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Intragraft gene expression profile associated with the induction of tolerance.

Doki T, Mello M, Mock D, Evans JM, Kearns-Jonker M - BMC Immunol. (2008)

Bottom Line: The expression of the incompatible carbohydrate antigen (alphaGal) was reduced by 2 months post-transplant when compared with the expression of this gene at Day 10 post-transplant.Our study suggests that tolerance is associated with intragraft gene expression changes that render the heart resistant to immune-mediated rejection.Genes associated with stress and immunity are up-regulated, however cytoprotective genes HO-1, Bcl2 and A20 were not up-regulated.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Cardiothoracic Surgery Research, Transplantation Biology Research Laboratory, Childrens Hospital Los Angeles, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA. tdoki@chla.usc.edu

ABSTRACT

Background: Xenotransplantation holds the promise of providing an unlimited supply of donor organs for terminal patients with organ failure. Pre-existing natural antibodies to the Galalpha1,3Galbeta1,4GlcNac-R (alphaGal) carbohydrate xenoantigen, however, bind rapidly to the graft endothelium and initiate hyperacute rejection of wild type pig grafts in humans. Experimental procedures designed to prevent xenoantibody-mediated rejection have been tested in gal knockout mice. These mice produce anti-gal xenoantibodies and are widely used as small animal models for xenotransplantation research. In this model, chimerism for cells expressing the gal carbohydrate can be achieved by transplantation of mixed cells or by transduction of bone marrow cells with viral vectors expressing a functional alpha1,3 galactosyltransferase gene. Chimerism induces tolerance to heart grafts expressing alphaGal. The mechanisms by which tolerance is achieved include systemic changes such as clonal deletion and/or anergy. Intragraft changes that occur during the early stages of tolerance induction have not been characterized.

Results: Cytoprotective genes heme oxygenase-1 (HO-1), Bcl2, and A20 that have been reported to contribute to long-term graft survival in various models of accommodation were not expressed at high levels in tolerant heart grafts. Intragraft gene expression at both early (Day 10) and late (>2 month) time points after heart transplant were examined by real-time PCR and microarray analysis was used to identify changes associated with the induction of tolerance. Intragraft gene expression profiling using microarray analysis demonstrated that genes identified in the functional categories of stress and immunity and signal transduction were significantly up-regulated in early tolerant grafts compared with syngeneic control grafts. Biological process classification showed lower binomial p-values in the categories of "response to biotic stimulus, defense response, and immune response" suggesting that up-regulated genes identified in these grafts promote survival in the presence of an immune response. The expression of the incompatible carbohydrate antigen (alphaGal) was reduced by 2 months post-transplant when compared with the expression of this gene at Day 10 post-transplant. These results suggest that the gal carbohydrate antigen is downmodulated over time in grafts that demonstrate tolerance.

Conclusion: Our study suggests that tolerance is associated with intragraft gene expression changes that render the heart resistant to immune-mediated rejection. Genes associated with stress and immunity are up-regulated, however cytoprotective genes HO-1, Bcl2 and A20 were not up-regulated. The expression of the gal carbohydrate, the key target initiating an immune response in this model, is down-regulated in the post-transplant period.

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Validation of microarray results by real-time PCR. Selected genes (Ptger4, Tnsls10, Il2rg, Stat3, Cfh, Cxcl12, Hfe) in the early tolerance group (GalT BMT) compared to syngeneic controls were analyzed by real-time PCR to determine whether the data obtained by microarray analysis could be validated using an alternative technique. Relative cDNA expression levels were normalized with respect to GAPDH gene expression as internal control. Results are shown as the logarithmic value of mean fold-change of gene expression.
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Figure 5: Validation of microarray results by real-time PCR. Selected genes (Ptger4, Tnsls10, Il2rg, Stat3, Cfh, Cxcl12, Hfe) in the early tolerance group (GalT BMT) compared to syngeneic controls were analyzed by real-time PCR to determine whether the data obtained by microarray analysis could be validated using an alternative technique. Relative cDNA expression levels were normalized with respect to GAPDH gene expression as internal control. Results are shown as the logarithmic value of mean fold-change of gene expression.

Mentions: Real-time PCR was used to confirm the results obtained from the gene expression profiling studies. Four up-regulated genes and 3 down-regulated genes were selected for quantification of gene expression by real-time PCR. RNA from transplanted hearts isolated from GalT BMT groups (n = 4) and syngeneic control groups (n = 4) was used for this experiment. Real-time PCR results were found to correlate with the differential gene expression data obtained by the microarray analysis (Fig. 5). The sequences of primers used for real-time PCR validation are listed in Table 3.


Intragraft gene expression profile associated with the induction of tolerance.

Doki T, Mello M, Mock D, Evans JM, Kearns-Jonker M - BMC Immunol. (2008)

Validation of microarray results by real-time PCR. Selected genes (Ptger4, Tnsls10, Il2rg, Stat3, Cfh, Cxcl12, Hfe) in the early tolerance group (GalT BMT) compared to syngeneic controls were analyzed by real-time PCR to determine whether the data obtained by microarray analysis could be validated using an alternative technique. Relative cDNA expression levels were normalized with respect to GAPDH gene expression as internal control. Results are shown as the logarithmic value of mean fold-change of gene expression.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2275216&req=5

Figure 5: Validation of microarray results by real-time PCR. Selected genes (Ptger4, Tnsls10, Il2rg, Stat3, Cfh, Cxcl12, Hfe) in the early tolerance group (GalT BMT) compared to syngeneic controls were analyzed by real-time PCR to determine whether the data obtained by microarray analysis could be validated using an alternative technique. Relative cDNA expression levels were normalized with respect to GAPDH gene expression as internal control. Results are shown as the logarithmic value of mean fold-change of gene expression.
Mentions: Real-time PCR was used to confirm the results obtained from the gene expression profiling studies. Four up-regulated genes and 3 down-regulated genes were selected for quantification of gene expression by real-time PCR. RNA from transplanted hearts isolated from GalT BMT groups (n = 4) and syngeneic control groups (n = 4) was used for this experiment. Real-time PCR results were found to correlate with the differential gene expression data obtained by the microarray analysis (Fig. 5). The sequences of primers used for real-time PCR validation are listed in Table 3.

Bottom Line: The expression of the incompatible carbohydrate antigen (alphaGal) was reduced by 2 months post-transplant when compared with the expression of this gene at Day 10 post-transplant.Our study suggests that tolerance is associated with intragraft gene expression changes that render the heart resistant to immune-mediated rejection.Genes associated with stress and immunity are up-regulated, however cytoprotective genes HO-1, Bcl2 and A20 were not up-regulated.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Cardiothoracic Surgery Research, Transplantation Biology Research Laboratory, Childrens Hospital Los Angeles, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA. tdoki@chla.usc.edu

ABSTRACT

Background: Xenotransplantation holds the promise of providing an unlimited supply of donor organs for terminal patients with organ failure. Pre-existing natural antibodies to the Galalpha1,3Galbeta1,4GlcNac-R (alphaGal) carbohydrate xenoantigen, however, bind rapidly to the graft endothelium and initiate hyperacute rejection of wild type pig grafts in humans. Experimental procedures designed to prevent xenoantibody-mediated rejection have been tested in gal knockout mice. These mice produce anti-gal xenoantibodies and are widely used as small animal models for xenotransplantation research. In this model, chimerism for cells expressing the gal carbohydrate can be achieved by transplantation of mixed cells or by transduction of bone marrow cells with viral vectors expressing a functional alpha1,3 galactosyltransferase gene. Chimerism induces tolerance to heart grafts expressing alphaGal. The mechanisms by which tolerance is achieved include systemic changes such as clonal deletion and/or anergy. Intragraft changes that occur during the early stages of tolerance induction have not been characterized.

Results: Cytoprotective genes heme oxygenase-1 (HO-1), Bcl2, and A20 that have been reported to contribute to long-term graft survival in various models of accommodation were not expressed at high levels in tolerant heart grafts. Intragraft gene expression at both early (Day 10) and late (>2 month) time points after heart transplant were examined by real-time PCR and microarray analysis was used to identify changes associated with the induction of tolerance. Intragraft gene expression profiling using microarray analysis demonstrated that genes identified in the functional categories of stress and immunity and signal transduction were significantly up-regulated in early tolerant grafts compared with syngeneic control grafts. Biological process classification showed lower binomial p-values in the categories of "response to biotic stimulus, defense response, and immune response" suggesting that up-regulated genes identified in these grafts promote survival in the presence of an immune response. The expression of the incompatible carbohydrate antigen (alphaGal) was reduced by 2 months post-transplant when compared with the expression of this gene at Day 10 post-transplant. These results suggest that the gal carbohydrate antigen is downmodulated over time in grafts that demonstrate tolerance.

Conclusion: Our study suggests that tolerance is associated with intragraft gene expression changes that render the heart resistant to immune-mediated rejection. Genes associated with stress and immunity are up-regulated, however cytoprotective genes HO-1, Bcl2 and A20 were not up-regulated. The expression of the gal carbohydrate, the key target initiating an immune response in this model, is down-regulated in the post-transplant period.

Show MeSH
Related in: MedlinePlus