ObjectiveTo investigate the association of serum exosomal microRNAs （miRNAs） with hepatic inflammatory injury and histological outcomes in patients with chronic hepatitis B （CHB）. MethodsPeripheral serum samples were collected from six healthy adults and six patients with CHB， and size exclusion chromatography was used to extract exosomes. Small RNA sequencing and transcriptomic analysis were used to identify the serum exosomal miRNAs associated with liver inflammatory injury and fibrosis， and quantitative real-time PCR was used for validation in a mouse model of acute liver injury induced by lipopolysaccharide/D-galactosamine， a rat model of liver fibrosis induced by carbon tetrachloride， and 84 CHB patients undergoing liver biopsy twice before and after treatment. The independent-samples t test was used for comparison of normally distributed continuous data between two groups； an analysis of variance was used for comparison between multiple groups， and the Tukey test was used for further comparison between two groups. The Mann-Whitney U test was used for comparison of non-normally distributed continuous data between two groups； the Kruskal-Wallis H test was used for comparison between multiple groups， and the Dunn test was used for further comparison between two groups. The chi-square test or the Fisher’s exact test was used for comparison of categorical data between groups. The univariate and multivariate Logistic regression analyses were used to investigate influencing factors. ResultsAbnormal expression of serum exosomal miR-122-5p was observed in patients with CHB， and it was downregulated in patients with confluent necrosis and advanced fibrosis. In the mouse model of acute liver injury and the rat model of liver fibrosis， compared with the control group， the model group had a significant reduction in the expression level of miR-122-5p in the liver （P=0.048 and 0.014）， and compared with the patients with mild liver injury， the patients with severe confluent necrosis and advanced fibrosis showed a significant reduction in the expression level of miR-122-5p in liver tissue （P<0.05）. Among the 84 CHB patients， the patients with severe hepatic confluent necrosis or advanced liver fibrosis had a significantly lower expression level of serum exosomal miR-122-5p than those with mild liver injury （P<0.001 and P=0.003）. The multivariate Logistic regression analysis showed that the expression level of miR-122-5p was an independent influencing factor for confluent necrosis （odds ratio ［OR］=0.001， 95% confidence interval ［CI］： 0.000‍ ‍—‍ ‍0.037， P=0.005） and liver fibrosis degree （OR=0.568， 95%CI： 0.331‍ ‍—‍ ‍0.856， P=0.019）. In addition， compared with the patients with low expression of miR-122-5p， the patients with high expression of miR-122-5p before treatment had a significantly higher reversal rate of liver fibrosis after 72 weeks of antiviral therapy （64.3% vs 38.1%， P=0.029）. ConclusionSerum exosomal miR-122-5p in CHB patients is closely associated with the progression of hepatic confluent necrosis and fibrosis， and the reduction in the expression level of miR-122-5p may aggravate hepatic confluent necrosis， promote the progression of fibrosis， and affect the histological outcome of CHB patients after antiviral therapy.

BACKGROUND: Several studies have demonstrated the occurrence of secondary tumors as a rare but significant complication of chimeric antigen receptor T (CAR-T) cell therapy, underscoring the need for a detailed investigation. Given the limited variety of secondary tumor types reported to date, a comprehensive characterization of the various secondary tumors arising after CAR-T therapy is essential to understand the associated risks and to define the role of the immune microenvironment in malignant transformation. This study aims to characterize the immune microenvironment of a newly identified secondary tumor post-CAR-T therapy, to clarify its pathogenesis and potential therapeutic targets. METHODS: In this study, the bone marrow (BM) samples were collected by aspiration from the primary and secondary tumors before and after CD19 CAR-T treatment. The CD45 + BM cells were enriched with human CD45 microbeads. The CD45 + cells were then sent for 10× genomics single-cell RNA sequencing (scRNA-seq) to identify cell populations. The Cell Ranger pipeline and CellChat were used for detailed analysis. RESULTS: In this study, a rare type of secondary chronic myelomonocytic leukemia (CMML) were reported in a patient with diffuse large B-cell lymphoma (DLBCL) who had previously received CD19 CAR-T therapy. The scRNA-seq analysis revealed increased inflammatory cytokines, chemokines, and an immunosuppressive state of monocytes/macrophages, which may impair cytotoxic activity in both T and natural killer (NK) cells in secondary CMML before treatment. In contrast, their cytotoxicity was restored in secondary CMML after treatment. CONCLUSIONS This finding delineates a previously unrecognized type of secondary tumor, CMML, after CAR-T therapy and provide a framework for defining the immune microenvironment of secondary tumor occurrence after CAR-T therapy. In addition, the results provide a rationale for targeting macrophages to improve treatment strategies for CMML treatment.
Humans ; Lymphoma, Large B-Cell, Diffuse/therapy* ; Tumor Microenvironment/genetics* ; Antigens, CD19/metabolism* ; Leukemia, Myelomonocytic, Chronic/genetics* ; Immunotherapy, Adoptive/adverse effects* ; Male ; Single-Cell Analysis/methods* ; Female ; Sequence Analysis, RNA/methods* ; Receptors, Chimeric Antigen ; Middle Aged

T-cell acute lymphoblastic leukemia (T-ALL) is a highly aggressive hematologic malignancy with a poor prognosis, despite advancements in treatment. Many patients struggle with relapse or refractory disease. Investigating the role of the super-enhancer (SE) regulated gene ubiquitin-specific protease 20 (USP20) in T-ALL could enhance targeted therapies and improve clinical outcomes. Analysis of histone H3 lysine 27 acetylation (H3K27ac) chromatin immunoprecipitation sequencing (ChIP-seq) data from six T-ALL cell lines and seven pediatric samples identified USP20 as an SE-regulated driver gene. Utilizing the Cancer Cell Line Encyclopedia (CCLE) and BloodSpot databases, it was found that USP20 is specifically highly expressed in T-ALL. Knocking down USP20 with short hairpin RNA (shRNA) increased apoptosis and inhibited proliferation in T-ALL cells. In vivo studies showed that USP20 knockdown reduced tumor growth and improved survival. The USP20 inhibitor GSK2643943A demonstrated similar anti-tumor effects. Mass spectrometry, RNA-Seq, and immunoprecipitation revealed that USP20 interacted with hypoxia-inducible factor 1 subunit alpha (HIF1A) and stabilized it by deubiquitination. Cleavage under targets and tagmentation (CUT&Tag) results indicated that USP20 co-localized with HIF1A, jointly modulating target genes in T-ALL. This study identifies USP20 as a therapeutic target in T-ALL and suggests GSK2643943A as a potential treatment strategy.

Objective To investigate the expression of disulfidptosis-related genes in hepatocellular carcinoma(HCC)and the prognostic value of disulfidptosis in HCC,to construct a prognostic model,and to analyze its impact on the biological processes of HCC and sorafenib resistance.Methods The TCGA-LIHC database was used to collect the mRNA expression profiles and corresponding clinical data of HCC patients,and the LASSO-Cox regression algorithm was used to construct a four-gene predictive model for prognosis in the TCGA cohort.The external datasets ICGC and GSE14520 were used to validate the prognostic efficacy of the model,and the Cancer Drug Sensitivity Genomics(GDSC)data were used to investigate the value of the disulfidptosis model in predicting sorafenib treatment response,and gene ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)analyses were used to investigate the biological functions of disulfidptosis-related genes.The independent-samples t test was used for comparison of continuous data between two groups,and the chi-square test was used for comparison of categorical data between two groups.The Kaplan-Meier curve and the log-rank test were used to evaluate the difference in prognosis,and univariate and multivariate Cox regression analyses were used to investigate whether risk score was an independent influencing factor for patient prognosis.Results The univariate Cox regression analysis in the TCGA cohort showed that seven known disulfidptosis-related genes were significantly associated with overall survival(OS)in HCC(all P<0.05).The LASSO-Cox regression analysis was used to construct a prognostic model based on disulfidptosis-related genes(DRG),and the risk score RS-DRG was calculated as RS-DRG=(0.061 6)×GYS1 expression level+(0.152 8)×LRPPRC expression level+(0.268 3)×RPN1 expression level+(0.183 5)×SLC7A11 expression level.The log-rank test showed that the patients with a high risk score based on the disulfidptosis model had a significantly lower OS than those with a low risk score(P<0.001).Based on the results of the multivariate Cox regression analysis,risk score was an independent predictive factor for OS in both TCGA and ICGC cohorts(TCGA:hazard ratio[HR]=1.869,P=0.002;ICGC:HR=3.469,P=0.004).The Spearman correlation analysis showed that RS-DRG was significantly positively correlated with the infiltration level of various immune cells(including B lymphocytes,CD4+T lymphocytes,neutrophils,macrophages,and dendritic cells)in tumor microenvironment(all P<0.05).The patients in the high-risk score group had a significantly lower IC50 value of sorafenib and were more sensitive to sorafenib(P<0.001).The KEGG/GO enrichment analysis showed that the differentially expressed disulfidptosis-related genes were significantly enriched in various mitosis-related molecular functions.Conclusion This study constructed a novel prognostic model based on disulfidptosis-related genes,which has a potential clinical value in predicting the prognosis of HCC,and targeting disulfidptosis-related genes may provide a promising approach for HCC treatment.

ObjectiveTo investigate the effect of transplantation of bone marrow mesenchymal stem cells （BMSCs） co-cultured with bone marrow-derived M2 macrophages （M2-BMDMs）， named as BMSC^M2， on a rat model of liver cirrhosis induced by carbon tetrachloride （CCl₄）/2-acetaminofluorene （2-AAF）. MethodsRat BMDMs were isolated and polarized into M2 phenotype， and rat BMSCs were isolated and co-cultured with M2-BMDMs at the third generation to obtain BMSC^M2. The rats were given subcutaneous injection of CCl₄ for 6 weeks to establish a model of liver cirrhosis， and then they were randomly divided into model group （M group）， BMSC group， and BMSC^M2 group， with 6 rats in each group. A normal group （N group） with 6 rats was also established. Since week 7， the model rats were given 2-AAF by gavage in addition to the subcutaneous injection of CCl₄. Samples were collected at the end of week 10 to observe liver function， liver histopathology， and hydroxyproline （Hyp） content in liver tissue， as well as changes in the markers for hepatic stellate cells， hepatic progenitor cells， cholangiocytes， and hepatocytes. A one-way analysis of variance was used for comparison of continuous data between multiple groups， and the least significant difference t-test was used for further comparison between two groups. ResultsCompared with the N group， the M group had significant increases in the activities of serum alanine aminotransferase （ALT） and aspartate aminotransferase （AST） （P<0.01）； compared with the M group， the BMSC and BMSC^M2 groups had significant reductions in ALT and AST （P<0.01）， and the BMSC^M2 group had significantly better activities than the BMSC group （P<0.05）. Compared with the N group， the M group had significant increases in Hyp content and the mRNA and protein expression levels of alpha-smooth muscle actin （α-SMA） in the liver （P<0.01）； compared with the M group， the BMSC and BMSC^M2 groups had significant reductions in Hyp content and the expression of α-SMA （P<0.05）， and the BMSC^M2 group had a significantly lower level of α-SMA than the BMSC group （P<0.01）. Compared with the N group， the M group had significant increases in the mRNA expression levels of the hepatic progenitor cell markers EpCam and Sox9 and the cholangiocyte markers CK7 and CK19 （P<0.01） and significant reductions in the expression levels of the hepatocyte markers HNF-4α and Alb （P<0.01）； compared with the M group， the BMSC and BMSC^M2 groups had significant reductions in the mRNA expression levels of EpCam， Sox9， CK7， and CK19 （P<0.05） and significant increases in the mRNA expression levels of HNF-4α and Alb （P<0.05）， and compared with the BMSC group， the BMSC^M2 group had significant reductions in the mRNA expression levels of EpCam and CK19 （P<0.05） and significant increase in the expression level of HNF-4α （P<0.05）. ConclusionM2-BMDMs can enhance the therapeutic effect of BMSCs on CCl₄/2-AAF-induced liver cirrhosis in rats， which provides new ideas for further improving the therapeutic effect of BMSCs on liver cirrhosis.

B-cell lymphoma is a group of hematological malignancies characterized by variable genetic and biological features and clinical behaviors. The tumor microenvironment (TME) is a complex network in tumors, which consists of surrounding blood vessels, extracellular matrix, immune and non-immune cells, and signaling molecules. Increasing evidence has shown that the TME, especially immune cells within, is a double-edged sword, acting either as a tumor killer or as a promoter of tumor progression. These pro-tumor activities are driven by subpopulations of immune cells that express typical markers but have unique transcriptional characteristics, making tumor-associated immune cells good targets for human anti-cancer therapy by ablating immunosuppressive cells or enhancing immune-activated cells. Thus, exploring the role of immune cells in the TME provides distinct insights for immunotherapy in B-cell lymphoma. In this review, we elucidated the interaction between immune cells and tumor cells and their function in the initiation, progression, and prognosis of B-cell lymphoma, from preclinical experiments to clinical trials. Furthermore, we outlined potential therapeutic approaches and discussed the potential clinical value and future perspectives of targeting immune cells in patients with B-cell lymphoma.

The advent of chimeric antigen receptor (CAR)-T cell immunotherapies has led to breakthroughs in the treatment of hematological malignancies. However, their success in treating solid tumors has been limited. CAR-natural killer (NK) cells have several advantages over CAR-T cells because NK cells can be made from pre-existing cell lines or allogeneic NK cells with a mismatched major histocompatibility complex (MHC), which means they are more likely to become an "off-the-shelf" product. Moreover, they can kill cancer cells via CAR-dependent/independent pathways and have limited toxicity. Macrophages are the most malleable immune cells in the body. These cells can efficiently infiltrate into tumors and are present in large numbers in tumor microenvironments (TMEs). Importantly, CAR-macrophages (CAR-Ms) have recently yielded exciting preclinical results in several solid tumors. Nevertheless, CAR-T, CAR-NK, and CAR-M all have their own advantages and limitations. In this review, we systematically discuss the current status, progress, and the major hurdles of CAR-T cells, CAR-NK cells, and CAR-M as they relate to five aspects: CAR structure, therapeutic mechanisms, the latest research progress, current challenges and solutions, and comparison according to the existing research in order to provide a reasonable option for treating solid tumors in the future.

Background::Although significant advances have been made in the treatment of multiple myeloma (MM), leading to unprecedented response and survival rates among patients, the majority eventually relapse, and a cure remains elusive. This situation is closely related to an incomplete understanding of the immune microenvironment, especially monocytes/macrophages in patients with treatment-na?ve MM. The aim of this study was to provide insight into the immune microenvironment, especially monocytes/macrophages, in patients with treatment-na?ve MM.Methods::This study used the single-cell RNA sequencing (scRNA-seq) data of both patients with MM and heathy donors to identify immune cells, including natural killer (NK) cells, T cells, dendritic cells (DCs), and monocytes/macrophages. Transcriptomic data and flow cytometry analysis of monocytes/macrophages were used to further examine the effect of monocytes/macrophages in treatment-na?ve MM patients.Results::A significant difference was observed between the bone marrow (BM) immune cells of the healthy controls and treatment-na?ve MM patients through scRNA-seq. It is noteworthy that, through an scRNA-seq data analysis, this study found that interferon (IFN)-induced NK/T cells, terminally differentiated effector memory (TEMRA) cells, T-helper cells characterized by expression of IFN-stimulated genes (ISG +Th cells), IFN-responding exhausted T cells, mannose receptor C-type 1 (MRC1) + DCs, IFN-responding DCs, MHCII + DCs, and immunosuppressive monocytes/macrophages were enriched in patients with treatment-na?ve MM. Significantly, transcriptomic data of monocytes/macrophages demonstrated that "don’t eat me" -related genes and IFN-induced genes increase in treatment-na?ve MM patients. Furthermore, scRNA-seq, transcriptomic data, and flow cytometry also showed an increased proportion of CD16 + monocytes/macrophages and expression level of CD16. Cell-cell communication analysis indicated that monocytes/macrophages, whose related important signaling pathways include migration inhibitory factor (MIF) and interleukin 16 (IL-16) signaling pathway, are key players in treatment-na?ve MM patients. Conclusions::Our findings provide a comprehensive and in-depth molecular characterization of BM immune cell census in MM patients, especially for monocytes/macrophages. Targeting macrophages may be a novel treatment strategy for patients with MM.

Objective:To investigate the interventional effect of bone marrow mesenchymal stem cell (BMMSC) transplantation with different doses of X-ray irradiation induced hepatic injury in mice.Methods:Eighteen female C57BL/6J mice were randomly divided into 0, 2, and 3 Gy irradiation groups and 0, 2, and 3 Gy transplantation groups. The irradiation group was used as the control and injected with an equal volume of culture medium. The mice in the transplantation group were irradiated with different doses of X-ray irradiation, and BMMSCs were intravenously infused into the bone marrow. The mice were sacrificed for sampling at the end of the 21st day. Mice body weight changes were recorded daily. The changes in the content of peripheral blood lymphocytes, red blood cells, platelets, and hemoglobin were detected by an automatic blood tester. The morphological changes in mice liver tissues were observed by hematoxylin-eosin staining. The serum activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were detected by a biochemical analyzer. The reduced glutathione contents in liver tissue were detected by the microplate method. The malondialdehyde content in liver tissue was detected by thiobarbituric acid. The content of total superoxide dismutase (T-SOD) in liver tissue was detected by the hydroxylamine method. The expression of the F4/80 protein in liver tissue was detected by the immunohistochemistry method. The protein expression of nuclear transcription factor erythroid 2 related factor 2 (Nrf2) and heme oxygenase 1 (HO-1) in liver tissue was determined by the western blotting method. The mRNA expression of NLRP3, IL-6, and Nrf2 in liver tissue was detected by a real-time quantitative polymerase chain reaction. The multiple-group comparisons were analyzed by factorial analysis of variance. The inter-group comparisons were analyzed by the LSD method for statistical analysis.Results:The contents of peripheral blood lymphocytes, erythrocytes, platelets, and hemoglobin were significantly decreased in the 3 Gy irradiation group than the 0 Gy irradiation group ( P<0.05), while the activities of serum ALT and AST were significantly increased ( P<0.05). The malondialdehyde content, F4/80 protein expression level, nucleotide-binding domain and leucine-rich repeats, nucleotide oligomerization domain-like receptor family, pyrin domain containing 3 (NLRP3), and interleukin 6 mRNA expression levels were significantly increased in liver tissue, while the contents of T-SOD and glutathione, Nrf2 and HO-1 protein expression levels, and Nrf2 mRNA expression level in liver tissue were significantly decreased ( P<0.05). The contents of peripheral blood lymphocytes, red blood cells, platelets, and hemoglobin were significantly increased in the 3 Gy transplantation group than the 3 Gy irradiation group ( P<0.05), while the activities of serum ALT and AST were significantly decreased ( P<0.05). The malondialdehyde content, F4/80 protein expression level, NLRP3 and interleukin-6 mRNA expression levels in liver tissue were significantly decreased ( P<0.05), while the content of T-SOD and glutathione, Nrf2 and HO-1 protein expression levels, and Nrf2 mRNA expression level in liver tissue were significantly increased ( P<0.05). Conclusion:X-ray irradiation at a dose of 3 Gy can induce liver oxidative damage in mice. BMMSC transplantation can improve X-ray irradiation-induced liver oxidative damage in mice, and its mechanism of action may be related to the regulation of the Nrf2/HO-1 pathway.

Objective To explore the effect of LIMK1 on the progression of cervical cancer(CC).Methods HeLa and C-33A human cervical cancer cells overexpressing LIMK1 were established and injected subcutaneously into nude mice.The tumor volume was measured and expression of NOX2,NOX4,p-Src,p-RUNX3,RUNX3,and MMP-9 proteins in tumor cells was detected by Western blot assays.LIMK1-overexpressing HeLa and C-33A cells were cultured in 5％O2 with antioxidants.The protein expression of LIMK1,NOX4,p-Src,p-RUNX3,RUNX3 and MMP-9 in the cells was detected by Western blot assays.Cell migration was assessed by a scratch assay.Transwell assays were used to assess cell migration and invasion.A monoclonal proliferation assay was used to assess cell proliferation.Results The tumor volume in nude mice injected with LIMK1-overexpressing HeLa cells was increased significantly,and NOX2,NOX4,p-Src,p-RUNX3 and MMP-9 protein levels were increased,while RUNX3 protein expression was decreased.In LIMK1-overexpressing HeLa and C-33A cells,the protein expression of LIMK1,NOX4,p-Src,p-RUNX3,and MMP-9 was increased,RUNX3 protein expression was decreased,and cell migration,invasion,and proliferation were increased.However,after adding antioxidants,the expression levels of NOX4,p-Src,p-RUNX3,RUNX3 and MMP-9 proteins,and cell migration,invasion,and proliferation were not different from those of control cells.Conclusions LIMK1 promotes the progression of cervical cancer by enhancing the ROS/Src pathway,thereby promoting the migration,invasion,and proliferation of cervical cancer cells.