Immunophenotyping of leukocytes in bovine placenta / Imunofenotipagem de leucócitos na placenta bovina

Lymphocytes and macrophages are the main white cells involved in fetal-maternal tolerance. Little is known about these leukocytes in bovine placenta, such as the quantity and location of these cells. Thus, the objective of this study was to identify lymphocyte and macrophage populations in bovine placenta using specific markers and flow cytometry. This study analyzed samples of placentomes and intercaruncular regions of cows in the three quarters of pregnancy. In the placentomes, during the first quarter of pregnancy, mean percentage of labeled CD3+ cells was 2.34%; CD8+, 1.28%; CD14+, 1.66%; and CD335+, 0.96%. For the intercaruncular region, percentage of CD3+ cells was 0.71%; CD8+, 1.63%; CD14+, 2.81%; and CD335+, 2.81%. In the second quarter, placentomes showed 0.94% CD3+ cells; 0.77% CD8+; 0.72% CD14+; and 0.51% CD335+. In the intercaruncular region, percentage of labeled CD3+ cells was 0.50%; CD8+, 1.81%; CD14+, 2.64%; and CD335+, 0.51%. In the third quarter, placentomes showed labeling of 0.88% CD3+; 0.66% CD8+; 1.06% CD14+; and 0.74% CD335+ cells. In the intercaruncular region, percentage of labeled CD3+ cells was 0.19%; CD8+, 2.23%; CD14+, 2.43%; and CD335+, 0.16%. The results showed that there was a greater immunomarking of leukocytes CD3+ and CD335+ in the placentome when compared to the intercaruncular region during the third trimester. It can be concluded that leukocytes populations in bovine placenta is reduced, probably because of the syndesmochorial characteristic of bovine placenta. This represents a significant barrier for the immunological system of the mother, sharply decreasing the exposure of the conceptus to the mother's immune system.(AU)

Linfócitos e macrófagos são os principais leucócitos envolvidos na tolerância materno-fetal. Pouco se sabe sobre esses leucócitos na placenta bovina, como por exemplo, a quantidade e localização dessas células. Assim, o objetivo desse estudo foi identificar populações de linfócitos e macrófagos na placenta bovina utilizando marcadores específicos e citometria de fluxo. Este estudo analisou amostras de placentônios da região intercaruncular de bovinos nos três trimestres da gestação. No primeiro trimestre, nos placentônios, a porcentagem média de células CD3+ foi 2,34%; CD8+, 1,28%; CD14+, 1,66%; e CD335+, 0,96%. Na região intercaruncular, a porcentagem de células CD3+ foi 0,71%; CD8+, 1,63%; CD14+, 2,81%; e CD335+, 2,81%. No segundo trimestre, os placentônios apresentaram 0,94% de células CD3+; 0,77% de CD8+; 0,72% de CD14+e 0,51% de CD335+. Na região intercaruncular, a porcentagem de células CD3+ foi 0,50%; CD8+, 1,81%; CD14+, 2,64%; e CD335+, 0,51%. No terceiro trimestre, os placentônios apresentaram 0,88% de células marcadas CD3+; 0,66% de CD8+; 1,06% de CD14+ e 0,74% de CD335+. Na região intercaruncular, a porcentagem de células CD3+ foi 0,19%; CD8+, 2,23%; CD14+, 2,43% e CD335+, 0,16%. Os resultados mostraram que a imunomarcação de leucócitos na região do placentônio foi maior do que na região intercaruncular no terceiro trimestre. Concluiu-se que a população de leucócitos CD3+ e CD335+ na placenta bovina está reduzida, provavelmente devido à sua característica sindesmocorial. Essa característica representa uma barreira significante para o sistema imunológico da mãe, o que diminui drasticamente a exposição do concepto ao sistema de defesa da mãe.(AU)

Efeitos de um minuto e dez minutos de deambulação em ratos com artrite induzida por adjuvante completo de Freund sobre os sintomas de dor e edema / Effects of one minute and ten minutes of walking activity in rats with arthritis induced by complete Freund's adjuvant on pain and edema symptoms

Este estudo avaliou o efeito de dois protocolos de exercício na nocicepção, edema e migração celular em ratos com artrite induzida por CFA. Ratos Wistar fêmeas (200 - 250 g, n = 50) foram induzidos à monoartrite por adjuvante completo de Freund (CFA, Mycobacterium butyricum; 0,5 mg/mL; 50 μL) na articulação do joelho direito (TF; n = 24) ou tornozelo direito (TT; n = 26). A incapacitação articular foi mensurada pelo tempo de elevação da pata (TEP; s) em 1 minuto de avaliação. O edema do joelho ou tornozelo foi avaliado pela medida do diâmetro articular (AD, cm) e pelo edema de pata (EP, mL), respectivamente. Ambos foram avaliados durante 10 dias consecutivos. Dois protocolos de exercício foram realizados: (a) exercício constante (TF, n = 6; TT, n = 6), realizando 1 minuto diário de exercício no cilindro (3 r.p.m.); (b) exercício variável (TF, n = 6; TT, n = 7), exercício com aumento de 1 minuto por dia, totalizando 10 minutos no último dia. Os grupos-controle (TF, n = 12; TT, n = 13) não realizaram exercício. Após 10 dias, os animais foram eutanasiados para contagem total (células/mm3) e diferencial (mononucleares e polimorfos nucleares; células/mm3) de leucócitos do tecido inflamado. O exercício variável inibiu a incapacitação e o edema em ambas as articulações. Entretanto, reduziu a migração total de leucócitos apenas na articulação TF. O exercício constante inibiu o edema nas duas articulações e reduziu a migração total de leucócitos da articulação TT. Porém, não reduziu a incapacitação. O exercício variável pareceu ser mais efetivo em reduzir os parâmetros inflamatórios em comparação com o exercício constante.

This study evaluated the effects of two protocols of exercise on nociception, edema and cell migration in rats with CFA-induced arthritis. Female Wistar rats (200 - 250 g, n = 50) was monoarthritis-induced by complete Freund's adjuvant (CFA; Mycobacterium butyricum, 0.5 mg/mL; 50 μL) into the right knee joint (TF; n = 24) or right ankle joint (TT; n = 26). Incapacitation was measured by the paw elevation time (TEP; s) in 1-min periods of observation. The edema of the knee or ankle joints was evaluated by the variation of the articular diameter (DA, cm) and by the paw volume variation (EP, mL), respectively. Both were measured during 10 consecutive days. Two protocols of exercise were performed: (a) in the constant exercise group (TF, n = 6; TT, n = 6) performing 1 minute of daily exercise on the cylinder; (b) variable exercise group (TF, n = 6; TT, n = 7), the exercise increased by 1 minute per day. The control groups (TF, n = 12; TT, n = 13) didn´t perform the exercise. After 10 days, the animals were euthanized for total (CT; cells/mm3) and differential leukocyte counts (mononuclear - MON, and polymorphonuclear - PMN, cells/mm3) of the articular inflammatory exudate. The variable exercise protocol inhibited incapacitation and edema for both joints. However, cell migration decreased only in the TF.The constant exercise reduced edema in both joints, and cell migration was decreased in the TT. However, the incapacitation was not reduced. Variable exercise seemed to be more effective in reducing the inflammatory parameters than constant exercise.

Gender differences in microcirculation: Observation using the hamster cheek pouch

OBJECTIVES: Estrogen has been shown to play an important protective role in non-reproductive systems, such as the cardiovascular system. Our aim was to observe gender differences in vivo with regard to the increase in macromolecular permeability and leukocyte-endothelium interaction induced by ischemia/reperfusion as well as in microvascular reactivity to vasoactive substances using the hamster cheek pouch preparation. METHODS: Thirty-six male and 36 female hamsters, 21 weeks old, were selected for this study, and their cheek pouches were prepared for intravital microscopy. An increase in the macromolecular permeability of post-capillary venules was quantified as a leakage of intravenously injected fluorescein-labeled dextran, and the leukocyte-endothelium interaction was measured as the number of fluorescent rolling leukocytes or leukocytes adherent to the venular wall, labeled with rhodamin G, during reperfusion after 30 min of local ischemia. For microvascular reactivity, the mean internal diameter of arterioles was evaluated after the topical application of different concentrations of two vasoconstrictors, phenylephrine (α1-agonist) and endothelin-1, and two vasodilators, acetylcholine (endothelial-dependent) and sodium nitroprusside (endothelial-independent). RESULTS: The increase in macromolecular permeability induced by ischemia/reperfusion was significantly lower in females compared with males [19 (17-22) leaks/cm2 vs. 124 (123-128) leaks/cm2, respectively, p<0.001), but the number of rolling or adherent leukocytes was not different between the groups. Phenylephrine-induced arteriolar constriction was significantly lower in females compared with males [77 (73-102)% vs. 64 (55-69)%, p<0.04], but there were no detectable differences in endothelin-1-dependent vasoreactivity. Additionally, arteriolar vasodilatation elicited by acetylcholine or sodium nitroprusside did not differ between the groups. CONCLUSION: The ...

Leukocytes respiratory burst activity as indicator of innate immunity of pacu Piaractus mesopotamicus / Atividade respiratória de leucócitos como indicador de imunidade inata de pacu (Piaractus mesopotamicus)

The present study evaluated the assay to quantify the respiratory burst activity of blood leukocytes of pacu as an indicator of the innate immune system, using the reduction of nitroblue tetrazolium (NBT) to formazan as a measure of the production of reactive oxygen species (ROS). In order to assess the accuracy of the assay, fish were challenged by Aeromonas hydrophila and sampled one week after challenge. The A. hydrophila infection increased the leukocyte respiratory burst activity. The protocol showed a reliable and easy assay, appropriate to determine the respiratory burst activity of blood leukocytes of pacu, a neotropical fish, in the present experimental conditions.

O presente estudo avaliou o ensaio para quantificar a atividade respiratória dos leucócitos do sangue de pacu como um indicador do sistema imune inato, usando a redução do nitroazul tetrazólio (NBT) a formazan como medida da produção de espécies reativas de oxigênio (EROs). Para avaliar a precisão do ensaio, peixes foram desafiados por Aeromonas hydrophila e amostrados uma semana após o desafio. A infecção com A. hydrophila aumentou a atividade respiratória dos leucócitos. O protocolo se mostrou confiável e de fácil aplicação, apropriado para determinar a atividade respiratória de leucócitos do sangue do pacu, peixe neotropical, nas condições experimentais apresentadas.

Os leucócitos e a resposta inflamatória na lesão de isquemia-reperfusão / Leukocytes and the inflammatory response in ischemia-reperfusion injury

Os eventos de isquemia-reperfusão desencadeiam uma resposta inflamatória sistêmica que pode levar a lesões celulares e até falência de órgãos. Tais repercussões são notadas no pós-operatório de cirurgias, em especial, com o uso de circulação extracorpórea. Sabe-se, atualmente, que os leucócitos exercem importante papel neste processo. Assim, este estudo aborda o papel dos leucócitos na fisiopatologia das lesões de isquemia-reperfusão e a ativação das cascatas inflamatórias por esse processo e procura auxiliar na compreensão destes mecanismos assim como trazer contribuições acerca das abordagens terapêuticas que possam atenuá-los. Esta revisão bibliográfica retrospectiva foi realizada a partir de documentos científicos publicados nos últimos dez anos, em português e inglês, indexados em bases de dados internacionais Medline e SciELO e de textos clássicos relacionados. Os descritores pesquisados foram: isquemia-reperfusão, leucócitos, resposta inflamatória, circulação extracorpórea, efeitos adversos e apoptose.

The events of ischemia-reperfusion injury triggers a systemic inflammatory response and can lead to cellular injury and organ failure. Such effects are noted in the postoperative recovery, especially with the use of cardiopulmonary bypass. Nowadays, it is known that leukocytes play an important role in this process. Therefore, this study addresses the role of leukocytes in the physiopathology of ischemia-reperfusion injuries and activation of inflammatory cascades through this process and seek to help in the understanding of these mechanisms as well as to bring contributions on the therapeutic approaches that can mitigate them. This retrospective review was performed from indexed scientific papers published over the last ten years in Portuguese and English in international databases MEDLINE and SciELO and related classic texts. The descriptors investigated were: ischemia-reperfusion, leukocytes, inflammatory response, cardiopulmonary bypass, adverse effects and apoptosis.

Leukocytes respiratory burst and lysozyme level in pacu (Piaractus mesopotamicus Holmberg, 1887) / Atividade respiratória de leucócitos e nível de lisozima em pacu (Piaractus mesopotamicus)

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.

Flow cytometry as a tool in the evaluation of blood leukocyte function in Chelonia mydas (Linnaeus, 1758) (Testudines, Cheloniidae) / A citometria de fluxo como ferramenta para avaliar a função celular de leucócitos sangüíneos de Chelonia mydas (Testudines, Cheloniidae)

Chelonia mydas is a sea turtle that feeds and nests on the Brazilian coast and a disease called fibropapillomatosis is a threat to this species. Because of this, it is extremely necessary to determine a methodology that would enable the analysis of blood leukocyte function in these sea turtles. In order to achieve this aim, blood samples were collected from C. mydas with or without fibropapillomas captured on the São Paulo north coast. Blood samples were placed in tubes containing sodium heparin and were transported under refrigeration to the laboratory in sterile RPMI 1640 cell culture medium. Leukocytes were separated by density gradient using Ficoll-PaqueTM Plus, Amershan Biociences®. The following stimuli were applied in the assessment of leukocyte function: Phorbol Miristate-Acetate (PMA) for oxidative burst activity evaluation and Zymosan A (Saccharomyces cerevisiae) Bio Particles®, Alexa Fluor® 594 conjugate for phagocytosis evaluation. Three cell populations were identified: heterophils, monocytes and lymphocytes. Monocytes were the cells responsible for phagocytosis and oxidative burst.

Chelonia mydas é uma tartaruga marinha que freqüenta o litoral brasileiro para alimentação e nidificação e uma doença denominada fibropapilomatose é uma das mais importantes ameaças à sobrevivência dessa espécie. Desta forma, a definição de uma metodologia que permita analisar a função dos leucócitos sangüíneos torna-se extremamente necessária. Foram utilizadas amostras sangüíneas de C. mydas com e sem fibropapilomas capturadas no litoral norte do estado de São Paulo. As amostras sangüíneas foram colocadas em tubos contendo heparina sódica e transportadas em meio de cultura celular RPMI 1640 estéril e sob refrigeração. Os leucócitos foram obtidos por gradiente de densidade usando Ficoll-PaqueTM Plus, Amershan Biociences®. Os estímulos aplicados foram Miristato Acetato de Phorbol (PMA) para avaliação de burst oxidativo e Zymosan A (Saccharomyces cerevisiae) Bio Particles®, Alexa Fluor® 594 conjugate para avaliação de fagocitose. Foram identificadas três populações celulares: heterófilos, monócitos e linfócitos. Os monócitos foram as células responsáveis pela fagocitose e pelo burst oxidativo.

The importance of the interaction between leukocyte integrin Mac-1 and platelet glycoprotein Ib-a for leukocyte recruitment by platelets and for the inflammatory response to vascular injury / Importância da interação entre a integrina Mac-1 dos leucócitos e a glicoproteína Iba das plaquetas para o recrutamento de leucócitos pelas plaquetas e para a resposta inflamatória à lesão vascular

OBJETIVO: Avaliar a importância da interação entre a integrina Mac-1 dos leucócitos (a Mb 2) e a glicoproteína (GP) Iba das plaquetas para o recrutamento de leucócitos após a lesão vascular e o efeito da neutralização da interação Mac-1-GPIba sobre a proliferação celular e a hiperplasia neointimal desencadeadas por lesão vascular. MÉTODOS: Um peptídeo denominado M2 ou anticorpo anti-M2 foi desenvolvido para bloquear a interação Mac-1-GPIba . Esse peptídeo foi injetado e comparado com anticorpo-controle em camundongos C57B1/6J submetidos a lesão vascular da artéria femoral com corda-guia. Um, cinco ou 28 dias após a lesão vascular, as artérias femorais foram retiradas para a realização de morfometria e imuno-histoquímica. RESULTADOS: O bloqueio da interação Mac-1-GPIba promoveu uma redução estatisticamente significativa do número de leucócitos na camada média no primeiro dia após a lesão vascular (controle: 7,9±5,0 por cento do total de células versus anti-M2: 2,0±1,6 por cento, p=0,021), bem como determinou uma diminuição estatisticamente significativa do acúmulo de leucócitos na neoíntima em cinco e 28 dias (controle: 42,3±12,9 por cento versus anti-M2: 24,6±10,8 por cento, p=0,047 e controle: 7,9±3,0 por cento versus anti-M2: 3,3±1,3 por cento, p=0,012; respectivamente). A proliferação celular na camada média do vaso em cinco dias pós-lesão foi reduzida com o bloqueio da interação Mac-1-GPIba (controle: 5,0±2,9 por cento do total de células versus anti-M2: 1,8±0,5 por cento; p=0,043), assim como houve diminuição significativa da proliferação celular na camada íntima do vaso em 28 dias (controle: 3,8±1,7 por cento versus anti-M2: 2,0±1,2 por cento; p=0,047). O bloqueio da interação Mac-1-GPIba também determinou uma redução estatisticamente significativa do espessamento intimal em 28 dias pós-lesão (controle: 10.395±3.549 µm² versus anti-M2: 4.561±4.915 ...

OBJECTIVE: To assess the importance of the interaction between leukocyte integrin Mac-1 (a Mb 2) and platelet glycoprotein (GP) Ib-a for leukocyte recruitment after vascular injury and the effect of the neutralization of the Mac-1-GPIba interaction on cell proliferation and the neointimal hyperplasia triggered by the vascular injury. METHODS: A peptide called M2 or anti-M2 antibody was developed to block the Mac-1-GPIba interaction. This peptide was injected and compared to a control-peptide in C57B1/6J mice submitted to vascular injury of the femoral artery with a guide wire. One, five or 28 days after the vascular injury, the femoral arteries were removed for morphometric and immunohistochemical analyses. RESULTS: The blocking of the Mac-1-GPIba interaction promoted a statistically significant reduction in the number of leukocytes in the neointimal layer on the first day after the vascular injury (control: 7.9±5.0 percent of the cell total versus anti-M2: 2.0±1.6 percent, p=0.021), as well as determined a statistically significant decrease in leukocyte accumulation in the neointimal layer on days 5 and 28 (control: 42.3±12.9 percent versus anti-M2: 24.6±10.8 percent, p=0.047 and control: 7.9±3.0 percent versus anti-M2: 3.3±1.3 percent, p=0.012; respectively). Cell proliferation in the neointimal layer of the vessel five days post-injury was reduced with the blocking of the Mac-1-GPIba interaction (control: 5.0±2.9 percent of the cell total versus anti-M2: 1.8±0.5 percent; p=0.043), along with a significant decrease in cell proliferation in the vessel neointimal layer 28 days post-injury (control: 3.8±1.7 percent versus anti-M2: 2.0±1.2 percent; p=0.047). The blocking of the Mac-1-GPIba interaction also determined a statistically significant decrease of the intimal thickening 28 days post-injury (control: 10,395±3,549 µm² versus anti-M2: 4,561±4,915 µm²; ...

In vivo observation of mesenteric leukocyte-endothelial interactions after cecal ligation/puncture and surgical sepsis source control

PURPOSE: Cecal ligation and puncture (CLP) has been used as a useful model for the induction of polymicrobial sepsis. Necrotic tissue resection and peritoneal lavage (REL) are the surgical procedures for controlling perforated appendicitis. The aim of this study was to evaluate leukocyte-endothelial interactions in the rat mesentery in vivo after CLP and REL. METHODS: Thirty-seven male Wistar rats (250-300 g) underwent laparotomy and were randomly assigned to the following groups: 1) SHAM; 2) CLP: animals submitted to CLP, 3) CLP+REL: animals submitted to CLP and REL. Mesenteric leukocyte-endothelial interactions were studied by intravital microscopy assessed once in each animal (3-5 postcapillary venules, 15-25 æm diameter) 24 hours after intervention. Follow-up was performed in all animals; this included analysis of glycemia, lactate, hematocrit, white blood cell count as well as a functional score that was the sum of scoring on the following parameters: alertness, mobility, piloerection, diarrhea, encrusted eyes, and dirty nose and tail. RESULTS: None of the animals showed significant changes in body weight (265 ± 20 g) or in hematocrit levels (46 percent ± 2 percent) during the experimental protocol. Compared to SHAM animals, CLP animals showed an increased number of rolling (2x), adherent, and migrating leukocytes (7x) in the mesenteric microcirculation, an increase in blood glucose (136 ± 8 mg/dL), lactate (3.58 ± 0.94 mmol/L), white cell count (23,570 ± 4,991 cells/mm³) and functional alterations (score 11 ± 1), characterized by impaired alertness and mobility, and presence of piloerection, diarrhea, encrusted eyes, and dirty nose and tail. The REL procedure normalized the number of rolling, adherent, and migrated leukocytes in the mesentery; glycemia; lactate; and white blood cell count. The REL procedure also improved the functional score (7 ± 1). CONCLUSION: Local and systemic inflammation was induced by CLP, while REL completely...

OBJETIVO: O procedimento de ligadura cecal e perfuração (CLP) tem sido usado como um modelo útil de indução de sepse polimicrobiana. A ressecção do tecido necrosado e lavagem peritoneal (REL) são procedimentos cirúrgicos freqüentemente utilizados para controlar uma apendicite perfurada. O objetivo desse estudo foi avaliar in vivo as interações leucócito-endotélio no mesentério de ratos após a CLP e REL. MÉTODOS: Trinta e sete ratos Wistar machos (250-300 g) foram submetidos à laparotomia e aleatoriamente divididos em grupos: 1) SHAM, 2) CLP: ratos submetidos à CLP, 3) CLP+REL: animais submetidos à CLP e REL. As interações leucócito-endotélio no mesentério foram estudadas através de microscopia intravital somente uma vez em cada animal (3-5 vênulas pós-capilares, 15-25 æm diâmetro), 24-horas após as intervenções. A evolução clínica foi realizada em todos os animais, incluindo glicemia, lactato, hematócrito, número total de células brancas e um escore funcional, o qual foi considerado como a somatória dos seguintes parâmetros: estado de alerta, mobilidade, piloereção, diarréia, olhos encrustados, e nariz e cauda sujos. RESULTADOS: Os animais não apresentaram alterações significantes no peso (265 ± 20 g) e hematócrito (46 ± 2 por cento) ao longo do estudo. Comparados ao SHAM, os animais CLP apresentaram aumento no número de leucócitos em rolamento (2x), aderidos (7x) e migrados (7x) na microcirculação mesentérica, aumentos da glicemia (136 ± 8 mg/dL), lactato (3,58 ± 0,94 mmol/L), leucocitose (23.570 ± 4.991 células/mm³) e alterações clínicas (escore 11±1), caracterizadas por comprometimento do estado de alerta e mobilidade, e presença de piloereção, diarréia, olhos encrustados, nariz e cauda sujos. REL normalizou o número de leucócitos em rolamento, aderidos e migrados no mesentério, a glicemia, o lactato e o número de leucócitos circulantes. REL também melhorou o escore clínico (7 ± 1). CONCLUSÃO: A CLP induziu inflamação local e sistêmica. A...

Indole ring oxidation by activated leukocytes prevents the production of hypochlorous acid

Hypochlorous acid (HOCl) released by activated leukocytes has been implicated in the tissue damage that characterizes chronic inflammatory diseases. In this investigation, 14 indole derivatives, including metabolites such as melatonin, tryptophan and indole-3-acetic acid, were screened for their ability to inhibit the generation of this endogenous oxidant by stimulated leukocytes. The release of HOCl was measured by the production of taurine-chloramine when the leukocytes (2 x 10(6) cells/mL) were incubated at 37°C in 10 mM phosphate-buffered saline, pH 7.4, for 30 min with 5 mM taurine and stimulated with 100 nM phorbol-12-myristate acetate. Irrespective of the group substituted in the indole ring, all the compounds tested including indole, 2-methylindole, 3-methylindole, 2,3-dimethylindole, 2,5-dimethylindole, 2-phenylindole, 5-methoxyindole, 6-methoxyindole, 5-methoxy-2-methylindole, melatonin, tryptophan, indole-3-acetic acid, 5-methoxy-2-methyl-3-indole-acetic acid, and indomethacin (10 æM) inhibited the chlorinating activity of myeloperoxidase (MPO) in the 23-72 percent range. The compounds 3-methylindole and indole-3-acetic acid were chosen as representative of indole derivatives in a dose-response study using purified MPO. The IC50 obtained were 0.10 ± 0.03 and 5.0 ± 1.0 æM (N = 13), respectively. These compounds did not affect the peroxidation activity of MPO or the production of superoxide anion by stimulated leukocytes. By following the spectral change of MPO during the enzyme turnover, the inhibition of HOCl production can be explained on the basis of the accumulation of the redox form compound-II (MPO-II), which is an inactive chlorinating species. These results show that indole derivatives are effective and selective inhibitors of MPO-chlorinating activity.

Disintegrins: integrin selective ligands which activate integrin-coupled signaling and modulate leukocyte functions

Extracellular matrix proteins and cell adhesion receptors (integrins) play essential roles in the regulation of cell adhesion and migration. Interactions of integrins with the extracellular matrix proteins lead to phosphorylation of several intracellular proteins such as focal adhesion kinase, activating different signaling pathways responsible for the regulation of a variety of cell functions, including cytoskeleton mobilization. Once leukocytes are guided to sites of infection, inflammation, or antigen presentation, integrins can participate in the initiation, maintenance, or termination of the immune and inflammatory responses. The modulation of neutrophil activation through integrin-mediated pathways is important in the homeostatic control of the resolution of inflammatory states. In addition, during recirculation, T lymphocyte movement through distinct microenvironments is mediated by integrins, which are critical for cell cycle, differentiation and gene expression. Disintegrins are a family of low-molecular weight, cysteine-rich peptides first identified in snake venom, usually containing an RGD (Arg-Gly-Asp) motif, which confers the ability to selectively bind to integrins, inhibiting integrin-related functions in different cell systems. In this review we show that, depending on the cell type and the microenvironment, disintegrins are able to antagonize the effects of integrins or to act agonistically by activating integrin-mediated signaling. Disintegrins have proven useful as tools to improve the understanding of the molecular events regulated by integrin signaling in leukocytes and prototypes in order to design therapies able to interfere with integrin-mediated effects.

The horse eosinophil as a model leucocyte for morphological and cytochemical studies

Mammalian eosinophil leucocytes contain specific lysosome-and peroxisome-like cytoplasmic granules that have important implications in inflammation, allergy and the bactericidal response to microorganisms. Several highly cationic proteins are responsible for the intense acidophilia of eosinophil granules, which also have a characteristic ultrastructure consisting of a dense core and an external matrix. The granules of horse eosinophils are large in size and are easily recognizable as individual elements by light microscopy. These characteristics make these cells an adequate model for cytochemical analyses and precise light-electron microscopical correlations. In this work, the selective and cytochemical staining and fluorescence reactions applied to horse blood smears are reviewed, and show that eosinophil granules are very suitable structures for testing new light microscopical methods. Transmission electron microscopy has shown that horse eosinophil granules have an electron-dense, non-crystalline core surrounded by a less dense external matrix, although there is considerable heterogeneity in their ultrastructural morphology. Cytochemical results show a ring-like pattern for some staining and fluorescence reactions (glutaraldehyde-oxidized hematoxylin, fast green FCF, 1-hydroxy-3,6,8-pyrene-trisulfonate at pH 12, Timm sulfide-silver method), indicating that the external matrix of horse eosinophil granules contains metal cations and cationic proteins with high isoelectric points.

Peripheral genotype-phenotype correlations in Asian Indians with type 2 diabetes mellitus.

OBJECTIVE: A genome-wide scan of gene expression in leucocytes in Asian Indians with type 2 diabetes was performed and correlated with their known phenotype. METHODS: Microarray gene profiling of 13,474 sequence-verified, non-redundant human cDNAs was done to study leukocyte gene expression in Asian Indians with type 2 diabetes (DM: n=3) and matched controls (n=3). RESULTS: Significant differential expression (fold change <0.3 or >3) was noted for 897 genes in DM vs. controls. The 147 known genes in this category belonged to following broad functional groups (%): enzyme (32), nucleic acid binding (22), ligand binding or carrier (10), signal transducer (9), transporter (7), structural protein (6), cell adhesion (3), tumor suppressor (3), transcription factor binding (2), enzyme inhibitor (2), chaperone (2), cell cycle regulator (1), and defense/immunity protein (1). The 20 genes with at least a 3-fold change, annotated with known phenotypic associations in the current gene databank (phenotype association, fold change) were aspartoacylase (Canavan disease, 9.96), growth hormone receptor (Laron dwarfism, idiopathic short stature, 8.25), lipoprotein lipase (familial chylomicronemia syndrome, lipoprotein lipase deficiency, 8.00), vitamin D (1,25- dihydroxyvitamin D3) receptor (involutional osteoporosis, vitamin D resistant rickets, 7.94), intercellular adhesion molecule 1 human rhinovirus receptor (cerebral malaria susceptibility, 7.16), peroxisomal membrane protein 3 35-kDa (Refsum disease, infantile form, Zellweger syndrome-3, 6.00), Bardet-Biedl syndrome 2 (Bardet-Biedl syndrome, 5.87), ribosomal protein S19 (Diamond Blackfan anemia, 5.85), apolipoprotein C-III (hypertriglyceridemia, 5.44), argininosuccinate lyase (argininosuccinicaciduria, 5.22), myosin VA (Griscelli syndrome-type pigmentary dilution with mental retardation, 4.92), lysozyme (renal amyloidosis, 4.17), SAM domain, SH3 domain and nuclear localisation signals 1 (Cherubism, 4.12 ), von Hippel-Lindau syndrome (hemangioblastoma, cerebellar, somatic, von Hippel-Lindau syndrome, 3.94), early-onset breast cancer 1 (BRCA1, papillary serous carcinoma of the peritoneum, 3.73), UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase (inclusion body myopathy, autosomal recessive, sialuria, 3.53), apolipoprotein A-I (amyloidosis, 3 or more types, hypoalphalipoproteinemia, 3.29), midline 1 Opitz/BBB syndrome (Opitz G syndrome, type I, 3.28), ATPase, Na+/K+ transporting, alpha 2 (+) polypeptide (familial hemiplegic migraine, 3.05). Canavan disease, Zellweger syndrome, infantile Refsum disease, Griscelli syndrome, cherubism, breast cancer, peritoneal papillary serous carcinoma, Opitz G/BBB syndrome, and familial hemiplegic migraine (FHM) are phenotypes not previously reported in association with type 2 DM, but whose underlying genes were up-regulated in this peripheral genome scan of Asian Indians. CONCLUSION: Rare and/or previously unknown phenotypes linked to known genes with significant differential expression in type 2 DM are reported. Further testing of heterogeneity in diabetes phenotype syndromes may reveal common pathogenic mechanisms and potential candidate genes responsible for type 2 DM.

Mechanisms of leukocyte lipid body formation and function in inflammation

An area of increasingly interest for the understanding of cell signaling are the spatio-temporal aspects of the different enzymes involved in lipid mediator generation (eicosanoid-forming enzymes, phospholipases and their regulatory kinases and phosphatases) and pools of lipid precursors. The compartmentalization of signaling components within discrete and dynamic sites in the cell is critical for specificity and efficiency of enzymatic reactions of phosphorilation, enzyme activation and function. We hypothesized that lipid bodies - inducible non-membrane bound cytoplasmic lipid domains - function as specialized intracellular sites of compartmentalization of signaling with major roles in lipid mediator formation within leukocytes engaged in inflammatory process. Over the past years substantial progresses have been made demonstrating that all enzymes involved in eicosanoid synthesis localize at lipid bodies and lipid bodies are distinct sites for eicosanoid generation. Here we will review our current knowledge on the mechanisms of formation and functions of lipid bodies pertinent to inflammation.

Effect of fatty acids on leukocyte function

Fatty acids have various effects on immune and inflammatory responses, acting as intracellular and intercellular mediators. Polyunsaturated fatty acids (PUFAs) of the omega-3 family have overall suppressive effects, inhibiting lymphocyte proliferation, antibody and cytokine production, adhesion molecule expression, natural killer cell activity and triggering cell death. The omega-6 PUFAs have both inhibitory and stimulatory effects. The most studied of these is arachidonic acid that can be oxidized to eicosanoids, such as prostaglandins, leukotrienes and thromboxanes, all of which are potent mediators of inflammation. Nevertheless, it has been found that many of the effects of PUFA on immune and inflammatory responses are not dependent on eicosanoid generation. Fatty acids have also been found to modulate phagocytosis, reactive oxygen species production, cytokine production and leukocyte migration, also interfering with antigen presentation by macrophages. The importance of fatty acids in immune function has been corroborated by many clinical trials in which patients show improvement when submitted to fatty acid supplementation. Several mechanisms have been proposed to explain fatty acid modulation of immune response, such as changes in membrane fluidity and signal transduction pathways, regulation of gene transcription, protein acylation, and calcium release. In this review, evidence is presented to support the proposition that changes in cell metabolism also play an important role in the effect of fatty acids on leukocyte functioning, as fatty acids regulate glucose and glutamine metabolism and mitochondrial depolarization

Evaluation of leukergy test as an indicator of infection in hip joint in children.

This new blood test (leukergy) for infection is based on the fact that white cells agglomerate in peripheral blood of patients with inflammatory diseases. We evaluated leukergy in 25 children with proven septic arthritis of hip. It was found to be the efficient and earliest indicator of septic arthritis than the erythrocyte sedimentation rate (ESR), total leucocyte count (TLC), polymorphs and C-reactive protein (CRP). It also correlated well with the clinical severity of infection and the prognosis of disease. Thus leukergy is a simple, rapid and inexpensive slide test which was found as the best indicator profile for the presence of septic arthritis.

A antibioticoterapia prévia sobre o infiltrado inflamatório pulmonar após isquemia e reperfusäo intestinal: estudo experimental em ratos / Previous antibiotic therapy on the lung inflamatory infiltrate after ischemia and intestinal reperfusion: experimental study in rats

A isquemia seguida de reperfusão intestinal (I/R) é um evento clínico comum que favorece a translocação bacteriana em órgãos à distância, como os pulmões. A antibioticoterapia prévia pode ser uma conduta eficaz para atenuar o desenvolvimento desse processo. Este esudo tem por finalidade reconhecer a concentração dos polimorfonucleares (PMN) nos septos alveolares em ratos, após indução de isquemia seguida de reperfusão intestinal, com e sem antibioticoterapia prévia. Utilizaram-se 48 ratos divididos, aleatoriamente em 6 grupos (A,B,C,D,E,F)com 8 animais cada um. Os grupos A,C e E não receberam antibiótico,e os grupos B, D e F receberam 500mg/Kg de eritromicina por via oral. Os grupos A e B serviram de controle; C e D induziu-se isquemia intestinaL por 30 min; e os grupos E e F seguiu-se à isquemia, reperfusão por 30 min. Em seguida, abriu-se o tórax e ressecaram-se os pulmões, sendo encaminhados para análise histopatológica e histométrica, obtendo-se uma média de PMN nos septos alvéolos...

Impaired anti-microbial function of leukocytes from patients with type 1 diabetes Mellitus

Twenty patients with non-ketoacidotic and non-insulin-dependent diabetes mellitus with adequate glycemic control were selected for this study in addition to ten age and sex ratio matched healthy control subjects without family history of diabetes mellitus. No one of the patients or controls had any evidence of cardiac, liver, renal or infectious disease. Complete blood count [CBC], oral glucose tolerance test [OGTT], determination of glycosylated hemoglobin [HbA1], fasting insulin, proinsulin and the sum of -30-60 minute incremental insulin response to OGTT were conducted for the total cohort. Neutrophils [PMNs] of all patients and controls were prepared in cell suspension samples and subjected for in vitro assessment of their chemotactic migration and phagocytic functions using the agarose plate technique and Candida albicans suspension. The results showed that the mean [SD] chemotactic and phagocytic indices [CI and PI] of PMNs from the insulin dependent diabetic group were significantly decreased compared with controls. Both indices did not show any significant correlation with either blood glucose levels or Hb Al- levels. The results of CHC showed a significant total leukocytosis and neutrophilia in the diabetic group compared with controls

The effects of peripheral leukocytes on the hippocampal neuronal changes in transient global ischemia and unilateral cerebral hemispheric infarction

The participation of activated leukocytes and subsequent production of chemical mediators has been well accepted in the pathophysiology of hypoxic-ischemic injury. This study was performed to see the effects of leukocytes on hippocampal neuronal damage in transient global ischemia induced by 10-min occlusion of bilateral common carotid arteries (CCAs) with reperfusion for various times, and in complete unilateral ischemia induced by 24-hr ligation of left CCA. Leukopenia was induced by intraperitoneal injection of cyclophosphamide for 4 days. The results showed that hippocampal neuronal damages were worse at 6-hr reperfusion in leukopenic experimental group than in the control group. In comparison, 24-hr and 3-day reperfusion leukopenic groups showed less numbers of damaged neurons and milder changes. The 5-day reperfusion group showed inconsistent changes. Unilateral CCA occlusion showed extensive infarction in 83.3% of gerbils in the control group, compared to 25% of gerbils in the experimental group (p<0.05). These results strongly suggest that the number of peripheral leukocytes were closely related to the development of delayed neuronal damage of hippocampus in transient global ischemia and the incidence of infarction induced by 24-hr unilateral CCA ligation.