Plasma viral RNA load is widely accepted as the most relevant parameter to assess the status and progression of Simian immunodeficiency virus (SIV) infections. To accurately measure RNA levels of the virus, a one-step fluorescent quantitative assay was established based on the SYBR green Real-time reverse transcription-polymerase chain reaction (RT-PCR). The lower detection limit of the assay was 10 copies per reaction for the virus. This method was successfully applied to quantify SIVmac251 and SIVmac239 viruses produced in CEM×174 cells. Additionally, the performance of the SYBR green RT-PCR was assessed in a SIVmac251 infected rhesus macaque. The result demonstrated that the method could detect as little as 215 copies per milliliter of plasma and the dynamic pattern of viral load was highly consistent with previous results. With regard to convenience, sensitivity and accuracy our assay represents a realistic alternative to both branched-chain DNA (b-DNA) assays or real-time PCR assays based on TaqMan probes.

Objective To investigate the association between single nucleotide polymorphisms (SNPs) of ATP-binding cassette B1 (ABCB1),ATP-binding cassette C2 (ABCC2) and solute carrier organic anion transporter 1B1 (SLCO1 B1) genes with high dose methotrexate (HDMTX)-induced toxicity in children with acute lymphoblastic leukemia (ALL).Methods This study was designed as a casecontrol.From September of 2005 to December of 2011,the blood samples were randomly collected from 142ALL patients from Nanjing Children's Hospital,Enzyme-multiplied immunoassay technique (EMIT) was used to measure the plasma concentration of MTX,Seven SNPs in ABCB1 (rs1045642,rs2032582,rs1128503),ABCC2 (rs717620,rs2273697) and SLCO1 B1 (rs4149081,rs11045879) genes were detected by polymerase chain reaction-ligase detection reaction (PCR-LDR).Results A significantly increased risk of MTX-induced toxicity was observed in patients with MTX elimination delay (OR ＝ 2.828,95％ CI:1.217-6.571,P ＜ 0.05).Two SNPs in SLCO1B1,rs4149081 and rs11045879 were linkage disequilibrium (LD) with each other (R2 =0.979,P ＜ 0.05).Multivariate analysis revealed that individuals with SLCO1B1 rs4149081 AA genotype or SLCO1B1 rs11045879 CC genotype showed increased incidence of MTX elimination delay (OR =4.41,95％ CI:1.537-12.654,P =0.042),and the two genotypes were also associated with significantly increased risk of MTX-induced toxicity (OR =4.118,95％ CI:1.135-14.944,P =0.022).No association of MTX elimination delay or MTX-induced toxicity with the other SNPs analyzed was found.Conclusions SLCO1B1 rs4149081 AA or SLCO1B1 rs11045879 CC genotypes might be a risk factor for the susceptibility to MTX-induced toxicity in children with ALL.

Objective To investigate the influences of the functional polymorphisms of cytochrome P450 isozymes 2A6 (CYP2A6),2B6 (CYP2B6),2C9 (CYP2C9),and 2C19 (CYP2C19) on plasma concentration of sodium valproate.Methods A total of 131 Chinese children with epilepsy receiving sodium valproate after a period of more than 5 half-time were recruited.The genotypes of CYP2A6 were detected by multiplex polymerase chain reaction (PCR),and the genotypes of CYP2B6,CYP2C9,and CYP2C19 were detected by PCR-ligase detection reaction.Enzyme-multiplied immunoassay technique was used to measure the plasma concentration of sodium valproate.The association between the polymorphisms and the plasma concentration of sodium valproate were analyzed by one-way ANOVA or Student' s t-test.Results Patients were divided into 4 groups according to the genotyping results of CYP2C9 and CYP2C19 (G1:extensive metabolizers in both CYP2C9 and CYP2C19; G2:CYP2C19 intermediate metabolizers; G3:CYP2C19 poor metabolizers; G4:CYP2C9 poor metabolizers),the mean normalized steady-state sodium valproate concentrations were significantly higher in G3 (3.70 ± 0.95) and G4 (4.35 ± 1.48) patients when compared to those in G 1 (2.57 ± 1.30,t =3.056,4.490,both P ＜ 0.01) and G2 (2.76 ± 1.19,t =2.827,4.462,both P ＜ 0.01) patients.The daily doses (mg/d) of sodium valproate received by G3 (19.46 ± 5.20) and G4 (19.30 ±7.67) patients were significantly lower than that of G1 patients(24.10 ±6.97,t =2.359,2.297,both P ＜ 0.05).There were no differences in daily doses or normalized steady-state concentrations of sodium valproate among the CYP2A6* 4 or CYP2B6* 6 genotypic groups.Conclusions The CYP2C9 and CYP2C19 polymorphisms have dramatic effects on the plasma concentration of sodium valproate.The daily doses of sodium valproate in G3 and G4 patients should be lower than usual.

Vitamin K(VK)occurs naturally in two forms, including vitamin K 1(VK 1)and vitamin K 2(VK 2). The function of VK is related to the number of its side chains.In addition to the function of coagulation, VK is also gradually found to be involved in bone metabolism, which is closely related to the occurrence of adult osteoporosis.The rapid growth and development in childhood is a period of extremely vigorous bone metabolism.The role of VK in children′s bone metabolic diseases has been gradually recognized in recent years.However, there are still few clinical studies on the level of VK in children of different ages and how VK participates in children′s bone metabolic diseases.This article reviews the types and metabolism of VK, the mechanism of VK regulating bone metabolism, the level of VK and its role in bone metabolic diseases, so as to provide new understanding and ideas for the prevention and treatment of bone health in children.

OBJECTIVETo observe the clinical characteristics of infections in adult acute leukemia （AL）patients during chemotherapy in hospital, and identify the risk factors for infections.

METHODSA retrospective study of patients with AL who underwent chemotherapy between July 2010 and Dec 2014 in the First Affiliated Hospital of Xiamen University was conducted. Clinical features and risk factors for infections were analyzed.

RESULTS191 patients with AL received a total of 728 courses of chemotherapies. During these admissions, 385（52.9%) infections episodes occurred. The common infections sites were lower respiratory tract infection（36.3%，153/374）, bloodstream infection（17.1%, 64/374）, oral infection（13.6%，51/374）, and perianal infection（13.4%, 50/374）. 164 strains of pathogenic bacteria were detected. Gram- negative bacteria were recorded in 59.1% of documented pathogens, and Gram- positive bacteria were responsible for 32.9% of infections. Multivariate unconditioned logistic analysis of factors identified consistent independent risk factors for no completely remission（OR=0.142, P< 0.001）, duration of neutropenia longer than 7 days（OR=12.764, P<0.001）, general wards（OR=1.821, P< 0.001）, and hospitalization interval longer than 10 days（OR=0.720, P=0.039）.

CONCLUSIONInfections after chemotherapy for AL continues to be common. AL patients with induction chemotherapy or severe neutropenia faced an increased risk of infections by multivariate analysis. And patients with short-term stay or laminar flow wards seem to be less susceptible to infections.

Acute Disease ; Bacterial Infections ; complications ; Gram-Negative Bacteria ; Gram-Positive Bacteria ; Hospitals ; Humans ; Leukemia ; complications ; drug therapy ; microbiology ; Multivariate Analysis ; Neutropenia ; complications ; Remission Induction ; Retrospective Studies ; Risk Factors

Objective:To analyze the clinical characteristics of lung cancer associated acute ischemic stroke (LCA-AIS) and atrial fibrillation associated acute ischemic stroke (AFA-AIS).Methods:From January 1, 2014 to December 31, 2018, 46 patients diagnosed with LCA-AIS (LCA-AIS group)in Hainan Hospital of Chinese PLA General Hospital were selected, and 46 patients diagnosed with AFA-AIS (AFA-AIS group) were matched according to age and sex.The general situation, laboratory test results and imaging results of the two groups were analyzed.Results:(1) The neurological deficit symptoms in AFA-AIS group were more serious than those in LCA-AIS group; there was significant difference in National Institutes of Health Stroke Scale (NIHSS) score and the Modified Rankin Scale (mRs) score between the two groups ( P=0.001, P=0.003). (2)The D-D polymer concentration in LCA-AIS group was significantly higher than that in AFA-AIS group ( P<0.001), but the hemoglobin, erythrocyte count and hematocrit were significantly lower than those in AFA-AIS group (all P<0.001). (3)There was no significant difference in imaging classification and the number of infarct basins between LCA-AIS group and AFA-AIS group ( P>0.05). LCA-AIS patients was more likely to have poly-period acute ischemic lesions ( P=0.015), while AFA-AIS had significantly larger infarct diameter and more likely to be complicated with acute hemorrhagic stroke or bleeding ( P<0.001). Conclusions:The clinical characteristics of LCA-AIS and AFA-AIS are similar, so it is necessary to distinguish LCA-AIS from AFA-AIS in combination with neurological impairment, laboratory tests and imaging findings to avoid misdiagnosis.

OBJECTIVETo discover the techniques for ex vivo generation and cryopreservation of erythroid progenitor cells (EPCs)derived from umbilical cord blood (UCB)mononuclear cells (MNCs).

METHODSUCB was chosen as the source of EPCs. Erythrocytes were precipitated by hydroxyethyl starch (HES). MNCs were separated by Ficoll density gradient centrifugation. Erythroid progenitor cell were generated from MNC ex vivo in suspension culture supplemented with stem cell growth factor, insulin growth factor, erythropoietin, Fms- liketyrosinekinase ligand, transferrin and dexamethasone. Cell maturation was evaluated by morphologic analysis and CD71/CD235a expression profiling. In vitro induced cells were cryopreserved using different cryopreservation media. The cell survival rate, phenotype and proliferation curves were detected after cell thawing.

RESULTSWith the extension of culture time, the total number of cells increased significantly accompanied with the elevation of CD71 and CD235 positive populations. After 14- day inducing, the cells reached to approximately 110 times of the starting number with the cell viability as (88.92±0.95)%. The percentages of cell surface markers were (86.77±9.11)% for CD71 and (64.47±16.67)% for CD71/CD235, respectively. With the extension of inducing time, wright- Giemsa staining showed that the middle erythroblasts appeared mostly at day 10, and the late erythroblasts were seen at day 14. The red pellets were present at day 14, which indicated the more production of hemoglobin. Colony forming assay showed that erythroid colonies at induction day 7 were higher than that for non-induced cells (326.00±97.96vs 61.60±20.03 per 2 000 cells). With the extension of culture time, the number of erythroid colonies decreased. Induced EPCs were preserved with different cryopreservation solutions, in which 10% DMSO were better than 5% DMSO. Additionally, 10% DMSO + 2% HSA showed no different with 10% DMSO + 5% HSA. Combined 50% plasma with 2% HSA was more effective.

CONCLUSIONSThis non- serum culture media could effectively induced and expanded EPCs, and 10% DMSO + 2% HSA + 50% plasma appeared to be a desirable cryopreservation solution for EPCs from UCB.

Cell Culture Techniques ; Cell Differentiation ; Cell Survival ; Cells, Cultured ; Cryopreservation ; methods ; Erythroblasts ; cytology ; Erythroid Precursor Cells ; cytology ; Fetal Blood ; cytology ; Humans ; Leukocytes, Mononuclear ; cytology ; Umbilical Cord

Abstract OBJECTIVE Oxcarbazepine(OXC) is an antiepileptic drug, which is metabolized to the active 10-monohydroxy derivative(MHD) after oral administration. The half-life period of MHD in children is significantly shorter than that in adults, and the clearance is increased by 30% to 160% compared with that in adults, which indicates that the pharmacokinetics(PK) of MHD in children is obviously different from that in adults, while adults and children exhibit different levels of expression of metabolism enzymes and transporter proteins with the same genotype. At present, there is no study describing the influence of genetic polymorphism of PK-related enzymes on MHD plasma concentrations in children with epilepsy. This study investigates whether the polymorphism of metabolic enzymes and transporter genes have significant effects on MHD plasma concentrations in children with epilepsy in China, so as to provide the reference for individualized application of OXC in pediatric patients. METHODS The plasma samples from pediatric patients with epilepsy aged 0-14 years old at the First Affiliated Hospital of Fujian Medical University who received OXC were prospective collected from June 2021 to June 2023. The MHD blood concentrations of the patients were measured using enzyme amplified immunoassay, and the metabolic enzyme genes UGT2B7 802T>C, UGT1A9 I399C>T, as well as the transporter genes ABCB1 3435C>T and ABCB2 1249G>A polymorphism were detected using dideoxy chain-termination method in epilepsy children. According to Hardy Weinberg's law of genetic balance, the theoretical values of genotype frequency of the patients were calculated, and a Chi-Square test method was used to compare whether there was a significant difference between the theoretical value and the measured value, to examine whether the genotype of the patients included in the study is accordance with the law of genetic balance. One-way ANOVA statistical method was used to analyze the correlation of the four single nucleotide polymorphisms, daily maintenance dosage of OXC, and MHD blood concentrations. Subsequently, Fisher's least significant difference(LSD) test was performed. LSD test is a pairwise comparison of the differences between the mean values of each group, calculated based on the standard error and degrees of freedom to obtain the minimum significant difference between each two groups, while P<0.05 indicated that the difference was significant. RESULTS In this study, 161 trough concentrations were collected from children with epilepsy. The genotype of the included population conformed to the genetic balance law, which indicated that the included patients were representative for the population. Unite analysis of variance showed a significant correlation between the transporter gene ABCB1 3435C>T and MHD blood drug concentration(P<0.05). Subsequently, Fisher's minimum significant difference test was conducted, and MHD plasma concentrations of patients carrying the ABCB1 3435C>T mutation allele were significantly higher than that of non-carriers. No significant association was found between the four single nucleotide polymorphisms and the daily maintenance dosage of OXC, and no significant impact of the other metabolic enzyme and transporter genetic polymorphisms on MHD plasma concentrations was found. CONCLUSION The results of research shows that the ABCB1 3435C>T polymorphism significantly affect the MHD blood concentration of pediatric patients with epilepsy, and the effects of UGT2B7 802T>C, UGT1A9 I399C>T and ABCB2 1249G>A genetic polymorphisms on MHD blood concentration and daily maintenance dosage of OXC are not found. The results suggest that MHD blood concentrations are significantly increased by affecting the expression of the encoded MDR1 transporter protein after ABCB1 3435C>T site mutation, which also may increase the risk of adverse reactions of OXC. The genetic polymorphisms of ABCB1 3435C>T can be detected in children with epilepsy when taking OXC, and the dosage can be adjusted appropriately for patients with genetic mutations. The results of this study can provide the reference for the individualized administration of OXC in clinic.

OBJECTIVETo build a protocol of separation and induction of megakaryocytes derived from cord blood mononuclear cells.

METHODSRed blood cells were precipitated by hydroxyethyl starch (HES). Mononuclear cells were obtained by density gradient centrifugation with Ficoll. The inducing efficiencies of megakaryocytes by using of different cytokine cocktails and culture media were analyzed.

RESULTSThe best choice for erythrocyte sedimentation and high efficiency of nucleated cells retrieving were obtained by using of 1.5% HES. The isolated cord blood mononuclear cells were cultured with domestic serum-free medium supplemented with 116t (IL-11, IL-6, TPO), st36(SCF, TPO, IL-3, IL-6), pt36 (PDGF,TPO,IL-3,IL-6) or pst36 for 7 days. St36 group (50 ng/ml SCF, 50 ng/ml TPO, 20 ng/ml IL-3 and 50 ng/ml IL-6) yielded the most CD41/CD61 positive [(6.79±1.97)×10⁴]. The cell viability [(82.85 ± 0.64)%] of st36 group by using of imported serum-free medium was better than [(60.90±6.93)%] that in domestic medium on day 7 after induction, and CD41/CD61 positive cells count [(18.60±1.97)×10⁴] were more than domestic serum-free medium group. Therefore, we chose imported serum-free medium containing st36 to induce cord blood mononuclear cells. After a prolonged culture, the total cell numbers increased accompanied with an elevated percentage of CD41/CD61 positive cells, which reached (54.27 ± 6.31)% on day 14. Wright-Giemsa staining showed that different phase cells, such as megakaryoblast, promegakaryocyte and granular megakaryocyte, occurred after 10 days'culture. Clone forming unit-megakarocytes (CFU-MK) assay showed that the colonies count increased with the prolonged incubation. CFU-MK colonies were [1 236.0±32.9] on day 14, which was higher than that in medium without induction (P<0.01). Platelets from megakaryocytes showed agglutination function after 10 days'culture.

CONCLUSION1.5% HES was the best solution to precipitate erythrocytes. The combination of an imported serum-free medium with IL-3, IL-6, SCF and TPO showed better induction efficiency than domestic medium or other cytokine cocktails. Meanwhile, induced megakaryocytes produced functional platelets.

Cell Culture Techniques ; Cell Differentiation ; Cell Division ; Cell Separation ; methods ; Cells, Cultured ; Culture Media, Serum-Free ; Fetal Blood ; cytology ; Humans ; Megakaryocyte Progenitor Cells ; cytology