Identification of key genes and pathways associated with gender difference in osteonecrosis of the femoral head based on bioinformatics analysis.

OBJECTIVE: To identify different key genes and pathways between males and females by studying differentially expressed genes (DEGs). METHODS: The gene expression data of GSE123568 were downloaded from GEO database, including osteonecrosis of the femoral head (ONFH) samples from 3 females and 7 males, and DEGs between different gender were identified with R software. Protein-protein interaction (PPI) network was constructed to further analyze the interactions between overlapping DEGs, and finally, GO, KEGG and gene set enrichment analysis (GSEA) were conducted for enrichment analysis. RESULTS: 131 DEGs were identified between ONFH females and ONFH males, including 76 up-regulated genes and 55 down-regulated genes. And 10 hub genes were identified in PPI network, including SLC4A1, GYPA, CXCL8, IFIT1, GBP5, IFI44, IFI44L, IFIT3, KEL and AHSP. Functional enrichment analysis revealed that these genes were mainly enriched in cGMP-PKG signaling pathway, Fatty acid degradation, Non-alcoholic fatty liver disease, Systemic lupus erythematosus, Hematopoietic cell lineage and NO-cGMP-PKG signaling. CONCLUSIONS: NO-cGMP-PKG signaling may play an important role in the occurrence and development of ONFH. SLC4A1, GYPA, CXCL8, GBP5 and AHSP may be key genes associated with gender difference in the progression of ONFH, which may be ideal targets or prognostic markers for the treatment of ONFH.

Pcv-aCO2 and procalcitonin levels for the early diagnosis of bloodstream infections caused by gram-negative bacteria.

BACKGROUND: The central venous-to-arterial carbon dioxide difference (Pcv-aCO2) is a biomarker for tissue perfusion, but the diagnostic value of Pcv-aCO2 in bacteria bloodstream infections (BSI) caused by gram-negative (GN) bacteria remains unclear. This study evaluated the expression levels and diagnostic value of Pcv-aCO2 and procalcitonin (PCT) in the early stages of GN bacteria BSI. METHODS: Patients with BSI admitted to the intensive care unit at Guangdong Provincial People's Hospital between August 2014 and August 2017 were enrolled. Pcv-aCO2 and PCT levels were evaluated in GN and gram-positive (GP) bacteria BSI patients. RESULTS: A total of 132 patients with BSI were enrolled. The Pcv-aCO2 (8.32 ± 3.59 vs 4.35 ± 2.24 mmHg p = 0.001) and PCT (30.62 ± 34.51 vs 4.92 ± 6.13 ng/ml p = 0.001) levels were significantly higher in the GN group than in the GP group. In the diagnosis of GN bacteria BSI, the area under the receiver operating characteristic curve (AUROC) for Pcv-aCO2 was 0.823 (95% confidence interval (CI): 0.746-0.900). The sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) were 71.90%, 88.00%, 74.07% and 78.21%, respectively. The AUROC for PCT was 0.818 (95% CI: 0.745-0.890). The sensitivity, specificity, PPV and NPV were 57.90%, 94.67%, 71.93% and 74.67%, respectively. CONCLUSIONS: Pcv-aCO2 and PCT have similar and high diagnostic value for the early diagnosis of BSI caused by GN bacteria.

2B4 costimulatory domain enhancing cytotoxic ability of anti-CD5 chimeric antigen receptor engineered natural killer cells against T cell malignancies.

BACKGROUND: Chimeric antigen receptor engineered T cells (CAR-T) have demonstrated extraordinary efficacy in B cell malignancy therapy and have been approved by the US Food and Drug Administration for diffuse large B cell lymphoma and acute B lymphocytic leukemia treatment. However, treatment of T cell malignancies using CAR-T cells remains limited due to the shared antigens between malignant T cells and normal T cells. CD5 is considered one of the important characteristic markers of malignant T cells and is expressed on almost all normal T cells but not on NK-92 cells. Recently, NK-92 cells have been utilized as CAR-modified immune cells. However, in preclinical models, CAR-T cells seem to be superior to CAR-NK-92 cells. Therefore, we speculate that in addition to the short lifespan of NK-92 cells in mice, the costimulatory domain used in CAR constructs might not be suitable for CAR-NK-92 cell engineering. METHODS: Two second-generation anti-CD5 CAR plasmids with different costimulatory domains were constructed, one using the T-cell-associated activating receptor-4-1BB (BB.z) and the other using a NK-cell-associated activating receptor-2B4 (2B4.z). Subsequently, BB.z-NK and 2B4.z-NK were generated. Specific cytotoxicity against CD5+ malignant cell lines, primary CD5+ malignant cells, and normal T cells was evaluated in vitro. Moreover, a CD5+ T cell acute lymphoblastic leukemia (T-ALL) mouse model was established and used to assess the efficacy of CD5-CAR NK immunotherapy in vivo. RESULTS: Both BB.z-NK and 2B4.z-NK exhibited specific cytotoxicity against CD5+ malignant cells in vitro and prolonged the survival of T-ALL xenograft mice. Encouragingly, 2B4.z-NK cells displayed greater anti-CD5+ malignancy capacity than that of BB.z-NK, accompanied by a greater direct lytic side effect versus BB.z-NK. CONCLUSIONS: Anti-CD5 CAR-NK cells, particularly those constructed with the intracellular domain of NK-cell-associated activating receptor 2B4, may be a promising strategy for T cell malignancy treatment.

Induced CD20 Expression on B-Cell Malignant Cells Heightened the Cytotoxic Activity of Chimeric Antigen Receptor Engineered T Cells.

CD20 is an effective immunotherapy target for CD20+ B-cell malignant cells. Monoclonal antibody, especially rituximab, has been a conventional strategy in the treatment of B-cell malignancies such as non-Hodgkin's lymphoma. However, treatment with monoclonal antibodies has not been enough to overcome the refractory/relapse problems. Chimeric antigen receptor engineered T (CAR-T) cells have exhibited excellent therapeutic effect on lymphocytic leukemia in recent years. In this study, a CD20-specific CAR was constructed and the cytotoxic efficacy of CD20 CAR-T cells on B-cell malignant cells was evaluated by CD107a degranulation, pro-inflammation cytokine production, and true lytic ability in vitro and in vivo. It was found that CD20 CAR-T cells possessed stronger cytotoxic ability against CD20 highly expressed cells. Furthermore, when histone deacetylase inhibitor was used to enhance the expression of CD20 antigen on the surface of B-cell malignant cells via inducing acetylation of H3K9 on CD20 promoter site, it revealed that the cytotoxicity of CD20 CAR-T cells against histone deacetylase inhibitor-treated B-cell malignant cells was significantly enhanced.

CD33-Specific Chimeric Antigen Receptor T Cells with Different Co-Stimulators Showed Potent Anti-Leukemia Efficacy and Different Phenotype.

Acute myeloid leukemia (AML) is a kind of a malignant hematologic tumor caused by uncontrolled repopulation of myeloid hematopoietic stem cells (HSCs). Current therapeutic effects for AML patients are unsatisfactory. In particular, relapsed and refractory AML still have a poor prognosis. T cells modified by chimeric antigen receptor (CAR) was an immunotherapeutic strategy for malignancies, which has a broad developing prospect. Most AML cells overexpress the myeloid antigen CD33. Therefore, CD33-specific CAR-T cells with different co-stimulators (CD28, 4-1BB, or both, referred to as CD33 28z.CAR-T cells, CD33 BBz.CAR-T cells, or CD33 28BBz.CAR-T cells, respectively) were developed to evaluate their efficacy against AML. The effectiveness of three types of CD33 CAR-T cells against AML was verified by specific killing effect to AML cells and prolonged survival of a xenograft mouse model. In terms of CAR-T cell efficacy, especially when transfused into human bodies, the persistence of T cells is also an important index, as it is closely associated with the long-term effect of CAR-T cells. Therefore, the characteristics of three types of CD33 CAR-T cells related to the persistence of T cells were examined. It was found that during expansion, CD33 BBz.CAR-T cells had an increased central memory compartment, while CD33 28z.CAR-T cells were predominantly effector memory T cells. In addition, CD33 28z.CAR-T cells were more inclined to become exhausted. The study suggests that incorporation of 4-1BB in CARs may endow T cells with long-lasting survival ability, thus improving the long-term anti-leukemia effect of CAR-T cells, especially when transfused to the human body.

N-acetylcysteine and allopurinol confer synergy in attenuating myocardial ischemia injury via restoring HIF-1α/HO-1 signaling in diabetic rats.

OBJECTIVES: To determine whether or not the antioxidants N-acetylcysteine (NAC) and allopurinol (ALP) confer synergistic cardioprotection against myocardial ischemia/reperfusion (MI/R) injury by stabilizing hypoxia inducible factor 1α (HIF-1α)/heme oxygenase 1 (HO-1) signaling in diabetic myocardium. METHODS: Control or diabetic [streptozotocin (STZ)-induced] Sprague Dawley rats received vehicle or NAC, ALP or their combination for four weeks starting one week after STZ injection. The animals were then subjected to thirty minutes of coronary artery occlusion followed by two hours reperfusion in the absence or presence of the selective HO-1 inhibitor, tin protoporphyrin-IX (SnPP-IX) or the HIF-1α inhibitor 2-Methoxyestradiol (2ME2). Cardiomyocytes exposed to high glucose were subjected to hypoxia/re-oxygenation in the presence or absence of HIF-1α and HO-1 achieved by gene knock-down with related siRNAs. RESULTS: Myocardial and plasma levels of 15-F2t-isoprostane, an index of oxidative stress, were significantly increased in diabetic rats while cardiac HO-1 protein and activity were reduced; this was accompanied with reduced cardiac protein levels of HIF-1α, and increased post-ischemic myocardial infarct size and cellular injury. NAC and ALP given alone and in particular their combination normalized cardiac levels of HO-1 and HIF-1α protein expression and prevented the increase in 15-F2t-isoprostane, resulting in significantly attenuated post-ischemic myocardial infarction. NAC and ALP also attenuated high glucose-induced post-hypoxic cardiomyocyte death in vitro. However, all the above protective effects of NAC and ALP were cancelled either by inhibition of HO-1 or HIF-1α with SnPP-IX and 2ME2 in vivo or by HO-1 or HIF-1α gene knock-down in vitro. CONCLUSION: NAC and ALP confer synergistic cardioprotection in diabetes via restoration of cardiac HIF-1α and HO-1 signaling.

A simple modification results in greater success in the model of coronary artery ligation and myocardial ischemia in mice.

Mouse models of myocardial ischemic preconditioning (IPC) and ischemic postconditioning (IPD) have proven to be very useful models of cardiovascular diseases. In 2010, Gao described a novel procedure without the aid of mechanical ventilation. However, the technique of heart externalization could not be applied to mouse models of IPC or IPD due to the limited time frame of the technique. We proposed a modified simple and safe method using lung recruitment and short-term ventilation to perform the procedure in mice with IPC or IPD. The mice were randomly divided into 4 groups: the modified groups, M-IPC and M-IPD, and the conventional groups, C-IPC and C-IPD. In the 2 modified groups, the mice were removed from the ventilator and allowed to resume breathing spontaneously upon completion of the lung recruitment and the rapid closure of the thorax. Our study demonstrated that the postoperative recovery time was significantly reduced for the modified groups compared with the 2 conventional groups. Moreover, the inflammatory damages were attenuated by the modified method compared with the conventional method. In addition, the modified method significantly increased the survival rates of mice with IPC or IPD. The modified method improved the survival rates of mouse models of myocardial ischemia.

Endothelial nitric oxide synthase enhancer for protection of endothelial function from asymmetric dimethylarginine-induced injury in human internal thoracic artery.

OBJECTIVES: Endogenous nitric oxide synthase inhibitor asymmetric dimethylarginine is a cardiovascular risk factor that is elevated in patients with coronary artery disease. We hypothesized that novel endothelial nitric oxide synthase enhancer AVE3085 might improve the endothelial function altered by asymmetric dimethylarginine in the human internal thoracic artery. METHODS: Cumulative concentration-relaxation curves to acetylcholine (-11 to -5 log mol/L) were established in left internal thoracic artery rings (n = 65) from 27 patients undergoing coronary artery bypass grafting in precontraction induced by U46619 (-8 log mol/L) in the absence or presence of asymmetric dimethylarginine (100 µmol/L) or AVE3085 (30 µmol/L). Protein expressions of endothelial nitric oxide synthase and levels of superoxide anion production were detected. RESULTS: Maximal relaxation induced by acetylcholine was significantly attenuated by asymmetric dimethylarginine (12.7% ± 2.3% vs 35.3% ± 5.0% in control; P < .05) and significantly restored by AVE3085 (23.4% ± 2.8%; P < .05). AVE3085 also markedly restored endothelial nitric oxide synthase expression (0.29 ± 0.008; P = .012) reduced by asymmetric dimethylarginine (0.05 ± 0.04 vs 0.36 ± 0.03 in control; P = .014). Increased superoxide anion production by asymmetric dimethylarginine (2.97 ± 0.25 vs 0.51 ± 0.10 relative light units/[s/mg] in control; P < .05) was inhibited by AVE3805 (0.62 ± 0.104 relative light units/[s/mg]; P < .05). CONCLUSIONS: AVE3085 may restore endothelium-dependent relaxation reduced by asymmetric dimethylarginine through upregulation of endothelial nitric oxide synthase expression and inhibition of production of superoxide anion in human internal thoracic artery. These findings provide new insights into endothelial protection of coronary bypass grafting vessels to improve long-term patency of grafts.

Remote ischaemic preconditioning reduces myocardial injury in patients undergoing heart valve surgery: randomised controlled trial.

OBJECTIVE: To determine whether remote ischaemic preconditioning (RIPC) is cardioprotective in patients undergoing heart valve replacement. DESIGN: Single-blinded, randomised controlled trial. SETTING: Tertiary referral hospital in China. PATIENTS: Adult patients (31-72 years) undergoing mitral valve, aortic valve or tricuspid valve surgery. INTERVENTIONS: Patients were randomised to either the RIPC (n=38) or control (n=35) group. After induction of anaesthesia, patients in the RIPC group underwent three 5 min cycles of right upper limb ischaemia, induced by an automated cuff-inflator placed on the upper arm and inflated to 200 mm Hg. Each cycle was interrupted by a 5 min period of reperfusion during which time the cuff was deflated. The control group had only a deflated cuff placed on the upper arm for 30 min. MAIN OUTCOME MEASURES: Serum troponin I concentration was measured before surgery and at 6, 12, 24, 48, and 72 h postoperatively. The cardiac function of all patients was followed postoperatively. RESULTS: Troponin I concentration was reduced in the RIPC group (398.7±179.3 µg/l) compared with the control group (708.4±242.5 µg/l). Mean difference was 309.7±50.8 (95% CI 210.1 to 409.3, p<0.0001). A greater improvement in postsurgical cardiac function was noted in the RIPC group than in the control group. CONCLUSIONS: These data indicate that RIPC reduces myocardial injury and improves cardiac function in patients undergoing heart valve surgery. TRIAL REGISTRATION NUMBER: NCT01175681.

[Construction, expression and functional characterization of single chain variable fragments (scFv) against human CD33 antigen].

AIM: To construct and express the single chain variable fragments (scFv) gene against human CD33 antigen, and characterize its bioactivity. METHODS: The genes encoding the light and heavy chain variable regions were cloned by RT-PCR from a murine hybridoma cell line, which could produce monoclonal antibody(mAb) against human CD33 antigen. Then the light and heavy chain variable regions were fused together by a short peptide linker containing 15 amino acid (Gly(4)Ser)(3) using splice-overlap extensive PCR. The recombinant anti-CD33 scFv was subcloned into the expression vector pET28a(+) and expressed in E.coli Rosetta after induction by IPTG. RESULTS: SDS-PAGE and Western blot analysis showed that the recombinant anti-CD33 scFv gene was expressed in the form of inclusion body in E.coli Rosetta, and the purified fusion protein was obtained after a series of purification steps including cell lysis, inclusion body solubilization, Ni(2+) metal affinity chromatography and protein refolding. Flow cytometry(FCM) analysis showed that the scFv could react with human CD33 antigen. CONCLUSION: Recombinant anti-CD33 scFv gene has been successfully constructed and expressed in E.coli Rosetta, which could provide foundation for the future target therapy to the myeloid leukemia.

Identification of a novel isoform of iASPP and its interaction with p53.

iASPP is an inhibitory member of ASPP (apoptosis stimulating protein of p53, or Ankyrin repeats, SH3 domain and proline-rich region contain Protein) family. As reported previously, it at least includes two isoforms, one is iASPP/RAI (351 amino acids, aa) and the other is iASPP (828 aa).Here, we identified a novel open reading frame of human iASPP, which encodes a 407 aa protein and highly matches with the C terminus of iASPP (828 aa, CAI60219). Hereafter, iASPP (407 aa) will be referred to as iASPP-SV (iASPP splice variant). In further study, we found that iASPP-SV is a nuclear protein, and is capable of binding to p53 in vivo. Moreover, overexpression of iASPP-SV can inhibit the transcriptional activity of p53 on the promoters of both Bax and p21.

HI44a, an anti-CD44 monoclonal antibody, induces differentiation and apoptosis of human acute myeloid leukemia cells.

CD44 is a cell surface antigen that expresses on leukemia blasts from most acute myeloid leukemia (AML) patients. It has been reported that ligation of CD44 with some specific anti-CD44 monoclonal antibodies can reverse the differentiation blockage of leukemia cell lines. In this study, the differentiation and apoptosis-inducing effects of HI44a, another anti-CD44 monoclonal antibody (IgG2a), were investigated on leukemia cells obtained from 31 patients with AML-M2, AML-M3, AML-M4 or AML-M5. When the AML cells were treated with HI44a, the percentage of nitroblue tetrazolium (NBT)+ cells was significantly increased. The expression of CD11b, CD14 and CD15 on treated AML cells was also increased compared to control AML cells. In addition, HI44a was found to induce apoptosis of leukemia cells, as evidenced by an annexin-V assay. The mean percentage of apoptotic cells in HI44a-treated AML cells was significantly increased compared to that in control AML cells. Moreover, the level of c-myc transcript expression on AML cells was found to be obviously decreased in all detected patients. These results indicate that HI44a effectively induces both differentiation and apoptosis of AML cells and suggest that this activity of the anti-CD44 antibody may be associated with its inhibitory effect on c-myc transcript expression.