[The experience of surgical treatment for Ebstein anomaly in 141 children].

Objective: To explore the surgical strategy for Ebstein anomaly in children. Methods: From January 2003 to December 2015, a total of 141 cases of Ebstein anomaly were treated at Department of Pediatric Cardiothoracic Surgery, Shanghai Children's Medical Center, School of Medicine, Shanghai Jiaotong University. There were 65 male and 76 female patients, with age of (6.9±1.6) years (ranging from 10 months to 15 years), weight of (19.6±4.7) kg (ranging from 6.5 to 59.0 kg). All patients were diagnosed by 2 dimensional Doppler echocardiography and the septal leaflet and posterior leaflet displaced downward from 1.0 to 5.0 cm. The tricuspid valve regurgitation (TR) were mild in 26 cases, moderate in 46 cases and severe in 69 cases. Tricuspid valvuloplasty were performed in 131 cases (94 cone reconstruction, 37 valve hoist), tricuspid valve replacement in 2 cases and tricuspid valve closed in 8 cases. Surgical strategy were divided into biventricular heart function in 77 cases, one and a half ventricular heart function in 56 cases, and single ventricular heart function in 8 cases. Results: Three patients were changed to one and a half ventricular repair from biventricular repair due to unstable hemodynamics in the early postoperative period. One case died in biventricular group. The complete atrioventricular block were occurred in 3 patients and pacemaker were applied. One hundred and forty cases discharged from hospital. There were mild TR in 118 cases, moderate in 14 cases and closed in 8 cases. One hundred and thirty-seven cases were followed up regularly in 18 to 172 months. Ninety-one cases were treated by cone reconstruction (mild TR in 75 cases, moderate in 15 cases and severe in 1 case). Thirty-six cases were operated by tricuspid valve hoist (mild TR in 21 cases, moderate in 12 cases and severe in 3 cases). In the patients with severe TR (4 cases), 3 cases were reoperated by cone reconstruction. One case's valve was closed because of the dysplasia of the anterior valve and then from one and a half ventricular heart function to single ventricular function, the oxygen saturation was increased. Two patients underwent tricuspid valve replacement, 1 died and the other's mechanical valve was removed, and changed to single ventricular function repair. Conclusions: Although tricuspid cone reconstruction can achieve good results, the stable hemodynamic of early postoperative can be effectively maintained by using the surgical strategy of one and a half ventricular repair. To the patients with severe tricuspid regurgitation and hypoxemia due to severe tricuspid valve dysplasia, transforming to a functional single ventricle may be the only choice when there comes to the unstable hemodynamic.

KNOCKDOWN OF CASEIN KINASE 1e INHIBITS CELL PROLIFERATION AND INVASION OF COLORECTAL CANCER CELLS VIA INHIBITION OF THE Wnt/ß-CATENIN SIGNALING.

Deregulation of casein kinase 1 epsilon (CK1ε) is involved in the development of multiple pathological disorders such as cancer, however the function and molecular mechanism of CK1εin cancer are still unclear. In the present study, we aimed to investigate the role of CK1ε in human colorectal cancer (CRC). The expression of CK1ε was examined by immunohistochemical assay using a tissue microarray procedure. A loss-of-function experiment was performed to observe the effects of lentivirus-mediated CK1ε shRNA (Lv-shCK1ε) on cell proliferation and invasive potential by MTT and Transwell assays in CRC cell line (SW480). As a result, we found that the expression of CK1ε protein was significantly increased in CRC tissues compared with that in adjacent non-cancerous tissues (ANCT) (68.9% vs 42.2%, P=0.017) and was correlated with the Duke’s staging and depth of invasion in CRC patients (P=0.012; P=0.015). Knockdown of CK1ε reduced cell proliferation and invasion of CRC cells followed by the downregulation of wnt3α, ß-catenin, PCNA and MMP-9. In conclusion, our findings show that high expression of CK1ε is positively associated with the Duke’s staging and depth of invasion in CRC patients, and knockdown of CK1ε suppresses the growth and invasion of CRC cells through inhibition of the wnt/ß-catenin signaling, suggesting that CK1ε may serve as a promising therapeutic target for the treatment of CRC.

[Observation on specific IgG4 antibody of schistosomiasis patients before and after treatment].

OBJECTIVE: To observe the alteration of specific IgG4 antibody of schistosomiasis patients before and after treatment. METHODS: ELISA. RESULTS: The SEA-IgG4 and AWA-IgG4 positive rates of 27 schistosomiasis cases were 96.3% and 100%, respectively, their average OD values were 1.62 and 0.72. 6 months post treatment 18 cases were followed up, the positive rates were 94.4% and 100%, respectively, their average OD values were 1.06 and 0.56, respectively. 12 months post treatment all cases were followed up, the positive rates of SEA-IgG4 and AWA-IgG4 were 96.3% and 92.6%, respectively, their average OD values were 0.99 and 0.58, respectively. CONCLUSION: No obvious changes were found in the SEA-IgG4 and AWA-IgG4 positive rates of 27 schistosomiasis cases before and after treatment, whereas the antibody level of specific IgG4 was decreased.

[Circulating antigen detection in schistosomiasis japonica patients by sandwich ELISA using polyclonal and monoclonal antibodies].

OBJECTIVE: To detect circulating antigen in the patients with schistosomiasis. METHODS: Polyclonal antibody(PcAb)-monoclonal antibody(McAb) sandwich ELISA. RESULTS: Sera from 150 patients with schistosomiasis were detected, the average sensitivity was 84.7%(72.0%-91.0%). No false positive reaction was detected in 40 normal controls and none but 2 patients(2/10) with paragonimiasis showed cross reactions among 74 cases with other parasitic infections. CONCLUSION: PcAb-McAb sandwich ELISA is a relatively ideal method for circulatling antigen detection.

Specificity of the solid phase alkaline phosphatase immunocapture assay for the diagnosis of human Schistosoma mansoni infection.

The species specificity of the solid phase alkaline phosphatase immunocapture assay (APIA) for the immunological detection of human immunoglobulin G antibodies to the alkaline phosphatase of adult Schistosoma mansoni was evaluated. Sera from schistosomiasis patients from South America, West Africa, south-east Asia and uninfected control subjects were compared. Only the sera of patients infected with S. mansoni gave positive results. There was no apparent difference between 2 populations infected with S. mansoni, one from South America and the other from West Africa. The results with sera from various regions of West Africa were also indistinguishable. Although the APIA was not able to discriminate the geographical origin of the S. mansoni-infected subjects, the method appeared to be specific for S. mansoni and suitable for use in the immunodiagnosis of schistosomiasis mansoni, particularly in endemic areas where mixed infections of Schistosoma spp. occur.

Strain complex of Schistosoma japonicum in the mainland of China.

The present paper deals with studies on the characteristics of Schistosoma japonicum isolated from five localities in the mainland of China. The following items were observed and compared including morphometric data, susceptibility of six mammalian hosts, prepatent period, compatibility between larvae and snail hosts, size of hepatic granuloma produced by eggs, immunoreactions in experimental animals, sensitivity to praziquantel, SDS-PAGE protein pattern and its antigenicity analysis, DNA hybridization and genetic variation and differentiation by analysis with multilocus enzyme electrophoresis. By means of these multidisciplinary methods, from morphological to molecular level, the following conclusions may be drawn from our results. The evidence indicates firstly that S. japonicum in the mainland of China comprises a strain complex with several components of geographically distributed strains. At least four distinct strains exist, ie Yunnan, Guangxi, Sichuan and Anhui-Hubei. Characteristics of each strain are distinct and the results of these studies lead to discussion on the problem of the intraspecific and interstrain differentiation of S. japonicum in the mainland of China.

SDS-PAGE protein pattern and its antigenicity analysis of different isolates of Schistosoma japonicum in China.

Homogenates prepared from S. japonicum adult worms of different isolates from Anhui, Hubei, Guangxi, Yunnan and Sichuan Provinces were analyzed by SDS-PAGE and enzyme linked immunoelectrotransfer blot (EITB) tested with rabbit anti-snails antibody. The results of SDS-PAGE indicated that with silver staining both male and female worms of Guangxi isolate showed some definite differences in their protein profile, namely, absence of one band between 50-75 kDa in male worms and marked reduction in quantity of > 110 and 30 kDa bands in female worms. There was no obvious difference among other isolates both in male and female worms. The EITB patterns were similar in S. japonicum of Anhui and Hubei, and it was also the case with isolates from Yunnan and Sichuan, except that Yunnan female worms had a distinct band at 84 kDa which could hardly be seen in EITB pattern of Sichuan female worms; female worms of Guangxi isolates also showed a distinct 84 kDa band. The EITB pattern of male worms from Guangxi isolates showed 2 main bands of MW > 130 kDa against anti-Anhui snail antiserum which corresponded with the result of male worms of Anhui isolates. But these bands could not be seen with male worms from isolates of Yunnan and Sichuan.

[Characterization of Echinococcus granulosus and Echinococcus multilocularis by DNA probe].

Cloned DNA fragments pHD5, pSM889 and pEG18 have been used as DNA probes in the restriction endonuclease analysis and southern blot hybridization to characterize E. granulosus and E. multilocularis protoscolices from China. Southern blot hybridization method is sensitive, specific and has the advantage in identification over microscopic examination. The authors deem that it can be used in the base-line epidemiological survey and surveillance of hydatid disease to provide data for hydatid disease control.

[Studies on the strain differences of Schistosoma japonicum in the mainland of China. XII. DNA hybridization of five isolates].

The nonradioactive labelled probe pSM 889 was hybridized to Southern blots of genomic DNA of five isolates of Schistosoma japonicum from Anhui, Hubei, Guangxi, Sichuan and Yunnan in the mainland of China. The major bands of hybridization of DNA digested by restriction endonucleases EcoRI and BamHI are same among the five isolates, but the minor bands of hybridization of DNA digested by EcoRI show some differences between 4.4-9.6 kb and between 2.3-4.4 kb among the five isolates. The result shows that the five isolates are of some genetic variation. It provides, at molecular level, evidence of the existence of different strains of S. japonicum in the mainland of China.

[Schistosoma japonicum adult worm RNA isolation with Chomczynski's method].

A rapid method of total RNA of Schistosoma japonicum adult worm isolation by single step extraction with an acid guanidinium thiocyanate-phenol-chloroform mixture is described. The method provides a pure preparation of intact RNA and can be completed within several hours. It is particularly useful for isolation of RNA from minute quantity of parasite material. Analysis of agarose gels electrophoresis reveals that the RNA preparations show a distinct band comigrating with 18S rat brain RNA and without large rRNA species. The large rRNA is in vivo nicked. The absence of a band at approximately 26S presumably reflects post transcriptional processing of the large rRNA.

[RFLP analysis of DNA from Echinococcus granulosus collected from four provinces/autonomous region in China].

A fragment of the ribosomal RNA gene of Schistosoma mansoni pSM889 and two DNA fragments specific to Echinococcus granulosus pHD5 and pEG18 have been used as DNA probes to assess the extent of genetic variability within E. granulosus. The DNA analysis, including restriction endonuclease digestion and Southern blot hybridization with the probes, did not demonstrate any genetic variation among E. granulosus collected from sheep in Xinjiang, Qinghai, Gansu and Ninxia. Similarly, there was no genetic variation among E. granulosus isolates collected from yak in Qinghai and Gansu provinces. The authors deemed that the yak isolates and the sheep isolates of E. granulosus appear to belong to a same strain.

[Studies on the strain differences of Schistosoma japonicum in the mainland of China. XI. Enzyme linked immunoelectrotransfer blot analysis].

Antigens prepared from S. japonicum adult worms of different isolates i. e. Anhui, Hubei, Guangxi, Yunnan and Sichuan by origin were analyzed by enzyme linked immunoelectrotransfer blot (EITB) probed with rabbit anti-snail antibody (Figs 1,2). Anti-sera against Oncomelania h. hupensis from Anhui, Hubei, Yunnan and Sichuan localities were prepared separately and used in the tests. The EITB patterns were similar in S. j. isolates of Anhui and Hubei, and it was also the case among S. j. isolates of Yunnan and Sichuan except Yunnan female worms with a marked band of 84 kDa but it was almost invisible in EITB pattern of Sichuan female worms. Like Yunnan isolate, female worms of Guangxi isolate also showed marked 84 kDa bands. The EITB pattern of male worms from Guangxi isolate showed 2 main bands of mw > 130 kDa against anti-Anhui snail anti-serum, which corresponded with the male worms of Anhui isolate whereas the color of the bands was darker and denser in the former isolates, and these bands can not be seen in the male worms from isolates of Yunnan and Sichuan. Based on the above mentioned results in connection with the information about the susceptibility between different isolates of schistosomes and their snail hosts which we have reported before, some preliminary analysis and discussion were made.

[Studies on the strain differences of Schistosoma japonicum in the mainland of China. X. Comparison of SDS-page protein patterns].

The purpose of the present study was to investigate the SDS-PAGE separation of proteins of the 5 different isolates of Schistosoma japonicum from the mainland of China to determine their similarities and/or differences and to gain additional data which could give an added insight into the degree of relationship. Soluble proteins of freshly sonicated adult worm extract of the 5 different isolates (i.e., Anhui, Hubei, Guangxi, Sichuan and Yunnan) were compared by SDS-polyacrylamide gel electrophoresis. The results indicated that no marked difference could be observed among the isolates after gels were stained by Coomassie blue white both male and female worms of Guangxi isolate showed some definite differences in their protein profile, i.e., lack of 50-75 kDa band in male worms and a marked reduction in the quantity of > 110 and 30 kDa bands in female worms as shown by silver stain.

[Detection of circulating membrane antigen of schistosome in schistosomiasis by dot-ELISA and idiotype/antiidiotype interaction inhibition test].

A monoclonal antibody (designated 8SE) against membrane antigen of adult Schistosoma japonicum labeled with HRP was used in dot-ELISA (direct method) to detect schistosomal circulating membrane antigen. Sera from 48 parasitologically confirmed schistosomiasis cases were tested, 39 (81.3%) were positive. No false positive reaction was found in sera from 24 healthy controls. No cross reaction was detected in sera from clonorchiasis or paragonimiasis in 18 cases each. This results suggest that circulating membrane antigen does exist in patients with schistosomiasis and it might be used as a complementary method for diagnosis of schistosomiasis. A preliminary result of idiotype/antiidiotype interaction inhibition reaction for detecting circulating membrane antigen was also presented.

[Construction of Echinococcus granulosus genomic library].

Using EMBL3 phage DNA as a vector, a perfect genomic library of Echinococcus granulosus from sheep from Xinjiang has been constructed, which contains 1.2 x 10(6) independent recombinants. The main procedure of construction comprised: 1. extraction of Echinococcus granulosus genomic DNA, 2. partial digestion of the extracted E. granulosus genomic DNA with restriction enzyme Sau3Al, 3. harvest of 15-23 kb DNA fragments by electric elution equipment, 4. preparation of EMBL3 phage vector DNA, 5. cleavage of EMBL3 vector DNA with two restriction enzymes Bam HI and Eco RI to remove the central fragment, 6. ligation of the harvested 15-23 kb E. granulosus genomic DNA and the cleavaged vector DNA with T4 DNA ligase, 7. the package reaction of ligated recombinant DNA in vitro with the package protein and with Q359 strain (Spi-) to screen recombinants, 8. identification of the genomic library, 9. hybridization in situ by pSM889 probe, obtaining 6 positive recombinant clones. It was estimated that the E. granulosus genome was about 1.5 x 10(8) bp. A perfect E.g. genomic library is expected to contain 4.6 x 10(4) independent recombinants at minimum. The constructed genomic library has enough independent recombinants (1.2 x 10(6)). This is the first time to establish an E. granulosus genomic library in China. Previous work showed that there existed differences in many aspects, including pathogenicity, among various isolates of E. granulosus from different hosts and areas. We plan to employ this library to screen the E. granulosus intraspecies DNA probe by hybridization in situ. This kind of probe is envisaged to be of advantage for epidemiological investigation of the hydatid disease in China. It also provides a base for researching E. granulosus at the molecular level.

[Detection of circulating antigen and/or antigen-antibody complex by using McAb against surface membrane antigen of adult Schistosoma japonicum].

In the present paper, the results of detecting circulating antigen and/or antigen-antibody complexes by McAb against surface membrane antigen of adult Schistosoma japonicum were reported. The McAb, coded as 8SE4, was prepared by fusion of SP2/0 myeloma cells with spleen cells of the BALB/c mice immunized with the saline extract of adult S. japonicum. The 8SE4- directed antigen was proved to be located on the surface of the adult worm. After being purified by a DE52 column, 8SE4 was labelled with HRP and the conjugate (HRP-8SE4) was used in the test. For testing, the serum sample was first incubated with HRP-8SE4, then PEG (mw. 6,000) was added to precipitate the antigen-antibody complex. Upon centrifugation, OPD was added to the precipitate. Results were read by ELISA reader at 492nm. The OD value was found to be proportional to the amount of circulating antigen and/or antigen-antibody complexes. Results from 5 heavily infected (1,500-2,000 cercariae) rabbits showed that the OD values were raised significantly at the 6th week post infection, being 1.9-4.5 times higher than those before infection. The OD values of the 5 rabbits each lightly infected with 10-500 cercariae were also markedly raised 6 weeks post infection and reached the peak at the 8th week, then maintained in high levels until 11th week post infection. The worm burden of the 5 lightly infected rabbits were 4-326. No obvious correlations between OD values and worm loads were observed. The results suggested the existence of surface membrane-related antigen and/or antigen-antibody complexes in the circulation of infected rabbits.

[Immunological humoral response in mice experimentally infected with Echinococcus granulosus, preliminary result of kinetic observation].

5 serological methods included latex agglutination test (LA), enzyme-linked immunoelectrotransfer blot (EITB), enzyme-linked immunosorbent assay (ELISA), dot-enzyme-linked immunosorbent assay (Dot-ELISA) and immunofluorescence antibody test (IFA) were used to trace the kinetic changes of the humoral response in NIH mice experimentally infected with protoscolex of Echinococcus granulosus. Results showed that Dot-ELISA and IFA were able to detect antibodies 2 wk after infection while ELISA, EITB and LA gave positive reactions 4 wk, 2 mos and 3 mos after infection respectively. Therefore Dot-ELISA and IFA were considered to be the most sensitive ones among the 5 methods used. All infected mice gave positive reactions at 8 and 12 mos but none of the normal control mice showed any reaction throughout the period of observation (Fig. 1).