Publications by authors named "Kobra Mokhtarian"

14 Publications

  • Page 1 of 1

Evaluation of Gelatinolytic and Collagenolytic Activity of Fasciola hepatica Recombinant Cathepsin-L1.

Iran J Biotechnol 2020 Jan 1;18(1):e2357. Epub 2020 Jan 1.

Department of Medical Parasitology, School of Medicine, Ardabil University of Medical Sciences.

Background: Cysteine proteases of the liver fluke, Fasciola hepatica, participate in catabolism of proteins, migration of the fluke through host tissues and combat host immune system.

Objectives: In this study, we evaluated proteolytic activity of F. hepatica recombinant cathepsin L1 (rCL1) against gelatin and collagen as common substrates.

Material And Methods: The coding sequences of F. hepatica CL1 were cloned and expressed in E. coli, in our previous study. The rCL1 was purified by nickel affinity chromatography with a HisTrap Column. The protein concentrations of the purified fractions were determined by Bradford assay. Rat collagen type-1 was treated with distinct amounts of rCL1 at 37 °C, overnight, and the byproduct was analyzed by SDS-PAGE. Furthermore, we used bovine skin gelatin as zymography substrate to evaluate the gelatinolytic activity of the purified rCL1.

Results: Recombinant CL1 was capable to digest intact type-1 collagen within 24 h and the gelatinlytic activity of rCL1 was visible at approximately 37 kDa region, with optimal activity at acidified conditions (pH 4).

Conclusion: Findings provide a possible mechanism by which a major secretory molecule of F. hepatica could be involved in parasite survival as well as its pathogenesis.
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http://dx.doi.org/10.30498/IJB.2020.143160.2357DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7461709PMC
January 2020

Morphological Description, Phylogenetic and Molecular Analysis of Isolated from Dogs in the Northwest of Iran.

Iran J Parasitol 2020 Jan-Mar;15(1):57-66

Department of Parasitology and Mycology, School of Medicine, Iran University of Medical Sciences, Tehran, Iran.

Background: Dirofilariasis is a globally distributed arthropod-borne parasitic disease of mainly canids and felids. We evaluated to extend the knowledge of morpho-molecular characteristics and outer ultrastructure of isolated from Northwest of Iran.

Methods: Overall, 67 filarial worms including 41 females and 26 males parasites were collected from the cardiovascular system of the 43 stray dogs in Meshkinshar, Ardebil Province, Northwest of Iran in 2017, and subjected to light and scanning electron microscopy (SEM) as well as carmine alum staining for morpho-molecular and identification. Molecular methods were used for confirmation of morphological findings by sequencing of Cyto-chrome c oxidase subunit I () gene.

Results: The partial DNA sequencing of gene of adult parasites showed considerable homology and close proximity to the previously isolated from Kerman and Meshkinshahr, Iran. The lowest genetic variation and the highest intra-species variability was found in and , respectively. No similarity was identified between nucleotide sequence and species as its endosymbiont bacteria.

Conclusion: The SEM technique is an excellent tool for differential recognition of the parasite surface morphology and molecular techniques could differentiate and identify spp.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7244830PMC
June 2020

Molecular Characterization of spp. from Some Parts of Iran.

Iran J Public Health 2020 Jan;49(1):157-166

Department of Parasitology and Mycology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran.

Background: Identification of liver flukes, , and by morphometric parameters is not always reliable due to the overlapping measurements. This study aimed to characterize the liver flukes of animals from different parts of Iran by the genetic markers, ITS1, and .

Methods: We collected flukes from infected livestock in six provinces of Iran from Sep to Nov 2016. The flukes were identified by amplification of a 680 bp sequence of ITS1 locus followed by a restriction fragment polymorphism (RFLP) assay. The genetic diversity among isolates was evaluated by amplification and sequencing of a 493 bp fragment of the gene.

Results: We obtained 38 specimens from Khuzestan, 22 from Tehran, 10 from Isfahan, 10 from Mazandaran, 4 from Kurdistan, and 3 from Ardabil provinces. PCR-RFLP analysis revealed two patterns, representing , and . Fifty specimens from cattle and sheep exhibited pattern and 37 from the cattle, sheep, buffalo, and goat that of . The phylogeny based on revealed two distinct clades separating from . In our phylogeny, the Iranian isolates showed a distinct separation from the African flukes, while grouped with the East Asia specimens demonstrating a common ancestor. The isolates clustered with the flukes from different parts of the world, including East Asia, Europe, and South America.

Conclusion: The present study revealed a substantial genetic difference between populations of Asia and Africa, while isolates from different parts of the world shared high similarities.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7152647PMC
January 2020

Fatty acid and retinol-binding protein: A novel antigen for immunodiagnosis of human strongyloidiasis.

PLoS One 2019 22;14(7):e0218895. Epub 2019 Jul 22.

Immunology Research center, Iran University of Medical Science, Tehran, Iran.

The tenacious human parasitic helminth Strongyloides stercoralis is a significant health problem worldwide. The current lack of a definitive diagnostic laboratory test to rule out this infection necessitates designing more specific diagnostic methods. Fatty acid and retinol-binding protein (FAR) plays a crucial role in the development and reproduction of nematodes. We generated a recombinant form of this protein and determined its applicability for immunodiagnosis of S. stercoralis. The L3 form of S. stercoralis was harvested and used for RNA extraction and cDNA synthesis. The coding sequence of S. stercoralis FAR (SsFAR) was cloned into pET28a(+) vector, expressed in E. coli BL21 and purified. ELISA and immunoblotting were employed to determine the specificity and sensitivity of rSsFAR using a set of defined sera. In addition, we analyzed the phylogenetic relationship of SsFAR with different FAR sequences from other nematodes. The cloned SsFAR had an open reading frame of 447 bp encoding 147 amino acids, with a deduced molecular mass of 19 kD. The SsFAR amino acid sequence was 93% identical to FAR of S. ratti. For differential immunodiagnosis of strongyloidiasis, rSsFAR exhibited 100% sensitivity and 97% specificity. However, cross-reactivity with FAR proteins of other parasites, namely Toxocara canis and Echinococcus granulosus, was noted. Our results provide a novel approach for immunodiagnosis of S. stercoralis infections using rSsFAR with reliable sensitivity and specificity.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0218895PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6645452PMC
February 2020

Production of recombinant 14-3-3 protein and determination of its immunogenicity for application in serodiagnosis of strongyloidiasis.

Trans R Soc Trop Med Hyg 2019 06;113(6):326-331

Department of Parasitology and Mycology, School of Medicine, Iran University of Medical Sciences, Tehran, Iran.

Background: Strongyloides stercoralis is the fourth most important intestinal nematode worldwide. The parasite load and larvae count are often low, thus conventional methods are not sufficiently sensitive to detect the infection. In this study we developed an immunoglobulin G-based enzyme-linked immunosorbent assay (ELISA) method to detect antibodies against S. stercoralis 14-3-3 protein in patients' sera.

Methods: S. stercoralis RNA was extracted and following complementary DNA synthesis, the 708-bp fragment of 14-3-3 protein was amplified by polymerase chain reaction and cloned into the pET28a+ expression vector. The 30-kDa recombinant 14-3-3 protein was expressed in Escherichia coli BL21 (DE3) cells and purified by affinity chromatography. Finally, its immunoreactivity was assessed by indirect ELISA and western blotting.

Results: The S. stercoralis 14-3-3 gene was successfully amplified and cloned into an expression vector. The 30-kDa recombinant protein was purified by affinity chromatography. An ELISA developed in-house detected infected patients' sera with 96% sensitivity.

Conclusions: We concluded that the recombinant 14-3-3 protein has enough sensitivity and specificity for detection of strongyloidiasis in human sera and could be applied for serodiagnosis.
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http://dx.doi.org/10.1093/trstmh/trz006DOI Listing
June 2019

Application of Dirofilaria immitis immunoreactive proteins in serodiagnosis.

Parasite Immunol 2019 01 15;41(1):e12598. Epub 2018 Nov 15.

Department of Parasitology and Mycology, School of Medicine, Iran University of Medical Sciences, Tehran, Iran.

Dirofilariasis is a zoonotic global vector-borne disease caused by Dirofilaria immitis. The present study focuses on the somatic and excretory/secretory (E/S) proteins released from adult D. immitis. We aimed to fractionate and identify adult D. immitis immunoreactive proteins. Somatic and E/S extracts were immunoblotted to identify the immunoreactive proteins. In the current study, we used matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF/MS) to characterize the immunogenic proteins. Additionally, we used fast protein liquid chromatography (FPLC) to fractionate and evaluate the immunogenicity of the D. immitis secretome. The most immunoreactive proteins were between 10 and 48 kDa. Six proteins including polyprotein antigen, P22u, pepsin inhibitor Dit33, neutrophil chemotactic factor (DiNCF) precursor, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and heat-shock protein 70 (HSP70) were found in both somatic and E/S extracts. Eluting the FPLC column with NaCl resolved two peaks in which the immunoreactivities of the purified proteins were conserved. Characterization of these proteins could provide a novel perspective for understanding the pathogenesis and diagnosing of this disease.
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http://dx.doi.org/10.1111/pim.12598DOI Listing
January 2019

Cloning, expression, and spectral analysis of mouse betatrophin.

Med J Islam Repub Iran 2017 18;31:102. Epub 2017 Dec 18.

Immunology Research Center, Iran University of Medical Sciences, Tehran, Iran.

Betatrophin, a novel secretory protein from liver and fatty tissues, is believed to be involved in lipid and glucose metabolism. However, its precise physiological role remains unclear. Here, we report the cloning, expression, and purification steps of mouse betatrophin in a prokaryotic system, followed by its structural analysis. Specific cloning primers were used to amplify the coding sequence of mouse liver betatrophin. The product was cloned into pET28 and expressed in E.coli BL21 (DE3) cells. The suitability of the refolding procedure was assessed by determining secondary structures of the initial and refolded proteins using circular dichroism spectroscopy. The polymerase chain reaction resulted in a 549 bp nucleotide sequence, encoding a 183 amino acid polypeptide, with an apparent molecular weight of 21 kDa, which was expressed in an inclusion body. Following an optimization and refolding procedure, the recombinant protein was purified by anion exchange and metal affinity chromatography. CD spectra revealed that the refolded protein has suitable configuration. We believe that the produced betatrophin is suitable for further biochemical studies on glucose and lipid metabolism.
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http://dx.doi.org/10.14196/mjiri.31.102DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6014755PMC
December 2017

Construction of a recombinant B-cell epitope vaccine based on a Der p1-derived hypoallergen: a bioinformatics approach.

Immunotherapy 2018 06 23;10(7):537-553. Epub 2018 Mar 23.

Department of Medical Biotechnology, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences - International Campus (IC-TUMS) Tehran, Iran.

Aim: House dust mite (HDM) allergens are important elicitors of IgE-mediated allergies. This study was aimed at constructing and characterizing a recombinant fusion protein, DpTTDp, which was based on carrier-bound Der p 1-derived peptides for HDM allergen immunotherapy.

Methods: Using the Immune Epitope Database (IEDB), we identified from Der p 1, a 34-mer hypoallergenic peptide. Two copies of the hypoallergen were then fused to a partial fragment of a tetanus toxoid molecule's N-and C terminus and expressed in Escherichia coli. After purification to homogeneity, the protein was evaluated for allergenicity and its ability to induce blocking antibodies upon immunization.

Results: Upon immunization of mice, DpTTDp induced high levels of protective IgG-antibodies that blocked allergic patients' IgE reactivity to HDM. In addition, DpTTDp lacked relevant IgE-reactivity, induced low T-cell proliferation and IFN-γ in peripheral blood mononuclear cells of HDM-allergic patients' sera.

Conclusion: The protein represents a promising HDM-allergy immunotherapy candidate vaccine.
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http://dx.doi.org/10.2217/imt-2017-0163DOI Listing
June 2018

Comparative assessment of recombinant and native immunogenic forms of Fasciola hepatica proteins for serodiagnosis of sheep fasciolosis.

Parasitol Res 2018 Jan 4;117(1):225-232. Epub 2017 Dec 4.

Immunology Research Center, Iran University of Medical Sciences, Tehran, Iran.

Laboratory diagnosis of sheep fasciolosis is commonly performed by coprological examinations; however, this method may lead to false negative results during the acute phase of the infection. Furthermore, the poor sensitivity of coprological methods is considered to be a paradox in the chronic phase of the infection. In this study, we compared the immunoreactivity of native and recombinant forms of Fasciola hepatica excretory/secretory antigens and determined their capabilities for the development of F. hepatica-specific immunoassays. Immunoreactivity and specificity of recombinant and native forms of F. hepatica antigens, including fatty acid binding protein (FABP), glutathione-S-transferase (GST), and cathepsin L-1 (CL1), in parallel with native forms of FABP and GST, were studied for serodiagnosis of the chronic form of sheep fasciolosis, individually or in combination with each other by enzyme-linked immunosorbent assays (ELISA). The correlation of the findings was assessed by receiver-operator characteristic (ROC); furthermore, the specificity and sensitivity were assessed by Youden's J. Serologic cross-reactivity was evaluated using samples from healthy sheep (n = 40), Fasciola-infected sheep (n = 30), and sheep with other parasitic infections (n = 43). The FABPs were determined to be greater than 95% sensitive for F. hepatica serodiagnosis. The most desirable diagnostic recombinant antigen was rCL1, which showed 100% sensitivity and 97% specificity in ELISA and was capable of discriminating the positive and negative samples by maximum Youden's J results. We conclude that rCL1 can be used for routine serodiagnosis of chronic fasciolosis. Thus, it could be advantageous in development of immunoassays for screening of ovine herds in fasciolosis-endemic areas and as a reliable agent for detection of fasciolosis in non-endemic regions.
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http://dx.doi.org/10.1007/s00436-017-5696-3DOI Listing
January 2018

Combine effect of Chondroitinase ABC and low level laser (660nm) on spinal cord injury model in adult male rats.

Neuropeptides 2017 Oct 14;65:90-99. Epub 2017 Jul 14.

Physiology Research Center, Department of Physiology, Faculty of Medicine, Iran University of Medical Sciences, Tehran, Iran. Electronic address:

After spinal cord injury (SCI) there are many recoveries inhibiting factors such as chondroitin sulfate proteoglycan (CSPG) and inflammation. The present study investigated the combinational effect of low level laser therapy (LLLT) as anti-inflammatory agent and Chondroitinase ABC (ChABC) enzyme as CSPG digesting factor on spinal cord after injury. This study performed on 44 male Wistar rats, spinal cord injury induced by a clip compression injury. Animals received two-weeks treatment of 660nm low level laser (LLL) and intraspinal injection of 1μg ChABC. Functional recovery, cavity size, myelination, axonal projections around the cavity, fibroblast invasion and expression of glycogen synthase kinase-3β (GSk 3β), CSPG and aquaporin 4 (AQP4) expression were evaluated. In statistical evaluation p<0.05 considered significant. Result showed the combination of LLLT and ChABC have more effect on reduction of cavity size, improvement of myelination and number of axons around the cavity and decreasing the expression of GSK3β, CSPG and AQP4 expression compared to LLLT and ChABC alone. In the laser and laser+enzyme groups AQP4 expression decreased significantly after SCI. Functional recovery, improved in LLLT and ChABC treated animals, but higher recovery belonged to the combination therapy group. The current study showed combination therapy by LLLT and ChABC is more efficient than a single therapy with each of them.
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http://dx.doi.org/10.1016/j.npep.2017.06.002DOI Listing
October 2017

Identification and Characterization of Main Allergic Proteins in Cooked Wolf Herring Fish.

Iran J Allergy Asthma Immunol 2016 Oct;15(5):363-371

Department of Medical Biotechnology, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran, Iran AND Immunology, Asthma and Allergy Research Institute, Tehran University of Medical Sciences, Tehran,Iran.

Our aim in this study was to identify and characterize allergic proteins in cooked wolf herring fish. We heated the crude extract alternatively at 50, 60, 70, 80, 90, and 100°C for one hour and results were compared by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Also, proteins were immunoblotted with fish-sensitive patients' sera. The major allergenic proteins were identified via mass spectrometry. These allergenic proteins were then purified by anion exchange chromatography and the IgE-immunoreactivity of the fractions was compared with the crude extracts via disk enzyme-linked immunosorbent assay (ELISA). SDS-PAGE of the crude extract showed more than 15 distinct protein bands. Five of these proteins, with apparent molecular weights of 12, 18, 24, 38, and 51 kDa, were only observed in the 100°C heated extract. Immunoblotting of the heated extract revealed that the 12 and 51 kDa proteins were IgE-immunoreactive with 88 percent of fish-sensitive patient sera while the 24 and 38 kDa proteins reacted with 33.3 and 55.5 percent of fish-sensitive patient sera, respectively. Mass spectrometry of the 12, 38, and 51 kDa proteins revealed that all three were parvalbumin oligomers. Disk ELISA results showed that 20 of 25 and 14 of 25 fish-allergic patients' sera were IgE-reactive with purified oligomeric parvalbumin-coated and crude extract-coated disks, respectively. Parvalbumin and its oligomers are the main allergenic molecules in cooked fish. Therefore, an enriched or purified fraction containing this protein could be a useful source of allergen for applications in ELISA-based immunoassays and could discriminate fish-allergic patients who can tolerate cooked fish from those who cannot.
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October 2016

Evaluation of anti-Cathepsin L1: a more reliable method for serodiagnosis of human fasciolosis.

Trans R Soc Trop Med Hyg 2016 09 19;110(9):542-550. Epub 2016 Oct 19.

Immunology Research center, Iran University of Medical Sciences, Tehran, Iran

Background: Coprological examinations are commonly used for diagnosis of fasciolosis. However, these methods are not useful during the acute phase of the infection and also show poor sensitivity during its chronic phase. In this study we compared the immunoreactivity of the native and recombinant forms of Fasciola hepatica excretory/secretory antigens and determined the most appropriate one for development of F. hepatica-specific immunoassays.

Methods: The coding sequences of previously-determined immunogenic proteins including cathepsin L1 (CL1), fatty acid binding protein (FABP) and glutathione-S-transferase (GST) were cloned and expressed in E. coli BL-21 cells. Native forms of FABP and GST were also purified. We evaluated the immunoreactivity of the native and recombinant proteins by ELISA using sera from 40 healthy individuals, 15 fasciolosis patients, and 57 patients with other infectious diseases.

Results: All of the studied proteins showed high sensitivity and specificity for F. hepatica serodiagnosis. However, CL1 was more sensitive and specific (100%) than the others for the detection of F. hepatica-specific antibodies. Notably, both FABP and GST showed significant cross-reactivity with hydatidosis patients' sera while CL1 did not.

Conclusions: Cathepsin L1 has acceptable sensitivity and specificity for serodiagnosis of F. hepatica and its application could be advantageous in immunoassay development.
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http://dx.doi.org/10.1093/trstmh/trw063DOI Listing
September 2016

Serodiagnosis of fasciolosis by fast protein liquid chromatography-fractionated excretory/secretory antigens.

Parasitol Res 2016 Aug 30;115(8):2957-65. Epub 2016 Apr 30.

Immunology Research Center, Iran University of Medical Science (IUMS), Tehran, Iran.

In several studies, different antigenic preparations and diverse immunological tests were applied for serodiagnosis of Fasciola hepatica infections. Most of these preparations showed cross-reactivity with proteins of other parasites. Application of purified antigens might reduce these cross-reactivities. Here, we used fast protein liquid chromatography (FPLC)-fractionated extracts of F. hepatica excretory/secretory antigens (E/S Ags) for serodiagnosis of human and sheep fasciolosis. To develop an improved diagnostic method, we fractionated F. hepatica E/S Ags by anion exchange chromatography on a Sepharose CL-6B column and then tested the serodiagnostic values of the fractions. We used sera from F. hepatica-infected human and sheep as positive controls. Sera from patients with hydatidosis and strongyloidiasis were used for cross-reactivity studies. Enzyme-linked immunosorbent assays (ELISA) of the second FPLC peak, containing 20, 25, and 70 kDa proteins, discriminated between F. hepatica-infected and uninfected human and sheep samples. Fractionation of F. hepatica E/S Ags by FPLC is a fast and reproducible way of obtaining antigens useful for serodiagnosis of human and sheep fasciolosis with acceptable sensitivity and specificity. Graphical abstract ᅟ.
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http://dx.doi.org/10.1007/s00436-016-5049-7DOI Listing
August 2016

Dicrocoelium dendriticum found in a Bronze Age cemetery in western Iran in the pre-Persepolis period: The oldest Asian palaeofinding in the present human infection hottest spot region.

Parasitol Int 2015 Oct 6;64(5):251-5. Epub 2015 Mar 6.

Departamento de Parasitología, Facultad de Farmacia, Universidad de Valencia, Av. Vicente Andrés Estellés s/n, 46100 Burjassot, Valencia, Spain. Electronic address:

Dicrocoeliasis of animals and humans is caused by trematode species of the genus Dicrocoelium, mainly Dicrocoelium dendriticum in ruminants of the Holarctic region. D. dendriticum may be considered an old parasite, probably related to the appearance and diversification of Eurasian ovicaprines, occurred 14.7-14.5 million years ago. The oldest palaeoparasitological findings of Dicrocoelium in domestic animals and humans date from more than 5000 years BC in Europe. Eggs of D. dendriticum have been found in a burial of a Bronze Age cemetery (2600-2200 BC) close to Yasuj city, southwestern Iran. This is the oldest finding of D. dendriticum in the Near East, where present human infection reports are more numerous than in other world regions where human dicrocoeliasis is rare and sporadic. This palaeofinding in the Zagros mountainous chain area is of interest by its location close to Persepolis, suggesting a narrow relationship between humans and herbivorous animals in these highlands. Domestic ruminant populations of these highlands were following a repeated contact with those of the western flat lowlands of the Fertile Crescent thanks to annual altitudinal transhumance migrations of the nomadic pastoral tribes with their herds living throughout Zagros Mountains in the several millennium period BC. It is concluded that D. dendriticum spread together with sheep and goats westward throughout Europe from the Fertile Crescent during the 8000-6000 year BC period and somewhat later southward into Africa, both spreads facilitated by the low specificity of that trematode species regarding the snail and ant intermediate hosts.
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http://dx.doi.org/10.1016/j.parint.2015.02.007DOI Listing
October 2015