OBJECTIVE: To investigate and analyze the effect of CXC chemokine receptor 1/2 (CXCR1/2) targeting inhibitor Reparixin combined with cytarabine (Ara-C) on the malignant biological behaviors of acute myeloid leukemia cells and its effect on the expression of the CXCR family, while exploring the accompanying molecular mechanism, providing scientific basis and reference for new molecular markers and targeted therapy for AML. METHODS: Acute myeloid leukemia U937 cells were treated with different concentrations of Reparixin, Ara-C alone or in combination, and the cell morphology was observed under an inverted microscope; Wright-Giemsa staining was used to detect cell morphological changes; CCK-8 method was used to detect cell proliferation; the ability of cell invasion was detected by Transwell chamber method; the ability of colony formation was detected by colony formation assay; cell apoptosis was detected by Hoechst 33258 fluorescent staining and Annexin V/PI double-staining flow cytometry; monodansylcadaverine(MDC) staining was used to detect cell autophagy; the expression of apoptosis, autophagy and related signaling pathway proteins was detected by Western blot and the expression changes of CXCR family were detected by real-time quantitative polymerase chain reaction (qRT-PCR). RESULTS: Reparixin could inhibit the proliferation, invasion, migration and clone formation ability of U937 cells. Compared with the single drug group, when U937 cells were intervened by Reparixin combined with Ara-C, the malignant biological behaviors such as proliferation, invasion and colony formation were significantly decreased, and the levels of apoptosis and autophagy were significantly increased (P<0.01). After Reparixin combined with Ara-C intervenes in U937 cells, it can up-regulate the expression of the pro-apoptotic protein Bax and significantly down-regulate the expression of the anti-apoptotic protein Bcl-2, and also hydrolyze and activate Caspase-3, thereby inducing cell apoptosis. Reparixin combined with Ara-C could up-regulate the expressions of LC3Ⅱ and Beclin-1 proteins in U937 cells, and the ratio of LC3Ⅱ/LC3Ⅰ in cells was significantly up-regulated compared with single drug or control group (P<0.01). MDC result showed that the green granules of vesicles increased significantly, and a large number of broken cells were seen (P<0.01). Reparixin combined with Ara-C can significantly inhibit the phosphorylation level of PI3K, AKT and NF-κB signaling molecule, inhibit the malignant biological behavior of cells by inhibiting the activation of PI3K/AKT/NF-κB pathway, and induce programmed cell death. Ara-C intervention in U937 cells had no effect on the expression of CXCR family (P>0.05). The expression of CXCR1, CXCR2, and CXCR4 mRNA could be down-regulated by Reparixin single-agent intervention in U937 cells (P<0.05), and the expression of CXCR2 was more significantly down-regulated than the control group and other CXCRs (P<0.01). When Reparixin and Ara-C intervened in combination, the down-regulated levels of CXCR1 and CXCR2 were more significant than those in the single-drug group (P<0.01), while the relative expressions of CXCR4 and CXCR7 mRNA had no significant difference compared with the single-drug group (P>0.05). CONCLUSION Reparixin combined with Ara-C can synergistically inhibit the malignant biological behaviors of U937 cells such as proliferation, invasion, migration and clone formation, and induce autophagy and apoptosis. The mechanism may be related to affecting the proteins expression of Bcl-2 family and down-regulating the proteins expression of CXCR family, while inhibiting the PI3K/AKT/NF-κB signaling pathway.
Humans ; U937 Cells ; Cytarabine/therapeutic use* ; Receptors, Interleukin-8A ; NF-kappa B ; Proto-Oncogene Proteins c-akt ; Phosphatidylinositol 3-Kinases ; Leukemia, Myeloid, Acute/genetics* ; Apoptosis ; Cell Proliferation ; Apoptosis Regulatory Proteins ; Proto-Oncogene Proteins c-bcl-2 ; RNA, Messenger ; Cell Line, Tumor

Objective: To produce chimeric antigen receptor T cells (CAR-T) targeting human hepatocyte growth factor/c-Met (HGF/c-Met) protein and detect its cytotoxicity against non-small cell lung cancer (NSCLC) cells H1975 in vitro. Methods: The whole gene sequence of c-Met CAR containing c-Met single-chain fragment variable was synthesized and linked to lentiviral vector plasmid, plasmid electrophoresis was used to detect the correctness of target gene. HEK293 cells were transfected with plasmid and the concentrated solution of the virus particles was collected. c-Met CAR lentivirus was transfected into T cells to obtain second-generation c-Met CAR-T and the expression of CAR sequences was verified by reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) and western blot, and the positive rate and cell subtypes of c-Met CAR-T cells were detected by flow cytometry. The positive expression of c-Met protein in NSCLC cell line H1975 was verified by flow cytometry, and the negative expression of c-Met protein in ovarian cancer cell line A2780 was selected as the control. The cytotoxicity of c-Met CAR-T to H1975 was detected by lactate dehydrogenase (LDH) cytotoxicity assay at 1∶1, 5∶1, 10∶1 and 20∶1 of effector: target cell ratio (E∶T). Enzyme-linked immunosorbent assay (ELISA) was used to detect the release of cytokines such as TNF-α, IL-2 and IFN-γ from c-Met CAR-T co-cultured with H1975. Results: The size of band was consistent with that of designed c-Met CAR, suggesting that the c-Met CAR plasmid was successfully constructed. The results of gene sequencing were consistent with the original design sequence and lentivirus was successfully constructed. CAR molecules expression in T cells infected with lentivirus was detected by western blot and RT-qPCR, which showed c-Met CAR-T were successfully constructed. Flow cytometry results showed that the infection efficiency of c-Met CAR in T cells was over 38.4%, and the proportion of CD8(+) T cells was increased after lentivirus infection. The NSCLC cell line H1975 highly expressed c-Met while ovarian cancer cell line A2780 negatively expressed c-Met. LDH cytotoxicity assay indicated that the killing efficiency was positively correlated with the E∶T, and higher than that of control group, and the killing rate reached 51.12% when the E∶T was 20∶1. ELISA results showed that c-Met CAR-T cells released more IL-2, TNF-α and IFN-γ in target cell stimulation, but there was no statistical difference between c-Met CAR-T and T cells in the non-target group. Conclusions: Human NSCLC cell H1975 expresses high level of c-Met which can be used as a target for immunotherapy. CAR-T cells targeting c-Met have been successfully produced and have high killing effect on c-Met positive NSCLC cells in vitro.
Humans ; Female ; Receptors, Chimeric Antigen/genetics* ; Carcinoma, Non-Small-Cell Lung ; CD8-Positive T-Lymphocytes ; Interleukin-2/pharmacology* ; Tumor Necrosis Factor-alpha ; Cell Line, Tumor ; HEK293 Cells ; Lung Neoplasms ; Ovarian Neoplasms ; Immunotherapy, Adoptive

OBJECTIVE: To investigate the expression and clinical significance of soluble interleukin-2 receptor(sIL-2R) in patients with multiple myeloma(MM). METHODS: 54 newly diagnosed MM patients in the Second Affiliated Hospital of Fujian Medical University from February 2020 to December 2021 were selected as the observation group, and 60 healthy people in our hospital in the same period were selected as the control group. The expression levels of sIL-2R in the serum of the two groups were detected by enzyme-linked immunosorbent assay. The differences of sIL-2R expression level among different clinical parameter groups in MM patients were compared. The clinical parameters include:gender, age, ISS stage, hemoglobin, albumin, serum creatinine, lactate dehydrogenase and β₂-microglobulin, blood calcium, bone marrow plasma cell ratio and treatment response. The relationship between sIL-2R expression level and progression-free survival(PFS) and overall survival(OS) in MM patients were analyzed. RESULTS: The expression of serum SIL-2R in MM patients was significantly higher than that in healthy control group (P<0.05). The expression of sIL-2R in MM patients who did not achieve complete remission(CR) was significantly higher than those of CR patients (P=0.037). There was no significant difference in the expression of serum sIL-2R between the groups of different sex, age, ISS stage, hemoglobin concentration, albumin content, serum creatinine level, lactate dehydrogenase level, the content of β₂-microglobulin, the concentration of blood calcium, and the proportion of bone marrow plasma cells(P>0.05). The PFS of sIL-2R high expression group(15 months) was shorter than that of sIL-2R low expression group (22 months), which was significant difference (P=0.041). But there was no significant difference in OS between sIL-2R high expression group and sIL-2R low expression group (P=0.124). Univariate analysis results showed that the high expression of serum sIL-2R was associated with poor PFS in MM patients. Multivariate analysis results showed that the high expression of serum sIL-2R was still an independent adverse prognostic factor for PFS in MM patients, However, the expression of serum sIL-2R was not statistically significant in evaluating OS in MM patients by univariate and multivariate analysis. CONCLUSION The expression of serum sIL-2R in MM patients was significantly higher than that in healthy people. Serum sIL-2R is an independent prognostic factor of PFS in MM patients.
Humans ; Calcium ; Clinical Relevance ; Creatinine ; Lactate Dehydrogenases ; Multiple Myeloma ; Receptors, Interleukin-2

The inhibition of the host's natural immune response by tumor cells was widely reported in the early phases of the development of oncology therapy, and the concept of employing the host's immune system to treat cancer, i.e. tumor immunotherapy, is not new. However, as a result of early theoretical constraints, clinical application of immunotherapy did not go smoothly and lagged significantly behind radiation and chemotherapy. The path has been winding, but the future now seems promising. Immunotherapy research has advanced enormously as a result of the maturing of immuno-editing theory and the creation of numerous technologies, despite a number of unsuccessful endeavors and clinical studies. Since around 1998, the US Food and Drug Administration (FDA) has approved a variety of tumor immunotherapies, including cytokines (interleukin-2, interferons), cancer vaccines (Provenge), immune checkpoint inhibitors (ipilimumab), and cellular therapies (chimeric antigen receptor-T (CAR-T)), signaling a boom in the field.
Cancer Vaccines/therapeutic use* ; Humans ; Immune Checkpoint Inhibitors ; Immunotherapy ; Interferons ; Interleukin-2/therapeutic use* ; Ipilimumab ; Neoplasms/pathology* ; Receptors, Chimeric Antigen

OBJECTIVE: To investigate the clinical value of expression level of interleukin-2 receptor (IL-2R) and interleukin-8 (IL-8) in the fever patients with hematological malignancies. METHODS: A total of 121 inpatients in the First Affiliated Hospital of Anhui Medical University from April 2018 to October 2019 were enrolled in this study. The patients were separated into infection group (61 cases) and non-infection group (60 cases). In the meantime, 40 healthy people without fever or infection in the hospital for physical examination were set as matched group. C-reactive protein (CRP), procalcitonin (PCT), and cytokines were detected in all the patients with fever after admission and infection control. While, blood samples were taken from healthy people during physical examination. RESULTS: The expression levels of IL-2R in infection group were higher than those in the control group (P<0.001), and the level of serum IL-2R in infection group was also higher than that in the non-infection group (P<0.05). Based on Spearman analysis, in patients with malignant hematologic disease, serum IL-2R level was positively correlated with CRP (r=0.557, P<0.001) and IL-8 (r=0.479, P<0.001), and IL-8 level was positively correlated with CRP (r=0.318, P<0.001). Compared with the non-infection group, the area under the curve (AUC) for the level of CRP, PCT, and IL-2R of the infection group was 0.714 (95%CI: 0.623-0.806), 0.765 (95%CI: 0.680-0.851), and 0.761 (95%CI: 0.686-0.836), the sensitivity was 0.705, 0.852, and 0.705, and the specificity was 0.717, 0.70, and 0.60, respectively. While, AUC of CRP+PCT, CRP+IL-2R, PCT+IL-2R, and CRP+PCT+IL-2R was 0.789 (95%CI: 0.712-0.866), 0.702 (95%CI: 0.623-0.782), 0.757 (95%CI: 0.677-0.838), and 0.789 (95%CI: 0.712-0.866), the sensitivity was 0.738, 0.934, 0.705, and 0.738, and the specificity was 0.840, 0.470, 0.810, and 0.840, respectively. CONCLUSION CRP, PCT, IL-2R, and IL-8 are useful parameters for diagnosis of the infectious fever in patients with hematological malignancies, which provides the basis of initial diagnosis and rational use of antibioties for clinician.
Biomarkers ; C-Reactive Protein ; Calcitonin ; Calcitonin Gene-Related Peptide ; Hematologic Neoplasms ; Humans ; Interleukin-8 ; Protein Precursors ; Receptors, Interleukin-2 ; Sepsis

The aim of this study was to investigate the relationship between the effects of different doses of X-rays on DNA damage and JAK/STAT signaling pathway activation in A549 cells. The A549 cells were radiated with X-rays at doses of 2, 4, and 8 Gy. The proliferation of A549 cells was detected by CCK8 method. The content of interleukin 6 (IL-6) in culture medium at different time points after irradiation was detected by enzyme-linked immunoassay, and the expression levels of IL-6 receptor (IL-6R) and p53 binding protein 1 (53BP1) were detected by immunofluorescent staining. The expression levels of JAK2, p-JAK2, STAT3 and p-STAT3 were detected by Western blot. The results showed that, compared with the control group, X-ray irradiation reduced the cellular proliferation, up-regulated the expression of 53BP1, increased the IL-6 content in the medium supernatant, and up-regulated the protein expression levels of IL-6R, JAK2, p-JAK2, STAT3, and p-STAT3. The above effects of X-ray irradiation were dose-dependent. These results suggest that the mechanism by which X-rays cause DNA damage in A549 cells may involve activation of the JAK/STAT signaling pathway.
A549 Cells ; DNA Damage ; radiation effects ; Humans ; Janus Kinase 2 ; metabolism ; Receptors, Interleukin-6 ; metabolism ; STAT3 Transcription Factor ; metabolism ; Signal Transduction ; Tumor Suppressor p53-Binding Protein 1 ; metabolism ; X-Rays

OBJECTIVE: Schizophrenia is a disabling disorder of unknown aetiology, lacking definite diagnostic method and cure. A reliable biological marker of schizophrenia is highly demanded, for which traceable immune mediators in blood could be promising candidates. We aimed to gather the best findings of neuroinflammatory markers for first-episode psychosis (FEP). METHODS: We performed an extensive narrative review of online literature on inflammation-related markers found in human FEP patients only. RESULTS: Changes to cytokine levels have been increasingly reported in schizophrenia. The peripheral levels of IL-1 (or its receptor antagonist), soluble IL-2 receptor, IL-4, IL-6, IL-8, and TNF-α have been frequently reported as increased in FEP, in a suggestive continuum from high-risk stages for psychosis. Microglia and astrocytes establish the link between this immune signalling and the synthesis of noxious tryptophan catabolism products, that cause structural damage and directly hamper normal neurotransmission. Amongst these, only 3-hydroxykynurenine has been consistently described in the blood of FEP patients. CONCLUSION: Peripheral molecules stemming from brain inflammation might provide insightful biomarkers of schizophrenia, as early as FEP or even prodromal phases, although more time- and clinically-adjusted studies are essential for their validation.
Astrocytes ; Biomarkers ; Encephalitis ; Humans ; Interleukin-1 ; Interleukin-4 ; Interleukin-6 ; Interleukin-8 ; Metabolism ; Methods ; Microglia ; Polytetrafluoroethylene ; Psychotic Disorders ; Receptors, Interleukin-2 ; Schizophrenia ; Synaptic Transmission ; Tryptophan

PURPOSE: The chemical structure of tubulosine has been known since the mid-1960s. However, little is known about its biological and pharmacological functions. The aim of this study was to investigate the novel functions of tubulosine in cancer treatment, specifically in breast cancer. METHODS: An Unpaired (Upd)-induced Drosophila cell line and interleukin (IL)-6-stimulated human breast cancer cell lines were used to investigate the biological and pharmacological activities of tubulosine in vitro. To investigate the activities of tubulosine, we performed molecular and cellular experiments such as Western blot and reverse transcription polymerase chain reaction analyses, immunoprecipitation and terminal deoxynucleotidyl transferase dUTP nick end labeling assays, and immunofluorescence staining using breast cancer cell lines. RESULTS: Tubulosine exhibited anticancer activity in IL-6-stimulated human breast cancer cells. Moreover, tubulosine reduced the tyrosine phosphorylation level and transcriptional activity of signal transducer and activator of transcription (STAT) protein at 92E in Upd-induced Drosophila cells. Additionally, tubulosine suppressed IL-6-induced Janus kinase 2 (JAK2)/STAT3 signaling, resulting in decreased viability and induction of apoptotic cell death in breast cancer cells. Interestingly, inhibition of IL-6-induced JAK2/STAT3 signaling by tubulosine was associated with the blocking of IL-6 receptor (IL-6R) and glycoprotein 130 (gp130) binding. CONCLUSION: Tubulosine exhibits anticancer activity through functional inhibition of IL-6-induced JAK2/STAT3 signaling by targeting IL-6Rα/gp130 binding in breast cancer cells. These findings suggest that tubulosine may hold promise for the treatment of inflammation-associated cancers, including breast cancer.
Blotting, Western ; Breast Neoplasms ; Cell Death ; Cell Line ; DNA Nucleotidylexotransferase ; Drosophila ; Fluorescent Antibody Technique ; Glycoproteins ; Humans ; Immunoprecipitation ; In Vitro Techniques ; Interleukin-6 ; Interleukins ; Janus Kinase 2 ; Phosphorylation ; Phosphotransferases ; Polymerase Chain Reaction ; Receptors, Interleukin-6 ; Reverse Transcription ; STAT3 Transcription Factor ; Transducers ; Tyrosine

Immunosuppressive regulatory T lymphocytes (Treg) expressing the transcription factor Foxp3 play a vital role in the maintenance of tolerance of the immune-system to self and innocuous non-self. Most Treg that are critical for the maintenance of tolerance to self, develop as an independent T-cell lineage from common T cell precursors in the thymus. In this organ, their differentiation requires signals from the T cell receptor for antigen, from co-stimulatory molecules, as well as from cytokine-receptors. Here we focus on the cytokines implicated in thymic development of Treg, with a particular emphasis on the roles of interleukin-2 (IL-2) and IL-15. The more recently appreciated involvement of TGF-β in thymic Treg development is also briefly discussed. Finally, we discuss how cytokine-dependence of Treg development allows for temporal, quantitative, and potentially qualitative modulation of this process.
Animals ; Cell Differentiation ; genetics ; Cytokines ; immunology ; Forkhead Transcription Factors ; genetics ; immunology ; Gene Expression Regulation ; Immune Tolerance ; genetics ; Interleukin-15 ; genetics ; immunology ; Interleukin-2 ; genetics ; immunology ; Mice ; Receptors, Antigen, T-Cell ; genetics ; immunology ; T-Lymphocytes, Regulatory ; immunology ; Transforming Growth Factor beta ; genetics ; immunology

Thyroid diseases, including autoimmune thyroid diseases and thyroid cancer, are known to have high heritability. Family and twin studies have indicated that genetics plays a major role in the development of thyroid diseases. Thyroid function, represented by thyroid stimulating hormone (TSH) and free thyroxine (T4), is also known to be partly genetically determined. Before the era of genome-wide association studies (GWAS), the ability to identify genes responsible for susceptibility to thyroid disease was limited. Over the past decade, GWAS have been used to identify genes involved in many complex diseases, including various phenotypes of the thyroid gland. In GWAS of autoimmune thyroid diseases, many susceptibility loci associated with autoimmunity (human leukocyte antigen [HLA], protein tyrosine phosphatase, non-receptor type 22 [PTPN22], cytotoxic T-lymphocyte associated protein 4 [CTLA4], and interleukin 2 receptor subunit alpha [IL2RA]) or thyroid-specific genes (thyroid stimulating hormone receptor [TSHR] and forkhead box E1 [FOXE1]) have been identified. Regarding thyroid function, many susceptibility loci for levels of TSH and free T4 have been identified through genome-wide analyses. In GWAS of differentiated thyroid cancer, associations at FOXE1, MAP3K12 binding inhibitory protein 1 (MBIP)-NK2 homeobox 1 (NKX2-1), disrupted in renal carcinoma 3 (DIRC3), neuregulin 1 (NRG1), and pecanex-like 2 (PCNXL2) have been commonly identified in people of European and Korean ancestry, and many other susceptibility loci have been found in specific populations. Through GWAS of various thyroid-related phenotypes, many susceptibility loci have been found, providing insights into the pathogenesis of thyroid diseases and disease co-clustering within families and individuals.
Autoimmunity ; Genes, Homeobox ; Genetics ; Genome-Wide Association Study* ; Graves Disease ; Hashimoto Disease ; Humans ; Leukocytes ; Neuregulin-1 ; Phenotype ; Protein Tyrosine Phosphatase, Non-Receptor Type 22 ; Receptors, Interleukin-2 ; T-Lymphocytes, Cytotoxic ; Thyroid Diseases* ; Thyroid Gland* ; Thyroid Neoplasms* ; Thyrotropin ; Thyroxine