Structural characterization and immunostimulatory activity of a water-soluble polysaccharide from abalone(Haliotis discus hannai Ino)muscle

A water-soluble polysaccharide from abalone muscle(AMPP)was isolated.The contents of carbohydrate,protein,uronic acid,and sulfate in AMPP were 83.5%,0.5%,2.7%,and 2.6%,respectively.High-performance liquid chromatography analysis indicated that AMPP was homogeneous and had an average molecular weight of approximately 3.2 kDa.The main monosaccharides of AMPP were glucose(Glc)and mannose with a molar ratio of 99.7:0.3.The structural characteristics of AMPP were elucidated through methylation analysis,Fourier transform infrared spectroscopy,and nuclear magnetic resonance spectroscopy.The linkages of AMPP consisted of terminal,1,4-linked,1,6-linked,and 1,4,6-linked Glcp with a molar ratio of 3.1:7.2:1.0:2.5.In one repeat unit of the proposed AMPP structure,the backbone chain was composed of eight 1→4 glycosidic bonds and one 1→6 glycosidic bond,with three branch chains linked by 1→6 glycosidic bond.In addition,AMPP was found to possess potent immunostimulatory activity via rising phagocytosis of RAW264.7 cells and promoting secretion of TNF-α.

Enhanced Immunostimulatory Properties in CpG-Oligonucleotides Modified by Gold Nanoparticles

Whether the immunostimulatory effects of CpG-oligonucleotides （CpG ODNs） could be enhanced by the use of gold nanoparticles （Au-NP） was investigated.The CpG ODNs were modified by the Au-NP （CpG /Au-NP） and their uptake and distribution in murine N9 microglial cells were studied.The immunostimulatory effects of CpG /Au-NP on N9 cells,human B cells and plamacytoid dendritic cells （pDCs） were examined.Results showed that the uptake of CpG /Au-NP in N9 cells was much higher than that of CpG ODNs and CpG /Au-NP localized in the cytoplasm of N9 cells.The amount of TNF-α and IL12p40 in N9 cells was increased greatly by the use of Au-NP.And the amount of IL-6 in B cells and IFN-α in pDCs was also significantly increased,while the activation of B cells was not changed.These results reveal that the Au-NP can be used as a delivery media for the oligonucleotides-based therapeutics.

Immunostimulatory eff ect of quaternary degree and acetyl group of quaternized chitosan on macrophages RAW 264.7

Hydroxypropyltrimethyl ammonium chloride chitosan(HACC)and hydroxypropyltrimethyl ammonium chloride fully deacetylated chitosan(De-HACC)were synthesized with various degrees of substitution by altering the ratio of chitosan to glycidyl trimethyl-ammonium chloride(GTMAC).The effects of the quaternary ammonium degree and the acetyl group of these polymers on immunostimulatory activities were detected in RAW 264.7 cells.The expression levels of nitrogen oxide(NO),interleukin-6(IL-6)and tumor necrosis factor(TNF-α)were compared.Results show that the removal of acetyl groups in chitosan obviously improved the degree of substitution of quaternary ammonium salts.In addition,HACC and De-HACC were capable of promoting immunological activity in a substitution-dependent manner;HACC was positively correlated,and De-HACC was negatively correlated.Among tested ratios,HACC-30%and De-HACC-54%performed better than the others,and De-HACC-54%performed the best.Generally,quaternized chitosan possesses immunostimulatory activity,which is related to the degree of quaternization and the acetyl group.

In vivo immunostimulatory effect of the amoebocyte lysate and plasma of Asian horseshoe crab,Tachypleus gigas in a piscine model

Antimicrobial proteins/peptides are becoming a new generation of immunostimulants for prevention and disease control in human and animals,including aquatic animals.As the haemolymph of horseshoe crabs(Tachypleus)contains broad ranges of bioactive compounds,we have explored the in vivo immunostimulating potential of amoebocyte lysate and plasma using a fish model.Indian major carp,Labeo rohita,yearlings were injected intraperitoneally with two doses of lysate and plasma at 50 and 100µg protein per fish.No abnormalities and/or mortalities were recorded in any group.L.rohita injected with 50µg lysate and 100µg plasma protein showed significant enhancement(P<0.01)of various haematological and immunological parameters.There was a significant rise in the total protein and globulin content,myeloperoxidase and respiratory burst activity following injection with 50µg lysate and 100µg plasma protein.The agglutinating and haemagglutinating activities were increased albeit not significantly(P>0.01)in any groups.On the contrary,a significantly high hemolysin titre was recorded in fish that received 100µg plasma protein.Following challenge with Aeromonas hydrophila,both lysate and plasma protein(s)cross protected the fish after 30 days.The highest survival(50%)was recorded in group injected with 50µg lysate protein,followed by 45%in both 100µg lysate and plasma protein injected groups.

A Sensitive and Simple Method to Assess NK Cell Activity by RT-qPCR for Granzyme B Using Spleen and Blood

A new assay method for natural killer (NK) cell activity was established using quantitative RT-PCR (RT-qPCR) to determine the gene expression of granzyme B (GzmB) and perforin 1 (Prf1). The RT-qPCR method was compared to a conventional cytotoxic assay. Upregulated expression of GzmB and Prf1 mRNA and enhanced cytotoxic activity were observed in splenocytes from lipopolysaccharide (LPS)-treated rats. A high correlation, R2 = 0.71, was observed between the gene expression of GzmB and the cytotoxic activity of splenocytes from these rats, indicating that GzmB RT-qPCR is a reliable alternative method to assess NK cell activity/activation. Remarkably, 12.6- to 59.7-fold upregulation of GzmB mRNA expression was observed in leukocytes, the spleen, and splenocytes from LPS-injected rats. Its upregulation appeared to be dose-dependent on the LPS concentration in the range of 0.01 - 0.1 mg/kg. Whereas, only 1.3- to 1.9-fold increase of cytotoxic activity was detected in splenocytes from the rats treated with LPS in the same range. From these, it is evident that, to assess NK cell activity/activation, the GzmB RT-qPCR method is highly sensitive compared with the conventional cytological assay. Furthermore, this GzmB RT-qPCR method is advantageous, as it does not require freshly prepared splenocytes and cell culture procedures. The convenience of GzmB RT-qPCR enables the use of whole blood, leukocytes, the spleen, and/or their frozen samples to evaluate NK cell activity/activation.

An aptamer-driven DNA nanodevice for improved delivery of synthetic immunostimulants

Efficient delivery of therapeutics to immune cells remains a formidable challenge for cancer immunotherapy.In this work,we demonstrate that an aptamer-driven DNA nanodevice,constructed through linkage of a synthetic immunostimulant(Toll-like receptor 9 agonist:CpG motif)to an aptamer,could significantly enhance the immunostimulatory activity by facilitating the uptake and retention of therapeutics in macrophages.Systemic administration of the DNA nanodevice results in efficient tumor growth inhibition in both breast cancer and melanoma mouse models.Our studies suggest that the DNA nanodevice leads to reeducation of tumor-associated macrophages and ultimately to reversing the tumor immune microenvironment.The strategy for aptamer-mediated and vehicle-free delivery of immunostimulatory oligonucleotides provides a potential platform for cancer immunotherapy.

Convergent synthesis and immunological study of oligosaccharide derivatives related to galactomannan from Antrodia cinnamomea

The galactomannan from Antrodia cinnamomea(AC)is characterized as one of the important bioactive components that exhibits potential immunostimulatory propriety.The biological function of its corresponding oligosaccharide fragments has not been revealed yet.In this study,we reported the first chemical synthesis of the series of oligosaccharide fragments related to AC galactomannan via the convergent glycosylation strategy.The preliminary immunological evaluation of these synthesized AC oligosaccharides disclosed that the backbone tetrasaccharide 1d showed the best immunomodulatory ability on enhancing proliferation,phagocytosis and cytokines secretion of Raw264.7 macrophages in vitro,indicating its immense potential as an immunostimulant candidate.

Myeloid-derived suppressor cells in cancer

Numerous recent studies have shown that myeloid-derived suppressor cells(MDSCs),a strongly heterogeneous population of immunosuppressive cells,are dysregulated in the presence of many cancers.MDSCs present different phenotypes and play prominent roles in the tumor microenvironment.To date,gene therapies targeting MDSCs are the most innovative and flexible methods to specifically modify the tumor microenvironment.Here,we summarize current studies related to the phenotypes,functions,and mechanisms of MDSCs and explore the therapeutic landscape of chemokines that affect the balance between subpopulations of MDSCs.