Immunohistochemical Monitoring of Wound Healing in Antibiotic Treated Buruli Ulcer Patients Arianna Andreoli 1,2. , Marie-The ´re `se Ruf 1,2. , Ghislain Emmanuel Sopoh 3 , Peter Schmid 1,2 , Gerd Pluschke 1,2 * 1 Swiss Tropical and Public Health Institute, Basel, Switzerland, 2 University of Basel, Basel, Switzerland, 3 Centre de Depistage et de Traitement de l’Ulcere de Buruli d’Allada, Allada, Benin Abstract Background: While traditionally surgery has dominated the clinical management of Buruli ulcer (BU), the introduction of the combination chemotherapy with oral rifampicin and intramuscular streptomycin greatly improved treatment and reduced recurrence rates. However management of the often extensive lesions after successful specific therapy has remained a challenge, in particular in rural areas of the African countries which carry the highest burden of disease. For reasons not fully understood, wound healing is delayed in a proportion of antibiotic treated BU patients. Therefore, we have performed immunohistochemical investigations to identify markers which may be suitable to monitor wound healing progression. Methodology/Principal findings: Tissue specimens from eight BU patients with plaque lesions collected before, during and after chemotherapy were analyzed by immunohistochemistry for the presence of a set of markers associated with connective tissue neo-formation, tissue remodeling and epidermal activation. Several target proteins turned out to be suitable to monitor wound healing. While a-smooth muscle actin positive myofibroblasts were not found in untreated lesions, they emerged during the healing process. These cells produced abundant extracellular matrix proteins, such as pro- collagen 1 and tenascin and were found in fibronectin rich areas. After antibiotic treatment many cells, including myofibroblasts, revealed an activated phenotype as they showed ribosomal protein S6 phosphorylation, a marker for translation initiation. In addition, healing wounds revealed dermal tissue remodeling by apoptosis, and showed increased cytokeratin 16 expression in the epidermis. Conclusion/Significance: We have identified a set of markers that allow monitoring wound healing in antibiotic treated BU lesions by immunohistochemistry. Studies with this marker panel may help to better understand disturbances responsible for wound healing delays observed in some BU patients. Citation: Andreoli A, Ruf M-T, Sopoh GE, Schmid P, Pluschke G (2014) Immunohistochemical Monitoring of Wound Healing in Antibiotic Treated Buruli Ulcer Patients. PLoS Negl Trop Dis 8(4): e2809. doi:10.1371/journal.pntd.0002809 Editor: Christian Johnson, Fondation Raoul Follereau, France Received February 5, 2014; Accepted March 10, 2014; Published April 24, 2014 Copyright: ß 2014 Andreoli et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the Stop Buruli Initiative funded by the UBS-Optimus Foundation and by the Medicor Foundation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: gerd.pluschke@unibas.ch . These authors contributed equally to this work. Introduction Buruli ulcer (BU) is a necrotizing skin disease caused by M. ulcerans. It is primarily affecting the subcutaneous tissue and can, if untreated, lead to extensive tissue destruction and ulceration. The disease has been reported from more than 30 mainly tropical countries [1] around the world with the highest incidence in West Africa. The distribution of the disease is very focal and typically associated with rural wetlands in close proximity to stagnant or slow flowing water bodies [1,2]. The mode of transmission and the environmental reservoir of M. ulcerans are still not fully characterized. The disease can affect all age groups, but the highest incidence is in children aged between 5 and 15 years and in the elderly [3,4]. Most of the lesions occur on the limbs, but all parts of the body can be affected. The currently recommended treatment consists of daily administration of oral rifampicin (10 mg/kg) and intramuscular streptomycin (15 mg/kg) for 8 weeks under regular supervision. BU presents with a variety of clinical forms including nodules, plaques, edema and ulcers and in more severe cases multiple lesions as well as osteomyelitis have been observed. The disease often starts as a painless swelling or an area of induration which eventually may develop the characteristic features of BU such as large ulcers with undermined edges [5]. In particular in remote areas of Africa, patients tend to report late to the treatment centers and therefore often with very extensive and severe lesions. Long recovery periods are common and in the case of large lesions skin transplantation is required and permanent morbidities including functional limitations may be observed [6]. While mycolactone causes massive local immune suppression in active BU lesions, vigorous local immune responses are observed during anti- mycobacterial chemotherapy [7]. Paradoxical reactions including the enlargement of ulcers, progression of non-ulcerated plaques and edemas to ulcerative lesions, and the emergence of new lesions are frequently observed during chemotherapy [8,9]. However, PLOS Neglected Tropical Diseases | www.plosntds.org 1 April 2014 | Volume 8 | Issue 4 | e2809