Aims. Bacterial infection activates neutrophils to release neutrophil extracellular traps (NETs) in bacterial biofilms of periprosthetic joint infections (PJIs). The aim of this study was to evaluate the increase in NET activation and release (NETosis) and haemostasis markers in the plasma of patients with PJI, to evaluate whether such plasma induces the activation of neutrophils, to ascertain whether increased NETosis is also mediated by reduced DNaseI activity, to explore novel therapeutic interventions for NETosis in PJI in vitro, and to evaluate the potential diagnostic use of these markers. Methods. We prospectively recruited 107 patients in the preoperative period of prosthetic surgery, 71 with a suspicion of PJI and 36 who underwent arthroplasty for non-septic indications as controls, and obtained citrated plasma. PJI was confirmed in 50 patients. We measured NET markers, inflammation markers, DNaseI activity, haemostatic markers, and the thrombin generation test (TGT). We analyzed the ability of plasma from confirmed PJI and controls to induce NETosis and to degrade in vitro-generated NETs, and explored the therapeutic restoration of the impairment to degrade NETs of PJI plasma with recombinant human DNaseI. Finally, we assessed the contribution of these markers to the diagnosis of PJI. Results. Patients with confirmed PJI had significantly increased levels of NET markers (cfDNA (p < 0.001), calprotectin (p < 0.001), and neutrophil elastase (p = 0.022)) and inflammation markers (IL-6; p < 0.001) in plasma. Moreover, the plasma of patients with PJI induced significantly more neutrophil activation than the plasma of the controls (p < 0.001) independently of tumour necrosis factor alpha. Patients with PJI also had a reduced DNaseI activity in plasma (p < 0.001), leading to a significantly impaired degradation of NETs (p < 0.001). This could be therapeutically restored with recombinant human DNaseI to the level in the controls. We developed a model to improve the diagnosis of PJI with cfDNA, calprotectin, and the start tail of TGT as predictors, though cfDNA alone achieved a good prediction and is simpler to measure. Conclusion. We confirmed that patients with PJI have an increased level of NETosis in plasma. Their plasma both induced NET release and had an impaired ability to degrade NETs mediated by a reduced DNaseI activity. This can be therapeutically restored in vitro with the approved Dornase alfa, Pulmozyme, which may allow novel methods of treatment. A combination of NETs and haemostatic biomarkers could improve the diagnosis of PJI, especially those patients in whom this diagnosis is uncertain. Cite this article: Bone Joint J 2024;106-B(9):1021–1030

Biomaterials with mechanical or biological competence are ubiquitous in musculoskeletal disorders, and understanding the inflammatory response they trigger is key to guide tissue regeneration. While macrophage role has been widely investigated, immune response is regulated by other immune cells, including neutrophils, the most abundant leukocyte in human blood. As first responders to injury, infection or material implantation, neutrophils recruit other immune cells, and therefore influence the onset and resolution of chronic inflammation, and macrophage polarization. This response depends on the physical and chemical properties of the biomaterials, among other factors. In this study we report an in vitro culture model to describe the most important neutrophil functions in relation to tissue repair. We identified neutrophil survival and death, neutrophils extracellular trap formation, release of reactive oxygen species and degranulation with cytokines release as key functions and introduced a corresponding array of assays. These tests were suitable to identify clear differences in the response by neutrophils that were cultured on material of different origin, stiffness and chemical composition. Overall, substrates from biopolymers of natural origin resulted in increased survival, less neutrophil extracellular trap formation, and more reactive oxygen species production than synthetic polymers. Within the range of mechanical properties explored (storage modulus below 5 k Pa), storage modulus of covalently crosslinked hyaluronic acid hydrogels did not significantly alter neutrophils response, whereas polyvinyl alcohol gels of matching mechanical properties displayed a response indicating increased activation. Additionally, we present the effect of material stiffness, charge, coating and culture conditions in the measured neutrophils response. Further studies are needed to correlate the neutrophil response to tissue healing. By deciphering how neutrophils initiate and modulate the immune response to material implantation, we aim at introducing new principles to design immunomodulatory biomaterials for musculoskeletal disorders. Acknowledgments. This work was supported by the AO Foundation, AO CMF, grant AOCMF-21-04S

Aims. This study aimed to explore the diagnostic value of synovial fluid neutrophil extracellular traps (SF-NETs) in periprosthetic joint infection (PJI) diagnosis, and compare it with that of microbial culture, serum ESR and CRP, synovial white blood cell (WBC) count, and polymorphonuclear neutrophil percentage (PMN%). Methods. In a single health centre, patients with suspected PJI were enrolled from January 2013 to December 2021. The inclusion criteria were: 1) patients who were suspected to have PJI; 2) patients with complete medical records; and 3) patients from whom sufficient synovial fluid was obtained for microbial culture and NET test. Patients who received revision surgeries due to aseptic failure (AF) were selected as controls. Synovial fluid was collected for microbial culture and SF-WBC, SF-PNM%, and SF-NET detection. The receiver operating characteristic curve (ROC) of synovial NET, WBC, PMN%, and area under the curve (AUC) were obtained; the diagnostic efficacies of these diagnostic indexes were calculated and compared. Results. The levels of SF-NETs in the PJI group were significantly higher than those of the AF group. The AUC of SF-NET was 0.971 (95% confidence interval (CI) 0.903 to 0.996), the sensitivity was 93.48% (95% CI 82.10% to 98.63%), the specificity was 96.43% (95% CI 81.65% to 99.91%), the accuracy was 94.60% (95% CI 86.73% to 98.50%), the positive predictive value was 97.73%, and the negative predictive value was 90%. Further analysis showed that SF-NET could improve the diagnosis of culture-negative PJI, patients with PJI who received antibiotic treatment preoperatively, and fungal PJI. Conclusion. SF-NET is a novel and ideal synovial fluid biomarker for PJI diagnosis, which could improve PJI diagnosis greatly. Cite this article: Bone Joint Res 2023;12(2):113–120

Aims. Prosthetic joint infection (PJI) remains a major clinical challenge. Neutrophil CD64 index, Fc-gamma receptor 1 (FcγR1), plays an important role in mediating inflammation of bacterial infections and therefore could be a valuable biomarker for PJI. The aim of this study is to compare the neutrophil CD64 index in synovial and blood diagnostic ability with the standard clinical tests for discrimination PJI and aseptic implant failure. Methods. A total of 50 patients undergoing revision hip and knee arthroplasty were enrolled into a prospective study. According to Musculoskeletal Infection Society (MSIS) criteria, 25 patients were classified as infected and 25 as not infected. In all patients, neutrophil CD64 index and percentage of polymorphonuclear neutrophils (PMN%) in synovial fluid, serum CRP, ESR, and serum CD64 index levels were measured preoperatively. Receiver operating characteristic (ROC) curves and the area under the curve (AUC) were analyzed for each biomarker. Results. Serum CD64 index showed no significant difference between the two groups (p = 0.091). Synovial fluid CD64 index and PMN% discriminated good differentiation between groups of PJI and aseptic failure with AUC of 0.946 (95% confidence interval (CI) 0.842 to 0.990) and 0.938 (95% CI 0.832 to 0.987) separately. The optimal threshold value of synovial CD64 index for the diagnosis of PJI was 0.85, with a sensitivity of 92.00%, a specificity of 96.00%, and diagnostic odds ratio (DOR) of 227.11. Conclusion. The present study demonstrates that CD64 index in synovial fluid could be a promising laboratory marker for screening PJI. The cut-off values of 0.85 for synovial CD64 index has the potential to distinguish aseptic failure from PJI. Cite this article: Bone Joint J 2020;102-B(4):463–469

Aims. Histology is an established tool in diagnosing periprosthetic joint infections (PJIs). Different thresholds, using various infection definitions and histopathological criteria, have been described. This study determined the performance of different thresholds of polymorphonuclear neutrophils (≥ 5 PMN/HPF, ≥ 10 PMN/HPF, ≥ 23 PMN/10 HPF) , when using the European Bone and Joint Infection Society (EBJIS), Infectious Diseases Society of America (IDSA), and the International Consensus Meeting (ICM) 2018 criteria for PJI. Methods. A total of 119 patients undergoing revision total hip (rTHA) or knee arthroplasty (rTKA) were included. Permanent histology sections of periprosthetic tissue were evaluated under high power (400× magnification) and neutrophils were counted per HPF. The mean neutrophil count in ten HPFs was calculated (PMN/HPF). Based on receiver operating characteristic (ROC) curve analysis and the z-test, thresholds were compared. Results. Using the EBJIS criteria, a cut-off of ≥ five PMN/HPF showed a sensitivity of 93% (95% confidence interval (CI) 81 to 98) and specificity of 84% (95% CI 74 to 91). The optimal threshold when applying the IDSA and ICM criteria was ≥ ten PMN/HPF with sensitivities of 94% (95% CI 79 to 99) and 90% (95% CI 76 to 97), and specificities of 86% (95% CI 77 to 92) and 92% (95% CI 84 to 97), respectively. In rTKA, a better performance of histopathological analysis was observed in comparison with rTHA when using the IDSA criteria (p < 0.001). Conclusion. With high accuracy, histopathological analysis can be supported as a confirmatory criterion in diagnosing periprosthetic joint infections. A threshold of ≥ five PMN/HPF can be recommended to distinguish between septic and aseptic loosening, with an increased possibility of detecting more infections caused by low-virulence organisms. However, neutrophil counts between one and five should be considered suggestive of infection and interpreted carefully in conjunction with other diagnostic test methods. Cite this article: Bone Joint Res 2021;10(8):536–547

Aims. The aim of this study was to evaluate the diagnostic accuracy of the absolute synovial polymorphonuclear neutrophil cell (PMN) count for the diagnosis or exclusion of periprosthetic joint infection (PJI) after total hip (THA) or knee arthroplasty (TKA). Methods. In this retrospective cohort study, 147 consecutive patients with acute or chronic complaints following THA and TKA were included. Diagnosis of PJI was established based on the 2018 International Consensus Meeting criteria. A total of 39 patients diagnosed with PJI (32 chronic and seven acute) and 108 patients with aseptic complications were surgically revised. Results. Using receiver operating characteristic curves and calculating the area under the curve (AUC), an optimal synovial cut-off value of 2,000 PMN/µl was determined (AUC 0.978 (95% confidence interval (CI) 0.946 to 1)). Using this cut-off, sensitivity and specificity of absolute synovial PMN count for PJI were 97.4% (95% CI 91.2 to 100) and 93.5% (95% CI 88.9 to 98.1), respectively. Positive and negative predictive value were 84.4% (95% CI 72.7 to 93.9) and 99.0% (95% CI 96.7 to 100), respectively. Exclusion of 20 patients with acute complications improved specificity to 97.9% (95% CI 94.6 to 100). Different cut-off values for THA (< 3,600 PMN/µl) and TKA (< 2,000 PMN/µl) were identified. Absolute synovial PMN count correlated strongly with synovial alpha-defensin (AD) (r = 0.759; p < 0.001). With a positive AD result, no additional PJI could be identified in any case. Conclusion. Absolute synovial PMN count is a widely available, rapid, cost-effective, and accurate marker in PJI diagnostics, whereas synovial AD appears to be a surrogate parameter of absolute synovial PMN count. Despite limitations in the early postoperative phase, wear, and rheumatic diseases in confirming PJI, an absolute synovial PMN count below 2,000/µl is highly suitable for ruling out PJI, with specific cut-off values for THA and TKA. Cite this article: Bone Joint J 2023;105-B(4):373–381

Aims. Aseptic loosening is a leading cause of uncemented arthroplasty failure, often accompanied by fibrotic tissue at the bone-implant interface. A biological target, neutrophil extracellular traps (NETs), was investigated as a crucial connection between the innate immune system’s response to injury, fibrotic tissue development, and proper bone healing. Prevalence of NETs in peri-implant fibrotic tissue from aseptic loosening patients was assessed. A murine model of osseointegration failure was used to test the hypothesis that inhibition (through Pad4-/- mice that display defects in peptidyl arginine deiminase 4 (PAD4), an essential protein required for NETs) or resolution (via DNase 1 treatment, an enzyme that degrades the cytotoxic DNA matrix) of NETs can prevent osseointegration failure and formation of peri-implant fibrotic tissue. Methods. Patient peri-implant fibrotic tissue was analyzed for NETs biomarkers. To enhance osseointegration in loose implant conditions, an innate immune system pathway (NETs) was either inhibited (Pad4-/- mice) or resolved with a pharmacological agent (DNase 1) in a murine model of osseointegration failure. Results. NETs biomarkers were identified in peri-implant fibrotic tissue collected from aseptic loosening patients and at the bone-implant interface in a murine model of osseointegration failure. Inhibition (Pad4-/-) or resolution (DNase 1) of NETs improved osseointegration and reduced fibrotic tissue despite loose implant conditions in mice. Conclusion. This study identifies a biological target (NETs) for potential noninvasive treatments of aseptic loosening by discovering a novel connection between the innate immune system and post-injury bone remodelling caused by implant loosening. By inhibiting or resolving NETs in an osseointegration failure murine model, fibrotic tissue encapsulation around an implant is reduced and osseointegration is enhanced, despite loose implant conditions. Cite this article: Bone Joint J 2021;103-B(7 Supple B):135–144

Introduction: Patients with multiple skeletal injuries are susceptible to Systemic Inflammatory Response Syndrome (SIRS) and consequently Acute Respiratory Distress Syndrome (ARDS). Fracture haematoma contains pro-inflammatory mediators. The aim of our study was to show in vitro that fracture haematoma is implicated in neutrophil mediated injury, SIRS, ARDS and MOF. Methods: Fracture haematoma was isolated from 10 patients at the time of surgery. Neutrophils (PMN) were isolated from 10 healthy volunteers. PMN were exposed to the fracture haematoma supernatant and PMN activation in both primed and unprimed neutrophils were examined (CD11b and CD18 adhesion receptor expression and respiratory burst). PMN phagocytosis and apoptosis were also assessed using flow cytometry. Transmigration across an endothelial barrier was also measured following exposure to fracture haematoma. Results: Fracture haematoma had a marked effect on respiratory burst in primed PMNs (control = 100% vs 20% fracture haematoma = 1044% ± 405, p=0.04). CD11b and CD18 adhesion receptor expression were not upregulated in the fracture haematoma group. PMN phagocytosis of E coli was increased following treatment with fracture haematoma (control = 100% vs fracture haematoma = 171% ± 6SE, p=0.0001). Transendothelial migration of treated neutrophils was unaffected. Treatment of endothelial monolayers with fracture haematoma did not result in upregulated ICAM1 expression but was observed to induce significant endothelial cell death. PMN apoptosis was significantly delayed following exposure to fracture haematoma (control = 46% ± 5 vs fracture haematoma = 8% ±2, p=0.0005). Discussion: We have shown that fracture haematoma activates neutrophils, increases phagocytosis and respiratory burst whilst delaying apoptosis. These effects, whilst beneficial at the site of injury, may cause neutrophil mediated tissue injury systemically

To determine the effect of normal human ageing on neutrophil function and to assess the contribution that any decline may play in the increased susceptibility of elderly patients to bacterial infections following minor trauma. Furthermore, to determine any contribution, of trauma, to further neutrophil decline in these elderly patients. Phagocytic index, CD16 (FcγRIIIB) and CD11b (CR3) expression were determined in neutrophils isolated from the peripheral blood of 15 healthy young (average age 26. 5 yrs, range 23–35 yrs; 8 male, 7 female) and elderly (average age 72. 9 yrs, range 65–71 yrs; 8 male, 7 female) volunteers. CD11b levels were unaltered, but phagocytic index and CD16 expression were both significantly reduced (p< 0. 05 and p< 0. 001 respectively) in the elderly group. CD16 levels were monitored in a large volunteer group and were found to correlate with phagocytic index. To determine whether trauma produces additional compromise to neutrophil function in the elderly, peripheral blood neutrophils from individuals (average age 82. 5 yrs, range 65–96 yrs; 7 male, 21 female) during neutrophilia, post-trauma, due to fracture of the femur, were analysed as described above. Patients with chronic inflammatory disease, diabetes or kidney disease, or who were receiving steroid medication, were excluded. The data showed that neutrophil CD16 expression was significantly reduced in the elderly group (p< 0. 05), furthermore following fracture of the neck of femur superoxide generation is significantly reduced. Patient follow up revealed that 17 (60. 8 %) of these patients subsequently acquired bacterial infections (including wound), within 4 weeks of trauma. Normal human ageing was accompanied by a decline in neutrophil phagocytic ability and this may be in part due to reduced levels of the Fcγ receptor CD16. The reduced neutrophil CD16 expression accompanied by reduced superoxide generation in the elderly trauma patients may significantly undermine their ability to combat bacterial infections and contribute to increased incidence of post-traumatic infections in the elderly

Introduction. Patients with aseptic loosening, a cause of failure in uncemented total joint arthroplasty (TJA), often present with fibrous tissue at the bone-implant interface. 1. In this study, we characterize the presence of neutrophil extracellular traps (NETs) in the intramedullary fibrotic membrane of aseptic loosening patients. We further explore the role of NETs, mediated by peptidyl arginine deiminase (PAD4), in peri-implant fibrosis and osseointegration failure through a murine model of unstable tibial implantation. 2–4. Methods. Peri-implant membrane was retrieved from five patients during total hip revision surgery and analyzed for the presence of NETs (citH3+ with extracellular DNA) via immunofluorescence. A Ti-6Al-4V implant was inserted in an oversized drill-hole in the right proximal tibia of 8-week-old C57BL/6J and PAD4 knockout mice (n=3 per group). Fourteen days later, all mice were euthanized, and implanted tibias were dissected. Fibrosis and osseointegration at the bone-implant interface were assessed by micro-computed tomography (microCT) and hematoxylin and eosin (H&E) staining. H&E samples were scored blindly by the investigator and another observer for signs of poor (score=0) to excellent osseointegration (score=3) using a rubric established in our lab. Results. NETs were found in peri-implant membrane collected from aseptic loosening patients (Figure 1a) and at the bone-implant interface in a murine model (Figure 1b). Unstable implants in wild type mice failed to osseointegrate, indicated by presence of fibroblast-like cells (dashed arrow), immature bone matrix (Figure 1c), low bone volume fraction (BV/TV) and bone surface area (BS) (Figure 1e). Unstable implants in PAD4. −/−. mice showed signs of good osseointegration such as mature trabeculae (solid arrow) (Figure 1d), higher BV/TV (p<0.10) and BS (p<0.05) (Figure 1f). Histological osseointegration scoring indicated wildtype mice exhibited an average score of 0.83 and PAD4. −/−. exhibited an average score of 2.5 (p<0.05, weighted Cohen's kappa = 0.714) (Figure 1g). Conclusion. NETs were characterized in fibrotic tissue in both aseptic loosening patients and in a murine model of unstable tibial implantation. NET inhibition was able to successfully prevent peri-implant fibrosis and osseointegration failure, leading the way for a potential novel non-invasive therapeutic approach for the treatment of aseptic loosening. For any figures, tables, or references, please contact the authors directly

We examined the usefulness of neutrophil CD64 expression in detecting local musculoskeletal infection and the impact of antibiotics on its expression. Of 141 patients suspected of musculoskeletal infection, 46 were confirmed by microbiological culture to be infected and 95 had infection excluded. The median CD64 count of patients with localised infection was 2230 molecules per cell (interquartile range (IQR) 918 to 4592) and that of the patients without infection was 937 molecules per cell (IQR 648 to 1309) (p < 0.001). The level of CD64 correlated with the CRP level in patients with infection, but not in those without infection (r = 0.59, p < 0.01). Receiver operator characteristic curve analysis revealed that CD64 was a good predictor of local infection. When the patients were subdivided into two groups based on the administration of antibiotics at the time of CD64 sampling, the sensitivity for detecting infection was better in those who had not received antibiotics. These results suggest that measurement of CD64 expression is a useful marker for local musculoskeletal infection

The purpose of this study was to determine the effect of positioning (lateral vs. supine) on pulmonary patho-physiology following pulmonary contusion and fat embolism in a canine model of polytrauma. Platelet and neutrophil activation were assessed using flow-cytometry. There were no significant differences between groups in CD62P and CD11/18 MCF (markers of platelet and neutrophil activation, respectively) following fat embolism. However, only animals in the lateral position displayed significant increases in both measures as compared to baseline values. Lateral positioning may exert an early effect on proinflammatory and coagulation activation, and may play a role in the development of acute lung injury. It has previously been suggested that acute lung injury can be influenced by patient positioning, be it lateral or supine. The purpose of this study was to determine the effect of positioning on pulmonary pathophysiology associated with concomitant pulmonary contusion and fat embolism in a canine model of polytrauma. Twelve dogs were randomly assigned to one of two surgical positioning groups, lateral and supine. The dogs were subjected to pulmonary contusion by application of force between 200–250 N/m. 2. for thirty seconds in three areas of one lung. Two hours later, fat embolism was induced via reaming of the ipsilateral femur and tibia and cemented nailing. Two hours later, the dogs were sacrificed. For flow-cytometric evaluation of platelet and neutrophil activation, venous blood samples were stained with fluorescence-conjugated antibodies against CD62P and CD11/18, respectively. There were no significant differences between the groups in CD62P and CD11/18 mean channel fluorescence (MCF) following pulmonary contusion and fat embolism. However, only animals in the lateral positioning group displayed significant increases in CD62P and CD11/18 MCF at two hours following fat embolism as compared to baseline values. Our findings suggest that lateral positioning, autoregulation and preferential blood flow to the contused non-dependent lung may render lung tissue more susceptible to congestion and lead to activation of both platelets and neutrophils. Lateral positioning may have an early effect on activation of the inflammatory and coagulation cascades and may be significant in the development of posttraumatic acute lung injury

The purpose of this study was to determine the effect of positioning (lateral vs. supine) on pulmonary pathophysiology following pulmonary contusion and fat embolism in a canine model of polytrauma. Platelet and neutrophil activation were assessed using flow-cytometry. There were no significant differences between groups in CD62P and CD11/18 MCF (markers of platelet and neutrophil activation, respectively) following fat embolism. However, only animals in the lateral position displayed significant increases in both measures as compared to baseline values. Lateral positioning may exert an early effect on proinflammatory and coagulation activation, and may play a role in the development of acute lung injury. It has previously been suggested that acute lung injury can be influenced by patient positioning, be it lateral or supine. The purpose of this study was to determine the effect of positioning on pulmonary pathophysiology associated with concomitant pulmonary contusion and fat embolism in a canine model of polytrauma. Twelve dogs were randomly assigned to one of two surgical positioning groups, lateral and supine. The dogs were subjected to pulmonary contusion by application of force between 200–250 N/m. 2. for thirty seconds in three areas of one lung. Two hours later, fat embolism was induced via reaming of the ipsilateral femur and tibia and cemented nailing. Two hours later, the dogs were sacrificed. For flow-cytometric evaluation of platelet and neutrophil activation, venous blood samples were stained with fluorescence-conjugated antibodies against CD62P and CD11/18, respectively. There were no significant differences between the groups in CD62P and CD11/18 mean channel fluorescence (MCF) following pulmonary contusion and fat embolism. However, only animals in the lateral positioning group displayed significant increases in CD62P and CD11/18 MCF at two hours following fat embolism as compared to baseline values. Our findings suggest that lateral positioning, autoregulation and preferential blood flow to the contused non-dependent lung may render lung tissue more susceptible to congestion and lead to activation of both platelets and neutrophils. Lateral positioning may have an early effect on activation of the inflammatory and coagulation cascades and may be significant in the development of posttraumatic acute lung injury

The purpose of this study was to determine the effect of positioning (lateral vs. supine) on pulmonary pathophysiology following pulmonary contusion and fat embolism in a canine model of polytrauma. Platelet and neutrophil activation were assessed using flow-cytometry. There were no significant differences between groups in CD62P and CD11/18 MCF (markers of platelet and neutrophil activation, respectively) following fat embolism. However, only animals in the lateral position displayed significant increases in both measures as compared to baseline values. Lateral positioning may exert an early effect on proinflammatory and coagulation activation, and may play a role in the development of acute lung injury. It has previously been suggested that acute lung injury can be influenced by patient positioning, be it lateral or supine. The purpose of this study was to determine the effect of positioning on pulmonary pathophysiology associated with concomitant pulmonary contusion and fat embolism in a canine model of polytrauma. Twelve dogs were randomly assigned to one of two surgical positioning groups, lateral and supine. The dogs were subjected to pulmonary contusion by application of force between 200–250 N/m. 2. for thirty seconds in three areas of one lung. Two hours later, fat embolism was induced via reaming of the ipsilateral femur and tibia and cemented nailing. Two hours later, the dogs were sacrificed. For flow-cytometric evaluation of platelet and neutrophil activation, venous blood samples were stained with fluorescence-conjugated antibodies against CD62P and CD11/18, respectively. There were no significant differences between the groups in CD62P and CD11/18 mean channel fluorescence (MCF) following pulmonary contusion and fat embolism. However, only animals in the lateral positioning group displayed significant increases in CD62P and CD11/18 MCF at two hours following fat embolism as compared to baseline values. Our findings suggest that lateral positioning, autoregulation and preferential blood flow to the contused non-dependent lung may render lung tissue more susceptible to congestion and lead to activation of both platelets and neutrophils. Lateral positioning may have an early effect on activation of the inflammatory and coagulation cascades and may be significant in the development of posttraumatic acute lung injury

Aims: Pharmocological modulation of skeletal muscle reperfusion injury after an ischaemic insult may improve limb salvage rates and prevent the associated systemic sequelae. Activated Protein c (APC) is an endogenous anti-coagulant with anti-inflammatory properties. The purpose of our study was to evaluate the effects of APC on skeletal muscle ischaemia reperfusion injury and to examine the direct effects of APC on neutrophil activation. Methods: Adult male Sprague Dawley rats (n=30) were randomised into three groups: control group, I/R group treated with normal saline and I/R treated with APC. Bilateral hind-limb ischaemia was induced by rubber ban application proximal to the level of the greater trochanters for two hours. Treatment groups received either normal saline or APC prior to tourniquet release. Following twelve hours reperfusion, the tibialis anterior was dissected and muscle function assessed electrophysiologically by electrical field stimulation. The animals were then sacrificed and skeletal muscle harvested for evaluation. Skeletal muscle injury was assessed based on myeloperoxidase content, wet-to-dry ratio and histological analysis. The effect of APC on TNF-α stimulated human peripheral blood neutrophils was also examined by measuring CD 18 expression and reactive oxygen species (ROS) generation. Results: APC significantly attenuated skeletal muscle reperfusion injury as shown by reduced myeloperoxidase content, wet-to-dry ratio and electrical properties of skeletal muscle. These findings were supported by our histological findings. Our in-vitro work demonstrated a reduction in CD 18 expression and ROS generation. Conclusion: Activated Protein C may have a protective role in the setting of skeletal muscle ischaemia reperfusion injury and this is in part mediated by a direct inhibitory effect on neutrophil activation

The formation of bacterial biofilms is increasingly recognised as the leading cause of chronic infections. It limits the application of implant materials including catheters, heart valves, or orthopaedic prostheses. It is generally assumed that the infection persists because bacteria organised as biofilms escape the host defence mechanisms. Nevertheless, when studying patients with infected implants, we found a massive infiltration of leukocytes particularly polymorphonuclear neutrophils, PMN, into the site of infection, which led to the question, whether the PMN interact with the bacterial biofilm or not. The interaction of human PMN with Staphylococcus aureus biofilms was studied in vitro. S.aureus was cultivated on glass cover slips for various times under conditions allowing formation of biofilms. Adherence of PMN to biofilms and phagocytosis of the bacteria were observed by confocal laser scan microscopy and time lapse video microscopy. Migration of PMN on and into the biofilm was identified as being phagocytosis, apparent as uptake of bacteria into the cell. Concominantly, in the wake of migrating PMN bacteria depleted zones appeared, which increased in size with time. In addition to phagocytosis, release from PMN of DNA and also of elastase was seen, suggesting the formation of neutrophil extracellular traps (NETs). So far, the signal for DNA release and NET formation has not been identified; of note is, however, that they occurred preferentially on established “old” biofilms and in the absence of the opsonising human serum, while phagocytosis was most efficient with developing “young” biofilms. Taken together, our data provide evidence that bacteria in biofilms are not entirely protected against host defence but that phagocytosis is still possible, especially when the biofilm is opsonised with human serum. Whether NET formation also contributes to bacteria killing in biofilms cannot be decided as yet but remains an attractive alternative

Aims: Neutrophil (PMN) dysfunction is implicated in both acute respiratory distress syndrome (ARDS) and sepsis. We aimed to determine the PMN response following isolated long-bone/pelvic fracture by investigating temporal changes in PMN migration and surface receptor expression (CXCR1, PECAM- 1, & CD18/ CD11b) following injury. Methods: Of the 20 patients consented to enter the study, 14 underwent reamed nailing/ORIF within 24 hours, and 6 were treated with an Ex-Fix or conservatively. 11 normal volunteers (NLV) were used as controls. Blood samples were obtained within 2 hours of admission, at 24 hours, at day 3 and day 5. PMN were isolated and the number of PMN migrating across porous collagen IV coated tissue culture inserts, in response to IL-8 were quantitated by myeloperoxidase activity. PMN surface receptor expression was assessed by whole blood FACScan analysis. Results: Signiþcantly greater numbers of fracture patient PMN migrated on admission as compared with NLV. In the Ex-Fix group the numbers migrating declined steadily and showed a hypo-response on day 5. In the reamed nailing group there was a further elevation in the PMN numbers migrating post-operatively. CXCR1 & CD18 expression was signiþcantly increased on admission. PECAM-1 was signiþcantly down-regulated on admission. Conclusions: Following isolated long-bone/pelvic fracture PMN are primed for increased migration in response to IL-8. This is associated with up-regulation of CXCR1 and CD18, and down-regulation of PECAM-1. Treatment by reamed nailing and ORIF confers a Ç second hit È manifest as a further increase in IL-8 mediated PMN migration

Background: Neutrophil (PMN) infiltration of the lung is characteristic of ARDS. Interleukin-8 (IL-8) plays a central role in the recruitment of PMN to the lung and their subsequent activation. This study examines PMN migratory activity in response to IL-8, over the first 24 hours of admissions following major trauma. Methodology: Study Population: Adult blunt trauma victims with ISS> /=18. PMN Migraoty Activity: PMN were isolated from citrated blood at admission, 8 and 24 hours later. The number of PMN migrating across porous tissue culture inserts in response to defined concentrations of IL-8 (zero, 10, 30 & 100ng/ml) were quantitated by peroxidase assay. Results: Significantly greater numbers of trauma patients PMN migrated to concentrations of IL-8 (30& 100ng/ml) at each time point, when compared to normal volunteers (Mann-Whitney-U Test p< 0.05). At admissions, and 8 hours later, PMN from those who later developed ARDS exhibit an enhanced migratory response to high concentrations of IL-8, in contrast to the noraml physiological attenuation of migration seen in both the remaining trauma patients (NAD) and normal volunteers (NLV). Discussion: These data indicate that major trauma fundamentally alters the migratory capacity of circulating PMN. Within 2 hours of admission, PMN show a unique pattern of activation in those who later develop ARDS, possibly due to alteration in IL-8 receptor expression, affinity or downstream signalling. These findings suggest that limiting PMN sequestration in the lung may represent a novel therapeutic target

Objective: It has been observed in previous studies of autologous blood transfusion in total knee arthroplasty, that this technique is associated with a lower infection rate, though studies have not been sufficiently large to demonstrate a significant difference. We hypothesised that autologous salvage blood contained high levels of pro-inflammatory mediators which may prime or augment the patients’ inflammatory response, and, in particular, the function of the polymorphonuclear leukocyte (PMN).

Methods: Patients were randomised after consent to those receiving autologous transfusion and those having conventional treatment which was homologous transfusion only if clinically indicated. PMN were then isolated from the patients pre-admission immediately after the operation and 24 hours post-total knee arthroplasty. Three different aspects of PMN activity were measured: 1) The respiratory burst activity; 2) the ability to phagocytose microbes using radiolabelled staphylococcus epidermis; 3) and the ability to migrate across a collagen coated porous inserts.

Results: 80 patients were studied, of whom 43 were in the autologous transfusion group and 37 in the first group. Of these 37, 6 had homologous transfusion. The study showed no significant difference between the phagocytic ability of the PMN from both groups. The main stimulus to transmigration is surgery itself. There was a significant increase in the production of reactive oxygen species by the PMN of those patients who received an autologous transfusion post-operatively.

Conclusions: Using molecular biology techniques to study the PMN directly, our study indicates that autologous transfusion alters the activity of PMN and this indicates a possible mechanism whereby the immune response to infection could be enhanced. This, in turn, would explain the observed differences in infection rate in previous studies.

Although it has long been appreciated that a healthy balanced diet improves health, there is a growing understanding of the way in which certain nutrients can actually improve immune function. Boosting immune function by the use of “immunonutrition” has been shown to improve outcomes, in particular rates of infective complications, in certain groups of surgical patients.

In this study we examine the immune status of elderly patients who have suffered a hip fracture and are known to be vulnerable to infection and poor post-operative outcomes to identify specific immune defects associated with this particular cohort. This may allow us to explore the potential benefits of immunonutrition in this group of patients in the future.

This was a cohort observational study, in which a series of 16 patients who underwent surgery for hip fractures were followed. The patients were female patients with an age of 60 to 85 years and a mental status questionnaire score of at least 8 out of 10.

Immune function was evaluated prior to surgery, on the day following surgery and then at between days 4 and 7 post-operatively. Samples were tested directly ex-vivo using a variety of flow cytometric assays.

We report profound loss of innate immune function related specifically to monocyte and granulocyte ability to generate a respiratory burst in response to E.coli uptake persisting up to day 7 post-operatively. In addition, serum cytokine levels indicated very poor T cell function, in identifying these patients as particularly vulnerable to infections.