OBJECTIVETo investigate the effect of lead-exposed astrocyte conditioned medium (ACM) on the synaptic formation of neurons and to provide reference for the mechanism of lead neurotoxicity.

METHODSAstrocytes were cultured in the medium containing 50, 100, 200, 400, and 800 µmol/L lead acetate for 72 h. Alamar Blue was used to assess the cell viability of astrocytes, and then ACM was collected. Primarily cultured neurons were divided into six groups: pure culture group, non-glutamic acid (Glu)-induced ACM treatment group, Glu-induced lead-free ACM treatment group, and Glu-induced 50, 100, and 200 µmol/L lead acetate-exposed ACM treatment groups. Neurons were collected after being cultured in ACM for 24, 48, or 72 h. The content of synaptophysin (SYP) in neurons was determined by Western blot. The SYP expression in neurons was measured by immunofluorescence after being cultured in ACMfor 72 h.

RESULTSIn all lead-exposed groups, the cell viability of astrocytes declined with increasing concentration of lead (P < 0.05). The Western blot showed that compared with the pure culture group, the non-Glu-induced ACM treatment group and Glu-induced lead- free ACM treatment group had significantly increased content of SYP in neurons (P < 0.01); compared with the non-Glu-induced ACM treatment group, the Glu-induced ACM treatment groups had significantly reduced SYP expression in neurons (P < 0.05); compared with the Glu-induced lead-free ACM treatment group, all lead-exposed ACM treatment groups had the content of SYP in neurons significantly reduced with increasing concentration of lead after 72-h culture (P < 0.01), the 200 µmol/L lead-exposed ACM treatment group had significantly reduced content of SYP in neurons after 48-h culture (P < 0.01), and all lead-exposed ACM treatment groups showed no significant changes in the content of SYP in neurons after 24-h culture. Double-labeling immunofluorescence of SYP showed that all lead-exposed ACM treatment groups had a significant decrease in the number of SYP-fluorescent particles after 72-h culture (P < 0.05).

CONCLUSIONAstrocytes promote synaptic formation of neurons, which may be inhibited during lead exposure.

Astrocytes ; drug effects ; physiology ; Cell Survival ; drug effects ; Cells, Cultured ; Culture Media, Conditioned ; metabolism ; Glutamic Acid ; metabolism ; Lead ; toxicity ; Neurons ; drug effects ; Synapses ; drug effects ; physiology

OBJECTIVETo prepare and characterize monoclonal antibodies (mAb) and polyclonal antibodies against nucleocapsid (N) protein of severe acute respiratory syndrome (SARS)-associated coronavirus (SARS-CoV) and to establish antibodies-based sandwich ELISA for detecting N protein of SARS-CoV, which might apply to early diagnosis of patients with SARS-CoV infection.

METHODSBALB/c mice were immunized with purified recombinant N protein of SARS-CoV for producing mAbs, and New Zealand white rabbits were immunized for producing polyclonal antibodies. The identification of antibodies was performed using indirect enzyme-linked immunosorbent assay (ELISA), indirect fluorescent-antibody assay (IFA), and Western immunoblotting. Capturing and detecting antibodies were selected by pairing the mAbs and polyclonal antibodies one by one and an antibodies-based sandwich antigen capture ELISA was used for detecting N antigen of SARS-CoV.

RESULTSNine mAbs and hyperimmune rabbit polyclonal antibodies, specifically against SARS-CoV nucleocapsid protein were obtained. Using paired ELISA assay, three mAbs N1E8, N8E1 and N10E4 were selected as capturing antibody and rabbit polyclonal antibodies as detecting antibody then triple antibodies-based sandwich ELISA was established following horseradish peroxidase (HRP)-conjugated goat anti-rabbit immunoglobulin G. The recombinant N protein was used as a standard to establish a detection sensitivity of approximated 50 pg/ml with this assay. When tested with 420 serum specimens from serologically confirmed SARS patients, the positive rates of serum N protein were 90.1%, 23% and 0%, in which sera collected from 1 to 10 days, 11 to 20 days and beyond 21 days respectively after the onset of symptoms. The specificity of the assay was 99.86% in 715 control serum specimens. There was no cross-reaction with other respiratory viruses and coronaviruses.

CONCLUSIONSpecific and high affinity mAbs and rabbit polyclonal antibodies were obtained. By paired and optimized sandwich ELISA, a sensitive and specific antigen capture ELISA was established for detecting N antigen of SARS-CoV, which might apply to early diagnosis, source tracing and epidemiological studies of SARS.

Animals ; Antibodies, Monoclonal ; biosynthesis ; Antibodies, Viral ; blood ; Enzyme-Linked Immunosorbent Assay ; Humans ; Mice ; Mice, Inbred BALB C ; Nucleocapsid ; immunology ; Rabbits ; SARS Virus ; immunology ; isolation & purification ; Sensitivity and Specificity ; Severe Acute Respiratory Syndrome ; virology

This study was purposed to investigate the role of post-thaw infused donor cells for predicting engraftment and hematopoietic reconstitution after unrelated cord blood transplantation (UCBT). The retrospective analysis was performed on clinical data of 97 children with malignant or non-malignant diseases received single unit UCBT from August 1999 to April 2010. The impact of pre-freezing and post-thaw cell dose of total nucleated cells (TNC), CD34(+) cells and colony-forming units-granulocyte/macrophage (CFU-GM) on engraftment and hematological recovery after UCBT was analyzed. Unrelated donors were from Guangzhou cord blood bank (GZCBB) entirely. The results indicated that the pre-freezing TNC (/kg) (mean ± SD: 7.65 × 10⁷ ± 4.26 × 10⁷; median: 6.34 × 10⁷), CD34(+)cells (/kg) (mean ± SD: 4.64 × 10(5) ± 4.47 × 10⁵; median: 3.03 × 10⁵) and CFU-GM (/kg) (mean ± SD: 0.79 × 10⁵ ± 1.09 × 10⁵; median: 0.57 × 10⁵) showed a good correlation with their post-thaw counterparts including TNC(/kg) (mean ± SD: 6.98 × 10⁷ ± 4.12 × 10⁷; median: 6.00 × 10⁷), CD34(+)cells (/kg)(Mean ± SD: 6.86 × 10⁵ ± 8.56 × 10⁵; Median: 4.17 × 10⁵), and CFU-GM (/kg) (mean ± SD: 0.52 × 10⁵ ± 0.52 × 10⁵; median: 0.39 × 10⁵) (r = 0.952, p < 0.001; r = 0.794, p < 0.001; r = 0.478, p < 0.001). Either the pre-freezing or post-thaw number of infused CFU-GM was significant higher in patients who achieved engraftment (n = 70) than those who suffered graft failure (n = 22) (p = 0.023 and 0.011, respectively), but no significant difference of TNC and CD34(+) cells dose (pre-freezing or post-thaw) were found between these two groups. Pre-freezing CFU-GM, TNC, CD34(+) cell dose negatively correlated with the time of neutrophil engraftment (r = -0.285, p = 0.018; r = -0.396, p = 0.002; r = -0.373, p = 0.002), as well as the post-thaw number of TNC and CD34(+) cells (r = -0.260, p = 0.031; r = -0.483, p < 0.001), whereas only pre-freezing CD34(+) cells showed a significant correlation with platelet engraftment time (r = -0.352, p = 0.013). It is concluded that the CFU-GM amount is useful for predicting engraftment of UCBT, while pre-freezing hematopoietic cell doses show superior correlation with the speed of engraftment and hematopoietic reconstitution than their post-thaw counterparts in pediatric recipients, suggesting that it is essential to perform hematopoietic potency assay on each cord blood unit prior to listing or release for administration.
Adolescent ; Antigens, CD34 ; blood ; Blood Banks ; Child ; Child, Preschool ; Cord Blood Stem Cell Transplantation ; methods ; Female ; Fetal Blood ; cytology ; Graft Survival ; Granulocyte-Macrophage Progenitor Cells ; Humans ; Infant ; Male ; Retrospective Studies ; Tissue Donors

Umbilical cord blood (UCB) is an alternative source of hematopoietic stem cells for transplantation with success being associated with the total nucleated cell (TNC) count, CD34(+) cells and colony-forming unit-granulocyte-macrophage (CFU-GM) content infused. This study was purposed to clarify the impact of maternal and neonatal factors on hematopoietic potential of UCB product. UCB samples were screened, processed, tested and cryopreserved according to the Standard Operation Procedure (SOP) of Guangzhou cord blood bank (GZCBB). Relationship of hematopoietic cell parameters with maternal and neonatal characteristics for 4615 UCB units was analyzed retrospectively. The results showed that both collected volume (Mean ± SD: 95.23 ± 22.42 ml; Median: 91.85 ml) and initial TNC [Mean ± SD: (1.34 ± 0.49) × 10(9); Median: 1.25 × 10(9)] correlated well with postprocessed TNC [Mean ± SD: (1.21 ± 0.42) × 10(9); Median: 1.14 × 10(9); p < 0.001], CD34(+)count [Mean ± SD: (5.14 ± 4.55) × 10(6); Median: 4.08 × 10(6); p < 0.001] and CFU-GM content [Mean ± SD: (9.72 ± 8.66) × 10(5); Median: 7.53 × 10(5); p < 0.001]. As for donor factors, only infant birth weight correlated strongly with volume collected and all hematopoietic cell parameters (p < 0.001). UCB samples from bigger babies had higher collected volume, TNC, CD34(+) count and CFU-GM content (p < 0.001). Mother's age had no correlation with all the above parameters. Gestational age correlated positively with initial/postprocessed TNC (p < 0.001) and negatively with CD34(+) count (p = 0.04), but no relation with collected volume and CFU-GM content. Cesarean section produced superior volume (Mean ± SD: 97.05 ± 22.23 ml vs 92.53 ± 22.43 ml; Median: 94.08 ml vs 88.82 ml; p < 0.001), but inferior cell count than vaginal delivery (p < 0.001). Male infants had more initial volume and CD34(+) count (Mean ± SD: 96.41 ± 22.31 ml vs 93.95 ± 22.47 ml; Median: 93.27 ml vs 90.14 ml; p < 0.001); [Mean ± SD: (5.28 ± 5.04) × 10(6) vs (5.00 ± 3.94) × 10(6); Median: 4.18 × 10(6) vs 3.94 × 10(6); p < = 0.042], but lower initial and postprocessed TNC than female ones [Mean ± SD: (1.31 ± 0.50) × 10(9) vs (1.37 ± 0.47) × 10(9); Median: 1.22 × 10(9) vs 1.28 × 10(9); p < 0.001]; [Mean ± SD: (1.18 ± 0.42) × 10(9) vs (1.24 ± 0.41) × 10(9); Median: 1.10 × 10(9) vs 1.17 × 10(9); p < 0.001], while no significant difference of CFU-GM were found between male and female infants. It is concluded that these data may be helpful to optimize the UCB donor selection and improve cost efficiency of UCB bank resource. The heavier infants after vaginal delivery should be selected and large-volume units with higher TNC should be chosen at first.
Adult ; Birth Weight ; Blood Banks ; methods ; Cord Blood Stem Cell Transplantation ; methods ; Delivery, Obstetric ; Donor Selection ; Female ; Fetal Blood ; cytology ; immunology ; Gestational Age ; Hematopoietic Stem Cells ; Humans ; Infant, Newborn ; Male ; Maternal Age ; Pregnancy ; Young Adult

OBJECTIVETo characterize the incidence, epidemiologic features, etiologic agents and sequelae of bacterial meningitis in children under 5 years of age in Nanning, Guangxi.

METHODSA population-based surveillance was conducted to evaluate children with signs and symptoms of meningitis. All hospitals, township health centers and village clinics in the surveillance area were structured to participate in the case referral and evaluation. Cerebrospinal fluid (CSF) and blood specimens were obtained and processed using standardized microbiologic methods.

RESULTSDuring the 26-month surveillance period, among the children under 5 years old, a total of 1272 cases who met the screening criteria of meningitis were studied. 265 of 1272 cases were identified as clinically diagnosed meningitis, with an incidence rate of 86.36 per 100 000 population. The annual incidence rate under the 38 cases of confirmed bacterial meningitis appeared to be 12.38/100 000. Staphylococcus species accounted for the largest proportion of laboratory-confirmed bacterial meningitis, followed by E. coli and S. pneumoniae. The highest attack rate occurred in neonates < 1 month, followed by children aged 1 - 12 months in the confirmed patients. Meningitis caused by Sp and Hi mainly occurred in children aged 1 - 12 months. All cases of meningitis due to Hi and Sp were children aged 1 - 24 months. 13.16% and 0.00% of the cases survived with complications and sequelae, and the case-fatality rate was 18.42%. 40 bacterial isolates were identified from 1193 blood cultures and 23 from 1211 cerebrospinal fluid samples, but no Neisseria meningitidis was found.

CONCLUSIONMeningitis due to Hi was first confirmed in Guangxi with the incidence of 0.98 per 100 000 population. The annual incidence rate of confirmed bacterial meningitis was 12.38 per 100 000, which was considered an important public health problem in children. Staphylococci was the predominant pathogen in confirmed bacterial meningitis.

Child, Preschool ; China ; epidemiology ; Female ; Humans ; Incidence ; Infant ; Infant, Newborn ; Male ; Meningitis, Bacterial ; epidemiology ; microbiology ; Meningitis, Escherichia coli ; epidemiology ; Meningitis, Haemophilus ; epidemiology ; Population Surveillance ; Staphylococcal Infections ; epidemiology

Objective To investigate the effect of Echinococcus granulosus cyst fluid(EgCF) on the cytoskeletal rearrangement and phagocytosis and the migration of macrophages induced by lipopolysaccharide(LPS). Methods Peritoneal macrophages of C57BL/6 mice were isolated and cultured in vitro, and divided into control group and LPS group and LPS combined with EgCF group. After 48 hours of treatment, filamentous actin (F-actin) changes were observed with rhodamine-labelled phalloidin staining and fluorescence microscopy; Transwell^TM chamber was used to test cell migration ability and flow cytometry to test cell phagocytosis. After 1 hour of treatment, PI3K and AKT, phosphorylated AKT (p-AKT), Rac1, guanosine triphospho-Rac1 (GTP-Rac1), WASP and Arp2 protein expressions were detected with Western blot analysis. Results Compared with the control group, after LPS stimulation, macrophages were deformed significantly; pseudopodia increased; actin cytoskeleton increased and was more distributed in pseudopodia; the ability of migration and phagocytosis were significantly improved, and the expression of PI3K, p-AKT, GTP-Rac1, WASP and Arp2 proteins significantly increased. EgCF treatment caused cell shrinkage and disappearance of pseudopodia protrusions of LPS-activated cells, and led to the reduced phagocytic and migratory of cells; the protein expression of PI3K, p-AKT, GTP-Rac1, WASP and Arp2 decreased significantly compared with the LPS group. Conclusion LPS induces the migration and enhances phagocytosis of macrophages while EgCF inhibits these effects, which is related to actin cytoskeleton rearrangement.
Mice ; Animals ; Lipopolysaccharides/pharmacology* ; Echinococcus granulosus/metabolism* ; Proto-Oncogene Proteins c-akt ; Cyst Fluid/metabolism* ; Mice, Inbred C57BL ; Macrophages/metabolism* ; Phagocytosis ; Actins/metabolism* ; Phosphatidylinositol 3-Kinases/metabolism* ; Guanosine Triphosphate/pharmacology*

OBJECTIVEOur research was aim to investigate the effect of microRNA-328 (miR-328) on proliferation of chronic myeloid leukemia(CML) cell K562 and the mediated effect of C/EBPα.

METHODSThe eukaryotic expression vectors of miR-328 targeting gene and suppressor gene (hsa-miR-328 and hsa-miR-328-inhibitor) were constructed, and transfected into K562 cells respectively. The mRNA expression levels of miR-328 and C/EBP α were detected by real-time fluorescence quantitative RT-PCR; C/EBP α protein expression was detected by Western blot; CCK-8 was used to estimate the cell viability.

RESULTSThe recombinant genes of hsa-miR-328 and hsa-miR-328-inhibitor were successfully constructed and transfected into K562 cells. Fluorescent cells were observed after 24 h, and the visible fluorescence cells were gradually increased after 48 h or 72 h, the miR-328 showed no effect on the mRNA expression of C/EBPα detected by RT-PCR. Meanwhile, miR-328 showed recovering effect on C/EBPα translation and inhibition of K562 cells proliferation.

CONCLUSIONmiR-328 has been successfully constructed and transfected into K562 cells, miR-328 inhibits the proliferation of K562 cells by up-regulation of C/EBPα.

CCAAT-Enhancer-Binding Protein-alpha ; Cell Proliferation ; Cell Survival ; Humans ; K562 Cells ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; MicroRNAs ; Transfection ; Up-Regulation

OBJECTIVE: To investigate the feasibility and clinical significance of high resolution melting(HRM) curve analysis to detect SLC4A1 gene D38A and K56E mutations in the patients with hereditary spherocytosis(HS). METHODS: Peripheral blood was collected from 23 cases of HS for routine tests and their genomic DNA was extracted by routine technique. Specific primers of mutation sites D38A and K56E of SLC4A1 gene were designed. The HRM method was used to analyze all the samples, and then the results of HRM were verified with DNA sequencing technology. RESULTS: Among 23 specimens of HS patients, 6 cases of heterozygous mutant gene were detected by HRM technology, including 3 cases of D38A mutation and 3 cases of K56E mutation, which were confirmed by DNA sequencing. CONCLUSION The HRM technology can correctly detect 2 common mutation sites including D38A and K56E in SLC4A1 gene in an efficient, fast, and reliable way, which not only can be used for clinical diagnosis, but also expected to be a new method for clinical researchers to define gene mutation spectrum in HS patients.
Anion Exchange Protein 1, Erythrocyte ; genetics ; Base Sequence ; DNA Mutational Analysis ; DNA Primers ; Heterozygote ; Humans ; Mutation ; Spherocytosis, Hereditary ; genetics

OBJECTIVES: To study the association of β2-drenergic receptor ( METHODS: A total of 143 children with asthma who attended the hospital from October 2016 to October 2020 were enrolled as the asthma group, among whom 61 children had mild symptoms (mild group) and 82 children had moderate-to-severe symptoms (moderate-to-severe group). A total of 137 healthy children were enrolled as the control group. Peripheral venous blood samples were collected from the two groups. The SNaPshot SNP technique was used to analyze the SNP and haplotypes of the RESULTS: Polymorphisms were observed in the CONCLUSIONS SNP/haplotype of the
Asthma/genetics* ; Case-Control Studies ; Child ; Genetic Predisposition to Disease ; Genotype ; Haplotypes ; Humans ; Polymorphism, Single Nucleotide ; Receptors, Adrenergic, beta-2/genetics* ; Regulatory Sequences, Nucleic Acid

Objective To analyze the death status and main causes of death among children under 5 years old in Changsha from 2016 to 2021, and to provide a scientific basis for formulating preventive measures for children's health care. Methods The data of 1 761 deaths of children under 5 years old in Changsha City from 2016 to 2021 were collected, and the mortality trend, the order of causes of death and the utilization of pre-death medical care services were retrospectively analyzed. Results The 7-day neonatal mortality, 28-day neonatal mortality, 0-1-year-old neonatal mortality, and the mortality rate of children under 5 years old (U5MR) in Changsha City from 2016 to 2021 were 0.76‰, 1.28‰, 2.41‰, and 3.86‰, respectively. All the mortality rates showed a decreasing trend (P<0.05). U5MR in males was significantly higher than that in females (P<0.05), and U5MR in rural areas was significantly higher than that in urban areas (P<0.05). The top five causes of U5MR were drowning, premature delivery or low birth weight, pneumonia, other congenital anomalies, and accidental asphyxia, respectively. The death places of children under 5 years old were mainly medical and health institutions, and 81.72% of them were treated in hospitals before death. Conclusion From 2016 to 2021, the mortality rate of children under the age of 5 in Changsha City has gradually decreased. Preventing congenital malformations, reducing preterm birth or low birth weight, improving the treatment level of pneumonia, and preventing accidents such as drowning and accidental suffocation are the key to reducing the mortality rate of children under 5 years old.