High serum proteinase-3 levels predict poor progression-free survival and lower efficacy of bevacizumab in metastatic colorectal cancer.

BACKGROUND: To improve the prognosis of patients with metastatic colorectal cancer (mCRC), investigating predictive biomarkers of their prognosis and chemotherapeutic responsiveness is necessary. This study aimed to analyze the clinical significance of serum proteinase-3 (PRTN3) as a predictor for prognosis and chemosensitivity, especially to bevacizumab therapy, in mCRC. METHODS: This single-center retrospective observational study enrolled 79 patients with mCRC in our hospital and 353 patients with colorectal cancer in the TCGA database. Preoperative serum PRTN3 levels were measured using an enzyme-linked immunosorbent assay. The clinicopathological characteristics and prognosis according to serum PRTN3 levels were then evaluated. PRTN3 expression in tumor and stromal cells was evaluated immunohistochemically. The impact of PRTN3 levels on angiogenesis and bevacizumab sensitivity was evaluated using the tube formation assay. RESULTS: Serum PRTN3 levels were an independent poor prognostic factor for progression-free survival (PFS) (hazard ratio, 2.082; 95% confidence interval, 1.118-3.647; P=0.010) in patients with mCRC. Similarly, prognostic analysis with TCGA data sets showed poorer overall survival in patients with PRTN3 expression than that in patients without PRTN3 expression, especially in patients with stage IV. Immunohistochemical analysis of resected specimens revealed that stromal neutrophils expressed PRTN3, and their expression level was significantly correlated with serum PRTN3 levels. Interestingly, the effectiveness of first-line chemotherapy was significantly poorer in the high serum PRTN3 level group. High serum PRTN3 was significantly associated with poor PFS (hazard ratio, 3.027; 95% confidence interval, 1.175-7.793; P=0.0161) in patients treated with bevacizumab, an anti-angiogenic inhibitor. The tube formation assay revealed that PRTN3 administration notably augmented angiogenesis while simultaneously attenuating the anti-angiogenic influence exerted by bevacizumab therapy. CONCLUSIONS: Serum PRTN3 levels could be a novel predictive biomarker of PFS of first-line chemotherapy, especially for bevacizumab therapy, in patients with mCRC.

The Course of AαVal541 as a Proteinase 3 Specific Neo-Epitope after Alpha-1-Antitrypsin Augmentation in Severe Deficient Patients.

In alpha-1-antitrypsin deficiency (AATD), neutrophil serine proteases such as elastase and proteinase 3 (PR3) are insufficiently inhibited. A previous study in AATD patients showed a higher plasma level of the specific PR3-generated fibrinogen-derived peptide AαVal541, compared with healthy controls. Here, we analyzed the course of AαVal541 plasma levels during 4 weeks after a single iv dose of 240 mg/kg AAT in ten patients with genotype Z/Rare or Rare/Rare. To this end, we developed an immunoassay to measure AαVal541 in plasma and applied population pharmacokinetic modeling for AAT. The median AαVal541 plasma level before treatment was 140.2 nM (IQR 51.5-234.8 nM)). In five patients who received AAT for the first time, AαVal541 levels decreased to 20.6 nM (IQR 5.8-88.9 nM), and in five patients who already had received multiple infusions before, it decreased to 26.2 nM (IQR 22.31-35.0 nM). In 9 of 10 patients, AαVal541 levels were reduced to the median level of healthy controls (21.4 nM; IQR 16.7-30.1 nM). At 7-14 days after treatment, AαVal541 levels started to increase again in all patients. Our results show that fibrinopeptide AαVal541 may serve as a biochemical footprint to assess the efficacy of in vivo inhibition of PR3 activity in patients receiving intravenous AAT augmentation therapy.

Proteinase 3 contributes to endothelial dysfunction in an experimental model of sepsis.

In sepsis-induced inflammation, polymorphonuclear neutrophils (PMNs) contribute to vascular dysfunction. The serine proteases proteinase 3 (PR3) and human leukocyte elastase (HLE) are abundant in PMNs and are released upon degranulation. While HLE's role in inflammation-induced endothelial dysfunction is well studied, PR3's role is largely uninvestigated. We hypothesized that PR3, similarly to HLE, contributes to vascular barrier dysfunction in sepsis. Plasma PR3 and HLE concentrations and their leukocyte mRNA levels were measured by ELISA and qPCR, respectively, in sepsis patients and controls. Exogenous PR3 or HLE was applied to human umbilical vein endothelial cells (HUVECs) and HUVEC dysfunction was assessed by FITC-dextran permeability and electrical resistance. Both PR3 and HLE protein and mRNA levels were significantly increased in sepsis patients (P < 0.0001 and P < 0.05, respectively). Additionally, each enzyme independently increased HUVEC monolayer FITC-dextran permeability (P < 0.01), and decreased electrical resistance in a time- and dose-dependent manner (P < 0.001), an effect that could be ameliorated by novel treatment with carbon monoxide-releasing molecule 3 (CORM-3). The serine protease PR3, in addition to HLE, lead to vascular dysfunction and increased endothelial permeability, a hallmark pathological consequence of sepsis-induced inflammation. CORMs may offer a new strategy to reduce serine protease-induced vascular dysfunction.

The relationship of neutrophil elastase and proteinase 3 with risk factors, and chronic complications in type 2 diabetes: A Fenofibrate Intervention and Event Lowering in Diabetes (FIELD) sub-study.

INTRODUCTION: Neutrophil elastase (NE) and proteinase 3 (PR3) are novel inflammation biomarkers. We investigated their associations with chronic complications, determinants of biomarker levels and effects of fenofibrate in patients with type 2 diabetes mellitus (T2DM) from Fenofibrate Intervention and Event Lowering in Diabetes study. METHODS: Plasma NE and PR3 levels were quantified at baseline (n = 2000), and relationships with complications over 5-years assessed. Effects of fenofibrate on biomarker levels (n = 200) were determined at four follow-up visits. RESULTS: Higher waist-to-hip ratio, homocysteine and C-reactive protein and lower apoA-II were determinants of higher NE and PR3 levels. Higher NE levels were associated with on-trial stroke and cardiovascular mortality, and higher PR3 levels with on-trial stroke, but associations were not significant after adjustment for confounding factors. Although higher NE and PR3 levels were associated with baseline total microvascular disease, only NE levels were associated with on-trial neuropathy or amputation. These associations were not significant after adjusting for multiple comparisons. NE and PR3 levels did not change with fenofibrate. CONCLUSIONS: In T2DM plasma NE and PR3 levels are associated with vascular risk factors, and total microvascular disease at baseline, but on rigorous analyses were not associated with on-trial complications. Levels were not changed by fenofibrate.

Enhanced Expression of Autoantigens During SARS-CoV-2 Viral Infection.

Immune homeostasis is disturbed during severe viral infections, which can lead to loss of tolerance to self-peptides and result in short- or long-term autoimmunity. Using publicly available transcriptomic datasets, we conducted an in-silico analyses to evaluate the expression levels of 52 autoantigens, known to be associated with 24 autoimmune diseases, during SAR-CoV-2 infection. Seven autoantigens (MPO, PRTN3, PADI4, IFIH1, TRIM21, PTPRN2, and TSHR) were upregulated in whole blood samples. MPO and TSHR were overexpressed in both lung autopsies and whole blood tissue and were associated with more severe COVID-19. Neutrophil activation derived autoantigens (MPO, PRTN3, and PADI4) were prominently increased in blood of both SARS-CoV-1 and SARS-CoV-2 viral infections, while TSHR and PTPRN2 autoantigens were specifically increased in SARS-CoV-2. Using single-cell dataset from peripheral blood mononuclear cells (PBMCs), we observed an upregulation of MPO, PRTN3, and PADI4 autoantigens within the low-density neutrophil subset. To validate our in-silico analysis, we measured plasma protein levels of two autoantigens, MPO and PRTN3, in severe and asymptomatic COVID-19. The protein levels of these two autoantigens were significantly upregulated in more severe COVID-19 infections. In conclusion, the immunopathology and severity of COVID-19 could result in transient autoimmune activation. Longitudinal follow-up studies of confirmed cases of COVID-19 could determine the enduring effects of viral infection including development of autoimmune disease.

Reduced neutrophil granular proteins and post-treatment modulation in tuberculous lymphadenitis.

BACKGROUND: Neutrophils are important for host innate immune defense and mediate inflammatory responses. Pulmonary tuberculosis (PTB) is associated with increased neutrophil granular protein (NGP) levels in the circulation. However, the systemic levels of neutrophil granular proteins were not examined in tuberculous lymphadenitis (TBL) disease. METHODS: We measured the systemic levels of NGP (myeloperoxidase [MPO], elastase and proteinase 3 [PRTN3]) in TBL and compared them to latent tuberculosis (LTB) and healthy control (HC) individuals. We also measured the pre-treatment (Pre-T) and post-treatment (Post-T) systemic levels of neutrophil granular proteins in TBL individuals upon anti-tuberculosis treatment (ATT) completion. In addition, we studied the correlation and discriminatory ability of NGPs using receiver operating characteristic (ROC) analysis. RESULTS: Our data suggests that systemic levels of NGPs (MPO, PRTN3, elastase) were significantly reduced in TBL individuals compared to LTB and HC individuals. Similarly, after ATT, the plasma levels of MPO and elastase but not PRTN3 were significantly elevated compared to pre-treatment levels. NGPs (except PRTN3) were positively correlated with absolute neutrophil count of TBL, LTB and HC individuals. Further, NGPs were able to significantly discriminate TBL from LTB and HC individuals. CONCLUSION: Hence, we conclude reduced neutrophil granular protein levels might be associated with disease pathogenesis in TBL.

Antibody subtypes and titers predict clinical outcomes in ANCA-associated vasculitis.

The objective of this study is to evaluate the association between antineutrophil cytoplasmic autoantibody (ANCA) subtype and ANCA titers on clinical outcomes and disease activity among a cohort of patients from Central Appalachia diagnosed with ANCA-associated vasculitis (AAV) over a 3-decade period. This is a retrospective chart review of all patients diagnosed with AAV. ANCA subtypes (myeloperoxidase (MPO) and proteinase 3 (PR3)) and titers at the time of diagnosis and at the time of relapse or last follow-up were evaluated along with patient outcomes. Outcomes of interest included relapse, development of end-stage renal disease (ESRD) and mortality. Sensitivity analysis and multivariable analysis were performed. Of the 202 patients, 111 patients were MPO-ANCA positive and 91 patients were PR3-ANCA positive. Relapse was more frequent among patients with PR3-ANCA compared to MPO-ANCA (35% vs 12%, p < 0.001). In both ANCA subgroups, the strongest predictor of relapse was an increase in titers prior to relapse, HR 8.1 (95% CI 1.6-40), p 0.009. Patients who achieved serological remission had a lower risk of ESRD [sub-HR 0.31 (95% CI 0.11-0.89)] and mortality [HR (95% CI) 0.24 (0.07-0.7)]. PR3-ANCA was associated with higher risk of ESRD [sub-HR 3.1 (95% CI 1.1-8.5)]. There was no difference in mortality between patients with MPO-ANCA and PR3-ANCA. Our study supports the use of both ANCA subtypes and titer levels for predicting clinical outcomes in patients receiving treatment for AAV. Monitoring of ANCA antibody titers may be useful since both serological remission and increase in titers provide prognostic information.

The Inflammatory Factors Associated with Disease Severity to Predict COVID-19 Progression.

Coronavirus disease 2019 (COVID-19) is associated with immune dysregulation and cytokine storm. Exploring the immune-inflammatory characteristics of COVID-19 patients is essential to reveal pathogenesis and predict progression. In this study, COVID-19 patients showed decreased CD3+, CD4+, and CD8+ T cells but increased neutrophils in circulation, exhibiting upregulated neutrophil-to-lymphocyte and neutrophil-to-CD8+ T cell ratio. IL-6, TNF-α, IL-1ß, IL-18, IL-12/IL-23p40, IL-10, Tim-3, IL-8, neutrophil extracellular trap-related proteinase 3, and S100A8/A9 were elevated, whereas IFN-γ and C-type lectin domain family 9 member A (clec9A) were decreased in COVID-19 patients compared with healthy controls. When compared with influenza patients, the expressions of TNF-α, IL-18, IL-12/IL-23p40, IL-8, S100A8/A9 and Tim-3 were significantly increased in critical COVID-19 patients, and carcinoembryonic Ag, IL-8, and S100A8/A9 could serve as clinically available hematologic indexes for identifying COVID-19 from influenza. Moreover, IL-6, IL-8, IL-1ß, TNF-α, proteinase 3, and S100A8/A9 were increased in bronchoalveolar lavage fluid of severe/critical patients compared with moderate patients, despite decreased CD4+ T cells, CD8+ T cells, B cells, and NK cells. Interestingly, bronchoalveolar IL-6, carcinoembryonic Ag, IL-8, S100A8/A9, and proteinase 3 were found to be predictive of COVID-19 severity and may serve as potential biomarkers for predicting COVID-19 progression and potential targets in therapeutic intervention of COVID-19.

The diagnostic role and clinical association of serum proteinase 3 anti-neutrophil cytoplasmic antibodies in Chinese patients with inflammatory bowel disease.

Background and aim: Accurate differentiation of patients with ulcerative colitis (UC) or Crohn's disease (CD) is important for appropriate therapy and prognosis. This study was designed to explore the utility of proteinase 3 anti-neutrophil cytoplasmic antibodies (PR3-ANCA) in the diagnosis of Chinese patients with inflammatory bowel disease (IBD).Methods: Blood samples were collected from 216 Chinese patients, including 175 IBD and 41 colorectal polyps (disease control). Clinical characteristics were extracted from electronic medical records.Results: Serum PR3-ANCA were increased in UC patients compared to those with CD or colorectal polyps (p < .0001). PR3-ANCA was negative in colorectal polyps and there was no significant difference between CD and colorectal polyps (p > .05). Using the cut-off value of 20 chemiluminescent units (CU) provided by manufacturer, the positive rate of PR3-ANCA was higher in UC than CD (41.7% vs. 1.1%; p < .0001). Receiver operating characteristic (ROC) analysis demonstrated an area under the curve (AUC) of 0.89 (95% CI: 0.84-0.95; p < .0001) for differentiating UC from CD and suggested an optimized cutoff of 7.3 CU which improved sensitivity from 41.7% to 57.1%, while maintaining a specificity of 98.9%. PR3-ANCA in severe UC patients were higher than those with moderate UC (p < .05), no difference was found between those in remission or with mild or moderate activity (p > .05).Conclusions: Serum PR3-ANCA is a potentially useful clinical biomarker in Chinese patients with IBD. A modified cut-off value of 7.3 CU improves the performance for distinguishing UC from CD.

Novel Insights Into the Effects of Interleukin 6 Antagonism in Non-ST-Segment-Elevation Myocardial Infarction Employing the SOMAscan Proteomics Platform.

Background Interleukin 6 concentration is associated with myocardial injury, heart failure, and mortality after myocardial infarction. In the Norwegian tocilizumab non-ST-segment-elevation myocardial infarction trial, the first randomized trial of interleukin 6 blockade in myocardial infarction, concentration of both C-reactive protein and troponin T were reduced in the active treatment arm. In this follow-up study, an aptamer-based proteomic approach was employed to discover additional plasma proteins modulated by tocilizumab treatment to gain novel insights into the effects of this therapeutic approach. Methods and Results Plasma from percutaneous coronary intervention-treated patients, 24 in the active intervention and 24 in the placebo-control arm, drawn 48 hours postrandomization were randomly selected for analysis with the SOMAscan assay. Employing slow off-rate aptamers, the relative abundance of 1074 circulating proteins was measured. Proteins identified as being significantly different between groups were subsequently measured by enzyme immunoassay in the whole trial cohort (117 patients) at all time points (days 1-3 [7 time points] and 3 and 6 months). Five proteins identified by the SOMAscan assay, and subsequently confirmed by enzyme immunoassay, were significantly altered by tocilizumab administration. The acute-phase proteins lipopolysaccharide-binding protein, hepcidin, and insulin-like growth factor-binding protein 4 were all reduced during the hospitalization phase, as was the monocyte chemoattractant C-C motif chemokine ligand 23. Proteinase 3, released primarily from neutrophils, was significantly elevated. Conclusions Employing the SOMAscan aptamer-based proteomics platform, 5 proteins were newly identified that are modulated by interleukin 6 antagonism and may mediate the therapeutic effects of tocilizumab in non-ST-segment-elevation myocardial infarction.

PR3 levels are impaired in plasma and PBMCs from Arabs with cardiovascular diseases.

Cardiovascular disease (CVD) risks persist in patients despite treatment. CVD susceptibility also varies with sex and ethnicity and is not entirely explained by conventional CVD risk factors. The aim of the present study was to identify novel CVD candidate markers in circulating Peripheral blood mononuclear cells (PBMCs) and plasma from Arab obese subjects with and without CVD using proteomic approaches. Human adults with confirmed CVD (n = 208) and matched non-CVD controls (n = 152) living in Kuwait were examined in the present cross-sectional study. Anthropometric and classical biochemical parameters were determined. We employed a shotgun proteomic profiling approach on PBMCs isolated from a subset of the groups (n = 4, each), and differentially expressed proteins selected between the two groups were validated at the mRNA level using RT-PCR (n = 6, each). Plasma levels of selected proteins from the proteomics profiling: Proteinase-3 (PR3), Annexin-A3 (ANX3), Defensin (DEFA1), and Matrix Metalloproteinase-9 (MMP9), were measured in the entire cohort using human enzyme-linked immunosorbent assay kits and were subsequently correlated with various clinical parameters. Out of the 1407 we identified and quantified from the proteomics profiling, 47 proteins were dysregulated with at least twofold change between the two subject groups. Among the differentially expressed proteins, 11 were confirmed at the mRNA levels. CVD influenced the levels of the shortlisted proteins (MMP9, PR3, ANX3, and DEFA1) in the PBMCs and plasma differentially. Despite the decreased levels of both protein and mRNA in PBMCs, PR3 circulating levels increased significantly in patients with CVD and were influenced by neither diabetes nor statin treatment. No significant changes were; however, observed in the DEFA1, MMP9, and ANX3 levels in plasma. Multivariate logistic regression analysis revealed that only PR3 was independently associated with CVD. Our results suggest that the dysregulation of PR3 levels in plasma and PBMCs reflects underlying residual CVD risks even in the treated population. More prospective and larger studies are required to establish the role of PR3 in CVD progression.

Proteinase 3-antineutrophil cytoplasmic antibody-positive necrotizing crescentic glomerulonephritis complicated by infectious endocarditis: a case report.

BACKGROUND: Proteinase 3-antineutrophil cytoplasmic antibody has been reported to be positive in 5-10% of cases of renal injury complicated by infective endocarditis; however, histological findings have rarely been reported for these cases. CASE PRESENTATION: A 71-year-old Japanese man with a history of aortic valve replacement developed rapidly progressive renal dysfunction with gross hematuria and proteinuria. Blood analysis showed a high proteinase 3-antineutrophil cytoplasmic antibody (163 IU/ml) titer. Streptococcus species was detected from two separate blood culture bottles. Transesophageal echocardiography detected mitral valve vegetation. Histological evaluation of renal biopsy specimens showed necrosis and cellular crescents in glomeruli without immune complex deposition. The patient met the modified Duke criteria for definitive infective endocarditis. On the basis of these findings, the patient was diagnosed with proteinase 3-antineutrophil cytoplasmic antibody-positive necrotizing crescentic glomerulonephritis complicated by Streptococcus infective endocarditis. His renal disease improved, and his proteinase 3-antineutrophil cytoplasmic antibody titer normalized with antibiotic monotherapy. CONCLUSION: Few case reports have described histological findings of proteinase 3-antineutrophil cytoplasmic antibody-positive renal injury complicated with infective endocarditis. We believe that an accumulation of histological findings and treatments is mandatory for establishment of optimal management for proteinase 3-antineutrophil cytoplasmic antibody-positive renal injury complicated with infective endocarditis.

Granulomatosis polyangiitis.

A 22-year-old female patient was admitted to hospital after being referred from the oral medicine clinic where she had been seen for persistent gingivitis and mouth ulcers. She described an insidious history of persistent fevers, dry cough and unexplained weight loss over 4-6 weeks. Imaging showed extensive bilateral pulmonary nodules with mediastinal lymphadenopathy and two lesions in the pancreas. MRI revealed these lesions to be well-defined fluid-filled cysts in the tail of the pancreas, without features of malignancy. Core biopsies taken from her lung nodules demonstrated features of vasculitis with granulomata. This was consistent with her positive immunology for c-antinuclear cytoplasmic antibodies and proteinase-3, which were sent after her fever failed to settle with antibiotic treatment. In keeping with a diagnosis of vasculitis, the patient showed a significant clinical and biochemical response to intravenous methylprednisolone and high-dose daily prednisolone thereafter.

Prevalence and Clinical Significance of Antineutrophil Cytoplasmic Antibodies in North American Patients With Idiopathic Pulmonary Fibrosis.

BACKGROUND: Antineutrophil cytoplasmic antibodies (ANCAs) have been reported to occur in 7% to 10% of patients with idiopathic pulmonary fibrosis (IPF), but their clinical relevance remains unclear. The aim of this study was to estimate the prevalence of ANCAs in a North American population with IPF and evaluate their clinical significance. METHODS: This was a retrospective study of two independent cohorts of patients diagnosed with IPF at the University of California San Francisco (discovery cohort) and the University of Chicago (replication cohort). Myeloperoxidase (MPO) and proteinase 3 (PR3) ANCAs were measured in all patients. Prevalence and associations of ANCAs with clinical characteristics and transplant-free survival were evaluated. RESULTS: A total of 14 of 353 (4.0%; 95% CI, 2.2-6.5) and 20 of 392 (5.1%; 95% CI, 3.1-7.8) patients with IPF were positive for ANCAs at the time of diagnosis in the discovery and replication cohorts, respectively. Among those positive for MPO antibodies, two of six (33%) in the discovery cohort and three of 12 (25%) in the replication cohort developed vasculitis. None of the patients who were PR3-positive developed vasculitis. Patients who were ANCA-positive were more likely to be women than patients who were ANCA-negative, and were more likely to have some ground-glass opacities on CT scan. In the combined cohort of 745 patients, median transplant-free survival was not significantly different in patients who were ANCA-positive vs ANCA-negative (P = .57). CONCLUSIONS: ANCA positivity is uncommon in North American patients with IPF and not associated with baseline disease severity or transplant-free survival; however, a significant proportion of patients who are MPO-positive with IPF develop clinical vasculitis.

Increased proteinase 3 and neutrophil elastase plasma concentrations are associated with non-alcoholic fatty liver disease (NAFLD) and type 2 diabetes.

INTRODUCTION: Non-alcoholic fatty liver disease (NAFLD) is becoming a major health problem worldwide. Inflammation plays an important role in disease pathogenesis and recent studies have shown a potential role for the neutrophil serine proteases (NSPs) proteinase-3 (PR3) and neutrophil elastase (NE) in NAFLD as well as an imbalance between NSPs and their natural inhibitor alpha-1 antitrypsin (AAT). The aim of this study was to investigate whether PR3 and NE plasma concentrations are associated with NAFLD and/or type 2 diabetes. METHODS: To explore this hypothesis we used several cohorts: a cohort of 271 obese individuals with liver steatosis, a cohort of 41 patients with biopsy-proven NAFLD, a cohort of 401 obese type 2 diabetes patients and a cohort of 205 lean healthy controls; and measured PR3 and NE plasma concentrations. In addition, we measured AAT plasma concentrations in order to investigate if the ratios between NSPs and their natural inhibitor were altered in NAFLD and type 2 diabetes when compared to healthy controls. RESULTS: Our data shows an increase in PR3 and NE concentrations and a decrease in AAT concentrations in obese patients when compared to controls. Moreover, PR3 plasma concentrations are increased in patients with liver steatosis. Furthermore, PR3 and NE concentrations in the liver are associated with the advanced stages of NAFLD characterized by NASH and/ or liver fibrosis. Additionally, PR3 and NE concentrations were up-regulated in patients with type 2 diabetes when compared to lean and obese controls. CONCLUSION: We conclude that circulating levels of NSPs associate with obesity-related metabolic disorders. Further research is needed to clearly establish the role of these proteases and investigate whether they could be used as non-invasive markers for NAFLD and/or type 2 diabetes.

PR3 vasculitis presenting with symptomatic splenic and renal infarction: a case report and literature review.

BACKGROUND: ANCA-associated vasculitis is a life-threatening, systemic autoimmune disease. There is an increased risk of organ infarction but in many cases this is asymptomatic. We described here the first reported case of PR3 vasculitis presenting with symptomatic bilateral renal wedge infarction. CASE PRESENTATION: A 19-year old Caucasian woman with no past medical history presented on a number of occasions over a number of weeks with progressively more severe back pain, fevers and arthralgia. On the final presentation she was noted to have developed splinter haemorrhages and her blood tests revealed impaired renal function along with elevated inflammatory markers. She was subsequently found to have high titres of serum PR3 antibodies and focal necrotising glomerulonephritis on renal biopsy, consistent with a diagnosis of PR3 ANCA-associated vasculitis. Cross-sectional imaging revealed multiple wedge infarcts of her spleen and both kidneys, confirmed on contrast-enhanced ultrasound. Large vessel, cardiac and thrombophilic causes of thromboembolism were excluded. She was treated with high-dose corticosteroids and CD20 monoclonal antibodies (rituximab) and at time of writing, 4 months after initial presentation, has entered clinical remission. CONCLUSIONS: Here we describe the first reported case of PR3 vasculitis presenting with symptomatic renal wedge infarction. In patients with vasculitis who present with flank or back pain, infarction of abdominal organs should be considered in the differential. Both splenic and renal infarctions are likely underdiagnosed in the setting of ANCA-associated vasculitis but may have clinical impact in contributing to infection risk and the degree or renal recovery, respectively.

Evaluation of PR3-ANCA Status After Rituximab for ANCA-Associated Vasculitis.

INTRODUCTION: The value of antineutrophil cytoplasmic antibody (ANCA) measurements among patients with an established diagnosis of ANCA-associated vasculitis (AAV) to assess disease activity or predict relapse remains controversial, but recent evidence suggests a possible role for rituximab-treated patients. PATIENTS AND METHODS: All patients with active vasculitis and positive proteinase 3 (PR3)-ANCA who were starting a 2-year treatment course of rituximab for induction of remission at Addenbrooke's Hospital between January 2011 and January 2016 were included in this study. Common department practice consists of 6 g of rituximab given over 2 years, concomitant corticosteroids (0.5-1.0 mg/kg) with rapid taper over 3 months, and cessation of oral maintenance immunosuppressive agents at time of first rituximab dose. Clinical and laboratory data were collected retrospectively using electronic patient records. RESULTS: Fifty-seven patients with current PR3-ANCA positivity were included in the analysis. Median follow-up was 59 months. PR3-ANCA negativity was achieved in 25 patients (44%) with a median time of 14 months. Clinical remission was achieved in 53 patients (93%) with a median time of 3 months. Among the 53 patients who achieved remission during follow-up, 24 (45%) relapsed with a median time to relapse of 36 months from remission. Both PR3-ANCA-negative status and 50% reduction in PR3-ANCA from baseline (as time-varying covariates) were significantly associated with a longer time to relapse (PR3-ANCA-negative status: hazards ratio, 0.08 [95% confidence interval, 0.01-0.63, p = 0.016]; 50% reduction in PR3-ANCA: hazards ratio, 0.25 [95% confidence interval, 0.18-0.99, p = 0.046]). CONCLUSIONS: Achieving and maintaining PR3-ANCA negativity after rituximab was associated with longer-lasting remission.

Serum levels of interleukin-32 and interleukin-6 in granulomatosis with polyangiitis and microscopic polyangiitis: association with clinical and biochemical findings.

BACKGROUND: Antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis is an autoimmune disorder of unknown etiology with dysregulated cytokines levels. OBJECTIVES: The main aim of this study was to assess the clinical correlation between antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis, granulomatosis with polyangiitis (GPA) serum levels of the microscopic polyangiitis (MPA), serum levels of the proinflammatory cytokines, interleukin (IL)-32 and interleukin-6. METHODS: Study included 71 patients, 47 with GPA and 24 with MPA. Serum IL-32 and IL-6 concentrations were analyzed in all patients, and compared with levels observed in 10 controls. IL-32 and IL-6 were evaluated using DuoSet and Quantikine HS ELISA, respectively. IL-32 and IL-6 concentrations were correlated with disease-related clinical and laboratory findings. RESULTS: IL-32 and IL-6 levels were significantly higher in GPA and MPA than in controls, especially IL-32 levels in GPA were elevated. IL-32 concentrations correlated positively with anti-proteinase 3 - ANCA (PR3-ANCA) levels in GPA (P < 0.0001), and with anti-myeloperoxidase ANCA (MPO-ANCA) in MPA (P = 0.049). IL-32 levels correlated positively with disease activity in GPA and MPA (P < 0.0001). GPA patients with pulmonary, cutaneous, and musculoskeletal involvement presented the highest IL-6 serum levels. Cutaneous manifestations correlated positively with IL-6 levels in MPA patients (P = 0.05). ANCA-positive patients with GPA expressed significantly high IL-6 levels (P = 0.036). No significant difference in IL-32 values was observed between ANCA-positive and ANCA-negative patients. CONCLUSIONS: Patients with GPA and MPA present higher serum IL-32 and IL-6 levels than controls. IL-32 levels correlate positively with disease activity.

PR3-ANCA and panel diagnostics in pediatric inflammatory bowel disease to distinguish ulcerative colitis from Crohn's disease.

BACKGROUND: Accurate classification of patients with inflammatory bowel disease into the subtypes ulcerative colitis (UC) and Crohn's disease (CD) is still a challenge, but important for therapy and prognosis. OBJECTIVES: To evaluate the diagnostic utility of anti-neutrophil cytoplasmic antibodies specific for proteinase-3 (PR3-ANCA) for ulcerative colitis (UC) and the value of an antibody panel incorporating PR3-ANCA to differentiate between Crohn's disease (CD) and UC. STUDY DESIGN: In this cohort study, 122 pediatric and adolescent individuals were retrospectively included (61 IBD patients of two clinical centers, 61 non-IBD controls). All subjects had a comprehensive antibody profile done from stored sera taken close to time of diagnosis. By employing quasi-exhaustive logistic regression the best discriminative model for UC and CD,subjects was determined in a training cohort and confirmed in a validation cohort. RESULTS: PR3-ANCA was specifically associated with UC (odds ratio (OR), 17.6; 95% confidence interval (CI); 3.6, 87); P < .001). A four antibody-panel including PR3-ANCA had an AUC of 90.81% (95%CI; 81.93, 99.69) to distinguish between UC and CD in the training cohort. In a smaller external validation cohort, the AUC was 84.13% (95%CI; 64.21, 100) for accurate diagnosis of CD and UC. CONCLUSION: PR3-ANCA is highly specific for UC. The differentiating capability of a panel, which contains PR3-ANCA and weighs broadly available antibodies, is superior and utilization of the panel can support accurate classification in the work-up of pediatric and adolescent patients with IBD patients.

Local enrichment of fatty acid-binding protein 4 in the pericardial cavity of cardiovascular disease patients.

BACKGROUND: The pericardial fluid may be representative of the interstitium of the heart. The aim of this study was to discriminate in cardiovascular disease patients between adipocytokines that are produced locally by the heart and those supplied by the circulation. METHODS: Enzyme-linked immunosorbent assays (ELISA) were used to determine levels of N-terminal pro-brain natriuretic peptide (NT-pBNP), fatty acid-binding protein 4 (FABP4), leptin, lipocalin-2, neutrophil elastase, proteinase-3, high sensitivity C-reactive protein (hsCRP) and adiponectin in venous plasma and pericardial fluid harvested during elective cardio-thoracic surgery (n = 132-152). RESULTS: In pericardial fluid compared to plasma, the levels were significantly smaller (p < 0.001) for leptin, lipocalin-2, neutrophil elastase, proteinase-3, hsCRP and adiponectin. For these biomarkers, the ratio of pericardial fluid-to-plasma level ([PF]/[P], median (interquartile range)) was 0.65 (0.47-1.01), 0.78 (0.56-1.09), 0.23 (0.11-0.60), 0.17 (0.09-0.36), 0.14 (0.08-0.35), and 0.25 (0.15-0.34), respectively. In contrast, pericardial fluid was significantly enriched (p < 0.001) in NT-pBNP ([PF]/[P]: 1.9 (1.06-2.73)) and even more so for FABP4 ([PF]/[P]: 3.90 (1.47-9.77)). Moreover, in pericardial fluid, the adipocytokines interrelated all significantly positive and correlated negative to hsCRP, whereas for NT-pBNP only a significantly positive correlation with adiponectin was found. These interrelations were distinct from those in the plasma, as were the correlations of the pericardial biomarkers with patient characteristics compared to plasma. CONCLUSIONS: In cardiovascular disease patients, the pericardial cavity is a distinct adipocytokine microenvironment in which especially FABP4 is mainly derived from the heart.