Transcript Slide 1
Laboratory of immunochemistry of glycoconjugates and Laboratory of lignin, Institute of Chemistry, SAS Preparation and immunological characterization of glycoconjugates based on cell-surface polysaccharide antigens from the yeasts of Candida genus – potential vaccines Yeasts of Candida genus represent serious social and medical problem. Our laboratory belongs to those few departments in the world where the potential vaccines against diseases caused by the Candida yeasts are being developed. Our vaccine constructs are based on polysaccharide from the cell of yhe yeasts, which are responsible for the antigenicity of the yeasts. Children < 2 yrs, elderly people and immunocompromised individuals can produce sufficient level of IgG antibodies only against T-cell dependent antigens (polysaccharides are T-cell independent). Conversion of polysaccharides to T-cell dependence antigens can be achieved via conjugation with protein carrier. General strategy of glycoconjugate vaccine preparation: Yeast cultivation (most frequently present human pathogens: C. albicans, C. tropicalis, C. parapsilosis, C. glabrata), isolation and purification of mannans Mannan derivatization Conjugation with protein Immunization of animal model Immunological characterization of the conjugate activity Study of the structure and biological activity of fungal polysaccharides at the Laboratory of microbial polymers By means of acidic and alkaline extractions, cellular β-D-glucan was isolated from the cell walls of yeasts Saccharomyces cerevisiae and Candida albicans. Using 13C-NMR spectroscopy and by observing the complex formation with Congo Red dye, it has been proven that β-D-glucans form helical structures depending on the pH value (single and triple helices). Water-soluble derivatives of S. cerevisiae glucan – carboxymethyl- and sulfoethyl glucans were prepared by a chemical modification, and by means of the ultrasonic treatment, fraction precipitation, and liquid chromatography, their defined fractions with different degrees of derivatization and molecular weight values were obtained. Water-soluble derivatives of S. cerevisiae glucan exterted protective effect in mice with experimental infection caused by bacteria Klebsiella pneumoniae. Carboxymethyl glucan inhibited lipid peroxidation in liposomes and revealed antioxidant activity comparable to that of the established antioxidants such as D-mannitol, α-tocopherol, and ascorbic acid. Carboxymethyl glucan and sulfoethyl glucan prevented growth and metastasis of Lewis lung carcinoma and lymphosarcoma tumors and showed synergistic antitumor effect with a known cytostatic drug cyclophosphamide, and moreover suppressed its toxic side-effects. Water-soluble derivatives of S. cerevisiae glucan demonstrated antimutagenic and antigenotoxic effect and inhibited the oxidative degradation of DNA in lung cells. The ability of carboxymethyl glucan to scavenge free radicals was for the first time directly proven by a spin-trap EPR spectroscopy. Water-soluble derivatives of S. cerevisiae glucan revealed protective anti-arthritic activity in an experimental model of adjuvant arthritis in mice. Antioxidant and antimutagenic properties were manifested also by other fungal polysaccharides: glucomannan from the yeast Candida utilis and glucan-chitin complex from the filamentous fungus Aspergillus niger. Synthesis and characterization of neoglycoconjugates based on Vibrio Cholerae O1 LPS. Derivatization of LPS carboxylic groups using different linkers and conjugation with proteins - disease caused by Gram negative bacteria Vibrio Cholerae, every year over 100 000 dead - prevention: strict sanitation and vaccination - cell surface of bacteria contains lipopolysaccharides (LPS). Immunity against these LPS prevents against disease. - isolation of antigen (extraction of cell mass and LPS purification), separation or destruction of lipidic part Lipid A (whole LPS is responsible for septic shock) - conjugate synthesis (conversion to T-cell dependent antigen). Conjugate consists of antigen covalently bound to protein carrier (different geometry, valency etc.) - conjugate characterization and in vivo testing (animal model) LPS Lignin belongs among main biomass components - chemical treatment of wood worldwide yields about 50x106 t/ year. In Lignin Laboratory under the supervising of professor Ing. B. Košíková DrSc. novel methods were developed for use of lignin biomass component: H2COH HC 1. application of lignin in polymer blends O H2COH HCOH H2COH CH2OH HC O H3CO CH O CH HC CH HCOH H2COH OH OCH3 CH H3CO HCOH H3CO H2COH H3CO OH H H2COH HCOH OCH3 HC O H2COH HCOH H3CO OCH3 H2COH O C H H3CO HC CH HC CH2 HCOH OCH3 OH HOCH2 CH OCH3 HCOH O HOCH2 H2COH CH O CH O H3CO HC O H2C H3CO HC HCOH C OH OH O O OCH3 H3CO - isolation and modification of various types of lignins - characterisation of rheological and physicomechanical properties of lignin-polyolefin blends OCH3 HOCH2 H3CO CH OH C HC O H C C O HOH2C CH O 2. preparation of novel lignin-based antimutagenic and anticarcinogenic compounds O H3CO - examination of the stability of polyolefin blends during processing and life service O - influence of lignin concentration on vulcanization, rheological and mechanical properties of rubber composites - determination of antioxidative effect of lignin samples on rubber blends Reduction of oxidative damage of DNA by lignin preparations Cells non-pretreated with lignin 0 – 20 % of tail DNA 20 – 40 % of tail DNA 40 – 60 % of tail DNA 60 – 80 % of tail DNA 80 –100 % of tail DNA 0 Cells pretreated with lignin 75 was prooved on: - cells on hamster cells V79 - human cells VH10 0 10 21 4 60 0 30 0 - human carcinoma cells Caco-2 - primary testicular cells and lymphocytes isolated from the rats in vitro - lymphocytes and testicular cells isolated from the rats fed by diet containing lignin ex vivo