2004年 · 第117卷第12期

Background The vaccination of mice with DNA encoding single candidate antigens has failed to induce significant protection against Schistosoma japonicum (S. japonicum) challenge infections. In this study, we evaluated the feasibility of using a multivalent DNA vaccine which co-expressed S. japonicum integral membrane protein Sj23 and murine cytokine IL-12 to induce protective immune responses. Methods The plasmid pVlVO2-IL12-Sj23, a eukaryotic expression vector expressing Sj23 and murine IL-12 simultaneously, was constructed, identified, and tested for expression in vitro. Its ability to protect against S. japonicum challenge infections was analyed according to worm reduction rate and egg reduction rate after vaccination of BALB/c mice. The serum levels of specific IgG antibody were determined by enzyme-linked-immuno sorbent assay (ELISA) and Western blot analysis. Using cultured spleen cells, IFN-γand IL-4 post-stimulation were quantified by ELISA. The phenotypes of splenocyte populations were analyzed by flow cytometry (FCM). Results The plasmid DNA pVlVO2-IL 12- Sj23 was proven to express well in vitro by transient transfection of HEK-293 cells. Immunization re suited in a worm reduction rate of 45. 53 %and egg reduction rate of 58. 35 %. ELISA and We stern blot analysis indicated that immunized mice generated specific IgG against Sj23. Spleen cells showed significant increases in IFN-γbut decreases in L-4. No significant differences in CD4+and CD8+ subgroup ratios were observed after the challenges. Conclusions The multivalent DNA vaccine pV1VO2-IL12-Sj23 is sufficient to elicit moderate but highly significant levels of protective immunity against challenge infections. Cytokine IL-12, as a gene adjuvant, was able to enhance the Thl responses and, hence, the protective immunity. Chin Med J 2004; 117 (12): 1842-1846

Background It is known that ultraviolet irradiation can affect cellular function through a number of signaling pathways. (-) - epigallocatechin-3-gallate (EGCG) is the major effective component in green tea and can offer protection from ultraviolet- indue ed damage. In this study, we investigated the protective mechanism of EGCGon human dermal fibroblasts damaged by ultraviolet A (UVA) in vitro. Methods Transcription factor Jun protein levels were measured by Western blot. Matrix metalloproteinase 1 (MMP-1) and tissue inhibitor of metalloproteinase-1 (TMP-1) mRNA were studied by reverse transcription-polymerase chain reaction (RT-PCR) analysis in conjunction with computer-assisted image analysis. MMP-1 and TIMP-1 proteins were quantified by enzyme-linked immunosorbent assay(ELISA). Results EGCG decreased transcription activity of Jun protein after induction by UVA. Both the mRNA and protein levels of MMP-1 were increased by UVA irradiation, while no significant changes were observed in TIMP-1 levels. The ratio of MMP-1 to TIMP-1 showed statistically significant differences compared with the control. EGCG decreased the ratio of MMP-1 to TIMP-1 by inhibiting UVA-induced MMP-1 expression (P< 0.05). Conclusion EGCG can protect human fibroblasts against UVA damage by downregulating the transcription activity of Jun protein and the expression of MMP-1. The ratio of MMP-1 to TIMP-1, rather than the levels of MMP-1 or TIMP-1 alone, may play a significant role in human skin photo damage. Chin Med J 2004; 117 (12): 1838-1841

Background Children with nonverbal learning disabilities (NLD) usually manifest defective attention function. This study sought to investigate the neuropsychological characteristics of selective attention, such as attention control, working memory, and attention persistence of the frontal lobe in children with NLD. Methods Using the auditory detection test (ADT), Wisconsin card sorting test (WCST), and C-WISC, 27 children with NLD and 33 normal children in the control group were tested, and the results of C-WISC subtests were analyzed with factor analysis. Results Compared with the control group, the correct response rate in the auditory detection test in the NLD group was much lower (P <0. 01), and the number of incorrect responses was much higher (P < 0.01); NLD children also scored lower in WCST categories achieved (CA) and perseverative errors (PE)(P <0. 05). Factor analysis showed that perceptual organization (PO) related to visual space and freedom from distractibility (FD) relating to attention persistence in the NLD group were obviously lower than in the control group (P <0. 01). Conclusions Children with NLD have attention control disorder and working memory disorder mainly in the frontal lobe. We believe that the disorder is particularly prominent in the right frontal lobe. Chin Med J 2004; 117 (12): 1834-1837

Background Single fiber electromyography (SFEMG) is a sensitive technique for detecting abnormalities in neuromuscular transmission and is mainly used in the diagnosis of neuromuscular junction disorders, such as myasthenia gravis. While the process of denervatioin-reinnervation in amyotrophic lateral sclerosis (ALS) can also result in immature collateral nerve terminals and instability of neuromuscular transmission, the purpose of this study was to investigate the changes and clinical values of SFEMG in patients with ALS. Methods Volitional SFEMGwas performed on the extensor digitorum communis (EDC) of 78 patients with ALS (men 52, women 26) who had been previously diagnosed by history, clinical features, and neurophy siological studies. The mean jitter, the percentage of jitter>55 μs, the impulse blocking percentage, and fiber density (FD) were determined. These results were conpared to normal controls. In addition, the SFEMG indices were analyzed for correlations with the duration of ALS, the EDC strength score on the Medical Research Council (MRC) scale, and spontaneous activity detected by EMG studies. Results SFEMG indices were abnormal in all patients with AL S. Mean jitter ranged from 30 to 178 μs(mean 80.2 μs); the percentage of jitter> 55 μs ranged from 5 % to 100 %(mean 60. 5 %). In addition, the inpulse blocking percentage ranged from 0 %to 90 %(mean 28. 1 %) and FD ranged from 1. 4 to 4. 1(mean 2. 6). Mean jitter, the percentage of jitter > 55 μs, and the blocking percentage in 57 patients with definite or probable ALS were significantly higher than in patients with possible or suspected ALS. MRC scores of the EDC negatively correlated with mean jitter, the percentage of jitter>55 μs, blocking percentage, and FD. Conclusions SFEMGis the most sensitive tool for diagnosing definite or probable ALS. Increased jitter, blocking percentage, and FD can indicate the degree of immature collateral sprouts and motor end plates re suiting from the progressive denervation and reinnervation associated with ALS, and may be helpful in evaluating prognosis. Chin Med J 2004; 117 (12): 1830-1833

Background Transforming growth factor beta (TGF-β) and matrix metalloproteinases-9 (MMP-9) have been implicated in the pathogenesis of human atherosclerosis but their relationship during lesion progression are poorly understood. The objective of this study was to investigate the expressionof MMP-9, TGF-βl and TGF-β receptor I (IβR- I) in human atherosclerotic plaque and their relationship and plaque stability. Methods Specimens of human coronary artery atherosclerotic plaques were obtained from 41 patients undergoing coronary endarterectomy, and were paraffin embedded, sectioned at 4 μm intervals then stained with haematoxylin and eo sin. They were divided into stable (with no or only little lipid core) and unstable plaque groups (with lipid core size > 40 %): the immuno histochemical staining were performed for MMP-9,TGF-β1 and TβR- I. Results The expression of MMP-9 in the unstable plaques was much higher than in the stable ones, but the expression of TGF-β1 was higher in the stable plaques. There was no similar significant difference for IβR- I. Correlation analysis showed that there was a negative correlation between the expre ssion of MMP-9 and TGF-β1 (r=- 0. 332, P=0.034 for average areal density; r= - 0.373, P=0.016 for average optical density). Conclusions There were close relationships between MMP-9, TGF-βl and plaque stability. Enhanced production of MMP-9 may participate in the formation of unstable plaque, while TCF-βl maybe an inportant stabilizing factor in preventing transition into an unstable plaque phenotype. Chin Med J 2004; 117 (12): 1825-1829

Background Neuroblastoma, one of the common tumors in children, possesses the feature of natural regression that might be related to apoptosis caspase-3 and survivin are believed to respectively induce and inhibit apoptosis. We investigated the expression of caspase-3 and survivin in pediatric neuroblastoma and the role that these genes played in apoptosis. Methods The expression of caspase-3 and survivin in pediatric neuroblastoma tissue samples was detected using in situ hybridization, ter mintuesal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (IUNEL), and immunohistochemical staining. The role that these genes played in apoptosis was then evaluated. Results A converse correlation was observed between the expression of survivin and caspase-3. When survivin was expressed at high levels in neuroblastoma samples, caspase-3 expression was downregulated, and the apoptotic index decreased simultaneously. Conclusion There is a converse correlation between the expression of caspase-3 and the expression of survivin in neuroblastoma cells, indicating that caspase-3 might induce apoptosis, and survivin may inhibit this process. Chin Med J 2004; 117 (12): 1821-1824

Background Although there were several clinical and experimental studies discussing the pathogenesis of dural arteriovenous fistula (DAVF), the pathological process leading to intracranial DAVF so far remains unknown. In this study, we investigated the expression of vascular growth factors in order to elucidate the possible role of these factors for the development of DAVF and to study the biological activity of this unco mmonlesion. Methods We examined the histological features, proliferative and angiogenic capacities of the tissue specimens obtained from 6 patients who underwent surgery at our institution. Immunohistochemical staining for vascular endothelial growth factor (VEGF), its receptors Flk-1 and Flt-l, ephrin-B2, MIB-1 and proliferating cell nuclear antigen (PCNA) was performed using standard immuno histo chemical techniques. Results A positive immuno staining was found for all antibodies studied except MB-1, whereas nuclear endothelial expression of PCNA was observed in only 3/6 cases. VECF stained positive in all of the available specimens (6/6) . Flk-1 showed a positive immuno re action in only 2/6 cases and Flt-1 in 4/6 cases. Ephrin-B2 was expressed in the majority (5/6) of the cases. Conclusions These results support the hypothesis that DAVFs might be acquired dynamic vascular malformations with low biological activity. Vascular growth factors like VEGF and ephrin-B2 might play a pivotal role in the formation of DAVF. Chin Med J 2004; 117 (12): 1815-1820

Background Inhibition of tumor growth by endostatin has been shown to be an effective strategy in cancer therapy in mice. However, its widespread application has been hampered by difficulties in a largescale production of the recombinant endostatin protein, rapid loss bio activity of the protein, and the cumbersome daily administration. These limitations could be resolved by in vivo delivery and expression of the endostatin gene. In this study, we observed the effect and advantage of endostatin gene therapy mediated by a recombinant adenoviral vector (Ad/hEndo) on the growth of hepatocellular carcinoma BEL-7402 xenografted tumors, comparison with recombinant endo statin protein. Methods Hepatocellular carcinoma BEL-7402 cells were inoculated subcutaneously in the flank of Balb/ c nude mice. Nine days after tumor cell inoculation, animals were given a cycle of four courses of intra-tumoral injections of Ad/hEndo of 5 X108 pfu (low-dose group) and 1 ×109 pfu (high-dose group) at intervals of six days, respectively. Recombinant human endostatin protein (rhEndo) was administrated daily subcutaneously at a dose of 10 mg ·kg-1 d-1 at a site nearby the tumor for ten days. The expression of endo statin mRNA in tumor tissue was analyzed by reverse transcription- polymerase chain reaction (RT-PCR) after Ad/hEndo injection. Dynamic changes of concentration of endostatin protein in tumor tissue were quantitated by enzyme-linked immunosorbent assay (ELISA). Results After 4 courses of treatment, the tumor growth rates of high-dose treated group with 1 X109 pfu of Ad/ hEndo were inhibited by 42. 26 % conpared with the Ad/ LacZ control group (P=0. 001) and by 46. 26 % compared with the NIH buffer control group (P=0. 003), respectively. However, in this study, Ad/ hEndo at low dose of 5 ×108 pfu failed to demonstrate significant inhibition of tumor growth, compared with control groups. After daily administration of recombinant human endostatin protein (rhEndo) for 9 days, the ratio of T/C (rhEndo group versus PBS group) was less than 47 %. However, two days after rhEndo treatment ceased, the ratio of T/C was more than 50 %. The peak of expression of endo statin mRNA in tumor tissue was at 2 or 3 days after administration intratumorally with Ad/ hEndo of 1 ×109 pfu and gradually dropped undetectable by day 7. Dynamic analysis of endo statin concentration in tumor tissue showed that the highest level of mRNA is up at the third day after injection, and dropped to basal level three weeks later. Conclusions Endo statin gene therapy mediated by a recombinant adenoviral vector had significantly inhibited the growth of hepatocellular carcinoma BEL-7402 xenografted tumors at a high dose of 1 ×109 pfu compared with other groups. The analysis of dynamic expression of endo statin in vivo indicated that Ad/ hEndo had acquired a high-level,relatively long-term expression in vivo and bio activity capability. Chin Med J 2004; 117 (12): 1809- 1814

Background Complement receptor type 2 (CR2) is the receptor for C3d and C3dg and for Epstein-Barr virus. The aim of our study was to explore whether CR2 can independently mediate the activation of mitogen-activated protein kinases (MAPKs, including ERK, JNK, and p38MAPK), and to highlight the molecular mechanism of CD4+ cell deletion in AIDS. Methods HOS cells (HOS-CR2) and HOS-CD4 cells (HOS-CD4CR2) stably expressing CR2 were established and then identified by FACS and We stern blotting. Activation and blocking tests of MAPKs were assessed by We stern blot. Cell proliferation was determined using Cell Titer Aqueous One Solution Reagent. Results FACS results showed that the positive rates of HOS-CR2 and HOS-CD4CR2 cells were greater than 96 %, and Western blot showed that the CR2 expression levels on HOS-CR2 and HOS-CD4CR2 cells were high. Activation and blocking tests of MAPKs (ERK, JNK, and p38MAPK) were carried out in HOS-CR2, HOS-CD4, and HOS-CD4CR2 cells. The activation of MAPKs in HOS-CR2 cells stimulated with PMA (100 ng/ml) and NHS (10%) was identical. The activation of MAPKs increased at 5 minutes, reached a peak at 10 minutes, and decreased to baseline within 30 minute s, all in a time-dependent manner; the activation of MAPKs was blocked by anti-CR2 McAb, PD98059 (inhibitor of ERK), and Wortmanin(inhibitor of PI-3K), respectively. In HOS-CD4 cells, MAPKs were activated by HIV-gp 160. In HOS-CD4CR2 cells, MAPK activation was induced by HlV-gpl60, 10%NHS, and HIV-gp160 + 10 %NHS;phosphorylation of p38MAPKwas dramatically induced by HIV-gp160 + NHS, and lasted for 1 hour. The cell proliferation results showed that HlV-gpl60 inhibited the proliferation of HOS-CD4 and HOS-CD4CR2 cells(P<0.01) and that NHS enhanced the effect of HIV-gp160 (P<0.01). Conclusions The activation of MAPKs is independently mediated by CR2 and that anti-CR2 McAb, PD98059, and Wortmanin block the activation of MAPKs, respectively. The results of the signal transduction and cell proliferation assays of HOS-CD4CR2 cells show that CR2 plays a role in the pathogenesis of HIV infection, especially in the inhibition of CD4+ cell proliferation. Chin Med J 2004; 117 (12): 1802- 1808

Background Mutations in GJB2 gene are a major cause of autosomal recessive congenital hearing loss and the cause in some rare cases of the autosomal dominant form. The purpose of this study was to investigate the frequency and the features of GB2 mutations in the Chine se patients with congenital sensorineural deafness. Methods Using PCR anplifying the entire coding region of GJB2 gene and direct DNA sequencing to analyze mutations in this gene among unrelated 69 cases with autosomal recessive congenital nonsyndromic deafness and 27 cases of dominant congenital deafness and 35 sporadic cases. We also detected mutations in GJB2 in 100 control subjects with normal hearing. Results 17. 4 %(12/69) of the probands in the autosomal recessive, 7. 4 %(2/ 27) of dominant families and 5.7%(2/ 35) of the sporadic congenital deafness patients had deafness-causing mutations in GB2, respectively. Nine types of the mutations in GB2 were detected in the recessive and sporadic group. They consisted of five types of polymorphism, and four types of deafness-causing mutation with homozygous 35del Gin 1 sporadic (1/35), and 235delC frame shift mutation in 1 sporadic (homozygotes) and 10 recessive patients (2 heterozygotes and 8 homozygotes), and homozygous 442G→missense mutation and homozygous 465T→A nonsense mutation in 1 different recessive proband, respectively. The 465T→A that related to recessive deafness was a novel mutation found by this study. The homozygous (10/69, 14. 5 %) and the heterozygous (2/69, 2. 9 %) GJB2 mutation in the recessive patients (12/69, 17. 4 %)and the homozygotes in the sporadic patient (2/35, 5.7 %) all had congenital severe to profound sensorineural hearing loss. 511G→A missense mutation and 299 - 300delATframeshift mutation were found intwo autosomal dominant congenital deafness families (2/27, 7.4 %) . The total mutation frequency of GJB2 was 12. 2 %(16/131) in the Chinese patients with congenital sensorineural deafness and 235delC was the most common deafness-causing mutation. Six types of mutation —5 types of polymorphism and 1 type of heterozygous deletion (235delC) mutation were found in the 100 control subjects. The carry rate of the most frequent type of mutation 235delC was 0. 5 %in the controls (1/200 alleles). 109G→A was the most frequent (15/ 100, 15 %) and 79G→A was the second common (8/ 100, 8 %) polymorphism in this population. Conclusions The general mutation rate of GJB2 is 12. 2 %(16/ 131) and the 235delC is the most common type of deafness-causing mutation in Chine se patients with congenital hearing loss. 465T →A nonsense mutation that is associated to autosomal recessive deafness is a novel mutation found by this screening. 511G→A and 299 - 300delAT mutations contribute to autosomal dominant hearing loss. The study further supports the view that the common types of mutation in GJB2 according to different ethnic background and that the mutation prevalence in the East Asian deafness population is lower than that in the white population. Chin Med J 2004; 117 (12): 1797-1801