La leishmaniose cutanée (LC) est une infection parasitaire classifiée par l’Organisation de Santé Mondiale (WHO) comme étant une des maladies tropicales négligées non-contrôlées. Dans la région du Moyen Orient, la LC est généralement endémique en Syrie et elle est causée principalement par Leishmania tropica et Leishmania major. La LC a été récemment introduite à des pays non endémiques, suite au déplacement intense des réfugiés Syriens échappant à la crise. Les interventions thérapeutiques contre la LC incluent des traitements locaux, systémiques et physiques. En revanche, le risque élevé de sélection et de résistance des parasites aux traitements actuels suscitent une quête sérieuse, pour trouver de nouvelles approches thérapeutiques. L’Imiquimod est un composé immunomodulateur approuvé pour utilisation clinique, et présente une efficacité vis-à-vis de certaines espèces de Leishmania. Dans cette étude, notre intérêt s’est focalisé sur l’efficacité d’un analogue de l’Imiquimod, l’EAPB0503, contre les stades promastigotes et amastigotes de L.tropica et L.major.Nos résultats montrent que l’Imiquimod et particulièrement l’EAPB0503 affectent les deux espèces. L’Imiquimod affecte majoritairement la motilité des promastigotes des deux espèces, alors que l’EAPB0503 affecte la motilité des promastigotes de L. major mais surtout l’invasion des promastigotes de L. tropica dans les macrophages. Les deux composés réduisent la réplication des amastigotes, avec un effet plus prominent de l’EAPB0503. Cet effet est médié par l’augmentation de l’expression du récepteur toll-Like-7 (TLR7), particulièrement pour l’Imiquimod et d’une manière moins importante pour l’EAPB0503. Les deux composés induisent l’activation de la voie de signalisation canonique de NF-κB. Ceci conduit à une production des cytokines pro-inflammatoires, et une diminution des cytokines anti-inflammatoires expliquant l’activité leishmanicide des deux composés. L’EAPB0503 semble agir via un autre TLR que l’imiquimod, comme il induit une expression plus élevée des transcrits TLR8 et TLR9, conférant une protection contre l’infection.Collectivement, nos résultats montrent l’effet de l’Imiquimod contre l’espèce la plus aggressive, L. tropica, et souligne l’activité plus puissante de l’EAPB0503 contre les deux espèces. De plus, cette étude montre le mécanisme d’action de ces deux composés, qui vraisemblablement activent des TLRs différents, mais finissent par induire la voie NF-κB et la réponse immunitaire correspondante. Ces résultats soulignent l’importance des drogues immuno-modulatrices contre la LC et ouvrent des perspectives sur des études précliniques puis cliniques de ces composés. / Cutaneous Leishmaniasis (CL) is a parasitic infection classified by the WHO as one of the most uncontrolled spreading neglected diseases. In the Middle East Region, CL is mostly endemic in Syria where it is mainly caused by Leishmania tropica and Leishmania major. CL has been lately introduced to under endemic countries, following the large-scale displacement of refugees from Syria fleeing the crisis. Therapeutic interventions against CL include local, systemic and physical treatments. However, the high risk for selection and spread of drug-resistant parasites is high; consequently new therapeutic approaches are still needed. Imiquimod is an FDA approved immunomodulatory compound with a tested efficacy against some leishmania species. In this study, our interest was to investigate the efficacy of an Imiquimod analog, EAPB0503 in comparison to the original compound, against promastigote and amastigote stages of L.tropica and L.major.We showed that Imiquimod affects the motility of promastigotes of both strains. EAPB0503 affected L. major promastigotes’ motility and impaired the invasion of L.tropica promastigotes into macrophages. Both drugs reduced amastigote replication, with a higher potency of EAPB0503. This activity is due to the upregulation of Toll-Like Receptor-7 (TLR7), mainly by Imiquimod, and to a lesser extent by EAPB0503. Importantly, both drugs activated the NF-κB canonical pathway leading to production of pro-inflammatory cytokines and upregulation of i-NOS levels. A decrease of anti-inflammatory cytokines secretion was obtained, explaining the leishmanicidal activity of both drugs. Importantly, EAPB0503 led to a prominent increase in TLR8 and TLR9 transcripts, presumably conferring protection against the infection.Collectively, our findings show the effect of Imiquimod against both strains especially, the aggressive L. tropica strain. We also show that EAPB0503 displays a more potent in vitro leishmanicidal activity than Imiquimod. These drugs seemingly activate different TLRs, but both activate the canonical NF-κB pathway and its subsequent mediated immune response. These results highlight the promising effect of immunomodulatory drugs against CL and warrant an in depth in vivo preclinical then clinical studies of these compounds.

El carcinoma basocelular (CBC) es el tumor maligno más frecuente y en nuestro país su incidencia ha aumentado progresivamente en los últimos años. El tratamiento del CBC se basa en la cirugía, pero ante el envejecimiento de la población ha surgido la necesidad de poder tratar los pacientes con terapias menos agresivas. Imiquimod es un potente inductor de citocinas, principalmente el interferón, con propiedad inmunomoduladora sobre el sistema inmune innato y adquirido. La inmunoterapia con imiquimod crema al 5% permite tratar los pacientes de modo ambulatorio y ha demostrado ser un tratamiento eficaz en el CBC. El mecanismo de acción de imiquimod en el CBC no es bien conocido y el presente trabajo ha estudiado los efectos de la aplicación tópica de imiquimod al 5% en la apoptosis del CBC, en la expresión de las proteínas bcl2, p53 y ki67 del CBC, y en el infiltrado inflamatorio peritumoral del CBC.En el 2001 se diseñó un ensayo clínico abierto con 55 CBC tratados con imiquimod crema al 5%. En todos los CBC se obtuvieron 3 biopsias, una antes del tratamiento, otra durante y otra 6 semanas después del mismo. A partir de muestras congeladas se estudió el índice apoptótico (Tunel) del CBC, y a partir de muestras parafinadas se estudió la expresión de bcl2, p53 y ki67 del CBC. En 25 CBC también se estudió el infiltrado inflamatorio peritumoral: células totales, CD3 +, CD8 +, CD56 +, Grancima B +, CD20 +, CD68 + y S-100 +. Todos los pacientes fueron seguidos durante 18 meses para evaluar la tasa de recidiva de CBC después del tratamiento. Entre 2001 y 2002 se diseñó un ensayo clínico randomizado con imiquimod crema al 5% y su excipiente en 30 CBC. En todos los CBC se obtuvieron 3 biopsias, una antes del tratamiento y dos durante el mismo. A partir de muestras congeladas se estudió el índice apoptótico (Tunel) del CBC, y a partir de muestras parafinadas se estudió la expresión de bcl2, p53 y ki67 del CBC. En 12 CBC también se estudió la densidad del infiltrado inflamatorio peritumoral y se realizaron estudios ecográficos con Dermascan C. Los principales resultados del estudio abierto fueron que imiquimod aumentó de modo significativo el índice apoptótico del CBC, la densidad del infiltrado inflamatorio peritumoral, el número de células CD3 +, CD8 +, CD20 +, CD68 +, S-100 + y grancima B +. Por el contrario imiquimod diminuyó de modo significativo la expresión de bcl2 del CBC. Imiquimod no modificó de modo significativo la expresión de p53 y ki67 del CBC. Después de 18 meses de seguimiento 41 CBC (75%) no mostraron signos de recidiva y fueron consideraros curados. Los resultados del estudio randomizado confirmaron que imiquimod crema al 5%, a diferencia de su excipiente, aumentó de modo significativo el índice apoptótico del CBC y la densidad del infiltrado inflamatorio peritumoral, y diminuyó de modo significativo la expresión de bcl2 del CBC. La expresión de p53 y ki67 del CBC no se modificaron de modo significativo ni con imiquimod ni con su excipiente. Como conclusión se puede afirmar que imiquimod aumenta el índice apoptótico del CBC, disminuye la expresión de bcl2 del CBC e induce una respuesta inmune antitumoral frente al CBC mediada por células CD3 +, CD8+, CD20 +, CD68 +, S-100 + y grancima B +. / Basal cell carcinoma (BCC) is the most frequent type of malignant tumor and its incidence has gradually increased during the last few years. The treatment of BCC is based on surgery. However, the aging of the population has made it necessary to treat patients with less aggressive therapies. Imiquimod is a potent cytokine inducer (especially of interferon) with immunomodulating properties on the innate and acquired immune systems. Immunotherapy with imiquimod 5% cream enables outpatient treatment and has been proved to be an efficient treatment for BCC. The mechanism of action of imiquimod on BCC is quite unknown. The purpose of this study was to assess the effects of imiquimod 5% external application on BCC apoptosis, on the expression of bcl2, p53 and ki67 proteins in BCC and on the peritumoral inflammatory infiltrate of BCC.An open trial was designed in 2001 with 55 BCCs treated with imiquimod 5% cream. Three biopsies were performed on each BCC: one before the treatment, another one during the treatment and a final one 6 weeks after its completion. The BCC apoptotic indexes (Tunel) were studied using frozen samples. Moreover, bcl2, p53 and ki67 expression in the BCC was studied using paraffin embedded samples. The peritumoural inflammatory infiltrate was studied in 25 BCCs, as well: total number of cells, CD3+, CD8+, CD56+, granzyme B+, CD20+, CD68+ and S-100+. All patients were followed-up for 18 months to evaluate the index of BCC recurrence after the treatment. A randomized clinical trial was carried out between 2001 and 2002 to evaluate imiquimod 5% cream and its excipient in 30 BCC. Three biopsies were performed on each BCC, one before the treatment and two during the treatment. The BCC apoptotic indexes (Tunel) were studied using frozen samples. Furthermore, bcl2, p53 and ki67 expression in the BCC was studied using paraffin-embedded samples. The density of the peritumoural inflammatory infiltrate was assessed in 12 BCCs. In addition to this, ultrasound studies were performed with Dermascan C. The main results obtained in the open study were the following: imiquimod significantly increased the BCC apoptotic index, the density of the peritumoural inflammatory infiltrate, the number of CD3+, CD8+, CD20+, CD68+, S-100+ and granzyme B+ cells. On the contrary, imiquimod remarkably decreased the expression of bcl2 in the BCC. Imiquimod did not modify significantly the expression of p53 and ki67 in the BCC. After 18 months of follow-up, 41 BCCs (75%) did not show evidence of recurrence, and were therefore considered healed of BCC. The results of the randomized study proved that, unlike its excipient, imiquimod 5% cream significantly increased the apoptotic index of the BCC and the density of the peritumoural inflammatory infiltrate, as well as decreased the expression of bcl2 in the BCC. The expression of p53 and ki67 in the BCC was not modified significantly neither using imiquimod nor its excipient. Thus, it may be concluded that imiquimod increases the apoptotic index of BCC, decreases the bcl2 expression and induces an antitumoural immune response against the BCC thanks to the intervention of CD3+, CD8+, CD20+, CD68+, S-100+ and granzyme B + cells.

La goutte est une maladie inflammatoire particulièrement prévalente causée par la formation de dépôts articulaires et péri-articulaires de cristaux d’urate monosodique (UMS ou MSU) et dépendante de la cytokine interleukine-1β (IL-1β). En 2006, l’inflammasome NLRP3 a été montré comme nécessaire pour la maturation de l’IL-1β, in vitro, en réponse aux cristaux de MSU. Néanmoins, sa nécessité in vivo est un sujet de controverse. Mon travail de thèse a porté sur la caractérisation d’un modèle murin d’inflammation aiguë uratique et l’analyse de la contribution des inflammasomes dans cette pathologie. J’ai d’abord montré que notre modèle par injection sous-cutanée de cristaux de MSU donne lieu une forte inflammation des tissus mous comme cela est souvent observé lors des crises de goutte chez l’Homme. L’emploi de souris invalidées génétiquement et d’inhibitions pharmacologiques m’a permis de décrire son indépendance vis-à-vis de plusieurs composants des inflammasomes et confirme le rôle majeur de l’IL-1β. De manière intéressante, j’ai ensuite montré qu’il est possible de réduire fortement l’inflammation dans ce modèle par un traitement topique à base d’imiquimod (crème ALDARA®), un ligand synthétique de TLR7. Des expériences réalisées in vivo et in vitro m’ont permis de relier l’effet de l’imiquimod à une baisse importante de l’Il1b au niveau transcriptionel, via une signalisation faisant probablement intervenir les interférons de type I et possiblement le facteur RUNX3. Mes données montrent donc que la production d’IL-1β, dans ce modèle, est visiblement indépendante de NLRP3 mais peut être fortement abaissée par l’application topique d’imiquimod. L’imiquimod pourrait ainsi représenter une piste thérapeutique attractive. / Gout is a prevalent inflammatory disease caused by the deposition of monosodium urate crystals (MSU) in articular/periarticular areas, which strongly depends on interleukine-1β (IL-1β). In 2006, the NLRP3 inflammasome has been shown to perform IL-1β maturation in vitro after MSU crystal exposure. However, its in vivo dependence is still matter of controversy. In my thesis project, I focused on the characterization of a murine acute uratic inflammation and analysed the contribution of inflammasome components. I first showed that the subcutaneous injection of MSU crystals in mice generate a strong soft tissue inflammation as observed in human gouty crises. Then, by using genetically-modified mouse lines and pharmacological inhibitions, I demonstrated that this model is inflammasome-independent, while still requiring IL-1β secretion. Interestingly, I observed that the topical application of imiquimod (ALDARA® cream), which is a synthetic TLR7 ligand, strongly dampens inflammation. In vivo and in vitro experiments further demonstrated that this effect is linked to reduced Il1b gene expression, which linkely involves type I interferon signaling and eventually the transcription factor RUNX3. Altogether, my results show that IL-1β production is NLRP3-independent in this mouse model but can be strongly decreased by topical application of imiquimod. Therefore, imiquimod might be an attractive therapeutic option for gouty patients.

Un réseau de cytokine complexe a été décrit dans le psoriasis mettant en évidence le rôle central des cytokines proinflammatoires dans la physiopathologie de cette maladie. Notre tentative de modéliser l'inflammation cutanée a montré que la combinaison de l'IL-17A, IL-22, IL-1α, oncostatine M (OSM) et le TNFα, augmente de manière synergique l'expression de chimiokines et de peptides antimicrobiens, reflétant certaines caractéristiques du psoriasis. D'autres caractéristiques de cette maladie sont l'acanthose et le blocage de la différenciation des kératinocytes. Notre premier objectif était d'étudier le rôle respectif de ces cytokines sur la différenciation des kératinocytes en comparaison avec les lésions de patients psoriasiques. Toutes ces cytokines inhibent l'expression des marqueurs de différentiation des kératinocytes, parmi lesquelles l’IL-22 et l’OSM sont les plus puissantes et le mélange M5 présente des effets synergiques. Si l'IL-22 et l'OSM déclenchent plus spécifiquement l'hyperplasie épidermique et le blocage de la différenciation, l’IL-1α, IL-17A et le TNFα sont plutôt impliqués dans l'activation de l'immunité innée. Le rôle fonctionnel de chacune de ces cytokines in vivo a été étudié dans un modèle d'inflammation cutanée de type psoriasique induit par l'imiquimod (IMQ), un agoniste TLR7, en utilisant des souris déficientes en cytokines. L'absence de l'OSM ou de l'OSMRβ n'a pas modifié le développement des lésions inflammatoires induites par l’IMQ. Une hypothèse est que d'autres cytokines peuvent avoir des effets redondants avec l'OSM. L'absence de l'IL-22 chez la souris diminue partiellement les lésions cutanées induites par l'IMQ, démontrant que son retrait du réseau cytokinique rompt une partie des effets synergiques des cytokines in vivo comme présenté dans le modèle M5. L'absence de l'IL-1α ou de l'IL-1β ne modifie pas l'inflammation cutanée induite par l'IMQ, ce qui n'est pas surprenant au vu des activités redondantes de ces deux cytokines. La diminution partielle de l'inflammation en absence d'IL-1α ET d'IL-1β OU de la chaine réceptrice commune IL-1RI confirme ces observations. A long terme ces études devraient permettre de proposer des stratégies anti-cytokine ciblées et combinées pour tenter de rompre la synergie et diminuer ainsi toutes les composantes de la réponse inflammatoire cutanée. / A complex cytokine network has been described in psoriasis and highlighted a central role of proinflammatory cytokines produced by infiltrated immune cells. Our attempt to model skin inflammation showed that the combination of IL-17A, IL-22, IL-1α, OSM and TNFα (Mix M5) synergistically increases chemokines and antimicrobial-peptides expression, recapitulating some features of psoriasis. Other characteristics of psoriasis are acanthosis and down-regulation of keratinocyte differentiation markers. Our aim was to characterize the specific roles of these cytokines on keratinocyte differentiation, and to compare with psoriatic lesion features. All cytokines decrease keratinocytes differentiation markers expression, but IL-22 and OSM were the most powerful, and the M5 strongly synergized the effects. If IL-22 and OSM more specifically drive epidermal hyperplasia and differentiation loss, IL-1α, IL-17A and TNFα were more involved in the activation of innate immunity. Functional role of these cytokines in vivo was studied in imiquimod-induced psoriasis-like skin inflammation by using knockout mice. Imiquimod (IMQ) is a TLR7 ligand. The absence of OSM or OSMRβ did not affect skin lesions after IMQ treatment. We supposed that other cytokines might be redundant with the OSM effects. The absence of IL-22 in mice partially reduced skin lesions induced by IMQ, demonstrating that removal of IL-22 in cytokine network break synergistic effects of cytokines in vivo as observed in in vitro with M5. The absence of IL-1α or IL-1β did not affect skin lesions after IMQ treatment, supporting the potential redundant activity of these cytokines, since the response is attenuated in mice deficient for both IL-1α and IL-1β or for IL-1RI. In the long term these studies should propose strategies targeted and combined anti-cytokine in order to break the synergy and thus reduce all components of the inflammatory skin response.

Durant ce travail de thèse j’ai étudié des mécanismes de contrôle de la réponse inflammatoire qui, lorsqu’ils sont dérégulés, peuvent mener à une pathologie autoimmune/autoinflammatoire sévère, la Polyarthrite Rhumatoïde (PR). J’ai essentiellement analysé deux aspects de ces mécanismes : en premier lieu, j’ai pu participer à la démonstration de l’importance de l’endonucléase DICER (impliquée dans la biogénèse des microARNs - miRs), dans le contexte de la PR, particulièrement au niveau des synoviocytes type fibroblastique (FLS), des cellules résidentes de la cavité synoviale. J’ai aussi initié une étude visant à identifier, in vivo, l’intégralité des transcrits dont l’expression est régulée par des miRs dans les FLS de patients atteints de PR. En plus de fournir une vision globale de la régulation miR-dépendante dans ces cellules, ce travail permettra aussi d’identifier des cibles de miRs d’intérêt dans la PR en s’affranchissant des prédictions bio-informatiques qui peuvent s’avérer incorrectes. Un second axe de mon projet de thèse avait pour objectif de fournir une meilleure description du dialogue intercellulaire dans la cavité articulaire. Pour cela, je me suis plus particulièrement intéressé au rôle des cellules dendritiques plasmacytoïdes (pDCs) dans la physiopathologie de la PR. Ainsi j’ai pu démontrer un rôle protecteur de ces cellules (initialement décrites pour leurs fonctions dans la défense antivirale), dans le contexte de l’arthrite inflammatoire dans plusieurs modèles murins. Ces travaux m’ont permis de proposer une stratégie thérapeutique innovante, basée sur le recrutement articulaire des pDCs. Cette approche, non invasive (par application topique de crème contenant 5% d’imiquimod), s’est montrée efficace aussi bien pour la réduction des symptômes cliniques de l’inflammation que pour l’amélioration des marqueurs biologiques comme l’érosion osseuse. / During my PhD, I studied the mechanisms that control inflammation which, when disturbed, can lead to a severe autoimmune/ auto inflammatory disease, rheumatoid arthritis (RA). My work was focused on the analysis of two aspects in these mechanisms: first, I participated to an analysis of the roles of the endonuclease DICER (involved in the biogenesis of microRNAs – miRs) in the pathogenesis of RA, specifically in fibroblast-like synoviocytes (FLS), which are resident cells of the synovial cavity. I also initiated a study aiming at the identification of the FLS transcriptome which is regulated by miRs in RA patients. This approach will provide an overview of the miR-dependent regulation in these cells and enable the identification of in vivo validated miR-targeted mRNAs in RA. A second axis of my thesis project aimed at providing a better description of the intercellular dialogue in the joint cavity. For this, I was particularly interested in the role of plasmacytoid dendritic cells (pDCs) in the pathophysiology of RA. I demonstrated a protective role of these cells (initially described for their functions in antiviral defense) in the context of inflammatory arthritis in several mouse models. During this work, I had the opportunity to try an innovative therapeutic strategy based on the recruitment and activation of pDCs in the joints. This noninvasive and painless approach (topical application of cream containing 5% imiquimod) was effective in reducing inflammatory clinical symptoms and also improved biological markers such as bone erosion.

Esta tese se fundamenta na necessidade de novos tratamentos para o câncer do colo de útero visando o aumento da adesão dos pacientes aos tratamentos, assim como à qualidade de vida dos mesmos. Nesse sentido, formulações nanotecnológicas foram desenvolvidas com o objetivo de carrear o fármaco imiquimode para um local específico – a mucosa vaginal – esperando gerar melhores desempenhos nesse tratamento quando comparados com a formulação comercial. Três nanoestruturas com morfologias distintas foram propostas visando potencializar o efeito do fármaco em células de câncer cervical (SiHa). As formulações desenvolvidas compreenderam: nanoemulsões (NEimiq), nanocápsulas poliméricas (NCimiq) e nanocápsulas poliméricas revestidas com quitosana (NCimiq-chit). Observou-se que nanocápsulas poliméricas produzidas com poli(ε-caprolactona) apresentaram efeito mais pronunciado frente às células SiHa. Para tanto, essas formulações (NCimiq e NCimiq-chit) foram incorporadas em hidrogéis de quitosana e de hidroxietilcelulose a fim de possibilitar uma melhor futura aplicação para o paciente. Estudos envolvendo mucosa vaginal suína demonstraram que ambas as formulações são mucoadesivas e permeiam a mucosa vaginal. Porém, a formulação produzida com hidrogel de quitosana (NCimiq) apresentou maior desempenho. Esta foi a formulação escolhida para dar continuidade aos estudos deste trabalho, sendo objeto de estudo posterior em cultura de células SiHa a fim de elucidar o mecanismo de ação da mesma. Esses estudos demonstraram que há uma ocorrência de processos combinados de diminuição da viabilidade celular de maneira tempo-dependente e que mecanismos como apoptose, autofagia e parada de ciclo celular estão presentes. Essa formulação (NCimiq) apresentou porcentagens de morte celular significativas, mesmo utilizando baixas concentrações do fármaco. Portanto, os achados desta tese constataram que nanoestruturas modulam efetivamente a interação do fármaco com as células. / This thesis deals with the need of new treatments for cervical cancer in order to increase the adherence of patients to the treatment as well as to improve their quality of life. In this sense, nanotechnological formulations were developed to carry imiquimod to a specific site – the vaginal mucosa – expecting to obtain better performance than the commercial drug in the cervical cancer treatment. Three nanostructures with different morphologies were proposed to potentilize the drug effect on cervical cancer cells (SiHa). The developed formulations are: nanoemulsions (NEimiq), polymeric nanocapsules (NCimiq) and polymeric nanocapsules coated with chitosan (NCimiq-chit). It was observed that polymeric nanocapsules produced with poly(ε-caprolactone) presented a stronger effect against SiHa cells. Therefore, formulations NCimiq and NCimiq-chit were incorporated into hydrogels of chitosan and hydroxyethylcellulose to enable a better future application on patients. The studies of this thesis involving porcine vaginal mucosa demonstrated that both formulations are mucoadhesive and that they provided a good drug permeation. However, the formulation produced with chitosan hydrogel (NCimiq) showed a better performance. This formulation was therefore chosen to follow the next steps of this work, conducted in SiHa cell culture to elucidate its action mechanism. This study demonstrated that there is an occurrence of combined processes of decreasing cell viability in a time-dependent type. The study also showed that mechanisms such as apoptosis, autophagy and cell cycle arrest are simultaneously present. The formulation NCimiq presented a significantly percentage of cellular death, even when low concentrations of the drug were used. Consequently, the findings of this thesis indicate that nanostructures effectively modulate the interaction of the drug with the cancer cells.

Στόχος της παρούσας ερευνητικής εργασίας είναι να διερευνηθεί η επίδραση της ιμικουϊμόδης, ενός τοπικώς εφαρμοζόμενου στο δέρμα αγωνιστού των Toll-like 7 υποδοχέων (Τoll-like receptors, ΤLRs), στην συστηματική ανοσία σε ασθενείς με δερματικές HSV και HPV λοιμώξεις. Πιο αναλυτικά θέλουμε να διερευνήσουμε μήπως η τοπική χορήγηση ιμικουϊμόδης μπορεί να επηρεάσει τις κυτταροκίνες που συμμετέχουν στους μηχανισμούς φυσικής και επίκτητης ανοσίας και τους κυτταρικούς πληθυσμούς και όπως είναι τα Τ κύτταρα (βοηθητικά, κυτταροτοξικά, παρθενικά, μνήμης, ενεργοποιημένα και ρυθμιστικά), τα Β κύτταρα και τα φυσικά φονικά κύτταρα (ΝΚ). Οι TLRs είναι ο συνδετικός κρίκος μεταξύ φυσικής και επίκτητης ανοσίας εφόσον είναι οι υποδοχείς που αναγνωρίζουν και προσδένουν μοριακές δομές των παθογόνων (pathogen associated molecular patterns-PAMPs). Η έκφραση TLRs έχει εντοπιστεί σε πολλούς κυτταρικούς όπως ουδετερόφιλα, μακροφάγα, δεντριτικά κύτταρα, ενδοθηλιακά κύτταρα της δερμίδας, επιθηλιακά κύτταρα των βλεννογόνων, Β και Τ κύτταρα. Η ενεργοποίηση των TLRs οδηγεί σε έκκριση κυτταροκινών και χημειοκινών που έχουν σαν στόχο την ενεργοποίηση της φυσικής ανοσίας, που προκαλεί τελικά την δημιουργία της επίκτητης ανοσίας. Οι ιμιδαζοκινολίνες συνιστούν μία κατηγορία νουκλεοσιδικών αναλόγων που αναπτύχθηκαν σαν αντιϊκοί παράγοντες. Η ιμικουϊμόδη είναι ένας εκπρόσωπος των ιμιδαζοκινολινών που χρησιμοποιείται για την θεραπεία των κονδυλωμάτων και των επιθηλιακών καρκίνων του δέρματος. Η ιμικουϊμόδη (imiquimod) είναι προσδέτης των TLRs - πιο ειδικά του TLR-7 και χορηγείται τοπικά υπό την μορφή αλοιφής. Έχει χαρακτηριστεί σαν ανοσορρυθμιστικός παράγοντας, γιατί μέσω της πρόσδεσης της στον TLR-7 προκαλεί ένα σύνολο αντιδράσεων όπως η αύξηση της κυτταροτοξικότητας των φυσικών φονικών κυττάρων (ΝΚ), η ενεργοποίηση των μακροφάγων να εκκρίνουν κυτταροκίνες, η ενεργοποίηση και η μετανάστευση των κυττάρων του Langerhans από το δέρμα στους λεμφαδένες και η επαγωγή του πολλαπλασιασμού και της διαφοροποίησης των Τ και Β κυττάρων. Επί του παρόντος παραμένει άγνωστο αν η τοπικώς εφαρμοζόμενη ιμικουϊμόδη είναι σε θέση να εξασκεί επιπλέον της τοπικής και συστηματική ανοσοτροποποιητική δράση. Για τον λόγο αυτό στην παρούσα εργασία μελετήσαμε την επίδραση της ιμικουϊμόδης στο ανοσοποιητικό σύστημα υγιών μαρτύρων και ασθενών με HSV και HPV λοιμώξεις τόσο σε επίπεδο κυτταροκινών με την μέθοδο Cytometric bead array (CBA) όσο και σε επίπεδο κυκλοφορούντων κυτταρικών πληθυσμών στο περιφερειακό αίμα με κυτταρομετρία ροής (FACS). Τα αποτελέσματα μας υποδεικνύουν ότι η τοπική εφαρμογή της ιμικουϊμόδης είναι ικανή να προκαλέσει συστηματική ανοσοτροποποίηση και αυτό αποτελεί μια νέα και αποτελεσματική εναλλακτική θεραπεία. / In this study we investigate the influence of imiquimod, a topical applicable agonist of Toll-like receptor 7 (TLR7) in skin, on systemic immunity of patients with herpes simplex virus (HSV) and human papilloma virus (HPV) infections. We investigated whether the topical application of imiquimod affects cytokines which participate in mechanisms of innate and adaptive immune responses and cell populations, such as T-cells (helper, cytotoxic, naïve, memory, activated and regulatory), B-cells and natural killer cells (NK cells). TLRs help to bridge the gap between innate and adaptive immunity, since they recognize and bind pathogen associated molecular patterns of pathogens. The expression of TLRs has been detected on neutrophils, macrophages, dendritic cells, dermal endothelial cells, mucosal epithelial cells, T and B cells. Activation of TLRs mediates the release of cytokines and chemokines that recruit innate immune responses, which lead to the formation of adaptive immunity. Imidazoquinolines represent a group of nucleoside analogues that have been used as antiviral agents. Imiquimod belongs to imidazoquinolines and it has been used for the treatment of genital warts (caused by HPV) and cutaneous cancers. Imiquimod is a ligand of TLR7 and a patient-applied cream. It has been characterized as an immune response modifier, because through its binding to TLR7 it generates a cascade of reactions such as upregulation of cytotoxicity in NK cells, activation of macrophages to secrete cytokines, activation and migration of Langerhans cells from skin to lymph nodes and induction of proliferation and differentiation of T and B cells. Currently it is unknown if the topical application of imiquimod is able to exert, apart from a local, also a systemic immunomodulatory action. For this reason we tried to elucidate the effects of imiquimod on the immune system of patients with HSV and HPV infections at two levels. First we have measured the circulating cell populations in whole blood using flow cytometry and secondly we have determined the serum levels of IL-2, IL-4, IL-6, IL-10, TNF-α, IFN-α and IFN-γ with cytometric bead array. The results demonstrate that our hypothesis is correct and imiquimod could be an alternative and effective treatment of cutaneous HSV and HPV infections.

As Ceratoses Actínicas (CA) são lesões hiperceratóticas, displásicas de pele. Estão comumente localizadas em áreas expostas ao sol como couro cabeludo, face e antebraços. Acredita-se que a radiação ultravioleta (RUV) cumulativa seja o maior fator etiológico, considerando-se, também, a imunossupressão e infecção pelo papilomavírus humano(HPV) fatores contribuintes importantes. Existe o potencial de uma CA se transformar em Carcinoma Espinocelular (CEC) e, dessa forma, indica-se tratamento para as referidas lesões. Terapias tradicionais como crioterapia, curetagem e eletrocoagulação, medicações tópicas como 5-fluorouracil (5-FU) são ainda habituais. Porém novas opções como terapia fotodinâmica, diclofenaco e imiquimod 5% creme apresentam boa eficácia e perfil menor de efeitos colaterais, embora possam representar custo maior. A Terapia Fotodinâmica (TFD) envolve o uso de um agente fotossensibilizante, oxigênio e luz de comprimento de onda específico para causar morte celular. O fotossensibilizante geralmente utilizado é o ALA (ácido aminodeltalevulínico) ou seu éster metilaminolevulinato (MAL). No tecido lesionado, esses são convertidos em porfirinas fotoativas (PFAs) por enzimas da via biossintética do heme. A ativação é realizada por meio de luzes de comprimentos de onda que variam de 405 nm a 635 nm. Células displásicas ou neoplásicas produzem maior quantidade de porfirinas que os queratinócitos normais, sendo destruídas durante a aplicação da luz. O imiquimod 5% creme é um imunomodulador que estimula a resposta imune inata através da indução, síntese e liberação de citocinas. Isso resulta em efeitos antitumorais e antivirais indiretos. Seu uso tópico é eficaz e liberado para o tratamento de CA, Carcinoma Basocelular (CBC) superficial, Doença de Bowen (DB) e verrugas vulgares. Este trabalho tem como objetivo comparar duas recentes opções de tratamento para CA, a Terapia Fotodinâmica com metilaminolevulinato e o imiquimod 5% creme, por não existirem, na literatura atual, estudos comparativos dessas duas opções de tratamento. Foram selecionadas 12 pacientes com CAs que foram submetidas primeiramente à 1 sessão de TFD com MAL num lado da face e, 1 mês após, iniciaram o tratamento com imiquimod 5% creme aplicado no lado contralateral, duas vezes na semana, durante 16 semanas. A randomização foi realizada para determinar a hemiface para cada tratamento. Na primeira semana após a realização da TFD e mensalmente durante o tratamento com imiquimod, as pacientes foram avaliadas em relação aos efeitos colaterais dos tratamentos. Seis meses após entrarem no estudo, ambos os tratamentos foram analisados por um investigador cego para sua eficácia, tolerabilidade e seu resultado cosmético. Previamente ao tratamento as pacientes apresentaram um total de 245 lesões de CAs, sendo 120 lesões no lado submetido à TFD e 125 no lado tratado com imiquimod. Após o tratamento o número total de lesões diminuiu para 34 no lado tratado com TFD e para 30 no lado tratado com imiquimod, respectivamente. Não foram observadas diferenças estatisticamente significativas na eficácia de ambos os tratamentos e na frequência de efeitos colaterais. Entretanto, os pacientes, significativamente preferiram o tratamento com a terapia fotodinâmica, talvez pela rapidez do método, comparado com a aplicação tópica do creme de imiquimod a 5%. / Background: Actinic keratosis (AK) represents an initial process that may lead to in situ or invasive squamous cell carcinoma. The importance of its early diagnosis and treatment is well-established. There are several effective options available for the treatment of actinic keratosis, including topical imiquimod 5% cream and photodynamic therapy (PDT). Objetive: To compare the efficacy and patient preference between topical MAL-PDT and imiquimod 5% cream for the treatment of AK. Methods: Twelve patients, with a total of 245 lesions, underwent treatment with MAL-PDT and imiquimod 5% cream. Randomization was performed to determine the hemiface (right or left) for each therapy. First, patients were submitted to MAL-PDT. After one month, they started to use imiquimod on the opposite side of the face, twice a week, for 16 weeks. Six months after entering the study, both treatments were analyzed by a blinded investigator for their effectiveness, tolerability and cosmetic result. Results: Both treatments showed a good therapeutic response. 72% of the lesions treated with MAL-PDT were completely cleared, and 76% of those treated with imiquimod. The mean size of the residual lesions after the treatments was similar. Ten patients (83%) preferred MAL-PDT rather than imiquimod. (p: 0.03). Conclusions: Both MAL-PDT and imiquimod are effective in clearing AKs. Our results showed similar efficacy and good cosmetic outcomes with both treatments. However, a significant percentage of the subjects preferred MAL – PDT.

Esta tese se fundamenta na necessidade de novos tratamentos para o câncer do colo de útero visando o aumento da adesão dos pacientes aos tratamentos, assim como à qualidade de vida dos mesmos. Nesse sentido, formulações nanotecnológicas foram desenvolvidas com o objetivo de carrear o fármaco imiquimode para um local específico – a mucosa vaginal – esperando gerar melhores desempenhos nesse tratamento quando comparados com a formulação comercial. Três nanoestruturas com morfologias distintas foram propostas visando potencializar o efeito do fármaco em células de câncer cervical (SiHa). As formulações desenvolvidas compreenderam: nanoemulsões (NEimiq), nanocápsulas poliméricas (NCimiq) e nanocápsulas poliméricas revestidas com quitosana (NCimiq-chit). Observou-se que nanocápsulas poliméricas produzidas com poli(ε-caprolactona) apresentaram efeito mais pronunciado frente às células SiHa. Para tanto, essas formulações (NCimiq e NCimiq-chit) foram incorporadas em hidrogéis de quitosana e de hidroxietilcelulose a fim de possibilitar uma melhor futura aplicação para o paciente. Estudos envolvendo mucosa vaginal suína demonstraram que ambas as formulações são mucoadesivas e permeiam a mucosa vaginal. Porém, a formulação produzida com hidrogel de quitosana (NCimiq) apresentou maior desempenho. Esta foi a formulação escolhida para dar continuidade aos estudos deste trabalho, sendo objeto de estudo posterior em cultura de células SiHa a fim de elucidar o mecanismo de ação da mesma. Esses estudos demonstraram que há uma ocorrência de processos combinados de diminuição da viabilidade celular de maneira tempo-dependente e que mecanismos como apoptose, autofagia e parada de ciclo celular estão presentes. Essa formulação (NCimiq) apresentou porcentagens de morte celular significativas, mesmo utilizando baixas concentrações do fármaco. Portanto, os achados desta tese constataram que nanoestruturas modulam efetivamente a interação do fármaco com as células. / This thesis deals with the need of new treatments for cervical cancer in order to increase the adherence of patients to the treatment as well as to improve their quality of life. In this sense, nanotechnological formulations were developed to carry imiquimod to a specific site – the vaginal mucosa – expecting to obtain better performance than the commercial drug in the cervical cancer treatment. Three nanostructures with different morphologies were proposed to potentilize the drug effect on cervical cancer cells (SiHa). The developed formulations are: nanoemulsions (NEimiq), polymeric nanocapsules (NCimiq) and polymeric nanocapsules coated with chitosan (NCimiq-chit). It was observed that polymeric nanocapsules produced with poly(ε-caprolactone) presented a stronger effect against SiHa cells. Therefore, formulations NCimiq and NCimiq-chit were incorporated into hydrogels of chitosan and hydroxyethylcellulose to enable a better future application on patients. The studies of this thesis involving porcine vaginal mucosa demonstrated that both formulations are mucoadhesive and that they provided a good drug permeation. However, the formulation produced with chitosan hydrogel (NCimiq) showed a better performance. This formulation was therefore chosen to follow the next steps of this work, conducted in SiHa cell culture to elucidate its action mechanism. This study demonstrated that there is an occurrence of combined processes of decreasing cell viability in a time-dependent type. The study also showed that mechanisms such as apoptosis, autophagy and cell cycle arrest are simultaneously present. The formulation NCimiq presented a significantly percentage of cellular death, even when low concentrations of the drug were used. Consequently, the findings of this thesis indicate that nanostructures effectively modulate the interaction of the drug with the cancer cells.

As Ceratoses Actínicas (CA) são lesões hiperceratóticas, displásicas de pele. Estão comumente localizadas em áreas expostas ao sol como couro cabeludo, face e antebraços. Acredita-se que a radiação ultravioleta (RUV) cumulativa seja o maior fator etiológico, considerando-se, também, a imunossupressão e infecção pelo papilomavírus humano(HPV) fatores contribuintes importantes. Existe o potencial de uma CA se transformar em Carcinoma Espinocelular (CEC) e, dessa forma, indica-se tratamento para as referidas lesões. Terapias tradicionais como crioterapia, curetagem e eletrocoagulação, medicações tópicas como 5-fluorouracil (5-FU) são ainda habituais. Porém novas opções como terapia fotodinâmica, diclofenaco e imiquimod 5% creme apresentam boa eficácia e perfil menor de efeitos colaterais, embora possam representar custo maior. A Terapia Fotodinâmica (TFD) envolve o uso de um agente fotossensibilizante, oxigênio e luz de comprimento de onda específico para causar morte celular. O fotossensibilizante geralmente utilizado é o ALA (ácido aminodeltalevulínico) ou seu éster metilaminolevulinato (MAL). No tecido lesionado, esses são convertidos em porfirinas fotoativas (PFAs) por enzimas da via biossintética do heme. A ativação é realizada por meio de luzes de comprimentos de onda que variam de 405 nm a 635 nm. Células displásicas ou neoplásicas produzem maior quantidade de porfirinas que os queratinócitos normais, sendo destruídas durante a aplicação da luz. O imiquimod 5% creme é um imunomodulador que estimula a resposta imune inata através da indução, síntese e liberação de citocinas. Isso resulta em efeitos antitumorais e antivirais indiretos. Seu uso tópico é eficaz e liberado para o tratamento de CA, Carcinoma Basocelular (CBC) superficial, Doença de Bowen (DB) e verrugas vulgares. Este trabalho tem como objetivo comparar duas recentes opções de tratamento para CA, a Terapia Fotodinâmica com metilaminolevulinato e o imiquimod 5% creme, por não existirem, na literatura atual, estudos comparativos dessas duas opções de tratamento. Foram selecionadas 12 pacientes com CAs que foram submetidas primeiramente à 1 sessão de TFD com MAL num lado da face e, 1 mês após, iniciaram o tratamento com imiquimod 5% creme aplicado no lado contralateral, duas vezes na semana, durante 16 semanas. A randomização foi realizada para determinar a hemiface para cada tratamento. Na primeira semana após a realização da TFD e mensalmente durante o tratamento com imiquimod, as pacientes foram avaliadas em relação aos efeitos colaterais dos tratamentos. Seis meses após entrarem no estudo, ambos os tratamentos foram analisados por um investigador cego para sua eficácia, tolerabilidade e seu resultado cosmético. Previamente ao tratamento as pacientes apresentaram um total de 245 lesões de CAs, sendo 120 lesões no lado submetido à TFD e 125 no lado tratado com imiquimod. Após o tratamento o número total de lesões diminuiu para 34 no lado tratado com TFD e para 30 no lado tratado com imiquimod, respectivamente. Não foram observadas diferenças estatisticamente significativas na eficácia de ambos os tratamentos e na frequência de efeitos colaterais. Entretanto, os pacientes, significativamente preferiram o tratamento com a terapia fotodinâmica, talvez pela rapidez do método, comparado com a aplicação tópica do creme de imiquimod a 5%. / Background: Actinic keratosis (AK) represents an initial process that may lead to in situ or invasive squamous cell carcinoma. The importance of its early diagnosis and treatment is well-established. There are several effective options available for the treatment of actinic keratosis, including topical imiquimod 5% cream and photodynamic therapy (PDT). Objetive: To compare the efficacy and patient preference between topical MAL-PDT and imiquimod 5% cream for the treatment of AK. Methods: Twelve patients, with a total of 245 lesions, underwent treatment with MAL-PDT and imiquimod 5% cream. Randomization was performed to determine the hemiface (right or left) for each therapy. First, patients were submitted to MAL-PDT. After one month, they started to use imiquimod on the opposite side of the face, twice a week, for 16 weeks. Six months after entering the study, both treatments were analyzed by a blinded investigator for their effectiveness, tolerability and cosmetic result. Results: Both treatments showed a good therapeutic response. 72% of the lesions treated with MAL-PDT were completely cleared, and 76% of those treated with imiquimod. The mean size of the residual lesions after the treatments was similar. Ten patients (83%) preferred MAL-PDT rather than imiquimod. (p: 0.03). Conclusions: Both MAL-PDT and imiquimod are effective in clearing AKs. Our results showed similar efficacy and good cosmetic outcomes with both treatments. However, a significant percentage of the subjects preferred MAL – PDT.