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.

Production and Evaluation of Ag85B:HspX:hFcγ1 Immunogenicity as an Fc Fusion Recombinant Multi-Stage Vaccine Candidate Against Mycobacterium tuberculosis.

An urgent need is to introduce an effective vaccine against Mycobacterium tuberculosis (M.tb) infection. In the present study, a multi-stage M.tb immunodominant Fcγ1 fusion protein (Ag85B:HspX:hFcγ1) was designed and produced, and the immunogenicity of purified protein was evaluated. This recombinant fusion protein was produced in the Pichia pastoris expression system. The HiTrap-rPA column affinity chromatography purified and confirmed the fusion protein using ELISA and Western blotting methods. The co-localisation assay was used to confirm its proper folding and function. IFN-γ, IL-12, IL-4, and TGF-ß expression in C57BL/6 mice then evaluated the immunogenicity of the construct in the presence and absence of BCG. After expression optimisation, medium-scale production and the Western blotting test confirmed suitable production of Ag85B:HspX:hFcγ1. The co-localisation results on antigen-presenting cells (APCs) showed that Ag85B:HspX:hFcγ1 properly folded and bound to hFcγRI. This strong co-localisation with its receptor can confirm inducing proper Th1 responses. The in vivo immunisation assay showed no difference in the expression of IL-4 but a substantial increase in the expression of IFN-γ and IL-12 (P ≤ 0.02) and a moderate increase in TGF-ß (P = 0.05). In vivo immunisation assay revealed that Th1-inducing pathways have been stimulated, as IFN-γ and IL-12 strongly, and TGF-ß expression moderately increased in Ag85B:HspX:hFcγ1 group and Ag85B:HspX:hFcγ1+BCG. Furthermore, the production of IFN-γ from splenocytes in the Ag85B:HspX:hFcγ1 group was enormously higher than in other treatments. Therefore, this Fc fusion protein can make a selective multi-stage delivery system for inducing appropriate Th1 responses and is used as a subunit vaccine alone or in combination with others.

Evaluation of Kynu, Defb2, Camp, and Penk Expression Levels as Psoriasis Marker in the Imiquimod-Induced Psoriasis Model.

Background: Psoriasis is a noncontagious auto-inflammatory chronic skin disease. So far, some of the inflammatory genes were upregulated in mouse model of psoriasis. This study examined changes in skin mRNA expression of L-kynureninase (Kynu), cathelicidin antimicrobial peptide (Camp), beta-defensin 2 (Defb2), and proenkephalin (Penk) in a mouse model of imiquimod-induced psoriasis. Materials and Methods: Tree groups of C57BL/6 female mice were allocated. The imiquimod (IMQ) cream was administered to the mice dorsal skin of the two groups to induce psoriatic inflammation. In the treatment group, IMQ was administered 10 min after hydrogel-containing M7 anti-IL-17A aptamer treatment. Vaseline (Vas) was administered to the negative control group. The psoriatic skin lesions were evaluated based on the psoriasis area severity index (PASI) score, histopathology, and mRNA expression levels of Kynu, Camp, Defb2, and Penk using real-time PCR. In order to assess the systemic response, the spleen and lymph node indexes were also evaluated. Results: The PASI and epidermal thickness scores were 6.01 and 1.96, respectively, in the IMQ group, and they significantly decreased after aptamer administration to 1.15 and 0.90, respectively (P < 0.05). Spleen and lymph node indexes showed an increase in the IMQ group, followed by a slight decrease after aptamer treatment (P > 0.05). Additionally, the mRNA expression levels of Kynu, Defb2, Camp, and Penk genes in the IMQ-treated region showed a significant 2.70, 4.56, 3.29, and 2.61-fold increase relative to the Vas mice, respectively (P < 0.05). The aptamer-treated region exhibited a significant decrease in these gene expression levels (P < 0.05). A positive correlation was found between Kynu, Penk, and Camp expression levels and erythema, as well as Camp expression with PASI, scaling, and thickness (P < 0.05). Conclusion: According to our results, it seems that Kynu, Camp, and Penk can be considered appropriate markers for the evaluation of psoriasis in IMQ-induced psoriasis. Also, the anti-IL-17 aptamer downregulated these important genes in this 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.

Evaluation of sperm chromatin/DNA integrity, morphology, and Catsper expression on diabetic C57BL/6 mice.

BACKGROUND: Diabetes is associated with reproductive impairment on the male reproductive system and causes complications such as decreased libido, fertility, spermatogenesis, sperm motility, and morphology. High levels of blood sugar may affect sperm quality and reduce the potential for male fertility. Increased levels of sperm DNA damage is often associated with reduced count and motility or abnormal morphology. METHODS: This experimental study was conducted in Mashhad University of Medical Sciences. In this work, 40 mice (C57BL/6) were divided randomly into 4 groups: 1) Control, 2) Diabetic, 3) Diabetic + Insulin, and 4) Sham. After 35 days, the right epididymis of all specimens was used for Real-Time PCR and left epididymis for evaluation of sperm parameters using Aniline blue, Toluidine blue, Papanicolaou, and immunohistochemical study. Also, testes were applied for immunohistochemical, TUNEL studies, and biochemical assay. RESULTS: Results of this study showed that chromatin integrity, morphology, cation channels of sperm (Catsper) expression, and biochemical factors level were significantly changed in diabetic mice in comparison to other groups (P<0.05) and treatment with insulin improved these parameters. CONCLUSION: Our findings showed that the sperm parameters such as DNA integrity, morphology, and Catsper expression change in diabetic mice.

The overview and perspectives of biosensors and Mycobacterium tuberculosis: A systematic review.

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.

Preventive cancer vaccination with P5 HER-2/neo-derived peptide-pulsed peripheral blood mononuclear cells in a mouse model of breast cancer.

This study compared the prophylactic effects from vaccines based on dendritic cells (DCs) and peripheral blood mononuclear cells (PBMCs) by pulsing the cells in-vitro with p5 peptide. The different test groups of mice were injected with free peptide or with peptide pulsed with DCs or PBMCs. Two weeks after the last booster dose, immunological tests were performed on splenocyte suspensions from three mice in each group and the remaining mice (5/each group) were evaluated for tumor growth and survival time. The levels of IFN-γ, granzyme B, and IL-10 were detected in T cells. Additionally, IFN-γ and perforin as well as mRNA levels of some genes associated with immune responses were assessed after challenging the splenocytes with TUBO cells. A significant increase was observed in frequency of CD4+ IFN-γ+, CD8+ IFN-γ+, and CD8+ granzyme B+ T cells, and the perforin of supernatants from mice in the DC and PBMC treatment groups. Significant expression levels of Fas ligand (FasL) and forkhead box P3 (Foxp3) were observed in the DC and PBMC groups. These responses led to smaller tumors and longer survival time in our mouse model of breast cancer. The efficacy of the PBMC-based vaccine in improving the protective immune response makes it a simpler and less expensive candidate vaccine compared with DC-based vaccines.

Nanotechnology-driven advances in the treatment of diabetic wounds.

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.

Comparative assessment of proliferation and immunomodulatory potential of Hypericum perforatum plant and callus extracts on mesenchymal stem cells derived adipose tissue from multiple sclerosis patients.

BACKGROUND: Mesenchymal stem cells-derived adipose tissue (AT-MSCs) are recognized for the treatment of inflammatory diseases including multiple sclerosis (MS). Hypericum perforatum (HP) is an anti-inflammatory pharmaceutical plant with bioactive compounds. Plant tissue culture is a technique to improve desired pharmacological potential. The aim of this study was to compare the anti-inflammatory and proliferative effects of callus with field-growing plant extracts of HP on AT-MSCs derived from MS patients. MATERIALS AND METHODS: AT-MSCs were isolated and characterized. HP callus was prepared and exposure to light spectrum (blue, red, blue-red, and control). Total phenols, flavonoids, and hypericin of HP callus and plant extracts were measured. The effects of HP extracts concentrations on proliferation were evaluated by MTT assay. Co-culture of AT-MSCs: PBMCs were challenged by HP plant and callus extracts, and Tregs percentage was assessed by flow cytometry. RESULTS: Identification of MSCs was performed. Data showed that blue light could stimulate total phenols, flavonoids, and hypericin. MTT test demonstrated that plant extract in concentrations (0.03, 1.2, 2.5 and 10 µg/ml) and HP callus extract in 10 µg/ml significantly increased. Both HP extracts lead to an increase in Tregs percentage in all concentrations. In particular, a comparison between HP plant and callus extracts revealed that Tregs enhanced 3-fold more than control groups in the concentration of 10 µg/ml callus. CONCLUSIONS: High concentrations of HP extracts showed effectiveness on AT-MSCs proliferation and immunomodulatory properties with a certain consequence in callus extract. HP extracts may be considered as supplementary treatments for the patients who receiving MSCs transplantation.

Analysis of different signal peptides for the secretory production of Ama r 2 in gram-positive systems (Lactococcus lactis).

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.

Diabetes up-regulated collagen IV and laminin α5 genes in mRNA and protein levels in seminiferous tubules of C57BL/6 adult mice.

Diabetes is a disease associated with impairment of the male reproductive system that causes complications such as decreased testosterone, the diameter of the seminiferous tubule, libido, and fertility. Extracellular matrix (ECM) molecules are involved in testicular development and spermatogenesis. Laminin and collagen are key proteins in seminiferous tubule basement membrane and play an important role in spermatogenesis. The present study was conducted to investigate the effect of diabetes on collagen IV and laminin α5 changes in mice testis. In this experimental study, 40 mice (C57BL/6) were divided randomly into 4 groups: 1) Control group: without intervention, 2) Diabetic group: treated mice with 50 mg/kg streptozotocin (STZ), 3) Diabetic + Insulin group: treated mice with STZ and insulin, and 4) Sham group: received citrate buffer. After 35 days, the left testes of all specimens were used for Real-Time PCR while their right testes were applied for immunohistochemical study and Periodic acid-Schiff (PAS) staining. This study showed that gene expression and immunoreactivity of laminin α5 and collagen IV were significantly increased in diabetic mice compared to other groups (P<0.05). Also, PAS staining showed the thickness of seminiferous tubule basement membrane in the Diabetic group compared to other group increased significantly (p<0.05). In Diabetic + Insulin compared to Diabetic group, gene expression, the intensity of immunoreactivity and thickness of seminiferous tubule basement membrane decreased significantly (P<0.05). Our findings indicated that diabetes causes up-regulation of collagen IV and laminin α5 in mRNA and protein levels in the seminiferous tubule basement membrane and may cause disorder in spermatogenesis in mice.

The immunomodulatory role of probiotics in allergy therapy.

The increased incidence of allergic disorders may be the result of a relative fall in microbial induction in the intestinal immune system during infancy and early childhood. Probiotics have recently been proposed as viable microorganisms for the prevention and treatment of specific allergic diseases. Different mechanisms have been considered for this probiotic property, such as generation of cytokines from activated pro-T-helper type 1 after bacterial contact. However, the effects of its immunomodulatory potential require validation for clinical applications. This review will focus on the currently available data on the benefits of probiotics in allergy disease.

Antagonistic activity of recombinant Lactococcus lactis NZ1330 on the adhesion properties of Escherichia coli causing urinary tract infection.

Death from infectious diseases has caused concerns about increases in the resistance of pathogens, impelling researchers to create novel therapeutic solutions. The management of intestinal tract problems has been the advance use of probiotics in medicine. The aim of this study was evaluate the physicochemical cell surface and adhesion properties of recombinant Lacotococcus lactis NZ1330 containing Ama r 2 gene, followed by the assessment of the antagonistic activity of this strain against the Escherichia coli causing urinary tract infection (UTI) in humans. For this purpose, cloning and expression of Ama r 2 gene were done. Afterwards, acid and bile resistance, which are the primary characteristics of any probiotic, were evaluated. The r-L. lactis NZ1330 was examined for the physicochemical properties of cell surfaces and the adhesion properties against Escherichia coli. Furthermore, the potential of the recombinant strain to adhere to adenocarcinoma intestinal cell line, Caco-2 cells, as well as the antagonistic properties of r-L. lactis NZ1330 against E. coli was investigated. r-L. lactis NZ1330 was capable of surviving at low pH and different concentrations of bile salts. 40.1% hydrophobicity, 36.5% auto-aggregation and 14.4% co-aggregation were observed for this strain. The adhesion level of r-L. lactis NZ1330 was 5.7% which was also confirmed by scanning electron microscopy (SEM). r-L. lactis NZ1330 was able to compete, inhibit and displace the adhesion of Escherichia coli to Caco-2 cells. r-L. lactis NZ1330 was considered to be a reliable probiotic alternative by showing these desirable properties. Results revealed that Ama r 2 gene expression had no effect on the positive probiotic properties of L. lactis NZ1330, proving this strain could be a suitable probiotic host for the expression of this allergen.

Mutation analysis of genes related to methylmalonic acidemia: identification of eight novel mutations.

Methylmalonic acidemia (MMA), an inherited metabolic disease, results from genetic defects in methylmalonyl-CoA mutase or any of the proteins involved in adenosylcobalamin synthesis. This enzyme is classified into several complementation groups and genotypic classes. In this work we explain the biochemical, structural and genetic analysis of 25 MMA patients, from Iran. The diagnosis was established by the measurement of propionylcarnitine in blood using tandem mass spectrometry and confirmed using a gas chromatography-flame ionization detector. Using clinical, biochemical, structural and molecular analyses we identified 15 mut MMA, three cblA, one cblB, and four cblC-deficient patients. Among mutations identified in the MUT gene (MUT) only one, the c.1874A>C (p.D625A) variant, is likely a mut- mutation. The remaining mutations are probably mut0. Here, we present the first molecular analysis of MMA in Iranian patients and have identified eight novel mutations. Four novel mutations (p.D625A, p.R326G, p.V157F, p.F379L) were seen exclusively in patients from northern Iran. One novel splice site mutation (c.2125-3C>G) in MUT and two novel mutation (p.N225M and p.A99P) in the MMAA gene were associated with patients from eastern Iran. The rs184829210 SNP was recognized only in patients with the novel c.958G>A (p.A320T) mutation. This study confirms pathogenesis of deficient enzyme activity in MUT, MMAA, MMAB, and MMACHC as previous observations. These results could act as a basis for the performance of pharmacological therapies for increasing the activity of proteins derived from these mutations.

Preparation allergenic pollen extracts; the points should be considered to make high-quality products.

Atopic diseases have an increasing trend worldwide during the last two decades. Determining the main cause of allergic diseases, allergens, is the first step in managing and improving the issue, usually is done by Skin Prick tests (SPTs). Having allergenic extract in high quality is desired to perform a reliable SPT. Several parameters of extracts are considered including composition, stability, potency, preservation conditions, and unit definition. In this review, these factors have been explained pointing to factors might have profitable points or harmful drawback in the quality of allergen extracts.

Type IV chitinase quantification in four different grape cultivars (Vitis vinifera) in northeast of Iran by an indirect sandwich enzyme-linked immunosorbent assay.

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 <5% and <15%, respectively. The recovery of the designed ELISA is 64 ± 0.9 %. The assessments showed that the highest level of type IV chitinase was 39.7 ± 2.3 µg/g in Peykani grape, whereas in the Sultana cultivar, it was 1.76 ± 0.1 µg/g. According to the data, the level of type IV chitinase is variable in different cultivars, and hence, it will be helpful for clinicians to recommend a less allergenic variety to the patient.

Cloning, Expression, and Refolding of PPE17 Protein of Mycobacterium Tuberculosis as a Promising Vaccine Candidate.

BACKGROUND: Tuberculosis as a global health problem requires special attention in the contexts of prevention and control. Subunit vaccines are promising strategies to protect burdens of tuberculosis via increasing the BCG protection. The present study aimed to design a vaccine study by means of high-throughput expression and correct refolding of recombinant protein PPE17. METHODS: We aimed to clone, express, and refold PPE17 protein of Mycobacterium tuberculosis in bacterial systems as a promising vaccine candidate. The PPE17 (Rv1168c) gene was PCR amplified and inserted into pET-21b(+) vector, cloned in E. coli TOP10, and finally expressed in E. coli BL21(DE3). RESULTS: The expressed recombinant protein was entirely found in insoluble form (inclusion bodies). The insoluble protein was denatured in 6M guanidine-HCl and refolded by descending denaturant concentration dialysis. Moreover, the recombinant protein was purified by Ni-NTA column chromatography. The changing temperature had no effects on solubilizing protein and the maximum expression was achieved at 0.5 mM concentration of isopropyl-D-thiogalactopyranoside (IPTG) induction. CONCLUSION: Bacterial expression system is a timesaving tool and refolding by descending denaturant concentration dialysis is a rapid and reliable method.

The Most Common Allergenic Tree Pollen Grains in the Middle East: A Narrative Review.

Allergy is becoming a major disease burden globally. Pollens are considered as the main component of aeroallergens that lead to rhinitis and asthma. Due to the lack of a comprehensive investigation on most allergic pollens of trees in the Middle East, the present study aimed to conduct a comprehensive literature review on this topic. The main goal of the study was to provide a checklist for allergists and patients to easily identify the commonest allergic pollens in their locality. The present review provides a broad range of information on the types and geographic locations of the most common allergic pollens of trees in each studied country. In general, among the 23 studied countries, palm and mesquite trees were the common producers of pollen allergen in the Persian Gulf region. Olive tree is common in Turkey, Palestine, and Israel, whereas sycamore tree is the common allergen pollen in Iran. Considering the uneven geographical distribution of these trees in the world, allergists are unable to accurately select the appropriate extracts for the skin prick test based on the information from the neighboring countries. This scenario becomes more complicated if one adds the imported ornamental trees in the picture.

Modified genome comparison method: a new approach for identification of specific targets in molecular diagnostic tests using Mycobacterium tuberculosis complex as an example.

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.