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FUNDAÇÃO OSWALDO CRUZ
INSTITUTO OSWALDO CRUZ
Doutorado em Biologia Celular e Molecular
Rio de Janeiro
CÉLULAS DE DOADORES NORMAIS PRIMADAS IN VITRO
RESPONDEM AO ANTÍGENO OVULAR SOLÚVEL E AO PEPTÍDEO
SINTÉTICO MAP4 DERIVADO DA TRIOSE FOSFATO ISOMERASE DO
SCHISTOSOMA MANSONI
ELIANA ALMEIDA GOMES REIS
Salvador-Bahia
2007
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INSTITUTO OSWALDO CRUZ
Ministério da Saúde
Fundação Oswaldo Cruz
Ministério da Saúde
Fundação Oswaldo Cruz
Ministério da Saúde
Fundação Oswaldo Cruz
Ministério da Saúde
Fundação Oswaldo Cruz
Pós-Graduação em Biologia Celular e Molecular
ELIANA ALMEIDA GOMES REIS
CÉLULAS DE DOADORES NORMAIS PRIMADAS IN VITRO RESPONDEM AO
ANTÍGENO OVULAR SOLÚVEL E AO PEPTÍDEO SINTÉTICO MAP4 DERIVADO
DA TRIOSE FOSFATO ISOMERASE DO SCHISTOSOMA MANSONI
Tese submetida à coordenação de Pós-Graduação em
Biologia Celular e Molecular do Instituto Oswaldo Cruz
para a obtenção do Título de Doutor em Biologia Celular e
Molecular na área de concentração em Imunologia.
Orientador: Dr. Mitermayer Galvão dos Reis
Co-orientador: Dr. Donald Harn Jr
Salvador-Bahia
2007
ads:
Ficha Catalográfica elaborada pela Biblioteca do
Centro de Pesquisas Gonçalo Moniz / FIOCRUZ - Salvador - Bahia
.
Reis, Eliana Almeida Gomes
R375c Células de doadores normais primadas in vitro respondem ao antígeno ovular solúvel
e ao peptídeo sintético MAP4 derivado da triose fosfato isomerase do Schistosoma mansoni
/ Eliana Almeida Gomes Reis. - 2007.
43 f.; 30 cm
Datilografado (fotocópia).
Tese (doutorado) – Instituto de Pesquisa Oswaldo Cruz. 2007.
Orientador: Prof. Mitermayer Galvão dos Reis.
1. Schistosoma mansoni. 3.Citocinas. 4. Imunidade celular
I.Título.
CDU 616.995.122
INSTITUTO OSWALDO CRUZ
Ministério da Saúde
Fundação Oswaldo Cruz
Ministério da Saúde
Fundação Oswaldo Cruz
Ministério da Saúde
Fundação Oswaldo Cruz
Ministério da Saúde
Fundação Oswaldo Cruz
Pós-Graduação em Biologia Celular e Molecular
ELIANA ALMEIDA GOMES REIS
CÉLULAS DE DOADORES NORMAIS PRIMADAS IN VITRO RESPONDEM AO
ANTÍGENO OVULAR SOLÚVEL E AO PEPTÍDEO SINTÉTICO MAP4 DERIVADO
DA TRIOSE FOSFATO ISOMERASE DO SCHISTOSOMA MANSONI
Orientador: Dr. Mitermayer Galvão dos Reis
Co-orientador: Dr. Donald Harn Jr
Aprovada em: 23 / 11 / 2007
Revisor: Dr. Zilton de Araujo Andrade
Banca Examinadora: Dra. Euzenir Nunes Sarno IOC/FIOCRUZ (Presidente)
Dr. Edgar Marcelino de Carvalho Filho HUPES/UFBA (Membro)
Dr. Manoel Barral Netto CPqGM/FIOCRUZ (Membro)
Novembro / 2007
A Mitermayer meu esposo, Mitermayer Almeida nosso filho,
pela compreensão e paciência ao longo do meu trabalho.
Aos meus pais e irmaõs, pelo apoio na minha vida.
AGRADECIMENTOS
AGRADECIMENTO ESPECIAL a Mitermayer, pelo incentivo e ensinamentos ético, científico
e social, pela compreensão, paciência, generosidade, simplicidade e objetividade que
caracterizam um bom educador e pesquisador.
AGRADECIMENTO ESPECIAL a Timothy Flanigan na Case Western University, John
David, Roberta David, Donald Harn, Kinje Hirayama e Charles Shoemaker professores e
pesquisadores da Harvard, pelo incentivo à pesquisa e pela oportunidade de tê-los como os
meus primeiros mestres na iniciação científica na área biológica, e também pelo importante
apoio e sugestões.
A Dr. Milton Osorio Moraes, coordenador do Curso de Pós-Graduação em Biologia
Celular e Molecular da FIOCRUZ-(IOC), pela paciência e compreensão.
A Isabel Miranda, pela sua atenção e orientação no meu treinamento na Harvard na
padronização do sistema PIV.
A Theomira, minha amiga e irmã, pela paciência, colaboração e contribuição em todas as
etapas deste trabalho.
Agradeço aos Drs. Neil Simonsen, Edgar Carvalho, John Ho, Lain Carvalho e Alan McBride,
pela contribuição e sugestões importantes na construção dos manuscritos.
Agradeço aos Drs. Manoel Barral-Netto e Ronald Blanton pela orientação em outros estudos
durante o meu período de doutorado.
A Dr. Albert Ko, pelo incentivo, amizade e contribuições preliminares na construção do
segundo manuscrito.
A Rodrigo Athanazio, pela participação na realização das análises estatísticas do segundo
manuscrito.
A Glenda Meira, pelo apoio, amizade e aprendizagem.
A Daniel Athanazio, pela disponibilidade em colaborar, além da amizade e atenção.
A Luciano Kalabric, pela disponibilidade em resolver todos os problemas, além da amizade,
e aprendizagem ao longo desses anos.
Aos doadores voluntários, pela importante participação na realização deste trabalho.
A Jorge Clarêncio, pelo apoio na citometria de fluxo.
A Martha Berbert, pela ajuda na reformatação da ABNT no EndNote.
A Maria Celia Zarife, pela gentil presteza na revisão do portugues.
A todos os colegas do Laboratório de Patologia e Biologia Molecular (LPBM), pela
convivência agradavel ao longo desses anos.
Aos colegas de todos os outros laboratórios do CPqGM-Fiocruz, pelo carinho e amizade.
“A Deus, por estar ao meu lado em todos os momentos da minha vida”.
SUPORTE FINANCEIRO
NIH–National Institutes of Health–AI30639 USA
Fundação Oswaldo Cruz-RJ&BA
CNPq -Conselho Nacional de Desenvolvimento Científico e Tecnológico
“O futuro pertence àqueles que
acreditam na beleza dos seus sonhos”
Eleanor Roosevel.
RESUMO
Dados da literatura demonstram que os antígenos do Schistosoma mansoni induzem
uma resposta imune de células T auxiliadora (Th) CD4
+
. Os eventos iniciais da resposta
imune na esquistossomose são bem caracterizados no modelo murino. Entretanto, ainda são
poucos os estudos que avaliam a resposta imune inicial desenvolvida em humanos,
especialmente aquelas contra os antígenos mais relevantes do parasito. Para estudar esses
eventos iniciais que ocorrem na resposta imune humana após a oviposição na
esquistossomose mansoni, foi realizado o “priming” a estimulação primaria in vitro (PIV), um
sistema que simula as condições in vivo. Este sistema pode ser usado para estudar os eventos
iniciais in vitro à semelhança de uma infecção natural. No estudo in vitro, foram utilizadas
células dendríticas profissionais como células apresentadoras de antígeno. Células
mononucleares do sangue periférico (PBMCs) de doze doadores normais saudáveis sem
historia prévia de contacto com S. mansoni ou com qualquer de seus antígenos, foram
primadas com antígeno ovular solúvel (SEA) do S. mansoni ou múltiplos peptídeos antigênico
(MAP4), contendo epitopos de células T e B derivado da triose fosfato isomerase. As células
dendríticas foram previamente geradas, caracterizadas e em seguida incubadas na presença ou
ausência de antígenos, posteriormente cultivadas na presença de PBMCs total por um período
de sete dias para simular a infecção in vivo. As células primadas foram coletadas, lavadas e
reestimuladas duas vezes. Em cada etapa do PIV foram determinadas as concentrações de
citocinas nos sobrenadantes de culturas e a expressão dos marcadores de superfície das
subpopulações de linfócitos, durante o “prime” e após as reestimulações da primeira e
segunda chamada. As PBMCs primadas com antígenos SEA apresentaram um perfil de
células semelhante do tipo Th2, exceto 2/12 doadores que apresentaram uma resposta
semelhante do tipo Th1. Diferente do observado para PBMCs primadas com SEA, PBMCs de
todos doadores primados com MAP4 desenvolveram uma resposta semelhante do tipo Th1.
Ambos os antígenos demonstraram semelhança com a resposta imune induzida em
camundongo in vivo e in vitro. Os resultados sugerem que as DCs sensibilizadas com SEA ou
MAP4 foram capazes de induzir proliferação e expansão clonal específica dos linfócitos.
ABSTRACT
It has been well established that Schistosoma mansoni induces an initial CD4
+
T-cell
response. The early events in the immune response to schistosomiasis have been evaluated in
mice but few studies have focused on the early response in humans, especially in regard to
parasite antigens that are potential vaccine candidates. To study the early immune response in
humans, we performed an in vitro assay mimicking the probable in vivo conditions. Naïve
peripheral blood mononuclear cell (PBMCs) were exposed to S. mansoni soluble egg antigen
(SEA) or a synthetic multiple antigen peptide containing B and T cell epitopes from Triose
Phosphate Isomerase (MAP4) in a priming in vitro assay. Priming in vitro has been used
recently as an important tool to reproduce in vitro the conditions of natural infection.
Dendritic cells were used as antigen presenting cells. PBMCs from twelve healthy volunteers
with no previous history of exposure to S. mansoni were stimulated by SEA or MAP4.
Dendritic cells were generated, characterized and incubated in the presence or absence of
antigen, and cultured with total PBMCs for seven days corresponding to a three day period of
natural infection. Primed cells were collected and restimulated a further two times. At each
step cytokine secretion was evaluated in the culture supernatant. The phenotypes of the
lymphocyte subsets were determined after priming, first and second recall. Donor SEA-
primed PBMCs developed a Th2-like response except for 2/12 donors who exhibited an
atypical Th1-like response. In contrast to the effects of SEA stimulation, MAP4-primed cells
developed a Th1-like response. Both antigens reproduced the effects previously observed in
vitro and in vivo in the murine model. The present results suggest that SEA and MAP4
sensitized dendritic cells are able to induce specific lymphocyte proliferation and clonal
expansion.
LISTA DE ABREVIATURAS
Ag: Antígeno
APC: Célula apresentadora de antígeno
B7: Molécula co-estimulatória ligante de CD28
CD: Grupos de diferenciação (“Cluster of differentiation”), nomenclatura usada para as
diversas moléculas de superfície celular
CD3: Marcador de superficie célular da população de linfócitos T
CD4: Marcador de superficie célular da subpopulação de linfócitos T auxiliar
CD8: Marcador de superficie célular da subpopulação de linfócitos T citotóxico
CD14: Moléculas expressa na superfície de monócitos
CD19: Marcador de superficie célular da subpopulação de linfócitos B
CD25: Cadeia α do receptor para a citocina IL-2
CD28: Moléculas de superfície celular para co-estimulação
CD45RO: Moléculas de superfície celular de memória expressa em linfócitos
CD80: Moléculas de superfície celular co-estimulatoria B7-1, ligante de CD28
CD86: Moléculas de superfície celular co-estimulatoria B7-2, ligante de CD28
DCs: Células dendriticas
ELISA: Ensaio de Imunoabsorbância Ligado a Enzima
FACS: Fluorescence – activated cell Sorter
FCS: Soro Fetal Bovino
FITC: Isotiocianato de fluoresceína, tipo FL-1
FL1: Fluorescência do tipo 1 (verde)
FL2: Fluorescência do tipo 2 (laranja)
Flow cytometry: Método de análise da população celular
FSC: Difração do raio laser, proporcional ao tamanho celular (Tamanho)
HLA-DR: Antígeno Leucocitário Humano, do complexo principal de histocompatibilidade
ICAM-1: Moléculas de adesão intercelular 1
IFN-γ: Interferon-gamma
IgG: Imunoglobulinas G total
IL-2: Fator de crescimento para os linfócitos T
IL-4: Interleucina 4
IL-5: Interleucina 5
IL-10: Interleucina 10
IL-12: Interleucina 12
LPS: Lipopolissacaridio
MAP4: Múltiplos peptídeos antigênico, contendo epitopos de células T e B derivado da triose
fosfato isomerase do Schistosoma mansoni
MHC: Complexo principal de histocompatibilidade
MO: Monócito
PBMC: Células mononucleares do sangue periférico
PE: Ficoeritrina, tipo FL-2
PIV: Estimulação primária in vitro (“priming” in vitro)
RPMI: Meio de cultura de células
SBF: Soro Bovino Fetal
SEA: Antígeno solúvel do ovo do Schistosoma mansoni
SSC: Refração e reflexão do raio laser, proporcional à granulosidade e complexidade interna
de uma célula (Granulosidade).
TCR: Molécula expressa por linfócitos T com a função de reconhecer o complexo MHC-Ag
Th: Linfócitos T auxiliadores
Th0: Linfócitos T auxiliadores que secretam citocinas do tipo 1 e do tipo 2
Th1: Linfócitos T auxiliadores que secretam citocinas do tipo 1
Th2: Linfócitos T auxiliadores que secretam citocinas do tipo 2
TNF-α: Fator de Necrose Tumoral alfa
WHO: Organização Mundial da Saúde
SUMÁRIO
1. INTRODUÇÃO....................................................................................................................12
1.1. ASPECTOS EPIDEMIOLOGICOS, ETIOLÓGICOS, E CLÍNICOS DA
ESQUISTOSSOMOSE MANSONI.............................................................................12
1.2. A RESPOSTA IMUNE NA ESQUISTOSSOMOSE MANSONI..................................15
1.3. A RESPOSTA IMUNE CELULAR E HUMORAL NA ESQUISTOSSOMOSE
MANSONI HUMANA AGUDA..................................................................................18
1.4. A RESPOSTA IMUNE CELULAR E HUMORAL NA ESQUISTOSSOMOSE
MANSONI HUMANA CRÔNICA..............................................................................19
1.5. PAPEL DAS CÉLULAS APRESENTADORAS DE ANTÍGENOS NA DEFINIÇÃO
DA RESPOSTA IMUNE NA ESQUISTOSSOMOSE..................................................20
2. OBJETIVOS.......................................................................................................................23
3. RESULTADOS ..................................................................................................................24
MANUSCRITO 1 ................................................................................................................24
Naive Donor Responses to Schistosoma mansoni Soluble Egg Antigens. ..................... 24
MANUSCRITO 2 ................................................................................................................26
Schistosoma mansoni Triose Phosphate Isomerase peptide MAP4 is able to trigger naïve
donor immune response toward a Type-1 cytokine profile..............................................26
4. DISCUSSÃO GERAL........................................................................................................27
5. CONCLUSÕES GERAIS ..................................................................................................31
6. REFERÊNCIAS BIBLIOGRÁFICAS ...............................................................................32
ANEXO A.................................................................................................................................41
ANEXO B.................................................................................................................................42
12
1. INTRODUÇÃO
1.1. ASPECTOS EPIDEMIOLOGICOS, ETIOLÓGICOS, E CLÍNICOS DA
ESQUISTOSSOMOSE MANSONI.
A esquistossomose é uma doença presente em todos os continentes com 200 milhões de
indivíduos infectados, dos quais 120 milhões são assintomáticos e 20 milhões têm doença
grave, sendo que 600 milhões vivem em áreas de risco (CHITSULO, 2000; GRYSEELS,
2006). Ela é causada pelo trematódeo do gênero Schistosoma presente em todos os
continentes sendo que cinco espécies são patogênicas para o homem. Destas, S. mansoni, S.
japonicum e S. haematobium são as mais importantes, enquanto que o S. intercalatum e S.
mekongi têm distribuição restrita e as espécies S. bovis e S. matteei ocasionalmente infectam
humanos. No Brasil e em outros países da América, a esquistossomose humana é causada
pelo S. mansoni, na África e Oriente Médio, pelos S. mansoni e S. haematobium e na Ásia
(China, Japão, Filipinas, e Indonésia) pelo S. japonicum. No Brasil, de acordo com os dados
do Ministério da Saúde, estima-se entre 3 milhões de pessoas infectadas e 25 milhões vivendo
em área de risco (FNS 1997, (CHITSULO et al., 2000), principalmente, nas pequenas cidades
e na zona rural (BINA; PRATA, 2003). Recentemente tem sido observada uma expansão da
esquistossomose para a periferia das grandes cidades, em função da migração de populações
do interior para as grandes metrópoles que crescem de forma desorganizada na periferia, sem
saneamento adequado, com populações desassistidas, sem educação e sem informação. No
presente trabalho, trataremos principalmente do S. mansoni, única espécie presente no Brasil.
A transmissão da esquistossomose mansônica depende da presença de hospedeiros
definitivos, indivíduos ou animais infectados eliminando ovos, e do caramujo hospedeiro
intermediário. Os ovos eliminados nas fezes, quando em contato com coleções de água doce,
eclodem liberando os miracídios que infectam caramujos. Estes, após trinta e cinco dias,
eliminam cercárias, formas infectivas para os hospedeiros definitivos. As cercárias infectam o
homem através da penetração na pele quando perdem a cauda e se transformam em
esquistossômulos. Estes, nas primeiras 24 horas, produzem e secretam enzimas proteolíticas
que destroem a matriz intersticial, facilitando sua penetração em vasos sanguíneos da
circulação periférica. A partir do quarto dia, os esquistossômulos alimentam-se de eritrócitos
do hospedeiro, iniciando um rápido período de crescimento e desenvolvimento. Entre 6-12
dias da infecção, os esquistossômulos se transformam em vermes imaturos, passam pelos
pulmões e migram para o fígado onde os vermes imaturos começam aparecer a partir do 16º
dia (ANDRADE, 1987). Aproximadamente após o 28º dia da infecção os vermes adultos
migram para as veias mesentéricas inferiores do intestino, onde se acasalam e, em torno do
13
35º dia de vida, iniciam a oviposição. Existem evidências que os ovos presentes na superfície
da mucosa induzem uma reação granulomatosa, destacam-se em direção à luz do intestino,
sendo carreados pelas fezes e eliminados no ambiente para manutenção do ciclo. Por outro
lado, ovos presentes nas veias mesentéricas são carreados pelo sangue em direção ao fígado,
ficando retidos nos pequenos ramos da veia porta. Estes ovos retidos eliminam antígenos que
destroem o tecido circunjacente, induzindo, por parte do hospedeiro, uma resposta
inflamatória crônica granulomatosa (ANDRADE, 1964; WARREN; DOMINGO, 1970). Em
adição, no tegumento do verme são incorporados antígenos do hospedeiro incluindo
substâncias do grupo sanguíneo ABO humano e produtos do MHC classe I e classe II
(GOLDRING et al., 1976; SHER et al., 1978).
Figura 1. Ciclo de vida do S. mansoni (fonte: adaptado da internete).
Os indivíduos infectados pelo S. mansoni podem desenvolver uma forma aguda
toxêmica mais freqüentemente observada nos indivíduos que não residem em áreas endêmicas
e nunca tiveram contato com o antígeno do Schistosoma, sendo raramente observada nos
indivíduos residentes nas áreas endêmicas que, na grande maioria das vezes, se apresentam
com a forma crônica (ANDRADE; BINA, 1983).
Na forma aguda da esquistossomose, observa-se uma reação de hipersensibilidade
sistêmica induzida pela migração dos esquistossômulos, que pode ocorrer nas primeiras
semanas antes do aparecimento dos ovos nas fezes (GRYSEELS et al., 2006). Essa forma
14
caracteriza-se pela presença de dermatite local e vermelhidão na pele, febre irregular, fadiga,
mialgia, tosse não produtiva, eosinofilia e dor de cabeça. Sintomas abdominais podem ocorrer
mais tarde, provocados pela migração e posicionamento dos vermes maduros. A maioria dos
pacientes recupera-se espontaneamente depois de 2-10 semanas, mas alguns desenvolvem
uma doença mais grave em decorrência da perda de peso, congestão pulmonar, bronquite,
dispnéia, diarréia, dor abdominal difusa e hepatoesplenomegalia. Em geral, nos indivíduos
residentes nas áreas endêmicas, os sintomas e sinais desaparecem em poucos dias (BOROS,
1989). Entretanto, nos turistas e migrantes, as lesões podem persistir por dias, especialmente
depois da infecção primária (BOTTIEAU et al., 2006). As alterações laboratoriais na forma
aguda caracterizam-se pelo aumento de eosinófilos e de células mononucleares do sangue
periférico, presença de citocinas pró-inflamatoria TNF-α no plasma, IL-1β e IL-6 e pouca
produção de citocinas do tipo Th2 (BARBOSA et al., 2004; DE JESUS et al., 2002; HIATT et
al., 1979).
A forma crônica, presente na quase totalidade dos indivíduos infectados residentes nas
áreas endêmicas, é dependente da resposta inflamatória do hospedeiro, que se desenvolve em
torno dos ovos retidos nos tecidos dos diferentes órgãos. Estudos têm demonstrado que a
depender do tempo e da intensidade de infecção o indivíduo pode apresentar diferentes formas
clínicas, subdivididas de acordo com a gravidade da doença, em intestinal, hepatointestinal e a
hepatoesplênica (ANDRADE; BINA, 1983), caracterizadas conforme descrito a seguir:
A forma intestinal na maioria das vezes é assintomática, sendo a principal lesão a
inflamação intestinal, evoluindo com úlceras necróticas hemorrágicas na mucosa, ás vezes
sanguinolenta, dor abdominal difusa, baço e fígado não palpáveis, podendo haver eosinofilia.
A forma hepatointestinal além das alterações descritas na forma intestinal apresenta
aumento do lobo esquerdo do fígado e baço não palpável.
E a forma hepatoesplênica caracteriza-se pelo aumento do fígado, especialmente do lobo
esquerdo, e aumento do baço. Os indivíduos com a forma hepatoesplênica apresentam um
quadro de hipertensão portal conseqüente ao bloqueio do sistema porta no fígado decorrente
da destruição dos ramos da veia porta e da fibrose portal (BOGLIOLO, 1957; CHEEVER;
ANDRADE, 1967). Este quadro de hipertensão pode levar ao aparecimento de varizes no
esôfago, que em alguns casos podem romper, levando à morte por hemorragia digestiva
(ANDRADE, 1987; BINA; PRATA, 2003). A forma hepatoesplênica pode ser subdividida
em forma compensada e descompensada. A forma compensada caracteriza-se pela
hepatomegalia, tipicamente com aumento desproporcional do lobo esquerdo do fígado,
esplenomegalia, além da sintomatologia intestinal apresentada acima. A forma
descompensada caracteriza-se por uma esplenomegalia densa com fígado pequeno, na
15
presença de ascite, circulação colateral e hematêmese, estado geral precário, anemia
progressiva pelos sangramentos e redução na taxa de eosinofilia, quando comparado às outras
formas da doença.
A evolução para formas graves parece depender de vários fatores atuando de forma
isolada ou simultânea, entre eles densidade e intensidade de infecção dos caramujos
(COOPER et al., 1992), fatores ambientais, como grau de exposição a águas contaminadas
(BOOTH et al., 2004; DUNNE; PEARCE, 1999), fatores do hospedeiro, como estado
nutricional (COUTINHO et al., 1992), fatores genéticos associados com a fibrose grave,
como expressão de genes de citocinas que codifica IFN-γ (CHEVILLARD et al., 2003;
DESSEIN et al., 1999; HENRI et al., 2002; MARQUET et al., 1996), de HLA classe I (A1 e
B5) e HLA classe II (DQB1*0201) (CABELLO et al., 1991; SECOR et al., 1996) associado a
hepatoesplenomegalia. Em adição, o papel da carga parasitaria, a suscetibilidade a infecção
(QUINNELL, 2003) e a intensidade da infecção (DESSEIN et al., 1999; MARQUET et al.,
1996) no desenvolvimento das formas graves. A re-infecção é considerada ainda como um
importante fator de risco no desenvolvimento da fibrose periportal de Symmers (CHEEVER,
1972; CHEEVER; ANDRADE, 1967). Por outro lado, chama atenção que entre os indivíduos
com alta taxa de infecção alguns não desenvolvem a forma grave hepatoesplênica (COLLEY
et al., 1978).
O controle da esquistossomose mansônica requer medidas de saneamento básico, rede
de esgoto e de água, educação e informação dos indivíduos, tratamento dos criadouros e dos
indivíduos infectados. Todas as formas da esquistossomose são comumente tratadas com
praziquantel, com uma dosagem única de 40 mg/Kg de peso corporal, ou com oxamniquine a
15 ou 20 mg por kg nos adultos e crianças respectivamente. Corticóide e anticonvulsivos são
necessários como possíveis adjuvantes para o tratamento da neuroesquistossomose com
praziquantel (GRYSEELS et al., 2006). Todos os cuidados acima devem ser implementados
enquanto não se tem uma vacina eficaz disponível (BERGQUIST, N. R. et al., 2005;
BERGQUIST, R., 1990).
1.2. A RESPOSTA IMUNE NA ESQUISTOSSOMOSE MANSONI
A resposta imune contra o parasita é principalmente mediada pelos linfócitos T CD4
+
.
Os linfócitos T estão divididos dentro de duas classes em função do tipo de receptores de
células T (TCR) que eles expressam; a maioria de células T expressa o TCR αβ, e uma
pequena população de linfócitos T expressam o TCR γδ. As células T imaturas (timócitos),
derivadas de células precursores da medula óssea, expressam genes específicos como as
moléculas CD4 e CD8 (VON BOEHMER et al., 2003).
16
Com base no perfil de citocinas secretadas, os linfócitos CD4
+
foram classificados
inicialmente em tipos Th1/Th2 como demonstrado no modelo murino (MOSMANN et al.,
2005; MOSMANN; COFFMAN, 1989) e subseqüentemente em humanos (MOSMANN;
SAD, 1996). Os linfócitos T CD4
+
do tipo-1 produzem IL-2, IFN-γ e linfotoxinas (TNF-β), e
media a resposta do tipo de hipersensibilidade tardia enquanto os linfócitos T CD4
+
do tipo-2
produzem IL-4, IL-5, IL-6, IL-10 e IL-13 e estimulam produção de anticorpos como IgE, que
potencializam proliferação e atividade funcional de eosinófilos. Algumas citocinas como,
TNF-α, GM-CSF e IL-3 são produzidas por ambos os tipos celulares Th1/Th2. As respostas
do tipo Th1/Th2 são reguladas reciprocamente. Citocinas do tipo-2 regulam negativamente a
produção de citocinas do tipo-1 e vice versa. Existem evidências de que, antes da polarização
para os padrões Th1 ou Th2, a resposta imune apresenta um padrão intermediário tipo Th0,
quando são liberadas simultaneamente citocinas Th1/Th2 (HIRAYAMA et al., 1994;
VELLA; PEARCE, 1994), e atualmente, já foram descritos no mínimo três principais subtipos
de células T reguladoras (Treg) (Th3, Tr1 e células T CD4
+
CD25
+
). Existem duas populações
de células T reguladoras as naturais ou constitutivas, e as induzidas (MILLS, 2004). As
células Treg naturais são geradas no timo e constituem 5 a 10 % das células Treg periféricas
expressando CD4
+
CD25
+
e FOXP3 em animais e humano. Enquanto, a população de células
Treg induzida tem no mínimo três subtipos: Th3, Tr1 e células T CD4
+
CD25
+
. Tr1 secretam
altos níveis de IL-10 muito pouco IFN-γ e ausência de IL-4. Th3 secretam altos níveis de
TGF-β (MILLS, 2004), entretanto, o papel dessas células nas doenças infecciosas ainda não
está bem definido (LIU et al., 2003; ZACCONE et al., 2006).
17
Figura 2. O verme do S. mansoni e seus antígenos modificam a resposta imune inata através
da interação com TLRs e CLRs, induzindo a liberação de mediadores inflamatórios como a
IL-10.
O estudo da resposta imune nas doenças infecciosas e parasitárias tem contribuído
enormemente para o entendimento do papel de subpopulações de linfócitos na resposta imune.
Por exemplo, para patógenos intracelulares, a resposta imune induzida nos hospedeiros é, na
grande maioria das vezes, uma resposta fortemente Th1, enquanto que nas infecções com
helminto extracelular geralmente a resposta é predominantemente Th2. Além disso, o fenótipo
susceptível ou resistente dos indivíduos infectados parece ter correlação com o perfil de
citocinas produzidas e secretadas após a infecção. Um dos melhores modelos para o
entendimento do paradigma da polarização da resposta imune para o tipo Th1 ou Th2 é o
modelo murino Leishemania major (BOGDAN et al., 1993; MILON et al., 1995).
Em relação ao paradigma Th1/Th2 na esquistossomose, foi demonstrado no modelo
murino que, na fase inicial da infecção, predomina uma resposta imune do tipo Th1, com
produção de IL-2 e IFN-γ e, após a oviposição, uma resposta imune do tipo Th2, com
produção de IL-4 , IL-5 e IL-10 (GRZYCH, J. M. et al., 1991; PEARCE et al., 1991) que é
antecedido por um padrão Th0 com produção de citocinas do tipo Th1/Th2 (VELLA;
PEARCE, 1994). Mais tardiamente, na forma crônica da esquistossomose, predomina um
padrão de resposta imune tipo Th2, com produção de IL-4, IL-5 e IL-13 sendo que IL-13 pelo
menos no modelo murino, parece estar envolvida no desenvolvimento da fibrose hepática
18
(CHIARAMONTE et al., 1999; HOFFMANN et al., 2002; WYNN, T. A. et al., 1995).
Acredita-se que a mudança do padrão da resposta imune após oviposição seja dependente da
produção de IL-10 produzida por clones de células B estimuladas por oligossacarídeos
presentes no antígeno ovular solúvel (VELUPILLAI; HARN, 1994), ou por citocinas IL-6 e
IL-10 (LA FLAMME et al., 2000) ou por fosfatidilserina específica do Schistosoma (VAN
DER KLEIJ et al., 2002).
Recentes estudos também sugerem que populações específicas de células T
regulad
em humano quanto em
camund
1.3. A RESPOSTA IMUNE CELULAR E HUMORAL NA ESQUISTOSSOMOSE
Os es pacientes com a forma aguda realizados com PBMCs
oras CD4
+
CD25
+
natural com alta produção de IL-10 contribuam para a inibição da
resposta Th1 (HESSE et al., 2004; MCKEE; PEARCE, 2004). IL-10 foi originalmente
descrita como fator inibitório de síntese de citocinas (CSIF), uma citocina produzida pelos
clones Th2 murino capaz de inibir a produção de IFN-γ pelos clones Th1 através da ação das
células apresentadoras de antígenos (APC) (FIORENTINO et al., 1989). IL-10 é produzida
pelas células T CD4
+
e CD8
+
, células B, monócitos, mastócitos, queratinócitos e células
dendríticas (MOORE et al., 1993). A inibição da síntese de IFN-γ exercida pela IL-10 parece
agir mediada via APCs, inibindo nestas a expressão de moléculas MHC
classe II, CD40, e de
moléculas co-estimulatórias como CD80 e CD86 (FIORENTINO et al., 1991) o que levaria a
não ativação das células T pela ausência da apresentação das APCs.
IL-10 é elevada durante a infecção pelo Schistosoma
tanto
ongos. Na esquistossomose parece que o principal papel da IL-10 é controlar a
polarização Th1 e Th2 durante a infecção (CHEEVER et al., 2000; KING et al., 1996;
MONTENEGRO et al., 1999; WYNN, T., A; et al., 1998), e regular a progressão da doença
(BOSSHARDT et al., 1997; DE WAAL MALEFYT et al., 1991; WYNN, T., A; et al., 1998).
A inibição da síntese do IFN-γ pela IL-10 é, em parte, dependente do papel de IL-10 na
regulação da produção de IL-12, que é predominantemente produzida pelas células
dendríticas, monócitos, e macrófagos (GATELY et al., 1998; TRINCHIERI et al., 1992). IL-
12, foi originalmente descoberta 1989 é um heterodímero de 70 kDa compreendendo de
subunidade p35 e p40 ligadas covalentemente (PARHAM et al., 2002) tem sido largamente
aceito como um importante regulador da resposta de células Th1.
MANSONI HUMANA AGUDA.
tudos in vitro da resposta imune de
estimuladas com antígenos SEA ou SWAP apresentaram um perfil de citocinas misto do tipo
Th0, com produção de citocinas Th1/Th2 simultaneamente (DE JESUS et al., 2002;
MONTENEGRO et al., 1999). Em geral, poucos indivíduos com a forma aguda produziram
19
IL-10 e a maioria produziram mais IFN-γ em resposta para o SEA in relação aos pacientes
infectados cronicamente. Além disso, pacientes em estágio mais prolongado da fase aguda
com mais ou menos 3 meses após a infecção parecem exibir uma regulação cruzada entre
IFN-γ e IL-10, com a participação da IL-10 na supressão da resposta Th1. IL-10 tem um papel
importante no controle da infecção aguda, e essencial no processo da modulação na infecção
da esquistossomose crônica (RABELLO et al., 1995; WYNN, T., A; et al., 1998).
O estudo da resposta imune humoral na fase inicial da infecção tem demonstrado que
os esq
1.4. A RESPOSTA IMUNE CELULAR E HUMORAL NA ESQUISTOSSOMOSE
clínicas, tem sido observado que os indivíduos
assint
e de pacientes com forma grave têm demonstrado
resultados controversos. Por exemplo, Mwatha demonstrou que PBMCs de pacientes
uistossômulos ativam a resposta imune pela via alternativa do complemento e pela
indução da produção de anticorpos IgM, IgA, IgG e IgE (BOROS, 1989) sendo que IgE atua
como um mecanismo efetor contra o parasito, através da citotoxicidade celular dependente de
anticorpos (ADCC) mediada pelos mastócitos, basófios e eosinófilos (BUTTERWORTH et
al., 1985) e tem sido demonstrado uma correlação entre IgE e resistência a re-infecção
(CAPRON; CAPRON, 1994). Lembrando que outras subclasses de Igs, IgM e IgG2 foram
chamadas bloqueadores e seriam em parte responsáveis pela susceptibilidade dos indivíduos
mais jovens, a re-infecção após o tratamento, considerando que níveis elevados de IgM e
IgG2 direcionados para epitopos de carboidratos expressos em ovos do parasita e na
superfície das larvas, correlacionaram fortemente com susceptibilidade a re-infecção
(BUTTERWORTH; HAGAN, 1987; KHALIFE et al., 1986) enquanto níveis elevados IgA se
correlacionaram com imunidade (GRZYCH, J. M; et al., 1993).
MANSONI HUMANA CRÔNICA
Em relação às diferentes formas
omáticos com a forma intestinal (I) secretam baixos níveis de citocinas Th1 e Th2 para
ambos os antígenos SEA e SWAP, enquanto os pacientes caracterizados com forma
hepatoesplênica (HE) no estagio inicial da doença respondem para os mesmos antígenos com
produção de citocinas do tipo Th2: IL-5, IL-10 e IL-13 (ARAUJO et al., 1994; DE JESUS et
al., 2004; MWATHA et al., 1998). Também tem sido demonstrado uma correlação entre
níveis elevados de IL-5 e IL-13 com fibrose hepática baseado em exames de ultra-sonografia
(DE JESUS et al., 2004). Nas formas HE, a resposta imune parece ser regulada com o
agravamento da doença, seguida de uma resposta do tipo Th1 com produção de altos níveis de
citocinas TNF-α e IFN-γ e baixo nível de IL-5 e receptores solúveis de TNF (sTNFR-I,II), e
(ICAM-1) (MWATHA et al., 1998).
Os estudos da resposta imun
20
hepat
iferentes
graus
s, porém com baixa
carga
E ANTÍGENOS NA DEFINIÇÃO
DA RESPOSTA IMUNE NA ESQUISTOSSOMOSE.
muito PCs) contra patógenos ou seus
própr
oesplenicos estimuladas in vitro com antígenos SEA ou SWAP produzem citocinas tipo
Th1, enquanto outro grupo trabalhando na região nordeste do Brasil demonstrou associação
com perfil Th2 com produção de citocinas IL-5 e IL-13 (DE JESUS et al., 2004). Em adição,
outro estudo com pacientes exibindo alta taxa de infecção com S. mansoni produzem altos
níveis de IL-10 e baixo nível de IFN-γ (SILVEIRA et al., 2004). Contudo tem sido observado
que indivíduos sem infecção, residentes em áreas endêmicas considerados como normal
endêmico, produzem IFN-γ em resposta ao SEA ou a SWAP (VIANA et al., 1994).
É comum a observação nas áreas endêmicas de que indivíduos infectados com S.
mansoni, possivelmente vivendo sob as mesmas condições ambientais, apresentam d
de morbidade (DOEHRING-SCHWERDTFEGER et al., 1990; KARDORFF et al.,
1994), indicando que fatores genéticos do hospedeiro definitivo quanto dos hospedeiros
intermediários do parasito podem ter importância na imunopatogênese da esquistossomose
(BOOTH et al., 2004; CARTON et al., 2005; DUNNE; PEARCE, 1999).
Os estudos da resposta imune humoral na forma crônica têm demonstrado que
indivíduos de área endêmica, tratados e curados, ou tratados e não curado
parasitária, exibem alto títulos de IgE e baixos de IgG4 associados à resistência a re-
infecção (BUTTERWORTH et al., 1992; HAGAN, P; et al., 1991; RIHET et al., 1991).
Enquanto que os indivíduos que se re-infectaram mais cedo, com carga parasitária elevada,
apresentaram baixo títulos de IgE e alto título de IgG4, associados à susceptibilidade a re-
infecção (BUTTERWORTH et al., 1992), provavelmente devido à competição com
receptores de IgE pelo sítio de ligação da célula efetora (HAGAN, P; et al., 1991). Também
outros anticorpos estariam envolvidos nesse processo, inibindo a ativação do sistema de
complemento e impedindo a ligação de receptores na superfície de monócitos, ou competindo
com receptores de IgE se ligando na superfície de mastócitos e basófilos, bloqueando a
ligação dos isotipos efetores, a IgE (HAGAN, P., 1993).
1.5. PAPEL DAS CÉLULAS APRESENTADORAS D
A habilidade para responder apropriadamente a uma infecção ou vacinação depende em
da resposta das células apresentadoras de antígenos (A
ios antígenos. As células dendríticas são as mais potentes e eficientes células na ativação
e apresentação de antígenos para as células T virgem (BANCHEREAU; STEINMAN, 1998;
MARTIN-FONTECHA et al., 2003; PERONA-WRIGHT et al., 2006; SHORTMAN; LIU,
2002; STEINMAN, 1991), sendo as principais iniciadoras da resposta primária específica
21
contra antígenos, ambos in vitro (AUFFERMANN-GRETZINGER et al., 2001) e in vivo
(ANGELI et al., 2001; FOUCRAS et al., 2000).
A resposta imune inata exercida pelas APCs determina se a sucessão da resposta
adapt
r dos microambientes, antígenos, subpopulações de
DCs
ulas
valiado a resposta imune humana utilizando PBMCs de indivíduos
infect
ativa de células T auxiliadora (Th) CD4
+
será do tipo Th1 ou do tipo Th2 (INABA;
INABA, 2005; INGULLI et al., 1997; LEVIN et al., 1993; MCCULLOUGH;
SUMMERFIELD, 2005; MELLMAN, 2005; SHANKAR; TITUS, 1997; STEINMAN, 1991).
As células dendríticas são ativadas quando patógenos ou antígenos se ligam a receptores na
superfície celular, incluindo numerosos receptores de lecitina tipo-C, receptores Toll-like
(TLRs), manose DEC 205 (MAJEWSKA; SZCZEPANIK, 2006; SALLUSTO et al., 1995),
moléculas de MHC classe II, adesão ICAM-1, co-estimulatórias B7-1 e B7-2 (CD80 e CD86)
e CD40, (BERNHARD et al., 2000; DUBEY et al., 1995; GELDHOF et al., 1998; KRONIN
et al., 2000; MOSER; MURPHY, 2000).
Existem evidências que, a depende
podem expressar diferentes citocinas e através de estímulos são capazes de direcionar a
resposta imune de clones de células Th1 ou Th2 ou T reguladoras (BERNHARD et al., 2000;
KAPSENBERG, 2003; MOSER; MURPHY, 2000; O'GARRA; ARAI, 2000) e que na
dependência da presença de diferentes quimiocinas podem influenciar a maturação de células
Th0 CD4
+
virgem (SALLUSTO et al., 2000). Alem disso, a natureza do antígeno pode
influenciar no tipo de resposta induzida pelas células dendríticas nas células (Th) CD4
+
efetora, polarizando para uma resposta tipo Th1 ou Th2 (VAN DER KLEIJ et al., 2002).
Estudos experimentais têm demonstrado que a indução de uma resposta tipo Th2 pelas cél
dendríticas na esquistossomose é dependente do tipo de antígeno (MACDONALD et al.,
2001), ou seus componentes tais como o lacto-N-fucopentaose III (OKANO et al., 2001;
THOMAS et al., 2003; VELUPILLAI; HARN, 1994) glican do ovo S. mansoni,
especialmente Lewis X que são reconhecidos pelas DCs através de receptores de superfície
para lectinas tipo-C, tais como DC-SIGN (VAN DIE et al., 2003) ou fosfatidilserina (VAN
DER KLEIJ et al., 2002).
Vários estudos têm a
ados (ARAUJO et al., 1996; MALAQUIAS et al., 1997; VELUPILLAI et al., 2000) ou
células dendríticas (AGRAWAL et al., 2003; DE JONG et al., 2002; VAN DEN
BIGGELAAR et al., 2000), mas poucos foram realizados com células de doadores normais
explorando os eventos iniciais da resposta imune através do sistema “priming” in vitro (PIV),
estimulação primária in vitro tendo as células dendríticas profissionais como células
apresentadoras de antígeno (VAN LIEMPT et al., 2007). No presente estudo, foi demonstrado
que células dendríticas humanas geradas in vitro sensibilizadas com SEA ou MAP4 foram
22
capazes de induzir a proliferação e expansão de clones de subpopulações de linfócitos T
conforme estão apresentados nos dois manuscritos parte desta tese.
23
2. OBJETIVOS
Geral
Avaliar os eventos iniciais da resposta imune de células mononucleares do sangue
periférico (PBMCs) humano à antígenos do S. mansoni através do sistema “priming” in vitro
(PIV).
Específicos
1. Determinar o perfil fenotípico das células dendríticas geradas in vitro com IL-
4/GM-CSF obtida de monócitos do sangue periférico de doadores normais: CD1a
+
CD14
-
, CD40, CD39, CD54, CD80, CD86 e HLA-DR;
2. Determinar o perfil fenotípico de células mononucleares do sangue periférico
(PBMCs) de doadores normais, primadas in vitro e reestimuladas com SEA ou
MAP4 após cada estimulação. Avaliando a expressão das subpopulações de
linfócitos (T) CD4
+
, CD8
+
, (B) CD19
+
, CD23
+
, e moléculas de ativação, adesão, co-
estimulatórias e memória (CD25, HLA-DR, CD54, CD45RO e B7-1 9CD80), B7-2
(CD86);
3. Determinar o perfil de citocinas (IL-5, IL-6, IL10, IL-12, IFN-γ e TNF-α) em
sobrenadante de culturas de células mononucleares do sangue periférico (PBMCs)
de doadores normais, primadas in vitro e reestimuladas com SEA ou MAP4 após
cada estimulação.
24
3. RESULTADOS
MANUSCRITO 1
E. A. G. Reis, T. M. Azevedo, A. J. A. McBride, D. A. Harn Jr and M. G. Reis. Naive Donor
Responses to Schistosoma mansoni Soluble Egg Antigens. Scand J. Immunol, v. 66, n. 6, p.
662-670, 2007
Os resultados avaliados após o “priming” in vitro (PIV) de PBMCs de 12 doadores
normais não residentes em área endêmica ao antígeno ovular solúvel (SEA) do S. mansoni
demostraram uma polarização na resposta imune.
As células dendriticas geradas in vitro com GM-CSF/IL-4 expressaram fenotípicamente
CD1a
+
/CD14
-
, moléculas de ativação HLA-DR, adesão ICAM-1 e co-estimulatorias B7-1/B7-
2, e CD40. Também foi avaliada a produção de citocinas após a diferenciação, tendo sido
observado um grande aumento na secreção de IL-8.
As células dendríticas foram sensibilizadas com ou sem antígenos e mantidas em cultura
com PBMCs autológas de doadores normais. Os linfócitos ativados foram reestimulados por
duas vezes com e sem o antígeno e caracterizados através dos marcadores de superfície
celular e do perfil de citocinas no sobrenadante de cultura em cada reestimulação. As
subpopulações de linfócitos estudada consistiram de linfócitos (T) CD4
+
64%, CD8
+ high
17%,
(B) CD19
+
12% e CD23
+
7%. As subpopulações de linfócitos T CD4
+
expressaram altos
níveis de moléculas ativadas para CD25
+
, HLA-DR
+
, CD54
+
e CD45RO
+
um marcador de
células de memória. As moléculas CD80 e CD86 foram expressas nas subpopulações de
linfócitos B após o “priming” e mantiveram a expressão fenotípica durante as reestimulações.
A maioria dos doadores 10/12 desenvolveu uma resposta de células (Th) CD4
+
do tipo Th2, e
2 doadores uma resposta do tipo Th1 inclusive mantendo o mesmo padrão após a repetição do
experimento. Novos experimentos devem ser realizados para elucidar essa questão.
CONCLUSÕES DO MANUSCRITO 1:
1) No PIV com PBMCs de doadores normais estimuladas com SEA foi predominantemente
de linfócitos (Th) CD4
+
com um aumento significativo na percentagem de células CD4
+
expressando CD25
+
, CD54
+
, HLA-DR
+
CD45RO
+
e linfócitos B CD19
+
expressando CD80
+
e
CD86
+
em relação ao controle PBMCs ex vivo;
25
2) O perfil de citocinas secretadas pelas PBMCs primadas e reestimuladas (primeira chamada)
com SEA foram do tipo Th0 (IFN-γ, IL-5, IL-6, IL-10 e TNF-α) e subseqüentemente na
segunda reestimulação (segunda chamada) a resposta foi predominantemente do tipo Th2 (IL-
5 e IL-10); exceto para dois doadores que desenvolveram uma resposta do tipo Th1 com
elevada produção de IFN-γ.
26
MANUSCRITO 2
E. A. G. Reis, T. M. Azevedo, R. Athanazio, M. G. Reis and D. A. Harn Jr. Schistosoma
mansoni Triose Phosphate Isomerase peptide MAP4 is able to trigger naïve donor immune
response toward a Type-1 cytokine profile. Scand J. Immunol, 2008.
A metodologia deste trabalho foi a mesma empregada no manuscrito 1. Células
dendríticas geradas in vitro com GM-CSF e IL-4 expressaram fenotipicamente CD1a
+
e
CD14
-
moléculas de ativação e adesão HLA-DR, ICAM-1, co-estimulatórias B7-1 e B7-2,
CD40.
As células dendríticas foram sensibilizadas na presença ou ausência de MAP4 e
cultivadas com PBMCs autológas de doadores normais. Os linfócitos ativados foram
reestimulados duas vezes com e sem o antígeno e avaliadas através de marcadores fenotípico
de superficie celular e do perfil de citocinas secretadas no sobrenadante de cultura após cada
reestimulação (veja no material e método dos manuscritos). As subpopulações de linfócitos
estudada foram composta de linfócitos (T) CD4
+
68,5%, CD8
+high
16% e B CD19
+
10%. As
células CD4
+
expressaram altos níveis de moléculas ativadas para CD25
+
, HLA-DR
+
, CD54
+
e CD45RO
+
. Foi observado também um aumento significante da expressão de CD80 nos
linfócitos B CD19
+
na avaliação da primeira chamada. CD86 também foi observado com um
incremento, porém, não foi significante quando comparado com o controle PBMCs ex vivo. O
PIV de PBMCs primadas com MAP4 apresentaram uma resposta do tipo Th1 após o
“priming”, primeira e segunda reestimulação (primeira e segunda chamada).
CONCLUSÕES DO MANUSCRITO 2:
1) No PIV com PBMCs de doadores normais primadas com MAP4 foi observado um
aumento significativo na percentagem das subpopulações de linfócitos T CD4
+
expressando
CD25
+
, HLA-DR
+
, CD54
+
e CD45RO
+
e linfócitos B CD19
+
expressando CD80
+
em relação
ao controle PBMC ex vivo;
2) O perfil de citocinas secretadas pelas PBMCs primadas e reestimuladas (primeira e
segunda chamada) com MAP4 foi do tipo Th1 com elevada produção de IFN-γ.
3) MAP4 é um indutor de IFN-γ, capaz de alterar a resposta de PBMCs primadas com SEA,
estimulando a produção de IFN-γ e inibindo a secreção de IL-10.
Naı
¨
ve Donor Responses to Schistosoma mansoni
Soluble Egg Antigens
E. A. G. Reis*, T. M. Azevedo*, A. J. A. McBride*, D. A. Harn Jr & M. G. Reis*,à
Introduction
Schistosoma mansoni is a helminth parasite infecting
approximately 200 million people world-wide, with over
600 million people living in endemic areas at risk of
acquiring the infection [1]. In the mouse model, S. man-
soni infection is characterized by a brief, early pro-inflam-
matory immune response, with production of IL-2 and
IFN-c [2]. The pro-inflammatory response to early egg
deposition shifts to Th2-type with production of IL-4,
IL-5 and IL-10 [2, 3]. Prio r to the Th2 response, a tran-
sient Th0-like response occurs, characterized by mixed
cytokine production [4, 5]. Carbohydrates [6] and other
ligands in soluble egg antigen (SEA) stimulate produc-
tion of IL-10 [7] and IL-6, contributing to the downre-
gulation of Th1 responses [8].
Many studies have documented early immune events
during experimental schistosomiasis in murine models
[5, 9–11]. However, there are few reports desc ribing the
development of the immune response in humans and it
is important to note that the majority of studies using
human cells were performed with total peripheral blood
mononuclear cells (PBMC) [12–14] or dendritic cells
(DC) [15–17] from infected individuals. Human DC
stimulated with SEA show similar responses to those
observed in mice, there is Th2 polarization and the DC
do not undergo maturation [18, 19]. The results pre-
sented here are the first based on priming in vitro (PIV)
of naı
¨
ve human PBMC stimulated with SEA using DC
as antigen presenting cells (APC) to evaluate the early
events in the immune response during a schistosomiasis
infection. DC are potent APC [20–22], being major ini-
tiators of primary antigen-specific responses both in vitro
[23] and in vivo [24, 25]. DC express several markers
including class II MHC, ICAM-1, DEC-205 and B7
co-stimulatory molecules [26–28]. Moreover, there is
evidence that DC subsets can produce different cytokine
microenvironments that may differentially direct the
immune response [29, 30]. In this study, we determined
whether SEA could induce cytokine patterns in naı
¨
ve
*Oswaldo Cruz Foundation (FIOCRUZ),
Salvador, Bahia, Brazil; Department of
Immunology and Infectious Diseases, Harvard
School of Public Health, Boston, MA, USA; and
àDepartment of Anatomical Pathology and
Legal Medicine, Faculty of Medicine of Federal
University of Bahia, Salvador,Bahia, Brazil
Received 13 June 2007; Accepted in revised
form 15 August 2007
Correspondence to: Dr M. G. Reis, Fundac¸a
˜
o
Oswaldo Cruz, Rua Waldemar Falca
˜
o, 121,
Candeal, CEP 40296-710, Salvador, Bahia,
Brazil. E-mail: miter@bahia.fiocruz.br
Abstract
Schistosome infection induces profound Th-biasing and immune suppression.
Although muc h has been examined in mice, few studies have examined
responses of naı
¨
ve humans to schistosome antigens. In this study, we examined
the response of naı
¨
ve human peripheral blood mononuclear cells (nPBMC) to
stimulation with Schistosoma mansoni soluble egg antigen (SEA) using a prim-
ing in vitro (PIV) assay. We found that SEA induced a pronounced CD4
+
T-helper cell res ponse based on cytokine secretion and phenotyping markers.
SEA-stimulated nPBMC (SEA cells) at day 7 post-priming and after the first
recall consisted predominantly of Th0-like CD4
+
T cells. Following the second
recall, the majority of donor (10 ⁄12) responses were Th2-like. The cell popula-
tion consisted of approximately 64% CD4
+
, 17% CD8
+high
, 12% CD19
+
, and
7% CD23
+
cells. The CD4
+
population also expressed HLA-DR
+
, CD54
+
,
CD45RO
+
and CD25
+
whereas the CD19
+
cells expressed CD80 and CD86.
Following priming, we detected high levels of IL-6, IFN-c, IL-12p40, IL-10
and IL-5. Upon restimulation, SEA cells secreted IL-5 and high levels of
IL-10, typical of a Th2-like response. The data presented herein shows that the
majority of naı
¨
ve donor dendritic cells, following stimulation with SEA, prime
and clonally expand SEA-specific T cells towards a Th2-type response. However,
two donors responded with an atypical response, producing IFN-c coincident
with low levels of IL-10. Whether this d ifferential response was due to HLA or
other genes was not determined but is currently under investigation.
doi: 10.1111/j.1365-3083.2007.02024.x
..................................................................................................................................................................
Ó 2007 The Authors
662 Journal compilation Ó 2007 Blackwell Publishing Ltd. Scandinavian Journal of Immunology 66, 662–670
human PBMC similar to those described in the murine
model. A PIV assay was established using naı
¨
ve PBMC
(nPBMC)-derived DC that were subsequently pulse-
stimulated with SEA. The immune response of nPBMC
after priming, first and second recalls was evaluated.
We demonstrated that the majority (10 ⁄12) of SEA-
stimulated PBMC CD4
+
T cells polarized towards a
Th2-like response after the second recall, based on cyto-
kine profile and surface antigen immunophenotyping.
However, SEA-stimulated PBMC from two individuals
showed evidence of a shift towards a Th1-like immune
response.
Materials and methods
Human donors. Peripheral blood mononuclear cells were
obtained from 12 healthy, schistosome naı
¨
ve, Brazilian
blood donors between 19 and 28 years of age and living
in a schistosomiasis non-endemic area. The donors were
screened for the presence of antibodies to HIV, HTLV
I ⁄ II, HBsAg, HCV, VDRL and Chagas disease. Individu-
als positive for any of these infections were excluded from
the study. The donors had no previous history of schisto-
somiasis and all were negative for S. mansoni infection by
stool examination using the Kato–Katz method [31] and
for antibodies to SEA by ELISA. The Ethics Committee
of the Oswaldo Cruz Foundation approved the study pro-
tocol and all donors gave informed consent prior to the
collection of blood.
Schistosoma mansoni antigen. Soluble egg antigen was pro-
duced and tested for endotoxin activity as previously
described [32]. The SEA preparation did not include
significant levels of non-specific contaminants, such as
lipopolysaccharide, verified by the lack of response to
SEA in nPBMC controls (data not shown). A flowchart of
the experimental design is presented in Fig. 1.
Isolation and preparation of human PBMC. Peripheral
blood mononuclear cells were isolated from heparinized
blood (30–40 ml) by centrifugation on a Ficoll–Hypaque
gradient (GE Healthcare, Piscataway, NJ, USA) at 400 g,
30 min, at 15 °C. PBMC were washed in Ca
2+
,Mg
2+
-
free Hanks’ balanced salt solution (HBSS) containing
penicillin (100 U ⁄ml) and streptomycin (100 lg ⁄ml)
(pen-strep; Invitrogen, Carlsbad, CA, USA). PBMC were
incubated in 24-well plates (Cornin g Inc., Lowell, MA,
USA) in AIM-V medium (serum free lymphocyte med-
ium; Invitrogen) at 2–3 · 10
6
cells ⁄ ml and cultured
with and without Con A (5 lg ⁄ml) and purified protein
derivative (PPD) (5 lg ⁄ml) for 72 h at 37 °C, 5% CO
2
.
Culture supernatants were harvested and stored at
)70 °C. Con A or PPD were used as positive controls for
the PIV assay.
Preparation of DC. Dendritic cells were prepared from
PBMC as described previously [33] with the following
modifications. Monocytes were obtaine d from PBMC
(6 · 10
7
cells ⁄ml) following spontaneous sedimenta tion
and incubated in 75-cm
2
cell culture flasks (Corning
Inc.) containing serum-free RPMI-1640 for 2 h at 37 °C,
5% CO
2
. Non-adherent cells were removed by washing
and the remaining cells were cultured for 6–8 days in
10 ml of DC complete medium (DCC-medium) contain-
ing RPMI-1640, 1 m
M sodium pyruvate, 2 mML-gluta-
mine, 1% pen-strep and 10% ultra-low IgG fetal bovine
serum (FBS; Invitrogen), rhGM-CSF 50 ng ⁄ml and
rhIL-4 1000 U ⁄ml (BD Biosciences, San Jose, CA, USA).
Every 2 days, 500 ll of culture medium was removed
and replaced with the same volume of fresh DCC-med-
ium. DC diffe rentiation was monitored by light micros-
copy, after 6–8 days, the DC were harvested and analysed
for cytokine secretion. The expression of surface markers
was determined by FACSort analysis (BD Biosciences).
PBMC PIV assay. Irradiated autologous DC (3000 rads)
were placed in 24-well plates and stimulated with SEA
(25 lg ⁄ml) or RPMI (control) and incubated in complete
medium (C-medium) containing AIM-V medium, 1%
pen-strep and 5% human serum AB (Invitrogen). After
2 h, 2–3 · 10
6
cells ⁄ml fresh nPBMC were added to the
cultures giving a DC:nPBMC ratio of 1:15 and cultured
Figure 1 Experimental design scheme: nPBMC from healthy donors were used in the priming in vitro assay (PIV). DC were differentiated with IL-4
and GM-CSF for 6–8 days. PBMC were cultured with (priming) or without SEA for 7 days in the presence of irradiated autologous DC. SEA or
RPMI cells were restimulated with (first recall) or without SEA (Ag) in the presence of irradiated PBMC as feeder cells and cultured for 7 days. SEA
or RPMI cells (secondary recall) were cultured with and without antigen for 72 h in the presence of irradiated autologous PBMC. Following SEA-
stimulation cytokine production was evaluated by ELISA and cell phenotypes were determined by FACS analysis.
E. A. G. Reis et al. Naı
¨
ve Human PBMC Stimulated by SEA 663
..................................................................................................................................................................
Ó 2007 The Authors
Journal compilation Ó 2007 Blackwell Publishing Ltd. Scandinavian Journal of Immunology 66, 662–670
for 7 days at 37 °C, 5% CO
2.
Culture supernatants were
collected and stored at )70 °C. Cells were harvested and
purified on Ficoll–Hypaque gradient s and surface markers
were identified by FACSort analysis. Freshly isolated
unstimulated nPBMC were used as controls.
Following priming, the PIV SEA-stimulated PBMC
(SEA cells) or RPMI-stimulated PBMC (RPMI cells) were
restimulated (first recall) in the presence of irradiated
(2,500 rads) autologous PBMC (2 · 10
6
cells ⁄ ml, 1:1
ratio) previously pulsed with SEA (20 lg ⁄ml) or RPMI in
24 well plates for 2 h. The cells were cultured in C-med-
ium for 7 days. IL-2 (20 U ⁄ml) (R&D Systems Inc., Min-
neapolis, MN, USA) was added to the cultures every
second and third day. Supernatants were collected on day 7
to measure cytokine production. Cells were harvested,
purified on Ficoll–Hypaque gradients and analysed by
FACSort. Cells from the first recall (SEA or RPMI cells)
were then restimulated a second time (second recall) and
cultured in AIM-V medium in the presence of irradiated
autologous PBMC (0.5–1 · 10
6
cells ⁄ ml, 1:1 ratio) previ-
ously pulsed with and without SEA (10 lg ⁄ ml), Con A
(5 lg ⁄ml) or PPD (5 lg ⁄ml) in 48-wel l plates for 72 h at
37 °C, 5% CO
2
. Supernatants were collected to measure
cytokine production and the cells were harvested to deter-
mine the phenotypes.
Flow cytometry analysis. Cells were stained using the fol-
lowing conjugated mouse anti-human mAbs in two or
three-colour immunocytometric assays: CD3-FITC
(HIT3a), CD14-FITC (M5E2), CD40-FITC (5C3),
CD45R0-FITC (UCHL1), CD80-FITC (BB1), CD86-
FITC (FUN-1), HLA-DR, DP, DQ-PE (TU39), CD25-
PE (IL-2Ra) (M-A251), CD54 (ICAM-1)-PE (HA58),
CD39-PE (TU66), CD1a-PE (HI149), CD19-PE
(HIB19), CD23-PE (M-L233), CD4-Cy (RPA-T4), CD8-
Cy (RPA-T8) and their isotype-matched negative-control
antibodies IgG
1
-PE, -FITC or -Cy (MOPC-21), mouse
IgG
2a
-PE (G155-178), and mouse IgM-FITC (G155-228)
(BD Biosciences).
Dendritic cells and lymphocyte phenotyping was
performed according to the manufacturers’ instructions
with the following modifications. In 12 · 75 mm
polystyrene tubes, 10
5
cells ⁄ml were incubated on ice
for 30 min with human serum (diluted 1:20). The
cells were resuspended in 40 ll of FACS buffer
[HBSS, 10% FCS, 0.01% sodium azide (Sigma-Aldrich,
St. Louis, MO, USA), pH 7.2, 5 ll of each mAb] and
incubated for 30–45 min at 4 °C in the dark. The
cells were washed three times in FACS buffer and
resuspended in 200 ll FACS buffer. Ten-thousand
events were acquired on the FACSort flow cytometer
(BD Biosciences) and the data analysed using CellQuest
Software (BD Biosciences). Cells were gated on FSC
versus SSC dot plots and further characterized by their
fluorescence profile with two colour dot plots or single
colour histograms. The results are expressed as either
the percentage of positive cells within the selected gate
or as proportions.
Cytokine assays. Sandwich ELISAs for IL-6, IL-8,
IL-12p40 and TNF-a were performed using the Duo-set
kit (R&D System, Inc.) and IFN-c using the Intertest
ELISA kit (Genzyme, Cambridge, MA, USA). IL-5 and
IL-10 were assayed using the Human ELISA Set kits (BD
Biosciences) following the manufacturers’ instructions.
Statistical analysis. Statistical analyses were performed
using the Student’s t and non-parametric Mann–Whitney
tests using the GraphPad Prism 5.0 software package
(San Diego, CA, USA). Differences were considered sig-
nificant when the P-value was <0.05 between SEA cells
and fresh unstimulated PBMC cont rols for phenotype
analysis and between SEA cells and control RPMI cells
for cytokine production.
Results
Phenotype analysis and cytokine secretion in DC
The successful generation of DC from naı
¨
ve PBMC in
the presence of GM-CSF and IL-4 was determined by the
expression of CD1a and the absence of CD14 surface
markers as characterized by high forward and side light
scatter (Fig. 2A). Monocyte-derived DC exhibited typical
morphology as confirmed by light microscopy (data not
shown). After 7 days, DC were analysed by FACS to
determine the percentage of cells expressing CD39
+
,
CD40
+
, CD54
+
(ICAM-1), HLA-DR
+
, CD86
+
(B7-2)
and CD80
+
(B7-1) molecules (Fig. 2B). Most of the DC
population expressed HLA-DR
+
and CD54
+
, while
62.5% of the DC expressed CD86
+
. In addition,
increased expression of CD39
+
, CD40
+
and modest
expression of CD80
+
was observed. The levels of
IL-12p40, IL-8, IL-6, TNF-a and IL-10 secreted during
DC culture differentiation prior to SEA stimulation were
also determined and are shown in Fig. 2C. Of these cyto-
kines, IL-8 was secreted at high levels whereas IL-12p40
production was low. In additi on, we were unable to
detect IL-2 (data not shown).
Phenotype analysis of the lymphocyte populations from
SEA-stimulated PBMC
Following priming and recall, SEA-stimulated PBMC or
nPBMC controls were counted and the frequency of sur-
face marker antigens of the lymphocyte subs ets were eval-
uated using anti-CD4, -CD8, -CD19 and -CD23 mAbs.
Throughout this period, there was an overall 10-fold
decrease in the total number of viable cells (Fig. 3). Spe-
cifically, there was a 50% drop post-priming, followed
by a 23% reduction after the first recall and a further 9%
reduction after the second recall. This decline was
observed with and without SEA stimulation indicating
664
Naı
¨
ve Human PBMC Stimulated by SEA E. A. G. Reis et al.
..................................................................................................................................................................
Ó 2007 The Authors
Journal compilation Ó 2007 Blackwell Publishing Ltd. Scandinavian Journal of Immunology 66, 662–670
that it was not due to SEA toxicity. The viable cell
population consisted of approximately 64% CD4
+
,
17% CD8
+high
, 12% CD19
+
and 7% CD23
+
cells. The
SEA-stimulated PBMC CD4
+
T-cell subs et increased
significantly post-priming (P < 0.005), and after the first
(P < 0.0001) and second recall responses (P < 0.05) com-
pared to the nPBMC control (Fig. 3).
Phenotype analysis of SEA-stimulated PBMC lymphocyte
subsets
The percentage of lymphocyte subsets post-priming and
after the first and second recall responses were analysed by
double-label immunophenotyping using CD4-CY in com-
bination with CD25-PE (Fig. 4A); or HLA-DR-FITC
(Fig. 4B); CD4-CY with CD54-PE (Fig. 4C) or
CD45RO-FITC (Fig. 4D); and CD19-PE with CD80
FITC (Fig. 4E) or CD86 FITC (Fig. 4F). The mean num-
ber of CD4
+
CD25
+
⁄CD4
+
T cells increased significantly
post-priming (8.6–20%, P < 0.001) and after the first
(42.5%, P < 0.0001) and second recall responses (60%,
P < 0.0001) compared to the unstimulated nPBMC con-
trol (Fig. 4A). Additionally, the population of
CD4
+
HLA-DR
+
⁄CD4
+
T cells increased significantly
post-priming (2.8–19.3%, P < 0.01) after the first recall
(38.8%, P < 0.001) and the second recall respons es (38%,
P < 0.001) compared to the nPBMC control (Fig. 4B).
Using anti-CD54 (ICAM-1), the percentage of
CD4
+
CD54
+
⁄CD4
+
T cells increased significantly post-
priming (18.2–61.8%, P < 0.01) and after the first
(84.1%, P < 0.001) and second recall responses (85.4%,
A
B
C
1000800600
Side scatter
4002000
504030
Counts
Counts
20100
50403020100
0
10
0
10
1
10
2
CD1A
M1
M1
10
3
10
4
10
0
10
1
10
2
CD14
10
3
10
4
200
R1
400 600
Forward scatter
800 1000
Figure 2 FACS analysis of DC surface markers and cytokine produc-
tion. A representative light scatter plot (A) shows the gate selected for
subsequent analyses by fluorescent antibody staining. Each histogram
represents an overlay of CD1a or CD14 (light line) and an isotype
matched control (bold line). DC surface marker-specific fluorochrome-
labeled mAbs: CD14, CD1a, CD39, CD40, CD54, HLA-DR, CD80
and CD86 (B). Each point represents the percentage of positive DC
within the selected gate in a single donor sample (n = 12). The mean
percentage (±95% CI) is also shown. Following DC differentiation, cul-
ture supernatants were analysed for IL-12p40, IL-8, IL-6, TNF-a and
IL-10 production (C). Each symbol represents an individual donor and
the vertical bars represent the standard error of the mean (±SEM).
Figure 3 FACS analysis was used to identify the lymphocyte popula-
tion present in SEA-stimulated PBMC following priming and recall
responses. The lymphocytes were identified, using antibodies for CD4
+
and CD8
+
T cells or CD19
+
and CD23
+
B-cell subsets. The results are
expressed as the percentage of positive cells within the lymphocyte pop-
ulation, the mean values (+95% CI) from 12 representative donors are
presented. The unstimulated naı
¨
ve PBMC control (nPBMC) or PIV cells
after priming, first recall (I-recall) and secondary recall (II-recall) are
shown. The total number of viable cells following each SEA stimulation
was determined. Significant differences in the lymphocyte populations
are indicated (*).
E. A. G. Reis et al. Naı
¨
ve Human PBMC Stimulated by SEA 665
..................................................................................................................................................................
Ó 2007 The Authors
Journal compilation Ó 2007 Blackwell Publishing Ltd. Scandinavian Journal of Immunology 66, 662–670
P < 0.001) compar ed to the nPBMC control (Fig. 4C).
In addition, CD45RO
+
was used as a marker for memory
T cells. The percentage of CD4
+
CD45RO
+
⁄CD4
+
T cells
increased significantly after the first recall (43.6 –65.7%,
P < 0.001) and second recall responses (73.7%, P < 0.01)
when compared to the nP BMC control (Fig. 4D).
CD19
+
CD80
+
and CD19
+
CD86
+
expression on B
cells derived from SEA-stimulated PBMC (Fig. 4E,F)
was determined. It was notable that despite the lack
of a significant difference in the total number of
CD19
+
B cells (Fig. 3), the CD19
+
CD80
+
⁄CD19
+
B-cell population increased significantly post-priming
(P < 0.01) and after the second recall (P < 0.01) whe n
compared to the nPBMC control (Fig. 4E). Expression
of CD19
+
CD86
+
⁄CD19
+
on B cells was significantly
upregulated following priming and after both recalls
Figure 4 FACS analyses of SEA-stimulated PBMC following priming and recall responses. The unstimulated naı
¨
ve PBMC control (nPBMC), or PIV
SEA-stimulated PBMC following priming, first recall (I-recall) and second recall responses (II-recall) are shown. (A) double-label staining with anti-
CD4 and anti-CD25-PE was used to identify CD4
+
CD25
+
⁄ CD4
+
cells; (B) CD4
+
HLA-DR
+
⁄ CD4
+
cells; (C) CD4
+
CD54
+
⁄ CD4
+
cells; and
(D) D4
+
CD45RO
+
⁄ CD4
+
T-cell subsets or (E) CD19
+
CD80
+
⁄ CD19
+
and (F) CD19
+
CD86
+
⁄ CD19
+
on B-cell subsets. Each symbol represents an
individual response. The mean percentage (+95% CI) values are also shown. The results from the Th-1 prone individuals are circled.
666 Naı
¨
ve Human PBMC Stimulated by SEA E. A. G. Reis et al.
..................................................................................................................................................................
Ó 2007 The Authors
Journal compilation Ó 2007 Blackwell Publishing Ltd. Scandinavian Journal of Immunology 66, 662–670
(P < 0.0001) when compared to the nPBMC control
(Fig. 4F).
Th-like patterns of cytokine secretion in SEA-stimulated
PBMC
Cytokine production of SEA-stimulated PBMC (SEA
cells) or RPMI-stimulated PBMC (RPMI cells) was deter-
mined to identify the Th CD4
+
cell subset response asso-
ciated with SEA stimulation. SEA cells secreted moderate
to high levels of IL-12p40, IL-5, IL-6, IL-10, IFN-c and
TNF-a post-priming and after the first recall response
compared to control RPMI cells (Fig. 5A,B), indicative
of a Th0-like response. After the first recall, SEA cells
maintained a similar Th0-like response, as seen post-
priming, with elevated levels of pro-inflammatory cyto-
kines IL-6, TNF-a and IFN-c. After the first recall, there
was a moderate but non-significant decrease observed in
the secretion of all cytokines except TNF-a.
In SEA cells from 10 ⁄ 12 donors there was a shift from
the Th0-like response to a Th2-like response after the
second recall, with high levels of IL-10 and low levels of
IFN-c detected (Fig. 5C). Unexpectedly, CD4
+
T cells
from two individuals secreted high levels of IFN-c and
low levels of IL-10, more typical of a Th1-like response.
To confirm the findings, the experiments were repeated
using cells from three donors from the 10 that had Th2-
like responses (Fig. 6A,B) and cells from one donor of
two that had evidence of polarization towards a Th1-like
response (Fig. 6C,D). These repeat assays yielded similar
results to the initial PIV assays (Fig. 6).
Con A and PPD activation of SEA-stimulated PBMC
after the second recall response
To confirm that the T-lymphocyte subsets resulting from
SEA-stimulated PBMC (SEA cells) were the result of a
SEA-specific clonal expansion, both stimulated and non-
stimulated PBMC were treated with either PPD or Con
A. ELISAs were used to determine the secretion levels of
IFN-c and IL-5 in the supernatant and both PPD and
Con A were found to increase IFN-c production in the
nPBMC control (Fig. 7A). However, SEA-stimulated
PBMC only secreted high levels of IFN- c in response to
Con A (Fig. 7B), demonstrating that this cell population
had lost the ability to recognize PPD and sig nifying that
the T-cell subset had clonally expanded in an SEA-spe-
cific event. IL-5 expression was detected at low levels
under all of the conditions tested.
Discussion
In this study, we employed a PIV assay to investigate
early immune events induced following treatment with
SEA in naı
¨
ve human PBMC from 12 healthy donors.
This is the first report of a PIV assay using DC as APC
to induce SEA-specific responses in naı
¨
ve PBMC, thereby
mimicking a schistosomiasis infection. Our findings
Figure 5 Cytokine production by SEA-stimulated PBMC following
priming and recall responses. Post-priming cell supernatants were
assayed for: IL-5, IL-10, IFN-c (A); IL-6, IL-12p40 and TNF-a (B).
(C) SEA-stimulated PBMC after the second recall response exhibited
different cytokine profiles, 10 donors secreted IL-10, IL-5 and low levels
of IFN-c in (open bars) while two donors secreted high levels of IFN-c
(solid bars). Each symbol represents an individual response. The mean
percentage (+SEM) values are also shown. The results from the Th-1
prone individuals are circled.
E. A. G. Reis et al. Naı
¨
ve Human PBMC Stimulated by SEA 667
..................................................................................................................................................................
Ó 2007 The Authors
Journal compilation Ó 2007 Blackwell Publishing Ltd. Scandinavian Journal of Immunology 66, 662–670
demonstrate that SEA activated and primed DC induce
naı
¨
ve PBM C T cells to initially differentiate into IL-6,
IL-12p40, IFN- c , IL-5 and IL-10 secreting T cells,
similar to what is observed during the acute infection
phase in mice [4, 5]. As expected, after the second recall
event, the immune response of most (10 ⁄12) donor
PBMC polarized towards a strong Th2-like response,
consistent with previous reports [22, 34, 35]. In contrast,
the response of two of 12 donors PBMC in the second
recall response to SEA was Th1-like. Th is unusual
response to SEA stimulation is similar to what was
described as the predominant response in endemic areas
[36]. Deposition of eggs leads to strong Th2 responses
along with increased IL-5 and IL-10 secretion in mice.
Imbalances in these responses led to severe lesions in
mice however, in humans the effect of polarization of the
immune response is not clear [37, 38].
Characterization of DC surface antigen and cytokine
production demonstrated that the DC produced high lev-
els of IL-8 and were associated with upregulated expres-
sion of CD86 and low expression of CD80 co-stimulatory
molecules (Fig. 2). HLA-DR, CD54 surface antigen
expression and levels of the pro-inflammator y cytokines
IL-6 and IL-12p40 were similar in all donors. Based on
this data, there did not appear to be phenotypic differ-
ences in the donor DC that, upon subsequent SEA stimu-
lation, resulted in either a Th2 or Th1-like immune
response. However, this observation does not excl ude the
possibility that DC could respond differently to SEA,
resulting in either Th1- or Th2-like responses.
In the majority of individuals, the composition of the
lymphocyte population in SEA-stimulated PBMC was
predominantly CD4
+
T cel ls secreting IL-5, IL-6 and
high levels of IL-10 (Fig. 5C) indicating a polarization
towards a Th2-like response as previously observed [2, 3,
22]. Conversely, SEA cells from two individuals secreted
high levels of IFN-c (Fig. 5C), indicating a Th1-like
phenotype. Further investigations into naı
¨
ve individual
donor cytokine and chemokine microenvironments fol-
lowing exposure to SEA should be carried out to deter-
mine which of these fac tors contribute to driving DC1
versus DC2 development and subsequently Th1 or Th2
maturation.
The SEA-stimulated PBMC CD4
+
T-cell population
showed a dramatic increase in the expression of HLA-DR
and CD54-ICAM-1, together with expression of
CD45RO, the memory cell marker. Additionally, acti-
vated CD4
+
CD25
+
T cells appear to be involved in the
response to SEA as we observed a higher frequency of
CD4
+
CD25
+
T cells after the first and second recall
responses (Fig. 4A). Although the cells expressing
CD4
+
CD25
+
could be regulatory T cells, the source
could also be activated cells as CD25 is the IL-2 receptor
a-chain. In addition, we also observed a higher frequenc y
of CD4
+
CD25
high
regulatory T cells following SEA stim-
ulation of PBMC (data not shown) as described prev i-
ously in Chagas disease [39]. CD4
+
CD25
high
regulatory
T cells express high and low CD25 molecules but only
CD4
+
CD25
high
T cells have a regulatory function in
Figure 6 IL-10 and IFN-c secretion by SEA-stimulated PBMC follow-
ing secondary recall. SEA cells after secondary recall demonstrating the
reproducibility of the Th1- and Th2-like responses presented in Fig. 5.
IL-10 and IFN-c production in SEA-stimulated PBMC from three Th2-
like donors (A–B); compared to two Th1-like donors (C–D). These
experiments were performed and repeated under the same conditions.
6000
nPBMC Source
SEA cell Source
Con A
PPD
Con A
PPD
(n = 12) (n = 13)(n = 7)
IL-5
IFN-
(n = 8)
(n = 12) (n = 13)(n = 8)
IL-5
(n = 9)
4000
pg/ml (10
6
cell)pg/ml (10
6
cell)
2000
0
6000
4000
2000
0
A
B
γ
IFN-
γ
Figure 7 Cytokine levels by SEA-stimulated PBMC following exposure
to Con A and PPD after the secondary recall response. (A) The naı
¨
ve
PBMC control (nPBMC) and (B) SEA-stimulated PBMC (SEA cells)
were stimulated with PPD or Con A for 72 h. Culture supernatants
were analysed for IL-5 and IFN-c secretion. Each symbol represents an
individual response. The mean values (+SEM) are also shown. The val-
ues from the Th-1 prone individuals are circled.
668 Naı
¨
ve Human PBMC Stimulated by SEA E. A. G. Reis et al.
..................................................................................................................................................................
Ó 2007 The Authors
Journal compilation Ó 2007 Blackwell Publishing Ltd. Scandinavian Journal of Immunology 66, 662–670
humans and mice [40]. This reinforces the idea that addi-
tional immunoregulatory events may be important in
driving CD4
+
T-cell type activity in the selection of
clones and suppressing the proliferation of Th1-like
responses following exposure to SEA. The ability of
CD4
+
CD25
high
regulatory T cells to secrete IL-10, which
inhibits IL-12 and consequently IFN-c production during
Th2 polarization in schistosomiasis [41], is in agreement
with the results reported herein, whereby low levels of
IFN-c were observed in the major ity of donors SEA cells.
In summary, the data support the hypothesis that
human DC activated with SEA could prime CD4
+
T lymphocytes in the early immune events driving SEA-
specific Th1- or Th2-like responses. Importantly, differ-
entiation of the immune response may require more than
just an initial stimulation event, as the tendency towards
a particular immune response was unclear after the first
recall. The polarization towards a Th1- or Th2-like
response was observed only after the second recall event.
The results described in this study suggest that naı
¨
ve
individuals can develop distinct Th1- or Th2-like
immune responses based on the results from the PIV
assay. However, it remains unclear whether this is due to
an individuals genetic background or to environmental
factors such as co-infections. For the first time, we dem-
onstrate that naı
¨
ve human PBMC can develop distinct
immune responses to SEA using a PIV system. This sys-
tem will be a useful tool towards understanding the early
responses of T cells during a schistosomiasis infection.
PIV with SEA could also provide insights into the
immunopathogenesis of schistosomiasis and in evaluating
potential vaccine candidates.
Acknowledgment
This work was supported by Grant AI 30639 to M.G.R.
and AI0546484 to D.A.H . from the National Institutes
of Health, grant 524170/1996-7 from the Brazilian
National Resource Center and by the Oswaldo Cruz
Foundation, Brazil. We thank Jorge Clarencio S. Andrade
for assistance with the flow cytometry.
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Journal compilation Ó 2007 Blackwell Publishing Ltd. Scandinavian Journal of Immunology 66, 662–670
Schistosoma mansoni Triose Phosphate Isomerase
Peptide MAP4 is Able to Trigger Naı
¨
ve Donor Immune
Response Towards a Type-1 Cytokine Profile
E. A. G. Reis*, T. A. Mauadi Carmo*, R. Athanazio*, M. G. Reis*, & D. A. Harn Jrà
Introduction
The ability to properly respond to infection or immuni-
zation depends largely on the response of antigen- present-
ing cells (APC) to pathogens or their antigens. This
innate APC response dictates whether the ensuing adap-
tive CD4
+
T helper (Th) cell responses will be pro-
inflammatory and Th1 type or anti-inflammatory and
Th2 type [1–7]. APC are activated when pathogens or
antigens bind to cell surface receptors, which include
numerous C-type lectin receptors, the Toll-like receptors,
scavenger receptors and others [8, 9]. Pathogen- or anti-
gen-activated APC upregulate the expressio n of DEC 205
[10], major histocompatibility complex (MHC) antigens,
ICAM-1, and co-stimulatory molecules B7 and CD40
[11]. They also secrete cytokines [12] and chemokines,
which induce the maturation of naı
¨
ve Th0 CD4
+
T cells
into IFN-c secreting Th1 or IL-4 ⁄IL-13 Th2 CD4
+
T
cells [13]. The nature of the response of activated and
mature effector CD4
+
Th cells is critical in determining
whether the response to a given pathogen or vaccine will
be protective or exacerbative [14].
Schistosomes are helminth parasites that infect approx-
imately 200 million people worldwide. Although pra-
ziquantel is an excellent drug that eliminates parasites,
the majority of individuals in endemic areas are re-
infected. Unfortunately, while the use of praziquantel
does reduce overall morbidity from disease, schistosomia-
sis remains uncontrolled in a large number of countries
[15], indicating an urgen t need for the development of a
vaccine [16]. The synthet ic schistosome multiple antigen
peptide (MAP4) derived from triose phosphate isomerase
(TPI) has been evaluated as a potential vaccine candidate
in mice and in infected individuals. TPI is expressed dur-
ing all stages of the parasite life cycle and therefore rep-
resents a valid target for vaccines [17]. TPI induces
partial protection in mice when formulated as a MAP
preparation and in mice and pigs when administered as a
SJI
2131
B
Dispatch: 19.5.08 Journal: SJI
CE: Krishna Sarma
Journal Name Manuscript No.
Author Received: No. of pages: 8 PE: Shyamala
*Oswaldo Cruz Foundation (FIOCRUZ),
Salvador, Bahia, Brazil; Faculty of Medicine,
Federal University of Bahia; and àDepartment
of Immunology and Infectious Diseases,
Harvard School of Public Health, Boston, MA,
USA
Received 30 March 2008; Accepted in revised
form 2 May 2008
Correspondence to: Dr M. G. Reis
1
, Fundac¸a
˜
o
Oswaldo Cruz, Rua Waldemar Falca
˜
o, 121,
Candeal, CEP: 40296-710, Salvador, Bahia,
Brazil. E-mail: miter@bahia.fiocruz.br
Abstract
We evaluated the ability of naı
¨
ve monocyte-derived dendritic cells (DC) to
sensitize autologous peripheral blood mononuclear cells (PBMC) to the schisto-
some vaccine candidate MAP4 using a priming in vitro (PIV) assay. MAP4 is a
multiple antigen peptide containing B- and T-cell epitopes derived from the
glycolytic enzyme triose phosphate isomerase. PBMC primed and restimulated
with MAP4 first and secondary recalls (MAP4 PIV cells) were examined for
cell phenotype and cytokine production. We found that after the first recall
stimulation with MAP4, the major cell population was predominantly CD4
+
T-cell subsets (68.5%), CD8
+high
(16%) and CD19
+
(10%). Additionally,
MAP4 PIV cells significantly expressed CD4
+
-HLA-DR
+
, -CD54
+
,
-CD45RO
+
(P < 0.0001) and -CD25
+
(P < 0.0004) together with significant
expression of CD80
+
on CD19
+
B cells (P < 0.007). Cytokine production
from activated MAP4 PIV cells was predominantly Th1-like, consisting
mainly of IFN-c. Interestingly, IFN-c production was suppressed when Schisto-
soma mansoni-soluble egg antigen (SEA) was added to a MAP4 PIV cell cul-
ture. Furthermore, addition of MAP4 to a SEA PIV cell culture significantly
reduced secretion of IL-10. The present findings add to the knowledge gained
from studies in the mouse model, and our results show that naı
¨
ve donor DC,
sensitized with MAP4, were able to prime and clonally expand MAP4-specific
T cells towards a Th1-type response.
BASIC IMMUNOLOGY doi: 10.1111/j.1365-3083.2008.02131.x
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Ó 2008 The Authors
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plasmid DNA vaccine [18]. MAP4 includes immuno-
genic B- and T-cell epitopes from TPI that have low
homology with human TPI. Independent of the MHC
background in mice, MAP4 activates IFN-c -producing
Th1 cells [19] and this cytokine is known to play an
important role in Schistosoma mansoni, both in protective
immunity and in pathology [20, 21].
In this study, we determined whether the stimulation
of human naı
¨
ve PBMC could induce a Th1-like response
similar to that described for the murine model [19].In
order to address this question, we performed priming
in vitro (PIV) assays using naı
¨
ve human monocyte-derived
dendritic cells (DC) as APC. We conclude that MAP4
can induce the expansion of Ag-specific CD4
+
T cells in
humans in a PIV assay, driving a Th1-type immune
response.
Materials and methods
Human donors. PBMC were obtained from 10 healthy,
schistosome naı
¨
ve, Brazilian blood donors living in a schis-
tosomiasis non-endemic area. The donors were screened for
the presence of antibodies to HIV, HTLV I ⁄II, HBsAg,
HCV and Chagas disease, and individuals positive for any
of these infections were excluded from the study. The
donors that met the inclusion criteria had no previous his-
tory of schistosomiasis, and all were negative for both S.
mansoni infection as determined by stool examination using
the Kato–Katz method [22] and for antibodies to S. man-
soni-soluble egg antigens (SEA) as determined by ELISA
[optical density (OD) = 0.006 ± 0.001][23]. Pooled
serum from infected individuals with an average
OD = 0.46 ± 0.043 and from non-endemic healthy indi-
viduals, average OD = 0.01 ± 0.003, were used as positive
and negative controls respectively. The Ethics Committee
of the Oswaldo Cruz Foundation approved the study proto-
col, and all donors provided informed consent prior to the
collection of blood.
Antigen preparations. MAP4 containing T- and B-cell
epitopes derived from TPI (MAP4) and SEA were pre-
pared and tested for endotoxin activity as previously
described [23, 24]. Neither antigen preparation contained
significant amounts of contaminants, such as lipopolysac-
charide, as indicated by the lack of response to SEA in
naı
¨
ve PBMC controls (data not shown) or an RPMI-
primed PBMC control (Fig. 5). The experimental design
is presented as a flowchart in Fig. 1.
DC generation. PBMC were isolated from heparinized
blood (20–30 ml) by density gradient centrifugation on
Ficoll–Hypaque (GE Healt hcare
2
) at 400 g, 30 min, at
15 °C. PBMC were washed in Ca
2+
,Mg
2+
-free Hank’s
balanced salt solution (HBSS) plus penicillin (100 U ⁄ ml)
and streptomycin (100 lg ⁄ml) (pen-strep; Invitrogen).
Monocyte-derived DC were prepared from PBMC as
described previously [25]. Briefly, monocytes were
obtained from PBMC (6 · 10
7
cells ⁄ml) following spon-
taneous sedimentation and incubated in serum-free
RPMI-1640 for 2 h at 37 °C, 5% CO
2
. Non-adherent
cells were removed by washing and adherent cells were
cultured for 6–8 days in 10 ml DC complete medium
(DCC-medium) RPMI-1640, supplemented with 1 mM
sodium pyruvate, 2 mML-glutamine, 1% pen-strep, 10%
ultra-low IgG fetal bovine serum (Invitrogen), (rGM-CSF
50 ng ⁄ml) and (rIL-4 1000 U ⁄ml (Pharmingen BD).
Every 2 days, 500 ll of culture medium was removed
and replaced with the same volume of fresh DCC-med-
ium. After 6 days, supernatants were collected and stored
at )70 °C until use. DC were harvested to determine the
expression of surface markers by FACSort analysis (Bec-
ton Dickinson, Mountain View, CA, USA or Grenoble,
France).
Priming in vitro of human naı
¨
ve PBMC to MAP4.
PBMC were primed in vitro by autologous DC (irradi-
ated with 3000 rads) pulsed with either MAP4
(75 lg ⁄ml), SEA (25 lg ⁄ml) or medium alone (RPMI
control) in 24-well plates in complete medium (C-med-
ium) containing AIM-V medium (serum-free lymphocyte
medium; Invitrogen), 1% pen-strep and 5% human
serum AB (Invitrogen) for 2 h. Later, PBMC (2 · 10
6
cells ⁄ml at a ratio of 1:15 DC:PBMC) were added to
each well and cultured for 7 days at 37 °C, 5% CO
2
.
RPMI-sensitized DC, the negative control, was cultured
Figure 1 Experimental design scheme: naı
¨
ve PBMC from healthy donors were used in the priming in vitro (PIV) system. Non-adherent cells were dif-
ferentiated into DC using IL-4 and GM-CSF for 6–8 days. PBMC (priming) were performed with irradiated (irrad) autologous DC previously pulsed
with or without MAP4 for 2 h and cultured for 7 days. MAP4 or RPMI PIV cells were restimulated (first recall) in the presence of autologous PBMC
(irrad) previously pulsed with and without MAP4 and cultured for 7 days. Those cells were restimulated again (secondary recall) as above for an addi-
tional 72 h. Cytokine production of culture supernatant was measured by ELISA and cell phenotypes were determined by FACS analysis.
2 MAP4 PIV Response in Naı
¨
ve Donor PBMC E. A. G. Reis et al.
..................................................................................................................................................................
Ó 2008 The Authors
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in parallel with PBMC under the same conditions. Cul-
ture supernatants were collected to measure cytokine
production and stored at )70 °C until use. About
20 ng ⁄ml of TNF-a (R&D Systems) was added to the
culture over a period of 20 h.
MAP4, SEA or RPMI-primed PBMC (MAP4, SEA or
RPMI PIV cells respectively) were restimulated (first
recall) in the presence of irradiated autologous PBM C
(2500 rads) at a 1:1 ratio (2 · 10
6
cells ⁄ ml) previously
pulsed with MAP4 (50 lg ⁄ml), SEA (20 lg ⁄ml) or
RPMI in C-medium and cultured for 7 days at 37 °C,
5% CO
2
. IL-2 (20 U ⁄ml) (R&D) was added to the cul-
tures every second and third day. Supernatants were col-
lected to measure cytokine production. PIV cells were
harvested and purified on Ficoll–Hypaque gradients, and
surface antigens were identified by FACSort analysis.
Naı
¨
ve PBMC (ex vivo) were used as controls. MAP4, SEA
or RPMI PIV cells were collected after the first recall
restimulated again (secondary recall) in the presence of
irradiated autologous PBMC (2500 rads) at a 1:1 ratio
(0.5–1 · 10
6
cells ⁄ ml) previously pulsed with MAP4 or
SEA (10 lg ⁄ml) or RPMI in AIM-V medium and
cultured for 72 h at 37 °C, 5 % CO
2
. Supernatants were
collected to measure cytokine production.
Flow cytometry. Cells were stained using the follow-
ing conjugated mouse anti-human monoclonal antibody
(MoAb): CD3-FITC (HIT3a), CD14-FITC (M5E2),
CD40-FITC (5C3), CD45R0-FITC (UCHL 1), CD80-
FITC (BB1), CD86-FITC (FUN-1), HLA-DR, DP,
DQ-PE (TU39), CD25-PE (IL-2Ra) (M-A251), CD54
(ICAM-1)-PE (HA58), CD39-PE (TU66) , CD1a-PE
(HI149), CD19-PE (HIB19), CD4-Cy (RPA-T4) and
CD8-Cy (RPA-T8), and their isotype-matched negative-
control antibodies were purchased from Pharmigen
(BD). Staining was performed as described previously
[25]. Briefly, 10
5
cells ⁄ ml were incubated on ice for
30 min with human serum (diluted 1:20). The cells
were resuspended in FACS buffer [containing HBSS,
10% FCS and 0.01% sodium azide (Sigma-Aldrich, St
Louis, MO, USA) ] at pH 7.2, and each MoAb was
incubated for 30–45 min at 4 °C in the dark. The
cells were washed and resuspended in FACS buffer and
immediately acquired in 10,000 events using a FAC-
Sort flow cytometer (BD Biosciences) and the data were
analysed using CellQuest Software (BD). Cells were
gated via forward and side scatter parameters by a
combination of two- or three-colour dot plots or sin-
gle-colour histograms. The results are expressed as
either the percentage of positive cells within the
selected gate or as proportions.
Cytokine assay. Culture supernatants were collected
after each stimulation, and cytokine concentrations were
determined by an enzyme-linked immunosorbent assay
(ELISA) using the Intertest ELISA kit (Genzyme) for
IFN-c and the IL-5 and IL-10 Human ELISA Set kits
(Pharmingen), according to the manufactur er’s instruc-
tions.
Statistical analysis. Statistical analyses were performed
using the non-p arametric Mann–Whitney test and
GraphPad Prism, version 5.0 (GraphPad Software, San
Diego, CA, USA). Differences were considered significant
when the P-value was <0.05.
Results
DC characterization
DC generated from human monocyte donors exhibited a
typical morphology as confirmed by light microsco py
(data not shown). The successful generation of monocyte-
derived DC was confirmed by the presence of CD1a and
the absence of CD14 surface markers by high forward
and side light scatter (Fig. 2A). In addition, DC were
characterized by FACS to determine the percentage of
cells expressing HLA-DR
+
, CD54
+
(ICAM-1), CD39
+
,
CD40
+
, CD80
+
(B7-1) and CD86
+
(B7-2) molecules
(Fig. 2B). The majority (96.5 %) of the DC population
expressed HLA-DR
+
and CD54
+
molecules, 56.9%
expressed CD86
+
, 30.2% CD40
+
, 21.3% CD39
+
and
modest expression of 3.0% of CD80
+
was observed. We
also evaluated the cytokine production at the basal state
during DC culture differentiation, prior to antigen stim-
ulation (dat a not shown) .
Expansion of a specific T-cell subset by PIV with MAP4
The frequency of the activated PIV cell subset after the
first recall stimulation with MAP4 and a PBMC ex vivo
control was determined by staining with anti-CD3
(Fig. 3A), anti-CD4 (Fig. 3B), anti-CD8 (Fig. 3C) and
anti-CD19 (Fig. 4A) MoAb. Analyses of CD3
+
T cells
after the first reca ll showed a significant percentage
(P < 0.0001) of activated T lymphocytes compared to
the PBMC ex vivo control. The viable cell population was
composed of 68.5% CD4
+
, 16% CD8
+high
T-cell
(Fig. 3B,C) and 10% CD19
+
B-cell subsets (Fig. 4A). A
significant increase in the mean percentage of CD4
+
T
cells (P < 0.0001), as well as a significant decrease in
CD8
+
T cells (P=0.02) and CD19
+
B cells (P=0.002,
Fig. 4A) was observed, compared to a PBMC ex vivo con-
trol. Additionally, there was a significant increase in the
CD4
+
:CD8
+
cell ratio in MAP4 PIV cells compared to a
PBMC ex vivo control (Fig. 3D). The total number of
cells was reduced following priming, first and second
recalls; however, this occurred with and without antigen
stimulation, indicating that it was not due to
antigen toxicity (data not shown). Analyses of gated
lymphocytes demonstrated activated T-lymphocyte
populations co-expressing CD54, HLA-DR, CD45
isoforms (CD45RO) and CD25 (Fig. 3E –H). There was
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significant upregulation of activated T cells expressing
CD4
+
CD54
+
(20.1–82.2%, Fig. 3E), CD4
+
HLA-DR
+
(3.6–22.0%, Fig. 3F), CD4
+
CD45RO
+
(47.4–59.7%,
Fig. 3G) and CD4
+
CD25
+
(6.0–22.0%, Fig. 3H). Fol-
lowing the first recall response of gated lymphocytes sub-
sets, there was a significant positive correlation between
activation markers CD4
+
CD25
+
⁄CD4
+
and CD4
+
HLA-
DR
+
⁄CD4
+
on T cells (Spearman r = 0.8333 and
P = 0.0154, Fig. 3I).
MAP4 stimulation leads to an increased T:B cell ratio
Analyses of T and B cells were perfor med on MAP4 PIV
cells after the first recall stimulation and a PBMC ex vivo
control (Fig. 4). The mean percentage of CD19
+
B cells
decreased significantly following MAP4 PIV stimulation
(Fig. 4A). These results were associated with a significant
increase in the CD3
+
⁄CD19
+
T:B cell ratio and
CD4
+
HLA-DR
+
⁄CD19
+
CD40
+
T:B cell ratio in MAP4
PIV cells when compared to the PBMC ex vivo control
(Fig. 4B,C). In addition, a significantly lower percentage
of CD19
+
CD40
+
B lymphocytes (10–5.5%, P < 0.002)
was observed in MAP4 PIV cells compared to a PBMC
ex vivo control (Fig. 4D). It was interesting to note that
although there we re no differences in the total percentage
of CD19
+
B cells following MAP4 stimulation (Fig. 4A),
CD19
+
CD80
+
B lymphocytes, within the CD19
+
cell
subset, increased significantly (4.9–17%, P < 0.007;
Fig. 4E) after the first recall response compared to a
PBMC ex vivo control, indicating a MAP4-specific expan-
sion of B lymphocytes during PIV.
IFN-c secreted by MAP4 PIV cells
By using the PIV approach, we demonstrated that human
PBMC primed with MAP4 secreted large amounts of
IFN-c after priming, first and secondary recall stimula-
tions (Fig. 5A,B). All donor PBMC demonstrated signifi-
cant, but variable, Th1-type responses following priming
and first recall stimulations (P<0.05, Fig. 5A). How-
ever, a more consistent expression pattern was observed
following the secondary recall response (P<0.0008,
Fig. 5B) when compared to the RPMI PIV cell control.
Similar to the priming and first recall data presented ear-
lier, IL-10 and IL-5 were not detected in the supernatant
of MAP4 PIV cells.
Effect of MAP4 on SEA PIV cell cytokine production
In order to determine if the Th2-like respons e induced
by SEA PIV cells could be altered or down modulated
upon addition of MAP4, we stimulated SEA-primed
PBMC (SEA PIV cells) with SEA or MAP4 (Fig. 6).
MAP4 PIV cells stimulated with SEA, a known Th2
inducer, continued to produce significant levels of IFN-c
(P < 0.05), albeit at lower levels compared to MAP4
PIV cells stim ulated with MAP4. Furthermore, MAP4
PIV cells stimulated with SEA maintained a similar level
of IFN-c production compared to the unstimulated
MAP4 PIV cell control. The level of IFN-c production
in the unstimulated MA P4 PIV cells was significant
(P < 0.01) compared to the RPMI control. In addition,
SEA stimulated MAP4 PIV cells did not produce IL-5 or
IL-10 (Fig. 6). As expected , SEA PIV cells stimulated
with SEA resulted in the production of significant levels
of IL-10 ( P < 0.001) but no IFN-c production. We
found that SEA PIV cells stimulated with MAP4 signifi-
cantly downregulated the expression of IL-5 (P < 0.05)
and IL-10 (P < 0.01) and stimulated the production of
IFN-c, although not significantly, compared to unstimu-
lated SEA PIV cells. Together, these results suggest that
A
B
Figure 2
3
Flow cytometry analysis of DC surface markers. A representa-
tive light scatter plot shows the gate selected for subsequent analyses by
fluorescent antibody staining (A). Each histogram represents an overlay
of CD1a or CD14 (grey line) and an isotype-matched control (bold
line). DC surface marker-specific fluorochrome-labelled MoAb: CD14,
CD1a, CD39, CD40, CD54, HLA-DR, CD80 and CD86 (B). The bars
represents the percentage of positive DC within the selected gate in
donor sample (n = 10). The mean percentage (±95% CI) is also shown.
LOW POOR QUALITY
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MAP4 is a potent Th1 driver, capable of altering the
response of human PBMC to SEA, stimulating IFN-c
production while inhibiting IL-10 secretion.
Discussion
The murine model has contributed significantly to the
understanding of the immune response to schistosomiasis
[26]. Yet the extrapolation of the results from the mouse
to those observed in humans is necessarily limited [27].
The mouse is not wholly representative of the human
infective state, and infected individuals cannot, by defini-
tion, be evaluated in a controlled environment. In an
attempt to study the various factors involved in the ini-
tial immune responses to a schistosome infection, PIV,
using human PBMC, represents a validated approach [25]
Figure 3 Frequencies of T-lymphocyte populations obtained from the PBMC ex vivo control (d) and MAP4 PIV cells after first recall stimulation
(
). Double-label staining was used to identify CD3
+
T cells (A), CD3
+
CD4
+
(B), CD3
+
CD8
+
T cells (C) and CD4
+
⁄ CD8
+
cell ratio (D) or anti-
CD4 Cy to identify CD4
+
CD54
+
(E) or CD4
+
HLA-DR
+
(F) or D4
+
CD45RO
+
(G) or CD4
+
CD25
+
cells within gated lymphocyte (H) or
CD4
+
CD25
+
⁄ CD4
+
versus CD4
+
DR
+
⁄ CD4
+
cells within the CD4
+
-lymphocyte population (I). There was significant positive correlation (r=)0.84,
P < 0.001) by the Spearman’s rank test. The results are expressed as individual values of gated lymphocytes. Significant differences are stated within
each individual analysis.
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whereby PBMC from individuals can be stimulated in a
controlled setting, albeit in vitro, which is perhaps more
representative of the disease. In the present study, we
characterized in vitro the human immune responses to
schistosome MAP4, a synthetic peptide previously shown
to drive Th1-cytokine production in mice [19].
Initial examination demonstrated that PBMC from
S. mansoni chronically infected individuals stimulated
with MAP4 induced low levels of cytokines, including
IFN-c production [28, 29]. To investigate MAP4-specific
immune response in naı
¨
ve human PBMC outside of the
context of schistosome infection, we employed a priming
in vitro assay using human monocyte-derived DC as APC
[25]. We first observed MAP4-pulsed DC induced naı
¨
ve
PBMC to differentiate into Th1-type IFN-c producers
(Fig. 5B). Our study is the first to analyse naı
¨
ve human
PBMC primed by monocyte-DC and pulsed with MAP4.
DC and lymphocytes were characterized by immunophe-
notyping and cytokine profiling in healthy individuals.
DC expressing co-stimulatory molecules, specifically
CD40 and CD80 on B cells, may account for the higher
levels of IFN-c production observed in this study
(Figs. 2B and 4E). In addition, previous studies have
implicated CD80 in stimulating a Th 1 response [30, 31].
The results reported here also support the concept previ-
ously suggested by Janeway and Medzhitov [32], whereby
the cytokines generated by DC in response to a pathogen
are important factors in determining the nature of the
ensuing adaptive responses. In addition, low production
of IFN-c in PBMC from chronic schistosomiasis individ-
uals, stimulated with MAP4 and other antigens [28, 33 ],
is possibly due to low expression of activated and co-
stimulatory molecules, such as MHC class II and B7-1
and B7-2. Indeed, this was observed in the mouse model
Figure 4 Frequencies of the B-lymphocyte population obtained from
the PBMC ex vivo control (d) and MAP4 PIV cells after first recall
stimulation (
). Double-label staining was used to identify CD19
+
B-
cell (A), CD3
+
CD19
+
cell ratio (B) and CD4
+
HLA-DR
+
⁄ C19
+
CD40
+
cell ratio (C), or anti-CD19 PE to identify C19
+
CD40
+
cells within
gated lymphocytes (D) and C19
+
CD80
+
cells within the CD19
+
-lym-
phocyte population (E). The results are expressed of individual values of
gated lymphocytes. Significant differences are stated in each individual
analysis.
Figure 5 Levels of IL-5, IL-10 and IFN-c obtained from MAP4-primed
PBMC with the PIV system after priming (d), first recall (
) and sec-
ondary recall response (r). RPMI controls are represented by the corre-
sponding open symbols and the horizontal bars signify the median
values (A). Pos-priming cell supernatants were assayed for: IL-5, IL-10,
IFN-c (A) and MAP4-primed PBMC after secondary recall response (B).
Cell concentration from the secondary recall was adjusted to (2 · 10
6
cells). Supernatants were harvested and cytokine levels were determined
by ELISA assays. The data represent values from eight representative
donors; significant differences are indicated (*P < 0.05, **P < 0.01,
***P < 0.001).
6 MAP4 PIV Response in Naı
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[30, 34]. However, we observed high expression of HLA-
DR
+
, ICAM-1
+
and CD25
+
(Fig. 3) on MAP4 PIV cells
in the CD4
+
T-cell population after the first recall
response indicated that these cells were activated and
could proliferate. Additionally, increased expression of
CD45RO
+
memory cells after the first recall responses
suggest a MAP4-specific T-cell proliferation (Fig. 3).
Following priming and first recall with MAP4, the
cytokine profile was mainly consistent with a Th1-like
response, except for IL-5 secretion, which may have been
produced as part of the transient Th0 phase as described
previously [35]. Unexpectedly, IFN-c production did not
increase after first recall stimula tion with MAP4, indeed
the first recall response appeared to be lower than that
seen in primed cells, although the difference was not sig-
nificant (P = 0.5, Fig. 5A). IFN-c production increased
significantly following priming and first recall with
MAP4, although only a minority of donor PBMC
produced high levels of IFN-c (Fig. 5A). However,
following secondary recall all donors differentiated to a
Th1-type immune response (Fig. 5B). Interestingly, when
SEA, a strong Th2 inducer, was used to stimulate MAP4
PIV cells, there was no alteration in the production of
IL-10 or IL-5 and both remained undetectable; however,
significant IFN- c levels were maintained even in the
presence of SEA (Fig. 6). In the reverse situation when
SEA PIV cells were stimulated with MAP4, IFN-c pro-
duction increased, albeit not significantly, and IL-5 and
IL-10 production were completely inhibited, suggesting
that MAP4 could be the major factor in driving a Th1-
type response. Interestingly, several donor PBMC failed
to produce IFN-c, a situatio n similar to that seen with
naı
¨
ve PBMC following priming and the first recall to
MAP4 (Fig. 5A), suggesting that further stimulation
with MAP4 is required as, following the second recall
stimulation with MAP4, all donor PBMC produced IFN-
c (Fig. 5B). Therefore, MAP4 is probably capable of
altering the response of cells previously exposed to SEA.
A possible explanation is that copolymerization of B- and
T-cell epitopes enhances MAP immunogenicity and
induces mice to respond to previously unrecognized epi-
topes [36].
In conclusion, our findings suggest that MAP4
appears to be capable of driving a CD4
+
Th1 immune
response. Furthermore, this PIV assay, using cultured
human DC as APC, may be useful in the analysis of the
early events associated with T-cell sensitization and in
the development of in vitro predictive tests to detect the
components involved.
Acknowledgment
This work was supported by Grant AI 30639 to MGR
and AI0546484 to DAH from the National Institutes of
Health and by the Oswaldo Cruz Foundation, Brazil. We
thank Jorge Clarencio S. Andrade for assistance with the
flow cytometry. We also thank Dr Alan McBride for a
critical review of the manuscript.
Figure 6 Effect of MAP4 or SEA on IL-5, IL-10 and IFN-c cytokine
production. Using the method described for Figure 5, we determined
the cytokine levels from RPMI control ( ), MAP4 PIV cells stimulated
with MAP4 (
), MAP4 PIV cells stimulated with SEA (h), unstimu-
lated MAP4 PIV cell control [cytotoxic T lymphocyte (CTL), e], SEA
PIV cells stimulated with SEA (
), SEA PIV cells stimulated with
MAP4 (D) and unstimulated SEA PIV cell control (CTL, r). The hori-
zontal bars signify the median values. PIV cell supernatants were har-
vested after 72 h, and cytokine levels were determined by ELISA assays.
The data represent values from eight representative donors; significant
differences are indicated (*P < 0.05, **P < 0.01, ***P < 0.001).
E. A. G. Reis et al. MAP4 PIV Response in Naı
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4. DISCUSSÃO GERAL
No presente estudo, foi avaliada a resposta imune in vitro de PBMCs de doadores
normais ao antígeno ovular solúvel e ao peptídeo sintético MAP4 derivado da triose fosfato
isomerase do Schistomsoma mansoni, presente em todos os estágios do S. mansoni. Esses
estudos resultaram em dois manuscritos:
1) Naive donors Responses to Schistosoma mansoni Soluble Egg Antigens (REIS et al.,
2007);
2) Schistosoma mansoni Triose Phosphate Isomerase peptide MAP4 is able to trigger
naive donor immune response toward a Type-1 cytokine profile (REIS et al., 2008).
Na realização desses estudos, foi empregado o PIV para investigar eventos que ocorre
na resposta imune humana, utilizando células humanas de doadores normais. Inicialmente,
monócitos do sangue periférico foram obtidos para gerar células dendríticas na presença de
citocinas GM-CSF e IL-4, e posteriormente caracterizadas baseadas na expressão de
marcadores de superfície e produção de citocinas. As células dendríticas foram sensibilizadas
com e sem antígenos SEA ou MAP4 e cultivadas com PBMCs autológas. Os linfócitos
ativados foram reestimulados duas vezes com os mesmos antígenos na primeira e segunda
chamada após o prime.
Os resultados observado no primeiro manuscrito, mostrou que as células dendríticas
apresentaram alta expressão de moléculas de ativação, adesão e co-estimulatórias HLA-DR,
ICAM-1, CD40, B7-2 (CD86) e baixa expressão de B7-1 (CD80). Além disso, as células
dendriticas produziram elevada concentração de citocinas IL-8, IL-6, TNF-α e baixa
concentração de IL-12p40. A expressão dessas moléculas de ativação e a producão das
citocinas pró-inflamatórias IL-6 e IL-12p40 foram semelhantes no “priming” com o antígeno
bruto SEA e com o peptideo MAP4 como demostrados nos manuscritos acima 1 e 2.
PBMCs de doadores normais primadas com SEA (REIS et al., 2007), 80%
desenvolveram uma resposta de células T auxiliadora (Th) CD4
+
do tipo Th2 e 20% do tipo
Th1. Esses achados estão de acordo com os dados da literatura confirmando que SEA é um
forte indutor da resposta imune Th2 in vivo no modelo murino e in vitro com células
dendríticas sensibilizadas com SEA (MACDONALD et al., 2001; OKANO et al., 2001;
THOMAS et al., 2003), assim como seus componentes como glican, especialmente, Lewis X,
reconhecidos pelas células dendríticas através de receptores de superfície para lecitinas tipo-C
tais como DC-SIGN (VAN DIE et al., 2003) e fosfatadilserina através de TLR2 (VAN DER
KLEIJ et al., 2002). No nosso sistema in vitro A expansão específica de células T CD4
+
28
primadas contra o antígeno SEA foi observado um aumento gradual na expressão de
marcadores de superfície HLA-DR, CD25, ICAM-1 e células de memória CD45RO durante o
“priming”, primeira e segunda reestimulação, ocorrendo o mesmo para linfócitos B
CD19
+
CD86
+
. Estes resultados indicam que moléculas co-estimulatórias são essenciais para
determinar o tipo da resposta imune, conforme previamente sugerido (DUBEY et al., 1995).
Células dendríticas, sensibilizadas com SEA e cultivadas com PBMCs virgens por um
período de sete dias, induziram uma resposta imune inicial compatível com o padrão Th0,
semelhante ao observado no modelo murino (VELLA; PEARCE, 1992;1994), com produção
simultânea de citocinas do tipo Th1/Th2 como IFN-γ, IL-5, IL-6, IL-10 e IL-12. Acredita-se
que a mudança da fase temporaria Th0 para a Th2 se deva aos componentes presentes no ovo
do parasita, tais como açúcares (OKANO et al., 2001; THOMAS et al., 2003; VELUPILLAI;
HARN, 1994), exercendo um papel importante na polarização da imunidade adquirida criando
um microambiente rico em IL-10, com diminuição da síntese de IL-12 e, conseqüentemente,
da síntese de IFN-γ, favorecendo uma resposta do tipo Th2. Esses dados indicam que pelo
menos in vitro, parece ser necessárias mais de uma estimulação antigenica após o prime, para
a definição de um padrão semelhante de resposta Th2/Th1, considerando que a polarização da
resposta imune em Th2 ou Th1 só foi observada depois da segunda reestimulação ou segunda
chamada. Diferentemente, ocorreu em PBMCs primadas com MAP4 (REIS et al., 2008),
onde as estimulações (durante o PIV) foi observado o padrão de resposta do tipo Th1 com
produção de IFN-γ e baixa concentração de IL-6 (dado não mostrado).
Interessante, PBMCs primadas com SEA de dois doadores neste estudo responderam
diferentemente com um padrão de resposta semelhante a do tipo Th1, com elevada
concentração de IFN-γ e ausência de IL-10 após a segunda reestimulação ou segunda
chamada do PIV. Dados na literatura também evidenciam que indivíduos caracterizados como
normais endêmico (VIANA et al., 1994) e indivíduos com a forma grave da esquistossomose
(MWATHA et al., 1998) produzem IFN-γ em resposta ao SEA. Os nossos resultados,
somados aos dados da literatura (VIANA et al., 1994; MWATHA et al., 1998; ABATH et al.,
2006; DE JESUS et al., 2002), apontam para a necessidade de estudos adicionais para o
esclarecimentos da polarização da resposta imune na esquistossomose.
Adicionalmente, avaliamos as caracteristicas fenotípicas de células T CD4
+
denominadas de reguladoras por apresentarem expressão elevada de CD25 (Treg
CD4
+
CD25
high
) presente em PBMCs primadas com SEA. A expansão e ativação especifica
de PBMCs primadas com SEA após a primeira e segunda chamada apresentaram aumento
significativo no percentual das células T CD4
+
CD25
high
, em relação as células não primadas
PBMCs ex vivo (dados não mostrados), sugerindo que esta subpopulação de linfócitos podem
29
desempenhar um papel importante no controle da resposta imune desenvolvida contra o SEA
e, consequentemente, na imunopatogênese da esquistossomose. A análise dessas células T reg
co-expressando altos níveis de CD25 foi feita segundo protocolo proposto por (BAECHER-
ALLAN et al., 2001). CD25 é uma molécula de superficíe também presente em células T
ativadas, portanto, a presença dessa molécula não é suficiente para determinar o perfil
fenotípico das células Treg. Também foi observado pelos autores REIS et al., 2007 um
aumento significante na freqüência de células ativadas expressando CD4
+
CD25
+
(figura 4A).
Esses achados sugerem que evento imuno-regulatório adicional seja importante na
participação de clones de células T CD4
+
regulando a proliferação de clones Th1 após a
exposição ao SEA. A habilidade de células T reguladoras CD4
+
CD25
high
de produzir IL-10
que inibe a produção de IL-12, principalmente nas células apresentadoras de antígenos
(FIORENTINO et al., 1991) assim como regulação da expressão da molécula co-estimulatória
B7 (DING et al., 1993) explicam em parte a diminuição da produção de IFN-γ durante a
polarização Th2 na esquistossomose (MCKEE; PEARCE, 2004), fato este também
demonstrado no presente trabalho pelos autores REIS et al., quando foi observado baixa
concentração de IFN-γ após o prime, primeira e segunda reestimulação ou primeira e segunda
chamada figura 5C e figura 7, manuscrito 1 (REIS et al., 2007). Contudo, à metodologia
utilizada no presente trabalho, não foi possível assegurar que altos níveis de IL-10 seja em
parte das células T reguladoras CD4
+
CD25
high
. Entretanto, esses dados sugerem que fatores
genéticos humano tenham papel importante em determinar o tipo de resposta imune na
esquistossomose.
Os nossos achados indicam que, no PIV, células dendriticas humanas sensibilizadas
com SEA têm um papel importante na resposta imune inicial e na diferenciação de linfócitos
T CD4
+
virgens. Finalmente, os dados do presente trabalho somados aos dados da literatura
sugerem que a polarização da resposta imune parece depender de múltiplos fatores, tais como
tipo de antígeno, natureza da população das células dendríticas, citocinas presentes no
microambiente e de provavelmente fatores genéticos do hospedeiro.
No segundo manuscrito, diferente do observado com SEA, foi demonstrado um padrão
de resposta imune do tipo Th1 durante o PIV de PBMCs primadas com MAP4, com produção
de IFN-γ, semelhante aos dados observados no modelo murino (REYNOLDS et al., 1994) e
nos estudos com pacientes infectados, que em geral também produziram citocinas incluindo
IFN-γ (AL-SHERBINY et al., 2003; RIBEIRO DE JESUS et al., 2000).
Durante os primeiros sete dias da estimulação, o padrão de citocinas foi direcionado
para Th1 exceto pela presença de IL-5, o qual talvez tenha sido produzido como parte da fase
transitória Th0 (VELLA; PEARCE, 1994). Entretanto, após a reestimulação (primeira
30
chamada), foi observado um aumento da produção de citocinas características da resposta
Th1, com predomínio de linfócitos T CD4
+
ativados expressando HLA-DR e CD25 e
aumento na expressão de células de memória (CD45RO). Além disso, as células T CD4
+
,
linfócitos B CD19
+
expressaram moléculas co-estimulatórias CD80 e CD86. A expressão de
CD40 nas células dendríticas e de CD80 nos linfócitos B, em parte pode explicar a alta
produção de IFN-γ com base nas evidências que demonstram associação entre expressão de
CD80 e geração de resposta imune tipo Th1 (KELLEHER; KNIGHT, 1998;
SUBRAMANIAN et al., 1997).
Para a comparação da resposta de PBMCs primadas com SEA ou MAP4 pelas células
dendríticas obtidas do mesmo doador, os experimento foram realizados em paralelo, sob as
mesmas condições. Demonstramos que PBMCs primadas com SEA e reestimuladas com SEA
(segunda chamada) secretaram IL-5, IL-10 e IL-6 e baixa concentração de IFN-γ enquanto
PBMCs primadas com MAP4 reestimuladas com MAP4 secretaram IFN-γ. IL-5 e IL-10
estavam ausentes nesta população reestimuladas com MAP4, demonstrando que PBMCs de
doadores não foram anteriormente favorecidos e que a resposta para o MAP4 e SEA foram
distintas figura 6, manuscrito 2 (REIS et al., 2008). A reestimulação na segunda chamada das
células primadas com MAP4 determinou uma produção de citocinas do tipo Th1, mesmo
quando essas células foram estimuladas na presença de SEA um forte indutor de resposta tipo
Th2 considerando que essas células foram capazes de secretar uma quantidade consideravel
de IFN-γ figura 6 (REIS et al., 2008). Além do IFN-γ, também tem sido observado moderada
produção de TNF-α e IL-6, não tendo sido detectado produção de IL-10 (dados não
mostrados). A forte resposta induzida por MAP4 pode ser explicada com base nas
observações realizadas por Cox et al. (COX et al., 1988), quando demonstraram que co-
polimerização de epitopos de células B e T potencializam a imunogenicidade e induzem cepas
de camundongos a responderem a epitopos desconhecidos.
Concluindo, os achados dos dois trabalhos demonstraram que o sistema de PIV utilizando
células dendríticas sensibilizadas e cultivadas com células mononucleares do sangue
periférico pode ser útil para o estudo dos eventos inicias da reposta imune a um dado
antígeno, e para avaliar antígenos com potencial de vacina.
31
5. CONCLUSÕES GERAIS
Apartir dos dados apresentados neste trabalho podemos concluir que no sistema
“priming” in vitro (PIV), com PBMC humano foi possível avaliar a resposta específica aos
antígenos SEA e MAP4 do S. mansoni.
Com base nos nossos achados podemos inferir que o sistema de PIV pode ser utilizado
para determinar a imunogenicidade de antígenos assim como para estudar os eventos iniciais
da imunopatogênese na esquistossomose humana e avaliar potenciais antígenos candidatos a
vacina.
32
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41
ANEXO A - MANUSCRITOS RELACIONADOS COM S. MANSONI:
Manuscritos publicados e ou aceitos para publicação durante o período de doutorado,
relacionados com esquistossomose mansoni.
41
Please cite this article in press as: Reis, E.A.G., et al., Influence of the HLA-DQB1*0201 allele on the immune response in a Schistosoma mansoni
infection, Acta Trop. (2008), doi:10.1016/j.actatropica.2008.04.004
ARTICLE IN PRESS
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ACTROP-2096; No. of Pages4
Acta Tropica xxx (2008) xxx–xxx
Contents lists available at ScienceDirect
Acta Tropica
journal homepage: www.elsevier.com/locate/actatropica
Influence of the HLA-DQB1*0201 allele on the immune response
in a Schistosoma mansoni infection
Eliana A.G. Reis
a
, Daniel A. Athanazio
a,b
, Alan J.A. McBride
a
, Theomira Mauadi Carmo Azevedo
a
,
Isis F. Magalh
˜
aes-Santos
a
, Donald Harn
c
, Mitermayer G. Reis
a,b,∗
a
Oswaldo Cruz Foundation (FIOCRUZ), Salvador, Bahia, Brazil
b
Federal University of Bahia, Bahia, Brazil
c
Department of Immunology and Infectious Diseases, Harvard School of Public Health, Boston, MA, USA
article info
Article history:
Available online xxx
Keywords:
HLA
Schistosomiasis
Schistosoma mansoni
Immunoglobulin isotype
abstract
We previously reported the association of the major histocompatibility complex class II HLA-DQB1*0201
allele with hepatosplenic schistosomiasis. The aim of this study was to evaluate the cytokine responses of
peripheral blood mononuclear cells (PBMCs) and the serum levels of immunoglobulin isotypes. The study
population was selected from a schistosomiasis endemic area. No significant differences in cytokine pro-
files were detected in PBMCs stimulated with Schistosoma mansoni soluble egg antigen (SEA), regardless of
the subjects DQB1*0201 genotype or infection status. However, previously infected DQB1*0201 positive
individuals had significantly lower levels of IgG4 compared to DQB1*0201 negative individuals (P < 0.05).
© 2008 Elsevier B.V. All rights reserved.
The major outcome of a Schistosoma mansoni infection is
chronic hepatosplenic schistosomiasis in which granulomatous
inflammation and fibrosis of the liver leads to potentially fatal
portal hypertension (Andrade, 2004). Approximately 5% of indi-
viduals living in S. mansoni endemic areas develop hepatosplenic
disease despite treatment (Jos
´
e C. Bina, personal communica-
tion). The investigation of host genetic determinants for disease
severity is a developing field in schistosomiasis research, and cer-
tain major histocompatibility complex (MHC) alleles have been
associated with schistosomal hepatosplenomegaly. Among Egyp-
tian individuals, positive associations were reported for human
leukocyte antigen (HLA) A1 and B5 (Salam et al., 1979), A2
and B12 (Hafez et al., 1991), B5 and DR3 (Assaad-Khalil et
al., 1993) and a gamma interferon (IFN-␥) gene polymorphism
(Blanton et al., 2005). In a study in Southern Brazil, however,
no association was observed between HLA-A1 or B5 expression
and hepatomegalic disease (Cabello et al., 1991). In northeast-
ern Brazil, we previously studied three loci (DRB1, DQA1, and
DQB1) and detected an association between DQB1*0201 and hep-
atosplenic disease (Secor et al., 1996). The same allele, especially
in combination with DRB1*0301, was subsequently associated
with post-schistosomal hepatic fibrosis caused by Schistosoma
∗
Corresponding author at: Oswaldo Cruz Foundation-Ba, Centro de Pesquisas
Gonc¸ alo Moniz–CPqGM, Rua Waldemar Falc
˜
ao, 121, Candeal, CEP: 40296-710, Sal-
vador, Bahia, Brazil. Tel.: +55 71 31762200; fax: +55 71 31762326.
E-mail address: miter@bahia.fiocruz.br (M.G. Reis).
japoni (S. japonicum) infection in Chinese patients (Hirayama et al.,
1999).
It is possible that these genetic determinants may interfere
in schistosomal pathogenesis and the host immune response. For
instance, latestage human infection is characterized by polarization
from a potentially protective Th1-type immune response to a Th2-
type response (Pearce and MacDonald, 2002). In addition, there is
strong evidence from animal models to support the involvement
of a Th2-type response in the severity of granulomatous inflam-
mation and fibrosis of the liver (Farah et al., 2000; Reiman et al.,
2006). However, the association is unclear in humans as peripheral
blood mononuclear cell (PBMC) responses have been associated
with higher levels of either tumor necrosis factor alpha (TNF-␣)
(Mwatha et al., 1998) or interleukin (IL)-13, IL-4, IL-5 and IL-10 that
have been linked to progressive or end-stage liver disease (de Jesus
et al., 2004; Montenegro et al., 1999; Alves Oliveira et al., 2006).
An additional mechanism may involve the expression of differ-
ent immunoglobulin isotypes as they have been associated with
protection and susceptibility to re-infection (Demeure et al., 1993;
Caldas et al., 2000). The present report focuses on the influence of
the HLA-DQB1*0201 allele on immune response-related cytokine
production and immunoglobulin isotype production.
The study population was selected from a schistosomiasis
endemic area (Itaquara, Bahia, Brazil). Field studies have been car-
ried out at this location since 1989 when the overall prevalence
of S. mansoni infection was 90%. Annual surveys are carried out
in this region for schistosomiasis and other helminth infections
and any infected individuals are subsequently treated. Thirty-three
0001-706X/$ – see front matter © 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.actatropica.2008.04.004
Please cite this article in press as: Reis, E.A.G., et al., Influence of the HLA-DQB1*0201 allele on the immune response in a Schistosoma mansoni
infection, Acta Trop. (2008), doi:10.1016/j.actatropica.2008.04.004
ARTICLE IN PRESS
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2 E.A.G. Reis et al. / Acta Tropica xxx (2008) xxx–xxx
individuals resident in the endemic region and previously treated
for a S. mansoni infection were selected for inclusion in this study.
Upon re-evaluation, 23 volunteers were negative by stool sam-
ple for S. mansoni eggs (egg-negative), while 10 individuals had
been reinfected (egg-positive) by S. mansoni. The HLA-DQB1*0201
status of the egg-negative (previously infected) group was deter-
mined as negative (n = 14) or positive (n = 9) as described previously
(Secor et al., 1996). Egg-positive (re-infected) individuals included
seven positive individuals and three negative individuals for the
HLA-DQB1*0201 genotype. All individuals provided informed writ-
ten consent and samples were collected according to protocols
approved by the Human Ethical Committee of the Oswaldo Cruz
Foundation. All infected individuals in the study were subsequently
treated for schistosomiasis and other parasitic infections.
Peripheral blood mononuclear cells (PBMCs) were isolated from
20 ml heparinized blood by density gradient centrifugation on
Ficoll Histopaque (Sigma–Aldrich, St. Louis, MO, USA) as described
previously (Velupillai et al., 2000). PBMCs were cultured with sol-
uble egg antigen (SEA) from S. mansoni (5 ␮g/ml), Concanavalin
A(5␮g/ml), or medium alone as a control. SEA was prepared as
described elsewhere (Harn et al., 1989). Supernatants were col-
lected at 24 h for IL-10 and TNF-␣ and at 72 h for IL-5 and IFN-␥.
Sandwich ELISAs were performed following the manufacturer’s
instructions for TNF-␣, Duo-set kit (R&D System, Minneapolis, MN,
USA); IFN-␥ kit from Genzyme (Cambridge, MA, USA) and IL-5 and
IL-10 kits from PharMingen (BD, Mountain View, CA, USA).
Specific levels of IgM, IgA, IgE, IgG1, IgG2, IgG3 and IgG4 against
S. mansoni SEA were analyzed by ELISA, as described previously
(Li et al., 1995). Briefly, microtiter plates were incubated with the
following antibodies diluted in PBS-Tween 80 (0.05%): monoclonal
anti-human IgA (1:15,000), anti-human IgE (1:2500), purified anti-
human IgM (1:15,000) conjugated to peroxidase and monoclonal
anti-human IgG1 (1:2000), IgG2 (1:15,000), IgG3 (1:2000) and IgG4
(1:15,000) conjugated to biotin (Sigma–Aldrich). Tetramethylben-
zidine (TMB) and peroxidase substrate solution B (1:1) were used
for detection of peroxidase activity (Kirkegaard & Perry Laborato-
ries, Gaithersburg, MD, USA). The reaction was stopped with 2N
H
2
SO
4
and read at 450 nm by a spectrophotometer linked to MDS-
Softmax software. Cut-off values for the ELISA were calculated
using sera collected from a group of individuals (n = 17) resident
in the city of Salvador, a non-endemic region for schistosomia-
sis. The cut-off value was calculated as the mean optical density
(OD
450
) plus three standard deviations and was determined to
be 0.14. Statistical analyses were performed using non-parametric
Mann–Whitney and Kruskal–Wallis tests using GraphPad Prism 4.0
software (GraphPad Software, San Diego, CA, EUA), P-values <0.05
were considered significant in this study.
No significant differences in cytokine production were detected
following SEA stimulation of PBMCs from egg-positive or egg-
negative individuals (Fig. 1) regardless of their DQB1*0201 status.
In addition, no significant differences were observed after Con-
canavalin A stimulation.
In the present study, SEA stimulated PBMCs from previously
infected DQB1*0201 positive individuals produced significantly
lower levels of anti-SEA IgG4 compared to previously infected
DQB1*0201 negative individuals (P < 0.05, Fig. 2). Among this group,
57.1% (8/14) of DQB1*0201 negative individuals had detectable
levels of IgG4 above the cut-off compared to only 11.1% (1/9) of
DQB1*0201 positive individuals, although this difference was not
significant (P < 0.05). No differences in IgG4 levels were observed
between individuals with an active infection (egg-positive). There
were no significant differences in the levels of the other antibody
isotypes evaluated, either in egg-positive or egg-negative subjects
nor with regard to their DQB1*0201 status.
High IgG4 serum levels have been related to advanced stage
liver fibrosis in S. mansoni infections (Bonnard et al., 2004; Silveira
et al., 2002). In addition, Hagan and colleagues reported epi-
demiological evidence that low serum IgE and high serum IgG4
Fig. 1. Cytokine production in PBMCs from HLA-DQB1*0201 positive and negative individuals actively or previously infected with S. mansoni. The cytokine profiles of PBMCs
stimulated with SEA, Concanavalin A or RPMI medium were evaluated. HLA refers to presence (+) or absence (−) of the HLA-DQB1*0201 allele and egg refers to the presence
(+, active infection) or absence (−, previous infection) of eggs in stool samples of individuals in the study population. The vertical bars represent the mean ± SEM.
Please cite this article in press as: Reis, E.A.G., et al., Influence of the HLA-DQB1*0201 allele on the immune response in a Schistosoma mansoni
infection, Acta Trop. (2008), doi:10.1016/j.actatropica.2008.04.004
ARTICLE IN PRESS
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E.A.G. Reis et al. / Acta Tropica xxx (2008) xxx–xxx 3
Fig. 2. Serum immunoglobulin isotype profiles from HLA-DQB1*0201 positive and negative individuals actively (Egg +) or previously (Egg −) infected with S. mansoni. The
vertical bars represent the mean ± SEM and the * indicates a statistically significant difference (P < 0.05) between the compared groups.
levels correlated with re-infection by Schistosoma haematobium (S.
haematobium)(Hagan et al., 1991). Subsequently, similar observa-
tions were reported for S. mansoni (Demeure et al., 1993; Caldas et
al., 2000). This is likely a consequence of early excessive production
of IgG4 antagonizing the potentially protective IgE immunological
pathways (Hagan et al., 1991). The influence of HLA polymorphisms
on antibody isotypes in schistosomiasis was recently reported. The
DRB1*13 allele was associated with higher, post-treatment, lev-
els of IgE against various schistosoma antigens and with a lower
frequency of re-infection (Booth et al., 2006). The majority of stud-
ies on IgG4 levels have been hampered by the high frequency of
undetectable levels of this isotype in young individuals, especially
children (Herrod, 1993; Moss et al., 1992). In the present report the
mean age of the egg-negative group was 35 ± 14 and 32 ± 08 for
DQB1*0201 positive and negative individuals, respectively.
In the present work we could not find a plausible relationship
between the HLA-DQB1*0201 allele and its effect on the immune
response and, more specifically, on cytokine profiles. Low levels of
IgG4 among previously infected DQB1*0201 carriers suggest that
individuals with a previous history of infection are less suscep-
tible to re-infection and this may be an important factor in the
development of hepatosplenic disease (Demeure et al., 1993; Caldas
et al., 2000). Since the presence of HLA-DQB1*0201 is associated
with hepatosplenic disease (Secor et al., 1996) and high serum
IgG4 is indicative of progression to liver fibrosis (Bonnard et al.,
2004; Silveira et al., 2002), the results of our study imply that the
susceptibility of DQB1*0201 positive individuals to re-infection is
independent of IgG4 levels.
In the present study,we did not detect anysignificant differences
in the immune response during active infection and DQB1*0201
status, however, our study population may not have been large
enough to detect such an association due to the small num-
ber (n = 10) of egg-positive (re-infected) patients. Further studies
involving larger numbers of actively infected individuals will be
required to clarify the interactions between host genetic back-
ground, immune response and susceptibility to severe forms of
disease.
Acknowledgments
This work was supported by grants AI-30639 and AI-0546484
from the National Institutes of Health NIH-USA, and from
FINEP/PRONEX 4196086200. We thank Claudio Roberto Santos for
technical support at the Itaquara field site.
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BIOCHEMICAL AND IMMUNOLOGIC PREDICTORS OF EFFICACY OF
TREATMENT OR REINFECTION RISK FOR SCHISTOSOMA MANSONI
ELIANA A. G. REIS, MITERMAYER G. REIS, RITA DE CÁSSIA R. SILVA, THEOMIRA M. A. CARMO,
ANA MARLÚCIA O. ASSIS, MAURÍCIO L. BARRETO, ISABEL M. PARRAGA, MÔNICA LEILA P. SANTANA, AND
RONALD E. BLANTON*
Oswaldo Cruz Foundation, Fiocruz, Salvador, Bahia, Brazil; School of Nutrition, Federal University of Bahia, Salvador, Bahia,
Brazil; Institute of Collective Health, Federal University of Bahia, Salvador, Bahia, Brazil; Department of Nutrition, Case Western
Reserve University, Cleveland, Ohio; Center for Global Health and Diseases, Case Western Reserve University, Cleveland, Ohio
Abstract. Most Schistosoma mansoni infections are egg-negative after a single dose of oxamniquine. A cohort of 661
infected children was treated at 6-month intervals and assessed for nutritional and parasitological status. Initial bio-
chemical and immunologic markers were measured in a subset of 84 children. All were treated at the start of therapy
and at 6 months. Immunoglobulins only served as markers for active infection. No markers were predictive of cure or
reinfection, except initial infection intensity and serum low-density lipoprotein. Ten percent were persistently infected
and had no change in infection intensity at any time-point. Several factors suggest that this group was biologically
different. In addition to failing to reduce their worm burden, they had significantly higher initial intensity of infection
(100 versus 65 eggs/g, P ס 0.001) and significantly lower initial serum low-density lipoprotein (72 versus 104 mg/dL,
P ס 0.045). The biologic plausibility of this observation is discussed.
INTRODUCTION
Control of schistosomiasis at present depends on repeated
rounds of chemotherapy at intervals of 1 or 2 years. On a
national scale, such as the programs in Brazil or Egypt, fol-
low-up stool surveys at 6 weeks after treatment are costly and
not usually performed. Therefore, in practical terms, the ef-
ficacy of annual or biennial chemotherapy for control of schis-
tosomiasis is the combined effects of the drug, the intensity of
transmission, and the rapidity of reinfection. The two drugs
most recently used in the treatment of Schistosoma mansoni,
oxamniquine and praziquantel, have similar profiles for effi-
cacy as single-dose therapies.
1
A consistent finding for both is
that 10–20% of those treated fail to clear their infection, al-
though nearly 100% experience a reduction in the intensity of
infection.
1,2
The factors associated with this fairly constant
level of treatment “failure” have been primarily associated
with very heavy infection and intense transmission,
3,4
as well
as some level of noncompliance with taking these drugs. Be-
cause the drug does not affect immature stages of the parasite,
drug efficacy may seem to be lower where many individuals
have experienced a new infection within 4–6 weeks of treat-
ment. Reinfection after treatment also has some well-
described associations. In addition to the intensity of trans-
mission, age < 14
5
and sexual immaturity in humans have
been associated with increased reinfection with S. mansoni.
6–8
Male sex has also been associated with increased susceptibil-
ity and severity of schistosomiasis, both in laboratory ani-
mals,
9
and population studies conducted in endemic areas.
10
To study whether other host factors, such as immune respon-
siveness or nutritional status, might be related to treatment
outcome, we conducted a prospective cohort study of 106
infected children. We analyzed initial parasite burden, socio-
economic status, age, sex, anthropometric indices, a panel of
biochemical markers, and immunoglobulin isotypes and as-
sessed their ability to predict parasitological outcome in the
treated children at 6 months and 1 year.
MATERIALS AND METHODS
Study site and population. The study was performed in the
Brazilian town of Jequié, which is situated in a semi-arid re-
gion of the State of Bahia. There are ∼ 135,000 inhabitants
some of whom rely on a major river that borders the town for
washing, sanitation, fishing, and recreation. A total of 13,771
children 7.0–17.9 years of age were surveyed for infection by
school-based and house-to-house examinations in neighbor-
hoods previously identified as at risk for schistosomiasis by
the Brazilian National Foundation of Health (Fundação Na-
cional de Saude [FNS]). Of the 1,766 children infected, we
elected to include all of those from the most heavily infected
neighborhoods (661) in a prospective cohort study of deter-
minants of response to oxamniquine. Some 19.3% of children
were excluded for stool egg counts > 400/g of stool (400 epg).
Those included were further evaluated for anthropometrics,
dietary intake, hemoglobin level, and socio-economic status.
Serum was collected for measurement of biochemical mark-
ers of nutrition, tumor necrosis factor (TNF), and immuno-
globulins. This number was determined by the resources
available for this part of the study. Complete data were ob-
tained on 84 of those selected for serologic and biochemical
studies. Informed consent was obtained for all study subjects
before clinical and parasitological study and blood collection.
This study was approved by the Human Investigation Com-
mittees of University Hospitals and the Oswaldo Cruz Foun-
dation, Salvador, Bahia, Brazil.
Parasitology and treatment. Stools were collected in pre-
labeled plastic containers delivered to the children’s homes.
Two stool samples obtained on different days were examined
for each participant. For each stool sample, two slides were
made and examined using the quantitative method of Katz
and others.
11
The slides were prepared and read within the
first 2 hours, and every 10th slide was re-examined by an
independent technician for quality control. Even three con-
secutive Kato-Katz thick smears may fail to detect up to 7%
of infections
12
; thus, those in whom no eggs were detected in
feces will be referred to as “egg-negative” rather than “cured”
or “uninfected.”
Oxamniquine (20 mg/kg) was administered to all partici-
* Address correspondence to Ronald Blanton, Center for Global
Health and Disease, Case Western Reserve University, Cleveland,
OH 44106. E-mail [email protected]
Am. J. Trop. Med. Hyg., 75(5), 2006, pp. 904–909
Copyright © 2006 by The American Society of Tropical Medicine and Hygiene
904
pants under direct observation at the start of the study and
again 6 months later. Only those still found to have schisto-
some eggs in stool were treated at 1 year.
Anthropometric studies. All measurements were carried
out using standard anthropometric methods as described pre-
viously.
13,14
For each child measurement of weight, height,
skinfold thicknesses, and arm circumference were made. In-
dices were calculated and converted to Z-scores for analysis.
Socioeconomic survey. To control for the effects of other
covariates such as environmental factors, an index was con-
structed from data collected by questionnaire during home
visits. To construct the index for environmental conditions, all
of the variables were dichotomized and coded as 1 (poor) or
0 (good). Variables were selected to make up the index ac-
cording to their association with anemia by a logistic regres-
sion model, because this is a significant morbidity associated
with helminthic infection.
15
Sex, age, and degree of education
of the head of household were used as covariates to adjust the
regression model. Variables that remained in the model were
source of water, regularity of water availability, place where
water is kept, care taken with drinking water, presence and
location of toilet, frequency of garbage collection, presence of
trash around the house, the presence of an open sewer, type
of flooring in the home, and composition of the house. The
included variables were summed and classified as adequate
(0–6) or inadequate (7–13) based on Reichenheim and Har-
pham.
16
Biochemical assays. Morning blood was collected in min-
eral-free tube from all participants by venipuncture. Hemo-
globin concentration was measured with a portable hemoglo-
binometer (HemoCue, Lagoon Hills, CA) from a drop of
finger stick blood. All other analyses were made in Pediatric
Gastroenterology Laboratory of the Paulista School of Medi-
cine, Federal University of São Paulo using commercial kits
for clinical laboratories. Assays performed were serum iron,
total iron binding capacity (TIBC), transferrin saturation,
zinc, albumin, and total protein. Total plasma cholesterol and
triglycerides were measured using standardized kits that use
enzymatic techniques (Synermed, Quebec, Canada).
17,18
High-density lipoprotein (HDL)-cholesterol was similarly
measured after heparin manganese precipitation of very-low-
density lipoprotein (VLDL) and low-density lipoprotein
(LDL)-cholesterol.
19
LDL-cholesterol was calculated using
the formula of Friedewald and others.
20
Measurement of TNF␣ and immunoglobulins. Free TNF␣
was determined in serum by enzyme-linked-immunosorbent
assay (ELISA), according to the manufacturer’s directions
(R&D Systems, Minneapolis, MN). Specific levels of IgM,
IgA, IgE, IgG1, IgG2, IgG3, and IgG4 against soluble egg
antigen (SEA) from S. mansoni were analyzed by ELISA, as
described
21
with minor modifications. Normal human serum
was obtained from individuals from a non-endemic area of
Brazil with no history of helminth infection. Cut-off values
were calculated using the mean for the endemic normal con-
trols plus 3 SD.
Analytic approach. Statistical tests for intensity of infection
were performed on log-transformed egg counts. Demo-
graphic and socio-economic differences of the study popula-
tion and subsample were compared by ␹
2
tests. ANACOVA
was used to test the association between treatment outcome
and baseline values for nutritional, biochemical, and immu-
nologic indicators. In the final model, these parameters were
adjusted for the confounders sex, age, and socio-economic
status. ANACOVA was selected for this analysis, because
there are both continuous and categorical independent vari-
ables. Student’s t test was used to compare intensities of in-
fection at 6 and 12 months with baseline. A significance level
of 0.05 was used throughout. Statistical calculations were per-
formed with the program SPSS (version 10.0).
RESULTS
Study population. Within the city as a whole the prevalence
of infection among school children was 18.9% and the geo-
metric mean intensity of infection was 109.5 eggs/g of feces
(epg). Because one of the criteria was an epg < 400, 19.3% of
children were excluded from the study on this basis. There
were a total of 661 children between the ages of 7.0 and 17.9
entered in the study. Of these, serum was collected from 106
for measurement of immunologic and biochemical indicators.
Slightly less than one half of the children were male, their
mean age was 11.7 ± 2.7, and one half were considered to live
in inadequate economic and environmental conditions (Table
1). There were no significant differences in sex, age, or socio-
economic status between the total study group and those for
whom biochemical and immunologic studies were performed.
In the first 6 months, 4.4% were lost to follow-up and an
additional 16.2% were lost in the final 6 months of the study.
Similar losses were observed in the subsample. In all, 79.4%
(525) of the total and 77.4% (84) of the subsample had com-
plete parasitological follow-up. The characteristics of those
lost to follow-up for repeat anthropometrics, stool examina-
tion, and/or blood studies did not differ significantly from the
remaining population (data not shown).
Reinfection rates and characteristics of children. Of the to-
tal number of children examined, the prevalence of S. man-
soni infection was 18.9%. All children were infected with S.
mansoni at the start of the study. Six months after treatment,
27.5% (172/625) were still positive for S. mansoni eggs in their
stool (Table 2). All children were treated again at that time
and at 12 months. The proportion of those who were again
egg-positive at the end of the study decreased to 21.1% (113/
525, P < 0.187 by ␹
2
compared with 6 months). The reinfec-
tion rate between 6 and 12 months was 15.6%. This rate was
not significantly different for those included in the subsample.
Pretreatment factors associated with resistance to reinfec-
tion. For the whole sample, 10% were egg positive at both the
TABLE 1
Demographic of study population and subsample
Study Subsample*
PN Percent N Percent
Sex
Male 389 58.9 56 52.8 0.269
Female 272 41.1 50 47.2
Age (years)
7–10 152 23.0 25 23.6 0.852
10–14 388 58.7 64 60.4
14–18 121 18.3 17 16.0
Socio-economic conditions
Adequate† 299 49.0 43 43.0 0.264
Inadequate 311 51.0 57 57.0
* Blood collection group for biochemical and immunoglobulin studies.
† According to a socio-economic and environmental index (see Methods).
SCHISTOSOME CHEMOTHERAPY AND REINFECTION RISK 905
6- and 12-month periods. The initial intensity of infection was
a strong factor in treatment outcome. Those still infected at 6
months had significantly higher mean geometric egg counts
than those who were egg negative (Figure 1). Furthermore,
the initial egg counts of those who became egg-negative after
the second treatment were significantly lower than those per-
sistently positive (Figure 2), but no different than those who
were negative at 6 months. In those persistently positive, the
intensity of infection was not significantly different at any
time period before or after the initial treatment. A relatively
higher initial egg count (in this case > 10 SD), therefore, was
predictive only of those who would not respond despite only
moderate intensities of infection.
Before treatment we measured immunoglobulin class, iso-
type, HDL, triglycerides, VLDL, LDL, cholesterol, total pro-
tein, albumin, globin, and hemoglobin. We evaluated how
each was associated with outcome of treatment at 12 months
in the 84 children with complete data. Variables that most
closely correlate with nutritional status, such as total protein,
albumin, and hemoglobin, did not differ between groups
(Table 3). However, baseline cholesterol and LDL were sig-
nificantly associated with negative stool examinations for S.
mansoni at the end of 1 year (P ס 0.011 and P ס 0.008,
respectively). Higher cholesterol and LDL were associated
with a better outcome at 1 year. Comparing the cholesterol
and LDL levels of those with the best response (negative at 6
months and 1 year) with the worst (positive at 6 months and
1 year), the difference between the cholesterol and LDL mea-
surements remained significant or borderline significant (cho-
lesterol, 150.6 ± 34.9 versus 118.0 ± 118.00; P ס 0.052; LDL:
104.0 ± 32.4 versus 72.0 ± 27.2, P ס 0.045). There were no
differences between those who were positive at only 6 months
or only 12 months. By Pearson’s correlation, there was no
significant correlation between LDL and initial or 6-month
intensity of infection, but LDL was significantly negatively
correlated with intensity of infection at 12 months (r ס −0.30,
P ס 0.029). No differences in socio-economic, anthropomet-
rics, or lipid intake were noted between those persistently
egg-positive and the other groups.
Changes in S. mansoni–specific immunoglobulins and re-
sistance to reinfection. Most immunoglobulin isotypes show
no pre-treatment differences between those who were egg-
negative and those still positive at 6 or 12 months. The change
in immunoglobulin class and IgG isotype between 0 and 12
months was consistent only with the presence of detectable
eggs in the stool (Table 4). Parasite-specific IgG4, IgG3, and
IgM at each time-point were elevated in those who were in-
fected and not in those who were egg negative. A direct mea-
surement of TNF␣ as well as VLDL concentration, an indi-
rect measure of TNF␣ activity,
22,23
failed to show an associa-
tion with cure at any time-point (Table 3).
DISCUSSION
In this study, ∼ 10% of the cohort either failed treatment or
were rapidly reinfected. Considering those that were negative
at 6 months and became positive at 12 months, we know that
the reinfection rate for some of the children is ∼ 16% over a
6-month period. Because the percent of positives at 6 months
was 27–26%, the drug “failure-to-cure” rate was ∼ 10%. This
is expected and only confirms that this group and this treat-
ment were not extreme in any way. There were several find-
ings, however, that suggest that the group that failed to cure
despite two rounds of treatment were not merely unlucky, but
differed significantly in their behavior or their biology. This
TABLE 2
Infection characteristics of study and subsample
Infection characteristics
Study (n ס 512) Subsample* (n ס 84)
Stool egg + Percent Stool egg + Percent
Positive at 6 months 172 27.5 27 26.5
Positive at 12 months 113 21.5 20 23.3
Reinfected at 12 months 57 15.6† 10 16.9†
Persistently infected‡ 51 10.0 8 9.5
* Blood collection group for biochemical and immunoglobulin studies.
† Egg negative at 6 months and positive at 12 months.
‡ Egg positive at 6 and 12 months. Persistently egg negative at 6 and 12 months. Study
group: 308 negative/512 total; subsample: 49 negative/84 total. Between-group comparison
for all infection characteristics, P > 0.05.
FIGURE 1. Informed consent was obtained for all study subjects
before clinical and parasitological study and blood collection. This
study was approved by the Human Investigation Committees of Uni-
versity Hospitals and the Oswaldo Cruz Foundation, Salvador, Bahia,
Brazil. Geometric mean egg counts (epg) according to infection status
at the 6- and 12-month interventions. The study population was ret-
rospectively stratified according to their infection status at 6 and 12
months and the geometric mean epg of each group calculated. Thus,
those infected still infected at 6 months started the study with a
statistically higher epg (77.9) than those who were egg negative (64.4)
at 6 months (P ס 0.023).
FIGURE 2. Geometric mean egg counts (epg) according to infec-
tion status comparing the 12-month intervention only. The study
population was retrospectively stratified according to their infection
status at 12 months and the geometric mean of each group calculated.
Thus, the starting epg of those who would eventually be egg negative
at the end of 12 months (99.8) were well separated from than those
who were egg negative (69.0) at 12 months (P ס 0.020).
REIS AND OTHERS906
group had a significantly higher intensity of infection from all
other groups at the start of the study, at 6 months after one
round of chemotherapy, and at 12 months after two rounds of
chemotherapy. Whereas most infections, even when the mean
intensity of infection is > 1,000 epg, will respond with nearly
a 90% reduction in intensity,
24
there was no significant dif-
ference between the starting intensity and the final intensity
for the persistently positive individuals.
In addition, while all nutritional parameters and markers
including anthropometrics and measurement of macro- and
micronutrients showed no differences between outcome
groups, the lowest mean total cholesterol and LDL were as-
sociated with those who failed to clear their infection or even
reduce its intensity at any time. This effect was essentially
isolated to LDL and was not merely a marker for the ad-
equacy of nutrition. Other and better markers of nutritional
status showed no differences, and the concentrations of other
types of cholesterol were similar for those egg-negative com-
pared with those egg-positive at 1 year. Furthermore, LDL
was not a marker for the initial intensity of infection, the
other factor associated with drug efficacy, because there was
no correlation between these variables. For the ages studied
here, the normal range for LDL cholesterol is 50–170 mg/dL.
No values were above the upper limit, and only two individu-
als were below the lower limit; therefore, the difference be-
tween the groups was not the result of pathologic lipid con-
centrations.
The trivial explanation for persistence in this group would
be that these children consistently failed to take the oxam-
niquine. This is highly unlikely because the medicine was ad-
ministered under direct observation as a suspension and not a
pill. Resistance to oxamniquine might also explain persis-
tence, but true resistance is not common and rarely clinically
relevant at the scale of mass treatment or public health. In the
Robert Toll outbreak in Senegal, “pseudo” resistance was
observed because of the high level of transmission.
4
In this
outbreak, many people probably presented with recent infec-
tions at the time of treatment. Because the parasite is only
susceptible praziquantel at 4–6 weeks after infection, these
individuals seemed not to respond. Furthermore, transmis-
sion was alarmingly intense when the mean infection intensity
was near 1,000 epg and the prevalence was 91%. Thus, rapid
reinfection was probably the rule. Neither of these factors
would seem to have been operative in Jequié. First, the mean
intensity of infection of all of those infected was relatively low
as was the prevalence, which suggests a low rate of transmis-
sion. Second, oxamniquine in contrast to praziquantel seems
to be able to affect immature stages of S. mansoni.
25
Differences in host immune response or host behavior
could also account for differences in apparent drug efficacy.
The only immunologic markers tested (immunoglobulins and
TNF␣) did not show any differences between outcome
groups. Although behavior cannot be entirely ruled out as a
factor in the persistence of the infection, it should be noted
that the prevalence and intensity of infection in this commu-
nity suggests a relatively low level of transmission. True ex-
tremes in behavior such as occupational exposure to contami-
nated waters would be expected to observe such a constant
high level of infection, and there is no direct evidence for such
behavior among children attending school.
While multiple factors were tested, many of them were
correlated or mutually dependent. The problem of multiple
testing is usually addressed by correcting. While it is difficult
to say exactly what value should be used for adjustment, we
estimate that 12 factors were independent (height, weight,
albumin/total protein, LDL, HDL, IgM, IgG1, IgG2, IgG3,
IgG4/IgE, and IgA). Using the Bonferroni correction, P <
0.004 would be considered significant under these conditions.
This correction is known to be very stringent and was taken as
a guideline for significance. The P value obtained for differ-
ences in initial LDL concentration for those who remained
negative compared with those who did not respond or became
reinfected was highly significant before correction (P ס
0.009), and there are multiple biologic mechanisms by which
LDL could be associated with drug efficacy and/or resistance
to reinfection.
Two potential mechanisms by which even normal LDL lev-
els might affect drug efficacy are by LDL’s influence on im-
mune responses and by modification of drug metabolism and
delivery. The immune response is an important component of
the mechanism of action for praziquantel.
26–29
Immunosup-
pression is associated with decreased efficacy of oxamniquine
TABLE 4
Immunoglobulins at 12 months post-treatment
Immunoglobulin
class/isotype
Egg negative
(n ס 52)
Egg positive
(n ס 14) P* P†‡
IgM 0.45 ± 0.32 0.62 ± 0.25 0.070 0.013
IgA 0.06 ± 0.063 0.05 ± 0.03 0.416 0.532
IgE 0.010 ± 0.020 0.01 ± 0.02 0.862 0.935
IgG1 0.61 ± 0.61 0.88 ± 0.65 0.231 0.257
IgG2 0.01 ± 0.04 0.09 ± 0.01 0.423 0.386
IgG3 0.05 ± 0.01 0.05 ± 0.05 0.951 0.780
IgG4 0.07 ± 0.15 0.38 ± 0.35 < 0.001 < 0.001
Values are mean ± SD.
* Unadjusted P value.
† P value adjusted for sex, age, and socio-economic level.
‡ No differences were observed between the groups at the start of the study.
TABLE 3
Mean pre-treatment values for egg negative and egg positive individuals at 12 months
Egg negative Egg positive
P* P†Mean ± SD n Mean ± SD n
Total protein (g/dL) 7.39 ± 0.64 45† 7.38 ± 0.63 8 0.996 0.848
Albumin (g/dL) 4.38 ± 0.57 45 4.17 ± 0.48 8 0.348 0.295
Total cholesterol (mg/dL) 156.31 ± 41.50 49 123.90 ± 21.52 9 0.027 0.011
LDL (mg/dL) 108.05 ± 41.44 41 72.89 ± 23.68 9 0.018 0.008
VLDL (mg/dL) 17.55 ± 9.08 49 19.33 ± 12.81 9 0.614 0.374
TNF␣§ (mg/dL) 8.34 ± 2.85 62 7.86 ± 2.60 16 0.546 0.763
* Unadjusted P values.
† P values adjusted for sex, age, and socio-economic status.
‡ Measurements of some variables was limited by quantities of serum available.
§ No differences were observed for immunoglobulin isotypes, globulin, hemoglobulin, HDL, or triglycerides, and anthropometric status.
SCHISTOSOME CHEMOTHERAPY AND REINFECTION RISK 907
in experimental mice, but reconstitution of these animals with
immune serum fully restores effectiveness. While much has
been written about the deleterious effect of immune re-
sponses to LDL-cholesterol,
30
the pro-inflammatory proper-
ties of this form of lipid might be responsible for the effects
we see here by altering the immune response to interluminal
tissues including those of the parasite.
Because LDL is a major carrier of lipid-soluble or lipid-
associated drugs, a second major possibility is that LDL levels
correlate with the availability of this carrier in the blood
stream. Oxamniquine is a weak amphipathic base that incor-
porates into liposomes with high efficiency (> 85%),
31
and the
liposomal preparation of the drug has a higher experimental
efficacy than the unmodified form. The incorporation of the
drug into liposomes or its association with LDL may protect
it from metabolism and earlier elimination leading to higher
levels and or a longer half-life. Lipid-associated drug may also
tend to be targeted to the liver or parasite membranes.
Because schistosomes are completely dependent on the
host for cholesterol and fatty acids, they require a transport
system for lipids. Multiple studies have shown the ability of
parasite membranes to bind host LDL.
32,33
This is mediated
at least in part by an abundant and efficient VLDL/LDL-
binding protein.
34
The requirement for host lipids obligates
transport of the drug as well. This may explain a mechanism
by which the lipid status of the host influences this process.
For trypanosomes, a lipid-associated protein, apolipoprotein-
I, is responsible for parasite lysis.
35
It is possible that some
component of the human lipid profile is also responsible for
natural resistance to schistosomes or cooperates with oxam-
niquine in parasite killing. Such a component might be low,
defective, or less efficient in those with lower LDL concen-
tration.
Ancestry, diet, and metabolic diseases, such as diabetes
mellitus, are the major determinants of lipid status in this
population and this age group. Severely malnourished chil-
dren were excluded from the study, and no differences in
socio-economic status, anthropometrics, or lipid intake were
noted between those persistently egg-positive and the other
groups, so that diet is unlikely to explain differences in LDL.
Diabetes in this age group in Brazil is very uncommon. The
differences in LDL concentrations we observed here are most
likely do to intrinsic biologic differences, perhaps genetics.
Several authors have correlated susceptibility to reinfection
or response to chemotherapy to pre-treatment, parasite-
specific antibody levels.
36
In this study, however, immuno-
globulin levels were clearly correlated with actual infection
status and were not predictive of response. Thus, IgM and
IgG4 serve as markers of ongoing infection but do not par-
ticipate in resistance or efficacy of drug treatment.
Received August 10, 2005. Accepted for publication April 12, 2006.
Financial support: This work was supported by a grant from the
Nestle Foundation, CNPq (Brazilian National Research Counsel),
and Pronex (Centers of Excellence Program–Brazilian Ministry of
Science and Technology).
Authors’ addresses: Eliana A. G. Reis, E-mail: ereis@cpqgm
.fiocruz.br. Mitermayer G. Reis, E-mail: [email protected].
Rita de Ca´ssia R. Silva, E-mail: [email protected]. Theomira M. A.
Carmo, Oswaldo Cruz Foundation, Fiocruz, Rua Waldemar Falca˜o,
121 Brotas, Salvador, Bahia, Brazil, CEP–40296-710. Telephone: 71-
3176-2205, Fax: 71-3176-2289. E-mail: [email protected]. Ana
Marlu´cia O. Assis, E-mail: [email protected]. Maurı´cio L. Barreto, In-
stitute of Collective Health, Federal University of Bahia, Rua Basilio
da Gama s/N°, Canela, Salvador-Bahia, Brasil Salvador, Bahia, Brazil
40-110-170. Telephone: 71-3263-7445, Fax: 71-3263-7460, E-mail:
[email protected]. Isabel M. Parraga, Department of Nutrition,
Case Western Reserve University, 10900 Euclid Avenue, Cleveland,
Ohio 44106-4906. Telephone: 216-368-6626, Fax: 216-368-6644,
E-mail: [email protected]. Moˆnica Leila P. Santana, School of
Nutrition, Federal University of Bahia, Rua Padre Feijo´ , 29/4° andar,
Canela, Salvador, Bahia, Brazip CEP–40.110-170. Telephone: 71-
3245-0544, Fax: 71-3237-5856, E-mail: [email protected]. Ronald E.
Blanton, Center for Global Health and Diseases, Case Western Re-
serve University, 2103 Cornell Road, 4th floor, Wolstein Research
Building, Cleveland, Ohio 44106-7286. Telephone: 216-368-4814, Fax:
216-368-4825, E-mail: [email protected].
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SCHISTOSOME CHEMOTHERAPY AND REINFECTION RISK 909
Page 1 of 28
Accepted Manuscript
1
Potential Immunomodulatory Effects of Plant Lectins in Schistosoma mansoni1
Infection. 2
3
Eliana A.G. Reis
1
; Daniel A. Athanazio
1
; Benildo Sousa Cavada
2
; Edson Holanda Teixeira
3
, 4
Vicente de Paulo Teixeira Pinto
3
; Theomira M.A. Carmo
1
; Alice Reis
1
; Graziela Trocolli
1
; Julio 5
Croda
1
; Donald Harn
4
; Manoel Barral-Netto
1
and Mitermayer G. Reis
1*
.6
7
1
Fundação Oswaldo Cruz (FIOCRUZ), Bahia, Brazil.8
2
Departamento de Bioquímica e Biologia Molecular da Universidade Federal do Ceará, Brazil.9
3
Faculdade de Medicina, UFC, Sobral-CE10
4
Department of Immunology and Infectious Diseases, Harvard School of Public Health, 665 11
Huntington Avenue, Boston, MA 02115, USA 12
13
14
15
*Corresponding author: Tel: (71) 3176-2200 Fax: (71) 3176-2326.16
E-mail address: [email protected] 17
Fundação Oswaldo Cruz - FIOCRUZ18
Rua Waldemar Falcão, 121, Candeal. CEP: 40296-710 - Salvador - Bahia - Brasil19
20
21
* 3. Manuscript
Page 2 of 28
Accepted Manuscript
2
Abbreviations used in this paper: SEA, S. mansoni soluble egg antigen; Ag, antigen; PBMC, 22
peripheral blood mononuclear cells; Th, T helper cell; IL, Interleukin; FCS, fetal calf serum; C-23
RPMI, complete medium; ELISA, enzyme linked immunosorbent assay.24
Page 3 of 28
Accepted Manuscript
3
25
ABSTRACT26
Lectins are sugar-binding glycoproteins that can stimulate, in a non antigen-specific fashion, 27
lymphocytes, leading to proliferation and cytokine production. Some lectins are utilized as in vitro28
mitogenic lymphocyte stimulators and their use as immunomodulators against infectious diseases29
has been evaluated experimentally. In the experimental murine model, the immune response to 30
schistosomiasis is Th1-like during the initial stage of infection, with a shift towards a Th2-like31
response after oviposition. We report the response of schistosomiasis patients´ (n= 37) peripheral 32
blood mononuclear cells (PBMC) to stimulation by lectins, including newly-isolated lectins from 33
Brazilian flora, and by Schistosoma mansoni soluble egg antigens (SEA). Cytokine production upon 34
lectin stimulation ex vivo was assessed in PBMC supernatants, collected at 24 and 72 hours, by 35
sandwich ELISA to IL-5, IL-10, TNF-α and IFN-. In PBMC from infected patients all but one of 36
the lectins induced a Th2-like cytokine response, characterized by elevated IL-5 production that was37
higher than that induced by SEA stimulation alone. Our results show that the Th2 environment 38
present during schistosomiasis is not affected and that it may be further stimulated by the presence 39
of lectins.40
41
42
Key Words: Lectins, Schistosoma mansoni, Cytokines 43
Page 4 of 28
Accepted Manuscript
4
INTRODUCTION44
45
Schistosomiasis is a tropical human disease with 200 million people infected worldwide and almost 46
600 million at risk (W.H.O, 2006). The infection, caused by the trematode Schistosoma mansoni, is 47
related to poverty and poor sanitation in contexts where people are exposed to snail infested water. 48
The most severe human pathology follows granulomatous inflammation of the liver portal tracts 49
leading to hepatic fibrosis, portal hypertension and related complications (Andrade, 2004). Late 50
stage human infection is characterized by a shift from potentially protective Th1-like immune 51
responses to Th2-like responses (Pearce and MacDonald, 2002). There is strong evidence from 52
animal models to support a role for Th2 cytokines in the severity of liver granulomatous 53
inflammation and fibrosis (Cheever et al., 2002, Farah et al., 2000, Reiman et al., 2006). The role of 54
Th2 cytokines in human disease is not so clear : peripheral blood mononuclear cell (PBMC) 55
responses with high production tumor necrosis factor (TNF-) and gamma-interferon (IFN-) 56
have been associated with the hepatosplenic form (Mwatha et al., 1998) while production of IL-13, 57
IL-4, IL-5 and IL-10 were related to progressive liver disease by others (de Jesus et al., 2004, 58
Montenegro et al., 1999, Alves Oliveira et al., 2006). 59
60
Glycan binding proteins (lectins) are a structurally heterogeneous group of reversible carbohydrate61
binding proteins that are ubiquitous in animals, plants and microorganisms (Van Damme et al., 62
1998, Gabius and Gabius, 1997, Cavada et al., 2001). Some plant lectins share the ability to act as 63
lymphocyte mitogens and enhance protein-specific immune responses, such as Concanavalin A 64
(ConA) from Jackbean (Canavalia ensiform). ConA has long been used in immunological studies 65
and recently additional plant lectins have been characterized as potent stimulators of immune 66
Page 5 of 28
Accepted Manuscript
5
responses. For instance, ConBr (from C. brasliensis) activates lymphocytes in vivo (Barbosa et al., 67
2001) and reduces the size of lesions caused by leishmaniasis in BALB/c mice. Although this lectin 68
is a potent stimulator of IFN- production, the protective mechanism seems to be independent of 69
this cytokine (Barral-Netto et al., 1996). Recently, ConBr and another two lectins were evaluated as 70
Th1-stimulating adjuvants for specific leishmanial antigen immunization and all three proved to be 71
potent stimulators of IFN-. ConBr resulted in lower parasite numbers and a higher inflammatory 72
response in the air pouch model while KM+ (from Arcocarpus integrifolia) was associated with 73
lower parasite counts and reduced lesions after challenge (Teixeira et al., 2006). Thus, although 74
different plant lectins may yield diverse results as in vivo Th1 driving immunostimulants, these 75
observations highlight the potential use of these molecules in conditions in which Th2 polarization 76
are implicated in disease. Importantly, plant lectins with highly conserved DNA sequences and 77
similar binding properties can exhibit distinct biologic effects in different models (Cavada et al., 78
2001).79
80
The general concept that hepatosplenic schistosomiasis is associated with Th2 response related 81
immunopathology supports the evaluation of Th1-like response stimulants in order to control or 82
revert chronic liver involvement. In this regard, vaccines combining egg antigens and IL-12 resulted 83
in less granuloma formation and reduced tissue fibrosis in BL/6 mice (Sher et al., 1996, Wynn et al., 84
1995). In contrast, immunization with egg antigen and Freund’s adjuvant, in the same mouse strain,85
resulted in earlier death and larger granulomas in survivors (Rutitzky et al., 2001). In the present 86
work, we report on the effect of lectins, isolated from Brazilian flora, in cytokine production by 87
PBMC of individuals from an endemic area with and without active S. mansoni infection. Our 88
objective was to evaluate the potential immunomodulatory effects of plant lectins from Brazilian 89
flora on S. mansoni infection.90
Page 6 of 28
Accepted Manuscript
6
91
92
MATERIALS AND METHODS93
94
Subjects95
The individuals included in this study were resident in a schistosomiasis endemic area (Itaquara, 96
Bahia, Brazil). We evaluated 37 individuals aged from 12 to 55 years old with a history of treatment 97
for schistosomiasis. The infection load was determined by quantitative parasitological stool 98
examinations and an estimation of egg number was performed using the modified Kato-Katz 99
method (Katz et al., 1972). Three stool samples from each patient were collected on consecutive 100
days and the geometric mean number of eggs per gram of stool (EPG) was calculated for each 101
individual. All volunteers were informed as to the nature of the research and the samples were 102
collected with the written consent of the subjects, according to a protocol approved by the Human 103
Ethical Committee of the Oswaldo Cruz Foundation. All patients were subsequently treated for 104
schistosomiasis and other parasitic infections.105
106
PBMC Separation and Cytokine Assay107
Blood (20 ml) was collected from each individual in heparinized Vacutainer tubes (Sodium heparin 108
BD). PBMC were isolated by centrifugation on a Ficoll-paque gradient from Pharmacia Biotech 109
(Piscataway, NJ, USA) at 400 X g, 30 min, at 15
0
C. The cells were washed in RPMI 1640 or HBSS 110
(Hank's Balanced Salt Solution) Ca
2+
Mg
2+
free, and 2-3 x 10
6
/ ml were resuspended in RPMI 111
1640 supplemented with 5% normal human serum AB, (Sigma-Aldrich), 1% sodium pyruvate, 100 112
Page 7 of 28
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7
U/ml penicillin and 100 g/ml streptomycin (pen-strep), L-glutamine (4mM), (GIBCO, Grand 113
Island, NY ) with or without 10 g/ml of SEA, 5 g/ml of each lectins, in 24 well/plates (Costar). 114
The cells were incubated at 37
0
C in 5% CO
2
. The supernatants were collected at 24 h for IL-10 and 115
TNF- , and at 72 h for IL-5 and IFN- cytokine production. 116
117
Antigens and Lectins.118
Soluble egg antigen (SEA) from S. mansoni was prepared as described elsewhere (Harn et al., 119
1989). The lectins employed here were derived from the seeds of Brazilian plants belonging to the 120
Diocleinae subtribe (like ConA) and including three genera not used as a food source, Canavalia, 121
Cratylia and Dioclea, as previously described (Dam et al., 1998). Each lectin was isolated 122
according to previously reported procedures (as indicated below). All these lectins have a very close 123
structural identity and share primary carbohydrate recognition specificity for D-mannose and D-124
glucose and stimulate human lymphocytes in vitro. ConA was used as a control for lymphocyte125
activation. The plant lectins used in this study were: Cfl from Cratylia floribunda (Oliveira et al., 126
1991), ConBr from C. braziliensis (Moreira and Cavada, 1984), Dgl from D. grandiflora, (Moreira 127
et al., 1996), Dguil from D. guianensis (Vasconselos et al., 1991), Dros from D. rostrata (Cavada 128
et al., 1996a), Dviol from D. violacea (Moreira et al., 1996), DvirL from D. virgata (Cavada et al., 129
1996b).130
131
ELISA Assay132
Cytokine levels were measured at 24 and 72h in culture supernatants according to the 133
manufacturer’s instructions. TNF- from Duo-set kits (R&D System); IFN- from Genzyme 134
Page 8 of 28
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8
(Cambridge, Mass.) and IL-5 and IL-10 were purchased from (PharMingen San Diego CA). 135
Briefly, polystyrene 96 well EIA/RIA plates 3590 from Corning Costar Corp. (NY, USA) were 136
coated with specific mAb, blocked, and incubated with culture supernatants and recombinant 137
cytokines in 96 well/plates, washed then incubated with biotinylated anti-human IgG antibodies. 138
Bound antibodies were detected using an avidin-peroxidase system, and the colored product was 139
measured at 450 nm in a UV Sofmax automated plate reader and each assay was performed in 140
triplicate. 141
142
Statistical analysis143
All data in this paper are expressed as a mean ± SEM unless otherwise described. To compare 144
levels of cytokine production in different groups, the Kruskal-Wallis tested was used for non-145
parametric analysis. Correlation coefficients between variables were determined by Spearmann rank 146
correlation. Differences with a p < 0.05 were considered significant. The software used in the 147
present work was GraphPad Prism 4.0 (GraphPad Software, San Diego, CA, EUA).148
Page 9 of 28
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9
RESULTS149
150
Demographic and parasitilogic characteristics of subjects151
152
Subjects were initially separated in two groups: positive for S. mansoni eggs (n = 24; 65%) or 153
negative for schistosomal eggs (n = 13; 35%) in their stools. The mean and standard deviation for 154
age in egg positive and negative groups were, respectively, 20.3 ± 10.4 and 37.0 ± 11.1 years (P < 155
0.001). Males represented 13/24 (54%) and 7/13 (54%) among the egg positive and negative 156
groups, respectively. The mean EPG of feces was 691 ± 959 in the egg positive group. Stratification 157
by infection load resulted in the following groups: < 100 eggs (n = 6; 25%), 100-400 eggs (n = 6; 158
25%) and >400 eggs (n = 12; 50%). Out of 30 patients, where the stools were also evaluated for 159
other parasites, all but one (in the S. mansoni egg positive group) were infected with other 160
intestinal helminthes, the most common being Ascaris lumbricoides (n = 17; 57% of all subjects) 161
and Trichiurus trichiura (n = 13; 43% of all subjects). A triple infection by S. mansoni, A. 162
lumbricoides and T. trichiura was observed in seven patients, the S. mansoni egg negative group did 163
not contain individuals infected with both A. lumbricoides and T. trichiura. 164
165
Influence of Schistosoma infection on immune response to lectins from Brazilian flora. 166
167
As shown in figure 1A, there was no difference in IL-5 production between unstimulated PBMC 168
and those stimulated by Cfl or SEA. Stimulation with Cfl or SEA resulted in a modest IL-5 169
production, not statistically significant, from unstimulated cultures. In contrast, significant higher 170
Page 10 of 28
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10
cytokine levels for the other seven lectins tested were detected in egg positive patients (P <0.05). 171
While there was no difference upon SEA stimulation between egg positive and negative individuals, 172
seven lectins induced higher IL-5 responses when compared to SEA stimulation: ConA (5.7x , 173
P<0.001), ConBr (6.0x, P<0.001), Dgl (7.3x , P<0.001 ), Dguil (4.5x , P < 0.001), Dros (4.7x , P < 174
0.01), Dviol (4.9 , P < 0.05) and DvirL (5.6x , P < 0.001).175
176
In contrast to the results of IL-5, there was a trend towards higher IFN-γ production in egg negative 177
individuals albeit those differences between egg-positive and egg-negative individuals were not 178
statistically significant (figure 1B). The evaluation of IL-5/ IFN-γ ratio confirmed higher values for 179
the seven lectins (ConA, ConBr, Dgl, Dguil, Dros, Dviol and DvirL) among egg positive patients 180
(figure 1E).181
182
With regard to IL-10 production, SEA induced higher cytokine production compared to all other 183
stimulants in both egg positive and negative groups. IL-10 production by PBMC did not differ 184
according to the presence of S. mansoni eggs (figure 1C). 185
186
As shown in figure 1D, the highest levels of TNF- were obtained following SEA stimulation. 187
PBMC from egg positive patients exhibited lower TNF- production upon SEA stimulation when 188
compared to the egg negative individuals (P < 0.05). There were no differences in TNF-189
production following stimulation by any of the lectins. When the TNF- response was compared 190
among the stimulants, SEA induced higher levels compared to all lectins except for ConA, both in 191
egg positive and negative groups. 192
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11
193
Influence of Schistosoma infection load on IL-5 production194
195
The relationship between parasite load and IL-5 production was studied. As shown in figure 2A, 196
individuals infected with 1-100 eggs exhibited higher IL-5 levels for all stimulants except SEA, 197
when compared to egg negative individuals. In contrast, there was trend in the group infected with 198
>400 eggs towards lower IL-5 production and, in these individuals, there was no significant 199
difference upon stimulation by Dguil, Dros, Dviol and SEA among egg positive and negative 200
individuals. As shown in figure 2B, no particular trend or significant difference was observed 201
regarding IFN- production. 202
203
All stimulants exhibited a trend towards a negative correlation between infection load and IL-5 204
production. The strongest (and only significant) negative trend correlation was observed with the 205
Dgl lectin (figure 3, Spearmann coefficient = - 0.4351, P < 0.05).206
207
Influence of gender and age on cytokine production208
209
In the group of individuals with active S. mansoni infection, males had a mean egg count similar to 210
females (548 ± 177 vs 860 ± 378) and were in the same age range (21.7 ± 3.7 vs 18.5 ± 1.7 years). 211
There was a trend among males towards a higher IL-5 production in egg positive individuals. The 212
difference between males and females was significant upon ConA (P < 0.05) and ConBr (P < 0.05) 213
Page 12 of 28
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12
stimulation (figure 4A). Conversely, there was a trend among males towards lower IFN- levels 214
although the difference was not significant for any one lectin (figure 4B). 215
216
Gender was not relevant in the production of other cytokines, nor was there a significant correlation 217
between age and cytokine production among the different groups and stimuli used. We did not 218
observe any influence of the helminth co-infections in cytokine production upon stimulation albeit 219
that this analysis was limited due to the low number of subgroups and non-infected individuals 220
(data not shown). 221
222
Influence of other worm infections on IL-5 production223
224
As all but one individual enrolled in this study exhibited at least one geohelminth infection, we 225
were not able to study a negative control group for all parasites in endemic area. We investigated 226
the potential influence of other helminthiasis by comparing individuals with any other helminths 227
with and without concurrent S. mansoni infection, individuals with Ascaris lumbricoides infection 228
with and without detectable S. mansoni eggs, and individuals with double or triple infections 229
including S. mansoni. No changes in cytokine response were observed when stratifications for other 230
helminthiasis were performed. As seen in figure 5, most features described above for IL-5 231
production were maintained. When considering individuals with one helminth other than S. 232
mansoni, IL-5 production persisted higher in S. mansoni infected subjects for all tested stimulants 233
except for SEA (figure 5A). When considering individuals with A. lumbricoides infection and no 234
other non S. mansoni parasite, the trend towards higher IL-5 persisted (even if only seven patients 235
Page 13 of 28
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13
remained as the egg positive group) but significance were reached only for ConBr and Dvirl (figure 236
5B). Moreover, the presence of double or triple infection in S. mansoni infected individuals did not 237
affect cytokine production upon any stimulation (figure 5C).238
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14
DISCUSSION239
240
In this study, subjects from an endemic area exhibited a high prevalence of intestinal parasites and, 241
in the case of S. mansoni infection, a higher egg count was observed. Individuals with detectable S. 242
mansoni eggs were younger, which is in accordance with the established higher prevalence of 243
severe disease in adolescents and young adults. As expected, individuals with active S. mansoni244
infection exhibited lower TNF- and IFN-γ responses to SEA, but the difference for the latter was 245
not significant. Although the difference in IL5 production was not significant among egg positive 246
and negative groups upon SEA stimulation, it was clear that activation by the lectins induced high 247
IL-5 levels. SEA was a potent stimulator of IL-10 production regardless of an individuals S. 248
mansoni egg status while lectins induced lower responses. Previous studies using PBMC from 249
subjects resident in endemic areas from the state of Bahia, Brazil (de Jesus et al., 2004), and other 250
areas (Silveira et al., 2004) confirmed the immunomodulatory properties of SEA on Th1-cytokine 251
downregulation and upregulation of IL-5 and IL-10. As all negative individuals enrolled in our 252
study had a previous history of treated schistosomiasis, the similar patterns of IL-5 and IL-10 253
production upon SEA stimulation in egg positive and negative groups probably reflects a memory 254
response to SEA. On the other hand, active S. mansoni infection appeared to shift the immune 255
response to an unspecific Th2 pattern of cytokine production which could be further induced by 256
lymphocyte stimulants. The active infection includes the exposure to egg and adult worm antigens 257
and their immunomodulatory effects may induce a Th2 polarizing state that is not observed in 258
individuals with past infection.259
260
Strikingly, IL-5 production was clearly higher in egg positive individuals but, in this group, there 261
was a negative correlation between egg counts and IL-5 levels. Recent findings from other endemic 262
Page 15 of 28
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15
areas in Brazil report a positive correlation between egg counts and IL-5 levels (Corrêa-Oliveira, 263
manuscript in preparation). There is no clear explanation for this discrepancy. Previously, we 264
showed that an egg sugar, Lacto-N-fucopentaose III (LNFPIII), stimulates PBMC regardless of the 265
infection load, although higher IL-10 responses were related to lower egg counts (Velupillai et al., 266
2000). Egg sugars are targets of lectin binding (Zelck and Becker, 1990, Yoshino et al., 1977) but it 267
is not clear whether lectin activation and the Th2 response induction share similar mechanisms with 268
that of immunomodulation by egg carbohydrates. 269
270
In conclusion, we studied the potential role of lectins from Brazilian flora in order to evaluate 271
whether they could polarize PBMC towards a Th1-like cytokine response. Our results indicate that 272
the lectins tested were not able to change the Th2 shift typical of a S. mansoni infection. This 273
precludes their use as potential adjuvants or immunomodulatory agents in experimental models of 274
immunization or treatment, in contrast to the beneficial effects seen in experimental leishmaniasis. 275
276
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16
277
ACKNOWLEDGEMENTS278
279
This work was supported by Grant AI0546484 from the National Institutes of Health NIH-USA, 280
CNPq 350052/95-6 (NV) and FINEP/PRONEX 4196086200. B.S. Cavada, M. Barral Netto and M.G. 281
Reis are senior investigators of CNPq (Brazil).282
283
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17
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380
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Legends381
382
Figure 1. Cytokine production in PBMCs after stimulation with soluble egg antigen (SEA) or 383
lectins. Unst (unstimulated cell cutures). Results are expressed as mean ± SEM. Groups were 384
separated according to presence or absence of S. mansoni eggs in their stools: “egg pos” – egg 385
positive individuals; “egg neg” – egg negative. Asterix indicates statistical difference between 386
groups.387
388
Figure 2. IL-5 (A) and IFN- (B) production in PBMCs in groups stratified by number of S. 389
mansoni eggs per gram of feces. Unst (unstimulated cell cutures). Results are expressed as mean ± 390
SEM.391
392
Figure 3. Negative correlation between IL-5 production and egg count upon Dgl stimulation. 393
394
Figure 4. Effect of gender on IL-5 and IFNγ production. Unst (unstimulated cell cutures). Results 395
are expressed as mean ± SEM. Mal (males), Fem (females, pos (presence of S.mansoni eggs in their 396
stools), neg (negative for S.mansoni eggs). Asterix indicates statistical difference between groups.397
398
Figure 5. Effect of other worm infections on the IL-5 production stimulated by soluble egg antigen 399
(SEA) or lectins. (A) Comparison between cases of S. mansoni infection associated with one other 400
worm (double infection: “DI with Schisto”) with cases of monoinfection by any helminth other than 401
S. mansoni (monoinfection: “MI without Schisto”). (B) Comparison between cases of Ascaris402
lumbricoides monoinfection with double infections by A. lumbricoides and S. mansoni. (C) 403
Page 23 of 28
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23
Comparison between cases of S. mansoni infection associated with one other worm (“DI with 404
Schisto”) with cases of triple infection by S. mansoni and two other helminths (“TI with Schisto”).405
406
Page 24 of 28
Accepted Manuscript
Unst Cfl ConA Conbr Dgl Dguil Dros Dviol Dvirl SEA
0
100
200
300
400
500
600
*
*
*
*
*
*
*
A
IL-5 (pg/ml)
Unst Cfl ConA Conbr Dgl Dguil Dros Dviol Dvirl SEA
0
300
600
900
1200
B
Egg pos
Egg neg
IFN-
(pg/ml)
Unst Cfl ConA Conbr Dg l Dguil Dros Dviol Dvirl SEA
0
1000
2000
3000
4000
5000
C
IL-10 (pg/ml)
Unst Cfl ConA Conbr Dgl Dguil Dros Dviol Dvirl SEA
0
1000
2000
3000
4000
5000
*
D
TNF-
(pg/ml)
ConA Conbr Dgl Dguil Dros Dv iol Dv irl SEA
0
1
2
3
4
E
*
*
*
*
*
*
*
IL-5 / IFN-
ratio
5. Figure(s)
Page 25 of 28
Accepted Manuscript
Unst Cfl ConA Conbr Dgl Dguil Dros Dviol Dvirl SEA
0
250
500
750
1000
< 100 eggs
100-400 eggs
> 400 eggs
Egg neg
IL-5 (pg/ml)
Unst Cfl ConA Conbr Dgl Dgvil Drl Dvl Dvirl SEA
0
500
1000
1500
2000
IFN-
(pg/ml)
5. Figure(s)
Page 26 of 28
Accepted Manuscript
0 1500 3000 4500
0
500
1000
1500
Egg count
IL-5 (pg/ml)
5. Figure(s)
Page 27 of 28
Accepted Manuscript
Unst Cfl ConA Conbr Dgl Dguil Dros Dviol Dvirl SEA
0
1000
2000
3000
B
IFN-
(pg/ml)
Unst Cfl ConA Conbr Dgl Dguil Dros Dviol Dvirl SEA
0
100
200
300
400
500
600
Fem pos
Mal pos
Fem neg
Mal neg
*
*
A
IL-5 (pg/ml)
5. Figure(s)
Page 28 of 28
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5. Figure(s)
42
ANEXO B - MANUSCRITOS RELACIONADOS COM OUTRAS DOENÇAS:
Manuscritos publicados durante o doutorado relacionado a outras doenças.
42
SHORT REPORT
Genetic ancestry and income are associated with
dengue hemorrhagic fever in a highly admixed
population
Ronald E Blanton*
,1
, Luciano K Silva
2
, Vanessa G Morato
3
, Antonio R Parrado
4
,
Juarez P Dias
5
, Paulo RS Melo
2
, Eliana AG Reis
2
, Katrina AB Goddard
4
,Ma
´
rcio RT Nunes
6
,
Sueli G Rodrigues
6
, Pedro FC Vasconcelos
6
, Jesuina M Castro
5
, Mitermayer G Reis
2
,
Maurı
´
cio L Barreto
3
and M Glo
´
ria Teixeira
3
1
Case Western Reserve University, Center for Global Health and Diseases, Cleveland, OH, USA;
2
Fundac¸a
˜
o Oswaldo
Cruz, Centro de Pesquisas Gonc¸alo Moniz, Laboratory of Pathology and Molecular Biology, Rua Waldemar Falca
˜
o,
121, Salvador, Bahia, Brazil;
3
Universidade Federal da Bahia, Institute of Collective Health, Salvador, Bahia, Brazil;
4
Case Western Reserve University, Division of Genetic Epidemiology, Department of Epidemiology & Biostatistics,
Wolstein Research Building, Cleveland, OH, USA;
5
Secretaria da Sau
´
de do Estado da Bahia, 4a Avenida do CAB, no
400, Lado B, CAB, Salvador, Bahia, Brazil;
6
Instituto Evandro Chagas, Department of Arbovirology and Hemorrhagic
Fever, Bele
´
m, Para
´
, Brazil
To test whether African ancestry is protective for severe dengue, we genotyped 49 hospitalized cases of
dengue hemorrhagic fever (DHF) as well as 293 neighborhood cases of dengue fever and 294
asymptomatic controls in Salvador, Bahia, Brazil. Ancestry-informative markers and 282 unlinked SNPs not
associated with the clinical presentation of dengue were used to estimate ancestry. After controlling for
income, both self-defined Afro-Brazilian ethnicity and African ancestry were protective for DHF (P ¼ 0.02,
OR ¼ 0.28 and P ¼ 0.02, OR ¼ 0.13, respectively). Income or an index of income indicators, however, was
also independently associated with the diagnosis of DHF.
European Journal of Human Genetics advance online publication, 13 February 2008; doi:10.1038/ejhg.2008.4
Keywords: ethnicity; race; population structure; socioeconomic status; DENV3
Introduction
The epidemiology of dengue hemorrhagic fever (DHF)
suggests that some ethnically defined populations differ in
their susceptibility. In the Cuban dengue epidemics of
1981 and 2001, African ancestry appeared to be relatively
protective for DHF and dengue shock syndrome, but not
for classic dengue fever (DF).
1
In Haiti, from 1994 to 1999,
no cases of DHF were reported despite intense circulation
of three serotypes,
2
and although dengue virus circulates
sporadically in Africa, there have been no reported
epidemics of DHF.
3
Socioeconomic factors also may
influence the severity of dengue. In Asia, ethnic Chinese
in Singapore show a higher attack rate for DHF compared
to ethnic Malays and Asian Indians.
4
In Trinidad, however,
it was the Pakistani/Asian Indian population that appeared
to disproportionately suffer severe disease.
5
In each case,
the economically more advantaged group was more
affected. As income and ethnic background are inter-
related, we tested these associations using self-declared
Received 24 April 2007; revised 15 December 2007; accepted 6 January
2008
*Correspondence: Dr R Blanton, Center for Global Health and Disease,
Case University, Wolstein Research Building, 2103 Cornell Road, Cleveland,
OH 44106-7286, USA.
Tel: þ 1 216 368 4814; Fax: þ 1 216 368 4825;
E-mail: [email protected]
European Journal of Human Genetics (2008), 1–4
&
2008 Nature Publishing Group All rights reserved 1018-4813/08
$30.00
www.nature.com/ejhg
11
11
1Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 102(8): 000-000, December 2007
The dynamics of dengue virus serotype 3 introduction and
dispersion in the state of Bahia, Brazil
Paulo Roberto Santana de Melo/*, Eliana Almeida Gomes Reis, Isolina Allen Ciuffo**,
Mônica Góes, Ronald Edward Blanton***, Mitermayer Galvão dos Reis/
+
Centro de Pesquisa Gonçalo Moniz-Fiocruz, R. Valdemar Falcão 121, 40295-001 Salvador, BA, Brasil *Universidade Estadual de
Santa Cruz, BA, Brasil **Laboratório Central do Estado da Bahia, Salvador, BA, Brasil ***Case University, Cleveland, OH, USA
By 2002, dengue virus serotype 1 (DENV-1) and DENV-2 had circulated for more than a decade in Brazil. In
2002, the introduction of DENV-3 in the state of Bahia produced a massive epidemic and the first cases of
dengue hemorrhagic fever. Based on the standardized frequency, timing and location of viral isolations by the
state’s Central Laboratory, DENV-3 probably entered Bahia through its capital, Salvador, and then rapidly
disseminated to other cities, following the main roads. A linear regression model that included traffic flow,
distance from the capital and DENV-1 circulation (r
2
= 0.24, p = 0.001) supported this hypothesis. This pattern
was not seen for serotypes already in circulation and was not seen for DENV-3 in the following year. Human
population density was another important factor in the intensity of viral circulation. Neither DENV-1 nor DENV-
2 fit this model for 2001 or 2003. Since the vector has limited flight range and vector densities fail to correlate
with intensity of viral circulation, this distribution represents the movement of infected people and to some
extent mosquitoes. This pattern may mimic person-to-person spread of a new infection.
Keywords: dengue - emerging diseases - surveillance - Aedes aegypti - public health - modeling
Dengue viruses are mosquito-borne RNA viruses of
the Flaviviridae family. The four closely related sero-
types (DENV-1-4) are thought to represent at least three
independent introductions into human populations from
sylvatic primates, the most recent (DENV-1) occurring
within the last century (Wang et al. 2000). Dengue is
one of the most important re-emergent infectious dis-
eases and one of the most serious health problems af-
fecting tropical and subtropical regions of the Americas
(McBride & Bielefeldt-Ohmann 2000, Guzman & Kouri
2002). All four serotypes cause dengue fever (DF), a
febrile illness characterized by severe joint and muscle
pains, and in some cases, the potentially fatal dengue
hemorrhagic fever (DHF) and dengue shock syndrome
(DSS). The wide distribution of the mosquito vector
Aedes aegypti in the tropics and subtropics results in
the exposure of approximately 2.5 billion individuals to
dengue infection yearly (Mackenzie et al. 2004).
Consistent with this, dengue viruses are the most
common, important Flavivirus causing human disease
in Brazil (Figueiredo 2000). In the last 14 years, Brazil
has accounted for nearly 70% of the 3,141,850 reported
cases of DF in the Americas in the last five years (PAHO
2003). Some viral serotypes and the mosquito vector
are now distributed throughout the country, and dengue
outbreaks occur in all regions, especially the most popu-
lated areas, such as in the state of Bahia (BA). By the
end of 2001, BA had only experienced epidemics with
DENV-1 and DENV-2. Both of which were actively cir-
culating when DENV-3 was introduced in 2002 and pro-
duced the first cases of DHF in BA. In 1987, DENV-1
was isolated for the first time in BA in the city of
Ipupiara, located in the center of the state (the Chapada
Diamantina region) (Vasconcelos et al. 2000). A second
serotype, DENV-2, was introduced in 1995 in the ex-
treme south of the state, although its exact point of in-
troduction was not identified. During these initial epi-
demics in BA, the dynamics of introduction and subse-
quent spread of the infection could not be followed state-
wide, since the necessary public health protocols and
the mechanism for viral isolation were not available.
These were introduced in 1995 with development of a
special unit at the state of Bahia’s Central Laboratory
(LACEN) for DENV isolation. In this study, we used the
information from viral isolations at the LACEN from
2001 to 2003 to assess the dynamics of circulation of
DENV serotypes in BA before, during and after the first
introduction of DENV-3.
MATERIALS AND METHODS
Study site - BA is the fifth-largest Brazilian state in
territorial extension and comprises 6.64% of the total
area of the country. BA has 13,323,212 inhabitants with
nearly 25% concentrated in the metropolitan area close
to Salvador, the state's capital. Approximately 69% of
the state is considered arid or semi-arid. By contrast,
the 1,183 km long coastline is generally tropical and hu-
mid. Urbanization in BA is marked by concentration of
the overwhelming part of the urban population in the capi-
tal, thus making Salvador the only city in the state whose
population exceeds 500,000. Nevertheless, the state is
also characterized by the dispersion of the population in
hundreds of smaller urban centers. In the last two de-
Financial support: Fiocruz (PDTSP), CNPq, NIH (AI056263-01)
+
Corresponding author: [email protected]
Received 29 May 2007
Accepted 28 November 2007
Acta Tropica 100 (2006) 256–260
Short communication
High serum nitric oxide levels in patients with severe leptospirosis
Elves A.P. Maciel
a
, Daniel A. Athanazio
a,b
, Eliana A.G. Reis
a
, Fernando Q. Cunha
c
,
Adriano Queiroz
a,b
, Deusdelia Almeida
a,b
, Alan J.A. McBride
a
,
Albert I. Ko
a,d
, Mitermayer G. Reis
a,b,∗
a
Gon¸calo Moniz Research Centre, Oswaldo Cruz Foundation, Ministry of Health, Salvador, Brazil
b
Federal University of Bahia, Salvador, Brazil
c
Ribeirao Preto Faculty of Medicine, University of Sao Paulo, Ribeirao Preto, Brazil
d
Division of International Medicine and Infectious Disease, Weill Medical College of Cornell University, New York, USA
Received 14 July 2006; received in revised form 7 November 2006; accepted 19 November 2006
Available online 2 January 2007
Abstract
Leptospirosis is a globally distributed zoonosis of major public health importance and is associated with severe disease man-
ifestations such as acute renal failure and pulmonary haemorrhage syndrome. However, the extent to which the pathogenesis of
leptospirosis mimics sepsis caused by Gram-negative bacteria remains unknown. The aim of this study was to evaluate serum levels
of nitric oxide (NO) in patients diagnosed with severe leptospirosis. Sera from 35 confirmed cases of severe leptospirosis and 13
healthy subjects were analysed. Patients with severe leptospirosis had significantly higher NO levels compared to healthy individuals
(30.82 ± 10.90 ␮M versus 3.86 ± 1.34 ␮M, P < 0.001), indicating that this immune mediator plays a role in the underlying systemic
inflammatory response.
© 2006 Elsevier B.V. All rights reserved.
Keywords: Leptospirosis; Nitric oxide; Acute renal failure; Pulmonary haemorrhage
Leptospirosis is a widespread zoonosis caused by
pathogenic spirochetes from the Leptospira genus.
Transmission to humans is accidental and typically
occurs through contact with mud or water contaminated
with urine of infected animals, principally rodents. Tra-
ditionally a sporadic disease confined to a rural setting,
leptospirosis now causes yearly epidemics in urban liv-
ing conditions associated with extreme poverty (Johnson
et al., 2004; Karande et al., 2003; Ko et al., 1999;
LaRocque et al., 2005; Sarkar et al., 2002). Outbreaks
∗
Corresponding author at: Centro de Pesquisas Gonc¸alo Moniz -
CPqGM, Rua Waldemar Falc
˜
ao, 121, Candeal, CEP: 40296-710 Sal-
vador, Bahia, Brazil. Tel.: +55 71 3176 2200; fax: +55 71 3176 2326.
E-mail address: miter@cpqgm.fiocruz.br (M.G. Reis).
are also associated with water sports and recreational
activities that result in exposure to pathogenic Leptospira
(Haake et al., 2002). The clinical spectrum ranges from
a mild anicteric disease to the more severe forms such as
Weil’s syndrome (jaundice, haemorrhagic diathesis and
acute renal failure), associated with a 10% mortality and
severe pulmonary haemorrhage syndrome (SPHS), for
which the case fatality rate can be >50% (McBride et
al., 2005).
The underlying pathogenic mechanisms associated
with the severe manifestations of leptospirosis are poorly
understood but it is assumed that a generalized endothe-
lial dysfunction, as observed in experimental and human
leptospirosis, is the main pathogenic mechanism of tis-
sue damage (De Brito et al., 1979; Nicodemo et al.,
1997). A similar process occurs in sepsis and is mediated
0001-706X/$ – see front matter © 2006 Elsevier B.V. All rights reserved.
doi:10.1016/j.actatropica.2006.11.006
Acta Tropica 98 (2006) 176–182
Domestic swine in a visceral leishmaniasis endemic area produce
antibodies against multiple Leishmania infantum antigens but
apparently resist to L. infantum infection
Evandro Moraes-Silva
a,b
, Fabiana Rodrigues Antunes
a
, Marcio Silva Rodrigues
a
,
Fred da Silva Juli
˜
ao
b
, Artur Gomes Dias-Lima
a
, Valderez Lemos-de-Sousa
a
,
Adriano Costa de Alcantara
b
, Eliana Almeida Gomes Reis
a
,
Maria Nakatani
c
, Roberto Badar
´
o
c
, Mitermayer Galv
˜
ao Reis
a
,
Lain Pontes-de-Carvalho
a
, Carlos Roberto Franke
b,∗
a
Centro de Pesquisas Gon¸calo Moniz (CPqGM), Funda¸c˜ao Oswaldo Cruz, Rua Waldemar Falc˜ao 121, Brotas,
CEP: 40295-001, Salvador, Bahia, Brazil
b
Laborat´orio de Infectologia Veterin´aria, Escola de Medicina Veterin´aria, Universidade Federal da Bahia,
Av. Adhemar de Barros 500, Ondina, CEP: 40.170-110, Salvador, Bahia, Brazil
c
Laborat´orio de Doen¸cas Tropicais, Hospital Universit´ario Professor Edgard Santos, Universidade Federal da Bahia,
Rua Jo˜ao das Botas s/n, Canela, CEP: 40110-160, Salvador, Bahia, Brazil
Received 11 May 2005; received in revised form 20 March 2006; accepted 12 April 2006
Abstract
In order to investigate whether pigs can be infected by Leishmania infantum, a serological and parasitological study was carried
out on swine in the Jequi
´
e municipality, Northeast of Brazil. Anti-Leishmania infantum antibodies were detected in 37 out of 92
swine (40.2%), by two different assays: an anti-L. infantum lysate and an anti-K39 recombinant protein ELISA. An experimental
study was also carried out to verify the susceptibility of domestic pigs to L. infantum infection. Three sows inoculated with 10
8
stationary-phase infective L. infantum promastigotes (26% metacyclic promastigotes) per kilogram of body weight produced anti-
Leishmania antibodies until the end of the experiment, 11 months later. No parasites, however, could be visualized through optical
microscopy of spleen, liver and bone marrow or by in vitro culture of these organs. Homogenates of these organs were also inoculated
in hamsters, without producing infection. No Leishmania DNA was detected by polymerase chain reaction (PCR) in sand flies fed
on these animals. The results indicate that domestic pigs bitten by L. infantum-infected vectors in the endemic area do not display
a full infection pattern, and the positive association in endemic areas between the presence of swine and infection in canines may
not be ascribable to the former acting as a parasite reservoir.
© 2006 Published by Elsevier B.V.
Keywords: Visceral leishmaniasis; Pig; Immune response; Experimental infection
∗
Corresponding author. Tel.: +55 71 33315042;
fax: +55 71 33315042.
E-mail address: [email protected] (C.R. Franke).
1. Introduction
American visceral leishmaniasis (AVL) is defined
as a zoonotic disease caused by Leishmania infantum,
although it was considered to be caused by the Leish-
0001-706X/$ – see front matter © 2006 Published by Elsevier B.V.
doi:10.1016/j.actatropica.2006.04.002
613613
613613
613Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 101(6): 613-615, September 2006
Schizodeme analysis of Trypanosoma cruzi Colombian strain
clones isolated from the acute phase of murine infection
Edson LP Camandaroba* , Eliana AG Reis, Mitermayer G Reis, Sonia G Andrade/
+
Laboratório de Chagas Experimental, Auto-imunidade e Imunidade Celular, Laboratório de Patologia e Biologia Molecular,
Centro de Pesquisas Gonçalo Moniz-Fiocruz, Rua Valdemar Falcão 121, 40295-001 Salvador, BA, Brasil *Laboratório de
Histologia e Embriologia Animal Comparada , Universidade Estadual de Feira de Santana, Feira de Santana, BA, Brasil
Colombian strain of Trypanosoma cruzi, biodeme Type III (T. cruzi I), has been cloned by micromanipulation at
two phases of the acute infection: early (10 days ) and advanced (30 days). Twelve clones were obtained therefrom.
Characterization by their biological and biochemical behavior showed an identity among the several clones and
their parental strain, albeit with different degrees of virulence. Molecular characterization of the kinetoplast DNA
(kDNA) after amplification by polymerase chain reaction revealed identical profiles of the bands from the kDNA
minicircle by the analysis of restriction fragment lenght polymorphism for the isolated clones, their parental strain,
and to the clones isolated at two different phases of the infection. Results suggest the predominance of a “principal
clone”, in the composition of the Colombian strain, responsible for the biological and biochemical behavior.
However, no relationship was detected between the molecular profile of kDNA and the degree of virulence pre-
sented by the several clones.
Key words: Trypanosoma cruzi - Colombian strain clones - schizodeme
Molecular characterization of strains and clones of
Trypanosoma cruzi was first performed by Morel et al.
(1980), through electrophoretic separation of restriction
endonuclease-generated fragments of the kinetoplast
DNA, by the restriction fragment length polymorphism
technique (RFLP), in order to obtain the electrophoretic
profiles or schizodeme, also known as “restriction finger-
prints” (Morel et al. 1986). The polymerase chain reaction
(PCR) can be used to amplify the fragments of kDNA
minicircles of T. cruzi, allowing its direct detection and
genotype classification (Avila et al. 1990). Studies on the
clonal composition of several strain maintained for pro-
longed time in laboratory have allowed the identification
of either strains with an homogeneous clonal structure or
the presence of heterogeneous clones, as has been dem-
onstrated for the Y and CL strains (Deane et al. 1984, Morel
et al. 1986). Previous studies have demonstrated a clonal
homogeneity for the 21 SF strain (T. cruzi II), prototype of
Biodeme Type II (Andrade & Magalhães 1997), by both
the biological and isoenzymic characterization (Campos
& Andrade 1996), corresponding to a high degree of simi-
larity by RFLP analysis for characterization of kDNA (Cam-
pos et al. 1999). Clones isolated from the Colombian strain,
representing the Type III Biodeme (T. cruzi I), also dem-
onstrated similarity among them (Camandaroba et al. 2001).
However, they presented variable degrees of virulence,
regardless of their main biological characters, and isoen-
zyme profiles. The hypothesis that the several clones,
isolated either from the early or advanced phases of acute
infection, could differ in their molecular structure of the
+
Corresponding author: [email protected]
Received 25 January 2006
Accepted 19 July 2006
kDNA is investigated in the present study by the molecu-
lar characterization of its clones at the level of kDNA vari-
able portions of the minicircles, to further clarify the rela-
tionship between the parasite biological behavior and its
clonal structure (Andrade 1999).
MATERIALS AND METHODS
Parental strains and clones of T. cruzi strains - Seven
clones of the Colombian strain, isolated at the 30th day of
infection (C1, C2, C3, C4, C5, C6, C7), and five clones
isolated at the 10th day of infection (C8, C10, C13, C14,
C15), were obtained by micromanipulation (Dvorak 1985),
by isolating one single parasite from citrated mouse blood;
after centrifugation at 900 g, the supernatant was exam-
ined in a Neubauer chamber for evaluation of parasite
concentration, diluted with PBS, pH 7.2, and drops of 1µl
were distributed in the wells of titration plaques and ex-
amined on an inverted microscope. By means of microma-
nipulation a single trypomastigote form, isolated with 1
ml syringe was inoculated into one 8-day-old mouse.
Clones and parental strains were inoculated into groups
of mice for biological characterization: evolution of para-
sitemia, cumulative mortality and isoenzymic profiles of
parasites. Clones isolated either at the 30th or 10th day of
infection varied in virulence, as evaluated by the levels
of parasitemia and mortality, as follows: (1) seven clones
isolated at the 30th day of infection: high virulence - clones
C1, C3, C4 , C6; low virulence - clones C2, C5,C7; (2)
five clones isolated at the 10th day of infection: high viru-
lence - clones C13, C14; low virulence - clones C8, C10,
C15. The parental strain (Col-P), disclosed high viru-
lence.
Molecular characterization by the schizodeme analy-
sis - Isolation of total kDNA from the parental Colombian
strain ant its clones was obtained from axenic culture,
using the Genomic kit (GFX
tm)
from Pharmacia. Parasites
were cryopreserved and 100 µl were transferred to a filtra-
Clin Rheumatol
DOI 10.1007/s10067-006-0281-7
ORIGINAL ARTICLE
Fernando Luiz Barros Edington
.
Maria Olívia Amado Ramos Bacellar
.
Paulo Roberto Machado
.
Lúcio Barbosa
.
Eliana Reis
.
Mitermayer Reis
.
Mittermayer Barreto Santiago
Anti-neutrophil cytoplasmic antibodies in leprosy
Received: 15 February 2006 / Revised: 2 March 2006 / Accepted: 3 March 2006
# Clinical Rheumatology 2006
Abstract Introduction: Anti-Neutrophil Cytoplasmic
Antibodies (ANCA) are auto-antibodies directed to intra-
cellular components of neutrophils and used to be
considered as present almost exclusively in granulomatous
vasculitis. Recently, these auto-antibodies have been found
in other autoimmune disorders as well as infectious
diseases. Materials and methods: We studied patients
with leprosy confirmed by bacilloscopy and/or skin biopsy,
in reaction phase from the Ambulatório de Hanseníase do
Hospital Universitário Professor Edgar Santos. ANCA
and Antinuclear antibodies (ANA) were determined by
indirect immunofluorescence using commercially available
kits. Results: Twenty patients were enrolled in our study,
nine males and 11 females. The mean age was 36.9±
18.2 years. ANCA were present only in one patient, with a
perinuclear staining pattern (p-ANCA), and no patient
tested positive for ANA. Discussion: Although other
studies have shown the presence of ANCA in leprosy,
the low frequency of these antibodies in leprosy sera
demonstrated in the present study illustrates the high
specificity of ANCA for the diagnosis of Wegener
granulomatosis.
Keywords Auto-antibodies
.
Hansen disease
.
Leprosy
.
Vasculitis
Introduction
The interest in studying anti-neutrophil cytoplasmic
antibodies (ANCA) emerged in the 1960s, together with
the discovery of auto-antibodies detection techniques by
indirect immunofluorescence [1]. However, only in the
1980s ANCA started to be used for diagnosis and follow-
up of Wegener’s granulomatosis (WG) [1]. Classically,
there are two major immunofluorescence staining patterns:
(1) c-ANCA, a cytoplasmic predominant one, which
correlates with the presence of antibodies against protein-
ase 3 (PR3) and (2) a perinuclear predominant staining
pattern, p-ANCA, which correlates with antibodies direct-
ed against other proteins, particularly, myeloperoxidase
(MPO). Until recently, ANCA, especially c-ANCA, were
thought to be exclusively associated with granulomatous
vasculitic syndromes, especially, WG. However, their
presence has been demonstrated in other diseases, such
as rheumathoid arthritis [2, 3], systemic lupus erythema-
tosus [4], inflammatory bowel disease [5], occupational
exposure to silica [ 6], HIV infection [7], subacute bacterial
endocarditis [8], and leprosy [9, 10], suggesting that
ANCA could not be so specific for WG as previously
thought.
The aim of the present study was to determine the
prevalence of ANCA in patients with leprosy in reactive
phase, when there is a hyperactivity of the immune system
F. L. B. Edington
Universidade Federal da Bahia,
Salvador, Bahia, Brazil
M. O. A. R. Bacellar
Laboratório de Imunologia do Hospital Universitário Professor
Edgar Santos, Universidade Federal da Bahia,
Salvador, Bahia, Brazil
P. R. Machado
Escola Baiana de Medicina e Saúde Pública and Universidade
Federal da Bahia,
Salvador, Bahia, Brazil
L. Barbosa
.
E. Reis
.
M. Reis
Fundação Osvaldo Cruz Laboratório de Patologia e Biologia
Molecular, Fiocruz,
Salvador, Bahia, Brazil
M. B. Santiago
Escola Baiana de Medicina e Saúde Pública and Serviço de
Reumatologia do Hospital Santa Izabel,
Salvador, Bahia, Brazil
M. B. Santiago (*)
Serviço de Reumatologia do Hospital Santa Izabel. Praça
Almeida Couto,
500, Nazaré,
Salvador, Bahia, CEP 40.000-000, Brazil
e-mail: [email protected]
ORIGINAL ARTICLE
Mittermayer Santiago Æ Reinaldo Martinelli
Mitermayer G. Reis Æ Eliana Al meida Reis Æ Albert Ko
Roberto Dias Fontes Æ Moacir Paranhos Silva
Eliane Goes Nascimento Æ Roge
´
rio de Jesus
Silvia Pierangeli Æ Ricardo Espinola Æ Azzudin Gharavi
Diagnostic performance of anti-b2 glycoprotein I and anticardiolipin
assays for clinical manifestations of the antiphospholipid syndrome
Received: 2 October 2003 / Accepted: 15 March 2004 / Published online: 28 April 2004
Ó Clinical Rheumatology 2004
Abstract The objective of the present study was to
analyse the performance of the tests for detection of
anti-b2 glycoprotein I (b2 GP I) and anticardiolipin
(aCL) antibodies for identification of clinical manifes-
tations of the antiphospholipid syndrome (APS). Pa-
tients with systemic lupus erythematosus (SLE) as well
as carriers of infectious diseases such as Kala-azar,
syphilis and leptospirosis were studied. Particular inter-
est was given to the presence of clinical complications
related to APS. Anticardiolipin and anti-b2 GP I anti-
bodies were searched using an enzyme-linked immuno-
sorbent assay (ELISA) assay. Clinical manifestations of
APS were observed in 34 of the 152 patients (22.3%)
with SLE and no patient with infectious disease had
such manifestations. Antibodi es to cardiolipin in mod-
erate or high levels and anti-b2 GP I were detected in 55
of 152 (36.1%) and 36 of 152 (23.6%) patients with SLE,
respectively, and in 2 of 30 (6.6%) and 16 of 30 (53.3%)
patients with Kala-azar, in 9 of 39 (23%) and 6 of 34
(17.6%) patients with leptospirosis, and 14 of 74
(18.9%) and 8 of 70 (11.4%) cases of syphilis, respec-
tively. The sensitivity, spec ificity, positive predictive va-
lue (PPV), negative predictive value (NPV) and
likelihood ratio (LR) of the anti-b2 GP I test for the
identification of the clinical manifestation of APS were,
respectively, 29% [95% confidence interval (CI)=24%–
34%], 78% (95% CI=73–83%), 15% (95% CI=11–
19%), 89% (95%CI=85–93%) and 1.38. Regarding the
aCL assay, the figure was 29% (95% CI=24–34%) ,
76% (95% CI=71–81%), 14% (95% CI=10–18%),
89% (95% CI=86–92%) and 1.26. As the validity and
performance of the anti-b2 GP I assay were similar to
the aCL in demonstrating the presence of clinical phe-
nomena associated with APS and due to the difficulties
in performing as well as the lack of standardisation of
the anti-b2 GP I test, we suggest that the test for aCL
should continue to be the first one performed when the
presence of APS is suspected.
Keywords Anticardiolipin Æ Antiphospholipid Æ
Diagnostic test Æ Systemic lupus erythematosus
Introduction
The development of the Wassermann reaction for the
diagnosis of syphilis [1] and the posterior characterisa-
tion of the antigen as cardiolipin [2] were the first steps
for the study of the antiphospholipid antibodies (aPL).
Also very important was the observation that some
individuals with a false positive serologic test for syphilis
could develop autoimmune diseases such as systemic
lupus erythematosus (SLE) [3], establishing a connection
between the aPL antibodies and immunological disor-
ders. Another landmark in the study of these antibodies
M. Santiago (&)
Escola Bahiana de Medicina e Sau´ de Pu´ blica,
Nu´ cleo de Reumatologia do Servic¸ o de Clı
´
nica Me
´
dica
do Hospital Santa Izabel, Prac¸ a Almeida Couto, 500,
CEP 40.000–000 Nazare
´
, Salvador, Bahia, Brazil
E-mail: [email protected]
R. Martinelli Æ R. de Jesus
Faculdade de Medicina da UFBa,
Salvador, Bahia, Brazil
M. G. Reis Æ E. A. Reis Æ A. Ko
Centro de Pesquisas Gonc¸ alo Muniz, Fundac¸ a
˜
o Osvaldo Cruz,
Salvador, Bahia, Brazil
A. Ko
Weill Medical College, New York, NY, USA
R. D. Fontes
COAS/DST/SESAB, Salvador, Bahia, Brazil
M. P. Silva Æ E. G. Nascimento
PIEJ/SESAB, Jequie
´
, Brazil
S. Pierangeli Æ R. Espinola Æ A. Gharavi
Morehouse School of Medicine, Atlanta, GA, USA
Clin Rheumatol (2004) 23: 485–489
DOI 10.1007/s10067-004-0924-5
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