Janos Fischer

Characterization of glycoconjugates of human gastrointestinal mucosa by lectins. I. Histochemical distribution of lectin binding sites in normal alimentary tract as well as in benign and malignant gastric neoplasms

Labeled lectins with binding specificity to the hexose components of mucus glycoproteins (HPA, RCA I, PNA, Con A, WGA, and UEA I) were used to demonstrate structural differences in the glycoprotein composition of various cell types of the normal, benign and malignant gastrointestinal mucosa. While in the RCA I, UEA I, and WGA binding of normal mucus secreting cell types only quantitative differences were observed, the mucus in the surface epithelial cells of gastric mucosa and in the colonic goblet cells was characterized by the absence of PNA, Con A, and PNA, HPA binding sites, respectively. These lectins, however, showed a strong binding to the supranuclear, Golgi-region in the undifferentiated or activated forms of these cells. Even the staining intensity of the luminal membrane surfaces of the non mucinous parietal and chief cells was often stronger by PNA, HPA, and RCA I lectins than that of the mucus secretions in the highly differentiated mucus cells. These results indicate the existence of either heterogeneous glycoprotein components or mucus molecules with variations in the degree of glycosylation of their oligosaccharide chains in the different cells. The latter seems more likely since in benign and malignant alterations lectin binding sites appear in great density, which were found to be characteristic of the undifferentiated mucus cells or for the non mucinous cells of the normal gastric mucosa. Similarly in some gastric cancers which do not stain with the periodic acid-Schiff reaction at all, large amount of free or neuraminic acid substituted PNA binding sites can be detected.

Effect of a Recombinant Lectin, Viscum album Agglutinin on the Secretion of Interleukin-12 in Cultured Human Peripheral Blood Mononuclear Cells and on NK-Cell-Mediated Cytotoxicity of Rat Splenocytes in vitro and in vivo

A plant lectin, Viscum album agglutinin-I (VAA-I) has been shown to increase the number and cytotoxic activity of natural killer (NK) cells in animal models, but the mechanisms underlying these effects are poorly understood. We investigated the effects of the recombinant form of this lectin (rVAA) on secretion of interleukin (IL)-12 and on NK-mediated cytotoxicity against K562 target cells in cultures of human peripheral blood mononuclear cells (PBMC) as well as against YAC-1 target cells in cultured rat spleen cells. In 24-hour cultures of PBMC, 10 ng/ml plant VAA-I and 50 ng/ml rVAA induced significant increases in the secretion of total IL-12. Its biologically active heterodimeric form, p70, was also significantly induced by rVAA. Preincubation of PBMC or splenocytes for 48 h with rVAA in concentrations ranging between 10 pg/ml and 100 pg/ml resulted in moderate enhancements of NK-mediated cytotoxicity. However, coincubation of a low dose of rVAA (100 pg/ml) together with IL-2 and IL-12 (60 U/ml and 2 U/ml, respectively) led to additive stimulation of NK activity. In in vivo experiments, rVAA showed an enhancing effect on NK activity with a bell-shaped curve of efficacy. Forty-eight hours after a single intravenous injection of its most effective doses, 0.5 and 1 ng/kg, into Wistar rats, the NK cytotoxicity of splenocytes against YAC-1 targets doubled, and the frequency of large granular lymphocytes in peripheral blood showed 2.1- and 3-fold increases as compared to control animals. Twenty-four hours following these low lectin doses, the number of large granular lymphocytes was also significantly elevated. After 48 h, 0.5 ng/kg rVAA induced a significant augmentation in the percentage of peripheral Mac-1+ mononuclear cells, including activated monocytes and NK cells. The present results suggest that rVAA augments the secretion of an active form of IL-12 and potentiates the cytokine-induced NK activation. These effects of rVAA may be related to its stimulatory effects on MHC-unrestricted cytotoxicity in vivo.

Immunomodulatory effects of Viscum album agglutinin-I on natural immunity.

In 24 h cultured human peripheral blood mononuclear cells, treated with various (1 µg/ml to 1 ng/ml) concentrations of Viscum album agglutinin-l, quantitative assessment of DNA breaks labelled with terminal deoxynucleotidyl transferase revealed a dose-dependent Viscum album agglutinin-l-induced apoptosis above a lectin concentration of 10 ng/ml. After 24 h incubation of peripheral blood mononuclear cells with non-cytotoxic concentrations of Viscum albumagglutinin- l (10 and 1 ng/ml), messenger (m)RNA expression and secretion of a panel of cytokines were evaluated by reverse polymerase chain reaction and by enzyme-linked immunosorbent assay (ELISA), respectively. The lectin induced expression of interleukin-1α interleukin-1 β, interleukin-6, tumour necrosis factor-α, interferon-γ, granulocyte-monocyte colony stimulating factor and interleukin-10 genes, but no expression of interleukin-2 or interferon-y production could be detected. In addition, cellular components of the natural immune system (such as monocytes and granulocytes) bound Viscum album agglutinin-l molecules to a higher degree than lymphocytes.To establish the modulatory potency of Viscum album agglutinin-l on the natural immunity of human subjects, four randomized, double-blind crossover trials were performed on healthy volunteers. In contrast to the significant lectin-induced increases in number and activity of natural killer cells observed in animal models, in the first and second trial human healthy individuals showed no significant differences between their natural killer responses following an injection of lectin-enriched preparation or saline. Due to considerable intrinsic fluctuation of these parameters, a third and fourth double-blind trial with freshly isolated Viscum album agglutinin-l was performed using a more rapidly detectable parameter, the priming of granulocytes. Here, significant lectin-induced increases were found.

Hit-to-lead optimization of pyrrolo[1,2-a]quinoxalines as novel cannabinoid type 1 receptor antagonists.

Hit-to-lead optimization of a novel series of N-alkyl-N-[2-oxo-2-(4-aryl-4H-pyrrolo[1,2-a]quinoxaline-5-yl)-ethyl]-carboxylic acid amides, derived from a high throughput screening (HTS) hit, are described. Subsequent optimization led to identification of in vitro potent cannabinoid 1 receptor (CB1R) antagonists representing a new class of compounds in this area.

Chemical and Biological Investigation of Cyclopropyl Containing Diaryl-pyrazole-3-carboxamides as Novel and Potent Cannabinoid Type 1 Receptor Antagonists

Obesity is a major clinical problem in the western world, and many molecular targets have been explored in the search for effective therapeutic agents. One of these, antagonism of the cannabinoid 1 (CB1) receptor, rose to prominence following reports demonstrating the positive modulation of food intake by the CB1 antagonist, rimonabant (3) (SR141716A). In the present study, various diaryl-pyrazole derivatives containing cycloalkyl building blocks were synthesized and tested for CB1 receptor binding affinities. Thorough structure-activity relationship (SAR) studies to optimize the pyrazole substituents led to several novel CB1 antagonists with K(i) <or= 5 nM and with acceptable metabolic stability with human liver microsomes. Among these analogues, we identified 5-(4-cyclopropylphenyl)-1-(2,4-dichlorophenyl)-4-ethyl-N-pyrrolidin-1-yl-1H-pyrazole-3-carboxamide (11r), which exhibited a favorable pharmacological profile with outstanding efficacy in reducing serum lipid parameters of metabolic syndrome compared to clinical references.

Characterization of glycoconjugates of human gastrointestinal mucosa by lectins. II. Lectin binding to the isolated glycoproteins of normal and malignant gastric mucosa.

Using affinity chromatography on HPA-, PNA-, Con A, and WGA-agarose columns only a part (10-30%) of the high molecular weight mucous glycoproteins could be isolated from the Triton X-100 solubilized components of normal as well as carcinomatous gastric mucosa. The main part of the mucus was not bound by the lectins, which corresponds to our earlier lectin histochemical observations on paraffin-embedded tissue sections. The lectin-bound mucous glycoproteins had a relatively lower molecular weight, ranging from about 250-1,000 kilodaltons, as indicated by polyacrylamide gradient gel electrophoresis and by gel filtration on Biogel A 1.5 m column. In gas chromatographic analysis the molar ratio of aminohexoses to galactose was found to be much higher (3:1) in the lectin-bound mucous substances than in the whole high molecular weight mucus (1:1). This finding indicates that lectins have a higher affinity to the hexosamine rich components of mucus, which may be special forms of mucous glycoprotein molecules or the incompletely glycosylated core and backbone regions of the oligosaccharide chains of mucus. Extremely high hexosamine values (10:1) were found in the PNA isolated mucus of gastric adenocarcinoma. Since it is known that PNA binds to the terminal disaccharide, beta-galactose-(1-3)-N-acetylgalactosamine, which is localized at the reducing end of the oligosaccharide chains of mucus, it is highly probable that the elongation of the oligosaccharide side chains is disturbed in gastric cancer cells.

Pharmacological comparison of traditional and non-traditional cannabinoid receptor 1 blockers in rodent models in vivo

Abstract Cannabinoid receptor 1 (CB1R) antagonists have been proven to be effective anti‐obesity drugs; however, psychiatric side effects have halted their pharmaceutical development worldwide. Despite the emergence of next generation CB1R blockers, a preclinical head to head comparison of the anti‐obesity and psychiatric side effect profiles of the key compounds has not been performed. Here, we compared classical CB1R antagonists (rimonabant, taranabant, otenabant, ibipinabant, and surinabant) and non‐traditional CB1R blockers (the partial agonist O‐1269, the neutral antagonists VCHSR and LH‐21 and the peripherally acting inverse agonist JD‐5037) using an in vivo screening cascade. First, the potencies of these compounds to reduce CB1R agonist‐induced hypothermia and decrease fasting‐induced food intake were determined. Then, equipotent doses of the non‐toxic compounds were compared in a diet‐induced obesity (DIO) test, which includes measurements of metabolic syndrome markers. Psychiatric side effects were assessed by measuring anxiogenicity in an ultrasonic vocalization test. All classical CB1R blockers were centrally acting appetite suppressants and decreased body weight and food intake in an obesity‐dependent manner, with only slight effects on metabolic syndrome markers. In addition, all classical CB1R blockers increased ultrasonic vocalization. Surprisingly, none of the non‐classical CB1R blockers was eligible for the DIO comparison and side effect profiling. O‐1269 and LH‐21 induced convulsive behavior, whereas VCHSR and JD‐5037 were devoid of any in vivo activity. The classical CB1R blockers displayed similar therapeutic and side effect profiles in vivo, whereas the available non‐traditional CB1R blockers were not appropriate tools for testing the therapeutic potential of alternative CB1R inhibitors. HighlightsNine cannabinoid receptor 1 blockers were profiled in preclinical in vivo tests.The non‐traditional blockers VCHSR and JD 5037 were inactive, and LH‐21 and O‐1269 were toxic.Classical CB1 antagonists decreased appetite and body weight.Rimonabant, surinabant, ibipinabant and taranabant displayed anxiogenic effects.