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The role of the epididymis in post-testicular maturation of spermatozoa is to gain the potential to fertilize the ovum. It is well known that thyroid malfunction has a negative impact on the male reproductive system. Hypothyroidism (underactive thyroid gland) causes marked structural and functional changes in the epididymis, as well as being able to influence extracellular matrix (ECM) proteins secretion and cell death. This study was designed to characterize the effects of hypothyroidism on epididymis’ structure. Twenty Balb/C mice were randomly divided into control, and hypothyroid group which received 0.05% 6-n-propyl-2-thiouracil (PTU) for 35 days. At the end of the experiment, to confirm the hypothyroidism, T4 and thyroid-stimulating hormone (TSH) levels were measured using chemiluminescent immunoassay kit. Real-time PCR, immunohistochemistry, periodic acid-Schiff (PAS) staining, TUNEL assay, and biochemical measurements were carried out. Based on the results, T4 level was significantly lower in the hypothyroid group compared to the control group, whereas TSH level increased in hypothyroid group compared to the control group (p < 0.05). Laminin α5 and collagen IV mRNA levels were upregulated in the hypothyroid group compared to the control group (p < 0.05). However, no significant difference was observed in the immunoreactivity of laminin α5 and collagen IV proteins between the two groups. Also, in PAS staining, no significant differences were found in the basement membrane (BM) staining intensity between the two groups. Hypothyroidism markedly increased epididymal epithelium apoptosis (p < 0.05). Besides, hypothyroidism reduced superoxide dismutase (SOD) enzyme activity while increasing the malondialdehyde (MDA) level (p < 0.05). Collectively, data indicated a possible relationship between alterations in the BM components and biochemical factors and increased apoptosis in the epididymal cells. These results suggest a regulatory role of thyroid hormones on the BM and structure of epididymis.

Closer attention should be paid to vitamin D status in patients with mycobacterial diseases.

Background: Interferon-gamma [IFN-γ) is the most important cytokine in the immune system. This protein has been expressed in bacterial cells. However, bacterial cloning is not an easy task. We aimed to clone, express, and purify recombinant mouse IFN-γ and overcome problems in favor of commercial purposes. Materials and Methods: To amplify the gene product for cloning, we primarily designed two specific primers for the target gene. Following PCR amplification, the amplicon was inserted into the pET-21b[+) vector. The E. coli BL21 [DE3) CodonPlus strain was chosen for the expression of the target gene. Finally, the expressed recombinant mouse IFN-γ was assessed through the western blotting method. Results: We performed a cloning process and produced recombinant mouse IFN-γ in an optimal condition. We also noticed that monomeric protein could be transformed to a homodimeric structure which can be observed using the SDS PAGE [SDS-polyacrylamide gel electrophoresis) and western blotting. Conclusion: Experimental conditions strongly affect the large-scale cloning procedures required to be optimized in each laboratory. The expressed recombinant mouse IFN-γ described here is appropriate for commercial purposes.

Capacity and ability of Lactiplantibacillus plantarum and Lacticaseibacillus rhamnosus probiotic strains isolated from some human and food sources were evaluated for modulating and regulating the immune system against hypersensitivity type 1 (dependent on IgE). In this study, given the probiotic properties of the strains and the use of mouse models, the strains were orally administered (1×10

This study was conducted to evaluate the effects of recombinant probiotic bacteria as a candidate for oral vaccine with the potential of treating allergy to Amaranthus retroflexus pollens. The main gene of this allergen, Ama r 2, was cloned into the food grade plasmid pNZ7025 and then was electrotransformed into the food grade Lactococcus lactis NZ1330. No expression was observed in the primary structure due to the distance between the ribosome binding site and the start codon. Therefore, the vector structure was corrected using the site-directed mutagenesis (SDM) technique. The cell extract of this strain was used for assessing the expression of the recombinant allergen in western blot analysis, and the existence of this protein with a molecular weight of 14.2 kDa was confirmed. To evaluate the efficacy of this strain in the treatment of allergies as an oral vaccine, probiotic ice cream was prepared. After the sensitization of mice, the treatment was performed by oral immunotherapy for 4 weeks, 4 to 5 times per week. 20 μl of functional ice cream with 1012 CFU/ml of r-L. lactis NZ1330 significantly reduced the serum IgE level. The levels of IFN-γ and TGF-β cytokines increased in the 20 μl ice cream treatment group as well as 40 μg/ml pure allergen compared with the PBS-treated group, and IL-4 cytokine levels decreased compared with the PBS-treated group. Overall, 20 μl ice cream with 1012 CFU/ml of the recombinant bacteria resulted in the best performance in terms of improving allergies to Th1 and Treg responses.

Tuberculosis (TB) is referred to as a "consumption" or phthisis, which has been a fatal human disease for thousands of years. Mycobacterium tuberculosis (M. tb) might have been responsible for the death of more humans than any other bacterial pathogens. Therefore, the rapid diagnosis of this bacterial infection plays a pivotal role in the timely and appropriate treatment of the patients, as well as the prevention of disease spread. More than 98% of TB cases are reported in developing countries, and due to the lack of well-equipped and specialized diagnostic laboratories, development of effective diagnostic methods based on biosensors is essential for this bacterium. In this review, original articles published in English were retrieved from multiple databases, such as PubMed, Scopus, Google Scholar, Science Direct, and Cochrane Library during January 2010-October 2019. In addition, the reference lists of the articles were also searched. Among 109 electronically searched citations, 42 articles met the inclusion criteria. The highest potential and wide usage of biosensors for the diagnosis of M. tb and its drug resistance belonged to DNA electrochemical biosensors (isoniazid and rifampin strains). Use of biosensors is expanding for the detection of resistant strains of anti-TB antibiotics with high sensitivity and accuracy, while the speed of these sensory methods is considered essential as well. Furthermore, the lowest limit of detection (0.9 fg/ml) from an electrochemical DNA biosensor was based on graphene-modified iron-oxide chitosan hybrid deposited on fluorine tin oxide for the MPT64 antigen target. According to the results, the most common methods used for M. tb detection include acid-fast staining, cultivation, and polymerase chain reaction (PCR). Although molecular techniques (e.g., PCR and real-time PCR) are rapid and sensitive, they require sophisticated laboratory and apparatuses, as well as skilled personnel and expertise in the commentary of the results. Biosensors are fast, valid, and cost-efficient diagnostic method, and the improvement of their quality is of paramount importance in resource-constrained settings.

Background: The engineered chimeric peptides including functional multi-epitope structures fused by various peptide linkers are widely applied in biotechnological research to improve the expression level and biological activity of chimera. Objective: The aim of our study was to evaluate the effect of helical and flexible linkers on solubility, expression level and folding of multi-epitope chimera containing four epitopes of Human T Lymphotropic Virus Type 1 (HTLV-1). Methods: For this purpose, the chimera sequences connected by the helical or flexible linker were inserted into different plasmid vectors and expressed in E. coli strains. The expressed products were analyzed using SDS-PAGE and Western blot techniques. Additionally, the molecular modeling study of the chimera with helical or flexible linker was performed using iterative threading assembly refinement (I-TASSER) to attain their three-dimensional structures. Results: Comparison of the chimera expression indicated that the insertion of a flexible (GGGGS)3 linker among chimera epitopes could significantly enhance the level of expression, whereas, the low-level of chimera expression was observed for chimera containing the contiguous helical (EAAAK)5 linker. According to the results of sequence alignment and plasmid stability test, the structure and function of a consecutive helical linker among chimera epitopes were similar to porins as the outer-membrane pore-forming proteins. The molecular modeling results confirmed our experimental study. Conclusion: This investigation illustrated the key role of linker design in determining the expression level of multi-epitope chimera and conformational folding.

Background: The production of recombinant proteins in Escherichia coli is one of the most valuable achievements in biotechnology, with many therapeutic and diagnostic applications; however, the aggregation and misfolding of proteins that result in the formation of insoluble inclusion bodies is a disruptive factor in this process. Various solubilization and refolding methods can be used to improve recombinant protein conformation. In this study, we applied a dilution method with a refolding buffer to produce a native form of soluble immature mouse TGF-β1. Materials and Methods: The TGF-β1 cDNA which encodes the protein without the signal peptide, was cloned into the pET21-b (+) vector. The target protein was expressed by the transformation of E. coli BL21 cells with the plasmid. The resulting inclusion bodies were solubilized and then diluted in the refolding buffer to make a protein with native structure. Results: Following PCR of the recombinant plasmid with T7 primers, electrophoresis and sequencing of the amplified product indicated 100% identity of the target sequence with the murine TGF-β1 gene. Finally, the protein solubility and immuno-reactivity were confirmed a 44 kDa protein which conducted with the anti-TGF-β1-specific polyclonal antibody on a western blot. Conclusion: Our dilution method and refolding buffer effectively converted aggregated immature mouse TGF-β1 to a soluble and immuno-reactive form.

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.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-1β, and S100a9, were assessed with quantitative reverse transcriptase-polymerase chain reaction in the mice back skin.Application of anti-IL-17A aptamers significantly ameliorated IMQ-induced keratinocyte proliferation, psoriatic lesions cumulative PASI score, IL-17A, IL-β, and S100a9 inflammatory factors mRNA expression levels (p 0.05).According to our results, it seems that M2 in high concentration and M7 in low concentration can be appropriate candidates to alleviate psoriasis lesions.

Tuberculosis affects one-third of the world's population and leads to a high rate of morbidity and mortality. Bacillus Chalmette-Guerin (BCG) as the only approved vaccine for theThe main aim of this study was to prepare a self-assembled nanomicelle composed from a di-block polymer in which, a di-fusion peptide was the hydrophobic block and polyethylene glycol (PEG) was the hydrophilic block. The micelles were characterizedThe micellar nanovaccine was able to elicit good dendritic cell maturation. Nanomicelles could efficiently induce systemic cytokines as well as nasal secretory predominant antibody titers (sIgA). The expression pattern of cytokines indicated the superiority of cellular immunity. Nasal administration of two doses of nanomicelles after a prime subcutaneous administration of BCG induced the highest mucosal and systemic immune responses.Based on our results PEG-HspX/EsxS self-assembled nanomicelle is highly immunogenic and can be considered a potential vaccine candidate against

Psoriasis is a common inflammatory skin disorder, which is mediated by the immune system and affects 1-4% of the world's population. Psoriasis is caused by a complex interaction between the immune system, autoantigens, psoriasis-associated genetic factors, and various environmental factors. As a chronic disease requiring long-term treatment, psoriasis is associated with follow-up costs and an economic burden on the patients, their families, and healthcare systems. The current treatments for moderate-to-severe plaque psoriasis include topical therapy, phototherapy, and systemic drugs consisting of biological/non-biological drugs. Within the past two decades, recent biological therapies for psoriasis have rapidly advanced. Moreover, new bispecific agents have the potential for better disease control, while small molecule drugs offer a future alternative to biological drugs and the more cost-effective, long-term treatment of the disease. The present study aimed to review updated data regarding the inhibitors used to improve plaque psoriasis that contain biologics, bispecific agents, small molecules, and aptamers (either approved or in the research phase).

Allergen-specific sublingual immunotherapy (SLIT), a safe and efficient route for treating type I hypersensitivity disorders, requires high doses of allergens. SLIT is generally performed without adjuvants and delivery systems. Therefore, allergen formulation with appropriate presentation platforms results in improved allergen availability, targeting the immune cells, inducing regulatory immune responses, and enhancing immunotherapy's efficacy while decreasing the dose of the allergen. In this review, we discuss the adjuvants and delivery systems that have been applied as allergen-presentation platforms for SLIT. These adjuvants include TLRs ligands, 1α, 25-dihydroxy vitamin D3, galectin-9, probiotic and bacterial components that provoke allergen-specific helper type-1 T lymphocytes (TH1), and regulatory T cells (Tregs). Another approach is encapsulation or adsorption of the allergens into a particulate vector system to facilitate allergen capture by tolerogenic dendritic cells. Also, we proposed strategies to increasing the efficacy of SLIT via new immunopotentiators and carrier systems in the future.

BackgroundTuberculosis (TB), a contagious disease caused by Mycobacterium tuberculosis (M. tuberculosis), remains a health problem worldwide and this infection has the highest mortality rate among bacterial infections. Current studies suggest that intranasal administration of new tuberculosis vaccines could enhance the immunogenicity of M. tuberculosis antigens. Hence, we aim to evaluate the protective efficacy and immunogenicity of HspX/EsxS fusion protein of M. tuberculosis along with ISCOMATRIX and PLUSCOM nano-adjuvants and MPLA through the intranasal administration in mice model.MethodsIn present study, the recombinant fusion protein was expressed in Escherichia coli and purified and used to prepare different nanoparticle formulations in combination with ISCOMATRIX and PLUSCOM nano-adjuvants and MPLA. Mice were intranasally vaccinated with each formulation three times at an interval of 2 weeks. Finally, IFN-γ, IL-4. IL-17 and TGF-β concentration in supernatant of cultured splenocytes of vaccinated mice as well as serum titers of IgG1 and IgG2a and sIgA titers in nasal lavage were determined.ResultsAccording to obtained results, intranasally vaccinated mice with formulations containing ISCOMATRIX and PLUSCOM nano-adjuvants and MPLA could effectively induced IFN-γ and sIgA responses. Moreover, both HspX/EsxS/ISCOMATRIX/MPLA and HspX/EsxS/PLUSCOM/MPLA and their BCG booster formulation could strongly stimulate the immune system and enhance the immunogenicity of M. tuberculosis antigens.ConclusionThe results demonstrate the potential of HspX/EsxS-fused protein in combination with ISCOMATRIX, PLUSCOM and MPLA after nasal administration in enhancing immune response against of M. tuberculosis antigens. So, nasal immunization with these formulations, could induce immune responses and considered as new TB vaccine or as BCG booster.

To increase the efficiency of aptamers to their targets, a simple and novel method has been developed based on aptamer oligomerization. To this purpose, previously anti-human TNF-α aptamer named T1-T4 was trimerized through a trimethyl aconitate core for neutralization of in vitro and in vivo of TNF-α. At first, 54 mer T1-T4 aptamers with 5'-NH2 groups were covalently coupled to three ester residues in the trimethyl aconitate. In vitro activity of novel anti-TNF-α aptamer and its dissociation constant (Kd ) was done using the L929 cell cytotoxicity assay. In vivo anti-TNF-α activity of new oligomerized aptamer was assessed in a mouse model of cutaneous Shwartzman. Anchoring of three T1-T4 aptamers to trimethyl aconitate substituent results in formation of the 162 mer fragment, which was well revealed by gel electrophoresis. In vitro study indicated that the trimerization of T1-T4 aptamer significantly improved its anti-TNF-α activity compared to non-modified aptamers (P < 0.0001) from 40% to 60%. The determination of Kd showed that trimerization could effectively enhance Kd of aptamer from 67 nM to 36 nM. In vivo study showed that trimer aptamer markedly reduced mean scar size from 15.2 ± 1.2 mm to 1.6 ± 0.1 mm (P < 0.0001), which prevent the formation of skin lesions. In vitro and in vivo studies indicate that trimerization of anti-TNF-α aptamer with a novel approach could improve the anti-TNF-α activity and therapeutic efficacy. According to our findings, a new anti-TNF-α aptamer described here could be considered an appropriate therapeutic agent in treating several inflammatory diseases.

Background Early detection of tuberculosis is one of the crucial steps for TB control. Although, the sensitivity of conventional methods like Lowenstein Jensen (LJ) culture and direct staining is quite low, molecular techniques like polymerase chain reaction (PCR) are more sensitive and be considered as useful tools for rapid detection of tuberculosis. Various genes like IS6110 and mpb64 have been used as target for detection of M. tuberculosis, but more research is needed to find the most specific targets. The short-chain dehydrogenases/reductases family (SDR) is one of a very large family of NAD- or NADP-dependent oxidoreductase enzymes which is present in all M. tuberculosis strains. The large part of SDR sequences in tuberculosis is completely conserved and different from non-tuberculosis mycobacterium. The aim of the study was to develop an in-house PCR assay using the SDR target for rapid detection of M. tuberculosis from clinical specimens. Method M. tuberculosis-specific sequences were found using modified genome comparison method and the primers were designed by the Primer Premier 5.0 software. A PCR assay was developed targeting the nucleotide sequences within the SDR gene. A total of 50 cultivated specimens and 120 clinical specimens were evaluated by PCR. Results The clinical evaluation of SDR PCR assay showed high specificity (100%) and high sensitivity (88.5%). The analytical sensitivity was 10 fg of template DNA which is theoretically equivalent to 2 copy of genomic DNA per microliter. The SDR is a new specific target of M. tuberculosis and no cross-reactivity was observed to non-tuberculosis mycobacterium and other pathogenic bacteria. Conclusions Based on our results, the SDR gene can be considered as a useful target for detection of M. tuberculosis complex from clinical specimens.

The imbalance between pro and anti-inflammatory cytokines plays an important role in the pathogenesis of multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE). Thymus vulgaris (thyme) as a traditional medicinal plant has been reported to exert antimicrobial, antioxidant, and anti-inflammatory effects. Therefore, this study evaluated the modulatory effects of Thymus vulgaris on the clinical symptoms, histopathological scores, and the production of some anti-inflammatory (TGF-β, IL-4, and IL-10) and pro-inflammatory (IFN-γ, IL-6 and IL-17) cytokines in EAE model. EAE was induced by MOG35–55 peptide and mice were treated intra-peritoneally (i.p) with phosphate buffered saline (PBS) in the control group or thyme extract (50 or 100 mg/kg of body weight, every other day) in thyme-treated EAE groups, from day 0 to +21 of post MOG immunization. Mice were sacrificed at day 22, and splenocytes were isolated and re-stimulated in vitro with MOG in order to measure the cytokine production and proliferation of re-stimulated cells by enzyme linked immunosorbent assay (ELISA) method and WST-1 reagent, respectively. The clinical symptoms and histopathological scores of the CNS were lower in thyme-treated than EAE control group. Furthermore, the production of IFN-γ and IL-6 by splenocytes was lower in thyme-treated EAE than in the control group. The production of IL-10 and TGF-β increased in mice treated with thyme extract compared to the control group. In this study, we showed for the first time that the immunomodulatory effects of Thymus vulgaris in EAE model. Thus, the possible therapeutic potential of thyme for treatment of MS could be considered in future research.

Incidence of Mycobacterium simiae pulmonary infection is increasing and diagnosis and treatment are challenging. We surveyed the clinical features, risk factors, diagnosis, and management in 20 patients from northeastern Iran diagnosed by line probe assay and confirmed by sequencing the ITS (16S‐23S) rRNA region and carried out a literature review using the keywords “pulmonary infection” and “Mycobacterium simiae.” The mean age of patients was 55.1 years, with 80% female and 90% diagnosed by sputum. Clinical symptoms included severe cough (90%), sputum production (70%), haemoptysis (50%), and chest pain (35%). Comorbidities included a history of tuberculosis (60%), smoking (40%), or chronic obstructive pulmonary disease (20%). Patients were treated with levofloxacin, clarithromycin, and co‐trimoxazole. Except for two patients, the clinical symptoms improved. Mycobacterium simiae pulmonary infection is increasing in people with underlying diseases. Although choosing the most appropriate treatment remains a challenge, combining successful treatments could be useful in treating these patients., In the current study, we surveyed the clinical features, risk factors, diagnosis, and treatment of Mycobacterium simiae pulmonary infection in Mashhad, Iran.

Mycobacterium simiae is an emerging and spreading pathogen in Iran and little data about its drug susceptibility test (DST) and no standard treatment regimen are available. We report a case of multidrug‐resistant M. simiae respiratory infection in a 65‐year‐old woman with a history of previous Mycobacterium tuberculosis infection. The patient was treated with clarithromycin, levofloxacin, and cotrimoxazole for one year and eventually died while still suffering from respiratory problems. For DST, broth microdilution method was used according to the Clinical and Laboratory Standards Institute guidelines as well as molecular DST in clinical isolate. Mycobacterium simiae was resistant to streptomycin, moxifloxacin, clarithromycin, and cotrimoxazole antibiotics and was sensitive to clofazimine and amikacin antibiotics. Inappropriate use of antibiotics without determining the pattern of antibiotic resistance increases the likelihood of resistance and, for resistant specimens, the need to review the treatment protocol and replace antibiotics. Effectiveness based on antibiotic resistance pattern is essential., Mycobacterium simiae is an emerging and spreading pathogen in Iran and little data about its drug susceptibility test (DST) and no standard treatment regimen are available. We report a case of multidrug‐resistant M. simiae respiratory infection in a 65‐year‐old woman with a history of previous Mycobacterium tuberculosis infection.

High expression of p32 in certain tumors makes it a potential target for immunotherapy. In the present study, the first goal was to design multi-epitope peptides from the P32 protein and the second goal was to compare the prophylactic effects of DCs- and PBMCs- based vaccines by pulsing them with designed peptides. For these purposes, 160 BALB/c mice were vaccinated in 5 different subgroups of each 4 peptides using PBS (F1-4a), F peptides alone (F1-4b), F peptides with CpG-ODN (F1-4c), F peptides with CpGODN and DCs (F1-4d), and F peptides with CpG-ODN and PBMCs (F1-4e). We found a significantly higher interferon-γ (IFN-γ) and granzyme B levels in T cells of F4d and F4e subgroups compared to control (p ≤ 0.05). The result of challenging spleen PBMCs of vaccinated mice with 4T1 cells showed significant up- and down- regulation of Fas ligand (FasL) and forkhead box P3 (Foxp3) gene expression between F4d and F4e subgroups with control, respectively. In addition, a significant change was seen in Caspase3 gene expression of F4d subgroup compared to control (p ≤ 0.05). Supernatant levels of IFN-γ and perforin were significantly increased in F4d and F4e subgroups compared to control. Consequently, significantly lower tumor sizes and prolonged survival time were detected in F4d and F4e subgroups compared to control after challenging mice with 4T1 cells. Accordingly, these results demonstrated that PBMCs pulsed F4 peptide-based vaccine could induce a protective immune response while it is a simple and less expensive vaccine.

AimsTuberculosis (TB) is one of the life-threatening infectious diseases, caused by Mycobacterium tuberculosis (M.tb). In the present study, a multi-stage M.tb immunodominant Fcγ1 fusion protein (Ag85B:HspX:hFcγ1) was produced and its immunogenicity as an hFcγRI targeted delivery systems for selective antigen presentation was evaluated in a mouse model. Methods and ResultsThe novel Ag85B:HspX:hFcγ1 recombinant fusion protein was designed and expressed in the Pichia pastoris (P. pastoris). After affinity chromatography purification, the purity of Ag85B:HspX:hFcγ1 was confirmed by ELISA, SDS-PAGE, and Western blotting methods. The immunogenicity of the construct was evaluated by assessing interferon-γ (IFN-γ) and transforming growth factor-beta (TGF-β) in a mouse model. Co-localization results of Ag85B:HspX:hFcγ1 with hFcγRI (CD64) confirmed its function for binding with its receptor and inducing Th1 selective responses. There was a significant difference in the expression of both IFN-γ, (P≤0.02) and TGF-β, (P=0.05).ConclusionsThe co-localization assay confirmed functionally the binding of the Ag85B:HspX:hFcγ1 to CD64 (FcγRI). Furthermore, in vitro assay showed that Ag85B:HspX:hFcγ1 can stimulate a modulated immune response in favor of anti-intracellular microbes, as IFN-γ increased, and also TGF-β as an immune-modulatory cytokine prevented the induction of hypersensitivity reactions.Significance and Impact of StudyThe combination of Ag85B as the most immunodominant M.tb Ag with HspX, as an Ag and adjuvant, could open a new venue for more studies for the design of multi-stage subunit vaccines for TB. Of note, an Fcγ1 fusion protein can be considered as a functional approved selective delivery vehicle for targeting antigen-presenting cells (APCs) and inducing cross-presentation.

Diabetic foot ulcers (DFUs) are chronic severe complications of diabetes disease and remain a worldwide clinical challenge with social and economic consequences. Diabetic wounds can cause infection, amputation of lower extremities, and even death. Several factors including impaired angiogenesis, vascular insufficiency, and bacterial infections result in a delayed process of wound healing in diabetic patients. Treatment of wound infections using traditional antibiotics has become a critical status. Thus, finding new therapeutic strategies to manage diabetic wounds is urgently needed. Nanotechnology has emerged as an efficient approach for this purpose. This review aimed to summarize recent advances using nanotechnology for the treatment of diabetic wounds.

An important issue in the prognosis of tuberculosis (TB) is a short period between correct diagnosis and start the suitable antibiotic therapy. So, a rapid and valid method for detection of Mycobacterium tuberculosis (M. tb) complex is considered as a necessity. Herein, a rapid, low-cost, and PCR-free DNA biosensor was developed based on multi-walled carbon nanotubes (MWCNTs), polypyrrole (PPy), and hydroxyapatite nanoparticles (HAPNPs) for highly sensitive and specific recognition of M.tb. The biosensor consisted of M.tb ssDNA probe covalently attached to the HANPs/PPy/MWCNTs/GCE surface that hybridized to a complementary target sequence to form a duplex DNA. The M.tb target recognition was based on the oxidation signal of the electroactive Methylene Blue (MB) on the surface of the modified GCE using differential pulse voltammetry (DPV) method. It is worth to mention that for the first time Plackett-Burman (PB) screening design and response surface method (RSM) based on central composite design (CCD) was applied as a powerful and an efficient approach to find optimal conditions for maximum M.tb biosensor performance leading to simplicity and rapidity of operation. The proposed DNA biosensor exhibits a wide detection range from 0.25 to 200.0 nM with a low detection limit of 0.141 nM. The performance of designed biosensor for clinical diagnosis and practical applications was revealed through hybridization between DNA probe-modified GCE and extracted DNA from sputum clinical samples.

Background: CATSPER 1 (Cation Channel Sperm Associated 1) and CATSPER2 channels have an important role in sperm motility. In this study, the effects of hyperthyroidism on Catsper1 and 2 genes of seminiferous tubules in mice testes were investigated. Objective: The present study was conducted to investigate the effect of hyperthyroidism on the expression of CATSPER1 and CATSPER2 genes in the seminiferous tubules of mice. Materials and Methods: This study was conducted on 20 BALB/C male mice divided into two groups - experimental and control. The experimental group was administered with 500 mg/l levothyroxine (L-thyroxine) liquid solution for two months for inducing hyperthyroidism, which was confirmed by radioimmunoassay. On the other hand, the control group was kept in animal houses under a normal condition. The implementation of real-time polymerase chain reaction and immunohistochemical studies was accomplished after the removal of the testes of the mice under anesthesia induced by chloroform. Results: Results showed that there was no significant difference in CATSPER1 (p = 0.45) and CATSPER2 (p = 0.34) gene expression between groups. At the same time, the color intensity showed no significant enhancement in the hyperthyroidism group (CATSPER1 p = 0.17 and CATSPER2 p = 0.22) as compared to the control group. Conclusion: Considering the key role of CATSPER in the molecular structure of the sperm, our findings showed that the hyperactivity of the thyroid gland has no significant effects on the function of these components. Therefore, it might be concluded that hyperthyroidism has no considerable effects on the seminiferous tubules. Key words: CATSPER1, CATSPER2, Hyperthyroidism, Mice, Sperm.

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.

Background Sublingual immunotherapy (SLIT) was introduced to deliver allergens in an effective and non-invasive route, which can be considered as an alternative for allergen-specific subcutaneous immunotherapy (SCIT). On the other hand, the use of gold nanoparticles (AuNPs) in allergen delivery has beneficial effects on sublingual immunotherapy. In addition, the molecular targeting agents like aptamers (Apt), have been widely applied for targeted drug delivery. Therefore, the current study aimed to evaluate the effects of dendritic cells (DCs)-specific Aptamer-modified AuNPs coated with ovalbumin (OVA) on the improvement of the SLIT outcome in the mouse model of allergy. Material and methods AuNPs with approximately 15 nm diameter were prepared by citrate reduction of HAuCl4. Afterward, Apt-modified AuNP complex was prepared and OVA was then loaded onto this complex. Following sensitization of Balb/c mice to OVA, SLIT was performed with Apt-AuNPs containing 5 µg OVA twice a week for a 2-month period. Allergen-specific IgE in serum, as well as cytokines secretion of spleen cells, were analyzed using ELISA. Also, nasopharyngeal lavage Fluid (NALF) was collected for total and eosinophil counts. Moreover, the lungs were removed for histopathological examination. Results SLIT with Apt-modified AuNPs complex containing 5 μg OVA, decreased the IgE levels compared to the other groups. Also, IL-4 production has significantly decreased in spleen cells, while TGF-β and IFN-γ have significantly increased. The assessment of NALF in the group treated by this complex showed a decrease in total cell as well as in eosinophil count. Also, the examination of lung tissues revealed that, in the group treated by this complex, inflammation and perivascular infiltration were lesser than the other groups, which were observed in only one vessel of tissue. Conclusion It was shown that, Sublingual immunotherapy with DC specific Apt-modified AuNPs containing 5 μg OVA can improve the Th1 and Treg immunomodulatory responses.

Prokaryotic systems have been considered the most affordable and simplest hosts which are being employed to express recombinant proteins such as allergens; nevertheless, without appropriate signal peptide (SP), these systems cannot be used for secretory proteins. Recently, a lot of effort has been put into assessing the potential of gram-positive strains such as lactic acid bacteria for new applications in the production of heterologous proteins. Ama r 2 is a respiratory allergen from Amaranthus retroflexus, whose recombinant production in the probiotic host could be introduced as a specific and effective way to rapid diagnosis and immunotherapy of this allergy. Consequently, the production of this recombinant protein using the prokaryotic system, requires a suitable SP to protect disulfide bonds and to prevent misfolding. This study was designed to predict the best SPs for the expression of Ama r 2 protein in Lactococcus lactis as the host. In this study, 42 signal sequences were selected from SP databases and the most important features of them were evaluated. First, n, h and c regions of the SPs and their probabilities were investigated by signalP software version 4.1. Then, their physicochemical properties were evaluated by Portparam and SOLpro. Moreover, the secretion sorting and sub-cellular localization sites were evaluated by PRED-TAT and ProtcompB software programs. The results revealed that yjgB, entC2 (Entrotoxine type C-2), ent B (Entrotoxine type), blaZ (Beta lactamase), dex (number 21), blm (Beta lactamase 2), dex (Dextranase; number 20) and number 26 were introduced theatrically as the best SPs to express Ama r 2 in Lactococcus lactis.

Objectives: Dysfunction of the thyroid gland has a negative effect on the male reproductive system. Studies also show that extracellular matrix (ECM) components play an essential role in testicular development and function. In hypothyroidism, there is a significant disruption in the ECM structure of mammalian tissues. In addition, notable changes have been reported in the germ cell population under a hypothyroid state. This study aimed to investigate the relationship between ECM proteins and apoptosis of testicular germ cells due to hypothyroidism. Materials and Methods: In the present experimental study, 20 male Balb/C mice were divided into control and hypothyroid groups. The hypothyroid group received 0.05% 6-n-propyl-2-thiouracil (PTU) through drinking water for 35 days. Finally, real-time polymerase chain reaction, immunohistochemistry, periodic acid-Schiff (PAS) staining, terminal transferase-mediated dUTP nick-end labelling (TUNEL) assay, and biochemical measurements were performed after hypothyroidism confirmation. Results: Laminin α5 and collagen IV mRNA levels were upregulated in the hypothyroid group compared to the controls (P

The incidence of grape (Vitis vinifera) allergy in the northeast of Iran is second to melon allergy. Type IV chitinase is one of the major grape allergens. The current study investigates the level of type IV chitinase in four grape variants for the first time in Khorasan Razavi Province using a highly sensitive sandwich enzyme-linked immunosorbent assay (ELISA). This assay was developed using a polyclonal antibody as a capture antibody and monoclonal antibody as a secondary one. Finally, the amount of type IV chitinase was measured by the validated ELISA test. The sensitivity of the developed sandwich ELISA is 16 ± 0.05 ng/ml, and its mean coefficients of intraday and interday variations are

Background The first step of designing any genome-based molecular diagnostic test is to find a specific target sequence. The modified genome comparison method is one of the easiest and most comprehensive ways to achieve this goal. In this study, we aimed to explain this method with the example of Mycobacterium tuberculosis complex and investigate its efficacy in a diagnostic test. Methods A specific target was identified using modified genome comparison method and an in-house PCR test was designed. To determine the analytical sensitivity and specificity, 10 standard specimens were used. Also, 230 specimens were used to determine the clinical sensitivity and specificity. Results The identity and query cover of our new diagnostic target (5KST) were ≥ 90% with M. tuberculosis complex. The 5KST-PCR sensitivity was 100% for smear-positive, culture-positive and 85.7% for smear-negative, culture-positive specimens. All of 100 smear-negative, culture-negative specimens were negative in 5KST-PCR (100% clinical specificity). Analytical sensitivity of 5KST-PCR was approximately 1 copy of genomic DNA per microliter. Conclusions Modified genome comparison method is a confident way to find specific targets for use in diagnostic tests. Accordingly, the 5KST-PCR designed in this study has high sensitivity and specificity and can be replaced for conventional TB PCR tests. Electronic supplementary material The online version of this article (10.1186/s12879-018-3417-x) contains supplementary material, which is available to authorized users.

Protein or peptide-based antigens are the most promising forms to generate custom protective immune responses for clinical applications. Over the last decades, poly(lactic-co-glycolic acid) (PLGA) as a biodegradable polymer has gained more attention for delivery of protein and peptide. Besides many appropriate characteristics, to improve its properties to overcome some obstacles such as release profile and it is important instability of antigen during both encapsulation and storage. Therefore, optimized procedures conditions require to be used to maintain the integrity of protein structure under several stress factors in formulation process. In this review article, the properties of PLGA particles, their preparation techniques and strategies for improvement of protein stability during encapsulation into PLGA, release from particle and storage as well as stabilization approaches were summarized. © 2018 Wiley Periodicals, Inc. J. Biomed. Mater. Res. Part A: 106A: 2540-2551, 2018.