Systemic suppression of interferon-
 responses in Buruli
ulcer patients resolves after surgical excision of the
lesions caused by the extracellular pathogen
Mycobacterium ulcerans
Dorothy Yeboah-Manu,*,† Elisabetta Peduzzi,† Ernestina Mensah-Quainoo,‡
Adwoa Asante-Poku,* David Ofori-Adjei,* Gerd Pluschke,† and Claudia A. Daubenberger†,1
*Noguchi Memorial Institute for Medical Research, University of Ghana, Legon; †Swiss Tropical Institute, Molecular
Immunology, Basel, Switzerland; and ‡Ghana Health Service, Amasaman, Ga District, Ghana
Abstract: Buruli ulcer (BU), caused by Mycobac- ation of subcutaneous (s.c.) tissues and the overlying skin. The
terium ulcerans, is the third most common myco- disease starts as a s.c. nodule, papule, or plaque, which even-
bacterial infection in immunocompetent humans tually ulcerates and progresses over weeks to months until
besides tuberculosis and leprosy. We have com- surgical excision or spontaneous healing occurs [1]. After
pared by ex vivo enzyme-linked immunospot anal- tuberculosis and leprosy, BU is the third most common myco-
ysis interferon-
 (IFN-
) responses in peripheral bacterial infection in immunocompetent humans [2]. The main
blood mononuclear cells (PBMC) from BU pa- burden of disease falls on children living in sub-Saharan
tients, household contacts, and individuals living in Africa, but healthy people of all ages, races, and socioeco-
an adjacent M. ulcerans nonendemic region. nomic class are susceptible [3]. The effectiveness of antimy-
PBMC were stimulated with purified protein deriv- cobacterial drug therapy has not been proven [3]. Conse-
ative (PPD) and nonmycobacterial antigens such as quently, surgery is presently the recommended treatment op-
reconstituted influenza virus particles and isopen- tion [4]. In BU lesions, clumps of extracellular, acid-fast
tenyl-pyrophosphate. With all three antigens, the organisms surrounded by areas of necrosis are found, particu-
number of IFN-
 spot-forming units was reduced larly in s.c. fat tissue [5]. M. ulcerans produces a family of
significantly in BU patients compared with the con- macrolide toxin molecules, the mycolactones, which are asso-
trols from a nonendemic area. This demonstrates ciated with tissue destruction and local immunosuppression
for the first time that M. ulcerans infection-associ- [6]. In cell culture experiments, mycolactones produce apopto-
ated systemic reduction in IFN-
 responses is not sis and necrosis in many human cell types [7, 8]. The toxin
confined to stimulation with live or dead mycobac- appears to play a role in inhibiting the recruitment of inflam-
teria and their products but extends to other anti- matory cells to the site of infection, which explains at least in
gens. Interleukin (IL)-12 secretion by PPD-stimu- part why inflammatory responses are poor in BU lesions [5].
lated PBMC was not reduced in BU patients, indi- However, intralesional influx of leukocytes and granulomatous
cating that reduction in IFN-
 responses was not responses in the dermis and panniculus has been reported in
caused by diminished IL-12 production. Several late stages of the disease [9, 10]. Spontaneous healing can
months after surgical excision of BU lesions, occur and is often accompanied by a conversion of the Burulin
IFN-
 responses of BU patients against all anti-
gens used for stimulation recovered significantly, (M. ulcerans sonicate) skin test from negative to positive.
indicating that the measured systemic immuno- However, the immune mechanisms involved in protection
suppression was not the consequence of a genetic against BU are largely unknown.
defect in T cell function predisposing for BU but The importance of interferon-
 (IFN-
) for immunity against
is rather related to the presence of M. ulcerans mycobacterial infections in humans is demonstrated by the
bacteria. J. Leukoc. Biol. 79: 1150–1156; increased susceptibility of children carrying complete IFN-

2006. receptor 1 (IFN-
R1) chain deficiency to environmental my-
cobacterial infection [11]. Apart from CD4 T cells, 
 T cells,
natural killer cells (NK), and CD8 T cells are potent sources
Key Words: ex vivo ELISpot analysis 
 immunosuppression 
 in-
terleukin-12 of IFN-
. CD4 T cells can be differentiated into T helper cell
INTRODUCTION 1 Correspondence: Swiss Tropical Institute, Molecular Immunology, Socin-
strasse 57, CH 4002 Basel, Switzerland. E-mail: Claudia.Daubenberger@
unibas.ch
Buruli ulcer (BU) caused by Mycobacterium ulcerans is an Received October 14, 2005; revised February 3, 2006; accepted February 6,
infectious disease characterized by chronic, necrotizing ulcer- 2006; doi: 10.1189/jlb.1005581.
1150 Journal of Leukocyte Biology Volume 79, June 2006 0741-5400/06/0079-1150 © Society for Leukocyte Biology
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type 1 (Th1) and Th2, distinguished by their patterns of cyto- or guardians before enrollment. For ethical reasons, the age range of the
kine production after antigen activation. Apart from other nonexposed controls, who were included into the analysis, was higher than that
cytokines, Th1 cells preferentially secrete IFN-
, and Th2 of the BU patients. Ethical approval for the study was obtained from the local
ethical review board of the Noguchi Memorial Institute for Medical Research,
cells preferentially secrete interleukin (IL)-4 and IL-5. Th1 or University of Ghana (Legon). Clinical diagnosis of BU was reconfirmed as
Th2 development is determined by the cytokine environment described [22, 23] by one or more laboratory verification tests (Table 2),
during T cell activation in the primary response to antigen, and including culture of M. ulcerans, microscopic detection of acid-fast bacilli
IL-12 and IFN-
 are implicated in the decision to adopt a Th1 (AFB), or IS2404 polymerase chain reaction (PCR). The clinical pictures of the
phenotype [12]. IFN-
 binds to the IFN-
R1/IFN-
R2 com- patients ranged from nodules or plaques to severe ulcerative forms (Table 2).
In the BU group, nine persons and in the other two groups, 10 persons each
plex and stimulates innate cell-mediated immunity through NK with a BCG scar were recruited (Table 1). PBMC were isolated from venous
cells and activation of bactericidal mechanisms in macro- peripheral blood using Ficoll-Hypaque gradient centrifugation following stan-
phages. The central role of IFN-
 in major histocompatibility dard procedures and cryopreserved prior to the analysis.
complex class I- and class II-restricted antigen processing and
presentation is well-documented [13]. At present, the contri- Antigens
bution of IFN-
 in immunity to extracellular M. ulcerans
remains to be established. After thawing, PBMC were directly stimulated with 50 M IPP (Sigma Chem-
ical Co., St. Louis, MO), 10 g/ml tuberculin PPD derivative of M. tuberculosis
Peripheral blood mononuclear cells (PBMC) of BU patients (Statens Seruminstitut, Denmark), 10 g/ml phytohemagglutinin (PHA; Sigma
with active disease showed significantly reduced lymphopro- Chemical Co.), or 20 g/ml immunopotentiating reconstituted influenza viro-
liferation and IFN-
 secretion in response to stimulation with somes (IRIV; Berna Biotech, Switzerland). These influenza virosomes are
live or killed preparations of Mycobacterium bovis, M. ulcerans, spherical, unilamellar vesicles prepared by detergent removal from influenza
M. tuberculosis, and the recombinant protein Ag85 of M. tu- surface glycoproteins and mixtures of natural and synthetic phospholipids
berculosis [14–18]. Here, we have determined in a cross- containing H1N1 from influenza virus strain A/Singapore/6/86 [24]. Cell
culture medium consisted of RPMI 1640, 10% heat-inactivated human AB
sectional study the frequency of IFN-
-secreting cells in serum, 2 mM glutamine, 100 U/ml penicillin, and 100 g/ml streptomycin
PBMC from BU patients, their household contacts, and controls (Gibco-BRL, Grand Island, NY).
from BU nonendemic areas by ex vivo enzyme-linked immu-
nospot (ELISpot) analysis. The antigens used for stimulation Ex vivo IFN-
 ELISpot analysis
included isopentenyl pyrophosphate (IPP), reconstituted influ-
6
enza virus particles (virosomes), and tuberculin-purified pro- PBMC were thawed, washed, and suspended at a concentration of 2  10
tein derivative (PPD) of M. tuberculosis, which stimulate dis- cells/ml in complete cell culture medium. The cells were then stimulated with
the different antigens at the final concentrations indicated above and incubated
tinct T cell subsets, such as V
2V2 T cells [19], CD4 T cells for 24 h at 37°C, 5% CO2 humidified atmosphere. A 96-well nitrocellulose-
[20], and CD4 and V
2V2 T cells [21], respectively. Results bottomed plate (Millipore, Beford, MA) was coated overnight at 4°C with 10
demonstrate that BU-associated reductions in IFN-
 responses g/ml anti-human IFN-
 primary antibody (Clone1-D1K; Mabtech, Sweden).
are not confined to stimulation with live or dead mycobacteria The plates were then washed five times with phosphate-buffered saline (PBS)
and mycobacterial antigens. Furthermore, it is shown for the and blocked with cell culture medium for 1 h at room temperature. The
medium was decanted, and preincubated cells (2105 or 1105) were added
first time that in individual BU patients, this suppression in to each well in triplicate and incubated at 37°C for another 20 h. Assays were
IFN-
 secretion improved in a time interval of 5–10 months. terminated by washing plates three times with PBS-Tween 80 (0.05%) followed
by PBS another three times. Secondary antibody (biotin-labeled anti-human-
IFN-
; Clone 7-B6-1) was added to each well at 1 g/ml, and the plate was
MATERIALS AND METHODS incubated for 2 h at room temperature (RT). Plates were washed again six times
with PBS before application of streptavidine-alkaline phosphatase (1:1000
Study population dilution in PBS, 0.5% fetal calf serum, 100 l/well) for 1 h at RT. After
washing wells seven times with PBS, distinct spots were developed by the
Thirteen BU patients, 19 clinically healthy household contacts who never had incubation of plates at RT for 4–10 min following the addition of the devel-
clinical BU, and 18 healthy persons living in a M. ulcerans nonendemic district oping buffer (5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium, di-
in the Greater Accra region of Ghana were enrolled for the study (Table 1). All luted 1:100, Bio-Rad, Hercules, CA). Spot development was stopped by
BU patients enrolled were residents of the BU-endemic Ga District and washing the plates extensively with water and left to dry. Plates were later
presented with preulcerative or ulcerative lesions at the Amasaman Health evaluated using the ELISpot Reader system (AID, Germany) to determine the
Centre. Informed consent was obtained from study participants or their parents number of spot-forming units (SFU).
TABLE 1. Characteristics of BU Patients, Household Contacts, and Controls from a BU Nonendemic Area Enrolled in the Study
BU patient Household contacts Controls from nonendemic area
Characteristic (n  13) (n  19) (n  18)
Sex
Male 5 (39%) 6 (32%) 8 (44%)
Female 8 (61%) 13 (68%) 10 (56%)
Median age (years) 15 15 29.5
Age range (years) 6–45 6–56 24–65
BCG scar present (%)a 69 52 55
a Percent values calculated for 13 patients, 14 household contacts, and 14 controls, respectively. Five household contacts and four nonexposed controls remained
with uncertain scar status. BCG, Bacillus Calmette-Guerin.
Yeboah-Manu et al. Disease-associated changes of IFN-
 secretion in M. ulcerans 1151
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TABLE 2. Clinical Data of Patients Presenting with Lesions As a Result of Confirmed M. ulcerans Infection
BCG First blood Second blood
Patient Age Gender Clinical forma statusb AFBc Cultured PCRe samplef samplef
P03 45 f ulcerative –    3 months 11 months
P04 9 f plaque  –   0 8 months
P05 10 m nodule     0 8 months
P08 16 m ulcerative –    4 months 14 months
P09 5 f ulcerative   –  3 months 12 months
P11 20 f ulcerative     7 days 7 months
P12 16 m ulcerative  – –  3 months 12 months
P13 11 f ulcerative –    7 days 7 months
P14 15 m plaque – – –  7 days 8 months
P15 7 f ulcerative     0 9 months
P19 6 m nodule     0 6 months
P21 17 f nodule   –  0 5 months
P22 17 f nodule    – 0 5 months
a Clinical forms of BU disease were graded according to the World Health Organization case definition [1]. b BCG status was determined by confirmation of the
presence of a BCG scar by two persons. c AFB detection was performed by direct smearing of tissue exudates followed by Ziehl-Neelsen staining [1]. d Culture of
M. ulcerans according to ref. [22]. e IS2404 PCR analysis according to ref. [23]. f Time of blood sample collection. Time 0 is time of surgical treatment.
Enzyme-linked immunosorbent assay (ELISA) RESULTS
quantification of IL-12 in cell culture
supernatants Frequency reduction of IFN-
-secreting SFU in
Levels of total IL-12 in culture supernatant of PBMC incubated with PHA (10 PBMC from BU patients
g/ml) and PPD (10 g/ml) for 96 h were determined by ELISA using a
commercial kit (Mabtech). Samples were analyzed in triplicates, and results Thirteen patients with laboratory-reconfirmed M. ulcerans in-
were expressed as the average of the three readings in an ELISA reader at 450 fection, 19 clinically healthy household contacts, and 18 indi-
nm with reference to curves generated using serially diluted recombinant viduals from a neighboring BU nonendemic area were enrolled
human IL-12. The sensitivity of the assay was 30 pg/ml for total IL-12.
for this study (Tables 1 and 2). The frequency of immediate
Statistical analysis IFN-
-secreting cells in PBMC upon stimulation with IPP,
Data were analyzed using the STATA program (Stata Corporation, College IRIV, and PPD was analyzed using an ex vivo ELISpot assay.
Station, TX). Comparisons among the paired samples were performed using the Figure 1 shows that the mean of SFU upon stimulation with
Wilcoxon signed-rank test, and the Wilcoxon rank-sum test was used to PPD, IPP, and IRIV was significantly lower (P0.0086,
analyze the significance of observed difference in IL-12 secretion between P0.001, and P0.0002, respectively) in BU patients com-
patients and unexposed controls. For comparing the frequencies of antigen- pared with nonexposed controls. In household contacts, the
specific IFN-
-secreting cells, the data were transformed to normality using
Box-Cox transformation before being analyzed by linear regression. Data were mean of SFU after IPP and IRIV stimulation was significantly
considered statistically significant when P  0.05. higher compared with BU patients (P0.005 and P0.001,
Fig. 1. Quantification of ex vivo IFN-
-secreting 1200
cells by ELISpot analysis after stimulation of PBMC
with IPP (
), IRIV (), and PPD (). PBMC de-
rived from BU patients, household contacts, and 1000
controls from a BU nonendemic area were thawed
into cell culture medium and kept overnight in the
presence or absence of the different stimuli. For the 800
detection of IFN-
-producing cells, 2 105 PBMC/
well were plated in triplicate wells, and spots were
developed after 24 h. Delta SFU  Mean of SFU of
600
stimulated triplicate cultures – mean SFU of un-
stimulated triplicate cultures. Data from each indi-
vidual analyzed are shown separately. Mean SFU/
106 PBMC were 26 (6–102), 137 (57–327), and 262 400
(157–436) after stimulation with IPP in BU pa-
tients, household contacts, and nonendemic con-
trols, respectively. In IRIV-stimulated cells, the 200
mean SFU/106 cells were 22 (7–70), 135 (83–221),
and 149 (56–394) in BU patients, household con-
tacts, and nonendemic controls, respectively. After
PPD stimulation, 88 (35–223), 39 (14–106), and
212 [100–445] SFU/106 PBMC were recorded in BU patients, household contacts, and nonendemic controls, respectively. The values given in brackets are the
95% confidence intervals of the geometric means of SFU. Data of Patients P03, P08, P09, and P12 are shown by shaded symbols.
1152 Journal of Leukocyte Biology Volume 79, June 2006 http://www.jleukbio.org
Delta SFU/106 PBMC
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respectively), and in PPD-stimulated cultures, no significant IPP and IRIV stimulation. PHA-stimulated control wells
difference was observed (P0.82). showed only a slight (1.7-fold) median increase between the
two different time-points analyzed (P0.17), and in PPD-
Recovery from systemic immunosuppression stimulated cultures, the boost was not significant (P0.09).
after surgical treatment One representative example of an ELISpot analysis is shown in
All patients enrolled for the study were treated by wide surgical Figure 2B, and the numbers of SFU detectable after IPP, IRIV,
excision of the BU lesions. Blood samples were collected and PPD stimulation rose between the first and second sam-
several months after the first sampling (Table 2), and PBMC pling. In contrast, medium and PHA control wells remained at
from both time-points were analyzed in parallel using the same a comparable level between the two analyses.
IFN-
 ELISpot assay as above. A 3.9-, 3.7-, and 3.6-fold IL-12 production in PPD-stimulated PBMC is not
median increase in cellular responses against IPP, IRIV, and affected in BU patients
PPD stimulation, respectively, was observed when PBMC,
taken at the two time-points, were compared (Fig. 2A). Sta- Next, we wanted to determine whether the systemic suppres-
tistical analysis confirmed this increase of responses as highly sion of IFN-
 responses in BU patients is related to a dimin-
significant with P values of 0.021 and 0.003, respectively, after ished capacity to secrete IL-12. PBMC of nine patients and 10
Fig. 2. (A) Frequency increase of antigen-specific
ex vivo IFN-
-secreting cells in BU patients. PBMC
of 13 BU patients sampled at two time-points (Table
2) were thawed into cell culture medium and kept
overnight in the presence or absence of IPP (
),
IRIV (e), PPD (), and PHA (). For the detection
of IFN-
-producing cells, 2 105 PBMC/well were
plated in triplicate wells, and spots were developed
after 24 h. Delta SFU were calculated as described
in Figure 1. Given is the ratio of SFU obtained after
the second sampling/first sampling. Data of Patients
P03, P08, P09, and P12 are shown by shaded
symbols. (B) Representative IFN-
 ELISpot pat-
terns of BU Patient P21. PBMC of BU Patient P21
were obtained immediately before surgical excision
of the BU lesion (first sampling) and 5 months later
(second sampling). Parallel analysis of both sam-
ples was conducted in triplicates arranged horizon-
tally with 2  105 cells/well plated. The different
stimulators used are given on the right, and the
numbers of SFU/well are shown in the lower-left
corners.
Yeboah-Manu et al. Disease-associated changes of IFN-
 secretion in M. ulcerans 1153
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persons from BU nonendemic areas were stimulated with PHA disposing for mycobacterial infections [11]. Therefore, we an-
or PPD, and the total IL-12 concentration in cell culture alyzed PBMC of BU patients sampled at two consecutive
supernatants was measured by ELISA (Fig. 3). It is interesting time-points. When the paired PBMC samples were analyzed in
that the IL-12 concentrations in PPD-stimulated PBMC of parallel by ELISpot analysis, the numbers of IFN-
-secreting
individuals living in M. ulcerans nonendemic regions were cells after IPP, IRIV, and PPD stimulation increased signifi-
statistically lower compared with BU patients (P0.011), and cantly after surgical treatment. Comparable numbers of SFU in
in PHA-stimulated cultures, no significant difference was ob- PHA-stimulated PBMC in paired samples excluded a general
served (P0.57; Fig. 3). The mean IL-12 concentrations in suppression of cellular immune responses in BU and variations
supernatants of PPD- and PHA-stimulated cultures of PBMC in quality of PBMC sample cryopreservation. To our knowl-
obtained before or after surgical treatment showed no differ- edge, this is the first report demonstrating that antigen-specific
ence (Fig. 3). IFN-
 production in BU patients is coming back to normal
levels after surgical treatment. Hence, confounding genetic
defects do not seem to be responsible for the observed immu-
DISCUSSION nosuppression. The question of whether IFN-
 production in
patients undergoing spontaneous healing of BU will also im-
An ex vivo ELISpot assay for detection of IFN-
 secretion prove over time is not addressed here.
permitting the direct detection of individual antigen-specific T IL-12 induces T and NK cells to produce several cytokines,
cells at low frequencies was used in the current study [25]. The including IFN-
 [26]. Total IL-12 concentrations in culture
48-h antigen challenge of PBMC suffices to engage cytokine supernatants of PPD-stimulated PBMC were statistically
production in the memory/effector T lymphocyte but not in higher in BU patients (treated and untreated) than in the
naı̈ve T cells and allows determination of frequencies and controls. This may reflect a compensatory mechanism of the
cytokine signatures of recirculating, antigen-specific T cells immune system to restore IFN-
 production and indicates that
[25]. The frequencies of systemic IFN-
-producing SFU after the observed reduction in systemic IFN-
 responses is not
stimulation with IPP, IRIV, or PPD were reduced significantly caused by diminished IL-12 production. Different cytokine
in BU patients compared with individuals living in a neigh- expression profiles in PBMC and skin lesions were described
boring BU nonendemic area. This result is consistent with in patients suffering from the nodular and ulcerative forms of
other reports demonstrating that PBMC from subjects with past BU. Nodules were associated with higher IFN-
 and lower
or current M. ulcerans disease had reduced IFN-
 production IL-10 production and BU, with lower IFN-
 and higher IL-10
in response to PPD of M. tuberculosis and M. bovis or whole- production [17]. Within the limited number of BU patients
killed M. bovis BCG or M. ulcerans [14–18]. However, our data enrolled in our study, a relationship between different stages of
strongly indicate that reduced IFN-
 production is not confined BU disease and reduction of IFN-
 secretion was not observed.
to immune responses specific for mycobacterial antigens or V
2V2 T cells compose the majority of human 
 T cells
whole mycobacteria but extends to CD4 T cell responses in circulation and among their defined ligands, is IPP, a
specific for influenza virus [20] and V
2V2 T cells [19]. metabolite found in prokaryotic and eukaryotic cells including
Reduced IFN-
 production in BU patients could be the mycobacteria [27]. Studies in rhesus monkeys provided evi-
consequence of a genetic abnormality in T cell function pre- dence that V
2V2 T cells contribute to adaptive immune
BU patients BU patients Non-exposed
(1st sampling) (2nd sampling) controls
3000
Fig. 3. Total IL-12 concentrations in cell culture 2500
supernatants of PHA- and PPD-stimulated PBMC of
nine BU patients obtained at the two different sam-
pling time-points (Table 2). Mean of total IL-12 2000
concentration (given in pg/ml) in PPD-stimulated
cultures was 543 and 750 after the first and second
sampling, respectively, and in PHA-stimulated cul- 1500
tures, 348 and 472, respectively. PBMC of 10 indi-
viduals living in a neighboring M. ulcerans nonen-
demic region were treated similarly, and the mean 1000
total IL-12 concentrations after PPD and PHA stim-
ulation were 276 and 402, respectively.
500
PHA PPD PHA PPD PHA PPD
1154 Journal of Leukocyte Biology Volume 79, June 2006 http://www.jleukbio.org
total IL-12 (pg/ml)
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ACKNOWLEDGMENTS pyrophosphate. Eur. J. Immunol. 27, 952–956.20. Schumacher, R., Adamina, M., Zurbriggen, R., Bolli, M., Padovan, E.,
Zajac, P., Heberer, M., Spagnoli, G. C. (2004) Influenza virosomes en-
This study was supported in part by the Stanley Thomas hance class I restricted CTL induction through CD4 T cell activation.
Vaccine 22, 714–723.
Johnson Foundation, the Ghana Government, and a stipend 21. Green, A. E., Lissina, A., Hutchinson, S. L., Hewitt, R. E., Temple, B.,
from the Amt für Ausbildungsbeiträge of the county Basel- James, D., Boulter, J. M., Price, D. A., Sewell, A. K. (2004) Recognition
Stadt for D. Y-M. We thank Samuel Owusu, Dr. Kwasi Addo, of non-peptide antigens by human V 
 9V  2 T cells requires contact with
cells of human origin. Clin. Exp. Immunol. 136, 472–482.
John Tetteh, and Charles Atiogbe from Noguchi Memorial 22. Yeboah-Manu, D., Bodmer, T., Mensah-Quainoo, E., Owusu, S., Ofori-
Institute for Medical Research (Legon, Ghana); the BU team Adjei, D., Pluschke, G. (2004) Evaluation of decontamination methods
nurses at Amasaman Health Centre (Ghana) for technical and and growth media for primary isolation of Mycobacterium ulcerans from
surgical specimens. J. Clin. Microbiol. 42, 5875–5876.
field support; and all the participants involved in the study for 23. Noeske, J., Kuaban, C., Rondini, S., Sorlin, P., Ciaffi, L., Mbuagbaw, J.,
their time. We are grateful to Dr. Penelope Vounatsou for Portaels, F., Pluschke, G. (2004) Buruli ulcer disease in Cameroon redis-
support in statistical analysis. covered. Am. J. Trop. Med. Hyg. 70, 520–526.
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1156 Journal of Leukocyte Biology Volume 79, June 2006 http://www.jleukbio.org
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