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Research paper thumbnail of Recombinant Staphylococcal Enterotoxin Type A Stimulate Antitumoral Cytokines

Technology in Cancer Research & Treatment, 2016

Background: About 20 different types of staphylococcal enterotoxins are produced by Staphylococcu... more Background: About 20 different types of staphylococcal enterotoxins are produced by Staphylococcus aureus, in which type A is more common in food poisoning syndrome. Also staphylococcal enterotoxin A superantigen is a potent inducer of cytotoxic T lymphocyte activity and cytokine production and could stimulate T cells containing T-cell receptor beta chain domains when binding to major histocompatibility complex class II molecules. Hence, it is an important reagent in cancer immunotherapy. Methods: For the construction of pET-21a/ entA cassette, the staphylococcal enterotoxin type A gene was isolated from S aureus strain HN2, cloned into pET-21a, and introduced into Escherichia coli strain BL-21(DE3). Consequently, Western blot analysis showed pET-21a/ entA cassette expression inserted entA gene successfully. It is the first prompt using a pET-21a as a cloning vector for entA gene and expression of construct in BL-21(DE3). In addition, this study examined the ability of standard stap...

Research paper thumbnail of Cloning, high level expression and immunogenicity of 1163-1256 residues of C-terminal heavy chain of C. botulinum neurotoxin type E

Research paper thumbnail of Recombinant Staphylococcal Enterotoxin Type A Stimulate Antitumoral Cytokines

Background: About 20 different types of staphylococcal enterotoxins are produced by Staphylococcu... more Background: About 20 different types of staphylococcal enterotoxins are produced by Staphylococcus aureus, in which type A is more common in food poisoning syndrome. Also staphylococcal enterotoxin A superantigen is a potent inducer of cytotoxic T lymphocyte activity and cytokine production and could stimulate T cells containing T-cell receptor beta chain domains when binding to major histocompatibility complex class II molecules. Hence, it is an important reagent in cancer immunotherapy. Methods: For the construction of pET-21a/entA cassette, the staphylococcal enterotoxin type A gene was isolated from S aureus strain HN2, cloned into pET-21a, and introduced into Escherichia coli strain BL-21(DE3). Consequently, Western blot analysis showed pET-21a/entA cassette expression inserted entA gene successfully. It is the first prompt using a pET-21a as a cloning vector for entA gene and expression of construct in BL-21(DE3). In addition, this study examined the ability of standard staphylococcal enterotoxin A and cloned staphylococcal enterotoxin A to activate T cells in vitro. Lymphocyte cells derived from lymph node BALB/c mice were exposed to standard staphylococcal enterotoxin A and cloned staphylococcal enterotoxin (1.10, 102,103, and 104 ng/mL) in order to evaluate the magnitude of proliferation, activation, and apoptosis of lymphocyte cells based on MTT and apoptosis assays, respectively. Results: Our investigation showed that the function of cloned staphylococcal enterotoxin A was same as standard staphylococcal enterotoxin A, and the optimal concentration for the activation of lymphocyte cells and induction of apoptosis was 100 ng/mL and 1000 ng/mL (P < .05), respectively. Quantification of cytokines clearly showed that lymphocyte cells exposed to standard staphylococcal enterotoxin A and cloned staphylococcal enterotoxin A significantly secreted higher interferon g and tumor necrosis factor a compared to control. Conclusion: According to our results, the biological activity of standard staphylococcal enterotoxin A and cloned staphylococcal enterotoxin A is identical; therefore, these procedures may be approved as an efficient method to express and purify this protein in a large scale.

Research paper thumbnail of Evaluation of the pathogenesis of pseudomonas aeruginosa's flagellum before and after flagellar gene knockdown by small interfering RNAs (siRNA)

Background: Pseudomonas aeruginosa possesses a polar flagellum made up of flagellar subunits, whi... more Background: Pseudomonas aeruginosa possesses a polar flagellum made up of flagellar subunits, which are encoded by fliC gene. Flagella have important roles in the motility, chemotaxis, and establishment of P. aeruginosa in the acute phase of infections. The inhibition of flagellar expression may be a promising therapeutic approach to prevent the pathogenesis. The gene-silencing effect of siRNA may be useful for this strategy. Objectives: The current study investigated the efficacy of siRNA on the expression of flagellin, because it is an important protein in the initial stages of P. aeruginosa infections.

Research paper thumbnail of Cloning, high level expression and immunogenicity of 1163-1256 residues of C-terminal heavy chain of C. botulinum neurotoxin type E

Biologicals, 2010

Botulinum neurotoxins (BoNTs) inhibit neurotransmitter release from peripheral cholinergic synaps... more Botulinum neurotoxins (BoNTs) inhibit neurotransmitter release from peripheral cholinergic synapses. BoNTs consist of a toxifying light chain and a heavy chain (HC) linked through a disulfide bond. In the present study we explored the immunogenicity and protective capability of the most effective part corresponding to 1163-1256 residues of botulinum type E neurotoxin HC gene. DNA encoding the 93 C-terminal amino acid of HC residues was synthesized with optimal codon usage for expression. These DNA fragments were ligated into a pLivSelect vector and subcloned into expression vector pET32a. Recombinant plasmids were then transformed into Escherichia coli strain BL21 DE3. The recombinant protein was purified by nickel affinity gel column chromatography. The HC1163-1256 was identified by antibodies raised against BoNT/E. HC1163-1256 was shown to bind with synaptotagmin and gangliosides, indicating that the expressed and purified HC1163-1256 protein retains a functionally active conformation. The immunization with recombinant protein induced a protection level in mice. The immunization with 2 mg of the recombinant protein induced a significant protection level in mice. In conclusion, availability of the recombinant protein provides an effective system to study the biochemical and physical interactions involved during BoNT binding to nerve cells and protection against its toxicity.

Conference Presentations by Reza Agheli

Research paper thumbnail of Proteomic profiling of fatty acid induced steatosis in human hepatoma cell lines reveal new insight into mechanisms of NAFLD

Goal: Search and identification of regulated proteins in fatty liver disease. Method: The hepat... more Goal:
Search and identification of regulated proteins in fatty liver disease.
Method:
The hepatoma cell lines Huh-7 and HepG2 were treated with a 0.5 mM mixture of oleic and palmitic acid and/or 5ng/ml TNFα to mimic steatohepatitis. Cell cultures were inspected by phase contrast microscopy and hepatic steatosis was confirmed with the Oro Red and/or MDP fluorescent stain. Protein extracted from hepatoma cells were separated by Two-Dimensional Gel Electrophoresis. Protein spots were analyzed using a MALDI-TOF/TOF spectrometer and identified with the MASCOT software. Some of the regulated proteins were validated by Western immunoblotting as detailed previously (Gazzana and Borlak, 2008).
Result:
Mapping of steatosis/NASH regulated proteins identified a total of 170 significantly regulated proteins (p < 0.05) of which 35 are novel candidates. Based on ontology mapping, the identified proteins could be grouped into lipid and carbohydrate metabolism, protein metabolism, oxidoreductases and apoptosis. By employing the DAVID’s functional enrichment tool, a high score cluster (score 10.88) for mitochondrial and peroxisomal enzyme activity were obtained and involved metabolism of reactive oxygen species, β-oxidation of fatty acids, glycolysis and gluconeogenesis. STRING analysis revealed protein-protein interactions of >70% steatosis/NASH regulated proteins. Specifically perilipin 2 a marker protein for LD growth was significantly upregulated. Equally, peroxiredoxin-6 and catalase, which participate in detoxification of reactive oxygen species were upregulated possibly as a result of enhanced FA-ß oxidation. Likewise, chloride intracellular channel 1, a protein involved in redox homeostasis and superoxide metabolism was upregulated. Furthermore, glycolytic enzymes such as phosphoglycerate mutase 1, which catalyzes the reaction 3P-Glycerate to 2P-Glycerate and alpha-enolase, which catalyzes 2P-Glycerate to PEP were significantly upregulated. Besides the mitochondrial translation elongation factor TU involved in synthesis of mitochondrial proteins was also upregulated. Taken collectively the proteome mapping of steatotic hepatocytes revealed major changes in metabolism affecting both lipid and carbohydrate catabolic and anabolic process.
Conclusion:
The data obtained are highly interesting and provide important clues on possible pathogenic mechanisms related to fatty liver disease. It also provides a stimulus for future research to explore the utility of the identified proteins for therapeutic interventions strategies and an identification/validation of mechanistically linked disease biomarker candidates.
Keywords: non-alcoholic fatty liver disease; steatosis; steatohepatitis

Research paper thumbnail of Recombinant Staphylococcal Enterotoxin Type A Stimulate Antitumoral Cytokines

Technology in Cancer Research & Treatment, 2016

Background: About 20 different types of staphylococcal enterotoxins are produced by Staphylococcu... more Background: About 20 different types of staphylococcal enterotoxins are produced by Staphylococcus aureus, in which type A is more common in food poisoning syndrome. Also staphylococcal enterotoxin A superantigen is a potent inducer of cytotoxic T lymphocyte activity and cytokine production and could stimulate T cells containing T-cell receptor beta chain domains when binding to major histocompatibility complex class II molecules. Hence, it is an important reagent in cancer immunotherapy. Methods: For the construction of pET-21a/ entA cassette, the staphylococcal enterotoxin type A gene was isolated from S aureus strain HN2, cloned into pET-21a, and introduced into Escherichia coli strain BL-21(DE3). Consequently, Western blot analysis showed pET-21a/ entA cassette expression inserted entA gene successfully. It is the first prompt using a pET-21a as a cloning vector for entA gene and expression of construct in BL-21(DE3). In addition, this study examined the ability of standard stap...

Research paper thumbnail of Cloning, high level expression and immunogenicity of 1163-1256 residues of C-terminal heavy chain of C. botulinum neurotoxin type E

Research paper thumbnail of Recombinant Staphylococcal Enterotoxin Type A Stimulate Antitumoral Cytokines

Background: About 20 different types of staphylococcal enterotoxins are produced by Staphylococcu... more Background: About 20 different types of staphylococcal enterotoxins are produced by Staphylococcus aureus, in which type A is more common in food poisoning syndrome. Also staphylococcal enterotoxin A superantigen is a potent inducer of cytotoxic T lymphocyte activity and cytokine production and could stimulate T cells containing T-cell receptor beta chain domains when binding to major histocompatibility complex class II molecules. Hence, it is an important reagent in cancer immunotherapy. Methods: For the construction of pET-21a/entA cassette, the staphylococcal enterotoxin type A gene was isolated from S aureus strain HN2, cloned into pET-21a, and introduced into Escherichia coli strain BL-21(DE3). Consequently, Western blot analysis showed pET-21a/entA cassette expression inserted entA gene successfully. It is the first prompt using a pET-21a as a cloning vector for entA gene and expression of construct in BL-21(DE3). In addition, this study examined the ability of standard staphylococcal enterotoxin A and cloned staphylococcal enterotoxin A to activate T cells in vitro. Lymphocyte cells derived from lymph node BALB/c mice were exposed to standard staphylococcal enterotoxin A and cloned staphylococcal enterotoxin (1.10, 102,103, and 104 ng/mL) in order to evaluate the magnitude of proliferation, activation, and apoptosis of lymphocyte cells based on MTT and apoptosis assays, respectively. Results: Our investigation showed that the function of cloned staphylococcal enterotoxin A was same as standard staphylococcal enterotoxin A, and the optimal concentration for the activation of lymphocyte cells and induction of apoptosis was 100 ng/mL and 1000 ng/mL (P < .05), respectively. Quantification of cytokines clearly showed that lymphocyte cells exposed to standard staphylococcal enterotoxin A and cloned staphylococcal enterotoxin A significantly secreted higher interferon g and tumor necrosis factor a compared to control. Conclusion: According to our results, the biological activity of standard staphylococcal enterotoxin A and cloned staphylococcal enterotoxin A is identical; therefore, these procedures may be approved as an efficient method to express and purify this protein in a large scale.

Research paper thumbnail of Evaluation of the pathogenesis of pseudomonas aeruginosa's flagellum before and after flagellar gene knockdown by small interfering RNAs (siRNA)

Background: Pseudomonas aeruginosa possesses a polar flagellum made up of flagellar subunits, whi... more Background: Pseudomonas aeruginosa possesses a polar flagellum made up of flagellar subunits, which are encoded by fliC gene. Flagella have important roles in the motility, chemotaxis, and establishment of P. aeruginosa in the acute phase of infections. The inhibition of flagellar expression may be a promising therapeutic approach to prevent the pathogenesis. The gene-silencing effect of siRNA may be useful for this strategy. Objectives: The current study investigated the efficacy of siRNA on the expression of flagellin, because it is an important protein in the initial stages of P. aeruginosa infections.

Research paper thumbnail of Cloning, high level expression and immunogenicity of 1163-1256 residues of C-terminal heavy chain of C. botulinum neurotoxin type E

Biologicals, 2010

Botulinum neurotoxins (BoNTs) inhibit neurotransmitter release from peripheral cholinergic synaps... more Botulinum neurotoxins (BoNTs) inhibit neurotransmitter release from peripheral cholinergic synapses. BoNTs consist of a toxifying light chain and a heavy chain (HC) linked through a disulfide bond. In the present study we explored the immunogenicity and protective capability of the most effective part corresponding to 1163-1256 residues of botulinum type E neurotoxin HC gene. DNA encoding the 93 C-terminal amino acid of HC residues was synthesized with optimal codon usage for expression. These DNA fragments were ligated into a pLivSelect vector and subcloned into expression vector pET32a. Recombinant plasmids were then transformed into Escherichia coli strain BL21 DE3. The recombinant protein was purified by nickel affinity gel column chromatography. The HC1163-1256 was identified by antibodies raised against BoNT/E. HC1163-1256 was shown to bind with synaptotagmin and gangliosides, indicating that the expressed and purified HC1163-1256 protein retains a functionally active conformation. The immunization with recombinant protein induced a protection level in mice. The immunization with 2 mg of the recombinant protein induced a significant protection level in mice. In conclusion, availability of the recombinant protein provides an effective system to study the biochemical and physical interactions involved during BoNT binding to nerve cells and protection against its toxicity.

Research paper thumbnail of Proteomic profiling of fatty acid induced steatosis in human hepatoma cell lines reveal new insight into mechanisms of NAFLD

Goal: Search and identification of regulated proteins in fatty liver disease. Method: The hepat... more Goal:
Search and identification of regulated proteins in fatty liver disease.
Method:
The hepatoma cell lines Huh-7 and HepG2 were treated with a 0.5 mM mixture of oleic and palmitic acid and/or 5ng/ml TNFα to mimic steatohepatitis. Cell cultures were inspected by phase contrast microscopy and hepatic steatosis was confirmed with the Oro Red and/or MDP fluorescent stain. Protein extracted from hepatoma cells were separated by Two-Dimensional Gel Electrophoresis. Protein spots were analyzed using a MALDI-TOF/TOF spectrometer and identified with the MASCOT software. Some of the regulated proteins were validated by Western immunoblotting as detailed previously (Gazzana and Borlak, 2008).
Result:
Mapping of steatosis/NASH regulated proteins identified a total of 170 significantly regulated proteins (p < 0.05) of which 35 are novel candidates. Based on ontology mapping, the identified proteins could be grouped into lipid and carbohydrate metabolism, protein metabolism, oxidoreductases and apoptosis. By employing the DAVID’s functional enrichment tool, a high score cluster (score 10.88) for mitochondrial and peroxisomal enzyme activity were obtained and involved metabolism of reactive oxygen species, β-oxidation of fatty acids, glycolysis and gluconeogenesis. STRING analysis revealed protein-protein interactions of >70% steatosis/NASH regulated proteins. Specifically perilipin 2 a marker protein for LD growth was significantly upregulated. Equally, peroxiredoxin-6 and catalase, which participate in detoxification of reactive oxygen species were upregulated possibly as a result of enhanced FA-ß oxidation. Likewise, chloride intracellular channel 1, a protein involved in redox homeostasis and superoxide metabolism was upregulated. Furthermore, glycolytic enzymes such as phosphoglycerate mutase 1, which catalyzes the reaction 3P-Glycerate to 2P-Glycerate and alpha-enolase, which catalyzes 2P-Glycerate to PEP were significantly upregulated. Besides the mitochondrial translation elongation factor TU involved in synthesis of mitochondrial proteins was also upregulated. Taken collectively the proteome mapping of steatotic hepatocytes revealed major changes in metabolism affecting both lipid and carbohydrate catabolic and anabolic process.
Conclusion:
The data obtained are highly interesting and provide important clues on possible pathogenic mechanisms related to fatty liver disease. It also provides a stimulus for future research to explore the utility of the identified proteins for therapeutic interventions strategies and an identification/validation of mechanistically linked disease biomarker candidates.
Keywords: non-alcoholic fatty liver disease; steatosis; steatohepatitis