ABSTRACT
The indirect immunofluorescence (IIF) technique for antineutrophil cytoplasmic antibodies (ANCA) detection is subject to substantial differences across laboratories. This study aimed to assess the impact of improvements in the IIF-ANCA technique on the positivity rate of ANCA tests. A cross-sectional study was performed with serum samples from patients with ANCA-associated vasculitis (AAV), autoimmune hepatitis (AIH), and ulcerative colitis (UC). A paired analysis was performed for IIF-ANCA results using the traditional method and a modified protocol after a series of specific adjustments in the technique based on the protocol of IIF-ANCA test performed at a nation-wide private laboratory in Brazil. ANCA specificity was assessed by ELISA for anti-proteinase 3 (PR3) and anti-myeloperoxidase (MPO) antibodies. Sixty-one patients were evaluated. The positivity rate of IIF-ANCA tests at disease presentation performed at the University reference laboratory was 32.3% in AAV, AIH, and UC patients, whereas the positivity rates of IIF-ANCA and ELISA tests in other laboratories were 75.0 and 72.7%, respectively. After modifications in the IIF-ANCA technique, there was a significant increase in the positivity rate (14.8 vs 34.3%; P=0.0002) and in median titers [1/40 (1/30-1/160) vs 1/80 (1/40-1/80); P=0.0003] in AAV, AIH, and UC patients. UC had the highest increment in positive results from 5.3 to 36.8%. There was poor agreement between MPO- or PR3-ANCA and both IIF-ANCA techniques. In conclusion, modifications in the IIF-ANCA protocol led to a significant improvement in its positivity rate and titers.
ABSTRACT
Objective:To design a modified S1PR3 specific agonist, GPS-725.017, and investigate its protective effect on acute lung injury by promoting macrophage clearance of bacteria.Methods:A short peptide derived from the intracellular region of S1PR3 receptor was named GPS725.017, which was modified with norleucine (Nle) and myristicacid (myr) at its N terminus. Mice were divided into the sham operation group, solvent group and GPS-725.017 treatment group. The acute lung injury model was induced by endotracheal injection of E. coli (5×10 6 CFU), and the experimental group was treated with GPS-725.017 (10 mg/kg). The 48-h survival rate of mice was recorded. After 5 h of modeling, the bacterial load and inflammatory cytokines in peripheral blood and lung were detected, and Vps34 protein content in alveolar macrophages was determined by Western blot. After 12-h of modeling, lung tissues were collected for H&E staining and pathological scores. Results:Compared with the solvent group, the survival rate of mice in the GPS-725.017 treatment group was significantly improved ( P<0.01), the bacterial CFU in blood and alveolar lavage fluid was significantly lower than that in the solvent group ( P<0.001), and the levels of TNF-α and IL-1β in blood and alveolar lavage fluid were significantly lower than those in the solvent group ( P<0.001). Western blot showed that the expression level of Vps34 protein in alveolar macrophages was significantly higher than that in the solvent group ( P<0.01). Histopathology result showed that the pathological damage of lung in the treatment group was significantly less than that in the solvent group ( P<0.001). Conclusions:The modified synthetic S1PR3 specific agonist GPS-725.017 could specifically activate the S1PR3 receptor on the membrane of alveolar macrophages and up-regulate the expression level of intracellular Vps34 protein, which can promote the removal of bacteria in alveolar macrophages, significantly reduce the degree of lung injury and improve the survival rate in ALI mice.
ABSTRACT
La endocarditis bacteriana con hemocultivo negativo constituye un dilema diagnóstico. Tanto Bartonella como Coxiella pueden causarla, con presentaciones clínicas similares que pueden simular una vasculitis sistémica no infecciosa. Sin embargo, difieren en el tipo y la duración del tratamiento, por lo que es fundamental identificar el agente etiológico. Presentamos un caso de endocarditis por Bartonella henselae asociada a glomerulonefritis y neurorretinitis, con hemocultivo negativo, anticuerpos anticitoplasma de neutrófilos y antiproteinasa 3 positivos, y serología positiva para Bartonella con reacción cruzada para Coxiella burnetti. El diagnóstico etiológico fue confirmado a posteriori mediante amplificación y secuenciación parcial del gen ribC a partir de tejido de la válvula cardíaca. El paciente recibió tratamiento antibiótico e inmunosupresor seguido de recambio valvular aórtico y presentó evolución favorable.
Blood-culture negative endocarditis is a diagnostic challenge. Both Bartonella and Coxiella can cause it with similar clinical presentations mimicking a systemic vasculitis. The identification of the etiologic agent is essential because they differ in treatment type and duration. We present a case of blood-culture negative endocarditis caused by Bartonella henselae, associated with glomerulonephritis and neuroretinitis, with negative blood culture, positive anti-neutrophil cytoplasmic and antiproteinase 3 antibodies. The serology was positive for Bartonella with cross-reactivity to Coxiella burnetti. The etiological diagnosis was achieved by polymerase chain reaction amplification and sequencing of a ribC gene fragment. The patient received antibiotic and immunosuppressive treatment followed by replacement of the aortic valve with favorable medium-term evolution.
Subject(s)
Humans , Male , Adult , Retinitis/microbiology , Bartonella henselae/isolation & purification , Endocarditis, Bacterial/microbiology , Glomerulonephritis/microbiology , Retinitis/complications , Endocarditis, Bacterial/complications , Glomerulonephritis/complicationsABSTRACT
Objective To investigate the pathogenesis of diseases with antineutrophil cytoplasmic antibody (ANCA)and its diagnostic value in primary small vessel vasculitis.Methods 57 patients with serum ANCA positive were involved in this study,and ELISA was employed to assay anti-MPO.Patients with ANCA positive,anti-MPO positive and/or anti-PR3 positive were involved in group A.Patients with ANCA positive,anti-MPO negative and anti-PR3 negative were involved in group B.X2 was used to analyze the differences between the two groups.Results The etiology of 57 ANCA positive patients included primary small vessel vasculitis (20 cases,35.1%),non inflam-matory connective tissue disease(19 cases,33.3%),non connective tissue disease(18 cases,31.6%).A group of primary small vessel vasculitis accounted for 58.6%,which was significantly higher than 10.7% of the B group (χ2 =14.354,P<0.01);while the B group of non inflammatory connective tissue disease accounted for 50%,which was significantly higher than 17.2%of the A group (χ2 =6.879,P<0.01).Conclusion ANCA should be found in many kinds of diseases,so combined detection of anti-MPO and anti-PR3 should be employed to improve the diag-nosis specificity in primary small vessel vasculitis.Furthermore,non vessel vasculitis connective tissue disease should be excluded in patients with ANCA positive,anti-MPO and anti-PR3 negative.
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Objective To study the effects of the myristoyl-glycine modified peptide which derived from the second intracellular loop of sphingosine 1-phosphate receptor 3 (S1PR3) on activation of mitogenactivated protein kinases (MAPKs) pathway.Methods The phosphorylation levels of JNK and ERK in THP-1 cells were detected by western blot after GPS-725.017 stimulation.Statistical data analysis was conducted by multivariate analysis of variance.Results Western blot showed that 10 min after 30 μmol/L or 50 μmol/L GPS-725.017 stimulated,phosphorylation of ERK significantly increased in comparison with the solvent-treated group [30 μmol/L group:(3.10 ± 0.27) vs.(7.98 ± 0.45),P ＜ 0.01;50 μmol/L group:(4.78 ±0.44) vs.(25.98 ±2.32),P ＜0.01];after 50 μmol/L GPS-725.017 stimulated THP-1 cells for 5 min,10 min,20 min or 30 min,p-ERK or p-JNK level raised at different time points (P ＜0.01vs.solvent group).Conclusions GPS-725.017,a kind of myristoyl-glycine modified peptide derived from S1 PR3,could traverse cytomembrane and activate MAPKs pathway.This study provides an implication of targeting S1PR3 for clinical therapy on inflammatory diseases or sepsis.
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Objective To observe the effect of S1PR2/3 on heart during myocardial ischemia-reperfusion (I/R) in rats. Methods Healthy adult male Sprague-Dawley rats were randomly divided into 7 groups: control group, sham operation group, IR group, IR group treated with DMSO, IR group treated with Cym5541( agonist of S1P3), IR group treated with Cay10444 (antagonist of S1P3), IR group treated with Cay10444/Jte-013 (antagonist both S1P3 and S1P2). In vivo model of myocardial ischemia-reperfusion was established. The hemodynamics, infarction area and mortality was recorded. Results Compared with IR, the S1PR3 antagonist group and S1PR2/3 antagonist group showed signiifcantly reduction of heart rate(HR) and increament left ventricular end-diastolic pressure(LVEDP)(P<0.05). In addition, the infarction area was increased in the S1PR3 antagonist group and S1PR2/3 antagonist treated group (55.7%:28.8%, 51.6%:28.8%), respectively. Treatment with S1PR3 agonist reduced the infarct size compared with IR group(18.6%:28.8%). Blocking S1P2/3 receptors increased IR-induced mortality signiifcantly (53%:22%, P<0.05). Conclusion S1PR2/3 have a beneifcial effect on heart. S1PR2 and S1PR3 were involved in the IR-induced SCD.
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Objective:To compare the performance of chemiluminescent microparticle immunoassay ( CMIA) and enzyme-linked immunosorbent assay ( ELISA) for the determination of Anti-PR3 and Anti-MPO.Methods:Concentration of Anti-PR3 and Anti-MPO in serum samples from 166 patients with autoimmune diseases and 50 healthy donors were determined by using CMIA (Method A) and ELISA(Method B),respectively.The results from both assays were analyzed and compared by statistical methods .Results:Method A showed better intra-assay reproducibility and inter-assay reproducibility than Method B for the determination of high ,medium and low levels of control serum .Both methods met the accuracy requirement .The correlation coefficient of Anti-PR3 and Anti-MPO were 0.987 8 and 0.989 6 for Method A and Method B ,respectively.And the Kappa coefficients were 0.897 and 0.882 for Method A and Method B,respectively.Conclusion:The performance of Method A is superior to Method B for the deter-mination of Anti-PR3 and Anti-MPO, which makes Method A to be a potentially better choice for clinical application .
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Objective To observe the effects of Xianfu Wenyang Tongluo Drink (XFWYTLD) on ANCA associated antigen in rats with thromboangiitis obliterans;To discuss its mechanism. Methods Totally 72 SPF male Wistar rats were randomized into sham-operation group, model group, Mailuoning Granule group and XFWYTLD low, medium, and high dose groups. Method of femoral artery injecting sodium laurate was used to duplicate models. From the second day after modeling, the rats in sham-operation group and model group were fed with distilled water, while other groups received gavage with relevant medicine. 15 days later, the activity of MPO in serum was detected through the method of ultraviolet spectrophotometry;the protein expressions of PR3 and LAMP-2 in femoral artery and the surrounding tissues were detected through the method of immunohistochemisty. Results Compared with sham-operation group, the activity of MPO in serum and the protein expressions of PR3 and LAMP-2 in rats of model group were significantly higher;Compared with model group, the activity of MPO in serum and the protein expressions of PR3 and LAMP-2 in rats of all medication administration groups decreased, among which the XFWYTLD medium dose group showed the most obvious decrease. Conclusion XFWYTLD may lower levels of ANCA associated antigen, and further inhibit humoral immune function.