[A retrospective analysis of medication in children with SARS-CoV-2 infection in Wuhan, China].

OBJECTIVE: To study the medication in children with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in Wuhan, China, and to provide a reference for rational drug use in clinical practice. METHODS: A retrospective analysis was performed on the medical data of the children who were diagnosed with SARS-CoV-2 infection from January 26 to March 5, 2020. The children were divided into an asymptomatic group with 41 children and a symptomatic group with 73 children. A subgroup analysis was performed to investigate the effect of different antiviral regimens (monotherapy, double therapy, or triple therapy) and whether interferon α-1b was used in combination with azithromycin on the length of hospital stay and the clearance time of SARS-CoV-2 nucleic acid. RESULTS: A total of 114 children with SARS-CoV-2 infection (72 boys and 42 girls) were enrolled. The median age of the children was 7.1 years. The median length of hospital stay was 10 days and the clearance time of SARS-CoV-2 nucleic acid was 6 days. In either group, the subgroup analysis showed no significance differences in the length of hospital stay and the clearance time of SARS-CoV-2 nucleic acid between the subgroups treated with different combinations of antiviral drugs and the subgroups treated with interferon α-1b alone or in combination with azithromycin (P > 0.05). CONCLUSIONS: It is not recommended to use the routine combinations of antiviral drugs for children with SARS-COV-2 infection or combine with azithromycin for the purpose of antiviral therapy.

Risk factors and clinical characteristics of tacrolimus-induced acute nephrotoxicity in children with nephrotic syndrome: a retrospective case-control study.

PURPOSE: Acute nephrotoxicity is a common adverse reaction of tacrolimus therapy; however, its risk factors in pediatric nephrotic syndrome (NS) remain to be evaluated. The objective of this study was to investigate the risk factors and characteristics of tacrolimus-induced acute nephrotoxicity in children with NS. METHODS: Past records of children with NS admitted to our hospital from 2014 to 2018 were reviewed. The incidence and characteristics of nephrotoxicity were analyzed. Multivariate logistic regression analysis was used to identify the risk factors of nephrotoxicity. A clinically applicable risk score was developed and validated. RESULTS: Tacrolimus-induced nephrotoxicity occurred in 25 of 129 patients, 13 patients were grade 1, and the renal function was recovered in 22 patients. Multivariate regression analysis showed that the maximum trough concentrations (C12h) of tacrolimus (OR, 1.48; 95% CI, 1.16 to 1.88; P < 0.001), huaiqihuang granules (OR, 0.095; 95% CI, 0.014 to 0.66; P = 0.017), and diarrhea (OR, 22.00; 95% CI, 1.58 to 306.92; P = 0.022) were independently associated with tacrolimus-induced nephrotoxicity. The maximum C12h were significantly higher in patients with nephrotoxicity (median 9.0 ng/ml) and the cut-off value for acute nephrotoxicity was 6.5 ng/ml. The area under the receiver operating characteristic curve was 0.821 for the proposed model based on the observations used to create the model and 0.817 obtained from k-fold cross-validation. CONCLUSIONS: High trough concentration of tacrolimus and diarrhea can potentiate the risk of tacrolimus-induced acute nephrotoxicity in children with NS, while huaiqihuang granules can protect this condition.

Population pharmacokinetics modeling of oxcarbazepine to characterize drug interactions in Chinese children with epilepsy.

AIM: To develop a population pharmacokinetics model of oxcarbazepine in Chinese pediatric patients with epilepsy, and to study the interactions between oxcarbazepine and other antiepileptic drugs (AEDs). METHODS: A total of 688 patients with epilepsy aged 2 months to 18 years were divided into model (n=573) and valid (n=115) groups. Serum concentrations of the main active metabolite of oxcarbazepine, 10-hydroxycarbazepine (MHD), were determined 0.5-48 h after the last dosage. A population pharmacokinetics (PPK) model was constructed using NLME software. This model was internally evaluated using Bootstrapping and goodness-of-fit plots inspection. The data of the valid group were used to calculate the mean prediction error (MPE), mean absolute prediction error (MAE), mean squared prediction error (MSE) and the 95% confidence intervals (95% CI) to externally evaluate the model. RESULTS: The population values of pharmacokinetic parameters estimated in the final model were as follows: Ka=0.83 h-1, Vd=0.67 L/kg, and CL=0.035 L·kg(-1)·h(-1). The enzyme-inducing AEDs (carbamazepine, phenytoin, phenobarbital) and newer generation AEDs (levetiracetam, lamotrigine, topiramate) increased the weight-normalized CL value of MHD by 17.4% and 10.5%, respectively, whereas the enzyme-inhibiting AED valproic acid decreased it by 3%. No significant association was found between the CL value of MHD and the other covariates. For the final model, the evaluation results (95% CI) were MPE=0.01 (-0.07-0.10) mg/L, MAE=0.46 (0.40-0.51) mg/L, MSE=0.39 (0.27-0.51) (mg/L)(2). CONCLUSION: A PPK model of OXC in Chinese pediatric patients with epilepsy is established. The enzyme-inducing AEDs and some newer generation AEDs (lamotrigine, topiramate) could slightly increase the metabolism of MHD.

[The effect of stimulation of dendritic cell on HBV-epitopic cytotoxic T-lymphocyte].

OBJECTIVE: To observe the enhancement of cytotoxic T lymphocyte responses in patients with chronic hepatitis B following vaccination with dendritic cell stimulated by Poly (I:C) in vitro. METHODS: Peripheral blood mononuclear cells (PBMC) were isolated from whole blood by density gradient centrifugation on Ficoll-Hypaque, the adherent cells were cultured in the medium AIM-V contained recombinant human IL-4 and recombinant human GM-CSF. On day 7, one part of wells were added with Poly (I:C). On day 9, mature DCs (mDCs) were harvested and used to phenotype analysis. Both of the immature DCs and mature DCs were concultured with the autologous T cells for another two-three days. According to the source of dendritic cells concultured with the T cells, the subjects were divided into three groups: the T cells isolated from CHB group; the T cells stimulated by dendritic cells pulsed with HBcAg18-27 CTL epitope peptide group; the T cells stimulated by dendritic cells concultured with Poly (I:C) and pulsed with HBcAg18-27 CTL epitope peptide group. The function and frequency of HBV specific CTL were detected by Elispot (Enzyme-linked immunospot assay, Elispot) and tetramer staining. RESULTS: The average of percentage of HBV specific CD8(+) cells of total CD8(+) cells was 0.77% (0.45% approximately 1.74%) in the T cells isolated from 22 chronic hepatitis B patients and the average of the spots of antigen-specific IFN-gamma-releasing effector cells was 16 (9 approximately 28); The percentage of HBV specific CD8(+) cells of the T cells stimulated by dendritic cells pulsed with HBcAg18-27 CTL epitope peptide group rose to 1.92% (1.36% approximately 2.65%) and the average of the spots of antigen-specific IFN-gamma-releasing effector cells was 46 (30 approximately 67); while stimulated by dendritic cells added with Poly (I:C) and pulsed with HBcAg18-27 CTL epitope peptide, the percentage of HBV specific CD8(+) cells of total CD8(+) cells rose to 3.49% (2.02% approximately 4.60%) and the average of the spots was 98 (59 approximately 130). There was statistical difference between the results of Elispot and tetramer of the three groups (P < 0.01). CONCLUSION: The T lymphocyte of patients with chronic hepatitis B stimulated by the autologous dendritic cells may result in obtaining high percentage and functional antigen-specific T lymphocyte. The addition of Poly (I:C) which was used as maturation promoting factor in DC culture can enhance the function of DC significantly and can get even higher percentage antigen-specific T lymphocyte with enhanced function.

[Dynamic expression of hepatitis B virus covalently closed circular DNA in 2.2.15 cell].

BACKGROUND: To determine the presence of covalently closed circular DNA (cccDNA), and to investigate the expression kinetics of HBV DNA, HBsAg and HBeAg in 2.2.15 cell. METHODS: HBV cccDNA was assessed by polymerase chain reaction, HBV DNA was measured by Taqman quantitative PCR and HBsAg and HBeAg was measured by EIA. RESULTS: HBV cccDNA was found in both intracellular and extracellular space. There was a good correlation between HBsAg, HBeAg and HBV DNA in the supernatant of 2.2.15 cell (r= 0.833, P < 0.05 and r= 0.939, P < 0.01 for HBsAg and HBeAg, respectively), whereas there was no significant correlation between intracellular HBV DNA levels and virus antigen levels (r= 0.024, P= 0.955 and r= 0.177; P= 0.625 for HBsAg and HBeAg, respectively). CONCLUSION: HBV cccDNA was detectable in the culture medium and intracellularly in 2.2.15 cells, and these data provided an indication of HBV replication in 2.2.15 cell.

Cloning, expression, and purification of HLA-A2-BSP and beta-2m in Escherichia coli.

Tetramer analysis is a novel technique in immunological research that has dramatically changed our knowledge of the immune response to pathogens, tumors and autoimmune disease. Through the formation of major histocompatibility complex (MHC)-peptide tetrameric complexes, it can provide accurate counts of antigen-specific T-cells and it allows their phenotypical and functional analysis. The tetramer is composed of the human leukocyte antigen (HLA) heavy chain, beta-2 microglobulin (beta-2m), the nominal peptide, and streptavidin. The HLA heavy chain and the beta-2m are expressed in Escherichia coli. But up to now, all laboratories have been expressing these two proteins by using isopropyl beta-d-thiogalactopyranoside IPTG. IPTG is very expensive, and it is tedious and laborious to induce expression protein. So it is difficult to scale up to express the objective protein. To address this problem, extracellular fractions of HLA-A0201 and beta-2m (absent signal peptide) genes were cloned from peripheral blood mononuclear cells (PBMCs) by RT-PCR. DNA coding for a Gly-Ser linker and a BSP (15-amino acid substrate peptide for BirA-dependent biotinylation) was added to the COOH-terminus of the extracellular fraction of HLA-A0201 by PCR, using an HLA-A0201 as the template. Then the HLA-A0201-BSP and beta-2m genes were cloned into pBV220 vector and expressed, respectively. The expressed proteins were purified and detected by ELISA and Western blot analyses. High-efficient expressions of HLA-A0201-BSP and beta-2m proteins lay a good foundation for further expression and purification in prokaryotic system and constructing MHC class I-peptide tetramer complexes to study the function of CTLs.