Identification of molecular mechanism of necroptosis mediated by PI3K / 军事医学

Objective To identify the molecular mechanism of phosphatidylinositol-3-kinase (PI3K) in mediating necroptosis induced by tumor necrosis factor alpha (TNFα).Methods RNA interference mediated by lentivirus short hairpin RNA(shRNA) was used to downregulate p110α in L929 cells and Western blotting was used to determine the knockdown efficiency.In addition,Western blotting was used to detect the phosphorylation level of RIP1,RIP3 and MLKL in L929 cells treated with or without TNFα plus Z-VAD.Duolink assay kit was used to detect the interactions between different proteins or the same proteins.Results p110α knockdown was efficiently mediated by the lentivirus shRNA,which significantly suppressed the phosphorylation of RIP1,RIP3 and MLKL in the absence or presence of TNFα plus Z-VAD stimulation.Moreover,the interactions between p110α and RIP3,but not RIP1,increased in a time-dependent manner during the process of necroptosis,and p110α knockdown significantly inhibited the formation of necrosome and RIP1 homodimer or RIP3 homodimer.Conclusion PI3K mediates the TNFα-induced necroptosis by promoting the activation of RIP1/RIP3/MLKL signaling pathway.Given the increasing direct interactions between p110α and RIP3 during the process of necrosome formation,PI3K may regulate RIP3 kinase activity via direct phosphorylation of RIP3.

Transformation activity and antigenicity of the human papillomavirus type 58 E6E7 fusion gene mutant / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To develop a prophylactic and therapeutic vaccine against human papillomavirus (HPV) type 58-associated cervical carcinoma, and explore its transformation activity and antigenicity.</p><p><b>METHODS</b>The E6 and E7 three amino acid codons in the HPV 58 virus were modified respectively and fused. The modified and fused gene was named HPV58 mE6E7. The recombinant HPV58 mE6E7 gene was inserted into pIRES-neo vector to generate plasmid pIRES-neo-HPV58 mE6E7. Then NIH/3T3 cell line was transfected with plasmid pIRES-neo-HPV58 mE6E7. The pIRES-neo-HPV58 mE6E7-transfected cells were the experimental group, pIRES-neo-HPV58 E6E7-transfected cells were the positive control group, and pIRES-neo empty vector-transfected cells were the negative control group. The expression of HPV58 mE6E7 protein in the experimental cells was detected by flow cytometry, immunofluorescence and Western blot. The transformation activity of HPV58 mE6E7 was tested by soft agar colony formation assay and subcutaneously tumors in nude mice. Finally, DNA vaccine was constructed with HPV58 mE6E7 fusion antigen and used to immunize C57BL/6 mice with the vaccine plasmids. The specific serum antibodies were detected by EIISA, and the number of splenic specific CD8(+) T cells secreting IFN-γ of the immunized mice was detected by ELISPOT assay.</p><p><b>RESULTS</b>Sequencing confirmed the expected mutation and a 100% homogeneity of the HPV58 E6E7 fusion gene. Stable transfected NIH/3T3 cells expressing HPV58 mE6E7 and HPV58 E6E7 gene were 70.3% and 84.1%, respectively. The relative expressions of HPV58 mE6E7 and HPV58 E6E7 fusion protein in 3T3-HPV58 mE6E7 experimental cells and 3T3-HPV58 E6E7 positive control cells were 2.1 ± 1.7 and 3.8 ± 1.4, respectively, and were negative in the negative control group. No colony formation was found in the experimental and 3T3-neo negative control cell groups, and 31 colonies were found in the positive control cell group, among them 10 colonies were consisted of more than 50 cells. No tumor mass was formed within 4 weeks in the nude mice of experimental and negative control groups, but among the 10 mice of positive control group tumor was formed in 6 mice. Using HPV58 mE6E7 fusion gene as target antigen of DNA vaccine, the antibody titer was 25 600, and specific immunity spots were 218.8 ± 34.4, significantly higher than that in the control group.</p><p><b>CONCLUSIONS</b>The fused and modified HPV58 E6E7 amino acid codons can abolish the transformation activity but preserve its antigenicity. HPV58 mE6E7 is a potential target gene for the development of therapeutic DNA vaccine against HPV58-associated cervical cancer.</p>

Construction of a replicative anti-tumor DNA vaccine PSCK-2PFcGB and its expression in vivo and in vitro / 南方医科大学学报

<p><b>OBJECTIVE</b>To construct a replicative anti-tumor DNA vaccine PSCK-2PFcGB based on Semliki Forest Virus (SFV) replicon vector and observe its expression in vivo and in vitro.</p><p><b>METHODS</b>The plasmid pVAX1-2PFcGB was digested with Nhe I, and the digestion product was blunted prior to further digestion with BssH II to obtain the fragment 2PFcGB, a fusion gene containing the multitarget complex antigen 2PAG encoding both the most cytotoxic T lymphocyte epitopes of human survivin and chorionic gonadotropin β chain-CTP37 of human and monkey. The 2PFcGB fragment was inserted into the PSCK vector digested by Sma I. The products with the expected size were extracted and ligated, and the positive clones were screened by kanamycin and amplified. The recombinant PSCK-2PFcGB, following identification by colony PCR and restriction endonuclease Nde I, was transfected into 293T cells via lipofectamine 2000 and its expression was detected. The recombinant plasmid was also transfected into mouse quadriceps femoris muscle to observe its expression in vivo by immunohistochemistry.</p><p><b>RESULTS</b>Nde I digestion resulted in a fragment of the expected size. Transfection with the recombinant plasmid PSCK-2PFcGB resulted in successful expression of the antigen and adjuvant molecular protein in 293T cells, with the positivity rates of 5.70% and 19.75%, respectively. The fusion tumor antigen survivin and hCGβ-CTP37 were also detected in the muscular tissues of the mice.</p><p><b>CONCLUSION</b>A novel replicative anti-tumor DNA vaccine PSCK-2PFcGB has been successfully constructed and can be expressed in 293T cells and in the muscular tissues of immunized mice, which provide a basis for further studies of the antitumor activity and immunological mechanism of the DNA vaccine.</p>

Atrophy of Multifidus Muscle on Low Back Pain Patients

OBJECTIVE: To investigate the correlation of multifidus muscle atrophy on MRI findings with clinical findings in low back pain patients. METHOD: Medical records of 80 patients presenting with low back pain were retrospectively reviewed. Their MR images were visually analysed to know lumbar multifidus muscle atrophy, disc herniation, disc degeneration, spinal stenosis and nerve root compression. RESULTS: Multifidus muscle atrophy increased from the upper lumbar level to the most caudal intervertebral level. It was bilateral in the majority of the cases. Multifidus muscle atrophy was well correlated with patient's age, referred leg pain, and disc degeneration. However, duration of low back pain, disc herniation, spinal stenosis, nerve root compression, sex, weight, height and BMI had no correlation with multifidus muscle atrophy. CONCLUSION: Examination of multifidus muscle atrophy should be considered when assessing MR images of lumbar spine. It may help for further evaluation and planning the treatment modalities of low back pain.

Off-pump CABG for Unstable Angina Complicated With COPD / 대한흉부외과학회지

In an attermpt to aviod the deleterious effects of cardiopulmonary by pass such as pulmonary complication neurologic complication and renal failure off-pump CABG has been rediscovered and developed. We experienced off-pump CABG in 2 cases with unstable angina complicated with COPD and report herein the cases with review of literature.

Sequential changes of Interleukin-6 , Tumor Necrosis Factor-alpha, and Troponin-T During Open Heart Surgery with Cardiopulmonary Bypass / 대한흉부외과학회지

BACKGROUND: Immunologic and inflammatory responses of cardiopulmonary bypass(CPB) influence postoperative mortality and morbidity with multiple organ injury. It has been reported that ischemia/reperfusion induced-myocardial injury during CPB is causative of release of inflammatory cytokines such as interleukin-6(IL-6) and tumor necrosis factor-alpha (TNF-alpha). The purpose of this study was to detect the time course of the activated cytokine and troponin-T(TnT), and to examine the correlation between such parameters during CPB. MATERIAL AND METHOD: The serial samples were collected from arterial blood via radial arterial catheter in 23 patients who are underwent open heart surgery (OHS) with CPB, the IL-6, TNF-alpha and TnT were checked. RESULT: (1) IL-6, TNFalpha- and TnT concentration increased significantly during CPB with a peaking level of CPB-off (p 0.05). (2) IL-6 had highly positive correlation with aortic cross clamping time and total bypass time(r=0.80, 0.78; p 0.05, respectively). (3) There was no correlation among IL-6, TNF-alpha and TnT. CONCLUSION: In conclusion, these data showed that elevated production of serum IL-6 during CPB was attributable to ischemia/reperfusion induced-myocardial damage. IL-6 will become a new and sensitive biological marker in assessment of myocardial damage during OHS with CPB. However, further studies will be needed to apply IL-6 in more patient population.

Clinical Experiences of Open Heart Surgery / 대한흉부외과학회지

BACKGROUND: From Sept. 1985 to Sept. 1997, 2,000 cases of open heart surgery (OHS) were performed in the Department of Thoracic & Cardiovascular Surgery, Pusan Paik Hospital, College of Medicine, Inje University. MATERIAL AND METHOD: Among the total of 2,000 cases of OHS, 1532 cases were congenital heart disease (CHD) and 468 cases were acquired heart disease (AHD). The age distribution was 9 days (4.0kg) to 68 years in CHD and 11 to 66 years in AHD. In 1532 cases of CHD, there were 1403 acyanotic cases and 129 cyanotic cases. RESULT: The CHD cases consisted of 940 ventricular septal defects (61.4%), 324 atrial septal defects (21.1%), 112 tetralogy of Fallot (7.3%), 46 pulmonary stenosis (3%), 38 endocardial cushion defects (2.5%), 15 valsalva sinus ruptures (1%), 4 transposition of great arteries (0.3%), 4 double outlet right ventricles (0.3%), and etc. Corrective operations were applied for congenital heart disease with a result of 3.1% hospital mortality. Of 468 AHD, 381 cases were valvular heart diseases, 48 ischemic heart diseases, 12 cardiac tumors, 8 annuloaortic ectasias, 16 dissecting aortic aneurysms and etc. In the 381 valvular heart diseases, there were 226 single valve replacements (36 aortic valve replacements (AVR), 188 mitral valve replacements (MVR), and 2 tricuspid valve replacements (TVR), among these were 71 cases of double valve replacements (AVR & MVR), 54 cases of MVR with tricuspid valve annuloplasty (TVA), and 18 cases of AVR, MVR with TVA. The total implanted prosthetic valves were 466. In MVR, 123 St. Jude Medical valves, 90 Carpentier-Edwards valves, 65 CarboMedics valves, 42 Sorin valves and 16 other valves were used. In AVR, 68 St. Jude Medical valves, 36 CarboMedics valves, 14 Carpentier-Edwards valves and 9 other valves were used. Coronary Artery Bypass Surgery (CABG) were performed in 48 cases. The patterns of bypass graft were 14 patients of single vessel graft, 21 patients of two vessels graft, 10 patients of three vessels graft and 3 patients of four vessels graft. CONCLUSION: The hospital operation mortality rate of congenital acyanotic, cyanotic and acquired heart diseases were 2.0%, 15.5%, and 5.1% respectively. The overall mortality rate was 3.6% (72/2,000).

Video Assisted Thoracoscopic Thoracic Sympathectomy for Palmar Hyperhidrosis / 대한흉부외과학회지

Hyperhidrosis is one of abnormalities in autonomic nervous system, it has been treated with dermatologic principles or thoracic sympathectomy via thoracotomy. But these techniques were rather ineffective or invasive. Recently, Video Assisted Thoracoscopic Surgery (VATS) is widely applided in thoracic surgical area, and palmar & axillary hyperhidrosis is not the exception. From August 1995 to February 1997, 52 patients with bilateral palmar hyperhidrosis underwent bilateral thoracic sympathectomy with VATS in the department of thoracic & cardiovascular surgery, Inje university, Pusan Paik Hospital. There were 27 men and 25 women and the mean age was 22 years. Mean operating time was 172 min and unilateral sympathectomy via minithoracotomy was applied in one patient due to severe pleural adhesion. Mean postoperative hospital stay was 2.6 days. During mean 12.5 months follow-up, there was no recurrence of sweating in the both hands. Thirty patients (57.7%) complained moderate degree of compensatory sweating, but the discomfort was decreased in severity. 83.8% of all patients were satisfied with the result of operation.

Primary Malignant Lymphoma of Lung: A Case Report / 대한흉부외과학회지

Primary malignant lymphoma of lung is a very rare disease, only 0.34% of all malignant lymphomas. In our case, a 76 year old male patient had a solitary mass without lympha-denopathy at chest CT scan. He underwent right middle lobectomy through a posterolateral thoracotomy incision. Pathologic study confirmed a diagnosis of malignant lymphoma and chemotherapy was started by an oncologist.