UNIVERSIDADE ESTADUAL DE CAMPINAS FACULDADE DE...
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UNIVERSIDADE ESTADUAL DE CAMPINAS FACULDADE DE ODONTOLOGIA DE PIRACICABA CINTHIA VERÓNICA BARDÁLEZ LÓPEZ DE CÁCERES DISTRIBUIÇÃO DAS CÉLULAS DENDRÍTICAS EM SARCOMA DE KAPOSI ORAL ASSOCIADO À AIDS DISTRIBUTION OF DENDRITIC CELLS IN ORAL AIDS-ASSOCIATED KAPOSI’S SARCOMA Piracicaba 2017
Transcript of UNIVERSIDADE ESTADUAL DE CAMPINAS FACULDADE DE...
UNIVERSIDADE ESTADUAL DE CAMPINAS
FACULDADE DE ODONTOLOGIA DE PIRACICABA
CINTHIA VERÓNICA BARDÁLEZ LÓPEZ DE CÁCERES
DISTRIBUIÇÃO DAS CÉLULAS DENDRÍTICAS
EM SARCOMA DE KAPOSI ORAL ASSOCIADO
À AIDS
DISTRIBUTION OF DENDRITIC CELLS IN
ORAL AIDS-ASSOCIATED KAPOSI’S
SARCOMA
Piracicaba
2017
CINTHIA VERÓNICA BARDÁLEZ LÓPEZ DE CÁCERES
DISTRIBUIÇÃO DAS CÉLULAS DENDRÍTICAS EM
SARCOMA DE KAPOSI ORAL ASSOCIADO À AIDS
DISTRIBUTION OF DENDRITIC CELLS IN ORAL
AIDS-ASSOCIATED KAPOSI’S SARCOMA
Dissertação apresentada à Faculdade de
Odontologia de Piracicaba da
Universidade Estadual de Campinas
como parte dos requisitos exigidos
para a obtenção do título de Mestra
em
Estomatopatologia, na Área de Patologia.
Dissertation presented to the
Piracicaba Dental School of the
University of Campinas in partial
fulfillment of the requirements for
the degree of Master in
Estomatopathology, in Pathology
area.
Orientador: Prof. Dr. Felipe Paiva Fonseca
Coorientador: Prof. Dr. Pablo Agustin Vargas ESTE EXEMPLAR CORRESPONDE À VERSÃO
FINAL DA DISSERTAÇÃO DEFENDIDA PELA
ALUNA CINTHIA VERÓNICA BARDÁLEZ
LÓPEZ DE CÁCERES ORIENTADA PELO
PROF. DR. FELIPE PAIVA FONSECA E
COORIENTADA PELO PROF. DR.
PABLO AGUSTIN VARGAS.
Piracicaba
2017
DEDICATÓRIA
Dedico este trabalho à minha pequena família, meus dois amores, meu esposo
Andrés e meu filho Rafael, por serem minha força e o motivo para tentar dar o melhor no
dia a dia.
Dedico também este trabalho à minha mãe e aos meus irmãos; apesar da distância
o amor de vocês está comigo.
AGRADECIMENTOS
À Faculdade de Odontologia de Piracicaba da Universidade Estadual de
Campinas, nas pessoas do seu diretor Prof. Dr. Guilherme Elias Pessanha Henriques e
do seu diretor associado Prof. Dr. Francisco Haiter Neto.
À Coordenadoria Geral dos cursos de Pós-graduação da Faculdade de
Odontologia de Piracicaba - UNICAMP, na pessoa da Profa. Dra. Cinthia Pereira
Machado Tabchoury.
Ao Coordenador do Programa de Pós-graduação em Estomatopatologia da
Faculdade de Odontologia de Piracicaba - UNICAMP Prof. Dr. Márcio Ajudarte Lopez.
À Fundação CAPES por ter concedido uma bolsa de estudos e por ter fornecido
suporte econômico para o desenvolvimento desta pesquisa.
Ao meu orientador Prof. Dr. Felipe Paiva Fonseca, pela orientação, pelo apoio
e dedicação não somente no desenvolvimento deste trabalho, assim como também em
meu crescimento como professora e pesquisadora, sem descuidar em momento nenhum
da parte pessoal. Agradeço pela confiança e boa disposição para me ajudar em tudo o
que eu precisei.
Ao meu coorientador Prof. Dr. Pablo Agustín Vargas agradeço ao senhor pela
confiança e por ter acreditado na minha pessoa para fazer parte da sua equipe.
À Dra. Belinda Bunn e ao Dr. Willie F. P. van Heerden, por suas valiosas
contribuições no desenvolvimento deste trabalho.
À Celeste Sanchez Romero por toda ajuda no desenvolvimento da parte
laboratorial do trabalho, obrigada por todos os dias nos quais sempre tinhas alguma
coisa para ensinar, pelas discussões de casos, aprendi muito com você, e por último,
obrigada pela amizade.
Ao Prof. Dr. Alan Roger dos Santos Silva pelo recebimento no programa e por
sempre manter as portas abertas.
Ao Prof. Dr. Oslei Paes de Almeida pela abertura e acesso aos laboratórios
permitindo meu crescimento profissional e o desenvolvimento da pesquisa.
À Fabiana Casarotti pela ajuda, introdução e dicas dobre o funcionamento do
laboratório e sobre a vida.
Aos meus colegas do programa pela convivência, troca de conhecimento e boa
disposição para me ajudar tanto nas atividades da Faculdade quanto fora dela.
À Faculdade de Odontologia da Universidade de San Martin de Porres por ter
me formado como cirurgiã-dentista e ter me introduzido no caminho da docência
proporcionando-me a oportunidade como professora por um ano na disciplina de
Anatomia Patológica.
Ao Prof. Dr. Juvenal Sanchez Lihon por ter me iniciado no mundo da pesquisa,
por ser meu orientador no meu trabalho de graduação e por ser meu primeiro vínculo
com a patologia oral.
À Profa. Dra. Erika Alberca Ramos por ter me acolhido e guiado na prática
laboral, além de ser a melhor chefe. É uma das melhores pessoas que tive a sorte de
conhecer, e me ensinou a importância da dedicação e perseverança.
Ao meu amigo e esposo Andrés Humberto Cáceres Barreno, por esses doze anos
juntos cheios de amor, de experiências boas e algumas poucas ruins que só reforçaram
e confirmaram os nossos sentimentos, obrigado pelo apoio incondicional.
Ao meu filho Rafael, minha mais linda surpresa, você que chegou em nossas vidas
para colocá-las de cabeça para baixo, tudo o que nós passamos e que ainda temos para
enfrentar não é nada comparado com ter você por perto, eu não sabia o que faltava em
minha vida até chegar você e completá-la. Seu sorriso no final do dia é o melhor incentivo
para continuar e tentar dar o melhor de min.
À minha mãe por ser meu exemplo de sacrifício, trabalho, renúncia e recomeço;
nunca é tarde para ser feliz e encontrar o que nos faz sentir bem, o único fato necessário é
tomar a decisão.
Aos meus irmãos Cristhian e Ivan por todos os momentos vividos; mais que irmãos
vocês foram filhos emprestados, pois ficaram sob meu cuidado desde pequenos. Sei que
não fui perfeita, mas me orgulho de vocês por serem boas pessoas que estão abrindo seus
caminhos ao futuro.
Aos meus sogros por serem meus exemplos de pessoas e exemplo de família,
obrigada pelo apoio e por estarem sempre cuidando de nós.
Também gostaria de agradecer a todos os amigos que fiz desde que cheguei
obrigada por serem o suporte e o apoio para estudar fora do meu país, diminuindo a
saudade.
RESUMO
O sarcoma de Kaposi (SK) é considerado uma neoplasia endotelial associada ao
vírus HHV-8 e que pode ser classificada em 4 tipos clinico-epidemiológicos: clássico,
endêmico, iatrogênico e associado a AIDS. As células dendríticas (CD) participam
ativamente no sistema imunológico da pele e mucosas, exercendo um importante papel
na apresentação de antígenos. Nos últimos anos, tem-se dado muita atenção à sua
importância para o desenvolvimento neoplásico, principalmente em contextos de
imunossupressão. Assim, este estudo objetivou avaliar se a distribuição das células de
Langerhans (CL) e as células dendríticas intersticiais (CDI) maduras e imaturas está
alterada no sarcoma de Kaposi (SK) oral associado a AIDS, quando comparado com
lesões orais altamente vascularizadas em pacientes HIV negativos (LOAV). As amostras
estudadas foram compostas por 51 casos de SK orais associados a AIDS emblocados em
parafina, recuperados dos arquivos do Departamento de Patologia Oral da Faculdade de
Odontologia da Universidade de Pretória (Pretória / África do Sul), e por 20 casos de
LOAV (10 hemangiomas e 10 granulomas piogênicos) pertencentes ao Departamento
de Diagnóstico Oral da Faculdade de Odontologia de Piracicaba da Universidade de
Campinas (Piracicaba/Brasil). Os dados clínicos relativos ao sexo, idade e localização
das lesões foram obtidos a partir dos laudos histopatológicos. Foram realizadas reações
imunoistoquímicas com o anticorpo CD207 para identificação de CD imaturas e
anticorpo CD83 para avaliar as CD maduras. A quantificação das CD foi realizada por
um observador previamente treinado, que contou para as CL o número de células
positivas no epitélio de superfície (10 campos sequenciais (40x) para cada anticorpo) e,
para estudo das CDI, o número de células positivas no estroma das lesões, distante das
áreas de ulcerações (10 campos sequenciais (40x) para cada anticorpo). Os resultados
foram expressos em número total de células positivas por caso. Os valores foram
apresentados em média ± desvio padrão (SD) para cada grupo de lesões. A distribuição
da normalidade dos resultados foi investigada pelos testes de Kolmogorov-Smirnov e
Shapiro-Wilk e a homocedasticidade dos grupos pelo teste de Levene. A comparação da
distribuição das células entre o SK associado à AIDS e as LOAV foi realizada utilizando
o teste t de Student e o teste de MannWhitney, com valor p ≤0,05 sendo considerado
estatisticamente significativo. O software SPSS versão 22.0 foi utilizado para as análises
estatísticas. Como resultado, foi observado que as células CD207+ predominaram no
epitélio das lesões, enquanto que as células CD83+ predominaram no estroma das lesões.
O SK associado à AIDS revelou uma contagem de células CL (CD207+)
significativamente menor (p = 0.02), e um aumento de CDI (CD207+) no estroma em
relação às LOAV (p < 0.001). Além disso, o SK associado à AIDS também mostrou um
aumento no número de CDI maduras (CD83+) em relação às LOAV (p < 0.001).
Portanto, o presente estudo demonstrou alterações significativas na distribuição das CL
e das CDI em SK associado à AIDS quando comparado as LOAV em pacientes HIV
negativos, sugerindo que mudanças na distribuição dessas células podem desempenhar
um papel na patogênese do sarcoma de Kaposi.
Palavras-chave: Sarcoma de Kaposi, AIDS, Células de Langerhans, Células dendríticas
intersticiais, Imunoistoquímica.
ABSTRACT
Kaposi's sarcoma (KS) is considered an endothelial neoplasm associated with the HHV-
8 virus and it can be classified into four clinical-epidemiological types: classic, endemic,
iatrogenic and associated with AIDS. Dendritic cells (DC) actively participate in the
immune system of the skin and mucous membranes, playing an important role in the
presentation of antigens. In recent years, much attention has been paid to its importance
for neoplastic development, especially in immunosuppressive settings. The aim of this
study was to determine whether the distribution of mature and immature Langerhans
cells (LC) and interstitial dendritic cells (IDC) were altered in AIDS-associated oral
Kaposi's sarcoma (SK) if compared to HIV-negative highly vascular oral lesions. The
samples were composed of 51 cases of paraffin-embedded AIDS-associated oral
Kaposi’s sarcoma retrospectively retrieved from the files of the Department of Oral
Pathology of the School of Dentistry of the University of Pretoria (Pretoria/South
Africa), and by 10 cases of hemangioma and 10 cases of pyogenic granuloma retrived
from the Department of Oral Diagnosis (Pathology) of the Piracicaba Dental School of
the University of Campinas (Piracicaba/Brazil). Clinical data regarding sex, age and
tumor location were obtained from pathology reports. Immunohistochemichal reactions
were done using CD207 antibody to identify immature dendritic cells and CD83 to
evaluate mature dendritic cells. Counting of dendritic cells was performed by one
previously trained observer that quantified the number of positive cells in 10 sequential
fields (40x) for each antibody. The quantification was first done in the surface epithelium
to study Langerhans cells and then inside the lesions, distant from ulcerated areas, to the
study f interstitial dendritic cells. The results were expressed as total number of positive
cells per case. The values were presented as mean ± standard deviation (SD) for each
group of lesions. The normality distribution of the results was investigated with
Kolmogorov-Smirnov and Shapiro-Wilk tests, while the Levene test was used to
determine the homoscedasticity of the groups. Comparison of cell distribution between
Kaposi's sarcoma and highly vascular oral lesions (hemangioma and pyogenic
granuloma) was performed using the Student t test and Mann-Whitney test, with a p
value < 0.05 being considered statistically significant. The software SPSS version 22.0
was used to statistical analyses. As result, it was observed that CD207+ cells
predominated in the epithelium of the lesions, whereas CD83+ cells predominated in
their stroma. Kaposi's sarcoma revealed a significantly lower CD207+ Langerhans cells
count (p = 0.02), and an increased CD207+ interstitial dendritic cell count in the stroma
than benign vascular lesions (p < 0.001). In addition, Kaposi's sarcoma also showed an
increase in the number of mature CD83+ interstitial dendritic cells than highly vascular
oral lesions (p <0.001). Therefore, the current study demonstrated significant changes
in the distribution of Langerhans cells and interstitial dendritic cells in AIDSassociated
oral Kaposi's sarcoma when compared to HIV-negative highly vascular oral lesions,
suggesting that changes in the concentration of these cells may play a role in the
pathogenesis of Kaposi's sarcoma.
Keywords: Kaposi's sarcoma, AIDS, Langerhans cells, Interstitial dendritic cells,
immunohistochemistry.
LISTA DE ILUSTRAÇÕES
Figure 1 – Microscopic and immunohistochemical aspects of oral Kaposi’s sarcoma.
Figure 2 – Microscopic aspect of highly vascular oral lesions.
Figure 3 – Immunohistochemical reactivity against CD207 and CD83 in the epithelium and
stroma of oral Kaposi’s sarcoma and of highly vascular oral lesions.
LISTA DE TABELAS
Tabela 1 – Distribution of LC and IDC in different compartments (epithelium and
stroma of the lesions) of oral Kaposi’s sarcoma and in highly vascular oral lesions
(hemangioma and pyogenic granuloma).
Tabela S1 – Summary of the available studies that investigated the importance of
different dendritic cells in the pathogenesis of Kaposi’s sarcoma.
LISTA DE ABREVIATURAS E SIGLAS
SK: Sarcoma de Kaposi
AIDS: Síndrome da imunodeficiência adquirida.
CMV: Citomegalovírus
HHV: Herpesvírus Humano.
HPV: Papilomavírus humano
KSHV: Herpesvírus associado ao sarcoma de Kaposi
HAART: Terapia Antiretroviral Altamente Ativa
CL: Células de Langerhans
CD: Células dendríticas
CDI: Células dendríticas intersticiais
APC: Células apresentadoras de antígeno
MHC: Complexo principal de
histocompatibilidade moDC: Células
dendríticas derivadas dos monócitos
EMPs: Progenitores eritromieloides primitivos
HSC: Células tronco hematopoiéticas
SUMÁRIO
1. INTRODUÇÃO 17
2. ARTIGO: DISTRIBUTION OF DENDRITIC CELLS IN
AIDS-ASSOCIATED ORAL KAPOSI’S SARCOMA
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3. CONCLUSÃO
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REFERÊNCIAS BIBLIOGRÁFICAS
49
ANEXOS
ANEXO 1 – Certificado do comitê de ética
52
ANEXO 2 – Certificado de submissão 53
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1 INTRODUÇÃO
Sarcoma de Kaposi
Em 1872, Moricz Kaposi descreveu pela primeira vez cinco casos de um tumor que
aparecia em homens idosos, principalmente na pele das extremidades inferiores, multifocal,
de curso lento e progressivo, sendo foi denominado “Sarcoma pigmentado de pele,
idiopático, múltiplo”, recebendo posteriormente a denominação de sarcoma de Kaposi (SK)
(Kaposi, 1872).
Durante muitos anos foi considerada uma neoplasia pouco comum, de pouco interesse
na comunidade médica, no entanto, entre os anos de 1950 e 1960 foi observado um segundo
padrão de SK na África Central e Ocidental, considerado endêmico, acometendo
principalmente crianças e adultos jovens de raça negra, envolvendo as extremidades e com
um bom prognóstico geral (Arruda, et al., 2014).
Nos anos 70 com o início dos transplantes de órgãos e a consequente imunossupressão
decorrente do uso de fármacos (corticosteroides e imunossupressores), observou-se o
desenvolvimento de SK neste grupo de pacientes, sendo então identificado o terceiro padrão
de SK que viria a ser designado SK iatrogênico (Arruda, et al., 2014).
A pouca atenção atribuída até então ao SK sofreu uma grande mudança a partir dos anos
de 1980, quando se observou um grande número de pacientes afetados, predominantemente
compostos por homens adultos jovens e homossexuais e, em menor frequência, usuários de
drogas administradas por via parenteral. O incremento foi tal que alcançou proporções
epidêmicas e acabou sendo um dos principais indicadores clínicos para esta nova doença
que foi conhecida como a síndrome de imunodeficiência adquirida (AIDS), estabelecendo-
se assim um novo padrão, o SK associado à AIDS (Pantanowitz et al., 2013).
Etiologia
Diferentes vírus foram investigados quanto aos seus potenciais no desenvolvimento do SK,
incluindo o citomegalovírus (CMV), os herpesvírus humanos tipos 6 e 7 (HHV-6 e
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HHV-7) e o papilomavírus humano (HPV). No entanto, em 1994 foi isolado um novo
subtipo do herpesvírus a partir da análise de diferentes amostras biológicas de pacientes
com AIDS, sendo denominado herpesvírus associado ao sarcoma de Kaposi (KSHV) ou
herpesvírus humano tipo 8 (HHV-8). Atualmente sabe-se que o HHV-8 é detectado em
todos os casos de SK, independentemente da forma clínica, podendo também ser detectado
no sangue periférico antes da aparição da doença (Lee, et al., 2015).
O HHV-8 pertence à família Herpesviridae, subfamília Gamaherpesvirinae e ao gênero
Radnovírus, sendo o único desse gênero capaz de infectar células humanas. Possui um
capsídeo viral, que é sintetizado na célula hospedeira durante a fase lítica de seu ciclo de
vida e um envelope no qual se inserem proteínas do hospedeiro e virais. Ele contém genes
que desempenham funções anti-apoptóticas, angiogênicas, de controle do ciclo celular, de
proliferação e transformação celular, além de genes associados à evasão do sistema
imunológico (Schulz et al., 2015).
Sabe-se que o HHV-8 é capaz de infectar alguns tipos celulares como macrófagos, células
dendríticas, células endoteliais, monócitos, células epiteliais, fibroblastos e linfócitos B.
Assim como os demais herpes vírus humanos, o ciclo biológico do HHV-8 apresenta duas
fases distintas, denominadas ciclo latente e ciclo lítico (ou produtivo). O HHV-8 é capaz de
infectar um grande número de células e se manter latente por um longo período. A
reativação do vírus desencadeia a produção e liberação de novas partículas virais prontas
para infectar novas células (Lee et al., 2015; Schulz et al., 2015).
Aspectos clínicos, microscópicos e imunoistoquímicos
As lesões na pele e mucosas podem ser observadas em três diferentes estágios
clínicos como manchas, placas e nódulos de coloração arroxeada que podem variar em
tamanho. Estas lesões usualmente assintomáticas e múltiplas podem coalescer e atingir
grandes dimensões e as lesões podem acometer a mucosa oral, tecidos moles, nódulos
linfáticos e órgãos viscerais (Simonart, et al., 2000).
As características microscópicas dos quatro tipos clínico-epidemiológicos do SK não
diferem. As lesões iniciais (manchas) demonstram uma proliferação vascular sutil e,
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conforme a lesão evolui para o estágio clínico de placa, observa-se uma maior proliferação
de vasos sanguíneos que permeiam as fibras colágenas da derme e submucosa (Simonart,
et al., 2000).
As células endoteliais que revestem os vasos sanguíneos são achatadas, com pouca
atipia. Um intenso infiltrado inflamatório crônico está usualmente presente, exibindo
também extravasamento de hemácias e depósitos de hemossiderina ao redor das estruturas
vasculares (Bunn et al., 2013).
No estágio nodular, todas as características da fase do estágio de placa encontramse
exacerbadas. Há uma maior angio-proliferação, com espaços vasculares de contornos
irregulares, intenso infiltrado inflamatório e evidente proliferação de células fusiformes
que demonstram leve grau de atipia e número variado de figuras de mitose (Simonart, et
al., 2000).
Os quatro tipos clínico-epidemiológicos podem apresentar outros subtipos
histológicos, sendo mais comum deles a variante sólida; outras menos comuns são:
telangiectásica, desmoplásica, linfangioma-like, linfangiectásica, equimótica e anaplásica,
as quais não estão correlacionadas com a evolução do SK (Bunn et al., 2013).
Reações imunoistquímicas demonstram intensa positividade para marcadores
vasculares sanguíneos (CD34 e CD31) e linfáticos (D2-40 e PROX-1) (Benevenuto de
Andrade et al., 2014). Como mencionado anteriormente, todos os casos são positivos para
HHV-8. O índice de proliferação celular medido por Ki-67 encontra-se em torno de 30%
(Pantanowitz, et al. 2014).
Sarcoma de Kaposi oral associado à AIDS
As manifestações da AIDS na região de cabeça e pescoço estão entre as complicações
mais comuns presentes neste grupo de pacientes. Algumas destas manifestações
representam sinais iniciais da infecção pelo HIV (vírus da imunodeficiência humana), e
outras estão associadas ao desenvolvimento da AIDS. As manifestações mais comuns são
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as infecções por fungos, protozoários, bactérias, vírus e algumas neoplasias (Pantanowitz,
et al. 2014; Berberi, et al. 2014).
O SK associado à AIDS é uma das manifestações mais frequente destes pacientes.
Em 22% dos casos de SK, a cavidade oral é o sítio primário de acometimento. A razão
disso pode ser pelo fato de a saliva possuir altos níveis de HHV-8 em relação ao sangue,
fluidos genitais e região anal (Bunn et al., 2013),
Os estudos em regiões endêmicas do SK, mostram que a saliva pode ser fonte de
infecção para o HHV-8 e que as células epiteliais da mucosa oral mostram tropismo pelo
HHV-8, o que as torna susceptíveis a infecção, no entanto (Braz-Silva et al., 2017; Tozetto-
Mendoza TR et al., 2018) mostraram que na população brasileira considerada não
endêmica de SK, o HHV-8 tem um potencial limitado para a transmissão oral, já que ao
contrário de outros herpes vírus, o HHV-8 não é excretado na saliva de indivíduos
saudáveis, sendo que nas amostras o HHV-8 estava presente na saliva dos pacientes HIV
positivos .
Pacientes acometidos por SK associado à AIDS demonstravam um prognóstico
pobre, apresentando-se como uma neoplasia agressiva, devido à potencialização do agente
viral, favorecendo a progressão da doença e levando a consequências potencialmente fatais.
Entretanto, com o surgimento do tratamento com antirretrovirais, principalmente a terapia
antirretroviral altamente ativa (HAART), o prognóstico desses pacientes melhorou
significativamente, assim como a incidência da neoplasia a qual revelou um importante
declínio, sendo os casos atualmente diagnosticados majoritariamente associados com
pacientes que exibem imunodeficiência grave (Arruda et al., 2014).
Células dendríticas
As células dendríticas (CD) participam ativamente no sistema imunológico da pele e
mucosas, agindo como sentinelas e apresentadoras de antígenos de natureza tanto endógena
como exógena, expressando altos níveis de MHC (complexo principal de
histocompatibilidade), principalmente MHC-II. Estas células são capazes de capturar os
antígenos para posteriormente migrar em direção aos gânglios linfáticos, dando início a
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uma resposta imune do tipo celular mediante a ativação e proliferação dos linfócitos T
(Hertoghs et al., 2017).
As CD podem ser classificadas como CD convencionais, CD plasmocitoides, e CD
derivadas de monócitos (moDCs), o qual este último grupo aparece apenas quando há
infecção ou um processo inflamatório mas, de acordo com sua localização no organismo,
estas células podem ainda ser divididas em outros subtipos com características próprias,
por exemplo as células de Langerhans (CL), as quais se encontram no epitélio da pele e
mucosas, e as células dendríticas intersticiais (CDI) que se encontram no tecido conjuntivo
da derme e no interstício dos órgãos; além disso, funcionalmente podem ser divididas em
dois estágios: o estágio imaturo e maduro, com as CD imaturas sendo especializadas na
captura e processamento de antígenos, e as CD maduras tornando-se especialistas na
apresentação de antígenos (Austyn, 2016; Collin, Milne, 2016).
As CL foram descritas pela primeira vez em 1868 por um estudante de medicina
chamado Paul Langerhans, que descreveu uma população de células localizada na camada
suprabasal do epitélio escamoso, utilizando a técnica de impregnação por ouro. Entretanto,
apenas no final do século passado estas células foram aceitas como parte do sistema de CD,
sendo anteriormente consideradas como células neuronais, células ectodérmicas ou até
mesmo simples artefatos (Austyn, 2016).
Atualmente sabe-se que as CL originam-se na hematopoiese primária do saco
vitelino, derivando dos progenitores eritro-mieloides primitivos (EMPs) e que migram
através do tecido embrionário, entrando na pele rudimentar onde irão proliferar e darão
origem às populações auto renováveis que persistirão durante a vida pós-natal e adulta. No
entanto, Ginhoux et al., (2010) mostraram que as CL podem ter uma origem secundária,
que foi atribuída a progenitores EMPs do fígado fetal, além da existência de um terceiro
grupo de CL derivado de células tronco hematopoiéticas (HSC) pós-natal (Ginhoux et al.,
2010).
Pela localização na pele e mucosas, as CL, agem como a primeira linha de defesa e
a sua configuração as torna uma maquinaria de captura de antígenos, além de contarem
com uma ampla capacidade de movimentação que permite a rápida migração para os órgãos
secundários, onde realizam a apresentação de antígenos para os linfócitos T (Boltjes et al.,
2014). No tecido conjuntivo, estas células são denominadas de células dendríticas
intersticiais (CDI). Estruturalmente, as CD têm um aparelho de Golgi desenvolvido,
apresentam um núcleo lobulado e, como característica especial, as células de Langerhans
22
possuem os grânulos de Birbeck que apresentam a forma de raquete de tênis quando
observadas na microscopia eletrônica. A morfologia das células dendríticas encontra-se
ligada ao grau de maturação; a forma imatura é caracterizada por seu aspecto irregular,
estrelado e com numerosos prolongamentos citoplasmáticos e são altamente endocíticas.
Já a célula madura é mais regular, de formato mais arredondado e com poucos ou nenhum
prolongamento citoplasmático (Austyn, 2016).
Perfil imunoistoquímico das células de Langerhans e células dendríticas intersticiais
O anticorpo anti-MHC-II foi durante muito tempo o marcador mais comumente
utilizado para a identificação das CL/CDI. Os marcadores anti-CD1a e anti-S100 também
foram utilizados frequentemente para identificação destas populações de CD. Entretanto,
nos últimos anos, tem surgido uma gama de novos marcadores moleculares que favorecem
a identificação destas populações celulares e que permitem reconhecê-las em diferentes
estágios de maturação. A progressão para os estágios de maturação das CL/CDI envolve
mudanças na expressão de receptores presentes na membrana celular, o que afeta a sua
capacidade de interagir com o ambiente extracelular e com outras células. Neste sentido,
as CD imaturas são caracterizadas por uma alta expressão de moléculas envolvidas na
captura de antígenos: FcyR1, CD1, CD205, CD207, CD209 e CLEC-9, enquanto que as
características de maturação das CD incluem um aumento na expressão de MHCI/MHCII
e moléculas co-estimuladoras, tais como CD70, CD80, CD83, CD86 e CCR7. O marcador
anti-CD207 é capaz de identificar a molécula Langerina (lecitina transmenbrana ligadora
de manose tipo C), que é um dos principais componentes dos grânulos de Birbeck; desse
modo, uma vez que os grânulos de Birbeck são exclusivos das CL, nas células dendríticas
intersticiais esta estrutura não está presente, no entanto, as CDI podem expressar níveis
baixos de Langerina (Collin, Milne., 2016; Austyn, 2016), tornando-se um marcador
específico destas células quando estão em um estágio imaturo (de Witte et al., 2007).
Relação das células dendríticas com o HIV e o Sarcoma de Kaposi
A AIDS caracteriza-se pela redução dos números de linfócitos T CD4+, mas sabe-se
também que o HIV tem outras células alvo, dentre elas os monócitos, macrófagos e as CD
(de Witte et al., 2007).
23
Devido a localização das CL nas membranas mucosas, incluindo a mucosa da
cavidade oral, especula-se que elas sejam uma das primeiras células a entrar em contato
com o vírus, acreditando-se que a ação da Langerina restringiria a atividade do vírus em
um primeiro momento, destruindo-o no interior das CL (de Witte et al., 2007). No entanto,
com a evolução da infecção, o vírus parece se tornar capaz de utilizá-las para se transportar
até as células T nos linfonodos (Cunningham et al, 2010; Lee et al., 2015, Botting et al.,
2014).
As CL têm sido estudadas em diferentes neoplasias, sendo que a presença desta
linhagem celular está correlacionada a um melhor prognóstico em alguns tumores humanos
(Boltjes et al., 2014) e a um pior prognóstico em outras neoplasias (Pileri, et al., 2017). No
contexto do SK, apesar de haver alguns estudos investigando diferentes tipos de células
dendríticas, em especial as células dendríticas plamocitoides (Karouni, et al 2016, West et
al., 2011), muito pouco se conhece acerca da importância das CL e das CDI na patogênese
do SK; escassos estudos prévios demonstraram que as CL são afetadas diretamente pela
infecção por HHV-8 (Knowlton et al., 2013), o que somado com a presença do HIV no
mesmo contexto clínico, leva-se a supor que uma alteração na função e/ou na distribuição
das CL e CDI favoreceria o desenvolvimento do SK. Portanto, o objetivo deste trabalho é
determinar o perfil de distribuição das CL e das CDI em SK oral associado à AIDS, e
compará-lo com aquele observado em lesões altamente vascularizada da cavidade oral em
paciente HIV negativos.
24
2 ARTIGO: DISTRIBUTION OF DENDRITIC CELLS IN AIDS-ASSOCIATED
ORAL KAPOSI’S SARCOMA
Artigo submetido ao Oral Surgery, Oral Medicine, Oral Pathology, Oral Radiology. (Anexo
2)
25
DISTRIBUTION OF DENDRITIC CELLS IN AIDS-ASSOCIATED ORAL KAPOSI’S
SARCOMA
Cinthia Verónica Bardalez Lopez1, Pablo Agustin Vargas1,2, Celeste Sánchez Romero1, Belinda
K. Bunn2, Willie F. P. van Heerden2 and Felipe Paiva Fonseca1,3
1. Department of Oral Diagnosis - Piracicaba Dental School – University of Campinas
(Piracicaba/Brazil).
2. Department of Oral Pathology and Oral Biology, School of Dentistry, Faculty of Health
Sciences, University of Pretoria (Pretoria/South Africa).
3. Department of Oral Surgery and Pathology – School of Dentistry – Universidade Federal
de Minas Gerais (Belo Horizonte/Brazil).
Conflict of interest statement: There is no financial disclosure associated with this study. The
results of this study were partially presented as a poster format in the 42o Brazilian Congress of
Oral Pathology and Medicine in Manaus/Brazil.
Word count for the abstract: 201 words Manuscript word count: 2808 words Number of
references: 30 words Number of figures/tables: 4 figures and 2 tables
Corresponding Author:
Prof. Felipe Paiva Fonseca
Department of Oral Surgery and Pathology, School of Dentistry
Universidade Federal de Minas Gerais
Address: Av. Antônio Carlos 6627, Pampulha, 31270-901, Belo Horizonte - Brazil. E-mail:
26
Abstract
Objective: To determine if the distribution of Langerhans cells (LC) and interstitial dendritic
cells (IDC) is altered in AIDS-associated oral Kaposi’s sarcoma when compared to
HIVnegative highly vascular oral lesions.
Study design: Fifty-one cases of AIDS-associated oral Kaposi’s sarcoma and 20 of highly
vascular oral lesions were retrospectively retrieved. All cases of Kaposi’s sarcoma were
confirmed with immunoreactions against CD34 and HHV-8. Clinical data regarding sex, age
and lesions location were obtained from pathology reports. Immunohistochemistry against
CD207 (immature dendritic cells) and CD83 (mature dendritic cells) were done. LC were in the
epithelium and IDC in the stroma.
Results: CD207+ cells predominated in the epithelium of the lesions, whereas CD83+ cells
predominated in their stromal compartment. Kaposi’s sarcoma had a lower CD207+ immature
LC count (p = 0.02) and an increased CD207+ IDC than highly vascular oral lesions (p <
0.001). Moreover, Kaposi’s sarcoma also showed an increased number of mature CD83+ IDC
than highly vascular oral lesions (p < 0.001).
Conclusion: There were significant alterations in the distribution of LC and IDC in
AIDSassociated Kaposi’s sarcoma when compared to HIV-negative highly vascular oral
lesions, suggesting that changes in their concentrations may play a role in the pathogenesis of
Kaposi’s sarcoma.
Keywords: Kaposi’s sarcoma, AIDS. Dendritic cells. Langerhans cells. CD207. CD83.
27
Introduction
Kaposi’s sarcoma was first described by Dr. Moritz Kaposi and is defined as an
intermediate-grade vascular malignancy always associated with HHV-8 infection1. Incidence
of Kaposi’s sarcoma had a significant increase after the Acquired Immunodeficiency Syndrome
(AIDS) burden early in the 1980’s, followed by a decrease with HAART development. Kaposi’s
sarcoma is classified according to its epidemiological features as classic, endemic,
immunodeficiency- and AIDS-associated disease, as well as according to its clinical stage of
development as macule, patch or nodule Kaposi’s sarcoma2,3. In all these scenarios, the
neoplasm is microscopically characterized by an intense vascular proliferation with a spindle
cell component and chronic inflammatory infiltrate. However, microscopic findings may also
give rise to different histological subtypes recognized in both cutaneous and oral mucosa
presentations and include solid, lymphangioma-like, telangiectatic, desmoplastic,
lymphangiectatic, ecchymotic, and anaplastic variants 4.
Kaposi’s sarcoma is considered the most common neoplasm in the HIV infection
context; nevertheless, in spite of the important improvements in the understanding of its
pathogenesis, some authors still consider Kaposi’s sarcoma as a reactive lesion, especially
because of its potential to disappear after HAART. Although the complex inflammatory
background of the disease is considered a major component in its behavior, the importance of
dendritic cells (DC) remains elusive and very few studies investigated the participation of these
cells in Kaposi’s sarcoma development.
In the skin and oral mucosa, Langerhans cell (LC) represents the most important antigen
presenting cells (APC) located in the overlying epithelium, responsible to capture and exhibit
foreign antigens to T-cells and, therefore, activating immune responses. When LC shifts to
connective tissue during their movement to regional lymph nodes, they present morphological
changes and are termed interstitial dendritic cells (IDC), which are also stromal resident cells 5.
Our group has previously demonstrated that HIV-positive patients had a decreased
concentration of LC and IDC in the mucosa of the oral tongue, predisposing these patients to a
myriad of opportunistic infections6,7. In addition, recent evidences pointed towards different DC
subtypes as important agents for HHV-8 infection, which in turn would cause disturbances in
the maturation, proliferation and function of these APC.
Current data on the importance of DC in the pathogenesis of KS are mostly derived from
cutaneous lesions and from in vitro assays. In addition, plasmacytoid DC is the most
28
investigated DC in the context of Kaposi’s sarcoma and HHV-8 infection. Therefore,
considering the previous reports observing DC disturbances in Kaposi’s sarcoma and the lack
of studies investigating this cellular population in the oral manifestation of the neoplasm, in this
research we aimed to determine if the distribution of LC and IDC is altered in AIDSassociated
oral Kaposi’s sarcoma when compared to HIV-negative highly vascular oral lesions.
Material and Methods
Samples
In a 30 year-period from 1985 to 2015, 51 formalin-fixed, paraffin-embedded cases of
Kaposi’s sarcoma were retrospectively retrieved from the files of the Oral Pathology
Department of the School of Dentistry of the University of Pretoria (Pretoria/South Arica).
New 5μm histological sections stained with hematoxylin and eosin were jointly revised by two
oral pathologists to confirm the original diagnoses that were further confirmed with positive
immunohistochemical reactions for HHV-8 and CD34. Clinical data regarding age, sex and
tumor location were retrieved from the patients’ pathology reports. As a comparative group, 10
cases of oral pyogenic granuloma and 10 oral hemangiomas were retrieved from the files of the
Oral Diagnosis Department (Pathology) of the Piracicaba Dental School of the University of
Campinas (Piracicaba/Brazil). The inclusion criteria were the presence of integral epithelial
tissue just above the lesions. Clinical data regarding age, sex and lesions location were also
retrieved from the pathology reports.
Immunohistochemistry
Immunohistochemical reactions for HHV-8 and CD34; samples were dewaxed with
xylene and then hydrated in an ethanol series. Antigen retrieval was performed using EDTA
/TRIS (pH 9.0) in a pressure cooker for 3 minutes, and the endogenous peroxidase activity was
blocked using 10% hydrogen peroxide. After washing in PBS buffer (pH7.4), slides were
incubated overnight with primary antibodies against HHV-8 LNA (Clone: 13B10,
NOVACASTRA, Nussloch, Germany, diluted 1:50); for the CD83 was made antigen retrieval
was performed using citrate solution (pH 6.0) in a pressure cooker for 3 minutes, and the
endogenous peroxidase activity was blocked using 10% hydrogen peroxide. After washing in
PBS buffer (pH7.4), slides were incubated overnight with primary antibodies against CD34
29
(Clone: QBEnd-10, DakoCytomation, Carpinteria, CA, USA, diluted 1:50). All slides were
subsequently exposed to avidin-biotin complex, horseradish peroxidase reagents (LSAB
KitDakoCytomation), and diaminobenzidine tetrahydrochloride (DAB, Sigma, St. Louis, MO,
USA), and subsequently counter-stained with Carazzi hematoxylin. As positive controls,
histological sections of Intestine (blood vessel endothelium) were used for CD34 and
Kaposi’s sarcoma were used for HHV-8 LNA. Negative controls were obtained by omitting the
primary antibodies.
Immunohistochemical reactions for CD83 and CD207; were done in 3µm histological
sections of all cases. Samples were dewaxed with xylene and then hydrated in an ethanol series.
Antigen retrieval was performed using citrate solution (pH 6.0) in a pressure cooker for 3
minutes, and the endogenous peroxidase activity was blocked using 10% hydrogen peroxide.
After washing in PBS buffer (pH7.4), slides were incubated overnight with primary antibodies
against CD83 (Polyclonal, Santa Cruz Technology, California, USA, diluted 1:100) and CD207
(Polyclonal, Cell Marque, diluted 1:50). All slides were subsequently exposed to avidin-biotin
complex, horseradish peroxidase reagents (LSAB KitDakoCytomation), and diaminobenzidine
tetrahydrochloride (DAB, Sigma, St. Louis, MO, USA), and subsequently counter-stained with
Carazzi hematoxylin. As positive controls, histological sections of normal skin were used for
CD207 and lymph node were used for CD83. Negative controls were obtained by omitting the
primary antibodies.
Immunohistochemical quantification
Quantification of LC was performed by a previously trained observer that counted the
number positive cells in the surface epithelium and for IDC, positive cells were counted in the
stroma of the lesions, far from the areas of ulcerations. In both counting, 10 sequential fields
(40x) for each antibody were selected. Because of the ramified aspect of LC in the epithelium,
morphological appearance of stained cells were also considered during the quantification
process in order to avoid that one same cell was counted twice. The results were expressed as
number of positive cells per case.
30
Statistics
The values were expressed as mean ± standard deviation (SD) and as median (minimum
and maximum values) for each group of lesions. Normality distribution of the results was
investigated with Kolmogorov-Smirnov and Shapiro-Wilk tests, whereas Levene test was used
to determine the homoscedasticity of the groups. The comparison of the cells distribution
between Kaposi’s sarcoma vs highly vascular oral lesions (hemangioma + pyogenic granuloma)
was done using Student t-Test and Mann-Whitney test. A p-value < 0.05 was considered
statistically significant. The software SPSS version 22.0 was used for the analyses. This study
was approved by the Ethics Committee of the Piracicaba Dental School, University of Campinas
(protocol 2.164.568/2017).
Results
In this study 51 cases of AIDS-associated oral Kaposi’s sarcoma were used. Men and
women were affected in the same proportion (M:F ratio 1:1) with a mean age of 31 years-old
(range from 21 to 61 years-old) and a site predilection for the palate (21 cases), followed by the
tongue (15 cases) and the gingiva (15 cases). Highly vascular oral lesions were retrieved to be
used as a comparative group. Hemangioma comprised 10 cases, more commonly affecting
females (M:F ratio 3:1) with a mean age of 57 years-old (range from 46 to 78 yearsold) affecting
the tongue (4 cases), lips (5 cases) and cheek mucosa (1 cases). Pyogenic granuloma accounted
for 10 cases mostly affecting males (M:F ratio 2:1) with a mean age of 45.6 years-old (range
from 18 to 87 years-old) always affecting the gingiva. There was no AIDS or HIV-positive
patient in the highly vascular oral lesions group.
Microscopically, all 71 cases used in this study had the overlying epithelium present.
Oral Kaposi’s sarcoma was predominantly composed of proliferating spindle-cells with the
presence of variably sized blood vessels. Erythrocytes extravasation was commonly found,
whereas mitosis figures were scarce. All cases were positive for CD34 and HHV-8 (Figure 1).
Hemangiomas exhibited blood vessels with different sizes being classified as either capillary or
cavernous hemangioma. Pyogenic granulomas were characterized by the presence of the
granulation tissue with intense chronic inflammatory infiltrate and a large number of blood
vessels (Figure 2).
31
Immunohistochemically, both markers (CD207 and CD83) were predominantly found
in the periphery of the lesions and more present in ulcerated areas, therefore, the cell counting
was done distant from these regions. The expression of CD207 identified immature DC mostly
in the overlying epithelium of all lesions, whereas only a few cells were found inside the lesions
(Figure 3). An opposite expression pattern was seen for CD83, which stained mature DC
predominantly in the middle of the lesions, with only scattered cells in the epithelium (Figure
4).
Immature CD207+ LC located in the epithelium presented their characteristic cytoplasm
extensions and were found in all layers, whereas mature CD83+ LC in the epithelium, CD207+
immature IDC and CD83+ mature IDC in the middle of the lesions, lost their extensions and
usually presented as round to oval cells.
Data on the mean (±standard deviation) and median (range) values obtained for
CD207 in the epithelium and stroma of oral Kaposi’s sarcoma and highly vascular oral lesions
are summarized in Table 1. In the epithelium, the number of immature CD207+ LC was
significantly higher in highly vascular oral lesions than in Kaposi’s sarcoma (p = 0.02), whereas
in the stroma of the lesions the number of immature IDC in Kaposi’s sarcoma was higher than
in benign lesions (p < 0.001). Regarding the distribution of mature CD83+ LC and IDC, it was
observed that Kaposi’s sarcoma presented slightly more LC in the epithelium than its benign
vascular counterparts (p = 0.373), however, the presence of mature CD83+
IDC in the stroma of the lesions were higher in Kaposi’s sarcoma than in highly vascular oral
lesions (p < 0.001).
Discussion
Kaposi’s sarcoma is a low-grade vascular malignancy considered an AIDS-defining
disease. Its incidence revealed a dramatic increase in the early 1980’s following HIVinfection
outburst worldwide and it is still overrepresented in this population, although the development
of the highly active anti-retroviral therapy (HAART) usually lead to the involution of the
neoplasm. Kaposi’s sarcoma is also known to be tightly associated with HHV-8 infection, which
is considered the main etiologic factor for the malignancy, despite the exact mechanisms that
cause tumor growth remains to be fully established 1. Disruptions of systemic
immunosurvilance are critical for Kaposi’s sarcoma onset, but the importance of changes in the
32
distribution and function of immune components in the local environment of the disease need
to be more investigated. Therefore, in this study we demonstrated significant differences in the
distribution of LC and IDC in AIDS-associated oral Kaposi’s sarcoma when compared to HIV-
negative highly vascular oral lesions, suggesting that alterations in the presence of these DC
may play an important role in the pathogenesis of Kaposi’s sarcoma.
LC are bone marrow derived DC found in the epithelium of normal skin and mucosas
that encounter invading foreign pathogens in these surfaces, transfer to connective tissue where
they are known as IDC and move toward regional lymph nodes where antigens are presented to
naïve T lymphocytes8. There is a growing number of evidences supporting the hypothesis that
different subtypes of DC play important roles in the immunosurveillance against neoplastic cells
and that the distribution and/or the regular function of these cellular populations are altered in
many human tumors including oral cancer, lip cancer, mycosis fungoids, cutaneous squamous
cell carcinomas, and others 8–11. Moreover, different authors have attributed a significant
prognostic importance for DC in several of these malignant neoplasms12,13, speculating that
changes in these cells would not only predispose to neoplastic development, but it would also
represent potential therapeutic targets for future chemotherapy schemes.
Our group has previously demonstrated that AIDS patients reveal important changes in
the distribution of LC and IDC in the oral mucosa, predisposing these individuals to local
opportunistic infections6,7. Moreover, both cells are important for certain virus infections,
including human immunodeficiency virus type 1, herpes simplex virus and cytomegalovirus14.
In addition, due to the strategic localization of these DC in the overlying oral mucosa and the
well known association of HHV-8 infection with the pathogenesis of Kaposi’s sarcoma, we
speculate that local alterations in the distribution of DC could be associated with the onset of
this malignant vascular neoplasm. Further supporting this hypothesis, Rappocciolo et al. 14
(2017) recently showed that HHV-8 was capable of infecting three different DC types,
monocyte-derived DC, LC and and interstitial dermal dendritic cells, resulting in a reduced
ability of these cells to stimulate allogeneic CD4+ T cells. The authors stated that HHV-8 caused
DC infections by different receptors, ultimately altering their function. This same group
previously demonstrated that the dendritic cell-specific ICAM-3 grabbing nonintegrin (DC-
SIGN; CD209) would be one of the important receptors for HHV-8 infection of myeloid DCs
and activated B lymphocytes 15,16. Therefore, the effects of HHV-8 on antigen presenting DC,
may be the cause of the weak T cell responses against HHV-8 antigens 17.
33
In this study, we observed a significant decrease in the number of CD207+ LC in the
epithelium of AIDS-associated oral kaposi’s sarcoma when compared to the epithelium of non-
AIDS highly vascular oral lesions, suggesting that not only the function of these cells is altered
by the viral infection, but also the concentration of this cellular population might be decreased.
However, it is also possible to speculate that the number of these immature LC is not disrupted,
but only its ability to express CD207, therefore, other LC immature- or panmarker would be
recommended to confirm these possibilities. Nevertheless, no matter what option is taking
place, our result is per se important to demonstrate the loss of CD207 protein, given its known
importance as a protective barrier against viral infections, like against HIV118,19; on the other
hand, CD207 role in HHV-8 immune processing remains obscure and needs to be investigated
since the effect of different viruses on LC may exhibit highly different results 20.
We have also obseerved that Kaposi’s sarcoma patients exhibited a higher number of
CD207+ and CD83+ IDC than benign lesions, we speculate that it possibly indicates a host
response against HIV and/or HHV-8 infections, with a higher concentration of immature and
activated stromal DC being recruted, supposedly from circulating monocytes 5. IDC may
complement the role of LC by adopting an immunostimulatory activity20.
Although our study represents an original contribution to the understanding of LC and
IDC importance for Kaposi’s sarcoma pathogenesis, several authors have previously attempted
to investigate the role played by different DC subsets in the development of this malignancy.
We reviewed the available literature searching for studies dealing with DC and
Kaposi’s sarcoma, and Table S1 summarizes the investigations encountered. The diversity of
molecules expressed by each DC not only makes their mechanisms more complex, but also
impair appropriate comparisons among different studies that used different markers to identify
DC. The first investigations of DC in Kaposi’s sarcomas used the unspecific proteins HLADR
21 and factor XIIIa to stain dendrocytes, but CD122,23 has been the most used DC marker,
although it does not discrimínate between mature and immature subpopulations. Using CD1a
Jivan et al. 24 (2016) recently showed a lower count of LC in non-Candida infected oral kaposi’s
sarcoma than in uninfected normal mucosa. Interestingly, plasmocytoid DC (pDC) has been the
most studied subpopulation in Kaposi’s sarcoma given its ability to recognize single-stranded
RNA and unmethylated CpG motifs associated with viral infection, inducing an antiviral
response within the host and a high production of interferon25–27. Similar to our study, Stebbing
et al. 28 (2003) using HIV-1 infected patients, also showed a depletion of pDC in Kaposi’s
sarcoma patients compared with Kaposi’s sarcoma-negative individuals, whereas Karouni et al
34
29 (2016) observed a significantly lower pDC count and a lower interferon production in classic
kaposi’s sarcoma than in mollusccum contagiosus cases.
Although we did not correlate the distribution of LC and IDC observed in Kaposi’s
sarcoma patients with their respective CD4 count since this data were unavailable for
consultation, potentially representing a limitation of our study, Grulich et al. 30 (2007) stated
that CD4 count could be an insensitive indicator of immune deficiency, because its count might
not be an accurate or unbiased measure of immune function. It is also important to consider that
the distribution of LC may vary according to different intraoral subsites, therefore, it remains
to be determined if Kaposi’s sarcomas biopsied in different oral locations could have interfered
in our DC count.
In conclusion, we observed a reduction of CD207+ immature LC and an increase of
CD207+ and CD83+ IDC in AIDS-associated Kaposi’s sarcoma than in HIV-negative highly
vascular oral lesions, suggesting that alterations in the distributions of these DC subsets, either
because HIV and/or HHV-8 effect, may play a role in the pathogenesis of this malignant
vascular neoplasm.
35
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38
Table 1. Distribution of LC and IDC in different compartments (epithelium and stroma of the lesions) of oral Kaposi’s sarcoma and in highly vascular oral
lesions (hemangioma and pyogenic granuloma).
Lesions CD207 CD83
Epithelium Stroma Epithelium Stroma
Mean (±SD) Median (range) Mean (±SD) Median (range) Mean (±SD) Median (range) Mean (±SD) Median (range)
Kaposi’s sarcoma 44.2† (37.1) 35 (0 – 142) 17.9 (15.3)† 13 (0 – 62) 8.31 (8.0) 6 (0 – 37) 37.2 (20.9)† 35 (0 – 100)
Highly vascular oral lesions * 65.7 (26.4) 63 (18 – 109) 4.2 (2.4) 4 (1 – 11) 6.9 (6.9) 4 (0 – 21) 17.6 (11.0) 16.0 (4 – 46)
*Hemangioma and pyogenic granuloma. †Statistically significant differences.
39
Table 2. Summary of the available studies that investigated the importance of different dendritic cells in the pathogenesis of Kaposi’s sarcoma. Authors Country KS clinical
subtype Sample
source No. cases KS location HIV status DC investigated Lab technique Markers
used Main results
Current study, 2017. Brazil/South Africa HIV-
associated KS Tissue
51 cases of KS 10 cases of pyogenic granuloma 10 cases of hemangioma
Oral mucosa 100%
positive Langerhans cells IHC
CD83 +
CD207 +
KS revealed a
significantly lower
CD207+ immature LC
count and an increased
CD207+ IDC than
benign vascular oral
lesions. Moreover, KS
also showed an
increased number of
mature CD83+ IDC
than benign vascular
lesions.
Rappocciolo et al., 2017 USA NA Neonatal
cord blood NA NA
Langerhans cells, Interstitial Dermal Dentritic cell,
monocyte-derived dendritic
cells.
Flow Cytometry Cell culture Immunofluoresc ence PCR
HLA-DR CD1a CD11b CD14 CD91 CD80 CD86 CD83 CD209 CD207
HHV-8 can target both
LC and iDDC for
productive infection
via different receptors
and alter their
function, supporting
their potential role in
HHV-8 pathogenesis
and KS
40
Jivan et al., 2016. South Africa HIV- associated KS
Tissue
Study groups: 15 cases of
KS without
Candida
albicans co- infection 17 cases of KS with
Candida albicans co-infection Control
groups: 23 cases of
noncandida and
nonHIV infected
mucosa AP. 19 cases of oral
candidosis
from healthy patients
Oral mucosa 100% positive
Langerhans cells IHC CD1a +
Significant lower
count of LC in
nonCandida infected
KS than in uninfected
normal mucosa.
Karouni et al., 2016. Lebanon Classic KS Tissue
20 cases of KS 20 cases of mollusccum
contagiosun
Cutaneous 100% negative
Plasmacytoid DC IHC
BDCA-2 + MxA + (to evaluate
interferon
production)
pDCs are recruited
into the skin lesions of
classic KS, but there is
a significantly lower
plasmacytoid cells and
lower interferon
production in cKS
than in mollusccum
cases.
Ramdial et al., 2011. UK/South Africa HIV- associated KS
Tissue 9 cases of anaplastic KS
Cutaneous 100% positive
Langerhans cells IHC S100+ CD1a+ CD207+
Presence of an
extensive amount of intratumoral Langerhans cells.
41
West et al., 2011. USA NA Peripheral
blood
Health subjects Laboratorial HHV8
infected DC cells NA NA Plasmacytoid DC
Flow cytometry Cell culture
BCDA4+ BDCA2+ CD303+ CD123+ CD83-/+ CD86-/+
KSHV can infect and activate human pDCs,
as measured by
upregulation of CD83 and CD86, and by IFN- secretion;
induction of IFN-α
occurs through
activation of TLR9
signaling.
Bella et al., 2006. Italy Classic KS Peripheral
blood 76 KS 72
controls NA
100% negative
Myeloid cell Plasmacytoid cell Monocytes (DC precursor)
Flow cytometry Cell culture
HLA-DR + CD3 - CD19 - CD20 - CD16 - CD14 - CD11c
+ CD123 + CD80 -/+ CD83 -/+ CD86 -/+ CD49c -/+ CD91 -/+ CD34 -/+ CD14 -/+ CD1a + CD40+ ILT2, 3, 4 +
PBDC (both myeloid, plasmacytoid and
monocytes) in cKS
patients were
significantly lower
than controls, and was
more severe in more advanced cases;
PBDC expressing the HHV8 receptor CD49c was higher in
cKS patients than in
healthy control;
42
Stebbing et al., 2003. UK HIV- associated KS
Peripheral
blood
12 cases of HIV- positive KS patients 6 cases of HIV- positive patients
without KS.
NA 100% positive
Myeloid cell Plasmacytoid cell Monocytes (DC precursor)
Flow cytometry Cell culture
CD3 - CD14 - CD16 - CD19 - HLA-DR + CD11c -/+ CD1a CD80 CD83 CD40 CD91
Despite the impaired functional capacity of
DC in patients with
KS, they retained the ability of activating
and stimulating CD8 cytotoxic T cells; Plasmacytoid DCs
were depleted in
KSpositive compared
with KS-negative
HIV-1– infected
patients.
Simonart et al., 2000. Belgium
Iatrogenic,
sporadic and
HIV- associated KS
Cell
cultures
derived
from
tissue
4 KS patients Cutaneous 50% positive
Dendritic cell IHC Flow cytometry Cell culture
CD1a
Less than 1% of the
spindle cells cultured were positive for
CD1a; Spindle cell cultures have nearly always
given rise to cells with
fibroblastic and/or
smooth muscle cell
differentiation; KS spindle cells lack
endothelial and/or
leucocyte markers.
Tabata et al., 1993. Germany/Japan HIV- associated KS
Tissue
15 cases of HIV+ KS 19 cases of oral mucosa
of HIV+ patients 22 cases of oral mucosa
of HIV- patients
Oral mucosa 100% positive
Dendritic cells IHC HLA-DR +
The number of HLA- DR+ cells was increased in patchlike
and nodular KS
compared to HIV+
and HIV- normal
mucosa.
43
Kanitakis et al., 1992. France
Classic KS
Immunossupr
essionassociated
KS
Tissue 13 classic KS 16
Immunes. KS Cutaneous
13 HIV+ patients
Dermal dendrocytes IHC Factor XIIIa +
Antigen H -
Dermal dendrocytes density tended to be decreased in immunosuppression-
associated
Kaposi's
sarcoma when compared with
classical form; The results do not support dermal
dendrocytes as being
the cells of origin of Kaposi's sarcoma;
Decreased density of
dermal dendrocytes
could be related with a
more aggressive
clinical course.
NA: Not available.
44
Figures
Figure 1. Microscopic and immunohistochemical aspects of oral Kaposi’s sarcoma.
A) All cases included in this study were categorized as solid Kaposi’s sarcoma and exhibited a
spindle cell component with irregular blood vessels with different shapes and sizes (H&E;
50X). B) Higher magnification demonstrating the spindle cell component of Kaposi’s sarcomas
(H&E; 100X). C) All cases were positive for the vascular marker CD34 (DAB; 200X) and D)
were confirmed to be associated with HHV-8 infection, demonstrated as a nuclear staining in
the neoplastic cells (DAB; 200X).
45
Figure 2. Microscopic aspect of highly vascular oral lesions.
A) Oral pyogenic granuloma exhibiting a fibrinous membrane in the ulcerated surface of the
lesion (H&E; 50X). B) Intense mononuclear inflammatory infíltrate and a dense proliferation
of blood vessels characterized the granulation tissue of pyogenic granulomas used in this study
(H&E; 100X). C) Oral hemangiomas exhibited blood vessels of different sizes and shapes full
of blood cells (H&E; 100X). D) Higher magnification of a cavernous oral hemangioma
demonstrating the presence of ectatic blood vessels (H&E; 200X).
46
Figure 3. Immunohistochemical reactivity against CD207 in the epithelium and stroma of oral
Kaposi’s sarcoma and of benign vascular oral lesions.
A) CD207 stained immature LC in the overlying epithelium of oral Kaposis’s sarcoma,
revealing the dendritic characteristic of these cells (DAB; 100X). B) In the stroma of Kaposi’s
sarcomas, CD207+ IDC were very scarce and were observed as round cells without dendritic
prolongations (DAB; 100X). C) A significantly higher count of CD207+ immature LC were
observed in the epithelium of HIV-negative benign vascular oral lesions (DAB; 100X), D)
whereas a lower count of CD207+ IDC was obtained inside the benign vascular oral lesions
(DAB; 200X).
47
Figure 4. Immunohistochemical reactivity against CD207 and CD83 in the epithelium and
stroma of oral Kaposi’s sarcoma and of highly vascular oral lesions.
A) CD83 stained mature LC, few cells were observed in the epithelium of AIDS-associated oral
Kaposi’s sarcomas (DAB; 100X). B) In the stroma of Kaposi’s sarcomas, whereas a higher
number of CD83+ IDC (DAB; 100X). C) In benign vascular oral lesions, the number of
CD83+ LC was lower than in Kaposi’s sarcoma (DAB; 200X)., and G) a significantly lower
number of CD83+ IDC was observed in benign vascular oral lesions (DAB; 200X).
48
3 CONCLUSÃO
Observou-se uma diminuição na concentração de CL imaturas CD207+ no epitélio e um
aumento de células dendríticas intersticiais CD207+ e CD83+ no sarcoma de Kaposi oral
associado à AIDS quando comparado com as lesões orais altamente vascularizadas em
pacientes HIV-negativos, sugerindo que alterações na distribuição destas subpopulações de
células dendríticas, seja pelo efeito da infecção pelo HIV e/ou do HHV8, podem desempenhar
um papel importante na patogênese desta neoplasia vascular maligna.
49
* De acordo com as normas da UNICAMP/FOP, baseadas na padronização do International Committee of Medical Journal Editors - Vancouver Group. Abreviatura dos periódicos em conformidade com o PubMed.
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52
ANEXOS
ANEXO 1 – Certificado do comitê de ética
53
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