Objective The Broccoli in Osteoarthritis (BRIO Study) was conducted to determine whether dietary sulforaphane (SFN), consumed as broccoli, improves pain and/or physical function in participants with knee osteoarthritis (OA). This was a proof of principle study to test the feasibility of the trial to optimise the design of an appropriately powered study. Design Two-centre, double-blind, two-arm parallel, randomised placebo-controlled, dietary intervention feasibility trial. Patients with radiographic knee osteoarthritis (Kellgren-Lawrence score 2-3), with pain of at least 4 on a scale of 0-10 were recruited. The intervention was a high glucoraphanin broccoli, (source of SFN), or a matched placebo (no SFN) soup. Pain and measures of physical function were measured at baseline, 6 and 12 weeks. Results The mean WOMAC pain score (scale 0 - 20) was decreased by 4.2 (95% CI: 1.03,7.38) following intervention, Similar patterns of improvement were observed for other pain and function outcome measures. Study data, sample collections and intervention adherence were 100% compliant except where COVID restrictions applied. Acceptability for randomisation was 100% and acceptability for the intervention was 92%. There were three related adverse events, two of which were expected. Conclusions High glucosinolate broccoli soup is a novel approach to managing OA that is widely accessible and can be used on a large scale. This study shows that it is an acceptable way of delivering dietary bioactives and has potential for therapeutic benefit. The primary outcome of pain improved in the intervention group compared to the placebo and the confidence interval encompassed the minimal clinically important difference. The data provide justification for proceeding to a large scale, appropriately powered intervention trial.### Competing Interest StatementDisclosure of interest: Rose Davidson: None declared, Laura Watts: None declared, Gemma Beasy: None declared, Shikha Saha: None declared, Paul Kroon: None declared, Aedin Cassidy: None declared, Allan Clark: None declared, William Fraser Speakers bureau: Roche, Incstar/Diasorin, IDS, Sanofi, Siemens, Menarini, Abbott, Entera Bio, NPS pharmaceuticals and Alexis, Consultant of: Roche, Incstar/Diasorin, IDS, Sanofi, Siemens, Menarini, Abbott, Entera Bio, NPS pharmaceuticals and Alexis, Grant/research support from: Roche, Incstar/Diasorin, IDS, Sanofi, Siemens, Menarini, Abbott, Entera Bio, NPS pharmaceuticals and Alexis., Iain McNamara: None declared, Sarah Kingsbury: None declared, Philip G Conaghan Speakers bureau: AbbVie, Novartis, Consultant of: AbbVie, AstraZeneca, Biosplice, BMS, Eli Lilly, Galapagos, Genascence, GSK, Janssen, Merck, Novartis, Pfizer, Regeneron, Stryker, and UCB, Ian Clark: None declared, Alex MacGregor: None declared### Clinical TrialISRCTN 11629849, CPMS 40910, ClinicalTrials.gov [NCT03878368][1]### Funding StatementThis work was supported by grants from Versus Arthritis (Ref: 21772) and Action Arthritis.### Author DeclarationsI confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained.YesThe details of the IRB/oversight body that provided approval or exemption for the research described are given below:The procedures followed were in accordance with the ethical standards of NHS Health Research Authority and Health and Care Research Wales (HCRW) and with the Helsinki Declaration of 1975, as revised in 2000. Ethical approval for the study was granted by East of England - Cambridge East Research Ethics Committee (ref 19/EE/0007), IRAS: 250371. All patients gave their full informed and written consent to participate in the study. All patient data was anonymised.I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals.YesI understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance).YesI have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable.YesAll data produced in the present study are available upon reasonable request to the authors [1]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT03878368&atom=%2Fmedrxiv%2Fearly%2F2024%2F06%2F21%2F2024.06.20.24309233.atom

Background Osteoarthritis (OA) accounts for more than a third of chronic moderate to severe pain in the UK. Sulforaphane (SFN) is a naturally occurring phytochemical derived from eating cruciferous vegetables, particularly broccoli. It has several biological activities that promote health, including anti-inflammatory properties. SFN has a potential role in limiting pain and cartilage destruction in OA. Objectives In a two centre, placebo-controlled, double-blind, two-arm parallel, RCT, proof of principle study with a primary outcome we evaluated whether dietary SFN (from consumption of broccoli) improves pain in participants with knee OA. Secondary objectives were to determine whether food containing SFN improves knee function, and the feasibility of the study design for an appropriately powered trial. Methods Participants with symptomatic and radiographic knee OA were recruited from regions of Norfolk and Leeds in the UK, were over 50 years of age with moderate to severe knee pain (at least 4 on 0-10 numeric rating scale), and radiological evidence of OA (Kellgren-Lawrence score 2-3). The intervention was a sensory-matched soup. Patients received either the intervention soup (300g high glucoraphanin soup (containing broccoli, and base vegetables)) or placebo soup (300g no glucoraphanin soup (base vegetables only)), once daily on 4 days per week. The study duration was 12 weeks with follow-up visits at 6 and 12 weeks. Knee pain and function outcome measures were obtained using WOMAC, an 11-point NRS, the ICOAP questionnaire and use of rescue analgesics/NSAIDS. Creatinine, metabolites of glucoraphanin, matrix metalloproteinase-3 and C-reactive protein were measured in plasma or 24-hour urine samples. Results Recruitment was severely curtailed due to the Covid-19 pandemic. In total n= 37 consented and n= 24 met screening criteria with one drop-out at week 12 (control n=17 and intervention n= 7), the recruitment aim was n= 64. Control group was 43% female, and mean age was 70.14 yrs [7.69 SD] (control), and 61.88 yrs [8.58 SD] (intervention). Mean BMI was 26.4[2.91 SD] (control) and 28.2[4.68 SD] (intervention). The intervention resulted in a trend towards a decrease in pain scores for each subscale of WOMAC, ICOAP and NRS measures: total WOMAC median difference from baseline (9.65 [CI:-0.78,20.09]), ICOAP constant (wk 12, 4.83 [CI: -1.99,11.64]) and intermittent pain (wk 12, 2.96 [CI: -0.31,6.24]), NRS overall pain (wk 12, 1.86 [CI: 0.13,3.58]), while a trend for increased satisfaction for knee function (wk12, -1.55 [CI: -3.55,0.44]) was also observed for this group. No change in rescue analgesics/NSAIDS for knee pain was seen in either group. Metabolites of glucoraphanin were detected in plasma and urine samples in the intervention group. Study compliance and intervention adherence was high (>70%-100%) while acceptability for randomisation was 100%. Conclusion Patient acceptability, adherence and retention was high and biomarkers and metabolites objectively confirming compliance with the intervention were detected. In this study, although underpowered to observe significant differences for pain, improving trends over time were observed across a range of clinical pain measures including the primary outcome, where the CI encompassed the minimal clinically important difference, requisite for progress to full trial. Given COVID impact on recruitment, the pilot achieved its aims, and we conclude that the intervention was feasible, and a full trial is justified. Acknowledgements Our thanks to the study participants, study team, research nurses, and doctors; Fiona Brudenell-Straw, Lisa Cook, Lizzy Daniel, Asim Ghouri, Robert Hindmarsh, Kiran Khokar, Teja Kodali, Angela Nauth, Iraklis Papageorgiou, Tracey Swingler, Rabia Thompson, Nicola Ward, and Celia Whitehouse, and Trial committee Simon Donell (Independent Chair), Sam Norton (Independent Biostatistician) and Trish Phillips (Independent member) for their tenacity and team spirit throughout such an unprecedented time. This work is supported by grants from Versus Arthritis and Action Arthritis. Disclosure of Interests Rose Davidson: None declared, Laura Watts: None declared, Gemma Beasy: None declared, Shikha Saha: None declared, Paul Kroon: None declared, Aedin Cassidy: None declared, Allan Clark: None declared, William Fraser Speakers bureau: Roche, Incstar/Diasorin, IDS, Sanofi, Siemens, Menarini, Abbott, Entera Bio, NPS pharmaceuticals and Alexis, Consultant of: Roche, Incstar/Diasorin, IDS, Sanofi, Siemens, Menarini, Abbott, Entera Bio, NPS pharmaceuticals and Alexis, Grant/research support from: Roche, Incstar/Diasorin, IDS, Sanofi, Siemens, Menarini, Abbott, Entera Bio, NPS pharmaceuticals and Alexis., Iain McNamara: None declared, Sarah Kingsbury: None declared, Philip G Conaghan Speakers bureau: AbbVie, Novartis, Consultant of: AbbVie, AstraZeneca, Biosplice, BMS, Eli Lilly, Galapagos, Genascence, GSK, Janssen, Merck, Novartis, Pfizer, Regeneron, Stryker, and UCB, Ian Clark: None declared, Alex MacGregor: None declared.

The COVID-19 pandemic continues to expand globally, with case numbers rising in many areas of the world, including the Eastern Mediterranean Region. Lebanon experienced its largest wave of COVID-19 infections from January to April 2021. Limited genomic surveillance was undertaken, with just 26 SARS-CoV-2 genomes available for this period, nine of which were from travellers from Lebanon detected by other countries. Additional genome sequencing is thus needed to allow surveillance of variants in circulation. In total, 905 SARS-CoV-2 genomes were sequenced using the ARTIC protocol. The genomes were derived from SARS-CoV-2-positive samples, selected retrospectively from the sentinel COVID-19 surveillance network, to capture diversity of location, sampling time, sex, nationality and age. Although 16 PANGO lineages were circulating in Lebanon in January 2021, by February there were just four, with the Alpha variant accounting for 97% of samples. In the following 2 months, all samples contained the Alpha variant. However, this had changed dramatically by June and July 2021, when all samples belonged to the Delta variant. This study documents a ten-fold increase in the number of SARS-CoV-2 genomes available from Lebanon. The Alpha variant, first detected in the UK, rapidly swept through Lebanon, causing the country's largest wave to date, which peaked in January 2021. The Alpha variant was introduced to Lebanon multiple times despite travel restrictions, but the source of these introductions remains uncertain. The Delta variant was detected in Gambia in travellers from Lebanon in mid-May, suggesting community transmission in Lebanon several weeks before this variant was detected in the country. Prospective sequencing in June/July 2021 showed that the Delta variant had completely replaced the Alpha variant in under 6 weeks.

BackgroundPhase 2 of the Norwich Testing Initiative (NTI2) was a SARS-CoV-2 PCR testing programme at the University of East Anglia, which ran from September to December 2020. It aimed to identify asymptomatic COVID-19 infections and limit outbreaks on campus. The NTI2 evaluation explored testing uptake, positivity rates, isolation compliance and the links between viral load and symptoms.MethodsAll staff and students (21,762) were eligible for testing. Users registered for the programme using a web application, which collected personal, demographic and location data. Users collected tests from a central location on campus, self-administered a nasopharyngeal swab and returned tests to a drop-off point. PCR testing was conducted by two laboratories. All those with a positive result were contacted by telephone for self-isolation advice and support. At this point symptom, isolation and location data were collected. The programme used a broader definition of COVID-19 symptoms than that used by NHS Test and Trace. Data were encrypted and stored in a data warehouse. Microsoft Excel was used to collate, clean and analyse the data.Results188 of 6537 (2.9%) users tested positive for COVID-19. The majority (82%) were students aged 18–24 years. Positivity rates were highest in students (3.5%), those living on campus (6.5%) and BAME groups (4%). There was clustering of cases in university halls: positivity rates in halls varied between 0% and 31%, with 18% of halls containing over half the cases on campus. Positivity rates spiked near the beginning of the programme and then declined over the course of the term. 99/187 (53%) cases reported symptoms of COVID-19. 37/99 (35%) symptomatic cases were not isolating. There was no significant relationship between PCR Ct values and self-reported symptoms.ConclusionNTI2 may have contributed to a decline in cases of COVID-19 during the testing period, as the incidence of COVID-19 in the local area increased during this period. Transmission within halls of residence may have contributed to high positivity rates on campus. A high proportion of users reported symptoms of COVID-19 infection, despite this being an asymptomatic testing programme. Over a third of symptomatic cases were not isolating prior to receiving their result . These findings suggest that testing and isolating messaging may not be reaching the student population. Those with symptoms had a similar viral load to those without and may be similarly infectious.

Background Phase 2 of the Norwich Testing Initiative (NTI2) was a SARS-CoV-2 PCR testing programme at the University of East Anglia, which ran from September to December 2020. It aimed to identify asymptomatic COVID-19 infections and limit outbreaks on campus. The NTI2 evaluation explored testing uptake, positivity rates, isolation compliance and the links between viral load and symptoms. Methods All staff and students (21,762) were eligible for testing. Users registered for the programme using a web application, which collected personal, demographic and location data. Users collected tests from a central location on campus, self-administered a nasopharyngeal swab and returned tests to a drop-off point. PCR testing was conducted by two laboratories. All those with a positive result were contacted by telephone for self-isolation advice and support. At this point symptom, isolation and location data were collected. The programme used a broader definition of COVID-19 symptoms than that used by NHS Test and Trace. Data were encrypted and stored in a data warehouse. Microsoft Excel was used to collate, clean and analyse the data. Results 188 of 6537 (2.9%) users tested positive for COVID-19. The majority (82%) were students aged 18–24 years. Positivity rates were highest in students (3.5%), those living on campus (6.5%) and BAME groups (4%). There was clustering of cases in university halls: positivity rates in halls varied between 0% and 31%, with 18% of halls containing over half the cases on campus. Positivity rates spiked near the beginning of the programme and then declined over the course of the term. 99/187 (53%) cases reported symptoms of COVID-19. 37/99 (35%) symptomatic cases were not isolating. There was no significant relationship between PCR Ct values and self-reported symptoms. Conclusion NTI2 may have contributed to a decline in cases of COVID-19 during the testing period, as the incidence of COVID-19 in the local area increased during this period. Transmission within halls of residence may have contributed to high positivity rates on campus. A high proportion of users reported symptoms of COVID-19 infection, despite this being an asymptomatic testing programme. Over a third of symptomatic cases were not isolating prior to receiving their result . These findings suggest that testing and isolating messaging may not be reaching the student population. Those with symptoms had a similar viral load to those without and may be similarly infectious.

The COVID-19 pandemic has spread rapidly throughout the world. In the UK, the initial peak was in April 2020; in the county of Norfolk (UK) and surrounding areas, which has a stable, low-density population, over 3200 cases were reported between March and August 2020. As part of the activities of the national COVID-19 Genomics Consortium (COG-UK) we undertook whole genome sequencing of the SARS-CoV-2 genomes present in positive clinical samples from the Norfolk region. These samples were collected by four major hospitals, multiple minor hospitals, care facilities and community organizations within Norfolk and surrounding areas. We combined clinical metadata with the sequencing data from regional SARS-CoV-2 genomes to understand the origins, genetic variation, transmission and expansion (spread) of the virus within the region and provide context nationally. Data were fed back into the national effort for pandemic management, whilst simultaneously being used to assist local outbreak analyses. Overall, 1565 positive samples (172 per 100 000 population) from 1376 cases were evaluated; for 140 cases between two and six samples were available providing longitudinal data. This represented 42.6 % of all positive samples identified by hospital testing in the region and encompassed those with clinical need, and health and care workers and their families. In total, 1035 cases had genome sequences of sufficient quality to provide phylogenetic lineages. These genomes belonged to 26 distinct global lineages, indicating that there were multiple separate introductions into the region. Furthermore, 100 genetically distinct UK lineages were detected demonstrating local evolution, at a rate of ~2 SNPs per month, and multiple co-occurring lineages as the pandemic progressed. Our analysis: identified a discrete sublineage associated with six care facilities; found no evidence of reinfection in longitudinal samples; ruled out a nosocomial outbreak; identified 16 lineages in key workers which were not in patients, indicating infection control measures were effective; and found the D614G spike protein mutation which is linked to increased transmissibility dominates the samples and rapidly confirmed relatedness of cases in an outbreak at a food processing facility. The large-scale genome sequencing of SARS-CoV-2-positive samples has provided valuable additional data for public health epidemiology in the Norfolk region, and will continue to help identify and untangle hidden transmission chains as the pandemic evolves.

Osteoarthritis (OA) is a degenerative joint disease for which there are no disease-modifying drugs. It is a leading cause of disability in the UK. Increasing age and obesity are both major risk factors for OA and the health and economic burden of this disease will increase in the future. Focusing on compounds from the habitual diet that may prevent the onset or slow the progression of OA is a strategy that has been under-investigated to date. An approach that relies on dietary modification is clearly attractive in terms of risk/benefit and more likely to be implementable at the population level. However, before undertaking a full clinical trial to examine potential efficacy, detailed molecular studies are required in order to optimise the design. This review focuses on potential dietary factors that may reduce the risk or progression of OA, including micronutrients, fatty acids, flavonoids and other phytochemicals. It therefore ignores data coming from classical inflammatory arthritides and nutraceuticals such as glucosamine and chondroitin. In conclusion, diet offers a route by which the health of the joint can be protected and OA incidence or progression decreased. In a chronic disease, with risk factors increasing in the population and with no pharmaceutical cure, an understanding of this will be crucial.

AbstractWe evaluated the FDA approved SARS-CoV-2 immunoassay (developed at Mount Sinai, by Krammer and colleagues) for the identification of COVID-19 seroconversion and potential cross-reactivity of the assay in a United Kingdom (UK) National Health Service (NHS) hospital setting. In our ‘set up’ cohort we found that the SARS-CoV-2 IgG was detectable in 100% of patients tested 14 days post positive COVID-19 nucleic acid test. Serum samples taken from pregnant women in 2018 were used as a negative control group with zero false positives. We also analysed samples from patients with non-COVID-19 viral infections, paraproteinaemia or autoantibodies and found false positive results in 6/179. Modification of the sensitivity threshold to five standard deviations from the mean of the control group eliminated all false positive result in the ‘set up’ cohort. We confirmed the validity of the test with a revised threshold on an independent prospective ‘validation cohort’ of patient samples. Taking data from both cohorts we report a sensitivity of the Mount Sinai assay of 96.6% (28/29) and specificity of 100% (299/299) using a revised threshold cut-off, at a time point at least 14 days since the diagnostic antigen test. Finally, we conducted a health economic probabilistic sensitivity analysis (PSA) on the costs of producing the tests, and the mean cost we estimate to be 13.63 pounds sterling (95%CI 9.63 - 18.40), allowing its cost effectiveness to be tested against other antibody tests. In summary, we report that the Mount Sinai IgG ELISA assay is highly sensitive test for SARS-Cov-2 infection, however modification of thresholding was required to minimise false positive results.

An abstract is not available for this content. As you have access to this content, full HTML content is provided on this page. A PDF of this content is also available in through the 'Save PDF' action button.

Changes of serum concentrations of glycated, oxidized, and nitrated amino acids and hydroxyproline and anticyclic citrullinated peptide antibody status combined by machine learning techniques in algorithms have recently been found to provide improved diagnosis and typing of early-stage arthritis of the knee, including osteoarthritis (OA), in patients. The association of glycated, oxidized, and nitrated amino acids released from the joint with development and progression of knee OA is unknown. We studied this in an OA animal model as well as interleukin-1β-activated human chondrocytes in vitro and translated key findings to patients with OA.

Osteoarthritis (OA) is a multifactorial disease and nutrition is a modifiable factor that may contribute to disease onset or progression. A detailed understanding of mechanisms through which diet-derived bioactive molecules function and interact in OA is needed. We profiled 96 diet-derived, mainly plant-based bioactives using an in vitro model in chondrocytes, selecting four candidates for further study. We aimed to determine synergistic interactions between bioactives that affected the expression of key genes in OA. Selected bioactives, sulforaphane, apigenin, isoliquiritigenin and luteolin, inhibited one or more interleukin-1-induced metalloproteinases implicated in OA (MMP1, MMP13, ADAMTS4, ADAMTS5). Isoliquiritigenin and luteolin showed reactive oxygen species scavenging activity in chondrocytes whereas sulforaphane had no effect and apigenin showed only a weak trend. Sulforaphane inhibited the IL-1/NFκB and Wnt3a/TCF/Lef pathways and increased TGFβ/Smad2/3 and BMP6/Smad1/5/8 signalling. Apigenin showed potent inhibition of the IL-1/NFκB and TGFβ/Smad2/3 pathways, whereas luteolin showed only weak inhibition of the IL-1/NFκB pathway. All four bioactives inhibited cytokine-induced aggrecan loss from cartilage tissue explants. The combination of sulforaphane and isoliquiritigenin was synergistic for inhibiting MMP13 gene expression in chondrocytes. We conclude that dietary-derived bioactives may be important modulators of cartilage homeostasis and synergistic relationships between bioactives may have an anti-inflammatory and chondroprotective role.

Background Glycation, oxidation and nitration of proteins are reactions involved in accelerated ageing of tissues. The products of these reactions are used as biomarkers of chronic pathologies such as diabetes or chronic inflammatory states. Objectives In this work, we studied by mass spectrometry the levels of amino acids and glycated, oxidised or nitrated proteins in culture media of chondrocytes cultivated in multi-layers and in the blood of guinea pigs or osteoarthritis patients. Methods Sixty male, 3-week-old Dunkin-Hartley guinea pigs were used in this work. At 4-weeks-old and 8 week intervals until week 36, twelve animals were sacrificed and histological severity of knee osteoarthritis evaluated and cartilage rheological properties. Human patients with early and advanced osteoarthritis and healthy subjects were recruited. Human chondrocytes cultured in multilayers were treated for 10 days with interleukin (IL) −1β. Amino acids and glycated, oxidised and nitrated proteins were analysed in the serum of guinea pigs, osteoarthritis patients and in the culture medium conditioned by chondrocytes by stable isotopic dilution analysis liquid chromatography-tandem mass spectrometry using the Acquity UPLC system. Results Severity of osteoarthritis increased progressively in guinea pigs with age. Glycated, oxidised and nitrated amino acids were increased markedly at week 36. Glucosepane and dityrosine increased progressively from weeks 20 and 28, respectively. Glucosepane was positively correlated with the OA histological severity (r=0.58, p<0.0001) and the Young’s modulus (r=0.52–0.56, p<0.0001). Oxidation free adducts were positively correlated with OA severity (p<0.0009–0.0029) and hydroxyproline with cartilage thickness (p<0.0003–0.003). In the clinical study, plasma glucosepane was increased 38% in patients with early osteoarthritis (p<0.05) and 6-fold in patients with advanced osteoarthritis (p<0.001) compared to healthy subjects. IL-1β increased the release of glycated, oxidised and nitrated products from chondrocytes in vitro. Conclusions The glycation, oxidation and nitration of proteins are reactions related to the severity of osteoarthritis. The products of these reactions are measurable in blood by mass spectrometry and could be biomarkers of osteoarthritis. More specifically, glucosepane is an advanced glycation product very strongly increased in the severe form of the disease. In conclusion, serum glucosepane is a potential biomarker for diagnosis and progression of osteoarthritis. Disclosure of Interest None declared

Purpose: A number of plant derived phytochemicals have been proposed to have a beneficial effect on joint health and osteoarthritis (OA). Research into the prevention or slowing of osteoarthritis through dietary intervention is important since there is little current high quality data in this area. We have shown previously that sulforaphane (SFN) can protect against cartilage damage in the DMM murine model of OA and alter the gene expression of a number of matrix-degrading enzymes in cartilage. We have also shown that dietary SFN can be detected in joint tissues in man. Future human dietary intervention trials need to be underpinned with strong mechanistic data to inform and better their design. The aim of this study is to identify dietary compounds with differing mechanisms of action in chondrocytes that may in combination act together synergistically. Methods: RT-qPCR was used to measure gene expression in C28/I2 and SW1353 cell lines and primary chondrocytes in response to pro-inflammatory cytokine treatment following pre-incubations with eleven bioactive compounds individually. Phosphorylation events were detected using a medium throughput phospho-kinase array. Transcriptional activity was measured using luciferase reporters for NF-κB, canonical Wnt, Smad2/3 and Smad1/5/8 signalling pathways. Oxidative stress response was measured using DCF assay. Synergism was assessed using the median effect equation and the CompuSyn software with MMP13 gene expression as an outcome measure in primary chondrocytes. In vitro cartilage degradation assays using bovine nasal cartilage are ongoing. Results: The initial gene expression studies identified sulforaphane, apigenin (API), isoliquiritigenin (ISO) and luteolin (LUT) as the most potent modifiers of metalloproteinase gene expression and were used in downstream studies. Reporter assays showed SFN and API to be potent inhibitors of NF-κB signalling (p<0.01 at 3μM and p<0.0001 at 0.75 μM respectively) with some weaker inhibition seen with LUT (p<0.05 at 6 μM). API was a strong inhibitor of Smad2/3 signalling (p<0.0001 at 0.75 μM). Conversely ISO and LUT inhibited tBHP-induced ROS production (p<0.05 at 3 μM and p<0.01 at 1.4 μM) but no inhibition was seen with SFN or API. Further gene expression analyses in primary chondrocytes indicated a strong synergistic interaction for SFN and ISO inhibition of IL-1-induced MMP13 mRNA. A weak synergism was observed with API and ISO. Conclusions: SFN and API are potent modifiers of gene expression and transcriptional activity whereas ISO and LUT can modulate oxidative stress in chondrocytes. Synergism for the inhibition of MMP13 gene expression was seen for SFN and ISO independent of effect size, whereas synergistic interactions of SFN and API or API and ISO were effect size dependent. We are continuing to investigate the mechanistic activities of these four chondroprotective bioactives to identify effective combinations of bioactives for chondroprotection.

Osteoarthritis is a major cause of disability and there is no current pharmaceutical treatment which can prevent the disease or slow its progression. Dietary advice or supplementation is clearly an attractive option since it has low toxicity and ease of implementation on a population level. We have previously demonstrated that sulforaphane, a dietary isothiocyanate derived from its glucosinolate precursor which is found in broccoli, can prevent cartilage destruction in cells, in in vitro and in vivo models of osteoarthritis. As the next phase of this research, we enrolled 40 patients with knee osteoarthritis undergoing total knee replacement into a proof-of-principle trial. Patients were randomised to either a low or high glucosinolate diet for 14 days prior to surgery. We detected ITCs in the synovial fluid of the high glucosinolate group, but not the low glucosinolate group. This was mirrored by an increase in ITCs and specifically sulforaphane in the plasma. Proteomic analysis of synovial fluid showed significantly distinct profiles between groups with 125 differentially expressed proteins. The functional consequence of this diet will now be tested in a clinical trial.

Osteoarthritis (OA) is a degenerative joint disease characterised in part by destruction of articular cartilage. There are currently no disease-modifying drugs to treat OA, with joint replacement the only treatment offered to patients at end-stage disease. With age the major risk factor for OA, the number of patients is predicted to double by 2030. An understanding of the role of bioactive molecules from the habitual diet on joint health offers a novel way in which to prevent the onset or slow the progression of OA. Our research has indicated that sulforaphane (SFN), gained from the consumption of cruciferous vegetables, particularly broccoli, could impact upon articular cartilage in laboratory models of OA because (1) it decreased the cytokine-induced expression of cartilage-degrading proteinases from chondrocytes (cartilage cells); (2) it prevented the cytokine-induced degradation of cartilage explants; and (3) it attenuated cartilage destruction in a murine model of OA. The major mechanism of action for SFN in human articular chondrocytes was inhibition of NF kappa B, not activation of Nrf2 nor inhibition of histone deacetylases. A proof-of-principle human trial was performed to measure uptake of SFN, or its metabolites, in the human knee joint following a broccoli-rich diet, and the expression or levels of several genes and proteins in cartilage, fat and synovial fluid were also measured. Data from this trial are about to be published. Overall, these findings support the utility of SFN in the prevention or treatment of OA. The proof of this requires an appropriately designed clinical trial of pain and function which we are currently pursuing.

Purpose: To determine whether isothiocyanates obtained from dietary glucosinolates in broccoli can be detected in synovial fluid or impact on tissues of the knee joint. Methods: A randomised parallel-design proof-of-principle trial enrolled 40 patients with knee osteoarthritis undergoing total knee replacement. Following a washout period, where participants consumed a low glucosinolate diet for 7 days, participants were randomised to either a low or high glucosinolate diet for a further 14 days prior to surgery. Blood samples were taken at baseline (following washout) and post intervention immediately prior to surgery. Synovial fluid, fat and cartilage tissue samples were obtained at surgery. Primary endpoints were isothiocyanate (ITC) detection in plasma and synovial fluid by HPLC and HPLC-MS. Secondary outcomes were proteomic analysis of the synovial fluid (HPLC-MS/MS), in vitro cartilage degradation rate, gene expression in cartilage and fat tissues (RT-qPCR), glutathione S-transferase genetic variation, and biomarker analyses. Results: Although plasma ITCs were detected in both groups, total ITC levels were significantly higher in the high glucosinolate group (p < 0.0001) as was sulforaphane (p < 0.0001). ITCs were only detected in the synovial fluid of the high glucosinolate group. Proteomic analysis of synovial fluid showed distinct profiles for high and low glucosinolate groups (p < 0.05). Calreticulin (p < 0.0001), adiponectin (p < 0.01) and extracellular superoxide dismutase (p < 0.01) protein levels were decreased in synovial fluid of the high glucosinolate group. Tubulin alpha-1B chain (p < 0.01) and transforming growth factor beta-induced protein (p < 0.01) protein levels were increased in the synovial fluid of the high glucosinolate group. No differences were observed for in vitro cartilage degradation rates, gene expression or biomarkers between groups. No association was observed between genotype and plasma levels of total ITC or SFN. Conclusions: ITCs obtained from the diet can be detected in the synovial fluid of the knee joint within 24 hours of consumption and alters the protein composition of synovial fluid. Together with data from cell and animal models where sulforaphane shows chondroprotective effects, this suggests that a high broccoli diet may help prevent or slow the progression of osteoarthritis. An appropriately powered clinical trial to test this is currently being designed.

OBJECTIVE:Dickkopf-3 (Dkk3) is a non-canonical member of the Dkk family of Wnt antagonists and its upregulation has been reported in microarray analysis of cartilage from mouse models of osteoarthritis (OA). In this study we assessed Dkk3 expression in human OA cartilage to ascertain its potential role in chondrocyte signaling and cartilage maintenance. METHODS:Dkk3 expression was analysed in human adult OA cartilage and synovial tissues and during chondrogenesis of ATDC5 and human mesenchymal stem cells. The role of Dkk3 in cartilage maintenance was analysed by incubation of bovine and human cartilage explants with interleukin-1β (IL1β) and oncostatin-M (OSM). Dkk3 gene expression was measured in cartilage following murine hip avulsion. Whether Dkk3 influenced Wnt, TGFβ and activin cell signaling was assessed in primary human chondrocytes and SW1353 chondrosarcoma cells using qRT-PCR and luminescence assays. RESULTS:Increased gene and protein levels of Dkk3 were detected in human OA cartilage, synovial tissue and synovial fluid. DKK3 gene expression was decreased during chondrogenesis of both ATDC5 cells and humans MSCs. Dkk3 inhibited IL1β and OSM-mediated proteoglycan loss from human and bovine cartilage explants and collagen loss from bovine cartilage explants. Cartilage DKK3 expression was decreased following hip avulsion injury. TGFβ signaling was enhanced by Dkk3 whilst Wnt3a and activin signaling were inhibited. CONCLUSIONS:We provide evidence that Dkk3 is upregulated in OA and may have a protective effect on cartilage integrity by preventing proteoglycan loss and helping to restore OA-relevant signaling pathway activity. Targeting Dkk3 may be a novel approach in the treatment of OA.

Osteoarthritis (OA) is a major social and economic burden that continues to grow globally with no effective disease modifying therapies in the pipeline. Current therapeutic strategies to address pain are largely insufficient and joint replacement for end stage disease is unsustainable. Drug development is particularly difficult and expensive due to the complexity of balancing efficacy and toxicity for chronic diseases. The main risk factors for developing OA are increasing age and obesity. Diet has a clear link to the latter, but also impacts strongly on the ageing process. There is no paucity of pre-clinical data investigating dietary bioactives and their impact on OA disease models. However clinical trials that test these data using relevant trial design and validated outcome measures are scarce. Current trials focus on vitamin D, fish oil omega fatty acids and curcumin. This review examines recently completed, current or proposed clinical trials of dietary bioactives and their utility in developing an OA therapeutic strategy either prophylactically or as a means of OA management. The number of scientifically high quality trials in the pipeline is low. There is an unmet need for investment in clinical trials of dietary intervention, for developing disease biomarkers (both biochemical and imaging) and refining the definition of OA so that clinically relevant outcome measures can be improved.

Purpose: Sulforaphane (SFN) is an isothiocyanate (ITC) obtained from the diet, and is particularly abundant after consumption of the mustard family of Brassicaceae. ITCs are purported to have many health benefits and SFN has been shown to prevent cartilage degradation in laboratory models of osteoarthritis. This study examined whether ITCs obtained from the diet could impact on tissues of the human synovial joint. Methods: Forty patients scheduled for total knee replacement due to osteoarthritis were recruited to the Norwich Broccoli and Osteoarthritis Study (NBOS). Following a washout period (a low ITC diet), the patients were randomly allocated to continue the low ITC diet or to consume 100g of high glucosinolate broccoli for a further 2 weeks. Tissue levels of ITCs were measured by cyclocondensation reaction and HPLC. In vitro rates of cartilage degradation were assessed. Plasma was analysed for lipids, the Coll2-1 degradation marker and hsCRP levels. Cytokine expression was measured by FACS analysis and gene expression was measured by RT-qPCR. Data were analysed for the effect of glutathione transferase (GST) genotype. Results: ITCs were detected in plasma and synovial fluid (SF) and these were significantly higher in the broccoli groups compared to control (plasma control 0.89μM±0.45 vs broccoli 2.19μM±1.42, p<0.0001). ITCs were not detected in SF from the control group. Mean total ITC levels in SF measured in the broccoli group were 496.nM±140. Plasma triglycerides were significantly decreased within the broccoli group at post-intervention compared to washout (p=0.0046) but were not significantly different in the broccoli group compared to control. No significant differences were seen between groups for SF cytokine protein expression or in vitro cartilage degradation experiments. Conclusions: The high glucosinolate broccoli diet was well tolerated. ITCs obtained from the diet can be detected in the tissues of the synovial joint. A short term high glucosinolate diet was not sufficient to impact on the rate of cartilage destruction in vitro. Studies of gene expression in cartilage and infrapatellar fat pad tissues are ongoing.