Objective: To observe the neuro-protective effect of Levocarnitine on severe hand, foot and mouth disease (HFMD) after enterovirus 71(EV71) infection, to preliminarily explore the possible mechanism preliminarily. Methods: One hundred and thirty-two children with EV71 infection and HFMD combined with serum S100 protein and neuronspecific enolase (NSE) abnormalities who were admitted to Children′s Hospital Affiliated to Zhengzhou University from March 2015 to July 2016 were enrolled in the study.They were divided into the routine group and the Levocarnitine group by the random number grouping method.The routine group (66 cases, including 32 males and 34 females, median age of 2 years and 3 months) was given symptomatic treatment such as antiviral therapy while the Levo-carnitine group (66 cases, including 36 males and 30 females, median age of 2 years and 5 months) was treated with Levocarnitine for neuroprotection on the basis of routine group.Forty healthy children (23 males and 17 females, median age of 2 years and 6 months) who were examined at the Children′s Hospital Affiliated to Zhengzhou University during the same period were selected as the healthy control group.The levels of S100, NSE, soluble apoptosis-related factors (sFas), soluble apoptosis-related factor ligands (sFasL), malondialdehyde (MDA), superoxide dismutase (SOD) in serum were compared between the healthy control group and children with HFMD.The levels of above-mentioned indexes in cerebrospinal fluid and serum, efficacy-related indicators such as duration of fever, white blood cell count on the 3^rd day of treatment, time to remission of nervous system symptoms, time of disease progression and critical conversion rate were compared between 2 groups of children with HFMD.The correlation between sFas, sFasL, MDA, SOD and S100, NSE was performed Results: (1) The levels of S100 [(0.38±0.16) μg/L vs. (0.06±0.23) μg/L], NSE [(43.70±8.80) μg/L vs. 10.10±3.60) μg/L], sFas [(6.61±1.86) μg/L vs. (3.88±1.22) μg/L], sFasL[(101.40±20.7) μg/L vs. (54.4±13.3) μg/L] and MDA[(11.98±2.54) nmol/L vs. (4.08±1.45) nmol/L] in serum of HFMD group were significantly higher than those of the healthy control group (t=-12.245, -22.895, -8.273, -12.803, -17.960, all P<0.05), while the SOD level [(57.10±10.40) kU/L vs. (70.3±14.4) kU/L] was significantly lower (t=5.457, P<0.05). (2) With the extension of treatment time for HFMD children in the two groups, S100 and NSE in cerebrospinal fluid, S100, NSE, sFas, sFasL and MDA in serum decreased, while SOD level increased.On the 3^rd and 7^th day after treatment, S100 (t₃=3.491, t₇=14.434), NSE (t₃=2.920, t₇=23.490) in cerebrospinal fluid, S100 (t₃=5.277, t₇=3.614), NSE (t₃=4.652, t₇=10.525), sFas (t₃=6.399, t₇=7.514), sFasL (t₃=11.155, t₇=8.804) and MDA (t₃=6.348, t₇=7.499) in serum of Levocarnitine group were significantly lower than those of routine group (all P<0.05), while SOD (t₃=3.162, t₇=-3.529) was significantly higher than that of routine group (P<0.05). (3) The relief time of neurological symptom in levocarnitine group was significantly shorter than that in the routine group [(1.23±0.65) d vs. (1.84±0.47) d], and WBC on the 3^rd day after treatment [(9.14±2.93)×10⁹/L vs. (7.12±2.58)×10⁹/L] and the progression time of the disease [(29.74±7.85) h vs. (17.36±8.73) h] were significantly better than the those in the routine group (t=-6.178, 4.204, 8.567, all P<0.05). The critical conversion rates of Levocarnitine group and the routine group were 7.58% and 18.18%, respectively, and the difference in critical conversion rate was not statistically significant (χ²=2.316, P>0.05). (4)There was a positive correlation between S100 and sFas, sFasL, MDA in children with HFMD (r=0.373, 0.735, 0.334, P<0.05). NSE was positively correlated with sFas and sFasL (r=0.479, 0.601, all P<0.05), while SOD and S100 were negatively correlated with NSE (r=-0.425, -0.460, all P<0.05). Conclusions Levocarnitine has good curative effect on severe HFMD in children infected by enterovirus EV71, which can effectively protect the cranial nerves.The mechanism may be related to scavenging oxygen free radicals and blocking nerve cell apoptosis.

Objective:To explore the clinical significance of serum Chromogranin A (CGA) level in predicting the prognosis of children with severe hand, foot, and mouth disease (HFMD) and complicating neurogenic pulmonary edema (NPE).Methods:A total of 162 patients with HFMD admitted in our hospital from January 2017 to December 2019 were enrolled in the study; and 40 age-matched healthy children were selected as controls. According to the disease severity and complication the patients were divided into three groups: mild group ( n=88), severe without NPE group ( n=46) and severe with NPE group ( n=28). In 72 severe HFMD patients 16 cases died (fatal group) and 56 cases survived (survival group) within 28 days of hospitalization. The serum CGA, LAC, GLU, WBC, PCT, IL-6, cTnT were measured in all subjects. SPSS 23.0 software was used for data analysis, and the receiver operating characteristic (ROC) curve was used to evaluate the various indicators for predicting the prognosis of severe HFMD combined with NPE. Results:The serum CGA, GLU, LAC, IL-6 and cTnT levels in severe HFMD group with NPE significantly higher than those in the other three group ( H=61.554, 79.031, 86.994, 36.477, 75.021, all P<0.05 ). The serum CGA, LAC, GLU and IL-6 levels in the fatal group were significantly higher than those in survival group ( Z=-6.094, -4.621, -4.283, -5.504, all P<0.05). There was no significant difference in the levels of WBC, PCT and cTnT between the survival group and the fatal group ( P>0.05). The area under the receiver operating curve (AUC) of serum CGA was 0.890 (95% CI: 0.833-0.947) for predicting the prognosis of patients and the best cut-off value was 120.59 μg/L. Conclusion:The detection of serum CGA levels may be beneficial for the early diagnosis of severe HFMD with NPE, and can be used as one of the predictors of death from severe HFMD.

BACKGROUND:Primary cortical neurons and microglial cells play a crucial role in exploring cell therapies for neurological disorders,and most of the current methods for obtaining the two types of cells are cumbersome and require separate extraction.It is therefore crucial to find a convenient and rapid method to extract both types of cells simultaneously. OBJECTIVE:To explore a novel method for simultaneous extraction of primary cortical neurons and microglial cells. METHODS:Newborn suckling SD rats were taken within 24 hours.The brain was removed and placed in a dish with DMEM,and the pia mater was removed for later use.Primary neurons were extracted from the same brain tissue,and then the remaining brain tissue was used to extract microglial cells.The whole process was performed on ice.Extraction and culture steps of primary cortical neurons:The cerebral cortex was taken 2.0-3.0 mm with forceps,and the tissue was digested with papain for 20 minutes.After aborting digestion,the blown tissue presented an adherent tissue suspension.The supernatant cell suspension was obtained,filtered,and dispensed into 15 mL centrifuge tubes.After centrifugation and re-suspension,the cells were inoculated onto 6-well plate crawls coated with L-polylysine.Neuronal morphology was observed at 1-day intervals,and staining could be performed for identification using immunofluorescence staining of MAP2 and β-Tubulin by day 7.Microglia extraction and culture steps:The remaining brain tissue at 8-10 mm thick was subjected to microglial cell extraction,digested by trypsin for 20 minutes.After digestion was stopped,the tissue was blown to a homogenate,and then the homogenate was transferred to the culture bottle for culture.On day 14,the culture flasks were sealed and subjected to constant temperature horizontal shaking for 2 hours.Microglial cells were shed in the supernatant.Purified microglial cells were taken and continued to be cultured for 3 days for identification by Iba1 immunofluorescence staining. RESULTS AND CONCLUSION:(1)After 24 hours of culture,the neurons were adherent to the wall,the cytosol was enlarged,and some neurons developed synapses.After 3 and 5 days of culture,the cytosol was further enlarged,and most of the neurons were in the form of synapses,and some neurons were growing in clusters.On day 7,neuronal synapses were prolonged and thickened,and they were connected with each other to form a network.The neurons were identified by β-Tubulin and MAP2 immunofluorescence staining.(2)The cells grew close to the wall on day 1 of culture.On days 3,5,and 7,the density of microglial cells was small,and the cell morphology was bright oval or round,but the cells basically grew in clumps on the upper layer of other cells.On day 10,the density of microglial cells increased significantly.On day 14,microglial cells grew in dense clumps on the upper layer of other cells,and then they could be isolated and purified.The isolated and purified cells were taken and re-cultured to day 3 and identified as microglial cells by Iba1 immunofluorescence;their purity was greater than 95%.(3)The results show that primary cortical neurons and microglial cells obtained by this method after extraction and culture are of high purity,good morphology,and high viability.

Objective To investigate the significance of matrix metalloproteinase-9 (MMP-9) and tissue inhibitor of metalloproteinase-1 (TIMP-1) in serum and cerebrospinal fluid for evaluation of severe hand ,foot ,and mouth disease (HFMD) complicated with neurogenic pulmonary edema (NPE).Methods A total of 140 patients diagnosed with HFMD in Henan Children′s Hospital were enrolled and divided into three groups including mild group ,severe HFMD group without NPE ,severe HFMD group with NPE .These severe HFMD patients were also divided into survival group and death group according to the 28-day prognosis .Meanwhile ,50 age-matched healthy children were selected as controls .Serum MMP-9 and TIMP-1 levels were measured in all enrolled children .At the same time ,MMP-9 ,TIMP-1 and ratio of MMP-9／TIMP-1 in cerebrospinal fluid were measured in the severe HFMD group with and without NPE .Quantitative data were compared using one-way analysis of variance , and means comparisons between samples were conducted using LSD-t test .Results Among 140 children with HFMD ,66 were in mild group ,42 in severe HFMD without NPE group ,and 32 in severe HFMD with NPE group .And 50 healthy children were in control group .After 28 days ,14 cases died in severe HFMD groups .MMP-9 , TIMP-1 and MMP-9／TIMP-1 in serum of severe HFMD group with NPE increased significantly greater than those in the other three groups (F＝269 .356 ,121 .301 and 101 .502 ,respectively ,all P ＜0 .05). MMP-9 ,TIMP-1 and MMP-9／TIMP-1 in cerebrospinal fluid of severe HFMD group with NPE were (57 .24 ± 8 .92) μg／L ,(35 .26 ± 8 .14) μg／L and (1 .66 ± 0 .23) μg／L ,respectively ,while those in cerebrospinal fluid of severe HFMD group without NPE were (30 .57 ± 3 .89) μg／L ,(26 .25 ± 0 .32) μg／L and (1 .17 ± 0 .61) μg／L ,respectively .The differences between the two groups were all statistically significant (t＝62 .485 ,37 .680 and 169 .387 ,respectively ,all P＜0 .01).MMP-9 ,TIMP-1 and MMP-9／TIMP-1 in serum and cerebrospinal fluid of death group increased significantly greater than those in survival group ,the difference were statistically significant (all P＜0 .01).The maximum area under curve (AUC) was reached when the MMP9／TIMP-1 ratio in cerebrospinal fluid was 0 .890 (95% CI :0 .801 －0 .978).Conclusions MMP-9 and TIMP-1 may be involved in the pathogenesis of HFMD complicated with NPE .The detection of MMP-9 and TIMP-1 levels may be beneficial for the early diagnosis of severe HFMD with NPE .The imbalance of MMP-9／TIMP-1 ratio can be used as one of the predictors of severe HFMD combined with NPE.

Objective:To examine the effect of short-term regular aerobic exercise on physical fitness of children with pulmonary with atresia ventricular septal defect after radical biventricular treatment.Methods:This was a prospective self pre-and post-control observation study. The subjects performed regular aerobic exercise for 10 days according to the exercise prescription. Body composition measurement and cardiopulmonary exercise test[lung ventilation function, maximum oxygen uptake(VO 2max), maximum oxygen pulse(O 2/HR max), ventilation oxygen uptake efficiency(OUES), exercise load time], 6 min walking distance(6MWD), sports psychometric test, motor function screening test and fitness test, were collected. The changes of test parameters and scale scoring before and after exercise were analyzed and compared. Results:A total of 7 children with PA/VSD after biventricular surgery were enrolled. The age ranged 8.2-16.2 years old, and there were 2 males and 5 females. VO 2max[(1 196.71±395.31)ml/min vs.(1 297.43±425.73)ml/min, P=0.031], O 2/HRmax[(82.43±7.53)ml/beat vs.(91.57±6.95)ml/beat, P<0.001]increased after exercise. The exercise load time was significantly increased compared with that before intervention[(476.43±35.73)s vs.(531.43±45.76)s, P=0.002]. Resting heart rate before exercise( P=0.013) and peak respiration exchange ratio(PeakRER, P=0.021) were significantly lower. Body composition tests suggest weight, intracellular water, protein and muscle content of lower limb were higher( P<0.05). The motor function score was higher than before( P=0.015); the score of sports fear was lower than before( P=0.009). There was no significant difference in lung capacity and 6-minute walking distance before and after exercise( P>0.05). There were no cardiovascular events during the study period. Conclusion:Short-term regular aerobic exercise for children with PA/VSD after biventricular surgery can improve exercise tolerance, increase lower limb muscle content, improve exercise fear and exercise function, and has good safety and feasibility.

Objective: To investigate the value of abnormal expression of HLA-DR on peripheral blood monocytes in evaluating the immune function status, clinical prognosis and severity of patients with hand, foot and mouth disease (HFMD). Methods: From June 2017 to October 2018, 100 cases of mild HFMD, 80 cases of severe HFMD, 32 cases of critical HFMD and 40 healthy children (control group) were recruited in this study. The patients were divided into two groups, lower DR group (DR-L, HLA-DR expression<30%) and normal DR group (DR-N, HLA-DR expression>30%) according to the HLA-DR expression on monocytes. Flow cytometry was used to detect the CD14⁺ monocytes expressing HLA-DR and the absolute count of lymphocyte subsets. Immunoturbidimetry was used to detect the levels of IgG, IgM and IgA in plasma samples. Enzyme-linked immunosorbent assay (ELISA) was performed to detect the levels of IFN-γ and IL-10 in plasma samples. Pediatric critical illness score (PCIS) and the pediatric risk of mortality Ⅲ (PRISM Ⅲ) were used to estimate the severity of HFMD. Results: ① There were significant differences in HLA-DR expression on monocytes among children with mild, severe and critical HFMD (F=47.102, P<0.05). Patients with critical HFMD had the lowest HLA-DR expression (P<0.05). ② The numbers of CD14⁺ monocytes, CD3⁺ T cells, CD4⁺ T cells, CD8⁺ T cells, B cells and NK cells in peripheral blood of the DR-L group were significantly lower than those of the DR-N group and the normal group, especially in patients with severe or critical HFMD (P<0.05). ③ There was no significant difference in the level of IgG, IgA or IgM among the DR-L, DR-N and control groups (P>0.05). ④ Compared with the DR-N group, the DR-L group showed decreased IFN-γ level and increased IL-10 level in plasma (P<0.05). The ratio of IFN-γ/IL-10 of the DR-L group was lower than that of the DR-N group and control group (P<0.05). HLA-DR expression was negatively correlated with the concentration of IL-10 in plasma (r=-0.704, P<0.05), and positively correlated with the IFN-γ/IL-10 ratio (r=0.773, P<0.05). ⑤ Compared with the DR-N group, the DR-L group showed lower PCIS and higher PRISM Ⅲ. HLA-DR expression was positively correlated with PCIS (r=0.715, P=0.00) and negatively correlated with PRISM Ⅲ (r=-0.610, P=0.00). ⑥ The incidence of pulmonary edema, pulmonary hemorrhage and cardiopulmonary failure and the mortality of HFMD patients in the DR-L group were significantly higher than those in the DR-N group (P<0.05). Conclusions Patients with severe or critical HFMD had cellular immune dysfunction and abnormal HLA-DR expression on CD14⁺ monocytes. Assessing the expression of HLA-DR on monocytes could be used to evaluate the cellular immunity of patients with severe or critical HFMD. Lower expression of HLA-DR on CD14⁺ monocytes might be associated with severe HFMD and poor prognosis.