Feline mammary carcinoma‑derived extracellular vesicle promotes liver metastasis via sphingosine kinase‑1‑mediated premetastatic niche formation / 한국실험동물학회지

Background@#Feline mammary carcinoma (FMC) is one of the most prevalent malignancies of female cats. FMC is highly metastatic and thus leads to poor disease outcomes. Among all metastases, liver metastasis occurs in about 25% of FMC patients. However, the mechanism underlying hepatic metastasis of FMC remains largely uncharacterized. @*Results@#Herein, we demonstrate that FMC-derived extracellular vesicles (FMC-EVs) promotes the liver metastasis of FMC by activating hepatic stellate cells (HSCs) to prime a hepatic premetastatic niche (PMN). Moreover, we provide evidence that sphingosine kinase 1 (SK1) delivered by FMC-EV was pivotal for the activation of HSC and the formation of hepatic PMN. Depletion of SK1 impaired cargo sorting in FMC-EV and the EV-potentiated HSC activation, and abol‑ ished hepatic colonization of FMC cells. @*Conclusions@#Taken together, our findings uncover a previously uncharacterized mechanism underlying liver-metas‑ tasis of FMC and provide new insights into prognosis and treatment of this feline malignancy.

Systemic reactions to subcutaneous immunotherapy for bronchial asthma and/or allergic rhinitis in children and their risk factors / 中国当代儿科杂志

OBJECTIVE@#To investigate the incidence of systemic reactions (SR) to subcutaneous immunotherapy (SCIT) for bronchial asthma and/or allergic rhinitis in children and their risk factors.@*METHODS@#A retrospective analysis was performed on 198 children with bronchial and/or allergic rhinitis. According to the presence or absence of SR and local reactions (LR) during SCIT, the patients were divided into two groups: SR (with SR and LR, n=31) and control (without SR or LR, n=142). A multivariate logistic regression analysis was used to determine the risk factors associated with SR.@*RESULTS@#Among the 198 patients who received 8 157 injections of SCIT, 25 (12.6%) experienced SR (31 times, 0.38%), including grade I SR (18 times, 58%), grade II SR (10 times, 32%), grade III SR (3 times, 10%), and no grade IV SR. The multivariate logistic regression analysis showed that multiple sensitization with both food and inhaled allergens, specific IgE to dust mites (grade 6), total IgE (grade 6), and a history of LR were independent risk factors for SR (P<0.05).@*CONCLUSIONS@#SCIT is a safe treatment for bronchial asthma and/or allergic rhinitis in children, with a low incidence of SR. Children with multiple sensitization with both food and inhaled allergens, a hypersensitive state (specific IgE to dust mites, grade 6; total IgE, grade 6), and a history of LR have an increased risk of SR to SCIT.

miRNA -100 promotes proliferation of human leukemia cells HL -60 by targeting carboxy -terminal domain small phosphatase-like protein / 中华实用儿科临床杂志

Objective To investigate the effect of miRNA -1 00 on the proliferation of human leukemia cells HL -60,and to explore the mechanism of this action.Methods The bioinformatics software and database were applied to predict and analyze target genes of miRNA -1 00.The vector contained the target gene 3′UTR portion cloned into a luciferase reporter construct.A luciferase reporter assay was performed following co -transfection of small molecular miRNA -1 00 mimics and target gene wild -type or mutant plasmid into HEK -293T cells.HL -60 cells were trans-fected with miRNA -1 00 mimics or anti -miRNA -1 00.After transfection,Western blot was applied to validate the expression of carboxy -terminal domain small phosphatase -like protein (CTDSPL),and the viability of HL -60 was measured by using cell counting kit (CCK -8)assay at 24 h,48 h,72 h,96 h.Results Online software predicted that CTDSPL was likely to be the target gene of miRNA -1 00.Dual luciferase reporter gene assay system showed that miRNA -1 00 could significantly suppress the activity of reporter gene containing CTDSPL 3′-UTR which decreased by about 57.1 %(P =0.000 7).Western blot showed that the expression of CTDSPL was increased after being trans-fected with miRNA -1 00 antisense oligonucleotides and decreased after being transfected with miRNA -1 00 mimics.At the same time,the growth rate of cells treated with miRNA -1 00 mimics or CTDSPL miRNA -1 00 was increased com-pared with that in control by CCK -8 test (P <0.05 ).Conclusions CTDSPL is a downstream target gene of miRNA -1 00.miRNA -1 00 can promote leukemia cell proliferation by inhibiting the expression of CTDSPL.

The expression and functional study of miR-181a in pediatric acute lymphoblastic leukemia / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the expression of miR-181a in bone marrow (BM) samples of pediatric acute lymphoblastic leukemia (ALL) and explore the mechanism of miR-181a on ALL cell line CCRF-CEM and drug resistance cell line CEM-C1.</p><p><b>METHODS</b>BM samples were obtained from 18 patients where matched samples at initial diagnosis and first BM relapse or complete remission were available. BM samples and cord blood samples (normal controls) were used to confirm the differential expression of miRNA-181a by quantitative real-time polymerase chain reaction (qRT-PCR). The expressions of miR-181a in both CCRF-CEM and its mutidrug-resistant counterpart CEM-C1 cells were also detected. Then, CCK-8 assay was performed to quantify the effects of miR-181a on CEM-C1 and CCRF-CEM cells growth and viability.</p><p><b>RESULTS</b>Up-regulated miR-181a with higher fold changes in both initial diagnosis (4.84 ± 2.71, 7.58 ± 2.50) and relapsed samples (6.53 ± 2.20) compared to normal controls (1.41 ± 0.53) (P=0.017, 0.000, 0.001, respectively) were observed, whereas the miR-181a expression in the samples of CR (1.35 ± 0.35) compared to normal control showed no significant difference (P=0.863). The miR-181a expression level was higher in CEM-C1 cells (-4.39 ± 0.08) than of in CCRF-CEM cells (-2.32 ± 0.03) (P=0.000). CCK-8 assay revealed that suppression of miR-181a in CEM-C1 cells by transfecting the specific inhibitor of miR-181a led to significantly higher cellular proliferation inhibition rate than negative control cells (P<0.05), IC50 were 30.61 ng/ml and 2 255.00 ng/ml with RI as 73.67. While increased miR-181a in CCRF-CEM cells led to significantly lower CPIR than negative control cells (P<0.01), IC50 were 126.60 ng/ml and 1.34 ng/ml with RI as 94.26.</p><p><b>CONCLUSION</b>Upregulation of miR-181a might play an important role in the development of drug resistance in CEM-C1 cells, and knockdown of miR-181a could sensitize CEM-C1 cells to camptothecin; Meanwhile increased expression of miR-181a could promote CCRF-CEM drug resistance. These results suggested that suppression of miR-181a expression might provide a promising therapeutic in drug resistance of leukaemia.</p>

Expression of EB virus in peripheral blood mononuclear cells in children with systemic lupus erythematosus / 中华实用儿科临床杂志

Objective To discuss the role of EB virus (EBV)in the pathogenesis of systemic lupus erythematosus(SLE) in children through investigating the copies of EBV DNA and expression of EBV genes in peripheral blood mononuclear cells(PBMCs).Methods (1)PBMCs were isolated from 30 patients with SLE and 12 healthy normal controls respectively and DNA was extracted from PBMCs.(2) PBMCs were co-cultured with EBV for 12 days and RNA was extracted from PBMCs.(3)Real-time fluorescence quantitative PCR(Real-time PCR) was applied to detect the copies of EBV DNA in PBMCs.(4)Reverse transcription PCR was applied to detect expression of EBV genes.Results (1) Compared with the healthy control group [(40.1 ± 11.6) copies/μg],a significant increase of EBV DNA copies was observed in SLE group[(658.6 ± 183.6) copies/μg] (P ＜0.05).The EBV DNA copies in the active SLE group [(785.2 ± 179.2) copies/μg] were significantly higher than those in the non-active SLE group [(586.0 ± 193.1) copies/μg] (P ＜ 0.05).(2)There was no correlation between EBV DNA copies and systemic lupus erythematosus disease activity index (r =0.03,P ＞ 0.05).(3) After PBMCs got co-cultured with EBV,expression of latent EBV genes and lytic genes were both increased in the patients and healthy controls.The latent EBV genes including latent membrane protein 1 (LMP1),LMP2,EBV nuclear antigen 1 and the lytic genes including BCRF1,BLLF1 were all increased significantly in the patients compared with the healthy controls (all P ＜ 0.05).Conclusions There is a significant increase of EBV DNA copies and aberrant expression of EBV genes in SLE patients,which suggests that EBV may contribute to the pathogenesis of SLE.

Aberrant expression of Epstein-Barr virus genes in children with systemic lupus erythematosus / 中华皮肤科杂志

Objective To investigate the expression and significance of Epstein-Barr virus (EBV) genes in children with systemic lupus erythematosus (SLE).Methods Peripheral blood mononuclear cells (PBMCs) were isolated from 20 children with SLE and 12 healthy human controls.Enzyme-linked immunosorbent assay (ELISA) was conducted to detect anti-EBV viral capsid antigen (VCA) IgG/IgM antibodies.The culture supernatants of cells from patients with anti-EBV VCA IgG/IgM antibodies were collected,and PBMCs from the patients and controls were co-cultured with the supernatants respectively for 12 days.RNA was extracted from PBMCs before and after the coculture,and reverse transcription-PCR was performed to detect the expression of EBV genes,including LMP1,LMP2,EBNA1,BCRF1,BLLF1 and BILF1 genes.Results LMP1 gene was detected in fresh PBMCs from 10 out of 20 patients and 1 out of 12 controls (P ＜ 0.05).No significant differences were observed between the patients and controls in the detection rate of LMP2 gene (4/20 vs.1/12),EBNA1 gene (13/20 vs.3/12),BCRF1 gene (3/20 vs.1/12) or BLLF1 gene (5/20 vs.2/12) in fresh PBMCs.After co-culture with the supernatants of cells from patients with anti-EBV VCA IgG/IgM antibodies,the expressions of EBV genes in these PBMCs were increased to different degrees,and there was a significant difference in the expressions of EBV latent genes LMP1,LMP2 and EBNA-1 as well as EBV replicative genes BCRF1 and BLLF1 between the patient-derived and control-derived PBMCs (all P ＜ 0.05).Conclusions There is an aberrant expression of EBV genes in children with SLE,and EBV genes may contribute to the development of SLE.

Elucidating the role of ApxI in hemolysis and cellular damage by using a novel apxIA mutant of Actinobacillus pleuropneumoniae serotype 10

Exotoxins produced by Actinobacillus (A.) pleuropneumoniae (Apx) play major roles in the pathogenesis of pleuropneumonia in swine. This study investigated the role of ApxI in hemolysis and cellular damage using a novel apxIA mutant, ApxIA336, which was developed from the parental strain A. pleuropneumoniae serotype 10 that produces only ApxI in vitro. The genotype of ApxIA336 was confirmed by PCR, Southern blotting, and gene sequencing. Exotoxin preparation derived from ApxIA336 was analyzed for its bioactivity towards porcine erythrocytes and alveolar macrophages. Analysis results indicated that ApxIA336 contained a kanamycin-resistant cassette inserted immediately after 1005 bp of the apxIA gene. Phenotype analysis of ApxIA336 revealed no difference in the growth rate as compared to the parental strain. Meanwhile, ApxI production was abolished in the bacterial culture supernatant, i.e. exotoxin preparation. The inability of ApxIA336 to produce ApxI corresponded to the loss of hemolytic and cytotoxic bioactivity in exotoxin preparation, as demonstrated by hemolysis, lactate dehydrogenase release, mitochondrial activity, and apoptosis assays. Additionally, the virulence of ApxIA336 appeared to be attenuated by 15-fold in BALB/c mice. Collectively, ApxI, but not other components in the exotoxin preparation of A. pleuropneumoniae serotype 10, was responsible for the hemolytic and cytotoxic effects on porcine erythrocytes and alveolar macrophages.

Vibroacoustography for the assessment of total hip arthroplasty

OBJECTIVES: This paper proposes imaging with 3-dimensional vibroacoustography for postoperatively assessing the uncovered cup area after total hip arthroplasty as a quantitative criterion to evaluate implant fixation. METHODS: A phantom with a bone-like structure covered by a tissue-mimicking material was used to simulate a total hip arthroplasty case. Vibroacoustography images of the uncovered cup region were generated using a two-element confocal ultrasound transducer and a hydrophone inside a water tank. Topological correction based on the geometry of the implant was performed to generate a 3-dimensional representation of the vibroacoustography image and to accurately evaluate the surface. The 3-dimensional area obtained by the vibroacoustography approach was compared to the area evaluated by a 3-dimensional motion capture system. RESULTS: The vibroacoustography technique provided high-resolution, high-contrast, and speckle-free images with less sensitivity to the beam incidence. Using a 3-dimensional-topology correction of the image, we accurately estimated the uncovered area of the implant with a relative error of 8.1% in comparison with the motion capture system measurements. CONCLUSION: Measurement of the cup coverage after total hip arthroplasty has not been well established; however, the covered surface area of the acetabular component is one of the most important prognostic factors. The preliminary results of this study show that vibroacoustography is a 3-dimensional approach that can be used to postoperatively evaluate total hip arthroplasty. The favorable results also provide an impetus for exploring vibroacoustography in other bone or implant surface imaging applications. .

miR-125b promotes proliferation of human acute myeloid leukemia cells by targeting Bak1 / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate miR- 125b regulation mechanism by identifying miR-125b target genes and its function in acute myeloid leukemia (AML).</p><p><b>METHODS</b>The bioinformatics software and database were applied to predict and analyze target genes of miR-125b. The vector contained the target gene 3'-UTR portion cloned into a luciferase reporter construct. A luciferase reporter assay was performed following co-transfection of small molecular miR-125b mimics and target gene wild-type or mutant plasmid into HEK-293T cells. Further in leukemia cell lines NB4 and HL-60, the protein level of target gene was measured by Western blot after overexpression miR-125b. Finally, the viabilities of NB4 and HL-60 cells were measured by CCK-8 assay at 24 h, 48 h, 72 h, 96 h after electroporation.</p><p><b>RESULTS</b>Bcl-2-antagonist/killer 1 (Bak1), a pro-apoptotic gene, was a target gene of miR-125b by software predicts. Reporter vector containing the 3'-UTR Bak1 wild and mutation sites were co-transfected with small molecule analogues of miR-125b in HEK-293T cells. Dual luciferase reporter gene assay system showed that miR-125b significantly suppresses the reporter gene activity containing Bak1 3'-UTR by about 53.8% (P<0.05), but it didn't suppresses the reporter gene activity containing 3'-UTR Bak1 mutation. Western blot showed that miR-125b mimics significantly down-regulated the expression of Bak1 in human leukemia cell lines NB4 and HL-60. Meanwhile, the growth rate of cells treated with miR-125b obviously increased compared with that in control by CCK-8 test (P<0.05).</p><p><b>CONCLUSION</b>Our findings strongly indicated that BAK1 was a downstream target gene of miR-125b, and miR-125b promoted proliferation in human AML cells at least partially by targeting Bak1, so we speculated that miR-125b as an oncogene could be a potential therapeutic target for treating AML.</p>