Objective To investigate the diagnostic and prognostic value of serum soluble CD163 (sCD163 )and the positive rate of membrane -bound CD163 (mCD163 )in peripheral blood mononuclear cells (PBMC)in children with infection -associated hemophagocytic syndrome (IAHS).Methods Between July 2012 and June 2016,26 pediatric patients with IAHS (IAHS group)and 28 pediatric patients with sepsis(sepsis group)admitted to Children′s Hospital Affiliated to Shanghai Jiaotong University were selected,and 20 healthy children were taken as healthy control group. Sandwich enzyme linked immunosorbent assay was used to detect serum sCD163 .The population of circulating mCD163 positive monocytes was determined by using flow cytometry.Receiver operating characteristic (ROC)curves were used to evaluate the diagnostic and prognostic values of sCD163 and mCD163 in children with IAHS compared with the diagnos-tic and prognostic values of plasma ferritin,and so on.Results The serum levels of sCD163 in patients of IAHS group, sepsis group and healthy control group were (1264 ±538)mg/L,(862 ±332)mg/L,(610 ±316)mg/L,respective-ly.And the population of mCD163 -positive PBMC in patients of IAHS group,sepsis group and healthy control group was (88.3 ±9.7)%,(68.5 ±18.3)%,(28.9 ±5.2)%,respectively.Both serum sCD163 and the population of mCD163 -positive PBMC were significantly higher in IAHS group compared with those of sepsis group (t =2.031 ,P =0.048;t =3.191 ,P =0.002,respectively).The serum sCD163 and population of mCD163 -positive PBMC in sepsis group were higher than controls (t =3.848,P =0.002;t =4.049,P =0.000,respectively).Moreover,the areas under the ROC curve (AUC)for the mCD163 ,sCD163 ,were 0.853(P =0.013),0.762(P =0.004),0.755(P =0.049),respec-tively.mCD163 at a cutoff of 83.7% had a high diagnosis sensitivity (81 .8%)and specificity (72.4%).The optimal cutoff values of sCD163 and ferritin for predicting IAHS was 888 mg/L (sensitivity 66.7% and specificity 63.3%)and 2880 μg/L (sensitivity 80.0% and specificity 54.5%).In addition,the serum level of sCD163 and the population of mCD163 -positive PBMCs were significantly increased in acute phase and decreased in recovery phase[(1553 ±542) mg/L vs.(866 ±92)mg/L,(91 .0 ±6.4)% vs.(79.0 ±4.6)%,t =2.450,χ2 =3.419,P =0.036,0.007]in IAHS group.Furthermore,subgroup analysis indicated that the serum level of sCD163 and the population of mCD163 -positive PBMCs were significantly higher in dead patients than those in survived patients [(1748.91 ±518.17)mg/L vs. (909.69 ±171 .35)mg/L,t =3.070,P =0.011 ;(93.50 ±8.42)% vs.(77.30 ±3.28)%,χ2 =3.005,P =0.024, respectively].Conclusion Serum sCD163 and the population of mCD163 -positive PMSCs are specific and validity bio-markers for early diagnosis of IAHS,which also are associated with treatment response assessment and prognostic analy-sis in IAHS.

OBJECTIVETo investigate the prevalence and main influences on sleep disorder among Chinese children aged 0 to 23 months, as to providing scientific interventions for infant sleep disorder.

METHODSAll 7601 children under two years old were selected by stratifying samples from twelve cities in China. The objects' parents were surveyed with questionnaire. All data were analyzed with SPSS statistical software.

RESULTSThe total incidence of sleep disorders at 0 to 23 months was 21.94%. The main problems were difficulty falling asleep, nighttime waking and snoring. Feeding manner, sleep environment, sleep-associated habits and medical conditions were all influences on infant's sleep disorder.

CONCLUSIONSEnhancing sleep health education to change parents' nurturing modes should be an important role in preventing infant sleep disorders.

China ; epidemiology ; Cross-Sectional Studies ; Female ; Humans ; Infant ; Infant, Newborn ; Male ; Prevalence ; Risk Factors ; Sleep Wake Disorders ; epidemiology ; Surveys and Questionnaires ; Urban Population ; statistics & numerical data

OBJECTIVETo develop an effective method for screening recombinant hairpin RNA expression plasmids using single restriction endonuclease analysis.

METHODSThe double-strand DNA fragment containing a ClaI site (the flanking sequences of which were not complementary) was annealed and ligated into small hairpin RNA (shRNA) expression vector pSilencer-4.1 that did not contain ClaI site to construct the circular pSilencer-4.1-ClaI vector. With BamHI and HindIII, the pSilencer-4.1-ClaIwas digested and ligated with the DNA template of green fluorescence protein (GFP) shRNA that did not include a ClaI site. The plasmid DNA of the positive clones was extracted and digested with ClaI, and the inserted DNA sequence of the non-linearized plasmid was identified by sequence analysis.

RESULT AND CONCLUSIONDNA sequencing showed that pSilencer-4.1-ClaI was correctly constructed and the plasmids resistant to ClaI digestion were all recombinant vectors encoding GFP shRNA. The constructed pSilencer-4.1-ClaI can be used as a universal vector to construct the shRNA expression plasmid, and the incorporated ClaI sites may allow efficient screening of recombinant shRNA expression vectors.

Base Sequence ; Gene Expression ; Genetic Engineering ; methods ; Genetic Vectors ; genetics ; Green Fluorescent Proteins ; genetics ; Inverted Repeat Sequences ; Molecular Sequence Data ; Plasmids ; genetics ; RNA, Small Interfering ; genetics ; Restriction Mapping ; methods ; Sequence Analysis, DNA ; Time Factors

OBJECTIVETo investigate the effect of bone marrow stem cell transplantation (BMT) on the diaphragm muscles of mdx mice, a mouse model of Duchenne muscular dystrophy (DMD).

METHODSThe bone marrow-derived stem cells form male SD rats was transplanted through the tail vein into 18 female 8-week-old mdx mice, which were sacrificed at 4, 8 and 12 weeks after BMT (6 at each time point), respectively. The diaphragm muscles of the mice were subjected to HE staining, immunofluorescence detection of dystrophin, reverse transcription (RT)-PCR analysis of dystrophin mRNA transcripts and PCR analysis of Sry (sex-determining region on the Y chromosome) gene, with age-matched female C57 mice and untreated mdx mice as the controls.

RESULTSThe proportion of centrally nucleated fibers (CNF) in the diaphragm muscle of the recipient mdx mice was (15.58+/-0.91) %, (12.50+/-1.87) % and (10.17+/-1.17) % at 4, 8 and 12 weeks after BMT, respectively, significantly smaller than that of untreated mdx mice [(19.5+/-1.87) %], and the fibers after BMT showed less inflammatory infiltration. Compared with the untreated mice, the recipient mdx mice showed green fluorescence on significantly more diaphragm muscle cell membranes [with the proportion of dystrophin-positive fibers of (1.00+/-0.32) %, (6.00+/-1.05) % and (11.92+/-1.11) % at 4, 8, and 12 weeks after BMT]. RT-PCR of dystrophin mRNA also demonstrated significantly higher relative levels of dystrophin in the recipient mdx mice (0.19+/-0.05, 0.26+/-0.06 and 0.36+/-0.04 at 4, 8 and 12 weeks after BMT) than in untreated mdx mice, and Sry gene was present in the recipient mice.

CONCLUSIONBMT can partially restore dystrophin expression and ameliorate the pathology in the diaphragm muscles of mdx mice, and has great potential to produce general therapeutic effect in patients with DMD.

Animals ; Bone Marrow Transplantation ; methods ; Diaphragm ; metabolism ; pathology ; Dystrophin ; biosynthesis ; genetics ; Female ; Male ; Mice ; Mice, Inbred C57BL ; Mice, Inbred mdx ; Muscular Dystrophy, Duchenne ; metabolism ; pathology ; surgery ; Rats ; Rats, Sprague-Dawley ; Transplantation, Heterologous

The use of stem cells will lead to novel treatments for a wide range of diseases due to their properties of self-renewing, pluripotent, and undifferentiated state, and the stem cells are usually genetically modified for cell and gene therapy. If the baculovirus, as a new gene vector, can be effectively transduced into various mammalian bone marrow-derived mesenchymal stem cells (BMSCs) in vitro, it will be a better gene vector to genetically modify the stem cells. The aim of the present study is to investigate the transduction efficiency of recombinant baculovirus (BacV-CMV-EGFP), which expressed a reporter gene encoding enhanced green fluorescent protein (EGFP) under a cytomegalovirus immediate early (CMV-IE) promoter, into various mammalian BMSCs. The BMSCs of mouse, rat, porcine, rhesus, and human were cultured primarily in vitro. After more than three passages, the mammalian BMSCs were seeded into dishes and cultured in a humidified incubator at 37 °C with 5% CO(2). When the cells reached about 80% confluence, the complete medium was removed by aspiration. The cells were transduced with recombinant baculovirus at a multiplicity of infection (MOI) of 200 vector genomes/cell with 500 μL PBS at 25 °C for 4 h. At the end of baculovirus transduction, cells were washed and incubated with 2 mL complete medium, and baculovirus-transduced mammalian BMSCs were cultured in a humidified incubator for 2 d. Then, the inverted fluorescent microscope was used to observe GFP expressions in different mammalian BMSCs, and flow cytometry was used to detect the transduction efficiency of baculovirus in various mammalian BMSCs. After more than three passages, the BMSCs of mouse, rat, porcine, rhesus, and human showed a homogeneous spindle-shaped morphology. Compared with the BMSCs of mouse, rat and porcine, the inverted fluorescent microscope observations showed that there were more BMSCs expressing GFP and greater mean fluorescence intensity in rhesus and human transduced with baculovirus. The baculovirus could efficiently transduce into the BMSCs of mouse, rat, porcine, rhesus and human, and the transduction efficiency was (20.21±3.02)%, (22.51±4.48)%, (39.13±5.79)%, (71.16±5.36)% and (70.67±3.74)%, respectively. In conclusion, baculovirus displays different transduction efficiency into various mammalian BMSCs. Due to the high transduction efficiency for primate and human BMSCs, baculovirus is possibly a more suitable gene vector to genetically modify BMSCs of human and primates.
Animals ; Baculoviridae ; Bone Marrow Cells ; cytology ; Genes, Reporter ; Genetic Vectors ; Green Fluorescent Proteins ; genetics ; Humans ; Macaca mulatta ; Mesenchymal Stromal Cells ; cytology ; Mice ; Promoter Regions, Genetic ; Rats ; Swine ; Transduction, Genetic

OBJECTIVETo analyze the clinical characteristics of living-related kidney transplantation (LRKT).

METHODSFrom January, 2004 to December, 2008, 175 LRKT were performed including 63 cases (36%) of parent-child relations and 49 cases (28%) of sibling relations between the recipients and donors. Out of 175 donors, 52 were 50 years old or above, 4 had microscopic hematuria (including 2 with also hypertension), 2 had kidney stone, and 2 had high body mass index (BMI). Zero-point graft biopsy was performed in 59 donors, and abnormalities were found in 15 of them. The recipients were at the age of 33-/+10.5 years, and the primary diseases are mainly dominant glomerular nephritis (72.6%, 127/175), and with a few cases of diabetes (4%, 7/175) and hypertensive nephropathy (4%, 7/175).

RESULTSSerum creatinine of the donors was 102-/+22.5 micromol/L at 7 days postoperatively, and 92-/+19.1 micromol/L at one month. One recipient died of severe pulmonary infection. Two recipients underwent graft nephrectomy due to anastomotic stenosis with concomitant acute graft rejection and renal arterial embolism. The one-year survival rates of the patients and grafts were 99.3% and 98.2%, respectively. The incident rates of accelerated rejection and acute rejection were 1.1% and 14.9%, respectively. Other complications included impaired liver function (22.3%), infection (9.7%) and leucopenia (4.6%). The renal arterial stenosis occurred in 2.3% (4/175) of the recipients.

CONCLUSIONSThe recipients of living-related and cadaveric kidney transplant have different primary kidney disease spectrums. Differential diagnosis and treatment of acute rejection and renal artery or anastomotic stenosis can be of vital importance. Marginal donor kidneys with appropriate inclusion criteria can be safely used for transplantation. With good short-term patient and graft survival, LRKT needs further study to evaluate its long-term effect.

Adolescent ; Adult ; Aged ; Child ; Child, Preschool ; China ; epidemiology ; Family ; Female ; Glomerulonephritis ; surgery ; Graft Rejection ; epidemiology ; Humans ; Kidney Transplantation ; adverse effects ; Living Donors ; Male ; Middle Aged ; Treatment Outcome ; Young Adult

OBJECTIVETo investigate the resection range of mesorectum and rectum below the inferior margin of tumor for the total mesorectum excision (TME) in middle-low rectal cancer.

METHODSSixty patients were enrolled in the study. After TME operation, serial 5 mm interval sections were made in specimens of middle-low rectal cancer. The retrograde metastasis of rectal cancer was observed by routine HE staining.

RESULTSThe phenomena of retrograde metastasis in mesorectum were found in 15 cases, and the distance of retrograde metastasis was 0.5-4.0(2.47+/-1.06) cm, which was correlated with Dukes stage, lymph node metastasis and histological differentiation. The retrograde metastases in bowel were found in 11 cases, and the distance of retrograde metastasis was 0.5-4.0 (1.64+/-1.16) cm, which was correlated with histological differentiation.

CONCLUSIONSThe distal mesorectum should be resected at least 4 cm when TME is carried out, and the distal bowel at least 2.5 cm. More than 5 cm mesorectum and bowel should be resected when advanced Dukes stage, extensive lymph node metastasis and poor histological differentiation occurred.

Adult ; Aged ; Aged, 80 and over ; Female ; Humans ; Male ; Middle Aged ; Neoplasm Metastasis ; Neoplasm Staging ; Rectal Neoplasms ; pathology ; Rectum ; pathology

OBJECTIVETo establish an efficient method for screening effective small interference RNA (siRNA) using dual-luciferase reporter assay system.

METHODSBased on the siRNA expression vector pSilencer-4.1, 3 candidate green fluorescence protein (GFP) gene siRNA expression plasmids, namely pSi-GFPsiRNA1, pSi-GFPsiRNA2, and pSi-GFPsiRNA3, along with the negative control pSi-Negative, were constructed. Using the pGL3-promoter vector, the GFP-luciferase (GFP-LUC) expression plasmid pGL3-GFPf was constructed with the same Kozak consensus translation initiation site and start codon ATG for GFP-LUC coding sequence. The GFP fragment containing the target sequences of 3 GFP siRNAs was introduced into the 3' untranslate region of LUC in the modified pGL3-promoter vector to construct the plasmid pGL3-GFPp. The GFP siRNAs expression plasmids and Renilla luciferase reporter vector pRL-TK were co-transfected with pGL3-GFPf or pGL3-GFPp into the HEK293 cells, respectively. The luciferase activities were determined by dual-luciferase reporter assay, and the GFP mRNA expressions were detected by real-time quantitative PCR.

RESULTSIn the groups cotransfected with GFP siRNAs expression plasmids and pGL3-GFPf, the luciferase activities were reduced obviously, and the reduction was more significant in cells transfected with GFPsiRNA1 compared with the control cells (P<0.01).GFP mRNA levels were also markedly lowered in cells transfected with GFPsiRNA1 as shown by real-time PCR (P<0.01). In addition, the results of dual-luciferase reporter assay and real-time PCR showed that among the groups cotransfected with GFP siRNAs expression plasmids and pGL3-GFPp, the GFP expression was inhibited most obviously by GFPsiRNA1 (P<0.01).

CONCLUSIONThe dual-luciferase reporter assay system provides a useful method for screening effective siRNAs targeting specific genes.

Animals ; Cell Line ; Genes, Reporter ; Green Fluorescent Proteins ; genetics ; Luciferases ; genetics ; Plasmids ; genetics ; RNA Interference ; RNA, Small Interfering ; genetics ; Transfection

OBJECTIVEThis was a nationwide study of sleep circadian in term infants. The aim was to understand the development characteristics of infants' sleep/wake patterns longitudinally in their own home environments over the first 12 months of life.

METHODTotally 524 healthy term infants from 9 urban districts took part in this project Their sleep/wake patterns over 24 h were recorded using parental sleep diaries, from the 2nd day to 12 months old.

RESULTThe results showed that infant daytime sleep changed significantly at 0-2, 3-4, 5-6, and 8-9 months after birth, and the change was the fastest in the first month, the mean percentage of daytime sleep decreased from 82.4% at Day 2 to 62.8% at 1 month old. Also, the average number of naps reduced from 3.7 to 2 across the infancy. The ability of continuous sleep throughout the night gradually enhanced from 1 month old, and the nocturnal longest sleep time extended to 6.8 h at 4 months of age as well as the nighttime awakening frequency less than 0.5 over 6 months old. Additionally, the nighttime sleep increased significantly at 4 and 9 months after birth, where the proportion of nighttime sleep increased from 55.8% at Day 2 to 64.3% of 4 months and 71.2% of 9 months respectively. In general, the total sleep time over a 24 h period presented a downward trend as the infant aged.

CONCLUSIONThe periods 0-6 and 8-9 months after birth were the key periods for the development of infant sleep.

Child Development ; China ; Circadian Rhythm ; Female ; Humans ; Infant ; Infant, Newborn ; Male ; Pregnancy ; Pregnancy Trimester, Third ; Sleep ; Wakefulness

The effects of ketamine on transient outward potassium current (I(to)) of isolated human atrial myocytes were investigated to understand the mechanism of part of its effects by whole-cell patch-clamp. Atrial myocytes were enzymatically isolated from specimens of human atrial appendage obtained from patients under going cardiac valve displacing. Ito is recorded in voltage-clamp modes using the patch-clamp technique at room temperature. Currents signals were recorded by an Axopatch 200B amplifier with the Digidata 1322A-pClamp 9.0 data acquisition system. Ketamine decreased I(to) of human atrial myocytes in a dose-dependent manner. The current-voltage curve was significantly lowered, 30, 100, 300, and 1000 micromol x L(-1) ketamine decreased respectively I(to) current density about (13.62 +/- 0.04)%, (38.92 +/- 0.05)%, (72.24 +/- 0.10)% and (83.84 +/- 0.05)% at the potential of 50 mV, with an IC50 of 121 micromol x L(-1). The I(to) activation curve, inactivation curve and the recovery curve were not altered by ketamine. So, ketamine concentration-dependently decreased I(to) of human atrial myocytes.
Adolescent ; Adult ; Aged ; Anesthetics, Dissociative ; administration & dosage ; pharmacology ; Dose-Response Relationship, Drug ; Female ; Heart Atria ; cytology ; Humans ; Ketamine ; administration & dosage ; pharmacology ; Male ; Middle Aged ; Myocytes, Cardiac ; cytology ; drug effects ; physiology ; Patch-Clamp Techniques ; Potassium Channels ; drug effects ; Young Adult