans la transmission de l'infection par le Virus de l'Immunodéficience Humaine (VIH). Dans une seconde partie, nous avons examiné les événements précoces dans la transmission du VIH à travers la muqueuse vaginale ainsi que le rôle des CL dans ce mécanisme. Pour cela, nous avons développé, à partir de cellules primaires humaines, un modèle de muqueuse vaginale reconstruite intégrant des CL. Nos travaux ont permis de mieux caractériser les populations de DC de la peau et des muqueuses ainsi que leur potentiel rôle dans l'immunité périphérique, ce qui devrait être utile pour le développement de futures thérapies anti-infectieuses dans lesquelles de telles DC sont ciblées.

Transmissible spongiform encephalopathies (TSEs), or prion diseases, are fatal neurodegenerative diseases that affect several species, such as scrapie in sheep or goats and CJD in humans. In several species, neurological disease is preceded by TSE agent accumulation in lymphoid tissues prior to neuroinvasion. While oral transmission is considered the most common route for scrapie, transmission can also occur through lesions to the skin or mucosa, for example in the mouth or gastrointestinal tract due to rough feed, or birth associated skin damage. Scrapie has also been experimentally transmitted through skin scarification in mice. Following scrapie infection via skin scarification, PrPSc accumulates in the draining lymph node (LN) before spreading to other organs in the lymphoreticular system. It is not yet known by what means the scrapie agent is transported from the skin to the draining LN. Dendritic cells (DCs) in the skin have been found to transport viruses, such as HIV or Dengue, from the skin, thereby raising the question whether DCs or Langerhans cells (LCs), located within the epidermis, play a role in the uptake and transport of the TSE agent from the skin to the draining LN. CD11c is a cell surface marker traditionally used to identify or isolate DCs from other cell types. Mice and rats are naturally resistant to Diphtheria toxin (DTX). A transgenic mouse line was created where the Diphtheria toxin receptor (DTR) was expressed on CD11c+ cells. The presence of this receptor on CD11c+ cells allowed for the temporary conditional depletion of CD11c+ cells following a single injection of DTX. The cells repopulate the tissues within a time frame specific to the tissues the cells are located in. These mice were used to determine whether the absence of CD11c+ cells at the time of scrapie infection via the skin had an effect on the early accumulation of PrPSc within the lymphoid tissues and on disease progression. Immunohistochemical analysis demonstrated that early PrPSc accumulation in the draining LNs was delayed following depletion of CD11c+ cells, indicating that their potential role in the transport of the scrapie agent from the skin. Scrapie incubation period was not affected by the absence of the CD11c+ cells at the time of infection. Recent findings show that CD11c is not exclusive to DCs and is also expressed on macrophage populations. Following DTX-mediated depletion, DCs repopulate the tissues much faster than CD11c+ macrophages. Scrapie infection was carried out in the skin in DTX treated mice after DCs had repopulated the tissues but before macrophage numbers had returned, to determine whether macrophages rather than DCs played a role in the early accumulation of PrPSc in the draining LNs. No differences in PrPSc accumulation were observed in mice depleted of macrophages compared to controls and there was no effect on disease incubation period. Another transgenic mouse line was used, where DTX expression on langerin+ cells (LCs and langerin+ DCs in the dermis), allowed for their temporary depletion through DTX treatment. Following langerin+ cell depletion, increased PrPSc accumulation was observed in the draining LNs 7 weeks post infection, but did not affect the incubation period of disease. These results indicate that the absence of LCs somehow accelerated PrPSc accumulation, and that LCs might play a preventative role in early stages after infection. Histopathological analysis was used to complement microarray studies aimed to determine what immune responses were associated with scarification and DTXmediated depletion of cells within the skin and whether these responses might be linked to disease transmission. DCs and LCs in the skin appear to play different roles in the early stages following scrapie infection via the skin, but the lack of effect on incubation period does not rule out the involvement of other cell types or cell-free mechanisms of scrapie agent spread from the skin.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.