Alfredo Mijares

Trypanosoma evansi: Effect of experimental infection on the osmotic fragility, lipid peroxidation and calcium-ATPase activity of rat red blood cells

Trypanosoma evansi is the causative agent of equine trypanosomoses. The disease is characterized by fever, anemia, and cachexia. Peroxidative damage of the red blood cells caused by the parasite, may contribute to the pathogenesis of the anemia seen in trypanosomoses. Consequently, we evaluated the hematocrit, the osmotic fragility of the red blood cells, the level of lipid peroxidation and the activity of the Ca-ATPase of red blood cell ghosts from rats experimentally infected with T. evansi. After 72 h inoculation, the hematocrit decreased from 49.5% to 33%; the osmotic fragility of the red blood cells was approximately 40% higher as compared to the healthy animals; and the red blood cell ghosts showed a higher level of lipid peroxidation and a lower Ca-ATPase activity than the red cell ghosts from the healthy animals. In vitro incubations of red blood cells from healthy animals with T. evansi, produced also a significant increase of the osmotic fragility of the red blood cells.

Age-dependent changes in diastolic Ca2+ and Na+ concentrations in dystrophic cardiomyopathy: Role of Ca2+ entry and IP3

Duchenne muscular dystrophy (DMD) is a lethal X-inherited disease caused by dystrophin deficiency. Besides the relatively well characterized skeletal muscle degenerative processes, DMD is also associated with a dilated cardiomyopathy that leads to progressive heart failure at the end of the second decade. The aim of the present study was to characterize the diastolic Ca(2+) concentration ([Ca(2+)]d) and diastolic Na(+) concentration ([Na(+)]d) abnormalities in cardiomyocytes isolated from 3-, 6-, 9-, and 12-month old mdx mice using ion-selective microelectrodes. In addition, the contributions of gadolinium (Gd(3+))-sensitive Ca(2+) entry and inositol triphosphate (IP3) signaling pathways in abnormal [Ca(2+)]d and [Na(+)]d were investigated. Our results showed an age-dependent increase in both [Ca(2+)]d and [Na(+)]d in dystrophic cardiomyocytes compared to those isolated from age-matched wt mice. Gd(3+) treatment significantly reduced both [Ca(2+)]d and [Na(+)]d at all ages. In addition, blockade of the IP3-pathway with either U-73122 or xestospongin C significantly reduced ion concentrations in dystrophic cardiomyocytes. Co-treatment with U-73122 and Gd(3+) normalized both [Ca(2+)]d and [Na(+)]d at all ages in dystrophic cardiomyocytes. These data showed that loss of dystrophin in mdx cardiomyocytes produced an age-dependent intracellular Ca(2+) and Na(+) overload mediated at least in part by enhanced Ca(2+) entry through Gd(3+) sensitive transient receptor potential channels (TRPC), and by IP3 receptors.

Trypanosoma evansi kDNA minicircle found in the Venezuelan nectar-feeding bat Leptonycteris curasoae (Glossophaginae), supports the hypothesis of multiple origins of that parasite in South America

Trypanosoma evansi is a mammal generalist protozoon which causes negative effects on health and productivity in bovine and equine herds in South America, Europe, Asia and Africa. By molecular methods, we screened the presence of that parasite together with other trypanosome species in 105 bats of 10 species collected in arid zones of northern Venezuela. The first molecular approach was fluorescent fragment length barcoding (FFLB), which relies on amplification of relative small regions of rRNA genes (four loci) and fluorescence detection. By FFLB, 17 samples showed patterns of possible trypanosomatid infections. These samples were used to test presence of trypanosomes by PCR using the following DNA markers: V7-V8 SSU rRNA, gGAPDH and kDNA minicircle regions. Only in one individual of the nectar-feeding bat, Leptonycteris curasoae, we were able to amplify 1000bp of the trypanosome kDNA minicircle. That PCR product was sequenced and the parasite species was determined by NCBI-BLAST and phylogenetic analysis. Both analyses showed that the minicircle sequence corresponds to Trypanosoma evansi. The phylogenetic analysis of the sequence obtained in this study clustered with a T. evansi sequence obtained in a Venezuelan capybara, Hydrochoerus hydrochaeris, and distant of others two T. evansi sequences obtained in a Colombian capybara and horse. This result supports the hypothesis of multiple origins of T. evansi in South America.

Evaluation of the Presence of a Thapsigargin-Sensitive Calcium Store in Trypanosomatids Using Trypanosoma evansi as a Model

Ca2+ plays an important role in the regulation of several important activities in different trypanosomatids. These parasites possess a Ca2+ transport system in the endoplasmic reticulum (ER) involved in Ca2+ homeostasis, which has been reported to be insensitive to thapsigargin, a classical inhibitor of the sarcoplasmic–ER Ca2+ adenosine triphosphatase (ATPase) (SERCA) in most eukaryotic cells. However, currently there is a controversy regarding the existence of a thapsigargin-sensitive ER Ca2+ store in these parasites. Therefore, we decided to explore the effect of this inhibitor using different methodological approaches. First, we selected Trypanosoma evansi as a parasite model to warrant the homogeneity of the population because this parasite has only a single life cycle, i.e., bloodstream-form trypomastigotes. Second, we compared the thapsigargin effect on Ca2+ homeostasis by spectrophotometrical Ca2+ measurements using 3 different approaches: whole-cell populations, cells that have been permeabilized by treatment with digitonin, and intact single cells. Our results demonstrate that a low concentration of thapsigargin induces Ca2+ release from intracellular Ca2+ stores in this parasite, which can be observed independently of the method used. Furthermore, the addition of thapsigargin before or after nigericin did not abolish its effect, showing that thapsigargin acts specifically on the ER. In conclusion, our results indicate the presence of a nonmitochondrial thapsigargin-sensitive Ca2+ store in T. evansi.

l-carnitine prevents increase in diastolic [Ca2+] induced by doxorubicin in cardiac cells

Doxorubicin is a highly effective anticancer chemotherapeutic agent that produces a dose-dependent cardiomyopathy that limits its clinical usefulness. We investigated the acute effects of doxorubicin on diastolic free Ca2+ concentrations ([Ca2+]) and the cardioprotective action of L-carnitine in isolated cardiomyocytes loaded with fura-2/AM (acetoxymethyl ester). Exposure to 10(-6)-10(-4) M doxorubicin induced an elevation of diastolic calcium concentration ([Ca2+]) that was concentration dependent. Nitrendipine failed to prevent the doxorubicin-induced elevation of [Ca2+]. Incubation with L-carnitine (10(-8) to 10(-6) M) did not modify [Ca2+]. Pretreatment of cardiomyocytes with L-carnitine 10(-8)-10(-7) M did not prevent the doxorubicin effect on [Ca2+]. However, L-carnitine 10(-6) M fully inhibited the increase in [Ca2+] induced by this anthracycline derivative. These results indicate that acute exposure to doxorubicin impairs intracellular Ca2+ handling and that L-carnitine exerts a cardioprotective effect, in part by preventing the doxorubicin-induced increase in diastolic Ca2+ concentration.

Detection of Theileria equi and Babesia caballi infections in Venezuelan horses using Competitive-Inhibition ELISA and PCR.

The focus of this study was the detection of equine piroplasmosis in Distrito Capital, Miranda, Aragua, Guárico and Apure States from Venezuela, using two methods: Competitive-Inhibition ELISA and multiplex PCR and the analysis of the possible differences in occurrence in relation to the primary purpose of the horses, which is related to varied degrees of exposure to tick. Antibody levels to Babesia caballi and Theileria equi were assessed in 694 equine serum samples using Competitive-Inhibition ELISA, while PCR assays were performed in 136 horses, using two sets of oligonucleotides to establish the presence of T. equi, B. caballi or both. The overall seroprevalence of equine piroplasmosis was 50.2%, antibodies to B. caballi were found in 161 horses (23.2%), whereas 97 (14.0%) were seropositive to T. equi and 90 (13.0%) were positives to both parasites (mixed infections). PCR determinations (n=136) showed a prevalence of 66.2%, distributed in 84 (61.8% positives) for T. equi and, 6 (4.4%) were positive to both parasites. The cELISA showed higher levels of prevalence of B. caballi and mixed infections, as compared to the PCR method. This discrepancy can be explained by the different parameters that are evaluated by each technique, PCR detect the parasite itself, while cELISA detects antibodies to the parasite. By PCR, the highest prevalence was found in Apure state, where 92.3% of the samples were positive to T. equi infections. In this locality, free grazing animals are used for livestock management. This high prevalence may be linked to the tick species present in that area. More epidemiological studies will be necessary to assess the epidemiological status of equine piroplasmosis in Venezuela.

Disfuncion de la (Ca 2+ ) diastolica en cardiomiocitos aislados de pacientes chagasicos

INTRODUCTION AND OBJECTIVES Chagas is an endemic disease in Latin America, caused by the parasite Trypanosoma cruzi, which usually affects the functioning of the heart. We have studied the regulation of intracellular calcium in cardiomyocytes isolated from chagasic patients with different degrees of heart dysfunction. METHODS Calcium selective microelectrodes were used to simultaneously measure diastolic calcium concentration ([Ca²⁺](d)) and resting membrane potential in endomyocardial biopsies obtained from chagasic patients and controls. RESULTS The [Ca²⁺](d) increased by 123%, 295%, and 738% in chagasic patients in functional class I, II, and III, respectively, in relation to controls. Membrane potential showed a partial depolarization of 6% in functional class I, 10% in functional class II, and 22% in functional class III, compared to control values. Alteration in the [Ca²⁺](d) was partially reverted by 1-[6-[[(17ß)-3-metoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione (U-73122), a β-phospholipase C antagonist, and by 2-aminoethoxydiphenyl-borate (2-APB), an inositol 1,4,5-trisphosphate receptor blocker. Phenylephrine, an agent that induces a rapid transient increase in 1,4,5-trisphosphate intracellular content, produced a rise in [Ca²⁺](d), higher in chagasic cardiomyocytes than in controls, and its effect was fully inhibited by 2-APB. CONCLUSIONS In cardiomyocytes from chagasic patients there is a dysfunction of the regulation of the [Ca²⁺](d), which correlates with the cardiac abnormalities observed in the different stages of the disease. This disturbance in the regulation of intracellular calcium appears to be associated with alterations in the regulation of intracellular messenger inositol 1,4,5-trisphosphate.

Trypanosoma evansi: pharmacological evidence of a nicotinic acetylcholine receptor.

The role of calcium and its relevance have been deeply revised with respect to trypanosomatids, as the mechanism by which calcium enters trypanosomes was, until now, not well understood. There is evidence supporting the presence of a nAChR in another member of the trypanosomatidae family, Trypanosoma cruzi, these receptors being one entry path to calcium ions. The aims of this work were to determine if there was a nicotinic acetylcholine receptor (nAChR) in Trypanosoma evansi, and to subsequently perform a partial pharmacological characterization of this receptor. After being loaded with FURA-2AM, individual cells of T. evansi, were exposed to cholinergic compounds, and the cells displayed a dose-dependent response to carbachol. This observation indicated that a cholinergic receptor may be present in T. evansi. Although a dose-dependent response to muscarine could not be demonstrated, nicotine could promote an incremental dose-dependent response. The relative potency of this specific agonist of nAChR is in agreement with previous reports. The estimated affinity values were a Kd1 value of 29.6+/-5.72 nM and a Kd2 value of 315.9+/-26.6 nM, which is similar to the Kd value reported for the alpha4 nicotinic receptor. The Hill coefficients were determined to be an n1 of 1.2+/-0.3 and an n2 of 4.2+/-1.3. Finally, our calculations indicated that there are about 1020 receptors in each T. evansi parasite, which is approximately 15-fold lower than the number reported in Torpedo californica electric cells. These results suggest the presence of a nAChR in T. evansi, which is able to bind nicotinic ligands and induce calcium signals.

Immune detection of acetylcholinesterase in subcellular compartments of Trypanosoma evansi

Trypanosoma evansi is a worldwide distributed hemoparasite with a strong economic impact in veterinary activities. Despite widespread knowledge about the etiology of the disease caused by T. evansi, there are few detailed studies about the metabolism of this parasite. The aim of this study was to determine the presence of Acetylcholinesterase (AChE) in T. evansi through a strategy of subcellular localization and confocal microscopy. The localization of the AChE by differential and isopycnic centrifugation strategy showed that this enzyme has a predominant localization in the glycosome, similar to hexokinase, and it is not present in either the cytosol or the plasma membrane. This study shows novel data that help to understand the non-neuronal role of AChE in the Trypanosomatidae family.

Whole Body Periodic Acceleration Improves Muscle Recovery after Eccentric Exercise.

INTRODUCTION The aim of this study was to determine whether whole body periodic acceleration (pGz) could improve muscle recovery after unaccustomed eccentric exercise (EE). METHODS Downhill treadmill running was used to elicit EE-induced muscle damage in mice, and pGz treatment (480 cycles per minute, 1 h·d) was applied daily for 10 d after the initial EE bout (day 0). Every 2 d during the pGz treatment course starting at day 0, we 1) assessed intracellular Ca and Na concentrations and membrane potential (as indicators of intracellular ion dysfunction) in vivo in gastrocnemius muscle from anesthetized animals and 2) quantified creatine kinase (CK), tumor necrosis factor α (TNF-α), monocyte chemoattractant protein-1 (MCP-1), interleukin-6 (IL-6), and interleukin-10 (IL-10) concentrations in plasma or muscle lysates (as indicators of muscle damage and inflammation). RESULTS EE significantly increased intracellular Ca and Na, CK, TNF-α, MCP-1, IL-6, and IL-10, all of which peaked on day 2 with the exception of IL-10 and declined slowly over 10 d of recovery. pGz treatment after the EE bout mitigated ion dyshomeostasis and expedited recuperation to control values after 6 d of treatment. pGz treatment also accelerated the normalization of CK, TNF-α, MCP-1, and IL-6 while further increasing IL-10 concentrations. The nitric oxide synthase inhibitor L-N-nitroarginine methyl ester, administered in drinking water before and maintained throughout the treatment course, was sufficient to abrogate the salutary effects of pGz after EE. CONCLUSIONS The present study demonstrates whole body periodic acceleration as an effective therapeutic strategy to accelerate muscle recovery after EE-induced skeletal muscle injury, as indicated by a faster normalization of all the studied parameters.