Adolescent ; Adult ; Aged ; Aged, 80 and over ; Child ; Child, Preschool ; Female ; Humans ; Infant ; Intraoperative Complications ; Male ; Middle Aged ; Postoperative Complications ; Retrospective Studies ; Tracheotomy ; adverse effects ; methods ; Young Adult

OBJECTIVETo explore the values of tissue factor (TF) and vascular endothelial growth factor (VEGF) expressions on peripheral CD14+ monocytes in disease assessment, prognosis, and short-term efficacy evaluation of non-Hodgkin lymphoma (NHL) patients.

METHODSTF and VEGF expressions on CD14+monocytes in 47 NHL patients (disease group) before chemotherapy and after 4 chemotherapy cycles and in 30 healthy subjects (control group) were detected by flow cytometry, and the potential relationship among TF, VEGF, International Prognostic Index (IPI), and short-term efficacy were analyzed.

RESULTSTF and VEGF expressions on CD14 + monocytes in disease group were significantly higher than those in control group ( all P <0. 01) and positive correlation was showed between them (r = 0. 708, P = 0.00). TF and VEGF expressions in Ann Arbor stage III and IV (n = 22 and 19) , symptomatic (n = 22) , lactate dehydrogenase (LDH) increased (n = 21) , Eastern Cooperative Oncology Group (ECOG) score 2-4 (n = 12) and extranodal lesions >1 (n = 16) groups were significantly higher than those in Ann Arbor stage II (an = 6) , asymptomatic (an =25) , LDH normal (n = 26) , ECOG score 0-1 ( n = 35) and extranodal lesions ~1 ( na = 31) groups, respectively (all P <0.05). The expressions of TF and VEGF on CD14 + monocytes in high-risk (n = 7) or high-middle-risk (n = 11) groups were significantly increased compared with low-risk (n = 15) or low-middle-risk(n = 14) groups, respectively (all P <0. 01). TF and VEGF expressions in non-remission group before chemotherapy (n = 11) were both obviously higher than those in remission group (an = 36, all P <0. 01) , and after chemotherapy their expressions in remission group were significantly lower than those before chemotherapy (all P <0. 01) , while such significant changes were not observed in the non-remission group ( all P > 0. 05).

CONCLUSIONThe high expressions of TF and VEGF on peripheral CD14 + monocytes can be useful markers in dis-ease assessment, prognosis evaluation and short-term efficacy observation of NHL patients.

Adolescent ; Adult ; Aged ; Aged, 80 and over ; Case-Control Studies ; Child ; Female ; Humans ; Lipopolysaccharide Receptors ; Lymphoma, Non-Hodgkin ; blood ; Male ; Middle Aged ; Monocytes ; metabolism ; Prognosis ; Thromboplastin ; metabolism ; Vascular Endothelial Growth Factor A ; blood ; Young Adult

OBJECTIVETo sort and identify side population (SP) cancer stem cells (CSC) in human prostate cancer (PCa) cell lines.

METHODSStem-like cells were isolated from five PCa cell lines Du145, IA8, LNCaP, TSU-Pr and PC-3 using FACS based on CD133+ CD44+ immunophenotype and SP in Hoechst staining. The in vitro growth pattern and tumorigenicity of SP stem cells were verified by soft agar colony-formation trial. LNCaP/SP cells were selected for further identification of stem cell properties using immunostaining, proliferation and invasion assay. Eventually, tumorigenicity and metastasis ability of LNCaP/SP were confirmed by xenograft experiments.

RESULTSThe percentages of CSCs of the CD133 CD44 + immunophenotype were extremely low in the five PCa cell lines. On the contrary, the percentages of the isolated SP cells were significantly higher in Du145 ([0.15 +/- 0.02]%), IA8 ([0.60 +/- 0.07 ]%), LNCaP ([0.8 +/- 0.1]%) and TSU-PrL ([2.0 +/- 0.4]%), but none was detected in PC-3. Besides, IA8/SP, LNCaP/SP and TSU-PrL/SP cells showed a significantly greater colony-forming efficiency than non-side population (NSP) cells (P < 0.05). Compared with LNCaP/NSP cells, LNCaP/SP cells exhibited high expressions of integrin alpha2, Nanog, CD44, OCT4 and ABCG2, remarkably enhanced invasive and proliferative potentials in vitro, and markedly increased tumorigenicity and metastasis (P < 0.01).

CONCLUSIONSP sorting is more suitable than CD133+ CD44+ selection for enriching CSCs from PCa cell lines, and LNCaP/ SP represents a typical CSC population.

Cell Line, Tumor ; cytology ; Cell Separation ; Humans ; Male ; Neoplastic Stem Cells ; cytology ; Prostatic Neoplasms ; Side-Population Cells ; cytology

Objective of this study was to establish a method for simultaneous detection of FLT3/ITD and NPM1 gene mutations in AML. A double PCR was firstly designed and optimized to amplify both exon 12 of NPM1 and exon 14-intron 14-exon 15 of FLT3, with the aim of detecting almost all reported mutations. After optimization, a touchdown PCR was chosen for the multiplex PCR procedure, with the primer concentrations of NPM1 and FLT3-ITD being 200 nmol/L and 152 nmol/L respectively. The PCR amplicons were separated by capillary electrophoresis and the presence of mutants was recognized by the size difference between the mutants and wild-type products. The areas of mutant peak and wild-type peak were used to calculate the mutant/wild-type ratio. All the positive mutated samples were confirmed by sequencing. The results showed that 17 patients with NPM1 mutation, 15 patients with FLT3-ITD mutation, 6 patients with both NPM1 and FLT3-ITD mutations were found among 93 patents. 7 patients with M₂, 4 patients with M₄, 5 patients with M₅ and 1 patients with M₆ were found out of 17 patients with NPM1 mutation, in which 10 patients were male and 7 patients were female, 15 patients were with type A, 1 patients was with type B and 1 patients was with type Nm, strikingly 1 CML patient in blast crisis was found to carry a type A mutation. Among 15 patients with FLT3-ITD mutation 1 patient with M₁, 8 patients with M₂, 2 patients with M₂, 2 patients with M₃, 1 patient with M₄, 3 patients with M₅ were found, in which 5 patients were male and 10 patients were female. Sequencing results further confirmed the accuracy and reliability of this method. It is concluded that a novel method with the ability to detect both FLT3-ITD and NPM1 mutations has been developed when genomic DNA was templated. This method is fast, easy, accurate and capable to calculate the mutant/wild-type ratio.
Exons ; Female ; Genotype ; Humans ; Karyotyping ; Leukemia, Myeloid, Acute ; diagnosis ; genetics ; Male ; Mutation ; Nuclear Proteins ; genetics ; Polymerase Chain Reaction ; methods ; fms-Like Tyrosine Kinase 3 ; genetics

This study was purposed to explore the expressions of platelet-activated markers PAC-1 and CD62p in peripheral blood of malignant lymphoma patients and the influence of dipyridamole on their expression. 32 lymphoma patients were divided into simple chemotherapy group (simple group) and chemotherapy plus dipyridamole group (combined group) randomly, and 15 healthy peoples were selected as control group. The dipyridamole of 100 mg/day was given to the patients in combined group. The expression levels of PAC-1, CD62p and fibrinogen (Fib) were detected by flow cytometry and magnetic bead method on day 0, 3, 7 and 14 of chemotherapy respectively. The results showed that the levels of PAC-1, CD62p and Fib in lymphoma patients were significantly higher than those in control group (p < 0.01, 0.05), moreover there was positive correlation between levels of PAC-1 and Fib (r = 0.549, p < 0.01). PAC-1 expression on day 0 and 3 of chemotherapy in simple group was higher than that on day 14 (p < 0.05, 0.01) and CD62p expression on day 3 of chemotherapy was higher than that on day 0, 7 and 14 (p < 0.05, 0.01). PAC-1 expression in combined group on day 14 of chemotherapy was lower than than on day 0 and 3 (p < 0.05, 0.01), and CD62p on day 14 was lower than that on day 3 of chemotherapy (p < 0.05); PAC-1 and CD62p expressions in combined group on day 3, 7 and 14 of chemotherapy were decreased than those in simple group, but Fib level was not changed significantly. It is concluded that the patients with malignant lymphoma usually accompany with platelet activation and hyperfibrinogenemia in peripheral blood. Applying dipyridamole routine dosage in chemotherapy can efficiently restrain platelet activation.
Adolescent ; Adult ; Aged ; Dipyridamole ; therapeutic use ; Dual Specificity Phosphatase 2 ; metabolism ; Female ; Fibrinogen ; metabolism ; Humans ; Lymphoma ; blood ; drug therapy ; Male ; Middle Aged ; P-Selectin ; metabolism ; Platelet Activation ; Young Adult

OBJECTIVETo develop an alternative method for assessment of gene delivery systems in vivo.

METHODSMouse primary spleen lymphocytes were genetically modified in vitro by a retroviral vector harboring a Gaussia luciferase (Gluc) expression cassette. After implantation of these cells into recipient mice, the expression of Gluc was detected in whole blood or plasma collected.

RESULTSAs little as 10 muL whole blood drawn from the recipient mice could guarantee prompt reading of Gluc activity with a luminometer. And the reading was found in good correlation with the number of genetically modified spleen lymphocytes implanted to the mice.

CONCLUSIONSGluc may be useful as an in vivo reporter for gene therapy researches, and Gluc blood assay could provide an alternative method for assessment of gene delivery systems in vivo.

Animals ; Arecaceae ; enzymology ; Cell Line ; Gene Transfer Techniques ; Genes, Reporter ; Humans ; Luciferases ; genetics ; Mice

The study was aimed to investigate the clinical significance of coagulation function changes in lymphoma patients and to analyze the relationship between their changes and international prognostic index (IPI). The prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin time (TT) and fibrinogen (FIB) were detected by magnetic bead method in 75 lymphoma patients and 20 healthy persons. The dehydrogenase (LDH) level was detected by rate method in all lymphoma patients and healthy persons. The results showed that (1) the APTT and FIB more obviously increased in lymphoma patients which displayed as hyperfibrinogenemia, as compared with control group (p < 0.05, p < 0.01); no obvious changes of coagulation indexes presented in patients with different ages and extranodal lesions (p > 0.05, p < 0.01). (2) APTT and FIB levels in stage III and IV patients were much higher than those in the stage II (p < 0.05 and < 0.01), and FIB level in stage IV group was significantly higher than those in the stage III (p < 0.05). FIB level in symptomatic group was significantly higher than that in asymptomatic group (p < 0.01). (3) APTT and FIB in increased LDH group were obviously higher than those in control group (p < 0.05, p < 0.01). Furthermore, FIB in increased LDH group was higher than that in normal LDH group (p < 0.05). FIB in performance status (PS) 2 - 4 groups increased significantly as compared with those in PS 0-1 group (p < 0.01). (4)FIB levels in the low-middle-risk, high-middle-risk and high-risk groups were significantly higher than those in control group (p < 0.01), while FIB levels in high-middle-risk and high-risk groups were higher than those in low-risk group (p < 0.05). (5) the number of FIB increased patients in symptomatic group, increased LDH group, PS 2 - 4 group and Ann Arbor stage III-IV group were much higher than those in counterparts (p < 0.05 or 0.01).There were positive correlations between FIB and LDH level, PS grades, Ann Arbor stages as well as risk grades respectively (p < 0.05 or 0.01). It is concluded that lymphoma patients usually accompany with hyperfibrinogenemia which may be influenced by Ann Arbor stage, systemic symptom, LDH level and PS grade. FIB is supposed to be an effective indication of prognosis in lymphoma patients.
Adolescent ; Adult ; Aged ; Aged, 80 and over ; Blood Coagulation ; Case-Control Studies ; Female ; Humans ; Lymphoma ; diagnosis ; pathology ; physiopathology ; Male ; Middle Aged ; Neoplasm Staging ; Partial Thromboplastin Time ; Prognosis ; Prothrombin Time ; Young Adult

BACKGROUNDSufentanil is a suitable choice for target-controlled infusion (TCI) because of its shorter context-sensitive half-time. The current study was to estimate the pharmacokinetics of sufentanil TCI in Chinese patients using the two-stage analysis.

METHODSTwelve adult patients with American Society of Anesthesiologists (ASA) physical status I or II undergoing elective surgery under general anesthesia were included. Anesthesia was induced with propofol, rocuronium and sufentanil administered by TCI lasting for 30 minutes, with target effect-site concentration of sufentanil 4 or 6 ng/ml. Frequent arterial blood samples (1.5 ml) were taken during and up to 24 hours after sufentanil TCI. Before the end of surgery, another arterial blood sample (1.0 ml) was drawn for the blood-gas analysis. Plasma sufentanil concentrations were determined by liquid chromatography-tandem mass spectrometry (limit of quantitation was 5 pg/ml). The data were analyzed with the two-stage approach, linear regression and correlation analysis.

RESULTSThe pharmacokinetics of sufentanil TCI were adequately described by a three-compartment model. The variables were derived as follows: the volume of central compartment (V(1)) was 5.4 L, volume of distribution at steady-state (Vdss) was 222.6 L, metabolic clearance (Cl(1)) was 0.84 L/min and elimination half-life (t(1/2Y)) was 389 minutes. Patients' age, gender and PaCO2 correlated significantly with the pharmacokinetic parameters. The Vdss, volume of slowly equilibrating compartment (V(3)) and t(1/2Y) increased, and rapid distribution clearance (Cl(2)) decreased with increasing patient age. Male patients had larger values of Vdss, volume of rapidly equilibrating compartment (V(2)) and V(3) than female patients. The Vdss and V(3) increased with higher PaCO2 values. There were no significant correlations between the pharmacokinetic variables and body weight, height, lean body mass, plasma albumin, sufentanil dose, duration of surgery, pH or base excess of blood (BE-B).

CONCLUSIONSThe pharmacokinetics of sufentanil TCI in Chinese patients can be optimally described by a three-compartment model. The pharmacokinetic analysis technique may affect the pharmacokinetic parameters and correlations.

Adult ; Aged ; Anesthetics, Intravenous ; administration & dosage ; pharmacokinetics ; Asian Continental Ancestry Group ; Chromatography, Liquid ; Female ; Humans ; Infusions, Intravenous ; methods ; Male ; Middle Aged ; Sufentanil ; administration & dosage ; pharmacokinetics ; Young Adult

The forkhead family members of transcription factors (FoxOs) are expected to be potential cancer-related drug targets and thus are being extremely studied recently. In the present study, FoxO3a, one major member of this family, was identified to be down-regulated in colorectal cancer through micro-array analysis, which was confirmed by RT-PCR and Western blot in 28 patients. Moreover, immunohistochemistry (IHC) showed that the expression levels of FoxO3a were remarkably reduced in 99 cases of primary colorectal cancer, liver metastasis, and even in metaplastic colorectal tissue. IHC also revealed an exclusion of FoxO3a from the nucleus of most cells of tumor-associated tissues. Silencing FoxO3a by siRNA led to elevation of G2-M phase cells. We conclude that the downregulation of FoxO3a may greatly contribute to tumor development, and thus FoxO3a may represent a novel therapeutic target in colorectal cancer.
Cell Cycle Checkpoints ; Colon ; metabolism ; pathology ; Colorectal Neoplasms ; metabolism ; pathology ; Down-Regulation ; Female ; Forkhead Box Protein O3 ; Forkhead Transcription Factors ; metabolism ; Humans ; Liver Neoplasms ; metabolism ; pathology ; secondary ; Male ; Metaplasia ; metabolism ; pathology ; Rectum ; metabolism ; pathology ; Tumor Cells, Cultured

Morphine is a frequently used analgesic that activates the mu-opioid receptor(MOR),which has prominent side effects of tolerance.Although the inefficiency of morphine in inducing the endocytosis of MOR underlies the development of morphine tolerance,currently,there is no effective therapy to treat morphine tolerance.In the current study,we aimed to develop a monoclonal antibody(mAb)precisely targeting MOR and to determine its therapeutic efficacy on morphine tolerance and the underlying molecular mechanisms.We successfully prepared a mAb targeting MOR,named 3A5C7,by hybridoma technique using a strategy of deoxyribonucleic acid immunization combined with cell immunization,and identified it as an immunoglobulin G mAb with high specificity and affinity for MOR and binding ability to antigens with spatial conformation.Treatment of two cell lines,HEK293T and SH-SY5Y,with 3A5C7 enhanced morphine-induced MOR endocytosis via a G protein-coupled receptor kinase 2(GRK2)/β-arrestin2-dependent mechanism,as demonstrated by immunofluorescence staining,flow cytometry,Western blotting,coimmunoprecipitation,and small interfering ribonucleic acid(siRNA)-based knock-down.This mAb also allowed MOR recycling from cytoplasm to plasma membrane and attenuated morphine-induced phosphorylation of MOR.We established an in vitro morphine tolerance model using differentiated SH-SY5Y cells induced by retinoic acid.Western blot,enzyme-linked immunosorbent assays,and siRNA-based knockdown revealed that 3A5C7 mAb diminished hyperactivation of adenylate cyclase,the in vitro biomarker of morphine tolerance,via the GRK2/β-arrestin2 pathway.Furthermore,in vivo hotplate test demonstrated that chronic intrathecal administration of 3A5C7 significantly alle-viated morphine tolerance in mice,and withdrawal jumping test revealed that both chronic and acute 3A5C7 intrathecal administration attenuated morphine dependence.Finally,intrathecal electroporation of silencing short hairpin RNA illustrated that the in vivo anti-tolerance and anti-dependence efficacy of 3A5C7 was mediated by enhanced morphine-induced MOR endocytosis via GRK2/β-arrestin2 pathway.Collectively,our study provided a therapeutic mAb,3A5C7,targeting MOR to treat morphine tolerance,mediated by enhancing morphine-induced MOR endocytosis.The mAb 3A5C7 demonstrates promising translational value to treat clinical morphine tolerance.