Interleukin-18 and Soluble Interleukin-2 Receptor Are Useful Markers for Enhanced Diagnosis of Intravascular Lymphoma

To investigate the characteristics of patients with adult-onset Still's disease (AOSD), serum cytokines and chemokines were measured to examine their associations with systemic manifestations of AOSD, especially hemophagocytic syndrome (HPS). Nineteen patients diagnosed with AOSD were enrolled. Serial serum samples were obtained from patients with AOSD in both active and inactive stages and controls. The concentrations of cytokines and chemokines, including IL-18, soluble IL-2 receptor (sIL-2R), CX3CL1, CXCL8, CXCL10, CCL2, and CCL3, were determined using enzyme-linked immunosorbent assay. Multivariate analysis was used to evaluate the correlations among serum chemokine levels, disease activity, and the clinical features of AOSD. Significantly higher serum levels of all cytokines and chemokines were observed in patients with active, untreated AOSD than in controls. The level of CX3CL1, but not other chemokines, was elevated in AOSD patients and was positively correlated with clinical activity and the levels of CRP, ferritin, IL-18, and sIL-2R. Among the 19 patients with AOSD, four patients also had HPS. Serum CX3CL1 and ferritin were significantly higher in AOSD patients with HPS than in those without HPS. The serum CX3CL1 level may be used as a clinical marker to assess the disease activity of AOSD, and high serum CX3CL1 and ferritin in patients with AOSD reflected the presence of HPS. The association between the chemokine profile and distinct clinical manifestations or various patterns of disease progression indicates that the pathogenesis of AOSD is heterogeneous.

Monocyte distribution width (MDW) correlates with volume modifications of circulating monocytes upon activation. Given the crucial role of monocyte activation in the pathogenesis of adult-onset Still's disease (AOSD), we aimed to examine the associations between MDW and disease activity or inflammatory parameters in this disease. In 58 AOSD patients and 95 other patients with coronavirus disease 2019 (COVID-19) as disease control, MDW and complete blood count were determined using a UniCel DxH800 analyser. C-reactive protein (CRP) levels were measured by nephelometry, and ferritin levels by chemiluminescent immunoassay. AOSD activity was assessed using a modified Pouchot score. MDW was significantly higher in active AOSD patients (median 28.3, interquartile range [IQR] 23.3-32.1) compared with inactive AOSD (19.2, IQR 18.0-20.6, p<0.001) or non-severe COVID-19 patients (23.2, IQR 21.0-25.2, p<0.01). MDW was positively correlated with AOSD activity scores, CRP, and ferritin levels (all p<0.001). Longitudinal follow-up evaluation revealed that median MDW significantly declined (28.3 versus 18.5, p<0.001) along with disease activity, paralleling a decrease in CRP and ferritin levels. Severe COVID-19 and sepsis patients had elevated MDW, which were not different from active AOSD patients. Multivariate analysis revealed MDW as a significant predictor of active AOSD, and MDW threshold at 21.7 could predict an active status with a high sensitivity of 91.3% and specificity of 94.3%. Elevated MDW and its positive correlation with inflammatory parameters in AOSD patients indicate MDW as a novel activity indicator, with a high MDW value above 21.7 linked to a high probability of active AOSD.

Objective To summarize the clinical data of macrophage activation syndrome (MAS) in adult-onset Still's disease(AOSD) patients and provide evidence for clinical diagnosis and treatment. Methods We retrospectively reviewed the clinical data of AOSD with MAS patients in the First Affiliated Hospital of Zhengzhou University from January 2012 to August 2018, and compared with patients with AOSD alone. Data were analyzed by t-test, Mann-Whitney U test, χ2 test or Fisher exact test. Results A total of 14 AOSD with MAS patients were enrolled, accounting for 7.6%(14/185) of AOSD patients at the same period, including 2 males and 12 females. The median duration of AOSD in MAS was 1.3(0.25, 4) months. Compared with the AOSD group, the age of onset was younger in the MAS group (t=-2.038, P=0.037), and the proportion of splenomegaly (t=9.020, P=0.003), pericardial effusion (t=8.663, P=0.003), pleural effusion (t=4.754, P=0.029) was higher. The white blood cell count (t=-4.171, P 2 000 μg/L (t=7.833, P=0.005) was significantly increased. Fourteen patients with AOSD complicated with MAS were treated with glucocorticosteroids, 5 with methylprednisolone, 8 with cyclosporine A, 8 with intravenous immunoglobulin (IVIG), 2 with etoposide, and 1 with tocilizumab. After treatment, 11 cases recovered and 3 cases died. Conclusion Younger AOSD patients tend to complicated with MAS, especially at the early course of the disease, and splenomegaly occur more frequently clinically compared to patients without MAS. When blood cell count, fibrinogen and ESR decreases, triglyceride and ferritin levels increases in AOSD patients, the occurrence of MAS is indicated. Timely treatment can improve the prognosis of patients. Key words: Still's disease, adult-onset; Macrophage activation syndrome; Glucocorticosteroids

AB0067 Association of CXCL10 AND CXCL13 in Active Adult Onset Still's Disease: Expression in Serum and Skin, and Disease Activity

IntroductionThe objective of this study was to investigate the potential role of the Toll-like receptor 7 (TLR7) signaling pathway in the pathogenesis of adult-onset Still's disease (AOSD).MethodsFrequencies of TLR7-expressing precursor of myeloid dendritic cells (pre-mDCs) and mDCs in 28 AOSD patients, 28 patients with systemic lupus erythematosus (SLE) and 12 healthy controls (HC) were determined by flow cytometry analysis. Transcript and protein levels of TLR7 signaling molecules in peripheral blood mononuclear cells (PBMCs) were evaluated by quantitative PCR and western blotting respectively. Serum cytokines levels were measured by ELISA.ResultsSignificantly higher median frequencies of TLR7-expressing pre-mDCs and mDCs were observed in AOSD patients (65.5% and 14.9%, respectively) and in SLE patients (60.3% and 14.4%, respectively) than in HC (42.8% and 8.8%, respectively; both P <0.001). Transcript and protein levels of TLR7-signaling molecules, including MyD88, TRAF6, IRAK4 and IFN-α, were upregulated in AOSD patients and SLE patients compared with those in HC. Disease activity scores were positively correlated with the frequencies of TLR7-expressing mDCs and expression levels of TLR7 signaling molecules in both AOSD and SLE patients. TLR7 ligand (imiquimod) stimulation of PBMCs resulted in significantly enhanced levels of interleukin (IL)-1β, IL-6, IL-18 and IFN-α in AOSD and SLE patients. Frequencies of TLR7-expressing mDCs and expression levels of TLR7 signaling molecules significantly decreased after effective therapy.ConclusionsElevated levels of TLR7 signaling molecules and their positive correlation with disease activity in AOSD patients suggest involvement of the TLR7 signaling pathway in the pathogenesis of this disease. The overexpression of TLR7 MyD88-dependent signaling molecules may be a common pathogenic mechanism for both AOSD and SLE.

SP0141 Adult Onset Still's Disease

Clinical characteristics and outcome of elderly onset adult-onset Still's disease: A 10-year retrospective study

Human neutrophil lipocalin (HNL) has been used extensively to differentiate acute bacterial infection from febrile diseases as a biomarker to reflect the activation of the neutrophil. The serum HNL levels in the adult-onset Still's disease (AOSD) patients with and without infection, as well as the healthy controls (HCs), were analyzed statistically in this study to evaluate the value of HNL for the diagnosis of AOSD. A total of 129 AOSD patients were enrolled, from whom blood samples were drawn and the AOSD diagnosis was confirmed through the review of the medical records, where the systemic score, demographic characteristics, clinical manifestations, and laboratory parameters were also collected for the patients; in addition, a total of 40 HCs were recruited among the blood donors from the healthcare center with the relevant information collected. The HNL test was done for the blood samples with the enzyme-linked immunosorbent assay and the analyses were done for the correlations of HNL with clinical manifestations and diagnostic effectiveness. The serum HNL increased significantly in the patients with only AOSD as compared with that in the HCs (139.76 ± 8.99 ng/mL vs . 55.92 ± 6.12 ng/mL; P < 0.001). The serum HNL level was correlated with the white blood cell (WBC) count ( r = 0.335, P < 0.001), neutrophil count ( r = 0.334, P < 0.001), erythrocyte sedimentation rate ( r = 0.241, P = 0.022), C-reactive protein ( r = 0.442, P < 0.0001), and systemic score ( r = 0.343, P < 0.0001) in the AOSD patients significantly. Patients with fever, leukocytosis ≥15,000/mm 3 , and myalgia in the HNL-positive group were observed relatively more than those in the HNL-negative group ( P = 0.009, P = 0.023, and P = 0.007, respectively). HNL was a more sensitive indicator than ferritin and C-reactive protein (CRP) to differentiate the AOSD patients with bacterial infection from AOSD-only patients, and the Youden index was 0.6 for HNL and 0.29 for CRP. Serum HNL can be used as a biomarker for the diagnosis of the AOSD, and HNL is also observed to be associated with the disease activity.

To investigate the potential role of Th type 17 (Th17) cells and Th17-related cytokines in the pathogenesis of adult-onset Still's disease (AOSD). The frequencies of circulating Th17 cells in 24 patients with active untreated AOSD, 16 patients with active SLE and 12 healthy volunteers were determined using intracellular cytokine staining and flow cytometry. Serum levels of Th17-related cytokines, including IL-1β, IL-6, IL-17, IL-18, IL-21 and IL-23 were measured by ELISA. Significantly higher median frequencies of circulating Th17 cells were found in active untreated AOSD patients (1.01%) and active SLE patients (1.26%) than in healthy volunteers (0.12%, both P<0.001). The frequencies of circulating Th17 cells were positively correlated with activity score (r=0.527, P<0.01) and serum ferritin levels (r=0.724, P<0.001) in AOSD patients, and correlated with SLEDAI (r=0.663, P<0.01) in SLE patients. Additionally, the frequencies of circulating Th17 cells were positively and significantly correlated with serum levels of IL-1β, IL-6, IL-17, IL-18, IL-21 and IL-23 in both AOSD and SLE patients. The frequencies of circulating Th17 cells and serum IL-17 levels significantly decreased after effective therapy in AOSD patients (both P<0.001). Elevated frequencies of circulating Th17 cells and a positive correlation with disease activity in our AOSD patients suggest that Th17 cells contribute to the pathogenesis of this disease. Dysregulation of Th17 cells may be a common pathogenic mechanism that underlies the development of both AOSD and SLE.

BackgroundCOVID-19 and autoinflammatory diseases, such as Adult-onset Still’s Disease (AOSD), are characterized by a massive production and uncontrolled secretion of pro-inflammatory cytokines. The specialized pro-resolving lipid mediators (SPMs) family is...

IL-18 is a pro-inflammatory cytokine of the IL-1 family that is naturally inhibited by IL-18 binding protein (IL-18BP). High levels of IL-18 have been described in the serum of adult-onset Still's disease (AOSD) patients, but only total IL-18 levels (including inactive IL-18 bound to IL-18BP) have been measured. With a specific immunoassay, we aimed to measure free IL-18 serum levels in AOSD patients and other rheumatic diseases. An ELISA was developed to measure free IL-18. Its sensitivity and specificity were tested by spiking recombinant IL-18 or IL-18BP in serum and PBS supplemented with 5% BSA. The binding affinity of IL-18 to IL-18BP was calculated by titration experiments using the ELISA and by Biacore analysis. Sera of 37 AOSD patients and 138 controls (40 healthy controls, 30 RA, 29 SLE, 21 AS and 18 PsA) were assayed for free IL-18, IL-18BP, total IL-18 and other cytokines. Correlations were performed between free IL-18 and markers of disease activity in AOSD patients. Free IL-18 serum levels were significantly higher in AOSD patients (median 8.89 pg/ml) than in healthy and disease controls (1.37 pg/ml; P < 0.01). Free IL-18 serum levels correlated with AOSD activity. The affinity of IL-18 to IL-18BP was found to be much higher than previously described, with a dissociation constant ranging from 30 to 50 pM. Free IL-18 levels are specifically elevated in AOSD compared with other inflammatory diseases, suggesting that IL-18 represents a potential target for the treatment of AOSD.

Objective To analyze the clinical features of patients with hemophagocytic lymphohiscytosis(HLH)in adult-onset Still's disease(AOSD). Methods We retrospectively reviewed the clinical data of patients with AOSD complicated with HLH in Peking Union Medical College Hospital from August 2012 to September 2014, and we conducted a case-control study from the cohort of AOSD patients seen during the same period. Data were analyzed by using t-test or Mann-Whitney test and χ2 test(Fisher exact test). The treatments and outcomes of AOSD with HLH were also retrospectively analyzed. Results Twelve AOSD with HLH patients were included, and the prevalence of HLH in AOSD patients was 13%(12/96). Twelve AOSD with HLH patients were all female. The age at HLH onset was 19.7-52.3(33±12)years, usually during the 1-48(20±24)months following AOSD onset. HLH was associated with the activation of AOSD(n=9)and virus infection(n=3). Disseminated intravascular coagulation and shock were more frequently observed in patients with HLH than those without. The neutrophil count [3.14(0~21.98)×109/L vs 12.46(1.28 ~57.42)×109/L, Z= -4.309, P<0.01], hemoglobin levels [(81 ±27)g/L vs(107±21)g/L, t=3.815, P<0.01], platelet count [48(8~199)× 109/L vs 316(81 ~1 016)×109/L, Z=-5.351, P<0.01], fibrinogen levels [(1.3±0.6)g/L vs(5.0±2.1)g/L, t=12.987, P<0.01]were all lower in the group of HLH patients compared with the non-HLH patients, while the serum ALT [338(18~5 290)U/L vs 56(9~3 227)U/L, Z=-3.097, P=0.002]; AST [352(35~15 964)U/L vs 50(10~ 4 147)U/L, Z=-4.083, P=0.000]; LDH [2 074(381~18 815)U/L vs 518(91~2 131)U/L, Z=-3.855, P=0.000]and ferritin levels [16 990(6 321~59 952)U/L vs 2 092(118~44 169)U/L, Z=-4.708, P=0.000]were higher in the group of HLH patients compared with the non-HLH patients. After treated with large dose glucocortico-steroids, combined with immunosuppressants, intravenous immunoglobulin(IVIG)and treating underlying infections, 11 patients recovered, 1 patient died. Conclusion HLH usually occurs at the early course of AOSD. Infection and activation of AOSD are potential triggers of HLH. HLH should be considered when AOSD patients manifested with cytopenia, progressive elevation of ALT, AST serum LDH or ferritin levels, hypofi-brinogenemia, DIC and shock. Large dose glucocorticosteroids combined with immunosuppressants and IVIG may improve the outcome of HLH, while anti-infection therapy is necessary for patients complicated with active infection. Key words: Still's disease, adult-onset; Lymphohistiocytosis, hemophagocytic; Glucocorticoids

Adult onset Still's disease (AOSD) in the era of biologic therapies: Dichotomous view for cytokine and clinical expressions

THU0406 Histological Findings of Spleen Affected by Adult Onset Still's Disease: an Analysis of 3 Cases

Adult-onset Still's disease (AOSD) is a systemic inflammatory disease characterized by cytokine storm. However, a diagnostic test for AOSD in clinical use is yet to be validated. The aim of our study was to identify non-invasive biomarkers with high specificity and sensitivity to diagnosis of AOSD. MicroRNA (miRNA) profiles in PBMC from new-onset AOSD patients without any treatment and healthy controls (HCs) were analyzed by miRNA deep sequencing. Plasma samples from 100 AOSD patients and 60 HCs were used to validated the expression levels of miRNA by qRT-PCR. The correlations between expression levels of miRNAs and clinical manifestations were analyzed using advanced statistical models. We found that plasma samples from AOSD patients showed a distinct miRNA expression profile. Five miRNAs (miR-142-5p, miR-101-3p, miR-29a-3p, miR-29c-3p, and miR-141-3p) were significantly upregulated in plasma of AOSD patients compared with HCs both in training and validation sets. We discovered a panel including 3 miRNAs (miR-142-5p, miR-101-3p, and miR-29a-3p) that can predict the probability of AOSD with an area under the receiver operating characteristic (ROC) curve of 0.8250 in training and validation sets. Moreover, the expression levels of 5 miRNAs were significantly higher in active AOSD patients compared with those in inactive patients. In addition, elevated level of miR-101-3p was found in AOSD patients with fever, sore throat and arthralgia symptoms; the miR-101-3p was also positively correlated with the levels of IL-6 and TNF-α in serum. Furthermore, five miRNAs (miR-142-5p, miR-101-3p, miR-29c-3p, miR-29a-3p, and miR-141-3p) expressed in plasma were significantly higher in AOSD patients than in sepsis patients (P < 0.05). The AUC value of 4-miRNA panel (miR-142-5p, miR-101-3p, miR-29c-3p, and miR-141-3p) for AOSD diagnosis from sepsis was 0.8448, revealing the potentially diagnostic value to distinguish AOSD patients from sepsis patients. Our results have identified a specific plasma miRNA signature that may serve as a potential non-invasive biomarker for diagnosis of AOSD and monitoring disease activity.