Selection of soybean F3 populations for agronomic and physiological traits and vegetation indices using multivariate approaches

The objective of this work was to select F3 segregating populations for agronomic and physiological traits and vegetation indices. The experiment was carried out in the 2018/2019 crop seasons in a randomized block design with three replicates and 10 F3 populations (P1, P2, P3, P4, P5, P6, P7, P8, P9, and P10). The evaluated traits were: number of days to maturity; number of pods per plant; number of nodes per plant; number of lateral stems per plant; grain yield, photosynthesis; stomatal conductance; internal CO2 concentration; transpiration; NIR, Red-edge, Red and Green wavelength; and NDVI and NDRE vegetation indices. In the physiological traits evaluated in soybean F3 populations, the P8 and P6 populations presented the highest averages for photosynthesis, while the P3, P10 and P9 populations presented the highest concentration values, CO2 (Ci), transpiration (E,) and stomatal conductance (gs). Therefore, the vegetation indices evaluated in soybean F3 populations, the populations P7, P2 and P1 presented higher averages for near and red infrared wavelengths. P5 and P4 showed a higher NDRE vegetation index and P8 showed a higher red wavelength. For agronomic traits, population P2 showed better results for the traits number of nodes per plant (NNP) and the number of lateral stems per plant (NHL). Populations P1 and P3 had a higher number of days for physiological maturation (NDM). The P7 population presented the highest number of pods per plant (NVP), and the P6 population the highest grain yield (PROD). Highlights Soybean F3 populations were selected for agronomic and physiological traits and vegetation indices. Principal components were used to select these populations. Factor analysis allowed us to understand the relationship between the traits evaluated.The objective of this work was to select F3 segregating populations for agronomic and physiological traits and vegetation indices. The experiment was carried out in the 2018/2019 crop seasons in a randomized block design with three replicates and 10 F3 populations (P1, P2, P3, P4, P5, P6, P7, P8, P9, and P10). The evaluated traits were: number of days to maturity; number of pods per plant; number of nodes per plant; number of lateral stems per plant; grain yield, photosynthesis; stomatal conductance; internal CO2 concentration; transpiration; NIR, Red-edge, Red and Green wavelength; and NDVI and NDRE vegetation indices. In the physiological traits evaluated in soybean F3 populations, the P8 and P6 populations presented the highest averages for photosynthesis, while the P3, P10 and P9 populations presented the highest concentration values, CO2 (Ci), transpiration (E,) and stomatal conductance (gs). Therefore, the vegetation indices evaluated in soybean F3 populations, the populations P7, P2 and P1 presented higher averages for near and red infrared wavelengths. P5 and P4 showed a higher NDRE vegetation index and P8 showed a higher red wavelength. For agronomic traits, population P2 showed better results for the traits number of nodes per plant (NNP) and the number of lateral stems per plant (NHL). Populations P1 and P3 had a higher number of days for physiological maturation (NDM). The P7 population presented the highest number of pods per plant (NVP), and the P6 population the highest grain yield (PROD). Highlights Soybean F3 populations were selected for agronomic and physiological traits and vegetation indices; Principal components were used to select these populations; Factor analysis allowed us to understand the relationship between the traits evaluated

Efficient Human Cytomegalovirus Replication in Primary Endothelial Cells Is SOCS3 Dependent

Background: In immunocompromised patients, human cytomegalovirus (HCMV) infection is a major cause of morbidity and mortality. Suppressor of cytokine signaling (SOCS) proteins are very potent negative regulators of the janus kinase/signal transducer and activator of transcription (JAK/STAT) pathways. We hypothesized that HCMV exploits SOCS1 and/or SOCS3 to its advantage. Methods: All experiments were carried out with primary human lung-derived microvascular endothelial cells (HMVEC). SOCS1 and SOCS3 were silenced by transfecting the cells with siRNA. HCMV was propagated and titered on human lung-derived fibroblasts MRC5. Real-time PCR and Western blot were used to detect mRNA and protein levels, respectively. Results: The data presented show that an efficient replication of HCMV in HMVEC is dependent on SOCS3 protein. Time course analysis revealed an increase in SOCS3 protein levels in infected cells. Silencing of SOCS3 (siSOCS3) resulted in inhibition of viral immediate early, early, and late antigen production. Consistently, HCMV titers produced by siSOCS3 cultures were significantly decreased when compared to control transfected cultures (siCNTRs). STAT1 and STAT2 phosphorylation was increased in siSOCS3-infected cells when compared to siCNTR-treated cells. Conclusion: These findings indicate the implication of SOCS3 in the mechanism of HCMV-mediated control of cellular immune responses

DESENVOLVIMENTO E VALIDAÇÃO DE MÉTODO ANALITICO PARA QUANTIFICAÇÃO DO VORICONAZOL EM CÓRNEA

Introdução e objetivos: O voriconazol (VOR) é um novo antifúngico da classe tiazol de amplo espectro1 e, sua administração tópica visa o tratamento de ceratites fúngicas. Entretanto, o desenvolvimento de novas formulações se faz necessário para aumentar a biodisponibilidade ocular do fármaco. Para este propósito, é necessário o desenvolvimento de métodos de quantificação e extração do fármaco em córnea suína. Desta forma, o objetivo foi desenvolver e validar um método analítico capaz de quantificar o VOR na córnea. Metodologia: VOR foi quantificado por CLAE -UV (255 nm), com fase móvel constituída por acetonitrila e água (50:50) e fluxo de 1,0 mL/min. O método foi validado de acordo com os parâmetros do FDA2. Para a recuperação do VOR, as amostras brancas da córnea formam contaminadas com diferentes soluções de VOR, secas e extraídas com metanol, após agitação 60 min em chapa magnética. Resultados e discussões: O método se mostrou especifico (não apresentando picos interferentes no tempo de retenção do VOR), linear (y=19,084x + 2,107, r2= 0,999), preciso e exato (precisão e exatidão inferior a 5%). A recuperação do VOR na córnea foi de 84,79% (± 1,34%) para concentração teórica de 20 mg/mL e 86,09% (± 6,20%) para concentração de 5 mg/mL. Conclusões: O método analítico foi validado e o método de recuperação mostrou-se eficaz para quantificação do VOR na córnea

Erratum: Global, regional, and national comparative risk assessment of 84 behavioural, environmental and occupational, and metabolic risks or clusters of risks for 195 countries and territories, 1990–2017: a systematic analysis for the Global Burden of Disease Study 2017

Interpretation: By quantifying levels and trends in exposures to risk factors and the resulting disease burden, this assessment offers insight into where past policy and programme efforts might have been successful and highlights current priorities for public health action. Decreases in behavioural, environmental, and occupational risks have largely offset the effects of population growth and ageing, in relation to trends in absolute burden. Conversely, the combination of increasing metabolic risks and population ageing will probably continue to drive the increasing trends in non-communicable diseases at the global level, which presents both a public health challenge and opportunity. We see considerable spatiotemporal heterogeneity in levels of risk exposure and risk-attributable burden. Although levels of development underlie some of this heterogeneity, O/E ratios show risks for which countries are overperforming or underperforming relative to their level of development. As such, these ratios provide a benchmarking tool to help to focus local decision making. Our findings reinforce the importance of both risk exposure monitoring and epidemiological research to assess causal connections between risks and health outcomes, and they highlight the usefulness of the GBD study in synthesising data to draw comprehensive and robust conclusions that help to inform good policy and strategic health planning

Global, regional, and national comparative risk assessment of 84 behavioural, environmental and occupational, and metabolic risks or clusters of risks for 195 countries and territories, 1990-2017: a systematic analysis for the Global Burden of Disease Study 2017

Background The Global Burden of Diseases, Injuries, and Risk Factors Study (GBD) 2017 comparative risk assessment (CRA) is a comprehensive approach to risk factor quantification that offers a useful tool for synthesising evidence on risks and risk–outcome associations. With each annual GBD study, we update the GBD CRA to incorporate improved methods, new risks and risk–outcome pairs, and new data on risk exposure levels and risk–outcome associations. Methods We used the CRA framework developed for previous iterations of GBD to estimate levels and trends in exposure, attributable deaths, and attributable disability-adjusted life-years (DALYs), by age group, sex, year, and location for 84 behavioural, environmental and occupational, and metabolic risks or groups of risks from 1990 to 2017. This study included 476 risk–outcome pairs that met the GBD study criteria for convincing or probable evidence of causation. We extracted relative risk and exposure estimates from 46 749 randomised controlled trials, cohort studies, household surveys, census data, satellite data, and other sources. We used statistical models to pool data, adjust for bias, and incorporate covariates. Using the counterfactual scenario of theoretical minimum risk exposure level (TMREL), we estimated the portion of deaths and DALYs that could be attributed to a given risk. We explored the relationship between development and risk exposure by modelling the relationship between the Socio-demographic Index (SDI) and risk-weighted exposure prevalence and estimated expected levels of exposure and risk-attributable burden by SDI. Finally, we explored temporal changes in risk-attributable DALYs by decomposing those changes into six main component drivers of change as follows: (1) population growth; (2) changes in population age structures; (3) changes in exposure to environmental and occupational risks; (4) changes in exposure to behavioural risks; (5) changes in exposure to metabolic risks; and (6) changes due to all other factors, approximated as the risk-deleted death and DALY rates, where the risk-deleted rate is the rate that would be observed had we reduced the exposure levels to the TMREL for all risk factors included in GBD 2017. Findings In 2017, 34·1 million (95% uncertainty interval [UI] 33·3–35·0) deaths and 1·21 billion (1·14–1·28) DALYs were attributable to GBD risk factors. Globally, 61·0% (59·6–62·4) of deaths and 48·3% (46·3–50·2) of DALYs were attributed to the GBD 2017 risk factors. When ranked by risk-attributable DALYs, high systolic blood pressure (SBP) was the leading risk factor, accounting for 10·4 million (9·39–11·5) deaths and 218 million (198–237) DALYs, followed by smoking (7·10 million [6·83–7·37] deaths and 182 million [173–193] DALYs), high fasting plasma glucose (6·53 million [5·23–8·23] deaths and 171 million [144–201] DALYs), high body-mass index (BMI; 4·72 million [2·99–6·70] deaths and 148 million [98·6–202] DALYs), and short gestation for birthweight (1·43 million [1·36–1·51] deaths and 139 million [131–147] DALYs). In total, risk-attributable DALYs declined by 4·9% (3·3–6·5) between 2007 and 2017. In the absence of demographic changes (ie, population growth and ageing), changes in risk exposure and risk-deleted DALYs would have led to a 23·5% decline in DALYs during that period. Conversely, in the absence of changes in risk exposure and risk-deleted DALYs, demographic changes would have led to an 18·6% increase in DALYs during that period. The ratios of observed risk exposure levels to exposure levels expected based on SDI (O/E ratios) increased globally for unsafe drinking water and household air pollution between 1990 and 2017. This result suggests that development is occurring more rapidly than are changes in the underlying risk structure in a population. Conversely, nearly universal declines in O/E ratios for smoking and alcohol use indicate that, for a given SDI, exposure to these risks is declining. In 2017, the leading Level 4 risk factor for age-standardised DALY rates was high SBP in four super-regions: central Europe, eastern Europe, and central Asia; north Africa and Middle East; south Asia; and southeast Asia, east Asia, and Oceania. The leading risk factor in the high-income super-region was smoking, in Latin America and Caribbean was high BMI, and in sub-Saharan Africa was unsafe sex. O/E ratios for unsafe sex in sub-Saharan Africa were notably high, and those for alcohol use in north Africa and the Middle East were notably low. Interpretation By quantifying levels and trends in exposures to risk factors and the resulting disease burden, this assessment offers insight into where past policy and programme efforts might have been successful and highlights current priorities for public health action. Decreases in behavioural, environmental, and occupational risks have largely offset the effects of population growth and ageing, in relation to trends in absolute burden. Conversely, the combination of increasing metabolic risks and population ageing will probably continue to drive the increasing trends in non-communicable diseases at the global level, which presents both a public health challenge and opportunity. We see considerable spatiotemporal heterogeneity in levels of risk exposure and risk-attributable burden. Although levels of development underlie some of this heterogeneity, O/E ratios show risks for which countries are overperforming or underperforming relative to their level of development. As such, these ratios provide a benchmarking tool to help to focus local decision making. Our findings reinforce the importance of both risk exposure monitoring and epidemiological research to assess causal connections between risks and health outcomes, and they highlight the usefulness of the GBD study in synthesising data to draw comprehensive and robust conclusions that help to inform good policy and strategic health planning

Global, regional, and national comparative risk assessment of 84 behavioural, environmental and occupational, and metabolic risks or clusters of risks for 195 countries and territories, 1990-2017: a systematic analysis for the Global Burden of Disease Study 2017.

BACKGROUND: The Global Burden of Diseases, Injuries, and Risk Factors Study (GBD) 2017 comparative risk assessment (CRA) is a comprehensive approach to risk factor quantification that offers a useful tool for synthesising evidence on risks and risk-outcome associations. With each annual GBD study, we update the GBD CRA to incorporate improved methods, new risks and risk-outcome pairs, and new data on risk exposure levels and risk-outcome associations. METHODS: We used the CRA framework developed for previous iterations of GBD to estimate levels and trends in exposure, attributable deaths, and attributable disability-adjusted life-years (DALYs), by age group, sex, year, and location for 84 behavioural, environmental and occupational, and metabolic risks or groups of risks from 1990 to 2017. This study included 476 risk-outcome pairs that met the GBD study criteria for convincing or probable evidence of causation. We extracted relative risk and exposure estimates from 46 749 randomised controlled trials, cohort studies, household surveys, census data, satellite data, and other sources. We used statistical models to pool data, adjust for bias, and incorporate covariates. Using the counterfactual scenario of theoretical minimum risk exposure level (TMREL), we estimated the portion of deaths and DALYs that could be attributed to a given risk. We explored the relationship between development and risk exposure by modelling the relationship between the Socio-demographic Index (SDI) and risk-weighted exposure prevalence and estimated expected levels of exposure and risk-attributable burden by SDI. Finally, we explored temporal changes in risk-attributable DALYs by decomposing those changes into six main component drivers of change as follows: (1) population growth; (2) changes in population age structures; (3) changes in exposure to environmental and occupational risks; (4) changes in exposure to behavioural risks; (5) changes in exposure to metabolic risks; and (6) changes due to all other factors, approximated as the risk-deleted death and DALY rates, where the risk-deleted rate is the rate that would be observed had we reduced the exposure levels to the TMREL for all risk factors included in GBD 2017

Global, regional, and national comparative risk assessment of 84 behavioural, environmental and occupational, and metabolic risks or clusters of risks for 195 countries and territories, 1990–2017: a systematic analysis for the Global Burden of Disease Study 2017

Stanaway JD, Afshin A, Gakidou E, et al. Global, regional, and national comparative risk assessment of 84 behavioural, environmental and occupational, and metabolic risks or clusters of risks for 195 countries and territories, 1990–2017: a systematic analysis for the Global Burden of Disease Study 2017. Lancet. 2018;392(10159):1923-1994.Background The Global Burden of Diseases, Injuries, and Risk Factors Study (GBD) 2017 comparative risk assessment (CRA) is a comprehensive approach to risk factor quantification that offers a useful tool for synthesising evidence on risks and risk outcome associations. With each annual GBD study, we update the GBD CRA to incorporate improved methods, new risks and risk outcome pairs, and new data on risk exposure levels and risk outcome associations. Methods We used the CRA framework developed for previous iterations of GBD to estimate levels and trends in exposure, attributable deaths, and attributable disability-adjusted life-years (DALYs), by age group, sex, year, and location for 84 behavioural, environmental and occupational, and metabolic risks or groups of risks from 1990 to 2017. This study included 476 risk outcome pairs that met the GBD study criteria for convincing or probable evidence of causation. We extracted relative risk and exposure estimates from 46 749 randomised controlled trials, cohort studies, household surveys, census data, satellite data, and other sources. We used statistical models to pool data, adjust for bias, and incorporate covariates. Using the counterfactual scenario of theoretical minimum risk exposure level (TMREL), we estimated the portion of deaths and DALYs that could be attributed to a given risk. We explored the relationship between development and risk exposure by modelling the relationship between the Socio-demographic Index (SDI) and risk-weighted exposure prevalence and estimated expected levels of exposure and risk-attributable burden by SDI. Finally, we explored temporal changes in risk-attributable DALYs by decomposing those changes into six main component drivers of change as follows: (1) population growth; (2) changes in population age structures; (3) changes in exposure to environmental and occupational risks; (4) changes in exposure to behavioural risks; (5) changes in exposure to metabolic risks; and (6) changes due to all other factors, approximated as the risk-deleted death and DALY rates, where the risk-deleted rate is the rate that would be observed had we reduced the exposure levels to the TMREL for all risk factors included in GBD 2017. Findings In 2017,34.1 million (95% uncertainty interval [UI] 33.3-35.0) deaths and 121 billion (144-1.28) DALYs were attributable to GBD risk factors. Globally, 61.0% (59.6-62.4) of deaths and 48.3% (46.3-50.2) of DALYs were attributed to the GBD 2017 risk factors. When ranked by risk-attributable DALYs, high systolic blood pressure (SBP) was the leading risk factor, accounting for 10.4 million (9.39-11.5) deaths and 218 million (198-237) DALYs, followed by smoking (7.10 million [6.83-7.37] deaths and 182 million [173-193] DALYs), high fasting plasma glucose (6.53 million [5.23-8.23] deaths and 171 million [144-201] DALYs), high body-mass index (BMI; 4.72 million [2.99-6.70] deaths and 148 million [98.6-202] DALYs), and short gestation for birthweight (1.43 million [1.36-1.51] deaths and 139 million [131-147] DALYs). In total, risk-attributable DALYs declined by 4.9% (3.3-6.5) between 2007 and 2017. In the absence of demographic changes (ie, population growth and ageing), changes in risk exposure and risk-deleted DALYs would have led to a 23.5% decline in DALYs during that period. Conversely, in the absence of changes in risk exposure and risk-deleted DALYs, demographic changes would have led to an 18.6% increase in DALYs during that period. The ratios of observed risk exposure levels to exposure levels expected based on SDI (O/E ratios) increased globally for unsafe drinking water and household air pollution between 1990 and 2017. This result suggests that development is occurring more rapidly than are changes in the underlying risk structure in a population. Conversely, nearly universal declines in O/E ratios for smoking and alcohol use indicate that, for a given SDI, exposure to these risks is declining. In 2017, the leading Level 4 risk factor for age-standardised DALY rates was high SBP in four super-regions: central Europe, eastern Europe, and central Asia; north Africa and Middle East; south Asia; and southeast Asia, east Asia, and Oceania. The leading risk factor in the high-income super-region was smoking, in Latin America and Caribbean was high BMI, and in sub-Saharan Africa was unsafe sex. O/E ratios for unsafe sex in sub-Saharan Africa were notably high, and those for alcohol use in north Africa and the Middle East were notably low. Interpretation By quantifying levels and trends in exposures to risk factors and the resulting disease burden, this assessment offers insight into where past policy and programme efforts might have been successful and highlights current priorities for public health action. Decreases in behavioural, environmental, and occupational risks have largely offset the effects of population growth and ageing, in relation to trends in absolute burden. Conversely, the combination of increasing metabolic risks and population ageing will probably continue to drive the increasing trends in non-communicable diseases at the global level, which presents both a public health challenge and opportunity. We see considerable spatiotemporal heterogeneity in levels of risk exposure and risk-attributable burden. Although levels of development underlie some of this heterogeneity, O/E ratios show risks for which countries are overperforming or underperforming relative to their level of development. As such, these ratios provide a benchmarking tool to help to focus local decision making. Our findings reinforce the importance of both risk exposure monitoring and epidemiological research to assess causal connections between risks and health outcomes, and they highlight the usefulness of the GBD study in synthesising data to draw comprehensive and robust conclusions that help to inform good policy and strategic health planning. Copyright (C) 2018 The Author(s). Published by Elsevier Ltd

Comparison of human cytomegalovirus entry mechanisms into porcine and human edothelial cells

Over the past decades, allotransplantation has evolved from an experimental procedure to a very successful, often life-saving therapy and has emerged as the best therapy to treat patients with end-stage organ failure. The success of this surgical intervention has led to an increasing demand limited by the availability of organs. To overcome the acute shortage of human organs, xenotransplantation has become a focus of interest and a very important area of transplantation research. Miniature pigs have evolved as the most suitable source of organs for several reasons including: housing in a pathogen-free environment, the capacity to breed and have a big number of offspring and last but not least some similarities shared with human organs regarding size and physiology. Currently, clinical pig-to-human trials involving porcine islet cells are performed with encouraging results since patients have seen their daily dose of insulin reduced and one of the patients is said to be insulin-free. However, because of a high immunosuppression treatment to prevent graft rejection, the transplanted organ will inevitably be in contact with pathogens. Concern has mainly been focused on donor-derived xenogeneic infection, neglecting host-derived infection with their potentially severe consequences for the xenograft including rejection and loss of function. Primary infection and reactivation of human cytomegalovirus (HCMV) is the most important viral complication post- transplantation and has been associated with allograft rejection. Our laboratory has previously demonstrated that HCMV cross-species infection of porcine cells induces atypical cytopathic effect and induces apoptosis, which may eventually result in porcine xenograft rejection. The goal of my thesis was to investigate potential HCMV entry pathways in porcine endothelial cells, in order to develop strategies for prevention and to compare it with human endothelial cells, yielding potentially new insights on the mechanisms of HCMV entry. My findings demonstrate that similarly to entry into human cells, entry of both the endotheliotropic (TB40/E) and the fibrotropic (TB40/F) (except for microvascular porcine endothelial cells) strains into porcine endothelial cells (pEC) requires the expression of the β1 integrin subunit. In contrast, the expression of platelet-derived growth factor receptor alpha (PDGFRα) is required for entry of both strains in pEC only. I demonstrate that internalization of TB40/E viral particles by human endothelial cells and pEC occurs with a similar mechanism, i.e. a dynamin-2-, lipid rafts-, actin- and pH-dependent mechanism, whereas internalization of TB40/F viral particles by pEC occurs mainly by a dynamin-2-dependent, clathrin-, lipid rafts- independent mechanism and in a pH dispensable manner. Besides, when actin polymerisation is prevented, TB40/F enters pEC in an actin-independent fashion, suggesting an entry by fusion or the involvement of another component of the cellular cytoskeleton. I demonstrate for the first time that after internalization, TB40/E viral particles translocate to the nucleus of hEC through the classical endolysosomal pathway going from early endosomes to late endosome / lysosomes. Similar results were observed for pEC. In contrast, in TB40/F-infected hEC, viral particles located only in early endosome, suggesting a sequestration of virions in this compartment, whereas in pEC, viral particles were able to transfer from early endosomes to late endosomes / lysosomes. Finally, I demonstrate that nuclear translocation differs between hEC and pEC and viral strains, as observed by a nuclear accumulation of the tegument protein pp65 only in TB40/F-infected pEC. This differential nuclear accumulation is correlated with a higher accumulation of pp65 in TB40/F-infected EC compared to TB40/E-infected EC, which results in an earlier initiation of replication of TB40/F compared to TB40/E in pEC. In conclusion, my findings suggest that HCMV uses distinct entry pathways that are dependent on both the strain and the origin of the target cells, underlying the capacity of HCMV to adapt to its environment, and its ability, following entry, to induce a successful infection of pEC challenges the traditional paradigm of species specificity. Besides its fundamental research significance, investigation of HCMV cross species infection has an important implication when applied to xenotransplantation. Determining the early events of infection will help in developing strategies to prevent HCMV infection by blocking entry into porcine cells or by rendering swine resistant to HCMV infection, which is essential for xenotransplantation to become a clinical reality. Die Allotransplantation hat sich von einem experimentellen Verfahren zu einer sehr erfolgreichen, häufig lebensrettenden Behandlung von Patienten mit terminalem Organversagen entwickelt. Dieser Erfolg hat zu einer zunehmenden Verknappung von Organen geführt. Als Alternative ist die Xenotransplantation in den Blickpunkt des Interesses gerückt. Miniaturschweine werden als die aktuell besten Spender angesehen, weil diese verschiedene Voraussetzungen erfüllen: Pathogen-freies Züchten und Halten der Tiere, eine relative kurze Tragzeit und Nachkommen in genügender Anzahl. Weitere Vorteile sind eine ähnliche Physiologie und Grösse der Organe. Aktuell gibt es weltweit klinische Versuche mit Transplantation porziner Inselzellen in Menschen zur Behandlung der Diabetes; diese Versuche sind vielversprechend. Für die Organtransplantation ist davon auszugehen, dass für die Verhinderung der Abstossung eine erhebliche immunsuppressive Therapie notwendig sein wird. Dabei steht das transplantierte Organ in engem Kontakt mit potentiellen Pathogenen. Bisher waren die Bedenken auf eine Uebertragung von vom Spender ausgehenden Infektionen ausgerichtet, sog. xenogenen Infektionen. Dabei wurde zu wenig berücksichtigt, dass humane Pathogene das transplantierte Organ ebenfalls befallen und schädigen können, mit dem Resultat eines Organversagens. Die Infektion mit Zytomegalievirus spielt in diesem Zusammenhang eine wichtige Rolle, ist sie doch die häufigste virale Komplikation nach Allotransplantation. Unser Labor konnte in früheren Studien darlegen, dass menschliche Zytomegalieviren porzine Zellen infizieren können. Diese Infektion führte zu Veränderungen des Phänotyps mit einer alterierten Expression verschiedener Marker und zu zytopathischen Effekten und Induktion der Apoptose. Diese Veränderungen haben das Potential, in einem transplantierten Organ eine Abstossung oder eine Funktionsverlust zu induzieren. Ziel dieser Arbeit war es, die Mechanismen des Eindringens von Zytomegalieviren in porzine Endothelzellen zu studieren. Ein besseres Verständnis würde es erlauben, Strategien zur Verhinderung einer solchen Infektion zu entwickeln. Meine Resultate demonstrieren, dass wie im menschlichen Modell mit humanen Endothelzellen sowohl endotheliotrope (TB40/E) als auch fibrotrope (TB40/F) Stämme für eine erfolgreiche Infektion den Korezeptor β1 Integrin benötigen. Auf der anderen Seite ist PDGFRα (platelet-derived growth factor receptor alpha) notwendig in porzinen, nicht aber in humanen Zellen. Internalisierung von TB40/E erfolgt sowohl in menschlichen als auch porzinen Zellen durch ähnliche Mechanismen, so ist dieser Prozess Dynamin-2-, lipid rafts (Cholesterin-reiche Mikrodomänen in Zellmembranen)-, Aktin- und pH-abhängig. Beim fibrotropen Stamm (TB40/F) erfolgt dieser Prozess in porzinen Zellen mittels eines Dynamin-2- abhängigen, aber Clathrin-, lipid raft-unabhängigen Mechanismus, auch schient der pH keine Rolle zu spielen. Aktin ist für das Eindringen von TB40/F in porzine Zellen nicht notwendig. Ich konnte ebenfalls zum ersten Mal im menschlichen Modell zeigen, das TB40/E nach Eindringen den klassischen endolysosomalen Weg zum Zellkern nimmt, und dass dies auch für porzine Zellen gilt. Ein anderes Verhalten wies der fibrotrope Stamm TB40/F auf: In menschlichen Ednothelzellen konnte dieser nur in einem frühen Lysosm nachgewiesen werden, während in porzinen Zellen der ganze Transportweg nachverfolgt werden konnte. Mittels des Nachweises der Akkumulation eines viralen Tegumentproteins, pp65, konnten wir nachweisen, dass der Transfer zum Nukleus differiert zwischen humanen und porzinen Endothelzellen, was zu einer früheren und gesteigerten Replikation von TB40/F im Vergleich zu TB40/E in porzinen Endothelzellen führt. Zusammenfassend zeigen meine Resultate auf, dass Zytomegalieviren eine Vielzahl verschiedener Möglichkeiten aufweisen, Zellen zu infizieren. Dabei sind die Unterschiede nicht primär zwischen Zellen verschiedener Arten sichtbar, sondern vielmehr abhängig von der jeweiligen anatomischen Abstammung der Zelle und vom Virusstamm. Diese Erkenntnisse zeigen die enorme Adaptationsfähigkeit von Zytomegalieviren auf, sich in verschiedenen Zellen zu replizieren und infektionsfähige Nachkommen zu generieren. Damit ist auch das lang-gehegte Paradigma der Spezies-Spezifität in Frage gestellt. Diese Resultate haben neben der biologischen Bedeutung auch die praktische Konsequenz, dass nach Transplantation eines porzinen Organs im Menschen mit einer Infektion durch humane Zytomegalieviren gerechnet werden muss. Der Verhinderung einer solchen Infektion kommt dabei zentrale Bedeutung zu, bevor Xenotransplantation eine klinische Realität wird

Statins demonstrate a broad anti-cytomegalovirus activity in vitro in ganciclovir-susceptible and resistant strains

Vasculoprotective and cholesterol-lowering properties are hallmarks of statins. Recently, statins have been found to exhibit antiviral activity. Little is known about the potential of statins against human cytomegalovirus (HCMV), a risk factor in the pathogenesis of atherosclerosis. In this study, the in vitro anti-CMV activity of four statins (atorva-, fluva-, prava-, and simvastatin) was explored in human aortic endothelial cells (HAEC) and fibroblasts. All statins dose-dependently reduced HCMV titers in both cell types. Whereas atorva-, fluva-, and simvastatin showed comparable EC50 and EC90 within a low micromolar range in HAEC, pravastatin exhibited only limited effects. In metabolite rescue experiments, mevalonate almost completely abrogated the anti-CMV activity of all statins, whereas cholesterol failed to counteract the effects. Geranylgeranyl-pyrophosphate partially reversed the anti-CMV activity of most statins, suggesting an involvement of the non-sterol isoprenoid arm of the mevalonate pathway as the mode-of-action. The accumulation of immediate early viral antigens was blocked after 1 dpi onwards, and early and late antigen expression was completely abolished in HAEC. The antiviral activity of statins was comparable to ganciclovir and was retained in a ganciclovir-resistant HCMV strain. These findings provide new insight into the beneficial effects of statins, adding antiviral activity against HCMV to their list of pleïotropic properties, and support further clinical investigations on combined therapy for the management of active HCMV disease. J. Med. Virol. 87: 141-153, 2015. © 2014 Wiley Periodicals, Inc