The influence of heparin on histone induced aggregation of low-density lipoprotein.

<p><b>A:</b> Titration of histones into LDL (final concentration 1.25 mg/ml by protein) was carried out for 1 hr in PBS at 21°C, and protein aggregation measured by spectrophotometric absorbance at 680 nm. <b>B:</b> Rapid aggregation of LDL was observed over the first 180 seconds of incubation of LDL (75 µl of 1.33 mg/ml by protein in PBS) with histones (25 µl of 0, 0.5, 1 and 2 mg/ml in PBS). <b>C:</b> Inclusion of varying concentrations of low molecular weight heparin (LMWH) and unfractionated heparin (UFH) with LDL (1.25 mg/ml) and histone (0.25 mg/ml) caused a general decrease in protein aggregation, relative to uninhibited incubations. At 0.1 mg/ml, UFH was not effective. <b>D:</b> LDL (100 µl of 0.5 mg/ml in PBS) was incubated with 100 µl of heparin agarose slurry, to which 100 µl of 0, 1, 2 or 4 mg/ml calf thymus histones in PBS had previously been bound. The apolipoprotein B content in the unbound (FT) and SDS-eluted (E) fractions was determined by SDS-PAGE (inset) and quantified by densitometry. This indicated that LDL was selectively pulled down onto histone-charged heparin agarose. This experiment was performed three times with similar results.</p

The influence of heparin on histone induced aggregation of high-density lipoprotein.

<p>A: Titration of histones into HDL (final concentration 1 mg/ml by protein) was carried out for 1 hr in PBS at 21°C, and protein aggregation measured by spectrophotometric absorbance at 680 nm. B: Aggregation of HDL was observed over the first 180 seconds of incubation of HDL (50 µl of 1 mg/ml by protein in PBS) with histones (50 µl of 0, 0.5 and 1 mg/ml in PBS). C: HDL (100 µl of 1 mg/ml in PBS) was incubated with 100 µl of heparin agarose slurry, to which 100 µl of 0, 1, 2 or 4 mg/ml calf thymus histones in PBS had previously been bound. The ApoA1 content in the unbound (FT) and SDS-eluted (E) fractions was determined by SDS-PAGE (inset) and quantified by densitometry (see Materials & <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009884#s2" target="_blank">Methods</a>). Graph shows mean ± SEM for an experiment run in duplicate. This indicated that HDL was selectively pulled down onto histone-charged heparin agarose. This experiment was performed twice with similar results.</p

Patient recruitment: 120 patients with an initial diagnosis of a PUL were recruited to the study and grouped according to final pregnancy outcomes.

<p>Patient recruitment: 120 patients with an initial diagnosis of a PUL were recruited to the study and grouped according to final pregnancy outcomes.</p

ADAM12 levels in sera collected from women at first presentation with a PUL, categorised according to final pregnancy outcome.

<p>Definite ectopic pregnancy (dEP: n = 17), probable ectopic pregnancy (pEP: n = 8), definite viable intrauterine pregnancy (dVIUP: n = 28), definite nonviable intrauterine pregnancy (dNVIUP: n = 26), spontaneously resolving PUL (srPUL: n = 27), treated persistent PUL (tpPUL: n = 3) and not pregnant (NP: n = 11). A ROC curve was generated (‘ROC of ADAM12’) to compare serum ADAM12 concentrations in patients with a dEP versus all other outcomes. The analysis was repeated (‘ROC of ADAM12 -PUL Data’) after ‘ambiguous’ pregnancy outcomes (srPUL, tpPUL and pEP) were excluded.</p

All TGF-β ligands are expressed in peritoneal fluid from women with and without endometriosis.

<p>TGF-β1 was significantly increased in peritoneal fluid from women with endometriosis compared to women without. Levels of TGF-β2 do not change between women with and without endometriosis. TGF-β3 levels appear to increase in women with endometriosis however this is not a significant change. Cultured HPMCs secreted TGF-β1 protein in-vitro. <i>n = 12 peritoneal fluid, n = 3 HMPC (*p<0.05).</i></p

Venn diagram displaying significant changes in gene expression of TGF-β signalling target in the 3 comparisons made between women with and without endometriosis.

<p>Venn diagram displaying significant changes in gene expression of TGF-β signalling target in the 3 comparisons made between women with and without endometriosis.</p

Serum MIC-1 levels in women at first presentation with a PUL, categorised according to final pregnancy outcome.

<p>Serum MIC-1 levels &gt;1000 ng/mL exclude EP.</p

Primers used for qRT-PCR including primer sequence.

<p>All primers were pre-validated and supplied by Primer Design.</p><p>Primers used for qRT-PCR including primer sequence.</p

TGF-β1, TGF-βR1, TGF-βR2, and pSmad 2/3, are localized to the mesothelial cells of the peritoneum of women without endometriosis at contol sites and prone sites.

<p>TGF-β1, TGF-βR1, TGF-βR2, and pSmad 2/3, are localized to the mesothelial cells of the peritoneum of with endometriosis at sites disteal and ajacent to endometriosis lesions. No staining was observed in the negative control sections. <i>n = 8.</i></p

Table displays gene expression including fold change and p value of TGF-β signalling target genes in peritoneal biopsies from women with endometriosis at sites ajacent compared to sites distal to endometriosis lesions.

<p><i>n = 6.</i></p><p>Table displays gene expression including fold change and p value of TGF-β signalling target genes in peritoneal biopsies from women with endometriosis at sites ajacent compared to sites distal to endometriosis lesions.</p