DNA sequencing of 13 cytokine gene fragments of Aotus infulatus and Saimiri sciureus, two non-human primate models for malaria.

Aotus and Saimiri are non-human primate models recommended by the World Health Organization for experimental studies in malaria, especially for vaccine pre-clinical trials. However, research using these primates is hindered by the lack of specific reagents to evaluate immune responses to infection or vaccination. As a step toward developing molecular tools for cytokine expression studies in these species, primer pairs for 18 cytokine gene fragments were designed based on human DNA sequences and used to amplify the corresponding genes in Aotus infulatus and Saimiri sciureus genomic DNA samples. IFNγ, TNFα, LTA, IL2, IL3, IL4, IL5, IL6, IL10, IL12, IL13, CSF2 and TGFß2 gene fragments were amplified and sequenced. Primer pairs for IL8, IL17, IL18, IL27 and MIF failed to generate amplification products. When compared to the available corresponding human and non-human primate sequences, most--except IL3 and IL4--showed identity degrees above 90%. Small variations in sequence can help to explain the failure to amplify certain genes or the amplification only at lower annealing temperatures as compared to human DNA samples for several primer pairs. The sequences made available provide the basis for designing molecular tools such as primers for real time PCR specific for A. infulatus and/or S. sciureus. The nucleotide sequences reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned accession numbers DQ985386 to DQ985389, DQ989356 to DQ989369, FJ89020 to FJ89024, and FJ89029.

Spatial distribution of acaricide profiles (Boophilus microplus strains susceptible or resistant to acaricides) in southeastern Mexico.

The ability of Boophilus microplus strains to be susceptible (-) or resistant (+) to amidines (Am), synthetic pyrethroids (SP), and/or organo-phosphates (OP) (or acaricide profiles) was investigated in 217 southeastern Mexican cattle ranches (located in the states of Yucatán, Quintana Roo, and Tabasco). Three questions were asked: (1) whether acaricide profiles varied at random and, if not, which one(s) explained more (or less) cases than expected, (2) whether the spatial distribution of acaricide profiles was randomly or non-randomly distributed, and (3) whether acaricide profiles were associated with farm-related covariates (frequency of annual treatments, herd size, and farm size). Three acaricide profiles explained 73.6% of the data, representing at least twice as many cases as expected (P<0.001): (1) Am-SP-, (2) Am+SP+, and (3) (among ranches that dispensed acaricides > or = 6 times/year) Am-OP+SP+. Because ticks collected in Yucatán ranches tended to be susceptible to Am, those of Quintana Roo ranches displayed, predominantly, resistance to OP/SP, and Tabasco ticks tended to be resistant to Am (all with P < or = 0.05), acaricide profiles appeared to be non-randomly disseminated over space. Across states, two farm-related covariates were associated with resistance (P < or = 0.02): (1) high annual frequency of acaricide treatments, and (2) large farm size. Findings supported the hypothesis that spatial acaricide profiles followed neither random nor homogeneous data distributions, being partially explained by agent- and/or farm-specific factors. Some profiles could not be explained by these factors. Further spatially explicit studies (addressing host-related factors) are recommended.

Stage-specific gene expression during Trypanosoma cruzi metacyclogenesis

The process of Trypanosoma cruzi metacyclogenesis involves the transformation of noninfective epimastigotes into metacyclic trypomastigotes, which are the pathogenic form. The analysis of stage-specific genes during T. cruzi metacyclogenesis may provide insight into the mechanisms involved in the regulation of gene expression in trypanosomatids. It may also improve the understanding of the mechanisms responsible for the pathology of Chagas disease, and could lead to the identification of new targets for chemotherapy of this disease. We have demonstrated that during metacyclogenesis the expression of several genes is controlled at the translational level by an alternative regulatory mechanism. This mechanism may involve the mobilization of mRNA to the translation machinery. We have been using self-made T. cruzi microarrays to investigate the role of polysomal mobilization in modulating gene expression during metacyclogenesis

Expression and cellular localization of Trypanosoma cruzi type II DNA topoisomerase.

Topoisomerases are enzymes that participate in many cellular functions involving topological manipulation of DNA strands. There are two types of topoisomerases in the cell: (a) type I topoisomerases; and (b) type II topoisomerases (topo II). Previously we have cloned and sequenced the gene encoding Trypanosoma cruzi topo II (TcTOP2). This study group has raised an antiserum against recombinant type II DNA topoisomerase (TctopoII) to study the expression of this gene during T. cruzi differentiation and to determine the cellular location of the enzyme. Western blot analysis showed that T. cruzi TctopoII is expressed in the replicative epimastigotes but not in the infective and non-replicative trypomastigotes. However, slot blot analysis of RNAs extracted from epimastigotes and metacyclic trypomastigotes showed that the mRNA encoding the enzyme is present in both developmental stages of the parasite. Confocal laser microscopy using the antiserum raised against recombinant TctopoII showed that the enzyme is located exclusively in the nucleus of the parasite. Similar results were obtained by immunofluorescence analysis of Crithidia fasciculata. However, monoclonal antisera against the corresponding enzyme extracted from C. fasciculata recognizes a kinetoplast protein in both T. cruzi and Crithidia.

Trypanosoma cruzi topoisomerase II: a target enzyme for the chemotherapy of Chagas' disease? Cloning and characterization of its encoding gene.

The study of Trypanosoma cruzi type II DNA-topoisomerase should provide new clues for the rational development of new drugs for the chemotherapy of Chagas' disease. This enzyme is very likely involved in the processes leading to T. cruzi replication and differentiation since both processes are blocked by bacterial type II DNA topoisomerase inhibitors. In this article, we review and discuss our recent data related to the cloning, sequencing, and expression of T. cruzi type II topoisomerase.

Cloning and characterization of the gene encoding Trypanosoma cruzi DNA topoisomerase II.

The gene encoding Trypanosoma cruzi type II topoisomerase (TcTOP2) was isolated from a genomic library with a heterologous probe corresponding to part of the Trypanosoma brucei type II topoisomerase (TBrTOP2) gene. Nucleotide sequencing of TcTOP2 showed that the gene consists of an open reading frame of 3696 nucleotides (1232 amino acids), predicting a polypeptide product of 138,413 Da. Comparison of the amino acid sequence with that of type II topoisomerases from T. brucei (TBrTOP2) and Crithidia fasciculata (CfaTOP2), shows a high degree of conservation with estimated identities of 78% and 69%, respectively. TcTOP2 is a single copy gene in the genome of T. cruzi Dm28c and is expressed as a 4.5-kb mRNA. PCR mapping showed two distinct mini-exon addition sites at positions 225 and 203 upstream from the initiator AUG.

Peculiar sequence organization of kinetoplast DNA minicircles from Trypanosoma cruzi.

The sequences of two minicircles from the kinetoplast DNA of the CL strain and one of the Y strain of Trypanosoma cruzi are reported. These 1.4 kb molecules have a peculiar sequence organization, the most distinctive feature being the occurrence of a 120 bp sequence repeated four times, located at 0, 90, 180 and 270 degrees along each circle. We have termed these conserved regions in this species 'minirepeats'. Minirepeats have a 3-fold higher concentration of cytosine residues in comparison with the variable regions and contain the universal 12-mer motif GGGGTTGGTGTA present in all sequenced minicircles and which was shown to be involved in DNA replication. A consensus sequence of T. cruzi minirepeats was determined using the 20 minirepeats present in five known T. cruzi minicircle sequences. This consensus sequence contains regions which have been remarkably well preserved in strains which show great biological diversity. In addition a low level of intraminicircle sequence similarity was also observed within the variable region, but this similarity did not extend between strains. The abundance of conserved minirepeat sequences containing invariant restriction sites in T. cruzi cells may prove valuable for the development of new direct diagnostic methods for Chagas' disease based on DNA probe technology.

Diatesis hemorragica autoinmunitaria. / Autoimmune hemorragic diathesis

En un paciente con diatesis hemorragica como principal manifestacion clinica, se identificaron simultaneamente un anticoagulante circulante de semiologia compleja, anticuerpos antiplaquetarios, alteraciones propias de la enfermedad de von Willebrand adquirida y deficiencia del factor XII, acompanantes del lupus eritematoso generalizado y el sindrome de Sjogren. En esta comunicacion se describen los procedimientos usados para identificar las multiples anormalidades encontradas en la paciente, su reaccion al tratamiento y se discuten sus mecanismos fisiopatologicos

El diagnostico de la leucemia aguda por medio de la biometria hematica. / Diagnosis of acute leukemia by hematic biometry

Se presentan los resultados de la biometria hematica inicial de 188 pacientes con diagnostico de leucemia aguda. Los resultados mostraron que los hallazgos mas frecuentes fueron trombocitopenia, anemia y presencia de blastos en la sangre periferica. Ademas, algunos de los resultados mostraron que tenian valor pronostico