Scaffold design for artificial tissue with bone marrow stem cells.

OBJECTIVE: The aim of this study was to test polymeric materials (collagen, fibrin, polyimide film, and polylactic acid) for single- and multi-layer scaffold formation. MATERIALS AND METHODS: In our study, we used rabbit bone marrow stem cells (rBMSCs) and human mesenchymal stem cells (hMSCs) with materials of a different origin for the formation of an artificial scaffold, such as a collagen scaffold, fibrin scaffold produced from clotted rabbit plasma, electrospun poly(lactic acid) (PLA) mats, polyimide film (PI), and the combination of the latter two. Cell imaging was performed 3-14 days after cell cultivation in the scaffolds. Time-lapse imaging was used to determine hMSC mobility on the PI film. RESULTS: Cell incorporation in collagen and clotted fibrin scaffolds was evaluated after 2-week cultivation in vitro. Histological analysis showed that cells penetrated only external layers of the collagen scaffold, while the fibrin clot was populated with rBMSCs through the entire scaffold thickness. As well, cell behavior on the laser micro-structured PI film was analyzed. The mobility of hMSCs on the smooth PI film and the micro-machined surface was 20±2µm/h and 18±4µm/h, respectively. After 3-day cultivation, hMSCs were capable of spreading through the whole 100±10µm-thick layer of the electrospun PLA scaffold and demonstrated that the multilayer scaffold composed of PI and PLA materials ensured a suitable environment for cell growth. CONCLUSIONS: The obtained results suggest that electrospinning technology and femtosecond laser micro-structuring could be employed for the development of multi-layer scaffolds. Different biopolymers, such as PLA, fibrin, and collagen, could be used as appropriate environments for cell inhabitation and as an inner layer of the multi-layer scaffold. PI could be suitable as a barrier blocking cell migration from the scaffold. However, additional studies are needed to determine optimal parameters of inner and outer scaffold layers.

Effect of (1→3),(1→6)-ß-glucan on in vitro production of cytokines by leukocytes of patients with periodontitis.

BACKGROUND. The aim of the study was to investigate the effect of (1→3), (1→6)-ß-glucan on the production of interleukin 10 (IL-10) and tumor necrosis factor α (TNF-α) in vitro by peripheral blood leukocytes of patients with periodontitis. MATERIAL AND METHODS. In total, 20 patients suffering from untreated severe chronic generalized periodontitis were enrolled in this study. Periodontitis was confirmed by clinical and radiologic examination. Besides, 20 periodontally healthy patients served as a control group. Peripheral venous blood was sampled from the patients, and isolated leukocytes were treated with (1→3),(1→6)-ß-glucan from yeast at different concentrations. The levels of IL-10 and TNF-α secreted by the leukocytes unstimulated and stimulated with unopsonized E. coli in vitro were determined by the enzyme amplified sensitivity immunoassay method. RESULTS. Our data showed that (1→3),(1→6)-ß-glucan induced a significant decrease (P<0.05) in the TNF-α level and a significant increase (P<0.001) in the IL-10 level in the media of unstimulated and stimulated leukocytes of the patients with periodontitis in comparison with those of the healthy subjects. CONCLUSIONS. The present in vitro study showed that (1→3),(1→6)-ß-glucan modulated the response of leukocytes of the patients with periodontitis differently in comparison with those of the healthy subjects. It increased the release of IL-10, which is protective of the tooth-supporting tissues in patients with periodontal disease, but decreased the release of TNF-α, which is mainly responsible for the destruction of the tooth-supporting tissues during periodontal disease.

Combined Fc-protein- and Fc-glyco-engineering of scFv-Fc fusion proteins synergistically enhances CD16a binding but does not further enhance NK-cell mediated ADCC.

Protein- or glyco-engineering of antibody molecules can be used to enhance Fc-mediated effector functions. ScFv-Fc fusion proteins (scFv-Fc) represent interesting antibody derivatives due to their relatively simple design and increased tissue penetration. Here, the impact of protein- and glyco-engineering on ADCC potency of a panel of human IgG1-based scFv-Fc was tested. Three matched sets of scFv-Fc variants targeting CD7, CD20 or HLA class II and optimized for CD16a binding by mutagenesis, lack of core-fucose, or their combination, were generated and functionally tested in comparison to the corresponding wild type scFv-Fc. Antigen binding activity was not compromised by altered glycosylation or Fc mutagenesis, whereas Fc binding to CD16a was significantly enhanced in the order: non-core fucosylated/Fc-mutated double-engineered≫Fc-mutated≥non-core-fucosylated>wild-type IgG1-Fc. All engineered variants triggered potent ADCC with up to 100-fold reduced EC50 values compared to non-engineered variants. Interestingly, double-engineered variants were similarly effective in triggering ADCC compared to single-engineered variants irrespective of their 1 log greater CD16a binding affinity. Thus, these data demonstrate that protein- and glyco-engineering enhances NK-cell mediated ADCC of scFv-Fc similarly and show that enhancing CD16a affinity beyond a certain threshold does not result in a further increase of NK-cell mediated ADCC.

Potentiating effect of beta-glucans on photodynamic therapy of implanted cancer cells in mice.

Photodynamic therapy (PDT) combines a drug or photosensitizer with a specific type of light to kill cancer cells. The cellular damage induced by PDT leads to activation of the DNA damage repair, which is an important factor for modulating tumor sensitivity to this treatment. beta-Glucans are natural polysaccharides that bind complement receptor 3 on the effector cells, thereby activating them to kill tumor cells during PDT. The hypothesis of the present study was that adjuvant therapy with beta-glucans would increase the efficacy of PDT. C57BL/6 female mice were subcutaneously implanted with Lewis lung carcinoma cells. Ten days after implantation, the mice were administered intravenously sodium porfimer (10 mg/kg) 24 h prior to laser irradiation, with or without oral administration of beta-glucan (400 microg/d/mouse, 5 days) from either barley, baker's yeast, or marine brown algae that contains the storage glucan, laminarin. Tumor volume and necrotic area in excised tumors were measured. The expression of proliferating cell nuclear antigen (PCNA) was determined as an indicator of the activity of the DNA damage repair system. PDT in combination with each beta-glucan significantly reduced tumor growth (P < 0.05, n = 10) and expression of PCNA (P < 0.001, n = 9), and increased necrosis in tumor tissues (P < 0.001, n = 9). Furthermore, each structurally different

Treatment of Lewis lung carcinoma by photodynamic therapy and glucan from barley.

OBJECTIVE: During the photodynamic treatment, complement system is activated and tumor cells are opsonized with iC3b fragment. beta-glucans can enhance cytotoxicity of iC3b-opsonized cells due to their specific interaction with complement receptor 3 (CR3; CD11b/CD18) on the surface of the effector cells. In contrast to microorganisms, tumor cells lack beta-glucan as a surface component and cannot trigger complement receptor 3-dependent cellular cytotoxicity and initiate tumor-killing activity. This mechanism could be induced in the presence of beta-glucans. This study aimed at determining the influence of coadministration of beta-glucan from barley on the efficacy of photodynamic tumor therapy (PDT). MATERIAL AND METHODS: C57 Bl/6 female mice bearing Lewis lung carcinoma were used throughout the study. Mice were randomized into groups (15 in each group) and exposed to the treatment with intravenous Photofrin injection (dose, 10 mg/kg) and after 24 h following laser illumination, or with oral administration of beta-glucan from barley at a dose of 400 microg/mouse per day up to 5 days, or with their combination. Tumor growth dynamics and survival of the treated and untreated mice were monitored. RESULTS: Tumor volume in all treated groups was significantly lower (P<0.001) than that in the control group. The most effective tumor growth suppression (P=0.033) was achieved in mice treated with combination of PDT and beta-glucan from barley as compared with PDT alone. The best survival was achieved in the same group, but difference was not significant as compared to the control group (P=0.143) and to PDT alone group (P=0.319). CONCLUSIONS: The present study demonstrates that coadministration of beta-glucan from barley can enhance efficacy of photodynamic therapy.

Effects of beta-glucans on the immune system.

Beta-glucans are naturally occurring polysaccharides. These glucose polymers are constituents of the cell wall of certain pathogenic bacteria and fungi. The healing and immunostimulating properties of mushrooms have been known for thousands of years in the Eastern countries. These mushrooms contain biologically active polysaccharides that mostly belong to group of beta-glucans. These substances increase host immune defense by activating complement system, enhancing macrophages and natural killer cell function. The induction of cellular responses by mushroom and other beta-glucans is likely to involve their specific interaction with several cell surface receptors, as complement receptor 3 (CR3; CD11b/CD18), lactosylceramide, selected scavenger receptors, and dectin-1 (betaGR). beta-Glucans also show anticarcinogenic activity. They can prevent oncogenesis due to the protective effect against potent genotoxic carcinogens. As immunostimulating agent, which acts through the activation of macrophages and NK cell cytotoxicity, beta-glucan can inhibit tumor growth in promotion stage too. Anti-angiogenesis can be one of the pathways through which beta-glucans can reduce tumor proliferation, prevent tumor metastasis. beta-Glucan as adjuvant to cancer chemotherapy and radiotherapy demonstrated the positive role in the restoration of hematopiesis following by bone marrow injury. Immunotherapy using monoclonal antibodies is a novel strategy of cancer treatment. These antibodies activate complement system and opsonize tumor cells with iC3b fragment. In contrast to microorganisms, tumor cells, as well as other host cells, lack beta-glucan as a surface component and cannot trigger complement receptor 3-dependent cellular cytotoxicity and initiate tumor-killing activity. This mechanism could be induced in the presence of beta-glucans.