Peptidoglycan-based immunomodulation.

Peptidoglycan (PGN) is a unique component in the cytoderm of prokaryotes which can be recognized by different pathogen-associated molecular patterns (PAMPs) in eukaryotes, followed by a cascade of immune responses via different pathways. This review outlined the basic structure of PGN, its immunologic functions. The immunomodulation pathways mediated by PGN were elaborated. PGN induces specific immunity through stimulating different cytokine release and Th1/Th2-dominated immune responses during humoral/cellular immune response. The nonspecific immunity activation by PGN involves immunomodulation by different pattern recognition receptors (PRRs) including PGN recognition proteins (PGRPs), nucleotide oligomerization domain (NOD)-like receptors (NLRs), Toll-like receptors (TLRs), and C-type lectin receptors (CLRs). The sources and classification of PGRPs were summarized. In view of the stimulating activities of PGN and its monomers, the potential application of PGN as vaccine or adjuvant was prospected. This review provides systematic information on PGN functionalities from the point of immunoregulation, which might be useful in the deep exploitation of PGN.Key points. The immunological functions of PGN were illustrated. Cellular and humoral immunomodulation by PGN were outlined. The use of PGN as vaccine or adjuvant was prospected.

Hydrogel Encapsulation of Lactobacillus casei by Block Charge Modified Pectin and Improved Gastric and Storage Stability.

Lactobacillus casei (L. casei W8) was encapsulated in pectin methylesterase (PME) charge modified pectin hydrogels; stability and in vitro release were evaluated under simulated gastrointestinal (GI) conditions. PME, 355 U/mL, de-esterified citrus pectin to 35% from 72% degree of esterification (DE). Pectin ζ-potential decreased to about -37 mV and molecular weight decreased from 177 kDa to 143 kDa during charge modification. More than 99% L. casei W8 were encapsulated in block charged, low methoxy pectin (35 mLMP) hydrogels by calcium ionotropic gelation. The integrity of the hydrogels was maintained under simulated GI conditions, and no release of L. casei W8 was observed. Microbial counts of encapsulated L. casei ranged from 6.94 log CFU/g to 10.89 log CFU/g and were 1.23 log CFU/g higher than for unencapsulated L. casei W8. The viability of encapsulated L. casei W8 in wet hydrogels remained the same for 2 weeks, but nearly all flora died after 4 weeks storage at 4 °C. However, freeze dried hydrogels of L. casei W8 were viable for 42 days at 4 °C and 14 days at room temperature. Charge modified pectin hydrogels are potentially good vehicles for colon-targeted delivery carrier for probiotics and longer stability of L. casei W8.

Isolation and hypoglycemic effects of water extracts from mulberry leaves in Northeast China.

Diabetes is the main chronic disease that greatly affects human life. Up to now, many measures have been taken to cope with the disease, among which natural products with hypoglycemic effects have aroused great interest. The objective of this study was to evaluate the hypoglycemic effects of Morus abla L. cv. longsang 1 leaf-derived water extract in vitro and in vivo. These leaves were firstly subjected to water extraction, and the obtained products were further isolated for polysaccharides, flavonoids and alkaloids. The α-glucosidase activity and anti-protein glycosylation activity of the aqueous extracts were examined in vitro. Hyperglycemic mouse models were used to evaluate the hypoglycemic effects of the aqueous extract by blood biochemical parameters, intestinal microbiota, and pathological changes to the kidneys. The results showed that the main hypoglycemic components in the aqueous extracts were flavonoids and alkaloids and their inhibition rates against α-glucosidase activity were 86.12 ± 1.79% and 87.29 ± 1.32%, respectively. High-dose mulberry leaf water extracts can reduce the blood glucose of diabetic mice by 28.17% and improve glucose tolerance by 19.02%. Furthermore, mulberry leaf water extracts could reduce the serum free fatty acid (FFA), tumor necrosis factor-α (TNF-α), insulin and glycated serum protein content, while alleviating kidney damage and improving intestinal microbiota. These results indicated that the synergistic effects among the different components of mulberry leaves might explain their alleviating effects on diabetic syndrome and thus provide a simple, convenient way to obtain the hypoglycemic components from mulberry leaves.

L. plantarum, L. fermentum, and B. breve Beads Modified the Intestinal Microbiota and Alleviated the Inflammatory Response in High-Fat Diet-Fed Mice.

This paper aims to study the effects of compound microbe-based beads on changes in the intestinal microbiota and alleviation of high-fat (HF) diet-induced inflammatory responses. Forty-eight mice were fed base chow or a high-fat diet for 4 weeks and then randomly separated into six groups: normal diet (group A), high-fat diet (group B), high-fat positive control (fed with high-fat chow plus Tetrahydrolipstatin, group C), high-fat chow plus B. breve beads (group D), high-fat chow plus L. plantarum-L. fermentum beads (group E), and high-fat chow plus L. plantarum-L. fermentum-B. breve beads (group F). The body weights were measured. The serum cytokine and lipid levels were determined by ELISA, and high-throughput sequence analysis of the fecal microbiota was conducted. Beads with cell encapsulation rates higher than 99% decreased the body weight from 50.97 ± 3.44 g in group B to 42.64 ± 2.63 g in group F at the end of the experiment (p = 0.00019). The total cholesterol content in group F was 80.14 ± 9.37 mmol/L, which was significantly lower than that in group A (96.13 ± 24.07 mmol/L) (p = 0.02765), group B (102.52 ± 12.20 mmol/L) (p = 0.00196), and group C (98.99 ± 11.32 mmol/L) (p = 0.00804). In addition, the serum IL-6 level showed no significant difference between group F and the base chow control group. The microbial cell-loaded bead intervention led to increased abundances of Bifidobacterium and Lactobacillus in mouse feces. Oral administration of three strain-based beads led to alleviation of inflammatory reactions in high-fat diet-fed mice.

Effects of a probiotic intervention on Escherichia coli and high-fat diet-induced intestinal microbiota imbalance.

Obesity is often associated with intestinal microbiota imbalance and increased Gram-negative bacteria characterized by higher endotoxin levels. Therefore, a study on the joint effects of a high-fat diet and Gram-negative bacteria prechallenge might provide further information on the interactive effects of intestinal microbiota and obesity as well as the effects of a probiotic intervention on these processes. This study focused on the joint effects of a high-fat diet and Escherichia coli on mouse inflammatory cytokines, intestinal microbiota, hepatic pathological changes, and the alleviating capacity of probiotics (Lactobacillus plantarum, Bifidobacterium breve, and Lactobacillus fermentum). Our results showed that E. coli administration and the high-fat diet exacerbated the inflammatory syndrome by increasing the visceral fat content, the inflammatory cell infiltration, and intestinal microbiota disorder in mice. E. coli administration caused a decrease in short-chain fatty acids in mouse feces, and probiotics effectively improved this phenomenon. Compound probiotic intervention reduced LPS and IL-1ß levels, while increased IL-10 levels in mice improved the degeneration and inflammatory infiltration of mouse liver cells. The intestinal microbiota showed great differences at 3 weeks and 6 weeks post-administration. High fat and E. coli alone or in combination caused intestinal microbiota disorder, with increased harmful bacteria, and the probiotics effectively improved the intestinal flora structure and increased the fecal short-chain fatty acid (SCFA) content. In conclusion, a high-fat diet and Gram-negative bacteria challenge exacerbated the inflammatory syndrome, which can be alleviated by compound probiotic intervention.

Beneficial Effects of Non-Encapsulated or Encapsulated Probiotic Supplementation on Microbiota Composition, Intestinal Barrier Functions, Inflammatory Profiles, and Glucose Tolerance in High Fat Fed Rats.

Development of obesity-associated comorbidities is related to chronic inflammation, which has been linked to gut microbiota dysbiosis. Thus, modulating gut microbiota composition could have positive effects for metabolic disorders, supporting the use of probiotics as potential therapeutics in vivo, which may be enhanced by a microencapsulation technique. Here we investigated the effects of non-encapsulated or pectin-encapsulated probiotic supplementation (Lactobacillus paracasei subsp. paracasei L. casei W8®; L. casei W8) on gut microbiota composition and metabolic profile in high-fat (HF) diet-fed rats. Four male Wistar rat groups (n = 8/group) were fed 10% low-fat, 45% HF, or HF with non-encapsulated or encapsulated L. casei W8 (4 × 107 CFU/g diet) diet for seven weeks. Microbiota composition, intestinal integrity, inflammatory profiles, and glucose tolerance were assessed. Non-encapsulated and pectin-encapsulated probiotic supplementation positively modulated gut microbiota composition in HF-fed male rats. These changes were associated with improvements in gut barrier functions and local and systemic inflammation by non-encapsulated probiotics and improvement in glucose tolerance by encapsulated probiotic treatment. Thus, these findings suggest the potential of using oral non-encapsulated or encapsulated probiotic supplementation to ameliorate obesity-associated metabolic abnormalities.

Preparation of Bifidobacterium breve encapsulated in low methoxyl pectin beads and its effects on yogurt quality.

Yogurt is a popular product worldwide partly because of the health-promoting effects of the probiotics that it contains. Probiotics with high survivability constitute a promising direction for fortified yogurt products. This study aimed to prepare Bifidobacterium breve-loaded yogurt with the bacteria surviving transit to the lower part of small intestine or colon. Bifidobacterium breve beads were prepared through an ion-crosslinking method using low methoxyl pectin as the encapsulating material. Features such as encapsulation efficiency and stability during storage and passage through the simulated gastrointestinal tract were studied in vitro. A commercial starter was used for yogurt fermentation, and B. breve with or without encapsulation was added as a probiotic supplement with the starter or 3 to 4 h after fermentation. The effects of B. breve beads on yogurt characteristics were evaluated after different fermentation processes: BC, milk fermented with marketed yogurt starter; UBFF, unencapsulated B. breve added to fresh milk and then fermented; EBFF, encapsulated B. breve added to fresh milk and then fermented; UBAF, unencapsulated B. breve added after fermentation with the starter; and EBAF, encapsulated B. breve beads added 3 to 4 h after fermentation with the starter. Evaluation was based on texture, electronic nose, and electronic tongue analyses. The particle size analysis of B. breve beads showed that they were uniform, mostly spherical, 1 to 1.5 mm in diameter with encapsulating efficiency higher than 99%. Following treatment with the simulated gastrointestinal tract conditions, the number of B. breve decreased by 1.76 and 4.82 log cfu/g for B. breve beads and unencapsulated B. breve, respectively. The EBAF group showed the lowest viscosity (2,235.67 cP) at d 0, and the lower postfermentation degree was reflected by the slow increase in yogurt viscosity. All groups kept a relatively stable pH during storage. The cohesiveness values of the EBAF and UBAF groups were significantly higher than those of the other groups. The trends in texture changes within the BC, UBFF, and EBFF groups were similar, and the UBAF and EBAF groups showed similar trends. In conclusion, B. breve beads showed good stability in vitro and improved yogurt characteristics by increasing the survival rate of the encapsulated cells. Good compatibility of low methoxyl pectin beads with yogurt was also observed.

The regulatory effects of L. plantarum peptidoglycan microspheres on innate and humoral immunity in mouse.

OBJECTIVE: To study the effects of Lactobacillus Plantarum cell wall peptidoglycan (LPG) microspheres on mouse intestinal flora changes, peptidoglycan recognitions protein (PGRP) and cytokines expression levels. METHOD: Plate counting was used for enumeration of the intestinal flora. Real-time PCR was used for quantification PGRP in different tissues. Cytokines content were determined by ELISA kits. RESULT: The mouse administered orally with LPG microspheres showed significantly higher number of Lactobacillus and Bifidobacterium in caecum contents (p < 0.01). The amount of PGRP expression in different organs was highest in LPG microspheres-treated group. IL-4, 12, IFN-γ, TNF-α contents in serum from LPG microspheres-treated mouse were significantly higher than those in normal saline-treated group (p < 0.01). CONCLUSIONS: This study shows that the LPG microspheres can regulate intestinal flora imbalance and improve systemic immunity, improve both Th1 and Th2 immune response, which provide some basis for the use of LPG as potential adjuvants.

Study on the effects of microencapsulated Lactobacillus delbrueckii on the mouse intestinal flora.

OBJECTIVE: To evaluate the protective effects of microencapsulation on Lactobacillus delbrueckii by random, parallel experimental design. MATERIALS AND METHODS: Lincomycin hydrochloride-induced intestinal malfunction mouse model was successfully established; then the L. delbrueckii microcapsule was given to the mouse. The clinical behaviour, number of intestinal flora, mucous IgA content in small intestine, IgG and IL-2 level in peripheral blood were monitored. The histological sections were also prepared. RESULTS: The L. delbrueckii microcapsule could have more probiotic effects as indicated by higher bifidobacterium number in cecal contents. The sIgA content in microcapsule treated group was significantly higher than that in non-encapsulated L. delbrueckii treated group (p < 0.05). Intestine pathological damage of the L. delbrueckii microcapsule-treated group showed obvious restoration. CONCLUSION: The L. delbrueckii microcapsules could relieve the intestinal tissue pathological damage and play an important role in curing antibiotic-induced intestinal flora dysfunction.

Synthesis, characterization, and immune efficacy of layered double hydroxide@SiO2 nanoparticles with shell-core structure as a delivery carrier for Newcastle disease virus DNA vaccine.

Layered double hydroxide (LDH)@SiO2 nanoparticles were developed as a delivery carrier for the plasmid DNA expressing the Newcastle disease virus F gene. The LDH was hydrotalcite-like materials. The plasmid DNA encapsulated in the LDH@SiO2 nanoparticles (pFDNA-LDH@SiO2-NPs) was prepared by the coprecipitation method, and the properties of pFDNA-LDH@SiO2-NPs were characterized by transmission electron microscopy, zeta potential analyzer, Fourier transform infrared spectroscopy, and X-ray diffraction analysis. The results demonstrated that the pFDNA-LDH@SiO2-NPs had a regular morphology and high stability with a mean diameter of 371.93 nm, loading capacity of 39.66%±0.45%, and a zeta potential of +31.63 mV. A release assay in vitro showed that up to 91.36% of the total plasmid DNA could be sustainably released from the pFDNA-LDH@SiO2-NPs within 288 hours. The LDH@SiO2 nanoparticles had very low toxicity. Additionally, their high transfection efficiency in vitro was detected by fluorescent microscopy. Intranasal immunization of specific pathogen-free chickens with pFDNA-LDH@SiO2-NPs induced stronger cellular, humoral, and mucosal immune responses and achieved a greater sustained release effect than intramuscular naked plasmid DNA, and the protective efficacy after challenge with the strain F48E9 with highly virulent (mean death time of chicken embryos ≤60 hours, intracerebral pathogenicity index in 1 -day-old chickens >1.6) was 100%. Based on the results, LDH@SiO2 nanoparticles can be used as a delivery carrier for mucosal immunity of Newcastle disease DNA vaccine, and have great application potential in the future.

Evaluation of toxicity and adjuvant effects of peptidoglycan microspheres orally administered to mice.

In this study, peptidoglycan microspheres were evaluated for their toxicity and adjuvant effects after oral administration to mice. The liver and spleen indexes, CD cell content in peripheral blood and spleen, and immunoglobulin content in peripheral blood were measured by flow cytometry and indirect ELISA, respectively. Peptidoglycan microspheres with a loading capacity of 46.41 ± 0.83 g/100 g were prepared. In vivo tests showed that peptidoglycan microspheres revealed an immuno-enhancing profile as indicated by the slow increase of IgG content in peripheral blood compared with that of the untreated peptidoglycan group. In conclusion, peptidoglycan microspheres may be used as a new oral adjuvant in the host.

Preparation and characterization of chitosan microsphere loading bovine serum albumin.

To optimize the preparation process of chitosan microspheres and study its loading capacity, chitosan microsphere was prepared by crosslinking with glutaraldehyde, and bovine serum albumin (BSA) was absorbed onto chitosan microsphere. Scanning electron microscope (SEM), Fourier transform infrared spectroscopy (FITR), TA instruments and zeta potentiometer analyzer were used to characterize the parameters with respect to size, thermal characters, morphology, and zeta potential of the microspheres. The loading capability and in vitro release tests were carried out. The results showed that chitosan microsphere with particle size less than 10 µm and positively charged (+25.97±0.56 mV) can be obtained under the aldehyde group to amino group ratio at 1:1. A loading capacity of BSA at 28.63±0.15 g/100 g with corresponding loading efficiency at 72.01±1.44% was obtained for chitosan microsphere. In vitro test revealed a burst release followed by sustained-release profile.

Preparation and immunological effectiveness of a swine influenza DNA vaccine encapsulated in chitosan nanoparticles.

Preparation conditions of a DNA vaccine against swine influenza encapsulated in chitosan nanoparticles were determined. The nanoparticles were prepared according to a complex coacervation method using chitosan as a biodegradable matrix forming polymer. Under the preparation conditions, chitosan nanoparticles containing the DNA vaccine were produced with good morphology, high encapsulation rate and high stability. Transfection test indicated that the vaccine could be expressed as an antigen in cells, and maintained good bioactivity. In addition, better immune responses of mice immunized with the chitosan nanoparticles containing the DNA vaccine were induced and prolonged release of the plasmid DNA was achieved compared to the DNA vaccine alone. These results laid a foundation for further development of DNA vaccines in nanoparticles before ultimate industrial application.

Tablets based on compressed zein microspheres for sustained oral administration: design, pharmacokinetics, and clinical study.

In our previous study, we reported a novel tablet based on compressed zein microspheres as a universal drug delivery system using the hydrophobic protein zein, which shows zero-order release in the presence of pepsin. However, this formulation had difficulty with disintegration under physiological conditions within 48 h, and thus could not be used directly for oral administration. In the present study, a formulation of ivermectin (IVM) tablets based on compressed zein microspheres was improved as a new dosage form. The plasma disposition pharmacokinetics of IVM tablets based on compressed zein microspheres after oral administration was studied over a 7-day period with six dogs (Canis familiaris), using a commercial IVM tablet (5 mg/piece, Yilijia(®) ) as a control. Clinical efficacy was tested using 270 dogs presented as veterinary patients for the treatment of demodicidosis. A formulation with disintegration time within 15 min could be obtained. The acquired C( max), T(max), and AUC were 9.89 ± 0.34 ng/mL, 11.33 ± 2.63 h, and 883.87 ng h/mL for IVM tablets based on compressed zein microspheres and 9.64 ± 1.05 ng/mL, 7.26 ± 2.09 h, and 666.30 ng h/mL for Yilijia(®), respectively. The bioavailability of the tablets based on compressed zein microspheres was 132.65% that of Yilijia( ®). Efficacy for the dogs in all the IVM tablets based on compressed zein microspheres-treated groups reached 100% at 7, 14, and 21 days post administration.

Characterization and evaluation of the Ag+-loaded soy protein isolate-based bactericidal film-forming dispersion and films.

UNLABELLED: This study aims to prepare bactericidal films developed from soy protein isolate (SPI) based film-forming dispersions (FFDs) for use in the food and medical fields. The FFD and films were prepared after the incorporation of different concentrations of AgNO3 as a bactericidal agent. The transparency, tensile strength, and antimicrobial features were evaluated. Structural characterizations were also performed by Fourier transform infrared spectroscopy, scanning electron microscope, and atomic force microscopy analysis. Results showed that the opacity of these FFD was greatly decreased after the incorporation of AgNO3. The SPI-5 film has the largest tensile strength (P < 0.05) compared with that of the other ones. Micro structural imaging analysis showed an increase in the surface irregularities with the addition of AgNO3. The minimum inhibitory concentration of AgNO3 was 336 µg/mL FFD for both Escherichia coli ATCC 25923 and Staphylococcus aureus ATCC 25922. The SPI-AgNO3 films developed from the FFD with the minimal AgNO3 concentration at 336 µg/mL FFD also showed bactericidal effects for both the strains. These results may prove promising for the use of SPI-AgNO3 films in the food or medical industries. PRACTICAL APPLICATION: The films prepared in this study are biodegradable and will be used in medical and food fields.

Preparation and immunological effectiveness of a Swine influenza DNA vaccine encapsulated in PLGA microspheres.

Optimal preparation conditions of DNA vaccine against swine influenza encapsulated in Poly (D,L)-lactic-co-glycolic acid (PLGA) microspheres were determined. The microspheres were prepared by an emulsion-evaporation method using PLGA as the biodegradable matrix forming polymer. Using the optimal preparation conditions, PLGA microspheres containing the DNA vaccine were produced with good morphology as evident from scanning electron micrographs, high encapsulation rate and high stability. The transfection test indicated that the vaccine could be expressed as an antigen in cells and maintained good bioactivity. Moreover, these results demonstrated that the PLGA microspheres containing DNA vaccine can be used to achieve prolonged release of plasmid DNA. These results have laid a foundation for further development before ultimate industrial application.

Mechanical properties and in vitro biocompatibility of porous zein scaffolds.

A porous scaffold utilizing hydrophobic protein zein was prepared by the salt-leaching method for tissue engineering. The scaffolds possessed a total porosity of 75.3-79.0%, compressive Young's modulus of (28.2+/-6.7)MPa-(86.6+/-19.9)MPa and compressive strength of (2.5+/-1.2)MPa-(11.8+/-1.7)MPa, the percentage degradation of 36% using collagenase and 89% using pepsin during 14 days incubation in vitro. The morphology of pores located on the surface and within the porous scaffolds showed good pore interconnectivity by scanning electron microscopy (SEM). Rat mesebchymal stem cells (MSCs) could adhere, grow, proliferate and differentiate toward osteoblasts on porous zein scaffold. With the action of dexamethasone, the cells showed a relative higher activity of alkaline phosphatase (ALP) and a higher proliferating activity (p<0.05) than those of MSCs without dexamethasone.

Comparison of cytocompatibility of zein film with other biomaterials and its degradability in vitro.

UNLABELLED: Cytocompatibility of particle zein (Pzein) and film zein (Fzein) was evaluated and compared with polyhydroxybutyrate (PHB), its copolymer poly(hydroxybutyrate-co-hydroxyvalerate) (PHBV), polylactic acid (PLA), and collagen, using HL-7702 cells, in terms of cell attachment rate within 3 h, and cell viabilities at 3 and 6 days determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) method. The zein degradation test was carried out using collagenase and trypsin, and the degradation product was added to the culture medium at different concentrations in order to examine the concentration-dependent cytotoxic effect. RESULT: The adhesion rate of the HL-7702 cells on both Pzein and Fzein was higher than that on collagen film. Cell viabilities were higher on both Pzein and Fzein than on films of PLA, PHB, PHBV, and collagen from fish skin. Zein can be degraded by both trypsin and collagenase, and the degradation product can enhance cell viability within a certain range of concentrations.

Microspheres of corn protein, zein, for an ivermectin drug delivery system.

A novel microsphere drug delivery system of ivermectin (IVM) using hydrophobic protein zein was prepared by the phase separation method and characterized by a scanning electron microscope and laser light scattering particle size analyzer. Releases of model drug IVM from zein microspheres, tabletted microspheres and pepsin degradation of tabletted microspheres were also performed in vitro to investigate the mechanism of model drug release. The results show that the zein microspheres and tabletted microspheres are suitable for use as a sustained-release form of IVM. The microspheres may also be useful in drug targeting system since the diameter of the microspheres is appropriate for phagocytosis by macrophages. Moreover, the release of IVM from enzymatic degraded tabletted microspheres shows a zero-order release, implying a potential application in tissue engineering for preparing scaffold, which is composed of microspheres encapsulating bioactive components for stimulating cell differentiation and proliferation.

Basic study of corn protein, zein, as a biomaterial in tissue engineering, surface morphology and biocompatibility.

The zein films, were prepared for culturing human liver cells (HL-7702) and mice fibroblast cells (NIH3T3), while the Corning microplate and polylactic acid (PLA) were chosen as controls. The surface morphology of zein films prepared by two different methods was studied by scanning electron microscope (SEM), which revealed that the zein films were composed of particles of diameter 100-500 and 500-2500 nm, respectively. The biocompatibility of zein films was assessed by attachment, extensibility and proliferation of cells on them. Our study indicated that over 60% of both HL-7702 cells and NIH3T3 cells could attach to the Corning microplate, zein films and PLA at 3h after seeding. The concentration and particle sizes for preparing zein films did not seem to affect the proliferation of the cells tested. There were no significant differences in the proliferation of both HL-7702 cells and NIH3T3 cells between the Corning microplate and two kinds of zein films, except that the zein film composed of smaller particles at the lowest concentration exhibited a very good ability for proliferation of both the cells, while PLA was a poor matrix in the latter period of the cell proliferation. This preliminary study demonstrates that zein is a promising biomaterial with good biocompatibility for the development of tissue engineering.