Objective:To express recombination human interleukin-13(rhIL-13)in methylotropic yeast Kchia pastoris and study its effect on the induction of DC from PBMC in vitro. Methods: An artificial gene with the optional condon usage of Kchia pastoris for IL-13 was designed and synthesized by T4 DNA ligase and PCR.The expression vector pPICZoA-IL-13 was constracted and introduced in Kchia pastoris GS115 by electroporation after linearized with Sacl.Recombinants were selected by plating cells on YPD/Zeocine plates.The recombinant protein secreted by the yeast was identified by 15%SDS-PAGE,ELISA and Western blot.The abilities to induce DC differentiation of IL-13 and IL-4 were compared. Results: Based on the result of the 15% SDS-PAGE and Western blot assay, an about 13 kD recommbinant protein expressed in the yeast supernatant was identified to be recombinant human IL-13. Adherent PBMC cultured in GM-CSF,TNFa and IL-13 displayed morphological characteristic of DC generated in media containing IL-4. They formed cellular clumps and had extensive dendrites sharp. Fluorescence-activated cell sorter analysis showed that they expressed a high level of MHC class II ,CDla,CD80,CD83,CD86. The purity and yield of both DC populations are not significantly different with IL-13 or IL-4. The phagocytosis of immature DC induced by IL-13 is potent. They were also equally potent stimulators of allogeneic lymphocytes in the mixed lymphocyte reaction. Conclusion: rhIL-13 can substitute for IL-4 in the proliferation and development of dendritic cells.

To investigate the immunological activities of the recombinant human phosphatase D2 (rhPLD2) in vitro and in vivo, especially its ability to reduce inflammatory reactions, the cDNA fragment encoding rhPLD2 was cloned into prokaryotic expression vector pET30a by RT-PCR and the recombinant protein rhPLD2 expressed in E.coli was purified from the inclusion bodies, while the anti inflammatory activity of rhPLD2 was determined by the amount of eosinophils in bronchoalveolar fluid(BALF) and blood and the expression of IL-5 and MMP-9 in lung tissues of guinea pig model of chronic asthma. It was found that the rhPLD2 recombinant protein was obtained from human Daudi cells by cloning to E.coli, which contained no membrane-binding site and signal peptide. The cDNA sequence encoded 631 amino acid residues (GenBank Accession Number: AY178289). The purity of the rhPLD2 approached up to 76% with a bioactivity of 50.9745 units/L (0.9212 g/L). In addition, the anti inflammatory effect of rhPLD2 protein could be demonstrated in the guinea pig model of chronic asthma after treatment with rhPLD2 protein, such as down regulation in the expression of the inflammatory cytokine IL-5. It is concluded that the anti-inflammator activity of the recombinant human truncated PLD2 protein produced from the E.coli plasmid can be demonstrated both in vitro and in vivo.

Objective To analyse CT diagnosis of unusual closed retroperitoneal trauma so that to improve its diagnostic accuracy.Methods CT features of unusual closed retroperitoneal trauma confirmed by clinical data and surgery in 13 cases were analyzed.Results There were adrenal hematomas in 11 cases,among them,2 cases accompanied with renal arterial occlusion,appeared as renal arterial ruptured suddenly and "interface sign".The hematomas appeared as round,similar round or mass hyperdense shadows and in combination with periadrenal tissue injury;pancreatic rupture in 2 cases,appeared as linear area of low attenuation inside pancreas.Conclusion CT scan is an effective modality for diagnosing the unusual closed retroperitoneal trauma.Correct CT diagnosis is very important for the effective surgical treatment as well as reduction of severe complications and mortality of unusual closed retroperitoneal trauma.

Objective To clone and express the fused gene fragment coding rhoptry protein ROP2 and major surface protein P30 from Toxoplasma gondii as a preparation for the construction of the complex ROP2,P30 antigen by gene engineering.Methods The gene fragment encoding P30 was amplified by PCR from T.gondii RH strain and subcloned into the recombinant plasmid pUC119/ROP2 already constructed.The recombinant plasmid pUC119/ROP2,P30 was digested by SacⅠ/HindⅢ and inserted into the same site of expression vector pET28b.The recombinant plasmid of pET28b/ROP2,P30 was transformed to E.coli and expressed under the induction of IPTG.Results The gene fragment 700 bp encoding P30 was obtained from the total DNA of T.gondii by PCR.The recombinant plasmid pET28b/ROP2,P30 was successfully constructed,which was highly expressed in E.coli,a fusion protein with molecular weight of 69 000.Conclusion The fusion gene encoding the rhoptry protein ROP2 and the major surface protein P30 of T.gondii has been successfully cloned and expressed to be an expected recombinant fusion protein ROP2,P30 with molecular weight 69 000.

Objective Trichophyton rubrum strains can cause superficial infection and also deep tissue infection, but relevant animal model has not been reported yet.The aim of this study was to construct an animal model of granuloma infected by T.rubrum. Methods Three T.rubrum strains isolated from clinical granuloma tissues, 2 T.rubrum strains isolated from tinea infection and a standard strain of ATCCMYA4438 were selected.Corticosteroids were given to the Balb/C mice before and after the injection of the T. rubrum and mucin suspension and the mice model of granuloma was established.Direct microcopy, culture and histopathologic method were adopt to verify the infection effects. Results The mice inoculated with the T.rubrum granuloma strains with mucin suspension were examined after 21 days in the condition of applying appropriate dose of glucocorticoids.Direct microscopic examination showed the slender mycelium, fungal culture showed the growth of colony and histopathology showed excessive keratinization of foot tissue, formation of granuloma in the dermis with inflammatory cell infiltration of neutro-philic granulocyte and lymphocytes.However, the mice inoculated with the T.Rubrum tinea strains with mucin suspension showed the negative result. Conclusion The rubrum granuloma mice model can be es-tablished using the clinical isolates of T.rubrum granuloma strains with the mucin and glucocorticoids interventions.

Objective To evaluate the efficacy and safety of a superpulse-mode fractional carbon dioxide(CO2) laser for the treatment of onychomycosis. Methods Patients with typical clinical manifestations of onychomycosis and positive for direct microscopic examinations of fungi were enrolled into this study, and treated with a superpulse-mode fractional CO2 laser for eight sessions. The scoring clinical index for onychomycosis (SCIO)and onychomycosis severity index (OSI)were calculated according to patients′ age, clinical type of onychomycosis, thickness of nails, area and length of nail involvement before the treatment, at the end of treatment, 1 month and 3 months after completion of treatment. Mycological clearance was also evaluated according to direct microscopy and fungal culture results. Adverse reactions to laser therapy were recorded. Statistical analysis was carried out by using the chi-square test and Wilcoxon signed-rank sum test with the SPSS 17.0 software. Results Totally, 20 patients with onychomycosis were enrolled into this study, and 75 affected nails were treated. Finally, 18 patients with 71 target nails completed the treatment and follow-up. The SCIO and OSI were 13.07 ± 6.47 and 21.11 ± 11.94 in these patients at baseline respectively, both significantly different from those at the end of treatment(9.03 ± 6.14 and 13.63 ± 12.10, respectively, both P < 0.05), 1 month((8.51 ± 6.99 and 14.18 ± 13.65, respectively, both P < 0.05)and 3 months(7.89 ± 7.26 and 13.70 ± 13.93 respectively, both P <0.05)after completion of treatment. No significant differences were observed in mycological clearance rates between the posttreatment time points(57.75%(41/71)at the end of treatment vs. 59.15%(42/71)at 1 month vs. 61.97%(44/71) at 3 months after completion of treatment, P > 0.05). The SCIO and OSI decreased from 12.48 ± 5.41 and 16.44 ± 9.89 at the baseline to 5.01 ± 5.56 and 6.44 ± 8.26 at 3 months after the treatment, respectively, in patients with distal and lateral subungual onychomycosis (DLSO), and from 17.86 ± 3.98 and 34.05 ± 2.56 to 15.88 ± 4.10 and 31.00 ± 7.28 respectively in patients with total dystrophic onychomycosis (TDO). During the treatment, several patients felt transient mild pain, but no subungual hemorrhage or other adverse reactions occurred. Conclusions The fractional CO2 laser in superpulse mode shows a reliable efficacy for the treatment of mild to moderate onychomycosis such as DLSO, especially when the nail plate is superficially invaded and grows rapidly. It directly inhibits and kills fungi, and treatment duration should be prolonged according to conditions.

Objective: Methylotropic yeast pichia pastoris system was used to express recombinant human soluble 4 1BBL protein with biological activity. Methods: According to the nuclear acid sequence coding human soluble 4 1BBL, we cloned the genes with PCR from XG 4 1BBL transfection cell line,then the gene fragment for extracellar domain was subcloned into the PUCm T vector and sequence of s4 1BBL cDNA was confirmed by sequencing. The s4 1BBL gene was inserted into the pPICZ?A , which was transformed into Pichia pastoris GS115 by linearized electroportion.The recombinant protein was identified by the assay of SDS PAGE and Western blot. Costimulating activity of rhs4 1BBL on T cell proliferation in vitro was evidenced by 3 H TdR incorporation assay. Results: The s4 1BBL cDNA was successfully obtained and insected into pPICZ?A. The protein molecular weight of hs4 1BBL in the yeast supernamant was about 21 kD by SDS PAGE analyses,and the specificitity was identified by western blot. Finally, rhs4 1BBL protein could costimulate the proliferation of T cells in vitro. Conclusion: The rhs4 1BBL protein was efficiently expressed in Pichia pastoris (GS115)and showed natural biological activities. And it may provide a valuable materials for further study of 4 1BB/4 1BBL.

Objective To identify the genetic locus for disseminated superficial actinic porokeratosis(DSAP).Methods Genome DNA was extracted from the whole blood of the family members of a pedigree of DSAP.Genotyping on chromosome12q that had been identified was performed by using7microsatellite mark-ers to scan the family members of DSAP and analysed with LINKAGE(5.1Version).Results A maximum2-point lod score of5.15with marker D12S79at a recombination fraction(?)=0.00was found.Conclusion Our study supports that DSAP gene localizes at the long arm of chromosome12,which was first reported in the literature.

Objective To investigate the effects of Sacubitril/Valsartan on amino terminal probrain natriuretic peptide (NT-proBNP),high sensitivity C-reactive protein (hs-CRP),soluble suppression of tumorigenicity 2(sST2)levels and on left ventricular(LV)structure in NYHA Ⅳ heart failure with reduced ejection fraction(HFrEF) patients.Methods A total of 67 HFrEF patients with NYHA Ⅳ were randomly divided into the control group (n =30)receiving conventional medical treatment,and the observation group(n=32)receiving Sacubitril/Valsartan instead of ACEI(or ARB if ACEI induced cough) in conventional medical treatment.NT-proBNP levels were determined by fluorescer-enhanced chemiluminescence.hs CRP levels were detected by latecx enhanced immunoturbidimetric assay.sST2 levels were determined by enzyme-linked immunosorbent assay (ELISA).The modified Simpson method was used to detect left ventricular end-diastolic diameter (LVEDD),LV posterior wall(LVPW)and LV ejection fraction(LVEF).Two groups of patients were treated and followed-up for 6 months.Results Clinical efficacy was better in the observation group than in the control group(effective rate,20 cases or 61.3％ vs.8 cases or 26.7％,P＜0.05).As compared with the control group,the observation group of patients had an increased LVEF[(46.7±9.2) ％ vs.(41.8±8.0)％,P＜0.05]and a decreased LVEDD[(52.6±6.7)mm vs.(58.8±7.5)mm,P＜0.05].After vs.before treatment,NT-proBNP,hs-CRP and sST2 levels were decreased in both control and observation groups [(1 427 ± 219) μg/L vs.(2 615 ± 273)μg/L,(1.14 ± 1.02) mg/L vs.(1.55±1.38)mg/L,(0.30±0.12)μg/L vs.(0.41±0.10)μg/L,all P＜0.05],and the decrements were much more in the observation group than in the control group (P＜0.05).The annual accumulated frequence and duration of hospitalization were less in the observation group than in the control group[(0.8±0.6)times vs.(1.8±1.0) times,(10.2±5.8)d vs.(16.5±7.2)d,P＜0.05].The maintenance dose of tolasemide was lower in the observation group than in the control group [(15.2±8.4)mg vs.(20.6±10.8)mg,P＜0.05].Conclusions Sacubitril/valsartan therapy is safe and effective and it can reduce hs-CRP and sST2 levels and improve the ventricular remodeling in HFrEF patients of HYHA Ⅳ.

Objective: To improve the diagnostic accuracy of transthoracic echocardiography (TTE) by analyzing its limitations in diagnosing partial anomalous pulmonary venous drainage (PAPVD). Method: This was a retrospective analysis of PAPVD patients seen at the Children′s Hospital of Fudan University from October 1 2006 to October 1 2016. The echocardiographic data were compared to findings on multi-slice spiral CT (MSCT), cardiac catheterization or surgery. The echocardiography machines used were Philip IE33, GE Vivid 7 and Vivid i with frequency ranging from 5.0 MHz to 7.5 MHz. The cardiac structure was analyzed according to Van Praagh segments. Result: A total of 43 cases of PAPVD were enrolled, male∶ female ratio 20∶23 with average age (27.9±21.4) months. Among them, 3 cases were simple PAPVD and 40 cases had other associated congenital heart diseases. TTE was successful in diagnosing 29 cases (67%) while 14 cases were missed. The diagnostic rate for right pulmonary vein drainage into superior vena cava, right atrium, inferior vena cava were 5/10, 17/20, and 3/5 respectively while left pulmonary vein drainage into left innominate vein was only 1/4. Added TTE images to re-exam the 9 of the 14 missed cases, 5 cases of abnormal drainage from right superior pulmonary vein were diagnosed, while 4 cases of drainage from right lower or left pulmonary vein were only picked up by indirect signs. Conclusion The distance of the pulmonary veins from the routine ultrasound view and the possibility of branch number variation may limit the accuracy of TTE in diagnosing PAPVD, especially for drainage from right lower and left pulmonary vein. But TTE is still the preferred diagnostic method. The diagnostic rate could be increased by paying special attention to non-routine views including the suprasternal fossa, the right parasternal and subcostal area.