A Hypothetical Model Suggesting Some Possible Ways That the Progesterone Receptor May Be Involved in Cancer Proliferation.

Cancer and the fetal-placental semi-allograft share certain characteristics, e.g., rapid proliferation, the capacity to invade normal tissue, and, related to the presence of antigens foreign to the host, the need to evade immune surveillance. Many present-day methods to treat cancer use drugs that can block a key molecule that is important for one or more of these characteristics and thus reduce side effects. The ideal molecule would be one that is essential for both the survival of the fetus and malignant tumor, but not needed for normal cells. There is a potential suitable candidate, the progesterone induced blocking factor (PIBF). The parent 90 kilodalton (kDa) form seems to be required for cell-cycle regulation, required by both the fetal-placental unit and malignant tumors. The parent form may be converted to splice variants that help both the fetus and tumors escape immune surveillance, especially in the fetal and tumor microenvironment. Evidence suggests that membrane progesterone receptors are involved in PIBF production, and indeed there has been anecdotal evidence that progesterone receptor antagonists, e.g., mifepristone, can significantly improve longevity and quality of life, with few side effects.

Biologia futura: embryo-maternal communication via progesterone-induced blocking factor (PIBF) positive embryo-derived extracellular vesicles. Their role in maternal immunomodulation.

Paternal antigens expressed by the foetus are recognized as foreign. Therefore,-according to the rules of transplantation immunity-the foetus ought to be "rejected". However, during normal gestation, maternal immune functions are re-adjusted, in order to create a favourable environment for the developing foetus. Some of the mechanisms that contribute to the altered immunological environment, for example, the cytokine balance and NK cell function, with special emphasis on the role of progesterone and the progesterone-induced blocking factor (PIBF) will be reviewed.

Progesterone-induced blocking factor-mediated Th1/Th2 balance correlates with fetal arrest in women who underwent in vitro fertilization and embryo transfer.

The role of progesterone-induced blocking factor (PIBF)-mediated Th1/Th2 balance in delivery outcomes of in vitro fertilization and embryo transfer (IVF-ET) has not been fully elucidated. In this study, 73 infertile women with successful IVF-ET were enrolled (16 fetal arrests and 57 live births). PIBF and IL-4 levels were significantly lower in the fetal arrest group than in the live birth group (p < 0.05). TNF-α level and Th1/Th2 ratios were significantly higher in the fetal arrest group than in the live birth group (p < 0.05). High TNF-α level and Th1/Th2 ratios were risk factors for fetal arrest, whereas increased PIBF and IL-4 levels were protective factors (P < 0.05). Increased TNF-α/IL-4 exhibited relatively strong predictive value for fetal arrest (AUC, 0.855; sensitivity, 93.8%; specificity, 71.9%). In summary, the PIBF-mediated Th1/Th2 balance is closely correlated with delivery outcomes of IVF-ET. TNF-α/IL-4 may be a predictive marker of fetal arrest.

Neutrophils recognize and amplify IFNT signals derived from day 7 bovine embryo for stimulation of ISGs expression in vitro: A possible implication for the early maternal recognition of pregnancy.

Previously, we reported that the presence of multiple day 7 (D7) bovine embryos in the uterus induces systemic immune responses in circulating polymorphonuclear neutrophils (PMNs), but with unknown mechanism. Thus, this study aimed to investigate the direct impact of D7 bovine embryo on PMNs' immune responses in vitro and whether these PMNs can amplify and transfer embryo signals further to another PMN population. PMNs were directly stimulated by embryo culture media (ECM) or interferon tau (IFNT) (10 ng/ml) followed by evaluating mRNA expression by real-time PCR and phenotypic analysis by flow cytometry. To test whether PMNs can transfer embryo signals to a new PMN population, PMNs triggered by ECM or IFNT, were thoroughly washed and diluted to remove any media components, and again were incubated in fresh culture media for 3 h, from which culture supernatants were collected and used as PMN conditioned media (CM) to stimulate a new PMN population. Similar to ECM, IFNT directly stimulated expressions of IFNs (IFNA, IFNG), interferon-stimulated genes (ISGs; OAS1, ISG15, MX1), STAT1, TGFB and IL8, and downregulated TNFA in PMNs. Flow cytometrical analyses demonstrated that IFNT stimulated expressions of pregnancy-related phenotypic markers, CD16 and arginase-1 (ARG1), in PMNs. Most importantly, PMN CM induced ISGs and STAT1 mRNA in fresh PMNs. Since IFNT directly upregulated IFNA expression in PMNs, the impact of IFNA on PMNs' immune responses was further tested. Stimulation of PMNs with IFNA, especially at a low level (1 pg/ml), induced IFNT-like immune responses comparable to those induced by PMN CM. Together, these findings indicated that D7 bovine embryos induce direct anti-inflammatory responses with upregulation of ISGs expressions in PMNs mainly via IFNT. Additionally, PMNs can amplify and transfer embryo signals to a new PMN population in a cell-to-cell communication mechanism possibly mediated in part by IFNA. Such a novel immunological crosstalk might contribute to embryo tolerance and pregnancy establishment in cattle.

Inhibitory effects of Vitamin D on inflammation and IL-6 release. A further support for COVID-19 management?

The ongoing worldwide pandemic of Coronavirus disease 2019 (COVID-19), raised the urgency to address knowledge gaps and to establish evidence for improving management and control of this viral infection. Throughout a keen analysis of the World Health Organization (WHO) most updated data, a gender-specific difference in the occurrence of infection was determined, which seems to correlate with patient's vitamin D status. Therefore, our purpose is to provide insights into the nutritional importance of vitamin D for its immunomodulatory effect, in order to help counteracting the COVID-19 pandemic. Novel interesting findings suggest that vitamin D, by inducing progesterone-induced blocking factor (PIBF), might regulate the immune response and also modulate cytokine IL-6, which appears to be increased in COVID-19 infections. Therefore, in addition to the standard recommendations to prevent the infection, supplementation of vitamin D might be considered an approach to help counteracting this global epidemic.

Preimplantation Factor (PIF): a peptide with various functions.

Preimplantation Factor (PIF) is a novel fifteen amino acid linear peptide (MVRIKPGSANKPSDD), which has different biological functions in mammalian species e.g. its role in neuron restoration, pregnancy and related disorders, and also in autoimmune diseases. Since all clinical studies have shown that PIF has both local and systemic effects, it can be considered as an integrated therapy for the treatment of inflammation conditions, along with the prevention of advanced disease. The synthetic PIF (sPIF) analog is a good representative of native PIF action, and it regulates peripheral immune cells to achieve endurance without immune suppression - an effective agent in nonpregnant autoimmune models. This study provides information, from evidence-based studies so far about PIF's different functional aspects.

Syncytin-1/HERV-W envelope is an early activation marker of leukocytes and is upregulated in multiple sclerosis patients.

Syncytin-1 is the envelope protein of the human endogenous retrovirus W (HERV-W). It has been related to multiple sclerosis (MS) but its role in cellular immunity and its pathogenic mechanism in the autoimmune context are not fully understood. We analyzed syncytin-1 levels in peripheral blood mononuclear cells (PBMC) subsets from healthy donors, MS patients in relapse or remission, and patients with acute infections by flow cytometry. PBMC cultures were also prepared to analyze protein expression kinetics. MS patients had higher levels of syncytin-1 levels than controls. We found that syncytin-1 is elevated in monocytes during MS relapses and infections. Cells expressing syncytin-1, including monocytes, T and B lymphocytes, and NKs presented mainly an activated phenotype and, upon stimulation with LPS, its levels increased rapidly on antigen-presenting cells. Syncytin-1 ligation promoted the activation of monocytes, as demonstrated by the upregulation of CD80 and the nonclassical subset CD14low CD16+ . Our results suggest an important role for syncytin-1 in the activation of leukocytes. Given that the expression of syncytin-1 is upregulated in MS patients, this protein might be contributing to the autoimmune cascade in the disease.

In silico Evaluation of PLAC1-fliC As a Chimeric Vaccine against Breast Cancer

Background: Breast cancer is one of the most prevalent cancers among women. Common cancer treatment methods are not effective enough, and there is a need for a more efficient treatment procedure. Cancer vaccine is a novel immunotherapy method that stimulates humoral and/or cellular immunity against cancer. Placenta-specific protein 1 (PLAC1) is a cancer/testis antigen, prevalent in breast cancer and rarely found in normal tissues. FliC, as a bacterial adjuvant, when fused to PLAC1 can elicit humoral and cellular responses. Therefore, PLAC1-fliC is a chimeric protein, which can be considered a suitable candidate against breast cancer. Methods: ProtParam was used to evaluate the physicochemical properties of PLAC1-fliC. Second structures were determined using the GOR V server. PLAC1-fliC 3D structure was modeled by Phyre2, and it was refined using GalaxyWEB. The refined model was submitted to RAMPAGE, PROCHECK, and ProSA-web for validation. Antigenicity and allergenicity of the construct were predicted by ANTIGENpro, VaxiJen, AllergenFP, and SDAP databases. Then MHC-I- and MHC-II-binding epitopes of PLAC1-fliC were forecasted by NetMHC 4.0 and NetMHCII 2.3 Servers. Finally, Ellipro and CTLpred were employed to predict B-cell and cytotoxic T lymphocyte epitopes. Results: The construct was evaluated as a stable fusion protein, which could be antigenic and could stimulate B and T cells against breast cancer. Conclusion: PLAC1-fliC, as a cancer vaccine candidate, might be suitable and specific for breast cancer, which could evoke humoral and cellular immunity against this type of tumor.

Antibody response against HERV-W in patients with MOG-IgG associated disorders, multiple sclerosis and NMOSD.

Increased expression of the retroviruses of HERV-W family has been linked to multiple sclerosis (MS) pathophysiology; nothing is known at the moment about MOG-IgG associated disorders. We compared antibody response against HERV-W peptides among patients with MOG-IgG associated disorders, multiple sclerosis (MS) and aquaporin-4 (AQP4)-IgG positive neuromyelitis optica spectrum disorder (NMOSD). A total of 102 serum samples were retrospectively analyzed. Antibody reactivity against HERV-W env peptides was similar in MOG-IgG associated disorders and MS, but different from AQP4-IgG positive NMOSD. Our findings expand the diagnostic role of HERV-W antibodies to the spectrum of demyelinating disorders associated with MOG-IgG.

IFITM proteins inhibit placental syncytiotrophoblast formation and promote fetal demise.

Elevated levels of type I interferon (IFN) during pregnancy are associated with intrauterine growth retardation, preterm birth, and fetal demise through mechanisms that are not well understood. A critical step of placental development is the fusion of trophoblast cells into a multinucleated syncytiotrophoblast (ST) layer. Fusion is mediated by syncytins, proteins deriving from ancestral endogenous retroviral envelopes. Using cultures of human trophoblasts or mouse cells, we show that IFN-induced transmembrane proteins (IFITMs), a family of restriction factors blocking the entry step of many viruses, impair ST formation and inhibit syncytin-mediated fusion. Moreover, the IFN inducer polyinosinic:polycytidylic acid promotes fetal resorption and placental abnormalities in wild-type but not in Ifitm-deleted mice. Thus, excessive levels of IFITMs may mediate the pregnancy complications observed during congenital infections and other IFN-induced pathologies.

Therapy Aimed to Suppress the Production of the Immunosuppressive Protein Progesterone Induced Blocking Factor (PIBF) May Provide Palliation and/or Increased Longevity for Patients With a Variety of Different Advanced Cancers - A Review.

Progesterone induced blocking factor (PIBF) is a unique protein that is not present in normal cells, but is found predominantly in rapidly growing cells of the fetal placental unit or cancer cells. There is a larger "parent" form that is a nuclear protein involved in cell to cell regulation, allowing tumor cells to proliferate and invade tissues. The parent compound is cleaved into smaller intracytoplasmic isoforms that can suppress cellular immune response, especially, but not limited to natural killer cells. The progesterone receptor antagonist mifepristone can suppress messenger RNA for PIBF, but can also suppress the intracytoplasmic protein. Treating cancer cell lines, intact animals with a variety of spontaneous cancers, and people with various cancers with mifepristone, has been found to inhibit cancer growth, and provide both palliation of symptoms and longevity possibly by suppressing this unique immunomodulatory protein.

PLAC1: biology and potential application in cancer immunotherapy.

The emergence of immunotherapy has revolutionized medical oncology with unprecedented advances in cancer treatment over the past two decades. However, a major obstacle in cancer immunotherapy is identifying appropriate tumor-specific antigens to make targeted therapy achievable with fewer normal cells being impaired. The similarity between placentation and tumor development and growth has inspired many investigators to discover antigens for effective immunotherapy of cancers. Placenta-specific 1 (PLAC1) is one of the recently discovered placental antigens with limited normal tissue expression and fundamental roles in placental function and development. There is a growing body of evidence showing that PLAC1 is frequently activated in a wide variety of cancer types and promotes cancer progression. Based on the restricted expression of PLAC1 in testis, placenta and a wide variety of cancers, we have designated this molecule with new terminology, cancer-testis-placenta (CTP) antigen, a feature that PLAC1 shares with many other cancer testis antigens. Recent reports from our lab provide compelling evidence on the preferential expression of PLAC1 in prostate cancer and its potential utility in prostate cancer immunotherapy. PLAC1 may be regarded as a potential CTP antigen for targeted cancer immunotherapy based on the available data on its promoting function in cancer development and also its expression in cancers of different histological origin. In this review, we will summarize current data on PLAC1 with emphasis on its association with cancer development and immunotherapy.

Anti-HERV-WEnv antibodies are correlated with seroreactivity against Mycobacterium avium subsp. paratuberculosis in children and youths at T1D risk.

Recent evidence points at the role that human endogenous retroviruses (HERVs) may play through the activation of genes integrated across the human genome. Although a variety of genetic/epigenetic mechanisms maintain most HERVs silenced, independent environmental stimuli including infections may transactivate endogenous elements favoring pathogenic conditions. Several studies associated exposures to Mycobacterium avium subsp. paratuberculosis (MAP) with increased anti-MAP seroreactivity in T1D patients. Here, we assessed humoral responses against HERV envelope antigens (HERV-KEnv and HERV-WEnv) and four MAP-derived peptides with human homologs in distinct populations: Sardinian children at T1D risk (rT1D) (n = 14), rT1D from mainland Italy (n = 54) and Polish youths with T1D (n = 74) or obesity unrelated to autoimmunity (OB) (n = 26). Unlike Sardinian rT1D, youths displayed increased anti-HERV-WEnv Abs prevalence compared to age-matched OB or healthy controls (24.32 vs. 11.54%, p = 0.02 for Polish T1D/OB and 31.48 vs. 11.90%, p = 0.0025 for Italian rT1D). Anti-HERV-KEnv responses showed variable trends across groups. A strong correlation between Abs levels against HERV-WEnv and homologous peptides was mirrored by time-related Abs patterns. Elevated values registered for HERV-WEnv overlaped with or preceded the detection of T1D diagnostic autoantibodies. These results support the hypothesis of MAP infection leading to HERV-W antigen expression and enhancing the production of autoantibodies in T1D.

Involvement of ILR4α and TLR4 in miscarriages.

BACKGROUND: The purpose of this study was to analyze the involvement of signaling via Interleukin-4-Receptor α (IL4Rα) and Toll like receptor (TLR) 4 at the fetomaternal interface in the process of early pregnancy. PATIENTS AND METHODS: Placenta specimens of 46 patients in early pregnancy were analyzed (normal pregnancy (n = 15), spontaneous (n = 15) and habitual abortion (n = 16)). TLR4 and IL4Rα were analyzed by immunohistochemistry, immunofluorescence and real time PCR. Statistical analysis was carried out using SPSS 23 and Microsoft Excel. RESULTS: IL4Rα could be detected in trophoblast cells of all groups. It was significantly downregulated in the syncytiotrophoblast of spontaneous and recurrent abortions (p = 0.001), and in decidual tissue of spontaneous abortions (p = 0.001). Expression of TLR4 was decreased in the intermediate villous trophoblast (IVT) and decidua of spontaneous abortions (p = 0.04 & 0.003, respectively). On mRNA level expression of IL4Rα and TLR4 was significantly decreased in the group of recurrent miscarriages (IL4Rα p = 0.002, TLR4 p = 0.004). CONCLUSION: This study contributes new findings to the understanding of the complex molecular interplay at the fetomaternal interface in normal pregnancy and miscarriages. For the first time signaling via IL4Rα being involved at the very beginning of the generation of new life could demonstrated. Moreover, new evidence was provided regarding TLR4 playing a pivotal role in early pregnancy.

Pro- and anti-inflammatory effects of sulforaphane on placental cytokine production.

Placental inflammation increases the risk of adverse pregnancy outcomes and possibly neurodevelopmental disorders in the offspring. Previous research suggests it may be possible to modulate the placental immune response to bacteria to favor an anti-inflammatory phenotype with dietary factors. Sulforaphane (SFN) is a dietary supplement with known anti-inflammatory activities, however, its effects on placental cytokine production are unclear. Therefore, we evaluated the effects of SFN on biomarkers of inflammation and neurodevelopment under basal conditions and a setting of mild infection. Placental explant cultures were established and treated with up to 10 µM SFN in the presence and absence of 107 CFU/ml heat-killed E. coli. Concentrations of IL-1ß, TNF-α, IL-6, sgp130, HO-1 and BDNF in conditioned medium were quantified by immunoassay. SFN increased antioxidant HO-1 expression in the absence, but not the presence, of infection. SFN inhibited IL-1ß and IL-10, but tended to promote, TNF-α production by bacteria-stimulated cultures. IL-6 and BDNF were inhibited by SFN irrespective of co-treatment with E.coli. A negative regulator of IL-6 signaling, sgp130, was increased by SFN under basal conditions, but not in E. coli-stimulated cultures. These results suggest that SFN has mixed effects on the placenta inhibiting both pro-inflammatory (IL-1ß) and anti-inflammatory factors (IL-10) but promoting regulators of oxidative stress and inflammation (HO-1 and sgp130) in an infection-dependent manner.

Expression of Plet1 controls interstitial migration of murine small intestinal dendritic cells.

Under homeostatic conditions, dendritic cells (DCs) continuously patrol the intestinal lamina propria. Upon antigen encounter, DCs initiate C-C motif chemokine receptor 7 (CCR7) expression and migrate into lymph nodes to direct T cell activation and differentiation. The mechanistic underpinnings of DC migration from the tissues to lymph nodes have been largely elucidated, contributing greatly to our understanding of DC functionality and intestinal immunity. In contrast, the molecular mechanisms allowing DCs to efficiently migrate through the complex extracellular matrix of the intestinal lamina propria prior to antigen encounter are still incompletely understood. Here we show that small intestinal murine CD11b+ CD103+ DCs express Placenta-expressed transcript 1 (Plet1), a glycophoshatidylinositol (GPI)-anchored surface protein involved in migration of keratinocytes during wound healing. In the absence of Plet1, CD11b+ CD103+ DCs display aberrant migratory behavior, and accumulate in the small intestine, independent of CCR7 responsiveness. RNA-sequencing indicated involvement of Plet1 in extracellular matrix-interactiveness, and subsequent in-vitro migration assays revealed that Plet1 augments the ability of DCs to migrate through extracellular matrix containing environments. In conclusion, our findings reveal that expression of Plet1 facilitates homeostatic interstitial migration of small intestinal DCs.

Induction of Proinflammatory Multiple Sclerosis-Associated Retrovirus Envelope Protein by Human Herpesvirus-6A and CD46 Receptor Engagement.

The aberrant expression of human endogenous retrovirus (HERV) elements of the HERV-W family has been associated with different diseases, including multiple sclerosis (MS). In particular, the expression of the envelope protein (Env) from the multiple sclerosis-associated retrovirus (MSRV), a member of HERV-W family and known for its potent proinflammatory activity, is repeatedly detected in the brain lesions and blood of MS patients. Furthermore, human herpesvirus 6 (HHV-6) infection has long been suspected to play a role in the pathogenesis of MS and neuroinflammation. We show here that both HHV-6A and stimulation of its receptor, transmembrane glycoprotein CD46, induce the expression of MSRV-Env. The engagement of extracellular domains SCR3 and SCR4 of CD46-Cyt1 isoform was required for MSRV-env transactivation, limiting thus the MSRV-Env induction to the CD46 ligands binding these domains, including C3b component of complement, specific monoclonal antibodies, and both infectious and UV-inactivated HHV-6A, but neither HHV-6B nor measles virus vaccine strain. Induction of MSRV-Env required CD46 Cyt-1 singling and was abolished by the inhibitors of protein kinase C. Finally, both membrane-expressed and secreted MSRV-Env trigger TLR4 signaling, displaying thus a proinflammatory potential, characteristic for this viral protein. These data expand the specter of HHV-6A effects in the modulation of the immune response and support the hypothesis that cross-talks between exogenous and endogenous viruses may contribute to inflammatory diseases and participate in neuroinflammation. Furthermore, they reveal a new function of CD46, known as an inhibitor of complement activation and receptor for several pathogens, in transactivation of HERV env genes, which may play an important role in the pathogenesis of inflammatory diseases.

Exosomes from human placenta purified by affinity chromatography on sepharose bearing immobilized antibodies against CD81 tetraspanin contain many peptides and small proteins.

Exosomes are nanovesicles (40-100 nm) containing various RNAs and different proteins. Exosomes are involved in intracellular communication and immune system function. Exosomes from different sources are usually isolated using standard methods-centrifugation and ultracentrifugations. Exosomes isolated by these procedures were reported to contain from a few dozen to thousands of different proteins. Here crude vesicle preparations from five placentas (normal pregnancy) were first obtained using standard centrifugation procedures. According to electron-microscopic studies, these preparations contained vesicles of different size (30-225 nm), particles of round shape of average electron density ("nonvesicles" 20-40 nm) (A), structured clusters of associated proteins and shapeless aggregations (B), as well as ring-shaped 10-14 nm structures formed by ferritin (C). After additional purification of the vesicle preparations by gel filtration on Sepharose 4B, the main part of protein structures was removed; however, the preparations still contained small admixtures of components A-C. Further purification of the preparations by affinity chromatography on Sepharose bearing immobilized antibodies against exosome surface protein CD81 led to isolation of highly purified exosomes (40-100 nm). These exosomes according to electron microscopy data contained tetraspanin embedded in the membrane, which was stained with antibodies against CD81 conjugated with 10-12 nm gold nanoparticles. SDS-PAGE and MALDI MS and MS/MS mass spectrometry of tryptic hydrolysates of proteins contained in these exosomes revealed eleven major proteins (>10 kDa): hemoglobin subunits, CD81, interleukin-1 receptor, annexin A5, cytoplasmic actin, alpha-actin-4, alkaline phosphatase, human serum albumin, serotransferrin, and lactotrasferrin. Using MALDI mass analysis of the highly purified exosomes, we for the first time found that in addition to the large proteins (>10 kDa), exosomes having affinity to CD81 contain more than 27 different peptides and small proteins of 2-10 kDa. This finding can be useful for revealing biological functions of pure exosomes. © 2018 IUBMB Life, 70(11):1144-1155, 2018.