Artificial intelligence promotes shared decision-making through recommending tests to febrile pediatric outpatients.

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.

Signaling pathways and intervention therapies in sepsis.

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.

Pre-Consultation System Based on the Artificial Intelligence Has a Better Diagnostic Performance Than the Physicians in the Outpatient Department of Pediatrics.

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.

The Clinical Characteristics of ARDS in Children With Hematological Neoplasms.

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.

The clinical outcomes and biomarker features of severe sepsis/septic shock with severe neutropenia: a retrospective cohort study.

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.

Peg-Asparaginase-Associated Pancreatitis in Chemotherapy-Treated Pediatric Patients: A 5-Year Retrospective Study.

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.

The Comparative Immunological Characteristics of SARS-CoV, MERS-CoV, and SARS-CoV-2 Coronavirus Infections.

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.

Postoperative Risk Factors and Outcome of Patients With Liver Transplantation Who Were Admitted to Pediatric Intensive Care Unit: A 10-Year Single-Center Review in China.

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.

Treatment of Neuroblastoma with an Engineered "Obligate" Anaerobic Salmonella typhimurium Strain YB1.

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. ResultsIn 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.

Clinical characteristics of children with hemolytic uremic syndrome in Hangzhou, China.

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.

Clinical application of extracorporeal membrane oxygenation in children with refractory cardiopulmonary failure.

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.

Genetically modified "obligate" anaerobic Salmonella typhimurium as a therapeutic strategy for neuroblastoma.

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.

Construction and expression of a novel anti-CD14 human-mouse chimeric antibody Hm2F9.

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.

Pathogenic analysis of sputum from ventilator-associated pneumonia in a pediatric intensive care unit.

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.

[Establishment of a transgenic cell line with stable expression of human CD14].

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.

Establishment of the cell line, HeLa-CD14, transfected with the human CD14 gene.

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.

[Targeted killing of the Nalm-6 cells with 2E8-Genistein immunotoxin and its mechanism].

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.

[Eukaryotic expression and determination of ZCH-7-2F9 single chain antibody against human CD14].

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.