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Background Tocilizumab blocks pro-inflammatory activity of interleukin-6 (IL-6), involved in pathogenesis of pneumonia the most frequent cause of death in COVID-19 patients. Methods A multicenter, single-arm, hypothesis-driven trial was planned, according to a phase 2 design, to study the effect of tocilizumab on lethality rates at 14 and 30 days (co-primary endpoints, a priori expected rates being 20 and 35%, respectively). A further prospective cohort of patients, consecutively enrolled after the first cohort was accomplished, was used as a secondary validation dataset. The two cohorts were evaluated jointly in an exploratory multivariable logistic regression model to assess prognostic variables on survival. Results In the primary intention-to-treat (ITT) phase 2 population, 180/301 (59.8%) subjects received tocilizumab, and 67 deaths were observed overall. Lethality rates were equal to 18.4% (97.5% CI: 13.6–24.0, P = 0.52) and 22.4% (97.5% CI: 17.2–28.3, P < 0.001) at 14 and 30 days, respectively. Lethality rates were lower in the validation dataset, that included 920 patients. No signal of specific drug toxicity was reported. In the exploratory multivariable logistic regression analysis, older age and lower PaO2/FiO2 ratio negatively affected survival, while the concurrent use of steroids was associated with greater survival. A statistically significant interaction was found between tocilizumab and respiratory support, suggesting that tocilizumab might be more effective in patients not requiring mechanical respiratory support at baseline. Conclusions Tocilizumab reduced lethality rate at 30 days compared with null hypothesis, without significant toxicity. Possibly, this effect could be limited to patients not requiring mechanical respiratory support at baseline. Registration EudraCT (2020-001110-38); clinicaltrials.gov (NCT04317092).

SUMMARYTissue damage during cold storage and reperfusion remains a major obstacle to wider use of transplantation. Vascular endothelial cells and complement activation are thought to be involved in the inflammatory reactions following reperfusion, so endothelial targeting of complement inhibitors is of great interest. Using an in vitro model of human umbilical vein endothelial cells (HUVEC) cold storage and an animal model of ex vivo liver reperfusion after cold ischaemia, we assessed the effect of C1-INH on cell functions and liver damage. We found that in vitro C1-INH bound to HUVEC in a manner depending on the duration of cold storage. Cell-bound C1-INH was functionally active since retained the ability to inhibit exogenous C1s. To assess the ability of cell-bound C1-INH to prevent complement activation during organ reperfusion, we added C1-INH to the preservation solution in an animal model of extracorporeal liver reperfusion. Ex vivo liver reperfusion after 8 h of cold ischaemia resulted in plasma C3 activation and reduction of total serum haemolytic activity, and at tissue level deposition of C3 associated with variable level of inflammatory cell infiltration and tissue damage. These findings were reduced when livers were stored in preservation solution containing C1-INH. Immunohistochemical analysis of C1-INH-treated livers showed immunoreactivity localized on the sinusoidal pole of the liver trabeculae, linked to sinusoidal endothelium, so it is likely that the protective effect was due to C1-INH retained by the livers. These results suggest that adding C1-INH to the preservation solution may be useful to reduce complement activation and tissue injury during the reperfusion of an ischaemic liver.

Background The influence of genetics on variation in DNA methylation (DNAme) is well documented. Yet confounding from population stratification is often unaccounted for in DNAme association studies. Existing approaches to address confounding by population stratification using DNAme data may not generalize to populations or tissues outside those in which they were developed. To aid future placental DNAme studies in assessing population stratification, we developed an ethnicity classifier, PlaNET (Placental DNAme Elastic Net Ethnicity Tool), using five cohorts with Infinium Human Methylation 450k BeadChip array (HM450k) data from placental samples that is also compatible with the newer EPIC platform. Results Data from 509 placental samples were used to develop PlaNET and show that it accurately predicts (accuracy = 0.938, kappa = 0.823) major classes of self-reported ethnicity/race (African: n = 58, Asian: n = 53, Caucasian: n = 389), and produces ethnicity probabilities that are highly correlated with genetic ancestry inferred from genome-wide SNP arrays (> 2.5 million SNP) and ancestry informative markers ( n = 50 SNPs). PlaNET’s ethnicity classification relies on 1860 HM450K microarray sites, and over half of these were linked to nearby genetic polymorphisms ( n = 955). Our placental-optimized method outperforms existing approaches in assessing population stratification in placental samples from individuals of Asian, African, and Caucasian ethnicities. Conclusion PlaNET provides an improved approach to address population stratification in placental DNAme association studies. The method can be applied to predict ethnicity as a discrete or continuous variable and will be especially useful when self-reported ethnicity information is missing and genotyping markers are unavailable. Electronic supplementary material The online version of this article (10.1186/s13072-019-0296-3) contains supplementary material, which is available to authorized users.

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Background5,10-Methylenetetrahydrofolate reductase (MTHFR) is a key enzyme in one-carbon metabolism that ensures the availability of methyl groups for methylation reactions. Two single-nucleotide polymorphisms (SNPs) in the MTHFR gene, 677C>T and 1298A>C, result in a thermolabile enzyme with reduced function. These variants, in both the maternal and/or fetal genes, have been associated with pregnancy complications including miscarriage, neural tube defects (NTDs), and preeclampsia (PE), perhaps due to altered capacity for DNA methylation (DNAm). In this study, we assessed the association between MTHFR 677TT and 1298CC genotypes and risk of NTDs, PE, or normotensive intrauterine growth restriction (nIUGR). Additionally, we assessed whether these high-risk genotypes are associated with altered DNAm in the placenta.ResultsIn 303 placentas screened for this study, we observed no significant association between the occurrence of NTDs (N = 55), PE (early-onset: N = 28, late-onset: N = 20), or nIUGR (N = 21) and placental (fetal) MTHFR 677TT or 1298CC genotypes compared to healthy pregnancies (N = 179), though a trend of increased 677TT genotype in PE/IUGR together was observed (OR 2.53, p = 0.048). DNAm was profiled in 10 high-risk 677 (677TT + 1298AA), 10 high-risk 1298 (677CC + 1298CC), and 10 reference (677CC + 1298AA) genotype placentas. Linear modeling identified no significantly differentially methylated sites between high-risk 677 or 1298 and reference placentas at a false discovery rate < 0.05 and Δβ ≥ 0.05 using the Illumina Infinium HumanMethylation450 BeadChip. Using a differentially methylated region analysis or separating cytosine-guanine dinucleotides (CpGs) by CpG density to reduce multiple comparisons also did not identify differential methylation. Additionally, there was no consistent evidence for altered methylation of repetitive DNA between high-risk and reference placentas.ConclusionsWe conclude that large-scale, genome-wide disruption in DNAm does not occur in placentas with the high-risk MTHFR 677TT or 1298CC genotypes. Furthermore, there was no evidence for an association of the 1298CC genotype and only a tendency to higher 677TT in pregnancy complications of PE/IUGR. This may be due to small sample sizes or folate repletion in our Canadian population attenuating effects of the high-risk MTHFR variants. However, given our results and the conflicting results in the literature, investigations into alternative mechanisms that may explain the link between MTHFR variants and pregnancy complications, or in populations at risk of folate deficiencies, are warranted.Electronic supplementary materialThe online version of this article (10.1186/s13148-018-0468-1) contains supplementary material, which is available to authorized users.

The placenta is a multifunctional organ that regulates key aspects of pregnancy maintenance and fetal development. As the placenta is in direct contact with maternal blood, cellular products (DNA, RNA, proteins, etc.) from the placenta can enter maternal circulation by a variety of ways. The application of serum proteins and circulating placental derived DNA has been well demonstrated for the diagnosis of aneuploidy, and there is great interest in exploring the use of placental biomarkers for the prediction of a range of fetal health parameters. In this review, we discuss how placental biomarkers might be used for the diagnosis and early detection of preeclampsia, fetal growth restriction and inflammation associated with preterm birth. We emphasize how increased understanding of the underlying placental biology can aid in the interpretation of such approaches and development of new biomarkers that can help predict the onset of pregnancy and neonatal health concerns before they manifest.