1. CITOKYNES AND IMMUNOGLOBULINS IN ACTINOMYCETOMA PATIENTS 1 Luis J. MÉNDEZ TOVAR Hospital de Especialidades Centro Médico Nacional, IMSS. México, D. F.

2. MYCETOMA 2

3. 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 3

4. HOST AGENT ENVIROMENT

5. (1910-1984) (1902-1989) 5 Dr. Fernando Latapí Dr. A. González-Ochoa

6. 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 6

7. 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 7

8. 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 8

9. 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) 9

10. 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. 10

11. 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. 11

12. ANTIGENIC FRACTIONS NB1, NB2, …, NB20) 12

13. Proliferation assay IMMUNOGLOBULIN TITRES INTERLEUKINS DETERMINATION 13 SUPERNATANTS ELISA

14. RESULTS 14

15. INTRADERMAL RESPONSE (n = 25) P value in both groups was p = 0.000 15 Induration diameter (mm)

16. Antigenic fractions 16

17. ANTIGENSCONTROL PATIENTS PHA2588 ± 676 4725 ± 2330 PPD 1685 ± 660 2629 ± 2533 NB 1230 ± 531 2145 ± 2376 NB2 1733 ± 1043 2343 ± 2899 NB4 1924 ± 918 2021 ± 1154 NB6 1053 ± 674 1921 ± 1001 NB8 891 ± 359 1336 ± 776 NB10 1056 ± 551 1075 ± 812 Proliferation assay 17

18. IFN-γ production (n = 25) PATIENTS CONTROL 0 200 400 600 800 1000 1200 1400 PHAPPDNBNB2NB4NB6NB8NB10 ANTIGEN pg/ml P = 0.001 P = 0.011P = 0.000 18

19. PATIENTS CONTROL TNF-α production (n = 25) 0 500 1000 1500 2000 2500 3000 PHAPPDNBNB2NB4NB6NB8NB10 ANTIGEN pg/ml P = 0.000 P = 0.035P = 0.001P = 0.009 19

20. 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 0.0 20

21. 0 100 200 300 400 500 600 700 800 pg/ml CONTROL PATIENTS PHAPPDNBNB2NB4NB6NB8NB10 ANTIGEN IL-10 production (n = 25) P = 0.035P = 0.004P = 0.035 21

22. IgG subclass 0 0.02 0.04 0.06 0.08 0.1 0.12 0.14 0.16 PPD NB NB2 NB4 NB6 NB8 NB10 ANTIGENOS D.O. a 490 nm P = 0.861 P=0.483P=0.781 P=0.087 P=0.000 CONTROLPATIENTS IgG1IgG2 IgG3 IgG4 22

23. Summary TESTCONTROLPATIENTS CandidinNormal (+)Increased PPDNormal (+)± (some of theme neg.) Cell proliferationNormal IFN-γNormalLow with NB2, NB4 y NB6 TNF-αPresent with all antigens Higher than in control (PPD, NB y NB2) IL-10Low concentrations Higher than in control (PPD, NB y NB2) 1L-4AbsentPresent IgG1, IgG2Present with all antigensHigher than in control IgG3, IgG4Present with all antigens2 - 4 fold higher 23

24. CONCLUSIONS The patients’ PBMC exhibited a normal antigenic recognizing. The cytokine profile in patients corresponded to T H 2 type response. The immunoglobulin titres (mainly IgG3 and IgG4) are also compatible with a T H 2 response. 24

25. 25