Effect of aliphatic, monocarboxylic, dicarboxylic, heterocyclic and sulphur-containing amino acids on Leishmania spp. chemotaxis.

In the sand-fly mid gut, Leishmania promastigotes are exposed to acute changes in nutrients, e.g. amino acids (AAs). These metabolites are the main energy sources for the parasite, crucial for its differentiation and motility. We analysed the migratory behaviour and morphological changes produced by aliphatic, monocarboxylic, dicarboxylic, heterocyclic and sulphur-containing AAs in Leishmania amazonensis and Leishmania braziliensis and demonstrated that L-methionine (10-12 m), L-tryptophan (10-11 m), L-glutamine and L-glutamic acid (10-6 m), induced positive chemotactic responses, while L-alanine (10-7 m), L-methionine (10-11 and 10-7 m), L-tryptophan (10-11 m), L-glutamine (10-12 m) and L-glutamic acid (10-9 m) induced negative chemotactic responses. L-proline and L-cysteine did not change the migratory potential of Leishmania. The flagellum length of L. braziliensis, but not of L. amazonensis, decreased when incubated in hyperosmotic conditions. However, chemo-repellent concentrations of L-alanine (Hypo-/hyper-osmotic conditions) and L-glutamic acid (hypo-osmotic conditions) decreased L. braziliensis flagellum length and L-methionine (10-11 m, hypo-/hyper-osmotic conditions) decreased L. amazonensis flagellum length. This chemotactic responsiveness suggests that Leishmania discriminate between slight concentration differences of small and structurally closely related molecules and indicates that besides their metabolic effects, AAs play key roles linked to sensory mechanisms that might determine the parasite's behaviour.

Fructose 2,6 bisphosphate promotes the monomer-tetramer conservation of Leishmania mexicana amazonensis pyruvate kinase type two.

The enzyme pyruvate kinase of Leishmania mexicana amazonensis presents two forms with different kinetic properties and behavior for the heterotrophic activator fructose 2,6 bisphosphate. Pyruvate kinase 1, which is isolated as a tetramer, is inhibited by this metabolite. The second activity, Pyruvate kinase 2, is activated by fructose 2,6 bisphosphate, which promotes the monomer-tetramer conversion of this enzyme.

Leishmania major lipophosphoglycan modulates the phenotype and inhibits migration of murine Langerhans cells.

Langerhans cells (LC), members of the dendritic cell family, play a central role in the initiation and regulation of the immune response against the protozoan parasite Leishmania major. LC take up antigens in the skin and transport them to the regional lymph nodes for presentation to T cells. However, it is not known whether LC functions are modulated by parasite antigens. In the present study, we examined the effect of a major parasite surface molecule, L. major lipophosphoglycan (LPG), on the maturation of LC and their migratory properties. The results show that exposure to LPG did not affect the expression of major histocompatibility complex (MHC) class II and B7, but induced an up-regulation of CD25, CD31 and vascular endothelial (VE)-cadherin expression and a down-regulation of Mac-1 expression, by LC. Importantly, LPG treatment inhibited the migratory activity of LC, as it reduced their efflux from skin explants and their migration in transwell cultures. These results suggest that Leishmania LPG impairs LC migration out of the skin and thus may modulate their immunostimulatory functions, which require LC translocation from skin to lymph nodes.

Experimental leishmaniasis: the glibenclamide-triggered decrease in parasite growth correlates with changes in macrophage features.

Previous studies from our laboratories revealed the susceptibility of Leishmania sp. to glibenclamide (GLIB), a potassium channel blocker which selectively interacts with adenosine-binding-cassette transporters. In the present work, we analyzed whether the drug sensitivity of intracellular amastigotes correlates with changes in macrophage features that are related to their function as antigen-presenting cells. We provide evidence that in BALB/c murine macrophages, GLIB induced a decrease in the interferon-gamma-stimulated expression of major histocompatibility complex class II molecules and the co-stimulatory molecule CD86 (B7-2). Furthermore, it caused a decrease in the interleukin-1 secretion by macrophages. The data indicate that the treatment with GLIB inhibits the Th2 development and polarizes macrophage functions towards the induction of a protective Th1 response.

Cell differentiation and infectivity of Leishmania mexicana are inhibited in a strain resistant to an ABC-transporter blocker.

We analysed whether markers of cell differentiation and infectivity differed when compared to the parental sensitive strain [NR(Gs)] in an in vitro selected Leishmania strain [NR(Gr)] resistant to Glibenclamide, an ATP-binding-cassette (ABC)-transporter blocker. The data show that the cell body area was larger in NR(Gr) compared to NR(Gs) and that functional characters associated with an infective metacyclic phenotype, such as resistance to the lytic effect of the alternative complement pathway and expression of the Meta-1 protein, were reduced. The infectivity of NR(Gr) to J774.1 macrophages was also significantly reduced. These results suggest that resistance in Leishmania against Glibenclamide, a general blocker of P-glycoproteins, could produce functional modifications that may be relevant for Leishmania differentiation, infectivity and survival.

Experimental leishmaniasis: synergistic effect of ion channel blockers and interferon-gamma on the clearance of Leishmania major by macrophages.

We have previously shown that cultured Leishmania promastigotes are sensitive to drugs blocking K+ and Na+ channels and Na+/H+ transport systems and that the percentage of parasite-infected macrophages decreases significantly in the presence of the drugs. In the present work, we analyzed whether this drug susceptibility of intracellular amastigotes was associated with the activation of macrophage microbicidal mechanisms. Pretreatment of the cells with glibenclamide (GLIB) increased their resistance to infection with Leishmania, an effect that may be mediated by calcium fluxes since it was reversed by ethylene glycol bis-(beta-aminoethyl ether)-N,N,N',N-tetraacetic acid (EGTA). It was noteworthy that in infected macrophages post-treated with the drugs the clearance of parasites was strongly enhanced when the cells were treated simultaneously with GLIB and interferon-gamma; this effect correlated with an increased production of reactive nitrogen intermediates. In conclusion, the data suggest that GLIB treatment increases the resistance of macrophages to infection with Leishmania and potentiates the interferon-gamma-stimulated clearance of parasites via the induction of nitric oxide.

[Graduate Program on Physiological Sciences of the School of Medicine of the Central University of Venezuela: XXV years of history]. / Postgrado en Ciencias Fisiológicas de la Facultad de Medicina de la Universidad Central de Venezuela: XXV años de historia.

The Graduate Program of Physiological Sciences, the first Master's and Doctor degrees of the Faculty of Medicine from the Central University of Venezuela reached its XXV anniversary in 1998. These pages are devoted to describe and analyze the main subjects related to its growth and its pioneer role on the development of the 4th level studies in the Faculty of Medicine. Also, we discuss in these pages the plans for the future of this program.

Changes in the infectivity, pyruvate kinase activity, acid phosphatase activity and P-glycoprotein expression in glibenclamide-resistant Leishmania mexicana.

We previously demonstrated susceptibility of Leishmania sp. to glibenclamide, a K+ -ATP transport blocker which interacts with members of the superfamily of adenosine 5' triphosphate-binding cassette transporters. In order to characterize the molecular differences between a sensitive Leishmania strain, NR(Gs), and an experimentally selected glibenclamide-resistant strain, NR(Gr), specific biochemical and functional parameters have been evaluated both in the wild type and in the resistant strain. Most noteworthy, NR(Gr) exhibit an increased expression of P-glycoprotein and a decreased activity of functional key enzymes such as acid phosphatase, a prominent virulent factor of the parasite, and pyruvate kinase, a key control enzyme for both carbohydrate and protein metabolism. The specific biochemical, metabolic and functional changes observed in the resistant strain correlated with a reduced infectivity of stationary phase NR(Gr) in J774 macrophages and suggested a mechanism to overcome the effect of glibenclamide.

Sensitivity of Leishmania spp. to glibenclamide and 4-aminopyridine: a tool for the study of drug resistance development.

We have demonstrated that Leishmania spp. grown as promastigotes, are sensitive to the K+ channel inhibitors 4-aminopyridine and glibenclamide. Their host cells, the macrophages, are not affected by similar concentrations of the drugs. We have also initiated the molecular characterization of the mechanisms involved in the development of drug resistance to glibenclamide by the parasite. Therefore, we have selected experimentally and begun to characterize the Venezuelan Leishmania (Leishmania) strain, NR resistant to glibenclamide [NR(Gr)]. The analysis of genomic DNA evidenced the existence of a fragment which apparently is amplified in NR(Gr). The fragment recognized by the pgpA probe, related to the Leishmania P-glycoprotein family and which was originally isolated from L. tarentolae, showed a size polymorfism between the sensitive and the resistant strain. These results suggest that the development of resistance to glibenclamide in the strain NR(Gr) might be associated with the amplification of the ltpgpA or related gene(s).

Leishmania sp.: growth and survival are impaired by ion channel blockers.

In the present work we examined the effect of ion transport blockers on the growth and viability of Leishmania sp. and on the infection of macrophages by the parasite. 4-aminopyridine and glibenclamide block voltage-dependent and K+ ATP channels, respectively; amiloride is used to detect Na+ channels and Na+/H+ antiporters; and anthracene-9-carboxylic acid affects chloride channels. The EC50 for promastigote cultures of three strains of the Leishmania subgenus, namely, Leishmania (Leishmania) NR, Leishmania (Leishmania) amazonensis LTB0016, and Leishmania (Leishmania) major, at their stationary phase of growth, were, respectively, 39, 46, and 464 microM for 4-aminopyridine; 7, 0.8, and 10 microM for glibenclamide and 66, 170, and 10 microM for anthracene-9-carboxylic acid. The amiloride EC50 for NR was 264 microM and 10 microM for L. (L.) major, but was never reached for LTB0016. Higher concentrations of the drugs impaired the exponential growth of Leishmania promastigotes. These results suggest the susceptibility of Leishmania sp. to blockers associated with K+ and Cl- and to Na+ or Na+/H+ transport systems. Blockade of such systems might have impaired the survival of the parasites as promastigotes. In addition, it affected the persistence of parasites in host cells. Although the infection of the macrophage cell line J774 and peritoneal-exudate macrophages was not significantly decreased by concentrations of the drugs around the promastigotes' EC50, the survival of intracellular parasites decreased significantly in the presence of these drugs without affecting the viability of the macrophages. Some blockers consistently gave small EC50 and significantly decreased the infection process as well as the survival of intracellular parasites. Thus, elucidation of their mechanism of action in Leishmania is relevant, since they could represent a potential subject for the development of leishmanicidal drugs.

Isolation of two pyruvate kinase activities in the parasitic protozoan Leishmania mexicana amazonensis.

Using phosphocellulose affinity chromatography we were able to separate two pyruvate kinase (EC 2.7.1.40) activities in the parasitic protozoan Leishmania mexicana amazonesis. One activity (PYK1) showed hyperbolic kinetics and was decreased by fructose 2,6-bisphosphate, whereas the second activity (PYK2) showed sigmoidal kinetics for the substrate phosphoenolpyruvate and was activated by fructose 2,6-bisphosphate. Molecular sieve chromatography (Sephacryl S-400) of PYK1 produced a single peak of apparent molecular mass around 200,000, while PYK2 eluted at a position corresponding to M(r) 55,000.

ABC proteins in leishmania mexicana: modulation of parasite-host cell interaction

Previamente hemos demostrado la sensibilidad de cultivos de leishmania a substratos de transportadores ABC y hemos desarrollado una línea de parásitos resistente a este tipo de compuestos, [NR(Gr)]. En el presente trabajo estudiamos la influencia del desarrollo de resistencia a substratos de transportadores ABC en la interacción entre el parásito y su célula hospedera, el macrófago. Los resultados demuestran que la incorporación de [(NR(Gr)] está incrementada en macrófagos tratados con glibenclamida (GLIB) y que su sensibilidad intracelular está significativamente aumentada. La reversión en la sensibilidad de los parásitos resistentes a la GLIB pueden ser el resultado de una inestabilidad de la resistencia en el ámbito intracelular o de la modificación de la expresión de proteínas de superficie indispensables para la supervivencia del parásito en el medio intracelular. En su conjunto los resultados enfatizan la importancia de la compresión de la modulación de la quimio-resistencia a lo largo del ciclo de vida del parásito

Modulación de la expresión de moléculas co-estimulatorias y de adhesión de las células de Langerhans por el lipofosfoglicano (LPG) de leishmania major / Modulation of the molecular expression co-stimulatory and the langerhans cell adherence by lipophosfoglicane (LPG) of leishmania major

Aunque el rol inmunológico central jugado por las LC durante la infección con Leishmania se conoce ampliamente, no se sabe si el LPG afecta su maduración y migración a los nódulos linfáticos. En el trabajo aquí referido presentamos evidencias de que la exposición de LG de ratones BALB/c al LPG tiene consecuencias en su maduración y fenotipo. Luego de una incubación de 24 h en presencia de LPG 3 ug ml-1 ocurre un aumento en la expresión de móleculas de superficie como la cadena alfa del receptor de interlukina-2 (CD25), la cual ha sido implicada en procesos de activación celular y las moléculas de adhesión CD31 (PECAM-1) y VE-cadherin (VE-CAD) implicadas en cambios de la migración celular. Estos cambios ocurren sin alteraciones en la expresión del MHC-clase II. Los efectos observados pueden ser mediados por factores solubles liberados por L. major al cultivo, como es el caso del dominio de carbohidratos de LPG ya que los mismos ocurren de forma similar cuando las LC se incuban en LCM

Molecular pharmacology of chemo-resistant leishmania

Infectious diseases leishmaniosis among them, constitute a leading cause of death world wide, especially in the developing world, where they remain as an important cause of concern and has become a serious problem because of the everyday enhanced risk of co infection with HIV and the increasing frequency of resistance development of the parasites to the drug agents. Emergence of drug resistance is usually associated with changes in the expression of an specific membrane P-glycoprotein, but also includes physiological responses with high complexity. In the present review we summarize results which emphasize that the comprehesion of the molecular pharmacology of drug-resistant phenotype must include, as a way for identifying new strategies for the control of the disease, the understanding of the multiple biochemical and functional parasite mechanisms involved