Bioprinting in vascularization strategies

Three-dimensional [3D] printing technology has revolutionized tissue engineering field because of its excellent potential of accurately positioning cell-laden constructs. One of the main challenges in the formation of functional engineered tissues is the lack of an efficient and extensive network of microvessels to support cell viability. By printing vascular cells and appropriate biomaterials, the 3D printing could closely mimic in vivo conditions to generate blood vessels. In vascular tissue engineering, many various approaches of 3D printing have been developed, including selective laser sintering and extrusion methods, etc. The 3D printing is going to be the integral part of tissue engineering approaches; in comparison with other scaffolding techniques, 3D printing has two major merits: automation and high cell density. Undoubtedly, the application of 3D printing in vascular tissue engineering will be extended if its resolution, printing speed, and available materials can be improved

Cyclic Stretch Effects on Adipose-Derived Stem Cell Stiffness, Morphology and Smooth Muscle Cell Gene Expression

Recent investigations consider adipose-derived stemcells (ASCs) as a promising source of stemcells for clinical therapies. To obtain functional cells with enhanced cytoskeleton and aligned structure, mechanical stimuli are utilized during differentiation of stem cells to the target cells. Since function of muscle cells is associated with cytoskeleton, enhanced structure is especially essential for these cells when employed in tissue engineering. In this study by utilizing a custom-made device, effects of uniaxial tension (1Hz, 10% stretch) on cytoskeleton, cell alignment, cell elastic properties, and expression of smooth muscle cell (SMC) genes in ASCs are investigated.Due to proper availability ofASCs, results can be employed in cardiovascular engineeringwhen production of functional SMCs in arterial reconstruction is required. Results demonstrated that cells were oriented after 24 hours of cyclic stretch with aligned pseudo-podia. Staining of actin filaments confirmed enhanced polymerization and alignment of stress fibers. Such phenomenon resulted in stiffening of cell body which was quantified by atomic force microscopy (AFM). Expression of SM α-actin and SM22 α-actin as SMC associated genes were increased after cyclic stretch while GAPDH was considered as internal control gene. Finally, it was concluded that application of cyclic stretch on ASCs assists differentiation to SMC and enhances functionality of cells.

Low Levels of Extensively Drug-resistant Tuberculosis among Multidrug Resistant Tuberculosis Isolates and Their Relationship to Risk Factors: Surveillance in Tehran, Iran; 2006 to 2014

OBJECTIVES: Extensively drug-resistant tuberculosis (XDR-TB) is more expensive and difficult to treat than multidrug-resistant tuberculosis (MDR-TB), and outcomes for patients are much worse; therefore, it is important that clinicians understand the magnitude and distribution of XDR-TB. We conducted a retrospective study to compare the estimated incidence of and risk factors for M/XDR-TB with those of susceptible TB controls. METHODS: Sputum culture and drug susceptibility testing (DST) were performed in patients with known or suspected TB. Strains that were identified as MDR were subjected to DST for second-line drugs using the proportion method. RESULTS: Among 1,442 TB patients (mean age, 46.48 ± 21.24 years) who were culture-positive for Mycobacterium tuberculosis, 1,126 (78.1%) yielded isolates that were resistant to at least one first-line drug; there were 33 isolates (2.3%) of MDR-TB, of which three (0.2%) were classified as XDR-TB. Ofloxacin resistance was found in 10 (0.7%) isolates. Women were 15% more likely than men to yield M/XDR-TB isolates, but this difference was not significant. In a multivariate analysis comparing susceptible TB with X/MDR-TB, only one variable—the number of previous treatment regimens—was associated with MDR (odds ratio, 1.06; 95% confidence interval, 1.14–21.2). CONCLUSION: The burden of M/XDR-TB cases is not sizeable in Iran. Nonetheless, strategies must be implemented to identify and cure patients with pre-XDR-TB before they develop XDR-TB. Our results provide a greater understanding of the evolution and spread of M/XDR-TB in an environment where drug-resistant TB has a low incidence.

Construction of various gamma 34.5 deleted fluorescent-expressing oncolytic herpes simplex type 1 [oHSV] for generation and isolation of HSV-based vectors

Background: Oncolytic herpes simplex virus [oHSV]-based vectors lacking gamma 34.5 gene, are considered as ideal templates to construct efficient vectors for [targeted] cancer gene therapy. Herein, we reported the construction of three single/dually-flourescence labeled and gamma 34.5-deleted, recombinant HSV-1 vectors for rapid generation and easy selection/isolation of different HSV-Based vectors

Methods: Generation of recombinant viruses was performed with conventional homologous recombination methods using green fluorescent protein [GFP] and BleCherry harboring shuttle vectors. Viruses were isolated by direct fluorescence observation and standard plaque purifying methods and confirmed by PCR and sequencing and flow cytometry. XTT and plaque assay titration were performed on Vero, U87MG, and T98 GBM cell lines

Results: We generated three recombinant viruses, HSV-GFP, HSV-GR [Green-Red], and HSV-Red. The HSV-GFP showed two log higher titer [1010 PFU] than wild type [108 PFU]. In contrast, HSV-GR and HSV-Red showed one log lower titer [107 PFU] than parental HSV. Cytotoxicity analysis showed that HSV-GR and HSV-Red can lyse target tumor cells at multiplicity of infection of 10 and 1 [P<0.001]. Moreover, HSV-GFP showed higher infection potency [98%] in comparison with HSV-GR [82%]

Conclusion: Our oHSVs provide a simple and an efficient platform for construction and rapid isolation of 2[nd] and 3[rd] generation oHSVs by replacing the inserted dyes with transgenes and also for rapid identification via fluorescence activated cell sorting. These vectors can also be used for tracing the efficacy of therapeutic agents on target cells, imaging of neural or tumoral cells in vitro/in vivo and as oncolytic agents in cancer therapy

Effects of electromagnetic stimulation on gene expression of mesenchymal stem cells and repair of bone lesions

Objective: Most people experience bone damage and bone disorders during their lifetimes. The use of autografts is a suitable way for injury recovery and healing. Mesenchymal stem cells [MSCs] are key players in tissue engineering and regenerative medicine. Their proliferation potential and multipotent differentiation ability enable MSCs to be considered as appropriate cells for therapy and clinical applications. Differentiation of stem cells depends on their microenvironment and biophysical stimulations. The aim of this study is to analyze the effects of an electromagnetic field on osteogenic differentiation of stem cells

Materials and Methods: In this experimental animal study, we assessed the effects of the essential parameters of a pulsatile electromagnetic field on osteogenic differentiation. The main purpose was to identify an optimum electromagnetic field for osteogenesis induction. After isolating MSCs from male Wistar rats, passage-3 [P3] cells were exposed to an electromagnetic field that had an intensity of 0.2 millitesla [mT] and frequency of 15 Hz for 10 days. Flow cytometry analysis confirmed the mesenchymal identity of the isolated cells. Pulsatile electromagnetic field-stimulated cells were examined by immunocytochemistry and real-time polymerase chain reaction [PCR]

Results: Electromagnetic field stimulation alone motivated the expression of osteogenic genes. This stimulation was more effective when combined with osteogenic differentiation medium 6 hours per day for 10 days. For the in vivo study, an incision was made in the cranium of each animal, after which we implanted a collagen scaffold seeded with stimulated cells into the animals. Histological analysis revealed bone formation after 10 weeks of implantation

Conclusion: We have shown that the combined use of chemical factors and an electromagnetic field was more effective for inducing osteogenesis. These elements have synergistic effects and are beneficial for bone tissue engineering applications

Expression, Purification and Functional Assessment of Smallest Isoform of Human Interleukin-24 in Escherichia coli

ABSTRACT Interleukin-24 (IL-24) is a novel tumor-suppressor gene that has different alternative splice isoforms. It has been shown that new smallest isoform of human IL-24 gene, lacking three exons, induces higher levels of cytotoxicity than all the isoforms, indicating shortest isoform of IL-24 may be a new promising anti-cancer agent. In this study, we aimed to provide a reproducible method for recombinant production of the smallest isoform of IL-24 (sIL-24). The Structure of sIL-24 was analyzed using bioinformatics tools (I-TASSER, Prosa, RAMPAGE and SPDBV version 4.1). The DNA sequence encoding sIL-24 was chemically synthesized and sub-cloned into the pET-32a (+) vector for further protein expression in Escherichia coli BL21 (DE3) strain. Upon IPTG induction, sIL-24 peptide was expressed as a thioredoxin fusion protein. The recombinant sIL-24 was released from the fusion by TEV protease cleavage followed by nickel affinity chromatography. The yield of the purified sIL-24 was estimated about 380 μg/ml. MTT assay showed that sIL-24 peptide inhibited the proliferation of PC-3 cancer cells more effectively than full length IL-24 protein, while none affect the survival of MRC-5 normal cells. These results indicate that the presented expression system is an efficient system for the production of small functional recombinant sIL-24 peptide.This functional peptide may have cancer therapeutic application.

Expression of COLLAGEN 1 and ELASTIN genes in mitral valvular interstitial cells within microfiber reinforced hydrogel

Objective: The incidence of heart valve disease is increasing worldwide and the number of heart valve replacements is expected to increase in the future. By mimicking the main tissue structures and properties of heart valve, tissue engineering offers new options for the replacements. Applying an appropriate scaffold in fabricating tissue-engineered heart valves [TEHVs] is of importance since it affects the secretion of the main extracellular matrix [ECM] components, collagen 1 and elastin, which are crucial in providing the proper mechanical properties of TEHVs

Materials and Methods: Using real-time polymerase chain reaction [PCR] in this experimental study, the relative expression levels of COLLAGEN 1 and ELASTIN were obtained for three samples of each examined sheep mitral valvular interstitial cells [MVICs]-seeded onto electrospun poly [glycerol sebacate] [PGS]-poly [?-caprolactone] [PCL] microfi-brous, gelatin and hyaluronic acid based hydrogel-only and composite [PGS-PCL/hydrogel] scaffolds. This composite has been shown to create a synthetic three-dimensional [3D] microenvironment with appropriate mechanical and biological properties for MVICs

Results: Cell viability and metabolic activity were similar among all scaffold types. Our results showed that the level of relative expression of COLLAGEN 1 and ELASTIN genes was higher in the encapsulated composite scaffolds compared to PGS-PCL-only and hydrogel-only scaffolds with the difference being statistically significant [P<0.05]

Conclusion: The encapsulated composite scaffolds are more conducive to ECM secretion over the PGS-PCL-only and hydrogel-only scaffolds. This composite scaffold can serve as a model scaffold for heart valve tissue engineering

Head and neck metastatic tumors: a retrospective survey of Iranian patients

Statement of the Problem: The head and neck region is an uncommon site for metastatic involvement, but it can be the first and only symptom of primary cancer. The incidence of these tumors and their primary origins are limited in Iranian patients

Purpose: Therefore, this retrospective study aimed to investigate the frequency and the common related clinical manifestations, as well as, the most common types of cancers and the prevalent sites of the primary tumor

Materials and Method: All medical records related to patients with history of head and neck tumors between 1991 and 2011 at Iran Cancer Institute were evaluated and the essential information was statistically analyzed

Results: Sixty cases of cervical lymph node metastasis [0.36%] and 26 cases of head and neck metastatic tumors [0.16%] including 17 cases of distant cancer [0.10%] were recorded among all 16232 registered cancers. Out of all distant head and neck metastatic tumors, 4 cases were related to oral and maxillofacial area. Pain, swelling of neck, oral mucosa ulcer and dryness were the chief complaints. Squamous cell carcinoma and adenocarcinoma were the most frequent types of cancers. The most common metastatic sites were cervical musculature, scalp and parotid gland, and the most prevalent sites of primary tumor in females were breast and lung in males

Conclusion: According to these cases, the incidence rate of head and neck metastatic tumors seems to be low. However, feasible similarity of clinical presentation of oral metastatic lesions to benign lesions might result in misdiagnosis. Hence, biopsy is mandatory in any case with unusual clinical presentation, especially in patients with a known malignant disease

Genipin cross-linked electrospun chitosan-based nanofibrous mat as tissue engineering scaffold

To improve water stability of electrospun chitosan/ Polyethylene oxide [PEO] nanofibers, genipin, a biocompatible and nontoxic agent, was used to crosslink chitosan based nanofibers. Different amounts of genipin were added to the chitosan/PEO solutions, chitosan/PEO weight ratio 90/10 in 80% acetic acid, and the solutions were then electrospun to form nanofibers. The spun nanofibers were exposed to water vapor to complete crosslinking. The nanofibrous membranes were subjected to detailed analysis by scanning electron microscopy [SEM], Fourier transform infrared-attenuated total reflection [FTIR-ATR] spectroscopy, swelling test, MTT cytotoxicity, and cell attachment. SEM images of electrospun mats showed that genipin-crosslinked nanofibers retained their fibrous structure after immerging in PBS [pH=7.4] for 24 hours, while the uncrosslinked samples lost their fibrous structure, indicating the water stability of genipincrosslinked nanofibers. The genipin-crosslinked mats also showed no significant change inswelling ratio in comparison with uncrosslinked ones. FTIR-ATR spectrum of uncrosslinked and genipin-crosslinked chitosan nanofibers revealed the reaction between genipin and amino groups of chitosan. Cytotoxicity of genipin-crosslinked nanofibers was examined by MTT assay on human fibroblast cells in the presence of nanofibers extraction media. The genipincrosslinked nanofibers did not show any toxic effects on fibroblast cells at the lowest and moderate amount of genipin. The fibroblast cells also showed a good adhesion on genipincrosslinked nanofibers. This electrospun matrix would be used for biomedical applications such as wound dressing and scaffold for tissue engineering without the concern of toxicity

In vitro therapeutic effects of low level laser at mrna level on the release of skin growth factors from fibroblasts in diabetic mice

Numerous in vitro reports suggest that Low Level Laser Therapy [LLLT] affects cellular processes by biostimulation, however most of them emphasize on using visible light lasers which have low penetration. The aim of this study was to determine the effect of infrared laser light [which is more useful in clinic because of its higher penetration] on secretion of Fibroblast Growth Factor [FGF], Platelet Derived Growth Factor [PDGF] and Vascular Endothelial Growth Factor [VEGF], as important growth factors in wound healing. Fibroblasts were extracted from the skin of 7 diabetic and 7 nondiabetic mice and cultured. Cell cultures of experimental group were irradiated with single dose of LLLT [energy density of 1 J/ cm[2]] using an 810 nm continuous wave laser and the control group was not irradiated. Secretion of growth factors by skin fibroblasts were quantified through real time polymerase chain reaction. Diabetic irradiated group showed significant increase in FGF [p=0.017] expression, although PDGF increased and VEGF decreased in both diabetic and nondiabetic irradiated groups, but these variations were not statistically significant. These results suggest that LLLT may play an important role in wound healing by stimulating the fibroblasts

Expression enhancement in trastuzumab therapeutic monoclonal antibody production using genomic amplification with methotrexate

Trastuzumab [Herceptin] is a humanized monoclonal antibody [mAb] which is used for specific treatment of metastatic breast cancer in patients with overexpression of HER2/neu receptor. In this study, we have attempted to develop a biosimilar version of trastuzumab mAb. According to in silico studies, the heavy and light chains of trastuzumab mAb were designed and constructed. The recombinant constructs were co-transfected in CHO DG44 cell line. Stable transformants were selected on a semi solid medium. Genomic amplification with methotrexate was achieved for heavy chain gene amplification. Biological activity of produced antibody in comparison with Herceptin was tested by flow cytometry method. Three folds of amplification were obtained after seven rounds of methotrexate treatments. The results indicated the equal expression level of heavy and light chains. The yield of purified mAb was between 50 to 60 mg/l /day. According to the results, the produced mAb had similar affinity to HER2[+] tumor cells to that of Herceptin. High-level recombinant protein expression can be achieved by amplification of the recombinant gene with a selectable marker, such as Dihydrofolate Reductase [DHFR]. It is usually accepted that DHFR gene can be amplified in DHFR CHO cells, which consequently leads to amplification of the co-linked target gene, and finally amplification of recombinant protein. In this research, with the aim of producing a biosimilar version of herceptin, the effect of genomic amplification was investigated on the increasing the gene copy number using quantitative real-time PCR

Effect of heavy metals on silencing of engineered long interspersed element-1 retrotransposon in nondividing neuroblastoma cell line

L1 retrotransposons are the most active mobile DNA elements in human genome. Unregulated L1 retrotransposition may have deleterious effect by disrupting vital genes and inducing genomic instabilities. Therefore, human cells control L1 elements by silencing their activities through epigenetic mechanisms. It has been shown that cell division and heavy metals stimulate the frequency of L1 activities. Removal of silencing by L1 motivators may restart L1 element functions. Here, we have proposed that weather neurotoxic environmental heavy metals [as L1 stimulating factors] have a role in removing L1 silencing and restating its activities in nondividing neuronal cells. L1-RP green fluorescent protein [GFP]-tagged knock-in human neuroblastoma clones were prepared. Single-cell clone was treated with mitomycin-c combined with nontoxic and toxic concentrations of iron [Fe], copper [Cu], and mercury [Hg]. Silencing status of engineered L1 elements in dividing and nondividing cells was determined through measuring the amount of GFP expressing cells with flow cytometry. The cytotoxic effect of mitomycin-c combined with metals was measured by MTT assay. Hg in nondividing cells and Fe, Cu, and Hg in dividing neuroblastoma cells could significantly remove L1 silencing. Also, mitomycin-c treatment did not have any effect on metal toxicity status in neuroblastoma cells.Totally, our findings have shown that cell division has a role in removing L1 silencing as well as L1 retrotransposition induced by environmental heavy metals. It has been also indicated that Hg at all concentrations could remove silencing of engineered L1 element regardless of cell cycle state

[Effect of mechanical stimulations on the fate of stem cells - a review]

The provision of an adequate quantity of cells with proper function and purity is one of the main challenges of tissue engineering studies. Stem cells, with their self-renewal and differentiation capacity, are considered one of the main cell sources in the field of tissue engineering. Previously, the use of chemical factors seemed to be the only possible way for stem cell differentiation. However, scientists have recently realized that physiological processes of the human body are composed of chemical, mechanical and electrical signals. Mechanical stimulation is one of the current methods that produce cells with proper morphology and alignment in the scaffold. Specific differentiation, a higher rate of cell growth, proliferation and differentiation, and lower experiment costs can be achieved using mechanical stimulation. Different parameters such as the chemical environment, physical environment that surrounds the cell [including geometry, stiffness and topology of scaffold surface], amplitude, frequency, and duration of mechanical stimulation can affect the stem cell fate. In this study we have investigated the impact of all types of mechanical stimulations under different loading regimes on the fate of stem cells with respect to the target tissue. The result has been reflected in the design of a proper bioreactor

Mesenchymal stem cells as an alternative for schwann cells in rat spinal cord injury

Spinal cord has a limited capacity to repair; therefore, medical interventions are necessary for treatment of injuries. Transplantation of Schwann cells has shown a great promising result for spinal cord injury [SCI]. However, harvesting Schwann cell has been limited due to donor morbidity and limited expansion capacity. Furthermore, accessible sources such as bone marrow stem cells have drawn attentions to themselves. Therefore, this study was designed to evaluate the effect of bone marrow-derived Schwann cell on functional recovery in adult rats after injury. Mesenchymal stem cells were cultured from adult rats' bone marrow and induced into Schwann cells in vitro. Differentiation was confirmed by immunocytochemistry and RT-PCR. Next, Schwann cells were seeded into collagen scaffolds and engrafted in 3 mm lateral hemisection defects. For 8 weeks, motor and sensory improvements were assessed by open field locomotor scale, narrow beam, and tail flick tests. Afterwards, lesioned spinal cord was evaluated by conventional histology and immunohistochemistry. In vitro observations showed that differentiated cells had Schwann cell morphology and markers. In this study, we had four groups [n = 10 each]: laminectomy, control, scaffold and scaffold + Schwann cells. Locomotor and sensory scores of cell grafted group were significantly better than control and scaffold groups. In histology, axonal regeneration and remyelination were better than control and scaffold groups. This study demonstrates that bone marrow-derived Schwann cells can be considered as a cell source for Schwann cells in SCI treatment

[Preparation, characterization and biological assessment of polycaprolactone/starch composites for bone tissue engineering applications]

Biodegradable polycaprolactone/starch composites can be used for bone tissue engineering applications. The effect of the ratio of components on composite properties is of tremendous importance. Polycaprolactone/starch composite of 80/20 and 70/30 ratios were fabricated by dissolving them in chloroform followed by evaporation of the solvent. The composites were characterized by fourier transform infrared spectroscopy. Their bioactivity was evaluated by studying the apatite formation ability after immersing the specimens in simulated body fluid. The results of compressive test on samples showed that the composite's modulus and strength approximated that of human trabecular bone. Mass loss in distilled water and starch degradation rate in PBS was evaluated, which showed that the starch ratio was effective in composite degradation. MTT analysis and alkaline phosphatase levels showed that this composite had no toxicity and could increase G-299 cell line osteoblastic activities. The increase in cellular osteoblastic activities and the ability for apatite formation on the composite surface, in addition to the polycaprolactone/starch samples' mechanical properties shows their capability to be used as substitutes for bone. Because this composite degradation rate is controlled by changing the starch ratio, it has the potential for use in bone tissue engineering applications. Samples that have a 70/30 ratio are considered optimal due to their enhanced cellular response and better mechanical properties

Healing potential of mesenchymal stem cells cultured on a collagen-based scaffold for skin regeneration

Wound healing of burned skin remains a major goal in public health. Previous reports showed that the bone marrow stem cells were potent in keratinization and vascularization of full thickness skin wounds. In this study, mesenchymal stem cells were derived from rat adipose tissues and characterized by flowcytometry. Staining methods were used to evaluate their differentiation ability. A collagen-chitosan scaffold was prepared by freeze-drying method and crosslinked by carbodiimide-based crosslinker. The results of immunecytochemistry and PCR experiments confirmed the adipose-derived stem cells [ASC] in differentiation to the keratinocytes under the treatment of keratinocyte growth factor. The isolated ASC were seeded on the scaffolds and implanted at the prepared wounds. The scaffolds without cells were considered as a control and implanted on the other side of the rat. Histopathological analyses confirmed the formation of new tissue on the scaffold-cell side after 14 days with the formation of dermis and epidermis. These results indicated the capacity of ASC in differentiation to keratinocytes and also wound healing in vivo

Hepatitis B virus genome asymmetry in Hepatocellular Carcinoma

The association between hepatitis B virus [HBV] mutations and hepatocar-cinogenesis were reported in the literature. Preference for G over C in the leading DNA strand has been reported to account for the asymmetry in nucleotide [nt] composition. The aim of this study was to analyze the complete genome sequence and compositional asymmetry of HBV in different stages of hepatitis B. Full genome sequencing of 24 patients with chronic hepatitis B, some of whom also had cirrhosis and hepatocellular carcinoma [HCC] was performed. Mutations analysis was implemented in a comparison with a HBV genotype D reference from an international DNA database. CpGProD, a web-based application, was used to evaluate CG content and predict CpG islands. All strains were 3182 base pairs [bp] in length, except for two cases of HCC in which 9 and 21 nt, respectively, were deleted in preS2. The genetic relatedness of these isolates was 97%-100%. There were common CpG-rich regions in all 24 isolated full genome sequences, however a strong negative GC skew for forming a CpG island in the minus strand were exhibited in overlap with enhancer I in three HCC patients, a cirrhotic patient and three with chronic hepatitis. The high percentage of sequence identity between HBV isolates in our patients demonstrates that genomic factors, except for genotype, are involved in hepatocarcinogenesis. Variations in GC content which were caused by a different spectrum of mutations may affect DNA compositional asymmetry and epigenetic modification of HBV DNA in HCC

HESA-A exerts its cytoprotective effects through scavenging of free radicals: an in vitro study

Natural medicines have been recently considered more reasonable for human use most notably due to their safety and tolerance. HESA-A is a marine-originated herbal medicine with a variety of healing effects. However, its exact biological mechanism is not clear. The present study aimed at the evaluation of the HESA-A antioxidant effect. Chinese hamster ovary [CHO] and human embryonic kidney [HEK293T] cells were treated with different concentrations of HESA-A and H2O2 followed by cell proliferation assays. The antioxidant effect of the HESA-A preparations was evaluated by an antioxidant assay kit. The viability of CHO and HEK293T cells were about 89% following their incubation with 100 and 200 ng/ml HESA-A, respectively for 1.5 hrs. However, when the cells were incubated with concentrations of 300 ng/ml or more, the cell viability significantly decreased to 48% compare to the control cells. The cytotoxic effects of H2O2 were observed after 2 hrs of incubation of the HEK293T or CHO cells with 10 mM or 16 mM H2O2, respectively, while in the presence of HESA-A the cytotoxicity was significantly decreased. Antioxidant assay revealed that HESA-A scavenges free radicals. The findings indicate that HESA-A had cytoprotective effects in vitro, and that such an effect might be due to antioxidant properties

Functional Recombinant Extra Membrane Loop of Human CD20, an Alternative of the Full Length CD20 Antigen

Targeting of CD20 antigen with monoclonal antibodies has become the mainstay in the treatment of non-Hodgkin's lymphomas and immunotherapeutic depletion of malignant B cells. Accessibility of antigen is one of the crucial factors in development of monoclonal antibodies against this antigen. One major problem in expression of full length CD20 is aggregation and misfolding. Therefore, production of an alternative polypeptide is easer and favorable comparing to that of a full length transmembrane protein CD20. In this study, we expressed the extra membrane loop of hCD20 [exCD20] consisting of a non-glycosylated 47-amino acids region. The exCD20 coding sequence was amplified by PCR and cloned in pET32a[+] expression vector. The desired protein was expressed in fusion with thioredoxin and 6 His tag in E. coll Origami strain. ELISA and Western-blotting data were performed to indicate the functionality of this protein. We have obtained the exCD20 recombinant protein which can be detected in ELISA and Western-blot experiments. This recombinant fusion protein was soluble and stable without aggregation and misfolding problems. Conclusion: The recombinant extra membrane loop of human CD20 protein in fusion with thioredoxin [exCD20] can be used in function assays and some applications such as ELISA, immuneblotting, affinity purification, immunization, screening, and development of anti-CD20 antibodies