Alpha-defensins: risk factor for thrombosis in COVID-19 infection.

The inflammatory response to SARS/CoV-2 (COVID-19) infection may contribute to the risk of thromboembolic complications. α-Defensins, antimicrobial peptides released from activated neutrophils, are anti-fibrinolytic and prothrombotic in vitro and in mouse models. In this prospective study of 176 patients with COVID-19 infection, we found that plasma levels of α-defensins were elevated, tracked with disease progression/mortality or resolution and with plasma levels of interleukin-6 (IL-6) and D-dimers. Immunohistochemistry revealed intense deposition of α-defensins in lung vasculature and thrombi. IL-6 stimulated the release of α-defensins from neutrophils, thereby accelerating coagulation and inhibiting fibrinolysis in human blood, imitating the coagulation pattern in COVID-19 patients. The procoagulant effect of IL-6 was inhibited by colchicine, which blocks neutrophil degranulation. These studies describe a link between inflammation and the risk of thromboembolism, and they identify a potential new approach to mitigate this risk in patients with COVID-19 and potentially in other inflammatory prothrombotic conditions.

Kynurenic Acid Analog Attenuates the Production of Tumor Necrosis Factor-α, Calgranulins (S100A 8/9 and S100A 12), and the Secretion of HNP1-3 and Stimulates the Production of Tumor Necrosis Factor-Stimulated Gene-6 in Whole Blood Cultures of Patients With Rheumatoid Arthritis.

Objectives: Rheumatoid arthritis (RA) is a chronic, inflammatory joint disease with complex pathogenesis involving a variety of immunological events. Recently, it has been suggested that kynurenic acid (KYNA) might be a potential regulator of inflammatory processes in arthritis. KYNA has a definitive anti-inflammatory and immunosuppressive function. The aim of the present study is to investigate the complex effects of a newly synthesized KYNA analog-SZR72 on the in vitro production of tumor necrosis factor-α (TNF-α), tumor necrosis factor-stimulated gene-6 (TSG-6), calprotectin (SA1008/9), SA100 12 (EN-RAGE), and HNP1-3 (defensin-α) in the peripheral blood of patients with RA and the various effects of the disease. Methods: Patients with RA (n = 93) were selected based on the DAS28 score, medication, and their rheumatoid factor (RF) status, respectively. Peripheral blood samples from 93 patients with RA and 50 controls were obtained, and activated by heat-inactivated S. aureus. Parallel samples were pretreated before the activation with the KYNA analog N-(2-N, N-dimethylaminoethyl)-4-oxo-1H-quinoline-2-carboxamide hydrochloride. Following the incubation period (18 h), the supernatants were tested for TNF-α, TSG-6, calprotectin, S100A12, and HNP1-3 content by ELISA. Results: SZR72 inhibited the production of the following inflammatory mediators: TNF-α, calprotectin, S100A12, and HNP1-3 in whole blood cultures. This effect was observed in each group of patients in various phases of the disease. The basic (control) levels of these mediators were higher in the blood of patients than in healthy donors. In contrast, lower TSG-6 levels were detected in patients with RA compared to healthy controls. In addition, the KYNA analog exerted a stimulatory effect on the TSG-6 production ex vivo in human whole blood cultures of patients with RA in various phases of the disease. Conclusion: These data further support the immunomodulatory role of KYNA in RA resulting in anti-inflammatory effects and draw the attention to the importance of the synthesis of the KYNA analog, which might have a future therapeutic potential.

Increased Gene Copy Number of DEFA1A3 Is Associated With the Severity of Ulcerative Colitis.

INTRODUCTION: DEFA1A3 encodes human neutrophil peptides (HNPs) 1-3 and has multiple copy number variations (CNVs). HNPs are associated with innate immunity. Ulcerative colitis (UC), a chronic inflammatory gastrointestinal disorder, is a life-threatening condition, and predictive markers of UC severity are needed. This study investigated the relationship between DEFA1A3 CNV and UC severity. METHODS: This study enrolled 165 patients with UC. The relationship between DEFA1A3 CNV and disease severity was analyzed based on Mayo score, patient characteristics, and treatment methods. In addition, serum and stimulated neutrophil-derived HNP concentrations were also measured in patients with high and low DEFA1A3 CNV. RESULTS: DEFA1A3 CNV was significantly correlated with Mayo score and white blood cell count (R = 0.46, P < 0.0001; R = 0.29, P = 0.003, respectively), and only high copy numbers of DEFA1A3 were independent factors for severe UC (P < 0.001, odds ratio: 1.88, 95% confidence interval, 1.34-2.61). The number of severe UC patients with high DEFA1A3 CNV was significantly greater than those with low CNV. We confirmed the associations between DEFA1A3 and UC severity using a validation cohort. In addition, the HNP concentration in high-copy number patients was significantly higher after neutrophil stimulation than that in low-copy number patients. DISCUSSION: This study demonstrated that there is a correlation between DEFA1A3 copy number and severity in patients with UC. In addition, neutrophils from UC patients with higher DEFA1A3 CNV had high reactivity of secretion of HNPs after stimulation. DEFA1A3 CNV may be a novel severity marker and a potential therapeutic target for UC.

Human neutrophil peptides 1-3 level in patients with acute myocardial infarction and its relation with coronary artery disease severity.

OBJECTIVE: Inflammation plays a crucial role in the pathogenesis and clinical outcome of atherosclerosis. Among the various inflammatory factors, antimicrobial peptides, such as alpha-defensins, seem to contribute to the development and progression of atherosclerosis. The aim of this study was to evaluate the plasma levels of human neutrophil peptide-1, -2, and -3 (HNP1-3) in patients with acute myocardial infarction (AMI) and to assess its relationship with the severity of coronary artery disease. METHODS: lasma HNP1-3 levels in patients with AMI and controls with angiographically normal coronary arteries were measured by solid-phase enzyme-linked immunosorbent assay. In the patient group, coronary artery disease severity was assessed using the SYNergy between percutaneous intervention with TAXus and cardiac surgery score (SS). RESULTS: HNP1-3 levels were significantly higher in the group with AMI than in the controls (6.5±5.8 ng/mL vs. 2.8±2.5 ng/mL, p<0.001). The receiver operator characteristic (ROC) analysis yielded a cut-off value of 3.13 ng/mL for differentiating patients with AMI from the controls (area under the curve: 0.739, 95% confidence interval: 0.629-0.831, p<0.001). HNP1-3 levels in the high SS tertile (≥33) were slightly but statistically nonsignificantly higher than that in the low (≤22) and intermediate SS tertiles (high SS: 7.0±6.1 ng/mL, intermediate SS: 5.9±6.2 ng/mL, low SS: 5.3±3.8 ng/mL; p=0.639). CONCLUSION: Patients with AMI had higher plasma HNP1-3 levels than the controls, but this did not show a significant correlation with angiographic disease severity. The nonsignificant trend toward higher SS in patients with higher HNP1-3 levels warrants future studies on larger populations.

Whole blood RNA sequencing reveals a differential transcriptomic profile associated with cervical insufficiency: a pilot study.

BACKGROUND: The uterine cervix is a mechanical and immunological barrier against ascending infection during pregnancy. Cervical insufficiency (CI), a painless cervical dilation that occurs in the mid-trimester, is an important cause of extremely preterm birth. We hypothesized that women with CI have a differential transcriptomic profile. Therefore, we compared the transcriptomic profile of peripheral blood in women with CI and that of controls. METHODS: RNA sequencing was used to generate the global gene expression profiles of 11 women with CI and 4 controls, and differential expression analysis was performed to identify genes showing significant expression changes between the CI (n = 11) and control (n = 4) groups as well as between the CI-preterm (n = 7) and CI-term (n = 4) groups. Gene set enrichment was assessed in terms of Gene Ontology processes, and a subset of differentially expressed genes in CI was validated in a different sample-set by qRT-PCR and ELISA. RESULTS: Thirty genes were differentially expressed between the CI and control groups. Differentially upregulated genes in the CI group included neutrophil-mediated immunity-associated (DEFA3 and ELANE) and bicarbonate transport-related genes. The serum concentration of alpha defensin 3 was significantly higher in women with CI than in controls (P = 0.014). Analysis of differential gene expression according to pregnancy outcomes revealed 338 differentially expressed genes between the CI-term and CI-preterm groups. Immune and defense response to organism-associated genes and influenza A and NOD-like receptor signaling pathways were upregulated in the CI-term group. CONCLUSIONS: Our results revealed significant differences in the whole blood transcriptomic profiles of women with CI compared to those of controls. Different immune responses in women with CI may affect pregnancy outcomes.

Evaluation of alpha defensin, IL-1 receptor antagonist, and IL-18 levels in COVID-19 patients with macrophage activation syndrome and acute respiratory distress syndrome.

BACKGROUND: Many laboratory parameters have been associated with morbidity and mortality in SARS-CoV-2 (COVID-19), which emerged in an animal market in Wuhan, China in December 2019 and has infected over 20 million people. This study investigated the relationship between serum interleukin (IL)-18, IL-1 receptor antagonist (IL-1Ra), and alpha defensin levels and the clinical course and prognosis of COVID-19. MATERIALS AND METHODS: This study included 100 patients who were admitted to the chest diseases department and intensive care unit of our hospital and diagnosed with COVID-19 by real-time polymerase chain reaction (PCR) of nasopharyngeal swab samples between March 24 and May 31, 2020. The control group consisted of 50 nonsymptomatic health workers with negative real-time PCR results in routine COVID-19 screening in our hospital. RESULTS: Serum alpha defensin, IL-1Ra, and IL-18 levels were significantly higher in patients who developed macrophage activation syndrome (MAS) and acute respiratory distress syndrome (ARDS) compared to patients who did not (p < .001 for all). Alpha defensin, IL-1Ra, and IL-18 levels were significantly higher in COVID-19 patients with and without MAS or ARDS when compared to the control group (p < .001 for all). When the 9 patients who died were compared with the 91 surviving patients, IL-1Ra and IL-18 levels were found to be significantly higher in the nonsurvivors (p < .001). CONCLUSION: Our findings of correlations between alpha defensin and levels of IL-1Ra and IL-18, which were previously shown to be useful in COVID-19 treatment and follow-up, indicates that it may also be promising in treatment.

The diagnostic effect of α-defensin, D-dimer, and IL-6 in periprosthetic joint infection: A systematic review and diagnostic meta-analysis.

BACKGROUND: Periprosthetic joint infection (PJI) is the most common complication after artificial joint replacement as previously reported. However, the main problem at present is its difficulty in diagnosis. This systematic review and meta-analysis aimed to compare the diagnostic accuracy of α-defensin, D-dimer, and interleukin-6 (IL-6) in clinical practice. METHOD: Online databases were systematically searched until June 18th, 2020 with keywords and medical sub-headings terms. Studies mentioned the sensitivity and specificity of biological markers in detecting PJI were included in our study. The sensitivity, specificity, and diagnostic odds ratios (DORs) were obtained after integration. RESULTS: A total of 34 studies with 1036 patients diagnosing as PJI were included for comparing α-defensin, D-dimer, and IL-6. The sensitivity and specificity of α-defensin for PJI were 0.88 and 0.96, and the DOR was 189 (95% CI 72-496), respectively. The sensitivity and specificity of D-dimer (0.82 and 0.72) and IL-6 (0.80 and 0.89) were lower than α-defensin. CONCLUSION: The detection of α-defensin is a promising biomarker for diagnosing PJI. The optional cut-off needs to be curtained when using other biomarkers.

Does the Alpha Defensin ELISA Test Perform Better Than the Alpha Defensin Lateral Flow Test for PJI Diagnosis? A Systematic Review and Meta-analysis of Prospective Studies.

BACKGROUND: Periprosthetic joint infection (PJI) following total joint arthroplasty is a serious complication that causes severe morbidity and adds a major financial burden to the healthcare system. Although there is plenty of research on the alpha-defensin (AD) test, a meta-analysis consisting of only prospective studies investigating AD's diagnostic efficacy has not been performed. Additionally, some important subgroups such as THA and TKA have not been separately analyzed, particularly regarding two commonly used versions of the AD test, the laboratory-based (ELISA) and lateral-flow (LF). QUESTIONS/PURPOSES: (1) Does the AD ELISA test perform better in the detection of PJI than the AD LF test, in terms of pooled sensitivity and specificity, when including prospective studies only? (2) Are there differences in sensitivity or specificity when using AD ELISA and AD LF tests for PJI diagnosis of THA or TKA PJI separately? METHODS: Following the Preferred Reporting Items for Systematic reviews and Meta-Analyses guidelines, we included prospective studies describing the use of either AD test in the workup of pain after total joint arthroplasty (primary or revision, but not after resection arthroplasty). Fifteen studies (AD ELISA: 4; AD LF: 11) were included, with 1592 procedures. Subgroup data on THA and TKA could be retrieved for 1163 procedures (ELISA THA: 123; LF THA: 257; ELISA TKA: 228; LF TKA: 555). Studies not describing THA or TKA, those not using Musculoskeletal Infection Society (MSIS) criteria as the standard for determining the presence or absence of PJI, those not clearly reporting data for the AD test for the total cohort, and those describing data published in another study were excluded. Studies were not excluded based on follow-up duration; the MSIS criteria could be used within a few weeks, when test results were available. Quality was assessed using the Quality Assessment of Diagnostic Accuracy Studies-2 criteria. Study quality was generally good. The most frequent sources of bias were related to patient selection (such as unclear inclusion and exclusion criteria) and flow and timing (uncertainty in place and time of aspiration, for example). Heterogeneity was moderate to high; a bivariate random-effects model therefore was used. To answer both research questions, sensitivity and specificity were calculated for AD ELISA and LF test groups and THA and TKA subgroups, and were compared using z-test statistics and meta-regression analysis. RESULTS: No differences were found between the AD ELISA and the AD LF for PJI diagnosis in the pooled cohorts (THA and TKA combined), in terms of sensitivity (90% versus 86%; p = 0.43) and specificity (97% versus 96%; p = 0.39). Differences in sensitivity for PJI diagnosis were found between the THA and TKA groups for the AD ELISA test (70% versus 94%; p = 0.008); pooled AD LF test sensitivity did not differ between THA and TKA (80% versus 87%; p = 0.20). No differences in specificity were found in either subgroup. CONCLUSIONS: Both the AD ELISA and AD LF test can be used in clinical practice because both have high sensitivity and very high specificity for PJI diagnosis. The lower sensitivity found for diagnosis of PJI in THA for the AD ELISA test must be carefully interpreted because the pooled data were heterogenous and only two studies for this group were included. Future research should analyze TKAs and THAs separately to confirm or disprove this finding. LEVEL OF EVIDENCE: Level II diagnostic study.

Alpha-defensin lateral flow test does not appear to be useful in predicting shoulder periprosthetic joint infections.

PURPOSE: Periprosthetic joint infections (PJIs) remain a challenging complication after shoulder arthroplasty. The antimicrobial peptide α-defensin has been proposed as a new synovial fluid biomarker in diagnosing PJIs. To date, only little data are available on the diagnostic accuracy of α-defensin in shoulder PJIs; thus, we aimed to evaluate its diagnostic value in a cohort of patients with a suspected shoulder PJI. METHODS: Between June 2016 and June 2018, we prospectively enrolled patients with a diagnostic shoulder aspiration due to painful shoulder arthroplasty or planned revision surgery. PJI diagnostics were performed according to the Musculoskeletal Infection Society (MSIS) criteria. All patients with an antibiotic therapy within two  weeks before enrollment, insufficient amount of synovial aspirate, or bloody aspiration were excluded. α-Defensin was measured in the synovial fluid using the α-defensin lateral flow (ADLF) test (Synovasure®). RESULTS: Out of 60 patients, we could include 29 (59% female) patients with a mean age of 70 (range, 50-92) years. A shoulder PJI was detected in five cases (Staphylococcus aureus, n = 2; Staphylococcus epidermidis, n = 2; Cutibacterium acnes, n = 1). The ADLF test was positive in seven out of 29 cases. According to the MSIS criteria, the ADLF test was false-negative in two patients and false-positive in four patients, resulting in sensitivity, specificity, and positive and negative predictive value of 60%, 83%, 43%, and 91%, respectively. The overall accuracy was 79%. CONCLUSION: The ALDF test does not appear to be useful in predicting shoulder PJIs but may be used as an additional diagnostic factor in rejecting these infections.

Current Clinical Methods for Detection of Peri-Prosthetic Joint Infection.

Background: Currently, one of the most pressing problems in the field of orthopedic surgery is peri-prosthetic joint infection [PJI]. While there are numerous ways to detect PJI, current clinical detection methods differ across institutions and have varying criteria and protocols. Some of these methods include the Modified Musculoskeletal Infection Society system, culturing, polymerase chain reaction, the determination of the presence of certain biomarkers, testing for the presence of alpha defensin peptides, and leukocyte level testing. Methods: This review summarizes the most recent publications in the field of PJI detection to highlight current strengths as well as provide future directions to find the system for the quickest, cost-effective, and most accurate way to diagnose these types of infections. Results: The results of this literature review suggest that, while each method of diagnosis has its advantages, each has various drawbacks as well. Current methods can be expensive, take days to weeks to complete, be prone to contamination, and can produce ambiguous results. Conclusions: The findings in this review emphasize the need for a more comprehensive and accurate system for diagnosing PJI. In addition, the specific comparison of advantages and drawbacks can be useful for researchers and clinicians with goals of creating new diagnostic tests for PJIs, as well as in clinical scenarios to determine the correct treatment for patients.

Upregulated Expression of Intestinal Antimicrobial Peptide HD5 Associated with Renal Function in IgA Nephropathy.

PURPOSE: It was reported that gut-kidney axis may play an important role in IgA nephropathy (IgAN). Previous five GWASs of different populations for IgAN have discovered several genes related to intestinal immunity, including DEFA gene. However, the roles of the encoded proteins of DEFA5/6 which were called intestinal antimicrobial peptides HD5 and HD6 were not clear in kidney disease, such as IgAN. The purpose of this study was to clarify the association of HD5 and HD6 with IgAN. METHODS: We measured HD5 and HD6 in serum, urine, and kidney of IgAN patients and normal controls by ELISA, Western blot, and immunofluorescence. The association of HD5 or HD6 levels with clinical and pathologic phenotypes was analyzed. RESULTS: Serum levels of HD5 and HD6 were significantly higher in IgAN patients than those in normal controls. Baseline serum HD5 levels were significantly associated with eGFR (P = 0.002) and tubular atrophy/interstitial fibrosis (P = 0.002) and tubular atrophy/interstitial fibrosis (P = 0.002) and tubular atrophy/interstitial fibrosis (P = 0.002) and tubular atrophy/interstitial fibrosis (. CONCLUSIONS: In IgAN patients, an elevated serum HD5 level at the time of renal biopsy was associated with poor renal outcomes. HD5 rather than HD6 was probably associated with renal function of IgAN patients.

Human defensins and Th-1 cytokines in hepatitis C viral infection.

INTRODUCTION: active or chronic exacerbated forms of hepatitis C virus (HCV) infection subsequently progress to liver disease and human defensins has been determined to have some level of anti-viral properties invitro whilst the expression of T helper-1 cytokines is known to promote complete recovery from acute HCV infection. The study sought to determine relationship between these immune responses. METHODS: a cross sectional descriptive study design was employed. Hundred and thirty-two individuals were assessed were assessed for to anti-HCV, HCV RNA, serum levels of human alpha defensins 1 (HAD-1) and human beta defensins 1 (HBD-1). T helper 1 cytokines (IL-2, IFN gamma, TNF alpha) secreted in serum were also analyzed using commercial ELISA assay. The study was conducted in Kumasi, Obuasi and Daboya in Ghana. RESULTS: the serum mean concentrations of HAD-1, HBD-1, IL-2, IFN gamma and TNF alpha showed no significant difference in concentrations among participants with chronic, spontaneously recovered or negative to HCV infection (p>0.05). Persons with hepatitis B co-infection were more likely to develop chronic HCV infection (p=0.039). HAD-1 and HBD-1 showed significant positive association with IL-2 (p=0.000) whilst only HAD-1 positively correlated with IL-2 (p<0.000). CONCLUSION: the immunological markers determined had no association with the status of HCV infection. HAD-1 increased with increasing levels of IL-2. These findings suggest that during HCV infection, inflammatory response through the production of cytokines by IL-2 cells may affect the release of HAD-1 and HBD-1.

Diagnosis of Shoulder Arthroplasty Infection: New Tests on the Horizon.

Periprosthetic shoulder infection (PSI), although less common than prosthetic hip and knee infections, continues to be a devastating complication of shoulder arthroplasty. Unlike its counterparts in the hip and knee, infection with nonsuppurative bacteria is more common than infection with more virulent bacteria in periprosthetic shoulder infection. The diagnosis of PSI can be challenging because the traditional clinical and laboratory findings are not always present. The authors present a narrative review of the current methods used in the diagnosis of PSI, as well as recently developed tests that may hold promise for the diagnosis of PSI. [Orthopedics. 2020; 43(2):76-82.].

Serum defensin levels in patients with systemic sclerosis

Abstract Background: Systemic sclerosis (SSc) is an autoimmune disease characterized by fibrosis of skin and lung as well as involvement of kidney, gastrointestinal system and heart. Aetiology and exact mechanism of disease is poorly understood. The association between antimicrobial peptides (AMPs) and other diseases such as idiopathic pulmonary fibrosis, diffuse panbronchiolitis, pulmoner alveolar proteinosis and psoriasis have been reported. A small number of studies have examined the role of AMPs on autoimmune diseases which has not been studied in scleroderma yet. We aimed to investigate AMP serum levels and their association with disease characteristics of SSc. Methods: Forty-two patients (40 female, mean age 42 years) and 38 healthy subjects (32 female, mean age 38 years) were enrolled. For SSc patients, the following data were recorded: disease subset (limited/diffuse), autoantibodies (antinuclear, anti-centromere (ACA), and anti-SCL-70), blood tests, erythrocyte sedimentation rate (ESR), and C-reactive protein (CRP), modified Rodnan skin score, presence and history of digital ulcers, kidney, gastrointestinal disease and lung involvement assessed by computed tomography and pulmonary function tests. Association between serum AMPs and disease characteristics were analysed. Results: Twenty-nine of the patients had diffuse (69%) and 13 of the patients had limited (31%) systemic sclerosis. Average disease duration was 5.5 years. Pulmonary involvement was detected in 20 patients (47.6%). Serum concentration of alpha defensin was higher than healthy subjects (563 ± 415 vs 377 ± 269 ng/mL, p = 0.02). However, no difference was observed for beta-1 and beta-2 defensins in SSc patients and healthy controls. In sub-group analysis patients with interstitial lung disease had higher levels of alpha defensin than those without lung involvement (684 ± 473 vs 430 ± 299 ng/ml, p = 0.04). There was also correlation between alfa defensin serum concentrations and CRP (r = 0.34). Conclusions: Alpha defensin levels are increased in scleroderma patients and correlated with lung involvement indicating a role in the pathogenesis of disease. Trial registration: This study is not a clinical trial study.(AU)

Neutrophil Extracellular Traps May Contribute to the Pathogenesis in Adult-onset Still Disease.

OBJECTIVE: Release of neutrophil extracellular traps (NET) has been described as an effector mechanism of polymorphonuclear neutrophils in several inflammatory diseases. Thus, this study was performed to evaluate the role of NET in the pathogenesis of adult-onset Still disease (AOSD). METHODS: We determined the serum levels of NET molecules and investigated their associations with clinical disease activities in patients with AOSD. Further, we analyzed the differences in the NETosis response in AOSD patients compared to healthy controls (HC). To explore the in vivo involvement of NET in AOSD, we performed immunohistochemical analysis of skin and lymph node (LN) biopsies for proteins related to NET in patients with active AOSD. RESULTS: Serum levels of cell-free DNA, myeloperoxidase (MPO)-DNA complex, and α-defensin were significantly increased in patients with AOSD compared to HC. Serum levels of the NET molecules, cell-free DNA, MPO-DNA, and α-defensin were correlated with several disease activity markers for AOSD. In followup of patients with AOSD after treatment with corticosteroid, the levels of cell-free DNA and α-defensin decreased significantly. On immunohistochemistry, neutrophil elastase-positive and MPO-positive inflammatory cells were detected in skin and LN of patients with AOSD, and were expressed in fiber form in the lesions. The serum from patients with active AOSD induced NETosis in neutrophils from HC. NET molecules induced interleukin 1ß production in monocytes, representing a novel mechanism in the pathogenesis of AOSD. CONCLUSION: The findings presented here suggest that NET may contribute to the inflammatory response and pathogenesis in AOSD.

What Is the Diagnostic Accuracy of Alpha-Defensin and Leukocyte Esterase Test in Periprosthetic Shoulder Infection?

BACKGROUND: The diagnosis of periprosthetic joint infection (PJI) after total shoulder arthroplasty (TSA) is challenging, especially in patients with Cutibacterium (formerly Propionibacterium) acnes infection. Despite the increasing number of patients with PJI of the shoulder, there are still no robust data regarding diagnostic tests in detecting shoulder PJI. QUESTIONS/PURPOSES: (1) What are the sensitivity, specificity, and negative- and positive-predictive values for the alpha-defensin enzyme-linked immunosorbent assay test in detecting PJI after TSA? (2) What are the diagnostic accuracies in detecting shoulder PJI for synovial alpha-defensin, leukocyte esterase Test, and serum C-reactive protein (CRP)? METHODS: All patients with painful TSA, who underwent joint aspiration to validate or exclude a PJI, between July 2015 and February 2018 were enrolled in this single-center study. Further indications for aspiration were as follows: planned revision arthroplasty, early loosening and clinical signs of infections, especially serum CRP elevation. A total of 121 patients were aspirated to exclude or verify a PJI, and 16 patients were excluded. In all, 105 patients with a mean age of 68 years (± 12 years) were included for analysis. Patients who underwent TSA were considered aseptic or septic according to the Musculoskeletal Infection Society criteria. Twenty-four patients had a PJI, and the remaining 81 patients were in the aseptic group. The microbiologic evaluation including polymicrobial infection showed C. (formerly P.) acnes in 15 patients (63%). Synovial fluid was then analyzed using microbiology cultures, alpha-defensin immunoassay, and leukocyte esterase. The specificity, sensitivity, and positive-predictive and negative-predictive values were calculated for each test. RESULTS: The overall accuracy for alpha-defensin was 91% (95% confidence interval [CI], 84.4-96); sensitivity was 75% (95% CI, 53-90), specificity was 96% (95% CI, 90-99), negative predictive value was 93% (95% CI, 85-97), and positive predictive value was 86% (95% CI, 64-97). In contrast, the overall accuracy for leukocyte esterase was 76% (95% CI, 61-88), sensitivity was 50% (95% CI, 21-79), specificity was 87% (95% CI, 69-96), positive predictive value 60% (95% CI, 26-88) and negative predictive value was 81% (95% CI, 64-93). CONCLUSIONS: Summarizing the study results, the alpha-defensin ELISA and leukocyte esterase tests had less sensitivity in detecting shoulder PJI than previously reported TKA or THA results. The quality and low amount of joint fluid is the difficult part of the diagnostic. C. (formerly P.) acnes was the most common cause of PJI. Focusing on low-grade infections, alpha-defensin has shown its advantages in diagnosing PJI regardless pathogen virulence. Since the diagnostic of a PJI is always a synopsis of findings, the alpha-defensin and leukocyte esterase test can be used as adjunct diagnostic tool in patients with painful TSA. We propose further prospective studies to improve the diagnostic and confirm the results. LEVEL OF EVIDENCE: Level III, diagnostic study.

Positive Alpha-defensin at Reimplantation of a Two-stage Revision Arthroplasty Is Not Associated with Infection at 1 Year.

BACKGROUND: Diagnosing periprosthetic joint infection (PJI) represents a challenge that relies on multiple clinical and laboratory criteria that may not be consistently present. The synovial alpha-defensin-1 (AD-1) test has been shown to correlate accurately with the Musculoskeletal Infection Society (MSIS) criteria for the diagnosis of PJI, however, its association with persistent PJI has not been elucidated in the setting of patients receiving antibiotic spacers during second-stage reimplantation. Applying a Delphi-based consensus to define successful eradication of PJI offers an opportunity to test the utility of AD-1 in this setting. QUESTIONS/PURPOSES: (1) Can the AD-1 test determine whether infection has been controlled using the Delphi criteria for persistent PJI as a surrogate for infection eradication during two-stage revision for PJI treatment with a spacer? (2) How does the performance of the AD-1 test compare with the MSIS criteria? METHODS: This was a multicenter analysis of retrospectively collected data on patients who underwent a two-stage revision arthroplasty between May 2014 and July 2016. We included patients who had a previously confirmed PJI and received a cement spacer, underwent the second stage, had MSIS criteria data and a synovial fluid AD-1 test, and had a minimum followup of 1 year. We were unable to determine for all study sites how many patients had the test but did not meet all the criteria and so could not be studied; however, we were able to identify 69 patients (43 knees, 26 hips) who met all criteria. During the period in question, indications for use of AD-1 varied by surgeon; however, during that time, in general if a surgeon ordered it as part of the initial workup, the test would have been repeated before the second-stage reimplantation procedure. To assess the validity of AD-1 against persistence of PJI criteria at 1 year, the following were calculated using the Delphi criteria for persistent PJI as the gold standard: sensitivity, specificity, positive and negative predictive values, accuracy, and area under the curve (AUC) with 95% confidence intervals (CIs). Concordance index (c-index) and its Wald 95% CI with receiver operating characteristic (ROC) curve were calculated in relation to Delphi criteria for persistent PJI using AD-1 and then MSIS criteria. The two c-indices of AD-1 and MSIS were compared using the DeLong nonparametric approach. RESULTS: The AD-1 test showed poor sensitivity (7%; 95% CI, 0.2-34), and poor overall accuracy (73%; 95% CI, 60-83; AUC = 0.5; 95% CI, 0.3-0.6) in detecting infection eradication at 1 year. The c-index for AD-1 versus Delphi criteria for persistent PJI was 0.519 (95% CI, 0.44-0.60), and the c-index for MSIS criteria versus Delphi criteria for persistent PJI was 0.518 (95% CI, 0.49-0.54), suggesting the weak diagnostic abilities of these models. The contrast estimate between MSIS criteria and AD-1 were not different from one another at -0.001 (95% CI%, -0.09 to 0.09; p = 0.99). CONCLUSIONS: We found that a positive synovial fluid AD-1 test correlated poorly with the presence of persistent infection 1 year after two-stage revision arthroplasty for PJI. For this reason, we recommend against the routine use of AD-1 in patients with cement spacers, until or unless future studies demonstrate that the test is more effective than we found it to be. LEVEL OF EVIDENCE: Level IV, diagnostic study.

Relative Deficiency of Plasma A Disintegrin and Metalloprotease with Thrombospondin Type 1 Repeats 13 Activity and Elevation of Human Neutrophil Peptides in Patients with Traumatic Brain Injury.

Traumatic microvascular injury (tMVI) is a universal endophenotype of traumatic brain injury (TBI) that is responsible for significant neurological morbidity and mortality. The mechanism underlying tMVI is not fully understood. The present study aims to determine plasma levels of von Willebrand factor (VWF), a disintegrin and metalloprotease with thrombospondin type 1 repeats (ADAMTS) 13 activity, and human neutrophil peptides (HNP) 1-3 and to correlate these biomarkers with functional outcomes after moderate-severe TBI. Thirty-one consecutive TBI patients (Glasgow Coma Scale [GCS] range, 3-12) were enrolled into the study between February 2010 and November 2014. Blood samples were collected on 0, 1, 2, 3, and 5 days after admission and analyzed for plasma levels of VWF antigen (VWFAg), collagen-binding activity (VWFAc), ADAMTS13 activity, and HNP1-3 proteins. Mean values of plasma VWFAg, VWFAc, and HNP1-3 were significantly increased in TBI patients compared to those in healthy controls (n = 30). Conversely, mean plasma values of ADAMTS13 activity in TBI patients were significantly decreased during the first 2 days after admission. This resulted in a dramatic reduction in the ratio of ADAMTS13 activity to VWFAg or ADAMTS13 to VWFAc in all 5 post-TBI days. Cluster analysis demonstrated that high median plasma levels of VWFAg and HNP1-3 were observed in the cluster with a high mortality rate. These results demonstrate that a relative deficiency of plasma ADAMTS13 activity, resulting from activation of neutrophils and endothelium, may contribute to the formation of microvascular thrombosis and mortality after moderate-severe TBI.

Neutrophil α-defensins promote thrombosis in vivo by altering fibrin formation, structure, and stability.

Inflammation and thrombosis are integrated, mutually reinforcing processes, but the interregulatory mechanisms are incompletely defined. Here, we examined the contribution of α-defensins (α-defs), antimicrobial proteins released from activated human neutrophils, on clot formation in vitro and in vivo. Activation of the intrinsic pathway of coagulation stimulates release of α-defs from neutrophils. α-Defs accelerate fibrin polymerization, increase fiber density and branching, incorporate into nascent fibrin clots, and impede fibrinolysis in vitro. Transgenic mice (Def++) expressing human α-Def-1 developed larger, occlusive, neutrophil-rich clots after partial inferior vena cava (IVC) ligation than those that formed in wild-type (WT) mice. IVC thrombi extracted from Def++ mice were composed of a fibrin meshwork that was denser and contained a higher proportion of tightly packed compressed polyhedral erythrocytes than those that developed in WT mice. Def++ mice were resistant to thromboprophylaxis with heparin. Inhibiting activation of the intrinsic pathway of coagulation, bone marrow transplantation from WT mice or provision of colchicine to Def++ mice to inhibit neutrophil degranulation decreased plasma levels of α-defs, caused a phenotypic reversion characterized by smaller thrombi comparable to those formed in WT mice, and restored responsiveness to heparin. These data identify α-defs as a potentially important and tractable link between innate immunity and thrombosis.

Detection of α-defensin in eosinophils in helminth-infected mouse model.

α-defensin is a potent antimicrobial peptide secreted from intestinal mucosal epithelial cells, such as Paneth cells, and affects not only bacteria but also parasites and fungi. Recently, human eosinophils have also been shown to produce α-defensin, but no studies have been done on other animals. In this study, we attempted to detect α-defensin protein in mouse eosinophils infiltrating the intestinal mucosa during a helminth infection using Zamboni fixation and immunohistochemistry. Most of the eosinophils infiltrating the intestinal mucosa during helminth infection were positive for α-defensin. The expression level of α-defensin mRNA was 50 fold that in the control. Meanwhile, the number of Paneth cells was doubled, and their α-defensin fluorescence intensity was increased. These results suggested that eosinophils are also important producers of α-defensin, such as Paneth cells in mice, and that α-defensin produced from eosinophils might be involved in defensive mechanisms against helminths. Moreover, the experimental system used in this study is a good model to study the generation of α-defensin by eosinophils.