Animals ; Humans ; Leptin ; genetics ; physiology ; Liver Cirrhosis ; etiology ; RNA, Messenger ; analysis ; Receptors, Cell Surface ; analysis ; physiology ; Receptors, Leptin ; Transforming Growth Factor beta ; genetics ; Transforming Growth Factor beta1

OBJECTIVETo construct the multi-probe ribonuclease protection assay (RPA) template set to be used for detecting expression patterns of MD-2, TLR4, CD14 mRNAs in human peripheral blood mononuclear cells.

METHODSThe designed cDNA fragments of the three genes were generated by polymerase chain reaction (PCR) using specific primers and directionally cloned into EcoR I and Hind III sites of expression plasmid pSP72 containing the T7 promoter, the linearized plasmids was used as template to synthesize anti-sense RNA probes. Then we extracted total RNA from peripheral blood mononuclear cells (PBMC) and detected the dynamic expression patterns of the three genes with RPA method.

RESULTSThe proper sequence and orientation of the template set were confirmed by sequencing and the template set was successfully used to assay TLR4, MD-2 and CD14 mRNAs in human PBMC. The results showed that the three detected genes decreased transiently 1-3 hours after 100 ng/ml LPS stimulation.

CONCLUSIONSThese new RPA multi-probe set provided valuable tool for the simultaneous quantitative determination of expression of TLR4, CD14 and MD-2 mRNAs in both constitutive and inducible types.

Antigens, Surface ; analysis ; Base Sequence ; Biological Assay ; Cells, Cultured ; DNA ; analysis ; genetics ; Gene Expression Profiling ; methods ; Humans ; Lipopolysaccharide Receptors ; analysis ; Lymphocyte Antigen 96 ; Membrane Glycoproteins ; analysis ; Molecular Probe Techniques ; Molecular Sequence Data ; Monocytes ; metabolism ; RNA Probes ; analysis ; genetics ; Receptors, Cell Surface ; analysis ; Receptors, Immunologic ; analysis ; Ribonucleases ; Toll-Like Receptor 4 ; Toll-Like Receptors

OBJECTIVETo study the clinical significance of CD163 in the diagnosis and the evaluation of severity and prognosis of childhood hemophagocytic lymphohistiocytosis (HLH).

METHODSNinety-four children were classified into Epstein-Barr virus (EBV)-positive (n=55) and EBV-negative groups (n=39; control group). The EBV-positive group was subgrouped into infectious mononucleosis (IM; n=47) and HLH (n=8). Serum levels of soluble CD163 were measured using ELISA. Expression of CD163 on mononuclear cells was detected by flow cytometry.

RESULTSThe serum levels of soluble CD163 were>10 000 ng/mL in all eight HLH patients (>30 000 ng/mL in 3 cases). The mean serum levels of soluble CD163 in the HLH group were significantly higher than in the control and IM groups (P<0.05). The serum levels of soluble CD163 in EBV-positive children were positively correlated with EBV-DNA copies and serum levels of ferritin and LDH, but were negatively correlated with white blood cell count, neutrophil count, hemoglobin and platelet count. The follow-up after treatment for three HLH patients showed that serum levels of soluble CD163 were significantly reduced, but the soluble CD163 levels rebounded in one patient who was complicated by fungal pneumonia infection.

CONCLUSIONSThe levels of serum soluble CD163 may be related to the severity in children with HLH. The EBV-positive children with soluble CD163 levels >10 000 ng/mL should be considered the possibility of HLH.

Adolescent ; Antigens, CD ; analysis ; Antigens, Differentiation, Myelomonocytic ; analysis ; Child ; Child, Preschool ; Epstein-Barr Virus Infections ; immunology ; Female ; Humans ; Infant ; Male ; Receptors, Cell Surface ; analysis

Radioiodinated oxytocin prepared by the lactoperoxidase method exhibited a substantial biologic activity in uterotonic assay of the rat uterus. 125I-oxytocin was bound to the uterine membrane particulate fraction, but the unlabelled oxytocin did not inhibit the binding of 125I oxytocin to the membrane fraction of rat uterus. Cold iodinated oxytocin, however, inhibited the 125I-oxytocin binding to the membrane fraction of rat uterus in proportion to its concentration. These results suggest that 125I-oxytocin is not a suitable radioligand for oxytocin receptor binding study.
Animal ; Binding Sites ; Cell Membrane/metabolism ; Female ; Iodine Radioisotopes/metabolism* ; Oxytocin/metabolism* ; Radioligand Assay ; Rats ; Receptors, Cell Surface/analysis* ; Uterus/metabolism*

OBJECTIVETo investigate PTCH gene mutations in odontogenic keratocysts (OKC).

METHODSPCR-SSCP and DNA sequencing were used to analyze the PTCH gene mutations in 12 OKCs, including 10 sporadic and 2 nevoid basal cell carcinoma syndrome (NBCCS) associated OKC.

RESULTSFour mutations were identified in 4 cysts, among which two germline mutations were associated with NBCCS and 2 somatic mutations were in 2 unrelated sporadic cases. In addition, eight previously reported polymorphisms in the PTCH gene were also found in 10 cases.

CONCLUSIONThe present study indicated that both sporadic and NBCCS-related OKCs could carry PTCH gene mutation. Thus, mutational inactivation of PTCH gene may play a significant role in the pathogenesis of OKC.

Basal Cell Nevus Syndrome ; Female ; Humans ; Mutation ; Odontogenic Cysts ; Polymerase Chain Reaction ; Polymorphism, Genetic ; Polymorphism, Single-Stranded Conformational ; Receptors, Cell Surface ; Sequence Analysis, DNA

To study the biological characteristics of mesenchymal stem cells (MSC) from patients with myelodysplastic syndrome (MDS) and their supportive capacity for hematopoiesis in vitro, MSCs from bone marrow samples of MDS patients were isolated, cultured and expanded. Morphology, immunophenotype, osteoblasts differentiative and proliferative property of MSC and colony forming unit-fibroblast (CFU-F) were measured and analyzed. Mononuclear cells (MNC) of cord blood were plated onto a feeder layer formed by MSC of MDS patient, cells count and CFU-GM production were observed. The results showed that the culture-expanded cells from MDS patients presented a typical fibroblast-like morphology. Cells were positive for SH2 (CD105), SH3 (CD73), Thy-1 (CD90), but negative for CD34 and CD45. After induction, these cells could differentiate into osteoblasts. Their proliferative capacity and CFU-F number were similar to those of MSC from healthy donors. The total cell count and CFU-GM yield in supernatants after culture for 2 weeks were significantly lower than those of control in hematopoiesis supportive experiments in vitro (P < 0.05). It is concluded that the biological characteristics of MSC from bone marrow of MDS patients are not different from those of MSC isolated from bone marrow of normal donors, however, their capacity of hematopoiesis support in vitro are significantly weaker.
5'-Nucleotidase ; analysis ; Adult ; Aged ; Antigens, CD ; analysis ; Antigens, CD34 ; analysis ; Bone Marrow Cells ; cytology ; immunology ; Cell Differentiation ; Endoglin ; Female ; Hematopoiesis ; Humans ; Male ; Mesenchymal Stromal Cells ; cytology ; immunology ; Middle Aged ; Myelodysplastic Syndromes ; blood ; Receptors, Cell Surface ; analysis

OBJECTIVEIn order to explore pathogenic mechanism of laryngeal carcinoma, the involved genes were identified in larynx carcinogenesis by comparing the gene expression profile in matched primary normal epithelial cells and primary laryngeal carcinoma cells from the same patients.

METHODSA cDNA microarray analysis consisting of 11,431 human genes revealed significant changes in the expression of 35 genes, with 8 genes being up-regulated and 27 being down-regulated. The epithelial membrane protein-1 (EMP-1) is one of the down-regulated genes. EMP-1 expression in various kinds of laryngeal carcinoma was determined by semi-quantitative reverse transcriptase polymerase chain reaction (qRT-PCR).

RESULTSThe EMP-1 mRNA levels in all laryngeal carcinoma cells was significantly lower than that in the matched primary normal epithelial cells (P < 0.05) and were not correlated to the stage and differentiation of laryngeal carcinoma (P > 0.05).

CONCLUSIONSThe EMP-1 expression was correlated to larynx carcinogenesis and may be helpful to elucidate the pathogenic mechanism in laryngeal carcinoma.

Adult ; Aged ; Female ; Humans ; Laryngeal Neoplasms ; genetics ; metabolism ; Male ; Middle Aged ; Neoplasm Proteins ; Oligonucleotide Array Sequence Analysis ; Receptors, Cell Surface ; biosynthesis ; genetics ; Reverse Transcriptase Polymerase Chain Reaction

OBJECTIVETo detect the "high pathogenicity island" of Yersinia enterocolitica WA in E. coli and the to provide evidence for theory base of bacterial evolution process and the different structures in different E. coil.

METHODSPolymerase chain reaction (PCR), nucleic acid hybridization in situ were used to detect and identify HPI. DNA sequencing was used to compare the gene homology of HPI among E. coli with Yersinia enterocolitica (Yen).

RESULTSThe irp2 and fyua genes of Yen HPI were investigated in E. coli strains. Among them, 30 strains were isolated from 93 Enterotoxigenic E. coli (ETEC) strains and 3 strains were positive in 10 strains Enteropathogenic (EPEC). HPI was also detected in Enteroaggregative E. coli (EAggEC) strain. In most of these isolates, HPI was bordered by an asntRNA locus, as in Yersinia sp. Through sequential comparison, the gene sequence homology was higher between in EPEC and EAggEC than ETEC and Yersinia enterocolica.

CONCLUSIONSETEC, EPEC and EAggEC were pathogenicity bacterias and many of them harboring HPI of Yen and the HPI had the same position in E. coli chromosome as Yersinia enterocolitica but the diversity of structure and sequence in these E. coli might suggest that the HPI of these different serotype E. coli were from different ancient bacterias. At the same time, the high positivity rate of HPI in E. coli might be crucial to virulence change, virulence evolution and virulence regulation in E. coli.

Bacterial Proteins ; analysis ; genetics ; Enterotoxins ; genetics ; Escherichia coli ; genetics ; pathogenicity ; Escherichia coli Infections ; microbiology ; Genes, Bacterial ; Receptors, Cell Surface ; genetics ; Virulence ; genetics ; Yersinia enterocolitica ; genetics ; pathogenicity

OBJECTIVE: To detect potential mutations of the PKHD1 gene in two pedigrees affected with infantile polycystic kidney disease. METHODS: Clinical data and peripheral venous blood samples were collected from the probands and their parents as well as fetal amniotic fluid cells. Genome DNA was extracted from the peripheral blood samples and amniotic fluid cells. Exons 32 and 61 of the PKHD1 gene were amplified with PCR and subjected to direct sequencing. RESULTS: The proband of pedigree 1 was found to carry c.4274T>G (p.Leu1425Arg) mutation in exon 32 and c.10445G>C (p.Arg3482Pro) mutation in exon 61 of the PKHD1 gene, which were inherited from her father and mother, respectively. The fetus has carried the c.4274T>G (p.Leu1425Arg) mutation. In pedigree 2, the wife and her husband had respectively carried a heterozygous c.5979_5981delTGG mutation and a c.9455delA mutation of the PKHD1 gene. No chromosomal aberration was found in the umbilical blood sample, but the genetic testing of their fetus was failed. Based on software prediction, all of the 4 mutations were predicted to be pathogenic. CONCLUSION PKHD1 c.4274T>G (p.Leu1425Arg), c.10445G>C (p.Arg3482Pro), c.5979_5981delTGG and c.9455delA were likely to be pathogenic mutations. The results have facilitated genetic counseling and prenatal diagnosis for the two pedigrees.
DNA Mutational Analysis ; Female ; Genetic Counseling ; Humans ; Mutation ; Pedigree ; Polycystic Kidney Diseases ; diagnosis ; genetics ; Pregnancy ; Prenatal Diagnosis ; Receptors, Cell Surface ; drug effects

OBJECTIVE: To study the cytokine patterns in patients with rheumatoid arthritis (RA) and healthy individuals and identify candidate serum biomarkers for clinical diagnosis of RA. METHODS: This study was conducted among 59 patients diagnosed with RA in our hospital from 2015 to 2019 with 46 age- and gender-matched healthy subjects who received regular physical examinations in our hospital as the control group. Serological autoimmune profiles of 5 RA patients and 5 healthy control subjects were obtained from human cytokine microarrays. We selected 4 differentially expressed cytokines (LIMPII, ROBO3, Periostin and IGFBP-4) and 2 soluble cytokine receptors of interest (2B4 and Tie-2) and examined their serum levels using enzyme-linked immunosorbent assay in 54 RA patients and 41 healthy control subjects. Spearman correlation test was performed to assess the correlation of serum cytokine and soluble receptor expression levels with the clinical features including rheumatoid factor (RF), erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), disease activity score (DAS28) and health assessment questionnaire (HAQ). Receiver operating characteristic (ROC) curve was used to evaluate the diagnostic capability of these cytokines. RESULTS: We identified 6 dysregulated cytokines and soluble receptors (2B4, LIMPII, Tie-2, ROBO3, periostin and IGFBP-4) in RA patients (P < 0.01). The serum levels of LIMPII, ROBO3 and periostin were significantly correlated with the disease activity indicators including RF (P < 0.001), CRP (P < 0.001), DAS28 (P < 0.001) and HAQ (P < 0.001) in RA patients. Among the 6 candidate cytokines, 2B4 showed the largest area under the curve (AUC) of 0.861 for RA diagnosis (P < 0.001), followed then by LIMPII, ROBO3, periostin, Tie-2 and IGFBP-4. CONCLUSION Serum levels of LIMPII, ROBO3 and periostin can be indicative of the disease activity of RA, and serum 2B4, LIMPII, periostin, ROBO3, IGFBP-4 and Tie-2 levels may serve as biomarkers for the diagnosis of RA.
Arthritis, Rheumatoid/diagnosis* ; Biomarkers ; C-Reactive Protein ; Cytokines ; Humans ; Insulin-Like Growth Factor Binding Protein 4 ; Protein Array Analysis ; Receptors, Cell Surface