Metalloproteinase and inhibitor expression profiling of resorbing cartilage reveals pro-collagenase activation as a critical step for collagenolysis

Excess proteolysis of the extracellular matrix (ECM) of articular cartilage is a key characteristic of arthritis. The main enzymes involved belong to the metalloproteinase family, specifically the matrix metalloproteinases (MMPs) and a group of proteinases with a disintegrin and metalloproteinase domain with thrombospondin motifs (ADAMTS). Chondrocytes are the only cell type embedded in the cartilage ECM, and cell-matrix interactions can influence gene expression and cell behaviour. Thus, although the use of monolayer cultures can be informative, it is essential to study chondrocytes encapsulated within their native environment, cartilage, to fully assess cellular responses. The aim of this study was to profile the temporal gene expression of metalloproteinases and their endogenous inhibitors, the tissue inhibitors of metalloproteinases (TIMPs), reversion-inducing cysteine-rich protein with Kazal motifs (RECK), and α(2)-macroglobulin (α(2)M), in actively resorbing cartilage. The addition of the pro-inflammatory cytokine combination of interleukin-1 (IL-1) + oncostatin M (OSM) to bovine nasal cartilage induces the synthesis and subsequent activation of pro-metalloproteinases, leading to cartilage resorption. We show that IL-1+OSM upregulated the expression of MMP-1, -2, -3, -9, 12, -13, -14, TIMP-1, and ADAMTS-4, -5, and -9. Differences in basal expression and the magnitude of induction were observed, whilst there was no significant modulation of TIMP-2, -3, RECK, or ADAMTS-15 gene expression. IL-1+OSM downregulated MMP-16,TIMP-4, and α(2)M expression. All IL-1+OSM-induced metalloproteinases showed marked upregulation early in the culture period, whilst inhibitor expression was reduced throughout the stimulation period such that metalloproteinase production would be in excess of inhibitors. Moreover, although pro-collagenases were upregulated and synthesized early (by day 5), collagenolysis became apparent later with the presence of active collagenases (day 10) when inhibitor levels were low. These findings indicate that the activation cascades for pro-collagenases are delayed relative to collagenase expression, further confirm the coordinated regulation of metalloproteinases in actively resorbing cartilage, and support the use of bovine nasal cartilage as a model system to study the mechanisms that promote cartilage degradation

Effect of a Perioperative, Cardiac Output-Guided Hemodynamic Therapy Algorithm on Outcomes Following Major Gastrointestinal Surgery A Randomized Clinical Trial and Systematic Review

Importance: small trials suggest that postoperative outcomes may be improved by the use of cardiac output monitoring to guide administration of intravenous fluid and inotropic drugs as part of a hemodynamic therapy algorithm.Objective: to evaluate the clinical effectiveness of a perioperative, cardiac output–guided hemodynamic therapy algorithm.Design, setting, and participants: OPTIMISE was a pragmatic, multicenter, randomized, observer-blinded trial of 734 high-risk patients aged 50 years or older undergoing major gastrointestinal surgery at 17 acute care hospitals in the United Kingdom. An updated systematic review and meta-analysis were also conducted including randomized trials published from 1966 to February 2014.Interventions: patients were randomly assigned to a cardiac output–guided hemodynamic therapy algorithm for intravenous fluid and inotrope (dopexamine) infusion during and 6 hours following surgery (n=368) or to usual care (n=366).Main outcomes and measures: the primary outcome was a composite of predefined 30-day moderate or major complications and mortality. Secondary outcomes were morbidity on day 7; infection, critical care–free days, and all-cause mortality at 30 days; all-cause mortality at 180 days; and length of hospital stay.Results: baseline patient characteristics, clinical care, and volumes of intravenous fluid were similar between groups. Care was nonadherent to the allocated treatment for less than 10% of patients in each group. The primary outcome occurred in 36.6% of intervention and 43.4% of usual care participants (relative risk [RR], 0.84 [95% CI, 0.71-1.01]; absolute risk reduction, 6.8% [95% CI, ?0.3% to 13.9%]; P?=?.07). There was no significant difference between groups for any secondary outcomes. Five intervention patients (1.4%) experienced cardiovascular serious adverse events within 24 hours compared with none in the usual care group. Findings of the meta-analysis of 38 trials, including data from this study, suggest that the intervention is associated with fewer complications (intervention, 488/1548 [31.5%] vs control, 614/1476 [41.6%]; RR, 0.77 [95% CI, 0.71-0.83]) and a nonsignificant reduction in hospital, 28-day, or 30-day mortality (intervention, 159/3215 deaths [4.9%] vs control, 206/3160 deaths [6.5%]; RR, 0.82 [95% CI, 0.67-1.01]) and mortality at longest follow-up (intervention, 267/3215 deaths [8.3%] vs control, 327/3160 deaths [10.3%]; RR, 0.86 [95% CI, 0.74-1.00]).Conclusions and relevance: in a randomized trial of high-risk patients undergoing major gastrointestinal surgery, use of a cardiac output–guided hemodynamic therapy algorithm compared with usual care did not reduce a composite outcome of complications and 30-day mortality. However, inclusion of these data in an updated meta-analysis indicates that the intervention was associated with a reduction in complication rate

Marine exploitation and the arrival of farming: resolving the paradox of the Mesolithic-Neolithic transition in Denmark

The transition to farming in the coastal environments of southern Scandinavia remains a key conundrum in European prehistory. This region was heavily exploited by Late Mesolithic communities of the Ertebølle culture, complex hunter-fisher-gatherers who flourished for some 1500 years prior to the arrival of farming at around 4000 BCE marking the start of the Neolithic period. Extensive genetic and isotopic analyses of skeletal remains suggests that the arrival of farming is marked by a rapid demographic change and that incoming populations of ‘farmers’ had little reliance on marine resources. In contrast, frequent archaeological finds of shell middens and fishing gear in the Early Neolithic supports evidence for continuity in the use of marine resources across the transition. To assess this apparent paradox, focusing on the Danish evidence, we explore the spatiotemporal trends in the density of some 1500 radiocarbon dates using new informatics tools and modelling strategies. We indeed find strong archaeological indicators of sustained and even intensified patterns of coastal exploitation across and beyond the transition; shell middens, fishing implements, and aquatic residues in ceramics continue well into the Neolithic. Using an agent-based demographic model, we demonstrate how small differences in fertility could rapidly dilute signals of coastal resource use in the context of a growing Neolithic population. More broadly, we suggest that complex palimpsests of archaeological remains and biological information from human remains can only usefully be interpreted through the lens of demography

Selective digestive tract decontamination to prevent healthcare associated infections in critically ill children:the PICNIC multicentre randomised pilot clinical trial

Healthcare-associated infections (HCAIs) are a major cause of morbidity and mortality in critically ill children. Data from adult studies suggest Selective Decontamination of the Digestive tract (SDD) may reduce the incidence of HCAIs and improve survival. There are no data from randomised clinical trials in the paediatric setting. An open label, parallel group pilot cRCT and mixed-methods perspectives study was conducted in six paediatric intensive care units (PICUs) in England. Participants were children (&gt; 37 weeks corrected gestational age, up to 16 years) requiring mechanical ventilation expected to last for at least 48 h. Sites undertook standard care for a period of 9 weeks and were randomised into 3 sites which continued standard care and 3 where SDD was incorporated into infection control practice for eligible children. Interviews and focus groups were conducted for parents and staff working in PICU. 434 children fulfilled eligibility criteria, of whom 368 (85%) were enrolled. This included 207 in the baseline phase (Period One) and 161 in the intervention period (Period Two). In sites delivering SDD, the majority (98%) of children received at least one dose of SDD and of these, 68% commenced within the first 6 h. Whilst admission swabs were collected in 91% of enrolled children, consent for the collection of additional swabs was low (44%). Recruited children were representative of the wider PICU population. Overall, 3.6 children/site/week were recruited compared with the potential recruitment rate for a definitive cRCT of 3 children/site/week, based on data from all UK PICUs. Parents (n = 65) and staff (n = 44) were supportive of the aims of the study, suggesting adaptations for a larger definitive trial including formulation and administration of SDD paste, approaches to consent and ecology monitoring. Stakeholders identified preferred clinical outcomes, focusing on complications of critical illness and quality-of-life. A definitive cRCT in SDD to prevent HCAIs in critically ill children is feasible but should include adaptations to ecology monitoring along with the dosing schedule and packaging into a paediatric specific format. A definitive study is supported by the findings with adaptations to ecology monitoring and SDD administration. Trial Registration: ISRCTN40310490 Registered 30/10/2020.</p

Whole-genome sequencing reveals host factors underlying critical COVID-19

Critical COVID-19 is caused by immune-mediated inflammatory lung injury. Host genetic variation influences the development of illness requiring critical care1 or hospitalization2–4 after infection with SARS-CoV-2. The GenOMICC (Genetics of Mortality in Critical Care) study enables the comparison of genomes from individuals who are critically ill with those of population controls to find underlying disease mechanisms. Here we use whole-genome sequencing in 7,491 critically ill individuals compared with 48,400 controls to discover and replicate 23 independent variants that significantly predispose to critical COVID-19. We identify 16 new independent associations, including variants within genes that are involved in interferon signalling (IL10RB and PLSCR1), leucocyte differentiation (BCL11A) and blood-type antigen secretor status (FUT2). Using transcriptome-wide association and colocalization to infer the effect of gene expression on disease severity, we find evidence that implicates multiple genes—including reduced expression of a membrane flippase (ATP11A), and increased expression of a mucin (MUC1)—in critical disease. Mendelian randomization provides evidence in support of causal roles for myeloid cell adhesion molecules (SELE, ICAM5 and CD209) and the coagulation factor F8, all of which are potentially druggable targets. Our results are broadly consistent with a multi-component model of COVID-19 pathophysiology, in which at least two distinct mechanisms can predispose to life-threatening disease: failure to control viral replication; or an enhanced tendency towards pulmonary inflammation and intravascular coagulation. We show that comparison between cases of critical illness and population controls is highly efficient for the detection of therapeutically relevant mechanisms of disease

Whole-genome sequencing reveals host factors underlying critical COVID-19

Critical COVID-19 is caused by immune-mediated inflammatory lung injury. Host genetic variation influences the development of illness requiring critical care1 or hospitalization2,3,4 after infection with SARS-CoV-2. The GenOMICC (Genetics of Mortality in Critical Care) study enables the comparison of genomes from individuals who are critically ill with those of population controls to find underlying disease mechanisms. Here we use whole-genome sequencing in 7,491 critically ill individuals compared with 48,400 controls to discover and replicate 23 independent variants that significantly predispose to critical COVID-19. We identify 16 new independent associations, including variants within genes that are involved in interferon signalling (IL10RB and PLSCR1), leucocyte differentiation (BCL11A) and blood-type antigen secretor status (FUT2). Using transcriptome-wide association and colocalization to infer the effect of gene expression on disease severity, we find evidence that implicates multiple genes—including reduced expression of a membrane flippase (ATP11A), and increased expression of a mucin (MUC1)—in critical disease. Mendelian randomization provides evidence in support of causal roles for myeloid cell adhesion molecules (SELE, ICAM5 and CD209) and the coagulation factor F8, all of which are potentially druggable targets. Our results are broadly consistent with a multi-component model of COVID-19 pathophysiology, in which at least two distinct mechanisms can predispose to life-threatening disease: failure to control viral replication; or an enhanced tendency towards pulmonary inflammation and intravascular coagulation. We show that comparison between cases of critical illness and population controls is highly efficient for the detection of therapeutically relevant mechanisms of disease

Duration of androgen deprivation therapy with postoperative radiotherapy for prostate cancer: a comparison of long-course versus short-course androgen deprivation therapy in the RADICALS-HD randomised trial

Background Previous evidence supports androgen deprivation therapy (ADT) with primary radiotherapy as initial treatment for intermediate-risk and high-risk localised prostate cancer. However, the use and optimal duration of ADT with postoperative radiotherapy after radical prostatectomy remains uncertain. Methods RADICALS-HD was a randomised controlled trial of ADT duration within the RADICALS protocol. Here, we report on the comparison of short-course versus long-course ADT. Key eligibility criteria were indication for radiotherapy after previous radical prostatectomy for prostate cancer, prostate-specific antigen less than 5 ng/mL, absence of metastatic disease, and written consent. Participants were randomly assigned (1:1) to add 6 months of ADT (short-course ADT) or 24 months of ADT (long-course ADT) to radiotherapy, using subcutaneous gonadotrophin-releasing hormone analogue (monthly in the short-course ADT group and 3-monthly in the long-course ADT group), daily oral bicalutamide monotherapy 150 mg, or monthly subcutaneous degarelix. Randomisation was done centrally through minimisation with a random element, stratified by Gleason score, positive margins, radiotherapy timing, planned radiotherapy schedule, and planned type of ADT, in a computerised system. The allocated treatment was not masked. The primary outcome measure was metastasis-free survival, defined as metastasis arising from prostate cancer or death from any cause. The comparison had more than 80% power with two-sided α of 5% to detect an absolute increase in 10-year metastasis-free survival from 75% to 81% (hazard ratio [HR] 0·72). Standard time-to-event analyses were used. Analyses followed intention-to-treat principle. The trial is registered with the ISRCTN registry, ISRCTN40814031, and ClinicalTrials.gov , NCT00541047 . Findings Between Jan 30, 2008, and July 7, 2015, 1523 patients (median age 65 years, IQR 60–69) were randomly assigned to receive short-course ADT (n=761) or long-course ADT (n=762) in addition to postoperative radiotherapy at 138 centres in Canada, Denmark, Ireland, and the UK. With a median follow-up of 8·9 years (7·0–10·0), 313 metastasis-free survival events were reported overall (174 in the short-course ADT group and 139 in the long-course ADT group; HR 0·773 [95% CI 0·612–0·975]; p=0·029). 10-year metastasis-free survival was 71·9% (95% CI 67·6–75·7) in the short-course ADT group and 78·1% (74·2–81·5) in the long-course ADT group. Toxicity of grade 3 or higher was reported for 105 (14%) of 753 participants in the short-course ADT group and 142 (19%) of 757 participants in the long-course ADT group (p=0·025), with no treatment-related deaths. Interpretation Compared with adding 6 months of ADT, adding 24 months of ADT improved metastasis-free survival in people receiving postoperative radiotherapy. For individuals who can accept the additional duration of adverse effects, long-course ADT should be offered with postoperative radiotherapy. Funding Cancer Research UK, UK Research and Innovation (formerly Medical Research Council), and Canadian Cancer Society

Naturally occurring canine osteosarcoma in the dog animal model for research of targeted radiotherapy using beta-emitting radioisotopes with various ligands

Metastatic and primary bone cancers are devastating diseases of paediatric and adult humans because of the morbidity associated with bone pain. Controlling bone pain from multiple metastatic sites can be difficult in end-stage cancers using conventional therapies. Bone-seeking radiopharmaceuticals have been successful in this area as radiation can be delivered with moderate selectivity to the target. Unfortunately, targeted radiotherapy using radiopharmaceuticals have been relegated to palliative therapy as myelosuppression largely limits the radiation dose to sub-therapeutic levels. Efforts to overcome this therapeutic limitation include autologous bone marrow transplants in combination with chemotherapy-radiosensitization and the development of new radiopharmaceuticals. Development work using laboratory rodent models has been complicated by dosimetric limitations because of size and inherent problems with human xenografted tumour models in rodents. To address this need we studied naturally occurring canine osteosarcoma as a translational model for human bone cancer. Central to our hypothesis was that naturally occurring canine osteosarcoma would serve as an investigational model for comparing the pharmacokinetics (biodistribution), dosimetry, toxicity, and therapeutic effect of 153Sm-EDTMP, 188Re-HEDP, 186Re-HEDP, and a novel ligand, polyethyleneiminomethyl phosphonic acid (PEI-MP). Data collected from existing radiopharmaceuticals was then compared to PEI-MP labelled with 99mTc, 153Sm, and 186Re. This innovative and unique study allowed for the evaluation of various radiopharmaceuticals in a naturally occurring animal model of bone cancer, documenting the pharmacokinetics and dosimetry of a novel radiolabelled-ligand (PEI-MP). Benefits resulting from the successful completion of the study would allow more rapid transfer of rodent preclinical data into a naturally occurring cancer model more resembling to the human diseases and would thus more likely identify problems with pharmacokinetics and toxicity before proceeding to expensive clinical trials. The expected outcomes of the study were originally formulated based on limited previous published data in dogs. For instance, no data exists describing the pharmacokinetics or toxicity of 188Re-HEDP and 186Re-HEDP in the dog. The study was conducted in two phases. The first phase deals with the evaluation of 153Sm-EDTMP, 188Re-HEDP, and 186Re-HEDP in the dog model. Phase-two was the development of a novel ligand (PEI-MP) in the dog model of osteosarcoma, which has the characteristics of an ideal ligand. Pharmacokinetic results for 153Sm-EDTMP in normal dogs (n=4) for blood were similar to published reports for dogs and human. When compared statistically to human data the majority of results were the same, lending credence to the hypothesis that dogs could serve as models for human radiopharmaceutical research. Normal dogs and the osteosarcoma dog did differ from human pharmacokinetics in the urine elimination phase (t½-â). This can most likely be explained by the tumour burden in the human research populations or due to the fact that most humans were aged and likely to have some renal disease. Certainly, the trend in dogs with osteosarcoma was to have a prolonged urine elimination phase (t½-â) compared to normal dogs which supported the hypothesis that the biodistribution and pharmacokinetics results from dogs were similar to human published data. Statistical comparisons were also made between normal dogs receiving 188Re-HEDP and 153Sm-EDTMP. The prolonged urine elimination phase (t½-â) and increased blood retention of 188Re-HEDP was most likely a reflection of prolonged bone washout and soft issue retention. This could be attributed to the differences between the antiresorptive capability of bisphosphonate ligands e.g., EDTMP (lexidronam) with a greater than 100-fold antiresorptive capability than HEDP (etidronate). Additional observational biodistribution studies using macro- and micro-autoradiography techniques were also performed in canine tissue and Sprague-Dawley rats. Results from the studies showed heterogeneous uptake within tumours in dogs. In rats, localization of 153Sm-EDTMP in red marrow areas would lead to a high radiation dose to blood producing elements. In addition, high uptake was documented at the metaphyseal growth plate confirming the likelihood of a delay or cessation of growth if 153Sm-EDTMP were used in growing children. Phase-one of the clinical trial in dogs with naturally occurring osteosarcoma identified only mild toxicity at the dosage rate of 37 MBq/kg (1 mCi/kg) for both 153Sm-EDTMP and188Re-HEDP. In addition, a pilot trial was conducted in dogs receiving 153Sm-EDTMP which also received a carboplatin infusion at the time of the radiopharmaceutical administration followed by another 3 cycles of carboplatin at 3 weekly intervals. No differences in toxicity were noted between the carboplatin group and dogs receiving only 153Sm-EDTMP. As a part of the 188Re-HEDP clinical trial, 3 dogs with osteosarcomas received weekly dose of 188Re-HEDP at 37MBq/kg for 4 weeks in which only mild toxicity was noted. Unfortunately, there was no cessation in growth of the tumours, with all dogs showing progression. The median survival time for both radiopharmaceuticals was 4 months, significantly shorter than the 10-month median survival time for amputation and chemotherapy. Interestingly six dogs that had 99mTc-MDP and 153Sm-EDTMP showed scans of tumours that had consistently lower 99mTc-MDP uptake ratios (normal bone compared to cancerous bone) compared to solely 153Sm-EDTMP. In contrast, this was not evident for uptake ratios between 188Re-HEDP and 99mTc-MDP scans. Once again, this finding highlights the differences between the antiresorptive capabilities of the bisphosphonates ligands. Interestingly, another three dogs were scanned with 99mTc-MDP , 153Sm-EDTMP, and 99mTc-PEI-MP (10-30 kDa) showed a variation in uptake between scans of the same tumours. More importantly, the uptake ratios of 99mTc-MDP and 153Sm-EDTMP scans showed wide variation with a coefficient of variance of 52% and 39% respectively. However, the range in uptake from the 99mTc-PEI-MP (10-30 kDa) scan was narrow with a coefficient of variance of only 6%. This could be attributed to more consistent uptake ratio of the unique ligand PEI-MP and its hypothesized mechanism of action: enhanced permeability and retention (EPR) in tumour vasculature. This requires further investigation with larger groups. In phase-two, the pharmacokinetic result for the novel ligand PEI-MP was initially studied labelled with 99mTc. Various molecular weights were tested in normal dogs and compared to previously published results in baboons. Results from the dog studies were found to be similar to those from the primate study. As in the primate study, molecular weight and charge played a significant role in 99mTc-PEI-MP pharmacokinetics. Increasing the size of the macromolecules and altering their charge resulted in marked changes in their pharmacokinetics and biodistribution. The PEI-MP molecular weight of 10-30 kDa and 20-30 kDa were the most promising and fulfilled the hypothesized criteria of an ideal radiopharmaceutical. In keeping with the aims of the study, the 20-30kDa polymer was considered more desirable because of its faster clearance. However, because of the limitations imposed by the percentage yield of the different molecular weights of the ligand during filtration, we decided to label the 10-30kDa molecular weight MW-fraction with 153Sm. Unexpectedly, the 153Sm-PEI-MP 10-30 kDa had a prolonged urine elimination phase (t½-â) associated with increased liver uptake when compared to 99mTc-PEI-MP10-30 kDa. To explain this, computer modelling for blood plasma (ECCLES) was done which predicted that there would be some chemical dissociation of the 153Sm from the PEI-MP polymer in blood. This is due to interaction between the radiopharmaceutical and citrate, forming 153Sm-citrate. The ECCLES model for blood plasma also predicted that the anionic MW-fraction, PEI-MP 10-30kDa, would be a poor ligand complexed to 166Ho, 212Pb, 213Pb, and 89Sr, but was expected to be effective when complexed to 186Re or 188Re, based on their close proximity to 99mTc on the periodic table. As a preliminary study 186Re was complexed to 20-30 kDa (n=2) and 30-50 kDa (n=1) MW-fractions and tested in dogs. The results were similar to 99mTc-PEI-MP 10-30 kDa. The biodistribution data and pharmacokinetic data were also used to do comparative dosimetry between radiopharmaceuticals. Not surprisingly, the dosimetry data confirmed the high red marrow dose for 153Sm-EDTMP and the increased soft-tissue dose of the radionuclides complexed to HEDP. The radiation dose to the tumour for all radiopharmaceuticals fell within the range of 26Gy to 44Gy. This is well within the range used to treat canine osteosarcoma using external beam radiotherapy. When compared to external beam radiotherapy, the probable lack of tumour response in our clinical trial relates to the heterogenous distribution of the radiopharmaceutical in the tumour and the inherent resistance of osteosarcoma cells to continuous low-dose radiation delivery (CLDR) inherent in radionuclide -particle decay. The study met the majority of outcomes with the exception of labelling PEI-MP with 153Sm. This was due to the interaction of the 153Sm-PEI-MP complex with citrate ions in blood. Rapid deterioration of the Rhenium-188 generator also led to earlier than expected curtailment of the 188Re-HEDP therapeutic trial although sufficient data was available to be used in a comparative study.Thesis (PhD)--University of Pretoria, 2013.Internal Medicineunrestricte