Abstract

Introduction

Materials & Methods

Results

Discussion & Conclusion

References

Discussion
Board

Immunomodulators used in the present study

OK-432, a streptococcal preparation, and an M-protein derived from streprococcus pyogenes were kindly provided from Chugai Pharmaceutical Co., Ltd, Tokyo, Japan. OK-PSA, a lipoteichoic acid preparation, was purified from the butanol extract of OK-432 by an affinity chromatography on CNBr-activated Sepharose 4B bound the TS-2 MAb which neutralizes IFN-g-inducing activity of OK-432 (20).

Treatment of PBMC with OK-432, OK-PSA or M-protein

PBMC were isolated from heparinized venous blood by Ficoll-Hypaque gradient density centrifugation according to standard procedures. PBMC (1 x 106/ml) were cultured in RPMI 1640 medium containing 10% FCS in the presence or absence of OK-432 (10-3, 10-2, 10-1 and 1 KE/ml), OK-PSA (10-3, 10-2, 10-1 and 1 mg/ml) or M-protein (0.1 or 10 mg/ml) for 0 to 48 h at 37 oC. In some experimenst, anti-IL-6 (10 mg/ml, Santa Cruz Biotechnology Inc., Santa Cruz, CA, U.S.A), anti-IL-10 (10 mg/ml, R & D Systems Inc., Minneapolis, MN, U.S.A) neutralizing antibody or recombinant IL-10 (rIL-10, 10 ng/ml, Genzyme Diagnostics, Cambridge, MA, U.S.A) was added into the above cultures.

Cytokines in the supernatants of these cultures were measured by the following techniques. The PBMC stimulated by OK-PSA were assayed for NK and LAK activities as follows. In addition, cytoplasmic RNAs were isolated from the PBMC stimulated by OK-PSA and expression of mRNA for several cytokines was analysed by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) method as follows.

Cytokine Assay

The assay for cytokines was performed using ELISA kits. The ELISA kits for TNF-a, -b, IL-1b and IL-6 were purchased from Genzyme (Cambridge, MA, U.S.A) and the kits for IFN-g, IL-2, -4, -10, -12 and -15 from BioSource International, Inc. (Camarillo, CA, U.S.A).

Assay for NK and LAK Activities

The cytotoxic activity of human PBMC was assayed against K-562 cells, markedly sensitive target cells for human NK cells, and Daudi cells, sensitive target cells for human LAK cells but not destroyed by human NK cells, in a 51Cr-release test.

Isolation of total RNA and semiquantitative RT-PCR

PBMC grown in RPMI 1640 medium containing 10% FCS in the presence or absence of OK-PSA (0.1 mg/ml) for 0 to 48 h were harvested and then total RNA was extracted using ISOGEN RNA extracting mixture (Nippon Gene, Toyama, Japan). Expression of several cytokines and GAPDH mRNA, a house-keeping gene, was detected by the semiquantitative RT-PCR method. One mg of total RNA was reverse-transcribed by Molony murine leukemia virus reverse transcriptase (Life Technologies Inc.) at 42 oC for 60 min in a 20 ml mixture with random primer (Life Technologies Inc.). Two ml of reverse-transcribed mixture was subjected to PCR in a 20 ml mixture [10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl2, 0.01% gelatin, 20 mM each dNTP (A,G,T,C), 0.5 U Taq polymerase (Takara, Otsu, Japan), and 0.25 pmol primer].

Twenty-four or 30 cycles of reaction at 94, 55, and 72°C for 60, 90, and 150 seconds, respectively, were carried out in a DNA Thermal Cycler. Amplified cDNA was subjected to electrophoresis in 1.5% agarose gels containing 100 ng/ml ethidium bromide. At the completion of electrophoresis, gels were viewed and photographed under UV light illumination (Polaroid type 667 film, Polaroid Corp., Cambridge, MA).

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