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R Buitrago

Publications and source records attributed to R Buitrago.

9 recordsLinked to original sources
PubMed2003

Human mixed infections of Leishmania spp. and Leishmania-Trypanosoma cruzi in a sub Andean Bolivian area: identification by polymerase chain reaction/hybridization and isoenzyme.

Parasites belonging to Leishmania braziliensis, Leishmania donovani, Leishmania mexicana complexes and Trypanosoma cruzi (clones 20 and 39) were searched in blood, lesions and strains collected from 28 patients with active cutaneous leishmaniasis and one patient with visceral leishmaniasis. PCR-hybridization with specific probes of Leishmania complexes (L. braziliensis, L. donovani and L. mexicana) and T. cruzi clones was applied to the different DNA samples. Over 29 patients, 8 (27.6%) presented a mixed infection Leishmania complex species, 17 (58.6%) a mixed infection Leishmania-T. cruzi, and 4 (13.8%) a multi Leishmania-T. cruzi infection. Several patients were infected by the two Bolivian major clones 20 and 39 of T. cruzi (44.8%). The L. braziliensis complex was more frequently detected in lesions than in blood and a reverse result was observed for L. mexicana complex. The polymerase chain reaction-hybridization design offers new arguments supporting the idea of an underestimated rate of visceral leishmanisis in Bolivia. Parasites were isolated by culture from the blood of two patients and lesions of 10 patients. The UPGMA (unweighted pair-group method with arithmetic averages) dendrogram computed from Jaccard's distances obtained from 11 isoenzyme loci data confirmed the presence of the three Leishmania complexes and undoubtedly identified human infections by L. (V.) braziliensis, L. (L.) chagasi and L. (L.) mexicana species. Additional evidence of parasite mixtures was visualized through mixed isoenzyme profiles, L. (V.) braziliensis-L. (L.) mexicana and Leishmania spp.-T. cruzi. The epidemiological profile in the studied area appeared more complex than currently known. This is the first report of parasitological evidence of Bolivian patients with trypanosomatidae multi infections and consequences on the diseases' control and patient treatments are discussed.

Animals↗
PubMed2002

First evidence of transmission of Leishmania (Viannia) lainsoni in a Sub Andean region of Bolivia.

Using ubiquitous primers which amplify the variable parts of kDNA minicircle of all Leishmania spp, we obtained for Leishmania (viannia) lainsoni a major band of 605 bp (band 1) shared with L. V. braziliensis and a minor 524 bp band (band 2) specific of L. V. lainsoni. The specificity of the two bands was examined through Southern blot hybridization of kDNA PCR obtained from reference strains belonging to L. braziliensis, L. mexicana, L. donovani complexes with L. V. lainsoni species. Band 1 was not specific of L. V. lainsoni since it hybridized with some isolates belonging to L. braziliensis complex. In contrast, band 2 was L. V. lainsoni specific. PCR-based detection followed by hybridization with the new L. V. lainsoni probe (Band 2) and L. V. braziliensis probe (564 bp), was assayed using sample from a pool of 25 females of Lutzomiya nuneztovari anglesi, blood, skin and liver samples of 18 mammals, spinal cords of four mammals and blood and cutaneous ulcers aspirates from 95 patents from Sub Andean region of La Paz, Bolivia. We observed a ositive hybridization of four patients lesions and the pool of L. nuneztovari anglesi with the L. V. lainsoni probe. It is the first time that L. V. lainsoni is observed in a cycle of transmission in Bolivia. PCR products of three patients lesions and the pool of L. nuneztovari anglesi were also hybridized with the specific probe of L. V. braziliensis suggesting mixed infection in this focus.

Animals↗
PubMed1999

Polymerase chain reaction-based identification of New World Leishmania species complexes by specific kDNA probes.

Here we define a new approach for the detection and characterisation of Leishmania complexes by polymerase chain reaction (PCR) and specific hybridisation. The first step consists of PCR amplification of kDNA minicircles using general kinetoplastid primers, which generate a polymorphic multi-banding pattern for all Leishmania species and other Kinetoplastidae. The second step is the identification of the Leishmania species complexes by hybridisation of the PCR products with specific kDNA probes. Polymorphic PCR-products from a genetically diverse set of Leishmania species were analysed by electrophoresis and the banding patterns compared with multi-locus enzyme electrophoresis (MLEE) data. The banding patterns produced by Leishmania species were very heterogeneous, making kDNA-PCR useful for determining closely related strains and for fingerprinting individual strains. The degree of kDNA-PCR and MLEE polymorphism was compared using UPGMA dendrograms. Three complex-specific probes were generated from major PCR bands of reference stocks belonging to the Leishmania mexicana, Leishmania donovani and Leishmania braziliensis complexes, and hybridisation of these probes to membrane-bound PCR products could reliably identify the strain to a complex level. A combination of kDNA-PCR fingerprinting and hybridisation with kDNA probes was found to be useful for both sensitive detection and direct identification of Leishmania species complexes.

Animals↗
PubMed1998

Interaction of diabetes and hypertension on determinants of endothelial adhesiveness.

Epidemiological studies have established that diabetes mellitus and hypertension are independent risk factors for atherosclerosis. One of the earliest abnormalities seen in atherogenesis is enhanced monocyte adherence to the endothelium. The mechanisms by which diabetes mellitus or hypertension enhances monocyte-endothelial cell interactions are incompletely characterized. It is not known whether there are additive interactions between these risk factors on endothelial adhesiveness for monocytes. Male Wistar-Kyoto (WKY) and spontaneously hypertensive (SHR) rats were fed a normal or fructose-enriched diet. In some cases, animals were injected with streptozotocin (35 mg/kg body weight) to induce diabetes. After 2 weeks, plasma was drawn for biochemical measurements, and thoracic aortas were harvested, opened longitudinally, and exposed to fluorescently labeled mouse monocytoid cells (WEHI 78/24, 2 x 10(6)/mL) for 30 minutes on a rocking platform. Adherent cells were counted by epifluorescence microscopy. WEHI 78/24 binding to aortic segments from SHR animals was elevated compared with segments from WKYs. Fructose feeding alone had no effect on endothelial adhesiveness. When WKYs were made hyperglycemic by STZ injection, monocyte binding was 160% of the control value. Elevated monocyte binding was also observed in aortas derived from SHR animals injected with STZ, indicating an additive effect of hypertension and hyperglycemia. To determine whether alterations in oxidative state played a role in the endothelial adhesiveness, aortic segments were exposed to lucigenin (250 micromol/L) for measurement of superoxide anion. Aortic segments from SHR elaborated 120% more superoxide anion than did controls. Elevated free-radical production was also observed in aortas from diabetic WKYs. Furthermore, thoracic aortas derived from diabetic SHR animals elaborated more superoxide anion than did any of the other groups (374%, P<0.05). Immunohistochemical staining for monocyte chemotactic protein-1 demonstrated increased expression in aortas isolated from diabetic WKY and SHR compared with control vessels. These studies demonstrate that both diabetes and hypertension lead to increased monocyte adherence to the endothelium. This abnormality is associated with increased vascular superoxide production and monocyte chemotactic protein-1 expression. Furthermore, there appears to be an additive interaction between hyperglycemia and hypertension in their effects on endothelial adhesiveness and its determinants.

Animals↗
PubMed1997

Nitric oxide regulates monocyte chemotactic protein-1.

BACKGROUND: Monocyte chemotactic protein-1 (MCP-1) is a 76-amino-acid chemokine thought to be the major chemotactic factor for monocytes. We and others have demonstrated that NO inhibits monocyte-endothelial cell interactions and atherogenesis. We hypothesize that the antiatherogenic effect of NO may be due in part to its inhibition of MCP-1 expression. METHODS AND RESULTS: Smooth muscle cells (SMCs) were isolated from normal rabbit aortas by the explant method. Cells were then exposed to LPS (10 microg/mL), native LDL, or oxidized LDL (30 microg/mL) for 6 hours. The expression of MCP-1 in SMCs and chemotactic activity in the conditioned medium were induced by lipopolysaccharide (LPS) or by oxidized LDL but not native LDL. The induction of MCP-1 by cytokines or oxidized lipoproteins was associated with an increased generation of superoxide anion by the SMCs and increased activity of the transcriptional protein nuclear factor-kappaB (NFkappaB). The induced expression of MCP-1 and activation of NFkappaB were reduced by previous exposure of the SMCs to the NO donor DETA-NONOate (100 micromol/L) (P<.05). To determine whether NO exerted its effect at a transcriptional level, SMCs and COS cells were transfected with a 400-bp fragment of the MCP-1 promoter. Promoter activity was enhanced by oxidized LDL, and LPS was inhibited by DETA-NO. Nuclear run-on assays confirmed that the effect of NO occurred at a transcriptional level. To investigate the role of endogenous NO in the regulation of MCP-1 in vivo, New Zealand White rabbits were fed normal chow, normal chow plus nitro-L-arginine (LNA), high-cholesterol diet (Chol), or high-cholesterol diet supplemented with L-arginine (Arg). After 2 weeks, thoracic aortas were harvested and total RNA was isolated. Northern analysis using full-length MCP-1 cDNA demonstrated increased expression in Chol and LNA aortas; this expression was decreased in aortas from Arg animals. CONCLUSIONS: These studies indicate that the antiatherogenic effect of NO may be mediated in part by its inhibition of MCP-1 expression.

Animals↗
PubMed1997

Cell cycle inhibition preserves endothelial function in genetically engineered rabbit vein grafts.

We have recently shown that ex vivo gene therapy of rabbit autologous vein grafts with antisense oligodeoxynucleotides (AS ODN) blocking cell cycle regulatory gene expression inhibits not only neointimal hyperplasia, but also diet-induced, accelerated graft atherosclerosis. We observed that these grafts remained free of macrophage invasion and foam cell deposition. Since endothelial dysfunction plays an important role in vascular disease, the current study examined the effect of this genetic engineering strategy on graft endothelial function and its potential relationship to the engineered vessels' resistance to atherosclerosis. Rabbit vein grafts transfected with AS ODN against proliferating cell nuclear antigen (PCNA) and cell division cycle 2 (cdc2) kinase elaborated significantly more nitric oxide and exhibited greater vasorelaxation to both calcium ionophore and acetylcholine than did untreated or control ODN-treated grafts. This preservation of endothelial function was associated with a reduction in superoxide radical generation, vascular cell adhesion molecule-1 (VCAM-1) expression, and monocyte binding activity in grafts in both normal and hypercholesterolemic rabbits. Our data demonstrate that AS ODN arrest of vascular cell cycle progression results in the preservation of normal endothelial phenotype and function, thereby influencing the biology of the vessel wall towards a reduction of its susceptibility to occlusive disease.

Animals↗
PubMed1996

Fluid flow inhibits endothelial adhesiveness. Nitric oxide and transcriptional regulation of VCAM-1.

BACKGROUND: In the arterial tree, regions exposed to reduced shear stress (low and/or disturbed flow) are predisposed to atherogenesis. Fluid flow is a potent stimulus for the release of endothelium-derived nitric oxide (NO). Because NO inhibits monocyte-endothelial cell interaction, we speculated that the effects of flow in inhibiting atherogenesis might be mediated in part by NO. METHODS AND RESULTS: Confluent monolayers of human aortic endothelial cells were exposed to static or fluid flow conditions for 4 hours. The medium was replaced, and cells were then incubated with native LDL (50 micrograms/mL), oxidized LDL (30 micrograms/mL), or lipopolysaccharide (LPS) (10 ng/mL)+tumor necrosis factor-alpha (TNF-alpha) (10 U/mL) for an additional 4 hours. Functional binding assays using THP-1 monocytes were then performed. Superoxide production by human aortic endothelial cells was monitored by lucigenin chemiluminescence, and expression of the adhesion molecules vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 were quantified by flow cytometry. Whereas native LDL had little effect, incubation with either oxidized LDL or LPS/TNF-alpha significantly increased superoxide production, nuclear factor-kappa B activity, VCAM-1 expression, and endothelial adhesiveness for monocytes. Previous exposure to fluid flow inhibited these sequelae of exposure to cytokines or oxidized lipoprotein. The effect of fluid flow appears to be due in part to shear-induced release of NO, because coincubation with nitro-L-arginine completely abolished these effects of flow. Furthermore, the NO donor PAPA-NONO-ate and 8-Br-cGMP (but not 8-Br-cAMP) mimicked the effects of flow. CONCLUSIONS: Previous exposure to fluid flow decreased cytokine- or lipoprotein-stimulated endothelial cell superoxide production, VCAM-1 expression, and monocyte binding; the effects of flow appear to be due to NO. Flow-mediated NO-dependent regulation of oxidant-responsive transcription may influence the site of a lesion.

Animals↗
PubMed1996

Regression or progression. Dependency on vascular nitric oxide.

We have shown that chronic administration of the nitric oxide (NO) precursor L-arginine inhibits atherogenesis in the hypercholesterolemic rabbit. However, the effect of supplemental arginine on preexisting lesions is not known and was the focus of the present study. New Zealand White rabbits received normal chow or 0.5% cholesterol chow for 10 weeks. Subsequently, L-arginine (2.25% in drinking water; ARG group) or vehicle (CHOL group) was administered for an additional 13 weeks, while the high-cholesterol diet was continued. Thoracic aortae were harvested at weeks 10, 14, 18, or 23. Rings of aorta were used to assess NO-dependent vasodilation to acetylcholine. Maximal relaxation to acetylcholine in the CHOL rabbits became progressively attenuated from 53.4% (at week 10) to 17.4% (by week 23). Planimetry of the luminal surface of the aortae from CHOL animals revealed a progressive increase in lesion surface area from 30.3% (at week 10) to 56.5% (by week 23). By contrast, animals in the ARG groups manifested improved endothelium-dependent relaxation associated with a reduction of lesion surface area at 14 and 18 weeks. The arginine-induced improvement in endothelium-dependent relaxation was associated with an increased generation of vascular NO and a reduced generation of vascular superoxide anion. By 23 weeks, 3 of 7 ARG animals had persistent improvement in NO-dependent vasodilation and exhibited a further reduction of lesion surface area tc 5.4%. We conclude that hypercholesterolemia induces a progressive loss of NO-dependent vasodilation associated with progressive intimal lesion formation. Administration of L-arginine to animals with preexisting intimal lesions augments vascular NO elaboration, reduces superoxide anion generation, and is associated with a reduction in lesion surface area. This is the first demonstration that restoration of NO activity can induce regression of preexisting intimal lesions and provides evidence that L-arginine therapy may be of potential clinical benefit.

Acetylcholine↗