Peritoneal carcinomatosis (PC), the intra-abdominal dissemination of malignancy, is equated with a 5-year survival of 15%, depending on the source. Appendiceal PC is a challenge to treat as cancer cells are embedded in copious amounts of mucin and are difficult to target. Oncolytic viruses (OVs) preferentially replicate and lyse cancer cells and present a targeted, novel strategy for PC. The hypothesis of this study is that appendiceal PC will show variable susceptibility to OVs and that protein expression in these tumours will predict OV replication efficiency. Human appendiceal PC infected ex-vivo with 4 different OVs displayed variable infectivity and replication by fluorescence microscopy and plaque assay. Immunohistochemistry analysis revealed differential expression of IRF3, pERK and TK in tumour compared to normal appendix. No correlation of protein expression with viral replication was observed. Personalizing OV therapy will be critical in the optimization of future care of patients treated with this modality.

A fototerapia com lasers em baixa intensidade de lesões de herpes simples tem sido demonstrada clinicamente ora prevenindo a formação de vesículas, ora cicatrizando rapidamente as lesões e até aumentando o espaço de tempo entre o aparecimento dessas manifestações recorrentes. No entanto, os mecanismos básicos de ação dos lasers nessas situações são desconhecidos. Dessa forma, o objetivo do trabalho foi realizar ensaios in vitro utilizando células epiteliais em cultivo e culturas do vírus HSV-1 para estudar a interferência do laser em baixa intensidade na infecção do HSV-1. Material e Métodos: Culturas de vírus HSV-1 e de células epiteliais de macaco (linhagem Vero) infectadas ou não infectadas, crescidas em déficit nutricional (2 % de soro fetal bovino - sfb) foram utilizadas. As irradiações foram realizadas com um laser de GaAlAs (660 e 780 nm, área focal de 3,6 mm2). Uma, duas e três irradiações com intervalos de 6 h foram realizadas. Os grupos experimentais foram: Controle: não-irradiadas; 660 nm/ 3 J/cm2 (28 s); 660 nm/ 5 J/cm2 (38 s); 780 nm/ 3 J/cm2 (19 s) e, 780 nm/ 5 J/cm2 (25 s). Os efeitos citopáticos do HSV-1 e a viabilidade celular de culturas irradiadas e controles foram analisadas em 4 condições: 1) irradiação das células epiteliais não infectadas; 2) células epiteliais irradiadas antes da infecção; 3) irradiação dos vírus antes da infecção; 4) irradiação das células previamente infectadas pelo HSV-1. A viabilidade celular foi obtida pelo teste da redução do MTT e os efeitos citopáticos por observação em microscopia de luz. Resultados: A viabilidade celular de culturas irradiadas crescidas em déficit nutricional, independentemente do número de irradiações, foi sempre significantemente menor que aquela de culturas não-irradiadas e crescidas nas condições ideais de concentração de sfb (10 %). A viabilidade celular de culturas não infectadas foi similar em todos os grupos. O número de irradiações influenciou o crescimento celular positiva e proporcionalmente ao número de irradiações, exceto para o grupo 660 nm/ 3 J/cm2. Nenhuma diferença nos efeitos citopáticos foi observada entre os grupos, independentemente do número de irradiações nas 3 condições do estudo. A viabilidade celular de todos os grupos não mudou nem pela irradiação das células nem do vírus antes da inoculação nas células. A viabilidade de células infectadas antes da irradiação foi significantemente maior que o controle quando 2 irradiações foram realizadas. Conclusão: Nas condições deste estudo a radiação laser em baixa intensidade é capaz de aumentar o crescimento de células Vero crescidas em déficit, no entanto, não o suficiente para atingir o crescimento característico dessas células crescidas nas suas condições ideais. O número de irradiações influencia o crescimento das células de forma positiva e proporcional ao número de irradiações, exceto para o parâmetro 660 nm/ 3 J/cm2. A radiação laser não altera nem a susceptibilidade das células à infecção, nem a virulência do HSV-1. No entanto, ela prolonga a viabilidade das células infectadas pelo HSV-1. Efeitos positivos da fototerapia que tem sido relatados clinicamente parecem ser devido a efeitos no hospedeiro não relacionados com a replicação viral nas células infectadas. / Purpose: The clinical effects attributed to phototherapy relative to Herpes simplex lesions have included prevention of lesion formation, speeding the healing of lesions, and decreasing the frequency of recurrent lesions. The mechanisms underlying these findings have not been established yet. The aim of this in vitro study was to analyze the effect of phototherapy on epithelial cells, on HSV-1, and on infected epithelial cells in culture. Material and Methods: Cultures of HSV-1 and infected or non-infected monkey epithelial cells (Vero cell line) grown in deficient media (2 % fetal bovine serum-fbs) were used. The laser irradiation was delivered using a GaAlAs laser (660 and 780 nm, focal spot of 3.6 mm2). One, two and three irradiations with 6 hourintervals were done. The experimental groups were: Control: non-irradiated; 660 nm/3 J/cm2 (28 sec); 660 nm/5 J/cm2 (38 sec); 780 nm/3 J/cm2 (19 sec), and 780 nm/5 J/cm2 (25 sec). The HSV-1 cytopatic effects and the cell viability of irradiated cultures and controls were analyzed in four different conditions: 1) irradiation of noninfected epithelial cells; 2) epithelial cells irradiated prior infection; 3) virus irradiated prior infection; and 4) irradiation of HSV-1 infected cells. The cell viability was assessed by the reduction of the MTT test and the cytopatic effects by the light microscopy observation. Results: The cell viability of irradiated cultures grown in nutritional deficit, independently of the irradiation numbers, was always significantly smaller than that of non-irradiated cultures grown at the ideal serum concentration condition (10 %). The cell viability of non-infected cells was similar amongst the groups. The number of irradiations influenced the cell growth positively and proportionally to the number of irradiations, except for the 660 nm/3J/cm2 group. Any variation in cytopatic effects was observed amongst the experimental groups, independently of the irradiation numbers at the 3 conditions analyzed. The cell viability of all experimental groups were not altered either by irradiation of the cells or of the virus prior infection. The viability of infected cells prior irradiation was significantly higher than that of non-irradiated cultures when 2 irradiations were done. Conclusion: The experimental conditions for this study demonstrate that the phototherapy is capable of enhancing the growth of Vero cells grown under nutritional deficit conditions, however, not enough to reach the characteristic cell growth of cells grown at the ideal serum concentration condition. The number of irradiations influences the cell growth positive and proportionally, except when the parameter 660 nm and 3 J/cm2 was used. The laser radiation does not change either the susceptibility of the Vero cell to the HSV-1 infection or the HSV-1 virulence; however, prolongs the cell viability of HSV-1 infected cells. Positive benefits of phototherapy that have been reported clinically would appear to be due to host effects unrelated to viral replication in infected cells.

Natural killer (NK) cells are non-specific killer cells of the innate immune system that eliminate target cells based on discrimination between self and non-self. Activation is carefully regulated through integration of signals received through both activating and inhibitory receptors. During the course of a herpes simplex virus type-1 (HSV-1) infection, NK cells can influence host susceptibility to infection with severe infections occurring in individuals with genetic defects in the NK cell response. In response to HSV infection, NK cells are recruited to the inflammatory tissue where ensuing reciprocal interactions with accessory cells and proinflammatory cytokines induce NK cell activation, cytolytic activity, and cytokine production, contributing to innate immune response and ultimately influencing the adaptive immune response. The objective of this study was to elucidate the multiple roles of NK cells during the numerous steps in anti-HSV immune induction. Accordingly, we have demonstrated that NK cells are novel helpers that assist and influence an anti-HSV immune response via the secretion of cytokines that enhance HSV-specific CD8+ T cell effector function and cytokine production. Taken together, data from this study presented the critical importance of NK cells in mounting an essential and efficient anti-HSV immunity. The key findings of our study were: 1. In the absence of NK cells, dendritic cells have decreased capacity to prime HSV-specific T cells. 2. HSV infected NK cells can be directly activated via toll-like receptor (TLR) in a MyD88-dependent mechanism; however, interaction with HSV infected dendritic cells yields optimal NK cell activation and function (CD69 and IFNγ). 3. TRAIL-expressing NK cells eliminate antigen-bearing immature dermal DCs (CD11c+CD8α-DR5+), that migrate to draining lymphoid organs, to facilitate antigen transfer to lymphoid resident CD8α+ DC for T cell cross priming. 4. 'Helpless' CD8+ T cell function, generated in the absence of CD4+ T cells, can be partially restored to wild-type levels by NK cell supplementation. 5. Treatment of NK cells with anti-CD69 antibody results in a heightened NK activated state and augments the adaptive immune response, without increasing NK cell numbers. These findings may contribute to the potential revelation of avenues to manipulate NK cells for anti-viral therapies.

The obligate intracellular bacterium, Chlamydia trachomatis, and Herpes Simplex Virus Type-2 (HSV-2) are the leading sexually transmitted pathogens in the world. These infections are usually asymptomatic and clinically mild, but complications can be severe. Reports of dual detection of Chlamydia and HSV within the genital tracts of humans led our laboratory to develop an in vitro Chlamydia/HSV co-infection model. Little is known regarding the specific pathogenesis of Chlamydia and HSV co-infections, but HSV-super-infection of Chlamydia-infected cells caused the chlamydiae to deviate from their normal developmental cycle into a non-replicative state termed persistence, or the chlamydial stress response. Interactions between HSV envelope protein, gD with host cell junction protein, nectin-1, were enough to stimulate the departure from normal chlamydial development. Additional data also suggested that there might be differences between single infection and co-infection outcomes in vivo. Thus, two diverging hypotheses were investigated here: i) that host nectin-1 is required for normal chlamydial development; and ii) that pathogen shedding and/or disease progression in Chlamydia and HSV-2 co-infected animals will differ from that observed in singly-infected animals. Chlamydial infection of nectin-1 knockdown cell lines revealed no inhibition of chlamydial entry, but significant reductions in inclusion size and production of infectious chlamydiae. Additionally, nectin-1 knockout mice shed fewer Chlamydia compared to wild type mice. In other studies, we developed a novel in vivo Chlamydia and HSV-2 intravaginal super-infection model in BALB/c mice. Infection with Chlamydia muridarum, followed up to 9 days later by HSV-2 super-infection, both reduced HSV shedding and protected mice from HSV-induced fatal neurologic disease compared to HSV singly-infected animals. Protection is lost when: i) infected animals are no longer shedding C. muridarum; ii) when mice are inoculated with UV-inactivated C. muridarum; or iii) when viable chlamydiae are eliminated from the genital tract using antibiotics prior to HSV-2 super-infection. Altogether, we have determined that host nectin-1 is required for chlamydial development both in vitro and in vivo, and that chlamydial pre-infection protects mice from subsequent HSV infection. We predict that these observations may lead to novel approaches to prevent human infection by these two common sexually transmitted pathogens.

Le virus Herpes Simplex de type 1 (HSV-1) possède un mode d'infection particulier dit bimodal. Il peut soit se répliquer de manière active lors d'une phase dite lytique soit migrer dans les neurones et rester en latence. Il peut réactiver pour rétablir une infection lytique. Une protéine virale majeure dans la réactivation de HSV-1 est ICP0. C'est une protéine nucléaire à activité E3 ubiquitine ligase, qui possède la particularité d'induire la dégradation par le protéasome de plusieurs protéines centromériques constitutives, ce qui provoque une déstabilisation du centromère interphasique. Mon équipe a découvert une réponse cellulaire à l'instabilité centromérique, induite par la protéine ICP0, et nommée iCDR (pour interphase Centromere Damage Response.). L'objectif général de ma thèse est de déterminer les modifications structurales que subissent les centromères endommagés par ICP0 à l'origine de l'iCDR et probablement de la réactivation. J'ai pu démontrer qu'ICP0 affectait toute la structure protéique étroitement associée aux centromères durant l'interphase. Suite à ces résultats, j'ai pu démontrer, par des analyses de digestion de chromatine à la nucléase microccocale (MNAse), que l'occupation nucléosomique de la chromatine centromérique suite à l'activité d'ICP0 était affectée de façon significative. Une étude in vivo effectuée à partir de tissus nerveux provenant de souris infectées de manière latente, a permis de démontrer une co-localisation entre les génomes HSV-1 latents et les centromères. Cette co-localisation est associée à une répression transcriptionnelle du virus. Les résultats de ma thèse montrent donc que les effets d'ICP0 sur la déstabilisation des centromères sont en relation avec un rôle de ces centromères durant la latence. Ceci suggère fortement une implication de la déstabilisation des centromères dans le processus de réactivation contrôlé par ICP0.

Thesis (Ph.D.)--University of Alberta, 2010. / A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Virology, Medical Microbiology and Immunology. Title from pdf file main screen (viewed on January 10, 2010). Includes bibliographical references.

Thesis (Ph. D.)--University of Washington, 2001. / Vita. Includes bibliographical references (leaves 106-125).

Thesis (Ph. D.)--University of Texas Health Science Center at Houston, School of Public Health, 2007. / "December 2007." Source: Dissertation Abstracts International, Volume: 68-11, Section: B, page: 7222. Adviser: Zuber D. Mulla. Includes bibliographical references (leaves 157-169).

Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (leaves 46-53).

Submitted by Marcos Anselmo (marcos.anselmo@unipampa.edu.br) on 2016-09-22T19:30:32Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) JESSICA BRANDAO REOLON.pdf: 405938 bytes, checksum: 02d1f792d3137ced3fc728e0e11dcb3e (MD5) / Approved for entry into archive by Marcos Anselmo (marcos.anselmo@unipampa.edu.br) on 2016-09-22T19:34:00Z (GMT) No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) JESSICA BRANDAO REOLON.pdf: 405938 bytes, checksum: 02d1f792d3137ced3fc728e0e11dcb3e (MD5) / Made available in DSpace on 2016-09-22T19:34:01Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) JESSICA BRANDAO REOLON.pdf: 405938 bytes, checksum: 02d1f792d3137ced3fc728e0e11dcb3e (MD5) Previous issue date: 2016 / O herpesvírus simples (HSV) é um importante problema de saúde pública em vários países provocando ulceração genital. Dentre o arsenal terapêutico para o tratamento da infecção por HSV destaca-se o aciclovir (ACV). No entanto, este apresenta limitações no seu uso como um tempo de meia-vida curto e uma baixa solubilidade aquosa. A curcumina (CUR) é um composto natural que vem demonstrando diversas atividades terapêuticas, dentre estas a atividade antiviral, porém o seu uso por via oral também é comprometido pela baixa solubilidade em água. A encapsulação em micropartículas poliméricas (MPs) é uma abordagem farmacotécnica que podem superar as dificuldades do uso terapêutico destes dois compostos. Em vista disto, o presente trabalho visou desenvolver e caracterizar MPs pelo método de aspersão compostas por HPMC e Eudragit® RS100 como materiais poliméricos, além de manitol como adjuvante de secagem. Os resultados demonstraram que o método de preparo por aspersão (spray drying) apresentou um rendimento em torno de 50% para todas as formulações desenvolvidas, mostrando um fluxo tecnológico mais vantajoso para as partículas sem manitol. O doseamento demonstrou teores de 82 a 99% e de 81 a 94%, para ACV e CUR, respectivamente. A análise granulométrica mostrou micropartículas de tamanho micrométrico entre 8,7 e 15,3 μm. A análise morfológica evidenciou o formato esférico e confirmou o resultado das análises de tamanho. A espectroscopia na região do infravermelho mostrou uma sobreposição dos espectros obtidos pelos componentes isolados indicando boa compatibilidade entre os materiais. O perfil de liberação in vitro permitiu observar que as MPs foram capazes de controlar a liberação e melhorar a solubilidade dos compostos, destacando-se, neste quesito, as MPs compostas por HPMC. A avaliação preliminar da atividade antiviral in vitro demonstrou que a associação de ACV e CUR possui um efeito sinérgico frente ao vírus BoVH-1, sendo que as MPs foram capazes de potencializar este efeito. Neste contexto, as MPs mostraram-se sistemas promissores para a veiculação de ACV e CUR por via oral, com elevado potencial para constituir um tratamento alternativo para o herpes viral. / The herpes simplex virus (HSV), also known as human herpes virus, infects humans causing mainly genital and labial ulcerations. Among the drugs available for the treatment of HSV infection the acyclovir (ACV) is an effective antiviral drug. However this drug presents limitations in its use due to the short half-life and low water solubility. Curcumin (CUR) has demonstrated several therapeutic activities, including antiviral activity, but the oral administration of this natural compound is also compromised by low solubility. The polymeric microparticles (MP) are a pharmacotechnical approach that modifies the pharmacokinetics of the compounds and offers a possibility to overcome the difficulties of the therapeutic use. In view of this, this study aimed to develop MPs by spray drying method composed of HPMC and Eudragit® RS100 as polymeric materials, and mannitol as drying material. The results showed a yield around 50% for all developed formulations and a better technological flow rate for the particles without mannitol. The assay showed levels 82-99 and 81% to 94%, to ACV and CUR, respectively. The particle size analysis showed microparticles micrometric size between 8.7 and 15.3 micrometers. Morphological analysis showed the spherical shape and confirmed the results of size analysis. Spectroscopy in the infrared showed an overlap of the spectra obtained by the individual components indicating good compatibility between the materials. The in vitro release profile observed that the PMs were able to control the release and improved the solubility of the compounds, especially when composed of HPMC. Besides, an in vitro preliminary assessment of antiviral activity demonstrated that the combination of ACV and CUR presented a synergistic effect against BoVH-1 virus, and the MPs were able to enhance this effect. The MPs showed to be promising systems for the oral administration of ACV and CUR and could be an improved alternative treatment for viral herpes.