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The effect of Lectranal® on the immunoregulation of inflammatory processes in mice
The objective of this research was to ascertain the effect of Lectranal® on the immunoregulation of inflammatory processes by verifying its function and the expression of the ikbkg, relb, and cd3e genes using Real Time PCR (RT-PCR) technology.

METHODS

After provoking a long-term inflammatory process by a subcutaneous application of Freund’s adjuvant (FA) and Lectranal® treatment or of Freund’s adjuvant and calcium for 14 days, the RNA was isolated from the mouse spleen cells. The expression of the ikbkg, relb, and cd3e genes was analyzed using the Real Time PCR method.
RESULTS

The results of the cd3e gene activity analysis:
In the case of the cd3e gene, RT-PCR technology showed that, after the injection of FA, a significant increase in the activity of this gene occurs, and that treatment with Ca2+ ions and Lectranal® attenuates this reaction, but only Lectranal® provides statistically significant results.

Relative quantification relation of cd3e gene expression during the therapy in comparison with the preliminary control value
Relative quantification relation of cd3e gene expression during the therapy in comparison with the preliminary control value

These results showed that the applicated FA activated an immune reaction and that it initiated processes of antigen recognition by the APC throughout the CD3 complex gene. However, after the administration of Lectranal® during the next 14 days, it was noted that the immune reaction induced by the FA was regulated and that the activity of this gene was attenuated.
The results of the relb gene activity analysis:

After the injection of FA, a significant decrease in relb gene activity is noticed, i.e., the inflammatory process inhibits the activity of this gene. Lectranal® treatment, on the other hand, encourages its activity.
Relative quantification relation of relb gene expression during the therapy in comparison with the preliminary control value
Relative quantification relation of relb gene expression during the therapy in comparison with the preliminary control value

Lectranal® is involved throughout the relb gene in the activation of the Th1 cells and it stimulates T lymphocytes to secrete IFNγ. Former results of the gene chip analysis in the case of the irf1 gene, which codes the regulation factor of the interferon γ, confirm these new results.
The results of the ikbkg gene activity analysis:

The RT-PCR method showed that ikbkg gene activity, after the injection of FA, did not grow significantly. In the group which was given Lectranal®, the activity of this gene grew, and it finally reached 1.7 of the relative value.
Relative quantification relation of the ikbkg gene expression during the therapy in comparison with the preliminary control value
Relative quantification relation of the ikbkg gene expression during the therapy in comparison with the preliminary control value


Ikbkg is a gene that encodes a protein of a signal transduction, while its expression regulates NFκB activity. The induced ikbkg activity recruits the Th cells (Th0 and Th1), regulates the clonal expansion of Th1 cells and its differentiation, and the production of IFN-γ gamma by the Th1 lymphocytes. Lectranal® directly regulates the relationship of Th1/Th2 cells via the ikbkg and NFκB genes.
CONCLUSION

Based on the results of this research, we conclude that Lectranal® gives a very powerful stimulus to the immune system, which is obvious from the noted divisions and activation of the Th1 cells, synthesis of an IFN-gamma which is actively involved as a cytokine in the recruitment of B lymphocytes and IgG production. These processes divert the immune reaction toward the blocking of the allergic response.
Besides all that, Lectranal® provokes the NFκB translocation in B lymphocytes of the spleen, which leads to clonal expression and plasma cell differentiation.

AUTHORS

Hadžija M, Popović Hadžija M., Korolija M., Križanac S.
Ruđer Bošković Institute, Zagreb 2006
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