Transgenesis and animal welfare : implications of transgenic procedures for the well-being of the laboratory mice

Transgenic animals play an important role in biomedical research. Their use as animal model is still increasing. Although the process of transgenesis may contribute to refinement of animal use, the application of the biotechnological procedures that are involved in the production of transgenic animals may also cause unexpected, uncontrolled, and even undetected animal suffering. Therefore, the aim of the present thesis was to study the effects of the biotechnological procedures involved in genetic modification on the welfare of the resulting offspring. The two most frequently used techniques for inserting new genetic material into the mammalian germ line, pronuclear microinjection and embryonic stem (ES) cell-mediated gene transfer, have been studied. Different groups of mice were generated, each of which had undergone different aspects of the microinjection technique (Chapters 2 and 3) or of the gene targeting technique (Chapters 4 and 5), in order to determine whether the specific manipulations have any effect on the development or behaviour of the progeny. In this way, the effects of microinjection, electroporation, embryo-culture or embryo transplantation on the development of (chimeric) mice could be studied separately. All mice of the different groups were tested in their pre-weaning (0-3 weeks of age) and post-weaning period (4-30 weeks of age) for behavioural and morphological/physiological development and for clinical appearance. Thereafter, post-mortem examinations were performed. In addition, the feasibility of the use of score sheets for monitoring the welfare of transgenic mice, as part of the daily care of animals, has been explored (Chapter 6). With both techniques, the biotechnological procedures increased (perinatal) pup mortality and body weight. In addition, with the gene targeting technique, 8% of the chimeric animals was hermaphrodite. The results so far indicate that, under the present conditions, the biotechnological procedures (microinjection; in vitro culture; embryo transfer) mainly affect the viability of the embryos and seem to have no major effects on the development and behaviour of mice that survive the first 2-3 days after birth. However, before drawing general conclusions, more and different transgenic lines should be studied in a comparable way. A scoring system has been developed, containing a limited number of sensitive, easy to determine and non-invasive parameters, selected from our previous studies on implications of transgenesis for the well-being of mice. The feasibility of this scoring system has been tested, to assess the use of score sheets for monitoring the welfare of transgenic mice on a practical basis, as part of the animal technicians daily routine in a transgenic unit. It has been found to be both practical and useful. Therefore, in the production of transgenic animals, the use of score sheets is recommended, in order to detect both the intended and the non-intended (side) effects of the introduced or mutated gene at an early stage of development

Effect of adipose tissue quantity and dysfunction on the risk of cancer in individuals with and without type 2 diabetes

Objective: To determine the role of waist circumference and metabolic dysfunction in the risk of cancer in individuals with type 2 diabetes (T2D) and to compare this to individuals without T2D. Methods: Individuals with (n = 1925) and without T2D (n = 10,204) were included from the UCC-SMART cohort. Incident cancer diagnoses were obtained by linkage with the Netherlands Cancer Registry. Metabolic dysfunction was defined as ≥ 3 adapted NCEP ATP-III metabolic syndrome criteria. The effects of waist circumference and metabolic dysfunction on cancer were assessed using Cox proportional hazards models, adjusted for confounders. Results: During a median follow-up of 8.3 years (IQR 4.2–13.1), 1740 individuals were diagnosed with cancer. Incidence rates of total cancer were 19.3 and 15.5/1000 person-years for individuals with and without T2D, respectively. In individuals without T2D, a higher waist circumference was associated with an increased risk of colorectal (per standard deviation: HR 1.23; 95%CI 1.03–1.46), urinary tract (HR 1.28; 95%CI 1.05–1.56) and total cancer (HR 1.06; 95%CI 1.02–1.13). Metabolic dysfunction was related to an increased risk of colorectal (HR 1.35; 95%CI 1.01–1.82), lung (HR 1.37; 95%CI 1.07–1.75) and total cancer (HR 1.13; 95%CI 1.01–1.25) in individuals without T2D. In individuals with T2D, no significant associations were found. Conclusion: Incidence rates of cancer are higher among individuals with T2D. However, higher waist circumference and metabolic dysfunction are only associated with an increased cancer risk in patients without T2D. These findings provide novel insights into the role of metabolic dysfunction in the occurrence of cancer

BTK inhibition sensitizes acute lymphoblastic leukemia to asparaginase by suppressing the amino acid response pathway

Asparaginase (ASNase) therapy has been a mainstay of acute lymphoblastic leukemia (ALL) protocols for decades and shows promise in the treatment of a variety of other cancers. To improve the efficacy of ASNase treatment, we used a CRISPR/Cas9-based screen to identify actionable signaling intermediates that improve the response to ASNase. Both genetic inactivation of Bruton’s tyrosine kinase (BTK) and pharmacological inhibition by the BTK inhibitor ibrutinib strongly synergize with ASNase by inhibiting the amino acid response pathway, a mechanism involving c-Myc–mediated suppression of GCN2 activity. This synthetic lethal interaction was observed in 90% of patient-derived xenografts, regardless of the genomic subtype. Moreover, ibrutinib substantially improved ASNase treatment response in a murine PDX model. Hence, ibrutinib may be used to enhance the clinical efficacy of ASNase in ALL. This trial was registered at www.clinicaltrials.gov as # NCT02884453

Histone deacetylase inhibition sensitizes p53-deficient B-cell precursor acute lymphoblastic leukemia to chemotherapy

In pediatric acute lymphoblastic leukemia (ALL), mutations/deletions affecting the TP53 gene are rare at diagnosis. However, at relapse about 12% of patients show TP53 aberrations, which are predictive of a very poor outcome. Since p53-mediated apoptosis is an endpoint for many cytotoxic drugs, loss of p53 function frequently leads to therapy failure. In this study we show that CRISPR/Cas9-induced loss of TP53 drives resistance to a large majority of drugs used to treat relapsed ALL, including novel agents such as inotuzumab ozogamicin. Using a high-throughput drug screen, we identified the histone deacetylase inhibitor romidepsin as a potent sensitizer of drug responsiveness, improving sensitivity to all chemotherapies tested. In addition, romidepsin improved the response to cytarabine in TP53-deleted ALL cells in vivo. Together, these results indicate that the histone deacetylase inhibitor romidepsin can improve the efficacy of salvage therapies for relapsed TP53-mutated leukemia. Since romidepsin has been approved for clinical use in some adult malignancies, these findings may be rapidly translated to clinical practice

Histone deacetylase inhibition sensitizes p53-deficient B-cell precursor acute lymphoblastic leukemia to chemotherapy

In pediatric acute lymphoblastic leukemia (ALL), mutations/deletions affecting the TP53 gene are rare at diagnosis. However, at relapse about 12% of patients show TP53 aberrations, which are predictive of a very poor outcome. Since p53-mediated apoptosis is an endpoint for many cytotoxic drugs, loss of p53 function frequently leads to therapy failure. In this study we show that CRISPR/Cas9-induced loss of TP53 drives resistance to a large majority of drugs used to treat relapsed ALL, including novel agents such as inotuzumab ozogamicin. Using a high-throughput drug screen, we identified the histone deacetylase inhibitor romidepsin as a potent sensitizer of drug responsiveness, improving sensitivity to all chemotherapies tested. In addition, romidepsin improved the response to cytarabine in TP53-deleted ALL cells in vivo. Together, these results indicate that the histone deacetylase inhibitor romidepsin can improve the efficacy of salvage therapies for relapsed TP53-mutated leukemia. Since romidepsin has been approved for clinical use in some adult malignancies, these findings may be rapidly translated to clinical practice

International Research Infrastructure Landscape 2019 : A European Perspective

The report is the final product of the RISCAPE project, funded by the European Commission H2020 programme

International Research Infrastucture Landscape 2019: A European Perspective

The book 'International Research Infrastucture Landscape 2019: A European Perspective' provides the final report of the RISCAPE-project, supported by the European Commission's Horizon 2020-project. The RISCAPE-project aims to provide a systematic, focused, high-quality, comprehensive, consistent and peer-reviewed international landscape analysis report on the position and complementarities of the major European RIs in the international Research Infrastructure landscape.University of Turku has contributed with the domain report on international Energy Research Infrastructures, which forms chapter 6 of the final book.</p

Identification of regulatory variants associated with genetic susceptibility to meningococcal disease.

Non-coding genetic variants play an important role in driving susceptibility to complex diseases but their characterization remains challenging. Here, we employed a novel approach to interrogate the genetic risk of such polymorphisms in a more systematic way by targeting specific regulatory regions relevant for the phenotype studied. We applied this method to meningococcal disease susceptibility, using the DNA binding pattern of RELA - a NF-kB subunit, master regulator of the response to infection - under bacterial stimuli in nasopharyngeal epithelial cells. We designed a custom panel to cover these RELA binding sites and used it for targeted sequencing in cases and controls. Variant calling and association analysis were performed followed by validation of candidate polymorphisms by genotyping in three independent cohorts. We identified two new polymorphisms, rs4823231 and rs11913168, showing signs of association with meningococcal disease susceptibility. In addition, using our genomic data as well as publicly available resources, we found evidences for these SNPs to have potential regulatory effects on ATXN10 and LIF genes respectively. The variants and related candidate genes are relevant for infectious diseases and may have important contribution for meningococcal disease pathology. Finally, we described a novel genetic association approach that could be applied to other phenotypes

Plasma lipid profiles discriminate bacterial from viral infection in febrile children

Fever is the most common reason that children present to Emergency Departments. Clinical signs and symptoms suggestive of bacterial infection are often non-specific, and there is no definitive test for the accurate diagnosis of infection. The 'omics' approaches to identifying biomarkers from the host-response to bacterial infection are promising. In this study, lipidomic analysis was carried out with plasma samples obtained from febrile children with confirmed bacterial infection (n = 20) and confirmed viral infection (n = 20). We show for the first time that bacterial and viral infection produces distinct profile in the host lipidome. Some species of glycerophosphoinositol, sphingomyelin, lysophosphatidylcholine and cholesterol sulfate were higher in the confirmed virus infected group, while some species of fatty acids, glycerophosphocholine, glycerophosphoserine, lactosylceramide and bilirubin were lower in the confirmed virus infected group when compared with confirmed bacterial infected group. A combination of three lipids achieved an area under the receiver operating characteristic (ROC) curve of 0.911 (95% CI 0.81 to 0.98). This pilot study demonstrates the potential of metabolic biomarkers to assist clinicians in distinguishing bacterial from viral infection in febrile children, to facilitate effective clinical management and to the limit inappropriate use of antibiotics

Plasma lipid profiles discriminate bacterial from viral infection in febrile children

Fever is the most common reason that children present to Emergency Departments. Clinical signs and symptoms suggestive of bacterial infection are often non-specific, and there is no definitive test for the accurate diagnosis of infection. The 'omics' approaches to identifying biomarkers from the host-response to bacterial infection are promising. In this study, lipidomic analysis was carried out with plasma samples obtained from febrile children with confirmed bacterial infection (n = 20) and confirmed viral infection (n = 20). We show for the first time that bacterial and viral infection produces distinct profile in the host lipidome. Some species of glycerophosphoinositol, sphingomyelin, lysophosphatidylcholine and cholesterol sulfate were higher in the confirmed virus infected group, while some species of fatty acids, glycerophosphocholine, glycerophosphoserine, lactosylceramide and bilirubin were lower in the confirmed virus infected group when compared with confirmed bacterial infected group. A combination of three lipids achieved an area under the receiver operating characteristic (ROC) curve of 0.911 (95% CI 0.81 to 0.98). This pilot study demonstrates the potential of metabolic biomarkers to assist clinicians in distinguishing bacterial from viral infection in febrile children, to facilitate effective clinical management and to the limit inappropriate use of antibiotics