Disaccharide compositional analysis of chondroitin sulphate using WAX HILIC-MS with pre-column procainamide labelling; application to the placenta in pre-eclampsia.

Chondroitin sulphate (CS) and dermatan sulphate are negatively charged linear heteropolysaccharides. These glycosaminoglycans (GAG) are involved in cellular signalling via binding to growth factors. CS is expressed in a range of tissue and biological fluids and is highly expressed in the placenta. There is evidence that decorin; a CS proteoglycan is significantly decreased in pre-eclampsia and fetal growth restriction. It is considered that GAG chain composition may influence cellular processes that are altered in pre-eclampsia. The goal of the present study was to develop an LC-MS method with precolumn procainamide labelling for the disaccharide compositional analysis of CS. The method was used to investigate whether the disaccharide composition of placenta-extracted CS is altered in pre-eclampsia. The study revealed differential disaccharide compositions of placental chondroitin sulphate between pre-eclampsia and other pregnancy conditions. This suggests that the method may have diagnostic potential for pregnancy disorders. Furthermore, the findings suggest that CS sulphation might play a significant role in maternal labour.

Effect of hot water immersion on acute physiological responses following resistance exercise.

Purpose: Hot water immersion (HWI) is a strategy theorised to enhance exercise recovery. However, the acute physiological responses to HWI following resistance exercise are yet to be determined. Methods: The effect of HWI on intramuscular temperature (IMT), muscle function, muscle soreness and blood markers of muscle cell disruption and inflammatory processes after resistance exercise was assessed. Sixteen resistance trained males performed resistance exercise, followed by either 10 min HWI at 40°C or 10 min passive recovery (PAS). Results: Post-intervention, the increase in IMT at all depths was greater for HWI compared to PAS, however this difference had disappeared by 1 h post at depths of 1 and 2 cm, and by 2 h post at a depth of 3 cm. There were no differences between groups for muscle function, muscle soreness or any blood markers. Conclusion: These results suggest that HWI is a viable means of heat therapy to support a greater IMT following resistance exercise. Recovery of muscle function and muscle soreness is independent of acute changes in IMT associated with HWI.

A Survey Into the Use of Priming Techniques Implemented by Athletes and Coaches to Improve Athletic Performance.

ABSTRACT: Collins, J, Bishop, C, Hills, F, Spiegelhalter, A, Cohen, R, and Turner, A. A survey into the use of priming techniques implemented by athletes and coaches to improve athletic performance. J Strength Cond Res 37(1): 107-113, 2023-This study aimed to examine the frequency and modes of psychological priming techniques and strategies being implemented by athletes of a variety of performance levels. A 15-question, anonymous questionnaire was developed and shared via social media sites. The survey implemented a quantitative method approach to collect background information (e.g., demographics, competition, and training history), the prevalence of priming, and the methods used. Ninety subjects met the inclusion criteria (71 men, 18 women, and 1 subject did not identify their sex), with a median age of 28 ± 7.47 years (range, 24-33 years) and training experience of 11 ± 7.57 years (range, 8-18 years). Self-selected participation level accounted for 11 professional, 17 semiprofessional, and 54 amateur-level athletes. Priming strategies were implemented by 79% of subjects without the use of a coach, 10% used strategies with their coach, and 11% did not prime. For athletes, music was the preferred choice (27%), followed by instructional self-talk (24%), motivational self-talk (23%), applied physical actions (20%), and watching videos clips (6.3%). Coaches preferred motivational statements with 55% implementing this technique, followed by 27% using inspiring team talks, and only 18% playing music. Of those who implemented a priming strategy, 66% found them to be either "very" or "extremely" effective," With 38% of subjects feeling that priming accomplished this through increased motivation, 22% felt that it reduced their fear and anxiety, 21% thought that it improved their intensity, 15% felt that it increased strength and power, and 2% felt that it improved endurance. The chi-square test also found a significant (φc = 0.27; p = 0.011) relationship with the use of priming to increase motivation. These results demonstrate that priming strategies are being used irrespective of coach intervention; therefore, educating coaches and athletes on the implementation of priming techniques has its place when aiming to improve athlete performance.

Establishment and Characterization of Single and Triple-Agent Resistant Osteosarcoma Cell Lines.

Two human osteosarcoma cell lines (MG-63 and HOS-143B) are developed into drug-resistant models using a short-term drug exposure and recovery in drug-free media. Cisplatin, doxorubicin, and methotrexate are used as single agents and in triple combination. The highest level of resistance to cisplatin is observed in MG-63/CISR8, doxorubicin in HOS-143B/DOXR8, and methotrexate in HOS-143B/MTXR8. The MG-63/TRIR8 and HOS-143B/TRIR8 triple-resistance models show lower levels of resistance to combination treatment and are not resistant to the drugs individually. Apoptosis assays suggest that the resistance in MG-63/TRIR8 isfrom cisplatin and methotrexate and not doxorubicin. In contrast, the resistance in HOS-143B/TRIR8 is from doxorubicin and methotrexate instead of cisplatin. Upregulation of P-glycoprotein is seen in all resistant models except those developed with single-agent methotrexate. However, P-glycoprotein is not causing resistance in all cell lines as the inhibitor elacridar only reverses the resistance of doxorubicin on MG-63/DOXR8 and HOS-143B/TRIR8. The migration of the MG-63 resistant models is significantly increased, their invasion rate tends to increase, and RT-PCR shows a switch from epithelial to mesenchymal gene signaling. In contrast, a significant decrease in migration is seen in HOS-143B resistant models with their invasion rate tending to decrease and a switch from mesenchymal to epithelial gene signaling.

A combination of cherry juice and cold water immersion does not enhance marathon recovery compared to either treatment in isolation: A randomized placebo-controlled trial.

Purpose: Cherry juice (CJ) and cold water immersion (CWI) are both effective recovery strategies following strenuous endurance exercise. However, athletes routinely combine recovery interventions and less is known about the impact of a combined CJ and CWI protocol. Therefore, this study investigated the effects of combining CWI and CJ (a "cocktail" (CT)) on inflammation and muscle damage following a marathon. Methods: A total 39 endurance trained males were randomly assigned to a placebo (PL), CWI, CJ, or CT group before completing a trail marathon run. Muscle damage (creatine kinase (CK)), muscle function (maximal voluntary isometric contraction (MVIC)), and inflammation (interleukin-6 (IL-6); C-reactive protein (CRP)) were measured at baseline, immediately after marathon (only IL-6), 24 h, and 48 h after marathon. Results: There were no statistically significant differences between groups and no group × time interaction effects for any of the dependent variables. Confidence intervals (CI) illustrated that CT had unclear effects on inflammation (IL-6; CRP) and MVIC, but may have increased CK to a greater extent than PL and CJ conditions. Conclusion: There is no evidence of an additive effect of CJ and CWI when the treatments are used in conjunction with each other. On the contrary, combining CJ and CWI may result in slightly increased circulating CK.

Cold Water Immersion Offers No Functional or Perceptual Benefit Compared to a Sham Intervention During a Resistance Training Program.

ABSTRACT: Wilson, LJ, Dimitriou, L, Hills, FA, Gondek, MB, van Wyk, A, Turek, V, Rivkin, T, Villiere, A, Jarvis, P, Miller, S, Turner, A, and Cockburn, E. Cold water immersion offers no functional or perceptual benefit compared to a sham intervention during a resistance training program. J Strength Cond Res 35(10): 2720-2727, 2021-Cold water immersion (CWI) is regularly used by athletes as a postexercise recovery strategy, but relatively little is understood about potential training adaptations associated with habitual use. The aim of this study was to investigate the influence of repeated CWI or a sham intervention on adaptations to a lower body resistance training program. Thirteen men (26 ± 6 years; 83.6 ± 15.7 kg) familiar with resistance training were allocated into a CWI (10 minutes at 10° C) or sham group and completed 2 × 4-week blocks of lower body resistance training. Subjects completed a total of 16 training sessions (2 × session·week-1), with each session immediately followed by their allocated recovery intervention. Measures of perceptual markers, muscle function, and muscle architecture were recorded at baseline, midpoint, and post-training. Data were analyzed using factorial analysis of variances. The training program resulted in significant increases in muscle fibre pennation angle (p = 0.009), isometric peak force (p = 0.018), and 1/4 squat (p < 0.001) with no differences between groups (all p > 0.05). There were no differences in perceptual responses between groups. Despite the popularity of CWI as a postexercise recovery intervention, the findings from the present study demonstrated no functional or perceptual benefit compared with a sham intervention during progressive strength and power training. Furthermore, there was no detrimental impact of CWI on morphological adaptations after 16 exposures. These findings are important for athletes and practitioners wishing to use CWI as an acute recovery strategy after training, without blunting potential training adaptations.

Whole body cryotherapy, cold water immersion, or a placebo following resistance exercise: a case of mind over matter?

PURPOSE: The use of cryotherapy as a recovery intervention is prevalent amongst athletes. Performance of high volume, heavy load resistance exercise is known to result in disturbances of muscle function, perceptual responses and blood borne parameters. Therefore, this study investigated the influence of cold water immersion (CWI), whole body cryotherapy (WBC) or a placebo (PL) intervention on markers of recovery following an acute resistance training session. METHODS: 24 resistance trained males were matched into a CWI (10 min at 10 °C), WBC (3- and 4 min at - 85 °C) or PL group before completing a lower body resistance training session. Perceptions of soreness and training stress, markers of muscle function, inflammation and efflux of intracellular proteins were assessed before, and up to 72 h post exercise. RESULTS: The training session resulted in increased soreness, disturbances of muscle function, and increased inflammation and efflux of intracellular proteins. Although WBC attenuated soreness at 24 h, and positively influenced peak force at 48 h compared to CWI and PL, many of the remaining outcomes were trivial, unclear or favoured the PL condition. With the exception of CRP at 24 h, neither cryotherapy intervention attenuated the inflammatory response compared to PL. CONCLUSION: There was some evidence to suggest that WBC is more effective than CWI at attenuating select perceptual and functional responses following resistance training. However, neither cryotherapy intervention was more effective than the placebo treatment at accelerating recovery. The implications of these findings should be carefully considered by individuals employing cryotherapy as a recovery strategy following heavy load resistance training.

Urate crystals induce macrophage PAF­AH secretion which is differentially regulated by TGFß1 and hydrocortisone.

The aim of the present study was to establish the role of platelet­activating factor acetyl hydrolase (PAF­AH) in the resolution phase of gout using an established in vitro mononuclear cell model. The effects of signalling pathway inhibitors on PAF­AH secretion, as well as the effects of the common treatments hydrocortisone and colchicine, an antibody against the anti­inflammatory cytokine transforming growth factor ß1 (TGFß1), and the transcriptional inhibitor actinomycin D, were also investigated. The effect of recombinant PAF­AH on cytokine secretion by these cells was also determined. Human peripheral blood­derived monocytes were isolated and differentiated into macrophages. Monocytes and macrophages were stimulated with monosodium monohydrate urate (MSU) crystals or lipopolysaccharide in the presence or absence of AEG3482 [a c­Jun N­terminal kinase (JNK) inhibitor], MG132 (a proteasome inhibitor), hydrocortisone or colchicine. Cultures were then analysed for PAF­AH secretion using ELISA. A 6­fold upregulation of PAF­AH secretion was observed following macrophage exposure to MSU crystals for 24 h (29.3±6 vs. 5.4±0.3 ng/ml unstimulated; P<0.05). Following 72 h, PAF­AH levels decreased significantly (11.1±1.8; P<0.01). Secretion was further enhanced following pre­treatment with the JNK protein kinase inhibitor AEG3482 prior to MSU crystal stimulation (P<0.05) and was abrogated when cells were preincubated with actinomycin D or the proteasome inhibitor MG132 (50, 100 and 200 µM). The addition of recombinant PAF­AH (2.5­10 ng/ml) to MSU crystal­stimulated immature monocyte cultures significantly decreased pro­inflammatory interleukin (IL)­1ß (unstimulated 687±124 vs. stimulated 113±30 pg/ml) and IL­6 secretion (unstimulated 590±50 vs. stimulated 182±19 pg/ml). Treatment of MSU crystal­stimulated macrophages with hydrocortisone (2 µM) also significantly decreased PAF­AH release (P<0.05). Neutralising anti­TGFß1 addition decreased PAF­AH dose­dependently with the highest inhibition observed at 1 µg/ml (P<0.05). The results implicated that PAF­AH may have an anti­inflammatory role in the resolution phase of gout.

Recovery following a marathon: a comparison of cold water immersion, whole body cryotherapy and a placebo control.

PURPOSE: Cryotherapy is an increasingly popular recovery strategy used in an attempt to attenuate the negative impact of strenuous physical activity on subsequent exercise. Therefore, this study aimed to assess the effects of whole body cryotherapy (WBC) and cold water immersion (CWI) on markers of recovery following a marathon. METHODS: Thirty-one endurance trained males completed a marathon. Participants were randomly assigned to a CWI, WBC or placebo group. Perceptions of muscle soreness, training stress and markers of muscle function were recorded before the marathon and at 24 and 48 h post exercise. Blood samples were taken at baseline, post intervention and 24 and 48 h post intervention to assess inflammation and muscle damage. RESULTS: WBC had a harmful effect on muscle function compared to CWI post marathon. WBC positively influenced perceptions of training stress compared to CWI. With the exception of C-reactive protein (CRP) at 24 and 48 h, neither cryotherapy intervention positively influenced blood borne markers of inflammation or structural damage compared to placebo. CONCLUSION: The findings show WBC has a negative impact on muscle function, perceptions of soreness and a number of blood parameters compared to CWI, contradicting the suggestion that WBC may be a superior recovery strategy. Further, cryotherapy is no more effective than a placebo intervention at improving functional recovery or perceptions of training stress following a marathon. These findings lend further evidence to suggest that treatment belief and the placebo effect may be largely responsible for the beneficial effects of cryotherapy on recovery following a marathon.

The effect of milk on recovery from repeat-sprint cycling in female team-sport athletes.

The consumption of milk following eccentric exercise attenuates the effects of muscle damage in team-sport athletes. However, participation in team sport involves both concentric-eccentric loading and metabolic stress. Therefore, the aim of this study was to investigate the effects of postexercise milk consumption on recovery from a cycling protocol designed to simulate the metabolic demands of team sport. Ten female team-sport athletes participated in a randomised crossover investigation. Upon completion of the protocol participants consumed 500 mL of milk (MILK) or 500 mL of an energy-matched carbohydrate (CHO) drink. Muscle function (peak torque, rate of force development, countermovement jump, 20-m sprint), muscle soreness and tiredness, serum creatine kinase, high-sensitivity C-reactive protein, and measures of oxidative stress (protein carbonyls and reduced glutathione/oxidized glutathione (GSH/GSSG) ratio) were determined at pre-exercise and 24 h, 48 h, and 72 h postexercise. MILK had a possible beneficial effect in attenuating losses in peak torque (180°/s) from baseline to 24 h (3.2% ± 7.8% vs. -6.2% ± 7.5%, MILK vs. CHO) and a possible beneficial effect in minimising soreness (baseline-48 h; baseline-72 h) and tiredness (baseline-24 h; baseline-72 h). There was no change in oxidative stress following the exercise protocol, though a likely benefit of milk was observed for GSH/GSSG ratio at baseline-24 h (0.369 ×/÷ 1.89, 1.103 ×/÷ 3.96, MILK vs. CHO). MILK had an unclear effect on all other variables. Consumption of 500 mL of milk after repeat sprint cycling had little to no benefit in minimising losses in peak torque or minimising increases in soreness and tiredness and had no effect on serum markers of muscle damage and inflammation.

Analysis of procainamide-derivatised heparan sulphate disaccharides in biological samples using hydrophilic interaction liquid chromatography mass spectrometry.

Glycosaminoglycans (GAGs) are a family of linear heteropolysaccharides made up of repeating disaccharide units that are found on the surface and extracellular matrix of animal cells. They are known to play a critical role in a wide range of cellular processes including proliferation, differentiation and invasion. To elucidate the mechanism of action of these molecules, it is essential to quantify their disaccharide composition. Analytical methods that have been reported involve either chemical or enzymatic depolymerisation of GAGs followed by separation of non-derivatised (native) or derivatised disaccharide subunits and detection by either UV/fluorescence or MS. However, the measurement of these disaccharides is challenging due to their hydrophilic and labile nature. Here we report a pre-column LC-MS method for the quantification of GAG disaccharide subunits. Heparan sulphate (HS) was extracted from cell lines using a combination of molecular weight cutoff and anion exchange spin filters and digested using a mixture of heparinases I, II and III. The resulting subunits were derivatised with procainamide, separated using hydrophilic interaction liquid chromatography and detected using electrospray ionisation operated in positive ion mode. Eight HS disaccharides were separated and detected together with an internal standard. The limit of detection was found to be in the range 0.6-4.9 ng/mL. Analysis of HS extracted from all cell lines tested in this study revealed a significant variation in their composition with the most abundant disaccharide being the non-sulphated ∆UA-GlcNAc. Some structural functional relationships are discussed demonstrating the viability of the pre-column method for studying GAG biology. Graphical abstract Extraction and HILIC UPLC-MS analysis of procainamide-labelled heparan sulphate disaccharides.

Competition Intensity and Fatigue in Elite Fencing.

Turner, AN, Kilduff, LP, Marshall, GJG, Phillips, J, Noto, A, Buttigieg, C, Gondek, M, Hills, FA, and Dimitriou, L. Competition intensity and fatigue in elite fencing. J Strength Cond Res 31(11): 3128-3136, 2017-As yet, no studies have characterized fencing competitions. Therefore, in elite male foilists and across 2 competitions, we investigated their countermovement jump height, testosterone (T), cortisol (C), alpha-amylase (AA), immunoglobulin A (IgA), heart rate (HR), blood lactate (BL), and rating of perceived exertion (RPE). Average (±SD) scores for RPE, BL, and HR (average, max, and percentage of time ≥80% HRmax) were highest in the knockout bouts compared with poules (8.5 ± 1.3 vs. 5.7 ± 1.3, 3.6 ± 1.0 vs. 3.1 ± 1.4 mmol·L, 171 ± 5 vs. 168 ± 8 b·min, 195 ± 7 vs. 192 ± 7 b·min, 74 vs. 68%); however, only significant (p ≤ 0.05) for RPE. Countermovement jump height, albeit nonsignificantly (p > 0.05), increased throughout competition and dropped thereafter. Although responses of C, AA, and IgA showed a tendency to increase during competition and drop thereafter (T and T:C doing the opposite), no significant differences were noted for any analyte. Results suggest that fencing is a high-intensity anaerobic sport, relying on alactic energy sources. However, some bouts evoke BL values of ≥4 mmol·L and thus derive energy from anaerobic glycolysis. High HRs appear possible on account of ample within- and between-bout rest. The small competition load associated with fencing competitions may explain the nonsignificant findings noticed.

Heparan sulfate disaccharide measurement from biological samples using pre-column derivatization, UPLC-MS and single ion monitoring.

Glycosaminoglycans are a heterogeneous family of linear polysaccharides comprised of repeating disaccharide subunits that mediate many effects at the cellular level. There is increasing evidence that the nature of these effects is determined by differences in disaccharide composition. However, the determination of GAG disaccharide composition in biological samples remains challenging and time-consuming. We have developed a method that uses derivatization and selected ion recording and RP-UPLCMS resulting in rapid separation and quantification of twelve heparin/heparin sulfate disaccharides from 5 µg GAG. Limits of detection and quantitation were 0.02-0.15 and 0.07-0.31 µg/ml respectively. We have applied this method to the novel analysis of disaccharide levels extracted from heparan sulfate and human cancer cell lines. Heparan sulfate disaccharides extracted from biological samples following actinase and heparinase incubation and derivatized using reductive amination with 2-aminoacridone. Derivatized disaccharides were analyzed used UPLC-MS with single ion monitoring. Eight HS disaccharide subunits were separated and quantified from HS and cell lines in eleven minutes per sample. In all samples the most abundant subunits present were the unsulfated ΔUA-GlcNAc, ΔUA-GlcNAc,6S and ΔUA,2S-GlcNS,6S. There was considerable variation in the proportions and concentrations of disaccharides between different cell lines. Further studies are needed to examine the significance of these differences.

Detection of the tau protein in human serum by a sensitive four-electrode electrochemical biosensor.

This study presents a novel approach based on a four-electrode electrochemical biosensor for the detection of tau protein - one of the possible markers for the prediction of Alzheimer's disease (AD). The biosensor is based on the formation of stable antibody-antigen complexes on gold microband electrodes covered with a layer of a self-assembled monolayer and protein G. Antibodies were immobilized on the gold electrode surface in an optimal orientation by protein G interaction. Electrochemical impedance spectroscopy was used to analyze impedance change, which revealed a linear response with increasing tau concentrations. The assay is fast (<1h for incubation and measurement) and very sensitive. The limit of quantification for the full-length 2N4R tau protein is 0.03pM, a value unaltered when the assay was processed in bovine serum albumin or human serum. This technology could be adapted for the detection of other biomarkers to provide a multiple assay to identify AD progression in a point of care setting.

Placental syndecan-1 and sulphated glycosaminoglycans are decreased in preeclampsia.

Glycosaminoglycans are found in extracellular matrix and on the cell surface in the form of proteoglycans. There is evidence that these molecules regulate biological processes, including cell survival, migration and angiogenesis. Preeclampsia is a common pregnancy disorder associated with insufficient placental development. This study aimed to determine the concentrations of glycosaminoglycans and the proteoglycan syndecan-1 within villous trophoblast and to investigate changes associated with preeclampsia. Seventy-five placental samples collected from third trimester singleton pregnancies were divided into term placentas following labour onset, gestational age-matched placentas prior to labour onset and preterm placentas. Preterm placentas were divided into three gestational age-matched groups, spontaneous preterm labour, preterm premature rupture of membranes (PPROM) and preterm preeclampsia. Sulphated glycosaminoglycan (sGAG) concentrations in placental extracts were quantified using a modified 1,9-dimethylmethylene blue assay. Syndecan-1 expression was localised using immunohistochemistry and concentrations in placental extracts determined by immunoassay. Preterm placentas had significantly lower sGAG concentrations compared to term tissues and concentrations were significantly lower in preeclampsia compared to spontaneous preterm labour (medians 5.80 and 10.0 µg/mg protein respectively, P<0.05). Syndecan-1 expression was localised to syncytiotrophoblast and median concentrations were lower in preeclampsia compared to PPROM material (preeclampsia median = 41.7, PPROM 74.4 ng/mg tissue) but not significantly different to concentrations in spontaneous preterm labour. Multivariate analysis revealed that decreased sGAG and syndecan-1 in preeclampsia were independent of labour, gestational age and birthweight centile. These findings may provide insights into a role for these molecules in the pathophysiology of preeclampsia.

Differential proteolysis of insulin-like growth factor binding protein-1 (IGFBP-1) in pregnancy.

The insulin-like growth factors and their binding proteins are important for placental and foetal growth. In this study, we have investigated the presence of proteolytic activity directed against insulin-like growth factor binding protein-1 (IGFBP-1) in pregnancy. In addition, the effect of protease activity on IGFBP-1 immunoreactivity and IGF binding was characterised. 125I-IGFBP-1 was incubated with maternal and foetal serum, amniotic fluid and placental extracts. Breakdown of 125I-IGFBP-1 was determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and autoradiography. The size distribution of endogenous IGFBP-1 was determined by Western immunoblotting. Protease inhibitor studies characterised the proteolytic activity, and Western ligand blotting with 125I-IGF-I was used to determine IGF binding capacity of proteolysed IGFBP-1. Amniotic fluid samples collected after labour onset contained proteolytic activity that generated 12- and 19-kDa IGFBP-1 fragments that did not bind to 125I-IGF-I. This activity was not detected in amniotic fluid collected prior to labour onset or in other tissues. Activity was blocked by aprotinin, leupeptin, phenyl methyl sulphonyl fluoride, and Kunitz soybean trypsin inhibitor but not by ethylene diamine tetraacetic acid or pepstatin. Incubation of IGFBP-1 with trypsin generated fragments of a similar size to the amniotic fluid protease. In conclusion, we have demonstrated the presence in vivo of a trypsin-like proteolytic activity that alters the IGF-binding function of IGFBP-1 in pregnancy.

Insulin-like growth factor-II and heparin are anti-apoptotic survival factors in human villous cytotrophoblast.

OBJECTIVE: This study aimed to determine the effects of insulin-like growth factors (IGF-I and IGF-II), heparin, aspirin and vitamin C on the proliferation and apoptosis of human villous cytotrophoblast from first trimester and term placentae. STUDY DESIGN: Villous cytotrophoblast cells were isolated from uncomplicated first trimester (n=12) and term placental tissues (n=12) using negative immunoselection with an antibody to HLA class I antigens. Cells were incubated with IGF-I, IGF-II, heparin, aspirin and vitamin C either alone, or in combination with either TNF-α/IFN-γ or staurosporine. Proliferation was determined by measurement of Ki67 expression using immunocytochemistry. Trophoblast apoptosis was determined by TUNEL staining. Finally RT-PCR was carried out to identify IGF-binding insulin receptor isoforms. Data were expressed as means±SEM. One way analysis of variance (ANOVA) with Bonferroni correction was used to determine if differences between groups were statistically significant. RESULTS: Following negative immunoselection >98% of cells were positively stained for cytokeratin 7, a marker for cytotrophoblasts, and <1% were vimentin positive. First trimester and term trophoblasts underwent spontaneous apoptosis which was inhibited by approximately 50% in the presence of IGF-II or heparin. Apoptosis was significantly increased following incubation with a combination of TNF-α and IFN-γ or staurosporine. Apoptosis was decreased to basal levels following coincubation with IGF-II or heparin. Incubation with IGFs or heparin resulted in a small, but significant increase in Ki67 expression. Insulin receptor isoform A, which binds IGF-II with high affinity, was present in all trophoblast samples tested. CONCLUSION: These results suggest that heparin and IGF-II, but not IGF-I are important regulators of villous cytotrophoblast survival in early and late pregnancy.

Reciprocal imprinting of human GRB10 in placental trophoblast and brain: evolutionary conservation of reversed allelic expression.

Genomic imprinting may have evolved not only to regulate fetal growth and development, but also behaviour. The mouse Grb10 gene provides a remarkable model to explore this idea because it shows paternal expression in brain, whereas in the placenta and most other embryonic tissues, expression is from the maternal allele. To assess the biological relevance of this reciprocal pattern of imprinting, we explored its conservation in humans. As in mice, we find the human GRB10 gene to be paternally expressed in brain. Maternal allele-specific expression is conserved only in the placental villous trophoblasts, an essential part of the placenta involved in nutrient transfer. All other fetal tissues tested showed equal expression from both alleles. These data suggest that the maternal GRB10 expression in placenta is evolutionarily important, presumably in the control of fetal growth. As in the mouse, the maternal transcripts originate from several kilobases upstream of the imprinting control region (ICR) of the domain, from a promoter region at which we find no allelic chromatin differences. The brain-specific paternal expression from the ICR shows mechanistic similarities with the mouse as well. This conserved CpG island is DNA-methylated on the maternal allele and is marked on the paternal allele by developmentally regulated bivalent chromatin, with the presence of both H3 lysine-4 and H3 lysine-27 methylation. The strong conservation of the opposite allelic expression in placenta versus brain supports the hypothesis that GRB10 imprinting evolved to mediate diverse roles in mammalian growth and behaviour.

Heparin prevents programmed cell death in human trophoblast.

Heparin is used clinically for the prevention of pregnancy complications associated with prothrombotic disorders, especially antiphospholipid antibody syndrome. Recent studies have suggested that heparin may exert direct effects on placental trophoblast, independently of its anticoagulant activity. We now demonstrate that heparin abrogates apoptosis of primary first trimester villous trophoblast in response to treatment with the pro-inflammatory cytokines interferon (IFN)-gamma and tumour necrosis factor (TNF)-alpha. This multifunctional glycosaminoglycan also inhibited apoptosis induced by other agents, including staurosporin, broad-spectrum kinase inhibitor and thrombin. Furthermore, heparin attenuated caspase-3 activity, a hallmark of apoptosis, in human first trimester villous and extravillous trophoblast cell lines treated with peptidoglycan, a Toll-like receptor-2 agonist isolated from Staphylococcus aureus. The ability of heparin to antagonize cell death induced by such diverse apoptotic signals suggested that it acts as a survival factor for human trophoblast. We demonstrate that heparin, like epidermal growth factor (EGF) and heparin-binding EGF (HB-EGF), elicits phosphorylation of the EGF receptor and activation of the phosphatidyl inositol 3-kinase (PI3K)-, the extracellular signal-related kinase 1/2 (ERK1/2)- and the c-Jun NH2 terminal kinase (JNK)-signal transduction pathways in primary villous trophoblast. In summary, we have demonstrated that heparin activates multiple anti-apoptotic pathways in human trophoblast. Our results suggest that heparin may be useful in the management of at-risk patients, even in the absence of an identifiable thrombophilic disorder.