Objective: Effects of Shenmai injection and aminophylline alone or in combination on transdiaphragmatic pressure and electrical activity of diaphragm were observed, so as to evaluate their effecfs on diaphragmatic fatigue. Methods: The diaphramatic fatique (DiF) model was estab- lished by stimulating bilateral phrenic nerves for 40 minutes. The animals were allocated to three groups: Shenmai group (2mL/kg, N=7), aminophylline group (20mg/kg, N=7) and combi- nation group (Shenmai injection 2mL/kg+aminophylline 20mg/kg, N=7) The transdi- aphragmatic pressure (Pdi) and diaphragmatic electromyogram (EMGdi) were recorded before and after treatment. EMGdi was analysed by computer and then the high/low frequency ratio (H/L) and the central frequency (Fc) were caculated. The diaphragm evoked potential (DEP) was record- ed simutaneously. All data was analysed by analysis of variance and q test. Results: Pdi, H/L, Fc and amplitude of DEP were markedly increased after the treatment with aminophyline and Shen- mai injection alone (Compared with DiF. P

Experiments were performed on spontaneously breathing rabbits anesthetized with urethan. The diaphragmatic fatigue (DiF) was reproduced by the phrenic nerve stimulation (30 Hz, 10 V,0. 2 ms). The transdiaphragmatic pressure (Pdi) and diaphragm evoked potential (DEP) were measured before injury and after injury 30, 60, 90, and 120 min. Pdimax and the amplitude of DEP obviously decreased and the latency of DEP obviously delayed after injury 30min. The amplitude of Pdi and DEP were quickly promoted by introvenous injection of neostig-mine after DiF. The latency of DEP was partially recovered by introvenous injection of neostig-mine. It is concluded that neostigmine can improve the generation of the fatigued diaphragm. and promote its recovery.

OBJECTIVETo test the effect of bifunctional molecule IL2-GMCSF in promoting the activation of dendritic cells (DCs) cultured in tumor conditioned medium.

METHODSWe prepared a tumor conditioned medium using mouse melanoma cell line B16F10 supplemented with IL2-GMCSF, GM-CSF, IL-2, or the combination of the latter two. After culturing mouse DC cell line DC2.4 in the conditioned medium for 24 h, the DCs were examined for phagocytosis, proliferation, maturation phenotype, cytokine secretion, and signal pathway activation.

RESULTSDC2.4 cells displayed characteristics of immature DCs. After cell culture in the conditioned medium, the cells showed enhanced phagocytosis but significantly suppressed cell proliferation activity. Culture in the conditioned medium also promoted DC cell maturation and secretion of macrophage-derived chemokine (MDC), but inhibited IL-12 secretion. Supplementation of the conditioned medium with IL2-GMCSF promoted phagocytosis, proliferation, maturation, and cytokine (including both IL-12 and MDC) secretion of DC2.4 cells. Compared with GM-CSF, IL2-GMCSF induced a higher level of NF-κB signal pathway activation but suppressed STAT3 activation.

CONCLUSIONCompared with GM-CSF, IL2-GMCSF can better promote DC activation in the context of tumor-induced immune suppression, and thus shows potentials in anti-tumor therapy.

Animals ; Cell Differentiation ; Cell Line, Tumor ; drug effects ; Cell Proliferation ; Chemokine CCL22 ; metabolism ; Culture Media, Conditioned ; chemistry ; Dendritic Cells ; cytology ; drug effects ; Gene Expression Regulation, Neoplastic ; Granulocyte-Macrophage Colony-Stimulating Factor ; pharmacology ; Immune Tolerance ; Interleukin-12 ; metabolism ; Interleukin-2 ; pharmacology ; Melanoma, Experimental ; pathology ; Mice ; NF-kappa B ; metabolism ; Phagocytosis ; STAT3 Transcription Factor ; metabolism ; Signal Transduction

Objective To test the effect of bifunctional molecule IL2-GMCSF in promoting the activation of dendritic cells (DCs) cultured in tumor conditioned medium. Methods We prepared a tumor conditioned medium using mouse melanoma cell line B16F10 supplemented with IL2-GMCSF, GM-CSF, IL-2, or the combination of the latter two. After culturing mouse DC cell line DC2.4 in the conditioned medium for 24 h, the DCs were examined for phagocytosis, proliferation, maturation phenotype, cytokine secretion, and signal pathway activation. Results DC2.4 cells displayed characteristics of immature DCs. After cell culture in the conditioned medium, the cells showed enhanced phagocytosis but significantly suppressed cell proliferation activity. Culture in the conditioned medium also promoted DC cell maturation and secretion of macrophage-derived chemokine (MDC), but inhibited IL-12 secretion. Supplementation of the conditioned medium with IL2-GMCSF promoted phagocytosis, proliferation, maturation, and cytokine (including both IL-12 and MDC) secretion of DC2.4 cells. Compared with GM-CSF, IL2-GMCSF induced a higher level of NF-κB signal pathway activation but suppressed STAT3 activation. Conclusion Compared with GM-CSF, IL2-GMCSF can better promote DC activation in the context of tumor-induced immune suppression, and thus shows potentials in anti-tumor therapy.

Objective To test the effect of bifunctional molecule IL2-GMCSF in promoting the activation of dendritic cells (DCs) cultured in tumor conditioned medium. Methods We prepared a tumor conditioned medium using mouse melanoma cell line B16F10 supplemented with IL2-GMCSF, GM-CSF, IL-2, or the combination of the latter two. After culturing mouse DC cell line DC2.4 in the conditioned medium for 24 h, the DCs were examined for phagocytosis, proliferation, maturation phenotype, cytokine secretion, and signal pathway activation. Results DC2.4 cells displayed characteristics of immature DCs. After cell culture in the conditioned medium, the cells showed enhanced phagocytosis but significantly suppressed cell proliferation activity. Culture in the conditioned medium also promoted DC cell maturation and secretion of macrophage-derived chemokine (MDC), but inhibited IL-12 secretion. Supplementation of the conditioned medium with IL2-GMCSF promoted phagocytosis, proliferation, maturation, and cytokine (including both IL-12 and MDC) secretion of DC2.4 cells. Compared with GM-CSF, IL2-GMCSF induced a higher level of NF-κB signal pathway activation but suppressed STAT3 activation. Conclusion Compared with GM-CSF, IL2-GMCSF can better promote DC activation in the context of tumor-induced immune suppression, and thus shows potentials in anti-tumor therapy.

Objective To establish adapted strain of seasonal influenza virus influenza A virus [A/ Hong Kong/1968 (H3N2)] in mice and explore the molecular mechanism of adaption preliminary.Methods The mice model was induced by nose dropping with wild type H3N2 influenza virus,by continuous passage in the lung of mice and detection of the clinical manifestation,viral load,virus titer,pathology,cytokines,to get the adapted strain of seasonal influenza A virus [A/Hong Kong/1968 (H3N2)].Results After 7 times continuous passage in the lung of mice,virulence was increased,viral load and virus titer were increased either.Genome sequencing and alignment indicated that the HA and NA gene was mutated.Conclusion The influenza virus H3N2 of mice lung adaption can be acquired from wild type seasonal H3N2 by continuous passage in the lung tissues of mice.Mutation of Ile to Val at residue 248 of HA and Val to Ile at residue 444of NA protein may be the key virulence determinant.

AIM:To analyze the expression profiles of long non-coding RNAs(lncRNAs)in the aortas of ath-erosclerotic mice,and explore the role and mechanism of lncRNA AI662270 in regulating macrophage inflammation and lipid phagocytosis.METHODS:Twenty 8-week-old male apolipoprotein E gene knockout(ApoE-/-)mice were randomly divided into experimental and control groups,with 10 mice in each group.The experimental group was fed a high-fat diet for 12 weeks to induce atherosclerosis,while the control group received a normal diet.Body weight and blood lipid levels were measured,and atherosclerotic lesions were detected using Oil Red O staining.High-throughput RNA sequencing(RNA-seq)was used to analyze the lncRNA expression profiles in mouse aortas,and differentially expressed lncRNAs were validated by RT-qPCR.Antisense oligonucleotides(ASO)were used to knock down the expression of lncRNA AI662270 in mouse macrophage RAW264.7 cells.Six treatment groups were established:blank(no treatment)group,ox-idized low-density lipoprotein(oxLDL)group(80 mg/L oxLDL),negative control ASO(ASO-NC)group(transfected with ASO-NC),oxLDL+ASO-NC group(transfected with ASO-NC and treated with 80 mg/L oxLDL),ASO-AI662270 group(transfected with ASO-AI662270),and oxLDL+ASO-AI662270 group(transfected with ASO-AI662270 and treated with 80 mg/L oxLDL).Tumor necrosis factor-α(TNF-α)and interleukin-6(IL-6)levels were measured using Enzyme-linked immunosorbent assay(ELISA).NF-κB p65,pNF-κB p65,IκBα and pIκBα levels were detected by Western blot,and NF-κB p65 in the cell nucleus was examined using fluorescent probes.RESULTS:The atherosclerosis mouse model showed significant differences in body weight,serum lipids,and aortic plaque area compared to the control group(P<0.01).A total of 29 differentially expressed lncRNAs were identified involved in metabolic pathways,autophagosome for-mation,and cell adhesion molecule expression(P<0.05).LncRNA AI662270 was significantly upregulated in lesions and oxLDL-stimulated macrophages(P<0.05).Knockdown of AI662270 significantly reduced TNF-α and IL-6 expression in oxLDL-induced RAW264.7 cells(P<0.01),suppressed lipid phagocytosis,and inhibited pNF-κB p65 and pIκBα ex-pression(P<0.05).Additionally,the knockdown of AI662270 reduced the nuclear content of NF-κB p65.CONCLU-SION:The lncRNA expression profiles in the aortas of atherosclerotic mice were significantly altered.LncRNA AI662270 is crucial in modulating inflammation and lipid phagocytosis in mouse macrophages through the NF-κB signaling pathway.

Objective To explore the outcome of allogeneic hematopoietic stem cell transplantation (allo-HSCT) for relapsed/refractory acute myeloid leukemia (AML) patients in nonremission (NR) status and its related risk factors.Methods Thirty-nine relapsed/refractory AML patients in NR status who received allo-HSCT between Jan.2006 and Dec.2016 were retrospectively analyzed.Major end points of study included overall survival (OS),disease free survival (DFS),and relapse rate.Results All patients achieved hematopoietic reconstitution.Median time to neutrophil and platelet engraftment was 13 (11 20) and 16 (10-58) days,respectively.Acute graft-versus-host disease (aGVHD) occurred in 25 (64.1％) patients,and 11 (31.4％) patients developed chronic GVHD.During a median follow-up period of 9.1 (1.6-93.8) months,11 (28.9％) cases survived,10(26.3％) survived without leukemia,and 21 (53.8％) relapsed.The estimated 2 year OS and DFS were 30.0％ ± 8.0％ and 26.7％ ± 7.7％,and cumulative incidence of relapse and transplantation related mortality at 2 years was 56.63％ (95％ CI 37.84％-71.71％),19.7％ (95％ CI 8.3％-34.5％),respectively.The multivariate analysis revealed that the number of bone marrow blasts≥25％ or any level of blasts in peripheral blood was significantly associated with worse OS (HR =11.91,P=0.003),DFS (HR =10.75,P =0.002) and higher rate of relapse (70.83％ versus 20.22％,P =0.002).In addition,the development of grade Ⅱ-Ⅳ aGVHD also predicted an inferior OS (HR =3.18,P =0.039).Conclusion Salvage therapy with allo-HSCT can induce long-term survival in part refractory/relapsed AML patients.Decrease in the pre-transplant disease burden is the key to reduce relapse and improve survival.

Objective To label granulocytes in a state of differentiation in mouse bone marrow (BM) with EdU (5-ethynyl-2′-deoxyuridine) for further understanding the changes in granulocyte produc-tion at different stages of differentiation during inflammation. Methods C57BL/6 mice were intraperitoneal-ly (i.p.) injected with EdU and heat-inactivated Escherichia coli(HI E.coli). BM cells were harvested at different time points after HI E.coli injection and then stained with fluorescent-conjugated antibodies(Abs). Myeloblasts,promyelocytes,myelocytes, metamyelocytes and band and segmented neutrophils were identi-fied by fluorescence-activated cell sorting(FACS). The percentage of EdU-positive cells in each population was recorded. Results The percentage of EdU-positive myeloblasts in mice increased by 10.0% at 24 h af-ter intraperitoneal injection with HI E.coli,but decreased by 75.0% and 23.0% at 48 h and 72 h,respec-tively. The percentage of EdU-positive promyelocytes declined by 23.0%,54.5%,64.3% and 77.8% at 24 h,48 h,72 h and 96 h,respectively. The percentage of EdU-positive myelocytes increased by 60.0% and 10.0% at 24 h and 48 h,but decreased by 80.0% and 90.0% at 72 h and 96 h. The percentage of EdU-positive metamyelocytes increased by 50.0% at 24 h,but decreased by 33.3%,61.5% and 66.7% at 48 h,72 h and 96 h. The percentage of EdU-positive band and segmented cells increased by 14.0% at 24 h,but decreased by 50.0%, 77.8% and 88.0% at 48 h, 72 h and 96 h. Conclusion Emergency granulopoiesis occurred 24 h after the establishment of HI E.coli-induced model of acute peritonitis, which meant that the proliferation of myeloid precursor cells,especially that of myelocytes and metamyelocytes,was accelerated and resulted in increasing number of mature neutrophils immigrating to sites of inflammation.

OBJECTIVE: To explore the protective effects of hydrogen sulfide (H2S) on diaphragmatic muscle of Type 1 diabetic rats and its anti-apoptotic mechanism.
 METHODS: Thirty male Sprague Dawley rats were randomly divided into a control group, a diabetes group and a treatment group (n=10 per group). Streptozotocin (i.p.) was utilized to establish a rat model of Type 1 diabetes mellitus (DM). The DM rats were treated with NaHS solution (i.p.). After 8 weeks, the diaphragmatic muscle contractility was assessed by isolated diaphragmatic strips experiments. The peak twitch tension (Pt), maximum tetanic tension (Po), time to peak contraction (CT), half relaxation time (1/2RT) and maximal rates of contraction/relaxation (±dT/dtmax) were measured. The alterations of diaphragmtic ultrastructure were observed by electron microscopy. The content of malondialdehyde (MDA), the activities of superoxide dismutase (SOD) and caspase-3 were analyzed by spectrophotometric method. The expressions levels of Bcl-2 and Bax mRNA in diaphragmatic muscle were detected by RT-PCR.
 RESULTS: Compared with the control group, in the diabetic group, the Pt, Po and ±dT/dtmax were significantly reduced (all P<0.01), while CT and 1/2RT were significantly increased (both P<0.01); ultrastructure in the diaphragmatic muscle were obviously changed; the content of MDA and the activity of caspase-3 were increased (both P<0.01), while the activity of SOD was decreased (P<0.01); the ratio of Bcl-2/Bax at mRNA level was decreased (P<0.01). Compared with the diabetes group, in the treatment group, the diaphragm contractility and ultrastructural damage were improved; the content of MDA and the activity of caspase-3 were decreased (P<0.05, P<0.01 respectively), while the activity of SOD was increased (P<0.01), the ratio of Bcl-2/Bax at mRNA level was also increased (P<0.01). 
 CONCLUSION The exogenous H2S can protect diaphragmatic muscle of Type 1 diabetic rats, which is related to reducing oxidative damage and suppressing cell apoptosis.
Animals ; Apoptosis ; drug effects ; Caspase 3 ; metabolism ; Diabetes Mellitus, Experimental ; Diaphragm ; drug effects ; Hydrogen Sulfide ; pharmacology ; Male ; Malondialdehyde ; metabolism ; Muscle Contraction ; drug effects ; Oxidative Stress ; Rats ; Rats, Sprague-Dawley ; Sulfides ; Superoxide Dismutase ; metabolism