citokynes and immunoglobulins in actinomycetoma patients 1 luis j. mÉndez tovar hospital de...
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CITOKYNES AND IMMUNOGLOBULINS IN ACTINOMYCETOMA PATIENTS
1
Luis J. MÉNDEZ TOVARHospital de Especialidades
Centro Médico Nacional, IMSS. México, D. F.
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MYCETOMA
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• Mariat F. Bull Soc Path Exot 1963;56:35-45
• López-Martínez R, Méndez-Tovar LJ, Lavalle P, Welsh O, Saul A, Macotela Ruíz E. Gac Med Mex 1992;128:477-82
MYCETOMA
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MYCETOMAMYCETOMA
HOST
AGEN
TEN
VIRO
MEN
T
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(1910-1984)(1902-1989) 5Dr. Fernando Latapí Dr. A. González-Ochoa
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Intradermal reaction (+)Low titres of IgG
Intradermal reaction (-)High titres of IgG
Good
prognosis
Bad
prognosis
González-Ochoa A, Baranda F, Bojalil LF, Bastarrachea F Shibayama H, Félix D
1960 - 1980
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• Macrophages and neutrophil cells don’t destroy N. asteroides
• Injection of sensibilized T lymphocytes is protective in athymic mice inoculated with N. asteroides.
• Superoxido dismutase (SDO) and catalase are present in virulent strains, mutants (SDO-) are not virulent.
• Athymic mice inoculated with N. asteroides developed disseminated infections with similar lesions observed in immunosuppresed human patients.
Beaman BL, Beaman L, Deem F, Richard L, Doughty A, Gershwin G, Steve M, Bourgeois L, Maslan S, Scates M,
Nocardia asteroides and Nocardiosis
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• Amikacina treatment
• Purified antigens from Nocardia brasiliensis.
• Diagnosis by ELISA.
• Identification of catalase gene
Vera-Cabrera L, Welsh O, Casillas S,, Rodríguez A, Torres-López E, Ramos A, Licón-Trillo A, González-Spencer D.
Contributions in 90s
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Salinas-Carmona M, Torres-López E, Ramos AI, et al. Infec Immun 1999;67:2428
Interleukines and Immunoglobulines in actinomycetoma murine model
• IFN-γ increasing (10 folds)
• Increase of IL-4, IL-6, IL-10
• Increase of IgM (7 day)
• Increase of IgG (45 day)
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OBJECTIVES
• By the cytokine profile obtained in proliferation assay of peripheral blood monocyte cell, to know the type of adaptative immune response (Th1 or Th2) exhibited by actinomycetoma patients.
• To demonstrate that this response is different to the response elicited in healthy people.
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METHODS
1. N. brasiliensis antigen obtention
2. Intradermal application of PPD y candidine
3. Proliferation assay of peripherical blood monocyte cell (PBMC)
4. Quantification of: IFN-γ, TNF-α, IL-4 and IL-10. using supernatants of proliferation assay
5. Seric levels of IgG1, IgG2, IgG3, IgG4.
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ANTIGENIC FRACTIONSNB1, NB2, …, NB20)
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Proliferation assay
IMMUNOGLOBULIN TITRES
INTERLEUKINSDETERMINATION
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SUPERNATANTS
ELISA
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RESULTS
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INTRADERMAL RESPONSE(n = 25)
0
4
8
12
16
20
CAND. CONT CAND. PAC. PPD CONT. PPD. PAC.
Di‡
me
tro
de
ind
ura
ci—
n (
mm
)
P value in both groups was p = 0.000
15
Ind
ura
tion
dia
me
ter
(mm
)
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Antigenic fractions
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ANTIGENS CONTROL PATIENTS
PHA 2588 ± 676 4725 ± 2330PPD 1685 ± 660 2629 ± 2533NB 1230 ± 531 2145 ± 2376NB2 1733 ± 1043 2343 ± 2899NB4 1924 ± 918 2021 ± 1154NB6 1053 ± 674 1921 ± 1001NB8 891 ± 359 1336 ± 776NB10 1056 ± 551 1075 ± 812
Proliferation assay
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IFN-γ production (n = 25)
PATIENTS
CONTROL
0
200
400
600
800
1000
1200
1400
PHA PPD NB NB2 NB4 NB6 NB8 NB10
ANTIGEN
pg
/ml
P = 0.001 P = 0.011 P = 0.000
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PATIENTS
CONTROL
TNF-α production (n = 25)
0
500
1000
1500
2000
2500
3000
PHA PPD NB NB2 NB4 NB6 NB8 NB10
ANTIGEN
pg
/ml
P = 0.000
P = 0.035 P = 0.001 P = 0.009
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IL-4 production(n = 25)
ANTIGEN PATIENTS DS
(pg/ml)
CONTROL
PHA 10.14 4.5 0.0
PPD 4.72 2.9 0.0
NB 5.80 1.6 0.0
NB2 5.58 3.4 0.0
NB4 5.93 2.2 0.0
NB6 7.37 4.9 0.0
NB8 6.86 4.8 0.0
NB10 6.14 3.6 0.020
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0
100
200
300
400
500
600
700
800
pg
/ml
CONTROL
PATIENTS
PHA PPD NB NB2 NB4 NB6 NB8 NB10
ANTIGEN
IL-10 production (n = 25)
P = 0.035 P = 0.004 P = 0.035
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IgG subclass
0
0.01
0.02
0.03
0.04
0.05
0.06
0.07
0.08
0.09
0.1
PPD PPD NB NB NB2 NB2 NB4 NB4 NB6 NB6 NB8 NB8 NB10 NB10 0
0.02
0.04
0.06
0.08
0.1
0.12
0.14
PPD PPD NB NB NB2 NB2 NB4 NB4 NB6 NB6 NB8 NB8 NB10 NB10
ANTÍGENOS
D. O
. a 4
90 n
m
0
0.02
0.04
0.06
0.08
0.1
0.12
0.14
0.16
PPD PPD NB NB NB2 NB2 NB4 NB4 NB6 NB6 NB8 NB8 NB10 NB100
0.02
0.04
0.06
0.08
0.1
0.12
0.14
0.16
PPD PPD NB NB NB2 NB2 NB4 NB4 NB6 NB6 NB8 NB8 NB10 NB10
ANTIGENOS
D.O
. a
490
nm
P = 0.861 P=0.483 P=0.781
P=0.087 P=0.000
CONTROL PATIENTS
IgG1 IgG2
IgG3 IgG4
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Summary
TEST CONTROL PATIENTS
Candidin Normal (+) Increased
PPD Normal (+) ± (some of theme neg.)
Cell proliferation Normal Normal
IFN-γ Normal Low with NB2, NB4 y NB6
TNF-α Present with all antigensHigher than in control (PPD, NB y NB2)
IL-10 Low concentrationsHigher than in control (PPD, NB y NB2)
1L-4 Absent Present
IgG1, IgG2 Present with all antigens Higher than in control
IgG3, IgG4 Present with all antigens 2 - 4 fold higher
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CONCLUSIONS
• The patients’ PBMC exhibited a normal antigenic recognizing.
• The cytokine profile in patients corresponded to TH2 type response.
• The immunoglobulin titres (mainly IgG3 and IgG4) are also compatible with a TH2 response.
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