Synthetic Preimplantation Factor Research Articles

Immune regulatory and neuroprotective properties of preimplantation factor: From newborn to adult.

Embryonic-maternal interaction from the earliest stages of gestation has a key, sustained role in neurologic development, persisting into adulthood. Early adverse events may be detrimental in adulthood. Protective factors present during gestation could significantly impact post-natal therapy. The role of PreImplantation Factor (PIF) within this context is herein examined. Secreted by viable early embryos, PIF establishes effective embryonic-maternal communication and exerts essential trophic and protective roles by reducing oxidative stress and protein misfolding and by blunting the nocive let-7 microRNA related pathway. PIF's effects on systemic immunity lead to comprehensive immune modulation, not immune suppression. We examine PIF's role in protecting embryos from adverse maternal environment, which can lead to neurological disorders that may only manifest post-nataly: Synthetic PIF successfully translates endogenous PIF features in both pregnant and non-pregnant clinically relevant models. Specifically PIF has neuroprotective effects in neonatal prematurity. In adult relapsing-remitting neuroinflammation, PIF reverses advanced paralysis while promoting neurogenesis. PIF reversed Mycobacterium smegmatis induced brain infection. In graft-vs.-host disease, PIF reduced skin ulceration, liver inflammation and colon ulceration while maintaining beneficial anti-cancer, graft-vs.-leukemia effect. Clinical-grade PIF has high-safety profile even at supraphysiological doses. The FDA awarded Fast-Track designation, and university-sponsored clinical trials for autoimmune disorder are ongoing. Altogether, PIF properties point to its determining regulatory role in immunity, inflammation and transplant acceptance. Specific plans for using PIF for the treatment of complex neurological disorders (ie. traumatic brain injury, progressive paralysis), including neuroprotection from newborn to adult, are presented.

Intralipid Infusion is the Current Favorite of Gynecologists for Immunotherapy.

Aspects of the immunological relationship between mother and conceptus still remain a mystery, although the recent advances in molecular biology have enlightened some of the parameters that participate in feto-maternal cross-talk during implantation. The atypical expression of major histocompatibility complex (MHC), the specific roles of some hormones and cytokines, as well as the temporal and spatial distributions of uterine natural killer (uNK) cells, represent substantive parameters of feto-maternal immunotolerance during implantation [1]. Although human maternal and fetal immunology is difficult to investigate, aberrant immune responses and an imbalanced cytokine network may be related to infertility, implantation failures after IVF, and recurrent pregnancy losses [2]. Patients with recurrent implantation failure (RIF) should be tested for inherited and acquired thrombophilias. Each patient should be individually assessed and counseled regarding management with low-molecular-weight heparin (LMWH). Empirical treatment with LMWH, aspirin, or corticosteroids is not effective for women with RIF who have negative thrombophilic tests [3]. If thrombophilic tests are normal, patients should be tested for immunological causes. The findings of a recent study suggest that increases in the percentage of CD56(dim) cells and NK cytotoxicity in peripheral blood may be important contributing factors for both RSA and IVF failure [4]. Human leukocyte antigen (HLA)-DQA1*0505 sharing or the maternal killer immunoglobulin-like receptor (KIR) repertoire is associated with recurrent spontaneous abortion (RSA) or repeated implantation failure (RIF) [5] and if abnormal, the patient might then benefit from intravenous immunoglobulin (IVIg) therapy [3]. IVIg has been successful in the treatment of recurrent miscarriage and recurrent implantation failure among women with elevated anti-phospholipid antibodies (APA) and/or NK cell activity [6]. When the pregnancy outcomes of women with a history of reproductive failure and elevated NK cell cytotoxicity treated with intralipid were compared with women treated with IVIg, no differences were seen [6]. Side-by-side comparison showed that synthetic pre-implantation factor (sPIF) is equally effective to inhibit NK cell toxicity at a lower dose than intravenous gamma immunoglobulin or intralipid treatment currently used [7]. sPIF is not yet available commercially, but intralipid infusions are available globally. Intralipid (IL) is a synthetic product composed of 10 % soybean oil, 1,2 % egg yolk phospholipids, 2.25 % glycerin, and water. When indicated, IL is infused 7–10 days prior to embryo transfer (ET), and one more time again after a positive pregnancy in women whose NKa is due to an autoimmune causes (antiphospholipid antibodies and/or antithyroid antibodies) [8]. In cases of alloimmune implantation dysfunction (DQa and/HLA matching between the embryo recipient and the male partner), the same applies, but in this situation, the infusion is repeated at 2–4 week intervals until the 24th week of pregnancy [8]. IL costs about 10 times less than IVIg, is not a blood product, and is without significant side effects [8].

106: Synthetic preimplantation factor (sPIF*) promotes neuroprotection by modulating PKA/PKC kinases

OBJECTIVE: Survivors of premature birth suffer from long term disabilities. Synthetic PreImplantation Factor (sPIF*) modulates inflammatory responses and reverses neuroinflammation. Proteinkinase A (PKA) and protein kinase C (PKC) are crucial signaling molecules. PKA up-regulates IL-10 and brain-derived neurotrophic factor (BDNF) expression, which exert neuroprotective effects. Anti-apoptotic phosphorylation of Bad is mediated by PKA. PKC phosphorylates GAP-43, a marker for neuronal plasticity and structural recovery. We explored sPIF protective role in neuronal (N2a) cells and in a rat model of encephalopathy of prematurity. *proprietary. STUDY DESIGN: Cells were subjected to LPS and treated with sPIF or scrambled sPIF. Neonatal rats (postnatal day 3: P3) were subjected to LPS, ligation of carotid artery, and hypoxia (8% O2, 65min; n¼ 30). sPIF (0.75mg/kg twice daily) was injected (P6-13) and brains harvested at P13. sPIF’s potential and mechanisms were evaluated using immunohistochemistry, ELISA, Western Blot, and qRT-PCR. Data were analyzed using two-tailed Student’s t-test. P<0.05 wasconsidered statistically significant. RESULTS: In vitro sPIF increased PKA/PKC activity in time dependent manner (Fig. 1A). sPIF induced higher IL-10, BDNF, and GAP-43 and lower CASP3, BAD, and TNF-a mRNA levels (Fig. 1B,C). sPIF increased pGap-43/Gap-43 and decreased pBad/Bad ratio while decreasing Bad (Fig. 1 D,E). In brain tissue sPIF treatment resulted in rescued neuronal number (NeuN positive cells) and reduced apoptosis (Casp-3 positive cells) with decreased glial (Iba-1 positive cells) activation (Fig. 2A,B). The Iba-1 morphology changed from predominantly amoeboid to ramified state. Additionally sPIF increased IL-10 mRNA levels (Fig. 2C) and pGap-43/Gap-43 ratio (Fig. 2D). CONCLUSION: sPIF modulates PKA/PKC pathways reducing apoptosis and inflammatory responses while increasing neuronal plasticity and survival. The identified PKA/PKC regulatory axis strengthens the potential of sPIF in reducing the burden of prematurity.

PreImplantation factor (PIF*) promotes embryotrophic and neuroprotective decidual genes: effect negated by epidermal growth factor.

BackgroundIntimate embryo-maternal interaction is paramount for pregnancy success post-implantation. The embryo follows a specific developmental timeline starting with neural system, dependent on endogenous and decidual factors. Beyond altered genetics/epigenetics, post-natal diseases may initiate at prenatal/neonatal, post-natal period, or through a continuum. Preimplantation factor (PIF) secreted by viable embryos promotes implantation and trophoblast invasion. Synthetic PIF reverses neuroinflammation in non-pregnant models. PIF targets embryo proteins that protect against oxidative stress and protein misfolding. We report of PIF’s embryotrophic role and potential to prevent developmental disorders by regulating uterine milieu at implantation and first trimester.MethodsPIF’s effect on human implantation (human endometrial stromal cells (HESC)) and first-trimester decidua cultures (FTDC) was examined, by global gene expression (Affymetrix), disease-biomarkers ranking (GeneGo), neuro-specific genes (Ingenuity) and proteins (mass-spectrometry). PIF co-cultured epidermal growth factor (EGF) in both HESC and FTDC (Affymetrix) was evaluated.ResultsIn HESC, PIF promotes neural differentiation and transmission genes (TLX2, EPHA10) while inhibiting retinoic acid receptor gene, which arrests growth. PIF promotes axon guidance and downregulates EGF-dependent neuroregulin signaling. In FTDC, PIF promotes bone morphogenetic protein pathway (SMAD1, 53-fold) and axonal guidance genes (EPH5) while inhibiting PPP2R2C, negative cell-growth regulator, involved in Alzheimer’s and amyotrophic lateral sclerosis. In HESC, PIF affects angiotensin via beta-arrestin, transforming growth factor-beta (TGF-β), notch, BMP, and wingless-int (WNT) signaling pathways that promote neurogenesis involved in childhood neurodevelopmental diseases—autism and also affected epithelial-mesenchymal transition involved in neuromuscular disorders. In FTDC, PIF upregulates neural development and hormone signaling, while downregulating genes protecting against xenobiotic response leading to connective tissue disorders.In both HESC and FTDC, PIF affects neural development and transmission pathways. In HESC interactome, PIF promotes FUS gene, which controls genome integrity, while in FTDC, PIF upregulates STAT3 critical transcription signal. EGF abolished PIF’s effect on HESC, decreasing metalloproteinase and prolactin receptor genes, thereby interfering with decidualization, while in FTDC, EGF co-cultured with PIF reduced ZHX2, gene that regulates neural AFP secretion.ConclusionsPIF promotes decidual trophic genes and proteins to regulate neural development. By regulating the uterine milieu, PIF may decrease embryo vulnerability to post-natal neurodevelopmental disorders. Examination of PIF-based intervention strategies used during embryogenesis to improve pregnancy prognosis and reduce post-natal vulnerability is clearly in order.

Synthetic PreImplantation Factor (sPIF)# neuroprotective role in intracranial stem cell transplantation: Encephalopathy of prematurity rat model

Background: Premature birth is a major cause of neonatal morbidity and mortality. Although improvements in perinatal and neonatal care reduce mortality, morbidity remains a serious challenge. Survivors of premature birth will confront enormous neurological problems. Wharton's jelly-derived mesenchymal stem cells (WJ-MSC) are a promising source for cell transplant. Embryo-derived PIF modulates systemic maternal immunity without suppression. PIF also reverses neuroinflammation (severe paralysis) while imparting neuroprotective/neurogenerative activity (multiple sclerosis models) and prevents juvenile diabetes while preserving islet function, by reducing oxidative stress and protein misfolding and controlling macrophages.

PreImplantation factor (PIF) potentiates static magnetic field (SMF) effect to decrease tumor burden (melanoma murine model)

Background and objectives PreImplantation factor (PIF) secreted by viable embryos exerts essential regulatory role on global systemic immune response. Synthetic PIF (PIF) translates endogenous effects to immune disorder models. Metastatic melanoma displays tumor-immunological behaviour. Static magnetic field (SMF) affects inflammatory reactions. There is increased interest toward SMF’s potential antitumor effects. Herein examined a novel anti-melanoma strategy using combined physical and immune-based therapy. Methods Daily whole-body SMF exposure, combined with subcutaneous PIF administration was examined in engrafted HT199 melanoma cells’ progression, transplanted into NSG mice. PIF effect on unique tumor-associated antigen expression relevant for tumor proliferation/ invasion was examined in vitro using specific antibodies. Direct PIF anti-proliferative effects on several cancer cell lines were tested using MTT. Results PIF potentiates SMF beneficial effect by reducing tumor volume vs. control (Mmax=96%) on day 34. Metastatic spleen mass is reduced by SMF alone (M=59%) or combined with PIF (M=62%). Daily SMF exposure alone inhibits tumor outgrowth (Mmax=60%, F5.32 (P<0.002) =21.16) while in combination with PIF, effect is considerably potentiated (M=80%), F5.32(P<0.0004)=34.84). PIF did not impair tumor antigen expression nor reduced significantly cultured tumor cell lines’ proliferation. Conclusions Collectively, results indicate that PIF’s potentiating antitumoral effect is mainly immune-regulatory, synergizing with SMF’s pro-tumor necrosis properties. The preserved tumor-associated antigen expression is important for the maintained antitumor immune activity. Overall, combined physico / immune regulatory treatment represents a useful, promising novel avenue for anti-cancer strategy.

Preimplantation and postimplantation therapy for the treatment of reproductive failure

Treatment of patients with recurrent pregnancy losses and recurrent implantation failure can be instituted only when the underlying etiology is determined. Embryo-secreted preimplantation factor (PIF) is essential for implantation and adequate trophoblastic invasion. Deficiency of PIF affects the outcome of the pregnancy leading to recurrent pregnancy losses. Synthetic PIF modulates the outcome of the pregnancy decreasing the incidence of recurrent implantation failure and recurrent pregnancy losses. In this article a thorough search is done regarding the data published for diagnoses of reproductive failure and its treatment. The effect of immunoglobulin (Ig), intralipid, heparin, aspirin, progesterone, estrogen, and granulocyte colony stimulating factor (G-CSF) is taken into consideration. Heparin, aspirin, and progesterone have successfully shown to decrease the incidence of recurrent pregnancy loses; whereas G-CSF, intralipids, estrogen, and Igs have shown success in the treatment of the recurrent implantation failure and recurrent pregnancy failure. The pregnancies treated with Igs and intralipids showed equal outcome when evaluated and compared. The place of intralipid in reducing natural killer (NK) cells has been discussed.

PreImplantation Factor Reduces Graft-versus-Host Disease by Regulating Immune Response and Lowering Oxidative Stress (Murine Model)

Bone marrow transplantation (BMT) to treat severe hematologic malignancies often leads to potentially fatal acute graft-versus-host disease (GVHD), despite attempts at better donor–recipient matching and/or use of immunosuppressive agents. We report that embryo-derived PreImplantation Factor (PIF) plays a determining role in developing maternal/host tolerance toward the semiallogeneic or total allogeneic embryo and in regulating systemic immune response. Synthetic PIF treatment has proven effective in preventing immune attacks in nonpregnant models of autoimmunity. In this study, we tested the capability of PIF to prevent the development of acute GVHD in semiallogeneic or totally allogeneic murine BMT models. We examined the regulatory effect of PIF both in vivo and in vitro to control deleterious GVHD while maintaining its ability to preserve the beneficial graft-versus-leukemia (GVL) effect. Bone marrow and spleen cells from C57BL/6 donors were transplanted in semiallogeneic (C57BL/6xBALB/c) F1 or allogeneic (BALB/c) mice, which were then treated with PIF 1 mg/kg/day for 2 weeks. Short-term PIF administration reduced acute GVHD in both models and increased survival for up to 4 months after semiallogeneic or totally allogeneic BMT. This effect was coupled with decreased skin inflammation (semiallogeneic model) and decreased liver inflammation (both models), as well as reduced colon ulceration (allogeneic model). GVHD-associated cytokine and chemokine gene expression were decreased in the liver. PIF further lowered circulating IL-17 levels, but not IFN-γ levels. Both in vivo and in vitro, PIF treatment was demonstrated to lead to decreased inducible nitric oxide synthase expression and decreased lipopolysaccharide-activated macrophages to lower nitric oxide secretion. Significantly, PIF did not diminish the beneficial GVL effect in the B cell leukemia model. PIF acts primarily by inducing the regulatory phenotype on monocytes/antigen-presenting cells, which controls T cell proliferation. Overall, our data demonstrate that PIF protects against semiallogeneic and allogeneic GVHD long term by reducing both target organ and systemic inflammation and by decreasing oxidative stress, while preserving the beneficial GVL effect.

Preimplantation factor inhibits circulating natural killer cell cytotoxicity and reduces CD69 expression: implications for recurrent pregnancy loss therapy

Embryo-secreted preimplantation factor (PIF) is necessary for, and its concentration correlates with, embryo development in humans by promoting implantation and trophoblast invasion. Synthetic PIF (sPIF) modulates systemic immunity and is effective in autoimmune disease models. sPIF binds monocytes and activated T and B cells, leading to immune tolerance without suppression. This study examined the effect of sPIF on natural killer (NK) cell cytotoxicity in 107 consecutive nonselected, nonpregnant patients with recurrent pregnancy loss (RPL) and 26 infertile IVF patients (controls). The effects of sPIF, intravenous gamma immunoglobulin (Ig), Intralipid and scrambled PIF (PIFscr; negative control) on NK cell cytotoxicity to peripheral-blood cells were compared by flow cytometry of labelled-K562 cell cytolysis. The effects of sPIF and PIFscr on whole-blood NKCD69+ expression were also compared. In patients with RPL, sPIF inhibited NK cell cytotoxicity at doses of 2.5 and 25ng/ml (37% and 42%) compared with PIFscr (18%; P<0.001), regardless of the proportion of peripheral-blood NKCD56+ cells to lymphocytes. Pre-incubation of blood from infertile patients with sPIF for 24h decreased NKCD69+ expression versus incubatino with PIFscr (P<0.05). In conclusion, sPIF inhibits NK cell cytotoxicity by reducing NKCD69 expression, suggesting a significant role in RPL patients.There is a continuous search to identify safe and effective agents to counteract recurrent pregnancy loss (RPL). Preimplantation factor (PIF) secreted by the embryo at the 2-cell stage is present throughout viable pregnancy but absent in nonviable pregnancy. Its immunomodulatory (not suppressive) effects promote embryo acceptance and maintenance by mother/host, control inflammation, facilitate uterine environment and placental embedding. Synthetic PIF (sPIF) was used to complete PIF’s role as a targeted, safe treatment for immune-based RPL. Previous reports showed sPIF’s significant protective systemic effect against maternal factors present in RPL serum. Herein is examined sPIF’s ability to inhibit the local protective toxicity induced by natural killer (NK) immune cells in a representative number of RPL patients. When elevated in blood, NK cells are associated with RPL. Low-dose physiological sPIF was highly effective to inhibit NK cell toxicity. Side-by-side comparison showed that sPIF is equally effective at a lower dose than intravenous gamma immunoglobulin or Intralipid treatment currently used. The sPIF effect on NK cells was targeted, indicating specific action. Overall, sPIF may represent a safe, effective and nontoxic immune-based therapy against RPL.

PreImplantation Factor (PIF) orchestrates systemic antiinflammatory response by immune cells: effect on peripheral blood mononuclear cells

Embryo-derived PreImplantation Factor (PIF) is essential for pregnancy immune modulation and synthetic PIF (sPIF), reverses neuroinflammation, and prevents diabetes mellitus through its immune modulatory properties. Herein, we explore sPIF's systemic effects on peripheral blood mononuclear cells (PBMCs). sPIF's effects on PBMCs and subset populations from nonpregnant patients (n = 7) and male patients were evaluated by the assessment of binding characteristics, mixed lymphocyte reaction, proliferation, cytokine secretion, and associated gene expression. Data analysis was by analysis of variance (P < .05). Fluorescein isothiocyanate-sPIF bound all myelomonocytic cells; binding was 30-fold up-regulated in mitogen-activated T and B cells (P < .05). sPIF decreased mixed lymphocyte reaction by 70% and blocked anti-CD3 antibody stimulated-PBMC proliferation by approximately 80% (P < .05). In naïve PBMCs, sPIF reduced interleukin (IL)-10 and -2; in activated PBMCs, sPIF increased IL-4, -5, -10, and -2, tumor necrosis factor-α, interferon-γ, and granulocyte-macrophage colony-stimulating factor (P < .05). Physiologic concentrations of PIF exert potent systemic antiinflammatory effects on nonpregnant activated immune cells.

Preimplantation factor negates embryo toxicity and promotes embryo development in culture

Preimplantation factor (PIF) is secreted by viable mammalian embryos and promotes implantation and trophoblast invasion. Whether PIF also has a direct protective or promoting effect on the developing embryo in culture is unknown. This study examined the protective effects of synthetic PIF (sPIF) on embryos cultured with embryo toxic serum (ETS) from recurrent pregnancy loss patients ( n = 14), by morphological criteria at 72 h of culture, and determined sPIF-promoting effect on singly cultured bovine IVF embryo development. sPIF negated the ETS-induced effect by increasing mouse blastocyst rate versus other embryonic stages (odds ratio (OR) 2.01, 95% confidence intervals (CI) 1.14–3.55, chi-squared = 12.74, P = 0.002), increased blastocyst rate (39.0% versus 23.7% ETS alone) and lowered embryo demise rate (11.0% versus 28.8%, OR 0.24, 95% CI 0.11–0.54), which was not replicated by scrambled PIF or the control. sPIF added to bovine embryos for 3 days promoted development at day 7 of culture (11% versus 0%, chi-squared = 4.0, P = 0.045). In conclusion, sPIF prevented embryo demise caused by exposure to ETS and promoted development of singly cultured bovine IVF embryos following short-term exposure. sPIF-based therapy for reducing recurrent pregnancy loss and improving lagging cultured IVF embryo development should be explored. Preimplantation factor (PIF) is a novel peptide, secreted by the embryo at the 2-cell stage. It is present throughout viable pregnancy but absent in non-viable pregnancy. PIF has essential immune-modulatory, acceptance and maintenance effects on the maternal system for embryo implantation, creating the necessary environment in the uterus and helping better embedding of the placenta. Herein we investigate in two complementary models whether PIF can help embryos survive under adverse conditions, protect against recurrent pregnancy loss and promote lagging embryo development. PIF’s synthetic analogue (sPIF) is available at suitable purity for human use and its use is proposed in physiological range doses. sPIF administration strongly suggested a targeted and safe treatment regimen, successfully negating the toxicity present in the blood (serum) of patients suffering from recurrent miscarriages in the relevant pooled mouse embryo model used. sPIF showed an equally important beneficial effect to prevent suboptimally developed embryo demise and improve their development. In a (single bovine) IVF model, highly relevant for low-responder human embryos that produce low-quality but viable embryos, sPIF increased embryo survival under adverse conditions. To document sPIF’s specific effect, an inactive, same-composition (scrambled) protein was tested, which showed no effect. To evidence sPIF’s safety and unique needs-based action, high concentrations added to optimally cultured embryos equally showed no effect. We conclude that sPIF administration may specifically reduce cases of repeat miscarriage and improve viable but suboptimal embryo survival. These added observations help complete PIF’s overall crucial function in accommodating and facilitating successful pregnancy.

Preimplantation factor promotes first trimester trophoblast invasion

Preimplantation factor is a novel embryo-derived peptide that influences key processes in early pregnancy implantation, including immunity, adhesion, remodeling, and apoptosis. Herein, we explore the effects of synthetic preimplantation factor on trophoblast invasion. Invasion patterns of immortalized cultured HTR-8 trophoblast cells were analyzed through Matrigel extracellular matrix ± synthetic preimplantation factor (25-100 nM) in a transwell assay. Effects were compared with epidermal growth factor 10 μg/mL, scrambled aminoacid sequence of preimplantation factor, or media alone as controls. Synthetic preimplantation factor enhances trophoblast invasion at physiologic doses (at 50 nM, 260%; 95% confidence interval [CI], 174-346%; P = .05; 100 nM ,178%; 95% CI, 170-184%; P < .02), compared with scrambled amnioacid sequence preimplantation factor or control media. Epidermal growth factor added to synthetic preimplantation factor does not further enhance trophoblast invasion (synthetic preimplantation factor 50 nM + epidermal growth factor, 238%; 95% CI, 237-239%; P < .03; synthetic preimplantation factor 100 nM + epidermal growth factor 269%; 95% CI, 265-273%; P < .04). Preimplantation factor should be further investigated as it shows a potential preventative or therapeutic role for pregnancy complications associated with inadequate trophoblast invasion.

A genomic and proteomic investigation of the impact of preimplantation factor on human decidual cells

Preimplantation factor (PIF) is a novel, 15 amino acid peptide, secreted by viable embryos. This study aims to elucidate PIF's effects in human endometrial stromal cells (HESC) decidualized by estrogen and progestin, which mimics the preimplantation milieu, and in first-trimester decidua cultures (FTDC). HESC or FTDC were incubated with 100 nmol/L synthetic PIF or vehicle control. Global gene expression was analyzed using microarray and pathway analysis. Proteins were analyzed using quantitative mass spectrometry, and PIF binding by protein array. Gene and proteomic analysis demonstrate that PIF affects immune, adhesion, and apoptotic pathways. Significant up-regulation in HESC (fold change) include: nuclear factor-k-beta activation via interleukin-1 receptor-associated kinase binding protein 1 (53); Toll-like receptor 5 (9); FK506 binding protein 15, 133kDa protein (2.3); and Down syndrome cell adhesion molecule like 1 (16). B-cell lymphoma protein 2 was down-regulated in HESC (21.1) and FTDC (27.1). Protein array demonstrates PIF interaction with intracellular targets insulin-degrading enzyme and beta-K+ channels. PIF displays essential multitargeted effects, of regulating immunity, promoting embryo-decidual adhesion, and regulating adaptive apoptotic processes.