Performance metrics for models designed to predict treatment effect.

BACKGROUND: Measuring the performance of models that predict individualized treatment effect is challenging because the outcomes of two alternative treatments are inherently unobservable in one patient. The C-for-benefit was proposed to measure discriminative ability. However, measures of calibration and overall performance are still lacking. We aimed to propose metrics of calibration and overall performance for models predicting treatment effect in randomized clinical trials (RCTs). METHODS: Similar to the previously proposed C-for-benefit, we defined observed pairwise treatment effect as the difference between outcomes in pairs of matched patients with different treatment assignment. We match each untreated patient with the nearest treated patient based on the Mahalanobis distance between patient characteristics. Then, we define the Eavg-for-benefit, E50-for-benefit, and E90-for-benefit as the average, median, and 90th quantile of the absolute distance between the predicted pairwise treatment effects and local-regression-smoothed observed pairwise treatment effects. Furthermore, we define the cross-entropy-for-benefit and Brier-for-benefit as the logarithmic and average squared distance between predicted and observed pairwise treatment effects. In a simulation study, the metric values of deliberately "perturbed models" were compared to those of the data-generating model, i.e., "optimal model". To illustrate these performance metrics, different modeling approaches for predicting treatment effect are applied to the data of the Diabetes Prevention Program: 1) a risk modelling approach with restricted cubic splines; 2) an effect modelling approach including penalized treatment interactions; and 3) the causal forest. RESULTS: As desired, performance metric values of "perturbed models" were consistently worse than those of the "optimal model" (Eavg-for-benefit ≥ 0.043 versus 0.002, E50-for-benefit ≥ 0.032 versus 0.001, E90-for-benefit ≥ 0.084 versus 0.004, cross-entropy-for-benefit ≥ 0.765 versus 0.750, Brier-for-benefit ≥ 0.220 versus 0.218). Calibration, discriminative ability, and overall performance of three different models were similar in the case study. The proposed metrics were implemented in a publicly available R-package "HTEPredictionMetrics". CONCLUSION: The proposed metrics are useful to assess the calibration and overall performance of models predicting treatment effect in RCTs.

Factor XII truncation accelerates activation in solution.

Essentials During contact system activation, factor XII is progressively cleaved by plasma kallikrein. We investigated the role of factor XII truncation in biochemical studies. Factor XII contains naturally occurring truncating cleavage sites for a variety of enzymes. Truncation of factor XII primes it for activation in solution through exposure of R353. SUMMARY: Background The contact activation system and innate immune system are interlinked in inflammatory pathology. Plasma kallikrein (PKa) is held responsible for the stepwise processing of factor XII (FXII). A first cleavage activates FXII (into FXIIa); subsequent cleavages truncate it. This truncation eliminates its surface-binding domains, which negatively regulates surface-dependent coagulation. Objectives To investigate the influence of FXII truncation on its activation and downstream kallikrein-kinin system activation. Methods We study activation of recombinant FXII variants by chromogenic assays, by FXIIa ELISA and western blotting. Results We demonstrate that FXII truncation primes it for activation by PKa in solution. We demonstrate this phenomenon in three settings. (i) Truncation at a naturally occurring PKa-sensitive cleavage site, R334, accelerates FXIIa formation in solution. A site-directed mutant FXII-R334A displays ~50% reduced activity when exposed to PKa. (ii) A pathogenic mutation in FXII that causes hereditary angioedema, introduces an additional plasmin-sensitive cleavage site. Truncation at this site synergistically accelerates FXII activation in solution. (iii) We identify new, naturally occurring cleavage sites in FXII that have so far not been functionally linked to contact system activation. As examples, we show that non-activating truncation of FXII by neutrophil elastase and cathepsin K primes it for activation by PKa in solution. Conclusions FXII truncation, mediated by either pathogenic mutations or naturally occurring cleavage sites, primes FXII for activation in solution. We propose that the surface-binding domains of FXII shield its activating cleavage site, R353. This may help to explain how the contact system contributes to inflammatory pathology.

Hereditary angioedema: the plasma contact system out of control.

The plasma contact system contributes to thrombosis in experimental models. Even though our standard blood coagulation tests are prolonged when plasma lacks contact factors, this enzyme system appears to have a minor (if any) role in hemostasis. In this review, we explore the clinical phenotype of C1 esterase inhibitor (C1-INH) deficiency. C1-INH is the key plasma inhibitor of the contact system enzymes, and its deficiency causes hereditary angioedema (HAE). This inflammatory disorder is characterized by recurrent aggressive attacks of tissue swelling that occur at unpredictable locations throughout the body. Bradykinin, which is considered to be a byproduct of the plasma contact system during in vitro coagulation, is the main disease mediator in HAE. Surprisingly, there is little evidence for thrombotic events in HAE patients, suggesting mechanistic uncoupling from the intrinsic pathway of coagulation. In addition, it is questionable whether a surface is responsible for contact system activation in HAE. In this review, we discuss the clinical phenotype, disease modifiers and diagnostic challenges of HAE. We subsequently describe the underlying biochemical mechanisms and contributing disease mediators. Furthermore, we review three types of HAE that are not caused by C1-INH inhibitor deficiency. Finally, we propose a central enzymatic axis that we hypothesize to be responsible for bradykinin production in health and disease.

Extracellular vesicles from human saliva promote hemostasis by delivering coagulant tissue factor to activated platelets.

Essentials Human salivary extracellular vesicles (EVs) expose coagulant tissue factor (TF). Salivary EVs expose CD24, a ligand of P-selectin. CD24 and coagulant TF co-localize on salivary EVs. TF+ /CD24+ salivary EVs bind to activated platelets and trigger coagulation. SUMMARY: Background Extracellular vesicles (EVs) from human saliva expose coagulant tissue factor (TF). Whether such TF-exposing EVs contribute to hemostasis, however, is unknown. Recently, in a mice model, tumor cell-derived EVs were shown to deliver coagulant TF to activated platelets at a site of vascular injury via interaction between P-selectin glycoprotein ligand-1 (PSGL-1) and P-selectin. Objectives We hypothesized that salivary EVs may deliver coagulant TF to activated platelets via interaction with P-selectin. Methods We investigated the presence of two ligands of P-selectin on salivary EVs, PSGL-1 and CD24. Results Salivary EVs expose CD24 but PSGL-1 was not detected. Immune depletion of CD24-exposing EVs completely abolished the TF-dependent coagulant activity of cell-free saliva, showing that coagulant TF and CD24 co-localize on salivary EVs. In a whole blood perfusion model, salivary EVs accumulated at the surface of activated platelets and promoted fibrin generation, which was abolished by an inhibitory antibody against human CD24. Conclusions A subset of EVs in human saliva expose coagulant TF and CD24, a ligand of P-selectin, suggesting that such EVs may facilitate hemostasis at a site of skin injury where the wound is licked in a reflex action.

Amplified endogenous plasmin activity resolves acute thrombotic thrombocytopenic purpura in mice.

Essentials Plasmin is able to proteolyse von Willebrand factor. It was unclear if plasmin influences acute thrombotic thrombocytopenic purpura (TTP). Plasmin levels are increased during acute TTP though suppressed via plasmin(ogen) inhibitors. Allowing amplified endogenous plasmin activity in mice results in resolution of TTP signs. SUMMARY: Background Thrombotic thrombocytopenic purpura (TTP) is an acute life-threatening pathology, caused by occlusive von Willebrand factor (VWF)-rich microthrombi that accumulate in the absence of ADAMTS-13. We previously demonstrated that plasmin can cleave VWF and that plasmin is generated in patients during acute TTP. However, the exact role of plasmin in TTP remains unclear. Objectives Investigate if endogenous plasmin-mediated proteolysis of VWF can influence acute TTP episodes. Results In mice with an acquired ADAMTS-13 deficiency, plasmin is generated during TTP as reflected by increased plasmin-α2-antiplasmin (PAP)-complex levels. However, mice still developed TTP, suggesting that this increase is not sufficient to control the pathology. As mice with TTP also had increased plasminogen activator inhibitor 1 (PAI-1) levels, we investigated whether blocking the plasmin(ogen) inhibitors would result in the generation of sufficient plasmin to influence TTP outcome in mice. Interestingly, when amplified plasmin activity was allowed (α2-antiplasmin-/- mice with inhibited PAI-1) in mice with an acquired ADAMTS-13 deficiency, a resolution of TTP signs was observed as a result of an increased proteolysis of VWF. In line with this, in patients with acute TTP, increased PAP-complex and PAI-1 levels were also observed. However, neither PAP-complex levels nor PAI-1 levels were related to TTP signs and outcome. Conclusions In conclusion, endogenous plasmin levels are increased during acute TTP, although limited via suppression through α2-antiplasmin and PAI-1. Only when amplified plasmin activity is allowed, plasmin can function as a back-up for ADAMTS-13 in mice and resolve TTP signs as a result of an increased proteolysis of VWF.

Factor XII: form determines function.

Factor XII is a mysterious plasma protein without a clear physiologic function. It was identified as a clotting factor, but has no clear role in hemostasis. However, FXII also contributes to the production of bradykinin, a short-lived inflammatory peptide. A growing body of mechanistic research from animal models indicates that FXII contributes to thrombotic disease by triggering excessive coagulation. FXII is evolutionarily conserved, suggesting that this molecule does have a physiologic function. This leads to intriguing questions: What does FXII really do? Is it even a real clotting factor at all? Before the groundbreaking discovery of a role for FXII in thrombotic disease, many studies investigated the biochemical properties of FXII and its activators. In this review, we highlight several biochemical studies that reveal much about the natural behavior of FXII. On the basis of these findings, it is possible to draft a conceptual model to explain how FXII reacts to surface materials. We then discuss how this model applies to the activities of FXII in its natural environment. There are two tentative physiologic functions of FXII that can operate exclusively: (i) maintenance of thrombus stability; (ii) local regulation of vascular permeability. Either, or both, of these natural functions may explain the evolutionary development and maintenance of FXII.

Tracking down contact activation - from coagulation in vitro to inflammation in vivo.

The contact system is a volatile and versatile enzyme system in blood plasma that responds to the presence of nonphysiological surface materials by spontaneous generation of enzymatic activity. In subsequent steps, it can trigger blood coagulation and is responsible for the generation of the proinflammatory peptide bradykinin. The physiological role of the contact system is presently unknown, but it is commonly used to trigger coagulation in a diagnostic setting. In this three-part review, we will first describe the molecular mechanisms that drive contact activation on nonphysiological materials. Next, we will summarize and compare a number of bioassays, which are commonly used to investigate the contact system in health and disease. Finally, we will discuss recent findings from both fundamental and clinical studies on the contributions of contact system to cardiovascular, infectious, and inflammatory disease.

CLL cells are resistant to smac mimetics because of an inability to form a ripoptosome complex.

In the lymph node (LN) environment, chronic lymphocytic leukemia (CLL) cells display increased NF-κB activity compared with peripheral blood CLL cells, which contributes to chemoresistance. Antagonists of cellular inhibitor of apoptosis proteins (cIAPs) can induce apoptosis in various cancer cells in a tumor necrosis factor-α (TNFα)-dependent manner and are in preclinical development. Smac-mimetics promote degradation of cIAP1 and cIAP2, which results in TNFR-mediated apoptosis via formation of a ripoptosome complex, comprising RIPK1, Fas-associated protein with death domain, FLICE-like inhibitory protein and caspase-8. CD40 stimulation of CLL cells in vitro is used as a model to mimic the LN microenvironment and results in NF-κB activation and TNFα production. In this study, we investigated the response of CLL cells to smac-mimetics in the context of CD40 stimulation. We found that treatment with smac-mimetics results in cIAP1 and cIAP2 degradation, yet although TNFα is produced, this did not induce apoptosis. Despite the presence of all components, the ripoptosome complex did not form upon smac-mimetic treatment in CLL cells. Thus, CLL cells seem to possess aberrant upstream NF-κB regulation that prevents ripoptosome formation upon IAP degradation. Unraveling the exact molecular mechanisms of disturbed ripoptosome formation may offer novel targets for treatment in CLL.

A nanobody-based method for tracking factor XII activation in plasma.

The physiological role of the plasma protein factor XII (FXII), as well as its involvement in human pathology, is poorly understood. While FXII is implicated in thrombotic pathology as a coagulation factor, it can contribute to inflammatory conditions without triggering coagulation. We recently generated nanobodies against the catalytic domain of activated FXII (FXIIa). Here, we describe two of these nanobodies, A10 and B7, both of which do not recognise FXII. Nanobody A10 recognises the catalytic domain of purified α-FXIIa (80 kDa), but not that of purified ß-FXIIa (28 kDa), whereas nanobody B7 recognises both. This suggests minute differences in the catalytic domain between these isoforms of FXIIa. The detection of FXIIa by these nanobodies in plasma can become compromised through inactivation by serine protease inhibitors. This effect can be efficiently countered through the addition of the small-molecular protease inhibitor PPACK. Finally, we show that our nanobody-based assays in vitro distinguish various activation products of FXII that differ with the type of activator present: whereas procoagulant activators solely trigger the formation of a species that is captured by B7, proinflammatory activators first generate a species that is recognised by B7, which is later converted into a species that is recognised by A10. These findings suggest that a progressive proteolysis of FXIIa results in the generation a non-procoagulant form of FXIIa, whereas retention of intermediate forms triggers coagulation. Moreover, our findings indicate the development of nanobodies against activated enzymes offers improved opportunities to investigate their contribution to health and disease.

A historic cohort study on accelerated advancement of enteral feeding volumes in very premature infants.

BACKGROUND: The optimal rate of enteral feeding (EF) advancement in very low birth weight infants is under debate. OBJECTIVES: To evaluate the effects of accelerated EF advancement on the time to full enteral feeds, on early postnatal growth as well as on the frequency of necrotizing enterocolitis (NEC) and focal intestinal perforation (FIP) in very premature infants. METHODS: In a retrospective single-center historic cohort study, infants with a gestational age <32 weeks at birth and birth weight <1,500 g, born between January 1, 2006, and December 31, 2007 (n = 136), were compared with infants born between January 1, 2010, and December 31, 2010 (n = 88). In 2006/2007, enteral feeds were initiated on day 1 with 10-15 ml/kg/day and advanced by 15-20 ml/kg/day. In 2010, enteral feeds were initiated with 20 ml/kg/day on day 1 and advanced by 25-30 ml/kg/day. Full enteral feeds were defined as ≥ 140 ml/kg/day. Data are presented as median (P25-P75). RESULTS: The time to establish full enteral feeds was shorter in 2010: 8 (7-11) days in 2006/2007 versus 6 (5-9) days in 2010. The incidences of NEC and FIP were 2.7 and 4.1% in 2006/2007 and 3.3 and 2.2% in 2010, respectively. Weight gain was not affected by the rate of EF advancement. Higher parenteral protein intake during week 1 in 2006/2007 was associated with better head circumference growth. CONCLUSIONS: The new approach was associated with a significantly shorter period to establish full enteral feeds. No difference in the incidence of FIP or NEC was observed; however, the study was underpowered to detect small but possibly important differences.

[Prevention of bone mineral deficiency in premature infants: review of the literature with focus on monitoring of urinary calcium and phosphate]. / Knochenmineralmangel bei Frühgeborenen: Prävention mittels Urinmonitoring von Kalzium und Phosphat.

BACKGROUND: Bone mineral deficiency of prematurity (BMDoP) is caused by the lack of simultaneous availability of calcium (Ca) and anorganic phosphate (P) during rapid skeletal growth. METHODS: Review of the literature on the prevention of BMDoP, with specific attention to the limitations of the monitoring of urinary calcium and phosphate concentrations. RESULTS: Intrauterine bone mineral accretion (BMA) can be achieved in preterm infants if urinary concentrations of Ca and P continuously show that the supplementation with these ions slightly exceeds the actual need. An individually adjusted supplementation with Ca and P appears rational because both growth velocity and enteral Ca absorption are highly variable and determine the need for enteral Ca and P administration. If, however, urinary concentrations of Ca and P are used to determine whether Ca and P supplementation is adequate, mechanisms affecting the urinary excretion of these ions other than nutrition have to be taken into account. Specifically, methylxanthines and diuretics increase the renal Ca losses, and the renal P threshold may be lowered in premature infants. A positive effect of physical activity on BMA has been shown in several studies. CONCLUSIONS: An individualized Ca and P supplementation in preterm infants aiming for supplementation in a slight excess of the actual need and guided by urinary Ca and P concentrations appears able to prevent BMDoP. Monitoring of urinary Ca and P concentrations needs to take into account non-nutritional factors affecting these concentrations. BMA may further be improved by physical activity.

Bid can mediate a pro-apoptotic response to etoposide and ionizing radiation without cleavage in its unstructured loop and in the absence of p53.

BH3-only protein Bid is a key player in death receptor-induced apoptosis, because it provides the link with the mitochondrial route for caspase activation. In this pathway, Bid is activated upon cleavage by caspase-8. Its BH3 domain-containing carboxy-terminal fragment subsequently provokes mitochondrial outer membrane permeabilization by Bak/Bax activation. Bid has also been implicated in the apoptotic response to ionizing radiation (IR) and the topoisomerase inhibitor etoposide, anti-cancer regimens that cause double-strand (ds)DNA breaks. We confirm the existence of this pathway and show that it is p53-independent. However, the degree of Bid participation in the apoptotic response to dsDNA breaks depends on the nature of cell transformation. We used Bid-deficient mouse embryonic fibroblast (MEF) lines that were reconstituted with Bid to control the cellular background and demonstrated that the Bid-dependent apoptotic pathway induced by IR and etoposide operates in MEFs that are transformed by SV40, but is not evident in E1A/Ras-transformed MEFs. The Bid-dependent apoptotic response in p53-deficient SV40-transformed MEFs contributed to clonogenic execution of the cells, implying relevance for treatment outcome. In these cells, Bid acted in a conventional manner in that it required its BH3 domain to mediate apoptosis in response to IR and etoposide, and triggered apoptotic execution by indirect activation of Bak/Bax, mitochondrial permeabilization and caspase-9 activation. However, the mechanism of Bid activation was unconventional, because elimination of all known or suspected cleavage sites for caspases or other proteolytic enzymes and even complete elimination of its unstructured cleavage loop left Bid's pro-apoptotic role in the response to IR and etoposide unaffected.

Acute scrotum in a neonate caused by renal vein thrombosis.

The authors report on a rare case of neonatal scrotal oedema occurring concurrently with pain upon palpation of the spermatic cord on the first day of life. An ultrasound examination showed poor perfusion of the left testicle and a thrombosis of the left renal vein; intraoperative exploration indicated necrosis of the left testicle without signs of torsion. Gorged vessels with paravasal bleeding were found in the spermatic cord. The authors hypothesise that necrosis of the testicle may result from haemorrhagic infarction caused by renal venous thrombosis. Acute scrotal discolouration with pain upon palpation in neonates is usually attributed to testicular torsion. The authors report a case where these symptoms had a different cause.

Survey of practices regarding utilization of inhaled steroids in 223 German neonatal units.

BACKGROUND: No survey has been published in recent years which primarily focuses on the prescription of inhaled corticosteroids in neonatal practice. Thus, the utilization rate of inhaled corticosteroids is unknown. OBJECTIVES: To elucidate the current utilization rate of inhaled corticosteroids in the prevention and therapy of bronchopulmonary dysplasia (BPD). METHODS: We developed an 18-item questionnaire that was distributed in March 2009, via electronic mail, to the pediatricians-in-chief of all the 343 German pediatric hospitals with a neonatal unit (all levels of neonatal care). We sent electronic reminders after 4 and 8 weeks. RESULTS: 223 hospitals (65%) returned the questionnaire. Of these, 102 (46%) administered inhaled corticosteroids to premature infants either as prophylaxis or treatment for BPD. Predominantly, treatment with inhaled steroids was seen as a 'rescue therapy' and used only if other therapeutic approaches had failed. Of the hospitals not administering inhaled steroids, the most frequently stated reason was 'insufficient robust evidence to support benefit of therapy' (57%). In the majority of hospitals (81%), the active substance of choice was budesonide. CONCLUSIONS: Of the responders, approximately 50% administer inhaled corticosteroids to premature infants either as a prophylaxis or treatment for BPD. Lack of beneficial evidence was the main reason for not administering inhaled steroids in about half of the units which took this approach. Future trials should address this discrepancy by aiming to establish a clear benefit-risk ratio of inhaled corticosteroids.

HIV-1 vertical transmission in Rio Grande, Southern Brazil.

The aim of this study was to determine the rate and risk factors of HIV-1 mother-to-child transmission (MTCT), the timing of transmission and the transmitted subtype in a population where subtypes B and C co-circulate. One hundred and forty-four babies born to HIV-1-infected mothers were studied. Subtype and timing of transmission were determined by a nested polymerase chain reaction of the gp41 gene. Seven children were infected (4.9%): four were infected intrautero and one intrapartum. The higher frequency of intrautero transmission was statistically significant (P = 0.001). Use of antiretrovirals (ARVs) in the three stages of gestation was a protective risk factor for MTCT (PR = 0.42; CI: 0.21-0.83; P = 0.013). A higher HIV viral load at delivery was the only independent risk factor for MTCT. Early and universal access to ARVs during pregnancy are the most important measures to decrease vertical HIV-1 transmission even in areas where HIV clade distribution differs.

Smac/DIABLO release from mitochondria and XIAP inhibition are essential to limit clonogenicity of Type I tumor cells after TRAIL receptor stimulation.

Death receptors, such as Fas/CD95 and TRAIL receptors, engage the extrinsic pathway for caspase activation, but also couple to the intrinsic mitochondrial route. In so-called Type II cells, death receptors require the mitochondrial pathway for apoptotic execution, whereas in Type I cells they reportedly do not. For established tumor cell lines, the Type I/Type II distinction is based on short-term apoptosis assays. We report here that the mitochondrial pathway is essential for apoptotic execution of Type I tumor cells by death receptors, when long-term clonogenicity is taken into account. A blockade of the mitochondrial pathway in Type I tumor cells - by RNA interference for Bid or Bcl-2 overexpression - reduced effector caspase activity and mediated significant clonogenic resistance to TRAIL. Downstream from the mitochondria, Caspase-9 did not contribute to clonogenic death of TRAIL-treated Type I cells. Rather, the release of Smac/DIABLO and the inhibition of XIAP activity proved to be crucial for full effector caspase activity and clonogenic execution. Thus, in Type I cells the intrinsic pathway downstream from death receptors is not redundant, but limits clonogenicity by virtue of Smac/DIABLO release and XIAP inhibition. This finding is relevant for cancer therapy using death receptor agonists.