1.Effect of chronic lead exposure on expression of autophagy-associated proteins in rat hippocampus.
Wei-feng YE ; Yun TIAN ; Ji-yun HUANG ; Mei-hua LIAO ; Rong-rong TAO ; Gen-sheng ZHANG ; Yi-jia LOU ; Feng HAN
Journal of Zhejiang University. Medical sciences 2012;41(4):402-409
OBJECTIVETo investigate the effects of chronic lead exposure on expression of autophagy-associated proteins in rat hippocampus.
METHODSSD rats were randomly divided into three groups: control group was given distilled water, lead-exposed groups were given 0.5 g/L (low-dose) or 2.0 g/L(high-dose) lead acetate solution in drinking water. The rat pups started to drink the lead content water until 60 d maturity. The lead contents in blood and brain samples were analyzed by graphite furnace atomic absorption spectrophotometry. The expressions of Beclin 1, LC3, LAMP2 and cathepsin B proteins were detected by Western blot and immunohistochemistry.
RESULTSCompared with control group, the contents of lead were significantly higher in blood and hippocampus samples in chronic lead-exposed rats (P<0.01). Western blot showed that the expression of Beclin 1 and LC3-II/LC3-I increased significantly in high dose lead-exposed group compared with control group (P<0.05 or P<0.001). The confocal laser immunostaining results demonstrated that increased immunofluorescence staining of cathepsin B in hippocampal neurons compared with control animals.
CONCLUSIONThe disturbance of autophagy-lysosome signaling molecules might be partially contribute to neurotoxicity of chronic lead exposure.
Animals ; Apoptosis Regulatory Proteins ; metabolism ; Autophagy ; drug effects ; physiology ; Beclin-1 ; Cathepsin B ; metabolism ; Chronic Disease ; Disease Models, Animal ; Female ; Hippocampus ; drug effects ; metabolism ; pathology ; Lead Poisoning ; metabolism ; pathology ; Lysosomal-Associated Membrane Protein 2 ; metabolism ; Male ; Microtubule-Associated Proteins ; metabolism ; Rats ; Rats, Sprague-Dawley ; Signal Transduction ; drug effects
2.Screening for cytotoxic defects with flow cytometric detection of CD107α on natural killer cells and cytotoxic lymphocyte cells.
Jing WANG ; Zheng LIU ; Li-ping JIANG ; Yun-fei AN ; Xiao-dong ZHAO
Chinese Journal of Pediatrics 2012;50(5):386-391
OBJECTIVETo establish a novel flow cytometry-based assay for measuring the expression of lysosomal-associated membrane protein 1 (LAMP-1, CD107α) on the cell surface of natural killer (NK) cells and cytotoxic T lymphocyte (CTL) and evaluate the screening value of this assay for cytotoxic defects-related diseases such as familial hemophagocytic lymphopro-liferative (FHL) syndrome.
METHODThree suspected Chediak-Higashi Syndrome (CHS) patients, three suspected FHL patients and 10 healthy children were enrolled in the study from October 2010 to June 2011. Their PBMCs were separated and activated overnight with IL-2. After the granule release of NK cells activated by phytohemagglutinin (PHA) and CD8+T cells by anti-CD3, the CD107α expression were analyzed by flow cytometry. The peripheral blood DNA and RNA of the patients were extracted to analyze the pathogenic genes via DNA-PCR/RT-PCR and direct sequencing.
RESULTThe CD107α expression on CTL in the ten healthy children significantly increased after activation by anti-CD3 [(0.18 ± 0.07)% vs. (4.47 ± 2.36)%, P < 0.05] and NK cells after activation by PHA [(0.27 ± 0.07)% vs. (5.80 ± 2.83)%, P < 0.05]. The frequency of CD107α-expression NK cells in three suspected CHS after activation was significantly elevated when compared with the healthy control [0.5%, 0.6% vs. (5.80 ± 2.83)%] except patient 2. After the anti-CD3 activation, the frequency of CD107α expression on CTL cells also showed no significant difference [0.3%, 0.9%, 0.2% vs. (4.47 ± 2.36)%] in three patients. All of their mean fluorescence intensity (MFI) showed the same trend. Patient 1 and 3 were identified to have LYST mutations (Patient 1: c.5411-5414 del TTTC, L1741fsX1758 and c.7975 C > T, R2596X; Patient 3: c.4863G > A, R1563H and c.5392-5393delAA, E1739fsX1756). There was no mutation identified in the LYST gene for patient 2. CD107α expression of NK cells and CTL in the suspected FHL patients and in mirror of these findings, no underlying gene variation of PRF, MUNC13-4 and STX11 were identified.
CONCLUSIONWe developed a method to quantitatively assess cytotoxicity of the NK cells and CTL by measuring the expression of CD107α on the cell membrane, which appeared to be an effective and rapid screening test for cytotoxic defects-related diseases such as FHL and other HLH secondary to primary immunodeficiency.
Case-Control Studies ; Cell Degranulation ; immunology ; Cell Membrane ; metabolism ; Chediak-Higashi Syndrome ; diagnosis ; genetics ; immunology ; metabolism ; Child, Preschool ; Cytotoxicity, Immunologic ; Female ; Flow Cytometry ; methods ; Humans ; Infant ; Interleukin-2 ; metabolism ; Killer Cells, Natural ; immunology ; metabolism ; Lymphohistiocytosis, Hemophagocytic ; diagnosis ; genetics ; immunology ; metabolism ; Lysosomal-Associated Membrane Protein 1 ; metabolism ; Male ; Mutation ; Phytohemagglutinins ; metabolism ; T-Lymphocytes, Cytotoxic ; immunology ; metabolism
3.Construction of hu-PBL/SCID chimeras and development of EBV-related lymphomas.
Run-liang GAN ; Ke LAN ; Zhi-hua YIN ; Li-jiang WANG ; Ying SONG ; Kai-tai YAO
Chinese Medical Sciences Journal 2005;20(1):16-22
OBJECTIVETo construct hu-PBL/SCID chimeras and to investigate the development of lymphoma and oncogenicity of the Epstein-Barr virus (EBV).
METHODSHuman peripheral blood lymphocytes (PBLs) were isolated from healthy adult donors and transplanted intraperitoneally into severe combined immunodeficient (SCID) mice. Mice with hu-PBL engraftment from healthy EBV seronegative donors were injected intraperitoneally with EBV-containing supernatant from suspension culture of B95-8 cell line (active infection), whereas mice receiving lymphocytes from healthy EBV seropositive donors were not re-infected with B95-8 derived EBV (latent infection). Pathological examination and molecular analysis were performed on experimental animals and induced neoplasms.
RESULTSIn the early stage of this experiment, 12 mice died of acute graft-versus-host disease, mortality was 34.3% (12/35 mice) with an average life span of 17.5 days. In 19 survival hu-PBL/SCID chimeric recipients from 12 healthy donors, tumor incidence was 84.2% (16/19 mice). The average survival time of tumor-bearing mice was 65.5 days. EBV-related neoplasms in SCID mice were nodular tumors with aggressive and fatal features. Histological morphology of tumors exhibited diffuse large cell lymphomas. Immunohistochemistry revealed that LCA (CD45) and L26 (CD20) were positive, but both PS1 (CD3) and UCHL-1 (CD45RO) were negative, and EBV products ZEBRA, LMP1, and EBNA2 were expressed in a small number of tumor cells. EB virus particles were seen in the nuclei of some tumor cells by electron microscopy, and EBV DNA could be amplified in the tumor tissues by PCR. In situ hybridization indicated that the nuclei of tumor cells contained human-specific Alu sequence.
CONCLUSIONSEBV-induced tumors were human B-cell malignant lymphomas. We obtained direct causative evidence dealing with EBV-associated tumor deriving from normal human cells.
Adult ; Animals ; Antigens, CD20 ; metabolism ; Chimera ; Epstein-Barr Virus Infections ; immunology ; virology ; Graft vs Host Disease ; prevention & control ; virology ; Herpesvirus 4, Human ; physiology ; Humans ; Leukocyte Common Antigens ; metabolism ; Leukocyte Transfusion ; methods ; Lymphoma, B-Cell ; immunology ; virology ; Lysosomal-Associated Membrane Protein 1 ; metabolism ; Mice ; Mice, SCID