Based on the different permeability of DNA-intercalant dyes YO-PRO-1(YP) and propidium iodide (PI) to the membrane of viable, apoptotic and necrotic cells, cell samples were stained with 4?mol/L YP and 4?g/ml PI for 10 min, and the fluorescence intensity of both YP and PI were measured by fluorometer at Ex/Em wavelength of 485/538nm and 530/590nm, respectively. The correlation between YP fluorescence intensity and the apoptotic cell number was confirmed by fluorescence microscope and linear regression(r=0.999,P

The synthetic gene with 1640bp encoding for the anti-CD3/anti-CD20 bispecific single-chain antibody was designed and obtained by SOE (splicing by overlap extension) PCR. The cDNA was cloned into Flp-In expression vector pcDNA5/FRT and transfeced into Flp-In CHO cells to generate a stable expression cell line with a capacity for expressing anti-CD3/anti-CD20 bispecific single-chain antibody at 300 microg/L. The protein, which had a molecular weight of about 70 kD,was purified by Ni-NTA affinity chromatography and identified by SDS-PAGE and Western-blot analysis. Immunofluorescence assay and cellular rosetting showed that it can react specifically on Jurkat (CD3+) and Ramous (CD20+) cells. The lysis of human PBL against CD20-positive lymphoma Ramous cells in the presence of the anti-CD3/anti-CD20 bispecific single-chain antibody can observed by microscope. All these results would lighten the further study of its biological functions in vitro and in vivo.
Animals ; Antibodies, Bispecific ; biosynthesis ; genetics ; immunology ; Antibodies, Monoclonal ; immunology ; Antigens, CD20 ; immunology ; CD3 Complex ; immunology ; CHO Cells ; Cricetinae ; Cricetulus ; Humans ; Immunoglobulin Variable Region ; biosynthesis ; genetics ; immunology ; Lymphoma, B-Cell ; metabolism ; Recombinant Proteins ; Transfection ; Tumor Cells, Cultured

By using the cell density, cell viability, size distribution of cell aggregates,specific consumption rate of glucose (q_ glc ), specific production rate of lactate (q_ lac ), lactate transform rate (Y_ lac/glc ) and amino acids utilization as the evaluation indexes, the growth and metabolism of HEK293 cells under carrier-free immobilization culture mode were examined and compared with those of HEK293 cells cultured in static tissue flasks. It was found that HEK293 cells grown as suspended cell aggregates in spinner flasks maintained the basic growth and metabolism characteristics of HEK293 cells in stationary anchored culture, and HEK293 cells under carrier-free immobilization culture mode as suspended aggregates in stirred bioreactor facilitate perfusion performance and increase unit productivity. Cultivation of HEK293 cells in carrier-free immobilization culture mode has potential for further improving mammalian cells culture technique.

BACKGROUND AND OBJECTIVERadiotherapy is effective in treating nasopharyngeal carcinoma (NPC). This study evaluated the treatment efficacy, toxicity, and prognostic factors of intensity-modulated radiotherapy (IMRT) in the treatment NPC.

METHODSBetween September 2003 and September 2006, 305 patients with NPC were treated with IMRT in Fujian Provincial Cancer Hospital. IMRT was delivered as follows: gross tumor volume (GTV) received 66.0-69.8 Gy in 30-33 fractions, high-risk clinical target volume (CTV-1) received 60.0-66.65 Gy, low-risk clinical target volume (CTV-2) and clinical target volume of cervical lymph node regions (CTV-N) received 54.0-55.8 Gy. Patients with stages III or IV disease also received cisplatin-based chemotherapy. All patients were assessed for local-regional control, survival, and toxicity.

RESULTSWith a median follow-up of 35 months (range, 5-61 months), there were 16, 8, and 39 patients who had developed local, regional, and distant recurrence, respectively. The 3-year rates of local control, regional control, metastasis-free survival, disease-free survival, and overall survival were 94.3%, 97.7%, 86.1%, 80.3%, and 89.1%, respectively. Multivariate analyses revealed that T-classification had no predictive value for local control and survival, whereas N-classification was a significant prognostic factor for overall survival (P < 0.001), metastasis-free survival (P < 0.001), and disease-free survival (P = 0.003). For stages III-IV disease, concurrent and adjuvant chemotherapy did not influence prognosis. The most severe acute toxicities included Grade III mucositis in 14 patients (4.6%), Grade III skin desquamation in 90 (29.5%), and Grades III-IV leucocytopenia in 20 (6.5%). There were 7% patients with Grade II xerostomia after 2 years of IMRT, no Grades 3 or 4 xerostomia was detected.

CONCLUSIONSIMRT provided favorable locoregional control and survival rates for patients with NPC, even in those with locally advanced disease. The acute and late toxicities were acceptable. N-classification was the main factor of prognosis. Further study is needed on chemotherapy for patients with NPC.

Adolescent ; Adult ; Aged ; Aged, 80 and over ; Antineoplastic Combined Chemotherapy Protocols ; therapeutic use ; Child ; Cisplatin ; administration & dosage ; Combined Modality Therapy ; Disease-Free Survival ; Female ; Follow-Up Studies ; Humans ; Leukopenia ; etiology ; Lymphatic Metastasis ; Male ; Middle Aged ; Mucositis ; etiology ; Nasopharyngeal Neoplasms ; drug therapy ; pathology ; radiotherapy ; Neoplasm Recurrence, Local ; Neoplasm Staging ; Radiotherapy Dosage ; Radiotherapy, Intensity-Modulated ; adverse effects ; methods ; Retrospective Studies ; Survival Rate ; Xerostomia ; etiology ; Young Adult

By using the size distribution of cell aggregates, viable cell density, cell viability, specific consumption rate of glucose (q(glc)), specific production rate of lactate (q(lac)) and lactate transform rate (Y(lac/glc)) as the evaluation indexes, the effects of hydrodynamic on aggregates formation, growth and metabolism of HEK293 cells in suspension culture were examined in 250mL spinner-flasks by setting the agitation rates at 25, 50, 75 and 100r/min, respectively. It was found that agitation plays an important role in HEK293 cell aggregates formation and cell aggregates size distribution. After 7d cultivation in spinner-flasks operated at 50r/min and 75r/min, the average diameter of HEK293 cell aggregates was 201 microm and 175 microm, respectively, with the fraction of aggregates larger than 225 microm less than 10%. The cell viability was kept above 90% with the metabolic indexes, including q(glc), q(lac) and Y(lac/glc) kept constant. These results demonstrated that hydrodynamic derived from the proper agitation play a decisive role in controlling the formation and size distribution of HEK293 cell aggregates, and provided sufficient mass transfer to support the normal growth and metabolism of HEK293 cells in suspended aggregates.
Bioreactors ; Cell Aggregation ; Cell Culture Techniques ; instrumentation ; Cell Line ; Cell Proliferation ; Humans ; Kinetics

After having successfully constructed and expressed the gene of the anti-CD3/anti-CD20 bispecific single-chain antibody (bscCD3 x CD20), here we analyzed its in vitro bioactivity of mediating the lysis of Ramous human B-lymphoma cells in the presence of T-enriched human peripheral blood lymphocytes (PBL). Obvious opoptosis characters were observed by Annexin V/PI(AV/PI) stained and scanning electron microscope. As evaluated by non-radioactive cytotoxity assay, the bscCD3 x CD20 showed potent bioactivity of mediating human B-lymphoma cells lysis in the presence of T-enriched human PBL. The potency of cytotoxicity depended on the ratios of effect cells to target cells (E:T) used. Further, the antibody showed a dose and time-dependent effect on mediating Ramous cells lysis. The specific lysis reached about 87.3% at an antibody concentration of 5microg/mL and E:T used at 10:1. Clear changes in apoptogenes expression profiles were detected by apoptosis gene array after Ramous cells were treated with the antibody and PBL. Among the upregulated apoptogenes, ATM and P53 showed an increase of 187 times and 15 times respectively, which suggested that ATM-p53 pathway may be the main apoptosis way of Ramous cells induced by T cells in the presence of the bscCD3 x CD20.
Antibodies, Bispecific ; immunology ; Antigens, CD20 ; immunology ; Apoptosis ; immunology ; CD3 Complex ; immunology ; Humans ; Lymphoma, B-Cell ; immunology ; pathology ; T-Lymphocytes, Cytotoxic ; immunology ; Tumor Cells, Cultured

Using the amino acids 1-147 of the yeast transcriptional activator GAL4 as the DNA-binding domain and four tandem repeats of the 12-aa peptide (DALDDFDLDMLG) of the herpesvirus as the activation domain, an artificial transcription factor, GVP4,was constructed via the linkage of the nuclear localization signal sequence of SV40. And then, GVP4 was cloned into expression vector pcDNA3 . 1/Hygro ( + ) . Various amounts of targeting sites of artificial transcription factor were linked to the upstream of promoter CMV in exogenous gene expression vector pcDNA3.1 ( + ) that separately harbored EGFP cDNA and t-PA cDNA.The CHO cells were then co-transfected with GVP4 expression vector and EGFP or t-PA expression vector. The effect of GVP4 on exogenous gene expression was evaluated by measuring the fluorescence intensity of EGFP in CHO cells and the concentration of t-PA in the supernatant. GVP4 showed positive effect on the enhancement of exogenous gene expression in CHO cells integrated with targeting sites of artificial transcription factor. And, CHO cells integrated with 10 targeting sites of GVP4 was more favorable to foreign gene expression, which resulted in 2-3-fold increase in both EGFP and t-PA expressions. These results indicated that artificial transcription factor is potent in the enhancement of exogenous gene expression in mammalian cells.
Amino Acid Sequence ; Animals ; CHO Cells ; Cricetinae ; Cricetulus ; Flow Cytometry ; Gene Expression Regulation ; Genetic Vectors ; genetics ; Green Fluorescent Proteins ; genetics ; metabolism ; Tissue Plasminogen Activator ; genetics ; metabolism ; Transcription Factors ; genetics ; metabolism ; Transcriptional Activation ; Transfection

This study was purposed to investigate the effects of N-Arachidonoylethanolamine (ANA) on the quality of platelets (Plt) stored in Plt M-sol preservative solution at 22 ± 2°C. Samples taken from collecting apheresis Plt by the Amicus instrument and splited into two equal parts were stored in Plt M-sol preservative solution on a shaker at 22 ± 2°C. Different working concentrations of ANA (from 0.1 to 50 µmol/L) were then added into one part of stored Plt as the experimental group, the other without ANA was used as the control group. The viability of Plts stored at 22 ± 2°C for 7 days was evaluated by MTT colorimetric assay. The most effective concentration of ANA was selected and added to the subsequent experimental group. Plt count (BPC), mean Plt volume (MPV), Plt distribution width (PDW), phosphatidyl serine (PS) and soluble P-selectin were detected on the 1(st), 5(th), 7(th), 9(th) and 11(th) day of storage. The results showed that the most effective working concentration of ANA was 0.5 µmol/L, which showed significant increasing Plt viability (91.23 ± 5.44%) compared to the control group (62.54 ± 4.79%). Thus, ANA concentration at 0.5 µmol/L was choose to perform subsequent experiments. During 11 days of storage, the BPC, MPV and PDW were not changed significantly between the experimental group and control group, although there was decreasing trend in the BPC and increasing trends in MPV and PDW in the two groups. The rate of Plt PS positive was enhanced during the storage period: the rate of PS positive in experimental group increased from 7.69 ± 1.82% to 10.74 ± 1.78% while it in control group increased from 11.21 ± 2.03% to 15.37 ± 1.95%, with significant differences between the two groups (P < 0.05) on the 9(th) and 11(th) day of storage, respectively. Soluble P-selectin contents in experimental group on the 9(th) and 11(th) day of storage were 30.19 ± 2.03 ng/ml and 34.52 ± 2.64 ng/mL, respectively, while those in control group were 39.18 ± 2.66 ng/ml and 43.23 ± 2.58 ng/ml, respectively, with significant differences between the two groups (P < 0.05). It is concluded that the extended storage of Plt in M-sol treated with low concentration ANA can potentially alleviate Plt storage lesions.
Adult ; Blood Platelets ; drug effects ; Blood Preservation ; Endocannabinoids ; pharmacology ; Female ; Humans ; Male

OBJECTIVETo study the relationship between the levels of erythropoietin (EPO) in serum and brain injury in preterm infants.

METHODSThree hundred and four preterm infants (gestational age: 28-34 weeks) born between October 2014 and September 2015 were enrolled in this study. Brain injury was diagnosed using cerebral ultrasound and MRI. The levels of EPO, S100 protein, neuron-specific enolase (NSE) and myelin basic protein (MBP) in serum were detected using ELISA. To compare the incidence of brain injury in different serum EPO levels in preterm infants, and the relationship between brain injury and serum EPO levels was analyzed.

RESULTSThe incidence rate of brain injury in preterm infants was 41.1% (125/304). The incidence rate of brain injury in the low EPO level group was significantly higher than that in the middle-high EPO level groups (P<0.01). The serum levels of S100 protein, NSE, and MBP in the brain injury groups were significantly higher than in the control group (P<0.01). The serum EPO levels were negatively correlated with serum S100 protein concentration and NSE levels (P<0.05). According to the multiple logistic regression analysis, low gestational age, low birth weight, asphyxia, prolonged mechanical ventilation, anemia and low serum EPO levels were the risk factor for brain injury in preterm infants.

CONCLUSIONSThere is a higher incidence rate of brain injury in preterm infants with lower serum EPO levels. The serum EPO levels may be correlated with brain injury in preterm infants.

Brain Injuries ; blood ; epidemiology ; Erythropoietin ; blood ; Female ; Humans ; Infant, Newborn ; Infant, Premature ; blood ; Male ; Myelin Basic Protein ; blood

OBJECTIVETo evaluate the efficacy and safty of the humanized anti-epidermal factor receptor monoclonal antibody h-R3 in combination with radiotherapy for locoregionally advanced nasopharyngeal carcinoma.

METHODSTotally, 137 patients from 7 medical center around China were randomly divided into combined therapy group or control group. There was no difference in Karnofsky performance score between two groups. All patients in both groups received radical conventionally fractionated radiotherapy to the total dose of D(T) 70-76 Gy. For the combined therapy group, h-R3 was added at a dose of 100 mg i.v. weekly for 8 weeks started at the beginning of radiotherapy.

RESULTSOf the 137 eligilbe patients, 70 were in the combined therapy group treated by h-R3 plus radiotherapy and 67 in the control group by radiotherapy alone. The intent-to-treat (ITT) population consisted of 130 patients, while the per-protocol (PP) population was composed of 126 patients. The efficacy was assessed respectively at three point of time: the end of treatment, the 5th- and 17th-week after treatment. The complete response (CR) of the combined therapy group was significantly higher than that of the control group in both ITT and PP (ITT: 65.63%, 87.50%, 90.63% versus 27.27%, 42.42%, 51.52%; PP: 67.21%, 90.16%, 93.44% versus 27.69%, 43.08%, 52.31%; P < 0.05, respectively). The most common h-R3-related adverse reactions were fever (4.3%), hypotension (2.9%), nausea (1.4%), dizziness (2.9%) and rash (1.4%), which could be reversible if treated properly. Radiotherapy combined with 100 mg h-R3 i. v. weekly was tolerable and did not aggravate the side effects of radiation. The quality of life in the combined therapy group was comparable to that in the control group.

CONCLUSIONThis phase 1 multicenter clinical trial shows that h-R3 in combination with radiotherapy is effective and well-tolerated for the treatment of locoregionally advanced nasopharyngeal carcinoma.

Adult ; Aged ; Antibodies, Monoclonal ; adverse effects ; therapeutic use ; Carcinoma, Squamous Cell ; pathology ; therapy ; Combined Modality Therapy ; Female ; Fever ; etiology ; Humans ; Hypotension ; etiology ; Male ; Middle Aged ; Nasopharyngeal Neoplasms ; pathology ; therapy ; Neoplasm Staging ; Quality of Life ; Radiotherapy ; adverse effects ; methods ; Receptor, Epidermal Growth Factor ; immunology ; Remission Induction