Stephen Tong

TLR7 is involved in sequence-specific sensing of single-stranded RNAs in human macrophages.

Human TLR7 and 8 (hTLR7/8) have been implicated in the sequence-dependent detection of RNA oligonucleotides in immune cells. Although hTLR7 sequence-specific sensing of short RNAs has been inferred from studies of murine TLR7, this has yet to be established for hTLR7. We found that different short ssRNA sequences selectively induced either TNF-α or IFN-α in human PBMCs. The sequence-specific TNF-α response to ssRNAs observed in PBMCs could be replicated in activated human macrophage-like (THP-1) cells pretreated with IFN-γ. Surprisingly, suppression of hTLR7 expression by RNA interference in this model reduced sensing of all immunostimulatory ssRNAs tested. Modulation of the relative expression ratio of hTLR7 to hTLR8 in THP-1 cells correlated with differential sensing of immunostimulatory sequences. Furthermore, the sequence-specific IFN-α induction profile in human PBMCs was accurately modeled by a sequence-specific activation of murine TLR7 in mouse macrophages. Thus, we demonstrate for the first time that hTLR7 is involved in sequence-specific sensing of ssRNAs. We establish a novel cell model for the prediction of TNF-α induction by short RNAs in human macrophages. Our results suggest that differential sequence-specific sensing of RNA oligonucleotides between human and mouse macrophages is due to the modulation of TLR7 sensing by human TLR8.

Parity is a major determinant of success rate in medical abortion: a retrospective analysis of 3161 consecutive cases of early medical abortion treated with reduced doses of mifepristone and vaginal gemeprost.

The antiprogesterone mifepristone in combination with a suitable prostaglandin provides an effective method for induction of abortion in early pregnancy up to 63 days of gestation. The combination of 600 mg mifepristone followed by 1 mg of gemeprost vaginal pessary 48 h later is one of the standard regimens in practice, which is registered in several countries in Europe. In 1995, we reduced the doses for both mifepristone and gemeprost to 200 mg and 0.5 mg respectively, as this was shown to decrease significantly the incidence of side effects whilst maintaining a high efficacy. In this article, we report our experience with this regimen in routine clinical practice by analysing 3161 consecutive medical abortions retrospectively. Twelve case notes (0.4%) were not available, and for 310 (9.8%) women, the outcome was not known with certainty as they did not return for their follow up visit. Of the remaining 2839 women, 2732 (96.2%) had a complete abortion following their treatment. One-hundred-two (3.6%) women required an evacuation of the uterus: for incomplete abortion in 63 (2.2%) and ongoing pregnancy in 39 (1.4%). Three women had to undergo surgery for ectopic pregnancies. The surgical intervention rate was significantly higher at gestation of >49 days compared to < or = 49 days (5.7% vs. 2.6%, p = 0.002) and at >56 days than among those at < or = 56 days (6.7% vs. 3.1%; p <0.001). However, for incomplete abortion a significant increase was only seen at gestation >49 days compared to < or = 49 days (3% vs. 1.6%, p = 0.017). The incidence of ongoing pregnancies increased significantly only after 56 days of gestation compared to < or = 56 days (3.8% vs. 0.9%; p <0.001). Parity was related to the outcome with parous women having significantly more incomplete/ongoing abortions compared to nulliparous women (5.4% vs. 2.0%; p <0.001), although parous women did present earlier in pregnancy for termination than nulliparous women (p = 0.01). The incidence of complications was low: 165 (5.8%) women were given antibiotics for presumed genital infection and severe haemorrhage occurred in 11 (0.4%) women, of whom only two required blood transfusion. In summary, the recommended regimen with the reduced doses of mifepristone and gemeprost is highly effective, meeting the anticipated efficacy with a complete abortion rate of >95%. We have concluded from the data that gestation and parity are strong predictors for clinicians to anticipate the probability of a successful medical termination of pregnancy.

Effects of Pravastatin on Human Placenta, Endothelium, and Women With Severe Preeclampsia

&NA;Preeclampsia is a major pregnancy complication where excess placental release of soluble fms-like tyrosine kinase-1 (sFlt-1) and soluble endoglin causes maternal endothelial and multisystem organ injury. Clinical trials have commenced examining whether pravastatin can be used to treat preeclampsia. However, the preclinical evidence supporting pravastatin as a treatment is limited to animal models, with almost no studies in human tissues. Therefore, we examined whether pravastatin reduced sFlt-1 and soluble endoglin secretion and decreased endothelial dysfunction in primary human tissues. Pravastatin reduced sFlt-1 secretion from primary endothelial cells, purified cytotrophoblast cells, and placental explants obtained from women with preterm preeclampsia. It increased soluble endoglin secretion from endothelial cells but did not change secretion from placental explants. The regulation of sFlt-1 by pravastatin seemed to be mediated via the 3-hydroxy-3-methylglutaryl-coenzyme A reductase cholesterol synthesis pathway. Pravastatin also reduced markers of endothelial dysfunction, including vascular cell adhesion molecule-1 expression and leukocyte adhesion on endothelial cells and increased endothelial cell migration and invasion. We also treated 4 patients with preterm preeclampsia presenting at <30 weeks of gestation with daily pravastatin. Pravastatin seemed to stabilize blood pressure, proteinuria, and serum uric acid levels. Furthermore, serum sFlt-1 levels decreased. We collected the placentas at delivery and found that pravastatin reduced sFlt-1 secretion. These results indicate that pravastatin reduced sFlt-1 and soluble endoglin production and decreased endothelial dysfunction in primary human tissues. We also present pilot data, suggesting that pravastatin can stabilize clinical and biochemical features of preterm preeclampsia. Our data obtained in human tissues support the concept that pravastatin is a candidate therapeutic for preeclampsia. Clinical Trial Registration—URL: http://www.anzctr.org.au. Unique identifier: ACTRN12613000268741.

Rational Design of Immunostimulatory siRNAs

Short-interfering RNAs (siRNAs) have engendered much enthusiasm for their ability to silence the expression of specific genes. However, it is now well established that siRNAs, depending on their sequence, can be variably sensed by the innate immune system through recruitment of toll-like receptors 7 and 8 (TLR7/8). Here, we aimed to identify sequence-based modifications allowing for the design of bifunctional siRNAs with both proinflammatory and specific silencing activities, and with potentially increased therapeutic benefits. We found that the introduction of a micro-RNA (miRNA)-like nonpairing uridine-bulge in the passenger strand robustly increased immunostimulatory activity on human immune cells. This sequence modification had no effect on the silencing efficiency of the siRNA. Increased immunostimulation with the uridine-bulge design was specific to human cells, and conserved silencing efficiency required a Dicer-substrate scaffold. The increased cytokine production with the uridine-bulge design resulted in enhanced protection against Semliki Forest virus (SFV) infection, in viral assays. Thus, we characterize a design scaffold applicable to any given siRNA sequence, that results in increased innate immune activation without affecting gene silencing. Our data suggest that this sequence modification coupled with structural modification differentially recruits human TLR8 over TLR7, and could have potential application in antiviral therapies.

Use of dizygotic to monozygotic twinning ratio as a measure of fertility

BACKGROUND Monozygotic (Mz, identical) twinning occurs at a rate of around three per 1000 maternities in all populations, whereas dizygotic (Dz, fraternal) twinning is highly heritable, and varies with age and race. The Dz/Mz twinning ratio reflects the frequency of twin ovulations, and can provide a useful measure of human fertility. METHODS 1625 pairs of twins of known sex were born in Isan Yuk Obstetric Hospital in Hong Kong during the period 1960-95. The yearly Dz/Mz ratio was calculated, and trends were analysed by chi 2 test with the Bonferroni correction. Ages of mothers of all opposite-sexed twins were recorded and trends analysed by ANOVA and linear regression. FINDINGS The Dz/Mz ratio declined significantly from 1.12 in 1960 to 0.05 in 1978 (p < 0.001), and then rose significantly to an average of 0.86 in 1994-95 (p < 0.003). There was a significant declining trend in age of mothers of opposite-sexed twins from 1960 to 1978 (p < 0.001), but there were no significant changes in maternal age after 1978 (p = 0.38). INTERPRETATION If we are correct in assuming that the frequency of Mz twinning remained constant during the study period, the declining Dz/Mz ratio from 1960 to 1978, which also occurred in many developed countries, could reflect some adverse environmental effect on human fertility. The increasing proportion of Dz twins in the past two decades is probably due to increasing use of ovulation-inducing drugs such as clomiphene citrate, which could mask a serious and continuing decline in human fertility. It is therefore important to continue to monitor the Dz/Mz ratio in the future in this and other subpopulations, after exclusion of any women who have taken drugs that stimulate fertility.

MMP-14 Is Expressed in Preeclamptic Placentas and Mediates Release of Soluble Endoglin

Soluble endoglin is an anti-angiogenic protein that is released from the placenta and contributes to both maternal endothelial dysfunction and the clinical features of severe preeclampsia. The mechanism through which soluble endoglin is released from the placenta is currently unknown; however, recent work in colorectal cancer identified matrix metalloproteinase 14 (MMP-14) as the cleavage protease of endoglin. To determine whether this is also the mechanism responsible for soluble endoglin release in preeclampsia, we investigated the expression of MMP-14 within the placenta and the effects of its inhibition on soluble endoglin release. Placentas were obtained from severe, early onset preeclamptic pregnancies (n = 8) and gestationally matched preterm controls (n = 8). MMP-14 was predominately localized to the syncytiotrophoblast. Results from a proximity ligation assay showed protein interactions between endogenous MMP-14 and endoglin within the preeclamptic placenta. To demonstrate that this interaction produces soluble endoglin, we treated trophoblastic BeWo cells with either a broad-spectrum MMP inhibitor (GM6001) or MMP-14 siRNA. Both treatments produced a decrease in soluble endoglin (P ≤ 0.05). Treatment of mice bearing BeWo xenografts with GM6001 decreased circulating soluble endoglin levels in mouse serum (P ≤ 0.05). These findings indicate that MMP-14 is the likely cleavage protease of endoglin in the setting of preeclampsia. This approach provides a novel method for the development of potential therapeutics to reduce circulating soluble endoglin and ameliorate the clinical features of severe preeclampsia.

Metformin as a prevention and treatment for preeclampsia: effects on soluble fms-like tyrosine kinase 1 and soluble endoglin secretion and endothelial dysfunction

BACKGROUND Preeclampsia is associated with placental ischemia/hypoxia and secretion of soluble fms-like tyrosine kinase 1 and soluble endoglin into the maternal circulation. This causes widespread endothelial dysfunction that manifests clinically as hypertension and multisystem organ injury. Recently, small molecule inhibitors of hypoxic inducible factor 1α have been found to reduce soluble fms-like tyrosine kinase 1 and soluble endoglin secretion. However, their safety profile in pregnancy is unknown. Metformin is safe in pregnancy and is also reported to inhibit hypoxic inducible factor 1α by reducing mitochondrial electron transport chain activity. OBJECTIVE The purposes of this study were to determine (1) the effects of metformin on placental soluble fms-like tyrosine kinase 1 and soluble endoglin secretion, (2) to investigate whether the effects of metformin on soluble fms-like tyrosine kinase 1 and soluble endoglin secretion are regulated through the mitochondrial electron transport chain, and (3) to examine its effects on endothelial dysfunction, maternal blood vessel vasodilation, and angiogenesis. STUDY DESIGN We performed functional (in vitro and ex vivo) experiments using primary human tissues to examine the effects of metformin on soluble fms-like tyrosine kinase 1 and soluble endoglin secretion from placenta, endothelial cells, and placental villous explants. We used succinate, mitochondrial complex II substrate, to examine whether the effects of metformin on soluble fms-like tyrosine kinase 1 and soluble endoglin secretion were mediated through the mitochondria. We also isolated mitochondria from preterm preeclamptic placentas and gestationally matched control subjects and measured mitochondrial electron transport chain activity using kinetic spectrophotometric assays. Endothelial cells or whole maternal vessels were incubated with metformin to determine whether it rescued endothelial dysfunction induced by either tumor necrosis factor-α (to endothelial cells) or placenta villous explant-conditioned media (to whole vessels). Finally, we examined the effects of metformin on angiogenesis on maternal omental vessel explants. RESULTS Metformin reduced soluble fms-like tyrosine kinase 1 and soluble endoglin secretion from primary endothelial cells, villous cytotrophoblast cells, and preterm preeclamptic placental villous explants. The reduction in soluble fms-like tyrosine kinase 1 and soluble endoglin secretion was rescued by coadministration of succinate, which suggests that the effects of metformin on soluble fms-like tyrosine kinase 1 and soluble endoglin were likely to be regulated at the level of the mitochondria. In addition, the mitochondrial electron transport chain inhibitors rotenone and antimycin reduced soluble fms-like tyrosine kinase 1 secretion, which further suggests that soluble fms-like tyrosine kinase 1 secretion is regulated through the mitochondria. Mitochondrial electron transport chain activity in preterm preeclamptic placentas was increased compared with gestation-matched control subjects. Metformin improved features of endothelial dysfunction relevant to preeclampsia. It reduced endothelial cell messenger RNA expression of vascular cell adhesion molecule 1 that was induced by tumor necrosis factor-α (vascular cell adhesion molecule 1 is an inflammatory adhesion molecule up-regulated with endothelial dysfunction and is increased in preeclampsia). Placental conditioned media impaired bradykinin-induced vasodilation; this effect was reversed by metformin. Metformin also improved whole blood vessel angiogenesis impaired by fms-like tyrosine kinase 1. CONCLUSION Metformin reduced soluble fms-like tyrosine kinase 1 and soluble endoglin secretion from primary human tissues, possibly by inhibiting the mitochondrial electron transport chain. The activity of the mitochondrial electron transport chain was increased in preterm preeclamptic placenta. Metformin reduced endothelial dysfunction, enhanced vasodilation in omental arteries, and induced angiogenesis. Metformin has potential to prevent or treat preeclampsia.

Effects of Maternal Obstructive Sleep Apnoea on Fetal Growth: A Prospective Cohort Study

Objective The objective of this study is to determine whether obstructive sleep apnea (OSA) is associated with reduced fetal growth, and whether nocturnal oxygen desaturation precipitates acute fetal heart rate changes. Study Design We performed a prospective observational study, screening 371 women in the second trimester for OSA symptoms. 41 subsequently underwent overnight sleep studies to diagnose OSA. Third trimester fetal growth was assessed using ultrasound. Fetal heart rate monitoring accompanied the sleep study. Cord blood was taken at delivery, to measure key regulators of fetal growth. Results Of 371 women screened, 108 (29%) were high risk for OSA. 26 high risk and 15 low risk women completed the longitudinal study; 14 had confirmed OSA (cases), and 27 were controls. The median (interquartile range) respiratory disturbance index (number of apnoeas, hypopnoeas or respiratory related arousals/hour of sleep) was 7.9 (6.1–13.8) for cases and 2.2 (1.3–3.5) for controls (p<0.001). Impaired fetal growth was observed in 43% (6/14) of cases, vs 11% (3/27) of controls (RR 2.67; 1.25–5.7; p = 0.04). Using logistic regression, only OSA (OR 6; 1.2–29.7, p = 0.03) and body mass index (OR 2.52; 1.09–5.80, p = 0.03) were significantly associated with impaired fetal growth. After adjusting for body mass index on multivariate analysis, the association between OSA and impaired fetal growth was not appreciably altered (OR 5.3; 0.93–30.34, p = 0.06), although just failed to achieve statistical significance. Prolonged fetal heart rate decelerations accompanied nocturnal oxygen desaturation in one fetus, subsequently found to be severely growth restricted. Fetal growth regulators showed changes in the expected direction- with IGF-1 lower, and IGFBP-1 and IGFBP-2 higher- in the cord blood of infants of cases vs controls, although were not significantly different. Conclusion OSA may be associated with reduced fetal growth in late pregnancy. Further evaluation is warranted to establish whether OSA may be an important contributor to adverse perinatal outcome, including stillbirth.

Predicting perinatal outcome through changes in umbilical artery Doppler studies after antenatal corticosteroids in the growth-restricted fetus.

OBJECTIVE: To investigate whether persistently absent umbilical artery end-diastolic flow in the intrauterine growth-restricted fetus after betamethasone administration is associated with altered perinatal outcomes. METHODS: This is a retrospective cohort study of 92 pregnancies complicated by intrauterine growth restriction (IUGR) and absent end-diastolic flow in which antenatal betamethasone was given. Predefined maternal outcomes (maternal age, gestational age at diagnosis of absent end-diastolic flow, gestational age at delivery, preexisting medical conditions) and neonatal outcomes (including birth weight; perinatal mortality; duration of neonatal intensive care unit admission; requirement for intubation, assisted ventilation, inotropic support; duration of supplemental oxygen, assisted ventilation; respiratory distress syndrome, necrotizing enterocolitis, intraventricular hemorrhage) were analyzed. RESULTS: Betamethasone administration was associated with a transient return of end-diastolic umbilical artery flow in 58 pregnancies (63%) and persistent absent end-diastolic flow in 34 (37%). Persistent absent end-diastolic flow was seen more frequently in women with prepregnancy medical disorders (59% compared with 24%, P<.001). Neonates from the persistent absent end-diastolic flow subgroup were more likely to require assisted ventilation (93.1% compared with 73.5%, P=.03) and to have longer durations of assisted ventilation (median time 30 days compared with 4 days, P=.03) and supplemental oxygen (median time 45 days compared with 4 days, P=.04). CONCLUSION: Betamethasone administration is associated with a transient return of end-diastolic flow in two thirds of pregnancies complicated by IUGR and umbilical artery absent end-diastolic flow. Persistent absent end-diastolic flow in the umbilical artery after betamethasone administration may identify a subgroup of fetuses with IUGR at further heightened perinatal risk that, as neonates, are more likely to require assisted ventilation and a longer duration of ventilation and supplemental oxygen. LEVEL OF EVIDENCE: III

The use of miRNA microarrays for the analysis of cancer samples with global miRNA decrease

Recent studies have established that mutations or deletions in microRNA (miRNA) processing enzymes resulting in a global decrease of miRNA expression are frequent across cancers and can be associated with a poorer prognosis. While very popular in miRNA profiling studies, it remains unclear whether miRNA microarrays are suited or not to accurately detecting global miRNA decreases seen in cancers. In this work, we analyzed the miRNA profiles of samples with global miRNA decreases using Affymetrix miRNA microarrays following the inducible genetic deletion of Dicer1. Surprisingly, up to a third of deregulated miRNAs identified upon Dicer1 depletion were found to be up-regulated following standard robust multichip average (RMA) background correction and quantile normalization, indicative of normalization bias. Our comparisons of five preprocess steps performed at the probe level demonstrated that the use of cyclic loess relying on non-miRNA small RNAs present on the Affymetrix platform significantly improved specificity and sensitivity of detection of decreased miRNAs. These findings were validated in samples from patients with prostate cancer, where conjugation of robust normal-exponential background correction with cyclic loess normalization and array weights correctly identified the greatest number of decreased miRNAs, and the lowest amount of false-positive up-regulated miRNAs. These findings highlight the importance of miRNA microarray normalization for the detection of miRNAs that are truly differentially expressed and suggest that the use of cyclic loess based on non-miRNA small RNAs can help to improve the sensitivity and specificity of miRNA profiling in cancer samples with global miRNA decrease.