OBJECTIVE: To explore the molecular bases of Chinese medicine (CM) syndrome classification in chronic hepatitis B (CHB) patients in terms of DNA methylation, transcription and cytokines. METHODS: Genome-wide DNA methylation and 48 serum cytokines were detected in CHB patients (DNA methylation: 15 cases; serum cytokines: 62 cases) with different CM syndromes, including dampness and heat of Gan (Liver) and gallbladder (CHB1, DNA methylation: 5 cases, serum cytokines: 15 cases), Gan stagnation and Pi (Spleen) deficiency (CHB2, DNA methylation: 5 cases, serum cytokines: 15 cases), Gan and Shen (Kidney) yin deficiency (CHB3, DNA methylation: 5 cases, serum cytokines: 16 cases), CHB with hidden symptoms (HS, serum cytokines:16 cases) and healthy controls (DNA methylation: 6 cases). DNA methylation of a critical gene was further validated and its mRNA expression was detected on enlarged samples. Genome-wide DNA methylation was detected using Human Methylation 450K Assay and furthered verified using pyrosequencing. Cytokines and mRNA expression of gene were evaluated using multiplex biometric enzyme-linked immunosorbent assay (ELISA)-based immunoassay and reverse transcription-quantitative polymerase chain reaction (RT-qPCR), respectively. RESULTS: Totally 28,667 loci, covering 18,403 genes were differently methylated among CHB1, CHB2 and CHB3 (P<0.05 and |Δβ value| > 0.17). Further validation showed that compared with HS, the hg19 CHR6: 29691140 and its closely surrounded 2 CpG loci were demethylated and its mRNA expressions were significantly up-regulated in CHB1 (P<0.05). However, they remained unaltered in CHB2 (P>0.05). Levels of Interleukin (IL)-12 were higher in CHB3 and HS than that in CHB1 and CHB2 groups (P<0.05). Levels of macrophage inflammatory protein (MIP)-1α and MIP-1β were higher in CHB3 than other groups and leukemia inhibitory factor level was higher in CHB1 and HS than CHB2 and CHB3 groups (P<0.05). IL-12, MIP-1α and MIP-1β concentrations were positively correlated with human leukocyte antigen F (HLA-F) mRNA expression (R²=0.238, P<0.05; R²=0.224, P<0.05; R=0.447, P<0.01; respectively). Furthermore, combination of HLA-F mRNA and differential cytokines greatly improved the differentiating accuracy among CHB1, CHB2 and HS. CONCLUSIONS: Demethylation of CpG loci in 5' UTR of HLA-F may up-regulate its mRNA expression and HLA-F expression was associated with IL-12, MIP-1α and MIP-1β levels, indicating that HLA-F and the differential cytokines might jointly involve in the classification of CM syndromes in CHB. REGISTRATION NO ChiCTR-RCS-13004001.
Chemokine CCL3/genetics* ; Chemokine CCL4/genetics* ; Cytokines/genetics* ; DNA Methylation/genetics* ; HLA Antigens ; Hepatitis B, Chronic/genetics* ; Histocompatibility Antigens Class I ; Humans ; Interleukin-12/genetics* ; Medicine, Chinese Traditional ; RNA, Messenger ; Syndrome

PURPOSE: The purpose of this study was to identify novel plasma biomarkers for distinguishing nasopharyngeal carcinoma (NPC) patients from healthy individuals who have positive Epstein-Barr virus (EBV) viral capsid antigen (VCA-IgA). MATERIALS AND METHODS: One hundred seventy-four plasma cytokines were analyzed by a Cytokine Array in eight healthy individuals with positive EBV VCA-IgA and eight patients with NPC. Real-time polymerase chain reaction, Western blotting, enzyme-linked immunosorbent assay (ELISA), and immunohistochemistry were employed to detect the expression levels of macrophage migration inhibitory factor (MIF) and CC chemokine ligand 3 (CCL3) in NPC cell lines and tumor tissues. Plasma MIF and CCL3 were measured by ELISA in 138 NPC patients, 127 EBV VCA-IgA negative (VN) and 100 EBV VCA-IgA positive healthy donors (VP). Plasma EBV VCA-IgA was determined by immunoenzymatic techniques. RESULTS: Thirty-four of the 174 cytokines varied significantly between the VP and NPC group. Plasma MIF and CCL3 were significantly elevated in NPC patients compared with VN and VP. Combination of MIF and CCL3 could be used for the differential diagnosis of NPC from VN cohort (area under the curve [AUC], 0.913; sensitivity, 90.00%; specificity, 80.30%), and combination of MIF, CCL3, and VCA-IgA could be used for the differential diagnosis of NPC from VP cohort (AUC, 0.920; sensitivity, 90.00%; specificity, 84.00%), from (VN+VP) cohort (AUC, 0.961; sensitivity, 90.00%; specificity, 92.00%). Overexpressions of MIF and CCL3 were observed in NPC plasma, NPC cell lines and NPC tissues. CONCLUSION: Plasma MIF, CCL3, and VCA-IgA combination significantly improves the diagnostic specificity of NPC in high-risk individuals.
Biomarkers* ; Blotting, Western ; Capsid* ; Cell Line ; Chemokine CCL3 ; Cohort Studies ; Cytokines ; Diagnosis ; Diagnosis, Differential ; Enzyme-Linked Immunosorbent Assay ; Herpesvirus 4, Human* ; Humans ; Immunoglobulin A* ; Immunohistochemistry ; Macrophages* ; Plasma* ; Real-Time Polymerase Chain Reaction ; Sensitivity and Specificity ; Tissue Donors

OBJECTIVETo observe the effects of recombinant fusion protein interleukin (IL)-18 on the expression of immune-inflammatory factors in the mice infected with Staphylococcus aureus (SA), and to investigate the mechanism of action of IL-18 in defense of SA infection in vivo.

METHODSA total of 40 specific pathogen-free female BLAB/c mice were randomly divided into four groups: control, SA infection, immunized, and intervention. A mouse model of SA infection was established by nasal inoculation with SA liquid. The immunized group and the intervention group were intranasally given IL-18 before SA modeling, and then the SA infection group and the intervention group received the nasal inoculation with SA liquid; the control group was treated with phosphate buffered saline instead. The levels of IL-4, interferon (IFN)-γ, tumor necrosis factor (TNF), granulocyte colony-stimulating factor (G-CSF), IgM in the serum and bronchoalveolar lavage fluid (BALF) of mice were measured by enzyme-linked immunosorbent assay. The expression of macrophage inflammatory protein (MIP)-1α mRNA and MIP-2β mRNA in the lung tissue of mice were determined by real-time fluorescent quantitative PCR.

RESULTSCompared with the control group, the SA infection group and the immunized group had significantly higher levels of IL-4, G-CSF, and IgM in the serum and BALF and expression of MIP-1α mRNA and MIP-2β mRNA in the lung tissue (P<0.05); the SA infection group had a significantly lower level of IFN-γ and a significantly higher level of TNF in the serum and BALF (P<0.05); the immunized group had a significantly higher level of IFN-γ in the serum and BALF (P<0.05). Compared with the SA infection group, the intervention group had significantly higher levels of IL-4, IFN-γ, G-CSF, and IgM in the serum and BALF and expression of MIP-1α mRNA in the lung tissue. In contrast, the intervention group showed a significantly lower level of TNF in the serum and BALF and expression of MIP-2β mRNA in the lung tissue (P<0.05). All the above indicators in the intervention group were significantly higher than those in the control group (P<0.05), except the serum level of IFN-γ.

CONCLUSIONSIn the mice infected with SA, the recombinant fusion protein IL-18 by mucosal immunity can affect inflammatory factors in the serum and BALF and the expression of MIP-1α mRNA and MIP-2β mRNA in the lung tissue to promote the anti-infective immune response and enhance the ability to clear pathogens.

Animals ; Chemokine CCL3 ; analysis ; Female ; Granulocyte Colony-Stimulating Factor ; blood ; Interferon-gamma ; blood ; Interleukin-18 ; therapeutic use ; Interleukin-4 ; blood ; Mice ; Mice, Inbred BALB C ; Recombinant Fusion Proteins ; pharmacology ; therapeutic use ; Staphylococcal Infections ; drug therapy ; immunology

UNLABELLEDBACKGROWND: Macrophage inflammatory protein-1α (MIP-l α/CCL3) belongs to the C-C chemokine family (CCL3), which can be secreted by macrophages, other types of hematopoietic cells and bone marrow stromal cells. Higher levels of MIP-1α were found to be associated with several kinds of hematologic malignancies, including multiple myeloma (MM), chronic lymphocytic leukemia (CLL) and chronic myeloid leukemia (CML). Moreover, MIP-1α has been reported to be an adverse prognostic factor for CLL. However, the impact of MIP-1α on acute myeloid leukemia (AML) has been poorly investigated.

OBJECTIVETo investigate the influence of MIP-1α on proliferction of AML cells.

METHODSUsing MLL-AF9 induced AML mouse model, the expression of MIP-1α was measured by real time quantitative RT-PCR. AML cell proliferation was examined by cell counting and colony forming assay (CFC). The influence of blocking the MIP-1α action on the growth and pathogenic ability of AML cells was explored by using the small molecule antagonist for interfering interaction of MIP-1α with its receptor CCR1.

RESULTSThe MIP-1α could promote the proliferation and colony formation of AML cells, the blocking MIP-1a could inhibit the growth of AML cells and delay onset of AML.

CONCLUSIONThe MIP-1a promotes the occurence and progression of AML, therefore blocking the MIP-1α signal pathway may be served as a strategy to inhibit the growth of AML cells, and MIP-1α can be a potential target for treatment of AML.

Animals ; Cell Line, Tumor ; Cell Proliferation ; Chemokine CCL3 ; Chemokine CCL4 ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; Leukemia, Myeloid, Acute ; Macrophage Inflammatory Proteins ; Mice ; Multiple Myeloma ; Receptors, CCR1

OBJECTIVETo detect the inflammatory factors of induced sputum (IS) and whole lung lavage fluid in pneumonoconiosis patients and to explore the correlation between the inflammatory factors with pulmonary function.

METHODSThe records of 45 cases of pneumonoconiosis patients were observed. All patients underwent lung function examination, sputum induction and massive whole lung lavage (WLL) sequentially through advance. IS and whole lung lavage fluid were collected respectively. Inflammatory factors of the two specimens were detected by using enzyme linked immunosorbent assay. The correlation of inflammatory factors between the two specimens was analyzed. The relationship between the inflammatory factor and lung function index was observed. The statistical analysis is performed with SPSS 17.0 for Windows. P < 0.05 is considered to be statistically significant.

RESULTSCytokines (MCP-1, TNF-α MIP-1α, NO(2)(-)/NO(3)(-) and IL-16) were significantly associated between IS and whole lung lavage fluid (P < 0.05), while TNF-α, MCP-1, NO(2)(-)/NO(3)(-) and IL-16 were no significantly associated with lung function index (P > 0.05). MIP-1α was significantly associated with FEV(1.0)/VCmax and MEF(25), respectively (P < 0.05).

CONCLUSIONSInflammatory factors were significantly associated between IS and whole lung lavage fluid, which could indicate early lung injury in pneumonoconiosis patients.

Bronchoalveolar Lavage Fluid ; chemistry ; Chemokine CCL2 ; Chemokine CCL3 ; Cytokines ; analysis ; Humans ; Interleukin-16 ; Lung ; Respiratory Function Tests ; Silicosis ; Sputum ; chemistry ; Tumor Necrosis Factor-alpha

Coal workers' pneumoconiosis (CWP) is characterized as a chronic inflammation of the lung associated with activation of macrophages and endothelial cells in the lung. The aim of the present study was to compare the levels of serum interleukin-8 (IL-8), macrophage inflammatory protein-1alpha (MIP-alpha), and intercellular adhesion molecule-1 (ICAM-1) as biomarkers for progressive massive fibrosis (PMF) in 106 subjects (27 non-CWP and 79 CWP patients). The levels of serum IL-8 (P<0.001) and ICAM-1 (P=0.001) of subjects with PMF were higher than those of non-CWP subjects. The IL-8 levels of PMF subjects were also higher than those of simple CWP subjects (P=0.003). Among the subjects without PMF, IL-8 levels in the subjects with International Labour Organization (ILO) category II or III were higher than those in the subjects with ILO category 0 (P=0.006) and with category I (P=0.026). These results suggest that high serum levels of IL-8 and ICAM-1, which are important as neutrophil attractants and adhesion molecules, are associated with PMF.
Aged ; Anthracosis/*blood/pathology ; Biological Markers/blood ; Chemokine CCL3/*blood ; Coal Mining ; Humans ; Intercellular Adhesion Molecule-1/*blood ; Interleukin-8/*blood ; Lung/pathology ; Male ; Middle Aged ; Occupational Diseases/blood/pathology ; Pulmonary Fibrosis/*blood/pathology

Coal workers' pneumoconiosis (CWP) is characterized as a chronic inflammation of the lung associated with activation of macrophages and endothelial cells in the lung. The aim of the present study was to compare the levels of serum interleukin-8 (IL-8), macrophage inflammatory protein-1alpha (MIP-alpha), and intercellular adhesion molecule-1 (ICAM-1) as biomarkers for progressive massive fibrosis (PMF) in 106 subjects (27 non-CWP and 79 CWP patients). The levels of serum IL-8 (P<0.001) and ICAM-1 (P=0.001) of subjects with PMF were higher than those of non-CWP subjects. The IL-8 levels of PMF subjects were also higher than those of simple CWP subjects (P=0.003). Among the subjects without PMF, IL-8 levels in the subjects with International Labour Organization (ILO) category II or III were higher than those in the subjects with ILO category 0 (P=0.006) and with category I (P=0.026). These results suggest that high serum levels of IL-8 and ICAM-1, which are important as neutrophil attractants and adhesion molecules, are associated with PMF.
Aged ; Anthracosis/*blood/pathology ; Biological Markers/blood ; Chemokine CCL3/*blood ; Coal Mining ; Humans ; Intercellular Adhesion Molecule-1/*blood ; Interleukin-8/*blood ; Lung/pathology ; Male ; Middle Aged ; Occupational Diseases/blood/pathology ; Pulmonary Fibrosis/*blood/pathology

OBJECTIVETo investigate the changes of cytokines in induced sputum at different stages of silicosis patients.

METHODSA total of 200 workers from one of the Shandong Province gold mine were chosen as object of observation. Among which 40 patients at silicosis stage I and 40 patients at silicosis stage II were divided into silicosis observed object group, silicosis stage I group, silicosis stage II group, and another 80 workers exposed to silica dust without suffering from silicotic Clinical symptoms, however, were chosen as group of dust exposed, and 40 logistical workers without being exposed and history of silicosis's illness were chosen as control group. And ask their basic information by questionnaire. Then, spray-inhalation the induced sputum and apply the ELISA to assess the level of tumor necrosis factor (TNF), interleukin (IL), macrophage inflammatory protein-1 (MIP-1α), monocyte chemotactic factor-1 (MCP-1), metalloproteinases (MMP), transforming growth factor-β (TGF-β), platelet derived growth factor (PDGF) in induced sputum from subjects.

RESULTSThe level of TGF-β [(901.60 ± 30.09) ng/L] in the induced sputumof patients in silicosis stage I group is lower than that in the observed object group [(913.02 ± 20.51) ng/L], and the level of MMP-9 [(212.49 ± 5.97) ng/L], MCP-1 [(129.91 ± 4.30) ng/L] has various degrees of increase than that in control group, observed object group and dust exposed group. All the differences have statistical significances (P < 0.05). The level of TNF-α [(85.76 ± 3.78) ng/L] in the induced sputum of patients in silicosis stage I group reaches the maximum, there are significant differences comparing with that level in the silica dust exposure group and the control group, whose differences are statistically significant (P < 0.05). Compared with the control group, the level of MMP-2 (427.95 ± 23.64) in the induced sputum of patients in silicosis stage I group has increased, whose differences also have statically significant (P < 0.05). Compared with the control group, silica dust exposed group, the observation group of objects, the pneumosilicosis patients of IL-16 in induced sputum IL-16 (21.40 ± 9.24) decreased, the content of PDGF [(5.96 ± 0.51) ng/L], MMP-2 [(447.86 ± 27.10) ng/L], MMP-9 [(223.91 ± 12.28) ng/L], MCP-1 [(122.87 ± 6.08) ng/L] increased, the differences are statistically significant (P < 0.05).

CONCLUSIONAs silicosis biomarkers, TNF-alpha, TGF-beta, IL-16, PDGF, MMP-2, MMP-9 and MCP-1 have certain significance, further suggesting that early detection rate of patients with silicosis can be improved by employing the multiple indexes discriminate equation.

Biomarkers ; metabolism ; Case-Control Studies ; Chemokine CCL2 ; metabolism ; Chemokine CCL3 ; metabolism ; Cytokines ; metabolism ; Discriminant Analysis ; Dust ; Humans ; Interleukin-16 ; metabolism ; Matrix Metalloproteinase 2 ; metabolism ; Matrix Metalloproteinase 9 ; metabolism ; Platelet-Derived Growth Factor ; metabolism ; Silicosis ; diagnosis ; Sputum ; chemistry ; Transforming Growth Factor beta ; metabolism ; Tumor Necrosis Factor-alpha ; metabolism

This study was purposed to explore the mechanism of central nervous system (CNS) leukemia resulting from brain metastasis of human acute T-cell leukemia (T-ALL) cells and the role of MIP-1α in migration of Jurkat cells through human brain microvascular endothelial cells (HBMEC). The real-time PCR, siRNA test, transendothelial migration test, endothelial permeability assay and cell adhesion assay were used to detect MIP-1α expression, penetration and migration ability as well as adhesion capability respectively. The results showed that the MIP-1α expression in Jurkat cells was higher than that in normal T cells and CCRF-HSB2, CCRF-CEM , SUP-T1 cells. The MIP-1α secreted from Jurkat cells enhanced the ability of Jurkat cells to penetrate through HBMEC, the ability of Jurkat cells treated by MIP-1α siRNA to adhere to HBMEC and to migrate trans endothelial cells decreased. It is concluded that the MIP-1α secreted from Jurkat cells participates in process of penetrating the Jurkat cells through HBMEC monolayer.
Brain Neoplasms ; pathology ; secondary ; Cell Adhesion ; Cell Movement ; Chemokine CCL3 ; metabolism ; Endothelial Cells ; pathology ; Endothelium, Vascular ; pathology ; Humans ; Jurkat Cells ; Precursor T-Cell Lymphoblastic Leukemia-Lymphoma ; pathology

This study aimed to investigate the inflammatory changes and their main indicators according to the time-period of postischemic reperfusion injury confirmed by analyzing changes of both pro-inflammatory and anti-inflammatory cytokines in the skeletal muscle and serum. By using 12-week-old male ICR strain mice were grouped into sham control and 8 different time-periods of reperfusion groups (0, 0.5, 1, 2, 4, 8, 16, 24 hours). Left common iliac artery of each mice in the reperfusion group was devascularized by a vascular clamp for 2 hours. Once anesthesia was applied to the experimental animals, blood serum was obtained from right heart atrium on the difference time-period of reperfusion (0-, 0.5-, 1-, 2-, 4-, 8-hour, respectively). Then, tissue fluid was collected in calf muscles (gastrocnemius muscle) after the mice were sacrificed by cervical dislocation. By using these serum and tissue fluids, enzyme-linked immunosorbent assay (ELISA) was used to analyze both pro-inflammatory cytokines (Eotaxin, IFNgamma, IL-1alpha, IL-1beta, IL-2, IL-3, IL-5, IL-6, MCP-1, MDC, MIP-1alpha, RANTES, TARC, TCA-3) and anti-inflammatory cytokines (IL-4, IL-10). Consequently, there were significant differences of pro-inflammatory cytokines levels in the skeletal muscle of 0-hour reperfusion group (p<.05) and those in the serum of 0-, 1-, 2-, 4-, 8-, 16-hour reperfusion groups (p<.05). In the serum of 4-hour reperfusion group, the presence of anti-iflammatory cytokines was significant from other groups (p<.05). By the comparison with the control group, furthermore, pro-inflammatory cytokines in the serum of 2-, 4-, 16-hour reperfusion group and anti-inflammatory cytokines in the serum of 4-hour reperfusion group were considerably different (p<.05). To sum up, changes of cytokine levels according to the time-period of reperfusion were considerably different in the serum rather than the tissue fluids from the skeletal muscle. In particular, IL-6 and MCP-1 in the serum showed higher density in 4- and 16-hour reperfusion groups so that they could be considered as the main indicator of pro-inflammatory cytokines.
Anesthesia ; Animals ; Chemokine CCL3 ; Chemokine CCL5 ; Cytokines* ; Dislocations ; Enzyme-Linked Immunosorbent Assay ; Heart Atria ; Humans ; Iliac Artery ; Interleukin-2 ; Interleukin-3 ; Interleukin-5 ; Interleukin-6 ; Ischemia ; Male ; Mice* ; Muscle, Skeletal* ; Muscles ; Reperfusion ; Reperfusion Injury* ; Serum