MOLECULAR DIAGNOSTICS AND GENOMICS

A. Eljaafari¹, P. Petruzzo², J. Kanitakis³, L. Badet⁴, E. Morelon⁵, A. Gazarian⁶, C. Dagot⁵, J. Beziat⁷, S. Testelin⁸, B. Devauchelle⁸, X. Martin², J.M. Dubernard², P. Miossec^9
1Immunogenomics Unit, Hopital E Herriot, Pav P, Lyon/FRANCE, ²Urology Deppartment, Hopiatl E Herriot, Lyon/FRANCE, ³Dermatology Department, Hopital E Herriot, Lyon/FRANCE, ⁴Service De Chirurgie Urologique Et De La Transplantation, Hôpital E Herriot HCL, Lyon/FRANCE, ⁵Dept Transplantation, Hopital Edouard Herriot, Lyon/FRANCE, ⁶8. chirurgie De La Main Et Du Membre Supérieur, Polyclinique Orthopédique de Lyon, Lyon/FRANCE, ⁷Maxillofacial Surgery, Hospices Civils de Lyon, Lyon/FRANCE, ⁸Maxillofacial Surgery, University Hospital, Amiens/FRANCE, ⁹Immunogenomics, Hopital E Herriot, Lyon/FRANCE

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Introduction: 5 patients were bilaterally hand grafted and two were face grafted in Lyon and Amiens. One or more rejection episodes were observed for most of them, whereas systemic viral infectionswith CMV and EBV occurred in only 2 among the 7 grafted patients. To investigate the local immune responses , biopsies from skin or face mucosae were performed upon putative graft rejection episodes , but also at 2 weeks, 1 month, 2, 3, 6 and 12 months post-transplantation. Concommittantly, blood samples were harvested at the same periods. Methods: One biopsy fragment was analyzed by histology, while the second was cut in two pieces. The former served to extract mRNA, whereas the latter was cultured in the presence of 25 UI/ml rhIL-2 to isolate T cells. After 2 weeks of culture, cytofluorometry, and/or cytolytic assays were performed. In parallel, PBMCs were isolated by Ficoll and mRNA were extracted. RT-PCR were performed to measure tissues and PBMC mRNA levels of the following genes: IL-2, IFNg, Perforine, Granzyme B, FoxP3, CD4 CD8 and CD25. Results: During the course of the study, the various rejection episodes were histologically attested by infiltration of mononuclear cells . However, the specificity of these infiltrates could only be determined by cellular assays, able to demonstrate anti-donor specific CTL responses. These responses were associated with an increase of Granzyme B, IFNg, IL2 and CD8 mRNA levels at the sites of rejection. FoxP3 was oftenly increased as well. However, during rejection episodes, no significant increase of these markers was observed in blood. In contrast, in systemic viral infections, they were up-regulated in both blood and tissues, which allowed the subsequent diagnosis of EBV, or CMV infections in the two patients who have been infected. Conclusion: we have shown herein that in our tissue composite allo-grafted patients, immunomonitoring of blood versus tissues helped to differentially diagnose rejection , versus systemic viral infection episodes. Indeed, during rejection episodes, mRNA markers of cytolysis increased in situ in composite tissues, but not in peripheral blood, while the concomitant increase of these markers in both composite tissues and peripheral blood was observed during systemic viral infection episodes. Finally, culture of biopsy fragments may also participate in the diagnosis of rejection by demonstrating anti-donor specific CTL responses.

Disclosure: All authors have declared no conflicts of interest.