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Background: Monoclonal nonspecific suppressor factor β (MNSFβ), a ubiquitously expressed member of the ubiquitin-like protein family, is associated with diverse cell regulatory functions. It has been implicated in glycolysis regulation and cell proliferation enhancement in the macrophage-like cell line Raw264.7. This study aims to show that HIF-1α regulates MNSFβ-mediated metabolic reprogramming., Methods and Results: In Raw264.7 cells, MNSFβ siRNA increased the oxygen consumption rate and reactive oxygen species (ROS) production but decreased ATP levels. Cells with MNSFβ knockdown showed a markedly increased ATP reduction rate upon the addition of oligomycin, a mitochondrial ATP synthase inhibitor. In addition, MNSFβ siRNA decreased the expression levels of mRNA and protein of HIF-1α-a regulator of glucose metabolism. Evaluation of the effect of MNSFβ on glucose metabolism in murine peritoneal macrophages revealed no changes in lactate production, glucose consumption, or ROS production., Conclusion: MNSFβ affects both glycolysis and mitochondrial metabolism, suggesting HIF-1α involvement in the MNSFβ-regulated glucose metabolism in Raw264.7 cells., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Proper placental development in early pregnancy ensures a positive outcome later on. The developmental relationship between the placenta and embryonic organs, such as the heart, is crucial for a normal pregnancy. However, the mechanism through which the placenta influences the development of embryonic organs remains unclear. Trophoblasts fuse to form multinucleated syncytiotrophoblasts (SynT), which primarily make up the placental materno-fetal interface. We discovered that endogenous progesterone immunomodulatory binding factor 1 (PIBF1) is vital for trophoblast differentiation and fusion into SynT in humans and mice. PIBF1 facilitates communication between SynT and adjacent vascular cells, promoting vascular network development in the primary placenta. This process affected the early development of the embryonic cardiovascular system in mice. Moreover, in vitro experiments showed that PIBF1 promotes the development of cardiovascular characteristics in heart organoids. Our findings show how SynTs organize the barrier and imply their possible roles in supporting embryogenesis, including cardiovascular development. SynT-derived factors and SynT within the placenta may play critical roles in ensuring proper organogenesis of other organs in the embryo., (© 2024. The Author(s).)

Problem: Recurrent pregnancy loss (RPL) is the spontaneous loss of two or more consecutive pregnancies prior to 20 weeks of gestation, occurring in 1% of the reproductive-age population. It is a major cause of infertility in India with a staggering 7.46% prevalence rate., Method of Study: Blood and product of conception (POCs) from RPL cases (n = 65) were enrolled for this study, along with cases of medically terminated pregnancy (MTP, n = 80) and term delivery cases (n = 90) as control. ELISA for progesterone and progesterone induced blocking factor (PIBF) levels was carried out, followed by mRNA expression analysis of progesterone receptor isoform B (PR-B) and its downstream immunomodulatory effectors, namely, PIBF, IL-10 and IL-12. Screening of PROGINS haplotype of PR gene and PIBF polymorphism were also conducted to correlate with their respective gene expression profiles., Results: Serum progesterone level was found to be comparable in the RPL and MTP cases. Although the mRNA expression of PR-B was found to be downregulated in the RPL cases, no significant PROGINS haplotype was observed. Presence of a single nucleotide polymorphism (SNP) in the PIBF gene (rs1372000) was more in healthy controls compared to RPL cases. Serum PIBF levels were found to be lower in the RPL cases with a resultant increase in IL-12 and a decrease in IL-10 mRNA expression in these cases., Conclusions: This study indicates that progesterone, acting through PIBF, modulates the immunological state of pregnancy to be Th1-biased in RPL, indicative of a pro-inflammatory, labour-like state not desired for a healthy pregnancy., (© 2023 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background: Quercetin is a flavonol that modifies many cellular processes. Monoclonal nonspecific suppressor factor β is a member of the ubiquitin-like family of proteins that are involved in various biological processes. It has been demonstrated that quercetin regulates the effect of MNSFβ on tumor necrosis factor-α secretion in lipopolysaccharide (LPS)-stimulated macrophages. This study found that quercetin and the heat shock protein HSC70 coregulate the action of MNSFβ., Methods and Results: Quercetin dose-dependently suppressed the LPS/interferon γ-induced nitric oxide production without cytotoxicity in the macrophage-like cell line Raw264.7. SiRNA knockdown experiments showed that quercetin inhibited the MNSFβ and HSC70 siRNA-mediated enhancement of TNFα and the production of RANTES, a member of C-C chemokine superfamily, in LPS-stimulated Raw264.7 cells. Western blot analysis showed that quercetin and HSC70 regulated ERK1/2 activation and LPS-stimulated IκBα degradation by affecting the complex formation of MNSFβ and the proapoptotic protein Bcl-G. Moreover, MNSFβ is implicated in TLR4/MyD88 signaling but not in TLR3 signaling., Conclusions: HSC70 is an important chaperone that facilitates the stabilization of MNSFβ. Quercetin may negatively control the function of MNSFβ by regulating the action of the molecular chaperone HSC70. MNSFβ mediates TLR4/Myd88 signaling but not TLR3 signaling., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)

Objectives: Success in pregnancy in mammals predominantly depends on a well-developed placenta. The differentiation of invasive trophoblasts is a fundamental process of placentation, the abnormalities of which are tightly associated with pregnancy disorders including preeclampsia (PE). Monoclonal nonspecific suppressor factor beta (MNSFβ) is an immunosuppressive factor. Its conventional knockout in mice induced embryonic lethality, whereas the underlying mechanism of MNSFβ in regulating placentation and pregnancy maintenance remains to be elucidated., Methods: Trophoblast-specific knockout of MNSFβ was generated using Cyp19-Cre mice. In situ hybridization (ISH), haematoxylin and eosin (HE), immunohistochemistry (IHC) and immunofluorescence (IF) were performed to examine the distribution of MNSFβ and insulin-like growth factor 2 mRNA-binding protein 2 (IGF2BP2) at the foeto-maternal interface. The interaction and expression of MNSFβ, IGF2BP2 and invasion-related molecules were detected by immunoprecipitation (IP), immunoblotting and quantitative real-time polymerase chain reaction (qRT-PCR). The cell invasion ability was measured by the Transwell insert assay., Results: We found that deficiency of MNSFβ in trophoblasts led to embryonic growth retardation by mid-gestation and subsequent foetal loss, primarily shown as apparently limited trophoblast invasion. In vitro experiments in human trophoblasts demonstrated that the conjugation of MNSFβ with IGF2BP2 and thus the stabilization of IGF2BP2 essentially mediated the invasion-promoting effect of MNSFβ. In the placentas from MNSFβ-deficient mice and severe preeclamptic (PE) patients, downregulation of MNSFβ was evidently associated with the repressed IGF2BP2 expression., Conclusions: The findings reveal the crucial role of MNSFβ in governing the trophoblast invasion and therefore foetal development, and add novel hints to reveal the placental pathology of PE., (© 2021 The Authors. Cell Proliferation published by John Wiley & Sons Ltd.)

Purpose: Progesterone-induced blocking factor (PIBF) is a protein secreted by lymphocytes exposed to progesterone (P4). P4 and PIBF have immunomodulatory effects on peripheral CD4 + T cells during normal pregnancy. Membrane progesterone receptors (mPRs) may correlate with the immunomodulatory properties of P4 on T cells. Variation in expression of mPRs may influence P4 regulatory performance during pregnancy. On the other hand, PIBF increases in pregnant normal women compared to women who have experienced abortion. The present study aimed to determine whether PIBF, in addition to having a direct influence on the immune system, can affect P4 performance through its effect on mPR expression. Such novel research findings demonstrate the importance of PIBF in the maintenance of pregnancy., Methods: Isolated peripheral blood mononuclear cells (PBMCs) from 30 healthy women were stimulated with the mitogen phytohemagglutinin (PHA). Cells were either exposed to various concentrations of PIBF or had no exposure at all in a culture medium at 37 °C for 3 days. The mean fluorescence intensity (MFI) of mPRα and mPRβ was evaluated using polyclonal and monoclonal antibodies on CD4 + T cells., Results: PIBF was able to significantly increase mPR expression on the surface of peripheral CD4 + T cells (p ≤ 0.05)., Conclusion: This study characterized the effects of PIBF on mPR expression on peripheral CD4 + T cells of healthy fertile women. Thus, a decrease in PIBF concentration during abnormal pregnancy can modulate mPR expression and regulatory performance of P4 on T cells. Future research into this issue is likely to open up a new understanding of the etiology of abortion., (© 2021. Hellenic Endocrine Society.)

Human mitochondrial chaperonin mHsp60 is essential for mitochondrial function by assisting folding of mitochondrial proteins. Unlike the double-ring bacterial GroEL, mHsp60 exists as a heptameric ring that is unstable and dissociates to subunits. The structural dynamics has been implicated for a unique mechanism of mHsp60. We purified active heptameric mHsp60, and determined a cryo-EM structure of mHsp60 heptamer at 3.4 Å. Of the three domains, the equatorial domains contribute most to the inter-subunit interactions, which include a four-stranded β sheet. Our structural comparison with GroEL shows that mHsp60 contains several unique sequences that directly decrease the sidechain interactions around the β sheet and indirectly shorten β strands by disengaging the backbones of the flanking residues from hydrogen bonding in the β strand conformation. The decreased inter-subunit interactions result in a small inter-subunit interface in mHsp60 compared to GroEL, providing a structural basis for the dynamics of mHsp60 subunit association. Importantly, the unique sequences are conserved among higher eukaryotic mitochondrial chaperonins, suggesting the importance of structural dynamics for eukaryotic chaperonins. Our structural comparison with the single-ring mHsp60-mHsp10 shows that upon mHsp10 binding the shortened inter-subunit β sheet is restored and the overall inter-subunit interface of mHsp60 increases drastically. Our structural basis for the mHsp10 induced stabilization of mHsp60 subunit interaction is consistent with the literature that mHsp10 stabilizes mHsp60 quaternary structure. Together, our studies provide structural bases for structural dynamics of the mHsp60 heptamer and for the stabilizing effect of mHsp10 on mHsp60 subunit association., (© 2021. The Author(s).)

Regulatory T (T reg ) cells, although vital for immune homeostasis, also represent a major barrier to anti-cancer immunity, as the tumour microenvironment (TME) promotes the recruitment, differentiation and activity of these cells 1,2 . Tumour cells show deregulated metabolism, leading to a metabolite-depleted, hypoxic and acidic TME 3 , which places infiltrating effector T cells in competition with the tumour for metabolites and impairs their function 4-6 . At the same time, T reg cells maintain a strong suppression of effector T cells within the TME 7,8 . As previous studies suggested that T reg cells possess a distinct metabolic profile from effector T cells 9-11 , we hypothesized that the altered metabolic landscape of the TME and increased activity of intratumoral T reg cells are linked. Here we show that T reg cells display broad heterogeneity in their metabolism of glucose within normal and transformed tissues, and can engage an alternative metabolic pathway to maintain suppressive function and proliferation. Glucose uptake correlates with poorer suppressive function and long-term instability, and high-glucose conditions impair the function and stability of T reg cells in vitro. T reg cells instead upregulate pathways involved in the metabolism of the glycolytic by-product lactic acid. T reg cells withstand high-lactate conditions, and treatment with lactate prevents the destabilizing effects of high-glucose conditions, generating intermediates necessary for proliferation. Deletion of MCT1-a lactate transporter-in T reg cells reveals that lactate uptake is dispensable for the function of peripheral T reg cells but required intratumorally, resulting in slowed tumour growth and an increased response to immunotherapy. Thus, T reg cells are metabolically flexible: they can use 'alternative' metabolites in the TME to maintain their suppressive identity. Further, our results suggest that tumours avoid destruction by not only depriving effector T cells of nutrients, but also metabolically supporting regulatory populations.

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., (© 2021. The Author(s).)

Aims: The invasion of extravillous trophoblast (EVT) cells into maternal decidua is essential for the establishment and maintenance of pregnancy. Derangement of EVT cell invasion might cause pregnancy complications including recurrent miscarriage (RM). We previously reported that deficiency of monoclonal nonspecific suppressor factor beta (MNSFβ) led to the early pregnancy failure in mice and the decidual MNSFβ expression level in RM patients was significantly decreased, but the underlying molecular mechanism of the role that MNSFβ played at the maternal-fetal interface remains unclear. Thus, in the present study, we determined effects of downregulated MNSFβ expression on human EVT cell activities., Methods: The MNSFβ expression in first-trimester human decidual and placental villus tissues was detected, respectively, by immunofluorescence or immunohistochemical analyses. The MNSFβ expression level in the immortalized first-trimester human EVT cell line HTR8/SVneo was downregulated by transfecting the small interfering RNA against MNSFβ and upregulated by transfecting the recombinant pDsRed-MNSFβ plasmids. The proliferation, migration, invasion, and apoptosis activities of HTR8/SVneo cells were, respectively, determined by cytometry assay, scratch test, transwell assay, and FITC/PI staining. The expression levels of P53, RhoA, Bcl-2, Bax, and MMP-9 in HTR8/SVneo cells, as well as the expression levels of MNSFβ and RhoA in placental villi of RM patients and physically normal pregnant women (NP), were examined by Western blot analysis., Results: MNSFβ protein signals were observed in first-trimester human villus and extravillous trophoblast cells. The downregulated MNSFβ expression significantly attenuated the proliferation, migration, and invasion abilities of HTR8/SVneo cells, accompanied with the obviously decreased expression levels of P53, RhoA, Bcl-2, Bax, and MMP-9, whereas the upregulated MNSFβ expression in HTR8/SVneo cells represented the inverse effects. Furthermore, expression levels of MNSFβ and RhoA in first-trimester human placental villus tissues of RM patients were significantly decreased compared to that of NP women., Conclusion: These data suggested that MNSFβ promotes proliferation and migration of human EVT cells, probably via the P53 signaling pathway, and the deficiency of MNSFβ in placental villi might lead to early pregnancy loss by reducing proliferation and invasion activities of EVTs., (© 2020 S. Karger AG, Basel.)

Pancreatic cancer (PC) is one of the most deadly cancers worldwide. To uncover the unknown novel biomarker used to indicate early diagnosis and prognosis in the molecular therapeutic field of PC is extremely of importance. Accumulative evidences indicated that aberrant expression or activation of immunoinhibitors is a common phenomenon in malignances, and significant associations have been noted between immunoinhibitors and tumorigenesis or progression in a wide range of cancers. However, the expression patterns and exact roles of immunoinhibitors contributing to tumorigenesis and progression of pancreatic cancer (PC) have not yet been elucidated clearly. In this study, we investigated the distinct expression and prognostic value of immunoinhibitors in patients with PC by analyzing a series of databases, including TISIDB, GEPIA, cBioPortal, and Kaplan-Meier plotter database. The mRNA expression levels of IDO1, CSF1R, VTCN1, KDR, LGALS9, TGFBR1, TGFB1, IL10RB, and PVRL2 were found to be significantly upregulated in patients with PC. Aberrant expression of TGFBR1, VTCN1, and LGALS9 was found to be associated with the worse outcomes of patients with PC. Bioinformatics analysis demonstrated that LGALS9 was involved in regulating the type I interferon signaling pathway, interferon-gamma-mediated signaling pathway, RIG-I-like receptor signaling pathway, NF-kappa B signaling pathway, cytosolic DNA-sensing pathway, and TNF signaling pathway. And TGFB1 was related to mesoderm formation, cell matrix adhesion, TGF-beta signaling pathway, and Hippo signaling pathway. These results suggested that LGALS9 and TGFBR1 might serve as potential prognostic biomarkers and targets for PC., Competing Interests: The authors declare that they have no competing interests., (Copyright © 2020 Yue Fan et al.)

Introduction: Recurrent implantation failure (RIF) is a challenging problem of assisted reproductive technology that arises mainly due to inadequate endometrial receptivity and its pathogenesis is still unclear., Objectives: In this study, we conducted the first investigation of the effect of decreased PIBF1 expression in mid-secretory phase on endometrial receptivity in patients with RIF., Methods: Microarray assay, reverse transcriptase-quantitative polymerase chain reaction, western blot, and in-vitro experiments were conducted., Results: The results showed that progesterone-induced blocking factor 1 (PIBF1) expression was highest in the mid-secretory endometrium in control subjects, but was significantly lower in RIF patients. In Ishikawa and human endometrial stromal cells (HESCs), rather than human endometrial epithelial cells, PIBF1 knockdown significantly downregulated cell proliferation and the levels of interleukin 6 (IL6) and phosphorylated signal transducer and activator of transcription-3 (p-STAT3). Besides, in HESCs, the levels of IL6, p-STAT3, prolactin and insulin-like growth factor binding-protein-1 (IGFBP1) decreased after PIBF1 knockdown during in-vitro decidualization. All these cellular changes could be notably restored by PIBF1 or IL6 overexpression. Consistent with our findings with PIBF1, the levels of IL6, p-STAT3, ki-67, prolactin, and IGFBP1 in the mid-secretory endometrium were notably lower in patients with RIF compared with controls., Conclusion: In summary, in the mid-secretory phase, decreased expression of PIBF1, IL6, and p-STAT3 inhibited HESC proliferation and decidualization, which is of theoretical and clinical importance for future research and clinical-treatment strategies., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2020 The Authors. Published by Elsevier B.V. on behalf of Cairo University.)

Purpose: This study evaluates the oncogenic role of PIBF1 in triple-negative breast cancer (TNBC). TNBC is considered to have a poorer prognosis than other types of breast cancer and is associated with high risk of recurrence and distant metastasis. Currently, there are no effective therapies for the TNBC patients with distant metastasis due to the lack of targeted therapeutic options., Methods: The effects of PIBF1 knockdown on the cell viability and motility of TNBC cell lines were investigated. Effects of PIBF1 overexpression on tumorigenicity and cell motility were confirmed using Ba/F3 cell line and xenograft study on BALB/c nude mice., Results: In TNBC cell lines that highly express PIBF1, knockdown of PIBF1 induces apoptosis and suppresses cell viability and motility with activation of the ATR/CHK1 signaling pathway. Moreover, the oncogenic function of PIBF1 was confirmed using the Ba/F3 cell line., Conclusion: For the first time, these findings clarify the role of PIBF1 in regulating ATR/CHK1 signaling pathway and inhibiting the proliferation and migration of TNBC cell lines. These results demonstrate the oncogenic roles of PIBF1 and provide new insights into the function and the molecular mechanism of PIBF1 in malignant TNBC.

Parkinson's disease (PD) is a common dyskinesia disease, the mitochondrial unfolded protein response (mtUPR) may be directly or indirectly involved in the occurrence and development of PD, although the exact mechanism is unclear. We established a dopaminergic neuronal-like cell model of PD, by overexpression of PGC-1α to detect evaluate the expression of proteases and molecular chaperones of involved in the mtUPR, as well as the expression of PGC-1α and LRPPRC, illustrated the distribution of LRPPRC. Remarkably, the mtUPR activation reached maximal at 24 h after MPP + treatment in SH-SY5Y cells, which the protein and transcription levels of the proteases and molecular chaperones reached maximal. The proteases and molecular chaperones were significantly increased when overexpressed PGC-1α, which indicated that PGC-1α overexpression activated the mtUPR, and PGC-1α had a protective effect on SH-SY5Y cells. The expression levels of PGC-1α and LRPPRC were significantly improved in the PGC-1α overexpression groups. LRPPRC was markedly reduced in the nucleus, suggesting that PGC-1α overexpression may play a protective role to the mitochondria through LRPPRC. Our finding indicates that overexpression of PGC-1α may activate mtUPR, reducing the oxidative stress injury induced by MPP + through LRPPRC signaling, thus maintain mitochondrial homeostasis.

Monoclonal nonspecific suppressor factor β (MNSFβ) is a ubiquitously expressed ubiquitin-like protein known to be involved in various biological functions. Previous studies have demonstrated that MNSFβ covalently modify its target proteins including Bcl-G, a proapoptotic protein. In this study, we purified a 65 kDa MNSFβ adduct from mouse liver lysates by sequential chromatography on DEAE and glutathione S-transferase (GST)-fusioned MNSFβ immobilized on glutathione-Sepharose beads in the presence of ATP. MALDI-TOF mass spectrometry fingerprinting revealed that this MNSFβ adduct consists of an 8.5 kDa MNSFβ and heat shock protein 60 (HSP60), a mitochondrial protein involved in protein folding. Fingerprinting analysis of the MNSFβ adduct demonstrates that MNSFβ conjugates to HSP60 with a linkage between the C-terminal Gly74 and Lys481. HSP60 siRNA neutralized the inhibition of apoptosis by MNSFβ siRNA in LPS/IFNγ-stimulated Raw264.7, a murine macrophage cell line. HSP60 siRNA also down-regulated the enhancement of TNFα production by MNSFβ siRNA in LPS-stimulated Raw264.7 cells. Here, we firstly report that MNSFβ activity is negatively regulated by molecular chaperone.

Pregnancy represents a unique immunological situation. Though paternal antigens expressed by the conceptus are recognized by the immune system of the mother, the immune response does not harm the fetus. Progesterone and a progesterone induced protein; PIBF are important players in re-adjusting the functioning of the maternal immune system during pregnancy. PIBF expressed by peripheral pregnancy lymphocytes, and other cell types, participates in the feto-maternal communication, partly, by mediating the immunological actions of progesterone. Several splice variants of PIBF were identified with different physiological activity. The full length 90 kD PIBF protein plays a role in cell cycle regulation, while shorter splice variants are secreted and act as cytokines. Aberrant production of PIBF isoforms lead to the loss of immune-regulatory functions, resulting in and pregnancy failure. By up regulating Th2 type cytokine production and by down-regulating NK activity, PIBF contributes to the altered attitude of the maternal immune system. Normal pregnancy is characterized by a Th2-dominant cytokine balance, which is partly due to the action of the smaller PIBF isoforms. These bind to a novel form of the IL-4 receptor, and induce increased production of IL-3, IL-4, and IL-10. The communication between the conceptus and the mother is established via extracellular vesicles (EVs). Pre-implantation embryos produce EVs both in vitro , and in vivo . PIBF transported by the EVs from the embryo to maternal lymphocytes induces increased IL-10 production by the latter, this way contributing to the Th2 dominant immune responses described during pregnancy.

Interferon-tau (IFNT) is the main signal for the maternal recognition of pregnancy in ruminants, and exerts its effects by stimulating the expression of interferon-stimulated genes, including the expression of interferon-stimulated gene15 kDa protein (ISG15). Progesterone (P4) exerts significant immune effects on the uterus during early pregnancy in ruminants that are partly mediated by progesterone-induced blocking factor (PIBF). The thymus is necessary for the normal development of immunologic function. In this study, thymuses were obtained on day 16 of the estrous cycle and on days 13, 16 and 25 of pregnancy (n = 6 for each group) from ewes. Our results showed that the expression of ISG15, P4 receptor (PGR) and PIBF mRNA and the expression of ISG15 and ISG15-conjugated proteins were upregulated in the thymuses during early pregnancy, and the 89-kDa PGR isoform and the 80-kDa PIBF variant were expressed constantly in the thymuses. However, there was no expression of the 60-kDa PGR isoform and the 62-kDa PIBF variant on day 16 of the estrous cycle. ISG15 and ISG15-conjugated proteins were limited to the epithelial reticular cells, capillaries and thymic corpuscles. This paper reports for the first time that early pregnancy exerts its effects on the thymus through IFNT and P4 in sheep., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Background: Heat shock proteins (HSP) might be useful as biomarkers for bipolar disorder (BD) which would be clinically valuable since no reliable biomarker for BD has so far been identified. The purpose of this study was to assess the heat shock proteins CPN10, CPN60, and CPN70 as potential biomarkers of BD., Methods: The study included 100 BD patients recruited from a hospital during 2012 and 2013. The study also included 94 healthy controls. Among the BD patients, 33 had abnormal hypothalamic-pituitary-adrenal (HPA) axis activity. Blood samples were obtained from the patients and controls. The chemiluminescence method, mass spectrometry, and flow cytometry were used for analysis., Results: The BD patients compared with the controls had a significantly lower level of CPN10 and significantly higher levels of CPN60 and CPN70. The BD patients with abnormal HPA axis activity had a significantly lower level of CPN60 compared with the normal HPA axis activity group of BD patients. The CPN60 level significantly inversely correlated with adrenocorticotropic hormone (ACTH) level in patients with bipolar depression and in patients with bipolar hypomania, and CPN70 significantly correlated with ACTH level in patients with bipolar depression and hypomania., Conclusions: Our findings suggest that the heat shock proteins CPN10, CPN60, and CPN70 might have potential as biomarkers for BD and CPN60 blood level might distinguish patients with abnormal HPA axis activity from those with normal HPA axis activity.

In addition to being immunomodulatory, Progesterone-Induced Blocking Factor (PIBF) plays a role in cell cycle regulation and invasion. The full length protein is associated with the pericentriolar satellites and as such, it is crucial for maintaining the integrity of spindle poles during mitosis. Another suggestive evidence for the involvement of PIBF in tumour progression is the fact that the PIBF gene has been identified on chromosome 13 in the region associated with breast cancer susceptibility. Earlier we showed that PIBF differentially regulates the invasiveness of trophoblast and tumour cell lines. The aim of the present study was to further investigate the role of PIBF in tumour development, using primary ovarian- (OC) and primary lung carcinoma (LC) cell cultures, and JEG-3 choriocarcinoma cell line. In the cultured cells PIBF was knocked down by siRNA treatment, and the impact of PIBF deficiency on MMP-9 activity and E-cadherin expression as well as on invasive and migratory capacity of the cells was tested. In conditioned media of PIBF-deficient JEG-3 cells, LC cells and OC cells MMP-9 activity was reduced to 36% 35%, and 65% respectively compared to controls. Though PIBF knock down did not affect migration, in JEG-3 cells, LC primary cells and OC primary cells PIBF deficiency resulted 20%, 50% and 50% decrease of invasion respectively. PIBF silencing resulted in increased E-cadherin expression, suggesting that by down regulating E-cadherin expression, PIBF might interfere with the cell-cell adhesion mechanisms and by increasing MMP activity induced extracellular matrix degradation, facilitates the invasion of tumour cells., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Progesterone (P4) regulates reproductive and immune functions through binding to the progesterone receptor (PGR), and the effects of P4 are partly mediated by a progesterone-induced blocking factor (PIBF). Bone marrow (BM) is a key component of the lymphatic system and has an important role in immune response. In this study, BM was harvested from femurs on days 13, 16 and 25 of pregnancy and day 16 of the estrous cycles without mated by intact rams, and a qRT-PCR assay, Western blot and an immunohistochemistry analysis were used to analyze the expression of PGR and PIBF genes in BM. The results showed that there was an increase in relative abundance of PGR and PIBF mRNA in BM during early pregnancy, and PGR-B and the full-length PIBF genes were up-regulated in pregnant ewes. Immunohistochemistry results confirmed that the PGR and PIBF proteins were localized in both the cytoplasm and nuclei of adipocytes and the cells in the stroma and capillaries. This is the first study reporting an up-regulated expression of PGR-B and full-length PIBF genes in BM during early pregnancy in sheep. It is suggested that the conceptus exerted its effects on the adipocytes and the cells in the stroma and capillaries in BM, which were involved in the immunoregulation of BM through both cytosolic and nuclear pathways in ewes., (Copyright © 2017 Elsevier B.V. All rights reserved.)

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., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Progesterone-induced blocking factor (PIBF) is a progesterone (P 4 ) regulated protein expressed in different types of high proliferative cells including astrocytomas, the most frequent and aggressive brain tumors. It has been shown that PIBF increases the number of human astrocytoma cells. In this work, we evaluated PIBF regulation by P 4 and the effects of PIBF on proliferation, migration, and invasion of U87 and U251 cells, both derived from human glioblastomas. PIBF mRNA expression was upregulated by P 4 (10 nM) from 12 to 24 h. Glioblastoma cells expressed two PIBF isoforms, 90 and 57 kDa. The content of the shorter isoform was increased by P 4 at 24 h, while progesterone receptor antagonist RU486 (10  μ M) blocked this effect. PIBF (100 ng/mL) increased the number of U87 cells on days 4 and 5 of treatment and induced cell proliferation on day 4. Wound-healing assays showed that PIBF increased the migration of U87 (12-48 h) and U251 (24 and 48 h) cells. Transwell invasion assays showed that PIBF augmented the number of invasive cells in both cell lines at 24 h. These data suggest that PIBF promotes proliferation, migration, and invasion of human glioblastoma cells., Competing Interests: The authors declare that they have no competing interests.

Preterm delivery (PTD) is one of the potent contributor of neonatal mortality and morbidity, and the underlying cause in some situation is elusive. This study attempts to delineate the association of deregulation in progesterone receptor (PR) pathway and deleterious immune responses in predisposing patients to PTD in Northeast India, a region with high rate of PTD cases. A total of 109 cases of PTD and 100 term delivery cases were enrolled with all clinical details. The PTD cases were stratified based on gestation age at delivery. The differential expression of PR and key downstream effectors and cytokines were evaluated for correlation with PTD susceptibility, gestational period, and pregnancy outcome. The results indicated a sharp downregulation in PR expression is associated with PTD susceptibility, lower gestational period and negative pregnancy outcome. The PR downstream effector PIBF was also found to be downregulated in PTD, and is associated with gestational period and negative pregnancy outcome. The downregulation of PR and PIBF expression was found to correlate with a predominant Th1 state with higher CD56+NK cell counts and pro-inflammatory burst lead by hyper TNF-α, NF-kB and IFNγ expression, and complicated by lower IL10 expression, contributing to PTD as well as negative pregnancy outcome in the PTD cases. TNF-α expression in placenta inversely correlated with placental PR expression. To conclude, deregulation in PR pathway is a hallmark of preterm delivery and negative pregnancy outcome. Differential expression of several markers such as PR, PIBF and TNF-α has prognostic significance, and hence is of clinical significance., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

MNSFβ, a ubiquitin-like protein, covalently binds to various target proteins including proapoptotic Bcl-G. During the course of isolation of MNSFβ-conjugating enzyme(s), we identified a novel target protein for MNSFβ. MALDI-TOF MS fingerprinting revealed that the MNSFβ-interacting protein is HSPA8 (heat shock 70-kDa protein 8). We observed that MNSFβ noncovalently binds to HSPA8 in the presence of ATP in vitro. Double knockdown of MNSFβ and HSPA8 strongly inhibited RANKL-induced osteoclastogenesis from Raw264.7 macrophage-like cells. The same treatment inhibited RANKL-induced ERK1/2 and p38 phosphorylation and TNFα production, suggesting that the association of MNSFβ with HSPA8 may promote RANKL-induced osteoclastogenesis. This is the first report that MNSFβ binds to a protein substrate via the noncovalent association and exerts biological effects.

Precise modulation of histone gene transcription is critical for cell cycle progression. As a direct substrate of Cyclin E/CDK2, nuclear protein ataxia-telangiectasia (NPAT) is a crucial factor in regulating histone transcription and cell cycle progression. Here we identified that Cpn10/HSPE, a 10-kDa heat shock protein, is a novel interacting partner of NPAT. A pool of Cpn10 is colocalized with NPAT foci during G1 and S phases in nuclei. Gain- and loss-of-function experiments unraveled an essential role of Cpn10 in histone transcription. A conserved DLFD motif within Cpn10 was critical for targeting NPAT and modulating histone transcription. More importantly, knockdown of Cpn10 disrupted the focus formation of both NPAT and FADD-like interleukin-1β-converting enzyme-associated huge protein without affecting Coilin-positive Cajal bodies. Finally, Cpn10 is important for S phase progression and cell proliferation. Taken together, our finding revealed a novel role of Cpn10 in the spatial regulation of NPAT signaling and disclosed a previously unappreciated link between the heat shock protein and histone transcription regulation., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Murine tumor models that closely reflect human diseases are important tools to investigate carcinogenesis and tumor immunity. The transgenic (tg) mouse strain tg(Grm1)EPv develops spontaneous melanoma due to ectopic overexpression of the metabotropic glutamate receptor 1 (Grm1) in melanocytes. In the present study, we characterized the immune status and functional properties of immune cells in tumor-bearing mice. Melanoma development was accompanied by a reduction in the percentages of CD4(+) T cells including regulatory T cells (Tregs) in CD45(+) leukocytes present in tumor tissue and draining lymph nodes (LNs). In contrast, the percentages of CD8(+) T cells were unchanged, and these cells showed an activated phenotype in tumor mice. Endogenous melanoma-associated antigen glycoprotein 100 (gp100)-specific CD8(+) T cells were not deleted during tumor development, as revealed by pentamer staining in the skin and draining LNs. They, however, were unresponsive to ex vivo gp100-peptide stimulation in late-stage tumor mice. Interestingly, immunosuppressive myeloid-derived suppressor cells (MDSCs) were recruited to tumor tissue with a preferential accumulation of granulocytic MDSC (grMDSCs) over monocytic MDSC (moMDSCs). Both subsets produced Arginase-1, inducible nitric oxide synthase (iNOS), and transforming growth factor-β and suppressed T-cell proliferation in vitro. In this work, we describe the immune status of a spontaneous melanoma mouse model that provides an interesting tool to develop future immunotherapeutical strategies.

Soon after the discovery of T suppressor cells by Gershon in 1970, it was demonstrated that one subpopulation of these lymphocytes induced by i.v. hapten injection suppresses contact sensitivity response mediated by effector CD4+ or CD8+ T cells in mice through the release of soluble T suppressor factor (TsF) that acts antigen specifically. Our experiments showed that biologically active TsF is a complex entity consisting of two subfactors, one antigen specific and other non-specific, produced by differently induced populations of cells. In following years, we found that the antigen-specific subfactor is a light chain of IgM antibody that is produced by B1a lymphocytes. However, the exact nature of non-specific part remained a mystery for about 30 years. Our current studies characterized TsF as regulatory miRNA-150 carried by T suppressor cell-derived exosomes that are antigen specific due to a surface coat of IgM antibody light chains produced by B1a cells. The present communication briefly summarizes our studies on TsF that led to discovery of regulating miRNA that acts antigen specifically to suppress immune response.

MNSFβ is a ubiquitously expressed member of the ubiquitin-like family that has been involved in various biological functions. Previous studies have demonstrated that MNSFβ covalently binds to various target proteins including Bcl-G, a proapoptotic protein. In this study, we purified a 115 kDa MNSFβ adduct from murine liver lysates by sequential chromatography on DEAE and anti-MNSFβ IgG-conjugated Sepharose in the presence of ATP. MALDI-TOF MS fingerprinting revealed that this MNSFβ adduct consists of an 8.5 kDa MNSFβ and 10-formyltetrahydrofolate dehydrogenase (FDH), an abundant enzyme of folate metabolism. Interestingly, MNSFβ preferably binds to cytosolic but not mitochondrial FDH. Fingerprinting analysis of the MNSFβ adduct demonstrate that MNSFβ conjugates to cytosolic FDH with a linkage between the C-terminal Gly74 and Lys72. The 115 kDa MNSFβ/FDH complex was not expressed in any of the tissues examined, indicating that this adduct formation is not ubiquitous. We found that MNSFβ/FDH complex formation was induced by dexamethasone in thymocytes. Double knockdown of MNSFβ and FDH strongly reduced dexamethasone-induced apoptosis. Collectively, MNSFβ/FDH complex formation may positively regulate apoptosis in thymocytes., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Defects in primary cilium biogenesis underlie the ciliopathies, a growing group of genetic disorders. We describe a whole-genome siRNA-based reverse genetics screen for defects in biogenesis and/or maintenance of the primary cilium, obtaining a global resource. We identify 112 candidate ciliogenesis and ciliopathy genes, including 44 components of the ubiquitin-proteasome system, 12 G-protein-coupled receptors, and 3 pre-mRNA processing factors (PRPF6, PRPF8 and PRPF31) mutated in autosomal dominant retinitis pigmentosa. The PRPFs localize to the connecting cilium, and PRPF8- and PRPF31-mutated cells have ciliary defects. Combining the screen with exome sequencing data identified recessive mutations in PIBF1, also known as CEP90, and C21orf2, also known as LRRC76, as causes of the ciliopathies Joubert and Jeune syndromes. Biochemical approaches place C21orf2 within key ciliopathy-associated protein modules, offering an explanation for the skeletal and retinal involvement observed in individuals with C21orf2 variants. Our global, unbiased approaches provide insights into ciliogenesis complexity and identify roles for unanticipated pathways in human genetic disease.

Objective: To explore progesterone-induced blocking factor (PIBF) expression in the placenta and blood of patients with severe preeclampsia and its relationship with immune tolerance imbalance., Methods: Forty-seven patients admitted between January and December, 2012 were enrolled in this study, including 25 patients with early-onset severe preeclampsia (EOPE) and 22 with late-onset severe preeclampsia (LOPE), with 25 women with normal pregnancy serving as control group. The antenatal blood and postpartum placenta were collected for immunohistochemical staining to detect PIBF expression in the placenta and for testing serum PIBF level using ELISA. Flow cytometry was used to detect the percentage of circulating Th1 and Th2 cells and the Th1/Th2 ratio was calculated., Results: PIBF was expressed in decidual cells, syncytiotrophoblasts and partial cytotrophablasts. The serum PIBF levels were 213.58 ± 44.93 ng/ml in EOPE group, 243.00∓61.19 ng/ml in LOPE group and 273.91 ± 48.57 ng/ml in control group. There were significant differences in serum PIBF, blood Th1/Th2 and placenta PIBF-IOD among the 3 groups (P<0.05). EOPE group had significantly lower serum PIBF, lower llacental PIBF quantity (PIBF-IOD) and higher blood Th1/Th2 than the control group (P<0.05). Serum PIBF in women with severe preeclampsia was positively correlated with placenta PIBF-IOD and negatively with blood Th1/Th2 ratio (P<0.05), but a negative correlation between serum PIBF and 24-hour urinary protein was found only in EOPE group (P<0.05)., Conclusion: The immune tolerance imbalance mediated by PIBF may participate in the pathogenesis of severe preeclampsia. PIBF, the immune suppressor secreted by lymphocytes of pregnancy women, is also a protective factor against severe preeclampsia, which is expected to be a new target in therapy.

Early pregnancy factor (EPF) is an immunosuppressant that promotes maternal immune system tolerance of the allogenic fetus. Little is known about localization of this factor in different tissues and nothing has been reported about localization in swine reproductive and placental tissues. We determined the concentration of EPF in serum of gilts and porcine placenta conditioned medium (PPCM). We also analyzed the expression of EPF in different reproductive tissues of pregnant gilts at 10, 30, 60 and 90 days of pregnancy. EPF concentration in serum and PPCM was determined by western blot and densitometry. EPF expression in reproductive tissue was assessed by immunohistochemistry. The highest concentration of EPF was observed at 30 days in serum and PPCM; the concentration was higher in PPCM than in serum at the stages we evaluated. All reproductive tissues from the gestational stages analyzed showed specific labeling of EPF, but this labeling did not appear in non-pregnant gilts. At 30 days pregnancy, the EPF expression in the ovary was predominantly in follicular lutein cells, probably owing to its function as a luteotrophic factor. In the oviduct, EPF was expressed in unciliated secretory epithelial cells and in the cilia of ciliated cells. In the placenta, EPF was expressed in the fetal portion (mesoderm chorioallantois and epithelium of endoderm). EPF acts as an autocrine and paracrine growth factor for the trophoblast during the peri-implantation period.

Monoclonal non-specific suppressor factor β (MNSFβ) is a ubiquitously expressed member of the ubiquitin-like family that is involved in various biological functions. Previous studies have demonstrated that MNSFβ covalently binds to intracellular pro-apoptotic protein Bcl-G and regulates apoptosis in macrophages. In this study, we demonstrate that MNSFβ negatively regulates T cell function. In murine T-helper type 2 clone, D10.G4.1 (D10) cells transfected with MNSFβ cDNA, CD3/CD28-induced ERK1/2 phosphorylation leading to IL-4 production was significantly inhibited. The formation of MNSFβ-Bcl-G complex was induced by the CD3/CD28 stimulation. Co-transfection with MNSFβ and Bcl-G greatly enhanced CD3/CD28-induced apoptosis in D10 cells. Similarly, co-over-expression of MNSFβ and Bcl-G caused a marked enhancement of apoptosis in purified splenic T cells. Interestingly, this MNSFβ adduct was also induced in T cells derived from DO11.10 mice stimulated with antigen. Collectively, CD3/CD28-inducible MNSFβ-Bcl-G complex may be involved in the regulation of T cell function and survival.

Pregnancy, parity, and circulating steroid hormone levels are associated with risk of breast cancer, but little is known about hormone concentrations during pregnancy and subsequent breast cancer risk. We evaluated early pregnancy (<140 days gestation) serum estradiol, estrone, progesterone, and testosterone and breast cancer risk in a nested case-control study in the Finnish Maternity Cohort. The cohort includes 98% of pregnancies registered in Finland since 1983. Individuals with samples collected in the first pregnancy leading to a live birth were eligible. Breast cancer cases (n = 1,199) were identified through linkage with the Finnish Cancer Registry; 2,281 matched controls were selected using incidence density sampling. ORs were calculated using conditional logistic regression. Hormone concentrations were not associated with breast cancer overall. Estradiol was positively associated with risk of breast cancer diagnosed age <40 [4th vs. 1st quartile OR 1.60 (1.07-2.39); Ptrend = 0.01], and inversely associated with breast cancer diagnosed at age ≥40 [4th vs. 1st quartile OR 0.71 (0.51-1.00); Ptrend = 0.02]. Elevated concentrations of the steroid hormones were associated with increased risk of estrogen receptor (ER)- and progesterone receptor (PR)-negative tumors in women age <40 at diagnosis. We observed no association between steroid hormones and ER(+)/PR(+) disease. These data suggest a positive association between high concentrations of early pregnancy steroid hormones and risk of ER(-)/PR(-) breast cancer in women diagnosed age <40, and an inverse association for overall breast cancer diagnosed age ≥40. Further research on pregnancy hormones and risk of steroid receptor-negative cancers is needed to further characterize this association., (©2014 American Association for Cancer Research.)

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., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

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.

Problem: Progesterone induced blocking factor (PIBF) is required for successful pregnancy. Alternative splicing produces PIBF isoforms with different functions. The full-length (90 kDa) PIBF is involved in cell cycle regulation, whereas smaller secreted forms act as cytokines. We aim to examine the PIBF exon pattern and protein isoform profile in normal and failed murine pregnancies., Method of Study: Pregnant Balb/c mice were killed on gestation days 12-14 or 17-19. Normal and resorbed fetuses, placentae, and uterine tissue were used for RNA and protein analysis with RT-PCR and Western blot, respectively., Results: Late pregnancy and resorption were associated with lower expression of the N-terminal exons, together with significantly reduced production of the full-length protein., Conclusion: Reduced production of the full-length PIBF protein might result in disturbed cell cycle regulation and dysregulated trophoblast invasion, while the absence of PIBF isoforms containing exon 2-4 coded sequences might lead to the loss of local immunosuppression., (© 2013 John Wiley & Sons Ltd.)

Receptor-ligand binding is an essential interaction for biological function. Oxidative stress can modify receptors and/or membrane lipid dynamics, thus altering cell physiological functions. The aim of this study is to analyze how oxidative stress may alter receptor-ligand binding and lipid domain distribution in the case of progesterone-induced blocking factor/progesterone-induced blocking factor-receptor. For membrane fluidity regionalization analysis of MEC-1 lymphocytes, two-photon microscopy was used in individual living cells. Lymphocytes were also double stained with AlexaFluor647/progesterone-induced blocking factor and Laurdan to evaluate -induced blocking factor/progesterone-induced blocking factor-receptor distribution in the different membrane domains, under oxidative stress. A new procedure has been developed which quantitatively analyzes the regionalization of a membrane receptor among the lipid domains of different fluidity in the plasma membrane. We have been able to establish a new tool which detects and evaluates lipid raft clustering from two-photon microscopy images of individual living cells. We show that binding of progesterone-induced blocking factor to progesterone-induced blocking factor-receptor causes a rigidification of plasma membrane which is related to an increase of lipid raft clustering. However, this clustering is inhibited under oxidative stress conditions. In conclusion, oxidative stress decreases membrane fluidity, impairs receptor-ligand binding and reduces lipid raft clustering., (© 2013.)

MNSFbeta (Monoclonal nonspecific suppressor factor beta) is a natural immunosuppressive factor which has been reported to be involved in various biological processes, such as immune responses, cell division, stress response, cell apoptosis, and nuclear transport. However, study on porcine MNSFbeta has been rarely reported. In this study, the full-length sequence of porcine MNSFbeta (GenBank accession number: KF77642500) was predicted in silicon and its cDNA sequence was obtained through RT-PCR from porcine spleen. The nucleic acid and protein sequences were analyzed. Then, the gene was subcloned into pEGFP-C1 to construct a recombinant plasmid pEGFP-MNSFbeta which was transfected into swine umbilical vein endothelial cells (SUVECs) using Lipofectamine 2000. The expression of GFP was detected by fluorescence microscopy, Western blot, and laser confocal fluorescence microscopy. The spatial expression patterns of porcine MNSFbeta were detected by real-time qPCR. Results showed that the full length of porcine MNSFbeta was 402 bp encoding 133 amino acids with only one exon. Bioinformatics analysis showed that porcine MNSFbeta protein was a stable protein consisting of a ubiquitin-like domain fused to the ribosomal protein S30 with no signal peptide. The analyses of homology and phylogenetic tree of porcine MNSFbeta and its homologs in other 18 species showed that the identities of MNSFbeta protein sequence were higher than 91% among different species and the evolutionary distance was less than 0.05. It indicates that MNSFbeta is highly conserved in the process of evolution. Fluorescence signal showed that the fusion protein GFP-MNSFbeta was successfully expressed in SUVECs which was then confirmed by Western blot. Laser confocal fluorescence microscopy showed that MNSFbeta was expressed in both nucleus and cytoplasm. Analysis of spatial expression patterns showed that procine MNSFbeta was widely expressed in immune tissues, but not in lung, suggesting that MNSFbeta may play an important role in immune response.

Invasiveness is a common feature of trophoblast and tumors; however, while tumor invasion is uncontrolled, trophoblast invasion is strictly regulated. Both trophoblast and tumor cells express high levels of the immunomodulatory progesterone-induced blocking factor (PIBF), therefore, we aimed to test the possibility that PIBF might be involved in invasion. To this aim, we used PIBF-silenced or PIBF-treated trophoblast (HTR8/Svneo, and primary trophoblast) and tumor (HT-1080, A549, HCT116, PC3) cell lines. Silencing of PIBF increased invasiveness as well as MMP-2,-9 secretion of HTR8/SVneo, and decreased those of HT-1080 cells. PIBF induced immediate STAT6 activation in both cell lines. Silencing of IL-4Rα abrogated all the above effects of PIBF, suggesting that invasion-related signaling by PIBF is initiated through the IL-4Rα/PIBF-receptor complex. In HTR-8/SVneo, PIBF induced fast, but transient Akt and ERK phosphorylation, whereas in tumor cells, PIBF triggered sustained Akt, ERK, and late STAT3 activation. The late signaling events might be due to indirect action of PIBF. PIBF induced the expression of EGF and HB-EGF in HT-1080 cells. The STAT3-activating effect of PIBF was reduced in HB-EGF-deficient HT-1080 cells, suggesting that PIBF-induced HB-EGF contributes to late STAT3 activation. PIBF binds to the promoters of IL-6, EGF, and HB-EGF; however, the protein profile of the protein/DNA complex is different in the two cell lines. We conclude that in tumor cells, PIBF induces proteins, which activate invasion signaling, while-based on our previous data-PIBF might control trophoblast invasion by suppressing proinvasive genes.

Objectives: We sought to investigate the expression of cells with immunosuppressive/protumorigenic phenotypes in oral lichen planus (OLP), such as M2-tumor-associated macrophages (TAM2), myeloid-derived suppressive cells (MDSCs), and regulatory T cells (Tregs) in association with clinical parameters., Materials and Methods: Cases of hyperkeratotic (HK)-OLP (n = 23) and erosive (E)-OLP (n = 26) were immunohistochemically stained to determine the percentages of CD163-TAM2, CD80-MDSCs, and FOXP3-Tregs of proinflammatory CD121a-Th17, CD4 and CD8 lymphocytes, and of cells positive for nuclear factor kappa B (NF-κB) and transforming growth factor beta. Clinical parameters included symptoms, treatment approach, treatment response, and others., Results: The inflammatory infiltrate in HK-OLP and E-OLP contained immunosuppressive cells; however, their pattern of expression was compatible with a proinflammatory response [membranous CD163-TAM2 staining (not extracellular), CD80+ lymphocytes (not macrophages), and a few Tregs]. The presence of CD4+, CD8+, and CD121a+ T lymphocytes was extensive. TAM2 were more frequent in E-OLP than in HK-OLP (P = 0.017). A higher frequency of CD80+ lymphocytes was associated with partial to no response to treatment (P = 0.028). Nuclear expression of NF-κB in the inflammatory cells was absent., Conclusions: The pattern of expression of the immunosuppressive cells, together with numerous CD4+, CD8+, and Th17-CD121a+ lymphocytes, suggest an extensive proinflammatory response rather than an immunosuppressive/protumorigenic response., Clinical Relevance: The frequency of selective types of inflammatory cells calls for individual profile analyses of inflammatory infiltrates and individually adjusted treatment.

Monoclonal non-specific suppressor factor β (MNSFβ) is a ubiquitously expressed member of the ubiquitin-like family that is involved in various biological functions. Previous studies have demonstrated that MNSFβ covalently binds to intracellular pro-apoptotic protein Bcl-G and regulates the extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) cascade in the mouse macrophage cell line Raw264.7. In this study, we demonstrate that MNSFβ promotes lipopolysaccharide (LPS)/interferon γ (IFNγ)-induced apoptosis of Raw264.7 macrophages. In Raw264.7 cells treated with MNSFβ small interfering RNA (siRNA), LPS/IFNγ- or NO donor S-nitrosoglutathione-induced apoptosis was inhibited. siRNA-mediated knockdown of MNSFβ did not affect inducible nitric-oxide synthase (iNOS) expression in LPS/IFNγ-stimulated Raw264.7 cells. Conversely, co-transfection with MNSFβ and Bcl-G greatly enhanced LPS/IFNγ- induced apoptosis in Raw264.7 cells, accompanied by increased expression of p53 and decreased Cox-2 activity. Unlike co-transfection with wild-type MNSFβ, co-transfection of a mutant MNSFβ (G74A) and Bcl-G did not result in enhancement of LPS/IFNγ-induced apoptosis. Co-over-expression of MNSFβ and Bcl-G reduced S-nitrosoglutathione-induced ERK1/2 phosphorylation. Furthermore, electrophoretic mobility shift assay experiments revealed that MNSFβ down-regulates the ERK/activator protein 1 (AP-1) signaling cascade which leads to Cox-2 activation. We also observed that MNSFβ-Bcl-G promotes LPS/IFNγ-induced apoptosis of mouse peritoneal macrophages, together with a decrease in Cox-2 expression. Taken together, our data indicate an apoptosis-enhancing effect of MNSFβ-Bcl-G is due in part to down-regulation of Cox-2 activation in macrophages., (© 2013 The Authors Journal compilation © 2013 FEBS.)

Background and Purpose: Ghrelin, a stomach-derived hormone, stimulates growth hormone secretion and appetite, and inhibits excessive inflammatory response. Plasma ghrelin might affect the inflammatory response to stressful surgical interventions. The aim of this study was to investigate the relationship between serial changes in plasma ghrelin concentrations and the postoperative clinical course after esophagectomy., Methods: The prospective cohort study subjects were 20 patients with esophageal cancer, who underwent esophagectomy with gastric tube reconstruction. Blood samples were taken six times perioperatively during the course of esophagectomy., Results: The plasma ghrelin level decreased to 33 % (range 15-90 %) on postoperative day (POD) 1, relative to the preoperative level, then recovered to about 50 % by POD 3-10. The duration of systemic inflammatory response syndrome (SIRS) was significantly longer in patients with a marked ghrelin reduction to <33 % on POD 1, than in those with less marked reduction of ≥ 33 % (6.1 ± 1.3 vs. 2.1 ± 0.6 days, P = 0.019). On POD 1, the only inflammatory marker that correlated with the duration of SIRS was the % ghrelin, whereas C-reactive protein, leukocyte count, and IL-6 did not., Conclusion: An early postoperative drop in plasma ghrelin correlated with prolonged SIRS after esophagectomy. Thus, the supplementation of low plasma ghrelin may help minimize excess inflammatory response in these patients.

Background: Measles virus (MV) causes T cell suppression by interference with phosphatidylinositol-3-kinase (PI3K) activation. We previously found that this interference affected the activity of splice regulatory proteins and a T cell inhibitory protein isoform was produced from an alternatively spliced pre-mRNA., Hypothesis: Differentially regulated and alternatively splice variant transcripts accumulating in response to PI3K abrogation in T cells potentially encode proteins involved in T cell silencing., Methods: To test this hypothesis at the cellular level, we performed a Human Exon 1.0 ST Array on RNAs isolated from T cells stimulated only or stimulated after PI3K inhibition. We developed a simple algorithm based on a splicing index to detect genes that undergo alternative splicing (AS) or are differentially regulated (RG) upon T cell suppression., Results: Applying our algorithm to the data, 9% of the genes were assigned as AS, while only 3% were attributed to RG. Though there are overlaps, AS and RG genes differed with regard to functional regulation, and were found to be enriched in different functional groups. AS genes targeted extracellular matrix (ECM)-receptor interaction and focal adhesion pathways, while RG genes were mainly enriched in cytokine-receptor interaction and Jak-STAT. When combined, AS/RG dependent alterations targeted pathways essential for T cell receptor signaling, cytoskeletal dynamics and cell cycle entry., Conclusions: PI3K abrogation interferes with key T cell activation processes through both differential expression and alternative splicing, which together actively contribute to T cell suppression.

Background: Although melanoma can elicit robust tumor antigen-specific immune responses, advanced melanoma is associated with immune tolerance. We have previously described several mechanisms of melanoma-induced immunosuppression, including the skewing of the immune response towards a Th2 cytokine profile and the induction of regulatory T cells. Since dendritic cells (DCs) are potentially important players that can direct other cells of the immune system towards a cytotoxic, humoral, or regulatory phenotype, we hypothesized that melanoma-produced factors directly affect the maturation and function of DCs, influencing the nature and magnitude of the resulting immune response., Materials and Methods: To test this hypothesis, immature myeloid-derived DCs (mdDCs) were derived with cytokines from CD14+ peripheral blood mononuclear cells (PBMCs) and exposed to 20% melanoma-conditioned media (MCM). After 2 d, the expression of maturation markers and the function of these mdDCs, measured by cytokine production, the amount of endocytosis, expression of the inhibitory molecule indoleamine 2,3-dioxygenase (IDO), and the ability to stimulate T cells were determined., Results: We found that incubation with MCM did not inhibit the expression of maturation markers or IDO, the production of cytokines, the amount of antigen uptake, or the ability to induce T cell proliferation in mixed-lymphocyte reactions by mdDC., Conclusions: These results suggest that the immunosuppressive effects of melanoma-produced factors are independent of directly measurable changes in mdDC function or maturation in vitro., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Background & Aims: Patients with autoimmune hepatitis (AIH) have reduced numbers and function of CD4+CD25(high)FOXP3+ T regulatory cells (Tregs). Tregs can be generated from CD25⁻ (ngTreg) cells, which suppress the immune response less efficiently than Tregs. We investigated whether their differentiation into T-helper (Th)17 cells, an effector subset that has the same CD4+ progenitors as Tregs, accounts for the reduced suppressive functions of ngTregs. We investigated whether blocking interleukin (IL)-17 increased the immunosuppressive activity of Tregs., Methods: ngTregs were generated from 36 patients with AIH and 23 healthy subjects (controls). During Treg differentiation, expression of IL-17 was inhibited by physical removal of IL-17-secreting cells, exposure to recombinant transforming growth factor β or neutralizing antibodies against IL-6 and IL-1β (to promote differentiation of ngTregs vs Th17 cells), small inhibitory RNAs specific for the Th17 transcription factor RORC, or a combination of all these approaches., Results: ngTregs from patients with AIH contained greater proportions of IL-17+ and RORC+ cells than Tregs from controls. All approaches to inhibit IL-17 increased expression of FOXP3 by ngTregs and their suppressive functions. Inhibition of IL-17 led to development of ngTregs that were phenotypically stable and did not acquire proinflammatory properties after exposure to IL-6 and IL-1β., Conclusions: Blocking Th17 allows ngTregs to differentiate into functionally stable immune inhibitory cells; this approach might be developed for therapy of patients with AIH., (Copyright © 2012 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Post-translational modification by monoclonal nonspecific suppressor factor β (MNSFβ) has been involved in the regulation of a variety of cellular processes. Previous studies have demonstrated that MNSFβ covalently binds to the intracellular pro-apoptotic protein Bcl-G and regulates TLR-4-mediated signal transduction. Recently, we found that MNSFβ also covalently conjugates to endophilin II, a member of the endophilin A family, and inhibits the signal pathway upstream of IKK activation, but not downstream of TLR-2 signaling. In this study, we further examined the mechanism of action of MNSFβ in TLR-2-mediated signal transduction in macrophage-like cell line Raw264.7 cells. Although MNSFβ siRNA enhanced Pam(3)CDK(4) (TLR-2-specific ligand)-stimulated TNFα production, Bcl-G siRNA did not affect. MNSFβ cDNA inhibited the Pam(3)CDK(4)-stimulated TNFα production. High-molecular weight (130 kDa) MNSFβ-adduct was induced in Pam(3)CDK(4)-stimulated Raw264.7 cells. This MNSFβ-adduct was not induced by LPS, indicative of the specificity of TLR-2-mediated signal transduction. Similar observations were seen in BALB/c peritoneal macrophages. Interestingly, 40-kDa MNSFβ-adduct was tyrosine phosphorylated by Pam(3)CDK(4) stimulation. Collectively, novel MNSFβ-adducts may regulate TLR-2 signaling pathway in macrophages.

Dogs with naturally occurring cancer represent an important large animal model for drug development and testing novel immunotherapies. However, poorly defined immunophenotypes of canine leukocytes have limited the study of tumor immunology in dogs. The accumulation of myeloid derived suppressor cells (MDSCs) is known to be a key mechanism of immune suppression in tumor-bearing mice and in human patients. We sought to identify MDSCs in the blood of dogs with cancer. Peripheral blood mononuclear cells (PBMCs) from dogs with advanced or early stage cancer and from age-matched healthy controls were analyzed by flow cytometry and microscopy. Suppressive function was tested in T cell proliferation and cytokine elaboration assays. Semi-quantitative RT-PCR was used to identify potential mechanisms responsible for immunosuppression. PBMCs from dogs with advanced or metastatic cancer exhibited a significantly higher percentage of CD11b(+)CD14(-)MHCII(-) cells compared to dogs diagnosed with early stage non-metastatic tumors and healthy dogs. These CD11b(+) CD14(-)MHCII(-) cells constitute a subpopulation of activated granulocytes that co-purify with PBMCs, display polymorphonuclear granulocyte morphology, and demonstrate a potent ability to suppress proliferation and IFN-γ production in T cells from normal and tumor-bearing donors. Furthermore, these cells expressed hallmark suppressive factors of human MDSC including ARG1, iNOS2, TGF-β and IL-10. In summary our data demonstrate that MDSCs accumulate in the blood of dogs with advanced cancer and can be measured using this three-marker immunophenotype, thereby enabling prospective studies that can monitor MDSC burden.

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., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)