Effects of HOXB4-transfecting directly and HOXB4-transfected HUCMSC on in vitro expansion of human umbilical cord blood CD34+ cells / 中国实验血液学杂志

This study was purposed to investigate the difference of nucleated cell (NC) count, CD34(+) cell ratio and expansion multiple, cell cycle and colony formation capability in in vitro expanded human umbilical cord blood CD34(+) cells from HOXB4-transfecting directly and HOXB4-transfected human umbilical cord mesenchymal stem cells (HUCMSC) by means of prepared feeder layers of HUCMSC. The HUCMSC were divided into 2 groups:first group, in which HOXB4 gene was transfected into HUCMSC by using lentiviral vecfor, and feeder layers were set up; and second group in which feeder layers for HUCMSC of non-transfected HOXB4 gene were set up. The CD34(+) cells were separated from HUCB by magmatic activated cell sorting(MACS). After culture in medium with cytokines for 2 days, CD34(+) cells were divided into 5 groups, including control group and experimental group. The control groups included CD34(+) cells as group A (blank control group) and GFP-CD34(+) cells as group B (negative control group) and experimental groups included HOXB4-CD34(+) cells as group C, HUCMSC+CD34(+) cells as group D, HOXB4-HUCMSC+ CD34(+) cells as group E and cells in all groups were cultured in vitro. The number of nucleated cells were counted at day 6, 10, 14 of culture and CD34 immunophenotypes, cell cycle and colony forming capability were measured at day 10 of culture in different conditions. The results indicated that HOXB4 gene could be transfected into HUCMSC by lentiviral vector and feeder layers were set up successfully. After culture for 14 days, the nucleated cells in 5 groups could be amplified effectively, and the expansion levels in 5 groups were in order HOXB4-HUCMSC+CD34(+) cell group> HOXB4-CD34(+) cell group>HUCMSC+CD34(+) cell group> control groups (P < 0.05). At day 10 of in vitro expansion the CD34(+) cell percentage decreased significantly in all groups, while the number of CD34(+) cell increased in experiment groups, which were in order HOXB4-CD34(+) cells group> HOXB4-HUCMSC+CD34(+) cell group>HUCMSC+CD34(+) cell group>control groups (P < 0.05). The cell cycle detection showed that the percentage of cells in S+G2/M phase in experiment groups were higher than that in control groups (P < 0.05), and percentage of cells in HOXB4-HUCMSC+CD34(+) cells group was higher (41.57%) than that in HOXB4-CD34(+) cells group(37.87%) and HUCMSC+CD34(+) cell group (28.65%) (P < 0.05). There was no statistical difference in the CFU number between HOXB4-HUCMSC+CD34(+) cell group and HOXB4-CD34(+) cell group, which were both higher than that in HUCMSC+CD34(+) cell group and control groups (P < 0.05).It is concluded that the CD34(+) cells cultured on HOXB4-HUCMSC feeder layers can be amplified significantly and kept the characteristics of stem cells, The feeder lager of HOXB4-HUCMSC is relative safe for amplification of CD34(+) cells in vitro, it possesses the potential useful value.

Clinical application of cardiac output monitoring in children with severe hand-foot-mouth disease / 中国当代儿科杂志

<p><b>OBJECTIVE</b>Significant cardiac dysfunction has been found in children with severe hand-foot-mouth disease and heart failure is the major cause of death in these patients. Evaluation of cardiac function is essential for the treatment of severe cases. This study evaluated the clinical value of cardiac output monitoring in children with severe hand-foot-mouth disease.</p><p><b>METHODS</b>A total of 107 children with severe hand-foot-mouth disease admitted to the pediatric intensive care unit from April 2011 to September 2011 were enrolled and divided into three groups by clinical stage: 73 cases in stage 2, 23 cases in stage 3 and 11 cases in stage 4. Cardiac output and stroke volume were measured by ultrasonic cardiac output monitors (USCOM). Ninety-five children received MRI scanning and were grouped according to the results of MRI: 41 cases (medulla oblongata involvements in 9 cases) in abnormal MRI group and 54 cases in normal MRI group. Cardiac output was compared between the children in different clinical stages and between different MRI results.</p><p><b>RESULTS</b>Compared with children in clinical stages 2 and 3, cardiac output in children in clinical stage 4 decreased significantly (P<0.05). There was no differences in cardiac output between the normal and abnormal MRI groups, however cardiac output was significantly lower in children with medulla oblongata involvement than in those with other involvements and normal MRI.</p><p><b>CONCLUSIONS</b>Significant decrease in cardiac output suggests critical conditions and medulla oblongata cardiovascular center involvement in children with severe hand-foot-mouth disease. Dynamic measurement of cardiac output is valuable for treatment of the disease.</p>

Changes of immune function in patients with 2009 influenza A (H1N1) / 中华儿科杂志

<p><b>OBJECTIVE</b>To investigate the alteration of immune function and possible immunopathogenesis in the children with 2009 influenza A (H1N1) infection.</p><p><b>METHOD</b>Sixty patients with 2009 influenza A (H1N1) infection hospitalized in Shenzhen Children's Hospital between November 1, 2009 and January 10, 2010 and 20 age-matched healthy children were enrolled in this study. The patients were divided into two groups according to the severity of influenza A infection: 35 mild cases (mild pneumonia) and 25 severe cases (severe pneumonia, acute encephalopathy associated with influenza A, and 3 died from acute necrotizing encephalopathy with influenza A infection). Real-time PCR was used to evaluate the expression levels of pattern recognition receptor (PRRs), retinoic acid induced gene I/melanoma differentiation associated gene 5 (RIG/MDA5), Toll-like receptors (TLRs) and TLRs signaling molecules, and negative-regulator. Three color fluorescent and flow cytometry were used to investigate the apoptosis of CD3(+), CD4(+), CD8(+) and CD19(+) cells. Plasma cytokines (IL-1β, IL-6, TNF-α, IFN-γ, IFN-α, IL-10) concentrations were measured by enzyme-linked immunosorbent assay (ELISA).</p><p><b>RESULT</b>(1) The expression levels of RIG/MDA5, TLR2, 4 were much higher in the patients with influenza A infection, especially severe cases [TLR2 (9.69 ± 3.15) × 10(-2) vs. (3.96 ± 0.83) × 10(-2), t = 10.16, P < 0.05; TLR4 (10.23 ± 2.85) × 10(-2) vs. (7.46 ± 2.18) × 10(-2), t = 3.76, P < 0.05]. The expression levels of TLRs signal transduction molecules like MyD88 and TRAM also increased. (2) The cell counts of CD3(+), CD4(+), CD8(+) T cells and NK cells were markedly lower in the patients with influenza A infection compared to the NC group [CD3(+)(1.22 ± 0.38) × 10(9)/L vs.(3.59 ± 1.10) × 10(9)/L, t = 9.21, P < 0.05]. (3) Plasma concentrations and the mRNA expression of TNF-α, IL-6, and IL-1β were elevated in mild cases, while declined in severe cases [TNF-α (6.42 ± 1.76) × 10(-2) vs. (9.05 ± 2.51) × 10(-2), t = 4.55, P < 0.05]. Plasma concentrations of IFN-α/IFN-β were up-regulated gradually with the aggravation of the disease, especially in severe cases. Compared with healthy controls, the expression of IFN-I inducible gene IP-10, RANTES, or iNOS was significantly higher in children with mild [IP-10 (20.52 ± 6.09) × 10(-2) vs.(1.18 ± 0.34) × 10(-2), t = 18.74, P < 0.05], and relatively lower in severe cases. (4) The apoptosis of CD3(+), CD4(+), CD8(+) and NK cells significantly increased in the patients with influenza A infection than those in NC group [CD3(+)(32.90 ± 7.66)% vs. (20.21 ± 6.58)%, t = 6.21, P < 0.05]. Compared with healthy controls, the expression levels of apoptosis-related gene like TRAIL and CASPASE-3 significantly increased in the patients with influenza A infection. (5) The expression levels of negative regulator of SOCS1, SOCS3, IRAK-M, TRAF4 and FLN29 were significantly increased in the patients with influenza A, especially in severe cases than those in NC group (P < 0.05).</p><p><b>CONCLUSION</b>Immune function changed with the severity of the disease. The mild cases presented systemic immune activation status, while critically ill cases presented mixed immune activation and immunosuppression status.</p>

Characteristics of severely and critically ill children with 2009 influenza A (H1N1) virus infection / 中华儿科杂志

<p><b>OBJECTIVE</b>To analyze the clinical characteristics of severely and critically ill children with 2009 influenza A (H1N1) infection.</p><p><b>METHOD</b>Clinical data of 150 cases with 2009 influenza A (H1N1) virus infection confirmed with the use of a real-time polymerase-chain-reaction assay on nasopharyngeal swab specimens were analyzed.</p><p><b>RESULT</b>Among 150 severely and critically ill children with 2009 influenza A (H1N1) virus infection, 103 were male, 47 were female; the median age was 5 years, 81(55%) were 5 years of age or older; 21 (14%) had underlying chronic diseases. The most common presenting symptoms were fever (95%), cough (89%), vomiting (23%), wheezing (19%), abdominal pain (16%), lethargy (7%), seizures (6%), myalgia (6%), and diarrhea (6%). The common laboratory abnormalities were increased or decreased white blood cells counts (40%), elevated of CRP (33%), LDH (29%), CK (25%) and AST (19%). Clinical complications included pneumonia (65%), encephalopathy (12%), myocarditis (5%), encephalitis (1%) and myositis (1%). All patients had received antibiotics before admission or on admission; 73% of patients had received oseltamivir treatment, 23% of patients had received corticosteroids; 32 (21%) were admitted to an ICU, 13 patients were intubated and mechanically ventilated. Fourteen patients with dyspnea who were irresponsive to the treatment experienced bronchoalveolar lavage with flexible bronchoscopy, and the branching bronchial casts were removed in 5 patients. Totally 145 (97%) patients were discharged, five (3%) died, three previously healthy patients died from severe encephalopathy, one patient died from ARDS, one previously healthy patient died from secondary fungal meningitis.</p><p><b>CONCLUSION</b>Severely and critically ill children with 2009 influenza A (H1N1) virus infection may occur mainly in older children without underlying chronic disease. The clinical spectrum and laboratory abnormality of the patients can have a wide range. Neurologic complications may be common and severe encephalopathy can lead to death in previously healthy children. Early use of bronchoalveolar lavage with flexible bronchoscopy may reduce death associated with pulmonary complications.</p>

Critical care and therapy based different illness state of 80 patients with severe hand-foot-and-mouth disease seen in Shenzhen / 中华儿科杂志

<p><b>OBJECTIVE</b>To discuss the treatment strategy of severe hand-foot-and-mouth disease (HFMD) cases, prevent the severe cases from progressing to fatal condition and enhance the survival rate of critically ill patients.</p><p><b>METHODS</b>Eighty HFMD cases were divided into four groups, A, B, C and D, according to the severity of patients' nervous system manifestation and other system involved. Different intensive care and treatments were used and the effect and outcome were analyzed for each group. All statistical analyses were performed by using SPSS software 13.0. One-way ANOVA and Chi-square test were used for data analysis.</p><p><b>RESULTS</b>The most common symptoms were continuous fever (69/70) and myoclonic jerk (67/70). The fewer the rashes were, the more severe the patient's condition was, heart rate >200/min, hypertension, increase of white blood cells in peripheral blood and hyperglycemia were common in patients with lesions in brain stem and pulmonary edema. There were no relations between patient's conditions and CSF white blood cells and CRP. CNS involvement was highly associated with EV71 infection. There were 69 cases in group A, B and C in total and all recovered. Of 11 patients in group D, 6 got complicated neurogenic pulmonary edema and circulatory failure, 2 cases died and 9 cases survived, 8 cases recovered without sequelae while one case had sequelae of mental retardation and dyscinesia.</p><p><b>CONCLUSION</b>Administration of mannitol, methylprednisolone, IVIG and other supportive treatments in time and reasonably might have advantages in avoiding aggravation of the condition and enhancing the rate of successful rescue in patients with nervous system involvement.</p>

Changes of immune function in patients with enterovirus 71 infection / 中华儿科杂志

<p><b>OBJECTIVE</b>To investigate the association of changes in immune function with enterovirus 71 (EV71) cases with different severity of the disease.</p><p><b>METHOD</b>Forty-six EV71-infected patients and 12 age-matched healthy children were enrolled in this study. The patients were divided into four groups according to critical degree of enterovirus 71 infection: hand-foot-and-mouth disease (HFMD); central nervous system disease (CNSD); autonomic nervous system dysregulation (ANSD) and pulmonary edema (PE). We analyzed CD14+ monocyte HLA-DR expression, lymphocyte immunophenotypes, the proportion of CD4+CD25+ Foxp3high regulatory T cells (Treg cells) and Th17 cells, cytokines (IL-1beta, TNF-alpha, IL-10, TGF-beta, IL-6, IL-17A), evaluated the mRNA levels of Foxp3 and ROR-gammat, and serum immunoglobulin and complements.</p><p><b>RESULT</b>(1) Serum concentrations of IL-1beta and TNF-alpha elevated in mild cases, while declined in severe cases, and were lower in PE group (P<0.05). Serum concentrations of IL-10 and IL-10/TNF-alpha ratio gradually raised with the aggravation of the disease, and higher in PE group (P<0.05). (2) Circulating CD14+ monocyte HLA-DR expression, CD3+T cells, CD4+T cells, CD8+T cells, and NK cells gradually decreased, and lower in PE group (P<0.05). There was no significant difference in B cells, immunoglobulin and complement among the four groups. (3) The proportion of CD4+CD25+ Foxp3high Treg cells, mRNA level of Foxp, and serum concentrations of TGF-beta gradually decreased with the aggravation of the disease, while the proportion of Th17 cells, serum concentrations of IL-17A, mRNA level of ROR-gammat, and IL-6 gradually increased with the aggravation.</p><p><b>CONCLUSION</b>Immune function changed with different illness phases. The mild cases presented systemic inflammatory response syndrome status, while critically ill cases presented compensatory anti-inflammatory response syndrome or mixed antagonist response status. Immunoregulatory treatment of patients with EV71 infection should emphasize different methods at different stage and individualization.</p>

Study of the clinical and laboratory features of hand-foot-mouth disease / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To study the clinical and laboratory features of the mild and severe hand-foot-mouth diseases (HFMD) in Shenzhen in 2008.</p><p><b>METHODS</b>145 cases were observed in East-Lake Hospital and Shenzhen Children's Hospital. Of the 145 cases, 124 mild cases and 21 severe cases were involved.All the clinical data and laboratory findings were collected and summarized. After collection of the acute and convalescent consecutive stools and peripheral bloods from the patients with HFMDI, EV71 genes were amplified from these samples by RT-PCR. Enterovirus 71 were cultured and isolated using Vero cell line and R&D cell line.</p><p><b>RESULTS</b>The WBC counts and blood glucose levels of the severe cases were significantly elevated, but the ages of the severe ones significantly decreased compared with those of the mild cases (P < 0.05). EV71 genes could be detected by RT-PCR with 35% positive rate in mild cases and 67% in severe cases. The EV71 gene detection rate of the severe cases was significantly increased in contrast to that of the mild ones. The EV71 were isolated and cultured from the stools of 9 patients, one specimens from the dead's stool. Two severe cases died of neurogenic pulmonary edema and brain-stem encephalitis.</p><p><b>CONCLUSIONS</b>EV71 mainly contributes to HFMD and is responsible for death of some severe cases. High fever, less rash, elevated white blood cell counts and blood glucose concentrations as well as age less than 4 years old should be used for prediction of severe cases.</p>

Comprehensive Assessment of Septic Encephalopathy in Children / 实用儿科临床杂志

Objective To investigate the clinical features and characteristics of imaging,cerebral hemodynamics and electrophysiology of septic encephalopathy in children.Methods The clinical data and examination result of computerized tomography(CT),ultrasound and electroencephalogram(EEG) on encephalon of the 44 consecutive children with septic encephalopathy from Jan.2003 to Jun.2006 were reviewed retrospectively.According to the prognosis and the etiology,the totally 44 patients were divided into survival group,death group and pneumonia group,enteritis group,septicemia group,appendicular perforation group,skin gangrene group as well as EBV infection group,respectively.Fi-sher′s exact test was used to compare the differences of ratio.Results 1.Clinical manifestations:loss of consciousness was observed in all 44 cases,convulsions in 29 cases,upper-right-limb paralysis in 1 case,cerebral hernia in 1 case,and 14 cases resulted in death.2.Forteen cases were detected by CT scanning.Multiple local and diffuse lower density in cerebral parenchyma was observed in 3 cases and 2 cases respectively.Intracranial hemorrhage was found in the latter 2 cases.3.Color doppler ultrasound was detected in 11 cases.Diffuse brain edema was found in 5 cases,and among them intracranial hemorrhage was found in 1 case.4.EEG was detected in 10 cases.Flat-to-isoelectric EEG was found in 3 cases.Diffuse slow wave activities and sharp wave with sharp-slow wave complex were found in 5 cases and 2 cases,respectively.5.Between the sharp wave group and the flat-to-isoelectric EEG group,the number of cases who was less than 7 with Glasgow coma scale was significant(P