- Email: [email protected]
Immunological disorders in reproductive failure
60
Abstracts / Journal of Reproductive Immunology 101–102 (2014) 40–60
P43
P42 Immunological disorders in reproductive failure Szilvia Zeher 2
Vad 1,∗ ,
A.
Szanto 2 ,
T.
Tarr 2 ,
J.
Vegh 2 ,
G.
Nagy 3 ,
M.
Structural design-based preimplantation factor (PIF*) fusion peptide synthetic DNA cloning and eukaryote expression aimed for functional proteomic studies and possible chronic immune disorders therapy
1
Department of Obstetrics and Gynecology, Medical and Health Science Center, University of Debrecen, Hungary 2 Department of Internal Medicine, Division of Clinical Immunology, Medical and Health Science Center, University of Debrecen, Hungary 3 Regional Immunological Laboratory, Medical and Health Science Center, University of Debrecen, Hungary Reproductive failure is a major health issue in all developed countries. Almost 10–25% of couples may suffer of reproductive failure despite extensive clinical investigations 10–15% of these cases remain unexplained. It is known that immunological disorders may play a significant role in recurrent pregnancy loss and infertility. The aim of this study was to investigate the potential immunological disorders among women with unexplained recurrent pregnancy loss and infertility. Between 2008 and 2010 95 female patients referred by gynecologists to immunology outpatient offices were investigated for autoimmune disorders. The patients were screened for the presence of: anti-TPO, anticardiolipin, anti-b2GP1, lupus anticoagulant, rare autoantibodies (against annexin V, prothrombin, phosphatidyl serine), aSS-A, aCCP, aSS-B, ANF, anti-sperm and anti-endothelial antibodies. The results were statistically evaluated using the SPSS software package. Results: The mean age of patients was 32.9 ± 4.1 years. 50 out of 95 patients had recurrent pregnancy loss, 45 women had infertility problem. In 25 cases elevated anti-TPO was found, in 11 cases out of 95 elevated anti-phospholipid antibodies were proven (anticardiolipin: 2/95, antib2GP1: 1/95, anti-annexin V: 7/95, anti-phosphatidyl serine: 1/95), while in 14 women other elevated antibody titers (aSS-A: 4/95, aSS-A + aCCP: 2/95, aSS-A + aSS-B: 1/95, ANF: 4/95, antisperm antibody: 2/95, anti-endothelial antibody: 1/95) were found. The disorders were treated with levothyroxine, methyl-prednisolone, LMWH or salicylates. Eighty-two of the 95 patients were followed up for 25 months. They conceived 25 pregnancies and delivered 28 newborns. Despite the high number of pregnancies no significant difference was found in the number of successful pregnancies between women who had immunological disorder and those who did not. Discussion: Although statistically significant differences were not found regarding the number of successful pregnancies, the proportion of pregnancies is still higher in the immunologically affected and treated group, which suggests the importance of conducting further studies. http://dx.doi.org/10.1016/j.jri.2013.12.073
PIF* proprietary Soren B. Hayrabedyan 1 , Krassimira O. Todorova 1 , Eytan R. Barnea 2,3,∗ 1
Bulgarian Academy of Science, Sofia, Bulgaria The Society for the Investigation of Early Pregnancy (SIEP), Cherry Hill, NJ, USA 3 BioIncept, LLC, Cherry Hill, NJ, USA 2
E-mail address: [email protected] (E.R. Barnea). Secreted by viable embryos, PIF is critical for embryo development, and promotes implantation and trophoblast invasion. Synthetic PIF administration translates endogenous efficiency to pregnant and non-pregnant autoimmunity and transplantation models: short-term low-dose PIF achieves long-term effects. So far, PIF has required subcutaneous in vivo administration. However, both autoimmune diseases and post-transplantation require life-long therapy. An expression vehicle that continuously releases PIF would enable chronic exposure, obviating even episodic long-term injections. So far, PIF sequence direct encoding by DNA has remained elusive as it may derive from a small ORF. First reverse encoded putative PIF nucleotide sequence by codon optimization techniques was created following several algorithms, which enabled peptide in vitro expression and functional studies. Bioinformatics was used to reverse-engineer the PIF sequence by using either human proteins sharing partial homology or software optimization. Using molecular modeling, FLAG and HA tags using both an N- and C-terminal flanking strategy were added and putative PIF conformations were functionally assessed in silico. The model was validated by synthetic cDNA In-Fusion HD-based seamless cloning produced constructs. We documented that PIF is expressed by transiently transfected HEK293 cells using both immunofluorescent confocal microscopy and flow cytometry. This novel method enabled successful ribosometolerant strategy to express tagged PIF (synthetic gene) and a design expression vehicle for small peptides. PIF transfection will permit the conducting of in vitro and in vivo studies using transgenes. http://dx.doi.org/10.1016/j.jri.2013.12.074
Abstracts / Journal of Reproductive Immunology 101–102 (2014) 40–60
P43
P42 Immunological disorders in reproductive failure Szilvia Zeher 2
Vad 1,∗ ,
A.
Szanto 2 ,
T.
Tarr 2 ,
J.
Vegh 2 ,
G.
Nagy 3 ,
M.
Structural design-based preimplantation factor (PIF*) fusion peptide synthetic DNA cloning and eukaryote expression aimed for functional proteomic studies and possible chronic immune disorders therapy
1
Department of Obstetrics and Gynecology, Medical and Health Science Center, University of Debrecen, Hungary 2 Department of Internal Medicine, Division of Clinical Immunology, Medical and Health Science Center, University of Debrecen, Hungary 3 Regional Immunological Laboratory, Medical and Health Science Center, University of Debrecen, Hungary Reproductive failure is a major health issue in all developed countries. Almost 10–25% of couples may suffer of reproductive failure despite extensive clinical investigations 10–15% of these cases remain unexplained. It is known that immunological disorders may play a significant role in recurrent pregnancy loss and infertility. The aim of this study was to investigate the potential immunological disorders among women with unexplained recurrent pregnancy loss and infertility. Between 2008 and 2010 95 female patients referred by gynecologists to immunology outpatient offices were investigated for autoimmune disorders. The patients were screened for the presence of: anti-TPO, anticardiolipin, anti-b2GP1, lupus anticoagulant, rare autoantibodies (against annexin V, prothrombin, phosphatidyl serine), aSS-A, aCCP, aSS-B, ANF, anti-sperm and anti-endothelial antibodies. The results were statistically evaluated using the SPSS software package. Results: The mean age of patients was 32.9 ± 4.1 years. 50 out of 95 patients had recurrent pregnancy loss, 45 women had infertility problem. In 25 cases elevated anti-TPO was found, in 11 cases out of 95 elevated anti-phospholipid antibodies were proven (anticardiolipin: 2/95, antib2GP1: 1/95, anti-annexin V: 7/95, anti-phosphatidyl serine: 1/95), while in 14 women other elevated antibody titers (aSS-A: 4/95, aSS-A + aCCP: 2/95, aSS-A + aSS-B: 1/95, ANF: 4/95, antisperm antibody: 2/95, anti-endothelial antibody: 1/95) were found. The disorders were treated with levothyroxine, methyl-prednisolone, LMWH or salicylates. Eighty-two of the 95 patients were followed up for 25 months. They conceived 25 pregnancies and delivered 28 newborns. Despite the high number of pregnancies no significant difference was found in the number of successful pregnancies between women who had immunological disorder and those who did not. Discussion: Although statistically significant differences were not found regarding the number of successful pregnancies, the proportion of pregnancies is still higher in the immunologically affected and treated group, which suggests the importance of conducting further studies. http://dx.doi.org/10.1016/j.jri.2013.12.073
PIF* proprietary Soren B. Hayrabedyan 1 , Krassimira O. Todorova 1 , Eytan R. Barnea 2,3,∗ 1
Bulgarian Academy of Science, Sofia, Bulgaria The Society for the Investigation of Early Pregnancy (SIEP), Cherry Hill, NJ, USA 3 BioIncept, LLC, Cherry Hill, NJ, USA 2
E-mail address: [email protected] (E.R. Barnea). Secreted by viable embryos, PIF is critical for embryo development, and promotes implantation and trophoblast invasion. Synthetic PIF administration translates endogenous efficiency to pregnant and non-pregnant autoimmunity and transplantation models: short-term low-dose PIF achieves long-term effects. So far, PIF has required subcutaneous in vivo administration. However, both autoimmune diseases and post-transplantation require life-long therapy. An expression vehicle that continuously releases PIF would enable chronic exposure, obviating even episodic long-term injections. So far, PIF sequence direct encoding by DNA has remained elusive as it may derive from a small ORF. First reverse encoded putative PIF nucleotide sequence by codon optimization techniques was created following several algorithms, which enabled peptide in vitro expression and functional studies. Bioinformatics was used to reverse-engineer the PIF sequence by using either human proteins sharing partial homology or software optimization. Using molecular modeling, FLAG and HA tags using both an N- and C-terminal flanking strategy were added and putative PIF conformations were functionally assessed in silico. The model was validated by synthetic cDNA In-Fusion HD-based seamless cloning produced constructs. We documented that PIF is expressed by transiently transfected HEK293 cells using both immunofluorescent confocal microscopy and flow cytometry. This novel method enabled successful ribosometolerant strategy to express tagged PIF (synthetic gene) and a design expression vehicle for small peptides. PIF transfection will permit the conducting of in vitro and in vivo studies using transgenes. http://dx.doi.org/10.1016/j.jri.2013.12.074