Objective To identify the mutational genotype in seven Chinese families with X-linked adrenoleukodystrophy (X-ALD). Methods The coding region of ABCD1 gene of seven patients was amplified in 4 segments by PCR after reverse transcription using RT-PCR technology. The PCR products were purified and directly sequenced. To confirm the mutations, the genomic DNA was analyzed by PCR-restrictive digestion or direct sequencing of purified PCR products. Results Six base substitutions (709CA, 807GA, 1161CT, 2065CT, 2113TC and 2235CT), one base deletion (1801delAG) and one base insertion (1126 ins GCCATCG) were identified in seven X-linked adrenoleukodystrophy pedigrees, resulting in five missense mutations (A141T, R259W, P560L, L576P and R617C), two frame shift mutations (fs I246 and fs E471) and one nonsense mutation (S108X), respectively. Conclusion Four novel ABCD1 mutations, namely S108X, fs I246, R259W and L576P, were detected in Chinese X-linked adenoleukodystrophy patients. There was different ABCD1 gene mutation in different pedigree and no obvious correlation between the type of mutation and phenotype was found.

Objective To identify hardly recognizable victims of an accident. Methods Tissues of muscle and cartilage were obtained from the dead bodies. Some of the tissues were checked by routine pathological microscopy. Genomic DNA was isolated from the tissues and subjected to STR profiling of 16 sites via multiple fluorescent PCR analysis with ABI’s AmpFLSTR Amplification Kit. Individual identification of the victims was carries out by matching the STR profiles of the victims with those of the parents. Results Routine pathological microscopy showed that the structure of some of the muscle tissues was totally destroyed, while the structure of all cartilage tissues was basically intact. Three patterns of genomic DNA isolated from victims’ muscle tissues could be seen in gel electrophoresis, i.e. basically undegraded, partially degraded and totally degraded. STR profiling failed due to the degradation of genomic DNA of some of the muscle tissues, while all samples of the cartilage genomic DNA could be used for STR typing. Conclusion Paternity identification based on STR genotyping was an effective way to identify victims of accidents, and cartilage tissue from the victims was the first choice for that purpose.

T),one dinucleotide deletion(1801-02 del AG) and one base insertion(1125 ins GCCATCG),which resulted in eight missense mutations,two nonsense mutations and two frame shift mutations,namely P534R,G343V,R259W,A141T,R401Q,K276E,Y174C,A314P,S108X,Q177X,fs E471 and fs A247.Conclusion The combined DHPLC and sequencing approach may act as a rapid and efficient method for ABCD1 gene mutation analysis in patients and carriers of X-linked adrenoleukodystrophy families.There exist different ABCD1 gene mutations in different pedigrees,and no obvious correlation between the genotype and phenotype has been found.

Objective To investigate antibiotic resistance of Klebsiella pneumoniae and Escherichia coli in old patients with lower respiratory tract infections. Methods Kirby Bauer agar diffusion method was used to evaluate the drug sensitivity in 240 strains of Klebsiella pneumoniae and Escherichia coli isolated from patients with lower respiratory tract infection. Phenotypic confirmatory test recommended by NCCLS1999 was used to detect extended spectrum beta lactamases(ESBLs). Results The resistant rates of Klebsiella pneumoniae and Escherichia coli to 14 antibiotics in old patients and in non old patients with lower respiratory tract infections were amoxicillin 93 2% vs 87 3%, piperacillin 57 1% and 42 9%, cefuroxime 51 4% and 33 3%, cefotaxime 40 1% and 17 5%, ceftazidime 13 6% and 3 2%, ceftriaxone 39 0% and 17 5%, cefoperazone 37 3% and 15 9%, cefepime 10 2% and 3 2%, amikacin 47 5% and 34 9%, ciprofloxacin 54 2% and 38 1%, imipenem 0, cefoperazone/sulbactam 0, piperacillin/tazobactam 1 1% vs 0, and cefmetazole 9 6% and 4 8% respectively. Out of 240 clinical strains of Klebsiella pneumoniae and Escherichia coli, 78(32 5%) were considered ESBLs producers by phenotypic confirmatory test. The prevalence of ESBLs in old patients was 38 4%, which was much higher than that in non old patients(15 9%). The resistant rate of ESBLs producing strains to imipinem, cefoperazone/sulbactam, piperacillin/tazobactam and cefmetazole was the lowest, being 0, 0, 2 6% and 12 8%. Conclusions The resistant rates of Klebsiella pneumoniae and Escherichia coli to most antibiotics and the prevalence of ESBLs in old patients with lower respiratory tract infection were higher than that in non old patients. Imipinem, cefoperazone/sulbactam, piperacillin/tazobactam and cefmetazole were the effective antibiotics to infections caused by ESBLs producing strains.

T2DM+LIRA group and T2DM+LIRA+UC-MSCs group (P<0. 05). The ratio of insulin positive area in pancreas tissue was obviously rised, while the ratio of glucagon positive area in pancreas tissue was clearly descended in T2DM+LIRA+UC-MSCs group. And the same difference in valuating islet cells apoptosis by TUNEL could be observed ( P<0. 05). The expression of NF-κB and TLR4 protein in pancreas tissue of T2DM+LIRA+UC-MSCs group were the least amongthefourgroups[(0.75±0.10)vs(0.60±0.08),(0.47±0.08),(0.31±0.04),P<0.05]and[(1.24± 0. 12) vs (0. 93 ± 0. 10), (0. 95 ± 0. 09), (0. 74 ± 0. 07), P<0. 05 ]. Conclusion The combined treatment of liraglutide with umbilical cord mesenchymal stem cells is superior over a single treatment of liraglutide or umbilical cord mesenchymal stem cells in improving the islet function in type 2 diabetes mellitus models, which may be related to the down modulating the TLR4/NF-κB inflammatory signaling pathway.

Objective To investigate the effect of liraglutide combined with bone marrow mesenchymal stem cells (BM-MSCs) on glucose metabolism in experimental type 1 diabetic (T1DM) rats.Methods T1 DM rats were established by injecting 60 mg/kg streptozotocin.According to random number table,they were divided into T1DM group (n =10),BM-MSCs group (n =10),LIRA group (n =10),and LIRA+BM-MSCs group (n =10),and were treated for 8 weeks respectively.Random blood glucose,24 h urine volume and protein excretion were tested.Serum concentrations of insulin,C-peptide,glucagon,gastrin,cholecystokinin,and glucagon-like peptid 1 (GLP-1) were assayed by ELISA.Expressions of insulin and glucagon in pancreas were measured by immunohistochemistry.Results After 8 weeks,the levels of random blood glucose,HbA1C,24 h urine volume and 24 h urinary protein excretion in group 4 were significantly decreased compared to the other three groups (P＜0.05).Compared to T1DM group and BM-MSCs group,the levels of insulin,C-peptide,gastrin,cholecystokinin and GLP-1 in LIRA+BM-MSCs group were significantly increased,while glucagon was decreased (P＜0.05).Compared to group LIRA,the levels of insulin,C-peptide,gastrin,and cholecystokinin in LIRA + BM-MSCs group were increased (P ＜ 0.05),there was no significantly difference in glucagon or GLP-1 (P＞0.05).The analysis revealed that the level of insulin was positively correlated with gastrin (r =0.544,P＜0.01),cholecystokinin (r =0.710,P＜0.01) and GLP-1 (r =0.669,P＜ 0.01),but was negatively correlated with glucagon (r =-0.506,P＜0.01);the level of glucagon was negatively correlated with gastrin (r =-0.364,P＜0.05),cholecystokinin (r =-0.433,P＜0.01) and GLP-1 (r =-0.591,P＜ 0.01).Compared to the other three groups respectively,immunohistochemistry displayed that the positive area of insulin in pancreas was significantly increased in LIRA + BM-MSCs group,while that of glucagon was decreased (P＜ 0.05).Conclusions By means of regulating gastrointestinal hormones efficiently,combination of liraglutide withBM-MSCs may improve glucose metabolism more efficaciously than treatment with a single agent in T1DM rats.

Objective To construct a recombinant vector of sperm-specific human lactate dehydrogenase ( hLDH-C4 ), express it in Escherichia coli ( E. coli ) BL21 ( DE3 ) and utilize it in the detection of anti-sperm antibody. Methods The coding sequence of hLDH-C4 was amplified from human testis λTripIEx cDNA library, and inserted into pET-28a( + ) after restriction enzyme digestion with Hind Ⅲ and Xho Ⅰ. The resultant recombinant vector was used to transform E. coli BL21 ( DE3 ) and the His-Tag fused hLDH-C4 was expressed after induction with IPTG. Western blot was used to analyzed the recombinant protein and LDH activity of bacterial lysates was determined. An indirect ELISA method for the detection of anti-sperm antibody was established by using the recombinant hLDH-C4 as antigen matrix. Results pET-28a( + )-hLDHC was successfully established. The protein with size of 35kD could be induced by IPTG when the recombinant plasmid was transfected into E. coli BL21 ( DE3 ). Western blot showed that the recombinant protein could be specifically recognized beth by anti-His tag monoclonal antibody and by rabbit anti-human LDH-C4 antibody. In addition, the recombinant protein showed high-level LDH activity when the bacterial lysate after IPIG induction was used to check LDH activity. The recombinant hLDH-C4 was confirmed when it was used in indirect EL1SA to detect anti-hLDH-C4 antibody. Conclusions The coding sequence of hLDH-C4 is cloned into the vector pET-28a( + ) and recombinant hLDH-C4 was expressed at a high level in E. coli. The recombinant hLDH-C4 is useful in the detection of anti-sperm antibody.

Objective To establish a new technique of isolating pancreatic islet of langerhans and glueoeortieoid-free immunosuppressive regimen and to evaluate the clinical efficacy and safety of simultaneous adult islet-kidney transplantation in the treatment of type 1 diabetes mellitus with endstage renal failure.Methods Pancreases were stored using the"2-layer method"of the oxygenated perfluoroehemieal and UW solution.The pancreases were digested by Liberase collagenase enzyme and purified using continuous gradients of Ficoll-diatrizoic acid on a refrigerated COBE 2991 centrifuge to separate the islets.Cadaver kidney was transplanted by conventional method and cultured islets were infused by surgical approach to the liver via portal vaseulature using glucocorticoid-free immunosuppressive regimen.Clinical metabolic data such as blood glucose,dose of insulin,C-peptide,HbAlc,liver function and renal function,were determined and compared with the pre-transplant data.ResuitsIslets of langerhans were isolated successfully in 23 pancreases.The average islet yield was 300000 islet equivalents(IEQ).Islet purity and viability were 91.6%,94.6%,respectively.The stimulation index as assessing function of human islet was 3.16 and etiology results in vivo were negative.Twelve islet transplant infusions were carried out in 7 patients after kidney transplantation.Three recipients received 2 islet infusions,1 patient had 3 transplants,and 3 patients received 1 transplant only.The average islet mass for infusion was 1 1 820 IEQ/kg.The immunosuppressive regimen glucocorticoid.During 18 months to 3 yearg follow-up,4 recipients had insulin independence,the dosage of insulin decreased by 70%in 3 patients.The level of blood glucose and H bAlc,liver and renal function were normal throughout follow-up period.C-peptide of all patients was positive after islet transplantation.No adverse effects and complications related to islet infusion procedure were found.Conclusions New technique has proved tO be suitable for isolating pancreatic islet of langerhans.Simuhaneous adult islet-kidney transplantation could be used as an effective and safe way for treating type 1 diabetes mellitus with end-stage renal failure.

Objective To investigate the effect of human umbilical cord mesenchymal stem cells (hUC-MSCs) on autophagy and the secretion of chemokine receptor CXCR4 induced by low-dose immunosuppressive durgs.Methods Flow cytometry was used to detect the changes of hUC-MSCs surface markers after treatment with low-dose tacrolimus and rapamycin.The effect of treatment with tacrolimus and rapamycin on proliferation of hUC-MSCs was analyzed with WST-1 assay.Regular RT-PCR was applied to analyze the mRNAs expression of ligands such as LC3B,Atg5 and Beclin1 in hUC-MSCs.Western blotting was carried out to detect the expression of LC3B,Atg5,Beclin1 and p-ULK1 in hUC-MSCs after treatment with tacrolimus and rapamycin.The secretion of chemokine receptor CXCR4 in hUC-MSCs was analyzed under the state of autophay by flow cytometry.Results Flow cytometry analysis confirmed low-dose immunosuppressive drugs tacrolimus and rapamycin did not cause changes in hUC-MSCs phenotypes significantly.Low-dose tacrolimus had no cytotoxic effect on hUC-MSCs,while,rapamycin could inhibit the proliferation of hUC-MSCs after 24 h or 48 h,with survival rate being 73.66％ and 68.81％ (P＜0.05) of controls,respectively.Moreover,both tacrolimus and rapamycin could inhibit PI3K/AKt/mTOR signaling pathway to activate hUC-MSCs autophagy,and the related proteins of LC3B,Atg5 and Beclin1 increased significantly and induced the up-regulation of CXCR4 secretion.Conclusion Our results here demonstrated that low-dose tacrolimus and rapamycin induce autophagy in hUC-MSCs and promote the secretion of CXCR4.

OBJECTIVETo study the status of oral pharyngeal carriage and characteristics of Haemophilus influenzae (Hi) in healthy preschool children in Fuzhou.

METHODSSix hundred and three healthy children in two representative kindergartens in Fuzhou were studied as research subjects, and the rates of oral pharyngeal carriage of Hi were studied in four seasons. All Hi strains were serotyped and biotyped.

RESULTSThe oral pharyngeal carriage of Hi in day care nursery healthy children were 36.7% in winter, 18.0% in autumn, 12.4% in summer and 10.9% in spring respectively. Serotype Hib was preponderant in autumn (6.9%). In winter, the carriage rates of NTHi and Hib were 17.1%, 5.4% respectively. The carriage rates of other serotypes were low. Biotype VII and VIII were preponderant in autumn, spring and summer but biotype VII and VIII were decreasing evidently in winter.

CONCLUSIONThere was evident seasonal difference in the rates of oral pharyngeal carriage and type of Hi in healthy preschool children. The carriage rate of Hi strains was high in autumn and winter. Results suggested that while the inoculation of Hib-binding bacterial vaccine was expanded the study on new bacterial vaccine of Hi still needs to be augmented.

Carrier State ; epidemiology ; Child ; Child, Preschool ; China ; epidemiology ; Female ; Haemophilus influenzae ; classification ; isolation & purification ; Humans ; Male ; Pharynx ; microbiology ; Seasons ; Serotyping