Proefschrift Universiteit Maastricht. / Met bibliogr., lit. opg. - Met samenvatting in het Nederlands.

Human Cytomegalovirus (HCMV) is the most common cause of viral congenital infection in the world. Around 5-10% of HCMV infected children are symptomatic at birth, and 50-90% of these develop severe manifestations with a 30% mortality rate. Among the asymptomatic children at birth, 10-15% develop late sequelae. The major cell entry glycoproteins of HCMV form three complexes: gC-I containing gB; gC-II containing gM &amp; gN; and gC-III containing gH, gL, and gO (or UL128-131). These entry glycoproteins are polymorphic, producing different glycoprotein genotypes. The polymorphic nature of the glycoproteins as well as their ability to elicit neutralizing antibodies made them of interest in correlating them with the severity and outcome of the disease. This study aimed to develop a robust system to identify clusters of glycoprotein genotypes and to correlate them with disease manifestation. PCR assays of high sensitivity were used to identify all six glycoproteins. The PCR products were digested using restriction enzymes (RFLP) to identify the glycoprotein genotypes. Available laboratory strains (AD169, Towne, Davis, Toledo, and Merlin) as well as 112 clinical samples were amplified and genotyped using the assay, and their glycoprotein genotype profile was determined. A reliable sensitive assay was successfully developed to identify all glycoprotein genotypes including a novel gM assay using PCR/RFLP. The clinical samples were grouped according to disease manifestation (Group 1: congenital/postnatal patients (subgroup 1A: confirmed congenital patients &amp; subgroup 1B: patients with either congenital or postnatal infection), Group 2: immunocompetent patients, Group 3: immunocompromised patients (subgroup 3A: immunocompromised patients with primary infection, subgroup 3B: immunocompromised patients with recurrent infection &amp; subgroup 3C: immunocompromised patients with unconfirmed primary or recurrent infection)). Genotype gB1 was found predominantly prevalent in congenital/postnatal and immunocompromised patients, while gB3 was the most common genotype in immunocompetent patients. This result along with the phylogenetic analysis performed in this study suggest a relationship between gB genotypes and the immune response of the patients, where gB3 may be positively selected by host immune pressure. The novel gM assay genotyped the highly conserved gene (UL100) into three distinct genotypes; gM3 genotype associated with the congenital/postnatal group; which may provide an insight into understanding viral attachment and spread into the host cell. In congenital/postnatal infection, gH1 (72.7%) and gL4 (65.1%) were the most prevalent genotypes (gH1= 32/44, gL4= 28/43; P=0.000). In immunocompetent patients, mixed gH and mixed gL genotypes significantly correlated with the group, and in the immunocompromised group gH2 and mixed gL genotype were the most common genotypes (51.1% and 46.9% respectively). Glycoproteins gO, gH and gL are components of gC-III complex and gO1 was found to be the most prevalent gO genotype in all infection types (Group 1= 32.1%, Group 2= 85.7%, Group 3= 18.8%; P < 0.05). Also, in congenital/postnatal infection gN and gO were found to significantly link with each other and this is expected since both glycoproteins are highly polymorphic and are located on adjacent gene loci in HCMV genome (gN1+gO1a (P=0.000), gN3a+gO4 (P= 0.000)). The specific gN-gO linkages found here could be potential indicators for congenital/postnatal infection. In congenital/postnatal infection group, gH had significant linkages to gN and gO (gH1+gN1 (P=0.023, gH1+gO1a (P=0.013)) suggesting that interlinked selection of glycoprotein genotypes in the gC-II and gC-III complexes is involved in the development of congenital infection. High viral loads were found trending with immunocompromised patients, while low viral loads were significantly associated with mixed infected patients. This study has shown significant associations between a number of glycoproteins and congenital infection. Previously ignored glycoproteins gM and gL have been shown to be potentially of significant interest in this study and a larger study to confirm this is needed. In most cases the pattern of glycoprotein genotypes in congenital infection is more similar to that of immunocompromised than immunocompetent patients and it is possible that immune pressure is selecting for or against particular glycoprotein genotypes. The relationship between mixed infection and sample type may offer opportunities for development of prognostic biomarkers for congenital disease and further work is warranted.

Swiss white mice and C3H/HeJ mice were used occasionally. It was shown that in vitro infection resulted in the formation of infectious centers in only a small percentage of the spleen cell population even at high multiplicities of infection, when a proportionately higher number of virus particles were being taken up by the cells. Virus could be rescued from some of these infected cells by co-cultivation with susceptible mouse embryo fibroblasts, and the emerging infectious virus particles were detectable 40-50 hours after the start of co-cultivation. It appeared that allogeneic stimulation was not essential to virus rescue in our in vitro system, for both syngeneic and allogeneic mouse embryo fibroblasts were equally efficient in effecting virus reactivation from infected spleen cells. A fraction of the infected spleen cells was capable of supporting spontaneous MCMV replication. Such replication was not affected by incubation with the mitogens Con A or LPS prior to or after infection with the virus. However, the presence of an increased number of activated T cells due to Con A stimulation possibly inhibited virus replication to a certain extent. Virus replication was also reduced in the absence of some heat-labile factor in the fetal calf serum. Cell separation techniques such as nylon wool column adherence, plastic adherence, anti-serum treatment and y-xay irradiation, showed that macrophage-like cells were probably involved in harboring MCMV in a latent state although spontaneous replication did take place to a limited extent in such cells. Another cell fraction, with B-cell-like properties, was capable of supporting spontaneous MCMV replication. Another effect of in vitro MCMV infection on spleen cells was their suppressive effect on the mitogenic responses of such cells. Preliminary evidence suggested that the defective con A response might be mediated by macrophages exposed to MCMV. Moreover, the immunosuppressive effect could only be observed after exposure to infectious virus particles, and the degree of suppression of mitogen responses could be increased by using higher multiplicities of infection. In certain cases, a slight stimulatory effect on the spleen cells was observed about 30 hours after infection. In summary, the spleen cell population contained cell fractions that were capable of harboring MCMV in a latent state and supporting spontaneous viral replication. In addition, the mitogenic responses of infected cells were impaired. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

OBJECTIVES: To determine the effect of murine cytomegalovirus (MCMV) infection on the growth and proliferation of glioblastoma using murine models of differing genetic backgrounds, as well as to determine whether the observed effects are dependent on the mutational background of the glioblastoma models. METHODS: An experimental orthotopic murine glioblastoma model was established using latently MCMV infected C57BL/6 mice, with non-infected mice as controls. Mouse brains were harvested at the end-point and subject to histological analysis using immunostaining and visualization by confocal microscopy. Viral infectivity and protein expression levels were assayed using time-lapse microscopy and western blotting. RESULTS: Our results indicate that while the degree of viral infection may differ between experimental cell lines, MCMV-infected tumor-bearing mice consistently demonstrate accelerated tumor growth and worsened survival relative to the uninfected controls. Additionally, our results provide further evidence to support the previously reported findings implicating CMV as a driver of angiogenesis in vivo, as sections from MCMV-infected mice showed a greater density of blood vessels and more complete blood-brain barrier formation. Finally, our results show that the addition of antiviral agents as an adjunct therapy to first-line chemotherapeutics effectively reduced tumor sphere size and partially reversed viral infection by MCMV. CONCLUSIONS: Here we demonstrate the tumor-promoting effects of MCMV infection are consistently observed in different glioblastoma mouse models suggesting that these effects are independent of the tumor genetic background of our tested tumor models. Moreover, the success of preliminary in vitro studies using a combination treatment of antiviral and chemotherapy strengthen the case for targeting CMV therapeutically in the treatment of GBM.

No description available.

As doenças respiratórias estão entre as maiores causas de prejuízo para a indústria suinícola, seja pelo retardo no crescimento e ganho de peso, mortalidade de animais ou pelos gastos com vacinas, medicamentos e assistência veterinária. Neste contexto os quadros de rinite têm apresentado uma contribuição significativa. O presente estudo propõe a determinação dos perfis de eliminação de agentes envolvidos em rinite nos suínos avaliando diferentes faixas etárias em nove propriedades de ciclo completo com histórico de lesão em cornetos e que utilizem diferentes formas de prevenção e controle destas manifestações. Foram avaliados suabes de tonsilas de 12 animais, nas seguintes faixas etárias: matrizes, leitões de 20, 40 e 60 dias, suínos de 90, 110 e 140 dias, totalizando 84 animais por propriedade e 756 amostras em todo o estudo. As amostras foram submetidas à pesquisa de P. multocida tipo capsular A e D, gene codificador de toxina dermonecrótica de P. multocida, B. bronchiseptica e cytomegalovirus suíno através da reação em cadeia pela polimerase (PCR). Apesar do histórico de lesões em corneto em todas as propriedades apenas um animal foi positivo para presença de P. multocida tipo A e todos foram negativos para a presença do gene codificador da toxina dermonecrótica. Dentre os 756 animais 22 (2,9%) foram positivos para presença de B. bronchiseptica e 198 (26,1%) para detecção cytomegalovirus suíno. A presença B. bronchiseptica apresentou associação estatisticamente significativa com as fases de maternidade e terminação. A maior freqüência de cytomegalovirus suíno apresentou associação estatisticamente significativa com a fase de creche. Observaram-se matrizes eliminando B. bronchiseptica nos três tipos de granjas avaliadas, indicando que a fêmeas tem participação ativa na infecção dos leitões pelo agente. O mesmo não foi detectado na disseminação do cytomegalovirus suíno. Maiores estudos devem ser realizados para esclarecer a baixa eliminação de P. multocida e o verdadeiro impacto do cytomegalovirus nos rebanhos suínos. / Respiratory diseases are one of the largest cause of economic losses in swine industry, it is related with grown and weight gain reduction, mortality, vaccines and medicaments costs, veterinary assistance. In that context, rinithis cases have been a major contribution. The present study propose the determination of elimination profile of agents related with rhinitis evaluating different ages in nine swine herds with history of cornet lesions and that uses different ways to control and prevent this problem. There were examined tonsils swabs from 12 animals in the following ages: sows, piglets of 20, 40 60 days and pigs of 90, 110 and 140 days, totalizing 84 pigs for farm. The swabs were searched to P. multocida capsular type A and D, dermonecrotic toxin gene from P. multocida, B. bronchiseptica and porcine cytomegalovirus through polymerase chain reaction (PCR). Despite de turbinate bones lesions present in all herds P. multocida type A was detected in only one pig and none were positive to dermonecrotic toxin gene. From 756 animals, 22 (2.9%) were positive to B. bronchiseptica and 198 (26.1%) to porcine cytomegalovirus detection. The presence of B. bronchiseptica presented statistical association with the farrowing and finishing times. Larger number of animals positive to cytomegalovirus show statistical association with the post weaning pigs. Sows carrying B. bronchiseptica in the three types of herds examined, suggesting that sows have an active participation in piglet infection by this agent. The same was not observed in porcine cytomegalovirus spread. More projects were need to clarify the low detection of P. multocida and to understand the impact of cytomegalovirus in swine production.

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 5 uppsatser.

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

As doenças respiratórias estão entre as maiores causas de prejuízo para a indústria suinícola, seja pelo retardo no crescimento e ganho de peso, mortalidade de animais ou pelos gastos com vacinas, medicamentos e assistência veterinária. Neste contexto os quadros de rinite têm apresentado uma contribuição significativa. O presente estudo propõe a determinação dos perfis de eliminação de agentes envolvidos em rinite nos suínos avaliando diferentes faixas etárias em nove propriedades de ciclo completo com histórico de lesão em cornetos e que utilizem diferentes formas de prevenção e controle destas manifestações. Foram avaliados suabes de tonsilas de 12 animais, nas seguintes faixas etárias: matrizes, leitões de 20, 40 e 60 dias, suínos de 90, 110 e 140 dias, totalizando 84 animais por propriedade e 756 amostras em todo o estudo. As amostras foram submetidas à pesquisa de P. multocida tipo capsular A e D, gene codificador de toxina dermonecrótica de P. multocida, B. bronchiseptica e cytomegalovirus suíno através da reação em cadeia pela polimerase (PCR). Apesar do histórico de lesões em corneto em todas as propriedades apenas um animal foi positivo para presença de P. multocida tipo A e todos foram negativos para a presença do gene codificador da toxina dermonecrótica. Dentre os 756 animais 22 (2,9%) foram positivos para presença de B. bronchiseptica e 198 (26,1%) para detecção cytomegalovirus suíno. A presença B. bronchiseptica apresentou associação estatisticamente significativa com as fases de maternidade e terminação. A maior freqüência de cytomegalovirus suíno apresentou associação estatisticamente significativa com a fase de creche. Observaram-se matrizes eliminando B. bronchiseptica nos três tipos de granjas avaliadas, indicando que a fêmeas tem participação ativa na infecção dos leitões pelo agente. O mesmo não foi detectado na disseminação do cytomegalovirus suíno. Maiores estudos devem ser realizados para esclarecer a baixa eliminação de P. multocida e o verdadeiro impacto do cytomegalovirus nos rebanhos suínos. / Respiratory diseases are one of the largest cause of economic losses in swine industry, it is related with grown and weight gain reduction, mortality, vaccines and medicaments costs, veterinary assistance. In that context, rinithis cases have been a major contribution. The present study propose the determination of elimination profile of agents related with rhinitis evaluating different ages in nine swine herds with history of cornet lesions and that uses different ways to control and prevent this problem. There were examined tonsils swabs from 12 animals in the following ages: sows, piglets of 20, 40 60 days and pigs of 90, 110 and 140 days, totalizing 84 pigs for farm. The swabs were searched to P. multocida capsular type A and D, dermonecrotic toxin gene from P. multocida, B. bronchiseptica and porcine cytomegalovirus through polymerase chain reaction (PCR). Despite de turbinate bones lesions present in all herds P. multocida type A was detected in only one pig and none were positive to dermonecrotic toxin gene. From 756 animals, 22 (2.9%) were positive to B. bronchiseptica and 198 (26.1%) to porcine cytomegalovirus detection. The presence of B. bronchiseptica presented statistical association with the farrowing and finishing times. Larger number of animals positive to cytomegalovirus show statistical association with the post weaning pigs. Sows carrying B. bronchiseptica in the three types of herds examined, suggesting that sows have an active participation in piglet infection by this agent. The same was not observed in porcine cytomegalovirus spread. More projects were need to clarify the low detection of P. multocida and to understand the impact of cytomegalovirus in swine production.

Class of 2012 Abstract / Specific Aims: To compare and evaluate the therapies prescribed, the incidence of adverse drug events, and the time to clinical cure in transplant patients with a cytomegalovirus (CMV) infection at an academic medical center before and during the foscarnet nationwide shortage. Methods: This study was a retrospective chart review to compare CMV treatment prescribed and clinical outcomes in pediatric and adult transplant patients at an academic medical center. Transplant patients were evaluated over a 16 month time period between December 2009 and March 2011. The average dose (mg/kg) and prevalence ganciclovir, foscarnet, and cidofovir prescribed in transplant patients with CMV infection were evaluated. Additionally, the incidence of adverse drug events including acute renal dysfunction and myelosuppression were characterized. Main Results: There were 30 subjects diagnosed with CMV disease during the evalutaion period. Of all of the patients treated for CMV before the shortage, 79% received ganciclovir, 43% received foscarnet, and 21% received cidofovir. Following the shortage in September 2010, the usage of the antiviral agents changed to 100%, 25%, and 13% respectively. Overall the usage of ganciclovir increased while the usage of foscarnet decreased when there was a shortage of medication. Conclusions: The antiviral prescribing patterns changed significantly during the foscarnet shortage. The average dose and incidence of ganciclovir increased which likely contributed to serious adverse events. Due to the limited amount of patients treated for CMV and the short time frame, clinical cure could not be determined at this time. Drug shortages are a serious problem and significantly influence patient outcomes.