*3.5. EGb Treatment Down-Regulates Inflammatory-Associated Genes Expression*

To detect and respond to viral infection host cell activates multiple cellular signaling networks. Viral proteins and nucleic acids ultimately drive an antiviral response and activating transcription factors. Therefore, to study the influence of EGb on innate immune mechanisms (PBLs resistance to VSV infection/level of innate immunity and cytokine production), we investigated the expression of *IRFs-3* and -*7* (interferon regulatory factors) mRNA as well as ISGs (IFN-stimulated genes)—*tetherin* (encoding bone marrow stromal cell antigen 2) and *MxA* (encoding myxovirus resistance protein 1). *NF-κB* transcription factors and *SOCS* (suppressor of the cytokine signaling proteins) were also examined.

As was suspected, VSV infection resulted in upregulation in all investigated genes in AD patients and controls. However, this effect was more pronounced in AD patients. From Table 6, we can see that *MxA* was the most highly expressed gene with over 40 relative fold change in AD, while in controls, it was over 16 relative fold change. Similarly, after VSV infection in AD, a marked induction (3–8.5 relative fold change) of *IRF-7*, *tetherin,* and *SOCS1* was demonstrated. As presented in Figure 4 and Table 6, in both groups (AD and controls), EGb treatment decreased expression of all investigated genes (average effect < 0) in uninfected and VSV-infected PBLs. In uninfected PBLs from AD patients, average reduction in all investigated genes expression after EGb treatment was about *EGb ef f ectPBLs all genes*;*AD* ≈ −0.4; i.e., the average level of expression was

1 *<sup>e</sup>*−0.4 = 1.5 times lower compared to the expression before EGb treatment. In the case of controls, it was *EGb ef f ectPBLs all genes*;*Controls* ≈ −1.1; i.e., average reduction in genes expression in controls after EGb treatment was <sup>1</sup> *<sup>e</sup>*−1.1 = <sup>3</sup> times lower compared to the expression before EGb treatment. Interestingly, in VSV-infected PBLs the effect of EGb treatment was stronger in AD group for all genes than for controls. In PBLs from AD patients, average reduction of all investigated genes expression was about *EGb ef f ectPBLs*+*VSV all genes*;*AD* ≈ −1; i i.e., the average level of expression was <sup>1</sup> *<sup>e</sup>*−<sup>1</sup> = 2.7 times lower compared, to the genes expression before EGb treatment. In PBLs from control group it was *EGb ef f ectPBLs*+*VSV all genes*;*Controls* ≈ −0.7; i.e., the average expression in VSV-infected leukocytes was <sup>1</sup> *<sup>e</sup>*−0.7 = 2 times lower compared to genes expression before EGb treatment. Table 6 presents level of investigated genes expressions before and after EGb treatment, EGb effects, and comparison of EGb effects in both groups. Confidence intervals (CI95) in Figure 4 and in Table 6 show greater precision of expression measurement than testing for statistical significance.

**Table 6.** Genes expression before and after EGb treatment and measure of EGb effect in AD patients and control group. Median is measure of average level. For every individual person and for every gene change of expression level after, treatment equals d = log *a f ter bef ore* = log(*a f ter*) − log(*bef ore*). EGb effect is average of all *d*s in a group. EGb effect is the average of ratios *a f ter bef ore* expressed in logs. Delta Δ is measure of difference in EGb effects between AD and controls, and this statistic is the average difference between a randomly selected person from AD group and a randomly selected person from control group.



**Table 6.** *Cont.*

**Figure 4.** Changes in relative expression of inflammatory-associated genes after EGb treatment in uninfected and VSV-infected PBLs from AD patients and healthy age-matched controls. EGb effect was measured as natural logarithm of ratio after/before gene expression. Zero means no effect. Points represent average change after treatment with confidence interval CI95% for the average.
