Zofia Błach-Olszewska

Flavones from Root of Scutellaria Baicalensis Georgi: Drugs of the Future in Neurodegeneration?

Flavonoids are natural, plant-derived compounds which exert diverse biological activities, also valuable neuroprotective actions within the brain and currently are intensively studied as agents able to modulate neuronal function and to prevent age-related neurodegeneration. Among them, flavones isolated from Scutellaria baicalensis root exhibit strong neuroprotective effects on the brain and are not toxic in the broad range of tested doses. Their neuroprotective potential has been shown in both oxidative stress-induced and amyloid-beta and alpha-synuclein-induced neuronal death models. Baicalein, the main flavone present in Scutellaria baicalensis root, strongly inhibited aggregation of neuronal amyloidogenic proteins in vitro and induces dissolution of amyloid deposits. It exerts strong antioxidative and anti-inflammatory activities and also exhibits anti-convulsive, anxiolytic, and mild sedative actions. Importantly, baicalein, and also another flavone: oroxylin A, markedly enhanced cognitive and mnestic functions in animal models of aging brains and neurodegeneration. In the preliminary study, wogonin, another flavone from Scutellaria baicalensis root, has been shown to stimulate brain tissue regeneration, inducing differentiation of neuronal precursor cells. This concise review provides the main examples of neuroprotective activities of the flavones and reveals their potential in prevention and therapyof neurodegenerative diseases.

Baicalin from the extract of Scutellaria baicalensis affects the innate immunity and apoptosis in leukocytes of children with acute lymphocytic leukemia.

Scutellariae Radix (root of Scutellaria baicalensis) has a long history of application in traditional and in modern herbal medications. The major components of Scutellariae Radix are baicalin, baicalein, wogonoside and wogonin. Accumulating evidence demonstrates that Scutellaria has immunomodulatory effects and possesses compelling anticancer potential. Treatment of peripheral blood leukocytes (PBLs) with Scutellaria extract (SBE) enriched in baicalin, reduced viability of PBLs obtained from patients with acute lymphoblastic leukemia (ALL). SBE had no impact on the survival of healthy, control leukocytes. The immune system modulation by SBE resulted in increased production of IFNγ in PBLs, and reduced TNFα and IL-10 production in bone marrow cells (BMC), in ALL patients. SBE stimulated the nonspecific antiviral immunity, assessed by resistance of PBLs and BMC to vesicular stomatitis virus (VSV) infection. SBE showed pro-apoptotic activity in NALM-6 cell line (B-type human leukemia). The number of cells expressing annexin V increased from 6% in control cultures to 29% and 52% after treatment with 100 μg/ml and 200 μg/ml respectively. Increased percentage of apoptotic cells was observed when cells were treated with corresponding concentration of baicalin. SBE enhanced apoptosis of PBLs in BMC of leukemic children. The percentage of PBLs that underwent apoptosis and mean annexin V expression increased from 11% in the control to 17% and 24% for the doses of 100 μg/ml and 200 μg/ml respectively. Importantly, SBE did not induce apoptosis of PBLs in the healthy, control group.

Effect of donepezil on innate antiviral immunity of human leukocytes

BACKGROUND The effect of donepezil on two mechanisms of innate immunity: leukocyte resistance to viral infection and cytokine production was studied. METHODS The degree of natural resistance of human peripheral blood leukocytes (PBLs) was determined by studying the kinetics of vesicular stomatitis virus (VSV) replication. A titer of 0-1 log TCID(50) indicated complete resistance, 2-3 log partial resistance, and >4 lack of resistance. Cytokine levels were determined with use of ELISA test. NFkappaB activation was assayed by immunocytochemical staining. RESULTS Preliminary study of VSV replication in the PBLs of Alzheimers disease patients showed a high sensitivity to infection, except of PBL those under donepezil therapy. The PBL resistance stimulated us to study the effect of donepezil on innate immunity. Donepezil inhibited VSV replication in the leukocytes of healthy blood donors but influence on infection in L929 and A549 cells was not shown. The effect was dose dependent and individually differentiated. The production of TNFalpha and IFNs was reduced in infected leukocytes in a dose-dependent manner in the PBLs of the healthy blood donors and of AD patients. NFkappaB activation was also reduced by donepezil. CONCLUSIONS Donepezil regulate two mechanisms of innate immunity of leukocytes: resistance to viruses and cytokine production.

Age- and disease-related innate immunity of human leukocytes ex vivo

Two mechanisms of innate immunity, i.e. resistance to viral infection and the production of cytokines by leukocytes, were compared in blood isolated from four groups of donors: healthy young (19-35 years old), healthy elderly (over 60), elderly Alzheimers disease (AD) patients, and elderly patients with alimentary tract cancer (CA). Peripheral blood leukocytes (PBLs) were isolated by gradient centrifugation in Gradisol G. The degree of resistance was calculated from the kinetics of vesicular stomatitis virus (VSV) replication in the PBLs. Cytokine (TNFα, IFNα, IFNγ, IL-12, and IL-10) levels were determined by ELISA. The antiviral resistance of the PBLs varied, but a difference was observed only between the young and elderly groups and not between the healthy elderly controls and those with AD or cancer. Differences observed in all the groups concerned the ability and intensity of cytokine production. The most impressive results were obtained for spontaneous TNF and IFNα release. While TNF was released spontaneously by the PBLs of the elderly CA patients and the young healthy group, it was usually undetected in the AD and only sometimes in the healthy elderly group. Leukocytes isolated from the elderly groups responded to VSV infection with more intense IFNα and IFNγ production than the younger group.

Cytokine production by human leukocytes with different expressions of natural antiviral immunity and the effect of antibodies against interferons and TNF

Abstract.Introduction:Two activities of innate antiviral immunity were studied: the resistance of human peripheral blood mononuclear cells (PMBCs) ex vivo to viral infection and the production of cytokines.Materials and Methods:Samples of blood were taken from healthy blood donors and from persons with frequent infections of the upper respiratory system. PMBCs were isolated by gradient centrifugation. Vesicular stomatitis virus (VSV) was used as the indicatory virus to infect PMBCs. The cytokines: IFN, TNF, and IL-6 were titrated by biological methods and IL-10 by ELISA.Results:Blood donors were divided for two groups: those with VSV-resistant and those with VSV-sensitive PMBCs and secretion of cytokines by them was compared. The resistant PMBCs produced more cytokines than the sensitive ones. A statistically significant difference, was found only in the case of the IFNs. To examine the contribution of IFNs and TNF in maintaining resistance, leukocytes from both groups were treated with specific anti-cytokine antibodies. The authors’ previous study showed that the elimination of spontaneous IFN-±, IFN-β, IFN-γ, and TNF-± from resistant leukocytes resulted in increased VSV replication This indicates the important role of cytokines. In VSV-sensitive PMBCs, anti-IFN-± showed the opposite effect (decreased virus replication). In the absence of spontaneous IFN-±, disturbances in cytokine production were observed.Conclusions:Complete resistance of PMBC to VSV infection is accompanied by higher cytokine release, The paradoxical effect of anti-IFN-± on virus replication in leukocytes sensitive to viral infection may be attributed to changes in the cytokine profile balance, i.e. high TNF production by VSV-infected leukocytes and a complete reduction of IL-6 production.

Resistance of human leukocytes to vesicular stomatitis virus infection as one of the innate antiviral immune activities; participation of cell subpopulations.

Among reactions of innate immunity, resistance of human peripheral blood leukocytes (PBL) to viral infection seems important. The purpose of our study was to find, which of the subpopulations of PBL is the most responsible for the innate antiviral immunity of these cells. The innate immunity was measured by using the direct method of infection of leukocytes with vesicular stomatitis virus (VSV). The lack of VSV replication by infected leukocytes (0-1 log TCID50) was taken as an indicator for complete immunity; a low level of VSV (2-3 log) for partial immunity; and high VSV titer (more than 4 log) for no immunity. The resistance/innate immunity of whole PBL and subpopulations such as: adherent cells, fractions enriched in lymphocytes T, and lymphocytes B (separated on column with nylon wool), NK(+) and NK(-) (separated by microbeads activated cell sorting MACS) differ from each other. All fractions express higher resistance/innate immunity than the whole PBL. NK(+) cells were found the most resistant fraction of PBL to VSV infection. The results indicate that among the leukocytes in PBL the regulation mechanisms of innate immunity exist. The study on the mechanism of innate immunity regulation as well as the role of NK in innate immunity of PBL must be continued.

P-095: Effect of donepezil on innate antiviral immunity of human leukocytes

Background: Peripheral blood leukocytes of Alzheimer Disease (AD) patients after donepezil therapy were previously found resistant to viral infection.These results contrasted with those obtained in control age group.The leukocytes were very sensitive to vesicular stomatitis virus (VSV) infection. Objective(s): In view of that donepezil effect on innate immunity reactions, i.e. production of cytokines and deficiency of resistance of ex vivo leukocytes to viral infections was studied. Methods: Results: ELISA test was used to determine the level of IFN-alfa, IFNgamma, TNF-alfa, and IL-10. Leukocyte resistance was determined by infection with VSV and study of kinetics of viral replication.NF-kB activation was measured by immunocytochemical method using anti-p65 antibodies (Chemicon). Donepezil (Pfizer) was used in nontoxic concentrations 10-50 ug/ml. Among healthy blood donors (n 30) two groups were distinguished: one with resistant leukocytes (n 15) and the second (n 15) with leukocytes sensitive to VSV.The resistant leukocytes produced about 10 times more cytokines than the sensitive ones, except IFN-alfa, which was produced by VSV-infected sensitive leukocytes only. Both groups differed also in cell percentage with constitutively activated NK-kB: 1523% in resistant, and 1-8% in sensitive leukocytes. Donepezil used in a concentration 10-50ug/ml reduced production of TNF-alfa in both groups, reduced production of IFN-alfa in sensitive cells, increased production of IL-10 and sometimes IFN-gamma in leukocytes producing these cytokines, but did not stimulate the cytokines. In the same concentration donepezil was able to stimulate resistance of leukocytes previously very sensitive to VSV. Conclusions: Donepezil exhibited two activities: ability to reduce TNF-alfa and IFN-alfa level, which may potentially be useful in a case of overproduction of these two cytokines in inflammatory, neurodegenerative and autoimmune disorders, and stimulation of leukocyte resistance, which may take advantage in viral diseases.