Inhibition of hippocampal melatonin synthesis by siRNA induced learning and memory deficits in male rats.

Melatonin, the multi-functional neurohormone, is synthesized in the extra-pineal tissues such as the hippocampus. The key enzyme in hippocampal melatonin synthesis is arylalkylamine-N-acetyltransferase (AANAT). The importance of melatonin synthesis in the hippocampus has not yet been determined. We investigated hippocampal AANAT role in cognitive function using gene silencing small interference RNA (siRNA) technology. The hippocampal local melatonin synthesis was inhibited by AANAT-siRNA injection. The time-gene silencing profile of AANAT-siRNA was obtained by RT-PCR technique. The cytotoxicity of siRNA dose was determined by MTT assay on the B65 neural cells. Animals received the selected dosage of AANAT-siRNA. Then, the spatial working memory (Y maze), object recognition memory and spatial reference memory (Morris's water maze, MWM) were evaluated. The anxiety-like behaviors were evaluated by the elevated plus maze. After one week, following the probe test of MWM, the rats were sacrificed for histological analysis. The hippocampal melatonin levels were measured using the liquid chromatography-mass spectrometry technique. The hippocampal melatonin levels in the AANAT-siRNA group decreased. Animals receiving the AANAT-siRNA showed deficits in spatial learning and working memory which were verified by increased escape latency and reduced spontaneous alternations, respectively. There was an increase in anxiety-like behaviors as well as a deficit in recognition memory in the AANAT-siRNA group. The Nissl staining and immunohistochemistry of activated caspase-3 showed the neuronal loss and cell apoptosis in hippocampal tissue of the AANAT-siRNA group. The 18F-FDG-PET imaging displayed lower glucose metabolism following the reduction in AANAT mRNA. Data suggest that the AANAT mRNA and hippocampal melatonin synthesis might be an essential factor for learning, memory and some aspects of cognition, as well as homeostasis of hippocampal cells.

Curcumin-loaded human endometrial stem cells derived exosomes as an effective carrier to suppress alpha-synuclein aggregates in 6OHDA-induced Parkinson's disease mouse model.

Parkinson disease (PD) is considered as one of the most worldwide neurodegenerative disorders. The major reasons associated to neurodegeneration process of PD pathogenesis are oxidative stress. Many studies reported that natural antioxidant molecules, especially, curcumin can suppress inflammatory pathways and preserve dopaminergic neurons damage in PD. Further, the poor pharmacokinetics, instability of chemical structure because of fast hydrolytic degradation at physiologic condition and especially, the presence of the blood brain barrier (BBB) has regarded as a considerable restriction factor for transfer of neurotherapeutic molecules to the brain tissue. The present research aims to the fabrication of nanoformulated curcumin loaded human endometrial stem cells derived exosomes (hEnSCs EXOs-Cur) to study on enhancing curcumin penetration to the brain across BBB and to improve anti- Parkinsonism effects of curcumin against neural death and alpha-synuclein aggregation. hEnSCs EXOs-Cur characterization results demonstrated the accurate size and morphology of formulated curcumin loaded exosomes with a proper stability and sustained release profile. In vivo studies including behavioral, Immunohistochemical and molecular evaluations displayed that novel formulation of hEnSCs EXO-Cur is able to cross BBB, enhance motor uncoordinated movements, suppress the aggregation of αS protein and rescue neuronal cell death through elevation of BCL2 expression level as an anti-apoptotic protein and the expression level reduction of BAX and Caspase 3 as apoptotic markers.

miR-219 overexpressing oligodendrocyte progenitor cells for treating compression spinal cord injury.

Oligodendrocyte progenitor cells (OPCs) transplantation has been considered a promising treatment for spinal cord injury, according to previous studies. Recent research shed light on the importance of microRNA 219 (miR-219) in oligodendrocyte development, so here miR-219-overexpressing OPCs (miR-219 OPCs) were transplanted in animal models of spinal cord injury to evaluate the impact of miR-219 on oligodendrocyte differentiation and functional recovery in vivo. Our findings demonstrate that transplanted cells were distributed in the tissue sections and contributed to reducing the size of cavity in the injury site. Interestingly, miR-219 promoted OPC differentiation into mature oligodendrocyte expressing MBP in vivo whereas in absence of miR-219, less number of cells differentiated into mature oligodendrocytes. An eight week evaluation using the Basso Beattie Bresnahan (BBB) locomotor test confirmed improvement in functional recovery of hind limbs. Overall, this study demonstrated that miR-219 promoted differentiation and maturation of OPCs after transplantation and can be used in cell therapy of spinal cord injury.

Decellularization of Small Intestinal Submucosa.

Small intestinal submucosa (SIS) is the most studied extracellular matrix (ECM) for repair and regeneration of different organs and tissues. Promising results of SIS-ECM as a vascular graft, led scientists to examine its applicability for repairing other tissues. Overall results indicated that SIS grafts induce tissue regeneration and remodeling to almost native condition. Investigating immunomodulatory effects of SIS is another interesting field of research. SIS can be utilized in different forms for multiple clinical and experimental studies. The aim of this chapter is to investigate the decellularization process of SIS and its common clinical application.

Derivation of preoligodendrocytes from human-induced pluripotent stem cells through overexpression of microRNA 338.

MicroRNAs (miRNAs) control gene expression at the posttranscriptional level and have a critical role in many biological processes such as oligodendrocyte differentiation. Recent studies have shown that microRNA 338 (miR-338) is overexpressed during the oligodendrocyte development process in the central nervous system; this finding indicates a potentially important role for miR-338 in oligodendrocyte development. To evaluate this assumption, we studied the effect of miR-338 overexpression on promoting the differentiation of oligodendrocyte progenitor cells (OPCs), derived from human-induced pluripotent stem cells (hiPSC), into preoligodendrocyte. hiPSCs were differentiated into OPCs after treating for 16 days with basic fibroblast growth factor (BFGF), epidermal growth factor (FGF), and platelet-derived growth factor (PDGF)-AA. Bipolar OPCs appeared and the expression of OPC-related markers, including Nestin, Olig2, Sox10, PDGFRα, and A2B5 was confirmed by real-time polymerase chain reaction (PCR) and immunofluorescence. Then, OPCs were transduced by miR-338 expressing lentivirus or were treated with triiodothyronine (T3) for 6 days. Data obtained from real-time PCR and immunofluorescence experiment indicated that preoligodendrocyte markers such as Sox10, O4, and MBP were expressed at higher levels in transduced cells with miR-338 in comparison with the T3 group. So, the overexpression of miR-338 in iPSC-derived OPCs can promote their differentiation into preoligodendrocyte which can be used in cell therapy of myelin-related diseases.

Serum Levels of miR-155, miR-326, and miR-133b as Early Diagnostic Biomarkers for the Detection of Human Acute Heart Allograft Rejection in Comparison with Serum Cardiac Troponin T.

BACKGROUND: Acute heart allograft rejection occurs as a result of antibody-mediated rejection that presents during the first month after transplantation. Finding a non-invasive biomarker is essential for diagnosis of heart allograft rejection. In this research, we intended to compare expression levels of several microRNAs across cardiac troponin T levels between rejected patients (who died before one month following transplantation), non-rejected patients (who survived for at least one month after transplantation), and non-transplanted patients (CABG surgery patients). METHODS: Serum levels of miR-155, miR-326, and miR-133b were evaluated by the q-RT-PCR method. Furthermore, cardiac troponin T levels were measured by a highly sensitive electrochemiluminescence assay. Finally, the data were analyzed by independent sample t-test using SPSS 21® computer software. Results: It was observed that miR-326 and miR-155 expression levels increased after 24h and 72h of surgery in rejected patients compared with the two other groups, but these increases were not statistically significant. Moreover, the decrease in miR-133b expression level was non-significant after transplantation in the rejected group compared with the non-rejected group. However, cTnT levels in rejected patients increased significantly compared with the other groups (P < .05). After ROC curve analysis, the cTnT marker with the most area under the curve (AUC = 1.00, 95% confidence interval, 1.00 to 1.00; P = .006), had the best discriminatory power, and among microRNAs, miR-326 had the largest area under curve (AUC = 0.81), and consequently the highest discriminatory power. CONCLUSIONS: We demonstrated that troponin T can be a more efficient biomarker than miRNAs for early prediction of human death caused by acute heart rejection, and the ROC curves analysis verified this finding.

Comparative evaluation of probiotics effects on plasma glucose, lipid, and insulin levels in streptozotocin-induced diabetic rats.

BACKGROUND: Diabetes is a chronic disorder caused by the relative decrease in insulin production from the pancreas, tissue resistance to insulin, or both. Daily consumption of probiotics, particularly Lactobacilli, has been proposed as a new strategy for prevention or control of diabetes. METHODS: In the current study, the effects of various probiotics including Lactobacillus reuteri (L reuteri), Lactobacillus crispatus (L crispatus), and Bacillus subtilis on the levels of blood glucose, lipid, and insulin as well as haemoglobin A1c (HbA1c ) were investigated in rat models of streptozotocin (STZ)-induced diabetes and compared with metformin. In addition, GLUT-4 and PPAR-γ transcript levels have been analysed in adipose tissues. RESULTS: A significant decrease in plasma glucose and HbA1c levels and a dramatic increase in insulin levels have been detected after consumption of probiotics. Furthermore, lipid profiles have been improved. The expression of Glut-4 and Ppar-γ genes in adipose tissues also increased after treatment period. CONCLUSION: The results of this experiment showed that daily consumption of probiotics can be effective in control of STZ-induced diabetes and its complications.

Application of immuno-PCR assay for the detection of serum IgE specific to Bermuda allergen.

In vivo and in vitro tests are the two major ways of identifying the triggering allergens in sensitized individuals with allergic symptoms. Both methods are equally significant in terms of sensitivity and specificity. However, in certain circumstances, in vitro methods are highly preferred because they circumvent the use of sensitizing drugs in patients. In current study, we described a highly sensitive immuno-PCR (iPCR) assay for serum IgE specific to Bermuda allergens. Using oligonucleotide-labelled antibody, we used iPCR for the sensitive detection of serum IgE. The nucleotide sequence was amplified using conventional PCR and the bands were visualized on 2.5% agarose gel. Results demonstrated a 100-fold enhancement in sensitivity of iPCR over commercially available enzyme-linked immunosorbent assay (ELISA) kit. Our iPCR method was highly sensitive for Bermuda-specific serum IgE and could be beneficial in allergy clinics.

A novel recombinant anti-epidermal growth factor receptor peptide vaccine capable of active immunization and reduction of tumor volume in a mouse model.

Over-expression of epidermal growth factor receptor (EGFR) has been reported in a number of human malignancies. Strong expression of this receptor has been associated with poor survival in many such patients. Active immunizations that elicit antibodies of the desired type could be an appealing alternative to conventional passive immunization. In this regard, a novel recombinant peptide vaccine capable of prophylactic and therapeutic effects was constructed. A novel fusion recombinant peptide base vaccine consisting of L2 domain of murine extra-cellular domain-EGFR and EGFR mimotope (EM-L2) was constructed and its prophylactic and therapeutic effects in a Lewis lung carcinoma mouse (C57/BL6) model evaluated. Constructed recombinant peptide vaccine is capable of reacting with anti-EGFR antibodies. Immunization of mice with EM-L2 peptide resulted in antibody production against EM-L2. The constructed recombinant peptide vaccine reduced tumor growth and increased the survival rate. Designing effective peptide vaccines could be an encouraging strategy in contemporary cancer immunotherapy. Investigating the efficacy of such cancer immunotherapy approaches may open exciting possibilities concerning hyperimmunization, leading to more promising effects on tumor regression and proliferation.

Application of immuno-PCR for the detection of early stage cancer.

Cancer detection in premalignant stage is directly related with increase survival rate. Several biomarkers have been investigated and characterized for monitoring changes inside the cancerous cells. Although enzyme-linked immunosorbent assay (ELISA) is the method of choice in clinical practice for detecting biomarkers in serum/urine samples. However, in certain malignancies the amount of biomarkers before reaching metastasis are too low to be detected by conventional ELISA. The seminal work of Sano et al. led to the development of highly sensitive and powerful detection method, the immuno-PCR (iPCR), which can detect very small amount of antigens/biomarkers. In spite of, several publications on iPCR sensitivity, it has not been recommended for clinical use and is limited to the scientific community only. In order to evaluate the importance of iPCR, we have made an effort to collect published studies, supporting the use of iPCR in detecting premalignant cancer.

MicroRNAs in lung diseases: Recent findings and their pathophysiological implications.

Lung diseases are one of the leading causes of mortality and morbidity worldwide and effective therapies are imperfect. Nonetheless, recently some novel strategies have been developed to treat and curtail their debilitating impact. Some of the treatments include the role of MicroRNAs (miRNAs) in stemming the spread of lung morbidities. Micro RNAs are small non-coding RNAs which are known as important players in the posttranscriptional regulation of gene expression in mammalian cells by regulating translation. MiRNAs are involved in basic regulatory mechanisms of cells including influencing inflammation. MiRNA dysregulation, resulting in aberrant expression of a gene, is suggested to play a key role in susceptibility of diseases. MiRNAs are involved in the pathogenesis of lung diseases such as cystic fibrosis, lung cancer, asthma, chronic obstructive pulmonary disease, and Idiopathic pulmonary fibrosis. A better understanding of the involvement of miRNAs in pathogenesis of these diseases could result in the development of new therapeutic and diagnostic tools. In this review, we provide an overview of the current understanding of miRNA biogenesis and role as well as recent insights into role of some miRNAs in different pulmonary diseases.

Human endometrial stem cells differentiation into functional hepatocyte-like cells.

In spite of certain clinical limitations, such as teratoma formation, the use of stem cells is considered as an appropriate source in cell therapy and tissue engineering. This study shows human endometrial stem cells (hEnSCs) has exceptional differentiation ability in hepatocyte formation. hEnSCs have high purification rate and immune-tolerance, and can be used as an appropriate substitute for hepatocytes in liver disorders. Differentiation required hepatogenic medium. Quantitative reverse transcription-polymerase chain reaction and immunofluorescent staining of hepatic genes and proteins including cytokeratin 18 (ck18), alpha-fetoprotein (afp), and albumin (alb) were used to assess differentiation. Cells differentiated with a hepatocyte-like morphology and expressed hepatic markers on 30 days of differentiation. The Periodic Acid-Schiff (PAS) reaction showed storage of glycogen, and albumin and afp secretions were also detected. In vitro hEnSCs behave like hepatocyte after differentiation and may be a suitable source of cells in liver regeneration.

Biomolecular markers in assessment and treatment of asthma.

Asthma is a chronic and heterogeneous inflammatory disorder with several different phenotypes. Whereas clinical features of asthma are non-specific and pulmonary function tests are often insensitive, further development is needed for efficient treatment or even early diagnosis. Recently, several airway inflammatory biomarkers have emerged as valuable tools in diagnosis and management of asthma. The analysis of molecular markers of airways inflammation has provided promising and non-invasive techniques that facilitate the detection of disease phenotypes as well as measurement of therapeutic efficacy. Although conventional treatments remain the preferred therapy, they do not adequately control some severe cases of asthma. Novel therapeutic agents have been developed to target various biomarkers involved in the inflammatory responses and have been investigated in patients with asthma. In this article, we summarized the most studied asthma biomarkers, derived from a variety of biological sources including exhaled gases, induced sputum, serum and urine. Likewise, the effects of current anti-inflammatory asthma treatments on inflammatory biomarkers and some promising biomarkers for developing new targeted therapies are also discussed.

Targeted expression of a designed fusion protein containing BMP2 into the lumen of exosomes.

BACKGROUND: Exosomes are 30-150 nm membrane vesicles, originating from the endocytic pathway. By acting as natural carriers of biomolecules, they can transfer various materials to recipient cells. Therefore, discovering novel strategies for cargo packaging into exosomes is crucial. METHODS: The fusion constructs, consisting of protein of interest (BMP2) along with the targeting motif, linkers, tracking proteins, and enzyme cleavage sites, were computationally designed. Following the homology modeling, the best structure was selected and subjected to molecular dynamics (MD) simulation and docking analyses. The fusion protein gene was expressed in the HEK-293LTV cell line. The high-efficiency transfected and transduced cells were screened and their exosomes were isolated. Finally, cell and exosome lysates were evaluated for expression of the fusion protein. RESULTS: A total of 12 constructs with lengths ranging from 483 to 496 were designed. The top three templates, 1REW, 2H5Q, and 2MOF were screened. MD simulation and docking analyses of the structures revealed their stability and functionality. In the protein expression analyses, three bands at sizes of approximately 60, 25, and 12.5 kDa were observed, consistent with the sizes of the complete fusion protein, dimeric, and monomeric BMP2 protein. The presence of a 12.5 kDa band at exosome lysate analysis might suggest that it was loaded and cleaved inside exosomes. CONCLUSION: In summary, these findings revealed that the proposed idea for cargo sorting within the exosome lumen through incorporating an appropriate cleavage site was effective, thus providing further insight into the potential of exosomes as nano-shuttles bearing therapeutic biomolecules.

Distinctive Expression of Metastamirs in Breast Cancer Mesenchymal Stem Cells Isolated from Solid Tumor.

BACKGROUND: MSCs are a part of the tumor microenvironment, which secrete cytokines and chemokines. They can affect metastasis and the growth of tumors. metastamiRs are newly recognized regulatory elements of the metastasis pathway which are involved in epithelial-to-mesenchymal transition (EMT). OBJECTIVE: In the present study, we aimed to assess the expression profile of metastamiRs in the context of MSCs in correlation with their invasion and migration power. METHODS: tumor-isolated BC-MSCs and normal human mammary epithelial cells (HMECs) along with MCF-7, MDA-MB231, and MCF-10A cells were prepared and confirmed for their identity. The cells were assessed for CD44+CD24¯ percentage, Oct-4, and Survivin expression. GEO, KEGG, and TCGA databases were investigated to detect differential miR-expressions. Real-time PCR for 13 miRs was performed using LNA primers. Ultimately, Transwell-Matrigel assays as used to assess the level of migration and invasion. RESULTS: Our results indicated that some oncomiRs like miR-10b were upregulated in BC-MSCs, while the levels of miR-373 and miR-520c were similar to the MCF-10A. Generally, miR-200 family members were on lower levels compared to the other miR-suppressor (miR-146a, 146b, and 335). miR-31 and 193b were up-regulated in MCF-10A. The most invasiveness was observed in the MDA-MB231 cell line. CONCLUSION: We have demonstrated that the miR-expression levels of BC-MSCs are somewhat in between MCF-7 and MDA-MB231 miR-expression levels. This could be the logic behind the moderate level of invasion in BC-MSCs. Therefore, miR-therapy approaches such as miR-mimic or antagomiRs could be used for BC-MSCs in clinical cancer therapy.

Structural evolution of Delta lineage of SARS-CoV-2.

One of the main obstacles in prevention and treatment of COVID-19 is the rapid evolution of the SARS-CoV-2 Spike protein. Given that Spike is the main target of common treatments of COVID-19, mutations occurring at this virulent factor can affect the effectiveness of treatments. The B.1.617.2 lineage of SARS-CoV-2, being characterized by many Spike mutations inside and outside of its receptor-binding domain (RBD), shows high infectivity and relative resistance to existing cures. Here, utilizing a wide range of computational biology approaches, such as immunoinformatics, molecular dynamics (MD), analysis of intrinsically disordered regions (IDRs), protein-protein interaction analyses, residue scanning, and free energy calculations, we examine the structural and biological attributes of the B.1.617.2 Spike protein. Furthermore, the antibody design protocol of Rosetta was implemented for evaluation the stability and affinity improvement of the Bamlanivimab (LY-CoV55) antibody, which is not capable of interactions with the B.1.617.2 Spike. We observed that the detected mutations in the Spike of the B1.617.2 variant of concern can cause extensive structural changes compatible with the described variation in immunogenicity, secondary and tertiary structure, oligomerization potency, Furin cleavability, and drug targetability. Compared to the Spike of Wuhan lineage, the B.1.617.2 Spike is more stable and binds to the Angiotensin-converting enzyme 2 (ACE2) with higher affinity.

A proteomics approach to the study of the molecular consequence of IgE-mediated cell signalling in RBL-2H3.1 cells and 2D reference map preparation for the RBL-2H3.1 cell line.

A highly reproducible 2D (two-dimensional) map for the proteome and a pattern of protein phosphorylation of high secretory variant of RBL-2H3 cells (RBL-2H3.1) (a model cell in allergy studies) in resting and treated cells with IgE or IgE+Ag are presented. Major molecular changes were seen in the proteome of 3 h-activated cells with IgE+Ag, especially for proteins of ∼17 kDa compared with the control. We have identified 13 proteins on 11 corresponding spots as up-regulated proteins in response to IgE+Ag activation. Also, protein identification on 55 spots with MALDI-TOF (matrix-assisted laser-desorption ionization-time-of-flight) and ESI-MS (electrospray ionization mass spectrometry) resulted in a reliable 2D reference map and an opportunity for the subsequent use of a 1 min-activated cell map for a phosphoproteomics study.

Effect of Paclitaxel/etoposide co-loaded polymeric nanoparticles on tumor size and survival rate in a rat model of glioblastoma.

The aim of this work is to co-load paclitaxel (PTX) and etoposide (ETP) in methoxy poly(ethylene glycol)-poly(lactic-co-glycolic acid) nanoparticles (mPEG-PLGA NPs) to overcome pharmacokinetics and physiological limitations and enhance therapeutic efficacy for treating intracranial glioblastoma. Both drugs were loaded into mPEG-PLGA NPs by a nano-precipitation method. The resultant NPs demonstrated an enhanced cytotoxic effect indicated by lower IC50 values and augmented cell apoptosis to U87 and C6 glioma cell lines compared to both free drugs. Additionally, blood compatibility assays showed that the PTX/ETP co-loaded mPEG-PLGA NPs did not induce blood hemolysis, blood clotting, or platelet aggregation. In vivo anti-glioma efficacy evaluation in rats bearingintracranialC6glioma revealed a superior anti-glioma activity for the treatment with PTX/ETP co-loaded mPEG-PLGA NPs compared to other formulations, particularly a significantly longer median survival, 76 days compared to 36 days for free PTX and 37 days for free ETP treatment, respectively, and higher tumor regression, proved by magnetic resonance imaging (MRI).

Additive effect of metastamiR-193b and breast cancer metastasis suppressor 1 as an anti-metastatic strategy.

BACKGROUND: It has been reported that enhancing the cellular levels of miR-193b as well as breast cancer-metastasis-suppressor-1 (BRMS1) protein is associated with diminished metastatic characteristics in breast cancer. In view of these facts, as a new therapeutic intervention, we employed a restoration-based strategy using both miR-193b-3p mimic and optimized BRMS1 in the context of a chimeric construct. METHODS: miR-193b-3p and BRMS1 genes were cloned and the resulting plasmids were transfected into the MDA-MB231, MCF-7 and MCF-10A cell lines. microRNA expression levels were assessed by rea time PCR using LNA-primer and protein expression was confirmed by western blot method. Then, apoptosis, MTT, colony formation and invasion assays were carried out. RESULTS: The expression levels of miR-146a, miR-146b and miR-373 were up-regulated, while the miR-520c, miR-335 and miR-10b were down-regulated following the exogenous BRMS1 expression. The exogenous over-expression of BRMS1 was associated with higher amounts of endogenous miR-193b-3p expression and enabled more efficient targeting of the 3'UTR of uPA. Although, miR-193b-3p and BRMS1 are individually capable of suppressing breast cancer cell growth, migration and invasion abilities, their cistronic expression was capable of enhancing the ability to repress the breast cancer cells invasion. CONCLUSIONS: Our results collectively indicated the existence of an additive anti-metastatic effect between miR-193b-3p and BRMS1. Moreover, it has been hypothesized that the exogenous expression of a protein can effect endogenous expression of non-relevant microRNA. Our findings provide new grounds for miR-restoration therapy applications as an amenable anti-metastatic strategy.

Functional Deimmunization of Interferon Beta-1b by Identifying and Silencing Human T Cells Epitopes.

Interferonbeta-1b (IFNß-1b) developed as therapeutic protein for the treatment of multiple sclerosis (MS). Studies have been shown that Long-term usage of this protein can lead to the development of anti-drug antibodies (ADAs) and this phenomenon cause total loss or reduced efficacy of IFNß-1b. The aim of this study was to predict and silence IFNß-1b T-cells epitopes by in silico methods and genetic engineering. Based on bioinformatics studies we identified optimal sets of conservative point mutations for eliminating T-cells epitopes in IFNß-1b protein. Four synthetic genes with desirable mutation constructed and PET26b+ was used as an expression vector in E. coli. The expression of this proteins confirmed by SDS-PAGE and Western blotting, consequently, IFNß-1b proteins was purified by His-tag chromatography. To determined activity of mutants' variants anti-proliferative and anti-viral activity compared to wild form was evaluated using MTT assay in A549 and Vero cells lines respectively. Also the immunogenicity of mutant proteins compared with Betaseron measured in BALB/c mice. The in vitro bioactivity analysis demonstrated that functional activities of all mutant proteins were maintained and is the same as biological activity of Betaseron. Pharmacokinetic studies suggest that, in engineered proteins that contain substitution of Histidine to Glutamic Acid at position 131 (mut 2 and mut 1+2) antibodies response reduced by about 50%, as compared to that for Betaseron. Computational analysis expedites identification and prediction of epitopes in therapeutic protein, therefore, we used immunoinformatic tools for modification of dominant T-cell epitope in IFNß-1b protein, and this strategy has capacity to create proteins which have naturally reduced immunogenicity.