ExperimentalLeishmania (L.) amazonensisLeishmaniasis: Characterization and Immunogenicity of Subcellular Fractions

Abstract

A technique developed in Trypanosoma cruzi biochemical studies was successfully used to fractionate Leishmania (Leishmania) amazonensis promastigotes. Ultrastructural analyses revealed a membrane fraction (MF) associated to subpellicular microtubules, a ribosomal-rich microsomal fraction (MicF), and a flagellar fraction (FF) free of associated membrane. All fractions proved to be immunogenic through delayed type hypersensitivity reaction assays. Therefore, a protocol was designed to test whether these fractions could elicit a protective response in mice infected by L. (L), amazonensis. The protocol consisted of a BCG injection (as cellular immunity inducer), followed by cyclophosphamide (once its cytotoxic effect is over, this immunosuppressor can increase the number of circulating leukocytes), then an injection with one of the fractions followed by a challenge. When compared to infected control animals, mice injected with any of the fractions presented a smaller footpad swelling, especially those injected with MicF or FF. Macroscopically, immunized mice under modulation by BCG presented no swelling. Histopathological studies performed on day 120 revealed fewer amastigotes and more intense inflammation in lesions of MicF and FF injected mice. Animals injected with MF presented an intermediate pattern. Parasite quantification corroborated these results. The results show that all fractions are potent immunostimulators, but MicF and FF have the strongest protective ability.

Sir2-Related Protein 1 fromLeishmania amazonensisis a glycosylated NAD+-dependent deacetylase

“…(c) Isolation of the microsomal fraction. Leishmania amazonensis promastigotes were submitted to subcellular fractioning in water/ice bath as previously described (Zaverucha do Valle et al 2007). Parasites were washed with PBS+2% glucose, re-suspended with 20 m m Tris-HCl, pH 7·4, 12·5 m m sucrose, incubated for 20 min, and mechanically disrupted by a Douncer homogenizer in the presence of 10% non-ionic detergent Lubrol PX to produce the total protein extract (ET).…”

“…Supernatant (6 ml) was layered on top of 2 ml of buffer containing 20% sucrose, 10 m m MgCl 2 , 25 m m KCl , and 10 m m Tris-HCl, pH 7·4, and centrifuged at 100 000 g for 30 min to produce the microsomal fraction (micF), which contains endoplasmic reticulum-derived vesicles, including ribosomes (Zaverucha do Valle et al . 2007).…”

A New Strategy for Mapping Epitopes of LACK and PEPCK Proteins of Leishmania amazonensis Specific for Major Histocompatibility Complex Class I

“…Immunological findings against Leishmania amazonensis infection in the murine model highlight is a strong tool for proposing immunology profiles and vaccines for cutaneous leishmaniasis [ 4 , 5 ]. In this context, previous studies with subcellular fractionation of L. amazonensis promastigote forms have shown that the microsomal fraction (FMic) associated with immunomodulators provides protection against experimental infection by this parasite [ 6 ]. Furthermore, analyses of the different components of the fraction allowed for the identification of Leishmania spp.…”