Le virus Herpes Simplex de type 1 (HSV1) est responsable chez l’homme, son seul hôte naturel, d’infections oculaires cornéennes (kératites) récurrentes, typiquement unilatérales, pouvant induire une perte majeure de la vision. Pendant toute la vie, le virus reste à l’état quiescent (latence) dans le système nerveux, en particulier dans les deux ganglions trigéminés (TG) qui sont responsables de l’innervation sensitive de la cornée. La réactivation du virus à partir de ces TG entraine la kératite. Jusqu’à présent, les seuls traitements disponibles contre HSV1 ne sont que curatifs, c’est à dire qu’ils permettent de contrôler la réactivation que lorsque est déclarée. Il n’existe pour l’instant aucune thérapeutique réellement préventive sur l’ensemble des récidives, en particulier aucun vaccin n’a fait la preuve de son efficacité.Notre équipe a caractérisé un modèle d’infection herpétique par primo-infection orale, qui reproduit chez la souris une grande partie de l’histoire naturelle de l’infection herpétique telle qu’elle est observée chez l’homme. Ce modèle reproduit aussi la latéralisation, puisque la kératite initiale (puis ses récidives éventuelles) n’est observée que du côté inoculé, alors que l’infection latente est retrouvée dans les deux TG. Cependant, cette latence bilatérale n’est pas parfaitement symétrique à l’échelle moléculaire: alors que la charge virale latente (nombre de copies de génome) est similaire entre les deux TG, la production de LAT (Latency-Associated Transcripts) est plus importante du côté inoculé, de même que le nombre de neurones exprimant ces LAT (Cavallero et al., 2014; Maillet et al., 2006). Or, l’expression des LAT, marqueur classique de l’infection latente par HSV1, est associée, d’après la littérature scientifique, à la possibilité ultérieure de réactivation virale. A l’inverse, une infection herpétique sans expression des LAT est considérée comme peu réactivable (Perng et al., 2000). L’asymétrie biologique observée dans notre modèle pourrait donc expliquer la plus forte capacité de réactivation de HSV1 du côté inoculé seulement.L’objectif de l’ensemble de notre projet a été de tenter de contraindre une souche virale sauvage (à virulence normale) à une infection latente mais à capacité réduite de réactivation (sans expression de LAT), c’est à dire comme observé du côté non-inoculé de notre modèle. Pour cela, nous avons étudié l’effet de la primo-infection herpétique d’une souche de HSV1 sur la sensibilité des tissus à héberger l’infection latente par une autre souche virale, inoculée ultérieurement et dans un autre site.Notre avons montré que la primo-infection par une souche de HSV1 a inhibé la pathogénie (morbidité et mortalité) induite une autre souche de HSV1 virulente, inoculée quelques jours après. La primo-infection a contraint cette souche réinfectante à une mise en latence sans réplication au préalable, cette latence ne s’accompagnant pas d’expression de LAT. Cet effet inhibiteur a également été observé lors de l’utilisation d’une souche atténuée, non virulente dans le système nerveux, lors de la primo-infection. L’étude de la réactivation des différentes souches a révélé que lors de l’utilisation d’une souche neurovirulente et réactivable en tant que souche de primo-infection, la souche réinfectante pouvait également réactiver (aussi bien que son contrôle). En revanche, lors d’une primo-infection avec une souche non réactivable, la réactivation de la souche réinfectante était presque entièrement inhibée.La primo-infection par une souche non neurovirulente a contraint une souche, réellement virulente et inoculée secondairement, à une infection latente sans capacité de réactivation. Nous disposons ainsi des bases du développement d’une stratégie réellement préventive de l’infection herpétique récidivante, premier temps d’une éventuelle utilisation à des fins vaccinales. / The Herpes Simplex virus 1 (HSV1), whose only natural hosts are humans, can persist during the whole lifetime in a quiescent state (latent infection) in the nervous system, especially in both trigeminal ganglia (TGs, right and left), which innervate the cornea. The virus can reactivate in the TG, leading to recurrent corneal infections (keratitis) that are typically unilateral and can lead to major vision loss. To date, the only available therapies against HSV1 are curative, i.e. they control the reactivation process only after its onset. Until now, no efficient preventive treatment against HSV1 has been established, and more specifically no vaccine has been shown to be clinically effective.Our team has developed an oro-ocular murine model (based on viral inoculation in the lip), that mimics most of the aspects of the natural history of HSV1 infection in humans. In particular, lateralization is also found in this model, as only the eye ipsilateral to the inoculated lip develops keratitis (initial keratitis and recurrences), while latent virus is found in both TGs with similar levels of viral genome copies. However, the bilateral latency isn’t perfectly symmetrical at the molecular level, since the production of Latency-Associated Transcripts (LATs) and the number of LAT+ neurons are higher in the ipsilateral TG (Cavallero et al., 2014; Maillet et al., 2006). As LAT expression is associated with the capacity of the virus to reactivate, the asymmetry in LAT expression could explain the unilaterality of keratitis events.The aim of this project was to constraint a wild-type HSV1 strain to enter a non-reactivable state of latent infection in the both TGs. As this peculiar type of latent infection is observed only in the controlateral TG following a unilateral primary infection, we hypothesized that this phenomenon is linked to the kinetics of HSV1 infection in the both TGs, respectively. To test this, we studied the impact of a primary HSV1 infection on the behavior (acute phase, latency, LAT expression, capacity of reactivation) of a superinfecting HSV1 strain, inoculated at another anatomical site some days later.We have shown that the primary infection with a HSV1 strain can inhibit the pathogeny (morbidity and mortality) of a superinfecting virulent HSV1 strain, inoculated few days afterwards. Moreover, the superinfecting strain was found to be very rapidly driven in a latent state, with very poor LAT expression. This inhibitory effect also occurred when using a non-neurovirulent strain of HSV1 for the primary infection, with no further ability of the wild-type superinfecting strain to reactivate.These results clearly show that the onset of productive infection in the TGs and later on, latent infection with putative reactivation, is related to the kinetics of infection. These observations may have implications in the future for the potential development of innovative preventive strategies.

Chez l’homme, la primo-infection par le vírus Herpès simplex de type 1 (HSV-1) a lieu au niveau de la muqueuse oro-pharyngée. Après une phase de réplication, les virions pénètrent les terminaisons axonales et migrent vers le ganglion trijumeau ipsilatéral (TG), puis vers le TG controlatéral. Le virus entre alors dans un état de latence dans les 2 TG. Les réactivations d’HSV-1 dans ces neurones sont responsables des kératites herpétiques (KH), unilatérales et survenant toujours du même côté chez un patient donné.En utilisant un modèle murin oro-oculaire, qui reproduit les aspects essentiels de la maladie chez l’homme, nous avons précédemment démontré que l’inoculation virale latéralisée d’un côté de la bouche entraine une réplication virale dans la lèvre, suivie d’une KH ipsilatérale à l’inoculation 6 jours plus tard. De manière concomitante, le virus se propage aux 2 TG, mais les réactivations ne surviennent que du côté ipsilatéral à l’inoculation. Nous avons également observé qu’après une primo-infection dans une lèvre avec une souche d’HSV-1 non-neurovirulente, les souris étaient protégées contre les signes de la maladie et contre la réactivation d’une souche sauvage pleinement virulente inoculée secondairement dans l’autre lèvre (souche ré-infectante). Afin de comprendre les mécanismes immunitaires en jeu dans cet état de protection, nous avons combiné une analyse en cytométrie en flux multiparamétrique à des tests immunologiques, pour quantifier et définir l’infiltrat immunitaire hématopoïétique et les chémokines inflammatoires au site d’inoculation et dans les TG. Nous avons démontré qu’après une inoculation unique avec la souche sauvage virulente, un infiltrat immun riche en cellules pro-inflammatoires, survenaient de manière retardée dans les lèvres inoculées, et persistaient dans les TG. A l’opposé, l’infiltrat immunitaire était plus précoce dans les tissus réinfectés (après une primo-infection par la souche non neurovirulente), plus riche en cellules de l’immunité adaptative, et associé à des concentrations moindres de chémokines inflammatoires. En outre, cet infiltrat s’estompait plus rapidement, avec une disparition concomitante des chémokines inflammatoires. Ces données permettent de mieux comprendre la nature et la cinétique de la réponse immunitaire anti-HSV-1, et pourront être utiles pour le développement futur de stratégies vaccinales anti-HSV-1. / In humans, Herpes simplex virus type 1 (HSV-1), primary infection occurs in the oral mucocutaneous tissues. Virions replicated here penetrate sensitive neuronal axons, migrate to both trigeminal ganglion (TG) where it established a lifelong latency. Reactivations of HSV-1 in the TG neurons induce clinical recurrences in the connected peripheral tissues. This process is involved in herpes simplex keratitis (HSK), a condition that, strikingly, occurs almost exclusively in the same eye for a given patient. Based on an experimental oro-ocular (OO) model of HSV-1 infection, that recapitulates most of these human clinical features, we previously demonstrated that a virus inoculation on one side of the mouth, leads to viral replication in the lip, followed by HSK. Virus concomitantly disseminates to both TG, but reactivation only occurs in the TG ipsilateral to the inoculation site. We also observed that after a primary inoculation with a non-neurovirulent strain of HSV-1 in one lip, mice are protected against both acute phase disease and reactivation after a superinfection with a fully virulent wild-type strain of HSV-1 in the contralateral lip.In order to understand the underlying mechanisms involved in this state of protection, we combined high resolution flow cytometry and bead-based immunoassays, to quantify hematopoietic subsets and inflammatory chemokines in the site of inoculation and in the TG. We demonstrated that after a single inoculation with the wild-type strain, a delayed immune infiltrate, boasting more proinflammatory subsets, occurred in the lip and persisted in the TG. In contrast, the immune infiltrate occurred earlier in the superinfected lip and ipsilateral TG, with less inflammatory chemokines but more adaptive immune subsets. Moreover, cellular infiltrate resolved faster, correlating with nullification of inflammatory chemokines locally. These data show that immune response kinetics influence the development of natural immunity to HSV-1, and can be harnessed to protect against disease and reactivations.

Numerous factors that affect herpes simplex virus 1 (HSV1)-mediated pathogenesis have been identified. Such factors directly impact the replication of HSV1 as well as modulate host immune responses following HSV1 infection. In this work, I characterize how HSV1 replication is impacted by expression of the protein encoded by C21orf91, or “Cold Sore Susceptibility Gene” (CSSG1), that has been linked to HSV1 reactivation in humans. I investigated expression of CSSG1 mRNA expression in various tissues and found that CSSG1 mRNA was present in several tissues of importance in HSV1 disease, including brain, trigeminal ganglia (TG), cornea and spleen. Western blot analysis demonstrated that CSSG1 protein is expressed in human cells. Subcellular fractionation analysis reveals that CSSG1 is predominantly found in the cell nucleus, where it colocalizes with chromatin and with Tip60, a chromatin-binding histone modifying protein that has been shown to be essential for the replication of herpesviruses. I also discovered that CSSG1 is present in the cytosol of cells where it forms large cytosolic aggregates in presence of TRAF6, a downstream adapter that plays an important role in innate immune receptor signaling. To determine if CSSG1 directly impacts viral replication, I generated CSSG1 knockdown human cell lines. I found that HSV1 replication was reduced in CSSG1 knockdown cells compared to control cells, whereas replication of the unrelated virus, vesicular stomatitis virus (VSV), was not affected by knockdown of CSSG1. I demonstrate that CSSG1 was necessary for efficient expression of HSV1 viral proteins during infection. Western blot analysis and measurement of expression of HSV1 proteins expressed at various stages of viral replication illustrates that CSSG1 was required for HSV1 replication at very early stage of infection. I also noted that CSSG1 expression impacted the DNA damage response in HSV1 infected cells. Levels of H2AX phosphorylation, a marker of the DNA damage response, were increased in HSV1-infected CSSG1 knockdown cells compared to control cells. DNA damage responses are thought to promote HSV1 reactivation from latency and HSV1 gene expression, indicating a potential mechanism for role of CSSG1 in HSV1 replication through modulating the DNA damage response. Overall, my work demonstrates that CSSG1 affects HSV1 replication and provides insight on how CSSG1 polymorphisms in humans could affect HSV1 reactivation and replication to promote cold sores. These discoveries may also lead to a better understanding of pathogenesis of other herpesviruses in humans.

No description available.

L'étiologie de la forme sporadique de la maladie d'Alzheimer (AD) reste largement inconnue. Toutefois, une adéquation entre des facteurs environnementaux et génétiques est fortement probable. C'est ainsi que de nombreux arguments suggèrent que le virus herpes simplex de type 1 (HSV1) en infectant et en se répliquant dans le système nerveux central, puisse être un co-facteur impliqué dans le déclenchement et le développement de l'AD. Pour éprouver cette hypothèse, nous avons développé un modèle cellulaire constitué de neurones de rat infectés par HSV1 pour analyser les modifications viro-induites de leur expression génique. Il a été mis en évidence dans les neurones infectés : i) une augmentation de la production du peptide amyloïde Aβ42 et de Tau phosphorylée, ainsi que leur agrégation dans un agrésome intracellulaire ; ii) des variations du niveau de transcription de nombreux gènes très similaires à celles observées chez des patients AD. Par ailleurs, l'étude des mécanismes moléculaires de l'apoptose viro-induite dans ce modèle original a permis de mettre en évidence une corrélation entre l'activation des caspases et la production d'Aβ42 et une corrélation entre le phénomène d'apoptose avortée (abortosis) et la formation d'agrésome. De l'ensemble des ces résultats, il apparait que ce modèle cellulaire est représentatif de certains aspects des stades précoces de l'AD et conforte l'hypothèse qu'HSV1 serait un co-facteur dans la maladie d'Alzheimer / The origin of the sporadic form of the Alzheimer's disease (AD) remains still widely unknown. However, an adequacy between environmental and genetic factors is highly probable. Numerous arguments suggest that the virus herpes simplex of type 1 (HSV1) by infecting and replicating in the central nervous system, could be a co-factor involved in the AD process. To evaluate this hypothesis, we set up a model made of rat neurons infected by HSV1 in order to analyse the virally-induced modifications of their gene expression. Using this model we have shown: i) an over-production of the amyloid peptide Aß42 and of phosphorylated form of Tau accompanied by their concentration within an intracellular aggresome; ii) variations of the transcription levels of numerous genes equivalent to that observed in AD patients. Furthermore, the study of the molecular mechanisms underlying the virally-induced apoptosis allowed to point out a correlation between caspase activation and Aß42 production as well as a correlation between abortosis and aggresome formation. All together these results demonstrate that this cellular model represents, at least in part, some aspects of the early stages of AD and bring evidences that HSV1 could be a co-factor in the AD process

O melanoma maligno é um tipo de câncer com grande risco de produzir metástases e com altas taxas de mortalidade resultantes de diagnósticos tardios e falta de tratamentos eficazes. Ao longo dos últimos anos, a terapia gênica voltada para o câncer e o desenvolvimento de métodos capazes de visualizar processos moleculares e celulares ao longo da terapia, tem recebido especial atenção. Diante deste quadro, nossos objetivos foram utilizar o sistema de Tomografia por Emissão de Pósitrons (PET) para diagnosticar precocemente tumores e investigar a eficácia terapêutica de uma nova imunoterapia em um modelo animal de melanoma metastático. Visando atingir esses objetivos, padronizou-se a síntese e realizou-se o controle de qualidade do 9- [4-18F-fluoro-3-hidroximetil-butil) guanina, [18F] FHBG, considerado o padrão-ouro em estudos clínicos, para acompanhamento de terapia gênica por PET. Métodos: Sintetizou-se o [18F] FHBG, por substituição nucleofílica tipo 2 do precursor tosilato com [18F-] fluoreto de potássio /Kryptofix 2.2.2, seguido de desproteção com HCl 1 M e purificação por HPLC. A identidade química, pureza radioquímica e atividade específica do [18F] FHBG foram determinadas por Cromatografia Líquida de Alta Eficiência (CLAE). Introduziu-se o gene de timidina quinase (TK) com o vetor retroviral pCL-TK nas linhagens B16F10 (melanoma murino) e LLC (carcinoma de pulmão murino). Os estudos de captação in vitro dos radiotraçadores [18F] FHBG e [18F] FDG foram realizados nas linhagens celulares tumorais murinas transduzidas ou não com a proteína TK. Para os estudos in vivo, camundongos C57BL6 previamente inoculados intravenosamente com células de melanoma expressando a enzima TK, foram imageados subsequentemente utilizando os radiotraçadores [18F] FDG e [18F] FHBG. A eficácia da imunoterapia foi testada em modelo profilático e terapêutico animal de melanoma metastático. Resultados: O tempo de síntese total do [18F] FHBG variou entre 80-150 minutos. O rendimento radioquímico variou entre 1-4%, (n = 19) decaimento corrigido. A pureza radioquímica foi superior a 99% e a atividade específica variou entre 0,14GBq/μmoL-0,21GBq/μmoL. Com a introdução do gene timidina quinase (TK), obtiveram-se as linhagens repórter B16F10-TK e LLC-TK, para os estudos in vitro. As células B16F10 e LLC, expressando GFP foram utilizadas como linhagens controles. Estudos in vitro com o [18F] FHBG revelaram uma captação cerca de 4 vezes maior em células que expressam TK (B16-TK e LLC-TK) em comparação com as células controle GFP. O [18F] FDG apenas captou cerca de duas vezes mais em células TK do que em células que expressam GFP. A detecção de tumores em modelo animal de metástase pulmonar com o [18F] FDG ocorreu a partir de 15 dias do estabelecimento das lesões. No entanto, nos estudos in vivo com [18F] FHBG, houve captação apenas na região intestinal, durante as três semanas em que os animais foram acompanhados. A imunoterapia com células tratadas pela combinação de p19Arf e IFNβ, em camundongos C57BL6 com metástase pulmonar, conferiu redução do tamanho dos focos metastáticos aos animais tratados. Conclusões: Neste trabalho padronizou-se a síntese manual do [18F] FHBG, o qual foi avaliado em estudos in vitro e in vivo. Os estudos in vitro confirmaram a especificidade do [18F] FHBG no monitoramento da expressão de HSV1-tk em linhagens celulares. No entanto, o [18F] FHBG não se acumulou nas lesões metastáticas in vivo e estudos posteriores serão necessários para uma melhor caracterização utilizando o [18F] FHBG. O resultado do tratamento combinado de p19Arf e IFNβ foi promissor para o tratamento de lesões metastáticas. / Malignant melanoma is a type of cancer with a great risk of producing metastases and with high mortality rates resulting from late diagnosis and lack of effective treatments. Over the past few years, directed gene therapy for cancer and the development of methods to visualize molecular and cellular processes throughout therapy, have received special attention. In this context, our aim was to use the Positron Emission Tomography (PET) system, as a tool, for early detection of tumors and investigate the therapeutic efficacy of a new immunotherapy in an animal model of metastatic melanoma. To achieving these goals, the synthesis of [18F] FHBG, the gold standard in clinical studies for monitoring gene therapy by PET, was standardized and the quality control was performed. We also present the results of in vitro uptake and in vivo evaluation of [18F] FHBG, compared to [18F] FDG, the most commonly used radiopharmaceutical for diagnosis in oncology by PET. The vaccine was derived from transduced B16F10-TK cells with the adenoviral vectors AdRGDPGp19Arf and AdRGDPGIFNβ. Methods: [18F] FHBG was synthesized by type 2 nucleophilic radiofluorination of a tosylate precursor with [18F-] potassium fluoride / Kryptofix 2.2.2, followed by deprotection with 1N HCl and purification by HPLC. The chemical identity, radiochemical purity and specific activity of [18F] FHBG were determined by High Performance Liquid Chromatography (HPLC). The thymidine kinase (TK) gene was introduced with the pCL-TK retroviral vector into the B16F10 (murine melanoma) and LLC (murine lung carcinoma) lines. In vitro uptake studies of [18F] FHBG and [18F] FDG were performed on cell lines transduced or not with TK protein. For in vivo studies, C57BL6 mice, previously injected with HSV1tk expressing tumors, were subsequently imaged using the [18F] FDG and [18F] FHBG radiotracers. The efficacy of immunotherapy was tested in a prophylactic and therapeutic animal model of metastatic melanoma. Results: The total synthesis time of [18F] FHBG ranged from 80-150 min. The radiochemical yield ranged from 1-4%, (n = 19) corrected decay. Radiochemical purity was greater than 99% and the specific activity ranged from 0.14GBq / μmoL- 0.21GBq / μmoL. With the introduction of the thymidine kinase (TK) gene, the B16F10-TK and LLC-TK reporter lines were obtained for in vitro studies, B16F10 cells and LLC, expressing GFP, were used as controls. In vitro studies with [18F] FHBG revealed about 4-fold uptake in TK-expressing cells (B16-TK and LLC-TK) compared to GFP control cells. [18F] FDG binds only about twice as much in TK cells as in cells expressing GFP. The detection of tumors in an animal model of pulmonary metastasis with [18F] FDG occurred 15 days after lesion establishment. However, the in vivo studies with [18F] FHBG, the uptake was only found in the intestinal region, over the 3 weeks in which the mice were followed. Immunotherapy with cells treated by the combination of p19Arf and IFNβ, in C57BL6 mice with pulmonary metastasis, reduced the size of the metastatic foci in treated animals. Conclusions: In this study we demonstrate the standardization of [18F] FHBG synthesis and its use in in vitro and in vivo. The in vitro studies have confirmed the specificity of [18F] FHBG to monitor HSV1-tk expression in cell lines. However, [18F] FHBG did not accumulate in the metastatic lesions in vivo and further studies will be required for a better characterization using [18F] FHBG. The outcome of the combined treatment of p19Arf and IFNβ was promising for the treatment of metastatic lesions.

O melanoma maligno é um tipo de câncer com grande risco de produzir metástases e com altas taxas de mortalidade resultantes de diagnósticos tardios e falta de tratamentos eficazes. Ao longo dos últimos anos, a terapia gênica voltada para o câncer e o desenvolvimento de métodos capazes de visualizar processos moleculares e celulares ao longo da terapia, tem recebido especial atenção. Diante deste quadro, nossos objetivos foram utilizar o sistema de Tomografia por Emissão de Pósitrons (PET) para diagnosticar precocemente tumores e investigar a eficácia terapêutica de uma nova imunoterapia em um modelo animal de melanoma metastático. Visando atingir esses objetivos, padronizou-se a síntese e realizou-se o controle de qualidade do 9- [4-18F-fluoro-3-hidroximetil-butil) guanina, [18F] FHBG, considerado o padrão-ouro em estudos clínicos, para acompanhamento de terapia gênica por PET. Métodos: Sintetizou-se o [18F] FHBG, por substituição nucleofílica tipo 2 do precursor tosilato com [18F-] fluoreto de potássio /Kryptofix 2.2.2, seguido de desproteção com HCl 1 M e purificação por HPLC. A identidade química, pureza radioquímica e atividade específica do [18F] FHBG foram determinadas por Cromatografia Líquida de Alta Eficiência (CLAE). Introduziu-se o gene de timidina quinase (TK) com o vetor retroviral pCL-TK nas linhagens B16F10 (melanoma murino) e LLC (carcinoma de pulmão murino). Os estudos de captação in vitro dos radiotraçadores [18F] FHBG e [18F] FDG foram realizados nas linhagens celulares tumorais murinas transduzidas ou não com a proteína TK. Para os estudos in vivo, camundongos C57BL6 previamente inoculados intravenosamente com células de melanoma expressando a enzima TK, foram imageados subsequentemente utilizando os radiotraçadores [18F] FDG e [18F] FHBG. A eficácia da imunoterapia foi testada em modelo profilático e terapêutico animal de melanoma metastático. Resultados: O tempo de síntese total do [18F] FHBG variou entre 80-150 minutos. O rendimento radioquímico variou entre 1-4%, (n = 19) decaimento corrigido. A pureza radioquímica foi superior a 99% e a atividade específica variou entre 0,14GBq/μmoL-0,21GBq/μmoL. Com a introdução do gene timidina quinase (TK), obtiveram-se as linhagens repórter B16F10-TK e LLC-TK, para os estudos in vitro. As células B16F10 e LLC, expressando GFP foram utilizadas como linhagens controles. Estudos in vitro com o [18F] FHBG revelaram uma captação cerca de 4 vezes maior em células que expressam TK (B16-TK e LLC-TK) em comparação com as células controle GFP. O [18F] FDG apenas captou cerca de duas vezes mais em células TK do que em células que expressam GFP. A detecção de tumores em modelo animal de metástase pulmonar com o [18F] FDG ocorreu a partir de 15 dias do estabelecimento das lesões. No entanto, nos estudos in vivo com [18F] FHBG, houve captação apenas na região intestinal, durante as três semanas em que os animais foram acompanhados. A imunoterapia com células tratadas pela combinação de p19Arf e IFNβ, em camundongos C57BL6 com metástase pulmonar, conferiu redução do tamanho dos focos metastáticos aos animais tratados. Conclusões: Neste trabalho padronizou-se a síntese manual do [18F] FHBG, o qual foi avaliado em estudos in vitro e in vivo. Os estudos in vitro confirmaram a especificidade do [18F] FHBG no monitoramento da expressão de HSV1-tk em linhagens celulares. No entanto, o [18F] FHBG não se acumulou nas lesões metastáticas in vivo e estudos posteriores serão necessários para uma melhor caracterização utilizando o [18F] FHBG. O resultado do tratamento combinado de p19Arf e IFNβ foi promissor para o tratamento de lesões metastáticas. / Malignant melanoma is a type of cancer with a great risk of producing metastases and with high mortality rates resulting from late diagnosis and lack of effective treatments. Over the past few years, directed gene therapy for cancer and the development of methods to visualize molecular and cellular processes throughout therapy, have received special attention. In this context, our aim was to use the Positron Emission Tomography (PET) system, as a tool, for early detection of tumors and investigate the therapeutic efficacy of a new immunotherapy in an animal model of metastatic melanoma. To achieving these goals, the synthesis of [18F] FHBG, the gold standard in clinical studies for monitoring gene therapy by PET, was standardized and the quality control was performed. We also present the results of in vitro uptake and in vivo evaluation of [18F] FHBG, compared to [18F] FDG, the most commonly used radiopharmaceutical for diagnosis in oncology by PET. The vaccine was derived from transduced B16F10-TK cells with the adenoviral vectors AdRGDPGp19Arf and AdRGDPGIFNβ. Methods: [18F] FHBG was synthesized by type 2 nucleophilic radiofluorination of a tosylate precursor with [18F-] potassium fluoride / Kryptofix 2.2.2, followed by deprotection with 1N HCl and purification by HPLC. The chemical identity, radiochemical purity and specific activity of [18F] FHBG were determined by High Performance Liquid Chromatography (HPLC). The thymidine kinase (TK) gene was introduced with the pCL-TK retroviral vector into the B16F10 (murine melanoma) and LLC (murine lung carcinoma) lines. In vitro uptake studies of [18F] FHBG and [18F] FDG were performed on cell lines transduced or not with TK protein. For in vivo studies, C57BL6 mice, previously injected with HSV1tk expressing tumors, were subsequently imaged using the [18F] FDG and [18F] FHBG radiotracers. The efficacy of immunotherapy was tested in a prophylactic and therapeutic animal model of metastatic melanoma. Results: The total synthesis time of [18F] FHBG ranged from 80-150 min. The radiochemical yield ranged from 1-4%, (n = 19) corrected decay. Radiochemical purity was greater than 99% and the specific activity ranged from 0.14GBq / μmoL- 0.21GBq / μmoL. With the introduction of the thymidine kinase (TK) gene, the B16F10-TK and LLC-TK reporter lines were obtained for in vitro studies, B16F10 cells and LLC, expressing GFP, were used as controls. In vitro studies with [18F] FHBG revealed about 4-fold uptake in TK-expressing cells (B16-TK and LLC-TK) compared to GFP control cells. [18F] FDG binds only about twice as much in TK cells as in cells expressing GFP. The detection of tumors in an animal model of pulmonary metastasis with [18F] FDG occurred 15 days after lesion establishment. However, the in vivo studies with [18F] FHBG, the uptake was only found in the intestinal region, over the 3 weeks in which the mice were followed. Immunotherapy with cells treated by the combination of p19Arf and IFNβ, in C57BL6 mice with pulmonary metastasis, reduced the size of the metastatic foci in treated animals. Conclusions: In this study we demonstrate the standardization of [18F] FHBG synthesis and its use in in vitro and in vivo. The in vitro studies have confirmed the specificity of [18F] FHBG to monitor HSV1-tk expression in cell lines. However, [18F] FHBG did not accumulate in the metastatic lesions in vivo and further studies will be required for a better characterization using [18F] FHBG. The outcome of the combined treatment of p19Arf and IFNβ was promising for the treatment of metastatic lesions.

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Introduction: Cognitive impairment is converted for 15% to Alzheimer’s disease (AD), which is incurable. The aim of this study is to investigate whether Herpes simplex virus type 1 (HSV1) and its interaction with allele ε4 of Apolipoprotein E (ApoE-ε4) may be possible risk factors for cognitive impairment. Here, suggestions for implementation of Precision Public Health (PPH) is also presented; population studies of relevant biomarkers of infectious burden and data from health outcomes with the aim of remedying public health crises, monitoring diseases, anticipating risks and using public health initiatives adapted to risk groups to increase understanding of diseases, such as AD. Method: An analysis of 1013 people aged 75 from the cohort, Prospective Investigation of the Vasculature in Uppsala Seniors (PIVUS), was performed. Linear mixed models (LMM) were used to investigate whether anti-HSV1 IgG and ApoE-ε4 are associated with risk of developing cognitive impairment over 5 years. Anti-HSV1 IgG in serum was detected by Enzyme-linked immunosorbent assays and cognitive impairment was examined by Mini Mental State Examination. Results: Neither anti-HSV1 IgG positivity nor its interaction with ApoE-ε4 were associated with cognitive impairment. Discussion: Studies of HSV1 without HSV2 or HSV show mixed results. Therefore, we do not yet have sufficient evidence to implement PPH-interventions against these risks. Other cognitive tests that are more sensitive to early changes and adjustment for underrepresented groups may potentially contribute to a more accurate analysis. / Bakgrund: Kognitiv nedsättning övergår för 15 % till Alzheimers sjukdom (AD) som är obotlig. Denna studie har målsättningen att undersöka om Herpes simplex virus typ 1 (HSV1) och dess interaktion med riskvarianten allel ε4 av genen Apolipoprotein E (ApoE-ε4) kan vara möjliga riskfaktorer för kognitiv nedsättning. I arbetet diskuteras även hur man genom Precision Public Health (PPH) kan studera befolkningen med avseende på relevanta biomarkörer och data från hälsoutfall med syfte att avhjälpa folkhälsokriser, övervaka sjukdomar och förutse risker såsom sjunkande kognition och med folkhälsoinsatser anpassade till riskgrupper öka förståelsen av dessa tillstånd. Metod: En analys av 1013 personer 75 år ur kohorten ”Prospecive Investigation of the Vasculature in Uppsala Seniors” (PIVUS). Linear mixed models (LMM) användes för att undersöka om anti-HSV1 IgG och ApoE-ε4 är associerade med kognitiv nedsättning under 5 år. Anti-HSV1 IgG i serum detekterades med ”Enzyme-linked immunosorbent assays” och kognitiv nedsättning undersöktes med Mini Mental State Examination. Resultat: Anti-HSV1 IgG positivitet var inte associerat med kognitiv nedsättning. Anti-HSV1 IgG positivitet och samtidig ApoE-ε4 var inte heller associerat med kognitiv nedsättning. Diskussion: Andra studier av endast HSV1 utan HSV2 eller HSV visar blandade resultat, varför kunskapsläget idag inte stödjer tillämpning av PPH-interventioner mot dessa risker. Andra kognitiva mått som är känsligare för små förändringar nära intakt kognition samt justering för underrepresenterade grupper kan möjligen bidra till säkrare analys.