Development of an ostrich-derived single-chain variable fragment (scFv) against PTPRN extracellular domain.

In type 1 diabetes, the immune system destroys pancreatic beta cells in an autoimmune condition. To overcome this disease, a specific monoclonal antibody that binds to pancreatic beta cells could be used for targeted immunotherapy. Protein tyrosine phosphatase receptor N (PTPRN) is one of the important surface antigen candidates. Due to its high sequence homology among mammals, so far, no single-chain monoclonal antibody has been produced against this receptor. In this study, we developed a novel single-chain variable fragment (scFv) against the PTPRN extracellular domain. To this aim, ostrich species was used as a host is far phylogenetically birds from mammals to construct a phage display library for the first time. An ostrich-derived scfv phage display library was prepared and biopanning steps were done to enrich and screen for isolating the best anti-PTPRN binders. An scFv with appropriate affinity and specificity to the PTPRN extracellular domain was selected and characterized by ELISA, western blotting, and flow cytometry. The anti-PTPRN scFv developed in this study could be introduced as an effective tool that can pave the way for the creation of antibody-based targeting systems in cooperation with the detection and therapy of type I diabetes.

Candidate Biomarkers for Targeting in Type 1 Diabetes; A Bioinformatic Analysis of Pancreatic Cell Surface Antigens.

OBJECTIVE: Type 1 diabetes (T1Ds) is an autoimmune disease in which the immune system invades and destroys insulin-producing cells. Nevertheless, at the time of diagnosis, about 30-40% of pancreatic beta cells are healthy and capable of producing insulin. Bi-specific antibodies, chimeric antigen receptor regulatory T cells (CAR-Treg cells), and labeled antibodies could be a new emerging option for the treatment or diagnosis of type I diabetic patients. The aim of the study is to choose appropriate cell surface antigens in the pancreas tissue for generating an antibody for type I diabetic patients. MATERIALS AND METHODS: In this bioinformatics study, we extracted pancreas-specific proteins from two large databases; the Human Protein Atlas (HPA) and Genotype-Tissue Expression (GTEx) Portal. Pancreatic-enriched genes were chosen and narrowed down by Protter software for the investigation of accessible extracellular domains. The immunohistochemistry (IHC) data of the protein atlas database were used to evaluate the protein expression of selected antigens. We explored the function of candidate antigens by using the GeneCards database to evaluate the potential dysfunction or activation/hyperactivation of antigens after antibody binding. RESULTS: The results showed 429 genes are highly expressed in the pancreas tissue. Also, eighteen genes encoded plasma membrane proteins that have high expression in the microarray (GEO) dataset. Our results introduced four structural proteins, including NPHS1, KIRREL2, GP2, and CUZD1, among all seventeen candidate proteins. CONCLUSION: The presented antigens can potentially be used to produce specific pancreatic antibodies that guide CARTreg, bi-specific, or labeling molecules to the pancreas for treatment, detection, or other molecular targeted therapy scopes for type I diabetes.

Production and characterization of a camelid single domain anti-CD22 antibody conjugated to DM1.

Antibody drug conjugates (ADCs) with twelve FDA approved drugs, known as a novel category of anti-neoplastic treatment created to merge the monoclonal antibody specificity with cytotoxicity effect of chemotherapy. However, despite many undeniable advantages, ADCs face certain problems, including insufficient internalization after binding, complex structures and large size of full antibodies especially in targeting of solid tumors. Camelid single domain antibody fragments (Nanobody®) offer solutions to this challenge by providing nanoscale size, high solubility and excellent stability, recombinant expression in bacteria, in vivo enhanced tissue penetration, and conjugation advantages. Here, an anti-human CD22 Nanobody was expressed in E.coli cells and conjugated to Mertansine (DM1) as a cytotoxic payload. The anti-CD22 Nanobody was expressed and purified by Ni-NTA resin. DM1 conjugated anti-CD22 Nanobody was generated by conjugation of SMCC-DM1 to Nanobody lysine groups. The conjugates were characterized using SDS-PAGE and Capillary electrophoresis (CE-SDS), RP-HPLC, and MALDI-TOF mass spectrometry. Additionally, flow cytometry analysis and a competition ELISA were carried out for binding evaluation. Finally, cytotoxicity of conjugates on Raji and Jurkat cell lines was assessed. The drug-to-antibody ratio (DAR) of conjugates was calculated 2.04 using UV spectrometry. SDS-PAGE, CE-SDS, HPLC, and mass spectrometry confirmed conjugation of DM1 to the Nanobody. The obtained results showed the anti-CD22 Nanobody cytotoxicity was enhanced almost 80% by conjugation with DM1. The binding of conjugates was similar to the non-conjugated anti-CD22 Nanobody in flow cytometry experiments. Concludingly, this study successfully suggest that the DM1 conjugated anti-CD22 Nanobody can be used as a novel tumor specific drug delivery system.

Targeting of human fibroblast growth factor receptor 2 by a novel specific nanobody.

Inhibition of FGFR2 signaling is promising in targeted therapy of FGFR2-related tumors. In this study, anti-FGFR2 nanobodies (Nbs) were isolated through screening of an immune camelid phage display library. Four rounds of biopanning were carried out with commercial human FGFR2 antigen and enrichment was assessed by ELISA and phage titration. The gene of Nb was sub-cloned into the expression vector, and the recombinant vector was transformed into Escherichia coli WK6 cells. The recombinant protein was purified using Ni-NTA affinity chromatography. The anti-FGFR2 Nb (C13) was characterized by SDS-PAGE, western blotting, competitive inhibition ELISA, flow cytometry, MTT, and migration assay. C13 Nb recognized FGFR2 with high specificity and no cross-reactivity was observed with other tested antigens. The affinity of C13 Nb was calculated to be 1.5 × 10-9 M. Results of cytotoxicity showed that C13 Nb (10 µg/ml) inhibited 85% of the proliferation of T-47D cells (p < 0.001). In addition, C13 inhibited the migration of 68% of T-47D toward the source of the growth factor (p < 0.01). The flow cytometry showed that C13 Nb bound to the surface of FGFR2+ cells, T-47D cell line (96%). Results indicate the potential of anti-FGFR2 Nb for targeted therapy of FGFR2-overexpressing tumors after complementary investigations.

Site-specific transgene integration in chimeric antigen receptor (CAR) T cell therapies.

Chimeric antigen receptor (CAR) T cells and natural killer (NK) cells are genetically engineered immune cells that can detect target antigens on the surface of target cells and eliminate them following adoptive transfer. Recent progress in CAR-based therapies has led to outstanding clinical success in certain patients with leukemias and lymphomas and offered therapeutic benefits to those resistant to conventional therapies. The universal approach to stable CAR transgene delivery into the T/NK cells is the use of viral particles. Such approaches mediate semi-random transgene insertions spanning the entire genome with a high preference for integration into sites surrounding highly-expressed genes and active loci. Regardless of the variable CAR expression level based on the integration site of the CAR transgene, foreign integrated DNA fragments may affect the neighboring endogenous genes and chromatin structure and potentially change a transduced T/NK cell behavior and function or even favor cellular transformation. In contrast, site-specific integration of CAR constructs using recent genome-editing technologies could overcome the limitations and disadvantages of universal random gene integration. Herein, we explain random and site-specific integration of CAR transgenes in CAR-T/NK cell therapies. Also, we tend to summarize the methods for site-specific integration as well as the clinical outcomes of certain gene disruptions or enhancements due to CAR transgene integration. Also, the advantages and limitations of using site-specific integration methods are discussed in this review. Ultimately, we will introduce the genomic safe harbor (GSH) standards and suggest some appropriate safety prospects for CAR integration in CAR-T/NK cell therapies.

Development and Characterization of a Novel Single-Chain Antibody Against B-Cell Activating Factor.

As a member of the tumor necrosis factor (TNF) superfamily, the B-cell activating factor (BAFF) plays a crucial role in B-cell survival and differentiation. Overexpression of this protein has been closely linked to autoimmune disorders and some B-cell malignancies. Using monoclonal antibodies (mAbs) against the BAFF soluble domain appears to be a complementary treatment for some of these diseases. This study aimed to produce and develop a specific Nanobody (Nb), a variable camelid antibody domain, against the soluble domain of BAFF protein. After camel immunization with recombinant protein and preparing cDNA from total RNAs separated from camel lymphocytes, an Nb library was developed. Individual colonies capable of binding selectively to rBAFF were obtained by periplasmic-ELISA, sequenced, and expressed in a bacterial expression system. The specificity and affinity of selected Nb were determined and its target identification and functionality were evaluated using flow cytometry.

Positive effect of acellular amniotic membrane dressing with immobilized growth factors in skin wound healing.

The human amniotic membrane dressing has been shown to accelerate the wound healing process in the clinic. In this study, heparin was conjugated to a human Acellular Amniotic Membrane (hAAM) to provide affinity binding sites for immobilizing growth factors. To study the acceleration of the wound healing process, we bound epidermal growth factor and fibroblast growth factor 1 to heparinized hAAMs (GF-Hep-hAAMs). The heparinized hAAMs (Hep-hAAMs) were characterized by toluidine blue staining and infrared spectroscopy. The quality control of hAAM was performed by hematoxylin staining, swelling capacity test and biomechanical evaluation. The cytotoxicity, adhesion, and migration in vitro assays of GF-Hep-hAAMs on L-929 fibroblast cells were also studied by MTT assay, scanning electron microscopy, and scratch assay, respectively. Finally, in vivo skin wound healing study was performed to investigate the wound closure rate, re-epithelization, collagen deposition, and formation of new blood vessels. The results showed that GF-Hep-hAAMs enhance the rate of wound closure and epidermal regeneration in BALB/c mice. In conclusion, GF-Hep-hAAMs could accelerate the wound healing process, significantly in the first week.

In Vitro and In Vivo Studies of a Heminecrolysin Toxin-VEGF Fusion Protein as a Novel Therapeutic for Solid Tumor Targeting.

Angiogenesis, the formation of new vessels, is a critical step in the malignancy progression of solid tumors. Many investigations have demonstrated the usefulness of immunotoxins to halt angiogenesis in solid tumors. Pharmaceutically, Vascular Endothelial Growth Factor (VEGF) can deliver coupled toxins to the tumor vessels through VEGF Receptors. In the current study, we designed, expressed, and assessed the in vitro and in vivo toxicities of a novel immunotoxin consisting of mouse VEGF and heminecrolysin toxin (mVEGF-HNc). The fusion protein was expressed in E. coli and purified via Ni+2 affinity chromatography. The biological activity of immunotoxin was evaluated on NIH/3T3 cells and TC1-tumorized mouse model. The mVEGF-NHc showed significant cytotoxicity on the cells as VEGFR-expressing cells. Moreover, the size of the tumor in the mVEGF-HNc-treated group started to reduce after six injections, while it continued to grow in the PBS-received mice. Efficacious targeting of solid tumor cells via mVEGF-HNc suggests its prospective therapeutic potential for cancer therapy.

Enhancement of keratinocyte growth factor potential in inducing adipose-derived stem cells differentiation into keratinocytes by collagen-targeting.

Different growth factors can regulate stem cell differentiation. We used keratinocyte growth factor (KGF) to direct adipose-derived stem cells (ASCs) differentiation into keratinocytes. To enhance KGF bioavailability, we targeted KGF for collagen by fusing it to collagen-binding domain from Vibrio mimicus metalloprotease (vibrioCBD-KGF). KGF and vibrioCBD-KGF were expressed in Escherichia coli and purified to homogeneity. Both proteins displayed comparable activities in stimulating proliferation of HEK-293 and MCF-7 cells. vibrioCBD-KGF demonstrated enhanced collagen-binding affinity in immunofluorescence and ELISA. KGF and vibrioCBD-KGF at different concentrations (2, 10, and 20 ng/ml) were applied for 21 days on ASCs cultured on collagen-coated plates. Keratinocyte differentiation was assessed based on morphological changes, the expression of keratinocyte markers (Keratin-10 and Involucrin), and stem cell markers (Collagen-I and Vimentin) by real-time PCR or immunofluorescence. Our results indicated that the expression of keratinocyte markers was substantially increased at all concentrations of vibrioCBD-KGF, while it was observed for KGF only at 20 ng/ml. Immunofluorescence staining approved this finding. Moreover, down-regulation of Collagen-I, an indicator of differentiation commitment, was more significant in samples treated with vibrioCBD-KGF. The present study showed that vibrioCBD-KGF is more potent in inducing the ASCs differentiation into keratinocytes compared to KGF. Our results have important implications for effective skin regeneration using collagen-based biomaterials.

In vivo solid tumor targeting with recombinant VEGF-diphtheria immunotoxin.

Objectives: A variety of signaling molecules have been identified that play a role in angiogenesis, of prime importance, vascular endothelial growth factor (VEGF) and its resceptor (VEGFR), which is highly expressed in most human solid tumors. Targeting VEGF or/and VEGFR with immunotoxin may be a promising approach to directly affect cancer cells. Immunotoxins are for targeted treatment comprising two functional moieties, an antibody that binds to target cells along with toxin that kills molecules. Materials and Methods: In this study, an immunotoxin comprising domain of diphtheria toxin subunit A (DT386) genetically fused to mouse VEGF (mVEGF-DT) was developed. The second construct, which contains the DT386 domain, was made to investigate the action of the DT386 domain on tumor cells. Both gene constructs were cloned, expressed, and were further purified. The biological activity of mVEGF-DT and DT386 proteins was assessed on the TC1 cell line bearing mouse model. Proteins were injected intra-tumoral in mice, in separate groups. Results: Tumors in the mVEGF-DT group started to dwindle after six injections, but tumor size in both control groups (DT386 and PBS), continued to grow. Conclusion: Successful targeting of solid tumor cells by mVEGF-DT immunotoxin demonstrates the therapeutic potential utility of these conjugates for tumor targeting.

Extremely low frequency magnetic field enhances expression of a specific recombinant protein in bacterial host.

Expression of proteins in bacterial host cells, particularly E.coli, has gained much attention in recent years. Low expression outcome is the main technical drawback associated with this procedure, further restricting its largescale application in industry. Therefore, application of new amendments or reformations are required before further proceedings. Extremely low frequency magnetic fields (ELF-MFs) have shown to significantly affect biological processes, including gene expression, in E.coli. In current study, we investigated whether application of ELF-MF could result in overexpression of proteins in E.coli or not. Cluster of differentiation-22 (CD22), as a model protein, was expressed in E.Coli Rosetta (DE3) under continuous exposure to ELF-MF after applying various concentrations of Isopropyl ß-d-1-thiogalactopyranoside (IPTG) (0.25-1.25 mM) as inducer. The strength and frequency of electromagnetic fields (EMFs) ranged between 15 and 100 mT and 2.5-20 Hz respectively. Interestingly, application of 55 mT EMFs with frequencies ranging from 2.5 to 2.8 Hz significantly enhanced the yield of expression at all studied IPTG concentrations. Contrarily, EMFs with intensities other than 55 mT meaningfully declined protein expression at IPTG concentrations equal to 1 and 1.25 mM. In conclusion, application of specific range of ELF-MFs may be exploited as a new modification for enhancing heterologous expression of proteins in E.coli.

Tumor Suppression by PD-1/PD-L1 Interaction Blockage in Mice Model.

Background: Overexpression of programmed cell death ligand 1 (PD-L1) in tumor cells and subsequent interaction with the programmed cell death protein 1 (PD-1) in tumor-infiltrating T cells cause an immune evasion of the tumor from cytotoxic T-cells. Therefore, inhibiting such interaction by a recombinant PD-1 can hinder tumor growth and extend the survival rate. Methods: The mouse extracellular domain of PD-1 (mPD-1) was expressed in E. coli BL21 (DE3) strain and purified using nickel affinity chromatography. The binding ability of the purified protein to human PD-L1 was studied using ELISA. Finally, the tumor-bearing mice were used to evaluate the potential antitumor effect. Results: The recombinant mPD-1 showed a significant binding capacity to human PD-L1 at the molecular level. The tumor size significantly decreased in the tumor-bearing mice after the intra-tumoral injections of mPD-1. Moreover, the survival rate increased significantly after eight weeks of monitoring. The histopathology revealed the necrosis in the tumor tissue of the control group compared to the mPD-1 received mice. Conclusions: Our outcomes propose that interaction blockade between PD-1 and PD-L1 is a promising approach for targeted tumor therapy.

Expressing of Recombinant VEGFR2-specific Nanobody in Baculovirus Expression System.

BACKGROUND: Baculovirus expression system, introduced more than 20 years ago, is considered as a useful tool for large and complex eukaryotic recombinant protein production. A baculovirus expression vector is a recombinant virus which desired foreign protein coding sequences is under control of the virus gene promoter. Baculovirus only infects insect cells and do not normally infect vertebrates therefore, they possess no risk of biological risks for human. OBJECTIVES: The aim of this study was to recombinant expression of vascular endothelial growth factor (VEGF) reseptor-2 specific Nanobody in the baculovirus expression system. MATERIALS AND METHODS: Gene of specific Nanobody against the VEGF reseptor-2 that called 3VGR19 was cloned and expressed in baculovirus system. RESULTS: 3VGR19 Nanobody gene was amplified by Polymerase Chain Reaction (PCR) using the specific primers, and was cloned in pFastBac HTA plasmid. DH10Bac bacteria was transformed with resulted donor plasmid. The cultured Sf9 insect cell line was transfected with recombinant bacmid, and finally, the expression and purification of 3VGR19 was confirmed in insect cells. CONCLUSIONS: In conclusion, Transient infection of insect cells with baculovirus can be a promising technology for expression of antibody fragments.

Chemical inhibitor anticancer drugs regulate mechanical properties and cytoskeletal structure of non-invasive and invasive breast cancer cell lines: Study of effects of Letrozole, Exemestane, and Everolimus.

Regardless of their target and mechanism, anticancer drugs directly influence biological behavior of cancer cells by activating chemical signaling pathways. Due to the complex interaction between diverse signaling pathways, these drugs may profoundly impact the physical characteristics of cancer cells and regulate their mechanical properties. In this study, the effects of two Aromatase Inhibitor (Letrozole and Exemestane), and one mTOR Inhibitor (Everolimus) on cell mechanical properties, actin content/distribution, and nuclear areas of two invasive and non-invasive breast cancer cell line after 24 h treatment with concentrations previously reported were investigated. While metabolic activity of cell lines was highly affected by drug treatment, significant alterations in Young's modulus of cell bodies, nuclear areas, and actin content and distribution were reported with higher impact on invasive cells. It was concluded that regulation of mechanical behavior of cells by all three drugs emphasizes the cross talk between chemical and physical signaling cascades, and describes a correlation between biological and physical behaviors of cancer cells which might give an insight to a better understanding of mechanisms by which anti-cancer drugs function to enhance their performances.

Development of camelid monoclonal nanobody against SLC39A6 zinc transporter protein.

Objectives: SLC39A6 (solute carrier family 39) or LIV-1, is a zinc-transporter protein associated with estrogen-positive breast cancer and its metastatic spread. Significantly there is a direct relation between high zinc intake and unregulated cell proliferation and cancers. Blocking SLC39A6 protein may result in reduced metastasis and proliferation in many malignant tumors. This study aimed to develop an anti-SLC39A6 nanobody that is able to detect and block the SLC39A6 protein on the surface of cancerous cells. Materials and Methods: The recombinant SLC39A6 was expressed and used for camel immunization. The VHH library was constructed and screened for SLC39A6-specific nanobody. Then, the strength of nanobody in SLC39A6 detection was evaluated by Western blotting and flow cytometry. Results: We showed the ability of SLC39A6 specific Nanobody (C3) to detect SLC39A6 by Western blotting and flow cytometry. Furthermore, the C3 nanobody potently inhibits cell proliferation in MTT assay. Conclusion: These data show the potential of SLC39A6-specific nanobody for the blockade of zinc transporter protein and provide a basis for the development of novel cancer therapeutics.

Developing and characterizing a single-domain antibody (nanobody) against human cytotoxic T-lymphocyte-associated protein 4 (hCTLA-4).

OBJECTIVES: Cytotoxic T-lymphocyte-associated protein-4 (CTLA-4) is the most important human immune checkpoint that modulates T cells activity and brings about immune-homeostasis. Accordingly, checkpoint inhibitor cancer therapy has been approved as a growing method to block over-expressed immune checkpoints, such as CTLA-4 receptors. Considering the competitive characteristics of single-domain antibodies with monoclonal antibodies, we tried to develop a camelid Nanobody against human CTLA-4. MATERIALS AND METHODS: We have constructed the VHH gene library by using immunized-camel peripheral blood mononuclear cells and carrying out the Nested-PCR technique. VHH-library was screened by phage display technique and specific nanobodies against CTLA-4 protein were selected and amplified with bio-panning steps. Stronger binders were screened by Periplasmic Extract-ELISA, followed by estimating the complexity of the library. Specific anti-CTLA-4 Nanobody and 3hCTL55, with longer CDR3 and a higher binding rate, were selected for more assays. RESULTS: Results revealed the existence of two different clones in the library with 108 binders. In comparison with seven different antigens, using the ELISA technique confirmed the specificity of Nanobody 3hCTL55 against human CTLA-4 antigen. We calculated Nanobody 3hCTL55 affinity for human CTLA-4 antigen at 50×10-9 M, approximately. Performing western blot and Flow-cytometry techniques showed that Nanobody 3hCTL55 was able to specifically detect and attach both commercial human CTLA-4 protein and human CTLA-4 antigen on the cell surface and in the cell lysate. CONCLUSION: Taken together, this developed camelid-specific anti-CTLA-4 Nanobody 3hCTL55, selected from a high-quality immune library by phage display technique, may be effective for further study about cancer diagnosis and cancer-therapy purposes.

Selection and characterization of specific nanobody against neuropilin-1 for inhibition of angiogenesis.

Neuropilin-1 (NRP-1), non-tyrosine kinase receptor, was initially identified as axonal protein and later recognized as co-receptor for vascular endothelial growth factor (VEGF). Neuropilins (NRPs) are involved in vascular development and tumor angiogenesis. Over the last years, many studies have been accomplished to inhibit angiogenesis. In this study, the nanobody library was panned against immobilized NRP-1 antigen. High affinity and specificity nanobodies were selected through monoclonal ELISA. The selected nanobodies inhibited proliferation and tube formation of HUVEC and MCF-7 cells in vitro and ex vivo. The results highlight potential of anti-NRP1 nanobodies in inhibition of angiogenesis both in vitro and ex vivo and promises development of novel therapeutics against pathologic angiogenesis.

Development of a mono-specific anti-VEGF bivalent nanobody with extended plasma half-life for treatment of pathologic neovascularization.

Vascular endothelial growth factor (VEGF) plays a crucial role in angiogenesis within solid cancers. Thus, targeting VEGF might be part of a feasible therapy for treating pathological neovascularization, and nanobodies - derived from heavy chain-only antibodies occurring within Camelidae - are a novel class of nanometer-sized antibodies possessing unique properties that could be developed into a promising therapeutic. However, nanobodies have a very short half-life in vivo due to their small size. Development of a bivalent nanobody is one way to remediate the half-life problem of nanobodies. Two identical anti-VEGF nanobodies were connected using the hinge region of llama IgG2c. The recombinant plasmid (pHEN6c-bivalent nanobody) was transformed into E.coli WK6 cells and expression of the bivalent nanobody construct was induced with 1mM Isopropyl ß-D-1-thiogalactopyranoside (IPTG). Recombinant bivalent nanobody was purified using nickel affinity chromatography and its activity on human endothelial cells was assessed using 3-(4,5-Dimethylthiazol-2-yr)-2,5-diphenyltetrazolium bromide (MTT), tube formation, and cell migration assays. The pharmacokinetic study was performed after intravenous (i.v.) injection of recombinant bivalent nanobody into six-week-old C57BL/6 mice. Recombinant bivalent nanobody performed significantly better than monovalent nanobody in inhibiting proliferation, tube formation, and migration of human endothelial cells. Pharmacokinetic results showed a 1.8-fold longer half-life of bivalent nanobody in comparison with the monovalent nanobody. These results underscore the potential of recombinant anti-VEGF bivalent nanobody as a promising tool for development of a novel therapeutic with an extended plasma half-life for VEGF-related diseases.

Genotoxicity assessment of antiepileptic drugs (AEDs) in human embryonic stem cells.

Several antiepileptic drugs (AEDs) are administrated during pregnancy according to recent therapeutic protocols. Ten percent of pregnant women with epilepsy give birth to offspring with malformations and teratogenic defects. Since the mechanism of action of AEDs is not yet completely understood, therefore, it could be hypothesized that they may cause cyto- or genotoxicity in embryonic fetus cells. To investigate this hypothesis, the genotoxicity and cell survival of AEDs treated human embryonic stem cells (hESCs) were investigated by single-cell gel electrophoresis (Comet assay) and MTS assay, respectively. HESCs (Royan H6 cell line) were treated in-vitro with high therapeutic doses of Carbamazepine, Gabapentin, Lamotrigine, Levetiracetam or Topiramate as monotherapy or combination therapy of each drug with Folic acid. After hESCs pluripotency confirmation, the effect of AEDs on cellular DNA damage of hESCs was investigated. levetiracetam and topiramate were found to damage the DNA significantly compared to untreated cells. The amount of DNA damage produced by carbamazepine and lamotrigine was similar while for gabapentin, the amount of DNA migration was very low and produced less DNA damage than the others. A considerable reduction in DNA damages occurred in genotoxicity in the presence of Folic acid in comparison to AEDs monotherapies. According to our results, all mentioned AEDs caused DNA damage, while Levetiracetam and topiramate caused more extensive DNA damages than the others. Noticeably, the addition of Folic acid to the treated cells decreased the DNA damages considerably.

Evaluation of Laboratory Application of Camelid Sera Containing Heavy-Chain Polyclonal Antibody Against Recombinant Cytotoxic T-Lymphocyte-Associated Protein-4.

Cytotoxic T-lymphocyte-associated protein-4 (CTLA-4) is a critical negative immunomodulatory receptor that is normally expressed in activated T cells and noticeably, in many cancerous cells. Indeed, molecular detection of CTLA-4 protein is crucial in basic research. In this work, the extracellular domain of the human CTLA-4 (hCTLA-4) protein was cloned, expressed, and purified. Subsequently, this protein was used as an antigen for camel (Camelus dromedarius) immunization to obtain polyclonal camelid sera against this protein. Furthermore, we evaluated the benefits of applying camelid hyperimmune sera containing heavy-chain antibodies in different antibody-based techniques. Our results indicated that hCTLA-4 protein was successfully expressed in the prokaryotic system. The polyclonal antibody (pAb) that raised against recombinant hCTLA-4 protein was able to detect the CTLA-4 protein in antibody-based techniques, such as enzyme-linked immunosorbent assay, western blotting, flow cytometry and immunohistochemistry (IHC) staining. This study shows that, due to the advantages such as multi-epitope-binding ability, camelid pAbs are potent to efficiently apply for molecular detection of CTLA-4 receptors in fundamental antibody-based researches such as IHC.