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Interleukin-1 (IL-1) is a key mediator of inflammation and immunity. Naturally-occurring IL-1 receptor antagonist (IL-1Ra) binds and blocks the IL-1 receptor-1 (IL-1R1), preventing signaling. Anakinra, a recombinant form of IL-1Ra, is used to treat a spectrum of inflammatory diseases. However, anakinra is rapidly cleared from the body and requires daily administration. To create a longer-lasting alternative, PASylated IL-1Ra (PAS–IL-1Ra) has been generated by in-frame fusion of a long, defined-length, N-terminal Pro/Ala/Ser (PAS) random-coil polypeptide with IL-1Ra. Here, we compared the efficacy of two PAS–IL-1Ra molecules, PAS600–IL-1Ra and PAS800–IL-1Ra (carrying 600 and 800 PAS residues, respectively), with that of anakinra in mice. PAS600–IL-1Ra displayed markedly extended blood plasma levels 3 days post-administration, whereas anakinra was undetectable after 24 h. We also studied PAS600–IL-1Ra and PAS800–IL-1Ra for efficacy in monosodium urate (MSU) crystal-induced peritonitis. 5 days post-administration, PAS800–IL-1Ra significantly reduced leukocyte influx and inflammatory markers in MSU-induced peritonitis, whereas equimolar anakinra administered 24 h before MSU challenge was ineffective. The 6-h pretreatment with equimolar anakinra or PAS800–IL-1Ra before MSU challenge similarly reduced inflammatory markers. In cultured A549 lung carcinoma cells, anakinra, PAS600–IL-1Ra, and PAS800-IL-Ra reduced IL-1α–induced IL-6 and IL-8 levels with comparable potency. In human peripheral blood mononuclear cells, these molecules suppressed Candida albicans–induced production of the cancer-promoting cytokine IL-22. Surface plasmon resonance analyses revealed significant binding between PAS–IL-1Ra and IL-1R1, although with a slightly lower affinity than anakinra. These results validate PAS–IL-1Ra as an active IL-1 antagonist with marked in vivo potency and a significantly extended half-life compared with anakinra.

Unchecked inflammation can result in severe diseases with high mortality, such as macrophage activation syndrome (MAS). MAS and associated cytokine storms have been observed in COVID-19 patients exhibiting systemic hyper-inflammation. Interleukin-18 (IL-18), a proinflammatory cytokine belonging to the IL-1 family, is elevated in both MAS and COVID-19 patients, and its level is known to correlate with the severity of COVID-19 symptoms. IL-18 binds its specific receptor IL-1 Receptor 5 (IL-1R5, also known as IL-18 Receptor alpha chain), leading to the recruitment of the co-receptor, IL-1 Receptor 7 (IL-1R7, also known as IL-18 Receptor beta chain). This heterotrimeric complex then initiates downstream signaling, resulting in systemic and local inflammation. Here, we developed a novel humanized monoclonal anti-IL-1R7 antibody to specifically block the activity of IL-18 and its inflammatory signaling. We characterized the function of this antibody in human cell lines, in freshly obtained peripheral blood mononuclear cells (PBMCs), and in human whole blood cultures. We found that the anti-IL-1R7 antibody significantly suppressed IL-18-mediated NFκB activation, reduced IL-18-stimulated IFNγ and IL-6 production in human cell lines, and reduced IL-18-induced IFNγ, IL-6 and TNFα production in PBMCs. Moreover, the anti-IL-1R7 antibody significantly inhibited LPS- and Candida albicans-induced IFNγ production in PBMCs, as well as LPS-induced IFNγ production in whole blood cultures. Our data suggest that blocking IL-1R7 could represent a potential therapeutic strategy to specifically modulate IL-18 signaling, and may warrant further investigation into its clinical potential for treating IL-18-mediated diseases, including MAS and COVID-19.

Applications of small-interfering RNA (siRNA) call for specific and efficient delivery of siRNA into particular cell types. We developed a novel, non-viral targeting system to deliver siRNA specifically into inflammation-activated endothelial cells. This was achieved by conjugating the cationic amphiphilic lipid SAINT to antibodies recognizing the inflammatory cell adhesion molecule E-selectin. These anti-E-selectin-SAINT lipoplexes (SAINTarg) maintained antigen recognition capacity of the parental antibody in vitro, and ex vivo in human kidney tissue slices subjected to inflammatory conditions. Regular SAINT mediated transfection resulted in efficient gene silencing in human microvascular endothelial cells (HMEC-1) and conditionally immortalized glomerular endothelial cells (ciGEnC). However, primary human umbilical vein endothelial cells (HUVEC) transfected poorly, a phenomenon that we could quantitatively correlate with a cell-type specific capacity to facilitate siRNA uptake. Importantly, SAINTarg increased siRNA uptake and transfection specificity for activated endothelial cells. Transfection with SAINTarg delivered significantly more siRNA into activated HUVEC, compared to transfection with non-targeted SAINT. The enhanced uptake of siRNA was Corroborated by improved silencing of both gene- and protein expression of VE-cadherin in activated HUVEC, indicating that SAINTarg delivered functionally active siRNA into endothelial cells. The obtained results demonstrate a successful design of a small nucleotide carrier system with improved and specific siRNA delivery into otherwise difficult-to-transfect primary endothelial cells, which in addition reduced considerably the amount of siRNA needed for gene silencing. (C) 2009 Elsevier B.V. All rights reserved.

Leave patches, uncut areas in a harvested forest, may conserve herbaceous layer species that decline after forest clearcutting. They may also serve to maintain source populations for the recolonization of the harvest area. The main objective of this study is to characterize the short-term response of the herbaceous layer to clearcut harvesting disturbance within and adjacent to leave patches. Four experimental 1 ha patches and two uncut references were established. Within each patch or reference, belts of five 1 m2 quadrats were placed at 50 m and 5 m outside the patch, at 0 m (edge), and at 25 m, 35 m, and 50 m (centre) inside. These belts of quadrats were replicated at all four aspects (North, South, East, and West). Ground vegetation was sampled yearly for 1 year before harvest and 3 years after harvest in the experimental patches and for 2 consecutive years in the references. Some common residual species declined significantly in the clearcuts, but remained stable inside the patches in the short term (3 years after harvest). Overall species composition changed little in the patch interior and edge after harvest. Colonizing species (those that appeared in quadrats after harvest) in the patch exterior were predominantly early-seral species, whereas colonizers in the patch interior tended to be shade-tolerant forest species. Species were grouped a priori based on two habitat preferences (amounts of canopy cover and disturbed substrate). Species characteristic of a habitat with closed canopy and undisturbed substrates that decreased significantly over time in the patch exterior were negatively correlated with the amount of exposed mineral soil and open canopy. Species characteristic of a habitat with open canopy and disturbed substrates that increased significantly at the patch exterior showed a positive correlation with the amount of exposed mineral soil and open canopy. No species’ habitat preference groups declined significantly in the patch interior or edge over time. Based on species’ responses to forest operations, it appears that at least the common forest species are maintained in the short term within leave patches.

Introduction: A new technique was developed to prepare precision-cut slices from small intestine and colon with the object of studying the biotransformation of drugs in these organs. Methods: Rat intestinal slices were prepared in two different ways. In the first method, slices were punched out of the small intestine. In the second method, precision-cut slices were made from agarose-filled and -embedded intestines, using the Krumdieck tissue slicer. This method was also applied to colon tissue. Viability of the slices was determined by analysis of intracellular ATP and RNA levels and morphology. Drug metabolizing activity was studied using lidocaine, testosterone, and 7-ethoxycoumarin (7-EC) as phase I substrates, and 7-hydroxycoumarin (7-HC) as a phase II substrate. Results: Precision-cut slices made from agarose-filled and -embedded intestine better preserved ATP levels than tissue that was punched out of the intestinal wall. After 24 h of incubation, morphology in precision cut-slices showed was quite well preserved while punched out tissue was almost completely autolytic after incubation. In addition, total RNA amount and quality was much better maintained in precision-cut slices, when compared to punched out tissue. Both intestinal slices and punched-out tissue showed high, and comparable, phase I and phase II biotransformation activities. Discussion: It is concluded that preparing precision-cut 0.25 mm slices out of agarose-filled and -embedded intestine provides an improvement, compared with punched-out tissue, and that both intestinal and colon slices are useful preparations for in vitro biotransformation studies.

Cryopreservation of tissue slices greatly facilitates their use in drug metabolism research, leading to efficient use of human organ material and a decrease of laboratory animal use. In the present review, various mechanisms of cryopreservation such as equilibrium slow freezing, rapid freezing and vitrification, and their application to cryopreservation of tissue slices are discussed as well as the viability parameters often used to evaluate the success of cryopreservation. Equilibrium freezing prevents intracellular ice formation by inducing cellular dehydration, but (large) ice crystals are still formed in the interstitial space of the slices. Upon rapid freezing, (small) intra- and extracellular ice crystals are formed which slices from some tissues can resist. Vitrification prevents the formation of both intra- and extracellular ice crystals while an amorphous glass is formed of the slice liquid constituents. To vitrify, however, high molarity solutions of cryoprotectants are required that may be toxic to the slices. The use of mixtures of high molarity of cryoprotectants overcomes this problem. We conclude that vitrification is the approach that most likely will lead to the development of universal cryopreservation methods for tissue slices of various organs from various animal species. In the future this may lead to the formation of a tissue slice bank from which slices can be derived at any desirable time point for in vitro experimentation.

Precision-cut liver slices are to some extent resistant to ice formation induced by rapid freezing. Susceptibility to rapid freezing damage has been shown to be (partly) dependent on intrinsic properties of cells. In the present study an attempt was made to decrease the susceptibility of rat liver slices for rapid freezing damage: the slices were pre-incubated at 37 degrees C under oxygen, prior to cryopreservation to recover from low ATP levels, impaired ion regulation and cell swelling induced by their preparation. It was shown that, unexpectedly, recovery of cellular homeostasis prior to the cryopreservation procedure by the 37 degrees C pre-incubation markedly decreased viability of rapidly frozen slices (in which ice was formed), but not of vitrified slices (in which no ice was formed), in a time- and temperature-dependent manner. UW was found to protect slices from this 'warm pre-incubation phenomenon.' Apparently, pre-incubation prior to freezing causes certain cellular alterations that render slices more susceptible to rapid freezing damage.

This study examined whether tissue vitrification, promoted by partitioning within the tissue, could be the mechanism explaining the high viability of rat liver slices, rapidly frozen after preincubation with 18% Me2SO or VS4 (a 7.5 M mixture of Me2SO, 1,2-propanediol, and formamide with weight ratio 21.5:15:2.4). To achieve this, we first determined the extent to which crystallization or vitrification occurred in cryoprotectant solutions (Me2SO and VS4) and within liver slices impregnated with these solutions. Second, we determined how these events were related to survival of slices after thawing. Water crystallization was evaluated by differential scanning calorimetry and viability was determined by histomorphological examination of the slices after culturing at 37 degrees C for 4 h. VS4-preincubated liver slices indeed behaved differently from bulk VS4 solution, because, when vitrified, they had a lower tendency to devitrify. Vitrified VS4-preincubated slices that were warmed sufficiently rapid to prevent devitrification had a high viability. When VS4 was diluted (to 75%) or if warming was not fast enough to prevent ice formation, slices had a low viability. With 45% Me2SO, low viability of cryopreserved slices was caused by cryoprotectant toxicity. Surprisingly, liver slices preincubated with 18% Me2SO or 50% VS4 had a high viability despite the formation of ice within the slice. In conclusion, tissue vitrification provides a mechanism that explains the high viability of VS4-preincubated slices after ultrarapid freezing and thawing (800 degrees C/min). Slices that are preincubated with moderately concentrated cryoprotectant solutions (18% Me2SO, 50% VS4) and cooled rapidly (100 degrees C/min) survive cryopreservation despite the formation of ice crystals within the slice.

The glass transitions of zein, gliadin and glutenin have been studied as a function of moisture content using mechanical spectrometry and differential scanning calorimetry. All three cereal proteins are plasticizable by water, suggesting that their hydrophilic domains play an important role in determining their rheological properties at low water content. The plasticization effect can be predicted by the Gordon-Taylor equation. Temperature sweeps using small-amplitude oscillatory measurements suggest that all three proteins undergo crosslinking reactions in the temperature range 70–160°C; above 160°C, all proteins show characteristic softening. This information has been used to develop state diagrams for each protein, which can be used to predict behavior during processes such as baking and extrusion.

To get more insight into preschool language and hearing in high-risk very low birthweight (VLBW) children, we conducted a prospective study in a cohort of 79 children. The prevalence of language impairment and hearing loss at age 3–4 years, their relationship to each other as well as to perinatal conditions, neurodevelopmental outcome and the home environment are described. Mild hearing loss was found in 26%, moderate hearing loss in 13% and severe hearing loss in 3% of the children. None of the children was deaf. Abnormal tympanometry was found in 57% of the children. Hearing loss at age 4 years was related to a less optimal neonatal condition and was not related to the obstetrical condition or to neonatal cerebral ultrasound findings. Language impairment was found in 21% of the children at age 3.6 years. Receptive and expressive language was not related to perinatal conditions. There was no relation between the language assessments and the audiological assessments. Cognition and the home environment of the child were the only independent variables in the prediction of language in preschool VLBW children.

Data on the educational attainments and occupational statuses of all siblings from 1653 West German families, from three cohorts born respectively around 1930, 1940, and 1950, are analyzed in order to estimate effects of measured (father's and mother's schooling, father's occupation, and number of siblings) and unmeasured family background factors. The analysis shows that there are unmeasured family factors which influence schooling and occupational status. Further, we find some evidence of trends in family effects on schooling over cohorts in West Germany, in that the effect of father's occupational status is decreasing, and no different family effects for males and females. Analyses on cross- and like-sex siblings pairs do not lead to divergent results. Including controls for measurement error we find no evidence for a family bias in the effect of schooling on occupational status in West Germany.

This article discusses the interplay between social and human capital in the income attainment process of managers. A multivariate analysis of a 1986/1987 sample of 1359 top managers of larger companies in the Netherlands indicates that top managers find their jobs largely through informal channels and more so if they possess more social capital. Social capital (external work contacts, memberships) has a substantial independent influence on income, net of human capital (education, experience) and position level (number of subordinates). Human and social capital can act as substitutes for each other. Human and social capital interact in the income attainment process, but not as expected. Social capital helps at any level of human capital, but human capital does not make a difference at the highest levels of social capital.

Non-steroidal anti-inflammatory drugs (NSAIDs) are associated with high prevalence of gastro-intestinal side-effects. In vivo studies suggest that uncoupling of oxidative phosphorylation is an important cause of the toxicity and that the toxicity is aggravated by enterohepatic circulation. Precision-cut intestinal slices (PCIS) are a newly established in vitro model with intact architecture of the intestine, preserved viability and metabolism during incubation up to 24 h. In this study, this model is used to predict NSAID-induced intestinal toxicity. PCIS from rat jejunum were incubated for 5 h with a concentration range of 5 NSAIDs: diflunisal (0.05-1 mM), indomethacin (0.05-1 mM), diclofenac (0.05-1 mM), naproxen (0.1-5mM) and aspirin (0.1-10 mM). Toxicity was assessed by the ATP content. In vitro TC50 of these 5 compounds was calculated and compared with in vivo TC50 and the mitochondrial toxicity data as reported in the literature. Based on TC50 values, toxicity ranking in the present study appeared to be: aspirin (>10mM)> naproxen (2.96mM)> diclofenac (0.29mM)> indomethacin (0.29mM)> diflunisal (0.18 mM). This is very well in line with the reported toxicity ranking found with the isolated mitochondria. When compared with the in vivo TC50, only one exception of diflunisal was found, which appeared highly toxic in vitro whereas in vivo it's one of the least toxic compound. This discrepancy could not yet be explained by in vivo toxicokinetic factors (absorbtion, enterohepatic circulation, metabolism, excretion). In conclusion, both intrinsic (mitochondrial) toxicity as well as in vivo toxicity ranking are well predicted by the PCIS model.

labor are associated with operative delivery? Mariette Ruckert, Myrthe Peelen, Michelle van Vliet, Mieke ten Eikelder, Nikki van Desel, Katrien Oude Rengerink, Ben Willem Mol, Irene de Graaf Diakonessenhuis, Obstetrics and Gynecology, Utrecht, Netherlands, Academic Medical Center, Obstetrics and Gynecology, Amsterdam, Netherlands, Groene Hart Hospital, Obstetrics and Gynecology, Gouda, Netherlands, Kennemer Gasthuis, Obstetrics and Gynecology, Haarlem, Netherlands OBJECTIVE: At present, there is little scientific fundament behind the interpretation of physical examination during labor and delivery. We aimed to indentify which findings at the start of the second phase of labor were associated with instrumental vaginal delivery (IVD) and emergency caesarean section (ECS). STUDY DESIGN: We performed a prospective cohort study in five hospitals in the Netherlands. Consecutive laboring women, with a viable singleton pregnancy in cephalic presentation and a gestational age of at least 34 weeks, were included. We assessed the predictive value of antepartum and intrapartum characteristics and findings at digital examination prior to the second phase of labor (start pushing) for operative delivery (OD) using logistic regression analysis. We selected variables based on literature and clinical experience. Backward selection was used to develop a prediction model, predictors with a p 0.157 remained in the final model. The discriminative ability of the model was assessed using ROC analyses (perfect when AUC 1). The correspondence between the predicted probability for OD and the observed number of operative deliveries over groups was assessed with a calibration plot (perfect when all points are on the diagonal). RESULTS: Between December 2010 and October 2011, we included 473 women, of whom 74 (16%) had a VID and 22 (5%) an ECS. For 425 women, data on all relevant variables were available. Predictors for OD at start of the second phase of labor were 10 cm dilatation, Caucasian etnicity, nulliparity, epidural analgesia, spontaneous start, molding, engagement and position of the fetal head (Table). The model including these variables showed good discriminative ability: AUC ROC 0.82 (95% CI 0.77-0.87). The model calibrated well (Figure). CONCLUSION: At start of the second phase of labor, antepartum and intrapartum characteristics can predict the need for OD. The prediction model may be implied as a useful tool for clinical decision making and assembling logistics at the delivery ward. 776 The validity of the variable “NICU admission” as an outcome measure for neonatal morbidity Nora Danhof, Melanie Wiegerinck, Anton van Kaam, Ben Willem Mol Academic Medical Center, Obstetrics & Gynecology, Amsterdam, Netherlands OBJECTIVE: The variable “neonatal intensive care (NICU) admission” is a frequently used outcome measure in clinical studies, as can be illustrated by the fact that it is reported as such in nearly one hundred Cochrane reviews from the Pregnancy and Childbirth Group. We addressed the question whether NICU admission is a valid surrogate outcome measure for neonatal morbidity, specifically when comparing different birth settings with different access to the NICU. STUDY DESIGN: We performed a retrospective cohort study among neonates admitted at the NICU of a tertiary hospital in the Netherlands during a ten-year period. The inclusion was restricted to singletons born at term, who were admitted at the NICU for delivery-related morbidity within 24 hours of birth. Patient characteristics and admission data were compared for four groups based on the line of care during delivery, i.e. home birth (group Ia), midwive lead hospital delivery (Ib), secondary care (II) and tertiary care (III). We specifically looked at the mortality rate, defined as the percentage of children that died at any time during NICU admission. RESULTS: We studied 871 newborns of which 53 were born at home, 25 under supervision of a midwife in the hospital, 166 in secondary Calibration plot, showing the average observed operative delivery rate (Y-axis) against the average calculated operative delivery rate (X-axis)

Chilling injury is one of the limiting steps for successful vitrification of cells, organs and tissue. Chilling injury is known to be caused by the decrease of temperature per se. Although some research has been done to study chilling injury in cryopreserved oocytes and sperm cells amongst others, the exact mechanism especially in the intact organ is yet unknown. Precision-cut liver slices (PCLS), contain all liver cell types and microstructures and closely resemble the organ from which they are derived. Therefore, it is an ideal model to study the mechanism of chilling injury in the intact organ. Using PCLS as a tool we aim at identifying the critical genes and signaling pathways responding to chilling injury by microarray analysis to enhance insight into its mechanism and to facilitate development of vitrification methods for integrated tissue and organs. Chilling injury was induced in rat PCLS loaded with cryoprotectant solutions (Sol Y or Sol Z, consisting of M22 without N-methyl-formamide and polyvinylpyrrolidone, and without or with sucrose respectively) and chilled to −15 °C for 10 min. At this temperature, no ice formation occurred as was shown by differential scanning calorimetry. The cryoprotectant solutions induced little or no damage as indicated by ATP levels. Principle Component Analysis indicated that gene expression changes induced in chilled PCLS compared to untreated controls were clearly distinguishable from those induced by the cryoprotectants alone. Pathway analysis in metacore revealed that immune innate and defense responses as well as cell cycle G1/S phase transition, MAP Kinase and lipid/fatty acid biosynthesis and metabolism were affected by chilling. Among the up-regulated pathways, cell surface receptor linked signaling pathways accounted for 68% of all processes, indicating that the cell membrane could be the primary site and sensor for chilling injury. Furthermore, we also found some indication that chilling might cause DNA damage, since DNA-damage inducible genes were up-regulated. In addition, chilling decreased gene expression of a set of enzymes required for cholesterol biosynthesis. Besides, pathway analysis indicated decreased fatty acid oxidation and unsaturated fatty acid biosynthesis. These observations could explain the decreased ratio of unsaturated fatty acid to saturated fatty acid and the decreased cholesterol content in cells after cryopreservation that was reported by others. Most importantly, we were able to observe the responses from the different cell types such as the increased differentiation of stellate cells, and immune responses (e.g. MHC class II activation) in Kupffer cells suggesting the involvement of all liver cell types in the injury. In conclusion, a broad spectrum of changes in the tissue at the gene level was identified that were specifically due to chilling, as well as previously unknown changes in the different cell types. This is the first report of a systematic investigation on the mechanism of chilling injury investigated in integrated tissue by microarray analysis under conditions in which other sources of injury are minimal.

The time dependence of the remanent magnetization for the Mn 2+ and Co 2+ amorphous aluminosilicates below their spin glass freezing temperature is fitted by a t −α law on five decades of time. Its magnetic field behavior has been compared for the two compounds.