Basic science232. Certolizumab pegol prevents pro-inflammatory alterations in endothelial cell function

Background: Cardiovascular disease is a major comorbidity of rheumatoid arthritis (RA) and a leading cause of death. Chronic systemic inflammation involving tumour necrosis factor alpha (TNF) could contribute to endothelial activation and atherogenesis. A number of anti-TNF therapies are in current use for the treatment of RA, including certolizumab pegol (CZP), (Cimzia ®; UCB, Belgium). Anti-TNF therapy has been associated with reduced clinical cardiovascular disease risk and ameliorated vascular function in RA patients. However, the specific effects of TNF inhibitors on endothelial cell function are largely unknown. Our aim was to investigate the mechanisms underpinning CZP effects on TNF-activated human endothelial cells. Methods: Human aortic endothelial cells (HAoECs) were cultured in vitro and exposed to a) TNF alone, b) TNF plus CZP, or c) neither agent. Microarray analysis was used to examine the transcriptional profile of cells treated for 6 hrs and quantitative polymerase chain reaction (qPCR) analysed gene expression at 1, 3, 6 and 24 hrs. NF-κB localization and IκB degradation were investigated using immunocytochemistry, high content analysis and western blotting. Flow cytometry was conducted to detect microparticle release from HAoECs. Results: Transcriptional profiling revealed that while TNF alone had strong effects on endothelial gene expression, TNF and CZP in combination produced a global gene expression pattern similar to untreated control. The two most highly up-regulated genes in response to TNF treatment were adhesion molecules E-selectin and VCAM-1 (q 0.2 compared to control; p > 0.05 compared to TNF alone). The NF-κB pathway was confirmed as a downstream target of TNF-induced HAoEC activation, via nuclear translocation of NF-κB and degradation of IκB, effects which were abolished by treatment with CZP. In addition, flow cytometry detected an increased production of endothelial microparticles in TNF-activated HAoECs, which was prevented by treatment with CZP. Conclusions: We have found at a cellular level that a clinically available TNF inhibitor, CZP reduces the expression of adhesion molecule expression, and prevents TNF-induced activation of the NF-κB pathway. Furthermore, CZP prevents the production of microparticles by activated endothelial cells. This could be central to the prevention of inflammatory environments underlying these conditions and measurement of microparticles has potential as a novel prognostic marker for future cardiovascular events in this patient group. Disclosure statement: Y.A. received a research grant from UCB. I.B. received a research grant from UCB. S.H. received a research grant from UCB. All other authors have declared no conflicts of interes

Case Reports1. A Late Presentation of Loeys-Dietz Syndrome: Beware of TGFβ Receptor Mutations in Benign Joint Hypermobility

Background: Thoracic aortic aneurysms (TAA) and dissections are not uncommon causes of sudden death in young adults. Loeys-Dietz syndrome (LDS) is a rare, recently described, autosomal dominant, connective tissue disease characterized by aggressive arterial aneurysms, resulting from mutations in the transforming growth factor beta (TGFβ) receptor genes TGFBR1 and TGFBR2. Mean age at death is 26.1 years, most often due to aortic dissection. We report an unusually late presentation of LDS, diagnosed following elective surgery in a female with a long history of joint hypermobility. Methods: A 51-year-old Caucasian lady complained of chest pain and headache following a dural leak from spinal anaesthesia for an elective ankle arthroscopy. CT scan and echocardiography demonstrated a dilated aortic root and significant aortic regurgitation. MRA demonstrated aortic tortuosity, an infrarenal aortic aneurysm and aneurysms in the left renal and right internal mammary arteries. She underwent aortic root repair and aortic valve replacement. She had a background of long-standing joint pains secondary to hypermobility, easy bruising, unusual fracture susceptibility and mild bronchiectasis. She had one healthy child age 32, after which she suffered a uterine prolapse. Examination revealed mild Marfanoid features. Uvula, skin and ophthalmological examination was normal. Results: Fibrillin-1 testing for Marfan syndrome (MFS) was negative. Detection of a c.1270G > C (p.Gly424Arg) TGFBR2 mutation confirmed the diagnosis of LDS. Losartan was started for vascular protection. Conclusions: LDS is a severe inherited vasculopathy that usually presents in childhood. It is characterized by aortic root dilatation and ascending aneurysms. There is a higher risk of aortic dissection compared with MFS. Clinical features overlap with MFS and Ehlers Danlos syndrome Type IV, but differentiating dysmorphogenic features include ocular hypertelorism, bifid uvula and cleft palate. Echocardiography and MRA or CT scanning from head to pelvis is recommended to establish the extent of vascular involvement. Management involves early surgical intervention, including early valve-sparing aortic root replacement, genetic counselling and close monitoring in pregnancy. Despite being caused by loss of function mutations in either TGFβ receptor, paradoxical activation of TGFβ signalling is seen, suggesting that TGFβ antagonism may confer disease modifying effects similar to those observed in MFS. TGFβ antagonism can be achieved with angiotensin antagonists, such as Losartan, which is able to delay aortic aneurysm development in preclinical models and in patients with MFS. Our case emphasizes the importance of timely recognition of vasculopathy syndromes in patients with hypermobility and the need for early surgical intervention. It also highlights their heterogeneity and the potential for late presentation. Disclosures: The authors have declared no conflicts of interes

Land cover and NDVI are important predictors in habitat selection along migration for the Golden-crowned Sparrow, a temperate-zone migrating songbird

BackgroundMigrating passerines in North America have shown sharp declines. Understanding habitat selection and threats along migration paths are critical research needs, but details about migrations have been limited due to the difficulty of tracking small birds. Recent technological advances of tiny GPS-tags provide new opportunities to delineate fine-scale movements in small passerines during a life stage that has previously been inherently difficult to study.MethodsWe investigated habitat selection along migration routes for a temperate-zone migratory passerine, the Golden-crowned Sparrow (Zonotrichia atricapilla), given GPS tags on California wintering grounds. We used a resource selection function combined with conditional logistic regression to compare matched sets of known stopover locations and available but unused locations to determine how land cover class, vegetation greenness and climate variables influence habitat selection during migration. We also provide general migration descriptions for this understudied species including migration distance, duration, and elevation, and repeated use of stopover areas.ResultsWe acquired 22 tracks across 19 individuals, with a total of 541 valid spring and fall migration locations. Birds traveled to breeding grounds in Alaska and British Columbia along coastal routes, selecting for shrubland and higher vegetation greenness in both migration seasons as well as grasslands during fall migration. However, model interactions showed they selected sites with lower levels of greenness when in forest (both seasons) and shrubland (fall only), which may reflect their preference for more open habitats or represent a trade-off in selection between habitat type and productivity. Birds also selected for locations with higher daily maximum temperature during spring migration. Routes during spring migration were lower in elevation on average, shorter in duration, and had fewer long stopovers than in fall migration. For two birds, we found repeated use of the same stopover areas in spring and fall migration.ConclusionsUsing miniaturized GPS, this study provides new insight into habitat selection along migration routes for a common temperate-zone migrating songbird, contributing to a better understanding of full annual cycle models, and informing conservation efforts. Golden-crowned Sparrows selected for specific habitats along migration routes, and we found previously unknown behaviors such as repeated use of the same stopover areas by individuals across different migratory seasons

Land cover and NDVI are important predictors in habitat selection along migration for the Golden-crowned Sparrow, a temperate-zone migrating songbird

Abstract Background Migrating passerines in North America have shown sharp declines. Understanding habitat selection and threats along migration paths are critical research needs, but details about migrations have been limited due to the difficulty of tracking small birds. Recent technological advances of tiny GPS-tags provide new opportunities to delineate fine-scale movements in small passerines during a life stage that has previously been inherently difficult to study. Methods We investigated habitat selection along migration routes for a temperate-zone migratory passerine, the Golden-crowned Sparrow (Zonotrichia atricapilla), given GPS tags on California wintering grounds. We used a resource selection function combined with conditional logistic regression to compare matched sets of known stopover locations and available but unused locations to determine how land cover class, vegetation greenness and climate variables influence habitat selection during migration. We also provide general migration descriptions for this understudied species including migration distance, duration, and elevation, and repeated use of stopover areas. Results We acquired 22 tracks across 19 individuals, with a total of 541 valid spring and fall migration locations. Birds traveled to breeding grounds in Alaska and British Columbia along coastal routes, selecting for shrubland and higher vegetation greenness in both migration seasons as well as grasslands during fall migration. However, model interactions showed they selected sites with lower levels of greenness when in forest (both seasons) and shrubland (fall only), which may reflect their preference for more open habitats or represent a trade-off in selection between habitat type and productivity. Birds also selected for locations with higher daily maximum temperature during spring migration. Routes during spring migration were lower in elevation on average, shorter in duration, and had fewer long stopovers than in fall migration. For two birds, we found repeated use of the same stopover areas in spring and fall migration. Conclusions Using miniaturized GPS, this study provides new insight into habitat selection along migration routes for a common temperate-zone migrating songbird, contributing to a better understanding of full annual cycle models, and informing conservation efforts. Golden-crowned Sparrows selected for specific habitats along migration routes, and we found previously unknown behaviors such as repeated use of the same stopover areas by individuals across different migratory seasons

Macrophage Depletion Does Not Prevent Tissue Plasminogen Activator (tPA) Upregulation in Islets with Amyloid Deposition

peer reviewe

Loss of perlecan heparan sulfate glycosaminoglycans lowers body weight and decreases islet amyloid deposition in human islet amyloid polypeptide transgenic mice.

peer reviewedIslet amyloid is a pathologic feature of type 2 diabetes (T2D) that is associated with β-cell loss and dysfunction. These amyloid deposits form via aggregation of the β-cell secretory product islet amyloid polypeptide (IAPP) and contain other molecules including the heparan sulfate proteoglycan perlecan. Perlecan has been shown to bind amyloidogenic human IAPP (hIAPP) via its heparan sulfate glycosaminoglycan (HS GAG) chains and to enhance hIAPP aggregation in vitro. We postulated that reducing the HS GAG content of perlecan would also decrease islet amyloid deposition in vivo. hIAPP transgenic mice were crossed with Hspg2Δ3/Δ3 mice harboring a perlecan mutation that prevents HS GAG attachment (hIAPP;Hspg2Δ3/Δ3), and male offspring from this cross were fed a high fat diet for 12 months to induce islet amyloid deposition. At the end of the study body weight, islet amyloid area, β-cell area, glucose tolerance and insulin secretion were analyzed. hIAPP;Hspg2Δ3/Δ3 mice exhibited significantly less islet amyloid deposition and greater β-cell area compared to hIAPP mice expressing wild type perlecan. hIAPP;Hspg2Δ3/Δ3 mice also gained significantly less weight than other genotypes. When adjusted for differences in body weight using multiple linear regression modeling, we found no differences in islet amyloid deposition or β-cell area between hIAPP transgenic and hIAPP;Hspg2Δ3/Δ3 mice. We conclude that loss of perlecan exon 3 reduces islet amyloid deposition in vivo through indirect effects on body weight and possibly also through direct effects on hIAPP aggregation. Both of these mechanisms may promote maintenance of glucose homeostasis in the setting of T2D

Islet macrophage depletion does not prevent amyloid-induced upregulation of tissue plasminogen activator (tPA)

peer reviewedBackground and Aims: Islet amyloid deposition and islet inflammation are pathogenic hallmarks of type 2 diabetes. Aggregation of human islet amyloid polypeptide (hIAPP) increases pro-inflammatory gene expression in islet macrophages and results in beta-cell loss. We previously found the fibrinolysis activator tissue plasminogen activator (tPA) is specifically upregulated in islets with amyloid deposition and reduces amyloid fibril formation through plasmin generation. Since macrophages can produce tPA, we determined whether they contribute to hIAPP-induced tPA upregulation. Materials and Methods: To determine whether hIAPP increases tPA expression in macrophages, tPA mRNA levels were quantified in rat bone-marrow-derived macrophages (BMDMs) treated for 24 h with 10 µM hIAPP, non-amyloidogenic 10 µM mouse IAPP (mIAPP) or vehicle. Further, to determine the contribution of islet macrophages to tPA upregulation by amyloid deposition, isolated islets from hIAPP transgenic mice (that have the propensity to form islet amyloid) and wild-type mIAPP mice (that do not form islet amyloid) were treated for 48 h with clodronate-containing liposomes (CLOD-lip) to deplete macrophages or PBS-containing liposomes (PBS-lip) as control. Islets were then cultured for 48 h in 16.7 mM glucose, which induces amyloid deposition in hIAPP, but not mIAPP islets. Islet macrophage markers (Emr1 and Itgam) and tPA mRNA levels were quantified. Results: hIAPP but not mIAPP increased tPA gene expression in BMDMs (4.21.0 vs 1.50.6 vs 1.00.5 fold, hIAPP vs mIAPP vs vehicle respectively; n=4; p<0.05 hIAPP vs mIAPP and vehicle; p=0.25 mIAPP vs vehicle). As expected, tPA gene expression significantly increased by 2.1-fold in PBS-lip-treated hIAPP islets vs mIAPP islets treated with PBS-lip (Table 1). In both hIAPP and mIAPP islets, CLOD-lip treatment significantly abrogated Emr1 and Itgam gene expression vs PBS-lip treatment, confirming islet macrophages depletion in both genotypes (Table 1). CLOD-lip treatment also significantly reduced tPA expression (vs PBS-lip) in both hIAPP and mIAPP islets; however, tPA expression was still increased by 2.3-fold in CLOD-lip treated hIAPP islets vs mIAPP islets (Table 1). Conclusion: hIAPP treatment induces tPA expression in macrophages and islet macrophage depletion decreases, but does not prevent, upregulation of tPA in amyloid-laden islets. Thus, our data suggests that, in addition to macrophages, other islet cell types produce tPA in response to amyloid deposition

DSCAM gene triplication causes excessive GABAergic synapses in the neocortex in Down syndrome mouse models

Down syndrome (DS) is caused by the trisomy of human chromosome 21 (HSA21). A major challenge in DS research is to identify the HSA21 genes that cause specific symptoms. Down syndrome cell adhesion molecule (DSCAM) is encoded by a HSA21 gene. Previous studies have shown that the protein level of the Drosophila homolog of DSCAM determines the size of presynaptic terminals. However, whether the triplication of DSCAM contributes to presynaptic development in DS remains unknown. Here, we show that DSCAM levels regulate GABAergic synapses formed on neocortical pyramidal neurons (PyNs). In the Ts65Dn mouse model for DS, where DSCAM is overexpressed due to DSCAM triplication, GABAergic innervation of PyNs by basket and chandelier interneurons is increased. Genetic normalization of DSCAM expression rescues the excessive GABAergic innervations and the increased inhibition of PyNs. Conversely, loss of DSCAM impairs GABAergic synapse development and function. These findings demonstrate excessive GABAergic innervation and synaptic transmission in the neocortex of DS mouse models and identify DSCAM overexpression as the cause. They also implicate dysregulated DSCAM levels as a potential pathogenic driver in related neurological disorders. Developmental brain disorders are a hallmark of Down syndrome, but what cellular and molecular mechanisms underlie these disorders? This study shows that the excessive number of inhibitory synapses in the neocortex of Down syndrome mouse models is caused by increased levels of Down Syndrome Cell Adhesion Molecule (DSCAM)