guilherme oliveira

Schistosome parasites exhibit separate sexes and with the evolution of sex they have developed an intricate relationship between the male and female worms such that signals between the male and female that are initiated at the time of mating, regulate female reproductive development and subsequent egg production. As the egg stage is responsible for pathogenesis and transmission, understanding the molecular mechanisms of female reproductive development may identify novel targets for the control of transmission and morbidity of this major world public health problem. Recent data have demonstrated that the pairing process, proliferation, and differentiation of vitelline cells, expression of female-specific genes and egg embryogenesis are regulated by the TGFβ pathway and protein tyrosine kinases.

Towards germ-line transformation miracidia were biolistically transformed with GFP reporter gene constructs and successfully reintroduced into the schistosome cycle. By PCR and confocal microscopy the presence and the expression of GFP were confirmed in cercariae or adults of the F0 and F1 generations. This indicated the presence of the constructs in the germ-line, although no evidence for genome integration was obtained. About 3 kb of 5′ upstream sequences of the actin gene SmAct1 were identified by in silico analyses, and different fragments up to 1.5 kb subcloned for GFP-vector construction. A 445 bp fragment was sufficient for transcription initiation in larvae or adults as confirmed by confocal microscopy. An actin gene characteristic assembly of TATA, CArG, and CAAT boxes has been identified, which seems to be functionally conserved between vertebrates and invertebrates. However, a vertebrate-specific intron containing an additional regulatory CArG box was not found indicating that the regulation of SmAct1 transcription depends exclusively on its promoter region.

A PCR-RFLP based method was developed to diagnose and identify the Leishmania species causing American cutaneous leishmaniasis (ACL) in a panel of clinical samples obtained from an endemic region of Brazil. The comparison of the results obtained by PCR-RFLP and PCR-hybridization in the identification of Leishmania (Viannia) braziliensis and L. (Leishmania) amazonensis were highly concordant (κ=91.5%). The PCR-RFLP method was reliable, fast and easy to conduct on biopsies and presents potential value of utmost importance for the diagnosis and identification of Leishmania in clinical specimens, infected reservoirs and vectors.

Studies were performed on humoral and cellular immune responses of patients from areas in Brazil endemic for hookworm and Ascaris lumbricoides, and either endemic or non-endemic for Schistosoma mansoni. Humoral and cellular responses were evaluated by enzyme-linked immunosorbant assay (ELISA) and peripheral blood mononuclear cell (PBMC) proliferation assays against larval hookworm antigens, A. lumbricoides egg antigens, and soluble egg antigens (SEA) or soluble whole adult antigenic preparation (SWAP) from S. mansoni. Patients from S. mansoni-endemic areas, who currently had only hookworm or Ascaris infections, expressed lower humoral and cellular responses to hookworm or Ascaris antigens, respectively, than did their counterparts from areas not endemic for S. mansoni. Individuals from S. mansoni endemic area, although without detectable S. mansoni infection, do mount humoral and cellular responses to SEA and SWAP. This group of individuals has been probably in contact with S. mansoni antigens, since the groups harboring A. lumbricoides or hookworm infections from non-S. mansoni endemic areas do not have detectable anti-S. mansoni responses. PBMC proliferative responses discriminated well between patients with active hookworm infections versus ascariasis, if they were from areas not endemic for S. mansoni.

The saliva of the sand fly Lutzomyia longipalpis, a major vector of Leishmania, exhibits pharmacological and immunomodulatory activities that may facilitate entry and establishment of parasites into the vertebrate host. Salivary gland components of the sand fly are, therefore, potential candidates in the development of a vaccine against human leishmaniasis. With the objective of identifying sand fly saliva proteins that could be used to immunise animals against canine visceral leishmaniasis, we have evaluated anti-saliva antibody reactivity using serum samples collected from dogs naturally infected with Leishmania chagasi. Two proteins with molecular weights of 28.6 and 47.3 kDa were recognised by dog antibodies in Western blot assays. Protein bands were excised from an SDS-PAGE gel and the sequences determined by mass spectrometry. The proteins were identified as LuLo-D7 and Lulo YELLOW, respectively. The significance of these findings in the context of the development of multi-component vaccination experiments is discussed.

The identification and description of signal transduction molecules and mechanisms are essential to elucidate Schistosoma mansoni host-parasite interactions and parasite biology. This mini review focuses on recent advancements in the study of signalling molecules and transduction mechanisms in S. mansoni, drawing special attention to the recently identified and characterised protein tyrosine kinases of S. mansoni.

Protein Tyrosine Kinases (PTKs) are important molecules in intra- and inter-cellular communication, playing a major role in signal transduction processes. We have previously identified and characterized the molecular structure of a new PTK in Schistosoma mansoni, SmFes. SmFes exhibits the characteristic features of Fes/Fps protein tyrosine kinase subfamily of which it is the first member described in helminths. Herein, we show that genes orthologous to SmFes are also present in other Schistosoma species and the transcript is detected in Schistosoma japonicum. The SmFes protein was detected at all the main life-cycle stages and was most abundant in cercariae and newly-transformed schistosomula. However, no protein was detected in schistosomula maintained in vitro for 7 days. By immunolocalization assays we showed that SmFes is particularly concentrated at the terebratorium of miracidia and tegument of cercaria and schistosomula skin-stage. These findings suggest that SmFes may play a role in signal transduction pathways involved in larval transformation after penetration into intermediate and definitive hosts.

Large scale EST sequencing projects have been carried out for Schistosoma mansoni and Schistosoma japonicum. This update will briefly review the most recent accomplishments in the area and discuss the use of EST data for the purposes of gene discovery, gene model development, genome annotation and SNP analysis. In addition, the use of ESTs for studying other features of the transcriptome such as splice site and transcription initiation variants will be discussed as well as approaches to assigning function to unknown transcripts. Although EST sequencing has contributed much for schistosome research, other data mining possibilities exist, including the identification of putative drug and vaccine targets.