The study aims to explore the methods for preparing nanocomplexes of Prussian blue nanoparticles (PBNPs) with UNO peptide (UNO-PBNPs) and the functions of the nanocomplexes targeting M2-type macrophages in vitro. PBNPs were prepared by the hydrothermal synthesis method. Subsequently, the peptide UNO (CSPGAKVRC) targeting the mannose receptor was modified on their surface by a heterobifunctional coupling approach. The morphological characteristics of nanoparticles were observed by scanning and transmission electron microscopy. Additionally, their particle size, Zeta potential, and dispersion stability were assessed. The structural characteristics of nanoparticles were analyzed by X-ray diffraction and other techniques. The biological safety of the nanoparticles was evaluated by the CCK-8 assay and hemolysis experiments. Moreover, the targeting performance of UNO-PBNPs towards M2-type macrophages was assessed in vitro. The results showed that the synthesized UNO-PBNPs exhibited uniform cubic morphology, with an average particle size of (202.00±4.21) nm. They were negative charged, well dispersed, and stable. At concentrations ≤ 200 μg/mL, the synthesized UNO-PBNPs led to the hemolysis rate below 5%, demonstrating excellent biocompatibility. The laser confocal imaging results showed that after co-incubation with M2-type macrophages, the FITC-labeled UNO-PBNPs were effectively accumulated in the cells, presenting a distinct fluorescence signal. Quantitative analysis by flow cytometry showed that the intracellular mean fluorescence intensity (6 019.00±346.04) of UNO-PBNPs was higher than that (4 054.00±379.14) of unmodified PBNPs (P < 0.001). In summary, the UNO-PBNPs prepared in this study exhibited a targeting effect on M2-type macrophages, providing a potential method for targeted delivery of PBNPs in the tumor microenvironment and laying a foundation for the remodeling of the tumor immunosuppressive microenvironment.
Ferrocyanides/chemistry* ; Nanoparticles/chemistry* ; Macrophages/drug effects* ; Peptides/chemistry* ; Particle Size ; Animals ; Mannose Receptor ; Mice ; Lectins, C-Type ; Mannose-Binding Lectins ; Receptors, Cell Surface

OBJECTIVES: To study the predictive value of serum macrophage M1/M2 markers for the risk of cardiac lesions in obese children. METHODS: A total of 60 obese children (mild-to-moderate obesity, n=32; severe obesity, n=28) and 50 healthy controls who visited the Second Affiliated Hospital of Nanchang University from June 2024 to December 2024 were included. The baseline characteristics and the levels of laboratory indicators, echocardiographic parameters, and macrophage markers (MCP-1, Arg-1, CD206, and CD86) were compared among the three groups. The correlation between macrophage marker levels and echocardiographic parameters and the influencing factors of cardiac lesions in obese children were analyzed. The receiver operating characteristic curve analysis was used to evaluate the predictive performance of each influencing factor for cardiac lesions in obese children. RESULTS: Multiple echocardiographic parameters differed significantly among the mild-to-moderate obesity, severe obesity, and control groups (P<0.01). Significant differences were also observed in MCP-1 and Arg-1 levels, CD206 positivity rate, and the CD86/CD206 ratio among the three groups (P<0.05). In obese children, MCP-1 and Arg-1 levels, as well as CD86 and CD206 positivity rates, were correlated with echocardiographic parameters (P<0.05). Univariate logistic regression identified MCP-1, Arg-1, the CD86/CD206 ratio, and the CD206 positivity rate as factors associated with cardiac lesions (P<0.05). The combined prediction model based on these markers yielded an area under the receiver operating characteristic curve of 0.887 (P<0.01). CONCLUSIONS Combined measurement of macrophage markers can predict the risk of early cardiac lesions in obese children.
Humans ; Male ; Female ; Child ; Biomarkers/blood* ; Macrophages ; Obesity/blood* ; Chemokine CCL2/blood* ; ROC Curve ; Adolescent ; Child, Preschool ; Heart Diseases/diagnosis* ; Echocardiography

OBJECTIVES: To elucidate the underlying mechanisms of macrophage-mediated inflammation and tissue injury in diabetic foot ulcer (DFU). METHODS: Skin tissue samples were collected from patients with DFU and with non-DFU. A total of 79 272 high-quality cell transcriptomes were obtained using single-cell RNA sequencing. An unbiased clustering approach was employed to identify cell subpopulations. Seurat functions were used to identify differentially expressed genes between DFU and non-DFU groups, and gene ontology (GO) enrichment analysis was used to reveal gene function. Furthermore, cell-cell communication network construction and ligand-receptor interaction analysis were performed to reveal the mechanisms underlying cellular interactions and signaling regulation in the DFU microenvironment from multiple perspectives. RESULTS: The results revealed a significant expansion of myeloid cells in DFU tissues, alongside a marked reduction in structural cells such as endothelial cells, epithelial cells, and smooth muscle cells. Major cell types underwent functional reprogramming, characterized by immune activation and impaired tissue remodeling. Specifically, macrophages in DFU skin tissues exhibited a shift toward a pro-inflammatory M1 phenotype, with upregulation of genes associated with inflammation and oxidative stress. Cell communication analysis further demonstrated that M1 macrophages served as both primary signal receivers and influencers in the COMPLEMENT pathway mediated communication network, and as key signal senders and mediators in the secreted phosphoprotein 1 (SPP1) pathway mediated communication network, actively shaping the inflammatory microenvironment. Key ligand-receptor interactions driving macrophage signaling were identified, including C3-(ITGAM+ITGB2) and SPP1-CD44. CONCLUSIONS This study establishes a comprehensive single-cell atlas of DFU, revealing the role of macrophage-driven cellular networks in chronic inflammation and impaired healing. These findings may offer potential novel therapeutic targets for DFU treatment.
Humans ; Macrophages/immunology* ; Diabetic Foot/pathology* ; Single-Cell Analysis ; Transcriptome ; Gene Expression Profiling ; Inflammation ; Skin ; Cell Communication ; Signal Transduction ; Cellular Reprogramming

OBJECTIVE: To explore the significance of the plasma cell percentage and immature plasma cells in the prognosis of patients with multiple myeloma (MM). METHODS: The clinical data of 126 newly diagnosed MM patients in Gansu Provincial Hospital from June 2017 to November 2022 were retrospectively analyzed. The enrolled patients were divided into a higher plasma cell percentage group (group A) and a lower plasma cell percentage group (group B) according to the median plasma cell percentage (33.5%). The clinicopathological data of the two groups were compared, and the effect of plasma cell percentage on the prognosis of MM patients was analyzed using survival curves. On this basis, group A and group B were divided into subgroups with immature plasma cells (A1 group, B1 group) and subgroups without immature plasma cells (A2 group, B2 group), respectively, then the survival curves were used to analyze the effect of immature plasma cells on the prognosis of MM patients. RESULTS: Among the 126 patients with MM, the proportions of patients with ISS stage III, elevated β₂-microglobulin(β₂-MG) level, and immature plasma cells in Group A were significantly higher compared those in Group B ( P =0.015, P =0.028, P =0.010). The median overall survival(OS) and progression-free survival(PFS) of group A were 32 months and 10 months, respectively. The median OS of group B was not reached, and the median PFS was 32 months. The 3-year OS rates of patients in group A and group B were 46.7% and 62.2%, respectively ( P =0.021), and the 3-year PFS were 29.2% and 42.5%, respectively ( P =0.033). There were no significant differences in OS and PFS between group A1 and group A2, or between group B1 and group B2 ( P >0.05). Multivariate COX survival analysis showed that the plasma cell percentage ≥33.5%（HR=1.253, 95%CI : 0.580-2.889, P =0.018）, age ≥65 years (HR=2.206, 95%CI : 1.170-3.510, P =0.012), lactate dehydrogenase(LDH) ≥250 U/L (HR=1.180, 95%CI : 0.621-2.398, P =0.048) and β₂-MG ≥3.5 mg/L (HR=1.507, 95%CI : 0.823-3.657, P =0.036) were independent risk factors affecting OS in MM patients. CONCLUSION MM patients with a higher plasma cell percentage (≥33.5%) at the initial diagnosis have a later disease stage, poorer OS and PFS, compared to the patients with a lower percentage(<33.5%) of plasma cells. The presence or absence of immature plasma cells has no significant impact on the survival of MM patients.
Humans ; Multiple Myeloma/pathology* ; Prognosis ; Plasma Cells/cytology* ; Retrospective Studies ; Male ; Female ; Middle Aged ; Aged ; Bone Marrow

OBJECTIVE: To analyze the correlation between the expression levels of Tim-3, C-myc and the proportion of T lymphocyte subsets and prognosis in patients with acute lymphoblastic leukemia (ALL). METHODS: The research group selected 60 ALL patients admitted to our hospital from December 2019 to December 2021, while the control group selected 55 healthy volunteers who underwent physical examination in our hospital. The expression levels of Tim-3, C-myc mRNA and the proportion of T lymphocyte subsets in the two groups were detected. The mortality rate of ALL patients was calculated, and the correlation between the expression levels of Tim-3, C-myc, and the proportion of T lymphocyte subsets and pathological features and prognosis was analyzed. RESULTS: Compared with the control group, the levels of Tim-3, C-myc and CD8⁺ in the research group were increased, while the levels of CD3⁺ , CD4⁺ and CD4⁺ /CD8⁺ were decreased (all P < 0.001). The levels of Tim-3, C-myc mRNA, CD3⁺ , CD4⁺ , CD8⁺ , CD4⁺ /CD8⁺ were correlated with risk classification and extramedullary infiltration (all P < 0.05). The survival rate of patients with low expression of Tim-3, C-myc, and CD8⁺ was higher than that of patients with high expression, while the survival rate of patients with high expression of CD3⁺ , CD4⁺ , and CD4⁺ /CD8⁺ was higher than that of patients with low expression (all P < 0.05). Univariate analysis showed that the deceased patients had higher proportions of extramedullary infiltration and high-risk classification, as well as higher levels of Tim-3, C-myc, and CD8⁺ , while lower levels of CD3⁺ , CD4⁺ , and CD4⁺ /CD8⁺ compared with surviving patients (all P < 0.01). Multivariate logistic regression analysis showed that extramedullary invasion, risk classification, Tim-3, C-myc, CD3⁺ , CD4⁺ , CD8⁺ , CD4⁺ /CD8⁺ were the main factors affecting the prognosis of ALL patients (all P < 0.05). ROC curve analysis showed that the combination of Tim-3, C-myc, and T lymphocyte subsets had higher sensitivity and accuracy in predicting prognosis of ALL patients compared with the single diagnosis of Tim-3, C-myc, CD3⁺ , CD4⁺ , CD8⁺ , and CD4⁺ /CD8⁺ (P < 0.05). CONCLUSION ALL patients show higher levels of Tim-3, C-myc mRNA and CD8⁺ but lower levels of CD3⁺ , CD4⁺ and CD4⁺/CD8⁺. Moreover, the expression levels of Tim-3, C-myc, CD3⁺ , CD4⁺ , CD8⁺ and CD4⁺/CD8⁺ are correlated with extramedullary invasion, high-risk classification and prognosis.
Humans ; Hepatitis A Virus Cellular Receptor 2/metabolism* ; Prognosis ; Proto-Oncogene Proteins c-myc/metabolism* ; Precursor Cell Lymphoblastic Leukemia-Lymphoma/diagnosis* ; T-Lymphocyte Subsets ; Male ; Female ; Adult ; Middle Aged ; Adolescent ; RNA, Messenger

OBJECTIVE: To explore the expression and clinical significance of programmed death receptor 1 (PD-1), Th1, Th2, and Th17 cytokines in multiple myeloma (MM). METHODS: A total of 76 MM patients treated in the Tengzhou Central People's Hospital from May 2021 to May 2023 were collected as MM group, and 48 healthy individuals who underwent physical examination during the same period were included as control group. The expression of PD-1 on the surface of CD4⁺ and CD8⁺ T cells and the levels of serum Th1 cytokines ［interleukin (IL) -2, interferon γ (IFN-γ), tumor necrosis factor α (TNF-α)］, Th2 cytokines (IL-4, IL-6, IL-10) and Th17 cytokines (IL-17) were detected in the two groups. Spearman correlation was used to examine the relationship between PD-1, Th1, Th2 and Th17 cytokines and clinical stage and immune typing of MM patients. Multivariate logistic regression analysis was used to analyze the related factors affecting the efficacy of chemotherapy in MM patients, and the factors were tested for multicollinearity. Receiver operating characteristic (ROC) curve was drawn to analyze the predictive value of PD-1, Th1, Th2 and Th17 cytokines in chemotherapy efficacy of MM patients. RESULTS: The levels of CD4⁺T PD-1, CD8⁺T PD-1, IL-4, IL-6, IL-10, and IL-17 in the MM group were higher than those in the control group, while the levels of IL-2, IFN-γ, and TNF-α were lower (all P <0.001). The levels of CD4⁺T PD-1, CD8⁺T PD-1, IL-4, IL-6, IL-10, and IL-17 in R-ISS stage III patients were higher than those in stage II and I patients, and the levels in stage II patients were higher than those in stage I patients (all P <0.05). The IL-2 level in R-ISS stage III patients was lower than that in stage II and I patients, and IL-2 level in R-ISS stage II patients was lower than that in stage I patients (all P <0.05). The levels of CD4⁺T PD-1, CD8⁺T PD-1, IL-4, IL-6, IL-10, and IL-17 in IgG patients were higher than those in IgA, light chain, and non secretory patients, while the level of IL-2 was lower (all P <0.05). Correlation analysis showed that CD4⁺T PD-1, CD8⁺T PD-1, IL-4, IL-6, IL-10, and IL-17 were positively correlated with R-ISS staging in MM patients (r =0.623, 0.635, 0.728, 0.330, 0.742, 0.412), and negatively correlated with immune classification (r =-0.664, -0.756, -0.642, -0.479, -0.613, -0.323). IL-2 was negatively correlated with R-ISS staging in MM patients (r =-0.280), and positively correlated with immune classification (r =0.483). The levels of CD4⁺T PD-1, CD8⁺T PD-1, IL-4, IL-6, IL-10, and IL-17 in the non-remission group were higher than those in the remission group, while the level of IL-2 was lower (all P <0.001). Multivariate logistic regression analysis showed that the increased CD4⁺T PD-1, CD8⁺T PD-1, IL-4, IL-6, IL-10 and IL-17 were risk factors for the efficacy of chemotherapy in MM patients (OR >1, P <0.05), while the increased IL-2 was a protective factor (OR < 1, P <0.05). The results of multicollinearity test showed that the tolerance of the seven factors included was between 0.714-0.885, and the variance inflation factor was between 1.130-1.400. There was no multicollinearity. The ROC curve analysis results showed that the area under the curve for the combined prediction of chemotherapy efficacy in MM patients by the above 7 factors was 0.942, with specificity of 0.741 and sensitivity of 0.909. CONCLUSION The expression levels of PD-1 on the surface of CD4⁺ and CD8⁺ T cells and serum Th2 and Th17 cytokines in MM patients are high, while Th1 cytokines are low. PD-1, Th1, Th2, and Th17 cytokines are related to clinical stage and immune classification of MM patients. The combined detection of these indicators can help predict the chemotherapy efficacy of MM patients.
Humans ; Programmed Cell Death 1 Receptor/metabolism* ; Multiple Myeloma/blood* ; Cytokines/metabolism* ; Th17 Cells/metabolism* ; Th1 Cells/metabolism* ; Th2 Cells/metabolism* ; Female ; Male ; Interleukin-10 ; Interferon-gamma ; Middle Aged ; Interleukin-17 ; Interleukin-2 ; Interleukin-4 ; Tumor Necrosis Factor-alpha ; Interleukin-6 ; Aged

OBJECTIVE: To investigate the role of MK2 inhibitor MMI-0100 on inflammatory response after allogeneic hematopoietic stem cell transplantation (allo-HSCT) and related mechanisms. METHODS: An allo-HSCT mouse model was established. Recipient rats were randomly divided into BMT+NaCl group and BMT+MMI-0100 group, and were injected with NaCl and MMI-0100 every day after transplantation, respectively. Samples of the two groups were collected on d 7 and 14, femur paraffin sections were stained with HE, and pathological changes in the bone marrow cavity were observed under the light microscope. The gene and protein expression levels of pro-inflammatory cytokines IL-1β and IL-18 were detected by qPCR and Western blot. Macrophage typing was detected by flow cytometry. The expression levels of NLRP3 and Caspase-1 were detected by Western blot. RESULTS: Inflammatory cell infiltration in the bone marrow cavity was significantly reduced in the BMT+MMI-0100 group. Western blot results showed that the protein expression levels of IL-1β and IL-18 in the BMT+MMI-0100 group were decreased compared to the BMT+NaCl group on day 7 and day 14 (all P <0.01). The qPCR results showed that compared to the BMT+NaCl group, the IL-18 gene expression levels in the BMT+MMI-0100 group were significantly reduced on day 7 and day 14 (both P <0.01). In the BMT+MMI-0100 group, the expression level of IL-1β gene decreased on day 7 (P <0.05), but increased and was higher than that in the BMT+NaCl group on day 14 (P <0.05). Flow cytometry results showed that the expression of M1 macrophages and M1/M2 ratio decreased in the BMT+MMI-0100 group compared to BMT+NaCl group (all P <0.05). Western blot results showed that the protein expression levels of NLRP3 and Caspase-1 in the BMT+MMI-0100 group were lower than those in the BMT+NaCl group (all P <0.05). CONCLUSION MMI-0100 can ameliorate bone marrow inflammatory injury after allo-HSCT and may act by reducing NLRP3 expression to promote M2 polarization.
Animals ; Interleukin-1beta/metabolism* ; Rats ; Interleukin-18/metabolism* ; Hematopoietic Stem Cell Transplantation/adverse effects* ; Mice ; NLR Family, Pyrin Domain-Containing 3 Protein/metabolism* ; Inflammation ; Bone Marrow/pathology* ; Protein Serine-Threonine Kinases/metabolism* ; Intracellular Signaling Peptides and Proteins/antagonists & inhibitors* ; Caspase 1/metabolism* ; Macrophages ; Transplantation, Homologous

OBJECTIVE: To analyze the Epstein-Barr virus (EBV) load in different lymphocyte subsets, as well as clinical characteristics and outcomes in patients with hematologic malignancies experiencing EBV reactivation. METHODS: Peripheral blood samples from patients were collected. B, T, and NK cells were isolated sorting with magnetic beads by flow cytometry. The EBV load in each subset was quantitated by real-time quantitative polymerase chain reaction (RT-qPCR). Clinical data were colleted from electronic medical records. Survival status was followed up through outpatient visits and telephone calls. Statistical analyses were performed using SPSS 25.0. RESULTS: A total of 39 patients with hematologic malignancies were included, among whom 35 patients had undergone allogeneic hematopoietic stem cell transplantation (allo-HSCT). The median time to EBV reactivation was 4.8 months (range: 1.7-57.1 months) after allo-HSCT. EBV was detected in B, T, and NK cells in 20 patients, in B and T cells in 11 patients, and only in B cells in 4 patients. In the 35 patients, the median EBV load in B cells was 2.19×10⁴ copies/ml, significantly higher than that in T cells (4.00×10³ copies/ml, P <0.01) and NK cells (2.85×10² copies/ml, P <0.01). Rituximab (RTX) was administered for 32 patients, resulting in EBV negativity in 32 patients with a median time of 8 days (range: 2-39 days). Post-treatment analysis of 13 patients showed EBV were all negative in B, T, and NK cells. In the four non-transplant patients, the median time to EBV reactivation was 35 days (range: 1-328 days) after diagnosis of the primary disease. EBV was detected in one or two subsets of B, T, or NK cells, but not simultaneously in all three subsets. These patients received a combination chemotherapy targeting at the primary disease, with 3 patients achieving EBV negativity, and the median time to be negative was 40 days (range: 13-75 days). CONCLUSION In hematologic malignancy patients after allo-HSCT, EBV reactivation commonly involves B, T, and NK cells, with a significantly higher viral load in B cells compared to T and NK cells. Rituximab is effective for EBV clearance. In non-transplant patients, EBV reactivation is restricted to one or two lymphocyte subsets, and clearance is slower, highlighting the need for prompt anti-tumor therapy.
Humans ; Hematologic Neoplasms/virology* ; Herpesvirus 4, Human/physiology* ; Epstein-Barr Virus Infections ; Hematopoietic Stem Cell Transplantation ; Virus Activation ; Lymphocyte Subsets/virology* ; Flow Cytometry ; Killer Cells, Natural/virology* ; Male ; Female ; B-Lymphocytes/virology* ; Viral Load ; Adult ; T-Lymphocytes/virology* ; Middle Aged

OBJECTIVE: To investigate the molecular alterations of splenocytes associated with anti-factor Ⅷ (FⅧ) immune response and the underlying mechanisms based on hemophilia A (HA) murine model via RNA sequencing (RNA-seq) technology. METHODS: Severe HA mice were immunized with recombinant human factor Ⅷ (rhF8) weekly for 4 weeks to establish an FⅧ inhibitor model. High quality raw data were obtained by using bulk RNA-seq and CASAVA base identification technology, and the differentially expressed genes (DEGs) were identified. The DEGs were statistically classified by gene ontology (GO) annotation to obtain information on the major signaling pathways and biological processes involved in anti-FⅧ immune response in HA mouse splenocytes. The cell clusters, genes, and signaling pathway datasets were comprehensively analyzed by GO, Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis and single cell RNA-seq (ScRNA-seq) analysis, respectively. Flow cytometry analysis was used to verify the changes in T follicular helper cells (Tfh) and regulatory T cells (Treg). RESULTS: A total of 3731 DEGs was identified, including 2275 genes with up-regulated expression and 1456 genes with down-regulated expression. The DEGs were enriched in helper T cell differentiation, cytokine receptor, T cell receptor signaling pathway, ferroptosis, etc. Uniform Manifold Approximation and Project (UMAP) downscaling and visualization analysis yielded a total number of 11 T/NK cell subsets, visualizing the overall expression distribution of C-X-C chemokine-specific receptor gene cxcr5 among these T/NK cell subsets. Higher expression of cxcr5 was found in activated Tfh from FⅧ inhibitor mice, in comparison to the control group. The visualization using Upset plot R language showed a close interaction between Tfh and Treg. Moreover, the increased frequencies of Tfh and the decreased frequencies of Treg in inhibitor mouse splenocytes were further verified by flow cytometry analysis. CONCLUSION Multiple immune cell subsets, signaling pathways, and characteristic genes may be involved in the process of anti-FⅧ immune response in HA mouse splenocytes. The molecules involved in the regulation of Tfh/Treg may play key roles, which provide potential biological targets and therapeutic strategies for HA patients with inhibitors in the future.
Animals ; Hemophilia A/genetics* ; Mice ; Sequence Analysis, RNA ; Disease Models, Animal ; Spleen/cytology* ; T-Lymphocytes, Regulatory/immunology* ; Humans ; Signal Transduction ; Factor VIII/immunology* ; T-Lymphocytes, Helper-Inducer/immunology*

OBJECTIVE: To investigate the regulatory effect of Wip1 phosphatase on hematopoietic function in the mouse spleen. METHODS: Wip1 knockout mice were bred, and the effect of Wip1 deletion on the proportion and number of hematopoietic stem/progenitor cells, as well as their mature subsets in mouse spleen was detected by flow cytometry. The Proteome Profiler^TM antibody array was used to analyze the role of Wip1 deletion on the expression of inflammatory cytokines in CD45^highCD11b⁺ myeloid cells sorted from mouse spleen. RESULTS: Wip1 deletion resulted in smaller size and significant reduction of cell number in the mouse spleen. The absolute numbers of hematopoietic stem/progenitor cells were decreased. Meanwhile, the absolute number of T and B lymphocytes also significantly declined. However, the proportion of erythroid progenitors and erythroid cells at various stage significantly increased, but the number of mature erythroid cells decreased. Furthermore, the myeloid cells and their subsets neutrophils, monocytes, CD45^highCD11b⁺ and CD45^lowCD11b⁺ were all reduced. CD45^highCD11b⁺ myeloid cells displayed proinflammatory phenotype in the spleen. CONCLUSION Wip1 gene deletion impairs normal hematopoietic function in the mouse spleen, leading to a significant reduction of mature hematopoietic cells of various lineages, and proinflammatory phenotype in CD45^highCD11b⁺ myeloid cells.
Animals ; Mice ; Spleen/cytology* ; Mice, Knockout ; Hematopoietic Stem Cells/cytology* ; Myeloid Cells/cytology* ; Protein Phosphatase 2C ; Hematopoiesis ; Flow Cytometry