Vliv polysacharidů na imunitní buňky a hojení ran

Mgr. Jana Franková, Ph.D.

Doctoral thesis

The influence of polysaccharides on immune cells and wound healing process

Abstract:

Polysaccharides are high molecular weight condensation polymers with tens and often hundreds of monosaccharides residues per polysaccharide chain. These biomolecules perform a number of different functions in living organisms. Some polysaccharides act as structural components, for example, in the cell walls of plants, some are food reserve and some serve as lubricating agents in joints. In this thesis we studied two types of polysaccharides: hyaluronan and D-glucans. Hyaluronan is an acidic polysaccharide consisting of a disaccharide repeat unit of D-glucuronic acid and N-acetyl-D-glucosamine. It is a water solution, which is very viscous even at low concentration. In spite of its high viscosity it is capable reacting with a wide range of biomolecules including tissue components, proteins, proteoglycans and growth factors. It is the ability of hyaluronan to interact and to recognise and to be recognised by other biological molecules, which makes it a potential use in the area of tissue repair. On the other hand D-glucans belong to neutral polysaccharides and are composed of D-glucopyranose. There are two possible configuration of D-glucan: alfa and beta. Its structure variability caused their wide distribution among higher plants, lower plants, animals and microorganisms. Glucans may have a linear or branched structure. The structural diversity is a reason for a wide range of solubility among the glucan class members, some being totally insoluble and some absolutely soluble. Biologically active molecules contain (1-3)-beta-D-glucan chains and glucans with this structure are known for their immunomodulating and antitumour activity. In the first part of the thesis we focused on 1. the effect of Hyiodine (combination of hyaluronan with KI3 complex) on cell lines in vitro 2. the effect on cells isolated from tissue and 3. the effect on wound healing process in patients with non-healing wound, leg ulcers and diabetic wounds. We tested the toxic effect of Hyiodine (0.5mg/ml and 1mg/ml) using XTT methods and detection of LDH release in cells lines (U937 and HL60) and cells isolated from human blood and tissue sections (keratinocytes). Non-toxic properties of Hyiodine were proved in these tests. We also studied the influence of Hyiodine on the production of pro-inflammatory cytokines (IL-6, IL-8, IL-12 and TNF-alfa). The production of IL-6 and TNF-alfa by lymphocytes was increased for both studied concentration of Hyiodine after 24 hours. The production by U937 and keratinocytes was decreased after 24 hours but increased after 48 hours for all measured cytokines. The production of pro-inflammatory cytokines by HL60 was close to the detection limit. The expression of K1, K5, K10 and K14 by keratinocytes was also detected. The expression of K5 was significantly decreased and the expression of K14 was significantly increased after 24 hours of co-incubation with Hyiodine. No differences were detected after 48 hours in comparison with non-treated cells. The K1 and K10 were not expressed. We also measured the expression of cell surface molecules (CD11b, CD62L and CD69) on leukocytes isolated from human peripheral blood using flow cytometry. The expression of these molecules was not changed. Monitoring the production of free radicals of blood fagocytes using methods of spontaneous and induced chemiluminiscence discovered that during induced chemiluminiscence the production of free radicals significantly decreased in comparison with non-treated cells. This part of the thesis was completed with a clinical study conducted under the direction of Prof. MUDr. L. Sobotka, CSc. at the Department of Metabolic Care and Gerontology (The Faculty Hospital in Hradec Kralove). Excellent positive effect of Hyiodine on wound healing was observed mainly in healing of large infection wounds, non-healing wounds, leg ulcers and mainly diabetic wounds. Main function of Hyiodine is capability of hyaluronan to bind water and the following hydratation of the wound a

Abstract:

nd prevention of the bandage adherence to the wounds. The KI3 complex ensured disinfection of the wounds. Hyiodine was applied on sterile gauze and then put on the wound. The bandage was changed every two days. Hyaluronan is a polysaccharide, which is abundant in connective tissue, cartilages and skin. It has function groups, which can attachment of specific ligands or act as sites for chemical derivatisation. It can be modified to change its physical characteristics and improve its applicability for a specific application. Typical widely used modifications include polymerisation, esterification, oxidation and cross-linking. HA is degraded in organisms by hyaluronidase to low-molecular weight products, which can act as a promoter of early inflammation and angiogenesis. In order to eliminate hyaluronidase digestive activity, we prepared a chemically derivative HA (HA-DR) and compared the response of human blood leukocytes to HA-DR and HA. The following parameters were measured: i) spontaneous, FMLP (N-formylmethionyl-leucyl-phenylalanine) and PMA (phorbol myristate acetate) induced oxidative burst of blood phagocytes, ii) expression of CD25 on lymphocytes, iii) CD11b and CD62L expression on PMNL on monocytes, and iv) release of cytokines by blood leukocytes. No significant differences were found between the concentrations of pro-inflammatory cytokines IL-6, IL-8, IL-10 produced by non-treated leukocytes and leukocytes treated with HA and HA-DR. HA (1mg/ml, 0.5mg/ml) induced significant decrease of free radical production by phagocytes activated with PMA. HA-DR did not induce the significant effect. The expression of surface receptors CD11b and CD25 was significantly increased after a treatment with HA or HA-DR (1mg/ml) on PMNL, monocytes and for CD25 also on lymphocytes. On the other hand expression of CD62L on PMNL and monocytes was significantly decreased. It can be concluded from the presented results that chemical modification of HA-DR did not change significantly the biological potential of HA. (1-3)-beta-D-glucans are another important polysaccharides with biological properties. They are known because of their immunomodulating activities, the ability to suppress the development of infections and to inhibit tumour growth. They are isolated from cell wall of fungi, algae and plants. In the third part of the thesis, the immunomodulatory activity of two (1-3)-beta-D-glucans: carboxymethylglucan (CMG) and schizophyllan (SPG) was determined and compared on human leukocytes in vitro. Both SPG and CMG activated blood phagocytes and lymphocytes what was demonstrated by increased of whole blood production of reactive oxygen species (ROS), by an increased production of pro-inflammatory cytokines IL-6, IL-8 and TNF-alfa, by an increased surface expression of CD69 on lymphocytes, and by an altered expression of CD11b on PMNL and monocytes and by decreasing of the expression of CD62L on PMNL and monocytes. SPG demonstrated a significantly higher potential to stimulate blood phagocytes and the production of selected pro-inflammatory cytokines in comparison with CMG. The higher potency of SPG to stimulate human blood phagocytes in vitro could be caused by factors such as higher branching frequencies or neutral polymer charge of SPG or different conformation in solution if compared with CMG.

Keywords

hyaluronan, (1-3), beta- D-glucans, wound healing, leukocytes

Language used: Czech

Date on which the thesis was submitted / produced: 6. 6. 2006

Thesis defence

• Date of defence: 6. 6. 2006
• Supervisor: doc. RNDr. Antonín Lojek, CSc.

Masaryk University

Faculty of Science

Doctoral programme / field:
Biology / Immunology