Significance of observed changes was determined using Student’s t-test and 95% confidence intervals of the difference expressed. meet our criteria as a diagnostic biomarker for cGVHD. These data are not able to confirm that induction of CD13 reactive antibodies is a mechanism for cGVHD in children nor are part of the pathogenesis of cGVHD associated with elevated soluble CD13. The exact role of CD13 in cGVHD remains to be determined. Keywords: anti-CD13 antibodies, CD13, aminopeptidase N, biomarkers, chronic graft-versus-host disease, blood and marrow transplantation Introduction Chronic graft-versus-host disease (cGVHD) is a multisystem alloimmune and autoimmune disorder complicating 40-70% of patients following allogeneic blood and marrow transplant (allo-BMT) [1-6]. The Children’s Oncology Group (COG) trial ASCT0031 (Phase III Trial of Hydroxychloroquine plus Standard Therapy for Chronic Graft-Versus-Host Disease) a newly discovered plasma biomarker, elevated soluble CD13 (aminopeptidase N) in subjects developing cGVHD early after allo-BMT [7, 8]. CD13 is an integral membrane protein with enzymatic activity, ubiquitously expressed on a number of tissue types affected by cGVHD including smooth muscle, endothelial, epithelial, and fibroblast cells [9, 10]. The mechanism by which soluble CD13 enzyme activity becomes elevated in cGVHD is unclear. One mechanistic possibility is that soluble CD13 becomes immunogenic once in the plasma, producing cytotoxic anti-CD13 antibodies that incite further tissue damage. Cytotoxic anti-CD13 antibodies have previously been described in a small series of patients following allo-BMT in AZD5582 association with cGVHD and cytomegalovirus (CMV) infection [11, 12]. Similar cGVHD mechanisms have also been reported for antibodies reactive with H-Y minor antigens [13] and platelet-derived growth factor receptor [14]. We sought to determine whether (1) soluble CD13 was immunogenic after allo-BMT, leading to the production of anti-CD13 antibodies, and (2) whether anti-CD13 antibodies met our previously defined criteria [7] as a biomarker of pediatric extensive cGVHD. Materials and Methods Subjects Peripheral blood samples were collected and evaluated prospectively from subjects enrolled on the COG trial ASCT0031, a phase III randomized, placebo-controlled, double blind trial evaluating two treatment regimens for pediatric subjects with newly diagnosed extensive cGVHD as previously described [7]. Subjects enrolled and treated on ASCT0031 formed the experimental group (n=52). To control for immune reconstitution following allo-BMT, a time matched comparison to the control group (n=28) was performed for the subjects with cGVHD, with days after allo-BMT at the time of cGVHD diagnosis used to divide the experimental group into early onset cGVHD (3-8 months after transplantation) or late onset cGVHD (9 months after transplantation) and compared to control BMT patients without cGVHD from 6 (early) and 12 months (late) after BMT. Healthy volunteer blood donor controls (n=6) not undergoing transplantation were evaluated AZD5582 for comparison. Samples Evaluated Peripheral blood was collected Rabbit Polyclonal to CDC7 from subjects with cGVHD at study entry (time of cGVHD diagnosis) and in control subjects collected at 6 and 12 months (after transplantation). The sample was separated into cells and plasma after centrifugation and stored at ?80C. Frozen plasma was defrosted in a waterbath at 37C and ultracentrifuged (15,000 rpm for 5 minutes) to rid of precipitate when ready for use. Following our previous analysis of biomarkers [7], a minority of subjects did not have adequate volume of plasma remaining for anti-CD13 antibody analysis and were not included with this analysis. AZD5582 Adequate plasma was available for anti-CD13 antibody analysis (45 cGVHD samples and 26 controls by ELISA; 42 cGVHD samples and 23 controls by Cellomics). Enzymatic Assay of Soluble CD13 (Aminopeptidase N) Plasma samples were tested for soluble CD13 (Aminopeptidase N) activity as we had previously described [7]. Results of soluble CD13 activity previously reported by our group [7] were used for correlative studies against anti-CD13 antibodies. ELISA for Anti-CD13 100 g of purified porcine aminopeptidase N / CD13 (Sigma-Aldrich, 83% amino acid homology with human CD13) was diluted in 10 ml of 0.1 M carbonate-bicarbonate buffer (pH 9.5). One hundred l was added to each well of a polystyrene flat-bottom ELISA plate, incubated overnight at 4C, washed three times with Tris buffered saline containing 0.05% Tween 20, pH 7.4. The nonspecific binding of sera proteins was prevented by adding 200 l of 1 1.5% bovine serum albumin in PBS followed by 2 hr incubation at room temperature. Plates were washed as above and 100 l of 1 1:100 diluted serum samples were added in duplicate and incubated for 2 hours at.

Significance of observed changes was determined using Student’s t-test and 95% confidence intervals of the difference expressed