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Friday 26 October 2012

Dr G. Cazander
The identification and isolation of the complement activation-reducing substrate in maggot excretions

Project
Maggot therapy is an ancient and very successful method that can heal severe, infected acute and chronic wounds. In current times, the therapy is widely used and it was approved by the US Food and Drug Administration in 2004 as an official wound treatment method, although it was not known what the underlying mechanisms of actions were. The last 6 years, microbiological and immunological research was performed to investigate the mechanisms of action of maggot therapy (LUMC, Leiden and VUmc, Amsterdam). One of the important findings of the study is that a substrate in the excretions of maggots reduces an essential part of our innate immune system, which is the complement system. The complement system plays an important role in activation of the inflammatory response to injury, pathogens or immune complexes. However, inappropriate complement activation can also cause injury and can contribute to severe tissue damage, caused by the same mediators. A recent example of inappropriate complement activation is the severe (kidney) disease that is caused by the Enterohemorrhagic E.Coli (EHEC) bacterium. EHEC cannot be destroyed by antibiotics, but a complement inhibitor can improve illness of patients and currently Eculuzimab (an antibody against one of the complement factors) proved to be effective against infection with EHEC. Antibiotic resistance is increasing rapidly and therefore, new strategies against infectious and immune-mediated diseases (such as complement inhibition) must be investigated. In this study, you will investigate the effective molecule(s) in the excretions of maggots that inhibit complement activation. What kind of substrate(s) are these? Do the molecule(s) exist? Can we synthesize them? You will also examine the exact point of interaction in the complement cascade.

Materials and Methods
Sterile maggot excretions are collected, following a standardized method and protein concentrations are measured, performing an ELISA. Complement activation is investigated using the enzyme immunoassays from WieslabTM, performed according to the manufacturer’s instructions. Briefly, the individual complement tests constituted of a 96-well microtiter plate coated with specific activators for every pathway of complement activation. Serum samples are obtained from healthy individuals. For the experiments, serum is diluted in diluent containing specific blockers to certify that only the tested pathway is activated in the well. Membrane Attack Complex formation (the end product of complement activation) in the absence or presence of maggot excretions was subsequently measured using a sandwich-ELISA principle. Separation and purification of compounds in maggot excretions is initially performed by high-performance liquid chromatography (HPLC) and gel electrophoresis. Maggot excretions are fractionated and the fraction in which the effective substrate is founded, will be determined. Furthermore, we will perform additional experiments in which we add specific substrates to eliminate chemical bonds in the excretions. Simultaneously experiments are done to determine complement activation products, such as C3bc, C4bc and C3d, using standardized and specific ELISA’s (Sanquin) to investigate at which point in the complement cascade maggot excretions interfere.

Research questions
What are the effective molecule(s) in the excretions of maggots that inhibit complement activation? What is the exact point of interaction of maggot excretions in the complement cascade?

Practical Information
The aim of the study is to investigate the effective complement inhibitor(s) in the maggot excretions. Furthermore, at the end of the study, you must be motivated to write a publication about your results. Experiments will be done at the department of Infectious Diseases at the LUMC and the immunological laboratory in Utrecht. Preferably, you can start your study in November 2012 and you are available during a minimum of 6 months. Part-time research is possible. For any questions, do not hesitate to send me an email.

Duration
20 or 28 weeks

Project at least available until
January 2016

Similar projects available thereafter
Yes

Date of submission of this project
October 2012

Contact Information
Dr G. Cazander, surgical recident
Address: LUMC, Albinusdreef 2, 2333 ZA, Den Haag
Phonenumber: +31 6 13616350
Faxnumber: +31 70 3124355
Email: gwendolyn_cazander@hotmail.com

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