Vaccination has proven a cost-effective method of managing infectious diseases, but attempts to develop an effective fetal vaccine have proven difficult due to the immaturity of the immune system and the propensity of the developing immune system to induce tolerance to immunizing antigens. This thesis is concerned with the induction of specific immunity in the second trimester ovine fetus using the oral DNA immunization method. In utero oral delivery of naked DNA plasmid was selected as the method of immunization due to previous successes in the third trimester ovine fetus and the immunostimulatory properties of the bacterial DNA backbone, which may help overcome developmental tolerance. Transfection and expression studies in the third trimester ovine fetus revealed the oral mucosal epithelium as the primary site of transgene expression and functionally active antigen was also localized to lymph nodes draining the oral cavity. Efficient transfection and expression of plasmid following oral delivery was specific to the fetus and correlated with a lesser degree of epithelial differentiation. Oral DNA delivery in the second trimester resulted in detection of transgene activity in 100% of treated fetuses and the level of transgene activity was greater than in fetuses treated in the mid-third trimester. Using a plasmid encoding the gene for bovine herpesvirus-1 truncated glycoprotein D (tgD), immunization studies were then conducted in the second trimester fetus. A new lower age limit for fetal immunization was established at 55-60 days gestation (gestation period is 148 days), which coincides with the appearance of lymphocytes in peripheral tissues. Antigen-specific antibody, interferon-× responses and/or neonatal anamnestic responses were detected in 66% of fetuses immunized between 55 and 84 days gestation. The duration of fetal primary immune responses was equivalent to that achieved in young lambs following optimized DNA vaccination, but the magnitude of fetal immune responses was limited. The persistence of immune memory from the second trimester to birth was consistent with experimental data which showed that the duration of immune memory had a stronger correlation to the duration, as compared to the magnitude, of the primary antibody response. Overall, the experiments within showed that oral DNA immunization of the early second trimester fetus is feasible and not associated with the induction of tolerance. These findings suggest that it may be possible to protect against mother-to-child transmission of infectious pathogens by targeting protection at the level of the fetus.

Vaccination has proven a cost-effective method of managing infectious diseases, but attempts to develop an effective fetal vaccine have proven difficult due to the immaturity of the immune system and the propensity of the developing immune system to induce tolerance to immunizing antigens. This thesis is concerned with the induction of specific immunity in the second trimester ovine fetus using the oral DNA immunization method. In utero oral delivery of naked DNA plasmid was selected as the method of immunization due to previous successes in the third trimester ovine fetus and the immunostimulatory properties of the bacterial DNA backbone, which may help overcome developmental tolerance. Transfection and expression studies in the third trimester ovine fetus revealed the oral mucosal epithelium as the primary site of transgene expression and functionally active antigen was also localized to lymph nodes draining the oral cavity. Efficient transfection and expression of plasmid following oral delivery was specific to the fetus and correlated with a lesser degree of epithelial differentiation. Oral DNA delivery in the second trimester resulted in detection of transgene activity in 100% of treated fetuses and the level of transgene activity was greater than in fetuses treated in the mid-third trimester. Using a plasmid encoding the gene for bovine herpesvirus-1 truncated glycoprotein D (tgD), immunization studies were then conducted in the second trimester fetus. A new lower age limit for fetal immunization was established at 55-60 days gestation (gestation period is 148 days), which coincides with the appearance of lymphocytes in peripheral tissues. Antigen-specific antibody, interferon-× responses and/or neonatal anamnestic responses were detected in 66% of fetuses immunized between 55 and 84 days gestation. The duration of fetal primary immune responses was equivalent to that achieved in young lambs following optimized DNA vaccination, but the magnitude of fetal immune responses was limited. The persistence of immune memory from the second trimester to birth was consistent with experimental data which showed that the duration of immune memory had a stronger correlation to the duration, as compared to the magnitude, of the primary antibody response. Overall, the experiments within showed that oral DNA immunization of the early second trimester fetus is feasible and not associated with the induction of tolerance. These findings suggest that it may be possible to protect against mother-to-child transmission of infectious pathogens by targeting protection at the level of the fetus.

名古屋大学博士学位論文 学位の種類 : 博士(医学)(課程) 学位授与年月日:平成25年1月31日 Tevfik Tolga SAHIN氏の博士論文として提出された

Human Herpes Simplex viruses and Simian Herpes Simplex viruses share a high degree of genome homology, but despite this, important differences arise when the viruses are compared at the level of gene expression and virulence in non-host primates. In Human Herpes viruses (HSV-1 and HSV-2); 5 genes (RL02, US01, RS01, UL54 and US12) are expressed with an immediate early kinetics, i.e. their transcriptional activation does not require de novo synthesis of host or viral factors. The five immediate early (IE) genes regulate the cascade of expression of the other early and late HSV genes. Literature indicates that in HSV-1 infections, ICP4, ICP27 and to a lesser extent, ICP0, are mandatory for the full expression of the early and late gene classes. In contrast, our data on the Simian simplexviruses SA8, HVP-2 and B virus indicate that ICP0 (RL2) is the only gene with true IE kinetics. It is possible that in Simian Herpes viruses, ICP0 is necessary for the expression of all other viral genes, and to test this hypothesis I have cloned and expressed in Vero cells the ICP0 protein for the simian simplexvirus SA8 and studied its effect on the SA8 genes that are homologous to the immediate early genes in HSV. Results demonstrate that ICP0 does not appear to be sufficient to activate the transcription of the other IE genes but it is likely that ICP0 functionality is a necessary component in the activation process.

Herpetic stromal keratitis (HSK) is an immunopathological and tissue destructive corneal lesion caused by herpes simplex virus (HSV) infection, which induces an intense inflammatory response and finally leads to blindness. Accumulating evidence using the murine model has shown that Th-1 phenotype CD4+ T cells orchestrating the inflammation mainly contribute to the immunopathological reaction in HSV-1 infected cornea. Initially various innate immune cells recruit and produce numerous inflammatory and angiogenic molecules into the corneal stroma those in turn drive the corneal immunopathology. While the basic principles of immunity to the influenza A viruses (IAV) are probably similar for all vertebrates, detailed understanding is based largely on experiments in laboratory mice. Virus clearance is normally mediated via CD8+ effector T cells but, in their absence, the class-switched antibody response can ultimately achieve the same goal. Influenza virus-specific plasma cells and CD8+ T cells persist in the long term and the recall of the CD8+ T cell response can lead to earlier virus clearance. The first part (Part I) of this dissertation focuses on the understanding of HSV-1 induced immunoinflammatory processes in the cornea and the secondary lymphoid tissues and the involvement of immuno-modulatory mechanisms following acute viral infections such as HSV and IAV. The next three parts (Part II-IV) focus on different inflammatory and counter-inflammatory mechanisms that are activated following acute viral infections. Results in Part II evaluate the role of small molecule inhibitors of VEGFR2/src kinase inhibitors in controlling the progression of the inflammatory lesions after ocular HSV infection. Results of the third section show that the host counter inflammatory mechanisms inhibit tissue damage but these may also act to constrain the effectiveness of immunity to acute infections. The fourth section describes the functional significance of HVEM expression on regulatory T cell in their expansion following HSV-1 infection. In this study, experiments were designed to understand the mechanisms involved in the regulation of immunity and resultant immunopathology using HSV-1 and IAV as the model systems and that modulation of these processes can enhance immune response and diminish immunopathology following acute infections.

Kaposi�s sarcoma (KS) is a peculiar vascular neoplasm that occurs mainly in elderly Mediterranean men and patients with acquired immunodeficiency syndrome (AIDS). The current literature indicates that KS is initiated by the human herpes virus 8 (HHV8) as a reactive polyclonal process but with deregulation of oncogene and tumour suppressor genes, it can progress to a true malignancy with monoclonality. Clinically, classical KS often presents as an indolent disease affecting mainly the lower extremities whereas AIDS-related KS has no site predilection and can progress rapidly with systemic involvement. Histologically, KS can be classified into patch, plaque and nodular stages. Interestingly, classical and AIDS-related KS are indistinguishable histologically and this suggests that AIDS-related KS and classical KS might be initiated by a common aetiology but given their different clinical courses, they may progress through different mechanisms. In view of the importance of the cell cycle proteins in the development and progression of many human malignancies, this thesis aims to examine the role of these proteins in the progression of the two main clinical subtypes of KS. The cell cycle protein expressions in a cohort of 47 patients with KS with welldocumented clinical and histological features were studied. Using a monclonal antibody against the latent nuclear antigen-1 molecule of HHV8, HHV8 was detected in 78% of the cases. The more advanced nodular lesions were found to have a higher level of proliferative activity as measured by the proliferation x marker, Ki-67. This suggests it is valid to use the histological specimens as a tumour progression model of KS. The role of the Rb/cyclin D1/p16 pathway was examined. The more advanced nodular stage KS lesions were more likely to be positive for cyclin D1, suggesting that cyclin D1 is important in the progression from patch stage to nodular stage. p16 acts as a tumour suppressor and it has an inhibitory effect on cyclin D1. The p16 expression rate was low in early stage KS but high in the more advanced lesions. It seems that reduced p16 expression occurs early in KS and may be important in its development. The rate of Rb expression, on the other hand, did not differ significantly among the histological subtypes. The results revealed the significant role of the Rb/cyclin D1/p16 pathway in the progression of KS. Of the mitotic cyclins examined, cyclin A expression was correlated with the advanced tumor stage. The rate of p34cdc2 expression was high in the lesions and there was no correlation with histological stage. This suggests that p34cdc2 is important in the early development of the tumour but not necessarily in its progression. Along the p53-apoptotic pathway, mutant p53 expression was significantly more common in the nodular stage. The cyclin G1 (a protooncogene, one of the target genes of p53) expression also paralleled that of mutant p53 with the majority of the KS lesions showing cyclin G1 expression and significant xi correlation between advanced histological stage and increasing rate of cyclin G1 expression. These findings suggest that progression along the p53 pathway may be important in the advanced stage development of KS. On the other hand, expression of the CDK inhibitor, p27, a protein that normally negatively regulates cyclin G1, was reduced in nodular KS. These findings suggest that some KS lesions may progress through a deregulated or abnormal p53 pathway. There were correlations between cyclin D1, cyclin A, cyclin G1, mutant p53 and negative HIV status. The findings suggest that components of both the Rb/cyclin D1/p16 and p53-apoptotic pathways are important in the progression of classical KS. Rb protein was the only cell cycle protein whose rate of expression correlated significantly with HHV8 status in KS. The majority of HHV8 positive lesions were also positive for Rb protein, unlike HHV8 negative lesions. This suggests that some of the HHV8 negative lesions can progress through a defective Rb pathway whereas the role of Rb in the progression may not be as important in the HHV8 positive lesions. This was an unexpected finding given that one of the postulated mechanisms of tumour initiation by the HHV8 virus is via the viral cyclin it produces. The viral cyclin produced by HHV8 acts through the Rb pathway much the same as cyclin D1 and one would have expected that HHV8 positive cases are less likely to be positive for the Rb protein. In summary, the majority of the KS lesions examined in this thesis show HHV8 infection. The Rb/cyclin D1/p16 pathway appears to be important in the progression of the different stages of KS and expression of the proteins involved in the p53 pathway were found to be important in the advanced stages of the development of KS. There were differential expressions of cell cycle proteins between AIDS-related and classical KS, and between HHV8 positive and HHV8 negative lesions. The findings also provided some clues to the possible mechanisms of development in KS lesions that were not initiated by HHV8.

Orientador: Tereza Cristina Cardoso / Coorientador: Alício Martins Júnior / Banca: Fernanda da Cruz Landim e Alvarenga / Banca: Eunice Oba / Resumo: Este estudo foi delineado com objetivo de verificar os efeitos da exposição experimental do sêmen fresco bovino ao BoHV-5, previamente à congelação, através de análises in vitro e de produção de embriões. Os seguintes testes foram avaliados no Grupo I (sêmen não exposto ao vírus) e II (sêmen exposto ao vírus): motilidade e movimento espermático, através de análise computadorizada, integridade da membrana plasmática/acrossomal (IMPA) e função mitocondrial (FM), utilizando sondas fluorescentes, translocação da fosfatidilserina (PS) na membrana do espermatozóide, com Anexina-V, desenvolvimento embrionário in vitro, evidenciado pela proporção de embriões gerados, detecção viral em espermatozóides e embriões, através do emprego da técnica de hibridização in situ (ISH) e PCR. Nenhuma diferença significativa foi observada para IMPA e FM entre os grupos. Entretanto, resultados da translocação da PS no Grupo II foram significativamente diferentes (P=0,00005) dos encontrados no Grupo I; valores maiores (P<0,05) para velocidade média (VAP), velocidade curvilínea (VCL) e amplitude de deslocamento lateral da cabeça (ALH) e menores para a retilinearidade (STR) e linearidade (LIN) foram obtidos no Grupo II quando comparados com o Grupo I. Concluindo, a ISH revelou a presença de DNA viral nos espermatozóides e embriões infectados; além disso, o BoHV-5 levou à alterações funcional e morfológica nos espermatozóides, sem comprometer o desenvolvimento embrionário, mesmo após ser carreado para os embriões, através da infecção dos oócitos na fertilização in vitro / Abstract: This study was designed to verify the effects of experimental exposure of fresh bull semen to BoHV-5, previously to the freezing process, on in vitro sperm evaluations and embryo production. The following tests were performed in groups I (semen not exposed to virus) and II (semen exposed to virus): sperm motility and movement by computerized analysis (CASA), plasma/acrosomal membrane integrity (PAMI) and mitochondrial function (MF) by fluorocromes dyes, sperm membrane phosphatidylserine (PS) transposition using Annexin-V, in vitro embryonic development evidenced by the proportion of yielded embryos, and viral detection in sperm and embryos performed by in situ hybridization (ISH) assay and PCR. No significant difference was observed for PAMI and MF between groups. However, results of PS transposition in group II were significantly different (P=0.00005) from those found in group I; higher values (P<0.05) for mean velocity (VAP), curvilinear velocity (VCL) and lateral head displacement (ALH) and lower values for straightness (STR) and linearity (LIN) were obtained in group II when compared to those observed in group I. In conclusion, the ISH assay showed the presence of viral DNA on spermatozoa and embryos; besides, BoHV-5 induced functional and morphological sperm alterations, but did not compromise the embryonic development even after being carried to the embryos through the IVF infected oocytes / Mestre

Made available in DSpace on 2014-06-11T19:27:17Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-03-29Bitstream added on 2014-06-13T18:31:15Z : No. of bitstreams: 1 souza_dg_me_araca.pdf: 1858727 bytes, checksum: 571362381118ee9810c1695198ec90f5 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Este estudo foi delineado com objetivo de verificar os efeitos da exposição experimental do sêmen fresco bovino ao BoHV-5, previamente à congelação, através de análises in vitro e de produção de embriões. Os seguintes testes foram avaliados no Grupo I (sêmen não exposto ao vírus) e II (sêmen exposto ao vírus): motilidade e movimento espermático, através de análise computadorizada, integridade da membrana plasmática/acrossomal (IMPA) e função mitocondrial (FM), utilizando sondas fluorescentes, translocação da fosfatidilserina (PS) na membrana do espermatozóide, com Anexina-V, desenvolvimento embrionário in vitro, evidenciado pela proporção de embriões gerados, detecção viral em espermatozóides e embriões, através do emprego da técnica de hibridização in situ (ISH) e PCR. Nenhuma diferença significativa foi observada para IMPA e FM entre os grupos. Entretanto, resultados da translocação da PS no Grupo II foram significativamente diferentes (P=0,00005) dos encontrados no Grupo I; valores maiores (P<0,05) para velocidade média (VAP), velocidade curvilínea (VCL) e amplitude de deslocamento lateral da cabeça (ALH) e menores para a retilinearidade (STR) e linearidade (LIN) foram obtidos no Grupo II quando comparados com o Grupo I. Concluindo, a ISH revelou a presença de DNA viral nos espermatozóides e embriões infectados; além disso, o BoHV-5 levou à alterações funcional e morfológica nos espermatozóides, sem comprometer o desenvolvimento embrionário, mesmo após ser carreado para os embriões, através da infecção dos oócitos na fertilização in vitro / This study was designed to verify the effects of experimental exposure of fresh bull semen to BoHV-5, previously to the freezing process, on in vitro sperm evaluations and embryo production. The following tests were performed in groups I (semen not exposed to virus) and II (semen exposed to virus): sperm motility and movement by computerized analysis (CASA), plasma/acrosomal membrane integrity (PAMI) and mitochondrial function (MF) by fluorocromes dyes, sperm membrane phosphatidylserine (PS) transposition using Annexin-V, in vitro embryonic development evidenced by the proportion of yielded embryos, and viral detection in sperm and embryos performed by in situ hybridization (ISH) assay and PCR. No significant difference was observed for PAMI and MF between groups. However, results of PS transposition in group II were significantly different (P=0.00005) from those found in group I; higher values (P<0.05) for mean velocity (VAP), curvilinear velocity (VCL) and lateral head displacement (ALH) and lower values for straightness (STR) and linearity (LIN) were obtained in group II when compared to those observed in group I. In conclusion, the ISH assay showed the presence of viral DNA on spermatozoa and embryos; besides, BoHV-5 induced functional and morphological sperm alterations, but did not compromise the embryonic development even after being carried to the embryos through the IVF infected oocytes

B virus diagnosis presents a challenge largely complicated by the asymptomatic infection of rhesus macaques, and extremely pathogenic fatal infections in humans. Humoral detection of antibodies is generally performed using whole virus antigen for which preparation requires strict biosafety measures and specialized BSL-4 facilities. As an alternative to utilizing B virus antigen, we describe the production of a truncated form of B virus envelope glycoprotein D, gD 287, in a baculovirus expression system, and evaluate its diagnostic potential as an antigen in recombinant ELISA. After purification and characterization, gD 287 was tested using 22 negative and 72 positive macaque sera samples previously classified using the traditional method. We find that sensitivity and specificity of the recombinant ELISA are dependent on antibody titer of tested serum and gD 287 shows good to excellent predictive potential for identification of positive sera with titers higher than 500.

Diss. (sammanfattning) Stockholm : Karol. inst., 2005. / Härtill 5 uppsatser.