Iranian Journal of Pharmaceutical Research, Feb 10, 2012
Our previous studies revealed an inhibitory effect of ICD-85 (Venom derived peptides) on breast c... more Our previous studies revealed an inhibitory effect of ICD-85 (Venom derived peptides) on breast cancer cell line MDA-MB231. ICD-85 was also confirmed by in-vivo studies to suppress the breast tumor in mice. However, the exact mechanism of ICD-85 was unknown. Hence, the present study was undertaken to assess the mechanism of ICD-85 effect as an anti-proliferative agent of cancer cells. The effect of ICD-85 on proliferation of HL-60 cancer cells was determined by using the MTT assay. The morphological changes of ICD-85 treated HL-60 cells were observed under transmission electron microscope (TEM). DNA fragmentation analysis was also carried out using gel electrophoresis. ICD-85 induced the marked inhibition of HL60 cell proliferation with an IC50-value of 0.04 μg/mL following 24 h of incubation. ICD-85 treated cells when compared with untreated cells, showed nuclear material condensation, endoplasmic reticulum dilation, mitochondria swelling or degradation, increased cytoplasmic vacuoles, reduction or disappearance in cytoplasmic process and decreased nuclear/cytoplasmic ratio was observed. The characteristic DNA ladder formation of ICD-85-treated cells in agarose gel electrophoresis confirmed the results obtained through the electron microscopy. The results of the present study indicated that ICD-85 inhibited the cancer cell proliferation by inducing cell apoptosis.
Bovine viral diarrhea (BVD) is an economically important cattle disease with a worldwide distribu... more Bovine viral diarrhea (BVD) is an economically important cattle disease with a worldwide distribution. Detection and elimination of animals persistently infected (PI) with bovine viral diarrhea virus (BVDV) is essential for the control of BVD and eradication of BVDV. There are usually no pathognomonic clinical signs of BVDV infection. Diagnostic investigations therefore rely on laboratory-based detection of the virus, or virus-induced antigens or antibodies. Erns as an immunogenic protein of BVDV, is genetically and antigenically conserved among different isolates and therefore, is a candidate antigen for development of the enzyme linked immunosorbent assay (ELISA) for serological studies or identification of PI animals. The aim of this study was to produce a monoclonal antibody (MAb) against recombinant Erns. For this purpose, recombinant maltose-binding protein (MBP)-Erns protein was expressed in Escherichia coli and purified using amylose resin chromatography column and used as an antigen in MAb production. Spleen cells of the immunized mice with the recombinant antigen were fused with SP2/0 myeloma cells. Next, culture supernatants of primary clones of fused cells were screened by indirect ELISA. After three rounds of cloning, the reactivity of the MAbs with recombinant and natural antigen was established by Western blotting. Based on our results, MAb against recombinant Erns was produced and reacted successfully with recombinant and natural antigens. With regards to the role of Erns in the identification of PI animals, it appears that Erns recombinant antigen and the specific monoclonal antibodies produced against it may be suitable for developing BVDV laboratory diagnostic assays.
Iranian journal of pharmaceutical research : IJPR, 2012
Our previous studies revealed an inhibitory effect of ICD-85 (Venom derived peptides) on breast c... more Our previous studies revealed an inhibitory effect of ICD-85 (Venom derived peptides) on breast cancer cell line MDA-MB231. ICD-85 was also confirmed by in-vivo studies to suppress the breast tumor in mice. However, the exact mechanism of ICD-85 was unknown. Hence, the present study was undertaken to assess the mechanism of ICD-85 effect as an anti-proliferative agent of cancer cells. The effect of ICD-85 on proliferation of HL-60 cancer cells was determined by using the MTT assay. The morphological changes of ICD-85 treated HL-60 cells were observed under transmission electron microscope (TEM). DNA fragmentation analysis was also carried out using gel electrophoresis. ICD-85 induced the marked inhibition of HL60 cell proliferation with an IC50-value of 0.04 μg/mL following 24 h of incubation. ICD-85 treated cells when compared with untreated cells, showed nuclear material condensation, endoplasmic reticulum dilation, mitochondria swelling or degradation, increased cytoplasmic vacuo...
Six consecutive overlapped coding regions (F1-F6) of whole NS3 molecule of bovine viral diarrhea ... more Six consecutive overlapped coding regions (F1-F6) of whole NS3 molecule of bovine viral diarrhea virus (BVDV) were cloned into pMAL-c2X plasmid vector and expressed in Escherichia coli cells (BL21 strain). The recombinant proteins were then purified by amylose resin to determine the most immunogenic domain(s) of the NS3 molecule. Evaluation of the recombinant proteins was carried out by indirect ELISAs using several bovine sera (previously characterized by virus neutralization test, a commercial ELISA kit, and a newly developed NS3-ELISA) and 6 monoclonal antibodies. The experiments showed that the most immunogenic domain of the NS3 protein was the fourth designed fragment (F4), a 122 amino-acid (AA) region of about 13.5 kDa (nucleotide 1003-1368; residue 335-456). Purified recombinant F4 was also evaluated as single ELISA antigen (F4-ELISA) for the detection of anti-BVDV antibodies in sera of infected cattle. Although this small recombinant fragment of NS3 protein was almost completely soluble and expressed more efficient respect to whole NS3 molecule, it did not show enough sensitivity and specificity to be a proper substitute for NS3 as ELISA antigen to detect specific antibodies against BVDV. However, statistical analyses showed a medium correlation between the results of the developed F4-ELISA and virus neutralization test (kappa coefficient=0.63, P<0.001), with the relative sensitivity and specificity of 78.05% and 84.91%, respectively, suggesting the potential use of this fragment as an ELISA antigen along with other antigens or monoclonal antibody(s) in a competitive ELISA.
The direct transmission of avian influenza viruses to human and increasing drug resisted strains ... more The direct transmission of avian influenza viruses to human and increasing drug resisted strains posing new threats for public health. Therefore, development of efficient vaccines is needed to generate protective and persistent immunity to the viruses. Three motifs of Mx protein sequence in human, mouse and poultry located in interferon induced (GTP ase) domain were candidate as biologic adjuvant for enhancing the immune responses against influenza virus. Chimera proteins composed with the conserved HA2 subunit of influenza virus and the Mx motifs named HA2/Mx were modeled and evaluated by in silico analysis includes bioinformatics algorithms in order to explore biological characteristics of these peptides. Amongst the predicted models, HA2/Mx1 peptide showed the better results following protein structures prediction, antigenic epitopes determination and model quality evaluation. Comparative homology modeling was performed with Swiss Model and the model was validated using ProSA. Epitope predictions revealed the construct could induce both B and T cell epitopes that expect a high immune response. Taken together, these data indicate that the HA2/Mx1 chimera peptide can be potentiated for developing an adjuvant-fused influenza vaccine capable of stimulating effective immune response.
Iranian Journal of Pharmaceutical Research, Feb 10, 2012
Our previous studies revealed an inhibitory effect of ICD-85 (Venom derived peptides) on breast c... more Our previous studies revealed an inhibitory effect of ICD-85 (Venom derived peptides) on breast cancer cell line MDA-MB231. ICD-85 was also confirmed by in-vivo studies to suppress the breast tumor in mice. However, the exact mechanism of ICD-85 was unknown. Hence, the present study was undertaken to assess the mechanism of ICD-85 effect as an anti-proliferative agent of cancer cells. The effect of ICD-85 on proliferation of HL-60 cancer cells was determined by using the MTT assay. The morphological changes of ICD-85 treated HL-60 cells were observed under transmission electron microscope (TEM). DNA fragmentation analysis was also carried out using gel electrophoresis. ICD-85 induced the marked inhibition of HL60 cell proliferation with an IC50-value of 0.04 μg/mL following 24 h of incubation. ICD-85 treated cells when compared with untreated cells, showed nuclear material condensation, endoplasmic reticulum dilation, mitochondria swelling or degradation, increased cytoplasmic vacuoles, reduction or disappearance in cytoplasmic process and decreased nuclear/cytoplasmic ratio was observed. The characteristic DNA ladder formation of ICD-85-treated cells in agarose gel electrophoresis confirmed the results obtained through the electron microscopy. The results of the present study indicated that ICD-85 inhibited the cancer cell proliferation by inducing cell apoptosis.
Bovine viral diarrhea (BVD) is an economically important cattle disease with a worldwide distribu... more Bovine viral diarrhea (BVD) is an economically important cattle disease with a worldwide distribution. Detection and elimination of animals persistently infected (PI) with bovine viral diarrhea virus (BVDV) is essential for the control of BVD and eradication of BVDV. There are usually no pathognomonic clinical signs of BVDV infection. Diagnostic investigations therefore rely on laboratory-based detection of the virus, or virus-induced antigens or antibodies. Erns as an immunogenic protein of BVDV, is genetically and antigenically conserved among different isolates and therefore, is a candidate antigen for development of the enzyme linked immunosorbent assay (ELISA) for serological studies or identification of PI animals. The aim of this study was to produce a monoclonal antibody (MAb) against recombinant Erns. For this purpose, recombinant maltose-binding protein (MBP)-Erns protein was expressed in Escherichia coli and purified using amylose resin chromatography column and used as an antigen in MAb production. Spleen cells of the immunized mice with the recombinant antigen were fused with SP2/0 myeloma cells. Next, culture supernatants of primary clones of fused cells were screened by indirect ELISA. After three rounds of cloning, the reactivity of the MAbs with recombinant and natural antigen was established by Western blotting. Based on our results, MAb against recombinant Erns was produced and reacted successfully with recombinant and natural antigens. With regards to the role of Erns in the identification of PI animals, it appears that Erns recombinant antigen and the specific monoclonal antibodies produced against it may be suitable for developing BVDV laboratory diagnostic assays.
Iranian journal of pharmaceutical research : IJPR, 2012
Our previous studies revealed an inhibitory effect of ICD-85 (Venom derived peptides) on breast c... more Our previous studies revealed an inhibitory effect of ICD-85 (Venom derived peptides) on breast cancer cell line MDA-MB231. ICD-85 was also confirmed by in-vivo studies to suppress the breast tumor in mice. However, the exact mechanism of ICD-85 was unknown. Hence, the present study was undertaken to assess the mechanism of ICD-85 effect as an anti-proliferative agent of cancer cells. The effect of ICD-85 on proliferation of HL-60 cancer cells was determined by using the MTT assay. The morphological changes of ICD-85 treated HL-60 cells were observed under transmission electron microscope (TEM). DNA fragmentation analysis was also carried out using gel electrophoresis. ICD-85 induced the marked inhibition of HL60 cell proliferation with an IC50-value of 0.04 μg/mL following 24 h of incubation. ICD-85 treated cells when compared with untreated cells, showed nuclear material condensation, endoplasmic reticulum dilation, mitochondria swelling or degradation, increased cytoplasmic vacuo...
Six consecutive overlapped coding regions (F1-F6) of whole NS3 molecule of bovine viral diarrhea ... more Six consecutive overlapped coding regions (F1-F6) of whole NS3 molecule of bovine viral diarrhea virus (BVDV) were cloned into pMAL-c2X plasmid vector and expressed in Escherichia coli cells (BL21 strain). The recombinant proteins were then purified by amylose resin to determine the most immunogenic domain(s) of the NS3 molecule. Evaluation of the recombinant proteins was carried out by indirect ELISAs using several bovine sera (previously characterized by virus neutralization test, a commercial ELISA kit, and a newly developed NS3-ELISA) and 6 monoclonal antibodies. The experiments showed that the most immunogenic domain of the NS3 protein was the fourth designed fragment (F4), a 122 amino-acid (AA) region of about 13.5 kDa (nucleotide 1003-1368; residue 335-456). Purified recombinant F4 was also evaluated as single ELISA antigen (F4-ELISA) for the detection of anti-BVDV antibodies in sera of infected cattle. Although this small recombinant fragment of NS3 protein was almost completely soluble and expressed more efficient respect to whole NS3 molecule, it did not show enough sensitivity and specificity to be a proper substitute for NS3 as ELISA antigen to detect specific antibodies against BVDV. However, statistical analyses showed a medium correlation between the results of the developed F4-ELISA and virus neutralization test (kappa coefficient=0.63, P<0.001), with the relative sensitivity and specificity of 78.05% and 84.91%, respectively, suggesting the potential use of this fragment as an ELISA antigen along with other antigens or monoclonal antibody(s) in a competitive ELISA.
The direct transmission of avian influenza viruses to human and increasing drug resisted strains ... more The direct transmission of avian influenza viruses to human and increasing drug resisted strains posing new threats for public health. Therefore, development of efficient vaccines is needed to generate protective and persistent immunity to the viruses. Three motifs of Mx protein sequence in human, mouse and poultry located in interferon induced (GTP ase) domain were candidate as biologic adjuvant for enhancing the immune responses against influenza virus. Chimera proteins composed with the conserved HA2 subunit of influenza virus and the Mx motifs named HA2/Mx were modeled and evaluated by in silico analysis includes bioinformatics algorithms in order to explore biological characteristics of these peptides. Amongst the predicted models, HA2/Mx1 peptide showed the better results following protein structures prediction, antigenic epitopes determination and model quality evaluation. Comparative homology modeling was performed with Swiss Model and the model was validated using ProSA. Epitope predictions revealed the construct could induce both B and T cell epitopes that expect a high immune response. Taken together, these data indicate that the HA2/Mx1 chimera peptide can be potentiated for developing an adjuvant-fused influenza vaccine capable of stimulating effective immune response.
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Papers by Mohsen Lotfi