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ABSTRACT: Repeated implantation failure (RIF) is a worldwide health problem that imposes a great deal of cost on patients and health care system. Vitamin D(3) has been proposed to have positive impact on the process of implantation. The present study was performed to compare the effect of 1,25-dihydroxy vitamin D(3) (1,25(OH)(2)D(3)) on cytokine production by endometrial cells of women with RIF and healthy fertile controls. Whole endometrial cells (WECs) and endometrial stromal cells (ESCs) from RIF and normal fertile women were treated with 1,25(OH)(2)D(3). The levels of IL-10, TGF-β, IFNγ, Il-6, IL-8 and IL-17 in culture supernatants were assayed by ELISA. Also, ability of the cells from both groups to produce 1,25(OH)(2)D(3) was evaluated and compared. 1,25(OH)(2)D(3) down-regulated cytokine production in WECs from both groups except for IL-8 which was upraised. Similar trends were also observed in ESCs except up-regulation of TGF-β in RIF group. Endometrial cells of both groups had comparable capacity to produce 1,25(OH)(2)D(3). Based on the minimal differential immunoregulatory effect of vitamin D(3) on endometrial cells from RIF and control women, it may be suggested that circulating levels of maternal vitamin D(3) be the subject of further investigation.
Gynecological Endocrinology 05/2012; 28(11):906-11. · 1.58 Impact Factor
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ABSTRACT: Menstrual blood stromal stem cells (MBSCs) have been demonstrated to exhibit stem cell properties such as the capability for self-renewal and multipotency, allowing for multilineage differentiation. In addition, this cell type has various immunomodulatory effects. In this study, we examined the potential effect of MBSCs on proliferation of peripheral blood mononuclear cells (PBMCs) in allogeneic mixed lymphocyte reaction (MLR).
Menstrual blood was collected from healthy donors after menstrual blood flow initiated and its mononuclear cell fraction was separated. Cells were subsequently cultured and adherent cells were allowed to propagate and used as stem cells. Flowcytometric immunophenotyping was performed using a panel of monoclonal antibodies including CD44, CD45, CD34, CD9, CD29, CD10, CD38, CD105, CD73, CD133, STRO-1 and Oct-4A. For functional analysis, PBMCs were co-cultured with MBSCs, collected after 4 days and added to allogeneic PBMCs. 2,3-Bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) assay was carried out to evaluate cell proliferation.
MBSCs showed surface and intracellular markers of mesenchymal stem cells with the exception of the high expression of Oct-4A. MBSCs affected the proliferative response of PBMC in a dose-dependent manner. At ratio of 1:1 to 1:2, MBSCs inhibited, while at lower ratios (1:32 to 1:64) stimulated the proliferative capacity of allogeneic PBMCs.
According to the present study, MBSCs exert their immunoregulatory effects on allogeneic PBMCs in a dose-dependent manner. This finding can be considered as a valuable point in future cell therapy strategies, when this cell population is used.
Journal of Obstetrics and Gynaecology Research 03/2012; 38(5):804-9. · 0.94 Impact Factor
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ABSTRACT: Expression of receptor tyrosine kinase Ror1 in a wide variety of cancers has emerged as a new era focusing on targeting this receptor in cancer therapy. Our preliminary re-sults indicate the presence of a truncated transcript of Ror1 in tumor cells. The trun-cated Ror1 encompasses extracellular and transmembrane domains, lacking catalytic kinase domain (Ror1-ECD). As enzyme activity is highly dependent on the catalytic domain, we were wondering how this transcript and its encoded protein could play a possible role in tumorigenesis. To understand the function of this truncated transcript and whether or not the encoded protein translocates to the cell surface, we construct-ed a mammalian expression vector containing exon 1 to exon 8 of human Ror1 gene as a model system. The encoded protein by this construct covers the entire extracellular and transmembrane domains of Ror1. The Chinese Hamster Ovary Cell line (CHO) was used for transfection. Our results showed that this construct could express Ror1-ECD at protein level and also the protein could effectively translocate to the surface of transfected cells. Such model may suggest that a proportion of Ror1 molecules ex-pressed by tumor cells are not full-length Ror1. This notion may be considered when applying flow cytometry using antibodies against Ror1 for screening of tumor cells in order to avoid any miscalculation in the number of Ror1 molecules expressed by tumor cells. Furthermore, such expression may bring about assumptions on functional roles of Ror1-ECD in tumorigenesis, which requires extensive functional studies.
Avicenna Journal of Medical Biotechnology 03/2012; 4((1)).
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ABSTRACT: Chronic lymphocytic leukemia (CLL) remains an incurable disease that requires innovative new approaches to improve therapeutic outcome. Many Ferula species, including F. asa-foetida, synthesize terpenyloxy coumarins. One of these coumarins is umbelliprenin, which has been implicated with induction of apoptosis in some cancer cell lines. In this study induction of apoptosis by umbelliprenin on Jurkat T-CLL and Raji B-CLL cell lines was studied. In this regard, cells were incubated with various concentrations of umbelliprenin in-vitro for different times and assayed for apoptosis with annexin V–FITC/PI double staining flowcytometry method. Results showed that umbelliprenin induced apoptosis in leukemic cells in a dose-and time-dependent manner and that CLL cells were more susceptible to umbelliprenin induced cell death than normal peripheral blood mononuclear cell (PBMCs). Moreover, we study the induction of apoptosis in Jurkat cells by umbelliprenin in the presence of interleukin 4 (IL-4) as an agent that causes resistance to apoptosis in CLL cells, was also student. We showed that IL-4 can not reduce apoptotic effect of umbelliprenin. The preferential toxicity of umbelliprenin for CLL cells, supports the hypothesis that oral administration of umbelliprenin in the form of foods or folk medicines containing this coumarin, might enhance protection against the development of CLL in man with little side effects. In conclusion, umbelliprenin may be an effective therapeutic agent in the treatment of CLL, and thus clinical studies with umbelliprenin may be appropriate.
Iranian journal of pharmaceutical research (IJPR) 01/2012; 11(2-11):653-659. · 0.64 Impact Factor
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ABSTRACT: Expression of receptor tyrosine kinase Ror1 in a wide variety of cancers has emerged as a new era focusing on targeting this receptor in cancer therapy. Our preliminary results indicate the presence of a truncated transcript of Ror1 in tumor cells. The truncated Ror1 encompasses extracellular and transmembrane domains, lacking catalytic kinase domain (Ror1-ECD). As enzyme activity is highly dependent on the catalytic domain, we were wondering how this transcript and its encoded protein could play a possible role in tumorigenesis. To understand the function of this truncated transcript and whether or not the encoded protein translocates to the cell surface, we constructed a mammalian expression vector containing exon 1 to exon 8 of human Ror1 gene as a model system. The encoded protein by this construct covers the entire extracellular and transmembrane domains of Ror1. The Chinese Hamster Ovary Cell line (CHO) was used for transfection. Our results showed that this construct could express Ror1-ECD at protein level and also the protein could effectively translocate to the surface of transfected cells. Such model may suggest that a proportion of Ror1 molecules expressed by tumor cells are not full-length Ror1. This notion may be considered when applying flow cytometry using antibodies against Ror1 for screening of tumor cells in order to avoid any miscalculation in the number of Ror1 molecules expressed by tumor cells. Furthermore, such expression may bring about assumptions on functional roles of Ror1-ECD in tumorigenesis, which requires extensive functional studies.
Avicenna journal of medical biotechnology. 01/2012; 4(1):41-5.
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ABSTRACT: Background: Repeated Implantation Failure (RIF) is one of the most intricate obstacles in assisted reproduction. The cytokine and chemokine composition of uterine cavity seem to play important roles in the implantation process. Objective: To compare the cytokine profile in the endometrium of normal fertile women and those with repeated implantation failure. Methods: After enzymatic digestion of endometrial tissues, whole endometrial cells and endometrial stromal cells from RIF and normal fertile women were cultivated and stimulated for cytokine secretion. The levels of IL-10, TGF-β, IFN-γ, IL-6, IL-8 and IL-17 in culture supernatants of the two groups were assayed by ELISA and compared together. Results: Endometrial stromal cells and whole endometrial cells of normal fertile women produced higher levels of IL-6, IL-8 and TGF-β compared to RIF group, although this difference was statistically significant only in endometrial stromal cells (p=0.005, 0.002 and 0.001, respectively). In addition, endometrial stromal cells of normal fertile women produced lower levels of IL-10 in comparison with RIF group (p=0.005). Conclusion: Disturbances in cytokine production at the feto-maternal interface could be a cause of implantation failure. A pro-inflammatory cytokine milieu seems to be pivotal for successful implantation.
Iranian journal of immunology: IJI 12/2011; 8(4):201-8.
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ABSTRACT: In many acute leukemias, normal differentiation does not occur. However, in many cell lines derived from hematologic malignancies, differentiation or apoptosis can be induced by variety of agents. Despite advances in the treatment of Acute Lymphoblastic Leukemia (ALL), in most patients long-term survival rates remain unsatisfactory, especially in T-cell derived ALL. Thus we studied the anti-cancer effects of fenretinide, 1α,25(OH)(2)D(3), and bryostatin-1 in CCRF-CEM (T-cell derived) and Nalm-6 (B-cell derived) ALL cell lines. Using MTT assays, both cell lines were shown to exhibit increased inhibition of proliferation at micro (fenretinide) and nanomolar (1α,25(OH)(2)D(3), bryostatin-1) concentrations. These anti-cancer agents were shown to induce apoptosis and activate caspase-3 pathway in both ALL cell lines. Furthermore, for the first time we are reporting consistent anti-proliferative and apoptotic effects of Bryostatin-1 in ALL T-cell derived cell line with the lowest ED(50) (ranging 4.6-7.4 nM). To evaluate the differentiation induction by fenretinide, 1α,25(OH)(2)D(3), and bryostatin-1 in ALL cell lines, we assayed for the expressions of CD19, CD38 markers on Nalm-6 and CD7 marker on CCRF-CEM cell line. The flow cytometric analysis showed a significant increase in expression of CD markers in response to anti-cancer drug treatments. To assay the effects of anti-cancer drugs on cell cycle distribution, cell cycle analysis using flow cytometry was employed. These anti-cancer drugs appear to affect the CCRF-CEM and Nalm-6 cell cycles differently (G0/G1 and G2/M arrest, respectively). Overall results demonstrate that the anti-cancer agents used in this study are strong inhibitors of ALL cell proliferation and inducers of apoptosis and differentiation in vitro. These findings may be quite helpful if these drugs are to be used for differentiation therapy of ALL patients in clinics in the future. Further studies are warranted to establish the in vivo effect of these drugs particularly in patients with T-cell derived ALL.
Avicenna journal of medical biotechnology. 10/2011; 3(4):177-93.
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ABSTRACT: To investigate immunomodulatory effect of 1,25(OH)2 vitamin D3 (1,25(OH)2D3) on cytokine production by endometrial cells of women with unexplained recurrent spontaneous abortion (URSA).
In vitro study.
Academic research center.
Patients with URSA and healthy controls.
Treatment with 1,25(OH)2D3.
Production of interferon γ (IFN-γ), interleukin-10 (IL-10), transforming growth factor β (TGF-β), IL-17, IL-6, and IL-8 by whole endometrial cells (WECs) and endometrial stromal cells in the presence and absence of 1,25(OH)2D3 and 1α-hydroxylase activity of these cell populations were measured in patients with URSA and healthy controls.
1,25(OH)2D3 interfered with production of cytokines by WECs of the control and URSA groups, except IL-8 which was increased in URSA group. In endometrial stromal cells, 1,25(OH)2D3 down-regulated cytokine production as well with stimulatory effect on the production of TGF-β in patients with URSA. Cytokine profile of WECs from patients with URSA skewed toward TH2 phenotype after treatment with 1,25(OH)2D3. Endometrial cells of both groups had comparable capacity to produce 1,25(OH)2D3.
Considering the complex network of immunoregulation at the fetomaternal interface, potential beneficial effects of vitamin D3 in patients with URSA need to be investigated in clinical practice. Comparable levels of 1,25(OH)2D3 production and similar trend of cytokine expression by WECs of URSA and control groups after vitamin D3 treatment reflect the same local metabolic machinery of this hormone.
Fertility and sterility 09/2011; 96(3):751-7. · 3.97 Impact Factor
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ABSTRACT: HER2 proto-oncogene encodes a transmembrane receptor tyrosine kinase overexpressed in a variety of solid tumors. Several mouse monoclonal antibodies (MAbs) have been developed that recognize the extracellular part of HER2; of them two MAbs were humanized and employed for targeted immunotherapy. In this study we aimed to produce murine MAbs that specifically recognize the extracellular domain of human HER2. BALB/c mice were first primed with HER2-transfected NIH-3T3 cells and then boosted with recombinant extracellular part of HER2. Splenocytes from hyperimmunized mice were fused with myeloma cells and growing hybridomas were selected and screened for HER2 reactivity by an indirect ELISA. HER2-specific hybridomas were selected, cloned by limiting dilution assay, and further characterized by Western blotting and flow cytometry techniques. All clones showed positive reactivity to HER2 with binding affinity, ranging from 1.9×10(8) to 5×10(9), and stained HER2-transfected cells and malignant cells overexpressing HER2. None of the MAbs inhibited the binding of trastuzumab (Herceptin(®)) to HER2, indicating recognition of distinct epitopes by these MAbs. Based on these findings, our MAbs could be potentially used for selective targeting of HER2-expressing malignancies.
Hybridoma (2005) 08/2011; 30(4):347-53. · 0.42 Impact Factor
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Mahdi Shabani,
Hossein Asgarian Omran,
Mohammad Hojjat Farsangi,
Parvaneh Vossough,
Ramazan A Sharifian,
Gholam R Toughe,
Seyed Mohsen Razavi,
Jalal Khoshnoodi, Mahmood Jeddi-Tehrani,
Hodjatallah Rabbani,
Fazel Shokri
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ABSTRACT: It has recently been shown that ROR1, a member of the receptor tyrosine kinase family, is overexpressed in leukemic B cells of Chronic Lymphocytic Leukemia (CLL) and a subset of Acute Lymphoblastic Leukemia (ALL). In this comparative study the expression profile of ROR1 mRNA was investigated in Iranian patients with CLL and Acute Myelogenous Leukemia (AML) and the results were compared with those previously reported in our Iranian ALL patients. RT-PCR was performed on bone marrow and/or peripheral blood samples of 84 CLL and 12 AML patients. CLL samples were classified into immunoglobulin heavy chain variable region (IGHV) gene mutated (n = 55) and unmutated (n = 29) and also indolent (n = 42) and progressive (n = 39) subtypes. ROR1 expression was identified in 94% of our CLL patients, but none of the AML patients expressed ROR1. No significant differences were observed between different CLL subtypes for ROR1 expression. Taken together the present data and our previous results on ROR1 expression in ALL, our findings propose ROR1 as a tumor-associated antigen overexpressed in a large proportion of lymphoid (CLL and ALL), but not myeloid (AML) leukemias. Expression of ROR1 seems to be associated to lineage and differentiation stages of leukemic cells with a potential implication for immunotherapy.
Avicenna journal of medical biotechnology. 07/2011; 3(3):119-25.
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ABSTRACT: To investigate the effect of 5'-(N-ethylcarboxamido) adenosine (NECA), an adenosine agonist, on triggering angiogenesis in transplanted human ovarian tissue, the expression of angiopoietin-1 (Ang1), Ang2, vascular endothelial growth factor 121 (VEGF-121) and VEGF-189 at both gene and protein levels as well as the density of vasculature were studied in tissue fragments grafted to NECA-treated and control groups of nude mice. The results showed that NECA treatment triggered down-regulation of Ang1, induced VEGF-189 expression, and stimulated neovascularization, highlighting the beneficial effect of NECA on the process of angiogenesis.
Fertility and sterility 06/2011; 95(8):2560-3.e1-5. · 3.97 Impact Factor
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ABSTRACT: Despite recent attempts to take advantage of dendritic cell (DC)-based vaccines for cancer immunotherapy, the results of clinical studies have been disappointing. This is mainly as a result of the diverse immune escape mechanisms used by the tumor together with the insufficient ability of DCs to mount an effective immune response against these mechanisms. In this regard, several approaches have been devised to improve the efficacy of DC-based vaccines. However, the application of each individual approach per se might not be sufficient to overwhelm the diverse immune escape mechanisms. In this review, we focus on current strategies for the ex vivo potentiation of DC-based vaccines, with an emphasis on combinational therapy methods as a promising alternative for tumor immunotherapy.
Drug discovery today 05/2011; 16(15-16):733-40. · 6.63 Impact Factor
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ABSTRACT: Objective: Synthetic fluorescent dyes that are conjugated to antibodies are useful tools to
probe molecules. Based on dye chemical structures, their photobleaching and photostability
indices are quite diverse. It is generally believed that among different fluorescent dyes,
Alexa Fluor family has greater photostability than traditional dyes like fluorescein isothiocyanate
(FITC) and Cy5. Alexa Fluor 568 is a member of Alexa Fluor family presumed to
have superior photostability and photobleahing profiles than FITC.
Materials and Methods: In this experimental study, we conjugated Alexa Fluor 568 and
FITC dyes to a mouse anti-human nestin monoclonal antibody (ANM) to acquire their photobleaching
profiles and photostability indices. Then, the fluorophore/antibody ratios were
calculated using a spectrophotometer. The photobleaching profiles and photostability indices
of conjugated antibodies were subsequently studied by immunocytochemistry (ICC).
Samples were continuously illuminated and digital images acquired under a fluorescent
microscope. Data were processed by ImageJ software.
Results: Alexa Fluor 568 has a brighter fluorescence and higher photostability than
FITC.
Conclusion: Alexa Fluor 568 is a capable dye to use in photostaining techniques and it
has a longer photostability when compared to FITC.
Keywords: Fluorescein Isothiocyanate, Alexa Fluor 568, Photostability, Photobleaching
Yakhteh 04/2011; 13(3).
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ABSTRACT: Mycoplasma genitalium (M.genitalium) is a sexually transmitted pathogen. Detection of this microorganism in clinical specimens by culture is rather difficult and time consuming. The aim of this study was to produce polyclonal antibody against a synthetic peptide from P110 protein of M.genitalium in order to develop a diagnostic tool for detection of this microorganism in clinical specimens. A synthetic peptide from P110 protein was conjugated to Keyhole Limpet Hemocyanin (KLH) and used for immunization of a white New Zealand rabbit. The produced antibody was purified by affinity chromatography and its specific interaction with immunizing peptide was determined by ELISA. Immunoreactivity of the antibody was also tested by Western blotting in bacterial cell lysate prepared from M.genitalium G-37. To confirm its application as a diagnostic tool, indirect immunofluorescent staining method was performed on M.genitalium-infected PBMC using anti-P110 as the primary antibody. The results showed that produced antibody has excellent reactivity with immunizing peptide and also detected a single band of 110 kDa corresponding to P110 protein. M.genitalium-infected PBMC showed a bright fluorescent signal in IF staining. This antibody might be used as a tool in diagnostic applications.
Avicenna journal of medical biotechnology. 04/2011; 3(2):79-85.
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Mahmood Jeddi-Tehrani,
Raheleh Torabi,
Amir H. Zarnani,
Afsaneh Mohammadzadeh,
Soheila Arefi,
Hojjat Zeraati,
Mohammad M. Akhondi,
Leili Chamani-Tabriz,
Farah Idali,
Shaghayegh Emami,
Saeed Zarei
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ABSTRACT: Citation Jeddi-Tehrani M, Torabi R, Zarnani AH, Mohammadzadeh A, Arefi S, Zeraati H, Akhondi MM, Chamani-Tabriz L, Idali F, Emami S, Zarei S. Analysis of plasminogen activator inhibitor-1, integrin beta3, beta fibrinogen and methylenetetrahydrofolate reductase polymorphisms in Iranian women with recurrent pregnancy loss. Am J Reprod Immunol 2011; 66: 149–156Problem To identify the associations of the plasminogen activator inhibitor-1 (PAI-1) −675 4G/5G, beta fibrinogen (BF) −455G/A, integrin beta 3 (ITGB3) 1565T/C, and methylenetetrahydrofolate reductase (MTHFR) 677C/T and 1298A/C polymorphisms with recurrent pregnancy loss (RPL).Method of study Polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) were performed to assess the frequency of five candidate genetic risk factors for RPL, and the frequencies of the polymorphisms were calculated and compared between case and control groups.Results The BF −455G/A, MTHFR 677C/T, and 1298A/C polymorphisms were found to be positively, and ITGB3 1565T/C polymorphism negatively, associated with RPL. Homozygosity but not heterozygosity for PAI-1 −675 4G/5G polymorphism was significantly higher in patients with RPL than in the control group. The presence of both mutations of MTHFR genes highly increased the risk of RPL.Conclusion The data highlight the importance of thrombophilia screening in patients with RPL.
American Journal Of Reproductive Immunology 01/2011; 66(2):149 - 156. · 2.17 Impact Factor
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[show abstract]
[hide abstract]
ABSTRACT: Synthetic fluorescent dyes that are conjugated to antibodies are useful tools to probe molecules. Based on dye chemical structures, their photobleaching and photostability indices are quite diverse. It is generally believed that among different fluorescent dyes, Alexa Fluor family has greater photostability than traditional dyes like fluorescein isothiocyanate (FITC) and Cy5. Alexa Fluor 568 is a member of Alexa Fluor family presumed to have superior photostability and photobleahing profiles than FITC.
In this experimental study, we conjugated Alexa Fluor 568 and FITC dyes to a mouse anti-human nestin monoclonal antibody (ANM) to acquire their photobleaching profiles and photostability indices. Then, the fluorophore/antibody ratios were calculated using a spectrophotometer. The photobleaching profiles and photostability indices of conjugated antibodies were subsequently studied by immunocytochemistry (ICC). Samples were continuously illuminated and digital images acquired under a fluorescent microscope. Data were processed by ImageJ software.
Alexa Fluor 568 has a brighter fluorescence and higher photostability than FITC.
Alexa Fluor 568 is a capable dye to use in photostaining techniques and it has a longer photostability when compared to FITC.
Cell Journal 01/2011; 13(3):169-172.
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Maryam Hormozi,
M.Sc.a,
Saeed Talebi,
M.D.b,
Amir Hassan Zarnani,
Ph.D, Mahmood Jeddi-Tehrani,
Ladan Hosseini Gohari,
Haleh Soltanghoraei,
M.D,
Mina Jafarabadi,
Mohammad Mehdi Akhondi
[show abstract]
[hide abstract]
ABSTRACT: To investigate the effect of 50-(N-ethylcarboxamido) adenosine (NECA), an adenosine agonist, on triggering angiogenesis
in transplanted human ovarian tissue, the expression of angiopoietin-1 (Ang1), Ang2, vascular endothelial
growth factor 121 (VEGF-121) and VEGF-189 at both gene and protein levels as well as the density of
vasculature were studied in tissue fragments grafted to NECA-treated and control groups of nude mice. The results
showed that NECA treatment triggered down-regulation of Ang1, induced VEGF-189 expression, and stimulated
neovascularization, highlighting the beneficial effect of NECA on the process of angiogenesis. (Fertil Steril�
2011;95:2560–3. �2011 by American Society for Reproductive Medicine.)
Fertility and sterility 01/2011; 95(8):2560-2563. · 3.97 Impact Factor
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Mahmood Jeddi-Tehrani,
Raheleh Torabi,
Amir H Zarnani,
Afsaneh Mohammadzadeh,
Soheila Arefi,
Hojjat Zeraati,
Mohammad M Akhondi,
Leili Chamani-Tabriz,
Farah Idali,
Shaghayegh Emami,
Saeed Zarei
[show abstract]
[hide abstract]
ABSTRACT: To identify the associations of the plasminogen activator inhibitor-1 (PAI-1) -675 4G/5G, beta fibrinogen (BF) -455G/A, integrin beta 3 (ITGB3) 1565T/C, and methylenetetrahydrofolate reductase (MTHFR) 677C/T and 1298A/C polymorphisms with recurrent pregnancy loss (RPL).
Polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) were performed to assess the frequency of five candidate genetic risk factors for RPL, and the frequencies of the polymorphisms were calculated and compared between case and control groups.
The BF -455G/A, MTHFR 677C/T, and 1298A/C polymorphisms were found to be positively, and ITGB3 1565T/C polymorphism negatively, associated with RPL. Homozygosity but not heterozygosity for PAI-1 -675 4G/5G polymorphism was significantly higher in patients with RPL than in the control group. The presence of both mutations of MTHFR genes highly increased the risk of RPL.
The data highlight the importance of thrombophilia screening in patients with RPL.
American Journal Of Reproductive Immunology 01/2011; 66(2):149-56. · 2.17 Impact Factor
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ABSTRACT: Background: The receptor tyrosine kinase (RTK) Ror1 is the first described RTK in chronic lymphocytic leukemia (CLL). RTKs have been shown not only to be key regulators of normal cellular processes but also to have a critical role in the development and progression of many types of cancer. Monoclonal antibodies (MAb) against RTKs (eg. EGFR, Her2/neu) have been successful in the treatment of various malignancies. Here we report the generation of six MAbs against the immunoglobulin (Ig), cysteine rich (CRD), and kringle (KNG) domains respectively of the extracellular part of Ror1.Aims: To generate cytotoxic anti-Ror1 monoclonal antibodies for a novel therapeutic strategy in CLL.Methods: A peptide-based mouse monoclonal antibody generation method was used for producing MAbs against the three extracellular domains of Ror1. Twenty CLL patients (10 with progressive and 10 with non-progressive disease) were enrolled in this study. Flow cytometry was used for surface staining of Ror1. Annexin V and propidium iodide (flow cytometry) as well as PARP cleavage (Western blot) was used for detection of apoptosis.
Results: Six monoclonal antibodies of different isotypes (IgG, IgM) were produced against Ror1. The frequency of Ror1 positive cells were in the range of 24-89%. Patients with progressive disease had a significantly higher number of Ror1 positive cells as compared to those with non-progressive disease as well as in patients with unmutated compared to mutated IgVH genes. All six antibodies alone induced apoptosis (Annexin V and PARP cleavage) of CLL cells. A higher frequency of apoptotic CLL cells was induced by the antibodies against the CRD region (ligand binding site for Wnt proteins) as well as one antibody against the kringle domain (also a binding region for regulatory proteins) . Apoptosis induced by these three antibodies alone was significantly higher than that induced by Rituximab (p<0.001). Apoptosis induction by all antibodies was statistically significantly augmented by crosslinking of the Ror1 antibodies with anti-Fc F(ab’)2 fragment antibodies. None of the antibodies induced apoptosis of PBMC of healthy individuals and normal PBMC did not express Ror1. Conclusion: Monoclonal antibodies alone against the RTK, Ror1, were shown to selectively kill CLL cells. Development of MAb targeting Ror1 might be a novel therapeutic approach complementary to existing therapies.
52th American Society of Hematology meeting (ASH), Orlando, FL, USA; 12/2010
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ABSTRACT: Background: The receptor tyrosine kinase Ror1 is a tumor-associated molecule over-expressed in chronic lymphocytic leukemia (CLL) and a variety of other malignancies with potential implication for targeted immunotherapy. Little is known about the functional role of Ror1 in the leucomogenesis of CLL. Aims: To assess spontaneous T cell response against Ror1 in CLL patients.
Methods: Autologous T cell response against Ror1 was studied in 9 CLL patients and 6 healthy subjects expressing the HLA-A2 allele. Four synthetic 9-mer HLA-A2 restricted peptides selected from different parts of the Ror1 molecule were loaded on dendritic cells and co-cultured with enriched autologous T cells. T cell response was determined by enumeration of the frequency of IFN-γ, IL-5 and IL-17A secreting T cells by ELISPOT. Cell proliferation was determined by 3H-thymidine incorporation. Results: Our results demonstrated a significantly higher frequencies of IFN-γ producing T cells (p<0.05-0.0001)
and to a lesser extent IL-17A secreting autologous T cells (p<0.05) in response to Ror1 peptides in CLL patients compared to healthy controls. No IL-5 secreting T cells were noted. The frequency of IFN-γ secreting T cells was significantly higher in non-progressive as compared to progressive CLL patients (P<0.05). No differences were observed between patients with mutated and unmutated immunoglobulin heavy chain variable region (IGHV) genes. Conclusion: Ror1 may spontaneously induce
mainly a type 1 T cell response in CLL patients, particularly in those with non-progressive disease as compared to progressive disease. Ror1 may be an immunodominant antigen in CLL which, has also been suggested by Fakuda et al. PNAS, 105, 3047, 2008. Active immunotherapy using Ror1 as a target might be an interesting approach to test in the clinic.
52th American Society of Hematology meeting (ASH), Orlando, FL, USA; 12/2010
