Progesterone-induced Blocking Factor Expression Research Articles

Early pregnancy modulates the expression of interferon-stimulated genes, progesterone receptors, and progesterone-induced blocking factor in ovine duodenum

In ruminants, the signal (interferon-tau, IFNT) for the maternal recognition of pregnancy has effects on maternal extrauterine organs, and progesterone plays a key role during pregnancy. However, it is unclear that early pregnancy has effects on the expression of interferon-stimulated genes (ISGs), progesterone receptors (PGRs), and progesterone-induced blocking factor (PIBF) in ovine small intestine. In this study, small intestines were obtained at day 16 of the estrous cycle, days 13, 16, and 25 of pregnancy, and RT-qPCR assay, Western blot, and immunohistochemistry were used to analyze the effects of early pregnancy on the expression of ISGs, PGR, and PIBF in the small intestine. The results indicated that expression levels of ISG15-conjugated protein, signal transducer and activator of transcription 1 (STAT1), myxovirus resistance protein 1, and interferon-gamma-inducible protein 10 (IP-10) at day 13 of pregnancy were the lowest. However, expression levels of PGR isoforms (60 kDa and 40 kDa), PIBF, STAT1 and 2′,5′-oligoadenylate synthetase 1 upregulated, but the expression value of IP-10 downregulated at day 25 of pregnancy compared with day 16 of the estrous cycle. In conclusion, early pregnancy modulated the expression of ISGs, PGR, and PIBF in ovine duodenum, which was implicated in immunoregulation and nutrient absorption of the small intestine in ewes.

Pregnancy influences expression of interferon-stimulated genes, progesterone receptor and progesterone-induced blocking factor in ovine thyroid.

Embryonic interferon-tau (IFNT) and progesterone affect expression of interferonstimulated genes (ISGs), progesterone receptor (PGR) and progesterone-induced blocking factor (PIBF) in the ovine thyroid. Thyroids of ewes were sampled at day 16 of nonpregnancy, days 13, 16, and 25 of pregnancy, and real-time quantitative polymerase chain reaction assay, western blot and immunohistochemistry were used to detect expression of ISGs, PGR, and PIBF. Free ISG15 protein was undetected, but ISG15 conjugated proteins upregulated at day 16 of pregnancy, and expression levels of ISG15 conjugated proteins, PGR isoform (70 kDa), PIBF, interferon-gamma-inducible protein 10 and myxovirusresistance protein 1 peaked, but expression level of signal transducer and activator of transcription 1 was the lowest at day 16 of pregnancy. In addition, the expression levels of PGR isoform (70 kDa) and signal transducer and activator of transcription 1 (STAT1) decreased, but levels of PGR isoform (43 kDa), 2',5'-oligoadenylate synthetase, IP-10 and MX1 increased at day 25 of pregnancy comparing with day 16 of the estrous cycle. Early pregnancy affects expression of ISGs, PGR, and PIBF in maternal thyroid through IFNT and progesterone, which may regulate thyroid autoimmunity and thyroid hormone secretion in ewes.

Имплантационная несостоятельность эндометрия в циклах экстракорпорального оплодотворения у пациенток с хроническим эндометритом

Introduction. Chronic endometritis is one of the most frequent gynecological diseases which negatively impacts the main pathogenetic links in the morphogenesis of secretory and gestational endometrial transformation. These links determine implantation failure. The study aimed to verify the expression of estrogen receptors (ER) and progesterone receptors (PR), progesterone-induced blocking factor (PIBF) and stromal cell-derived factor-1 (SDF-1) in the endometrium of patients having chronic endometritis with ineffective IVF cycles or undeveloped pregnancies occurred after assisted reproductive technologies in their histories. Materials and methods. We formed 3 study groups: group I included samples of the endometrium of patients with ineffective IVF cycles (n=50); group II contained samples received from patients with a history of undeveloped pregnancy after IVF (n=50); and group III (the control group) included endometrial tissue from healthy patients (n=25). We carried out routine histological evaluation followed by an immunohistochemical assay with ER, PR, PIBF, and SDF-1 in the endometrial glands and stroma in the middle secretion phase. esults. The middle stage of the secretion phase of the menstrual cycle in endometrial samples of groups I and II was detected in only 46% and 42% of cases, respectively. We showed a decrease in the ER and PR expression in more than 92% of cases. Statistically significant decrease was detected in PIBF and SDF-1 expression in the endometrial glands and stroma within the implantation window in patients with a history of reproductive loss. Conclusion. Violation of implantation viability with an imbalance of chemokines, cytokines, cellular factors, and structural and functional characteristics of the endometrium was found in patients with revealed chronic endometritis and history of ineffective IVF cycles and miscarriage after ART. Keywords: endometrium,IVF, missed abortion after IVF, estrogen receptor, progesterone receptor, progesterone-induced blocking factor, stromal cell-derived factor-1

MORPHOLOGICAL FEATURES OF THE ENDOMETRIUM TRANSFORMATION IN RECURRENT MISCARRIAGE

The problems of pathological diagnosis of recurrent miscarriage are relevant in modern reproductive medicine. One of the important molecular markers of endometrium is the progesterone-induced blocking factor, which is induced by progesterone under the influence of activated lymphocytes and has an immunomodulatory effect on the implantation characteristics of the endometrium. In the process of trophoblast invasion, a special role is also played stromal cell factor molecules synthesized by endometrial and trophoblast cells, as a mechanism that potentiates the susceptibility of the endometrium to the onset and development of pregnancy, trophoblast invasion and embryogenesis in general. The purpose of the study was the histological and immunohistochemical features of the transformation of the endometrium in recurrent miscarriage. It were studied 100 samples of endometrium in case of non-developing pregnancy at a development period of 5-8 weeks, 85 samples in habitual miscarriage and 15 samples in pregnancy interrupted surgically at the request of the woman. The presence of 2 non-developing pregnancies was verified in 57 cases (67,1%), the presence of 3 non-developing pregnancies in 28 cases (32,9%). A histological examination of the abortion material stained with hematoxylin and eosin was performed to verify the morphological transformation of the endometrium. Immunohistochemical study included detection of estrogen and progesterone receptors, stromal cell factor (SDF1), progesterone-induced blocking factor (PIBF). The results of histological and immunohistochemical studies showed that with a complete morphological transformation of the endometrium in the group of patients with habitual miscarriage, there is a violation of the receptor profile. The decrease in PIBF expression in the glands and the stromal component and the expression of SDF1 in the glands of the compact layer of the endometrium is a reflection of the immunological imbalance in the endometrium in recurrent miscarriage. The development of a unified morphological algorithm, taking into account the basic indicators of the processes of transformation of the endometrium with an assessment of its receptor profile and diagnostically significant immunological factors, makes it possible to verify the pathology of the endometrium at the molecular level and justify the need for using pathogenetically substantiated therapy at recurrent miscarriage.

Экспрессия PIBF в гравидарном эндометрии при репродуктивных потерях первого триместра после экстракорпорального оплодотворения

Introduction. Demographic decline is one of the main challenges to modern society. Thus, the problem of early pregnancy loss retains medical and social significance regardless of the way of conception. The endometrium continues to be an underestimated component in the reproduction process, while studies ofthe gravid endometrium are few. The aim of the paper was to evaluate expression of progesterone-induced blocking factor (PIBF) in the gravid endometrium in early pregnancy loss following IVF methods. Materials and methods. Histological and immunohistochemical studies were carried out on samples of products of conception (POCs). Seventy-seven POCs with normal chorionic villi karyotype were collected from women with missed miscarriage of 5–8 weeks of embryohistological term after IVF; the other 15 samples of elective POCs were collected at the same term. We evaluated PIBF expression in glands and stroma of the gravid endometrium compact layer using immunohistochemical method. Results. Morphological features of missed miscarriage after IVF were characterized by violated transformation and decidualization of the gravid endometrium. Immunohistochemistry showed decreased PIBF expression in the experimental groups regardless of endometrial transformation options. It might indicate disturbances in the gravid endometrium and implantation compared to the control group (p<0.001). Conclusion. A comprehensive morphological evaluation of POCs with PIBF expression assessment will allow to verify violation of transformational potential of glands and stroma of the gravid endometrium in cases of early pregnancy loss regardless of the way of conception. Keywords: missed abortion, infertility, IVF, progesterone-induced blocking factor, gravid transformation of the endometrium

High Immune Expression of Progesterone-Induced Blocking Factor in Epithelial Ovarian Cancer.

Background:Progesterone-induced blocking factor, which is released from maternal lymphocytes during pregnancy mediates the immune effect of progesterone. According to new reports, it is suggested that proliferating cells, such as human trophoblasts, mesenchymal stem cells, and malignant tumors, can excrete progesterone-induced blocking factor at high ratio to escape from maternal immunity. It is shown in recent studies that progesterone-induced blocking factor is overexpressed in many malignant tumors such as breast, cervical, lymphoma, and leukemia. There are no data about progesterone-induced blocking factor expression in ovarian cancer cells. Hence, it is aimed to determine the progesterone-induced blocking factor expression levels in epithelial ovarian cancer.Methods:The study which was a retrospective cross-sectional study was conducted in a University Hospital. Twenty tissue specimens of patients with epithelial ovarian cancer and 20 tissue specimens of patients with healthy ovary were included in the study. Primary rabbit polyclonal anti- progesterone-induced blocking factor antibody was used to incubate the sections at a ratio of 1:300.Results:When the tissue sections were compared based on immunostaining with progesterone-induced blocking factor, we detected high stromal progesterone-induced blocking factor expression in the epithelial ovarian cancer group as check against to the normal ovarian group (P = .007). Similarly, we found high glandular progesterone-induced blocking factor expression in the epithelial ovarian cancer group as check against to the normal ovarian group (P < .001).Conclusion:Proving the existence of progesterone-induced blocking factor expression in epithelial ovarian cancer cells may lead new visions or new studies for epithelial ovarian cancer immunotherapy. As a result, epithelial ovarian cancer cells have greater levels of expression of progesterone-induced blocking factor protein than normal ovarian tissue according to immunohistochemistry. Further research is needed to understand the clinical importance of this finding, to learn outcomes of high levels of progesterone-induced blocking factor, and to investigate its underlying mechanisms.

Changes in T Cell and Dendritic Cell Phenotype from Mid to Late Pregnancy Are Indicative of a Shift from Immune Tolerance to Immune Activation.

During pregnancy, the mother allows the immunologically distinct fetoplacental unit to develop and grow. Opinions are divided as to whether this represents a state of fetal-specific tolerance or of a generalized suppression of the maternal immune system. We hypothesized that antigen-specific T cell responses are modulated by an inhibitory T cell phenotype and modified dendritic cell (DC) phenotype in a gestation-dependent manner. We analyzed changes in surface markers of peripheral blood T cells, ex vivo antigen-specific T cell responses, indoleamine 2,3-dioxygenase (IDO) activity (kynurenine/tryptophan ratio, KTR), plasma neopterin concentration, and the in vitro expression of progesterone-induced blocking factor (PIBF) in response to peripheral blood mononuclear cell culture with progesterone. We found that mid gestation is characterized by reduced antigen-specific T cell responses associated with (1) predominance of effector memory over other T cell subsets; (2) upregulation of inhibitory markers (programmed death ligand 1); (3) heightened response to progesterone (PIBF); and (4) reduced proportions of myeloid DC and concurrent IDO activity (KTR). Conversely, antigen-specific T cell responses normalized in late pregnancy and were associated with increased markers of T cell activation (CD38, neopterin). However, these changes occur with a simultaneous upregulation of immune suppressive mechanisms including apoptosis (CD95), coinhibition (TIM-3), and immune regulation (IL-10) through the course of pregnancy. Together, our data suggest that immune tolerance dominates in the second trimester and that it is gradually reversed in the third trimester in association with immune activation as the end of pregnancy approaches.

A study of altered cytokine rhythms associated with successful implantation in cows

Implantation is a critical process wherein the dam accepts the semi-allogenic embryo, characterized by certain vital immunological changes involving certain factors and cytokines. The current study was undertaken in Sahiwal cows to analyze the changes in certain selective immunological components of the body during peri-implantation period. Post-insemination the study was performed for a period till day 40 and the plasma was used to estimate the levels of progesterone, interleukin 1β (IL-1β), IL-2, IL-4, IL-6, and leukemia inhibiting factor (LIF) using Bovine specific ELISA Test Kits. The phagocytic activity and mRNA expression of progesterone-induced blocking factor (PIBF) were also analyzed. It was observed that in pregnant cows, the expression of PIBF increased from day 16th onward and remained high throughout the study period whereas the phagocytic activity was higher in non-pregnant cows all through the study period. The IL-1β, IL-2, and IL-6 were significantly higher in non-pregnant cows from 16th to 18th day post-AI as compared to P cows. On the other hand, IL-4 and LIF showed a significantly higher concentration in pregnant cows during peri-implantation period. Hence, it can be concluded that early embryonic survival is dependent on interplay of certain critical immunological factors in the body.

Serum levels of the immunomodulatory protein, the progesterone induced blocking factor (PIBF) which is found in high levels during pregnancy is not higher in women with progesterone (P) receptor (R) positive vs. negative breast cancer

To determine if serum levels of the immunomodulatory protein, the progesterone induced blocking factor (PIBF), which is present in high levels during normal pregnancy, is present in higher levels in women with breast cancer positive for progesterone receptors. The study would also determine whether the presence or absence of the estrogen receptor in any way modifies PIBF expression. PIBF using a research ELISA was evaluated in the follicular phase in 21 women with receptor status as follows: seven with estrogen receptor (ER)+ and progesterone receptor (PR)+, seven with ER- and PR+, and seven with ER+ and PR. The results showed no differences in serum PIBF in the three groups. The serum PIBF levels were no different than historical controls in the follicular phase. Measurement of serum PIBF does not seem to be an important marker to use to either detect women with breast cancer or to help determine tumor virulence or potential specific therapies. If PIBF plays a role in helping cancer cells to escape immune surveillance, it seems that the intracytoplasmic PIBF would be the form most likely operative.

The interplay between intracellular progesterone receptor and PKC plays a key role in migration and invasion of human glioblastoma cells

Intracellular progesterone receptors (PRs) and protein kinases C (PKCs) are known regulators of cancer cell proliferation and metastasis. Both PRs and PKCs are found overexpressed in grade IV human astrocytomas, also known as glioblastomas, which are the most frequent and aggressive brain tumors. In the present study, we investigated whether PR activation by PKC induces the migration and invasion of glioblastoma derived cell lines and if PKCα and δ isoforms are involved in PR activation. We observed that PKC activation with tetradecanoylphorbol acetate (TPA) increases the migration and invasion capacity of two human glioblastoma derived human cell lines (U251 MG and U87) and that the treatment with the PR receptor antagonist RU486 blocks these processes. Interestingly, the pharmacological inhibition of the isoenzymes PKCα and PKCδ also resulted in a blocked PR transcriptional activity. Also, TPA-dependent PR activation increases the expression of progesterone-induced blocking factor (PIBF), a known PR target gene. These results hint to an existing cross-talk between PKCs and PRs in regulating the infiltration process of human glioblastomas.

Progesterone treatment of threatened abortion, maternal serum levels of progesterone-induced blocking fac-tor and its clinical significance

Objective To investigate the treatment of threatened abortion progesterone maternal serum pro-gesterone-induced blocking factor(PIBF)levels and clinical significance, in order to be able to provide theoretical references of progesterone treatment of threatened abortion. Methods Threatened abortion women(study group)and non-threatened abortion women(control group),50 patients in the observation group given progesterone therapy, enzyme-linked immunosorbent assay(ELISA)was used to detect serum progesterone and PIBF levels of the obser-vation group before treatment and two weeks later, the control group was selected and named after two weeks. Results Observation group after treatment 47 patients progesterone tocolysis successful, success rate was 94.00%, tocolysis bleeding stop time 8.42d, abdominal pain time 11.32d.PIBF of the observation group was(313.52±90.43)mmol／L, which was lower than that of the control group(398.47±104.25)mmol／L, the difference was statistically significant (t＝10.90,P＜0.05). The progesterone of the observation group after 2 weeks of treatment was(92.35±20.85)mmol／L, which was higher than that before treatment(74.49±14.22)mmol／L, compared with progesterone levels of the con-trol group at the same time(83.26±20.14)mmol／L and(78.05±15.38)mmol／L, the difference was statistically significant(t＝7.96,8.11,P＜0.05),PIBF after 2 weeks of treatment in the observation group was(510.73± 87.49)mmol／L, which was higher than that of the control group at the same time(498.26±69.88)mmol／L, the difference was statistically significant(t＝4.35,P＜0.05).Follow-up observation group miscarriage successful term delivery rate 95.74%of pregnant women, childbirth fetal malformations seen in the control group of pregnant women at term birth rate was 94.00%, no delivery fetal malformation, the difference of full-term delivery rate was not statis-tically significant(P＞0.05). Conclusion Reduced PIBF during early pregnancy may be one of the reasons of threatened abortion, the use of progesterone therapy can increase PIBF expression, possibly through this pathway plays a role in tocolysis, so when using progesterone tocolysis monitoring PIBF levels helps evaluate the efficacy of tocolysis. Key words: Abortion, Threatened; Progesterone-induced blocking factor; Progesterone

P33. Decreasing of placental progesterone induced blocking factor expression and spiral artery remodeling disturbance in mice preeclampsia model

Introduction Preeclampsia is a major cause of morbidity and mortality in our developing country. The biggest obstacle in the high prevalence of preeclampsia and its management is due to lack of knowledge in preeclampsia pathologic mechanism. Progesterone is an important pregnancy hormone that has immunomodulatory effect by inducing progesterone induced blocking factor (PIBF) release, due to binding of progesterone with its receptor in lymphocyte. PIBF inhibits cytolytic NK cells activity and inducing T-helper 2 cytokine dominant while on contrary, preeclampsia has a condition of T-helper 1 cytokine dominant and increase of NK cells cytolytic activity. This was the first study to evaluated PIBF expression directly on the placenta when the pathogenesis of preeclampsia occurred after completion of vascular remodeling in animal models. Objective The objectives of this study were to compare placental PIBF expression, spiral artery wall thickness as characteristic of vascular remodeling disturbance and investigated correlation between both in mice preeclampsia model. Methods This experimental study used 32 pregnant mus musculus mice and randomly divided to normal group and preeclampsia model group. Our preeclampsia model was created by injecting anti-Qa2 (anti HLA-G) 10 ng from 1st until 4th day of pregnancy, to reduced HLA-G expression. Termination of both groups were performed after completion of spiral artery remodeling in mice by day 16th of pregnancy followed by immunohistochemistry examination for PIBF expression using immunoreactive score and spiral artery wall thickness measurement. Ethical clearance was taken from Medical Veterinary Faculty ethics commission. Difference and correlation were analysed using SPSS 20. Probability values Result In the result, PIBF were expressed in trophoblast, decidual and lymphocyte cells in placental tissue. The placental PIBF expression on preeclampsia model (2.01 ± 1.14) was significantly reduced (p ± 0.01) compared with control (3.99 ± 2.53). There was a significant increase of spiral artery wall thickness (p ± 0.001) in preeclampsia model (20.70 ± 5.93 micrometers) compared with control (9.98 ± 3.36 micrometers). There was a significant association (p ± 0.001) with negative correlation (rho/Pearson correlation = −0.623) between placental PIBF expression and the spiral artery wall thickness. Conclusion We concluded a decreased expression of placental PIBF and the increase of spiral artery wall thickness as a characteristic of spiral artery remodeling disturbance in mice model of preeclampsia, with negative correlation between placental PIBF expression and spiral artery wall thickness.

Progesterone-induced blocking factor is hormonally regulated in human astrocytoma cells, and increases their growth through the IL-4R/JAK1/STAT6 pathway

Astrocytomas are the most frequent and aggressive primary brain tumors in humans and constitute the leading cause of brain cancer related deaths. There are reports indicating that progesterone (P4) participates in the growth of astrocytomas through the interaction with its intracellular receptor (PR). Recently, it has been found that P4 induces the growth of several tumors through the up-regulation of progesterone-induced blocking factor (PIBF), a protein that has been related to the immunologic and proliferative actions of P4. U373 cells derived from a human astrocytoma grade III were used to study the role of P4 in PIBF expression and the effects of the latter in cell number. By using RT-PCR and Western blot techniques, we found that U373 cells express PIBF mRNA and protein. P4 (10nM and 100nM) increased PIBF mRNA expression after 1 and 3h of treatment, respectively, and this increase lasted 24h. This effect was blocked by the PR antagonist, RU486. Two PIBF isoforms were detected: one of 57kDa and the predominant one of 90kDa. The content of the 90kDa isoform increased after 12h of P4 treatment, and RU486 also blocked this increase. We observed that PIBF was released into the extracellular medium, being the 57kDa isoform the most abundant in this compartment. Immunofluorescence analysis showed that PIBF was localized in both the cytoplasm and nucleus. The effects of PIBF on cell number were analyzed for five consecutive days. PIBF (200ng/mL) significantly increased the number of U373 cells on days 2–5. Co-immunoprecipitation and Western blot assays revealed that PIBF associates to IL-4 receptor, and increases JAK1 and STAT6 phosphorylation at 20min. Our results suggest that P4 regulates PIBF expression in U373 cells through PR, and that PIBF increases cell number through IL-4 receptor/JAK1/STAT6 signaling pathway.

Expression of progesterone-induced blocking factor (PIBF) in preimplantation mouse embryos

There is an urgent need for the management of the population of stray dogs as they act as a carrier of the deadly rabies virus. Contraceptive vaccines based on either spermatozoa or egg-specific proteins aiming to interfere in sperm–egg interactions have been proposed as one of the strategies for controlling the population of various animal species. To this end, a fusion protein TT-KK-ZP3; encompassing promiscuous T cell epitope of tetanus toxoid (TT), adilysine linker (KK)anddogzonapellucidaglycoprotein-3 (ZP3)without any affinity tag has been expressed and purified from E. coli. Production of the recombinant protein has been successfully up-scaled at fermenter level. Refolded TT-KK-ZP3 showed the presence of the defined secondary structure in CD and fluorescent spectroscopy. Female FvB/J mice immunized with TT-KK-ZP3 showed a dosedependent increase in antibody titre that correlated well with reduction in fertility. Antibodies against TT-KK-ZP3 showed binding with mouse and dog ZP matrix in indirect immunofluorescence. Additionally, a chimeric polypeptide comprising TT, KK, C-terminus fragment of ZP domain of dog ZP3 (ZP3c-term), a triglycine spacer (GGG) and a fragment of dog spermatozoa-specific protein, Izumo (Iz), was expressed and purified from E. coli (TT-KK-ZP3-GGG-Iz) without any affinity tag. Female mice immunized with TTKK-ZP3-GGG-Iz showed specific antibody response against ZP3c-term as well as Izumo in ELISA. However, antibody titre against Izumo was higher compared with ZP3c-term. Antibodies reacted with mouse and dog ZP matrix and acrosome reacted mouse and dog spermatozoa. A physical mixture of individually expressed ZP3c-term and Izumo also elicited a similar kind of immune response, but antibody titre was low compared with chimeric protein. Groups of mice were also immunized with Izumo and ZP3c-term individually. All the groups showed varying degrees of curtailment in fertility compared with the controls. Mouse in vitro fertilization was inhibited significantly in the presence of immune serum from mice immunized with any of the above recombinant proteins. It is likely that these endeavours might help us to propose a candidate vaccine for the management of the population of the street dogs that may eventually lower the burden of rabies infection.

Cells Isolated from Human Glioblastoma Multiforme Express Progesterone-Induced Blocking Factor (PIBF)

Glioblastoma multiforme (GBM) is the most common and malignant tumor in the central nervous system. One of the contemporary hypotheses postulates that its pathogenesis is associated with the cancer stem cells (CSCs) which originate from mutations in the normal neural stem cells residing in their specific "niches." Simultaneously with its aggressive development the tumor suppresses the local immune system by different secreted and/or cell expressed factors. Progesterone-induced blocking factor (PIBF) is an immunomodulatory protein with known role in the regulation of the immune response in the reproductive system. Expression of PIBF has been described in some tumors as one of the factors suppressing the anti-tumor immunity. The aim of the present study was to check for the expression of PIBF from cells isolated from six GBMs. To characterize the cultured cells and to study the PIBF expression confocal microscopy, flow cytometry, ELISA, and real-time PCR were used. The results obtained showed expression of markers typical for cancer CSCs and secretion of interleukin 6 by the GBM-derived cultured cells. The results convincingly prove that PIBF is intracellularly expressed by the cultured cells from the all six GBM samples, and this fact is confirmed by three different methods-flow cytometry, confocal microscopy, and real-time PCR. This paper reports for the first time the expression of PIBF by GBM-derived cells cultured in vitro and reveals a new aspect of the immunosuppressive mechanism used by GBM in escaping the immune control.

Abstract 485: Evidence that mifepristone, a progesterone receptor antagonist, can cross the blood brain barrier and provide palliative benefits for glioblastoma multiforme grade IV.

Abstract The prognosis of patients with malignant glioma remains extremely poor despite surgery and improvement in radio and chemo therapies. Unfortunately few drugs cross the blood-brain barrier. Therapeutic treatment with chemotherapy agents, e.g., temozolomide, 1,3 bis (2-chloroethinyl)-1 nitrosurea carmustine wafers or tipifarnib have not significantly improved patient survival. Mifepristone has been found to provide significant palliative benefit to both mice with various types of malignancies and also humans with a wide variety of advanced cancers no longer or unresponsive to chemotherapy. The hypothesized mechanism of action is that tumor cells either directly secrete or cause gamma delta T cells in the tumor microenvironment to secrete a similar immunomodulatory protein as in normal pregnancies which inhibits natural killer (NK) cell cytotoxicity and causes a shift from thymic helper (TH)1 cytokines which stimulate cellular immune responses to TH2cytokines which evoke a humoral response. This protein is called the progesterone induced blocking factor (PIBF). The hypothesis suggests that also similar to pregnancy, progesterone or a P-like substance secreted by the tumor up-regulates PIBF response. Mifepristone has been found to down-regulate PIBF expression from human leukemia cell lines. Whether this is the precise mechanism of action or not, the progesterone receptor antagonist seems to inhibit tumor growth and provide extended and better quality of life. The objective of the present study was to determine if mifepristone may cross the blood brain barrier and provide a palliative benefit to patients with stage IV glioblastoma multiforme. A 43 year old male with a large glioblastoma multiforme grade IV malignant brain tumor arising originally from the temporal lobe with metastases to the spinal cord was told that he was no longer a candidate for anymore chemo or radiation therapy. He was advised that death should occur within 2 months. Indeed 2 months later death was considered imminent. He slept most of the day and was unable to converse. He was completely paralyzed from the neck down and his hands were in a clenched position. After 2 weeks of taking 200mg/day of mifepristone he became alert and was able to carry out intelligent conversations. He was now able to open his hand. He remained alert for 3 months, but paralysis had spread making swallowing difficult. He could not longer ingest the mifepristone. He died 2 weeks later. This case proves that mifepristone can cross the blood brain barrier and provide palliative benefits for grade IV glioblastoma multiforme. It is well tolerated with minimal or no side effects at this dosage. In view of the paucity of effective therapy for this type of cancer, mifepristone should be given consideration for treatment earlier than this extremely advanced stage. Citation Format: Jerome H. Check, Carrie Wilson, Diane Check. Evidence that mifepristone, a progesterone receptor antagonist, can cross the blood brain barrier and provide palliative benefits for glioblastoma multiforme grade IV. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 485. doi:10.1158/1538-7445.AM2013-485

Abstract 490: Clinical improvement of symptomatic advancing chronic lymphocytic leukemia following mifepristone therapy despite normal serum levels of the immunomodulatory protein the progesterone induced blocking factor (PIBF).

Abstract Recently a sensitive ELISA assay been created to detect serum levels of the immunomodulatory protein, the PIBF. One of the 34 kDa forms of PIBF increases markedly after exposure to progesterone (P) and inhibits natural killer (NK) cytolytic activity by stabilizing perforin granules and causes of shift from thymic helper (TH)1 to TH2 dominance thus diminishing the activity of the cellular immune system. The 34 kDa form is secreted by gamma/delta T cells and is considered a vital step in preventing immune destruction of the fetal semi-allograft. Evaluation of mRNA for PIBF and PIBF protein found that 29 of 29 human leukemia cell lines over-expressed mRNA for PIBF but only 3 of the lines expressed the PIBF protein. Adding P to the culture up-regulated PIBF protein expression whereas adding the P receptor antagonist mifepristone caused down-regulation of PIBF expression. Mifepristone treatment has been demonstrated to show significant palliative effects on a wide variety of murine and human cancers many of which are not known to be P receptor positive. The hypothesized mechanism is that these tumors either secrete PIBF or direct secretion by gamma/delta T cells in the tumor microenvironment and thus inhibit NK cell cytolytic activity. The inhibition is abrogated by inhibiting P receptors and thus a local form of P secretion by the tumor is hypothesized. The objective of this study was to determine if mifepristone will only help inhibit cancer growth in those individuals who have an increased PIBF level present in their serum. An 81 year old woman with chronic lymphocytic leukemia (CLL) started more rapid advancement and became very symptomatic. She refused chemotherapy when she almost had a lethal complication to oral chlorambucil. She requested mifepristone. Her serum PIBF level of 34.9 ng/mL was in the same range as controls without cancer. However within a month of taking 200mg daily of mifepristone orally she showed a dramatic improvement in her CLL with her white blood cell count dropping from 28,000 to 8,000. Her platelet count increased from 40,000 to 240,000 and 2 lung nodules thought to represent possible primary lung cancer (but possibly nodules from her CLL) completely disappeared on repeat CT scan. Also her energy markedly improved and her persistent cough stopped. The serum PIBF did not decrease with mifepristone therapy - 48.3 ng/mL one month later. These data demonstrated that failure to see a marked increase in serum PIBF in a patient with cancer does not predict failure to respond to mifepristone. Though mifepristone has ameliorated murine leukemia in AKR mice this is the first case report of this drug improving human leukemia. The 34-36 kDa PIBF found in cytoplasm of cancer cells may either not be secreted in the serum or may be immunologically different from serum PIBF found after P exposure. Citation Format: Jerome H. Check, Ann DiAntonio, Diane Check, Alex Jaffe, Rachael Cohen, Mojirayo Sarumi, Maya Srivastava. Clinical improvement of symptomatic advancing chronic lymphocytic leukemia following mifepristone therapy despite normal serum levels of the immunomodulatory protein the progesterone induced blocking factor (PIBF). [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 490. doi:10.1158/1538-7445.AM2013-490

Progesterone induced blocking factor expression in decidual stromal cells as a potential marker for successful pregnancy

Progesterone induced blocking factor expression in decidual stromal cells as a potential marker for successful pregnancy

Progesterone-induced blocking factor (PIBF) and trophoblast invasiveness

Controlled trophoblast invasion is a key process during human placentation and a prerequisite for successful pregnancy. Progesterone is one of the factors to regulate trophoblast invasiveness. Progesterone-induced blocking factor (PIBF) is a progesterone-induced molecule expressed by the trophoblast, and also by tumors. The distribution of PIBF within the first-trimester decidua coincides with sites of trophoblast invasion. Another molecule that has been implicated in the control of trophoblast invasiveness is placental leptin. Leptin inhibits the secretion of progesterone by cytotrophoblast. The aim of this work was to investigate the possible interaction of PIBF and leptins in regulating trophoblast invasion. Paraffin-embedded sections from normal first-trimester placentae, partial moles, complete moles, and choriocarcinomas were reacted with PIBF, leptin, and leptin receptor specific antibodies. PIBF-deficient trophoblast cells were generated using siRNA and leptin receptor was detected on Western blot analysis. The lysates of PIBF-treated cells were used for detecting leptin expression in a protein array. PIBF was expressed in both normal first-trimester villous trophoblast and in partial mole. Compared with this, PIBF expression was markedly decreased in complete mole and absent in choriocarcinoma. Neither leptinR nor leptin were detected in partial mole, whereas both of these molecules were present in complete mole and choriocarcinoma. Leptin receptor expression was upregulated in PIBF-deficient cells, while leptin expression was decreased in PIBF-treated cells. These data suggest that PIBF affects the expression of leptin and its receptor, and that PIBF expression is inversely related to trophoblast invasiveness.

Endometrial expression of progesterone-induced blocking factor and galectins-1, -3, -9, and -3 binding protein in the luteal phase and early pregnancy in cattle

Progesterone-induced blocking factor (PIBF) and galectins modulate the maternal immune response during pregnancy. We hypothesized that the relative transcript abundance of the above genes would be different during the luteal phase/early pregnancy and would be affected by progesterone supplementation. To further test this, hypothesis protein expression analyses were carried out to evaluate the abundance and localization of LGALS9 and PIBF. Following estrus synchronization, heifers were inseminated (n = 140) or not (n = 70). Half the heifers in each status (cyclic or potentially pregnant) were randomly assigned to receive a progesterone-releasing intravaginal device (PRID) on day 3 after estrus, which elevated progesterone concentrations from day 3.5 to 8 (P < 0.05), resulting in four treatment groups: cyclic and pregnant heifers, each with normal and high progesterone. After confirmation of pregnancy status in inseminated animals, uterine tissue was collected on days 5, 7, 13, or 16 of the luteal phase of the cycle/pregnancy. Gene and protein expression was determined using Q-RT-PCR and IHC, respectively, on 5 heifers per treatment per time point (i.e., 80 in total). Progesterone concentrations did not affect expression of any of the genes (P > 0.05). LGALS9 and LGALS3BP were expressed at low levels in both cyclic and pregnant endometria until day 13. On day 16, expression increased only in the pregnant heifers (P < 0.0001). LGALS1 and LGALS3 decreased on day 7 (P < 0.0001) and remained low until day 16. Pregnancy had no effect on the expression of LGALS1, LGALS3, and PIBF. Additionally, LGALS9 and PIBF proteins were expressed in distinct uterine cell types. These results indicate that the galectins may be involved in uterine receptivity and/or implantation in heifers.