Matrix metalloproteinases 2 and 9 (gelatinases A and B) expression in malignant mesothelioma and benign pleura

Matrix metalloproteinases (MMPs), in particular the gelatinases (MMP-2 and -9), play a significant role in tumour invasion and angiogenesis. The expression and activities of MMPs have not been characterised in malignant mesothelioma (MM) tumour samples. In a prospective study, gelatinase activity was evaluated in homogenised supernatants of snap frozen MM (n = 35), inflamed pleura (IP, n = 12) and uninflammed pleura (UP, n = 14) tissue specimens by semiquantitative gelatin zymography. Matrix metalloproteinases were correlated with clinicopathological factors and with survival using Kaplan-Meier and Cox proportional hazard models. In MM, pro- and active MMP-2 levels were significantly greater than for MMP-9 (P = 0.006, P<0.001). Active MMP-2 was significantly greater in MM than in UP (P=0.04). MMP-2 activity was equivalent between IP and MM, but both pro- and active MMP-9 activities were greater in IP (P=0.02, P=0.009). While there were trends towards poor survival with increasing total and pro-MMP-2 activity (P=0.08) in univariate analysis, they were both independent poor prognostic factors in multivariate analysis in conjunction with weight loss (pro-MMP-2 P = 0.03, total MMP-2 P = 0.04). Total and pro-MMP-2 also contributed to the Cancer and Leukemia Group B prognostic groups. MMP-9 activities were not prognostic. Matrix metalloproteinases, and in particular MMP-2, the most abundant gelatinase, may play an important role in MM tumour growth and metastasis. Agents that reduce MMP synthesis and/or activity may have a role to play in the management of MM. © 2003 Cancer Research UK.

School children’s personal exposure to ultrafine particles in the urban environment

There has been considerable scientific interest in personal exposure to ultrafine particles (UFP). In this study, the inhaled particle surface area doses and dose relative intensities in the tracheobronchial and alveolar regions of lungs were calculated using the measured 24-hour UFP time series of school children personal exposures for each recorded activity. Bayesian hierarchical modelling was used to determine mean doses and dose intensities for the various microenvironments. Analysis of measured personal exposures for 137 participating children from 25 schools in the Brisbane Metropolitan Area showed similar trends for all the participating children. Bayesian regression modelling was performed to calculate the daily proportion of children's total doses at different microenvironments. The proportion of alveolar doses in the total daily dose for \emph{home}, \emph{school}, \emph{commuting} and \emph{other} were 55.3\%, 35.3\%, 4.5\% and 5.0\%, respectively, with the \emph{home} microenvironment contributing a majority of children's total daily dose. Children's mean indoor dose was never higher than the outdoor's at any of the schools, indicating there were no persistent indoor particle sources in the classrooms during the measurements. Outdoor activities, eating/cooking at home and commuting were the three activities with the highest dose intensities. Personal exposure was more influenced by the ambient particle levels than immediate traffic.

‘We’ve always cooked kangaroo. We still cook kangaroo. Although sometimes we use cookbooks now’ : Aboriginal Australians and cookbooks

Aboriginal Australians have a long history of eating native animals and plants. Food preparation techniques were handed down through the generations, without any need for cookbooks. But colonisation changed the diets of Aboriginal Australians, introducing us to a processed diet high in salt, sugar and fat, and causing a wide range of diet-related health problems. Over the years, many Aboriginal Australians lost their connections to traditional food preparation practices. In this paper, the authors provide a brief overview of Aboriginal food history and describe a newly-emerging focus on reintroducing native foods. They describe the work of an Aboriginal chef, Dale Chapman, who is actively promoting native foods and creating a native-Western food fusion. Chapman has developed native food recipes and a cookbook, in an effort to make native foods accessible to all Australians. She promotes a future when native foods are part of the identity of all Australians – both Aboriginal and non-Aboriginal.

Food literacy : what is it and does it influence what we eat?

"Food literacy" has emerged as a term to describe the everyday practicalities needed for healthy eating. It is increasingly used in policy, practice, research and in the public arena. This thesis empirically defined the term, identified its components, and developed models of its relationship to nutrition and health. Food literacy was examined from two perspectives; that of food experts and that of individuals using the case study of young people experiencing disadvantage. The research provides a common language and conceptualisation of food literacy which is being used by governments, policy-makers and practitioners to guide investment and practice.

Defining food literacy and its components

Food literacy has emerged as a term to describe the everyday practicalities associated with healthy eating. The term is increasingly used in policy, practice, research and by the public; however, there is no shared understanding of its meaning. The purpose of this research was to develop a definition of food literacy which was informed by the identification of its components. This was considered from two perspectives: that of food experts which aimed to reflect the intention of existing policy and investment, and that of individuals, who could be considered experts in the everyday practicalities of food provisioning and consumption. Given that food literacy is likely to be highly contextual, this second study focused on disadvantaged young people living in an urban area who were responsible for feeding themselves. The Expert Study used a Delphi methodology (round one n = 43). The Young People’s Study used semi-structured, life-course interviews (n = 37). Constructivist Grounded Theory was used to analyse results. This included constant comparison of data within and between studies. From this, eleven components of food literacy were identified which fell into the domains of: planning and management; selection; preparation; and eating. These were used to develop a definition for the term “food literacy”.

A comparison between bagasse and water efficient alternatives using KOH/AQ pulping

Numerous crops grow in sugar regions that have the potential to increase the amount of biomass available to a small bagasse-based pulp factory. Arundo donax and Sorghum offer unique advantages to farmers compared to other agricultural crops. Sorghum bicolour requires only 1/3 of the water of sugarcane. Arundo donax is a very high yield crop, it can also grow with little water but it has the further advantage in that it is also highly stress tolerant, making it suitable for land which is unsuited to other crops. Pulps produced from these crops were benchmarked against sugarcane bagasse pulp. Arundo, sorghum and bagasse were pulped using KOH and anthraquinone to 20 Kappa number so as to produce a bleachable pulp which is suitable for making photocopier paper and tissue products. The unbleached sorghum pulp has better tensile strength properties than the unbleached Arundo pulp (43.8 Nm/g compared to 21.4 Nm/g) and the bleached sorghum pulp tensile strength was similar to bagasse (28.4 Nm/g). At 20 Kappa number, sorghum pulp had acceptable yield for a non-wood fibre (45% c.f. 55% for bagasse), Arundo donax pulp had low tensile strength, and relatively low yield (38.7%), even for an agricultural fibre and required severe cooking conditions to achieve similar delignification to sugarcane bagasse or sorghum. Sorghum and Arundo donax produced thicker handsheets than bagasse (>160 µm c.f. 122 µm for bagasse). In preliminary experiments sorghum and bagasse responded slightly better to Totally Chlorine Free peroxide bleaching (QPP), although none achieved a satisfactory brightness level and further improvement would be required to produce a bleached pulp.

Osteogenic differentiation of murine embryonic stem cells is mediated by fibroblast growth factor receptors

The mechanisms involved in the control of embryonic stem (ES) cell differentiation are yet to be fully elucidated. However, it has become clear that the family of fibroblast growth factors (FGFs) are centrally involved. In this study we examined the role of the FGF receptors (FGFRs 1-4) during osteogenesis in murine ES cells. Single cells were obtained after the formation of embryoid bodies, cultured on gelatin-coated plates, and coaxed to differentiate along the osteogenic lineage. Upregulation of genes was analyzed at both the transcript and protein levels using gene array, relative-quantitative PCR (RQ-PCR), and Western blotting. Deposition of a mineralized matrix was evaluated with Alizarin Red staining. An FGFR1-specific antibody was generated and used to block FGFR1 activity in mES cells during osteogenic differentiation. Upon induction of osteogenic differentiation in mES cells, all four FGFRs were clearly upregulated at both the transcript and protein levels with a number of genes known to be involved in osteogenic differentiation including bone morphogenetic proteins (BMPs), collagen I, and Runx2. Cells were also capable of depositing a mineralized matrix, confirming the commitment of these cells to the osteogenic lineage. When FGFR1 activity was blocked, a reduction in cell proliferation and a coincident upregulation of Runx2 with enhanced mineralization of cultures was observed. These results indicate that FGFRs play critical roles in cell recruitment and differentiation during the process of osteogenesis in mES cells. In particular, the data indicate that FGFR1 plays a pivotal role in osteoblast lineage determination.

Sharpening our tools – translating research into practice

Providing culturally appropriate health communication tools at a community level, whilst meeting funding objectives set by Government led initiatives, can be challenging. Literature states that a translational research framework fostering community communication can encourage the development of appropriate communication tools to facilitate transfer of health information between community and researchers. Reflections from initial Need for Feed cooking and nutrition education program trials in remote Indigenous communities across Cape York indicated program resources were neither meeting community nor researchers needs. In response, a translational research framework was modelled with collaborative partnerships formed between researchers and community with the aim of modifying current resources. Local working groups were established to facilitate communication and guide continual remodelling and retrial of resources for successive programs. Feedback from working groups indicated community members wanted resources with more pictures and less words. Partnership with Chronic Disease Resources Online (CDRO) led to the development of pictorial resources including 3 evaluation tools, 27 recipe sets and 10 education support materials. Between June to December 2012 resources were trialled across 4 Cape York communities with 69 school aged children and 4 community elders. Qualitative data has indicated high satisfaction with modified pictorial resources, proving pictorial resources to be an effective and culturally appropriate method to both communicate health messages to community and facilitate flow of evaluation data to researchers. A translational research framework fostering communication between community and researchers can potentially enhance the quality of health communication tools.

Survival of rat functional dental pulp cells in vascularized tissue engineering chambers

Regenerative endodontics aims to preserve, repair or regenerate the dental pulp tissue. Dental pulp stem cells, have a potential use in dental tissue generation. However, specific requirements to drive the dental tissue generation are still obscured. We established an in vivo model for studying the survival of dental pulp cells (DPC) and their potential to generate dental pulp tissue. DPC were mixed with collagen scaffold with or without slow release bone morphogenic protein 4 (BMP-4) and fibroblast growth factor 2 (FGF2). The cell suspension was transplanted into a vascularized tissue engineering chamber in the rat groin. Tissue constructs were harvested after 2, 4, 6, and 8 weeks and processed for histomorphological and immunohistochemical analysis. After 2 weeks newly formed tissue with new blood vessel formation were observed inside the chamber. DPC were found around dentin, particularly around the vascular pedicle and also close to the gelatin microspheres. Cell survival, was confirmed up to 8 weeks after transplantation. Dentin Sialophosphoprotein (DSPP) positive matrix production was detected in the chamber, indicating functionality of dental pulp progenitor cells. This study demonstrates the potential of our tissue engineering model to study rat dental pulp cells and their behavior in dental pulp regeneration, for future development of an alternative treatment using these techniques.

Collagen induced MMP-2 activation in human breast cancer

Matrix metalloproteinase-2 (MMP-2), a zymogen requiring proteolytic activation for catalytic activity, has been implicated broadly in the invasion and metastasis of many cancer model systems, including human breast cancer (HBC). MMP-2 has been immunolocalized to carcinomatous human breast, where the degree of activation of MMP-2 correlates well with tumor grade and patient prognosis. Using Matrigel assays, we have stratified HBC cell lines for invasiveness in vitro, and compared this to their potential for metastatic spread in nude mice. HBC cell lines expressing the mesenchymal marker protein vimentin were found to be highly invasive in vitro, and tended to form metastases in nude mice. We have further discovered that culture on collagen-I gels (Vitrogen(TM): Vg) induces MMP-2-activator in highly invasive but not poorly invasive HBC cell lines. As seen for other MMP-2-activator inducing regimens, this induction requires protein synthesis and an intact MMP-2 hemopexin-like domain, appears to be mediated by a cell surface activity, and can be inhibited by metalloproteinase inhibitors. The induction is highly specific to collagen I, and is not seen with thin coatings of collagen I, collagen IV, laminin, or fibronectin, or with 3-dimensional gels of laminin, Matrigel, or gelatin. This review focuses on collagen I and MMP- 2, their localization and source in HBC, and their relationship(s) to MMP-2 activation and HBC metastasis. The relevance of collagen I in activation of MMP-2 in vivo is discussed in terms of stromal cell: tumor cell interaction for collagen I deposition, MMP-2 production and MMP-2-activation. Such cooperativity may exist in vivo for MMP-2 participation in HBC dissemination. A more complete understanding of the regulation of MMP-2-activator by type I collagen may provide new avenues for improved diagnosis and prognosis of human breast cancer.

Collagen-induced activation of the Mr. 72,000 type IV collagenase in normal and malignant human fibroblastoid cells

Although the Mr. 72,000 type IV collagenase (matrix metalloproteinase 2) has been implicated in a variety of normal and pathogenic processes, its activation mechanism in vivo is unclear. We have found that fibroblasts from normal and neoplastic human breast, as well as the sarcomatous human Hs578T and HT1080 cell lines, activate endogenous matrix metalloprotease 2 when cultured on type I collagen gels, but not on plastic, fibronectin, collagen IV, gelatin, matrigel, or basement membrane-like HR9 cell matrix. This activation is monitored by the zymographic detection of Mr 59,000 and/or Mr 62,000 species, requires 2-3 days of culture on vitrogen to manifest, is cycloheximide inhibitable, and correlates with an arborized morphology. A similar activation pattern was seen in these cells in response to Concanavalin A but not transforming growth factor β or 12-O-tetradecanoylphorbol-13-acetate. The interstitial matrix may thus play an important role in regulating matrix degradation in vivo.

Substrate choice of membrane-type 1 matrix metalloproteinase is dictated by tissue inhibitor of metalloproteinase-2 levels

Although tissue inhibitor of metalloproteinase-2 (TIMP-2) is known to be not only an inhibitor of matrix metalloproteinases (MMP) but also a cofactor for membrane-type 1 MMP (MT1-MMP)-mediated MMP-2 activation, it is still unclear how TIMP-2 regulates MMP-2 activation and cleavage of substrates by MT1-MMP. In the present study we examined the levels of cell-surface MT1-MMP, MMP-2 activation and cleavage of MT1-MMP substrates in 293T cells transfected with the MT1-MMP and TIMP-2 genes. Co-expression of TIMP-2 at an appropriate level increased the level of cell-surface MT1-MMP, both the TIMP-2-bound and free forms, and generated processed MMP-2 with gelatin-degrading activity. In contrast, MT1-MMP substrates testican-1 and syndecan-1 were cleaved by the cells expressing MT1-MMP, which was inhibited by TIMP-2 even at levels that stimulate MMP-2 activation. These results suggest that TIMP-2 environment determines MT1-MMP substrate choice between direct cleavage of its own substrates and MMP-2 activation.

Characterization and novel activation of 72-kDa metalloproteinase in retinal interphotoreceptor matrix and Y-79 cell culture medium

Analysis of bovine interphotoreceptor matrix and conditioned medium from human Y-79 retinoblastoma cells by gelatin SDS-PAGE zymography reveals abundant activity of a 72-kDa M(r) gelatinase. The 72-kDa gelatinase from either source is inhibited by EDTA but not aprotinin or NEM, indicating that it is a metalloproteinase (MMP). The 72-kDa MMP is converted to a 62-kDa species with APMA treatment after gelatin sepharose affinity purification typical of previously described gelatinase MMP-2. The latent 72-kDa gelatinase from either bovine IPM or Y-79 media autoactivates without APMA in the presence of calcium and zinc after 72 hr at 37°C, producing a fully active mixture of proteinase species, 50 (48 in Y-79 medium), 38 and 35 kDa in size. The presence of inhibitory activity was examined in both whole bovine IPM and IPM fractions separated by SDS-PAGE. Whole IPM inhibited gelatinolytic activity of autoactivated Y-79-derived MMP in a dose-dependent manner. Inhibitory activities are observed in two protein fractions of 27-42 and 20-25 kDa. Western blots using antibodies to human tissue inhibitor of metalloproteinase 1 and 2 (TIMP-1 and -2) reveal the presence of two TIMP-1-like proteins at 21 and 29 kDa in inhibitory fractions of the bovine IPM. TIMP-2 was not detected in the inhibitory IPM fractions, consistent with the observed autoactivation of bovine IPM 72-kDa gelatinase. Potential roles for this IPM MMP-TIMP system include physiologic remodelling of the neural retina-RPE cell interface and digestion of shed rod outer segment as well as pathological processes such as retinal detachment, PE cell migration, neovascularization and tumor progression. Cultured Y-79 cells appear to be a good model for studying the production and regulation of this proteinase system.

Scleral matrix metalloproteinases, serine proteinase activity and hydrational capacity are increased in myopia induced by retinal image degradation

In the avian model of myopia, retinal image degradation quickly leads to ocular enlargement. We now give evidence that regionally specific changes in ocular size are correlated with both biomechanical indices of scleral remodeling, e.g. hydration capacity and with biochemical changes in proteinase activities. The latter include a 72 kDa matrix metalloproteinase (putatively MMP-2), other gelatin-binding MMPs, an acid pH MMP and a serine protease. Specifically, we have found that increases in scleral hydrational capacity parallel increases in collagen degrading activities. Gelatin zymography reveals that eyes with 7 days of retinal image degradation have elevated levels (1.4-fold) of gelatinolytic activities at 72 and 67 kDa M(r) in equatorial and posterior pole regions of the sclera while, after 14 days of treatment, increases are no longer apparent. Lower M(r) zymographic activities at 50, 46 and 37 kDa M(r) are collectively increased in eyes treated for both 7 and 14 days (1.4- and 2.4-fold respectively) in the equator and posterior pole areas of enlarging eyes. Western blot analyses of scleral extracts with an antibody to human MMP-2 reveals immunoreactive bands at 65, 30 and 25 kDa. Zymograms incubated under slightly acidic conditions reveal that, in enlarging eyes, MMP activities at 25 and 28 kDa M(r) are increased in scleral equator and posterior pole (1.6- and 4.5-fold respectively). A TIMP-like protein is also identified in sclera and cornea by Western blot analysis. Finally, retinal-image degradation also increases (~2.6-fold) the activity of a 23.5 kDa serine proteinase in limbus, equator and posterior pole sclera that is inhibited by aprotinin and soybean trypsin inhibitor. Taken together, these results indicate that eye growth induced by retinal-image degradation involves increases in the activities of multiple scleral proteinases that could modify the biomechanical properties of scleral structural components and contribute to tissue remodeling and growth.

Adipose tissue engineering based on the controlled release of fibroblast growth factor-2 in a collagen matrix

Adipose tissue forms when basement membrane extract (Matrigel™) and fibroblast growth factor-2 (FGF-2) are added to our mouse tissue engineering chamber model. A mouse tumor extract, Matrigel is unsuitable for human clinical application, and finding an alternative to Matrigel is essential. In this study we generated adipose tissue in the chamber model without using Matrigel by controlled release of FGF-2 in a type I collagen matrix. FGF-2 was impregnated into biodegradable gelatin microspheres for its slow release. The chambers were filled with these microspheres suspended in 60 μL collagen gel. Injection of collagen containing free FGF-2 or collagen containing gelatin microspheres with buffer alone served as controls. When chambers were harvested 6 weeks after implantation, the volume and weight of the tissue obtained were higher in the group that received collagen and FGF-2 impregnated microspheres than in controls. Histologic analysis of tissue constructs showed the formation of de novo adipose tissue accompanied by angiogenesis. In contrast, control groups did not show extensive adipose tissue formation. In conclusion, this study has shown that de novo formation of adipose tissue can be achieved through controlled release of FGF-2 in collagen type I in the absence of Matrigel.