Evaluation of the prophylactic effect of egg yolk antibody (IgY) produced against the recombinant protein containing IpaD, IpaB, StxB, and VirG proteins from Shigella.

INTRODUCTION: Shigellosis is a gastrointestinal disease causes high morbidity and mortality worldwide, however, there is no anti-Shigella vaccine. The use of antibiotics in shigellosis treatment exacerbates antibiotic resistance. Antibodies, particularly egg yolk antibody (IgY), offer a promising approach to address this challenge. This study aimed to investigate the prophylactic effect of IgY produced against a recombinant chimeric protein containing the immunogens IpaD, IpaB, StxB, and VirG from Shigella. METHODS: The chimeric protein, comprising IpaD, IpaB, StxB, and VirG, was expressed in E. coli BL21 and purified using the Ni-NTA column. Following immunization of chickens, IgY was extracted from egg yolk using the PEG-6000 method and analyzed through SDS-PAGE and ELISA techniques. Subsequently, the prophylactic efficacy of IgY was assessed by challenging of mice with 10 LD50 of S. dysenteriae and administering different concentrations of IgY (1.25, 2.5, 5, and 10 mg/kg) under various time conditions. RESULTS: The recombinant protein, weighing 82 kDa, was purified and confirmed by western blotting. The IgY concentration was determined as 9.5 mg/ml of egg yolk and the purity of the extracted IgY was over 90 %. The results of the ELISA showed that at least 19 ng of pure antibody identified recombinant protein and reacts with it. The challenge test employing IgY and Shigella demonstrated a direct correlation between the survival rate and antibody concentration, with increased concentrations leading to decreased mortality rates. Treatment of mice with 10 mg/kg IgY leads to 80 % survival of the mice against 10 LD50 S. dysenteriae. CONCLUSION: Our findings suggest that IgY may offer therapeutic potential in treating Shigella infections and combating antibiotic resistance.

Synthesis of BLF1-containing trimethyl chitosan nanoparticles and evaluation of its immunogenicity and protection in syrian mice by oral and subcutaneous injections.

The bacterium Burkholderia pseudomallei is the cause of melioidosis infectious disease. In this bacterium, the BLF1 protein wide inhibits the synthesis of proteins in human cells. This disease is reported to cause a death rate of 40% in some parts of the world. Currently, no effective vaccine is available against this bacterial infection. In this study, therefore, a Nano vaccine was synthesized based on the trimethyl chitosan (TMC) polymer containing the BLF1 recombinant protein, and its immunogenicity and protection in Syrian mice were evaluated by oral and subcutaneous injections. The BLF1 recombinant protein expression was induced in Escherichia coli Bl21 (DE3) and purified by the affinity chromatography technique. Recombinant protein-containing nanoparticles (NPs) were then synthesized by the ionotropic gelation method. After oral and subcutaneous injections, antibody titration was assessed by the indirect ELISA assay. Finally, murine groups were challenged using the BLF1 toxin. The results indicated that the immune system showed more antibody titration in subcutaneous injection than in the oral form. However, the results were reversed in the challenge results, and the survival rate was more significant in the oral injection.

Cellulose nanocrystals-reinforced dual crosslinked double network GelMA/hyaluronic acid injectable nanocomposite cryogels with improved mechanical properties for cartilage tissue regeneration.

Improvement of mechanical properties of injectable tissue engineering scaffolds is a current challenge. The objective of the current study is to produce a highly porous injectable scaffold with improved mechanical properties. For this aim, cellulose nanocrystals-reinforced dual crosslinked porous nanocomposite cryogels were prepared using chemically crosslinked methacrylated gelatin (GelMA) and ionically crosslinked hyaluronic acid (HA) through the cryogelation process. The resulting nanocomposites showed highly porous structures with interconnected porosity (>90%) and mean pore size in the range of 130-296 µm. The prepared nanocomposite containing 3%w/v of GelMA, 20 w/w% of HA, and 1%w/v of CNC showed the highest Young's modulus (10 kPa) and excellent reversibility after 90% compression and could regain its initial shape after injection by a 16-gauge needle in the aqueous media. The in vitro results demonstrated acceptable viability (>90%) and migration of the human chondrocyte cell line (C28/I2), and chondrogenic differentiation of human adipose stem cells. A two-month in vivo assay on a rabbit's ear model confirmed that the regeneration potential of the prepared cryogel is comparable to the natural autologous cartilage graft, suggesting it is a promising alternative for autografts in the treatment of cartilage defects.

PLGA nanoparticles containing Intimin-Flagellin fusion protein for E. coli O157:H7 nano-vaccine.

Escherichia coli O157:H7 is a foodborne pathogen that can lead to severe gastrointestinal diseases in humans. Vaccination is a promising strategy for preventing E. coli O157:H7 infections, which offers socio-economic benefits and provides the possibility of stimulating both humoral and cellular immune responses at systemic and mucosal sites. In this study, we developed a needle-free vaccine candidate against E. coli O157:H7 using poly(lactic-co-glycolic acid) (PLGA) nanoparticles entrapping a chimeric Intimin-Flagellin (IF) protein. The IF protein was expressed and verified using SDS-PAGE and western blot analysis, with a yield of 1/7 mg/L and a molecular weight of approximately 70 kDa. The prepared nanoparticles showed uniformly shaped spherical particles in the 200-nm range, as confirmed by SEM and DLS analysis. Three different routes of vaccine administration were used, including intranasal, oral, and subcutaneous, and the groups vaccinated with NPs protein had a higher antibody response compared to those receiving free protein. Subcutaneous administration of IF-NPs resulted in the highest level of IgG antibody titer, while oral administration of IF-NPs produced the highest amount of IgA antibody titer. Finally, all mice in the nanoparticle- intranasal and oral administered groups challenged with 100LD50 survived, while all control mice died before day 5. Based on these findings, we conclude that the PLGA-encapsulated IF protein has the potential to serve as a promising needle-free vaccine candidate against E. coli O157:H7.

In silico and in vivo approaches to recombinant multi-epitope immunogen of GroEL provides efficient cross protection against S. Typhimurium, S. flexneri, and S. dysenteriae.

OBJECTIVES: Stress or Heat Shock Proteins (HSPs) have been included in various operations like protein folding, autophagy, and apoptosis. HSP families recognize as protective antigens in a wide range of bacteria because they have been conserved through evolution. Due to their homology as well as antigenicity they are competent for applying in cross-protection against bacterial diseases. METHODS: In the present study, bioinformatics approaches utilized to design epitope-based construction of Hsp60 (or GroEL) protein. In this regard, potential B-cell and T-cell epitopes except for allergenic sequences were selected by immunoinformatic tools. The structural and functional aspects of the DNA, RNA, and protein levels were assessed by bioinformatics software. Following in silico investigations, recombinant GroEL multi-epitope of Salmonella typhi was expressed, purified, and validated. Mouse groups were immunized with recombinant protein and humoral immune response was measured by enzyme linked immunosorbent assay (ELISA). Animal challenge against Salmonella Typhimurium, Shigella flexneri, and Shigella dysenteriae was evaluated. RESULTS: recombinant protein expression and purification with 14.3 kilodaltons (kDa) was confirmed by SDS-PAGE and western blotting. After animal administration, the immunoglobulins evaluated increase after each immunization. Immunized antisera exhibited 80%, 40%, and 40% protection against the lethal dose infection by S. Typhimurium, S. flexneri, and S. dysenteriae respectively. Passive immunization conferred 50%, 30%, and 30% protection in mice against S. Typhimurium, S. flexneri and S. dysentery respectively. In addition, bacterial organ load had exhibited a significant decrease in colony forming unit (CFU) in the liver and spleen of the immunized mice compared to the control. CONCLUSION: Our study demonstrates the efficacy of S. Typhi recombinant GroEL multi-epitope to consider as a universal immunogen candidate versus multiple bacterial pathogens.

Preparation of internally-crosslinked alginate microspheres: Optimization of process parameters and study of pH-responsive behaviors.

In this study, the effects of various parameters of the water-in-oil emulsification/internal gelation method on the properties of calcium-alginate microparticles were evaluated and optimized. Results showed that the spherical-shaped microparticles with the highest circularity and high production yield can be produced by alginate solution with a concentration of 2 wt.%, calcium carbonate/alginate ratio of 10/1 (w/w), water/oil volume ratio of 1/20, emulsifier concentration of 5 % (v/v), and emulsification speed of 1000 rpm. Two model drugs including simvastatin lactone and simvastatin ß-hydroxyacid were loaded into the microspheres with promising encapsulation efficiencies of 73 % and 69 %, respectively. The microspheres showed a pH-responsive swelling behavior with a percentage of 10.60 %, 352.65 %, 690.03 %, and 1211.46 % at the pH values of 2.0, 4.5, 7.4, and 8.5, respectively. The microspheres showed an increasing trend of release rate in direct proportion to pH. These findings would be useful for therapeutic applications which need pH-responsive drug carriers.

Fabrication of Nanofibrous PVA/Alginate-Sulfate Substrates for Growth Factor Delivery.

Providing affinity sites on alginate (ALG) matrix enables specific binding of growth factors to the polymer backbone and allows their release in a controlled fashion. In this study, we used a blend of alginate sulfate (ALG-S) and polyvinyl alcohol (PVA) to fabricate electrospun scaffolds capable of delivering a heparin-like growth factor, transforming growth factor-beta1 (TGF-ß1). The alginate was sulfated with different degrees of sulfation (DS, from 0.8, 3.4 to 12.4%) by a simple process. The success of sulfation was determined by Fourier-transform infrared spectroscopy (FTIR), nuclear magnetic resonance spectroscopy (NMR), elemental analysis, ultraviolet (UV) spectroscopy and staining with dimethylmethylene blue. The physical-mechanical properties of nanofibrous mats were characterized by scanning electron microscopy (SEM), FTIR, energy-dispersive X-ray spectroscopy (EDX), tensile strength and mass loss analysis. Additionally, the release kinetics of transforming growth factor-ß1 (TGF-ß1) from PVA/ALG-S and PVA/ALG scaffolds were compared. The results showed that the binding and entrapment of TGF-ß1 to the nanofibrous scaffolds are improved by the addition of sulfate group to alginate. In conclusion, our results support that nanofibrous scaffold based on PVA/ALG-S can deliver growth factors in tissue engineering application. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 403-413, 2019.

An LTB-entrapped protein in PLGA nanoparticles preserves against enterotoxin of enterotoxigenic Escherichia coli.

OBJECTIVES: Enterotoxigenic Escherichia coli (ETEC) is known as the most common bacterial causes of diarrheal diseases related to morbidity and mortality. Heat-labile enterotoxin (LT) is a part of major virulence factors in ETEC pathogenesis. Antigen entrapment into nanoparticles (NPs) can protect them and enhance their immunogenicity. MATERIALS AND METHODS: In the present study, recombinant LTB protein was expressed in E. coli BL21 (DE3) and purified by an Ni-NTA agarose column. The protein was entrapped in PLGA polymer by the double emulsion method. NPs were characterized physicochemically and the protein release from the NPs was evaluated. ELISA assay was performed for investigation of raised antibody against the recombinant protein in mice. The anti-toxicity and anti-adherence attributes of the immune sera against ETEC were also evaluated. RESULTS: It showed the successful cloning of a 313 bp DNA fragment encoding LTB protein in the pET28a vector. Over-expression in BL21 (DE3) led to the formation of corresponding 15.5 kDa protein bands in the SDS-PAGE gel. Western blotting by using anti-CTX confirmed the purified LTB. Protein-entrapped NPs had a spherical shape with the size of 238 nm mean diameter and 85% entrapment efficiency. Immunological analyses showed the production of a high titer of specific IgG antibody in immunized animals. The neutralizing antibody in the sera of immunized animals was approved by GM1 binding and Ileal loop assays. CONCLUSION: The results indicate the efficacy of the entrapped LTB protein as an effective immunogen which induces the humoral responses.

Electrospun nanofibers comprising of silk fibroin/gelatin for drug delivery applications: Thyme essential oil and doxycycline monohydrate release study.

In this study, a nanofibrous electrospun substrate based on the silk fibroin (SF) and gelatin (GT) polymers were prepared and evaluated. The SF/GT blended solutions were prepared with various ratios of GT in formic acid and electrospun to obtain bead-free fibers. Results showed that addition of GT to SF increased nanofiber's diameter, bulk hydrophilicity, surface wettability, mass loss percentage, but decreased Young's modulus, tensile strength, and porosity of the SF/GT mats. According to the obtained results, the mat containing 10% of GT was selected as the optimized mat for further studies and loaded with thyme essential oil (TEO) and doxycycline monohydrate (DCMH) as the antibacterial agents. Release studies showed a burst release of TEO from the mat within the first 3 h, while the DCMH had a sustained release during 48 h. In comparison to the TEO-loaded mat, the DCMH-loaded one showed larger inhibition zones against Staphylococcus aureus and Klebsiella pneumoniae bacteria. Meanwhile, cellular studies using mouse fibroblast L929 cells showed excellent cell-compatibility of TEO- and DCMH-loaded mats. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 1092-1103, 2018.

Preparation and characterization of poly (hydroxy butyrate)/chitosan blend scaffolds for tissue engineering applications.

BACKGROUND: Poly (hydroxy butyrate) (PHB) is a biodegradable and biocompatible polymer with good mechanical properties. This polymer could be a promising material for scaffolds if some features improve. MATERIALS AND METHODS: In the present work, new PHB/chitosan blend scaffolds were prepared as a three-dimensional substrate in cartilage tissue engineering. Chitosan in different weight percent was added to PHB and solved in trifluoroacetic acid. Statistical Taguchi method was employed in the design of experiments. RESULTS: The Fourier-transform infrared spectroscopy test revealed that the crystallization of PHB in these blends is suppressed with increasing the amount of chitosan. Scanning electron microscopy images showed a thin and rough top layer with a nodular structure, supported with a porous sub-layer in the surface of the scaffolds. In vitro degradation rate of the scaffolds was higher than pure PHB scaffolds. Maximum degradation rate has been seen for the scaffold with 90% wt. NaCl and 40% wt. chitosan. CONCLUSIONS: The obtained results suggest that these newly developed PHB/chitosan blend scaffolds may serve as a three-dimensional substrate in cartilage tissue engineering.

Adipose-derived stem cells combined with beta-tricalcium phosphate: a novel possible strategy for periodontal defects regeneration.

Regeneration of destroyed periodontal tissue is a complicated problem in periodontal therapy. Conventional strategies such as surgical techniques, occlusive barrier membranes, and bone grafts have disadvantages that limit their application. Tissue engineering is a novel approach to regenerate periodontal tissue overcoming limitations of current strategies. In recent studies, researchers reported the potential of adipose-derived stem cells (ADSCs) to use in periodontal tissue regeneration. Unfortunately, in vivo studies indicated that small amount of alveolar bone formed using ADSCs implantation in the periodontal defects, which would be overcome by inducing osteogenic differentiation of ADSCs using osteoinductive biomaterials combined with ADSCs. Beta-tricalcium phosphate (beta-TCP) is an osteoinductive material which reported to induce osteogenic differentiation of adipose-derived stem cells. Based on the performed studies, we hypothesize that ADSCs from Wistar rat are isolated, induced by appropriate signaling molecules, seeded by beta-TCP, and then the complex was implanted into the periodontal defects in the same Wistar rat to observe the regeneration of periodontal tissue in vivo. This study will explore the possibility and application of ADSCs/beta-TCP complex for reconstructing periodontal defects. We believe it is especially useful to future clinical study and application in this field.