Objective To investigate cytokine levels in serum and culture medium of peripheral blood mononuclear cells(PBMC) from patients with hepatitis B virus(HBV)infection.Methods PBMCs isolated from fresh heparinized blood were cultured and stimulated with rHBcAg.After 72h at 37℃ 5% CO2 in air,the culture supernatant was collected.Levels of interferon-gamma(IFN-?) and interleukin(IL)-4 in blood serum in spontaneous and supernatant were measured by enzyme-linked immunosorbent assay(ELISA).Results Serum IFN-? levels in patients with acute self-limited hepatitis B(AH) and chronic hapetiti B(CHB) were significantly higher than those in normal control(NC)(P

Objective To investigate the effects of HBcAg-specific CD8+T cells on inhibiting HBV replication in vitro,and to search the cytokine of noncytolytic mechanisms in viral clearance. Methods By the method of coculture of HepG2.2.15 cell (target cells) with HLA-A2 matched HBcAg-specific CD8+T cell clone (effector cells) at E:T ratios of 1:50,and monitoring HBV production (HBsAg,HBeAg,and HBV-DNA)in coculture supernatants at 24h,48h and 72h,the percentage of decrease in HBV replication level was observed. Furthermore,blocking experiment with neutralizing mAbs to IFN-? was performed to evaluate the effect of this cytokine. Results CD8+T clone produced high levels of IFN-?following coculture with 2.2.15 cells. HBsAg,HBeAg and HBV-DNA in coculture supernatants were significantly reduced,and the greatest effect was observed at 72h by 54.55%,50.36% and 74.55%,respectively. The reduction of HBV DNA was decreased followed by using neutralizing mAbs to IFN-?. The maximum activity of cytotoxicity of target cells was at 24h by 15.66%. Conclusion ①HBV-specific CD8+T cells inhibit HBV replication by cytolytic and noncytolytic mechanisms.②The effect of noncytolytic mechanisms is mainly mediated by IFN-?.

Objective To generate HLA - A * 0201 and A * 2403 restricted HBcAg - specific cytotoxic T lymphocyte (CTL) clones. Methods Peripheral blood mononuclear cells (PBMCs) were derived from a HLA - A * 0201/2403 - positive patient with chron- ic hepatitis B . PBMCs were stimulated respectively using two synthetic peptides( HBc18 ～ 27 and HBc 117 ～ 125) , and epitope -specific CTL clones were generated by limiting dilution technique with PHA alone or combined with synthetic peptides. The cell clones were then characterized by IF staining,FCM and LDH release. Results After 2 weeks of in vitro stimulating PBMCs, specific CTL lines were established. 29 T clones were generated from those CTL lines using HBc18 ～27 stimulating. Among the 29 clones ,28 clones belonged to CD8~+ T cells and all displayed cytolytic activity. 12 CD8 +T clones were generated from those CTL lines using HBc117 ～ 125 stimulating. Specific cytotoxic activity was observed in 9 of those clones. The other three displayed less cytolytic activity. In the process of cloning, PHA was used alone or combined with synthetic peptides,and the achievement showed a rate of 15.62% and 14.58% . Conclusion HBc18 ～ 27 and HBc117 ～ 125 are capable of activating CD8~+ T cells in PBMCs of HLA - A * 0201 /2403 patient with chronic hepatitis B. In the process of CD8~+T cloning, synthetic peptides could not increase the rate of successful cloning.

Objectives: To express HBeAg in prokaryotic and eukaryotic cells and compare the two types of HBeAg in the anti-HBeAg testing. Methods: HBeAg was expressed both in E.coli cells and in silk worm cells, purified by Sephacryl S-200.HBeAg protein concentration and antigenic titer were determined respectively by ultraviolet-spectroscopy and EIA. Results: HBeAg produced by E.coli cells: Activation ratio was 10 000/mg, HBeAg/HBcAg = 50; The specificity in testing anti-HbeAg was 96%;HBeAg produced by silk worm cells: Activation ratio was 160 000/mg, HBeAg/HBcAg = 5 000, The specificity in testing anti-HbeAg was 100%. Conclusions: HBeAg produced by eukaryotic cells contained much lower proportion of HbcAg and higher activation ratio, which therefore bring about a possibility to improve the quality of the kit for testing Anti-HBe.

We have observed and studied the immune response, ultrastructure and phagocytosis of peritoneal macrophages (M?) of mice in protein deficiency by means of indirect fluorescent antibody test (IFAT), immunoenzymatic staining technique (IEST),fluorescence isothiocyanate antibody (FITC-Ab) quantitative assay, M? phagocytosis test, scanning electron microscopy (SEM) and im-munoelectron microscopy (IEM).The results showed that the body weight of mice was continuously declined after fed protein deficient diet. In the same time fluorescence reaction and enzyme stain on the M? surface was retarded. The amount of FITC-Ab on the M? membrane was decreased. The villi on the M? surface were shortened, the positive rates and positive degree of cells were lowered,the reaction of cell membrane and nuclear membrane was retarded in SEM and IEM.The phagocytic function of M? was inhibited.The results showed that in protein deficiency, the immune reaction, structure and function of peritoneal M? of mice were markedly affected.

OBJECTIVE: To explore the influencing factors of perioperative blood transfusion in the treatment of elderly femoral intertrochanteric fractures with proximal femoral nail antirotation(PFNA). METHODS: The clinical data of 109 elderly patients with intertrochanteric fractures who received PFNA treatment from July 2018 to January 2020 were retrospectively analyzed. Both pelvic hip X-rays and CT plain scans were performed before surgery. All patients were diagnosed by X-ray and CT plain scan of pelvis and hip before operation. Through the statistical analysis of the basic data of patients before and during operation, the risk factors of perioperative blood transfusion were explored. RESULTS: Logistic regression analysis showed that age ( CONCLUSION Age, fracture type, diabetes history, and preoperative hemoglobin are independent risk factors for perioperative blood transfusion in the treatment of elderly intertrochanteric fractures with PFNA. The older the patient, the history of diabetes, the more unstable the fracture, and the lower preoperative hemoglobin, the more likely it is to require a blood transfusion, which may provide a reference for clinical perioperative blood transfusion decisions.
Aged ; Blood Transfusion ; Bone Nails ; Fracture Fixation, Intramedullary/adverse effects* ; Hip Fractures/surgery* ; Humans ; Retrospective Studies ; Risk Factors ; Treatment Outcome

Objective:To investigate the pathogenic spectrum of children with severe infection by metagenomic next generation sequencing (mNGS).Methods:This study was a cross-sectional study. We collected 212 cases of severely infected pediatric patients admitted to the Intensive Care Unit (ICU) of the Women and Children′s Hospital of Ningbo University from January 2022 to June 2023, and performed metagenomic next-generation sequencing (mNGS) on 249 samples to analyze the pathogenic distribution characteristics.Results:Among the 249 samples of 212 children, the positive detection rate was 49.80% (124/249), including 14 cases of mixed infections, accounting for 6.60% (14/212). According to the mNGS technology, the pathogen distribution of severely infected children showed that the most common Gram-positive bacteria were Staphylococcus aureus (3.61%, 9/249), Streptococcus pneumoniae (2.81%, 7/249), and Staphylococcus epidermidis (2.41%, 6/249); the most common Gram-negative bacteria were Klebsiella aerogenes (2.41%, 6/249), Klebsiella pneumoniae (2.41%, 6/249), and Haemophilus parainfluenzae (2.01%, 5/249). The most common fungus was Candida parapsilosis (2.01%, 5/249). The most common virus was Human Cytomegalovirus (HCMV) (6.02%, 15/249), Human Herpesvirus 1 (HHV-1) (1.61%, 4/249), and Epstein-Barr virus (EBV) (1.61%, 4/249). The most common atypical pathogen was Mycoplasma pneumoniae (3.21%, 8/249). Conclusions:This study explored the pathogen spectrum in severely infected pediatric patients through mNGS, contributing to the diagnosis of mixed infections or infections caused by uncommon or rare pathogens, which enables rapid and efficient identification of pathogens.

OBJECTIVE To investigate the efficacy and safety of doxycycline in the treatment of macrolide-unresponsive Mycoplasma pneumoniae pneumonia （MUMPP） in children under 8 years of age. METHODS The medical records of children with MUMPP admitted to the Women and Children’s Hospital Affiliated to Ningbo University were collected from January 1st， 2023 to December 31st， 2023. They were divided into doxycycline group （44 cases）， doxycycline combined with methylprednisolone group （35 cases）， and azithromycin combined with methylprednisolone group （35 cases） according to the treatment methods. Doxycycline group was given Doxycycline hyclate enteric-coated capsules； doxycycline combined with methylprednisolone group was given Doxycycline hyclate enteric-coated capsules and Methylprednisolone sodium succinate for injection； azithromycin combined with methylprednisolone group was given Azithromycin for injection and Methylprednisolone sodium succinate for injection. Treatment courses of 3 groups lasted for 10 d. The fever reduction rate， the time of fever reduction and improvement rate of lung infection absorption were compared among the three groups. The occurrence of adverse drug reactions was recorded during their hospitalization and followed up within 5 months after discharge. RESULTS The fever reduction rats 48， 72 h after treatment and improvement rate of lung infection absorption in doxycycline group and doxycycline combined with methylprednisolone group were significantly higher than azithromycin combined with methylprednisolone group； the time of fever reduction was significantly shorter than azithromycin combined with methylprednisolone group （P＜0.05）； there was no statistical significance in the difference between the doxycycline group and the doxycycline combined with methylprednisolone group （P＞0.05）. There was no statistical significance in the incidence of rash， vomiting， abdominal pain， diarrhea， and elevated transaminases among the three groups during hospitalization and within 5 months after discharge （P＞0.05）. None of the children treated with doxycycline suffered from tooth discoloration or enamel hypoplasia. CONCLUSIONS Doxycycline has good efficacy and safety in therapy of MUMPP in children under 8 years of age； adjunctive coadministration of low-dose glucocorticoids does not necessarily result in significant additional efficacy.

Objective:To explore potential predictors of refractory Mycoplasma pneumoniae pneumonia (RMPP) in early stage. Methods:The prospective multicenter study was conducted in Zhejiang, China from May 1 st, 2019 to January 31 st, 2020. A total of 1 428 patients with fever >48 hours to <120 hours were studied. Their clinical data and oral pharyngeal swab samples were collected; Mycoplasma pneumoniae DNA in pharyngeal swab specimens was detected. Patients with positive Mycoplasma pneumoniae DNA results underwent a series of tests, including chest X-ray, complete blood count, C-reactive protein, lactate dehydrogenase (LDH), and procalcitonin. According to the occurrence of RMPP, the patients were divided into two groups, RMPP group and general Mycoplasma pneumoniae pneumonia (GMPP) group. Measurement data between the 2 groups were compared using Mann-Whitney U test. Logistic regression analyses were used to examine the associations between clinical data and RMPP. Receiver operating characteristic (ROC) curves were used to analyse the power of the markers for predicting RMPP. Results:A total of 1 428 patients finished the study, with 801 boys and 627 girls, aged 4.3 (2.7, 6.3) years. Mycoplasma pneumoniae DNA was positive in 534 cases (37.4%), of whom 446 cases (83.5%) were diagnosed with Mycoplasma pneumoniae pneumonia, including 251 boys and 195 girls, aged 5.2 (3.3, 6.9) years. Macrolides-resistant variation was positive in 410 cases (91.9%). Fifty-five cases were with RMPP, 391 cases with GMPP. The peak body temperature before the first visit and LDH levels in RMPP patients were higher than that in GMPP patients (39.6 (39.1, 40.0) vs. 39.2 (38.9, 39.7) ℃, 333 (279, 392) vs. 311 (259, 359) U/L, both P<0.05). Logistic regression showed the prediction probability π=exp (-29.7+0.667×Peak body temperature (℃)+0.004×LDH (U/L))/(1+exp (-29.7+0.667×Peak body temperature (℃)+0.004 × LDH (U/L))), the cut-off value to predict RMPP was 0.12, with a consensus of probability forecast of 0.89, sensitivity of 0.89, and specificity of 0.67; and the area under ROC curve was 0.682 (95% CI 0.593-0.771, P<0.01). Conclusion:In MPP patients with fever over 48 to <120 hours, a prediction probability π of RMPP can be calculated based on the peak body temperature and LDH level before the first visit, which can facilitate early identification of RMPP.