Objective To explore the alterations in macrophage polarization and the expression of decorin (DCN) protein in the decidua of patients with missed abortion (MA), as well as to elucidate the regulatory effect of DCN on macrophage polarization. Methods Flow cytometry was employed to assess the polarization ratio of decidual macrophages in MA, recurrent spontaneous abortion (RSA) and normal pregnancy (NP); The expression and localization of DCN and hypoxia-inducible factor 1α (HIF-1α) in decidua and villi were assessed by immunohistochemistry staining, while their protein levels were measured by Western blot. Primary trophoblasts and peripheral blood mononuclear cell (PBMC)-derived macrophages were isolated and cultured. ELISA was conducted to quantify DCN levels in the culture supernatant of primary trophoblast and PBMC-derived macrophages. Additionally, flow cytometry was applied to evaluate the polarization ratio of PBMC-derived macrophages. Immunofluorescence cytochemical staining was conducted to examine HIF-1α expression in macrophages. Western blot was performed to detect the expression of proteins related to the gene associated with retinoid-IFN-induced mortality 19 (GRIM-19)/signal transducer and activator of transcription 3 (STAT3)/HIF-1α signaling pathway in macrophages. Results The polarization ratio of M1 macrophages in the decidua of abortion patients was significantly higher than that of NP, whereas the ratio of M2 macrophages was significantly lower. The expression of DCN and HIF-1α protein were significantly evaluated in abortion patients compared to NP. The supernatant DCN content and HIF-1α protein expression of primary trophoblast and PBMC-derived macrophages cultured under 10 mL/L O₂ for 24 hours were markedly increased compared to cells treated with 210 mL/L O₂. Compared with the PBS group, the proportion of M1 macrophage and GRIM-19 protein expression were significantly reduced in the DCN group, while phosphorylated STAT3 (p-STAT3) and HIF-1α protein expression were significantly increased. Conclusion The expression of DCN in decidua and villi of MA is higher than that of NP. DCN exhibits an inhibitory effect on the M1 polarization of PBMCs-derived macrophages, which is likely mediated through the GRIM-19/STAT3/HIF-1α signaling pathway.
Humans ; Female ; Decidua/metabolism* ; Macrophages/cytology* ; Decorin/genetics* ; Pregnancy ; Hypoxia-Inducible Factor 1, alpha Subunit/metabolism* ; Adult ; Leukocytes, Mononuclear/cytology* ; Abortion, Missed/genetics* ; STAT3 Transcription Factor/metabolism* ; Cells, Cultured ; Young Adult

Leukocyte-specific protein 1 (LSP1) is an F-actin binding protein expressed in various leukocytes, including lymphocytes, mononuclear macrophages, and neutrophils. LSP1 is highly conserved across different species. Human LSP1 protein contains 339 amino acids, featuring a Ca²⁺ binding site in the acidic NH2-terminal region and multiple F-actin binding domains along with phosphorylatable sites in the basic COOH-terminal region. Under Ca²⁺ regulation, the COOH-terminal domain of LSP1 binds to F-actin to regulate cell movement and signal transduction. Additionally, LSP1 activates the mitogen-activated protein kinase (MAPK) signaling pathway through phosphorylation mediated by protein kinase C (PKC) and MAPK-activated protein kinase-2, thereby regulating leukocyte proliferation and chemotaxis. The main effects of LSP1 on leukocytes are as follows: LSP1 plays important roles in neutrophil and macrophage migration, affecting cell adhesion, polarization and movement. LSP1 also functions in endothelial cells to regulate leukocyte transendothelial migration. In addition, LSP1 regulates macrophage phagocytosis through interaction with myosin 1e. Moreover, LSP1 regulates leukocyte proliferation and differentiation and plays significant roles in the development of leukemia and other tumors. In summary, LSP1 regulates leukocyte morphology, movement and function through interactions with cytoskeletal and signaling proteins. This review provides a comprehensive summary of these aspects.
Humans ; Leukocytes/cytology* ; Animals ; Cytoskeleton/metabolism* ; Microfilament Proteins/physiology* ; Cell Movement ; Signal Transduction

OBJECTIVE: To detect the expression level of suppressors of cytokine signaling 3 （SOCS3） in acute lymphoblastic leukemia (ALL), and to observe the effect of over-expresson of SOCS3 in Jurkat cells on the cytotoxicity of NK cells. METHODS: The expression levels of SOCS3 mRNA in peripheral blood mononuclear cells of 20 children with ALL and 20 healthy children (normal control group) were detected by RT-PCR. The peripheral blood NK cells from healthy subjects were selected by immunomagnetic technique, and the purity was detected by flow cytometry. SOCS3 was overexpressed in Jurkat cells infected with lentivirus vector, and SOCS3 mRNA expression was detected by RT-PCR after lentivirus infection. The NK cells were co-cultured with the infected Jurkat, and LDH release method was used to detect the cytotoxicity of NK cells on the infected Jurkat cells. The concentrations of TNF-α and IFN-γ were determined by ELISA. The expression of NKG2D ligands MICA and MICB on the surface of Jurkat cells were detected by flow cytometry. Western blot was used to detect the effect of SOCS3 overexpression on STAT3 phosphorylation in Jurkat cells. RESULTS: Compared with the control group, the mRNA expression of SOCS3 in the peripheral blood mononucleated cells of ALL children was significantly decreased. The purity of NK cells isolated by flow cytometry could reach more than 70%. The expression of SOCS3 mRNA in Jurkat cells increased significantly after lentivirus infection. Overexpression of SOCS3 in Jurkat cells significantly promoted the killing ability of NK cells and up-regulated the secretion of TNF-α and IFN-γ from NK cells. The results of flow cytometry showed that the expression of NKG2D ligands MICA and MICB on Jurkat cells increased significantly after SOCS3 overexpression. Western blot results showed that overexpression of SOCS3 significantly reduced the phosphorylation level of STAT3 protein in Jurkat cells. CONCLUSION SOCS3 mRNA expression was significantly decreased in ALL patients, and overexpression of SOCS3 may up-regulate the expression of MICA and MICB of NKG2D ligands on Jurkat cell surface through negative regulation of JAK/STAT signaling pathway, thereby promoting the cytotoxic function of NK cells.
Child ; Histocompatibility Antigens Class I/metabolism* ; Humans ; Killer Cells, Natural/cytology* ; Leukocytes, Mononuclear/cytology* ; Ligands ; NK Cell Lectin-Like Receptor Subfamily K/metabolism* ; Precursor Cell Lymphoblastic Leukemia-Lymphoma/genetics* ; RNA, Messenger/genetics* ; Suppressor of Cytokine Signaling 3 Protein/metabolism* ; Tumor Necrosis Factor-alpha/metabolism*

BACKGROUND: Owing to the multifactorial nature of the pathogenesis of diabetic peripheral neuropathy (DPN), conventional drug therapies have not been effective. The application of stem cells transplantation may be useful for the treatment of DPN. This study was designed to assess the safety and therapeutic effects of autologous bone marrow mononuclear cells (BMMNCs) transplantation on the treatment of refractory DPN. METHODS: One hundred and sixty-eight patients with refractory DPN were recruited and enrolled in the study. They received intramuscular injection of BMMNCs and followed at 1, 3, 6, 12, 18, 24, and 36 months after the transplantation. Clinical data, Toronto Clinical Scoring System (TCSS), and nerve conduction studies (NCSs) were compared before and after the transplantation. RESULTS: The signs and symptoms of neuropathy were significantly improved after BMMNCs transplantation. The values of the TCSS scores at 1 month (9.68 ± 2.49 vs. 12.55 ± 2.19, P < 0.001) and 3 months (8.47 ± 2.39 vs. 12.55 ± 2.19, P < 0.001) after the treatment reduced significantly compared with the baseline value. This decrement remained persistent until the end of the study. The conduction velocity and action potential and sensory nerves were significantly improved after transplantation (3 and 12 months after the treatment vs. the baseline: motor nerve conduction velocity, 40.24 ± 2.80 and 41.00 ± 2.22 m/s vs. 38.21 ± 2.28 m/s, P < 0.001; sensory nerve conduction velocity, 36.96 ± 2.26 and 39.15 ± 2.61 m/s vs. 40.41 ± 2.22 m/s, P < 0.001; compound muscle action potential, 4.67 ± 1.05 and 5.50 ± 1.20 μV vs. 5.68 ± 1.08 μV, P < 0.001; sensory nerve action potential, 4.29 ± 0.99 and 5.14 ± 1.26 μV vs. 5.41 ± 1.14 μV, P < 0.001). No adverse event associated with the treatment was observed during the follow-up period. CONCLUSIONS Autologous transplantation of BMMNCs may be an effective and promising therapeutic strategy for the treatment of refractory DPN.
Adult ; Aged ; Aged, 80 and over ; Bone Marrow Transplantation ; methods ; Diabetic Neuropathies ; therapy ; Female ; Humans ; Leukocytes, Mononuclear ; cytology ; physiology ; Male ; Middle Aged ; Transplantation, Autologous ; methods

Astragalus membranaceus may be a potential therapy for childhood asthma but its driving mechanism remains elusive. The main components of A. membranaceus were identified by HPLC. The children with asthma remission were divided into two combination group (control group, the combination of budesonide and terbutaline) and A. membranaceus group (treatment group, the combination of budesonide, terbutaline and A. membranaceus). The therapeutic results were compared between two groups after 3-month therapy. Porcine peripheral blood mononuclear cells (PBMCs) were isolated from venous blood by using density gradient centrifugation on percoll. The levels of FoxP3, EGF-β, IL-17 and IL-23 from PBMCs and serum IgE were measured. The relative percentage of Treg/Th17 cells was determined using flow cytometry. The main components of A. membranaceus were calycosin-7-O-glucoside, isoquercitrin, ononin, calycosin, quercetin, genistein, kaempferol, isorhamnetin and formononetin, all of which may contribute to asthma therapy. Lung function was significantly improved in the treatment group when compared with a control group (P < 0.05). The efficacy in preventing the occurrence of childhood asthma was higher in the treatment group than the control group (P < 0.05). The levels of IgE, IL-17 and IL-23 were reduced significantly in the treatment group when compared with the control group, while the levels of FoxP3 and TGF-β were increased in the treatment group when compared with the control group (P < 0.05). A. membranaceus increased the percentage of Treg cells and reduced the percentage of Th17 cells. A. membranaceus is potential natural product for improving the therapeutic efficacy of combination therapy of budesonide and terbutaline for the children with asthma remission by modulating the balance of Treg/Th17 cells.
Animals ; Asthma ; drug therapy ; immunology ; Astragalus propinquus ; chemistry ; Budesonide ; administration & dosage ; Cells, Cultured ; Child ; Child, Preschool ; Cytokines ; metabolism ; Drugs, Chinese Herbal ; administration & dosage ; pharmacology ; Female ; Humans ; Immunologic Factors ; administration & dosage ; pharmacology ; Leukocytes, Mononuclear ; drug effects ; metabolism ; Lung ; drug effects ; physiology ; Male ; Swine ; T-Lymphocytes, Regulatory ; cytology ; drug effects ; Terbutaline ; administration & dosage ; Th17 Cells ; cytology ; drug effects ; Treatment Outcome

OBJECTIVE: To identify differentially expressed genes in peripheral blood mononuclear cells between patients with continuous mild-to-moderate asthma and healthy controls using mRNA microarray in order to explore the underlying signaling pathways and clarify the roles of CD4+ T cells in the pathogenesis of asthma. METHODS: Global transcriptomic profiles of the CD4+ T cells were defined by using Agilent Sure Print G3 Human GE 8×60K microarray. Enrichment pathways were analyzed with Ingenuity Pathway Analysis (IPA) software. RESULTS: Compared with controls, 805 genes were up-regulated, 192 were down-regulated in asthma patients. Among these, the expression of 38 annotated genes have varied by 4 times or more. Expression of CD300A was inversely proportional to the absolute value of eosinophils (r=-0.89, P=0.02) as well as the proportion of eosinophils (r=-0.94, P=0.004), while CSF1R was inversely proportional to PD20 (r=-0.83, P=0.04) and AQLQ (r=-0.88, P=0.02) by correlation analysis. CONCLUSION Numerous pathophysiological pathways may be involved in the pathogenesis of asthma. Above findings have provided a basis for the delineation the pathogenesis of asthma.
Antigens, CD ; genetics ; Asthma ; immunology ; CD4-Positive T-Lymphocytes ; cytology ; Case-Control Studies ; Eosinophils ; Gene Expression Profiling ; Humans ; Leukocytes, Mononuclear ; Oligonucleotide Array Sequence Analysis ; Receptors, Granulocyte-Macrophage Colony-Stimulating Factor ; genetics ; Receptors, Immunologic ; genetics ; Transcriptome

Leukocyte differentiation antigens (LDAs) play important roles in the immune system, by serving as surface markers and participating in multiple biological activities, such as recognizing pathogens, mediating membrane signals, interacting with other cells or systems, and regulating cell differentiation and activation. Data mining is a powerful tool used to identify novel LDAs from whole genome. LRRC25 (leucine rich repeat-containing 25) was predicted to have a role in the function of myeloid cells by a large-scale "omics" data analysis. Further experimental validation showed that LRRC25 is highly expressed in primary myeloid cells, such as granulocytes and monocytes, and lowly/intermediately expressed in B cells, but not in T cells and almost all NK cells. It was down-regulated in multiple acute myeloid leukemia (AML) cell lines and bone marrow cells of AML patients and up-regulated after all-trans retinoic acid (ATRA)-mediated granulocytic differentiation in AML cell lines and acute promyelocytic leukemia (APL; AML-M3, FAB classification) cells. Localization analysis showed that LRRC25 is a type I transmembrane molecule. Although ectopic LRRC25 did not promote spontaneous differentiation of NB4 cells, knockdown of LRRC25 by siRNA or shRNA and knockout of LRRC25 by the CRISPR-Cas9 system attenuated ATRA-induced terminal granulocytic differentiation, and restoration of LRRC25 in knockout cells could rescue ATRA-induced granulocytic differentiation. Therefore, LRRC25, a potential leukocyte differentiation antigen, is a key regulator of ATRA-induced granulocytic differentiation.
Antigens, Differentiation ; immunology ; metabolism ; Cell Differentiation ; drug effects ; Cell Line, Tumor ; Granulocytes ; cytology ; drug effects ; immunology ; metabolism ; Humans ; Leukocytes ; cytology ; drug effects ; immunology ; metabolism ; Membrane Proteins ; antagonists & inhibitors ; immunology ; metabolism ; RNA, Small Interfering ; pharmacology ; Tretinoin ; pharmacology

To investigate the effects of adenosine triphosphate (ATP) on expression of inflammatory factors induced by lipopolysaccharide (LPS) in endothelial progenitor cells (EPCs), and to elucidate the possible mechanisms.
 Methods: Mononuclear cells were isolated from human umbilical cord blood by density gradient centrifugation, RT-PCR was performed to detect the expression of inflammatory factors induced by LPS (1 mg/mL) in EPCs, the effect of low concentration (5 μmol/L) of ATP on expression of IL-1β, MCP-1 and ICAM-1, and the effect of different concentrations (5, 50 μmol/L) of ATP on the expression of Toll-like receptor (TLR) 4, myeloid differentiation primary response protein 88 (MyD88) and CD14. Western blot was performed to detect expression of TLR4 regulated proteins MyD88 and CD14 or to detect the low concentration (1, 5 μmol/L) of ATP on the expression of TLR4, MyD88 and CD14 and the NF-κB signaling pathway.
 Results: EPCs highly expressed TLR4, and its ligand LPS (1 mg/mL) significantly upregulated mRNA expression of IL-1β, MCP-1 and ICAM-1 and protein expression of MyD88 and CD14 in a time-dependent manner (P<0.01), accompanied by activation of ERK and NF-κB signal pathway. ATP at low concentration (5 μmol/L) significantly inhibited LPS-induced mRNA expression of IL-1β, MCP-1 and ICAM-1(P<0.05), downregulated the LPS-induced protein expression of TLR4, MyD88 and CD14 in EPCs (P<0.05), and suppressed LPS-induced activation of NF-κB signaling pathway (P<0.05).
 Conclusion: ATP at low concentration may suppress LPS-induced expression of inflammatory factors in EPCs through negative regulation of the TLR4 signaling pathway.
Adenosine Triphosphate ; pharmacology ; Endothelial Progenitor Cells ; drug effects ; Gene Expression Regulation ; drug effects ; Humans ; Leukocytes, Mononuclear ; cytology ; Lipopolysaccharide Receptors ; genetics ; Lipopolysaccharides ; pharmacology ; Myeloid Differentiation Factor 88 ; genetics ; NF-kappa B ; metabolism ; Signal Transduction ; drug effects ; Toll-Like Receptor 4 ; genetics

OBJECTIVEThis study observed attenuating effect of hydroxysafflor yellow A (HSYA), an effective ingredient of aqueous extract of Carthamus tinctorius L, on lipopolysaccharide (LPS)-induced endothelium inflammatory injury.

METHODSEahy926 human endothelium cell (EC) line was used; thiazolyl blue tetrazolium bromide (MTT) test was assayed to observe the viability of EC; Luciferase reporter gene assay was applied to measure nuclear factor-κB (NF-κB) p65 subunit nuclear binding activity in EC; Western blot technology was used to monitor mitogen activated protein kinase (MAPKs) and NF-κB activation. Reverse transcription polymerase chain reaction (RT-PCR) method was applied to observe intercellular cell adhesion molecule-1 (ICAM-1) and E-selectin mRNA level; EC surface ICAM-1 expression was measured with flow cytometry and leukocyte adhesion to EC was assayed with Rose Bengal spectrophotometry technology.

RESULTSHSYA protected EC viability against LPS-induced injury (P <0.05). LPS-induced NF-κB p65 subunit DNA binding (P <0.01) and nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor α (IκBα) phosphorylation was inhibited by HSYA. HSYA attenuated LPS triggered ICAM-1 and E-selectin mRNA levels elevation and phosphorylation of p38 MAPK or c-Jun N-terminal kinase MAPK. HSYA also inhibited LPS-induced cell surface ICAM-1 protein expression P <0.01) and leukocyte adhesion to EC (P <0.05).

CONCLUSIONHSYA is effective to protect LPS-induced high expression of endothelium adhesive molecule and inflammatory signal transduction.

Cell Adhesion ; drug effects ; Cell Nucleus ; drug effects ; metabolism ; Cell Survival ; drug effects ; Chalcone ; analogs & derivatives ; chemistry ; pharmacology ; therapeutic use ; E-Selectin ; genetics ; metabolism ; Endothelium, Vascular ; drug effects ; pathology ; Gene Expression Regulation ; drug effects ; Human Umbilical Vein Endothelial Cells ; drug effects ; metabolism ; pathology ; Humans ; I-kappa B Proteins ; metabolism ; Inflammation ; drug therapy ; pathology ; Intercellular Adhesion Molecule-1 ; genetics ; metabolism ; Leukocytes ; cytology ; drug effects ; Lipopolysaccharides ; MAP Kinase Signaling System ; drug effects ; NF-KappaB Inhibitor alpha ; Phosphorylation ; drug effects ; Protective Agents ; pharmacology ; Protein Binding ; drug effects ; Quinones ; chemistry ; pharmacology ; therapeutic use ; RNA, Messenger ; genetics ; metabolism

Enterovirus 71 frequently involves the central nervous system and may present with a variety of neurologic manifestations. Here, we aimed to describe the clinical features, magnetic resonance imaging (MRI) findings, and cerebrospinal fluid (CSF) profiles of patients presenting with neurologic complications of enterovirus 71 infection. We retrospectively reviewed the records of 31 pediatric patients hospitalized with acute neurologic manifestations accompanied by confirmed enterovirus 71 infection at Ulsan University Hospital between 2010 and 2014. The patients' mean age was 2.9 ± 5.5 years (range, 18 days to 12 years), and 80.6% of patients were less than 4 years old. Based on their clinical features, the patients were classified into 4 clinical groups: brainstem encephalitis (n = 21), meningitis (n = 7), encephalitis (n = 2), and acute flaccid paralysis (n = 1). The common neurologic symptoms included myoclonus (58.1%), lethargy (54.8%), irritability (54.8%), vomiting (48.4%), ataxia (38.7%), and tremor (35.5%). Twenty-five patients underwent an MRI scan; of these, 14 (56.0%) revealed the characteristic increased T2 signal intensity in the posterior region of the brainstem and bilateral cerebellar dentate nuclei. Twenty-six of 30 patients (86.7%) showed CSF pleocytosis. Thirty patients (96.8%) recovered completely without any neurologic deficits; one patient (3.2%) died due to pulmonary hemorrhage and shock. In the present study, brainstem encephalitis was the most common neurologic manifestation of enterovirus 71 infection. The characteristic clinical symptoms such as myoclonus, ataxia, and tremor in conjunction with CSF pleocytosis and brainstem lesions on MR images are pathognomonic for diagnosis of neurologic involvement by enterovirus 71 infection.
Acute Disease ; Brain/diagnostic imaging ; Central Nervous System Diseases/etiology/*pathology ; Child ; Child, Preschool ; Encephalitis/pathology ; Enterovirus A, Human/genetics/*isolation & purification ; Enterovirus Infections/drug therapy/*pathology/virology ; Feces/virology ; Female ; Humans ; Immunoglobulins/administration & dosage ; Infant ; Injections, Intravenous ; Leukocytes/cytology ; Leukocytosis/cerebrospinal fluid/pathology ; Magnetic Resonance Imaging ; Male ; RNA, Viral/genetics/metabolism ; Real-Time Polymerase Chain Reaction ; Republic of Korea ; Retrospective Studies ; Seasons