Computer navigation-aided joint-preserving resection and custom-made endoprosthesis reconstruction for bone sarcomas: long-term outcomes / 中华医学杂志(英文版)

BACKGROUND@#Computed tomography (CT) and magnetic resonance imaging (MRI) data can be fused to identify the tumor boundaries. This enables surgeons to set close but tumor-free surgical margins and excise the tumor more precisely. This study aimed to report our experience in performing computer navigation-aided joint-preserving resection and custom-made endoprosthesis reconstruction to treat bone sarcoma in the diaphysis and metaphysis of the femur and tibia.@*METHODS@#Between September 2008 and December 2015, 24 patients with bone sarcomas underwent surgical resection and joint-sparing reconstruction under image-guided computer navigation. The cohort comprised 16 males and eight females with a median age of 19.5 years (range: 12-48 years). The tumor location was the femoral diaphysis in three patients, distal femur in 19, and proximal tibia in two. The tumors were osteosarcoma (n = 15), chondrosarcoma (n = 3), Ewing sarcoma (n = 3), and other sarcomas (n = 3). We created a pre-operative plan for each patient using navigation system software and performed navigation-aided resection before reconstructing the defect with a custom-made prosthesis with extracortical plate fixation.@*RESULTS@#Pathological examination verified that all resected specimens had appropriate surgical margins. The median distance from the tumor resection margin to the joint was 30 mm (range: 13-80 mm). The median follow-up duration was 62.5 months (range: 24-134 months). Of the 24 patients, 21 remain disease free, one is alive with disease, and two died of the disease. One patient developed local recurrence. Complications requiring additional surgical procedures occurred in six patients, including one with wound hematoma, one with delayed wound healing, one with superficial infection, one with deep infection, and two with mechanical failure of the prosthesis. The mean Musculoskeletal Tumor Society score at the final follow-up was 91% (range: 80%-100%). The 5- and 10-year implant survival rates were 91.3% and 79.9%, respectively.@*CONCLUSIONS@#Computer navigation-aided joint-preserving resection and custom-made endoprosthesis reconstruction with extracortical plate fixation is a reliable surgical treatment option for bone sarcoma in the diaphysis and metaphysis of the femur and tibia.

Influences of scutellarin on ATP-induced inflammasome activation and pyroptosis in J774A.1 macrophages / 中国药理学通报

Aim To explore the influences of scutellarin on ATP-induced NLRP3 inflammasome activation and pyroptosis,using LPS-primed murine macrophages J774A.1 as an inflammatory cell model,and to explore the underlying mechanism.Methods The effects of scutellarin on ATP-induced pyroptosis in murine J774A.1 macrophages were analyzed by propidium iodide (PI) staining assay.The levels of IL-1β,caspase-1 and HMGB1 in cell lysates and culture supernatants were analysed using Western blot.The levels of IL-1β in cell culture supernatants were measured by cytometric beads array (CBA).Results ATP significantly induced caspase-1 activation and mature IL-1β and HMGB1 release into the culture supernatants in LPS-primed murine J774A.1 macrophages,and induced pyroptosis.Scutellarin treatment dose-dependently inhibited ATP-induced caspase-1 activation,mature IL-1β and HMGB1 release,and pyroptosis.Notably,scutellarin's inhibitory effects on ATP-induced pyroptosis were markedly reversed by the adenylate cyclase inhibitor MDL12330A and selective protein kinase A (PKA) inhibitor H89.Conclusion Scutellarin inhibits NLRP3 inflammasome activation and pyroptosis by modulating the PKA activity in macrophages,thereby exhibiting anti-inflammatory activities.

Application of psychological intervention combined with gemcitabine combined with tiggio chemotherapy in patients with pancreatic cancer after radical operation / 中国生化药物杂志

Objective To discuss the application effect of psychological intervention combined with gemcitabine combined with S-1 chemotherapy in patients with radical resection of pancreatic cancer in. Methods Randomly selected from February 2015 to December 2016 in our hospital 76 cases of patients with pancreatic cancer and pancreatic cancer radical surgery, were randomly divided into experimental group 38 cases, 38 cases in control group were given psychological intervention combined with gemcitabine combined with S-1 chemotherapy after surgery in the experimental group, the control group was given gemcitabine combined with oxaliplatin in the treatment of adverse reactions of the two scheme. Incidence rate and benefit rate were compared and analyzed and the clinical effective rate of treatment. Results The adverse reaction rate and clinical benefit rate of the experimental group were obviously better than that of the control group (P<0.05), and there was no significant difference in the treatment efficiency between the two groups. Conclusion The combined chemotherapy with S-1 can improve the clinical benefit rate in radical resection of pancreatic cancer after psychological intervention combined with gemcitabine, reduce adverse reactions, it is worthy of promotion and application.

Application of psychological intervention combined with gemcitabine combined with tiggio chemotherapy in patients with pancreatic cancer after radical operation / 中国生化药物杂志

Objective To discuss the application effect of psychological intervention combined with gemcitabine combined with S-1 chemotherapy in patients with radical resection of pancreatic cancer in. Methods Randomly selected from February 2015 to December 2016 in our hospital 76 cases of patients with pancreatic cancer and pancreatic cancer radical surgery, were randomly divided into experimental group 38 cases, 38 cases in control group were given psychological intervention combined with gemcitabine combined with S-1 chemotherapy after surgery in the experimental group, the control group was given gemcitabine combined with oxaliplatin in the treatment of adverse reactions of the two scheme. Incidence rate and benefit rate were compared and analyzed and the clinical effective rate of treatment. Results The adverse reaction rate and clinical benefit rate of the experimental group were obviously better than that of the control group (P<0.05), and there was no significant difference in the treatment efficiency between the two groups. Conclusion The combined chemotherapy with S-1 can improve the clinical benefit rate in radical resection of pancreatic cancer after psychological intervention combined with gemcitabine, reduce adverse reactions, it is worthy of promotion and application.

Protection and Mechanism of Qingyuan Shenghua Decoction on Multiple Organs of Sepsis Patients after Bone Trauma / 中国中西医结合杂志

<p><b>OBJECTIVE</b>To observe the protection of Qingyuan Shenghua Decoction (QSD) on multiple organs of sepsis patients after bone trauma, and to preliminarily explore its mechanism.</p><p><b>METHODS</b>Totally 60 sepsis patients after bone trauma were randomly assigned to the treatment group and the control group according to random digit table, 30 in each group. All patients received routine Western medical treatment. Patients in the treatment group additionally took QSD or were nasally fed with QSD, one dose per day for 1 week. Changes of WBC, oxygenation index (PaO2/FiO2), serum creatinine (SCr), total bilirubin (TBIL), aspartate aminotransferase (AST), fibrinogen (FIB), D-dimer (DD), activated partial thromboplastin time (APTT), pro-calcitonin (PCT), C-reactive protein (CRP), heart rate (HR), mean arterial pressure (MAP), intra-abdominal pressure, scores for Acute Physiology and Chronic Health Evaluation II (APACHE II), sequential organ failure assessment (SOFA) scores were observed before treatment and on day 1, 3 and 7 after treatment.</p><p><b>RESULTS</b>Compared with the control group at the same time point, MAP increased at post-treatment day 1 and 3; CRP, APTT, HR, SCr, TBIL, AST, intra-abdominal pressure at post-treatment day 3 obviously decreased in the treatment group (P < 0.05, P < 0.01). WBC, SOFA scores, PCT, CRP, APACHE II, APTT, D-D, HR, SCr, TBIL, AST and intra-abdominal pressure significantly decreased; FIB, MAP and PaO2/FiO2 obviously increased at post-treatment day 7 (P < 0.05, P < 0.01).</p><p><b>CONCLUSION</b>QSD had good protective effect on multiple organ function in sepsis patients after bone trauma, and its mechanism might be related with effectively clearing endotoxin, alleviating inflammatory reactions, and fighting against coagulation dysfunction.</p>

Diagnosis and treatment of giant cell tumor of bone in skeletal immature patients / 中华外科杂志

<p><b>OBJECTIVE</b>To demonstrate the common characteristics of giant cell tumor of bone in immature skeletons.</p><p><b>METHODS</b>From 1989 to 2009, the 8 skeletal immature patients were pathologically diagnosed with giant cell tumor (GCT) in our department, which accounted for 1.3% (8/621) of all GCT patients in an extremity. All patients were identified with an open epiphyseal plate by retrospective review of the radiograph, CT or MRI by senior consultants. Oncological and functional outcome were followed for a mean 44.1 months. There were 5 boys and 3 girls. The mean age was 13.8 years. All cases had a primary lesion. The distal femur is the most common site involved (3 cases), followed by the proximal tibia (2 cases). The proximal humerus, the distal tibia and the distal radius accounted for 1 case respectively. Oncological and functional outcome are followed for a mean 44.1 months.</p><p><b>RESULTS</b>All lesions were lytic. Six lesions involved both the epiphysis and metaphysis. Two lesions located in the metaphysis area. Six lesions were treated with extended curettage and were reconstructed with allograft and (or) bone cement. Internal fixations were used in 2 cases. Two cases were treated with segmental resection. And one was reconstructed with cement spacer and the other one with segmental allograft and internal fixation. One patient (1/6) developed a bone recurrence after extended curettage. No extremity deformity and discrepancy were found during the follow up after the curettage. No metastasis was found during the follow up.</p><p><b>CONCLUSION</b>Histologically GCT occurs in skeletal immature bone has the same pathological appearance but radiologically has its unique features. These lesions share same behavior as that in adults. A low local recurrence rate and good function can be achieved after a proper surgery.</p>

Preparation and identification of human soluble sPD-L1 and its antibodies / 生物工程学报

This study reports the preparation and identification of soluble programmed death-1 (PD-1) ligand-1 (sPD-L1) and its antibodies of mouse origin. Immobilized metal ion affinity chromatography was used to perform on-column refolding with simultaneous purification of denatured sPD-L1, and soluble sPD-L1 with purity of 95% was obtained. The purified sPD-L1 was verified by immunoblotting using a commercial goat-anti-human PD-L1 antibody. An ELISA-based assay showed that it also had high binding activity for its cognate receptor PD-1. Furthermore, mouse anti-sPD-L1 antiserum of high titer was raised using the purified sPD-L1 as an immunogen, and the specific IgG antibodies were purified using sPD-L1-HiTrap affinity chromatography. In addition, a sensitive sandwich ELISA was established using the purified IgG antibodies together with the commercial goat antibodies. In conclusion, the preparation of soluble sPD-Ll and its antibodies provide the basis for detection of the potential anti-PD-L1 antibodies and soluble PD-L1 in humans as well as for further investigation of its in vivo bioactivities and characterization of its potential receptors.

Improved expression of HLA-A* 2402-BSP in Escherichia coli and its tetramer preparation / 生物工程学报

HLA-A* 2402 is one of the most frequently encountered HLA-A alleles in East Asian populations. In order to study the CD8+ T cell responses in Chinese populations, we have described the generation and functional test of HLA-A* 2402 tetramer loaded with HCMV pp65(341-349) peptide (QYDPVAALF, QYD). The cDNA of HLA-A* 2402 heavy chain was cloned by RT-PCR from one of the donors. DNA fragment encoding the ectodomain of HLA-A* 2402 heavy chain fused at its carboxyl-terminal a BirA substrate peptide (BSP) was amplified by PCR with the cloned heavy chain cDNA as a template. The wild-type gene of HLA-A* 2402-BSP was not expressed in Escherichia coli (E. coli), while mutant HLA-A* 2402-BSP gene with optimized codons was overexpressed as inclusion bodies in E. coli. Furthermore, the soluble HLA-A* 2402-QYD monomers were generated by in vitro refolding of washed inclusion bodies in the presence of beta2-microglobulin and QYD peptide. The tetramer was subsequently formed by mixing HLA-A* 2402-QYD monomers with streptavidin-PE at a molar ratio of 4:1. Flow cytometry analysis indicated that this tetramer possessed binding activity with specific CTL from HLA-A24+ donors and the frequencies of tetramer-binding CTL were 0.09% - 0.37% within total CD8+ T cells. This tetrameric agent provides a powerful tool to explore the secrets of CTL responses against HCMV antigens in HLA-A* 2402 individuals.

Preparation and identification of soluble trimeric CD40-isoleucine zipper fusion protein / 生物工程学报

The interaction of CD40 with its cognate ligand, CD40L (CD154), plays important roles in immune responses. Blockade of CD40-CD40L signal pathway can protect the progression of antibody- and cell-mediated autoimmune diseases, and reduce allograft rejection thus prolonging graft survival, even engendering long-lived antigen-specific tolerance. The present study aims to enhance the binding activity of CD40 by incorporating an isoleucine zipper (IZ) trimeric motif into CD40 ectodomain to promote the formation of soluble CD40 trimers, which would be useful for blocking CD40-CD40L interaction. A prokaryotic expression vector for soluble human CD40 ectodomain fused with an IZ motif and a hexa-histidine (His6) tag at its carboxyl terminus (sCD40IZ) was constructed by multiple round PCR using cloned CD40 cDNA as a template. The recombinant sCD40IZ protein was expressed highly in Escherichia coli (E. coli) with a molecular weight of 27kD, which is consistent with its theoretical value. It mainly existed in inclusion bodies. After refolding from inclusion bodies, soluble sCD40IZ protein was purified by gel filtration. Its molecular weight in solution was about 91kD when determined by gel filtration, suggesting that it most probably existed in the form of trimers. Moreover, this protein could bind to CD40L expressed on Jurkat T cells and its binding activity was significantly higher than that of soluble CD40 without an IZ motif. These results suggest that incorporation of an IZ motif at the carboxyl terminus of soluble CD40 can facilitate the formation of trimers and enhance its binding activity with CD40L. Thus, the trimeric CD40 protein may be used to block CD40-CD40L signal pathway, suggesting that it may have potential application in preventing autoimmune diseases and transplantation rejection.

On-column refolding and purification of human EGF receptor L2 domain inclusion body overexpressed in Escherichia coli / 生物工程学报

The human epidermal growth factor receptor (EGFR) extracellular region (residues 1-621) consists of four subdomains, i.e. L1, S1, L2, and S2. The L2 domain (EGFR-L2) is composed of residues 311-479 and plays a major role in ligand-binding. Due to the high content of cysteine residues (42 cysteines) in the S1 and S2 domains, it is quite difficulty to get a correctly refolded product of the complete EGFR extracellular domain. In contrast, only 4 cysteine residues are present in EGFR-L2 domain. The aim of the present study is to prepare a soluble EGFR-L2 domain from the recombinant protein inclusion body overexpressed in Escherichia coli (E. coli). DNA fragment encoding EGFR-L2 containing a polyhistidine-tag at the carboxyl terminus was amplified by PCR from the cDNA of EGFR extracellular region, and was inserted into pET-3c to construct the prokaryotic expression vector. The target protein was highly expressed in E. coli BL21 (DE3) strain and was only present in the inclusion body as revealed by immunoblotting analysis. No soluble product could be refolded through dilution or stepwise dialysis strategies. However, on-column refolding of denatured EGFR-L2 bound to Ni2+ -NTA produced a soluble one. Furthermore,the soluble EGFR-L2 was simultaneously purified to high purity (>95%) through eluting from the same Ni2+ -NTA column with a linear imidazole gradient. The refolded EGFR-L2 had specific binding activity with the cognate ligand EGF, although its affinity was low. These results suggest that a polyhistidine-tag fused with a recombinant protein facilitate not only the purification but also the renaturation of the target product through on-column refolding. Besides, this refolding strategy may be suitable for the preparation of those recombinant proteins which are hard to refold through conventional approaches.

Immune-functional epitopes and inflammation-inducing effects of the major outer envelope proteins of Leptospira interrogans / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To investigate the immune-functional epitopes and inflammation-inducing effects of the major outer envelope proteins of Leptospira interrogans.</p><p><b>METHODS</b>Ni-NTA affinity chromatography was established to extract the target recombinant proteins rOmpL1/1 and OmpL1/2, LipL32/1 and rLipL32/2, LipL41/1 and rLipL41/2 expressed by the different genotypes. By using Signal P-NN software in Signal P3.0 prediction server, EMBOSS software in propred MHC class-II binding peptide prediction-ProPred prediction server, the possible signal peptides, MHC-II binding peptides and lymphocyte B epitopes were analyzed. The IL-1, IL-8 and TNF-alpha secretion in human umbilical vein endothelial cell line EVC-304 induced by target recombinant proteins were measured by ELISA.</p><p><b>RESULTS</b>Under the inducement of IPTG, the constructed prokaryotic systems efficiently expressed rOmpL1/1 and rOmpL1/2, rLipL32/1 and rLipL32/2, and rLipL41/1 and rLipL41/2 with outputs of 30% and 15%, 40% and 35%, and 15% and 10% of the total bacterial proteins, respectively. Each of the purified target recombinant proteins showed a single protein band in SDS-PAGE. The signal peptides of OmpL1s, LipL32/1 and LipL32/2, and LipL41s were located at the N ends of 1-24, 1-21 and 1-24, and 1-24 amino acid residuals, respectively. OmpL1s, LipL32s and LipL41s displayed 2,2 and 1 same major epitopes of MHC-II binding peptides and lymphocyte B and OmpL1/2 had another one (59-78). The different dosages of rOmpL1s, rLipL32s and rLipL41s increased the secretion of IL-1alpha , IL-8 and TNF-alpha (P<0.05) in EVC-304 cells. The IL-1alpha levels reached the highest at the 24 h and then declined,while the IL-8 and TNF-alpha levels after 48 h treatment were higher that those after 24 h.</p><p><b>CONCLUSION</b>The expression products in ompL1/1, lipL32 or lipL41 genotypes of L.interrogans contain similar immune functional epitopes. rOmpL1/1 and rOmpL1/2, rLipL32/1 and rLipL32/2, and rLipL41/1 and rLipL41/2 are able to directly induce inflammatory reaction in EVC-304 cells.</p>

Cloning of human beta-microglobulin gene and its high expression in Escherichia coli / 生物工程学报

Human beta2-microglobulin (beta2m) is the light chain of major histocompatibility complex (MHC) class I molecule. High-yield production of this protein is a prerequisite to the preparation of MHC I tetramer. The present study aims to obtain recombinant human beta2m expressed in Escherichia coli (E. coli), for the purpose of preparing MHC class I tetramers. For cloning of human beta2m gene, a pair of specific primers was designed based on the published sequence of this gene and the cDNA of full coding region for beta2m precursor was obtained by RT-PCR from the total RNA of human leukocytes. The amplified cDNA was subsequently cloned and its sequence was confirmed by DNA sequencing analysis (the sequence has been deposited in GenBank with accession number of AY187687). The prokaryotic expression vector containing a gene encoding mature beta2m was constructed by inserting the DNA fragment, which was generated by PCR reaction with the cloned beta2m gene as template, into an IPTG-inducible expression vector pET-3c plasmid. The first eight codons for N terminal amino acid residues of beta2m were optimized for its expression in E. coli. The complete sequence of beta2 m gene in the expression vector was verified by DNA sequencing analysis. High-yield expression of beta2m was achieved in E. coli transformed with the expression vector, and most of the recombinant beta2m existed in the inclusion body after IPTG induction. The inclusion body was washed extensively and beta2m in the inclusion body was solublized with 8 mol/L urea. The beta2m was refolded by dialysis and purified by ion-exchange chromatography (Q-Sepharose). Western blotting assay indicated that the polyclonal antibody against human native beta2m could react specifically with the recombinant protein. The purified protein appeared as a single band on both SDS-PAGE and Western blotting, indicating that it was chemical and antigenic pure. This work establishes a convenient approach for renaturation and purification of large quantity of recombinant beta2m which is identical to the native protein without any tags fused except for a methionine residue at the amino terminus. This provides the basis for the preparation of MHC tetramers.

Preparation and characterization of HLA-A * 0201 monomer and tetramer loaded with HCMV antigenic peptide / 生物工程学报

Quantification of cytotoxic T lymphocytes (CTL) is extremely important due to the pivotal role they play in controlling pathogen infection and anti-tumor actions. Previously used methods for detecting specific CTL are usually indirect. In recent years, tetramer technology has been developed to directly visualize antigen-specific CTL efficiently, and become the critical approach in studying T cell immune responses. A simplified procedure for preparing tetramers is reported here in this paper and a tetramer loaded with human cytomegalovirus (HCMV) peptide was successfully obtained using this procedure, which possessed binding activity with specific CTL. The heavy chain of HLA-A * 0201 gene was cloned by RT-PCR from HLA-A2+ donor. An expression vector, encoding the extracellular domain of HLA-A * 0201 heavy chain (A2) fused with a BirA substrate peptide (BSP) at its carboxyl terminus, was constructed by PCR with cloned A2 gene as the template. The A2 heavy chain was expressed in Escherichia coli mostly in the form of inclusion body and purified by washing inclusion body. The monomer of soluble A2 loaded with peptide was reconstructed by dilution from the heavy chain in the presence of light chain beta2-microglobulin and HLA-A2 restricted HCMV pp65(495-503) peptide (NLVPMVATV, NLV). Refolded A2-NLV monomer was biotinylated with a commercial BirA and purified by low pressure anion exchange chromatography on a Q-Sepharose (fast flow) column. The tetramer was then formed by mixing A2-NLV monomer with streptavidin-PE in a ratio of 4:0.8 leading to more than 85% multiplication as revealed by SDS-PADE under non-reducing conditions without boiling the sample. Flow cytometry analysis indicated that this tetramer could bind to specific CTL from HLA-A2+ donor. In conclusion, a simplified procedure is established to prepare HLA-A2 tetramer, which may not only facilitate the application of tetramer technology for studying specific T lymphocyte immune response but A2-NLV itself be applied clinically to monitor CMV-specific CTL in stem cell and organ transplantation.