Antioxidant and immunomodulatory properties of polysaccharides from Allanblackia floribunda Oliv stem bark and Chromolaena odorata (L.) King and H.E. Robins leaves.

BACKGROUND: Many plant polysaccharides have shown high antioxidant and immunostimulating properties and can be explored as novel molecules with biological properties that can potentially improve immune function. The objective of this work was to characterize soluble and cell wall polysaccharides isolated from the stem bark of Allanblackia floribunda and Chromolaena odorata leaves and to evaluate their antioxidant and immunomodulatory properties. METHODS: Three polysaccharide fractions: soluble polysaccharides (PoS), pectins (Pec) and hemicelluloses (Hem) were extracted from A. floribunda stem bark and C. odorata leaves. These samples were analysed for their proteins, phenolic compounds and total sugar contents. The monosaccharide composition was determined by gas chromatography and arabinogalactan proteins content in PoS was evaluated by rocket electrophoresis. The in vitro antioxidant activities were evaluated by 1, 1-diphenyl-2-picryl hydrazyl (DPPH) and 2,2'-azino-bis-3-éthylbenzylthiazoline-6-sulphonic acid (ABTS) radical scavenging assays and ferrous ions chelating activity. Immunomodulatory activities were performed on the peripheral blood mononuclear cells (PBMCs) using proliferation and enzyme linked immunospot (ELISPOT) method to determine the production of an interferon-gamma. RESULTS: The characterization of the various fractions showed varied metabolites in each plant. In PoS fractions, Ara and Gal were the major monosaccharides found, indicating that arabinogalactans are the primary macromolecules. Hem fractions contained predominantly Xyl and GalA for A. floribunda and Xyl (upto 80 %) for and C. odorata. A. floribunda Hem fraction and C. odorata PoS fraction showed significant DPPH and ABTS radical scavenging activities and immunostimulatory activity via stimulation of PBMC and production of IFN-γ in a dose-dependent manner. CONCLUSION: The results obtained from this study support the ethnomedicinal use of the stem bark of A. floribunda and leaves of C. odorata. Further research is necessary to have supporting evidence that the antioxidative and immunomodulative activities of these fractions are really connected to the polysaccharides and not polyphenols.

Effect of Omega-3 Fatty Acids on Low Density Lipoprotein Subfraction, Adiponectin and Apolipoprotein B in Type 2 Diabetic Patients

BACKGROUND: Omega-3 fatty acids derived from fish oil have been reported to exert a beneficial effect on reducing cardiovascular disease. Reports about their mechanism have generated several interesting findings, including a change in small dense low density lipoprotein (sdLDL) cholesterol proportion, adiponectin, and apolipoprotein B (apoB), in addition to changes in the lipid profile. The principal objective of our study was to evaluate the effects of omega-3 fatty acids on plasma sdLDL, adiponectin, apoB100, and B48 in type 2 diabetic patients with hypertriglyceridemia. METHODS: We randomized 28 type 2 diabetic patients in a placebo-controlled, double-blind trial to receive either omega-3 fatty acids or placebo, both administered at a dose of 4 g daily for 12 weeks. LDL subfractions prior to and after treatment were separated via low-speed ultracentrifugation and analyzed via immunoelectrophoresis. Adiponectin, apoB100, and B48 levels were measured using an ELISA kit. RESULTS: sdLDL proportions were reduced in the omega-3 fatty acids group by 11% after 12 weeks of treatment (n = 17, P = 0.001), and were reduced by 4% in the control group (n = 11, P = 0.096). The patients receiving the omega-3 fatty acids evidenced a significant reduction in the levels of triglyceride (P = 0.001), apoB100, and B48 after 12 weeks (P = 0.038 and P = 0.009, respectively) relative to the baseline. Omega-3 fatty acids supplementation increased fasting blood glucose (P = 0.011), but the levels of HbA1c in each group did not change to a statistically significance degree. The adiponectin value was not reduced in the omega-3 fatty acids group (P = 0.133); by way of contrast, the placebo group evidenced a significant reduction in adiponectin value after 12 weeks (P = 0.002). CONCLUSION: Omega-3 fatty acid treatment proved effective in the reduction of atherogenic sdLDL and apoB in type 2 diabetic patients (Clinical trials reg. no. NCT 00758927, clinicaltrials.gov).

Stress-induced signaling pathways in hyalin chondrocytes: inhibition by Avocado-Soybean Unsaponifiables (ASU).

OBJECTIVE: Avocado-Soybean Unsaponifiables (ASU) represent one of the most commonly used drugs for symptomatic osteoarthritis (OA). The mechanisms of its activities are still poorly understood. We investigate here the effects of ASU on signaling pathways in mouse or human chondrocytes. METHODS: Mouse or human chondrocytes stimulated with interleukin-1beta (IL1beta, 10 ng/ml) and cartilage submitted to a compressive mechanical stress (MS) were studied in the presence or absence of ASU (10 microg/ml). Nuclear factor kappaB (NF-kappaB) activation was assessed by immunoblot, using an I-kappa B alpha antibody, nuclear translocation of NF-kappaB using p65 antibody, and extra-cellular signal-regulated kinase (ERK)1/2 activation using phospho and ERK1/2 antibodies. The binding of the p50/p65 complex on DNA was studied by electrophoretic mobility shift assay. RESULTS: ASU decrease matrix metalloproteinases-3 and -13 expressions and Prostaglandin E(2) (PGE(2)) release in our model. The degradation of I-kappa B alpha is prevented in the presence of ASU as shown by the persistent expression of I-kappa B alpha protein in the cytosol when chondrocytes are stimulated by IL1beta or MS. Nuclear translocation of the NF-kappaB complex is shown by the decrease of the p65 protein from the cytosol, whereas p65 appears in the nucleus under IL1beta stimulation. This translocation is abolished in the presence of ASU. Moreover, bandshift experiments show an inhibition of the IL1beta-induced binding of p50/p65 complexes to NF-kappaB responsive elements in response to ASU. Finally, among the different mitogen-activated protein kinases known to be induced by IL1beta, ERK1/2 was the sole kinase inhibited by ASU. CONCLUSION: These results demonstrate that ASU express a unique range of activities, which could counteract deleterious processes involved in OA, such as inflammation.

[Glycosylation profile of selected acute phase proteins in children with chronic tonsillitis and allergic symptoms]. / Glikozylacja bialek ostrej fazy u dzieci z zapaleniem migdalków podniebiennych i objawami alergii.

INTRODUCTION: Acute phase proteins may be regarded as laboratory markers of inflammatory processes of various origin, but they also play several important biological roles. As majority of them are glycoproteins alterations in glycosylations profiles form additional sign of disturbances in the cytokines network during inflammation and allow to distinguish between acute and chronic inflammatory conditions. MATERIAL AND METHODS: A group of 25 children, aged from 6 to 13 years, admitted due to tonsillectomy was examined using skin tests towards specific allergens. Fifteen children out of the whole group showed reaction to pollens, whereas in ten children no allergen was detected despite clear allergic symptoms. In sera samples from every child concentrations of C-reactive protein, alpha1-acid glycoprotein (AGP) and alpha1-antichymotrypsin (ACT) were measured using rocket immunoelectrophoresis acc. to Laurell, and glycosylations profiles of AGP and ACT were determined, using crossed affino-immunoelectrophoresis acc. to Bøg-Hansen. RESULTS: Lower concentration of AGP and higher of ACT was shown for children allergic to pollens. Glycosylation profile of both proteins was altered towards higher reactivity with ConA for children allergic to pollens, whereas rather chronic image was observed in children allergic to unknown allergen. The latter image was similar to previously described in children with food allergies. CONCLUSIONS: The presence of allergic reaction may alter the cytokine network activity in children, thus affecting also the immune status, independently from chronic inflammatory process in tonsillitis.

Developmental expression of two Haliotis asinina hemocyanin isoforms.

Hemocyanins are large copper-containing respiratory proteins that play a role in oxygen transport in many molluscs. In some species only one hemocyanin isoform is present while in others two are expressed. The physiological relevance of these isoforms is unclear and the developmental and tissue-specific expression of hemocyanin genes is largely unknown. Here we show that two hemocyanin genes in the gastropod Haliotis asinina, which encode H. asinina hemocyanin (HaH1) and HaH2 isoforms, are developmentally expressed. These genes initially are expressed in a small number of mesenchyme cells at trochophore and pre-torsional veliger stages, with HaH1 expression slightly preceding HaH2. These cells largely are localized to the visceral mass, although a small number of cells are present in head and foot regions. Following metamorphosis the isoforms show overlapping as well as isoform-specific expression profiles, suggesting some degree of isoform-specific function.

Levels of common antigens in determining pathogenicity of Curvularia eragrostidis in different tea varieties.

AIMS: Pathogenicity of Curvularia eragrostidis, a foliar fungal pathogen of tea was studied in 24 commercially cultivated tea varieties by analysing the antigenic patterns of host and pathogen with the help of immunoserological techniques. METHODS AND RESULTS: Initial testing by cut shoot inoculation technique followed by whole plant inoculation technique showed that among the varieties tested, TV12 was the most susceptible and TV25 most resistant. Antigen preparations from tea varieties, fungal pathogens (C. eragrostidis and Lasiodiplodia theobromae) and a nonpathogen (Gliocladium virens) were compared by immunodiffusion, immunoelectrophoresis and indirect ELISA to detect common antigens shared by host and pathogen. Common antigens were detected by immunodiffusion and immunoelectrophoresis only among susceptible varieties and the pathogens. Such antigens were not found between the pathogens and the resistant varieties and also between nonpathogens and tea varieties. However, ELISA revealed the presence of low level of common antigens between all combinations. A certain minimum level of antigens was present for compatible host-pathogen interaction. Indirect labelling of antibodies with fluorescein isothiocyanate (FITC) showed that cross-reactive antigens were found to be concentrated mainly in the epidermal cells and also spread throughout the cortical cells. CONCLUSION: Pathogenicity of C. eragrostidis to different varieties of tea was found to be related to the level of common antigens present between host and pathogen. SIGNIFICANCE AND THE IMPACT OF THE STUDY: Indirect ELISA proved to be valuable in screening commercially cultivated varieties of tea for their susceptibility to C. eragrostidis.

Production of polyclonal antisera against feline immunoglobulin E and its use in an ELISA in cats with experimentally induced asthma.

Serum samples from six cats with experimentally induced asthma were used to purify feline IgE using gel filtration and affinity chromatography. The resultant IgE, evaluated for purity by immunoelectrophoresis (IEP) and reactivity by Prausnitz-Kustner (PK) testing, was used to develop polyclonal rabbit anti-feline IgE antisera. Using reverse cutaneous anaphylaxis (RCA), the antisera were determined to be specific for feline IgE. The polyclonal rabbit anti-feline IgE antiserum was then validated in an allergen-specific ELISA. Serum samples from an additional five asthmatic cats sensitized with Bermuda grass allergen (BGA) were evaluated prior to sensitization, after parenteral sensitization, and after aerosol sensitization and challenge. A significant increase in serum BGA-specific IgE was noted over time.

Activation of the alternative complement pathway by Agaricus blazei Murill.

Components of Agaricus blazei Murill have been demonstrated to have a wide range of immunopotentiating activities. The present study was designed to evaluate the effect of A. blazei Murill upon activation of the complement system in human serum in vitro. Additional studies were performed to determine the cytotoxic effect of complement-opsonized particles of A. blazei Murill against human tumor cells in culture. A fine particle of A. blazei Murill (ABP), prepared by mechanical disruption, was used throughout the experiments. ABP activated the human complement system via the alternative pathway in human serum. Activation of the alternative pathway was both time- and dose-dependent. When the particles from fruiting bodies of A. blazei Murill (ABP-F) were reacted with human serum, the formation of complement-opsonized ABP, iC3b-ABP-F complexes, and binding of the complexes to human peripheral blood monocytes, were demonstrated in vitro by immunofluorescence. Further, the resident human peripheral nucleated cells incubated in the presence of iC3b-ABP-F complexes inhibited the proliferation of human tumor cell line TPC-1 in vitro.

Dietary iron status has little effect on expression of ceruloplasmin but alters that of ferritin in rats.

Evidence supports a role for ceruloplasmin (ferroxidase I) in the release of iron to the blood from mammalian cells. However, recent studies with cultured cells have suggested that it has the opposite effect, and that iron deficiency enhances expression of ceruloplasmin. We therefore examined in rats how nutritional iron status would affect expression of ceruloplasmin. Groups of male Sprague-Dawley rats were reared on a low iron, starch-based diet for 6-8 wk; half were supplemented by injection of iron dextran. At killing, hematocrits of deficient rats were half normal. Supplemented rats had normal liver concentrations of ferritin and ferritin iron. No ferritin was detected in the livers of the deficient rats. Northern analysis showed that ferritin L and H mRNAs were present in the deficient livers, but expression was half that of the normal rats. There was also twice as much copper. Levels of circulating ceruloplasmin (measured by rocket immunoelectrophoresis) were not altered by iron deficiency, although p-phenylenediamine oxidase activity and plasma copper were reduced approximately 30%. In repeated studies, no differences in the expression of hepatic ceruloplasmin mRNA were detected. Treatment of rats of both sexes with additional iron (25 mg as iron dextran) 5-14 d before killing increased liver ferritin but did not alter liver ceruloplasmin mRNA expression or levels of circulating ceruloplasmin. We conclude that iron status is not an important factor in the expression of plasma ceruloplasmin made by the liver. However, it does have modest effects on steady-state levels of liver ferritin mRNA.

Antigenic relationship between four airborne palm pollen grains from Calcutta, India.

The pollen grains of Areca catechu, Borassus flabellifer, Cocos nucifera and Phoenix sylvestris, all belonging to the family Aracaceae (Palmae), are airborne and found to be potent in causing human respiratory allergy. The present study was undertaken to discover the antigenic relationship, if any, in the four relevant palm pollen grains. The study was conducted by using Borassus and Phoenix antisera raised in rabbit. These antisera were used in rabbit IgG specific ELISA-inhibition and rocket immunoelectro-phoresis (RIE) assays for all four palm pollen extracts. In ELISA-inhibition, a distinct inhibition was obtained with comparable amount of soluble pollen protein. The RIE precipitin bands also revealed the presence of common antigenic components in the palm pollen. After isolation and purification, such common antigens may be useful in allergen immunotherapy in asthmatics.

Physicochemical and immunologic characterization of low-molecular-weight allergoids of Dactylis glomerata pollen proteins.

BACKGROUND: Orchard grass (Dactylis glomerata) pollen proteins were chemically modified by means of acid anhydrides (maleic and succinic anhydride) to obtain low-molecular-weight allergoids. Chemical modification in both cases led to the replacement of one positive charge (epsilon amino group of Lys) by one negative charge, yielding proteins with changed physicochemical properties in comparison to the native orchard grass-pollen proteins. METHODS: Physicochemical characterization of derivatives was done by gel chromatography, SDS-PAGE, and isoelectric focusing. To examine the IgE-binding properties of these derivatives, we carried out immunoblotting. To examine the ability of derivatives to induce IgG production, we immunized rabbits. Skin prick testing with the allergoids was performed on 15 individuals allergic to orchard grass pollens and on two healthy subjects. RESULTS: It was shown that the modified proteins retain their original molecular weights, but change pI to more acidic values. In the case of allergoids, a strong reduction in IgE binding was found. Immunization of rabbits with allergoids showed that the derivatives retain the ability to induce IgG production, and that the antisera obtained in such a way react to native (unmodified) extract. The ability of derivatives to induce allergic reaction was significantly reduced. The patients (86.6%) included in our study exhibited less than 50% of native extract response. Among them, 53.3% had no response to one or both allergoids. CONCLUSIONS: These modification procedures yield allergoids with a reduced allergenic activity and preserved immunogenic potential suitable for use in immunotherapy.

Effect of selenium deficiency on cellular and extracellular glutathione peroxidases: immunochemical detection and mRNA analysis in rat kidney and serum.

To determine the effect of selenium (Se) deficiency on expression of glutathione peroxidase (GSH-Px) 1 and 2, we measured GSH-Px activity in rat serum, liver and kidneys, serum immunoreactive GSH-Px 2, and the mRNAs of kidney GSH-Px 1 and 2. We purified rat GSH-Px 2 and raised polyclonal antibodies. Immunoreactive GSH-Px 2 was measured by rocket immunoelectrophoresis. GSH-Px 2 was purified 1470-fold with a specific activity of 250 units/mg. Immunoblotting detected only GSH-Px 2 in rat serum, and much less GSH-Px 2 than GSH-Px 1 in kidney. Immunoblot signal of kidney GSH-Px 1 and 2 decreased progressively in Se deficient rats. Serum GSH-Px activity in Se deficient rats at 1, 2, 3, and 4 weeks declined to 33, 20, 10, and 9% of the control, while the serum level of immunoreactive GSH-Px 2 was 58, 24, 15, and 10% of the control, suggesting the presence of an inactive protein at week 1. GSH-Px activity declined to 4 and 11% of the control in the liver and kidney at 4 weeks. The mRNAs of kidney GSH-Px 1 and 2 showed similar decreases, and were 24 and 23% of the control at 4 weeks. GSH-Px mRNA levels were better preserved than GSH-Px activity, suggesting that GSH-Px expression was regulated at both pre-translational and translational levels.

Immunochemical distinction of Aloe vera, A. arborescens, and A. chinensis gels.

Verectin antiserum raised in white rabbits was immunoprecipitated with the Aloe vera nondialysable fraction. Analysis of the immunoprecipitation revealed that verectin accounted for about 1.25% of the total proteins in the nondialysable fraction of Aloe vera gel. The verectin antibody showed differential immunoreactivities against nondialysable fractions of A. arborescens, A. chinensis, and A. vera: 1) an immunopreciptin line was formed against the fraction of A. vera, but not against those of A. arborescens and A. chinensis gel in an Ouchterlony double immunodiffusion test and 2) an immunopositive band was detected in the A. vera and A. chinensis nondialysable fractions but not in that of A. arborescens in immunoblotting. These findings indicate that the verectin antibody can be used to distinguish Aloe materials.

Produçäo de albumina a partir de placentas humanas / Production of albumin from human placental

Albumina é a proteína mais abundante do plasma e suas funçöes säo: manter a pressäo osmótica e o transporte de Ca++, ácidos graxos e biliares, esteróides, bilirrubina, hematina, fármacos, etc. Principal uso clínico da albumina é a recuperaçäo do volume plasmático após hemorragia. As matérias-primas usadas para sua produçäo industrial säo plasma ou soro humano de doadores voluntários. Outra alternativa é o uso do sangue da placenta. O Centro de Biotecnologia do Instituto Butantan propôs o desenvolvimento de um processo industrial para a obtençäo de albumina de hemolisado de placentas humanas. O processo desenvolvido para a purificaçäo da albumina a partir de 50 Kg de placentas contém as seguintes etapas: 1) Extraçäo do hemolisado com salina e separaçäo sólido/líquido por centrífuga de cesto; 2) Precipitaçäo seletiva da hemoglobina com etanol/clorofórmio e remoçäo do precipitado por filtraçäo em filtro prensa; 3) Concentraçäo/diafiltraçäo do filtrado com membranas de filtraçäo tangencial de 30 kDa; 4)Termocoagulaçäo a 70ºC em presença de caprilato-Na/EDTA e posterior filtraçäo do material insolúvel...

Group 5 determination in Pooideae grass pollen extracts by monoclonal antibody-based ELISA. Correlation with biologic activity.

A solid-phase, monoclonal antibody-based ELISA was set up to quantitate group 5 allergens in pollen extracts of wild and cultivated Pooideae grasses. The method was able to evaluate group 5 concentration in mass units with a sensitivity in the ng/ml range and a practical working range of 1-100 ng/ml. The group 5 ELISA was compared with rocket immunoelectrophoresis for determination of allergen levels in several Phleum pratense extracts, and a very good quantitative correlation was found (r = 0.98; P < 0.0001). A highly significant correlation (r > 0.8) was also obtained in comparing allergenic potency determined by RAST inhibition to group 5 content in several wild and cultivated grass species. The results proved the usefulness of the method in the standardization of Pooideae pollen extracts employed in diagnosis and treatment.

Quantification in mass units of Bet v 1, the main allergen of Betula verrucosa pollen, by a monoclonal antibody based-ELISA.

BACKGROUND: Fagales pollens are considered among the main agents responsible for allergic diseases in many countries of the northern hemisphere and single major allergens have been shown to be responsible for these responses. OBJECTIVE: To develop a solid phase immunoassay for the quantification of Bet v 1, the main allergen from Betula verrucosa (birch), and to assess its suitability for quantitating the equivalent major allergen in other Fagales species as well. METHODS: The assay is based on the use of two different anti-Bet v 1 monoclonal antibodies which were immobilized on the solid phase and, as a primary standard, affinity purified Bet v 1, the protein content of which was determined by amino acid analysis. RESULTS: The ELISA proved to measure less than 0.2 ng/mL of Bet v 1 with a practical range of 0.4-40 ng/mL and could be suitable to quantify the equivalent major allergen in other Fagales species such as Corylus avellana (hazel), Carpinus betulus (horbeam) and Alnus glutinosa (alder). The method was compared with quantitative electrophoresis and rocket immuno-electrophoresis for the determination of the allergen content in several Betula verrucosa extracts, and a very good quantitative correlation was found. Likewise, the Bet v 1 content exhibited a good correlation (r = 0.87; P < 0.005) with the allergenic potency values obtained by RAST inhibition. CONCLUSIONS: The results indicate that the Bet v 1-assay could be useful for standardization purposes in Fagales pollen extracts intended for clinical use.

Heterogeneity of pollen proteins within individual Betula pendula trees.

To determine the variation of antigenic water soluble proteins in white birch (Betula pendula) pollen, extracts of pollen from different sides of individual trees were analyzed by isoelectric focusing (IEF), crossed immunoelectrophoresis, and crossed radioimmunoelectrophoresis. IgE-antibody-binding patterns were also studied in samples analyzed by IEF by probing with serum pooled from patients with birch pollen allergy, followed by radiolabelled anti-IgE. Antigenic proteins and allergens per unit weight of extracted protein were greatest in pollen extracts from the south side of the trees. Allergens decreased progressively in pollen from west-through east- to north- facing branches. Proteins with high isoelectric points (pI > 8.5) and proteins between pI 4.5 and 5.6 were infrequent in extracts from the north side. Extracts from branches facing north were poor in allergens: in general, only one or two precipitin lines were found, and in some cases they did not bind to IgE antibodies. Differences between numbers of proteins and allergens found in extracts from south, west and north branches were statistically significant for all methods used. The results indicate the need to collect birch pollen for allergen extract manufacture from south-facing branches.

Basic isoforms of Par o 1, the major allergen of Parietaria officinalis pollen.

We describe a group of basic isoforms of Par o 1 (cumulatively referred to as Par o 1b), purified by anion-exchange chromatography. The allergenic activity of Par o 1b was compared with that of the acidic isoform (Par o 1a) by RAST inhibition. Par o 1b showed a cathodic mobility in crossed immunoelectrophoresis. It was found to be homogeneous in SDS-PAGE and SE-HPLC (14.5 kDa), and heterogeneous in PAG-IEF, yielding five IgE-binding bands with pI ranging between 7.9 and 9.6 PAG-IEF individual components were isolated by cation-exchange HPLC. The N-terminal amino acid sequence of the main component (pI 8.8) was determined and found to be similar to that of Par o 1a.

Effect of resuscitation solutions on the immune status of dogs in hemorrhagic shock.

The purpose of this study is to examine the effects of three different types of fluid resuscitation on the immune system of dogs in hemorrhagic shock. Using a modified Wigger shock model, 18 conditioned male dogs were bled to mean arterial blood pressure of 60 mm Hg for 90 minutes and placed into three groups based on the resuscitative method. Group I: Crystalloid Resuscitation; Group II: Autotransfusion; Group III: Banked Blood. Laboratory methods for immune status evaluation included total lymphocyte count, T4/T8 ratio, total serum immunoglobulins, and immunoglobulin electrophoresis. These values were obtained pre-hemorrhagic shock, just before resuscitation, and subsequently on days 1, 4, and 7. Humoral immunity, represented by total serum immunoglobulin levels (IgA, IgG, IgM), was higher in Groups II and III when compared with group I on all post-resuscitation days. IgA and IgM levels were higher in Group III compared with Groups I and II. IgG level was higher in Group II compared with Groups I and III. Cellular immunity was also affected by transfusion. Total lymphocyte count was increased in Group II on Day 1; however, the three groups were similar with respect to this variable on subsequent days. The absolute T4 helper cell level in Group II was similar to Groups I and III until Day 7, at which time the level became higher in Group II.(ABSTRACT TRUNCATED AT 250 WORDS)