Of voles and men: novel Hantavirus in vitro models

Hantavirus-infection can cause severe disease in humans, with up to 40% case fatalities. Presently, no therapeutics or prophylaxis against hantaviral illness exists. The mechanisms underlying the onset of symptoms and the following pathogenesis are not fully known. In vitro research conducted on mono-layered human cell cultures with cell line adapted hantaviruses provides important knowledge for understanding the virus and the innate immune responses induced, but the results might not always correspond to infection in humans. Furthermore, neither the interactions between the virus and its natural animal host, nor why infections in them do not cause disease are well understood. To better understand hantavirus pathogenesis, new models and tools are needed. In paper I, genetic properties of hantavirus were investigated by analyzing two substrains of Puumala hantavirus (PUUV) derived from virus propagated on cells lacking parts of the innate antiviral response. From this study we could conclude that the differences in phenotype and replication were caused without mutations in the viral glycoproteins. Mutations were however observed in the nucleocapsid protein and in the RNA dependent RNA polymerase. We also observed that the phenotypic differences between the substrains and the parental strain were cell line specific. To be able to analyze hantavirus infection of cells from the natural host in vitro, vole embryonic fibroblasts derived from bank voles, the host for PUUV, were isolated in paper II. These cells were susceptible and permissive not only to PUUV, but to a range of other bank vole-borne viruses, indicating that these cells can be an important tool for studies of several zoonotic viruses. Regarding IFN-ß and Mx, two important antiviral proteins, infection of the vole fibroblasts with PUUV induced a different response compared to that observed in human fibroblasts, indicating a possible species difference in innate immune response against hantavirus infection. To better mirror the situation in human organs, complex in vitro models resembling human tissue might be valuable. In paper III we took the advantage of using a 3-dimensional organotypic model of human lung tissue to study early and long term infections of the highly pathogenic Andes virus (ANDV). With this model we could show that a peak in progeny virus production occurs more than a week after initial infection. We also observed increased extracellular levels of the pro-inflammatory cytokines IL-6 and IL-8, and of IP-10 and eotaxin-1 upon ANDVinfection, as well as suppression of RANTES-responses. These ANDV-induced effects were observed late after infection. VEGF-A are suggested to be involved in the pathogenesis of hantaviral illness, as it might be responsible for the increased vascular permeability observed in patients. We observed higherlevels of VEGF-A after ANDV-infection, suggesting that infection of lung tissue per se might be responsible for the increased VEGF-A levels observed in patients. The potential role of dendritic cells (DCs) during hantaviral illness is not known. In paper IV, we studied the effect DCs have on hantavirus-infection, by adding these cells to the organotypic human lung tissue model. We showed that DCs had an antiviral effect against hantavirus- infection, suggesting that DCs might be involved in limiting the infection. In this thesis, establishment of novel in vitro models, and studies of different aspects of hantavirus-infection were performed. Genetic properties of PUUV were investigated and by using different in vitro models, the cellular responses of voles and humans during hantavirus-infection were analyzed

Children of Unknown Fathers: Prevalence and Outcomes in Sweden

-Childhood conditions; family structure; child outcomes; father unknown

Human papillomavirus test and vaccination : impact on cervical cancer screening and prevention

Human papillomavirus (HPV) is the world’s most common sexually transmitted infection and its consequence cervical cancer is one of the world’s most common cancer forms. Revolutionary advances in HPV testing and HPV vaccination have the potential to radically change women’s health. However, several challenges remain before effective cervical cancer control can be reached. This thesis has exploited the excellent Swedish register and biobank infrastructure, for studies aiming to inform HPV-based prevention of cervical cancer. To this end, we have combined epidemiological, virological and biostatistical investigation of the use of HPV tests in organized cervical screening. We also investigated awareness of HPV and acceptability of HPV vaccination in the population. Study I was a molecular epidemiological case-control study, nested within the Swedish cervical screening program, including 515 women with cancer in situ (CIS), 315 women with invasive squamous cervical cancer (SCC), and matched control women. 2772 archival cervical smears were gathered and subjected to full HPV-typing. The median follow-up was 5-7 years. We provide prospective evidence that infection with non-16/18 high-risk HPV types, and persistent infection with HPV16, both confer increased risks for future invasive cervical cancer. In Study II, we extended this case-control study to include 621 women with CIS and 457 women with SCC; a uniquely large sample. Here, 5665 archival smears were tested for HPV, and HPV16 positive smears further analyzed for HPV16 viral load through realtime-PCR. The median follow-up was 6-8 years. We show that HPV16 viral load predicts risk for both CIS and SCC, but also that the risk functions differ per diagnosis and over time. Thus, HPV16 viral load appears highly complex which may limit its use in HPV-based cervical screening. We further show unexpectedly low viral loads early in invasive disease, which may carry implications for the weighing of sensitivity against specificity in HPV testing. In Study III, we report results from a cross-sectional population-based survey examining awareness and knowledge of HPV in 24,513 adult Swedish respondents. We show that awareness of condyloma and cervical cancer was high, but that awareness and understanding of their causal factor HPV was poor. The knowledge that men could contract HPV needs to be improved. Education campaigns on HPV should particularly target young men, and those of low education. Finally, Study IV was also a cross-sectional survey, examining acceptability of HPV vaccination among 10,567 young adults age 18-30 in Sweden. We show that willingness to vaccinate was quite high, but that information on the benefits of vaccinating before sexual debut is important. Few adults stated their health-care related behavior would change after vaccination, but a number were uncertain, suggesting an educational need when vaccinating this group. A perceived risk of side effects was the largest potential barrier to vaccination. Our findings should assist risk stratification in HPV-based screening, and design of HPV vaccination campaigns. Future research should include investigations of cervical screening attendance, HPV vaccine uptake, and acceptability of HPV-based screening

Green-Lean Synergy - Root-Cause Analysis in Food Waste Prevention

Purpose_The goal of this paper is to explore the possible synergetic effects between lean philosophy and green endeavors in improving resource efficiency in the food sector. To that end, it is investigated how a proper and tailor-made adaptation of the lean six sigma root cause analysis method could help in overcoming the complexities of increased resource efficiency in food production.Design/methodology/approach_The case study concerned reduction of waste at an industrial production line of a dough-based product, through the implementation of the lean six sigma tool.Findings_An achievement of a 50% reduction of waste on the studied process line was reached, thus exceeding the initial improvement goal.Research limitations/implications (if applicable)_While the explicit findings on the specific root causes of waste on this actual production line are not immediately transferrable to other cases, they show that applying this method to identifying and eliminating root causes of waste for other products and processes in the food sector could not only reduce costs but also contribute to more resource-efficient and sustainable industrial food production.Practical implications (if applicable)_ Political and public high interest in environmental and social sustainability associated with food waste render this an important development.Originality/value_ While the potential of linking green and lean efforts has been acknowledged, the application of the lean six sigma methodology for more sustainable food production has not yet been explored. This paper contributes to this researc

Human papillomavirus detection by whole-genome next-generation sequencing : importance of validation and quality assurance procedures

Funding: This work was funded by the Center for Innovative Medicine (CIMED grant number 613/06, to KS), the Swedish Medical Society (SLS, grant number 885941, to KS),and the Swedish Foundation for Strategic Research (grant number RB13-0011, supporting KS and SAM). The SHPVRL is supported by National Services Division of the National Health Service in Scotland.Next-generation sequencing (NGS) yields powerful opportunities for studying human papillomavirus (HPV) genomics for applications in epidemiology, public health, and clinical diagnostics. HPV genotypes, variants, and point mutations can be investigated in clinical materials and described in previously unprecedented detail. However, both the NGS laboratory analysis and bioinformatical approach require numerous steps and checks to ensure robust interpretation of results. Here, we provide a step-by-step review of recommendations for validation and quality assurance procedures of each step in the typical NGS workflow, with a focus on whole-genome sequencing approaches. The use of directed pilots and protocols to ensure optimization of sequencing data yield, followed by curated bioinformatical procedures, is particularly emphasized. Finally, the storage and sharing of data sets are discussed. The development of international standards for quality assurance should be a goal for the HPV NGS community, similar to what has been developed for other areas of sequencing efforts including microbiology and molecular pathology. We thus propose that it is time for NGS to be included in the global efforts on quality assurance and improvement of HPV-based testing and diagnostics.Publisher PDFPeer reviewe

Sodium content in processed food items in Sweden compared to other countries: a cross-sectional multinational study

BackgroundDietary sodium has a dose-response relationship with cardiovascular disease, and sodium intake in Sweden exceeds national and international recommendations. Two thirds of dietary sodium intake comes from processed foods, and adults in Sweden eat more processed foods than any other European country. We hypothesized that sodium content in processed foods is higher in Sweden than in other countries. The aim of this study was to investigate sodium content in processed food items in Sweden, and how it differs from Australia, France, Hong Kong, South Africa, the United Kingdom and the United States.MethodsData were collected from retailers by trained research staff using standardized methods. Data were categorized into 10 food categories and compared using Kruskal-Wallis test of ranks. Sodium content in the food items was compared in mg sodium per 100 g of product, based on the nutritional content labels on the packages.ResultsCompared to other countries, Sweden had among the highest sodium content in the “dairy” and “convenience foods” categories, but among the lowest in “cereal and grain products,” “seafood and seafood products” and “snack foods” categories. Australia had the overall lowest sodium content, and the US the overall highest. The highest sodium content in most analyzed countries was found in the “meat and meat products” category. The highest median sodium content in any category was found among “sauces, dips, spreads and dressings” in Hong Kong.ConclusionThe sodium content differed substantially between countries in all food categories, although contrary to our hypothesis, processed foods overall had lower sodium content in Sweden than in most other included countries. Sodium content in processed food was nonetheless high also in Sweden, and especially so in increasingly consumed food categories, such as “convenience foods”

Sex differences in the prognostic significance of KRAS codons 12 and 13, and BRAF mutations in colorectal cancer: a cohort study

Background: Activating KRAS and BRAF mutations predict unresponsiveness to EGFR-targeting therapies in colorectal cancer (CRC), but their prognostic value needs further validation. In this study, we investigated the impact of KRAS codons 12 and 13, and BRAF mutations on survival from CRC, overall and stratified by sex, in a large prospective cohort study. Methods: KRAS codons 12 and 13, and BRAF mutations were analysed by pyrosequencing of tumours from 525 and 524 incident CRC cases in The Malmö Diet and Cancer Study. Associations with cancer-specific survival (CSS) were explored by Cox proportional hazards regression, unadjusted and adjusted for age, TNM stage, differentiation grade, vascular invasion and microsatellite instability (MSI) status. Results: KRAS and BRAF mutations were mutually exclusive. KRAS mutations were found in 191/ 525 (36.4%) cases, 82.2% of these mutations were in codon 12, 17.3% were in codon 13, and 0.5% cases had mutations in both codons. BRAF mutations were found in 78/524 (14.9%) cases. Overall, mutation in KRAS codon 13, but not codon 12, was associated with a significantly reduced CSS in unadjusted, but not in adjusted analysis, and BRAF mutation did not significantly affect survival. However, in microsatellite stable (MSS), but not in MSI tumours, an adverse prognostic impact of BRAF mutation was observed in unadjusted, but not in adjusted analysis. While KRAS mutation status was not significantly associated with sex, BRAF mutations were more common in women. BRAF mutation was not prognostic in women; but in men, BRAF mutation was associated with a significantly reduced CSS in overall adjusted analysis (HR = 3.50; 95% CI = 1.41–8.70), but not in unadjusted analysis. In men with MSS tumours, BRAF mutation was an independent factor of poor prognosis (HR = 4.91; 95% CI = 1.99–12.12). KRAS codon 13 mutation was associated with a significantly reduced CSS in women, but not in men in unadjusted, but not in adjusted analysis. Conclusions: Results from this cohort study demonstrate sex-related differences in the prognostic value of BRAF mutations in colorectal cancer, being particularly evident in men. These findings are novel and merit further validation

The WID-CIN test identifies women with, and at risk of, cervical intraepithelial neoplasia grade 3 and invasive cervical cancer

BACKGROUND: Cervical screening is transitioning from primary cytology to primary human papillomavirus (HPV) testing. HPV testing is highly sensitive but there is currently no high-specificity triage method for colposcopy referral to detect cervical intraepithelial neoplasia grade 3 or above (CIN3+) in women positive for high-risk (hr) HPV subtypes. An objective, automatable test that could accurately perform triage, independently of sample heterogeneity and age, is urgently required. METHODS: We analyzed DNA methylation at ~850,000 CpG sites across the genome in a total of 1254 cervical liquid-based cytology (LBC) samples from cases of screen-detected histologically verified CIN1-3+ (98% hrHPV-positive) and population-based control women free from any cervical disease (100% hrHPV-positive). Samples were provided by a state-of-the-art population-based cohort biobank and consisted of (i) a discovery set of 170 CIN3+ cases and 202 hrHPV-positive/cytology-negative controls; (ii) a diagnostic validation set of 87 CIN3+, 90 CIN2, 166 CIN1, and 111 hrHPV-positive/cytology-negative controls; and (iii) a predictive validation set of 428 cytology-negative samples (418 hrHPV-positive) of which 210 were diagnosed with CIN3+ in the upcoming 1-4 years and 218 remained disease-free. RESULTS: We developed the WID-CIN (Women's cancer risk IDentification-Cervical Intraepithelial Neoplasia) test, a DNA methylation signature consisting of 5000 CpG sites. The receiver operating characteristic area under the curve (AUC) in the independent diagnostic validation set was 0.92 (95% CI 0.88-0.96). At 75% specificity (≤CIN1), the overall sensitivity to detect CIN3+ is 89.7% (83.3-96.1) in all and 92.7% (85.9-99.6) and 65.6% (49.2-82.1) in women aged ≥30 and <30. In hrHPV-positive/cytology-negative samples in the predictive validation set, the WID-CIN detected 54.8% (48.0-61.5) cases developing 1-4 years after sample donation in all ages or 56.9% (47.6-66.2) and 53.5% (43.7-63.2) in ≥30 and <30-year-old women, at a specificity of 75%. CONCLUSIONS: The WID-CIN test identifies the vast majority of hrHPV-positive women with current CIN3+ lesions. In the absence of cytologic abnormalities, a positive WID-CIN test result is likely to indicate a significantly increased risk of developing CIN3+ in the near future

Sodium content in processed food items in Sweden compared to other countries: a cross-sectional multinational study

Background Dietary sodium has a dose-response relationship with cardiovascular disease, and sodium intake in Sweden exceeds national and international recommendations. Two thirds of dietary sodium intake comes from processed foods, and adults in Sweden eat more processed foods than any other European country. We hypothesized that sodium content in processed foods is higher in Sweden than in other countries. The aim of this study was to investigate sodium content in processed food items in Sweden, and how it differs from Australia, France, Hong Kong, South Africa, the United Kingdom and the United States. Methods Data were collected from retailers by trained research staff using standardized methods. Data were categorized into 10 food categories and compared using Kruskal-Wallis test of ranks. Sodium content in the food items was compared in mg sodium per 100 g of product, based on the nutritional content labels on the packages. Results Compared to other countries, Sweden had among the highest sodium content in the “dairy” and “convenience foods” categories, but among the lowest in “cereal and grain products,” “seafood and seafood products” and “snack foods” categories. Australia had the overall lowest sodium content, and the US the overall highest. The highest sodium content in most analyzed countries was found in the “meat and meat products” category. The highest median sodium content in any category was found among “sauces, dips, spreads and dressings” in Hong Kong. Conclusion The sodium content differed substantially between countries in all food categories, although contrary to our hypothesis, processed foods overall had lower sodium content in Sweden than in most other included countries. Sodium content in processed food was nonetheless high also in Sweden, and especially so in increasingly consumed food categories, such as “convenience foods”