Innate immune receptor NOD2 promotes vascular inflammation and formation of lipid‐rich necrotic cores in hypercholesterolemic mice

Atherosclerosis is an inflammatory disease associated with the activation of innate immune TLRs and nucleotide‐binding oligomerization domain‐containing protein (NOD)‐like receptor pathways. However, the function of most innate immune receptors in atherosclerosis remains unclear. Here, we show that NOD2 is a crucial innate immune receptor influencing vascular inflammation and atherosclerosis severity. 10‐week stimulation with muramyl dipeptide (MDP), the NOD2 cognate ligand, aggravated atherosclerosis, as indicated by the augmented lesion burden, increased vascular inflammation and enlarged lipid‐rich necrotic cores in Ldlr^?/? mice. Myeloid‐specific ablation of NOD2, but not its downstream kinase, receptor‐interacting serine/threonine‐protein kinase 2, restrained the expansion of the lipid‐rich necrotic core in Ldlr^?/? chimeric mice. In vitro stimulation of macrophages with MDP enhanced the uptake of oxidized low‐density lipoprotein and impaired cholesterol efflux in concordance with upregulation of scavenger receptor A1/2 and downregulation of ATP‐binding cassette transporter A1. Ex vivo stimulation of human carotid plaques with MDP led to increased activation of inflammatory signaling pathways p38 MAPK and NF‐κB‐mediated release of proinflammatory cytokines. Altogether, this study suggests that NOD2 contributes to the expansion of the lipid‐rich necrotic core and promotes vascular inflammation in atherosclerosis.     

Effective Variant Detection by Targeted Deep Sequencing of DNA Pools: An Example from Parkinson's Disease

Next‐generation sequencing technologies will dominate the next phase of discoveries in human genetics, but considerable costs may still represent a limitation for studies involving large sample sets. Targeted capture of genomic regions may be combined with deep sequencing of DNA pools to efficiently screen sample cohorts for disease‐relevant mutations. We designed a 200 kb HaloPlex kit for PCR‐based capture of all coding exons in 71 genes relevant to Parkinson's disease and other neurodegenerative disorders. DNA from 387 patients with Parkinson's disease was combined into 39 pools, each representing 10 individuals, before library preparation with barcoding and Illumina sequencing. In this study, we focused the analysis on six genes implicated in Mendelian Parkinson's disease, emphasizing quality metrics and evaluation of the method, including validation of variants against individual genotyping and Sanger sequencing. Our data showed 97% sensitivity to detect a single nonreference allele in pools, rising to 100% where pools achieved sequence depth above 80x for the relevant position. Pooled sequencing detected 18 rare nonsynonymous variants, of which 17 were validated by independent methods, corresponding to a specificity of 94%. We argue that this design represents an effective and reliable approach with possible applications for both complex and Mendelian genetics.     

Asparaginase‐associated pancreatitis in children with acute lymphoblastic leukaemia in the NOPHO ALL2008 protocol

L‐asparaginase is an important drug in the treatment of childhood acute lymphoblastic leukaemia (ALL). Treatment is associated with several toxicities, including acute pancreatitis. Clinical course, presentation, re‐exposure to L‐asparginase after pancreatitis and risk of recurrent pancreatitis within an asparaginase‐intensive protocol has been poorly reported. Children (1–17 years) on the ongoing Nordic Society of Paediatric Haematology and Oncology (NOPHO) ALL2008 protocol with asparaginase‐associated pancreatitis (AAP) diagnosed between 2008 and 2012 were identified through the online NOPHO ALL toxicity registry. NOPHO ALL2008 includes eight or 15 doses of intramuscular pegylated L‐asparginase (PEG‐asparaginase) 1000 iu/m²/dose at 2–6 weeks intervals, with a total of 30 weeks of exposure to PEG‐asparaginase (clinicaltrials.gov no: NCT00819351). Of 786 children, 45 were diagnosed with AAP with a cumulative risk of AAP of 5·9%. AAP occurred after a median of five doses (range 1–13), and 11 d (median) from the latest administration of PEG‐Asparaginase. Thirteen patients developed pseudocysts (30%) and 11 patients developed necrosis (25%). One patient died from pancreatitis. Twelve AAP patients were re‐exposed to L‐asparginase, two of whom developed mild AAP once more, after four and six doses respectively. In conclusion, re‐exposure to PEG‐asparaginase in ALL patients with mild AAP seems safe.     

Flow disturbances generated by feeding and swimming zooplankton

Interactions between planktonic organisms, such as detection of prey, predators, and mates, are often mediated by fluid signals. Consequently, many plankton predators perceive their prey from the fluid disturbances that it generates when it feeds and swims. Zooplankton should therefore seek to minimize the fluid disturbance that they produce. By means of particle image velocimetry, we describe the fluid disturbances produced by feeding and swimming in zooplankton with diverse propulsion mechanisms and ranging from 10-µm flagellates to greater than millimeter-sized copepods. We show that zooplankton, in which feeding and swimming are separate processes, produce flow disturbances during swimming with a much faster spatial attenuation (velocity u varies with distance r as u ∝ r⁻³ to r⁻⁴) than that produced by zooplankton for which feeding and propulsion are the same process (u ∝ r⁻¹ to r⁻²). As a result, the spatial extension of the fluid disturbance produced by swimmers is an order of magnitude smaller than that produced by feeders at similar Reynolds numbers. The “quiet” propulsion of swimmers is achieved either through swimming erratically by short-lasting power strokes, generating viscous vortex rings, or by “breast-stroke swimming.” Both produce rapidly attenuating flows. The more “noisy” swimming of those that are constrained by a need to simultaneously feed is due to constantly beating flagella or appendages that are positioned either anteriorly or posteriorly on the (cell) body. These patterns transcend differences in size and taxonomy and have thus evolved multiple times, suggesting a strong selective pressure to minimize predation risk.Zooplankters move to feed, find food, and find mates, so moving is critical to the efficient execution of essential functions. However, moving comes at a predation risk: Swimming increases the predator encounter velocity (encounter rate increases with prey velocity to a power ≤1), and feeding and swimming generate fluid disturbances that may be perceived by rheotactic predators, thus increasing the predator’s detection distance (encounter rate increases with detection distance squared) (1–5). So, the advantages of moving and feeding must be traded off against the associated risks, and organisms should aim at moving and foraging in ways that reduce the predation risk and optimize the trade-off (6, 7). They may do so by moving in patterns that minimize encounter rates (8) and/or they may feed and propel themselves in ways that generate only small fluid disturbances (9). For example, theoretical models suggest that zooplankton that swim by a sequence of jumps may create a smaller fluid disturbance than similar-sized ones that swim smoothly (9), that a hovering zooplankter generates a larger fluid signal than one that cruises through the water (10, 11), and that a zooplankter moving at low Reynolds numbers will generate a relatively larger fluid signal than one moving at higher Reynolds numbers (11). Thus, motility patterns and propulsion modes may strongly influence predation risk and must be subject to strong selection pressure during evolution.Zooplankton span a huge taxonomic diversity and a large size range (from microns to centimeters) and their propulsion mechanisms vary substantially (12). Unicellular plankton may use one or more flagella or cilia, and the flagella may be smooth or plumose, which has implications for whether the cell is pulled or pushed by the beating flagellum (13). Ciliates may have the cilia rather evenly distributed on the cell surface or concentrated on certain parts of the cell, typically either anteriorly or as an equatorial band. Small animals may have an anterior “corona” of cilia (e.g., rotifers and many pelagic invertebrate larvae) to generate feeding currents and propulsion, or they may have beating or vibrating appendages that can be positioned anteriorly, ventrally, or laterally. The implications and potential adaptive value of this diversity of propulsion modes for feeding and survival are largely unexplored.Various idealized models, simplifying the swimming organisms to combinations of point forces acting on the water, have been used to describe the fluid disturbance generated by moving and feeding plankton. A self-propelled plankton is often described by a so-called stresslet (two oppositely directed point forces of equal magnitude), a hovering one by a stokeslet (a stationary point force), and a jumping animal by an impulsive stresslet (a stresslet working impulsively) (9, 11, 12). These highly idealized models yield very different predictions of the spatial attenuation of the fluid disturbance and, thus, of how far away the feeding and swimming animal can be detected. A few studies have compared observed flow patterns with those predicted from these simple models and in some cases found fair comparisons (4, 14–17). However, numerical simulations as well as observations of self-propelled microplankton have demonstrated that the distribution of propulsion forces, i.e., the position of flagella, cilia, or appendages on the (cell) body, may have a profound effect on the imposed fluid flow (18, 19). Also, most of the idealized models ignore the fact that swimming in most cases is unsteady, which leads to fluctuating flows at scales smaller than the Stokes length scale (ν/ω, where ν is the kinematic viscosity and ω is the beat frequency) (e.g., ref. 19). The simple, idealized models hitherto applied may be insufficient to represent the diverse propulsion modes observed in real organisms and to understand the associated trade-offs.Feeding and swimming are often part of the same process in zooplankton. Many zooplankton generate a feeding current that at the same time propels the animal through the water. In others, feeding and swimming are separate processes. For example, ambush feeding “sit-and-wait” zooplankters do not move as part of feeding but may swim to undertake vertical migration or to search for mates or patches of elevated food availability. Also, many of the plankton that generate a feeding current by vibrating appendages may in addition swim by using the same appendages in a different way (e.g., the nauplius larvae of most crustaceans) or by using other swimming appendages dedicated to propel themselves (most pelagic copepods and cladocerans).Whereas feeding and swimming may both compromise the survival of the organism, the trade-offs may be different. To get sufficient food, zooplankters need to daily clear a volume of water for prey that corresponds to about 10⁶ times their own body volume (20, 21) and hence, implicit in the feeding process is the need to examine or process large volumes of water. In contrast, dedicated swimming should translate the organism through the water as quietly as possible. Thus, we hypothesize that in microplankton, dedicated swimming produces flow fields that attenuate more readily and/or have a smaller spatial extension than the cases in which feeding and propulsion are intimately related.In this study we use particle image velocimetry (PIV) to describe the flow fields generated by micron- to millimeter-sized feeding and swimming zooplankton that use a variety of propulsion modes. We show that—across taxa and sizes—dedicated swimming produces flow fields with a much smaller spatial extension and a faster spatial attenuation than those produced by the plankton for which feeding and swimming are integrated, and we characterize the propulsion modes that minimize susceptibility to rheotactic predators.     

Pneumocystis jiroveci pneumonia prophylaxis during maintenance therapy influences methotrexate/6-mercaptopurine dosing but not event-free survival for childhood acute lymphoblastic leukemia

Trimethoprim-sulfamethoxazole (TMP/SMX) is used in children with acute lymphoblastic leukemia (ALL) to prevent Pneumocystis pneumonia (PCP). We explored to which extent TMP/SMX influenced methotrexate (MTX)/6-mercaptopurine (6MP) dosage, myelosuppression, and event-free survival (EFS) during maintenance therapy. Of 447 study patients treated by the NOPHO ALL92 protocol, 120 patients received TMP/SMX continuously for 2-7 d/wk (TMP/SMX(2-7) ) and 287 patients never received TMP/SMX (TMP/SMX(never) ). Ten patients (all TMP/SMX(never) ) developed PCP, eight of which occurred within 7 months from the start of maintenance therapy. The TMP/SMX(2-7) group received lower oral 6MP doses than TMP/SMX(never) patients (50.6 vs. 63.9 mg/m(2) /d; P<0.001) but had lower absolute neutrophil counts (ANC) (median 1.7 vs. 2.0 × 10(9) /L; P<0.001). In Cox multivariate analysis, higher ANC levels (P=0.04) and male gender (P=0.06) were related to reduced EFS. ANC had no effect on EFS among TMP/SMX(2-7) patients (P=0.40) but did for TMP/SMX(never) patients (P=0.02). The difference in the effect on EFS between TMP/SMX(2-7) and TMP/SMX(never) patients was not significant (P=0.46). EFS did not differ between TMP/SMX(2-7) and TMP/SMX(never) patients (0.83 vs. 0.83; P=0.82). These results suggest that TMP/SMX is effective in preventing PCP and may have an antileukemic effect. TMP/SMX should be given the entire duration of maintenance therapy.     

Fibroblast growth factor 23 and parathyroid hormone after treatment with active vitamin D and sevelamer carbonate in patients with chronic kidney disease stage 3b, a randomized crossover trial

ABSTRACT: BACKGROUND: Fibroblast growth factor 23 (FGF23) is a phosphaturic hormone that is secreted from bone and serum level increases as renal function declines. Higher levels of FGF23 are associated with increased mortality in hemodialysis-patients. The use of active vitamin D and phosphate binders as recommended in international guidelines, may affect the level of FGF23 and thereby clinical outcome. We investigated the effects of a phosphate binder and active vitamin D on the serum levels of intact FGF23 (iFGF23) and intact parathyroid hormone (iPTH) in patients with chronic kidney disease (CKD) stage 3b (glomerular filtration rate (GFR) 30-44 ml/min/1.73m2). Subjects Seven women and 14 men were included, mean age 65.6 +/- 12.2 years. They were randomized in a 1:1 ratio to receive one of two treatment sequences. Group-1 (the alphacalcidol-sevelamer carbonate group): alphacalcidol 0.25 ug once daily for two weeks followed by sevelamer carbonate 800 mg TID with meals for two weeks after a two-week washout period. Group-2 (the sevelamer carbonate-alphacalcidol group): vice versa. Nineteen patients completed the study. The 25-hydroxyvitamin D level at baseline was 97.6 +/- 25.0 nmol/l. RESULTS: There were no treatment effects on the iFGF23 and iPTH levels overall. In group-1 the iFGF23 level was higher after treatment with alphacalcidol compared with sevelamer carbonate (mean 105.8 +/- 41.6 vs. 79.1 +/- 36.5 pg/ml, p=0.047 (CI: 0.4-52.9), and the iPTH level was lower (median: 26.5, range: 14.6-55.2 vs. median 36.1, range 13.4-106.9 pg/ml, p=0.011). In group-2 the iFGF23 level increased non-significantly after treatment with sevelamer carbonate and throughout the washout period. CONCLUSIONS: In this crossover trial with alphacalcidol and sevelamer carbonate in patients with CKD stage 3b, the levels of iFGF23 were not significantly different after the two treatments. However, in the group of patients initiating therapy with sevelamer carbonate the iFGF23 levels seemed to increase while this response was mitigated in the group of patients given alphacalcidol followed by sevelamer carbonate. This may have therapeutic implications on choice of first line therapy. The number of patients is small and this conclusion is in part based on subgroup analysis. It is therefore important that these results are confirmed in larger studies. Trial Registration Number: European Clinical trial database (EudraCT) 2010-020415-36 and ClinicalTrials.gov NCT01231438.     

Magnetic Resonance Imaging Angiography in Patency Evaluation of Bronchial Artery Revascularization Grafts

In this report we present our experience of non-invasive magnetic resonance imaging (MR) angiography and selective catheter angiography in assessing the patency of bronchial artery revascularization grafts after an en bloc double-lung and heart-lung transplantation. We studied 8 patients who had undergone pulmonary transplantation with direct bronchial artery revascularization. Catheter angiography was performed 10 days to 63 months postoperatively. MR angiography was performed within 24 h of the catheter procedure and the results were compared with the findings from catheter angiography. Catheter angiography showed the bronchial revascularization graft to be patent in 6 patients and occluded in 2. At MR angiography, the patency of bronchial artery revascularization grafts was reliably identified in 7 of the 8 patients. One patient had inadequate image quality because of void artefacts caused by haemostatic clips. It is concluded that MR angiography is a reliable method for assessing the patency of bronchial artery revascularization grafts.     

Bronchial Artery Revascularization Improves Tracheal Anastomotic Healing after Lung Transplantation

The study aimed to clarify the role of direct bronchial artery revascularization (BAR) after en bloc double-lung (DLT) and heart-lung transplantation (HLT). Group I comprised eight patients with en bloc DLT or HLT and successful BAR, while group II included 14 DLT or HLT cases without BAR or with failed BAR. From these groups, 2 subgroups were extracted: group III, including 6 cases of en bloc DLT with successful BAR and group IV 10 HLT cases without or with failed BAR. Airway healing was evaluated at bronchoscopy and patency of BAR with angiography. Pulmonary viral, bacterial and fungal infections, rejections and bronchiolitis obliterans syndrome (BOS) were registered. Tracheal healing at 2 weeks and 3 months was better in group I than in group II (p = 0.003 and p = 0.05, respectively). Compared with group IV, tracheal anastomotic healing at 2 weeks was better in group III (p = 0.007) and tended to be better also after 3 months (p = 0.07). The incidence of infections, rejection or BOS did not differ between groups I and II. BAR thus improved healing of tracheal anastomosis.