Although locoregional recurrence (LRR) after mastectomy is regarded as a signal of disease progression,but it is different from distant metastasis.Some patients could have better prognosis and benefit from salvage treatments,especially for patients with isolated LRR.Time to recurrence,site of recurrence and number of recurrent nodules have great influence on the prognosis of patients with LRR.For suited patients with LRR,surgical resection and endocrine therapy are valuable in suitable cases,but the role of chemotherapy still need further study.

Purpose　The aim is to develop a method for the analysis of micro-scale amino acids in biological samples.Methods　After having been extracted and dried,the residues were dissolved in a running buffer or a distilled water and acetonitrile.The samples were loaded into column for 5,25 or 50s at a hydrodynamic injection and were separated by capillary electrophoresis.The effect of different dissolvent on the sample stacking was observed.Results　A 100-fold improvement in the amount of material that can be injected into a capillary column without loss of resolution was shown.Conclusion　The marked effect of sample stacking was achieved by the using distilled water and acetonitrile(1∶1,v/v) as dissolvent.

Objective To investigate the short-term clinical efficacy of CT-guided percutaneous implantation of 125I radioactive seeds in treating the postoperative lung metastases of malignant fibrous histiocytoma(MFH).Methods Eight patients with lung metastases after MFH surgery received CT-guided percutaneous implantation of 125I radioactive seeds.There are 28 metastasis lesions in total.Prescribed dose (PD) was 80 Gy and radioactivity was 0.7 mCi(2.59 × 107 Bq).Six months after implantation,chest CT-scan was performed and the changes were reviewed according to the international standards.ResultsAverage dose received by tumor was ( 172.9 ± 39.9 ) Gy,D100 ( 77.7 ± 10.2 ) Gy and D90 ( 97.2 ± 6.8 ) Gy.The overall response rate was 82.1％.Patients' survival period was 10.0 - 26.0 ( 14.6 ± 3.5 ) months with the median survival time of 13 months.ConclusionIn the treatment of postoperative lung metastases of MFH,implantation of 125I radioactive seeds produces a good short-term clinical efficacy.

Although two chain transfering separately could be used to overcome the volume limitation of adeno-associated virus vectors (AAV) in coagulation factor VIII (FVIII) gene delivery, it leads to chain imbalance for inefficient heavy chain secretion. In this study we aimed to improve the efficacy of two chain strategy in FVIII gene delivery through the degradation of glucose-regulated protein 78 (GRP78) known as a protein chaperone in endoplasmic reticulum (ER) by deoxynivalenol (DON) to decrease GRP78-bound FVIII heavy chain. By treating the two-chain gene transduced 293 cells with DON, the heavy chain (HC) secretion and FVIII bioactivity were observed. Data showed that 293 cells after three hours post-treatment with DON at a concentration of 500 ng mL(-1) resulted in obvious decrease the level of GRP78 but no effect on the cell proliferation. The HC secreted from DON-treated cells transfected with HC gene alone was 59 +/- 11 ng mL(-1), higher than that secreted by control cells (15 +/- 4 ng mL(-1)), and the HC secretion was further increasing to 146 +/- 34 ng mL(-1) in light chain (LC) gene co-transfected cells with an activity measured up to 0.66 +/- 0.15 U mL(-1), also greater than control cells (76 +/- 17 ng mL(-1) and 0.35 +/- 0.09 U mL(-1)). Taken together, these data suggest that DON-mediated GRP78 down-regulation could improve the efficacy of two-chain FVIII gene transfering by facilitating HC secretion, providing an experimental basis for in vivo dual-AAV application in FVIII gene delivery.

In our recent study by exploring an intein-based dual-vector to deliver a B-domain-deleted FVIII (BDD-FVIII) gene, it showed that covalently ligated intact BDD-FVIII molecules with a specific coagulant activity could be produced from expressed heavy and light chains by protein trans-splicing. Here, we assessed the hypothesis that the efficiency of trans-splicing may be increased by adding to the intein sequences a pair of leucine zippers that are known to bring about specific and strong protein binding. The intein-fused heavy and light chain genes were co-transferred into cultured COS-7 cells using a dual-vector system. After transient expression, the intracellular BDD-FVIII splicing was observed and the spliced BDD-FVIII and bioactivity secreted to culture media were quantitatively analyzed. An enhanced splicing of BDD-FVIII with decreased protein precursors from gene co-transfected cells was observed by Western blotting. The amount of spliced BDD-FVIII and bioactivity secreted to the culture media were 106 +/- 12 ng x mL(-1) and 0.89 +/- 0.11 U x mL(-1) analyzed by ELISA and Coatest method respectively, which was greater than leucine zipper free intein-fused heavy and light chain genes co-transfected cells (72 +/- 10 ng x mL(-1) and 0.62 +/- 0.07 U x mL(-1)). The activity of cellular mechanism-independent protein splicing was also improved, as showed by the increasing of spliced BDD-FVIII and bioactivity in culture media from combined cells separately transfected with heavy and light chain genes which was 36 +/- 11 ng x mL(-1) and 0.28 +/- 0.09 U x mL(-1). It demonstrated that the leucine zippers could be used to increase the efficiency of protein trans-splicing to improve the efficacy of a dual-vector mediated BDD-FVIII gene delivery by strengthening the interaction between the two intein-pieces fused to heavy and light chains. It provided evidence for further study in animal model using a dual-adeno-associated virus vector to deliver FVIII gene in vivo.

Heat shock protein gp96 is a highly conserved and monomorphic glycoprotein in the endoplasmic reticulum. It functions as molecular chaperone and can associate with a variety of antigenic peptides noncovalently in vivo and in vitro. Recent studies have indicated that gp96 molecules participate in major histocompatibility complex class I-restricted antigen presentation pathway. Immunization of mice with gp96 preparations isolated from cancer cells can elicit a cancer-specific protective T cell immune response that is recallable, which is a prerequisite for gp96 as a therapeutic vaccine against cancers. The immunogenicity of gp96 molecules has been attributed to the antigenic peptides associated with them. These phenomena provide a new pathway for cancer immunotherapy. The mechanism that the gp96-peptide complex induces specific immune response and the explorations for gp96-peptide complex as a therapeutic cancer vaccine are reviewed.
Animals ; Antigens, Neoplasm ; immunology ; therapeutic use ; Cancer Vaccines ; therapeutic use ; Humans ; Immunotherapy ; Membrane Glycoproteins ; immunology ; metabolism ; Molecular Chaperones ; immunology ; Neoplasms ; immunology ; therapy ; Peptides ; immunology ; metabolism

As synthesized by vascular endothelial cells and megakaryocytes, the von Willebrand factor (vWF) plays an important hemostatic role in the binding to and stabilizing blood coagulation factor VIII (FVIII) and preventing its enzymatic degradation. Our recent work demonstrated intein can efficiently ligate BDD-FVIII (B-domaim deleted FVIII) posttranslationally by protein trans-splicing after transfer of split BDD-FVIII gene by a dual-vector system. In this study we investigated the effect of vWF on secretion and activity of intein-ligated BDD-FVIII. We observed the levels of full-length BDD-FVIII antigen secreted into culture supernatant by ELISA and their activity by Coatest assay after transfection of cultured 293 cells with intein-fused BDD-FVIII heavy- and light-chain genes simultaneously with the vWF gene co-transfected. The data showed that the amount of full-length BDD-FVIII protein and their bioactivity in vWF gene co-transfected cell supernatant were 235 +/- 21 ng x mL(-1) and 1.98 +/- 0.2 u x mL(-1), respectively, greater than that of non-vWF co-transfected cell (110 +/- 18) ng x mL(-1) and 1.10 +/- 0.15 u x nL(-1)) or just BDD-FVIII gene transfected control cell (131 +/- 25 ng x mL(-1) and 1.22 +/- 0.18 u x mL(-1)) indicating the benefit of vWF gene co-transfection in the secretion and activity of intein-spliced BDD-FVIII protein. It provided evidence that vWF gene co-transfer may be useful to improve efficacy of gene therapy for hemophilia A in protein splicing-based split FVIII gene transfer.

This study is to construct a chimeric human/porcine BDD-FVIII (BDD-hpFVIII) containing the substituted porcine A1 and A3 domains which proved to have a pro-secretory function. By exploring Ssp DnaB intein's protein trans-splicing a dual-vector was adopted to co-transfer the chimeric BDD-hpFVIII gene into cultured COS-7 cell to observe the intracellular BDD-hpFVIII splicing by Western blotting and secretion of spliced chimeric BDD-hp FVIII protein and bio-activity using ELISA and Coatest assay, respectively. The dada showed that an obvious protein band of spliced BDD-hpFVIII can be seen, and the amount of spliced BDD-hpFVIII protein and bio-activity in the supernatant were up to (340 +/- 64) ng x mL(-1) and (2.52 +/- 0.32) u x mL(-1) secreted by co-transfected cells which were significantly higher than that of dual-vector-mediated human BDD-FVIII gene co-transfection cells [(93 +/- 22) ng x mL(-1), (0.72 +/- 0.13) u x mL(-1)]. Furthermore, a spliced BDD-hpFVIII protein and activity can be detected in supernatant from combined cells separately transfected with intein-fused BDD-hpFVIII heavy and light chain genes indicating that intein-mediated BDD-hpFVIII splicing occurs independently of cellular mechanism. It provided evidence for enhancing FVIII secretion in the research of animal models using intein-based dual vector for the delivery of the BDD-hpFVIII gene.

Objective To explore influential factors of local therapeutic effect in CT guided brachytherapy of 125I seeds for non-small-cell lung carcinoma (NSCLC).Methods Totally 141 primary NSCLC patients diagnosed by bronchoscope or puncture biopsy were treated with CT guided 125I seeds implantation treatment from 2003 January to 2005 January.Among them,26 patients were treated with seeds implantation only and remaining 115 combined with chemical therapy.Preplans were performed by using treatment planning system before the implantation.We took the implantation with the prescription dose of 80-110 Gy,1 seed per 1 cm3,under the guide of computed tomography.Six months after implantation treatment,CT graphs were taken to evaluate the therapeutic effect.Results All the patients were survival until 6 months after implantation,and 37 were complete remission,93 were partial remissions.The effective rate was 92.2％.Among all the observed factors,pathologic type (F =5.162,P =0.023),dose of cover 100％ tumor(D100) (F =100.713,P =0.000) and treatment methods (F =16.205,P =0.000) were the independent influent factors (P ＜ 0.05).Among these,D100 was the most important factor (P =0.000).Single factor analysis indicated that pathologic type (x2 =7.313,P =0.007),D100 (x2 =71.6,P =0.000)and treatment methods (x2 =20.5,P =0.000) were significant influent factors.Of all 141 cases,24 had complications during or after implantation treatment,while no severe complications were reported.There was no significant correlation between complication and local therapeutic effect (P ＞ 0.05).Conclusion CT guided implantation of 125I seeds for lung cancer has good clinical effects and few complications.D100 is the most important factor to influence the local therapeutic effect.Implantation treatment combined with chemotherapy is an ideal measure for NSCLC treatment.

Objective To evaluate the clinical effects of CT-guided 125I radioactive seed implantation in treatment of stage Ⅲ non-small cell lung cancer ( NSCLC ) and the influential factors of prognosis.Methods 247 patients of stage Ⅲa/Ⅲb NSCLC underwent CT-guided 125I radioactive seed implantation.The clinical effects and the factors affecting prognosis were analyzed by univariate and multivariate analyses.Results The 1-,3-,and 5- year overall survival rates were 82.8％,23.8％,and 11.5 ％,respectively.The median survival time was 24.8 months,and the local control rate was 92.2 ％,63.8％,and 25.7％,respectively.The 5- year overall survival rate was 14.7％,and the median survival time was 29.7 months of the stage Ⅲ,patients.And the 5- year overall survival rate was 11.2％,and the median survival time was 24.0 months at the stage Ⅲb.Univariate analysis showed that age,course of disease,hemoglobin before treatment,clinical stage,maximum diameter of tumor,prescribed dose (PD),post-operational mean dose,post-operational dose covering 100％ volume (D100),remedial model were the main prognostic factors; however,multivariate analysis revealed that hemoglobin ≥ 120 g/L before treatment,post-operational dose covering 100％ volume (D100) and maximum diameter of tumor were the independent risk factors for predicting the survival.Aerothorax was observed in 37 patients with an incidence rate of 14.9％,and hemothorax was observed in 22 patients with an incidence rate of 9％.Conclusions 125I radioactive seed implantation therapy is effective in the treatment of stage Ⅲ NSCLC.Hemoglobin level before treatment,post-operational dose covering 100％ volume (D100 ),and maximum diameter of tumor are the main prognostic factors for the NSCLC patients treated with radiotherapy for NSCLC.