In Vitro Analysis of Nanoparticle Effects on the Zymosan Uptake by Phagocytic Cells.

This chapter provides a protocol for analysis of nanoparticle effects on the function of phagocytic cells. The protocol relies on luminol chemiluminescence to detect zymosan uptake. Zymosan is an yeast particle which is typically eliminated by phagocytic cells via the complement receptor pathway. The luminol, co-internalized with zymosan, is processed inside the phagosome to generate a chemiluminescent signal. If a test nanoparticle affects the phagocytic function of the cell, the amount of phagocytosed zymosan and, proportionally, the level of generated chemiluminescent signal change. Comparing the zymosan uptake of untreated cells with that of cells exposed to a nanoparticle provides information about the nanoparticle's effects on the normal phagocytic function. This method has been described previously and is presented herein with several changes. The revised method includes details about nanoparticle concentration selection, updated experimental procedure, and examples of the method performance.

Chemiluminescence of non-differentiated THP-1 promonocytes: developing an assay for screening anti-inflammatory milk proteins and peptides.

Human leukemic THP-1 promonocytes are widely used as a model for peripheral blood monocytes. However, superoxide production during respiratory burst (RB) of non-differentiated THP-1 (nd-THP-1) cells is very low. Here we present a rapid and low-cost method for measuring the chemiluminescence (CL) of opsonized zymosan (OZ) induced RB which allows detection of Escherichia coli lipopolysaccharide (LPS) induced priming of nd-THP-1 cells on the basis of CL reaction kinetics. Maximum CL intensity obtained was 2.20 ± 0.25 and 1.30 ± 0.11 relative light units, while CL peak time was achieved at 18.1 ± 2.6 and 28.7 ± 1.3 min in primed and non-primed cells, respectively. The priming of nd-THP-1 cells with LPS evoked typical TNF-α and IL-6 production. We tested the effects of bovine lactoferrin and protein fractions from Lactobacillus helveticus BGRA43 fermented milk for potential anti-inflammatory effects on LPS primed nd-THP-1 cells. Four fractions were found to inhibit the OZ-induced CL in a dose-dependent manner (IC(50) 3-30 µg/mL), while lactoferrin inhibited CL to a lesser extent (IC(50) 270 µg/mL). These results suggest that measuring CL response of nd-THP-1 cells can serve as a method for screening anti-inflammatory compounds which could be used in reducing the risk of phagocyte-mediated inflammatory diseases.

Activation of toll-like receptor-mediated NF-kappa beta by zymosan-derived water-soluble fraction: possible contribution of endotoxin-like substances.

Zymosan is a well-known reagent for the examination of inflammatory response and is prepared from yeast, Saccharomyces cerevisiae. In the activation process, Toll-like receptor (TLR) 2 and TLR6 act as functional receptors for NF-kappaB activation. Although zymosan is primarily composed of beta-glucans, little is known about the active component of zymosan-mediated biological activities. The active moiety of zymosan was fractionated by its solubility in water, and its biological activity on macrophages and TLRs-transfectants examined. The macrophage cell line, RAW264.7, was treated with zymosan-derived preparations, and tumor necrosis factor alpha (TNF-alpha) produced in the culture supernatant was measured by ELISA. Increased TNF-alpha production was observed by stimulation with water-soluble (ZWS) or water-insoluble fraction (ZWIS). ZWS showed higher activity in TNF-alpha production. NF-kappaB activation via TLR2, TLR1/ TLR2, TLR2/TLR6, and TLR4/MD-2/CD14 also was enhanced by stimulation with ZWS and ZWIS. In particular, ZWS showed higher activity via TLR1/TLR2, TLR2/TLR6, and TLR4/MD-2/CD14 than other preparations. ZWS activity was decreased by treatment with polymyxin B, but not with lysozyme and zymolyase. Furthermore, ZWS contained significant more endotoxin than any other preparations. Therefore, we suggest that the active moiety of ZWS for the NF-kappaB activation has an endotoxin-like substance, that is abundantly observed in Gram-negative bacteria. These results imply that the inflammatory activity of zymosan is induced not only by beta-glucans, but also by other endotoxin-like water-soluble substances.

Functional comparison of the mouse DC-SIGN, SIGNR1, SIGNR3 and Langerin, C-type lectins.

The mouse (m) DC-SIGN family consists of several homologous type II transmembrane proteins located in close proximity on chromosome 8 and having a single carboxyl terminal carbohydrate recognition domain. We first used transfected non-macrophage cell lines to compare the polysaccharide and microbial uptake capacities of three of these lectins--DC-SIGN, SIGNR1 and SIGNR3--to another homologue mLangerin. Each molecule shares a potential mannose-recognition EPN-motif in its carbohydrate recognition domain. Using an anti-Tag antibody to follow Tag-labeled transfectants, we found that each molecule could be internalized, although the rates differed. However, mDC-SIGN was unable to take up FITC-dextran, FITC-ovalbumin, zymosan or heat-killed Candida albicans. The other three lectins showed distinct carbohydrate recognition properties, assessed by blocking FITC-dextran uptake at 37 degrees C and by mannan binding activity at 4 degrees C. Furthermore, only SIGNR1 was efficient in mediating the capture by transfected cells of Gram-negative bacteria, such as Escherichia coli and Salmonella typhimurium, while none of the lectins tested were competent to capture Gram-positive bacteria, Staphylococcus aureus. Interestingly, transfectants with SIGNR1 lacking the cytoplasmic domain were capable of binding FITC-zymosan in a manner that was abolished by EDTA or mannan, but not laminarin. In addition, resident peritoneal CD11b+ cells expressing SIGNR1 bound zymosan at 4 degrees C in concert with a laminarin-sensitive receptor. Therefore these homologous C-type lectins have distinct recognition patters for microbes despite similarities in the carbohydrate recognition domains.

[The phagocytosis by polymorphonuclear neutrophils in patients with systemic lupus erythematosus]. / Fagocitose por neutrófilos no Lúpus Eritematoso Sistêmico.

OBJECTIVE: To evaluate the presence of immune complexes and the phagocytes by polymorphonuclear neutrophils in patients with systemic lupus erythematosus, with and without disease activity. METHODS: The peripheral blood of 55 subjects was analyzed. Ten of those subjects had disease activity, 15 had not disease activity, and 30 were healthy. We used radial immune diffusion to detect immune complexes. The phagocytic function was estimated by the ingestion of zymosan by polymorphonuclear neutrophils. RESULTS: In this study we found the presence of immune complexes formatted of IgM, IgG, IgA, and complement component C3 and C4 in LES patients. The arithmetic average of zymosan particles ingested by the neutrophils incubated with homologous human serum and autologous human serum was significantly decreased (p<0.05) in the LES activity patients when we compare with the group without activity, and the control group. CONCLUSION: We conclude that there are immune complexes in the LES patients with and without disease activity, and there is a reduction in the digestive step of the phagocytes by polymorphonuclear neutrophils in patients with disease activity. The conclusions of the present study are according with the pathogenesis of the disease and with the high mortality in these patients.

Saccharomyces cerevisiae- and Candida albicans-derived mannan induced production of tumor necrosis factor alpha by human monocytes in a CD14- and Toll-like receptor 4-dependent manner.

The cytokine-inducing activities of fungal polysaccharides were examined in human monocytes in culture, with special reference to CD14 and Toll-like receptors (TLRs). Tumor necrosis factor alpha (TNF-alpha) production by monocytes was markedly induced in a dose-dependent manner upon stimulation with cell walls from Candida albicans and mannan from Saccharomyces cerevisiae and C. albicans, although relatively high concentrations (10 to 100 microg/ml) of stimulants were required for activation as compared with the reference lipopolysaccharide (LPS) (1 to 10 ng/ml). The yeast form C. albicans and its mannan and cell wall fractions exhibited higher TNF-alpha production than respective preparations from the hyphal form. Only slight TNF-alpha production was induced by the S. cerevisiae glucan. The TNF-alpha production triggered by reference LPS and purified fungal mannans required the presence of LPS-binding protein (LBP), and these responses were inhibited by anti-CD14 and anti-TLR4 antibodies, but not by anti-TLR2 antibody. In contrast to the activity of LPS, the activity of purified S. cerevisiae mannan was not inhibited by polymyxin B. These findings suggested that the mannan-LBP complex is recognized by CD14 on monocytes and that signaling through TLR4 leads to the production of proinflammatory cytokines in a manner similar to that induced by LPS.

[Interaction between immunomodulatory drugs in mice with special regard to changes of drug sensitivity and rate of bacterial translocation]. / Immunmoduláns anyagok kölcsönhatása egérkísérletekben különös tekintettel gyógyszerérzékenység változásra és a bakteriális transzlokáció alakulására.

In acute toxicity experiments the changes in drug sensitivity and in the rate of bacterial translocation (BT) were investigated in mice treated with immunomodulatory drugs: dianhydrogalactitol (DAG) in doses 20 and 30 mg/kg, chlorpromazine (CPZ) in doses 60 and 75 mg/kg and Mannozym (M) in dose 40 mg/kg for zymosan content. The drugs were used separately or in combination. The sensitivity of mice to immunosuppressive DAG or CPZ was higher in the case of combined treatment, than that of separately treated ones. The rate of BT was also higher in combined treated mice. The pretreatment with M that has immunostimulatory effect, influenced neither the sensitivity of mice to DAG or CPZ, nor the normal very low rate of BT. The present results reinforced the authors' earlier observations, that the effects of immunosuppressive drugs could cumulate and cause more serious damage of the organism. The authors suggest that the increase in drug sensitivity to immunosuppressive agents is in connection with increased rate of BT and effect of endotoxin.

Hypokalemic nephropathy in the rat. Role of ammonia in chronic tubular injury.

Chronic potassium deficiency results in progressive tubulointerstitial injury, associated with augmented renal ammoniagenesis. We investigated the role of elevated renal ammonia levels and the interaction of ammonia with the complement system in this injury. Potassium deficiency was induced in rats by feeding a low potassium diet. Experimental animals received 150 mM NaHCO3 or equimolar NaCl, as drinking water. After 3 wk, NaHCO3 supplemented rats demonstrated decreased ammonia production, less renal hypertrophy, less histologic evidence of injury, and less proteinuria. In in vitro studies on normal cortical tubular fragments, the addition of ammonia to serum in concentrations comparable to renal cortical levels in potassium-deficient animals significantly increased tubular deposition of C3 as quantitated by a radiolabeled antibody binding technique. Thus, alkali supplementation reduced chronic tubulointerstitial disease in a rat model of hypokalemic nephropathy. We propose that increased cortical ammonia levels contribute to hypokalemic nephropathy through ammonia-mediated activation of the alternative complement pathway.

Evaluation of elastase and alpha 1-proteinase inhibitor-elastase uptake by polymorphonuclear leukocytes and evidence of an elastase-specific receptor.

Neither resting nor stimulated isolated human polymorphonuclear leukocytes did bind or ingest preformed complexes of alpha 1-proteinase inhibitor and unlabeled/125I-labeled human leukocyte elastase. In contrast, granulocytes bound unlabeled/125I-labeled elastase and the extent of binding was reduced in the presence of respiratory burst stimulators, such as 4 beta-phorbol 12 beta-myristate 13 alpha-acetate, E. coli endotoxin, and N-formyl-L-methionyl-L-leucyl-L-phenylalanine. In association/dissociation and competition inhibition experiments it was demonstrated that granulocyte-elastase binding was specific and saturable. From Scatchard and non-linear regression analysis there was evidence of a two-class receptor model with independent binding sites. Calculated by the non-linear regression method assuming a two-class receptor model the characteristics of the high affinity/low capacity binding site were K1 = 216 +/- 129 X 10(6) l X mol-1 (means +/- s; n = 3) and R1 = 1.38 +/- 0.95 nmol X l-1 corresponding to 0.083 X 10(6) receptors per cell, whereas the low affinity/high capacity binding site had the characteristics K2 = 0.50 +/- 0.09 X 10(6) l X mol-1 and R2 = 237 +/- 103 nmol X l-1 corresponding to 14.3 +/- 6.2 X 10(6) receptors per cell.

[The chemical composition of "zymosans" from yeasts]. / Uber die chemische Zusammensetzung von "Zymosanen" aus Bäckerhefe.

The chemical composition of "Zymosanes" (complex cell-wall-polysaccharides), prepared by different methods from Baker's yeast, were analyzed by classical and modern techniques, e.g. GLC and TLC. They are very complex natural products consisting of a basic structure of polysaccharides, which proteins and different lipids are bound to. Higher purified zymosanes seem to be free from nucleic acids and do not contain sialic acid.

Preparation of zymosan from yeast cell walls.

Cell walls of the yeast Saccharomyces cerevisiae after disintegration and protoplasm removal by centrifugation and repeated washing were suspended in 0.5 M Na2HPO4, pH 7.8-8.0, as a 5% or 10% suspension, depending on the mode of heating. The suspension was boiled for 3 h, purified by repeated washing with water and ethanol and dried. The yield was approximately 1.8% of the starting amount of pressed commercial baker's yeast.

Localization of structural polymers in the cell wall of Neurospora crassa.

The distribution and localization of structural polymers in the cell wall of Neurospora crassa has been studied by selective removal and light and electron microscope examination. Observations with the light microscope indicated that each polymer by itself can provide structural integrity to the cell wall. Examination by electron microscopy showed that the cell wall consists of an outer layer of thick fibrils, identified chemically as a glucan-peptide-galactosamine complex, and an inner layer made up of beta-1,3 glucan and thin fibrils of chitin.