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Despite the widespread use of the Bacillus Calmette–Guérin (BCG) vaccine, Mycobacterium tuberculosis (MTB) continues to be a global burden. Vaccination has been proposed to prevent and treat tuberculosis (TB) infection, and several of them are in different phases of clinical trials. Though vaccine production is in progress but requires more attention. There are several TB vaccines in the trial phase, most of which are based on a combination of proteins/adjuvants or recombinant viral vectors used for selected MTB antigens. In this review, we attempted to discuss different types of TB vaccines based on the vaccine composition, the immune responses generated, and their clinical trial phases. Furthermore, we have briefly overviewed the effective delivery systems used for the TB vaccine and their effectiveness in different vaccines.

It was previously reported that crocin, a water-soluble carotenoid isolated from the Crocus sativus L. (saffron), has protective effects on cardiac cells and may neutralize and even prevent the formation of excess number of free radicals; however, functional mechanisms of crocin activity have been poorly understood. In the present research, we aimed to study the functional mechanism of crocin in the heart exposed to oxidative stress. Accordingly, oxidative stress was modeled in vitro on human umbilical vein endothelial cells (HUVECs) and in vivo in mice using cellular stressors. The beneficial effects of crocin were investigated at cellular and molecular levels in HUVECs and mice hearts. Results indicated that oral administration of crocin could have protective effects on HUVECs. In addition, it protects cardiac cells and significantly inhibits inflammation via modulating molecular signaling pathways TLR4/PTEN/AKT/mTOR/NF-κB and microRNA (miR-21). Here we show that crocin not only acts as a direct free radical scavenger but also modifies the gene expression profiles of HUVECs and protects mice hearts with anti-inflammatory action under oxidative stress.

ABSTRACT TcdA and TcdB are known as the major virulence attributes of Clostridioides difficile. Hence, neutralizing the TcdA and TcdB activities can be considered as an efficient therapeutic approach against C. difficile infection (CDI). In this work, we utilized phage display technique to select single-chain fragment variable (scFv) fragments as recombinant antibodies displayed on the surface of phages, which specifically target native TcdA, or TcdB (nTcdA and nTcdB), and their recombinant C-terminal combined repetitive oligopeptide (CROP) domains (rTcdA and rTcdB). After three rounds of biopanning, abundance of phage clones displaying high reactivity with TcdA or TcdB was quantified through enzyme-linked immunosorbent assay (ELISA). Furthermore, selected scFvs were characterized by cell viability and neutralization assays. The gene expression of immunological markers, IL-8 and TNF-α, was examined in treated Caco-2 cells by RT-qPCR. The epitopes of neutralizing scFvs were also identified by molecular docking. Totally, 18 scFv antibodies (seven for TcdA and 11 for TcdB) were identified by ELISA. Among selected scFvs, two clones for TcdA (rA-C2, A-C9) and three clones for TcdB (rB-B4, B-F5, B-F11) exhibited the highest neutralizing activity in Caco-2 and Vero cells. Moreover, the cocktail of anti-TcdA and anti-TcdB antibodies notably decreased the mRNA expression of TNF-α and IL-8 in Caco-2 cells. Molecular docking revealed that the interaction between scFv and toxin was mostly restricted to CROP domain of TcdA or TcdB. Our results collectively provided more insights for the development of neutralizing scFvs against C. difficile toxins using phage display. Further research is needed to meticulously evaluate the potential of scFvs as an alternative treatment for CDI using animal models and clinical trials. IMPORTANCE Targeting the major toxins of Clostridioides difficile by neutralizing antibodies is a novel therapeutic approach for CDI. Here, we report a panel of new anti-TcdA (rA-C2, A-C9) and anti-TcdB (rB-B4, B-F5, and B-F11) recombinant antibody fragments (scFvs) isolated from Tomlinson I and J libraries using phage display technique. These scFv antibodies were capable of neutralizing their respective toxin and showed promise as potential therapeutics against TcdA and TcdB of C. difficile in different in vitro models. In addition, in silico analysis showed that at least two neutralization mechanisms, including inhibiting cell surface binding of toxins and inhibiting toxin internalization can be proposed for the isolated scFvs in this work. These findings provide more insights for the applicability of specific scFvs toward C. difficile toxins at in vitro level. However, further research is required to evaluate the potential application of these scFvs as therapeutic agents for CDI treatment in clinical setting.

Introduction: Breast cancer is the most serious cause of women’s death throughout the world. Using nanocarrier vehicles to the exact site of cancer upgrades the therapeutic efficiency of the drugs. Capsulation of active proteins in the vesicular liposomes’ hydrophilic core is essential to develop a therapeutic protein carrier system. We aimed to encapsulate the medicinal leech saliva extract (LSE) and assess the inhibition of angiogenesis of breast cancer cells by targeting vascular endothelial growth factor A (VEGFA). Methods: In this research, enhanced formulation of liposomal protein was determined by zeta potential analysis, droplet size, drug release assay, and transmission electron microscopy (TEM). Furthermore, a cytotoxicity assay of liposomal LSE was performed to determine the cytotoxic activity of components. For assessing the expression of VEGFA, P53, and hypoxia-inducible factor subunit alpha (HIF1a) genes, Real-Time PCR was applied. Results: Nano liposome was chosen as an enhanced formulation due to its much smaller size (46.23 nm). Liposomal LSE had more practical actions on the MCF-7 cells. As noticed by DAPI staining, apoptosis was extensively greater in treated MCF-7 cells. Wound healing assay demonstrated that MCF-7 cells could not sustain growth at the presence of liposomal LSE and expression of the VEGFA gene was declined in treated cells. Downregulation of VEGFA was evaluated with western blotting technique. Conclusion: It can be concluded that our investigation of the tests confirmed the fact that nano liposomal LSE is a novel promising formulation for anticancer drugs and can significantly improve the penetration of protein drugs to cancer cells.

Cancer stem cells (CSCs) with their self-renewal ability are accepted as cells which initiate tumors. CSCs are regarded as interesting targets for novel anticancer therapeutic agents because of their association with tumor recurrence and resistance to conventional therapies, including radiotherapy and chemotherapy. Chimeric antigen receptor (CAR)-T cells are engineered T cells which express an artificial receptor specific for tumor associated antigens (TAAs) by which they accurately target and kill cancer cells. In recent years, CAR-T cell therapy has shown more efficiency in cancer treatment, particularly regarding blood cancers. The expression of specific markers such as TAAs on CSCs in varied cancer types makes them as potent tools for CAR-T cell therapy. Here we review the CSC markers that have been previously targeted with CAR-T cells, as well as the CSC markers that may be used as possible targets for CAR-T cell therapy in the future. Furthermore, we will detail the most important obstacles against CAR-T cell therapy and suggest solutions.

Background: In spite of Bacillus Calmette-Guerin (BCG) vaccination, still tuberculosis caused by Mycobacterium tuberculosis remains a problematic impediment that requires perspective management. The aim of this study was to evaluate the immunogenicity of chitosan nanoparticles containing recombinant mycobacterial proteins and adjuvant in the Balb/C mice through a non-invasive nasal inhalation delivery route and measure the level of cytokines interferon gamma (IFN-γ), interleukin-4 (IL-4), and IL-17. Methods: Thirty mice in five different groups were vaccinated through inhalation with compounds set in different combinations. Two weeks after the last nasal delivery, IFN-γ, IL-4, and IL-17 were measured in spleen cell culture supernatants. Results: The IFN-γ and IL-17 concentrations were found to increase in the groups that received chitosan nanoparticles containing protein and adjuvant alone or as a BCG booster. Our study showed that the chitosan nanoparticle containing protein and adjuvant induced a Th1 response. However, the groups that first received BCG and then chitosan nanoparticles containing protein and adjuvant had the greatest Th1 response in terms of IFN-γ and IL-17 production in all the groups. Conclusion: Our findings showed that the vaccine designed to be administered through the nasal mucosa well stimulates cellular immunity and enhances the BCG vaccine’s effectiveness.

Background and purpose: Colorectal cancer ( CRC ) is highly prevalent and conventional therapies are associated with side effects, therefore, application of novel complementary treatment such as probiotics (especially Saccharomyces cerevisiae) is necessary. The aim of this study was to investigate the effect of heat-killed form of S.cerevisiae on growing rate and apoptosis (expression levels of PTEN and RelA) in colon cancer cell line HT29. Materials and methods: In this experimental study, the cytotoxic effects of heat-killed form of S.cerevisiae and Fluorouracil (5-FU), as positive control, were tested by MTT assay. Apoptosis induction was investigated by determining the PTEN and RelA expression levels using qRT-PCR. Data were analyzed in GraphPad Prism Software. Results: Heat killed form of S.cerevisiae showed anti-proliferative and apoptotic effect by decreasing the expression levels of RelA and increasing PTEN expression levels in HT-29 cells. The expression levels were found to be significantly different between the intervention groups and controls. Conclusion: The yeast probiotics could decrease the growth rate of colorectal cancer cells and play key role in induction of apoptosis via regulation of RelA and PTEN.

Tumor microenvironment consists of malignant and non-malignant cells. The interaction of these dynamic and different cells is responsible for tumor progression at different levels. The non-malignant cells in TME contain cells such as tumor-associated macrophages (TAMs), cancer associated fibroblasts, pericytes, adipocytes, T cells, B cells, myeloid-derived suppressor cells (MDSCs), tumor-associated neutrophils (TANs), dendritic cells (DCs) and Vascular endothelial cells. TAMs are abundant in most human and murine cancers and their presence are associated with poor prognosis. The major event in tumor microenvironment is macrophage polarization into tumor-suppressive M1 or tumor-promoting M2 types. Although much evidence suggests that TAMS are primarily M2-like macrophages, the mechanism responsible for polarization into M1 and M2 macrophages remain unclear. TAM contributes cancer cell motility, invasion, metastases and angiogenesis. The relationship between TAM and tumor cells lead to used them as a diagnostic marker, therapeutic target and prognosis of cancer. This review presents the origin, polarization, role of TAMs in inflammation, metastasis, immune evasion and angiogenesis as well as they can be used as therapeutic target in variety of cancer cells. It is obvious that additional substantial and preclinical research is needed to support the effectiveness and applicability of this new and promising strategy for cancer treatment.

Proprotein convertase subtilisin/kexin type 9 (PCSK9), as a vital modulator of low-density lipoprotein cholesterol (LDL-C) , is raised in hepatocytes and released into plasma where it binds to LDL receptors (LDLR), leading to their cleavage. PCSK9 adheres to the epidermal growth factor-like repeat A (EGF-A) domain of the LDLR which is confirmed by crystallography. LDLR expression is adjusted at the transcriptional level through sterol regulatory element binding protein 2 (SREBP-2) and at the post translational stages, specifically through PCSK9, and the inducible degrader of the LDLR PCSK9 inhibition is an appealing new method for reducing the concentration of LDL-C. In this review the role of PCSK9 in lipid homeostasis was elucidated, the effect of PCSK9 on atherosclerosis was highlighted, and contemporary therapeutic techniques that focused on PCSK9 were summarized. Several restoration methods to inhibit PCSK9 have been proposed which concentrate on both extracellular and intracellular PCSK9, and they include blockage of PCSK9 production by using gene silencing agents and blockage of it’s binding to LDLR through antibodies and inhibition of PCSK9 autocatalytic processes by tiny molecule inhibitors.

Objectives: Cation Channel Spermia (CatSper) 1-4 are considered as a unique family of "sperm cation channels" and play an important role in the motility of the sperm and male fertility. The aim of this study was to compare and evaluate the effects of selenium (Se) and electrical stimulation (Es) on the expression of CatSper 1 and 2 genes in the spinal cord injury (SCI) rat model. Materials and Methods: The present experimental study was performed on 50 male Wistar rats. The animals were randomly divided into 5 groups of 10 which experienced SCI using an SCI impactor device. Forty-eight hours after injury, the Se group received Se for 6 consecutive days for 6 weeks, and Es was performed for 1 hour per day for 6 consecutive days during 6 weeks in the Es group. Finally, the real-time polymerase chain reaction (PCR) was performed for the expression of CatSper 1 and 2 genes, followed by using one-way ANOVA for the analysis of gene expression data. The results were considered statistically significant when P < 0.05. Results: Se treatment showed an increase in the relative expression of CatSper 1 and 2 genes compared to the control group, but this increase was higher for CatSper 2. Eventually, Es did not affect the relative intensity of CatSper 1 and 2 gene expression after SCI. Conclusions: Se, more than Es, could increase the relative intensity of CatSper1 and 2 expressions, and this increase was higher in CatSper2.

Objectives: Spinal cord injury (SCI) often occurs in young adult men. Factors such as erectile and ejaculation dysfunction and abnormal anomalies may contribute to the incidence of infertility. In healthy men, there is a balance between reactive oxygen species (ROS) and antioxidant scavenging systems and antioxidants have a positive impact on male fertility. Therefore, the present study examined the findings of different review studies on the role of oxidative stress (OS) and antioxidants in reducing the sperm function and treating the sperm components, respectively. Materials and Methods: The relevant keywords were used to search for related articles in different databases such as Medline, PubMed, ISI, Scopus, and Google Scholar, which resulted in retrieving 28 articles about OS, antioxidants, DNA damage, apoptosis, and sperm parameters. Results: Based on the findings of previous studies, antioxidant supplementation improved the number, motility, morphology, and the DNA integrity of the sperm. In addition, the combination of several antioxidants was found to perform better compared to using only one antioxidant. Conclusions: In general, the heightened levels of uncontrolled OS is modified through the antioxidant supplements or their combination. Therefore, antioxidant supplementation can be considered in the treatment of infertile men.

Abstract Cancer stem cells obtain energy demand through the activation of glycolysis and lipolysis. It seems that the use of approached targeting glycolysis and lipolysis could be an effective strategy for the inhibition of cancer stem cells. In the current experiment, we studied the potential effect of glycolysis and lipolysis inhibition on cancer stem cells differentiation and mesenchymal–epithelial-transition capacity. Cancer stem cells were enriched from human ovarian cells namely SKOV3 by using MACS technique. Cells were exposed to Lonidamine, an inhibitor of glycolysis, and TOFA, a potent inhibitor of lipolysis for 7 days in endothelial differentiation medium; EGM-2 and cell viability was studied by MTT assay. At the respective time point, the transcription level of genes participating in EMT such as Zeb-1, -2, Vimentin, Snail-1, -2 and VE-cadherin were measured by real-time PCR analysis. Our data noted that the inhibition of lipolysis and glycolysis could decrease cell viability compared to the control of cancer stem cells. The inhibition of glycolysis prohibited the expression of Zeb-1, Snails, and Vimentin while increased endothelial differentiation rate indicated by the expression of VE-cadherin. In contrast, the inhibition of lipolysis increased EMT associated genes and reduced endothelial differentiation rate by suppressing the transcription of VE-cadherin. Notably, the simultaneous inhibition of glycolysis and lipolysis had moderate effects on the transcription of EMT genes. We concluded that the modulation of the metabolic pathway of glycolysis in ovarian CSCs is more effective than the inhibition of lipolysis in the control of angiogenesis potential and stemness feature.

Purpose: The single-chain variable fragment (scFv) domain of antibodies is now considered asone of the therapeutic tools that can be produced by phage display technology (PDT). Antibodypurification is one of the most important steps in antibodies production. The aim of study waspurification and characterization of anti-VEGFR2 scFv antibody fragments.Methods: After the coating of vascular endothelial growth factor receptor 2 (VEGFR2) peptidein ELISA microplates, the phage display library of Tomlinson was used for antibody isolation.The targeted scFv was purified by chromatography using a zeolite-based column. The purity andfunctional assessment of purified scFv were evaluated by sodium dodecyl sulfate polyacrylamidegel electrophoresis (SDS-PAGE) and western blotting techniques, respectively. Affinity bindingwas evaluated by surface plasmon resonance (SPR).Results: The desired scFv was selected after four stages of biopanning. SDS-PAGE analysisshowed a 28 kDa scFv with high purity (>90%). The western bloting analysis confirmed thebinding of produced scFv antibody to the desired peptide. The affinity binding of scFv antibodyanalyzed by SPR was about 60 μM.Conclusion: In this study, the novel scFv antibody against VEGFR2 peptide was purified bychromatography column containing zeolite. Based on our findings the produced antibody maybe applied for diagnosis or targeting of VEGFR2 in antibody-based therapy strategies.

Heat shock protein 70.1 (Hsp70.1), also known as Hsp70, is a highly conserved member of the heat shock protein family that exists in all living organisms and determines the protein fate as molecular chaperones. Hsp70 basal expression is undetectable or low in most unstressed normal cells, however, its abundant presence in several types of human cancer cells is reported. Several studies support upregulated Hsp70 involved in tumor progression and drug resistance through modulation of cell death pathways and suppresses anticancer immune responses. However, numerous studies have confirmed that Hsp70 can also induce anticancer immune responses through the activation of immune cells in particular antigen-presenting cells (APCs). Regarding the significant and the promising role of vaccines in cancer immunotherapy, identification and characterization of the overexpressed Hsp70 as a potential immune stimulatory factor can pave the path for development of highly effective anticancer vaccines. In this review, we will discuss the interactions of Hsp70 with components of the immune system in cancers as well as possible strategies to harness Hsp70 for eliciting anticancer immune responses.

Introduction: Glomalin is known as a specific fungal glycoprotein belonging to the order Glomerales in phylum Glomeromycota and has been introduced as a heat shock protein. We hypothesised that increasing the level of Pb would lead to increase in glomalin production. Glomalin is usually determined by two methods, the Bradford protein dye-binding assay and the enzyme-linked immunosorbent assay (ELISA). Since many laboratories are not equipped to carry out the ELISA assay, many studies have measured glomalin-related soil protein using the Bradford colorimetric total protein assay. While, the ELISA method specifically measures glomalin by using monoclonal antibody MAb32B11. Materials and Methods: The pot experiment was conducted in the sterile free-glomalin sand with Trifolium repens L. mycorrhized by Rhizophagus irregularis fungus and treated with the Pb levels of 0, 150, 300 and 450 µM. Thus, in vitro experiment was performed in two-compartments plates containing of the transformed carrot roots (Daucus carota L.) mycorrhized with the same fungus in root compartment and hyphal compartment treated with the Pb levels of 0, 0.01, 0.1 and 1 mM as Pb(NO3)2. For glomalin extraction, hyphal or root samples were autoclaved at 121 ⁰C with 50 mM sodium citrate buffer for 60 min in three cycles. Protein concentrations in the extracted samples were determined using a modified Bradford protein assay. Also, glomalin content in the samples were determined by indirect ELISA using monoclonal antibody MAb32B11. The percentages of the total root length were colonised by mycorrhizal fungi in pot culture and both hyphal and spore densities in the metal-containing hyphal compartment were determined. Results and Discussion: In the in vitro culture the percentage of total hyphae and spore frequency decreased, while Bradford reactive total hyphal protein (BRHP) and Immunoreactive hyphal protein (IRHP) in hyphal compartment increased as the concentrations of Pb increased. Also, there was positive and significant correlation between immunoreactive hyphal protein (IRHP) and Bradford reactive total hyphal protein (BRHP) in hyphal compartment of in vitro culture (r= 0.941**). In the pot culture, the percentage of the total mycorrhized root length in all the treatments increased compared to the unleaded control as the concentrations of Pb raised. In general, Bradford reactive total protein and Immunoreactive protein in both the hyphal and root compartments of pot culture increased with increasing the Pb levels. Also, there were positive and significant correlations between immunoreactive hyphal protein (IRHP) with Bradford reactive total hyphal protein (BRHP) (r= 0.845 **) and immunoreactive root protein (IRRP) with Bradford reactive total root protein (BRRP) (r= 0.706 **) in pot experiment. Some previously researches had reported correlation between ELISA with Bradford contents, whether, Bradford and ELISA values were nearly the same, this means that the extraction process mostly separates glomalin. The results of non-mycorrhizal roots indicated that a small proportion of root protein is cross-reactive with the MAb32B11 antibody. There are some evidences that MAb32B11 is slightly cross-reactive with plant compounds, non-AMF species, and non-target proteins present in large concentration, such as BSA. Additionally, we found the increasing of BRRP contents of AMF-colonized root compared to the non-mycorhizal roots. This may be as a result of uptake and storage of arginine within AMF intraradical hyphae. Considering IRHP to BRHP ratio indicates that immunoreactivity percentage enhances by rising Pb levels. Immunoreactivity indicates a molecular configuration similar to the configuration of glomalin on hyphae. Our findings are in agreement with previous observations confirming that the toxicity-induced stress by metals may be enhancing glomalin production by AMF. The sequence of the glomalin gene revealed that is likely a 60-KDa heat-shock protein (Hsp) homolog. Glomalin relation with the heat shock proteins clarifies how stress is imposed by heavy metals may rapidly increase glomalin production by AMF and its concentrations in polluted soils. Conclusion: The high contents of glomalin along with the increasing of Pb concentrations may be explained by the overexpression of this protein. This suggests that under Pb-induced stress and the toxic effect of Pb, the fungus exerts a protective mechanism against toxicant. Therefore, glomalin as a heat shock protein can involve in the reduction of possible cytosolic damages and the transfiguration of proteins under Pb toxicity. We can conclude that glomalin may reduce toxic elements availability via their stabilization and decrease their toxicity risk to other microorganisms and plants in heavy metal polluted sites.

Peripheral nerve injuries with a poor prognosis are common. Evening primrose oil (EPO) has beneficial biological effects and immunomodulatory properties. Since electrical activity plays a major role in neural regeneration, the present study investigated the effects of electrical stimulation (ES), combined with evening primrose oil (EPO), on sciatic nerve function after a crush injury in rats. In anesthetized rats, the sciatic nerve was crushed using small haemostatic forceps followed by ES and/or EPO treatment for 4 weeks. Functional recovery of the sciatic nerve was assessed using the sciatic functional index. Histopathological changes of gastrocnemius muscle atrophy were investigated by light microscopy. Electrophysiological changes were assessed by the nerve conduction velocity of sciatic nerves. Immunohistochemistry was used to determine the remyelination of the sciatic nerve following the interventions. EPO + ES, EPO, and ES obviously improved sciatic nerve function assessed by the sciatic functional index and nerve conduction velocity of the sciatic nerve at 28 days after operation. Expression of the peripheral nerve remyelination marker, protein zero (P0), was increased in the treatment groups at 28 days after operation. Muscle atrophy severity was decreased significantly while the nerve conduction velocity was increased significantly in rats with sciatic nerve injury in the injury + EPO + ES group than in the EPO or ES group. Totally speaking, the combined use of EPO and ES may produce an improving effect on the function of sciatic nerves injured by a crush. The increased expression of P0 may have contributed to improving the functional effects of combination therapy with EPO and ES as well as the electrophysiological and histopathological features of the injured peripheral nerve.

As the most frequent plasma protein, albumin constitutes more than 50% of the serum proteins in healthy individuals. It has a key role in oncotic pressure maintenance and it is known as a versatile protein carrier for transportation of various endogenous and exogenous ligands. Reduced amounts of albumin in the body will lead to different kinds of diseases such as hypovolemia and hypoproteinemia. It also has various indications in shocks, burns, cardiopulmonary bypass, acute liver failure and etc. Further applications in research consist of cell culture supplement, drug delivery carrier and protein/drug stabilizer. So, the demand for albumin increased annually worldwide. Due to different applications of albumin, many efforts have been accomplished to achieve albumin during a long period of time. In this review, an overview of serum albumin and different purification methods are summarized.

Purpose: Albumin is an abundant protein of blood and has many biopharmaceutical applications. The aim of this study was to purify bovine serum albumin (BSA) using produced rabbit anti-BSA antibody. Methods: The polyclonal antibody was produced against the BSA in rabbits. Then, the pure BSA was injected to three white New Zealand rabbits. ELISA test was done to evaluate antibody production. After antibody purification,the purified antibody was attached to CNBr-activated sepharose and finally it was used for purification of albumin from bovine serum. Western blotting analysis was used for functional assessment of immunoaffinity purified BSA. Results: The titer of anti-bovine albumin determined by ELISA was obtained 1: 256000. The SDS-PAGE showed up to 98% purity of isolated BSA and western blotting confirmed the BSA functionality. Purified bovine serum albumin by affinity chromatography showed a single band with molecular weight of 66 KDa. Conclusion: Affinity chromatography using produced rabbit anti-BSA antibody would be an economical and safe method for purification of BSA.

Vascular endothelial growth factor receptor 2 (VEGFR-2) is known as one of the important antigens playing a vital role in angiogenesis. In this study, phage display technology (PDT) was used to produce a single-chain variable fragment (scFv) antibody against a region of the domain 3 in VEGFR-2 called kinase insert domain receptor 3 (KDR3). After designing the KDR3 peptide and biopanning, a colony was chosen for scFv antibody expression. Following expression and purification; western blotting, dot blotting and immunofluorescence (IF) were used to evaluate the antibody function. Surface plasmon resonance (SPR) was also employed to measure affinity of produced antibody. Once a colony was selected and transferred to the expression host, the scFv antibody was expressed in the expected range of 28 kDa. Using a designed chromatography column, antibody purification was found to be about 95%. In this study, a novel scFv with the capability of binding to KDR3 was isolated and purified and its intracellular function was investigated and verified.

Purpose: Recombinant human endostatin (rhEs) is an angiogenesis inhibitor which is used as a specific drug in the treatment of non-small-cell lung cancer. In the current research, we developed an efficient method for expressing soluble form of the rhEs protein in the periplasmic space of Escherichia coli via fusing with pelB signal peptide. Methods: The human endostatin (hEs) gene was amplified using synthetic (hEs) gene as a template; then, cloned and expressed under T7 lac promoter. IPTG was used as an inducer for rhEs expression. Next, the osmotic shock was used to extraction of protein from the periplasmic space. The presence of rhEs in the periplasmic space was approved by SDS-PAGE and Western blotting. Results: The results show the applicability of pelB fusion protein system usage for secreting rhEs in the periplasm of E. coli in the laboratory scale. The rhEs represents approximately 35 % (0.83mg/l) of the total cell protein. Conclusion: The present study apparently is the first report of codon-optimized rhEs expression as a fusion with pelB signal peptide. The results presented the successful secretion of soluble rhEs to the periplasmic space.

Background: The ability of polyclonal antibodies to react with many epitopes of an antigen makes them valuable reagents in research and diagnosis. The aim of this study was purification of mouse IgG2a and production of polyclonal antibody against purified mouse IgG2a subclass. Materials and Methods: Mouse IgG2a was purified by ProA affinity. Verification method of the purified antibody was SDS-PAGE and ELISA by a mouse isotyping Kit. Rabbit was immunized with purified IgG2a. The production of antibody in rabbit was investigated by direct ELISA method. Rabbit serum was collected and precipitated at the final concentration of 50% ammonium sulfate. Polyclonal antibody was purified by ion-exchange chromatography and labeled with HRP. The titre and cross reactivity of product was detected by direct ELISA method. Results: The results of SDS-PAGE in reduced and non-reduced conditions showed bands with 50-KDa, 25-30 KDa MW and a distinct band with 150 KDa MW. Isotype determination showed the presence of mouse IgG2a in related fraction. The titer of Anti-mouse polyclonal antibody was 200000. The optimum titer of prepared HRP conjugated IgG was 4000. Conjugated rabbit IgG has more cross reactivity with mouse IgG2b. Conclusion: Taking together, affinity chromatography and ion-exchange chromatography are appropriate techniques for purification of mouse IgG subclasses and rabbit IgG, respectively. .

Purpose: Mouse IgG subclasses containing IgG1, IgG2a, IgG2b and IgG3 have been defined and described both physiochemically and immunologically. Methods: Sepharose beads conjugated with protein A affinity chromatography was used for purification of mouse IgG2b. Sodium citrate buffer (0.1 M, pH: 3.5) was used for separation of mouse IgG2b. Verification of the purified fractions was monitored by SDS-PAGE (polyacrylamide gel electrophoresis) in reducing condition. Immunized rabbit serum was collected and precipitated at the final concentration of 50% ammonium sulfate. After dialysis against tris-phosphate buffer (pH: 8.1) ion exchange chromatography column was used for purification of rabbit anti-mouse IgG2b. The periodate method was performed for conjugation with some variations. After conjugation, direct ELISA was used to determine the titer of HRP conjugated rabbit IgG against mouse IgG2b. Results: The titer of rabbit anti-mouse IgG2b that determined by ELISA was 32000. The purity of rabbit anti-mouse IgG2b was about 95%. The optimum dilution of prepared HRP conjugated IgG was 1:10000. This study showed that ion-exchange chromatography and affinity chromatography could be appropriate techniques for purification of mouse IgG and IgG subclasses respectively. Conclusion: This study showed that affinity chromatography could be an appropriate method for purification of IgG2b antibodies.

Purpose: Monoclonal antibodies are potentially powerful tools used in biomedical research, diagnosis, and treatment of infectious diseases and cancers. The monoclonal antibody against Human IgA can be used as a diagnostic application to detect infectious diseases. The aim of this study was to improve an appropriate protocol for large-scale production of mAbs against IgA. Methods: For large-scale production of the monoclonal antibody, hybridoma cells that produce monoclonal antibodies against Human IgA were injected intraperitoneally into Balb/c mice that were previously primed with 0.5 ml Pristane. After ten days, ascitic fluid was harvested from the peritoneum of each mouse. The ELISA method was carried out for evaluation of the titration of produced mAbs. The ascitic fluid was investigated in terms of class and subclass by a mouse mAb isotyping kit. MAb was purified from the ascitic fluid by ion exchange chromatography. The purity of the monoclonal antibody was confirmed by SDS-PAGE, and the purified monoclonal antibody was conjugated with HRP. Results: Monoclonal antibodies with high specificity and sensitivity against Human IgA were prepared by hybridoma technology. The subclass of antibody was IgG1 and its light chain was the kappa type. Conclusion: This conjugated monoclonal antibody could have applications in designing ELISA kits in order to diagnose different infectious diseases such as toxoplasmosis and H. Pylori.

Purpose: It is estimated that one third of the world’s population were infected with M. tuberculosis, but only 10% of them have developed in to disease form. This subject refers to differences in host immune system activity against the tuberculosis. Vitamin D and its receptor (VDR) are important factors in the host innate immune system against the tuberculosis. In the present study VDR gene polymorphisms and its relationship with plasma vitamin D levels in susceptibility to tuberculosis have been investigated. Methods: The subjects were 84 patients with tuberculosis and 90 healthy controls. Vitamin D levels were measured in all study participants. DNA was isolated from the blood leukocytes of all groups and amplified by polymerase chain reaction (PCR). Then restriction fragment length polymorphism (RFLP) was performed on each PCR products to study the VDR gene polymorphisms. The statistical analyses were conducted using SPSS. Results: There was no statistically significant relationship between polymorphisms of FokI, BsmI, ApaI and TaqI in VDR gene and susceptibility to tuberculosis. Vitamin D deficiency and susceptibility to tuberculosis were closely related (95% CI -0.08 – 4.7, P = 0.059). Also the relationship between plasma vitamin D levels and frequency of FokI-ff gene polymorphism was significant in all study participants (P = 0.045). Conclusion: When the genotype frequencies of VDR gene polymorphisms were analyzed with respect to plasma vitamin D levels, a significant association was seen. As an enhancement in plasma vitamin D levels in individuals (with FokI-ff genotype and low levels of vitamin D) may protect them against active tuberculosis.

CD34 is a type I membrane protein with a molecular mass of approximately 110 kDa. This antigen is associated with human hematopoietic progenitor cells and is a differentiation stage-specific leukocyte antigen. In this study we have generated and characterized monoclonal antibodies (mAbs) directed against a CD34 marker. Mice were immunized with two keyhole lympet hemocyanin (KLH)-conjugated CD34 peptides. Fused cells were grown in hypoxanthine, aminopterine and thymidine (HAT) selective medium and cloned by the limiting dilution (L.D) method. Several monoclones were isolated by three rounds of limited dilutions. From these, we chose stable clones that presented sustained antibody production for subsequent characterization. Antibodies were tested for their reactivity and specificity to recognize the CD34 peptides and further screened by enzyme-linked immunosorbent assay (ELISA) and Western blotting analyses. One of the mAbs (3D5) was strongly reactive against the CD34 peptide and with native CD34 from human umbilical cord blood cells (UCB) in ELISA and Western blotting analyses. The results have shown that this antibody is highly specific and functional in biomedical applications such as ELISA and Western blot assays. This monoclonal antibodies (mAb) can be a useful tool for isolation and purification of human hematopoietic stem cells (HSCs).

Is Hair Lice Still a Public Health Problem?

In the publication of this article [1], there is an error in one of the contributing author names.

Purpose: Monoclonal antibodies and related conjugates are key reagents used in biomedical researches as well as, in treatment, purification and diagnosis of infectious and non- infectious diseases. Methods: Balb/c mice were immunized with purified human IgG. Spleen cells of the most immune mouse were fused with SP2/0 in the presence of Poly Ethylene Glycol (PEG). Supernatant of hybridoma cells was screened for detection of antibody by ELISA. Then, the sample was assessed for cross-reactivity with IgM & IgA by ELISA and confirmed by immunoblotting. The subclasses of the selected mAbs were determined. The best clone was injected intraperitoneally to some pristane-injected mice. Anti-IgG mAb was purified from the animals' ascitic fluid by Ion exchange chromatography and then, mAb was conjugated with HRP. Results: In the present study, over than 50 clones were obtained that 1 clone had optical density over than 3. We named this clone as supermonoclone which was selected for limiting dilution. The result of the immunoblotting, showed sharp band in IgG position and did not show any band in IgM&IgA position. Conclusion: Based on the findings of this study, the conjugated monoclonal antibody could have application in diagnosis of infectious diseases like Toxoplasmosis, Rubella and IgG class of other infectious and non- infectious diseases.

Purpose: In the present study cytotoxic effects of the alcoholic extract of Dorema Glabrum seed on viability of WEHI-164 cells, mouse Fibrosarcoma cell line and L929 normal cells were compared with the cytotoxic effects of Taxol (anticancer and apoptosis inducer drug). Methods: To find out the plant extract cytotoxic effects, MTT test and DNA fragmentation assay, the biochemical hallmark of apoptosis were performed on cultured and treated cells. Results: According to the findings the alcoholic extract of Dorema Glabrum seed can alter cells morphology and because of chromatin condensation and other changes they shrink and take a spherical shape, and lose their attachment too. So the plant extract inhibits cell growth albeit in a time and dose dependent manner and results in degradation of chromosomal DNA. Conclusion: Our data well established the anti-proliferative effect of methanolic extract of Dorema Glabrum seed and clearly showed that the plant extract can induce apoptosis and not necrosis in vitro, but the mechanism of its activities remained unknown. These results demonstrated that Dorema Glabrum seed might be a novel and attractive therapeutic candidate for tumor treatment in clinical practices.

Purpose: Monoclonal antibodies are important tools are used in basic research as well as, in diagnosis, imaging and treatment of immunodeficiency diseases, infections and cancers. The purpose of this study was to produce large scale of monoclonal antibody against CD20 in order to diagnostic application in leukemia and lymphomas disorders. Methods: Hybridoma cells that produce monoclonal antibody against human CD20 were administered into the peritoneum of the Balb/c mice which have previously been primed with 0.5 ml Pristane. After twelve days, approximately 7 ml ascetic fluid was harvested from the peritoneum of each mouse. Evaluation of mAb titration was assessed by ELISA method. In the present study, we describe a protocol for large scale production of MAbs. Results: We prepared monoclonal antibodies (mAbs) with high specificity and sensitivity against human CD20 by hybridoma method and characterized them by ELISA. The subclass of antibody was IgG2a and its light chain was kappa. Ascetic fluid was purified by Protein-A Sepharose affinity chromatography and the purified monoclonal antibody was conjugated with FITC and Immunofluorescence was done for confirming the specific binding. Conclusion: The conjugated monoclonal antibody could have application in diagnosis B-cell lymphomas, hairy cell leukemia, B-cell chronic lymphocytic leukemia, and melanoma cancer stem cells.

Purpose: Monoclonal antibodies or specific antibodies are now an essential tool of biomedical research and are of great commercial and medical value. The purpose of this study was to produce large scale of monoclonal antibody against CD34 in order to diagnostic application in leukemia and purification of human hematopoietic stem/progenitor cells. Methods: For large scale production of monoclonal antibody, hybridoma cells that produce monoclonal antibody against human CD34 were injected into the peritoneum of the Balb/c mice which have previously been primed with 0.5 ml Pristane. 5 ml ascitic fluid was harvested from each mouse in two times. Evaluation of mAb titration was assessed by ELISA method. The ascitic fluid was examined for class and subclasses by ELISA mouse mAb isotyping Kit. mAb was purified from ascitic fluid by affinity chromatography on Protein A-Sepharose. Purity of monoclonal antibody was monitored by SDS -PAGE and the purified monoclonal antibody was conjugated with FITC. Results: Monoclonal antibodies with high specificity and sensitivity against human CD34 by hybridoma technology were prepared. The subclass of antibody was IgG1 and its light chain was kappa. Conclusion: The conjugated monoclonal antibody could be a useful tool for isolation, purification and characterization of human hematopoietic stem cells.

Purpose: Recombinant tumor necrosis factor-alpha (TNF-α) has been utilized as an antineoplastic agent for the treatment of patients with melanoma and sarcoma. It targets tumor cell antigens by impressing tumor-associated vessels. Protein purification with affinity chromatography has been widely used in the downstream processing of pharmaceutical-grade proteins. Methods: In this study, we examined the potential of our produced anti-TNF-scFv fragments for purification of TNF-α produced by Raji cells. he Raji cells were induced by lipopolysaccharides (LPS) to express TNF-α. Western blotting and Fluorescence-activated cell sorting (FACS) flow cytometry analyses were used to evaluate the TNF-α expression. The anti-TNF-α scFv selected from antibody phage display library was coupled to CNBr-activated sepharose 4B beads used for affinity purification of expressed TNF-α and the purity of the protein was assessed by SDS-PAGE. Results: Western blot and FACS flow cytometry analyses showed the successful expression of TNF-α with Raji cells. SDS-PAGE analysis showed the performance of scFv for purification of TNF-α protein with purity over 95%. Conclusion: These findings confirm not only the potential of the produced scFv antibody fragments but also this highly pure recombinant TNF-α protein can be applied for various in vitro and in vivo applications.

Purpose: Salvia officinalis L., also known as Maryam Goli, is one of the native plants used to Persian medicinal herbs. Hence, the objective of this study was to examine the in vitro cytotoxic activities of a standardized crude methanol extracts prepared from Salvia officinalis L., on a non-Hodgkin’s B-cell lymphoma (Raji) and human leukemic monocyte lymphoma (U937), Human acute myelocytic leukemia (KG-1A) and Human Umbilical Vein Endothelial (HUVEC) cell lines. Methods: The effect of methanolic extract on the inhibition of cell proliferation and cytotoxic activity was evaluated by Dye exclusion and Micro culture tetrazolium test (MTT) cytotoxicity assay. Cell death ELISA was employed to quantify the nucleosome production result from nuclear DNA fragmentation during apoptosis and determined whether the mechanism involves induction of apoptosis or necrosis. Results: The present results demonstrated that methanolic extract at 50 to 800 μg/ml dose and time-dependently suppressed the proliferation of KG-1A, U937 and Raji cells by more than 80% (p800 Ag/ml). Nucleosome productions in KG-1A, Raji and U937 cells were significantly increased respectively upon the treatment of Salvia officinalis L. extract. Conclusion: The Salvia officinalis L. extract was found dose and time-dependently inhibits the proliferation of lymphoma and leukemic cells possibly via an apoptosis-dependent pathway.

Western blotting or immunoblotting commonly use for study of reaction between antigens and antibodies. Denaturation of many proteins in immunoblotting can affect greatly the reactivity of antibodies and outcome of the procedure. In this study proteins of Brucella abortus (S19) was extracted by a mild method and reaction of the extracted proteins with serum of infected human and goat and immunized rabbit compared by affinity chromatography and immunoblotting. Gamma globulin (mostly IgG) fraction of the sera was precipitated by half saturation of ammonium sulfate and linked to activated sepharose 4B. The extracted proteins were loaded on the affinity column. Attached proteins was eluted by low pH and analyzed by SDS-PAGE. Reaction of the total extract and eluted fractions with IgG fraction of sera was evaluated by Western blotting. Upon the results of affinity chromatography and immunoblotting , Brucella proteins can be classified in four groups: 1- The proteins that adsorbed to the affinity column and react with IgG in westernblotting. 2- Proteins that react with IgG in native state but no in denatured state. 3- Proteins that do not react with IgG in native state but react in denatured state. 4- Proteins that do not react with IgG in native and denatured state.