Objective To search for the changes of T cells subpopulations and immunoglobulin and their relation-ship in children patients with simple nephrotic syndrome. Design Case-control research. Patients aud Participants 39 patients with simple nephrotic syndrome were divided into two groups:the incipient group and relapse group (6 cases were determined at the incipient and relapse time) .Thereare 28 patients in incipient group, 19 males and 9 females, at the age of 2 to 10 years old. There are20 patients in relapse group, 12 males and 8 females, at the age of 3 to 13 years old. There are 35health children in control group, 21 males and 14 females, 2～13 years old. Interventions T cells subpopulations were determined by indirect immunofluorescence of OKT linesmonoclonal antibodies. The serum IgG was determined by routine simple agar immunodiffusion tests. Results and Conclusions The CD_3~+ and CD_4~+ cells are of no change in the children patients withsimple nephrotic syndrome, and the CD_8~+ and CD_(10)~+ cells are obviously increased, the Values of CD_4~+/CD_8~+ are obviously lower than those in the control qroup, there are no difference between the incipientand relapse groups. The levels of serum IgG were decreased in the 85.3% children patients, IgM were inc-reased in 29.4% of that. The values of CD_4~+/CD_8~+ have positive correlation and negative correlationwith the levels of serum IgG and IgM respectively.

This study was purposed to investigate the efficacy of different whole flow lysis reagents for lysis of red blood cells in flow cytometric analysis. The expression of immunocytes was detected by flow cytometry after lysis of red blood cells using commercial reagents (Optilyse C, FACS Lysing Solution) and self-made red blood cell lysis reagents (RBC Lysis Buffer), the detection results were analyzed comparatively. The results showed that there was no significant difference in the percentage of CD3e(+), CD3e(+)CD4(+), CD3e(+)CD8a(+), CD3e(-)CD19(+), CD3e(-)NK1.1(+) and Gr-1(+) cells between 3 different lysis reagent groups. However OptiLyse C solution was suitable to Gr-1(+) cell detection, but did not suit to Foxp3(+) Treg detection. The self-made RBC Lysis Buffer and FACS Lysing Solution were suited to Foxp3(+) Treg detection. It is concluded that the use of self-made RBC Lysis Buffer for flow cytometry can get the lysis efficiency of commercially available lysis solutions when samples are prepared in accordance with standardized procedure. The self-made RBC Lysis Buffer not only can satisfy experimental requirements, but also can reduce the experimental costs.
Animals ; Erythrocyte Count ; Erythrocytes ; immunology ; metabolism ; Flow Cytometry ; instrumentation ; methods ; Immune System ; immunology ; Indicators and Reagents ; analysis ; Mice ; Mice, Inbred C57BL

AIMTo study the effects of ginkgolides (Gin) on the expression of hypoxia-inducible factor-1alpha (H1F-1alpha) in primary cultured cortical neurons treated with CoCl2 and the relationship with ERK signal pathway.

METHODSWe observed the effects of Gin (37.5 mg/L) on morphology and viability on primary cultured cortical neurons with treatment of CoCl2 (125 micromol/L). The expression of HIF-1alpha and p-ERK of neurons induced by CoCl2 pretreated with Gin were assessed by Western-blot. We analyzed the relationship between HIF-1alpha expression activated by Gin and ERK signal pathway with treatment of PD98059 (100 micromol/L), a selective inhibitor of ERK.

RESULTSIt was shown that Gin had protective effects on CoCl2 damaged neurons by raising the neuronal viability. Some basic expression of HIF-1alpha and p-ERK were observed in normal cultured cortical neurons. The expression of HIF-1alpha and p-ERK increased strikingly when treated with CoCl2 for 4 h. The levels of HIF-1alpha and p-ERK increased even more in the neurons pretreated with Gin for 24 h before CoCl2. The levels of HIF-1alpha and p-ERK were notably inhibited with pretreatment of PD98059, while Gin could prevent this inhibition.

CONCLUSIONGin has protective effects on neurons damaged by CoCl2 which might be related to the increase of the level of HIF-1alpha and the activation of ERK signal pathway.

Animals ; Cell Hypoxia ; drug effects ; Cells, Cultured ; Ginkgolides ; pharmacology ; Hypoxia-Inducible Factor 1, alpha Subunit ; metabolism ; MAP Kinase Signaling System ; Mice ; Mice, Inbred ICR ; Neurons ; drug effects ; metabolism

OBJECTIVETo explore the relationship between gene expression of human telomerase reverse transcriptase (hTERT) and its clinical characteristics in leukemia.

METHODSThe protocol of RT-PCR was used to detect the hTERTmRNA expressing levels in peripheral blood samples from leukemic patients under primary treatment(n=42), in complete remission(n=21), with recurrent leukemia (n=4); and from normal subjects (n=5), respectively.

RESULTSThe positive percentage of hTERTmRNA expression was 73.81% for the primary treatment cases, and 19.05% for the complete remission cases. All of the recurrent cases gave positive results. One of the normal controls presented low level of hTERTmRNA expression. The expressing level of hTERTmRNA in primary treatment cases was 0.64+/-0.21, in complete remission leukemia 0.31+/-0.16, in recurrent cases 0.84+/-0.09, and in normal controls 0.10.

CONCLUSIONThe activation of telomerase may be an essential factor in the development of leukemia and usually be the late event in its progression. As an indicator of leukemia cell, the detection of hTERT mRNA may be used in clinical analysis, disease monitoring and prognosis judgement.

Acute Disease ; Adolescent ; Adult ; Child ; Child, Preschool ; DNA-Binding Proteins ; Female ; Gene Expression Regulation, Enzymologic ; Gene Expression Regulation, Neoplastic ; Humans ; Infant ; Leukemia ; genetics ; pathology ; Male ; Neoplasm Recurrence, Local ; RNA, Messenger ; genetics ; metabolism ; Remission Induction ; Reverse Transcriptase Polymerase Chain Reaction ; Telomerase ; genetics

This preject is to explore the reversal efficacy of calmodulin antagonist berbamine (BBM) on multidrug resistance (MDR) and its mechanism. Human erythroleukemic cell line K562 and its adriamycin-resistant counterpart K562/A02 were used in the study. The cells were co-cultured with ADR and BBM in different concentrations. MTT assay was used to analyze the effect of BBM on cell growth inhibition. According to the MTT assay, the 50% inhibitory concentration (IC(50)), the multiples of drug resistance and increased sensitivity of ADR were calculated. The concentration of intracellular ADR and expression level of P-gp were detected by flow cytometry (FCM). The expression level of mdr1 mRNA and survivin mRNA was detected by semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) with beta-actin as internal reference. The results showed that IC(50) of ADR in K562 and K562/A02 cells was 1.16 +/- 0.09 micro mol/L and 37.47 +/- 1.76 micro mol/L, respectively. The resistant multiple of K562/A02 cells to ADR was 32.30 higher than that of K562 cells. BBM increased the chemo-sensitivity of ADR in K562/A02 cells with dose-dependent relationship, i.e. when 5, 10 and 20 micro mol/L BBM was added in the culture the chemo-sensitivity of ADR was increased to 2.01-, 9.68-, and 41.18-fold (P < 0.01), respectively. After treating K562/A02 cells by 5 or 10 micro mol/L BBM for 2 hours the accumulation of intracellular ADR was increased to 1.41- and 1.52-fold (P < 0.01), respectively. Treating by BBM for 72 hours decreased 4.12% (P < 0.05) and 27.09% (P < 0.01) of P-gp expression, respectively, meanwhile down-regulated expression of mdr1 mRNA and survivin mRNA was found. In conclusion, BBM could increase intracellular concentration of ADR in K562/A02 that down-regulated expression level of mdr1 mRNA and P-gp and survivin so that the sensitivity of K562/A02 to ADR was increased significantly.
ATP-Binding Cassette, Sub-Family B, Member 1 ; analysis ; Alkaloids ; Benzylisoquinolines ; pharmacology ; Calmodulin ; antagonists & inhibitors ; Cell Division ; drug effects ; Doxorubicin ; pharmacokinetics ; pharmacology ; Drug Resistance, Multiple ; Drug Resistance, Neoplasm ; Genes, MDR ; Humans ; Inhibitor of Apoptosis Proteins ; K562 Cells ; Leukemia ; drug therapy ; Microtubule-Associated Proteins ; genetics ; Neoplasm Proteins ; RNA, Messenger ; analysis

0.05). The RBC folate level of birth defect group except the urinary defect was significantly lower compared with the control group(233-547 vs 689 nmol/L,P

OBJECTIVETo screen and diagnose fatty acid oxidation disorders (FAOD) in high risk children with inborn error of metabolism using tandem mass spectrometry.

METHODSThe study group consisting of 2941 high risk cases of suspected inborn error of metabolism was tested. The acylcarnitines in the dry blood filter papers of patients were tested by tandem mass spectrometry. The diagnosis of FAOD was according to the levels of the acylcarnitines, the clinical symptoms, and other biochemistry study.

RESULTSFourteen patients were diagnosed as FAOD. These patients included one carnitine palmitoyltransferase deficiency I, one carnitine palmitoyltransferase deficiency II, one short-chain acyl-CoA dehydrogenase deficiency, seven medium-chain acyl-CoA dehydrogenase deficiency, two very long-chain acyl-CoA dehydrogenase deficiency, and two multiple acyl-CoA dehydrogenase deficiency.

CONCLUSIONFAOD are not rare in China. Analysis of acylcarnitines levels tested by tandem mass spectrometry is helpful to diagnose FAOD.

Adolescent ; Amino Acid Metabolism, Inborn Errors ; diagnosis ; genetics ; Carnitine ; analogs & derivatives ; chemistry ; Child ; Child, Preschool ; Female ; Humans ; Infant ; Infant, Newborn ; Lipid Metabolism ; physiology ; Lipid Metabolism, Inborn Errors ; diagnosis ; Male ; Mass Spectrometry ; Tandem Mass Spectrometry ; methods

OBJECTIVETo investigate the development of differential diagnosis of tetrahydrobiopterin (BH4) deficiency among patients with hyperphenylalaninemia (HPA) in provinces or cities of China and to investigate the incidence of BH4 deficiency.

METHODSOf the thirteen hundreds and ninety-two patients with HPA received, the differential diagnosis for BH4 deficiency during 1993 - 2007 were enrolled in this study. Of which, 591 patients came from outpatient and 801 patients' samples from other provinces or cities were sent to author's laboratory to investigate the case number of differential diagnosis for BH4 deficiency in provinces or cities of China according to the data from both outpatient case histories and laboratory as to investigating the development of differential diagnosis in the whole country. To discuss the diagnostic criteria for BH4 deficiency was according to the results of urinary pterin analysis, determination of dihydropteridine reductase (DHPR) activity and the tetrahydrobiopterin loading test as well as to get the incidence of BH4 deficiency and find some provinces or cities with higher incidence of BH4 deficiency in China.

RESULTS(1) The number of HPA patients, who were performed by urinary pterin analysis and the determination of DHPR activity, were remarkably increased in last three years (2005 - 2007). The patient numbers of both urinary pterin analysis and DHPR activity determination were 217 and 198 respectively in 2005. And in 2007 they increased to 511 and 458, which was about 2.3 times than that in 2005. The patients came from 29 provinces or cities in 2007. (2) The urinary biopterin and biopterin percent were key marks for diagnosis of 6-pyruvoyl tetrahydropterin synthase (PTPS) deficiency. The less than 5% [(1.41 +/- 1.10)%] biopterin percent and very low biopterin level [(0.14 +/- 0.17) mmol/mol Cr] were found in 96.83% (61/63) patients with PTPS deficiency in this study. The blood phenylalanine level was remarkably decreased to normal range at 2 - 6 hours after BH4 loading test. The very low DHPR activity was a final diagnostic mark for DHPR deficiency. The very low DHPR activities of 0.27 nmol/(min x 5 mm disc) (6.11% - 7.00% of normal controls) were found in two patients with DHPR deficiency in this study. (3) The incidences of PTPS deficiency and DHPR deficiency among 1392 patients with hyperphenylalaninemia were 8.41% (117/1392) and 0.18% (2/1108) respectively. About 67.23% (80/119) patients with BH4 deficiency came from the south of Yangtze liver. The 80% (8/10) provinces or cities with higher incidence of BH4 deficiency are located in eastern and southern China. The incidence of PTPS deficiency among patients with HPA and normal newborns was 10.81% (8/74) and 0.007 per thousand (8/1,121,429) respectively in Shanghai, China according to data from neonatal screening.

CONCLUSIONThe awareness of differential diagnosis for BH4 deficiency from clinic pediatricians has been increased in most provinces or cities of China in last three years, but it should be more strengthened.

Biopterin ; analogs & derivatives ; deficiency ; China ; epidemiology ; Diagnosis, Differential ; Humans ; Incidence ; Infant, Newborn ; Neonatal Screening ; Phenylketonurias ; complications ; diagnosis ; epidemiology

OBJECTIVETo explore the molecular mechanisms of Glanzmann thrombasthenia caused by alpha II b L721R and Q860X compound heterozygous mutation.

METHODSAll exons and exon-intron boundaries of alpha II b and beta3 gene were amplified by PCR and analyzed by direct DNA sequencing. Gene polymorphisms were excluded by direct DNA sequencing. Alpha II b L721R and Q860X mutants expressing vectors were constructed by in vitro site-directed mutagenesis. The expression of alpha II b L721R and Q860X mutants on transfected cell membrane were analyzed by flow cytometry and the whole expression level was confirmed by Western blot. The subcellular localizations of alpha II b L721R and Q860X mutants were determined by immunofluorescent confocal scanning microscopy.

RESULTSThe alpha II b compound heterozygous mutations, T2255G (L721R) and C2671T (Q860X), were identified in the proband, the former being inherited from the maternal side and the latter the paternal side. The 293T cells cotransfected with mutated alpha II b L721R and wild-type beta3 expression plasmids expressed 2.1% of normal amount of alpha II b on the cell surface as shown by FACS, in contrast to 31.9% of normal amount of alpha II b on the cells cotransfected with cDNAs of mutated alpha II b Q860X and wildtype beta3 expression plasmids. Western blot of the cell lysates showed no detectable mature alpha II b in cells lysates with L721R mutant. While, truncated alpha II b protein was detected in cell lystes with Q860X mutant. Immunofluorescence studies demonstrated that both L721R and Q860X mutant pro-alpha II bbeta33 complex colocalized in endoplasmic reticulum, but a little in Golgi.

CONCLUSIONSThe L721R and Q860X mutations of alpha II b prevent transport of the pro-alpha II bbeta3 complex from the endoplasmic reticulum to the Golgi, hindering its maturation and surface expression. The impaired alpha II bbeta3 transport is responsible for the thrombasthenia.

Animals ; CHO Cells ; Child, Preschool ; Cricetinae ; Cricetulus ; Female ; Genetic Vectors ; Heterozygote ; Humans ; Integrin alpha2beta1 ; genetics ; metabolism ; Mutagenesis, Site-Directed ; Mutation ; Thrombasthenia ; genetics ; Transfection

OBJECTIVEAnti-influenza virus activity of "Benovoair Concentrate".

METHODSThe different dilution of samples were mixed with the same quantity of 100 TCID50 virus at 37 degrees C for 30 minutes. Add suitable quantity mixture in wells containing cells. Every 3 wells were the same mode. Viruses control, cells control and samples control of different dilution were performed and set in the CO2 incubator at 37 degrees C. CPE was observed every day. When CPE appears in viruses control as "++++", stopped testing and performed the hemagglutination titration.

RESULTS"Benovoair Concentrate" with dilution of 1:60, 1:120, 1:240 and 1:480 have 100% anti-influenza A and anti-influenza B activities. "Benovoair Concentrate" with dilution of 1:960 and 1:1920 have 25%-50% anti-influenza A and anti-influenza B activities.

CONCLUSIONThe test was the proof of anti-influenza virus activities which provided for the development of "Benovoair Concentrate".

Air Microbiology ; Animals ; Cell Line ; Dogs ; Drugs, Chinese Herbal ; pharmacology ; Oils, Volatile ; pharmacology ; Orthomyxoviridae ; drug effects ; Plant Oils ; pharmacology