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

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

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.

Identification of two novel pathogenic variants of PIBF1 by whole exome sequencing in a 2-year-old boy with Joubert syndrome.

BACKGROUND: Joubert syndrome (OMIM 213300) is an autosomal recessive disorder with gene heterogeneity. Causal genes and their variants have been identified by sequencing or other technologies for Joubert syndrome subtypes. CASE PRESENTATION: A two-year-old boy was diagnosed with Joubert syndrome by global development delay and molar tooth sign of mid-brain. Whole exome sequencing was performed to detect the causative gene variants in this individual, and the candidate pathogenic variants were verified by Sanger sequencing. We identified two pathogenic variants (NM_006346.2: c.1147delC and c.1054A > G) of PIBF1 in this Joubert syndrome individual, which is consistent with the mode of autosomal recessive inheritance. CONCLUSION: In this study, we identified two novel pathogenic variants in PIBF1 in a Joubert syndrome individual using whole exome sequencing, thereby expanding the PIBF1 pathogenic variant spectrum of Joubert syndrome.

A biallelic 36-bp insertion in PIBF1 is associated with Joubert syndrome.

Biallelic pathogenic variants in PIBF1 have been identified as one of the genetic etiologies of Joubert syndrome. We report a two-year-old girl with global developmental delay, facial dysmorphism, hypotonia, enlarged cystic kidneys, molar tooth sign, and thinning of corpus callosum. A novel homozygous 36-bp insertion in PIBF1 (c.1181_1182ins36) was identified by exome sequencing as the likely cause of her condition. This is the second publication demonstrating the cause and effect relationship between PIBF1 and Joubert syndrome.

Development of Phage-Based Antibody Fragment Reagents for Affinity Enrichment of Bacterial Immunoglobulin G Binding Proteins.

Disease and death caused by bacterial infections are global health problems. Effective bacterial strategies are required to promote survival and proliferation within a human host, and it is important to explore how this adaption occurs. However, the detection and quantification of bacterial virulence factors in complex biological samples are technically demanding challenges. These can be addressed by combining targeted affinity enrichment of antibodies with the sensitivity of liquid chromatography-selected reaction monitoring mass spectrometry (LC-SRM MS). However, many virulence factors have evolved properties that make specific detection by conventional antibodies difficult. We here present an antibody format that is particularly well suited for detection and analysis of immunoglobulin G (IgG)-binding virulence factors. As proof of concept, we have generated single chain fragment variable (scFv) antibodies that specifically target the IgG-binding surface proteins M1 and H of Streptococcus pyogenes. The binding ability of the developed scFv is demonstrated against both recombinant soluble protein M1 and H as well as the intact surface proteins on a wild-type S. pyogenes strain. Additionally, the capacity of the developed scFv antibodies to enrich their target proteins from both simple and complex backgrounds, thereby allowing for detection and quantification with LC-SRM MS, was demonstrated. We have established a workflow that allows for affinity enrichment of bacterial virulence factors.

Ubiquitin-like protein MNSFß negatively regulates T cell function and survival.

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

Progesterone-induced blocking factor differentially regulates trophoblast and tumor invasion by altering matrix metalloproteinase activity.

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.

[Cloning, eukaryotic expression and spatial expression patterns of porcine MNSFbeta].

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.

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.

Ubiquitin-like protein MNSFß regulates TLR-2-mediated signal transduction.

Post-translational modification by monoclonal nonspecific suppressor factor ß (MNSFß) has been involved in the regulation of a variety of cellular processes. Previous studies have demonstrated that MNSFß covalently binds to the intracellular pro-apoptotic protein Bcl-G and regulates TLR-4-mediated signal transduction. Recently, we found that MNSFß 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 MNSFß in TLR-2-mediated signal transduction in macrophage-like cell line Raw264.7 cells. Although MNSFß siRNA enhanced Pam(3)CDK(4) (TLR-2-specific ligand)-stimulated TNFα production, Bcl-G siRNA did not affect. MNSFß cDNA inhibited the Pam(3)CDK(4)-stimulated TNFα production. High-molecular weight (130 kDa) MNSFß-adduct was induced in Pam(3)CDK(4)-stimulated Raw264.7 cells. This MNSFß-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 MNSFß-adduct was tyrosine phosphorylated by Pam(3)CDK(4) stimulation. Collectively, novel MNSFß-adducts may regulate TLR-2 signaling pathway in macrophages.

High viral load and deregulation of the progesterone receptor signaling pathway: association with hepatitis E-related poor pregnancy outcome.

BACKGROUND & AIMS: Hepatitis E virus (HEV) infection is associated with high maternal and fetal mortalities. A prospective study was undertaken to evaluate the role of viral and host factors in HEV related pregnancy outcomes. METHODS: The study included HEV infected pregnancy cases; acute viral hepatitis (AVH), n=100 and fulminant hepatic failure (FHF), n=43, and healthy pregnancy cases, n=50. HEV genotypes and viremia were studied by nucleotide sequencing and real time PCR, respectively. Progesterone receptor (PR) gene mutations (PROGINS) were studied by PCR, PR expression at the mRNA and protein levels in the placenta were studied by semi-quantitative RT-PCR and immunohistochemistry, respectively. Progesterone induced blocking factor (PIBF) expression was studied by RT-PCR in blood. Serum interleukin-10 (IL-10) and interleukin-12 (IL-12) levels were assayed by ELISA. RESULTS: HEV viral load was significantly higher in FHF than AVH (p<0.001) and in cases with fetal mortality in AVH (p=0.001) and FHF (p=0.018). PROGINS were predominant in FHF compared to AVH (p=0.26) and showed reduced mRNA and protein expression. The risk of fetal mortality in AVH was two times higher (OR, 2.190; CI, 0.303-15.85) and maternal and fetal mortalities in FHF were 4-fold (OR, 4.0; CI, 0.363-44.113) increased in PROGINS carriers. PR and PIBF expression was lower in AVH and even lower in FHF compared to healthy controls. The higher IL-12/IL-10 ratio observed in FHF compared to other groups correlated with fetal mortality in AVH and FHF (p<0.001). CONCLUSIONS: In conclusion, reduced expression of PR and PIBF, a higher IL-12/IL-10 ratio, and a high viral load results in poor pregnancy outcome in Hepatitis E.

MNSFß Regulates TNFα Production by Interacting with RC3H1 in Human Macrophages, and Dysfunction of MNSFß in Decidual Macrophages Is Associated With Recurrent Pregnancy Loss.

Decidual macrophages (dMϕ) are the second largest population of leukocytes at the maternal-fetal interface and play critical roles in maintaining pregnancy. Our previous studies demonstrated the active involvement of monoclonal nonspecific suppressor factor-ß (MNSFß) in embryonic implantation and pregnancy success. MNSFß is a ubiquitously expressed ubiquitin-like protein that also exhibits immune regulatory potential, but its function in human dMϕ remains unknown. Here, we observed that the proportion of CD11chigh (CD11cHI) dMϕ was significantly increased in dMϕ derived from patients with recurrent pregnancy loss (RPL dMϕ) compared to those derived from normal pregnant women (Control dMϕ). The production of MNSFß and TNFα by RPL dMϕ was also significantly increased compared to that by Control dMϕ. Conditioned medium from RPL dMϕ exerted an inhibitory effect on the invasiveness of human trophoblastic HTR8/SVneo cells, and this effect could be partially reversed by a neutralizing antibody against TNFα. Bioinformatics analysis indicated a potential interaction between MNSFß and RC3H1, a suppressor of TNFα transcription. Immunoprecipitation experiments with human Mϕ differentiated from the human monocyte cell line Thp1 (Thp1-derived Mϕ) proved the binding of MNSFß to RC3H1. Specific knockdown of MNSFß in Thp1-derived Mϕ led to a marked decrease in TNFα production, which could be reversed by inhibiting RC3H1 expression. Interestingly, a significant decrease in the protein level of RC3H1 was observed in RPL dMϕ. Together, our findings indicate that aberrantly increased MNSFß expression in dMϕ may promote TNFα production via its interaction with RC3H1, and these phenomena could result in the disruption of the immune balance at the maternal-fetal interface and thus pregnancy loss.

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.

Structure and DNA binding of the human Rtf1 Plus3 domain.

The yeast Paf1 complex consists of Paf1, Rtf1, Cdc73, Ctr9, and Leo1 and regulates histone H2B ubiquitination, histone H3 methylation, RNA polymerase II carboxy-terminal domain (CTD) Ser2 phosphorylation, and RNA 3' end processing. We provide structural insight into the Paf1 complex with the NMR structure of the conserved and functionally important Plus3 domain of human Rtf1. A predominantly beta-stranded subdomain displays structural similarity to Dicer/Argonaute PAZ domains and to Tudor domains. We further demonstrate that the highly basic Rtf1 Plus3 domain can interact in vitro with single-stranded DNA via residues on the rim of the beta sheet, reminiscent of siRNA binding by PAZ domains, but did not detect binding to double-stranded DNA or RNA. We discuss the potential role of Rtf1 Plus3 ssDNA binding during transcription elongation.

A recurrent de novo HSPD1 variant is associated with hypomyelinating leukodystrophy.

Standardization of the use of next-generation sequencing for the diagnosis of rare neurological disorders has made it possible to detect potential disease-causing genetic variations, including de novo variants. However, the lack of a clear pathogenic relevance of gene variants poses a critical limitation for translating this genetic information into clinical practice, increasing the necessity to perform functional assays. Genetic screening is currently recommended in the guidelines for diagnosis of hypomyelinating leukodystrophies (HLDs). HLDs represent a group of rare heterogeneous disorders that interfere with the myelination of the neurons in the central nervous system. One of the HLD-related genes is HSPD1, encoding the mitochondrial chaperone heat shock protein 60 (HSP60), which functions as folding machinery for the mitochondrial proteins imported into the mitochondrial matrix space. Disease-causing HSPD1 variants have been associated with an autosomal recessive form of fatal hypomyelinating leukodystrophy (HLD4, MitCHAP60 disease; MIM #612233) and an autosomal dominant form of spastic paraplegia, type 13 (SPG13; MIM #605280). In 2018, a de novo HSPD1 variant was reported in a patient with HLD. Here, we present another case carrying the same heterozygous de novo variation in the HSPD1 gene (c.139T > G, p.Leu47Val) associated with an HLD phenotype. Our molecular studies show that the variant HSP60 protein is stably present in the patient's fibroblasts, and functional assays demonstrate that the variant protein lacks in vivo function, thus confirming its disease association. We conclude that de novo variations of the HSPD1 gene should be considered as potentially disease-causing in the diagnosis and pathogenesis of the HLDs.

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.

Molecular analysis of the hotspot of recombination in the murine major histocompatibility complex.

Biological and serological assays have been used to define four subregions for the I region of the major histocompatibility complex (MHC) in the order I-A, I-B, I-J, and I-E. The I-J subregion presumably encodes the I-J polypeptide of the elusive T-cell suppressor factors. Restriction enzyme site polymorphisms and DNA sequence analyses of the I region from four recombinant mouse strains were used to localize the putative I-B and I-J subregions to a 1.0-kilobase (kb) region within the E beta gene. Sequencing this region from E beta clones derived from the two mouse strains: B10.A(3R), I-Jb and B10.A(5R), I-Jk initially used to define the I-J subregion revealed that these regions are identical, hence the distinct I-Jb and I-Jk molecules cannot be encoded by this DNA. In addition, the DNA sequence data also refute the earlier mapping of the I-B subregion. Analysis of the DNA sequences of three parental and four I region recombinants reveals that the recombinant events in three of the recombinant strains occurred within a 1-kb region of DNA, supporting the proposition that a hotspot for recombination exists in the I region. The only striking feature of this hotspot is a tetramer repeat (AGGC)n that shows 80 percent homology to the minisatellite sequence which may facilitate recombination in human chromosomes.

Bradykinin-induced blood-tumor barrier opening is mediated by tumor necrosis factor-alpha.

Bradykinin has been shown to increase the permeability of blood-tumor barrier (BTB) selectively. This study was performed to determine whether tumor necrosis factor-alpha (TNF-alpha) was involved in the regulation of this biological process. We found that the levels of TNF-alpha mRNA and heat shock factor-1 (HSF1) protein in C6 cells were markedly up-regulated by bradykinin via real-time RT-PCR and Western blot methods. And the most obvious increase of HSF1 protein and TNF-alpha mRNA in C6 cells were observed at 5 min and 10 min of bradykinin perfusion, respectively. In addition, the radioactivity of TNF-alpha in C6 cells' culture fluid also mostly increased at 15 min of bradykinin perfusion. And the Evans blue content of brain tumor tissues in rats and the concentration of TNF-alpha reached the maximum at 15 min of bradykinin perfusion. Our results suggested that the bradykinin-mediated BTB permeability increase is due to accelerated release of TNF-alpha, which could cause the increase of BTB permeability by promoting to the release HSF1 from neurospongioma cells.

Support for the hypothesis that successful immunotherapy of various cancers can be achieved by inhibiting a progesterone associated immunomodulatory protein.

A hypothesis was proposed that cancer cells may utilize a pre-existing mechanism that pregnant mammals use to avoid natural killer cell immune surveillance of the fetus. The hypothesis suggested that those cancer cells that are able to proliferate may have found a way to cause the expression of the immunomodulatory protein known as the progesterone induced blocking factor (PIBF). The cancer cells could find an alternate pathway to make this protein that does not require progesterone secretion, or the cancer cells may actually utilize progesterone and thus make PIBF in a similar fashion to normal pregnancy. If the former mechanism was operational, then one could develop monoclonal antibody type therapy directed to this unique protein not needed for normal body functions. However, if the latter pathway involving progesterone secretion is operational, then there would be drugs already on the market, e.g., the progesterone receptor antagonist mifepristone that could be used to treat these cancers. In vitro data has shown that 100% of human leukemia cell lines express mRNA for the PIBF protein. Some leukemia cell lines have been found that actually express the PIBF protein. In fact adding progesterone to the culture media upregulated PIBF protein expression and mifepristone inhibited it. Controlled studies in various murine spontaneous cancers not known to be associated with progesterone receptors showed increased length and quality of life following mifepristone therapy. Anecdotal improvement in advanced widely metastatic human cancers has also been found. Thus there is now experimental data to support this hypothesis and a new door to a completely different type of cancer therapy has been opened.

Human trophoblast cells express the immunomodulator progesterone-induced blocking factor.

Progesterone-induced blocking factor (PIBF) is an immunomoduatory factor with anti-abortive properties. In this study, we present evidence that PIBF is synthesized in the human placenta and determine its cellular source. Expression of PIBF was analysed with polyclonal rabbit anti-human PIBF antibodies against recombinant N-terminal 48kDa PIBF in first trimester and term placental tissues and in the choriocarcinoma cell line JAR by means of immunohistochemistry, confocal laser scanning microscopy of double immunofluorescence labelling, and Western blotting; RT-PCR was performed for analysis of PIBF mRNA in isolated trophoblast cells. PIBF protein is present in human first trimester and term placenta. Double immunofluorescence labelling localised PIBF to the extravillous cytotrophoblast. PIBF is also expressed heterogeneously by syncytiotrophoblast and part of the villous cytotrophoblast. Full-length PIBF mRNA encoded by exons 1-18 is present in isolated first trimester and term villous trophoblast and in the choriocarcinoma cell line JAR. The corresponding 90kDa protein is expressed by JAR cells, first trimester and term villous trophoblast cells. In addition, these cells express PIBF proteins of 50 and 34kDa. Trophoblast is a source of PIBF; its tissue distribution suggests a role both in systemic and local (decidual) immunoregulation.