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General
Project Summary

Title: Identification of the Genetic Factors Which Control Tropism in Leishmania
Synopsis: A study of the parasite leishmania identifies if there is a gene(s) causing temperature resistance or sensitivity, that may influence the parasite to remain in the skin or move to the viscera (large interior organs of the body, especially the abdomen).
Overall Project Objective: Identify the gene(s) that control tropism in Leishmania and determine its (their) sequence and function.
Status/Results to Date: An in vitro promastigote temperature model was developed and the investigators determined that temperature sensitivity in vitro correlates with Leishmania tropism in vivo. There is no absolute temperature requirement for dermotropism, but there is a minimum temperature resistance requirement for visceralization. The parasite strains that have an unusual tropism in the human host (to include the viscerotropic L. tropica from Desert Storm) also show an unexpected temperature sensitivity in the model. Two animal models were developed where the tropism of the Leishmania strain is known, uniform, and reproducible (all organisms are found in one location either visceral or cutaneous) when inoculated in the skin (sc) to mimic a sand fly bite, including a cutaneous model with no visceralization, and a visceral model with no skin lesions. Leishmania temperature sensitive and resistant mutants have been obtained. Laboratory techniques were developed and standardized for transfection and for the Leishmania cosmid DNA library. An L. donovani and two L. tropica temperature-resistant strains were selected in vitro. An L. mexicana strain was chemically mutagenized and a tropism mutant was selected in our hamster model. Wild-type DNA cosmid libraries were made of all the Leishmania strains (4) to be mutagenized and selected for altered tropism in the hamster model. Both the in vitro and in vivo models developed to do the molecular biology studies have been published. The most relevant issue of viscetropic leishmaniasis for Desert Storm was that L. tropica which should be solely dermotropic, was found in viscera of veterans. The study identified an important question for further research prior to termination due to lack of funding: the identification of genetic factors which control tropism in leishmaniasis.
Project:DoD-9
Agency:Department Of Defense
Location:WRAIR, Brazilia
P.I. Name:Max Grogl, LTC, Ph.D.
Research Type:Mechanistic
Research Focus:Leishmaniasis
Focus Category:General Health & Physical Symptoms
Status:Complete
Study Start Date:July 01,1994
Estimated Completion Date:July 01,1998
Specific Aims: The goal of this research is to identify the gene(s) that control tropism in Leishmania. The identification of a "tropism" gene will enable the development of specific gene probes (primers) to be used in a patient screening program to identify those at risk of reactivation of latent infections; address the fundamental question of infectious disease pathophysiology, namely why an organism infects a particular cell; optimize treatment regimens according to Leishmania species and the immune status of the host. The identification of the genetic factor(s) involved in the visceralizaiton of Leishmania will require development in vitro and in vivo models of tropism to facilitate the study of viscerotropic leishmaniasis (diagnosis, therapy prevention) and Leishmania tropism.
Methodology: Develop in vitro and in vivo models of Leishmania tropism to use for the determination of the genetic factors controlling tropism: a promastigote temperature sensitivity model; and a cutaneous and a visceral animal model of Leishmania tropism. Create Leishmania mutants with altered tropism in the in vitro and in vivo models of tropism: temperature sensitive parasites changed to resistant and visa versa; and cutaneous parasites (in the animal model) changed to visceral and visa versa. Use recently developed molecular genetic techniques to restore the original (wild-type) phenotype to the Leishmania temperature and tropism mutants by transfection with the appropriate Leishmania cosmid DNA libraries. Identify the gene(s) controlling temperature sensitivity and tropism present in the cosmid(s) which restore(s) a wild-type phenotype to the Leishmania cells. Retest the function of these genes in both models using transfection methodology.
Most Recent Publications:

Kelly DJ, Chan CT, Paxton H, Thompson K, Howard R, Dasch GA. Comparative evaluation of a commercial enzyme immunoassay for the detection of human antibody to Rickettsia typhi. Clinical and Diagnostic Laboratory Immunology, 2 (3): 356-6, May 1995. Abstract

Callahan HL, Grogl M. Development of animal models to study Leishmania tropism at the molecular level. American Journal Tropical Medicine and Hygiene, In press, 1999. Article

Weddle JR, Chan CT, Thompson K, Paxton H, Kelly DJ, Dasch GA, Strickman D. Effectiveness of a dot-blot immunoassay of anti-Rickettsia tsutsugamushi antibodies for serologic analysis of scrub typhus. American Journal Tropical Medicine and Hygiene, 53 (1): 43-6, Jul 1995. Abstract

Callahan HL, Portal IF, Bensinger SJ, Grogl M. Leishmania spp: temperature sensitivity of promastigotes in vitro as a model for tropism in vivo. Exp Parasit, 84 (3): 400-9, Dec 1996. Abstract