Loss of mucosal tolerance to glycoprotein 2 isoform 1 is a potential novel diagnostic biomarker for cholangiocarcinoma.

BACKGROUND: Anti-glycoprotein 2 (anti-GP2) IgA and antineutrophil-cytoplasmic antibodies to proteinase 3 (PR3-ANCA) have been reported as predictive markers of cholangiocarcinoma (CCA) in patients with primary sclerosing cholangitis (PSC), but their prevalence in CCA patients without PSC remains unclear. METHODS: This study involved Asian discovery (n = 118) and European validation (n = 38) cohorts of CCA patients without PSC, alongside 49 Asian and 82 European pancreatic ductal adenocarcinoma (PDAC) patients, 21 with benign pancreatic neoplasms (BPN) and 45 with hepatocellular carcinoma (HCC), and 157 healthy controls (HC) from Asia and Europe. We analyzed the prevalence of PR3-ANCA, IgA and IgG against GP21 and GP24, and the CA19-9 levels. RESULTS: Anti-GP21 IgA was the most prevalent in both CCA cohorts (discovery: 55.1 %; validation: 42.1 %) and significantly higher than in other groups except PDAC (all p < 0.05). It demonstrated the best diagnostic performance in distinguishing CCA from disease controls and HC, outperforming tumor markers. No significant correlation was found between anti-GP21 IgA levels and CA19-9 levels. CONCLUSION: Our findings show that anti-GP21 IgA revealing the loss of mucosal tolerance is a potential novel diagnostic biomarker for CCA.

Clinical potential of serum prostaglandin A2 as a novel diagnostic biomarker for hepatocellular cancer.

BACKGROUND: Hepatocellular cancer (HCC) is one of the most harmful tumors to human health. Currently, there is still a lack of highly sensitive and specific HCC biomarkers in clinical practice. In this study, we aimed to explore the diagnostic performance of prostaglandin A2 (PGA2) for the early detection of HCC. METHODS: Untargeted metabolomic analyses on normal control (NC) and HCC participants in the discovery cohort were performed, and PGA2 was identified to be dysregulated in HCC. A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for detecting serum PGA2 was established and applied to validate the dysregulation of PGA2 in another independent validation cohort. Receiver operating characteristic (ROC), decision curve analysis (DCA) and some other statistical analyses were performed to evaluate the diagnostic performance of PGA2 for HCC. RESULTS: At first, PGA2 was found to be dysregulated in HCC in untargeted metabolomic analyses. Then a precise quantitative LC-MS/MS method for PGA2 has been established and has passed rigorous method validation. Targeted PGA2 analyses confirmed that serum PGA2 was decreased in HCC compared to normal-risk NC and high-risk cirrhosis group. Subsequently, PGA2 was identified as a novel biomarker for the diagnosis of HCC, with an area under the ROC curve (AUC) of 0.911 for differentiating HCC from the combined NC + cirrhosis groups. In addition, PGA2 exhibited high performance for differentiating small-size (AUC = 0.924), early-stage (AUC = 0.917) and AFP (-) HCC (AUC = 0.909) from the control groups. The combination of PGA2 and AFP might be useful in the surveillance of risk population for HCC and early diagnosis of HCC. CONCLUSION: This study establishes that PGA2 might be a novel diagnostic biomarker for HCC.

Altered CD226/TIGIT expressions were associated with NK phenotypes in primary antiphospholipid syndrome and affected by IL-4/JAK pathway.

Natural killer (NK) cells were reported to be involved in the pathogenesis of primary antiphospholipid syndrome (pAPS). Immunosuppressive receptor T-cell immunoreceptor with Ig and ITIM domains (TIGIT) and activating receptor cluster of differentiation 226 (CD226) are specifically expressed on NK cells with competitive functions. This study aims to investigate the expression diversities of CD226/TIGIT on NK subsets and their associations with NK subsets activation phenotypes and potential clinical significance, furthermore, to explore potential cause for CD226/TIGIT expression diversities in pAPS. We comparatively assessed the changes of CD56brightNK, CD56dimNK, and NK-like cells in 70 pAPS patients compared with control groups, including systemic lupus erythematosus, asymptomatic antiphospholipid antibodies carriers (asymp-aPLs carriers), and healthy controls and their expression diversities of CD226/TIGIT by flow cytometry. CD25, CD69, CD107α expression, and interferon gamma (IFN-γ) secretion levels of NK subsets were detected to determine the potential association of CD226/TIGIT expression with NK subsets phenotypes. CD226/TIGIT expression levels were compared among different subgroups divided by aPLs status. Moreover, in vitro cultures were conducted to explore the potential mechanisms of CD226/TIGIT expression imbalance. CD56brightNK and CD3+CD56+NK-like cells were significantly increased while CD56dimNK cells were obviously decreased in pAPS, and CD56brightNK and NK-like cells exhibited significantly higher CD226 but lower TIGIT expressions. CD226+CD56brightNK and TIGIT-CD56brightNK cells show higher CD69 expression and IFN-γ secretion capacity, and CD226+NK-like and TIGIT-NK-like cells showed higher expressions of CD25 and CD69 but lower apoptosis rate than CD226- and TIGIT+CD56brightNK/NK-like cells, respectively. The imbalanced CD226/TIGIT expressions were most significant in aPLs triple-positive group. Imbalanced expressions of CD226/TIGIT on CD56brightNK and NK-like cells were aggravated after interleukin-4 (IL-4) stimulation and recovered after tofacitinib blocking. Our data revealed significant imbalanced CD226/TIGIT expressions on NK subsets in pAPS, which closely associated with NK subsets phenotypes and more complicated autoantibody status. CD226/TIGIT imbalanced may be affected by IL-4/Janus Kinase (JAK) pathway activation.

SARS-CoV-2 antibody response in SARS survivors with and without the COVID-19 vaccine.

OBJECTIVES: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to spread worldwide. However, it remains unknown whether individuals with prior SARS-CoV-1 infection are protected from SARS-CoV-2 infection. This study assessed protective antibody levels in SARS survivors with and without the COVID-19 vaccine. METHODS: We recruited 17 SARS survivors infected with SARS-CoV-1 in 2003, including 8 not vaccinated with the COVID-19 vaccine and 9 vaccinated with two doses of inactivated whole-virion COVID-19 vaccine (Sinopharm). In addition, 105 healthy adult volunteers without SARS-CoV-1 and SARS-CoV-2 infections were used as controls. The relative concentrations of three protective antibodies including anti-SARS-CoV-2 spike IgG (nCoV S-IgG), anti-SARS-CoV-2 spike receptor-binding domain IgG (nCoV RBD-IgG), and anti-SARS-CoV-2 neutralizing antibodies (nCoV NAbs) were measured to evaluate humoral immunity. RESULTS: We found that the positive rates of these antibodies in unvaccinated SARS survivors were 37.5%, 37.5%, and 62.5%, respectively. In contrast, the corresponding positive rates were all 0% in controls before vaccination. In controls, the levels of protective antibodies reached a peak ca. 28 days after the second dose of vaccine and then started to decline. Surprisingly, the levels of these antibodies were maintained at very high levels even 166 days after the second dose of vaccine in SARS survivors. CONCLUSION: Our study suggests that there are protective antibodies cross-reacting with SARS-CoV-2 in recovered SARS patients and that SARS survivors can generate a much stronger antibody response induced by the COVID-19 vaccine than can controls. These initial findings show the feasibility of developing novel pan-sarbecovirus vaccines.

Altered Phenotypes of Colonic and Peripheral Blood Follicular Helper and Follicular Cytotoxic T Cells in Mice with DSS-Induced Colitis.

Background: Follicular helper T (Tfh), follicular regulatory T (Tfr), and follicular cytotoxic T (Tfc) cells play important roles in autoimmune diseases. Nevertheless, their changes of functional phenotypes in ulcerative colitis (UC), most importantly, their changes in colon tissue as the target-organ, have not been explored. Methods: DSS-colitis was induced in Balb/c mice and lymphocytes were collected from spleen, mesenteric lymph nodes, peripheral blood and colon. Tfh, Tfr, and Tfc cells were analyzed using flow cytometry based on their CD4+CXCR5+FOXP3-Tfh, CD4+CXCR5+FOXP3+Tfr and CD8+CXCR5+Tfc expressions. Various functional characterization markers including CD44, CD62L, TIGIT, CD226, PD-1, ICOS, Helios, CTLA-4 and Bcl6 were analyzed in the T cell subsets of the organs. Results: Tfh and Tfr cells in the colon were significantly increased in DSS-colitis mice. Additionally, the proportions of Tfr and Tfc cells in the peripheral blood were also increased, while Tfc cell proportions in the colon were decreased. The proportion of naïve cells in the Tfh, Tfr and Tfc cells in the colon and peripheral blood decreased, while the proportion of effector memory T cells increased. The TIGIT+CD226-Tfh and Tfc cells were upregulated in the colon of DSS-colitis mice. The PD-1+, ICOS+ and PD-1+ICOS+ Tfh cells were increased in both the colonic and peripheral blood Tfh and Tfc of DSS-colitis mice. The Bcl6+ proportions in the Tfh and Tfr were increased in the colon of DSS-colitis mice. Conclusion: The colonic and peripheral blood Tfh and Tfc cells of DSS-colitis mice have a significantly activated T cell phenotype, which may play a significant role in the pathogenesis of UC.

Antibodies against neutrophil extracellular traps (NETs) potentiate clinical performance of anti-double-stranded DNA antibodies in systemic lupus erythematosus.

Autoantibodies against NETs (ANETA) are present in SLE patients. We aimed to determine the clinical relevance of ANETA in SLE. Serum from 129 SLE patients, 161 patients with various rheumatoid diseases (DC), and 53 healthy controls (HC) were tested by a home-made ANETA ELISA platform. ANETA showed a sensitivity of 35.7% and a specificity of 92.5%, respectively, in the diagnosis of SLE. The combination of ANETA with anti-dsDNA antibody increased the diagnostic sensitivity from 49.6% to 62.8% for SLE. The presence of ANETA potentiates the clinical utility of anti-dsDNA antibodies in identifying a subset of SLE patients with higher disease activity and hematological abnormalities. The binding of ANETA to NETs did not inhibit the immunostimulatory effect of NETs. Our findings suggested that ANETA have potential as clinically relevant biomarkers that potentiate the clinical performance of anti-dsDNA antibodies in the diagnosis, risk stratification and subtyping of patients with SLE.

Immune phenotype changes in IgG4-related disease: CD161 + Treg and Foxp3 + Treg.

OBJECTIVE: We aimed to characterize the alterations in the immune phenotypes and explore the potential relevance to pathogenesis in IgG4-RD. METHODS: Forty-two IgG4-RD patients and thirty-eight healthy controls were recruited in this study. Peripheral immunocompetent cells including T cells, CD4 + T cells, CD8 + T cells, B cells, NK cells CD4 + CD45RA + T cells (naïve T cells), CD4 + CD25 - / + Foxp3 - T cells (Teff), CD4 + CD25hiCD127lowCD161 + T cells (CD161 + Treg), CD4 + CD25hiFoxp3 + T cells (Foxp3 + Treg), CD4 + CD4RA-CXCR5 + PD1 + CCR7low T cells (pTfh), T helper (Th) 1, Th2, and Th17 before and after treatment were immunophenotyped by flow cytometry. RESULTS: Compared with healthy controls, IgG4-RD patients showed higher proportions of NK (20.1% vs 13.6%, p < 0.01), Th1 (CD4 + IFN-γ + : 17.9% vs 14.2%, p = 0.061; TNF-α: 43.7% vs 36.7%, p < 0.05), Th2 (CD4 + IL-4 + : 2.4% vs 1.3%, p < 0.0001), CD161 + Treg (14.9% vs 11.6%, p < 0.01), pTfh (3.2% vs 2.4%, p < 0.05), and Foxp3 + Treg (8.3% vs 7.0%, p < 0.01) and lower proportions of B lymphocytes (8.4% vs 13.1%, p < 0.001), Teff (91.6% vs 92.6%, p < 0.01), and naïve Th cells (19.9% vs 32.1%, p < 0.01) before treatment. Foxp3 + Treg percentage decreased significantly after treatment (8.6% vs 6.9%, p < 0.05). Both serum C3 (r = - 0.6374, p < 0.01) and C4 (r = - 0.6174, p < 0.01) levels were in negative correlation with CD161 + Treg. The eosinophil percentage was positively correlated with Foxp3 + Treg (r = 0.5435, p < 0.05). Serum IgE level was positively correlated with Th2 (r = 0.5545, p < 0.05). There was a positive correlation between CD161 + Treg and pTfh (r = 0.4974, p < 0.05) while a negative correlation between Th2 and B cells (r = - 0.4925, p < 0.05). CONCLUSION: Immune phenotypes were altered in IgG4-RD. Treg/Teff balance was shifted toward Treg in IgG4-RD. CD161 + Treg was likely to be involved in the pathogenesis of IgG4-RD. Key Points •Immune phenotypes were altered in B cells, T cells, and NK cells in IgG4-RD. •Treg/Teff balance was shifted toward Treg in IgG4-RD. •CD161+ Treg maybe play a proinflammatory role in IgG4-RD.

IL-7 Promotes the Expansion of Circulating CD28- Cytotoxic T Lymphocytes in Patients With IgG4-Related Disease via the JAK Signaling.

Objectives: This study aimed to elucidate the changes and associated mechanisms of circulating CD28- cytotoxic T lymphocytes (CTLs) in patients with IgG4-related disease (IgG4-RD). Methods: Fifty IgG4-RD patients and 15 healthy controls (HCs) were recruited. Peripheral blood mononuclear cells (PBMCs) were isolated, the levels of circulating CD28- CTLs were detected by flow cytometry, and the proportions of CD127lo or GZMB+CD28- CTL subsets were analyzed in the meantime. Mechanistically, PBMCs isolated from IgG4-RD patients were stimulated with IL-7 in the presence or absence of the JAK inhibitor tofacitinib. Flow cytometry was used to analyze the proliferation of CD28- CTLs and the changes in related subpopulations. Results: Circulating CD4+CD28- CTLs and CD8+CD28- CTLs were significantly increased in IgG4-RD patients compared with HCs, accompanied by an elevation of CD127lo or GZMB+ CTL subsets. The ex vivo culture of PBMCs showed that IL-7 could induce the amplification of CD4+CD28- CTLs and CD8+CD28- CTLs in IgG4-RD. Furthermore, IL-7 promotes the proliferation and functional subset changes of these CD28- CTLs in this disease. The selective JAK inhibitor tofacitinib significantly inhibited the effects of IL-7 on CD4+CD28- CTLs and CD8+CD28- CTLs. Conclusion: IL-7 can affect the immune balance of IgG4-RD patients by promoting the expansion and function of CD4+CD28- and CD8+CD28- CTLs in IgG4-RD through the JAK pathway. Blockade of the IL-7 signaling pathway may be a new therapeutic strategy for IgG4-RD.

The altered HLA-DQ expression in peripheral blood T cells of chronic hepatitis B patients characterizes the function of T cells.

OBJECTIVE: This study aimed to clarify the expression of HLA-DQ and granulysin in peripheral blood T-cell subsets in patients with chronic hepatitis B virus (CHB) and to evaluate their significance in assisting CHB diagnosis and immune status assessment. METHODS: Peripheral blood from 34 CHB patients, 36 inactive HBsAg carriers and 33 healthy controls were collected, and HLA-DQ and granulysin in a series of T-cell subsets were analysed by flow cytometry. The ability to secrete IL-10 and IFN-γ and the functional T-cell subsets were measured in Treg and CD4 cells expressing HLA-DQ or not. Correlation analyses were further conducted between HLA-DQ/granulysin-related subsets and clinical indicators of HBV infection, and ROC curves were built to evaluate diagnosis efficiency of HLA-DQ-related subsets. RESULTS: HLA-DQ+ percentages in circulating CD4 T cells were downregulated in CHB patients. The proportions of HLA-DQ + Tfh in CHB were upregulated while HLA-DQ+ percentages in Treg were decreased. In terms of function, the IFN-γ secretion ability of CD4 + T cells and IL-10 secretion in Tregs were stronger in HLA-DQ+ than HLA-DQ- subsets. HLA-DQ + CD4 + T cells and HLA-DQ + Treg were negatively correlated with HBV-DNA, while HLA-DQ + Tfh and Tfc cells were positively correlated with HBV-DNA and ALT. HLA-DQ + Treg/Tfh/Tfc could help to distinguish CHB from inactive HBsAg carriers. CONCLUSION: HLA-DQ on T cells can characterize the function of T-cell subsets and analysis of HLA-DQ can help to evaluate immune status and assist in diagnosis of CHB.

Serum Human Epididymis Protein 4 as a Novel Biomarker in Identifying Patients With Interstitial Lung Disease in Rheumatoid Arthritis.

Objective: Human epididymis protein 4 (HE4) have been implicated in the pulmonary involvements. We aimed to investigate the clinical utility of HE4 in clinical stratification in patients with rheumatoid arthritis (RA). Methods: This study included a discovery cohort comprising 70 RA patients and 64 healthy controls (HCs), and a validation cohort comprising 98 RA patients and 75 HCs. Human epididymis protein 4 were determined by electrochemical luminescence analyzer. Results: The levels of HE4 were significantly elevated in patients with RA compared to HCs. The positive rates of HE4 in patients with RA and HCs were 50.0% and 0, respectively, in the discovery cohort and 53.1 and 1.3%, respectively, in the validation cohort. When RA patients were subgrouped according to HE4 status, HE4-positive group displayed higher prevalence of interstitial lung disease (ILD) compared to HE4-negative group (28.6 vs. 11.4% in discovery cohort and 57.7 vs. 8.7% in the validation cohort). A positive correlation between the levels of HE4 with the degree of lung impairment was identified. Receiver operating curve (ROC) analysis revealed an optimal cut-off value of 104.3 pmol/L in HE4 for distinguishing RA-ILD from RA-non ILD with the areas under the curve (AUC) of 0.790. Multivariate logistic regression analysis illustrated that high levels of HE4 independently identified patients with RA-ILD (OR, 9.080, p < 0.001). Conclusion: Our findings showed a novel role of HE4 in RA risk stratification, suggest that introducing HE4 to the current RA test panel may serve as an indicator in identifying RA patients for further RA-ILD workups, such as high-resolution computed tomography (HRCT).

Circulating CD4+ FoxP3- CXCR5- CXCR3+ PD-1hi cells are elevated in active rheumatoid arthritis and reflect the severity of the disease.

OBJECTIVE: To examine the expression and clinical significance of circulating CD4+  FoxP3-  CXCR5-  CXCR3+  PD-1hi cells in rheumatoid arthritis (RA). METHODS: CD4+  FoxP3-  CXCR5-  CXCR3+  PD-1hi cells in peripheral blood of 35 patients with active RA, 17 with RA in stable remission, and 24 healthy controls were analyzed by flow cytometry. Serum IgG and circulating plasmablast percentages were measured and correlations with CD4+  FoxP3-  CXCR5-  CXCR3+  PD-1hi cells were systematically analyzed. Disease Activity Scale 28 (DAS28) scores were also calculated and correlation analysis with CD4+  FoxP3-  CXCR5-  CXCR3+  PD-1hi cells was conducted. The levels of CD4+  FoxP3-  CXCR5-  CXCR3+  PD-1hi cells were compared before and after disease-modifying anti-rheumatic drug treatment. Cytokine levels in plasma and cytokine secretion in CD4 cells were measured and their correlations with CD4+  FoxP3-  CXCR5-  CXCR3+  PD-1hi cells were further analyzed. RESULTS: The levels of CD4+  FoxP3-  CXCR5-  CXCR3+  PD-1hi cells in the peripheral blood of patients with active RA were significantly increased compared with healthy controls. CD4+  FoxP3-  CXCR5-  CXCR3+  PD-1hi cells in patients with active RA were positively correlated with serum IgG and DAS28 scores. CD4+  FoxP3-  CXCR5-  CXCR3+  PD-1hi cells were significantly decreased in patients after treatment. Plasma interleukin-10 concentrations and interleukin-10-positive CD4 cell percentages were significantly positively correlated with CD4+  FoxP3-  CXCR5-  CXCR3+  PD-1hi cell levels. CONCLUSION: Circulating CD4+  FoxP3-  CXCR5-  CXCR3+  PD-1hi cells in patients with active RA are increased and could reflect the severity of the disease, which may play a potential role in the pathogenesis of RA.

Increased circulating PD-1hiCXCR5- peripheral helper T cells are associated with disease severity of active ulcerative colitis patients.

PURPOSE: A recently identified population of T cells, phenotypically CD4+PD-1hiCXCR5-, has been firstly termed as peripheral helper T cells (Tph) and found to be pathogenic in autoimmune diseases. However, the potential role of Tph in ulcerative colitis (UC) remains unclear. We aim to investigate the changes of circulating Tph in UC patients and their potential significance in the pathogenesis of UC. METHODS: Totally 68 UC patients and 34 age- and sex-matched healthy controls were enrolled. Circulating Tph and B cell subsets were analyzed by flow cytometry. Expressions of inducible T-cell co-stimulator (ICOS) on Tph cells were analyzed. Serum IL-4, IL-10, IL-12 and IL-21 were detected using ELISA. Correlation analyses were conducted between Tph cells and disease severity, functional B cell subsets and serum cytokines. RESULTS: Both the frequency and absolute number of Tph were significantly increased in active UC patients and ICOS levels in Tph cells were also elevated, compared with remission UC patients and healthy controls. Tph and ICOS expression were significantly positively correlated with Mayo score and serum CRP in active UC patients, and were significantly decreased when achieving remission after treatment. Tph levels were correlated with new memory B cells, plasmablasts, serum IL-4 and IL-21. Meanwhile, serum IL-10 showed negative correlation while IL-12 exhibited positive correlation with circulating Tph cells in UC patients. CONCLUSIONS: Circulating Tph cells are elevated in active UC patients and are associated with the disease activity, which may contribute to the pathogenesis of UC.

Elevated Granulocyte Colony-stimulating Factor Levels in Patients With Active Phase of Adult-onset Still Disease.

OBJECTIVE: Neutrophilia is a hallmark of adult-onset Still disease (AOSD). We aimed to investigate the levels of granulocyte colony-stimulating factor (G-CSF), an essential regulator of neutrophil production and function, in the pathogenesis of AOSD. METHODS: Sera were collected from 70 patients with AOSD and 20 healthy controls (HCs). The levels of G-CSF were determined by ELISA. Low-density granulocytes (LDGs) were quantified by flow cytometry. Correlations between G-CSF levels and disease activity, laboratory variables, and LDG levels in patients with AOSD were analyzed by Spearman correlation test. RESULTS: Patients with active AOSD presented significantly higher levels of G-CSF compared to inactive AOSD patients (P < 0.001) and HCs (P < 0.0001). The G-CSF levels were significantly decreased after active AOSD patients achieved disease remission (P = 0.0015). The G-CSF levels were significantly correlated with C-reactive protein, erythrocyte sedimentation rate, ferritin, and systemic score in AOSD (P < 0.0001). Significant correlations between the levels of G-CSF and circulating neutrophils (P < 0.0001), neutrophil-to-lymphocyte ratio (P < 0.0001), percentages of LDGs in the peripheral blood mononuclear cells (P = 0.004), as well as absolute numbers of circulating LDGs (P = 0.018) were identified. Patients with fever, evanescent rash, sore throat, arthralgia, myalgia, lymphadenopathy, or hepatomegaly/elevated liver enzymes displayed significantly higher levels of G-CSF compared to patients without these manifestations (P < 0.05). CONCLUSION: Our findings indicate that G-CSF is implicated in the pathogenesis of AOSD, and targeting G-CSF may have therapeutic potential for AOSD. In addition, introducing circulating G-CSF levels into the clinical assessment system may help to monitor disease activity.

Infection of Epstein-Barr Virus is Associated with the Decrease of Helios+FoxP3+Regulatory T Cells in Active Ulcerative Colitis Patients.

Background: Loss of immune homeostasis to enteric pathogens is considered to be involved in the pathogenesis of ulcerative colitis (UC), and regulatory T cells (Tregs) are key for this immune homeostasis. Helios exhibits an important effect on regulating the suppressive function of Tregs. Epstein-Barr virus (EBV) is more commonly detected in UC. However, whether there is an association between EBV infection and Helios+Tregs and its impact on disease activity of UC remain unclear. We aimed to explore the clinical significance of Helios+Tregs and their potential association with EBV infection in UC. Methods: Seventy-six UC patients and 38 controls were consecutively enrolled. Helios+FoxP3+Tregs were analyzed using flow cytometry and compared among groups. Eight active UC patients treated with 5-aminosalicylic acid were followed up. Correlation analyses were conducted between Helios+FoxP3+Tregs and disease activity indicators. In addition, EBV viral loads in the mucosal lesion were quantified in active UC by quantitative polymerase chain reaction and were comprehensively analyzed in subgroups of different disease severity, and their associations with Helios+FoxP3+Tregs were also analyzed. Results: Helios+FoxP3+Tregs were significantly decreased in active UC and were inversely correlated with serum C-reactive protein and Mayo score. Moreover, we observed the recovery of Helios+FoxP3+Tregs in followed-up active UC achieving remission after treatment. EBV loads were higher in active UC, and levels of Helios+FoxP3+Tregs in the EBV-positive subgroup were lower than the EBV-negative subgroup in moderate and severe active patients. Most importantly, we found that Helios+FoxP3+Tregs were significantly negatively correlated with EBV viral loads. Conclusion: Helios+FoxP3+Tregs are likely to play a pivotal role in disease activity of UC and may be influenced by EBV infection.

Clinical features of IgG4-related retroperitoneal fibrosis among 407 patients with IgG4-related disease: a retrospective study.

OBJECTIVES: IgG4-related disease (IgG4-RD) is recently recognized as a fibro-inflammatory condition featured by tumefactive lesions in multiple organs, and the retroperitoneum is one of the common involved sites. We undertook this study to compare detailed demographic, clinical and laboratory characteristics of IgG4-RD patients with retroperitoneum lesion (IgG4-RD RPF+) and retroperitoneum free IgG4-RD (IgG4-RD RPF-) in a large cohort. METHODS: We carried out a retrospective review of the medical records of 407 cases of IgG4-RD diagnosed at Peking University People's Hospital between March 2009 and May 2019. RESULTS: Among 407 patients, 58 had retroperitoneum affected. As compared with IgG4-RD RPF- patients, IgG4-RD RPF+ patients showed older age at disease onset and diagnosis. IgG4-RD RPF+ group involved more male patients. In terms of organ involvement, IgG4-RD RPF+ group was more frequently presented with kidney involvement, while salivary gland, lacrimal gland and pancreas were more prominent in the IgG4-RD RPF- group. In addition, the CRP, ESR level and creatinine level were significantly higher in IgG4-RD RPF+ patients, and hypocomplementemia were more common in this group. CONCLUSION: We have revealed demographic, clinical and laboratory differences between IgG4-RD RPF+ and RPF- patients, which indicated potential differences in pathogenesis and important implications for the diagnosis and management of these two phenotypes.

Elevated circulating pro-inflammatory low-density granulocytes in adult-onset Still's disease.

OBJECTIVES: Neutrophilia is a hallmark of adult-onset Still's disease (AOSD). This study aimed to investigate the role of a distinct subset of granulocytes, the low-density granulocytes (LDGs) in the pathogenesis of AOSD. METHODS: A total of 56 patients with AOSD were included in the study. LDGs were quantified by flow cytometry. Correlations between LDGs with disease activity and laboratory parameters were determined by Spearman's nonparametric test. The cellular sources of the pro-inflammatory cytokines in AOSD were determined by intracellular staining. RESULTS: Active AOSD patients displayed significantly higher levels of LDGs compared with inactive AOSD patients and healthy controls (HCs) (P<0.001). Circulating LDGs were significantly correlated with CRP, ESR and the modified Pouchot score in patients with AOSD (P<0.01). The levels of LDGs were significantly decreased after the active AOSD patients achieved disease remission (P=0.0391). CD14+ monocytes constituted over 90% IL-1ß+ peripheral blood mononuclear cells (PBMCs) and over 80% TNF-α+ PBMCs in both active AOSD patients and HCs, respectively. In active AOSD, CD14+ monocytes accounted for 24.6% to 75.0% of IL-6+ PBMCs, while LDGs comprised 22.8% to 72.2% of IL-6+ PBMCs. In contrast, over 90% IL-6+ PBMCs were CD14+ monocytes in HCs. A significant correlation was identified between the levels of LDGs and serum IL-6 levels in AOSD (P<0.0001). CONCLUSION: Active AOSD is associated with elevated levels of a pro-inflammatory subset of neutrophils, the LDGs that produce IL-6. Our data highlight an unappreciated role of LDGs in the aberrant innate immune responses in AOSD.

Increased Circulating Th1 and Tfh1 Cell Numbers Are Associated with Disease Activity in Glucocorticoid-Treated Patients with IgG4-Related Disease.

BACKGROUND: This study is aimed at exploring the changes and significance of circulating Th and Tfh cell subsets in glucocorticoid-treated IgG4-RD patients. METHODS: 39 glucocorticoid-treated IgG4-RD patients and 22 healthy controls (HC) were enrolled. Peripheral blood mononuclear cells were separated, and circulating Th and Tfh cell subsets were examined by flow cytometry according to the surface and intranuclear markers. Disease activity was accessed by the IgG4-RD responder index (RI) score. Correlation analyses were conducted between Th/Tfh subset numbers and clinical indicators. The receiver operating characteristic (ROC) curve was used to evaluate the efficacy of Th and Tfh subsets to distinguish active IgG4-RD patients from remission IgG4-RD patients. RESULTS: Circulating Th1, Th17, Tfh1, and Tfh17 cells were significantly increased in active IgG4-RD patients compared with HC. Th1 and Tfh1 numbers were positively correlated with serum IgG4 levels in patients with IgG4-RD. Meanwhile, the absolute numbers of circulating Th1 and Tfh1 cells were positively correlated with IgG4-RD RI scores. The areas under the curve (AUC) were 0.8276 for Th1 and 0.7310 for Tfh1, 0.5862 for Tfh2, and 0.6810 for Tfh17. CONCLUSION: Increased circulating Th1 and Tfh1 subsets are related to elevated serum IgG4 levels in active IgG4-RD patients during glucocorticoid treatment, which may play an important role in the course of IgG4-RD disease, and could be potential biomarkers for monitoring disease activity of IgG4-RD.

Use of the Nuclear Matrix Protein 22 BladderChek Test for the Detection of Primary and Recurrent Urothelial Carcinoma.

OBJECTIVE: To evaluate the performance of the nuclear matrix protein 22 (NMP22) BladderChek test in urothelial carcinoma (UC). METHODS: We retrospectively analyzed 1318 patients who performed the NMP22 BladderChek tests. Of them, 103 were primary UC patients, 90 were surgical treatment UC patients, and 1125 were benign disease patients. The performance of the NMP22 BladderChek test for the diagnosis of primary and recurrent UC was evaluated. Moreover, the performance of urine cytology and the NMP22 BladderChek test for the diagnosis of primary UC was compared in 90 available subjects including 48 primary UC patients and 42 benign disease patients. RESULTS: The sensitivity and specificity of the NMP22 BladderChek test were 37.9% and 95.8%, respectively, for the diagnosis of primary UC (n = 1228). The corresponding parameters of the NMP22 BladderChek test were 31.0% and 88.5%, respectively, for the diagnosis of recurrent UC (n = 90). The sensitivity and specificity of urine cytology were 54.2% and 97.6%, respectively, for the diagnosis of primary UC (n = 90); the corresponding parameters of the NMP22 BladderChek test were 41.7% and 83.3%, respectively; the corresponding parameters of the two tests combination were 64.6% and 83.3%, respectively. There was a significant difference in the performance between the NMP22 BladderChek test and urine cytology or the combination of two tests (P = 0.017 and 0.001, respectively). CONCLUSIONS: The NMP22 BladderChek test has a low sensitivity for detecting primary and recurrent UC. Urine cytology is superior to the NMP22 BladderChek test, and combined use of the two tests improves the sensitivity in the detection of primary UC.

The Imbalance of Circulating Follicular Helper T Cells and Follicular Regulatory T Cells Is Associated With Disease Activity in Patients With Ulcerative Colitis.

Ulcerative colitis (UC) is a chronic inflammatory bowel disease affecting the colon and rectum, in which the abnormality of B cells is involved in both its pathogenesis and progression. Follicular helper T cells (TFH) play an important role in assisting the immune function of human B cells in germinal centers, and follicular regulatory T cells (TFR) have the function of inhibiting TFH and germinal center B cell responses. The significance of circulating TFH and TFR in ulcerative colitis (UC) remains unclear. We analyzed peripheral blood of active and stable remission UC patients and found that circulating TFR was significantly decreased while TFH was increased in active UC patients. As to TFH subsets, TFH2 was elevated while TFH17 was decreased in active UC, with IL-4/IL-17A secretion enhanced. Helios+ and CD45RA-FoxP3high TFR cells were decreased while CD226+ and CD45RA+FoxP3int TFR cells were increased in active UC patients. The levels of new memory B cells, plasmablasts and serum IgG were significantly increased in active UC patients, and were positively correlated with TFH and TFH2, and negatively correlated with TFR. Serum CRP and Mayo Clinic scores were positively correlated with TFH and TFH2 but negatively correlated with TFR. Serum IL-12 and IL-21 were up-regulated while IL-10 was down-regulated in active UC. To conclude, an imbalance of circulating TFH and TFR cells is associated with disease activity in UC patients. Our results suggest a new mechanism for TFH and TFR imbalance in the pathogenesis of UC, providing a new perspective for theoretical research and therapeutic strategies for UC.

Relapse predictors and serologically unstable condition of IgG4-related disease: a large Chinese cohort.

OBJECTIVES: Patients with IgG4-related disease (IgG4-RD) typically respond well to initial glucocorticoid therapy, but always relapse with tapered or maintenance dosage of steroid. We aimed to identify the risk factors for relapse of IgG4-RD and explore the impact of active intervention on the serologically unstable condition. METHODS: We performed a retrospective study of 277 IgG4-RD patients at Peking University People's Hospital from February 2012 through February 2019. They were all followed for >4 months. The primary outcome was patient relapse. Data on recurrence of IgG4-RD symptoms, laboratory and image findings were recorded, along with information on treatment in the serologically unstable condition. RESULTS: The cumulative relapse rate was 12.86%, 27.84% and 36.1% at 12, 24 and 36 months, respectively. Younger age at onset, younger age at diagnosis, longer time from diagnosis to treatment and history of allergy were associated with relapse. Identified independent risk factors were longer time from diagnosis to treatment and history of allergy. When serum IgG4 level was 20%, 50% or 100% higher than that of the remission period, similar percentages of patients finally relapsed, regardless of whether they were in the immunosuppression intensified or non-intensified group. Median duration from serum IgG4 level instability to relapse in the intensified and non-intensified group was not statistically different. CONCLUSION: The risk factors of relapse were longer time from diagnosis to treatment and history of allergy. Intervention in the serologically unstable condition was not helpful for reducing relapse rate.