Bronchoalveolar lavage cell gene expression in acute lung rejection: development of a diagnostic classifier.

BACKGROUND: Acute lung rejection is a risk factor for chronic rejection, which jeopardizes long-term recipient survival. Presently, acute rejection is diagnosed with the use of transbronchial lung biopsies, which are invasive, expensive, and subject to sampling error. We seek to improve acute rejection diagnostics by identifying genes whose expression in bronchoalveolar lavage (BAL) cells best classifies acute rejection versus no rejection. METHODS: BAL samples were analyzed from 32 subjects whose concurrent histology showed acute rejection (n=14) or no rejection (n=18). Gene expression was measured with Affymetrix microarrays. Quantitative real-time polymerase chain reaction confirmed the microarray results for selected genes. The nearest shrunken centroid method with 10-fold cross validation defined the classification model. A total of 250 iterations of the algorithm were performed to determine the misclassification error rate and the most influential genes in determining classifiers. RESULTS: The estimated overall misclassification rate was <20%. Seven transcripts were present in every classifier, and 52 transcripts were present in >70% of classifiers; these transcripts are related to T-cell function, cytotoxic CD8 activity, and granulocyte degranulation. Eleven of the 52 genes were analyzed with quantitative real-time polymerase chain reaction; all were found to significantly different between the groups, with 10 of 11 increased in acute rejection samples. The proportions of lymphocytes and neutrophils in BAL samples increased in acute rejection but did not outperform the gene-based classifier. CONCLUSIONS: There is a prominent acute rejection-associated signature in BAL cells characterized by increased T-cell, CD8 cytotoxic cell, and neutrophil gene expression. These findings lay the foundation for development of rapid PCR-based assays of gene expression for clinical acute rejection diagnosis.

Novel insights into lung transplant rejection by microarray analysis.

Gene expression microarrays can estimate the prevalence of mRNA for thousands of genes in a small sample of cells or tissue. Organ transplant researchers are increasingly using microarrays to identify specific patterns of gene expression that predict and characterize acute and chronic rejection, and to improve our understanding of the mechanisms underlying organ allograft dysfunction. We used microarrays to assess gene expression in bronchoalveolar lavage cell samples from lung transplant recipients with and without acute rejection on simultaneous lung biopsies. These studies showed increased expression during acute rejection of genes involved in inflammation, apoptosis, and T-cell activation and proliferation. We also studied gene expression during the evolution of airway obliteration in a murine heterotopic tracheal transplant model of chronic rejection. These studies demonstrated specific patterns of gene expression at defined time points after transplantation in allografts, whereas gene expression in isografts reverted back to that of native tracheas within 2 wk after transplantation. These studies demonstrate the potential power of microarrays to identify biomarkers of acute and chronic lung rejection. The application of new genetic, genomic, and proteomic technologies is in its infancy, and the microarray-based studies described here are clearly only the beginning of their application to lung transplantation. The massive amount of data generated per tissue or cell sample has spawned an outpouring of invention in the bioinformatics field, which is developing methodologies to turn data into meaningful and reproducible clinical and mechanistic inferences.

Reduced recombination in maternal meiosis coupled with non-disjunction at meiosis II leading to recurrent 47,XXX.

We determined the meiotic origin and the stage of non-disjunction of the extra X chromosomes in two sisters with 47,XXX chromosomal complements. Segregation of the X chromosomes in all family members was analyzed using X-linked short tandem repeat polymorphic (STRP) markers. Densitometric analysis of two STRP markers confirmed that both sisters had three copies of the X chromosome and the extra X chromosomes were maternally derived. Both sisters did not share the same maternal homologue suggesting that the recurrent trisomy is non-homologous X chromosome-specific. Haplotype analysis demonstrated a reduction to homozygosity for markers examined, covering most of the length of the X chromosomes in both sisters. These findings suggested that the extra X chromosomes have derived from meiotic II non-disjunction following a nullitransitional meiosis I (MI). A lack of recombination in the X chromosomes of both sisters suggests a possible maternal genetic defect leading to an erratic recombination at MI. This information may contribute to further understanding of mechanisms leading to X chromosome non-disjunction and may assist in counseling of families with this chromosomal rearrangement.

Gene expression profiling of bronchoalveolar lavage cells in acute lung rejection.

Lung transplantation is effective for many diseases that are unresponsive to other therapy. However, long-term survival of recipients is limited by the development of bronchiolitis obliterans syndrome. Acute rejection is a major risk factor for bronchiolitis obliterans syndrome, but noninvasive biomarkers have not been identified. To address this deficiency, gene expression microarrays were performed using bronchoalveolar lavage cells of lung transplant recipients with acute rejection (n = 7) and with no rejection (n = 27). The cell and differential counts were similar. Signal values for genes between groups were compared using t tests. One hundred thirty-five genes were upregulated in the acute-rejection group, including genes involved in acute rejection, immune response genes with an unknown role in rejection, genes not known to have a role in rejection, and genes of unknown function. Two-dimensional hierarchical clustering grouped all acute rejection samples into one cluster and the majority of the no-rejection samples into a second cluster. The acute-rejection samples showed significant changes in gene expression for seven biological pathways. Bronchoalveolar lavage cells are a reliable RNA source for microarray analysis, which is powerful in identifying acute-rejection genes. The individual genes, patterns of gene expression, or biologic pathways identified may represent novel biomarkers for acute rejection.