A doença enxerto contra hospedeiro é uma complicação comum nos pacientes submetidos ao transplante de medula óssea alogênico. Com o objetivo de contribuir para o esclarecimento da participação das células de Langerhans na doença enxerto contra hospedeiro crônica (GVHDc) quando de sua ocorrência na mucosa bucal, foram analisados 40 pacientes oncohematológicos e hematológicos submetidos ao transplante de medula óssea alogênico no Hospital Amaral Carvalho, Jaú - SP. Cortes microscópicos de 3µm de espessura da mucosa jugal com padrão de normalidade (controle - 20 pacientes) e de pacientes transplantados com e sem GVHDc, foram avaliados em hematoxilina e eosina e pela técnica imuno-histoquímica padrão da estreptavidina-biotina-peroxidase utilizando-se o anticorpo monoclonal anti- CD1a. As células de Langerhans imunomarcadas foram quantificadas no epitélio da mucosa jugal, sendo o número médio destas células estatisticamente comparado entre os pacientes controle e os pacientes transplantados com e sem GVHDc. Os resultados demonstraram um maior número de células de Langerhans na mucosa jugal dos pacientes com GVHDc quando comparado aqueles sem GVHDc e ao grupo controle (p=0,001). Foi observado também, a presença de intenso infiltrado inflamatório crônico, justaepitelial, com desorganização das células da camada basal do epitélio, com vacuolização celular, satelitose, corpúsculos acidofílicos e ocorrência de clivagem entre e o epitélio e o tecido conjuntivo nos pacientes que desenvolveram GVHDc. Estes resultados sugerem que a células de Langerhans participa da doença enxerto contra hospedeiro crônica na mucosa jugal dos pacientes submetidos ao transplante de medula óssea alogênico, sendo provavelmente recrutadas pelo processo inflamatório e imunopatológico que caracteriza esta doença. / The graft versus host disease (GVHD) is a common complication in patients submited to alogeneic bone marrow transplantation. To understand the role of Langerhans cells in chronic GVHD (cGVHD) in oral mucosa, we analyzed 40 oncohematological or hematological patients who received alogeneic bone marrow transplantation at Hospital Amaral Carvalho, Jaú - SP. Slices of 3µm from normal oral mucosa (control - 20 patients) and transplanted patients with and without cGVHD were analyzed by hematoxylin-eosin technique and conventional immunohistochemistry of streptavidin-biotin peroxidase technique for monoclonal antibody anti-CD1a. The immunomarked Langerhans cells were quantified in the epithelium of oral mucosa; the average number of these cells was statistically significant when compared to the control group and patients with and without cGVHD. The results showed higher number of Langerhans cells in oral mucosa of cGVHD when we compared the control group and the group of patients with and without cGVHD (p=0,001). We also observed the presence of chronic juxtaepithelial inflammatory infiltrate, with basal layer epithelium desorganization, vacuolization, satellitosis, acidophilic bodies and presence of gap between epithelium and connective tissue of patients with cGVHD. These results suggest that Langerhans cells may have a role in cGVHD of oral mucosa in patients submited to alogeneic bone marrow transplantation, and they may be recruited by inflammatory and immunopathologic process that are characteristic in this disease.

A pele é o maior órgão do corpo humano e constitui a principal defesa do organismo contra agentes físicos e químicos, sendo também fundamental para evitar a perda de água por dessecação. Formada por três camadas distintas, mas complementares, sendo as duas principais denominadas derme e epiderme, contendo diferentes tipos celulares, como fibroblastos, queratinócitos, melanócitos, células de Merkel e células de Langerhans, sendo que estas últimas desempenham um papel fundamental na hipersensibilidade de contato. Devido à importância da manutenção da pele saudável para a vida humana, existe uma crescente necessidade da elaboração de substitutos de tecidos para o tratamento de feridos e doentes, assim como, há grande demanda de pele para testes químicos das áreas farmacêutica e cosmética. Outro fator de fundamental importância para o desenvolvimento de métodos alternativos in vitro, é a pressão mundial para que estes testes substituam os modelos animais. Esta abordagem vai de encontro aos novos conceitos de substituição, redução e refinamento na utilização de animais em estudos científicos, ditando o futuro da cultura celular e bioengenharia de tecidos. Graças ao grande desenvolvimento do cultivo celular e descoberta de que as células cultivadas podem ser reagrupadas de acordo com o delineamento experimental, se torna possível à criação de equivalentes dermoepidérmicos para estudos in vitro, como por exemplo, testes de cito e fototoxicidade ou avaliação da fase inicial da reação alérgica e processos de sensibilização da pele. Neste caso, se faz necessária a obtenção de grande quantidade de células de Langerhans imaturas. As células de Langerhans (CLs) são células dendríticas imaturas localizadas na epiderme e epitélio superficial que desempenham um papel central na imunidade da pele, agindo como verdadeiras sentinelas capazes de captar antígenos de contato. Desta forma, foram testados quatro diferentes protocolos para extração e criopreservação destas células, sendo ainda analisadas as suas características morfológicas e fenotípicas. Obtivemos resultados não expressivos quanto ao isolamento, pureza e marcação positiva para CD1a no Protocolo 2 (Expansor de Pele). Os Protocolo 1A (Coleta de Sobrenadante) e 3 (Epiderme + Gradiente de Ficoll Paque) ofereceram altos níveis de células marcadas positivamente para CD1a, apresentando a mesma qualidade de marcação. No entanto, o Protocolo 3 forneceu um maior número de células viáveis, e uma maior pureza da amostra, uma vez que só utiliza a epiderme para a obtenção da suspensão de células, o que o coloca como modelo a ser seguido em posteriores experimentos. Os métodos aqui apontados como mais promissores, podem ser reproduzidos em laboratórios de cultura celular convencionais, contribuindo para aumentar a reprodutibilidade e confiabilidade de resultados experimentais relativos às CLs. Da mesma forma, avaliamos a utilização dos equivalentes de pele humana para a realização de testes in vitro de cito e fototoxicidade, os quais podem de fato reduzir a utilização de modelos animais para identificação do perfil tóxico de uma substância ou de formulações mais complexas. / The skin is the largest organ from the human body and constitutes the main protection of the organism against physical and chemical agents and it is also fundamental to avoid water loss by desiccation. Formed by three distinct stratus, yet complementary, being the two main called dermis and epidermis, containing different cell types, as fibroblasts, keratinocytes, melanocytes, Merkel cells and Langerhans cells (LCs), being these latter fundamental in the contact hypersensitivity. Due to the importance of the healthy skin maintenance to the human´s life, there is a growing need of elaboration of skin models to the treatment of injured and diseased, as well as there is a big demand of skin models to chemical tests from the pharmaceutics and cosmetology fields. Another factor of fundamental importance to the development of alternative in vitro methods is the worldwide pressure for these tests to replace animal models. This approach meets new concepts of replacement, reduction and refinement in the use of animals in scientific studies, dictating the future of cell culture and bioengineering of skin models. Thanks to the large development of cell culture and the discovery that the cultured cells can be regrouped according to the experimental delineation, the creation of skin models to in vitro studies is made possible, as for instance, tests of cytotoxicity and phototoxicity or evaluation of the initial phase from the allergic reaction and processes of skin sensitization. In this case, it is necessary the achievement of a large amount of immature Langerhans cells. The LCs are immature dendritic cells located in the epidermis and superficial epithelium that perform a central role in the skin immunity, acting as real sentinels able to collect contact antigens. Accordingly, were tested four different protocols for extraction and cryopreservation of these cells, and further analyzed its morphological and phenotypic features. We obtained no significant results in relation to the isolation, purity and CD1a positive expression in the Protocol 2. The Protocols 1A and 3 offered high levels of CD1a positively marked cells, showing the same expression levels. However, the Protocol 3 provided a bigger number of viable cells and a high purity yield, since it only uses the epidermis to obtain the single cell suspension, which places it as a model to be followed in subsequent experiments. The methods appointed here as the most promising, can be reproduced in conventional cell culture labs, contributing to increase the reproducibility and reliability of experimental results related to the LCs. In the same way, we evaluated the use of the human skin equivalents to the accomplishment of in vitro tests of cyto and phototoxicity, which can in fact reduce the use of animal models to the identification of single substances toxicity or even complex formulations.

Com o propósito de colaborar na elucidação da fisiopatologia do pênfigo foliáceo endêmico (PFE), realizou-se titulação de anticorpos séricos e quantificação das células de Langerhans (CL) e células dendríticas dérmicas (CD) na pele de pacientes com PFE. Sangue e biopsia de pele de lesão ativa foram colhidos de 22 pacientes com PFE e, em 13 deles realizou-se também biopsia de pele normal não adjacente à lesão, em área não exposta ao sol. Dos 22 pacientes, 13 apresentavam a forma clínica localizada e 9, generalizada; 11 estavam em tratamento. O grupo controle constituiu-se de pele normal obtida da região torácica anterior de 8 cadáveres e de 12 mulheres submetidas à cirurgia plástica (mastoplastia). A imunofluorescência indireta (IFI) foi realizada com pele humana normal da região abdominal como substrato e anti-IgG humana. A identificação das CL e CD foi feita pela técnica imunohistoquímica da avidina-biotina-peroxidase com o anticorpo anti-CD1a e quantificação por análise morfométrica. Houve correlação entre a titulação de anticorpos séricos por IFI e a forma clínica do PFE, sendo esse maior na forma generalizada. O número de CL na lesão (60,18 CL/ mm2; 5,00 CL/ mm de membrana basal (MB); 3,55 CL/mm de camada córnea) e na pele normal do PFE (28,45 CL/ mm2; 2,50 CL/ mm de MB; 2,87 CL/mm de camada córnea) foi semelhante ao número de CL na pele dos grupos controles de cirurgia plástica (72,35 CL/ mm2; 4,53 CL/ mm de MB; 4,42 CL/mm de camada córnea) e de cadáveres (47,15 CL/ mm2; 2,53 CL/ mm de MB; 2,42 CL/mm de camada córnea). O número de CD dérmicas na pele lesada de PFE (0,98 CD/ mm de MB) foi semelhante ao do grupo controle de plástica de mama (0,48 CD/ mm de MB), porém maior do que o do grupo cadáver (0,13 CD/ mm de MB). A razão entre o número de CL e CD dérmicas foi menor na pele lesada do paciente com PFE comparada à dos grupos controles, confirmando maior número de CD na derme. Num mesmo paciente, as CL e CD da derme encontravam-se em maior número na lesão de PFE (61,50 CL/ mm2; 5,49 CL/ mm de MB; 6,64 CL/mm de camada córnea, 0,86 CD dérmicas/ mm de MB) quando comparadas à pele normal (28,45 CL/ mm2; 2,50 CL/ mm de MB; 2,87 CL/mm de camada córnea; 0,04 CD dérmicas/ mm de MB). Houve correlação direta entre o número de CD dérmicas na lesão de PFE e a titulação de anticorpos séricos por IFI (r=0,4779, p<0,05), indicativo de que as CD dérmicas poderiam estar participando da patogênese do PFE. Poder-se-ia supor que as CD estariam transitando pela derme em direção ao linfonodo regional, exercendo função estimuladora de linfócitos T na indução da resposta imune e produção de auto-anticorpos. / In order to contribute to the elucidation of pathophysiology of Endemic Pemphigus Foliaceus (EPF) titration of serum antibodies and quantification of Langerhans Cells (LC) and dermal dendritic cells (DC) in skin of patients with EPF were made. Blood and skin biopsies (lesional skin) of 22 EPF patients were collected and, in 13 of them, biopsies of normal sun-protected skin of non perilesional area were collected too. 13 patients had localized lesions and 9, generalized; 11 were in treatment. Control groups consisted of thoracic normal skin from 8 cadavers and 12 women submitted to breast plastic surgery. For indirect immunofluorescence (IFI), abdominal normal skin as substrate and anti-IgG were used. LC and dermal DC identification was done by immunohistochemistry with anti-CD1a antibody and quantification by morfometric analysis. It was found association between titration of serum antibodies by IFI and clinical form of EPF, with greater titration in the generalized one. LC number in lesion (60.18 LC/ mm2, 5.00 LC/ mm basement membrane (BM), 3.55 LC/mm stratum corneum) and normal skin of EPF patients (28.45 LC/ mm2, 2.50 LC/ mm BM, 2.87 LC/mm stratum corneum) was similar to LC number in skin of plastic surgery (72.35 LC/ mm2, 4.53 LC/ mm BM, 4.42 LC/mm stratum corneum) and cadaver controls (47.15 LC/ mm2, 2.53 LC/ mm BM, 2.42 LC/mm stratum corneum). Dermal DC number in lesional skin of EPF patients (0.98 DC/ mm BM) was similar to the DC number of plastic surgery controls (0.48 DC/ mm BM), but greater than DC number in cadaver controls (0.13 DC/ mm BM). The ratio LC number/ dermal DC number was smaller in lesional EPF skin than in controls, confirming the greatest DC number in dermis. In the same patient, LC and dermal DC were in greater amounts in EPF lesional skin (61.50 LC/ mm2, 5.49 LC/ mm BM, 6.64 LC/mm stratum corneum, 0.86 dermal DC/ mm BM) than in normal skin (28.45 LC/ mm2, 2.50 LC/ mm BM, 2.87 LC/mm stratum corneum, 0.04 dermal DC/ mm BM). It was found direct association between dermal DC number in lesional skin of EPF patients and titration of antibodies by IFI (r=0.4779, p<0.05), confirming that dermal DC could play an important role in EPF pathogenesis. It could be proposed that DC would be in transit through the dermis towards the regional lymph node, stimulating T lymphocytes to produce autoantibodies.

A doença enxerto contra hospedeiro é uma complicação comum nos pacientes submetidos ao transplante de medula óssea alogênico. Com o objetivo de contribuir para o esclarecimento da participação das células de Langerhans na doença enxerto contra hospedeiro crônica (GVHDc) quando de sua ocorrência na mucosa bucal, foram analisados 40 pacientes oncohematológicos e hematológicos submetidos ao transplante de medula óssea alogênico no Hospital Amaral Carvalho, Jaú - SP. Cortes microscópicos de 3µm de espessura da mucosa jugal com padrão de normalidade (controle - 20 pacientes) e de pacientes transplantados com e sem GVHDc, foram avaliados em hematoxilina e eosina e pela técnica imuno-histoquímica padrão da estreptavidina-biotina-peroxidase utilizando-se o anticorpo monoclonal anti- CD1a. As células de Langerhans imunomarcadas foram quantificadas no epitélio da mucosa jugal, sendo o número médio destas células estatisticamente comparado entre os pacientes controle e os pacientes transplantados com e sem GVHDc. Os resultados demonstraram um maior número de células de Langerhans na mucosa jugal dos pacientes com GVHDc quando comparado aqueles sem GVHDc e ao grupo controle (p=0,001). Foi observado também, a presença de intenso infiltrado inflamatório crônico, justaepitelial, com desorganização das células da camada basal do epitélio, com vacuolização celular, satelitose, corpúsculos acidofílicos e ocorrência de clivagem entre e o epitélio e o tecido conjuntivo nos pacientes que desenvolveram GVHDc. Estes resultados sugerem que a células de Langerhans participa da doença enxerto contra hospedeiro crônica na mucosa jugal dos pacientes submetidos ao transplante de medula óssea alogênico, sendo provavelmente recrutadas pelo processo inflamatório e imunopatológico que caracteriza esta doença. / The graft versus host disease (GVHD) is a common complication in patients submited to alogeneic bone marrow transplantation. To understand the role of Langerhans cells in chronic GVHD (cGVHD) in oral mucosa, we analyzed 40 oncohematological or hematological patients who received alogeneic bone marrow transplantation at Hospital Amaral Carvalho, Jaú - SP. Slices of 3µm from normal oral mucosa (control - 20 patients) and transplanted patients with and without cGVHD were analyzed by hematoxylin-eosin technique and conventional immunohistochemistry of streptavidin-biotin peroxidase technique for monoclonal antibody anti-CD1a. The immunomarked Langerhans cells were quantified in the epithelium of oral mucosa; the average number of these cells was statistically significant when compared to the control group and patients with and without cGVHD. The results showed higher number of Langerhans cells in oral mucosa of cGVHD when we compared the control group and the group of patients with and without cGVHD (p=0,001). We also observed the presence of chronic juxtaepithelial inflammatory infiltrate, with basal layer epithelium desorganization, vacuolization, satellitosis, acidophilic bodies and presence of gap between epithelium and connective tissue of patients with cGVHD. These results suggest that Langerhans cells may have a role in cGVHD of oral mucosa in patients submited to alogeneic bone marrow transplantation, and they may be recruited by inflammatory and immunopathologic process that are characteristic in this disease.

This project has provided a better understanding of the human conjunctiva, the glistening tissue covering the white of the eye, at the cellular level. The observations of this study may serve as a useful marker against which changes in conjunctival tissue due to disease, surgery, drug therapy or contact lens wear can be assessed. Laser scanning confocal microscopy was used to observe and measure characteristics the conjunctiva of healthy human volunteer subjects. It was concluded that this technique is a powerful tool for studying the human conjunctiva and assessing key aspects of the structure of this tissue. The effects of contact lens wear on the conjunctiva can be investigated effectively at a cellular level using this technology.

Submitted by Suelen Reis (suziy.ellen@gmail.com) on 2013-04-23T18:34:23Z No. of bitstreams: 1 Dissertacao-luciano-sec.pdf: 1411311 bytes, checksum: 683e953cb766c31b8aeb9633635ca645 (MD5) / Approved for entry into archive by Rodrigo Meirelles(rodrigomei@ufba.br) on 2013-05-08T11:40:25Z (GMT) No. of bitstreams: 1 Dissertacao-luciano-sec.pdf: 1411311 bytes, checksum: 683e953cb766c31b8aeb9633635ca645 (MD5) / Made available in DSpace on 2013-05-08T11:40:25Z (GMT). No. of bitstreams: 1 Dissertacao-luciano-sec.pdf: 1411311 bytes, checksum: 683e953cb766c31b8aeb9633635ca645 (MD5) Previous issue date: 2005 / Os Granulomas Dentários e Cistos Radiculares representam lesões periapicais crônicas que, frequentemente, acometem os ossos maxilares. As Células de Langerhans são células dendríticas, responsáveis pela apresentação de antígenos aos Linfócitos T, que desempenham importante função nos tecidos epiteliais, bem como na patogênese das lesões periapicais. O presente estudo analisou a expressão das Células de Langerhans, através da técnica imuno-histoquímica para o marcador CD1a em 18 casos de Granuloma Dentário (GD) e 26 casos de Cisto Radicular (CR). Essas células dendríticas foram observadas em 11,1% dos Granulomas Dentários e em 69,2% dos Cistos Radiculares, mostrando correlação estatisticamente significante (p-valor=0,000. Teste de Fisher). Nos Cistos Radiculares, as CLs exibiram tanto a forma arredondada quanto a dendrítica, em todas as camadas epiteliais. Já nos Granulomas Dentários, as CLs foram vistas apenas no tecido de granulação com densidade discreta de marcação. Apesar de termos encontrado uma correlação entre densidade de marcação e espessura de epitélio, bem como entre imunomarcação e intensidade inflamatória, não foi observada representatividade estatística entre essas correlações. Dos resultados obtidos conclui-se que as Células de Langerhans parecem influenciar na imunopatogênese das lesões periapicais aqui estudadas, principalmente nos Cistos Radiculares. / Salvador

Com o propósito de colaborar na elucidação da fisiopatologia do pênfigo foliáceo endêmico (PFE), realizou-se titulação de anticorpos séricos e quantificação das células de Langerhans (CL) e células dendríticas dérmicas (CD) na pele de pacientes com PFE. Sangue e biopsia de pele de lesão ativa foram colhidos de 22 pacientes com PFE e, em 13 deles realizou-se também biopsia de pele normal não adjacente à lesão, em área não exposta ao sol. Dos 22 pacientes, 13 apresentavam a forma clínica localizada e 9, generalizada; 11 estavam em tratamento. O grupo controle constituiu-se de pele normal obtida da região torácica anterior de 8 cadáveres e de 12 mulheres submetidas à cirurgia plástica (mastoplastia). A imunofluorescência indireta (IFI) foi realizada com pele humana normal da região abdominal como substrato e anti-IgG humana. A identificação das CL e CD foi feita pela técnica imunohistoquímica da avidina-biotina-peroxidase com o anticorpo anti-CD1a e quantificação por análise morfométrica. Houve correlação entre a titulação de anticorpos séricos por IFI e a forma clínica do PFE, sendo esse maior na forma generalizada. O número de CL na lesão (60,18 CL/ mm2; 5,00 CL/ mm de membrana basal (MB); 3,55 CL/mm de camada córnea) e na pele normal do PFE (28,45 CL/ mm2; 2,50 CL/ mm de MB; 2,87 CL/mm de camada córnea) foi semelhante ao número de CL na pele dos grupos controles de cirurgia plástica (72,35 CL/ mm2; 4,53 CL/ mm de MB; 4,42 CL/mm de camada córnea) e de cadáveres (47,15 CL/ mm2; 2,53 CL/ mm de MB; 2,42 CL/mm de camada córnea). O número de CD dérmicas na pele lesada de PFE (0,98 CD/ mm de MB) foi semelhante ao do grupo controle de plástica de mama (0,48 CD/ mm de MB), porém maior do que o do grupo cadáver (0,13 CD/ mm de MB). A razão entre o número de CL e CD dérmicas foi menor na pele lesada do paciente com PFE comparada à dos grupos controles, confirmando maior número de CD na derme. Num mesmo paciente, as CL e CD da derme encontravam-se em maior número na lesão de PFE (61,50 CL/ mm2; 5,49 CL/ mm de MB; 6,64 CL/mm de camada córnea, 0,86 CD dérmicas/ mm de MB) quando comparadas à pele normal (28,45 CL/ mm2; 2,50 CL/ mm de MB; 2,87 CL/mm de camada córnea; 0,04 CD dérmicas/ mm de MB). Houve correlação direta entre o número de CD dérmicas na lesão de PFE e a titulação de anticorpos séricos por IFI (r=0,4779, p<0,05), indicativo de que as CD dérmicas poderiam estar participando da patogênese do PFE. Poder-se-ia supor que as CD estariam transitando pela derme em direção ao linfonodo regional, exercendo função estimuladora de linfócitos T na indução da resposta imune e produção de auto-anticorpos. / In order to contribute to the elucidation of pathophysiology of Endemic Pemphigus Foliaceus (EPF) titration of serum antibodies and quantification of Langerhans Cells (LC) and dermal dendritic cells (DC) in skin of patients with EPF were made. Blood and skin biopsies (lesional skin) of 22 EPF patients were collected and, in 13 of them, biopsies of normal sun-protected skin of non perilesional area were collected too. 13 patients had localized lesions and 9, generalized; 11 were in treatment. Control groups consisted of thoracic normal skin from 8 cadavers and 12 women submitted to breast plastic surgery. For indirect immunofluorescence (IFI), abdominal normal skin as substrate and anti-IgG were used. LC and dermal DC identification was done by immunohistochemistry with anti-CD1a antibody and quantification by morfometric analysis. It was found association between titration of serum antibodies by IFI and clinical form of EPF, with greater titration in the generalized one. LC number in lesion (60.18 LC/ mm2, 5.00 LC/ mm basement membrane (BM), 3.55 LC/mm stratum corneum) and normal skin of EPF patients (28.45 LC/ mm2, 2.50 LC/ mm BM, 2.87 LC/mm stratum corneum) was similar to LC number in skin of plastic surgery (72.35 LC/ mm2, 4.53 LC/ mm BM, 4.42 LC/mm stratum corneum) and cadaver controls (47.15 LC/ mm2, 2.53 LC/ mm BM, 2.42 LC/mm stratum corneum). Dermal DC number in lesional skin of EPF patients (0.98 DC/ mm BM) was similar to the DC number of plastic surgery controls (0.48 DC/ mm BM), but greater than DC number in cadaver controls (0.13 DC/ mm BM). The ratio LC number/ dermal DC number was smaller in lesional EPF skin than in controls, confirming the greatest DC number in dermis. In the same patient, LC and dermal DC were in greater amounts in EPF lesional skin (61.50 LC/ mm2, 5.49 LC/ mm BM, 6.64 LC/mm stratum corneum, 0.86 dermal DC/ mm BM) than in normal skin (28.45 LC/ mm2, 2.50 LC/ mm BM, 2.87 LC/mm stratum corneum, 0.04 dermal DC/ mm BM). It was found direct association between dermal DC number in lesional skin of EPF patients and titration of antibodies by IFI (r=0.4779, p<0.05), confirming that dermal DC could play an important role in EPF pathogenesis. It could be proposed that DC would be in transit through the dermis towards the regional lymph node, stimulating T lymphocytes to produce autoantibodies.

Le follicule pileux (FP) est un appendice cutané animé par un cycle régénératif dynamique provoquant des modifications de son microenvironnement. Les cellules de Langerhans (CLs), sentinelles de l’épiderme, sont en partie localisées à proximité du FP. Cette association spatiale nous a conduit à explorer le possible impact du cycle pileux sur l’homéostasie des CLs. Durant mon doctorat, nous avons mis en évidence (1) une augmentation de la prolifération des CLs au cours de l’anagène (phase de pousse du poil), (2) le mécanisme moléculaire sous-jacent impliquant une variation d’expression folliculaire de l’IL-34, une cytokine cruciale dans l’homéostasie des CLs et (3) un départ accru des CLs vers les ganglions lymphatiques en catagène (phase de régression du FP) concomitant avec le recrutement de cellules susceptibles d’être des précurseurs des CLs.Par ailleurs, la structure de la peau ainsi que la densité et le type de FP peuvent varier selon la région corporelle considérée. Nous avons émis l’hypothèse de variations locales dans la composition du système immunitaire cutané. Notre étude, focalisée sur les cellules dendritiques cutanées, a démontré l’existence d’une hétérogénéité de ces cellules en fonction de la zone de peau considérée. / The hair follicle (HF) is a skin appendage endowed with a dynamic regenerating cycle. This renewal remodels the HF microenvironment. Langerhans cells (LCs) are epidermal immune sentinels, a part of which localizes close to the HF. This spatial association led us to explore whether the HF cycle could impact on LC homeostasis. During my doctorate, we uncovered an anagen (HF growing phase)-associated burst of LC proliferation with dividing cells associated with the HF. Using mouse models of HF loss and hair cycle manipulation, we showed that HFs are dispensable for initial formation of the LC network but critical for the proliferation burst. We correlated it to a cyclic variation of IL-34 expression, a crucial cytokine for LC homeostasis, by a specific subset of HF cells. In addition, catagen (HF regression phase) is characterized by the departure of LCs to draining lymph nodes and the concomitant recruitment of a potential LC precursor.The skin structure as well as the density and type of HFs vary across body areas. This observation led us to assess the possibility of local variations in skin immune cells composition. Our study, focused on cutaneous dendritic cells, highlighted an heterogeneity in those cells according to the skin area considered.

Abstract Both immunosuppressive and nifedipine medication have been associated with drug-induced gingival overgrowth. There are several hypothetical mechanisms for drug-induced gingival overgrowth, such as the influence of genetic predisposition, alterations in gingival tissue homeostasis, especially in the function of fibroblasts, and drug-induced action on growth factors. Clinical studies have also shown that, those with poor oral hygiene status drug-induced gingival overgrowth is more prevalent and severe than those with good oral hygiene status. The working hypothesis was that immunosuppressive medication and/or nifedipine medication affects inflammatory cell profile and mitotic activity of keratinocytes in human overgrown gingiva. We studied gingival samples, collected from nifedipine-medicated cardiac outpatients and immunosuppression-medicated organ-transplant recipients. Patients were placed into four groups: 1) the immunosuppression group, patients receiving cyclosporin-A (CsA), azathioprine (AZA) and prednisolone (Pred) 2) the immunosuppression plus nifedipine group, patients receiving CsA, AZA, Pred. and nifedipine 3) the nifedipine group patients receiving only nifedipine and 4) the non-medicated control group. All of the samples related to moderate to severe degrees of gingival overgrowth, covering half to two thirds of the clinical crown. The aim of the study was to investigate the occurrence of Langerhans cells, macrophages, mast cells and mitotic activity of keratinocytes in human drug-induced overgrown gingiva, and consequently to assess their possible role in the pathogenesis of drug-induced gingival overgrowth. We found that immunosuppressive medication increased the numbers of reparative macrophages (RM3/1) and decreased the numbers of tryptase- and chymase-positive mast cells (MCTC) cells. We have also shown that immunosuppressive and nifedipine medication decreased the numbers of Langerhans cells (CD1a) and increased the numbers of 27E10-macrophages parallelly. Additionally we found increase in the mitotic activity of gingival keratinocytes and even two-fold thickening of gingival epithelium in immunosuppressive and nifedipine medication-induced gingival overgrowth as compared with healthy gingiva. Immunosuppressive medication activated gingival epithelium (27E10 expression in gingival keratinocytes) more than nifedipine medication. In conclusion, our results suggest that gingival overgrowth among immunosuppressive- and nifedipine-medicated patients is related to alteration of tissue homeostasis. First, this suggestion is supported by changes found in the numbers of cells that directly affect connective tissue turnover, e.g. reparative macrophages (RM3/1) and mast cells. Changes in the numbers of these cells could alter the cytokine- and growth factor-profile, which affects fibroblast function. Secondly, we found changes in the numbers of cells involved in regulation of inflammation, e.g. Langerhans cells and monocytes as compared with healthy controls. Immunosuppressive medication could directly activate gingival keratinocytes. We suggest that our findings mainly reflect the effects of immunosuppressive medication, but the role of inflammation cannot be excluded. The changes observed above represent differences of the pathogenesis of drug-induced gingival overgrowth between immunosuppressive and nifedipine medication. It must be however remembered that drug-induced gingival overgrowth is a result of multicausal intrinsic and extrinsic factors. Age, gender, concomitant medication with multiple drugs, plaque accumulation, and genetic disposition are additional risk factors. The abnormal distribution of specific immune system cell subpopulations does not alone prove a functional relationship to gingival overgrowth.

Vitiligo is an autoimmune disease of the skin characterized by epidermal depigmentation that results from CD8+ T cell-mediated destruction of pigment producing melanocytes. Vitiligo affects up to 1% of the population and current treatments are moderately effective at facilitating repigmentation by suppressing cutaneous autoimmune inflammation to promote melanocyte regeneration. In order to cause disease, CD8+ T cells must overwhelm the mechanisms of peripheral tolerance in the skin and if we understand the suppressive mechanisms that are compromised during vitiligo, we can potentially use this information to improve existing treatments or engineer novel interventions. Therefore, my goal is to characterize the regulatory factors in the skin that suppress depigmentation during vitiligo. Our lab has developed a mouse model of vitiligo that accurately reflects human disease and I used this model to demonstrate that regulatory T cells suppress CD8+ T cell-mediated depigmentation and interact with CD8+ T cells in the skin during vitiligo. In this model of disease, I investigated the molecules involved in regulatory T cell function and observed that the chemokine receptors CCR5 and CCR6 play different roles in regulatory T cell suppression. While CCR6 facilitates regulatory T cell migration to the skin, CCR5 is dispensable for migration but required for optimal regulatory T cell function. Additionally, I used our mouse model to demonstrate that Langerhans cells suppress the incidence of disease during vitiligo. Taken together the results from these studies provide novel insights into the mechanisms of suppression during vitiligo.