Insights from computational modeling in inflammation and acute rejection in limb transplantation

Acute skin rejection in vascularized composite allotransplantation (VCA) is the major obstacle for wider adoption in clinical practice. This study utilized computational modeling to identify biomarkers for diagnosis and targets for treatment of skin rejection. Protein levels of 14 inflammatory mediators in skin and muscle biopsies from syngeneic grafts [n = 10], allogeneic transplants without immunosuppression [n = 10] and allografts treated with tacrolimus [n = 10] were assessed by multiplexed analysis technology. Hierarchical Clustering Analysis, Principal Component Analysis, Random Forest Classification and Multinomial Logistic Regression models were used to segregate experimental groups. Based on Random Forest Classification, Multinomial Logistic Regression and Hierarchical Clustering Analysis models, IL-4, TNF-α and IL-12p70 were the best predictors of skin rejection and identified rejection well in advance of histopathological alterations. TNF-α and IL-12p70 were the best predictors of muscle rejection and also preceded histopathological alterations. Principal Component Analysis identified IL-1α, IL-18, IL-1β, and IL-4 as principal drivers of transplant rejection. Thus, inflammatory patterns associated with rejection are specific for the individual tissue and may be superior for early detection and targeted treatment of rejection. © 2014 Wolfram et al

Abstracts from the 8th International Conference on cGMP Generators, Effectors and Therapeutic Implications

This work was supported by a restricted research grant of Bayer AG

Differentiation between Acute Skin Rejection in Allotransplantation and T-Cell Mediated Skin Inflammation Based on Gene Expression Analysis

Advances in microsurgical techniques and immunosuppressive medication have rendered transplantation of vascularized composite allografts possible, when autologous tissue is neither available nor sufficient for reconstruction. However, skin rejection and side effects of long-term immunosuppression still remain a major hurdle for wide adoption of this excellent reconstructive technique. Histopathologic changes during acute skin rejection in vascular composite allotransplantation often mimic inflammatory skin disorders and are hard to distinguish. Hence, the identification of diagnostic and therapeutic markers specific for skin rejection is of particular clinical need. Here we present novel markers allowing for early differentiation between rejection in hind limb allotransplantation and contact hypersensitivity. Assessment of Ccl7, Il18, and Il1b expression is most indicative of distinguishing skin rejection from skin inflammatory disorders. Gene expression levels varied significantly across skin types and regions, indicating localization specific mechanism of leukocyte migration and infiltration. Expression of Il12b, Il17a, and Il1b gene expression levels differed significantly between rejection and inflammation, independent of the skin type. In synopsis of the RNA expression profile and previously assessed protein expression, the Il1 family appears as a promising option for accurate skin rejection diagnosis and, as a following step, for development of novel rejection treatments

Similarity of sample groups and association between inflammatory mediators in rat limb transplantation models based on their profiles of mean levels in each condition.

<p>Heatmap as a result of complete linkage hierarchical clustering on log2-transformed and mean centered data. Log2-fold differences against the respective mean levels of each inflammatory mediator are color coded (red means higher inflammatory mediator levels and blue means lower inflammatory mediator levels than the mean levels of the respective inflammatory mediator according to the color scheme at the top). Dendrograms (trees) show similarity between different conditions and different inflammatory mediator profiles, respectively.</p

Histological evaluation of muscle biopsies from postoperative (POD) 3 to 9.

<p>Number of biopsies taken on each postoperative day (POD 3,5,7,9) in muscle according to their histological grading (Grad 0-III) based on H&E grading. The first biopsy was taken from the lateral proximal part of the thigh, the second one from the lateral distal thigh, the third one from the ventral thigh and the last one from the medial part of the thigh.</p

Appearance (Grading for skin rejection: I–IV) of each allograft on POD 1–11.

<p>*This animal died on POD 3 during the anesthesia. Bold numbers mark biopsy timepoints (skin and muscle) for each animal.</p

Results for multinomial logistic regression models in skin and muscle for classification of the rejection groups based inflammatory mediator/chemokine levels at early postoperative time points (POD 3/5) in rat transplantation models.

<p>Best prediction models for skin and muscle and their respective logistic prediction functions and classifier performance including confusion table (assessed by an 8-fold cross validation procedure) are summarized.</p

Results from Random Forest classification of the different rejection groups (ISO, TAC, ATC) using over the whole time course (POD 3, POD 5, POD 7, POD 9) measured inflammatory mediators in skin (A) and muscle (B) samples of rat limb transplantation models.

<p>Most important mediators for the decision trees based classification approach are evident by ranked mean decrease accuracy. Performances of the classifiers are indicated by the confusion table and the out-of-bag (OOB) error rate.</p