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

Objective: The current study was designed to investigate the relationship between the pattern of methylation of calcitonin (CT) gene and the activity of DNA methyltransferase (MTase). Methods: The methylation rate of 5′ region of CT gene (CTMR) and the activity of MTase in 6 solid tumor cell lines and 2 leukemia cell lines were determined. CTMR was detected by polymerase chain reaction(PCR) with inter and external references in combination with restriction endonuclease and laser scan technique. MTase activity was examined by isotope labeled microassay. Results:Both CTMR and MTase activity in all tumor cell lines were significantly higher than that of control cells. The increased MTase activity was relative to elevated CTMR. Conclusion: There is prevalence of hypermethylation of CT gene and elevated activity of MTase in malignant cells. The increased MTase activity is one of the possible reasons for CT gene hypermethylation in tumor cells.

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.

Objective To understand HFMD knowledge and behavior among parents of children with HFMD in Xuzhou,so as to provide scientific basis for effective control measures.Methods 1 125 parents of children with HFMD in Xuzhou children' s hospital from January 2010 to December 2010 were surveyed.Results Among 1 017 parents,90％ parents heard about HFMD and approved visiting a doctor immediately,but only 7.18％ to 41.89％ of them knew about the pathogen and susceptible population of HFMD.57.52％ of the children suck fingers,19.96％ of the children and parents washed hands before meals/after toilet in time,4.03％ of the families disinfected dinnerware and 3.64％ of them disinfected toys,which were all risk behaviors of HFMD infection.Awareness of HFMD among parents aged 25-40 years old(50.32％)was higher than that of parents younger than 25 years old(32.95％)and that of parents elder than 40 years old(23.70％).The difference was statistically significant(x2 =44.881,P ＜ 0.01).Awareness of HFMD was found positively correlated with education level.The difference was statistically significant(x2 =55.661,P ＜0.01).Suburban showed lower awareness(36.38％)than the citizens(57.90％).There was significant difference(x2 =47.179,P ＜ 0.01).61.32％ of the parents had needs for knowledge of HFMD prevention and control,53.75％for knowledge of living habits and 27.19％ for knowledge of HFMD treatment.Conclusions Popular and pertinent propaganda should be carried out among different population,so as to enrich the knowledge of HFMD of the parents.

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.

Objective To understand and master the situation in which enteroviros caused handfoot-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. Methods The researchers adopted fluorescence RT-PCR method to detect EV and EV71 as well as the CAI6 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. Results Among 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-lgM antibody of the 258 healthy children's serum samples is 2.71% (7/258).Conclusion The 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.