Acute cardiac allograft rejection and infection remain significant sources of morbidity and mortality after heart transplantation, accounting for nearly 50% of reported deaths. It is often difficult to clinically distinguish between rejection and infection because they are both inflammatory processes with similar, nonspecific symptoms. However, this differential is essential for determining therapy. Identifying laboratory methods that will permit safe and concise early differentiation between rejection and infection in the transplant patient will improve outcomes. We established an ACUC protocol that allowed us to study whether gene microarray analysis of peripheral blood mononuclear cells (PBMC) would reliably differentiate acute heart rejection from infection in the transplanted rat. The ACUC protocol also allowed us to do pilot studies necessary to support the main protocol. We established the surgical techniques necessary to successfully perform and maintain the rat transplant model. We determined the dose of cyclosporine (CSA) in this model that reliably suppresses rejection during its administration, but will permit the emergence of Grade 3 rejection upon its discontinuation. We have also determined the appropriate inoculant of intra-bronchial E. coli bacteria that is sufficient to cause a pneumonia and a systemic inflammatory response without being immediately lethal in transplanted rats receiving CSA. In addition, we have used gene microarray technology to study the impact of animal strain on gene expression during rejection (BMC Genomics 10:280, 2009) and the time course of post-surgical inflammatory changes in order to determine the most opportune time to harvest the transplanted hearts (i.e. when gene microarray signatures due to surgical inflammatory changes are dissipating). We have also completed the main study protocol. Our main protocol combines two well-established rat models, the first is a heterotopic heart transplantation model and the second is an E. coli pulmonary infection model. All rats underwent heart transplantation on day 0 in conjunction with daily CSA (10 mg/kg subcutaneous) to suppress rejection. After transplant, animals were randomized at day 6 to have CSA discontinued, in order to initiate rejection, or continued, in order to further suppress rejection. After discontinuing CSA the animals were again randomized on day 13 to receive intrabronchial E. coli inoculation or saline inoculation. Consequently, four groups (2 by 2 design) were studied: no rejection (i.e. receiving CSA) without infection, no rejection (i.e. receiving CSA) with infection, rejection (i.e. not receiving CSA) without infection, and rejection (i.e. not receiving CSA) with infection. On day 14, all animals were sacrificed and the blood and heart removed for gene microarray analysis. Other analytic tools that may be employed include: RT-PCR, western blot, in-situ hybridization, proteomics, histopathology, and immunehistochemistry. In addition, the animals' hearts, lungs, spleen, liver, and thymus were procured in the primary study and preserved for potential future analysis. A total of 124 rats were used over the duration of the protocol, which was closed in 2012. However, we are continuing to process and analyze data related to the metabolic effects of rejection on cellular energy metabolism (JHLT 36 (4S):S372-S373, 2017); the effects of surgical inflammation over time on gene expression; and the effects of acute cellular rejection and/or infection on gene expression. During the 2016-17 reporting period we entered into a collaboration with the Laboratory of Transplantation Genomics (LTG) of the NHLBI to develop a protocol for processing DNA derived from formalin-fixed tissue for methylatomic sequencing. As part of routine transplant care, lung and heart transplant recipients undergo scheduled biopsies to monitor for acute rejection with excess biopsy tissue being stored in formalin. We provided LTG with 10 paraffin blocks of formalin-fixed rat cardiac transplant tissues from this protocol. The plan was to extract DNA for bisulfite treatment and methylation sequencing and then compare the epigenetic landscape with known cardiac specific signatures. If this analysis yields a significant correlation, then it may be possible to use formalin fixed human allotransplant cardiac tissue for epigenetic profiling. During the 2017-18 reporting period one mg of this tissue from each of the provided samples was used for DNA isolation. While we were able to extract DNA, further analysis showed the DNA to be fragmented and of less than suitable quality for methylatomic analysis. As noted above the protocol was closed in 2012, but we continue to report on the protocol do to our ongoing collaborations and available library of samples.