TRAINEE EDUCATION

The Rheumatology Research Laboratory provides the opportunity for students in Biomedical Sciences (Health Science, Medical Biology) and HLO students to carry out a trainee program in one of the research programs. The main purpose of the trainee programs is to introduce students in the world of biomedical research by active participation in this field of research. Students are provided with a research project which can be carried out in the trainee period. Under the supervision of a senior scientist, both the practical and the theoretical part of the research project have to be covered by the trainee. All the research projects have to be presented orally and in a written report.

If you are interested to work as a trainee on the Rheumatology Research Laboratory Nijmegen please contact:

Dr Peter M. van der Kraan

Email: [email protected]

Tel: 024-3616568 (+31-24-3616568)

Subjects for trainees

Gene Therapy Program:

Supervisor: Dr Fons van de Loo

Short term practice (3-4 months):

  1. Cloning of IL-18AcPL (Accessory protein like): This protein forms a complex with the IL-18 receptor and is essential for signal transduction of IL-18. In this practice the student will clone a C-terminal truncated form (lacking the signal sequences) of IL-18AcPL and test the inhibitory potency against IL-18. Basic molecular biological techniques will be learned.
  2. Receptor interaction of cytokines: To introduce the newly developed technique of fluorescence Polarization to measure the ligand interaction with its membrane receptor. Using this technique the inhibitory action of truncated receptor forms (IL-6 receptor antagonist)/antibodies can also be determined. The student will learn fluorescence, protein tagging, and cell culture techniques.

Long term practice (6 months or more):

  1. IL-6 in chronicity of arthritis: In IL-6 knockout mice the acute joint inflammation does not become chronic. Our hypothesis is that IL-6 reduces apoptosis of inflammatory – and resident cells. To better understand the underlying mechanism the activation of STAT3 (IL-6 signaal transduction protein), expression of BCL-xl (anti-apoptotic factor), and PARP degradation (marker of apoptosis) will be studied. The student will learn biotechnological, immunohistological, and RT-PCR (molecular beacons) techniques.
  2. Disease regulated promoter activities: To improve the efficacy of anti-inflammatory protein therapy, the transgene in the adenoviral vector can be put under the control of a disease responding promoter construct. An alternative method is to interfere with the natural silencing of the viral promoters (e.g. CMV) used. The student will learn basic molecular biological, cell-culture, and protein expression techniques.
  3. Optimalisation of local transfection: Adenoviral vectors are widely used to deliver genes into tissues. In this study the expression of marker genes (luciferase, green-fluorescent protein) using modified adenoviral vectors will be studied in the murine knee joint. The student will learn biotechnological, histological, and viral (safety) techniques.

 

Program: Osteoarthritis and tissue engineering of cartilage

Supervisor: Dr Peter M. van der Kraan

Short term practice (3-4 months) and long term practice (6 months or more) in the following subjects:

  1. Tissue Engineering of articular cartilage: One of the main causes of the loss of articular cartilage is the limited repair capacity of this tissue. The formation of new cartilage tissue by tissue engineering is one of the most promising new techniques to overcome this problem. To reach the goal of tissue engineered cartilage, cartilage cells (chondrocytes) are cultured in defined biomaterials and the formation of cartilage is stimulated by the application of specific growth factors directly or by gene therapy. In this project it is studied which matrices and growth factors are optimal to stimulate the formation of new articular cartilage. The trainee will learn the following techniques; cell culture, RT-PCR, histology, immunohistochemistry and biochemical techniques.
  2. Role of cytokines and growth factors in osteoarthritis. Mesenchymal cells differentiate to chondrocytes and form the growth plate and the articular cartilage. The articular chondrocytes have a highly specialized function in the maintenance of the articular cartilage. Chondrocyte differentiation and metabolism is regulated by cytokines, growth factors and the extracellular matrix. Under pathological conditions, such as osteoarthritis, chondrocyte differentiation and metabolism is altered resulting in breakdown of articular cartilage and loss of joint function. The goal of this research line is to elucidate the regulatory role of cytokines and growth factors in cartilage homeostasis and pathology. Elucidation of the specific role of these factors during pathology will open new avenues for therapy such as preventing cartilage degeneration and the stimulation of cartilage repair. The trainee will learn the following techniques; Cell and tissue culture, molecular biology (PCR, gene cloning), histology, immunohistochemistry, apoptosis detection, biochemical techniques.
  3. Collagen degradation in osteoarthritis. Osteoarthritis results in the loss of articular cartilage and eventually in impaired joint function. Until now it is not known what is the cause of cartilage loss by osteoarthritis. Degradation of type II collagen, the most abundant collagen type in cartilage, is thought to be a crucial step in the degradation process. Therefore it is essential to know the exact mechanism of collagen breakdown, and the role of proteolytic enzymes, in osteoarthritis. Knowledge about the most important players in collagen breakdown will provide new targets for therapeutic interference with the breakdown process. The trainee will learn the following techniques; Cell and tissue culture, molecular biology (PCR), histology, immunohistochemistry, apoptosis detection, biochemical techniques.

Program: Mechanisms and modulation of chronic inflammation and joint tissue destruction during Rheumatoid Arthritis.

Supervisor: Dr Peter van Lent

Short term practice (3-4 months) and long term practice (6 months or more) in the following subjects:

  1. Macrophages: Regulators of tissue damage during rheumatic diseases. During rheumatic diseases like arthrosis, severe tissue damage develops in the joint. A characteristic feature of arthrosis joints is an excessive outgrowth of bone (called osteophyte formation). These osteophytes result in the visable deformations of hands and feet often seen in patients with arthrosis. The mechanism responsible for the formation of osteophytes is not yet known. Recently we found that macrophages which cover the inside of the diarthrodial joint are involved in the development of these osteophytes. The goal of this study is to investigate which factors derived from activated macrophages are responsible for osteophyt development. Knowledge of which proteins are involved in regulation of this pathology may focus therapy of this crippling disease. The trainee will learn the following techniques: Tissue or cell culture (macrophages/cartilage/bone). Immunology: measurements of cytokines, immunohistochemistry, autoradiography). Biochemistry : Functional tests for enzymes and oxygen radicals. Molecular biology: RNA isolation and PCR.
  2. Citrulin; The antigen that causes rheumatoid arthritis? The cause of Reumatoid Arthritis (RA) is not known. This severe crippling disease which develops worldwide in 2% of the population leads to severe joint damage. Many researchers believe that RA is a derailed immunological reaction against an up to now unknown antigen. As inflammation predominantly is found within joints, the "RA" antigen is thought to be expressed locally in the joint. In RA patients, antibodies circulate which are directed against an antigen which is present in the organelles of cells of the mucous membrane of the cheek. Recently this antigen was identified. The gene was cloned and the protein was expressed. It appeared to be the protein citrulin. This protein is detected in only limited amounts in the body. We have found first indications that this protein is found within the synovium of RA patients. Characterisation of the RA antigen will have large consequences for new therapeutical approaches. Using this antigen as a vaccine could efficiently combat this disease. The trainee will learn the following techniques; Immunology: measurement of humoral ( antibodies) and cellular (specific T cell responses) immunity. Measurement of cytokines (bioassay and ELISPOT). Molecular biology: RT-PCR and in situ hybridisation. Experimental arthritis: Induction and measurement of experimental arthritis.
  3. Metalloproteinases: Enzymes which cause unrepairable cartilage damage during rheumatoid arthritis. One of the main characteristics of Rheumatoid Arthritis is severe destruction of the cartilagenous layers within the joints. The cartilage matrix consists of a collagen network in which large proteoglycan molecules are embedded. Enzymes are thought to be mainly responsible for the breakdown of the cartilage matrix seen during this disease. One important group of enzymes are the metalloproteinases. These enzymes are characterised by a metal ion in their active centre and act at neutral pH. Many of these enzymes are released by chondrocytes in an inactive form and additonally stored in the cartilage matrix. During the disease, the inactive enzymes in the cartilage matrix are altered into their active form. The mechanisms which are involved in transformation of the latent enzymes into their active form are not yet known. We investigate which mediators released by macrophages and granulocytes, the main components of inflammation during RA, are involved in the activation of metalloproteinases. The trainee will learn the following techniques; Tissue/cell culture: cartilage/macrophages/PMN. Immunology: Isolation of immune cells/ Immunocytohistochemistry. Biochemistry: Measurements of enzymes and oxygen radicals.Molecular Biology: RNA isolation and RT-PCR.
  4. Rheumatoid arthritis a macrophage disease? Rheumatoid arthritis (RA) is a disease which leads to chronic inflammations in compartments of the body which are covered by a layer of macrophages. During RA, inflammation can be found in the cavities in which the heart or lungs are present. In RA, particularly the diarthrodial joints which also contain cavities surrounded by macrophages form the target of the disease. Macrophages probably are activated by immune complexes which are often found in large amounts within the RA joint. Macrophages communicate with immune complexes by receptors, the so called Fc gamma receptors. Three classes of Fc gamma receptors are described and each class mediates specific functions of the cell. The working hypothesis is that RA patients have an altered expression of Fc receptors on the macrophage and for this reason may react in a different manner to immune complexes thus causing severe inflammation and damage of joint structures. The trainee will learn the following techniques; Cell biology: isolation of immune cells/ FACS analysis. Immunology: Measurement of cytokines (bioassay/ELISA)/ Immunocytohistochemistry. Biochemistry: Measurements of enzymes and oxygen radicals.Molecular Biology: RNA isolation and RT-PCR, in situ hybridisation.

Supervisor: Dr Pilar Barrera

  1. Genetic contribution in rheumatoid artrhitis. Rheumatoid arthritis (RA) is a multifactorial disease involving genetic and environmental factors. The genetic predisposition for RA is only partly explained by the human leukocyte antigen (HLA) locus on chromosome 6. Most other genetic factors involved in its susceptibility (and/or severity) await further identification. Our department is member of the European Consortium on RA Families (ECRAF). The ECRAF has collected DNA from a large number of European families and performed a whole genome scan searching for new susceptibility loci. This genome scan has provided evidence for linkage with several regions located on other chromosomes. These have yet to be analyzed. The goal is to further analyze regions of interest potentially involved in the susceptibility for RA. Techniques: Some knowledge of molecular biology techniques. RT-PCR, Single stranded conformation polymorphism, designing primers. Note: This stage requires a stay in Paris with a duration of some weeks.
  2. Role of macrophages and dendritic cells in rheumatoid arthritis. Rheumatoid arthritis (RA) is a chronic disease characterized by severe joint inflammation leading to joint destruction. Macrophages and dendritic cells are believed to play an important role both in early and chronic stages of the disease. These cells, accumulate in inflamed joints were they are involved in processes such as antigen presentation and phagocytosis. Moreover, once in the synovial membrane, macrophages become activated and produce large amounts of soluble mediators including a large variety of proinflammatory cytokines and enzymes involved in cartilage destruction. These mediators, together with direct cell to cell interactions are involved in the homing and activation of other cells in the joint. This results in an unopposed proinflammatory milieu which leads to joint destruction. The goal of this study is to analyze the effect of current and new therapies on macrophage and dendritic cell markers and macrophage products at the synovial level. Techniques; Histological and immunohistological analysis, PCR, in situ hybridization.



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