Laser capture microdissection and molecular analysis of Plasmodium yoelii liver-stage parasites

Abstract

Understanding the liver stage development of Plasmodial parasites, and by extension the proteins expressed during that phase of the life cycle, has become increasingly more important as investigators have identified the pre-erythrocytic stage of the parasite as a target for effector T lymphocytes and their products. It has been clearly demonstrated that immunization with radiation-attenuated sporozoites can confer long lasting sterile protective immunity and that the immunity is stage-specific [1–3]. This form of immunization is effective against a sporozoite challenge, but not a blood stage challenge. Therefore, the protective immune response must be directed against the sporozoite and/or liver stage antigens. Experimental evidence, in mice, has clearly demonstrated that this protection requires the cellular immune system [4,5] and specifically CD8 T lymphocytes [6–8]. Malarial antigens expressed during the liver stage are processed and presented, in the context of class I MHC molecules, on the surface of infected hepatocytes [9,10]. Identifying these antigens, that may act as targets for cell mediated immunity, would significantly enhance the understanding of how the irradiated sporozoite vaccine model confers protection and ultimately lead to the identification of candidates for a subunit vaccine. Recent advances in tissue microdissection have provided a methodology to allow the isolation of a pure population of liver stage schizonts from heterogeneous liver tissue. In the past, microdissection was done manually or with the aid of a micromanipulator. The area of interest was scraped off of a tissue section using a needle and subsequent processing was performed with the tissue that was recovered. This was a slow, laborious and unreliable practice that frequently yielded an impure cell population. The development of Laser-capture microdissection (LCM) has turned microdissection into a fast, reliable and precise technique. LCM utilizes an infra red laser and a transparent thermoplastic film to selectively isolate cells or regions of interest [11,12]. Briefly, a slide containing a section of infected liver is placed on the microscope stage. A transport arm, holding the thermoplastic film, is then moved over the region of interest. The cell(s) to be isolated are moved to the center of the optical field and the laser is activated. The laser melts the film directly onto the tissue containing the targeted cell. The film rapidly cools and solidifies such that when the film is moved by the transport arm the film/tissue combination shears away from the surrounding tissue. The process can be repeated many times, utilizing areas of the film that have not been melted with the laser. The film, which is held in a plastic ring, the same dimensions as a miAbbre iations: AMA-1, apical membrane antigen 1; CS, circumsporozoite; LCM, laser capture microdissection; MSP1, merrozoite surface protein 1; NYLS3, navy yoelii liver stage 3; RT-PCR, reverse transcriptase-polymerase chain reaction. * Corresponding author. E-mail address: jsacc001@umaryland.edu (J.B. Sacci, Jr). 1 Present address: Celera Genomics, 45 West Gude Dr., Rockville, MD 20852, USA.