Development of fast-dissolving sublingual nanofibers containing allergen and curcumin for immune response modulation in a mouse model of allergic rhinitis.

Curcumin (CUR) has been considered a potential therapeutic agent for allergic reactions due to its antioxidant and anti-inflammatory activities. Nanofibers have attracted increasing attention in drug delivery. The aim of this study was to investigate the combined therapeutic effects of curcumin and allergen in nanofiber-based treatments in order to increase the effectiveness of sublingual immunotherapy (SLIT) efficacy in a mouse model of allergic rhinitis. Nanofibers containing CUR (1.25% and 2.5%) and ovalbumin 2% (OVA) as an allergen were prepared via electrospinning and characterized. BALB/c mice were sensitized with OVA to the induced allergic rhinitis model. SLIT with free and/or nanofibers was carried out. IL-4, INF-γ, and IgE serum levels were measured using ELISA. Splenocyte proliferation was evaluated by the MTT assay. Lung and nasal histological examinations and nasal lavage fluid (NALF) cell counting were carried out. Nanofibers containing 1.25% CUR and 2% OVA were chosen as the optimal formulations. SLIT treatment with the CUR and OVA nanofiber co-administration led to a significantly decreased serum IgE. Nanofiber containing 2.5 µg of CUR/mouse combined with OVA nanofiber showed a significant decrease in IL-4 and an increase in IFN-γ compared to other groups. NALF assessment showed a significant decrease in specific cell and eosinophil counts in the treated nanofiber groups. The histopathological results of NAL in the optimal formulations were near normal, with diminished cellular infiltration and inflammation. Our findings suggest that co-sublingual administration of allergen and CUR nanofibers can be considered as potential immunomodulatory agents.

High efficacy and economical procedure of oral vaccination against Lactococcus garvieae/Streptococcus iniae in rainbow trout (Oncorhynchus mykiss).

The present study was aimed to examine the efficacy of chitosan-alginate coated vaccines against pathogenicity of Lactococcus garvieae and Streptococcus iniae in rainbow trout. Fish were divided into four groups including: Group A: fish immunized by chitosan-alginate coated vaccine, Group B: fish immunized by non-coated vaccine, Group C: fish feed by chitosan-alginate coated pellets without vaccine and Group D: fish feed by basic diet (non-coated and without vaccine). In groups A and B, the vaccination was carried out for 14 days and after that supplemented with fundamental diet (control diet). Comparable to groups A and B, fish of group C were also fed 14 days with test diets and after that fed control food. On day 0, 20, 40 and 60 of the experiment, serum samples were given. Fish have been challenged with live L. garvieae and S. iniae after 60 days. The levels of bactericidal activity and complement activity among innate immunity components extended on day 20 of the research and after that decreased in group A and B (P < 0.05) all through the examination. The relative expression of IL-6 and IgM in groups A and B extended on examination day 20. The expression of these genes illustrated no advancements in different groups in during the examination (P > 0.05). In group A, the serum antibody titer against L. garvieae and S. iniae broadly raised on day 40 and 60 of examination, whereas in group B, the immune response titer against S. iniae and L. garvieae illustrated a significant elevation on day 60 of the trial (P < 0.05). After challenge with live bacteria, survival rate of 83 ± 9.1%(challenged with S. iniae) and 72.18 ± 9.8% (challenged with L. garvieae) were gotten independently in group A, which were higher than survival of other exploratory groups (P < 0.05). In conclusion, the results of the present examination appear that the orally vaccination of rainbow trout with chitosan-alginate covered vaccine stimulates immunity system and also efficiently protects rainbow trout against Lactococcus garvieae and Streptococcus iniae.

Incorporation of Silver Sulfadiazine into An Electrospun Composite of Polycaprolactone as An Antibacterial Scaffold for Wound Healing in Rats.

OBJECTIVE: Fabrication of an antibiotic-loaded scaffold with controlled release properties for wound dressing is one of tissue engineering challenges. The aim of this study was to evaluate the wound-healing effectiveness of 500-µm thick polycaprolactone (PCL) nanofibrous mat containing silver sulfadiazine (SSD) as an antibacterial agent. MATERIALS AND METHODS: In this experimental study, an electrospun membrane of PCL nanofibrous mat containing 0.3% weight SSD with 500 µm thickness, was prepared. Morphological and thermomechanical characteristics of nanofibers were evaluated. Drug content and drug release properties as well as the surface hydrophobicity of the nanofibrous membrane were determined. Antimicrobial properties and cellular viability of the scaffold were also examined. A full thickness wound of 400 mm2 was created in rats, to evaluate the wound-healing effects of PCL/SSD blend in comparison with PCL and vaseline gas used as the control group. RESULTS: SSD at a concentration of 0.3% improved physicochemical properties of PCL. This concentration of SSD did not inhibit the attachment of human dermal fibroblasts (HDFs) to nanofibers in vitro, but showed antibacterial activity against Gram-positive Staphylococcus aureus (ST) and Gram-negative Pseudomonas aeruginosa (PS). Overall, results showed that SSD improves characteristics of PCL nanofibrous film and improves wound-healing process in one-week earlier compared to control. CONCLUSION: Cytotoxicity of SSD in fabricated nanofibrous mat is a critical challenge in designing an effective wound dressing that neutralizes cellular toxicity and improves antimicrobial activity. The PCL/SSD nanofibrous membrane with 500- µm thickness and 0.3% (w/v) SSD showed applicable characteristics as a wound dressing and it accelerated wound healing process in vivo.

Formulation and Optimization of a New Cationic Lipid-Modified PLGA Nanoparticle as Delivery System for Mycobacterium tuberculosis HspX/EsxS Fusion Protein: An Experimental Design.

Polymeric particles and liposomes are efficient tools to overcome the low immunogenicity of subunit vaccines. The aim of the present study was formulation and optimization of a new cationic lipid-modified PLGA nanoparticles (NPs) as a delivery system for Mycobacterium tuberculosis HspX/EsxS fusion protein. The cationic lipid-modified PLGA NPs containing HspX/EsxS fusion protein were prepared using a modified double emulsion solvent evaporation method. Scanning electron microscopy and dynamic light scattering (DLS) tools were used to determine physical properties of hybrid NPs. A multi-level full factorial design was used to evaluate the influence of two factors of PLGA:DDA weight ratio (w/w) and PVA concentration (%) on size, surface charge, polydispersity index, encapsulation efficiency and yield. Finally, the optimal formulation was achieved based on desired responses. Mathematical models were obtained to indicate the relation between the studied factors and responses. The DDA concentration showed an increasing effect on surface charge and also a decreasing effect on particle size, encapsulation efficiency and yield. Higher amounts of DDA increased surface charge of NPs; however, the size, encapsulation efficiency and yield were decreased. The influence of various concentrations of PVA on different physical characteristics of PLGA:DDA hybrid NPs was variable. The optimal formulation consisted of 0.91 (55:5, w/w) weight ratio of PLGA:DDA and 0.5% PVA. The hybrid NPs showed acceptable particle size distribution, strong positive surface charge, prolonged antigen release and good encapsulation efficiency in comparison to PLGA alone. However, further preclinical and clinical studies are needed.

Valuable method for production of oral vaccine by using alginate and chitosan against Lactococcus garvieae/Streptococcus iniae in rainbow trout (Oncorhynchus mykiss).

The effectiveness of ionotropic gelation method (by combining alginate and chitosan) vaccine against Lactococcus garvieae and Streptococcus iniae was examined in rainbow trout. Fish were separated into four groups and fed the distinctive examined feeds. Our groups were included: A) fish immunized by chitosan-alginate coated vaccine, B) fish immunized by non-coated vaccine, C) fish feed by chitosan-alginate coated pellets without vaccine and D) fish feed by basic diet (non-coated and without vaccine). In groups A and B, the vaccination was carried out for 14 days. Fish of group C, like groups A and B were fed 14 days with pellets covered with chitosan-alginate without vaccine and a short time later they were fed with control diet. On day 0, 20, 40 and 60 of the trial, serum samples were extracted. Fish were challenged with L. garvieae and S. iniae after 60 days of research. Innate immunity components containing complement activity, total protein and IgM appeared no significant changes nearly in all groups during the 60 days that the examination finished. Although, bactericidal activity and lysozyme activity demonstrated a significant increase on days 20, 40 and 60 in group A compared to control groups (C and D) (P < 0.05) and similar results about the blood respiratory burst activity just on days 20 and 40 were obtained. Also, the relative expression of IL-6 of group A, was significantly higher compared to all of other groups (B, C and D) on days 20 and 60 of experiment (P < 0.05). The same results were obtained about the relative expression of IgM. The serum ELISA antibody titer against L. garvieae, increased significantly on days 20 and 40 of experiment in fish immunized by chitosan-alginate coated vaccine (Group A) compared to control groups (C and D)(P < 0.05) while the result of ELISA test against S. iniae was significantly higher on days 40 and 60 of experiment in group A compared to groups B, C and D (P < 0.05). After challenge with these two live bacteria (S. iniae and L. garvieae), a survival rates of 76.67 ±â€¯5.77% (challenged with S. iniae) and 66.67 ±â€¯5.77% (challenged with L. garvieae) were seen in group immunized with chitosan-alginate coated vaccine (Group A), which were higher than survival rates gotten in other trial groups (P < 0.05). The consequences of the present experiment show that the oral vaccination of rainbow trout with improved chitosan-alginate (via ionotropic procedure) (group A) properly secures this important fish against Lactococcus garvieae and Streptococcus iniae.

Efficacy of a Eudragit L30D-55 encapsulated oral vaccine containing inactivated bacteria (Lactococcus garvieae/Streptococcus iniae) in rainbow trout (Oncorhynchus mykiss).

The efficacy of a Eudragit L30D-55 encapsulated vaccine against Lactococcus garvieae and Streptococcus iniae was investigated in rainbow trout. Fish were divided into four groups and fed the different experimental feeds. Groups were: A) fish immunized by Eudragit-coated pellets containing vaccine, B) fish immunized by vaccine-coated pellets without Eudragit, C) fish fed Eudragit-coated pellets without vaccine and D) fish fed pellets without vaccine orEudragit (control group). In groups A and B, the vaccination was conducted for 14 days. Similar to groups A and B, fish of group C were fed 14 days with pellets coated with Eudragit and afterwards they were fed control diet. Serum samples were taken on day 0, 20, 40 and 60 of the experiment. After 60 days, fish were challenged with L. garvieae and S. iniae. In almost all groups, innate immunity components including alternative complement activity, lysozyme activity, bactericidal activity, IgM and total protein showed no significant changes during the 60 days that the experiment lasted. However, the blood respiratory burst activity and lysozyme activity showed a significant increase on day 20 of experiment in groups B and D respectively (P < 0.05). The relative expression of immune-related genes including IL-6 and IgM genes was higher in vaccinated fish, with the highest expression in those immunized by Eudragit-coated pellets (Group A). In addition, the relative expression of IL-6 and IgM peaked on day 20 but decreased on day 60 in vaccinated groups. The ELISA antibody titer against L. garvieae increased from day 20 and peaked on day 60 of experiment (P < 0.05). Also, the antibody titer against L. garvieae was higher in fish immunized by Eudragit-coated pellets (Group A) compared to fish of group C and control. After bacterial challenge, a survival percentages of % 85 ±â€¯7.07% (challenged with S. iniae) and % 72.21 ±â€¯7.8% (challenged with L. garvieae) were observed respectively in groups immunized with pellets coated with Eudragit L30D-55 (Group A), which were higher than survival percentages obtained in other experimental groups (P < 0.05). The results of the present study demonstrate that the oral administration of Eudragit L30D-55-encapsulated vaccine appropriately protects rainbow trout against Lactococcus garvieae and Streptococcus iniae.

Evaluation of ethylcellulose and its pseudolatex (Surelease) in preparation of matrix pellets of theophylline using extrusion-spheronization.

OBJECTIVES: This study evaluates the effect of substitution of microcrystalline cellulose (MCC) with ethylcellulose (EC) on mechanical and release characteristics of theophylline pellets. MATERIALS AND METHODS: The effect of addition of EC was investigated on characteristics of pellets with varying drug content prepared by extrusion-spheronization. Also the effect of type of granulating liquid (water or Surelease) was investigated on characteristics of selected pellets. The pellets were characterized for particle size (sieve analysis), mechanical strength, morphology (microscopy), thermal (DSC) and dissolution behaviors. RESULTS: The exrtudability of the wet mass was reduced upon inclusion of EC so that complete replacement of MCC was not possible. Increase in EC percentage led to lower production yield and formation of pellets with larger diameter and slightly rough surfaces. Inclusion of EC also affected the mechanical properties of pellets but had negligible effect on drug release profile. The surface of selected pellets became smoother and their production yield increased upon the use of Surelease as granulating liquid. In addition the rate of drug release decreased to some extent when Surelease was used. CONCLUSION: Preparation of theophylline pellets with EC alone was not possible in process of extrusion-spheronization. Partial replacement of MCC with EC changed physicomechanical properties of pellets but hardly affected drug release. Although the use of Surelease as granulation liquid slightly decreased the rate of drug release, desirable matrix pellets with sustained drug release could not be produced. Despite this outcome however, these pellets could benefit from reduced coating thickness for drug release control.

A dermal equivalent developed from adipose-derived stem cells and electrospun polycaprolactone matrix: an in vitro and in vivo study.

Polycaprolactone (PCL) is used as a material of choice for surgical sutures, wound dressings, contraceptives, fixation devices and dentistry in paramedical sciences. In addition, adipose-derived stem cells (ASCs) have been shown to be effective in the treatment of acute and chronic wounds. This study aimed to evaluate the effect of electrospun PCL fibers on keratinocyte differentiation of ASCs and wound healing. PCL solution was electrospun and characterized. Isolated and characterized ASCs were differentiated into keratinocyte-like cells on a tissue culture plate (TCP) and PCL matrices and compared. PCL nano-/microfibers cultured with ASCs (test group) or alone (control) were implanted as a dermal substitute for wound healing. There were significant increases in the proliferation rate and expression level of cytokeratin 14, filaggrin and involucrin in cells cultured on PCL matrices compared to TCP (p < 0.05). After histological and immunological evaluation of the reconstituted skin, a thick epidermal layer with several skin appendages was evidently observed in the ASC/PCL group, whereas no real and mature epidermis was formed, especially in the central area of the healing wound in the pure PCL group on day 14. Pure PCL, if possessing suitable properties including good adhesiveness, high proliferative capability, inductive elasticity and stiffness for migration and differentiation, could drive the keratinocyte differentiation of ASCs and act as an efficient dermal equivalent to promote wound healing.

Skin-derived precursors possess the ability of differentiation into the epidermal progeny and accelerate burn wound healing.

Skin-derived precursors (SKPs) are remnants of the embryonic neural crest stem cells that reside in the dermis until adulthood. Although they possess a wide range of differentiation potentials, their differentiation into keratinocyte-like cells and their roles in skin wound healing are obscure. The present study aimed to investigate the differentiation of SKPs into keratinocyte-like cells and evaluate their role in healing of third degree burn wounds. To this aim, SKPs were differentiated into keratinocyte-like cells on tissue culture plate and collagen-chitosan scaffold prepared by freeze-drying. Their differentiation capability was detected by real-time RT-PCR and immunofluorescence. Thereafter, they were cultured on scaffold and implanted in a rat model of burn wound. Histopathological and immunohistochemical analyses were employed to examine the reconstituted skin. The research findings revealed that SKPs were able to differentiate along the epidermal lineage and this ability can be enhanced on a suitable scaffold. Additionally, the results indicated that SKPs apparently promoted wound healing process and accelerate its transition from proliferating stage to maturational phase, especially if they were differentiated into keratinocyte-like cells. Regarding the results, it is concluded that SKPs are able to differentiate into keratinocyte-like cells, particularly when they are cultured on collagen-chitosan scaffold. Moreover, they can regenerate epidermal and dermal layers including thick collagen bundles, possibly through differentiation into keratinocyte-like cells.

Characterization of A Three-Dimensional Organotypic Co-Culture Skin Model for Epidermal Differentiation of Rat Adipose-Derived Stem Cells.

OBJECTIVE: The organotypic co-culture is a well-known technique to examine cellular interactions and their roles in stem cell proliferation and differentiation. This study aims to evaluate the effects of dermal fibroblasts (DFs) on epidermal differentiation of adipose-derived stem cells (ASCs) using a three-dimensional (3D) organotypic co- culture technique. MATERIALS AND METHODS: In this experimental research study, rat DFs and ASCs were isolated and cultured separately on electrospun polycaprolactone (PCL) matrices. The PCL matrices seeded by ASCs were superimposed on to the matrices seeded by DFs in order to create a 3D organotypic co-culture. In the control groups, PCL matrices seeded by ASCs were placed on matrices devoid of DFs. After 10 days, we assessed the expressions of keratinocyte-related genes by real-time reverse transcriptase-polymerase chain reaction (RT-PCR) and expression of pan-cytokeratin protein by immunofluorescence in the differentiated keratinocyte-like cells from co- culture and control groups. Keratinocyte-like cell morphologies were also observed by scanning electron microscopy (SEM). RESULTS: The early, intermediate, and terminal differentiation keratinocyte markers-Cytokeratin14, Filaggrin, and Involucrin significantly expressed in the co-culture groups com- pared to the control ones (P<0.05). We observed pan-cytokeratin in keratinocyte-like cells of both groups by immunofluorescence. SEM observation of the co-culture groups showed that the differentiated keratinocyte-like cells developed a polygonal cobblestone shape, considered characteristic of keratinocytes. CONCLUSION: The 3D organotypic co-culture bilayered construct that consisted of DFs and ASCs was an effective technique for epidermal differentiation of ASCs. This co-culture might be useful for epidermal differentiation of stem cells for future applications in skin regeneration.

Oral nicotinamide reduces serum phosphorus, increases HDL, and induces thrombocytopenia in hemodialysis patients: a double-blind randomized clinical trial.

BACKGROUND: Recently, nicotinamide has been suggested as an effective drug for hyperphosphatemia in hemodialysis patients. The authors assessed the efficacy and safety of nicotinamide in these patients with lower doses and longer duration than other studies. METHODS: Forty eight patients with fasting serum phosphorus >5 mg/dl enrolled in this randomized clinical trial study and were randomly assigned to two equal-sized groups of nicotinamide or placebo. The study lasted 8 weeks. In the first four weeks, nicotinamide was administered at 500 mg/day, and in the second four weeks at 1,000 mg/day. Blood samples were tested at baseline, week 4, and week 8. RESULTS: In nicotinamide group, the mean phosphorus level decreased from 5.9 ± 0.58 mg/dl to 4.77 ± 1.43 mg/dl in week 4 (P = 0.002) and to 4.66 ± 1.06 mg/dl in week 8 (P = 0.000). The mean calcium-phosphorus product decreased significantly with the same pattern as phosphorus. High-density lipoprotein level increased from 42.46 ± 8.01 mg/dl to 55.71 ± 11.88 mg/dl in week 4 (P = 0.000) and to 65.25 ± 20.18 mg/dl in week 8 (P = 0.000). Levels of serum calcium, uric acid, SGOT, SGPT, and iPTH didn't change significantly. Compared to baseline, the platelet counts were decreased in both week 4 and week 8. No significant changes were observed in placebo group. CONCLUSIONS: In our patients, nicotinamide effectively decreased phosphorus, increased high-density lipoprotein, and caused thrombocytopenia. Since nicotinamide lowered platelet counts and caused thrombocytopenia in lower doses than other studies in these patients, it is necessary to plan other studies for assessing the safety of the drug especially in different populations.