A Bioinformatic Analysis of Gut Microbiota Related with Immune Cell Infiltration in Colorectal Cancer.

OBJECTIVE: The composition of microbiota which correlates with infiltrating immune cells and clinical signatures is not clarified in CRC. METHODS: We applied 4 kinds of bioinformatic tools GSVA (version: 1.42.0), ESTIMATE (version: 1.0.13), CIBERSORT (version: 2.0), and immune-related genes. RESULTS: We found that a total of 8 types of microbiotas appeared in the three immune correlation analyses. Among these microbiotas, significant enrichments in relative abundances associated with immune cell infiltration can be found for the dominant phyla Proteobacteria, Firmicutes, and Actinobacteria. Moreover, there existed correlations between some of the 8 microbiotas and clinical-related indicators. CONCLUSION: We identified some novel microbiotas involved in immune regulation in CRC.

Uric acid-lowering effect of harpagoside and its protective effect against hyperuricemia-induced renal injury in mice.

Hyperuricemia (HUA) is caused by increased synthesis and/or insufficient excretion of uric acid (UA). Long-lasting HUA may lead to a number of diseases including gout and kidney injury. Harpagoside (Harp) is a bioactive compound with potent anti-inflammatory activity from the roots of Scrophularia ningpoensis. Nevertheless, its potential effect on HUA was not reported. The anti-HUA and nephroprotective effects of Harp on HUA mice were assessed by biochemical and histological analysis. The proteins responsible for UA production and transportation were investigated to figure out its anti-HUA mechanism, while proteins related to NF-κB/NLRP3 pathway were evaluated to reveal its nephroprotective mechanism. The safety was evaluated by testing its effect on body weight and organ coefficients. The results showed that Harp significantly reduced the SUA level and protected the kidney against HUA-induced injury but had no negative effect on safety. Mechanistically, Harp significantly reduced UA production by acting as inhibitors of xanthine oxidase (XOD) and adenosine deaminase (ADA) and decreased UA excretion by acting as activators of ABCG2, OAT1 and inhibitors of GLUT9 and URAT1. Moreover, Harp markedly reduced infiltration of inflammatory cells and down-regulated expressions of TNF-α, NF-κB, NLRP3 and IL-1ß in the kidney. Harp was a promising anti-HUA agent.

Severe gastrointestinal involvements in patients with adult dermatomyositis with anti-NXP2 antibody.

OBJECTIVE: Gastrointestinal (GI) involvements were scarcely reported in adult anti-nuclear matrix protein 2 (NXP2) dermatomyositis (NXP2+DM). In this study, we investigated the clinical, pathological and molecular features as well as treatment options of this rare yet life-threatening disease. METHODS: We retrospectively collected the data of the cohort of NXP2+ DM from 2012 to 2022 in our hospital. RNA sequencing was performed in intestinal samples of perforated patients compared with healthy controls data set. RESULTS: A total of 56 patients with adult NXP2+DM were collected including 10 cases with GI involvements. Abdominal pain and melena were the initial manifestations for GI involvements with a median 10-month time lag after the diagnosis of NXP2+DM when myositis largely subsided. Within weeks, GI perforation occurred in 8 of 10 patients, while five patients underwent eight surgical interventions subsequently. The short-term mortality was observed in four patients. NXP2+DM with GI involvements presented with more extramuscular systemic manifestations such as interstitial lung disease and subcutaneous calcinosis. The GI pathological features encompassed vasculitis/vasculopathy with high MxA expression, intestinal smooth muscle necrosis and serosal calcinosis. Gene expression profile validated the type-I interferon activation and revealed that epithelial mesenchymal transition and focal adhesion pathway may also contribute. Finally, vedolizumab, an anti-α4ß7-integrin monoclonal antibody, exhibited promising therapeutic signals which should be further investigated. CONCLUSIONS: GI involvement is a unique complication in patients with adult NXP2+DM. Timely recognition and targeted therapy may turn out to be lifesaving.

Single-Cell Profiling of Bone Marrow B Cells and Early B Cell Developmental Disorders Associated With Systemic Lupus Erythematosus.

OBJECTIVE: The peripheral B cell compartment is heavily disturbed in systemic lupus erythematosus (SLE), but whether B cells develop aberrantly in the bone marrow (BM) is largely unknown. METHODS: We performed single-cell RNA/B cell receptor (BCR) sequencing and immune profiling of BM B cells and classified patients with SLE into two groups: early B cell (Pro-B and Pre-B) normal (EBnor) and EB defective/low (EBlo) groups. RESULTS: The SLE-EBlo group exhibited more severe disease activity and proinflammatory status, overaction of type I interferon signaling and metabolic pathways within the B cell compartment, and aberrant BCR repertoires compared with the SLE-EBnor group. Moreover, in one patient with SLE who was initially classified in the SLE-EBlo group, early B cell deficiency and associated abnormalities were largely rectified in a second BM sample at the remission phase. CONCLUSION: In summary, this study suggests that early B cell loss in BM defines a unique pathological state in a subset of patients with SLE that may play an active role in the dysregulated autoimmune responses.

SMARCA4­deficient non­small cell lung cancer with an EGFR mutation: A case report.

SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily a, member 4 (SMARCA4)-deficient non-small cell lung cancer (dNSCLC) is a rare malignant tumor that originates in the lungs. It occurs more frequently in male smokers, and the epidermal growth factor receptor (EGFR) gene is often mutation-free. In the present study, the case of a 60-year-old, non-smoking female patient diagnosed with SMARCA4-dNSCLC is reported. Biopsy of the tumor showed solid flaky, nest-like infiltrating growth. Immunohistochemistry revealed the following: SMARCA4/BRG1(-), SMARCB1/INI-1(+), cytokeratin7 (+), cytokeratin 5.2 (+), CK5/6(+) and calretinin(+). The Ki-67 positivity index was 75%, and the thyroid transcription factor-1, NapsinA, p40, nuclear protein in testis, CD34, Sal-like protein 4, SRY-box transcription factor 2 and synaptophysin were negative. Molecular analysis showed mutations in both EGFR and TP53. The pathological diagnosis was SMARCA4-dNSCLC with an EGFR gene mutation. The present case report could be used for broadening the pathological diagnosis of SMARCA4-dNSCLC and for selecting appropriate treatment approaches.

Rare urachal mucinous cystic tumor of low malignant potential with peritoneal pseudomyxoma: A case report.

Mucinous cystic tumors of low malignant potential (MCTLMP) are rare urachal neoplasms. The morphological characteristics and clinical prognosis of MCTLMP is similar to that of mucinous cystic tumors occurring in the ovary and appendix. After complete resection, almost no cases of recurrence or metastasis have been reported. Because MCTLMP is rare, it may be missed in the clinic. MCTLMP can lead to the formation of pseudomyxoma peritonei (PMP), which manifests as the widespread production of mucus in the abdominal cavity and makes the disease complex or difficult to diagnose. At present, only 3 cases of MCTLMP with PMP have been reported in the literature. In the present study a fourth case of urachal MCTLMP in a 74-year-old male that resulted in widespread PMP is presented. Initially, a multilocular cystic lesion was revealed in the urachal duct area at the anterior upper margin of the bladder after a patient, experiencing lower abdominal pain, was imaged. As revealed using light microscopy, the cyst was lined with a mucous columnar epithelium, and part of the epithelium indicated pseudolamellar hyperplasia and papillary structures. The cells indicated mild atypia and low mitotic activity. There was no stromal infiltration of tumor cells, and a large amount of mucous exudate was observed. As preoperative computed tomography examination suggested the presence of a large amount of ascites and there were increased levels of blood tumor markers, carcinoembryonic antigen and carbohydrate antigen 125, clinicians considered that the diagnosis maybe a malignant tumor of the urachal gland with peripheral dissemination. However, the diagnosis of MCTLMP with PMP was confirmed by histopathological examination. The mass was completely removed, along with part of the peritoneum and bladder wall as these were within the tumor margin. The appendix appeared normal during surgery. A one off dose of intraperitoneal infusion chemotherapy with 1,000 mg 5-fluorouracil was performed after surgery. No recurrence was observed during the 8-month follow-up period.

Type I interferon score is associated with the severity and poor prognosis in anti-MDA5 antibody-positive dermatomyositis patients.

Objectives: To investigate the clinical significance of the interferon (IFN) score, especially the IFN-I score, in patients with anti-melanoma differentiation-associated gene 5 (MDA5) antibody-positive dermatomyositis (anti-MDA5+ DM). Methods: We enrolled 262 patients with different autoimmune diseases, including idiopathic inflammatory myopathy, systemic lupus erythematosus, rheumatoid arthritis, adult-onset Still's disease, and Sjögren's syndrome, as well as 58 healthy controls. Multiplex quantitative real-time polymerase chain reaction (RT-qPCR) using four TaqMan probes was used to evaluate type I IFN-stimulated genes (IFI44 and MX1), one type II IFN-stimulated gene (IRF1), and one internal control gene (HRPT1), which were used to determine the IFN-I score. The clinical features and disease activity index were compared between the high and low IFN-I score groups in 61 patients with anti-MDA5+ DM. The associations between laboratory findings and the predictive value of the baseline IFN-I score for mortality were analyzed. Results: The IFN score was significantly higher in patients with anti-MDA5+ DM than in healthy controls. The IFN-I score was positively correlated with the serum IFN-α concentration, ferritin concentration, and Myositis Disease Activity Assessment Visual Analogue Scale (MYOACT) score. Compared with patients with a low IFN-I score, patients with a high IFN-I score showed a higher MYOACT score, C-reactive protein concentration, aspartate transaminase concentration, ferritin concentration, plasma cell percentage, and CD3+ T-cell percentage, as well as lower lymphocyte, natural killer cell, and monocyte counts. The 3-month survival rate was significantly lower in patients with an IFN-I score of >4.9 than in those with an IFN-I score of ≤4.9 (72.9% vs. 100%, respectively; P = 0.044). Conclusion: The IFN score, especially the IFN-I score, measured by multiplex RT-qPCR is a valuable tool to monitor disease activity and predict mortality in patients with anti-MDA5+ DM.

Alpha-lipoic acid inhibits sodium arsenite-mediated autophagic death of rat insulinoma cells.

AIM: To investigate the protective effect of α-lipoic acid on sodium arsenite (NaAsO2) induced INS-1 cells injury and its mechanism. METHODS: The cell viability was measured by CCK-8 assay. The autophagosomes was observed under transmission electron microscopy. The autophagosomes in cells transfected with green fluorescent protein microtubule-associated protein light chain 3 (GFP-LC3) plasmids were observed under a laser scanning con-focal microscope. The expression of LC3-II, P62, PI3K, and mTOR proteins in INS-1 cells treated with a combination of chloroquine (CQ, autophagy inhibitor) and NaAsO2 were detected by Western blot assay. The expression of LC3-II, P62, PI3K, and mTOR proteins were detected in INS-1 cells treated with a combination of rapamycin (autophagy inducer, mTOR inhibitor) and α-LA. RESULTS: The cytotoxicity induced by NaAsO2 was reversed by α-LA, and the viability of NaAsO2-treated INS-1 cells increased. α-LA pretreatment decreased the autophagosome accumulation induced by NaAsO2. α-LA also reduced the fluorescence spot aggregation of GFP-LC3 in INS-1 cells exposed to NaAsO2 as observed under a laser scanning con-focal microscope. α-LA inhibited NaAsO2 induced autophagy by up-regulating PI3K and mTOR and down-regulating LC3-II and P62. CQ inhibited NaAsO2 induced autophagy by up-regulating PI3K, mTOR, P62 and down-regulating LC3-II. α-LA inhibited rapamycin-induced autophagy by up-regulating PI3K, mTOR and P62 and down-regulating LC3-II. The results showed that NaAsO2 could induce autophagy activation in INS-1 cells. The α-LA may inhibit autophagy activation by regulating the PI3K/mTOR pathway. CONCLUSION: The data indicated that α-LA might inhibit the NaAsO2-induced autophagic death of INS-1 cells by regulating the PI3K/mTOR pathway.

Longitudinal study of patients with antimelanoma differentiation-associated gene 5 antibody-positive dermatomyositis-associated interstitial lung disease.

OBJECTIVES: To describe the 8-year longitudinal study and long-term prognosis of a large inception cohort of anti-melanoma differentiation-associated gene 5 (MDA5) antibody-positive (MDA5+) DM-interstitial lung disease (ILD) patients. METHODS: In total, 216 patients diagnosed with MDA5+ DM-ILD were enrolled and followed up to analyse long-term survival rate. Demographic and clinical variables were collected at baseline and each temporal end point. Seventy patients who survived the first year were analysed for the long-term serological and respiratory outcomes. RESULTS: A total of 85 patients (39.3%) died during the follow-up period up to 96 months, with 89% of the deaths occurring in the first year after diagnosis. Long-term outcome was reported in 70 patients. Serological markers including anti-MDA5 antibody showed significant improvement with time. Radiographic findings and pulmonary function also improved notably in the follow-up period, especially in rapidly progressive ILD group, as measured by high-resolution computed tomography imaging scores, the estimated forced vital capacity, estimated diffusing capacity of lung carbon monoxide and dyspnoea scores. Early application of anti-fibrosis therapy helped to improve long-term pulmonary function. CONCLUSIONS: MDA5+ DM-ILD patients had a high mortality rate despite aggressive treatment. Patients who survived the first year usually showed a significant improvement in serological markers and pulmonary function during the long-term follow-up.

Single-cell profiling reveals distinct adaptive immune hallmarks in MDA5+ dermatomyositis with therapeutic implications.

Anti-melanoma differentiation-associated gene 5-positive dermatomyositis (MDA5+ DM) is an autoimmune condition associated with rapidly progressive interstitial lung disease and high mortality. The aetiology and pathogenesis of MDA5+ DM are still largely unknown. Here we describe the immune signatures of MDA5+ DM via single-cell RNA sequencing, flow cytometry and multiplex immunohistochemistry in peripheral B and T cells and in affected lung tissue samples from one patient. We find strong peripheral antibody-secreting cell and CD8+ T cell responses as cellular immune hallmarks, and over-stimulated type I interferon signaling and associated metabolic reprogramming as molecular immune signature in MDA5+ DM. High frequency of circulating ISG15+ CD8+ T cells at baseline predicts poor one-year survival in MDA5+ DM patients. In affected lungs, we find profuse immune cells infiltration, which likely contributes to the pro-fibrotic response via type I interferon production. The importance of type I interferons in MDA5+ DM pathology is further emphasized by our observation in a retrospective cohort of MDA5+ DM patients that combined calcineurin and Janus kinase inhibitor therapy show superior efficacy to calcineurin inhibitor monotherapy. In summary, this study reveals key immune-pathogenic features of MDA5+ DM and provides a potential basis for future tailored therapies.

Platelets control liver tumor growth through P2Y12-dependent CD40L release in NAFLD.

Platelets, the often-overlooked component of the immune system, have been shown to promote tumor growth. Non-alcoholic fatty liver disease (NAFLD) is a common disease in the Western world and rising risk for hepatocellular carcinoma (HCC). Unexpectedly, we observed that platelets can inhibit the growth of established HCC in NAFLD mice. Through pharmacological inhibition and genetic depletion of P2Y12 as well as in vivo transfusion of wild-type (WT) or CD40L-/- platelets, we demonstrate that the anti-tumor function of platelets is mediated through P2Y12-dependent CD40L release, which leads to CD8+ T cell activation by the CD40 receptor. Unlike P2Y12 inhibition, blocking platelets with aspirin does not prevent platelet CD40L release nor accelerate HCC in NAFLD mice. Similar findings were observed in liver metastasis models. All together, our study reveals a complex role of platelets in tumor regulation. Anti-platelet treatment without inhibiting CD40L release could be considered for liver cancer patients with NAFLD.

Leflunomide versus azathioprine for maintenance therapy of lupus nephritis: a prospective, multicentre, randomised trial and long-term follow-up.

OBJECTIVES: Previous studies have compared mycophenolate mofetil and azathioprine as maintenance therapy for lupus nephritis (LN). Leflunomide is an immunosuppressant widely used in the treatment of rheumatoid arthritis. The aim of this investigator-initiated study was to compare the efficacy and safety of leflunomide versus azathioprine as maintenance therapy for LN. METHODS: 270 adult patients with biopsy-confirmed active LN from 7 Chinese Rheumatology Centres were enrolled. All patients received induction therapy with 6-9 months of intravenous cyclophosphamide plus glucocorticoids. Patients who achieved complete response (CR) or partial response (PR) were randomised to receive prednisone in combination with leflunomide or azathioprine as maintenance therapy for 36 months. The primary efficacy endpoint was the time to kidney flare. Secondary outcomes included clinical parameters, extrarenal flare and adverse effects. RESULTS: A total of 215 patients were randomly allocated to the leflunomide group (n=108) and azathioprine group (n=107). Kidney flares were observed in 17 (15.7%) leflunomide-treated patients and 19 (17.8%) azathioprine-treated patients. Time to kidney flare did not statistically differ (leflunomide: 16 months vs azathioprine: 14 months, p=0.676). 24-hour proteinuria, serum creatinine, serum albumin, serum C3 and serum C4 improved similarly. Extrarenal flare occurred in two patients from the azathioprine group and one patient from the leflunomide group. The incidence of adverse events was similar in the 2 groups: leflunomide 56.5% and azathioprine 58.9%. CONCLUSIONS: The efficacy and safety profile of leflunomide are non-inferior to azathioprine for maintenance therapy of LN. Leflunomide may provide a new candidate for maintenance therapy in patients with LN. TRIAL REGISTRATION NUMBER: NCT01172002.

Two Distinct Immune Cell Signatures Predict the Clinical Outcomes in Patients With Amyopathic Dermatomyositis With Interstitial Lung Disease.

OBJECTIVE: Amyopathic dermatomyositis (ADM) is a heterogeneous and life-threatening autoimmune disease with a high mortality rate. In particular, anti-melanoma differentiation-associated protein 5 antibody-positive patients are at a high risk of developing rapidly progressive interstitial lung disease (RPILD). This study was undertaken to identify immunologic signatures among patients who have ADM with ILD (ADM-ILD) and to discover the biomarkers predicting prognosis. METHODS: The landscape of 42 immune cell phenotypes in the peripheral blood of 82 ADM-ILD patients and 82 age- and sex-matched healthy donors was assessed by multicolor flow cytometry. Patients were stratified using an unsupervised machine learning method (hierarchical clustering analysis) by immune cell subsets. Multiple Wilcoxon's signed rank tests and supervised machine learning methods were performed to identify important immune cell subsets. Kaplan-Meier survival analysis with log rank tests was used to create survival curves. RESULTS: We identified 2 distinct clusters correlating with different disease activities and clinical outcomes in ADM-ILD. Cluster 1 was enriched in the activated CD45RA+HLA-DR+CD8+ T cells with decreased CD56dim natural killer cell proportions and showed a higher prevalence of RPILD and higher mortality. In contrast, the other subgroup, cluster 2 (the nonactivated T cell-dominant cluster), displayed favorable clinical outcomes with high survival rates. Our data also revealed that immunophenotype was an independent risk factor associated with 1-year survival. CONCLUSION: Peripheral immunologic features may have the potential to stratify patients with ADM-ILD according to different disease severity and clinical outcomes, which may have implications for outcome prediction, pathogenesis study, and therapy selection.

Short-term, low-dose etoposide in refractory adult-onset Still's disease-associated macrophage activation syndrome.

INTRODUCTION: In this study, we modified the classical regimen of the hemophagocytic lymphohistiocytosis-04 protocol and evaluated the efficacy and safety of short-term, low-dose etoposide in patients with refractory macrophage activation syndrome (MAS) associated with adult-onset Still's disease (AOSD). METHODS: A total of 17 patients with refractory AOSD-associated MAS were enrolled and received short-term, low-dose etoposide (100 mg twice a week for four times). Another 11 patients, who were not treated with etoposide, were included as historical controls. Patient information, such as clinical manifestations, laboratory results, treatments, and short-term prognosis, were recorded and analyzed. RESULTS: In this case series, 88.24% of the patients with MAS who were treated with short-term, low-dose etoposide had a favorable response in 3 weeks, which was significantly higher (p = 0.017) than that in the patients with MAS who were treated without etoposide (45.45%). The 90-day survival rate after the onset of MAS was significantly higher (p = 0.0029) among the patients in the short-term etoposide group (16/17, 94.12%) than in the control group (5/11, 45.45%). CONCLUSION: The regimen of short-term (2 weeks), low-dose etoposide was highly effective in the treatment for patients with refractory AOSD-associated MAS with an acceptable safety profile. Key Points • There is no high level evidence to guide the management of refractory MAS-associated AOSD patients. • This study was the first to propose and confirm the efficacy and safety of short-term, low-dose etoposide in the treatment of refractory MAS-associated AOSD patients.

A Preliminary Study of Frequency and Clinical Relevance of Cytotoxic Peripheral CD4 and CD8 T Cells in Patients with Anti-MDA5 Positive Dermatomyositis.

Objectives: Anti-melanoma differentiation-associated gene 5-positive dermatomyositis (MDA5+DM) is an autoimmune disease frequently accompanied by rapidly progressive interstitial lung disease (RP-ILD) with high mortality. T cells are implicated in the pathogenesis of MDA5+DM and this study aims to measure the frequency and clinical relevance of cytotoxic CD4 and CD8 T cells in this disease. Methods: T cells expressing Perforin, Granzyme B (GZMB) and Granzyme K (GZMK) were analyzed by flow cytometry from peripheral blood of 19 patients with active MDA5+DM and 19 age- and sex-matched healthy donors (HDs). The frequency of CD4 and CD8 T cells and the cytotoxic subsets were compared between patients with MDA5+DM and HDs. Correlations within T cell subsets and between T cell subsets and clinical parameters of lactate dehydrogenase (LDH), ferritin, neutrophil-to-lymphocyte ratio (NLR), and Myositis Intention-to-Treat Index (MITAX) were evaluated. Results: Compared with HDs, patients with active MDA5+DM significantly had increased frequency of CD4 T cells, and reduced frequency of GZMK+GZMB- CD8 T cells. Furthermore, the frequency of GZMK+GZMB- CD8 T cells positively correlated with serum ferritin levels in active MDA5+DM patients. Notably, the patients in the Dead group of MDA5+DM had a significant higher frequency of GZMK+GZMB- CD4 and CD8 T cells. Conclusion: Substantial changes of cytotoxic T cell subsets are observed in active MDA5+DM patients. In addition, a high frequency of GZMK+GZMB- CD4 and CD8 T cells is associated with unfavorable prognosis in MDA5+DM. More studies are warranted to further explore the roles of cytotoxic T cells in MDA5+DM.

Gut Microbiome Directs Hepatocytes to Recruit MDSCs and Promote Cholangiocarcinoma.

Gut dysbiosis is commonly observed in patients with cirrhosis and chronic gastrointestinal disorders; however, its effect on antitumor immunity in the liver is largely unknown. Here we studied how the gut microbiome affects antitumor immunity in cholangiocarcinoma. Primary sclerosing cholangitis (PSC) or colitis, two known risk factors for cholangiocarcinoma which promote tumor development in mice, caused an accumulation of CXCR2+ polymorphonuclear myeloid-derived suppressor cells (PMN-MDSC). A decrease in gut barrier function observed in mice with PSC and colitis allowed gut-derived bacteria and lipopolysaccharide to appear in the liver and induced CXCL1 expression in hepatocytes through a TLR4-dependent mechanism and an accumulation of CXCR2+ PMN-MDSCs. In contrast, neomycin treatment blocked CXCL1 expression and PMN-MDSC accumulation and inhibited tumor growth even in the absence of liver disease or colitis. Our study demonstrates that the gut microbiome controls hepatocytes to form an immunosuppressive environment by increasing PMN-MDSCs to promote liver cancer. SIGNIFICANCE: MDSCs have been shown to be induced by tumors and suppress antitumor immunity. Here we show that the gut microbiome can control accumulation of MDSCs in the liver in the context of a benign liver disease or colitis.See related commentary by Chagani and Kwong, p. 1014.This article is highlighted in the In This Issue feature, p. 995.

Prognostic values of anti-Ro52 antibodies in anti-MDA5-positive clinically amyopathic dermatomyositis associated with interstitial lung disease.

OBJECTIVE: Anti-Ro52 antibody often co-occurs with anti-Jo1 antibody in antisynthetase syndrome and their co-occurrence correlates with a more aggressive clinical phenotype and poorer prognosis. The strong association of anti-Ro52 antibody with anti-melanoma differentiation-associated protein-5 (anti-MDA5) antibody has been indicated in juvenile myositis. The aim of this study was to assess the clinical significance of anti-Ro52 antibody in a cohort of adult patients with anti-MDA5-positive clinically amyopathic dermatomyositis with interstitial lung disease (CADM-ILD). METHODS: We assessed a cohort of 83 consecutive patients with anti-MDA5-positive CADM-ILD. Anti-MDA5 antibodies and anti-Ro52 antibodies were detected in immunoblotting and semi-quantitatively analysed by densitometry. Clinical features and the 24 month survival were compared between anti-MDA5-positive patients with and without anti-Ro52 antibodies. RESULTS: Anti-Ro52 antibodies were found in 74.7% of anti-MDA5-positive CADM-ILD patients and were associated with an increased frequency of rapidly progressive interstitial lung disease (RP-ILD; 54.8% vs 23.8%; P = 0.014) and cutaneous ulcerations (27.4% vs 4.8%; P = 0.033). The cumulative 24 month survival rate tended to be lower in patients with anti-Ro52 antibodies than patients without (59.9% vs 85.7%; P = 0.051). The combination of anti-Ro52 antibody status and anti-MDA5 antibody levels further stratified patients' survival rates, showing that the survival rate of patients who were dual positive for anti-MDA5 antibody and anti-Ro52 antibody was significantly lower than patients with mild positive anti-MDA5 antibody alone (59.9% vs 100%; P = 0.019). CONCLUSION: Anti-Ro52 antibody is highly prevalent in anti-MDA5-positive CADM-ILD patients and their coexistence correlates with a subgroup of patients with more aggressive phenotypes. The combination of anti-MDA5 antibody levels and anti-Ro52 antibody status could help to predict patients' prognosis and guide risk-based therapy.

Steatohepatitis Impairs T-cell-Directed Immunotherapies Against Liver Tumors in Mice.

BACKGROUND & AIMS: Nonalcoholic steatohepatitis causes loss of hepatic CD4+ T cells and promotes tumor growth. The liver is the most common site of distant metastases from a variety of malignancies, many of which respond to immunotherapy. We investigated the effects of steatohepatitis on the efficacy of immunotherapeutic agents against liver tumors in mice. METHODS: Steatohepatitis was induced by feeding C57BL/6NCrl or BALB/c AnNCr mice a methionine and choline-deficient diet or a choline-deficient l-amino acid-defined diet. Mice were given intrahepatic or subcutaneous injections of B16 melanoma and CT26 colon cancer cells, followed by intravenous injections of M30-RNA vaccine (M30) or intraperitoneal injections of an antibody against OX40 (aOX40) on days 3, 7, and 10 after injection of the tumor cells. We measured tumor growth and analyzed immune cells in tumor tissues by flow cytometry. Mice were given N-acetylcysteine to prevent loss of CD4+ T cells from liver. RESULTS: Administration of M30 and aOX40 inhibited growth of tumors from intrahepatic injections of B16 or CT26 cells in mice on regular diet. However, M30 and/or aOX40 did not slow growth of liver tumors from B16 or CT26 cells in mice with diet-induced steatohepatitis (methionine and choline-deficient diet or choline-deficient l-amino acid-defined diet). Steatohepatitis did not affect the ability of M30 to slow growth of subcutaneous B16 tumors. In mice with steatohepatitis given N-acetylcysteine, which prevents loss of CD4+ T cells, M30 and aOX40 were able slow growth of hepatic tumors. Flow cytometry analysis of liver tumors revealed reduced CD4+ T cells and effector memory cells in mice with vs without steatohepatitis. CONCLUSIONS: Steatohepatitis reduces the abilities of immunotherapeutic agents, such as M30 and aOX40, to inhibit tumor liver growth by reducing tumor infiltration by CD4+ T cells and effector memory cells. N-acetylcysteine restores T-cell numbers in tumors and increases the ability of M30 and aOX40 to slow tumor growth in mice.

The Roles of AMPK in Revascularization.

Coronary heart disease (CHD) is the most common and serious illness in the world and has been researched for many years. However, there are still no real effective ways to prevent and save patients with this disease. When patients present with myocardial infarction, the most important step is to recover ischemic prefusion, which usually is accomplished by coronary artery bypass surgery, coronary artery intervention (PCI), or coronary artery bypass grafting (CABG). These are invasive procedures, and patients with extensive lesions cannot tolerate surgery. It is, therefore, extremely urgent to search for a noninvasive way to save ischemic myocardium. After suffering from ischemia, cardiac or skeletal muscle can partly recover blood flow through angiogenesis (de novo capillary) induced by hypoxia, arteriogenesis, or collateral growth (opening and remodeling of arterioles) triggered by dramatical increase of fluid shear stress (FSS). Evidence has shown that both of them are regulated by various crossed pathways, such as hypoxia-related pathways, cellular metabolism remodeling, inflammatory cells invasion and infiltration, or hemodynamical changes within the vascular wall, but still they do not find effective target for regulating revascularization at present. 5'-Adenosine monophosphate-activated protein kinase (AMPK), as a kinase, is not only an energy modulator but also a sensor of cellular oxygen-reduction substances, and many researches have suggested that AMPK plays an essential role in revascularization but the mechanism is not completely understood. Usually, AMPK can be activated by ADP or AMP, upstream kinases or other cytokines, and pharmacological agents, and then it phosphorylates key molecules that are involved in energy metabolism, autophagy, anti-inflammation, oxidative stress, and aging process to keep cellular homeostasis and finally keeps cell normal activity and function. This review makes a summary on the subunits, activation and downstream targets of AMPK, the mechanism of revascularization, the effects of AMPK in endothelial cells, angiogenesis, and arteriogenesis along with some prospects.