Neutralizing anti-diphtheria toxin scFv produced by phage display.

BACKGROUND: Diphtheria can be prevented by vaccination, but some epidemics occur in several places, and diphtheria's threat is considerable. Administration of diphtheria antitoxin (DAT) produced from hyperimmunized animals is the most common treatment. Recombinant human antibody fragments such as single-chain variable fragments (scFv) produced by phage display library may introduce an interesting approach to overcome the limitations of the traditional antibody therapy. In the present study, B cells of immunized volunteers were used to construct a human single-chain fragment (HuscFv) library. MATERIALS AND METHODS: The library was constructed with the maximum combination of heavy and light chains. As an antigen, Diphtheria toxoid (DTd) was used in four-round phage bio-panning to select phage clones that display DTd bound HuscFv from the library. After panning, individual scFv clones were selected. Clones that were able to detect DTd in an initial screening assay were transferred to Escherichia coli HB2151 to express the scFvs and purification was followed by Ni metal ion affinity chromatography. Toxin neutralization test was performed on Vero cells. The reactivity of the soluble scFv with diphtheria toxin were done and affinity calculation based on Beatty method was calculated. RESULTS: The size of the constructed scFv library was calculated to be 1.3 × 106 members. Following four rounds of selection, 40 antibody clones were isolated which showed positive reactivity with DTd in an ELISA assay. Five clones were able to neutralize DTd in Vero cell assay. These neutralizing clones were used for soluble expression and purification of scFv fragments. Some of these soluble scFv fragments show neutralizing activity ranging from 0.6 to 1.2 µg against twofold cytotoxic dose of diphtheria toxin. The affinity constant of the selected scFv antibody was determined almost 107 M-1. CONCLUSION: This study describes the prosperous construction and isolation of scFv from the immune library, which specifically neutralizes diphtheria toxin. The HuscFv produced in this study can be a potential candidate to substitute the animal antibody for treating diphtheria and detecting toxins.

In vitro assessment of Thimerosal cytotoxicity and antimicrobial activity.

BACKGROUND: Thimerosal (Merthiolate) is a well-known preservative used in pharmaceutical products, the safety of which was a matter of controversy for decades. Thimerosal is a mercury compound, and there is a debate as to whether Thimerosal exposure from vaccination can contribute to the incidence of mercury-driven disorders. To date, there is no consensus on Thimerosal safety in Vaccines. In 1977, a maximum safe dose of 200 µg/ml (0.5 mM) was recommended for Thimerosal by the WHO experts committee on biological standardization. Up-to-date guidelines, however, urge national control authorities to establish their own standards for the concentration of vaccine preservatives. We believe such safety limits must be studied at the cellular level first. The present study seeks a safe yet efficient dose of Thimerosal exposure for human and animal cells and control microorganism strains. METHODS: The safety of Thimerosal exposure on cells was analyzed through an MTT cell toxicity assay. The viability of four cell types, including HepG2, C2C12, Vero Cells, and Peripheral blood mononuclear cells (PBMCs), was examined in the presence of different Thimerosal concentrations and the maximum tolerable dose (MTD) and the half maximal inhibitory concentration (IC50) values for each cell line were determined. The antimicrobial effectiveness of Thimerosal was evaluated on four control strains, including Pseudomonas aeruginosa, Staphylococcus aureus, Candida albicans, and Aspergillus brasiliensis, to obtain the minimum inhibitory concentration (MIC) of Thimerosal. The MIC test was performed in culture media and under optimal growth conditions of microorganisms in the presence of different Thimerosal concentrations. RESULTS: The viability of all examined cell lines was suppressed entirely in the presence of 4.6 µg/ml (12.5 µM) of Thimerosal. The MTD for HepG2, C2C12, PBMC, and Vero cells was 2, 1.6, 1, and 0.29 µg/ml (5.5, 4.3, 2.7 and 0.8 µM), respectively. The IC50 of Thimerosal exposure for HepG2, C2C12, PBMC, and Vero cells was 2.62, 3.17, 1.27, and 0.86 µg/ml (7.1, 8.5, 3.5 and 2.4 µM), respectively. As for antimicrobial effectiveness, the growth capability of Candida albicans and Staphylococcus aureus was suppressed entirely in the presence of 6.25 µg/ml (17 µM) Thimerosal. The complete growth inhibition of Pseudomonas aeruginosa in culture media was achieved in 100 µg/ml (250 µM) Thimerosal concentration. This value was 12.5 µg/ml (30 µM) for Aspergillus brasiliensis. CONCLUSION: According to our results Thimerosal should be present in culture media at 100 µg/ml (250 µM) concentration to achieve an effective antimicrobial activity. We showed that this amount of Thimerosal is toxic for human and animal cells in vitro since the viability of all examined cell lines was suppressed in the presence of less than 5 µg/ml (12.5 µM) of Thimerosal. Overall, our study revealed Thimerosal was 333-fold more cytotoxic to human and animal cells as compared to bacterial and fungal cells. Our results promote more study on Thimerosal toxicity and its antimicrobial effectiveness to obtain more safe concentrations in biopharmaceuticals.

The hepatoprotective effects of Pyrus biossieriana buhse leaf extract on tert-butyl hydroperoxide toxicity in HepG2 cell line.

OBJECTIVE: In present study, the effects of the leaf extract of Pyrus biossieriana Buhse on tert-Butyl hydroperoxide (t-BHP) induced toxicity in the HepG2 cell line were investigated. RESULTS: HepG2 cells were exposed to different concentrations of both extract (1.5, 2.0, and 2.5 mg/mL) and t-BHP (100, 150, and 200 µM). The total flavonoid and phenolic contents, the cell viability, lipid peroxidation, NO generation, and the total antioxidant capacity in cell media were assessed. The amount of arbutin was estimated 12.6% of the dry weight of leaves (equivalent to 126 mg/g). Additionally, the amounts of flavonoids and phenols in extract were estimated 119 mg/g and 418 mg/g, respectively. The cells incubated with t-BHP showed a significant decrease in survival (p < 0.001). Preincubation with extract (1.5 mg/mL and 2.0 mg/mL) attenuated the t-BHP toxicity and increased the cell viability in cells exposed even to the highest concentration of t-BHP (200 µM) (p value < 0.001, and p value = 0.035) respectively. Additionally, treatment with extract reduced the cell growth suppression caused by t-BHP. The P. biossieriana Buhse leaf extract at concentrations of 1.5 and 2.0 mg/mL is capable of attenuating t-BHP-induced cytotoxicity in HepG2 cells.

Therapeutic effect of resveratrol supplementation on oxidative stress: a systematic review and meta-analysis of randomised controlled trials.

BACKGROUND: Resveratrol is a naturally occurring polyphenol compound mainly found in grapes and red wine. The evidence has suggested that resveratrol has an antioxidant effect. However, the results are inconsistent and inconclusive. Thus, we conducted a systematic review and meta-analysis to evaluate the effect of resveratrol supplementation on markers of oxidative stress. METHODS: We searched PubMed, ISI Web of Science, EMBASE, Scopus and the Cochrane library up to December 2018 to identify randomised controlled trials (RCTs) assessing resveratrol supplementation effects on oxidative markers. Heterogeneity, publication bias, risk of bias and subgroup analysis were analysed. This meta-analysis was conducted in accordance with the guidelines of the Preferred ReportingItems for Systematic Reviews and Meta-Analysis (PRISMA). RESULTS: Meta-analysis of data from 12 RCTs did not support significant effect of resveratrol supplementation on circulating levels of superoxide dismutase (SOD) (standardized mean difference (SMD) (1.12), (95% CI -0.91 to 3.1), p=0.28), catalase (CAT) (SMD (-0.07), (95% CI -1.4 to 1.3), p=0.92) and glutathione peroxidase (GPx) (SMD (-0.76), (95% CI -2.56 to 1.04), p=0.40). Although, resveratrol supplementation increased significantly circulating total antioxidant capacity (TAC) concentrations (SMD (0.52), (95% CI -0.02 to 1.07), p=0.05). Severe heterogeneity was observed between studies, and no obvious publication bias was observed in included RCTs. CONCLUSION: Collectively, our findings of available RCTs did no show any benefit of resveratrol supplementation on SOD, CAT and GPx except for TAC. Well-designed RCTs are necessary to confirm these results.

Expression optimization, purification, and functional characterization of cholesterol oxidase from Chromobacterium sp. DS1.

Cholesterol oxidase is a bifunctional bacterial flavoenzyme which catalyzes oxidation and isomerization of cholesterol. This valuable enzyme has attracted a great deal of attention because of its wide application in the clinical laboratory, synthesis of steroid derived drugs, food industries, and its potentially insecticidal activity. Therefore, development of an efficient protocol for overproduction of cholesterol oxidase could be valuable and beneficial in this regard. The present study examined the role of various parameters (host strain, culture media, induction time, isopropyl ß-D-1-thiogalactopyranoside concentration, as well as post-induction incubation time and temperature) on over-expression of cholesterol oxidase from Chromobacterium sp. DS1. Applying the optimized protocol, the yield of recombinant cholesterol oxidase significantly increased from 92 U/L to 2115 U/L. Under the optimized conditions, the enzyme was produced on a large-scale, and overexpressed cholesterol oxidase was purified from cell lysate by column nickel affinity chromatography. Km and Vmax values of the purified enzyme for cholesterol were estimated using Lineweaver-Burk plot. Further, the optimum pH and optimum temperature for the enzyme activity were determined. This study reports a straightforward protocol for cholesterol oxidase production which can be performed in any laboratory.

A comparison of three strategies for biopanning of phage-scFv library against diphtheria toxin.

The biopanning process is a critical step in phage display for isolating peptides or proteins with specific binding properties. Conventional panning methods are sometimes not so effective and may result in nonspecific or low-yield positive results. In this study, three different strategies including soluble antibody-capturing, pH-stepwise elution, and conventional panning were used for enrichment of specific clones against diphtheria toxoid. The reactivity of the selected clones was evaluated using an indirect enzyme-linked immunosorbent assay. The positive clones were screened using Vero cell viability assay. The neutralizing clones were expressed in HB2151 strain of Escherichia coli and soluble single-chain fragment variable (scFv) fragments were purified by nickel-nitrilotriacetic acid affinity chromatography. Finally, the ability of scFv fragments for neutralizing diphtheria toxin (DT) were evaluated again using Vero cell viability assay. After four rounds of panning, the soluble antibody-capturing method yielded 15 positive phage-scFv clones against diphtheria toxoid. Conventional panning and pH-stepwise elution model resulted from nine and five positive phage-scFv clones, respectively. Among all positive clones, three clones were able to neutralize DT in Vero cell viability assay. Two of these clones belonged to a soluble antibody-capturing method and one of them came from conventional panning. Three neutralizing clones were used for soluble expression and purification of scFvs fragments. It was found that these soluble scFv fragments possessed neutralizing activity ranging from 0.15 to 0.6 µg against two-fold cytotoxic dose 99% of DT. In conclusion, the results of our study indicate that soluble antibody-capturing method is an efficient method for isolation of specific scFv fragments.

Expression optimization of recombinant cholesterol oxidase in Escherichia coli and its purification and characterization.

Cholesterol oxidase is a bacterial flavoenzyme which catalyzes oxidation and isomerization of cholesterol. This enzyme has a great commercial value because of its wide applications in cholesterol analysis of clinical samples, synthesis of steroid-derived drugs, food industries, and potentially insecticidal activity. Accordingly, development of an efficient protocol for overexpression of cholesterol oxidase can be very valuable and beneficial. In this study, expression optimization of cholesterol oxidase from Streptomyces sp. SA-COO was investigated in Escherichia coli host strains. Various parameters that may influence the yield of a recombinant enzyme were evaluated individually. The optimal host strain, culture media, induction time, Isopropyl ß-D-1-thiogalactopyranoside concentration, as well as post-induction incubation time and temperature were determined in a shaking flask mode. Applying the optimized protocol, the production of recombinant cholesterol oxidase was significantly enhanced from 3.2 to 158 U/L. Under the optimized condition, the enzyme was produced on a large-scale, and highly expressed cholesterol oxidase was purified from cell lysate by column nickel affinity chromatography. Km and Vmax values of the purified enzyme for cholesterol were estimated using Lineweaver-Burk plot. Further, the optimum pH and optimum temperature for the enzyme activity were also determined. We report a straightforward and easy protocol for cholesterol oxidase production which can be performed in any laboratory.

Immunomodulatory effects of M2000 (ß-D-Mannuronic acid) on TNF-α, IL-17 and FOXP3 gene expression in patients with inflammatory bowel disease.

INTRODUCTION: Inflammatory bowel diseases (IBD) are immune-mediated disorders that result from an aberrant immunological response to the gut luminal antigen in genetically susceptible patients. IBD is categorized into two serotype, Crohn's diseases (CD) and ulcerative colitis (UC), both subtype are important cause of gastrointestinal diseases. The increasing rate of hospitalization, with the high economic burden experienced by the IBD patients, calls for more concerted research efforts to design a potent and affordable treatment option for the treatment of IBD. AIMS/OBJECTIVE: This research was designed to test the efficacy and potency of ß-D Mannuronic acid (M2000) and assess if it could serve as a better therapeutic option in the treatment of IBD. METHODOLOGY: Ten (10)ml of blood was aseptically collected into an EDTA container, from 24 IBD patients and 24 normal healthy controls. PBMC was isolated and stimulated with 1µg/ml of LPS in cell culture plate and incubated for 4h. The cells were later treated with 10µg/ml and 50µg/ml of ß-D Mannuronic acid (M2000) and incubated for 24h at 37°C under 5% CO2 and 100% humidity. The RNA extractions, cDNA synthesis, and QRT-PCR were performed. RESULTS: Our findings showed a significant down-regulation of TNF-α and IL-17 gene expression, while the expression of FOXP3 gene was significantly up-regulated. CONCLUSION: This result has indicated that ß-D Mannuronic acid (M2000) have immunoregulatory and anti-inflammatory effects on these cytokines that are pivotal in the pathogenesis of IBD.

Production of Recombinant Human scFv Against Tetanus Toxin Heavy Chain by Phage Display Technology.

Tetanus, as a major cause of death in developing countries, is caused by tetanus neurotoxin. Recombinant antibodies against tetanus neurotoxin can be useful in tetanus management. Phage display of antibody fragments from immune human antibody libraries with single chain constructs combining the variable fragments (scFv) has been one of the most prominent technologies in antibody engineering. The aim of this study was the generation of a single chain fragment of variable region (scFv) library and selection of specific antibodies with high affinity against tetanus toxin. Immune human single chain fragment variable (HuscFv) antibody phagemid library was displayed on pIII of filamentous bacteriophage. Selection of scFv clones was performed against tetanus toxin antigens after three rounds of panning. The selected scFv clones were analyzed for inhibition of tetanus toxin binding to ganglioside GT1b. After the third round of panning, over 35 HuscFv phages specific for tetanus toxin were isolated from this library of which 15 clones were found to bind specifically to tetanus toxin. The selected HuscFv phages expressed as a soluble HuscFv peptide and some clones showed positive signals against tetanus toxin. We found that six HuscFv clones inhibit toxin binding to ganglioside GT1b. These selected antibodies can be used in the management of tetanus.

Pyrus biossieriana Buhse leaf extract: An antioxidant, antihyperglycaemic and antihyperlipidemic agent.

The antihyperglycaemic, antihyperlipidemic and antioxidant effects of wild pear (Pyrus biossieriana Buhse) leaf extract were investigated. An alloxan-induced rat model of hyperglycaemia was used to evaluate the antihyperglycaemic, antihyperlipidemic and antioxidant properties of the Pyrus biossieriana Buhse leaf extract. The arbutin content of Pyrus biossieriana Buhse leaves, measured by HPLC, was 12.6 dry weight percent. Administration of the Pyrus biossieriana Buhse leaf extract (at doses of 500 and 1000mg/kg/day) significantly reduced the increase in serum glucose concentration seen in alloxan-treated hyperglycaemic rats. Both concentrations of the extract enhanced serum insulin levels compared to the control group. Both high and low doses of the extract decreased serum triacylglycerol (TG) and cholesterol (CHOL) levels as compared to controls. Serum antioxidant levels were significantly higher in rats treated with low (500mg/kg/day) and high (1000mg/kg/day) doses of Pyrus biossieriana Buhse extracts at 24, 48 and 72h after alloxan injection than in control rats. This study demonstrated that Pyrus biossieriana Buhse leaf extract reduces blood glucose and lipid levels and increases antioxidant status in rats with alloxan-induced hyperglycaemia.