Objective To investigate expression and significance of decorin(DCN)in liver tissue and serum of liver transplant patients with chronic rejection(CR).Methods Immunohistochemistry(SP method)was used to detect expression of DCN in liver tissue of 16 normal controls, 20 patients with cirrhosis, 46 liver translantion patients without CR and 8 patients with CR.Enzyme-linked immunosorbent assay method(ELISA method)was used to determined the content of DCN in serum of all research subjects.Results The expression of DCN was negative in normal hepatic tissues and with/without CR, cirrhosis tissues showed strong expression of DCN.The positive expression rate and the average optical density value of DCN in liver transplant tissues with CR had significant difference comparing with Cirrhosis tissues(25% vs 55%, 0.1249 ±0.0039 vs 0.2357 ±0.0396, P <0.01,while no statistic siqnificance compared to normal liver tissues and those without CR.The level of DCN in serum was significantly higher in liver transplant patients with CR, with significant difference comparing with normal people, liver cirrhosis and transplant liver patients without CR(54.0833 ± 6.0325)μg/L vs(1.0232 ± 0.9105)μg/L,(12.6202 ± 1.5370)μg/L,(17.7102 ± 2.3562)μg/L, P < 0.01).The concentration of DCN in serum showed a positive correlation with the degree of CR.Conclusions DCN showed negative expression in liver tissue and increased significantly in serum of liver transplantation patients with CR.This suggests that DCN may be involved in occurrence and development of CR.At the same time the determination of DCN in serum maybe become an important indicator of the early diagnosis, development and prognosis of CR for liver transplant patients.

Objective Tissue kallikrein cleaves kininogen substrate to produce vasoactive kinin peptides that have been implicated in the proliferation of vascular smooth muscle cells. We investigated the effects of adenovirus-mediated human tissue kallikerin(Ad-hKLK1)gene delivery on the proliferation of vascular smooth muscle cells of SHR(VSMCsSHR)induced by platelet derived growth factor-BB(PDGF-BB). Methods Primary VSMCsSHR were isolated and cultured from thoracic aorta of male SHR. The VSMCsSHR proliferation induced by PDGF-BB was accessed by cell counting and methyl thiazolyl tetrazoliuin(MTT). Western blot was used to determine the protein expression of hKLK1, the cycle-independent kinase inhibitors p27Kip1 and p21Cip1 . The mRNA expressions of bradykinin B1 receptor and B2 receptor were detected by RT-PCR in VSMCsSHR. Results Proliferation of VSMCsSHR induced by PDGF-BB was significantly inhibited post transfection of Ad-hKLK1(20 - 100 MOI)in a MOI-dependent manner. The peak inhibition titer of Ad-hKLK1 was 100 MOI with peak inhibition rate of 39.3%(cell counting, n = 3,P ＜0.01), 30.2%(MTT, n-3 ,P ＜ 0.01)and 36.4%(peak stunning rate of cell-cycle in phase G0/G1). The inhibitory effects of proliferation and cell-cycle caused by hKLK1 gene delivery could be abolished by Hoe140, a bradykinin B2 receptor antagonist. The protein expression of p27Kip1 and p21Cip1 increased significantly after the hKLK1 gene delivery, whereas Hoe140 nearly completely blocked these effects(n = 3,P ＜0.001 ,respectively). PDGF-BB also significantly upregulated the mRNA expression of B2 receptor but not B1 receptor in VSMCsSHR. Conclusion The hKLK1 gene delivery could inhibit PDGF-BB induced proliferation in VSMCsSHR through Bradykinin B2 receptor and up-regulate expression of p27Kip1 and p21Cip1.

A 9-day-old male patient was admitted to the hospital because of cough, anhelation, feeding difficulty and lethargy. The diagnostic examinations indicated pulmonary infection, severe metabolic acidosis, hyperglycemia, hyperammonemia and pancytopenia in the patient. Blood and urine screening and isovaleryl-CoA dehydrogenase (IVD) gene detection for inherited metabolic diseases were performed to clarify the etiology. Tandem mass spectrometric screening for blood showed an elevated isovalerylcarnitine (C5) level. The organic acid analysis of urine by gas chromatography-mass spectrometry showed significantly increased levels in isovaleryl glycine and 3-hydroxyisovaleric acid. Homozygous mutations (c.1208A>G, p.Tyr403Cys) in the IVD gene were identified in the patient. His parents were heterozygous carriers. After the treatment with low-leucine diets and L-carnitine for 3 days, the patient showed a significant improvement in symptoms, but he died one week later. It is concluded that the neonates with pneumonia and metabolic decompensation of unknown etiology should be screened for genetic metabolic disease.
Amino Acid Metabolism, Inborn Errors ; diagnosis ; genetics ; Humans ; Infant, Newborn ; Isovaleryl-CoA Dehydrogenase ; deficiency ; genetics ; Male ; Mutation ; Pancytopenia ; etiology

This study aimed to analyze the clinical phenotype of chromosome 9p deletion or duplication and its relationship with karyotype. A patient, female, aged 6 months, visited the hospital due to motor developmental delay. Karyotype analysis identified abnormalities of chromosome 9 short arm, and high-throughput sequencing found 9p24.3-9p23 deletion and 9p23-9p13.1 duplication. Her parents had a normal karyotype. Karyotype analysis combined with high-throughput sequencing is of great significance for improving the efficiency of etiological diagnosis in children with motor developmental delay or multiple congenital deformities and mental retardation.

OBJECTIVETo study the gene mutation profile of primary carnitine deficiency (PCD) in neonates, and to provide a theoretical basis for early diagnosis and treatment, genetic counseling, and prenatal diagnosis of PCD.

METHODSAcylcarnitine profile analysis was performed by tandem mass spectrometry using 34 167 dry blood spots on filter paper. The SLC22A5 gene was sequenced and analyzed in neonates with free carnitine (C0) levels lower than 10 μmol/L as well as their parents.

RESULTSIn the acylcarnitine profile analysis, a C0 level lower than 10 μmol/L was found in 10 neonates, but C0 level was not reduced in their mothers. The 10 neonates had 10 types of mutations at 20 different sites in the SLC22A5 gene, which included 4 previously unreported mutations: c.976C>T, c.919delG, c.517delC, and c.338G>A. Bioinformatics analysis showed that the four new mutations were associated with a risk of high pathogenicity.

CONCLUSIONSTandem mass spectrometry combined with SLC22A5 gene sequencing may be useful for the early diagnosis of PCD. Identification of new mutations enriches the SLC22A5 gene mutation profile.

Cardiomyopathies ; diagnosis ; genetics ; Carnitine ; deficiency ; genetics ; Computational Biology ; Genetic Counseling ; Humans ; Hyperammonemia ; diagnosis ; genetics ; Infant, Newborn ; Muscular Diseases ; diagnosis ; genetics ; Mutation ; Solute Carrier Family 22 Member 5 ; genetics ; Tandem Mass Spectrometry

This study aimed to identify the type of carnitine palmitoyltransferase 2 (CPT2) gene mutation in the child with carnitine palmitoyltransferase II (CPT II) deficiency and her parents and to provide the genetic counseling and prenatal diagnosis for the family members. As the proband, a 3-month-old female baby was admitted to the hospital due to fever which had lasted for 8 hours. Tandem mass spectrometric analysis for blood showed an elevated plasma level of acylcarnitine, which suggested CPT II deficiency. The genomic DNA was extracted from peripheral blood of the patient and her parents. Five exon coding regions and some intron regions at the exon/intron boundaries of the CPT2 gene were analyzed by PCR and Sanger sequencing. Amniotic fluid was taken from the mother during the second trimester, and DNA was extracted to analyze the type of CPT2 gene mutation. Sanger sequencing results showed that two mutations were identified in the CPT2 gene of the proband: c.886C>T (p.R296X) and c.1148T>A (p.F383Y), which were inherited from the parents; the second child of the mother inherited the mutation of c.886C>T (p.R296X) and showed normal acylcarnitine spectrum and normal development after birth. It is concluded that the analysis of CPT2 gene mutations in the family suggested that the proband died of CPT II deficiency and that the identification of the mutations was helpful in prenatal diagnosis in the second pregnancy.
Carnitine O-Palmitoyltransferase ; deficiency ; genetics ; Female ; Humans ; Infant ; Metabolism, Inborn Errors ; diagnosis ; genetics ; Mutation ; Prenatal Diagnosis

Medium- and short-chain acyl-CoA dehydrogenase deficiency is a disorder of fatty acid β-oxidation. Gene mutation prevents medium- and short-chain fatty acids from entry into mitochondria for oxidation, which leads to multiple organ dysfunction. In this study, serum acylcarnitines and the organic acid profile in urea were analyzed in two children whose clinical symptoms were hypoglycemia and metabolic acidosis. Moreover, gene mutations in the two children and their parents were evaluated. One of the patients was a 3-day-old male who was admitted to the hospital due to neonatal asphyxia, sucking weakness, and sleepiness. The serum acylcarnitine profile showed increases in medium-chain acylcarnitines (C6-C10), particularly in C8, which showed a concentration of 3.52 μmol/L (reference value: 0.02-0.2 μmol/L). The analysis of organic acids in urea gave a normal result. Sanger sequencing revealed a reported c.580A>G (p.Asn194Asp) homozygous mutation at exon 7 of the ACADM gene. The other patient was a 3-month-old female who was admitted to the hospital due to cough and recurrent fever for around 10 days. The serum acylcarnitine profile showed an increase in serum C4 level, which was 1.66 μmol/L (reference value: 0.06-0.6 μmol/L). The analysis of organic acids in urea showed an increase in the level of ethyl malonic acid, which was 55.9 (reference value: 0-6.2). Sanger sequencing revealed a reported c.625G>A (p.Gly209Ser) homozygous mutation in the ACADS gene. This study indicates that screening tests for genetic metabolic diseases are recommended for children who have unexplained metabolic acidosis and hypoglycemia. Genetic analyses of the ACADM and ACADS genes are helpful for the diagnosis of medium- and short-chain acyl-CoA dehydrogenase deficiency.
Acyl-CoA Dehydrogenase ; deficiency ; genetics ; Carnitine ; analogs & derivatives ; blood ; Female ; Humans ; Infant ; Infant, Newborn ; Lipid Metabolism, Inborn Errors ; genetics ; Male ; Mutation ; Urea ; analysis

OBJECTIVETo detect the infection of human papillomavirus (HPV) 16/18 in patients with head and neck squamous cell carcinoma and explore the relationship between HPV infection and expressions of Ki-67 and P53 proteins in tumor tissue.

METHODThe level of HPV 16/18 DNA was measured by real time polymerase chain reaction, and Ki-67 and P53 proteins were measured by immunohistochemistry in tissues from head and neck squamous cell carcinoma.

RESULTSHPV 16/18 DNA was detected in 62.8% of our patients. In each cancer tissue sample, Ki-67 protein was expressed between 2% to 70%. P53 protein was expressed in 46.15% of our patients. No significant relation was found between HPV 16/18 DNA level and sex, smoking, drinking, and tumor clinical stages. However, level of HPV 16/18 DNA was found to have positive relation with tumor pathological grades and negative relation with P53 protein expression. No relation with Ki-67 protein expression was found.

CONCLUSIONHead and neck squamous cell carcinoma may be initiated by HPV 16/18 infection and the mechanism in carcinogenesis involves abnormal expression in P53 protein.

Adult ; Aged ; Aged, 80 and over ; Carcinoma, Squamous Cell ; metabolism ; virology ; DNA, Viral ; analysis ; Female ; Human papillomavirus 16 ; isolation & purification ; Human papillomavirus 18 ; isolation & purification ; Humans ; Ki-67 Antigen ; metabolism ; Male ; Middle Aged ; Tumor Suppressor Protein p53 ; metabolism ; Uterine Cervical Neoplasms ; metabolism ; virology

Objective To investigate the effects of human tissue kallikrein 1 (Ad-hKLK1) gene delivery on the neointima formation in carotid arteries of spontaneously hypertensive rats (SHRs). Methods Carotid artery restenosis was induced in male SHR rats by balloon-injury. Rats were randomly assigned into 4 groups: Sham-operated (n = 6); Angioplasty (phosphate buffered solution 50 μl, n = 8); Vector virus (control virus, 1×10~9 IU in 50 μl, n =8) and Ad-hKLK1 (Ad-hKLK1, 1×10~9 IU in 50 μl, n =8). Rats were sacrificed 4 weeks later. The wall-to-lumen area ratio and intima/media ratio in carotid artery were assessed by image analysis in HE stained sections. The mRNA bradykinin receptor(B1R and B2R) expressions were detected by RT-PCR. The protein expression of the cycle-independent kinase inhibitors p27Kip1 and p21Cip1 were determined by Western blot analysis. Results Wall-to-lumen area ratio reduced 35.6% and intima/media ratio reduced 38.8% in Ad-hKLK1 treated SHRs compared to angioplasty group (all P <0.001). The expression of p27Kip1 and p21Cip1 increased significantly in Ad-hKLK1 treated SHRs compared with angioplasty rats (all P <0.001). The mRNA expression of B2R was significantly upregulated in angioplasty rats compared with sham-operated rats (P <0.05) while mRNA expression of B1R was similar between the 2 groups. Conclusion hKLK1 gene delivery may effectively reduce neointimal formation via downregulating bradykinin B2R and up-regulating the expressions of p27Kip1, p21Cip1 signaling pathways in carotid arteries of SHRs after balloon injury.

To isolate and identify the influenza virus in Shandong Province in 2009-2010 and analyze the genetic characteristics of hemagglutinin and neuraminidase gene, further study the variation of gene. A total of 17 126 nasopharyngeal swabs from fever patients were collected and detected by real time quantitative RT-PCR method. The results showed 4004 samples were pandemic influenza A (H1N1) virus positive, with an overall positive rate as 23.38%. The positive samples were incubated and cultured in MDCK cells. The HA and NA genes of isolated pandemic influenza A(H1N1) virus were sequenced, the homology analysis of the HA and NA genes showed an average of 96.9%-99.3% and 99.1%-99.6% sequence identity, respectively, compared with WHO-recommended vaccine strain. The genetic evolution and amino acid substitutions were performed with Mega 4.0 Software. Twenty one amino acids were changed in HA protein, of which 11 were located in the antigenic site; Sixteen amino acids were changed in NA protein, which didn't lead to the changes of enzyme sites. Furthermore, one glycosylation site of HA protein and NA protein were changed respectively. No H275Y mutation in NA protein was found. The results showed that the HA and NA genes of the epidemic strains were highly homologous, some mutations in the HA and NA proteins were found, the antigenic site and glycosylation site of some strains were changed during the epidemic process. All the strains were sensitive to oseltamivir.
China ; epidemiology ; Hemagglutinin Glycoproteins, Influenza Virus ; chemistry ; Humans ; Influenza A Virus, H1N1 Subtype ; genetics ; isolation & purification ; Influenza, Human ; epidemiology ; virology ; Neuraminidase ; genetics ; Pandemics ; Phylogeny ; Time Factors