Plasma levels of IL-37 and correlation with TNF-α, IL-17A, and disease activity during DMARD treatment of rheumatoid arthritis.

The aim of this study was to assess the change of IL-37 concentrations in rheumatoid arthritis (RA) patients under Disease-modifying anti-rheumatic drug (DMARD) therapy, and to establish a correlation between Interleukin-37 and pro-inflammatory cytokines in plasma and disease activity. The plasma level of IL-37 was determined using ELISA in 50 newly diagnosed RA patients and 30 healthy controls (HC). Plasma levels of IL-17A, IL-6 and TNF-α were measured using flow a cytometric bead array assay. We found that the concentrations of IL-37, as well as IL-17A, IL-6 and TNF-α, were higher in plasma of RA patients compared to HCs. Compared to patients who did not respond to DMARD treatment, treatment of patients responsive to DMARDs resulted in down-regulation of IL-17A, IL-6 and TNF-α expression. The plasma level of the anti-inflammatory cytokine IL-37 was also decreased in drug responders after DMARD treatment. The plasma level of IL-37 in RA patients was positively correlated with pro-inflammatory cytokines (IL-17A, TNF-α) and disease activity (CRP, DAS28) in RA patients. IL-37 expression in RA and during DMARD treatment appears to be controlled by the level of pro-inflammatory cytokines. This results in a strong correlation between plasma levels of IL-37 and disease activity in RA patients.

Role of PLC-PIP2 and cAMP-PKA signal pathways in radiation-induced immune-suppressing effect.

OBJECTIVE: The purpose of the present study was to observe the changes in CD4+CD25+Nrp1+Treg cells after irradiation with different doses and explore the possible molecular mechanisms involved. METHODS: ICR mice and mouse lymphoma cell line (EL-4 cells) was used. The expressions of CD4, CD25, Nrp1, calcineurin and PKC-α were detected by flow cytometry. The expressions of TGF-ß1, IL-10, PKA and cAMP were estimated with ELISA. RESULTS: At 12 h after irradiation, the expression of Nrp1 increased significantly in 4.0 Gy group, compared with sham-irradiation group (P<0.05) in the spleen and thymus, respectively, when ICR mice received whole-body irradiation (WBI). Meanwhile the synthesis of Interleukin 10 (IL-10) and transforming growth factor-ß1 (TGF-ß1) increased significantly after high dose irradiation (HDR) (> or = 1.0 Gy). In addition, the expression of cAMP and PKA protein increased, while PKC-α, calcineurin decreased at 12h in thymus cells after 4.0 Gy X-irradiation. While TGF-ß1 was clearly inhibited when the PLC-PIP2 signal pathway was stimulated or the cAMP-PKA signal pathway was blocked after 4.0 Gy X-irradiation, this did not limit the up-regulation of CD4+CD25+Nrp1+Treg cells after ionizing radiation. CONCLUSION: These results indicated that HDR might induce CD4+CD25+Nrp1+Treg cells production and stimulate TGF-ß1 secretion by regulating signal molecules in mice.

Higher frequency of peripheral blood interleukin 21 positive follicular helper T cells in patients with ankylosing spondylitis.

OBJECTIVE: The role of follicular Th (TFH) cells remains unclear in the pathogenesis of ankylosing spondylitis (AS). Our study examined the frequency of different subsets of circulating CXCR5+CD4+ T cells in patients with AS before and after receiving therapy. METHODS: Percentages of peripheral blood inducible costimulator (ICOS)+, programmed death 1 (PD-1)+, and interleukin 21 (IL-21)+ CXCR5+CD4+ T cells in 26 patients with AS and 12 healthy controls (HC) were examined by flow cytometry, and the disease activity of individual patients was measured by Bath AS Disease Activity Index (BASDAI). The concentrations of serum IL-21, IgG, IgA, IgM, and C-reactive protein (CRP) were examined and the values of erythrocyte sedimentation rate (ESR) were measured. The potential association among these measures was analyzed. RESULTS: In comparison with that in HC, significantly increased percentages of CXCR5+CD4+, CXCR5+CD4+PD-1+, and CXCR5+CD4+IL-21+, but not CXCR5+CD4+ICOS+ and PD-1+ICOS+CXCR5+CD4+ T cells, and elevated concentrations of serum IL-21 were detected in patients with AS (p = 0.001, p = 0.012, p < 0.001, p = 0.233, p = 0.216, p < 0.001, respectively). Treatment with meloxicam, thalidomide, and etanercept for 1 month significantly reduced percentages of IL-21+CXCR5+CD4+ T cells and concentrations of serum IL-21 (p < 0.001, p < 0.001, respectively), accompanied by significantly minimized disease activity in drug responders, but not in the drug nonresponders. Further, percentages of IL-21+CXCR5+CD4+ T cells were positively correlated with BASDAI in patients (r = 0.6, p = 0.0012) and in the drug-responders 1 month after treatment (r = 0.68, p = 0.005), while the percentages of PD-1+CXCR5+CD4+ T cells were negatively correlated with BASDAI (r = -0.58, p = 0.0018). CONCLUSION: These data suggest that IL-21+CXCR5+CD4+ T cells may be associated with development of AS and that the frequency of IL-21+CXCR5+CD4+ T cells may be a biomarker for evaluation of disease activity and drug responses in patients with AS, particularly in drug-responding patients.

Peripheral B-cell activation and exhaustion markers in patients with ankylosing spondylitis.

AIMS: Ankylosing spondylitis (AS) is an autoimmune disease and is associated with abnormal B cell function. However, the roles of different B cell subsets are less clear. This study aimed to examine the frequency of different subsets of B cells in AS patients following standard therapies. MAIN METHODS: Eighteen newly diagnosed AS patients and 10 healthy controls (HC) were recruited in this study. The expression of CD27, CD38, CD86, CD95 and IgD on CD19(+) B cells was examined by flow cytometry. The disease activity was scored according to the Bath Ankylosing Spondylitis Disease Activity Index (BASDAI). Serum levels of C-reactive protein (CRP), rheumatoid factor (RF), IgG, IgA and IgM, and the erythrocyte sedimentation rate (ESR) were measured. KEY FINDINGS: The frequency of CD27(+) B cells was decreased in AS patients compared with HC (p=0.018), while CD86(+) and CD27(-)CD95(+) B cell subsets increased in AS patients (p<0.001). Meanwhile, the frequencies of CD38(+) and CD95(+) B cell positively correlated with BASDAI (r=0.6505, p=0.0035; r=0.6854, p=0.0017, respectively), while CD38(-)CD86(+) B cell negatively correlated with BASDAI (r=-0.7329, p<0.001). We also found that CD27(-) and CD95(+) B cell negatively correlated with RF levels (r=-0.5141, p=0.0290; r=-0.4944, p=0.0370, respectively), while CD27(+) B cell positively correlated with IgG levels (p=0.0148, r=0.5640). Moreover, CD86(+) and CD27(-)CD95(+) B cell subsets increased following treatment with Meloxicam and Etanercept for one month (p<0.001; p<0.001). SIGNIFICANCE: These findings suggest that CD27(-)CD95(+)CD19(+) and CD86(+)CD19(+) B cells may be reasonable cellular targets for the therapeutic intervention of AS.

Downregulation and altered function of natural killer cells in hepatitis B virus patients treated with entecavir.

The aim of the present study was to investigate the natural killer (NK) cell phenotype and function in chronic hepatitis B virus (HBV) patients and to study the effects of entecavir therapy (10 mg/day, p.o.) on these responses. Peripheral blood NK cells were collected from 18 chronic HBV patients and 14 healthy controls. The effect of entecavir therapy on the phenotype and function of NK cells in chronic HBV patients was characterized by flow cytometry analysis. Concentrations of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), HBV viral loads in both groups and potential associations between the frequency of peripheral NK cell subsets and clinical measures were determined. There was a significant reduction in the number of CD3(-)CD56(+) NK cells in chronic HBV patients compared with healthy controls. Furthermore, there were significant increases in the percentage of CD3(-)CD56(+)NKG2D(+) and CD3(-)CD56(+)NKP30(+) NK activating receptors in chronic HBV patients compared with healthy individuals, who exhibited downregulated expression following entecavir treatment. Spearman's correlation analysis revealed that there was a significant positive correlation between the percentage of NKG2D(+) and NKP30(+) NK cells and serum ALT levels. Characterization of NK cell degranulation indicated that the frequency of CD107a(+) NK cells in HBV patients (in response to K562 stimulation) was significantly greater than in healthy controls but decreased following entecavir treatment. Entecavir treatment of hepatitis B e antigen-positive chronic HBV-infected patients not only led to a reduction in HBV DNA loads and normalization of ALT and AST levels, but also resulted in the recovery of NK cell-mediated immunity.

Ionizing radiation stimulates secretion of pro-inflammatory cytokines: dose-response relationship, mechanisms and implications.

In previous studies we showed a marked increase in secretion of inflammatory cytokines TNFalpha and interleukin (IL)-1beta by mouse macrophages in response to different doses of ionizing radiation (IR). Here we show the stimulation of IL-12 and IL-18 secretion by mouse peritoneal macrophages after whole-body irradiation with exploration of the possible mechanisms and implications in cancer radiotherapy. Both low (0.075 Gy) and high (2 Gy) doses of IR were found to cause sustained stimulation of IL-12 and IL-18 secretion by mouse macrophages; this paralleled the activation of NF-kappaB as well as up-regulated expression of CD14 and TLR4-MD2 on the macrophage surface and MyD88 in the cytoplasm. The expression of CD14, TLR4-MD2 and MyD88 increased in a dose-dependent manner from radiation doses between 0.05 and 2 Gy. The secretion of IL-12 and IL-18 showed a dose-dependent increase from doses between 0.05 and 4 Gy. It is concluded that IR can stimulate the secretion of IL-12 and IL-18 presumably via activation of the Toll signaling pathway in macrophages. The potential harmful effect of repeated doses of radiation used in radiotherapy for certain cancers is discussed.

Labeling of human insulin-like growth factor-I eukaryotic expression vector with green fluorescent protein.

OBJECTIVE: To label human insulin-like growth factor-I (hIGF-I) eukaryotic expression vector with green fluorescent protein (GFP) for the repair of articular cartilage defects. METHODS: GFP cDNA was inserted into pcDNA(3.1)-hIGF-1 to construct the co-expression vector with two multiple cloning sites mammalian expression vector under two cytomegalovirus promoters/enhancers respectively. Recombinant pcGI was transfected into NIH 3T3 cells with the help of lipofectamine. RESULTS: Enzyme digestion and agarose gel electrophoresis analysis revealed that pcGI vector contained correct GFP and hIGF-I cDNA. Expression of hIGF-1 and GFP was confirmed in transfected NIH 3T3 cells by immunocytochemical analysis and fluorescence microscopy. CONCLUSIONS: hIGF-I eukaryotic expression vector has been successfully labeled with GFP.

[Influence of different mechanical environments on repair of cartilage defect with rabbit marrow mesenchymal stem cells].

OBJECTIVE: To study the influence of different mechanical environments on repair cartilage defect with marrow mesenchymal stem cells as seed cells. METHODS: The rabbit marrow mesenchymal stem cells were isolated and cultured. The cartilage defects were repaired by autologous tissue engineered cartilage with the marrow mesenchymal stem cells as seed cells. Fifteen rabbits with cartilage defect were divided into 3 groups: dislocation group with cell-free scaffold (control group), dislocation group with cartilaginous construct and normal mechanical environment group with cartilaginous construct. The repaired tissue was harvested and examined 6 weeks postoperatively. RESULTS: The repair tissue in normal mechanical environment group with cartilaginous construct showed cartilage-like tissue in superficial layer and subchondral bone tissue in deep layer 6 weeks postoperatively. The defect was filled with bone tissue in dislocation group with cartilaginous construct 6 weeks postoperatively. The surrounding normal cartilage tissue showed vascular invasion from subchondral area and the concomitant thinning of the normal cartilage layer. The cartilaginous construct left in the femoral trochlea groove formed hyaline cartilage-like tissue. The defect was repaired by fibrous tissue in control group. CONCLUSION: The repaired tissue by tissue engineered cartilage with marrow mesenchymal stem cells as seed cells showed the best result in normal mechanical environment group, which indicates that it will be essential for the formation and maintenance of tissue engineered cartilage to keep the normal mechanical stress stimulus.

Radiographic verification of pedicle screw pilot hole placement in thoracic spine using Kirschner wires versus spiral wires.

OBJECTIVE: To evaluate the feasibility of the pedicle screw pilot holes placement in thoracic spine using the spiral wires as the guide pin. METHODS: The pedicle screw pilot holes were drilled within the center of the pedicle and the lateral and medial pedicle walls were violated in 9 human dried thoracic vertebrae. Kirschner wires or spiral wires were separately placed in the holes, and then the posteroanterior and lateral radiographs were taken. The radiographs were evaluated by 3 experienced spine surgeons and 3 young orthopedists. After radiographs were shown to these observers, they combined the posteroanterior and lateral radiographs in each place and determined whether the pedicle screw pilot hole violated the pedicle cortex or not. The results were analyzed by a statistical software. RESULTS: Sensitivity, specificity and accuracy of the method using spiral wires to detect pedicle pilot hole placement were significantly higher than those of using Kirschner wires. With a true posteroanterior radiograph, the sensitivity, specificity and accuracy of the method using spiral wires approximated or attained 100%. CONCLUSIONS: The method of intrapedicular pilot hole placement verification using spiral wires is effective for guiding the accurate placement of pedicle screws.