Intranasal vaccination with influenza HA/GO-PEI nanoparticles provides immune protection against homo- and heterologous strains.

Intranasal (i.n.) immunization is a promising vaccination route for infectious respiratory diseases such as influenza. Recombinant protein vaccines can overcome the safety concerns and long production phase of virus-based influenza vaccines. However, soluble protein vaccines are poorly immunogenic if administered by an i.n. route. Here, we report that polyethyleneimine-functionalized graphene oxide nanoparticles (GP nanoparticles) showed high antigen-loading capacities and superior immunoenhancing properties. Via a facile electrostatic adsorption approach, influenza hemagglutinin (HA) was incorporated into GP nanoparticles and maintained structural integrity and antigenicity. The resulting GP nanoparticles enhanced antigen internalization and promoted inflammatory cytokine production and JAWS II dendritic cell maturation. Compared with soluble HA, GP nanoparticle formulations induced significantly enhanced and cross-reactive immune responses at both systemic sites and mucosal surfaces in mice after i.n. immunization. In the absence of any additional adjuvant, the GP nanoparticle significantly boosted antigen-specific humoral and cellular immune responses, comparable to the acknowledged potent mucosal immunomodulator CpG. The robust immune responses conferred immune protection against challenges by homologous and heterologous viruses. Additionally, the solid self-adjuvant effect of GP nanoparticles may mask the role of CpG when coincorporated. In the absence of currently approved mucosal adjuvants, GP nanoparticles can be developed into potent i.n. influenza vaccines, providing broad protection. With versatility and flexibility, the GP nanoplatform can be easily adapted for constructing mucosal vaccines for different respiratory pathogens.

The Polysaccharides from Pinellia ternata and Their Derivatives: Preparation, Structure Characteristics, and Activities in Vitro.

Three polysaccharides, PTC, PTH, and PTB, were extracted from Pinellia ternata using three different extraction conditions: room temperature water, hot water, and 2 % Na2CO3 solution. PTC and PTH were composed of rhamnose, glucose, galactose, mannose, glucuronic acid, galacturonic acid, and arabinose, which combine to form complex structures. PTB was composed solely of glucose and rhamnose. Further analysis indicated that PTC and PTB exhibited triple-helix structures. PTC showed the highest scavenging capacity against DPPH, superoxide anion, and hydroxyl radicals, with half maximal inhibitory concentrations (IC50) of 1004.1, 1584.1, and 1584.1 µg/mL, respectively. Additionally, PTC, PTH, and PTB were subjected to sulfation, phosphorylation, and selenization, resulting in the production of nine derivates. The distinctive absorptive bands of these derivates were determined through infrared spectroscopy. Selenized and sulfated derivates have shown significant antitumor and immunoenhancing properties. Our findings revealed that at 400 µg/mL, the inhibition rate of selenated PTB on HeLa cells was 54.2 % and that on HepG2 cells was 43.1 %. Additionally, selenized PTC displayed significant immunoenhancing activity, with a proliferation rate of 63.7 % at 400 µg/mL in RAW264.7 cells. These results provide valuable evidence supporting the consideration of polysaccharides from Pinellia ternata as a potential candidate for the development of antineoplastic drugs.

Structural Characterization and Immunoenhancing Properties of Polysaccharide CPTM-P1 from Taxus media.

Polysaccharides extracted from Taxus media hrough an aqueous method were further refined by removing proteins via the Sevag technique and purified by dialysis. The separation of these polysaccharides was accomplished using a DEAE-cellulose chromatog-raphy column, yielding two distinct fractions, named CPTM-P1 and CPTM-P2. Notably, CPTM-P1 emerged as the primary polysaccharide component within Taxus media. Consequently, a comprehensive analysis focusing exclusively on CPTM-P1 was undertaken. The molecular weight of CPTM-P1 was established through gel permeation chromatography (GPC), and its monosaccharide composition was deciphered using HPLC-MS. The structure was further elucidated through nuclear magnetic resonance (NMR) spectroscopy. The molecular weight of CPTM-P1 was determined to be 968.7 kDa. The monosaccharide composition consisted of galactose (Gal), arabinose (Ara), galacturonic acid (Gal-UA), glucose (Glc), rhamnose (Rha), xylose (Xyl), mannose (Man), fucose (Fuc), glucuronic acid (Glc-UA), and ribose (Rib). The proportional distribution of these components was 30.53%, 22.00%, 5.63%, 11.67%, 11.93%, 1.69%, 8.50%, 1.23%, 5.63%, and 1.17%, respectively. This confirmed CPTM-P1 as an acidic heteropolysaccharide with a glycuronic acid backbone. Moreover, CPTM-P1 showed immunoenhancing properties, effectively augmenting the secretion of nitric oxide and cytokines (TNF-α, IL-1ß, and IL-6). Additionally, it significantly enhances the phagocytic capacity of RAW264.7 cells. These findings underscore the potential application of these polysaccharides in functional foods and pharmaceuticals, providing a solid scientific basis for further exploration and utilization of Taxus media polysaccharides.

Tumor-Specific Immunoenhancing Effects after Local Cryoablation for Metastatic Bone Tumor in a Mouse Model.

We investigated the abscopal effect after cryoablation (CA) on bone metastasis using a mouse model. Breast cancer cells were implanted in the bilateral tibiae of mice. The left tumor was treated locally with CA, and the right abscopal tumor (AT) was left untreated. The mice were divided into four groups based on the combination of CA and intraperitoneal administration of anti-PD-1 antibody (PD) as treatment interventions (Control, CA, PD, and CA + PD). The reduction ratio of the size of AT, the quantitative immune effects at enzyme-linked immunospot (ELISPOT) assay, and the intensity of infiltration of immune-related cells to AT were compared among the groups. CA alone showed a significant immunoenhancing effect on the volume change ratio of AT from day 0 to day 14 (Control-CA: p < 0.05), ELISPOT assay (Control-CA: p < 0.01), and CD4+ cell count in immunostaining (Control-CA: p < 0.05). CA alone showed no significant immunoenhancing effect on CD8+ and Foxp3+ cell counts in immunostaining, but the combination of CA and PD showed a significant immunoenhancing effect (Control-CA + PD: p < 0.01 [CD8, Foxp3]). The results suggested that the abscopal effect associated with the local cryotherapy of metastatic bone tumors was activated by CA and enhanced by its combination with PD.

ß-1, 3 glucan binding protein based selenium nanowire enhances the immune status of Cyprinus carpio and protection against Aeromonas hydrophila infection.

In the present study, immunoenhancing effect of ß-1, 3 glucan binding protein based selenium nanowire (Phß-GBP-SeNWs) in common carp, Cyprinus carpio was assessed. Biological based selenium nanoform was synthesized, using crustacean immune molecule ß-GBP purified from the haemolymph of Paratelphusa hydrodromus. The morphological property of Phß-GBP-SeNWs was analyzed through TEM which reveals, the synthesized nanowire exhibits approximately 30-50 nm width with smooth surface. For this current study, fish were fed with experimental diet includes Phß-GBP, sodium selenite, selenomethionine and Phß-GBP-SeNWs supplemented diet at different concentrations (0.5 mg, 1 mg and 2 mg) for 30 days. The growth performance, cellular and humoral immune responses (myeloperoxidase, reactive oxygen species, alkaline phosphatase and lysozyme activity) and antioxidant enzymes (glutathione peroxidase and catalase activity) in the fish fed with Phß-GBP-SeNWs supplemented diet were significantly increased in dose-dependent manner, which was observed at two different interval period (15th and 30th day). Also, Phß-GBP-SeNWs supplemented diet fed fish gain resistant after challenged with aquatic pathogen Aeromonas hydrophila and the relative survival percentage was increased. Agar disc diffusion and BacLight assay clearly demonstrated the antibacterial property of plasma of fish fed with Phß-GBP-SeNWs supplemented diet against aquatic pathogen A. hydrophila, Vibrio parahaemolyticus and Vibrio alginolyticus. Moreover, confocal laser scanning microscopic analysis clearly showed that, Phß-GBP-SeNWs supplemented diet fed fish plasma was more efficient in disrupting the architecture of bacterial colonies and thereby reduced the thickness of biofilm. Thus, the present study indicates that, incorporation of Phß-GBP-SeNWs in the diet enhances the fish immune responses and disease resistance against aquatic pathogens.

Preparation Optimization and Immunological Activity Studies of Portulaca oleracea L. Polysaccharides Liposomes.

AIMS: This study combines traditional Chinese medicine polysaccharides with nanomaterials to enhance drug bioavailability and immunological activity. BACKGROUND: The study of polysaccharide preparation, structure identification, pharmacological activity, and mechanism of action is deepening, but the research combined with the new drug delivery system is relatively weak, so the application of polysaccharides is still facing great limitations. In order to prolong the action time of polysaccharides and improve their bioavailability, liposome has become the most promising delivery carrier. OBJECTIVES: The purpose of this study was to optimize the preparation process of Portulaca oleracea L. polysaccharides liposomes (POL-PL) and evaluate the immunoactivity in vitro. METHODS: POL-PL was prepared by reverse evaporation, and the preparation process was optimized using the response surface methodology. The characteristic analysis of POL-PL was detected by the indicators including morphology, particle size, zeta potential, encapsulation efficiency, release, and stability. The effects of POL-PL on the proliferation and immunological activity of mouse spleen lymphocytes and RAW264.7 cells were evaluated in vitro. RESULTS: POL-PL is highly homogeneous in morphology and particle size, and its sustained release improves the bioavailability of Portulaca oleracea L. polysaccharides (POL-P). Moreover, POL-PL treatment significantly enhanced the proliferation and phagocytic activity of RAW264.7 cells and increased the secretion of IL-6, TNF-α, IL-1ß, and NO. CONCLUSION: This study suggested that POL-PL were prepared successfully by reverse evaporation method, and POL-PL had immunoenhancing activity in vitro. The results provided a theoretical basis for further application of POL-PL.

In vitro and in vivo immuno-enhancing effect of fucoidan isolated from non-edible brown seaweed Sargassum thunbergii.

Fucoidan has been reported to have various biological activities, such as antioxidant, antitumor and anticoagulant, with various health benefits. However, few studies have been conducted to extract fucoidan from Sargassum thunbergii in terms of its immuno-enhancing activities. This aim of this study was to investigate the immuno-enhancing effect of fucoidan (S3) isolated from Sargassum thunbergii through water extraction and ethanol precipitation in RAW 264.7 macrophages and zebrafish. The results showed that S3 contained a relatively high content of fucose and sulfated polysaccharide. Fourier-transform infrared spectroscopy (FTIR) results show that the characteristic peaks at 845 cm-1 and 1220-1270 cm-1 indicate that S3 contains sulfate groups. In vitro, S3 effectively enhanced nitric oxide (NO) production and phagocytic activity. In addition, the results of the study demonstrated that the secretion of tumor necrosis factor-α, interleukin (IL)-6, IL-1ß, and IL-10 was upregulated by nuclear factor kappa B (NF-κB) signaling pathway in a dose-dependent manner. In vivo, S3 activates zebrafish immune responses by promoting secretion of NO and activating the NF-κB pathway. Overall, these results suggest that S3 could be used as a functional ingredient added to nutritional supplements and functional foods.

Chitosan modification and pharmaceutical/biomedical applications.

Chitosan has received much attention as a functional biopolymer for diverse applications, especially in pharmaceutics and medicine. Our recent efforts focused on the chemical and biological modification of chitosan in order to increase its solubility in aqueous solutions and absorbability in the in vivo system, thus for a better use of chitosan. This review summarizes chitosan modification and its pharmaceutical/biomedical applications based on our achievements as well as the domestic and overseas developments: (1) enzymatic preparation of low molecular weight chitosans/chitooligosaccharides with their hypocholesterolemic and immuno-modulating effects; (2) the effects of chitin, chitosan and their derivatives on blood hemostasis; and (3) synthesis of a non-toxic ion ligand--D-Glucosaminic acid from oxidation of D-Glucosamine for cancer and diabetes therapy.

Preparation, Characterization, and Immuno-Enhancing Activity of Polysaccharides from Glycyrrhiza uralensis.

Glycyrrhiza uralensis is a Chinese herbal medicine with various bioactivities. Three fractions (GUPS-I, GUPS-II and GUPS-III) of G. uralensis polysaccharides (GUPS) were obtained with molecular weights of 1.06, 29.1, and 14.9 kDa, respectively. The monosaccharide compositions of GUPS-II and GUPS-III were similar, while that of GUPS-I was distinctively different. The results of scanning electron microscopy, FT-IR, and NMR suggested that GUPS-II and GUPS-III were flaky with a smooth surface and contained α- and ß-glycosidic linkages, while GUPS-I was granulated and contained only α-glycosidic linkages. Moreover, GUPS-II and GUPS-III exhibited better bioactivities on the maturation and cytokine production of dendritic cells (DCs) in vitro than that of GUPS-I. An in vivo experiment showed that only GUPS-II significantly enhanced the maturation of DCs. These results indicate that GUPS-II has the potential to be used in combination with cancer immunotherapy to enhance the therapeutic effect.

Regulation of immune response by S-1-propenylcysteine through autophagy-mediated protein degradation.

Autophagy is a key event in cellular recycling processes due to its involvement in the intracellular degradation of proteins. It has been demonstrated that S-1-propenylcysteine (S1PC), a characteristic sulfur compound in aged garlic extract, induces the activation of autophagy. S1PC degrades the adaptor protein myeloid differentiation response protein 88 (MyD88) of downstream of Toll-like receptor (TLR) by activating autophagy in vitro and in vivo. The degradation of MyD88 inhibits the TLR signaling pathway, including the phosphorylation of interleukin 1 receptor associated kinase 4 (IRAK4) and nuclear factor (NF)-κB p65 in vitro, and eventually leads to the inhibition of interleukin (IL)-6 production in vitro and C-C motif chemokine ligand 2 (Ccl2) mRNA expression in vivo. S1PC also increases the level of intestinal immunoglobulin A (IgA) and the number of IgA-producing cells in Peyer's patches in vivo. In addition, S1PC triggers the mRNA expression of X-box binding protein 1 (Xbp1), an inducer of IgA-producing cell differentiation via the phosphorylation of extracellular signal-regulated kinase (ERK)1/2 and the degradation of paired box protein 5 (Pax5), a suppressor of Xbp1 mRNA expression. The present review summarizes the mechanisms through which the activation of autophagy by S1PC modulates the immune response.

Assessment of the Effect of Baicalin on Duck Virus Hepatitis.

BACKGROUND: Duck virus hepatitis (DVH) caused by duck hepatitis A virus type 1 (DHAV-1) is a malignant disease in ducklings, causing economic losses in the duck industry. However, there is still no antiviral drug against DHAV-1 in the clinic. OBJECTIVE: Our aim is to investigate the anti-DHAV-1 effect of baicalin, which is a flavonoid derived from the Chinese medicinal herb huangqin (Scutellaria baicalensis Georgi). METHODS: Here, we first detected its anti-DHAV-1 ability in vitro and in vivo. At the same time, the inhibition of baicalin on DHAV-1 reproduction was determined. Finally, we tested and verified the anti-oxidative and immuno-enhancing roles of baicalin on its curative effect on DVH. RESULTS: Baicalin possessed anti-DHAV-1 effect. It improved the cytoactive of DEH which was infected by DHAV-1 as well as reduced the DHAV-1 reproduction in DEH. Under baicalin treatment, mortality of ducklings infected by DHAV-1 decreased, additionally the DHAV-1 level and liver injury in such ducklings were significantly reduced or alleviated. The in vitro mechanism study indicated baicalin inhibited DHAV-1 reproduction via interfering the viral replication and release. Furthermore, the in vivo mechanism study manifested both the anti-oxidative and immuno-enhancing abilities of baicalin, which played crucial roles in its curative effect on DVH. CONCLUSION: This study may provide a scientific basis for developing baicalin as one or a part of the anti-DHAV-1 drugs.

Mycelial polysaccharides of Lentinus edodes (shiitake mushroom) in submerged culture exert immunoenhancing effect on macrophage cells via MAPK pathway.

A crude polysaccharide fraction (termed cLEP) and two derived fractions (termed LEP1 and LEP2) from Lentinus edodes mycelia were purified and characterized. LEP1/-2 were classified as α-type heteropolysaccharides with 1 → 2, 1 → 3, 1 → 4, 1 → 6 linkages. Their monosaccharide components were respectively Gal, Glc, Man, Ara, Fuc, and Rha (molar ratio 10.17:9.75:9.01:1.61:1.26:1), and Glc, Man, Fuc, Rha, and Gal (molar ratio 5.18:4.69:2.85:1.43:1). In vitro culture experiments with macrophage RAW264.7 cells showed no cytotoxic effects of the polysaccharides. Phagocytosis (neutral red uptake) was significantly enhanced by LEP1/-2. Levels of NO, TNF-α and IL-6 were higher in LEP1/-2-treated groups than in cLEP-treated group. qRT-PCR analysis showed that LEP1/-2 had greater enhancing effect on mRNA transcription of iNOS, TNF-α, and IL-6 genes. Western blotting analysis revealed that LEP1/-2 strongly promoted phosphorylation of kinases ERK and JNK, and suggested that they exert immunoenhancing effects via MAPK signaling pathway.

Alkaline extractive crude polysaccharide from Russula senecis possesses antioxidant potential and stimulates innate immunity response.

OBJECTIVES: Over past decades, investigation on bioactive polysaccharides extracted from mushroom by heated water reflux has been an emerging field of biomedicine especially in the area of immune stimulation. While studies with macromolecules isolated from remainder residue of aqueous extraction are scarce. In this context, crude polysaccharide from a traditionally edible macrofungus, Russula senecis, was prepared (RuseCap) by alkaline solvent using leftover residue of that conventional process and its structural along with therapeutic properties were evaluated. KEY FINDINGS: Investigation by FT-IR, HPTLC, GC-MS and spectrophotometry showed that the fraction was mainly consisted of carbohydrate with backbone of xylose, rhamnose, mannose and glucose (mostly ß-glucan). Besides, RuseCap exhibited strong antioxidant activity evident by radical scavenging (superoxide, hydroxyl, DPPH, ABTS), chelating ability and reduction power where EC50 values ranged from 257 to 4068 µg/ml concentration. In addition, it also exhibited immune-boosting potentiality as the treatment effectively induced proliferation, phagocytosis, nitric oxide production, intracellular reactive oxygen species generation, morphological changes and increased transcription level of iNOS, COX-2, TNF-α, IL-6 genes in macrophage cells. CONCLUSIONS: Overall, the study provided blueprint for extended utilization of R. senecis basidiocarps beyond hot water process and defined use of RuseCap as potent therapeutic agent against free radicals as well as deprived immunity.

Prothymosin Alpha and Immune Responses: Are We Close to Potential Clinical Applications?

The thymus gland produces soluble molecules, which mediate significant immune functions. The first biologically active thymic extract was thymosin fraction V, the fractionation of which led to the isolation of a series of immunoactive polypeptides, including prothymosin alpha (proTα). ProTα displays a dual role, intracellularly as a survival and proliferation mediator and extracellularly as a biological response modifier. Accordingly, inside the cell, proTα is implicated in crucial intracellular circuits and may serve as a surrogate tumor biomarker, but when found outside the cell, it could be used as a therapeutic agent for treating immune system deficiencies. In fact, proTα possesses pleiotropic adjuvant activity and a series of immunomodulatory effects (eg, anticancer, antiviral, neuroprotective, cardioprotective). Moreover, several reports suggest that the variable activity of proTα might be exerted through different parts of the molecule. We first reported that the main immunoactive region of proTα is the carboxy-terminal decapeptide proTα(100-109). In conjunction with data from others, we also revealed that proTα and proTα(100-109) signal through Toll-like receptor 4. Although their precise molecular mechanism of action is yet not fully elucidated, proTα and proTα(100-109) are viewed as candidate adjuvants for cancer immunotherapy. Here, we present a historical overview on the discovery and isolation of thymosins with emphasis on proTα and data on some immune-related new activities of the polypeptide and smaller immunostimulatory peptides thereof. Finally, we propose a compiled scenario on proTα's mode of action, which could eventually contribute to its clinical application.

Effects of Bush Sophora Root polysaccharide and its sulfate on immuno-enhancing of the therapeutic DVH.

Bush Sophora Root polysaccharide (BSRPS) and its sulfate, sulfated Bush Sophora Root polysaccharide (sBSRPS), possess the antiviral activities against duck hepatitis A virus. However their antiviral mechanisms are still not clear. This paper reported their immuno-enhancing roles in the therapeutic effects for duck virus hepatitis (DVH). The effects of BSRPS and sBSRPS on stimulating lymphocyte proliferation were investigated by MTT methods. After that, ducklings were challenged with DHAV and treated with BSRPS and sBSRPS. Meanwhile, the total antibody (Ab), cytokines including interferon gamma (IFN-γ), hepatocyte growth factor (HGF), interleukin (IL)-2, IL-6 and IL-8 were determined by enzyme-linked immuno sorbent assay methods. The results showed that BSRPS owned a fine hepatoprotective effect with stable HGF producing ability. Sulfated modification was able to increase the proliferation rates of B and T lymphocytes and the secretions of total Ab, IFN-γ and IL-2, as comparison with those of BSRPS group. In summary, both of them exhibited immuno-enhancing effects on the therapeutic effects for DVH, and the capacity of sBSRPS was stronger than that of BSRPS.

Anti-tumor activity and the mechanism of SIP-S: A sulfated polysaccharide with anti-metastatic effect.

Our previous studies demonstrated that SIP-S had anti-metastatic activity and inhibited the growth of metastatic foci. Here we report the anti-tumor and immunoregulatory potential of SIP-S. SIP-S could significantly inhibit tumor growth in S180-bearing mice, and the inhibition rates was 43.7% at 30 mg/kg d. Besides, SIP-S could improve the thymus and spleen indices of S180-bearing mice and the mice treated with CTX. The combination of SIP-S (15 mg/kg d) with CTX (12.5 mg/kg d) showed higher anti-tumor potency than CTX (25 mg/kg d) alone. These results indicated that SIP-S had immunoenhancing and anticancer activity, and the immunoenhancing activity might be one mechanism for its anti-tumor activity. Flow cytometry results showed that SIP-S could induce tumor cells apoptosis. Western blot analysis indicated that SIP-S could upregulate the expression of pro-apoptotic proteins, caspase-3, -8, -9 and Bax, and downregulate the expression of anti-apoptotic protein PARP-1 in tumor cells in a dose-dependent manner. In summary, SIP-S has anti-tumor activity, which may be associated with its immunostimulating and pro-apoptotic activity.

Evaluation of antioxidant and immuno-enhancing activities of Purslane polysaccharides in gastric cancer rats.

In the last three decades, numerous polysaccharides and polysaccharide-protein complexes have been isolated from plant or animal and used as a promising source of therapeutic agents for cancer. In this study, we examined the effects of Purslane polysaccharides (PPs) on the oxidative injury and immune status in N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced gastric cancer rats. PPs administration (200, 400 or 800mg/kg body weight) could not only increase the body weight, peripheral white blood cells (WBC) count, thymus and spleen indexes, but also remarkably promote splenocytes proliferation of gastric cancer rats. Furthermore, the production of serum cytokines in gastric cancer rats, such as interleukin-2 (IL-2), interleukin-4 (IL-4) and tumor necrosis factor-alpha (TNF-α) was enhanced by PPs treatment. Besides, treatment with PPs was found to provide a dose-dependent protection against MNNG-induced oxidative injury by enhancing SOD, CAT, GSH-Px activities of gastric cancer rats. Taken together, we concluded that enhancement of antioxidants and immune response might be responsible for the anticancer effect of PPs in gastric cancer.