Efficacy of continuous versus intermittent administration of nanoformulated benznidazole during the chronic phase of Trypanosoma cruzi Nicaragua infection in mice.

BACKGROUND: Benznidazole and nifurtimox are effective drugs used to treat Chagas' disease; however, their administration in patients in the chronic phase of the disease is still limited, mainly due to their limited efficacy in the later chronic stage of the disease and to the adverse effects related to these drugs. OBJECTIVES: To evaluate the effect of low doses of nanoformulated benznidazole using a chronic model of Trypanosoma cruzi Nicaragua infection in C57BL/6J mice. METHODS: Nanoformulations were administered in two different schemes: one daily dose for 30 days or one dose every 7 days, 13 times. RESULTS: Both treatment schemes showed promising outcomes, such as the elimination of parasitaemia, a reduction in the levels of T. cruzi-specific antibodies and a reduction in T. cruzi-specific IFN-γ-producing cells, as well as an improvement in electrocardiographic alterations and a reduction in inflammation and fibrosis in the heart compared with untreated T. cruzi-infected animals. These results were also compared with those from our previous work on benznidazole administration, which was shown to be effective in the same chronic model. CONCLUSIONS: In this experimental model, intermittently administered benznidazole nanoformulations were as effective as those administered continuously; however, the total dose administered in the intermittent scheme was lower, indicating a promising therapeutic approach to Chagas' disease.

Anti-Trypanosoma cruzi effects of cyclosporin A derivatives: possible role of a P-glycoprotein and parasite cyclophilins.

Cyclophilins are target molecules for cyclosporin A (CsA), an immunosuppressive antimicrobial drug. We have previously reported the in vitro anti-Trypanosoma cruzi activity of H-7-94 and F-7-62 non-immunosuppressive CsA analogues. In this work, we continue the study of the parasiticidal effect of H-7-94 and F-7-62 CsA analogues in vitro and in vivo and we analyse 3 new CsA derivatives: MeIle-4-CsA (NIM 811), MeVal-4-CsA (MeVal-4) and D-MeAla-3-EtVal-4-CsA, (EtVal-4). The most efficient anti-T. cruzi effect was observed with H-7-94, F-7-62 and MeVal-4 CsA analogues evidenced as inhibition of epimastigote proliferation, trypomastigote penetration, intracellular amastigote development and in vivo T. cruzi infection. This trypanocidal activity could be due to inhibition of the peptidyl prolyl cis-trans isomerase activity on the T. cruzi recombinant cyclophilins tested. Furthermore, CsA and F-7-62 derivative inhibited the efflux of rhodamine 123 from T. cruzi epimastigotes, suggesting an interference with a P-glycoprotein activity. Moreover, H-7-94 and F-7-62 CsA analogues were not toxic as shown by cell viability and by aminopyrine-N-demethylase activity on mammalian cells. Our results show that H-7-94, F-7-62 and MeVal-4 CsA analogues expressed the highest inhibiting effects on T. cruzi, being promissory parasiticidal drugs worthy of further studies.

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.

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.

[Myocardial cell response to Trypanosoma cruzi infection]. / Respuesta de las células musculares cardíacas a la infeccion con Trypanosoma cruzi.

The aim of this study is to establish the response of cardiac myocytes to the infection with Trypanosoma cruzi. The role of myocardial cell proliferation on heart remodelation and the ability of these cells to produce nitric oxide and control intracellular parasite growth during T. cruzi infection were evaluated. The presence of proliferating cell nuclear antigen (PCNA) was determined in myocardial cells of Wistar rats infected with T. cruzi, resulting in a significant increase of PCNA+ labelling in all stages of disease. The ability of myocardial cells to control growth of intracellular parasites and the production of nitric oxide were evaluated in cultures of cardiac myocytes obtained from neonatal rats. Different combinations of cytokines were added to culture media. The number of cardiac cells displaying intracellular amastigotes was lower in cultures supplemented with IL-1b, TNF-a and IFN-g than with other cytokine combinations and controls. The addition of cytokines resulted also in an increase of nitric oxide production in both infected and non-infected controls. These results demonstrate that myocardial cells participate actively in the response of the heart to the infection with T. cruzi.

Embryology of Triatoma infestans (Klug), (Hemiptera, Reduviidae), a Chagas' disease vector.

This study reports the embryogenesis of T. infestans (Hemiptera, Reduviidae). Morphological parameters of growth sequences from oviposition until hatching (12-14 d 28 degrees C) were established. Five periods, as percent of time of development (TD), were characterized from oviposition until hatching. The most important morphological features were: 1) formation of blastoderm within 7% of TD; 2) germ band and gastrulation within 30% of TD; 3) nerve cord, limb budding, thoracic and abdominal segmentation and formation of body cavity within 50% of TD; 4) nervous system and blastokinesis end, and development of embryonic cuticle within 65% of TD; 5) differentiation of the mouth parts, fat body, and malpighian tubules during final stage and completion of embryo at day 12 to day 14 around hatching. These signals were chosen as appropriate morphological parameters which should enable the evaluation of embryologic modifications due to the action/s of different insecticides.

Structures and properties of vitellin isolated from Triatominae species.

A comparative study was conducted on the molecular weight, chemical composition, and immunological identity of vitellin (VN) from Triatominae species. VN is a lipoglycophosprotein: 12.76% carbohydrates, 12.53% lipids, and 0.6% phosphorus of the protein mass. Native VN shows one band of 310 kDa by gel filtration. VN shows a main band of 175 kDa by SDS-PAGE coincident with a vitellogenin (VG) band. Amino acid composition was very similar in the four species tested: T.infestans, T.pallidipennis, R.prolixus and D.maximus. T.infestans VN-VG has a total immunological identity with D.maximus female haemolymph and egg homogenates.

Structures and properties of vitellin isolated from Triatominae species.

A comparative study was conducted on the molecular weight, chemical composition, and immunological identity of vitellin (VN) from Triatominae species. VN is a lipoglycophosprotein: 12.76

carbohydrates, 12.53

lipids, and 0.6

phosphorus of the protein mass. Native VN shows one band of 310 kDa by gel filtration. VN shows a main band of 175 kDa by SDS-PAGE coincident with a vitellogenin (VG) band. Amino acid composition was very similar in the four species tested: T.infestans, T.pallidipennis, R.prolixus and D.maximus. T.infestans VN-VG has a total immunological identity with D.maximus female haemolymph and egg homogenates.

Structures and properties of vitellin isolated from Triatominae species.

A comparative study was conducted on the molecular weight, chemical composition, and immunological identity of vitellin (VN) from Triatominae species. VN is a lipoglycophosprotein: 12.76

carbohydrates, 12.53

lipids, and 0.6

phosphorus of the protein mass. Native VN shows one band of 310 kDa by gel filtration. VN shows a main band of 175 kDa by SDS-PAGE coincident with a vitellogenin (VG) band. Amino acid composition was very similar in the four species tested: T.infestans, T.pallidipennis, R.prolixus and D.maximus. T.infestans VN-VG has a total immunological identity with D.maximus female haemolymph and egg homogenates.

[Biochemistry of the development cycle of Triatoma infestans (vinchuca). X. Hemolymph lipoproteins of females]. / Bioquímica del ciclo evolutivo del Triatoma infestans (vinchuca). X. Lipoproteinas hemolinfaticas de hembras.

A lipoprotein of high density (HDL) and three of very high density (VHDL-I, VHDL-f and VHDL-II) were separated by ultracentrifugation from T. infestans female hemolymph. Lipids were mainly transported by HDL, whereas VHDL-II presented the highest protein content. In all the lipoproteins, 1,2 and 1,3-diacylglycerols, triacylglycerols, free fatty acids, cholesterol, cholesterol esters and hydrocarbons, were present. Phosphatidylcholine and phosphatidylethanolamine were also identified. The main lipids were represented by phospholipids, 1,2 and 1,3-diacylglycerols and hydrocarbons. All lipoproteins were delipidated to study the corresponding apolipoproteins. They were examined by SDS-polyacrylamide gel electrophoresis in the presence of 2-mercaptoethanol. Apo-HDL was separated into four intense polypeptide bands of approximate MW 19,000; 45,000; 86,000 and 200,000. Apo-VHDL-I was fractionated into three polypeptide bands--a major one of MW 18,500 and two minor ones of 28,000 and 45,000. The apolipoproteins belonging to VHDL-f that only appeared in females, were separated into 10 bands; the major ones corresponded to MW of 58,000, 86,000 and 160,000. In these apolipoproteins, the specimens of 58,000 and 160,000 showed positive carbohydrate reaction. Yet, in the apo-VHDL-II the most intense protein subunits primarily corresponded to bands of MW 160,000 and 86,000. Apparently, the four lipoproteins would share two polypeptide chains.

Bioquímica del ciclo evolutivo del Triatoma infestans (vinchuca). X. Lipoproteinas hemolinfaticas de hembras. / [Biochemistry of the development cycle of Triatoma infestans (vinchuca). X. Hemolymph lipoproteins of females]

A lipoprotein of high density (HDL) and three of very high density (VHDL-I, VHDL-f and VHDL-II) were separated by ultracentrifugation from T. infestans female hemolymph. Lipids were mainly transported by HDL, whereas VHDL-II presented the highest protein content. In all the lipoproteins, 1,2 and 1,3-diacylglycerols, triacylglycerols, free fatty acids, cholesterol, cholesterol esters and hydrocarbons, were present. Phosphatidylcholine and phosphatidylethanolamine were also identified. The main lipids were represented by phospholipids, 1,2 and 1,3-diacylglycerols and hydrocarbons. All lipoproteins were delipidated to study the corresponding apolipoproteins. They were examined by SDS-polyacrylamide gel electrophoresis in the presence of 2-mercaptoethanol. Apo-HDL was separated into four intense polypeptide bands of approximate MW 19,000; 45,000; 86,000 and 200,000. Apo-VHDL-I was fractionated into three polypeptide bands--a major one of MW 18,500 and two minor ones of 28,000 and 45,000. The apolipoproteins belonging to VHDL-f that only appeared in females, were separated into 10 bands; the major ones corresponded to MW of 58,000, 86,000 and 160,000. In these apolipoproteins, the specimens of 58,000 and 160,000 showed positive carbohydrate reaction. Yet, in the apo-VHDL-II the most intense protein subunits primarily corresponded to bands of MW 160,000 and 86,000. Apparently, the four lipoproteins would share two polypeptide chains.

[Biochemistry of the growth cycle of Triatoma infestans (Vinchuca). VIII. Preliminary study of hemo-lymphatic apolipoproteins in the adult male]. / Bioquímica del ciclo evolutivo del Triatoma infestans (Vinchuca). VIII. Estudio preliminar de las apolipoproteinas hemolinfaticas de machos adultos.

The three lipoproteins of high (HDL) and very high density (VHDL-I and VHDL-II) were separated from the hemolymph of adult males of T. infestans. They were delipidated and the corresponding apolipoproteins examined by sodium dodecyl sulphate (SDS) gel electrophoresis in polyacrylamide in the presence of 2-mercaptoethanol. They were also iso-electro focused in a pH gradient of 5 to 8. All three groups of apolipoproteins were separated in several polypeptide chains, but all of them had in common a glycoproteic unit of PM 86 000. The VHDL-II presented the simplest composition with predominance of the 86 000 band. Apo-HDL was the most complicated and showed intense bands of PM as high as 210 000 and as low as 17 000 together with 44 000 and 86 000 and less intense bands. The apo-VHDL-I was separated in polypeptides chains in the range of 86 000 to 17 000. The different apoproteins are apparently formed by the association at least in part of common polypeptides.

Bioquímica del ciclo evolutivo del Triatoma infestans (Vinchuca). VIII. Estudio preliminar de las apolipoproteinas hemolinfaticas de machos adultos. / [Biochemistry of the growth cycle of Triatoma infestans (Vinchuca). VIII. Preliminary study of hemo-lymphatic apolipoproteins in the adult male]

The three lipoproteins of high (HDL) and very high density (VHDL-I and VHDL-II) were separated from the hemolymph of adult males of T. infestans. They were delipidated and the corresponding apolipoproteins examined by sodium dodecyl sulphate (SDS) gel electrophoresis in polyacrylamide in the presence of 2-mercaptoethanol. They were also iso-electro focused in a pH gradient of 5 to 8. All three groups of apolipoproteins were separated in several polypeptide chains, but all of them had in common a glycoproteic unit of PM 86 000. The VHDL-II presented the simplest composition with predominance of the 86 000 band. Apo-HDL was the most complicated and showed intense bands of PM as high as 210 000 and as low as 17 000 together with 44 000 and 86 000 and less intense bands. The apo-VHDL-I was separated in polypeptides chains in the range of 86 000 to 17 000. The different apoproteins are apparently formed by the association at least in part of common polypeptides.

[Biochemistry of the developmental cycle of Triatoma infestans (Vinchuca). VI. Identification and lipid composition of hemolymph lipoproteins of adult males]. / Bioquímica del ciclo evolutivo del Triatoma infestans (Vinchuca). VI. Reconocimiento y composición lipídica de las lipoproteínas hemolinfáticas de machos adultos.

Three lipoproteins were separated from the haemolymph of adult males of Triatoma infestans fed on hen blood. The densities were similar to the high density lipoprotein (HDL) and to two very high density lipoproteins (VHDL) isolated from a pool of adult male and female insects fasted during twelve days. The relative distribution and composition of the three lipoproteins were studied. The fatty acids were mainly carried by the 1.3 and 1.2 diacylglycerols of high density lipoprotein. Triacylglycerols were minor components. Similarly to fasted insects, the main fatty acids were oleic and palmitic. Linoleic was also present. Very high density lipoproteins (VHDL-II) (d 1.25-1.26) were found in the haemolymph of male insects. The relative distribution of HDL and VHDL on fed and fasted insects was different.

Isolation and characterization of the haemolymph lipoproteins of Triatoma infestans.

1. Three lipoproteins differing in hydrated densities were isolated by ultracentrifugal procedures from the haemolymph of adult and fasted Triatoma infestans. 2. They are the high density lipoprotein HDL (d = 1.115-1.152) and the two very high density lipoproteins VHDL-I (d = 1.190-1.231) and VHDL-II (d = 1.245-1.260). HDL was the predominant lipoprotein. 3. The total lipid content expressed as % of lipoprotein weight in HDL, VHDL-I an VHDL-II was 30.1, 7.8 ad 2.9% respectively. Diacylglycerols are the predominant lipids of HDL (38.7%) and VHDL-I (27.0%) but only amount of 6.2% in VHDL-II. 4. Triacylglycerols are minor components (about 6.0%) of all fractions. 5. The phospholipids (phosphatidylethanolamine and phosphatidylcholine), sterols, sterol esters and hydrocarbons were present in all lipoprotein fractions. 6. Phospholipids and hydrocarbons were the most abundant lipids of VHDL-II.

Bioquimica del ciclo evolutivo del Triatoma infestans (Vinchuca). VI. Reconocimiento y composicion lipidica de las lipoproteinas hemolinfaticas de machos adultos. / Biochemistry of the evolutional cycle of Triatoma infestans (Vinchuca).VI. Indentification and lipid composition of hemolymph lipoproteins of adult males

Se separaron tres lipoproteinas de la hemolinfa de machos adultos de Triatoma infestans alimentados con sangre de gallina. Las densidades son similares a la lipoproteinas de alta densidad (HDL) y a las dos lipoproteinas (VHDL) de muy alta densidad reconocidas en un conjunto de insectos machos y hembras ayunados. Se estudio la composicion de los lipidos de cada una de las lipoproteinas y resulto evidente que las lipoproteinas de alta densidad son tambien los principales transportadores de acidos grasos en los insectos alimentados. El transporte se realiza principalmente por l.3 y l.2 diacilgliceroles. El contenido de triacilgliceroles es bajo. Los acidos oleico y palmitico son los acidos grasos predominantes en todos los lipidos. Sin embargo, tambien se reconocio la presencia de acido linoleico de origen exogeno. Se hallaron lipoproteinas de muy alta densidad VHDL-II (d l.25-l.26) en la hemolinfa de insectos machos. La proporcion relativa de HDL y VHDL-I en animales alimentados fue diferente de la hallada en animales ayunados

Bioquimica del ciclo evolutivo del Triatoma infestans (Vinchuca). VI. Reconocimiento y composicion lipidica de las lipoproteinas hemolinfaticas de machos adultos. / Biochemistry of the evolutional cycle of Triatoma infestans (Vinchuca).VI. Indentification and lipid composition of hemolymph lipoproteins of adult males

Se separaron tres lipoproteinas de la hemolinfa de machos adultos de Triatoma infestans alimentados con sangre de gallina. Las densidades son similares a la lipoproteinas de alta densidad (HDL) y a las dos lipoproteinas (VHDL) de muy alta densidad reconocidas en un conjunto de insectos machos y hembras ayunados. Se estudio la composicion de los lipidos de cada una de las lipoproteinas y resulto evidente que las lipoproteinas de alta densidad son tambien los principales transportadores de acidos grasos en los insectos alimentados. El transporte se realiza principalmente por l.3 y l.2 diacilgliceroles. El contenido de triacilgliceroles es bajo. Los acidos oleico y palmitico son los acidos grasos predominantes en todos los lipidos. Sin embargo, tambien se reconocio la presencia de acido linoleico de origen exogeno. Se hallaron lipoproteinas de muy alta densidad VHDL-II (d l.25-l.26) en la hemolinfa de insectos machos. La proporcion relativa de HDL y VHDL-I en animales alimentados fue diferente de la hallada en animales ayunados

Bioquímica del ciclo evolutivo del Triatoma infestans (Vinchuca). VI. Reconocimiento y composición lipídica de las lipoproteínas hemolinfáticas de machos adultos. / [Biochemistry of the developmental cycle of Triatoma infestans (Vinchuca). VI. Identification and lipid composition of hemolymph lipoproteins of adult males]

Three lipoproteins were separated from the haemolymph of adult males of Triatoma infestans fed on hen blood. The densities were similar to the high density lipoprotein (HDL) and to two very high density lipoproteins (VHDL) isolated from a pool of adult male and female insects fasted during twelve days. The relative distribution and composition of the three lipoproteins were studied. The fatty acids were mainly carried by the 1.3 and 1.2 diacylglycerols of high density lipoprotein. Triacylglycerols were minor components. Similarly to fasted insects, the main fatty acids were oleic and palmitic. Linoleic was also present. Very high density lipoproteins (VHDL-II) (d 1.25-1.26) were found in the haemolymph of male insects. The relative distribution of HDL and VHDL on fed and fasted insects was different.