Objective: To predict and identify HLA-A * 0201 restricted CD8+ CTL epitopes in Mycobacterium tuberculosis (Mtb) antigen Ag85C, so as to provide evidence for epitope-based study for tuberculosis (TB) vaccine. Methods: The online database SYFPEITHI was applied to predict the potential HLA-A * 0201 restricted epitopes from Ag85C, an antigen of Mycobacterium tuberculosis. T2 cell line was used to assay the affinity between the predicted peptides and HLA-A * 0201 molecules. The specific CTL lines were induced from peripheral blood mononuclear cells (PBMCs) of HLA-A * 0201 positive TB patients and PPD+ healthy donors by peptides with high binding affinity to HLA-A * 0201 molecules. IFN-γ production, in vitro proliferation and cytotoxicity of peptide-induced CTL were determined to screen HLA-A * 0201 restricted CD8+ CTL epitopes from those candidates. Results: Fourteen potential epitopes were identified from the SYFPEITHI database. After binding affinity assay, 3 of the 14 peptides (170-178 aa, 317-325 aa, and 144-153 aa) were found to have high binding affinity to HLA-A * 0201 molecules. However, only one peptide (144-153 aa) stimulated its specific CTL to release IFN-γ, proliferate in vitro and produce specific cytotoxicity. Conclusion: We have successfully identified a HLA-A * 0201 restricted CD8+ CTL epitope of Mtb Ag85C-FLTREMPAWL(144-153 aa), which might be a candidate epitope for TB vaccine designing. Our findings provides a basis for developing novel and effective anti-TB vaccine.

Objective: To construct a recombinant multiepitope fusion polypeptide containing 6 HLA-A * 0201 restricted CTL epitopes derived from Mycobacterium tuberculosis (Mtb.) antigens and express it in E. coli. Methods: The full-length sequence encoding 6 HLA-A * 0201 restricted CTL epitopes were derived from Mtb. antigens Rv03O9, Rv0173, and Ag85A, Ag85C; pan DR Th epitope (PADRE), Trojan peptide, and furin-sensitive linker RVKR were synthesized, amplified by polymerase chain reaction(PCR), inserted into the expression vector pGEX-4T-1, and transformed into E. coli BL21 (DE3). After induced by isopropylthio-β-D-galactoside (IPTG), the recombinant protein expression was examined by SDS-PAGE and Western blot. Results: The recombinant multiepitope fusion polypeptide plasmid was successfully constructed and expressed(about 40 000) in E. coli BL21 (DE3). Conclusion: The recombinant multiepitope fusion polypeptide may provide a basis for developing novel TB vaccine.

OBJECTIVETo determine the expression of Skp2 in different prostate cancer (PCa) cell lines and tissues, and explore its influence on the androgen receptor (AR) signaling pathway and development of castration-resistant prostate cancer (CRPC).

METHODSThe expression levels of Skp2 and AR in different PCa cell lines were detected by Western blot. After knockdown of Skp2 in the C4-2 and 22RV1 cells transfected with shRNA, the expressions of AR and P27 were determined and the activity of ARR3-Luc measured by dual-luciferase reporter gene assay following treatment with dihydrotestosterone (DHT). The expressions of AR and Skp2 in human naïve PCa or CRPC specimens were detected by immunohistochemical staining followed by analysis of their differences and correlation.

RESULTSThe Skp2 protein expression level was significantly higher in the C4-2 or 22RV1 cells than in the LNCaP cells. DHT treatment increased the expression of Skp2 in the C4-2 cells, but knock-down of Skp2 significantly up-regulated the expression of the well-known downstream protein P27 and down-regulated that of AR. Consistently, DHT treatment increased the activity of ARR3-Luc, while knockdown of Skp2 remarkably decreased it in the C4-2 and 22RV1 cells (P < 0.05). In addition, significantly higher expressions of Skp2 and AR were observed in the CRPC than in the naïve specimens (P < 0.05), with a positive correlation between the two proteins (r = 0.658 1, P < 0.05).

CONCLUSIONSkp2 can enhance the expression and transcription activity of the AR protein in CRPC cells or tissues and is promising to be a critical molecular therapeutic target.

Androgens ; pharmacology ; Cell Line, Tumor ; Dihydrotestosterone ; pharmacology ; Disease Progression ; Gene Knockdown Techniques ; Humans ; Male ; Neoplasm Proteins ; genetics ; metabolism ; Prostatic Neoplasms, Castration-Resistant ; metabolism ; Receptors, Androgen ; genetics ; metabolism ; S-Phase Kinase-Associated Proteins ; physiology ; Transcriptional Activation ; Up-Regulation

Adolescent ; Adult ; Aged ; Child ; Child, Preschool ; China ; epidemiology ; Female ; Humans ; Infant ; Infant, Newborn ; Male ; Metapneumovirus ; Middle Aged ; Paramyxoviridae Infections ; epidemiology ; pathology

The high performance liquid chromatography-fluorescence micelle assay (HPLC-FMA) method for the content determination of polysorbate 80 in monoclonal antibody drugs was validated to study its applicability and transferability between various laboratories, and the feasibility to be included in the Chinese Pharmacopoeia. Both J.T. Baker and Nanjing Well-sourced polysorbate 80 was used in the collaborative validation of polysorbate 80 content analysis in seven different laboratories. The results show that when the protein concentration was no more than 20 mg·mL^-1 and the concentration of polysorbate 80 ranged from 0.05 to 0.5 mg·mL^-1, the method had good specificity. The recovery rates of the spiked samples ranged from 92.20% to 117.70% for J.T.Baker and from 93.90% to 117.20% for Nanjing Well. The intra-laboratory precision (%RSD) was less than 4.30% for J.T. Baker, and less than 2.60% for Nanjing Well, while the overall precision was less than 5.45% for J.T. Baker, and less than 6.70% for Nanjing Well. The linear correlation coefficient was more than 0.98 for J.T. Baker and more than 0.99 for Nanjing Well. The results of the collaborative validation prove that the HPLC-FMA method has good accuracy, precision, linearity, and specificity, and could be used for release control analysis of polysorbate 80 content in monoclonal antibodies across different laboratories.

Abstract: Mycobacterium senegalense is one of the major pathogens causing bovine farcy, and reports of its infection in human are rare. Here is a report on a woman who had been taking hormones and immunosuppressants for a long time for SLE and underwent abdominal soft tissue infection with Mycobacterium senegalense after abdominal liposuction, to provide reference for clinical diagnosis and treatment. The patient, female, 32 years old, has a history of SLE for more than 2 years, and currently takes "methylprednisolone, hydroxychloroquine, and mycophenolate mofetil" regularly. Nine months before the patient was admitted to the hospital, she once performed abdominal, waist and buttock liposuction in a medical beauty institution. One month after the operation, several masses gradually appeared on the abdominal wall, accompanied by tenderness, one of the masses had obvious fluctuation on palpation and purulent fluid could be drawn out. The location of the abdominal wall mass was consistent with the insertion site of the liposuction needle. After the onset of the disease, the patient went to the medical beauty institution for puncture of the abdominal wall mass, and 5 mL of purulent fluid was pierced and sent for bacterial culture, and cultured "Mycobacterium Senegalense", after 3 days of treatment with "cephalosporin" antibiotics (specifically unknown), the symptoms did not improve, so she went to the second affiliated hospital of hainan medical college. After completing the relevant examinations during the hospitalization in our hospital, in order to clarify the etiology, another abdominal puncture to extract pus was performed, the mycobacterial culture + identification results: Mycobacterium senegalense. Consistent with the out-of-hospital results, the diagnosis of Mycobacterium senegalense infection was confirmed. After 3 months of treatment with "cefoxitin, azithromycin, amikacin, and levofloxacin", the patient's abdominal wall soft tissue infection was cured. Trauma or invasive procedures can lead to skin, muscle, or bone infection with nontuberculous mycobacteria (NTM), which can manifest as chronic painless nodules that progress to purulent folliculitis and abscesses. NTM infection should be suspected when the patient's wound has been exposed to water, there is a history of surgery, and empirical anti-infection is ineffective. This is the first case of Mycobacterium senegalense infection caused by medical beauty, which tell people that they should be cautious when choosing medical aesthetic projects and medical aesthetic institutions.

Purpose: To evaluate the use of signal intensity index (SII) of skull-base invasion in nasopharyngeal carcinoma (NPC) using magnetic resonance imaging (MRI), select a best cut-off SII value to predict the outcome of NPC. Materials and Methods: One hundred and twenty-two NPC patients (92 men, 30 women) with skull-base invasion were included. All patients underwent MRI, signal intensities on T1-weighted imaging (T1WI) were measured for each invaded site and its contralateral normal counterpart. The SIIs were calculated, receiver operating characteristic curves were constructed. The optimal cut-off values were extracted. The overall survival (OS) rates of 5-year follow-up were performed. Results: Sensitivities for differentiating skull-base invasion from normal contralateral anatomy were 98.9%, 88.5% and 70.0%, and specificities were 98.9%, 96.0% and 74.4%, respectively. There were three cut-off values for differentiating invasion from normal anatomy of skull-base, 49%, 98%, and 60%. Significant difference in OS rates (84.2% vs. 57.1%, p=0.007) was seen for SII threshold values > 60% and those ≤ 60%. Conclusions: The SII might be a useful means of differentiating invasion from normal tissue at the skull-base in NPC. The cut-off value of quantitative SII at the skull-base may aid in monitoring the response to treatment of NPC patients.

OBJECTIVETo understand and master the situation in which enterovirus caused hand-foot-and-mouth disease (HFMD) in Xuzhou district in 2009 so as to provide scientific basis for the control and prevention of hand-foot-and-mouth disease.

METHODSThe researchers adopted fluorescence RT-PCR method to detect EV and EV71 as well as the CA16 specificity RNA from 222 samples of anal swabs and oropharyngeal swabs from the 240 cases who were diagnosed clinically as hand-foot-mouth disease infected by enterovirus. Also, the researchers conducted EV71-IgM antibody detection on 114 samples of acute phase serum with ELA method.

RESULTSAmong the 240 enterovirus infected patients, the total EV infection rate is 72.50% (174/240), among which EV71 infection rate is 57.92% (139/240), CoxA16 infection rate is 9.17% (22/240), and other EV infection rate is 5.42% (13/240). The EV71-RNA positive rate of the samples of 222 anus swabs among the 240 suspected enterovirus infected patients is 45.94% (102/222), the samples of swallow swab EV71-RNA positive rate is 25.68% (57/222) and the EV71-IgM antibody positive rate of 114 samples of acute phase serum is 86.84% (99/114). The EV71-RNA positive rate of oropharyngeal swabs of 254 healthy children is 1.57% (4/254) , and no CoxA16-RNA was detected. In the oropharyngeal swabs of 54 close contacts (medical personnel), the EV-RNA detected is negative. The positive rate of EV71-IgM antibody of the 258 healthy children's serum samples is 2.71% (7/258).

CONCLUSIONThe widespreading of hand-foot-mouth disease in Xuzhou district is caused mainly by type 71 enterovirus. Inapparent infection of type 71 enterovirus exists among children under the age of 3 during the time of widespreading period and IgM antibody develops in them. It is difficult for adults to be infected by EV71 even if they contact the contagion source closely. The positive rate of EV71-IgM antibody in the samples of acute phase serum of suspected cases is the highest (86.84%), and the second highest is the positive rate of RNA of EV71 of anal swabs (45.94%) and of the EV71 of oropharyngeal swabs (25.68%). ELA reagent kit is used in the early diagnosis of EV71 infection for it is easy to operate, fast and economic, so, it is worth popularizing in the grass-root medical units.

Child, Preschool ; China ; Enterovirus A, Human ; genetics ; isolation & purification ; Female ; Hand, Foot and Mouth Disease ; virology ; Humans ; Infant ; Male

OBJECTIVETo investigate the effect of gene transfection at different time on bone mineral density and strength of newly formed bone in mandibular distraction gap in rabbit, so as to explore the optimal time for gene therapy and enhance the therapeutic effect.

METHODS48 New-Zealand rabbits were employed to receive mandibular osteotomy and implantation of distraction devices bilaterly. Then the rabbits were randomly divided into 4 groups as group A, B and C and D. The animals in group A, B, and C were transfected with recombinant plasmids pIRES-hBMP2-hVEGF165 via electroporation-mediated approach at latency period, distraction period, consolidation period respectively. Group D was used as control group without gene transfection. After 3 days of latency period, the distraction devices were activated at the rate of 0.8 mm per day for 10 days. Three rabbits in each group were sacrificed at 1 wk, 2 wk, 4 wk and 8 wk of consolidation respectively. The mandibles were harvested and the left mandible received X-ray examination for bone healing, and quantitative computed tomography (QCT) dectection for the bone mineral density (BMD) of newly formed bone in the distraction gap. The biomechanical properties of the new generation bone at 4 th and 8 th week of consolidation in each group were detected by three point bending test.

RESULTSThe bone mineral density and the biomechanical strength of newly formed bone increased along the length of consolidation in each group. After 1 week of consolidation, there was no significant difference in BMD among group A (83.43 +/- 9.96), group B (92.29 +/- 11.25), group C (89.93 +/- 14.15), P > 0.05. But the BMD of group A, B and C was higher than that of group D (70. 31 +/- 3.30), P < 0.05. After 2wk, 4 wk and 8 wk of consolidation, the BMD of group B (137.54 +/- 7.20,492.93 +/- 17.57, 790.48 +/- 12.19) was significantly higher than those of group A (121.44 +/- 9.27, 396.15 +/- 15.70, 603.39 +/- 16.46), C (125.06 +/- 7.24, 464.15 +/- 15.45, 764.15 +/- 17.28), and D (98.86 +/- 8.13, 336.45 +/- 11.95, 577.89 +/- 18.43), P < 0.05. The biomechanical parameters were also higher in group B than those of group A, C and D after four and eight weeks of consolidation (P < 0.05).

CONCLUSIONIt is better to transfect gene at the beginning of distraction (distraction period) than at other stages of DO. In this way, more remarkable effect could be obtained on new bone formation. It suggests that the distraction stage is the optimal time for gene therapy.

Animals ; Bone Density ; genetics ; physiology ; Electroporation ; Genetic Therapy ; Mandible ; physiology ; surgery ; Osteogenesis, Distraction ; Osteotomy ; Rabbits ; Time Factors ; Transfection

OBJECTIVETo explore the effect of electroporation mediated gene therapy on bone mineral density and strength of new-formed bone in mandibular distraction gap, so as to enhance the osteogenesis and shorten the distraction term.

METHODSNew-Zealand rabbits were employed. The distraction began after 3 days of latency period at the rate of 0. 8 mm per day for 7 days. After distraction, the rabbits were randomly divided into 5 groups to receive injection in the distraction gap with recombinant plasmid 2 microg (0.1 microg/microl) pIRES-hVEGF165-hBMP2 in group A, with recombinant plasmid pIRES-hBMP2 in group B, with recombinant plasmid pIRES-hVEGF165 in group C, with pIRES in group D, and with normal saline (NS) in group E. After injection, electroporation was performed in all the groups. After 1 week, 2 weeks, 4 weeks and 8 weeks of consolidation, all the animals underwent X-ray and quantitative computed tomography (QCT). The new-formed bone in distraction gap was selected as regions of interest (ROI) to measure the bone mineral density(BMD). Then the rabbits were sacrificed and the new-formed bone samples were harvested to detect 3-point crushing strength.

RESULTSBMD of newly formed bone in group A, B and C was markedly higher than that in group D and E (P < 0.01). After 2 weeks of consolidation, BMD in group A was much higher than that in the other groups, but there was no difference between group B and C. After 4 weeks of consolidation, BMD in group A and B was markedly higher than that in group C, D and E (P < 0.01). After 8 weeks of consolidation, BMD in group A was markedly higher than that in the other groups. While the BMD was not significantly different between group B and C, but the BMD in group B and C was higher than that in group D and E (P < 0.01). After 4 weeks of consolidation, the 3-point crushing strength of newly formed bone in group A was markedly higher than that in group B,C, D and E (P < 0.01), which was still the same after 8 weeks of consolidation. And the crushing strength in group B was higher than that in group C, D and E (P < 0. 05).

CONCLUSIONSElectroporation-mediated transfection of recombinant plasmid pIRES-hVEGF165-hBMP2 could greatly enhance osteogenesis and calcification. A combination of VEGF and BMP may promote osteogenesis and angiogenesis simultaneously, so as to magnify the effect of each growth factor, resulting a synergetic effect.

Animals ; Bone Density ; Bone Regeneration ; Electroporation ; Genetic Therapy ; Mandible ; physiology ; surgery ; Osteogenesis, Distraction ; Rabbits