Assessment of cytokine expression has become crucial to understand host responses to infections as well as autoimmunity. Several approaches including Northern blot, RNase protection assay and enzyme-linked immunosorbent assay have been used for this purpose, but they are time consuming, labour intense, and relatively large quantity of the samples is usually required. Recently, a technique termed real-time reverse transcriptase-polymerase chain reaction (RT-PCR) has been developed to determine genetic expression with great sensitivity and specificity; however, specialized instrumentation and costly reagents are usually needed. We aimed at using low-cost reagents for real-time PCR. This was achieved by adapting a conventional RT-PCR protocol to the quantitative real-time format, by the addition of the SYBR 1 Green I reagent. We validated the approach by assessing the cytokine gene expression of murine splenocytes upon stimulation with phorbol 12-myristate 12-acetate (PMA)-ionomycin. The results using this technique were compared with those obtained with the well-established gene array method. We conclude that the use of the SYBR 1 Green I reagent during real-time RT-PCR provides a highly specific and sensitive method to quantify cytokine expression with accuracy and no post-PCR manipulation.

Many modified genes are products regulated by the Nuclear Factor-kappaB and Mitogen Activated Protein Kinase pathways, suggesting MLIF involvement with these two major pathways for the modulation of the inflammation and immune responses.

Human dialyzable leukocyte extracts (DLE) have been used for the last 50 years to successfully treat different diseases. They are obtained by disruption of cells in buffy coats and subsequent dialysis (<12 KDa) of the extracts. They can transfer cellular immune response from an immune donor to a naïve recipient, and despite their immunomodulatory effect, until now little is known about their mechanism of action. One possibility is that they may contain ligands that activate TLRs. In this work using a reporter gene assay and HEK293 cells transfected with either TLR-2 or TLR-4 plasmids, we showed that different concentrations of DLE (100, 10 and 1 microg/mL) resulted in the activation of NF-kappaB (luciferase reporter system) in cells TLR-2 transfected cells with 10 or 1 microg/mL DLE. Interesting none of the DLE concentrations used induced a significant luciferase activity in the TLR-4 transfected cells. Our result can explain in part the immunomodulatory effects produced by DLE both in vivo and in vitro. Work will now focus in the identification of this ligand(s). This work was supported in part by SIP/IPN. 1Are fellows of COFAA, EDI and SNI.

Surgery, radiotherapy and chemotherapy are the most widely used and well-established modalities for treating malignant diseases. However, all modalities have major drawbacks. An alternative approach is to expand and activate tumour-specific immune cells in vivo. Dialyzable spleen extracts (DSE) are obtained from freeze-thawed murine spleen cell suspensions and subsequent dialysis (<12 KDa), they have immunomodulatory activity and have shown to cure experimental murine tuberculosis. In this work a BALB/c DSE was used to examine in mice its effectiveness as a prophylactic anti-metastatic therapy, using the mouse B16-F10 melanoma model. 1 Unit DSE (equiv. 1x105 spleen cells) or 0.1 U, were orally given daily for 7 days to 6 and 4 mice respectively. A third group of mice (N=6) was treated with placebo. On day 8, all mice were bleed and each animal inoculated with 1x106 B16-F10 cells. On day 28 all animals were sacrificed, lungs were dissected and macroscopic metastatic nodules counted. An statistically decrease in metastatic nodules (P¡Ü0.01) was observed in both groups that received DSE when compared with the control group. These results indicate that DSE is a promising approach for inhibiting metastatic tumor growth. Further work will focus on the mechanism which elicit this antimetastatic activity. This work was supported in part by SIP/IPN. 1Are fellows of COFAA, EDI and SNI.