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A M Ruiz

Publications and source records attributed to A M Ruiz.

At least 19 recordsLinked to original sources
PubMed2006

Immunological and pathological responses in BALB/c mice induced by genetic administration of Tc 13 Tul antigen of Trypanosoma cruzi.

Tc13 is a trans-sialidase family protein of Trypanosoma cruzi, the aetiological agent of Chagas' disease. Recently, in vitro studies had suggested that Tc13 might participate in the pathogenesis of the disease. In order to study the role of Tc13 antigens in an in vivo model, we administered plasmid DNA encoding a Tc13 antigen from the Tulahuén strain (Tc13 Tul) to BALB/c mice and evaluated the immunological and pathological manifestations as well as the capacity of this antigen to confer protection against T. cruzi infection. Tc13 Tul immunization did not elicit a detectable humoral immune response but induced specific memory T-cells with no capacity to produce IFN-gamma. Five months after DNA-immunization with Tc13 Tul, signs of hepatotoxicity and reactive changes in the heart, liver and spleen were observed in 40-80% of mice. When Tc13 Tul DNA-immunized animals were challenged with trypomastigotes, a significant decrease in parasitaemia in early and late acute phase was observed without modification in the survival rate. Surprisingly, Tc13 Tul-immunized mice chronically infected with T. cruzi showed a decrease in the severity of heart damage. We conclude that, in BALB/c mice, genetic immunization with Tc13 Tul mainly induces immune responses associated with pathology.

Animals↗
PubMed2006

Analysis of the Trypanosoma cruzi cyclophilin gene family and identification of Cyclosporin A binding proteins.

The Trypanosoma cruzi cyclophilin gene family comprises 15 paralogues whose nominal masses vary from 19 to 110 kDa, namely TcCyP19, TcCyP20, TcCyP21, TcCyP22, TcCyP24, TcCyP25, TcCyP26, TcCyP28, TcCyP29, TcCyP30, TcCyP34, TcCyP35, TcCyP40, TcCyP42 and TcCyP110. Under the conditions used, only some of the T. cruzi cyclophilin paralogue products could be isolated by affinity chromatography. The 15 paralogues were aligned with 495 cyclophilins from diverse organisms. Analyses of clusters formed by the T. cruzi cyclophilins with others encoded in various genomes revealed that 8 of them (TcCyP19, TcCyP21, TcCyP22, TcCyP24, TcCyP35, TcCyP40, TcCyP42 and TcCyP110) have orthologues in many different genomes whereas the other 7 display less-defined patterns of their sequence attributes and their classification to a specific group of cyclophilin's orthologues remains uncertain. Seven epimastigote cDNA clones encoding cyclophilin isoforms were further studied. These genes were found dispersed throughout the genome of the parasite. Amastigote and trypomastigote mRNAs encoding these 7 genes were also detected. We isolated 4 cyclosporin A-binding proteins in T. cruzi epimastigote extracts, which were identified by mass spectrometry as TcCyP19, TcCyP22, TcCyP28 and TcCyP40. Cyclosporin A-binding to these cyclophilins might be of importance to the mechanism of action of Cyclosporin A and its non-immunosuppressive analogues, whose trypanocidal effects were previously reported, and therefore, of potential interest in the chemotherapy of Chagas' disease.

Amino Acid Sequence↗
PubMed2002

Trypanosoma cruzi: methoprene is a potent agent to sterilize blood infected with trypomastigotes.

The effects of methoprene, a juvenile hormone analogue (JHA), on Trypanosoma cruzi bloodstream trypomastigotes (Tulahuen strain, Tul 2 stock) were studied. It was observed that 150microM of methoprene in in vitro experiments cause cellular death of T. cruzi. In contrast, methoprene was not able to clear bloodstream trypomastigotes in in vivo experiments, but it was observed a decrease of parasitemia levels of infected mice treated with 200microg of methoprene/mouse/day during 5 days. According to these results and the low toxicity of methoprene, we suggest that this compound will serve as an effective agent to sterilize blood for transfusions.

Animals↗
PubMed2001

Characterisation of a cyclophilin isoform in Trypanosoma cruzi.

The immunosuppressive drug cyclosporin A (CsA) has shown antiparasitic activity against several protozoans and helminths, when complexed to proteins called cyclophilins (CyPs). In this paper, the molecular characterisation of one member of the CyP family in Trypanosoma cruzi is reported. TcCyP19 gene proved to be highly conserved compared to CyPs from other organisms and was highly homologous to a Trypanosoma brucei brucei CyPA. This gene was expressed in Escherichia coli and the purified recombinant protein exhibited a peptidyl prolyl cis-trans isomerase activity that was inhibited by CsA (IC(50) = 18.4 + /-0.8 nM). The TcCyP19 gene was located on two chromosomal bands in T. cruzi CL Brener clone.

Amino Acid Sequence↗
PubMed2000

The tyrosine aminotransferase from Trypanosoma rangeli: sequence and genomic characterization.

The complete sequence and genomic characterization of the tyrosine aminotransferase (TAT) gene from Trypanosoma rangeli is reported. The gene was found to be organized in a tandem multicopy gene array. A homologous mRNA species (2.5 kb) was identified in the epimastigote form of the parasite. From the deduced amino acid sequence, the gene encodes a protein of 420 amino acids with a predicted molecular mass of 46.4 kDa and a theoretical pI of 6.23. A high sequence identity was found with the Trypanosoma cruzi, human and rat enzymes. All the essential residues for TAT enzymatic activity are conserved, as well as a pyridoxal-phosphate attachment site typical of class-I aminotransferases. The recombinant enzyme was recognized by a monoclonal antibody against the T. cruzi enzyme. Additionally, the recombinant protein showed enzymatic activity when incubated with L-tyrosine and 2-oxoglutaric acid as substrates.

Amino Acid Sequence↗
PubMed2000

Molecular cloning, sequencing and expression of a serine proteinase inhibitor gene from Toxoplasma gondii.

A cDNA clone from a Toxoplasma gondii tachyzoite cDNA library encoding a serine proteinase inhibitor (serpin) was isolated. The 1376 bp cDNA sequence encodes a 294 amino acid protein with a putative signal peptide of 23 amino acids resulting in a mature protein with a predicted mass of 30,190 Da and a pI of 4.86. This protein has internal sequence similarity of residues 30-66, 114-150, 181-217 and 247-283 indicating a four-domain structure. The four domains exhibit high identity to serine proteinase inhibitors belonging to the non-classical Kazal-type family. The gene is single copy in the tachyzoite haploid genome of RH strain and was amplified by polymerase chain reaction (PCR). Several introns were identified. The sequence encoding the mature protein was amplified by PCR, cloned into the pQE30 vector and expressed in Escherichia coli. Specific antiserum generated against the recombinant protein was used in immunoblot assay and two bands of 38 and 42 kDa were detected in a whole parasite homogenate. The recombinant protein showed trypsin-inhibitory activity, one of the two potential specificities. We discuss the possible roles that T. gondii serpin(s) may play in the survival of the tachyzoites in the host.

Amino Acid Sequence↗
PubMed2000

Six-month treatment of obesity with sibutramine 15 mg; a double-blind, placebo-controlled monocenter clinical trial in a Hispanic population.

OBJECTIVE: To evaluate the safety and efficacy of sibutramine 15 mg by mouth once per day in obese patients over a period of 6 months. RESEARCH METHODS AND PROCEDURES: A monocenter, double-blind, placebo controlled, parallel, prospective clinical trial was carried out. Sixty-nine male and female obese patients (body mass index [BMI] > 30 kg/m2) aged 16 to 65 years entered the trial. RESULTS: 22 of 35 patients in the sibutramine group and 9 of 34 patients in the placebo group completed the trial. The high dropout rate in the sibutramine group was due to adverse events in 3 cases, lack of efficacy (as judged by patients) in 7, loss to follow-up in 2, and an orthopedic device being worn in 1; in the placebo group the dropouts were ascribed to lack of efficacy (as judged by patients) in 17 cases and to loss to follow-up in 8 cases. Using the method of last observation carried forward, the weight loss in the sibutramine group was 10.27 kg (95% confidence intervals [95% CI] 7.66; 13.07) and 1.26 kg (95% CI 0.3; 2.23) in the placebo group. The BMI loss was 4.17 kg/m2 (95% CI 3.11; 5.22) in the sibutramine group and 0.53 kg/m2 (95% CI 0.13; 0.92) in the placebo group. The waist circumference reduction was 12.51 cm (95% CI 9.25; 15.77) in the sibutramine group and 3.26 cm (95% CI 1.38; 5.14) in the control group (p<0.05 by paired Student's t test for all the intragroup comparisons). Twenty-three sibutramine patients had 34 adverse events, the most frequent adverse events in the sibutramine group were upper respiratory tract infections (n = 6) and constipation (n = 6); 16 placebo patients had 21 adverse events. Three sibutramine patients withdrew their informed consent when they had adverse events. DISCUSSION: The results show that sibutramine induces significant loss of body weight and waist circumference. Cardiovascular function was not significantly affected by sibutramine. Sibutramine was well tolerated by most of the patients.

Adolescent↗
PubMed1999

[Trypanosoma cruzi genome: transcriptional mapping and karyotype correlation; molecular characterization of a surface antigen from the Tc13 family].

We describe herein the genome mapping of Trypanosoma cruzi, the etiological agent of Chagas' disease, by hybridization of a cosmid library gridded in high density filters with cDNA from an epimastigote expression library as probes. We also show the correlation of some cosmid contigs with parasite chromosomal bands. With libraries from the T. cruzi Genome Project we have characterized a new member of the Tc13 family belonging to the superfamily of trypomastigote surface antigens. Starting with a Tulahuén strain (Tul 2 stock) clone, homologous to these antigens, we have sequenced and characterized the complete gene in the CL Strain (CL Brener clone). We have also found homologies with different ESTs which characterization would lead to further knowledge of this antigen family.

Animals↗
PubMed1998

Efficacy of chemotherapy with benznidazole in children in the indeterminate phase of Chagas' disease.

A double-blind, randomized, clinical field trial was designed to test the efficacy and tolerance of a specific drug treatment in children in the indeterminate phase of infection by Trypanosoma cruzi. Children were treated with benznidazole at a dose of 5 mg/kg/day for 60 days or placebo and followed-up for 48 months. The treated children showed a significant decrease in geometric mean titers of antibodies against T. cruzi measured by indirect hemagglutination, indirect immunofluorescence, and ELISA. After a four year follow-up, 62% of the benznidazole-treated children and no placebo-treated child were seronegative for T. cruzi when tested by an ELISA using a T. cruzi flagellar calcium-binding protein (F29). Xenodiagnosis carried out after 48 months of follow-up was positive in 4.7% of the benznidazole-treated children and in 51.2% of the placebo-treated children. These results show the tolerance to and efficacy of benznidazole against T. cruzi in seropositive children six to 12 years of age. We used an early serologic marker of cure after treatment, consisting of a recombinant antigen implemented in a rapid, conventional serologic procedure.

Chagas Disease↗
PubMed1998

Trypanocidal effect of SKF525A, proadifen, on different developmental forms of Trypanosoma cruzi.

SKF525A, an inhibitor and inducer of cytochrome P450, was tested on different developmental stages of Trypanosoma cruzi. Growth, motility and structure of epimastigotes, motility and infectivity of trypomastigotes, and infectivity of trypomastigotes to Vero cells in culture were abolished by the drug at 10-100 microM concentrations. When blood from infected mice was treated with the drug, and used to infect 8 day-old mice, no parasites were observed at 0.6-1 mM, and all animals survived. Blood cell morphology was well preserved, and the sleeping time of pentobarbital-treated mice inoculated with the same amount of drug was not increased. The present results suggest that SKF525A or other related inhibitors of cytochrome P450 coned be tested as an additive for blood sterilization in blood banks.

Animals↗
PubMed1997

Trypanosoma cruzi: specific detection of parasites by PCR in infected humans and vectors using a set of primers (BP1/BP2) targeted to a nuclear DNA sequence.

In the present work we evaluate Trypanosoma cruzi DNA detection by PCR using the nuclear oligonucleotides BP1/BP2 as primers. These primers are targeted to the 5' and 3' ends of the coding region for the flagellar protein F29. An amplification product of BP1/BP2 is a DNA band 692 bp long. Titration assays were performed to evaluate the minimum amount of parasite DNA that can be detected by this assay, resulting in 10 fg (equivalent to about 1/20 of the genome). The assay was also performed using T. cruzi DNA from different strains, clones, and human-derived isolates obtaining, in all cases, amplification products. No DNA amplification was observed when the PCR was performed using DNA from Leishmania braziliensis, but when T. rangeli DNA was used, a 615-bp-long fragment was amplified. Under appropriate gel conditions T. cruzi and T. rangeli DNA amplicons could be differentiated. When both conventional xenodiagnosis and PCR detection of parasite DNA in the feces of insect vectors fed with blood from infected patients were compared, 10 of 20 samples were positive by both techniques. However, 2 other samples with positive serology were also positive by PCR. When PCR was performed on blood samples from infected and uninfected individuals, 62 of 65 serologically positive human samples amplified the BP1/BP2 692-bp T. cruzi DNA fragment (sensitivity >95%). The 3 negative samples were positive when Southern blot hybridization was performed using the radiolabeled PCR amplification product as probe (sensitivity 100%).

Animals↗
PubMed1996

Trypanosoma rangeli and Trypanosoma cruzi: molecular characterization of genes encoding putative calcium-binding proteins, highly conserved in trypanosomatids.

Genes encoding a 29-kDa flagellar calcium-binding protein (F29) in Trypanosoma cruzi, strongly homologous to EF-hand calcium-binding protein-encoding genes previously reported in this parasite, were isolated by immunoscreening. F29 is encoded by a number of very similar genes, highly conserved among different T. cruzi isolates. The genes are located on a pair of homologous chromosomes, arranged in one or two clusters of tandem repeats. PCR amplification of Trypanosoma rangeli genomic DNA, using primers derived from the T. cruzi F29 sequence made it possible to isolate the homologous gene in T. rangeli, encoding a 23-kDa protein called TrCaBP. Gene sequence comparisons showed homology to EF-hand calcium-binding proteins from T. cruzi (82.8%), Trypanosoma brucei brucei (60.2%), and Entamoeba histolytica (28.4%). Northern blot analysis revealed that the TrCaBP gene is expressed in T. rangeli as a polyadenylated transcript. The TrCaBP-encoding genes are present in at least 20 copies per cell, organized in tandem arrays, on large T. rangeli chromosomes in some isolates and on two smaller ones in others. This gene, however, seems to be absent from Leishmania.

Amino Acid Sequence↗
PubMed1996

Elevated peripheral blood natural killer cells are effectively downregulated by immunoglobulin G infusion in women with recurrent spontaneous abortions.

PROBLEM: We investigated the hypothesis that elevated peripheral blood natural killer cells (NK) are decreased by immunoglobulin G infusion (IVIg) therapy in women with recurrent spontaneous abortions (RSA) and elevated NK cells. METHODS: Seventy-three women with RSA and elevated NK cells received IVIg therapy (400 mg/Kg/day for 3 days ever 4 wks) and anticoagulation treatment. Peripheral blood immunophenotype assay by flow cytometry was done prospectively prior to and 7 days after first IVIg therapy, every 2 wks until 20 wks gestation and then monthly. Controls were 95 women with RSA and normal NK cells who received anticoagulation treatment. RESULTS: (1) 86.3% of women with elevated NK cells who received the IVIg and anticoagulation therapy had a successful pregnancy outcome; (2) Peripheral blood CD56+ NK cells and CD56+/16+ NK cells were significantly suppressed 7 days post IVIg infusion (P < 0.0005); (3) Pre-IVIg infusion levels of other lymphocyte subsets were not different as compared with those of 7 days post-IVIg therapy; (4) Women who delivered a liveborn infant with IVIg therapy demonstrated downregulation of peripheral blood NK cells (CD56+, CD56+/16+) during early pregnancy when compared to women who miscarried the index pregnancy (P < 0.05); (5) Women with normal NK cells who miscarried while on anticoagulation therapy demonstrated significantly elevated CD56+ NK cells during early pregnancy as compared with that of women who delivered a liveborn infant (P < 0.05); (6) CD19+ B cells were significantly downregulated during pregnancy in women with anticoagulation and IVIg therapy when compared to women with anticoagulation therapy (P < 0.05). CONCLUSION: Downregulation of NK cells in women with RSA is associated with a favorable pregnancy outcome. Peripheral blood NK cells (CD56+, CD56+/16+) are effectively suppressed after IVIg therapy. Women with RSA and high NK cells benefit from IVIg therapy and experience suppression of CD56+ and CD56+/16+ NK cells.

Abortion, Habitual↗
PubMed1996

Impact of age on reproductive outcome in women with recurrent spontaneous abortions and infertility of immune etiology.

The objective of this paper is to determine whether age has any impact on conception rate, pregnancy outcome, or autoimmune status in women with recurrent spontaneous abortions (RSA) and infertility of immune etiology. One hundred twenty-four women with 3 or more RSA and 36 women with unexplained infertility were prospectively studied. Maternal antipaternal lymphocyte antibodies and autoantibodies to phospholipids and nuclear antigens were tested. All achieved an adequate alloimmune recognition after lymphocyte immunization and followed for 1 year with optimal preconception autoimmune treatment. Conception rate and pregnancy outcome were prospectively studied. 1) 10.1% of women with RSA and 30.6% of women with infertility failed to achieve a pregnancy after 1 year of trial (P = 0.0084); 2) in women with RSA, the number of previous fetal death after 28 weeks of gestation was significantly higher in women who failed to achieve a pregnancy within 1 year when compared to women who became pregnant (P = 0.0296). Conception rate was not different with advancing age; 3) women with infertility demonstrated significantly higher incidence of anti-phosphatidylethanolamine antibody when compared to women with RSA (P = 0.052); 4) in women with infertility, those who failed to achieve a pregnancy were significantly older (P = 0.0012) and demonstrated a higher incidence of autoantibodies to phosphatidic acid than women with infertility who became pregnant (P = 0.0339); 5) the subsequent spontaneous abortion rate while on optimal immune therapy was the same in the women with RSA (39.3%) and women with infertility (32%). Spontaneous abortion rate of women with RSA or infertility was not different among four age groups; 6) the presence of anticardiolipin antibody (P = 0.0055) and higher gravidity (P = 0.0354) correlated significantly with pregnancy failure in women with a history of infertility. The prevalence of auto-antibodies to phospholipids and nuclear components was not different among four age groups in women with RSA or infertility. Age does not affect pregnancy outcome or conception rate in women with RSA. In women with infertility of immune etiology, conception rate was significantly reduced over age 40 although pregnancy outcome was no different with advanced age.

Abortion, Habitual↗
PubMed1995

A recombinant tyrosine aminotransferase from Trypanosoma cruzi has both tyrosine aminotransferase and alanine aminotransferase activities.

Tyrosine aminotransferase purified from epimastigotes of Trypanosoma cruzi displays an additional activity of alanine aminotransferase, absent in all other tyrosine aminotransferases characterized so far. Since the parasite's genome contains a high number of copies of the tyrosine aminotransferase gene, we could not rule out the possibility that two very similar proteins, with changed specificity due to a few amino acid substitutions, might be responsible for the two activities. We have now expressed in Escherichia coli a recombinant tyrosine aminotransferase as a fusion protein with glutathione S-transferase. The purified fusion protein, intact or after thrombin cleavage, displays tyrosine aminotransferase and alanine aminotransferase activities with apparent Km values similar to those for the natural enzyme, thus proving that they belong to the same protein.

Alanine Transaminase↗
PubMed1995

Outcome of infection with different strains of Trypanosoma cruzi in mice lacking CD4 and/or CD8.

Mice lacking CD4 and/or CD8 gene expression, generated by embryonic stem-cell technology, were used to study the role of CD4+ and CD8+ cells in the resistance to the acute infection with virulent (Tulahuén and RA) or mild (CA-I) strains of Trypanosoma cruzi. The presence of both CD4+ and CD8+ cells contributed to the survival of mice infected with T. cruzi, and each T-cell subtype was able to sustain protective functions in the absence of the other one. However, in certain host-parasite combinations, CD8+ cell-independent mechanisms were able to control the parasite load. Moreover, CD8- mice chronically infected with a low virulent strain of T. cruzi were protected from an otherwise lethal challenge with the parasite. A different organ distribution of parasite nests was observed when mutant (but not wild type) animals infected with different parasite strains were compared. CD4- mice produced high levels of IgG antibodies against peptide antigens or a whole homogenate from the parasite after infection with CA-I strain. A dramatic enhancement of IgG1- and IgG2a-specific antibodies was observed.

Amino Acid Sequence↗