Involvement of PAF (Platelet-Activating Factor) in chloroquine-induced retinopathy]

Chloroquine retinopathy is a severe toxic retinal impairment which may result in loss of vision by alterations of the pigmentary epithelium and photoreceptors. Currently, there is no specific treatment for this retinopathy. In order to test the possible involvement of Platelet-Activating Factor (PAF) in chloroquine-induced retinopathy and the use of PAF antagonists for prevention of this condition, we have examined the effect of these substances on the electroretinogram (ERG) of isolated rat retina. When retinas from normal rats were perfused with chloroquine (10(-6) M), a marked and rapid decrease in ERG b-wave amplitude was observed. In contrast, chloroquine had no effect on the ERG of retina isolated from animals pretreated with the PAF antagonist, BN 50730 (30 mg/kg/day i.p., 5 days). The results obtained indicate that (i) chloroquine is a toxic drug for retinal function, (ii) PAF plays a key role in chloroquine retinopathy and (iii) PAF antagonists may constitute valuable agents for the treatment of this retinal impairment.     

Distinct Macrophage Fates after in vitro Infection with Different Species of Leishmania: Induction of Apoptosis by Leishmania (Leishmania) amazonensis,but Not by Leishmania (Viannia) guyanensis

Leishmania is an intracellular parasite in vertebrate hosts, including man. During infection, amastigotes replicate inside macrophages and are transmitted to healthy cells, leading to amplification of the infection. Although transfer of amastigotes from infected to healthy cells is a crucial step that may shape the outcome of the infection, it is not fully understood. Here we compare L. amazonensis and L. guyanensis infection in C57BL/6 and BALB/c mice and investigate the fate of macrophages when infected with these species of Leishmania in vitro. As previously shown, infection of mice results in distinct outcomes: L. amazonensis causes a chronic infection in both strains of mice (although milder in C57BL/6), whereas L. guyanensis does not cause them disease. In vitro, infection is persistent in L. amazonensis-infected macrophages whereas L. guyanensis growth is controlled by host cells from both strains of mice. We demonstrate that, in vitro, L. amazonensis induces apoptosis of both C57BL/6 and BALB/c macrophages, characterized by PS exposure, DNA cleavage into nucleosomal size fragments, and consequent hypodiploidy. None of these signs were seen in macrophages infected with L. guyanensis, which seem to die through necrosis, as indicated by increased PI-, but not Annexin V-, positive cells. L. amazonensis-induced macrophage apoptosis was associated to activation of caspases-3, -8 and -9 in both strains of mice. Considering these two species of Leishmania and strains of mice, macrophage apoptosis, induced at the initial moments of infection, correlates with chronic infection, regardless of its severity. We present evidence suggestive that macrophages phagocytize L. amazonensis-infected cells, which has not been verified so far. The ingestion of apoptotic infected macrophages by healthy macrophages could be a way of amastigote spreading, leading to the establishment of infection.     

Modulation of Leishmania (L.) amazonensis Growth in Cultured Mouse Macrophages by Prostaglandins and Platelet Activating Factor

The role of endogenously synthesized PAF and prostaglandins on the infection of mouse macrophages by Letsbmanta (L.) amazonensis was investigated, as well as the possible correlation between the effects of these inflammatory mediators with nitric oxide production. It was found that pretreatment of macrophages with 10(-5) M of the PAF antagonists, BN-52021 or WEB-2086, increased macrophage infection by 17 and 59%, respectively. The cyclooxygenase inhibitor, indomethacin (10 mug/ml), induced a significant inhibition which was reversed by addition of PGE (10-3 M) to the culture medium. These results suggested that the infection of macrophages by leisbmanla is inhibited by PAF and enhanced by prostaglandins and that these mediators are produced by macrophages during this infection. This was confirmed by addition of these mediators to the culture medium before infection; PAF (10(-6), 10(-9) and 10(-12)M) reduced significantly the infection whereas PGE(2) (10(-5) M) induced a marked enhancement. This effect of exogenous PAF on macrophage infection was reversed by the two PAF antagonists used in this study as well as by the inhibitor of nitric oxide synthesis, L-arginine methyl ester (100 mM). Taken together the data suggest that endogenous production of PAF and PGE(2) exert opposing effects on Lesbmana-macrophage interaction and that nitric oxide may be involved in the augmented destruction of parasites induced by PAF.     

Free Radicals Scavengers Attenuate Platelet-Activating Factor (PAF)-And Endotoxin-Induced Intestinal Myoelectric Disturbances in Rats

Pretreatment with radical scavengers significantly reduced the intestinal myoelectric disturbances following either E. coli endotoxin or platelet-activating factor (PAF) injection in the rat indicating that free radicals might be involved in the intestinal motor alterations observed in endotoxin shock and that PAF acts partially via free radical production. Moreover, dimethylsulfoxide (DMSO) was found to be more effective in inhibiting the endoxotin-induced intestinal motor alterations, than superoxide dismutase (SOD) and allopurinol. BN 52021, a specific PAF antagonist, was able to reduce the effects of endotoxin on intestinal motility, However, when BN 52021 was combined with free radical scavengers, no additive effect was observed. It is concluded that free radicals involved in endotoxin-induced intestinal motility alterations are at least in part produced in response to PAF.     

Active Transport of Glutamate in Leishmania (Leishmania) amazonensis

ABSTRACT. Leishmania spp. are the causative agents of leishmaniasis, a complex of diseases with a broad spectrum of clinical manifestations. Leishmania (Leishmania) amazonensis is a main etiological agent of diffuse cutaneous leishmaniasis. Leishmania spp., as other trypanosomatids, possess a metabolism based significantly on the consumption of amino acids. However, the transport of amino acids in these organisms remains poorly understood with few exceptions. Glutamate transport is an important biological process in many organisms. In the present work, the transport of glutamate is characterized. This process is performed by a single kinetic system (K_m=0.59±0.04 mM, V_max=0.123±0.003 nmol/min per 20 × 10⁶ cells) showing an energy of activation of 52.38±4.7 kJ/mol and was shown to be partially inhibited by analogues, such as glutamine, aspartate, α‐ketoglutarate and oxaloacetate, methionine, and alanine. The transport activity was sensitive to the extracellular concentration of H⁺ but not to Na⁺ or K⁺. However, unlike other amino acid transporters presently characterized, the treatment with specific ionophores confirmed the participation of a K⁺, and not H⁺ membrane gradient in the transport process.     

Characterization of Leishmania (Leishmania) amazonensis promastigotes resistant to pentamidine

Pentamidine is a second-line agent used in the treatment of leishmaniasis and its mode of action and mechanism of resistance is not well understood. It was previously demonstrated that transfection of promastigotes and amastigotes with the ABC transporter PRP1 gene confers resistance to pentamidine. To further clarify this point, we generated Leishmania amazonensis mutants resistant to pentamidine. Our results indicated that this ABC transporter is not associated with pentamidine resistance in lines generated by drug pressure through amplification or overexpression mechanisms of PRP1 gene.     

转移因子对小鼠腹腔巨噬细胞吞噬功能的影响和E玫瑰花形成的作用

本文报道了转移因子对小鼠腹腔巨噬细胞吞噬功能的影响和对脾细胞E玫瑰花形成的作用。转移因子为本单位从健康猪脾细胞提取的针剂,含多核苷酸和多肽等低分子生物活性物质,每支含量为3×10~3个脾细胞提取物,每天一次0.5ml剂量注入小鼠体内,连续5次后取动物腹腔巨噬细胞和脾细胞悬液,测定其吞噬功能并观察E玫瑰花形成作用。结果表明,转移因子对小鼠腹腔巨噬细胞吞噬的百分率和吞噬指数与对照组比有明显差异(P<0.01),对小鼠脾细胞E玫瑰花形成作用与对照组比差异也极显著(P<0.01)。从而看出,转移因子能使小鼠腹腔巨噬细胞吞噬功能增强,亦能使特异的玫瑰花形成细胞中T淋巴细胞增多,增强了机体的免疫功能。     

The leishmanicidal flavonols quercetin and quercitrin target Leishmania (Leishmania) amazonensis arginase

Polyamine biosynthesis enzymes are promising drug targets for the treatment of leishmaniasis, Chagas' disease and African sleeping sickness. Arginase, which is a metallohydrolase, is the first enzyme involved in polyamine biosynthesis and converts arginine into ornithine and urea. Ornithine is used in the polyamine pathway that is essential for cell proliferation and ROS detoxification by trypanothione. The flavonols quercetin and quercitrin have been described as antitrypanosomal and antileishmanial compounds, and their ability to inhibit arginase was tested in this work. We characterized the inhibition of recombinant arginase from Leishmania (Leishmania) amazonensis by quercetin, quercitrin and isoquercitrin. The IC(50) values for quercetin, quercitrin and isoquercitrin were estimated to be 3.8, 10 and 4.3 μM, respectively. Quercetin is a mixed inhibitor, whereas quercitrin and isoquercitrin are uncompetitive inhibitors of L. (L.) amazonensis arginase. Quercetin interacts with the substrate l-arginine and the cofactor Mn(2+) at pH 9.6, whereas quercitrin and isoquercitrin do not interact with the enzyme's cofactor or substrate. Docking analysis of these flavonols suggests that the cathecol group of the three compounds interact with Asp129, which is involved in metal bridge formation for the cofactors Mn(A)(2+) and Mn(B)(2+) in the active site of arginase. These results help to elucidate the mechanism of action of leishmanicidal flavonols and offer new perspectives for drug design against Leishmania infection based on interactions between arginase and flavones.     

A serine protease from a detergent-soluble extract of Leishmania (Leishmania) amazonensis

Proteases mediate important crucial functions in parasitic diseases, and their characterization contributes to the understanding of host-parasite interaction. A serine protease was purified about 43-fold with a total recovery of 60% from a detergent-soluble extract of promastigotes of Leishmania amazonensis. The purification procedures included aprotinin-agarose affinity chromatography and gel filtration high performance liquid chromatography. The molecular mass of active enzyme was 110 kDa by native gel filtration HPLC and by SDS-PAGE gelatin under non-reducing conditions. Under conditions of reduction using SDS-PAGE gelatin analyses the activity of enzyme was observed in two proteins of 60 and 45 kDa, suggesting that the enzyme may be considered as a dimer. The Leishmania protease was not glycosylated, and its isoelectric point (pI) was around 4.8. The maximal protease activity was at pH 7.0 and 28 degrees C, using a-N-o-tosyl-L-arginyl-methyl ester (L-TAME) as substrate. Assays of thermal stability indicated that this enzyme was totally denatured after pre-treatment at 42 degrees C for 12 min and preserved only 20% of its activity after pre-treatment at 37 degrees C for 24 h, in the absence of substrate. Hemoglobin, bovine serum albumin (BSA), ovalbumin and gelatin were hydrolyzed by Leishmania protease. Inhibition studies indicated that the enzyme belonged to a serine protease class because of a significant impediment by serine protease inhibitors such as benzamidine, aprotinin, and antipain. The activity of the present serine protease is negatively modulated by calcium and zinc and positively modulated by manganese ions. This is the first study that reports the purification of a protease from a detergent-soluble extract of Leishmania species.     

Genomic organization of telomeric and subtelomeric sequences of Leishmania (Leishmania) amazonensis

Telomeres are DNA-protein complexes that protect linear chromosomes from degradation and fusions. Telomeric DNA is repetitive and G-rich, and protrudes towards the end of the chromosomes as 3'G-overhangs. In Leishmania spp., sequences adjacent to telomeres comprise the Leishmania conserved telomere associated sequences (LCTAS) that are around 100 bp long and contain two conserved sequence elements (CSB1 and CSB2), in addition to non-conserved sequences. The aim of this work was to study the genomic organization of Leishmania (Leishmania) amazonensis telomeric/subtelomeric sequences. Leishmania amazonensis chromosomes were separated in a single Pulsed Field Gel Electrophoresis (PFGE) gel as 25 ethidium bromide-stained bands. All of the bands hybridized with the telomeric probe (5'-TTAGGG-3')3 and with probes generated from the conserved subtelomeric elements (CSB1, CSB2). Terminal restriction fragments (TRF) of L. amazonensis chromosomes were analyzed by hybridizing restriction digested genomic DNA and chromosomal DNA separated in 2D-PFGE with the telomeric probe. The L. amazonensis TRF was estimated to be approximately 3.3 kb long and the telomeres were polymorphic and ranged in size from 0.2 to 1.0 kb. Afa I restriction sites within the conserved CSB1 elements released the telomeres from the rest of the chromosome. Bal 31-sensitive analysis confirmed the presence of terminal Afa I restriction sites and served to differentiate telomeric fragments from interstitial internal sequences. The size of the L. amazonensis 3' G-overhang was estimated by non-denaturing Southern blotting to be approximately 12 nt long. Using similar approaches, the subtelomeric domains CSB1 and CSB2 were found to be present in a low copy number compared to telomeres and were organized in blocks of 0.3-1.5 kb flanked by Hinf I and Hae III restriction sites. A model for the organization of L. amazonensis chromosomal ends is provided.     

Analysis and chromosomal mapping of Leishmania (Leishmania) amazonensis amastigote expressed sequence tags

The characterization of expressed sequence tags (ESTs) generated from a cDNA library of Leishmania (Leishmania) amazonensis amastigotes is described. The sequencing of 93 clones generated new L. (L.) amazonensis ESTs from which 32% are not related to any other sequences in database and 68% presented significant similarities to known genes. The chromosome localization of some L. (L.) amazonensis ESTs was also determined in L. (L.) amazonensis and L. (L.) major. The characterization of these ESTs is suitable for the genome physical mapping, as well as for the identification of genes encoding cysteine proteinases implicated with protective immune responses in leishmaniasis.     

Leishmania (Leishmania) amazonensis: purification and characterization of a promastigote serine protease

Pathogenic protozoan proteases play crucial roles in the host-parasite interaction, and its characterization contributes to the understanding of protozoan disease mechanisms. A Leishmania amazonensis promastigote protease was purified 36-fold, using aprotinin-agarose affinity chromatography and gel filtration high performance liquid chromatography, yielding a total recovery of 49%. The molecular mass of active enzyme obtained from native gel filtration HPLC and SDS-PAGE under conditions of reduction and non-reduction was 68 kDa, suggesting that the enzyme may exist as a monomer. The protease isoelectric point (pI) was around 4.45 and, as demonstrated by deglycosylation assay, it did not have any carbohydrate content. The optimal pH and temperature of the enzyme were 8.0 and 28 degrees C, respectively, determined using alpha-N-rho-tosyl-L-arginyl-methyl ester (L-TAME) as substrate. Assays of thermal stability indicated that 50% of the enzymatic activity was preserved after 4 min of pre-treatment at 42 degrees C and after 24 h of pre-treatment at 37 degrees C, both in the absence of substrate. Hemoglobin, bovine serum albumin (BSA), ovalbumin, and both gelatin and peptide substrates containing arginine in ester bound were hydrolyzed by 68 kDa protease. The insulin beta-chain was also hydrolyzed by the protease, and four peptidic bonds (L11-V12, E13-A14, L15-Y16, and Y16-L17) were susceptible to the 68-kDa protease action. Inhibition studies suggested that the enzyme belonged to a serine protease class inhibited by calcium ions and activated by manganese ions. These findings demonstrate that the L. amazonensis 68-kDa serine protease differs from those of other protozoan parasites.     

Action of pentoxifylline on experimental cutaneous leishmaniasis due to Leishmania (Leishmania) amazonensis

In the animal model of leishmaniasis caused by Leishmania (Leishmania) amazonensis there is a complex mechanism of the host-parasite interaction. The present study was performed to interfere with the inflammatory reaction to the parasites, through immune modulation. Female C5BL/6 isogenic mice were used, some of which were inoculated on the right ear and others on the right footpad with 3.10(6) stationary phase promastigotes of the MHOM/BR/PH8 strain of L. (L.) amazonensis, and were allocated in three groups: the first received pentoxifylline 8mg/kg every 12 h, since the first day; the second one received the same dose since the 40th day of infection and a control group that did not receive any treatment. All the ears excised were analyzed to determine the variation in weight between both ears and for histopathological analyses. A quantification of the parasites was done using the limiting dilution assay. A significant reduction of the number of parasites, was observed among the animals treated which had an accordingly significant reduction on the weight of the ears. Pentoxifylline reduced the macrophages propensity to vacuolation and induced a more effective destruction of the parasites by these cells. Moreover, the group that began the treatment later did not show the same effectiveness.     

Platelet-Activating Factor Modulates a Secreted Phosphatase Activity of the Trypanosomatid Parasite Herpetomonas muscarum muscarum

In the present work we characterized the secreted phosphatase activity of the trypanosomatid parasite Herpetomonas muscarum muscarum. This housefly parasite hydrolyzed p-nitrophenylphosphate at a rate of 10.26 nmol Pi/mg protein/min. Classical inhibitors of acid phosphatases, such as sodium orthovanadate (N_aVO₃), sodium fluoride (NaF), and ammonium molybdate promoted a decrease in this phosphatase activity. When the parasites were assayed in the presence of sodium tartrate, an inhibitor of Leishmania spp-secreted acid phosphatases, this activity was drastically diminished. Cytochemical analysis showed the localization of this enzyme on the external surface and in the flagellar pocket of these parasites. Sodium tartrate inhibited this reaction, confirming the biochemical data. Platelet-activating factor (PAF) inhibited the phosphatase activity determined in the supernatant of living H. m. muscarum. Received: 2 February 2001 / Accepted: 5 March 2001     

Leishmania (Leishmania) amazonensis: experimental cutaneous leishmaniasis associated with systemic amyloidosis in mice

We infected Swiss and C57BL/6 female mice in the left hind footpad with 10⁴Leishmania (L.) amazonensis promastigotes in stationary phase. The macroscopic examination showed a nodular non-ulcerated lesion at the site of inoculation and hepatic and spleenic enlargement. Histopathologically, the primary lesion showed an extensive liquefactive necrosis and inflammatory infiltrate, mainly consisting of macrophages filled with amastigotes, and rare lymphocytes. The inflammatory reaction in liver, spleen and kidney showed amyloid deposits. Additionally, C57BL/6 had accentuated amyloidosis in both ovarian cortical and medullar region and inflammatory infiltrates in the pancreas and adrenal gland.     

Dolabelladienetriol, a Compound from Dictyota pfaffii Algae, Inhibits the Infection by Leishmania amazonensis

Background

Chemotherapy for leishmaniasis, a disease caused by Leishmania parasites, is expensive and causes side effects. Furthermore, parasite resistance constitutes an increasing problem, and new drugs against this disease are needed. In this study, we examine the effect of the compound 8,10,18-trihydroxy-2,6-dolabelladiene (Dolabelladienetriol), on Leishmania growth in macrophages. The ability of this compound to modulate macrophage function is also described.

Methodology/Principal Findings

Leishmania-infected macrophages were treated with Dolabelladienetriol, and parasite growth was measured using an infectivity index. Nitric oxide (NO), TNF-α and TGF-β production were assayed in macrophages using specific assays. NF-kB nuclear translocation was analyzed by western blot. Dolabelladienetriol inhibited Leishmania in a dose-dependent manner; the IC₅₀ was 44 µM. Dolabelladienetriol diminished NO, TNF-α and TGF-β production in uninfected and Leishmania-infected macrophages and reduced NF-kB nuclear translocation. Dolabelladienetriol inhibited Leishmania infection even when the parasite growth was exacerbated by either IL-10 or TGF-β. In addition, Dolabelladienetriol inhibited Leishmania growth in HIV-1-co-infected human macrophages.

Conclusion

Our results indicate that Dolabelladienetriol significantly inhibits Leishmania in macrophages even in the presence of factors that exacerbate parasite growth, such as IL-10, TGF-β and HIV-1 co-infection. Our results suggest that Dolabelladienetriol is a promising candidate for future studies regarding treatment of leishmaniasis, associated or not with HIV-1 infection.     

Leishmania amazonensis Infection Induces Changes in the Electrophysiological Properties of Macrophage-like Cells

Whole cell patch-clamp recordings were used to study the electrical properties of the macrophage-like cell line J774.1, after infection with Leishmania amazonensis. Infection induced a significant increase in cell size and membrane capacitance, suggesting that parasite invasion leads to the addition of plasma membrane to the host cell. By 24 hr after infection, the host cell membrane potential was significantly more hyperpolarized than control cells, and this difference remained for the subsequent 72 hr post-infection. The hyperpolarization was paralleled by an increase in the density of inward rectifying K⁺ currents. The shape of the conductance vs. voltage curve, the kinetic properties and the pharmacological profile of these currents were not significantly altered by infection. These results suggest that infection by L. amazonensis causes an increase in the number of functional inward rectifying K⁺ channels, leading to hyperpolarization of the host cell membrane. Received: 19 January 1999/Revised: 20 April 1999     

Experimental infection with Leishmania (Viannia) braziliensis and Leishmania (Leishmania) amazonensis in the marmoset, Callithrix penicillata (Primates:Callithricidae)

Fourteen marmosets (Callithrix penicillata) were inoculated intradermally with promastigotes and/or amastigotes of Leishmania (Viannia) braziliensis (L. (V) b.) strains MHOM/BR/83/LTB-300 MHOM/BR/85/LTB-12 MHOM/BR/81/LTB-179 and MHOM/BR/82/LTB-250. The evolution of subsequent lesions was studied for 15 to 75 weeks post-inoculation (PI). All but 3 of the L. (V) b. injected marmosets developed a cutaneous lesion at the point of inoculation after 3 to 9 weeks, characterized by the appearance of subcutaneous nodules containing parasites. Parasites were isolated by culture (Difco Blood Agar) from all 11 positive animals. The maximum size of the lesions was variable and ranged between 37 mm2 to 107 mm2. Ulceration of primary nodules became evident after 3 to 12 weeks in all infected marmosets, but was faster and larger in 5 of the 11 animals. The active lesions persisted in 9 out of 11 Callithrix until the end of the observation period, which varied from 15-75 weeks. In 3 animals spontaneous healing of their lesions (13 to 25 weeks, PI) was observed but with cryptic parasitism. In another 2 infected animals there was regression followed by reactivation of the cutaneous lesions. The appearance of smaller satellite lesions adjacent to primary ones, as well as metastatic lesions to the ear lobes, were documented in 2 animals. Promastigotes of L. (Leishmania) amazonensis (L. (L) a.) MHOM/BR/77/LTB-16 were inoculated in 1 marmoset. This animal remained chronically infected for 6 months and the lesion developed in a similar manner to L. (V) b. infected marmosets. No significant differences in clinical and parasitological behaviour were observed between promastigote or amastigote derived infections of the 2 species.(ABSTRACT TRUNCATED AT 250 WORDS)