Aberrations in the progesterone pathway and the Th1/Th2 cytokine dichotomy - An evaluation of RPL predisposition in the northeast Indian population.

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.

Quercetin and HSC70 coregulate the anti-inflammatory action of the ubiquitin-like protein MNSFß.

BACKGROUND: Quercetin is a flavonol that modifies many cellular processes. Monoclonal nonspecific suppressor factor ß 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 MNSFß 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 MNSFß. 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 MNSFß 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 MNSFß and the proapoptotic protein Bcl-G. Moreover, MNSFß is implicated in TLR4/MyD88 signaling but not in TLR3 signaling. CONCLUSIONS: HSC70 is an important chaperone that facilitates the stabilization of MNSFß. Quercetin may negatively control the function of MNSFß by regulating the action of the molecular chaperone HSC70. MNSFß mediates TLR4/Myd88 signaling but not TLR3 signaling.

MNSFß regulates placental development by conjugating IGF2BP2 to enhance trophoblast cell invasiveness.

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 (MNSFß) is an immunosuppressive factor. Its conventional knockout in mice induced embryonic lethality, whereas the underlying mechanism of MNSFß in regulating placentation and pregnancy maintenance remains to be elucidated. METHODS: Trophoblast-specific knockout of MNSFß 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 MNSFß and insulin-like growth factor 2 mRNA-binding protein 2 (IGF2BP2) at the foeto-maternal interface. The interaction and expression of MNSFß, 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 MNSFß 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 MNSFß with IGF2BP2 and thus the stabilization of IGF2BP2 essentially mediated the invasion-promoting effect of MNSFß. In the placentas from MNSFß-deficient mice and severe preeclamptic (PE) patients, downregulation of MNSFß was evidently associated with the repressed IGF2BP2 expression. CONCLUSIONS: The findings reveal the crucial role of MNSFß in governing the trophoblast invasion and therefore foetal development, and add novel hints to reveal the placental pathology of PE.

Decreased PIBF1/IL6/p-STAT3 during the mid-secretory phase inhibits human endometrial stromal cell proliferation and decidualization.

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.

Progesterone-induced blocking factor (PIBF) influences the expression of membrane progesterone receptors (mPRs) on peripheral CD4+ T lymphocyte cells in normal fertile females.

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 mPRß 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.

Metabolic support of tumour-infiltrating regulatory T cells by lactic acid.

Regulatory T (Treg) 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 cells1,2. Tumour cells show deregulated metabolism, leading to a metabolite-depleted, hypoxic and acidic TME3, which places infiltrating effector T cells in competition with the tumour for metabolites and impairs their function4-6. At the same time, Treg cells maintain a strong suppression of effector T cells within the TME7,8. As previous studies suggested that Treg cells possess a distinct metabolic profile from effector T cells9-11, we hypothesized that the altered metabolic landscape of the TME and increased activity of intratumoral Treg cells are linked. Here we show that Treg 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 Treg cells in vitro. Treg cells instead upregulate pathways involved in the metabolism of the glycolytic by-product lactic acid. Treg 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 Treg cells reveals that lactate uptake is dispensable for the function of peripheral Treg cells but required intratumorally, resulting in slowed tumour growth and an increased response to immunotherapy. Thus, Treg 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.

MNSFß Promotes the Proliferation and Migration of Human Extravillous Trophoblast Cells and the Villus Expression Level of MNSFß Is Decreased in Recurrent Miscarriage Patients.

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 (MNSFß) led to the early pregnancy failure in mice and the decidual MNSFß expression level in RM patients was significantly decreased, but the underlying molecular mechanism of the role that MNSFß played at the maternal-fetal interface remains unclear. Thus, in the present study, we determined effects of downregulated MNSFß expression on human EVT cell activities. METHODS: The MNSFß expression in first-trimester human decidual and placental villus tissues was detected, respectively, by immunofluorescence or immunohistochemical analyses. The MNSFß expression level in the immortalized first-trimester human EVT cell line HTR8/SVneo was downregulated by transfecting the small interfering RNA against MNSFß and upregulated by transfecting the recombinant pDsRed-MNSFß 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 MNSFß and RhoA in placental villi of RM patients and physically normal pregnant women (NP), were examined by Western blot analysis. RESULTS: MNSFß protein signals were observed in first-trimester human villus and extravillous trophoblast cells. The downregulated MNSFß 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 MNSFß expression in HTR8/SVneo cells represented the inverse effects. Furthermore, expression levels of MNSFß 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 MNSFß promotes proliferation and migration of human EVT cells, probably via the P53 signaling pathway, and the deficiency of MNSFß in placental villi might lead to early pregnancy loss by reducing proliferation and invasion activities of EVTs.

Comprehensive Analysis of Immunoinhibitors Identifies LGALS9 and TGFBR1 as Potential Prognostic Biomarkers for Pancreatic Cancer.

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.

PIBF1 suppresses the ATR/CHK1 signaling pathway and promotes proliferation and motility of triple-negative breast cancer cells.

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.

Overexpression of PGC-1α influences the mitochondrial unfolded protein response (mtUPR) induced by MPP+ in human SH-SY5Y neuroblastoma cells.

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.

Heat shock protein 60 negatively regulates the biological functions of ubiquitin-like protein MNSFß in macrophages.

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

The Role of Extracellular Vesicles and PIBF in Embryo-Maternal Immune-Interactions.

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.

The effect of the Progesterone-Induced Blocking Factor (PIBF) on E-cadherin expression, cell motility and invasion of primary tumour cell lines.

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.

Pregnancy-associated changes in expression of progesterone receptor and progesterone-induced blocking factor genes in bone marrow of ewes.

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.

Proliferative and Invasive Effects of Progesterone-Induced Blocking Factor in Human Glioblastoma Cells.

Progesterone-induced blocking factor (PIBF) is a progesterone (P4) 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 P4 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 P4 (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 P4 at 24 h, while progesterone receptor antagonist RU486 (10 µM) 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.

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

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

Preterm delivery and associated negative pregnancy outcome - A tale of faulty progesterone receptor signalling pathway and linked derailed immunomodulation: A study from Northeast India.

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.

Ubiquitin-like protein MNSFß noncovalently binds to molecular chaperone HSPA8 and regulates osteoclastogenesis.

MNSFß, a ubiquitin-like protein, covalently binds to various target proteins including proapoptotic Bcl-G. During the course of isolation of MNSFß-conjugating enzyme(s), we identified a novel target protein for MNSFß. MALDI-TOF MS fingerprinting revealed that the MNSFß-interacting protein is HSPA8 (heat shock 70-kDa protein 8). We observed that MNSFß noncovalently binds to HSPA8 in the presence of ATP in vitro. Double knockdown of MNSFß 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 MNSFß with HSPA8 may promote RANKL-induced osteoclastogenesis. This is the first report that MNSFß binds to a protein substrate via the noncovalent association and exerts biological effects.

An siRNA-based functional genomics screen for the identification of regulators of ciliogenesis and ciliopathy genes.

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.

Impaired gp100-Specific CD8(+) T-Cell Responses in the Presence of Myeloid-Derived Suppressor Cells in a Spontaneous Mouse Melanoma Model.

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-ß 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.