Objective:To generate Epstein-Barr virus(EBV) Latent Membrane Protein 2A(LMP2A) recombinant adenovirus,and provide for further investigation on the therapy vaccine against EBV associated malignancies.Methods:Full length cDNA of encoding LMP2A of EBV had been amplified by reverse transcription-PCR and cloned into pGEM-T vector.The encoding cDNA of LMP2A was inserted into E1,E3-substituted adenovirus vector pAX1CW,then the LMP2A recombinant adenovirus vector was contransfected into 293 cells togetherwith EcoT221 digested Ad5-TPC.The LMP2A recombinant adenovirus was generated by homologous recombination,and primarily identificated by ClaI enzyme digestion.The expression of LMP2A on CV1 cells infected with recombinant adenovirus analyzed by fluorescence-activated cell sorting(FACS) and confocal microscope.Results:The replication-deficient LMP2A recombinant adenovirus was generated efficiently with the titers of 2.3?10 8 pfu/ml.The LMP2A could be seen on CV1 cells membrane with confocal microscope 48 h post infected with recombinant adenovirus and the percentage of CV1 cells expressing LMP2A was 94.4% by means of FACS analysis.Conclusion:These suggested that LMP2A could be expressed efficiently by recombinant adenovirus mediated transfer,and it was the foundation of further researching in its function and developing the suitable genetic engineering vaccine against EBV associated malignancies.

Objective:Generation and functional analysis of EBV LMP2A specific CTL elicited by DC transfected with recombinant adenovirus in vitro .Methods:PBMC were isolated from healthy EBV carriers and NPC patients, and then cocultured with autologous mature Ad5 LMP2A transfected DC at the ratios of 20∶1. Cytotoxicity of LMP2A specific CTL was determined with LDH release assay, the populations of CTL were performed by FACS,the IFN ? secretion and FasL mRNA expression of the CTL were detected by biological activity assays and RT PCR, respectively.Results:The results showed that high cytotoxicity of LMP2A specific CTL could be elicited by autologous transfected DC. The cytotoxicity boosted with the increase of transfected DC stimulation times, but there were no significant changes between two and three stimulations.The phenotypic analysis demonstrated that the LMP2A specific CTL induced at day 14 consisted of a majority of CD4 + and CD8 +T cells, and only a small percentage of CD56 + cells. The IFN ? secreted in the supernatants of cell culture also increased with the stimulation times. In addition, the specific CTL at day 14 from EBV healthy carriers could express FasL mRNA.Conclusion:Strong and functional EBV specific CTL could be generated by autologous mature DC transfected with adenovirus encoding LMP2A, which could provide a rationale for the immunotherapy of EBV associated NPC. [

Biopsy ; Dissection ; Gastric Mucosa ; pathology ; surgery ; Gastroscopy ; methods ; Humans ; Neoplasm Invasiveness ; Neoplasm Staging ; Neoplastic Cells, Circulating ; Stomach Neoplasms ; pathology ; surgery ; Stomach Ulcer ; pathology

OBJECTIVE: To explore the possibility of far-lateral retro-condylar approach in an attempt to apply endoscope. METHOD: For anatomical information, the microneurosurgical anatomical dissection, observation and measurement had been performed under microscope and endoscope by mimicking the far-lateral retro-condylar approach on 10 adult cadaver heads and 10 adult dry skulls. RESULT: The complex relationship exists between the osseous jugular foramen and its adjacent structures. The exposed anatomic structures of jugular foramen region were observed under microscope and endoscope without drilling occipital condyle and jugular tubercle. CONCLUSION With the technology of modern microsurgery and endoscope, several diseases in jugular foramen region can be operated via far-lateral retro-condylar approach without drilling occipital condyle and jugular tubercle.
Adult ; Endoscopy ; Female ; Humans ; Jugular Veins ; anatomy & histology ; Male ; Occipital Bone ; anatomy & histology ; surgery

OBJECTIVETo study the reasons for the discrepancies in pathologic diagnosis of gastric dysplasia/early cancer in endoscopic submucosal dissection (ESD) specimens, and how to cope with the discrepancies.

METHODSThe pathologic diagnoses in 60 cases of ESD specimens according to the three currently used classification systems (namely Western criteria, Japanese criteria and Vienna classification) were compared. The diagnostic discrepancies were analyzed.

RESULTSFifteen of the 17 cases diagnosed as low-grade intraepithelial neoplasia according to the Western criteria were revised as adenoma by the Japanese criteria. Amongst the 43 cases of gastric intramucosal adenocarcinoma diagnosed according to the Japanese criteria, 23 cases had concordant diagnosis by the Western criteria. While the diagnosis of low-grade intraepithelial neoplasia/adenoma was basically similar irrespective of classification system used, there were significant differences in the interpretation of gastric early cancer.

CONCLUSIONSThe diagnostic discrepancies in the gastric dysplasia/early cancer are mainly related to the morphologic criteria applied in different classifications. In order to facilitate clinical and pathologic communication, a consensus using Vienna/WHO classifications, supplemented with Japanese system, is desirable.

Adenoma ; pathology ; Carcinoma in Situ ; pathology ; Dissection ; methods ; Gastroscopy ; Humans ; Hyperplasia ; pathology ; Stomach ; pathology ; Stomach Neoplasms ; pathology

OBJECTIVETo study the influence of heat-induced epitope retrieval (HIER) on endogenous avidin-binding activity (EABA) and to establish an effective way to block EABA in immunohistochemistry.

METHODSSystematically screening EABA in 164 (679 samples) formalin-fixed and paraffin-embedded human tissues including 76 (102 samples) normal tissues and 88 (577 samples) tumor tissues as well as 4 (80 samples) formalin-fixed and paraffin-embedded rat normal tissues using tissue array (tissue chip), HIER, immunohistochemistry and egg white solution blocking. In addition, EABA was also examined in 9 (15 samples) human frozen tissues.

RESULTS(1) EABA was detected in frozen tissues. (2) No staining for EABA was seen in formalin-fixed and paraffin-embedded tissues. (3) EABA was revealed after the tissues treated with microwave HIER. (4) The density of signal for EABA was variable from tissue to tissue and cell to cell. (5) The signals of EABA expressed in scatter or diffuse in tissues and in granular form in cytoplasm. (6) EABA was found in a wide range of epithelial tissues, especially in gland epithelia of normal and tumor tissues. These included kidney, adrenal cortex, liver, C cells of thyroid gland, oxyphil cells of parathyroid, fundal gland of stomach, sebaceous gland of skin, duct of salivary; oncocytoma and papillary adenocarcinoma of kidney and thyroid gland, adenolymphoma of parotid, carcinoma of liver cell, adenocarcinoma of stomach, colon, prostate, gall bladder and endometrium, and so on. (7) EABA was easier revealed by higher pH value buffer (EGTA pH 9.0) than that with lower pH value (EDTA pH 8.0 and citrate pH 6.0). (8) The revealed EABA could be effectively blocked using 20% egg white solution.

CONCLUSIONSHIER could unmask EABA in formalin-fixed and paraffin-embedded tissues. The unmasked EABA present in a wide range of human normal and tumor tissues as well as in rat normal tissues. The EABA could influence routine immunohistochemistry staining when using (strept)avidin-horseradish peroxidase detective system. The egg white solution could effectively block EABA and eliminate the influence of EABA on immunohistochemistry.

Animals ; Avidin ; antagonists & inhibitors ; metabolism ; Biotin ; metabolism ; Cells, Cultured ; Egg Proteins ; pharmacology ; Epithelial Cells ; metabolism ; Epitopes ; Female ; Hot Temperature ; Humans ; Immunohistochemistry ; Male ; Neoplasms ; metabolism ; pathology ; Rats

Objective To investigate the clinical features and treatment of solid pseudopapillary neoplasm (SPN) of the pancreas.Methods The retrospective cross-sectional study was conducted.The clinicopathological data of 69 patients with SPN of the pancreas who were admitted to the Qilu Hospital of Shandong University from January 2012 to July 2017 were collected.Serum tumor markers detection,enhanced computed tomography (CT) and magnetic resonance imaging (MRI) of abdomen were carried out preoperatively for all the patients,and a part of the patients received endoscopic ultrasonography (EUS).Surgery plans were formulated after completion of examinations.Observation indicators:(1) clinical features;(2) treatment situation;(3) results of pathological examination;(4)follow-up.All the patients were followed up via outpatient examination and telephone interview to detect the survival and tumor recurrence and metastasis till July 2017.Measurement data with normal distribution were presented by (x)±s and were compared by Student's t test.Count data were compared by chi-square test.Results (1) Clinical features:① epidemiologic features:the ratio of male to female was 1∶5.9;patients were aged between 9 and 65 years,including 40 under 30 years and 29 above or equal to 30 years.The onset age was (34± 15)years for male patients and (28 ± 11)years for female patients,respectively,with no statistically significant difference (t=1.364,P＞0.05).Of 69 patients,SPN was located at pancreatic uncinate process in 25 patients,at neck of pancreas in 12 patients,body and tail of pancreas in 32 patients.② Medical history:history of acute or chronic pancreatitis and abdominal trauma were denied by all the 69 patients.③ Clinical manifestation:26 patients had no obvious symptoms and were detected by physical examination;31 patients had discomfort in upper abdomen,nausea and vomiting;other patients were admitted to the hospital because of upper abdominal mass (10 patients),jaundice (1 patient) or nausea,constipation (1 patient).④ Laboratory examination:the levels of preoperative carcinoembryonic antigen (CEA) and CA19-9 were normal.⑤ Imaging examination:plane scan of the CT examination showed round or round-like low-density shadows in the 69 patients,including 51 of cystic solid lesion,13 of solid lesion and 5 of cystic lesion.Complete capsules were observed in 64 patients,blurred boundary between pancreas and adjacent viscera in 5 patients,calcified foci in the pancreatic parenchyma and capsules in 14 patients.Ten patients received MRI examination,and the T1-weighted images showed equal or slightly lower signal,T2-weighted images showed slightly higher signal in the plane scan,and T1-weighted and T2-weighted images of the tumor capsule showed continuous or non-continuous ring-like signal.The results of enhanced scan showed slightly heterogeneous enhancement of the capsule and the parenchyma of the pancreas in the arterial phase,and progressive enhancement in the venous and delayed phase,while the enhancement degree was lower than that of the normal pancreas parenchyma.The parenchyma was cloud-,papillaor mural nodule-like enhanced.Obvious enhancement was observed in capsule while not in the cystic components.The boundaries of the tumors in 5 patients were clear under EUS.Hypo-,iso-and hyperechoic regions were found in the masses,and the masses were confirmed as cyst-solidary type.Obvious calcified foci were found in 1 patient.(2) Treatment outcome:twenty-seven patients received laparoscopic surgery,including tumor expiration in 13 patients,distal pancreatectomy with preservation of spleen in 8 patients,distal pancreatectomy combined with splenectomy in 2 patients,middle pancreatectomy in 2 patients,pancreaticoduodenectomy with preservation of pylorus in 1 patient,pancreatic head resection with preservation of duodenum under the assistance of laparoscopy in 1 patient.Forty-two patients received open surgery,including tumor expiration in 12 patients,distal pancreateetomy with preservation of spleen in 10 patients,distal pancreatectomy combined with spleneetomy in 6 patients,middle pancreatectomy in 5 patients,pancreaticoduodenectomy in 7 patients (with preservation of pylorus in 2 patients) and pancreatic head resection combined with preservation of duodenum in 1 patient.One patient with SPN + hepatic metastasis received distal pancreatectomy+ metastatic foci resection in the lesser omental bursa,and then followed by radiofrequency ablation in the hepatic metastatic foci.Postoperative complications:21 of 69patients had postoperative complications,including 17 intestinal fistulas,2 abdominal bleedings,1 incomplete obstruction,1 pleural effusion + atelectasis,and all of them were cured by symptomatic treatment.(3)Pathological examination:the resection margins of 69 patients were negative.The mean diameter of the tumor was (7±4) cm (21 patients with tumor diameter ＜ 5 cm,and 48 with tumor diameter ≥5 cm).The tumor diameters of 4 in 10 male patients were above or equal to 5 cm,and the number was 44 in 59 female patients,with statistically significant difference (x2 =4.828,P＜0.05).The tumor diameters of 32 in 40 patients who aged under 30 years were above or equal to 5 cm,and the number was 16 in 29 patients who were aged above or equal to 30 years,with statistically significant difference (x2=4.895,P＜0.05).Solid,pseudo-papillary and cystic regions in the SPN tissues were seen under the light microscope.Tumor cells were surrounded the blood vessels and were arranged in the nest or sheet shape in the solid region;blood vessels were surrounded by one or multiple layers of tumor cells in the axis or pseudopapillary shape in the pseudopapillary region;large amount of mucus and clusters of blood cells were seen in the cystic regions.The result of immunohistochemistry showed that the positive rates of α1-antitrypsin,vimentin,β-catenin,progesterone receptor,CD10,synaptophysin and chromogranin A were 100.0％ (39/39),96.6％ (28/29),95.7％ (45/47),94.4％ (51/54),92.5％ (49/53),72.9％ (35/48) and 5.6％ (3/54),respectively.(4) Follow-up:63 of 69 patients were followed up for 1-68 months,with median time of 29 months.No SPN recurrence or metastasis was detected.One patient died of lung cancer at postoperative month 35 and other patients survived well.Conclusions SPN of pancreas is mostly detected in young female patients,and it could be solid or cystic.Abdominal enhanced CT or MRI examination could clarify the diagnosis.EUS-fine needle aspiration examination could provide pathological evidence for definitive diagnosis.Typical cellular morphology and pseudopapillary regions may provide hints for the diagnosis of SPN,and the diagnosis could be clarified when combined with the detection of vimentin,α 1-antitrypsin or other indexes.Complete resection of SPN and ensure negative resection margin are fundamental principles of treatment.

Objective: To investigate whether the cyclic adenosine monophosphate (cAMP) / exchange proteins directly activated by cAMP (Epac) / ras-related protein 1 ( Rap1 ) signalling pathway is involved in the intervening mechanisms of interleukin-1 β (IL-1 β) ，tumor necrosis factor-α (TNF-α) ，brain-derived neurotrophic factor (BDNF) and Jujuboside A(JuA) secretion by NG2 cells． Methods: NG2 cells were cultured in vitro and the experiment was divided into control group ，pertussis toxin ( PTX) group ，ESI-09 group，JuA group and positive drug group．The effect of different concentrations of JuA on the survival rate of NG2 cells was detected by CCK-8 method，and the expression of IL-1 β , TNF-α , BDNF，cAMP，Epac，Rap1 mRNA and protein in each group was detected by RT-PCR and Western blot. Results: Compared with the control group，the PTX group decreased the expression of IL-1 β and TNF-α mRNA and protein (P＜0. 01) and increased the expression of cAMP and BDNF mRNA and protein (P＜0. 01) ; the ESI-09 group increased the expression of IL-1 β and TNF-α mRNA and protein (P < 0. 05) and decreased the expression of BDNF，Epac and Rap1 mRNA and protein expression (P＜0. 01) ; the JuA group and positive drug group increased IL-1 β , TNF-α , BDNF，cAMP，Epac，Rap1 mRNA and protein expression (P＜0. 01) ． Conclusion The cAMP / Epac / Rap1 signaling pathway is involved in the secretion of IL-1 β , TNF- α , and BDNF by NG2 cells．JuA may act on cAMP / Epac / Rap1 signaling pathway to affect the secretion of BDNF by NG2 cells.