Experimental poisoning by Senecio brasiliensis in calves: quantitative and semi-quantitative study on changes in the hepatic extracellular matrix and sinusoidal cells

Extracellular matrix plays an important role in chronic hepatic lesions and has been studied in experimental intoxication models. However in cattle, studies on chronic disease have focused on the hepatocellular damage and extracellular matrix (ECM) changes are usually overlooked. There are no specific studies on the hepatic ECM in either normal or chronically damaged bovine liver. Thus an experimental model of hepatic toxicity model using Senecio brasiliensis poisoned calves was designed. Senecio brasiliensis contains pyrrolizidine alkaloids which cause either acute or chronic progressive dose dependent liver damage. Five calves were orally fed with 0.38g of dry leaves of S. brasiliensis/kg/day for 24 days. Liver needle biopsy specimens were obtained every 15 days for 60 days. Clinical signs of digestive complications appeared at 3rd week. One calf died on 45th day and four were evaluated up to 60th day. Biopsy samples were processed for routine light microscopy, immuno-histochemistry and transmission electron microscopy. From 30th day on progressive liver damage characterized by hepatocellular ballooning, necrosis, apoptosis and megalocytosis, centrilobular, pericellular and portal fibrosis were seen by light microscopy. Quantitative and semi-quantitative measurements of hepatic ECM components were performed before and after the onset of lesions. Morphometric analysis of total collagen and elastic fiber system was conducted. Total collagen and I and III collagen types progressively increased in throughout the liver of affected calves. Changes in location, amount and disposition of the elastic fiber system were also observed. Then numbers of Kupffer cells were significantly increased at 30th day and total numbers of sinusoidal cells were significantly increased at 45th and 60th days. Liver damage was progressive and irreversible even after the exposure to the plant was discontinued. Severe fibrotic lesions occurred mainly in portal tracts, followed by veno-occlusive and pericellular fibrosis. Collagen types I and III s were present in every normal and damaged liver, with predominance of type I. In affected calves the increase of total collagen and elastic fibers system paralleled the number of total sinusoidal cells.

Non-linear analysis of laminated metal matrix composites by an integrated micro/macro-mechanical model

The present paper describes an integrated micro/macro mechanical study of the elastic-viscoplastic behavior of unidirectional metal matrix composites (MMC). The micromechanical analysis of the elastic moduli is based on the Composites Cylinder Assemblage model (CCA) with comparisons also draw with a Representative Unit Cell (RUC) technique. These "homogenization" techniques are later incorporated into the Vanishing Fiber Diameter (VFD) model and a new formulation is proposed. The concept of a smeared element procedure is employed in conjunction with two different versions of the Bodner and Partom elastic-viscoplastic constitutive model for the associated macroscopic analysis. The formulations developed are also compared against experimental and analytical results available in the literature.

Regulation of pituitary hormones and cell proliferation by components of the extracellular matrix

The extracellular matrix is a three-dimensional network of proteins, glycosaminoglycans and other macromolecules. It has a structural support function as well as a role in cell adhesion, migration, proliferation, differentiation, and survival. The extracellular matrix conveys signals through membrane receptors called integrins and plays an important role in pituitary physiology and tumorigenesis. There is a differential expression of extracellular matrix components and integrins during the pituitary development in the embryo and during tumorigenesis in the adult. Different extracellular matrix components regulate adrenocorticotropin at the level of the proopiomelanocortin gene transcription. The extracellular matrix also controls the proliferation of adrenocorticotropin-secreting tumor cells. On the other hand, laminin regulates the production of prolactin. Laminin has a dynamic pattern of expression during prolactinoma development with lower levels in the early pituitary hyperplasia and a strong reduction in fully grown prolactinomas. Therefore, the expression of extracellular matrix components plays a role in pituitary tumorigenesis. On the other hand, the remodeling of the extracellular matrix affects pituitary cell proliferation. Matrix metalloproteinase activity is very high in all types of human pituitary adenomas. Matrix metalloproteinase secreted by pituitary cells can release growth factors from the extracellular matrix that, in turn, control pituitary cell proliferation and hormone secretion. In summary, the differential expression of extracellular matrix components, integrins and matrix metalloproteinase contributes to the control of pituitary hormone production and cell proliferation during tumorigenesis.

Dose-dependent in vitro inhibition of rabbit corneal matrix metalloproteinases by an extract of Pothomorphe umbellata after alkali injury

The in vitro ability of Pothomorphe umbellata ethanolic crude extract to inhibit matrix metalloproteinase (MMP) in normal cornea and in cornea after alkali injury was demonstrated. Corneas of albino rabbits were injured with 1 N NaOH for 20 s. After 48 h the corneas were excised, homogenized and analyzed for MMP-9 (92 kDa), pro-MMP-2 (72 kDa) and MMP-2 (67 kDa) activity by gelatin zymography. The activity was also measured in untreated corneas. After electrophoresis of 20 µg protein, gels were incubated with 50, 100, or 250 µg/mL lyophilized hydroethanolic (1:1) root crude extract of P. umbellata standardized for 4-nerolidylcatechol (7.09%). The activity of the enzymes was compared with that of untreated gel. At 48 h after injury, the activity of all MMPs was increased compared with untreated eyes. When the gels were incubated with P. umbellata extract the activity of MMP-2, pro-MMP-2 and MMP-9 decreased in a dose-dependent manner. MMP-9 activity decreased by approximately 50% after incubation with 50 µg/mL and was completely abolished at 100 and 250 µg/mL of the extract. After incubation with 50 µg/mL the activity of pro-MMP-2 and MMP-2 also decreased by 50%. The activity of pro-MMP-2 was almost completely abolished after incubation with 250 µg/mL of the extract. For MMP-2 the incubation with 100 or 250 µg/mL of the extract of P. umbellata promoted a 10-fold decrease in activity. In conclusion, P. umbellata root crude extract can be useful as an alternative therapy to control MMP activity after corneal injury.

Modulation of extracellular matrix by nutritional hepatotrophic factors in thioacetamide-induced liver cirrhosis in the rat

Nutritional substances associated to some hormones enhance liver regeneration when injected intraperitoneally, being denominated hepatotrophic factors (HF). Here we verified if a solution of HF (glucose, vitamins, salts, amino acids, glucagon, insulin, and triiodothyronine) can revert liver cirrhosis and how some extracellular matrices are affected. Cirrhosis was induced for 14 weeks in 45 female Wistar rats (200 mg) by intraperitoneal injections of thioacetamide (200 mg/kg). Twenty-five rats received intraperitoneal HF twice a day for 10 days (40 mL·kg-1·day-1) and 20 rats received physiological saline. Fifteen rats were used as control. The HF applied to cirrhotic rats significantly: a) reduced the relative mRNA expression of the genes: Col-α1 (-53%), TIMP-1 (-31.7%), TGF-β1 (-57.7%), and MMP-2 (-41.6%), whereas Plau mRNA remained unchanged; b) reduced GGT (-43.1%), ALT (-17.6%), and AST (-12.2%) serum levels; c) increased liver weight (11.3%), and reduced liver collagen (-37.1%), regenerative nodules size (-22.1%), and fibrous septum thickness. Progranulin protein (immunohistochemistry) and mRNA (in situ hybridization) were found in fibrous septa and areas of bile duct proliferation in cirrhotic livers. Concluding, HF improved the histology and serum biochemistry of liver cirrhosis, with an important reduction of interstitial collagen and increased extracelullar matrix degradation by reducing profibrotic gene expression.

The absorption of vitamin E is influenced by the amount of fat in a meal and the food matrix

Vitamin E absorption requires the presence of fat; however, limited information exists on the influence of fat quantity on optimal absorption. In the present study we compared the absorption of stable-isotope-labelled vitamin E following meals of varying fat content and source. In a randomised four-way cross-over study, eight healthy individuals consumed a capsule containing 150 mg H-2-labelled RRR-alpha-tocopheryl acetate with a test meal of toast with butter (17.5 g fat), cereal with full-fat milk (17.5 g fat), cereal with semi-skimmed milk (2.7 g fat) and water (0g fat). Blood was taken at 0, 0.5, 1, 1.5, 2, 3, 6 and 9 h following ingestion, chylomicrons were isolated, and H-2-labelled alpha-tocopherol was analysed in the chylomicron and plasma samples. There was a significant time (P<0.001) and treatment effect (P<0.001) in H-2-labelled alpha-tocopherol concentration in both chylomicrons and plasma between the test meals. H-2-labelled alpha-tocopherol concentration was significantly greater with the higher-fat toast and butter meal compared with the low-fat cereal meal or water (P< 0.001), and a trend towards greater concentration compared with the high-fat cereal meal (P= 0.065). There was significantly greater H-2-labelled α-tocopherol concentration with the high-fat cereal meal compared with the low-fat cereal meal (P< 0.05). The H-2-labelled alpha-tocopherol concentration following either the low-fat cereal meal or water was low. These results demonstrate that both the amount of fat and the food matrix influence vitamin E absorption. These factors should be considered by consumers and for future vitamin E intervention studies.

Direct observation of membrane insertion by enveloped virus matrix proteins by phosphate displacement

Enveloped virus release is driven by poorly understood proteins that are functional analogs of the coat protein assemblies that mediate intracellular vesicle trafficking. We used differential electron density mapping to detect membrane integration by membrane-bending proteins from five virus families. This demonstrates that virus matrix proteins replace an unexpectedly large portion of the lipid content of the inner membrane face, a generalized feature likely to play a role in reshaping cellular membranes.

Unpacking the mechanism by which psychological ownership manifests at the level of the individual: a dynamic model of identity and self

Increasing prominence of the psychological ownership (PO) construct in management studies raises questions about how PO manifests at the level of the individual. In this article, we unpack the mechanism by which individuals use PO to express aspects of their identity and explore how PO manifestations can display congruence as well as incongruence between layers of self. As a conceptual foundation, we develop a dynamic model of individual identity that differentiates between four layers of self, namely, the “core self,” “learned self,” “lived self,” and “perceived self.” We then bring identity and PO literatures together to suggest a framework of PO manifestation and expression viewed through the lens of the four presented layers of self. In exploring our framework, we develop a number of propositions that lay the foundation for future empirical and conceptual work and discuss implications for theory and practice.

Matrix Metalloproteinase Expression in Teeth with Apical Periodontitis Is Differentially Modulated by the Modality of Root Canal Treatment

Introduction: The objective of this study was to investigate the expression of matrix metalloproteinases (MM Ps) in apical periodontitis and during the periapical healing phase after root canal treatment. Methods: Apical periodontitis was induced in dog teeth, and root canal treatment was performed in a single visit or by using an additional calcium hydroxide root canal dressing. One hundred eighty days after treatment the presence of inflammation was examined, and tissues were stained to detect bacteria. Bacterial status was correlated to the degree of tissue organization, and to further investigate molecules involved in this process, tissues were stained for MMP-1, MMP-2, MMP-8, and MMP-9. Data were analyzed by using one-way analysis of variance followed by Tukey test or Kruskal-Wallis followed by Dunn test. Results: Teeth with apical periodontitis that had root canal therapy performed in a single visit presented an intense inflammatory cell infiltrate. Periapical tissue was extremely disorganized, and this was correlated with the presence of bacteria. Higher MMP expression was evident, similar to teeth with untreated apical periodontitis. In contrast, teeth with apical periodontitis submitted to root canal treatment with calcium hydroxide presented a lower inflammatory cell infiltrate. This group had moderately organized connective tissue, lower prevalence of bacteria, and lower number of MMP-positive cells, similar to healthy teeth submitted to treatment. Conclusions: Teeth treated with calcium hydroxide root canal dressing exhibited a lower percentage of bacterial contamination, a lower MMP expression, and a more organized extracellular matrix, unlike those treated in a single visit. This suggests that calcium hydroxide might be beneficial in tissue repair processes. (J Endod 2010;36:231-237)

High Matrix Metalloproteinase Activity Is a Hallmark of Periapical Granulomas

Introduction: The inability to distinguish periapical cysts from granulomas before performing root canal treatment leads to uncertainty in treatment outcomes because cysts have lower healing rates. Searching for differential expression of molecules within cysts or granulomas could provide information with regard to the identity of the lesion or suggest mechanistic differences that may form the basis for future therapeutic intervention. Thus, we investigated whether granulomas and cysts exhibit differential expression of extracellular matrix (ECM) molecules. Methods: Human periapical granulomas, periapical cysts, and healthy periodontal ligament tissues were used to investigate the differential expression of ECM molecules by microarray analysis. Because matrix metalloproteinases (MMP) showed the highest differential expression in the microarray analysis, MMPs were further examined by in situ zymography and immunohistochemistry. Data were analyzed by using one-way analysis of variance followed by the Tu-key test. Results: We observed that cysts and granulomas differentially expressed several ECM molecules, especially those from the MMP family. Compared with cysts, granulomas exhibited higher MMP enzymatic activity in areas stained for MMP-9. These areas were composed of polymorphonuclear cells (PMNs) in contrast to cysts. Similarly, MMP-13 was expressed by a greater number of cells in granulomas compared with cysts. Conclusion: Our findings indicate that high enzymatic MIMP activity in PMNs together with MMP-9 and MMP-13 stained cells could be a molecular signature of granulomas unlike periapical cysts. (J Endod 2009;35:1234-1242)

Human Stem Cell Cultures from Cleft Lip/Palate Patients Show Enrichment of Transcripts Involved in Extracellular Matrix Modeling By Comparison to Controls

Nonsyndromic cleft lip and palate (NSCL/P) is a complex disease resulting from failure of fusion of facial primordia, a complex developmental process that includes the epithelial-mesenchymal transition (EMT). Detection of differential gene transcription between NSCL/P patients and control individuals offers an interesting alternative for investigating pathways involved in disease manifestation. Here we compared the transcriptome of 6 dental pulp stem cell (DPSC) cultures from NSCL/P patients and 6 controls. Eighty-seven differentially expressed genes (DEGs) were identified. The most significant putative gene network comprised 13 out of 87 DEGs of which 8 encode extracellular proteins: ACAN, COL4A1, COL4A2, GDF15, IGF2, MMP1, MMP3 and PDGFa. Through clustering analyses we also observed that MMP3, ACAN, COL4A1 and COL4A2 exhibit co-regulated expression. Interestingly, it is known that MMP3 cleavages a wide range of extracellular proteins, including the collagens IV, V, IX, X, proteoglycans, fibronectin and laminin. It is also capable of activating other MMPs. Moreover, MMP3 had previously been associated with NSCL/P. The same general pattern was observed in a further sample, confirming involvement of synchronized gene expression patterns which differed between NSCL/P patients and controls. These results show the robustness of our methodology for the detection of differentially expressed genes using the RankProd method. In conclusion, DPSCs from NSCL/P patients exhibit gene expression signatures involving genes associated with mechanisms of extracellular matrix modeling and palate EMT processes which differ from those observed in controls. This comparative approach should lead to a more rapid identification of gene networks predisposing to this complex malformation syndrome than conventional gene mapping technologies.

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.