Salivary Proteinase 3 as a Biomarker for Caries Severity in Children: A Cross-sectional Study.

AIM: This study aims to evaluate the relation between salivary proteinase 3 (PR3) concentration and caries severity in children. MATERIALS AND METHODS: Six-to-eight-year age group children, from the Outpatient Department of Pediatric and Preventive dentistry at PMS Dental College were selected for the study. From these children, three groups each consisting of 28 children were selected according to the dental caries severity. Three groups were: (1) No Dental Caries group, (2) Low Dental Caries group with DMFT/DEFT score of 1-4, and (3) High Dental Caries group with DMFT/DEFT score of 5-15. Thus, a total of 84 children who satisfied the inclusion criteria were selected. The concentration of PR3 in saliva of the donors were analyzed using an ELISA kit. One way ANOVA was used for finding the relation of salivary PR3 concentration with caries severity. Pairwise comparison of PR3 concentration and caries severity were analyzed using post hoc Tukey test. RESULTS: Severity of caries and concentration of salivary PR3 showed an inverse relation. As the caries severity increases there was a decrease in PR3 concentration and vice versa. CONCLUSION: The children with high caries severity showed lower concentration of PR3 in their saliva compared with those with lower caries severity which indicates that PR3 can be used as a biomarker for assessing caries severity and also paves way to use PR3 as a caries vaccine in future. Nowadays, interest toward noninvasive and personalized dentistry has been increased. Molecular assays using salivary biomarkers can be an effective tool in detecting the caries in earlier stages and assessing a patient's caries risk. CLINICAL SIGNIFICANCE: Salivary PR3 can be used as prognostic biomarker for assessing caries severity and after treatment the value of PR3 can be used as a assessment tool to confirm its relation with caries. How to cite this article: Karthika S, George S, Soman A, et al. Salivary Proteinase 3 as a Biomarker for Caries Severity in Children: A Cross-sectional Study. J Contemp Dent Pract 2024;25(3):236-240.

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.

Co-occurrence of PLA2R-positive membranous nephropathy without crescents, and PR3-positive eosinophilic granulomatosis with polyangiitis
.

Primary membranous nephropathy (PMN) is a common cause of adult nephrotic syndrome, most commonly associated with autoantibodies against M-type phospholipase A2 receptor (PLA2R). Eosinophilic granulomatosis with polyangiitis (EGPA), previously known as Churg-Strauss syndrome, is a rare disorder characterized by asthma, eosinophilia, and multiorgan vasculitis. Here, we report the case of an adult who presented with typical nephrotic syndrome. Renal biopsy revealed PLA2R-positive PMN without crescents. He had a history of asthma, eczema, and eosinophilia, and testing revealed positive serological proteinase 3 (PR3) and antineutrophil cytoplasmic antibody (ANCA). Further skin and bone marrow biopsy revealed histologic eosinophilic infiltration, and a diagnosis of EGPA was made. The renal biopsy revealed a few eosinophils in glomerular capillary lumen and tubulointerstitial. Treatment with a glucocorticoid and cyclophosphamide was initiated. At 32 months after completing therapy, the patient was in complete clinical remission, and the PR3-ANCA result was negative.

In situ detection of PR3-ANCA+ B cells and alterations in the variable region of immunoglobulin genes support a role of inflamed tissue in the emergence of auto-reactivity in granulomatosis with polyangiitis.

Circulating anti-neutrophilic cytoplasmic autoantibodies targeting proteinase 3 (PR3-ANCA) are a diagnostic and pathogenic hallmark of granulomatosis with polyangiitis (GPA). It is, however, incompletely understood if inflamed tissue supports presence or emergence of PR3-ANCA+ B cells. In search of such cells in inflamed tissue of GPA, immunofluorescence staining for IgG and a common PR3-ANCA idiotype (5/7 Id) was undertaken. Few 5/7 Id+/IgG+ B cells were detected in respiratory and kidney tissue of GPA. To gain more insight into surrogate markers possibly indicative of an anti-PR3-response, a meta-analysis comprising IGVH and IGVL genes derived from respiratory tract tissue of GPA (231 clones) was performed. Next generation sequencing-based IGHV genes derived from peripheral blood of healthy donors (244.353 clones) and previously published IGLV genes (148 clones) served as controls. Additionally, Ig genes of three murine and five known human monoclonal anti-PR3 antibodies were analyzed. Primary and probably secondary rearrangements led to altered VDJ usage and an extended complementarity determining region 3 (CDR3) of IGHV clones from GPA tissue. Selection against amino acid exchanges was prominent in the framework region of IGHV clones from GPA tissue. The comparison of V(D)J rearrangements and deduced amino acid sequences of the CDR3 yielded no identities and few similarities between clones derived from respiratory tissue of GPA and anti-PR3 antibodies, arguing against a presence of B cells that carry PR3-ANCA-prone Ig genes among the clones. In line with the scarcity of 5/7 Id+ B lymphocytes in GPA tissue, the results suggest that with respect to a local anti-PR3 response, methods detecting rare clones are required.

Design, Synthesis, and Enzymatic Evaluation of Novel ZnO Quantum Dot-Based Assay for Detection of Proteinase 3 Activity.

Herein, the synthesis and application of functionalized quantum dot-based protease probes is described. Such probes are composed of nontoxic ZnO nanocrystals decorated by amino groups followed by linker and labeled peptide attachment. Spherical NH2-terminated ZnO quantum dots (QDs) with the average size ranging from 4 to 8 nm and strong emission centered at 530 nm were prepared using the sol-gel method. The fluorescence of ZnO QDs was quenched by the BHQ1 moiety present on the N-terminal amino group of the peptide. The enzymatic cleavage of the peptide mediated by the proteinase 3 (PR3) bond resulted in an increase in the QD probe fluorescence. This observation was verified using both model and biological systems; and the picomolar detection limit was found to be more than 30 times lower than that of the previously reported internally quenched peptide (a decrease in detection limit from 43 to 1.3 pmol was observed).

Testing and reporting antineutrophil cytoplasmic antibodies (ANCA) in treated vasculitis and non-vasculitic disease.

Testing for antineutrophil cytoplasmic antibodies (ANCA) is performed to diagnose or exclude small vessel vasculitis, and, in treated patients, to monitor disease activity. However testing is also undertaken to assist with the diagnosis of other autoimmune diseases and some infections. Most laboratories use the same assays for all sera regardless of the testing indications. The International Consensus Statement on ANCA Testing and Reporting recommended screening for ANCA by indirect immunofluorescence (IIF) and confirming IIF-positive sera in antigen-specific ELISAs for both proteinase 3 (PR3) and myeloperoxidase (MPO). These guidelines have been reviewed after many refinements of the assays, and the development of new testing methodologies. However the advances have focused largely on improving the diagnostic accuracy in new-onset vasculitis, and not on more accurately monitoring disease activity, nor increasing the diagnostic sensitivity for non-vasculitic conditions. The recently-revised guidelines for ANCA testing indicate that where new onset vasculitis is suspected, sera should be examined for both PR3- and MPO-ANCA using any highly sensitive and specific assay, rather than IIF. They further state that where sera are negative in one assay but the suspicion of vasculitis is high, that testing should be repeated using a different assay. The guidelines do not provide recommendations for treated vasculitis or non-vasculitic disease. However for a routine diagnostic laboratory where sera are tested for many different indications, or where the reasons are not known, IIF screening followed by confirmation of IIF-positive sera in antigen-specific assays remains a highly sensitive, specific and convenient method for detecting ANCA in "all-comers".

The Epidemiology of Antineutrophil Cytoplasmic Autoantibody-Associated Vasculitis in Olmsted County, Minnesota: A Twenty-Year US Population-Based Study.

OBJECTIVE: To estimate the annual incidence, prevalence, and mortality of antineutrophil cytoplasmic autoantibody (ANCA)-associated vasculitis (AAV) and its subsets, granulomatosis with polyangiitis (Wegener's) (GPA), microscopic polyangiitis (MPA), and eosinophilic granulomatosis with polyangiitis (Churg-Strauss) (EGPA), in a US-based adult population. METHODS: All medical records of patients with a diagnosis of, or suspicion of having, AAV in Olmsted County, Minnesota from January 1, 1996 to December 31, 2015 were reviewed. AAV incidence rates were age- and sex-adjusted to the 2010 US white population. Age- and sex-adjusted prevalence of AAV was calculated on January 1, 2015. Survival rates observed in the study cohort were compared with expected rates in the Minnesota population. RESULTS: Of the 58 incident cases of AAV in Olmsted County during the study period, 23 (40%) were cases of GPA, 28 (48%) were cases of MPA, and 7 (12%) were cases of EGPA. Overall, 28 (48%) of the patients with AAV were women and 57 (98%) were white. The mean ± SD age at diagnosis was 61.1 ± 16.5 years. Thirty-four patients (61%) had myeloperoxidase (MPO)-ANCAs, and 17 (30%) were positive for proteinase 3 (PR3)-ANCAs; 5 (9%) were ANCA-negative. The annual incidence of AAV was 3.3 per 100,000 population (95% confidence interval [95% CI] 2.4-4.1). The incidence rates of GPA, MPA, and EGPA were 1.3 (95% CI 0.8-1.8), 1.6 (95% CI 1.0-2.2), and 0.4 (95% CI 0.1-0.6), respectively. The overall prevalence of AAV was 42.1 per 100,000 (95% CI 29.6-54.6). The mortality rate among AAV patients overall, and among patients with EGPA, those with MPA, and those with MPO-ANCAs, was increased in comparison to the Minnesota general population (each P < 0.05), whereas mortality rates among patients with GPA, those with PR3-ANCAs, and ANCA-negative patients did not differ from that in the general population. CONCLUSION: The annual incidence of AAV in Olmsted County, Minnesota over the 20 years of the study was 3.3 per 100,000, with a prevalence of 42.1 per 100,000, which is substantially higher than the rates reported in other areas worldwide. The incidence of GPA was similar to that of MPA. Patients with MPA and those with EGPA, but not patients with GPA, experienced higher rates of mortality than that in the Minnesota general population. MPO-ANCAs were a marker of poor survival in this population of patients with AAV.

Comparison of enzyme-linked immunosorbent assay and rapid chemiluminescent analyser in the detection of myeloperoxidase and proteinase 3 autoantibodies.

Antibodies to myeloperoxidase (MPO) and proteinase 3 (PR3) are vital in the diagnosis and management of ANCA-associated vasculitis. A chemiluminescent immunoassay (CLIA; Quanta Flash) provides MPO and PR3 antibody results in 30 minutes, which is much faster than enzyme-linked immunosorbent assay (ELISA). We compared the performance of ELISA (Orgentec) and CLIA (Quanta Flash) for MPO and PR3 antibody quantitation on 303 samples, comprising 196 consecutive samples received in a single diagnostic laboratory over a 3 month period, and 107 samples collected from 42 known vasculitis patients over a 40 month period. We observed a correlation between both methods using spearman correlation coefficients (MPO, rs = 0.63, p < 0.01; PR3, rs = 0.69, p < 0.01). There was agreement between both methods in determining a positive or negative result. In the vasculitis cohort, CLIA performed well at clinically important stages of disease; diagnosis (eight samples all positive by both assays) and disease relapse (correlation for both MPO and PR3 antibody quantitation rs = 0.84, p = 0.03 and rs = 0.78, p < 0.01, respectively). Three samples were discordant at clinical relapse, testing positive by CLIA, including one high positive associated with relapse requiring a change in treatment. In summary, CLIA appears to be at least as accurate as ELISA for measurement of MPO and PR3 antibodies.

Gingival crevicular fluid levels of protease-activated receptors type 1 and type 2 in diabetic patients with periodontitis.

BACKGROUND AND OBJECTIVE: Protease activated receptor type 1 (PAR1 ) seems to play a role in periodontal repair, while PAR2 is associated with periodontal inflammation. As diabetes is a known risk factor for periodontal disease, the aim of this study was to evaluate the influence of type 2 diabetes on PAR1 and PAR2 mRNA expression in the gingival crevicular fluid of patients with chronic periodontitis before and after non-surgical periodontal treatment. MATERIAL AND METHODS: Gingival crevicular fluid samples and clinical parameters consisting of measuring probing depth, clinical attachment level, bleeding on probing and plaque index were collected from systemically healthy patients and patients with type 2 diabetes and chronic periodontitis, at baseline and after non-surgical periodontal therapy. PAR1 and PAR2 , as well as the presence of the proteases RgpB gingipain and neutrophil proteinase-3 were assessed by quantitative polymerase chain reaction in the gingival crevicular fluid. RESULTS: The periodontal clinical parameters significantly improved after periodontal therapy (p < 0.01). Diabetes led to increased expression of PAR1 in gingival crevicular fluid, and in the presence of chronic periodontitis, it significantly decreased the expression of PAR1 and PAR2 (p < 0.05). Moreover, non-surgical periodontal treatment in diabetics resulted in increased expression of PAR1 and PAR2 (p < 0.05), and decreased expression of RgpB gingipain and proteinase-3 (p < 0.05). CONCLUSION: The present data demonstrated that diabetes was associated with an altered expression of PAR1 and PAR2 in the gingival crevicular fluid cells of subjects with chronic periodontitis. Future studies are necessary to elucidate the effects of PAR1 upregulation in periodontally healthy sites and PAR2 downregulation in chronic periodontitis sites on the increased susceptibility and severity of periodontitis in diabetes.

Oral microbiota and host innate immune response in bisphosphonate-related osteonecrosis of the jaw.

Bacterial biofilms have emerged as potential critical triggers in the pathogenesis of bisphosphonate (BP)-related osteonecrosis of the jaw (ONJ) or BRONJ. BRONJ lesions have shown to be heavily colonized by oral bacteria, most of these difficult to cultivate and presents many clinical challenges. The purpose of this study was to characterize the bacterial diversity in BRONJ lesions and to determine host immune response. We examined tissue specimens from three cohorts (n=30); patients with periodontal disease without a history of BP therapy (Control, n=10), patients with periodontal disease having history of BP therapy but without ONJ (BP, n=5) and patients with BRONJ (BRONJ, n=15). Denaturing gradient gel electrophoresis of polymerase chain reaction (PCR)-amplified 16S rRNA gene fragments revealed less bacterial diversity in BRONJ than BP and Control cohorts. Sequence analysis detected six phyla with predominant affiliation to Firmicutes in BRONJ (71.6%), BP (70.3%) and Control (59.1%). Significant differences (P<0.05) in genera were observed, between Control/BP, Control/BRONJ and BP/BRONJ cohorts. Enzyme-linked immunosorbent assay (ELISA) results indicated that the levels of myeloperoxidase were significantly lower, whereas interleukin-6 and tumor necrosis factor-alpha levels were moderately elevated in BRONJ patients as compared to Controls. PCR array showed significant changes in BRONJ patients with downregulation of host genes, such as nucleotide-binding oligomerization domain containing protein 2, and cathepsin G, the key modulators for antibacterial response and upregulation of secretory leukocyte protease inhibitor, proteinase 3 and conserved helix-loop-helix ubiquitous kinase. The results suggest that colonization of unique bacterial communities coupled with deficient innate immune response is likely to impact the pathogenesis of ONJ.

Renal survival in proteinase 3 and myeloperoxidase ANCA-associated systemic vasculitis.

BACKGROUND AND OBJECTIVES: This study evaluated predictors for patient and renal survival in patients with ANCA-associated vasculitis (AAV) with and without renal involvement. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: There were 273 consecutive AAV patients from January 1990 until December 2007 who were followed until death, loss to follow-up, or December 2010. Based on organ involvement, patients were divided into renal (n=212) and nonrenal groups (n=61). The primary end point was ESRD requiring renal replacement therapy (RRT) or renal transplantation or death. RESULTS: Patient survival was significantly better in the nonrenal group compared with the renal group (hazard ratio, 0.55; 95% confidence interval, 0.33 to 0.92; P=0.02). In the renal group, renal survival was significantly worse in MPO-ANCA-positive patients (n=65) compared with PR3-ANCA-positive patients (n=138) (hazard ratio, 2.1; 95% confidence interval, 1.11 to 3.8; P=0.01). Of 48 patients who needed RRT at diagnosis, 11 patients (23%) died within 6 months and 14 patients (29%) did not regain renal function. Of all 23 patients who regained renal function after RRT, 7 patients (30%) were temporarily dialysis independent and needed dialysis later (range, 13-63 months). Five patients had a renal relapse in the 6 months before restart of RRT. Of all 203 PR3-ANCA-positive and MPO-ANCA-positive patients with renal involvement, 12 patients (6%) developed ESRD during follow-up. These patients were classified as CKD stage 4 or 5 after initial treatment and eight patients had a renal relapse before becoming dialysis dependent. CONCLUSIONS: AAV patients with renal involvement who needed RRT had the worst survival probability. In multivariate analysis, the only major determinants for long-term renal survival were renal function at 6 months and renal relapses.

Comparison of PR3-ANCA specific assay performance for the diagnosis of granulomatosis with polyangiitis (Wegener's).

BACKGROUND: PR3-ANCA, the serological marker of granulomatosis with polyangiitis (GPA), is usually detected by immunometric assays, with purified PR3 directly coated onto the solid-phase. Novel methods for PR3-ANCA detection have been developed to improve the performance of traditional PR3-ANCA specific assays, but little is known about their diagnostic performance in real-life clinical settings. This study aimed to compare the performance of nine different commercial PR3-ANCA specific assays, including traditional and newer ones, for the diagnosis of GPA. METHODS: The evaluated assays for PR3-ANCA detection were representative of the first, second, and third generation tests (direct, capture and anchor assays, respectively). A third-generation assay employing both human and recombinant PR3 was also evaluated. The study population consisted of 55 GPA patients, 175 disease controls (representing most diseases in differential diagnosis with primary small-vessel vasculitis) including 52 with microscopic polyangiitis, and 20 healthy subjects. We performed the primary evaluation of test sensitivity using cut-off points which provided adequate and identical specificity for each test. RESULTS: Although specificity and area under the ROC curve did not differ significantly between the different assays, substantial differences in sensitivity at 98%-specificity were found in some instances (p<0.001). Compared to first generation direct PR3-ANCA specific assays, some of the second and third generation tests increased the positive predictive value (PPV) for GPA diagnosis. CONCLUSIONS: Some of the newer PR3-ANCA specific assays have better PPV than traditional ones.

Coronary artery aneurysms in Wegener's granulomatosis.

Atherosclerosis is the main cause of coronary artery aneurysm, however they can be observed in connective tissue diseases such as systemic lupus erythematosus and vasculitis. Kawasaki's disease and polyarteritis nodosa (PAN) are the systemic vasculitis that more often present coronary artery aneurysms. There are descriptions in the literature that small vessel vasculitis such as microscopic polyangiitis and PAN could develop coronary artery aneurysm, which are infrequent in other ANCA-associated vasculitis. Here, we report the case of a 25-year-old man who developed an extensive anterior myocardial infarct. The coronary angiogram showed coronary artery aneurysms, on laboratory ANCA C positivity with elevated levels of anti-proteinase 3 antibodies were present. He was treated with high doses of corticosteroids and cyclophosphamide with resolution of the aneurysms.

A case of cerebral aquaporinopathy.

A 35-year-old woman was hospitalized due to impaired consciousness. Magnetic resonance imaging (MRI) revealed multiple parenchymal lesions in supra and infratentorial brain regions, which were considered responsible for her declining consciousness level. She was treated with intravenous methylprednisolone. Neurological symptoms improved and she was discharged. She was readmitted 14 months later due to intractable hiccups. A follow-up brain MRI revealed an abnormal signal near the area postrema in the dorsal medulla. Serum aquaporin-4 antibody levels were positive, but there were no visual manifestations or myelitis. Spinal MRI was negative for longitudinally extended transverse myelitis throughout the clinical course.

Protease-activated receptor-2 (PAR(2)) in human periodontitis.

No evidence for the role of protease-activated receptor-2 (PAR(2)) in human periodontal disease has been demonstrated so far. Thus, we sought to investigate the expression of PAR(2) mRNA in chronic periodontitis, and to examine whether its expression is related to the presence of PAR(2) potential activators. Microbiological and gingival crevicular fluid samples were collected from individuals with chronic periodontitis and control individuals, and the presence of neutrophil serine proteinase 3 (P3) and Porphyromonas gingivalis was evaluated. PAR(2) mRNA expression was higher (p < 0.001) in those with chronic periodontitis compared with control individuals, and it was statistically decreased (p = 0.0006) after periodontal treatment. Furthermore, those with chronic periodontitis presented higher (p < 0.05) levels of IL-1alpha, IL-6, IL-8, and TNF-alpha, total proteolytic activity, P. gingivalis prevalence, and P3mRNA expression compared with control individuals. We conclude that PAR(2) mRNA expression and its potential activators are elevated in human chronic periodontitis, therefore suggesting that PAR(2) may play a role in periodontal inflammation.

Respuesta a rituximab en un paciente con granulomatosis de Wegener refractaria a terapia convencional / Response to rituximab in a patient with Wegener's granulomatosis refractory to conventional therapies

Se presenta el caso de un paciente con granulomatosis de Wegener asociada a anticuerpos antiproteinasa 3 que tuvo mejoría inicial con dosis altas de glucocorticoides y ciclofosfamida, pero que posteriormente a una recaída no respondió a los mismos medicamentos, ni a metotrexate ni azatioprina. Por tal motivo, recibió tratamiento con rituximab en 4 dosis, además de glucocorticoides obteniendo mejoría del cuadro tanto clínica como radiográficamente(AU)

Male with diagnosis of Wegener's granulomatosis associated to anti-proteinase 3 antibodies that improved initially to the treatment with high dose glucocorticoids and ciclophosphamide, but in a relapse he did not have good response to glucocorticoid treatment, ciclophosphamide, methotrexate nor azathioprine. The patient received treatment with rituximab in 4 doses with clinical and radiographic improvement(AU)

EUROPLUS ANCA BIOCHIP mosaic: PR3 and MPO antigen microdots improve the laboratory diagnostics of ANCA-associated vasculitis.

INTRODUCTION: The consensus on anti-neutrophil cytoplasmic antibody (ANCA) testing requires screening with indirect immunofluorescence (IIF) and confirmation in MPO- and PR3-ANCA specific assays. The EUROPLUS system combines in one incubation the conventional cell substrates with microdots of single antigens, i.e., MPO and PR3. We evaluated the diagnostic applicability of this new system for ANCA-associated vasculitis (AAV). METHODS: To assess the diagnostic performance of the EUROPLUS Granulocyte Mosaic, sera from 249 AAV patients, 85 disease controls and 27 healthy controls were analysed. Results were compared with a reference multi-testing algorithm based on IIF with ethanol-fixed granulocytes, direct and capture ELISAs for both MPO- and PR3-ANCA. RESULTS: Based on the reference multi-testing algorithm 123 AAV patients were defined as having PR3-ANCA and 68 AAV patients as having MPO-ANCA (diagnostic sensitivity: 76.7%). For the EUROPLUS MPO and PR3 microdots the diagnostic sensitivity was 77.1% in the same AAV cohort. The concordance between both methods for PR3- and MPO-ANCA was 96.8% and 99.2%, respectively. In the control cohort the diagnostic specificity was 99.1% for the multi-testing algorithm and 98.2% for the EUROPLUS microdots. CONCLUSIONS: The combination of conventional cell substrates and single MPO and PR3 antigen microdots greatly facilitates the identification of ANCA reactivity clinically relevant for AAV. Since our results obtained after a single incubation in the EUROPLUS system are highly concordant with the reference multi-testing algorithm (based on IIF, direct and capture ELISAs) the EUROPLUS system is advocated as an efficient test system.

Coexpression of CD177 and membrane proteinase 3 on neutrophils in antineutrophil cytoplasmic autoantibody-associated systemic vasculitis: anti-proteinase 3-mediated neutrophil activation is independent of the role of CD177-expressing neutrophils.

OBJECTIVE: Wegener's granulomatosis (WG) is strongly associated with antineutrophil cytoplasmic autoantibodies (ANCAs) directed against proteinase 3 (PR3). Recent studies have shown that membrane-bound PR3 (mPR3) is differentially expressed and colocalizes with CD177/NB1 on circulating neutrophils. We undertook this study to assess the differential expression of CD177 on neutrophils from patients with ANCA-associated systemic vasculitis (ASV) in comparison with patients with systemic lupus erythematosus (SLE), patients with rheumatoid arthritis (RA), and healthy individuals, and to investigate whether colocalization of mPR3 and CD177 affects anti-PR3-mediated neutrophil activation. METHODS: Expression of CD177 and mPR3 was analyzed by flow cytometry on isolated neutrophils from patients with ASV (n=53), those with SLE (n=30), those with RA (n=26), and healthy controls (n=31). Neutrophil activation mediated by anti-PR3 antibodies was assessed by measuring the oxidative burst with a dihydrorhodamine assay. RESULTS: Percentages of CD177-expressing neutrophils were significantly higher in patients with ASV and those with SLE than in healthy controls. In 3 healthy donors, CD177 expression was not detected. After priming with tumor necrosis factor alpha, neutrophils remained negative for CD177 while mPR3 expression was induced. Neutrophils from CD177-negative donors or CD177- neutrophils sorted from donors with bimodal expression were susceptible to anti-PR3-mediated oxidative burst. Variation in the extent of anti-PR3-mediated neutrophil activation among different donors occurred independent of the percentage of CD177-expressing neutrophils. CONCLUSION: Membrane expression of CD177 on circulating neutrophils is increased in patients with ASV and in those with SLE, but not in RA patients. However, primed neutrophils from CD177-negative individuals also express mPR3 and are susceptible to anti-PR3-mediated oxidative burst, suggesting that recruitment of CD177-independent mPR3 is involved in anti-PR3-induced neutrophil activation.

The membrane proteinase 3 expression on neutrophils was downregulated after treatment with infliximab in patients with rheumatoid arthritis.

Proteinase 3 (PR3) expression on neutrophils was examined in rheumatoid arthritis (RA) patients before and after antitumor necrosis factor (TNF)-alpha therapy. Membrane PR3 expression from patients with either an infection or RA significantly increased. Membrane PR3 expression on neutrophils from RA patients treated with infliximab (anti-TNF-alpha antibody) therapy was less than in those without such treatment in a resting state, but the expression later increased after stimulation in vitro. Membrane PR3 expression increased because of the stimulation of TNFalpha, whereas it was significantly suppressed by plasma or alpha(1)-proteinase inhibitor. The condition of patients with RA improved after treatment with infliximab. Membrane PR3 expression on neutrophils in RA patients was downregulated by infliximab. As a result, PR3 might play an important role in the neutrophil-mediated inflammatory reaction in patients with either RA or an infection.