Accuracy of qPCR for quantifying Leishmania kDNA in different skin layers of patients with American tegumentary leishmaniasis.

OBJECTIVES: Superficial swab sampling of American tegumentary leishmaniasis (ATL) lesions shows higher amounts of Leishmania than those from biopsy. Subcutaneous involvement is also important in ATL, but parasite quantification according to lesion depth has not been evaluated. We aim to present the best depth at which sampling should be performed for molecular exams of ATL. METHODS: Patients with a clinical presentation compatible with ATL were allocated to ATL and control groups. Qualitative and quantitative qPCR assays were performed using SYBR Green and primers amplifying the kDNA minicircle of Leishmania spp. in different skin layers, including the epidermis, the superior dermis, the inferior dermis, and the hypodermis. RESULTS: Fifty-nine patients were included in this study, including 40 who had been diagnosed with ATL and 19 controls. The number of parasites was greater in samples of the epidermis and superior dermis (159.1 × 106, range 4.0-781.7, and 75.4 × 106, range 8.0-244.5, mean Leishmania parasite equivalents per µg of tissue DNA, respectively) than those in samples of the inferior dermis and hypodermis (54.6, range 8.0-256.6, and 16.8 × 106, range 8.0-24.1, mean Leishmania parasite equivalents per µg of tissue DNA, respectively). The best diagnostic accuracy was achieved in the superior dermis (77.9%) and was significantly greater than that in the hypodermis (63.3%; p 0.039). CONCLUSIONS: We conclude that superficial sampling can retrieve a greater quantity of parasites. Future studies of the role of transepidermal elimination as a mechanism of host defence in ATL must be performed as there is a considerable quantity of Leishmania kDNA in the epidermis.

Analysis of GATA1 mutations and leukemogenesis in newborns with Down syndrome.

It has been reported that patients with Down syndrome (DS) frequently develop transient myeloproliferative disorder (TMD) and less commonly myeloid leukemia in DS (ML-DS). We examined the pathogenetic relationship of these conditions with somatic mutations of the GATA1 gene in children with both TMD and ML-DS. To determine the incidence of GATA1 mutations in a cohort of DS patients and the applicability of these mutations as a clonal marker to detect minimal residual disease, we screened 198 samples of 169 patients with DS for mutations in GATA1 exon 2 by direct sequencing. Novel mutations were detected in four of the 169 DS patients (2 with TMD and 2 with ML-DS). We examined spontaneous remission and response to therapy in TMD and ML-DS patients and concluded that these mutations can be used as stable markers in PCR analysis to monitor these events.

TcZFP8, a novel member of the Trypanosoma cruzi CCHC zinc finger protein family with nuclear localization.

In a 17-kb genomic fragment of Trypanosoma cruzi chromosome XX, we identified three tandemly linked genes coding for CX(2)CX(4)HX(4)C zinc finger proteins. We also showed that similar genes are present in T. brucei and Leishmania major, sharing three monophyletic groups among these trypanosomatids. In T. cruzi, TcZFP8 corresponds to a novel gene coding for a protein containing eight zinc finger motifs. Molecular cloning of this gene and heterologous expression as a fusion with a His-tag were performed in Escherichia coli. The purified recombinant protein was used to produce antibody in rabbits. Using Western blot analysis, we observed the presence of this protein in all three forms of the parasite: amastigote, trypomastigote and epimastigote. An analysis of cytoplasmic and nuclear cell extracts showed that this protein is present in nuclear extracts, and indirect immunofluorescence microscopy confirmed the nuclear localization of TcZFP8. Homologues of TcZFP8 in T. brucei are apparently absent, while one candidate in L. major was identified.

TcZFP8, a novel member of the Trypanosoma cruzi CCHC zinc finger protein family with nuclear localization

In a 17-kb genomic fragment of Trypanosoma cruzi chromosome XX, we identified three tandemly linked genes coding for CX2CX4HX4C zinc finger proteins. We also showed that similar genes are present in T. brucei and Leishmania major, sharing three monophyletic groups among these trypanosomatids. In T. cruzi, TcZFP8 corresponds to a novel gene coding for a protein containing eight zinc finger motifs. Molecular cloning of this gene and heterologous expression as a fusion with a His-tag were performed in Escherichia coli. The purified recombinant protein was used to produce antibody in rabbits. Using Western blot analysis, we observed the presence of this protein in all three forms of the parasite: amastigote, trypomastigote and epimastigote. An analysis of cytoplasmic and nuclear cell extracts showed that this protein is present in nuclear extracts, and indirect immunofluorescence microscopy confirmed the nuclear localization of TcZFP8. Homologues of TcZFP8 in T. brucei are apparently absent, while one candidate in L. major was identified.

Molecular cloning and characterization of a gene encoding the 29-kDa proteasome subunit from Trypanosoma cruzi.

Antiserum raised against purified Trypanosoma cruzi proteasomes was used to isolate two cDNA clones, tcpr29 and tcpr29B, and the corresponding genomic sequence, termed tcpr29A. Both cDNAs and the gene contain a 798-bp ORF, coding for a 266-amino acid protein, with a predicted molecular mass of 29 kDa. Sequence comparisons show that the protein encoded by tcpr29 belongs to the alpha6 subfamily of proteasome subunits. Southern analysis indicated that tcpr29 subunit is encoded by a single-copy gene which maps to chromosome 20 of the CL Brener clone. Allelic variants were found in other T. cruzi isolates, suggesting heterozygosity for the gene in some and homozygosity in other strains. A spliced-leader addition site was identified 123 bp upstream from the start codon, generating a stable 1.5-kb transcript. Western analysis revealed that tcpr29A is constitutively expressed during the life cycle of the parasite.

Sequence-dependent in vivo importation of tRNAs into the mitochondrion of Leishmania tarentolae.

Sequence determinants for the importation of tRNAs into the mitochondrion of Leishmania tarentolae in vivo were investigated. tRNA(Ile)(UAU) is exclusively localized within the mitochondrion and tRNA(Gln)(CUG) exclusively in the cytosol (Lye LF, Chen DHT, Suyama Y, 1993, Mol Biochem Parasitol 58:233-246; Shi X, Chen DHT, Suyama Y, 1994, Mol Biochem Parasitol 65:23-37). L. tarentolae cells were transfected with plasmids encoding either tRNA(Ile) or tRNA (Gln) that were tagged with altered sequences in the D loop, permitting discrimination from the endogenous tRNAs. Primer extension analysis was used to show that the plasmid-encoded genes were expressed and that the tagged tRNAs showed a similar intracellular localization as the endogenous tRNAs. Exchange or deletion of the 5'-flanking genomic sequences had no effect on the expression or mitochondrial localization of the tagged tRNA(Ile) or on the expression or cytosolic localization of the tagged tRNA(Gln), suggesting that the signals for importation are localized within the tRNA itself. Swapping the D loop+stem from the exclusively cytosolic tRNA(Gln) with that from the tRNA(Ile) produced a partial mitochondrial localization of the plasmid-expressed mutated tRNA(Gln). However, D loop exchange did not eliminate the mitochondrial localization of the plasmid-expressed mutated tRNA(Ile), suggesting that tertiary structure or additional sequence elements may be involved in the importation signal.