Anna Fišerová

Effects of D2-dopamine and α-adrenoceptor antagonists in stress induced changes on immune responsiveness of mice

The involvement of catecholamine receptors (alpha-adrenergic, D2-dopamine (DA)) was investigated in restraint stress influenced immune responses with concomitant changes of G-protein signal transduction. Impairment of the spleen morphology, TH1/TH2 cytokine network and natural killer (NK) cell function was observed. In vivo administration of specific antagonists prior to restraint stress reversed the immunosuppression. These findings demonstrate that D2-type dopaminergic mechanism represents the dominant component in regulation of Galphas/Galphai(1,2)/Galphaq/11-protein signal transduction and contribute to cell responses at postreceptor level of both, central nervous and immune systems. G-protein-coupled receptors (GPCRs) can modulate cytokine production and may play a regulatory role in immune effector mechanisms.

Glycodendrimeric ligands of C-type lectin receptors as therapeutic agents in experimental cancer

The increased knowledge about the receptor-ligand relationships of the cytotoxic effector cells is suggesting modalities for new systems of immunomodulation. The lectin receptors on natural killer cells (NK) and their recognition of carbohydrate ligands are part of these new perspectives.

Association of human NK cell surface receptors NKR-P1 and CD94 with Src-family protein kinases

Abstract Human natural killer (NK) cells express on their surface several members of the C-type lectin family such as NKR-P1, CD94, and NKG2 that are probably involved in recognition of target cells and delivery of signals modulating NK cell cytotoxicity. To elucidate the mechanisms involved in signaling via these receptors, we solubilized in vitro cultured human NK cells by a mild detergent, Brij-58, immunoprecipitated molecular complexes containing the NKR-P1 or CD94 molecules, respectively, by specific monoclonal antibodies, and performed in vitro kinase assays on the immunoprecipitates. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, autoradiography, and phospho-amino acid analysis revealed the presence of in vitro tyrosine phosphorylated proteins that were subsequently identified by re-precipitation (and/or by western blotting) as the respective C-type lectin molecules and Src family kinases Lck, Lyn, and Fyn. The NKR-P1 and the CD94-containing complexes were independent of each other and both very large, as judged by Sepharose 4B gel chromatography. Crosslinking of NKR-P1 on the cell surface induced transient in vivo tyrosine phosphorylation of cellular protein substrates. These results indicate involvement of the associated Src-family kinases in signaling via the NKR-P1 and CD94 receptors.

N-Acetyl-D-glucosamine-coated polyamidoamine dendrimer modulates antibody formation via natural killer cell activation

N-Acetyl-D-glucosamine-coated polyamidoamine dendrimer (GlcNAc8) was shown previously to exhibit binding affinity to the rat recombinant NKR-P1 molecule (known in mice also as NK1.1) and to induce NK cell-mediated cytotoxicity. In this study, we investigated whether GlcNAc8 modulates antibody formation as activated NK cells were reported to participate in its regulation. C57BL/6 mice treated with GlcNAc8 and intact controls were immunized either with sheep red blood cells (SRBCs), 2,4-dinitrophenylated-lipopolysaccharide (DNP-LPS) or keyhole limpet hemocyanin (KLH) for evaluation of splenic antibody forming cell counts and serum immunoglobulin (Ig) levels. In vitro Ig formation was determined using supernatants of spleen mononuclear cells (SMCs) and CD49b or NK1.1-depleted SMC subpopulations. Serum antigen-specific IgG2a levels were also measured in DBA/2 and BALB/c mice (NK1.1-negative mouse strains on the basis of flow cytometric analysis) which possess different Nkr-p1c gene form than C57BL/6 ones. A significant increase in anti-SRBC IgG forming cells, serum levels of anti-KLH as well as anti-DNP IgG and IgG2a was observed after GlcNAc8 administration in C57BL/6 mice. IgM levels in supernatants of SMCs stimulated in vitro simultaneously with DNP-LPS and GlcNAc8 were significantly mounted compared with supernatants of SMCs primed with the antigen alone, but this enhancement was blocked after depletion of CD49b-positive or NK1.1-positive cells. In DBA/2 and BALB/c mice, GlcNAc8 influenced neither serum levels of anti-KLH nor anti-DNP IgG2a. These results indicate that GlcNAc8-induced upregulation of antibody formation is triggered by NK cell stimulation and depends on expressed NKR-P1 isoforms, particularly NKR-P1C.

Clustered ergot alkaloids modulate cell-mediated cytotoxicity

Dimers of agroclavine (1) and terguride (2), as well as a series of terguride oligomers, for example trimers (5, 6), tetramer (7), hexamer (8) and functionalized tergurides for further complex clustering were synthesized. Terguride oligomers were screened for their direct cellular toxicity on lymphoma cell lines in vitro and for their immunomodulating activities, represented by the natural killer (NK) cell-mediated cytotoxicity, as the most sensitive screening marker during immune responses. Dimers linked via aromatic spacer showed a high toxicity (1 microM) to lymphoma cells, which was not detected in other derivatives. In vitro and ex vivo experiments performed on mouse spleen lymphocytes in the presence of terguride oligomers demonstrated an immunosuppressive effect of dimers with aromatic spacer (4c-d) and NK cell stimulatory effect of terguride hexamer (8) and trimer with aliphatic spacer (5c). There is a considerable evidence that indolic part of molecule contributes to immunosuppressive action of terguride, which is potentiated in dimers carrying aromatic linker. This effect can be reversed by higher oligomerization of the respective alkaloids.

N-Acetyl-d-glucosamine-coated polyamidoamine dendrimer promotes tumor-specific B cell responses via natural killer cell activation

N-acetyl-D-glucosamine-coated polyamidoamine dendrimer (GN8P), exerting high binding affinity to rodent recombinant NKR-P1A and NKR-P1C activating proteins, was shown previously to delay the development of rat colorectal carcinoma as well as mouse B16F10 melanoma, and to potentiate antigen-specific antibody formation in healthy C57BL/6 mice via NK cell stimulation. In this study, we investigated whether GN8P also modulates tumor-specific B cell responses. Serum anti-B16F10 melanoma IgG levels, IgG2a mRNA expression, antibody dependent cell-mediated cytotoxicity (ADCC), and counts of plasma as well as antigen presenting B cells were evaluated in tumor-bearing C57BL/6 mice treated with GN8P and in respective controls. To reveal the mechanism of GN8P effects, the synthesis of interferon-gamma (IFN-γ) and interleukin-4 (IL-4), cytokines involved in regulation of immunoglobulin class switch, was determined. The GN8P treatment significantly elevated IgG, and particularly IgG2a, response against B16F10 melanoma, which led to augmented ADCC reaction. The significant increase in production of IFN-γ, which is known to support IgG2a secretion, was observed solely in NK1.1 expressing cell populations, predominantly in NK cells. Moreover, GN8P raised the number of plasma cells, and promoted antigen presenting capacity of I-A/I-E-positive B lymphocytes by up-regulation of their CD80 and CD86 co-stimulatory molecule expression. These results indicate that GN8P-induced enhancement of tumor-specific antibody formation is triggered by NK cell activation, and contributes to complexity of anticancer immune response involving lectin-saccharide interaction.

Glycosylation regulates NK cell-mediated effector function through PI3K pathway.

Aberrant glycosylation, which impairs recognition capability of NK cells or modifies recognition pattern of target cells, is associated with cancer. Synthetic glycoconjugates (GCs), which modulate cell glycosylation, increase the sensitivity of tumor cells to therapy or boost anti-cancer immune response. In the current study, we employed N-acetyl-D-glucosamine-calix[4]arene (GN4C) as a modulator of cell glycosylation of NK cells represented by the NK-92 cell line and fresh human NK cells. For the first time, we have demonstrated that calix[4]arene-based GC down-regulated the expression of glycosyltransferases MGAT3 and MGAT5 in NK-92 and fresh NK cells. GN4C increased the susceptibility of tumor cells to cytotoxicity by purified fresh NK cells or NK-92 cells. This functional activation of NK cells and the NK-92 cell line correlated with an increased expression of NKG2D mRNA. In the NK-92 cell line, GN4C induced the synthesis of IL-2, IFN-gamma and tumor necrosis factor-alpha as well. Cellular signaling triggered by GN4C engaged PI3-kinase/ERK but not phospholipase C-gamma/JNK pathways. Simultaneously, in transformed NK-92 cells, GN4C reduced the rate of proliferation and down-regulated the c-MYC, EGF-receptor 1 and REL-A molecules. In conclusion, the modulation of glycosyltransferases MGAT3 and MGAT5 by synthetic GN4C correlated with the improvement of NK cell effector functions and the augmentation of tumor cells sensitivity to NK cell-mediated cytotoxicity.

Antitumor activity of N-acetyl-d-glucosamine-substituted glycoconjugates and combined therapy with keyhole limpet hemocyanin in B16F10 mouse melanoma model

N-Acetyl-d-glucosamine-substituted glycoconjugates (GCJs) with the polyamidoamine (GN8P) or calix[4]arene (GN4C) scaffold represent ligands for NKR-P1 molecule and induce NK cell-mediated cytotoxicity in vitro. The in vivo effect of these GCJs on mouse melanoma model was determined when administered either alone or in combination with non-specific immunostimulator keyhole limpet hemocyanin (KLH). All types of treatment significantly reduced the tumor growth on day 23, while GN4C as well as KLH were effective continuously (from day 14). The GN4C also induced the longest mean survival time (46.3 ± 11.1 d), followed by KLH+GN4C (36.4 ± 12.1), KLH (35.6 ± 6.5), KLH+GN8P (35.6 ± 6.7), and GN8P (32.4 ± 7.0), compared to controls (29.8 ± 3.6). The B16F10 specific cytotoxicity of peripheral blood cells was significantly elevated by both KLH and GN8P, whereas not by GN4C. KLH increased the effect of the GN4C, but did not influence that of GN8P. GN4C was proved to exert anticancer activity in mouse melanoma model. The combination of KLH with GCJs did not generate synergism.

Colorectal carcinoma: Importance of colonic environment for anti-cancer response and systemic immunity

The intestinal environment is considered to play an important role both in colorectal tumor development and in the evolution and modulation of mucosal immunity. Studies in animals reared in germ-free (GF, without any intestinal microflora) versus conventional (CV, with regular microflora in bowel) conditions can aid in clarifying the influence of bacteria on carcinogenesis and anti-cancer immune responses in situ. The lower incidence of colon cancers and better immunological parameters in GF animals versus CV ones after chemically-induced carcinogenesis raises questions about specific characteristics of the immunological networks in each respective condition. Different levels of tolerance/regulatory mechanisms in the GF versus CV animals may influence the development of immune responses not only at the level of mucosal, but also at the systemic, immunity. We hypothesize that GF animals can better recognize and respond to evolving neoplasias in the bowel as a consequence of their less-tolerogenic immunity (i.e., due to their more limited exposure to antigens to become tolerated against at the intestinal level). In this paper, we review the role of bacteria in modulating gut environment and mucosal immunity, their importance in cancer development, and aspects of immune regulation (both at local and systemic level) that can be modified by bacterial microflora. Lastly, the use of GF animals in comparison with conventionally-raised animals is proposed as a suitable and potent model for understanding the inflammatory network and its effect on cancer immunity especially during colorectal cancer development.

Ergot alkaloid glycosides with immunomodulatory activities

New glycosides derived from ergot alkaloids elymoclavine and DH-lysergol were synthesized by chemoenzymatic methods. beta-Glucosides were obtained either by chemical method or by transglycosylation (glycosidase from Aspergillus oryzae), lactosides were prepared by further extension of carbohydrate chain using beta-1,4-galactosyltransferase (bovine milk) and alpha-5-N-acetylneuraminyl-(2-->6)-beta-D-galactopyranosyl-(l-->4)-2- acetamido-2-deoxy-beta-D-glucopyranosyl-(1-->O)-elymoclavine was prepared using alpha-2,6-sialyltransferase (rat liver). Immunomodulatory activity of elymoclavine and 9,10-dihydrolysergol and their glycosylated derivatives on natural killer (NK) cell-mediated cytotoxicity of human resting and activated human peripheral blood mononuclear cells (PBMC) was investigated. Addition of ergot alkaloid glycosides to the mixtures of effector and target cells potentiated the PBMC cytotoxicity against both NK-sensitive and -resistant target cells. The glycoconjugates of elymoclavine enhanced cytotoxicity of PBMC against NK-resistant target cells. The glycoconjugates of DH-lysergol potentiated NK cytotoxicity of PBMC against NK-sensitive target cells.