A total of 1 500 000 SEK was awarded in Grants from the Swedish Fund for Research without Animal Experiments for 2008-2009. It was divided between 13 projekts, 6 of which were new. The recipients shortly describe their projects below.
Torbjörn Bengtsson, Örebro University
”In vitro-studier av cell-cell interaktion och inflammatoriska och mitogena mekanismer i en human kärlmodell”
90 000 SEK
Atherosclerosis gives rise to serious cardiovascular complications and is the principal cause of morbidity and mortality in the industrialized world. Studies during the last decade emphasize that atherosclerosis is a chronic inflammatory fibroproliferative disease of the arterial wall. To understand what leads to the fatal events of cardiovascular disease we have to search for suitable models of atherosclerosis involving the major components of the blood-vascular compartment. This project aims to replace or limit the use of animal models by developing a dynamic in vitro model based on human vascular and blood cells and protein components, in order to study cellular and molecular processes related to atherosclerosis. The project has a high priority in a new-started strategic research programme "
A good human cell model would help us not only to understand the mechanisms of atherosclerosis but also to design and test different preventive, diagnostic and terapeutic approaches of cardiovascular disease.
Anna Forsby, Department of neurochemistry and neurotoxicology, Stockholm University
"Neuronal cell model for identification of mildly eye-irritating compounds"
The Draize’s eye irritation test has been used during decades for identification of irritating and tissue damaging chemicals. The test, which is performed on rabbits, is quite painful and the scientific value of the results from the test has been questioned. Therefore, extensive efforts to find alternative methods have resulted in several validation studies. These studies have generated alternatives to the Draize’s test for chemicals, which induce severe eye tissue damage (corrosion), but there is still no method that is able to identify chemicals, which only generate mild eye-irritation, e.g. smarting pain. Mild irritation occurs when specific pain-mediating receptors (nociceptors) are activated. We will therefore in this project develop neuronal cell clones, which express specific nociceptors, in order to investigate the possibility to use them for the identification of mild eye-irritating chemicals. If the neuronal clones fulfil the criteria, there is a good chance that the alternative test batteries will be significantly improved. Today, when the EU chemical test strategy proposes an extensive eye irritation screening programme by using the Draize’s test, an optimised alternative test battery can result in that thousands of rabbits will be saved from painful experiments.
Roland Grafström, Institute of Environmental Medicine, Karolinska Institute, Stockholm
" Culture of oral keratinocyte stem cells: gene expression and sensitivity to programmed cell death"
The overall aim is study the growth of the stem cells underlying epithelial regeneration in oral mucosa. Culture methods will be established, including variants without serum, for assessment of gene expression. Profiling of transcript and proteins will be made to determine expression differences between stem cells and epithelial cells of higher maturity. The utilization of stem cells in biomedical research are likely to provide new alternatives to work with laboratory animals, including for cancer research.
Magnus Ingelman-Sundberg, Karolinska institute
”Novel in vitro systems for prediction of drug hepatotoxicity”
The aim of this project is to develop better in vitro models being able to predict drug hepatotoxicity. The most common in vitro models used today are based on 2D monocultures of primary hepatocytes or hepatoma cell lines. The liver is made up of many different cell types and the general idea behind this project is to test if in vitro toxicity testing can be improved by introducing other cell types besides hepatocytes into the test system. It has been shown that drug induced liver injury often requires the participation of immune cells, primarily cells of the innate immune system such as the resident Kupffer cells (liver tissue macrophages) and infiltrating neutrophils/monocytes. The innate immune cells mediate different inflammatory processes when they are activated by e.g. infections or tissue injury.
In this project we will establish human cell culture systems based on co-cultures between human hepatoma cell lines and different human immune cells. In a first co-culture system that has been set up, a human monocytic cell line is plated in an insert keeping the cells separated from the human hepatoma cells by a semi-permeable membrane.
In parallel the project also aims to develop a 3-dimensional model, so-called bioreactors, where all the cells of the liver are incorporated creating a cellular architecture, i.e. biliary structures, which allows for nutrient and gaseous homeostasis. This may open up new principles for toxicity testing without the use of animals.
Maria Karlgren, Uppsala University
”In vitro models for prediction of drug-drug interactions"
In the cell membrane there are many proteins responsible for transporting drugs and other substances in and out of the cell. During drug development it is important to study these events and also to examine potential drug-drug interactions with regard to drug transport and drug metabolism. Today these studies are mainly performed in animals, however due to species differences the results are very difficult to translate into humans.
In this project we will develop new in vitro models were the individual proteins responsible for drug transport can be studied. When developing these models we will in particular focus on transport proteins in the liver. In addition we will also study a new promising cell line obtained from human liver. We think that this cell line has the potential to function as a complex model for human liver and allow us to simultaneously study drug transport and drug metabolism.
Our goal is that these different kinds of models will replace animal testing in the early phase of drug development and also reduce animal testing in later phases.
Maris Lapins, Uppsala University
"Design of selective inhibitors of protein kinases by the use of proteochemometrics”
Protein kinases comprise a large family of enzymes with more than five hundred genes identified in the human genome. Protein kinases act as regulatory switches for essentially all cellular processes, including metabolic pathways, cell growth, differentiation, survival, and apoptosis.
Abnormal function of protein kinases leads to development of many serious diseases, such as cancer, diabetes, inflammatory and autoimmune disorders, and cardiac diseases. In particular, many cancers may be associated to overexpression of specific growth-factor-receptor tyrosine kinases or to their increased activity due to mutations.
Great hopes have been placed that inhibition of kinases will lead to new highly effective treatments of cancer. It has, however, been difficult to design drugs that block specific kinases without interfering with multiple vital cellular pathways. Numerous anti-cancer drug design and development projects have been abandoned due to toxicity or lack of efficacy. Unfortunately, these negative consequences of lack of specificity are often assessed in painful animal experiments using animals, which are infected by tumor sells to develop cancer.
We have developed a completely new computational approach - proteochemometrics - to analyze drug interactions with multiple proteins and to design drugs with improved specificity. The technology is now applied to protein kinase-inhibitor interactions, aiming to inhibit only the malfunctioning kinases without blocking other vitally important kinases. The study will also aid the further development of technologies that can speed up design of selective drugs and replace animal experiments by computational modeling.
Malin Lindstedt, Lund University
"Studies of the effect of allergens on dendritic cells - basis for an in vitro test of the potential of chemicals and proteins to cause sensitization"
The objective of this project is to identify the mechanisms involved when allergens (chemicals and protein causing allergy or hypersensitivity) are recognized by the central regulators of the immune response; the dendritic cells (DCs). Understanding the cellular responses to allergens, and the specific markers involved, is of outmost importance for the development of novel strategies for in vitro prediction of allergenicity. We are part of an EU funded consortium within the 6th framework, consisting of 28 European universities, institutes, companies and organizations, focusing on the development of ‘Novel Testing Strategies for In Vitro Assessment of Allergens – Sens-it-iv’. Risk assessment concerning the allergenicity of compounds so far essentially depends on in vivo animal models. The main goal of the Sens-it-iv program is to design a human cell-based assay to test the propensity of new chemicals and proteins, used by the e.g. the cosmetic and pharmaceutical industries, to cause allergy.
During recent years, we have optimized protocols for culture of a myeloid cell line MUTZ-3, which is able to differentiate into in vitro-derived dendritic cells (DCs) mimicking the phenotype and transcriptional profile of human primary DCs. The cell line serves as the basis for our in vitro test assay and we have studied the response of MUTZ-3 cells to both protein and chemicals causing respiratory and skin sensitization. The effect of a panel of known sensitizers, as well as irritants as controls, has been screened on MUTZ-3 cells using protein microarrays covering 140 CD antigens. We have identified a group of biomarkers that can be used to distinguish between allergenic compounds and irritant controls.
During 2009, we will verify our results and expand the panel of compounds. The search for more biomarkers will be extended to include analytic tools such as transcriptomics using RNA microarrays, proteomics using MS, Luminex analysis of soluble proteins, as well as flow cytometric analysis of phenotypic alterations. In order to evaluate the robustness and reproducibility of our test system, we are also currently involved in a ring-trial consisting of three independent groups within Sens-it-iv which run parallel cultures and allergen-stimulations of MUTZ-3 cells with identical protocols. If our assay proves to be consistent in the ongoing pre-validation, and has potential to partially replace animal experimentation for sensitization, this project will most likely have large impact on future testing procedures.
Tommy Linné, SLU, BMC, Uppsala
"To develop methods for substitution of animal testing and straintyping of TSE agens (prions) with an alternative animal free cell-culture based system"
Prion diseases or TSE (transmissible spongiform encephalopathies) can affect humans (CJD, vCJD), cattle (BSE), cervids (CWD) and sheep (Scrapie). Both Scrapie and BSE have been diagnosed in Sweden and recently 13 cases of an atypical Scrapie named Nor98 have been demonstrated.
The definition of prion diseases as transmissible clearly differ prion diseases from other disorders due to protein aggregation as for ex Alzheimers- and Parkinson disease. In prion diseases as Scrapie in sheep and Chronic Wasting Disease (CWD) of cervids a horizontal transmission is evident with a direct spread in the population of concern, resembling a classic infectious disease.
The infectious prion, PrPSc, can be transmitted to an individual from a diseased source or can be generated spontaneously in the nervous system. In both cases PrPSc will directly affect the normal cellular prion protein and convert it into the diseased prion conformation. The newly formed PrPSc will by unknown mechanisms be spread to neighboring cells.
In vitro system based on cell cultures present many obvious advantages including a possibility to analyze the biological properties of PrPC and PrPSc both on a molecular level and on a cellular level. In addition, the determination of the infectious principle, identifying factors important for it's propagating and the mechanism of shedding and transmission between cells can be elucidated. The most used cell culture is the mouse neuroblastoma cell line; N2a and studies in this and other cell-lines have outlined various chapters of prion biology. However, common to all these cell lines are that they support propagation of selected mouse adapted TSE strains and not propagating natural TSE cases as sheep Scrapie strains or BSE. Multiple distinct natural strains of Scrapie in sheep have been isolated by propagation in inbred mice and been distinguished by incubation periods and specific neuropathology. In Sweden both classical Scrapie and atypical Scrapie Nor98 have been demonstrated. We have earlier differentiated these two natural strains by Western immunoblot electrophoretic profiling from naturally diseased sheep victims.
Our aim is to find cell culture systems instead of animal interventions, which will faithfully propagate prion strains and preserve their associated biochemical characteristics and biological properties on a cellular level permitting straintyping without animal involvements.
Hans-Gustaf Ljunggren, Karolinska institute,
"Development of a human liver-model allowing dynamic immunological studies in hepatitis C virus infection"
The WHO in 2001 named infectious diseases the most important health problem worldwide and reiterated the same statement in 2007. More than 130 million individuals are chronically infected with hepatitis C virus (HCV). Understanding how the human immune system counteracts HCV infection is considered a prerequisite for the design of new treatment protocols to archive efficient pathogen eradication. Man and chimpanzee are the only hosts susceptible to HCV infection. As a consequence of this, experimental immunological studies are to a high degree performed in chimpanzees.
Scientific and ethical considerations relating to research performed in chimpanzee substantiate the need for the development of human in vitro models that in detail mimic the in vivo human liver microenvironment. This development has been restrained due to the following reasons: cultivation of HCV in cell-culture based systems was until recently not possible, and primary human hepatocytes are notoriously hard to propagate ex vivo with sustained phenotypical and functional characteristics. Finally, scarcity of primary human tissue represents a major challenge.
The aim of the current project is to, by circumventing and resolving above presented issues, develop a human in vitro model that to a high degree resemble the human liver and the unique immune system found therein. This system is likely to in most situations replace the chimpanzee model for many of the questions scientists currently try to answer. Further, it is foreseeable that the proposed model can be used to address previously unresolved problems with respect to pathogenesis and resolution of clinical HCV infection. If successful, we anticipate a substantial animal-saving effect in a global perspective.
Matts Olovsson, Uppsala university
”Effekter av hormoner, kemikalier och hormonstörande ämnen på odlade celler från human livmoderslemhinna”
Project information to be added
Johan Rönnelid, Immune complex-mediated regulation of cytokine production in rheumatic diseases and cancer
Uppsala University, Uppsala firstname.lastname@example.org
Antibodies produced by the immune system can aggregate into immune complexes. Such immune complexes composed of antibodies and substances from the body are essential in the pathogenesis of rheumatic disorders and may have importance in the regulation of the immune defence against tumours. Today basic research in both these areas utilizes substantial numbers of laboratory animals.
Our aim is to understand how immune complexes influence the production of cytokines, signal substances of the immune system. The work is entirely based on in vitro research on human tissues and blood. For these purposes we develop new sensitive laboratory techniques to detect immune complexes and their immunological effects on human cells in vitro. Important parts are e.g. to establish totally human cell culture conditions without addition of animal sera, and to develop sensitive techniques for the detection of cytokine production in vitro.
Michael Stigson, Uppsala University
”Developmental toxicity testing based on toxicogenomic responses in embryo-derived pluripotent stem cell lines”
The use of cultured cells for the prediction of potential developmental toxicity would enable rapid and cost-effective high throughput screening
of large numbers of chemical compounds. Such screening tests could be
devised based on prior knowledge of molecular mechanisms and markers of toxicity.
Test methods using embryonic stem cells are currently available but are based on the evaluation of late endpoints of manifest toxicity and do not take into account the underlying mechanisms or pathways of toxicity. We use toxicogenomics approaches to investigate early gene expression changes in response to known teratogenic compounds in embryo-derived pluripotent stem cells. The goal is to identify molecular targets and early response markers of teratogen exposure that can be evaluated as predictive endpoints in a cell-based test system.
Erik Ullerås, SLU, Uppsala
Project information to be added