ObjectiveTo investigate the distribution and expression of y-secretase subunit (APH-1)in the central nervous system (CNS) of APP/PS1 double transgenic Alzheimer's disease (AD) adult mouse model,and to detect the expression difference of APH-1 in developmental brain between AD model mouse and wild-type littermates in order to further clarify the relationship between APH-1 and AD. MethodsOffspring bred by APP/PS1 double transgenic AD mice were genotyped.Immunohistochemical staining was used to detect APH-1 distribution and expression in the CNS of adult APP/PS1 double transgenic AD mouse model,in the brain of AD model mouse and its wild-type littermates on postnatal day 1,7,21 and 120.Results APH-1 was widely expressed in almost all regions of the CNS,especially in the cerebral cortex,hippocampus,olfactory bulb,hypothalamus,ventral striatum,caudate putamen,raphe magnus nucleus,cerebellum,brainstem and spinal cord of the adult APP/PS1 double transgenic mice.APH-1 expression was higher in the cortex of both AD and wild type mouse on postnatal day 1 than on postnatal day 7 and 21 with increased level of APH-1 protein in adult mouse brain.APH-1 expression in the brain of AD mice was higher than in its wild type littermates at any stage(P＜0.05).Conclusions Distribution of APH-1 is ubiquitous and region-dependent in the CNS.The different distribution and expression between APP/PS1 double transgenic mouse model and its wild type littermate indicate that APH-1 may be related to AD.

Objective To investigate whether ultrasound (US)-targeted microbubble (MB) destruction (UTMD) can influence the biofilm and bacteria in morphology.Methods Twenty-hour biofilms of Staphylococcus epidermidis RP62A were treated with US or UTMD.The acoustic intensity was 0.5-1.5W/cm2,the duty cycle was 50％ and the duration was 10 minutes.After treatment,the absorbance values (A570) of biofilms stained with the crystal violet were measured to assess the biofilm density.The biofilms were observed with macroscopy and light microscopy.The biofilms were examined by confocal laserscanning microscopy (CLSM) and scanning electron microscopy (SEM).Results A thick and compact biofilm was observed in the untreated control group,and there were no obvious micropores in biofilms under macrology and light micrology.Although there were no significant changes under macroscopy in both biofilms treated with US only and UTMD with 0.5 W/cm2 acoustic intensity,interestingly,many micropores could be found under microscopy.The diameters of micropores increased with increasing acoustic intensity,and the micropores in biofilms treated with UTMD were bigger than those treated with US-only in the same condition of acoustic intensity (P ＜0.05).The largest diameters of micropores were up to 1 mm in biofilms treated with UTMD using 1.5 W/cm2 (P ＜0.05).The biofilm density (A570 value) decreased with increasing of the acoustic intensity,and the values in UTMD group of 1.5 W/cm2 were the lowest (P ＜ 0.05).Micropores also could be observed under CLSM.There were no obvious dead bacteria in biofilms treated with US and UTMD compared with untreated control group (P ＞0.05).Under SEM,the shape of bacteria in biofilms treated with US and UTMD became irregular,and many rounded projection could be observed in the surface of the bacteria treated with UTMD.Conclusions US and UTMD can produce micropores in biofilms,which might help to promote antibiotic activity against biofilms

Objective To investigate the CD34~+ umbilical cord blood hematopoietic stem cells (CD34~+ UBSC) transfected with interleukin 21 (IL-21) against the ovarian cancer effect in tumor-bearing nude mice. Methods CD34~+ UBSC were obtained from the UBSC by a magnetically activated cell sorting technique and then transfected with recombinant plasmid plRES2-IL-21-EGFP after the CD34~+ UBSC were proliferated in vitro. The therapeutic effect was evaluated by the size of the tumor and the life span in nude mice treated with the CD34~+ UBSC-IL-21. The expression of IL-21 and its bioactivity in CD34~+ UBSC-IL-21 and in local neoplasitc tissues were respectively detected by reverse transcription-polymerase chain reaction (RT-PCR), immune fluorescence technique, ELISA, Western blot, immunohistochemistry and splenocyte proliferative activity. The NK cell cytotoxicity and the numbers of NK cells, serum level of IFN-γ and TNF-αwere simultaneouly detected by FCM and ELISA, respectively. Results CD34~+ UBSC were cultured and transfected with pIRES2-IL-21-EGFP successfully. CD34~+ UBSC-IL-21 could inhibit the tumor growth and extended nude mice life span compared with other groups (P < 0.01). The expression of IL-21 in the neo-plastic tissue, serum level of IFN-γ and TNF-α , NK cell activity and the numbers of NK cells of mice origin and of human origin in splenocytes were increased significantly in the nude mice treated with CD34~+ UBSC-IL-21 compared with other groups(P <0.01). Conclusion The CD34~+ UBSC transfected with IL-21 have competent against ovarian cancer in tumor-bearing nude mice. The findings may establish a foundation for gene therapy of the ovarian cancer by CD34~+ UBSC-IL-21 in clinic application.

Objective To investigate the effect of HCV RNA on virological and immunological response to highly active antiretroviral therapy (HAART),liver function and blood lipid levels in HIV/HCV co-infected patients.Methods In a cohort study,275 HIV/HCV co-infected former blood donors receiving HAART were followed up every six month in Henan province in China.HCV RNA,HIV RNA,CD+4 T cell counts,indexes of liver function and lipid levels were periodically tested.The differences of HIV viral load suppression,immunological response,liver injury and blood lipid levels between HCV RNA positive group and negative group were compared by x2 test and two independent-samples tests.Result There was no significant difference of HIV viral load suppression between HCV RNA positive group and HCV RNA negative group six-month treatment (45.6% vs.38.5% ,X2=1.150,P>0.05) and CD+4 T cell counts before (286 cells/μ1 vs.209 cells/μ1,Z=0.734,P=0.463)and after 6-month (310 cells/μ1 vs.362 cells/μl,Z=0.562,P=0.574) ,12-month(378 cells/μ1 vs.289 cells/μ1,Z=1.091,P=0.275),18-month(363 cells/μ1 vs.288 cells/μl,Z=1.435,P=0.151) ,24-month(413 cells/μ1 vs.348 cells/μ1,Z=0.939,P=0.348) HAART.The mean levels of serum ALT (55.0 U/L vs.29.5 U/L,Z=6.789,P<0.01),AST(46.0 U/L vs.33.0 U/L,Z=4.890,P<0.01)、TBIL(9.3 mmol/L vs.7.2 mmol/L,Z=3.748,P<0.01)were significantly higher in HCV RNA positive group than that in HCV RNA negative group.HCV RNA was the independent variables associated with liver injury after HAART (aOR=3.8,P<0.01).The serum triglyceride level was higher in HCV RNA positive group than that in HCV RNA negative group(1.2 mmoL/L vs.1.4 mmol/L,Z=1.936,P=0.043) .The serum HDL level was higher in HCV RNA positive group than that in HCV RNA negative group (1.5 mmol/L vs.1.3 mmol/L,Z=2.251,P=0.024).Conclusions HCV RNA does not affect HIV virological responses to HAART and CD+4 T recovery.HCV RNA is an independent risk factor associated with liver injury in HIV/HCV co-infected patients receiving HAART,but appears to provide significant protection against HAART-ieduced hyperlipidemia.

OBJECTIVETo investigate the expression of matrix metalloproteinase-2 (MMP-2) and insulin-like growth factor-1 (IGF-1) in gastric carcinoma and their clinicopathological significance.

METHODSExpressions of MMP-2 and IGF-1 were examined by using immunohistochemical SP staining and cross-compared with clinicopathological features of gastric carcinoma.

RESULTSHigh expression of MMP-2 and IGF-1 were observed in 70.4% (307/436) and 49.5% (216/436) of gastric carcinoma tissues respectively, significantly higher than those in non-tumor gastric mucosa (3.3% and 5.4%, respectively; all P < 0.05). The high expression rate of MMP-2 and IGF-1 were significantly associated with the patient age, tumor size, tumor location, Lauren classification, TNM staging, depth of tumor infiltration, presence of vessel invasion, lymph node and distant metastasis (all P < 0.05). In addition, the expression of MMP-2 was positively linked with the expression level of IGF-1 (P < 0.05). Univariate analysis showed that high expression of MMP-2, was significantly associated with poor prognosis of tumor of TNM stage I and II (all P < 0.05), high expression of IGF-1 was significantly correlated with poor prognosis of patients with TNM stage I, II and III tumor (all P < 0.05). Cox multivariate analysis indicated that the high expressions of MMP-2 and IGF-1 could be independent prognostic indices for gastric carcinoma.

CONCLUSIONSHigh expression of MMP-2 and IGF-1 proteins are significantly correlated with the invasion and metastasis of gastric carcinoma, it is helpful to simultaneously detect the expressions of MMP-2 and IGF-1 proteins in predicting prognosis of patients with gastric carcinoma.

Adolescent ; Adult ; Aged ; Aged, 80 and over ; Female ; Humans ; Insulin-Like Growth Factor I ; metabolism ; Lymphatic Metastasis ; Male ; Matrix Metalloproteinase 2 ; metabolism ; Middle Aged ; Neoplasm Invasiveness ; Prognosis ; Stomach Neoplasms ; metabolism ; pathology ; Young Adult

OBJECTIVETo evaluate the effect of alpha-galactosidase A (Gla) deficiency on FV Leiden (FVL) associated thrombosis in vivo.

METHODSTo generate the mice carrying mutations in Gla and FVL and analyze the tissue fibrin deposition in organs and thrombosis.

RESULTSIn the presence of FVL, Gla deficiency greatly increased tissue fibrin deposition compared with that in wild-type [Gla(-/0) FV(Q/Q) vs. Gla(+/0) FV(Q/Q) = (0.24 +/- 0.07)% vs. (0.086 +/- 0.049)%, P < 0.0001; Gla(-/-) FV(Q/Q) vs. Gla(+/+) FV(Q/Q) = (0.32 +/- 0.03)% vs. (0.06 +/- 0.005)%, P < 0.05]. With Gla deficiency, the number of thrombi on organ sections in FVL mice was significantly increased [(Gla(-/-) FV(Q/Q) and Gla(-/0) FV(Q/Q)) vs. (Gla(+/+) FV(Q/Q) and Gla(+/0) FV(Q/Q)) = 1.9 +/- 0.7 vs. 0.3 +/- 0.1, P < 0.05].

CONCLUSIONSGla deficiency could be an important genetic modifier for the enhanced thrombosis associated with FVL.

Animals ; Fabry Disease ; genetics ; Factor V ; genetics ; Genotype ; Male ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; Mutation ; Thrombosis ; genetics ; pathology

OBJECTIVE To study the differentiation of PC12 cells induced by total salvianolic acid (Tsa) and the mechanism. METHODS MTT assay was used to detect the effect of Tsa 0.01, 0.1 and 1.0 μg·L-1on proliferation of PC12 cells and on the cells damaged by oxygen and glucose deprivation (OGD).The number of projections of PC12 cells was statistically analyzed.Western blotting was applied to detect the levels of microtubule-associated protein2 (MAP-2), extracellular signal-regulated kinase1/2 (ERK1/2), phosphorylated ERK1/2(p-ERK1/2), mitogen-activated protein kinase kinase1/2(MEK1/2) and p-MEK1/2 proteins.MEK inhibitor U0126 was examined for its effect on expressions of p-ERK1/2 and ERK1/2 protein in PC12 cells induced by Tsa 1.0 μg·L-1.RESULTS Compared with normal control group, Tsa 1.0 μg·L-1could promote PC12 cell proliferation, and the survival rate was increased by 90%, but the survival rate of PC12 cells was not affected by Tsa 0.01 or 0.1 μg·L-1. Compared with OGD injured group,PC12 cells injured by OGD could be repaired by Tsa 0.1 or 1.0 μg·L-1,and the survival rate was increased to (47.7±1.8)% and (63.2±13.0)%, respectively (P<0.05, P<0.01). Compared with normal control group,Tsa 0.01,0.1 and 1.0 μg·L-1could promote the growth of PC12 cell projections (P<0.01). Western blotting results showed that Tsa could promote the expressions of MAP-2, p-ERK1/2 and p-MEK1/2 proteins, and this effect could be blocked by U0126 inhibitor (P<0.01). CONCLUSION Tsa can induce the proliferation and differentiation of PC12 cells, the mechanism of which is possibly the activation of p-MEK and p-ERK1/2.

This article reported the clinical characteristics and SRD5A2 gene mutation pattern of a child with steroid 5-α reductase type 2 deficiency. The 2-month-old boy showed hypospadias and short penis shortly after birth. DNA was extracted from the peripheral blood of the child and his parents. The endocrine disease-related genes were captured and sequenced by high-throughput sequencing technology, and the family DNA samples were verified by Sanger sequencing. The results showed that c.680G>A(p.R227Q) and c.608G>A(p.G203D) compound heterozygous mutations existed in the SRD5A2 gene of the child. The c.680G>A mutation inherited from his father, which was a known pathogenic mutation. The c.608G>A mutation originated from his mother, which was a novel mutation discovered in this study. These results provide molecular evidence for the etiological diagnosis of the child and genetic counseling for the family, as well as extend the mutation spectrum of SRD5A2 gene.
3-Oxo-5-alpha-Steroid 4-Dehydrogenase ; genetics ; Base Sequence ; Child ; Female ; Humans ; Hypospadias ; Infant ; Male ; Membrane Proteins ; genetics ; Mutation

OBJECTIVETo detect and study the types of Borrelia burgdorferi sensu lato in ticks and rodents from Da Xing-An Mountains Forest areas of China.

METHODSNested PCR was performed to amplify 5S-23S rRNA intergenic spacer of B. burgdorferi. Positive products were analysed by restriction fragment length polymorphism (RFLP) and single strand conformation polymorphism (SSCP), specimens showing unique RFLP profile were sequenced and analysed.

RESULTS1336 Ixodes persulcatus, 144 Dermacento silvarum, 144 Haemaphysalis concinna and 145 rodents of 9 species were collected from 16 sections of Da Xing-An Mountains Forest areas of China. Specific fragments were amplified from 293 I. persulcatus and 6 D. silvarum and 5 rodents of 4 species. B. burgdorferi was not detected in H. concinna. Among the positively tested I. persulcatus, 209 contained B. garinii genospecies and 45 contained B.afzelii genospecies based on RFLP. Moreover, B.garinii genospecies consisted of B. garinii 20047 and B. garinii NT29. 17 adult I. persulcatus were simultaneously infected with B. garinii 20047 and B. garinii NT29. Nine adult I. persulcatus were simultaneously infected with B. garinii 20047 and B. afzelii. Four adult I. persulcatus were simultaneously infected with B. garinii 20047 and B. garinii NT29 and B. afzelii. Two D. silvarum were infected with B. garinii 20047, 1 D. silvarum with B. garinii 20047, 2 D. silvarum with B. afzelii. 3 rodents were infected with B. garinii 20047 while 2 rodents were infected with B. garinii NT29. Mixed infection was not found in D. silvarum and rodents. In addition, nine I. persulcatus and one D. silvarum specimens showed unique RFLP pattern. Data from sequential analysis showed that they all belonged to B. garinii. PCR-SSCP profiles of 5S-23S rRNA intergenic spacer of B. burgdorferi in the positive specimens exceeded 36 types; B. garinii 20047 showed 16 types while B. garinii NT29 showing 11 types, B. afzelii showing 9 types. SSCP profiles of the specimens coinfected with multiple B. burgdorferi was relatively complex.

CONCLUSIONThe infection of B. burgdorferi was found in the ticks and rodents in Da Xing-An Mountains Forests areas. The infection rate of I. persulcatus was high. B. garinii was predominant genospecies, and the population of B. burgdorferi was heterogeneous in the area. Mixed infections of different B. burgdorferi genospecies in ticks were found. I. persulcatus and Clethrionomys rufocanus were possibly served as major vector and major host for B. burgdorferi, respectively, suggesting that further study is needed to confirm the coinfection in humans and animals in this region.

Animals ; Borrelia burgdorferi Group ; genetics ; isolation & purification ; China ; epidemiology ; Humans ; Lyme Disease ; epidemiology ; Polymorphism, Restriction Fragment Length ; Polymorphism, Single-Stranded Conformational ; RNA, Bacterial ; analysis ; Rodentia ; microbiology ; Ticks ; microbiology ; Trees

OBJECTIVETo detect the quantity, proportion and function of producing cytokines of Th1 and Th2 cells in aplastic anemia (AA) patients and their contribution to the hematopoietic failure.

METHODS(1) Eleven patients with severe aplastic anemia (SAA) at diagnosis were observed by Marsh's method for the CFU-E, BFU-E and CFU-GM before and after depletion of CD(4)(+) T lymphocytes from bone marrow mononuclear cells (BMMNC); (2) Th1 (CD(4)(+) IFN-gamma(+)) and Th2 (CD(4)(+) IL-4(+)) cells in peripheral blood mononuclear cells (PBMNC) of 21 SAA patients and 17 normal controls were counted by FACS. (3) mRNA expression of IFN-gamma and IL-4 gene in unstimulated BMMNC from 16 SAA patients, 11 chronic aplastic anemia (CAA) patients, 26 other hematological diseases patients and 11 normal controls were measured by reverse transcriptase polymerase chain reaction (RT-PCR).

RESULT(1) CFU-E, CFU-GM and BFU-E increased significantly after depletion of CD(4)(+) T lymphocytes from BMMNC of SAA patients. (2) The percentage of IFN-gamma producing CD(4)(+) T cell (Th1) of SAA patients was significantly higher than that of controls, the percentages of IL-4 producing CD(4)(+) T cells (Th2) had no difference between SAA patients and normal controls. (3) IFN-gamma mRNA was detected in unstimulated BMMNC in 13 of 16 SAA patients, 6 of 11 CAA patients and one of 6 paroxysmal nocturnal hemoglobinuria (PNH) patients. The IFN-gamma mRNA was not detected in unstimulated BMMNC of 11 normal controls and other hematological diseases patients.

CONCLUSIONSDisbalance of CD(4)(+) T lymphocytes subsets and increases in quantity and IFN-gamma producing function of Th1 cells might be important for the development of bone marrow failure in AA and in distinguishing AA from other kinds of pancytopenic diseases.

Adolescent ; Adult ; Anemia, Aplastic ; blood ; etiology ; Colony-Forming Units Assay ; Erythroid Precursor Cells ; cytology ; Female ; Granulocytes ; cytology ; Hematopoietic Stem Cells ; cytology ; Humans ; Interferon-gamma ; genetics ; Interleukin-4 ; genetics ; Macrophages ; cytology ; Male ; Middle Aged ; RNA, Messenger ; genetics ; metabolism ; Th1 Cells ; cytology ; metabolism ; physiology ; Th2 Cells ; cytology ; metabolism