Publications by authors named "Vahid Khalaj"

49 Publications

Rabies virus matrix protein targets host actin cytoskeleton: a protein-protein interaction analysis.

Pathog Dis 2021 Jan;79(1)

Biotechnology Research Center, Pasteur Institute of Iran, Tehran, 1316943551, Iran.

Multifunctional matrix protein (M) of rabies virus (RABV) plays essential roles in the pathogenesis of rabies infection. Identification of M protein interacting partners in target hosts could help to elucidate the biological pathways and molecular mechanisms involved in the pathogenesis of this virus. In this study, two-dimensional Far-western blotting (2D-Far-WB) technique was applied to find possible matrix protein partners in the rat brainstem. Recombinant RABV M was expressed in Pichia pastoris and was partially purified. Subsequently, 2D-Far-WB-determined six rat brainstem proteins interacted with recombinant M proteins that were identified by mass spectrometry. Functional annotation by gene ontology analysis determined these proteins were involved in the regulation of synaptic transmission processes, metabolic process and cell morphogenesis-cytoskeleton organization. The interaction of viral M protein with selected host proteins in mouse Neuro-2a cells infected with RABV was verified by super-resolution confocal microscopy. Molecular docking simulations also demonstrated the formation of RABV M complexes. However, further confirmation with co-immunoprecipitation was only successful for M-actin cytoplasmic 1 interaction. Our study revealed actin cytoplasmic 1 as a binding partner of M protein, which might have important role(s) in rabies pathogenesis.
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http://dx.doi.org/10.1093/femspd/ftaa075DOI Listing
January 2021

Complete neutralization of the lethality of Hemiscorpius lepturus crude venom by a novel anti-recombinant phospholipase D1 IgGs.

Toxicon 2020 Aug 21;183:36-43. Epub 2020 May 21.

Venom and Biotherapeutics Molecules Lab., Medical Biotechnology Department, Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran. Electronic address:

Treatment of scorpion envenomation is a challenging issue since serotherapy is implemented by administration of polyvalent equine antisera. In our previous study we discovered that recombinant phospholipase D1 (Hl-RecPLD1) is responsible for the lethality of Hemiscorpius lepturus (H. lepturus) venom in mice. Accordingly, this study was aimed to investigate the protectivity of purified anti-Hl-RecPLD1 IgG against the lethality or major complications of H. lepturus venom. The neutralization efficiency of purified anti-Hl-RecPLD1 IgGs against sphingomyelinase activities of the crude venom and Hl-RecPLD1 was also assessed. Anti-Hl-RecPLD1 IgGs at optimum amount of 3.7 mg completely neutralized one Lethal Dose 100 (LD) of crude venom in mice. The anti-Hl-RecPLD1 IgGs remarkably reduced the necrosis area from 6.5 to 1 cm in rabbit derma, induced by the crude venom. The anti-Hl-RecPLD1 IgGs remarkably reduced the sphingomyelinase and hemolytic activities of crude venom as well. In conclusion, a novel rabbit monovalent IgG against Hl-RecPLD1 was able to completely protect the mice against the lethality of H. lepturus crude venom and reduced its toxicity as well. Such monovalent anti-Hl-RecPLD1 IgGs may have potential applications in serotherapy of H. lepturus envenomation.
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http://dx.doi.org/10.1016/j.toxicon.2020.05.010DOI Listing
August 2020

Comparison of Linear Poly Ethylene Imine (LPEI) and Poly L-Lysine (PLL) in Fabrication of CHOK Cell-Loaded Multilayer Alginate Microcapsules.

Adv Pharm Bull 2020 Jun 18;10(2):290-296. Epub 2020 Feb 18.

Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran.

Poly l-lysine (PLL) has been introduced as a strengthening covering layer for alginate microcapsules which are the most convenient way for cell encapsulation. Some disadvantages of PLL such as high price and low biocompatibility have prompted scientists to find better alternatives. Linear poly ethylene imine (LPEI), thanks to its highly similar structure to PLL, could be considered as a proper cost-effective alternative. In this study LPEI and PLL were compared as covering layers of cell-loaded alginate-LPEI-alginate (cALA) and alginate-PLL-alginate (cAPA) microcapsules. In addition to the physico-mechanical properties, the encapsulation efficiency, cell survival post encapsulation, cell viability, and cellular metabolic activity within the microcapsules were evaluated using trypan blue, live/dead cell staining, and MTT test, respectively. Physico-mechanical evaluation of the microcapsules revealed that the cell microencapsulation process did not affect their shape, size, and mechanical stability. Although the encapsulation efficiency for cALA and cAPA was not different ( >0.05), cell survival post encapsulation was higher in cALA than in cAPA (<0.05) which could be the reason for the higher cell viability and also cellular metabolic activity within these microcapsules in comparison to cAPA. Here, based on these results, ALA could be introduced as a preferable alternative to APA for cell encapsulation.
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http://dx.doi.org/10.34172/apb.2020.035DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7191236PMC
June 2020

Targeting c-Met on gastric cancer cells through a fully human fab antibody isolated from a large naive phage antibody library.

Daru 2020 Jun 19;28(1):221-235. Epub 2020 Mar 19.

Medical Biotechnology Department, Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran.

Purpose: The aberrant Hepatocyte growth factor (HGF)/ mesenchymal-epithelial transition factor (c-Met) signaling pathway in various malignancies and its correlation with tumor invasion and poor prognosis has validated c-Met as a compelling therapeutic target. Up to now, several monoclonal antibodies and small molecule inhibitors targeting c-Met have been introduced with different outcomes, none are yet clinically approved. Toward the generation of novel fully human anti-c-Met molecules, we generated a large naïve Fab antibody library using phage display technology, which subsequently screened for novel Fabs against c-Met.

Methods: A phage library, with a functional size of 5.5 × 10 individual antibody clones, was prepared using standard protocols and screened for c-Met-specific Fabs by successive rounds of panning. A panel of Fabs targeting c-Met were isolated, from which four clones were selected and further characterized by DNA sequencing. The c-Met binding ability of our selected Fabs was evaluated by c-Met ELISA assay and flow cytometry techniques.

Results: Among the confirmed anti-c-Met Fabs, clone C16, showed the highest affinity (K: 0.3 × 10 M), and 63% binding to MKN45 cells (a human gastric adenocarcinoma cell-line) as compared to c-Met negative T47D cell-line (9.03%).

Conclusion: Together, our study presents a single-pot antibody library, as a valuable source for finding a range of antigen-specific Fab antibodies, and also, a fully human, high affinity and specific anti c-Met Fab antibody, C16, which has the potential of developing as a therapeutic or chemotherapeutic delivery agent for killing c-Met-positive tumor cells.
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http://dx.doi.org/10.1007/s40199-020-00334-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7238820PMC
June 2020

Absence of AfuXpot, the yeast Los1 homologue, limits Aspergillus fumigatus growth under amino acid deprived condition.

World J Microbiol Biotechnol 2020 Jan 30;36(2):28. Epub 2020 Jan 30.

Medical Biotechnology Department, Biotechnology Research Center, Pasteur Institute of Iran, Tehran, 1316943551, Iran.

In Saccharomyces cerevisiae, los1 encodes a nuclear tRNA exporter. Despite the non-essentiality, the deletion of los1 has been shown to extend replicative life span in yeast. Here, we characterized AfuXpot, the los1 homologue in human pathogen Aspergillus fumigatus and found that it is continuously expressed during fungal growth. Microscopic examination of an AfuXpot-GFP-expressing transformant confirmed the nuclear localization of the fusion protein. The targeted gene deletion affirmed the non-essential role of AfuXpot in hyphal growth and sporulation. However, the growth of the deletion mutant was affected by amino acid, but not glucose, deprivation. The susceptibility of the deletant strain to protein and DNA/RNA synthesis inhibitors was also altered. Using bioinformatics tools, some transcription factor binding sites were predicted in AfuXpot promoter. Expression analyses of potential AfuXpot-interacting genes showed a marked down-regulation of sfp1 and mtr10 homologues in ΔAfuXpot strain. Our data demonstrates some conserved aspects of AfuXpot as a tRNA exporter in A. fumigatus.
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http://dx.doi.org/10.1007/s11274-020-2805-8DOI Listing
January 2020

Surface display of uropathogenic Escherichia coli FimH in Lactococcus lactis: In vitro characterization of recombinant bacteria and its protectivity in animal model.

Microb Pathog 2020 Apr 8;141:103974. Epub 2020 Jan 8.

Department of Molecular Biology, Pasteur Institute of Iran, Pasteur Ave, Tehran, 13164, Iran. Electronic address:

Urinary tract infections (UTIs) caused by uropathogenic Escherichia coli (UPEC) are very common, leading to high patient morbidity and substantial medical costs. The development of non-antibiotic strategies such as food-grade lactic acid bacterium can be recognized as an attractive and safe alternative way against UTI. Here, we report the construction of Lactococcus lactis (L. lactis) strain genetically modified to produce FimH virulence factor of UPEC on the cell surface. We showed the FimH inserted into the pT1NX vector is actively synthesized on L. lactis. The L. lactis-pT1NX-FimH exhibited an auto-aggregation phenotype in liquid cultures and formed robust biofilm on abiotic surface compared to vector-only bacteria. Then, we developed protective biofilms with L. lactis strains and examined their inhibitory effect for exclusion of uropathogenic biofilm formation. In the natural protective biofilm assays, L. lactis-pT1NX-FimH resulted in significant reduction in the pathogen load when compared to the L. lactis-pT1NX. Evaluation of the colonization ability in the bladder showed that L. lactis expressing FimH survived better in the mice bladder than L. lactis harboring vector. Protection assay against UPEC infection was investigated using a UTI mouse model. L. lactis-pT1NX-FimH displayed high effectiveness in the protection of the bladder as compared to the control group after UPEC challenge. The results suggest that genetically engineered L. lactis-pT1NX-FimH can be used as a safe alternative way for control of biofilm formation in UPEC. Furthermore, the possibility of using L. lactis-pT1NX-FimH as a new promising strategy against UTIs caused by UPEC strains is proposed.
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http://dx.doi.org/10.1016/j.micpath.2020.103974DOI Listing
April 2020

AurH1: a new heptapeptide derived from Aurein1.2 antimicrobial peptide with specific and exclusive fungicidal activity.

J Pept Sci 2019 Jul 1;25(7):e3175. Epub 2019 Jul 1.

Drug Design and Bioinformatics Unit, Department of Medical Biotechnology, Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran.

Due to the increasing incidence of fungal opportunistic infections and emergence of antibiotic-resistant fungal strains, antimicrobial peptides (AMPs) are considered as ideal candidates for antifungal compounds. In silico methods can reduce the limitations of natural AMPs such as toxicity and instability and improve their antimicrobial properties and selectivity. In this study, we designed AurH1, a new truncated peptide, based on the six-amino acid sequence of Aurein1.2. Further, the antimicrobial activities and toxicity effects of AurH1 on human skin fibroblast cells and red blood cells were investigated. Finally, field emission scanning electron microscopy (FE-SEM) and flow cytometry were performed in order to study the mechanism of action of AurH1. The results indicated that AurH1 had only antifungal activity (at a minimal inhibitory concentration (MIC) of 7.3-125 μg/mL) without any antibacterial effects on the selected bacteria, while Aurein1.2 had both antifungal and antibacterial activities as positive control. Furthermore, AurH1 did not show any toxicity on Hu02 cells and human red blood cells at its MIC range. In conclusion, it became clear that AurH1 is a selective peptide against fungi with no toxic effects on the selected bacteria and human cells.
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http://dx.doi.org/10.1002/psc.3175DOI Listing
July 2019

Proteomics investigation of molecular mechanisms affected by EnBase culture system in anti-VEGF fab fragment producing E. coli BL21 (DE3).

Prep Biochem Biotechnol 2019 8;49(1):48-57. Epub 2019 Feb 8.

b Medical Biotechnology Department , Biotechnology Research Centre, Pasteur Institute of Iran , Tehran , Iran.

Aggregation of recombinant proteins, a major problem in E. coli expression system, is improved by using EnBase culture system based on slow release of glucose. In the present study, to understand the intracellular mechanisms involved in increased solubility of the target recombinant protein through EnBase system, the effect of this system was investigated on E. coli cells proteome profile. The proteome profile of E. coli cells cultured in EnBase and conventional batch mode was analyzed by two-dimensional gel electrophoresis. The proteins with significant expressional changes were identified through MALDI-TOF/TOF mass spectrometry. In EnBase system, the expressions of carbon metabolism-related proteins, sugar transport system-related proteins, and amino acids metabolism-related proteins were significantly altered. Furthermore, the expression of Thioredoxin 1 as the facilitator of protein folding was up-regulated in EnBase system that could be related to the increased solubility of recombinant protein. The proteomics analysis of E. coli cells cultured in EnBase system revealed that Thioredoxin 1 can be a potential candidate for future studies aiming at increased anti-VEGF fab fragment solubility. Studying proteomics is a valuable tool for revealing the target proteins that play the central role in EnBase culture system for increasing the solubility.
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http://dx.doi.org/10.1080/10826068.2018.1508037DOI Listing
April 2019

Sustained release of TGF-β1 via genetically-modified cells induces the chondrogenic differentiation of mesenchymal stem cells encapsulated in alginate sulfate hydrogels.

J Mater Sci Mater Med 2018 Dec 29;30(1). Epub 2018 Dec 29.

National Cell Bank Department, Pasteur Institute of Iran, Tehran, Iran.

Strategies based on growth factor (GF) delivery have attracted considerable attention in tissue engineering applications. Among different GFs, transforming growth factor beta 1 (TGF-β1) is considered to be a potent factor for inducing chondrogenesis. In the present study, an expression cassette encoding the TGF-β1 protein was prepared and transfected into the SP2/0-Ag14 cell line. The confocal microscopy of the transfected cells was performed to confirm the correct transfection process. The expression and in vitro release kinetics of the recombinant TGF-β1 were assessed by western blot analysis and ELISA, respectively. Moreover, the biological activity of the expressed protein was compared with that of a commercially available product. The chondrogenic effects of the sustained release of the recombinant TGF-β1 in an in vitro co-culture system were evaluated using a migration assay and real-time PCR. Results of confocal microscopy confirmed the successful transfection of the vector-encoding TGF-β1 protein into the SP2/0-Ag14 cells. The bioactivity of the produced protein was in the range of the commercial product. The sustained release of the TGF-β1 protein via SP2/0-Ag14 cells encapsulated in hydrogels encouraged the migration of adipose-derived MSCs. In addition, the expression analysis of chondrogenesis-related genes revealed that the pretreatment of encapsulated Ad-MSCs cells in alginate sulfate hydrogels through their exposure to the sustained release of TGF-β1 is an efficient approach before transplantation of cells into the body.
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http://dx.doi.org/10.1007/s10856-018-6203-9DOI Listing
December 2018

Molecular Characterization of a Fungus Producing Membrane Active Metabolite and Analysis of the Produced Secondary Metabolite

Iran Biomed J 2019 03 16;23(2):121-8. Epub 2018 Sep 16.

Drug Design and Bioinformatics Unit, Medical Biotechnology Department, Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran.

Background: The majority of studies on soil Aspergillus concern the isolation and characterization of the antimicrobial compounds produced by this organism. Our previous studies indicated an isolated Aspergillus strain soil to be of interest, and this subject is further investigated here.

Methods: Soil samples of various locations in Iran were collected. Extract from Aspergillus sp. culture was obtained using ethyl acetate fractionation. Antimicrobial activity testing was performed using broth microdilution assay against Escherichia coli, Candida albicans, and Staphylococcus aureus microorganisms. One metabolite PA3-d10 was isolated from these active extracts and identified using thin layer chromatography, preparative thin-layer chromatography, HPLC, 1HNMR (proton nuclear magnetic resonance), 2D NMR, and LC-MS (liquid chromatography-mass spectrometry).

Results: According to morphological and biochemical properties as well as ITS rDNA sequencing, we identified an isolate of Aspergillus flavus. The ethyl acetate fraction of the fermentation medium containing membrane active metabolites showed antimicrobial effects against different bacterial and yeast indicator strains. One metabolite from these active extracts was finally identified.

Conclusion: Membrane active fraction produced by Aspergillus strain in this research demonstrated antimicrobial activities against bacteria and yeast strains. Therefore, this metabolite can be considered as a potential antimicrobial membrane active agent.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6707112PMC
March 2019

Assessment of Recombinant A2-Latex Agglutination Test (RA2-LAT) and RA2-ELISA for Detection of Canine Visceral Leishmaniasis: A Comparative Field Study with Direct Agglutination Test in Northwestern Iran.

Iran J Parasitol 2018 Apr-Jun;13(2):172-179

Dept. of Immunology, Pasteur Institute of Iran, Tehran, Iran.

Background: This study aimed to set-up latex agglutination test (LAT) and ELISA based on recombinant A2 from Iranian strain of (rA2-Ag) and evaluated for detection of anti- antibodies in dogs compared to standard direct agglutination test (DAT).

Methods: The rA2-Ag was synthesized under a part of the A2 gene sequences which contain immune dominant sequences and less number of repetitive sequences. Latex beads, 0.8 μm (Sigma, USA) were sensitized with rA2-Ag. The tests were carried out on sera collected from 350 ownership dogs including symptomatic (n=67), asymptomatic (n=230) canine visceral leishmaniasis (CVL), and (n=53) uninfected domestic dogs as control group.

Results: Anti-leishmanial antibodies were detected in 97 (27.7%), 96 (27.4%) and 29 (%9) of the serum samples by using DAT, rA2-ELISA, and rA2-latex, respectively with ≥1:320 as a cut-off titer when DAT-confirmed cases were compared with the control groups. A combined sensitivity of 52% and specificity of 82.40% for rA2-ELISA and 23.8% and specificity 95.38%, respectively were found with ≥1:320 as a cut-off titer when DAT-confirmed cases were compared with the control groups. The concordance between rA2-ELISA and rA2 latex compared with DAT as a gold standard serological test for VL were found 73.7% and 77.5%, respectively.

Conclusion: A good degree of agreement was found between rA2-ELISA and DAT (73.7%). rA2-ELISA could detect more seropositive serum samples than rA2-LAT and it may be recommended as an alternative tool for the diagnosis of CVL.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6068373PMC
August 2018

Oral Administration of Recombinant Expressing Ovalbumin-CPE Fusion Protein Induces Antibody Response in Mice.

Front Microbiol 2018 13;9:723. Epub 2018 Apr 13.

Department of Medical Biotechnology, Pasteur Institute of Iran, Tehran, Iran.

, a subspecies of , is a well-known eukaryotic probiotic with many benefits for human health. In the present study, a recombinant strain of was prepared to use as a potential oral vaccine delivery vehicle. In this sense, a ura3 auxotroph strain of CNCM I-745 (known as HANSEN CBS 5926, Yomogi) was generated using CRISPR/Cas9 methodology. Then a gene construct encoding a highly immunogenic protein, ovalbumin (OVA), was prepared and transformed into the . To facilitate the transport of the recombinant immunogen across the intestinal barrier, a claudin-targeting sequence from enterotoxin (CPE) was added to the C-terminus of the expression cassette. The recombinant strain expressing the OVA-CPE fusion protein was then administered orally to a group of mice, and serum IgG and fecal IgA levels were evaluated by ELISA. Our results demonstrated that anti-OVA IgG in serum significantly increased in test group ( < 0.001) compared to control groups (receiving wild type or PBS), and the fecal IgA titer was significantly higher in test group ( < 0.05) than control groups. In parallel, a recombinant strain expressing the similar construct lacking C-terminal CPE was also administered orally. The result showed an increased level of serum IgG in group receiving yeasts expressing the CPE negative construct compared to control groups; however, the fecal IgA levels did not increase significantly. In conclusion, our findings indicated that the yeast , as a delivery vehicle with possible immunomodulatory effects, and c-CPE, as a targeting tag, synergistically assist to stimulate systemic and local immunity. This proposed recombinant system might be useful in the expression of other antigenic peptides, making it as a promising tool for oral delivery of vaccines or therapeutic proteins.
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http://dx.doi.org/10.3389/fmicb.2018.00723DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5908956PMC
April 2018

Multiplex serology of Helicobacter pylori antigens in detection of current infection and atrophic gastritis - A simple and cost-efficient method.

Microb Pathog 2018 Jun 14;119:137-144. Epub 2018 Apr 14.

HPGC Research Group, Department of Medical Biotechnology, Pasteur Institute of Iran, Tehran, Iran. Electronic address:

Introduction: Helicobacter pylori express a large array of antigens, each of which is duly responsible for successful colonization and pathogenesis. Here, we have studied host serum antibody responses to four of its immunodominant antigens in association with the infection status and the resulting clinical outcomes.

Methods: For this purpose, four individual H. pylori proteins (UreB, CagA, Tip-α and HP0175) were produced in recombinant forms. Serum antibody responses of 246 (75 GC and 171 NUD) patients, against the above antigens, were evaluated by multiplex immunoblotting. The associations between the resulting data and the infection status, as well as clinical outcomes were evaluated using logistic regression models.

Results: Serum antibodies to all four recombinant antigens increased the chances of detecting screening ELISA-positive subjects, in an escalating dose-dependent manner, ranging from 2.6 (1.5-4.7) for HP0175 to 14.3 for UreB (4.3-50.7), exhibiting the lowest and highest odds ratios, respectively (P ≤ 0.001), such that 98.2% of the subjects with antibodies to all four antigens, were also positive by the screening ELISA (P < 0.0001). Among the screening ELISA-positive subjects, the three antigens of CagA, Tip-α, and HP0175 were able to segregate current from past H. pylori infection (P < 0.05). Accordingly, subjects with antibodies to one or more antigen(s) were at 5.4 (95% CI: 1.8-16.4) folds increased chances of having current infection, as compared to triple negatives (P = 0.003). In reference to the clinical outcomes, those with serum antibodies to CagA were more prevalent among gastric cancer, as compared to NUD patients (OR: 5.4, 95% CI: 2.4-12.2, P < 0.0001). When NUD patients were categorized according to their histopathologic status, multiple antigen analysis revealed that subjects with serum antibodies to one or more of the 3 current infection-positive antigens (CagA, Tip-α, and HP0175) were at 9.7 (95% CI: 2.1-44.9, P = 0.004) folds increased risk of atrophic gastritis, in reference to triple negatives.

Conclusion: The non-invasive multiplex serology assay, presented here, was able to not only detect subjects with current H. pylori infection, it could also screen dyspeptic patients for the presence of gastric atrophy. This simple and cost-efficient method can supplement routine screening ELISAs, to increase the chances of detecting current infections as well as atrophic gastritis.
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http://dx.doi.org/10.1016/j.micpath.2018.04.018DOI Listing
June 2018

SCIENTISTS and SCIENCE ADVOCATES: Professor David W. Denning, An Extraordinary High Achiever in Clinical and Translational Science

Authors:
Vahid Khalaj

Iran Biomed J 2018 09 4;22(5):290-1. Epub 2018 Feb 4.

Medical Biotechnology Department, Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran

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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6058189PMC
September 2018

SHuffle™ T7 strain is capable of producing high amount of recombinant human fibroblast growth factor-1 (rhFGF-1) with proper physicochemical and biological properties.

J Biotechnol 2017 Oct 18;259:30-38. Epub 2017 Aug 18.

Department of Biochemistry, Pasteur Institute of Iran, Tehran, Iran. Electronic address:

Background: Human fibroblast growth factor-1 (FGF-1) has powerful mitogenic activities in a variety of cell types and plays significant roles in many physiological processes e.g. angiogenesis and wound healing. There is increasing demand for large scale production of recombinant human FGF-1 (rhFGF-1), in order to investigate the potential medical use. In the present study, we explored SHuffle™ T7 strain for production of rhFGF-1.

Methods: A synthetic gene encoding Met-140 amino acid form of human FGF-1 was utilized for expression of the protein in three different E. coli hosts (BL21 (DE3), Rosetta-gami™ 2(DE3), SHuffle™ T7). Total expressions and soluble/insoluble expression ratios of rhFGF-1 in different hosts were analyzed and compared. Soluble rhFGF-1 produced in SHuffle™ T7 cells was purified using one-step heparin-Sepharose affinity chromatography and characterized by a variety of methods for physicochemical and biological properties.

Results: The highest level of rhFGF-1 expression and maximum soluble/insoluble ratio were achieved in SHuffle™ T7 strain. Using a single-step heparin-Sepharose chromatography, about 1500mg of purified rhFGF-1 was obtained from one liter of the culture, representing purification yield of ∼70%. The purified protein was reactive toward anti-FGF-1 ployclonal antibody in immunoblotting. Mass spectrometry confirmed the protein had expected amino acid sequence and molecular weight. In reverse-phase high-performance liquid chromatography (RP-HPLC), the protein displayed the same retention time with the human FGF-1 standard, and purity of 94%. Less than 0.3% of the purified protein was comprised of oligomers and/or aggregates as judged by high-performance size-exclusion chromatography (HP-SEC). Secondary and tertiary structures of the protein, investigated by circular dichroism and intrinsic fluorescence spectroscopy methods, respectively, represented native folding of the protein. The purified rhFGF-1 was bioactive and stimulated proliferation of NIH 3T3 cells with EC50 of 0.84ng/mL.

Conclusion: Although SHuffle™ T7 has been introduced for production of disulfide-bonded proteins in cytoplasm, we herein successfully recruited it for high yield production of soluble and bioactive rhFGF-1, a protein with 3 free cysteine and no disulfide bond. To our knowledge, this is the highest-level of rhFGF-1 expression in E. coli reported so far. Extensive physicochemical and biological analysis showed the protein had similar characteristic to authentic FGF-1.
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http://dx.doi.org/10.1016/j.jbiotec.2017.08.015DOI Listing
October 2017

Expression Optimization of Anti-CD22 scFv-Apoptin Fusion Protein Using Experimental Design Methodology

Iran Biomed J 2018 01 10;22(1):66-9. Epub 2017 Jul 10.

Department of Pharmaceutical Chemistry, School of Pharmacy, Shahid Beheshti University of Medical Sciences, Tehran, Iran.

Background: Design of experiments is a rapid and cost-effective approach for optimization of recombinant protein production process. In our previous study, we generated a potent dual-acting fusion protein, anti-CD22 scFv-apoptin, to target B-cell malignant cell lines. In the present investigation, we report the effect of different variables on the expression levels of this fusion protein.

Methods: Four variables (cell optical density at induction, IPTG concentration, induction temperature, and induction time) were tested using experimental design.

Results: Our findings demonstrated that among the examined variables, only the induction time had a significant positive effect on the protein expression yield.

Conclusion: Experimental design was successfully applied in this study. The optimized condition obtained in the current study can be applied in future commercial production of this novel fusion protein.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5712387PMC
http://dx.doi.org/10.22034/ibj.22.1.66DOI Listing
January 2018

A novel anti-CD22 scFv-apoptin fusion protein induces apoptosis in malignant B-cells.

AMB Express 2017 Dec 2;7(1):112. Epub 2017 Jun 2.

Department of Medical Biotechnology, Biotechnology Research Center, Pasteur Institute of Iran, 13164, Tehran, Iran.

CD22 marker is a highly internalizing antigen which is located on the surface of B-cells and is being used as a promising target for treatment of B cell malignancies. Monoclonal antibodies targeting CD22 have been introduced and some are currently under investigation in clinical trials. Building on the success of antibody drug conjugates, we developed a fusion protein consisting of a novel anti-CD22 scFv and apoptin and tested binding and therapeutic effects in lymphoma cells. The recombinant protein was expressed in E. coli and successfully purified and refolded. In vitro binding analysis by immunofluorescence and flow cytometry demonstrated that the recombinant protein specifically binds to CD22 positive Raji cells but not to CD22 negative Jurkat cells. The cytotoxic properties of scFv-apoptin were assessed by an MTT assay and Annexin V/PI flow cytometry analysis and showed that the recombinant protein induced apoptosis preferentially in Raji cells with no detectable effects in Jurkat cells. Our findings indicated that the recombinant anti-CD22 scFv-apoptin fusion protein could successfully cross the cell membrane and induce apoptosis with high specificity, make it as a promising molecule for immunotherapy of B-cell malignancies.
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http://dx.doi.org/10.1186/s13568-017-0410-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5457376PMC
December 2017

Human-Yeast Hybrids: New Visions to Genetic Disorders and Drug Discovery

Iran Biomed J 2017 07 13;21(4):205. Epub 2017 Jun 13.

Medical Biotechnology Department, Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran

Yeast has been a very helpful organism for centuries, especially with respect to fermentation of sugars and production of bread. However, for an even longer time, yeast has been a distant relative of humans having diverged from a common ancestor, about one billion years ago. More than one third of the yeast genes have human counterparts, despite this evolutionary distance. Yeast and human orthologs perform the same or similar functions. Investigations have demonstrated that 9-92% of the amino acid sequences in similar human and yeast proteins overlap. However, even if two genes perform similar functions in two different organisms, it may not be possible to replace some of yeast genes with their human counterpart...
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July 2017

Acceleration of bone regeneration in bioactive glass/gelatin composite scaffolds seeded with bone marrow-derived mesenchymal stem cells over-expressing bone morphogenetic protein-7.

Mater Sci Eng C Mater Biol Appl 2017 Jun 24;75:688-698. Epub 2017 Feb 24.

Institute of Materials Physics and Engineering, Department of Applied Science and Technology (DISAT), Politecnico di Torino, Torino, Italy.

In this research, the osteoinduction effect of a novel variant of bone morphogenetic protein-7 (BMP-7), delivered through bone marrow mesenchymal stem cells (BM-MSCs) seeded on bioactive glass/gelatin nanocomposite scaffolds, was evaluated in a calvarial critical size defect in rats. After being harvested and characterized in vitro, BM-MSCs were infected by a plasmid vector containing BMP-7 encoding gene enriched with a heparin-binding site (B2BMP-7) to assess its osteogenic effects in vivo. The animals were randomly categorized into three groups receiving the scaffold alone (group I), the scaffold seeded with BM-MSCs (group II), and the scaffold seeded with manipulated BM-MSCs (group III). After 2, 4 and 12 postoperative weeks, the animals were sacrificed and the harvested specimens were analyzed using histological and immunohistochemical staining. The results of in vitro tests (preliminary screening) showed that the synthesized scaffolds were biocompatible constructs supporting cell attachment and expansion. The in vivo results revealed higher osteogenesis in the defects filled with the B2BMP-7 excreting BM-MSCs/scaffolds compared to the other two groups. After 12weeks of implantation, fully mature newly formed bone was detected throughout the damaged site, which indicates a synergistic effect of cells, scaffolds and growth factors in the process of tissue regeneration. Therefore, bioactive glass-containing scaffolds pre-seeded with manipulated BM-MSCs exhibit an effective combination to improve osteogenesis in bone defects, and the approach followed in this work could have a significant impact in the development of novel tissue engineering constructs.
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http://dx.doi.org/10.1016/j.msec.2017.02.097DOI Listing
June 2017

Characteristics and Lethality of a Novel Recombinant  Dermonecrotic Venom Phospholipase D from  Hemiscorpius lepturus.

Toxins (Basel) 2017 03 13;9(3). Epub 2017 Mar 13.

Venom and Biotherapeutics Molecules Lab., Medical Biotechnology Department, Biotechnology Research Center, Pasteur Institute of Iran, Tehran 13169-43551, Iran.

Hemoscorpius lepturus is the most medically important scorpion in Iran. The clinical signs of H. lepturus envenomation are remarkably similar to those reported for brown spiders, including dermonecrosis, hematuria, renal failure and even death. The lethality and toxicity of brown spiders' venom have been attributed to its phospholipase D activity. This study aims to identify a phospholipase D with possible lethality and dermonecrotic activity in H. lepturus venom. In this study, a cDNA library of the venom glands was generated by Illumina RNA sequencing. Phospholipase D (PLD) from H. lepturus was characterized according to its significant similarity with PLDs from brown spiders. The main chain designated as Hl-RecPLD1 (the first recombinant isoform of H. lepturus PLD) was cloned, expressed and purified. Sphingomyelinase, dermonecrotic and lethal activities were examined. Hl-PLD1 showed remarkable sequence similarity and structural homology with PLDs of brown spiders. The conformation of Hl-PLD1 was predicted as a "TIM beta/alpha-barrel". The lethal dose 50 (LD50) and dermonecrotic activities of Hl-RecPLD1 were determined as 3.1 μg/mouse and 0.7 cm2 at 1 μg respectively. It is the first report indicating that a similar molecular evolutionary mechanism has occurred in both American brown spiders and this Iranian scorpion. In conclusion, Hl-RecPLD1 is a highly active phospholipase D, which would be considered as the lethal dermonecrotic toxin in H. lepturus venom.
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http://dx.doi.org/10.3390/toxins9030102DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5371857PMC
March 2017

Evaluating the expression profile and stability of different UCOE containing vector combinations in mAb-producing CHO cells.

BMC Biotechnol 2017 02 22;17(1):18. Epub 2017 Feb 22.

Biotechnology Research Center, Pasteur Institute of Iran, Tehran, 1316943551, Iran.

Background: As the demand for monoclonal antibodies (mAb) increases, more efficient expression methods are required for their manufacturing process. Transcriptional gene silencing is a common phenomenon in recombinant cell lines which leads to expression reduction and instability. There are reports on improved antibody expression in ubiquitous chromatin opening element (UCOE) containing both heavy and light chain gene constructs. Here we investigate the impact of having these elements as part of the light chain, heavy chain or both genes during cell line development. In this regard, non-UCOE and UCOE vectors were constructed and stable Chinese hamster ovary (CHO) cell pools were generated by different vector combinations.

Results: Expression analysis revealed that all UCOE cell pools had higher antibody yields compared to non-UCOE cells, Moreover the most optimal expression was obtained by cells containing just the UCOE on heavy chain. In terms of stability, it was shown that the high level of expression was kept consistence for more than four months in these cells whereas the expression titers were reduced in the other UCOE pools.

Conclusions: In conclusion, UCOE significantly enhanced the level and stability of antibody expression and the use of this element with heavy chain provided more stable cell lines with higher production level.
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http://dx.doi.org/10.1186/s12896-017-0330-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5322649PMC
February 2017

Corrigendum to "The first report on transcriptome analysis of the venom gland of Iranian scorpion, Hemiscorpius lepturus" [Toxicon 125 (2017) 123-130].

Toxicon 2017 03 10;128:60. Epub 2017 Feb 10.

Venom & Biotherapeutics Molecules Laboratory, Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran. Electronic address:

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http://dx.doi.org/10.1016/j.toxicon.2017.01.012DOI Listing
March 2017

Identification and Evaluation of Novel Drug Targets against the Human Fungal Pathogen Aspergillus fumigatus with Elaboration on the Possible Role of RNA-Binding Protein.

Iran Biomed J 2017 03 21;21(2):84-93. Epub 2016 Dec 21.

Fungal Biotechnology Unit, Medical Biotechnology Department, Biotechnology Research Center, Pasteur Institute of Iran, Tehran 1316943551, Iran.

Background: Aspergillus fumigatus is an airborne opportunistic fungal pathogen that can cause fatal infections in immunocompromised patients. Although the current anti-fungal therapies are relatively efficient, some issues such as drug toxicity, drug interactions, and the emergence of drug-resistant fungi have promoted the intense research toward finding the novel drug targets.

Methods: In search of new antifungal drug targets, we have used a bioinformatics approach to identify novel drug targets. We compared the whole proteome of this organism with yeast Saccharomyces cerevisiae to come up with 153 specific proteins. Further screening of these proteins revealed 50 potential molecular targets in A. fumigatus. Amongst them, RNA-binding protein (RBP) was selected for further examination. The aspergillus fumigatus RBP (AfuRBP), as a peptidylprolyl isomerase, was evaluated by homology modeling and bioinformatics tools. RBP-deficient mutant strains of A. fumigatus were generated and characterized. Furthermore, the susceptibility of these strains to known peptidylprolyl isomerase inhibitors was assessed.

Results: AfuRBP-deficient mutants demonstrated a normal growth phenotype. MIC assay results using inhibitors of peptidylprolyl isomerase confirmed a higher sensitivity of these mutants compared to the wild type.

Conclusion: Our bioinformatics approach revealed a number of fungal-specific proteins that may be considered as new targets for drug discovery purposes. Peptidylprolyl isomerase, as a possible drug target, was evaluated against two potential inhibitors and the promising results were investigated mechanistically. Future studies would confirm the impact of such target on the antifungal discovery investigations.
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http://dx.doi.org/10.18869/acadpub.ibj.21.2.84DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5274715PMC
March 2017

The first report on transcriptome analysis of the venom gland of Iranian scorpion, Hemiscorpius lepturus.

Toxicon 2017 Jan 30;125:123-130. Epub 2016 Nov 30.

Venom & Biotherapeutics Molecules Laboratory, Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran. Electronic address:

Hemiscorpius lepturus scorpion is one of the most venomous members of the Hemiscorpiidae family. H. lepturus is distributed in Iran, Iraq and Yemen. The prevalence and severity of scorpionism is high and health services are not able to control it. Scorpionism in Iran especially in the southern regions (Khuzestan, Sistan and Baluchestan, Hormozgan, Ilam) is one of the main health challenges. Due to the medical and health importance of scorpionism, the focus of various studies has been on the identification of H. lepturus venom components. Nevertheless, until now, only a few percent of H. lepturus venom components have been identified and there is no complete information about the venom components of H. lepturus. The current study reports transcriptome analysis of the venom gland of H. lepturus scorpion. Illumina Next Generation Sequencing results identified venom components of H. lepturus. When compared with other scorpion's venom, the venom of H. lepturus consists of mixtures of peptides, proteins and enzymes such as; phospholipases, metalloproteases, hyaluronidases, potassium channel toxins, calcium channel toxins, antimicrobial peptides (AMPs), venom proteins, venom toxins, allergens, La1-like peptides, proteases and scorpine-like peptides. Comparison of identified components of H. lepturus venom was carried out with venom components of reported scorpions and various identities and similarities between them were observed. With transcriptome analysis of H. lepturus venom unique sequences, coding venom components were investigated. Moreover, our study confirmed transcript expression of previously reported peptides; Hemitoxin, Hemicalcin and Hemilipin. The gene sequences of venom components were investigated employing transcriptome analysis of venom gland of H. lepturus. In summary, new bioactive molecules identified in this study, provide basis for venomics studies of scorpions of Hemiscorpiidae family and promises development of novel biotherapeutics.
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http://dx.doi.org/10.1016/j.toxicon.2016.11.261DOI Listing
January 2017

Helicobacter pylori Peptidyl Prolyl Isomerase Expression Is Associated with the Severity of Gastritis.

J Gastrointest Cancer 2016 Dec;47(4):375-380

HPGC Group, Department of Medical Biotechnology, Biotechnology Research Center, Pasteur Institute of Iran, Tehran, 1316943551, Iran.

Purpose: Helicobacter pylori secretory peptidyl prolyl isomerase, HP0175, is progressively identified as a pro-inflammatory and pro-carcinogenic protein, which serves to link H. pylori infection to its more severe clinical outcomes. Here, we have analyzed host HP0175-specific antibody responses in relation to the severity of gastritis.

Methods: The HP0175 gene fragment was PCR-amplified, cloned, expressed and purified by Ni-NTA affinity chromatography. Serum antigen-specific antibody responses of non-ulcer dyspeptic patients (N = 176) against recombinant HP0175 were detected by western blotting. The infection status of these subjects was determined by rapid urease test, culture, histology, and serology. The grade of inflammation and stage of atrophy were scored blindly according to the OLGA staging system.

Results: The recombinant HP0175 (rHP0175) was expressed as a ~35 kDa protein and its identity was confirmed by western blotting using anti-6X His tag antibody and pooled H. pylori-positive sera. Serum IgG antibodies against rHP0175 segregated our patients into two similar-sized groups of sero-positives (90/176, 51.1 %) and sero-negatives (86/176, 48.9 %). The former presented with higher grades of gastric inflammation (OR = 4.4, 95 % CI = 1.9-9.9, P = 0.001) and stages of gastric atrophy (OR = 18.3, 95 %CI = 1.4-246.6, P = 0.028).

Conclusion: Our findings lend further support to the pro-inflammatory nature of H. pylori peptidyl prolyl isomerase (HP0175) and recommends this antigen as a non-invasive serum biomarker of the severity of H. pylori-associated gastritis.
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http://dx.doi.org/10.1007/s12029-016-9849-xDOI Listing
December 2016

Expression of granulocyte colony stimulating factor (GCSF) in Hansenula polymorpha.

Iran J Microbiol 2016 Feb;8(1):21-8

Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran.

Background And Objectives: During past decades Hansenula polymorpha has attracted global attention for the expression of recombinant proteins due to its high growth rate, minimal nutritional porequirements and use of methanol as a low cost inducer.

Materials And Methods: The corresponding nucleotide sequences for the expression of heterologous genes in Hansenula poylmorpha were extracted and assembled in an E. coli vector. The constructed expression cassette included formate dehydrogenase promoter (pFMD), a secretory signal sequence, a multiple cloning site (MCS) and methanol oxidase (MOX) terminator. Zeocin resistance gene fragment and complete cDNA encoding granulocyte colony stimulating factor (GCSF) were cloned downstream of the expression cassette in-frame with signal sequence. Restriction mapping and sequence analysis confirmed the correct cloning procedures. Final vector was transformed into Hansenula and recombinant host was induced for the expression of GCSF protein by adding methanol. SDS-PAGE and immuno-blotting were performed to confirm the identity of r-GCSF.

Results: The expression cassette containing gcsf gene (615bp) and zeocin resistance marker (sh-ble, 1200bp) was prepared and successfully transformed into competent Hansenula polymorpha cells via electroporation. Zeocin resistant colonies were selected and GCSF expression was induced in recombinant Hansenula transformants using 0.5% methanol and an approximately 19kDa protein was observed on SDS-PAGE. Western blot analysis using serum isolated from GCSF-treated rabbit confirmed the identity of the protein.

Conclusions: Molecular studies confirmed the designed expression cassette containing gcsf gene along with pFMD and signal sequence. The expressed 19kDa protein also confirmed the ability of designed vector in expressing heterologous genes in Hansenula cells.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4833737PMC
February 2016

T7-RNA polymerase dependent RNAi system in Aspergillus fumigatus: a proof of concept study.

FEMS Microbiol Lett 2016 Mar 5;363(5):fnw029. Epub 2016 Feb 5.

Medical Biotechnology Department, Biotechnology Research Center, Pasteur Institute of Iran, Pasteur Ave, 1316943551 Tehran, Iran

An RNAi system based on T7 RNA polymerase (TRNAP) was designed and examined in Aspergillus fumigatus. This system consists of two elements; an inducible T7RNAP expressing cassette and an AMA1-based episomal RNAi plasmid. These constructs were transformed into the A. fumigatus protoplasts and the efficiency of this system was tested in downregulation of alb1 gene. Upon the induction of T7RNAP expression, the recombinant T7RNAP was able to recognize T7 promoters, which were located on the episomal plasmid and in opposite direction. As a result, the bidirectional transcription of alb1 fragment led to the silencing of the target gene. However, our results demonstrated that this silencing system is unstable and may not be applicable in preparation of RNAi libraries.
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http://dx.doi.org/10.1093/femsle/fnw029DOI Listing
March 2016

Fungal annexins: a mini review.

Springerplus 2015 24;4:721. Epub 2015 Nov 24.

Medical Biotechnology Department, Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran.

The large family of annexins is composed of more than a thousand members which are typically phospholipid-binding proteins. Annexins act in a number of signalling networks and membrane trafficking events which are fundamental to cell physiology. Annexins exert their functions mainly through their calcium-dependent membrane binding abilities; however, some calcium-independent interactions have been documented in the literature. Although mammalian and plant annexins have been well characterized, little is known about this family in fungi. This mini review summarizes the available data on fungal annexins.
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http://dx.doi.org/10.1186/s40064-015-1519-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4656261PMC
December 2015

Comparison of recombinant A2-ELISA with rKE16 dipstick and direct agglutination tests for diagnosis of visceral leishmaniasis in dogs in Northwestern Iran.

Rev Soc Bras Med Trop 2015 Mar-Apr;48(2):188-93

Department of Parasitology, Pasteur Institute of Iran, Tehran, Iran.

Introduction: Various methods are used for the diagnosis of visceral leishmaniasis (VL), such as microscopic examination, culture and inoculation of laboratory animals; however, serological assays are commonly used for the detection of antibodies in serum samples with a wide range of specificity and sensitivity.

Methods: The purpose of this study was to compare three serological methods, including rA2-ELISA, the recombinant KE16 (rKE16) dipstick test and the direct agglutination test (DAT), for the detection of antibodies against VL antigens. The assays utilized 350 statistically based random serum samples from domestic dogs with clinical symptoms as well as samples from asymptomatic and healthy dogs from rural and urban areas of the Meshkinshahr district, northwestern Iran.

Results: Samples were assessed, and the following positive rates were obtained: 11.5% by rKE16, 26.9% by DAT and 49.8% by ELISA. The sensitivity among symptomatic dogs was 32.4% with rKE16, 100% with DAT and 52.9% with ELISA. Conversely, rA2-ELISA was less specific for asymptomatic dogs, at 46.5%, compared with DAT, at 88.9%.

Conclusions: This study recommends rA2-ELISA as a parallel assay combined with DAT to detect VL infection among dogs. Further evaluations should be performed to develop an inexpensive and reliable serologic test for the detection of Leishmania infantum among infected dogs.
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http://dx.doi.org/10.1590/0037-8682-0285-2014DOI Listing
February 2016

Effect of Cysteamine on Cell Growth and IgG4 Production in Recombinant Sp2.0 Cells.

Iran J Pharm Res 2015 ;14(1):177-87

Medical Biotechnology Department, Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran.

The manipulation of redox potential in secretory pathway by thiol reducing agents can be a strategy to improve the production levels of disulfide-bonded proteins including recombinant antibodies. Here we have studied the influence of cysteamine on viability and the production level of IgG4 in Sp2.0 cells. For this purpose, the recombinant Sp2.0 cells producing an anti CD33 IgG4, were subjected to different concentrations of cysteamine. At concentrations of 2, 4 and 5 mM cysteamine, the secreted levels of IgG4 did not change significantly. However, in concentration of 7 mM cysteamine, a significant decrease was observed in IgG4 levels which may indicate the cytotoxicity of this compound in higher concentrations. Our results show that the cysteamine treatment reduces the cell viability in a dose-dependent manner. Also it was observed that 2 mM cysteamine had no late effect on IgG4 production level and only at day 3, this concentration of cysteamine decreased the cell viability significantly. To test whether the addition of cysteamine can affect the expression level of protein disulfide isomerase, RT-PCR analysis was carried out. The results revealed that cysteamine does not affect the PDI transcription and expression level of IgG4 in this type of recombinant cells.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4277631PMC
January 2015