*7.3.1. Endothelial-associated transcripts (ENDATs) as a new marker for CAMR*

Recognizing the key role of endothelial changes in AMR, it was postulated by Sis and collea‐ gues (Sis et al., 2009) that altered expression of endothelial genes in biopsies from patients with alloantibody would identify kidneys incurring antibody-mediated damage and at risk for graft loss, whether they were C4d+ or negative. They explored whether expression of endothelial genes was increased in biopsies manifesting antibody-mediated graft injury, and whether such changes could be seen in C4d negative as well as C4d positive biopsies. They identified 119 endothelial-associated transcripts (ENDATs) from literature, and studied their expression by microarrays in 173 renal allograft biopsies for cause.

Mean ENDAT expression was increased in all rejection but was higher in AMR than in T-cell-mediated rejection and correlated with histopathologic lesions of AMR, and alloan‐ tibody. Many individual ENDATs were increased in AMR and predicted graft loss. Kid‐ neys with high ENDATs and antibody showed increased lesions of AMR and worse prognosis in comparison to controls. Only 40% of kidneys with high ENDAT expression and chronic AMR or graft loss were diagnosed by C4d positivity. High ENDAT expres‐ sion with antibody predicts graft loss with higher sensitivity (77% versus. 31%) and slightly lower specificity (71% vs. 94%) than C4d. The results were validated in inde‐ pendent set of 82 kidneys. They concluded that in patients with alloantibodies, abnor‐ malities in expression of endothelial genes identify not only C4d+ AMR but some kidney transplants developing antibody associated graft injury despite negative C4d staining and that ENDAT changes in renal transplants occur in rejection and in other forms of re‐ nal injury, and their impact on transplant glomerulopathy and graft loss is principally in patients with circulating HLA antibodies. The elevation of the ENDATs is of value in de‐ termining which biopsies for cause in patients with antibody may have antibody-mediat‐ ed injury, even when they are C4d negative. Based on their study, the combined burden of C4d+ and C4d negative AMR accounts for the majority of graft losses in kidney trans‐ plants biopsied for clinical indications (17 of 26, 65%). ENDAT expression in biopsy pro‐ vides a new tool for understanding the pathogenesis of late kidney graft loss and AMR, and for predicting graft outcomes and defining AMR even in C4d negative biopsies in patients with antibodies (Sis et al., 2009).

**8. Management of antibody mediated rejection**

humoral alloresponse after organ transplantation.

**8.1. Intravenous immunoglobulins (IVIG)**

4 patients.

Unfortunately, no immunosuppressive standard for the prevention or therapy of alloantibody production has been established yet. Although based on very limited evidence, acute humoral rejections are frequently treated with a switch to tacrolimus, plasmapheresis or immunoad‐ sorption, as well as T- and B-cell-depleting antibodies. However, the best therapeutic approach for C4d-positive, chronic humoral kidney rejection associated with an unfavourable prognosis remains completely unclear. Neither the dose nor the best drug combination for the therapy of an established humoral rejection is based on solid evidence. Although various immuno‐ suppressive drugs can reduce the number of acute rejection ns via inhibition of the T-cell response, only very few data are available regarding immunosuppressive drugs affecting the

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The immunomodulatory effects of IVIG are multiple, and the exact mechanisms are not elucidated. However, effective alloantibody inhibition by IVIG was shown in the context of desensitization protocols only relying on high dose IVIG treatment (Jordan et al., 2003). IVIG inhibits mixed lymphocyte reactions and induces apoptosis mainly in B cells (Toyoda et al., 2004). There are numerous proposed mechanisms how IVIG exerts its immunomodulatory action. They include modification of circulating alloantibody concentration through induction of antiidiotypic circuits, antigen binding through the Fab part of the immunoglobulin mole‐ cule, Fc receptor-mediated interaction with antigen-presenting cells to block T- and B-cell

In vivo, IVIG reduces the number of B cells and monocytes, and it reduces CD19, CD20 and CD40 expression by B cells, thereby modulating B-cell signaling (Jordan et al., 2003). IVIG inhibits binding of donor-reactive antibodies to target cells in ∼80% of patients, indicating that the presence of blocking antibodies might explain the efficacy of IVIG, although the mechanism is not known (Jordan et al., 2003). Billing and colleagues (Billing et al., 2008) studied Six pediatric renal transplant recipients with CAMR and gave them four weekly doses of IVIG (1 g/kg body weight per dose), followed by a single dose of rituximab (375 mg/m2 body surface area) 1 week after the last IVIG infusion. Median glomerular filtration rate during 6 months before intervention dropped by 25 (range, 11–26) mL/ min/1.73m2 (P<0.05) and increased in response to antihumoral therapy by 21 (-14 to+30) 6 months (P<0.05) and by 19 (-14 to+\_23) mL/min/1.73 m2 12 months (P=0.063) after start of treatment. Glomerular filtration rate improved or stabilized in 4 patients; the two non-responders had the highest degree of transplant glomerulopathy, the highest degree of C4d deposition in peritubular capillaries and pronounced interstitial inflammation. The treatment regimen was well tolerated. Another study conducted by Fehr and colleagues (Fehr et al., 2009) who reported four kidney allograft recipients suffering from chronic AMR 1 to 27 years post-transplant, who were treated with a combination of rituximab and intravenous immunoglobulin (IVIG) with improved kidney allograft function in all four patients, whereas donor-specific antibodies were reduced in 2 of

activation, and inhibition of complement activity (Jordan et al., 2006).

#### *7.3.2. TRIB1as a new non-invasive marker for CAMR*

The discovery of novel and less invasive surrogate biomarkers of acute cellular rejection, for which urine levels of Granzyme B and FOXP3 transcripts have been shown to have diagnostic and prognostic value (Muthukumar et al., 2005; Veale et al., 2006), has proved successful. Such an approach in the case of the different causes of late graft failure would facilitate the introduction of more targeted immunosuppression and thereby im‐ prove long-term outcome. Ashton-Chess and colleagues (Ashton-Chess et al., 2008) set out to discover novel minimally invasive biomarkers of more precise histologic diagno‐ ses of late graft scarring. Using a literature gene-set comparison approach for late graft injury, they identified TRIB1, a human homolog of Drosophila tribbles, (Grosshans & Wieschaus, 2000) as a potentially informative biomarker. TRIB1 is a scarcely character‐ ized member of the tribbles family that has been shown to be a potent regulator of cell signaling18 in various cells lines. It was determined that TRIB1 is expressed primarily by antigen-presenting cells (APC) and activated endothelial cells (EC). TRIB1 differs from the other minimally invasive biomarkers of transplant rejection described to date that are of T/NK cell origin, (Muthukumar et al., 2005; Seiler et al., 2007; Veale et al., 2006) in that it is expressed primarily by APC as well as EC.

They explored the potential of TRIB1 as a tissue, peripheral blood, and urine biomarker by measuring its mRNA profiles in graft biopsies, blood, and urine from healthy volunteers and kidney transplant recipients with different histologic and/or clinical diagnoses. For testing this, mRNA expression in 76 graft biopsies, 71 blood samples, and 11 urine samples were profiled from independent cohorts of renal transplant patients with different histologic diagnoses recruited at two European centers. TRIB1 but not TRIB2 or TRIB3 was found to be a potential blood and tissue (but not urine) biomarker of chronic antibody-mediated rejection. Moreover, TRIB1 mRNA in the blood was more specific and sensitive for diagnosing chronic AMR than TRIB1 mRNA in biopsies.

TRIB1 mRNA levels in peripheral blood mononuclear cells discriminated patients with chronic antibody-mediated rejection from those with other types of late allograft injury with high sensitivity and specificity, suggests TRIB1 to be a marker of an active immune response. Overall, these data support the potential use of TRIB1 as a biomarker of chronic antibodymediated allograft failure.
