Objective To develop anoverlap syndrome (OS)rat model by intermittent hypoxia (IH) exposure on the base of pre-existing emphysema, and to explore its characters of severe systemic inflammation and immune responses. Methods Sixty Wistar rats were put into four groups:control group, IH group, emphysema group and overlap (emphysema+IH) group. The peripheral blood samples were collected for detecting apoptosis of CD3+CD4+,CD3+CD8+T lymphocytes and neutrophils (PMN). Tumor necrosis factor (TNF)-αand interleukin (IL)-6 were evaluated by ELISA. The bronchoalveolar la-vage fluid (BALF) was taken to calculate the ratio of macrophages, neutrophils and lymphocytes under light microscope. Tis-sue blocks of lung, liver, pancreas, and right carotid artery were taken for pathologic scoring. Results The apoptotic rates of PMN and CD3 +CD8 +T cells were significantly lower in overlap group than those of other three groups (P<0.05). Pro-in-flammatory factor IL-6, TNF-αand peripheral blood CD3 +CD4 +T cell apoptosis were the highest in overlap group com-pared to those of other three groups (P<0.05). The ratio of PMN and macrophages in BALF were significantly higher in em-physema group than those of other three groups (P<0.05) and the pathology scores of lung, liver, pancreas, the ratio of carot-id artery intima-media thickness of whole thickness of vascular were significantly higher in overlap group than those of other three groups (P < 0.05).Conclusion In rat model of intermittent hypoxia-emphysema there are more serious systemic multi-organ inflammation and immune responses.

Objective:To obtain the prokaryotic expression product for the group 6 allergen of Dermatophagoides farine ( Der f 6) and detect its IgE-binding rates with sera from asthmatic children. Methods: By enzyme digestion of pET28a (+)-Der f 6 with BamHⅠ plus XhoⅠ,the target gene Der f 6 was obtained and linked into the vector pET32a (+) to construct the recombinant plasmid pET32a(+)-Der f 6, which was then transfected into E. coli BL21 cells for expression, induced with isopropyl-β-D-thiogalactoside ( IPTG) ,purified by affinity chromatography and identified by SDS-PAGE,Western blot and AMLDI-TOF,and tested by ELISA for IgE reactivity with sera from asthmatic children. Results:The plasmids pET32a(+)-Der f 6 were constructed,transformed into E. coli BL21 and expressed successfully. SDS-PAGE of the purification product showed a specific band,Western blot showed the successful binding between the purification product and the His-tag in the plasmids,and MALDI-TOF/TOF identified the identical structure to the allergen Der f 6. Using the ELISA method developed with the recombinant proteins as coating antigen,the positive rate was 41. 3% (19/46) in asthmatic children allergic to dust mite. Conclusion: The plasmids pET32a (+)-Der f 6 were constructed successfully,expressed in E. coli BL 21 (DE3). The recombinant fusion protein has a good reactivity with sera from asthmatic children.

Objective To investigate antimicrobial resistance and ceftriaxone resistance mechanisms in clinically isolated nontyphoidal Salmonella(NTS),and provide evidence for the prevention and control of NTS infection and rational use of antimicrobial agents.Methods 108 NTS isolates were isolated from stool specimens of outpatients with acute diarrhea in the Second Hospital of Tianjin Medical University and Tianjin Medical University General Hospital from May to October of 2014,NTS were performed antimicrobial susceptibility testing;non-ceftriaxone-susceptible isolates were typed by serological,multilocus sequence (MLST),and pulsed-field gel electrophoresis (PFGE)methods,extended-spectrumβ-lactamase (ESBL)detection and AmpC genes were detection.Results Among 108 NTS isolates,mono-drug resistance rate to 11 antimicrobial agents was 49.07% (n= 53),multidrug resistance rate was17.59% . Susceptibility rates to nalidixic acid,levofloxacin,ciprofloxacin,ceftriaxone,and ertapenem were 61.11% ,66.67% ,68.52% ,97.22% ,and 100.00% respectively. Three non-ceftriaxone-susceptible NTS isolates were detected,2 were ST11 Salmonellaenterica serotype (Sa8709,Sa8771),1 was ST34 Salmonellatyphimurium serotype(Sa8763). Cluster analysis of PFGE revealed that Sa8709 was highly similar to Sa8771 strains(91 .70% ), but the similarity to Sa8763 was low(55.80% );Sa8709 strain carried CTX-M gene,Sa8771 strain carried CTX-M and TEM genes,Sa8763 strain carried OXA gene. Conclusion Clinically isolated NTS in this area are low resistant to fluoroquinolones,multidrug resistant strains carrying ESBLs have emerged.

Objective To survey birth defects of neonates conceived by using various types of in vitro fertilization and embryo transfer (IVF-ET) between 1998 and 2007 in Shanghai. Methods From 1998 to 2007, 8507 neonates from 6551 pregnancies conceived through assistant reproductive technology (ART) from 7 reproductive medicine center in Shanghai were enrolled in this retrospective study, including Shanghai Ji-Ai Genetics and IVF Institute, Shanghai Jiaotong University School of Medicine affiliated Renji Hospital,Buijin Hospital, China Welfare Institute International Maternal and Infant Health Hospital, Shanghai First Maternity and Infant Health Hospital, Shanghai the Ninth People's Hospital and the Second Military Medical University affiliated Changhal Hospital. The clinical data about the type and incidence of birth defect were analyzed. Meanwhile, the factors associated with birth defect were investigated, such as various ART,gender, mother age, numbers of gestational sac, the source and quality of sperm. Results The birth defect rate was 1.08% (92/8507) and the most remarkable malformation occurred in circulation system [34% (31/92)]. The rates of major congenital malformations were 1.21% (34/2799) in fresh conventional IVF-ET, 1.07% (20/1871) in IVF-frozen-thawed embryo transfer(IVF-FET), 1.04% (23/2212) in fresh intracyto plasmic sperm injection (ICSI)-ET and 0.92% (15/1625) in ICSI-FET, which did not show statistical difference (P>0.05). There was also no significantly different incidence of malformations between 1.12% (49/4371) in male neonates and 1.02% (42/4136) in female neonates (P >0.05).However, the occurrence of congenital malformation is positively related with maternal age, the rates were 0.84% (41/4884) in mother's age less than 30 years and 1.77% (16/902) in mother' age more than 35 years, which reached statistical difference (P < 0.05). It also showed remarkable different incidence among 0.53% (25/4679) in singletons, 1.59% (57/3576) in twins and 3.97% (10/252) in triplets (P< 0.05). No remarkable difference of malformation rate among sperm sources used in fertilization were observed, including 1.09% (81/7419)in ejaculated sperm, 1.21% (7/579)in percutaneous epididymal aspiration (PESA) and 0. 79% (4/509) in donor sperm (P>0.05). Conclusions The overall incidence of birth defect in neonates from ART is similar to those conceived naturally and is not associated with different types of IVF-ET, embryo cryopreservation, sperm sources. However, maternal age and multiple pregnancies coder the higher possibility of birth defect.

Objective To investigate the antibiotic susceptibilities and the profiles of virulence genes of clinically isolated Salmonella enterica serovars Schwarzengrund ( S. Schwarzengrund) strains for bet-ter understanding the epidemiological trend of this type of non-typhoidal Salomonella and to provide guide-lines for the prevention and treatment of S. Schwarzengrund infection. Methods Stool samples and clinical data of patients with acute diarrhea who received treatment in the Second Hospital of Tianjin Medical Univer-sity during May, 2014 to October, 2014 were collected for this study. Enrichment culture and biochemical identification were used to isolate and identify the S. Schwarzengrund strains. The isolated strains were fur-ther analyzed with serotyping analysis, drug susceptibility test, pulsed field gel electrophoresis ( PFGE) and multiple locus sequence typing ( MLST ) . The representative genes carried by Salmonella pathogenicity islands (SPI) 1-5, SPI regulators and virulence plasmids were amplified by PCR. The coding genes of CdtB-islet, which were cdtB, pltA and pltB were amplified and sequenced. Results In total, 16 (14. 8%) out of 108 non-typhoidal Salmonella strains were identified as S. Schwarzengrund strains and all of them were sus-ceptible to 11 kinds of antibiotics such as fluoroquinolone, ampicillin, ceftriaxone and trimethoprim-sulfame-thoxazole. PFGE categorized the 16 S. Schwarzengrund strains into 3 clusters including A clone ( 14 strains), B clone (1 strain) and C clone (1 strain). The strains that isolated from 8 patients who ate the same food belonged to one cluster ( A clone ) , suggesting that it was an outbreak of infection. The 16 S. Schwarzengrund strains showed identical MLST type, which was ST241. The representative genes carried by SPI1-5 ( invA, sitC, hilA, sseL, sifA, mgtC, siiE and sopB) , the regulatory gene ( phoP) and the cytole-thal distending toxin islet (CdtB-islet) coding genes (cdtB, pltA and pltB) were positive, while the genes carried by virulence plasmids (pefA, prot6E and spvB) were negative. The similarities in CdtB-islet coding genes and amino acids sequences between Salmonella typhi and S. Schwarzengrund strains in this study were more than 97% and 98%, respectively. Conclusion In this study, polyclonal S. Schwarzengrund strains of ST241 type were isolated from the patients. They were susceptible to common antibiotics, but carried the virulence genes contained in SPI1-5 and CdtB-islet coding genes and might cause an outbreak of infection. Attention should be paid to the tendency and threat of clinical S. Schwarzengrund infection and continuous surveillance and investigation should be performed.

Objective:To investigate mucosal injury after high-dose methotrexate (HD-MTX) chemotherapy in treatment of children with acute lymphoblastic leukemia (ALL), and to analyze the association with methotrexate blood concentration, risk stratification and clinical efficacy.Methods:The data of 95 children with ALL who received 539 times of HD-MTX chemotherapy in the First Hospital of Harbin from November 2015 to October 2018 were retrospectively analyzed, and the association of mucosal injury with methotrexate blood concentration, disease risk degree and clinical efficacy was also analyzed.Results:Among 95 children who received 539 times of HD-MTX chemotherapy, the total incidence of mucosal injury was 8.4% (45/539); the incidence of mucosal injury was 4.6% (11/239), 7.6% (8/105), 13.3% (26/195), respectively in the low-risk group, middle-risk group and high-risk group. With the elevation of disease risk, the incidence of mucosal injury was increased ( χ2 = 10.787, P < 0.05). There was no correlation of the degree of mucosal injury with methotrexate blood concentration and disease risk degree (all P > 0.05), and the mucosal injury was not related with the clinical efficacy ( P > 0.05). Conclusion:After the application of HD-MTX in children with ALL, adjustment of the dose of rescue drug by monitoring of methotrexate blood concentration can improve the safety of therapeutic drugs.

Objective:To explore the therapeutic effect of low-dose arsenous acid/tretinoin with or without low-dose cytarabine induction regimen on myelodysplastic syndromes (MDS) with TET2 gene mutation in children.Methods:Nine children with MDS who were hospitalized at the First Hospital of Harbin from March 2009 to April 2018 were collected, including 3 cases of refractory cytopenia of childhood (RCC), 4 cases of MDS with excess of blasts type Ⅰ (MDS-EBⅠ) and 2 cases of MDS with excess of blasts type Ⅱ (MDS-EBⅡ). The next-generation gene sequencing method (NGS) was applied to detect the TET2 gene mutation in bone marrow cells of children with MDS. The low-dose arsenous acid/tretinoin with or without low-dose cytarabine induction regimen was adopted to conduct tiered therapy on children with MDS. The clinical efficacy was assessed in line with the MDS response evaluation criteria adopted by the MDS International Working Group (IWG) in 2006.Results:Among the 9 patients, the short-term clinical efficacy was observed in 8 cases. Two patients with TET2 gene mutation achieved the short-term clinical efficacy of bone marrow complete remission (mCR) and hematological improvement (HI). Among 7 patients without TET2 gene mutation, 4 patients obtained CR, 1 patients obtained mCR and HI, and 1 patients obtained stable disease (SD) in terms of the short-term clinical efficacy.Conclusion:The low-dose arsenous acid/tretinoin with or without low-dose cytarabine regimen may be beneficial to MDS children with TET2 gene mutation.

Objective:To establish a quantitative detection method for the main components of dust mite allergens Der p 1, Der p 2 specific immunoglobulin E (sIgE) by using the nano-magnetic particle chemiluminescence immunoassay.Methods:The performance indexes of the established method were evaluated after setting up and optimizing the chemiluminescence detection system and immune reaction conditions of sIgE for dust mite allergen. Serum sIgE levels of 50 suspected allergic patients with dust mite were determined by this chemiluminescence method. At the same time, this method was compared with the Phadia kit and the consistency was analyzed by Kappa test. Results:The optimal amount of magnetic beads was 25 μg, the optimal reaction buffer (pH=7.4) contained 0.1 mol/L Tris-HCl and 0.25%( W/ W) casein, the optimal coating solution contatined 20 mmol/L phosphate buffer (PB) and 1%( W/ W) bovine serum albumin (BSA), and the luminescence enhancement solution contained 0.05%( V/ V) Triton X-100. The two-step immunoreaction was adopted, and the detection could be completed with 20 μl sample at the optimal reaction temperature of 37℃. The limit of detection (LOD) of the established nano-magnetic particle chemiluminescence system in detecting Der p 1 and Der p 2 sIgE antibodies were both less than 0.01 kU/L, with the linear range of 0.2-100.0 kU/L, the precision of less than 7%, and the cross contamination rate of 0.19% and 0.21%. Compared with the Phadia system, the positive and negative coincidence rate of Der p 1 were 78.0%(32/41) and 9/9 with good consistency ( Kappa=0.65, P=0.008), and the positive and negative coincidence rate of Der P 2 were 93.3%(28/30) and 85.0%(17/20) with good consistency ( Kappa=0.79, P=0.003). Conclusion:The nano-magnetic particle chemiluminescence immunoassay is successfully established for detecting dust mite allergen sIgE, which has good detection performance and good consistency with Phadia system.

Although multifarious tumor-targeting modifications of nanoparticulate systems have been attempted in joint efforts by our predecessors, it remains challenging for nanomedicine to traverse physiological barriers involving blood vessels, tissues, and cell barriers to thereafter demonstrate excellent antitumor effects. To further overcome these inherent obstacles, we designed and prepared mycoplasma membrane (MM)-fused liposomes (LPs) with the goal of employing circulating neutrophils with the advantage of inflammatory cytokine-guided autonomous tumor localization to transport nanoparticles. We also utilized in vivo neutrophil activation induced by the liposomal form of the immune activator resiquimod (LPs-R848). Fused LPs preparations retained mycoplasma pathogen characteristics and achieved rapid recognition and endocytosis by activated neutrophils stimulated by LPs-R848. The enhanced neutrophil infiltration in homing of the inflammatory tumor microenvironment allowed more nanoparticles to be delivered into solid tumors. Facilitated by the formation of neutrophil extracellular traps (NETs), podophyllotoxin (POD)-loaded MM-fused LPs (MM-LPs-POD) were concomitantly released from neutrophils and subsequently engulfed by tumor cells during inflammation. MM-LPs-POD displayed superior suppression efficacy of tumor growth and lung metastasis in a 4T1 breast tumor model. Overall, such a strategy of pathogen-mimicking nanoparticles hijacking neutrophils in situ combined with enhanced neutrophil infiltration indeed elevates the potential of chemotherapeutics for tumor targeting therapy.