Heme oxygenase-1 protects cells from replication stress.

Heme oxygenase-1 (HO-1, HMOX1) degrades heme protecting cells from heme-induced oxidative damage. Beyond its well-established cellular functions, heme has emerged as a stabilizer of G-quadruplexes. These secondary DNA structures interfere with DNA replication. We recently revealed that nuclear HO-1 colocalizes with DNA G-quadruplexes and promotes their removal. Here, we investigate whether HO-1 safeguards cells against replication stress. Experiments were conducted in control and HMOX1-deficient HEK293T cell lines. Immunostaining unveiled that DNA G-quadruplexes accumulated in the absence of HO-1, the effect that was further enhanced in response to δ-aminolevulinic acid (ALA), a substrate in heme synthesis. This was associated with replication stress, as evidenced by an elevated proportion of stalled forks analyzed by fiber assay. We observed the same effects in hematopoietic stem cells isolated from Hmox1 knockout mice and in a lymphoblastoid cell line from an HMOX1-deficient patient. Interestingly, in the absence of HO-1, the speed of fork progression was higher, and the response to DNA conformational hindrance less stringent, indicating dysfunction of the PARP1-p53-p21 axis. PARP1 activity was not decreased in the absence of HO-1. Instead, we observed that HO-1 deficiency impairs the nuclear import and accumulation of p53, an effect dependent on the removal of excess heme. We also demonstrated that administering ALA is a more specific method for increasing intracellular free heme compared to treatment with hemin, which in turn induces strong lipid peroxidation. Our results indicate that protection against replication stress is a universal feature of HO-1, presumably contributing to its widely recognized cytoprotective activity.

Neutrophil Gelatinase-Associated Lipocalin and Hypertensive Disorders of Pregnancy: A Cohort Study in Twin Pregnancies.

Hypertensive disorders complicate more than 10% of twin pregnancies. Several studies showed increased neutrophil gelatinase-associated lipocalin (NGAL) values in women with singleton pregnancies and preeclampsia. This study aimed to assess NGAL values in twin pregnancies complicated by hypertensive disorders. We conducted a study of 242 consecutive twin pregnancies at the Medical University of Vienna. Serum NGAL was evaluated twice during pregnancy and once in the postpartum period. Furthermore, serum NGAL values were compared between women who developed hypertensive disorders and those who had normal blood pressure. In all twin pregnancies, mean NGAL values increased significantly from the first to the second visit (p = 0.004) and, further, after delivery (p < 0.001). NGAL was significantly higher in pregnancies that developed pregnancy hypertension or preeclampsia when compared to the control group at the first visit (109.2 ± 48.9 ng/mL vs. 91.9 ± 29.4 ng/mL, p = 0.04, respectively). The predictive power of first visit NGAL values for development of pregnancy hypertension or preeclampsia was evaluated. When using a cut-off value of 115 ng/mL, we obtained a sensitivity of 45% with a specificity of 77%. We conclude that women with twin pregnancies who develop hypertensive disorders of pregnancy showed increased NGAL values at 11−16 weeks.

Comparison of Ultrasound Descriptors of Abnormally Invasive Placenta (AIP) over the Course of the Second and Third Trimester-Is an Increase Verifiable?

Limited data exist regarding the course of abnormally invasive placentation (AIP) (=placenta accreta spectrum (PAS)) during the 2nd and 3rd trimester, although this knowledge would be important for optimal patient care. In this retrospective single-center longitudinal cohort study, potential aggravation of AIP was evaluated in 37 patients with ultrasound (US) pictures stored on a minimum of two visits. Five raters, blinded to diagnosis and gestational age, judged the degree of AIP as recommended by the International Society for PAS. The probability of invasiveness was estimated as absent, low, intermediate, severe (0-3 points), the extent as absent, focal, diffuse (0-2 points), and the presence and appearance of each US-sign as absent, mild, severe (0-3 points). None of the 10 judged signs appeared more severe (p ≥ 0.41) with progressing pregnancy. Neither the number of positively scored US-signs (earlier scan; 6.14 ± 2.06, later scan; 5.94 ± 2.16; p = 0.28), nor the estimated probability & extent of AIP rose (3.69 ± 1.15 vs. 3.67 ± 1.22; p = 1.0). Test-retest reliability corroborated excellent agreement between visits (mean number of positive US-signs ICC (3,1) = 0.94, 95% CI 0.91-0.97; p < 0.0001). Overall, there was no clinically detectable increase in invasiveness over the course of the 2nd and 3rd trimester. This should be further evaluated in prospective studies.

Fetal Survival in Second-Trimester Termination of Pregnancy Without Feticide.

OBJECTIVE: To evaluate the rate of live birth and the duration of survival after termination of pregnancy without feticide. METHODS: We conducted a retrospective analysis of 241 terminations of pregnancy without feticide for fetal anomalies or genetic abnormalities between 20 0/7 and 24 0/7 weeks of gestation at a single tertiary care referral center in Europe between February 2003 and May 2017. A multivariate binary regression model was used to evaluate factors associated with live birth. RESULTS: Pregnancies were terminated at a mean gestational age of 22.1±1.1 completed weeks of gestation. Median birth weight was 440 g (range 141-1,890 g). Live birth occurred in 122 cases (50.6%, 95% CI 44.4-56.8); median survival time was 32 minutes (range 1-267 minutes). Factors associated with live birth were gestational age at labor induction (odds ratio 1.41, 95% CI 1.01-2.01; P=.049) and fetal anomalies (P=.046). CONCLUSION: After termination of pregnancy without feticide between 20 0/7 and 24 0/7 weeks of gestation, the live birth rate was 50.6% (95% CI 44.4-56.8). A lower gestational age at labor induction and the presence of skeletal, cerebral, renal, or multiple fetal anomalies increased the chance of stillbirth.

Plasmatic tissue factor pathway inhibitor is a major determinant of clotting in factor VIII inhibited plasma or blood.

Tissue factor pathway inhibitor (TFPI) is a major inhibitor of coagulation. We therefore hypothesised that high plasmatic TFPI levels are associated with impaired ex vivo clotting in a model of acquired haemophilia. Blood samples were collected in a prospective clinical study from 30 healthy volunteers. Coagulation in normal or factor VIII (FVIII)-inhibited human blood or plasma was measured by the calibrated automated thrombogram (CAT) and rotational thromboelastometry (ROTEM). Both methods are global haemostatic assays that provide insight into the whole coagulation process. Monoclonal mouse antibodies raised against either the C-terminus or the Kunitz domain 2 of TFPI were used to determine full-length (fl-) and total TFPI by an enzyme-immunoassay. Clotting times and parameters of thrombin generation correlated with TFPI levels. Subjects with low fl-TFPI levels had significantly shorter clotting times and a higher endogenous thrombin potential (ETP) compared to those with high fl-TFPI levels (p≤0.005 for all). An even stronger effect was seen in FVIII-inhibited blood/plasma: ROTEM clotting time was 26% shorter (p=0.01) and the ETP assessed by CAT was >2-fold higher in subjects with low fl-TFPI levels (p≤0.0001). Plasmatic TFPI is a major determinant of coagulation in global haemostatic tests particularly when FVIII is missing. Thus, inhibition of TFPI might be a promising novel treatment approach, especially in haemophilia patients with FVIII inhibitors.

A dose ranging phase I/II trial of the von Willebrand factor inhibiting aptamer ARC1779 in patients with congenital thrombotic thrombocytopenic purpura.

Congenital thrombotic thrombocytopenic purpura (TTP) is a very rare but potentially life-threatening disorder. This phase I/II trial compared the pharmacokinetics and pharmacodynamics and safety of three different administration modes of the anti-von Willebrand factor (VWF) aptamer ARC1779. This was a prospective clinical trial with a partial cross-over design: three periods comprised subcutaneous injections of 50 mg of ARC1779 on seven subsequent days, a low-dose infusion of ARC1779 (0.002 mg/kg/min) for 24-72 hours and a high-dose infusion (0.004-0.006 mg/kg/min) up to 72 hours. ARC1779 concentrations were determined with high performance liquid chromatography, VWF inhibition was measured with enzyme immunoassay and platelet function was determined with the platelet function analyser (PFA-100) and impedance aggregometry. ARC1779 was well tolerated without any bleeding at concentrations spanning over three orders of magnitude. The daily s.c. injection yielded plasma levels (0.5 µg/ml) of the drug that were too low to sufficiently suppress VWF. The low-dose i.v. infusion increased platelet counts in one patient, whereas the high i.v. dose increased plasma concentrations up to 69 µg/ml, completely blocked free A1 domains, VWF-dependent platelet plug formation and enhanced platelet counts in 2/3 patients. In conclusion, infusion of ARC1779 dose-dependently inhibits VWF-dependent platelet function and during infusion ARC1779 increases or stabilises platelet counts in congenital TTP. However, the tested doses, particularly the daily s.c. injections, did not correct all clinical or laboratory features of TTP.

Inhibition of von Willebrand factor by ARC1779 in patients with acute thrombotic thrombocytopenic purpura.

Thrombotic thrombocytopenic purpura (TTP) can cause severe organ damage due to enhanced platelet aggregation by ultra-large von Willebrand factor (VWF) multimers. Thus inhibition of VWF by the anti-VWF ARC1779 might potentially be beneficial for TTP patients. This prospective trial tested the safety, pharmacokinetics and pharmacodynamics of the anti-VWF aptamer ARC1779 added to plasma exchange therapy (PEX) in patients with acute TTP. Seven patients received bolus primed continuous i.v. infusions of ARC1779 (1-2 µg/kg/min) in addition to PEX until remission of TTP was induced or for 14 days. Mean steady state ARC1779 plasma concentrations of 9.9 µg/ml reduced VWF activity to 5% (mean baseline activity was 125% in TTP patients compared to a reference plasma). PEX reduced ARC1779 levels by 50%, but steady state concentrations were restored rapidly with a mini-bolus. After discontinuation of PEX, ARC1779 alone further increased platelet counts in one patient. Stopping ARC1779 was associated with an immediate drop of platelet counts in this patient. This suggests that ARC1779 can block the progression of TTP in patients with severe ADAMTS13 is deficiency. ARC1779 was generally well tolerated without any signs of bleeding. Pharmacokinetics and pharmacodynamics of ARC1779 were well predictable and in agreement with those observed in a previous trial with healthy volunteers. Based on its mechanism of action and the observed effect on platelet counts, ARC1779 used as an adjunctive to PEX may help accelerate recovery from organ dysfunction.