[Analysis of APTT Mixing Test Results in Factor â § Inhibitor-Positive Hemophilia Patients].

OBJECTIVE: To analyze the results of activated partial thromboplastin time (APTT) mixing test in coagulation factor Ⅷ inhibitor-positive hemophilia patients, so as to increase the value of APTT mixing test in the screen of factor Ⅷ inhibitor. METHODS: Eighty plasmas samples with different titers of coagulation factor Ⅷ inhibitors had been collected and diluted for routine immediate APTT mixing test and at 37 ℃ 2 hours incubation APTT mixing test. Fifteen samples were selected for immediate and normal temperature incubation for 15 min, 30min, 1 hour, 2 hours and 37 ℃ for 30 min, 1 hour, 2 hours APTT mixing test. RESULTS: The results of APTT mixing test were significantly correlated with the titers of coagulation factor Ⅷ inhibitors. The ROC curve result showed that the best diagnostic cut-off value for 2 hours incubation APTT mixing test at 37 ℃ to determine the presence or absence of coagulation factor Ⅷ inhibitors was 43.8 s (sensitivity and specificity was 85.90% and 100%, respectively), while the best diagnostic cut-off value for distinguishing high-titer and low-titer Ⅷ inhibitors was 52.4 s (sensitivity and specificity was 98.18% and 95.65%, respectively). The critical coagulation factor Ⅷ inhibitor titer that could not be corrected by immediate APTT was 5.14 BU/ml, while that could not be corrected by 37 ℃ 2 hours incubation APTT was 1.31 BU/ml. Paired samples t -test was performed on the APTT mixing test results at different times and temperatures, and the differences were statistically significant (P < 0.05). CONCLUSIONS: The APTT mixing test can be used as a screening index for coagulation factor Ⅷ inhibitors. APTT mixing test result shows a significant time-temperature dependence with lower titers of coagulation factor Ⅷ inhibitor. Patients with hemophilia who cannot be corrected by immediate APTT mixing test should be alert to the possibility of high titer of coagulation factor Ⅷ.

[Effect of Cyr61 on Imatinib Resistance in Chronic Myeloid Leukemia and Its Mechanism].

OBJECTIVE: To investigate the effect of Cyr61 on imatinib (IM) resistance in chronic myeloid leukemia (CML) and its mechanism. METHODS: Cyr61 level in cell culture supernatant was determined by enzyme-linked immunosorbent assay. The expression of Cyr61 and Bcl-xL were measured by real-time PCR and Western blot. Cell apoptosis was analyzed using an Annexin V-APC Kit. Expression of signal pathways related proteins was determined by Western blot. RESULTS: The level of Cyr61 obviously increased in K562G cells (IM resistance to CML cell line K562). Down-regulating the expression of Cyr61 decreased the resistance of K562G cells to IM and promoted IM induced apoptosis. In CML mouse model, down-regulating the expression of Cyr61 could increase the sensitivity of K562G cells to IM. The mechanism studies showed that Cyr61 mediated IM resistance in CML cells was related to the regulation of ERK1/2 pathways and apoptosis related molecule Bcl-xL by Cyr61. CONCLUSION: Cyr61 plays an important role in promoting IM resistance of CML cells. Targeting Cyr61 or its related effectors pathways may be one of the ways to overcome IM resistance of CML cells.

Combination of Neutrophil-to-Lymphocyte Ratio and Red Cell Distribution Width With Serum Tumor Markers for the Differential Diagnosis of Breast Cancer and its Association With Pathological Features and Molecular Types.

OBJECTIVE: To explore the neutrophil-to-lymphocyte ratio (NLR), red cell distribution width (RDW) and serum tumor markers as differential diagnostic markers of breast cancer (BC) and breast fibroadenoma (FA) and their associations with histopathological indicators and molecular typing in BC patients. METHODS: We collected pathological and routine clinical test data [NLR, RDW, serum tumor markers (CEA, CA15-3, CA125, CA19-9)] of 653 patients with BC and 100 patients with FA. After identifying indicators with significant inter-group differences, we used ROC curves to determine clinically significant cutoff values. Binary logistic regression analyses and ROC curves were used to analyze combined models for the differential diagnosis of BC and FA. Ordinal multinomial logistic regression analysis was used to explore correlations between routine clinical test indicators and pathological BC features. RESULTS: The BC and FA groups had significantly different CEA, NLR, and CA19-9 levels (P < .05), with respective areas under the curve (AUC) of 0.799, 0.747, and 0.711 for differentiating BC from FA and respective optimal cutoff values of 1.35 ng/mL, 1.58, and 8.55 U/mL. Binary logistic regression and ROC curve analysis showed that CEA was superior to the other 2 factors for the differential diagnosis of BC and FA. whereas the combined use of all three indicators was diagnostically most effective (AUC = 0.886; 95% confidence interval: 0.838-0.933, P = .000; sensitivity and/or specificity: 76.5%/88.9%). Ordered multinomial logistic regression analysis showed that NLR, CEA, and CA15-3 correlated positively with BC TNM staging; RDW was negatively correlated with BC histological grade; RDW, CA15-3 and CA125 were obviously associated with BC molecular typing. CONCLUSION: The combination of CEA, NLR, and CA19-9 may be used to screen and diagnose BC. NLR, CEA and CA15-3 are related to the TNM staging of BC. RDW is related to BC histological grading. RDW, CA15-3 and CA125 are related to BC molecular typing.

An Integrated Bioinformatic Analysis of the S100 Gene Family for the Prognosis of Colorectal Cancer.

BACKGROUND: S100 family genes exclusively encode at least 20 calcium-binding proteins, which possess a wide spectrum of intracellular and extracellular functions in vertebrates. Multiple lines of evidences suggest that dysregulated S100 proteins are associated with human malignancies including colorectal cancer (CRC). However, the diverse expression patterns and prognostic roles of distinct S100 genes in CRC have not been fully elucidated. METHODS: In the current study, we analyzed the mRNA expression levels of S100 family genes and proteins and their associations with the survival of CRC patients using the Oncomine analysis and GEPIA databases. Expressions and mutations of S100 family genes were analyzed using the cBioPortal, and protein-protein interaction (PPI) networks of S100 proteins and their mutation-related coexpressed genes were analyzed using STRING and Cytoscape. RESULTS: We observed that the mRNA expression levels of S100A2, S100A3, S100A9, S100A11, and S100P were higher and the level of S100B was lower in CRC tissues than those in normal colon mucosa. A high S100A10 levels was associated with advanced-stage CRC. Results from GEPIA database showed that highly expressed S100A1 was correlated with worse overall survival (OS) and disease-free survival (DFS) and that overexpressions of S100A2 and S100A11 were associated with poor DFS of CRC, indicating that S100A1, S100A2, and S100A11 are potential prognostic markers. Unexpectedly, most of S100 family genes showed no significant prognostic values in CRC. CONCLUSIONS: Our findings, though still need to be ascertained, offer novel insights into the prognostic implications of the S100 family in CRC and will inspire more clinical trials to explore potential S100-targeted inhibitors for the treatment of CRC.

[Efficacy and Safety of Decitabine Combined with CAG (Cytarabine, Aclarubicin, G-CSF) for Patients with Intermediate or High Risk Myelodysplastic Syndrome and Acute Myeloid Leukemia: a Meta-Analysis].

OBJECTIVE: To systematically evaluate the efficacy and safety of DCAG regimen for treating the intermediate or high risk MDS and AML. METHODS: PubMed, EMbase, The Cochrane Library, WanFang Data and CNKI databases were searched to collect randomized controlled trials (RCTs) of decitabine combined with CAG regimen for intermediate or high risk MDS and AML from inception to March, 2018. The quality of each RCT was evaluated by the Cochrane collaboration´s tool for assessing the risk of bias.Then, the data were analyzed by using RevMan 5.3. RESULTS: Twenty-four RCTs were included in the meta-analysis, containing 1 557 patients with intermediate or high-risk MDS and AML, of whom 594 were AML patients and 590 were MDS patients. The patients treated with the DCAG regimen were enrolled in DCAG group, and the patients treated with single-agent decitabine or CAG regimen were enrolled in control group. RESULTS: The results of meta-analysis showed that compared with other therapies, the complete remission rate of DCAG regimen in patients with intermediate or high-risk MDS and AML was high (RR=1.63，95% CI=1.43-1.85，P＜0.000 01), and the overall response rate was also high (RR=1. 35，95% CI=1.24-1.46，P＜0.000 01); Subgroup analysis results showed that DCAG regimen was better than CAG regimen in the complete remission rate (RR=1.71，95% CI=1.49-1.97，P＜0.000 01), and slightly better than single-agent decitabine group (RR=1.43，95% CI=1.08-1.91，P=0.01). In terms of adverse reactions, there was no statistically significant difference in the rates of myelosuppression, pulmonary infection, gastrointestinal reactions, and bleeding events between the 2 groups (P＞0.05). CONCLUSION: DCAG regimen has significant efficacy in the treatment of intermediate or high-risk MDS and AML, and is superior to CAG regimen and single-agent dicitabine regimen. As compared with control group, there was no significant difference in adverse events. Due to limited quantity and quality of the included studies, more high quality studies are needed to verify above mentioned conclusion.

[Values of Hematological Indicators in the Screening of α-Thalassemia in Fujian Area of China].

OBJECTIVE: To analyze the genotypes and the hematological phenotypic characteristics of α-thalassemia in different areas of Fujian and to evaluate the values of mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), hemoglobin (Hb), RBC distribution width/red blood cell (RDW/RBC) for screening α-thalassemia in this area. METHODS: The Gap-PCR assay was applied for detecting 3 common deletional mutations of patients with α-thalassemia, and the reverse dot-blot (RDB) assay was adopted to detect the foci of 3 common non-deletional gene mutations.Then,the hematological parameters of individuals with α-thalassemia were analyzed. Finally, the optimal cut-off value in hematological indexes for screening α-thalassemia were determined by the ROC curve. RESULTS: Altogether 16 types of gene mutations were found in 772 patients with α-thalassemia. Among them, the -SEA/αα deletion mutation was the most common which was observed in 521 cases(67.49%). Compared with the control group, the differences in MCV, MCH, and Hb were statistically significant between the patients of the same sex but no same type. In male groups, the RDW/RBC ratio was statistically significant in individuals of light type and HbH disease as compared with the healthy control group. But in female groups, the statistical different of RDW/RBC ratio was found between only HbH disease group and control group. MCV<81.25 fl, MCH<27.30 pg, Hb(male)<128.5 g/L, and Hb(female) <123.5 g/L, with the highest specificity and the highest sensitivity, were the best cut-off points for screening α-thalassemia in the laboratory. CONCLUSION: Due to the difference of regional heterogeneity and hospital equipment environment, the different laboratories need to establish cut-off value for screening α-thalassemia suitable for its local region. In future, our laboratory can use MCV<81.25 fl, MCH<27.30 pg, Hb(male)<128.5 g/L, and Hb(female) <123.5 g/L for value for clinical screening, of α-thalassemia.

Serum markers in the differential diagnosis of Waldenstrom macroglobulinemia and other IgM monoclonal gammopathies.

BACKGROUND: IgM monoclonal gammopathy can be present in a broad spectrum of diseases. We evaluated the value of serum markers in the differential diagnosis of Waldenstrom macroglobulinemia (WM) and other types of IgM monoclonal gammopathies. METHODS: We included patients who were first admitted to hospital and identified as having IgM monoclonal gammopathy by serum immunofixation electrophoresis (sIFE). We evaluated basic clinical features, sIFE, diagnosis, and serum markers. Furthermore, we applied the receiver operating characteristic (ROC) curve to analyze the differential diagnosis value of serum markers for WM. Finally, we used logistic regression and ROC curve to analyze the differential diagnosis value of multimarker combinations to identify WM. RESULTS: IgM monoclonal gammopathy was most frequently found in patients with Waldenstrom macroglobulinemia, followed by monoclonal gammopathy of undetermined significance (MGUS), B-cell non-Hodgkin Lymphoma (B-NHL), and multiple myeloma (MM). Serum markers showed significant differences among the four diseases. The diagnostic markers LDH, IgM, IgG, IgA, and serum light chain К had higher diagnostic efficiency. Among these markers, serum IgM provided the highest diagnostic efficiency. Additionally, the combined use of all five serum markers provided the most effective diagnosis. CONCLUSIONS: The five serum markers, LDH, IgM, IgG, IgA, and К, each yielded a specific efficacy in differential diagnosis of WM. The single marker with the highest diagnostic efficiency was the serum IgM level. However, a combination of multiple serum markers was better than the use of a single marker in diagnosing WM. The combined use of all five serum markers provided the most effective diagnosis, with an AUC of .952 and sensitivity and specificity of 87.8% and 86.9%, respectively.

Molecular characteristic of mcr-1 producing Escherichia coli in a Chinese university hospital.

BACKGROUND: Colistin has been considered as a last-line treatment option in severe infections caused by multidrug-resistant (MDR) gram-negative pathogens. However, the emergence of the mobile colistin resistance gene (mcr-1) has challenged this viewpoint. The aim of this study is to explore the prevalence of mcr-1 in Escherichia coli (E. coli) in a Chinese teaching hospital, and investigate their molecular characteristics. METHODS: A total of 700 E. coli isolates were used to screen mcr-1 by PCR and sequencing in a Chinese university hospital from August 2014 to August 2015. Susceptibility test of mcr-1-producing isolates was determined by Vitek -2 Compact system. 26 virulence factors (VFs), phylogenetic groups, Multi-locus sequence typing (MLST), and DNA Fingerprinting (ERIC-PCR) of strains were investigated by PCR. RESULTS: Four (0.6%) mcr-1 producing E. coli isolates were found in this study. The results of antibiotic susceptibility test showed that all four isolates were resistant to colistin, ciprofloxacin, levofloxacin, cefazolin, and trimethoprim/sulfamethoxazole, and were susceptible to amikacin, ertapenem and imipenem. In addition, all 4 isolates exhibited high-level resistance to aztreonam, cefotaxime and gentamicin. The numbers of VFs contained in mcr-1 positive isolates were no more than 4 in our study. MLST result demonstrated that these isolates were assigned to two sequence types: ST156 and ST167. The result of phylogenetic analysis showed that four mcr-1-positive isolates belong to two phylogenetic groups: A and B1 group. ERIC-PCR showed that four mcr-1 positive strains were categorized into three different genotypes. CONCLUSIONS: Our study demonstrated a low prevalence of mcr-1 in E. coli clinical isolates in a Chinese teaching hospital, and we have gained insights into the molecular characteristics of these mcr-1-positive strains. Increasing the surveillance of these infections, as well as taking effective infection control measures are urgently needed to take to control the transmission of mcr-1 gene.

High prevalence of metallo-ß-lactamase among carbapenem-resistant Klebsiella pneumoniae in a teaching hospital in China.

The aim of this study was to characterize the carbapenemases in carbapenem-resistant Klebsiella pneumoniae (CR-KP) from a Chinese teaching hospital. A total of 40 CR-KPs were screened for the presence of carbapenemases. Minimum inhibitory concentrations were determined by agar dilution. The modified Hodge test was used for the detection of carbapenemase production. Carbapenemase, extended-spectrum ß-lactamase, and AmpC genes were detected using polymerase chain reaction (PCR) and sequencing. A conjugation test was performed using a broth culture mating method, transferred plasmids were typed by PCR-based replicon typing, and clonal relatedness was investigated by enterobacterial repetitive intergenic consensus sequences PCR (ERIC-PCR) and multilocus sequence typing (MLST). The results revealed that modified Hodge test was positive for 28 CR-KPs, and CR-KPs exhibited high resistance rates against various antibiotics, except colistin (5.0%) and tigecycline (22.5%). ERIC and MLST profiles showed no clonal outbreak. PCR demonstrated a high prevalence rate (55.0%, 22/40) of metallo-ß-lactamases (MBLs) in CR-KPs. IMP-4, IMP-8, NDM-1, and KPC-2 were identified in 14 (35.0%), 7 (17.5%), 2 (5.0%), and 7 (17.5%) isolates, respectively. Notably, 2 CR-KPs coproduced 2 carbapenemases simultaneously (IMP-8/NDM-1 and IMP-4/KPC-2). In vitro transfer of carbapenem resistance was successful for 11 MBL-producing CR-KPs. The extended spectrum ß-lactamase genes were detected in 30 (75.0%) of these CR-KPs. To the best of our knowledge, this is the first report focusing on carbapenem resistance in K. pneumoniae due to metalloenzymes in China. Screening and surveillance of MBLs in Enterobacteriaceae is urgently needed in this region to control and prevent the spread of these resistance determinants.

[Effects of fractalkine on the expression of inflammatory substances in LPS-activated microglia cells].

AIM: To investigate the effect of fractalkine on the expression of the tumor necrosis factor-α(TNF-α), interleukin-1ß (IL-1ß), and nitric oxide (NO) in LPS-activated murine microglia cells line N9. METHODS: In vitro LPS-activated microglia cells were treated for 24 h in the presence of fractalkine. The level of TNF-α and IL-1ß in the culture supernatants were measured by enzyme-linked immunosorbent assay (ELISA), The level of NO in the culture supernatants were quantitated by the NO test assay. RESULTS: The concentration of TNF-α, IL-1ß and NO in the culture supernatants evidently increased in LPS-activated microglia cells groups and prominently decreased by the fractalkine co-incubated. CONCLUSION: It is thus concluded that fractalkine has neuroprotective functions by inhibiting the expression of inflammatory factor in activated microglia cells.

[Isolation of OsFAD2, OsFAD6 and FAD family members response to abiotic stresses in Oryza sativa L.].

Unsaturated fatty acids are synthesized by fatty acid desaturase in plant, which play an important role in plant development and defense to abiotic stresses. The key enzymes for the conversion of oleic into linoleic acid were isolated from rice (Oryza sativa L.) and were designated OsFAD2 and OsFAD6, respectively. The open reading frame (ORF) of OsFAD2 was 1 167 bp in length, which encoded a 388 amino acids sequence with the isoelectric point of 8.17 and molecular mass of 52.24 kDa, the OsFAD2 protein contained a C-terminal ER retrieval motif. The ORF of OsFAD6 was 1 365 bp in length and the predicted OsFAD6 protein has 454 amino acids with an estimated molecular mass of 44.35 kDa and an isoelectric point of 9.24, the predicted OsFAD6 protein possessed a putative N-terminal plastidial signal peptide. Both of them had three his-boxes, which were peculiar to membrane integrated fatty acid desaturase by Clustal X analysis. RT-PCR analysis showed that both genes were expressed in all tissues rice seedlings, with the maximum transcript accumulation in leaves. Among FAD family members of Oryza sativa L., the mRNA abundance of OsFAD2 and OsFAD6 in leaves did not change under cold stress; however, the mRNA abundance of OsFAD7 and OsFAD8 increased in the same condition. It was also found that the expression of FAD family members had diurnal rhythm phenomena. Based on the results of this study, it suggested that diurnal rhythm expression of OsFAD6 and OsFAD7 was related to the change of NADPH abundance.

[Expression changes of genes related to calcium pathway in all-trans retinoic acid-induced differentiation of APL cells].

In order to investigate the role of calcium pathway in myeloid differentiation, the expression level of genes related to calcium pathway in all-trans retinoic acid (ATRA)-induced NB4 cell differentiation was detected by cDNA microarray, some of which were further confirmed by quantitative real time RT-PCR. At the same time, the expressions of these genes in NB4-R1 cells treated with ATRA and 8-CPT-cAM P alone or in combination, and in differentiation of primary cells from ATRA-induced newly diagnosed APL patients were detected by real time RT-PCR. The results showed that during differentiation of ATRA-induced NB4 cells, the expressions of genes related to calcium concentration had changed, the expression of downstream effectors in calcium pathway was up-regulated and confirmed by real time RT-PCR assay. The expression of genes related to calcium concentration did not change significantly when NB4-R1 cells were treated by ATRA or 8-CPT-cAMP alone, but expression changes of those genes were similar to the changes in ATRA-induced NB4 cell differentiation when NB4-R1 cells were treated by ATRA combined with 8-CPT-cAMP. In addition, the expression changes of those genes in ATRA-induced primary cells of patients with APL were also similar to changes in ATRA-induced NB4 cell differentiation. It is concluded that calcium pathway may be involved in ATRA-induced differentiation in APL cell.

[Impact of intrapleural hyperthermic perfusion on immunologic reaction state of cytokines TH1/TH2 of lung carcinoma patients with malignant pleural effusion].

BACKGROUND & OBJECTIVE: Intrapleural hyperthermic perfusion is the distinctive therapy of malignant pleural effusion (MPE) caused by lung carcinoma. The expression pattern of T helper type 1 (Th1)/Th2 is an important index that reflects antitumor immunologic function. This study was to evaluate the therapeutic effect of intrapleural hyperthermic perfusion on MPE, and to investigate the impact of intrapleural hyperthermic perfusion on the immunologic reaction state of lung carcinoma patients with MPE by observing the expression pattern of cytokines Th1/Th2. METHODS: A total of 24 lung carcinoma patients with MPE underwent intrapleural hyperthermic perfusion with 43 celsius warmed normal saline for 60 min under video-assisted thoracoscopic surgery (VATS). The responses of pleural effusion, adverse events, life quality and survival time were observed. The concentrations of Th1/Th2-related cytokines interferon-gamma (IFN-gamma), interleukin-2 (IL-2)/IL-4, and IL-10 in peripheral blood and pleural effusion were detected by ELISA, and their mRNA expression was detected by reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: Neither operation-related death nor postoperative complication occurred. The total response rate of pleural effusion control was 100%, including 23 cases of complete remission (CR) and 1 case of partial remission (PR). No recurrence of MPE occurred. The quality of life of all patients had been improved. The median survival time was 18.3 months. The 1-and 2-year survival rates were 91.7% and 16.7%. The concentrations and positive rates of IL-2 and IFN-gamma were significantly lower and those of IL-4 and IL-10 were significantly higher in peripheral blood and pleural effusion before hyperthermia than in those after hyperthermia (P<0.05). CONCLUSIONS: Intrapleural hyperthermic perfusion under VATS is a safe and effective treatment for MPE caused by lung carcinoma. It can convert the predominant state of Th2 cytokines in lung carcinoma patients with MPE to that of Th1 cytokines.

[Experimental study on the role of cytokines and keratinocytes in the survival mechanism of auto and allogeneic mixed skin grafting].

OBJECTIVE: To explore the role of cytokines and keratinocytes in the survival mechanism of mixed auto and allogeneic skin grafting. METHODS: Thirty-six SD rats were employed in the study. The rat model with mixed auto and allogeneic skin grafting and mixed human epithelial and lymphocytic culture (MELC) model were established. The change of IL-10 in the serum and the supernatant of the cultured tissue sample from the local wound was observed after the mixed skin grafting in scalded rats. And the role of epithelium in the induction of immunosuppression in vitro was monitored. RESULTS: The serum IL-10 content in the rats with mixed skin grafting (25.89 +/- 2.82 ng/L) at 7 postoperative day (POD) was evidently higher than that in normal rats (14.20 +/- 2.43 ng/L) (P < 0.05). The IL-10 content in the culture supernatant of rat tissue samples exhibited evident different during 4-14 PODs (P < 0.05-0.01), while which was no difference to that in normal rat on 21st and 28th POD. The inhibiting effects of autologous epithelia and keratinocytes in MELC system were correlated with their dosage. After the adding of autologous keratinocytes to MELC system the cytokines secreted from Th1 could induce the secretion of cytokines from Th2 by IL-10 mediation. This effect could be corrected by the addition of monoclonal antibody of IL-10. CONCLUSION: The keratinocytes inlayed in the autoskin during mixed grafting could increase the local IL-10 level by activating Th2 cells, which might be one of the important reasons of the survival of mixed skin grafting.