Expression of CC Chemokine Ligand 5 in Patients with Rheumatoid Arthritis and Its Correlation with Disease Activity and Medication

Objective To determine the levels of CC chemokine ligand 5 (CCL5) in serum and synovial fluid (SF) from patients with rheumatoid arthritis (RA) and their relations with disease activity and medication. Methods CCL5 in serum and SF was quantified by enzyme-linked immunosorbent assay (ELISA) in 28 RA patients and 21 osteoarthritis (OA) patients. In RA patients, the correlations of CCL5 levels in serum and SF with disease activity were analyzed. Meanwhile, the serum CCL5 levels among RA patients treated with disease-modifying antirheumatic drugs (DMARDs), Tripterygium Glucosides, and other Chinese herbs without disease-modifying effects were also compared. Results CCL5 levels in both serum and SF of RA patients were significantly higher than those of OA patients (P<0.05). Moreover, the level of CCL5 was higher in SF than that in serum of RA patients (P<0.01). Serum CCL5 level was correlated significantly with the number of swollen joints (r=0.3329, P<0.05), erythrocyte sedimentation rate (r=0.4001, P<0.05), and C reactive protein (r=0.3735, P<0.01). In addition, the level of CCL5 had a trend of lower in patients treated with DMARDs or Tripterygium Glucosides than those treated with other Chinese herbs, although the difference was not significant among those patients due to the small number of patients in each group. Conclusions In RA patients, the expression of CCL5 increases and correlates with some clinical and laboratory parameters of RA, which indicate that CCL5 plays an important role in RA and may serve as a useful marker of disease activity. DMARDs and Tripterygium Glucosides might exert their clinical effects through reducing CCL5 production in RA.

Expression of FLICE-inhibitory Protein in Synovial Tissue and Its Association with Synovial Inflammation in Juvenile Idiopathic Arthritis

Objective To examine the expression of FLICE-inhibitory protein (FLIP) in juvenile idiopathic arthritis (JIA) and analyze its correlation with synovial inflammation. Methods The expression of FLIP was assessed in 11 JIA and 3 normal synovial tissue samples by reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry. The cell types expressing FLIP were further characterized,and the correlation of FLIP expression with the degree of synovial inflammation,as well as the activity of caspase 8 was then analyzed. Results RT-PCR revealed the expression of FLIP mRNA in all 11 JIA samples,but not in 3 normal synovial tissues. In JIA,FLIP expression could be found in both the lining and sublining layers,mainly in the macrophage-like cells. Moreover,the expression of FLIP in JIA synovial tissues was positively correlated with the degree of synovial inflammation (r=0.563,P<0.05). Conclusion The expression of antiapoptotic FLIP in JIA synovial tissue and its correlation to accumulation of inflammatory cells in synovial tissue suggests that FLIP potentially extends the lifespan of synovial cells and thus contributes to the progression of joint destruction.

MRI-guided photothermal/photodynamic immune activation combined with PD-1 inhibitor for the multimodal combination therapy of melanoma and metastases

Non-invasive image-guided precise photothermal/photodynamic therapy(PTT/PDT)has been proven to be an effective local treatment modality but incompetent against metastases.Hence,the combination of local PTT/PDT and systemic immunotherapy would be a promising strategy for tumor eradication.Herein,a magnetic resonance imaging(MRI)-visualized PTT/PDT agent(SIDP NMs)was constructed,and the efficacy of its multimodal combination with a programmed cell death 1(PD-1)inhibitor in the treatment of melanoma and metastases was studied.Due to the hydrophobic encapsulation of indocyanine green within the micellar core,SIDP NMs exhibited excellent photothermal/photodynamic properties and stability under an 808 nm near-infrared laser.In vitro cell experiments showed that SIDP NMs had a good killing effect.After incubating with B16-F10 cells for 24 h and irradiating with an 808-nm laser for 10 min,cell viability decreased significantly.Magnetic resonance imaging experiments in melanoma-bearing mice have shown that the dynamic distribution of SIDP NMs in tumor tissue could be monitored by T2WI and T2-MAP non-invasively due to the presence of superparamagnetic iron oxide nanocrystal in SIDP NMs.When the 808 nm laser was irradiated at the maximum focusing time point shown by MRI,the temperature of the tumor area rapidly increased from 32℃to 60.7℃in 5 min.In mouse melanoma ablation and distant tumor immunotherapy studies,SIDP NMs provided excellent MRI-guided PTT/PDT results and,when combined with PD-1 inhibitor,have great potential to cure primary tumors and eradicate metastases.

Expression of C5aR ( CD88) of synoviocytes isolated from patients with rheumatoid arthritis and osteoarthritis

Objective To investigate the expression of anaphylatoxin receptor C5aR (CD88) in synoviocytes from patients with rheumatoid arthritis (RA) and osteoarthritis (OA).Methods The expression of C5aR was assessed in synoviocytes isolated from 27 RA and 12 OA patients using reverse transcription-polymerase chain reactions (RT-PCR), flow cytometry, and immunofluorescence analysis. The effects of C5a on the release of tumor necrosis factor a (TNFα) from synoviocytes were assayed using enzyme-linked immunosorbent assays (ELISA).Results C5aR mRNA was detected in 24 of 27 samples from RA patients, and 10 of 12 samples from OA patients. Flow cytometric analysis and immunofluorescence study demonstrated the cell surface expression of C5aR in a significant proportion of synoviocytes from both RA and OA patients, and the level of C5aR expression in synoviocytes was significantly correlated with joint swelling, erythrocyte sedimentation rate ( ESR) and C-reactive protein ( CRP) in RA patients. Finally, interaction of C5aR with its ligand C5a was shown to enhance lipopolysaccharide (LPS) -induced TNFa release from synoviocytes.Conclusions The expression of C5aR in synoviocytes from inflammatory joint diseases and also the induction of TNFa release in activated synovicytes by the interaction of C5a and C5aR suggest that the C5a/C5aR system may play an important role in joint inflammation process.

Expression of PYCARD Gene Transcript Variant mRNA in Peripheral Blood Mononuclear Cells of Primary Gout Patients with Different Chinese Medicine Syndromes

Objective： To study the expression level and role of apoptosis-associated speck-like protein containing a caspase recruitment domain （PYCARD） gene transcript variant mRNA in peripheral blood mononuclear cells （PBMCs） of primary gout （PG） patients with different Chinese medicine （CM） syndromes. Methods： The expressions of PYCARD gene transcript variant mRNA and interleukin-1β （IL-1β） mRNA in PBMCs were investigated in 96 PG patients with acute phase （APPG, 44 cases） and non-acute phase （NAPPG, 52 cases） and 30 healthy controls （HCs） by reverse transcription-polymerase chain reaction （PCR） and/or real-time quantitative PCR. PYCARD and nuclear factor-κB （p50） [NF-κB （p50）] protein was detected by Western blot in PBMCs respectively. IL-1β, IL-4 and IL-10 protein levels in plasma of HCs and PG patients were measured by enzyme-linked immuno sorbent assay. Results： The main CM syndromes in APPG patients were obstruction of dampness and heat syndrome （ODHS, 36.36%） and intermingled phlegm-blood stasis syndrome （IPBSS, 27.27%）, while in NAPPG patients were Pi （Spleen）-deficiency induced dampness syndrome （PDIDS, 40.38%） and qi-blood deficiency syndrome （QBDS, 26.92%）. It showed statistical significances of the expressions of PYCARD gene and its transcript variant mRNA, the protein of PYCARD and NF-κB （p50） and the plasma IL-1β, IL-4 and IL-10 in APPG, NAPPG, ODHS, IPBSS, PDIDS and QBDS groups, compared with the HC group respectively （P〈0.05 or P〈0.01）. There were also significant differences of mRNA expressions of PYCARD-1 and PYCARD-2 as well as protein expressions of IL-1β, IL-4 and IL-10 among the 4 CM syndromes groups （P〈0.05 or P〈0.01）. Correlation analysis showed positive correlation between the mRNA expressions of PYCARD-1 gene transcript variant and IL-1β in APPG patients （r=0.3088, P=0.0183）. Conclusion： PYCARD gene and its transcript variant may play a critical and regulative role in the inflammatory response of PG patients with different phases and CM syndromes.

Increased Macroautophagy in Interferon-Gamma-Producing T Cells from Patients with Newly Diagnosed Systemic Lupus Erythematosus

Background： Imbalance of interferon-gamma （IFN-γ）, interleukin （IL）-4, and IL-17 producing by T cells is confirmed to contribute to the pathogenesis of systemic lupus erythematosus （SLE）. Autophagy is now emerging as a core player in the development and the function of the immune system. Therefore, we investigated the autophagic behavior in I FN-γ, IL-4-, and IL-17-producing T cells from patients with SLE. Methods： Thirty patients with SLE and 25 healthy controls matched for gender and age were recruited between September 2016 and May 2017. The autophagic levels in IFN-γ T cells, IL-4＋ T cells, and IL-17＋ T cells fi＇om patients with newly diagnosed SLE and healthy controls were measured using flow cytometry. The plasma levels of IFN-y were determined by enzyme-linked immunosorbent assay in SLE patients and healthy controls. Unpaired t-tests and the nonparametric Mann-Whitney U-test were used to compare data from patients with SLE and controls. Spearman＇s rank correlation coefficient was applied for calculation of the correlation between parallel variables in single samples. Results： Our results showed increased percentage of atttophagy in IFN-γ＋ T cells from patients with SLE and healthy controls （[8.07 ± 2.72]% vs. [3.76 ± 1.671%, t=5.184, P 〈 0.001）, but not in IL-4 T cells or IL-17＋ T cells （P 〉 0.05） as compared to healthy donors. Moreover, the plasma levels of IFN-γ in SLE patients were significantly higher than those in healthy controls （[68.9 ± 29.1] pg/ml vs. [24.7 ± 17.6] pg/ml, t = 5.430, P 〈 0.001 ）. Moreover, in SLE patients, the percentage of autophagy in IFN-γ T cells was positively correlated with the plasma levels of IFN-y （r = 0.344, P = 0.046）, as well as the disease activity of patients with SLE （r = 0.379, P =0.039）. Conclusion： The restllts indicate that autophagy in IFN-γ＋ T cells from SLE patients is activated, which might contribute to the persistence ofT cells producing IFN-y, such as Thl cells, and consequently restly in the high plasma levels of I FN-γ, and then enhance the disease activity of SLE.