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Background: To promote the shared decision-making (SDM) between patients and doctors in pediatric outpatient departments, this study was designed to validate artificial intelligence (AI) -initiated medical tests for children with fever., Methods: We designed an AI model, named Xiaoyi, to suggest necessary tests for a febrile child before visiting a pediatric outpatient clinic. We calculated the sensitivity, specificity, and F1 score to evaluate the efficacy of Xiaoyi's recommendations. The patients were divided into the rejection and acceptance groups. Then we analyzed the rejected examination items in order to obtain the corresponding reasons., Results: We recruited a total of 11,867 children with fever who had used Xiaoyi in outpatient clinics. The recommended examinations given by Xiaoyi for 10,636 (89.6%) patients were qualified. The average F1 score reached 0.94. A total of 58.4% of the patients accepted Xiaoyi's suggestions (acceptance group), and 41.6% refused (rejection group). Imaging examinations were rejected by most patients (46.7%). The tests being time-consuming were rejected by 2,133 patients (43.2%), including rejecting pathogen studies in 1,347 patients (68.5%) and image studies in 732 patients (31.8%). The difficulty of sampling was the main reason for rejecting routine tests (41.9%)., Conclusion: Our model has high accuracy and acceptability in recommending medical tests to febrile pediatric patients, and is worth promoting in facilitating SDM., Competing Interests: Conflicts of interest: The authors declared no conflicts of interest., (Copyright: © World Journal of Emergency Medicine.)

Competing Interests: Conflicts of interests: The authors have no competing interests.

Sepsis is defined as life-threatening organ dysfunction caused by dysregulated host systemic inflammatory and immune response to infection. Over decades, advanced understanding of host-microorganism interaction has gradually unmasked the genuine nature of sepsis, guiding toward new definition and novel therapeutic approaches. Diverse clinical manifestations and outcomes among infectious patients have suggested the heterogeneity of immunopathology, while systemic inflammatory responses and deteriorating organ function observed in critically ill patients imply the extensively hyperactivated cascades by the host defense system. From focusing on microorganism pathogenicity, research interests have turned toward the molecular basis of host responses. Though progress has been made regarding recognition and management of clinical sepsis, incidence and mortality rate remain high. Furthermore, clinical trials of therapeutics have failed to obtain promising results. As far as we know, there was no systematic review addressing sepsis-related molecular signaling pathways and intervention therapy in literature. Increasing studies have succeeded to confirm novel functions of involved signaling pathways and comment on efficacy of intervention therapies amid sepsis. However, few of these studies attempt to elucidate the underlining mechanism in progression of sepsis, while other failed to integrate preliminary findings and describe in a broader view. This review focuses on the important signaling pathways, potential molecular mechanism, and pathway-associated therapy in sepsis. Host-derived molecules interacting with activated cells possess pivotal role for sepsis pathogenesis by dynamic regulation of signaling pathways. Cross-talk and functions of these molecules are also discussed in detail. Lastly, potential novel therapeutic strategies precisely targeting on signaling pathways and molecules are mentioned., (© 2021. The Author(s).)

Artificial intelligence (AI) has been deeply applied in the medical field and has shown broad application prospects. Pre-consultation system is an important supplement to the traditional face-to-face consultation. The combination of the AI and the pre-consultation system can help to raise the efficiency of the clinical work. However, it is still challenging for the AI to analyze and process the complicated electronic health record (EHR) data. Our pre-consultation system uses an automated natural language processing (NLP) system to communicate with the patients through the mobile terminals, applying the deep learning (DL) techniques to extract the symptomatic information, and finally outputs the structured electronic medical records. From November 2019 to May 2020, a total of 2,648 pediatric patients used our model to provide their medical history and get the primary diagnosis before visiting the physicians in the outpatient department of the Shanghai Children's Medical Center. Our task is to evaluate the ability of the AI and doctors to obtain the primary diagnosis and to analyze the effect of the consistency between the medical history described by our model and the physicians on the diagnostic performance. The results showed that if we do not consider whether the medical history recorded by the AI and doctors was consistent or not, our model performed worse compared to the physicians and had a lower average F1 score (0.825 vs. 0.912). However, when the chief complaint or the history of present illness described by the AI and doctors was consistent, our model had a higher average F1 score and was closer to the doctors. Finally, when the AI had the same diagnostic conditions with doctors, our model achieved a higher average F1 score (0.931) compared to the physicians (0.92). This study demonstrated that our model could obtain a more structured medical history and had a good diagnostic logic, which would help to improve the diagnostic accuracy of the outpatient doctors and reduce the misdiagnosis and missed diagnosis. But, our model still needs a good deal of training to obtain more accurate symptomatic information., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Qian, Dong, Yuan, Yin, Wang, Wang, Wang, Tian, Li, Zhang, Zhao and Ning.)

In order to explore the clinical characteristics of pediatric patients admitted to the pediatric intensive care unit (PICU) who suffered from hematological neoplasms complicated with acute respiratory distress syndrome (ARDS), we retrospectively analyzed 45 ARDS children with hematological neoplasms who were admitted to the PICU of Shanghai Children's Medical Center from January 1, 2014, to December 31, 2020. The 45 children were divided into a survival group and a non-survival group, a pulmonary ARDS group and an exogenous pulmonary ARDS group, and an agranulocytosis group and a non-agranulocytosis group, for statistical analysis. The main clinical manifestations were fever, cough, progressive dyspnea, and hypoxemia; 55.6% (25/45) of the children had multiple organ dysfunction syndrome (MODS). The overall mortality rate was 55.6% (25/45). The vasoactive inotropic score (VIS), pediatric critical illness scoring (PCIS), average fluid volume in the first 3 days and the first 7 days, and the incidence of MODS in the non-survival group were all significantly higher than those in the survival group ( P < 0.05). However, total length of mechanical ventilation and length of hospital stay and PICU days in the non-survival group were significantly lower than those in the survival group ( P < 0.05). The PCIS (OR = 0.832, P = 0.004) and the average fluid volume in the first 3 days (OR = 1.092, P = 0.025) were independent risk factors for predicting death. Children with exogenous pulmonary ARDS were more likely to have MODS than pulmonary ARDS ( P < 0.05). The mean values of VIS, C-reactive protein (CRP), and procalcitonin (PCT) in children with exogenous pulmonary ARDS were also higher ( P < 0.05). After multivariate analysis, PCT was independently related to exogenous pulmonary ARDS. The total length of hospital stay, peak inspiratory pressure (PIP), VIS, CRP, and PCT in the agranulocytosis group were significantly higher than those in the non-agranulocytosis group ( P < 0.05). Last, CRP and PIP were independently related to agranulocytosis. In conclusion, children with hematological neoplasms complicated with ARDS had a high overall mortality and poor prognosis. Children complicated with MODS, positive fluid balance, and high VIS and PCIS scores were positively correlated with mortality. In particular, PCIS score and average fluid volume in the first 3 days were independent risk factors for predicting death. Children with exogenous pulmonary ARDS and children with agranulocytosis were in a severely infected status and more critically ill., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Zhang, Hu, Yin, Qian, Wang, Li, Qian, Tang and Ning.)

Objective: To investigate the immunity markers related to nosocomial infection in children with sepsis. Methods: A retrospective study including 155 cases diagnosed as sepsis from September 2015 to June 2020 in children's intensive care unit (PICU) of Shanghai Children's Medical Center was conducted. According to the presence of nosocomial infection occurred in PICU, septic children were divided into two groups: no nosocomial infection and nosocomial infection group. The differences about helper T-cells 1 and 2 cytokines, T cells subgroup absolute count, the proportion of CD14 + human leukocyte antigen DR (CD14 + HLA-DR), the proportion of regulatory T cells, pediatric risk of mortality Ⅲ (PRISM-Ⅲ), the treatment and outcome between the two groups were compared. Through propensity score matching (PSM), the disease severity and treatment of the two groups were matched to analyze the differences between the above indicators. Chi-square test or U test was used for comparison between groups. Receiver operating characteristic (ROC) curve was used to predict the occurrence of nosocomial infection. Results: There were 104 cases in no nosocomial infection group and 51 cases in nosocomial infection group. The first PICU-acquired infections occurred at (12±7) days after PICU admission. The most common PICU-acquired infections were pneumonia (26 cases, 51.0%) and bloodstream infections (15 cases, 29.4%). PRISM-Ⅲ of nosocomial infection group was significantly higher than that in no nosocomial infection group (8 (0-31) vs. 4 (0-17), Z =3 913.00, P <0.01).The proportion of using vasoactive drugs and invasive mechanical ventilation of nosocomial infection group was significantly higher (35.3% (18/51) vs. 10.6% (11/104), χ²=13.77, P <0.01; 86.3% (44/51) vs. 38.5% (40/104), χ²=31.51, P <0.01).The PICU length of stay of nosocomial infection group was significantly longer (20 (3-94) vs. 7 (2-41) days, Z =4 585.50, P <0.01). The mortality of the nosocomial infection group was significantly higher than that of the group without nosocomial infection (29.4% (15/51) vs. 6.7% (7/104), χ²=14.45, P <0.01). Interleukin-6 and interleukin-10 of the nosocomial infection group were significantly higher than that in no nosocomial infection group (37.83 (2.23-7 209.99) vs. 13.45 (0.80~50 580.64) ng/L, Z =3 390.50, P =0.01; 10.42 (1.11-6 052.21) vs. 4.10 (0.16-409.28) ng/L, Z =3 212.00, P =0.03). CD4 + /CD8 + and the percentage of CD14 + HLA-DR were significantly lower in the nosocomial infection group compared with the no nosocomial infection group (1.16 (0.44-4.96) vs. 1.61 (0.15-6.37), Z =1 955.00, P =0.01; 0.48 (0.08-0.99) vs. 0.67 (0.09-0.98), Z =1 915.50, P <0.01). After PSM, the percentage of CD14 + HLA-DR of nosocomial infection group was significantly lower than that in no nosocomial infection group (0.44 (0.08-0.99) vs. 0.64 (0.09-0.98), Z =758.00, P =0.02). The ROC curve analysis of the percentage of CD14 + HLA-DR in predicting nosocomial infection showed that the area under the curve was 0.642, the cut-off value was 0.39, and the 95% CI was 0.528-0.755. Conclusion: The level of the percentage of CD14 + HLA-DR maybe is related to the occurrence of nosocomial infection in children with sepsis.

Background: Severe sepsis/septic shock with severe neutropenia often leads to poor prognosis. However, it is unknown if severe neutropenia is associated with different clinical outcomes and biomarker features in severe sepsis/septic patients., Methods: This retrospective cohort study enrolled 141 severe sepsis/septic shock patients admitted to intensive care unit of Shanghai Children's Medical Center between January 2015 and November 2019. Patients were followed up for the development of ventilation support, the use of vasoactive drugs, continuous renal replacement therapy (CRRT) procedure, and mortality. Biomarkers that reflect the level of inflammation in severe sepsis/septic shock patients with neutropenia were compared to that in patients without neutropenia., Results: Of 141 patients enrolled, 54 patients suffered from severe sepsis/septic shock with severe neutropenia. In patients with severe sepsis/septic shock, severe neutropenia as a complication was an independent risk factor for the use of vasoactive drugs (RR 9.796; 95% CI: 3.774, 25.429; P<0.001), but not for ventilation support (RR 0.157; 95% CI: 0.06, 0.414; P<0.001), CRRT procedure (RR 1.032; 95% CI: 0.359, 2.969; P=0.953) or 28-day mortality (RR 1.405; 95% CI: 0.533, 3.708; P=0.492). Severe sepsis/septic patients with severe neutropenia had a higher plasma level of the following biomarkers: c-reaction protein (CRP) (180.5 vs. 121 mg/mL, P<0.001), procalcitonin (PCT) (12.15 vs. 2.7 ng/mL; P=0.005), interleukin (IL)-6 (316.83 vs. 55.77 pg/mL, P<0.001), IL-10 (39.165 vs. 10.09 pg/mL, P<0.001), interferon (IFN)-γ (6.155 vs. 3.71 pg/mL, P=0.016), and the percentage of regulatory T cells (Tregs) (2.7% vs. 2.09%, P=0.003). Based on the receiver operating characteristic curves, IL-10 exhibited high specificity (79.4%) in evaluating the prognosis of septic patients with neutropenia., Conclusions: In patients with severe sepsis/septic shock, being complicated with severe neutropenia is associated with higher proportion of using vasoactive drugs, and those patients tend to have higher plasma levels of IL-6, IL-10, IFN-γ and percentage of Treg., Competing Interests: Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at http://dx.doi.org/10.21037/tp-20-230). The authors have no conflicts of interest to declare., (2021 Translational Pediatrics. All rights reserved.)

Introduction: The aim of this study was to present our 10-year experience of pediatric intensive care unit (PICU) management with pediatric liver recipients and to understand the importance of close interdisciplinary cooperation in 2 hospitals., Methods: A retrospective chart review study was performed according to our hospital's medical records and the pediatric liver transplant database of Renji hospital., Results: A total of 767 patients received liver transplantation (LT) performed in Renji hospital between October 2006 and December 2016, of which 97 of them were admitted to PICU in our center for various complications developed after transplantation. 8.8% (16/208) and 14.4% (81/559) of patients were transferred to PICU in stages I and II, respectively, and was comparable in the 2 stages ( P = .017). The majority of patients was late postoperative children (median 185 post-LT days) in stage I. More patients were transferred to PICU directly in stage II. PICU admitted more younger (median 8.2 months) and early postoperative patients in stage II. The median length of PICU stay was 11.0 (6.0-20.5) days. The median length of mechanical ventilation was 5.0 (0.0-12.0) days. The most frequent complications were pulmonary complications (52 [53.6%] patients), surgical complications (22 [22.7%] patients), sepsis (7 [7.2%]), and other miscellaneous complications (16 [16.5%] patients). The overall 28-day PICU mortality was 25.8% (n = 25) and 64.0% (n = 16) of the deaths happened in the early postoperative period. There was significant difference concerning mortality in children with surgical complications and medical problems (54.5% [12/22] vs 17.3% [13/75], P = .001). Multivariate analysis by regression showed that the pediatric risk of mortality III score was the only independent prognostic factor ( P = .031)., Conclusions: Multiple complications occur in children with LT. Although challenging, interdisciplinary cooperation between different hospitals is an effective mean to enable children to maximize the benefit gained from LT in China.

Background: Asparaginase-associated pancreatitis (AAP) is one of the most common complications occurring in patients with asparaginase-treated acute lymphoblastic leukemia (ALL). Peg-asparaginase (peg-asp), a chemically recombined asparaginase with lower hyposensitivity and better patient tolerance, is now approved as the first line asparaginase formulation in ALL chemotherapy regimens. Due to the differences in pharmacokinetic characteristics and administration procedure between l-asp and peg-asp, this study aimed to investigate the clinical manifestations of peg-asp-associated pancreatitis., Method: Patients with peg-asp-associated pancreatitis diagnosed within a 5-year period (July 2014 to July 2019) were identified and retrospectively studied. The clinical manifestations, laboratory findings, and imaging results of patients with AAP were analyzed. AAP patients were further classified into mild/moderate and severe groups based on criteria used in previous studies. Clinical outcomes were compared between groups., Results: A total of 38 patients were enrolled in this study. The underlying disease included ALL (n=35) and lymphoma (n=3). The majority of patients developed AAP during the first phase, called remission induction (n=26, 68.4%), after a median of 2 peg-asp doses (range: 1-11). The DVLP regimen (n=23) is the most common peg-asp regimen used in AAP patients. Abdominal pain occurred after a median of 14.5 days (range: 1-50) from the last peg-asp administration, accompanied by abdominal distension (n=14), nausea (n=17), vomiting (n=21), and fever (n=19). Serum amylase elevation was reported in all AAP patients, of whom 65.8% (n=25) exhibited an elevation in the level of this enzyme three times the upper normal level, fulfilling the Atlanta criteria. The level of serum lipase (median days of elevation=23 days, range: 4-75) was significantly elevated compared with that of serum amylase (median days of elevation=9 days, range: 2-71) and persisted at a markedly high level after the level of serum amylase returned to normal. Common local complications included abdominal ascites (n=10) and peripancreatic fluid collection (n=8). Approximately 42.1% (n=16) of patients with severe AAP experienced systemic complications (septic shock or hypovolemic shock) or severe local complications (pseudocyst), among whom 5 failed to recover. Approximately 84.8% (n=28/33) of the remaining patients resumed chemotherapy; among them, peg-asp formulation in 30.3% (n=10/33) of these patients was adjusted, while asparaginase treatment in 39.4% (n=13/33) was permanently discontinued. Five patients experienced an AAP relapse in later stages of asparaginase treatment. Comparison between mild/moderate and severe AAP patients showed a statistically significant difference in the number of pediatric intensive care unit stays (p=0.047), survival rate (p=0.009), AAP prognosis (p=0.047), and impacts on chemotherapy (p=0.024), revealing a better clinical outcome in mild/moderate AAP patients., Conclusion: Early recognition and management of AAP is essential in reversing the severity of AAP. The existing AAP criteria had a low strength in determining the severity of pediatric AAP. A well-defined AAP definition could help distinguish patients with high anticipated risk for redeveloping AAP and ALL relapse, in order to prevent unnecessary withdrawal of asparaginase. Our study could serve as a basis for conducting future large cohort studies and for establishing an accurate definition of pediatric AAP., (Copyright © 2020 Zhang, Yang, Qing, Li, Qian, Wang and Ning.)

Immune dysfunction and aberrant cytokine storms often lead to rapid exacerbation of the disease during late infection stages in SARS-CoV and MERS-CoV patients. However, the underlying immunopathology mechanisms are not fully understood, and there has been little progress in research regarding the development of vaccines, anti-viral drugs, and immunotherapy. The newly discovered SARS-CoV-2 (2019-nCoV) is responsible for the third coronavirus pandemic in the human population, and this virus exhibits enhanced pathogenicity and transmissibility. SARS-CoV-2 is highly genetically homologous to SARS-CoV, and infection may result in a similar clinical disease (COVID-19). In this review, we provide detailed knowledge of the pathogenesis and immunological characteristics of SARS and MERS, and we present recent findings regarding the clinical features and potential immunopathogenesis of COVID-19. Host immunological characteristics of these three infections are summarised and compared. We aim to provide insights and scientific evidence regarding the pathogenesis of COVID-19 and therapeutic strategies targeting this disease., (Copyright © 2020 Zhang, Li and Ning.)

Objective: To investigate the sedation weaning strategies in critically ill patients with mechanical ventilation in pediatric intensive care unit (PICU) and to explore the effect of different sedative weaning patterns on withdrawal syndrome. Methods: A single-center prospective cohort study was conducted from April 1, 2016 to April 30, 2017. One hundred and twelve patients who required mechanical ventilation and benzodiazepines and (or) opioids for at least 5 consecutive days in PICU of Shanghai Children's Medical Center were enrolled. Twenty patients (17.9%) had an intermittent weaning pattern, defined as a 50% or greater increase in daily benzodiazepine and (or) opioid dose after the start of weaning, and the remaining 92 cases (82.1%) had a steady weaning pattern. The demographic and clinical features, duration and dose of sedative and analgesics, and the incidence of withdrawal syndrome were evaluated. Mann-Whitney U test was used for comparison about clinical features between different weaning pattern groups and children with withdrawal syndrome or not. Logistic regression was used to explore the risk factors of withdrawal syndrome. Results: Among the 112 patients, 46 (41.1%) had withdrawal syndrome. The patients with the intermittent weaning pattern had a high score of pediatric risk of mortality Ⅲ (PRISM-Ⅲ) (10.0 (3.5, 12.0) vs. 6.0 (2.0, 10.0), U= 654.50, P= 0.043) and were prone to re-intubation (35.0% (7/20) vs. 7.6% (7/92), P= 0.003). The patients with withdrawal syndrome had longer duration of sedation (19.5 (16.8, 24.3) vs. 10.0 (7.0, 17.3) days, U= 743.50, P< 0.01), higher incidence of intermittent weaning pattern (32.6% (15/46) vs. 7.6% (5/66),χ(2)=11.58, P= 0.001), longer PICU hospitalization (19.0 (15.8, 25.3) vs. 12.0 (8.8, 17.0) days, U= 755.00, P< 0.01) and higher cost (89 (57,109) vs. 53 (32, 79) thousand yuan, U= 804.00, P< 0.01). Logistic regression showed that intermittent weaning pattern (odds ratio ( OR ) = 4.85, 95 % confidence interval ( CI ) 1.39-16.91, P= 0.013), perioperative period of liver transplantation ( OR= 6.97, 95 %CI 1.25-39.04, P= 0.027) and a cumulative dose of midazolam ≥ 34.7 mg/kg ( OR= 8.12, 95 %CI 3.09-21.37, P< 0.01) were risk factors of withdrawal syndrome. Conclusions: Withdrawal syndrome is more likely to occur in children who are intermittently weaned from sedation. Steady weaning strategy may help prevent iatrogenic withdrawal syndrome.

Objective: To investigate the safety, feasibility and operation key points of whole lung lavage in infants with pulmonary alveolar proteinosis. Methods: The clinical manifestations, genetic screening, therapeutic interventions and outcome of an infant with pulmonary alveolar proteinosis complicated with respiratory failure who received whole lung lavage in November 2018 in Shanghai Children's Medical Center Affiliated to Shanghai Jiaotong University School of Medicine were reported. Websites including PubMed, Springer Link, China National Knowledge Infrastructure (CNKI), Weipu Database, and Wanfang Database were searched using the key words of "whole lung lavage" "pediatric" and "pulmonary alveolar proteinosis" for articles published from their establishments to April 2019. Relevant literature was reviewed. Results: A 3-month-old boy had experienced cough, shortness of breath and cyanosis for 1 week prior to admission to pediatric intensive care unit. Physical examination showed hepatosplenomegaly. Complete blood cell count showed mild anemia (hemoglobin 96 g/L) and normal white blood cells. The patient had normal C-reactive protein and normal blood platelet. Biochemical panel showed hypoalbuminemia (31 g/L), mildly elevated glutamic oxaloacetic transaminase (115 U/L) and blood ammonia (165 μmol/L), extremely elevated lactate dehydrogenase (>6 600 U/L) and hyperferritinemia (>4 500 μg/L). Chest computed tomography (CT) revealed decreased transmittance of both lungs, patchy high density shadow and ground glass opacity. Genetic testing revealed a mutation of c.625+1G>A in SLC7A7. Schiff reaction (PAS staining) in bronchoalveolar lavage fluid was positive. The patient was diagnosed with severe pneumonia, respiratory failure, lysinuria urinary protein intolerance, and pulmonary alveolar proteinosis. The patient received sequential unilateral whole lung lavage in 2 days and was successfully weaned from ventilator. He was discharged home breathing room air. Eleven articles (11 in English and non in Chinese) were reviewed. Twenty-one patients were included. After whole lung lavage, 76% (16/21) of the patients had improvement in respiratory function. Conclusions: Whole lung lavage can effectively improve respiratory failure caused by pulmonary alveolar proteinosis in infant patients. The procedure is feasible and safe.

Purpose Neuroblastoma is an embryonic solid tumor derived from the progenitors of the sympathetic nervous system. More than half of the patients developed metastatic disease at the time of initial diagnosis and had poor outcome with current therapeutic approaches. In recent years, some obligate and facultative anaerobic bacteria were reported to target the hypoxic and necrotic region of solid tumor models and caused tumor regression. We recently successfully constructed an "obligate" anaerobic Salmonella strain YB1 that was applied in breast cancer nude mice model by us. Here, we report the application of YB1 in neuroblastoma treatment. Methods The anti-cancer effect and side-effects of YB1 was examined in both in vitro and in vivo experiment. Previous established orthotopic neuroblastoma SCID/beige murine model using SK-NLP/luciferase cell line was adopted. Results In vitro , YB1 induced apoptosis for up to 31.4% of the neuroblastoma cells under anaerobic condition, three times more than that under aerobic condition (10.9%). The expression of both Toll like Receptor 4 and 5 (TLR4 and TLR5) in cancer cells were significantly up-regulated ( p <0.05 , p <0.01 respectively) after the treatment of YB1 under anaerobic condition. In mouse model, YB1 preferentially accumulated inside the core of the tumors, rather than in normal tissues as our previous reported. This is suggestive of the hypoxic nature of tumor core. Tumor growth was significantly retarded in YB1 treatment group ( n=6, P<0.01 ). Furthermore, there was no long-term organ damage noted in all the organs examined including heart, lung, liver, spleen and brain in the YB1 treated mice. Conclusion The genetic modified Salmonella strain YB1 is a promising anti-tumor strategy against the tumor bulk for neuroblastoma. Future study can be extended to other common cancer types to verify the relative efficacy on different neoplastic cells., Competing Interests: Competing Interests: The authors have declared that no competing interest exists.

Background: Hemolytic uremic syndrome (HUS) is a main cause of acute renal failure in children. This study aimed to analyze the clinical characteristics of HUS., Methods: A retrospective analysis was performed in 46 children with sporadic HUS., Results: Of the 46 HUS patients, 20 (43.5%) were diarrhea-related HUS, and 26 (56.5%) were atypical HUS. Anemia, edema, oliguria, hemoglobinuria and hypertension were the most common manifestations. Thrombocytopenia, hyponatremia, hypocalcemia, hyperkalemia, metabolic acidosis, increased fibrinogen and hypocomplementemia were found in most patients. The age of onset (younger than 2 years or not, P=0.009), the duration of oliguria or anuria (more than one week or not, P=0.005), accompanied with extrarenal complications or not (P=0.005), dialysis and plasma exchange (P=0.04) were associated with the mortality rate., Conclusions: The age of onset younger than 2 years, oliguria/anuria more than 1 week, and associated with extrarenal complications were predictive factors of poor prognosis.

Acute pancreatitis (AP) has a fast onset and progression, which lead to an unfavorable prognosis. Therefore, the development of novel drugs for its treatment is critical. As a homologous derivative of resveratrol, pterostilbene exerts a variety of effects including anti-inflammatory, antioxidant, and antitumor effects. This study investigated the potential of pterostilbene for treatment of severe AP (SAP) and related mechanisms. Effects of pterostilbene were evaluated in a Wistar rat model of AP. Serum levels of amylase (AMY), creatinine (Cr), and alanine aminotransferase (ALT) were quantified. Furthermore, serum levels of tumor necrosis factor (TNF)-a and interleukin (IL)-1b were quantified using enzyme-linked immunosorbent assay. Nuclear factor (NF)-kB expression in pancreatic tissues was quantified by real-time PCR and western blotting. The production of reactive oxygen species (ROS) was determined using a spectrometer, while superoxide dismutase (SOD) activity was assayed. In the AP rat model, the expression of inflammatory markers TNF-a and IL-1b, expression of NF-kB, and serum indices (AMY, Cr, and ALT) increased compared to the corresponding levels in the control group (P < 0.05). Pterostilbene reduced serum levels of TNF-a and IL-1b; decreased NF-kB gene expression, serum indices, and ROS generation; and increased SOD activity in a dose-dependent manner. In conclusion, pterostilbene can alleviate SAP-induced tissue damage by decreasing the inflammatory response and by promoting antioxidation leading to the protection of pancreatic tissues., Competing Interests: The authors declare no conflict of interest.

Background: This study aimed to discribe the experience in supporting children with refractory cardiopulmonary failure with extracorporeal membrane oxygenation (ECMO)., Methods: We retrospectively reviewed 12 children with refractory cardiopulmonary failure supported with ECMO from February 2009 to August 2015 in the Pediatric Intensive Care Unit (PICU), Children's Hospital, Zhejiang University School of Medicine., Results: Seven of the 12 patients were weaned successfully from ECMO and dischaged from the hospital, with a survival rate of 58.3% (7/12). Among them, five patients had acute fulminant myocarditis (AFM). Complications during ECMO included hemorrhage, hemolysis, thrombosis, acute kidney injury, and secondary hematogenous infection. During 1-24 month follow-up, the seven surviving patients recovered with normal cardiopulmonary function., Conclusions: ECMO is useful for supporting children with refractory cardiopulmonary failure, especially for treatment of AFM.

Background: Neuroblastoma currently has poor prognosis, therefore we proposed a new strategy by targeting neuroblastoma with genetically engineered anaerobic Salmonella (Sal-YB1)., Methods: Nude and nonobese diabetic-severe combined immunodeficiency (NOD-SCID) orthotopic mouse models were used, and Sal-YB1 was administered via tail vein. The therapeutic effectiveness, bio-safety, and mechanisms were studied., Results: No mice died of therapy-related complications. Tumor size reduction was 70 and 30% in nude and NOD-SCID mice, respectively. No Salmonella was detected in the urine; 75% mice had positive stool culture if diaminopimelic acid was added, but all turned negative subsequently. Tumor tissues had more Sal-YB1 infiltration, necrosis, and shrinkage in Sal-YB1-treated mice. Significantly higher expression of TLR4, TNF-stimulated gene 6 protein (TSG6), and cleaved caspase 1, 3, 8, and 9 was found in the tumor masses of the Sal-YB1-treated group with a decrease of interleukin 1 receptor-associated kinase (IRAK) and nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor alpha (IκBα). There was a high release of TNFα both in human macrophages and mouse tumor tissues with Sal-YB1 treatment. The antitumor effect of the supernatant derived from macrophages treated with Sal-YB1 could be reversed with TNFα and pan-caspase inhibitors., Conclusions: This new approach in targeting neuroblastoma by bio-engineered Salmonella with the assistance of macrophages indirectly may have a clinical therapeutic impact in the future.

Anti-CD14 antibody can inhibit the lipopolysaccharide (LPS)-induced systemic inflammatory response syndrome in case of bacteremia or endotoxemia. To obtain chimeric anti-CD14 antibody, we constructed and expressed a novel chimeric antibody Hm2F9 composed of anti-CD14 single-chain fragment variable (scFv) and the Fc region (the hinge, CH2, and CH3 domains) of human IgG1 in Chinese hamster ovary (CHO) cells based on our previous study of scFv2F9. The Hm2F9 antibody, sized 150 kDa, retained the strong specific antigen-binding ability to the CD14 antigen with a comparable activity (the percentage of positive cells 99.07%) to its parental murine antibody 2F9 (the percentage of positive cells 98.86%). At the same time, Hm2F9 could manifestly block the binding of LPS to CD14, whose positive-cell percentage drops significantly with percentage of 98.63% (from 98.37% to 1.35%). The chimeric antibody Hm2F9 expressed in CHO cells retained high affinity to human CD14 and biological function to LPS.

Ventilator-associated pneumonia (VAP) is a common and sometimes fatal complication in pediatric intensive care units (PICU). The aim of our study was to characterize the distribution and drug susceptibility of the pathogens isolated from the sputum of patients with VAP in the PICU of our hospital and to provide support to the administration of antibiotics early and reasonably in the clinic. Our study was conducted between January 2007 and December 2011 at the PICU of the Children's Hospital of Zhejiang University School of Medicine. The endotracheal aspirates were collected and transported to a microbiology laboratory within 15 min. The pathogens were routinely analyzed and identified with Vitek 60 and Kirby-Bauer disk diffusion methods. Among the 121 VAP patients, 127 pathogenic strains were isolated from sputum specimens. Gram-negative and gram-positive bacteria and fungi accounted for 64.57% (82/127), 29.92% (38/127) and 5.51% (7/127), respectively. Acinetobacter baumannii (25.61%), Escherichia coli (20.27%), Stenotrophomonas maltophilia (20.27%), Klebsiella pneumoniae (16.22%) and Pseudomonas aeruginosa (9.46%) were frequently identified isolates among gram-negative bacteria. Staphylococci were susceptible to vancomycin and linezolid. All fungi were sensitive to the antimicrobial agents. The gram-negative bacteria were more prevalent than gram-positive bacteria and fungi in VAP and demonstrated a higher drug resistance. It is important to administer antimicrobial agents early and reasonably for children with VAP. Knowledge of antibiotic resistance and the characteristics of drug resistance is important for VAP prophylaxis and treatment.

Objective: To establish a transgenic cell line with stable expression of CD14., Methods: Total RNA extracted from peripheral blood mononuclear cells was treated with RNAase-free DNAase, the human CD14 gene was cloned and sequenced through the RT-PCR, T-A clone techniques and ABI PRISM377 machine. Eukaryotic expression vector pcDNA3.1(+)/CD14 was constructed by cleaving with double restriction endonucleases EcoR I/Xba I and ligating with T4 ligase. The human cervical cancer cell line Hela was transfected with the positive recombinant plasmid pcDNA3.1(+)/CD14 using superfect transfection reagent. Positive clones were selected by G418 at a concentration of 0.5 μg/μl and the expression of human CD14 on the transfected Hela cells was confirmed by quantitative PCR and immunofluorescent assay., Results: There was significantly difference om expression of CD14 mRNA between the blank pcDNA3.1(+) transfected cells and pcDNA3.1(+)/CD14 transfected cells (P<0.01). The fluorescence was significantly stronger on the stable cell line Hela-CD14 than that on the transiently transfected Hela cells,and no visible fluorescence was observed in blank vector transfected cells., Conclusion: The transfectant cell line Hela-CD14 with stable expression of human CD14 has been successfully established, which can be used to study human CD14 molecular and CD14-associated monocyte/macrophage cell diseases.

CD14 is the pivotal molecule in the diagnosis and therapy of CD14-associated diseases, and is important in bacteremia. The HeLa cell line is regarded as immortal due to its prolific character. The HeLa cell line is derived from human cervical cancer cells and has been widely used in cancer research and gene transfection. In the present study, we established the expression plasmid pcDNA3.1(+)-CD14, and transfected it into the human cervical cancer cell line HeLa to establish a stable cell line (HeLa-CD14) expressing human CD14 antigen on the membrane. After the human CD14 gene was cloned and sequenced through RT-PCR and T-A cloning techniques, the eukaryotic expression vector pcDNA3.1(+)-CD14 was constructed by cleaving with double restriction endonucleases and ligating with T4 ligase. HeLa cells were transfected with the pcDNA3.1(+)-CD14 recombinant plasmid using Superfect transfection reagent. The cells were selected using G418 and the expression of human CD14 on the transfectant was confirmed by RT-PCR and immunohistochemistry. The expression of CD14 mRNA was significantly different between the blank pcDNA3.1(+)-transfected cell group and the pcDNA3.1(+)-CD14-transfected cell group (p<0.01). The fluorescence was significantly stronger on the established stable cell line than on the transiently transfected HeLa cells, and no visible fluorescence was observed in blank pcDNA3.1(+)-transfected cells. In this study, the human CD14 transfectant, stable cell line HeLa-CD14, was successfully established, which may be used to study CD14 and cervical cancer in vitro and in vivo.

Objective: Leukemia is the most common hematopoietic malignancies in children. Chemotherapy is currently the primary modality of treatment for this fatal disease. Although chemotherapy is very effective in terms of cell killing, severe side effects such as severe infections, intracranial hemorrhage etc. are frequently encountered due to its poor selective damage between normal and malignant cells or tissues. Thus, a new therapy with highly selective killing of malignant cells which leaves the normal cells unaffected is desperately desired. The aim of this study was to investigate the targeting efficacy in vitro with a new clone of anti-human CD19 antibody immunotoxin 2E8-Genistein on B lineage leukemia cell line Nalm-6 cells and its mechanisms in order to provide the evidence of target therapy on B lineage leukemia and lymphoma., Methods: 2E8-Genistein immunotoxin was generated by conjugating Mab 2E8 with a tyrosine kinase inhibitor, Genistein (Gen) via the Sulfo-SANPAH, an ultra-violet sensitive reagent. Nalm-6, a CD19+ B cell leukemia cell line, was used as target cells, while Molt-3, a CD19-T cell leukemia cell line, was taken as the negative control. The morphology of the cells was observed under the reverted reversed light microscope and the viability was checked with either trypan blue exclusion or MTT methods. Two-color flow cytometry was applied to study the mechanism of cell killing., Results: After 24 hours of culture, 2E8-Genistein showed marked target killing on Nalm-6 cells at nine different concentrations from 20 nmol/L through 100 nmol/L with cell survival rates from (71.8 +/- 7.9)% down to (16.6 +/- 12.9)%, respectively (n = 3), which were all significantly lower than that of control group (100 +/- 13.9)% (P < 0.05). The killing effect was even more significant when the concentration was over 80 nmol/L. The growth inhibition rates of this immunotoxin on Nalm-6 cells were 82%, 84% and 94%, respectively at 24, 48 and 72 hours of culture in a time dependent manner. Significant difference was observed between the cell growth curve of Nalm-6 cultured with 100 nmol/L of 2E8-Gen and those of Nalm-6 cultured with medium (blank), PBS (negative control) or the same concentration of pure 2E8 antibody (negative control) groups (F = 152.15, P = 2.15 x 10(-7)), but there was no significant difference among the three control groups (F = 1.51, P = 0.29). When Molt-3 cells were used as target cells, the cell growth curves of Molt-3 cultured with 2E8-Gen (100 nmol/L) and with negative control of blank did not show any significant difference (F = 0.34, P = 0.59). PI/FITC Annexin V double staining analysis with flow cytometry showed that the positive rate (33.45 +/- 8.77)% of early apoptosis on Nalm-6 cells induced by 100 nmol/L of 2E8-Genistein was significantly higher than that of negative control of blank (10.44% +/- 1.28%, t = -4.39, P = 0.001) at 24 hours of culture., Conclusion: 2E8-Genistein immunotoxin can significantly target the Nalm-6 cells in vitro in a time response manner and the apoptosis induction is involved in the course of this killing effect.

Objective: Acute monocytic leukemia (AML)-M5 is the common type of acute myeloid leukemias in children. Studies have shown that there are abundant lipopolysaccharide (LPS) receptor (designated as CD14) molecules on the cell membrane of M5 cells and they play an important role in the diagnosis of M5, since they can be recognized and bound by mouse-anti-human CD14 monoclonal antibody (McAb). However, mouse-originated antibodies are largely not suitable for clinical application due to the severe side effects, thus "humanized antibody" is desired. As the first step for developing humanized antibody, the construction and expression of single chain antibody (scFv) with functional protein are necessary. The present study aimed to express ZCH-7-2F9 ScFv (scFv(2F9)) in eukaryotic cells and obtain the scFv(2F9) protein with a high biological activity., Methods: Four primers were synthesized to construct the eukaryotic expressional vector, which included SfiI and EcoRI enzyme cleaving site, 6 x His and stop code TGA sequences. scFv(2F9) gene was amplified through splicing by overlap extension (SOE) using the high fidelity Taq polymerase. Positive recombinants (pSectag2A/scFv(2F9)) were identified through enzyme cleaving and sequenced before expression and were transformed into Chinese hamster ovary (CHO) cells for expression. Western-Blot and flow cytometry (FCM) were carried out to determine the relative molecular mass (Mr) and binding activity of scFv(2F9)., Results: The cloned scFv(2F9) gene was identified to be functional by sequencing and expressing. The interested protein could be detected in the culture supernatant of transformed CHO cells with an Mr of 31 000. The blocking test showed that the positive cell percentages, the mean fluorescence intensity (MFI) and the peak of channel (peak Ch) were reduced by 90.02%, 63.30% and 63.38%, respectively after blocking with scFv(2F9), while those were 4.55%, 10.09% and 5.02% after blockage using the supernatant from the CHO cells transfected with blanked vector pSectag2A., Conclusions: The scFv(2F9) against human CD14 antigen was successfully expressed in eukaryotic cells and showed a high biological activity, which may be useful for the further studies on its humanized antibodies.

Objective: To construct a prokaryotic vector of ZCH-7-2F9 single chain antibody (ScFv2F9) and to obtain the ScFv2F9 protein with biological activity for further studies., Methods: Primers were synthesized according to the gene sequence of ScFv2F9, four tandem glycin and one serine (G4S) 3linker and multiple cloning site(MCS) of pIVEX2.3-MCS vector, which included NdeI and SmaI enzyme cleaving sites. ScFv2F9 gene was amplified through splicing by overlap extension (SOE) using the high fidelity Taq polymerase. Then the gene was cloned to pGEM-T easy and pIVEX2.3-MCS vectors. Positive recombinants (pIVEX2.3-MCS/ScFv2F9) were identified through enzyme cleaving and sequenced before expression. The recombinant plasmids were transfected into E.coli BL21star(DE3)plysS for expression. After purification with Ni+resin and renaturation in vitro, the relative molecular mass (Mr) and the binding activity of the interesting protein were determined by SDS-PAGE and flow cytometry (FCM), respectively., Result: The cloned ScFv2F9 gene was identified to be functional by sequencing and expressing. The interesting protein was detected in inclusion body with a Mr of 31 000. The blocking test showed that the positive cell percentage, the mean fluorescence intensity (MFI) and the peak of channel (peak Ch) were reduced by 11.73%, 11.96% and 31.57%, respectively after once blockage by scFv2F9 protein, and 26.44 %, 21.75 % and 42.11 % after blockage twice., Conclusion: The ScFv2F9 against human CD14 antigen has been successfully expressed in prokaryotic cells with partial biological activity, which lays the foundation for further studies on its immunotoxin and other kinds of engineered antibodies.

Objective: To acquire the genes of light chain variable region (VL) and heavy chain variable region (VH) of a novel clone ZCH-7-2F9(2F9) of anti-hCD14 for construction of anti-hCD14 single chain antibody(ScFv)., Methods: From the mouse hybridoma cell line 2F9 and its fusion partner murine myeloma cell line NS-1, total RNA was prepared. The VL and VH genes were amplified by RT-PCR with family specific primer pairs, respectively. The PCR products were cloned into pGEM(sound recording copyright sign)-T Easy vectors, then transfected into DH5alpha and the positive recombinants were identified and purified. After sequencing with automatic DNA sequencer the sequences were analyzed online., Results: VL gene of the new clone of CD14 monoclonal antibody (McAb) 2F9 consisted of 321 bps encoding a peptide of 107 amino acid residues, and VH gene of the 2F9 antibody contained 360 bps encoding a peptide of 120 amino acid residues. According to IMMUNOGENETICS online analysis by IMGT/V-QUEST, the VL and VH genes belonged to mouse IGKV and mouse IGHV subgroups, respectively. On the position 23/88 of the light chain and 22/96 of the heavy chain genes there were cysteines, which play the key role in forming disulfo-bond in each chain. Both VL and VH chains had definitely 4 frame regions (FR) and 3 complementary determinant regions (CDR)., Conclusion: The treatment of CML consisting of myeloablative Allo-SCT combined with Gleevec before and after transplantation is an effective and safe method for CML.

Objective: Leukemia is the most common malignancy in children. Combined chemotherapy is currently the primary treatment modality. During the past decade, very high cure rates of childhood acute lymphoblastic leukemia (ALL) have been reported both at home and abroad. However, the cure rates of children with acute myeloid leukemia (AML) remain low due to the multiple-drug resistance (MDR). P-glycoprotein (P-gp) is one of the most important mechanisms of MDR for leukemia cells. However, the function of the protein, the clinical application of its reversal agents and the efficacy of the combination of the reversal agents remain to be elucidated. The present study aimed to evaluate the P-gp pump function on leukemia cell membrane and the effects of the combined administration of the reversal agents cyclosporin A (CSA) and verapamil (VER) through the observation of Calcein-AM (C-AM) metabolism in the cell line K562 and its multi-drug resistant subline K562/VCR., Methods: The mean fluorescence intensity (MFI) of C-AM inside the cytoplasm was analyzed with flow cytometry (FCM). The events of K562 and K562/VCR cells treated and untreated with CSA, VER and CSA + VER were acquired at time points 0, 30, 60, 90 and 120 minutes, respectively, and the data obtained were analyzed with CellQuest software., Results: The C-AM in the K562 and K562/VCR varied more apparently in the fist 24 hours. In addition, the MFI of the C-AM in K562 was significantly higher than that in K562/VCR cells indicating that the P-gp pump molecules were functioning. The MFIs of the CSA, VER and CSA + VER groups co-cultured with K562/VCR cells were 4014 +/- 219, 3879 +/- 116 and 4158 +/- 302, respectively after 120 min of incubation, significantly higher as compared to that of control group (3251 +/- 107, P < 0.05). On the other hand, significant inhibition of the efflux from the K562/VCR cell line was also noticed after the same time period of incubation with the MFIs of 2237 +/- 155, 1932 +/- 233 and 2231 +/- 147, respectively in the three groups, which was significantly higher than that of control group (1622 +/- 191, P < 0.05). CSA, VER and CSA + VER could increase the uptake and inhibit the efflux of C-AM by K562/VCR cells, while no evident influence on those functions inside the parental cell line K562 cells was noticed., Conclusions: CSA, VER and CSA + VER could increase the uptake and reduce the efflux of C-AM by K562/VCR cells while no significant difference between the CSA + VER and CSA or VER was noticed. P-gp pump function and the effects of its reversal agents on leukemic cells can be rapidly and easily evaluated by using the C-AM and FCM.

Objective: Acute promyelocytic leukemia (APL) is a specific type of hematopoietic malignancy, accounting for 10% of the de novo acute myeloid leukemia (AML). The data on long-term outcome of APL in children are limited. The aim of this study was to investigate the clinical biological features, diagnosis, prognosis and long-term survival of childhood APL., Methods: A total of 46 children with newly diagnosed APL from April 1998 to October 2005 were enrolled into this study. Induction treatment containing all-trans retinoic acid (ATRA) plus daunorubicin (DNR) or pirarubicin (THP) was performed on these patients, followed by 6 courses of chemotherapy consolidation: DNR, homoharringtonine or etoposide plus Ara-C. A maintenance therapy was then administered once 3-6 months. The total period of treatment was 2.5 years., Results: Of the 39 patients who had completed the regular treatment, 36 (92.3%) achieved a complete remission. The 5-year cumulative incidence of relapse (CIR) was 28.6%. The estimated overall survival (OS) rates at 1, 3 and 5 years were (86.1 +/- 5.8)%, (76.1 +/- 7.5)% and (70.2 +/- 8.9)% respectively, while the event free survival (EFS) rates were (78.4 +/- 6.8)%, (63.6 +/- 8.7)% and (53.1 +/- 10.0)% respectively. The 5-year OS rate of patients with WBC less than or equal to 10.0 X 10(9)/L was (81.4 +/- 10.3)%, which was significantly higher than that with WBC greater than 10.0 X 10(9)/L[(51.6 +/- 14.7)%, P < 0.05]. Five patients with RT-PCR positive for PML/RARalpha S (short) subtype died eventually although all of them achieved CR, but none of the 13 patients with PML/RARalpha L (long) subtype died., Conclusions: Remission induction therapy with ATRA + DNR or THP is effective and safe for newly diagnosed childhood APL. The remission induction therapy combined with chemotherapy containing high/intermediate dose Ara-C can improve the long-term survival rates of APL patients. High WBC count and S subtype of PML-RARa are two poor prognostic factors for children with APL.

Our aim was to construct the eukaryotic expressional system of single-chain antibody (ScFv(2F9)) derived from a new clone of monoclonal antibody named ZCH-7-2F9 against human CD14 antigen, and to obtain the ScFv(2F9) protein with a high biological activity for further studies on ScFv(2F9) immunotoxin and other kinds of genetically engineered antibodies. Primers were synthesized according to the gene sequence of ScFv(2F9), 4 tandem glysines and 1 serine (Gly4Ser)3 linker and multiple cloning site(MCS) of pSectag2A vector, which included SfiI and EcoRI enzyme cleaving site, 6 x His and stop code TGA sequences. ScFv(2F9) gene was amplified through splicing by overlap extension (SOE) using the high fidelity Taq polymerase. Then the gene was cloned to pGEM-T easy and pSectag2A vectors. Positive recombinants (pSectag2A/ScFv(2F9)) were identified through enzyme cleaving and sequenced before expression. The recombinant plasmids were transfected into Chinese hamster ovary (CHO) cells for expression. The relative molecular weight (MW) and the binding activity of the interesting protein were determined by SDS-PAGE, Western-blot and flow cytometry (FCM), respectively. The scFv(2F9) V(H) is a member of the IgH gene family V(1) subgroup and the scFv(2F9) V(L) gene belongs to the Ig(kappa) gene family V(10) subgroup. The recombined ScFv(2F9) gene was identified to be functional by sequencing and expressing. The interesting protein could be detected in the culture supernatant of transformed CHO cells with a MW of 31 kDa. The blocking test showed that the positive cell percentages, the mean fluorescence intensity (MFI) and the peak of channel (peak Ch) were reduced by 90.02%, 63.3%and 63.38%, respectively after blockage of ScFv(2F9). The ScFv(2F9) gene was cloned and the interest protein against human CD14 antigen has been successfully expressed in eukaryotic cells with a high biological activity, which lays the foundation for further studies on its immunotoxin and other kinds of engineered antibodies.

Objective: To analyze the reactive pattern and its clinical significance of ZCH-7-2D3 monoclonal antibody on leukemia cells., Methods: Bone marrow samples from 100 leukemia patients (male 59: female 41, 34 cases of children and 66 adults, aged 11 months - 77 year) were collected. A CD45 gating strategy and multi-parameter flow cytometry were used to analyze the leukemia cells., Result: The positive rate (10/31) of 2D3 antibody on acute lymphocytic leukemia (ALL) patients was significantly lower than that (39/55) of acute myelogeneous leukemia (AML) (P <0.01). 2D3 was positive for all three cases of B/myeloid mixed lineage leukemia, but negative in 6 cases of chronic myelogeneous leukemia (CML), significantly lower than that in AML (39/55, P<0.01) patients. There was no difference between the positive rates of chronic lymphocytic leukemia (CLL, 3/5) and B lineage ALL (9/28, P = 0.2389). The antibody was not found to recognize the differentiation stages of either ALL and AML subtypes., Conclusion: 2D3 monoclonal antibody primarily recognizes AML cases. Further studies on cloning of gene encoding the antigen protein and its biological functions are warranted.

This study was aimed to construct the CD14 eukaryotic expression vector, establish the transgeneic CD14 positive cell line in order to facilitate the establishment of a mouse model of antibody targeting therapy for human acute monocytic leukemia (AML-M(5)). Total RNA extracted from peripheral blood mononuclear cells was treated with RNAase-free DNAase, the human CD14 gene was cloned and sequenced through the RT-PCR and T-A clone techniques. Eukaryotic expressional vector pcDNA3.1(+)/CD14 was constructed by cleaving with double restriction endonuleases and ligating with T4 ligase. A murine melanoma cell line B16 was transfected with the pcDNA3.1(+)/CD14 recombinant with Superfect transfection reagent. Positive clones were selected by G418 and the expression of human CD14 on the transfectant was confirmed by flow cytometry (FCM). The results indicated that the sequence of the human CD14 cDNA cloned was exact to be same as the one from GenBank database. The recombinant pcDNA3.1(+)/CD14 was identified with double-enzyme cleaving. The expression of the human CD14 on the transfectant (B16/CD14) was confirmed by FCM. In conclusion, the murine cell line B16/CD14 fransfected with human CD14 gene has been established which can be used for the study of human AML-M(5) antibody targeting therapy with mouse model.

In order to provide the evidences for CD19 as a better antibody targeting molecule for B lineage acute leukemias than CD20 through the multi-parameter flow-cytometry analysis of leukemia cells, the samples from 321 patients with acute leukemia (AL) were immunophenotyped by multi-color flow cytometry and CD45/SSC gating strategy followed by the analysis of CD19 and CD20 expression. The results showed that the positive rate of CD19 (115/116, 99.1%) in 116 cases with B lineage acute lymphoblastic leukemia (B lineage ALL) was significantly higher than that of CD20 (33/116, 28.4%) (P < 0.01); in 17 patients with B lineage/Myeloid (B/My) acute mixed lineage leukemia (AMLL), the former positive rate (17/17, 100%) was also higher than the latter (5/17, 29.4%) (P < 0.01). Both of the two antigens were negative in 29 patients with acute T lymphoblastic leukemia and 7 patients with T/My AMLL. The positive rates of CD19 and CD20 in 152 patients with acute myeloid leukemia (AML) were 7.2% and 2.0%, respectively. The difference of the fluorescence intensity between the two antigens on the cells from each patient with B lineage ALL or B/My AMLL was statistically significant (t = 20.68, P < 0.001). The specificity of CD19 and CD20 in B lymphocytic lineage was 92.3% (132/143) and 92.7% (38/41), respectively, while the sensitivity was 99.2% (132/133) and 28.6% (38/133), respectively, the former sensitivity was significantly higher than the latter (chi(2) = 144.018, P = 0.001). It is concluded that CD19 continuously and steadily express on almost all subtypes of B lineage leukemic cells with homogeneous pattern while only a small number of leukemias express CD20. Both the specificity and sensitivity of CD19 were very high with a much broader reaction pattern than that of CD20 on this group of diseases. These indicate that CD19 may be a better antibody targeting molecule than CD20 for patients with B-lineage acute leukemia.

Objective: To prepare fluorescein isothiocyanate (FITC) directly conjugated to monoclonal antibody (McAb) anti-human CD14, ZCH-7-2F9 (2F9-FITC)., Methods: After generation and purification, the purity and the murine immunoglobulin subtype of the antibody were evaluated with SDS-PAGE and multicolor flow cytometry (FCM). 2F9 McAb was directly labeled with FITC through modified Marsshall's method and the positive rate of the 2F9-FITC on different types of leukemic cells were compared with the standard CD14-FITC by FCM., Result: A large quantity of purified 2F9 McAb was prepared. The subtype of 2F9 was murine IgG1kappa. 2F9-FITC was successfully manufactured with A295/A280 ratio of 0.44. The positive cell percentages of 2F9-FITC and CD14-FITC on the monocytes were 84.50% and 90.08%, respectively, while those on lymphocytes were only 0.52% and 1.01%. There was no significant difference between the CD14 expressions with 2F9-FITC and CD14-FITC on each type of leukemia (n=23, t=0.922, P=0.367)., Conclusion: 2F9-FITC has been successfully prepared and it can be applied in diagnosis and differentiation of monoblastic leukemias.

Objective: To investigate the expression of CD19 on childhood acute leukemia (AL) and its significance, and to provide evidence for the diagnosis and differential diagnosis as well as monoclonal antibody-targeting treatment of leukemia., Methods: There were 210 cases of childhood AL, of which 130 cases were male and 80 were female with a mean age of 9 years old. Using a panel of 27 fluorochrome directly labeled monoclonal antibodies, 210 samples from the patients were analyzed with CD45/SSC double parameters and multi-color flow cytometry to determine the expression of CD19., Results: In the 93 cases of B lineage acute lymphoblastic leukemia (ALL), the positive rate (98.9%, 92/93) of CD19 was significantly higher than that of the other B cell related antigens, such as CD10 (88.2%, 82/93, P = 0.003), CD20 (24.7%, 23/93, P = 0.001) and CD22 (60.2%, 56/93, P = 0.001). CD19 was expressed on all 8 cases of B/myeloid (My) hybrid acute leukemia (HAL) and 1 case of B/T HAL, but was not expressed on all 24 cases of T lineage leukemia and 5 cases of T/My HAL. In the 79 cases of acute myeloid leukemia (AML), only 5 (6.3%) cases expressed CD19. The positive rate (6.3%) of CD19 on AML was significantly lower than that on B lineage ALL (98.9%, P = 0.001). The percentage of CD19 positive cells in B/My HAL (41.6% - 88.7% with a mean of 73.8%) was significant higher than that in CD19(+)-AML (21.4% - 50.4% with a mean of 24.2%; Run Sum test, P = 0.0084). Of the 210 cases, 102 were B lineage related AL including B lineage ALL, B/My HAL and B/T HAL. In B lineage related AL, the sensitivity and the specificity of CD19 was 99.0% (101/102) and 95.4% (13/108) while the positive predictive and the negative predictive values to B lineage were 95.3% (101/106) and 99.0% (103/104), respectively. Using CD19(+) as a single reagent to diagnose B lineage, the false positive rate was 4.6% (5/108) and the false negative rate was 1.0% (1/102) with a general diagnosis index (GDI) of 94.4% [GDI = 1-(false positive rate + false negative rate)]., Conclusion: CD19 is continuously and stably expressed on all stages of B lineage differentiation. It is a reliable cell membrane marker for diagnosing B lineage ALL and an ideal target for antibody-targeting treatment of leukemia as well; the expression degree of CD19 can be used to distinguish B/My HAL from CD19(+)-AML; CD19 didn't express on normal myeloid cells but did on some AML cells. Therefore it can be used to detect the minimal residual disease.

Objective: To define the immune phenotype of colon cancer cells., Methods: Using a panel of 40 anti-human monoclonal antibodies (MoAbs), the cells of colon cancer HR8348 were analyzed with three-color flow cytometry after direct immunofluorescent staining., Results: HR8348 cell line did not express CD2, CD3, CD4, CD5, CD7, CD8, TCR, CD10, CD11b, CD14, CD16, CD19, CD22, CD25, CD28, SmIg, CD33, CD35, CD36, CD41a, CD45, CD45RA, CD45RO, CD56, CD61, CD64, CD66b, CD69, CD71, CD117, CD122 and P-glycoprotein but expressed CD13, CD15, CD20, CD38, CD95 and HLA-DR., Conclusion: The results demonstrate that colon cancer cell line HR8348 shares some antigenic determinants with leucocyte lineage.