Effects of PDTC and caspase-8 inhibitor on MUTZ-1 cells.

<p>(<b>A</b>) Effects of PDTC on MUTZ-1 cell hTERT mRNA expression. Detection of hTERT mRNA from MUTZ-1 cells treated with 100 µM PDTC for the indicated times using Quantitative RT-PCR. (<b>B</b>) Effects of PDTC on MUTZ-1 cell caspase-8 protein level. Western blot analysis of caspase-8 protein from MUTZ-1 cells treated with different concentrations of PDTC for 10 h. The doses of PDTC used are expressed in µM. (<b>C</b>) Effects of caspase-8 inhibitor and As₂O₃ on NF-κB activity as evaluated by gel shift assays. C =  control; lane 1 =  treatment with 40 µM caspase-8 inhibitor; lane 2 =  treatment with 8.0 µM As₂O₃; lane 3 =  treatment with 40 µM caspase-8 inhibitor plus 8.0 µM As₂O₃. Marked inhibition of NF-κB activity by As₂O₃ alone, and by As₂O₃ plus caspase-8 inhibitor was observed, while caspase-8 inhibitor alone showed little effect on NF-κB activity when compared with controls (lane C, and lane 1).</p

Cleavage of caspase-8, caspase-3, and poly (ADP-ribose) polymerase (PARP) in MUTZ-1 and SKM-1 cells treated with As₂O₃ for 24 h.

<p>The concentrations of As₂O₃ used to treat MUTZ-1 and SKM-1 cells in the experiments are indicated on top of the figure. To control for equal loading, anti-β-actin and GAPDH antibody were used as probe in MUTZ-1and SKM-1 cells respectively.</p

Expression of FLIP, BCL-2, BCL-XL, XIAP in MUTZ-1 and SKM-1 cells treated with As₂O₃ for 24 h.

<p>The concentrations of As₂O₃ used to treat MUTZ-1 and SKM-1 cells in the experiments are indicated on top of the figure. To control for equal loading, a GAPDH antibody was used as a probe.</p

Gel shift assay of AP-1 and SP-1 in As₂O₃-treated MUTZ-1.

<p>Represents dose responses of AP-1 and SP-1 in MUTZ-1 cells treated with different concentration of As₂O₃. Results are representative of three independent experiments.</p

Effect of PDTC on MUTZ-1 and SKM-1 cells proliferation and Gel shift assay of NF-κB in As₂O₃-treated MUTZ-1 and SKM-1 cells.

<p>(<b>A</b>) Effect of PDTC on MUTZ-1 and SKM-1 cells proliferation. MUTZ-1 and SKM-1 cells were treated for 10 h with indicated concentrations of PDTC. The experiments were performed in triplicate and MTT assays were used to evaluate proliferation. Data are given as means ± standard deviation (SD) of three independent experiments. (<b>B</b>) and (<b>C</b>) Represents time and dose responses of NF-κB in MUTZ-1 and SKM-1 cells treated with different concentration of As₂O₃. (N =  negative control).</p

Detection of MUTZ-1 and SKM-1 cells apoptosis after treatment with As₂O₃.

<p>(<b>A</b>): Morphologic evaluation of apoptotic cells using Wright-Giemsa staining. (a) untreated cells; (b) apoptotic cells with nuclear karyorrhexis; (c) apoptotic cells with nuclear pyknosis; (d) apoptotic cell with apoptotic bodies. Magnification is 100×, using an Olympus microscope. (<b>B</b>): Detection of DNA ladder after 24 h of As₂O₃ treatment. Cellular DNA was isolated and separated by 2% agarose gel electrophoresis. M = 100 bp DNA marker. (<b>C</b>) and (<b>D</b>): Flow cytometry analysis of cell apoptosis. After treatment with As₂O₃, the cells were double stained with annexin V-FITC and PI and analyzed by flow cytometry. Annexin V positive cells are shown in the second and fourth quadrants of the plots. (<b>C</b>): Percent of apoptotic MUTZ-1 cells were 5.89%±1.56%, 29.62%±9.95%, 43.88%±15.08%, and 74.8%±7.97% after 24 h treatment of the cells with 0, 2.0, 4.0, and 8.0 µM of As₂O₃, respectively. (<b>D</b>): Percent of apoptotic SKM-1 cells were 2.6%, 15.8%, 30.8%, and 48.7% after 24 h treatment of the cells with 0, 2.0, 4.0, and 8.0 µM of As₂O₃, respectively. Values represent means ± standard deviation (SD) of three individual experiments.</p

<i>In vitro</i> inhibitory effect of As₂O₃ on MUTZ-1 cells and SKM-1 cells proliferation.

<p>(<b>A</b>): MUTZ-1 cells were incubated with the indicated concentrations of As₂O₃ for 24 h, 48 h, and 72 h. (<b>B</b>): SKM-1 cells were incubated with the indicated concentrations of As₂O₃ for 12 h, 24 h, and 48 h. The MTT assay was performed in triplicate. The results are given as the means ± standard deviation (SD) of three independent experiments.</p

Expressions of hTERT mRNA and protein of MUTZ-1 and SKM-1 cells evaluated by Quantitative RT-PCR and Western blotting.

<p>(<b>A</b>): Expressions of hTERT mRNA of MUTZ-1 and SKM-1 cells evaluated by Quantitative RT-PCR. (<b>B</b>): Expression of hTERT protein in MUTZ-1 cells treated with As₂O₃ evaluated by Western blot. (<b>C</b>): Expression of hTERT protein in SKM-1 cells treated with As₂O₃ evaluated by Western blot. The concentrations of As₂O₃ (µM) are indicated on the tops of the figures. To control for equal loading, anti-β-actin and GAPDH antibody were used as probe. Results are representative of three independent experiments.</p

Table_1_Risk of HBV reactivation in relapsed or refractory diffuse large B-cell lymphoma patients receiving Bruton tyrosine kinase inhibitors therapy.docx

BackgroundBruton tyrosine kinase inhibitors (BTKis) interrupt B-cell receptor signaling and thereby could potentially reactivate hepatitis B virus (HBV). However, data about the risk for HBV reactivation (HBVr) of BTKis in relapsed or refractory diffuse large B-cell lymphoma (R/R DLBCL) patients are sparse.MethodsA total of 55 R/R DLBCL patients receiving BTKis therapy in the Tongji Hospital of Tongji University were enrolled. Patient clinical characteristics, treatment outcomes and details of HBVr were collected and analyzed, aiming to demonstrate the risk of HBVr in R/R DLBCL patients post BTKis therapy and the efficacy of BTKis in HBV-associated R/R DLBCL patients.ResultsOf 55 R/R DLBCL patients treated with ibrutinib (N=38) and zanubrutinib (N=17), 4 were with chronic HBV infection (HBsAg positive), 26 with resolved HBV infection (HBsAg negative and HBcAb positive) and 25 without HBV infection (HBsAg negative and HBcAb negative). In resolved HBV infection group, 2 patients developed HBVr after the use of ibrutinib and zanubrutinib respectively. Neither of them developed HBV-related hepatitis. Our finding showed that the incidence of HBVr in resolved HBV infection group was 7.69% (95% CI, 0.9-25.1%). In this study, Overall response rate (ORR) was 70.9%. 1-year overall survival (OS) rate was 80.0%. Median progression-free survival (PFS) was 4 months (95% CI, 3-5 months). In addition, HBV infection was not associated with response rates or survival among R/R DLBCL patients post BTKis treatments.ConclusionOur study suggested that HBV infection do not affect the efficacy of BTKis’ treatment. However, R/R DLBCL patients with resolved HBV infection are at a moderate risk of developing HBVr throughout BTKis treatment. Patients should be screened for HBVr during BTKis therapy.</p

Image_1_Apremilast Ameliorates Experimental Arthritis via Suppression of Th1 and Th17 Cells and Enhancement of CD4+Foxp3+ Regulatory T Cells Differentiation.tif

<p>Apremilast is a novel phosphodiesterase 4 (PDE4) inhibitor suppressing immune and inflammatory responses. We assessed the anti-inflammatory effects of Apremilast in type II collagen (CII)-induced arthritis (CIA) mouse model. To determine whether Apremilast can ameliorate arthritis onset in this model, Apremilast was given orally at day 14 after CII immunization. Bone erosion was measured by histological and micro-computed tomographic analysis. Anti-mouse CII antibody levels were measured by enzyme-linked immunosorbent assay, and Th17, Th1 cells, and CD4⁺Foxp3⁺ regulatory T (Treg) cells were assessed by flow cytometry in the lymph nodes. Human cartilage and rheumatoid arthritis (RA) synovial fibroblasts (RASFs) implantation in the severe combined immunodeficiency mouse model of RA were used to study the role of Apremilast in the suppression of RASF-mediated cartilage destruction in vivo. Compared with untreated and vehicle control groups, we found that Apremilast therapy delayed arthritis onset and reduced arthritis scores in the CIA model. Total serum IgG, IgG1, IgG2a, and IgG2b were all decreased in the Apremilast treatment groups. Moreover, Apremilast markedly prevented the development of bone erosions in CIA mice by CT analysis. Furthermore, in the Apremilast treated group, the frequency of Th17 cells and Th1 cells was significantly decreased while Treg cells’ frequency was significantly increased. The high dose of Apremilast (25 mg/kg) was superior to low dose (5 mg/kg) in treating CIA. Apremilast treatment reduced the migratory ability of RASFs and their destructive effect on cartilage. Compared with the model group, Apremilast treatment significantly reduced the RASFs invasion cartilage scores in both primary implant and contralateral implant models. Our data suggest that Apremilast is effective in treating autoimmune arthritis and preventing the bone erosion in the CIA model, implicating its therapeutic potential in patients with RA.</p