Infants suffering from angiostrongylus eosinophilic meningitis (AEM) is rare, while AEM can cause severe consequences.The diagnostic value of high-throughput sequencing for AEM was studied by analyzing 2 AEM children (< 2 years old) in the Department of Neurology, Shenzhen Children′s Hospital in 2019.Case 1 mainly pre-sented intermittent fever, vomiting, mental fatigue and bregma bulge.Case 2 mainly manifested intermittent fever, cough, vomiting and convulsion.Due to hypereosinophils in patients′ peripheral blood and cerebrospinal fluid (CSF), and abundant DNA sequences from a cantonensis in CSF and positive antibody test, the patients were diagnosed with AEM.The patients were treated with albendazole to deworm, and small doses of methylprednisolone to reduce inflammation.The clinical characteristics of AEM infant are not typical, and high-throughput sequencing technology can assist the diagnosis of AEM.

Objective To compare clinical data of the death in different intensive care unit,in order to provide the medical strategies for patients in EICU.Methods The clinical data of lethal cases from January 1,2013 to December 31,2014 in EICU,SICU and MICU of the First Affiliated Hospital of Sun Yat-sen University were compared.EICU (252 cases),SICU (93 cases) and MICU (80 cases) were enrolled.The demographics of each patient,clinical condition such as critical score (APACHE Ⅱ score),length of stay,overall costs,and the patient families' different opinions to the treatment in each ICU were analyzed.The data was analyzed with SPSS 13.0 software,averaged value was presented as mean ± standard and the non-normal distributions were expressed as median (25％,75％).The one-way analysis of variance was followed by the Tukey post hoc test for pairwise comparisons and chi-square test was used for comparison of percentage between two groups.Results Two hundred and fifty-two cases in EICU had gender ration of 148/96 (male/female),92 cases in SICU 68/24,80 cases in MICU 56/24.Ages of the fatal were EICU 72 ± 17 years,SICU 56 ± 17 years,and MICU 63 ± 20 years,respectively.Age of the fatal in EICU was significantly older than that of the SICU (P ＜ 0.01) and the MICU (P ＜ 0.01).APACHE Ⅱscores were 33 ± 8 in EICU,34 ± 10 in SICU,29 ± 10 in MICU,respectively.The severity scores in EICU patients were higher than those in MICU patients and SICU patients (P =0.01 and 0.021).Lengths of stay were 2 days (1,46) in EICU,14 days (1,84) in SICU,12 days (1,77) in MICU,respectively.EICU hospitalization time was significantly shorter than that of SICU (P ＜ 0.01) and the MICU (P ＜ 0.01).Total costs of hospitalization were 9 777 yuan (400,164 126) yuan in EICU,100 628 yuan (13 639,964 783) yuan in SICU,119 463 yuan (5 650,590 903) yuan in MICU,and that in EICU was significantly less than the total cost of hospitalization in SICU (P ＜ 0.01) and in MICU (P ＜ 0.01).The opinion of patient families was proposed to give up treatment associated with 165 dead cases in EICU,18 death cases in SICU and 20 dead cases in MICU,and the rate of discontinuous treatment in EICU patients was significantly greater than that in SICU (P ＜ 0.01) and in MICU (P ＜ 0.01).There were no significant differences in invasive procedures,invasive hemodynamic monitoring,mechanical ventilation,blood purification and deep vein puncture among three groups.The 5 leading causes in EICU were severe sepsis,stroke,sudden cardiac arrest,acute myocardial infarction and advanced malignancy.Conclusions The death of patients were due to advanced age with severe disease,poor prognosis,and the request of patient family members to give up treatment.The 5 leading causes were severe sepsis,stroke,sudden cardiac arrest,acute myocardial infarction and advanced tumors suggesting the establishment of corresponding treatment scheme to be made and preparation of abundant medical resources to be ready.Timely communication with the patients' families and let them participate in end-stage treatment decisions was the best strategies to improve the successful rate of treating severe patients and use EICU resource effectively.

Objective To evaluate the application value of confocal laser scanning microscopy(CLSM)in the differentiation between seborrheic keratosis and Bowen′s disease. Methods CLSM was used to observe typical skin lesions in 88 patients clinically diagnosed with seborrheic keratosis and 18 patients clinically diagnosed with Bowen′s disease. Then, tissue specimens were resected from these lesions and subjected to histopathological examination. Results CLSM imaging of seborrheic keratosis lesions showed gyrus?like structures and keratin?filled inclusion cysts in the epidermis with trabecula?like extension of rete ridges in all the 88 cases, basal cells arranged in a cordike or radial pattern in 9 cases, and bright reflective structures in the basal layer and dermis in 6 cases. CLSM imaging of Bowen′s disease lesions revealed disorderly arrangement of large, irregularly shaped atypical cells in some areas in the middle and lower epidermis, and infiltration of scattered mononuclear cells in the superficial dermis. Conclusion CLSM images of seborrheic keratosis are different from those of Bowen′s disease, and CLSM may be helpful for their differential diagnosis.

Objective To investigate the impact of purinergic receptors P2Y12 gene inhibited by small interference RNA (siRNA) on the angiogenesis of hepatocellular carcinoma (HCC). Methods Human HCC cell line MHCC97H was infected using P2Y12-siRNA lentiviral expression vector and empty negative control vector to establish siRNA group and control group. The expression of P2Y12 protein in two groups was detected by Western blot assay. angiogenesis assay was used to deifne the angiogenesis potential of HCC cell. The microvessel density (MVD) of nude mice subcutaneous tumors was observed. The experimental data between two groups were compared using t test. Results The average relative expression of P2Y12 protein were 0.07±0.01 and 0.26±0.02 respectively in siRNA group and control group. The expression of P2Y12 protein in siRNA group decreased significantly compared with that in control group (t=-35.64, P<0.05). No continuous closed small tubular structure was observed in the human umbilical vein endothelial cell (HUVEC) stimulated by the supernatant of siRNA group, while large number of closed tubules was observed in the HUVEC stimulated by the supernatant of control group. A few microvessels of nude mice subcutaneous tumors were observed in siRNA group. The MVD was 5±1, which was signiifcantly lower than that in control group (23±6) (t=-17.01, P<0.05). Conclusion Purinergic receptors P2Y12 gene inhibited by siRNA can suppress the angiogenesis of HCC.

OBJECTIVETo monitor human cytomegalovirus (HCMV) drug resistance in recipients of hematopoietic stem cell transplantation by phenotypic and genotypic methods.

METHODSHCMV clinical isolates was isolated from the urine of hematopoietic stem cell transplantation recipients treated with GCV. Tissue cell infection median dose (TCID50) of the isolates was calculated using Reed-Muench method, and their drug susceptibility was determined by plaque reduction assay. We amplified the UL97 DNA fragment of the virus by nested PCR followed by automated DNA sequencing.

RESULTSHCMV clinical strain isolated from the urine samples of the recipients using a human fibroblast cell line showed a TCID50 value of 10(-4.618)/0.1 ml and a 50% inhibitory concentration (IC50) to GCV of 5.847 µmol/L, suggesting its sensitivity to GCV. Alignment with the AD169 DNA reference sequence identified 4 point mutations of the virus at 1509 (T-C), 1575 (C-T), 1794 (T-C), and 1815 (C-G), and only the last mutation resulted in one amino acid mutation to D605E. No gene mutation was found in relation to GCV resistance.

CONCLUSIONSPhenotypic and genotypic assays were established to examine antiviral drug resistance of HCMV in recipients of hematopoietic stem cell transplantation. We did not find any drug resistance of the clinical HCMV isolate.

Antiviral Agents ; pharmacology ; Cell Line ; Cytomegalovirus ; drug effects ; genetics ; isolation & purification ; Drug Resistance, Viral ; genetics ; Ganciclovir ; pharmacology ; Genes, Viral ; Genotype ; Hematopoietic Stem Cell Transplantation ; Humans ; Mutation ; Phosphotransferases (Alcohol Group Acceptor) ; genetics

Objective To analyze the clonality in Kaposi's sarcoma (KS) lesions by evaluating Xchromosome inactivation pattern in the human androgen receptor (HUMARA) gene.Methods Twenty-five paraffinembedded tissue specimens were collected from female patients with KS (n =15) or cutaneous hemangioma (n =10).DNA was extracted from these specimens,and digested with the methylation-sensitive restriction endonuclease Hpa Ⅱ.PCR was performed to amplify the HUMARA gene,and the amplicons were separated on a 10％ denaturing polyacrylamied gel and stained with ethidium bromide (EB).The loss of heterozygosity of the HUMARA gene was defined as the presence of two DNA fragments before and one fragment after the endonuclease digestion.The clonality in KS lesions was assessed based on the above results.Results Among the 15 patients with KS,13 (86.7％) were heterozygous for the HUMARA gene,of which,92.31％ (12/13) showed loss of heterozygosity of the HUMARA gene on X-chromosome,suggesting a monoclonal origin.Of the 10 patients with hemangioma,9 were heterozygous for the HUMARA gene,and only one lost heterozygosity of the HUMARA gene.The heterozygosity rate for HUMARA gene was significantly different between the patients with KS and hemangioma (P ＜ 0.01).No statistical difference was observed in the clonality status of KS between patients of different nationality,at different stages,or between patients with and without complicated human immunodeficiency virus (HIV) infection (all P ＞ 0.05).Conclusion KS is monoclonal in origin.

Objective To research the antiviral activity of artesunate (ART) in vitro fighting against both standard laboratory strains and ganciclovir(GCV)-resistance strains of human cytomegalovims(HCMV) and to explore whether fractionation dosage method can obviously enhance the antiviral effect of ART.Methods 1.Cytotoxicity assay to ART was performed by the use of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetry.The 0％ toxic concentration (TC0) were determined,and median cytotoxic concentration (TC50) was calculated with Probit regression method.2.Antiviral activity assays of ART against HCMV:human embryonic lung fibroblast cells (HELs) were infected with standard laboratory strains and GCV-resistance strains of HCMV,respectively,after which virus was removed and overlays of dulbecco's modified eagle medium(MEM) containing different antiviral drugs were added to the wells.All cells were cultured continuously at 37 ℃ in a 50 mL/L CO2 humidified atmosphere for 7-10 days and the cytopathic effect (CPE) was observed under a microscope.When the degree of CPE was clear (+ + +-+ + + +),the values of absorbency at 490 nm of all cell wells were measured by MTT colorimetry.The cell survival rate (CSR)and drug inhibitory rate (IR) for HCMV were calculated.By Probit regression method,the median inhibitory concentration (IC50) of 2 drugs was calculated respectively.3.To explore whether fractionation dosage method could obviously enhance the antiviral effect of ART against HCMV,the experiment was divided into 3 groups and compared with GCV group,respectively:Group 1:ART antiviral compounds were added to cell layers by one dosage.Group 2:Total drug dosage was divided into 3 parts,and each part was added to cell layers once a day for 3 days.Group 3:Total antiviral compounds were divided into 6 and delivery 2 times a day.The values of absorbency at 490 nm of all cell wells were measured by MTT colorimetry.The CSR and viral inhibitory rates were calculated.All data were statistically analyzed by One-Way ANOVA analyzing using SPSS 18.0 statistical software.P value of ＜0.05 was considered to indicate statistical significance.Results 1.Cytotoxicity assay showed that cytotoxicity was not found in the relevant range of ART concentrations under 62.5 μmol/L.TC0 and TC50 value of ART were 62.5 μmol/L and 171.7 μmol/L.2.In concentration of 5 μmol/L,15 μmol/L and 30 μmol/L,ART and GCV could obviously inhibit growth of HCMV AD169 strains.There was no significant difference between them.The value of GCV IC50 was 3.49μmol/L,and the value of ART IC50 was 2.17 μmol/L.Treatment index (TI) of ART was 28.8,and GCV was 716.3.ART could still obviously inhibit growth of HCMV resistant strains,but GCV couldn't.Differences between them were statistically significant.The value of GCV IC50 to HCMV resistant strains was 44.4 μmol/L,and the value of ART IC50 was 2.5 μmol/L.3.Fractionation dosage method (2 times a day) of ART could improve the inhibition rate of virus significantly compared to that used once a day and single dose method.Difference was statistically significant(P ＜ 0.01).GCV delivered as the same method had little different changes in virus suppression ratio(P ＞ 0.05).Conclusions 1.Cytotoxicity was not found in the relevant range of ART concentrations under 62.5 μmol/L.2.ART could obviously inhibit growth of HCMV resistant strains and standard laboratory strains.3.Fractionation dosage method (2 times a day) of ART could improve the inhibition rate of virus significantly compared to that used once a day and single dose method.4.Because the action mode of ART is different from other anti-HCMV drugs,and ART has a high biological activity and fewer side effects,it is expected to become a kind of new antiviral drugs for HCMV infections.

Objective To monitor human cytomegalovirus (HCMV) drug resistance in recipients of hematopoietic stem cell transplantation by phenotypic and genotypic methods. Methods HCMV clinical isolates was isolated from the urine of hematopoietic stem cell transplantation recipients treated with GCV. Tissue cell infection median dose (TCID50) of the isolates was calculated using Reed-Muench method, and their drug susceptibility was determined by plaque reduction assay. We amplified the UL97 DNA fragment of the virus by nested PCR followed by automated DNA sequencing. Results HCMV clinical strain isolated from the urine samples of the recipients using a human fibroblast cell line showed a TCID50 value of 10-4.618/0.1ml and a 50%inhibitory concentration (IC50) to GCV of 5.847 μmol/L, suggesting its sensitivity to GCV. Alignment with the AD169 DNA reference sequence identified 4 point mutations of the virus at 1509 (T-C), 1575 (C-T), 1794 (T-C), and 1815 (C-G), and only the last mutation resulted in one amino acid mutation to D605E. No gene mutation was found in relation to GCV resistance. Conclusions Phenotypic and genotypic assays were established to examine antiviral drug resistance of HCMV in recipients of hematopoietic stem cell transplantation. We did not find any drug resistance of the clinical HCMV isolate.

Objective To investigate the impact of micro ribonucleic acid -214 (miR-214) on the proliferation of hepatocellular carcinoma (HCC) and its mechanism. Methods MHCC97L cells were respectively transfected using miR-214 mimics and negative control mimics to establish M214 group, negative control (NC214) group. And untransfected control (Ctrl) group was established. The contents of miR-214 of MHCC97L cells in three groups were detected by fluorescence quantitative polymerase chain reaction (PCR). Cell counting kit (CCK)-8 assay was used to define the cell proliferation inhibition rate. Plate cloning formation assay was used to define the cell clonality. Cell apoptosis rate was detected by flow cytometery. The expressions of protein c-myc, Bax and B-cell lymphoma (Bcl)-2 were detected by Western blot assay. The comparison of three groups was conducted using one way analysis of variance and pairwise comparison using LSD-t test. Results The miR-214 content of MHCC97L cells in M214 group was (2 536±7) times of that in Ctrl group, where significant difference was observed (LSD-t=58.75, P<0.05). The cell proliferation inhibition rates were (15.33±0.62) %, (24.07±0.75) %, (41.02±0.91) % at the 24, 48, 72 h in M214 group, and significant difference was observed compared with that in Ctrl group (LSD-t =14.64, 19.87, 31.86; P<0.05). The clone formation quantity was (5.3±0.7) /well in M214 group, which was significantly lower compared with that in Ctrl group [(37.1±1.0)/well] (LSD-t =-12.51, P<0.05). The cell apoptosis rate was (42.1±3.2)% in M214 group, which was significantly higher compared with that in Ctrl group [(7.0±0.7) % ] (LSD-t=35.66, P<0.05). In M214 group, the expressions of protein c-myc, Bcl-2 decreased and Bax increased. Conclusion The miR-214 can inhibit the proliferation of human HCC cells through down-regulating the protein c-myc expression and Bcl-2/Bax ratio.

Objective To monitor human cytomegalovirus (HCMV) drug resistance in recipients of hematopoietic stem cell transplantation by phenotypic and genotypic methods. Methods HCMV clinical isolates was isolated from the urine of hematopoietic stem cell transplantation recipients treated with GCV. Tissue cell infection median dose (TCID50) of the isolates was calculated using Reed-Muench method, and their drug susceptibility was determined by plaque reduction assay. We amplified the UL97 DNA fragment of the virus by nested PCR followed by automated DNA sequencing. Results HCMV clinical strain isolated from the urine samples of the recipients using a human fibroblast cell line showed a TCID50 value of 10-4.618/0.1ml and a 50%inhibitory concentration (IC50) to GCV of 5.847 μmol/L, suggesting its sensitivity to GCV. Alignment with the AD169 DNA reference sequence identified 4 point mutations of the virus at 1509 (T-C), 1575 (C-T), 1794 (T-C), and 1815 (C-G), and only the last mutation resulted in one amino acid mutation to D605E. No gene mutation was found in relation to GCV resistance. Conclusions Phenotypic and genotypic assays were established to examine antiviral drug resistance of HCMV in recipients of hematopoietic stem cell transplantation. We did not find any drug resistance of the clinical HCMV isolate.