Differentiation Of Acute Rejection From Infection In Rat Heart Transplant Model

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Abstract

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 outcome substantially. 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. To date, we have established the surgical techniques necessary to successfully perform and maintain the rat transplant model. We have established a dose of cyclosporin (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 inocula 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 microarry technology to study the impact of animal strain on gene expression during rejection 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 microarry 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 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, immunohistochemistry, and histopathology. In addition, the animals' lungs, spleen, liver, and thymus were procured in the primary study and preserved for potential future analysis. There have been 124 animals used on this protocol to date. This protocol was closed in 2012; however we are continuing to process and analyze data related to the metabolic effects of rejection on cellular energy metabolism; the effects of surgical inflammation over time on gene expression; and the effects of acute cellular rejection and/or infection on gene expression.