Abstract: To develop novel live attenuated influenza vaccine, we explored the feasibility to attenuate influenza virus by codon deoptimization of NS1. According to the codon usage bias in influenza A virus, we designed and synthesized a condon-deoptimized NS gene by substituting codons of 110 amino acids in the NS1 gene of A/Puerto Rico/8/34(H1N1) with unpreferred synonymous codons. The influenza A virus with the codon deoptimized NS1 gene (deoNS virus) was rescued by reverse genetics. Plaque forming assay and virus growth curve showed that the growth of deoNS virus was reduced about 1000 times in MDCK cells compared to that of the wild-type virus. Intranasal inoculation with deoNS virus did not cause death or evident disease in infected BALB/c mice. Furthermore, the virus titer in the lungs of mice infected with deoNS virus was significantly lower (i.e. 100-1000 times) than that of wild-type virus. Our results indicated that influenza virus could be effectively attenuated by synonymous codon deoptimization of NS1 gene. This strategy will be useful to develop new attenuated candidates for the production of live attenuated influenza vaccines.
Animals ; Base Sequence ; Chick Embryo ; Codon ; genetics ; Influenza A virus ; genetics ; pathogenicity ; Influenza Vaccines ; immunology ; Mice ; Mice, Inbred BALB C ; Molecular Sequence Data ; Orthomyxoviridae Infections ; immunology ; prevention & control ; Recombination, Genetic ; Vaccines, Attenuated ; immunology ; Viral Nonstructural Proteins ; genetics ; Virulence ; genetics

Carcinoma, Hepatocellular ; pathology ; Humans ; Liver Neoplasms ; pathology ; Neoplasm Invasiveness ; Neoplasm Metastasis ; Peptide Fragments ; pharmacology ; Peptides ; pharmacology ; Tumor Cells, Cultured

BACKGROUNDTissue-engineered heart valves have the potential to overcome the limitations of present heart valve replacements. This study was designed to develop a tissue engineering heart valve by using human umbilical cord blood-derived endothelial progenitor cells (EPCs) and decellularized valve scaffolds.

METHODSDecellularized valve scaffolds were prepared from fresh porcine heart valves. EPCs were isolated from fresh human umbilical cord blood by density gradient centrifugation, cultured for 3 weeks in EGM-2-MV medium, by which time the resultant cell population became endothelial in nature, as assessed by immunofluorescent staining. EPC-derived endothelial cells were seeded onto the decellularized scaffold at 3 x 10(6) cells/cm(2) and cultured under static conditions for 7 days. Proliferation of the seeded cells on the scaffolds was detected using the MTT assay. Tissue-engineered heart valves were analyzed by HE staining, immunofluorescent staining and scanning electron microscopy. The anti-thrombogenic function of the endothelium on the engineered heart valves was evaluated by platelet adhesion experiments and reverse transcription-polymerase chain reaction (RT-PCR) analysis for the expression of endothelial nitric oxide synthase (eNOS) and tissue-type plasminogen activator (t-PA).

RESULTSEPC-derived endothelial cells showed a histolytic cobblestone morphology, expressed specific markers of the endothelial cell lineage including von Willebrand factor (vWF) and CD31, bound a human endothelial cell-specific lectin, Ulex Europaeus agglutinin-1 (UEA-1), and took up Dil-labeled low density lipoprotein (Dil-Ac-LDL). After seeding on the decellularized scaffold, the cells showed excellent metabolic activity and proliferation. The cells formed confluent endothelial monolayers atop the decellularized matrix, as assessed by HE staining and immunostaining for vWF and CD31. Scanning electron microscopy demonstrated the occurrence of tight junctions between cells forming the confluent monolayer. Platelets adhesion experiments suggested that the neo-endothelium was non-thrombogenic. The expression levels of eNOS and t-PA genes in the neo-endothelium were quite similar to those in human umbilical vein endothelial cells.

CONCLUSIONSEPCs isolated from the human umbilical cord blood can differentiate into endothelial cells in vitro and form a functional endothelium atop decellularized heart valve scaffolds. Thus, EPCs may be a promising cell source for constructing tissue-engineered heart valves.

Animals ; Cell Proliferation ; Endothelial Cells ; cytology ; metabolism ; Heart Valve Prosthesis ; Heart Valves ; cytology ; metabolism ; ultrastructure ; Humans ; Immunohistochemistry ; Microscopy, Electron, Scanning ; Nitric Oxide Synthase Type III ; genetics ; metabolism ; Platelet Aggregation ; Reverse Transcriptase Polymerase Chain Reaction ; Stem Cells ; cytology ; metabolism ; Swine ; Tissue Engineering ; methods ; Tissue Plasminogen Activator ; genetics ; metabolism ; Umbilical Cord ; cytology

To block tumor cell adhesion, inhibit tumor metastasis and recurrence, the anti-adhesion peptide-trimeric beta peptide (DLYYLMDLSYSMKGGDLYYLMDLSYSMKGGDLYYLMDLSYSMK, beta3) was designed. The DNA fragment of beta3 was cloned into expression vector pET-His and the fusion protein His-beta3 was expressed in E. coli. BL21(DE3)plysS. After 1.5 hours' induction with IPTG, His-beta3 peptide was expressed significantly amounting to 10% of the insoluble proteins and 4% of the total proteins. 20mg of beta3 peptide was obtained from one litter culture medium after purification by using metal-chelating sepharose 6B FF. The purity of beta3 is 92.2% according to Gel-Pro analysis. The anti-adhesion effects of beta3 peptide, beta1 peptide (DLYYLMDLSYSMK) and GRGDS on the hepatocellular carcinoma cell line SMMC-7721 and the high metastasis hepatocellular carcinoma cell line HCCLM6 were studied. The result showed the beta3 blocked the adhesion of HCCLM6 cells and SMMC-7721 cells to fibronectin (FN) specifically. The inhibition effect was dose-dependent and time-dependent and the inhibition rate of beta3 was higher than three times concentration of beta1 and GRGDS. This suggested that pET-His-beta3/BL21(DE3)plysS was a suitable expression system for beta3, and the expressed beta3 specially inhibited the adhesion of cancer cells.
Amino Acid Sequence ; Antineoplastic Agents ; pharmacology ; Cell Adhesion ; drug effects ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Molecular Sequence Data ; Peptide Fragments ; pharmacology ; Peptides ; genetics ; metabolism ; pharmacology ; Recombinant Proteins ; biosynthesis ; genetics ; Tumor Cells, Cultured

OBJECTIVETo study the feasibility and effect of substituting esophagus with stomach or colon without thoracotomy in the treatment of cervical esophageal carcinoma with laryngeal function preserved.

METHODSTwenty-four patients with cervical esophageal carcinoma were retrospectively reviewed. The esophagus was resected and substituted with 19 gastric pull-up and 5 colon interposition. Nineteen patients received radiotherapy postoperatively (dose 50 - 70 Gy).

RESULTSTwenty two patients were follow up over 3 years. The 3- and 5-year survival rates for T2 were 3 and 1, for T3, T4 8 and 3, respectively. The laryngeal function preservation rate was 77% (17/24) and the decannulation rate was 75% (12/16). The complication rate was 29%.

CONCLUSIONSurgical resection of cervical esophageal carcinoma with removal of the extraesophageal invaded tissues while preserving the laryngeal function is possible. The continuity of the esophagus is restored by stomach transposition and colon interposition. Combined with radiotherapy, the survival rate and life quality of the patient might be improved.

Adult ; Aged ; Esophageal Neoplasms ; mortality ; physiopathology ; surgery ; Esophagoplasty ; methods ; Female ; Follow-Up Studies ; Humans ; Larynx ; physiopathology ; Male ; Middle Aged ; Quality of Life ; Survival Rate ; Thoracotomy

OBJECTIVETo study the surgical treatment of tonsillar cancer.

METHODSTwenty-four patients with tonsillar cancer were treated with surgery and postoperative radiotherapy. The choice of surgical procedure was decided on the condition of the lesion. The tumor was resected through the transoral approach, mandibular swing approach, mandibular resection approach or hyoid approach. Surgical defect was repaired by pectoralis major myocutaneous flap, sternohyoid myofascial flap, tongue flap or soft palate flap.

RESULTSThe 3- and 5-year survival rates were 76.0% and 60.8%. Function of chewing, deglutition, respiration and speech was restored well.

CONCLUSIONMethod of total resection of the tonsillar carcinoma through the optimum approach is best chosen according to the condition of the lesion, while preserving the oropharyngeal function. When combined with postoperative radiotherapy, the survival rate and quality of life of patients can be improved.

Adult ; Aged ; Female ; Humans ; Male ; Middle Aged ; Palate, Soft ; surgery ; Reconstructive Surgical Procedures ; Survival Rate ; Tongue ; surgery ; Tonsillar Neoplasms ; mortality ; surgery

OBJECTIVETo review the experience of different surgical construction methods and comprehensive treatments for hypopharyngeal cancer.

METHODSThree hundred and fifty-two cases (According to UICC 2002 criteria, stage I, 3; II, 31; III, 134; IV, 184) with hypopharyngeal cancer were retrospectively reviewed from 1999 to 2005 in the Department of Otorhinolaryngology of Qilu Hospital of Shandong University, including 321 males and 31 females. The median age was 58 years old, ranged from 26 to 82 years old. All the tumors originated from the pyriform sinus (272), posterior pharyngeal wall (61), postcricoid area (19). There were no distant metastasis. Two hundred and fourty-one cases were surgically treated with laryngeal functions preserved and 111 cases without laryngeal functions preserved. All the patients received modified neck dissection, including both unilateral (247 patients) and bilateral (105 patients). Pharyngoesophageal defect reconstruction methods in cases with laryngeal functions preserved were: direct suture in 137, pectoralis major musculocutaneous flap in 62, split graft in 2, pectoralis major musculocutaneous flap combined with the split graft in 5, stomach pulling-up in 12, colon interposition in 23 patients. While in cases without laryngeal functions preserved the methods includes: direct suture in 54, laryngotracheal flap in 54 patients, pectoralis major musculocutaneous flap in 7, laryngotracheal flap combined with pectoralis major musculocutaneous flap in 8, stomach pulling-up in 22, colon interposition in 3 patients. All patients received radiotherapy postoperatively (dose 55 - 75 Gy).

RESULTSThe overall 3 and 5 year survival rates were 65.1% (229/352) and 53.6% (142/265), respectively. For stage I, the 5 year survival rate was 3/3, stage II, 80.6%(25/31), stage III, 65.0% (67/103), stage IV, 36.7% (47/128). The 3 and 5 year survival rates in functionally preserved group were 68.0% (164/241) and 59.7% (114/191), respectively, while in non-functionally preserved group were 58.6% (65/111) and 37.8% (28/74), respectively. The cervical lymph node metastasis was found in 239 sides. Pathologic findings showed that well, moderately and lower differentiated squamous cell carcinomas were 84, 163, 105 cases, respectively. Laryngeal functions (voice, respiration and deglutition) were completely restored in 169 patients and partially restored (voice and deglutition) in 72 patients.

CONCLUSIONSCombined surgery and radiotherapy are the best choice for hypopharyngeal cancer. The continuity of the pharyngoesophagus is restored and the laryngeal function is preserved as far as possible. The preservation of laryngeal function and the laryngeal and pharyngeal reconstruction are based on the premise that the tumor was excised completely.

Adult ; Aged ; Aged, 80 and over ; Carcinoma, Squamous Cell ; therapy ; Combined Modality Therapy ; Female ; Humans ; Hypopharyngeal Neoplasms ; therapy ; Laryngectomy ; Male ; Middle Aged ; Pharyngectomy ; Retrospective Studies

BACKGROUNDA major shortcoming in tissue engineered blood vessels (TEBVs) is the lack of healthy and easily attainable smooth muscle cells (SMCs). Smooth muscle progenitor cells (SPCs), especially from peripheral blood, may offer an alternative cell source for tissue engineering involving a less invasive harvesting technique.

METHODSSPCs were isolated from 5-ml fresh rat peripheral blood by density-gradient centrifugation and cultured for 3 weeks in endothelial growth medium-2-MV (EGM-2-MV) medium containing platelet-derived growth factor-BB (PDGF BB). Before seeded on the synthesized scaffold, SPC-derived smooth muscle outgrowth cell (SOC) phenotypes were assessed by immuno-fluorescent staining, Western blot analysis, and reverse transcription polymerase chain reaction (RT-PCR). The cells were seeded onto the silk fibroin-modified poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (SF-PHBHHx) scaffolds by 6x10(4) cells/cm2 and cultured under the static condition for 3 weeks. The growth and proliferation of the seeded cells on the scaffold were analyzed by 3-(4,5-dimethylthiazol-2-yl)-diphenyltetrazolium bromide (MTT) assay, scanning electron microscope (SEM), and 4,6-diamidino-2-phenylindole (DAPI) staining.

RESULTSSOCs displayed specific "hill and valley" morphology, expressed the specific markers of the SMC lineage: smooth muscle (SM) alpha-actin, calponin and smooth muscle myosin heavy chain (SM MHC) at protein and messenger ribonucleic acid (mRNA) levels. RT-PCR results demonstrate that SOCs also expressed smooth muscle protein 22alpha (SM22alpha), a contractile protein, and extracellular matrix components elastin and matrix Gla protein (MGP), as well as vascular endothelial growth factor (VEGF). After seeded on the SF-PHBHHx scaffold, the cells showed excellent metabolic activity and proliferation.

CONCLUSIONSPCs isolated from peripheral blood can be differentiated into the SMCs in vitro and have an impressive growth potential in the biodegradable synthesized scaffold. Thus, SPCs may be a promising cell source for constructing TEBVs.

3-Hydroxybutyric Acid ; chemistry ; Animals ; Blood Vessels ; cytology ; Caproates ; chemistry ; Cell Adhesion ; Cell Differentiation ; Cell Proliferation ; Immunophenotyping ; Microscopy, Electron, Scanning ; Muscle, Smooth, Vascular ; cytology ; Myocytes, Smooth Muscle ; cytology ; RNA, Messenger ; analysis ; Rats ; Tissue Engineering ; Vascular Endothelial Growth Factor A ; genetics

OBJECTIVETo review the experience of different surgical construction methods for hypopharyngeal cancer with cervical esophageal invasion.

METHODSFrom 1989 to 2000,forty-eight patients with advanced hypopharyngeal cancer and cervical esophageal invasion were retrospectively reviewed, including 38 males and 10 females. The median age was 54. 3 years old, ranged from 26 to 71 years old. According to UICC 1997 criteria, all the tumors were T4 stage and originated from the pyriform sinus (33), posterior pharyngeal wall (14), postcricoid area (1), there were 28 patients in cN0, 15 in cN1, 5 in cN2 and no distant metastasis. Precise preoperative evaluation was performed with computed tomography scan, barium swallow perspective and biopsy. All the patients received modified neck dissection, including both unilateral (38 patients) and bilateral (10 patients). Pharyngoesophageal defect reconstruction methods were: laryngotracheal flap in 11 patients, pectoralis major musculocutaneous flap in 13, laryngotracheal flap combined with pectoralis major musculocutaneous flap in 6, pectoralis major musculocutaneous flap combined with the split graft in 10, stomach pulling-up in 3, colon interposition in 5 patients. Total laryngectomy was carried out in 8 patients. All patients received radiotherapy postoperatively (dose 55 - 75 Gy).

RESULTSThe cervical lymph node metastasis was found in 20 patients. Pathologic findings showed that well, moderately and lower differentiated squamous cell carcinomas were 18, 24, 6 cases, respectively. The overall 3 and 5 year survival rates were 52.1% (25/48) and 27.3% (12/44), respectively. The 3 and 5 year survival rates in functionally preserved group were 65.2% (15/23) and 33.3% (7/21), while in non functionally preserved group were 40.0% (10/25) and 21.7% (5/23), respectively. Fifteen patients laryngeal functions (voice, respiration and deglutition) were completely restored and 8 patients partially restored (voice and deglutition). The decannulation rate was 65% (15/23). The complication included pharyngeal fistulas in 10 cases and splitting of chest wall in 1 cases.

CONCLUSIONSCombined therapy was the best choice for hypopharyngeal cancer with cervical esophageal invasion. The laryngeal function is preserved as far as possible. The continuity of the pharyngoesophagus was restored by pectoralis major musculocutaneous flap, laryngotracheal flap, or combined with the split graft. Stomach transposition or colon interposition was used while the defect of the esophagus was greater.

Adult ; Aged ; Carcinoma, Squamous Cell ; mortality ; pathology ; surgery ; Esophageal Neoplasms ; mortality ; secondary ; surgery ; Esophagus ; pathology ; Female ; Humans ; Hypopharyngeal Neoplasms ; mortality ; pathology ; surgery ; Lymphatic Metastasis ; Male ; Middle Aged ; Retrospective Studies ; Survival Rate

Purpose: Cavernous nerve injury induced erectile dysfunction (ED) is a refractory complication with high incidence in person under radical prostatectomy. Studies have shown that relaxin-2 (RLX-2) plays a vital role of endothelial protection, vasodilation, anti-fibrosis and neuroprotection in a variety of diseases. However, whether penile cavernous erection can benefit from RLX-2 remains unknown. The purpose of the experiment was to explore the effects of RLX-2 on ED in the rat suffering with bilateral cavernous nerve injury (BCNI). Materials and Methods: The rats were divided into three groups: Sham group was underwent sham operation, BCNI+RLX group or BCNI group was underwent bilateral cavernous nerve crush and then randomly treated with RLX-2 (0.4 mg/kg/d) or saline by continuous administration using a subcutaneously implanted micro pump for 4 weeks respectively. Then, erectile function was evaluated by electrical stimulation of cavernous nerves. Cavernous nerves and penile tissues and were collected for histological evaluation. Results: Erectile function of rats with BCNI was partially improved after RLX-2 treatment. The BCNI group had lower expression of relaxin family peptide receptor (RXFP) 1, p-AKT/AKT, p-eNOS/eNOS ratios than sham operation rats, but RLX-2 could partially reversed these changes. Histologically, the BCNI+RLX group had a significant effect on preservation of neurofilament, neuronal glial antigen 2 of penile tissue and nNOS of cavernous nerves when compared with BCNI group. RLX-2 could inhibited the lever of BCNI induced corporal fibrosis and apoptosis via regulating TGFβ1-Smad2/3-CTGF pathway and the expression of Bax/Bcl-2 ratio, caspase3. Conclusions RLX-2 could improve erectile function of BCNI rats by protecting cavernous nerve and endothelial function and suppressing corporal fibrosis and apoptosis via RXFP1 and AKT/eNOS pathway. Our findings may provide a promising treatment for refractory BCNI induced ED.