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Endoplasmic reticulum stress-mediated apoptosis involved in indirect recognition pathway blockade induces long-term heart allograft survival.

Xiang J, Gu X, Qian S, Chen Z - J. Biomed. Biotechnol. (2010)

Bottom Line: This approach could specifically and effectively knock down CD80 and CD86 expression.We also found a higher percentage of apoptotic T cells in lymph tissues and grafts than that detected in control group.Our results indicated that ERS-induced apoptosis may be involved in allogeneic T-cell apoptosis, and the ERS-mediated apoptosis pathway may be a novel target in clinical prevention and therapy of allograft rejection.

View Article: PubMed Central - PubMed

Affiliation: Department of General Surgery, Huashan Hospital, Fudan University, Shanghai 200040, China.

ABSTRACT
Implementation of dendritic cell- (DC-) based therapies in organ transplantation can reduce dependency on nonspecific immunosuppression. Despite extensive research, mechanisms of equipped DCs inducing transplant tolerance remain incomplete. Here, we applied RNA interference technique to inhibit CD80 and CD86 expression in host bone marrow-derived DCs. This approach could specifically and effectively knock down CD80 and CD86 expression. T cells primed by these DCs inhibited allogeneic responses. Administration of recipient DCs loaded with alloantigen after CD80 and CD86 blockade prolonged cardiac allograft survival. We also found a higher percentage of apoptotic T cells in lymph tissues and grafts than that detected in control group. In addition, these T cells expressed high expression of GRP78 than controls, indicating activation of unfolded protein responses. Upregulation of CHOP expression among these cells suggested that the endoplasmic reticulum stress (ERS) response switched to a proapoptotic response. Our results indicated that ERS-induced apoptosis may be involved in allogeneic T-cell apoptosis, and the ERS-mediated apoptosis pathway may be a novel target in clinical prevention and therapy of allograft rejection.

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Related in: MedlinePlus

(a) Schematic presentation of the plasmid pGCL-GFP, which encodes an HIV-derived lentiviral vector containing a multiple cloning site (MCS) for insertion of shRNA constructs to be driven by an upstream U6 promoter and a downstream cytomegalovirus promoter-GFP fluorescent protein (marker gene) cassette flanked by loxp sites. (b) The stem-loop-encoded shRNA has sequence identity to a 21-nt region of CD80 mRNA.
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fig1: (a) Schematic presentation of the plasmid pGCL-GFP, which encodes an HIV-derived lentiviral vector containing a multiple cloning site (MCS) for insertion of shRNA constructs to be driven by an upstream U6 promoter and a downstream cytomegalovirus promoter-GFP fluorescent protein (marker gene) cassette flanked by loxp sites. (b) The stem-loop-encoded shRNA has sequence identity to a 21-nt region of CD80 mRNA.

Mentions: The recombinant lentivirus was constructed as previously described [20], with some modifications. In brief, short hairpin RNA (shRNA) fragments (Table 1) were hybridized with synthesized sense and antisense oligonucleotides. The hybridized CD80 shRNA fragment was cloned into the plasmid pGCL-GFP (Figure 1). Recombinant lentiviruses were produced by the transduction of 293T cells. The 293T cells were cotransduced with 20 μg of pGCL-GFP, 15 μg of pHelper1.0, and 10 μg of pHelper2.0 by 100 μL of Lipofectamine 2000 (Invitrogen, Garlsbad, CA). This recombinant lentivirus was designated CD80 lenti. The titers of CD80 lenti were determined with a green fluorescent protein (GFP) assay. CD86 lenti and NC lenti were constructed by a similar process. NC lenti contained scrambled shRNA fragment, an irrelevant shRNA with random nucleotides, and a GC ratio close to above-mentioned shRNA.


Endoplasmic reticulum stress-mediated apoptosis involved in indirect recognition pathway blockade induces long-term heart allograft survival.

Xiang J, Gu X, Qian S, Chen Z - J. Biomed. Biotechnol. (2010)

(a) Schematic presentation of the plasmid pGCL-GFP, which encodes an HIV-derived lentiviral vector containing a multiple cloning site (MCS) for insertion of shRNA constructs to be driven by an upstream U6 promoter and a downstream cytomegalovirus promoter-GFP fluorescent protein (marker gene) cassette flanked by loxp sites. (b) The stem-loop-encoded shRNA has sequence identity to a 21-nt region of CD80 mRNA.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2871569&req=5

fig1: (a) Schematic presentation of the plasmid pGCL-GFP, which encodes an HIV-derived lentiviral vector containing a multiple cloning site (MCS) for insertion of shRNA constructs to be driven by an upstream U6 promoter and a downstream cytomegalovirus promoter-GFP fluorescent protein (marker gene) cassette flanked by loxp sites. (b) The stem-loop-encoded shRNA has sequence identity to a 21-nt region of CD80 mRNA.
Mentions: The recombinant lentivirus was constructed as previously described [20], with some modifications. In brief, short hairpin RNA (shRNA) fragments (Table 1) were hybridized with synthesized sense and antisense oligonucleotides. The hybridized CD80 shRNA fragment was cloned into the plasmid pGCL-GFP (Figure 1). Recombinant lentiviruses were produced by the transduction of 293T cells. The 293T cells were cotransduced with 20 μg of pGCL-GFP, 15 μg of pHelper1.0, and 10 μg of pHelper2.0 by 100 μL of Lipofectamine 2000 (Invitrogen, Garlsbad, CA). This recombinant lentivirus was designated CD80 lenti. The titers of CD80 lenti were determined with a green fluorescent protein (GFP) assay. CD86 lenti and NC lenti were constructed by a similar process. NC lenti contained scrambled shRNA fragment, an irrelevant shRNA with random nucleotides, and a GC ratio close to above-mentioned shRNA.

Bottom Line: This approach could specifically and effectively knock down CD80 and CD86 expression.We also found a higher percentage of apoptotic T cells in lymph tissues and grafts than that detected in control group.Our results indicated that ERS-induced apoptosis may be involved in allogeneic T-cell apoptosis, and the ERS-mediated apoptosis pathway may be a novel target in clinical prevention and therapy of allograft rejection.

View Article: PubMed Central - PubMed

Affiliation: Department of General Surgery, Huashan Hospital, Fudan University, Shanghai 200040, China.

ABSTRACT
Implementation of dendritic cell- (DC-) based therapies in organ transplantation can reduce dependency on nonspecific immunosuppression. Despite extensive research, mechanisms of equipped DCs inducing transplant tolerance remain incomplete. Here, we applied RNA interference technique to inhibit CD80 and CD86 expression in host bone marrow-derived DCs. This approach could specifically and effectively knock down CD80 and CD86 expression. T cells primed by these DCs inhibited allogeneic responses. Administration of recipient DCs loaded with alloantigen after CD80 and CD86 blockade prolonged cardiac allograft survival. We also found a higher percentage of apoptotic T cells in lymph tissues and grafts than that detected in control group. In addition, these T cells expressed high expression of GRP78 than controls, indicating activation of unfolded protein responses. Upregulation of CHOP expression among these cells suggested that the endoplasmic reticulum stress (ERS) response switched to a proapoptotic response. Our results indicated that ERS-induced apoptosis may be involved in allogeneic T-cell apoptosis, and the ERS-mediated apoptosis pathway may be a novel target in clinical prevention and therapy of allograft rejection.

Show MeSH
Related in: MedlinePlus