Peptidylarginine deiminase (PAD): A promising target for chronic diseases treatment.

In 1958, the presence of citrulline in the structure of the proteins was discovered for the first time. Several years later they found that Arginine converted to citrulline during a post-translational modification process by PAD enzyme. Each PAD is expressed in a certain tissue developing a series of diseases such as inflammation and cancers. Among these, PAD2 and PAD4 play a role in the development of rheumatoid arthritis (RA) by producing citrullinated autoantigens and increasing the production of inflammatory cytokines. PAD4 is also associated with the formation of NET structures and thrombosis. In the crystallographic structure, PAD has several calcium binding sites, and the active site of the enzyme consists of different amino acids. Various PAD inhibitors have been developed divided into pan-PAD and selective PAD inhibitors. F-amidine, Cl-amidine, and BB-Cl-amidine are some of pan-PAD inhibitors. AFM-30a and JBI589 are selective for PAD2 and PAD4, respectively. There is a need to evaluate the effectiveness of existing inhibitors more accurately in the coming years, as well as design and production of novel inhibitors targeting highly specific isoforms.

Designing of a new multi-epitope vaccine against Leishmania major using Leish-F1 epitopes: An In-silico study.

Cutaneous leishmaniasis (CL) is the most common form of the disease which can cause malignant lesions on the skin. Vaccination for the prevention and treatment of leishmaniasis can be the most effective way to combat this disease. In this study, we designed a novel multi-epitope vaccine against Leishmania major (L. major) using immunoinformatics tools to assess its efficacy in silico. Sequences of Leish-F1 protein (TSA, Leif, and LMSTI1) of L. major were taken from GenBank. The helper T (Th) and cytotoxic T (Tc) epitopes of the protein were predicted. The final multi-epitope consisted of 18 CTL epitopes joined by AAY linker. There were also nine HTL epitopes in the structure of the vaccine construct, joined by GPGPG linker. The profilin adjuvant (the toll-like receptor 11 agonist) was also added into the construct by AAY Linker. There were 613 residues in the structure of the vaccine construct. The multi-epitope vaccine candidate was stable and non-allergic. The data obtained from the binding of final multi-epitope vaccine-TLR11 residues (band lengths and weighted scores) unveiled the ligand and the receptor high score of binding affinity. Moreover, in silico assessment of the vaccine construct cloning achieved its suitable expression in E. coli host. Based on these results, the current multi-epitope vaccine prevents L. major infection in silico, while further confirmatory assessments are required.

Design of a novel multi-epitope vaccine candidate against Chlamydia trachomatis using structural and nonstructural proteins: an immunoinformatics study.

Chlamydia trachomatis (C. trachomatis) is an obligate intracellular bacterium which causes eye and sexually transmitted infections. During pregnancy, the bacterium is associated with preterm complications, low weight of neonates, fetal demise and endometritis leading to infertility. The aim of our study was design of a multi-epitope vaccine (MEV) candidate against C. trachomatis. After protein sequence adoption from the NCBI, potential epitopes toxicity, antigenicity, allergenicity, MHC-I and MHC-II binding, cytotoxic T lymphocytes (CTLs), Helper T lymphocytes (HTLs) and interferon-γ (IFN-γ)- induction were predicted. The adopted epitopes were fused together using appropriate linkers. In the next step, the MEV structural mapping and characterization, three-dimensional (3D) structure homology modeling and refinement were also performed. The MEV candidate interaction with the toll-like receptor 4 (TLR4) was also docked. The immune responses simulation was assessed using the C-IMMSIM server. Molecular dynamic (MD) simulation verified the structural stability of the TLR4-MEV complex. The Molecular Mechanics Poisson-Boltzmann Surface Area (MMPBSA) approach demonstrated the MEV high affinity of binding to the TLR4, MHC-I and MHC-II. The MEV construct was also stable and water soluble and had enough antigenicity and lacked allergenicity with stimulation of T cells and B cells and INF-γ release. The immune simulation confirmed acceptable responses of both the humoral and cellular arms. It is proposed that in vitro and in vivo studies are needed to evaluate the findings of this study.Communicated by Ramaswamy H. Sarma.

Advanced simulations and screening to repurposing a 3C protease inhibitor against the rupintrivir-resistant human norovirus-induced gastroenteritis.

Human norovirus (HuNoV) causes acute viral gastroenteritis in all age groups, and dehydration and severe diarrhea in the elderly. The World Health Organization reports ∼1.45 million deaths from acute gastroenteritis annually in the world. Rupintrivir, an inhibitory medicine against the human rhinovirus C3 protease, has been reported to inhibit HuNoV 3C protease. However, several HuNoV 3C protease mutations have been revealed to reduce the susceptibility of HuNoV to rupintrivir. The structural details behind rupintrivir-resistance of these single-point mutations (A105V and I109V) are not still clear. Hence, in this study, a combination of computational techniques were used to determine the rupintrivir-resistance mechanism and to propose an inhibitor against wild-type and mutant HuNoV 3C protease through structure-based virtual screening. Dynamic structural results indicated the unstable binding of rupintrivir at the cleft binding site of the wild-type and mutant 3C proteases, leading to its detachment. Our findings presented that the domain II of the HuNoV 3C protease had a critical role in binding of inhibitory molecules. Binding energy computations, steered molecular dynamics and umbrella sampling simulations confirmed that amentoflavone, the novel suggested inhibitor, strongly binds to the cleft site of all protease models and has a good structural stability in the complex system along the molecular dynamic simulations. Our in silico study proposed the selected compound as a potential inhibitor against the HuNoV 3C protease. However, additional experimental and clinical studies are required to corroborate the therapeutic efficacy of the compound.

Design of a novel multi-epitope vaccine candidate against hepatitis C virus using structural and nonstructural proteins: An immunoinformatics approach.

Hepatitis C virus (HCV) infects the liver and causes chronic infection. Several mutations in the viral genome have been associated with drug resistance development. Currently, there is no approved vaccine against the HCV. The employment of computational biology is the primary and crucial step for vaccine design or antiviral therapy which can substantially reduce the duration and cost of studies. Therefore, in this study, we designed a multi-epitope vaccine using various immunoinformatics tools to elicit the efficient human immune responses against the HCV. Initially, various potential (antigenic, immunogenic, non-toxic and non-allergenic) epitope segments were extracted from viral structural and non-structural protein sequences using multiple screening methods. The selected epitopes were linked to each other properly. Then, toll-like receptors (TLRs) 3 and 4 agonists (50S ribosomal protein L7/L12 and human ß-defensin 2, respectively) were added to the N-terminus of the final vaccine sequence to increase its immunogenicity. The 3D structure of the vaccine was modeled. Molecular dynamics simulations studies verified the high stability of final free vaccines and in complex with TLR3 and TLR4. These constructs were also antigenic, non-allergenic, nontoxic and immunogenic. Although the designed vaccine traits were promising as a potential candidate against the HCV infection, experimental studies and clinical trials are required to verify the protective traits and safety of the designed vaccine.

The Unspecific Primers of Nuclear Factor-kappa B (NF-κB) Signaling Mediators.

The article is a letter to editor and does not have the abstract.

Therapeutic Effects of Mesenchymal Stem Cells Expressing Erythropoietin on Cancer-Related Anemia in Mice Model.

BACKGROUND: Cancer-related anemia (CRA) negatively influences cancer patients' survival, disease progression, treatment efficacy, and quality of life (QOL). Current treatments such as iron therapy, red cell transfusion, and erythropoietin-stimulating agents (ESAs) may cause severe adverse effects. Therefore, the development of long-lasting and curative therapies is urgently required. OBJECTIVE: In this study, a cell and gene therapy strategy was developed for in vivo delivery of EPO cDNA by way of genetic engineering of human Wharton's jelly mesenchymal stem cells (hWJMSCs) to produce and secrete human EPO protein for extended periods after transplantation into the mice model of CRA. METHODS: To evaluate CRA's treatment in cancer-free and cancerous conditions, first, a recombinant breast cancer cell line 4T1 which expressed herpes simplex virus type 1 thymidine kinase (HSV1-TK) by a lentiviral vector encoding HSV1-TK was developed and injected into mice. After three weeks, all mice developed metastatic breast cancer associated with acute anemia. Then, ganciclovir (GCV) was administered for ten days in half of the mice to clear cancer cells. Meanwhile, another lentiviral vector encoding EPO to transduce hWJMSCs was developed. Following implantation of rhWJMSCs-EPO in the second group of mice, peripheral blood samples were collected once a week for ten weeks from both groups. RESULTS: Analysis of peripheral blood samples showed that plasma EPO, hemoglobin (Hb), and hematocrit (Hct) concentrations significantly increased and remained at therapeutic for >10 weeks in both treatment groups. CONCLUSION: Data indicated that rhWJMSCs-EPO increased the circulating level of EPO, Hb, and Hct in both mouse subject groups and improved the anemia of cancer in both cancer-free and cancerous mice.

Overexpression Effects of miR-424 and BMP2 on the Osteogenesis of Wharton's Jelly-Derived Stem Cells.

Recently, the translational application of noncoding RNAs is accelerated dramatically. In this regard, discovering therapeutic roles of microRNAs by developing synthetic RNA and vector-based RNA is attracting attention. Here, we studied the effect of BMP2 and miR-424 on the osteogenesis of Wharton's jelly-derived stem cells (WJSCs). For this purpose, human BMP2 and miR-424 DNA codes were cloned in the third generation of lentiviral vectors and then used for HEK-293T cell transfection. Lentiviral plasmids contained miR424, BMP-2, miR424-BMP2, green fluorescent protein (GFP) genes, and helper vectors. The recombinant lentiviral particles transduced the WJSCs, and the osteogenesis was evaluated by real-time PCR, Western blot, Alizarin Red staining, and alkaline phosphatase enzyme activity. According to the results, there was a significant increase in the expression of the BMP2 gene and secretion of Osteocalcin protein in the group of miR424-BMP2. Moreover, the amount of dye deposition in Alizarin Red staining and alkaline phosphatase activity was significantly higher in the mentioned group (p < 0.05). Thus, the current study results clarify the efficacy of gene therapy by miR424-BMP2 vectors for bone tissue engineering. These data could help guide the development of gene therapy-based protocols for bone tissue engineering.

Exploring membrane proteins of Leishmania major to design a new multi-epitope vaccine using immunoinformatics approach.

Leishmaniasis is one of the major global endemic diseases. Among all the different forms of the disease, cutaneous Leishmaniasis has the highest prevalence worldwide. Treatment with current drugs has not had a significant effect on the improvement of the disease. An attempt to replace an appropriate vaccine that can stimulate host cellular immunity and induce the response of Major histocompatibility complex I (MHCI) and Major histocompatibility complex II (MHCII) against Leishmania is essential. Vaccine production remains a challenge despite the use of different antigens for vaccination against Leishmania major. Hence, we were used the immunoinformatics approach to design a new multi-epitope vaccine against L. major using immunogenic outer membrane proteins. Helper T-lymphocyte (HTL) and Cytotoxic T lymphocyte (CTL) epitopes were predicted and for final confirmation of the selected epitopes, docking analysis, and molecular dynamics simulation was performed. Then, GDGDG linker and profilin adjuvant were added to enhance the immunity of vaccines. The designed vaccine was evaluated in terms of molecular weight, PI, immunogenicity, and allergenicity. Moreover, the secondary and three-dimensional structure of the final construct was identified. In silico cloning approach was carried out to improve expression of the vaccine construct. Finally, molecular docking, followed by molecular dynamic was performed to determine the interaction between multi-epitope vaccine and TLR11. We hope that the designed vaccine can be a good candidate for the development of cutaneous leishmaniasis vaccine. but its effectiveness should be assessed in vivo.

A lentiviral vaccine expressing KMP11-HASPB fusion protein increases immune response to Leishmania major in BALB/C.

Hydrophilic acylated surface protein B (HASPB) is an immunogenic Leishmania-specific protein that antibodies are produced against it in the sera of Leishmania-infected individuals. Kinetoplastid membrane protein 11 (KMP11) is another Leishmania antigen and considered as the suitable candidate for vaccine development Leishmaniasis. It is a highly conserved surface protein expressed in both promastigotes and amastigotes. In this study, KMP11 and HASPB coding sequences were cloned into a pCDH-cGFP lentiviral vector as a fusion protein to be used as a DNA vaccine against L. major. The KMP11-HASPB fusion protein was successfully expressed as evidenced by RT-PCR and Western blot assays. The effect of the vaccine was determined by evaluating the level of IFN-γ, IL-10, IgG1, and IgG2a performed using ELISA as well as determining the parasite load after challenge with L. major in vaccinated mice. The results revealed that IFN-γ, IL-10, IgG1, and IgG2a significantly increased after vaccination using KMP11-HASPB-expressing lentiviruses in BALB/c mice. It is noteworthy that the level of IFN-γ and IgG2a was higher than that of IL-10 and IgG1, respectively, which indicates the activation Th1 cells, macrophages, and cellular immunity. Moreover, the parasite load in the spleen and lymph node of vaccinated mice after challenge was significantly lower than that of controls.

Multiple Sclerosis Gene Therapy with Recombinant Viral Vectors: Overexpression of IL-4, Leukemia Inhibitory Factor, and IL-10 in Wharton's Jelly Stem Cells Used in EAE Mice Model.

OBJECTIVES: Immunotherapy and gene therapy play important roles in modern medicine. The aim of this study is to evaluate the overexpression of interleukin-4 (IL-4), IL-10 and leukemia inhibitory factor (LIF) in Wharton's jelly stem cells (WJSCs) in the experimental autoimmune encephalomyelitis (EAE) mice model. MATERIALS AND METHODS: In this experimental study, a DNA construction containing IL- 4, IL-10 and LIF was assembled to make a polycistronic vector (as the transfer vector). Transfer and control vectors were co-transfected into Human Embryonic Kidney 293 (HEK-293T) cells with helper plasmids which produced recombinant lentiviral viruses (rLV). WJSCs were transduced with rLV to make recombinant WJSC (rWJSC). In vitro protein and mRNA overexpression of IL-4, LIF, and IL-10 were evaluated using quantitative polymerase chain reaction (qPCR), enzyme-linked immunosorbent assay (ELISA) and western blot (WB) analysis. EAE was induced in mice by MOG-CFA and pertussis toxin. EAE mice were injected twice with 2×105 rWJSCs. The in vivo level of IL-4, LIF, IL-10 cytokines and IL-17 were measured by ELISA. Brain tissues were analyzed histologically for evaluation of EAE lesions. RESULTS: Isolated WJSCs were performed to characterize by in vitro differentiation and surface markers were analyzed by flow cytometry method. Cloning of a single lentiviral vector with five genes was done successfully. Transfection of transfer and control vectors were processed based on CaPO4 method with >90% efficiency. Recombinant viruses were produced and results of titration showed 2-3×107 infection-unit/ml. WJSCs were transduced using recombinant viruses. IL-4, IL-10 and LIF overexpression were confirmed by ELISA, WB and qPCR. The EAE mice treated with rWJSC showed reduction of Il-17, and brain lesions as well as brain cellular infiltration, in vivo. Weights and physical activity were improved in gene-treated group. CONCLUSIONS: These results showed that gene therapy using anti-inflammatory cytokines can be a promising approach against multiple sclerosis (MS). In addition, considering the immunomodulatory potential of WJSCs, an approach using a combination of WJSCs and gene therapy will enhance the treatment efficacy.

An in vitro assay to evaluate the immunomodulatory effects of unrestricted somatic stem cells.

BACKGROUND: Unrestricted somatic stem cells (USSC) are cord blood stem cells that have been considered as candidates for the regulation of immune responses. Therefore, potential exists for their use in the suppression of immune response after transplantation surgery. OBJECTIVE: The aim of this study was evaluation of the effect of USSC on mixed lymphocyte reaction (MLR) as a model for graft rejection. METHODS: USSC and mesanchymal stem cells (MSC) were isolated and cultured from cord blood and bone morrow, respectively. The immunophenotypes of USSC and MSC were evaluated by flow cytometery and USSC and MSC were co-cultured with peripheral blood lymphocytes (PBL) in an MLR to evaluate the immunomodulatory effect of these cells as a percentage of the control response. RESULTS: Current study demonstrated that proliferation of lymphocytes in the MLR was decreased after treatment with USSC, in a similar fashion to that seen with MSC. CONCLUSION: It can be concluded that USSC have similar regulatory effects as MSC on the MLR, which can be used as an indicator for potential organ rejection after transplantation. Therefore, the immunregulatory effect of these cells could be used in the clinic during organ transplantation and in the management of autoimmunity.

Interleukin-18 promoter polymorphism is associated with lung cancer: a case-control study.

BACKGROUND: Interleukin-18 (IL-18) is a multifunctional cytokine that augments IFN-gamma production and affects tumor immune response. In the present case-control study, we tested whether IL-18 promoter polymorphism contributes to lung cancer susceptibility in Iranian patients. MATERIAL AND METHODS: The study groups were 73 patients with lung cancer, including 53 with squamous carcinoma (SC) and 20 with small cell lung carcinoma (SCLC), and 97 healthy regional aged-matched individuals. The frequency of IL-18 promoter single nucleotide polymorphisms (SNPs) at positions -656 (G/T), -607 (C/A), and -137 (G/C) was determined by polymerase chain reaction analyses. RESULTS: There were significant differences in the IL-18 -607 allele and genotype distributions between the 73 lung cancer patients and controls. A significantly higher A allele frequency at position -607, which is associated with lower IL-18 production, was observed in lung cancer patients (48.6% vs. 35%; OR = 1.75; 95% CI 1.13-2.72). Also, patients with the -607 CA and the AA genotypes had a 2.60-fold (95% CI 1.26-5.36) and 3.15-fold (95% CI 1.16-8.55) increase in risk of lung cancer. Subdivision of the patients according to histological type revealed that SC was significantly associated with IL-18 -607 SNPs. Although the percentages of -607 alleles and genotypes in SCLC patients were similar to the results in SC patients, the differences compared to control individuals did not reach statistical significance. Analysis with Arlequin software identified eight haplotypes from three SNPs analyzed here. The distributions of IL-18 gene haplotypes were not significantly different between patients and controls after Bonferroni correction. DISCUSSION: This is the first report to investigate the association between IL-18 polymorphism and lung cancer. Our results suggest that IL-18 polymorphism contributes to the lung cancer risk, particularly among SC patients. Further studies with larger numbers of patients are required to determine the possible association between IL-18 polymorphisms and different histological types of lung cancer.