Hanseníase neural, aspectos diagnósticos da forma neural pura e mecanismos imunopatogênicos da lesão do nervo na doença. Participação de quimiocinas CCL2 e CXCL10 e metaloproteinases 2 e 9

O diagnóstico da hanseníase neural pura baseia-se em dados clínicos e laboratoriais do paciente, incluindo a histopatologia de espécimes de biópsia de nervo e detecção de DNA de Mycobacterium leprae (M. leprae) pelo PCR. Como o exame histopatológico e a técnica PCR podem não ser suficientes para confirmar o diagnóstico, a imunomarcação de lipoarabinomanana (LAM) e/ou Glicolipídio fenólico 1 (PGL1) - componentes de parede celular de M. leprae foi utilizada na primeira etapa deste estudo, na tentativa de detectar qualquer presença vestigial do M. leprae em amostras de nervo sem bacilos. Além disso, sabe-se que a lesão do nervo na hanseníase pode diretamente ser induzida pelo M. leprae nos estágios iniciais da infecção, no entanto, os mecanismos imunomediados adicionam severidade ao comprometimento da função neural em períodos sintomáticos da doença. Este estudo investigou também a expressão imuno-histoquímica de marcadores envolvidos nos mecanismos de patogenicidade do dano ao nervo na hanseníase. Os imunomarcadores selecionados foram: quimiocinas CXCL10, CCL2, CD3, CD4, CD8, CD45RA, CD45RO, CD68, HLA-DR, e metaloproteinases 2 e 9. O estudo foi desenvolvido em espécimes de biópsias congeladas de nervo coletados de pacientes com HNP (n=23 / 6 BAAR+ e 17 BAAR - PCR +) e pacientes diagnosticados com outras neuropatias (n=5) utilizados como controle. Todas as amostras foram criosseccionadas e submetidas à imunoperoxidase. Os resultados iniciais demonstraram que as 6 amostras de nervos BAAR+ são LAM+/PGL1+. Já entre as 17 amostras de nervos BAAR-, 8 são LAM+ e/ou PGL1+. Nas 17 amostras de nervos BAAR-PCR+, apenas 7 tiveram resultados LAM+ e/ou PGL1+. A detecção de imunorreatividade para LAM e PGL1 nas amostras de nervo do grupo HNP contribuiu para a maior eficiência diagnóstica na ausência recursos a diagnósticos moleculares. Os resultados da segunda parte deste estudo mostraram que foram encontradas imunoreatividade para CXCL10, CCL2, MMP2 e MMP9 nos nervos da hanseníase, mas não em amostras de nervos com outras neuropatias. Além disso, essa imunomarcação foi encontrada predominantemente em células de Schwann e em macrófagos da população celular inflamatória nos nervos HNP. Os outros marcadores de ativação imunológica foram encontrados em leucócitos (linfócitos T e macrófagos) do infiltrado inflamatório encontrados nos nervos. A expressão de todos os marcadores, exceto CXCL10, apresentou associação com a fibrose, no entanto, apenas a CCL2, independentemente dos outros imunomarcadores, estava associada a esse excessivo depósito de matriz extracelular. Nenhuma diferença na frequência da imunomarcação foi detectada entre os subgrupos BAAR+ e BAAR-, exceção feita apenas às células CD68+ e HLA-DR+, que apresentaram discreta diferença entre os grupos BAAR + e BAAR- com granuloma epitelioide. A expressão de MMP9 associada com fibrose é consistente com os resultados anteriores do grupo de pesquisa. Estes resultados indicam que as quimiocinas CCL2 e CXCL10 não são determinantes para o estabelecimento das lesões com ou sem bacilos nos em nervo em estágios avançados da doença, entretanto, a CCL2 está associada com o recrutamento de macrófagos e com o desenvolvimento da fibrose do nervo na lesão neural da hanseníase.

Delayed local inflammatory response induced by Thalassophryne nattereri venom is related to extracellular matrix degradation

Symptoms evoked by Thalassophryne nattereri fish envenomation include local oedema, severe pain and intense necrosis with strikingly inefficient healing, continuing for several weeks or months. Investigations carried out in our laboratory showed that, in the venom-induced acute inflammation, thrombosis in venules and constrictions in arterioles were highly visible, in contrast to a notable lack of inflammatory cell. Nevertheless, the reason that the venom toxins favour delayed local inflammatory response is poorly defined. In this study, we analysed the movement of leucocytes after T. nattereri venom injection in the intraplantar region of Swiss mice, the production of pro-inflammatory mediators and the venom potential to elicit matrix metalloproteinase production and extracellular matrix degradation. Total absence of mononuclear and neutrophil influx was observed until 14 days, but the venom stimulates pro-inflammatory mediator secretion. Matrix metalloproteinases (MMP)-2 and MMP-9 were detected in greater quantities, accompanied by tissue degradation of collagenous fibre. An influx of mononuclear cells was noted very late and at this time the levels of IL-6, IL-1 beta and MMP-2 remained high. Additionally, the action of venom on the cytoskeletal organization was assessed in vitro. Swift F-actin disruption and subsequent loss of focal adhesion was noted. Collectively these findings show that the altered specific interaction cell-matrix during the inflammatory process creates an inadequate environment for infiltration of inflammatory cells.

Matrix metalloproteinases with gelatinolytic activity induced by Paracoccidioides brasiliensis infection

P>Matrix metalloproteinases (MMPs) modulate extracellular matrix turnover, inflammation and immunity. We studied MMP-9 and MMP-2 in experimental paracoccidioidomycosis. At 15 and 120 days after infection (DAI) with virulent Paracoccidioides brasiliensis, MMP-9 was positive by immunohistochemistry in multinucleated giant cells, in mononuclear cells with macrophage and lymphocyte morphologies and also in fungal cells in the lesions of susceptible and resistant mice. Using gelatin zymography, pro- and active MMP-9 and active MMP-2 were detected in all infected mice, but not in controls. Gelatinolytic activity was not observed in P. brasiliensis extracts. Semiquantitative analysis of gelatinolytic activities revealed weak or absent MMP-2 and strong MMP-9 activity in both mouse strains at 15 DAI, declining at 120 DAI. Avirulent P. brasiliensis-infected mice had residual lesions with MMP-9-positive pseudoxantomatous macrophages, but no gelatinase activity at 120 DAI. Our findings demonstrate the induction of MMPs, particularly MMP-9, in experimental paracoccidioidomycosis, suggesting a possible influence in the pattern of granulomas and in fungal dissemination.

Expression of matrix metalloproteinases, type IV collagen, and interleukin-10 in rabbits treated with morphine after lamellar keratectomy

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

High levels of serum matrix metalloproteinases in dogs with natural visceral leishmaniosis: A preliminary report

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Effects of cyclosporin, phenytoin, and nifedipine on the synthesis and degradation of gingival collagen in tufted capuchin monkeys (Cebus apella): Histochemical and MMP-1 and -2 and collagen I gene expression analyses

Background: The purpose of this experimental study was to evaluate the collagen fiber distribution histologically after phenytoin, cyclosporin, or nifedipine therapy and to correlate it with collagen I and matrix metalloproteinase (MMP)-1 and -2 gene expression levels.Methods: Gingival samples from the canine area were obtained from 12 male monkeys (Cebus apella). The mesial part of each sample was assessed by reverse transcription-polymerase chain reaction, whereas the distal part was processed histologically for picrosirius red and hematoxylin and eosin stainings, as well as for collagen IV immunostaining. One week after the first biopsy, the animals were assigned to three groups that received daily oral dosages of cyclosporin, phenytoin, or nifedipine for 120 days. Additional gingival samples were obtained on days 52 and 120 of treatment from two animals from each group on the opposite sides from the first biopsies.Results: Picrosirius red staining showed a predominance of mature collagen fibers in the control group. Conversely, there was an enlargement of areas occupied by immature collagen fibers in all groups at days 52 and 120, which was not uniform over each section. There was a general trend to lower levels of MMP-1 gene expression on day 52 and increased levels on day 120. Phenytoin led to increased levels of MMP-2 and collagen I gene expression on day 120, whereas the opposite was observed in the nifedipine group.Conclusion: Cyclosporin, phenytoin, and nifedipine led to phased and drug-related gene expression patterns, resulting in impaired collagen metabolism, despite the lack of prominent clinical signs.

The Short-Term Effectiveness of Non-Surgical Treatment in Reducing Levels of Interleukin-1 beta and Proteases in Gingival Crevicular Fluid From Patients With Type 2 Diabetes Mellitus and Chronic Periodontitis

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Calcaneal Tendon Regions Exhibit Different MMP-2 Activation After Vertical Jumping and Treadmill Running

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Finasteride treatment alters MMP-2 and-9 gene expression and activity in the rat ventral prostate

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Proteinase-activated receptor-2 (PAR2) agonist causes periodontitis in rats

Proteinase-activated receptor-2 (PAR2) is a G-protein-coupled receptor that mediates cellular responses to extracellular proteinases. Since PAR2 is expressed by oral epithelial cells, osteoblasts, and gingival fibroblasts, where its activation releases interleukin-8, we hypothesized that PAR2 activation may participate in periodontal disease in vivo. We investigated the role of PAR2 activation in periodontal disease in rats. Radiographic and enzymatic (myeloperoxidase) analysis revealed that topical application of PAR2 agonist causes periodontitis but also exacerbates existing periodontitis, leading to significant alveolar bone loss and gingival granulocyte infiltration. Inhibition of matrix metalloproteinase (MMP) and cyclo-oxygenase (COX) decreased PAR2 agonist-induced periodontitis. More specifically, the overexpression of COX-1, COX-2, MMP-2, and MMP-9 in gingival tissues suggests that they are involved in PAR 2-induced periodontitis. In conclusion, PAR2 agonist causes periodontitis in rats through a mechanism involving prostaglandin release and MMP activation. Inhibition of PAR2 may represent a novel approach to modulate host response in periodontitis.

Effect of phosphoric acid on the degradation of human dentin matrix

This study determined if dentin proteases are denatured by phosphoric acid (PA) used in etch-and-rinse dentin adhesives. Dentin beams were completely demineralized with EDTA for 30 days. We acid-etched experimental groups by exposing the demineralized dentin beams to 1, 10, or 37 mass% PA for 15 sec or 15 min. Control beams were not exposed to PA but were incubated in simulated body fluid for 3 days to assay their total endogenous telopeptidase activity, by their ability to solubilize C-terminal crosslinked telopeptides ICTP and CTX from insoluble dentin collagen. Control beams released 6.1 ± 0.8 ng ICTP and 0.6 ± 0.1 ng CTX/mg dry-wt/3 days. Positive control beams pre-incubated in p-aminophenylmercuric acetate, a compound known to activate proMMPs, released about the same amount of ICTP peptides, but released significantly less CTX. Beams immersed in 1, 10, or 37 mass% PA for 15 sec or 15 min released amounts of ICTP and CTX similar to that released by the controls (p > 0.05). Beams incubated in galardin, an MMP inhibitor, or E-64, a cathepsin inhibitor, blocked most of the release of ICTP and CTX, respectively. It is concluded that PA does not denature endogenous MMP and cathepsin activities of dentin matrices. © 2013 International & American Associations for Dental Research.

Low level laser therapy increases angiogenesis in a model of ischemic skin flap in rats mediated by VEGF, HIF-1α and MMP-2

It is known that low level laser therapy is able to improve skin flap viability by increasing angiogenesis. However, the mechanism for new blood vessel formation is not completely understood. Here, we investigated the effects of 660 nm and 780 nm lasers at fluences of 30 and 40 J/cm2 on three important mediators activated during angiogenesis. Sixty male Wistar rats were used and randomly divided into five groups with twelve animals each. Groups were distributed as follows: skin flap surgery non-irradiated group as a control; skin flap surgery irradiated with 660 nm laser at a fluence of 30 or 40 J/cm2 and skin flap surgery irradiated with 780 nm laser at a fluence of 30 or 40 J/cm2. The random skin flap was performed measuring 10 × 4 cm, with a plastic sheet interposed between the flap and the donor site. Laser irradiation was performed on 24 points covering the flap and surrounding skin immediately after the surgery and for 7 consecutive days thereafter. Tissues were collected, and the number of vessels, angiogenesis markers (vascular endothelial growth factor, VEGF and hypoxia inducible factor, HIF-1α) and a tissue remodeling marker (matrix metalloproteinase, MMP-2) were analyzed. LLLT increased an angiogenesis, HIF-1α and VEGF expression and decrease MMP-2 activity. These phenomena were dependent on the fluences, and wavelengths used. In this study we showed that LLLT may improve the healing of skin flaps by enhancing the amount of new vessels formed in the tissue. Both 660 nm and 780 nm lasers were able to modulate VEGF secretion, MMP-2 activity and HIF-1α expression in a dose dependent manner. © 2013 Published by Elsevier B.V.

Platelet-Rich Plasma And Chronic Wounds: Remaining Fibronectin May Influence Matrix Remodeling And Regeneration Success

Background: Platelet-rich plasma has been largely used as a therapeutic option for the treatment of chronic wounds of different etiologies. The enhanced regeneration observed after the use of platelet-rich plasma has been systematically attributed to the growth factors that are present inside platelets' granules. Aim: We hypothesize that the remaining plasma and platelet-bound fibronectin may act as a further bioactive protein in platelet-rich plasma preparations. Methods: Recent reports were analyzed and presented as direct evidences of this hypotheses. Results: Fibronectin may directly influence the extracellular matrix remodeling during wound repair. This effect is probably through matrix metalloproteinase expression, thus exerting an extra effect on chronic wound regeneration. Conclusions: Physicians should be well aware of the possible fibronectin-induced effects in their future endeavors with PRP in chronic wound treatment. © 2013 International Society for Cellular Therapy.

Extracellular matrix remodeling of the testes through the male reproductive cycle in Teleostei fish

During the fish reproductive cycle, testes undergo morphological changes related to germinal epithelium and remodeling of extracellular matrix components (ECM). ECM is degraded mainly by action of matrix metalloproteinases (MMPs). Due to the natural renewal of ECM in fish testes, we choose Pimelodus maculatus to study remodeling of ECM throughout reproductive cycle, using picrosirius (to identify type I, II, III collagen) and reticulin (type III collagen), and to immunolocalize MT1-MMP (membrane type 1-matrix metalloproteinase) and MMP-2 in testis cells. Testes were classified in four reproductive phases: regenerating, development, spawning capable and regressing. Picrosirius and reticulin demonstrated a differential distribution of total collagen fibers during the reproductive cycle. Immunohistochemistry showed MT1-MMP only in acidophilic granulocyte cells mainly inside blood vessels, in connective tissue of capsule close to the germinal compartment, and also infiltrated in interstitial connective tissue. MMP-2 was detected in fibroblast and endothelial cells of interstitial and capsule blood vessels, in epithelial cells of capsule, and in acidophilic granulocyte cells at same description for MT1-MMP. The fish testes ECM were remodeled throughout reproductive cycle in according to morphophysiological alterations. During reproductive season (spawning capable), the interstitium increased in total collagen fibers (type I, II, III). After spermiation period (regression and regenerating), the amount of collagen fibers decreased in response to action of MMPs on collagen degradation and other interstitial components (not assessed in this study). MMPs seem to be indispensable components for natural cyclic events of ECM remodeling of fish testes and for guarantee tissue homeostasis throughout reproductive cycle.

Down-regulation of protease activated receptor 2, interleukin-17 and other pro-inflammatory genes by subantimicrobial doxycycline dose in a rat periodontitis model

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)