Elagab, E.A.M. (Ehab A.)

Fahal, A.H. (Ahmed)

Mukhtar, M.M. (Maowia)

Sande, W.W.J. (Wendy) van de

PubMed

Elagab, EAM

Mukhtar, MM

Fahal, AH

Laureijssen - van de Sande, Wendy

EUR Research Repository

ViewpointsPeripheral Blood Mononuclear Cells of MycetomaPatients React Differently to Madurella mycetomatisAntigens than Healthy Endemic ControlsEhab A. M. Elagab1,2, Maowia M. Mukhtar2, Ahmed H. Fahal1*, Wendy W. J. van de Sande31 Mycetoma Research Centre, University of Khartoum, Khartoum, Sudan, 2 Institute of Endemic Diseases, University of Khartoum, Khartoum, Sudan, 3 Erasmus MedicalCenter, Department of Medical Microbiology and Infectious Diseases, Rotterdam, the NetherlandsMycetoma is a chronic, specific, gran-ulomatous progressive, and destructiveinflammatory disease of mainly the footcaused by either fungi (eumycetoma) orbacteria (actinomycetoma) [1]. Mycetomais endemic in many tropical and subtrop-ical regions, and it prevails in what isknown as the mycetoma belt, whichstretches in bands between the latitudesof 15u South and 30u North of theequator. With massive international travel,it also has been occasionally reported fromdifferent temperate regions [1]. The triadof a painless subcutaneous mass, multiplesinus formation, and purulent or sero-purulent discharge that contains grains ischaracteristic of mycetoma. It usuallyspreads to involve the skin, deep struc-tures, and bone-producing massive defor-mities. Due to these deformities, myceto-ma has a high morbidity rate, with a hugeimpact on the patient, family, and thecommunity [1].Since mycetoma has been highly ne-glected, much is still not known regardingthe susceptibility, resistance, infectionroute, disease progression, and responseto medical treatment of mycetoma. Basedon antibody measurements in earlierstudies, it was demonstrated that althoughmost people living in mycetoma-endemicareas are exposed to the causative agent,only a few developed mycetoma [2,3].Therefore, the question arises of why somepeople develop mycetoma and others not,and when mycetoma is developed, whysome patients respond well to treatmentand others not. The answer to thesequestions could lie in the different reac-tions toward M. mycetomatis by the immunesystem between individuals who developmycetoma and individuals who do not.So far there has been limited data onimmune response toward mycetoma in-fection. The only data available werederived from animal models and actino-mycetoma patients. From mouse modelsit was evident that mycetoma only oc-curred when M. mycetomatis was inoculatedwith an adjuvant (either soil or Freundsincomplete adjuvant), both predisposing to-ward a Th2 response [4,5]. Remarkably, in1974, Cavanagh demonstrated thatM. mycetomatis emulsified in the Th1-predis-posing Freunds complete adjuvant (FCA)could not induce mycetoma in L20 mice[6,7]. Furthermore, when Peripheral BloodMononuclear Cells (PBMCs) from actinomy-cetoma patients were stimulated with anti-gens from Nocardia brasiliensis, a Th2-typecytokine profile was detected [8]. This couldindicate that the causative agent probablyinduces a Th2 response that facilitates furthergrowth and the development of a clinicallysignificant mycetoma lesion. In order todetermine if a Th2 cytokine profile is indeedassociated with mycetoma development anda Th1 cytokine profile with protection againstmycetoma, this prospective, cross-sectionalstudy was conducted at the MycetomaResearch Centre and the Institute of Endem-ic Diseases, University of Khartoum, Khar-toum, Sudan, after obtaining ethical clear-ance from the Soba University HospitalEthical Committee to determine the profileof four cytokines in both healthy endemiccontrols and mycetoma patients. The studyincluded 27 patients with confirmed eumy-cetoma due to Madurella mycetomatis treatedwith 800 mg/d ketoconazole and 21 normalindividuals from eumycetoma-endemic areas(Table 1). The present study was approved bythe Ethics Committee of Soba UniversityHospital, Khartoum, Sudan. Written in-formed consents were obtained from theparticipants prior to their enrollment inthe study. For each study, individual10 ml of venous blood were collected inheparinized blood collection tubes.From the collected blood, peripheralmononuclear cells were isolated usingFicoll-Hypaque gradient centrifugationas described previously [9]. Stimulationof cells was done using 50 ml of unchar-acterized culture filtrate of M. mycetoma-tis. The culture filtrate of M. mycetomatiswas prepared by culturing M. mycetomatisfor 3 wk at 37uC in RPMI-1640 medi-um supplemented with morpholinopro-panesulfonic acid (MOPS) and phenolred and by filtrating this through a0.45 micron pore size filter joined to a20 ml syringe to purify any mycelia anddebris. The filtered culture filtrate wasstored at 220uC until used. To stimu-late the isolated PBMCs, 16106 cellswere plated into a 24-well flat-bottomedcell culture plate. Stimulation of PBMCswas done using 50 ml of culture filtrateof M. mycetomatis. For each patient andhealthy control, some wells were leftunstimulated to serve as control. Theculture plates were incubated at 37uCwith 5% CO2. After 72 h, the cells wereharvested by centrifugation at1,200 rpm for 3 min, and the superna-tants were then collected for cytokinemeasurement by sandwich ELISAs us-ing BD OptEIA ELISA Set B (CatalogNo. 550534).Citation: Elagab EAM, Mukhtar MM, Fahal AH, van de Sande WWJ (2013) Peripheral Blood Mononuclear Cellsof Mycetoma Patients React Differently to Madurella mycetomatis Antigens than Healthy EndemicControls. PLoS Negl Trop Dis 7(4): e2081. doi:10.1371/journal.pntd.0002081Editor: Joseph M. Vinetz, University of California San Diego School of Medicine, United States of AmericaPublished April 25, 2013Copyright:  2013 Elagab et al. This is an open-access article distributed under the terms of the CreativeCommons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium,provided the original author and source are credited.Funding: Wendy van de Sande was funded by Veni-grant 916.11.178 of the Netherlands organisation for Scientificresearch (NWO) (http://www.nwo.nl/onderzoek-en-resultaten//programmas/vernieuwingsimpuls/toekenningen/alle+veni+toekenningen/Toekenningen+Veni+2010). The funders had no role in study design, data collection andanalysis, decision to publish, or preparation of the manuscript.Competing Interests: The authors have declared that no competing interests exist.* E-mail: ahfahal@hotmail.comPLOS Neglected Tropical Diseases | www.plosntds.org 1 April 2013 | Volume 7 | Issue 4 | e2081Table 1. Patient demographics.Eumycetoma Patients Healthy Endemic ControlsNumber (n) 27 21Gender (male/female) 22/5 19/2Mean age years (range) 23.5 (9–45) 33.9 (22–53)Lesion site Foot NALesion size (n) Large 18 NAMedium 5 NASmall 4 NAKetoconazole responding (yes/no) 11/8 NACausative agent M. mycetomatis NANA, not applicable.doi:10.1371/journal.pntd.0002081.t001PLOS Neglected Tropical Diseases | www.plosntds.org 2 April 2013 | Volume 7 | Issue 4 | e2081After stimulating the PBMCs of bothpatients and healthy endemic controlswith the M. mycetomatis antigen prepara-tion, it was noted that the PBMCs of thepatients reacted differently to this antigenthan the healthy controls. Statisticallysignificantly higher IL-10 concentrationswere detected in all the patients (mean458.2 pg/ml) compared to the healthycontrols (mean 30.7 pg/ml) (Figure 1A,Mann–Whitney, p = 0.004, GraphPadPrism 5.01). In contrast, the INF-c con-centrations were significantly higher in thecontrol population (mean 792.8 pg/ml)than in the patient group (mean 77.4 pg/ml) (Figure 1B, Mann–Whitney,p = 0.007). Generally it was noted thatwhen PBMCs produced INF-c uponstimulation with the antigen, no IL-10was produced and vice versa. There wereno significant differences in TNFa orTGF-b concentrations between patientsand control groups (Figure 1C and 1D,Mann–Whitney, p.0.05). In order todetermine if mycetoma patients withlarger lesions had higher levels of IL-10and lower levels of INF-c, the IL-10 andINF-c levels were stratified according tolesion size. Although in general higherconcentrations of Il-10 and lower levels ofINF-c were found in the massive lesions, itappeared that this was not statisticallydifferent from the smaller lesion sizes(Kruskall–Wallis, p.0.05; GraphPadPrism 5.01). There is only one other studywith which we could use to compare ourresults [10]. Castro–Matteotti used threedifferent Nocardia brasiliensis extracts tostimulate PBMCs from seven actinomyce-toma patients, seven tuberculosis patients,and seven healthy controls and measuredthe INF-c response [10]. None of theextracts was able to stimulate INF-cproduction in humans [10]. This is incontrast to our findings. When we stimu-lated the healthy controls in our study witha M. mycetomatis extract, the PBMCs of thehealthy controls produced INF-c. Clearly,the PBMC response toward eumycetomaantigens and actinomycetoma antigensdiffers.At the moment, eumycetoma is treatedwith surgery and prolonged antifungaltherapy with ketoconazole. Ketoconazoleis known to suppress the production ofTh2-type cytokines IL-4 and IL-5; there-fore, the working mechanism of this drugin mycetoma might be 2-fold: inhibitingfungal growth and reversing the immunesystem toward a Th1 response [11,12].Unfortunately not all patients respond wellto ketoconazole treatment. Many stillshow recurrent infections, even if thefungal species itself is susceptible towardthis drug [13]. Furthermore, it alsodetermined if patients responding toketoconazole had different cytokine pro-files than patients not responding to thisdrug. All patients were treated with800 mg/d of ketoconazole for at least6 mo. If a patient was responding to thistreatment, the size of the lesion decreased,discharging sinuses closed, and there was anoted pain relief. According to thesecriteria, the patients were further dividedinto two groups: patients who respondedto 800 mg/d ketoconazole (n = 11) andthose who did not respond (n = 8). PBMCsof patients who responded to ketoconazoletherapy produced similar levels of IL-10,INF-c, TNF-a, or TGF-b as patients whodid not respond to this therapy (Mann–Whitney, p = ns for all cytokines).In conclusion, this study showed thatmycetoma patients produce high concen-trations of IL-10 that correlate with thedisease severity. IFN-c seems to beassociated with protection against myceto-ma, and it may indicate past subclinicalinfections. The four cytokine concentra-tion levels were not affected by a medicaltreatment response. An important limita-tion of the present study is the smallnumber of patients included. Furtherstudies at the field level in endemic areasand on controls from nonendemic areasthat study cytokine profile in serum and inthe histopathological lesion are recom-mended for more in-depth understandingof the immune responses of the mycetomapatients.References1. Ahmed AO, van Leeuwen W, Fahal A, van deSande WWJ, Verbrugh H, et al. (2004) Myceto-ma caused by Madurella mycetomatis: a neglectedinfectious burden. Lancet Infect Dis 4: 566–574.2. de Klerk N, de Vogel C, Fahal A, van Belkum A, vande Sande WW (2012) Fructose-bisphosphate aldolaseand pyruvate kinase, two novel immunogens inMadurella mycetomatis. Med Mycol 50: 143–151.3. van de Sande WW, Janse DJ, Hira V, GoedhartH, van der Zee R, et al. (2006) Translationallycontrolled tumor protein from Madurella myceto-matis, a marker for tumorous mycetoma progres-sion. J Immunol 177: 1997–2005.4. Ahmed AO, van Vianen W, ten Kate MT, van deSande WW, van Belkum A, et al. (2003) A murinemodel of Madurella mycetomatis eumycetoma.FEMS Immunol Med Microbiol 37: 29–36.5. Zhang L, Mia MY, Zheng CL, Hossain MA,Yamasaki F, et al. (1999) The preventive effects ofincomplete Freund’s adjuvant and other vehicleson the development of adjuvant-induced arthritisin Lewis rats. Immunology 98: 267–272.6. Cavanagh LL (1974) Attempts to induce myceto-ma in monkeys and mice using Madurellamycetomi. Sabouraudia 12: 258–262.7. Stills HF, Jr. (2005) Adjuvants and antibody produc-tion: dispelling the myths associated with Freund’scomplete and other adjuvants. Ilar J 46: 280–293.8. Mendez-Tovar LJ, Mondragon-Gonzalez R,Vega-Lopez F, Dockrell HM, Hay R, et al.(2004) Cytokine production and lymphocyteproliferation in patients with Nocardia brasiliensisactinomycetoma. Mycopathologia 158: 407–414.9. English D, Andersen BR (1974) Single-stepseparation of red blood cells. Granulocytes andmononuclear leukocytes on discontinuous densitygradients of Ficoll-Hypaque. J Immunol Methods5: 249–252.10. Castro-Matteotti B, Vera-Cabrera L, Ocampo-Candiani J, Rendon A, Salinas-Carmona MC,et al. (2008) Immune response to Nocardiabrasiliensis extracellular antigens in patients withmycetoma. Mycopathologia 165: 127–134.11. Kanda N, Enomoto U, Watanabe S (2001) Anti-mycotics suppress interleukin-4 and interleukin-5production in anti-CD3 plus anti-CD28-stimulatedT cells from patients with atopic dermatitis. J InvestDermatol 117: 1635–1646.12. Kanda N, Watanabe S (2002) Ketoconazolesuppresses interleukin-4 plus anti-CD40-inducedIgE class switching in surface IgE negative B cellsfrom patients with atopic dermatitis. J InvestDermatol 119: 590–599.13. van de Sande WWJ, Luijendijk A, Ahmed AO,Bakker-Woudenberg IA, van Belkum A (2005)Testing of the in vitro susceptibilities of Madurellamycetomatis to six antifungal agents by using thesensititre system in comparison with a viability-based 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydrox-ide (XTT) assay and a modified NCCLS method.Antimicrob Agents Chemother 49: 1364–1368.Figure 1. Cytokine concentrations of stimulated PBMCs of eumycetoma patients and healthy controls. (A) IL-10 concentrations (pg/ml)measured after stimulating PBMCs from eumycetoma patients and healthy endemic controls with M. mycetomatis antigen. Statistically significantlydifferent concentrations were found between eumycetoma patients and healthy controls (p = 0.004) as determined by the Mann–Whitney U test. (B)IFN-c concentrations (pg/ml) measured after stimulating PBMCs from eumycetoma patients and healthy endemic controls with M. mycetomatisantigen. Statistically significantly different concentrations were found between eumycetoma patients and healthy controls (p = 0.007) as determinedby the Mann–Whitney U test. (C) TNF-a concentrations (pg/ml) measured after stimulating PBMCs from eumycetoma patients and healthy endemiccontrols with M. mycetomatis antigen. No statistically significantly different concentrations were found between eumycetoma patients and healthycontrols (p = 0.85) as determined by the Mann–Whitney U test. (D) TGF-b concentrations (pg/ml) measured after stimulating PBMCs fromeumycetoma patients and healthy endemic controls with M. mycetomatis antigen. No statistically significantly different concentrations were foundbetween eumycetoma patients and healthy controls (p = 0.89) as determined by the Mann–Whitney U test.doi:10.1371/journal.pntd.0002081.g001PLOS Neglected Tropical Diseases | www.plosntds.org 3 April 2013 | Volume 7 | Issue 4 | e2081

Undetermined

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

NARCIS 

EUR Research Information PortalPeripheral Blood Mononuclear Cells of Mycetoma Patients React Differently toMadurella mycetomatis Antigens than Healthy Endemic ControlsPublished in:PLoS Neglected Tropical Diseases (print)Publication status and date:Published: 01/01/2013DOI (link to publisher):10.1371/journal.pntd.0002081Document VersionPublisher's PDF, also known as Version of recordCitation for the published version (APA):Elagab, EAM., Mukhtar, MM., Fahal, AH., & Laureijssen - van de Sande, W. (2013). Peripheral Blood Mononuclear Cells ofMycetoma Patients React Differently to Madurella mycetomatis Antigens than Healthy Endemic Controls. PLoS NeglectedTropical Diseases (print), 7(4). https://doi.org/10.1371/journal.pntd.0002081Link to publication on the EUR Research Information PortalTerms and Conditions of UseExcept as permitted by the applicable copyright law, you may not reproduce or make this material available to any third partywithout the prior written permission from the copyright holder(s). Copyright law allows the following uses of this materialwithout prior permission:            • you may download, save and print a copy of this material for your personal use only;            • you may share the EUR portal link to this material.In case the material is published with an open access license (e.g. a Creative Commons (CC) license), other uses may beallowed. Please check the terms and conditions of the specific license.Take-down policyIf you believe that this material infringes your copyright and/or any other intellectual property rights, you may request itsremoval by contacting us at the following email address: openaccess.library@eur.nl. Please provide us with all the relevantinformation, including the reasons why you believe any of your rights have been infringed. In case of a legitimate complaint,we will make the material inaccessible and/or remove it from the website.ViewpointsPeripheral Blood Mononuclear Cells of MycetomaPatients React Differently to Madurella mycetomatisAntigens than Healthy Endemic ControlsEhab A. M. Elagab1,2, Maowia M. Mukhtar2, Ahmed H. Fahal1*, Wendy W. J. van de Sande31 Mycetoma Research Centre, University of Khartoum, Khartoum, Sudan, 2 Institute of Endemic Diseases, University of Khartoum, Khartoum, Sudan, 3 Erasmus MedicalCenter, Department of Medical Microbiology and Infectious Diseases, Rotterdam, the NetherlandsMycetoma is a chronic, specific, gran-ulomatous progressive, and destructiveinflammatory disease of mainly the footcaused by either fungi (eumycetoma) orbacteria (actinomycetoma) [1]. Mycetomais endemic in many tropical and subtrop-ical regions, and it prevails in what isknown as the mycetoma belt, whichstretches in bands between the latitudesof 15u South and 30u North of theequator. With massive international travel,it also has been occasionally reported fromdifferent temperate regions [1]. The triadof a painless subcutaneous mass, multiplesinus formation, and purulent or sero-purulent discharge that contains grains ischaracteristic of mycetoma. It usuallyspreads to involve the skin, deep struc-tures, and bone-producing massive defor-mities. Due to these deformities, myceto-ma has a high morbidity rate, with a hugeimpact on the patient, family, and thecommunity [1].Since mycetoma has been highly ne-glected, much is still not known regardingthe susceptibility, resistance, infectionroute, disease progression, and responseto medical treatment of mycetoma. Basedon antibody measurements in earlierstudies, it was demonstrated that althoughmost people living in mycetoma-endemicareas are exposed to the causative agent,only a few developed mycetoma [2,3].Therefore, the question arises of why somepeople develop mycetoma and others not,and when mycetoma is developed, whysome patients respond well to treatmentand others not. The answer to thesequestions could lie in the different reac-tions toward M. mycetomatis by the immunesystem between individuals who developmycetoma and individuals who do not.So far there has been limited data onimmune response toward mycetoma in-fection. The only data available werederived from animal models and actino-mycetoma patients. From mouse modelsit was evident that mycetoma only oc-curred when M. mycetomatis was inoculatedwith an adjuvant (either soil or Freundsincomplete adjuvant), both predisposing to-ward a Th2 response [4,5]. Remarkably, in1974, Cavanagh demonstrated thatM. mycetomatis emulsified in the Th1-predis-posing Freunds complete adjuvant (FCA)could not induce mycetoma in L20 mice[6,7]. Furthermore, when Peripheral BloodMononuclear Cells (PBMCs) from actinomy-cetoma patients were stimulated with anti-gens from Nocardia brasiliensis, a Th2-typecytokine profile was detected [8]. This couldindicate that the causative agent probablyinduces a Th2 response that facilitates furthergrowth and the development of a clinicallysignificant mycetoma lesion. In order todetermine if a Th2 cytokine profile is indeedassociated with mycetoma development anda Th1 cytokine profile with protection againstmycetoma, this prospective, cross-sectionalstudy was conducted at the MycetomaResearch Centre and the Institute of Endem-ic Diseases, University of Khartoum, Khar-toum, Sudan, after obtaining ethical clear-ance from the Soba University HospitalEthical Committee to determine the profileof four cytokines in both healthy endemiccontrols and mycetoma patients. The studyincluded 27 patients with confirmed eumy-cetoma due to Madurella mycetomatis treatedwith 800 mg/d ketoconazole and 21 normalindividuals from eumycetoma-endemic areas(Table 1). The present study was approved bythe Ethics Committee of Soba UniversityHospital, Khartoum, Sudan. Written in-formed consents were obtained from theparticipants prior to their enrollment inthe study. For each study, individual10 ml of venous blood were collected inheparinized blood collection tubes.From the collected blood, peripheralmononuclear cells were isolated usingFicoll-Hypaque gradient centrifugationas described previously [9]. Stimulationof cells was done using 50 ml of unchar-acterized culture filtrate of M. mycetoma-tis. The culture filtrate of M. mycetomatiswas prepared by culturing M. mycetomatisfor 3 wk at 37uC in RPMI-1640 medi-um supplemented with morpholinopro-panesulfonic acid (MOPS) and phenolred and by filtrating this through a0.45 micron pore size filter joined to a20 ml syringe to purify any mycelia anddebris. The filtered culture filtrate wasstored at 220uC until used. To stimu-late the isolated PBMCs, 16106 cellswere plated into a 24-well flat-bottomedcell culture plate. Stimulation of PBMCswas done using 50 ml of culture filtrateof M. mycetomatis. For each patient andhealthy control, some wells were leftunstimulated to serve as control. Theculture plates were incubated at 37uCwith 5% CO2. After 72 h, the cells wereharvested by centrifugation at1,200 rpm for 3 min, and the superna-tants were then collected for cytokinemeasurement by sandwich ELISAs us-ing BD OptEIA ELISA Set B (CatalogNo. 550534).Citation: Elagab EAM, Mukhtar MM, Fahal AH, van de Sande WWJ (2013) Peripheral Blood Mononuclear Cellsof Mycetoma Patients React Differently to Madurella mycetomatis Antigens than Healthy EndemicControls. PLoS Negl Trop Dis 7(4): e2081. doi:10.1371/journal.pntd.0002081Editor: Joseph M. Vinetz, University of California San Diego School of Medicine, United States of AmericaPublished April 25, 2013Copyright:  2013 Elagab et al. This is an open-access article distributed under the terms of the CreativeCommons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium,provided the original author and source are credited.Funding: Wendy van de Sande was funded by Veni-grant 916.11.178 of the Netherlands organisation for Scientificresearch (NWO) (http://www.nwo.nl/onderzoek-en-resultaten//programmas/vernieuwingsimpuls/toekenningen/alle+veni+toekenningen/Toekenningen+Veni+2010). The funders had no role in study design, data collection andanalysis, decision to publish, or preparation of the manuscript.Competing Interests: The authors have declared that no competing interests exist.* E-mail: ahfahal@hotmail.comPLOS Neglected Tropical Diseases | www.plosntds.org 1 April 2013 | Volume 7 | Issue 4 | e2081Table 1. Patient demographics.Eumycetoma Patients Healthy Endemic ControlsNumber (n) 27 21Gender (male/female) 22/5 19/2Mean age years (range) 23.5 (9–45) 33.9 (22–53)Lesion site Foot NALesion size (n) Large 18 NAMedium 5 NASmall 4 NAKetoconazole responding (yes/no) 11/8 NACausative agent M. mycetomatis NANA, not applicable.doi:10.1371/journal.pntd.0002081.t001PLOS Neglected Tropical Diseases | www.plosntds.org 2 April 2013 | Volume 7 | Issue 4 | e2081After stimulating the PBMCs of bothpatients and healthy endemic controlswith the M. mycetomatis antigen prepara-tion, it was noted that the PBMCs of thepatients reacted differently to this antigenthan the healthy controls. Statisticallysignificantly higher IL-10 concentrationswere detected in all the patients (mean458.2 pg/ml) compared to the healthycontrols (mean 30.7 pg/ml) (Figure 1A,Mann–Whitney, p = 0.004, GraphPadPrism 5.01). In contrast, the INF-c con-centrations were significantly higher in thecontrol population (mean 792.8 pg/ml)than in the patient group (mean 77.4 pg/ml) (Figure 1B, Mann–Whitney,p = 0.007). Generally it was noted thatwhen PBMCs produced INF-c uponstimulation with the antigen, no IL-10was produced and vice versa. There wereno significant differences in TNFa orTGF-b concentrations between patientsand control groups (Figure 1C and 1D,Mann–Whitney, p.0.05). In order todetermine if mycetoma patients withlarger lesions had higher levels of IL-10and lower levels of INF-c, the IL-10 andINF-c levels were stratified according tolesion size. Although in general higherconcentrations of Il-10 and lower levels ofINF-c were found in the massive lesions, itappeared that this was not statisticallydifferent from the smaller lesion sizes(Kruskall–Wallis, p.0.05; GraphPadPrism 5.01). There is only one other studywith which we could use to compare ourresults [10]. Castro–Matteotti used threedifferent Nocardia brasiliensis extracts tostimulate PBMCs from seven actinomyce-toma patients, seven tuberculosis patients,and seven healthy controls and measuredthe INF-c response [10]. None of theextracts was able to stimulate INF-cproduction in humans [10]. This is incontrast to our findings. When we stimu-lated the healthy controls in our study witha M. mycetomatis extract, the PBMCs of thehealthy controls produced INF-c. Clearly,the PBMC response toward eumycetomaantigens and actinomycetoma antigensdiffers.At the moment, eumycetoma is treatedwith surgery and prolonged antifungaltherapy with ketoconazole. Ketoconazoleis known to suppress the production ofTh2-type cytokines IL-4 and IL-5; there-fore, the working mechanism of this drugin mycetoma might be 2-fold: inhibitingfungal growth and reversing the immunesystem toward a Th1 response [11,12].Unfortunately not all patients respond wellto ketoconazole treatment. Many stillshow recurrent infections, even if thefungal species itself is susceptible towardthis drug [13]. Furthermore, it alsodetermined if patients responding toketoconazole had different cytokine pro-files than patients not responding to thisdrug. All patients were treated with800 mg/d of ketoconazole for at least6 mo. If a patient was responding to thistreatment, the size of the lesion decreased,discharging sinuses closed, and there was anoted pain relief. According to thesecriteria, the patients were further dividedinto two groups: patients who respondedto 800 mg/d ketoconazole (n = 11) andthose who did not respond (n = 8). PBMCsof patients who responded to ketoconazoletherapy produced similar levels of IL-10,INF-c, TNF-a, or TGF-b as patients whodid not respond to this therapy (Mann–Whitney, p = ns for all cytokines).In conclusion, this study showed thatmycetoma patients produce high concen-trations of IL-10 that correlate with thedisease severity. IFN-c seems to beassociated with protection against myceto-ma, and it may indicate past subclinicalinfections. The four cytokine concentra-tion levels were not affected by a medicaltreatment response. An important limita-tion of the present study is the smallnumber of patients included. Furtherstudies at the field level in endemic areasand on controls from nonendemic areasthat study cytokine profile in serum and inthe histopathological lesion are recom-mended for more in-depth understandingof the immune responses of the mycetomapatients.References1. Ahmed AO, van Leeuwen W, Fahal A, van deSande WWJ, Verbrugh H, et al. (2004) Myceto-ma caused by Madurella mycetomatis: a neglectedinfectious burden. Lancet Infect Dis 4: 566–574.2. de Klerk N, de Vogel C, Fahal A, van Belkum A, vande Sande WW (2012) Fructose-bisphosphate aldolaseand pyruvate kinase, two novel immunogens inMadurella mycetomatis. Med Mycol 50: 143–151.3. van de Sande WW, Janse DJ, Hira V, GoedhartH, van der Zee R, et al. (2006) Translationallycontrolled tumor protein from Madurella myceto-matis, a marker for tumorous mycetoma progres-sion. J Immunol 177: 1997–2005.4. Ahmed AO, van Vianen W, ten Kate MT, van deSande WW, van Belkum A, et al. (2003) A murinemodel of Madurella mycetomatis eumycetoma.FEMS Immunol Med Microbiol 37: 29–36.5. Zhang L, Mia MY, Zheng CL, Hossain MA,Yamasaki F, et al. (1999) The preventive effects ofincomplete Freund’s adjuvant and other vehicleson the development of adjuvant-induced arthritisin Lewis rats. Immunology 98: 267–272.6. Cavanagh LL (1974) Attempts to induce myceto-ma in monkeys and mice using Madurellamycetomi. Sabouraudia 12: 258–262.7. Stills HF, Jr. (2005) Adjuvants and antibody produc-tion: dispelling the myths associated with Freund’scomplete and other adjuvants. Ilar J 46: 280–293.8. Mendez-Tovar LJ, Mondragon-Gonzalez R,Vega-Lopez F, Dockrell HM, Hay R, et al.(2004) Cytokine production and lymphocyteproliferation in patients with Nocardia brasiliensisactinomycetoma. Mycopathologia 158: 407–414.9. English D, Andersen BR (1974) Single-stepseparation of red blood cells. Granulocytes andmononuclear leukocytes on discontinuous densitygradients of Ficoll-Hypaque. J Immunol Methods5: 249–252.10. Castro-Matteotti B, Vera-Cabrera L, Ocampo-Candiani J, Rendon A, Salinas-Carmona MC,et al. (2008) Immune response to Nocardiabrasiliensis extracellular antigens in patients withmycetoma. Mycopathologia 165: 127–134.11. Kanda N, Enomoto U, Watanabe S (2001) Anti-mycotics suppress interleukin-4 and interleukin-5production in anti-CD3 plus anti-CD28-stimulatedT cells from patients with atopic dermatitis. J InvestDermatol 117: 1635–1646.12. Kanda N, Watanabe S (2002) Ketoconazolesuppresses interleukin-4 plus anti-CD40-inducedIgE class switching in surface IgE negative B cellsfrom patients with atopic dermatitis. J InvestDermatol 119: 590–599.13. van de Sande WWJ, Luijendijk A, Ahmed AO,Bakker-Woudenberg IA, van Belkum A (2005)Testing of the in vitro susceptibilities of Madurellamycetomatis to six antifungal agents by using thesensititre system in comparison with a viability-based 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydrox-ide (XTT) assay and a modified NCCLS method.Antimicrob Agents Chemother 49: 1364–1368.Figure 1. Cytokine concentrations of stimulated PBMCs of eumycetoma patients and healthy controls. (A) IL-10 concentrations (pg/ml)measured after stimulating PBMCs from eumycetoma patients and healthy endemic controls with M. mycetomatis antigen. Statistically significantlydifferent concentrations were found between eumycetoma patients and healthy controls (p = 0.004) as determined by the Mann–Whitney U test. (B)IFN-c concentrations (pg/ml) measured after stimulating PBMCs from eumycetoma patients and healthy endemic controls with M. mycetomatisantigen. Statistically significantly different concentrations were found between eumycetoma patients and healthy controls (p = 0.007) as determinedby the Mann–Whitney U test. (C) TNF-a concentrations (pg/ml) measured after stimulating PBMCs from eumycetoma patients and healthy endemiccontrols with M. mycetomatis antigen. No statistically significantly different concentrations were found between eumycetoma patients and healthycontrols (p = 0.85) as determined by the Mann–Whitney U test. (D) TGF-b concentrations (pg/ml) measured after stimulating PBMCs fromeumycetoma patients and healthy endemic controls with M. mycetomatis antigen. No statistically significantly different concentrations were foundbetween eumycetoma patients and healthy controls (p = 0.89) as determined by the Mann–Whitney U test.doi:10.1371/journal.pntd.0002081.g001PLOS Neglected Tropical Diseases | www.plosntds.org 3 April 2013 | Volume 7 | Issue 4 | e2081

Ehab A. M. Elagab

Maowia M. Mukhtar

Ahmed H. Fahal

Wendy W. J. van de Sande

Crossref

PLoS Neglected Tropical Diseases

Erasmus University Digital Repository

ViewpointsPeripheral Blood Mononuclear Cells of MycetomaPatients React Differently to Madurella mycetomatisAntigens than Healthy Endemic ControlsEhab A. M. Elagab1,2, Maowia M. Mukhtar2, Ahmed H. Fahal1*, Wendy W. J. van de Sande31Mycetoma Research Centre, University of Khartoum, Khartoum, Sudan, 2 Institute of Endemic Diseases, University of Khartoum, Khartoum, Sudan, 3 Erasmus MedicalCenter, Department of Medical Microbiology and Infectious Diseases, Rotterdam, the NetherlandsMycetoma is a chronic, specific, gran-ulomatous progressive, and destructiveinflammatory disease of mainly the footcaused by either fungi (eumycetoma) orbacteria (actinomycetoma) [1]. Mycetomais endemic in many tropical and subtrop-ical regions, and it prevails in what isknown as the mycetoma belt, whichstretches in bands between the latitudesof 15u South and 30u North of theequator. With massive international travel,it also has been occasionally reported fromdifferent temperate regions [1]. The triadof a painless subcutaneous mass, multiplesinus formation, and purulent or sero-purulent discharge that contains grains ischaracteristic of mycetoma. It usuallyspreads to involve the skin, deep struc-tures, and bone-producing massive defor-mities. Due to these deformities, myceto-ma has a high morbidity rate, with a hugeimpact on the patient, family, and thecommunity [1].Since mycetoma has been highly ne-glected, much is still not known regardingthe susceptibility, resistance, infectionroute, disease progression, and responseto medical treatment of mycetoma. Basedon antibody measurements in earlierstudies, it was demonstrated that althoughmost people living in mycetoma-endemicareas are exposed to the causative agent,only a few developed mycetoma [2,3].Therefore, the question arises of why somepeople develop mycetoma and others not,and when mycetoma is developed, whysome patients respond well to treatmentand others not. The answer to thesequestions could lie in the different reac-tions toward M. mycetomatis by the immunesystem between individuals who developmycetoma and individuals who do not.So far there has been limited data onimmune response toward mycetoma in-fection. The only data available werederived from animal models and actino-mycetoma patients. From mouse modelsit was evident that mycetoma only oc-curred when M. mycetomatis was inoculatedwith an adjuvant (either soil or Freundsincomplete adjuvant), both predisposing to-ward a Th2 response [4,5]. Remarkably, in1974, Cavanagh demonstrated thatM. mycetomatis emulsified in the Th1-predis-posing Freunds complete adjuvant (FCA)could not induce mycetoma in L20 mice[6,7]. Furthermore, when Peripheral BloodMononuclear Cells (PBMCs) from actinomy-cetoma patients were stimulated with anti-gens from Nocardia brasiliensis, a Th2-typecytokine profile was detected [8]. This couldindicate that the causative agent probablyinduces a Th2 response that facilitates furthergrowth and the development of a clinicallysignificant mycetoma lesion. In order todetermine if a Th2 cytokine profile is indeedassociated with mycetoma development anda Th1 cytokine profile with protection againstmycetoma, this prospective, cross-sectionalstudy was conducted at the MycetomaResearch Centre and the Institute of Endem-ic Diseases, University of Khartoum, Khar-toum, Sudan, after obtaining ethical clear-ance from the Soba University HospitalEthical Committee to determine the profileof four cytokines in both healthy endemiccontrols and mycetoma patients. The studyincluded 27 patients with confirmed eumy-cetoma due to Madurella mycetomatis treatedwith 800 mg/d ketoconazole and 21 normalindividuals from eumycetoma-endemic areas(Table 1). The present study was approved bythe Ethics Committee of Soba UniversityHospital, Khartoum, Sudan. Written in-formed consents were obtained from theparticipants prior to their enrollment inthe study. For each study, individual10 ml of venous blood were collected inheparinized blood collection tubes.From the collected blood, peripheralmononuclear cells were isolated usingFicoll-Hypaque gradient centrifugationas described previously [9]. Stimulationof cells was done using 50 ml of unchar-acterized culture filtrate of M. mycetoma-tis. The culture filtrate of M. mycetomatiswas prepared by culturing M. mycetomatisfor 3 wk at 37uC in RPMI-1640 medi-um supplemented with morpholinopro-panesulfonic acid (MOPS) and phenolred and by filtrating this through a0.45 micron pore size filter joined to a20 ml syringe to purify any mycelia anddebris. The filtered culture filtrate wasstored at 220uC until used. To stimu-late the isolated PBMCs, 16106 cellswere plated into a 24-well flat-bottomedcell culture plate. Stimulation of PBMCswas done using 50 ml of culture filtrateof M. mycetomatis. For each patient andhealthy control, some wells were leftunstimulated to serve as control. Theculture plates were incubated at 37uCwith 5% CO2. After 72 h, the cells wereharvested by centrifugation at1,200 rpm for 3 min, and the superna-tants were then collected for cytokinemeasurement by sandwich ELISAs us-ing BD OptEIA ELISA Set B (CatalogNo. 550534).Citation: Elagab EAM, Mukhtar MM, Fahal AH, van de Sande WWJ (2013) Peripheral Blood Mononuclear Cellsof Mycetoma Patients React Differently to Madurella mycetomatis Antigens than Healthy EndemicControls. PLoS Negl Trop Dis 7(4): e2081. doi:10.1371/journal.pntd.0002081Editor: Joseph M. Vinetz, University of California San Diego School of Medicine, United States of AmericaPublished April 25, 2013Copyright:  2013 Elagab et al. This is an open-access article distributed under the terms of the CreativeCommons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium,provided the original author and source are credited.Funding: Wendy van de Sande was funded by Veni-grant 916.11.178 of the Netherlands organisation for Scientificresearch (NWO) (http://www.nwo.nl/onderzoek-en-resultaten//programmas/vernieuwingsimpuls/toekenningen/alle+veni+toekenningen/Toekenningen+Veni+2010). The funders had no role in study design, data collection andanalysis, decision to publish, or preparation of the manuscript.Competing Interests: The authors have declared that no competing interests exist.* E-mail: ahfahal@hotmail.comPLOS Neglected Tropical Diseases | www.plosntds.org 1 April 2013 | Volume 7 | Issue 4 | e2081Table 1. Patient demographics.Eumycetoma Patients Healthy Endemic ControlsNumber (n) 27 21Gender (male/female) 22/5 19/2Mean age years (range) 23.5 (9–45) 33.9 (22–53)Lesion site Foot NALesion size (n) Large 18 NAMedium 5 NASmall 4 NAKetoconazole responding (yes/no) 11/8 NACausative agent M. mycetomatis NANA, not applicable.doi:10.1371/journal.pntd.0002081.t001PLOS Neglected Tropical Diseases | www.plosntds.org 2 April 2013 | Volume 7 | Issue 4 | e2081After stimulating the PBMCs of bothpatients and healthy endemic controlswith the M. mycetomatis antigen prepara-tion, it was noted that the PBMCs of thepatients reacted differently to this antigenthan the healthy controls. Statisticallysignificantly higher IL-10 concentrationswere detected in all the patients (mean458.2 pg/ml) compared to the healthycontrols (mean 30.7 pg/ml) (Figure 1A,Mann–Whitney, p=0.004, GraphPadPrism 5.01). In contrast, the INF-c con-centrations were significantly higher in thecontrol population (mean 792.8 pg/ml)than in the patient group (mean 77.4 pg/ml) (Figure 1B, Mann–Whitney,p=0.007). Generally it was noted thatwhen PBMCs produced INF-c uponstimulation with the antigen, no IL-10was produced and vice versa. There wereno significant differences in TNFa orTGF-b concentrations between patientsand control groups (Figure 1C and 1D,Mann–Whitney, p.0.05). In order todetermine if mycetoma patients withlarger lesions had higher levels of IL-10and lower levels of INF-c, the IL-10 andINF-c levels were stratified according tolesion size. Although in general higherconcentrations of Il-10 and lower levels ofINF-c were found in the massive lesions, itappeared that this was not statisticallydifferent from the smaller lesion sizes(Kruskall–Wallis, p.0.05; GraphPadPrism 5.01). There is only one other studywith which we could use to compare ourresults [10]. Castro–Matteotti used threedifferent Nocardia brasiliensis extracts tostimulate PBMCs from seven actinomyce-toma patients, seven tuberculosis patients,and seven healthy controls and measuredthe INF-c response [10]. None of theextracts was able to stimulate INF-cproduction in humans [10]. This is incontrast to our findings. When we stimu-lated the healthy controls in our study witha M. mycetomatis extract, the PBMCs of thehealthy controls produced INF-c. Clearly,the PBMC response toward eumycetomaantigens and actinomycetoma antigensdiffers.At the moment, eumycetoma is treatedwith surgery and prolonged antifungaltherapy with ketoconazole. Ketoconazoleis known to suppress the production ofTh2-type cytokines IL-4 and IL-5; there-fore, the working mechanism of this drugin mycetoma might be 2-fold: inhibitingfungal growth and reversing the immunesystem toward a Th1 response [11,12].Unfortunately not all patients respond wellto ketoconazole treatment. Many stillshow recurrent infections, even if thefungal species itself is susceptible towardthis drug [13]. Furthermore, it alsodetermined if patients responding toketoconazole had different cytokine pro-files than patients not responding to thisdrug. All patients were treated with800 mg/d of ketoconazole for at least6 mo. If a patient was responding to thistreatment, the size of the lesion decreased,discharging sinuses closed, and there was anoted pain relief. According to thesecriteria, the patients were further dividedinto two groups: patients who respondedto 800 mg/d ketoconazole (n=11) andthose who did not respond (n=8). PBMCsof patients who responded to ketoconazoletherapy produced similar levels of IL-10,INF-c, TNF-a, or TGF-b as patients whodid not respond to this therapy (Mann–Whitney, p= ns for all cytokines).In conclusion, this study showed thatmycetoma patients produce high concen-trations of IL-10 that correlate with thedisease severity. IFN-c seems to beassociated with protection against myceto-ma, and it may indicate past subclinicalinfections. The four cytokine concentra-tion levels were not affected by a medicaltreatment response. An important limita-tion of the present study is the smallnumber of patients included. Furtherstudies at the field level in endemic areasand on controls from nonendemic areasthat study cytokine profile in serum and inthe histopathological lesion are recom-mended for more in-depth understandingof the immune responses of the mycetomapatients.References1. Ahmed AO, van Leeuwen W, Fahal A, van deSande WWJ, Verbrugh H, et al. (2004) Myceto-ma caused by Madurella mycetomatis: a neglectedinfectious burden. Lancet Infect Dis 4: 566–574.2. de Klerk N, de Vogel C, Fahal A, van BelkumA, vande SandeWW (2012) Fructose-bisphosphate aldolaseand pyruvate kinase, two novel immunogens inMadurella mycetomatis. Med Mycol 50: 143–151.3. van de Sande WW, Janse DJ, Hira V, GoedhartH, van der Zee R, et al. (2006) Translationallycontrolled tumor protein from Madurella myceto-matis, a marker for tumorous mycetoma progres-sion. J Immunol 177: 1997–2005.4. Ahmed AO, van Vianen W, ten Kate MT, van deSande WW, van Belkum A, et al. (2003) A murinemodel of Madurella mycetomatis eumycetoma.FEMS Immunol Med Microbiol 37: 29–36.5. Zhang L, Mia MY, Zheng CL, Hossain MA,Yamasaki F, et al. (1999) The preventive effects ofincomplete Freund’s adjuvant and other vehicleson the development of adjuvant-induced arthritisin Lewis rats. Immunology 98: 267–272.6. Cavanagh LL (1974) Attempts to induce myceto-ma in monkeys and mice using Madurellamycetomi. Sabouraudia 12: 258–262.7. Stills HF, Jr. (2005) Adjuvants and antibody produc-tion: dispelling the myths associated with Freund’scomplete and other adjuvants. Ilar J 46: 280–293.8. Mendez-Tovar LJ, Mondragon-Gonzalez R,Vega-Lopez F, Dockrell HM, Hay R, et al.(2004) Cytokine production and lymphocyteproliferation in patients with Nocardia brasiliensisactinomycetoma. Mycopathologia 158: 407–414.9. English D, Andersen BR (1974) Single-stepseparation of red blood cells. Granulocytes andmononuclear leukocytes on discontinuous densitygradients of Ficoll-Hypaque. J Immunol Methods5: 249–252.10. Castro-Matteotti B, Vera-Cabrera L, Ocampo-Candiani J, Rendon A, Salinas-Carmona MC,et al. (2008) Immune response to Nocardiabrasiliensis extracellular antigens in patients withmycetoma. Mycopathologia 165: 127–134.11. Kanda N, Enomoto U, Watanabe S (2001) Anti-mycotics suppress interleukin-4 and interleukin-5production in anti-CD3 plus anti-CD28-stimulatedT cells from patients with atopic dermatitis. J InvestDermatol 117: 1635–1646.12. Kanda N, Watanabe S (2002) Ketoconazolesuppresses interleukin-4 plus anti-CD40-inducedIgE class switching in surface IgE negative B cellsfrom patients with atopic dermatitis. J InvestDermatol 119: 590–599.13. van de Sande WWJ, Luijendijk A, Ahmed AO,Bakker-Woudenberg IA, van Belkum A (2005)Testing of the in vitro susceptibilities of Madurellamycetomatis to six antifungal agents by using thesensititre system in comparison with a viability-based 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydrox-ide (XTT) assay and a modified NCCLS method.Antimicrob Agents Chemother 49: 1364–1368.Figure 1. Cytokine concentrations of stimulated PBMCs of eumycetoma patients and healthy controls. (A) IL-10 concentrations (pg/ml)measured after stimulating PBMCs from eumycetoma patients and healthy endemic controls with M. mycetomatis antigen. Statistically significantlydifferent concentrations were found between eumycetoma patients and healthy controls (p=0.004) as determined by the Mann–Whitney U test. (B)IFN-c concentrations (pg/ml) measured after stimulating PBMCs from eumycetoma patients and healthy endemic controls with M. mycetomatisantigen. Statistically significantly different concentrations were found between eumycetoma patients and healthy controls (p=0.007) as determinedby the Mann–Whitney U test. (C) TNF-a concentrations (pg/ml) measured after stimulating PBMCs from eumycetoma patients and healthy endemiccontrols with M. mycetomatis antigen. No statistically significantly different concentrations were found between eumycetoma patients and healthycontrols (p= 0.85) as determined by the Mann–Whitney U test. (D) TGF-b concentrations (pg/ml) measured after stimulating PBMCs fromeumycetoma patients and healthy endemic controls with M. mycetomatis antigen. No statistically significantly different concentrations were foundbetween eumycetoma patients and healthy controls (p=0.89) as determined by the Mann–Whitney U test.doi:10.1371/journal.pntd.0002081.g001PLOS Neglected Tropical Diseases | www.plosntds.org 3 April 2013 | Volume 7 | Issue 4 | e2081

Ehab A M Elagab

Maowia M Mukhtar

Ahmed H Fahal

Wendy W J van de Sande

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Peripheral blood mononuclear cells of mycetoma patients react differently to Madurella mycetomatis antigens than healthy endemic controls.

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

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