Drying enhances immunoactivity of spent brewer's yeast cell wall ß-D-glucans.

Due to immunological activity, microbial cell wall polysaccharides are defined as 'biological response modifiers' (BRM). Cell walls of spent brewer's yeast also have some BRM activity. However, up to date there is no consensus on the use of spent brewer's yeast D-glucan as specific BRM in humans or animals. The aim of this paper is to demonstrate the potential of spent brewer's yeast ß-D-glucans as BRM, and drying as an efficient pretreatment to increase ß-D-glucan's immunogenic activity. Our results revealed that drying does not change spent brewer's yeast biomass carbohydrate content as well as the chemical structure of purified ß-D-glucan. However, drying increased purified ß-D-glucan TNF-α induction activity in the murine macrophage model. We presume drying pretreatment enhances purity of extracted ß-D-glucan. This is corroborated with FT-IR analyses of the ß-D-glucan spectra. Based on our results, we suggest that dry spent brewer's yeast biomass can be used as a cheap source for high-quality ß-D-glucan extraction. Drying in combination with carboxylmethylation (CM), endows spent brewer's yeast ß-D-glucan with the immunoactivity similar or exceeding that of a well-characterized fungal BRM pleuran.

Diquat-induced cytotoxicity on Vero and HeLa cell lines: effect of melatonin and dihydromelatonin.

Diquat dibromide is a moderately toxic contact herbicide belonging to the bipyridyl group of redox-active compounds that induce a strong oxidative damage. Melatonin (MEL) can protect against oxidative damage under in vivo conditions, probably through its anti-oxidative capacity and ability to induce expression of anti-oxidative enzymes. The objective of this study was to investigate effects of diquat on viability of Vero and HeLa cells and possible protective effects of MEL and its analogue 2,3-dihydromelatonin (DMEL). Cell viability was evaluated with the MTT test. First, we analyzed dose-dependent effects of diquat on cell viability using the concentration range of 0.1-100 µM. Second, we used the diquat dose which reduced cell viability by 50% and treated cells with either MEL or DMEL (both in the concentration range of 1-100 µM) in the presence or absence of diquat. In addition, effects of both diquat and MEL on oxidative stress in HeLa cells were measured by flow cytometry using 2',7'-dichlorofluorescin diacetate. We confirmed the expected negative effects of diquat on viability of Vero and HeLa cells. Melatonin and DMEL were able to prevent diquat reduced viability of Vero cells in rather low concentrations (1 µM) and DMEL exerted substantially stronger protective effects than MEL. However in HeLa cells, we did not find the same effects and MEL even reduced their viability. Moreover, treatment of HeLa cells with high concentrations of MEL (100 µM) exaggerated the pro-oxidative effects of diquat. The results suggest that in addition to the expected anti-oxidative effects, MEL exerts a pro-oxidative action which is cell type and dose dependent.

Vibrio cholerae O1 Ogawa detoxified lipopolysaccharide structures as inducers of cytokines and oxidative species in macrophages.

Multidrug resistance in several strains of Vibrio cholerae has encouraged anti-cholera vaccine developmental attempts using various subcellular moieties. In order to examine the immunological efficacy of detoxified LPS (dLPS)-derived saccharide immunogens, ex vivo activation of mouse peritoneal macrophages (MPhis) was investigated. The immunomodulatory effect was evaluated via induction of the pro-inflammatory cytokines tumour necrosis factor-alpha, interleukin (IL)-1 alpha and IL-6 and acceleration of nitric oxide (NO) and reactive oxygen species (ROS). Immunologically active structures triggered mouse peritoneal MPhis to secrete cytokines and release NO/ROS, even at concentrations as low as 12.5 microg ml(-1). It was found that the O-specific polysaccharide moiety was more immunologically efficient than the glycolipid one, probably due to the position of 3-deoxy-D-manno-octulosonic acid. The results revealed effective structure-immunomodulating relationships of dLPS-derived moieties that are desirable in subcellular anti-cholera vaccine design.

Anti-cytomegalovirus antibodies and other atherosclerosis risk factors in patients with cardiovascular diseases / 老年心脏病学杂志（英文版）

Objective To determine anti-cytomegalovirus (CMV) antibodies along with anti-Chlamydia pneumoniae (CP)antibodies in comparison with inflammatory markers and other risk factors of atherosclerosis in patients with selected cardiovascular diseases(CVD).Methods A total of 228 patients with coronary heart disease (CHD) and/or hypertension (HT), and those who underwent reconstructive vascular surgery (RVS) on carotids or abdominal aorta were tested for the presence of anti-CMV IgG and IgM antibodies as well as for anti-CP IgA antibodies, C-reactive protein (CRP),and interleukin-6 (IL-6). Other risk factors for atherosclerosis, namely age, gender,smoking, hypercholesterolemia, and diabetes mellitus were also analyzed. Results Anti-CMV IgG antibodies were found in 204 patients sera (89.5%),compared with 46 positive of 68 sera in the controls (67.6%), whereas anti-CMV IgM antibodies were detected in 4 of 54 sera of patients tested (7.4%), but not in the controls. The highest proportion of positive sera with not only anti-CMV IgG antibodies (95.6.7%),but also anti-CP IgA antibodies (78.3%), IL-6 (84.8%) and CRP (97.8%), was observed in patients with RVS. The results obtained corresponded to age, hypercholesterolemia, and diabetes. Conclusions The presence of anti-CMV antibodies together with antibodies to CP and markers of inflammation (CRP and IL-6) in our study was associated with CVD, primarily in elderly patients who underwent RVS.

The immunostimulatory effect of the recombinant apalbumin 1-major honeybee royal jelly protein-on TNFalpha release.

Apalbumin1 (Apa1) is the major royal jelly (RJ) and honey glycoprotein having various biological properties. We have previously demonstrated that Apa1 is a regular component of honey and honeybee pollen and stimulates macrophages to release tumor necrosis factor alpha (TNFalpha). The recombinant Apa1 (rApa1) and its four recombinant protein fragments derived on the basis of partial tryptic products of Apa1 were prepared by heterologous expression in Escherichia coli BL21-CodonPlus(DE3)-RIL. L-arginine at 50 mM concentration was used for improving the recombinant protein solubility. We report that the proteinous moiety of glycoprotein is responsible for stimulation of TNFalpha production by murine peritoneal macrophages. Moreover, we have shown that immunostimulatory effect is significantly increased after partial tryptic digestion of Apa1. It has been determined that recombinant N-terminal fragment of Apa1 is the most active elicitor of TNFalpha release in comparison to other three protein fragments of Apa1, as well as to the native Apa1 and rApa1. Furthermore, it was found that native honey was able to stimulate TNFalpha secretion from murine macrophages, whereas the deproteinized honey had no effect on the release of TNFalpha. This result suggests that immunostimulatory effect of honey is based on its RJ-protein content, primarily on its dominant protein Apa1.

Stimulation of TNF-alpha release by fungal cell wall polysaccharides.

Carboxymethylated derivatives were prepared from the (1-->3)-beta-D-glucan isolated from the cell wall of baker's yeast Saccharomyces cerevisiae and from the chitin-glucan complex of the mycelium of the industrial filamentous fungus Aspergillus niger. The polysaccharides were applied to peritoneal mouse macrophages and after a 2-h incubation the release of TNF-alpha by the stimulated macrophages was measured using an enzyme-linked immunosorbent assay. As the third polysaccharide stimulant, a water-soluble derivative of chitin was assayed and the observed cytokine release was compared with the control experiment. In three concentrations of the polysaccharides applied, carboxymethyl glucan revealed a dramatic increase in the TNF-alpha release, while addition of carboxymethyl chitin-glucan resulted only in a moderate enhancement, and carboxymethyl chitin was inactive. The results indicate that fungal polysaccharides, especially (1-->3)-beta-D-glucan, are potent macrophage stimulators and activators of TNF-alpha release, which implies their potential application in antitumor therapy.

Immunochemical approach to detection of adulteration in honey: physiologically active royal jelly protein stimulating TNF-alpha release is a regular component of honey.

The presence of royal jelly (RJ) proteins in honey collected from nectars of different plants, origin, and regions and in honeybee's pollen was detected by Western-blot analysis using polyclonal antibodies raised against water-soluble RJ-proteins. The most abundant RJ-protein in honeybee products corresponded to a 55 kDa protein. The N-terminal amino acid sequence of 55 kDa protein was N-I-L-R-G-E. This sequence is identical to the apalbumin-1, the most abundant protein of RJ. Apalbumin-1 is a regular component of honeybee products and thus is a suitable marker tool for proving adulteration of honey by means of immunochemical detection. Its presence in all tested samples of honeys and honeybee pollen was confirmed also by Western-blot analysis using polyclonal antibodies raised against recombinant apalbumin-1. It has been found that major RJ-proteins, apalbumin-1, and apalbumin-2, stimulate mouse macrophages to release TNF-alpha, which demonstrates that physiologically active proteins of honey could be used for its biological valuation.