Aim To ascertain whether defibrase has the protective effect against reperfusion injury after cerebral ischemia.Methods 70 renovascular hypertensive rats(RHR) were randomly divided into defibrase group, control group and sham-operated group.Reversible middle cerebral artery occlusion(MCAO) models were produced by the modified. Longa's method,and reperfusion was begun 2 hours after occlusion.Rats in the defibrase group were given defibrase 10 U?kg-1 body weight via femonal intraveneous injection, and in the control group with the same amount of saline. The brain pieces were processed by TTC and HE staining and the infarct size,brain microvessels damage and secondary bleeding were compared between the two groups. Results The volume of infarction in the defibrase group was obviously smaller than in the control group, the damage of brain microvessels was less severe, and the bleeding lesions under optical microscope were less than in the control group. Conclusion Defibrase has protective effect against reperfusion injury post cerebral ischemia.

OBJECTIVETo observe the correlation between constitution of yin deficiency syndrome (YDS) and polymorphism of HLA-DQA1/treatment response of Peg-lFNalpha therapy in HBeAg positive chronic hepatitis B (CHB) patients, and to explore constitution of Chinese medicine (CM) in response of interferon therapy.

METHODSTotally 120 HBeAg positive CHB patients who were treated with Peg-IFNalpha were enrolled, and assigned to YDS group (59 cases) and non-YDS group (61 cases) according to classification of CM constitutions. All patients were subcutaneously injected with Peg-IFNalpha-2b (1.0 microg/kg body weight) or Peg-IFNalpha-2a (180 microg), once per week. Effective efficacy was primarily judged when complete response (CR) or partial response (PR) was obtained at month 6. Those with CR or PR completed 1 year therapeutic course. HLA-DQA1 gene types were detected by polymerase chain reaction sequence specific primers (PCR-SSP). The distribution difference of CM constitutions in patients with CR or PR and their inter-group HLA-DQA1 allele frequency were compared.

RESULTSDifferent treatment responses of Peg-IFNalpha were observed in CHB patients of two different CM constitutions. The ratio of CR + PR was 61.0% (36/59) in YDS group, obviously lower than that in NYDS group [78.7% (48/61), P < 0. 05]. Patients with CR had a lower allele frequency of HLA-DQA1 * 0501 than those with no-response [14.8% (8/54) vs. 30.6% (22/72)] with statistical difference (P < 0.05). Patients with CR had a higher allele frequency of HLA-DQA1 * 0601 than those with no-response [18.5% (10/54) vs. 5.6% (4/72)] with statistical difference (P < 0.05). The allele frequency of HLA-DQA1 * 0301 was lower in YDS group than in non-YDS group [2. 5% (3/118) vs. 9.8% (12/122)] with statistical difference (P < 0.05). The allele frequency of HLA-DQA1 * 0501 was higher in YDS group than in non-YDS group [33.9% (40/118) vs. 18.9% (23/122)] with statistical difference (P < 0.05). Yet statistical significance was lost after adjustment (Pc > 0.05 for both).

CONCLUSIONSBoth constitutions of CM and HLA-DQA1 gene polymorphism af- fect HBeAg positive CHB patients' response to Peg-INFalpha. Constitutions of YDS and HLA-DQA1 * 0501 was not favorable to response, their association needed to be further studied.

Antiviral Agents ; therapeutic use ; Gene Frequency ; HLA-DQ alpha-Chains ; genetics ; Hepatitis B e Antigens ; blood ; Hepatitis B, Chronic ; drug therapy ; genetics ; Humans ; Interferon-alpha ; therapeutic use ; Medicine, Chinese Traditional ; Polyethylene Glycols ; therapeutic use ; Polymorphism, Genetic ; Recombinant Proteins ; therapeutic use ; Remission Induction ; Yin Deficiency ; genetics

To compare the DAAO expression level in different Pichia pastoris host strains, the gene encoding DAAO from Trigonopsis variabilis was cloned into plasmid pPIC3.5k and then transformed into P. pastoris GS115 and KM71 respectively. The positive transformants PDK13 (MutS) and PD27 (Mut+) were obtained by PCR analysis. Their optimal and different expression conditions were investigated. To compare with PD27, PDK13 was determined to poss a slower consumption of methanol, a longer induction time, a lower oxygen request and apparently higher expression of DAAO. The highest expression levels were reached up to 2700, 2500 IU/L in shaking flask and 10140, 8463.5 IU/L in fermentor respectively. The over-expression of DAAO can meet its large demand for production of 7-ACA, alpha-keto acid and L-amino acid. In addition, the phenylpyruvate and L-phenylalanine were obtained by crude DAAO reacting with DL-phenylalanine at 37 degrees C for 3h.
D-Amino-Acid Oxidase ; genetics ; Fermentation ; Methanol ; metabolism ; Phenylalanine ; metabolism ; Pichia ; genetics ; Polymerase Chain Reaction

Objective To analyze the clinicopathological features and prognostic factors of patients with breast invasive ductal carcinoma with extensive intraductal components (EIC).Methods The clinical data of 182 patients of early breast invasive ductal carcinoma undergoing breast-conserving surgery were analyzed retrospectively in the Second Affiliated Hospital of Soochow University,and the clinicopathological features and prognosis of two groups of breast cancer patients with EIC positive (n =23) and EIC negative (n =159) were compared.Results In the EIC positive group,ipsilateral breast tumor recurrence (IBTR) was 21.7％ (5/23) and the distant metastasis rate was 4.3％ (1/23);in the EIC negative group,ipsilateral breast tumor recurrence (IBTR) was 1.9％ (3/159) and the distant metastasis rate was 3.8％ (6/159).The Kaplan-Meier curve showed that the 5-year local recurrence-free survival rates in the EIC positive group was lower than that of the EIC negative group (78％ vs.97.7％) (P ＜ 0.001),and the difference was statistically significant,but the 5-year distant metastasis-free survival rates in the two groups was similar (95.7％ vs.96.2％) (P =0.892).COX multivariate analysis showed that EIC was an independent risk factor for local recurrence after breast-conserving surgery in breast invasive ductal carcinoma patients.Conclusions EIC increases the local recurrence rate in patients with breast invasive ductal carcinoma undergoing breast-conserving surgery.

BACKGROUNDOne of the major characteristics of the human immunodeficiency virus type 1 (HIV-1) is its unusually high degree of genetic variability, which involves in genetic diagnosis, subtyping, vaccine design, and epidemiology. HIV-1 CRF01_AE is a main prevalent HIV-1 recombinant strain in China. In this study, three full-length CRF01_AE genomes from Fujian Province, China were cloned, sequenced, and analyzed; and the further genetic diversity defining and epidemiologic analysis were carried out.

METHODSProviral DNA was extracted from non-cultured peripheral blood mononuclear cells, the near full-length HIV-1 genome was amplified and the PCR products were cloned into pCR-XL-TOPO vector and sequenced. 5'-long terminal repeat (LTR) and 3'-LTRs were amplified by additional independent PCR and cloned into pMD18T vector. Gene-based phylogenic tree was constructed and genetic distances were calculated by MEGA 3.1. Simplot was used for Bootscan analysis.

RESULTSThe phylogeny and genetic distance analysis of the three near full-length sequences confirmed that these three samples clustered with CRF01_AE isolates, more close to Thailand CRF01_AE strain CM240, and were distantly related to African CRF01_AE strain 90CF402. Analysis of their genomic organization revealed the presence of nine potential open reading frames. There were no major deletions, rearrangements, or insertions in the three sequences, but an in-frame stop codon was found in tat gene of Fj051. LTRs of the three sequences contained a few nucleotides mutation. We did not find new mosaic recombinant in the three sequences. The V3 motif was GPGQ in all the three sequences, and there were only few amino acids differences in all three V3 loop sequences.

CONCLUSIONThis report reveals the background of the three full-length CRF01_AE genomes, the most dominantly circulating HIV-1 strain in Fujian Province, China. The work is essential for the design and development of an effective AIDS vaccine for the region.

Adult ; Amino Acid Sequence ; Base Sequence ; DNA, Viral ; chemistry ; Female ; Genome, Viral ; HIV Long Terminal Repeat ; HIV-1 ; classification ; genetics ; Humans ; Male ; Molecular Sequence Data ; Phylogeny ; Recombination, Genetic

OBJECTIVETo assess the effects of different treatment complex on esophageal vascular structures in patients with portal hypertension.

METHODSPatients (142 cases) with esophageal varices received either endoscopic variceal ligation (EVL) alone (54 cases), pericardial devascularization procedure (PDP) alone (23 cases), a combination of EVL and partial splenic embolization (PSE) (34 cases), or a combination of EVL and PDP (31 cases) for variceal eradication. Esophageal vascular structures were examined with miniature ultrasonic probe. The recurrence and rebleeding of esophageal varices were investigated.

RESULTSEsophageal submucous varices were obliterated and collateral veins remained unchanged in patients treated by EVL or EVL combined with PSE; esophageal submucous varices were diminished in size and collateral veins were obliterated by PDP, and both esophageal submucous varices and collateral veins were obliterated by the combination of EVL and PDP.

CONCLUSIONSThe combination of EVL and Hassab's procedure can effectively shut off the portoazygous shunt, prevent esophageal varices from bleeding and recurrence. It's a simply and less cost procedure.

Cardia ; blood supply ; surgery ; Combined Modality Therapy ; Embolization, Therapeutic ; Endoscopy, Digestive System ; Esophageal and Gastric Varices ; diagnostic imaging ; etiology ; therapy ; Female ; Humans ; Hypertension, Portal ; complications ; Ligation ; methods ; Male ; Middle Aged ; Retrospective Studies ; Splenectomy ; Treatment Outcome ; Ultrasonography ; Vascular Surgical Procedures ; methods

This study was aimed to analyze the mRNA expression of cytokines (TGF-beta, IL-2, IL-6, IL-10, IFN-gamma, TNF-alpha, FAS-L) in five rhesus treated with haploidentical peripheral blood stem cell transplantation after nonmyeloablative preparative regimens and to explore the role of these cytokines in the development and pathology of acute graft-versus-host-disease (aGVHD). Five rhesus monkeys received nonmyeloablative haploidentical peripheral blood stem cells transplantation. Semi-quantitative reversed transcription polymerase chain reaction (RT-PCR) was used to analyze the kinetics of cytokine mRNA expression in the transplantation and aGVHD. The results showed that five rhesus monkeys acquired hematopoietic reconstitution successfully. The graft was rejected in one monkey which survived without disease, the other four achieved mixed chimerism and full donor chimerism. Chimerism of low centigrade in one monkey achieved high centigrade at 35 days after donor stem cell infusion. Intestinal aGVHD grade III developed in one monkey. Cytokines of Th1 and Th2 changed after transplantation. In period of aGVHD, expression of TGF-beta decreased but all others increased in various levels. When donor chimerism decreased, the cytokines decreased accordingly. It is concluded that the decrease of TGF-beta mRNA may be an indicator to predict aGVHD, and can be used as a differential diagnostic indicator for intestinal GVHD.
Animals ; Cytokines ; biosynthesis ; genetics ; Graft vs Host Disease ; diagnosis ; metabolism ; Haploidy ; Macaca mulatta ; Peripheral Blood Stem Cell Transplantation ; adverse effects ; RNA, Messenger ; biosynthesis ; genetics ; Transforming Growth Factor beta ; biosynthesis ; genetics

This study was aimed to detect the gene expression profile changes between human acute leukemia cell line HL-60 and VCR-resistance HL-60, and to investigate the underlying mechanisms of MDR by using genechip technology. In experiments, mRNA were harvested using TrizoL reagent from these two cell lines, through RT-PCR, the biotinylated nucleotide were incorporated into the cRNA during the in vitro transcription reaction. The high quality RNA was hybridized to the gene expression array--human genome U133A developed by Affymetrix. It was scanned by G2500A GeneArray Scanner and the acquired image was analysed by a series of softwares. The results showed that 5,507 genes were differentially expressed between human acute leukemia cell line HL-60 and VCR-resistant HL-60. Compared with HL-60, 3,100 genes were up-regulated and 2,407 genes were down-regulated in VCR-resistant cell line. These genes were involved in different cell activities such as growth regulation and signal transduction. Among the genes with remarkable differential expression between the two cell lines, 435 were up-regulated and 605 were down-regulated. It is concluded that many different kinds of genes are involved in the mechanism of MDR and there is an intricate molecular network that controls the sensitivity of leukemia cells to the chemotherapeutic agents. Genechip is an efficient tool for parallel gene expression analysis.
Drug Resistance, Neoplasm ; genetics ; Gene Expression Profiling ; Gene Expression Regulation, Neoplastic ; Genome, Human ; HL-60 Cells ; Humans ; Oligonucleotide Array Sequence Analysis ; Vincristine ; pharmacology

OBJECTIVETo establish rhesus haploidentical hematopoietic stem cell transplantation model by nonmyeloablative conditioning, and examine the effects of mesenchymal stem cells (MSC) in haploidentical transplantation.

METHODSThe recipient haploidentical rhesus monkeys were conditioned with a nonmyeloablative regimen consisted of fludarabine, cyclophosphamide, 200 cGy total body irradiation, and rabbit anti-human thymocyte globulin. Cyclosporine A, mycophenolate mofetil and anti CD25 antibody were used for graft versus host disease (GVHD) prophylaxis. Rhesus monkeys in one group were given hematopoietic stem cell (HSC) only, while in the other group HSC combined with MSC. The differences in hematopoiesis recovery, chimerism level, and GVHD between the two groups were evaluated.

RESULTSStable chimerism could be achieved in recipient monkeys. Hematopoiesis recovery was mainly related with chimerism level. MSC seemed capable of facilitating HSC engraftment, as there were more mixed chimerism and less GVHD occurrence in the HSC combined with MSC recipient group.

CONCLUSIONA rhesus haploidentical hematopoietic stem cell transplantation model is successfully established by nonmyeloablative conditioning. MSC was of great benefit to haploidentical transplantation.

Animals ; Chimerism ; Graft vs Host Disease ; prevention & control ; Haploidy ; Hematopoietic Stem Cell Transplantation ; Macaca mulatta ; genetics ; surgery ; Mesenchymal Stem Cell Transplantation ; Models, Animal ; Transplantation Conditioning

BACKGROUNDThere are no reports on exnografting cultured human fetal neocortical cells in this infracted cavities of adult rat brains. This study was undertaken to observe whether cultured human cortical neurons and astrocytes can survive and grow in the infarcted cavities of adult rat brains and whether they interconnect with host brains.

METHODSThe right middle cerebral artery was ligated distal to the striatal branches in 16 adult stroke-prone renovascular hypertensive rats. One week later, cultured cells from human embryonic cerebral cortexes were stereotaxically transferred to the infarcted cavity of 11 rats. The other 5 rats receiving sham transplants served as controls. For immunosuppression, all transplanted rats received intraperitoneal injection of cyclosporine A daily starting on the day of grafting. Immunohistochemistry for glial fibrillary acidic protein (GFAP), synaptophysin, neurofilament, and microtubule associated protein-2 (MAP-2) was performed on brain sections perfused in situ 8 weeks after transplantation.

RESULTSGrafts in the infarcted cavities of 6 of 10 surviving rats consisted of bands of neurons with an immature appearance, bundles of fibers, and GFAP-immunopositive astrocytes, which were unevenly distributed. The grafts were rich in synaptophysin, neurofilament, and MAP2-positive neurons with long processes. The graft/host border was diffuse with dendrites apparently bridging over to the host brain, into which neurofilament immunopositive fibers protruded.

CONCLUSIONCultured human fetal brain cells can survive and grow in the infarcted cavities of immunodepressed rats and integrate with the host brain.

Animals ; Astrocytes ; transplantation ; Brain ; pathology ; Cell Proliferation ; Cell Survival ; Cells, Cultured ; Cerebral Infarction ; metabolism ; pathology ; therapy ; Fetal Tissue Transplantation ; Glial Fibrillary Acidic Protein ; analysis ; Humans ; Microtubule-Associated Proteins ; analysis ; Neocortex ; cytology ; Neurons ; transplantation ; Rats ; Synaptophysin ; analysis