Role of thiamine in Huntington's disease pathogenesis: In vitro studies.

BACKGROUND: Oxidative stress accompanies neurodegeneration and also causes abnormalities in thiaminedependent processes. These processes have been reported to be diminished in the brains of patients with several neurodegenerative diseases. OBJECTIVES: The aim of this work was to conduct a comparative analysis of the impact of supplemented thiamine on the viability of human B lymphocytes with CAG abnormal expanded huntingtin gene (mHTT) (GM13509) and control, B lymphocytes without mHTT (GM14467) through the following studies: determination of the supplemented thiamine concentrations, which are effective for cell growth stimulation after incubation in thiamine deficit conditions; determination of cell capability to intake the exogenous thiamine; evaluation of exogenous thiamine influence on the profile of the genes related to thiamine and energy metabolism; determination of ATP synthesis and activities of thiamine-dependent enzymes, KGDHC and BCKDHC in the intact cells and upon the exogenous thiamine. MATERIAL AND METHODS: The following methods were used: EZ4U test for cell growth analysis; HPLC for determination of thiamine intake and ATP synthesis, qRT-PCR for evaluation of the gene profiles and spectrophotometric method for KGDHC and BCKDHC activities determination. RESULTS: Maximal cell growth stimulation was observed at 2.5 mM in GM14467 up to 135% of the control culture and at 5.0 mM in GM13509 cells up to 165% of the control culture. Native levels of total ATP and KGDHC and BCKDHC activities in both cell types were comparable and did not changed upon thiamine deficit or supplementation. GM13509 cells showed more of an increase in growth stimulation upon thiamine supplementation than GM14467 cells and this effect was reflected in the increase of intracellular thiamine concentration. CONCLUSIONS: The above results and reported changes in expression of GAPDH, IDH1 and SLC19A3 genes observed upon thiamine deficit conditions suggest that intracellular thiamine status and energy metabolism can have a role in HD pathogenesis.

Assessment of cytotoxic and genotoxic activity of alcohol extract of Polyscias filicifolia shoot, leaf, cell biomass of suspension culture and saponin fraction.

Some medicinal plants are the object of biotechnologists' special interest owing to their content of secondary metabolites, which have a strong pharmacological effect. Polyscias filicifolia is a plant known for long in traditional medicine of the Southeast Asia. Literature data suggest that it acts on the endocrine system, has adaptogenic and antiulcerative activity, shows bactericidal and insecticidal properties, restores the activity of the protein synthesis system in the conditions of long- and short-term anoxia, as well as reduces the effect of many mutagens in vitro. The purpose of the studies was to assess the cytotoxic and genotoxic effect of ethanol extracts from Polyscias filicifolia dry shoots and leaves obtained in vitro, as well as cell biomass from suspension culture. Saponin fraction from dried shoots was also tested. Initially, the cytotoxic effect was evaluated using the murine connective tissue cell line C3H/AN - L929. The genotoxic properties of the extracts were assessed using standard screening tests: the Ames test and the micronucleus test. Based on the obtained results it can be concluded that none of the extracts increases the number of revertants, both in tests with and without metabolic activation. The lack of in vitro genotoxic and mutagenic activity of tested shoot, dried leaf, cell biomass extracts, as well as the saponin fraction from dried shoots allows us to hope that Polyscias filicifolia could be used as a possible pharmaceutical raw material showing therapeutic properties.

Effects of bilberry fruit aqueous extract and selected antibiotics on immune response in mice.

It was demonstrated that an extract from the pharmacopoeial raw material bilberry fruit (Fructus Myrtilli) co-administered with selected antibiotics: cefuroxime, cefoperazone and doxycycline, positively affects the survival of mouse thymocytes in cultures with hydrocortisone (cytotoxicity test), increases the count of splenocytes forming spontaneous rosettes (E-rosette test) and increases the agglutination titre of mouse serum (active hemagglutination test). In the cytotoxicity test, the best protective effect has been obtained with the combination of doxycycline with bilberry fruit aqueous extract. The increased count of splenocytes capable of binding sheep red blood cells has been observed for cultures with cephalosporins (cefuroxime and cefoperazone). The highest agglutination titre has been demonstrated for mouse serum treated with the extract in combination with doxycycline and cefuroxime.

The influence of plant raw materials, containing ellagic acid and selected antibiotics on immunological response in mice.

It is demonstrated that the combined addition to a culture of aqueous extracts from tannin pharmacopoeial raw materials, containing elladic acid (Cortex Quercus, Folium Uvae ursi and Rhizoma Tormentillae) and the antibiotics: cefuroxime, cefoperazone and doxycycline influences the enhancement of survival of mouse thymocytes in cultures with supplementation of hydrocortisone. A cytotoxic test was used for the evaluation. The presented results confirmed that each of the applied aqueous extracts shows a better survival of mouse thymocytes after combined addition of an antibiotic to the culture. The extract from Cortex Quercus showed the highest activation when added to the culture with cefuroxime, whereas cefoperazone best stimulation demonstrated when combined with the extract from Rhizoma Tormentillae, and doxycycline with the extract from Folium Uvae ursi.

Determination of immunological activity in vitro of some plant raw materials.

Aqueous extracts of five tannin plant raw materials have been studied for the survivals of thymocytes from the thymus of Balb-c mice. The immunostimulation activity was compared with a preparation from calf thymi TFX and phenolic acids: ellagic and gallic acids. It was found that only the Fructus Myrtilli extract has revealed a statistically significant influence on the survival of thymocytes.

The effect of the Melissa officinalis extract on immune response in mice.

The effect of an extract from Melissa officinalis on immune response in mice was analysed using the cytotoxicity test in three dilutions (undiluted extract and extract diluted 10 and 100 times) and hemagglutination and rosette tests with various routes of administration (oral and subcutaneous). The immunostimulating activity of the extract was compared with that of a synthetic compound--levamisole, which influence on the immune system is well known. The present results confirm the effect of water extracts from leaves of Melissa on the immune system, in both humoral and cellular response.

Investigations of the thymomimetic activity of a selected phytopharmaceutical preparation--Reumaherb tablets by flow cytometry.

The aim of the present study was to investigate the influence of Reumaherb tablets, a plant preparation, on the maturation of mice thymocytes in the presence of glycocorticosteroid by the method of cytotoxicity test performed in vitro in two versions differing in methodology. The cell source for incubation was thymus of female Balb/c mice, four weeks old. Reumaherb tablets were added to the culture of thymocytes diluted by a factor of 10-400. The searched thymomimetic activity of the investigated Reumaherb preparation was found in both versions of the test, this being demonstrated by a 17% increase in the number of surviving cells in the presence of glycocorticosteroid added to the culture. This effect strongly depends on the applied doses. By a microscopic evaluation of the results a maximum stimulation was found at a 200-fold dilution of the examined preparation, while in the cytometric analysis a similar maximum effect was observed at a 20-fold dilution.

Estimation of humoral activity of Eleutherococcus senticosus.

The aim of the present work was an estimation of the influence of two plant pharmaceutical preparations containing an extract from the root of Eleutherococcus senticosus: Argoeleuter tablets and Immuplant tablets, on the humoral response of immunological system. Experiments were performed with female Balb/c mice six weeks old. In order to reveal the influence of taking preparations, containing an extract from Eleutherococcus senticosus on some elements of the immunological system, three ways of their administration have been compared: before illness, during illness and a combination of both. The obtained results allow formulating the following conclusions: - the pharmaceutical preparations, containing the extract from Eleutherococcus senticosus administered orally, influence on the increase of the level of immunoglobulins comprised in the mice's blood serum, - the pharmaceutical preparations act with different power, not fully dependent on the content of marker of the active substance - eleutheroside E, - dosage of the preparations containing the extract from Eleutherococcus senticosus should not be established basing only on the extract content, - best curative results, measured as the stimulation of humoral response of the organism were obtained when a given preparation was administered therapeutically, even though the combined administration - prophylactically with prolonged administration during illness also is correct.