Graciela L. Uzcanga

Bio: Graciela L. Uzcanga is an academic researcher from Simón Bolívar University. The author has contributed to research in topics: Trypanosoma evansi & Trypanosoma vivax. The author has an hindex of 10, co-authored 23 publications receiving 359 citations. Previous affiliations of Graciela L. Uzcanga include Central University of Venezuela & Universidad Simón Rodríguez.

Identification and Partial Characterization of Excretory/Secretory Products with Proteolytic Activity in Giardia intestinalis

Abstract: This report examines the presence of proteolytic activity detected in media collected from in vitro cultures of Giardia intestinalis, and the partial characterization by gelatin-substrate polyacrylamide gel electrophoresis and inhibition studies. Gelatin-substrate polyacrylamide gel electrophoresis revealed 6 bands with proteolytic activity, with estimated molecular weights of 36, 59, 63, 72, 103, and 175 kDa. These bands were not present in the control medium. On the other hand, G. intestinalis trophozoite lysates showed proteolytic bands at 16, 20, 66, 82, 108, and 120 kDa, thus indicating that intracellular proteases could be different from the excretory/secretory (E/S) products. Based on inhibition studies, 2 bands of 59 and 63 kDa were inhibited by iodoacetic acid, indicating the presence of cysteine proteases. Partial inhibition of a band of 36 kDa was found with EDTA, a metal-chelating agent, suggesting the possible presence of metalloproteases. The presence of aspartic and serine proteases were not detected under the assay conditions used. As G. intestinalis E/S may be involved in differentiation mechanisms of the parasite and also be responsible for the mucosal alterations that occur in giardiasis, the characterization of these proteases may facilitate their evaluation as targets in the therapy of the disease.

In Vitro Anti-Trypanosoma cruzi Activity of Dronedarone, a Novel Amiodarone Derivative with an Improved Safety Profile

Abstract: Amiodarone, a commonly used antiarrhythmic, is also a potent and selective anti-Trypanosoma cruzi agent. Dronedarone is an amiodarone derivative in which the 2,5-diiodophenyl moiety of the parental drug has been replaced with an unsubstituted phenyl group aiming to eliminate the thyroid toxicity frequently observed with amiodarone treatment. Dronedarone has been approved by the Food and Drug Administration (FDA), and its use as a safe antiarrhythmic has been extensively documented. We show here that dronedarone also has potent anti-T. cruzi activity, against both extracellular epimastigotes and intracellular amastigotes, the clinically relevant form of the parasite. The 50% inhibitory concentrations against both proliferative stages are lower than those previously reported for amiodarone. The mechanism of action of dronedarone resembles that of amiodarone, as it induces a large increase in the intracellular Ca 2 concentration of the parasite, which results from the release of this ion from intracellular storage sites, including a direct effect of the drug on the mitochondrial electrochemical potential, and through alkalinization of the acidocalcisomes. Our results suggest a possible future repurposed use of dronedarone for the treatment of Chagas’ disease.

Variant surface glycoprotein from Trypanosoma evansi is partially responsible for the cross-reaction between Trypanosoma evansi and Trypanosoma vivax.

Abstract: Salivarian trypanosomes use antigenic variation of their variant-specific surface glycoprotein (VSG) coat as a defense against the host immune system. Although about 1000 VSG and pseudo-VSG genes are scattered throughout the trypanosome genome, each trypanosome expresses only one VSG, while the rest of the genes are transcriptionally silent. A 64-kDa glycosylated cross-reacting antigen between Trypanosoma evansi and Trypanosoma vivax (p64), which was purified from the TEVA1 T. evansi Venezuelan isolate, was proven here to represent the soluble form of a VSG. Initially, a biochemical characterization of p64 was carried out. Gel filtration chromatography, sedimentation, and chemical cross-linking provided evidences of the dimeric nature of p64. The hydrodynamic parameters indicated that p64 is asymmetrical with a frictional ratio f/fo = 1.57. Isoelectric focusing and two-dimensional polyacrylamide gel electrophoresis revealed that p64 contained two isoforms with isoelectric points of 6.8-6.9 and 7.1-7.2. When p64 and three p64 Staphylococcus aureus V8 proteolytic fragments were sequenced, the same N-termini sequence was obtained: Ala-Pro-Ile-Thr-Asp-Ala-Asp-Leu-Gly-Pro-Ala-Gln-Ile-Ala-Asp, which displayed a significant homology with a putative Trypanosoma brucei VSG gene located on chromosome 4. Additionally, immunofluorescence microscopy on T. evansi and T. vivax established that p64 and its T. vivax homologue were confined to the surface of both parasites. An immunological characterization of this antigen was also carried out using several Venezuelan T. evansi isolates expressing different VSGs, which were obtained from naturally infected animals. Although sera from animals infected with the various T. evansi isolates recognized p64, only one isolate, besides TEVA1, contained polypeptides that were recognized by anti-p64 antibodies. All these results together with prior evidences [Uzcanga, G. et al. (2002) Parasitology 124, 287-299] confirmed that p64 is the soluble form of a T. evansi VSG, containing common epitopes recognized by sera from animals infected with T. evansi or T. vivax. Despite the huge repertoire of VSG genes existing on bloodstream trypanosomes, our data also demonstrated the potential use of a VSG variant from the TEVA1 T. evansi isolate as a diagnostic reagent.

Current trends in research into the waterborne parasite Giardia.

Abstract: The waterborne flagellated parasite Giardia intestinalis continues to be the most frequent protozoan agent of intestinal disease world-wide, causing an estimated 2.8 x 10(8) cases per annum. Severe symptoms of diarrhea and sickness can be persistent and even life threatening in the immunocompromised, in infants, and in the aged, although self-limiting in the majority of patients. Despite a growing awareness and intensified research many uncertainties remain, especially with respect to the risk of potential zoonotic transmission. Water supplies can be monitored for cysts using automated cytofluorimetric immunoassays, but this does not measure infectivity. Filtration provides the best protection, because cysts are highly resistant to chlorine and ozone. Other incompletely elucidated aspects include mechanisms of pathogenicity, host reaction to infection, immunity and parasite control using vaccines or antigiardial compounds; the 5-nitroimidazole metronidazole is the most effective of these. Molecular typing of various isolates indicates that most animal parasites are not infective to humans, but those that are can be genotypically classified as assemblage A or B. The phylogeny of the organism remains uncertain, but there is a growing opinion that Giardia is not an ancient primitive eukaryote, but that it is derived from a more complex mitochondria-containing protozoon.

Diagnosis and management of Chagas disease and cardiomyopathy.

Abstract: Chagas cardiomyopathy is the most severe and life-threatening manifestation of human Chagas disease--a 'neglected' tropical disease caused by the protozoan parasite Trypanosoma cruzi. The disease is endemic in all continental Latin American countries, but has become a worldwide problem because of migration of infected individuals to developed countries, mainly in Europe and North America. Chagas cardiomyopathy results from the combined effects of persistent parasitism, parasite-driven tissue inflammation, microvascular and neurogenic dysfunction, and autoimmune responses triggered by the infection. Clinical presentation varies widely according to the extent of myocardial damage, and manifests mainly as three basic syndromes that can coexist in an individual patient: heart failure, cardiac arrhythmia, and thromboembolism. NYHA functional class, left ventricular systolic function, and nonsustained ventricular tachycardia are important prognostic markers of the risk of death. Management of Chagas cardiomyopathy focuses on the treatment of the three main syndromes. The use of β-blockers in patients with Chagas disease and heart failure is safe, well tolerated, and should be encouraged. Most specialists and international institutions now recommend specific antitrypanosomal treatment of patients with chronic Chagas disease, even in the absence of evidence obtained from randomized clinical trials. Further research on the management of patients with Chagas cardiomyopathy is necessary.

Multi-target pharmacology: possibilities and limitations of the “skeleton key approach” from a medicinal chemist perspective

Abstract: Multi-target drugs have raised considerable interest in the last decade owing to their advantages in the treatment of complex diseases and health conditions linked to drug resistance issues. Prospective drug repositioning to treat comorbid conditions is an additional, overlooked application of multi-target ligands. While medicinal chemists usually rely on some version of the lock and key paradigm to design novel therapeutics, modern pharmacology has recognized that the long-term effects of a given drug on a biological system may depend not only on the specific ligand-target recognition events but also on the influence of the chronic administration of a drug on the cell gene signature. The design of multi-target agents also poses challenging restrictions on the either the topology or flexibility of the candidate drugs which are briefly discussed in the present article. Finally, computational strategies to approach the identification of novel multi-target agents are overviewed.

Experimental and Clinical Treatment of Chagas Disease: A Review.

Abstract: Chagas disease (CD) is caused by the protozoan parasite Trypanosoma cruzi that infects a broad range of triatomines and mammalian species, including man. It afflicts 8 million people in Latin America, and its incidence is increasing in nonendemic countries owing to rising international immigration and nonvectorial transmission routes such as blood donation. Since the 1960s, the only drugs available for the clinical treatment of this infection have been benznidazole (BZ) and nifurtimox (NFX). Treatment with these trypanocidal drugs is recommended in both the acute and chronic phases of CD. These drugs have low cure rates mainly during the chronic phase, in addition both drugs present side effects that may result in the interruption of the treatment. Thus, more efficient and better-tolerated new drugs or pharmaceutical formulations containing BZ or NFX are urgently needed. Here, we review the drugs currently used for CD chemotherapy, ongoing clinical assays, and most-promising new experimental drugs. In addition, the mechanism of action of the commercially available drugs, NFX and BZ, the biodistribution of the latter, and the potential for novel formulations of BZ based on nanotechnology are discussed.Taken together, the literature emphasizes the urgent need for new therapies for acute and chronic CD.