RAPSYN-mediated neddylation of BCR-ABL alternatively determines the fate of Philadelphia chromosome-positive leukemia.

Philadelphia chromosome-positive (Ph+) leukemia is a fatal hematological malignancy. Although standard treatments with tyrosine kinase inhibitors (TKIs) have achieved remarkable success in prolonging patient survival, intolerance, relapse, and TKI resistance remain serious issues for patients with Ph+ leukemia. Here, we report a new leukemogenic process in which RAPSYN and BCR-ABL co-occur in Ph+ leukemia, and RAPSYN mediates the neddylation of BCR-ABL. Consequently, neddylated BCR-ABL enhances the stability by competing its c-CBL-mediated degradation. Furthermore, SRC phosphorylates RAPSYN to activate its NEDD8 E3 ligase activity, promoting BCR-ABL stabilization and disease progression. Moreover, in contrast to in vivo ineffectiveness of PROTAC-based degraders, depletion of RAPSYN expression, or its ligase activity decreased BCR-ABL stability and, in turn, inhibited tumor formation and growth. Collectively, these findings represent an alternative to tyrosine kinase activity for the oncoprotein and leukemogenic cells and generate a rationale of targeting RAPSYN-mediated BCR-ABL neddylation for the treatment of Ph+ leukemia.

Chronic myeloid leukemia (CML for short) accounts for about 15% of all blood cancers diagnosed in adults in the United States. The condition is characterized by the overproduction of immature immune cells that interfere with proper blood function. It is linked to a gene recombination (a type of mutation) that leads to white blood cells producing an abnormal 'BCR-ABL' enzyme which is always switched on. In turn, this overactive protein causes the cells to live longer and divide uncontrollably. Some of the most effective drugs available to control the disease today work by blocking the activity of BCR-ABL. Yet certain patients can become resistant to these treatments over time, causing them to relapse. Other approaches are therefore needed to manage this disease; in particular, a promising avenue of research consists in exploring whether it is possible to reduce the amount of the enzyme present in diseased cells. As part of this effort, Zhao, Dai, Li, Zhang et al. focused on RAPSYN, a scaffolding protein previously unknown in CML cells. In other tissues, it has recently been shown to participate in neddylation ­ a process by which proteins receive certain chemical 'tags' that change the way they behave. The experiments revealed that, compared to healthy volunteers, RAPSYN was present at much higher levels in the white blood cells of CML patients. Experimentally lowering the amount of RAPSYN in CML cells led these to divide less quickly ­ both in a dish and when injected in mice, while also being linked to decreased levels of BCR-ABL. Additional biochemical experiments indicated that RAPSYN sticks with BCR-ABL to add chemical 'tags' that protect the abnormal protein against degradation, therefore increasing its overall levels. Finally, the team showed that SRC, an enzyme often dysregulated in emerging cancers, can activate RAPSYN's ability to conduct neddylation; such mechanism could promote BCR-ABL stabilization and, in turn, disease progression. Taken together, these experiments indicate a new way by which BCR-ABL levels are controlled. Future studies should investigate whether RAPSYN also stabilizes BCR-ABL in patients whose leukemias have become resistant to existing drugs. Eventually, RAPSYN may offer a new target for overcoming drug-resistance in CML patients.

Clinical prognostic value of different NPM1 mutations in acute myeloid leukemia patients.

BACKGROUND: Acute myeloid leukemia (AML) patients with various nucleophosmin 1 (NPM1) mutations are controversial in the prognosis. This study aimed to investigate the prognosis of patients according to types of NPM1 mutations (NPM1mut). METHODS: Bone marrow samples of 528 patients newly diagnosed with AML, were collected for morphology, immunology, cytogenetics, and molecular biology examinations. Gene mutations were detected by next-generation sequencing (NGS) technology. RESULTS: About 25.2% of cases exhibited NPM1mut. 83.5% of cases were type A, while type B and D were respectively account for 2.3% and 3.0%. Furthermore, 15 cases of rare types were identified, of which 2 cases have not been reported. Clinical characteristics were similar between patients with A-type NPM1 mutations (NPM1A - type mut) and non-A-type NPM1 mutations (NPM1non - A-type mut). Event-free survival (EFS) was significantly different between patients with low NPM1non - A-type mut variant allele frequency (VAF) and low NPM1A - type mut VAF (median EFS = 3.9 vs. 8.5 months, P = 0.020). The median overall survival (OS) of the NPM1non - A-type mutFLT3-ITDmut group, the NPM1A - type mutFLT3-ITDmut group, the NPM1non - A-type mutFLT3-ITDwt group, and the NPM1A - type mutFLT3-ITDwt group were 3.9, 10.7, 17.3 and 18.8 months, while the median EFS of the corresponding groups was 1.4, 5.0, 7.6 and 9.2 months (P < 0.0001 and P = 0.004, respectively). CONCLUSIONS: No significant difference was observed in OS and EFS between patients with NPM1A - type mut and NPM1non - A-type mut. However, types of NPM1 mutations and the status of FLT3-ITD mutations may jointly have an impact on the prognosis of AML patients.

Performance of a novel eight-color flow cytometry panel for measurable residual disease assessment of chronic lymphocytic leukemia.

BACKGROUND: Measurable residual disease (MRD) is an important prognostic indicator of chronic lymphocytic leukemia (CLL). Different flow cytometric panels have been developed for the MRD assessment of CLL in Western countries; however, the application of these panels in China remains largely unexplored. METHODS: Owing to the requirements for high accuracy, reproducibility, and comparability of MRD assessment in China, we investigated the performance of a flow cytometric approach (CD45-ROR1 panel) to assess MRD in patients with CLL. The European Research Initiative on CLL (ERIC) eight-color panel was used as the "gold standard." RESULTS: The sensitivity, specificity, and concordance rate of the CD45-ROR1 panel in the MRD assessment of CLL were 100% (87/87), 88.5% (23/26), and 97.3% (110/113), respectively. Two of the three inconsistent samples were further verified using next-generation sequencing. In addition, the MRD results obtained from the CD45-ROR1 panel were positively associated with the ERIC eight-color panel results for MRD assessment (R = 0.98, p < 0.0001). MRD detection at low levels (≤1.0%) demonstrated a smaller difference between the two methods (bias, -0.11; 95% CI, -0.90 to 0.68) than that at high levels (>1%). In the reproducibility assessment, the bias was smaller at three data points (within 24, 48, and 72 h) in the CD45-ROR1 panel than in the ERIC eight-color panel. Moreover, MRD levels detected using the CD45-ROR1 panel for the same samples from different laboratories showed a strong statistical correlation (R = 0.99, p < 0.0001) with trivial interlaboratory variation (bias, 0.135; 95% CI, -0.439 to 0.709). In addition, the positivity rate of MRD in the bone marrow samples was higher than that in the peripheral blood samples. CONCLUSIONS: Collectively, this study demonstrated that the CD45-ROR1 panel is a reliable method for MRD assessment of CLL with high sensitivity, reproducibility, and reliability.

[Comprehensive Diagnosis of Mantle Cell Lymphoma].

OBJECTIVE: To explore the value of multiple detection methods based on histopathology and supplemented by bone marrow or peripheral blood sample detections in the comprehensive diagnosis of mantle cell lymphoma (MCL). METHODS: The clinical, immunophenotypic, pathologic, cytogenetic and molecular features of 153 newly diagnosed MCL patients admitted to the hematology department of our hospital from May 2009 to September 2022 were analyzed. RESULTS: 144 (96.6%) of the 149 MCL patients who underwent marrow or peripheral blood IGH/CCND1 FISH detection at initial diagnosis were positive, of which 36 cases (24.2%) had a low proportion positive. The immunophenotypes in 115 patients were analyzed by flow cytometry (FCM), 89 cases (77.4%) conformed to MCL while 23 cases (20.0%) were initially diagnosed as B-cell lymphoproliferative disorders (B-LPD). Of the 75 cases who performed bone marrow biopsy, 50 cases (66.7%) had morphological and immunophenotypic characteristics consistent with MCL, 15 cases (20.0%) were classified as B-LPD, and 10 cases with no obvious abnormality. 77 patients underwent histopathology examination, of which 73 cases (94.8%) had typical clinicopathological features of MCL, including 2 CCND1 negative MCL, 2 pleomorphic variants, 5 pleomorphic variants and 4 cases diagnosed as other leukemia or lymphoma. Among 153 cases of MCL, 128 cases were classic MCL(cMCL), and another 25 cases (16.3%) were diagnosed as leukemic non-lymph node MCL (lnnMCL). The incidence of IGHV mutation, TP53 mutation and CD23 expression positive were significantly different between cMCL and lnnMCL. CONCLUSION: Histopathology is still the main standard for the diagnosis of cMCL, and detection based on bone marrow or peripheral blood samples is an important means for the diagnosis of lnnMCL. Single marker or examination can cause a certain proportion of misdiagnosis. The accurate diagnosis of MCL depends on a combination of multiple detection methods.

Enhancing morphological analysis of peripheral blood cells in chronic lymphocytic leukemia with an artificial intelligence-based tool.

BACKGROUND: Real-time monitoring is essential for the management of chronic lymphocytic leukemia (CLL) patients. Utilizing peripheral blood is advantageous due to its affordability and convenience. Existing methods of assessing peripheral blood films have limitations that include lack of automation, dependence on personal experience, and low repeatability and reproducibility. To overcome these challenges, we have designed an artificial intelligence-driven system that provides a clinical perspective to objectively evaluate morphologic features in CLL patients' blood cells. METHODS: Based on our center's CLL dataset, we developed an automated algorithm using a deep convolutional neural network to precisely identify regions of interest on blood films and used the well-established Visual Geometry Group-16 as the encoder to segment cells and extract morphological features. This tool enabled us to extract morphological features of all lymphocytes for subsequent analysis. RESULTS: Our study's lymphocyte identification had a recall of 0.96 and an F1 score of 0.97. Cluster analysis identified three clear, morphological groups of lymphocytes that reflect distinct stages of disease development to some extent. To investigate the longitudinal evolution of lymphocyte, we extracted cellular morphology parameters at various time points from the same patient. The results showed some similar trends to those observed in the aforementioned cluster analysis. Correlation analysis further supports the prognostic potential of cell morphology-based parameters. CONCLUSION: Our study provides valuable insights and potential avenues for further exploration of lymphocyte dynamics in CLL. Investigating morphological changes may help in determining the optimal timing for intervening with CLL patients, but further research is needed.

Clinical Significance of Methyltransferase-like 16 Expression in Epithelial Ovarian Cancer.

OBJECTIVE: To detect methyltransferase-like (METTL) 16 expression in epithelial ovarian cancer (EOC) by immunohistochemistry (IHC), and its relationship with clinicohistopathological parameters and prognosis. STUDY DESIGN: Observational study. PLACE AND DURATION OF STUDY: Department of Gynaecology and Pathology, The First Hospital of Wannan Medical College (Yijishan Hospital of Wannan Medical College), from February to June 2022. METHODOLOGY: METTL16 expression in 115 EOC patients was evaluated by IHC. According to the immunoreactive score (IRS), scores <6 represented low expressions and ≥6 high expressions. Clinicopathologic data and follow-up information were collected for statistical evaluation. RESULTS: METTL16 expression decreased in EOC (p = 0.001) and affected the poor prognosis of EOC patients. Low METTL16 patients expression had significantly higher frequencies of advanced FIGO stage, low grade, more lymph node metastasis, high CA125 levels, bilateral disease, distant metastasis, and high frequency of neural/vascular invasion compared to high METTL16 patients (p ≤0.001, <0.001, <0.001, 0.017, 0.027, <0.001, and 0.010, respectively). The survival analysis showed that the overall survival (p <0.0001) as well as the disease-free survival (p <0.0001) were remarkably shorter in low METTL16 patients compared to high METTL16 patients, suggesting worse survival. CONCLUSION: There was a clear association between the expression of METTL16, poor prognostic factors, and lower survival of EOC patients, suggesting that it might exert a vital effect on the malignant progression / prognosis of EOC. KEY WORDS: Epithelial ovarian cancer (EOC), METTL16, Immunohistochemistry, Prognosis.

Identification of clinical implications and potential prognostic models of chromatin regulator mutations in multiple myeloma.

BACKGROUND: With the rapid development of next-generation sequencing (NGS) technologies, researchers are making efforts to reveal the genomic landscape of multiple myeloma (MM). However, the clinical significance of many mutations remains poorly defined due to the genetic heterogeneity of MM. To systematically explore the clinical implications of gene mutations and build practical prognostic models, we performed DNA sequencing in newly diagnosed MM patients. METHODS: MM cells were purified from bone marrow aspirates using CD138 microbeads and subjected to sequencing with a 387-gene Panel. Nomogram was developed using Cox's proportional hazards model, and candidate variables were screened by stepwise regression. Internal validation was carried out by the bootstrap method. RESULTS: Between July 2016 and December 2020, a total of 147 patients were included in our study. We found patients with a higher mutational load had a significantly shorter progress-free survival (PFS) (19.0 vs. 32.0 months, P = 0.0098) and overall survival (OS) (3-year OS rates were 66.1% and 80.0%, P = 0.0290). Mutations in chromatin regulators (CRs) including KMT2C (14.3%), KMT2D (14.3%), EP300 (11.6%) and ARID gene family (31.3%) were highly frequent in newly diagnosed MM patients. Interestingly, proteins encoded by these genes could form a complex called KMT2C/D COMPASS (KCDCOMs). Patients with mutations of ARID gene family had a significantly shorter PFS (15.5 vs. 34.0 months, P = 0.0003) and OS (3-year OS rates were 64.9% and 81.0%, P = 0.0351) than patients without ARID gene mutations. Incorporating ARID gene mutations into the current staging system could successfully improve their prognostic performance. The PFS and OS nomogram models (including 1q21 copies, ARID gene mutations, extramedullary disease, mutational load and TP53 mutations) showed good predicting performance in both training and validation sets. CONCLUSION: Our findings emphasized the importance of CRs mutations in newly diagnosed MM patients and indicated the mutations affecting KCDCOMs might promote the development of MM. High mutational load and harboring mutations in the ARID gene family were novel predictors of adverse prognosis in MM. Prognostic models based on gene mutations were commendably prognostic evaluation methods that could provide a reference for clinical practices.

Single-cell profiling-guided combination therapy of c-Fos and histone deacetylase inhibitors in diffuse large B-cell lymphoma.

BACKGROUND: Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of non-Hodgkin lymphoma. Histone deacetylase inhibitors (HDACis) have been widely applied in multiple tumours, but the expected efficacy was not observed in DLBCL. Therefore, this study is aimed to explore superior HDACis and optimise a relative combinational therapeutic strategy. METHODS: The antitumour effects of the drug were evaluated by Cell Counting Kit-8 (CCK-8) assay and apoptosis analysis. Single-cell RNA sequencing (scRNA-Seq) was used to analyse the intratumoural heterogeneity of DLBCL cells. Whole-exome sequencing and RNA sequencing were performed to analyse the genetic and transcriptional features. Western blotting, qRT-PCR, protein array, immunohistochemistry, and chromatin immunoprecipitation assays were applied to explore the involved pathways. The antitumour effects of the compounds were assessed using subcutaneous xenograft tumour models. RESULTS: LAQ824 was screened and confirmed to kill DLBCL cells effectively. Using scRNA-Seq, we characterised the heterogeneity of DLBCL cells under different drug pressures, and c-Fos was identified as a critical factor in the survival of residual tumour cells. Moreover, we demonstrated that combinatorial treatment with LAQ824 and a c-Fos inhibitor more potently inhibited tumour cells both in vitro and in vivo. CONCLUSION: Altogether, we found an HDACi, LAQ824, with high efficacy in DLBCL and provided a promising HDACi-based combination therapy strategy.

Functional Evaluation of KEL as an Oncogenic Gene in the Progression of Acute Erythroleukemia.

Acute erythroleukemia (AEL) is an infrequent subtype of acute myeloid leukemia (AML) with worse prognosis. Though the last decade has seen major advances in the novel features and genomic landscape in AEL, there is still a lack of specific therapeutic targets and effective treatment approaches for this disease. Here, we found a novel oncogene KEL that specifically and aberrantly expressed in patients with AEL. In this study, we demonstrated that KEL promoted cell proliferation and the downregulation of KEL reversed drug resistance in AEL cells to JQ1. Our findings suggested that KEL contributed to gain of H3K27 acetylation and promoted erythroid differentiation induced by GATA1. Additionally, GATA1 and TAL1 as cotranscription factors (TFs) modulated the expression of KEL. Maintaining cell viability and differentiation, KEL also played parts in the immune evasion of tumor cells. Our work expands the current knowledge regarding molecular mechanisms involved in cancer onset and progression, offering promising therapeutic target to broaden the treatment options.

[A Retrospective Analysis of TKI Discontinuation in Patients with CML].

OBJECTIVE: To investigate the clinical characteristics of the patients with chronic myeloid leukemia (CML) discontinued tyrosine kinase inhibitors (TKI) therapy and the outcome of the patients. METHODS: 35 cases of CML patients experienced initiative discontinuation of TKI therapy in our hospital from June 1st 2015 to December 31th 2019 were retrospectively analyzed. The TFR of the patients and the factors affecting it were analyzed. RESULTS: The median duration of TKI administration was 72 (range 35-173) months in the 35 patients. Among these patients, 8 had experienced TKI dose reduction or suspension. All the enrolled patients have achieved at least MMR. The median time for these patients achieving MMR was 15 (range 3-75) months after administration of TKI, and for MMR maintenance before TKI suspension was 55 (range 13-164) months. After TKI withdrawal the median follow up time was 20.3 (range 3-57.9) months, 22 out of 35 patients kept TFR, among them, 2 （5.71%） patients restarted TKI after 12 month suspension, and maintained MMR during suspension. 13 （37.1%）patients lost MMR, among them, 9 patients restarted TKI treatment, and 5 of them achieved MR4.0 after the median duration of 3(2-5) month. No patients were found to have disease progression. The estimated TFR rate was 57.8% and 51.8% at 12 and 24 months after discontinuation, respectively. Other clinical characteristic related to relapse were also analyzed, including the cumulative TKI administration duration, cumulative MMR duration, time to achieve MMR, median age at diagnosis, risk stratification by Sokal score, TKI dose reduction and discontinuation history, and second-generation TKI administration before stopping TKI, however, no statistical difference was found. CONCLUSION: TKI discontinuation is practical for CML patients in our center.

Low prevalence and independent prognostic role of del(11q) in Chinese patients with chronic lymphocytic leukemia.

The 11q deletion (del(11q)) is a conventional cytogenetic aberration observed in chronic lymphocytic leukemia (CLL) patients. However, the prevalence and the prognostic value of del(11q) are still controversial. In this research, we retrospectively explored the prevalence, association, and prognostic significance of del(11q) in 352 untreated and 99 relapsed/refractory Chinese CLL patients. Totally 11.4% of untreated and 19.2% of relapsed/refractory patients harbored del(11q). Del(11q) was more common in patients with ß2-microglobulin > 3.5 mg/L, positive CD38, positive zeta-chain associated protein kinase 70, unmutated immunoglobulin heavy variable-region gene and ataxia telangiectasia mutated mutation. Kaplan-Meier method and univariate Cox regression indicated that del(11q) was an independent prognostic factor for overall survival (OS). Based on the results of univariate Cox regression analysis, two nomograms that included del(11q) were established to predict survival. Desirable area under curve of receiver operating characteristic curves was obtained in the training and validation cohorts. In addition, the calibration curves for the probability of survival showed good agreement between the prediction by nomogram and actual observation. In summary, the prevalence of del(11q) is relatively low in our cohort and del(11q) is an unfavorable prognostic factor for untreated CLL patients. Besides, these two nomograms could be used to accurately predict the prognosis of untreated CLL patients.

Co-occurrence of KIT and NRAS mutations defines an adverse prognostic core-binding factor acute myeloid leukemia.

Molecular abnormalities are frequent in core-binding factor (CBF) AMLs, but their prognostic relevance is controversial. Sixty-two patients were retrospectively analyzed and 47 harbored at least one gene mutation with a next-generation-sequencing assay. The most common molecular mutation was KIT mutation (30.6%), followed by NRAS (24.2%) and ASXL1 (14.5%) mutations, which was associated with a higher number of bone marrow blasts (p = .049) and older age (p = .027). The survival analysis showed KIT mutation adversely affected the overall survival (OS) (p = .046). NRAS mutation was associated with inferior OS (p = .016) and RFS (p = .039). Eight patients carried co-mutations of KIT and NRAS and had worse OS (p = .012) and RFS (p = .034). The multivariate analysis showed age ≥60 years and additional chromosomal abnormalities were significant adverse factors for OS. Thus, co-mutations of KIT and NRAS were significantly associated with a poor prognosis and should be taken into account when assessing for prognostic stratification in patients with CBF-AML.

Potential Effects of the FLT3-ITD Mutation on Chemotherapy Response and Prognosis of Acute Promyelocytic Leukemia.

PURPOSE: To evaluate the influence of FLT3-ITD mutations on the treatment response and long-term survival of newly-diagnosed patients with acute promyelocytic leukemia (APL) treated with all-trans retinoic acid and arsenic trioxide. METHODS: The long-term survival of 90 newly-diagnosed APL patients (age range 12-75 years) was retrospectively analyzed.The FLT3-ITD mutation rate was assayed by polymerase chain reaction (PCR) amplification and sequencing analysis. Its impact on the treatment response, event-free survival(EFS), or overall survival(OS) was investigated in patients with and without the mutations. RESULTS: The FLT3-ITD mutation rate in newly-diagnosed APL patients was 20% (18/90). The white blood cell (WBC) count at diagnosis in patients with mutations was significantly higher than that in patients without mutations while the FLT3-ITD mutation rate was higher in the high-risk group than in the low/intermediate-risk group. Patients with mutations had a significantly higher early death (ED) rate (16.67% vs 1.39%) for those lacking the mutation (P =0.024). However, the complete remission (CR) and differentiation syndrome (DS) rates in the two groups were similar. Kaplan Meier analysis for EFS and OS at five years showed a significant difference between the patients stratified by FLT3-ITD mutation status (log-rank P =0.010 and P =0.009, respectively). CONCLUSION: FLT3-ITD mutations can be related to high peripheral WBC counts in APL patients. APL patients with mutations displayed a higher ED rate compared to those without mutations. Patients carrying mutations had reduced five-year EFS and OS rates. Thus, reducing the overall death rate during induction treatment might be an effective way to improve the prognosis of patients with FLT3-ITD mutations.

Next-generation sequencing reveals gene mutations landscape and clonal evolution in patients with acute myeloid leukemia.

OBJECTIVES: The study aims to understand geneome diversification and complexity that developed in Acute myeloid leukemia (AML). METHODS: Next-generation sequencing (NGS) was used to identify the genetic profiles of 22 genes relevant to hematological malignancy in 204 patients with de novo non-M3 AML. RESULTS: At time of initial diagnosis, at least one mutation was identified in 80.9% of patients (165/204). The most commonly mutated gene was NPM1 (22.1%), followed by ASXL1 (18.1%), TET2 (18.1%), IDH2 (15.7%), CEBPA (14.7%), FLT3-ITD (13.2%) and DNMT3A (11.8%). Mutations landscape analysis indicated several patterns of co-occurring and mutual exclusive gene mutations. Some correlation was observed between gene mutations and clinicohematological features. Multivariate analysis showed that age >60 years, karyotypes, IDH2 and KIT mutations were the independent unfavorable prognostic factors for OS; NPM1-mut/ FLT3-ITD-wt was independently correlated with prolonged OS; whereas the independent poor risk factors for RFS were karyotypes, high WBC and RUNX1 mutation. According to different genotype demonstrated by multivariate analysis, 163 patients with intermediate-risk cytogenetics were classified into three subgroups: patients with NPM1-mut/ FLT3-ITD-wt or biallelic CEBPA mutation as favorable risk, patients with KIT, IDH2, TP53 or NRAS mutations as unfavorable risk, and the remaining was the intermediate risk. We also obtain information of clonal evolution during leukemia progression by observing five patients who underwent repeat NGS at relapse in our cohort. CONCLUSION: NGS techniques is a useful tool for discovering related gene mutations and clonal evolution in AML genomes, leading to novel targeted therapeutic approaches that could improve patients outcomes.

Management of Gout-associated MSU crystals-induced NLRP3 inflammasome activation by procyanidin B2: targeting IL-1ß and Cathepsin B in macrophages.

Gout, the most prevalent inflammatory arthritis worldwide, released interleukin-1ß (IL-1ß) and Cathepsin B inflammatory mediators that constitute the hallmark of the disease. Herein we aimed to investigate whether procyanidin B2 (PCB2), a natural dietary compound, can suppress MSU crystals-stimulated gouty inflammation. Treated with lipopolysaccharide (LPS) plus MSU, both mouse peritoneal macrophages (MPM) and mouse bone marrow-derived macrophages (BMDM) released a large amount of mature IL-1ß compared to those treated with MSU or LPS alone, while IL-1ß release was blocked by TLR4 and its downstream effector inhibitors. In two mouse models of gout, oral administration of PCB2 suppressed MSU crystals-induced increasing expression of IL-1ß, Cathepsin B and NLRP3 in the air pouch skin and paws, accompanied with the downregulation prostaglandin E2 (PGE2) in pouch exudates. Inflammatory immune cell infiltration including macrophages and neutrophils were significantly blocked by PCB2 in air pouch skin and paws of mice gout groups. PCB2 also suppressed the release of IL-1ß and Cathepsin B induced by MSU plus LPS in MPM. Our results suggest that the inhibitory effects of PCB2 on NLRP3 inflammasome may alleviate inflammatory response in gout, and this might be a promising anti-inflammatory mechanism of PCB2 against the inflammation in gout.

[Clinical Characteristics and Bone Marrow Histopathology Features in Essential Thrombocythaemia Patients with Different Gene Mutation in China].

OBJECTIVE: To investigate the clinical characteristics, laboratorial and bone marrow pathological features of primary thrombocytopenia (ET) patients with different mutations of CALR, JAK2 and MPL genes. METHODS: The chinical data of 120 cases of ET in Jiangsu provincial people's hospital/ The First Affiliated Hospital of Nanjing Medical University from January 2015 to December 2017 were collected and analyzed, including 76 cases with JAK2 gene mutation, 40 cases with CALR gene mutation, 2 cases with MPL gene mutations, 2 cases without gene mutation. RESULTS: Among the ET patients, compared with the JAK2 gene mutation, CALR gene mutation showed statistically significant deareament of white blood cells and hemoglobin (P=0.001, P=0.01) and the male platelets in CALR group showed significant increament (P=0.04). Fourthermore, the average number of megakaryocytes and its cluster numbers in each hight power field of vision showed statistically significant decreament in CALR group as compared with JAK2 group (P=0.001, P=0.001), and thrombotic events in CALR group were signicantly lower than those in JAK2 group (7.5% vs 18.4%) (P=0.03). CONCLUSION: Mutations of CALR, JAK2 have different clinical characteristics and blood pathological changes of Chinese ET patients, and their clinical significance is worth to explore.

P2X7R orchestrates the progression of murine hepatic fibrosis by making a feedback loop from macrophage to hepatic stellate cells.

HSCs (hepatic stellate cells) contribute to the excessive extracellular matrix (ECM) deposition, inflammatory pathways and crucial cell-cell interactions in hepatic disease leading to fibrosis. P2x7R is considered a potential orchestrater in liver fibrosis. For this reason, this work explored the role of P2x7R in liver fibrosis and the mechanism by which P2x7R in macrophages promotes fibrogenesis. In a model of liver fibrosis induced by administration of thioacetamide (TAA), inhibition of P2x7R with its selective inhibitor A438079 reversed TAA-induced liver damage and fibrosis. The mechanism was linked to inhibition of P2x7R-NLRP3 inflammasome activation and thereby infiltration of macrophages and neutrophils into the liver. This result indicated that the P2x7R-TLR4-NLRP3 axis is involved in the process of TGF-ß-mediated ECM deposition in HSCs. Ectopic overexpression of P2x7R lowered the threshold of extracellular matrix (ECM) deposition and maintained HSCs in an activated state. The culture medium of THP-1 macrophages stimulated by LPS/ATP aggravated ECM deposition in HSCs by activating P2x7R. Additionally, IL-1ß secreted by LPS / ATP activated macrophages amplified fibrosis. These data indicate that P2x7R plays a key regulative role in the activation and maintenance of HSCs promoted by macrophages. Thus, pharmacological inhibition of P2x7R could be a potential therapeutic mechanism to treat human liver fibrosis.

Flow Cytometric Pattern of TCRVδ Subtype Expression Rapidly Identifies γδT Cell Lymphoma.

Background: γδT cell lymphoma (γδ TCL) is a class of hematopoietic malignancy that expresses the γδ T cell receptor (TCR) with a low incidence. Determining the clonal proliferation of γδT cells is important for the diagnosis of such malignancies. Few studies have used flow cytometry to detect VδTCR and its subtypes (Vδ1 and Vδ2) at the protein level, although it is a practical method for determining the neoplastic γδT cells. Methods: A TCRVδ-based 10-color protocol was designed for the detection of malignant proliferation of γδT subtype cells by multiparameter flow cytometry, and the diagnostic results were compared with the gene rearrangement results. Results: All 19 cases of γδ TCL were positive for cluster of differentiation 3 (CD3) and TCR γδ and presented with abnormal distribution patterns of Vδ1 and Vδ2, of which 16 of the 19 cases showed a restricted Vδ1 staining pattern and the remaining three cases lacked the expression of either Vδ1 or Vδ2. Among the 10 normal controls and 11 patients with reactively higher CD4 and CD8 double-negative ratio, the percentage of Vδ2 positive events (range: 16.4-99.0%) was significantly higher than that of Vδ1 (range: 0-50.5%; p < 0.0001), and all cases had a normal Vδ distribution pattern. To detect clonality, there was no difference in the detection rate between the TCRVδ analysis and the gene scanning techniques (p = 1.000) with a high degree of coincidence (Kappa = 0.850, p < 0.001). The heteroduplex analysis was less sensitive than the other methods but was more specific (100%) than the gene scanning techniques, and the TCRVδ subtype analysis had the highest sensitivity, specificity, positive predictive value, and negative predictive value. Compared with molecular methods, immunophenotyping is able to distinguish the T cell lineage. Conclusion: The γδT panel, based on the TCRVδ antibody by flow cytometry, could be advantageous for the rapid identification of suspected γδTCL.