Chondroitin Sulphate/Dermatan Sulphate Proteoglycans: Potential Regulators of Corneal Stem/Progenitor Cell Phenotype In Vitro.

Chondroitin sulphate (CS) proteoglycans with variable sulphation-motifs along their glycosaminoglycan (GAG) chains are closely associated with the stem cell niche of articular cartilage, where they are believed to influence the characteristics of the resident stem cells. Here, we investigated the immunohistochemical distribution of hybrid CS/dermatan sulphate (DS) GAGs in the periphery of the adult chicken cornea, which is the location of the cornea's stem cell niche in a number of species, using a monoclonal antibody, 6C3, that recognises a sulphation motif-specific CS/DS GAG epitope. This revealed positive labelling that was restricted to the subepithelial corneal stroma, as well as nearby bony structures within the sclera, called ossicles. When cultivated on cell culture dishes coated with 6C3-rich CS/DS, corneal stromal cells (keratocytes) that had been isolated from embryonic chicken corneas formed circular colonies, which took several days to reach confluency. A flow cytometric analysis of these keratocytes revealed changes in their expression levels of the indicative stem cell markers, Connexin 43 (Cx43), Paired Box 6 (PAX6), B-lymphoma Moloney murine leukemia virus insertion region-1 (Bmi-1), and C-X-C Chemokine Receptor 4 (CXCR4) suggestive of a less-differentiated phenotype compared with expression levels in cells not exposed to CS/DS. These findings support the view that CS/DS promotes the retention of a stem cell phenotype in corneal cells, much as it has been proposed to do in other connective tissues.

Are physical performance and frailty assessments useful in targeting and improving access to adjuvant therapy in patients undergoing resection for pancreatic cancer?

BACKGROUND: Many patients fail to receive adjuvant chemotherapy following pancreatic cancer surgery. This study implemented a multimodal, multidisciplinary approach to improving recovery after pancreatoduodenectomy (the 'Fast Recovery' programme) and measured its impact on adjuvant chemotherapy uptake and nutritional decline. The predictive accuracies of a bundle of frailty and physical performance assessments, with respect to the recipient of adjuvant chemotherapy, were also evaluated. RESULTS: The N = 44 patients treated after the introduction of the 'Fast Recovery' programme were not found to have a significantly higher adjuvant chemotherapy uptake than the N = 409 treated before the pathway change (80.5 vs. 74.3%, p = 0.452), but did have a significantly lower average weight loss at six weeks post-operatively (mean: 4.3 vs. 6.9 kg, p = 0.013). Of the pre-operative frailty and physical performance assessments tested, the 6-min walk test was found to be the strongest predictor of the receipt of adjuvant chemotherapy (area under the ROC curve: 0.91, p = 0.001); all patients achieving distances ≥ 360 m went on to receive adjuvant chemotherapy, compared to 33% of those walking < 360 m. CONCLUSIONS: The multimodal 'Fast Recovery' programme was not found to significantly improve access to adjuvant chemotherapy, but did appear to have benefits in reducing nutritional decline. Pre-operative assessments were found to be useful in identifying patients at risk of non-receipt of adjuvant therapies, with markers of physical performance appearing to be the best predictors. As such, these markers could be useful in targeting pre- and post-habilitation measures, such as physiotherapy and improved dietetic support.

Changes in public support for alcohol policies in NSW, Australia, 2013-2019.

OBJECTIVES: Alcohol contributes to significant health, social and economic burdens worldwide, but evidence-based policy options can reduce the harm associated with alcohol use. The aim of this paper is to understand factors influencing public support for various alcohol policies in New South Wales (NSW), Australia, and to determine any change over time. METHODS: An online survey of adults in NSW, in 2013 (n = 2482), 2016 (n = 1585) and 2019 (n = 1601), assessed support for alcohol policies. Multivariable logistic regression models examined the change in support over time, adjusting for demographics, alcohol consumption, smoking status and knowledge of alcohol as a risk factor for cancer. RESULTS: Most participants (68-72%) supported policies preventing underage internet users from exposure to alcohol advertising, and banning alcohol sponsorship of underage music and sporting events. Fiscal policies and restrictions on the number of alcohol outlets were the least supported policies (<40% support). Compared with 2013, participants in 2016 and 2019 were less likely to support policies increasing price, applying a volumetric tax and reducing the number of alcohol outlets. In 2019, more than 55% of respondents were aware that alcohol was a cancer risk factor, and knowledge of that relationship was associated with an increased likelihood of support for alcohol policies. CONCLUSIONS: Support was greatest for alcohol harm-reduction policies that had less impact on an individual's drinking. Overall, support for alcohol policies in NSW is not increasing. Initiatives to raise awareness about the health consequences of alcohol use, together with effective alcohol policies, are needed to counter industry influence on decision makers and negative public discourse.

An ecological study of obesity-related cancer incidence trends in Australia from 1983 to 2017.

Background: Overweight and obesity is a growing public health issue as it contributes to the future burden of obesity-related diseases, including cancer, especially in high-income countries. In Australia, 4.3% of all cancers diagnosed in 2013 were attributable to overweight and obesity. Our aim was to examine Australian age-specific incidence trends over the last 35 years for obesity-related cancers based on expert review (colorectal, liver, gallbladder, pancreas, breast in postmenopausal women, uterine, ovary, kidney, thyroid, and multiple myeloma) individually and pooled. Methods: Australian incidence data for 10 obesity-related cancers among people aged 25-84 years, diagnosed from 1983 to 2017, were obtained from the Australian Cancer Database. We used age-period-cohort modelling and joinpoint analysis to assess trends, estimating incidence rate ratios (IRR) by birth-cohort for each individual cancer and pooled, and the annual percentage change. The analyses were also conducted for non-obesity-related cancers over the same period. Findings: The total number of cancers where some proportion is obesity-related, diagnosed from 1983-2017, was 1,005,933. This grouping was 34.7% of cancers diagnosed. The IRR of obesity-related cancers increased from 0.77 (95% CI 0.73, 0.81) for the 1903 birth-cohort to 2.95 (95% CI 2.58, 3.38) for the recent 1988 cohort relative to the 1943 cohort. The IRRs of non-obesity related cancers were stable with non-significant decreases in younger cohorts. These trends were broadly similar across sex and age groups. Interpretation: The incidence of obesity-related cancers in Australia has increased by birth-cohort across all age-groups, which should be monitored. Obesity, a public health epidemic, needs to be addressed through increased awareness, policy support and evidence-based interventions. Funding: This research received no specific funding.

Monitoring complaints about food marketing to children under the Australian industry Codes 2015-20: a qualitative analysis.

OBJECTIVE: To analyse the case reports of complaints regarding food marketing to children over six years and compare Australian marketing Codes to best practice recommendations. METHODS: Case reports on complaints about food marketing to children under the five industry Codes - the Responsible Children's Marketing Initiative, the Quick Service Restaurant Initiative and the Australian Association of National Advertisers Code of Ethics, Code of Advertising and Marketing to Children and Food and Beverages Code - were qualitatively analysed. Reports on the Ad Standards website in the food/beverage groceries and food/beverage venues categories from 2015-2020 were investigated. The most common clauses from the Codes were identified and quotes from reports used to illustrate the determinations. Codes were compared with World Cancer Research Fund recommendations on policy to protect children. RESULTS: Only 14 of 119 complaints resulted in a reported breach of industry Codes. The most common reason for dismissing complaints involved clauses requiring advertisements to be 'primarily' directed to children. The Codes did not align with best practice recommendations. CONCLUSIONS: Complaints by the public show concern for food advertising to children but the Australian industry Codes fall short of addressing those concerns. Implications for public health: Government regulation is required to protect children from unhealthy food marketing.

Monitoring changes in community support for policies on obesity prevention.

OBJECTIVE: To investigate the New South Wales (NSW) community's support for obesity prevention policies and concern for food marketing and promotion issues, and to determine any demographic differences or changes over time. METHODS: In 2013 (n=2474), 2016 (n=1602) and 2019 (n=1613) a sample of adults who were representative of the NSW population for age, gender, education and location was asked about support for policy initiatives that influence the food environment. Analysis identified the characteristics of those who supported policies and variation in support over time. RESULTS: There were limited changes in support over time; however, support for many policies was strong and sustained. In 2019, support was highest for regulation of claims about nutrition (77.2%), and health warning labels (75.7%). Support for a ban on unhealthy food advertising that targets children (64.6%) had decreased since 2013. Women, older people and those who were aware that obesity was a risk factor for cancer were generally more likely to support policies. Parents were more likely than non-parents to be concerned about positioning unhealthy food at supermarket checkouts (OR 1.32) and unhealthy outdoor advertisements (OR 1.22). Concern increased in 2019 for unhealthy marketing on the internet (OR 1.21). CONCLUSIONS: This study shows public support for policy options at moderate to high levels but not increasing in the six-year study period. Implications for public health: These results form part of a package that, along with the well-established evidence, makes the case for policy action in Australia.

Effects of HER Family-targeting Tyrosine Kinase Inhibitors on Antibody-dependent Cell-mediated Cytotoxicity in HER2-expressing Breast Cancer.

PURPOSE: Antibody-dependent cell-mediated cytotoxicity (ADCC) is one mechanism of action of the monoclonal antibody (mAb) therapies trastuzumab and pertuzumab. Tyrosine kinase inhibitors (TKIs), like lapatinib, may have added therapeutic value in combination with mAbs through enhanced ADCC activity. Using clinical data, we examined the impact of lapatinib on HER2/EGFR expression levels and natural killer (NK) cell gene signatures. We investigated the ability of three TKIs (lapatinib, afatinib, and neratinib) to alter HER2/immune-related protein levels in preclinical models of HER2-positive (HER2+) and HER2-low breast cancer, and the subsequent effects on trastuzumab/pertuzumab-mediated ADCC. EXPERIMENTAL DESIGN: Preclinical studies (proliferation assays, Western blotting, high content analysis, and flow cytometry) employed HER2+ (SKBR3 and HCC1954) and HER2-low (MCF-7, T47D, CAMA-1, and CAL-51) breast cancer cell lines. NCT00524303 provided reverse phase protein array-determined protein levels of HER2/pHER2/EGFR/pEGFR. RNA-based NK cell gene signatures (CIBERSORT/MCP-counter) post-neoadjuvant anti-HER2 therapy were assessed (NCT00769470/NCT01485926). ADCC assays utilized flow cytometry-based protocols. RESULTS: Lapatinib significantly increased membrane HER2 levels, while afatinib and neratinib significantly decreased levels in all preclinical models. Single-agent lapatinib increased HER2 or EGFR levels in 10 of 11 (91%) tumor samples. NK cell signatures increased posttherapy (P = 0.03) and associated with trastuzumab response (P = 0.01). TKI treatment altered mAb-induced NK cell-mediated ADCC in vitro, but it did not consistently correlate with HER2 expression in HER2+ or HER2-low models. The ADCC response to trastuzumab and pertuzumab combined did not exceed either mAb alone. CONCLUSIONS: TKIs differentially alter tumor cell phenotype which can impact NK cell-mediated response to coadministered antibody therapies. mAb-induced ADCC response is relevant when rationalizing combinations for clinical investigation.

Evaluation of the Cancer Council NSW Eat It To Beat It Healthy Lunch Box Sessions: A short intervention to promote the intake of fruit and vegetables among families of primary school children in NSW Australia.

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.

Tyrosine kinase inhibitors as modulators of trastuzumab-mediated antibody-dependent cell-mediated cytotoxicity in breast cancer cell lines.

BACKGROUND: Trastuzumab is an anti-HER2 monoclonal antibody (mAb) therapy capable of antibody-dependent cell-mediated cytotoxicity (ADCC) and used in the treatment of HER2+ breast cancer. Through interactions with FcÆ´R+ immune cell subsets, trastuzumab functions as a passive immunotherapy. The EGFR/HER2-targeting tyrosine kinase inhibitor (TKI) lapatinib and the next generation TKIs afatinib and neratinib, can alter HER2 levels, potentially modulating the ADCC response to trastuzumab. Using LDH-release assays, we investigated the impact of antigen modulation, assay duration and peripheral blood mononuclear cell (PBMC) activity on trastuzumab-mediated ADCC in breast cancer models of maximal (SKBR3) and minimal (MCF-7) target antigen expression to determine if modulating the ADCC response to trastuzumab using TKIs may be a viable approach for enhancing tumor immune reactivity. METHODS: HER2 levels were determined in lapatinib, afatinib and neratinib-treated SKBR3 and MCF-7 using high content analysis (HCA). Trastuzumab-mediated ADCC was assessed following treatment with TKIs utilising a colorimetric LDH release-based protocol at 4 and 12h timepoints. PBMC activity was assessed against non-MHC-restricted K562 cells. A flow cytometry-based method (CFSE/7-AAD) was also used to measure trastuzumab-mediated ADCC in medium-treated SKBR3 and MCF-7. RESULTS: HER2 antigen levels were significantly altered by the three TKIs in both cell line models. The TKIs significantly reduced LDH levels directly in SKBR3 cells but not MCF-7. Lapatinib and neratinib augment trastuzumab-related ADCC in SKBR3 but the effect was not consistent with antigen expression levels and was dependent on volunteer PBMC activity (vs. K562). A 12h assay timepoint produced more consistent results. Trastuzumab-mediated ADCC (PBMC:target cell ratio of 10:1) was measured at 7.6±4.7% (T12) by LDH assay and 19±3.2 % (T12) using the flow cytometry-based method in the antigen-low model MCF-7. CONCLUSIONS: In the presence of effector cells with high cytotoxic capacity, TKIs have the ability to augment the passive immunotherapeutic potential of trastuzumab in SKBR3, a model of HER2+ breast cancer. ADCC levels detected by LDH release assays are extremely low in MCF-7; the flow cytometry-based CFSE/7-AAD method is more sensitive and consistent for the determination of ADCC in HER2-low models.

Support for food policy initiatives is associated with knowledge of obesity-related cancer risk factors.

OBJECTIVES: To investigate community support for government-led policy initiatives to positively influence the food environment, and to identify whether there is a relationship between support for food policy initiatives and awareness of the link between obesity-related lifestyle risk factors and cancer. METHODS: An online survey of knowledge of cancer risk factors and attitudes to policy initiatives that influence the food environment was completed by 2474 adults from New South Wales, Australia. The proportion of participants in support of seven food policy initiatives was quantified in relation to awareness of the link between obesity, poor diet, insufficient fruit and vegetable consumption, and physical inactivity with cancer and other health conditions. RESULTS: Overall, policies that involved taxing unhealthy foods received the least support (41.5%). Support was highest for introducing a colour-coded food labelling system (85.9%), restricting claims being made about the health benefits of foods which are, overall, unhealthy (82.6%), displaying health warning labels on unhealthy foods (78.7%) and banning unhealthy food advertising that targets children (72.6%). Participants who were aware that obesity-related lifestyle factors are related to cancer were significantly more likely to support food policy initiatives than those who were unaware. Only 17.5% of participants were aware that obesity, poor diet, insufficient fruit and vegetable consumption, and physical inactivity are linked to cancer. CONCLUSIONS: There is strong support for all policies related to food labelling and a policy banning unhealthy food advertising to children. Support for food policy initiatives that positively influence the food environment was higher among those who were aware of the link between cancer and obesity-related lifestyle factors than among those who were unaware of this link. Increasing awareness of the link between obesity-related lifestyle factors and cancer could increase community support for food policy initiatives, which, in turn, support the population to maintain a healthy weight.

Do Health Claims and Front-of-Pack Labels Lead to a Positivity Bias in Unhealthy Foods?

Health claims and front-of-pack labels (FoPLs) may lead consumers to hold more positive attitudes and show a greater willingness to buy food products, regardless of their actual healthiness. A potential negative consequence of this positivity bias is the increased consumption of unhealthy foods. This study investigated whether a positivity bias would occur in unhealthy variations of four products (cookies, corn flakes, pizzas and yoghurts) that featured different health claim conditions (no claim, nutrient claim, general level health claim, and higher level health claim) and FoPL conditions (no FoPL, the Daily Intake Guide (DIG), Multiple Traffic Lights (MTL), and the Health Star Rating (HSR)). Positivity bias was assessed via measures of perceived healthiness, global evaluations (incorporating taste, quality, convenience, etc.) and willingness to buy. On the whole, health claims did not produce a positivity bias, while FoPLs did, with the DIG being the most likely to elicit this bias. The HSR most frequently led to lower ratings of unhealthy foods than the DIG and MTL, suggesting that this FoPL has the lowest risk of creating an inaccurate positivity bias in unhealthy foods.

Discussing alcohol and cancer with patients: Knowledge and practices of general practitioners in New South Wales and South Australia.

BACKGROUND: Alcohol is associated with several cancers; however, the Australian community has low awareness of the link between alcohol consumption and cancer. Little information exists regarding when and why general practitioners (GPs) discuss alcohol with patients. OBJECTIVE: The objective of this article is to explore GPs' attitudes and practices when discussing alcohol with patients. This includes awareness of alcohol recommendations and evidence of the alcohol-cancer link, and discussion around barriers and enablers to encouraging patients' alcohol behaviour change. RESULTS: GPs did not routinely ask patients about their alcohol consumption or advise on drinking recommendations. Many had a broad understanding of alcohol as a cancer risk factor, but knowledge of the causal mechanisms and current evidence was limited. DISCUSSION: GPs are trusted health advisers. Providing them with up-to-date evidence on the alcohol-cancer link and drinking recommendations may encourage routine patient screening of alcohol consumption and delivery of simple education on the harms of long-term drinking.

Time to address continued poor vegetable intake in Australia for prevention of chronic disease.

BACKGROUND: Australian and most international Dietary Guidelines recommend people consume more fruits and vegetables (F&V) to maintain a healthy weight and reduce chronic disease risk. Previous Australian and international surveys have shown sub-optimal consumption of F&V. OBJECTIVES: This study aimed to assess adults' F&V consumption, knowledge of recommended servings, readiness to change, barriers/enabling factors, so that this knowledge might be used for campaigns that support improved consumption. MATERIAL AND METHODS: An online survey of a representative sample of adults living in New South Wales, Australia (n = 2474) measuring self-reported F&V consumption; attitudes towards F&V consumption; stage of change for increasing F&V; barriers to consumption; and knowledge of cancer-health benefits. RESULTS: F&V consumption was below recommendations, with vegetable consumption notably low. Only 10% of participants ate at least five servings of vegetables/day (median intake was two daily servings), and 57% consumed two servings fruit/day. There was poor recognition that intake of vegetables was inadequate and this was a barrier to improving vegetable consumption; with preferences for other foods, habit and cost also important barriers. Key barriers to increasing fruit intake were habit, preferences for other foods, perishability, and cost. For vegetable consumption, 49% of participants were in the pre-contemplation stage of change, whereas for fruits 56% were in the action/maintenance stage. Sixty-four percent of respondents believed that eating F&V would protect against cancer, with 56% reporting they thought not eating enough F&V would cause cancer. IMPLICATIONS: Understanding what motivates and prevents people from consuming F&V is important for developing effective health promotion programs. Similar to previous surveys, there has been little shift in F&V consumption. Social marketing campaigns have been shown to improve health-related behaviours, and this study may assist in identifying audience segmentation for better targeted campaigns.

Can front-of-pack labelling schemes guide healthier food choices? Australian shoppers' responses to seven labelling formats.

There is evidence that easily accessible, comprehensible and consistent nutrient information on the front of packaged foods could assist shoppers to make healthier food choices. This study used an online questionnaire of 4357 grocery shoppers to examine Australian shoppers' ability to use a range of front-of-pack labels to identify healthier food products. Seven different front-of-pack labelling schemes comprising variants of the Traffic Light labelling scheme and the Percentage Daily Intake scheme, and a star rating scheme, were applied to nine pairs of commonly purchased food products. Participants could also access a nutrition information panel for each product. Participants were able to identify the healthier product in each comparison over 80% of the time using any of the five schemes that provided information on multiple nutrients. No individual scheme performed significantly better in terms of shoppers' ability to determine the healthier product, shopper reliance on the 'back-of-pack' nutrition information panel, and speed of use. The scheme that provided information about energy only and a scheme with limited numerical information of nutrient type or content performed poorly, as did the nutrition information panel alone (control). Further consumer testing is necessary to determine the optimal format and content of an interpretive front-of-pack nutrition labelling scheme.

Increased exposure to community-based education and 'below the line' social marketing results in increased fruit and vegetable consumption.

OBJECTIVE: To determine if localised programmes that are successful in engaging the community can add value to larger fruit and vegetable mass-media campaigns by evaluating the results of the Eat It To Beat It programme. DESIGN: The Eat It To Beat It programme is a multi-strategy intervention that uses community-based education and 'below the line' social marketing to increase fruit and vegetable consumption in parents. This programme was evaluated by a controlled before-and-after study with repeat cross-sectional data collected via computer-assisted telephone interviews with 1403 parents before the intervention (2008) and 1401 following intervention delivery (2011). SETTING: The intervention area was the Hunter region and the control area was the New England region of New South Wales, Australia. SUBJECTS: Parents of primary school-aged children (Kindergarten to Year 6). RESULTS: The programme achieved improvements in knowledge of recommended intakes for fruit and vegetables and some positive changes in knowledge of serving size for vegetables. Exposure to the programme resulted in a net increase of 0.5 servings of fruit and vegetables daily for those who recalled the programme compared with those who did not (P = 0.004). Increased intake of fruit and vegetables was significantly associated with increasing exposure to programme strategies. CONCLUSIONS: The Eat It To Beat It programme demonstrates that an increase in consumption of fruit and vegetables can be achieved by programmes that build on the successes of larger mass-media and social-marketing campaigns.This suggests that funding for localised, community-based programmes should be increased.

Histopathology of chondronecrosis development in knee articular cartilage in a rat model of Kashin-Beck disease using T-2 toxin and selenium deficiency conditions.

The objective of this study is to observe pathogenic lesions of joint cartilages in rats fed with T-2 toxin under a selenium deficiency nutrition status in order to determine possible etiological factors causing Kashin-Beck disease (KBD). Sprague-Dawley rats were fed selenium-deficient or control diets for 4 weeks prior to their being exposed to T-2 toxin. Six dietary groups were formed and studied 4 weeks later, i.e., controls, selenium-deficient, low T-2 toxin, high T-2 toxin, selenium-deficient diet plus low T-2 toxin, and selenium-deficient diet plus high T-2 toxin. Selenium deficiencies were confirmed by the determination of glutathione peroxidase activity and selenium levels in serum. The morphology and pathology (chondronecrosis) of knee joint cartilage of experimental rats were observed using light microscopy and the expression of proteoglycans was determined by histochemical staining. Chondronecrosis in deep zone of articular cartilage of knee joints was seen in both the low and high T-2 toxin plus selenium-deficient diet groups, these chondronecrotic lesions being very similar to chondronecrosis observed in human KBD. However, the chondronecrosis observed in the rat epiphyseal growth plates of animals treated with T-2 toxin alone or T-2 toxin plus selenium-deficient diets were not similar to that found in human KBD. Our results indicate that the rat can be used as a suitable animal model for studying etiological factors contributing to the pathogenesis (chondronecrosis) observed in human KBD. However, those changes seen in epiphyseal growth plate differ from those seen in human KBD probably because of the absence of growth plate closure in the rat.

The effect of beta-xylosides on the chondrogenic differentiation of mesenchymal stem cells.

Chondroitin/dermatan sulphate (CS/DS) sulphation motifs on cell and extracellular matrix proteoglycans (PGs) within stem/progenitor cell niches are involved in modulating cell phenotype during the development of many musculoskeletal connective tissues. Here, we investigate the importance of CS/DS chains and their motifs in the chondrogenic differentiation of bone marrow mesenchymal stem cells (bMSCs), using p-nitrophenyl xyloside (PNPX) as a competitive acceptor of CS/DS substitution on PGs. Comparison of cultures grown in control chondrogenic medium, with those grown in the presence of PNPX showed that PNPX delayed the onset of chondrogenesis, characterised by cell rounding and aggregation into spheroidal beads. PNPX reduced gene expression of SOX-9, aggrecan and collagen type II, and caused reduced levels of collagen type II protein. PNPX-treated cultures also showed delayed expression of a native CS/DS sulphation motif epitope recognised by antibody 6C3. This epitope appeared associated with a range of PGs, particularly biglycan, and its close association was lost after PNPX treatment. Overall our data show that perturbation of PG glycosylation with CS/DS GAGs using PNPX significantly delays the onset of chondrogenic differentiation of bMSCs, highlighting the importance of CS/DS during the initial stages of chondrogenesis. The delayed expression of the CS/DS sulphation motif recognised by 6C3 suggests that this motif, in particular, may have early involvement in chondrogenesis. The mechanism(s) by which CS/DS chains on PGs contribute to early chondrogenic events is unknown; however, they may be involved in morphogenetic signalling through the capture and cellular presentation of soluble bioactive molecules (e.g. growth factors).

Increased expression of chondroitin sulphate proteoglycans in rat hepatocellular carcinoma tissues.

AIM: To investigate the expression of chondroitin sulphate proteoglycans (CSPGs) in rat liver tissues of hepatocellular carcinoma (HCC). METHODS: Thirty male Sprague Dawley rats were randomly divided into two groups: control group (n = 10) and HCC model group (n = 20). Rats in the HCC model groups were intragastrically administrated with 0.2% (w/v) N-diethylnitrosamine (DEN) every 5 d for 16 wk, whereas 0.9% (w/v) normal saline was administered to rats in the control group. After 16 wk from the initiation of experiment, all rats were killed and livers were collected and fixed in 4% (w/v) paraformaldehyde. All tissues were embedded in paraffin and sectioned. Histological staining (hematoxylin and eosin and Toluidine blue) was performed to demonstrate the onset of HCC and the content of sulphated glycosaminoglycan (sGAG). Immunohistochemical staining was performed to investigate the expression of chondroitin sulphate (CS)/dermatan sulphate (DS)-GAG, heparan sulphate (HS)-GAG, keratan sulphate (KS)-GAG in liver tissues. Furthermore, expression and distribution of CSPG family members, including aggrecan, versican, biglycan and decorin in liver tissues, were also immunohistochemically determined. RESULTS: After 16 wk administration of DEN, malignant nodules were observed on the surface of livers from the HCC model group, and their hepatic lobule structures appeared largely disrupted under microscope. Toluidine blue staining demonstrated that there was an significant increase in sGAG content in HCC tissues when compared with that in the normal liver tissues from the control group [0.37 ± 0.05 integrated optical density per stained area (IOD/area) and 0.21 ± 0.01 IOD/area, P < 0.05]. Immunohistochemical studies demonstrated that this increased sGAG in HCC tissues was induced by an elevated expression of CS/DS (0.28 ± 0.02 IOD/area and 0.18 ± 0.02 IOD/area, P < 0.05) and HS (0.30 ± 0.03 IOD/area and 0.17 ± 0.02 IOD/area, P < 0.01) but not KS GAGs in HCC tissues. Further studies thereby were performed to investigate the expression and distribution of several CSPG components in HCC tissues, including aggrecan, versican, biglycan and decorin. Interestingly, there was a distinct distribution pattern for these CSPG components between HCC tissues and the normal tissues. Positive staining of aggrecan, biglycan and decorin was localized in hepatic membrane and/or pericellular matrix in normal liver tissues; however, their expression was mainly observed in the cytoplasm, cell membranes in hepatoma cells and/or pericellular matrix within HCC tissues. Semi-quantitative analysis indicated that there was a higher level of expression of aggrecan (0.43 ± 0.01 and 0.35 ± 0.03, P < 0.05), biglycan (0.32 ± 0.01 and 0.25 ± 0.01, P < 0.001) and decorin (0.29 ± 0.01 and 0.26 ± 0.01, P < 0.05) in HCC tissues compared with that in the normal liver tissues. Very weak versican positive staining was observed in hepatocytes near central vein in normal liver tissues; however there was an intensive versican distribution in fibrosis septa between the hepatoma nodules. Semi-quantitative analysis indicated that the positive rate of versican in hepatoma tissues from the HCC model group was much higher than that in the control group (33.61% and 21.28%, P < 0.05). There was no positive staining in lumican and keratocan, two major KSPGs, in either normal or HCC liver tissues. CONCLUSION: CSPGs play important roles in the onset and progression of HCC, and may provide potential therapeutic targets and clinical biomarkers for this prevalent tumor in humans.

Modification of palm oil for anti-inflammatory nutraceutical properties.

Palm oil is one of the most important edible oils in the world. Its composition (rich in palmitate and oleate) make it suitable for general food uses but its utility could be increased if its fatty acid quality could be varied. In this study, we have modified a palm olein fraction by transesterification with the n-3 polyunsaturated fatty acids, alpha-linolenate or eicosapentaenoic acid (EPA). Evaluation of the potential nutritional efficacy of the oils was made using chondrocyte culture systems which can be used to mimic many of the degenerative and inflammatory pathways involved in arthritis. On stimulation of such cultures with interleukin-1alpha, they showed increased expression of cyclooxygenase-2, the inflammatory cytokines tumour necrosis factor-alpha (TNF-alpha), IL-1alpha and IL-1beta and the proteinase ADAMTS-4. This increased expression was not affected by challenge of the cultures with palm olein alone but showed concentration-dependent reduction by the modified oil in a manner similar to EPA. These results show clearly that it is possible to modify palm oil conveniently to produce a nutraceutical with effective anti-inflammatory properties.