Oral arsenic plus retinoic acid versus intravenous arsenic plus retinoic acid for non-high-risk acute promyelocytic leukaemia: a non-inferiority, randomised phase 3 trial.

BACKGROUND: Intravenous arsenic trioxide plus all-trans retinoic acid (ATRA) without chemotherapy is the standard of care for non-high-risk acute promyelocytic leukaemia (white blood cell count ≤10 × 109 per L), resulting in cure in more than 95% of cases. However, a pilot study of treatment with oral arsenic realgar-Indigo naturalis formula (RIF) plus ATRA without chemotherapy, which has a more convenient route of administration than the standard intravenous regimen, showed high efficacy. In this study, we compare an oral RIF plus ATRA treatment regimen with the standard intravenous arsenic trioxide plus ATRA treatment regimen in patients with non-high-risk acute promyelocytic leukaemia. METHODS: We did a multicentre, non-inferiority, open-label, randomised, controlled phase 3 trial at 14 centres in China. Patients aged 18-70 years with newly diagnosed (within 7 days) non-high-risk acute promyelocytic leukaemia, and a WHO performance status of 2 or less were eligible. Patients were randomly assigned (2:1) to receive treatment with RIF-ATRA or arsenic trioxide-ATRA as the induction and consolidation therapy. Randomisation was done centrally with permuted blocks and stratification according to trial centre and was implemented through an interactive web response system. RIF (60 mg/kg bodyweight daily in an oral divided dose) or arsenic trioxide (0·15 mg/kg daily in an intravenous dose) and ATRA (25 mg/m2 daily in an oral divided dose) were used until complete remission was achieved. The home-based consolidation therapy was RIF (60 mg/kg daily in an oral divided dose) or intravenous arsenic trioxide (0·15 mg/kg daily in an intravenous dose) in a 4-week on 4-week off regimen for four cycles and ATRA (25 mg/m2 daily in an oral divided dose) in a 2-week on 2-week off regimen for seven cycles. Patients and treating physicians were not masked to treatment allocation. The primary outcome was event-free survival at 2 years. A non-inferiority margin of -10% was used to assess non-inferiority. Primary analyses were done in a modified intention-to-treat population of all patients who received at least one dose of their assigned treatment and the per-protocol population. This study was registered with the Chinese Clinical Trial Registry (ChiCTR-TRC-13004054), and the trial is complete. FINDINGS: Between Feb 13, 2014, and Aug 31, 2015, 109 patients were enrolled and assigned to RIF-ATRA (n=72) or arsenic trioxide-ATRA (n=37). Three patients in the RIF-ATRA and one in the arsenic trioxide-ATRA did not receive their assigned treatment. After a median follow-up of 32 months (IQR 27-36), 67 (97%) of 69 patients in the RIF-ATRA group and 34 (94%) of 36 in the arsenic trioxide-ATRA group had achieved 2-year event-free survival in the modified intention-to-treat population. The percentage difference in event-free survival was 2·7% (95% CI, -5·8 to 11·1). The lower limit of the 95% CI for the difference in event-free survival was greater than the -10% non-inferiority margin, confirming non-inferiority (p=0·0017). Non-inferiority was also confirmed in the per-protocol population. During induction therapy, grade 3-4 hepatic toxic effects (ie, increased liver aspartate aminotransferase or alanine transaminase concentrations) were reported in six (9%) of 69 patients in the RIF-ATRA group versus five (14%) of 36 patients in the arsenic trioxide-ATRA group; grade 3-4 infection was reported in 15 (23%) of 64 versus 15 (42%) of 36 patients. Two patients in the arsenic trioxide-ATRA group died during induction therapy (one from haemorrhage and one from thrombocytopenia). INTERPRETATION: Oral RIF plus ATRA is not inferior to intravenous arsenic trioxide plus ATRA for the treatment of patients with non-high-risk acute promyelocytic leukaemia. This study suggests that a completely oral, chemotherapy-free model might be an alternative to the standard intravenous treatment for patients with non-high-risk acute promyelocytic leukaemia. FUNDING: Foundation for innovative research group of the National Natural Science Foundation of China, the Beijing Municipal Science and Technology Commission, the National Key R&D Program of China, and the National Natural Science Foundation of China.

Cytotoxic T Lymphocyte Antigen-4 Down-Regulates T Helper 1 Cells by Increasing Expression of Signal Transducer and Activator of Transcription 3 in Acute Graft-versus-Host Disease.

Numerous previous studies have suggested that cytotoxic T lymphocyte antigen-4 (CTLA-4) plays an important role in acute graft-versus-host disease (GVHD). How CTLA-4 acts in regulating acute GVHD remains unknown, however. In the present study, we found that, compared with healthy controls, CTLA-4 plasma and relative mRNA levels in patients with acute GVHD were initially decreased and then markedly elevated after 28 days of treatment. CTLA-4 levels were higher in patients with grade I-II acute GVHD compared with those with grade III-IV acute GVHD both before and after treatment. Up-regulation of CTLA-4 significantly increased the luciferase activity and degree of phosphorylation of signal transducer and activator of transcription 3 (STAT3). Meanwhile, T cell activation was significantly inhibited, and levels of IFN-γ, IL-17, and IL-22 decreased. These findings suggest that CTLA-4 might be involved in the pathogenesis of acute GVHD, and may down-regulate T helper 1 cells by increasing STAT3 expression in acute GVHD.

Increased expression of T cell immune response cDNA 7 in patients with acute graft-versus-host disease.

Acute graft-versus-host disease (aGVHD) has become the important complication post-allogeneic hematopoietic stem cell transplantation. Abnormally activated T cells might play an important role in the pathogenesis of aGVHD. But its exact mechanism remains poorly understood. T cell immune response cDNA 7 (TIRC7) has been identified to be essential in T cell activation; however, the role of TIRC7 in aGVHD remains unclear. The purpose of this study was to measure the expression of TIRC7 and T helper (Th) cells in patients with aGVHD before and after treatment. We showed that TIRC7 levels in aGVHD patients were higher than those of healthy controls and markedly declined after treatment. The levels of IFN-γ (Th1), IL-17 (Th17), and IL-22 (Th22) were in accordance with the grade of aGVHD. In addition, TIRC7 levels were also associated with the severity of aGVHD. In conclusion, TIRC7 might be involved in the pathogenesis of aGVHD and TIRC7 level might be an indicator to evaluate the response of patients with aGVHD to treatment.

Celastrus orbiculatus Extract Inhibits Immune Inflammatory Thrombotic State of B-Lymphoma.

OBJECTIVE: To investigate the inhibitory effect of Celastrus orbiculatus extracts (COE) on the proliferation of lymphoma cells and the immune regulation ability on inflammation and thrombophilia in vivo. METHODS: The 38B9 lymphoma cells were treated with COE (160 µ g/mL) and CTX (25 µ mol/L). The apoptosis rate and cell cycle of each group were detected by flow cytometry. The secretion of inflammatory factors, including interleukin (IL)-6, IL-10, and tumor necrosis factor α (TNF-α), in cell supernatant was detected by enzyme-linked immunosorbent assay (ELISA). In vivo, BALB/c mice were subcutaneously injected with 38B9 lymphoma cells to establish lymphoma model. COE (3 mg·kg-1·d-1) and CTX (40 mg·kg-1·d-1) were administered to the model mice, respectively. The expression of plasma inflammatory factors (IL-6, IL-10 and TNF-α) and thrombus indexes, including D-dimer (D-D), von Willebrand factor (vWF) and tissue factor (TF), were detected by ELISA before tumor bearing (1 d), after tumor formation (14 d) and after intervention (21 d). PicoGreen dsDNA was used to detect the level of serum neutrophil extracellular traps (NETs). Flow cytometry was used to detect the expression of platelet activation marker calcium-dependent lectin-like receptor 2 (CLEC-2). The tumor growth and survival of mice were recorded. RESULTS: The 38B9 lymphoma cells were apoptotic after the intervention of COE and CTX. The ratio of G2-M phase cells decreased in COE intervented cells compared with the control cells (P<0.05), and S phase cells decreased in CTX intervented cells (P<0.05). Also, the secretion level of IL-6 was significantly reduced after COE or CTX intervention (P<0.05), and IL-10 was significantly increased (P<0.05). Furthermore, the tumor mass was reduced, and the median survival time was longer in COE and CTX intervented tumor-bearing mice than in non-intervented mice. The significantly lower levels of TNF-α, IL-6, NETs, TF, DD and CLEC-2, as well as higher IL-10 were observed in COE and CTX treatment mice in comparision with the control mice (P<0.05). CONCLUSION: COE has a mild and stable anti-tumor effect, which can reduce the secretion of inflammatory factors by lymphoma cells and regulate thrombophilic state caused by tumor inflammatory microenvironment.

[Prognostic Significance of Progression of Disease within 24 Months in Mantle Cell Lymphoma].

OBJECTIVE: To investigate the effect of progression of disease within 24 months (POD24) on overall survival (OS) in patients with mantle cell lymphoma (MCL), and compare the clinical characteristics between POD24 and non-POD24 patients. METHODS: A retrospective analysis was performed on 50 MCL patients with treatment indications and regular treatment who were admitted to the Affiliated Hospital of Xuzhou Medical University from January 2010 to August 2020. According to the occurrence of POD24, the patients were grouped for prognostic evaluation and clinical characteristics comparison. RESULTS: Univariate Cox regression analysis showed that POD24, PLT, albumin, MIPI score, ECOG PS score, LDH were the factors influencing OS in newly diagnosed MCL patients (all P < 0.05). The results of multivariate Cox regression analysis showed that POD24ï¼»HR=16.797(95%CI : 3.671-76.861),P < 0.001ï¼½, albumin<40 g/Lï¼»HR=3.238(95%CI :1.095-9.572),P =0.034ï¼½ and ECOG PS score≥2ï¼»HR=4.005(95%CI :1.033-15.521),P =0.045ï¼½ were independent risk factors influencing OS in MCL patients. The incidence of PLT<100×109/L (33.3% vs 5.9%, P =0.033) and ECOG PS score ≥2 (45.5% vs 5.9%, P =0.040) were significantly higher in POD24 patients than those in non-POD24 patients. CONCLUSION: POD24 is an independent poor prognostic factor affecting the OS of MCL patients, and the patients with PLT<100×109/L and ECOG PS score≥2 at diagnosis have a higher probability of POD24.

[The In Vivo Intervention and Relative Mechanism of Genistein on the Inflammation and Thrombophilia in Lymphoma-Bearing Mice].

OBJECTIVE: To investigate the in vivo intervention and relative mechanism of Genistein (GEN) on tumor-associated inflammatory and tumor thrombophilia in lymphoma-bearing mice. METHODS: Forty female Balb/c mice aged 5-6 weeks were injected with murine-derived Pro B-cell lymphoma cell line 38B9 to establish a lymphoma mouse model, which was randomly divided into control group, tumor-bearing group, GEN drug intervention group and cyclophosphamide (CTX）drug intervention group. Histopathologic was used to evaluate the tumorigenesis. Tumor formation was observed, and tumor tissues were collected of HE and immunohistochemical staining. ELISA and flow cytometry were used to detect the expression of inflammatory factors and the changes of thrombus indices in plasma after intervention of GEN and Cyclophosphamide (CTX) respectively. Immunohistochemistry method was used to detect the expression of CD19 in tomor tissues of tummor bearing mice. RESULTS: After 14 days of tumor bearing, the mice were tumorigenic. The lymphoma cells were diffusely distributed in the tumor tissue and the expression of CD19 in the tumor tissue was positive. The inflammatory factors such as IL-6, NETs and CLEC-2, and thrombotic indices such as TF, FIB and D-D in lymphoma-bearing mice were significantly higher than those before tumor-injection and lower than those after drug-intervention (all P<0.05). The levels of CLEC-2 and D-D in GEN group were significantly lower than those in CTX group (P<0.05). CONCLUSION: Tumor-associated inflammation and thrombophilia exist in lymphoma-bearing mice. GEN shows better anti-inflammatory and anti-thrombotic effects compared with CTX by interfering with tumor inflammatory factors.

[The Efficacy and Safety of Daratumumab-Based Regimen in Treatment of Multiple Myeloma Patients with Renal Impairment].

OBJECTIVE: To investigate the efficacy and safety of daratumumab in treatment of multiple myeloma (MM) patients with renal impairment (RI). METHODS: The clinical data of 15 MM patients with RI who received daratumumab-based regimen from January 2021 to March 2022 in three centers were retrospectively analyzed. Patients were treated with daratumumab or daratumumab combined with dexamethasone or daratumumab combined with bortezomib and dexamethasone and the curative effect and survival were analyzed. RESULTS: The median age of 15 patients was 64 (ranged 54-82) years old. Six patients were IgG-MM, 2 were IgA-MM,1 was IgD-MM and 6 were light chain MM. Median estinated glomerular filtration rate (eGFR) was 22.48 ml/(min·1.73 M2). Overall response rate of 11 patients with MM was 91% (≥MR), including 1 case of stringent complete response (sCR), 2 cases of very good partial response (VGPR), 3 cases of partial response (PR) and 4 cases of minor response (MR). The rate of renal response was 60%(9/15), including 4 cases of complete response (CR), 1 case of PR and 4 cases of MR. A median time of optimal renal response was 21 (ranged 7-56) days. With a median follow-up of 3 months, the median progression-free survival and overall survival of all patients were not reached. After treatment with daratumumab-based regimen, grade 1-2 neutropenia was the most common hematological adverse reaction. Non-hematological adverse reactions were mainly infusion-related adverse reactions and infections. CONCLUSION: Daratumumab-based regimens have good short-term efficacy and safety in the treatment of multiple myeloma patients with renal impairment.

[Construction of a Mouse Model for Myeloproliferative Neoplasms and an Evaluation System].

OBJECTIVE: To construct a myeloproliferative neoplasms (MPN) transplanted mouse model with JAK2-V617F, MPLW515L or CALR-Type I gene mutation, and establish a systematic evaluation system to verify the success of model construction. METHODS: The bone marrow c-kit+ cells of the mice were obtained by the following steps: The mice were killed by cervical dislocation, the femur, tibia and ilium were separated, and the bone marrow cells were collected. The c-kit+ cells were sorted after incubation with CD117 magnetic beads. The method of constructing mouse primary mutant cells is as follows: A gene mutation vector with a GFP tag was constructed by the retroviral system, and the retroviral vector was packaged into the Platinum-E cells to obtain the virus supernatant, and then used it to infect the c-kit+ cells of mice. The MPN mouse model was constructed as follows: the mouse primary c-kit+ cells containing the mutant genes were collected after infection, and then transplanted them via the tail vein into the female recipient mice of the same species which were irradiated with a lethal dose of gamma rays (8.0 Gy). The MPN mouse model was evaluated as follows: After transplantation, the peripheral blood of the mice was regularly collected from the tail vein to perform the complete blood count test, and the size of spleen and the degree of bone marrow fibrosis were estimated. RESULTS: The mouse c-kit+ cells with the mutant genes were successfully obtained from the bone marrow. MPN mouse model was successfully constructed: The peripheral blood cells of the MPN-transplanted mice carried exogenous implanted GFP-positive cells, and the white blood cells (WBC), platelet (PLT) and hematocrit (HCT) were all increased; the body weight loss, and the water and food intake were reduced in the transplanted mice; further pathological analysis showed that the transplanted mice displayed splenomegaly and bone marrow fibrosis. These results suggested that the MPN mouse model was successfully constructed. According to the common and different characteristics of the three MPN mouse model, a preliminary evaluation system for judging the success of MPN mouse model construction was summarized, which mainly included the following indicators, for example, the proportion of GFP-positive cells in the peripheral blood of mice; WBC, PLT and HCT; the degree of spleen enlargement and the bone marrow fibrosis. CONCLUSION: The MPN mouse model with JAK2-V617F, MPLW515L or CALR-Type I gene mutation is successfully established by retroviral system, which can provide an important experimental animal model for the research of MPN pathogenesis and drug-targeted therapy.

Molecular basis of JAK2 H608Y and H608N mutations in the pathology of acute myeloid leukemia.

Risk-stratification of acute myeloid leukemia (AML) based on (cyto)genetic aberrations, including hotspot mutations, deletions and point mutations have evolved substantially in recent years. With the development of next-generation sequence technology, more and more novel mutations in the AML were identified. Thus, to unravel roles and mechanism of novel mutations would improve prognostic and predictive abilities. In this study, two novel germline JAK2 His608Tyr (H608Y) and His608Asn (H608N) mutations were identified and the molecular basis of these mutations in the leukemiagenesis of AML was elucidated. Our results indicated that JAK2 H608Y and H608N mutations disrupted the hydrogen bond between Q656 and H608 which reduced the JH2 domain's activity and abolished interactions between JH1 and JH2 domains, forced JAK2 into the active conformation, facilitated the entrance of substrates and thus caused JAK2 hyperactivation. Further studies suggested that JAK2 H608Y and H608N mutations enhanced the cell proliferation and inhibited the differentiation of Ba/F3 and MV4-11 cells via activating the JAK2-STAT5 signaling pathway. Moreover, rescue experiments demonstrated that mutations repaired the hydrogen bond between Q656 and H608 displayed opposite results. Thus, this study revealed the molecular basis of JAK2 H608Y and H608N mutations in the pathology of AML.

[Effect of Hemoglobin on Efficacy of CAR-T Therapy in Patients with Multiple Myeloma].

OBJECTIVE: To investigate the effect of hemoglobin (Hb) on the efficacy of chimeric antigen receptor T cell therapy (CAR-T) in patients with multiple myeloma (MM). METHODS: From June 2017 to December 2020, 76 MM patients who received CAR-T therapy in the Department of Hematology, The Affiliated Hospital of Xuzhou Medical University, with complete clinical data and evaluable efficacy, were selected as the research objects. According to the receiver operating characteristic (ROC) curve, the best cut-off value was obtained. The patients were divided into groups on the basis of Hb 105.5 g/L as the cut-off value. The age, sex, serum calcium, ß2-microglobulin, serum creatinine, lactate dehydrogenase (LDH), and the influencing factors of CAR-T treatment efficacy in MM patients were analyzed. RESULTS: Hb was an influencing factor of efficacy. Univariate analysis showed that Hb, LDH, and albumin affected the efficacy of CAR-T therapy. Multivariate analysis showed that Hb ( OR=1.039, 95% CI: 1.002-1.078) and LDH ( OR=1.014, 95% CI: 1.000-1.027) were the influencing factors for the efficacy of CAR-T therapy. CONCLUSION: The efficacy of CAR-T therapy in MM patients with low Hb is poor, and Hb is a factor affecting the efficacy of CAR-T therapy.

[Novel CD19-KIRS2/Dap12-BB CAR-T Treatment for 3 Patients with Relapsed and Refractory B-Cell Tumors].

OBJECTIVE: To investigate the safety and efficacy of novel CD19-KIRS2/Dap12-BB chimeric antigen receptor T cells (CAR-T cells) in the treatment of relapsed/refractory B-cell malignancy (R/R BCM). METHODS: Three patients with R/R BCM treated with novel CD19-KIRS2/Dap12-BB CAR-T cells from June 2020 to November 2020 were enrolled, including 1 case of B-cell acute lymphoblastic leukaemia (B-ALL) and 2 cases of non-Hodgkin's lymphoma (NHL), and the efficacy and adverse reactions were observed. RESULTS: After CAR-T cells infusion, patient with B-ALL achieved complete remission (CR) and minimal residual disease (MRD) turned negative, and 2 patients with NHL achieved partial remission (PR). Grade 2 cytokine release syndrome (CRS) occurred in B-ALL patient, grade 1 CRS occurred in 2 NHL patients, and grade II to IV hematologic adverse reactions occurred in 3 patients, all of which were controllable and reversible. The progression-free survival (PFS) of the 3 patients was 143, 199, and 91 days, and overall survival (OS) was 282, 430, and 338 days, respectively. CONCLUSION: The novel CD19-KIRS2/Dap12-BB CAR-T cells in treatment of 3 patients with R/R BCM have significant short-term efficacy and controllable adverse reactions, but the long-term efficacy needs to be further improved.

Amino acid residue E543 in JAK2 C618R is a potential therapeutic target for myeloproliferative disorders caused by JAK2 C618R mutation.

Janus kinase 2 (JAK2) is an important mediator of cytokine receptor signaling and plays a key role in the hematopoietic and immune responses. The acquired JAK2 C618R somatic mutation is detected in a subset of myeloproliferative disorders (MPDs) patients and presumed to be a biomarker for MPDs. However, how JAK2 C618R mutation causes MPDs is still unclear. Our results indicate that the amino acid residue E543 in JAK2 C618R is indispensable for its constitutive activation. When the glutamic acid at this position was mutated to alanine (E543A) in the JAK2 C618R, its activity significantly decreased. However when the glutamic acid was mutated to the acidic amino acid, aspartic acid, JAK2 C618R activity changed little. These results suggest that there is an interaction between the amino acid residue R618 and E543, and that this interaction is crucial to sustain the constitutive activation of JAK2 C618R. More importantly, the E543 single mutation had no effects on the function of wild type JAK2 (WT JAK2). This study suggests that the amino acid residue E543 might be a potential target for specific inhibitors to treat MPDs caused by the JAK2 C618R mutation.

[Influence of the Ratio of Peripheral Hemoglobin-to-Red Cell Distribution width on the Prognosis of Patients with Diffuse Large B-cell Lymphoma].

OBJECTIVE: To investigate the influence of peripheral hemoglobin (Hb)-to-red cell distribution width (RDW) ratio (HRR) on the prognosis of patients with diffuse large B-cell lymphoma (DLBCL). METHODS: Data of 265 patients with diffuse large B-cell lymphoma (DLBCL) at the Affiliated Hospital of Xuzhou Medical University from January 2014 to December 2019 were retrospectively analyzed. 132 healthy people in the same period were used as normal control group. The best cut-off points of HRR was determined by receiver operating characteristics (ROC) curve; the chi-square test was used to analyze the correlation of clinical characteristics with HRR; the Kaplan-Meier method was used to compare the overall survival (OS) and progression-free survival (PFS) of HRR patients in different groups; the Cox proportional risk model was used for univariate and multivariate analysis. RESULTS: The best cut-off value of HRR was 0.936, which was divided into low HRR group and high HRR group. The low HRR group had a higher ECOG score, higher incidence of advanced Ann Arbor stage, higher NCCN-IPI score, and elevated LDH level. K-M survival analysis showed that OS (P<0.001) and PFS (P<0.001) in the low HRR group were significantly shorter than that in the higher HRR group. The multivariate analysis revealed that HRR was an independent predictor of OS（HR＝0.379，95％CI：0.237-0.605，P<0.001） and PFS （HR＝0.384，95％CI：0.241－0.614，P<0.001） in DLBCL patients. CONCLUSION: Low HRR（<0.936） in patients with DLBCL indicates a poor prognosis, which is an independent prognosis risk factor.

The efficacy and safety of cyclosporine A plus androgen versus androgen alone for adult patients with non-severe aplastic anemia in China: a meta-analysis of randomized controlled trials.

BACKGROUND: Whether combined CsA with androgen therapy was superior to androgen therapy alone in NSAA remains controversial. This study aimed to assess the efficacy and safety of combined therapy versus androgen therapy for NSAA patients using a meta-analytic approach. METHODS: An electronic database of PubMed, EmBase, Cochrane library, CNKI, VIP, and Wanfang was systematically searched for randomized controlled trials (RCTs) from their inception to February 2020. The primary endpoint was effective rate, while the secondary endpoints included white blood cell (WBC), hemoglobin, platelet, and potential adverse events. The pooled results from included trials were calculated with the random-effects model. RESULTS: Forty-three RCTs recruited 2610 NSAA patients for the final quantitative meta-analysis. We noted that combined therapy was associated with an increased incidence of effective rate than androgen therapy alone (relative risk [RR]: 1.35; 95% confidence interval [CI]: 1.29-1.41; P < 0.001). Moreover, patients treated with combined therapy were associated with higher WBC (weighted mean difference [WMD]: 1.22; 95%CI: 0.94-1.49; P < 0.001), hemoglobin (WMD: 12.93; 95%CI: 8.86-17.01; P < 0.001), and platelet (WMD: 8.65; 95%CI: 7.05-10.24; P < 0.001). Finally, the pooled incidence of hirsutism, handshake, gingiva hyperplasia, liver function damage, and renal function damage were 0.35 (95%CI: 0.22-0.48), 0.24 (95%CI: 0.15-0.32), 0.22 (95%CI: 0.10-0.35), 0.19 (95%CI: 0.14-0.25), and 0.06 (95%CI: 0.01-0.11), respectively. CONCLUSIONS: This study found that combined CsA with androgen therapy was superior to androgen therapy alone for Chinese patients with NSAA, and the most common adverse of combined therapy included hirsutism, handshake, gingiva hyperplasia, liver function damage, and renal function damage.

Keratinase improves the growth performance, meat quality and redox status of broiler chickens fed a diet containing feather meal.

The objective of this study was to assess the effects of dietary supplementation of keratinase on the production of broilers fed a diet containing feather meal. A total of 162 1-d-old Cobb 500 male broiler (n = 9 cages/diet with 6 chicks/cage) were randomly allocated to 3 dietary treatments. The broilers were fed a corn-soybean-feather meal based diet (BD), or BD supplemented with keratinase at 100,000 or 200,000 U/kg for 6 weeks. Compared to the control, dietary supplementation with 200,000 U/kg keratinase increased (P < 0.05) body weight gain (3.6-4.3%) and reduced feed conversion ratio (2.4-5.6%) during the various experimental periods, and also improved (P < 0.05) apparent total tract digestibility of ash and calcium by 45.0% and 8.8%, respectively. Meanwhile, dietary supplementation of keratinase at 100,000 U/kg reduced (P < 0.05) the drip loss (29.2%), while 200,000 U/kg keratinase supplementation increased (P < 0.05) the pH value (1.6%) at 45 min and decreased (P < 0.05) the lightness (L* value; 13.6%) and drip loss (22.1%) of pectoral muscle. Moreover, dietary supplementation of keratinase at both levels of 100,000 and 200,000 U/kg increased (P < 0.05) Glutathione peroxidase activity (82.5-87.5%) and decreased the Malondialdehyde concentration (14.5-18.3%) in the pectoral muscle. In conclusion, dietary supplementation of keratinase at 200,000 U/kg can improve the performance, meat quality, apparent total tract digestibility of nutrients, and redox status of broiler chickens fed a diet containing feather meal.

Effects of the PLK4 inhibitor Centrinone on the biological behaviors of acute myeloid leukemia cell lines.

Polo-like kinase 4 (PLK4), a key regulator of centriole biogenesis, is frequently overexpressed in cancer cells. However, roles and the mechanism of PLK4 in the leukemiagenesis of acute myeloid leukemia (AML) remain unclear. In this study, the PLK4 inhibitor Centrinone and the shRNA knockdown were used to investigate roles and the mechanism of PLK4 in the leukemiagenesis of AML. Our results indicated that Centrinone inhibited the proliferation of AML cells in a dose- and time-dependent manner via reduced the expression of PLK4 both in the protein and mRNA levels. Moreover, colony formation assay revealed that Centrinone reduced the number and the size of the AML colonies. Centrinone induced AML cell apoptosis by increasing the activation of Caspase-3/poly ADP-ribose polymerase (PARP). Notably, Centrinone caused the G2/M phase cell cycle arrest by decreasing the expression of cell cycle-related proteins such as Cyclin A2, Cyclin B1, and Cyclin-dependent kinase 1 (CDK1). Consistent with above results, knockdown the expression of PLK4 also inhibited cell proliferation and colony formation, induced cell apoptosis, and caused G2/M phase cell cycle arrest without affecting cell differentiation. All in all, this study suggested that PLK4 inhibited the progression of AML in vitro, and these results herein may provide clues in roles of PLK4 in the leukemiagenesis of AML.

Combination of RSK inhibitor LJH-685 and FLT3 inhibitor FF-10101 promoted apoptosis and proliferation inhibition of AML cell lines.

PURPOSE: FLT3 mutations occurred in approximately one third of patients with acute myeloid leukemia (AML). FLT3-ITD mutations caused the constitutive activation of the RAS/MAPK signaling pathway. Ribosomal S6 Kinases (RSKs) were serine/threonine kinases that function downstream of the Ras/Raf/MEK/ERK signaling pathway. However, roles and mechanisms of RSKs inhibitor LJH-685, and combinational effects of LJH-685 and FLT3 inhibitor FF-10101 on AML cells were till unclear. METHODS: Cell viability assay, CFSE assay, RT-qPCR, Colony formation assay, PI stain, Annexin-V/7-AAD double stain, Western blot, and Xenogeneic transplantation methods were used to used to investigate roles and mechanisms of LJH-685 in the leukemogenesis of AML. RESULTS: LJH-685 inhibited the proliferation and clone formation of AML cells, caused cell cycle arrest and induced the apoptosis of AML cells via inhibiting the RSK-YB-1 signaling pathway. MV4-11 and MOLM-13 cells carrying FLT3-ITD mutations were more sensitive to LJH-685 than that of other AML cell lines. Further studies suggested that LJH-685 combined with Daunorubicin or FF- 10101 synergistically inhibited the cell viability, promoted the apoptosis and caused cycle arrest of AML cells carrying FLT3-ITD mutations. Moreover, in vivo experiments also indicated that LJH-685 combined with FF-10101 or Daunorubicin prolonged the survival time of NSG mice and reduced the leukemogenesis of AML. CONCLUSION: Thus, these observations demonstrated combination of RSK inhibitor LJH-685 and FLT3 inhibitor FF-10101 showed synergism anti-leukemia effects in AML cell lines with FLT3-ITD mutations via inhibiting MAPK-RSKs-YB-1 pathway and provided new targets for therapeutic intervention especially for AML with FLT3-ITD mutations and Daunorubicin-resistant AML.

[An absence of donor TH17 differentiation ameliorates dermal tissue damage].

OBJECTIVE: To explore the functions of TH17 cell in cutaneous graft-versus-host disease (GVHD). METHODS: A model of acute GVHD (aGVHD) was established with a major histocompatibility complex class I/II-disparate allogeneic bone marrow transplantation (BMT). Bone marrow monocytes and splenic T cells from donor C57/BL6 were enriched. The recipient BABL mice were irradiated ((60)Co source) with 7.5 Gy total body irradiation (TBI) and injected with 5 × 10(6) marrow monocytes and 5 × 10(5) T cells. The experimental mice were divided into 3 groups: lethal total body irradiation (TBI); allogeneic bone marrow transplantation (BMT) and recipients of halofuginone (HF). The symptoms of aGVHD were observed daily and detailed histopathologic analyses of recipient skin were performed at Day 6 post-transplantation. And Tri-color flow cytometry (FCM) was performed at Day 6 post-transplantation to measure the levels of interleukin (IL)-17, interferon (IFN)-γ and TH1/TH17. RESULTS: Clinical GVHD symptoms were observed in recipient mice. The administration of HF to lethally irradiated recipients led to very modest GVHD-induced cutaneous changes manifested predominantly by fur loss. However, the experimental animals receiving only allogeneic BMT showed significant fur loss and pathologic skin conditions. Consistent with the clinical evaluations, the histopathologic results demonstrated significantly increased pathologic cutaneous lesions in recipients undergoing only BMT. The median ratios of TH1/TH17 cells were 17.57 and 5.31 in the HF and BMT groups respectively. The difference had statistical significance (P < 0.05). The serum levels of IL-17 were(1.47 ± 0.18) and (2.81 ± 0.19) pg/ml in the TBI and BMT groups respectively (P < 0.05). But IL-17 could not be detected in the HF group. The serum levels of IFN-γ were (3.86 ± 0.32), (42.97 ± 0.42) and (9.89 ± 0.51) pg/ml in the TBI, BMT and HF groups respectively. The inter-group differences had statistical significance (P < 0.05). CONCLUSION: An absence of TH17 cell may alleviate the cutaneous GVHD but exacerbate the systemic GVHD.

[Kinetics of Infiltrating CD3+ T Cells and Their Relationship with Target Organ Injury of Graft-Versus-Host Disease].

OBJECTIVE: To explore the kinetics of infiltrated T cell in murine acute graft-versus-host disease (aGVHD) target organs after allogeneic hematopoietic stem cell transplantation (allo-HSCT) and its relationship with tissue pathological damage and aGVHD progress. METHODS: Male C57BL/6 (H-2Kb) mice at age of 8-10 weeks were selected as donors, from which splenic cells and bone marrow cells were isolated. And 10-12 weeks of BALB/c (H-2Kd) male mice which received 7.5 Gy total body irradiation (TBI) were recipients to transplant. Recipients were randomly divided into allogeneic bone marrow transplantation (BMT) group and BMT+T group, which were transplanted bone marrow cells with or without splenic cells, respectively. All recipients were daily monitored and the dynamic changes of the body weights along with clinical scores of aGVHD were detected. HE staining was used to investigate the pathological damage and score of aGVHD target organs. The number of infiltrated CD3+ T cells in target organs was numerated and statistically analyzed after immunohistochemistry staining on day 7, 14, 28, 40 and 47 after transplantation. RESULTS: Compared with BMT group, the number of infiltrated T cells in aGVHD target organs including liver, lung and gut increased since day 7 in BMT+T group (P<0.05). On day 14, 28, 40 and 47 after transplantation, more infiltrated CD3+ T cells were detected in target tissues of mice in BMT+T group than those in BMT group (P<0.05). Higher clinical score and histopathological score of target organs in aGVHD mice were detected (P<0.05). Positive correlation was found in the number of liver infiltrated T cells and pathological damage, and the numbers of infiltrated CD3+ T cells in gut were positively related to aGVHD clinical scores. CONCLUSION: Pathological damage of aGVHD target organs is induced by CD3+ T cell infiltration, and the number of infiltrated T cell may be an important evaluated index of aGVHD severity.