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.

In vitro identification of single-stranded DNA aptamers targeting human IL-23 using the protein-SELEX strategy.

Interleukin (IL)-23 inhibitor monoclonal antibodies shown significant efficacy in treating autoimmune diseases. DNA or RNA aptamers exhibit comparable specificity to antibodies, are cost-effective, non-immunogenic, and do not have batch to batch variation. This study aimed to characterize a single-stranded DNA (ssDNA) aptamer targeting human IL-23. The alpha subunit of IL-23 (P19) and intact IL-23 were cloned, expressed, and the proteins finally were purified through Ni2+-iminodiacetic acid affinity chromatography. The selection and characterization of ssDNA aptamer against P19 were conducted using the protein-systematic evolution of ligands by exponential enrichment (SELEX). Dot blot assay was carried out to monitor binding of the aptamer output of SELEX rounds, to P19 protein. The dissociation constant (Kd) of aptamers with positive results in dot blot assay, determined based on their binding to IL-23 using an ELISA method. Recombinant P19 and IL-23 proteins were 26 and 72 kDa, respectively, observed on SDS-PAGE .12 %. The aptamers output from 7, 8, 9, 10, 11, and 12 rounds of the SELEX was monitored by dot blot assay, revealing that the aptamer from the round 8 has stronger luminescent signal and was selected for TA-cloning. After analyzing the biotinylated aptamers from clones, positive clones in dot blot assay and ELISA were sequenced. Finally, the Kd calculation revealed three aptamers with high affinity, named A23P3, A23P6, and A23P15 with Kd values of 1.37, 2.139, and 2.88 nM, respectively. Results of this study introduced three specific anti-IL-23 ssDNA aptamers with high affinity, which could be utilized for therapeutic and diagnostic purposes.

The Hydrogel Based Allergen-Coated Gold Nanoparticles for Topical Administration: A Possible Epicutaneous Immunotherapy in Pollen-Sensitized Mice?

BACKGROUND: The rapid uptake of antigens by antigen-presenting cells (APCs) and their migration to draining lymph nodes in the initial hours after antigen administration in epicutaneous allergen specific immunotherapy (EPIT) prompted us to investigate whether the topical administration of allergens without patch application could alleviate allergy in pollen-sensitized mice. We evaluated the immunotherapeutic effect of topically administering hydrogel-based Gold nanoparticles (AuNPs) loaded with a total extract of Platanus orientalis pollen (Pla. ext (50 µg)-AuNPs) on intact skin. METHODS: Mice sensitized to P. orientalis pollen were divided into three groups and treated with Pla. ext (50 µg)-AuNPs: 1) patch with Pla. ext (50 µg)-AuNPs, 2) patch with Pla. ext (50 µg)-AuNPs in combination with hydrogel, and 3) topical application of Pla. ext (50 µg)-AuNPs in combination with hydrogel. The immunotherapeutic effects were evaluated by measuring serum specific and total IgE antibodies, total cell and eosinophil count in nasopharyngeal lavage fluid, cytokines in the supernatants of re-stimulated splenocytes by the total extract, and histological examination of lung and nasal mucosa. RESULTS: Topical administration of Pla. ext (50 µg)-AuNPs, like patch-based administration, significantly downregulated specific and total IgE and IL-4 production, promoted secretion of IFN-γ and IL-10, markedly reduced the number of inflammatory cells, particularly eosinophils, in nasopharyngeal lavage fluid (p < .05), and inhibited inflammation and pathological damage in lung and nasal mucosa. CONCLUSION: Our results suggest that topical administration of AuNPs loaded with P. orientalis total pollen extract on intact skin could be a potential application for EPIT in the P. orientalis pollen -sensitized mice.

An epicutaneous therapeutic pollen-allergen extract delivery system in an allergic rhinitis mouse model: based on allergen loading on DC-specific aptamers conjugated nanogolds.

BACKGROUND: Gold nanoparticles (GNPs) have previously been suggested as appropriate carriers for allergen-specific immunotherapy (AIT). In this study, we assessed efficacy of GNPs and dendritic cells (DC)-specific aptamer-modified GNPs (Apts-GNP) for epicutaneous immunotherapy (EPIT) in the case of pollen allergen extracts containing a variety of allergenic and non-allergenic components. METHODS: BALB/c mice were sensitized to the total protein extract of Platanus orientalis pollen and epicutaneously treated in different groups either with free P. orientalis total pollen extract, naked GNPs, total extract loaded GNPs, and total extract loaded Apts-GNPs with and without skin-penetrating peptides (SPPs). Then, the specific IgE level (sIgE), total IgE concentration (tIgE) in the serum sample, IL-4, IL-17a, IFN-γ, and IL-10 cytokine concentrations in re-stimulated splenocytes with the total extract and mixture of recombinant allergens, nasopharyngeal lavage fluid (NALF) analysis, and histopathological analysis of lung tissue were evaluated. RESULTS: This study indicated the total extract-loaded GNPs, especially Pla. ext (50 µg)-GNPs, significantly decreased sIgE, tIgE, IL-17a, and IL-4 concentrations, immune cells and eosinophils infiltration in NALF, and increased IL-10 and IFN-γ concentrations compared with the PBS-treated group. In addition, the histopathological analysis of lung tissue showed a significant decrease in allergic inflammation and histopathological damage. The DC-targeted group revealed the most significant improvement in allergic-related immune factors with no histopathological damage compared with the same dose without aptamer. CONCLUSION: Loading total protein extract on the GNPs and the Apt-modified GNPs could be an effective approach to improve EPIT efficacy in a pollen-induced allergic mouse model.

Topical anti-TNF-a ssDNA aptamer decreased the imiquimod induced psoriatic inflammation in BALB/c mice.

BACKGROUND: Tumor Necrosis Factor-α (TNF-α) is a pro-inflammatory factor that plays a pivotal role in psoriasis. Due to limitations of monoclonal antibody-based therapies, it is needed to discover new anti-TNF-α factors instead of usual anti-TNF-α monoclonal antibodies. Compared to antibodies, single-stranded DNA or RNA molecules named aptamers, have advantages such as time-saving, less risk for immunogenicity and cost-effectiveness. Therefore, the aim of the present study was to assess the therapeutic effects of T1-T4 dimer anti-TNF-ɑ ssDNA aptamer topical treatment in the imiquimod (IMQ)-induced psoriasis animal model. METHODS: 5% IMQ cream was prescribed on the right ear of BALB/c to induce psoriasis model. The hydrogel-containing anti-TNF-ɑ aptamer or treatment control aptamer (anti- Interleukin (IL)17A) was topically prescribed to the mice's ears 10 min before IMQ cream treatment. The psoriasis area severity index (PASI) score was used to evaluate psoriasis intensity. Histopathology analysis was done for mice ears sections. Mass, size, and cell number of mice spleens were measured. The IL-17 level was determined in culture supernatants of axillary lymph node cells using ELISA. The mRNA expression levels of IL-17A, IL-1ß, STAT3, and S100a9, were evaluated in mice treated ear with quantitative Real Time-PCR. RESULTS: The anti-TNF-ɑ ssDNA aptamer lower doses had significant decrease in IMQ-induced PASI score (p < 0.05). In addition, in these groups, the IL-17A, STAT3, and S100a9 mRNA levels were significantly lower than the IMQ group (p < 0.05). CONCLUSION: According to our findings, this aptamer seems to be a prospective candidate for treating psoriatic inflammation especially in lower concentrations.

Selection and characterization of a new human Interleukin-17A blocking DNA aptamer using protein-SELEX.

BACKGROUND: Interleukin-17A (IL-17A) is an important pro-inflammatory cytokine observed in the development of many disorders, such as psoriasis, rheumatoid arthritis, and multiple sclerosis. The anti-IL-17A biological drugs, including Secukinumab, Ixekizumab, and Brodalumab, are monoclonal antibodies approved for several disease treatments. Due to the disadvantages of biological therapies, including their immunogenicity, difficulties in scale generation, and high production costs and time, it is necessary to find new alternative anti- IL-17A agents for these monoclonal antibodies. Our study aimed to identify ssDNA aptamers that block IL-17A activity using the protein-SELEX procedure. METHODS: The hIL-17A was expressed in codon plus E. coli, and after 14 rounds of the SELEX process, monitoring of aptamer pools was done using the dot blot method. Three families of aptamers were obtained from the selected round 9 aptamer pool, and seven truncates were created. Inhibitory effects of aptamer truncate on IL-17-induced CCL20 expression in HaCaT keratinocytes were evaluated. RESULTS: All aptamer truncates had a significant inhibitory effect compared to the library, but the inhibitory effect of M2 and M7 truncates was more than 80%. Moreover, we evaluated the potential binding site of selected aptamers by ELISA. CONCLUSIONS: We introduced a new small 17-nucleotide DNA aptamer that efficiently binds and blocks hIL-17A with a 0.3 nM kd, a potential anti-IL-17A therapeutic agent.

Influence of biologic and conventional disease-modifying antirheumatic drugs on COVID-19 incidence among rheumatic patients during the first and second wave of the pandemic in Iran.

Introduction: During the SARS-CoV-2 virus pandemic, immunosuppressive agents in treating chronic disease have become a concern, and rheumatic patients are not an exception. The controversies about the deteriorating effects of such medications led this study to evaluate the influence of biologic and conventional disease-modifying antirheumatic drugs (DMARDs) on the incidence of COVID-19 infection in rheumatic patients. Material and methods: In the present cohort-analytical study, 512 patients with rheumatic diseases were enrolled during the COVID-19 pandemic (2020-2021). The incidence of COVID-19 infection was diagnosed according to the definition of the Iranian Ministry of Health. The frequency of COVID-19 infection in patients treated with biological and conventional DMARDs and glucocorticosteroids were compared. Results: Among 512 rheumatic patients, 19.9% were definitely infected with COVID-19, and 23.3% of infected patients were hospitalized. Only one patient with vasculitis died during the two outbreaks. Our study showed that adding biologic DMARDs to conventional DMARDs did not increase the risk of COVID-19 infection. However, unlike biologic DMARDs, in conventional DMARDs, methotrexate increased, and hydroxychloroquine decreased COVID-19 infection. Regression analysis showed that prednisolone at a dosage higher than 10 mg/day increased the risk of COVID-19 infection 5-fold; hydroxychloroquine had a protective impact and reduced the risk of infection by 40%. Conclusions: Biologic DMARDs and the type of selected rheumatic diseases in our study did not influence the susceptibility to COVID-19 infection. Prednisolone raised the coronavirus infection, and hydroxychloroquine played a protective role in the current study. Most of our patients showed good adherence to the health protocols. Further studies after worldwide vaccination are now required to reevaluate the influence of rheumatic diseases and DMARDs on COVID-19 infection.

Anti-IL-17A ssDNA aptamer ameliorated psoriasis skin lesions in the imiquimod-induced psoriasis mouse model.

OBJECTIVES: IL-17 is an important player in the psoriasis pathogenesis, which recruits inflammatory cells to the psoriatic lesions, induced keratinocyte proliferation and plaque formation. Three monoclonal antibodies that block IL-17 have been approved for psoriasis treatment in the last decade. Compared to monoclonal antibodies, aptamers which are single-stranded DNA or RNA, bind with high affinity to proteins or other molecules and are more cost-effective. We previously showed that M2 and M7 anti-IL17A ssDNA aptamers could block IL-17 in vitro. The current study evaluated the therapeutic effects of M2 and M7 anti-IL17A ssDNA aptamers in the imiquimod (IMQ)-induced psoriasis mouse model. METHODS: IMQ cream and Vaseline (Vas) were administered on the back skin of C57BL/6 mice as IMQ-induced psoriasis and Vas control groups, respectively. In addition, hydrogel-containing aptamers were topically administered on the back skin of the mice, 10 min before IMQ treatment. Psoriatic lesions were evaluated by histology, clinical factors, and psoriasis area severity index (PASI) score. The mRNA expression levels of inflammatory factors, including IL-17A, IL-1ß, and S100a9, were assessed with quantitative reverse transcriptase-polymerase chain reaction in the mice back skin. RESULTS: Application of anti-IL-17A aptamers significantly ameliorated IMQ-induced keratinocyte proliferation, psoriatic lesions cumulative PASI score, IL-17A, IL-ß, and S100a9 inflammatory factors mRNA expression levels (p < 0.05). CONCLUSION: According to our results, it seems that M2 in high concentration and M7 in low concentration can be appropriate candidates to alleviate psoriasis lesions.

Introducing a Stabilizer Formulation for Allergenic Mold Extracts.

BACKGROUND: Sensitization to common mold allergens is one of the major causes of allergic rhinitis and asthma. Therefore, there is a critical need for standard sensitivity tests including skin prick tests to improve the stability of fungi extracts in traditional allergenic formulations. To address this concern, the present study aimed to develop a formulation to preserve allergenic activity of mold extracts. METHODS: 48 stabilizer formulations were designed and monitored for allergenic activity during a 40-days incubation period at 37 °C using an ELISA. Specifically, the IgE reactivity of allergenic A. alternata extracts were examined. After establishing the most effective stabilizer formulation, we evaluated whether it could protect the allergenic activity of Alt a1, A. fumigatus, and C. herbarum using an IgE inhibition ELISA after 40 days at 37 °C. RESULTS: We demonstrated that the most effective stabilizer formulation was a glycerol-based extract containing Arg and Glu. This formulation had an equal ratio of sucrose, sorbitol and protein and was able to preserve more than 95% of allergenic A. alternata extract activity during a 40-days incubation period at 37 °C. CONCLUSION: The present study reveals a novel formulation that is an efficient stabilizer of allergenic mold extract activity and has practical applications in mold skin prick tests, ELISAs, immunotherapies, and RAST.

Sublingual dendritic cells targeting by aptamer: Possible approach for improvement of sublingual immunotherapy efficacy.

The efficacy improvement of current sublingual immunotherapy (SLIT) for preventing and treating respiratory airway allergic diseases is the main purpose of many investigations. In this study, we aimed to assess whether ovalbumin (Ova) encapsulated poly (lactic-co-glycolic) acid nanoparticles (PLGA NPs) decorated with dendritic cells (DCs)-specific aptamer could be applied for this purpose.The nanoparticles containing Ova were synthesized by emulsion/solvent evaporation method and attached to DCs-specific aptamer. Ova-sensitized BALB/c mice have been treated in five ways: subcutaneously with free Ova (SCIT), sublingually either with free Ova, Ova-PLGA NPs (two doses), Apt-Ova-PLGA NPs (two doses) and placebo/control Apt-Ova-PLGA NPs. For assessment of immunologic responses, IL-4, IFN-γ, IL-17, IL10, and TGF-ß and IgE antibody levels were measured by ELISA and T cell proliferation were evaluated by MTT. In addition, lung and nasal histological examinations, NALF cells counting were carried out. Results declared that the lowest IgE and IL- 4 levels were observed in Apt-Ova-PLGA NPs (both doses). In the other hands, Apt-Ova-PLGA NPs (high dose) showed the highest increase of IFN- γ and TGF- ß, decrease of IL-17 levels, total cell count and T-cell proliferation. IL-10 levels showed more decrease in SCIT, Apt-Ova-PLGA NPs (high dose) and Ova-PLGA NPs (high dose) than other groups. Histopathological examinations also confirmed in vitro results. Our findings suggest SLIT with this functionalized delivery system could be a promising approach for promoting the SLIT efficiency by decreasing the required allergen doses through specific delivery of allergen to sublingual DCs and enhancing the suppression of allergic responses.

Dc-specific aptamer decorated gold nanoparticles: A new attractive insight into the nanocarriers for allergy epicutaneous immunotherapy.

Recently, the main goal of many allergy epicutaneous immunotherapy (EPIT) studies is to enhance the allergen delivery through the intact skin. Therefore, applying new strategies for tackling this issue are inevitable. For this purpose, ten groups of Che a 2-sensitized BALB/c mice were epicutaneously treated for a 6-week period with the rChe a 2-GNPs-Aptamer, rChe a 2-GNPs-Aptamer + skin-penetrating peptides (SPPs), rChe a 2-GNPs, rChe a 2, GNPs, and PBS. Afterward, the serum IgE and IFN-γ, TGF-ß, IL-10, IL-4, IL-17a cytokine production, NALF analysis, and lung/nasal histological examinations were performed. The present study results demonstrate that, EPIT in aptamer treated groups had a significant increase of IFN-γ, TGF-ß, and IL-10 concentrations and a significant decrease of IgE, IL-4, and IL-17a concentrations as well as NALF infiltrated immune cell count compared to the non-targeted ones. In addition, SPPs led to more significant improvement of immunoregulatory parameters, especially IL-10 cytokine. Accordingly, the targeted-GNPs with DC-specific aptamers could act as an efficient approach for the improvement of EPIT efficacy compared to the free allergen. Moreover, the application of SPPs might be considered as a useful tool in achieving a successful EPIT with lower doses of allergen at a shorter duration of the treatment.

The Influence of Gamma Radiation Processing on the Allergenicity of Main Pistachio Allergens.

BACKGROUND: Gamma irradiation is a form of processing with an array of applications in medical sciences such as microbial decontamination, viruses inactivation, cervical carcinoma and breast cancer treatment. One of the ways in which gamma irradiation has the potential to be used is in reducing the allergenicity of food allergens. METHODS: In the present study, pistachios were irradiated with either a 1, 10, or 100 kGy dose of gamma irradiation. The binding rate of mice and human antibodies to the allergens of the pistachio extracts were examined via Western blot analysis. RESULTS: Our findings show an inverse dose-response relationship between the binding rate of antibodies to the pistachio allergens and the gamma irradiation dose. Despite these promising findings, the results of our sensory evaluation indicate that gamma irradiation causes undesirable changes to the sensory characteristics of pistachios, especially at the dose of 100 kGy. CONCLUSION: Gamma irradiation appears to be an effective method in reducing the allergenicity of pistachios. Thus, this form of processing has the potential to prevent adverse allergic reactions to the major pistachio allergens in sensitized subjects. However, further research must be dedicated to examining the dose sufficient in reducing allergencity, while maintaining adequate sensory quality for satisfactory consumption.

Determination of Optimum Excipients for Platanus orientalis Pollen Extract by Accelerating Chemical Stability Test and Their Synergistic Effect.

BACKGROUND: The quality of extracts used in the skin prick test directly influences the interpretation of the test. Accordingly, the outcomes and effectiveness of immunotherapy for the management of IgE-mediated allergies depend on the quality of the extracts used. Excipients, which are pharmacologically inert ingredients, are intentionally added to the active ingredients. The aim of this study was to address optimum excipients for stability Platanus (P.) orientalis extract. METHODS: In this study the excipients examined were l-lysine (20 mM), l-cysteine (20 mM), albumin (0.5%), sorbitol (2%), sucrose (750 mM), trehalose (20 mM), D-mannitol (2% w/v), urea (100 mM) and Tween-20 (0.1%). Their effects on P. orientalis extract stability were analyzed using an inhibition enzyme linked immune assay at 37 °C. RESULTS: A mixture of lysine (20 mM), trehalose (20 mM), and D-mannitol (2% w/v) conferred the greatest stability on the P. orientalis extract. CONCLUSION: The P. orientalis extract stability was increased by a mixture of lysine (20 mM), trehalose (20 mM), and D-mannitol.

Sublingual Immunotherapy with Sal k1 Expressing Lactococcus lactis Down-regulates Th2 Immune Responses in Balb/c Mice.

Sublingual immunotherapy (SLIT) has been introduced as a noninvasive and safer approach for allergen-specific immunotherapies. In this study we investigated the efficacy of oral immunotherapy with recombinant Salsola kali 1 protein (Sal k 1) on Th1/Th2 balance in a mouse model of allergy. Female Balb/c mice were intraperitoneally sensitized with rSal k1, followed by a respiratory challenge with 1% (w/v) rSal k1. The sensitized mice were subjected to SLIT using rSal K1 expressing Lactobacillus lactis strain for three weeks. Each week the experimental group underwent SLIT protocol twice. Finally, serum levels of specific immunoglobulins including IgE, IgG1 and IgG2a, as well as secretion of different cytokines from splenocytes including IL-2, IL-4, IL-10, IFNγ and TGFß into culture media were measured by ELISA. Following immunotherapy, the levels of specific IgE and IgG1 in mice sera as well as IL-4 level in supernatant of splenocytes were significantly lower than allergic controls. While serum IgG2a, IgG2a/IgG1 ratio as well as concentration of IL-2, IL-10, IFNγ, and TGFß were higher in the SLIT group compared to the controls. The histopathological examination of intestinal tissues revealed no sign of inflammatory response following SLIT. This study revealed that Th2 immune responses are reduced in allergic mice after feeding them with allergen expressing probiotic bacteria as a SLIT approach. Since the safety of this procedure was previously approved, thus, it seems that a similar protocol using human based probiotics could be applied for Salsola kali sensitive patients.

Production of Recombinant Protein of Salsola Kali (Sal k1) Pollen Allergen in Lactococcus Lactis.

The Salsola kali pollen is considered the main cause of allergic sensitization in desert and semi-desert regions. We have constructed recombinant Lactococcus lactis producing Sal k1 protein with the aim of using it as a mucosal vaccine for specific immunotherapy. The Sal k1 gene was amplified, and transferred into a PNZ 8148 plasmid. The PNZ8148-Sal k1 recombinant plasmid was transformed into competent E.coli strain MC1061 for replication, and then was isolated and cloned into competent L. lactis by electroporation. The cloning was verified by PCR and gene sequencing. The production of recombinant Sal K1 (rSal K1) protein was induced by nisin. The rSal K1 protein was purified by affinity chromatography and dialysis, and confirmed by SDS-PAGE and western blot analyses. The recombinant L. lactis was successfully constructed. Production of a 40-kDa rSal k1 protein with the L. lactis was shown by sodium dodecyl sulfate-polyacrylamid gel electrophoresis (SDS-PAGE) analysis. In addition, western blot analysis using specific mouse anti-Sal k1 polyclonal antibodies and sensitive human sera verified the 40-kD protein as rSal k1 allergen. This study demonstrated that L. lactis may be used as a promising live delivery system for recombinant Sal k1 protein without altering its immunoreactivity; however, its efficacy in the context of the immune system is suggested to be pursued in future studies.

Interaction Between Air Pollutants and Pollen Grains: The Role on the Rising Trend in Allergy.

Asthma and allergic diseases cases have risen in recent decades. Plant pollen is considered as the main aeroallergen causing allergic reactions. According to available data, urban residents experience more respiratory allergies than rural residents mainly due to the interaction between chemical air pollutants and pollen grains.This interaction can occur through several mechanisms; chemical pollutants might facilitate pollen allergen release, act as adjuvants to stimulate IgE-mediated responses, modify allergenic potential, and enhance the expression of some allergens in pollen grains. This review focuses on the most recent theories explaining how air pollutants can interact with pollen grains and allergens.

Association between smoking and non-alcoholic fatty liver disease: A systematic review and meta-analysis.

BACKGROUND/AIMS: Non-alcoholic fatty liver disease is one of the most common chronic liver diseases. Some risk factors are known to influence the development of non-alcoholic fatty liver disease, but the effect of tobacco smoking on the progression of non-alcoholic fatty liver disease is controversial. The main goal of this systematic review and meta-analysis is to investigate the association between smoking and non-alcoholic fatty liver disease. METHOD: Electronic databases (PubMed, Scopus, and ISI Web of Science) were searched to find published articles on non-alcoholic fatty liver disease and smoking until December 2016. All relevant studies were screened by inclusion and exclusion criteria and compatible studies were chosen. The Newcastle-Ottawa Scale was used to assess the methodological quality of eligible articles. Subsequently, information was gathered based on the following: author, publication year, keywords, country, inclusion and exclusion criteria, main results, study design, conclusion, and confounder variables (age, body mass index, gender, ethnicity, and diabetes). Finally, analyses were performed using Comprehensive Meta-Analysis Software. RESULTS: Data were extracted from 20 observational studies (9 cross-sectional, 6 case-control, 4 cohort studies, and 1 retrospective cohort study). A significant association was observed between smoking and non-alcoholic fatty liver disease with a pooled odds ratio of 1.110 (95% confidence interval, 1.028-1.199), p-value = 0.008. The statistical heterogeneity was medium with an I2 of 40.012%, p-heterogeneity = 0.074. Also there was a significant relation between non-alcoholic fatty liver disease and passive smoking with a pooled odds ratio of 1.380 (95% confidence interval, 1.199-1.588; p-value = 0.001; I2 = 59.41; p-heterogeneity = 0.117). CONCLUSION: Our meta-analysis demonstrated that smoking is significantly associated with non-alcoholic fatty liver disease. Further prospective studies exploring the underlying mechanisms of this association should be pursued. Also passive smoking increases the risk of non-alcoholic fatty liver disease about 1.38-fold. The effects of smoking cigarettes on active smokers (current smoker, former smoker, and total smoker) are less than passive smokers. Further studies are needed to compare the of effects of passive and active smoking on non-alcoholic fatty liver disease.

Pulsed Dilution Method for the Recovery of Aggregated Mouse TNF-α.

BACKGROUND: The expression of mouse tumor necrosis factor alpha (TNF-α) in Escherichia coli is a favorable way to get high yield of protein; however, the formation of cytoplasmic inclusion bodies, which is the consequence of insoluble accumulated proteins, is a major obstacle in this system. To overcome this obstacle, we used a pulsed dilution method to convert the product to its native conformation. METHODS: Reducing agent and guanidine hydrochloride were used to solubilize inclusion bodies formed after TNF-(α) expression. Then, the refolding procedure was performed by pulsed dilution of the denatured protein into a refolding buffer. The properly-folded protein was purified by metal affinity chromatography. RESULTS: SDS-PAGE showed a 19.9 kDa band related to the mature TNF-(α) protein. The protein was recognized by anti-mouse TNF-(α) on western blots. The final concentration of the purified recombinant TNF-(α) was 62.5 µg/mL. CONCLUSIONS: Our study demonstrates the efficiency of this method to produce a high yield of folded mature TNF- (α).

Lung-derived innate cytokines: new epigenetic targets of allergen-specific sublingual immunotherapy.

OBJECTIVES: Sublingual allergen-specific immunotherapy is a safe and effective method for treatment of IgE-mediated respiratory allergies; however, the underlying mechanisms are not fully understood. This study was planned to test whether sublingual immunotherapy (SLIT) can exert epigenetic mechanisms through which the airway allergic responses can be extinguished. MATERIALS AND METHODS: BALB/c mice were sensitized intraperitoneally and challenged intranasally. Then, they received sublingual treatment with recombinant Che a 2 (rChe a 2), a major allergen of Chenopodium album. After SLIT, allergen-specific antibodies in sera, cytokine profiles of spleen cell cultures, mRNA and protein expression of lung-derived IL-33, IL-25, and TSLP (thymic stromal lymphopoietin), and histone modifications of these three genes were assessed. RESULTS: Following Immunotherapy, systemic immune responses shifted from Th2 to Th1 profile as demonstrated by significant decrease in IgE and IL-4 and substantial increase in IgG2a and IFN-γ. At local site, mRNA and protein levels of lung-derived pro-inflammatory cytokines IL-33 and TSLP were markedly down-regulated following SLIT that was associated with marked enrichment of trimethylated lysine 27 of histone H3 at promoter regions of these two cytokines. CONCLUSION: In our study, sublingual immunotherapy with recombinant allergen effectively attenuated allergic immune responses, at least partly, by induction of distinct histone modifications at specific loci. Additionally, the lung-derived pro-allergic cytokines IL-33 and TSLP could be promising mucosal candidates for either monitoring allergic conditions or therapeutic approaches.

The effects of WW2/WW3 domains of Smurf2 molecule on TGF-ß signaling and arginase I gene expression.

Smad ubiquitination regulatory factor 2 (Smurf2) consists of multiple WW domains which can interact with Smad7 molecule and inhibit signaling of transforming growth factor-beta (TGF-ß) cytokine. Arginase I (ArgI) is one of the main products of TGF-ß signaling that plays important roles in tumor escape and airway tissue fibrosis and remodeling in asthma. In this study, the effects of TAT fused to WW2/WW3 (TAT-WW2/WW3) recombinant protein on TGF-ß signaling and ArgI gene expression were evaluated on J774A.1 cell culture. For this purpose, interaction of TAT-WW2/WW3 with Smad7, mRNA expression of ArgI, and phosphorylated Smad3 (P-Smad3) were analyzed in TAT-WW2/WW3-treated J774A.1 cell. The results showed interaction of TAT-WW2/WW3 with Smad7, downregulation of ArgI gene expression (P < 0.05), and higher amount of P-Smad3 in the TAT-WW2/WW3-treated cells. In conclusion, we suggest that TAT-WW2/WW3 could interfere with TGF-ß signaling and reduce ArgI gene expression. Since, ArgI has important effects on tissue remodeling in asthma and cancer progression, so these findings could be used to develop a new approach in the treatment of asthma and cancers.