AIM: To establish a simplified method for generating peptide-major histocompatibility complex (MHC) class I tetramers.METHODS: cDNAs encoding the extracellular domain of human lymphocyte antigen (HLA)-A*0201 heavy cha...AIM: To establish a simplified method for generating peptide-major histocompatibility complex (MHC) class I tetramers.METHODS: cDNAs encoding the extracellular domain of human lymphocyte antigen (HLA)-A*0201 heavy chain (A2) and β2-microglobulin (β2m) from total RNA extracted from leukocytes of HLA-A2+ donors were doned into separate expression vectors by reverse transcription-polymerase chain reaction. The recombinant A2 and β2m proteins were expressed in Escherichia coli strain BL21(DE3) and recovered from the inclusion body fraction. Soluble A2 proteins loaded with specific antigen peptides were refolded by dilution from the heavy chain in the presence of light chain β2m and HLA-A2-restricted peptide antigens. The refolded A2monomers were biotinylated with a commercial biotinylation enzyme (BirA) and purified by low pressure anion exchange chromatography on a Q-Sepharose (fast flow) column.The tetramers were then formed by mixing A2 monomers with streptavidin-PE in a molar ratio of 4:1. Flow cytometry was used to confirm the expected tetramer staining of CD8+ T cells.RESULTS: Recombinant genes for HLA-A*0201 heavy chain (A2) fused to a BirA substrate peptide (A2-BSP) and mature β2m from HLA-A2+ donor leukocytes were successfully doned and highly expressed in E. coli. Two soluble monomeric A2-peptide complexes were reconstituted from A2-BSP in the presence of β2m and peptides loaded with either human cytomegalovirus pp65495-503 peptide (NLVPMVATV,NLV; designated as A2-NLV) or influenza virus matrix protein Mp58-66 peptide (GILGFVFTL, GIL; designated as A2-GIL). Refolded A2-NLV or A2-GIL monomers were biotinylated and highly purified by single step anion exchange column chromatography. The tetramers were then formed by mixing the biotinylated A2-NLV or A2-GIL monomers with streptavidin-PE, leading to more than 80% multiplication as revealed by SDS-PAGE under non-reducing, unboiled conditions. Flow cytometry revealed that these tetramers could specifically bind to CD8+ T cells from a HLA-A2+ donor,but failed to bind to those from a HLA-A2- donor.CONCLUSION: The procedure is simple and efficient for generating peptide-MHC tetramers.展开更多
AIM: To evaluate the surface expression of triggering receptor on myeloid cell 1 (TREM-1), class Ⅱ major histocompatibility complex molecules (HLA-DR), andthe expression of the splicing variant (svTREM-1) ofTREM-1 in...AIM: To evaluate the surface expression of triggering receptor on myeloid cell 1 (TREM-1), class Ⅱ major histocompatibility complex molecules (HLA-DR), andthe expression of the splicing variant (svTREM-1) ofTREM-1 in septic patients and those subjected to major abdominal surgery.METHODS: Using flow cytometry, we examined the surface expression of TREM-1 and HLA-DR in peripheral blood monocytes from 11 septic patients, 7 elective gastrointestinal surgical patients, and 10 healthy volunteers. svTREM-1 levels were analyzed by RT-PCR. RESULTS: Basal expression of TREM-1 and HLA-DR in healthy volunteers was 35.91±14.75 MFI and75.8±18.3%, respectively. In septic patients, TREM-1 expression was 59.9±23.9 MFI and HLA-DR expression was 44.39±20.25%, with a significant differencebetween healthy and septic groups (P＜0.05) for bothmolecules. In the surgical patients, TREM-1 and HLA-DR expressions were 56.8±20.85 MFI and 71±13.8% before surgery and 72.65±29.92 MlFI and 72.82±22.55% after surgery. TREM-1 expression was significantly different(P = 0.0087) between the samples before and aftersurgery and svTREM-1 expression was 0.8590±0.1451 MF1, 0.8820±0.1460 MF1, and 2.210±0.7873MF1 in the healthy, surgical (after surgery) and septic groups, respectively. There was a significant difference (P = 0.048) in svTREM-1 expression between the healthy and surgical groups and the septic group.CONCLUSION: TREM-1 expression is increased during systemic inflammatory conditions such as sepsis and the postoperative phase. Simultaneous low expression of HLA-DR molecules correlates with the severity of illness and increases susceptibility to infection. Additionally, TREM-1 expression is distinctly different in surgical patients at different stages of the inflammatory response before and after surgery. Thus, surface TREM-1 appears to be an endogenous signal during the course of the inflammatory response. svTREM-1 expression is significantly increased during sepsis, appearing to be an indicator of severity of illness. Together, these data indicate that TREM-1 may play an important role in establishing and amplifying the systemic inflammatory response. TREM-1, HLA-DR, and svTREM-1 expression analysis can provide useful diagnostic and prognostic indicators during SIRS, CARS, and sepsis.展开更多
基金Supported by the National Natural Science Foundation of China, No. 30230350 and No. 30371651Major State Basic Research Development Program of China, 973 Program, No. G2000057006
文摘AIM: To establish a simplified method for generating peptide-major histocompatibility complex (MHC) class I tetramers.METHODS: cDNAs encoding the extracellular domain of human lymphocyte antigen (HLA)-A*0201 heavy chain (A2) and β2-microglobulin (β2m) from total RNA extracted from leukocytes of HLA-A2+ donors were doned into separate expression vectors by reverse transcription-polymerase chain reaction. The recombinant A2 and β2m proteins were expressed in Escherichia coli strain BL21(DE3) and recovered from the inclusion body fraction. Soluble A2 proteins loaded with specific antigen peptides were refolded by dilution from the heavy chain in the presence of light chain β2m and HLA-A2-restricted peptide antigens. The refolded A2monomers were biotinylated with a commercial biotinylation enzyme (BirA) and purified by low pressure anion exchange chromatography on a Q-Sepharose (fast flow) column.The tetramers were then formed by mixing A2 monomers with streptavidin-PE in a molar ratio of 4:1. Flow cytometry was used to confirm the expected tetramer staining of CD8+ T cells.RESULTS: Recombinant genes for HLA-A*0201 heavy chain (A2) fused to a BirA substrate peptide (A2-BSP) and mature β2m from HLA-A2+ donor leukocytes were successfully doned and highly expressed in E. coli. Two soluble monomeric A2-peptide complexes were reconstituted from A2-BSP in the presence of β2m and peptides loaded with either human cytomegalovirus pp65495-503 peptide (NLVPMVATV,NLV; designated as A2-NLV) or influenza virus matrix protein Mp58-66 peptide (GILGFVFTL, GIL; designated as A2-GIL). Refolded A2-NLV or A2-GIL monomers were biotinylated and highly purified by single step anion exchange column chromatography. The tetramers were then formed by mixing the biotinylated A2-NLV or A2-GIL monomers with streptavidin-PE, leading to more than 80% multiplication as revealed by SDS-PAGE under non-reducing, unboiled conditions. Flow cytometry revealed that these tetramers could specifically bind to CD8+ T cells from a HLA-A2+ donor,but failed to bind to those from a HLA-A2- donor.CONCLUSION: The procedure is simple and efficient for generating peptide-MHC tetramers.
基金Supported by Fondo para el Fomento de la Investigacion Medica (FOFOI), Institute Mexicano del Seguro Social No. IMSS-2004/045
文摘AIM: To evaluate the surface expression of triggering receptor on myeloid cell 1 (TREM-1), class Ⅱ major histocompatibility complex molecules (HLA-DR), andthe expression of the splicing variant (svTREM-1) ofTREM-1 in septic patients and those subjected to major abdominal surgery.METHODS: Using flow cytometry, we examined the surface expression of TREM-1 and HLA-DR in peripheral blood monocytes from 11 septic patients, 7 elective gastrointestinal surgical patients, and 10 healthy volunteers. svTREM-1 levels were analyzed by RT-PCR. RESULTS: Basal expression of TREM-1 and HLA-DR in healthy volunteers was 35.91±14.75 MFI and75.8±18.3%, respectively. In septic patients, TREM-1 expression was 59.9±23.9 MFI and HLA-DR expression was 44.39±20.25%, with a significant differencebetween healthy and septic groups (P＜0.05) for bothmolecules. In the surgical patients, TREM-1 and HLA-DR expressions were 56.8±20.85 MFI and 71±13.8% before surgery and 72.65±29.92 MlFI and 72.82±22.55% after surgery. TREM-1 expression was significantly different(P = 0.0087) between the samples before and aftersurgery and svTREM-1 expression was 0.8590±0.1451 MF1, 0.8820±0.1460 MF1, and 2.210±0.7873MF1 in the healthy, surgical (after surgery) and septic groups, respectively. There was a significant difference (P = 0.048) in svTREM-1 expression between the healthy and surgical groups and the septic group.CONCLUSION: TREM-1 expression is increased during systemic inflammatory conditions such as sepsis and the postoperative phase. Simultaneous low expression of HLA-DR molecules correlates with the severity of illness and increases susceptibility to infection. Additionally, TREM-1 expression is distinctly different in surgical patients at different stages of the inflammatory response before and after surgery. Thus, surface TREM-1 appears to be an endogenous signal during the course of the inflammatory response. svTREM-1 expression is significantly increased during sepsis, appearing to be an indicator of severity of illness. Together, these data indicate that TREM-1 may play an important role in establishing and amplifying the systemic inflammatory response. TREM-1, HLA-DR, and svTREM-1 expression analysis can provide useful diagnostic and prognostic indicators during SIRS, CARS, and sepsis.
