OBJECTIVE: To construct a human phage display antibody library, which will help to develop new drugs and vaccines against respiratory syncytial virus (RSV) and solve many of the issues that have limited the progression and application of murine monoclonal antibodies (McAbs) in the clinic. This can provide a platform for human antibody preparation and diagnosis, prophylaxis and therapy of RSV infection in children. METHODS: Peripheral blood lymphocytes were isolated from 52 children with RSV infection. cDNA was synthesized from the total RNA of lymphocytes. The light and heavy chain Fd (VH-CH1) fragments of immunoglobulin gene were amplified by RT-PCR. The amplified products were cloned into phagemid vector pComb3x and the clone samples were electrotransformed into competent E.coli XL1-Blue. The transformed cells were then infected with M13K07 helper phage to yield recombinant phage antibody of Fabs. The plasmids extracted from amplified E.coli were digested with restriction endonucleases Sac I, Xba I,Spe I and Xho I to monitor the insertion of the light or heavy chain Fd genes. RSV virions were utilized as antigens to screen Fab antibodies. RESULTS: By recombination of light and heavy chain genes, an immune Fab phage display antibody library against RSV containing 2.08×107 different clones was constructed, in which 70% clones had light chains and heavy chain Fd genes. The capacity of Fab phage antibody gene library was 1.46×107 and the titre of the original Fab antibody library was about 1.06×1012 pfu/mL. The antibody library gained an enrichment in different degrees after the preliminary panning. CONCLUSIONS: Utilizing the technology of phage display, an immune Fab phage display antibody library against RSV was successfully constructed in this study, which laid a valuable experimental foundation for further study and created favorable conditions for preparing human McAbs. This may also contribute to the improvement in the diagnosis, therapy and prophylaxis of RSV infection in children.

OBJECTIVE: Sponsored by the Subspecialty Group of Neonatology of Pediatric Society, China Medical Association, more than 10 large-scale hospitals participated in the near two-year multicenter investigation for brain injuries in premature infants in China. This study presented the investigation result for the incidence of periventricular leukomalacia (PVL) in premature infants from 10 Third Class A Level hospitals. METHODS: The premature infants with a gestation age <37 weeks in the 10 hospitals were given routine cranial ultrasound scanning within seven days after birth, and then repeated every 3-7 days until discharge from January 2005 to August 2006. The severity of PVL was graded based on de Vries classification. RESULTS: A total of 4 933 premature infants were enrolled. The total incidence of PVL and the incidence of cystic PVL were 2.3% (112/4 933) and 0.3% (16/4 933), respectively. Of the 112 PVL cases, 96 (85.7%) were with grade I, 14 (12.5%) with grade II, and 2 (1.8%) with grade III. The incidence of PVL in 4 maternal and child health care hospitals were significantly lower than that in 6 general or children's hospitals (1.4% vs 2.8%) (χ2=10.284, P<0.01). Vaginal delivery and mechanical ventilation were possible high-risk factors for the development of cystic PVL. CONCLUSIONS: The data of the multicenter investigation can basically reflect the situation about the occurrence of PVL in premature infants in major big cities of China. It is important to improve the ability to recognize the sonogram of non-cystic periventricular white matter injury.

OBJECTIVE: To investigate the relationship of disease severity with angiotensin converting enzyme (ACE) gene insertion/deletion (I/D) polymorphism and serum ACE activity in preterm infants during the first 7 days of life. METHODS: ACE genotypes were determined in 85 preterm infants admitted to the neonatal intensive care unit (NICU). Serum ACE activity was measured and disease severity was evaluated by the Neonatal Critical Score (draft) 1, 3 and 7 days after birth. RESULTS: Of the 85 preterm infants, DD genotype was found in 19 cases, ID genotype in 34 cases and II genotype in 32 cases. On the 1st day of life, serum ACE activity in the DD genotype (33.42±7.93 U/L) and the ID genotype groups (31.53±7.56 U/L) were significantly higher than that in the II genotype group (25.53±7.56 U/L) (P<0.01). After 3 and 7 days of life, serum ACE activity decreased in the three groups, but the DD genotype group remained the highest ACE activity, followed by the ID genotype and the II genotype groups. On the 1st day of life, the critical score of the DD genotype group (87.37±8.30) was lower than the ID genotype (95.82±5.85) and the II genotype groups (95.88±6.85) (P<0.01). On the 3rd day, the critical score of the DD genotype group was still lower than the ID genotype group (92.95±7.10 vs 96.94±5.85) (P<0.05). CONCLUSIONS: ACE gene I/D polymorphism may be associated with the disease severity in preterm infants. The DD genotype carriers present more severe disease status, with higher serum ACE activity. Although the disease status can influence serum ACE activity, serum ACE activity is determined by the ACE genotype.

OBJECTIVE: To study the correlation between brainstem auditory evoked potential (BAEP) and serum neuron-specific enolase (NSE) in neonates with asphyxia and explore the role of NSE in the evaluation of hearing impairment following asphyxia. METHODS: Fifty-two term neonates with asphyxia, including 38 cases of simple asphyxia (mild: 23 cases; severe: 15 cases) and 14 cases of asphyxia complicated by hypoxic-ischemic encephalopathy (HIE), were enrolled. In the double-blind trial, BAEP and NSE were simultaneously detected 7 days after birth. The patients who did not pass BAEP test received another BAEP and NSE examinations 3 months after birth. Thirty healthy term neonates served as normal control group. RESULTS: Of the 52 neonates with asphyxia, 50.0% and 21.2% of patients failed the initial and the second BAEP tests, respectively. The detection rates of BAEP anomalies in the simple severe asphyxia group in the initial and the second tests (63.3% and 26.3%, respectively) were significantly higher than those in the simple mild asphyxia group (36.9% and 5.9%, respectively)(P<0.05). The neonates with asphyxia complicated by HIE showed a higher detection rate of BAEP anomalies in the second test compared with the asphyxiated neonates without HIE (31.3% vs 16.7%; P<0.05). Mean serum NSE levels in asphyxiated neonates were significantly higher than those in normal controls (P<0.01). There were significant differences in serum NSE levels between the neonates with mild and severe asphyxia (26.70±2.34 μg/L vs 17.18±3.16 μg/L;P<0.01). The asphyxiated neonates with HIE had serum NSE levels similar to the simple severely asphyxiated neonates. Serum NSE levels in patients who failed the initial BAEP test were significantly higher than those who passed the test (25.69±4.12 μg/L vs 17.15±3.09 μg/L; P<0.01). Serum NSE levels had a positive correlation with wave V reaction threshold detected in the BAFP test (P<0.05). CONCLUSIONS: The serum level of NSE is closely correlated with BAEP, and it may be useful to the evaluation of the hearing impairment and the outcome in neonates with asphyxia.

OBJECTIVE: To study the relationship between the growth rate of the corpus callosum and neurological motor development in premature infants. METHODS: Fifty infants whose gestational ages were less than 34 weeks and who were admitted to the neonatal intensive care unit from March 2007 to August 2007 were enrolled. From 0 to 6 weeks of postnatal age, the sagittal midline cranial sonography via anterior fontanel was performed, once weekly. The length and the morphology of the corpus callosum were measured. The 52-neuromotor examinations were performed at 3 months of corrected gestational age. RESULTS: The mean length of the corpus callosum was 39.16 mm at birth. The mean growth rate of the corpus callosum during the first 6 weeks of life was 1.05 mm/week. Fourteen infants showed abnormal neuromotor development and 36 had normal neuromotor function at 3 months of corrected gestational age. A decreased growth rate of the corpus callosum was observed in the abnormal nervimotion group between 2 and 3 weeks (0.68 mm/week vs 1.17 mm/week) and between 4 and 5 weeks (0.86 mm/week vs 1.12 mm/week) after birth compared with that in normal nervimotion group (P<0.05). The total growth rate of the corpus callosum from 2 to 6 weeks after birth in the abnormal nervimotion group was also lower than that in the normal nervimotion group (0.91 mm/week vs 1.15 mm/week; P<0.01). CONCLUSIONS: A neuromotor delay at 3 months of corrected gestational age may be associated with the decreased growth rate of the corpus callosum between 2 and 6 weeks of life in premature infants.

OBJECTIVE: To study the risk factors for the development of fungal pneumonia in neonates. METHODS: A total of 260 neonates with nosocomial infectious pneumonia were classified into two groups: fungal pneumonia (n=160) and bacterial pneumonia (n=100). Their medical data were retrospectively studied. RESULTS: Eighty-seven percent of patients in the fungal pneumonia group had a duration of hospital stay of >10 days, but 30.1% of patients in the bacterial pneumonia group (P<0.01). The preterm infants in the fungal pneumonia group accounted for 49.5%, which was significantly higher than that in the bacterial pneumonia group (22.0%) (P<0.01). In the fungal pneumonia group, 59.9% of patients showed a weight of <2 500 g, but 34.0% of patients in the bacterial pneumonia group (P<0.01). The duration of the combined use of more than two broad-spectrum antibiotics (P<0.05) and the total duration of antibiotics use in the fungal pneumonia group was significantly longer than that in the bacterial pneumonia group (P<0.05). Nosocomial infectious bacterial pneumonia was common as the primary disease of fungal pneumonia (n=145). CONCLUSIONS: Premature delivery, lower weight, longer duration of hospital stay and antibiotics use, and combined use of more than two broad-spectrum antibiotics may increase the risk of fungal pneumonia in neonates. The development of fungal pneumonia may be related to nosocomial infectious bacterial pneumonia in neonates.

OBJECTIVE: To study the effects of different models of mechanical ventilation on inflammatory cytokines, IL-6, IL-10 and TNF-α, in children after cardiopulmonary bypass (CPB). METHODS: Sixty patients who underwent CPB were randomly divided into group A and group B. After CPB, group A was ventilated with high tidal volume (VT, 10-12 mL/kg) /low positive end-expiratory pressure (PEEP, 3-5 cm H2O), while group B was ventilated with low VT (6-8 mL/kg) /high PEEP (6-9 cm H2O). Plasma levels of IL-6, IL-10and TNF-α were measured before operation, at the end of the operation, and 1 and 6 hrs after operation. RESULTS: Serum levels of IL-6, IL-10 and TNF-α in both groups increased significantly at the end of the operation and reached a peak by 1 hr after operation. Group B showed lower serum levels of IL-6, IL-10 and TNF-α than group A 1 and 6 hrs after operation. CONCLUSIONS: Mechanical ventilation with low VT /high PEEP may more effectively inhibit the release of inflammatory cytokines than that with high VT /low PEEP in children after CPB.

OBJECTIVE: To evaluate the efficacy and tolerability of levetiracetam (LEV) in the treatment of epilepsy as a monotherapy in children. METHODS: hirty-two children with epilepsy (age ranged from 8 months to 12 years) and who had received LEV monotherapy were investigated by a self-controlled and open-label research. LEV was administered at a dose of 10 mg/kg?d, and increased by 10 mg/kg?d per week till to the target dose (20-40 mg/kg?d), with a mean dose of 35 mg/kg?d. RESULTS: Thirty-one patients were followed up for more than three months. Twenty-five patients (80.6%) had at least 50% reduction in seizures, 22 cases (70.9%) became seizure-free, and LEV therapy was discontinued in 5 patients (16.1%) due to either an inadequate seizure control or aggravated seizures. The therapy-related adverse events included mood and behavioral changes (6/31, 19.4%), asthenia (2/31, 6.5%), somnolence (2/31, 6.5%), and skin rashes (1/31, 3.2%). The adverse effects were spontaneously disappeared or disappeared after reducing the LEV dose. CONCLUSIONS: LEV monotherapy is effective and safe for the control of partial and generalized tonic-clonic seizures in children with epilepsy. LEV appears to be a promising anti-epileptic drug for monotherapy in children with epilepsy.

OBJECTIVE: To study the clinical features of children with acute pancreatitis complicated by systemic inflammatory response syndrome (SIRS). METHODS: Clinical data of 71 children with acute pancreatitis from May 2005 to June 2007 were retrospectively studied. According to complications with or without SIRS, the patients were classified into two groups: SIRS (n=30) and non-SIRS (n=41). The SIRS group was subdivided into three groups based on the item number consistent with SIRS diagnostic criteria: S1 (2 items), S2 (3 items) and S3 (4 items). RESULTS: More patients had severe abdominal pain (n: 12 vs 3) and fever (n: 25 vs 15) in the SIRS group than in the non-SIRS group (P<0.01).The serum C-reactive protein (CRP) levels (49.61±8.09 mg/L) in the SIRS group were significantly higher than those in the non-SIRS group (7.98±2.82 mg/L) (P<0.01).The serum calcium level (1.93 mol/L) in the SIRS group was statistically lower than that in the non-SIRS group (2.81 mol/L)(P<0.01). Among the SIRS group, the S3 group showed the highest CRP level (120.40±10.04 mg/L), followed by the S2 group (75.78±9.50 mg/L) and S1 group (28.51±8.51 mg/L) (P<0.01). The highest sensitivity (50%) and negative predictive value (96.8%) for the diagnosis of severe acute pancreatitis was obtained for a CRP cut-off at 110 mg/L. The imaging changes were more severe in the SIRS group than in the non-SIRS group. CONCLUSIONS: Severe abdominal pain and fever were common clinical symptoms in children with acute pancreatitis complicated by SIRS. Serum CRP and calcium levels as well as imaging changes are important markers for the severity evaluation of acute pancreatitis. The patients with serum CRP level above 110 mg/L or with more than three items consistent with SIRS diagnostic criteria are at high risk for the development of severe acute pancreatitis.

OBJECTIVE: To study the clinical characteristics, therapeutic regimen and outcome of severe Mycoplasma pneumonia (MP) in children. METHODS: Clinical data of 79 children with MP, including 69 mild and 10 severe cases, were retrospectively analyzed. The 10 children with severe MP were followed-up. RESULTS: In severe MP cases, the fever duration prior to hospitalization and the total fever duration were more prolonged, peripheral blood leucocytes counts, C-reactive protein and erythrocyte sedimentation rate increased, and serum IgM and IgE levels increased as compared to mild MP cases. Of the 10 cases of severe MP, 4 manifested as pulmonary consolidation, 4 as pulmonary consolidation complicated by moderate to large pleural effusion and 2 as progressively worsening pulmonary radiographic findings. Nine severe MP cases were administered with glucocorticoid as well as antibiotics, and the therapeutic effect was satisfactory. In the convalescence stage, bronchofiberoscope lavages were used in 5 severe cases because of persistent pulmonary consolidation. CONCLUSIONS: Severe MP was characterized by rapid progression, pulmonary consolidation, moderate to severe pleural effusion, obviously increased inflammatory indexes, and poor therapeutic reaction to simple macrolide antibiotics. Besides antibiotics, glucocorticoid should be used for severe MP cases as soon as possible. For severe cases with persistent pulmonary consolidation, bronchofiberoscope lavages are recommended.

OBJECTIVE: To investigate the effect of high levels of fluoride on children's intelligence development by analyzing the literatures systematically. METHODS: The literatures on the relationship between endemic fluorosis and children's intelligence development published between 1995 and 2007 were retrieved electronically and manually. A total of 13 relevant literatures that met the inclusion criteria were included for Meta analysis. Meta analysis was performed using the Coorporative network software RevMan 4.3. RESULTS: The subjects of the 13 literatures included 2 508 children from the high fluoride group and 2 330 children from the control group. Meta analysis showed these literatures were inhomogenous (χ2=268.05, P<0.01). Therefore a random effect model was used. The average effect scale (OR=-7.14; 95%CI: -9.90, -4.37) suggested a negative correlation between high fluorosis exposure and children's intelligence development. CONCLUSIONS: Exposure to high levels of fluoride may adversely influence children's intelligence development.

OBJECTIVE: Attention deficit hyperactivity disorder (ADHD) is a common behavioral disorder in children. When the children fulfill cognition tasks, brain θ wave activity increases and β wave activity weakens. This study aimed to explore the efficacy of electroencephalographic (EEG) biofeedback therapy for ADHD in children by assessing the changes of the ratio of brain θ to β waves and the integrated visual and auditory continuous performance test (IVA-CPT). METHODS: EEG biofeedback therapy was performed in 30 children with ADHD. The ratio of brain θ to β waves was measured before and after therapy. IVA-CPT was used to assess the effectiveness of biofeedback therapy. RESULTS: After two courses of treatment, the mean ratio of brain θ to β waves in the 30 children with ADHD was significantly reduced from 12.32±4.35 (before treatment) to 6.54±1.27 (P<0.01). IVA-CPT demonstrated that the values of six indexes measured, including integrate reaction control quotient, integrate attention quotient, auditory and visual reaction control quotients, auditory and visual attention control quotients, were significantly increased after biofeedback therapy (P<0.01). CONCLUSIONS: EEG biofeedback can reduce the ratio of brain θ to β waves and lead to significant decreases in inattention and hyperactivity and it is effective for treatment of ADHD in children.

OBJECTIVE: This study investigated the life style and health status of preterm and term infants' mothers in order to explore the risk factors for preterm delivery. METHODS: A total of 600 matched cases, including 120 pairs of preterm infants and their mothers and 180 pairs of term infants and their mothers were recruited. All of the mothers participated in a questionnaire survey on life style and health status during or before pregnancy. General data of the infants were also collected. Logistics regression analysis was used for evaluating the risk factors for preterm delivery. RESULTS: The height, body weights and body mass index before pregnancy in preterm infants' mothers were significantly lower than those in term infants' mothers (P<0.05). The birth height and weights and Apgar scores in preterm infants were significantly lower than those in term infants (P<0.05). The whole nutritional Knowledge-Attitude-Practice (KAP) scores in preterm infants' mothers were significantly lower than those in term infants' mothers (P<0.05). Multiple factor regression analysis showed that recurrent abortions before gestation (OR=2.332, P<0.05) and premature rupture of membrane before delivery (OR=7.979, P<0.01) were risk factors for preterm delivery, while maternal nutritional KAP scores (OR=0.949, P<0.01) and body weights before pregnancy (OR=0.954, P<0.05) were protective factors. CONCLUSIONS: The women with low body weight, low nutritional KAP scores or recurrent abortions history before pregnancy or presenting premature rupture of membrane before delivery are at high risk for preterm delivery.

OBJECTIVE: To explore a new method of rapid and reliable diagnosis of bacterial infectious diseases such as purulent meningitis and septicemia. METHODS: A pair of universal primers and a set of probes (including universal fluorescence probe, Gram-positive probe and Gram-negative probe) were designed based on the bacterial highly conserved region of 16S rRNA gene. By using the FQ-PCR method, 12 standard strains, 23 clinical cultural isolations and the controls such as HBV, Cryptococcus histolyticus, Blastomyces albicans and human DNA were detected with the three kinds of probes. The correlation among the results of the three kinds of probes detection was analyzed. RESULTS: The determination of 16S rRNA gene with FQ-PCR was a highly specific and sensitive method and not cross-reactive with human DNA, virus or fungi. The least amount of 10 copies of 16S rRNA gene which corresponded to 2 bacteria could be detected with FQ-PCR. Twelve standard strains and 23 clinical cultural isolations were detected by FQ-PCR with the three kinds of probes mentioned above. All samples presented positive results using the universal probe. The results of 16S rRNA gene detected by the Gram-positive probe were positive to the 18 G+ strains. The results of 16S rRNA gene detected by the Gram-probe were positive to the 17 G- strains. CONCLUSIONS: The FQ-PCR technique was established for bacteria quantifying and typing using the universal primer and the double type probes. This method was convenient and rapid in detecting, quantifying and typing bacteria, with a high specificity and sensitivity.

OBJECTIVE: To investigate the protective effects of adenovirus-mediated vascular endothelial growth factor (Ad-VEGF)165 gene transfer against hypoxic-ischemic brain damage (HIBD) in neonatal rats. METHODS: Ad-VEGF recombinant adenovirus was constructed by bacterial homologous recombination technology. Seven-day-old Sprague-Dawley rats were randomly assigned to 4 groups: sham-operated (n=20), HIBD (n=25), buffer-treated (n=20）, and Ad-VEGF-treated (n=25). The HIBD model was prepared by permanent occlusion of left common carotid artery, followed by exposure to 8 % oxygen for 2 hrs. In the Ad-VEGF-treated and the Buffer-treated groups, 2 μL recombinant adenovirus suspension or buffer was injected into the left sensorimotor cortex of the rat brain 3 days after HIBD. Seven days after transplantation, VEGF165 mRNA expression was detected using RT-PCR. Neuronal apoptosis was detected by the terminal deoxynucleotidyl transferase-mediated biotinylated deoxyuridine triphosphate nickel end labeling (TUNEL). CD34 and VEGF protein were detected using immunohistochemistry. Microvascular density in the cerebral cortex was measured based on CD34 positive cells. A radial arm maze test was performed from 30 postnatal days to evaluate long-term learning and memory functions. At 35 postnatal days, the rats were sacrificed for cerebral histological examinations by hematoxylin and eosin. RESULTS: The expression of VEGF165 mRNA increased in the Ad-VEGF-treated group more than in the untreated HIBD and the buffer-treated groups (P<0.05). The number of apoptotic neurons was less in the Ad-VEGF-treated group compared with that in the untreated HIBD and the buffer-treated groups (P<0.05). Microvascular density and VEGF positive cells increased in the Ad-VEGF-treated group compared with that in the untreated HIBD and the buffer-treated groups (P<0.05). In the radial arm maze test, the Ad-VEGF-treated group had more improved achievements than the HIBD and the buffer groups (P<0.05). Neuronal degeneration and necrosis were lessened in the Ad-VEGF-treated group compared with the HIBD and the buffer groups. CONCLUSIONS: Ad-VEGF gene transfer can increase the expression of VEGF mRNA and VEGF protein, decrease neuronal apoptosis, and increase angiopoiesis in the brain. This attenuates brain damage and improves long-term learning and memory functions in neonatal rats after HIBD.