OBJECTIVE: To investigate the biological activity and antitumor effect of pegylated recombinant human interleukin 2 (PEG-rhIL-2) obtained by site-specific conjugation of polyethylene glycol (PEG) with non-natural amino acids, and to explore its antitumor mechanism. METHODS: The binding activities of PEG-rhIL-2 at three different sites (T41, Y45, and V91) to human interleukin 2 receptors α (IL-2R_α) and β (IL-2R_β) and were detected by surface plasmon resonance (SPR) technology. Western blot was used to detect the levels of the Janus kinase-signal transducer and activator of transcription 5 (JAK-STAT5) signaling pathway activated by different doses of rhIL-2 and PEG-rhIL-2 in CTTL-2 and YT cells. Blood was collected after a single administration in mice to detect the drug concentration at different time points and evaluate the pharmacokinetic parameters of Y45-PEG-rhIL-2. Mouse hepatoma cell line Hepa1-6, pancreatic cancer cell line Pan-02, and colon cancer cell line MC-38 were selected. Tumor models were constructed in C57BL/6 mice. Different doses of Y45-PEG-rhIL-2 and excipient control were administrated respectively to evaluate the tumor suppression effect of the drug. In the MC-38 colon cancer model, the tumor suppression effect of Y45-PEG-rhIL-2 combined with anti-programmed death-1 (PD-1) monoclonal antibody was evaluated. Hepa1-6 mouse tumor models were constructed and rhIL-2, Y45-rhIL-2 and Y45-PEG-rhIL-2 were administrated respectively. The proportion of tumor-infiltrating lymphocytes was analyzed by flow cytometry. RESULTS: The SPR detection results showed that the binding activities of PEG-rhIL-2 to IL-2R_α/IL-2R_β were both reduced. The affinity of Y45-PEG-rhIL-2 to IL-2R_α was reduced to approximately 1/250, and its affinity to IL-2R_β was reduced to 1/3. Western blot results showed that the activity of Y45-PEG-rhIL-2 in stimulating JAK-STAT5 signaling in CTLL-2 cells expressing heterotrimeric IL-2 receptor complex IL-2R_αβγwas reduced to approximately 1/300, while its activity in YT cells expressing heterodimeric IL-2 receptor complex IL-2R_βγwas reduced to approximately 1/3. The pharmacokinetic evaluation after a single dose in the mice showed that the elimination half-life of Y45-PEG-rhIL-2 was 17.7 h. Y45-PEG-rhIL-2 has pharmacokinetic characteristics superior to those of rhIL-2. Y45-PEG-rhIL-2 showed dose-dependent tumor suppression activity, and the combination of Y45-PEG-rhIL-2 and anti-PD-1 antibody had a better tumor-inhibiting effect than the single use of Y45-PEG-rhIL-2 or anti-PD-1 antibody. Flow cytometry analysis demonstrated that 72 h after the administration of Y45-PEG-rhIL-2, the proportion of tumor-infiltrating cytotoxic T lymphocytes (CD8⁺T cells) increased by 86.84%. At 120 h after administration, the ratio of CD8⁺T cells to regulatory T cells (Treg) increased by 75.10%. CONCLUSION Y45-PEG-rhIL-2 obtained by site-specific conjugation <i>viai> non-natural amino acids changed its receptor binding activity and inhibited tumor growth in dose-dependent manner in multiple tumor models by regulating CD8⁺T cells.
Interleukin-2/pharmacokinetics* ; Animals ; Mice ; Humans ; Recombinant Proteins/pharmacology* ; Polyethylene Glycols/chemistry* ; Cell Line, Tumor ; Antineoplastic Agents/pharmacokinetics* ; Signal Transduction/drug effects* ; STAT5 Transcription Factor/metabolism* ; Interleukin-2 Receptor alpha Subunit/metabolism* ; Interleukin-2 Receptor beta Subunit/metabolism*

OBJECTIVES: There is currently no consensus on whether extra-gastrointestinal stromal tumors (EGISTs) and gastrointestinal stromal tumors (GISTs) are the same type of tumor, and whether the diagnosis and treatment of EGISTs can directly replicate the current diagnostic and treatment standards for GISTs. This study aims to further elucidate the clinical and pathological characteristics, diagnosis, treatment, and prognosis of EGISTs by analyzing the research results of domestic scholars in the field of EGISTs in the past decade. METHODS: A review was conducted on original Chinese and English research articles published from 2013 to 2022 focusing on EGISTs. A descriptive approach was used to extract key information from the literature, including patient demographics, tumor location, tumor diameter, mitotic figures, risk stratification, immunohistochemical markers, cell type, and prognostic factors. The data were subjected to statistical analysis. RESULTS: A total of 12 articles containing 780 EGIST patients were included. The male-to-female incidence of EGISTs was 0.92꞉1. The most common sites of EGISTs were mesentery (30.96%), peritoneum or retroperitoneum (28.53%), omentum (20.32%), and pelvic cavity (12.52%). 52.77% of EGISTs had tumor diameters greater than 10 cm, and the proportions of EGISTs with nuclear fission patterns greater than 5/50 high power field (HPF) and greater than 10/50 HPF were 51.24% and 26.11%, respectively. The proportion of high-risk EGISTs was 79.05%. The positive rates of immune markers CD117, CD34, and DOG-1 in EGISTs were 82.3%, 69.0%, and 79.5%, respectively. The proportion of Ki-67 >5% was 49.2%, and the proportion of Ki-67 >10% was 24.8%. The proportions of EGISTs in spindle cells, epithelial cells, and mixed cells were 74.4%, 14.8%, and 13.1%, respectively. The diameter of the tumor, resection method, risk level, Ki-67 index, mitotic counts, presence of rupture/bleeding/necrosis/peripheral tissue invasion/recurrence and metastasis, as well as the use of imatinib treatment after surgery were important factors affecting the prognosis of EGISTs. CONCLUSIONS Current medical research is relatively well cognizant of GISTs with primary sites in the gastrointestinal tract. Compared with GISTs, EGISTs have large tumor diameters, high mitotic counts, a high percentage of high-risk grades, relatively unique molecular expression, and high aggressiveness. EGISTs differ from GISTs in clinicopathological characteristics. Whether EGISTs and GISTs share a common origin remains unclear. If they are distinct tumor entities, separate diagnostic and treatment guidelines for EGISTs should be established. If EGISTs are ultimately confirmed to be a special subtype of GISTs, then directly applying existing GIST-based standards to EGISTs may be inappropriate. A more scientific approach would involve subclassifying EGISTs based on anatomical location and then tailoring treatment strategies accordingly with reference to GIST guidelines.
Humans ; Gastrointestinal Stromal Tumors/epidemiology* ; Male ; Female ; Prognosis ; Proto-Oncogene Proteins c-kit/metabolism*

OBJECTIVE: To analyze the gene mutation characteristics and survival time of patients with newly diagnosed acute myeloid leukemia (AML) based on next-generation sequencing(NGS) gene detection. METHODS: A retrospective analysis was conducted on the clinical data of 92 patients with AML (non APL) admitted to our hospital from January 2018 to May 2022. AML related genes tested were using NGS, the mutation characteristics and survival time of AML patients were analyzed. RESULTS: Among the 92 patients, 41 were males and 51 were females. A total of 38 types of gene mutations were detected. Six-two patients carried at least one gere mutation, while no gene mutations were detected in 30 patients. In the group with favourable prognosis (<i>ni> =14), the frequencies of higher gene mutations were <i>NRAS, KITi> (21.43%, <i>ni> =3), <i>KRASi> (14.29%, <i>ni> =2). In the group with intermediate prognosis (<i>ni> =64), the gene mutation frequencies from high to low were <i>DNMT3Ai> (18.75%, <i>ni> =12), <i>NPM1i> (17.19%, <i>ni> =11), <i>IDH2, FLT3-ITD, CEBPAi> (12.50%, <i>ni> =8), <i>TET2i> (10.94%, <i>ni> =7). In the poor prognosis group (<i>ni> =14), <i>ASXL1, TP53, EZH2, NRASi> had higher gene mutation frequency than others(14.29 %, <i>ni> =2 ). Statistical analysis revealed that <i>KITi> had a relative hotspot of mutations in the intermediate-risk group, and <i>DNMT3Ai> had a relative hotspot of mutations in the high-risk group (<i>Pi> < 0.05). The correlation analysis of genes with high mutation rates in different prognostic groups, such as <i>NRAS, KIT, IDH2, DNMT3A, NPM1i>, and <i>FLT3-ITDi>, with prognosis found that <i>KITi> was a factor affecting OS (<i>Pi> < 0.05), while no significant differences were observed for the others(<i>Pi> >0.05). CONCLUSION The frequency of gene mutations is high in AML patients, 67.4% of the patients carried at least one gene mutation. The mutation frequency varies among different genes in patients with different karyotypes, and there are obvious dominant mutations. <i>KITi> and <i>DNMT3Ai> can be used as factors for evaluating the prognosis of AML.
Humans ; Leukemia, Myeloid, Acute/genetics* ; Nucleophosmin ; Mutation ; Prognosis ; Retrospective Studies ; Male ; Female ; High-Throughput Nucleotide Sequencing ; Middle Aged ; DNA Methyltransferase 3A ; Adult ; Aged ; Survival Analysis ; Proto-Oncogene Proteins c-kit/genetics*

OBJECTIVE: To investigate the predictive role of the modified Endothelial Activation and Stress Index (mEASIX) in the efficacy of chimeric antigen receptor T-cell (CAR-T) therapy and cytokine release syndrome (CRS). METHODS: The clinical data of 70 relapsed and refractory (R/R) B-cell tumor patients who were treated with CAR-T therapy from September 1, 2018 to February 28, 2023 in the Department of Hematology, Affiliated Hospital of Xuzhou Medical University, were retrospectively analyzed. The value of log-2 mEASIX before conditioning (-7 d) was calculated, and the patients were divided into a low-mEASIX group (42 patients) and a high-mEASIX group (28 patients) based on the cut-off value of 5.443 determined by the receiver operating characteristic (ROC) curve. Eventually, the predictive role of mEASIX before conditioning on the efficacy of CAR-T cell therapy and CRS was analyzed. RESULTS: The high-mEASIX group exhibited significantly worse median overall survival (OS) and median progression-free survival (PFS) in comparison to the low mEASIX group (OS: 3.2 months <i>vsi> not reached, <i>Pi> < 0.01; PFS: 1.3 months <i>vsi> 6.0 months, <i>Pi> =0.009). The incidence of grade ≥2 CRS in the high-mEASIX group was substantially higher than that in the low-mEASIX group (57.1% <i>vsi> 19.0%, <i>Pi> =0.007). The degree of remission after CAR-T therapy (<i>Pi> =0.001), whether CRS occurs or not (<i>Pi> =0.041), the lactate dehydrogenase (LDH) level before conditioning (<i>Pi> =0.046), and the mEASIX score before conditioning (<i>Pi> =0.047) were independent influencing factors for the OS of patients receiving CAR-T cell therapy. CONCLUSION The mEASIX score before conditioning can predict OS and the incidence of grade ≥2 CRS in patients with relapsed and refractory B-cell tumors who receive CAR-T cell therapy.
Cytokine Release Syndrome/therapy* ; Immunotherapy, Adoptive/methods* ; Humans ; Lymphoma, B-Cell/therapy* ; Retrospective Studies ; Hematology ; China ; Receptors, Chimeric Antigen/blood* ; Predictive Value of Tests

OBJECTIVE: To screen anti-tumor drugs that improve antigen processing and presentation in acute myeloid leukemia (AML) cells. METHODS: A TCR-like or TCR mimic antibody that can specifically recognize HLA-A*0201:WT1_126-134 ( <i>RMFPNAPYLi>) complex (hereafter referred to as HLA-A2:WT1) was synthesized to evaluate the function of antigen processing and presentation machinery (APM) in AML cells. AML cell line THP1 was incubated with increasing concentrations of IFN-γ, hypomethylating agents (HMA), immunomodulatory drugs (IMiD), proteasome inhibitors (PI) and γ-secretase inhibitors (GSI), followed by measuring of HLA-ABC, HLA-A2 and HLA-A2:WT1 levels by flow cytometry at consecutive time points. RESULTS: The TCR-like antibody we generated only binds to HLA-A*0201⁺WT1⁺ cells, indicating the specificity of the antibody. HLA-A2:WT1 level of THP-1 cells detected with the TCR-like antibody was increased significantly after co-incubation with IFN-γ, showing that the HLA-A2:WT1 TCR like antibody could evaluate the function of APM. Among the anti-tumor agents screened in this study, GSI (LY-411575) and HMA (decitabine and azacitidine) could significantly increase the HLA-A2:WT1 level. The IMiD lenalidomide and pomalidomide could aslo upregulate the expression of HLA-A2:WT1 complex under certain concentrations of the drugs and incubation time. As proteasome inhibitors, carfilzomib could significantly decreased the expression of HLA-A2:WT1, while bortezomib had no significant effect on HLA-A2:WT1 expression. CONCLUSION HLA-A2:WT1 TCR-like antibody can effectively reflect the APM function. Some of the anti-tumor drugs can affect the APM function and immunogenicity of tumor cells.
Humans ; Leukemia, Myeloid, Acute/immunology* ; Antineoplastic Agents/pharmacology* ; Antigen Presentation/drug effects* ; HLA-A2 Antigen/immunology* ; Receptors, Antigen, T-Cell/immunology* ; Cell Line, Tumor ; Interferon-gamma

BACKGROUND: Depression among older adults is an important public health issue, and air and noise pollution have been found to contribute to exacerbation of depressive symptoms. This study examined the association of exposure to air and noise pollutants with clinically-newly-diagnosed depressive disorder. The mediating role of individual pro-inflammatory markers was explored. METHODS: We linked National Health Insurance claim data with 2998 healthy community-dwellers aged 55 and above who participated in the Healthy Aging Longitudinal Study between 2009 and 2013. Newly diagnosed depressive disorder was identified using diagnostic codes from the medical claim data. Pollutants were estimated using nationwide land use regression, including PM_2.5 and PM₁₀, carbon monoxide, ozone, nitrogen dioxide, sulfur dioxide, and road traffic noise. Cox proportional hazard models were employed to examine the association between pollutants and newly developed depressive disorders. The mediating effect of serum pro-inflammatory biomarkers on the relationship was examined. RESULTS: Among the 2998 participants, 209 had newly diagnosed depressive disorders. In adjusted Cox proportional hazard models, one interquartile range increase in PM_2.5 (8.53 µg/m³) was associated with a 17.5% increased hazard of developing depressive disorders. Other air pollutants and road traffic noise were not linearly associated with depressive disorder incidence. Levels of serum tumor necrosis factor receptor 1 mediated the relationship between PM_2.5 and survival time to newly onset depressive disorder. CONCLUSION PM_2.5 is related to an increased risk of newly developed depressive disorder among middle-aged and older adults, and the association is partially mediated by the pro-inflammatory marker TNF-R1.
Humans ; Particulate Matter/analysis* ; Male ; Female ; Middle Aged ; Longitudinal Studies ; Aged ; Incidence ; Air Pollutants/analysis* ; Environmental Exposure/adverse effects* ; Taiwan/epidemiology* ; Receptors, Tumor Necrosis Factor, Type I/blood* ; Proportional Hazards Models ; Biomarkers/blood* ; Depression/epidemiology* ; Aged, 80 and over ; Depressive Disorder/chemically induced* ; Risk Factors ; Air Pollution/adverse effects*

OBJECTIVES: To study the therapeutic mechanism of <i>Tuihuangi> Mixture against cholestasis. METHODS: Forty-eight Wistar rats were randomized equally into blank group, model group, ursodeoxycholic acid group and <i>Tuihuangi> Mixture group. Except for those in the blank group, all the rats were given α‑naphthylisothiocyanate (ANIT) to establish rat models of cholestasis, followed by treatments with indicated drugs or distilled water. Serum levels of ALT, AST, ALP, γ-GT, TBA and TBIL of the rats were determined, and hepatic expressions IL-1β, IL-18, FXR, NLRP3, ASC, Caspase-1 and GSDMD were detected using q-PCR, ELISA or Western blotting. Histopathological changes of the liver tissues were observed using HE staining. RESULTS: The rat models of cholestasis had significantly increased serum levels of ALT, AST, ALP, γ-GT, TBA and TBIL with increased mRNA and protein expressions of IL-1β and IL-18, decreased protein and mRNA expressions of FXR, and increased protein expressions of NLRP3 and Caspase-1 and mRNA expressions of NLRP3, ASC, Caspase-1 and GSDMD in the liver tissue, showing also irregular arrangement of liver cells, proliferation of bile duct epithelial cells and inflammatory cells infiltration. Treatment of the rat models with <i>Tuihuangi> Mixture significantly decreased serum levels of ALT, AST, ALP, γ-GT, TBA and TBIL, lowered IL-1β and IL-18 and increased FXR protein and mRNA expressions, and reduced NLRP3, ASC, Caspase-1 and GSDMD proteins and NLRP3, ASC and Caspase-1 mRNA expressions in the liver tissue. <i>Tuihuangi> Mixture also significantly alleviated hepatocyte injury, bile duct epithelial cell proliferation and inflammatory cell infiltration in the liver of the rat models. CONCLUSIONS <i>Tuihuangi> Mixture can effectively improve cholestasis in rats possibly by inhibiting NLRP3 inflammatosome-mediated pyroptosis via regulating FXR.
Animals ; NLR Family, Pyrin Domain-Containing 3 Protein ; Rats ; Receptors, Cytoplasmic and Nuclear/metabolism* ; Cholestasis/drug therapy* ; Rats, Wistar ; Inflammasomes/metabolism* ; 1-Naphthylisothiocyanate ; Drugs, Chinese Herbal/therapeutic use* ; Male ; Interleukin-18/metabolism* ; Caspase 1/metabolism* ; Interleukin-1beta/metabolism* ; Liver/metabolism*

Taste buds relay taste sensory information to the primary taste neurons but depend on those same neurons for essential components to maintain function. While denervation-induced taste bud degeneration and subsequent regeneration were discovered decades ago, the mechanisms underlying these phenomena (e.g., heterogenous cellular responses to nerve injury and the signaling pathways involved) remain poorly understood. Here, using mouse genetics, nerve injury models, pharmacologic manipulation, and taste bud organoid models, we identify a specific subpopulation of taste cells, predominantly c-Kit-expressing sweet cells, that exhibit superior resistance to nerve injury. We found the c-Kit inhibitor imatinib selectively reduced the number of residual c-Kit-expressing sweet cells at post-operation week 2, subsequently attenuating the re-emergence of other type II cells by post-operation week 4. In taste bud organoids, c-Kit-expressing cells were resistant to R-spondin withdrawal but susceptible to imatinib, while other taste cell types showed the opposite behavior. We also observed a distinct population of residual taste cells that acquired stem-like properties, generating clonal descendent cells among suprabasal keratinocytes independent of c-Kit signaling. Together, our findings reveal that c-Kit signaling confers resilience on c-Kit-expressing sweet cells and supports the broader reconstruction of taste buds during the later regenerative stage following nerve injury.
Animals ; Taste Buds/metabolism* ; Proto-Oncogene Proteins c-kit/metabolism* ; Mice ; Signal Transduction ; Imatinib Mesylate/pharmacology* ; Mice, Inbred C57BL

BACKGROUND: Increasing evidence indicates that oxidative stress and interferon γ (IFNγ)-driven cellular immune responses are responsible for the pathogenesis of vitiligo. However, the connection between oxidative stress and the local production of IFNγ in early vitiligo remains unexplored. The aim of this study was to identify the mechanism underlying the production of IFNγ by mast cells and its impact on vitiligo pathogenesis. METHODS: Skin specimens from the central, marginal, and perilesional skin areas of active vitiligo lesions were collected to characterize changes of mast cells, CD8 + T cells, and IFNγ-producing cells. Cell supernatants from hydrogen peroxide (H 2 O 2 )-treated keratinocytes (KCs) were harvested to measure levels of soluble stem cell factor (sSCF) and matrix metalloproteinase (MMP)-9. A murine vitiligo model was established using Mas-related G protein-coupled receptor-B2 (MrgB2, mouse ortholog of human MrgX2) conditional knockout (MrgB2 -/- ) mice to investigate IFNγ production and inflammatory cell infiltrations in tail skin following the challenge with tyrosinase-related protein (Tyrp)-2 180 peptide. Potential interactions between the Tyrp-2 180 peptide and MrgX2 were predicted using molecular docking. The siRNAs targeting MrgX2 and the calcineurin inhibitor FK506 were also used to examine the signaling pathways involved in mast cell activation. RESULTS: IFNγ-producing mast cells were closely aligned with the recruitment of CD8 + T cells in the early phase of vitiligo skin. sSCF released by KCs through stress-enhanced MMP9-dependent proteolytic cleavage recruited mast cells into sites of inflamed skin (Perilesion vs . lesion, 13.00 ± 4.00/high-power fields [HPF] vs . 26.60 ± 5.72/HPF, P <0.05). Moreover, IFNγ-producing mast cells were also observed in mouse tail skin following challenge with Tyrp-2 180 (0 h vs . 48 h post-recall, 0/HPF vs . 3.80 ± 1.92/HPF, P <0.05). The IFNγ + mast cell and CD8 + T cell counts were lower in the skin of MrgB2 -/- mice than in those of wild-type mice (WT vs . KO 48 h post-recall, 4.20 ± 0.84/HPF vs . 0.80 ± 0.84/HPF, P <0.05). CONCLUSION Mast cells activated by MrgX2 serve as a local IFNγ producer that bridges between innate and adaptive immune responses against MCs in early vitiligo. Targeting MrgX2-mediated mast cell activation may represent a new strategy for treating vitiligo.
Vitiligo/metabolism* ; Mast Cells/immunology* ; Animals ; Interferon-gamma/metabolism* ; Mice ; Humans ; Melanocytes/metabolism* ; Receptors, G-Protein-Coupled/genetics* ; Mice, Knockout ; Mice, Inbred C57BL ; Male ; Female ; Matrix Metalloproteinase 9/metabolism* ; Stem Cell Factor/metabolism*

Objective To explore the effect of Evodiamine (Evo) on the immune function of allergic rhinitis (AR) rats and the regulatory mechanism on C-C motif chemokine ligand 2 (CCL2)/ C-C motif chemokine receptor 2 (CCR2) pathway. Methods The related targets of Evo-AR-immune function were screened by network pharmacology, and the protein interaction network diagram of intersecting targets was constructed. The AR rat model was established by ovalbumin (OVA) combined with aluminium hydroxide, and the rats were divided into six groups: a normal control (NC) group, a model group, a Loratadine (LOR) group, an Evodiamine low dose (Evo-L) group, a Evodiamine high dose (Evo-H) groups, and an Evo-H combined with CCL2 group. After the last administration, the symptoms of rats in each group were scored; ELISA was applied to detect the levels of histamine, immunoglobulin E (IgE), interleukin 4 (IL-4), IL-13 and interferon γ (IFN-γ); Diff-Quick staining solution was applied to detecte the number of cells in the nasal lavage fluid (NALF); hematoxylin eosin (HE) staining was applied to observe the pathological changes of nasal mucosa tissue; real-time quantitative PCR was applied to detect the levels of CCL2 and CCR2 mRNA in tissue; Western blot was applied to detect the expression levels of CCL2, CCR2 and CXC motif chemokine ligand 8 (CXCL8) proteins in nasal mucosa. Results There were eight intersection targets of EVo-AR-immune function, and protein interaction network diagram showed that CXCL8 was the core target. Compared with the NC group, the score of nasal symptoms, the levels of histamine, IgE, IL-4 and IL-13, the numbers of eosinophil, macrophages, neutrophils, lymphocytes and total cells, the mRNA and protein expression levels of CCL2 and CCR2, and the expression of CXCL8 protein in the model group were increased, while the level of IFN-γ was decreased. Compared with the model group, the score of nasal symptoms, the levels of histamine, IgE, IL-4 and IL-13, the numbers of eosinophil, macrophages, neutrophils, lymphocytes and total cells, the mRNA and protein expression levels of CCL2 and CCR2, and the expression of CXCL8 protein in LOR and Evo groups were decreased, while the level of IFN-γ was increased. Further use of CCL2 recombinant protein for compensatory experiments revealed that the improvement effect of Evo on immune function in AR rats was reversed by CCL2. Conclusion Evo can improve the immune function of AR rats, and its mechanism may be related to the inhibition of the CCL2/CCR2 pathway.
Animals ; Receptors, CCR2/immunology* ; Signal Transduction/drug effects* ; Chemokine CCL2/immunology* ; Rats ; Rhinitis, Allergic/metabolism* ; Immunoglobulin E/blood* ; Quinazolines/pharmacology* ; Male ; Interferon-gamma ; Rats, Sprague-Dawley ; Interleukin-13 ; Histamine ; Interleukin-4/immunology* ; Disease Models, Animal