A toxoplasmose é uma infecção mundial que é transmitida por carne contendo cistos teciduais, água ou legumes crus contaminados com oocistos, contato com felinos, ou transmissão vertical. A toxoplasmose é usualmente assintomática, mas pode causar doença severa no feto quando transmitida durante fase aguda na mãe. O rastreamento da toxoplasmose congênita é normalmente executado durante o pré-natal, pela detecção de anticorpos IgM específico ou pela demonstração de aumento dos títulos de anticorpos IgG para T.gondii. Neste trabalho, 160 mães IgM positivas em rastreamento externo foram avaliadas para IgM, IgG, IgA e avidez dos anticorpos IgG em ensaios usando Uréia e NH4SCN como agente caotrópico, e extrato salino e uma proteína recombinante ROP2 como antígeno. A maioria das mães com resultado positivo de IgM eram negativas (124) quando retestadas, 16 negativas para todos os testes e somente 36 casos de IgM e IgG positivos. Nossa reação foi convalidada pelo Kit VIDAS Toxo IgG (Biomerieux), as 15 amostras aleatórias foram concordantes na maioria das amostras, incluindo as 3 de baixa avidez. A maioria das mães com IgG e IgM positivas confirmadas apresentaram índices intermediário e alto de avidez, e apenas 3 com índice baixo de avidez, que é sugestivo de infecção aguda. O uso de proteína recombinante Rop2 como antígeno resultou em muitos falsos negativos. O rastreamento de toxoplasmose congênita em áreas de alta prevalência induz uma baixa eficiência da sorologia, altos índices de pacientes tratados e baixa detecção. Ensaios de avidez são promissores, mas a sua padronização é obrigatória. / Toxoplasmosis is a worldwide infection transmitted by meat containing tissue cysts, water or crude vegetables contaminated with oocysts, contact with feline or vertical transmission. Toxoplasmosis is usually asymptomatic, but it can cause severe disease to the fetus during acute mother infection. The screening of congenital toxoplasmosis is usually been performed during prenatal, by detecting specific IgM antibodies or by demonstration of a significant increase specific IgG antibodies. In this work, 160 confirmed IgM positive mothers by external screening were evaluated for IgM, IgG, IgA, and avidity by several approaches using Urea and NH4SCN as chaotropic agents, and saline extract and a recombinant protein Rop2 as antigen. Most mother (124) were IgM negative when retested, 16 negative for all T.gondii tests and only 36 IgM and IgG positive. Our avidity reaction was convalidated by Kit VIDAS Toxo IgG (Biomerieux), 15 aleatory samples were concordant in most samples, including the 03 low avidity. Most of confirmed IgG and IgM positive mothers presented intermediary or high avidity indexes, only 3 presenting low avidity index, sugestive of acute infection. Use of recombinant proteins Rop 2 as antigen resulted in high false negative. Screening congenital toxoplasmosis in high prevalent areas by IgM serology showed low efficiency, higher treatment rates and low detection of acute infection. Avidity assays are promising markers of recent infection, but false negatives could be frequent.

HCMV is a common viral pathogen that infects most of the world's population by early adulthood. It is typically asymptomatic in immunologically healthy individuals but causes severe disease in immunocompromised patients and congenitally infected infants. HCMV glycoproteins are highly polymorphic, and various types of associations have been suggested between glycoprotein types and the pathogenicity of the virus. Several studies on viruses other than HCMV have related the glycosylation of the viral glycoproteins to virulence. This project aimed to determine whether there is a robust relationship between the individual glycoprotein sequence and its glycosylation, how this influences the growth characteristic of the virus and whether this is related to its pathogenicity. Glycosylation patterns of 89 clinical specimens of different infection categories and specimen types were correlated with genetic sequence alterations of the virus glycoproteins (gB, gH, gL, gM, gN, gO), followed by determining whether mutation results in specific changes in glycosylation. The aim was approached using a cell culture model and a quantitative lectin-based assay (ELLA). A significantly increased glycosylation level for the following genotypes: mixed gH, gN4a, gO4, mixed gL was detected. Whereas a decreased pattern was found to be associated with gH1, gH2, gN3a, gO1a and gL2 genotypes (P < 0.05). Glycoproteins of strains isolated from respiratory specimens were significantly highly glycosylated compared to the blood and urine samples, and from blood specimens compared to the urine samples (P < 0.05). Furthermore, strains from congenitally infected infants and urine samples had a significantly higher growth rate than others tested. No direct association between the virus growth and its virulence was found. These findings demonstrate that glycosylation of glycoproteins in HCMV is affected by the glycoprotein polymorphisms and signifies a potentially important mechanism for avoidance of antibody-mediated neutralization, which, in turn, facilitates HCMV pathogenicity. This phenomenon requires further study and may have application for the selection of novel targets for diagnosis, vaccine development and other preventive measures to combat diseases caused by this virus.

La toxoplasmose est une anthropozoonose cosmopolite due à un protozoaire parasite intracellulaire obligatoire : Toxoplasma gondii. Cette maladie infectieuse est la plus souvent bénigne chez les personnes immunocompétentes mais revêt un caractère de gravité si l’atteinte concerne les femmes enceintes séronégatives ou les personnes immunodéprimées. En plus de cette incidence forte en médecine humaine, la toxoplasmose représente un important problème de santé vétérinaire. A l’heure actuelle, les seuls moyens de lutte contre ce parasite demeurent la chimiothérapie car il n’existe aucune stratégie prophylactique efficace. Le développement d’un vaccin efficace est d’une réelle nécessité et repose sur l’observation qu’une primo-infection par ce parasite confère à l’hôte immunocompétent une réponse immunitaire protectrice efficace à long terme et qui protège lors d’une réinfection et en particulier contre le risque d’une infection congénitale. / Toxoplasmosis is a cosmopolitan anthropozoonosis due to the obligate intracellular protozoan parasite Toxoplasma gondii. This infectious disease is most often benign in immunocompetent individuals but is particularly severe for pregnant women or immunocompromised patients. In addition to its significant impact on human medicine, toxoplasmosis is a major veterinary health problem. Currently, the only means to fight this parasite remain chemotherapy because there is no effective prophylactic strategy. The development of an effective vaccine is a real challenge and is based on the observation that a primary infection of immunocompetent hosts induces a effective and long-term protective immune response and protects during reinfection and in particular against the risk of congenital infection.

Os objetivos do estudo consistiram em verificar o impacto da infecção congênita por CMV ao nascer e avaliar potenciais preditores prognósticos de anormalidades tardias em uma coorte de criança s com esta infecção. Métodos: Por meio de uma triagem neonatal, 66 de 11.957 crianças foram identificadas como portadoras de infecção congênita por CMV. Todas as crianças infectadas sintomáticas e assintomáticas foram avaliadas ao nascimento por meio de exame físico detalhado, avaliação auditiva, fundoscopia ocular, neurossonografia transfontanelar (NTF) realizada por neurorradiologista infantil e quando alterada, foram submetidas à ressonância magnética de encéfalo (RMc). Análise de regressão logística foi realizada para verificar a associação de potenciais fatores de risco para ocorrência de surdez neurossensorial relacionada ao CMV e/ou presença de achados anormais em NTF e/ou RMc. A análise da Curva ROC foi utilizada para avaliar a associação da carga do DNA do CMV expresso em log10, com a presença de sintomas ao nascer, achados anormais em NTF e/ou RMc e ocorrência de surdez neurosensorial. Resultados: Em 66 crianças infectadas, os sinais clínicos sugestivos de infecção congênita ao nascer foram observados em 8/66 (12,12%; IC95%: 5,74-23,03), sendo considerados sintomáticos. As 58 crianças restantes foram considerados assintomáticos. A surdez neurossensorial foi observada em 8/66 (12,12%; IC95%: 5,74-23,03%) das crianças. Destas, 4/58 (6,9%; IC95%: 2,23- 17,54%) e 4/8 (50%; IC95%: 17,44-82,55%) crianças eram assintomáticas e sintomáticas, respectivamente. Achados de NTF e/ou RMc sugestivas de infecção congênita foram observadas em 7 de 8 crianças sintomáticas (87,5%; IC95%: 46,67-99,34%). Dentre as 58 assintomáticas, 53 (92,4%) completaram todas as avaliações e destas 29/52 apresentaram alterações sugestivas de infecção congênita em NTF e/ou RMc (55,7%; IC95%: 41,41 - 69,27%). O achado mais comum foi a vasculopatia lenticuloestriada acompanhado ou não de cistos subpendimários (13/29: 43.3%). Outros achados anormais foram os cistos subpendimários como achado isolado (11/29: 37.9%), calcificação única ou periventricular com ou sem áreas de gliose (4/29: 13,8%) e ventriculomegalia (4/29: 13,8%). Análise de regressão logística mostrou que apenas a presença de sintomas clínicos ao nascer foi preditivo para a ocorrência de surdez neurossensorial. Achados de neuroimagens (NTF e/ou RMc) foram observados em 3 de 4 crianças assintomáticas (75%) enquanto 26 (54,2%) de 48 crianças sem surdez tinham avaliações normais de NTF e/ou RMc (p=0,42; RR:2,38: IC95%: 0,26-21,39). Nenhum dos outros fatores de risco foram independentemente associados com surdez. A presença de plaquetopenia e/ou níveis altos de gama glutamil transferase (?GT) foi associado com a presença de achados anormais em NTF e/ou RMc uma análise univariada. O poder discriminatório pela determinação do ponto de corte do valor da carga do DNA do CMV foi avaliada pela área abaixo da curva ROC (AUC) e não houve associação entre a carga viral e a ocorrência de surdez e/ou achados anormais de NTF e/ou RMc. Conclusões: Embora a triagem neonatal da infecção congênita por CMV permita identificar a maioria das crianças infectadas que são clinicamente assintomáticas ao nascer, uma proporção significante destas crianças poderá ser beneficiada por uma avaliação do sistema nervoso central através de NTF, uma vez que, achados anormais são muito frequentes. Embora não tenha sido possível determinar fatores independentemente preditivos de ocorrência de surdez neurossensorial, os achados de NTF podem ser potenciais fatores preditivos de anormalidades tardias em crianças assintomáticas. / The objectives of this study were to verify the impact of congenital cytomegalovirus (CMV) infection at birth and to evaluate potential prognostic predictors of late abnormalities in a cohort of children with this infection. Methods: By means of a CMV neonatal screening, 66 of 11.957 infants were identified as congenitally infected. Infants with and without clinical abnormalities detectable at birth underwent physical examination, cranial ultrasound performed by a paediatric radiologist and/or cranial magnetic resonance imaging, ocular fundoscopy, and hearing evaluation using evoked otoacoustic emissions and auditory brainstem response. Logistic regression analysis was carried out to verify the association between the risk factors for occurrence of hearing loss related to CMV and/ or abnormal cranial ultrasound findings. ROC curve was plotted using the log10 value of CMV DNA load to evaluate the association between viral load and clinical symptoms at birth, abnormal cranial ultrasound findings and hearing loss. Results: Of all 66 infected children, the clinical signs suggestive of congenital infection at birth were observed in 8/66 (12.12%; IC95%: 5.74- 23.03) symptomatic infants. Sensorineural hearing loss was observed in 8/66 (12,12%; IC95%: 5.74-23.03%) children. Of these, 4/58 (6,9%; IC95%: 2.23-17.54%) and 4/8 (50%; IC95%: 17,44-82.55%) children were asymptomatic and symptomatic, respectively. Cranial ultrasound findings suggestive of congenital infection were observed in 7 of the 8 symptomatic children (87.5%; IC95%: 46.67-99.34%). Among the 58 asymptomatic infants, 53 underwent complete evaluation and 29/52 had abnormal cranial ultrasound results (55,7%; IC95%: 41.41 - 69,27%). The most prevalent findings was lenticulostriate vasculopathy with subependymal pseudocysts present in 13 of the /29 (43.3%) infants with cranial ultrasound. Other abnormal findings were isolated subependymal pseudocysts (11/29: 37.9%); single or periventricular calcifications and/or gliosis (4/29: 13.8%); and ventriculomegaly (4/29: 13,8%). Logistic regression analysis showed that only the presence of clinical findings predicted the occurrence of hearing loss. Cranial ultrasound findings were observed in 3 of asymptomatic infants (75.0%) while 26 (54.2%) of 48 infants with no hearing loss had abnormal imaging features (p=0,42; RR:2,38: IC95%: 0,26-21,39). None of the other factors risk were independely associated with development of hearing loss. The presence of thrombocytopenia and/or high level of gamma-glutamyltranspeptidase (?GT) was associated with cranial ultrasound findings on univariated analysis. No discrimination power was achieved using the area under the ROC curve to verify the association between CMV DNA load in the urine of the infected children and the developing of hearing loss, presence of cranial ultrasound findings and clinical signs at birth. Conclusions: Although a neonatal screening of cCMV will identify the majority of infected infants who are clinically asymptomatic, a significant proportion of them could benefit from a central nervous system image evaluation, since abnormal findings are frequent. Althoug it was not possible to determine risk factors that are independently associated to development of sensorineural hearing loss, cranial ultrasound findings could be a potential prognostic markers of adverse outcomes of congenital CMV in asymptomatic infants.

A toxoplasmose é uma infecção mundial que é transmitida por carne contendo cistos teciduais, água ou legumes crus contaminados com oocistos, contato com felinos, ou transmissão vertical. A toxoplasmose é usualmente assintomática, mas pode causar doença severa no feto quando transmitida durante fase aguda na mãe. O rastreamento da toxoplasmose congênita é normalmente executado durante o pré-natal, pela detecção de anticorpos IgM específico ou pela demonstração de aumento dos títulos de anticorpos IgG para T.gondii. Neste trabalho, 160 mães IgM positivas em rastreamento externo foram avaliadas para IgM, IgG, IgA e avidez dos anticorpos IgG em ensaios usando Uréia e NH4SCN como agente caotrópico, e extrato salino e uma proteína recombinante ROP2 como antígeno. A maioria das mães com resultado positivo de IgM eram negativas (124) quando retestadas, 16 negativas para todos os testes e somente 36 casos de IgM e IgG positivos. Nossa reação foi convalidada pelo Kit VIDAS Toxo IgG (Biomerieux), as 15 amostras aleatórias foram concordantes na maioria das amostras, incluindo as 3 de baixa avidez. A maioria das mães com IgG e IgM positivas confirmadas apresentaram índices intermediário e alto de avidez, e apenas 3 com índice baixo de avidez, que é sugestivo de infecção aguda. O uso de proteína recombinante Rop2 como antígeno resultou em muitos falsos negativos. O rastreamento de toxoplasmose congênita em áreas de alta prevalência induz uma baixa eficiência da sorologia, altos índices de pacientes tratados e baixa detecção. Ensaios de avidez são promissores, mas a sua padronização é obrigatória. / Toxoplasmosis is a worldwide infection transmitted by meat containing tissue cysts, water or crude vegetables contaminated with oocysts, contact with feline or vertical transmission. Toxoplasmosis is usually asymptomatic, but it can cause severe disease to the fetus during acute mother infection. The screening of congenital toxoplasmosis is usually been performed during prenatal, by detecting specific IgM antibodies or by demonstration of a significant increase specific IgG antibodies. In this work, 160 confirmed IgM positive mothers by external screening were evaluated for IgM, IgG, IgA, and avidity by several approaches using Urea and NH4SCN as chaotropic agents, and saline extract and a recombinant protein Rop2 as antigen. Most mother (124) were IgM negative when retested, 16 negative for all T.gondii tests and only 36 IgM and IgG positive. Our avidity reaction was convalidated by Kit VIDAS Toxo IgG (Biomerieux), 15 aleatory samples were concordant in most samples, including the 03 low avidity. Most of confirmed IgG and IgM positive mothers presented intermediary or high avidity indexes, only 3 presenting low avidity index, sugestive of acute infection. Use of recombinant proteins Rop 2 as antigen resulted in high false negative. Screening congenital toxoplasmosis in high prevalent areas by IgM serology showed low efficiency, higher treatment rates and low detection of acute infection. Avidity assays are promising markers of recent infection, but false negatives could be frequent.

Os objetivos do estudo consistiram em verificar o impacto da infecção congênita por CMV ao nascer e avaliar potenciais preditores prognósticos de anormalidades tardias em uma coorte de criança s com esta infecção. Métodos: Por meio de uma triagem neonatal, 66 de 11.957 crianças foram identificadas como portadoras de infecção congênita por CMV. Todas as crianças infectadas sintomáticas e assintomáticas foram avaliadas ao nascimento por meio de exame físico detalhado, avaliação auditiva, fundoscopia ocular, neurossonografia transfontanelar (NTF) realizada por neurorradiologista infantil e quando alterada, foram submetidas à ressonância magnética de encéfalo (RMc). Análise de regressão logística foi realizada para verificar a associação de potenciais fatores de risco para ocorrência de surdez neurossensorial relacionada ao CMV e/ou presença de achados anormais em NTF e/ou RMc. A análise da Curva ROC foi utilizada para avaliar a associação da carga do DNA do CMV expresso em log10, com a presença de sintomas ao nascer, achados anormais em NTF e/ou RMc e ocorrência de surdez neurosensorial. Resultados: Em 66 crianças infectadas, os sinais clínicos sugestivos de infecção congênita ao nascer foram observados em 8/66 (12,12%; IC95%: 5,74-23,03), sendo considerados sintomáticos. As 58 crianças restantes foram considerados assintomáticos. A surdez neurossensorial foi observada em 8/66 (12,12%; IC95%: 5,74-23,03%) das crianças. Destas, 4/58 (6,9%; IC95%: 2,23- 17,54%) e 4/8 (50%; IC95%: 17,44-82,55%) crianças eram assintomáticas e sintomáticas, respectivamente. Achados de NTF e/ou RMc sugestivas de infecção congênita foram observadas em 7 de 8 crianças sintomáticas (87,5%; IC95%: 46,67-99,34%). Dentre as 58 assintomáticas, 53 (92,4%) completaram todas as avaliações e destas 29/52 apresentaram alterações sugestivas de infecção congênita em NTF e/ou RMc (55,7%; IC95%: 41,41 - 69,27%). O achado mais comum foi a vasculopatia lenticuloestriada acompanhado ou não de cistos subpendimários (13/29: 43.3%). Outros achados anormais foram os cistos subpendimários como achado isolado (11/29: 37.9%), calcificação única ou periventricular com ou sem áreas de gliose (4/29: 13,8%) e ventriculomegalia (4/29: 13,8%). Análise de regressão logística mostrou que apenas a presença de sintomas clínicos ao nascer foi preditivo para a ocorrência de surdez neurossensorial. Achados de neuroimagens (NTF e/ou RMc) foram observados em 3 de 4 crianças assintomáticas (75%) enquanto 26 (54,2%) de 48 crianças sem surdez tinham avaliações normais de NTF e/ou RMc (p=0,42; RR:2,38: IC95%: 0,26-21,39). Nenhum dos outros fatores de risco foram independentemente associados com surdez. A presença de plaquetopenia e/ou níveis altos de gama glutamil transferase (?GT) foi associado com a presença de achados anormais em NTF e/ou RMc uma análise univariada. O poder discriminatório pela determinação do ponto de corte do valor da carga do DNA do CMV foi avaliada pela área abaixo da curva ROC (AUC) e não houve associação entre a carga viral e a ocorrência de surdez e/ou achados anormais de NTF e/ou RMc. Conclusões: Embora a triagem neonatal da infecção congênita por CMV permita identificar a maioria das crianças infectadas que são clinicamente assintomáticas ao nascer, uma proporção significante destas crianças poderá ser beneficiada por uma avaliação do sistema nervoso central através de NTF, uma vez que, achados anormais são muito frequentes. Embora não tenha sido possível determinar fatores independentemente preditivos de ocorrência de surdez neurossensorial, os achados de NTF podem ser potenciais fatores preditivos de anormalidades tardias em crianças assintomáticas. / The objectives of this study were to verify the impact of congenital cytomegalovirus (CMV) infection at birth and to evaluate potential prognostic predictors of late abnormalities in a cohort of children with this infection. Methods: By means of a CMV neonatal screening, 66 of 11.957 infants were identified as congenitally infected. Infants with and without clinical abnormalities detectable at birth underwent physical examination, cranial ultrasound performed by a paediatric radiologist and/or cranial magnetic resonance imaging, ocular fundoscopy, and hearing evaluation using evoked otoacoustic emissions and auditory brainstem response. Logistic regression analysis was carried out to verify the association between the risk factors for occurrence of hearing loss related to CMV and/ or abnormal cranial ultrasound findings. ROC curve was plotted using the log10 value of CMV DNA load to evaluate the association between viral load and clinical symptoms at birth, abnormal cranial ultrasound findings and hearing loss. Results: Of all 66 infected children, the clinical signs suggestive of congenital infection at birth were observed in 8/66 (12.12%; IC95%: 5.74- 23.03) symptomatic infants. Sensorineural hearing loss was observed in 8/66 (12,12%; IC95%: 5.74-23.03%) children. Of these, 4/58 (6,9%; IC95%: 2.23-17.54%) and 4/8 (50%; IC95%: 17,44-82.55%) children were asymptomatic and symptomatic, respectively. Cranial ultrasound findings suggestive of congenital infection were observed in 7 of the 8 symptomatic children (87.5%; IC95%: 46.67-99.34%). Among the 58 asymptomatic infants, 53 underwent complete evaluation and 29/52 had abnormal cranial ultrasound results (55,7%; IC95%: 41.41 - 69,27%). The most prevalent findings was lenticulostriate vasculopathy with subependymal pseudocysts present in 13 of the /29 (43.3%) infants with cranial ultrasound. Other abnormal findings were isolated subependymal pseudocysts (11/29: 37.9%); single or periventricular calcifications and/or gliosis (4/29: 13.8%); and ventriculomegaly (4/29: 13,8%). Logistic regression analysis showed that only the presence of clinical findings predicted the occurrence of hearing loss. Cranial ultrasound findings were observed in 3 of asymptomatic infants (75.0%) while 26 (54.2%) of 48 infants with no hearing loss had abnormal imaging features (p=0,42; RR:2,38: IC95%: 0,26-21,39). None of the other factors risk were independely associated with development of hearing loss. The presence of thrombocytopenia and/or high level of gamma-glutamyltranspeptidase (?GT) was associated with cranial ultrasound findings on univariated analysis. No discrimination power was achieved using the area under the ROC curve to verify the association between CMV DNA load in the urine of the infected children and the developing of hearing loss, presence of cranial ultrasound findings and clinical signs at birth. Conclusions: Although a neonatal screening of cCMV will identify the majority of infected infants who are clinically asymptomatic, a significant proportion of them could benefit from a central nervous system image evaluation, since abnormal findings are frequent. Althoug it was not possible to determine risk factors that are independently associated to development of sensorineural hearing loss, cranial ultrasound findings could be a potential prognostic markers of adverse outcomes of congenital CMV in asymptomatic infants.

Lymphocytic choriomeningitis virus (LCMV) infection during pregnancy severely injures the human fetal brain. Neonatal rats inoculated with LCMV are an excellent model of congenital LCMV infection, as they develop neuropathology, including cerebellar injuries, similar to those seen in humans. The goal of this thesis was to determine what underlies brain injury and the differential immune response and to determine the role of T-cells in LCMV induced pathology. First, I examined whether cytokine and chemokine expression after LCMV infection was higher in the cerebellum and olfactory bulbs, which undergo destruction, compared to the hippocampus and septum, which undergo no acute destruction. Second, I used T-cell deficient and T-cell competent animals to evaluate the role of T-lymphocytes in LCMV-induced cerebellar and hippocampus pathology. Finally, I characterized the migration abnormality that develops in the cerebellum after LCMV infection. My results showed that cytokine and chemokine expression is higher in the cerebellum and olfactory bulb than in the hippocampus and septum. Using astrocyte cultures, I determined that astrocytes isolated from the cerebellum have a more robust cytokine response to infection compared to astrocytes from the hippocampus. Furthermore, inoculation of congenitally athymic (rnu/rnu) rats, which are deficient in T-lymphocytes, demonstrated that cerebellar hypoplasia is T-cell independent while cerebellar destruction and abnormal neuron migration is T-cell dependent. In the hippocampus, T-cells protect against loss of dentate granule cells. A study of the migration abnormality determined that LCMV infection disrupts radial glia fibers and extends proliferation of granule cells in a T-cell dependent manner. The findings reported here support a pivotal role of the immune system in regional brain pathology as well as in the disruption of migration.

Vγ9Vδ2 T cells are the main circulating γδ T cells in human adult blood. They are known for their T cell receptor (TCR)-dependent recognition of microbe and endogenous-derived non-peptide pyrophosphate antigens (phosphoantigens, PAg). With the intrinsically biased type 1 immune responses, Vγ9Vδ2 T cells are an important force in the defense of infections and tumors. However, the immune responses of Vγ9Vδ2 T cells in early life infections and in immunotherapies are not clear yet. In this thesis, we explored Vγ9Vδ2 T cell immune responses in both aspects. Vγ9Vδ2 T cells are abundant in human fetal peripheral blood, but compared to their adult counterparts they have a distinct developmental origin, are hyporesponsive towards in vitro phosphoantigen exposure and they do not possess a cytotoxic effector phenotype. In order to obtain insight into the role of Vγ9Vδ2 T cells in the human fetus, we investigated in the first part of this thesis their responses upon in utero infection with the phosphoantigen-producing parasite Toxoplasma gondii (T. gondii). Most congenital infections are caused by viruses, T. gondii is one of the exceptions. The organelle apicoplast present in T. gondii can generate the most potent Vγ9Vδ2 T cell activator. Thus infection in utero with T. gondii makes it a good model to observe Vγ9Vδ2 T cell immune responses in early life. By comparing to age-matched controls, we found that fetal Vγ9Vδ2 T cells were highly expanded in congenital T. gondii infected newborns, and these expanded cells were highly differentiated towards potent cytotoxic effector cells. While the impact of congenital infection on Vγ9Vδ2 T cell expansion and function waned after birth, the Vγ9Vδ2 TCR repertoire of infected infants possessed a clear fetal footprint with public clonotypes, reflecting the Vγ9Vδ2 T cell response in utero. Indeed, verification of the antigen recognition related complementarity-determining region 3 (CDR3) of the TCR for γ and δ chain by high-throughput sequencing revealed the enrichment of three Vδ2 sequences in congenitally-infected infants that are already generated at 8 weeks of gestation. Vγ9Vδ2 T cells possess several characteristics, including MHC-independent recognition of tumor cells and potent killing potential, that make them attractive candidates for cancer immunotherapeutic approaches. In the second part of this thesis we investigated Vγ9Vδ2 T cell responses towards two kinds of hemiparasite plant Viscum album L. (European mistletoe) extract drugs in vitro. Mistletoe therapy is the most used complementary cancer therapy in European countries. Mistletoe extract drugs are considered to benefit for increasing the quality of life of cancer patients and modulate immune cells, but the mechanism of action is not clear. Here, we investigated in-depth the in vitro response of human T cells towards mistletoe extract drugs by analyzing their functional and TCR responses using flow cytometry and high-throughput sequencing respectively. Non-fermented mistletoe-extract drugs (AbnobaViscum), but not their fermented counterparts (Iscador), induced specific expansion of Vγ9Vδ2 T cells among T cells. Furthermore, AbnobaViscum rapidly induced the release of cytotoxic granules and the production of the cytokines IFNγ and TNFα in Vγ9Vδ2 T cells. This stimulation of anti-cancer Vγ9Vδ2 T cells was mediated by the butyrophilin BTN3A, did not depend on the accumulation of endogenous phosphoantigens and involved the same Vγ9Vδ2 TCR repertoire as those of phosphoantigen-reactive Vγ9Vδ2 T cells.In summary, in the first part of this thesis we showed that the human fetus intrinsically possesses a group of Vγ9Vδ2 T cells that are responding to congenital parasite infections that provide potential protective effects to the fetus. In the second part, we provided insight into the in vitro responses of Vγ9Vδ2 T cells towards mistletoe extract drugs, indicating that Vγ9Vδ2 T cells can be an important target in mistletoe therapy. / Doctorat en Sciences biomédicales et pharmaceutiques (Pharmacie) / info:eu-repo/semantics/nonPublished

<p>Cytomegalovirus (CMV) is a widespread infection affecting 50-90% of the human population. A typical silent primary infection is followed by life-long persistence in the host under control by virus-specific CD8 (“killer”) and CD4 (“helper”) T cells. Although harmless in most people, CMV may cause disease and sequelae in patients with deficient cellular immunity, such as AIDS patients, recipients of organ transplants and children who have acquired the virus before birth. In this thesis we have characterized the cellular immunity to CMV in immunocompetent subjects, in patients receiving transplants and in infants.</p><p>In healthy individuals with latent CMV, the frequencies of CMV-specific CD8 T cells varied considerably between the donors. Within the same individual, the changes over time were usually small. In patients with primary, symptomatic CMV infection, the frequencies of CMV-specific CD8 T cells peaked within the first month after the appearance of symptoms. The frequencies then declined to levels similar to those in latently infected CMV carriers. The CD4 T-cell function followed the same pattern, but with lower peak values.</p><p>Immunosuppressed renal transplant patients with latent CMV had CMV-specific CD4 cell function similar to healthy controls. The frequencies of CMV-specific CD8 T cells were also comparable, but their function was impaired. When renal transplant recipients were investigated longitudinally, we found that their CMV-specific T cells decreased rapidly after transplantation. Whereas the frequencies and function of CD8 T cells rebounded within 3 months, CD4 T-cell recovery was impaired during the entire first year after transplantation.</p><p>Finally, the frequencies and function of CMV-specific T-cells were investigated in children with congenital and postnatal CMV. CMV-specific CD8 T cells could be detected in even the youngest children, suggesting that these cells can develop early in life. In contrast, CMV specific CD4 T cells were low or absent in the youngest children but increased slowly with age.</p>

Cytomegalovirus (CMV) is a significant cause of mortality and morbidity in immunocompromised patients and an important cause of birth defects if acquired in utero. The licensed CMV antivirals, ganciclovir, cidofovir and foscarnet, all target the viral DNA polymerase. For each drug prolonged use is associated with significant toxicities and development of drug resistance. None are approved for use during pregnancy. Therefore, development of new anti-CMV drugs that target different pathways would be beneficial. All herpesviruses encode an alkaline nuclease. That genetic disruption of the CMV alkaline nuclease, UL98, reduces CMV replication by 1,000-fold suggests that UL98 may be a useful target for development of novel anti-CMV drugs. Moreover, using herpes simplex virus type 1 Hsiang and Ho found that the anthraquinone emodin inhibits activity of the viral alkaline nuclease, blocks viral replication in cell culture, and reduces viral pathogeneses in a mouse model (Brit. J. of Pharm., 2008). Earlier studies also showed that anthraquinone derivatives including emodin have anti-CMV activity (Barnard et al., Antiviral Research 1992 & 1995), although the mechanism of CMV inhibition has not been further studied. We therefore sought to confirm the anti-CMV activities of emodin and related anthraquinone derivatives, to characterize their mechanisms of action, and to determine specifically if they act through inhibition of UL98. Using a luciferase-based CMV yield reduction assay emodin inhibited CMV replication (IC50 = 4.9 μM); however, that the TD50 for cytotoxicity (determined using an luciferase-based cell viability assay) was only 2-fold higher suggested that emodin may act non-specifically. Two additional anthraquinone derivatives (acid blue 40 and alizarin violet R) inhibited CMV only at high concentrations (IC50 = 238; 265 μM) that were also cytotoxic. Atanyl blue PRL, however, exhibited anti-CMV activity (IC50 = 6.3 μM) with low cytotoxicity (TD50 = 216 μM). Thus, characterization of atanyl blue PRL (impact on gene expression, GFP expression, viral spread, infectivity, time of addition studies, and inhibition of UL98 nuclease activity) should be informative. Atanyl blue PRL appears to block immediate-early gene expression and reduce early and late gene expression. Atanyl blue PRL also blocked GFP expression, reduced viral spread, and also lowered the infectivity of CMV. Finally, atanyl blue PRL inhibits UL98 alkaline nuclease activity at an IC50 of 5.7 μM. This suggests that atanyl blue PRL may inhibit CMV through inhibition of UL98. Thus, atanyl blue PRL represents a novel class of anti-herpesvirals and provides a lead structure for structure based drug discovery.