Detection of peripheral CD14+CD277+monocyte-macrophage ratio with γδ TCR tetramer and analy-sis of its relationship with treatment outcome / 中华微生物学和免疫学杂志

Objective To investigate the role of peripheral CD14+monocyte-macrophages in the recognition of phosphorylated antigen by γδ T cells and its relationship with treatment outcome. Methods Three kinds of γδ TCR tetramers were used to stain PBMC collected from patients with tuberculosis ( TB) and neonatal umbilical cord blood samples. The proportions of various TB-specific antigen presenting cells (APC) in peripheral blood were analyzed, and their relationships with treatment outcome were assessed based upon clinical data. Results CD14+monocyte-macrophages both in tuberculosis patients′ peripheral blood and neonatal umbilical cord blood were the strongest binding cells to CD277 antibody and γδ TCR tet-ramers. The median (P50) of CD14+monocyte-macrophages reached the highest peak after taking anti-tu-berculosis treatment for about one month and patients′condition was improved obviously during this period. Conclusion This study elucidated that CD14+monocyte-macrophages accounted for the largest proportion of APC when γδ T cells recognized phosphorylated antigens, which provided reference data for further study on the mechanism of γδ T cells restrictively recognizing phosphorylated antigen and their significance in innate and adaptive immunity.

Prestin Expression in HEI-OC1 Cells With Oxidative Stress Damage / 听力学及言语疾病杂志

Objective To examine the effects of oxidative stress induced damage to the Prestin expression in HEI-OC1 cells,and to study the mechanism of sensory deafness.Methods We used different concentrations (50μM,100μM,200μM)of hydrogen peroxide canister to cultivate HEI-OC1 cells,and to detect the activity of su-peroxide dismutase(SOD).The quantitative real-time PCR and immunofluorescence were used to detect the prestin expression of mRNA.Results The SOD activity decreased in the HEI-OC1 cells damaged by oxidative stress.The high concentration of the infected group decreased more significantly(F= 9926.293,P<0.01).The expressions of Prestin mRNA and Prestin protein were decreased obviously in the HEI-OC1 cells.The high concentration of in-fected group decreased more significantly (F= 4065.046and7657.217,P<0.01).Conclusion Oxidative stress in-ducing damage inhibits the expression of prestin.Prestin protein may be used as a molecular marker of sensory deafness.

Detection of antigen-specific CD14+ monocytes in blood of tuberculosis patients by using CD4+ T-cell receptor tetramer-based flow cytometric analysis and cell climbing slice assay / 中国感染与化疗杂志

Objective To evaluate the utility of CD4+ TCR tetramers‐based flow cytometric analysis and cell climbing slice assay in detecting antigen‐specific CD14+ monocytes in the blood of tuberculosis (TB) patients .Methods CD4+ TCR tetramers were used to detect tetramer‐positive CD14+ monocytes in the peripheral blood (PBL ) samples of inpatients with advanced pulmonary TB (PTB) by flow cytometric analysis .The PBL samples obtained from non‐TB patients and umbilical cords were used as controls .These tetramers were also used to examine tetramer‐bound CD14+ monocytes and Mycobacterium tuberculosis (MTB) antigen‐specific and tetramer‐bound cells by cell climbing slice in situ staining .Results The median percentage of tetramer‐bound CD14+ monocytes in PBL samples from PTB patients ,non‐TB patients and umbilical cords were 1 .32% , 0 .50% and 0 .26% respectively by using CD4+ Vα21‐J39/Vβ29‐D1‐J2 tetramer , while the medians were 1 .05% , 0 .49% and 0 .19% respectively by using CD4+ Vα21‐J39/Vβ29‐D2‐J2 tetramer . The percentage of tetramer‐bound CD14+ monocytes in PTB patients group was significantly higher than the other two control groups .In cell climbing slice in situ staining ,tetramer‐bound CD14+ monocytes ,and MTB antigen‐specific and tetramer‐bound cells were positive in PTB tissue compared with negative in control tissues . Conclusions CD4+ TCR tetramers‐based flow cytometric analysis and cell climbing slice assay could be used to sensitively detect M TB antigen‐specific CD14+ monocytes in the blood of TB patients ,and more accurately evaluate the changing profile and clinical significance of these cells in TB patients .

The preliminary study of CD4+ Vα9-J27/Vβ29-D1-J2 tetramers in detecting Mycobacterium tuberculosis infections / 中华微生物学和免疫学杂志

ObjectiveTo investigate the specificity of CD4+ Vα9-J27/Vβ29-D1-J2 tetramer in detecting Mycobacterium tuberculosis(MTB) infections.MethodsThe above TCR tetramer by using biotinylated monomers expressed and purified from constructed stable Drosophila Schneider 2 cell( S2 cell) lines was prepared.The PE-labled TCR tetramer was used to costain with S2 cell lines expressing MTB prptide/HLA-DR complexes on the cell membrane,and also was used to detect tetramer-bound CD14+ monocytes and macrophages in the peripheral blood mononuclear cells (PBMC) of pulmonary tuberculosis (PTB) patients and three control groups by flow cytometric analysis.And the FITC-labled tetramer was used to examine tetramer-bound CD14+ monocytes and macrophages,and MTB antigen-specific and tetramer-bound cells by in situ staining.ResultsThe TCR tetramer was well binding with S2 cell lines expressing C14/HLA-DR *1504 on the cell membrane.By flow cytometric analysis,the percentage of tetramer-bound CD14+ monocytes and macrophages in PTB patients group was higher than the other three control groups( P＜0.001 ).By in situ staining,tetramer-bound CD14+ monocytes and macrophages,and MTB antigen-specific and tetramer-bound cells were positive in PTB tissue and negative in control pneumonia tissue.ConclusionThe spcificity of TCR tetramer in monitoring MTB infections by flow cytometric analysis and in situ staining could be seen,which laid a laboratory foundation in the diagnosis and immune mechanism research of TB by using TCR tetramers.

The regulating role of TNF-α with dexamethasone and shenfu separate and joint administration of the flap after ischemia-reperfusion injury / 中国基层医药

Objective To discuss the protective effects and mechanisms of dexamethasone and Shenfu sepa-rate and joint administration of flap after ischemia-reperfusion injury. Methods 40-month-old fairly healthy rats were randomly divided into 4 group as A, B, C, D, and to product the abdominal island flap, then blocking the flow of blood of the pedicle artery respectively before 30 minutes when injecting with normal saline (1 ml/kg), dexametha-sone (1 ml/kg), Senate (10 ml/kg), joint injection with dexamethasone (1 ml/kg) and Shenfu (10 ml/kg). 4 groups of animals' blood samples were collected from the pedicle vein before the time of I hour when blocking vascu-lar pedicle and reperfusion after the time of 1 h, 6 h, 12 h, 24 h, respectively. Then the plasma concentration of TNF-α was measured. Results The concentration of TNF-α in the treatment group was significantly lower than the blank group(P <0.01). And the group D and the group B、C has significant differences either(P <0.01). Conclu-sion Using Dexamethasone, Shenfu injection in early can reduce the concentration of TNFα in repeffusion injury of flap and has a protective effect on the flap, but make better effect in combined.

Construction and application of cell fines screening Mycobacterium tuberculosis-specific tetramers of CD4+α/β T cell receptor / 中华微生物学和免疫学杂志

Objective To construct and apply a cell line screening Mycobacterium tuberculosis (Mtb)-specific tetramers of CD4+α/β T cell receptor(TCR). Methods The β chains of HLA class Ⅱ (DR) were amplified from tuberculosis patients by PCR. The pMT-HLA-DRB expression vectors that carries the HLA-DR 13 chain and pMT-HLA-DRA-P expression vectors which carries the genes of HLA-DR α chain loaded with Mtb antigen were transfected into S2 cells with the method of calcium phosphate transfection. The expressed Mtb peptide/HLA-DR complexes were primarily identified by the method of cell immunohistochemistry. The cell lines expressing Mtb peptide/HLA-DR complexes were used to screen tetramers of CD4+ TCR by flow cytometry. Results S2 cell lines expressing Mtb peptide/HLA-DR complexes on the cell surface were obtained, two kinds of Mtb specific tetramers of CD4+α/β TCR were screened. Conclusion S2 cell lines expressing Mtb peptide/HLA-DR complexes on the cell surface provide the solid basis of the further research on the TCR tetramers and are helpful for exploring new diagnostic study methods about tuberculosis and developing new vaccines.

Analyses for the ?/? T Cell Receptor Gene Rearrangement and CDR3 Repertoire in Active Pulmonary Tuberculosis Patients / 中国免疫学杂志

Objective:To establish a method of multi-PCR to amplify the complete DNA sequence (CDS) of TCR ? and ? chain of the antigen-specific T lymphocytes in local pathologic specimen of active pulmonary tuberculosis patients, and to analyze ?/? T cell receptor gene rearrangement and CDR3 repertoire.Methods:The lymphocytes in bronchoalveolar lavage (BAL) of active pulmonary tuberculosis patients were separated. Following total RNA extraction, cDNA synthesis, Multi-PCR, recombinant clones construction, and sequencing, the CDS of TCR ? and ? chains from these lymphocytes were analyzed by using software of DNAstar and internet TCR resources.Results:24 of ? chain CDS and 13 of ? chain CDS from 3 samples of BAL were obtained. As for TCR ? chain, AV1S2 (54%), AV12S3 (41%), and AV12S2(5%) appeared frequently. BV2(38%), BV29S1(46%), BV14(3%), and BV4S2(3%) in TCR ? chain appeared more often. There were CDR3 diversities between samples and even in the same sample by amino acid sequence analysis, but there were a few identical or similar amino acid sequences. There was the same amino acid sequence of SVGTGTLHQETQY in CDR3 region of ? chain of BAL sample No.1 and No.2; The sequence of AVRDWAGNMLT appeared in two ? chains of BAL sample No.2 and No.3; Moreover, the sequence of AV…DNN…RLM appeared in ? chains of BAL sample No.2 and No.3.Conclusion:A method of Multi-PCR is used to amplify TCR ? and ? chain CDS of tuberculosis patients. There are characteristic T cell clones to proliferate,with TCR ? and ? chain repertiore skewing in local infective focus. The sequences of CDR3 in different TCR clones are mostly different but there are a few identical or similar sequences in the same patient or even between different patients. The identical amino acid sequences of CDR3 are possibly specific for recognizing MTB polypeptide.