Christopher E. Gonde

NITRIC OXIDE GENERATION : A PREDICTIVE PARAMETER OF ACUTE ALLOGRAFT REJECTION

The L-arginine:nitric oxide (NO) biosynthetic pathway has been proposed as an important mediator in host defense mechanisms and may therefore play a role in the acute allograft response. We have studied NO generation in liver allograft rejection and determined its value in immunological monitoring. Stable end products of this pathway have been determined serially in 50 primary liver recipients and compared with 2 known mediators and markers of acute allograft rejection (IL-2R positive lymphocytes and circulating TNF alpha). Plasma concentrations of acid-labile nitrosocompounds (NOx), which increased during acute allograft rejection (P < 0.0001), correlated with rejection severity and were reduced after administration of supplemental high dose glucocorticoids. Concentrations were significantly lower in nonrejection graft complications but were elevated during episodes of sepsis. Correlations between plasma NOx levels and circulating TNF-alpha (r = 0.451, P < 0.001) and IL-2R-positive lymphocytes in peripheral blood (r = 0.781, P < 0.001) were demonstrated. In a logistic analysis of these variables, plasma NOx was the most predictive parameter of an episode of acute cellular rejection. Nitric oxide generation in FK506-treated patients was lower compared with patients receiving a CsA-based immunosuppression regimen and was associated with a reduced frequency of acute rejection in the FK506 group. These data are consistent with a role for NO in the cellular alloantigen immune response and indicate that monitoring of plasma levels of NOx may be useful in the detection of acute allograft rejection.

Serial circulating markers of inflammation in biliary atresia—Evolution of the post‐operative inflammatory process

Biliary atresia (BA) may be characterized as an occlusive cholangiopathy affecting both intra‐ and extra‐hepatic parts of the biliary tree, together with a pronounced inflammatory response consisting of hepatic infiltration of (predominantly) CD4+ lymphocytes and macrophages. Soluble cellular adhesion molecules are also known to be raised at the time of portoenterostomy, presumably reflecting intrahepatic disease. We investigated this measurable inflammatory component longitudinally by studying a panel of cellular adhesion molecules (soluble intercellular adhesion molecule‐1 [sICAM‐1], soluble vascular cell adhesion molecule‐1 [sVCAM‐1]) and soluble proinflammatory mediators (T helper 1 [interleukin {IL}‐2 and interferonγ] and T helper 2 [IL‐4 and IL‐10]) cytokines and macrophage markers (tumor necrosis factor [TNF] α and IL‐18) in 21 consecutive infants with BA post‐Kasai portoenterostomy (KP). The levels of all adhesion molecules and cytokines (except IL‐10) increased progressively by 6 months post‐portoenterostomy. The response was non‐polarized but with 100‐fold increases in IL‐2, TNFα and IL‐18 particularly but only modest elevations in IL‐10. When proinflammatory profiles were related to outcome, we found poor discrimination if assessed as clearance of jaundice but markedly higher values for IL‐2, interferonγ, IL‐4, IL‐10, TNFα and sICAM‐1 for those who would be transplanted by 1 year. Using ROC curve analysis for sICAM‐1 levels at 1 month post‐KP, a cutoff level of 1,779 ng/ml was determined to predict the need for transplantation at 1 year with 92% specificity and 87% sensitivity. Conclusion: The early circulating inflammatory process in BA is persistent, progressive and involves a non‐polarized T cell, macrophage and cell adhesion molecule response only partially ameliorated by KP. (HEPATOLOGY 2007;46:180–187.)

An Investigation Into the Bias Between Liquid Chromatography-Tandem Mass Spectrometry and an Enzyme Multiplied Immunoassay Technique for the Measurement of Mycophenolic Acid

Mycophenolic acid is now the second most widely used immunosuppressant in solid organ transplantation. Overestimation of mycophenolic acid concentration is a recognized problem of immunoassay, and high-performance liquid chromatography with ultraviolet detection methods have long analysis times and a risk of analyte coelution which may compromise high sample throughput in a clinically meaningful time frame. A novel liquid chromatography-tandem mass spectrometry assay for mycophenolic acid was developed using very small (10 μL) sample volumes and evaluated in comparison with an established immunological assay. The enzyme mediated immunoassay showed a median positive bias compared with liquid chromatography-tandem mass spectrometry of 14.6%. Linear regression analysis showed a significant positive impact of bilirubin (r2 = 0.230) on bias with further increases of r2 to 0.261, 0.286, and 0.294 with the stepwise addition of creatinine, hematocrit, and γ-glutamyl transpeptidase, respectively. The impact of comedication and transplant type depended on the patient population: analysis of all samples showed opposing effects to analysis of those samples lacking data with biochemical variables above. The liquid chromatography-tandem mass spectrometry method described in this report is capable of measuring mycophenolic acid concentrations in very small sample volumes and in a timely fashion without the significant overestimates characterizing enzyme mediated immunoassay measurements in patients with serologic features characterizing liver or renal graft rejection.