Peripheral Blood Mononuclear Cells of Mycetoma Patients React Differently to Madurella mycetomatis Antigens than Healthy Endemic Controls

Mycetoma is a chronic, specific, granulomatous progressive, and destructive inflammatory disease of mainly the foot caused by either fungi (eumycetoma) or bacteria (actinomycetoma) [1]. Mycetoma is endemic in many tropical and subtropical regions, and it prevails in what is known as the mycetoma belt, which stretches in bands between the latitudes of 15u South and 30u North of the equator. With massive international travel, it also has been occasionally reported from different temperate regions [1]. The triad of a painless subcutaneous mass, multiple sinus formation, and purulent or seropurulent discharge that contains grains is characteristic of mycetoma. It usually spreads to involve the skin, deep structures, and bone-producing massive deformities. Due to these deformities, mycetoma has a high morbidity rate, with a huge impact on the patient, family, and the community [1]. Since mycetoma has been highly neglected, much is still not known regarding the susceptibility, resistance, infection route, disease progression, and response to medical treatment of mycetoma. Based on antibody measurements in earlier studies, it was demonstrated that although most people living in mycetoma-endemic areas are exposed to the causative agent, only a few developed mycetoma [2,3]. Therefore, the question arises of why some people develop mycetoma and others not, and when mycetoma is developed, why some patients respond well to treatment and others not. The answer to these questions could lie in the different reactions toward M. mycetomatis by the immune system between individuals who develop mycetoma and individuals who do not. So far there has been limited data on immune response toward mycetoma infection. The only data available were derived from animal models and actinomycetoma patients. From mouse models it was evident that mycetoma only occurred when M. mycetomatis was inoculated with an adjuvant (either soil or Freunds incomplete adjuvant), both predisposing toward a Th2 response [4,5]. Remarkably, in 1974, Cavanagh demonstrated that M. mycetomatis emulsified in the Th1-predisposing Freunds complete adjuvant (FCA) could not induce mycetoma in L20 mice [6,7]. Furthermore, when Peripheral Blood Mononuclear Cells (PBMCs) from actinomycetoma patients were stimulated with antigens from Nocardia brasiliensis, a Th2-type cytokine profile was detected [8]. This could indicate that the causative agent probably induces a Th2 response that facilitates further growth and the development of a clinically significant mycetoma lesion. In order to determine if a Th2 cytokine profile is indeed associated with mycetoma development and a Th1 cytokine profile with protection against mycetoma, this prospective, cross-sectional study was conducted at the Mycetoma Research Centre and the Institute of Endemic Diseases, University of Khartoum, Khartoum, Sudan, after obtaining ethical clearance from the Soba University Hospital Ethical Committee to determine the profile of four cytokines in both healthy endemic controls and mycetoma patients. The study included 27 patients with confirmed eumycetoma due to Madurella mycetomatis treated with 800 mg/d ketoconazole and 21 normal individuals from eumycetoma-endemic areas (Table 1). The present study was approved by the Ethics Committee of Soba University Hospital, Khartoum, Sudan. Written informed consents were obtained from the participants prior to their enrollment in the study. For each study, individual 10 ml of venous blood were collected in heparinized blood collection tubes. From the collected blood, peripheral mononuclear cells were isolated using Ficoll-Hypaque gradient centrifugation as described previously [9]. Stimulation of cells was done using 50 ml of uncharacterized culture filtrate of M. mycetomatis. The culture filtrate of M. mycetomatis was prepared by culturing M. mycetomatis for 3 wk at 37uC in RPMI-1640 medium supplemented with morpholinopropanesulfonic acid (MOPS) and phenol red and by filtrating this through a 0.45 micron pore size filter joined to a 20 ml syringe to purify any mycelia and debris. The filtered culture filtrate was stored at 220uC until used. To stimulate the isolated PBMCs, 1610 cells were plated into a 24-well flat-bottomed cell culture plate. Stimulation of PBMCs was done using 50 ml of culture filtrate of M. mycetomatis. For each patient and healthy control, some wells were left unstimulated to serve as control. The culture plates were incubated at 37uC with 5% CO2. After 72 h, the cells were harvested by centrifugation at 1,200 rpm for 3 min, and the supernatants were then collected for cytokine measurement by sandwich ELISAs using BD OptEIA ELISA Set B (Catalog No. 550534).