RESUMO
BACKGROUND: The phagocytic function in pulmonary tuberculosis (PTB) and Type 2 diabetes (T2D) has been explored mainly in macrophages but not in polymorphonuclears (PMN). The purpose of this study was to determine the functional status of PMN leukocytes in patients with pulmonary tuberculosis (PTB), Type 2 diabetes (T2D), and in patients with both diseases. METHODS: An observational, prospective, and comparative study was carried out. 30 ambulatory patients with T2D, 10 with PTB undergoing treatment and 10 patients with PTB and T2D, and 44 healthy subjects were studied. PMN leukocytes were separated, the capacity of these cells to produce hydrogen peroxide and to reduce nitroblue tetrazolium (NBT) in response to stimulus with the phorbolic ester of myristic acid (PMA) was measured; and the capacity of PMN leukocytes to adhere to surfaces was determined. RESULTS: Concerning the test for adherence, on comparing healthy subjects with patients with T2D+PTB, we observed a clear decrease in cellular adherence in the group of patients with both diseases; it was statistically significant (p = 0.007).With regard to phagocytic function, we observed that in NBT reduction as well as in hydrogen peroxide production, statistically significant differences were not obtained on comparing healthy subjects with any of the three groups of patients. CONCLUSIONS: We observed a clear decrease in cellular adherence when both diseases co-exist. These results could indicate the need for the co-existence of T2D and TB to cause deterioration in the cells' adherence activity. The microtechniques employed permit the evaluation in a practical manner of certain phagocytic-activity expressions.
Assuntos
Adesão Celular/fisiologia , Diabetes Mellitus Tipo 2/imunologia , Granulócitos/imunologia , Fagocitose , Tuberculose Pulmonar/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Diabetes Mellitus Tipo 2/complicações , Feminino , Granulócitos/metabolismo , Humanos , Peróxido de Hidrogênio/metabolismo , Masculino , Pessoa de Meia-Idade , Nitroazul de Tetrazólio/metabolismo , Estudos Prospectivos , Acetato de Tetradecanoilforbol/metabolismo , Tuberculose Pulmonar/complicaçõesRESUMO
Larrea divaricata Cav. (jarilla) is a plant with well-documented applications in folk medicine in Argentina. In this study, we aimed to evaluate functional parameters of peritoneal macrophages isolated from mice injected with three fractions (F1, F2 and F3) of L. divaricata. The response of macrophages against Candida albicans was evaluated. Cell viability was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test, apoptosis was evaluated using Giemsa, acridine orange/ethidium bromide and ladder assay, oxidative burst was assayed using nitroblue tetrazolium test and nitrite production using Griess assay. Cell stimulation and their ability to kill C. albicans in vitro were measured. The number and cell viability were similar to controls. However, we found that F1 induces pre-activation of macrophages, and this pre-activation is enhanced by C. albicans. The effects exerted by F1 make it more important than F2 and F3 for the treatment of disseminated candidiasis in patients with immunodeficiency diseases such as AIDS and chronic granulomatous disease, among others.
Assuntos
Candida albicans/imunologia , Fatores Imunológicos/farmacologia , Larrea/química , Macrófagos Peritoneais/efeitos dos fármacos , Macrófagos Peritoneais/imunologia , Extratos Vegetais/farmacologia , Animais , Apoptose , Sobrevivência Celular , Células Cultivadas , Fragmentação do DNA , Etídio/metabolismo , Feminino , Fatores Imunológicos/isolamento & purificação , Masculino , Camundongos , Nitritos/metabolismo , Nitroazul de Tetrazólio/metabolismo , Extratos Vegetais/isolamento & purificação , Explosão Respiratória , Coloração e Rotulagem/métodos , Sais de Tetrazólio/metabolismo , Tiazóis/metabolismoRESUMO
Innate immune responses are useful to determine the health status of fish and to evaluate the effect of immunomodulatory substances in fish farming. Leukocytes respiratory burst was measured in pacu (Piaractus mesopotamicus) using chemiluminescence assay and nitroblue tetrazolium (NBT) reduction assay. The nitroblue tetrazolium reduction seemed more adequate than chemiluminescence assay for leukocytes oxidative burst determination, since it was difficult to isolate the blood leucocytes for chemiluminescence assay. Plasma and serum lysozyme were measured using a turbidimetric assay. The heating of serum and plasma samples (56 masculineC for 30 minutes) for complement system inactivation darkened the plasma samples and interfered in the results. The lysozyme activity in serum was higher than in plasma, suggesting that serum samples are more appropriate for the analysis. This study established protocols that can be useful tools in the study of immune mechanisms of the tropical fish pacu.
Assuntos
Peixes/fisiologia , Leucócitos/fisiologia , Muramidase/metabolismo , Explosão Respiratória/fisiologia , Animais , Peixes/metabolismo , Medições Luminescentes , Muramidase/sangue , Nitroazul de Tetrazólio/metabolismoRESUMO
Innate immune responses are useful to determine the health status of fish and to evaluate the effect of immunomodulatory substances in fish farming. Leukocytes respiratory burst was measured in pacu (Piaractus mesopotamicus) using chemiluminescence assay and nitroblue tetrazolium (NBT) reduction assay. The nitroblue tetrazolium reduction seemed more adequate than chemiluminescence assay for leukocytes oxidative burst determination, since it was difficult to isolate the blood leucocytes for chemiluminescence assay. Plasma and serum lysozyme were measured using a turbidimetric assay. The heating of serum and plasma samples (56 ºC for 30 minutes) for complement system inactivation darkened the plasma samples and interfered in the results. The lysozyme activity in serum was higher than in plasma, suggesting that serum samples are more appropriate for the analysis. This study established protocols that can be useful tools in the study of immune mechanisms of the tropical fish pacu.
Respostas imunológicas inatas são úteis para determinar o estado de saúde de peixes e avaliar o efeito de substâncias imunomoduladoras no cultivo destes animais. A atividade respiratória de leucócitos foi medida em pacu (Piaractus mesopotamicus) através de ensaio de quimioluminescência e ensaio de redução do nitroblue tetrazolium (NBT). O ensaio de redução do nitroblue tetrazolium pareceu mais adequado que o ensaio de quimioluminescência para determinação da atividade respiratória de leucócitos, uma vez que foi difícil isolar com êxito os leucócitos do sangue para o ensaio de quimioluminescência. Lisozima sérica e plasmática foram medidas por meio de ensaio turbidimétrico. Com o objetivo de inativar as proteínas do sistema complemento, as amostras de soro e plasma foram aquecidas (56 ºC por 30 minutos). Porém, este procedimento provocou a turvação das amostras de plasma e interferiu nos resultados. A atividade de lisozima no soro foi maior que no plasma, sugerindo que amostras de soro são mais apropriadas para esta análise. Este estudo estabeleceu protocolos que podem ser utilizados como ferramentas no estudo de mecanismos imunológicos do peixe tropical pacu.
Assuntos
Animais , Peixes/fisiologia , Leucócitos/fisiologia , Muramidase/metabolismo , Explosão Respiratória/fisiologia , Peixes/metabolismo , Medições Luminescentes , Muramidase/sangue , Nitroazul de Tetrazólio/metabolismoRESUMO
Larval and adult stages of Taenia solium and Taenia crassiceps WFU strain were analyzed by histochemical and biochemical methods to determine the existence of steroid pathways. The presence of the key enzyme 3beta-hydroxisteroid-dehydrogenase (3beta-HSD) was examined in frozen sections of cysticerci obtained from mice and segments of tapeworms obtained from the intestine of hamsters. 3beta-HSD activity was detected by nitroblue-tetrazolium products after incubation with dehydroepiandrosterone, androstendiol, or pregnenolone. Tapeworm tissues exhibited 3beta-HSD activity in the subtegumentary areas of the neck and immature proglottids following incubation with androstendiol, as well as surrounding the testes in mature proglottids. T. solium cysticerci exhibited 3beta-HSD activity in the subtegumentary tissues. The synthesis of steroid hormones involving the activity of 3beta-HSD was studied in cysticerci or tapeworms incubated in the presence of tritiated steroid precursors. The culture media were analyzed by thin layer chromatography and showed synthesis of androstendiol, testosterone, and 17beta-estradiol by cysticerci, androstendiol, and 17beta-estradiol by tapeworms. The results strongly suggest the activity of 3beta-HSD in taeniid parasites that have at least a part of the enzymatic chain required for androgen and estrogen synthesis and that the enzymes are present in the larval stage and from the early strobilar stages to the mature proglottids.
Assuntos
3-Hidroxiesteroide Desidrogenases/metabolismo , Cysticercus/enzimologia , Taenia/enzimologia , Androstenodiol/metabolismo , Animais , Cromatografia em Camada Fina , Cricetinae , Meios de Cultura/química , Cysticercus/crescimento & desenvolvimento , Desidroepiandrosterona/metabolismo , Estradiol/análise , Intestinos/parasitologia , Camundongos , Camundongos Endogâmicos BALB C , Nitroazul de Tetrazólio/metabolismo , Pregnenolona/metabolismo , Coloração e Rotulagem , Taenia/crescimento & desenvolvimento , Testosterona/análiseRESUMO
The in vitro effect of different concentrations of beta-hydroxybutyric acid (betaHBA) on bovine milk leukocytes was examined. betaHBA level similar to those found in cows with clinical ketosis induced a significant inhibitory effect on the nitroblue tetrazolium reduction as a mean of assaying the metabolic integrity of macrophages after the phorbol-mirystate- acetate and opsonized zymosan stimulation. In the same way, the H2O2 production after stimulation with both soluble and particulate agents decreased significantly in 33 and 26%, respectively, compared with cells incubated without ketone bodies. This result suggests a possible fault in the microbicidal oxidative activity. The macrophage phagocytosis also decreased in cells treated with different betaHBA concentrations, in relation to that obtained from control cells. Neutrophils migration in agarose was determined, and the mean chemotactic response was higher when the cells were incubated with lower level or absence of ketone bodies. Considering the determined differences, we hypothesize that abnormally high levels of ketone bodies could produce a direct effect on leukocyte membranes. The induction of some modification on the receptor structure impairment the interaction ligand-receptor and this may be, in part, responsible for the higher susceptibility to local infections in mammary gland during subclinical and clinical ketosis.
Assuntos
Ácido 3-Hidroxibutírico/farmacologia , Leucócitos/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Leite/efeitos dos fármacos , Neutrófilos/efeitos dos fármacos , Fagocitose/efeitos dos fármacos , Animais , Bovinos , Feminino , Peróxido de Hidrogênio/metabolismo , Corpos Cetônicos/metabolismo , Cetose/metabolismo , Cetose/veterinária , Leucócitos/metabolismo , Macrófagos/metabolismo , Leite/metabolismo , Neutrófilos/metabolismo , Nitroazul de Tetrazólio/metabolismo , Fagocitose/fisiologia , Acetato de Tetradecanoilforbol/farmacologia , Zimosan/farmacologiaRESUMO
Dehydrocrotonin (DHC) is a diterpene lactone obtained from Croton cajucara (Sacaca). Dimethylamide-crotonin (DCR), a DHC derivative, has a similar inhibitory effect on leukemic HL60 cells than its parent compound evaluated by different endpoints of cytotoxicity. No cytotoxicity or morphological alterations associated with apoptosis were detected in human peripheral blood mononuclear cells (PBMC) after treatment with up to 400 micro M DCR in presence of phytohemaglutinin (5 micro g/ml). Based on morphological changes and the pattern of DNA fragmentation, DHC and DCR were found to induce apoptosis and terminal differentiation (assessed by nitro blue tetrazolium reduction) in HL60 cells, but these compounds did not show any toxic effect in PBMC. Thus, DCR and DHC inhibit HL60 cell growth in vitro partly by inducing apoptosis and cell differentiation, but does not cause serious damage to immune cells according to our experimental conditions.
Assuntos
Antineoplásicos Fitogênicos/toxicidade , Diterpenos Clerodânicos , Diterpenos/toxicidade , Lactonas/toxicidade , Leucócitos Mononucleares/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Divisão Celular/efeitos dos fármacos , Fragmentação do DNA/efeitos dos fármacos , Eletroforese em Gel de Ágar , Células HL-60 , Humanos , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/metabolismo , Nitroazul de Tetrazólio/metabolismo , Plantas MedicinaisRESUMO
New derivatives from dehydrocrotonin (DHC, compound I), with the same anti-ulcerogenic properties but less toxicity were synthesised by reducing the cyclohexenone moiety of DHC with NaBH(4) (compound II), by reducing the cyclohexenone and lactone moieties with LiAlH(4) (compound III) and by transforming the lactone moiety into an amide (compound IV) using dimethylamine. The cytotoxicity of these derivatives from DHC was assayed on V79 fibroblast cell line. Three independent endpoints for cytotoxicity were evaluated; namely, the nucleic acid content (NAC), tetrazolium reduction (MTT) and neutral red uptake (NRU). IC(50) values of 540 and 350 microM were obtained for compound II in the NRU and NAC tests, respectively. Compound III was less toxic than the other DHC derivatives (IC(50)=1800 microM) on V79 cells based on NAC assay. Compound IV showed an IC(50) ranging from 350 to 600 microM based on the three endpoints evaluated. The three compounds were less toxic on V79 cells than DHC. DHC, compounds II, III and IV did not change the respiration rate of Escherichia coli on the acute toxicity assay.
Assuntos
Diterpenos Clerodânicos , Diterpenos/toxicidade , Escherichia coli/efeitos dos fármacos , Pulmão/efeitos dos fármacos , Plantas Medicinais/toxicidade , Animais , Contagem de Células , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Cricetinae , Cricetulus , Relação Dose-Resposta a Droga , Escherichia coli/metabolismo , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Pulmão/citologia , Pulmão/metabolismo , Vermelho Neutro/metabolismo , Nitroazul de Tetrazólio/metabolismo , Ácidos Nucleicos/análise , Testes de ToxicidadeRESUMO
It is known that chicken macrophages derived in vitro from blood monocytes have the capacity to destroy Trypanosoma cruzi, but Toxoplasma gondii can survive within these cells. This study was performed to determine the involvement of nitric oxide (NO) in the killing of T. cruzi by chicken macrophages. Activated (by interferon-gamma and lipopolysaccharide) mouse peritoneal macrophages were used as controls. Macrophages were infected with T. cruzi and T. gondii; after 2, 24, and 48 h, NO was assayed using the Griess reagent. Respiratory-burst involvement, revealed by the reduction of nitroblue tetrazolium (NBT), was determined in chicken macrophages. Chicken macrophages did not produce NO; mouse macrophages were capable of producing NO with no multiplication of parasites. Reduction of NBT could be detected in chicken macrophages that interacted with T. cruzi but was absent in those that interacted with T. gondii. These results demonstrate that chicken macrophages do not use NO as a microbicidal agent when infected with T. cruzi or T. gondii.
Assuntos
Galinhas/parasitologia , Macrófagos Peritoneais/parasitologia , Óxido Nítrico/metabolismo , Trypanosoma cruzi/fisiologia , Animais , Células Cultivadas , Interferon gama/farmacologia , Lipopolissacarídeos/farmacologia , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos Peritoneais/metabolismo , Masculino , Camundongos , Nitroazul de Tetrazólio/metabolismo , Oxirredução , Toxoplasma/fisiologiaRESUMO
Neutrophil function in 32 workers occupationally exposed to anti-ChE insecticides, as measured by chemotaxis through the leading front method and nitroblue tetrazolium dye reduction, was investigated and compared to those of age- and sex-matched controls. The cholinesterase (ChE) activity was normal in all the workers studied, although decrease of chemotaxis and of nitroblue tetrazolium reduction was observed in the exposed population. These results suggest that the identified functional changes in polymorphonuclear neutrophils might be an early indicator of anti-ChE insecticides toxicity, even in those individuals with no impairment in the ChE activity.