2014

Grants awarded in 2014

A total of SEK 1 222 000 was awarded in grants from the Swedish Fund for Research without Animal Experiments in 2014. It was divided between 11 projects, three of which were new. Recipients and project titles below. More information about the projects will be added shortly.

New projects

Kristina Blom, Medibiome AB, “A novel alternative method to study wound healing in infected human living skin exposed to substances and biomaterials ” SEK 100 000″

“A unique method to analyze how different products affect wound healing in presence of bacteria has been developed where human living skin tissue is cultured in a bioreactor invented by Medibiome (PCT/EP2014/050899). This bioreactor allows the culture of explanted tissue for prolonged period owing to a continuous flow of nutrients which causes minimal stress to the cells. Preliminary results show that different wound dressings can be evaluated and wound healing processes analyzed in presence of bacteria. To confirm our preliminary promising results, verification and validation studies will be run.

The verification studies will evaluate wound healing when the skin is cultured in Medibiome bioreactor with continuous flow versus in a static system with and without presence of bacteria. Controls known to inhibit and stimulate wound healing, will be assessed for suitability. Optimal methodologies to analyze wound healing will be evaluated and established.

The validation includes: identification of critical parameters; definition of the criteria of the critical parameters; identification of references and controls; establishment of a validation plan and a validation protocol. In this process, validations of instruments are included as well as the practical work.”

Vesa Loitto, Linköpings University, Quantification of ER-stress in immortalized cells refines in vitro toxicity screening/prediction ” SEK 130 000

“In depth understanding and early recognition of cellular and whole organism cytotoxicity in the development of new drugs is essential for the wellbeing of humans and animals. To achieve these goals a large number of animals are sacrificed to identify failures among drug candidates. It is therefore both essential and urgent to find new tools that reduce and replace animal testing.

The purpose of this project is to develop genetically encoded, fluorescent biosensors for assessment of endoplasmic reticulum (ER) stress to be used during in vitro screening of chemicals and drug candidates, using changes in fluorescence intensity and distribution as diagnostic markers, together with other more common methodologies, such as Ca2+ and apoptosis.

The ER carries out fundamental functions, such as the synthesis of cholesterol and membranes, and the detoxification of hydrophobic drugs. Accumulation of unfolded or misfolded proteins in the ER initiates a series of self-defense mechanisms known as ER stress. ER stress is a plexus point in the pathways of many mechanisms of both inflammation and toxicity.

Using ER stress as a preclinical indicator for drug-induced toxicity has relevance for 3R by substantially reducing the need of animal tests. The ER is an extremely sensitive subcellular structure and an ER stress sensor would constitute a stringent and straightforward tool to early on identify compounds with toxic liabilities.”

Jan-Ingvar Jönsson, Linköpings university, “Identification and validation of anti-leukemic therapeutic targets by single cell CyTOF mass cytometry” SEK 100 000

“The main purpose with our research is to understand how leukemic cells escape drug treatment by conventional chemotherapy as well as novel inhibitors targeting genetic alterations. Due to multiple genetic alterations related to the disease, one problem with acute leukemia is the vast heterogeneity between patients but also within the same patients’ leukemic clones. This is likely an important reason to why it remains one of the most difficult cancers to cure. In addition, our hypothesis is that a rare population of leukemia-initiating cells hide in the bone marrow where the drugs cannot reach the cells in an efficient manner.

Recent years have led to the generation of many mouse models of leukemia to test novel drugs and to study disease progression. Although these mouse models are valuable tools to study leukemogenesis and ways to monitor therapy response, they do not mirror the individual variations of genotypes and diverse responses to chemotherapy between patients. Animal models are also lacking the adequate answers to the mechanisms of drug resistance that are associated with drug therapies in leukemia patients.

We propose ways to circumvent the use of animals in leukemic research by establishing the novel approach of CyTOF mass cytometry at Linköping university for the first time in Scandinavia. The technology provides a high-throughput insight of the protein signature in leukemic cells and to classify the drug response. This is possible due to the simultaneous analysis of a large number of proteins quantitatively and qualitatively in single cells within hundreds of cells in a few seconds. It allows us to test the efficacy of various cytostatic drugs by profiling a large number of signaling states in individual leukemic cells before and after treatment by conventional chemotherapy and novel inhibitors. Moreover, it will be possible to determine what signaling events that are connected to resistance in patients which cannot be determined in animals. While traditional chemotherapy research is focused on patients that do not answer to therapy, this new approach will identify why chemotherapy is actually working. The result will help us to understand the effect of therapy, choose the patients who will have most benefit, and eventually find new targets.”

Projects receiving continued funding

Anna Herland, Karolinska Institutet
Stem cell derived cholinergic neurons as in vitro models to reduce animal experiments within neurodegeneration and analgesia ” SEK 110 000

“The aim of our research is to create better model systems of human neuronal tissue. Such models are essential to increase knowledge in basic scientific questions as well as to facilitate pharmaceutical development. We are focused on research within analgesia, drugs that relieve pain, and neurodegeneration. These disease conditions are prevalent, it is estimated that one in five Europeans suffer from chronic pain and that about 5.4 million Americans are diagnosed with Alzheimer’s disease.

The available models systems for neuronal tissue used today are animals, especially rodents, and human, cancer derived, cell lines. Both have strikingly low predictivity, of both effects and side effects. The guiding hypothesis of this project is that neural differentiation of stem cells in combination with three-dimensional cell culture systems can provide highly relevant models of the human brain.

We are using human induced pluripotent cells (iPS cells) originating from human skin biopsies. These cells can be differentiated to virtually any specialized cell in the human body; we have developed efficient methods to make them into neurons. By culturing the differentiated neurons in three-dimensional, gel like systems we can recapitulate disease specific phenomena that do not arise in conventional cell culture systems.

We have furthermore derived iPS cells from skin biopsies from Alzheimer’s patients and are now studying on how neurons differentiated from these patients show signs of disease in our cell culture system. Ultimately our systems could decrease the demand to use animals or animal derived cells both in academic and industrial settings.”

Anna Forsby , Stockholm University
Evaluation of neural stem cell lines for estimation of acute, systemic toxicity “. SEK 100 000.

“Classification of chemicals’ systemic toxicity after acute exposure is determined by the use of mice or rats according to three OECD test guidelines.  Several attempts have been performed to find in vitro- and in silico-based test strategies as alternatives. Cell assays measuring the level of viable cells after exposure can give a rough estimate of toxicity, but special cell models are needed for estimation of chemicals that induce adverse effects through organ specific modes of action (MOA), e.g. in the nervous system.

In the EU-project “ACUTETOX” (acutetox.eu), it was found that a re-aggregate culture of embryonic rat brain tissue was the best model for estimation of toxicity for chemicals that have a specific MOA in brain. The aggregates consist of different cell types that are present in brain, i.e. different neuronal phenotypes, astrocytes and oligodendrocytes.
In the present project, which is supported by “Research without animal experiments”, we have established an alternative cell model to the primary aggregates. The C17.2 cells are multipotent neural progenitor cells that were cloned from a mouse in 1991. We have optimised the culture conditions for C17.2 cells so that neurons and astrocytes develop already after 10 days in differentiation medium. The neurochemical characterisation and phenotyping of the mature cells is in progress.

Eight natural ACUTETOX compounds with well-known MOA for acute toxicity have been tested at non-cytotoxic concentrations in the “mature” C17.2 cell culture. Cell specific genomic biomarkers and the effect on cell membrane potential were analysed after exposure. Nicotine, caffeine, strychnine and atropine were correctly identified as neurotoxic where as ethanol, digoxin, rifamipicin and acetylic salicylic acid were not identified as neurotoxic. More ACUTETOX compounds will be tested validate the C17.2

During the coming year, we will analyse a full gene expression profile of 700,000 targets of the 33,000 genes in the C17.2 cell cultures at the immature progenitor stage, during differentiation and at the stage when we have two distinct cell types, i.e. neurons and astrocytes. Specific biomarkers for neuronal differentiation will then be used for further studies on chemicals that are suspected to induce adverse effects in the developing nervous system, starting with acrylamide.”

Maria Karlgren , Uppsala University
A humanized cell model for accurate assessment of molecular transport across the blood-brain barrier
SEK 100 000.

“Developing drugs for treatment of CNS diseases is more difficult as compared to development of drugs for other therapeutic areas. This is due to the blood-brain barrier (BBB) which protects the brain from entry of drugs and other xenobiotics. In drug development in vitro and in vivo animal models are used for prediction of BBB permeability. However, these models don’t translate very well to humans. The reason for this is that the drug transport proteins, which are present in the BBB and are important for the barrier function, differs between species. Hence, today the role of human drug transporters in the BBB is often overlooked. This project aims to solve the problem by developing a predictive humanized BBB model with expression levels of the drug transporters mimicking those in human brain endothelial cells. After validation, this model will be used for quantification of delivery to the brain of CNS drug candidates for selection of compounds with desired BBB permeability. Previously we have successfully developed a similar in vitro model for human liver. We are convinced that this unique first-of-its-kind model will expand the technical platform and provide more predictive and high-throughput BBB models than currently available and thereby reduce the number of animal based experiments in CNS drug development.”

 

Sara Lindén, University of Gothenburg “Gastrointestinal bio-engineered mucosal surfaces for studying host-pathogen interactions at the mucosal interphase “. SEK 100 000.

“Of all the bacteria the body meet only a fraction is a threat, and despite that the body meets pathogenic bacteria several times a day , it is rare to get sick. The body surface is covered on the outside of the skin (approximately 2 m2) and on the inside of the mucous membranes, which together have a much larger surface area (approximately  400 m2) because they are pleated . Infection begins when one of these surfaces become colonized by bacteria. The body’s mucous membrane is protected by a mucus layer that is constantly renewed and thus  ” washes away ” unwanted guests,  and the surface of the cells underlying this protective mucus layer is also covered by a dense forest of carbohydrate chains. The carbohydrates attached to the mucus molecules and the surface of the mucosa has hundreds of different structures and these carbohydrate structures varies between individuals , among others depending on what blood type you have and depending on whether you have inflammation or not.

Both disease-causing germs and bacteria that live in harmony with humans  have structures on their surface  called adhesins, which they use to bind to the host structures. These adhesins often bind to specific carbohydrate structures. Using adhesins, bacteria can ” lock ” into specific structures in the same way as a space shuttle docked to a space station. As the bacteria binds to specific carbohydrate structures, the interaction between bacteria and humans differ between individuals. Some pathogens, such as Helicobacter pylori and the winter vomiting disease virus makes people with certain blood group structures on their stomach and intestinal mucosa sicker than others. Interactions between bacteria and human cells can be studied in the laboratory on cells grown outside the body. We have developed bio-engineered mucosal surfaces that behaves in a manner similar to the lining found in the intestinal tract. Now we will gastric mucosal surfaces, and also develop them further so that they may represent individuals with different blood group structures on their mucosa and also so that they can represent individuals with and without inflammation.”

Pär Matsson, Uppsala University “Models for predicting intracellular drug exposure ” SEK 100 000.

“For a pharmaceutical drug to give the desired effect it must reach the organ and the cells that need treatment. In most cases, the pharmacological effect results from that the drug molecule binds to a specific protein inside the target cell. To predict if a new drug will give the desired effect it is thus necessary to determine the amount of drug that is distributed into the different cells of the body.

We have developed a novel experimental method for measuring intracellular exposure to unbound drug – i.e., the concentration that can interact with intracellular targets and metabolizing enzymes. We have shown that our method accurately predicts drug binding in the human liver, and preliminary results indicate equally good predictivity for other human tissues.

We will now establish a unique database of high quality intracellular drug accumulation data. This will be used to develop computational models that will allow prediction of intracellular concentrations directly from a drug’s chemical structure. Drug-like molecules that do not reach their targets in sufficient amounts, or which result in dangerously high concentrations, can thereby be filtered out in the early stages of the drug development process, avoiding unnecessary experiments in laboratory animals and patients.

The developed models can be used to prioritize candidate drugs for further development, ultimately leading to significantly fewer animal experiments in the pharmacological and toxicological evaluation of new drugs.”

Malin Lindstedt, Lund University  “A novel testing strategy for assessing sensitizing capacity of chemicals and proteins “. SEK 150 000.

“The overall objective is to further develop a human cell-based assay for prediction of sensitization. >20% of the population in EU suffers from allergic contact dermatitis today and regulatory frameworks demand that chemical substances are investigated for their sensitizing capacity. A recent ban on the use of animal tests for cosmetic ingredients has led to an urgent need for alternative animal-free methods. We have during the recent years developed a test system for prediction of skin sensitizing chemicals, called GARD – Genomic Allergen Rapid Detection test. Building on this platform, we will in this project investigate the capacity of GARD to predict potency of sensitizers, necessary for accurate risk assessment, and to predict skin irritants. By studying the signaling pathways involved, we will gain important information about the process of sensitization, which is largely unknown today. Further, we will transfer the assay platform from complete genome arrays to focused, high-throughput RNA assays useful for future routine testing and validate the test using blinded samples. The project is expected to generate a state-of-the-art test strategy for in vitro sensitization testing, with high-throughput and predictive accuracy, which has the potential to replace animal experimentation for sensitization hazard evaluation.

Stina Oredsson, Lund University
Neurotoxicity, Genotoxicity and Metabolism of Salinomycin Analogs
with Cancer Stem Cell Inhibiting Properties
” SEK 162 000

Lena Palmberg, Karolinska Institutet
A three-dimensional model based on human cells from the airways to replace animals for controlled nanoparticles aerosol exposures “. SEK 100 000.

“Good reliable human airway wall models will most likely replace animal models in the study of nanoparticle exposures, a research field under intense growth. Our primary goal is to develop reproducible and reliable experimental methods with primary bronchial epithelial cells cultured in Air Liquid Interface (ALI) in order to study immunologic (both adaptive and innate), inflammatory and toxic reaction to different exposures in this airway wall model. Secondary, these models will then be combined with aerosol exposures to nanoparticles with a newly developed technique. The ALI culture system will enable much more realistic studies on the kinetics and dynamics of inhaled particles and solutes. The 3D-model enables further studies of cell- to cell interactions and cross-talk between the cells. Thirdly, the 3D-model will be modified to develop goblet cell hyperplasia and metaplasia, which are characteristic features in asthma and chronic bronchitis. This modified model will give us important information regarding the interaction between glucocorticosteroids and innate immune responses, which may have implications regarding the beneficial effects of glucocorticosteroid treatment of exacerbation in chronic lung disorders.”

Senast uppdaterad: 7 mars 2016