Optimization of Ultrasonic-Assisted Extraction Process of Polysaccharides from American Ginseng and Evaluation of Its Immunostimulating Activity

Ultrasonic-assisted extraction （UAE） of American ginseng polysaccharides （AGP） was investigated using response surface methodology. Three-factor-three-level Box-Behnken design was employed to optimize the ultrasonic power, extraction time and ratio of water to raw material to obtain a high AGP yield. The analysis of variance and response surface plots indicated that ultrasonic power was the most important factor affecting the extraction yield. The optimal conditions were ultrasonic power 400 W, extraction time 71 min, and ratio of water to raw material 33 mL g-1. Under these conditions, the yield of AGP was 8.09%, which was agreed closely to the predicted value. Gas chromatography （GC） analysis showed that AGP was composed of arabinose, rhamnose, galactose, glucose, and galacturonic acid. Fourier transform infrared spectra revealed the general characteristic absorption peaks of AGP. In addition, AGP exhibited good immunostimulating activities by up-regulating the production of nitric oxide and cytokines. Compared with hot water extraction, UAE required shorter extraction time and gave a higher extraction yield, without changing the structure and immunostimulating activity of AGP. The results indicated that UAE could be an effective and advisable technique for the large scale production of plant polysaccharides.

Purification,chemical modification and immunostimulating activity of polysaccharides from Tremella aurantialba fruit bodies

Ultrafiltration and a series of chromatographic steps were used to isolate and purify polysaccharides from Tremella aurantialba fruit bodies.Three crude fractions(TAP50w,TAP10-50w,and TAP1-10w),five semi-purified fractions(TAPA-TAPE),and one purified fraction(TAPA1) were obtained.A sulfated derivative of TAPA1(TAPA1-s) was prepared by chemical modification.The immunostimulating activity of the polysaccharide fractions in vitro was determined using the mouse spleen lymphocyte proliferation assay.Of the three crude fractions tested,cell prolifera-tion rates were increased most by TAP50w.Furthermore,TAPA1-s was markedly more stimulatory than TAPA1,indicating that sulfonation was an effective way to enhance the immunostimulating activity of polysaccharide.

Immunomodulatory therapies for acute pancreatitis

It is currently difficult for conventional treatments of acute pancreatitis (AP), which primarily consist of anti-inflammatory therapies, to prevent the progression of AP or to improve its outcome. This may be because the occurrence and progression of AP, which involves various inflammatory cells and cytokines, includes a series of complex immune events. Considering the complex immune system alterations during the course of AP, it is necessary to monitor the indicators related to immune cells and inflammatory mediators and to develop more individualized interventions for AP patients using immunomodulatory therapy. This review discusses the recent advances in immunomodulatory therapies. It has been suggested that overactive inflammatory responses should be inhibited and excessive immunosuppression should be avoided in the early stages of AP. The optimal duration of anti-inflammatory therapy may be shorter than previously expected (< 24 h), and appropriate immunostimulatory therapies should be administered during the period from the 3rd d to the 14th d in the course of AP. A combination therapy of anti-inflammatory and immune-stimulating drugs would hopefully constitute an alternative to anti-inflammatory drug monotherapy. Additionally, the detection of the genotypes of critical inflammatory mediators may be useful for screening populations of AP patients at high risk of severe infections to enable the administration of early interventions to improve their prognosis. (C) 2014 Baishideng Publishing Group Inc. All rights reserved.

Adjuvant effects of saponins on animal immune responses

Vaccines require optimal adjuvants including immunopotentiator and delivery systems to offer long term protection from infectious diseases in animals and man. Initially it was believed that adjuvants are responsible for promoting strong and sustainable antibody responses. Now it has been shown that adjuvants influence the isotype and avidity of antibody and also affect the properties of cell-mediated immunity. Mostly oil emulsions, lipopolysaccharides, polymers, saponins, liposomes, cytokines, ISCOMs (immunostimulating complexes), Freund’s complete adjuvant, Freund’s incomplete adjuvant, alums, bacterial toxins etc., are common adjuvants under investigation. Saponin based adjuvants have the ability to stimulate the cell mediated immune system as well as to enhance antibody production and have the advantage that only a low dose is needed for adjuvant activity. In the present study the importance of adjuvants, their role and the effect of saponin in immune system is reviewed.

Calcium phosphate nanoparticles show an effective activation of the innate immune response in vitro and in vivo after functionalization with flagellin

For subunit vaccines,adjuvants play a key role in shaping the magnitude,persistence and form of targeted antigen-specific immune response.Flagellin is a potent immune activator by bridging innate inflammatory responses and adaptive immunity and an adjuvant candidate for clinical application.Calcium phosphate nanoparticles are efficient carriers for different biomolecules like DNA,RNA,peptides and proteins.Flagellin-functionalized calcium phosphate nanoparticles were prepared and their immunostimulatory effect on the innate immune system,i.e.the cytokine production,was studied.They induced the production of the proinflammatory cytokines IL-8 （Caco-2 cells） and IL-1β（bone marrow-derived macrophages; BMDM） in vitro and IL-6 in vivo after intraperitoneal injection in mice.The immunostimulation was more pronounced than with free flagellin.

Screen and effect analysis of immunostimulants for sea cucumber,Apostichopus japonicus

Immunostimulants may improve disease resistance of aquaculture animals by promoting the nonspecific immunity response of the organisms. Five types of saccharides, including chitosan, yeast polysaccharide, burdock oligosaccharide, seaweed polysaccharide and lentinus edodes polysaccharide, were screened for potential use as immunostimulants by using spectrophotometry. The saccharides were injected into Apostichopusjaponicus, a sea cucumber, and the lysozyme and superoxide dismutase （SOD） activities of the coelomic fluid and epidermal slime were monitored in six consecutive days. The results show that the lysozyme activity of the animal＇s coelomic fluid was significantly stimulated on day 2, day 4 and day 6 after the injection of the saccharides （P〈0.05）. The effects of chitosan and yeast polysaccharide were the most notable. The lysozyme activity of the epidermal slime was significantly increased by chitosana, yeast polysaccharide, seaweed polysacchafide, and burdock oligosaccharide on day 1 and day 2 after the injection （P〈0.05）. The SOD activity of the coelomic fluid was significantly promoted by the saccharides on day 2 and day 4 post-injection （P〈0.05）, while the SOD activity of the epidermal slime increased on day 2. These findings indicate that chitosan and yeast polysaccharide are the most effective immunostimulants and potential healthy anti-disease feedstuff for A. japonicus.

A Review of Immune System Components, Cytokines, and Immunostimulants in Cultured Finfish Species

Aquaculture is a rapidly growing global agriculture sector and the importance of fish health has become of upmost importance as production levels and stocking densities increase. Over the past few decades, there have been a large number of immunological investigations on commonly cultured finfish species. Further, new technologies and strategies that embody use of fish immunostimulants, probiotics, and vaccinology rely heavily upon a comprehensive understanding of teleost immune system mechanics. The teleost immune system works in concert to properly recognize, control, and clear aquatic pathogens. Recent findings have exemplified the cooperative efforts of the nonspecific and adaptive branches, and have put forth an emphasis on the importance of the mucosal immune response in all aspects of a mounted immune response. This review provides a generalized overview of the innate and adaptive arms of the fish immune system, and provides highlights of recently published work in the areas of signaling networks and mucosal immune interactions.

Molecular Characterization, Expression Profiles, and Immunostimulation Responses of TRAF6 and TAK1 in Japanese Flounder(Paralichthys olivaceus)

Tumor necrosis factor receptor-associated factor 6(TRAF6) and transforming growth factor-β-activated kinase 1(TAK1) are two important adaptor molecules in Toll-like receptor(TLR) signaling pathway. In this study, TRAF6(PoTRAF6) and TAK1(PoTAK1) were cloned and characterized in Japanese flounder(Paralichthys olivaceus). The full-length cDNA sequence of Po TRAF6 is 1953 bp, with an open reading frame(ORF) of 1713 bp encoding a putative protein of 570 amino acids. PoTRAF6 contains one really interesting new gene(RING) domain, two zinc fingers, one coiled-coil region, and one meprin and TRAF homology(MATH) domain, which shows a high similarity to TRAF6 s in other species. The full-length PoTAK1 cDNA sequence is 2086 bp, with an ORF of 1728 bp that encodes a putative protein of 575 amino acids. PoTAK1 contains a conserved serine/threonine protein kinase catalytic domain and a coiled-coil region. The promoter regions of PoTRAF6 and PoTAK1 were also analyzed to predict several potential transcription factor-binding sites. In addition, the expression patterns of these two genes were examined in developmental stages, different tissues, and challenged samples. PoTRAF6 and PoTAK1 were expressed during the whole developmental stages, and the highest expressions were in intestine and heart, respectively. In challenged embryonic cells with LPS, CpG ODN, and poly I:C, the expressions of PoTRAF6 and PoTAK1 were both up-regulated significantly. These results suggest that PoTRAF6 and Po TAK1 play crucial roles in immune responses and may be involved in the developmental process of Japanese flounder.

Evaluation of in vitro and in vivo immunostimulatory activities of poly(lactic-co-glycolic acid) nanoparticles loaded with soluble and autoclaved Leishmania infantum antigens: A novel vaccine candidate against visceral leishmaniasis

Objective: To prepare and characterize poly lactic-co-glycolic acid(PLGA) nanoparticles loaded with soluble leishmanial antigen or autoclaved leishmanial antigen and explore in vitro and in vivo immunogenicity of antigen encapsulated nanoparticles. Methods: Water/oil/water double emulsion technique was employed to synthesize PLGA nanoparticles, and scanning electron microscopy, Fourier transform infrared spectroscopy and Zeta-potential measurements were used to identify the characteristics of nanoparticles. Cytotoxicity of synthetized nanoparticles on J774 macrophage were investigated by MTT assays. To determine the in vitro immunostimulatory efficacies of nanoparticles, griess reaction and ELISA was used to measure the amounts of NO and cytokines. During the in vivo analysis, Balb/c mice were immunized with vaccine formulations, and protective properties of nanoparticles were measured by Leishman Donovan unit in the liver following the infection. Cytokine levels in spleens of mice were determined by ELISA. Results: MTT assay showed that neither soluble leishmanial antigen nor autoclaved leishmanial antigen encapsulated nanoparticles showed cytotoxicity against J774 macrophage cells. Contrary to free antigens, both autoclaved leishmanial antigen-nanoparticle and soluble leishmanial antigen-nanoparticle formulations led to a 10 and 16-fold increase in NO amounts by macrophages, respectively. Leishman Donovan unit calculations revealed that soluble leishmanial antigen-nanoparticles and autoclaved leishmanial antigen-nanoparticles yielded 52% and 64% protection against visceral leishmaniasis in mouse models. Besides, in vitro and in vivo tests demonstrated that by increasing IFN-γ and IL-12 levels and inhibiting IL-4 and IL-10 secretions, autoclaved leishmanial antigen-nanoparticles and soluble leishmanial antigennanoparticles triggered Th1 immune response. Conclusions: Both autoclaved leishmanial antigen-nanoparticles and soluble leishmanial antigen-nanoparticles formulations provide exceptional in vitro and in vivo immunostimulatory activities. Hence, PLGA-based antigen delivery systems are recommended as potential vaccine candidates against visceral leishmaniasis.

Immunostimulatory Effect of Levamisole on the Immune Response of Goats to <i>Peste des petits ruminants </i>(PPR) Vaccination

The use of Levamisole hydrochloride as an immunostimulant has been successful in the control of certain diseases such as Derzsy’s disease in chicken as well as in increasing lymphocytes and weight gain in pigs. However, its use as immunostimulant in the prevention of Peste des petits ruminants (PPR) in goats has not been investigated. In this study, the use of Levamisole in enhancing immune response to PPR vaccination including its effects on weight gain was investigated among groups of goats (groups A, B, C and D). Virus neutralization test was used to determine the antibody profile in vaccinated goats while the total and differential leucocyte counts and body mass index (BMI) were determined using standard procedures. Goats in group A (Levamisole primed and PPR vaccinated) seroconverted to more than 3-folds of the initial pre-vaccination geometric mean titre (GMT) of neutralizing antibody beginning from second week post-vaccination and remained high throughout the period of the experiment. Similarly, there was significant increase (p 0.05) in BMI of animals in other groups throughout the period of the experiment. The results of this study further confirm the immunostimulatory effect of Levamisole when used in combination with a vaccine.

Effects of Several Immunostimulants on Phenoloxidase and Hemocytes of the Crab Charybdis japonica

To investigate the stimulating effects of immunostimulants on the autogenous immunocompetence of crabs and the possible mechanisms involved, the immunostimulating effects of β-1,3-glucan, lipopolysaccharide (LPS), inactivated Vibrio harveyi and Vibrio anguillarum on phenoloxidase (PO) and hemocytes of Charybdis japonica were investigated in this study. It was found that the yields and the enzymatic activities of purified PO in C. japonica increased significantly after the crabs were treated with immunostimulants, while the unit enzymatic activities remained almost the same. After treatment with β-1,3-glucan and LPS, the amount of rough endoplasmic reticulum (RER) and the number of mitochondria in both semigranular cells and granular cells increased greatly, and the number of cytoplasmic granules decreased but with enlarged volume. However, the corresponding characteristics of hyaline cells remained almost the same. On the other hand, the number of granules in semigranular cells decreased greatly, and the number of mitochondria of hyaline cells increased greatly, after treatment with inactivated vibrios. It may be concluded that the effect of polysaccharide immunostimulants on the innate immune system of C. japonica is different from that of inactivated vibrio immu-nostimulants. The immunity-enhancing mechanism of polysaccharides in crab autogenous immunocompetence is probably accomplished by the increased yields of PO and total PO activities, while that of inactivated vibrios is probably accomplished by the partially increased yields of PO and total PO activities as well as the significantly improved phagocytotic abilities of semigranular cells and hyaline cells.

Phytochemical bioprospecting, antioxidant, antimicrobial and cytotoxicity activities of saline extract from Tithonia diversifolia(Hemsl) A.Gray leaves

Objective:To evaluate antimicrobial and antioxidant properties of saline extract from Tithonia diversifolia leaves by phytochemical bioprospecting,and investigate its safety against animal cells.Methods:The saline extract was prepared,with NaCl(0.15 M),by constant stirring of the dried and pulverized leaves,followed by volume reduction by lyophilization.The extract was phytochemical characterized using ultra-performance liquid chromatography,and total phenol and flavonoid analysis also was performed.The antioxidant capacity was determined through DPPH*radical,the antimicrobial property was evaluated against standard bacteria and fungi,and the viability assays were performed against mice splenocytes.Results:Fifteen compounds were identified belonging to two main classes terpenoids and phenolics.The extract showed 22.185 mg GAE/g of total phenolic compounds and 3.220 mg QE/g of flavonoid.Moreover,extract showed higher antioxidant ability similar to butylated hydroxytoluene a standard molecule[(3.042±0.019)mg AAE/g and(4.12±0.10)mg AAE/g to saline extract and butylated hydroxytoluene,respectively].The antimicrobial assays demonstrated that the extract had a significant antifungal potential against Candida species and could be used with safety against mice splenocytes,in concentrations lower than 50μg/mL,promoting higher proliferation in these cells.Conclusions:Saline extract from Tithonia diversifolia leaves presents potential antioxidant,antifungal properties and induces immunostimulation in mice splenocytes.

Immunopotentiality of Ayurvedic polyherbal formulations “Saribadi” and “Anantamul Salsa” with augmentation of IgM production and lymphocytes proliferation:A preliminary study

Objective:To assess the immunopotentiality of Ayurvedic polyherbal preparations,"Saribadi" and "Anantamul Salsa".Methods: Freshly prepared BALB/c mice splenocytes were cultured with "Saribadi" or"Anantamul Salsa" treatment [doses of 0.25%, 0.50%, 0.75%, 1.00%, 1.50%, 2.00%,3.00% and 4.00%(v/v)] at 37C for 5 days. The immunoglobulin M(IgM) production and lymphocytes proliferation were determined by ELISA and MTT methods, respectively.Endotoxin contamination was assessed by treating the preparations with polymyxin B.Results: The doses of "Saribadi" [0.25%, 0.50%, 0.75% and 1.00%(v/v)] significantly increased IgM productions(0.966, 0.728, 0.695 and 0.615 mg/m L vs. control 0.265 mg/m L)and lymphocytes proliferation [absorbance 0.311, 0.394, 0.372 and 0.334 optical density(OD) vs. control 0.162 OD]. Similarly, the doses of "Anantamul Salsa" [0.50%, 0.75%,1.00% and 1.50%(v/v)] promoted IgM productions(0.933, 0.919, 0.917 and 0.892 mg/m L vs. control 0.502 mg/m L) and the doses of "Anantamul Salsa" [0.50%, 0.75%, 1.00%,1.50%, 2.00%, and 3.00%(v/v)] stimulated lymphocytes proliferation(absorbance 0.395,0.326, 0.440, 0.398, 0.452 and 0.355 OD vs. control 0.199 OD). The activity of "Saribadi"and "Anantamul Salsa" was not retarded by the treatment of preparations with polymyxin B.Conclusions: Immunomodulatory activity of "Saribadi" and "Anantamul Salsa" was unveiled for the first time. "Saribadi" and "Anantamul Salsa" possess immunostimulating potential acting through the induction of lymphocyte proliferation and IgM production.These preparations may be useful in strengthening immune responses. However, further cellular and in vivo studies are required.

Factors affecting effectiveness of vaccination against hepatitis B virus in hemodialysis patients

Hepatitis B virus(HBV)is a major global health problem.Despite the success of the general measures against blood transmitted infections in hemodialysis(HD)units,the prevalence of HBV infection among the HD patients is still high.Thus vaccination against HBV is indicating in this population.However,compared with the general population the seroprotection achieved in HD patients remains relatively low,at about 70%.In this review patient,HD procedure and vaccine-associated factors that affect the efficacy of HBV vaccination are analyzed.Also alternative routes of HBV vaccine administration as well as new and more immunogenic vaccine formulations are discussed.However,besides scientific progress,vigilance of HD physicians and staff regarding the general measures against the transmission of blood borne infections and the vaccination against HBV is also required for reducing the prevalence of this viral infection.

Immunostimulatory Properties of a Novel Polyphosphoramidate Based on Stearyl Tyrosine

The immunostimulatory property of a synthetic polyphosphoramidate based on stearyl tyrosine (PPST) is described. The immunostimulatory activity of this negatively charged polymer was investigated in vitro through examining the effect on the mitogen induced lymphocytes proliferation by a tetrazolium based colormetric assay and also through studying the primary plaque forming cells (IgM PFC) responses of Balb/c mice after the intraperitoneal (i.p.) injection of the polymer with sheep red blood cells (SRBC). PPST exhibits specific and high immunostimulatory activity in vitro by the stimulation on lipopolysaccharide(LPS) induced murine B cells proliferation, which is probably due to the interaction between the polymer with LPS responding B lymphocytes. This polymer also makes a significant increase of single antibody producing cells. The result shows that the stimulation rate to IgM PFC responses of Balb/c mice achieved 77.9% ( p <0.01) when 160 mg/kg of PPST was injected 4 h before with SRBC.

Dietary intake of bamboo vinegar and charcoal powder(BVC)enhances resistance of African catfi sh Clarias gariepinus to bacterial pathogen

Although the immunomodulatory activity of bamboo vinegar and charcoal powder(BVC)has been increasingly documented in domestic animals,the use of BVC as immunostimulant in fi sh aquaculture awaits verifi cation.In the present study,the immunostimulatory impacts of dietary BVC intake were investigated in an important aquaculture fi sh species,the African catfi sh Clarias gariepinus.Results show that the pathogen resistance of African catfi sh was signifi cantly improved by dietary BVC intake at the tested levels(0.5%-2%),as indicated by higher survival rates(approximately 1.52-1.85 times that without BVC supplementation)upon intraperitoneally injection of bacterial pathogen Aeromonas hydrophila.In addition,both the count and phagocytic activity of blood cells were signifi cantly up-regulated by a 45-day dietary supplementation of BVC.Moreover,compared to that of the control,fi sh individuals fed BVC containing diets exhibited signifi cant higher activities of superoxide dismutase(SOD),myeloperoxidase(MPO),and lysozyme(LYZ).The content of immunoglobulin M(IgM)in serum of African catfi sh was also induced by dietary BVC supplementation.Furthermore,the expression levels of interleukin-8(IL-8),toll-like receptor 1 and 5(TLR1 and TLR5),myeloid diff erentiation factor 88(MyD88),and nuclear factor kappa B(NFκB)in the head kidney were signifi cantly up-regulated.Our fi ndings indicate BVC could be an eff ective immunostimulant in teleost species,which may enhance fi sh immunity through improving hematic parameters,activating bioactive humoral molecules,and up-regulating immune related molecular pathways.

Applicability, Feasibility and Efficacy of Phytotherapy in Aquatic Animal Health Management

The use of chemotherapeutants, antibiotics, and pesticides in aqua farms and non-adherence to scientific management practices have resulted in adverse impact on aquacultural production, serious outbreaks of diseases, development of drug resistance in microbes, and accumulation of antibiotics and pesticide residues in finfish and shellfish and environmental pollution. As a result, a need has been felt by the aquaculturists as well as aquatic animal health management professionals to find a suitable alternative therapy in place of antibiotics and chemotherapy. Phytotherapy has come to be recognized as a handy and viable alternative to chemotherapy, as it is economical, effective, non-resistance forming, renewable, eco-friendly and farmer-friendly. Although the use of medicinal plants is known to humanity since the dawn of human civilization for the treatment and control of human and animal diseases but its importance in combating finfish and shellfish diseases has been realized only recently. The phytotherapy of aquacultural diseases is in its infancy in most part of the world except in China to some extent. However, many important contributions in this field by different workers during the last quarter of the 20th century and early 21st century have shown encouraging results and opened new vistas in phytotherapy of aquatic animals. The present review crtically evaluates the present status of knowledge of phytotherapy in the world combating various aquacultural diseases, identifying the bottlenecks and suggests remedial measures.

Influence of the Zerox Immunostimulant on Cotton Production

In this article, information about universal and triple contact actional ZEROX immunostimulant at 3 various norms and different periods when it affected to Bukhara 10 cotton on 3 numbers of seedlings is presented. Research has shown, when in pre-sowing of seed 2 l/t and 2 l/ha in vegetation periods of ZEROX immunostimulant in the number of left seedlings 80 - 90,000 plant/ha of Bukhara 10 cotton it was treated, it was determined that the highest yield was obtained.

Vitamin D supplementation for autoimmune hepatitis: A need for further investigation

Autoimmune hepatitis is a chronic liver disease harboring an autoimmune basis and progressive character.Despite still obscurity in etiology and pathogenesis,some evidence supports the importance of sustaining the immune system.Vitamin D is a lipo-soluble vitamin,which has been identified as decreased in our body.It is often due to the daily habit change and decrease of individual sun exposure due to the increase of the ultraviolet-induced potential melanocytic transformation.Here,we emphasize the importance of vitamin D supplementation in patients affected with liver disease.

Influence of Dietary Administration of β-hydroxy-β-methylbutyrate （HMB） on the Innate Immunity and Resistance against Bacterial Infections in Pikeperch （Sander lucioperca）

The present study examined the influence of leucine metabolite β-hydroxy-β-methylbutyrate （HMB） on the nonspecific cellular and humoral defence mechanisms and protection against Yersinia ruckeri and Aeromonas salmonicida in juvenile pikeperch （Sander lucioperca）. The fish was fed with a commercial trout pellets, containing either 0 （control group） or 50 mg HMB kg^-1 body weight day^-1 （HMB fed group）. After four weeks feeding, 20 pikeperch from each group were anaesthetised and blood and pronephros samples were taken. The levels of the following immunological parameters were measured： respiratory burst activity of phagocytes, potential killing activity of phagocytes, lymphocytes proliferation stimulated by concanavaline A or lipopolisaccharide, lysozyme activity in serum, ceruloplasmin activity in serum, total protein in serum and total immunoglobulin （Ig） level in serum. A disease challenge test using Yersinia ruckeri or Aeromonas salmonicida was conducted after 4 weeks of feeding. The levels of all immunological parameters excluding ceruloplasmin activity and total protein in serum were statistically significantly higher （P 〈 0.05） in the HMB treated group compared to the control group. Feeding with HMB has led to a significant decrease in the mortality after the challenge trial with Y. ruckeri （30% lower mortality than in the control group） and with theA. salmonicida （40% lower mortality than in the control group）. The current study strongly suggests that feeding pikeperch with HMB in dose 50 mg kg^-1 body weight day^-1 may improve the innate immunity and decrease the mortality rates after experimental infection with pathogenic bacteria Y. ruckeri and A. salmonicida.