Laminarin Research Articles

The involvement of C5a in the progression of experimental arthritis with Porphyromonas gingivalis infection in SKG mice

BackgroundEpidemiological evidence to suggest that periodontal disease (PD) is involved in the progression of rheumatoid arthritis (RA) is increasing. The complement system plays a critical role in immune responses. C5a has been implicated in chronic inflammatory diseases, including PD and RA. Porphyromonas gingivalis is the major causative bacteria of PD and can produce C5a. Therefore, it is hypothesized that P. gingivalis infection is involved in the progression of RA by elevating C5a levels. In the present study, P. gingivalis–infected RA model mice were established to investigate the involvement of C5a.MethodsSKG mice orally infected with P. gingivalis were immunized with intraperitoneal injection of laminarin (LA) to induce arthritis. Arthritis development was assessed by arthritis score (AS), bone destruction on the talus, histology, and serum markers of RA. In order to investigate the effects of serum C5a on bone destruction, osteoclast differentiation of bone marrow mononuclear cells was examined by using serum samples from each group of mice. The relationship between C5a levels and antibody titers to periodontal pathogens in patients with RA was investigated by enzyme-linked immunosorbent assay.ResultsP. gingivalis oral infection increased AS, infiltration of inflammatory cells, bone destruction on the talus, and serum markers of RA in mice immunized with LA. The addition of serum from LA-injected mice with the P. gingivalis oral infection promoted osteoclast differentiation, and the addition of a neutralization antibody against C5a suppressed osteoclast differentiation. C5a levels of serum in RA patients with positive P. gingivalis antibody were elevated compared with those in RA patients with negative P. gingivalis antibody.ConclusionsThese results suggest that P. gingivalis infection enhances the progression of RA via C5a.

Multivalent and multifunctional polysaccharide-based particles for controlled receptor recognition

Polysaccharides represent a versatile class of building blocks that are used in macromolecular design. By choosing the appropriate saccharide block, various physico-chemical and biological properties can be introduced both at the level of the polymer chains and the resulting self-assembled nanostructures. Here, we synthetized amphiphilic diblock copolymers combining a hydrophobic and helical poly(γ-benzyl-L-glutamate) PBLG and two polysaccharides, namely hyaluronic acid (HA) and laminarin (LAM). The copolymers could self-assemble to form particles in water by nanoprecipitation. In addition, hybrid particles containing both HA and LAM in different ratios were obtained by co-nanoprecipitation of the two copolymers. By controlling the self-assembly process, five particle samples with different morphologies and compositions were developed. The interaction between the particles and biologically relevant proteins for HA and LAM, namely CD44 and Dectin-1 respectively, was evaluated by surface plasmon resonance (SPR). We demonstrated that the particle-protein interaction could be modulated by the particle structure and composition. It is therefore suggested that this method based on nanoprecipitation is a practical and versatile way to obtain particles with controllable interactions with proteins, hence with the appropriate biological properties for biomedical applications such as drug delivery.

Laminarin improves developmental competence of porcine early stage embryos by inhibiting oxidative stress

Laminarin (LMA), a β-glucan mixture with good biocompatibility, improves the growth performance and immune response when used as food additives and nutraceuticals. The aim of the present research was to explore the effects of LMA on porcine early stage embryo development, as well as the underlying mechanisms. The results showed that the developmental competence of porcine early stage embryos was dramatically improved after LMA supplementation during the in vitro culture period. The presence of 20 μg/mL LMA during the in vitro culture period significantly improved cleavage rate, blastocyst formation rates, hatching rate, and total cell number in the blastocyst compared to that in the control group. Notably, LMA attenuated the intracellular reactive oxygen species generation induced by H2O2. Furthermore, LMA not only increased intracellular glutathione levels, but also ameliorated mitochondrial membrane potential. In addition, the expression of a zygotic genome activation related gene (YAP1), pluripotency-related genes (OCT4, NANOG, and SOX2), and hatching-related genes (COX2, GATA4, and ITGA5) were up-regulated following LMA supplementation during porcine early stage embryo development. These results demonstrate that LMA has beneficial effects on the development of porcine early stage embryos via regulation of oxidative stress. This evidence provides a novel method for embryo development improvement associated with exposure to LMA.

Laminarin enhances the quality of aged pig oocytes by reducing oxidative stress.

Laminarin (LAM) is a β-glucan oligomer known to possess biological activities such as anticancer and antioxidant effects. This study explored the influence of LAM supplementation on in vitro aged porcine oocytes and the underlying mechanisms behind this influence. We found that LAM delayed the aging process and improved the quality of aged oocytes. LAM supplementation enhanced the subsequent developmental competence of aged oocytes during the in vitro aging process. The blastocyst formation rate was significantly increased in aged oocytes treated with 20 µg/ml LAM compared to non-treated aged oocytes (45.3% vs. 28.7%, P < 0.01). The mRNA levels of apoptosis-related genes, B cell lymphoma-2-associated X protein (Bax) and Caspase-3, were significantly lower in blastocysts derived from the LAM-treated aged oocytes during the in vitro aging process. Furthermore, the level of intracellular reactive oxygen species was significantly decreased and that of glutathione was significantly increased in aged oocytes following LAM treatment. Mitochondrial membrane potential was increased, and the activities of caspase-3 and cathepsin B were significantly reduced in the LAM-treated aged oocytes compared with the non-treated aged oocytes. Taken together, these results suggest that LAM is beneficial for delaying the aging process in porcine oocytes.

Extracts of laminarin and laminarin/fucoidan from the marine macroalgal species Laminaria digitata improved growth rate and intestinal structure in young chicks, but does not influence Campylobacter jejuni colonisation

Promoting growth performance, while limiting the proliferation of bacteria such as Campylobacter jejuni, in the post-hatch period is a key goal of the broiler industry. Therefore, the objective of this study was to evaluate the effects of supplementing the diet with Laminarin and Fucoidan extracts on growth performance, small intestinal morphology and function, immune response and Campylobacter jejuni colonisation following an experimental challenge in young chicks following dietary supplementation. The experiment consisted of three diets: 1) basal diet; 2) basal diet+250ppm Laminarin (LAM); 3) basal diet+250ppm LAM+80ppm FUC (LAM/FUC). Day old Ross chicks (n=135), were housed in groups of three, with 15 replicates per treatment group. On day three, all chicks were orally gavaged with 0.1ml 106 colony forming units of C. jejuni. On day 13, caecal digesta samples were collected for quantification of C. jejuni and Lactobacillus. Ileal tissue was also collected post-slaughter in order to examine small intestinal morphology and the gene expression of targets related to gut health. The mean total intake for the chicks fed the LAM, LAM/FUC and basal diets during the experiment (day 0–13) were 411g, 411g and 373g respectively, with chicks fed the LAM and LAM/FUC supplemented diets having increased feed intake compared to the basal diet fed chicks (P<0.05). Chicks offered diets containing LAM and the LAM/FUC combination had higher (P<0.05) total weight gain (262g and 254g respectively) compared to the basal diet 243g. Dietary inclusion of LAM/FUC combination increased feed conversion ratio (FCR) (1.68g/g vs 1.77g/g) compared to the basal diet (P<0.05). Chicks offered the LAM and LAM/FUC diet had increased ileal villus width compared to chicks offered the basal diet, while chicks fed the LAM diet had increased villus height compared to chicks fed the basal diet (P<0.05). Chicks offered the both the LAM and LAM/FUC supplemented diets had increased gene expression of the tight junction protein CLDN1, compared to chicks fed the basal diet (P<0.05). The gene expression of the measured intestinal nutrient transporters was unaffected by dietary supplementation (P>0.10). There was no effect of LAM or LAM/FUC extracts on the proliferation of C. jejuni or on Lactobacilli numbers in the caeca. In conclusion, supplementation with LAM or LAM/FUC in the post-hatch period improved growth rate, positively modified small intestinal architecture and impacted the intestinal immune response, but did not impact the extent of C. jejuni proliferation. Supplementation of the diet with LAM provided beneficial effects over supplementation with LAM/FUC in relation to FCR, villus height and aspects of the immune response.

Dectin-1 Compromises Innate Responses and Host Resistance against Neospora caninum Infection.

Neospora caninum is an intracellular protozoan parasite that has drawn increasing interest due to its association with worldwide repetitive bovine abortions, which cause billionaire losses to the meat and dairy industries annually. Innate immunity plays an important role in infection control, and N. caninum activates the production of inflammatory mediators through toll-like receptors, NOD-like receptors, and mitogen-activated protein kinase signaling pathways. Advances in the knowledge of initial host–parasite interactions are desirable for the design of control measures against the infection, obliterating its pathogenesis. In that sense, we here aimed to describe the role of the innate C-type lectin receptor Dectin-1 during the infection by N. caninum. With that intent, we observed that the absence of Dectin-1, observed in genetically depleted (Dectin-1−/−) mice or competitively inhibited by an inert agonist [laminarin (LAM)], rescued 50% of the mice infected with lethal doses of N. caninum. Dectin-1−/− and LAM-treated mice also presented a reduction in the parasite load during acute and chronic phases, associated with decreased inflammatory scores in the central nervous system. Among all the cell phenotypes that migrated to the initial site of infection, dendritic cells and macrophages gained subpopulations with high Dectin-1 surface expression. The impairment of the receptor in these cells led to a decreased parasite burden, as well as augmented production of IL-12p40. We also found that Dectin-1+ cells produced less reactive oxygen species (ROS) at the initial site of the infection, while mice deficient in NADPH oxidase isoform 2 (NOX2−/−) were not able to control parasite replication and produce IL-12p40, even upon LAM treatment. Interestingly, the absence of functional Dectin-1 did not alter the susceptibility of mice against closely related Toxoplasma gondii. In conclusion, the gathered data suggest that Dectin-1 is involved in the parasite-induced downmodulation of ROS, and other key molecules triggered for the control of N. caninum infection and are a promising target for future development of protocols intended for intervention against neosporosis.

Maternal laminarin supplementation decreases Salmonella Typhimurium shedding and improves intestinal health in piglets following an experimental challenge with S. Typhimurium post-weaning

During the period immediate post-weaning (PW), piglets become vulnerable to pathogenic bacteria such as Salmonella Typhimurium, the most common Salmonella serovar. Laminarin (LAM), a β-glucan derived from seaweed, is known to have antimicrobial and anti-inflammatory activity when supplemented maternally or directly to the piglet. The aim of this study was to investigate the effects of supplementing sows and/or piglets post-weaning with LAM after an experimental challenge with S. Typhimurium. Gestating sows (n=24) were supplemented with LAM (0 vs. 1.0g/d) from day 107 of gestation until weaning (day 20). The lactation and PW diet were arranged in a 2×2 factorial to provide four treatment groups: (1) pigs weaned from basal fed sows and offered a basal diet post-weaning (n=12; BB); (2) pigs weaned from basal fed sows and offered a LAM supplemented diet post-weaning (n=12; BL); (3) pigs weaned from LAM supplemented sows and offered a basal diet post-weaning (n=12; LB); (4) pigs weaned from LAM supplemented sows and offered a LAM supplemented diet post-weaning (n=12; LL). The LAM supplemented post-weaning diets contained 300ppm LAM/t of feed. On day 10 PW, piglets were challenged with 108 CFU S. Typhimurium and euthanized on day 26 PW to facilitate sample collection for microbial populations, volatile fatty acid (VFA) and immune related gene expression analysis. Pigs were weighed at birth, at weaning, at challenge and at slaughter and feed intake post-weaning was recorded. Faeces were visually scored for diarrhoea. Piglets weaned from LAM supplemented sows had improved average daily gain (ADG) and feed efficiency post-challenge (P<0.05). The faecal score was improved when LAM was supplemented maternally or post-weaning (P<0.001). Maternal supplementation of LAM reduced faecal counts of S. Typhimurium by 0.35 log gene copy numbers post-challenge (measured on day 2, 5 and 7; P<0.05). Total VFA production in the colon was increased after maternal or post-weaning LAM supplementation (P<0.05). The expression of IL-22 in the colon was reduced after maternal LAM supplementation (P<0.05). It can be concluded that, after an experimental challenge with S. Typhimurium, maternal LAM supplementation resulted in improved feed efficiency, faecal scores and increased the VFA production in the colon, while reducing faecal counts of S. Typhimurium and gene expression of IL-22 in the colon.

Zinc methionine and laminarin have growth-enhancing properties in newly weaned pigs influencing both intestinal health and diarrhoea occurrence.

Three experiments were conducted to investigate the interaction between zinc methionine (ZnM) and laminarin (LAM) on piglet growth performance and intestinal health post-weaning. Experiment 1 was designed as 2×2 factorial with four treatments [n=8, weaning age (WA) 24days, live weight (LW) 7.15kg]: (i) basal diet (BD); (ii) BD+500mg/kg ZnM; (iii) BD+300mg/kg LAM; and (iv) BD+500mg/kg ZnM+300mg/kg LAM. There was an interaction (p<0.05) between LAM and ZnM. Pigs that were offered the LAM diet had a similar performance to the BD. However, when combining LAM with ZnM, pigs had reduced average daily gain (ADG), gain-to-feed ratio (G:F) and LW at slaughter at day 8 post-weaning compared to the ZnM. Both LAM and ZnM improved the small intestinal morphology of the pigs at day 8 post-weaning. Experiment 2 was designed as 2×2 factorial with four dietary treatments (n=9, WA 24days, LW 7.32kg): (i) BD; (ii) BD+500mg/kg ZnM; (iii) BD+175mg/kg LAM; and (iv) BD+500mg/kg ZnM+175mg/kg LAM. The ADG and average daily feed intake were improved between day 0 and 31 PW when pigs were offered a LAM diet (p<0.01). Faecal scores were reduced between day 0 and day 31 post-weaning with ZnM (p<0.001). Experiment 3 consisted of four dietary treatments (n=10, WA 24days, LW 7.32kg): (i) BD; (ii) BD+3300mg/kg zinc oxide (ZnO); (iii) BD+500mg/kg ZnM; and (iv) BD+175mg/kg LAM. Pigs that were offered the ZnO diet had an increased ADG compared to the BD or ZnM diets (p<0.01). Pigs that were offered the LAM diet had increased ADG compared to the ZnM diet (p<0.05). Faecal scores were reduced between day 0 and day 31 PW with ZnM or ZnO supplementation (p<0.001). In conclusion, the inclusion of 175mg/kg LAM and ZnO improved ADG while both ZnO and ZnM reduced the faecal scores post-weaning.

Porphyromonas gingivalis infection exacerbates the onset of rheumatoid arthritis in SKG mice.

Epidemiological studies have linked periodontitis to rheumatoid arthritis (RA). Porphyromonas gingivalis (Pg) was reported recently to produce citrullinated protein (CP) and increase anti-cyclic CP antibody (ACPA), both of which have been identified as causative factors of RA. In the present study, we determined the effects of Pg infection on the exacerbation of RA in a mouse model. RA model mice (SKG mice) were established by an intraperitoneal (i.p.) injection of laminarin (LA). Mice were divided into six groups, Ctrl (PBS injection), LA (LA injection), Pg/LA (Pg + LA injection), Pg (Pg injection), Ec/LA (Escherichia coli and LA injection) and Ec (E. coli injection). In order to evaluate RA, joint swelling by the arthritis score, bone morphology by microcomputed tomography (microCT), haematoxylin and eosin staining, ACPA, matrix metalloproteinase-3 (MMP-3) and cytokine level in serum by enzyme-linked immunosorbent assay were determined. Osteoclast differentiation from bone marrow mononuclear cells (BMCs) was examined to clarify the underlying mechanisms of RA. The presence of Pg and CP in joint tissue was also investigated. The arthritis score was threefold higher in the Pg/LA group than in the LA group. Severe bone destruction was observed in joint tissue of the Pg/LA group. A microCT analysis of the Pg/LA group revealed a decrease in bone density. ACPA, MMP-3, interleukin (IL)-2, IL-6, CXCL1 and macrophage inflammatory protein (MIP)-1α levels from the Pg/LA group were the highest. The osteoclastogenesis of BMCs was enhanced in the Pg/LA group. Furthermore, large amounts of Pg components and CP were detected in the Pg/LA group. In conclusion, Pg infection has the potential to exacerbate RA.

The effect of algal polysaccharides laminarin and fucoidan on colonic pathology, cytokine gene expression and Enterobacteriaceae in a dextran sodium sulfate-challenged porcine model.

The algal polysaccharides laminarin (LAM) and fucoidan (FUC) have potent anti-inflammatory activities in the gastrointestinal tract. Our objective was to examine the impact of prior consumption of LAM and/or FUC on pathology and inflammation following a dextran sodium sulfate (DSS) challenge in pigs. Pigs (n 7/group) were assigned to one of five experimental groups for 56 d. From 49-55 d, distilled water or DSS was administered intragastrically. The experimental groups were: (1) basal diet + distilled water (control); (2) basal diet + DSS (DSS); (3) basal diet + FUC + DSS (FUC + DSS); (4) basal diet + LAM + DSS (LAM + DSS); and (5) basal diet + LAM + FUC + DSS (LAMFUC + DSS). The DSS group had decreased body-weight gain (P < 0·05) and serum xylose (P < 0·05), and increased proximal colon pathology score (P < 0·05), diarrhoeal score (P < 0·001) and colonic Enterobacteriaceae (P < 0·05) relative to the control group. The FUC + DSS (P < 0·01), LAM + DSS (P < 0·05) and LAMFUC + DSS (P < 0·05) groups had improved diarrhoeal score, and the LAMFUC + DSS (P < 0·05) group had improved body weight relative to the DSS group. The FUC + DSS group (P < 0·001), LAM + DSS group (P < 0·05) and LAMFUC + DSS group (P < 0·001) had lower IL-6 mRNA abundance relative to the DSS group. The LAM + DSS group had reduced Enterobacteriaceae in proximal colon digesta relative to the DSS group (P < 0·05). In conclusion, FUC or a combination of FUC and LAM improved body-weight loss, diarrhoeal scores and clinical variables associated with a DSS challenge in pigs, in tandem with a reduction in colonic IL-6 mRNA abundance.

Dectin-1, a C-type lectin receptor, participates in the killing of Candida albicans by human neutrophils: an experimental study

Objective To investigate whether human neutrophils kill Candida albicans through recognition of insoluble β-glucan in cell walls of C.albicans (CalG) by dectin-1,a C-type lectin receptor.Methods Neutrophils were obtained from peripheral blood of healthy human subjects and cultured in vitro.Real-time PCR was carried out to quantify the mRNA expressions of dectin-1 and Toll-like receptor 2 (TLR2) in neutrophils challenged with CaIG of 100 mg/L for 1,6,and 24 hours.A Fluoro hydrogen peroxide (H2O2) detection kit was used to determine H2O2 levels in some neutrophils exposed to CaIG (100 mg/L) for 15 minutes,2 hours,6 hours,as well as in some neutrophils preincubated with laminarin (a dectin-1 inhibitor) of 100 mg/L and 50 mg/L for 30 minutes followed by challenge with CaIG of 100 mg/L for 2 hours.Colony forming units (CFUs) were counted after the incubation of C.albicans with neutrophils pretreated with laminarin of 100 mg/L and 50 mg/L for 30 minutes.Results The relative mRNA expression level of dectin-1 was 2.8195 + 0.1669,5.4859 + 0.7244 and 3.6041 + 0.5372 in neutrophils challenged with CaIG for 1,6 and 24 hours,respectively,significantly higher than that in unchallenged neutrophils at these corresponding time points (all P ＜ 0.01).The level of H2O2 was (64.55 + 15.67),(290.34 + 30.56),and (208.54 ± 26.88) μ mol/L respectively in neutrophils treated with CaIG for 15 minutes,2 hours,and 6 hours respectively,compared to (22.05 ± 3.99) μmol/L in untreated neutrophils (all P ＜ 0.01).The pretreatment with laminarin of 100 and 50 mg/L attenuated the release of H2O2 in CaIG-treated neutrophils by 73％ ((80.45 + 22.41) μ mol/L,P＜ 0.01) and 45％ ((130.42 + 44.55) μmol/L,P＜ 0.01),respectively,compared with neutrophils treated with CaIG only.The fungicidal activity of neutrophils against C.albicans was also significantly inhibited by pretreatment with laminarin of 50 and 100 mg/L (both P＜ 0.01).Conclusions Dectin-1 may be involved in the secretion of H2O2 as well as killing of C.albicans by human neutrophils,which may provide a preliminary evidence for adoptive transfer of neutrophils as an approach to the treatment of systemic C.albicans infection. Key words: Candida albicans; Neutrophils; beta-Glucans; Lectins, c-type; Hydrogen peroxide

Effect of supplementing varying inclusion levels of laminarin and fucoidan on growth performance, digestibility of diet components, selected faecal microbial populations and volatile fatty acid concentrations in weaned pigs

A 3×2 factorial experiment was conducted to investigate the effect of dietary supplementation of purified laminarin (LAM) and fucoidan (FUC), independently or in combination on growth performance, coefficient of total tract apparent digestibility (CTTAD), selected faecal microbial populations and volatile fatty acid concentrations (VFA) in weaned pigs. At weaning, 168 pigs (24 days of age, 6.8±0.6kg live weight) were assigned to one of six dietary treatments (n=14): (1) basal diet; (2) basal diet+240mg/kg FUC; (3) basal diet+150mg/kg LAM; (4) basal diet+150mg/kg LAM and 240mg/kg FUC; (5) basal+300mg/kg LAM; (6) basal diet+300mg/kg LAM and 240mg/kg FUC. The pigs were offered the diets ad libitum for 35 days post-weaning. Pigs offered 300mg/kg LAM supplemented diets had a higher average daily gain (ADG) throughout the entire experimental period (d 0–35) compared with pigs offered diets with 0mg/kg and 150mg/kg LAM supplementation (0.374kg/d vs. 0.346kg/d vs. 0.340kg/d, SEM±0.0094; P<0.05). There was an interaction between LAM and FUC supplementation on gain to feed (G:F) ratio (P<0.05) during the entire experimental period. Pigs offered the 300mg/kg LAM diet had a higher G:F ratio than pigs offered the basal diet. However, there was no effect of the 300mg/kg LAM on overall G:F ratio when combined with FUC. There was an interaction between LAM and FUC supplementation on the CTTAD of gross energy (GE) (P<0.05). Pigs offered the 150mg/kg or 300mg/kg LAM diets had an increased CTTAD of GE compared with pigs offered the basal diet. However, there was no further effect of LAM on the CTTAD of GE when combined with FUC. Pigs offered diets supplemented with FUC had an increased faecal Lactobacillus population compared with pigs offered diets without FUC supplementation (10.11 gene copy number/g vs. 9.62 gene copy number/g, SEM±0.173; P<0.05). Pigs offered the 150mg/kg and 300mg/kg LAM supplemented diets had an improved faecal consistency from d 7–14 compared with pigs offered diets without LAM supplementation (3.14 vs. 3.00 vs. 3.49, SEM±0.348; P<0.05). In summary, the inclusion of 300mg/kg LAM showed the greatest benefit in growth performance with improvements in ADG and G:F ratio partially due to an increased CTTAD of diet components and lower faecal score.

Effect of supplementing different ratios of laminarin and fucoidan in the diet of the weanling piglet on performance, nutrient digestibility, and fecal scoring

A 3 × 2 factorial experiment was conducted to investigate the effects of different ratios of dietary laminarin (LAM) and fucoidan (FUC) on growth performance and nutrient digestibility in newly weaned piglets. At weaning, 168 piglets (24 d of age; 6.8 kg live weight) were assigned to 1 of 6 dietary treatments (n = 14/treatment): (i) basal diet, (ii) basal diet + 240 mg/kg FUC, (iii) basal diet + 150 mg/kg LAM, (iv) basal diet + 150 mg/kg LAM and 240 mg/kg FUC, (v) basal diet + 300 mg/kg LAM, and (vi) basal diet + 300 mg/kg LAM and 240 mg/kg FUC. Pigs offered 300 mg/kg LAM-supplemented diets had a higher ADG (P < 0.05) throughout the entire experimental period (days 0-35) compared with pigs offered diets with 0 and 150 mg/kg LAM supplementation. There was a significant interaction between LAM and FUC supplementation on G:F (P < 0.05) during the entire experimental period. Pigs offered the 300 mg/kg LAM-supplemented diet had a higher G:F than pigs offered the basal diet. However, there was no effect of the high level LAM on G:F when combined with FUC. There was a significant interaction between LAM and FUC supplementation on the apparent total tract digestibility (ATTD) of GE (P < 0.05). Pigs offered the 150 and 300 mg/kg LAM diet had an increased ATTD of GE compared with pigs offered the basal diet. However, there was no effect of LAM on ATTD of GE when combined with FUC. Pigs offered the 150 and 300 mg/kg LAM-supplemented diets had an improved fecal consistency (P < 0.05) from day 7 to day 14 compared to pigs offered diets without LAM supplementation. In conclusion, the inclusion of LAM improved growth performance of pigs after weaning partially due to an increased ATTD of GE.

Effects of supplementing dietary laminarin and fucoidan on intestinal morphology and the immune gene expression in the weaned pig

Previous studies have shown the benefit of combining seaweed extracts laminarin (LAM) and fucoidan (FUC) on improving growth in piglets' performance after weaning. A 2 × 2 factorial experiment was conducted to investigate the interaction between FUC (0 and 240 mg/kg) and LAM (0 and 300 mg/kg) levels on gut morphology and colonic cytokine gene expression in the weaned pig. Twenty-eight piglets (6.9 kg BW) were assigned to 1 of 4 dietary treatments for 8 d after weaning and then sacrificed. The dietary treatments were as follows: (i) basal diet, (ii) basal diet + 240 mg/kg FUC, (iii) basal diet + 300 mg/kg LAM, and (iv) basal diet + 300 mg/kg LAM and 240 mg/kg FUC. There was an interaction (P < 0.01) between LAM and FUC supplementation on duodenal villous height and the villus height to crypt depth ratio. Pigs offered the LAM or FUC diet singularly had an increased villus height and villus height to crypt depth ratio compared with pigs offered the basal diet. However, there was no effect of the LAM and FUC combination diet on morphology. Pigs offered the LAM-supplemented diets had a lower IL-6 (P < 0.05), IL-17 (P < 0.001), and IL-1β (P < 0.001) mRNA expression in the colon compared with pigs offered diets without LAM supplementation. In conclusion, the enhancement in intestinal structure and downregulation of inflammatory cytokine gene expression obtained suggest that LAM may provide a dietary means to improve gut health in weaned pigs.

Characterization of a natural hemolysin in the serum of a hermit crab Clibanarius longitarsus (Crustacea: Decapoda)

A naturally occurring hemolysin (HL) was detected in the serum of the hermit crab Clibanarius longitarsus using mammalian RBC as indicator cells. The serum gave the highest HL titer with rabbit RBC. Cross adsorption of C. longitarsus serum with fixed rabbit RBC resulted in the complete removal of the lytic activity from the serum. The hemolytic activity in the serum of C. longitarsus was independent of divalent cations, reversibly sensitive to EDTA, thermolabile, stable between the pH 6-8. Further studies demonstrated that the HL activity is proteinaceous as it was precipitable by conventional deproteinizing agents and susceptible to the action of proteases and 2-mercapthoethanol. The HL-inhibition assay performed with several carbohydrates (mono, di, oligo, polysaccharides and Lipo polysaccharides) revealed that the serum HL was capable of specifically recognizing laminarin (a homopolymer of β 1, 3-glucan). Thus this hemolysin appears to be unique among all the known crustacean hemolysins.

EFFECT OF 1,3;1,6-β-D-GLUCAN ON THE FORMATION OF ABNORMAL TOBACCO MOSAIC VIRUS PARTICLES IN THE LEAVES OF NICOTIANA TABACUM cv. SAMSUN

The carbohydrate-containing polymer 1,3;1,6-s-Dglucan was obtained by transformation of laminaran from the alga Laminaria cichorioides with endo-s-1,3- glucanase from marine mollusks. In electron microscope observations of phosphotungstic acid-stained preparations from Nicotiana tabacum cv. Samsun leaves inoculated with a mixture of Tobacco mosaic virus (TMV) (1 µg/ml) and glucan (1 mg/ml) or with TMV alone, we found that such preparations contained, along with virus particles of normal diameter (about 18 nm), abnormal (swollen and thinner) particles. The highest number of thin viral particles was found in dips from leaves inoculated with TMV together with glucan. It is suggested that this may be caused by a glucan-mediated increase of TMV particle proteolysis in infected leaves.

Effects of abiotic and biotic elicitors on growth and isoflavonoid accumulation in Pueraria candollei var. candollei and P. candollei var. mirifica cell suspension cultures

This study demonstrates the effects of various concentrations of abiotic and biotic elicitors on the cell growth and isoflavonoid accumulation of P. candollei var. mirifica (PM) and P. candollei var. candollei (PC) cell suspension cultures. The two plant varieties exhibited different growth responses and varied isoflavonoid accumulation after the addition of elicitors. Copper sulfate, methyl jasmonate (MeJA), and yeast extract did not significantly affect the growth of either plant variety, whereas oligosaccharide and the biotic elicitors used in this study [i.e., 50 mg l−1 chitosan and all concentrations of laminarin (LAM)] suppressed the growth of PM. The addition of MeJA to the medium principally induced an effect on the isoflavonoid content in both PM and PC, with 2.0 μM MeJA inducing the highest isoflavonoid content, as indicated by the induction index—4.41 in PM and 9.62 in PC cells on the 12th and ninth day of culture, respectively. A maximum total isoflavonoid content of 40.49 mg g−1 dry weight was achieved in PM 21 days after elicitation with 2.0 μM MeJA. LAM elicited the PM cell suspension culture to produce puerarin, which was not found in the unelicited culture. The results of this study provide information that will be useful for enhancing the accumulation of isoflavonoids in P. candollei cell suspension cultures.

Dietary supplementation with laminarin, a fermentable marine β (1–3) glucan, protects against hepatotoxicity induced by LPS in rat by modulating immune response in the hepatic tissue

We tested the hypothesis that laminarin (LAM), a β (1–3) polysaccharide extracted from brown algae, can modulate the response to a systemic inflammation. Male Wistar rats ( n = 7 per group) were fed a standard diet (control) or a diet supplemented with LAM for 25 days (5% during 4 days followed by 10% during 21 days). Thereafter, Escherichia coli lipopolysaccharides (LPS; 10 mg/kg i.p.) were injected and the animals were sacrificed 24 h after LPS challenge. The hypothermia, hyperglycemia and hypertriglyceridemia occurring early after LPS administration were less pronounced in LAM-treated rats than in controls. The increase in serum alanine aminotransferase (ALT), aspartate aminotransferase (AST) and lactate dehydrogenase (LDH) activities – reflecting hepatic alterations – was lessened after LPS injection in LAM-treated rats compared to control rats. LAM treatment decreased serum monocytes number, nitrite (NO 2) and tumor necrosis factor-alpha (TNF-α). LAM also modulated intra-hepatic immune cells: it lowered the occurrence of peroxidase-positive cells (corresponding to monocytes/neutrophils) and, in contrast, it increased the number of ED2-positive cells, corresponding to resident hepatic macrophages, i.e. Kupffer cells. In conclusion, the hepatoprotective effect of marine β (1–3) glucan during endotoxic shock may be linked to its immunomodulatory properties. We propose that both lower recruitment of inflammatory cells inside the liver tissue and lower secretion of inflammatory mediators play a role in the tissue protective effect of LAM. These effects could be due to a direct effect of β-glucan on immune cells, or to an indirect effect through their dietary fibre properties (fermentation in the gut).

Cell Wall .BETA.-Glucan Derived from Candida albicans Acts as a Trigger for Autoimmune Arthritis in SKG Mice

SKG mice are a recently established experimental model for rheumatoid arthritis (RA). Although they spontaneously develop chronic autoimmune arthritis under conventional conditions, SKG mice failed to develop chronic arthritis in a strictly controlled specific pathogen-free (SPF) environment. Beta-glucan (BG) from Laminaria digitata, laminarin (LAM), induced arthritis under SPF conditions, thus BG would be a pathogenic factor for arthritis in SKG mice. Therefore, we prepared BG from Candida albicans, a pathogenic fungus and investigated whether BG from C. albicans induced arthritis in SKG mice under SPF conditions. SKG mice were injected intraperitoneally with particulate BG (oxidative-Candida albicans (OX-CA)), soluble BG (Candida soluble beta-glucan (CSBG)) from C. albicans and LAM as a positive control. In addition, schizophyllan (SPG) from Schizophyllum commune or Mycobacterium whole cells were injected into SKG mice to induce arthritis. Mice injected with OX-CA, CSBG and SPG had more severe arthritis than with LAM, and whole Mycobacterium cells. IL-6 concentration in sera from SKG mice injected with OX-CA or CSBG was high, whereas not detected in sera from mice treated with LAM. In histological analysis, infiltration of inflammatory cells was observed in SKG mice injected with BG. These results suggest that fungal infection may be a factor to induce and exacerbate autoimmune diseases such as RA.

Structure-activity relationship of (1-->3)-beta-D-glucans in the induction of cytokine production from macrophages, in vitro.

In a previous study, we reported that one of the gel-forming (1-->3)-beta-D-glucans, grifolan (from Grifola frondosa, GRN), stimulated cytokine production from macrophages in vitro. However, several other gel-forming (1-->3)-beta-D-glucans, such as sonifilan (SPG) and SSG, did not induce cytokine production from macrophages. The ultrastructure of gel-forming (1-->3)-beta-D-glucans, especially the triple- and single-helix, does not affect the cytokine-inducing activity. The action on tumor necrosis factor alpha (TNF alpha) release was correlated with the molecular weight of GRN, since the highest molecular weight fraction of GRN, Mr > or = 45000, exhibited the strongest activity. Although, native SSG (Mr > or = 2000000) did not induce cytokine production, chemical modification involving debranching of the side chain glucosyl residues of SSG resulted in TNF alpha inducing activity. These results suggest that the branching ratio and molecular weight of (1-->3)-beta-D-glucans are important factors for the production of cytokines from macrophages. GRN-inducible TNF alpha release was reduced by co-culturing with SPG, SSG, or the soluble beta-glucan, laminarin (LAM). Pretreatment alone with SPG or LAM was not sufficient for significant inhibition of GRN-inducible TNF alpha release. TNF alpha production induced with 50 micrograms/ml of zymosan (ZyM) was also reduced by addition of SPG, but TNF alpha production, stimulated with a higher concentration (100 micrograms/ml) of ZyM or with lipopolysaccharide (LPS), was not reduced significantly. The inhibitory effect of LAM on the uptake of GRN by RAW264.7 cells was not completely correlated with TNF alpha release. These results suggest that macrophages may incorporate beta-glucans through certain (1-->3)-beta-D-glucan-specific mechanisms and/or other endocytosis pathways, and that the beta-glucan-specific route is partially associated with cytokine production. In conclusion, TNF alpha release by macrophages is induced only by beta-glucans with high molecular weights and lower branching ratios, and the mechanism for the recognition of beta-glucans is multiple and assumed to be divided into several parts involving various cellular functions.