The Immunological Effect of Oxygen Carriers on Normothermic Ex Vivo Liver Perfusion.

Introduction: Normothermic ex vivo liver perfusion (NEVLP) is an organ preservation method that allows liver graft functional assessment prior to transplantation. One key component of normothermic perfusion solution is an oxygen carrier to provide oxygen to the liver to sustain metabolic activities. Oxygen carriers such as red blood cells (RBCs) or hemoglobin-based oxygen carriers have an unknown effect on the liver-resident immune cells during NEVLP. In this study, we assessed the effects of different oxygen carriers on the phenotype and function of liver-resident immune cells. Methods: Adult Lewis rat livers underwent NEVLP using three different oxygen carriers: human packed RBCs (pRBCs), rat pRBCs, or Oxyglobin (a synthetic hemoglobin-based oxygen carrier). Hourly perfusate samples were collected for downstream analysis, and livers were digested to isolate immune cells. The concentration of common cytokines was measured in the perfusate, and the immune cells underwent phenotypic characterization with flow cytometry and quantitative reverse transcription polymerase chain reaction (qRT-PCR). The stimulatory function of the liver-resident immune cells was assessed using mixed lymphocyte reactions. Results: There were no differences in liver function, liver damage, or histology between the three oxygen carriers. qRT-PCR revealed that the gene expression of nuclear factor κ light chain enhancer of activated B cells (NF-kB), Interleukin (IL-1ß), C-C motif chemokine ligand 2 (CCL2), C-C motif chemokine ligand 7 (CCL7), and CD14 was significantly upregulated in the human pRBC group compared with that in the naive, whereas the rat pRBC and Oxyglobin groups were not different from that of naive. Flow cytometry demonstrated that the cell surface expression of the immune co-stimulatory protein, CD86, was significantly higher on liver-resident macrophages and plasmacytoid dendritic cells perfused with human pRBC compared to Oxyglobin. Mixed lymphocyte reactions revealed increased allogeneic T-cell proliferation in the human and rat pRBC groups compared to that in the Oxyglobin group. Conclusions: Liver-resident immune cells are important mediators of rejection after transplantation. In this study, we show that the oxygen carrier used in NEVLP solutions can affect the phenotype of these liver-resident immune cells. The synthetic hemoglobin-based oxygen carrier, Oxyglobin, showed the least amount of liver-resident immune cell activation and the least amount of allogeneic proliferation when compared to human or rat pRBCs. To mitigate liver-resident immune cell activation during NEVLP (and subsequent transplantation), Oxyglobin may be an optimal oxygen carrier.

Positioning accuracy assessment of minimally invasive percutaneous injection techniques for the treatment of temporomandibular disorders.

OBJECTIVE: The aim of the present study was to evaluate the accuracy of an extraoral CBCT-planned 3D-printed surgical guide aimed to percutaneous injection of substances into the temporomandibular joint (TMJ) and the lateral pterygoid muscle (LPM). METHODS: Nine human cadaver heads were used. Pre-planning CBCT and facial scans were obtained and three percutaneous injection sites were planned: one for the lower compartment of the TMJ and two for the LPM. A digital surgical guide was then designed with small titanium sleeves and printed by a 3D printer. After the injections, new CBCT scans with the needles in place were obtained in order to assess the accuracy of the procedure in relation to the virtual planning. RESULTS: The mean values for angle deviation were very low (range 1.13o-4.08o), the same happening for the mean difference in the length reached (range 1.82-2.64 mm), as well as for the mean difference in the needle tip dislocation (range 0.94-2.03 mm). CONCLUSION: The guide seems to be a reliable tool for accurate percutaneous injection of drugs into the inferior compartment of the TMJ and the LPM. Further studies are necessary to test the efficacy and validate the method in an in vivo study.

Activated protein C targets immune cells and rheumatoid synovial fibroblasts to prevent inflammatory arthritis in mice.

OBJECTIVES: To investigate whether activated protein C (APC), a physiological anticoagulant can inhibit the inflammatory/invasive properties of immune cells and rheumatoid arthritis synovial fibroblasts (RASFs) in vitro and prevent inflammatory arthritis in murine antigen-induced arthritis (AIA) and CIA models. METHODS: RASFs isolated from synovial tissues of patients with RA, human peripheral blood mononuclear cells (PBMCs) and mouse thymus cells were treated with APC or TNF-α/IL-17 and the following assays were performed: RASF proliferation and invasion by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and cell invasion assays, respectively; cytokines and signalling molecules using ELISA or western blot; Th1 and Th17 phenotypes in human PBMCs or mouse thymus cells by flow cytometry. The in vivo effect of APC was evaluated in AIA and CIA models. RESULTS: In vitro, APC inhibited IL-1ß, IL-17 and TNF-α production, IL-17-stimulated cell proliferation and invasion and p21 and nuclear factor κB activation in RASFs. In mouse thymus cells and human PBMCs, APC suppressed Th1 and Th17 phenotypes. In vivo, APC inhibited pannus formation, cartilage destruction and arthritis incidence/severity in both CIA and AIA models. In CIA, serum levels of IL-1ß, IL-6, IL-17, TNF-α and soluble endothelial protein C receptor were significantly reduced by APC treatment. Blocking endothelial protein C receptor, the specific receptor for APC, abolished the early or preventative effect of APC in AIA. CONCLUSION: APC prevents the onset and development of arthritis in CIA and AIA models via suppressing inflammation, Th1/Th17 phenotypes and RASF invasion, which is likely mediated via endothelial protein C receptor.

Structured patient handoff on an internal medicine ward: A cluster randomized control trial.

BACKGROUND: The effect of a multi-faceted handoff strategy in a high volume internal medicine inpatient setting on process and patient outcomes has not been clearly established. We set out to determine if a multi-faceted handoff intervention consisting of education, standardized handoff procedures, including fixed time and location for face-to-face handoff would result in improved rates of handoff compared with usual practice. We also evaluated resident satisfaction, health resource utilization and clinical outcomes. METHODS: This was a cluster randomized controlled trial in a large academic tertiary care center with 18 inpatient internal medicine ward teams from January-April 2013. We randomized nine inpatient teams to an intervention where they received an education session standardizing who and how to handoff patients, with practice and feedback from facilitators. The control group of 9 teams continued usual non-standardized handoffs. The primary process outcome was the rate of patients handed over per 1000 patient nights. Other process outcomes included perceptions of inadequate handoff by overnight physicians, resource utilization overnight and hospital length of stay. Clinical outcomes included medical errors, frequency of patients requiring higher level of care overnight, and in-hospital mortality. RESULTS: The intervention group demonstrated a significant increase in the rate of patients handed over to the overnight physician (62.90/1000 person-nights vs. 46.86/1000 person-nights, p = 0.002). There was no significant difference in other process outcomes except resource utilization was increased in the intervention group (26.35/1000 person-days vs. 17.57/1000 person-days, p-value = 0.01). There was no significant difference between groups in medical errors (4.8% vs. 4.1%), need for higher level of care or in hospital mortality. Limitations include a dependence of accurate record keeping by the overnight physician, the possibility of cross-contamination in the handoff process, analysis at the cluster level and an overall low number of clinical events. CONCLUSIONS: Implementation of a multi-faceted resident handoff intervention did not result in a significant improvement in patient safety although did improve number of patients handed off. Novel methods to improve handoff need to be explored. TRIAL REGISTRATION: Registered at ClinicalTrials.gov: NCT01796756.

Therapeutic interventions for osteoarthritis of the wrist: a systematic review and meta-analysis.

Background: In order to evaluate the effectiveness of interventions for osteoarthritis of the wrist in adults we performed a systematic review and meta-analysis. Methods: The MEDLINE and EMBASE via OVID, CINAHL and SPORTDiscus via EBSCO databases were searched from inception to 25 th April  2018.All randomised controlled clinical trials (RCTs) and any prospective studies of adults with wrist osteoarthritis investigating any intervention with a comparator were included.  Data were extracted and checked for accuracy and completeness by pairs of reviewers. Primary outcomes were pain and function. Comparative treatment effects were analysed by random effects at all time points. Results: Three RCTs were identified for inclusion after screening and all had a high risk of bias. Two compared proximal row carpectomy (PRC) with four corner fusion (4CF) for post-traumatic osteoarthritis, while the other compared leather with commercial wrist splints in patients with chronic wrist pain, of which a small group had wrist osteoarthritis.  Conclusion: There is no prospective study comparing operative to non-operative treatment for wrist osteoarthritis, while there is a paucity of prospective studies assessing the effectiveness of both non-operative and operative interventions.  Further research is necessary in order to better define which patients benefit from which specific interventions. Registration: The review protocol was registered with PROSPERO under the registration number CRD42018094799.

Who to handover: a case-control study of a novel scoring system to prioritise handover of internal medicine inpatients.

BACKGROUND: Handover of patients between care providers is a critical event in patient care. There is, however, little evidence to guide the handover process, including determining which patients to handover. AIM: Compare the ability of gestalt-based handover with two structured scores, the modified early warning score (MEWS) and our novel iHAND clinical decision support system, to predict which patients will be assessed by a physician overnight. METHODS: This case-control study included 90 inpatients, comprising 32 patients assessed overnight (cases) and 58 patients not assessed overnight (controls) at a teaching hospital in British Columbia, Canada (May 2012). Gestalt, MEWS and iHAND scores were analysed against patients seen overnight using logistic regression and receiver-operating characteristic (ROC) curves. RESULTS: Neither current gestalt-based handover practice (odds ratio (OR) 1.50, 95% CI 0.89 to 3.83) nor MEWS (OR 0.96, 95% CI 0.75 to 1.24, area under the ROC curve (AUC) 0.61, 95% CI 0.49 to 0.73) were significantly associated with need to be seen overnight. The iHAND score was associated with need to be seen (OR 1.93, 95% CI 1.24 to 3.02, AUC 0.70, 95% CI 0.60 to 0.81). CONCLUSIONS: The iHAND score had moderate ability to predict which patients required assessment overnight, while MEWS score and current gestalt approach correlated poorly, suggesting the iHAND score may help prioritisation of patients likely to be seen overnight for handover.

Multifidus Muscle Changes After Back Injury Are Characterized by Structural Remodeling of Muscle, Adipose and Connective Tissue, but Not Muscle Atrophy: Molecular and Morphological Evidence.

STUDY DESIGN: Longitudinal case-controlled animal study. OBJECTIVE: To investigate putative cellular mechanisms to explain structural changes in muscle and adipose and connective tissues of the back muscles after intervertebral disc (IVD) injury. SUMMARY OF BACKGROUND DATA: Structural back muscle changes are ubiquitous with back pain/injury and considered relevant for outcome, but their exact nature, time course, and cellular mechanisms remain elusive. We used an animal model that produces phenotypic back muscle changes after IVD injury to study these issues at the cellular/molecular level. METHODS: Multifidus muscle was harvested from both sides of the spine at L1-L2 and L3-L4 IVDs in 27 castrated male sheep at 3 (n = 10) or 6 (n = 17) months after a surgical anterolateral IVD injury at both levels. Ten control sheep underwent no surgery (3 mo, n = 4; 6 mo, n = 6). Tissue was harvested at L4 for histological analysis of cross-sectional area of muscle and adipose and connective tissue (whole muscle), plus immunohistochemistry to identify proportion and cross-sectional area of individual muscle fiber types in the deepest fascicle. Quantitative polymerase chain reaction measured gene expression of typical cytokines/signaling molecules at L2. RESULTS: Contrary to predictions, there was no multifidus muscle atrophy (whole muscle or individual fiber). There was increased adipose and connective tissue (fibrotic proliferation) cross-sectional area and slow-to-fast muscle fiber transition at 6 but not 3 months. Within the multifidus muscle, increases in the expression of several cytokines (tumor necrosis factor α and interleukin-1ß) and molecules that signal trophic/atrophic processes for the 3 tissue types (e.g., growth factor pathway [IGF-1, PI3k, Akt1, mTOR], potent tissue modifiers [calcineurin, PCG-1α, and myostatin]) were present. CONCLUSION: This study provides cellular evidence that refutes the presence of multifidus muscle atrophy accompanying IVD degeneration at this intermediate time point. Instead, adipose/connective tissue increased in parallel with the expression of the genes that provide putative mechanisms for multifidus structural remodeling. This provides novel targets for pharmacological and physical interventions. LEVEL OF EVIDENCE: N/A.

Can proinflammatory cytokine gene expression explain multifidus muscle fiber changes after an intervertebral disc lesion?

STUDY DESIGN: Longitudinal case-controlled animal study. OBJECTIVE: To investigate the effect of an intervertebral disc (IVD) lesion on the proportion of slow, fast, and intermediate muscle fiber types in the multifidus muscle in sheep, and whether muscle fiber changes were paralleled by local gene expression of the proinflammatory cytokines tumor necrosis factor α (TNF-α) and interleukin 1-ß. SUMMARY OF BACKGROUND DATA: Structure and behavior of the multifidus muscle change in acute and chronic back pain, but the mechanisms are surprisingly poorly understood and the link between structure and behavior is tenuous. Although changes in muscle fiber types have the potential to unify the observations, the effect of injury on muscle fiber distribution has not been adequately tested, and understanding of possible mechanisms is limited. METHODS: The L1-L2, L3-L4, and L5-L6 IVDs of 11 castrated male sheep received anterolateral lesions. Six control sheep underwent no surgical procedures. Multifidus muscle tissue was harvested at L4 for muscle fiber analysis using immunohistochemistry and L2 for cytokine analysis with polymerase chain reaction for local gene expression of TNF-α and interleukin-1ß. RESULTS: The proportion of slow muscle fibers in multifidus was significantly less in the lesioned animals both ipsilateral and contralateral to the IVD lesion. The greatest reduction in slow fibers was in the deep medial muscle region. A greater prevalence of intermediate fibers on the uninjured side implies a delayed fiber-type transformation on that side. TNF-α gene expression in multifidus was greater on both sides in the lesion animals than in the muscle of control animals. Interleukin-1ß was increased only on the injured side. CONCLUSION: These data provide evidence of muscle fiber changes after induction of an IVD lesion and a parallel increase in TNF-α expression. Proinflammatory cytokine changes provide a novel mechanism to explain behavioral and structural changes in multifidus. LEVEL OF EVIDENCE: N/A.

An evaluation of inertial sensor technology in the discrimination of human gait.

Inertial sensors may provide the opportunity for broader and more cost effective gait analysis; however some questions remain over their potential use in this capacity. The aim of the study was to determine whether an inertial sensor could discriminate between normal walking, fast walking, and running. A single group crossover design was used to compare acceleration profiles between three gait conditions: normal walking, fast walking, and running. An inertial sensor was placed on the sacrum of 12 participants (6 male, 6 female) who performed 3 trials of each gait condition on both overground and treadmill settings. A significant difference (P < 0.001) in the occurrence of heel strike in the gait cycle was found between running and both walking conditions. No differences were seen between overground and treadmill in any condition or variable. The results indicate that a single sacral mounted inertial sensor can differentiate running from normal walking and fast walking using temporal gait event measures. This study indicates that inertial sensors can differentiate walking from running gait in healthy individuals which may have potential for application in the quantification of physical activity in the health and exercise industry.

Tranexamic acid reduces early post-operative blood loss after total knee arthroplasty: a prospective randomised controlled trial of 29 patients.

INTRODUCTION: Extensive blood loss related to knee arthroplasty is quite normal and many patients require blood transfusions. Surgery and the use of pneumatic tourniquets lead to an increase in the activity of the fibrinolytic system, which in turn may accentuate the blood loss. Drugs that inhibit the fibrinolytic system may thus be used to reduce blood loss. Tranexamic acid (TA) acts by binding to one of the enzymes at the start of the coagulation cascade, so inhibiting the fibrinolytic system. A concern is that this inhibition may have the side effect of increasing thromboembolic disease, a common complication of joint replacement surgery. We aimed to confirm the reductions in blood loss and to assess the impact of TA usage on clinical and sub-clinical DVT. METHOD: We performed a prospective, randomised, double blind, controlled trial, using patients due to undergo primary unilateral total knee arthroplasty. Patients were randomised to receive either 15 mg/kg of tranexamic acid or a similar volume of normal saline at the time of cementing of the prosthesis. Perioperative blood loss was recorded and patients were screened for DVT with duplex ultrasound assessment of both legs on the fifth post-operative day. RESULTS: A statistically significant (p=0.006) decrease in blood loss in the early post-operative period was noted in the group receiving tranexamic acid. This was not associated with a significant difference in total blood loss (p=0.55) or in transfusion requirements. There was no of evidence in DVT in either group on duplex ultrasound screening of the lower limbs. INTERPRETATION: One injection of 15 mg/kg of tranexamic given at the time of cementing the prosthesis in total knee arthroplasty, before deflation of the tourniquet, significantly decreases the amount of blood loss in the early post-operative period. The treatment was not associated with an increase in thromboembolic complications.

Products resulting from cleavage of the interglobular domain of aggrecan in samples of synovial fluid collected from dogs with early- and late-stage osteoarthritis.

OBJECTIVE: To investigate interglobular domain (IGD) cleavage of aggrecan in dogs with naturally developing osteoarthritis (OA). SAMPLE POPULATION: Samples of synovial fluid (SF) obtained from 3 cubital (elbow) joints and 3 stifle joints of 4 clinically normal dogs, 24 elbow joints of 12 dogs with early-stage OA, 8 stifle joints of 5 dogs with early-stage OA, and 10 stifle joints of 9 dogs with late-stage OA. PROCEDURE: Fractions of SF were assayed for total glycosaminoglycan (GAG) content and also subjected to Western blot analysis by use of monoclonal antibodies against neoepitopes generated by cleavage of the IGD of the aggrecan protein core by matrix metalloproteinase (MMP; BC-14) and aggrecanase (BC-3). RESULTS: Total GAG content of SF from joints of clinically normal dogs did not differ from that of dogs with early-stage OA. The GAG content of SF from joints of dogs with late-stage OA was significantly lower, compared with GAG content for other SF samples. Aggrecanase-generated fragments were detected in SF from all groups but not in all samples. Matrix metalloproteinase-generated fragments were not detected in any SF samples. In early-stage OA, high-molecular-weight aggrecanase-generated aggrecan catabolites were evident. CONCLUSIONS AND CLINICAL RELEVANCE: GAG content of SF obtained from dogs with late-stage OA is significantly decreased, suggesting proteoglycan depletion of cartilage. Aggrecanases, but not MMPs, are the major proteolytic enzymes responsible for IGD cleavage of aggrecan in canine joints. Analyses of SF samples to detect aggrecanase-generated catabolites may provide an early biomarker for discriminating early- and late-stage OA in dogs.

ADAMTS-1-knockout mice do not exhibit abnormalities in aggrecan turnover in vitro or in vivo.

OBJECTIVE: To determine the role of the proteinase ADAMTS-1 in normal and accelerated catabolism of aggrecan in articular and growth plate cartilage of mice. METHODS: Expression of ADAMTS-1 was determined using reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of RNA isolated from microdissected chondrocytes from different zones of mouse growth plate and articular cartilage. Real-time RT-PCR for ADAMTS-4, ADAMTS-5, and ADAMTS-9 was performed on femoral head cartilage of wild-type (WT) and ADAMTS-1-knockout (KO) mice. Histologic and immunohistologic evaluation of growth plate and articular cartilage was performed in WT and KO mice from birth to 12 weeks of age. The effect of ADAMTS-1 ablation on cartilage proteoglycan loss was studied in antigen-induced arthritis (AIA). Aggrecan catabolism in WT and KO mice was studied in an in vitro model of cartilage degradation, by quantitation of glycosaminoglycan loss and histologic, immunohistologic, and Western immunoblot analyses. RESULTS: ADAMTS-1 messenger RNA (mRNA) was expressed in normal mouse articular and growth plate cartilage and was up-regulated in terminal hypertrophic differentiation of growth plate chondrocytes. There was no difference in mRNA levels in the cartilage of WT compared with KO mice for the other potential aggrecanases ADAMTS-4, ADAMTS-5, or ADAMTS-9. ADAMTS-1-KO mice were significantly smaller than their WT littermates; however, no morphologic differences between the genotypes were evident in growth plate or articular cartilage from birth to skeletal maturity (12-16 weeks). Similarly, no difference in cartilage aggrecan content or presence of aggrecan degradation products was detected between WT and KO mice. There was no difference between WT and KO mice in the degree of synovial inflammation or depletion of cartilage aggrecan in AIA. There was no difference between WT and KO cartilage in either basal or stimulated aggrecan loss in vitro; however, subtle changes in the aggrecanase-generated aggrecan catabolites were observed in interleukin-1-treated cartilage. CONCLUSION: Although ADAMTS-1 is expressed in articular and growth plate cartilage and is able to cleave aggrecan at physiologically relevant sites, our results indicate that it does not play a significant nonredundant role in normal cartilage and bone development and growth. Similarly, ablation of ADAMTS-1 offered no protection from accelerated aggrecanolysis in an inflammatory model of arthritis or in an in vitro model of early cartilage degradation. ADAMTS-1 does not appear to be a viable target for treatment of cartilage destruction in arthritis.

Effects of n-3 fatty acids on cartilage metabolism.

Although the clinical benefits of dietary supplementation with n-3 polyunsaturated fatty acids (PUFA) has been recognised for a number of years, the molecular mechanisms by which particular PUFA affect metabolism of cells within the synovial joint tissues are not understood. This study set out to investigate how n-3 PUFA and other classes of fatty acids affect both degradative and inflammatory aspects of metabolism of articular cartilage chondrocytes using an in vitro model of cartilage degradation. Using well-established culture models, cartilage explants from normal bovine and human osteoarthritic cartilage were supplemented with either n-3 or n-6 PUFA, and cultures were subsequently treated with interleukin 1 to initiate catabolic processes that mimic cartilage degradation in arthritis. Results show that supplementation specifically with n-3 PUFA, but not n-6 PUFA, causes a decrease in both degradative and inflammatory aspects of chondrocyte metabolism, whilst having no effect on the normal tissue homeostasis. Collectively, our data provide evidence supporting dietary supplementation of n-3 PUFA, which in turn may have a beneficial effect of slowing and reducing inflammation in the pathogenesis of degenerative joint diseases in man.

Pathologic indicators of degradation and inflammation in human osteoarthritic cartilage are abrogated by exposure to n-3 fatty acids.

OBJECTIVE: To determine if n-3 polyunsaturated fatty acid (PUFA) supplementation (versus treatment with n-6 polyunsaturated or other fatty acid supplements) affects the metabolism of osteoarthritic (OA) cartilage. METHODS: The metabolic profile of human OA cartilage was determined at the time of harvest and after 24-hour exposure to n-3 PUFAs or other classes of fatty acids, followed by explant culture for 4 days in the presence or absence of interleukin-1 (IL-1). Parameters measured were glycosaminoglycan release, aggrecanase and matrix metalloproteinase (MMP) activity, and the levels of expression of messenger RNA (mRNA) for mediators of inflammation, aggrecanases, MMPs, and their natural tissue inhibitors (tissue inhibitors of metalloproteinases [TIMPs]). RESULTS: Supplementation with n-3 PUFA (but not other fatty acids) reduced, in a dose-dependent manner, the endogenous and IL-1-induced release of proteoglycan metabolites from articular cartilage explants and specifically abolished endogenous aggrecanase and collagenase proteolytic activity. Similarly, expression of mRNA for ADAMTS-4, MMP-13, and MMP-3 (but not TIMP-1, -2, or -3) was also specifically abolished with n-3 PUFA supplementation. In addition, n-3 PUFA supplementation abolished the expression of mRNA for mediators of inflammation (cyclooxygenase 2, 5-lipoxygenase, 5-lipoxygenase-activating protein, tumor necrosis factor alpha, IL-1alpha, and IL-1beta) without affecting the expression of message for several other proteins involved in normal tissue homeostasis. CONCLUSION: These studies show that the pathologic indicators manifested in human OA cartilage can be significantly altered by exposure of the cartilage to n-3 PUFA, but not to other classes of fatty acids.