Articular inflammation induced by an enzymatically-inactive Lys49 phospholipase A2: activation of endogenous phospholipases contributes to the pronociceptive effect

Abstract Background Arthritis is a set of inflammatory conditions that induce aching, stiffness, swelling, pain and may cause functional disability with severe consequences to the patients lives. These are multi-mediated pathologies that cannot be effectively protected and/or treated. Therefore, the aim of this study was to establish a new model of acute arthritis, using a Lys49-PLA2 (Bothrops asper myotoxin II; MT-II) to induce articular inflammation. Methods The articular inflammation was induced by MT-II (10 g/joint) injection into the left tibio-tarsal or femoral-tibial-patellar joints. Cellular influx was evaluated counting total and differential cells that migrated to the joint. The plasma extravasation was determined using Evans blue dye. The edematogenic response was evaluated measuring the joint thickness using a caliper. The articular hypernociception was determined by a dorsal flexion of the tibio-tarsal joint using an electronic pressure-meter test. The mediators involved in the articular hypernociception were evaluated using receptor antagonists and enzymatic inhibitors. Results Plasma extravasation in the knee joints was observed 5 and 15 min after MT-II (10 g/joint) injection. MT-II also induced a polymorphonuclear cell influx into the femoral-tibial-patellar joints observed 8 h after its injection, a period that coincided with the peak of the hyperalgesic effect. Hyperalgesia was inhibited by the pretreatment of the animals with cyclooxygenase inhibitor indomethacin, with type-2 cyclooxygenase inhibitor celecoxib, with AACOCF3 and PACOCF3, inhibitors of cytosolic and Ca2+-independent PLA2s, respectively, with bradykinin B2 receptor antagonist HOE 140, with antibodies against TNF, IL-1, IL-6 and CINC-1 and with selective ET-A (BQ-123) and ET-B (BQ-788) endothelin receptors antagonists. The MT-II-induced hyperalgesia was not altered by the lipoxygenase inhibitor zileuton, by the bradykinin B1 receptor antagonist Lys-(Des-Arg9,Leu8)-bradykinin, by the histamine and serotonin antagonists promethazine and methysergide, respectively, by the nitric oxide inhibitor LNMMA and by the inhibitor of matrix 1-, 2-, 3-, 8- and 9- metalloproteinases GM6001 (Ilomastat). Conclusion These results demonstrated the multi-mediated characteristic of the articular inflammation induced by MT-II, which demonstrates its relevance as a model for arthritis mechanisms and treatment evaluation.

Articular inflammation induced by an enzymatically-inactive Lys49 phospholipase A2: activation of endogenous phospholipases contributes to the pronociceptive effect

Background Arthritis is a set of inflammatory conditions that induce aching, stiffness, swelling, pain and may cause functional disability with severe consequences to the patient's lives. These are multi-mediated pathologies that cannot be effectively protected and/or treated. Therefore, the aim of this study was to establish a new model of acute arthritis, using a Lys49-PLA2 (Bothrops asper myotoxin II; MT-II) to induce articular inflammation. Methods The articular inflammation was induced by MT-II (10 μg/joint) injection into the left tibio-tarsal or femoral-tibial-patellar joints. Cellular influx was evaluated counting total and differential cells that migrated to the joint. The plasma extravasation was determined using Evans blue dye. The edematogenic response was evaluated measuring the joint thickness using a caliper. The articular hypernociception was determined by a dorsal flexion of the tibio-tarsal joint using an electronic pressure-meter test. The mediators involved in the articular hypernociception were evaluated using receptor antagonists and enzymatic inhibitors. Results Plasma extravasation in the knee joints was observed 5 and 15 min after MT-II (10 μg/joint) injection. MT-II also induced a polymorphonuclear cell influx into the femoral-tibial-patellar joints observed 8 h after its injection, a period that coincided with the peak of the hyperalgesic effect. Hyperalgesia was inhibited by the pretreatment of the animals with cyclooxygenase inhibitor indomethacin, with type-2 cyclooxygenase inhibitor celecoxib, with AACOCF3 and PACOCF3, inhibitors of cytosolic and Ca2+-independent PLA2s, respectively, with bradykinin B2 receptor antagonist HOE 140, with antibodies against TNFα, IL-1β, IL-6 and CINC-1 and with selective ET-A (BQ-123) and ET-B (BQ-788) endothelin receptors antagonists. The MT-II-induced hyperalgesia was not altered by the lipoxygenase inhibitor zileuton, by the bradykinin B1 receptor antagonist Lys-(Des-Arg9,Leu8)-bradykinin, by the histamine and serotonin antagonists promethazine and methysergide, respectively, by the nitric oxide inhibitor LNMMA and by the inhibitor of matrix 1-, 2-, 3-, 8- and 9- metalloproteinases GM6001 (Ilomastat). Conclusion These results demonstrated the multi-mediated characteristic of the articular inflammation induced by MT-II, which demonstrates its relevance as a model for arthritis mechanisms and treatment evaluation.(AU)

Efeito do dinitrato de isossorbida sobre a hiperalgesia corneana induzida por lipopolissacarídeo / Isosorbide dinitrate effect on corneal hyperalgesia induced by lipopolysaccharide

OBJETIVO: Avaliar o efeito do dinitrato de isossorbida (DNI) sobre a hiperalgesia corneana e a infiltração de neutrófilos em um modelo experimental de ceratite superficial induzida pelo lipopolissacarídeo (LPS). MÉTODOS: A hiperalgesia foi estudada através da indução de uma ceratite inflamatória em ratos (n = 60), pela exposição da córnea ao LPS (LPS +). Após a exposição, os olhos foram tratados durante quatro dias, com as soluções tópicas: DNI (200 mg, 65 mg e 20 mg), prednisolona 1 por cento (PRED) ou Veículo. Olhos controles foram expostos apenas à solução salina (LPS -). Para avaliar a dor ocular, contouse o número de piscadas em 40 segundos, após instilação de uma gota de capsaicina 0,01 mM. A análise histopatológica foi realizada para avaliação da infiltração neutrofílica. RESULTADOS: Foram observadas áreas esbranquiçadas no estroma corneano dos olhos com a ceratite induzida pelo LPS, entre os dias 3 e 15 dias. Esses olhos (LPS +) apresentaram um número significativamente maior de piscadas que os LPS - (P = 0,019) à estimulação química. O tratamento tópico com o DNI reduziu o número de piscadas observadas (P = 0,010). Da mesma forma, olhos expostos ao LPS e tratados com DNI demonstraram redução significativa na infiltração neutrofílica (P = 0,0031). CONCLUSÃO: Baixas doses de DNI reduziram a hiperalgesia corneana e a infiltração neutrofílica nesse modelo de ceratite. Dessa forma, doadores de óxido nítrico, como o DNI, poderão ser úteis no futuro ao tratamento clínico de diversas condições dolorosas da superfície ocular.

PURPOSE: To analyze effects of isosorbide dinitrate (DNI) on corneal hyperalgesia and neutrophil infiltration in an experimental model of superficial keratitis induced by lipopolysaccharide (LPS). METHODS: Hyperalgesia was studied through the induction of inflammatory keratitis in rats (n=60), by corneal exposure to LPS. Following exposure, the inflamed eye was treated for four days, with one of the following solutions: topical DNI (200 λg, 65 λg and 20 λg), prednisolone 1 percent (PRED), and vehicle. Saline-exposed eyes (LPS -) underwent the same protocol. To evaluate ocular pain, the number of blinks in 40 seconds was counted, after one drop of 0.01 λM capsaicin. Histopathological analysis was performed with evaluation of neutrophil infiltration. RESULTS: White clouding areas were observed in the corneal stroma of eyes with LPS-induced keratitis, between day 3 and day 15. Eyes exposed to LPS had a significantly higher number of blinks than LPS - (P=0.019). Topical treatment of LPS-induced keratitis eyes with DNI reduced capsaicin-induced blinks (P=0.010). Similarly, eyes exposed to LPS and treated with DNI also displayed reduced neutrophil infiltration (P=0.0031). CONCLUSION: Low doses of topical NO donors, like DNI, reduce corneal hyperalgesia and neutrophil infiltration in this keratitis model. NO donors may be useful in the clinical treatment of painful conditions associated with surgical procedures of the ocular surface.

The pattern of immune cell infiltration in chromoblastomycosis: involvement of macrophage inflammatory protein-1 alpha/CCL3 and fungi persistence

Chromoblastomycosis (CR) is a subcutaneous chronic mycosis characterized by a granulomatous inflammatory response. However, little is known regarding the pattern of leukocyte subsets in CR and the pathways involved in their recruitment. The objective of this study was to assess the cellular subsets, chemokine, chemokine receptors and enzymes in CR. The inflammatory infiltrate was characterized by immunohistochemistry using antibodies against macrophages (CD68), Langerhans'cells (S100), lymphocytes (CD3, CD4, CD8, CD45RO, CD20 and CD56) and neutrophils (CD15). The expression of MIP-1alpha (Macrophage inflammatory protein-1alpha), chemokine receptors (CXCR3 and CCR1) and enzymes (superoxide dismutase-SOD and nitric oxide synthase-iNOS) was also evaluated by the same method. We observed an increase in all populations evaluated when compared with the controls. Numbers of CD15+ and CD56+ were significantly lower than CD3+, CD4+, CD20+ and CD68+ cells. Statistical analysis revealed an association of fungi numbers with CD3, CD45RO and iNOS-positive cells. Furthermore, MIP-1alpha expression was associated with CD45RO, CD68, iNOS and CXCR3. Our results suggest a possible role of MIP-1alpha and fungi persistence in the cell infiltration in CR sites.

A cromomicose é micose subcutânea crônica sistêmica caracterizada por resposta inflamatória crônica granulomatosa. No entanto, existem poucos dados a respeito do padrão de subtipos de leucócitos na cromomicose e sobre as vias envolvidas no recrutamento destas células. O objetivo deste trabalho foi avaliar os tipos celulares, bem como a expressão de quimiocinas, receptores de quimiocinas e enzimas em lesões de cromomicose. O infiltrado inflamatório foi caracterizado por meio de técnica imuno-histoquímica utilizando os seguintes marcadores CD68 (macrófagos), S100 (células de Langerhans), CD3, CD4, CD8, CD45RO, CD20 e CD56 (linfócitos) e CD15 (neutrófilos). A expressão de MIP-1alfa (Proteína Inflamatória do Macrófago-1alfa), receptores de quimiocinas (CXCR3 e CCR1) e enzimas (superóxido dismutase-SOD e óxido nítrico sintase induzida-iNOS) foi avaliada pelo mesmo método. Observou-se um aumento de todas as populações celulares avaliadas em relação às amostras controle. As populações de células CD15+ e CD56+ foram significativamente menores que células CD3+, CD4+, CD20+ e CD68+. A análise estatística revelou uma correlação positiva entre o número de fungos com as células CD3, CD45RO e iNOS-positivas. A expressão de MIP-1alfa foi também associada às populações de células CD45RO, CD68, iNOS e CXCR3 positivas. Nossos resultados apontam para um possível papel de MIP-1alfa e da persistência fúngica na infiltração de células inflamatórias nos sítios de cromomicose.