OBJECTIVETo explore the role of platelet-activating factor receptor (PAFR) in adhesion and invasion of phospho- rylcholine (PC)-positive Aggregatibacter actinomycetemcomitans in cultured human umbilical vein endothelial cells (HUVEC).

METHDOSCultured HUVECs were pretreated with the PAFR antagonist CV3988 or anti-human PAFR monoclonal antibody for 30 min before infection with PC-positive or -negative A. actinomycetemcomitans strains. The bacterial adhesion and invasion and cytotoxicity in the cells were examined using MTT assay.

RESULTSPretreatment with PAFR antagonists at 100, 200 and 500 nmol/L significantly reduced the adhesion rate (36.29∓3.52)%, (19.04∓3.35)% and (7.69∓3.19%), respectively] and invasion rate [(12.12∓1.58)%, (7.08∓0.29)% and (2.60∓2.26)%, respectively] of PC-positive A.actinomycetemcomitans in HUVECs. Similarly, pretreatment with anti-PAFR antibody also significantly reduced A.actinomycetemcomitans adhesion and invasion in HUVECs [(50.05∓5.28)% and (39.09∓6.50)%, respectively]. Pretreatment with PAFR antagonist (200 and 500 nmol/L) and anti-PAFR antibody (25 µg/mL) significantly increased the viability of HUVECs incubated with PC-positive A.actinomycetemcomitans from (25.39∓9.33)% to (91.12∓3.14)%, (94.12∓2.15)% and (65.5∓1.87)%, respectively, but such pretreatments did not increase the viability of cells incubated with PC-negative A.actinomycetemcomitans.

CONCLUSIONSPAFR plays an important role in the adhesion, invasion, and cytotoxicity of PC-positive A.actinomycetemcomitans in cultured HUVECs.

Aggregatibacter actinomycetemcomitans ; pathogenicity ; Bacterial Adhesion ; Cells, Cultured ; Human Umbilical Vein Endothelial Cells ; microbiology ; Humans ; Platelet Membrane Glycoproteins ; metabolism ; Receptors, G-Protein-Coupled ; metabolism

The impact factors were explored to determine the horizontal positional relationship between the umbilicus and the 2nd lumbar spinal process in adults and to verify the accuracy of the localization of Shenshu (BL 23) via the umbilicus. The position of the umbilicus and the 2nd lumbar spinal process was measured in 100 participants and the data were analyzed through SPSS 20.0 software. It was found that the umbilicus and the 2nd lumbar process were not positioned horizontally. The positional relationship of these two sites was not apparently correlated with gender, age, body weight, body height, BMI, waistline and discomfort of lumbar region. The umbilicus was commonly and posteriorly projected on the site between the 4th and 5th lumbar vertebra. It is explained that the localization of Shenshu (BL23) via the umbilicus is not accurate.
Acupuncture Points ; Adolescent ; Adult ; Female ; Humans ; Lumbosacral Region ; anatomy & histology ; Male ; Meridians ; Middle Aged ; Umbilicus ; anatomy & histology ; Young Adult

Acoustic Stimulation ; Evoked Potentials, Auditory, Brain Stem ; Humans ; Speech

OBJECTIVETo investigate the association between children's and their parents' lifestyles, household environmental exposures and risk of childhood acute leukemia (AL).

METHODSA 1:2 matched case-control study of childhood AL was conducted in Shanghai between April 2011 and January 2014. The study enrolled 66 cases aged < 15, diagnosed with AL and 132 controls matched by age, gender and residence. All of the controls had no hematological diseases or previous history of malignancy. Children who had been adopted and had congenital genetic syndromes such as Down's syndrome or a positive HIV test result were not eligible as either cases or controls. Information was obtained from standardized face-to-face interviews of their parents/guardians with detailed questions on demographic characteristics, lifestyle, and household environment. Conditional logistic regression models were used to analyze effecting factors of childhood AL, odds ratios (OR) and their 95% confidence intervals (CI) were calculated.

RESULTSAmong 198 cases, 66 cases were aged (5.0 ± 3.7) years old, and 132 controls were aged (6.0 ± 3.8) years old (t = 0.48, P = 0.523). The paternal drink frequencies of cases and controls were 57.6% (38/66), and 31.1% (41/132), respectively (χ² = 4.91, P = 0.027). And the frequencies of household insecticides usage in the last year in the two groups were 78.8% (52/66), and 65.2% (86/132) (χ² = 3.87, P = 0.049). Chemical exposure during childhood (OR = 4.76, 95% CI: 1.34-16.89), maternal exposure to chemicals (OR = 4.51, 95% CI: 1.65-12.33), household insecticides use during 0-3 years of child (OR = 2.90, 95% CI: 1.31-6.39), and renovating after their children's birth (OR = 3.12, 95% CI: 1.26-7.74) were associated with an increased risk of childhood AL and these differences between the cases and the controls have statistical significance. Besides, we found that frequent contaction with other children during 0-3 years old (OR = 0.32, 95% CI: 0.15-0.69) and ventilation during sleeping in summer (OR = 0.43, 95% CI: 0.18-0.98) were associated with a decreased risk of childhood AL.

CONCLUSIONOur results support the association between children's and their parents' lifestyles, household environmental exposures and childhood AL.

Acute Disease ; Case-Control Studies ; Child ; Child, Preschool ; China ; Environmental Exposure ; Female ; Humans ; Insecticides ; Leukemia ; epidemiology ; Life Style ; Logistic Models ; Maternal Exposure ; Neoplasms ; Odds Ratio ; Parents ; Risk Factors

OBJECTIVETo investigate the clinicopathologic features and 19q13.42 gene changes in embryonal tumors with multilayered rosettes (ETMR).

METHODSImmunohistochemistry and fluorescence in situ hybridization (FISH) were performed in three ETMRs.

RESULTSThe average age of the patients were 34 months. Imaging revealed huge masses with inhomogeneous enhancement and two cases showed cystic lesions. Follow-up data showed 14 and 38 months survival in two children, the third had a recurrence 4 months after operation. Morphologically, the tumor was mainly composed of dense small primitive neuroepithelial cells in patchy or multilayer rosettes within a background of advanced neuronal differentiation, containing neurocytes, ganglion cells, and neuropil-like background. Immunohistochemical staining showed the neuronal marker, synaptophysin, was positive in differentiated areas. Nestin as a neural stem cell marker was immunoreactive in the primitive neuroepithelial cells including multilayered rosettes. Neurons with positive expression of NeuN were observed occasionally. Ki-67 index was up to 40%-80% in the undifferentiated cells and rosettes, but was only 1%-3% in the differentiated areas. CD99 was positive in perivascular papillary pattern areas in one case. 19q13.42 amplification was detected in more than 30% of tumor cells in all cases.

CONCLUSIONSETMR is a unique entity with distinctive clinical and pathological features. Chromosome 19q13.42 abnormality is valuable for confirming the diagnosis and for further treatment research.

Antigens, Nuclear ; genetics ; Child, Preschool ; Chromosomes, Human, Pair 19 ; genetics ; Genetic Testing ; Humans ; Immunohistochemistry ; In Situ Hybridization, Fluorescence ; Neoplasm Recurrence, Local ; Neoplasms, Germ Cell and Embryonal ; genetics ; pathology ; Nerve Tissue Proteins ; genetics ; Neuropil ; pathology ; Synaptophysin ; genetics

OBJECTIVETo investigate the effect of ERK1/2 inhibitor AZD8330 on human Burkitt's lymphoma cell line Raji cells and its mechanism.

METHODSRaji cells were treated with different concentrations of AZD8330. CCK-8 was used to detect the cell viability. The apoptosis rate of Raji cells was detected by flow cytometry using Annexin V/PI-staining. Real-time PCR was used to assess the expression of Bcl-2, Bcl-xl, caspase-3 and VEGF genes. The protein expression level of Bcl-2, Bcl-xl, caspase-3 and p-ERK1/2 was tested with Western blot.

RESULTSThe cell survival rate decreased to(62.09±0.86)%,(50.06±1.33)% and (39.13±2.34)% respectively after cells were treated with AZD8330 at 1.00 μmol/L in vitro for 24 h, 48 h and 72 h, and statistically significant differences were observed in groups with different time of treatment(P<0.05). Apoptosis of cells treated with AZD8330 at 0.10, 1.00, 10.00 μmol/L in vitro for 24 h, 48 h and 72 h was analyzed, and the statistically significant differences were observed in groups of different time and concentration treatment (P<0.05). AZD8330 induced Raji cell apoptosis and upregulated expression of Bcl-2, Bcl-xl, VEFG and decreased the expression of caspase-3 in a dose and time dependent manner, and statistically significant differences were observed in groups of different time and concentration treatment (P<0.05). At the same time, the Bcl-2, Bcl-xl and p-ERK1/2 proteins expression is suppressed obviously, but the expression of caspase-3 protein increased.

CONCLUSIONAZD8330 induces cell apoptosis by down-regulating the activation of ERK1/2 signal transduction pathway in Burkitt's lymphoma cell line Raji cells in a dose and time dependent manner.

Apoptosis ; Burkitt Lymphoma ; Caspase 3 ; Cell Line, Tumor ; Dihydropyridines ; Flow Cytometry ; Humans ; MAP Kinase Signaling System ; Protein Kinase Inhibitors ; Real-Time Polymerase Chain Reaction

BACKGROUNDNon-alcoholic fatty liver disease (NAFLD) is a complex disorder and has been closely linked to obesity. The fat mass and obesity-associated (FTO) gene is a newly discovered gene related to obesity, which enhances oxidative stress and lipogenesis in NAFLD. The forkhead transcription factor O1 (FoxO1) is another important gene involved in NAFLD, which causes lipid disorders when insulin resistance appears in the liver. However, the interactions between FTO and FoxO1 during the pathogenesis of NAFLD have not been fully elucidated. This study was designed to identify the relationship between these two factors that are involved in the development of NAFLD.

METHODSThis study includes two parts referred to as animal and cell experiments. Twelve female SPF C57BL/6 mice were fed a high-fat diet to serve as an NAFLD animal model. Aspartate aminotransferase (AST), alanine aminotransferase (ALT), total triglyceride (TG), total cholesterol (TC), alkaline phosphatase (ALP), high-density lipoprotein (HDL), and low-density lipoprotein (LDL) were measured. Immunohistochemical analysis was used to detect the expression and histological localization of FTO, FoxO1, and adenosine monophosphate (AMP)-activated protein kinase (AMPK). The L02 cells were exposed to high fat for 24, 48, or 72 hours. Oil red O staining was used to detect intracellular lipid droplets. Reverse transcription-polymerase chain reaction was used for analyzing the levels of FTO and FoxO1 mRNA.

RESULTSAt the end of 10 weeks, ALP, ALT, AST, and LDL were significantly increased (P < 0.01), while TC and TG were also significantly higher (P < 0.05). In addition, HDL was significantly decreased (P < 0.05). The FTO and FoxO1 proteins were weakly expressed in the control group, but both FTO and FoxO1 were expressed significantly higher (P < 0.01) in the experimental group, and the expression of the two factors was significantly correlated. AMPK in the high-fat group showed a low level of correlation with FTO, but not with FoxO1. Oil Red O staining results showed that the cells cultured in 50% fetal bovine serum for 24, 48, or 72 hours exhibited steatosis. FTO and FoxO1 mRNA were increased in the high-fat group compared with the normal group (P < 0.01). The expression levels of FTO and FoxO1 mRNA were the highest at 48 hours (P < 0.05).

CONCLUSIONSA high-fat diet leads to higher expression of FTO, phosphorylation of FoxO1, and decreased phosphorylation of AMPK. These results suggest that the interactions between FTO and FoxO1 are closely related to the pathogenesis of NAFLD.

Alanine Transaminase ; genetics ; metabolism ; Animals ; Aspartate Aminotransferases ; genetics ; metabolism ; Cholesterol ; metabolism ; Diet, High-Fat ; adverse effects ; Disease Models, Animal ; Female ; Forkhead Transcription Factors ; genetics ; metabolism ; Lipoproteins, HDL ; metabolism ; Lipoproteins, LDL ; metabolism ; Liver ; metabolism ; Mice ; Mice, Inbred C57BL ; Non-alcoholic Fatty Liver Disease ; metabolism ; pathology ; Obesity ; metabolism ; pathology ; Triglycerides ; metabolism

BACKGROUNDErythropoietin (EPO) and granulocyte colony-stimulating factor (G-CSF) are both potential novel therapeutics for use after myocardial infarction (MI). However, their underlying mechanisms remain unclear and the efficacy of monotherapy with EPO or G-CSF is also controversial. Therefore, we investigated the effects of combined treatment with EPO and G-CSF on neovascularization and cardiac function in post-infarction rats and explored the potential mechanisms.

METHODSFour groups of rats were used: control (saline injection after MI, i.h.), EPO (a single dose of 5 000 IU/kg after MI, i.h.), G-CSF (a dose of 50 µg× kg(-1)× d(-1) for 5 days after MI, i.h.), and both EPO and G-CSF (EPO+G-CSF, using the same regiment as above). Cardiac function was assessed by echocardiography before and 1 day, 7 days, 14 days and 21 days after MI. CD34(+)/Flk-1(+) cells in the peripheral blood were evaluated by flow cytometry before and 3 days, 5 days and 7 days after MI. The infarct area and angiogenesis in the peri-infarct area were analyzed. The mRNA and protein expression of vascular endothelial growth factor (VEGF) and stromal-derived factor-1a (SDF-1α) in the peri-infarct area were detected by real-time quantitative RT-PCR and Western blotting.

RESULTSCompared with the control and monotherapy groups, the EPO+G-CSF group had significantly increased CD34(+)/Flk-1(+) endothelial progenitor cells (EPCs) in the peripheral blood (P < 0.05), up-regulated VEGF and SDF-1α levels in the peri-infarct region (P < 0.05), enhanced capillary density (P < 0.05), reduced infarct size (P < 0.05) and improved cardiac structure and function (P < 0.05). G-CSF alone did not dramatically increase EPCs in the peripheral blood, enhance capillary density in the peri-infarct area or reduce infarct size compared with the control group.

CONCLUSIONSCombined treatment with EPO and G-CSF increased EPCs mobilization, up-regulated VEGF and SDF-1α levels in the post-infarction microenvironment, subsequently enhanced neovascularization in the peri-infarct region and reduced infarct size. All factors contributed to its beneficial effects on cardiac function in post-infarction rats.

Animals ; Blotting, Western ; Chemokine CXCL12 ; metabolism ; Echocardiography ; Erythropoietin ; therapeutic use ; Flow Cytometry ; Granulocyte Colony-Stimulating Factor ; therapeutic use ; Male ; Myocardial Infarction ; drug therapy ; metabolism ; Neovascularization, Physiologic ; drug effects ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo investigate MUC2 expression in rat colons induced by probiotics and its effects on the inhibition of E.coli K1 (E44) penetration of the intestinal barrier by probiotics.

METHODSSD rats were subjected to intragastric administration of probiotics, E44, or probiotics +E44 on a daily basis for 7 days, and MUC2 expression in the colons was determined by RT-PCR. MUC2-targeted shRNA (shRNA MUC2) and scrambled shRNA plasmids (shRNA NC) were respectively transfected into Lovo cells, and the efficiency of MUC2 knockdown was determined using qRT-PCR. Competitive exclusion assay was used to evaluate the effects of the probiotics against E44 adhesion and invasion.

RESULTSIntestinal MUC2 mRNA expression was up-regulated in the rats after intragastric administration of probiotics, while E44 administration caused significantly lowered MUC2 expression. MUC2 expression was down-regulated (by 66.7%) by transfection with shRNA MUC2 in Lovo cells as compared with the negative control and mock control cells. The inhibition of E44 adherence and invasion by probiotics was significantly attenuated in transfected Lovo cell culture (in which the relative adhesion and invasion rates of E44 were 56.64% and 66.64%, respectively) as compared with those in the control group.

CONCLUSIONThe up-regulation of MUC2 in rat colons can be one of the mechanisms of the probiotics in antagonizing the translocation of the pathogenic bacteria. Silencing MUC2 expression causes attenuated inhibitory effect of the probiotics on E. coli K1 penetration across human intestinal epithelial cells.

Animals ; Animals, Newborn ; Cell Line, Tumor ; Colon ; drug effects ; metabolism ; microbiology ; Escherichia coli ; pathogenicity ; Escherichia coli Infections ; genetics ; Female ; Gene Expression Regulation, Neoplastic ; Gene Silencing ; Humans ; Mucin-2 ; genetics ; Probiotics ; pharmacology ; RNA, Messenger ; genetics ; RNA, Small Interfering ; Rats ; Rats, Sprague-Dawley ; Transfection

OBJECTIVETo construct dengue virus-specific full-length fully human antibody libraries using mammalian cell surface display technique.

METHODSTotal RNA was extracted from peripheral blood mononuclear cells (PBMCs) from convalescent patients with dengue fever. The reservoirs of the light chain and heavy chain variable regions (LCκ and VH) of the antibody genes were amplified by RT-PCR and inserted into the vector pDGB-HC-TM separately to construct the light chain and heavy chain libraries. The library DNAs were transfected into CHO cells and the expression of full-length fully human antibodies on the surface of CHO cells was analyzed by flow cytometry.

RESULTSUsing 1.2 µg of the total RNA isolated from the PBMCs as the template, the LCκ and VH were amplified and the full-length fully human antibody mammalian display libraries were constructed. The kappa light chain gene library had a size of 1.45×10(4) and the heavy chain gene library had a size of 1.8×10(5). Sequence analysis showed that 8 out of the 10 light chain clones and 7 out of the 10 heavy chain clones randomly picked up from the constructed libraries contained correct open reading frames. FACS analysis demonstrated that all the 15 clones with correct open reading frames expressed full-length antibodies, which could be detected on CHO cell surfaces. After co-transfection of the heavy chain and light chain gene libraries into CHO cells, the expression of full-length antibodies on CHO cell surfaces could be detected by FACS analysis with an expressible diversity of the antibody library reaching 1.46×10(9) [(1.45×10(4)×80%)×(1.8×10(5)×70%)].

CONCLUSIONUsing 1.2 µg of total RNA as template, the LCκ and VH full-length fully human antibody libraries against dengue virus have been successfully constructed with an expressible diversity of 10(9).

Animals ; Antibodies, Viral ; CHO Cells ; Cell Surface Display Techniques ; Cricetinae ; Cricetulus ; Dengue Virus ; immunology ; Gene Library ; Genetic Vectors ; Humans ; Immunoglobulin Heavy Chains ; immunology ; Transfection