CITOKYNES AND IMMUNOGLOBULINS IN ACTINOMYCETOMA PATIENTS

1

Luis J. MÉNDEZ TOVARHospital de Especialidades

Centro Médico Nacional, IMSS. México, D. F.

MYCETOMA

2

• 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

MYCETOMAMYCETOMA

HOST

AGEN

TEN

VIRO

MEN

T

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

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

• 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

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

)

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

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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