Synovial calprotectin: a potential biomarker to exclude a prosthetic joint infection.

AIMS: Recently, several synovial biomarkers have been introduced into the algorithm for the diagnosis of a prosthetic joint infection (PJI). Alpha defensin is a promising biomarker, with a high sensitivity and specificity, but it is expensive. Calprotectin is a protein that is present in the cytoplasm of neutrophils, is released upon neutrophil activation and exhibits anti-microbial activity. Our aim, in this study, was to determine the diagnostic potential of synovial calprotectin in the diagnosis of a PJI. PATIENTS AND METHODS: In this pilot study, we prospectively collected synovial fluid from the hip, knee, shoulder and elbow of 19 patients with a proven PJI and from a control group of 42 patients who underwent revision surgery without a PJI. PJI was diagnosed according to the current diagnostic criteria of the Musculoskeletal Infection Society. Synovial fluid was centrifuged and the supernatant was used to measure the level of calprotectin after applying a lateral flow immunoassay. RESULTS: The median synovial calprotectin level was 991 mg/L (interquartile range (IQR) 154 to 1787) in those with a PJI and 11 mg/L (IQR 3 to 29) in the control group (p < 0.0001). Using a cut-off value of 50 mg/L, this level showed an excellent diagnostic accuracy, with an area under the curve of 0.94. The overall sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) was 89%, 90%, 81% and 95% respectively. The NPV was 97% in the nine patients with a chronic PJI. CONCLUSION: Synovial calprotectin may be a valuable biomarker in the diagnosis of a PJI, especially in the exclusion of an infection. With a lateral flow immunoassay, a relatively rapid quantitative diagnosis can be made. The measurement is cheap and is easy to use. Cite this article: Bone Joint J 2017;99-B:660-5.

The predictive power of serum S-100B for non-sentinel node positivity in melanoma patients.

BACKGROUND: Completion lymph node dissection (CLND) in sentinel node (SN) positive melanoma patients leads to substantial morbidity and costs, while only approximately 20% have a metastasis in non-sentinel nodes (NSNs). The aim of this study was to investigate if the biomarkers S-100B and Lactate Dehydrogenase (LDH) are associated with NSN positivity, to identify patients in whom CLND could safely be omitted. METHODS: All SN positive patients who underwent CLND at the University Medical Centre Groningen between January 2004 and January 2015 were analysed. Patient and tumor characteristics, and serum S-100B and LDH values measured the day before CLND were statistically tested for their association with NSN positivity. RESULTS: NSN positivity was found in 20.6% of the 107 patients undergoing CLND. Univariate analysis revealed male gender (p = 0.02), melanoma of the lower extremity (p = 0.05), Breslow thickness (p = 0.004), ulceration (p = 0.04), proportion of involved SNs (p = 0.045) and S-100B value (p = 0.01) to be associated with NSN positivity. LDH level was not significantly associated with positive NSNs (p = 0.39). In multivariable analysis, S-100B showed to have the strongest association with NSN positivity, within its reference interval of 0.20 µg/l (p = 0.02, odds ratio 5.71, 95% confidence interval 1.37-23.87). CONCLUSION: In this study, the preoperatively measured S-100B value is the strongest predictor for NSN positivity in patients planned for CLND. Fluctuations of the S-100B level within the reference interval might give important clues about residual tumor load. Although further validation will be needed, this new closer look of S-100B could be of value in patient selection for CLND in the future.

S-100B: a stronger prognostic biomarker than LDH in stage IIIB-C melanoma.

BACKGROUND: In melanoma patients with nodal macrometastases, the distinction between good and poor prognosis is based on the presence of primary melanoma ulceration or metastatic involvement of 4 or more lymph nodes in the 7th edition of the American Joint Committee on Cancer (AJCC) classification. We hypothesized that biomarkers would increase the accurateness of staging in these patients. The aim was to assess and compare the prognostic impact of biomarkers S-100B and LDH and to determine the best timing of their measurement in stage IIIB-C melanoma. METHODS: A total of 119 patients underwent therapeutic lymph node dissection (TLND) for nodal macrometastases with serum S-100B and LDH level measurements preoperatively. In 75 of them, S-100B and LDH were also measured on postoperative days 1 and 2. S-100B and LDH levels on days 0, 1, and 2 were compared for their association with disease-free survival (DFS) and disease-specific survival (DSS). RESULTS: At a median follow-up of 17 (range 1-89) months, S-100B levels at all time points were associated with DFS. In multivariable analysis, preoperative S-100B and S-100B measured on day 2 showed the strongest association with DFS (hazard ratio [HR] 2.55, P = 0.007 and HR 3.80, P = 0.01). For DSS, the preoperative S-100B level was the strongest independent predictor (HR 2.81, P = 0.01). LDH measurements showed a significant association with DSS in univariate analysis only when measured preoperatively (HR 2.46, P = 0.01). In multivariable analysis, LDH measurement was not associated with melanoma prognosis. CONCLUSIONS: The S-100B level measured preoperatively is, in contrast to LDH, one of the most important independent predictors of melanoma prognosis in patients undergoing TLND for nodal macrometastases.

The fibrosis marker galectin-3 and outcome in the general population.

OBJECTIVE: Galectin-3 is involved in fibrosis and inflammation and plays a role in heart failure, renal disease, obesity and cancer. We aimed to establish the relationship between galectin-3 and cardiovascular (CV) risk factors and mortality in the general population. DESIGN AND SUBJECTS: This study included 7968 subjects from the Prevention of REnal and Vascular ENd-stage Disease (PREVEND) cohort, with a median follow-up of approximately 10 years. Plasma galectin-3 was measured in baseline samples. MAIN OUTCOME MEASURES: We investigated the relationships between galectin-3 levels, demographic characteristics and risk factors of CV disease. We determined the prognostic value for all-cause, CV and cancer mortality. RESULTS: The mean age of the population was 50 ± 13 years. Mean blood pressure was 129/74 mmHg, mean cholesterol was 5.7 ± 1.1 mmol L(-1) and median galectin-3 was 10.9 ng mL(-1) [interquartile range (IQR) 9.0-13.1]. Galectin-3 levels correlated with a wide range of risk factors of CV disease, including blood pressure, serum lipids, body mass index, renal function and N-terminal pro-B-type natriuretic peptide (P < 0.0001). We observed a strong association between galectin-3 and age. Furthermore, we found a gender interaction, with female subjects (n = 4001) having higher median galectin-3 levels (11.0 ng mL(-1) , IQR 9.1-13.4 vs. men (n = 3967) 10.7 ng mL(-1) , IQR 8.9-12.8; P < 0.0001), and galectin-3 levels in women more strongly correlated with risk factors of CV disease. After correction for the classical CV risk factors (smoking, blood pressure, cholesterol and diabetes), galectin-3 levels independently predicted all-cause mortality (hazard ratio per SD galectin-3 1.09, 95% CI 1.01-1.19; P = 0.036), but not CV and cancer mortality separately. CONCLUSIONS: Galectin-3 is associated with age and risk factors of CV disease, with a strong gender interaction for these correlations. Galectin-3 predicts all-cause mortality in the general population.

Monocyte intracellular cytokine production during human endotoxaemia with or without a second in vitro LPS challenge: effect of RWJ-67657, a p38 MAP-kinase inhibitor, on LPS-hyporesponsiveness.

In the present study, we investigated the effect of RWJ-67657, a p38 MAP kinase inhibitor, upon in vivo LPS-induced monocyte cytokine production and upon monocyte LPS-hyporesponsiveness. Thirty minutes before a single injection of LPS (4 ng/kg BW), healthy male volunteers received a single oral dose of RWJ-67657 at increasing dosages (0-1400 mg). Blood samples (pre-medication, 3, 6 and 24 h after LPS) were immediately incubated with LPS (reflecting LPS-hyporesponsiveness) or without LPS (reflecting in vivo monocyte stimulation) for 4 h at 37 degrees C. Following red blood cells lysis and white blood cell permeabilization, cells were labelled with alpha-CD14-FITC and alpha-IL-1beta, alpha-IL-12 or alpha-TNFalpha (PE-labelled), fixed, and analysed using flow cytometry. In vivo LPS injection resulted in an increased percentage of circulating monocytes producing IL-1beta, TNFalpha and IL-12 only at 3 h after the LPS injection. This was dose-dependently inhibited by RWJ-67657 treatment. LPS-hyporesponsiveness to in vitro LPS treatment was most prominent at 3 and 6 h after the in vivo LPS injection; compared with pre-medication monocytes, at these intervals a reduced percentage of monocytes produced IL-1beta, TNFalpha or IL-12 after the in vitro LPS stimulus. At t = 6 h, this LPS-hyporesponsiveness could dose-dependently be inhibited by RWJ-67657 treatment of the volunteers. We therefore conclude that p38 MAP kinase inhibition with RWJ-67657 inhibited monocyte production of cytokines following in vivo LPS injection. Treatment with RWJ-67657 also reversed the LPS-hyporesponsiveness. Whether this result can be extended to the clinical situation remains to be elucidated. Patients with sepsis or an otherwise high risk for multi-organ failure are potential study groups.

Leukocyte activation in sepsis; correlations with disease state and mortality.

OBJECTIVE: The immune response in sepsis shows a bimodal pattern consisting of an early, frequently exaggerated inflammatory response followed by a state of hyporesponsiveness often referred to as the compensatory anti-inflammatory response syndrome (CARS). Insight into the disease state may be helpful in deciding whether to choose immune stimulatory or anti-inflammatory therapy in these patients and may determine clinical outcome. We hypothesized that poor outcome in patients with sepsis is related to the severity of CARS, as reflected in the degree of leukocyte activation. DESIGN: Prospective study. SETTING: Intensive and respiratory care unit at a university hospital. PATIENTS: Twenty consecutive patients with sepsis and 20 healthy age-matched volunteers. INTERVENTIONS: None. MEASUREMENTS AND RESULTS: Analysis of surface expression of HLA-DR, CD11b, ICAM-1, CD66b, CD63 and CD64 on neutrophils and monocytes by flow cytometry and determination of plasma concentrations of lactoferrin, interleukin 6 and neopterin by ELISA at the time of diagnosis. Patient data were related to those of controls; moreover patient data between survivors and non-survivors were compared. Increased expression of all markers, except HLA-DR, was observed on both neutrophils and monocytes from patients compared to healthy controls. HLA-DR expression on monocytes was significantly decreased in patients with sepsis (p < 0.01). Expression of CD11b and HLE on neutrophils, and ICAM-1 on monocytes, were lower in patients who died compared to those who survived (p < 0.05). CONCLUSION: In sepsis, both neutrophils and monocytes are activated compared to healthy controls. Poor prognosis is associated with a lower expression of activation markers on monocytes and neutrophils, suggesting that poor outcome in these patients may be due to the compensatory anti-inflammatory response.

Staphylococcal acid phosphatase binds to endothelial cells via charge interaction; a pathogenic role in Wegener's granulomatosis?

The majority of patients with Wegener's granulomatosis (WG) are chronic nasal carriers of Staphylococcus aureus. Chronic nasal carriage of S. aureus is associated with an increased risk of developing a relapse of the disease. The mechanism by which this occurs is still unknown. We hypothesized that a cationic protein of S. aureus, staphylococcal acid phosphatase (SAcP), acts as a planted antigen and initiates glomerulonephritis and vasculitis in patients with WG. In order to test the hypothesis that SAcP can act as a planted antigen in WG, we studied the ability of SAcP to bind to human umbilical vein endothelial cells (HUVEC) and human glomerular endothelial cells. We also studied whether this binding can be prevented by preincubation with an anionic protein, and whether binding of SAcP activates endothelial cells. We also evaluated whether antibodies in sera of patients with WG are able to bind to endothelial cell-bound SAcP. The results show that SAcP can act as a planted antigen by binding to both types of endothelial cells in a concentration-dependent manner. Binding of concentrations as low as 4 microg/ml can be detected on HUVEC within 5 min of incubation. Binding of SAcP to endothelial cells was charge-dependent but did not activate endothelial cells. Finally, endothelial cell-bound SAcP was recognized by sera of patients with WG. The data suggest a possible pathogenic role for SAcP by acting as a planted antigen thereby initiating glomerulonephritis and vasculitis in patients with WG.

The central role of monocytes in the pathogenesis of sepsis: consequences for immunomonitoring and treatment.

Despite important advances in critical care medicine during the last two decades, the mortality rate of sepsis has remained high, probably because the pathogenesis of sepsis is still incompletely understood. Recent studies have shown that sepsis is a bimodal entity. The first phase is characterized by the systemic release of pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNF-alpha), interleukin-1 (IL-1), and IL-8, and by activation of the complement and coagulation cascades. In the second phase, anti-inflammatory mediators such as transforming growth factor-beta (TGF-beta), IL-10 and prostaglandin E2 (PGE2) may be released in an effort to counteract ongoing inflammation. Depending whether the pro- or anti-inflammatory response predominates, sepsis results in a systemic inflammatory response syndrome (SIRS), or a compensatory anti-inflammatory response syndrome (CARS). So far, most efforts to intervene in the immunopathogenesis of sepsis have been directed at the pro-inflammatory response. None of these interventions has been shown to improve the prognosis of sepsis, possibly because many patients were already in a state in which anti-inflammatory responses dominated. Recently, it has been shown that decreased expression of HLA-DR on monocytes in patients with sepsis constitutes a marker for CARS. We suggest that HLA-DR expression on monocytes might constitute a useful indicator of the immunological status of the individual patient with sepsis and a guide for treatment. Patients with CARS, as manifested by low HLA-DR expression, might benefit from immunostimulants, while patients with SIRS and normal or high monocyte HLA-DR expression should receive treatment directed to interfere with pro-inflammatory pathways.

In vitro up-regulation of E-selectin and induction of interleukin-6 in endothelial cells by autoantibodies in Wegener's granulomatosis and microscopic polyangiitis.

OBJECTIVE: In patients with Wegener's granulomatosis (WG) or microscopic polyangiitis (MPA) autoantibodies to myeloid granule proteins (ANCA), particularly proteinase 3 (Pr3) and myeloperoxidase (MPO), and to endothelial cells (AECA) are frequently detected. The role of these autoantibodies in the development of vascular injury is incompletely understood. Since the expression of E-selectin and the production of interleukin 6 by endothelial cells is an early step in the sequence of events leading to vascular injury, we examined the capacity of IgG fractions from patients with WG and/or MPA to activate endothelial cells to the expression of E-selectin and the production of IL-6. We related those findings to the presence of ANCA and AECA in the IgG preparations. METHODS: Human umbilical vein endothelial cells (HUVEC) were incubated with immunoglobulin (IgG) preparations from 28 patients (17 positive for anti-Pr3, 10 for anti-MPO, and one for anti-Pr3/MPO) with active vasculitis and from 10 healthy volunteers. The final IgG concentration in the activation assay was 2 mg/ml. TNF alpha (10 ng/ml) and LPS (10 ng/ml) were used as positive controls for HUVEC activation. The extent of HUVEC activation was assessed by the measurement of E-selectin expression by flow cytometry (after 4 hours of incubation) and the production of interleukin 6 by ELISA (after 24 hours). RESULTS: We found that 11 of the 28 ANCA positive IgG samples were capable of activating endothelial cells: six samples induced IL-6 production alone, one sample upregulated E-selectin expression alone, and four samples induced both IL-6 production and E-selectin upregulation. Five of 17 anti-Pr3 positive samples (one of which was also positive for AECA) and 6 of 10 anti-MPO positive samples (all simultaneously positive for AECA) induced endothelial cell activation. AECA positive samples that induced endothelial cell activation (n = 7) had higher AECA titres than samples that did not induce endothelial cell activation (n = 6). CONCLUSION: Our data suggest that the activation of endothelial cells in patients with WG and MPA can be induced by circulating autoantibodies. Both ANCA and AECA can be responsible for this effect.

In vitro neutrophil activation by antibodies to proteinase 3 and myeloperoxidase from patients with crescentic glomerulonephritis.

Previously, it was found that patients with necrotizing crescentic glomerulonephritis (NCGN) and anti-neutrophil cytoplasmic autoantibodies (ANCA) directed against proteinase 3 (anti-PR3) had a faster deterioration of renal function and more active renal vasculitic lesions than patients with ANCA directed against myeloperoxidase (anti-MPO). Because ANCA-mediated neutrophil activation is thought to play an important role in the pathophysiology of this form of glomerulonephritis, this study was conducted to determine whether anti-PR3 are capable of inducing a more pronounced activation of neutrophils in vitro than anti-MPO. To test this hypothesis, the release of reactive oxygen radicals, as assessed by ferricytochrome c reduction and by dihydrorhodamine 123 oxidation, and the release of granule constituents from healthy donor neutrophils upon stimulation with IgG fractions were measured from 17 anti-PR3- and 14 anti-MPO-positive patients with active NCGN. Patients with anti-PR3 had a higher renal activity index (P < 0.05) compared with patients with anti-MPO. IgG fractions from anti-PR3-positive patients induced more oxygen radical release from tumor necrosis factor-alpha-primed neutrophils compared with IgG fractions from anti-MPO-positive patients, as assessed by ferricytochrome c reduction (P < 0.05) and dihydrorhodamine 123 oxidation (P < 0.01). In addition, IgG fractions from anti-PR3-positive patients generated more neutrophil degranulation of beta-glucuronidase (P < 0.01) than IgG fractions from anti-MPO-positive patients. In conclusion, IgG fractions from anti-PR3-positive patients with NCGN are more potent activators of the respiratory burst and degranulation in vitro than IgG fractions from anti-MPO-positive patients. These observations may be relevant in view of the clinical differences between anti-PR3- and anti-MPO-positive patients with NCGN.

Monocyte activation in patients with Wegener's granulomatosis.

OBJECTIVE: Wegener's granulomatosis (WG) is an inflammatory disorder characterised by granulomatous inflammation, vasculitis, and necrotising vasculitis and is strongly associated with anti-neutrophil cytoplasmic antibodies (ANCA). Activated monocytes/macrophages are present in renal biopsy specimens and participate in granuloma formation by synthesising and secreting a variety of chemoattractants, growth factors, and cytokines. In view of these findings, in vivo monocyte activation was evaluated in patients with WG and the findings related to parameters of clinical disease activity. METHODS: Monocyte activation was analysed by measuring plasma concentrations of soluble products of monocyte activation, that is neopterin and interleukin 6 (IL6), by ELISA, and by quantitating the surface expression of activation markers on circulating monocytes by flow cytometry. RESULTS: Twenty-four patients with active WG were included in this study. Ten of these patients were also analysed at the time of remission. Twelve patients with sepsis served as positive controls, and 10 healthy volunteers as negative controls for monocyte activation. Patients with active disease had increased monocyte activation compared with healthy controls as shown by increased concentrations of neopterin (p < 0.0001) and increased surface expression of CD11b (p < 0.05) and CD64 (p < 0.05). In those patients with increased concentrations of IL6 during active disease plasma concentrations of IL6 decreased during follow up when patients went into remission (p < 0.0001). In addition, neopterin (r = 0.37, r = 0.44), IL6 (r = 0.37, r = 0.60) and CD63 expression (r = 0.39, r = 0.45) correlated significantly with disease activity as measured by the Birmingham Vasculitis Activity Score and C reactive protein values, respectively. Compared with patients with sepsis, all markers of monocyte activation in patients with vasculitis were lower. CONCLUSION: It is concluded that disease activity in WG correlates with the extent of activation of monocytes, compatible with their role in the pathophysiology of this disease.

Are circulating neutrophils intravascularly activated in patients with anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitides?

Vascular injury in vasculitis may be due to activation of circulating neutrophils resulting in their increased adhesiveness to locally activated endothelium (Shwartzman phenomenon). Previously, we demonstrated up-regulation of endothelial intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) in biopsies from patients with ANCA-associated vasculitis. In the present study, we investigated the expression of adhesion molecules (CD11b, ICAM-1, VLA-4, L-selectin) and activation markers (CD66b, CD64, CD63) on circulating neutrophils from patients with ANCA-associated vasculitis in comparison with their expression on cells from healthy volunteers and patients with sepsis. We related these findings to parameters of disease activity. Surface marker expression was determined by using a non-activating whole blood flow cytometric assay. The expression of activation markers, but not the expression of adhesion molecules, was increased on neutrophils from patients with active vasculitis. The expression of CD63 and CD66b on neutrophils correlated with disease activity as determined by the Birmingham Vasculitis Activity Score (BVAS). In contrast to patients with active vasculitis, patients with sepsis showed up-regulation of all markers, including adhesion molecules, suggesting that circulating neutrophils are fully activated in sepsis. We conclude that in ANCA-associated vasculitis, circulating neutrophils are not fully activated, since they do not express increased levels of adhesion molecules as sepsis or in the Shwartzman reaction. These findings are compatible with the concept that in vivo vascular damage in ANCA-associated vasculitides does not occur due to a Shwarzman-like reaction but only after ANCA-induced neutrophil activation at the endothelial cell surface.

Levels of soluble Fc gammaRIII correlate with disease severity in sepsis.

Neutrophil activation is thought to play a crucial role in the pathogenesis of sepsis. During activation, neutrophils adhere to and migrate through the endothelium. Therefore, the amount of circulating neutrophils does not adequately reflect the total amount of neutrophils that are involved in the pathophysiologic process of this condition. In this study we test the hypothesis that the severity of sepsis is associated with the total body mass of neutrophils as reflected in the plasma concentration of soluble Fc gamma receptor type III (sFc gammaRIII). Nineteen patients with sepsis (12 male, seven female, median age of 69 years, range 29-87 years) were included in this study. Ten healthy volunteers served as controls. Plasma sFc gammaRIII concentrations were measured by ELISA. Other parameters that were studied were leucocyte count, plasma concentrations of lactoferrin and soluble L-selectin, and surface expression of CD11b and CD66b on circulating neutrophils. Disease activity was measured using the Acute Physiology and Chronic Health Evaluation (APACHE) II score. Soluble Fc gammaRIII levels were elevated in sepsis patients whereas soluble L-selectin levels were moderately decreased compared with healthy controls. Markers of cell activation were significantly increased in sepsis patients. Soluble Fc gammaRIII correlated with disease severity as measured by the APACHE score (P<0.05, r=0.53), whereas the other parameters did not correlate with the APACHE score. In conclusion, this study demonstrates that soluble Fc gammaRIII is a useful marker for disease severity in patients with sepsis.

Leucocyte membrane expression of proteinase 3 correlates with disease activity in patients with Wegener's granulomatosis.

Wegener's granulomatosis (WG) is an inflammatory disorder characterized by granulomatous inflammation and vasculitis, and is strongly associated with antineutrophil cytoplasmic antibodies (ANCA). ANCA in patients with WG are directed against proteinase 3 (Pr3) in most of the cases. In vitro, upon neutrophil priming, ANCA antigens are expressed on the cell surface, thereby becoming available for interaction with ANCA. Subsequently, these neutrophils become activated. Since ANCA can only interact with leucocytes when the ANCA antigens are present on the cell surface, we questioned whether Pr3 is already expressed on the membranes of circulating granulocytes and monocytes of patients with WG, and whether Pr3 expression is related to disease activity, so explaining the systemic nature and severity of the disease. The expression of Pr3, and other ANCA antigens, i.e. myeloperoxidase (MPO) and human leucocyte elastase (HLE), was analysed on circulating granulocytes and monocytes by flow cytometry, using a non-activating whole-blood method. Disease activity was quantitated using the Birmingham Vasculitis Activity Score (BVAS). Seventeen patients with active WG and anti-Pr3 antibodies were included in this study. Nine of these patients were also analysed at the time of remission. Twelve patients with sepsis served as positive controls, and 10 healthy volunteers as negative controls for granulocyte/monocyte activation. Pr3 expression on neutrophils was increased in patients with active WG compared to patients with quiescent disease and healthy controls. On monocytes, no differences in Pr3 expression were found between those groups. Furthermore, the expression of MPO and HLE did not differ between patient groups and healthy controls. Upon follow-up, the expression of Pr3 on neutrophils from patients with active WG decreased when patients went into remission. Pr3 expression on neutrophils correlated with the BVAS score (r = 0.40, P < 0.05). In conclusion, circulating neutrophils from patients with active WG have increased expression of Pr3. In addition, the expression of Pr3 correlates with disease activity, suggesting that the availability of Pr3 for interaction with ANCA plays a central role in the disease process.