Bilirubin ; blood ; Child Development ; Female ; Humans ; Infant ; Infant, Newborn ; Male ; Neuropsychological Tests

Objective To explore the change of nerve growth factors(NGF) through the blood brain barrier(BBB) after distal intravenous injection of mannitol into the experimental rats and the effect of exogenous NGF on the expression of growth associated protein-43 in hypoxic-ischemic brain.Methods One hundred cases of 7 days rats were divided into 2 units.One unit was divided into 3 groups:treatment group,control group and sham operated group,20 rats in each group.The other unit was divided into 4 groups:mannitol and NGF treated group,NGF treated group,control group,and sham operated group,there were 10 rats in each group.The model rats with perinatal hypoxic-ischemic brain damage(HIBD) rats were prepared by ligation of left common carotid artery with a temporary systemic hypoxia(inhaling 80 mL/L O2 and 920 mL/L N2).The sections of brains were processed by immunochemistry with antibodies against GAP-43,and the study and memory ability of rats were tested by maze test.The effect of osmotic opening of BBB on the facilitation of NGF′s passage was tested by ELISA.Results The expression of GAP-43 increased after NGF treatment,and the differences were remarkable(P

OBJECTIVETo study the role of N-methyl-D-aspartate-receptor (NMDAR) expression in the development of hearing damage in neonatal rats with hyperbilirubinemia.

METHODSSixty seven-day-old Sprague-Dawley rats were randomly injected with bilirubin of 100 microg/g (low-dose treatment group) or 200 microg/g (high-dose treatment group) or normal saline (control group). Auditory brainstem response (ABR) was examined. The concentrations of bilirubin in blood and brain were measured. NMDAR expression in the cochlear nucleus slices was examined by immunohistochemistry assay.

RESULTSABR reflecting threshold obviously increased, and I, II and III wave latency as well as I-II, II-III and I-III interval were more prolonged in the two bilirubin treatment groups when compared with the control group. The NMDAR expression in the cochlear nucleuse in the two bilirubin treatment groups was obviously lower than that in the control group. The NMDAR expression in the cochlear nucleuse was negatively correlated with the brain bilirubin content and the ABR reflecting threshold in the two bilirubin treatment groups.

CONCLUSIONSAn increased NMDAR activity may play an important role in hearing damage following hyperbilirubinemia.

Animals ; Animals, Newborn ; Bilirubin ; analysis ; Cochlear Nucleus ; chemistry ; Evoked Potentials, Auditory, Brain Stem ; Female ; Hearing Disorders ; etiology ; Hyperbilirubinemia ; complications ; metabolism ; Immunohistochemistry ; Male ; Rats ; Rats, Sprague-Dawley ; Receptors, N-Methyl-D-Aspartate ; analysis

OBJECTIVETo construct a three plasmids lentiviral vector containing canine coagulation factor IX (cFIX) gene with ubiquinone promoter (PUB) and observe the expression of cFIX gene.

METHODSLentivirus was generated by transient three-plasmid transfection, namely, the VSV-G envelope expression cassette, the delta NRF packaging plasmid and the PTK 164 plasmid. Viral particles were used to infect the target cell, third passage mesenchymal stem cells (MSCs) and 293T cell respectively at MOI 3: 1. The cFIX activity was detected in cultured cells with one-stage clotting assay.

RESULTSThe MSCs were obtained in vitro. The lentivirus infected MSCs and 293T cells all expressed the active factor IX with the activity of (26.30 +/- 2.10)% and (19.70 +/- 1.53)%, respectively, which are significantly higher than that of control (1.00 +/- 0.05)%.

CONCLUSIONSThe lentiviral vector of three plasmids with ubiquinone promoter (PUB) was constructed and can transfect the MSCs and 293T cells.

Animals ; Bone Marrow Cells ; metabolism ; Cells, Cultured ; Dogs ; Factor IX ; genetics ; metabolism ; Genetic Vectors ; Hemophilia B ; genetics ; metabolism ; Humans ; Lentivirus ; genetics ; Plasmids ; genetics ; Transfection

OBJECTIVETo establish strategies for preventing graft versus host disease (GVHD) and reducing treatment associated morbidity while preserving graft versus leukemia (GVL) effect in nonmyeloablative allogeneic bone marrow transplantation (allo-BMT), with or without donor lymphocyte infusion (DLI) after BMT.

METHODS3 x 10(7) bone marrow cells mixed with 1 x 10(7) spleen cells from the same BALB/c mouse were transplanted into the nonablative irradiated inbred 615 mouse which received a single subcutaneous injection of 1 x 10(6) L615 leukemia cells three days before. The experiments were designed as follows (ten mice in each group): myeloablative BMT control group (group A), nonmyeloablative conditioning without BMT group (group B), nonmyeloablative BMT group (group C), and nonmyeloablative BMT + DLI group (group D). GVL effects were assessed by survival time, white blood cell count and L615 cells in peripheral blood and histologic changes. GVHD was assessed by signs of weight loss, ruffled fur, diarrhea and histologic changes of skin, liver and small intestines. Chimerism was detected by cytogenetic analysis and PCR technique.

RESULTSThe survival time of group A, B, C and D was (20.3 +/- 13.4), (15.9 +/- 1.1), (21.6 +/- 1.7) and (37.8 +/- 2.0) days, respectively, being no significant difference between group A and group C (P > 0.05). The survival time of group C was longer than that of group B (P < 0.01). And among group B, C and D, group D had the longest survival time (P < 0.01). GVHD signs and histologic changes were observed in 60% of control group mice at + 14 day, but none of group C and group D. 40% of mice in group A died of treatment associated morbidity within two weeks, but none in group C and group D. Allogeneic chimerism was kept in group A, but excluded gradually in group C.

CONCLUSIONGVL effect seems preserved in nonmyeloablative BMT mice, but weaker than that in myeloablative BMT mice. GVL effect seems to be enhanced by DLI after nonmyeloablative BMT. GVHD and transplantation associated morbidity seems to be reduced in nonmyeloablative BMT.

Animals ; Bone Marrow Transplantation ; immunology ; methods ; Combined Modality Therapy ; Female ; Graft vs Host Disease ; prevention & control ; Graft vs Leukemia Effect ; Leukemia, Experimental ; therapy ; Leukemia, Lymphoid ; therapy ; Lymphocyte Transfusion ; Male ; Mice ; Mice, Inbred C57BL ; Mice, Inbred Strains ; Transplantation Conditioning ; methods ; Transplantation, Heterologous

The study was purposed to prepare the recombinant lentiviral vector pTK161 and pTK162 carrying B-domain-deleted canine factor (BDDcFVIII) gene, and to investigate whether the canine FVIII (cVIII) can be expressed in vitro. The BDDcFVIII gene was ligated behind PUB and 2OH1 promotors to create lentiviral vectors pTK161 and pTK162. Meantime lentiviral vectors pTK161' and pTK161' were produced by cloning a green fluorescent protein (GFP) into pTK151 and pTK152, which was driven by PUB and 2OH1 promotors respectively. Vector supernatant were prepared by using transfer calcium phosphate mediated-cotransfection of 293T cells. The virus vector, DeltaNRF packaging-plasmid, and VSV-G envelope-plasmid was assayed by titers and cFVIII activity in cell culture supernatant after infection into 293T cells. pTK161, pTK162, pTK161' and pTK161' were identified by restriction enzyme analyzing. The results showed that the lentiviral vectors pTK161, pTK162, pTK161' and pTK161' were successfully constructed, and the titers of pTK161' and pTK161' reached to 1.54 x 10(6) U/ml and 2.83 x 10(6) U/ml; the activity of cFVIII could be detected at 24 hours after infection of 293T cells by pTK161 and pTK162, and achieved the highest level at 72 hours later. The higher level of cFVIII activity was achieved by transfected with pTK162 than that of pTK161 (p < 0.05), which closed to the cFVIII activity in normal dog plasma. 1/4 of the highest level could be detected 6 weeks later. It is concluded that the prepared HIV1-based lentiviral vectors can infect 293T cells to express cFVIII effectively. The results provide the basis for further studying HIV-1-based lentiviral vector gene therapy for hemophilia A.
Animals ; Dogs ; Factor VIII ; biosynthesis ; genetics ; Genetic Vectors ; genetics ; HIV-1 ; genetics ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics

The present study was purposed to investigate the relation and difference of expression phase between class II transactivator (CIITA) and HLA-DR antigens after IFN-gamma induction, and the inhibition of CIITA and HLA-DR by STAT1-alpha antisense oligonucleotides (STAT1-alpha AS); and to explore the potential effect and significance of CIITA and STAT1-alpha AS in transplantation immunity. T lymphocytes from peripheral blood of healthy subjects were incubated with IFN-gamma at different doses. RT-PCR was used to detect CIITA mRNA and Western blot was used to analyze HLA-DR antigen. Then the optimum dose of IFN-gamma was chosen for the experiment. CIITA mRNA and HLA-DR antigen were detected at various time points. Different doses of STAT1-alpha AS and sense oligonucleotides (STAT1-alpha S) were added to T lymphocytes followed by IFN-gamma. After incubation with IFN-gamma, the expression of CIITA mRNA and HLA-DR was detected once again. The results showed that CIITA mRNA was detectable at 5 hours after IFN-gamma incubation and reached the peak at 14 hours, then declined, but the CIITA mRNA was still found at 23 hours. HLA-DR antigen was detectable at 28 hours after IFN-gamma incubation and reached a peak at 52 hours, then declined. CIITA mRNA expression was positively correlated to HLA-DR expression, and was earlier than the latter. The expression of CIITA mRNA in the AS groups was significantly lower than that in the control group after 5 micromol/L, 10 micromol/L and 20 micromol/L STAT1-alpha AS treatment (P < 0.01). The expression of CIITA mRNA in the S groups was higher than that in the AS groups (P < 0.01), but there was no significant difference between the S group and the control group. The expression of HLA-DR antigen was significantly inhibited by STAT1-alpha AS, and the expression level of HLA-DR protein in the AS group was about 64.3% of that in the control group (P < 0.01), while there was no significant difference in HLA-DR expression between the S group and the control group. The changes in HLA-DR expression were similar to those in CIITA expression after STAT1-alpha AS treatment. It is concluded that CIITA expression is positively correlated with HLA-DR expression, and was detectable earlier than that of latter after IFN-gamma incubation. Stat1-alpha antisense oligonucleotides may have a sequence-specific inhibiting effect on the expression of CIITA and HLA-DR antigen after IFN-gamma incubation in vitro culture, and can prevent T lymphocyte activation. CIITA may play an important role in pathogenesis of transplantation immunity.
Cells, Cultured ; HLA-DR Antigens ; biosynthesis ; genetics ; Humans ; Interferon-gamma ; pharmacology ; Nuclear Proteins ; biosynthesis ; genetics ; Oligonucleotides, Antisense ; antagonists & inhibitors ; RNA, Messenger ; biosynthesis ; genetics ; STAT1 Transcription Factor ; antagonists & inhibitors ; T-Lymphocytes ; cytology ; Trans-Activators ; biosynthesis ; genetics ; Transplantation Immunology ; immunology

This study was aimed to construct a lentiviral vector carrying human coagulant factor VIII (FVIII) and to investigate its expression in 293T cells. B-domain-deleted factor VIII gene fragment (BDDhFVIIIcDNA) was obtained by enzyme digestion and cloned into lentiviral vector pXZ208 to establish the expression vector pXZ208-BDDhFVIII. Recombinant viral particles were prepared by cotransfection with packaging plasmid delta NRF and envelope plasmid VSV-G using calcium phosphate precipitation method. 293T cells were transfected by viral supernatant. Coagulant activity of FVIII, BDDhFVIIImRNA and genome integration were assayed by one-step method, RT-PCR and PCR after transfection. The results showed that 293T cells could be transfected by recombinant virus. The transfection rate of 293T was 59.57%. After transfection, the cells expressed FVIII efficiently. Detection confirmed that the activity of FVIII was 12%, 43% and 87% respectively at 24, 48 and 72 hours after infection. BDDhFVIII transcription was detected by RT-PCR from the infected cells. The gene integration in the targeted cells was also observed. It is concluded that the successfully constructed lentiviral vector is able to generate high level expression of human FVIII in 293T cells, which may provide a potential application of gene therapy to haemophilia A.
Cell Line ; Factor VIII ; genetics ; metabolism ; Gene Expression ; Genetic Vectors ; Humans ; Lentivirus ; genetics ; metabolism ; Recombinant Proteins ; genetics ; metabolism

OBJECTIVETo study the urodynamic changes in patients with recent non-infective voiding dysfunction following radical hysterectomy and assess its significance.

METHODSNinety-six patients with cervical cancer, who were not found any abnormal representation of urodynamics before the operation, were selected into this study group. Eighty-three patients in the study group without urinary infection were detected by urodynamic examination following radical hysterectomy, in order to analyze the urodynamic reasons for the non-infective voiding dysfunction following the surgery.

RESULTSForty-two patients were found with non-infective voiding dysfunction after the operation. Low compliance bladder, bladder destrusor dysfunction and destrusor overactivity were the three leading types of postoperative bladder dysfunction. Moreover, the incidences of low compliance bladder (50.0% vs. 17.1%), bladder destrusor dysfunction (58.4% vs. 14.6%) and destrusor overactivity (31.0% vs. 4.9%) in the group with voiding dysfunction were significantly higher than the corresponding values in the group without voiding dysfunction (P < 0.01). Secondarily, forty-two patients with recent non-infective voiding dysfunction were divided into simple irritation sign group, simple obstruction sign group and mixed sign group according to their main symptoms. The incidence of bladder destrusor dysfunction in the simple obstruction sign group was significant higher than that in the simple irritation sign group, and the incidence of detrusor overactivity in the simple irritation sign group was significant higher than that in the other two groups (P < 0.05).

CONCLUSIONSThere were many different types of urodynamic disorder in the patients with recent non-infective voiding dysfunction after radical hysterectomy. Low compliance bladder, bladder destrusor dysfunction and detrusor overactivity caused by the damage of the pelvic autonomic nerve during the operation may be the main reasons for the recent non-infective voiding dusfunction after radical hysterectomy. Moreover, bladder destrusor dysfunction and detrusor overactivity may be the key points for the symptoms of bladder irritation and bladder obstruction. Urodynamic study is important for the etiology analysis and clinical treatment of recent non-infective voiding dysfunction postoperation.

Adult ; Female ; Humans ; Hysterectomy ; adverse effects ; methods ; Middle Aged ; Urinary Bladder ; physiopathology ; Urinary Bladder, Overactive ; etiology ; physiopathology ; Urination Disorders ; etiology ; physiopathology ; Urodynamics ; Uterine Cervical Neoplasms ; physiopathology ; surgery

OBJECTIVETo explore the impact of patient compliance on the long-term outcomes in hypertensive patients receiving hydrochlorothiazide (HCTZ) based combination therapy with spironolactone or captopril.

METHODSA total of 853 patients with mild to moderate hypertension were recruited and randomly divided into HCTZ group (HCTZ 12.5 mg q.d), spironolactone group (HCTZ 12.5 mg q.d and spironolactone 20 mg q.d), and captopril group (HCTZ 12.5 mg q.d and captopril 25 mg bid) after 2-week placebo washout period and 6-week loading period for HCTZ. Since the efficacy of combination therapy was proven to be better than monotherapy 1 year after therapy beginning, patients in HCTZ group were randomly assigned to spironolactone group or captopril group. The patients were followed up for 4 years. Patients were divided to compliance (n = 424) or non-compliance group (n = 429) according test drug taking questionnaire. During the follow-up time, the blood pressure and the outcomes were recorded monthly, and blood biochemical parameters were determined once a year.

RESULTSAt the end of follow up, incidence of cardio-cerebral vascular events was significantly lower in compliance group (2 fatal, 8 non-fatal) than that in noncompliance group (7 fatal, 21 non-fatal, P < 0.05). Systolic blood pressure [-(19.4 +/- 20.6) mm Hg, 1 mm Hg = 0.133 kPa] and diastolic blood pressure [-(10.7 +/- 13.5) mm Hg] were significantly reduced compared values at baseline and noncompliance group (all P < 0.001) while the reduction did not reach statistically significance in noncompliance group [-(7.3 +/- 18.2) mm Hg and -(3.5 +/- 10.2) mm Hg, all P > 0.05 vs. baseline]. The serum BUN, Cr and UA levels in the compliance group were significantly higher and the serum K(+), CHO, LDL-C level were significantly lower than baseline values. The serum BUN, UA levels in the compliance group were significantly higher while the serum K(+), cholesterol levels were significantly lower than those in the noncompliance group (all P < 0.05).

CONCLUSIONSThis study indicates that patient compliance could affect the long-term outcome and antihypertensive efficacy in hypertensive patients receiving HCTZ based combination therapy with spironolactone or captopril.

Aged ; Antihypertensive Agents ; therapeutic use ; Captopril ; therapeutic use ; Drug Therapy, Combination ; Female ; Follow-Up Studies ; Humans ; Hydrochlorothiazide ; therapeutic use ; Hypertension ; drug therapy ; Male ; Middle Aged ; Patient Compliance ; Spironolactone ; therapeutic use ; Treatment Outcome