N-Acetylcysteine alleviates gut dysbiosis and glucose metabolic disorder in high-fat diet-fed mice.

BACKGROUND: N-Acetylcysteine (NAC), an antioxidative reagent for clinical diseases, shows potential in the treatment of diabetes and other metabolic diseases. However, it is unknown how NAC modulates the gut microbiota of mice with metabolic syndrome. The aim of the present study was to demonstrate the preventive effect of NAC on intestinal dysbiosis and glucose metabolic disorder. METHODS: Mice (C57BL/6J strain) were fed either a normal chow diet (NCD), NCD plus NAC, a high-fat diet (HFD), or HFD plus NAC for 5 months, after which glucose levels, circulating endotoxins and key metabolism-related proteins were determined. Fecal samples were analyzed by 16S rRNA sequencing. A novel analysis was performed to predict functional changes in gut microbiota. In addition, Spearman's correlation analysis was performed between metabolic biomarkers and bacterial abundance. RESULTS: Treatment with NAC significantly reversed the glucose intolerance, fasting glucose concentrations, and gains in body weight and plasma endotoxin in HFD-fed mice. Further, NAC upregulated occludin and mucin glycoprotein levels in the proximal colon of HFD-treated mice. Noticeably, NAC promoted the growth of beneficial bacteria (i.e. Akkermansia, Bifidobacterium, Lactobacillus and Allobaculum) and decreased populations of diabetes-related genera, including Desulfovibrio and Blautia. In addition, NAC may affect the metabolic pathways of intestinal bacteria, including lipopolysaccharide biosynthesis, oxidative stress, and bacterial motility. Finally, the modified gut microbiota was closely associated with the metabolic changes in NAC-treated HFD-fed mice. CONCLUSIONS: N-Acetylcysteine may be a potential drug to prevent glucose metabolic disturbances by reshaping the structure of the gut microbiota.

Conjugation of chitosan oligosaccharides enhances immune response to porcine circovirus vaccine by activating macrophages.

Porcine circovirus type 2 (PCV2)-associated diseases have led to great economic losses to the pig industry. Our lab previously found that conjugation of chitosan oligosaccharides (COS) or via a carrier protein enhanced the immunogenicity of PCV2 vaccine against infectious pathogens. However, precise mechanisms and signal transduction pathways underlying the efficacy of COS conjugation remains poorly defined. In this study, to better understand the effects and mechanism of COS conjugates maintain the adjuvant potential in vivo, we investigated its augmentation of macrophage function, including cell activation, NO production, cytokine production and phagocytosis. Additionally, the role of Toll-like receptors (TLR) proteins in this process was also assessed. The results indicate that, as compared to the PCV and PCV/COS, conjugation of COS effectively enhanced the NO production, cytokines generation and phagocytosis activity of macrophages. Noticeably, the generation of NO and proinflammatory cytokines was closely related to the TLR2/4 signaling pathways, strongly suggesting that conjugation of COS regulates innate and adaptive immunity by activation of macrophages, resulting in immune enhancement. In summary, the present study provides a potential mechanism of COS conjugation as a novel adjuvant to improve immune responses against various diseases.

Conjugation of chitosan oligosaccharides via a carrier protein markedly improves immunogenicity of porcine circovirus vaccine.

Porcine circovirus type 2 (PCV2)-associated diseases have led to huge economic losses in pig industry. Our laboratory previously found that conjugation of chitosan oligosaccharides (COS) enhanced the immunogenicity of PCV2 vaccine against infectious pathogens. In this study, an effective adjuvant system was developed by covalent conjugation of COS via a carrier protein (Ovalbumin, OVA) to further increase the immunogenicity of vaccine. Its effect on dendritic cells maturation was assessed in vitro and its immunogenicity was investigated in mice. The results indicated that, as compared to the PCV2 and COS-PCV2, COS-OVA-PCV2 stimulated dendritic cells to express higher maturation markers (CD80, CD86, CD40 and MHC class II) and remarkably promoted both humoral and cellular immunity against PCV2 by enhancing the lymphocyte proliferation and inducing a mixed Th1/Th2 response, including the increased production of PCV2-specific antibodies and raised levels of inflammatory cytokines. Furthermore, it displayed better immune-stimulating effects than the physical mixture of vaccine and ISA206 (a commercialized adjuvant). In conclusion, conjugation of COS via a carrier protein might be a promising strategy to enhance the immunogenicity of vaccines.

Antrodia cinnamomea Oligosaccharides Suppress Lipopolysaccharide-Induced Inflammation through Promoting O-GlcNAcylation and Repressing p38/Akt Phosphorylation.

Antrodia cinnamomea (AC), an edible fungus growing in Taiwan, has various health benefits. This study was designed to examine the potential inhibitory effects of AC oligosaccharides on lipopolysaccharide (LPS)-induced inflammatory responses in vitro and in vivo. By trifluoroacetic acid degradation, two oligosaccharide products were prepared from AC polysaccharides at 90 °C (ACHO) or 25 °C (ACCO), which showed different oligosaccharide identities. Compared to ACCO, ACHO displayed better inhibitory effects on LPS-induced mRNA expression of pro-inflammatory cytokines including IL-6, IL-8, IL-1ß, TNF-α and MCP-1 in macrophage cells. Further, ACHO significantly suppressed the inflammation in lung tissues of LPS-injected C57BL/6 mice. The potential anti-inflammatory molecular mechanism may be associated with the promotion of protein O-GlcNAcylation, which further skewed toward the marked suppression of p38 and Akt phosphorylation. Our results suggest that the suppressive effect of AC oligosaccharides on inflammation may be an effective approach for the prevention of inflammation-related diseases.

Correction: Zhang, G.; Cheng, G.; Jia, P.; Jiao, S.; Feng, C.; Hu, T.; Liu, H.; Du, Y. The Positive Correlation of the Enhanced Immune Response to PCV2 Subunit Vaccine by Conjugation of Chitosan Oligosaccharide with the Deacetylation Degree. Marine Drugs 2017, 15, 236.

The authors wish to correct Figure 1 in this paper [1] to be as follows:[...].

The Positive Correlation of the Enhanced Immune Response to PCV2 Subunit Vaccine by Conjugation of Chitosan Oligosaccharide with the Deacetylation Degree.

Chitosan oligosaccharides (COS), the degraded products of chitosan, have been demonstrated to have versatile biological functions. In primary studies, it has displayed significant adjuvant effects when mixed with other vaccines. In this study, chitosan oligosaccharides with different deacetylation degrees were prepared and conjugated to porcine circovirus type 2 (PCV2) subunit vaccine to enhance its immunogenicity. The vaccine conjugates were designed by the covalent linkage of COSs to PCV2 molecules and administered to BALB/c mice three times at two-week intervals. The results indicate that, as compared to the PCV2 group, COS-PCV2 conjugates remarkably enhanced both humoral and cellular immunity against PCV2 by promoting lymphocyte proliferation and initiating a mixed T-helper 1 (Th1)/T-helper 2 (Th2) response, including raised levels of PCV2-specific antibodies and an increased production of inflammatory cytokines. Noticeably, with the increasing deacetylation degree, the stronger immune responses to PCV2 were observed in the groups with COS-PCV2 vaccination. In comparison with NACOS (chitin oligosaccharides)-PCV2 and LCOS (chitosan oligosaccharides with low deacetylation degree)-PCV2, HCOS (chitosan oligosaccharides with high deacetylation degree)-PCV2 showed the highest adjuvant effect, even comparable to that of PCV2/ISA206 (a commercialized adjuvant) group. In summary, COS conjugation might be a viable strategy to enhance the immune response to PCV2 subunit vaccine, and the adjuvant effect was positively correlated with the deacetylation degree of COS.

Enhanced immune response to inactivated porcine circovirus type 2 (PCV2) vaccine by conjugation of chitosan oligosaccharides.

This study aimed to investigate the effect of chitosan oligosaccharide (COS) conjugation on the immunogenicity of porcine circovirus type-2 (PCV2) vaccine. Two conjugates (PCV2-COS-1 and PCV2-COS-2) were designed by covalent conjugation of an inactivated PCV2 vaccine with COS, and administered to C57BL/6 mice three times at two-week intervals. The results indicate that, as compared to PCV2 alone group, the PCV2-COS conjugates remarkably enhanced both humoral and cellular immunity against PCV2 by promoting T lymphocyte proliferation and initiating a mixed Th1/Th2 response, including the elevated production of PCV-2 specific antibodies and up-regulated secretion of inflammatory cytokines. Noticeably, the immunization with PCV2-COS-1 conjugate displayed similar or even better immune-stimulating effects than that by PCV2/ISA206 (a commercialized adjuvant) and showed no infection or pathological signs at injection sites of the mice. Presumably, the covalent linkage of PCV2 vaccine to COS might be a viable strategy to increase the efficacy against PCV2-associated diseases.

The potential adjuvanticity of quaternized chitosan hydrogel based microparticles for porcine reproductive and respiratory syndrome virus inactivated vaccine.

Infectious diseases possess a big threat to the livestock industry worldwide. Currently, inactivated veterinary vaccines have attracted much attention to prevent infection due to their safer profile compared to live attenuated vaccine. However, its intrinsic poor immunogenicity demands the incorporation of an adjuvant. Mineral oil based adjuvant (Montanide™ ISA206) was usually used to potentiate the efficacy of veterinary vaccines. However, ISA206 could not induce robust cellular immune responses, which was very important in controlling virus replication and clearing the infected cells. Moreover, mineral oil would result in severe side effects. To improve both the humoral and cellular immune responses of porcine reproductive and respiratory syndrome virus (PRRSV) inactivated vaccine, we developed pH-sensitive and size-controllable quaternized chitosan hydrogel microparticles (Gel MPs) without using chemical cross linking agent. Gel MPs, ionic cross-linked with glycerophosphate (GP), were biocompatible and could efficiently adsorb the inactivated PRRSV vaccine with a loading capacity of 579.05µg/mg. After intramuscular immunization in mice, results suggested that Gel MPs elicited significantly higher cell-mediated immune responses and comparable humoral immune responses compared to ISA 206. Regarding the biocompatibility, safety and effectiveness, Gel MPs would be a promising candidate to enhance the efficacy of veterinary vaccine.

Monoclonal antibody, mAb 4C13, an effective detoxicant antibody against ricin poisoning.

Ricin is a glycoprotein produced in castor seeds and consists of two polypeptide chains named Ricin Toxin A Chain (RTA) and Ricin Toxin B Chain (RTB), linked via a disulfide bridge. Due to its high toxicity, ricin is regarded as a high terrorist risk for the public. However, antibodies can play a pivotal role in neutralizing the toxin. In this research, the anti-toxicant effect of mAb 4C13, a monoclonal antibody (mAb) established using detoxicated ricin as the immunized antigen, was evaluated. Compared with mAb 4F2 and mAb 5G6, the effective mechanism of mAb 4C13 was analyzed by experiments relating to its cytotoxicity, epitope on ricin, binding kinetics with the toxin, its blockage on the protein synthesis inhibition induced by ricin and the intracelluar tracing of its complex with ricin. Our result indicated that mAb 4C13 could recognize and bind to RTA, RTB and exert its high affinity to the holotoxin. Both cytotoxicity and animal toxicity of ricin were well blocked by pre-incubating the toxin with mAb 4C13. By intravenous injection, mAb 4C13 could rescue the mouse intraperitoneally (ip) injected with a lethal dose of ricin (20µg/kg) even at 6h after the intoxication and its efficacy was dependent on its dosage. This research indicated that mAb 4C13 could be an excellent candidate for therapeutic antibodies. Its potent antitoxic efficiency was related to its recognition on the specific epitope with very high affinity and its blockage of protein synthesis inhibition in cytoplasm followed by cellular internalization with ricin.

Combination of inverted pyramidal nanovoid with silver nanoparticles to obtain further enhancement and its detection for ricin.

We have obtained the surface-enhanced Raman scattering substrate by depositing silver nanoparticles on the surface of the inverted pyramidal nanovoid in order to improve the enhance effects. Experimental results showed that the combined substrate exhibited greater enhancement than the nanovoid substrate or nanoparticles. In order to test the SERS activity of the combined substrates, Rh6G and ricin toxin were used as Raman probes. Finite element method was employed to simulate electric field and induced charge distribution of the substrates, which have been used to explore the interaction between nanoparticles and nanovoid as well as mechanism of the great enhancement.

Absorption, distribution and pathological injury in mice due to ricin poisoning via the alimentary pathway.

The aim of this work was to investigate the potential interactions between intestinal absorbance and ricin poisoning. The Caco-2 cell monolayer and everted intestinal sac (VEIS) models were used. The distribution of ricin in CD-1 mice intoxicated with 0.1 mg/kg of ricin intragastrically was determined by immunohistochemistry. The results showed that ricin could not transfer across the healthy Caco-2 cell monolayer within three hours after poisoning. However, it could pass through the everted rat intestinal wall after 0.5 h of incubation. The toxin in the liver, spleen, lungs and kidneys of mice could be detected as early as 1 h after intoxication. The pathological results were in accordance with the cytotoxicities of ricin in Caco-2, HepG 2, H1299 and MDCK cells, indicating that though no significant symptom in mice could be observed within 3 h after ricin intoxication, important tissues, especially the kidneys, were being injured by the toxin and that the injuries were progressing.

An α-glucan isolated from root of Isatis Indigotica, its structure and adjuvant activity.

The root of Isatis indigotica is a traditional Chinese herbal medicine. An α-glucan (IIP-A-1) was firstly isolated from the roots. In this study we elucidated the chemical structure of IIP-A-1 and determined its adjuvant activity by co-immunizing mice with H1N1 influenza virus split and recombinant hepatitis B surface antigen (HBsAg), respectively. The polysaccharide was pretreated with periodate oxidation, Smith degradation and methylation in order to analyze its structure using GC, HPGPC, FT-IR, NMR and GC-MS. The adjuvant effect was evaluated by determining the antibody titers of serum against H1N1 influenza and HBsAg using ELISA. The proliferation and TNF-α secretion of macrophages administrated with different dose of IIP-A-1 were measured in vitro. The results of this study revealed that IIP-A-1 was an α-glucan with the molecular weight of 3,600 Da. The backbone was α-(1 → 4)-D-glucan with (1 → 6) branch chain. The α-glucan could significantly enhance the immune response of mice immunized with H1N1 influenza or HBsAg in vivo and exert good dose-dependent effects on the proliferation and the TNF-α secretion of macrophages in vitro. These results supported that IIP-A-1 was expected to be an efficacious adjuvant candidate for prophylactic and therapeutic vaccines.

The novel fusion proteins, GnRH-p53 and GnRHIII-p53, expression and their anti-tumor effect.

p53, one of the most well studied tumor suppressor factor, is responsible to a variety of damage owing to the induction of apoptosis and cell cycle arrest in the tumor cells. More than 50% of human tumors contain mutation or deletion of p53. Gonadotrophin-releasing hormone (GnRH), as the ligand of Gonadotrophin-releasing hormone receptor (GnRH-R), was used to deliver p53 into tumor cells. The p53 fusion proteins GnRH-p53 and GnRH iii-p53 were expressed and their targeted anti-tumor effects were determined. GnRH mediates its fusion proteins transformation into cancer cells. The intracellular delivery of p53 fusion proteins exerted the inhibition of the growth of H1299 cells in vitro and the reduction of tumor volume in vivo. Their anti-tumor effect was functioned by the apoptosis and cell cycle arrest induced by p53. Hence, the fusion protein could be a novel protein drug for anti-tumor therapy.

Carbon dots: a safe nanoscale substance for the immunologic system of mice.

We aimed at investigating the effect of carbon dots on the BALB/c mice immune system. Mice were respectively treated with different doses of carbon dots and saline. At 1 and 9 days after intravenous administration of carbon dots, splenocyte proliferation, subpopulation of the peripheral lymphocytes, and induction of primary immune responses in mice were investigated. The results showed that high dose of carbon dots could promote the percentages of CD3+ and interferon-γ (IFN-γ) secretion and decrease the proportions of CD4+/CD8+ on the first day after administration. At 9 days post exposure, the proliferation of splenocytes had a significant increase. IFN-γ secretion and proportions of CD3+/CD19+ were also found to have an obvious promotion, and both the percentages of CD4+ and CD8+ T lymphocytes were raised, whereas the expression of cytokines made little change in the treated groups, except for IL-12 which had a slight increase in the 50-mg/kg group. The weight coefficients and histological analysis of the spleen and thymus of the treated mice exerted fewer differences compared with those from the control mice. It suggests that carbon dots could influence the immune functions of normal BALB/c mice by inducing Th1 and Tc responses and that these effects were not enough to induce the morphological change of the immune organs.

Introduction of hypoxia-targeting p53 fusion protein for the selective therapy of non-small cell lung cancer.

Non-small cell lung cancer (NSCLC), which accounts for ~85% of lung cancer, is the major cause of malignancy mortality around the world. TP53 dysfunction and hypoxia are the typical biological features of the diverse solid tumors, including NSCLC. To develop an effective and low cytotoxic biological agent for targeted therapy, a p53 fusion protein, which was conjugated with the minimum motif of oxygen-dependent degradation domain (ODD) and the basic domain of TAT of HIV-1 named as TAT-ODD-p53, was evaluated for the treatment of NSCLC established by grafting H1299 cell line in which TP53 is homozygously deleted. We provide the evidence that this p53 fusion protein could significantly induce the cell-cycle arrest and/or apoptosis to inhibit H1299 cells' growth via p53-dependent pathways, including up-regulation of p21 expression and activation of pro-caspase-3, especially under hypoxia in vitro. The results in vivo indicated that this protein could selectively accumulate in the low oxygen tension areas of solid tumor tissues, inhibiting tumor growth via a similar mechanism to that in vitro. No obvious side effects were observed. Therefore, this recombinant p53 protein is likely to become a good candidate for targeted therapy of NSCLC.

[Cloning of human scFv against soman transition state analogues (P6) from a large phage antibody library].

AIM: To clone human anti-soman transition state analogues antibodies from a large single-chain phage antibody library. METHODS: An organophosphorus hapten P6 [O1-methyl-O2-(1, 2, 2-trimethylpropyl)-2-hydroxy-5-nitrophenyl methylphosphonic acid] was synthesized. Its chemical conjugate with bovine serum albumin (BSA) was used as antigen (P6-BSA) to screen antibodies against soman. Panning of a large single-chain phage antibody library was conducted to select specific antibodies against soman. The antigen binding characteristics were analyzed by ELISA. RESULTS: After 4 rounds of panning, 14 clones had specific binding ability to P6. DNA fingerprinting showed that diverse specific human scFvs against P6 was obtained from the library by biopanning. CONCLUSION: Human anti-soman transition state analogues scFvs have been cloned from large phage antibody library.

Detection of residual toxin in tissues of ricin-poisoned mice by sandwich enzyme-linked immunosorbent assay and immunoprecipitation.

This work aimed to evaluate a method to detect the residual ricin in animal tissues. Immunoprecipitation and sandwich enzyme-linked immunosorbent assay (ELISA) were used to detect ricin in the tissues of intoxicated mice. The monoclonal antibodies (Mabs) 4C13 and 3D74 were used to assay the whole ricin molecules via sandwich ELISA. Mab 4C13 was conjugated with Sepharose 4B to capture ricin or ricin A chain by immunoprecipitation. Mice injected intravenously with ricin at the dosage of 5 microg/mouse were killed at different time points after intoxication. The serum, liver, kidney, lung, and intestine were harvested. High levels of ricin were found in serum and liver samples at each poisoning time point by sandwich ELISA, suggesting the possibility of determining ricin intoxication by detecting residual ricin in the serum. However, this method turned out to be ineffective for examining ricin in the kidney, lung, and intestine of poisoned mice. Although the same tissue samples of intoxicated mice were analyzed by immunoprecipitation, positive bands were found. This indicated that some components in the kidney, lung, and intestine could bind with ricin and interfere in its binding activity with the coated antibody. Immunoprecipitation could be used to measure the existence of ricin in these samples.

An organophosphorus hapten used in the preparation of monoclonal antibody and as an active immunization vaccine in the detoxication of soman poisoning.

Soman is an organophosphorus neurotoxin which inhibits the activity of acetylcholinesterase (AChE). The goal of this work was to find out whether antibodies against an organophosphorus hapten could protect mice from soman toxicity. An organophosphorus hapten P6 was synthesized. Its chemical conjugates with limulus polyphemus hemocyanin and bovine serum albumin were used as immune antigen (P6-LPH) and detection antigen (P6-BSA), respectively. Eight hybridoma cell lines secreting monoclonal antibodies (Mabs) were established. The binding reactivities of Mabs with P6 and soman were determined by competitive inhibition enzyme immunoassay (CIEIA). All antibodies recognized P6 and four of them (2C10, 3G1, 3B9 and 3C11) combined with soman. The IC(50) was 10(-6.5) to 10(-5.3)mol/l for P6 and 10(-5) to 10(-3.5)mol/l for soman. Furthermore, Mab 3G1 reduced the inhibition of AChE activity by soman in vitro. When soman was pre-incubated with Mabs before being injected into mice, soman potency was reduced, indicating that Mabs could protect mice from soman toxicity. In an active immunization regimen, mice immunized with P6-LPH and challenged with 0.15mg/kg soman injected subcutaneously, had fewer signs of intoxication and a higher survival rate compared with control mice. These results demonstrate that the anti-soman antibodies have proper characteristics as scavengers in the detoxication of soman poisoning.

A recombinant cell-permeable p53 fusion protein is selectively stabilized under hypoxia and inhibits tumor cell growth.

More than 50% of human tumors contain a mutation in p53. Over 90% of tumors are solid tumors. Solid tumors have low oxygenated regions, called hypoxic regions where the tumor cells are more resistant to radio- and chemo-therapy than their well-oxygenated counterparts. In this study, we constructed a cell-permeable p53 fusion protein with selective stability in the hypoxic region. The fusion protein contained the TAT peptide for transduction across membranes, the oxygen-dependent degradation domain of hypoxia-inducible factor-1alpha and wild-type p53. This protein was effectively delivered into tumor cells where it exerted anticancer activity leading to the inhibition of cancer cell growth in vitro and the reduction of tumor weight in vivo. Hence, the fusion protein can be a novel protein drug for antitumor therapies, especially for hypoxic tumor cells.