Noninvasive discrimination of rejection in cardiac allograft recipients using gene expression profiling.

Rejection diagnosis by endomyocardial biopsy (EMB) is invasive, expensive and variable. We investigated gene expression profiling of peripheral blood mononuclear cells (PBMC) to discriminate ISHLT grade 0 rejection (quiescence) from moderate/severe rejection (ISHLT > or = 3A). Patients were followed prospectively with blood sampling at post-transplant visits. Biopsies were graded by ISHLT criteria locally and by three independent pathologists blinded to clinical data. Known alloimmune pathways and leukocyte microarrays identified 252 candidate genes for which real-time PCR assays were developed. An 11 gene real-time PCR test was derived from a training set (n = 145 samples, 107 patients) using linear discriminant analysis (LDA), converted into a score (0-40), and validated prospectively in an independent set (n = 63 samples, 63 patients). The test distinguished biopsy-defined moderate/severe rejection from quiescence (p = 0.0018) in the validation set, and had agreement of 84% (95% CI 66% C94%) with grade ISHLT > or = 3A rejection. Patients >1 year post-transplant with scores below 30 (approximately 68% of the study population) are very unlikely to have grade > or = 3A rejection (NPV = 99.6%). Gene expression testing can detect absence of moderate/severe rejection, thus avoiding biopsy in certain clinical settings. Additional clinical experience is needed to establish the role of molecular testing for clinical event prediction and immunosuppression management.

The binding site for Xenopus glucocorticoid receptor accessory factor and a single adjacent half-GRE form an independent glucocorticoid response unit.

In Xenopus laevis, transcription of the gamma-fibrinogen subunit gene is activated by glucocorticoids. Hormone induction is regulated by three glucocorticoid response element (GRE) half-sites and an additional DNA sequence which binds a novel hepatocyte nuclear protein, Xenopus glucocorticoid receptor accessory factor (XGRAF). The XGRAF binding site (GAGTTAA) is located directly upstream of the most distal half-GRE. The proximity of the binding sites for XGRAF and the glucocorticoid receptor (GR) led to the hypothesis that these two sites form a glucocorticoid response unit (GRU). By transfecting DNA into primary hepatocytes, we showed that this GRU confers hormone responsiveness in the absence of other half-GREs. The XGRAF binding site enhances function of the half-GRE without itself responding to glucocorticoids. The GRU retains efficacy in other locations relative to the gamma-fibrinogen gene promoter, further increases transcription when present in multiple copies, and activates a heterologous promoter. Despite the contiguity of the XGRAF binding site and half-GRE, the two sites can be occupied simultaneously in vitro. The binding characteristics correlate with function since mutations that disrupt concurrent XGRAF and GR binding also impair transcription. This novel GRU represents a new regulatory mechanism that may be applicable to other glucocorticoid responsive genes that lack a full GRE.

Overexpression of transforming growth factor beta1 in arterial endothelium causes hyperplasia, apoptosis, and cartilaginous metaplasia.

Uninjured rat arteries transduced with an adenoviral vector expressing an active form of transforming growth factor beta1 (TGF-beta1) developed a cellular and matrix-rich neointima, with cartilaginous metaplasia of the vascular media. Explant cultures of transduced arteries showed that secretion of active TGF-beta1 ceased by 4 weeks, the time of maximal intimal thickening. Between 4 and 8 weeks, the cartilaginous metaplasia resolved and the intimal lesions regressed almost completely, in large part because of massive apoptosis. Thus, locally expressed TGF-beta1 promotes intimal growth and appears to cause transdifferentiation of vascular smooth muscle cells into chondrocytes. Moreover, TGF-beta1 withdrawal is associated with regression of vascular lesions. These data suggest an unexpected plasticity of the adult vascular smooth muscle cell phenotype and provide an etiology for cartilaginous metaplasia of the arterial wall. Our observations may help to reconcile divergent views of the role of TGF-beta1 in vascular disease.

Analysis of the DNA-binding site for Xenopus glucocorticoid receptor accessory factor. Critical nucleotides for binding specificity in vitro and for amplification of steroid-induced fibrinogen gene transcription.

In addition to the glucocorticoid receptor, DNA-binding proteins called accessory factors play a role in hormone activation of many glucocorticoid-responsive genes. Hormonal regulation of the gamma-fibrinogen subunit gene from the frog Xenopus laevis requires a novel DNA sequence that binds a liver nuclear protein called Xenopus glucocorticoid receptor accessory factor (XGRAF). Here we demonstrate that the recognition site for XGRAF encompasses GAGTTAA at positions -175 to -169 relative to the start site of transcription. This sequence is not closely related to the binding sites for known transcription factors. The two guanosines make close contact with XGRAF, as shown by the methylation interference assay. Single-point mutagenesis of every nucleotide in the 9-base pair region from positions -177 to -169 showed an excellent correlation between ability to bind XGRAF in vitro and ability to amplify hormone-induced transcription from DNA transfected into Xenopus primary hepatocytes. Conversely, XGRAF had little or no effect on basal transcription of the gamma-fibrinogen gene. Maximal hormonal induction also requires three half-glucocorticoid response elements (half-GREs) homologous to the downstream half of the consensus GRE. Interestingly, the XGRAF-binding site is immediately adjacent to the most important half-GRE. This close proximity suggests a new mechanism for activation of a gene lacking a conventional full GRE.

Novel accessory factor-binding site required for glucocorticoid regulation of the gamma-fibrinogen subunit gene from Xenopus laevis.

Glucocorticoids induce gene expression by binding to an intracellular receptor that interacts with genomic DNA and stimulates transcription of specific genes. The consensus DNA-binding site for the glucocorticoid receptor, called a glucocorticoid response element (GRE), is GGTACAnnnTGTTCT. In the classical model, binding of the receptor as a dimer to the two halves of the GRE is required for activation of transcription. For some glucocorticoid-regulated genes, additional DNA-binding proteins called accessory factors are necessary for hormonal responsiveness. We have identified a new factor required for glucocorticoid-induced expression of the gamma-fibrinogen subunit gene from the frog Xenopus laevis. Transfection of cloned DNA fragments into primary Xenopus hepatocytes showed that the DNA between 163 and 187 bp upstream of the transcription initiation site is essential for hormonal activation. A single complex forms when this small region of DNA is incubated in vitro with Xenopus liver nuclear proteins. The protein recognition site has been narrowed to AAGAGTTAA, a sequence not previously described as a transcription factor-binding site. We have named the protein(s) bound to this sequence Xenopus glucocorticoid receptor accessory factor (XGRAF). In addition to the XGRAF-binding site, glucocorticoid regulation of the gamma-fibrinogen gene requires at least three nearby GREs, each of which is a poor match to the consensus GRE. The position of the binding site for XGRAF overlaps the putative upstream half of the most important GRE. Models are presented to show possible ways that the novel accessory factor and the glucocorticoid receptor could act through closely juxtaposed sites on the DNA.