Matrix assembly and homeostasis in collagen-rich tissues are mediated by interactions with proteoglycans (PGs) substituted with sulfated glycosaminoglycans (GAGs). The major GAG in cornea is keratan ...sulfate (KS), which is N-linked to one of three PG core proteins. To ascertain the importance of the carbohydrate chain sulfation step in KS functionality, we generated a strain of mice with a targeted gene deletion in Chst5 , which encodes an N -acetylglucosamine-6- O -sulfotransferase that is integral to the sulfation of KS chains. Corneas of homozygous mutants were significantly thinner than those of WT or heterozygous mice. They lacked high-sulfated KS, but contained the core protein of the major corneal KSPG, lumican. Histochemically stained KSPGs coassociated with fibrillar collagen in WT corneas, but were not identified in the Chst5 -null tissue. Conversely, abnormally large chondroitin sulfate/dermatan sulfate PG complexes were abundant throughout the Chst5 -deficient cornea, indicating an alteration of controlled PG production in the mutant cornea. The corneal stroma of the Chst5 -null mouse exhibited widespread structural alterations in collagen fibrillar architecture, including decreased interfibrillar spacing and a more spatially disorganized collagen array. The enzymatic sulfation of KS GAG chains is thus identified as a key requirement for PG biosynthesis and collagen matrix organization. collagen glycosaminoglycans proteoglycans
Issue addressed: Children and adults in Australia are not eating the recommended amounts of fruits and vegetables. Our objective was to assess the effectiveness of a health promotion intervention to ...improve fruit and vegetable intake among primary school children and their families in NSW.
Methods: The Cancer Council New South Wales Healthy Lunch Box sessions were a 25-minute session delivered to parents of primary school-aged children. The sessions provided information and resources about fruit and vegetables and healthy school lunch boxes. The evaluation is a quantitative uncontrolled pre-post design. Data were collected using three questionnaires, pre-intervention, 1 week post-intervention and 6 months post-intervention.
Results: A total of 204 parents completed all three evaluation questionnaires to 6 months. Knowledge of recommended intakes and serving sizes of fruit and vegetables improved significantly after the intervention. There was an increase in parents reporting packing vegetables (often/always) in the child's lunch box at 1 week (47%) and 6 months post-intervention (40%) compared to pre-intervention (32%). The proportion of parents reporting that they were confident in packing a healthy lunch box increased from 45% pre-intervention to 62% after the intervention.
Conclusion: The Healthy Lunch Box sessions were effective in improving parental knowledge and practices related to fruit and vegetables and parental confidence with packing a healthy lunch box.
So what? This short intervention could be a useful component of a portfolio of interventions to support parents with knowledge and resources to pack a healthy lunch box for their children.
Aggrecanases are considered to play a key role in the destruction of articular cartilage during the progression of arthritis. Here we report that the N-terminal inhibitory domain of tissue inhibitor ...of metalloproteinases 3 (N-TIMP-3), but not TIMP-1 or TIMP-2, inhibits glycosaminoglycan release from bovine nasal and porcine articular cartilage explants stimulated with interleukin-1α or retinoic acid in a dose-dependent manner. This inhibition is due to the blocking of aggrecanase activity induced by the catabolic factors. Little apoptosis of primary porcine chondrocytes is observed at an effective concentration of N-TIMP-3. These results suggest that TIMP-3 may be a candidate agent for use against cartilage degradation.
This study aims to review the literature regarding the barriers to sampling, recruitment, participation, and retention of members of socioeconomically disadvantaged groups in health research and ...strategies for increasing the amount of health research conducted with socially disadvantaged groups.
A systematic review with narrative synthesis was conducted. Searches of electronic databases Medline, PsychInfo, EMBASE, Social Science Index via Web of Knowledge and CINHAL were conducted for English language articles published up to May 2013. Qualitative and quantitative studies as well as literature reviews were included. Articles were included if they reported attempts to increase disadvantaged group participation in research, or the barriers to research with disadvantaged groups. Groups of interest were those described as socially, culturally or financially disadvantaged compared to the majority of society. Eligible articles were categorised according to five phases of research: 1) sampling, 2) recruitment and gaining consent, 3) data collection and measurement, 4) intervention delivery and uptake, and 5) retention and attrition.
In total, 116 papers from 115 studies met inclusion criteria and 31 previous literature reviews were included. A comprehensive summation of the major barriers to working with various disadvantaged groups is provided, along with proposed strategies for addressing each of the identified types of barriers. Most studies of strategies to address the barriers were of a descriptive nature and only nine studies reported the results of randomised trials.
To tackle the challenges of research with socially disadvantaged groups, and increase their representation in health and medical research, researchers and research institutions need to acknowledge extended timeframes, plan for higher resourcing costs and operate via community partnerships.
Immunohistochemistry is a powerful investigative tool that can provide researchers with important supplemental information to the routine morphological assessment of musculo-skeletal connective ...tissues in health and disease and also during tissue repair and regeneration. A wide variety of antibodies (both monoclonal and polyclonal) are now available from commercial and non-commercial sources that recognise the major structural and soluble components of cellular and extracellular matrix compartments. These include antibodies towards the major collagen and proteoglycan species and their metabolites, glycosaminoglycans, glycoproteins, enzymes, enzyme generated neo-epitopes, growth factors, cytokines and related signalling molecules. In addition, cell surface markers, cytoskeletal components and many other cytoplasmic and nuclear proteins, too numerous to mention, can also be detected. When allied with high resolution imaging modalities (e.g. confocal laser scanning microscopy) immunohistochemistry thus has the potential to reveal a wealth of macromolecular information about the complex three-dimensional composition and organisation of cellular and extracellular matrix compartments in many different connective tissue types. These technologies can also be used to quantify signal intensities and thereby facilitate numerical computation of image data.
We have previously described a large proteoglycan named superficial zone protein that was isolated and purified from culture medium of superficial slices of bovine articular cartilage. Monoclonal ...antibodies were raised against superficial zone protein and used as probes in Western blot analyses for immunohistochemical studies both to determine precisely which cells within the joint synthesize the proteoglycan and to isolate a cDNA fragment from a bovine chondrocyte lambdagt11 library that encodes part of the proteoglycan. The cDNA fragment that was obtained with use of monoclonal antibody 6-A-1 encodes the 3' end of the sequence for superficial zone protein. On Western blots, monoclonal antibody 3-A-4 recognized an epitope on native, but not reduced, superficial zone protein, whereas monoclonal antibody 6-A-1 reacted with both native and denatured antigen. The proteoglycan was immunolocalized with monoclonal antibody 3-A-4 in chondrocytes predominantly within the superficial zone of fetal and adult articular cartilage and in some cells of the synovial lining. However, the proteoglycan was not detected in chondrocytes deep in articular cartilage, in nasal septal cartilage, or in synovial stromal cells. The only matrix staining positively for superficial zone protein was at the articular surface bordering the synovial cavity in adult, but not fetal, joints. Isolated chondrocytes and synovial cells showed intracellular binding of monoclonal antibody 3-A-4, and flow-cytometric analysis with the antibody gave the following percentages of immunopositive cells: 37.4, 52.5, 3.4, and 7.5 from chondrocytes from the full-thickness, superficial, and deep zones and from synovial cells, respectively. Thus, both chondrocytes and synovial cells bordering the joint cavity synthesize superficial zone protein and substantiate its usefulness as a phenotypic marker of particular cellular species lining the articular cavity.
Cartilage superficial zone protein/proteoglycan (SZP) or proteoglycan 4 (PRG4), has been demonstrated to have the potential for several distinct biological functions including cytoprotection, ...lubrication and matrix binding. In the present study, we have examined both the immunolocalisation and the mRNA expression pattern of PRG4 in tissue harvested from the compressed and tensional regions of young and mature bovine tendons. Immunohistochemical analyses, utilizing monoclonal antibody 3-A-4 which recognizes a conformational-dependent epitope on native PRG4, demonstrated that PRG4 is present predominantly at the surface of fibrocartilaginous regions of tendon, with the intensity of immunoreactivity in this region increasing with age. RT-PCR analyses revealed that the expression of PRG4 mRNA can be modulated by exposure to cytokines and growth factors. In addition, analyses of human pathological tendon revealed that PRG4 may also be expressed as an alternatively spliced form lacking exons which encode part of the N-terminal matrix-binding and cell-proliferative domain; however, it remains to be determined whether such splice variants are a feature of human tendon, regardless of disease state. Taken together, these data indicate that PRG4 may play an important cytoprotective role by preventing cellular adhesion to the tendon surface as well as providing lubrication during normal tendon function, in a manner complimentary to cartilage PRG4. Structural modifications to SZP, together with a reduction in synthesis during tendon inflammation with injury and disease may account for the formation of tendon adhesions and contribute to the overall dysfunction of the tissue.
Objective： To identify the relationship between T-2 toxin and Kashin-Beck disease （KBD）, the effects of T-2 toxin on aggrecan metabolism in human chondrocytes and cartilage were investigated in ...vitro. Methods： Chondrocytes were isolated from human articular cartilage and cultured in vitro. Hyaluronic acid （HA）, soluble CD44 （sCD44）, IL-1β and TNF-α levels in supernatants were measured by enzyme-linked immunosorbent assay （ELISA）. CD44 content in chondrocyte membrane was determined by flow cytometry （FCM）. CD44, hyaluronic acid synthetase-2 （HAS-2） and aggrecanases mRNA levels in chondrocytes were determined using reverse transcription polymerase chain reaction （RT-PCR）. Immunocytochemical method was used to investigate expressions of BC-13, 3-B-3（-） and 2-B-6 epitopes in the cartilage reconstructed in vitro. Results： T-2 toxin inhibited CD44, HAS-2, and aggrecan mRNA expressions, but promoted aggrecanase-2 mRNA expression. Meanwhile, CD44 expression was found to be the lowest in the chondrocytes cultured with T-2 toxin and the highest in control plus selenium group. In addition, ELISA results indicated that there were higher sCD44, IL-1β and TNF-α levels in T-2 toxin group. Similarly, higher HA levels were also observed in T-2 toxin group using radioimmunoprecipitation assay （RIPA）. Furthermore, using monoclonal antibodies BC-13, 3-B-3 and 2-B-6, strong positive immunostaining was found in the reconstructed cartilage cultured with T-2 toxin, whereas no positive staining or very weak staining was observed in the cartilage cultured without T-2 toxin. Selenium could partly inhibit the effects of T-2 toxin above. Conclusion： T-2 toxin could inhibit aggrecan synthesis, promote aggrecanases and pro-inflammatory cytokines production, and consequently induce aggrecan degradation in chondrocytes. These will perturb metabolism balance between aggrecan synthesis and degradation in cartilage, inducing aggrecan loss in the end, which may be the initiation of the cartilage degradation.
Monoclonal antibody (MAb) technology was used to examine aggrecan metabolites and the role of aggrecanases and matrix metalloproteinases (MMPs) in proteolysis of the interglobular domain (IGD) and ...C-terminus of aggrecan. An in vitro model of progressive cartilage degradation characterized by early proteoglycan loss and late stage collagen catabolism was evaluated in conjunction with a broad-spectrum inhibitor of MMPs. We have for the first time demonstrated that IGD cleavage by MMPs occurs during this late stage cartilage degeneration, both as a primary event in association with glycosaminoglycan (GAG) release from the tissue and secondarily in trimming of aggrecanase-generated G1 metabolites. Additionally, we have shown that MMPs were responsible for C-terminal catabolism of aggrecan and generation of chondroitin sulfate (CS) deficient aggrecan monomers and that this aggrecan truncation occurred prior to detectable IGD cleavage by MMPs. The onset of this later stage MMP activity was also evident by the generation of MMP-specific link protein catabolites in this model culture system. Recombinant MMP-1, -3 and -13 were all capable of C-terminally truncating aggrecan with at least two cleavage sites N-terminal to the CS attachment domains of aggrecan. Through analysis of aggrecan metabolites in pathological synovial fluids from human, canine and equine sources, we have demonstrated the presence of aggrecan catabolites that appear to have resulted from similar C-terminal processing of aggrecan as that induced in our in vitro culture systems. Finally, by developing a new MAb recognizing a linear epitope in the IGD of aggrecan, we have identified two novel aggrecan metabolites generated by an as yet unidentified proteolytic event. Collectively, these results suggest that C-terminal processing of aggrecan by MMPs may contribute to the depletion of cartilage GAG that leads to loss of tissue function in aging and disease. Furthermore, analysis of aggrecan metabolites resulting from both C-terminal and IGD cleavage by MMPs may prove useful in monitoring different stages in the progression of cartilage degeneration.
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