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- جایگاه : پژوهشی
- مجله: Iranian Journal of Immunology
- نوع مقاله: Journal Article
- کلمات کلیدی: Breast cancer,HER2,Immunotherapy,Polyclonal antibody,subdomains of HER2
- چکیده:
- چکیده انگلیسی: Background: Human epidermal growth factor receptor 2 (HER2) has a crucial role in several malignancies. The extracellular domain of HER2 (HER2-ECD) has been extensively employed as an important target in passive and active immunotherapy. Isolated recombinant prokaryotic HER2-ECD subdomains were previously found to be ineffective in inducing anti-tumor antibody response. Objective: To employ recombinant eukaryotic HER2-ECD subdomains to raise anti-HER2 antibodies and determine their anti-tumor activity in vitro. Methods: Two paired subdomains of HER2-ECD (DI+II and DIII+IV), representing Pertuzumab and Trastuzumab binding domains, respectively, along with the full extracellular domain of HER2 were generated in CHO-K1 cells. Polyclonal antibodies were raised against these subdomains and characterized using ELISA, flow cytometry, and immunoblot and their anti-tumor activity was assessed by XTT assay. The cross-reactivity of these antibodies was specified along with other members of the human HER family. Results: Similar to Trastuzumab and anti-HER2-ECD antibody, anti-DI+II and DIII+IV polyclonal antibodies reacted with recombinant HER2-ECD and native HER2 expressed on tumor cells. These two polyclonal antibodies were able to inhibit the binding of Pertuzumab and Trastuzumab to HER2, respectively, and did not cross-react with other members of HER family. These antibodies were able to inhibit tumor cell growth in vitro, similar to Trastuzumab. Conclusion: The high immunogenicity of human HER2 DI+II and DIII+IV subdomains in rabbits and the tumor inhibitory activity of the purified specific antibodies imply that they might be suitable for active immunotherapy in formulation with appropriate adjuvants and in combination with other HER2 specific therapeutics.
- انتشار مقاله: 27-06-1396
- نویسندگان: Reza Hosseini-Ghatar,Tahereh Soltantoyeh,Motahareh Bahadori,Jalal Khoshnoodi,Forough Golsaz-Shirazi,Mahmood Jeddi-Tehrani,Mohammad Mehdi Amiri,Fazel Shokri
- مشاهده
- جایگاه : پژوهشی
- مجله: Iranian Journal of Immunology
- نوع مقاله: Journal Article
- کلمات کلیدی: Monoclonal antibody,"a" Determinant,HBs Ag
- چکیده:
- چکیده انگلیسی: Background: The antibody response to hepatitis B surface antigen (HBsAg) controls hepatitis B virus infection. The "a" determinant of HBsAg is the most important target for protective antibody response, diagnosis and immunoprophylaxis. Mutations in this area may induce immune escape mutants and affect the performance of HBsAg assays.
Objectives: To construct clinically relevant recombinant mutant forms of HBsAg and assessment of their reactivity with anti-HBs monoclonal antibodies (MAbs).
Methods: Wild type (wt) and mutant (mt) HBsAg genes were constructed by site directed mutagenesis and SEOing PCR. The amplified genes were inserted into pCMV6-neo plasmid and transfected in CHO cell line. The expression of wt- and mtHBsAg was assessed by commercial ELISA assays and stable cells were established and cloned by limiting dilution. The recombinant mutants were further characterized using a panel of anti-HBs monoclonal antibodies (MAbs) and the pattern of their reactivity was assessed by ELISA.
Results: Ten HBsAg mutants having single mutation within the "a" determinant including P120E, T123N, Q129H, M133L, K141E, P142S, D144A, G145R, N146S and C147S together with a wt form were successfully constructed and expressed in CHO cells. Reactivity of anti-HBs MAbs with mtHBsAgs displayed different patterns. The effect of mutations on antibody binding differed depending on the amino acid involved and its location within the ‘‘a’’ determinant. Mutation at amino acids 123 and 145 resulted in either complete loss or significant reduction of binding to all anti-HBs MAbs.
Conclusion: Our panel of mtHBsAgs is a valuable tool for assessment of the antibody response to HBV escape mutants and may have substantial implications in HBV immunological diagnostics.- انتشار مقاله: 15-05-1395
- نویسندگان: Forough Golsaz Shirazi,Mohammad Mehdi Amiri,Hamed Mohammadi,Ali Ahmad Bayat,Azam Roohi,Jalal Khoshnoodi,Amir Hassan Zarnani,Mahmood Jeddi-Tehrani,Gholam Ali Kardar,Fazel Shokri
- مشاهده
- جایگاه : پژوهشی
- مجله: Iranian Journal of Immunology
- نوع مقاله: Journal Article
- کلمات کلیدی: Peripheral blood mononuclear cells,atherosclerosis,Cytotoxicity,oxLDL
- چکیده:
- چکیده انگلیسی: Background: Atherosclerosis, a chronic inflammatory disease of the vessel wall is characterized by local and systemic immune responses to a variety of antigens. Oxidized low-density lipoprotein (oxLDL) is considered as an important determining factor in the pathogenesis of atherosclerosis.
Objective: The purpose of this study was to investigate the degree of peripheral blood mononuclear cells (PBMC) vulnerability to in vitro oxLDL-induced cytotoxicity from atherosclerotic patients in comparison to healthy individuals.
Methods: Thirty patients with atherosclerotic lesions, confirmed by angiography, and 30 matched healthy individuals were investigated. PBMC was prepared from individuals' blood samples which were further stimulated with low dose (1 μg/mL) and high dose (50 μg/mL) of extensively oxidized LDL. MTT assay was utilized to measure cell viability and proliferation. Stimulation index (SI) was calculated as mean ratio of optical density (OD) of the stimulated cells divided by OD of untreated cells.
Results: Low dose oxLDL treatment caused no significant proliferative or cytotoxic effect in the control group; however, similar treatment caused significant cytotoxic effect in the patient group compared to the controls (p=0.026). High dose oxLDL treatment induced more significant cytotoxicity in the patient compared to the control group (p=0.006). Comparison of the SI between the two groups of patients and controls showed significantly lower index by either the low (p=0.03) or the high dose (p<0.001) oxLDL in the patients compared to the controls.
Conclusions: PBMC from patients with atherosclerosis showed increased susceptibility to oxLDL-induced cytotoxicity. Our results imply that prolonged exposure to elevated levels of circulating oxLDL could weaken the cellular defense mechanisms by progressive depletion of the pool of antiapoptotic proteins, rendering the cells more vulnerable to oxLDL-induced cell death.- انتشار مقاله: 15-05-1395
- نویسندگان: Mohammad Jafar Mahmoudi,Maryam Mahmoudi,Fereydoon Siassi,Fazel Shokri,Mohammad Reza Eshraghian,Amir Hassan Zamani,Reza Chahardoli,Mona Hedayat,Jalal Khoshnoodi,Hashem Nayeri,Nima Rezaei,Ali-Akbar Saboor-Yaraghi
- مشاهده
- جایگاه : پژوهشی
- مجله: Iranian Journal of Immunology
- نوع مقاله: Journal Article
- کلمات کلیدی: Flow cytometry,ALL,Immunophenotyping,Pro-B,Pre-B
- چکیده:
- چکیده انگلیسی: Background: Immunophenotypic characterization of the leukemic cells has been widely used as a tool for diagnosis, classification, stratification and prognosis of leukaemia.
Objective: To investigate the immunophenotypic subtype profiles of Iranian patients with acute lymphoblastic leukemia (ALL) and its association to disease outcome.
Methods: In this study, a total of 60 Iranian patients with ALL were immunophenotyped by flow cytometry using a panel of monoclonal antibodies specific for CD2, CD3, CD5, CD10, CD13, CD14, CD19, CD20, CD33, CD34, CD45, HLA-DR and TdT molecules.
Results: The samples were initially categorized into T-ALL (n=9), B-ALL (n=50) and mixed lineage (n=1) based on the expression patterns of CD3 and CD19 molecules. B-ALL patients could further be classified into four subtypes, including Pro-B (n=7, 11.7%), Pre-B I (n=28, 46.7%), Pre-B II (n=13, 21.7%) and immature/mature B cells (n=2, 3.3%) on the basis of expression of CD10, CD19, CD20, HLA-DR and TdT. Clinical manifestations and laboratory findings of the patients did not reveal association with immunophenotypic sub-types of ALL, with the exception of mediastinal mass and WBC count at the time of diag-nosis which were found to be significantly higher in patients with T-ALL compared with B-ALL (p=0.001 and 0.014), respectively.
Conclusion: Our results indicate that overall the immunophenotypic profile of Iranian ALL patients is similar to previous reports and it might be used for monitoring of minimal residual disease and prognosis.- انتشار مقاله: 17-05-1395
- نویسندگان: Hossein Asgarian Omran,Mahdi Shabani,Tahereh Shahrestani,Abdolfattah Sarafnejad,Jalal Khoshnoodi,Parvaneh Vossough,Mohammad Faranoush,Ramzan A. Sharifian,Mahmood Jeddi-Tehrani,Hodjatallah Rabbani,Fazel Shokri
- مشاهده
- جایگاه : پژوهشی
- مجله: Iranian Journal of Immunology
- نوع مقاله: Journal Article
- کلمات کلیدی: RT-PCR,Antigen,Wilm’s Tumor Gene 1,Acute Myeloblastic Leukemia,Tumorassociated
- چکیده:
- چکیده انگلیسی: Background: The Wilm’s tumor gene 1 (WT1) encodes a zinc finger transcription factor that is inactivated in a subset of Wilm’s tumors. It plays a crucial role in growth, proliferation and development of some embryonic and adult organs. WT1 is expressed as a tumor associated antigen (TAA) in various types of solid and hematopoietic malignancies and can be employed as a useful marker for targeted immunotherapy and monitoring of minimal residual disease (MRD).
Objective: To investigate the profile of WT1 gene expression in Iranian patients with acute myeloblastic leukemia.
Methods: RT-PCR method was used to determine the WT1 gene expression in bone marrow (BM) and/or peripheral blood (PB) samples from 11 patients with AML and PB samples of 36 normal subjects. Isolated cells from all patients were immunophenotyped by flow cytometry.
Results: The leukemic cells from 10 patients (91%) were found moderately or strongly positive for WT1 expression whereas only 3 out of 36 normal subjects expressed WT1 at very low levels. A highly significant correlation was observed for WT1 expression between paired BM and PB samples of the AML patients.
Conclusion: Our results indicate that WT1 is expressed in the majority of Iranian AML patients and may be employed for screening and monitoring of minimal residual disease in these patients.- انتشار مقاله: 16-05-1395
- نویسندگان: Hossein Asgarian,Mahdi Shabani,Parvaneh Vosoogh,Ramazan Ali Sharifian,Soheila Gharagozlou,Jalal Khoshnoodi,Tahereh Shahrestani,Mahin Kordmahin,Abdolfattah Sarrafnejad,Mahmood Jeddi Tehrani,Hodjatallah Rabbani,Fazel Shokri
- مشاهده
- جایگاه : پژوهشی
- مجله: Iranian Journal of Immunology
- نوع مقاله: Journal Article
- کلمات کلیدی: ELISA,Immunoblotting,Monoclonal antibody,Human IgA2,Conformational Epitope,Linear Epitope
- چکیده:
- چکیده انگلیسی: Background: There are two subclasses of human IgA (IgA1 and IgA2) that differ in antigenic properties and in chemical composition. The constant domains of α1 and α2 heavy chains have >95% sequence homology though major structural differences exist in the hinge region. Quantitation of IgA subclass levels depends on the availability of monoclonal antibodies (MAbs) specific for conserved conformational or linear epitopes restricted to each subclass.
Objective: To produce, select and characterize monoclonal antibodies specific for human IgA2.
Methods: Splenocytes from BALB/C mice immunized with a human IgA2 myeloma protein were fused with SP2/0 myeloma cells. Fused cells were grown in hypoxanthine, aminopterine and thymidine (HAT) selective medium and cloned by limiting dilution assay. Antibody (Ab) secreting cells were screened by enzyme-linked immunosorbent assay (ELISA) and the specificity of secreted MAbs was further analyzed, using a panel of purified myeloma proteins and some animal sera by ELISA and immunoblotting. The affinity constant (Kaff) was also determined by ELISA.
Results: Four murine hybridoma clones designated 2F20G5, 2F20B5, 3F20E3 and 6F20H11 were obtained that secreted MAbs specific for the human IgA2. 2F20G5 and 6F20H11 MAbs react with linear epitope(s) while 2F20B5 and 3F20E3 react with conformational epitope(s) located to human IgA2 subclass. 2F20G5 MAb displays a weak cross-reactivity with monkey and rabbit sera and a strong cross-reactivity with cat and dog sera while the other three MAbs showed no cross-reactivity with the animal sera tested.
Conclusion: These MAbs, especially 6F20H11 with high affinity constant (6.03 ×109 M-1) are useful tools for quantitation of human IgA2 subclass levels in various diseases. Cross-reactivity of 2F20G5 MAb with some animalsera suggests phylogenic conservation ofthe epitope recognized by this MAb.- انتشار مقاله: 16-05-1395
- نویسندگان: Fatemeh Hajighasemi,Soheila Gharagozlou,Nasrin Moheghi,Roya Ghods,Jalal Khoshnoodi,Fazel Shokri
- مشاهده
- جایگاه : پژوهشی
- مجله: Iranian Journal of Immunology
- نوع مقاله: Journal Article
- کلمات کلیدی: Hepatitis B,neonate,Ant i-HBs antibody,Vaccinat ion,Immunogenicit y,Seroprotection
- چکیده:
- چکیده انگلیسی:
Objective: To compare immunogenicity of a recombinant hepatitis B (HB) vaccine in two groups of neonates born in two cities of Iran with different geographic and ethnic backgrounds. Materials and
Methods: Ten micrograms of a recombinant HB vaccine was administered under field condition to Iranian healthy neonates at 0, 1.5 and 9 months intervals. The subjects consisted of two groups of 290 and 231 neonates selected from two cities located at north-west (Urmia) and south-east (Kerman) of Iran, respectively. The level of anti-HBs antibody was quantitated in serum 2-4 weeks after administration of the last vaccine dose, by sandwich ELISA.
Results: A higher seroprotection rate (anti-HBs> 10 IU/L) (98.3% vs. 96.1%) and significantly increased serum anti- HBs antibody titer (11869 vs. 6104 IU/L) (P<0.001) were induced in vaccinated neonates from Urmia city, compared to those born in Kerman.
Conclusion: These findings suggest contribution of ethnic and/or environmental factors in the antibody response to recombinant HB vaccine in human.- انتشار مقاله: 15-05-1395
- نویسندگان: Abdollah Jafarzadeh,Jalal Khoshnoodi,Shayesteh Ghorbani,Saleh Mohaghegh Hazrati,Babak Faraj Mazaheri,Fazel Shokri
- مشاهده
- جایگاه : پژوهشی
- مجله: Iranian Journal of Immunology
- نوع مقاله: Journal Article
- کلمات کلیدی: Hepatitis B,neonate,Ant i-HBs antibody,Vaccinat ion,Immunogenicit y,Seroprotection
- چکیده:
- چکیده انگلیسی:
Objective: To compare immunogenicity of a recombinant hepatitis B (HB) vaccine in two groups of neonates born in two cities of Iran with different geographic and ethnic backgrounds. Materials and
Methods: Ten micrograms of a recombinant HB vaccine was administered under field condition to Iranian healthy neonates at 0, 1.5 and 9 months intervals. The subjects consisted of two groups of 290 and 231 neonates selected from two cities located at north-west (Urmia) and south-east (Kerman) of Iran, respectively. The level of anti-HBs antibody was quantitated in serum 2-4 weeks after administration of the last vaccine dose, by sandwich ELISA.
Results: A higher seroprotection rate (anti-HBs> 10 IU/L) (98.3% vs. 96.1%) and significantly increased serum anti- HBs antibody titer (11869 vs. 6104 IU/L) (P<0.001) were induced in vaccinated neonates from Urmia city, compared to those born in Kerman.
Conclusion: These findings suggest contribution of ethnic and/or environmental factors in the antibody response to recombinant HB vaccine in human.- انتشار مقاله: 15-05-1395
- نویسندگان: Abdollah Jafarzadeh,Jalal Khoshnoodi,Shayesteh Ghorbani,Saleh Mohaghegh Hazrati,Babak Faraj Mazaheri,Fazel Shokri
- مشاهده
- جایگاه : پژوهشی
- مجله: Iranian Journal of Immunology
- نوع مقاله: Journal Article
- کلمات کلیدی: Hepatitis B,neonate,Ant i-HBs antibody,Vaccinat ion,Immunogenicit y,Seroprotection
- چکیده:
- چکیده انگلیسی:
Objective: To compare immunogenicity of a recombinant hepatitis B (HB) vaccine in two groups of neonates born in two cities of Iran with different geographic and ethnic backgrounds. Materials and
Methods: Ten micrograms of a recombinant HB vaccine was administered under field condition to Iranian healthy neonates at 0, 1.5 and 9 months intervals. The subjects consisted of two groups of 290 and 231 neonates selected from two cities located at north-west (Urmia) and south-east (Kerman) of Iran, respectively. The level of anti-HBs antibody was quantitated in serum 2-4 weeks after administration of the last vaccine dose, by sandwich ELISA.
Results: A higher seroprotection rate (anti-HBs> 10 IU/L) (98.3% vs. 96.1%) and significantly increased serum anti- HBs antibody titer (11869 vs. 6104 IU/L) (P<0.001) were induced in vaccinated neonates from Urmia city, compared to those born in Kerman.
Conclusion: These findings suggest contribution of ethnic and/or environmental factors in the antibody response to recombinant HB vaccine in human.- انتشار مقاله: 15-05-1395
- نویسندگان: Abdollah Jafarzadeh,Jalal Khoshnoodi,Shayesteh Ghorbani,Saleh Mohaghegh Hazrati,Babak Faraj Mazaheri,Fazel Shokri
- مشاهده
- جایگاه : پژوهشی
- مجله: Iranian Journal of Biotechnology
- نوع مقاله: Journal Article
- کلمات کلیدی: Purification,Protein expression,Polyclonal antibody,FCRL,His-tag
- چکیده:
- چکیده انگلیسی: Background: The Fc receptor like (FCRL) molecules belong to the immunoglobulin (Ig) superfamily with potentially immunoregulatory function. Among the FCRL family FCRL2 and 4 are predominantly expressed on memory B cells and FCRL1 is a pan- B cell marker. To date, no ligand has been identifid for the human FCRL1, 2 and 4 molecules. Objectives: Cloning, expression, purifiation and structural analysis of the extracellular domain of human FCRL1, 2 and 4 proteins. Materials and Methods: In this study, the extracellular part of human FCRL1, 2 and 4 were subcloned into prokaryotic expression vectors pET-28b (+) and transformed into BL21-DE3 E.coli strain. Protein expression was optimized by fie adjustments such as induction time, incubation temperature and expression hosts. Recombinant FCRL proteins were purifid by metal affity chromatography using Ni-NTA resin. Purifid FCRL proteins were further characterized by SDS-PAGE and immunoblotting using His-tag and FCRL specifi polyclonal antibodies. Results: Our results demonstrated that FCRL1, 2 and 4 were successfully expressed in pET-28b (+) vector. Optimization of the expression procedure showed that IPTG induction at OD600 = 0.9 and overnight incubation at 37˚C resulted in the highest expression levels of FCRL proteins ranging from approximately 15% (FCRL1) to 25% (FCRL2 and 4) of the total bacterial lysate proteins. Conclusions: These purifid recombinant proteins are potentially a valuable tool for investigating the immunoregulatory function of FCRL molecules and the production of specifi mAbs for immunotherapeutic interventions.
- انتشار مقاله: 04-10-1391
- نویسندگان: Mahdi Shabani,Azam Hemmati,Mahdi Zandemami,Jalal Khoshnoodi,Mahmood Jeddi-Tehrani,Hodjatallah Rabbani,Zahra Amirghofran,Fazel Shokri
- مشاهده