Cell surface display of rabbit MCP1 on human embryonic kidney 293T cell line

284-288As atherosclerosis is a prevalent non-communicable disease, and yet, no definitive medical treatment found for it, Trapping MCP1 as a key factor in inflammation could be effective. Therefore, we decided to display rabbit MCP-1 (R-MCP1) on human embryonic kidney 293T cell line surface.Firstly, R-MCP1 plasmid (pR-MCP1) containing kappa chain signal sequence,  R-MCP1 sequence and PDGFR intra membrane domain was constructed. The delivered pR-MCP1 was transformed in E.coli TOP10F’, and the resulted clones were assessed by PCR and digestion. After linearizing pR-MCP1 by BglII, HEK cells were transfected by them. MCP1 gene integration and expression was confirmed at RNA and protein levels by real- time PCR and flow cytometry, respectively.PCR product gel electrophoresis on genomic DNA of transfected HEK cells showed a 737 bp band.Based on real- time PCR results, We observed R-MCP1 gene expression significantly increased in transfected cells (272.26±37.32) compare to untransfected HEK 293T cells (2.67±0.12) (p=0.001).The results of flow cytometry showed that about 85% of transfected cells were positive and express R-MCP1. Therefore, cell surface display of R-MCP1 has successfully been performed and the produced cells can be used in future research to prepare diagnostic and therapeutic agents like aptamers

Construction and Characterization of Recombinant HEK Cell Over Expressing α4 Integrin

Purpose: Integrins are heterodimeric membrane proteins, which are exposed to post translational modifications in eukaryotic cells in contrast to prokaryotic cells. These modifications provide advantages for production of proper nanobody, mono and polyclonal antibody against this surface protein and also in aptamer selection process. Since the majority of diagnostic and therapeutic antibodies, target the surface epitopes, eukaryotic membrane proteins provide an appropriate model for further investigation on therapeutic agents. Methods: Escherichia coli strain top 10, was used as host for ITGA-4 expression vector encoding the human integrin α4. The plasmid was extracted and consequently, ITGA-4 vector was digested to make a linear plasmid. Human Embryonic Kidney-293 (HEK-293) cell transfected with linear plasmid and subsequently screened for stable ITGA-4 expressing Cells. Three separated clones were isolated twenty one days after transfection. Chromosomal DNA was extracted from ITGA-4-transfected cells. The presence of ITGA-4 gene in HEK-293 genome was confirmed by PCR. The expression level of ITGA-4 on HEK-293 cells was also analyzed by Flow cytometry. Results: Flow cytometric analysis showed that HEK-293 cells have no expression of integrin α4 on their surface while 95% of transfected HEK-293 cells with ITGA4, expressed different levels of integrin α4 on their surfaces which correlates well with genomic DNA PCR amplification results. Conclusions: The results suggest that we have successfully constructed the integrin α4 expressing HEK293 cell, which will facilitate further research into the production of antibody, nanobody and aptamer against α4 integrin

Evaluation of Urokinase Plasminogen Activator Receptor, Soluble Urokinase Plasminogen Activator Receptor, and β1 Integrin in Patients with Hodgkin′s Lymphoma

Background: The objective of this study is to indicate the role of urokinase plasminogen activator receptor (uPAR), soluble uPAR (suPAR), and β1 integrin in tumor growth and invasion of lymph nodes from Hodgkin's lymphoma (HL) patients. Materials and Methods: In this study, 25 lymph nodes from HL patients were analyzed for the expression of β1 integrin and uPAR on mononuclear cells using two-color flow cytometry and immunohistochemical analysis. Moreover, the levels of suPAR in the serum samples of HL patients were measured and compared with 32 healthy controls. Results: Flowcytometry and immunohistochemical results indicated no significant association of uPAR expression with tumor size, different stages, or different histological subtypes of HL; however, an increased expression of β1 integrin was detected in the advanced stages of HL. Higher expression of β1 integrin was detected in nodular sclerosis compared to lymphocyte predominant. No significant difference was observed between the serum levels of suPAR in patients with different stages of HL and healthy controls. Moreover, the levels of suPAR were significantly higher in nodular sclerosis in comparison with other subtypes. Conclusions: This study showed that the levels of suPAR and β1 integrin varied between different histological subtypes of HL. Although uPAR may play only a minor role in the growth and metastasis of lymphoma, β1 integrin may be important in predicting prognosis and metastasis in HL

Crosstalk of Transcriptional Regulators of Adaptive Immune System and microRNAs: An Insight into Differentiation and Development

MicroRNAs (miRNAs), as small regulatory RNA molecules, are involved in gene expression at the post-transcriptional level. Hence, miRNAs contribute to gene regulation of various steps of different cell subsets’ differentiation, maturation, and activation. The adaptive immune system arm, which exhibits the most specific immune responses, is also modulated by miRNAs. The generation and maturation of various T-cell subsets concomitant with B-cells is under precise regulation of miRNAs which function directly on the hallmark genes of each cell subset or indirectly through regulation of signaling pathway mediators and/or transcription factors involved in this maturation journey. In this review, we first discussed the origination process of common lymphocyte progenitors from hematopoietic stem cells, which further differentiate into various T-cell subsets under strict regulation of miRNAs and transcription factors. Subsequently, the differentiation of B-cells from common lymphocyte progenitors in bone marrow and periphery were discussed in association with a network of miRNAs and transcription factors

Altered Th17/Treg Ratio in Recurrent Miscarriage after Treatment with Paternal Lymphocytes and Vitamin D3: a Double- Blind Placebo-Controlled Study

ABSTRACT Background: Recurrent miscarriage (RM) affects 2-5% of pregnant women. Paternal lymphocyte immunotherapy is a common treatment for RM patients but the outcome has not been consistent. Therefore, combined therapy with other immunosuppressive drugs such as 1a, 25-dihydroxy-vitamin-D3 (vitamin D3) may improve the outcome. Objectives: To investigate the effect of vitamin D3 on the balance of two essential T cells subsets, T helper (Th) 17 and T regulatory (Treg) cells, which contribute to the immune tolerance during pregnancy. Methods: The expression levels of CD4 and forkhead box protein 3 (FOXP3) in Treg cells, and the expression levels of CD4 and IL-17 in Th17 cells, were evaluated pre-and 3 months post-immunotherapy in RM patients treated with a combination of paternal lymphocytes and vitamin D3 compared with RM patients receiving lymphocyte immunotherapy alone. Results: Vitamin D3 therapy decreased the frequency of Th17 cells in addition to reducing the Th17/Treg ratio in peripheral blood of RM patients compared with the control group (p<0.05). Conclusion: Considering that RM patients have a higher Th17/Treg ratio in peripheral blood, vitamin D3 may be a candidate therapeutic approach in this disease. Rafiee M, et al. Iran J Immunol. 2015; 12(4):252-262

A Novel Prokaryotic Green Fluorescent Protein Expression System for Testing Gene Editing Tools Activity Like Zinc Finger Nuclease

Background: Gene editing technology has created a revolution in the field of genome editing. The three of the most famous tools in gene editing technology are zinc finger nucleases (ZFNs), transcription activator-like effector nucleases, clustered regularly interspaced short palindromic repeats (CRISPR), and CRISPR-associated systems. As their predictable nature, it is necessary to assess their efficiency. There are some methods for this purpose, but most of them are time labor and complicated. Here, we introduce a new prokaryotic reporter system, which makes it possible to evaluate the efficiency of gene editing tools faster, cheaper, and simpler than previous methods. Materials and Methods: At first, the target sites of a custom ZFN, which is designed against a segment of ampicillin resistance gene, were cloned on both sides of green fluorescent protein (GFP) gene to construct pPRO-GFP. Then pPRO-GFP was transformed into Escherichia coli TOP10F' that contains pZFN (contains expression cassette of a ZFN against ampicillin resistant gene), or p15A-KanaR as a negative control. The transformed bacteria were cultured on three separate media that contained ampicillin, kanamycin, and ampicillin + kanamycin; then the resulted colonies were assessed by flow cytometry. Results: The results of flow cytometry showed a significant difference between the case (bacteria contain pZFN) and control (bacteria contain p15A, KanaR) in MFI (Mean Fluorescence Intensity) (P < 0.0001). Conclusion: According to ZFN efficiency, it can bind and cut the target sites, the bilateral cutting can affect the intensity of GFP fluorescence. Our flow cytometry results showed that this ZFN could reduce the intensity of GFP color and colony count of bacteria in media containing amp + kana versus control sample

The Molecular Basis of COVID-19 Pathogenesis, Conventional and Nanomedicine Therapy

In late 2019, a new member of the Coronaviridae family, officially designated as “severe acute respiratory syndrome coronavirus 2” (SARS-CoV-2), emerged and spread rapidly. The Coronavirus Disease-19 (COVID-19) outbreak was accompanied by a high rate of morbidity and mortality worldwide and was declared a pandemic by the World Health Organization in March 2020. Within the Coronaviridae family, SARS-CoV-2 is considered to be the third most highly pathogenic virus that infects humans, following the severe acute respiratory syndrome coronavirus (SARS-CoV) and the Middle East respiratory syndrome coronavirus (MERS-CoV). Four major mechanisms are thought to be involved in COVID-19 pathogenesis, including the activation of the renin-angiotensin system (RAS) signaling pathway, oxidative stress and cell death, cytokine storm, and endothelial dysfunction. Following virus entry and RAS activation, acute respiratory distress syndrome develops with an oxidative/nitrosative burst. The DNA damage induced by oxidative stress activates poly ADP-ribose polymerase-1 (PARP-1), viral macrodomain of non-structural protein 3, poly (ADP-ribose) glycohydrolase (PARG), and transient receptor potential melastatin type 2 (TRPM2) channel in a sequential manner which results in cell apoptosis or necrosis. In this review, blockers of angiotensin II receptor and/or PARP, PARG, and TRPM2, including vitamin D3, trehalose, tannins, flufenamic and mefenamic acid, and losartan, have been investigated for inhibiting RAS activation and quenching oxidative burst. Moreover, the application of organic and inorganic nanoparticles, including liposomes, dendrimers, quantum dots, and iron oxides, as therapeutic agents for SARS-CoV-2 were fully reviewed. In the present review, the clinical manifestations of COVID-19 are explained by focusing on molecular mechanisms. Potential therapeutic targets, including the RAS signaling pathway, PARP, PARG, and TRPM2, are also discussed in depth

Gene Editing-Based Technologies for <i>Beta-hemoglobinopathies</i> Treatment

Beta (β)-thalassemia is a group of human inherited abnormalities caused by various molecular defects, which involves a decrease or cessation in the balanced synthesis of the β-globin chains in hemoglobin structure. Traditional treatment for β-thalassemia major is allogeneic bone marrow transplantation (BMT) from a completely matched donor. The limited number of human leukocyte antigen (HLA)-matched donors, long-term use of immunosuppressive regimen and higher risk of immunological complications have limited the application of this therapeutic approach. Furthermore, despite improvements in transfusion practices and chelation treatment, many lingering challenges have encouraged researchers to develop newer therapeutic strategies such as nanomedicine and gene editing. One of the most powerful arms of genetic manipulation is gene editing tools, including transcription activator-like effector nucleases, zinc-finger nucleases, and clustered regularly interspaced short palindromic repeat–Cas-associated nucleases. These tools have concentrated on γ- or β-globin addition, regulating the transcription factors involved in expression of endogenous γ-globin such as KLF1, silencing of γ-globin inhibitors including BCL11A, SOX6, and LRF/ZBTB7A, and gene repair strategies. In this review article, we present a systematic overview of the appliances of gene editing tools for β-thalassemia treatment and paving the way for patients’ therapy