Complement-fixing Activity Research Articles

Release and characterization of single side chains of white cabbage pectin and their complement‐fixing activity

A mixture of single side chains from white cabbage pectin were obtained by anion exchange chromatography after applying mild chemical conditions promoting beta-elimination. These pectin fragments were characterized by their molecular weight distribution, sugar composition, 13C-NMR, and MALDI-TOF-MS analysis. These analyses revealed that the large oligosaccharides released by beta-eliminative treatment were composed of alpha-1,5 linked arabinosyl residues with 2- and 3-linked alpha-arabinosyl side chains, and, or beta-1,4 linked galactosyl side chains. Fractions were tested for complement-fixing activity in order to determine their interaction with the complement system. These results strongly indicated that there was a minimal unit size responsible for the complement-fixing activity. Neutral pectin fragments (8 kDa) obtained from beta-elimination were inactive in the complement system, although they contained a sugar composition previously shown to be highly active. Larger pectin fragments (17 kDa) retained some activity, but much lower than polymers containing rhamnogalacturonan type 1 (RGI) structures isolated from the same source. This implied that structural elements containing multiple side chains is necessary for efficient complement-fixing activity.

Complement-fixing activity of fulvic acid from Shilajit and other natural sources.

Shilajit has been used traditionally in folk medicine for the treatment of a variety of disorders, including syndromes involving excessive complement activation. Extracts of Shilajit contain significant amounts of fulvic acid (FA), and it has been suggested that FA is responsible for many therapeutic properties of Shilajit. However, little is known regarding the physical and chemical properties of Shilajit extracts, and nothing is known about their effects on the complement system. To address this issue, extracts of commercial Shilajit were fractionated using anion exchange and size-exclusion chromatography. One neutral (S-I) and two acidic (S-II and S-III) fractions were isolated, characterized and compared with standardized FA samples. The most abundant fraction (S-II) was further fractionated into three sub-fractions (S-II-1 to S-II-3). The van Krevelen diagram showed that the Shilajit fractions are the products of polysaccharide degradation, and all fractions, except S-II-3, contained type II arabinogalactan. All Shilajit fractions exhibited dose-dependent complement-fixing activity in vitro with high potency. Furthermore, a strong correlation was found between the complement-fixing activity and carboxylic group content in the Shilajit fractions and other FA sources. These data provide a molecular basis to explain at least part of the beneficial therapeutic properties of Shilajit and other humic extracts.

The effect of antibody isotype and antigenic epitope density on the complement-fixing activity of immune complexes: a systematic study using chimaeric anti-NIP antibodies with human Fc regions

A systematic study has been carried out to investigate the role of immunoglobulin isotype, epitope density, and antigen/antibody ratio on the capacity of immune complexes to activate the classical and alternative pathways of human complement and for the complexes subsequently to bind to erythrocyte C3b-C4b receptors (CRI). For this purpose, a series of chimaeric monoclonal anti-NIP antibodies was used, which all shared the same combining site but had different human constant domains. Antigen epitope density was varied by coupling different numbers of NIP hapten molecules to bovine serum albumin. All three parameters affect complement fixation. In general, complement activation is better in antibody excess and at equivalence than it is in antigen excess, and better at high epitope density than at low epitope density, although the effects are variable for different immunoglobulin isotypes and for the two pathways. It has been confirmed that IgG1 and IgG3 are good activators of the classical pathway and are tolerant to variations in both epitope density and antigen/antibody ratio. IgG4 and IgA do not activate the classical pathway in any circumstances. IgG2 activates the classical pathway only at high epitope density and at equivalence or antibody excess. IgM activates the classical pathway well only at the higher epitope densities and at equivalence or antibody excess but, in addition, shows an interesting and unexpected prozone phenomenon where immune complex in antibody excess inhibits complement activation by the classical pathway. The results of the alternative pathway activation are strikingly different. IgA is by far the best activator of the alternative pathway and is relatively tolerant to epitope density and to antigen/antibody ratio. IgM, IgG1 and IgG3 do not significantly activate the alternative pathway in any circumstances. IgG2 is the best IgG subclass for alternative pathway activation but requires high epitope density and equivalence or antibody excess. Binding to CR1 in general parallels the amount of complement fixed independent to the pathway by which it is fixed. However, IgG1 and IgG3 complexes in antigen excess activate complement well but bind poorly to CR1. Nascently formed complexes seem to bind complement in a way that is similar to that bound by preformed complexes, but are then less able to bind to red cell CR1. These observations help to explain the pathogenesis of complement activation in various autoimmune and immune complex diseases such as systemic lupus erythematosus, autoimmune thyroiditis and others.

Fractionation and characterization of biologically-active polysaccharides from Artemisia tripartita

The leaves of Artemisia species have been traditionally used for prevention and treatment of a number of diseases. In this study, five polysaccharide fractions (designated A-I–A-V) were isolated from the leaves of Artemisia tripartita Rydb. by the sequential use of hot-water extraction, ethanol precipitation, ultra-filtration, and chromatography. The homogeneity and average molecular weight of each fraction were determined by high performance size-exclusion chromatography. Sugar composition analysis revealed that Artemisia polysaccharides consisted primarily of xylose, glucose, arabinose, galactose, and galactosamine. Moreover, all fractions contained at least 3.4% sulfate, and fractions A-II–A-V contained an arabinogalactan type II structure. All fractions exhibited macrophage-activating activity, enhancing production of intracellular reactive oxygen species and release of nitric oxide, interleukin 6, interleukin 10, tumor necrosis factor α, and monocyte chemotactic protein 1. In addition, all fractions exhibited scavenging activity for reactive oxygen species generated enzymatically or produced extracellularly by human neutrophils. Finally, fractions A-I and A-V exhibited complement-fixing activity. Taken together, our results provide a molecular basis to explain at least part of the beneficial therapeutic effects of Artemisia extracts, and suggest the possibility of using Artemisia polysaccharides as an immunotherapeutic adjuvant.

Polysaccharides with complement fixing and macrophage stimulation activity from Opilia celtidifolia, isolation and partial characterisation

Aim of the study The present study is aimed to determine the bioactivity and structure of polysaccharides present in the leaves from the Malian medicinal plant Opilia celtidifolia [Guill. & Perr. Endl. ex Walp (Opiliaceae)]. Materials and methods The polysaccharides from the leaves of Opilia celtidifolia were isolated from water extracts of the leaves using gelfiltration and anion exchange chromatography giving the fractions Oc50A 1 and Oc50A 2. Monosaccharide composition was determined by gas chromatography of the derived TMS-derivatives of the methyl-glycosides. Linkages were determined of the partly methylated, partly acetylated alditol acetates obtained after a process including reduction, methylation, hydrolysis, reduction and acetylation followed by GC-MS. Effects on the complement system and the macrophages were determined using specific methods aimed for studying those activities. Results The polysaccharide fractions isolated from the leaves of Opilia celtidifolia has high complement fixing activity and induce nitrite oxide release from macrophages in a dose dependent manner. The fractions had an ICH 50 of 0.5 and 0.9 μg/ml respectively in the complement fixing assay. They induced the release of 7.2 and 7.3 μM of nitrite oxide from macrophages respectively at a dose of 100 μg/ml. The monosaccharide composition in Oc50A 1 and Oc50A 2, analysed, showed the presence of arabinose (26.7 and 13.2%), galactose (31.5 and 28%) and galacturonic acid (5.3 and 7.8%) respectively. The Yariv test confirmed the presence of arabinogalactan type II in both fractions. Structural analyses did also show the presence of terminal and 1-4 linked galacturonic acid and terminal and 1-2 linked rhamnose. Endo-polygalacturonanase treatment was performed to isolate the heavily substituted parts of the polysaccharides. These parts contained the same monosaccharides in similar proportion, and showed stronger dose dependent complement fixing activity and also stimulated macrophages to release nitrite oxide. Conclusions The leaves of Opilia celtidifola contains polysaccharides of pectic type that have both complement fixing and macrophage stimulating activity.

Immunomodulatory activity of acidic polysaccharides isolated from Tanacetum vulgare L.

Tanacetum vulgare L. (Tansy) has been extensively used in folk medicine for treatment of a variety of medical disorders. In the present study, we isolated and purified four acidic polysaccharide fractions (designated T-I to T-IV) from Tansy florets by the sequential use of hot-water extraction, ethanol precipitation, ultra-filtration, anion-exchange, and size-exclusion chromatography. The average Mr of fractions T-I through T-IV was estimated to be 326, 151, 64 and 9 kDa, respectively, as determined by high performance size-exclusion chromatography analysis. Sugar composition analysis revealed that Tansy polysaccharides consisted primarily of galacturonic acid, galactose, arabinose, and rhamnose. Fractions T-II through T-IV contained an arabinogalactan type II structure, as determined by reaction with Yariv reagent. High Mr fractions T-I and T-II exhibited potent macrophage/monocyte-activating activity, enhancing production of reactive oxygen species (ROS), nitric oxide (NO), and tumor necrosis factor α (TNF-α) by J774.A1 murine macrophages, and activating nuclear factor κB (NF-κB) in THP-1 human monocytes. In addition, Tansy polysaccharides stimulated human neutrophil function by greatly enhancing neutrophil myeloperoxidase (MPO) release. Furthermore, the low Mr fraction T-IV had potent complement-fixing activity, which may also contribute to the anti-inflammatory and would-healing properties of Tansy extracts. Taken together, our results provide a molecular basis to explain at least part of the beneficial therapeutic effects of Tansy extracts, and support the concept of using Tansy polysaccharides as an immunotherapeutic adjuvant.

Immunological and Structural Properties of a Pectic Polymer from Glinus Oppositifolius

The aim of this paper was to further elucidate the structure and the immunomodulating properties of the pectic polymer GOA2, previously isolated from Glinus oppositifolius. Enzymatic treatment of GOA2 by endo-alpha-d-(1 --> 4)-polygalacturonase led to the isolation of three pectic subunits, GOA2-I, GOA2-II, and GOA2-III, in addition to oligogalacturonides. GOA2-I was shown to consist of 1,2-linked Rhap and 1,4-linked GalpA in an approximately 1:1 ratio, and NMR-analysis showed that the monomers were linked together in a strictly alternating manner. The galactose units in GOA2-I were found as terminal-, 1,3-, 1,6-, 1,4-, 1,3,4-, and 1,3,6-linked residues, while the arabinofuranosyl existed mainly as terminal- and 1,5-linked units. A rhamnogalacturonan-I type structure was suggested being the predominant part of GOA2-I. According to linkage analysis GOA2-II and GOA2-III contained glycosidic linkages characteristic for rhamnogalacturonan-II type structures. GOA2 was shown by sedimentation velocity in the analytical ultracentrifuge, to have a broad degree of polydispersity with a mode s(20,w) value of approximately 1.9 S, results reinforced by atomic force microscopy measurements. The polydispersity, as manifested by the proportion of material with s(20,w) > 3 S, decreased significantly with enzyme treatment. The abilities of GOA2, GOA2-I, GOA2-II, and GOA2-III to induce the proliferation of B cells, and to exhibit complement fixing activities were tested. In both test systems, GOA2-I showed significantly greater effects compared to its native pectin GOA2. GOA2-I was in addition shown to exhibit a more potent intestinal immune stimulating activity compared to GOA2. The ability of GOA2 to induce secretion of proinflammatory cytokines was examined. Marked upregulations in mRNA for IL-1beta from rat macrophages and IFN-gamma from NK cells were found.

Effects of extraction conditions on the chemical structure and biological activity of white cabbage pectin

The purpose of this study was to isolate and perform chemical analyzes as well as biological testing of pectic material from white cabbage isolated by sequential aqueous ionic solutions (SEQAIS) or a simple pure water extraction (PW). Water extraction was aimed at yielding water-soluble pectins only, while the harsher conditions in SEQAIS aimed at extracting proto pectin as well. The pectic material resulting from the various extraction steps was characterized and tested, in order to determine whether structural and biological activity were influenced through different isolation procedures. The SEQAIS fractions obtained were one water-soluble and six partly water-soluble extracts, whereas PW yielded two water-soluble extracts. Sugar composition analysis, linkage analysis, HPSEC molecular weight distribution, HPAEC and 13C NMR were run to obtain structural characteristics of the extracted material. Both extraction procedures resulted in degradation of pectin. Pectin containing highly methyl esterified Gal pA probably underwent β-elimination due to neutral pH during PW, while hydrolysis of Ara f occurred in the first step of SEQAIS in 50 mM acetic acid pH 4.5. Water-soluble extracts were tested for complement-fixing activity and acidic extracts with degraded side chains showed reduced activity. Authors suggest that extraction conditions at neutral pH should be used in order to withhold side chain structure and immuno-activity.

C5a Receptor‐Deficient Mice Are Protected from Thrombophilia and Endothelial Cell Activation Induced by Some Antiphospholipid Antibodies

Recent findings indicate that complement activation--involving specifically C3 and C5--contributes to antiphospholipid (aPL)-mediated thrombosis. Two complement effector pathways are initiated by the cleavage of C5, C5a and C5b, which leads to the formation of the C5b-9 membrane attack complex. To delineate and distinguish the role of C5a from the C5b-9 membrane attack complex seeded by C5b, we examined the in vivo effects (thrombosis) of aPL on C5a receptor-deficient (C5aR-/-) mice. C5aR-/- and C5aR+/+ mice were injected with IgM or with IgG from two different patients with APS (IgM-APS or IgG-APS) or with control IgM or IgG (IgM-NHS or IgG-NHS) twice. Complement fixing activity of the Ig fractions and anticardiolipin activity in the sera of the mice were determined by enzyme-linked immunosorbent assay. Surgical procedures to study thrombus dynamics were performed. IgM-APS but not IgG-APS fixed C1q to cardiolipin-coated plates. IgM-APS significantly enhanced thrombus size in C5aR+/+ mice compared to C5aR+/+ mice treated with IgM-NHS (3198 +/- 2361 microm2 versus 585 +/- 460 microm2). C5aR-/- mice treated with IgM-APS showed a significant reduction in thrombi size as compared with C5aR+/+ mice (676 +/- 690 microm2 versus 3198 +/- 2361 microm2; P = 0.001). IgG-APS enhanced thrombus formation significantly in C5aR+/+ when compared to IgG-NHS-treated mice (3507 +/- 965 microm2 versus 1321 +/- 798 microm2), and these effects were not altered in C5aR-/- mice (3400 +/- 1681 microm2). The data indicate that C5aR-/- mice are protected from the thrombogenic effects of some aPL antibodies.

An immunomodulating pectic polymer from Glinus oppositifolius

An immunomodulating pectic polymer, GOA1, obtained from the aerial parts of the Malian medicinal plant Glinus oppositifolius (L.) Aug. DC. (Aizoaceae) has previously been reported to consist of arabinogalactans type I and II, probably linked to a rhamnogalacturonan backbone. To further elucidate the structure of the polymer GOA1, enzymatic degradation studies and weak acid hydrolysis were performed. Five different glycosidases were used, endo-α- d-(1 → 4)-polygalacturonase, exo-α- l-arabinofuranosidase, endo-α- l-(1 → 5)-arabinanase, endo-β- d-(1 → 4)-galactanase and exo-β- d-galactosidase. It appears that GOA1 may contain a structural moiety consisting of a 1,3-linked galactopyranosyl (Gal p) main chain with 1,6-linked Gal p side chains attached to position 6 of the main chain. The 1,6-linked Gal p side chain may be branched in position 3 with arabinofuranosyl (Ara f) side chains. A 1,4-linked Gal p backbone which might carry side chains or glycosyl units attached to position 3 is also a structural element in the polymer. We further show that GOA1 induce proliferation of B cells and the secretion of IL-1β by macrophages, in addition to a marked increase of mRNA for IFN-γ in NK-cells. To elucidate structure–activity relations the native polymer and the digested fractions were tested for complement fixing activity and intestinal immune stimulating activity. The partial removal of Ara f residues after enzymatic degradations did not affect the bioactivities, while the acid hydrolysed fraction showed reduced complement fixing activity. A decrease in Ara f units, 1,3,6-linked Gal p units and a partial hydrolysed rhamnogalacturonan backbone, in addition to a reduction in molecular weight are factors that might have contributed to reduced bioactivity.

Structural Features and Complement-Fixing Activity of Pectin from Three Brassica oleracea Varieties: White Cabbage, Kale, and Red Kale

Leaves of different cabbage species are used both as food and as wound healing remedies in traditional medicine. This supposed wound healing activity might be connected to presence of immunomodulating water soluble polysaccharides. To study this, three different cabbage varieties, white cabbage (W), kale (K), and red kale (RK), were pretreated with 80% ethanol and then extracted with water at 50 degrees C and 100 degrees C for isolation of polysaccharide-containing fractions. The fractions were analyzed for monosaccharide composition, glycosidic linkages, Mw distribution, protein content, and phenolic compounds and then tested for complement-fixing activity. All fractions contained pectin type polysaccharides with linkages corresponding to homogalacturonan and hairy regions. Those extracted at 50 degrees C contained higher amounts of neutral side chains and were more active in the complement-fixation test than those extracted at 100 degrees C. The fractions can be ranged by decreasing activity: K-50 > RK-50 > W-50 approximately = K-100 > RK100 approximately = W-100. Studies on structure-activity relationships (SAR) employing multivariate statistical analysis strongly suggest that the magnitude of the measured activity is influenced by the content of certain side chains in the polymers. High activity correlates to large neutral side chains with high amounts of (1-->6)- and (1-->3,6)-linked Gal and low amounts of (1-->4)-linked GalA but not on molecular weight distribution of the polymers.

Immunoactive Polysaccharide-Rich Fractions from Panax notoginseng

Panax notoginseng is a commonly used medicinal plant in south-western China. In a previous study, a sequential solubilisation of P. notoginseng high-molecular-weight (HMW) polymers using phenol-acetic acid-water, hot water, weak and strong alkali was performed to determine the structure of the component polysaccharides and proteins. The effects of these extracted HMW fractions on the human complement system, polymorphonuclear neutrophils (PMN) and peripheral blood mononuclear cells (PBMC) are reported here. Fr (1MKOH), which was extracted with 1 M KOH, showed the strongest complement-fixing activity and priming of reactive oxygen species (ROS) production by PMNs, as well as a mitogenic effect. Fr (1MKOH) was further fractionated by anion-exchange chromatography followed by gel-permeation chromatography. 1MD3-G2, the fraction most strongly bound to the DEAE anion-exchange column with a molecular weight of 1140 kDa, showed the highest complement-fixing activity. It is composed of acidic polysaccharides [including glucuronoarabinoxylan (GAX), homogalacturonan (HGA), rhamnogalacturonan I (RG I)], neutral polysaccharides (4-galactan and arabinan), and some protein.

Pectin isolated from white cabbage – structure and complement-fixing activity

This study was done to investigate whether white cabbage contained polysaccharides with immunostimulatory activity using the complement-fixing test as an indicator. The main polysaccharide isolated was of pectin nature. Methanolysis and (13)C-NMR showed that the polymers consisted of highly esterified alpha-galactopyranoside (alpha-GalpA), significant amounts of alpha-arabinose furanoside (alpha-Araf), beta-Galp and lesser amounts of rhamnose in the pyranose form (Rhap) and xylose in the pyranose form (Xylp). Linkage analyses showed that the alpha-GalpA residues were mainly 1,4-linked with small amounts of 1,3,4-linkages. The alpha-Araf residues were mainly terminally (t)- and 1,5-linked, whereas beta-Galp was t-, 1,3-, 1,6-, and 1,3,6-linked. Positive Yariv reaction indicated polymers with arabinogalactan type 2 like structures. alpha-Rhap was mainly present as 1,2- and 1,2,4-linked residues and Xylp was t- and 1,4-linked. The molecular weight varied greatly and was from 10 to 150 kDa. Cabbage polymers had biological activity and this complement-fixing activity was greatly affected by hydrolytic removal of Araf from pectic side chains.

Complement-fixing activity of anticardiolipin antibodies in patients with and without thrombosis

We have analysed in vitro the complement-fixing activity of anticardiolipin antibodies (C-fix aCL) from patients with persistent and moderate/high titres IgG aCL antibodies: 21 with thrombosis and 11 without thrombosis. Titre and C-fix ability of aCL were measured by ELISA. APS and non-APS patients were similar with regard to mean levels of IgG aCL (46 +/- 24 versus 51 +/- 30 GPL, P = 0.7), frequency of IgM aCL (P = 0.7) and a comparable predominance of IgG2 aCL reactivity on ELISA (95% versus 100%, respectively, P = 1.0). Remarkably, a high frequency of C-fix aCL (71% versus 92%, P = 0.35) was observed in both groups. Similarly, no difference was observed in the mean level of C-fix aCL in APS and non-APS patients (7 +/- 6 versus 9 +/- 8 SDunits, P = 0.3). Analysis of 10 primary and 11 secondary APS also revealed a comparable IgG aCL mean titre (57 +/- 29 versus 37 +/- 11, P = 0.06), frequency of IgM aCL (P = 0.6) and of C-fix aCL (70% versus 73%, P = 0.99). Among APS patients six had exclusive arterial events and seven exclusive venous events. The IgG aCL mean titre (36 +/- 10 versus 36 +/- 11 GPL, P = 0.9) and the frequency of IgM aCL antibodies (P = 0.56) in these subgroups of patients were comparable. There was a trend of higher frequency of C-fix aCL in patients with exclusive venous events (100%) compared to 50% of those with exclusive arterial events (p = 0.07). Importantly, C-fix aCL titre was higher in the former group compared to the later one (8 +/- 5 SDunits versus 2 +/- 2 SDunits, P = 0.016). Our data support the notion of a high frequency of C-fix aCL in APS. Although it does not discriminate those patients without thrombotic events with persistent moderate/high levels of aCL, this property seems to be more relevant in venous events and may provide the basis for further understanding the distinct pathogenic mechanisms underlying arterial and venous occlusive disorders of APS.

DNA vaccine against the non-structural 1 protein (NS1) of dengue 2 virus

Dengue is one of the most important mosquito-borne viral disease causing dengue fever and/or dengue shock syndrome/haemorrhagic fever. In some reports, the non-structural protein 1 (NS1) has been identified as a promising antigen for the development of vaccines against dengue virus (DENV). Apparently, it can elicit a protective antibody response with complement-fixing activities. In order to investigate the potential of a DNA vaccine based on the NS1 protein against DENV, we used the plasmid pcTPANS1, which contains the secretory signal sequence derived from human tissue plasminogen activator (t-PA) fused to the full length of the DENV-2 NS1 gene. All Balb/c mice intramuscularly inoculated with the pcTPANS1 presented high levels of NS1-specifc antibodies. Vaccinated animals were challenged with intracerebral DENV-2 virus inoculations and a 100% survival was observed. In general, results demonstrate that the pcTPANS1 plasmid is able to induce protection in mice, and then may be used as a vaccination approach against DENV in further assays.

Macrophage immunomodulatory activity of polysaccharides isolated from Juniperus scopolorum

Extracts of cones and leaves of different species of the genus Juniperus have been used for centuries to treat a variety of medical problems; however, little is known about the active components conferring therapeutic properties to these extracts. To address this issue, we extracted water-soluble polysaccharides from Juniperus scopolorum cones and used ion exchange and size exclusion chromatography to separate them into five fractions, with estimated M r of 30, 60, 100, 200, and 680 kDa, respectively. All fractions contained type II arabinogalactan in their structure, as determined by reaction with Yariv reagent and structural analysis by proton nuclear magnetic resonance spectroscopy, but lacked complement fixing activity. Analysis of the effects of Juniper polysaccharides on murine peritoneal macrophages, cultured J774.A1 macrophages, and human mononuclear phagocytes demonstrated that the high molecular weight polysaccharide fractions (200 and 680 kDa) had potent immunomodulatory activity. These polysaccharide fractions primed macrophages for an enhanced respiratory burst, directly stimulated NO production via induction of nitric oxide synthase, and induced macrophages to secrete both inflammatory (IL-1, IL-6, TNF-α, and IL-12) and anti-inflammatory (IL-10) cytokines. These data suggest that at least part of the beneficial therapeutic effects reported for extracts of juniper cones are due to modulation of monocyte/macrophage immune functions.

W01-P-002 Comparative study of inflammatory cells in human arteries with clinically significant atherosclerotic sequelae

Tanacetum vulgare L. (Tansy) has been extensively used in folk medicine for treatment of a variety of medical disorders. In the present study, we isolated and purified four acidic polysaccharide fractions (designated T-I to T-IV) from Tansy florets by the sequential use of hot-water extraction, ethanol precipitation, ultra-filtration, anion-exchange, and size-exclusion chromatography. The average Mr of fractions T-I through T-IV was estimated to be 326, 151, 64 and 9 kDa, respectively, as determined by high performance size-exclusion chromatography analysis. Sugar composition analysis revealed that Tansy polysaccharides consisted primarily of galacturonic acid, galactose, arabinose, and rhamnose. Fractions T-II through T-IV contained an arabinogalactan type II structure, as determined by reaction with Yariv reagent. High Mr fractions T-I and T-II exhibited potent macrophage/monocyte-activating activity, enhancing production of reactive oxygen species (ROS), nitric oxide (NO), and tumor necrosis factor α (TNF-α) by J774.A1 murine macrophages, and activating nuclear factor κB (NF-κB) in THP-1 human monocytes. In addition, Tansy polysaccharides stimulated human neutrophil function by greatly enhancing neutrophil myeloperoxidase (MPO) release. Furthermore, the low Mr fraction T-IV had potent complement-fixing activity, which may also contribute to the anti-inflammatory and would-healing properties of Tansy extracts. Taken together, our results provide a molecular basis to explain at least part of the beneficial therapeutic effects of Tansy extracts, and support the concept of using Tansy polysaccharides as an immunotherapeutic adjuvant.

Structural and immunological studies of a pectin and a pectic arabinogalactan from Vernonia kotschyana Sch. Bip. ex Walp. (Asteraceae)

Two polysaccharides, a pectin (Vk100A2b) and a pectic arabinogalactan (Vk100A2a) with mean M w 2 × 10 4 and 1.15 × 10 6 Da, respectively, were isolated from the dried powdered roots of Vernonia kotschyana Sch. Bip. ex Walp. by hot water extraction followed by fractionation on DEAE-Sepharose fast flow and Sephacryl S-400 HR. The pectin showed low-complement fixation activity and no influence on proliferation of B or T cells, while the pectic arabinogalactan showed a potent, dose-dependent complement fixation activity and a T cell independent induction of B-cell proliferation. Both polysaccharides induced chemotaxis of human macrophages, T cells and NK cells. exo-α- l-arabinofuranosidase and exo-β- d-galactosidase digestion followed by component sugar and methylation analysis indicated that Vk100A2a consisted of a highly branched rhamnogalacturonan core with approximately 50% of the rhamnose 1,2,4-substituted, side chains rich in terminal-, 1,5-linked and 1,3,5-branched arabinose and terminal-, 1,4-, 1,6-linked and 1,3,6-branched galactose. The enzyme resistant part of Vk100A2a still showed strong complement fixating activity, suggesting that this activity may at least in part be expressed by carbohydrate structures present in the enzyme resistant, inner portion of the polymer.

The Complement—Fixing Activity of Immune Complexes Containing IgG Antibodies of Different Functional Affinities: Effects on Superoxide Production by Rabbit Neutrophils

When neutrophil phagocytes are stimulated by IgG containing immune complexes (IgG‐IC), with or without the participation of the complement system, they show a sharp increase in oxygen uptake and begin to release large quantities of superoxide anions (O2−) and hydrogen peroxide (H2O2) into the surrounding medium. The aim of the present investigation was to provide insights into the production and release of O2− by rabbit neutrophils activated with immune complexes (IC) containing IgG antibodies of different functional affinity, opsonized and not opsonized by complement system components. For this purpose, two populations of polyclonal anti‐ovalbumin (OVA) IgG antibodies with different functional affinity, 5 × 108 M− 1 and 2 × 107 M− 1, were prepared. The production of O2− was measured spectrophotometrically by a method using the superoxide dismutase‐inhibited reduction of ferricytochrome C to the ferrous form. The activation of complement by different IgG‐IC was determined by estimating the total residual haemolytic activity of the alternative and classical pathways in sera treated with different concentrations of anti‐OVA IgG/OVA immune complexes formed at equivalence. The results showed that: 1) antibody functional affinity influenced O2− production and the complement‐fixing activity induced by the IC. In general, the higher functional affinity antibodies were more efficient in stimulating the respiratory burst of neutrophils and in activating complement by the classical and alternative pathways than the lower functional affinity antibodies at all IC concentrations tested; 2) complement components incorporated into the immune complex lattice caused an increase in the stimulatory activity of both IgG antibodies to produce O2− (≅ 15% for the IC of IgG with Ka = 5 × 108 M− 1 and ≅ 7% for the IC of IgG with Ka = 2 × 107 M− 1). This effect was dependent on antibody affinity and concentration; 3) there was a direct relationship between the overall level of complement activation, antibody affinity and superoxide production by neutrophils. Thus, we conclude that antibody affinity influences immune complex lattice formation, modulating its three‐dimensional structure and the disposition of Fc fragments interfering with the antibody's biological properties. These results can help understand the precise role of antibody functional affinity in antigen‐antibody complex diseases and define the immunochemical characteristics of pathogenic complexes.

Isolation and partial characterization of a pentraxin-like protein with complement-fixing activity from snapper ( Pagrus auratus, Sparidae) serum

Pentraxin-like molecules have been isolated from a number of fish species. However, little is known about the function of these proteins in the teleosts. In this study we report the isolation and characterization of a pentraxin-like molecule from the serum of snapper ( Pagrus auratus) that has the ability to activate complement. This pentraxin-like protein was isolated from serum by calcium-dependent binding to agarose. SDS–PAGE analysis demonstrated an oligomeric protein of approximately 200 kDa consisting of non-covalently bound subunits of 26 and 23 kDa. Protein sequencing revealed significant (50%) sequence identity with pentraxins from both Atlantic salmon ( S. salar) and rainbow trout ( O. mykiss). However, polyclonal antibodies raised against snapper pentraxin did not recognise salmon or trout pentraxin in Western blot analysis. Following LPS injection, snapper pentraxin levels increased 2-fold before gradually returning to basal levels. Most significantly, the isolated pentraxin initiated complement-mediated lysis of ligand-coated sheep erythrocytes in a dose-dependent fashion. In view of the similarity between the known fish pentraxins, and their similarity to mammalian serum amyloid P-components we conclude that the isolated protein may be a snapper pentraxin homologue.