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Placenta specific-microRNAs in normal pregnancy and preeclampsia
Abstracts / Journal of Reproductive Immunology 86 (2010) 79–111
in favor of the labyrinth; (2) the number of Plf+ giant cells was reduced; (3) levels of progesterone in maternal urine were decreased and estradiol increased. Also, the immune environment at the fetomaternal interface was altered by stress, as mirrored by an increased frequency of CD8+ T cells and – although at an overall lower frequency compared to CD8+ cells – of CD11c+ CD11bneg dendritic cells. Strikingly, as subpopulation of CD8+ cells in uterusdraining lymph nodes, identified by CD122+ CD28neg and presumed to be CD8+ T regulatory (Treg)/suppressor cells, declined upon stress challenge. Further, CD8+ cells isolated from stressed dams showed an enhanced cytotoxicity and augmented production of the inflammatory cytokines, such as tumor necrosis factor-␣ and interferon-␥. Progesterone supplementation abrogated the stress-induced placental/endocrine and immune changes and restored a normal fetal development. Strikingly, depletion of CD8+ cells rendered the protective effect of progesterone supplementation on fetal development refractory. Conclusions: In pregnancy, progesterone orchestrates an intrauterine environment promoting a normal fetal development. However, levels of progesterone are subject to stress challenges and subsequently alter downstream immune markers critically involved in maintaining placental function, such as skewing the phenotype of CD8+ Treg/suppressor into cytotoxic cells. In turn, fetal development is impaired and long term negative repercussions on the health of the offspring can be programmed. Keywords: Fetal development; Progesterone; CD8+ cells; Placenta Funding source: German Academic Exchange Program (DAAD), Alexander von Humboldt Foundation and AllerGen. doi:10.1016/j.jri.2010.08.048 48 Disorders options
of
implantation—are
there
therapeutic
T. Strowitzki Department of Gynecologic Endocrinology and Fertility Disorders, Ruprecht-Karls University, Voßstr. 9, 69115 Heidelberg, Germany The implantation of the human embryo is regulated by a complex network of endometrial and embryonal factors. Although many particulars contributing to the implantation process have been described, their interactions are still poorly understood. Apposition of the embryo to the implantation site is influenced by pinopodes, mucins and the embryonal factor L-selectin. The most important factors during the adhesion process seem to be the integrins and their receptor osteopontin. More factors recently described, which might play a decisive role during the implantation process are glycodelin A, glucose transporters and galectins. Microarray analysis revels a large cohort of up- and down-regulated genes of endometrial origin during the implantation.
105
The translation of basic research into clinical routine is rather limited. Systemic therapeutic options with immunoglobulins, heparin or corticosteroids yielded disappointing results. Uterine gene therapy of single deficient factors is still experimental and studies of the intrauterine stimulation by seminal plasma are not yet finished. doi:10.1016/j.jri.2010.08.049 49 PIBF regulates trophoblast invasion J. Szekeres-Bartho ∗ , M. Halasz, E. Miko, B. Polgar, T. Palkovics Department of Medical Microbiolgy and Immunology, Medical School, Pecs University, Pecs, Hungary Introduction: A progesterone-induced gene PIBF encodes a protein which mediates several of the immunological effects of progesterone. Recent data revealed an inverse relationship between PIBF and leptin receptor expression in normal 1st trimester trophoblast, incomplete and complete mole as well as in choriocarcinoma. These findings suggest a role of PIBF in invasiveness. Methods: To investigate the involvement of PIBF in invasiveness, a matrigel invasion assay was performed on intact and PIBF knock down trophoblast (HT-80/SVneo and a tumor (HT1080) cell line; furthermore, invasion signaling was investigated in the PIBF treated cell lines. Results: Invasiveness of HTR-8/SVneo increased, whereas that of HT 1080 decreased after PIBF depletion. PIBF acted via the IL-4R in both cell lines, and activated the STAT6 pathway. In addition, in HTR8/SVneo cell PIBF treatment resulted in an immediate and transient Akt activation, while in HT 1080 cells the same treatment resulted in a late (6 and 24 h after the treatment) activation of STAT3, Akt, Erk and Wnt5. Conclusions: In HT-1080, but not in HTR8/SVneo cells PIBF activates genes that lead to increased invasiveness. Funding source: Hungarian National Research Fund OTKA 77717 doi:10.1016/j.jri.2010.08.050 50 Placenta specific-microRNAs in normal pregnancy and preeclampsia T. Takizawa a,∗ , M.M. Ali a , O. Ishibashi a , K. Kikuchi a , T. Kosuge a , S. Matsubara b , T. Takeshita c a
Department of Molecular Medicine and Anatomy, Nippon Medical School, Tokyo, Japan b Department of Obstetrics and Gynecology, Jichi Medical University, Tochigi, Japan c Department of Obstetrics and Gynecology, Nippon Medical School, Tokyo, Japan Introduction: MicroRNAs (miRNAs) are singlestranded non-coding RNAs of approximately 22 nucleotides that participate in posttranscriptional reg-
106
Abstracts / Journal of Reproductive Immunology 86 (2010) 79–111
ulation of their target genes. Recently, we identified human placenta-specific miRNAs by small RNA library sequencing. Most placenta-specific miRNAs are linked to the miRNA cluster on chromosome 19. Interestingly, some miRNAs, including the placenta-specific miRNAs, were upregulated in preeclampsia placentas, indicating that these miRNAs could be promising biomarkers for the diagnosis of preeclampsia. Although the placenta-specific miRNAs are abundant in the plasma of pregnant women, the rapid clearance of the miRNAs from the plasma occurs after delivery. To determine whether human chorionic villi, especially the surface-covering syncytiotrophoblast, could secrete miRNAs extracellularly via exosomes, which in turn enter into maternal circulation, miRNAs in exosomes derived from the human trophoblast cell line BeWo were examined. We demonstrated that miRNAs are indeed extracellularly released via exosomes in BeWo cells. However, little is known about the role[s] of placenta-specific miRNAs secreted via exosomes in maternal cells and tissues during pregnancy. Exosomes could be involved in cell-cell communication. Thus, we hypothesized that circulating placenta-specific miRNAs could be delivered to maternal lymphocytes via exosomes and could be functional in the recipient cells. We tested this hypothesis using an in vitro model system. Methods: Exosomes were isolated from the BeWo cell culture supernatant by ultracentrifugation. The isolated exosomes (BeWo-exosomes) were incubated with the human T cell lymphoblast-like cell line Jurkat. After incubation with the BeWo-exosomes, total RNAs from Jurkat cells were isolated. The expression levels of miRNAs and their target mRNAs were examined by real-time PCR. Results: First, we confirmed that exosomes were successfully isolated from the BeWo cell culture supernatant by Western blot analysis for CD63, a commonly used marker of exosomes. Next, we examined whether BeWoderived miRNAs were transferable to Jurkat cells via exosomes. BeWo cells were transfected with a well characterized miRNA (i.e., an exogenous miRNA whose target mRNA had been identified so far). Exosomes were isolated from their culture supernatant and then added to Jurkat cell culture. Real-time PCR analysis revealed that the exogenous miRNA was detected in the BeWo-exosomes. We also detected a significant level of the exogenous miRNA in Jurkat cells after treatment with the BeWo-exosomes. Furthermore, we demonstrated that the expression level of a target mRNA of the exogenous miRNA was significantly downregulated in the recipient cells. In parallel, we demonstrated that endogenous placenta-specific miRNAs in BeWo cells were also detected in both BeWo-exosomes and recipient Jurkat cells. Taken together, our findings suggest that BeWo-derived exosomal miRNAs are transferable to Jurkat cells and are functional after entering the cells. Conclusions: We demonstrated the miRNAs present in trophoblast-exosomes to be capable of modulating gene expression in T cells using in the vitro model system. We provide a new insight into cell-cell communication via exosomes between placenta and lymphocytes. Circulation of placenta-specific miRNAs via exosomes may participate in immune tolerance during pregnancy, although more detailed studies are necessary.
Keywords: Placenta; Micro-RNA; pregnancy Funding source: This work was supported in part by Grants-in-Aids and ‘Research Core’ Project for Private University: Matching Fund Subsidy from the Ministry of Education, Culture, Sports, Science and Technology, Japan, and the Maruyama Memorial Research Fund of Nippon Medical School, Japan. doi:10.1016/j.jri.2010.08.051 51 Autoimmune and oncological diseases in identical female twins (case report) Z. Ulcova-Gallova a,∗ , H. Brabcova b , V. a a Hradecky , Z. Novotny , Z. Rokyta a
Kokes a , L.
a
Department of Gynecology Obstetrics, Medical School of Charles University and University Hospital, Pilsen, Czech Republic b Department of Clinical Pharmacology, Medical School of Charles University and University Hospital, Pilsen, Czech Republic Introduction: We report a special case on the history of identical female twins born in 1977 suffered from autoimmune diseases (twin A—Sjogren’s syndrome, and twin B—systemic lupus erythematosus). Patients: Twins A—LR (2250 g/45 cm). At the age of 17 years, her menarche commenced, at 18 years of age the diagnosis of autoimmune disease, manifested as Sjogren’s syndrome was found. On June 2006, in her 37th week of pregnancy, she spontaneously delivered a daughter, 2670 g/48 cm, Apgar 9-10-10 with cheilognathopalatoschis. At the age of 31 years, loop electrosurgical excision procedure (LEEP) proved precancerosis of cervix uteri. Twins B—LI (2260 g/45 cm). Her menarche commenced at the age of 15 years. At the age of 23 years, systemic lupus erythematosus and chronic rheumatism were diagnosed. At the age of 28 years, carcinoma vaginae, with invasive proliferation (T2 NX Mx G2) appeared. Results: Both sisters suffered from autoimmune and gynecological diseases at the same time. Relationships between disease activities and severities in the female twins were similar and the treatments were directed according to clinical symptoms and laboratory results. Conclusion: Dramatic change, unfortunately, occurred with twin B. The reason may be the association between SLE activity (lupus nephritis), hematological complication (leukopenia) and oncological fatal vaginal recidivation. Funding source: This work was supported by grant MSM 002 162 0812. doi:10.1016/j.jri.2010.08.052
in favor of the labyrinth; (2) the number of Plf+ giant cells was reduced; (3) levels of progesterone in maternal urine were decreased and estradiol increased. Also, the immune environment at the fetomaternal interface was altered by stress, as mirrored by an increased frequency of CD8+ T cells and – although at an overall lower frequency compared to CD8+ cells – of CD11c+ CD11bneg dendritic cells. Strikingly, as subpopulation of CD8+ cells in uterusdraining lymph nodes, identified by CD122+ CD28neg and presumed to be CD8+ T regulatory (Treg)/suppressor cells, declined upon stress challenge. Further, CD8+ cells isolated from stressed dams showed an enhanced cytotoxicity and augmented production of the inflammatory cytokines, such as tumor necrosis factor-␣ and interferon-␥. Progesterone supplementation abrogated the stress-induced placental/endocrine and immune changes and restored a normal fetal development. Strikingly, depletion of CD8+ cells rendered the protective effect of progesterone supplementation on fetal development refractory. Conclusions: In pregnancy, progesterone orchestrates an intrauterine environment promoting a normal fetal development. However, levels of progesterone are subject to stress challenges and subsequently alter downstream immune markers critically involved in maintaining placental function, such as skewing the phenotype of CD8+ Treg/suppressor into cytotoxic cells. In turn, fetal development is impaired and long term negative repercussions on the health of the offspring can be programmed. Keywords: Fetal development; Progesterone; CD8+ cells; Placenta Funding source: German Academic Exchange Program (DAAD), Alexander von Humboldt Foundation and AllerGen. doi:10.1016/j.jri.2010.08.048 48 Disorders options
of
implantation—are
there
therapeutic
T. Strowitzki Department of Gynecologic Endocrinology and Fertility Disorders, Ruprecht-Karls University, Voßstr. 9, 69115 Heidelberg, Germany The implantation of the human embryo is regulated by a complex network of endometrial and embryonal factors. Although many particulars contributing to the implantation process have been described, their interactions are still poorly understood. Apposition of the embryo to the implantation site is influenced by pinopodes, mucins and the embryonal factor L-selectin. The most important factors during the adhesion process seem to be the integrins and their receptor osteopontin. More factors recently described, which might play a decisive role during the implantation process are glycodelin A, glucose transporters and galectins. Microarray analysis revels a large cohort of up- and down-regulated genes of endometrial origin during the implantation.
105
The translation of basic research into clinical routine is rather limited. Systemic therapeutic options with immunoglobulins, heparin or corticosteroids yielded disappointing results. Uterine gene therapy of single deficient factors is still experimental and studies of the intrauterine stimulation by seminal plasma are not yet finished. doi:10.1016/j.jri.2010.08.049 49 PIBF regulates trophoblast invasion J. Szekeres-Bartho ∗ , M. Halasz, E. Miko, B. Polgar, T. Palkovics Department of Medical Microbiolgy and Immunology, Medical School, Pecs University, Pecs, Hungary Introduction: A progesterone-induced gene PIBF encodes a protein which mediates several of the immunological effects of progesterone. Recent data revealed an inverse relationship between PIBF and leptin receptor expression in normal 1st trimester trophoblast, incomplete and complete mole as well as in choriocarcinoma. These findings suggest a role of PIBF in invasiveness. Methods: To investigate the involvement of PIBF in invasiveness, a matrigel invasion assay was performed on intact and PIBF knock down trophoblast (HT-80/SVneo and a tumor (HT1080) cell line; furthermore, invasion signaling was investigated in the PIBF treated cell lines. Results: Invasiveness of HTR-8/SVneo increased, whereas that of HT 1080 decreased after PIBF depletion. PIBF acted via the IL-4R in both cell lines, and activated the STAT6 pathway. In addition, in HTR8/SVneo cell PIBF treatment resulted in an immediate and transient Akt activation, while in HT 1080 cells the same treatment resulted in a late (6 and 24 h after the treatment) activation of STAT3, Akt, Erk and Wnt5. Conclusions: In HT-1080, but not in HTR8/SVneo cells PIBF activates genes that lead to increased invasiveness. Funding source: Hungarian National Research Fund OTKA 77717 doi:10.1016/j.jri.2010.08.050 50 Placenta specific-microRNAs in normal pregnancy and preeclampsia T. Takizawa a,∗ , M.M. Ali a , O. Ishibashi a , K. Kikuchi a , T. Kosuge a , S. Matsubara b , T. Takeshita c a
Department of Molecular Medicine and Anatomy, Nippon Medical School, Tokyo, Japan b Department of Obstetrics and Gynecology, Jichi Medical University, Tochigi, Japan c Department of Obstetrics and Gynecology, Nippon Medical School, Tokyo, Japan Introduction: MicroRNAs (miRNAs) are singlestranded non-coding RNAs of approximately 22 nucleotides that participate in posttranscriptional reg-
106
Abstracts / Journal of Reproductive Immunology 86 (2010) 79–111
ulation of their target genes. Recently, we identified human placenta-specific miRNAs by small RNA library sequencing. Most placenta-specific miRNAs are linked to the miRNA cluster on chromosome 19. Interestingly, some miRNAs, including the placenta-specific miRNAs, were upregulated in preeclampsia placentas, indicating that these miRNAs could be promising biomarkers for the diagnosis of preeclampsia. Although the placenta-specific miRNAs are abundant in the plasma of pregnant women, the rapid clearance of the miRNAs from the plasma occurs after delivery. To determine whether human chorionic villi, especially the surface-covering syncytiotrophoblast, could secrete miRNAs extracellularly via exosomes, which in turn enter into maternal circulation, miRNAs in exosomes derived from the human trophoblast cell line BeWo were examined. We demonstrated that miRNAs are indeed extracellularly released via exosomes in BeWo cells. However, little is known about the role[s] of placenta-specific miRNAs secreted via exosomes in maternal cells and tissues during pregnancy. Exosomes could be involved in cell-cell communication. Thus, we hypothesized that circulating placenta-specific miRNAs could be delivered to maternal lymphocytes via exosomes and could be functional in the recipient cells. We tested this hypothesis using an in vitro model system. Methods: Exosomes were isolated from the BeWo cell culture supernatant by ultracentrifugation. The isolated exosomes (BeWo-exosomes) were incubated with the human T cell lymphoblast-like cell line Jurkat. After incubation with the BeWo-exosomes, total RNAs from Jurkat cells were isolated. The expression levels of miRNAs and their target mRNAs were examined by real-time PCR. Results: First, we confirmed that exosomes were successfully isolated from the BeWo cell culture supernatant by Western blot analysis for CD63, a commonly used marker of exosomes. Next, we examined whether BeWoderived miRNAs were transferable to Jurkat cells via exosomes. BeWo cells were transfected with a well characterized miRNA (i.e., an exogenous miRNA whose target mRNA had been identified so far). Exosomes were isolated from their culture supernatant and then added to Jurkat cell culture. Real-time PCR analysis revealed that the exogenous miRNA was detected in the BeWo-exosomes. We also detected a significant level of the exogenous miRNA in Jurkat cells after treatment with the BeWo-exosomes. Furthermore, we demonstrated that the expression level of a target mRNA of the exogenous miRNA was significantly downregulated in the recipient cells. In parallel, we demonstrated that endogenous placenta-specific miRNAs in BeWo cells were also detected in both BeWo-exosomes and recipient Jurkat cells. Taken together, our findings suggest that BeWo-derived exosomal miRNAs are transferable to Jurkat cells and are functional after entering the cells. Conclusions: We demonstrated the miRNAs present in trophoblast-exosomes to be capable of modulating gene expression in T cells using in the vitro model system. We provide a new insight into cell-cell communication via exosomes between placenta and lymphocytes. Circulation of placenta-specific miRNAs via exosomes may participate in immune tolerance during pregnancy, although more detailed studies are necessary.
Keywords: Placenta; Micro-RNA; pregnancy Funding source: This work was supported in part by Grants-in-Aids and ‘Research Core’ Project for Private University: Matching Fund Subsidy from the Ministry of Education, Culture, Sports, Science and Technology, Japan, and the Maruyama Memorial Research Fund of Nippon Medical School, Japan. doi:10.1016/j.jri.2010.08.051 51 Autoimmune and oncological diseases in identical female twins (case report) Z. Ulcova-Gallova a,∗ , H. Brabcova b , V. a a Hradecky , Z. Novotny , Z. Rokyta a
Kokes a , L.
a
Department of Gynecology Obstetrics, Medical School of Charles University and University Hospital, Pilsen, Czech Republic b Department of Clinical Pharmacology, Medical School of Charles University and University Hospital, Pilsen, Czech Republic Introduction: We report a special case on the history of identical female twins born in 1977 suffered from autoimmune diseases (twin A—Sjogren’s syndrome, and twin B—systemic lupus erythematosus). Patients: Twins A—LR (2250 g/45 cm). At the age of 17 years, her menarche commenced, at 18 years of age the diagnosis of autoimmune disease, manifested as Sjogren’s syndrome was found. On June 2006, in her 37th week of pregnancy, she spontaneously delivered a daughter, 2670 g/48 cm, Apgar 9-10-10 with cheilognathopalatoschis. At the age of 31 years, loop electrosurgical excision procedure (LEEP) proved precancerosis of cervix uteri. Twins B—LI (2260 g/45 cm). Her menarche commenced at the age of 15 years. At the age of 23 years, systemic lupus erythematosus and chronic rheumatism were diagnosed. At the age of 28 years, carcinoma vaginae, with invasive proliferation (T2 NX Mx G2) appeared. Results: Both sisters suffered from autoimmune and gynecological diseases at the same time. Relationships between disease activities and severities in the female twins were similar and the treatments were directed according to clinical symptoms and laboratory results. Conclusion: Dramatic change, unfortunately, occurred with twin B. The reason may be the association between SLE activity (lupus nephritis), hematological complication (leukopenia) and oncological fatal vaginal recidivation. Funding source: This work was supported by grant MSM 002 162 0812. doi:10.1016/j.jri.2010.08.052