Scavenger receptors mediate increased uptake of irradiated T.gondii extracts by J774 macrophages.

PURPOSE: Protein extracts developed increased immunogenicity without the aid of adjuvants after gamma irradiation. Gamma irradiation of snake venom increased antivenin production by detoxification and enhanced immunity, probably due preferential uptake of irradiated venoms by macrophage scavenger receptors. We studied this uptake of irradiated soluble Toxoplasma gondii extract (STag) by the J774 macrophage cell line similar to antigen presenting cells. MATERIAL AND METHODS: We labeled STag by biosynthesis in living tachyzoites with radioactive amino acids before purification and irradiation or by adding labels as biotin or fluorescein in stored STag, for quantitative studies or subcellular distribution visualization. RESULTS: There was enhanced binding and uptake of irradiated STag into the cells compared to non-irradiated STag. Using fluorescein labeled antigens and morphological assays, we confirmed that cells avidly ingested both native and irradiated proteins but native STag were digested after ingestion while irradiated proteins remained in the cell, suggesting diverse intracytoplasmic pathways. Native or irradiated STag present the same in vitro sensitivity to three types of peptidases. Inhibitors of scavenger receptors (SRs) such as Dextran sulfate (SR-A1 blocker) or Probucol (SR-B blocker) affect the specific uptake of irradiated antigens, suggesting its association with enhanced immunity. CONCLUSIONS: Our data suggests that cell SRs recognize irradiated proteins, mainly SRs for oxidized proteins, leading to antigen uptake by an intracytoplasmic pathway with fewer peptidases that prolongs presentation to nascent major histocompatibility complex I or II and enhances immunity by better antigen presentation.

Radiation effects on Toxoplasma antigens: different immune responses of irradiated intact tachyzoites or soluble antigens in experimental mice models.

Purpose: Purpose: Protein irradiation causes aggregation, chain breakage, and oxidation, enhancing its uptake by antigen-presenting cells. To evaluate if irradiated proteins participate on the protection, we studied the immune response induced in mice immunized with irradiated soluble extracts of T. gondii tachyzoites (STag) or irradiated intact T. gondii RH tachyzoites (RH0.25 kGy).Material and Methods: Soluble extracts of Toxoplasma gondii tachyzoites (STag) were irradiated at different dose by Cobalt-60 source. By polyacrylamide gel electrophoresis (SDS-Page) we evaluated the effects on primary structures of protein STags induced by irradiation. By Enzyme-linked Immunosorbent Assay (ELISA) we evaluated the difference between humoral immune response induced by irradiated STag or RH tachyzoites in immunized mice from the detection of specific immunoglobulin G (IgG) antibodies in the serum of immunized mice. From challenge with viable RH strain of T. gondii we evaluated the protection induced in the immunized animals. By cytometry we performed the phenotyping of T and B lymphocytes in the peripheral blood of the immunized animals.Results: Irradiation dose of 1.5 kGy induced minimal changes in most proteins, without affecting their antigenicity or immunogenicity. Immunization showed saturation at the dose of 10 µg/mice, with worst response at higher doses. STag irradiated at 1.5 kGy (STag1.5 kGy) induced higher survival and protection similar to T. gondii RH strain irradiated at 0.25 kGy (RH0.25 kGy), with higher serum levels of high affinity IgG compared to STag native. Blood immune memory cells of mice immunized with STag1.5 kGy had higher proportions of CD19+ (cluster of differentiation 19) and CD4+ (cluster of differentiation 14) cells, whereas mice RH0.25 kGy had high proportion of memory CD8+ (cluster of differentiation 8) cells.Conclusions: Our data suggest that major histocompatibility complex type I (MHCI) pathway, appears seem to be used by RH0.25 kGy to generate cytotoxic cells while STag1.5 kGy uses a major histocompatibility complex type II (MHCII) pathway for B-cell memory, but both induce sufficient immune response for protection in mice without any adjuvant. Irradiation of soluble protein extracts enhances their immune response, allowing similar protection against T. gondii in mice as compared to irradiated intact parasites.

Pharmacokinetics of neutron-irradiated meglumine antimoniate in Leishmania amazonensis-infected BALB/c mice

Cutaneous leishmaniasis (CL) is a parasitic disease caused by the protozoan Leishmania spp. Pentavalent antimonial agents have been used as an effective therapy, despite their side effects and resistant cases. Their pharmacokinetics remain largely unexplored. This study aimed to investigate the pharmacokinetic profile of meglumine antimoniate in a murine model of cutaneous leishmaniasis using a radiotracer approach. Methods: Meglumine antimoniate was neutron-irradiated inside a nuclear reactor and was administered once intraperitoneally to uninfected and L. amazonensis-infected BALB/c mice. Different organs and tissues were collected and the total antimony was measured. Results: Higher antimony levels were found in infected than uninfected footpad (0.29% IA vs. 0.14% IA, p = 0.0057) and maintained the concentration. The animals accumulated and retained antimony in the liver, which cleared slowly. The kidney and intestinal uptake data support the hypothesis that antimony has two elimination pathways, first through renal excretion, followed by biliary excretion. Both processes demonstrated a biphasic elimination profile classified as fast and slow. In the blood, antimony followed a biexponential open model. Infected mice showed a lower maximum concentration (6.2% IA/mL vs. 11.8% IA/mL, p = 0.0001), a 2.5-fold smaller area under the curve, a 2.7-fold reduction in the mean residence time, and a 2.5-fold higher clearance rate when compared to the uninfected mice. Conclusions: neutron-irradiated meglumine antimoniate concentrates in infected footpad, while the infection affects antimony pharmacokinetics.(AU)

Antimonial drugs entrapped into phosphatidylserine liposomes: physicochemical evaluation and antileishmanial activity.

INTRODUCTION: Leishmaniasis is a disease caused by the protozoan Leishmania that resides mainly in mononuclear phagocytic system tissues. Pentavalent antimonials are the main treatment option, although these drugs have toxic side effects and high resistance rates. A potentially alternative and more effective therapeutic strategy is to use liposomes as carriers of the antileishmanial agents. The aims of this study were to develop antimonial drugs entrapped into phosphatidylserine liposomes and to analyze their biological and physicochemical characteristics. METHODS: Liposomes containing meglumine antimoniate (MA) or pentavalent antimony salt (Sb) were obtained through filter extrusion (FEL) and characterized by transmission electron microscopy. Promastigotes of Leishmania infantum were incubated with the drugs and the viability was determined with a tetrazolium dye (MTT assay). The effects of these drugs against intracellular amastigotes were also evaluated by optical microscopy, and mammalian cytotoxicity was determined by an MTT assay. RESULTS: Liposomes had an average diameter of 162nm. MA-FEL showed inhibitory activity against intracellular L. infantum amastigotes, with a 50% inhibitory concentration (IC50) of 0.9µg/mL, whereas that of MA was 60µg/mL. Sb-FEL showed an IC50 value of 0.2µg/mL, whereas that of free Sb was 9µg/mL. MA-FEL and Sb-FEL had strong in vitro activity that was 63-fold and 39-fold more effective than their respective free drugs. MA-FEL tested at a ten-times higher concentration than Sb-FEL did not show cytotoxicity to mammalian cells, resulting in a higher selectivity index. CONCLUSIONS: Antimonial drug-containing liposomes are more effective against Leishmania-infected macrophages than the non-liposomal drugs.

Antimonial drugs entrapped into phosphatidylserine liposomes: physicochemical evaluation and antileishmanial activity

Abstract: INTRODUCTION: Leishmaniasis is a disease caused by the protozoan Leishmania that resides mainly in mononuclear phagocytic system tissues. Pentavalent antimonials are the main treatment option, although these drugs have toxic side effects and high resistance rates. A potentially alternative and more effective therapeutic strategy is to use liposomes as carriers of the antileishmanial agents. The aims of this study were to develop antimonial drugs entrapped into phosphatidylserine liposomes and to analyze their biological and physicochemical characteristics. METHODS: Liposomes containing meglumine antimoniate (MA) or pentavalent antimony salt (Sb) were obtained through filter extrusion (FEL) and characterized by transmission electron microscopy. Promastigotes of Leishmania infantum were incubated with the drugs and the viability was determined with a tetrazolium dye (MTT assay). The effects of these drugs against intracellular amastigotes were also evaluated by optical microscopy, and mammalian cytotoxicity was determined by an MTT assay. RESULTS: Liposomes had an average diameter of 162nm. MA-FEL showed inhibitory activity against intracellular L. infantum amastigotes, with a 50% inhibitory concentration (IC50) of 0.9μg/mL, whereas that of MA was 60μg/mL. Sb-FEL showed an IC50 value of 0.2μg/mL, whereas that of free Sb was 9μg/mL. MA-FEL and Sb-FEL had strong in vitro activity that was 63-fold and 39-fold more effective than their respective free drugs. MA-FEL tested at a ten-times higher concentration than Sb-FEL did not show cytotoxicity to mammalian cells, resulting in a higher selectivity index. CONCLUSIONS: Antimonial drug-containing liposomes are more effective against Leishmania-infected macrophages than the non-liposomal drugs.

Biodistribution of meglumine antimoniate in healthy and Leishmania (Leishmania) infantum chagasi-infected BALB/c mice.

Pentavalent antimonials such as meglumine antimoniate (MA) are the primary treatments for leishmaniasis, a complex disease caused by protozoan parasites of the genus Leishmania . Despite over 70 years of clinical use, their mechanisms of action, toxicity and pharmacokinetics have not been fully elucidated. Radiotracer studies performed on animals have the potential to play a major role in pharmaceutical development. The aims of this study were to prepare an antimony radiotracer by neutron irradiation of MA and to determine the biodistribution of MA in healthy and Leishmania (Leishmania) infantum chagasi-infected mice. MA (Glucantime®) was neutron irradiated inside the IEA-R1 nuclear reactor, producing two radioisotopes, ¹²²Sb and ¹²4Sb, with high radionuclidic purity and good specific activity. This irradiated compound presented anti-leishmanial activity similar to that of non-irradiated MA in both in vitro and in vivo evaluations. In the biodistribution studies, healthy mice showed higher uptake of antimony in the liver than infected mice and elimination occurred primarily through biliary excretion, with a small proportion of the drug excreted by the kidneys. The serum kinetic curve was bi-exponential, with two compartments: the central compartment and another compartment associated with drug excretion. Radiotracers, which can be easily produced by neutron irradiation, were demonstrated to be an interesting tool for answering several questions regarding antimonial pharmacokinetics and chemotherapy.

Biodistribution of meglumine antimoniate in healthy and Leishmania (Leishmania) infantum chagasi-infected BALB/c mice

Pentavalent antimonials such as meglumine antimoniate (MA) are the primary treatments for leishmaniasis, a complex disease caused by protozoan parasites of the genus Leishmania . Despite over 70 years of clinical use, their mechanisms of action, toxicity and pharmacokinetics have not been fully elucidated. Radiotracer studies performed on animals have the potential to play a major role in pharmaceutical development. The aims of this study were to prepare an antimony radiotracer by neutron irradiation of MA and to determine the biodistribution of MA in healthy and Leishmania (Leishmania) infantum chagasi-infected mice. MA (Glucantime(r)) was neutron irradiated inside the IEA-R1 nuclear reactor, producing two radioisotopes, 122Sb and 124Sb, with high radionuclidic purity and good specific activity. This irradiated compound presented anti-leishmanial activity similar to that of non-irradiated MA in both in vitro and in vivo evaluations. In the biodistribution studies, healthy mice showed higher uptake of antimony in the liver than infected mice and elimination occurred primarily through biliary excretion, with a small proportion of the drug excreted by the kidneys. The serum kinetic curve was bi-exponential, with two compartments: the central compartment and another compartment associated with drug excretion. Radiotracers, which can be easily produced by neutron irradiation, were demonstrated to be an interesting tool for answering several questions regarding antimonial pharmacokinetics and chemotherapy.

A simple immune complex dissociation ELISA for leishmaniasis: standardization of the assay in experimental models and preliminary results in canine and human samples.

Visceral leishmaniasis, caused by Leishmania (Leishmania) chagasi, is a chronic parasitic disease of humans and dogs. Confirmation of the protozoal agent in bone marrow, lymph node or spleen aspirate is diagnostic, while specific-IgG serology is used mainly for epidemiology despite the general presence of high levels of serum immunoglobulin. Anecdotal reports of false-negative serology in active disease cases are known and are ascribed to the formation of immune complexes. Because dissociation of immune complexes can be accomplished by acid treatment, we devised a simple, routine enzyme immunoassay (ELISA) for the dissociation of immune complexes in serum samples using acid treatment in wells adsorbed with Leishmania antigen (dELISA). Confirmatory acid dot-blot was also developed for antigen detection by anti-Leishmania rabbit antiserum. In experimental L. chagasi hamster models, immune complexes interfered with ELISA mostly in the 30 and 60 days postinfection, according to both dELISA and antigen dot-blot results. In larger samples from endemic areas, dELISA was positive in 10% of seronegative dog samples (7/70) and 3.5% in negative human samples (3/88), showing that dELISA could be used in the serodiagnosis of visceral leishmaniasis. Moreover, dELISA could be used as an alternative approach to screening asymptomatic visceral leishmaniasis patients, instead of invasive confirmatory testing.

Immune response and neutralization capacity of antibodies produced in young sheep immunized with Crotalus durissus terrificus native or Cobalt-60 irradiated venom / esposta imune e capacidade de neutralização de anticorpos produzidos em ovinos jovens imunizados com veneno de Crotalus durissus terrificus nativo e irradiado com Cobalto 60

The ELISA technique was used to evaluate and compare young ovine humoral immune response during crotalic antiserum production. Animals were clinically evaluated throughout this process, and the neutralizing capacity of antisera raised against natural (NV) and Cobalt-60 irradiated (IrV) venoms of Crotalus durissus terrificus (C.d.t.) was verified by means of in vitro challenges. Three groups of six animals each were used: G1 received NV; G2 was inoculated with IrV; and G3 was used as control. Animals received six immunizations during 84 days at 14-day intervals. ELISA of antibody profile showed significant difference (p<5%) between experimental groups (G1

A técnica de Elisa foi utilizada para avaliar e comparar a resposta imune humoral de ovinos jovens para a produção de soro anticrotálico. Durante o processo de soroprodução, foi realizada a avaliação clínica dos animais. A capacidade de neutralização do soro produzido a partir de veneno de serpente Crotalus durissus terrificus, nativo (VN) e irradiado (VIr) com Cobalto–60 foi verificada por meio de desafios in vitro. Um grupo de seis animais recebeu veneno nativo, o segundo grupo recebeu veneno irradiado e o terceiro grupo foi o controle. Os animais receberam seis imunizações durante 84 dias com intervalo de14 dias. Houve diferença significativa (p<5%) no teste de ELISA do perfil de anticorpos produzidos pelos grupos experimentais (VN

Effects of gamma irradiation on mechanical behavior and calcification of glutaraldehyde-fixed bovine pericardium.

OBJECTIVE: To evaluate the effect of gamma irradiation on glutaraldehyde-fixed bovine pericardium. METHOD: Glutaraldehyde-fixed bovine pericardium was exposed to gamma radiation (doses from 0 to 10000 Gy). Six samples from each of nine groups were evaluated by optic microscopy, and shrinking and mechanical tests and the denaturation temperature was determined. Additionally, they were subcutaneously implanted in rats and after four months they were explanted and Ca2+ levels measured by atomic absorption spectroscopy. RESULTS: The Ca2+ levels were (in microg/mg): control (0 Gy) - 194.45; 50 Gy - 154.64; 100 Gy - 169.37; 200 Gy - 163.64; 500 Gy - 199.89; 1000 Gy - 184.02; 2000 Gy - 198.95; 5000 Gy - 227.95; 10000 Gy - 362.62. Gamma irradiation caused a significant effect on the biomechanical properties of the tissue. CONCLUSION: e-fixed bovine pericardium.

Efeitos da radiação gama no comportamento mecânico e na calcificação do pericárdio bovino fixado com glutaraldeído / Effects of gamma irradiation on mechanical behavior and calcification of glutaraldehyde-fixed bovine pericardium

OBJETIVO: Avaliar o comportamento mecânico e os efeitos no processo de calcificação pós-implante do pericárdio bovino tratado com glutaraldeído submetido a várias doses de radiação gama. MÉTODO: Pericárdios bovinos fixados com glutaraldeído foram submetidos a radiação gama, nas doses de 0 a 10000 Gy. Seis amostras de cada grupo foram avaliadas pela microscopia óptica, determinação da temperatura de desnaturação do colágeno e ensaio mecânico de tração e implantadas subcutaneamente em ratos. Após quatro meses do implante, as amostras foram explantadas e o conteúdo de Ca2+ determinado pela espectrometria de absorção atômica. RESULTADOS: Níveis de Ca2+ (em µg/mg): 0 Gy (controle) - 194,45; 50 Gy - 154,64; 100 Gy - 169,37; 200 Gy - 163,64; 500 Gy - 199,89; 1000 Gy - 184,02; 2000 Gy - 198,95; 5000 Gy - 227,95 e 10000 Gy - 362,62. Houve alteração significativa no comportamento mecânico do tecido irradiado, quando comparado ao grupo controle, mesmo com o emprego de baixas doses de radiação. CONCLUSÃO: O emprego da radiação gama no pericárdio bovino tratado com glutaraldeído não reduziu os níveis de Ca2+ em implantes subcutâneos em ratos por quatro meses e promoveu alteração significativa no comportamento mecânico do tecido, com redução na sua resistência, quando comparados ao grupo controle.

OBJECTIVE: To evaluate the effect of gamma irradiation on glutaraldehyde-fixed bovine pericardium. METHOD: Glutaraldehyde-fixed bovine pericardium was exposed to gamma radiation (doses from 0 to 10000 Gy). Six samples from each of nine groups were evaluated by optic microscopy, and shrinking and mechanical tests and the denaturation temperature was determined. Additionally, they were subcutaneously implanted in rats and after four months they were explanted and Ca2+ levels measured by atomic absorption spectroscopy. RESULTS: The Ca2+ levels were (in µg/mg): control (0 Gy) - 194.45; 50 Gy - 154.64; 100 Gy - 169.37; 200 Gy - 163.64; 500 Gy - 199.89; 1000 Gy - 184.02; 2000 Gy - 198.95; 5000 Gy - 227.95; 10000 Gy - 362.62. Gamma irradiation caused a significant effect on the biomechanical properties of the tissue. CONCLUSION: e-fixed bovine pericardium.

Tratamentos anticalcificantes do pericárdio bovino fixado com glutaraldeído: comparação e avaliação de possíveis efeitos sinérgicos / Anti-calcifying treatment of glutaraldehyde fixed bovine pericardium: comparisons and evaluation of possible synergic effects

OBJETIVO: Avaliar possível efeito sinérgico dos agentes anticalcificantes mais promissores investigados na literatura, por meio de tratamento seqüencial. MÉTODO: Pericárdios bovinos fixados com glutaraldeído foram tratados com: aquecimento a 50°C, éter dietílico 80 por cento, quitosana 0,125 por cento, heparina, NaBH4 e formaldeído 4 por cento. Amostras foram avaliadas pela microscopia óptica, determinação da temperatura de desnaturação do colágeno e ensaio mecânico de tração e implantadas subcutaneamente em ratos. Após quatro meses do implante, as amostras foram explantadas e o conteúdo de Ca2+ determinado pela espectrometria de absorção atômica. RESULTADOS: Níveis de Ca2+ (em æg/mg): Controle -194,45; aquecimento a 50°C - 6,87; éter dietílico - 3,69; quitosana - 68,89; quitosana+heparina - 6,81; formaldeído - 107,34; tratamento seqüencial - 0,17. O comportamento mecânico variou de acordo com os tratamentos empregados. CONCLUSÃO: O tratamento seqüencial foi efetivo na inibição da calcificação e o tecido apresentou comportamento mecânico adequado à confecção de biopróteses.

OBJECTIVE: To evaluate a possible synergic effect of the most promising anti-calcifying agents investigated in literature by sequential treatment. METHOD: Glutaraldehyde-fixed bovine pericardium was treated by: heating to 50°C, 80 percent ether, 0.125 percent chitosan, heparin, NaBH4 and 4 percent formaldehyde. Samples were evaluated by optic microscopy, shrinking and mechanical tests and implanted subcutaneously in rats. Four months later the samples were explanted and Ca2+ levels measured by atomic absorption spectroscopy. RESULTS: Ca2+ levels were (in æg/mg): control - 194.45; heating at 50°C - 6.87; ether - 3.69; chitosan - 68.89; heparin - 6.81; formaldehyde - 107.34; sequential treatment - 0.17. The mechanical characteristics changed according to the treatments employed. CONCLUSION: Sequential treatment was effective to inhibit calcification and the tissue showed an adequate mechanical structure for the manufacture of bioprosthesis.

In Vitro antileishmanial properties of neutron-irradiated meglumine antimoniate

Pentavalent antimony, as meglumine antimoniate (Glucantime® ) or sodium stibogluconate (Pentostam® ), is the main treatment for leishmaniasis, a complex of diseases caused by the protozoan Leishmania, and an endemic and neglected threat in Brazil. Despite over half a century of clinical use, their mechanism of action, toxicity and pharmacokinetic data remain unknown. The analytical methods for determination of antimony in biological systems remain complex and have low sensitivity. Radiotracer studies have a potential in pharmaceutical development. The aim of this study was to obtain a radiotracer for antimony, with suitable physical and biological properties. Meglumine antimoniate was neutron irradiated inside the IEA-R1 nuclear reactor, producing two radioisotopes 122Sb and 124Sb, with high radionuclidic purity and good specific activity. This compound showed the same antileishmanial activity as the native compound. The use of the radiotracers, easily created by neutron irradiation, could be an interesting tool to solve important questions in antimonial pharmacology.

Os antimoniais pentavalentes, como o antimoniato de meglumina (Glucantime® ) ou estibogluconato de sódio (Pentostam® ), são o principal tratamento para a leishmaniose, um complexo de doenças causadas pelo protozoário parasita Leishmania, uma doença endêmica e negligenciada no Brasil. Apesar do seu uso clínico por mais de meio século, seu mecanismo de ação, toxicidade e dados de farmacocinética permanecem desconhecidos. Os métodos analíticos para determinação de antimônio em sistemas biológicos são complexos e apresentam baixa sensibilidade. Estudos utilizando radiotraçadores têm papel potencial no desenvolvimento farmacológico. O objetivo deste estudo foi desenvolver um radiotraçador de antimônio, com propriedades físicas e biológicas adequadas. O antimoniato de meglumina foi irradiado por nêutrons no reator nuclear IEA-R1, produzindo dois radioisótopos: 122Sb e 124Sb, com alta pureza radionuclídica e boa atividade específica. Este composto mostrou atividade antileishmania similar ao fármaco não irradiado. O uso de radiotraçadores, facilmente produzidos por irradiação por nêutrons pode ser um importante instrumento para elucidar questões sobre a farmacologia dos antimoniais.