Objective To investigate the effects of DNA methylation on the expression of lympho-cyte activation gene 3 (lag3) in different human T cell lines.Methods A quantitative PCR and a flow cy-tometry analysis were performed to measure the expression of lag3 gene in various T cell lines at mRNA and protein levels.The distribution of CpG sites within the promoter and body of lag3 gene was detected to locate the potential regulatory region(s) (CpG island and CpG island shore).The levels of DNA methylation in each cell line were analyzed.The T cell lines were demethylated with 5-Aza-2′-deoxycytidine (5-Aza-2′-dc) for further investigation on the changes of lag3 gene expression and DNA methylation.Results Jurkat E6-1 cells showed the highest expression level of lag3 gene as compared with J.CaM1.6 and CEM cells.Hyperm-ethylated CpG islands were detected in cells of each cell line.The methylation levels of CpG island shore in J.CaM1.6 and CEM cells were higher than that in Jurkat E6-1 cells.Treatment of J.CaM1.6 and CEM cells with 5-Aza-2′-dc significantly promoted the expression of lag3 gene at mRNA and protein levels as well as the demethylation of CpG island shore.No significant differences with the expression of lag3 gene and the methylation of CpG island were observed in Jurkat E6-1 cells with or without 5-Aza-2′-dc stimulation.Con-clusion Methylation and demethylation of CpG island shore played important roles in regulating the tran-scription of lag3 gene.

Objective To investigate the relationship between low serum calcium concentration and hematoma volume in patients with intracerebral hemorrhage.Methods Between January 2012 and October 2014,870 consecutive patients with intracerebral hemorrhage admitted to the Department of Neurosurgery,West China Hospital,Sichuan University were enrolled prospectively.The patients completed laboratory serum calcium concentration and head CT examinations within 24 h after attack,and the baseline data and laboratory findings were collected.According to the normal reference value of laboratory serum calcium concentration,the patients were divided into a hypocalcemia calcium group (<2.1 mmol/L;n=193) and a normal calcium group (2.1-2.7 mmol/L;n=677).Spearman correlation analysis was used to analyze the correlation between the blood serum calcium concentration and the hematoma volume on admission.Results (1) The hypocalcemia group compared with normal calcium group,the proportion of male patients was high (73.6% [n=142] vs.66.0% [n=447]),the median score for Glasgow coma scale was lower (9 vs.11),and the median hematoma volume was larger (33.86 cm3 vs.21.69 cm3).The differences were statistically significant (all P<0.05).(2) Spearman correlation analysis showed that the lower serum calcium level on admission was weakly negatively correlated with the volume of hematoma in patients with intracerebral hemorrhage (r=-0.113,P<0.01).Conclusion The study suggested that the hypocalcemia on admission was mostly males in patients with intracerebral hemorrhage,the condition was serious,the volume of hematoma was larger,and the lower serum calcium concentration was negatively correlated with the hematoma volume.

0bjective To determine the immunogenicities of DNA vaccines expressing tat-rev-integrase(c-half)-vif-neffusion genes(TRIVN) derived from prevalent B', B'/C and AE recombinant subtypes of HIV-1 in China. Methods Two DNA vaccines were constructed by inserting the codon optimized tat-revintegrase(c-half)-vif-nef fusion genes derived from B' and B'/C subtype of HIV-1 into mammalian expression vector pSVI. 0. DNA vaccine containing tat-rev-integrase (c-half)-vif-nef fusion gene derived from HIV-1AE2f has been constructed previously. In vitro expression efficiencies of three DNA vaccines were determined by Western blot and their immunogenicities were compared by immunizing female BALB/c mice. IFN-γ ELISPOT assay was used to read out the specific T cell immunity. Results The constructed DNA vaccines were validated by restriction enzyme digestion and DNA sequencing. Western blot assay showed three constructed DNA vaccines could be expressed at a comparable level in vitro. After vaccination, AE-TRIVN mounted T cell immune responses at (948.0 ± 330.0) SFCs/106 splenocytes, followed by the mixed DNA vaccine[ (500.0 ± 155.0) SFCs/106 splenocytes ], RL-TRIVN r[ ( 195. 1 ± 44.0) SFCs/106 splenocytes ]and CN-TRIVN [ (89.5 ± 17.0) SFCs/106 splenocytes]. Interestingly, we observed that single DNA vaccination induced specific T cell responses predominantly targeting Integrase (C-half) and Vif, whereas the mixed DNA could significantly improve T cell responses against Nef. Conclusion AE-TRIVN was the most immunogenic among the three DNA vaccines and the mixed DNA vaccination could change the immunogenic hierarchy of T cell epitopes across the fusion genes vaccine.

Objective To investigate and compare the features of the HIV-1-specific CTL responses among three HIV-infected groups with varied infection history. Methods Three HIV-infeeted groups were enrolled in this study, including two groups infected by blood transmission (one group has been infected for more than 10 years and the other for 1-2 years) and one group of the man who have sex with man. The HIV-1-specific CTL responses were quantified by an IFN-γ based ELISPot assay with a peptide matrix system containing overlapping peptides spanning the entire HIV-1 Clade B genomic consensus sequences. Results The responding rate of CTL responses against all 17 peptide pools among the group that infected 1-2 years,the group infected more than 10 years and the group of MSM were 40% ,65% ,23%. One way ANOVO analysis showed that the responding rate of CTL responses against all 17 peptide pools were statistical significant among the three groups (F=19.96, P＜0.01);the magnitude of CTL responses of the three groups were 0-5 835 SFCs/106 PBMC, 0-7 225 SFCs/106PBMC, 0-9 740SFCs/106pBMC, Kruskal-Wallis test showed that the magnitude of CTL responses were statistical significant among the three groups( H = 101.90 , P ＜0.01);the breadth of CTL were 7 ( 2-11 ), 11(9-14) and 4 (2-6) respectively and Kruskal- Wallis test showed that the breadth of CTL had no statistical significant among the three groups( H = 34. 75 ,P ＜0. 01 ). The sequence of responding rate, magnitude and breadth of CTL from high to low was the group that had been infected for more than 10 years, the group infected 1-2 years and the sex transmission group. The common characteristics of the CTL response among the three groups were that the responding rate and the magnitude of the peptide Nef and Gag was higher than other peptide's. The magnitude of CTL responses among three different CD4count groups (CD4 ＜ 200/μl, CD4 200-500/μl, CD4 ≥500/μl,) was 0-18 475 SFCs/106pBMC, 350-34 095 SFCs/106pBMC, 490-21 550 SFCs/106 PBMC and had no statistic difference among the three different CD4 groups(H=2.93, P=0.23) while the breadth of CTL was 3(0-8), 10(2-17), 10 (1-17)respoctively and the breadth of CTL was lower in the group of CD4 count less than 200/μl than the other two groups( H = 14. 72, P ＜ 0. 01 ). The magnitude of CTL responses among three different viral load (VL)groups (VL＜ LDL, LDL ＜ VL ＜ 1 × 104 copys/ml, VL≥1 ×104 copys/ml) was 490-18 475 SFCs/106pBMC, 0-24 115 SFCs/106pBMC, 770-34 095 SFCs/106 pBMC and had no statistic difference among the three different viral load groups ( H = 0.79, P=0.67) and the breadth of the three different viral load groups CTL was 8( 1-17), 11 (0-17), 8 (1-16) and Kruskal-Wallis test showed that there was no statistic difference among the three different viral load groups (H =5.27, P =0. 07). Conclusions All groups predominantly develope T cell immune responses against Nef and Gag proteins. With the elapse of HIV infection, the CTL responses are increased in both magnitude and responding rate. This information is important for vaccine development.

Objective To construct two DNA vaccines encoding Gag-Env fusion protein and Tat-Rev-Integrase(C-half)-Vif-Nef fusion protein derived from the first HIV-1 CRF01_AE isolate(AE2f) in Chi-na and to evaluate the immunogenicity in mice. Methods Two DNA vaccines were constructed by inserting the codon optimized and synthesized gag-env fusion gene and tat-rev-integrase(c-half)-vif-nef fusion gene de-rived from AE2f into mammalian expression vector pDRVISV1. 0, the generated DNA vaccines were desig-nated as pSVAE/GE and pSVAE/TRIVN, respectively, and their in vitro expression were determined by Western blot with transfected 293T cells. Mice were i. m. immunized with either pDRVI1.0 as mock control, pSVAE/GE or pSYAE/TRIVN for 4 times at two-week interval. Two weeks following the final im-munization, cellular responses to pool of HIV-1 Env, Gag, Tat, Rev, Intergrase, Vif and Nef peptides were evaluated by ELISPOT assay. Results The construction of DNA vaccine pSVAE/GE and pSVAE/TRIVN was validated by restriction enzyme digestion and bidirectional sequencing. Western blot showed a specific band at molecular mass 220×10~3 in lane of pSVAE/GE transfeeted 293T cell and a specific band at 95×10~3 in the lane of pSVAE/TRIVN. Both DNA vaccines mounted significant specific T cell responses with (3010 ± 566) SFC/10~6 splenocytes for DNA vaccine pSV AE/GE and (948 ± 737) SFC/10~6 spleno-cytes for DNA vaccine pSVAE/TRIVN, whereas the mock control of pDRVISV1.0 only raised marginal T cell responses. Conclusion Both pSVAE/GE and pSVAE/TRIVN were capable of expressing the inserted fusion immunogen genes and able to elicit vigorous cellular immune responses, therefore, these DNA vac-cines are highly immunogenic.

Objective:To investigate the effect of continuous renal replacement therapy on sepsis complicated with acute renal injury and its influence on cytokines and renal function.Methods:From January 2017 to October 2019, 82 patients with sepsis and acute renal injury admitted to the People's Hospital of Jiangshan were divided into control group and observation group according to the random digital table, with 41 cases in each group.The control group was given routine treatment, and the observation group was treated with continuous veno venous hemofiltration(CVVH) on the basis of the control group.The course of treatment in both groups was 5 days.The time of ICU stay and mechanical ventilation, Marshall score, acute physiology and chronic health status scoreⅡ(APACHEⅡ), cytokines and renal function were compared before and after treatment.Results:The ICU stay time[(13.25±1.97)d] and mechanical ventilation time[(11.83±2.43)d] in the observation group were shorter than those in the control group[(11.83±2.43)d and (16.78±1.85)d]( t=9.092, 10.378, all P<0.05). After treatment, the Marshall organ dysfunction score[(5.78±0.81)points] and APACHEⅡ score[(15.40±1.62)points] in the observation group were lower than those in the control group[(7.19±0.67)points and (21.25±3.25)points]( t=8.589, 10.315, all P<0.05). The serum levels of PCT[(7.68±2.10)μg/L] and CRP[(45.41±10.20)mg/L] in the observation group were lower than those in the control group[(13.76±1.78)μg/L and (109.87±12.76)mg/L]( t=14.142, 11.320, 25.266, all P<0.05). The serum levels of BUN[(4.62±0.38)nmol/L] and Scr[(25.45±5.17)μmol/L] in the observation group were lower than those in the control group[(13.79±0.43)nmol/L and (68.79±4.15)μmol/L]( t=20.011, 18.421, all P<0.05). Conclusion:The effect of continuous renal replacement therapy on sepsis combined with acute renal injury is good, which can significantly reduce the inflammatory response of cells and improve the renal function of patients.

The correlation of single nucleotide polymorphism (SNP) rs10569304 on the second expressed region of hole gene and congenital heart disease (CHD) of human being, and the effect of hole gene on CHD were investigated. 179 patients with CHD as CHD group and 183 healthy people as control group were selected in the case-control study. DNA was abstracted from the peripheral blood by phenol-chloroform method. Primer was designed for the flanking sequence of SNP rs10569304 on the second expressed region of hole gene. The genotype was identified by PCR degenerative acrylamide electrophoresis with amplification products. Then the three amplification products received sequencing. By chi-square test, the genotype frequency and allele frequency in CHD group and control group were analyzed. There was insertion-deletion (GCC/-) of SNP rs10569304 which corresponded to alleles of A and B in Southern Chinese people. The genotype frequency and allele frequency in control group and CHD group were met the Hardy-Weinberg equilibrium. By chi-square test, in control group and CHD group, the genotype frequency of AA (insertion homozygous), AB (insertion-deletion heterozygous) and BB (deletion homozygous) was 21.31%, 54.09%, 24.59% and 16.75%, 46.36%, 36.87%, respectively. The distributional difference of genotype frequency had statistical significance (chi (2)=6.51, P<0.05); The allele frequency of A and B was 48.36% and 51.64% in control group, 39.94% and 60.06% in CHD group, respectively. The distributional difference of allele frequency had statistical significance (chi (2)=5.20, P<0.05). Meanwhile, by contrast with the control group, the BB genotype frequency and B allele frequency in CHD group was higher, but the AA and AB frequency was lower. There was higher risk to suffer from CHD involving B allele. BB genotype had 1.907-fold increased risk of developing CHD according to AA genotype (P<0.05). It is concluded that there is insertion-deletion (GCC/-) of SNP rs10569304 in the Southern Chinese people, and the people whose hole gene involving BB genotype have higher risk to suffering from CHD.

BACKGROUND:In recent years, the application of stem cel s to treat autoimmune diseases has become a hot spot. But, studies on umbilical cord blood mesenchymal stem cel s transplantation for the treatment of polymyositis/dermatomyositis are rarely reported. OBJECTIVE:To explore the immunologic mechanism of Th cytokines on the occurrence and development of polymyositis/dermatomyositis by observing the changes in serum interferon-γ, interleukin-4 and interleukin-17 in patients after umbilical cord blood mesenchymal stem cel s transplantation. METHODS:Eighty-one polymyositis/dermatomyositis patients were selected and divided into conventional therapy group (n=44) undergoing glucocorticoid and immunosuppressants therapy and cel transplantation group (n=37) undergoing intravenous infusion of umbilical cord blood mesenchymal stem cel s at a density of (3.5-5.2 )×107 . Dosing regimen was same in the two groups. After fol ow-up of 1, 3, 6 months, the changes of creatine kinase and myodynamia were evaluated;after fol ow-up of 3 and 6 months, lung imaging was evaluated;in the cel transplantation group, interferon-γ, interleukin-4 and interleukin-17 levels were detected before treatment and at 3 and 6 months after treatment. RESULTS AND CONCLUSION:At 1, 3, 6 months after treatment, the creatine kinase level was significantly decreased, and the muscle force grade was significantly increased in both groups (both P<0.001). Compared with the conventional therapy group, the creatine kinase level was lower and the muscle force grade was higher in the cel transplantation group (both P<0.001). Results from lung imaging test showed a remarkable improvement after cel transplantation, and it indicated that umbilical cord blood mesenchymal stem cel s transplantation had good stability. At 6 months after transplantation, the level of interferon-γwas significantly increased, while the interleukin-4 level was decreased significantly (both P<0.01);at 3, 6 months after cel transplantation, the levels of interleukin-17 were significantly decreased (P<0.01). Levels of interleukin-4 and interleukin-17 were positively correlated with the level of creatine kinase at 6 months after cel transplantation (r=0.467, 0.488, both P<0.05), but there was no obvious correlation between the levels of interferon-γand creatine kinase (r=0.213, P>0.05). These findings indicate umbilical cord blood mesenchymal stem cel s transplantation combined with glucocorticoid and immunosuppressants therapy can adjust immune network effects and improve the immune tolerance in polymyositis/dermatomyositis patients, which is safe and effective.

In 2013, Shanghai Medical College of Fudan University restarted the enrollment of the undergraduate students in directional pediatrics. To cultivate medical talents in pediatrics, a serious of educational innovations and practices have been carried out guided by competency training, including training a team of teachers with simulated teaching skills and establishing a teaching platform for simulation teaching. Medical students can practice medicine and gain experience through the risk-free simulated scenarios, that is helpful to enhance their confidence in clinical skills and communications and decrease medical errors in their future careers.

OBJECTIVETo summarize the clinicopathologic features of neuroblastic tumors (NT), and to explore the prognostic significance of MYCN amplification in NT.

METHODSThe clinicopathologic data of 267 NT were reviewed. MYCN gene amplification was detected by fluorescence in situ hybridization (FISH) in 119 cases and the relationship with pathological characteristics and prognostic significance were analyzed.

RESULTSThe study included 267 cases of children NT from patients aged from 1 day to 13 years (median 27 months). The male to female ratio was 1.43. There were 38 cases (14.2%), 43 cases (16.1%), 71 cases (26.6%), and 115 cases (43.1%) of INSS stages I, II, III and IV respectively.Favorable histology group had 157 cases (59.9%); unfavorable histology group had 110 cases (40.1%).Of the 119 NT cases with MYCN FISH performed, 18 cases (15.1%) showed amplification and the signal ratio of MYCN to CEP2 was 4.08-43.29. One hundred and one cases of non-amplified MYCN included MYCN gain in 79 cases (66.3%) and MYCN negative in 22 cases (18.5%). MYCN expression showed significant difference (P = 0.000) between ages, gender, NT type and MKI, but not INPC and clinical stage (P > 0.05).Of the 18 cases with MYCN amplification, 3 were undifferentiated, and 15 poorly differentiated; 17 had high MKI and one moderate MKI. All 18 cases were in unfavorable histology group; the overall survival rate was 3/18, with an average survival time of (17.9 ± 2.4) months.Of the 101 MYCN non-amplification cases, the overall survival rate was 68.3% (69/101), with an average survival time of (29.8 ± 1.3) months. Survival analysis showed the cases with MYCN amplification had worse prognosis (P < 0.05).

CONCLUSIONSNT were commonly diagnosed in early ages and easily to metastasize. Most of cases with favorable histology. The cases of MYCN amplification showed unfavorable histology, and the majority cases with high MKI; The patients with MYCN gene amplification had poor prognosis.

Adolescent ; Cell Differentiation ; Child ; Child, Preschool ; Female ; Gene Amplification ; Humans ; In Situ Hybridization, Fluorescence ; Infant ; Male ; N-Myc Proto-Oncogene Protein ; Neuroblastoma ; genetics ; mortality ; pathology ; Nuclear Proteins ; genetics ; Oncogene Proteins ; genetics ; Prognosis ; Survival Analysis ; Survival Rate