Objective The aim was to study the clinical characteristics, diagnostic standards and genetic features of MELAS and try to find the association between the A3243G mtDNA mutation rate and clinical phenotypes.Methods PCRRFLP for screening the heteroplasmic A to G transition at nucleotide 3243 of the mitochondrial tRNALeu(UUR) of blood cells and muscle, neuroradiological examination, blood level of lactic acid and muscle biopsy analyses were performed on the 272 patients suspected to be with MELAS in Beijing Children′s Hospital，the Capital Medicine University, during the years of 20012008. Eighteen patients with molecular genetic abnormalities of mitochondrial DNA A3243G mutation were taken as the gene diagnosis group for MELAS. Four patients with ragged red fibers on muscle biopsy specimens and with the absence of A3243G mutation were taken as the pathology diagnosis group for MELAS. Comparisons of clinical, laboratorial and neuroradiologic features between two groups were performed. The A3243G point mutation in the mtDNA of blood cells was also detected in some of their parents, maternal relatives(mother and siblings) by using PCRRFLP.ResultsEighteen patients showed heteroplasmy with a mutant load of mtDNA A3243G mutation ranging from 9.0% to 50.0% in blood cells and from 42.4% to 64.8% in muscle tissues in 4 of them. The main clinical features were characterized by epilepsy,exercise intolerance, progressive dementia, fever, vomiting,vision loss, short stature and hypertrichosis on back. Strokelike episodes were found in 7 patients. Laboratory studies revealed elevated serum lactate with levels at 4.2-10.8 mmol·L-1. Cranial CT scan showed calcifications in the bilateral basal ganglia in 9 cases and MRI showed infarctlike lesions in 11 cases, involving the lateral temporaloccipital and parietal lobes in 6 cases, the bilateral parietal occipital lobes in 4 cases, the frontal lobes in 2 cases. The A3243G point mutation in the mtDNA of blood cells was detected in 37 persons from 14 families. The study showed the A3243G mutation was found in 5/10 of the mothers, the mutation rates were 3.0％, 5.0%,11.8%, 21.3% and 26.9%, respectively, and in 4/7 of the siblings 19.3％,33.3%,37.5％ and 41.5%, respectively, all of them were asymptomatic.Conclusions MELAS was the most common maternally inherited mitochondrial disease with various clinical phenotypes and A3243G mutation of the mtDNA accounted for most MELAS in Chinese children. The proportion of mutant mtDNA was not related to the course of the disease but to the age.
Objective Hepatitis B virusassociated nephritis (HBV-GN) is one of the common secondary glomerular diseases in China. However, up to now, the optimal therapy is undefined although several approaches have been made. The aim of this study was to evaluate the efficacy of drug therapy for HBVGN by performing a systematic review of the literature with a metaanalysis method.Methods Medical electronic databases including PubMed, EMBASE, Cochrane Library and CNKI were searched in two languages (English and Chinese) till December 2007, and other sources were manually screened. Two investigators independently identified literatures according to eligibility and exclusion criteria, assessed quality by the Sackett assessment plus Jadadscale, and extracted data. The primary outcome was clinical response (remission of proteinuria), the secondary outcome was virological response (clearance of HBeAg). The fixed effect model or random effect model were used to pool the data, with heterogeneity and sensitivity analyses by software STATA 9.0 and RevMan 4.2. In addition, other data were reviewed and described.ResultsNine studies (8 in English, 1 in Chinese) met the predefined criteria, one of which was randomized controlled trial (RCT), and others were cohort studies. The efficacy of antiviral therapy was assessed by synthesizing 6 out of 9 articles. There were 1 RCT and 5 cohort studies (159 unique patients). Recombinant IFN-α (rIFN-α) was given in most (5/6) studies, and lamivudine therapy in two study. It was found by the metaanalysis that, compared with the control, antiviral therapy could significantly elevate the remission rate of proteinuria ［91.0% vs 56.0%, RR=1.69, 95%CI：1.08-2.65, P=0.02］, and the clearance rate of HBeAg ［59.7% vs 8.33%, RR=6.44, 95%CI：3.11-13.35, P<0.000 01］. Furthermore, Kappa analysis showed a significant link between proteinuria remission and HBeAg clearance after antiviral therapy (P=0.002). On the other hand, the efficacy of corticosteroid treatment was assessed by synthesizing 5 out of 9 articles. All of them were cohort studies (76 unique patients). Metaanalysis showed there was no significant difference between corticosteroid treatment group and control group in the remission rate of proteinuria (RR=1.45, 95%CI：0.68-3.11, P=0.34).Conclusions This study showed the efficacy and safety of antiviral therapy (including IFN and lamivudine) in HBVGN. IFN-α was efficacious in remission of proteinuria, clearance of HBeAg and delay of renal function deterioration. In addition, remission of proteinuria was accompanied by clearance of HBV replication marker, which demonstrated the benefit of antiviral therapy. However, corticosteroid treatment couldn′t improve renal outcome in patients with HBV-GN. There were some shortcomings in our meta-analysis duo to limited high-quality RCTs, thus welldesigned RCTs are still needed before final conclusions is made.
Objective Recently, a lot of papers have been published in abroad which recommended lowdose rapid ACTH1-24 stimulation test instead of largedose ACTH1-24stimulation test when evaluating adrenocortical insufficiency. There was no similar research report in China. This study used ACTH1-39to replace ACTH1-24 for performing lowdose rapid ACTH stimulation test. A self control design was also used to compare the results of lowdose rapid ACTH stimulation test with conventional 2hour ACTH stimulation test. Methods Two kinds of ACTH stimulation tests were performed in 18 patients with nephritic syndrome on alternate days. On day 1, lowdose rapid ACTH stimulation test was performed. 1 U ACTH was injected as an intravenous bolus after collection of a sample at 0 min. Another sample was drawn at 25 min for cortisol measurement. On day 3, conventional 2hour ACTH stimulation test was performed. 25 U ACTH was injected as an intravenous drip for 2 hours after collection of a sample at 0 min. The other sample was attained at 2 hour for cortisol measurement. ACTH1-24 was replaced by ACTH1-39 in both lowdose rapid ACTH stimulation test and conventional 2hour ACTH stimulation test. Student's test and Kappa analysis were used for statistical analysis.ResultsThe results of two kinds of tests were completely equal in 10 cases who took prednisone daily or prior treatment. 8 cases took prednisone every other day, 5 cases of them showed the same results in the two tests. Only 3 cases showed different results in the two tests, who took prednisone more than 1 mg·kg-1 every other day. The lowdose rapid ACTH stimulation test revealed a diminished adrenocortical reserve, while the traditional 2hour ACTH stimulation test was normal. Kappa analysis of paired results showed good concordance. Conclusions ACTH1-39 could substitute ACTH1-24 to perform lowdose rapid ACTH stimulation test. The two kinds of ACTH stimulation tests had good correlation. Compared with the traditional 2hour ACTH stimulation test，the lowdose rapid ACTH stimulation test was more convenient and could be performed in outpatient clinic. So the lowdose rapid ACTH stimulation test may replace the conventional 2-hour ACTH stimulation test for evaluating adrenocortical function.
Objective To explore the prevalence of SLC25A13 gene mutations in Chinese infants with intrahepatic cholestasis and to understand the clinical presentation,laboratory biochemistry and blood amino acids features of patients with mutations.Methods Blood amino acids were analyzed by using mass chromatography in 115 infants who were referred to our hospital for further investigations of intrahepatic cholestasis from December 2003 to December 2006. All exons and their neighbouring sequences of SLC25A13 gene were analysed in children whose plasma citrulline obviously elevated. Two most common mutations of SLC25A13 gene, 851del4 and 1638ins23 were screened in children without obvious citrullinemia. Mutation 851del4 was detected by realtime fluorescent quantitation PCR with double labelling probes, and mutation 1638ins23 was detected by electrophoresis of PCR product directly. If the children in whom only one heterozygous mutation was found, all exons and nearby sequences were analysed then after. Clinical presentation,laboratory biochemistry and blood amino acids examinations were analyzed.ResultsSLC25A13 gene mutations were found in 4 of 5 children with citrullinemia, including 1 patient with homozygous mutation 851del4/851del4, 1 patient with compound heterozygous mutation 851del4/1638ins23, and 2 patients with heterozygous mutation 851del4. SLC25A13 gene mutations were also found in 6 cases of the other 110 intrahepatic cholestasis patients, including 1 patient with homozygous mutation 851del4/851del4, 1 patient with compound heterozygous mutation 851del4/1638ins23, and 4 patients with heterozygous mutation 851del4. Totally, SLC25A13 gene mutations were detected in 10 of the 115 intrahepatic cholestasis patients. The prevalence rate was 8.7％. Liver biopsy was done in 7 of 10 children with SLC25A13 gene mutations, and significant steatosis was found in 4 of them. Laboratory biochemistry changes included obviously elevation of bilirubin,γglutamyl transpeptidase and alkaline phosphatase. The elevation of aspartate aminotransferase was more obvious than alanine aminotransferase. Characteristic amino acids such as citrulin,threonine and methionine elevation was found in 5 children with mutations.Conclusions SLC25A13 gene mutation was one of the important causes of Chinese infantal intrahepatic cholestasis. Liver biopsy, laboratory biochemistry, and blood amino acids examination were important for the diagnosis of SLC25A13 gene mutations, however, the final diagnosis must rely on gene detection.
Objective To explore the value of ATP stress echocardiography and selective coronary angiography(SCAG) for longterm followup in children with coronary artery lesions by Kawasaki disease.Methods The ATP stress echocardiography and SCAG were perfomed on 9 children (1 female and 8 males) with a history of Kawasaki disease(5 with giant coronary aneurysms,3 with coronary aneurysms ,and 1 with dilatation of bilateral coronary arteries in acute stage). The average age of the patients was (7.89±2.62) years(4-12 years) when SCAG was taken，the followup period was 1.5-7years［average（ 3.44±1.67）years］from onset.Results The wall motion abnormality in 6 cases, decreased coronary flow reserve in 5 cases were found by ATP stress echocardiography. SCAG showed : the multiple bilateral coronary aneurysms in 4 cases(1 of them with LAD stenosis; 2 of 4 with right coronary artery tortuous and stenosis); left anterior descending coronary aneurysms with right coronary artery occlusion and collateral branches in 1 case;dilatation of coronary arteries in 4 cases . Comparing with SCAG, 3 distal coronary aneurysms in 2 cases ; coronary stenosis in 3 cases, coronary collateral branches in 2 cases were found by ATP stress echocardiography. Comparing with ATP stress echocardiography, 5 of 6 cases with wall motion abnormalities were found to have coronary tortuous and stenosis in relative diferent arteries by SCAG. Conclusions The coronary artery lesions caused by Kawasaki disease may last for long time，it is nesscery to have longterm follow up for patients with kawasaki disease.The ATP stress echocardiography is safe and reliable for detecting the myocardial ischemia; The selective coronary angiography can show the coronary artery lesions in number, diferent form and site, especially in finding coronary stenosis,distal aneurysms and coronary collateral branches. It is very helpful to evaluate the coronary artery lesions caused by Kawasaki disease for longterm follow-up.
Objective Kawasaki disease (KD) is one of the most common acute vasculitis syndromes in children, and mostly affects arteries, particularly, coronary arteries. Currently, its etiology and etiopathology are still largely unknown. S100 A8, S100 A9 are members of calcium binding protein family, and mostly secreted by activated neutrophils and monocytes. Several studies have demonstrated that they play vital roles in acute inflammation. However, the expression of these two proteins in KD is rarely studied. This project was to investigate functional change of neutrophils in KD and its relevance to the progress of the disease.Methods According to the criteria set in Japan 2002, 32 patients with KD were enrolled in the study. Meanwhile, 20 surgery patients with similar age were enrolled as negative controls. Blood samples were collected at the time of diagnosis before the initiation of intravenous immune globulin(IVIG) treatment, and 1 week after IVIG treatment. To evaluate the activation of neutrophils, partial patients underwent DHR assay. 0.2 mL of heparin anticoagulated venous blood was collected, 50μL of which was incubated in phosphate buffer solution(PBS), phorbol myristate acetate(PMA) for 15 minutes, NFormylMetLeuPhe(fMLP) for 5 minutes at 37℃ respectively, then incubated with 25 μL of dihydrorhodamine(DHR) for 5 minutes at 37℃. After that erythrocytes were removed by adding 2 mL of hemolysis and cells were washed once with PBS, resuspended in 300-500 μL of PBS and then assayed by flow cytometry. Total RNA was extracted from neutrophils and preserved by Trizol reagent. The purity and quantity of RNA were determined according to ultraviolet absorbance at 260nm and 280nm.Gene expression of S100A8 and S100A9 in neutrophils was analyzed by realtime fluorescence quantitative polymerase chain reaction.ResultsAfter incubation with PBS and fMLP, the proportions of activated neutrophils in 11 cases before IVIG treatment were higher than those of the control (PBS: P=0.033; fMLP: P=0.000), decreased after IVIG treatment in 6 self control cases (PBS: from 33.5±21.4 to 16.0±11.7, P=0.109; fMLP: from 78.4±15.9 to 30.1±21.1, P=0.001), reached the control level (PBS:P=0.149; fMLP:P=0.867). Before IVIG treatment in KD patients, the expression of S100 A8,S100 A9 in neutrophils was significantly higher than that of the control (P=0.000). In patients without conronary artery lesions(CALs), the expression levels of S100 A8,S100 A9 mRNA in neutrophils were 6.92±0.59 and 6.80±0.51 respectively. After IVIG treatment, the expressions of these two genes decreased to 6.16±0.81 and 6.00±0.77 respectively (P=0.001 and 0.000). However, in patients with CALs, the expression levels of S100 A8,S100 A9 mRNA were 5.97±0.32 and 6.14±0.45 respectively. After IVIG treatment, the expressions of these two genes increased to 6.66±0.63 and 6.58±0.45 respectively(P=0.038 and 0.12). Conclusions In acute Kawasaki disease, neutrophils not only proliferated but were activated, which demonstrated that neutrophils may participate in the onset of KD. After IVIG treatment, the activity of neutrophils was inhibited in some patients, however, in patients with CALs, neutrophils were constantly activated, suggesting that neutrophils inhibition may be one of the mechanisms of IVIG. And neutrophils may play a valuable role in the development of coronary artery lesions in Kawasaki disease.
Objective To investigate whether the exogenous BDNF or the phosphorylation of cAMP response elementbinding protein(pCREB) influence the hippocampal BDNF expression and the apoptotic procedure in vivo.MethodsSeizures were induced in ARs with lithium and pilocarpine injected intraperitoneally. The rats were intraventricular (IV) injected with BDNF or antipCREB antibody after SC, and sacrificed 1 d later. The levels of BDNF in both hippocampi were determined by quantitative ELISA（pg·μg-1）. The apoptotic cells was quantified by the Annexin VFITC apoptosis detection. The expression of pCREB in hippocampus 30 min after SC was observed by semiquantitative immunohistochemistry.Results①In the normal control group, there was no significant alteration for BDNF levels in the lateral received IVI NS or antipCREB antibody, but the BDNF level in the lateral received IVIBDNF was much higher than that in the nonIVI group and NSIVI group（P＜0.01）.The expression of BDNF was obviously increased in rat hippocampus after SC（P＜0.01）. The administration of IVI NS did not cause marked change of BDNF levels in the lateral received IVI after SC. The level of BDNF in the hippocampus of the lateral received IVI BDNF (0.4 μg)was significantly increased in the normal group and SC group, and was much higher in SC group (55.40±4.11) compared with the normal group(P＜0.01）.Among the SC experimental groups, the BDNF levels were the highest in BDNF IVI group. The level of BDNF in the lateral received IVI antipCREB antibody( 400 μg) was dropped dramatically(P＜0.01）in the SC group compared with that in the other SC experimental group(nonIVI control group, NSIVI group, BDNFIVI group, P＜0.01)and that in the blank control group(P>0.05). In the lateral received IVI antipCREB antibody in the normal group , BDNF level was not significantly increased compared with the blank control (5.94±0.60, 5.91±1.63, respectively).The intraventricular injection(NS or BDNF) had no significant influence on BDNF expression in the contralateral in the normal group or SC group. The intraventricular injection of antipCREB antibody could induce the decrease of BDNF expression in the contralateral after SC. ② In the normal control group, the proportion of apoptotic cells in the lateral received IVI was remarkably higher than that in the the blank control （P＜0.01）. The neuronal apoptosis in hippocampus had no positive correlation with injection .Compared to the time point before SC, the proportion of apoptotic cells in the hippocampus was increased obviously at d1 after SC .After SC , the proportion of apoptotic cells in the lateral treated was remarkably higher than that in the non IVI control group （P＜0.01）. The proportions of apoptotic cells were different because of the three kinds of injectants. The proportion of apoptotic cells in the lateral received IVI was (8.36±0.61)% after IVI NS. The proportion of apoptotic cells was (4.10±1.00)% after IVI BDNF ,which was remardedly lower than that after IVI NS. The proportion of apoptotic cells was (9.37±2.50)% after IVI antipCREB antibody ,which was higher than that after IVI NS.The patterns of neuronal apoptosis in the contralateral were similar to that in the lateral treated . Whether in the normal condition or after SC, the severity degree of apoptosis in the contralateral was less than that in the lateral treated.Conclusions ①The exogenous BDNF could induce the expression of endogenous BDNF and partly inhibit neuronal apoptosis in the lateral hippocampus with intraventricular injection. ② The blockade of CREB phosphorylation induced the apoptotic procedure, probably through suppressing the BDNF expression.
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