Recommendation for validation and quality assurance of non-invasive prenatal testing for foetal blood groups and implications for IVD risk classification according to EU regulations.

BACKGROUND AND OBJECTIVES: Non-invasive assays for predicting foetal blood group status in pregnancy serve as valuable clinical tools in the management of pregnancies at risk of detrimental consequences due to blood group antigen incompatibility. To secure clinical applicability, assays for non-invasive prenatal testing of foetal blood groups need to follow strict rules for validation and quality assurance. Here, we present a multi-national position paper with specific recommendations for validation and quality assurance for such assays and discuss their risk classification according to EU regulations. MATERIALS AND METHODS: We reviewed the literature covering validation for in-vitro diagnostic (IVD) assays in general and for non-invasive foetal RHD genotyping in particular. Recommendations were based on the result of discussions between co-authors. RESULTS: In relation to Annex VIII of the In-Vitro-Diagnostic Medical Device Regulation 2017/746 of the European Parliament and the Council, assays for non-invasive prenatal testing of foetal blood groups are risk class D devices. In our opinion, screening for targeted anti-D prophylaxis for non-immunized RhD negative women should be placed under risk class C. To ensure high quality of non-invasive foetal blood group assays within and beyond the European Union, we present specific recommendations for validation and quality assurance in terms of analytical detection limit, range and linearity, precision, robustness, pre-analytics and use of controls in routine testing. With respect to immunized women, different requirements for validation and IVD risk classification are discussed. CONCLUSION: These recommendations should be followed to ensure appropriate assay performance and applicability for clinical use of both commercial and in-house assays.

Following targeted routine antenatal anti-D prophylaxis, almost half of the pregnant women had undetectable anti-D prophylaxis at delivery.

INTRODUCTION: In September 2016, a nationwide targeted routine antenatal anti-D prophylaxis program was implemented in Norway. The prophylaxis (anti-D immunoglobulin) aims to cover the whole third trimester and is administered in gestational week 28 to RhD-negative women who carry RhD-positive fetuses. However, in many women, antibody screening at delivery does not detect anti-D immunoglobulin. The goal of this study was to investigate the presumable role of dose and timing of antenatal anti-D immunoglobulin administration in non-detectable prophylaxis at the time of delivery. MATERIAL AND METHODS: In this retrospective observational study, RhD-negative pregnant women who gave birth at Oslo University Hospital and Akershus University Hospital between January 2017 and December 2019 were analyzed. Women who received antenatal anti-D immunoglobulin (1500 IU at Oslo University Hospital and 1250 IU at Akershus University Hospital) when fetal RHD genotyping at gestational week 24 predicted an RhD-positive fetus were included if an antibody screen at delivery was available. Data from the blood bank, maternity information systems, and electronic patient records were used. RESULTS: Analysis of the 984 RhD-negative women at the two hospitals revealed that 45.4% had non-detectable anti-D at delivery. A significant difference between the two hospitals was observed: 40.5% at Oslo University Hospital (n = 509) and 50.7% at Akershus University Hospital (n = 475) (p = 0.001). The proportion with non-detectable anti-D increased to 56.0 and 75.3%, respectively (p = 0.008) in the group of women who gave birth 12 weeks after routine antenatal anti-D prophylaxis. Significantly fewer women had detectable anti-D at delivery when the lower anti-D immunoglobulin dose (1250 IU) was administered antenatally. Multiple logistic regression indicated that the time interval between routine antenatal anti-D prophylaxis and delivery, in addition to anti-D dose, were significantly associated with detectable anti-D at delivery (p < 0.001). CONCLUSIONS: We do not know which RhD-negative pregnant women, despite antenatal anti-D prophylaxis, are at risk of RhD alloimmunization, when antibody screening is negative at delivery. Administration of antenatal prophylaxis should probably be moved closer to delivery, since the risk of fetomaternal hemorrhage is higher during the last weeks of the third trimester.

Quality of clinical management of cardiometabolic risk factors in patients with severe mental illness in a specialist mental health care setting.

PURPOSE: Cardiometabolic disease in patients with severe mental illness is a major cause of shortened life expectancy. There is sparse evidence of real-world clinical risk prevention practice. We investigated levels of assessments of cardiometabolic risk factors and risk management interventions in patients with severe mental illness in the Norwegian mental health service according to an acknowledged international standard. METHODS: We collected data from 264 patients residing in six country-wide health trusts for: (a) assessments of cardiometabolic risk and (b) assessments of levels of risk reducing interventions. Logistic regressions were employed to investigate associations between risk and interventions. RESULTS: Complete assessments of all cardiometabolic risk variables were performed in 50% of the participants and 88% thereof had risk levels requiring intervention according to the standard. Smoking cessation advice was provided to 45% of daily smokers and 4% were referred to an intervention program. Obesity was identified in 62% and was associated with lifestyle interventions. Reassessment of psychotropic medication was done in 28% of the obese patients. Women with obesity were less likely to receive dietary advice, and use of clozapine or olanzapine reduced the chances for patients with obesity of getting weight reducing interventions. CONCLUSIONS: Nearly nine out of the ten participants were identified as being at cardiometabolic high risk and only half of the participants were adequately screened. Women with obesity and patients using antipsychotics with higher levels of cardiometabolic side effects had fewer adequate interventions. The findings underscore the need for standardized recommendations for identification and provision of cardiometabolic risk reducing interventions in all patients with severe mental illness.

The Norwegian experience with nationwide implementation of fetal RHD genotyping and targeted routine antenatal anti-D prophylaxis.

OBJECTIVES: To reduce the risk of RhD alloimmunization during the last trimester of pregnancy, a targeted routine antenatal anti-D prophylaxis (RAADP) programme was implemented in Norway in 2016. Here, we present and discuss our experience with the nationwide implementation of the programme, and report sample uptake and preliminary data of de novo anti-D in pregnancy. BACKGROUND: The targeted RAADP was advised by the academic community and evaluated by the health authorities. A National Working Group has conducted the implementation in the transfusion services and contributed to organise the administration of the antenatal anti-D prophylaxis. Fetal RhD type is determined by non-invasive prenatal testing at gestational week 24, and anti-D prophylaxis is administrated at gestational week 28 only to women with RhD positive fetuses. METHODS: We describe the implementation process of targeted RAADP in Norway. The sample uptake is calculated by comparing the number of fetal RHD screens with the expected number of samples. RESULTS: The sample uptake shows regional variations: 88%-100% after 3 years. Promising decrease in de novo anti-D detected during pregnancy is observed. CONCLUSIONS: Nationwide targeted RAADP is implemented and included in the Norwegian maternity care programme. Compliance to sample uptake should further improve in some regions. A remaining issue to fulfil is the documentation of the accuracy of the fetal RHD-typing at all sites. Post-natal prophylaxis will then be guided by the fetal RHD result. Dedicated registries will ensure data to evaluate the expected reduction in pregnancy-related RhD immunisations, which is the final success criterion of the programme.

Multidisciplinary aortopathy clinics: A systematic scoping review of the literature and evaluation of patient experiences from a newly started clinic in Norway.

BACKGROUND: International guidelines recommend hereditary thoracic aortic diseases (HTADs) to be managed in multidisciplinary aorta clinics. AIM: To study HTAD patient's experiences with a aortopathy clinic in Norway and to review the literature on aortopathy clinics. METHODS: (a) A systematic scoping review of research on multidisciplinary clinics for HTADs. (b) A cross-sectional postal questionnaire study to investigate patient experiences with the health-services. Fifty consecutive patients from the aortopathy clinic and 50 controls in usual care were invited to participate. RESULTS: The review identified eight publications on aortopathy clinics. Although the papers were not judged for quality, these showed promising results from such clinics in terms of diagnostics and increased adherence to guideline-directed therapy. The survey constituted thirty-seven (74%) patients and 22 (44%) controls who responded to postal questionnaires. Both groups reported delays in diagnostics and follow-up appointments prior to the start of the clinic. Patients indicated high satisfaction with the aortopathy clinic, whereas controls reported poor coordination of medical follow-up. Individuals in both groups struggled with disease self-management. CONCLUSION: Norwegian patient experiences found the aortopathy clinic beneficial. According to studies included in the review, disease management in aortopathy clinics may improve patient satisfaction, diagnostics and follow-up. Effect studies may further document the benefits of clinic organization, treatment, cost-efficiency and patient experiences.

Marfan syndrome: Evolving organ manifestations-A 10-year follow-up study.

The age-dependent penetrance of organ manifestations in Marfan syndrome (MFS) is not known. The aims of this follow-up study were to explore how clinical features change over a 10-year period in the same Norwegian MFS cohort. In 2003-2004, we investigated 105 adults for all manifestations in the 1996 Ghent nosology. Ten years later, we performed follow-up investigations of the survivors (n = 48) who consented. Forty-six fulfilled the revised Ghent criteria. Median age: females 51 years, range 32-80 years; males 45 years, range 30-67 years. New aortic root dilatation was detected in patients up to 70 years. Ascending aortic pathology was diagnosed in 93 versus 72% at baseline. Sixty-five percent had undergone aortic surgery compared to 39% at baseline. Pulmonary trunk mean diameter had increased significantly compared to baseline. From inclusion to follow-up, two patients (three eyes) developed ectopia lentis, four developed dural ectasia, four developed scoliosis, three developed incisional or recurrent herniae, and 14 developed hindfoot deformity. No changes were found regarding protrusio acetabuli, spontaneous pneumothorax, or striae atrophicae. The study confirms that knowledge of incidence and progression of organ manifestations throughout life is important for diagnosis, treatment, and follow-up of patients with verified or suspected MFS.

Pichia pastoris yeast as a vehicle for oral vaccination of larval and adult teleosts.

Oral vaccination is of major interest because it can be used for mass vaccination of fish of various size and age. Given that their administration is relatively easy and stress-free, oral vaccines have both economic and animal welfare benefits. Yet, mostly due to their limited efficacy, only very few oral vaccines are available to aquaculture industry. Here we present a method for oral vaccine delivery based on the yeast Pichia pastoris. We could express a model antigen, green fluorescent protein (GFP), in this yeast and subsequently show delivery of the GFP protein to the intestine of juvenile flounder or adult carp and trout. We tested this approach in several commercially-relevant fish species, from juvenile to adult stage. To test the oral delivery of antigen to larval fish, the GFP-expressing Pichia pastoris was first fed to planktonic crustacean Daphnia or rotifers that served as 'bioencapsulation vehicles' and afterwards, fed to flounder larvae. Again, we could show delivery of intact GFP protein to the intestine. In rainbow trout, the orally-administered GFP-expressing yeast elicited a rapid local innate immune response in the intestine and a subsequent systemic response in the spleen. Our results show that Pichia pastoris is a good vehicle for oral antigen delivery and that it can be used in non-encapsulated form for older fish or in bioencapsulated form for larval fish. We discuss the immunomodulatory properties of the yeast itself, and its potential to enhance local immune responses and act as an adjuvant.

Determination of fetal RHD type in plasma of RhD negative pregnant women.

Alloimmunization against the RhD antigen is the most common cause of hemolytic disease of the fetus and newborn. Antenatal anti-D prophylaxis in addition to postnatal anti-D prophylaxis reduces the number of RhD-immunizations compared to only postnatal administration. Cell-free fetal DNA released from the apoptotic trophoblastic placental cells into the maternal circulation can be used to determine the fetal RHD type in a blood sample from an RhD negative mother. Based on this typing, antenatal anti-D prophylaxis can be recommended only to RhD negative women carrying an RhD positive fetus, since only these women are at risk of developing anti-D. The objective was to establish and validate a method for non-invasive fetal RHD typing. The fetal RHD genotype was studied in 373 samples from RhD negative pregnant women (median gestational week 24). DNA extracted from plasma was analyzed for the presence/absence of RHD exon 7 and 10 in a real-time PCR. The RHD genotype of the fetus was compared with the serological RhD type of the newborn. In 234 samples, the fetal RHD test was positive and in 127 samples negative. There was one false positive and no false negative results. In 12 samples, the fetal RHD type could not be determined, in all of them due to a maternal RHD gene. This method gives a reliable detection of fetal RHD positivity in plasma from RhD negative pregnant women. Antenatal anti-D prophylaxis based on the predicted fetal RhD type will avoid unnecessary treatment of pregnant women carrying an RhD negative fetus.

Semiautomated Magnetic Resonance Imaging Assessment of Carotid Plaque Lipid Content.

BACKGROUND: The composition of a carotid plaque is important for plaque vulnerability and stroke risk. The main aim of this study was to assess the potential of semiautomated segmentation of carotid plaque magnetic resonance imaging (MRI) in the assessment of the size of the lipid-rich necrotic core (LRNC). METHODS: Thirty-four consecutive patients with carotid stenosis of 70% or higher, who were scheduled for carotid endarterectomy, underwent a clinical neurological examination, Color duplex ultrasound, 3-T MRI with an 8-channel carotid coil, and blood tests. All examinations were performed less than 24 hours prior to surgery and plaques were assessed histologically immediately following endarterectomy. Plaques were defined as symptomatic when associated with ipsilateral cerebral ischemic symptoms within 30 days prior to inclusion. The level of agreement between the size of the LRNC and calcification on MRI to the histological estimation of the same tissue components, plaque echolucency on ultrasound, and symptoms was assessed. RESULTS: The size of the LRNC on MRI was significantly correlated to the percentage amount of lipid per plaque on histological assessment (P = .010, r = .5), and to echogenicity on ultrasound with echolucent plaques having larger LRNC than echogenic plaques (P = .001, r = -.7). CONCLUSIONS: In this study, we found that semiautomated MRI assessments of the percentage LRNC in carotid plaques were significantly correlated to the percentage LRNC per plaque on histological assessment, and to echogenicity on ultrasound with echolucent plaques having larger LRNC than echogenic plaques.

Interleukin 23 levels are increased in carotid atherosclerosis: possible role for the interleukin 23/interleukin 17 axis.

BACKGROUND AND PURPOSE: Interleukin (IL)-23 is a cytokine in the IL-12 family, mainly produced by antigen-presenting cells with a central role in inflammation. We hypothesize that IL-23 is also important in atherogenesis and investigate this in a population with carotid atherosclerosis. METHODS: Plasma levels of IL-23 were measured in patients with carotid artery stenosis and in healthy controls. The mRNA levels of IL-23 and its receptor, IL-23R, were measured in atherosclerotic plaques, nonatherosclerotic vessels, peripheral blood mononuclear cells, and plasmacytoid dendritic cells. RESULTS: Our findings were as follows: (1) patients with carotid atherosclerosis (n=177) had significantly raised plasma levels of IL-23 when compared with healthy controls (n=24) with particularly high levels in those with the most recent symptoms. (2) mRNA levels of IL-23 and IL-23R were markedly increased in carotid plaques (n=68) when compared with nonatherosclerotic vessels (n=8-10). Immunostaining showed colocalization to plaque macrophages. (3) Patients with carotid atherosclerosis had increased mRNA levels of both IL-23 and IL-23R in plasmacytoid dendritic cells, but not in peripheral blood mononuclear cells. (4) IL-23 increased IL-17 release in monocytes and particularly in peripheral blood mononuclear cells from patients with carotid atherosclerosis, but not in cells from healthy controls. (5) IL-23 gave a prominent tumor necrosis factor release in monocytes from patients with carotid atherosclerosis but not in cells from healthy controls. (6) High plasma levels of IL-23 were associated with increased mortality during follow-up. CONCLUSIONS: We have shown an association between IL-23 and disease progression in patients with carotid atherosclerosis, potentially involving IL-17-related mechanisms.

Ichthyotoxicity of the microalga Pseudochattonella farcimen under laboratory and field conditions in Danish waters.

Blooms of the marine dictyochophyte Pseudochattonella farcimen have been associated with fish kills, but attempts to verify ichthyotoxicity of this microalga under experimental conditions have not been successful. In the early spring of 2009 and 2011, P. farcimen bloomed in the inner Danish waters. The blooms occurred at a seawater temperature of ~2°C and correlated with extensive kills of farmed salmonid fish (2009) and wild populations (2011). Several strains of P. farcimen were isolated from the 2009 bloom. However, exposure of rainbow trout Oncorhynchus mykiss to laboratory-grown P. farcimen cultures did not reveal any toxic effects. During the 2011 bloom, fish were exposed to bloom water under both laboratory and field conditions. While no clinical effect was observed on fish incubated in bloom water in the laboratory trial, a remarkable difference was seen in the field trial between rainbow trout kept in tanks supplied with a continuous flow of filtered versus non-filtered bloom water. Histological examination of the gill tissue revealed karyorrhexis and epithelial loosening in the affected fish. Microscopy analysis of algal cell morphology suggested that mucocysts detected on the cell surface only in freshly sampled bloom water might be associated with ichtyotoxicity.

The use of segregation analysis in interpretation of sequence variants in SMAD3: A case report.

BACKGROUND: While representing a significant improvement, the introduction of next-generation sequencing in genetic diagnosis also prompted new challenges. Despite widely recognized consensus guidelines for the interpretation of sequence variants, many variants remain unclassified or are discordantly interpreted. In heritable thoracic aortic aneurysms with dissection (HTAAD), most cases are caused by a heterozygous, private missense mutation, possibly contributing to the relatively common reports of variants with uncertain significance in this group. Segregation analysis necessitates advanced likelihood-based methods typically inaccessible to non-experts and is hampered by reduced penetrance, possible phenocopies, and non-availability of DNA from deceased relatives. METHODS: In this report, challenges in variant interpretation and the use of segregation analyses were illustrated in two families with a suspected HTAAD disorder. The R package segregatr, a novel implementation of full-likelihood Bayes factor (FLB), was performed to explore the cosegregation of the variants in these families. CONCLUSION: Using the R package segregatr, cosegregation in the reported families concluded with strong and supporting evidence for pathogenicity. Surveillance of families in a multidisciplinary team enabling systematic phenotype description for standardized segregation analysis with a robust calculation method may be imperative for reliable variant interpretation in HTAAD.

High levels of S100A12 are associated with recent plaque symptomatology in patients with carotid atherosclerosis.

BACKGROUND AND PURPOSE: Atherosclerosis is a progressive chronic disease, in which inflammation plays a key role. The calcium-binding proteins calgranulins including S100A8, S100A9, and S100A12 are involved in many cellular activities and pathological processes including inflammation. We therefore hypothesized that calgranulins may be markers of plaque instability in patients with carotid atherosclerosis. METHODS: Plasma levels of S100A8/A9 and S100A10 were measured in 159 consecutive patients with high-grade carotid stenosis and in 22 healthy control subjects. The mRNA levels of calgranulins were also measured within the atherosclerotic carotid plaques, and their regulation was analyzed in vitro in monocytes. RESULTS: Our main findings were: (1) plasma levels of S100A12 were significantly higher in patients with carotid atherosclerosis compared with healthy control subjects with the highest levels in patients with the most recent symptoms (ie, within 2 months); (2) plasma levels of S100A8/S100A9 showed a modest increase in patients with symptoms in the previous 2 to 6 months but not in the other patients; (3) mRNA levels of S100A8, S100A9, and S100A12 showed increased expression in atherosclerotic carotid plaques from patients with the most recent symptoms compared with the remaining patients; (4) in THP-1 monocytes, activation of Toll-like receptors 2 and 4 increased mRNA levels of S100A8, S100A9, and S10012 and interleukin-1ß, interferon γ, and releasate from thrombin-activated platelets significantly enhanced the expression of S100A12. CONCLUSIONS: Our findings support a link between calgranulins and atherogenesis and suggest that these mediators, and in particular S100A12, may be related to plaque instability.

Genome-wide association scan and phased haplotype construction for quantitative trait loci affecting boar taint in three pig breeds.

BACKGROUND: Boar taint is the undesirable smell and taste of pork meat derived from some entire male pigs. The main causes of boar taint are the two compounds androstenone and skatole (3-methyl-indole). The steroid androstenone is a sex pheromone produced in the testis of the boars. Skatole is produced from tryptophan by bacteria in the intestine of the pigs. In many countries pigs are castrated as piglets to avoid boar taint, however, this is undesirable for animal welfare reasons. Genetic variations affecting the level of boar taint have previously been demonstrated in many breeds. In the study presented in this paper, markers and haplotypes, which can be applied to DNA-based selection schemes in order to reduce or eliminate the boar taint problem, are identified. RESULTS: Approximately 30,000 SNPs segregating in 923 boars from three Danish breeds; Duroc, Landrace, and Yorkshire, were used to conduct genome wide association studies of boar taint compounds. At 46 suggestive quantitative trait loci (QTL), 25 haplotypes and three single markers with effects were identified. Furthermore, 40% of the haplotypes mapped to previously identified regions. Haplotypes were also analysed for effects of slaughter weight and meat content. The most promising haplotype was identified on Sus scrofa chromosome 1. The gain in fixed effect of having this haplotype on level of androstenone in Landrace was identified to be high (1.279 µg/g). In addition, this haplotype explained 16.8% of the phenotypic variation within the trait. The haplotype was identified around the gene CYB5A which is known to have an indirect impact on the amount of androstenone. In addition to CYB5A, the genes SRD5A2, LOC100518755, and CYP21A2 are candidate genes for other haplotypes affecting androstenone, whereas, candidate genes for the indolic compounds were identified to be SULT1A1 and CYP2E1. CONCLUSIONS: Despite the small sample size, a total of 25 haplotypes and three single markers were identified including genomic regions not previously reported. The haplotypes that were analysed showed large effects on trait level. However, little overlap of QTL between breeds was observed.

Markers of extracellular matrix remodeling and systemic inflammation in patients with heritable thoracic aortic diseases.

Background: In approximately 20% of patients with thoracic aortic aneurysms or dissections a heritable thoracic aortic disease (HTAD) is suspected. Several monogenic connective tissue diseases imply high risk of aortic disease, including both non-syndromic and syndromic forms. There are some studies assessing inflammation and extracellular matrix remodeling in patients with non-hereditary aortic disease, but such studies in patients with hereditary diseases are scarce. Aims: To quantify markers of extracellular matrix (ECM) and inflammation in patients with vascular connective tissue diseases versus healthy controls. Methods: Patients with Loeys-Dietz syndrome (LDS, n = 12), Marfan syndrome (MFS, n = 11), and familial thoracic aortic aneurysm 6 (FTAA6, n = 9), i.e., actin alpha 2 (ACTA2) pathogenic variants, were recruited. Exome or genome sequencing was performed for genetic diagnosis. Several markers of inflammation and ECM remodeling were measured in plasma by enzyme immunoassays. Flow cytometry of T-cell subpopulations was performed on a subgroup of patients. For comparison, blood samples were drawn from 14 healthy controls. Results: (i) All groups of HTAD patients had increased levels matrix metalloproteinase-9 (MMP-9) as compared with healthy controls, also in adjusted analyses, reflecting altered ECM remodeling. (ii) LDS patients had increased levels of pentraxin 3 (PTX3), reflecting systemic inflammation. (iii) LDS patients have increased levels of soluble CD25, a marker of T-cell activation. Conclusion: Our data suggest that upregulated MMP-9, a matrix degrading enzyme, is a common feature of several subgroups of HTAD. In addition, LDS patients have increased levels of PTX3 reflecting systemic and in particular vascular inflammation.

Mitral annulus disjunction is associated with adverse outcome in Marfan and Loeys-Dietz syndromes.

AIMS: We aimed to assess the prevalence of mitral annulus disjunction (MAD) and to explore the association with aortic disease and mitral valve surgery in patients with Marfan syndrome (MFS) and Loeys-Dietz syndrome (LDS). METHODS AND RESULTS: We included consecutive MFS patients fulfilling Revised Ghent Criteria and LDS patients fulfilling Loeys-Dietz Revised Nosology. MAD was identified by echocardiography and was quantified as the longitudinal distance from the ventricular myocardium to the hinge point of the posterior mitral leaflet. Aortic events were defined as aortic dissection or prophylactic aortic surgery. We recorded the need of mitral valve surgery including mitral valve repair or replacement. We included 168 patients (103 with MFS and 65 with LDS). The prevalence of MAD was 41%. MAD was present in all age groups. Aortic events occurred in 112 (67%) patients (27 with dissections and 85 with prophylactic surgical interventions). Patients with MAD were younger at aortic event than those without MAD (log rank = 0.02) Patients with aortic events had greater MAD distance in posterolateral wall [8 (7-10) mm vs. 7 (6-8) mm, P = 0.04]. Mitral events occurred more frequently in patients with MAD (P < 0.001). CONCLUSION: MAD was highly prevalent in patients with MFS and LDS. MAD was a marker of severe disease including aortic events at younger age and need of mitral valve surgery. Screening patients with MFS an LDS for MAD may provide prognostic information and may be relevant in planning surgical intervention. Detection of MAD in patients with MFS and LDS may infer closer clinical follow-up from younger age.

Evolution and regulation of the Lotus japonicus LysM receptor gene family.

LysM receptor kinases were identified as receptors of acylated chitin (Nod factors) or chitin produced by plant-interacting microbes. Here, we present the identification and characterization of the LysM receptor kinase gene (Lys) family (17 members) in Lotus japonicus. Comprehensive phylogenetic analysis revealed a correlation between Lys gene structure and phylogeny. Further mapping coupled with sequence-based anchoring on the genome showed that the family has probably expanded by a combination of tandem and segmental duplication events. Using a sliding-window approach, we identified distinct regions in the LysM and kinase domains of recently diverged Lys genes where positive selection may have shaped ligand interaction. Interestingly, in the case of NFR5 and its closest paralog, LYS11, one of these regions coincides with the predicted Nod-factor binding groove and the suggested specificity determining area of the second LysM domain. One hypothesis for the evolutionary diversification of this receptor family in legumes is their unique capacity to decipher various structures of chitin-derived molecules produced by an extended spectrum of interacting organisms: symbiotic, associative, endophytic, and parasitic. In a detailed expression analysis, we found several Lotus Lys genes regulated not only during the symbiotic association with Mesorhizobium loti but also in response to chitin treatment.

Cholesterol crystals use complement to increase NLRP3 signaling pathways in coronary and carotid atherosclerosis.

BACKGROUND: During atherogenesis, cholesterol precipitates into cholesterol crystals (CC) in the vessel wall, which trigger plaque inflammation by activating the NACHT, LRR and PYD domains-containing protein 3 (NLRP3) inflammasome. We investigated the relationship between CC, complement and NLRP3 in patients with cardiovascular disease. METHODS: We analysed plasma, peripheral blood mononuclear cells (PBMC) and carotid plaques from patients with advanced atherosclerosis applying ELISAs, multiplex cytokine assay, qPCR, immunohistochemistry, and gene profiling. FINDINGS: Transcripts of interleukin (IL)-1beta(ß) and NLRP3 were increased and correlated in PBMC from patients with acute coronary syndrome (ACS). Priming of these cells with complement factor 5a (C5a) and tumour necrosis factor (TNF) before incubation with CC resulted in increased IL-1ß protein when compared to healthy controls. As opposed to healthy controls, systemic complement was significantly increased in patients with stable angina pectoris or ACS. In carotid plaques, complement C1q and C5b-9 complex accumulated around CC-clefts, and complement receptors C5aR1, C5aR2 and C3aR1 were higher in carotid plaques compared to control arteries. Priming human carotid plaques with C5a followed by CC incubation resulted in pronounced release of IL-1ß, IL-18 and IL-1α. Additionally, mRNA profiling demonstrated that C5a and TNF priming followed by CC incubation upregulated plaque expression of NLRP3 inflammasome components. INTERPRETATION: We demonstrate that CC are important local- and systemic complement activators, and we reveal that the interaction between CC and complement could exert its effect by activating the NLRP3 inflammasome, thus promoting the progression of atherosclerosis.

Cerebral microemboli and brain injury during carotid artery endarterectomy and stenting.

BACKGROUND AND PURPOSE: Cerebral microembolic signals detected by transcranial Doppler are frequent during carotid angioplasty with stenting and carotid endarterectomy (CEA). Their potential harmful effects on the brain are, however, unclear. The aim of this study was to relate the frequency and type of per-procedural microembolic signals to procedure-related ipsilateral ischemic strokes and new ipsilateral ischemic lesions on diffusion-weighted cerebral MRI. METHODS: Eighty-five patients who were prospectively treated with CEA (61) or carotid angioplasty with stenting (30) for high-grade (>/=70%) internal carotid artery stenoses were monitored during the procedures using multifrequency transcranial Doppler with embolus detection and differentiation. Pre- and postprocedural cerebral diffusion-weighted cerebral MRIs were performed on a subset of patients. RESULTS: Solid and gaseous microemboli were independently associated with procedure-related ipsilateral ischemic strokes (solid: P=0.027, gaseous: P=0.037) or new ipsilateral diffusion-weighted cerebral MRI lesions (solid: P=0.043, gaseous: P=0.026). Microembolic signals were detected during all procedures except one (CEA); 17% and 21% of all emboli were solid during carotid angioplasty with stenting and CEA, respectively. Patients undergoing carotid angioplasty with stenting had more solid (P<0.001) and gaseous (P<0.001) emboli and more new ipsilateral ischemic strokes (P=0.033) compared with patients undergoing CEA. Echolucent plaques (P=0.020) and preprocedural diffusion-weighted cerebral MRI ischemic lesions (P=0.002) were associated with increased numbers of solid emboli. CONCLUSIONS: Solid and gaseous microemboli were increased in patients with procedure-related ipsilateral ischemic strokes or new diffusion-weighted cerebral MRI lesions, which suggests that both solid and gaseous emboli may be harmful to the brain during CEA and carotid angioplasty with stenting.