sPIF treatment results in global gene changes including T cell receptor signalling.

<p><b>(A)</b> Gene set network showing the relations between the significant gene sets after sPIF treatment of epithelial ectopic cells. Every node represents a gene set and every edge reflects the intersection between two sets. Node size reflects the number of genes in each gene set; node fill color reflects the p-value corrected for intersections with the color going from blue to red with decreasing p-value; node border reflects network prominence, how important a particular gene set is compared to others. Edge width reflects the relative size of the intersection between two gene sets. Edge direction, as indicated by the arrow heads, is towards to more significant gene set. Repeated arrows indicate subset relations. (<b>B)</b> Detailed analysis of the T cell receptor signaling pathways showing multiple up (red) and down (blue) regulated genes (6 samples: 3 sPIF treated and 3 control samples).</p

PreImplantation Factor in endometriosis: A potential role in inducing immune privilege for ectopic endometrium

<div><p>Endometriosis is a chronic inflammatory condition characterised by the growth of endometrial epithelial and stromal cells outside the uterine cavity. In addition to Sampson’s theory of retrograde menstruation, endometriosis pathogenesis is facilitated by a privileged inflammatory microenvironment, with T regulatory FoxP3⁺ expressing T cells (Tregs) being a significant factor. PreImplantation Factor (PIF) is a peptide essential for pregnancy recognition and development. An immune modulatory function of the synthetic PIF analog (sPIF) has been successfully confirmed in multiple animal models. We report that PIF is expressed in the epithelial ectopic cells in close proximity to FoxP3⁺ stromal cells. We provide evidence that PIF interacts with FoxP3⁺ cells and modulates cell viability, dependent on cell source and presence of inflammatory mediators. Our finding represent a novel PIF-based mechanism in endometriosis that has potential for novel therapeutics.</p></div

sPIF treatment results in global gene changes including T cell receptor signalling.

<p><b>(A)</b> Gene set network showing the relations between the significant gene sets after sPIF treatment of epithelial ectopic cells. Every node represents a gene set and every edge reflects the intersection between two sets. Node size reflects the number of genes in each gene set; node fill color reflects the p-value corrected for intersections with the color going from blue to red with decreasing p-value; node border reflects network prominence, how important a particular gene set is compared to others. Edge width reflects the relative size of the intersection between two gene sets. Edge direction, as indicated by the arrow heads, is towards to more significant gene set. Repeated arrows indicate subset relations. (<b>B)</b> Detailed analysis of the T cell receptor signaling pathways showing multiple up (red) and down (blue) regulated genes (6 samples: 3 sPIF treated and 3 control samples).</p

PIF re-expresses in epithelial ectopic cells and modulates cell viability.

<p><b>(A)</b> PIF positive cells were not detected in tissues derived from healthy patients (upper panel). PIF positive cells were detected in epithelial compartment of ectopic endometrial tissues (lower panel). (<b>B)</b> Semi-quantitative analysis of PIF staining. We detected increased number of PIF positive cells with high HSCORE in epithelial compartment of ectopic tissues only. (<b>C) and (D)</b> Diverse influence of sPIF on cell survival. (<b>C)</b> sPIF treatment of epithelial ectopic cells (EM ‘osis −) resulted in a significant decrease of cell viability. However, treatment of epithelial eutopic cells (EM E6/E7 --) resulted in a significant increase of cell viability. (<b>D)</b> The viability of ectopic (−) and eutopic (--) stromal cells did not show a significant change in response to sPIF treatment. *p<0.05; **; p<0.01; ***p<0.001. Scale bar 20 μm.</p

Insight into PreImplantation Factor (PIF*) Mechanism for Embryo Protection and Development: Target Oxidative Stress and Protein Misfolding (PDI and HSP) through Essential RIPK Binding Site

<div><p>Background</p><p>Endogenous PIF, upon which embryo development is dependent, is secreted only by viable mammalian embryos, and absent in non-viable ones. Synthetic PIF (sPIF) administration promotes singly cultured embryos development and protects against their demise caused by embryo-toxic serum. To identify and characterize critical sPIF-embryo protein interactions novel biochemical and bio-analytical methods were specifically devised.</p><p>Methods</p><p>FITC-PIF uptake/binding by cultured murine and equine embryos was examined and compared with scrambled FITC-PIF (control). Murine embryo (d10) lysates were fractionated by reversed-phase HPLC, fractions printed onto microarray slides and probed with Biotin-PIF, IDE and Kv1.3 antibodies, using fluorescence detection. sPIF-based affinity column was developed to extract and identify PIF-protein interactions from lysates using peptide mass spectrometry (LC/MS/MS). <i>In silico</i> evaluation examined binding of PIF to critical targets, using mutation analysis.</p><p>Results</p><p>PIF directly targets viable cultured embryos as compared with control peptide, which failed to bind. Multistep Biotin-PIF targets were confirmed by single-step PIF-affinity column based isolation. PIF binds protein disulfide isomerases a prolyl-4-hydroxylase β-subunit, (PDI, PDIA4, PDIA6-like) containing the antioxidant thioredoxin domain. PIF also binds protective heat shock proteins (70&90), co-chaperone, BAG-3. Remarkably, PIF targets a common RIPK site in PDI and HSP proteins. Further, single PIF amino acid mutation significantly reduced peptide-protein target bonding. PIF binds promiscuous tubulins, neuron backbones and ACTA-1,2 visceral proteins. Significant anti-IDE, while limited anti-Kv1.3b antibody-binding to Biotin-PIF positive lysates HPLC fractions were documented.</p><p>Conclusion</p><p>Collectively, data identifies PIF shared targets on PDI and HSP in the embryo. Such are known to play a critical role in protecting against oxidative stress and protein misfolding. PIF-affinity-column is a novel utilitarian method for small molecule targets direct identification. Data reveals and completes the understanding of mechanisms involved in PIF-induced autotrophic and protective effects on the embryo.</p></div

BeATMuSiC server predicted in silico mutagens disrupting the interface of the PIF docking models with several targets.

<p>BeATMuSiC server predicted in silico mutagens disrupting the interface of the PIF docking models with several targets.</p

PIF-affinity chromatography identified PIF targets.

<p>PIF-affinity chromatography identified PIF targets.</p

Biotin-PIF binds the F6 fraction in mouse embryo extracts.

<p>Biotin-PIF binds the F6 fraction in mouse embryo extracts.</p

Biotin-PIF binds significantly to anti-KV1.3b positive fractions.

<p>Averaged normalized intensity data is represented. The highest intensity was found in the A9 fraction. The frequency of the fraction showing positive reactivity is indicated above the intensity bar.</p

PepSite 2 server prediction of PIF residues participating in targets binding site.

<p>PepSite 2 server prediction of PIF residues participating in targets binding site.</p