Acute and Repeated Administration of NLX-101, a Selective Serotonin-1A Receptor Biased Agonist, Reduces Audiogenic Seizures in Developing Fmr1 Knockout Mice.

Fragile XSyndrome (FXS) is a leading known genetic cause of Autism Spectrum Disorders (ASD) and intellectual disability. A consistent and debilitating phenotype of FXS is sensory hypersensitivity that manifests strongly in the auditory domain and may lead to delayed language and high anxiety. The mouse model of FXS, the Fmr1 KO mouse, also shows auditory hypersensitivity, an extreme form of which is seen as audiogenic seizures (AGS). The midbrain inferior colliculus (IC) is critically involved in generating audiogenic seizures and IC neurons are hyper-responsive to sounds in developing Fmr1 KO mice. Serotonin-1A receptor (5-HT1A) activation reduces IC activity. Therefore, we tested whether 5-HT1A activation is sufficient to reduce audiogenic seizures in Fmr1 KO mice. A selective and post-synaptic 5-HT1A receptor biased agonist, 3-Chloro-4-fluorophenyl-[4-fluoro-4-[[(5-methylpyrimidin-2-ylmethyl)amino]methyl]piperidin-1-yl] methanone (NLX-101, 0.6, 1.2, 1.8 or 2.4 mg/kg, i.p.) was administered to Fmr1 KO mice 15 min before seizure induction. Whereas the 0.6 mg/kg dose was ineffective in reducing seizures, the 1.2, 1.8 and 2.4 mg/kg doses of NLX-101 dramatically reduced seizures and increased mouse survival. Treatment with a combination of NLX-101 and 5-HT1A receptor antagonists prevented the protective effects of NLX-101, indicating that NLX-101 acts selectively through 5-HT1A receptors to reduce audiogenic seizures. NLX-101 (1.8 mg/kg) was still strongly effective in reducing seizures even after repeated administration over 5 days, suggesting an absence of tachyphylaxis to the effects of the compound. Together, these studies point to a promising treatment option targeting post-synaptic 5-HT1A receptors to reduce auditory hypersensitivity in FXS, and potentially across autism spectrum disorders.

NLX-112, a highly selective 5-HT1A receptor biased agonist, does not exhibit misuse potential in male rats or macaques.

NLX-112 (a.k.a. F13640 or befiradol) exhibits nanomolar affinity, exceptional selectivity and biased agonism at serotonin 5-HT1A receptors. NLX-112 displays robust analgesic activity in a number of rodent models of pain, and is currently developed as a treatment for l-DOPA-induced dyskinesia (LID) in Parkinson's disease (PD) patients. Noteworthy, PD patients can suffer from comorbid chronic pain, thus necessitating the use of analgesic drugs, such as opioids, which have potential for misuse. Additionally, dopamine agonists used to treat PD can produce cocaine-like effects in preclinical assays of misuse potential. The present study investigated whether NLX-112 possesses misuse potential of its own using two behavioural assays routinely used for this purpose: intracranial self-stimulation (ICSS) in rats, and cocaine discrimination in macaque monkeys. In rats, low doses of NLX-112 (0.03 and 0.1 mg/kg p.o.) did not alter ICSS frequency-rate curves, while higher doses (0.3 and 1.0 mg/kg) shifted the curve to the right and flattened it, i.e., reduced ICSS. As expected, cocaine (10 mg/kg i.p.) shifted the curve to the left, i.e., facilitated ICSS, but NLX-112 (0.03 and 0.1 mg/kg p.o.) did not further enhance cocaine-induced facilitation of ICSS. In monkeys trained to discriminate cocaine (0.4 mg/kg i.m.) from saline, NLX-112 (0.01-0.1 mg/kg p.o.) did not substitute for cocaine. Taken together, these results suggest that NLX-112, at doses displaying anti-dyskinetic activity in rat, marmoset and macaque models of LID, is free from misuse potential. From a translational perspective, this is a desirable property for a compound destined to be used in PD patients, who can suffer from comorbid chronic pain necessitating the use of potentially misused analgesic drugs.

Bell-shaped agonist activation of 5-HT1A receptor-coupled Gαi3 G-proteins: Receptor density-dependent switch in receptor signaling.

A previous study observed bell-shaped concentration-response isotherms for activation of Gαi3 G-protein subunits by high efficacy 5-HT1A receptor agonists in a Chinese hamster ovary (CHO) cell line expressing high levels of these receptors. This suggested that a signaling switch took place in that cell line (from Gαi3 to activation of other G-proteins) but it was unclear if such effects are observed for 5-HT1A receptors in other cellular environments. Here, using an antibody capture-based [35S]GTPγS binding assay for Gαi3 activation, we investigated whether efficacious 5-HT1A receptor agonists (5-HT, F13714, befiradol, NLX-101), prototypical agonists ((+) and (-)8-OH-DPAT), and partial agonist, antagonists, inverse agonists (pindolol, WAY100635, spiperone) produced similar effects on 5 cell lines expressing different levels of human 5-HT1A receptors. In membranes from cell lines (HeLa, C6-glia and CHO-low) expressing moderate receptor levels (between 1 and 4 pmol/mg of protein), 5-HT, F13714, befiradol and NLX-101 elicited classical sigmoid concentration-response isotherms. In contrast, in cell lines (CHO-high, HEK-293F) expressing high receptor levels (>9 pmol/mg) these agonists elicited bell-shaped concentration-response isotherms that peaked at nanomolar-range concentrations and then returned to baseline or below. Spiperone elicited inverse agonist inhibitory sigmoid isotherms in all membrane preparations while WAY100635 was mostly 'silent' for Gαi3 activation. The other compounds elicited diverse responses in the different cell lines suggesting that other factors, in addition to receptor expression levels, could be influencing Gαi3 activation. These data indicate that Gαi3 G-protein activation by 5-HT1A receptor ligands is highly dependent on receptor expression levels and on cellular background. Moreover, the induction of bell-shape concentration-response isotherms by 5-HT and other high-efficacy agonists is consistent with a switch in signaling to other G-protein-mediated signaling cascades, possibly elicited by receptor conformational changes.

Discovering the pharmacodynamics of conolidine and cannabidiol using a cultured neuronal network based workflow.

Determining the mechanism of action (MOA) of novel or naturally occurring compounds mostly relies on assays tailored for individual target proteins. Here we explore an alternative approach based on pattern matching response profiles obtained using cultured neuronal networks. Conolidine and cannabidiol are plant-derivatives with known antinociceptive activity but unknown MOA. Application of conolidine/cannabidiol to cultured neuronal networks altered network firing in a highly reproducible manner and created similar impact on network properties suggesting engagement with a common biological target. We used principal component analysis (PCA) and multi-dimensional scaling (MDS) to compare network activity profiles of conolidine/cannabidiol to a series of well-studied compounds with known MOA. Network activity profiles evoked by conolidine and cannabidiol closely matched that of ω-conotoxin CVIE, a potent and selective Cav2.2 calcium channel blocker with proposed antinociceptive action suggesting that they too would block this channel. To verify this, Cav2.2 channels were heterologously expressed, recorded with whole-cell patch clamp and conolidine/cannabidiol was applied. Remarkably, conolidine and cannabidiol both inhibited Cav2.2, providing a glimpse into the MOA that could underlie their antinociceptive action. These data highlight the utility of cultured neuronal network-based workflows to efficiently identify MOA of drugs in a highly scalable assay.

NLX-112, a highly selective 5-HT1A receptor agonist, mediates analgesia and antidepressant-like activity in rats via spinal cord and prefrontal cortex 5-HT1A receptors, respectively.

NLX-112 (a.k.a. F13640 or befiradol) possesses marked activity in a variety of animal models of pain and of neuropsychiatric disorders; it exhibits nanomolar affinity, exceptional selectivity and high agonist efficacy at 5-hydroxytryptamine1A (5-HT1A) receptors. Although NLX-112 has been shown to activate 5-HT1A postsynaptic heteroreceptors in the prefrontal cortex (PFC), a brain region involved in the control of depressive states, the influence of NLX-112 on spinal cord 5-HT1A receptors (implicated in the control of pain) has not been described. Here we report on the ability, in rats, of NLX-112 to elicit analgesia in the intraplantar formalin model of nociceptive pain following intrathecal (i.t.) administration, and its ability to produce antidepressant-like activity in the forced swim test (FST) following in situ PFC microinjection. NLX-112, injected i.t. (L5-L6 region) induced analgesic effects in the formalin model of tonic nociceptive pain. At 20 µg, it almost abolished the effect of formalin on both the paw licking and paw elevation measures, and in both the early (0-5 min after formalin administration, reflecting acute pain) and the late (22.5-27.5 min, reflecting inflammatory pain) phases. The effects of NLX-112 (20 µg i.t.) were reversed by co-administration of 20 µg i.t. of the 5-HT1A receptor antagonist, WAY100635. Furthermore, the analgesic effects of systemically administered NLX-112 (0.63 mg/kg i.p.) were reversed by i.t. administration of WAY100635 (20 µg), most notably on paw licking. Finally, microinjection of NLX-112, bilaterally in the PFC, dose-dependently (MED 4 µg) and markedly reduced immobility in the FST (circa 90% reduction at 32 µg). In conclusion, the present data demonstrate that activation of spinal cord-located 5-HT1A receptors is sufficient for NLX-112 to mediate its analgesic effects in a rat model of tonic nociceptive pain. The data also highlight the involvement of PFC 5-HT1A receptors in the antidepressant-like activity of NLX-112 in the FST. Overall, the study suggests that highly selective and high efficacy 5-HT1A receptors agonists, such as NLX-112, could be useful to treat painful conditions associated with depressive states, through activation of different sub-populations of 5-HT1A receptors.

Resolving incomplete single nucleotide polymorphism tagging of HLA-DQ2.2 for coeliac disease genotyping using digital droplet PCR.

A hallmark of coeliac disease (CD) is the exceptionally strong genetic association with HLA-DQ2.5, DQ8, and DQ2.2. HLA typing provides information on CD risk important to both clinicians and researchers. A method that enables simple and fast detection of all CD risk genotypes is particularly desirable for the study of large populations. Single nucleotide polymorphism (SNP)-based HLA typing can detect the CD risk genotypes by detecting a combination of six SNPs but this approach can struggle to resolve HLA-DQ2.2, seen in 4% of European CD patients, because of the low resolution of one negatively predicting SNP. We sought to optimise SNP-based HLA typing by harnessing the additional resolution of digital droplet PCR to resolve HLA-DQ2.2. Here we test this two-step approach in an unselected sample of Mexican DNA and compare its accuracy to DNA typed using traditional exon detection. The addition of digital droplet PCR for samples requiring negative prediction of HLA-DQ2.2 enabled HLA-DQ2.2 to be accurately typed. This technique is a simple addition to a SNP-based typing strategy and enables comprehensive definition of all at-risk HLA genotypes in CD in a timely and cost-effective manner.

NLX-112, a highly selective 5-HT1A receptor agonist: Effects on body temperature and plasma corticosterone levels in rats.

NLX-112 (a.k.a. F13640 or befiradol), exhibits nanomolar affinity, exceptional selectivity and high agonist efficacy at 5-hydroxytryptamine 5-HT1A receptors. It possesses marked activity in a variety of animal models of depression, pain and L-DOPA-induced dyskinesia. However, its influence on translational biomarkers of central 5-HT1A receptor activation has not been previously described. Here, we report on the activity, in rats, of NLX-112 to increase plasma corticosterone levels and produce hypothermia, two responses which are also elicited by 5-HT1A receptor agonists in humans. NLX-112 elicited dose-dependent hypothermia (minimal effective dose, MED: 0.31mg/kg p.o.) and also increased plasma corticosterone both by oral and intraperitoneal routes (MED: 0.63mg/kg in both cases). The increase in corticosterone induced by NLX-112 (0.63mg/kg p.o.) was abolished by co-administration of the selective 5-HT1A receptor antagonist, WAY100635. Additionally, NLX-112 also dose-dependently induced flat body posture, forepaw treading and lower lip retraction (MEDs 0.31-0.63mg/kg p.o.). The doses of NLX-112 which induce hypothermia or corticosterone release were similar to those inducing serotonergic behaviors but greater than those reported previously in models of therapeutic-like activity (range 0.04 to 0.16mg/kg). Overall, the present study provides information for clinical dose estimations of NLX-112 and suggests that therapeutic effects may occur at doses below those at which biomarker responses are observed.

CXCR1/2 Chemokine Network Regulates Melanoma Resistance to Chemotherapies Mediated by NF-κB.

BACKGROUND: Cancer-related inflammation is recognized as a driver for tumor progression and chemokines are important players in both inflammation and the progression of many cancer types. CXC chemokines, especially CXCL8, have been implicated in melanoma growth and metastasis, while less is known for their roles in drug resistance. METHODS: We generated drug-resistant cells by continuous exposure to chemotherapeutic drugs and analyzed the mechanism(s) of therapy resistance in malignant melanoma. RESULTS: We report chemotherapies induced upregulation of a variety of chemokines in the CXCR1/CXCR2 network by an NF-κB-dependent mechanism. Notably, analysis of the drug-resistant melanoma cell line selected after prolonged exposure to chemotherapeutic drug dacarbazine revealed higher levels of CXCL8 and CXCR2 compared with parent cells as a signature of drug resistance. CXCR2 neutralization markedly improved sensitivity to dacarbazine in melanoma cells. CONCLUSION: These data provide insights into what drives melanoma cells to survive after chemotherapy treatment, thus pointing to strategies for developing combined drug therapies for combating the problem of chemotherapy resistance in melanoma.

Epistasis between TIFAB and miR-146a: neighboring genes in del(5q) myelodysplastic syndrome.

This corrects the article DOI: 10.1038/leu.2016.276.

Emergency medicine providers' opioid prescribing practices stratified by gender, age, and years in practice / 世界急诊医学杂志（英文）

@#BACKGROUND: Emergency medicine providers (EMPs) prescribe about 25％ of opioids, but the effect of EMP risk perception on decisions to prescribe opioids is unknown. This study was undertaken to identify factors that influence EMP risk and opioid prescribing practices. METHODS: We distributed an anonymous questionnaire to EMPs at a military trauma and referral center. Response frequencies and distributions were assessed for independence using the Chi-square test. RESULTS: Eighty-nine EMPs completed the questionnaire (100％ response). Respondents were primarily younger male physicians (80％) in practice under five years (55％). Male EMPs were more likely to prescribe more opioid tablets than female ones both when and when not concerned for opioid misuse (P<0.001, P<0.007, respectively). Of the providers, 70％ stated that patient age would influence their prescribing decisions. Hydrocodone and oxycodone were the opioids prescribed most frequently. About 60％ of the providers reported changing their prescribing behavior would not prevent opioid misuse. Additionally, 40％ of the providers believed at least 10％ of patients seen at this military ED misused opioids. CONCLUSION: Female EM providers reported prescribing fewer opioid tablets. Patient age influenced prescribing behavior, but the effect is unknown. Finally, EM providers reported that altering their prescribing behavior would not prevent prescription opioid misuse.

NLX-112, a novel 5-HT1A receptor agonist for the treatment of L-DOPA-induced dyskinesia: Behavioral and neurochemical profile in rat.

L-DOPA is the gold-standard treatment for Parkinson's disease (PD), but induces troublesome dyskinesia after prolonged treatment. This is associated with the 'false neurotransmitter' conversion of L-DOPA to dopamine by serotonin neurons projecting from the raphe to the dorsal striatum. Reducing their activity by targeting pre-synaptic 5-HT1A receptors should thus be an attractive therapeutic strategy, but previous 5-HT1A agonists have yielded disappointing results. Here, we describe the activity of a novel, highly selective and potent 5-HT1A agonist, NLX-112 (also known as befiradol or F13640) in rat models relevant to PD and its associated affective disorders. NLX-112 (0.16 mg/kg, i.p.) potently and completely reversed haloperidol-induced catalepsy in intact rats and abolished L-DOPA-induced Abnormal Involuntary Movements (AIMs) in hemiparkinsonian rats, an effect that was reversed by the selective 5-HT1A antagonist, WAY100635. In microdialysis experiments, NLX-112 profoundly decreased striatal 5-HT extracellular levels, indicative of inhibition of serotonergic function. NLX-112 also blunted the L-DOPA-induced surge in dopamine levels on the lesioned side of the brain, an action that likely underlies its anti-dyskinetic effects. NLX-112 (0.16 mg/kg, i.p.) robustly induced rotations in hemiparkinsonian rats, suggesting that it has a motor facilitatory effect. Rotations were abolished by WAY100635 and were ipsilateral to the lesioned side, suggesting a predominant stimulation of the dopamine system on the non-lesioned side of the brain. NLX-112 also efficaciously reduced immobility time in the forced swim test (75% reduction at 0.16 mg/kg, i.p.) and eliminated stress-induced ultrasonic vocalization at 0.08 mg/kg, i.p., effects consistent with potential antidepressant- and anxiolytic-like properties. In other tests, NLX-112 (0.01-0.16 mg/kg, i.p.) did not impair the ability of L-DOPA to rescue forepaw akinesia in the cylinder test but decreased rotarod performance, probably due to induction of flat body posture and forepaw treading which are typical of 5-HT1A agonists upon acute administration. However, upon repeated administration of NLX-112 (0.63 mg/kg, i.p., twice a day), flat body posture and forepaw treading subsided within 4 days of treatment. Taken together, these observations suggest that NLX-112 could exhibit a novel therapeutic profile, combining robust anti-dyskinetic properties without impairing the therapeutic properties of L-DOPA, and with additional beneficial effects on non-motor (affective) symptoms.

Appropriate clinical use of human leukocyte antigen typing for coeliac disease: an Australasian perspective.

The past decade has seen human leukocyte antigen (HLA) typing emerge as a remarkably popular test for the diagnostic work-up of coeliac disease with high patient acceptance. Although limited in its positive predictive value for coeliac disease, the strong disease association with specific HLA genes imparts exceptional negative predictive value to HLA typing, enabling a negative result to exclude coeliac disease confidently. In response to mounting evidence that the clinical use and interpretation of HLA typing often deviates from best practice, this article outlines an evidence-based approach to guide clinically appropriate use of HLA typing, and establishes a reporting template for pathology providers to improve communication of results.

Activity of serotonin 5-HT(1A) receptor 'biased agonists' in rat models of Parkinson's disease and L-DOPA-induced dyskinesia.

Serotonin 5-HT1A receptor agonists reduce L-DOPA-induced dyskinesia (LID) in animal models of Parkinson's disease (PD). Here, we compared the effects of novel 5-HT1A receptor 'biased agonists' on LID in hemiparkinsonian rats. F13714 preferentially activates pre-synaptic 5-HT1A autoreceptors. F15599 preferentially activates cortical postsynaptic 5-HT1A heteroreceptors. The partial agonist, tandospirone, does not differentiate these receptor subpopulations. The drugs were also tested on rotational behavior, rotarod and cylinder test for evaluation of locomotor activity, motor coordination and forelimb akinesia. Finally, the effects of F13714 and F15599 on 5-HT, DA, glutamate, and GABA release were investigated by microdialysis. F13714 abolished L-DOPA-induced AIMs even at very low doses (0.02-0.04 mg/kg). This effect was reversed by the selective 5-HT1A receptor antagonist, WAY100635. F13714 also elicited ipsilateral rotations (which were blocked by WAY100635) and potentiated the rotational activity of a sub-threshold dose of L-DOPA (2 mg/kg). F13714 profoundly inhibited striatal 5-HT release on both sides of the brain, and slightly increased DA release on the intact side. F15599 inhibited the L-DOPA-induced AIMs only at a dose (0.16 mg/kg) that reduced 5-HT release. Tandospirone produced a modest attenuation of peak AIMs severity and did not elicit rotations. F13714, F15599 and tandospirone did not modify the action of L-DOPA in the cylinder test but impaired rotarod performance at the highest doses tested. Targeting 5-HT1A receptors with selective biased agonists exerts distinct effects in the rat model of PD and LID. Preferential activation of 5-HT1A autoreceptors could potentially translate to superior antidyskinetic and L-DOPA dose-sparing effects in PD patients.

Divergent effects of the 'biased' 5-HT1 A receptor agonists F15599 and F13714 in a novel object pattern separation task.

BACKGROUND AND PURPOSE: Pattern separation, that is, the formation of distinct representations from similar inputs, is an important hippocampal process implicated in cognitive domains like episodic memory. A deficit in pattern separation could lead to memory impairments in several psychiatric and neurological disorders. Hence, mechanisms by which pattern separation can be increased are of potential therapeutic interest. EXPERIMENTAL APPROACH: 5-HT1A receptors are involved in spatial memory. Herein we tested the 'biased' 5-HT1A receptor agonists F15599, which preferentially activates post-synaptic heteroreceptors, and F13714, which preferentially activates raphe-located autoreceptors, in rats in a novel spatial task assessing pattern separation, the object pattern separation (OPS) task. KEY RESULTS: The acetylcholinesterase inhibitor donepezil, which served as a positive control, significantly improved spatial pattern separation at a dose of 1 mg·kg(-1) , p.o. F15599 increased pattern separation at 0.04 mg·kg(-1) , i.p., while F13714 decreased pattern separation at 0.0025 mg·kg(-1) , i.p. The selective 5-HT1A receptor antagonist WAY-100635 (0.63 mg·kg(-1) , s.c.) counteracted the effects of both agonists. These data suggest that acute preferential activation of post-synaptic 5-HT1A heteroreceptors improves spatial pattern separation, whereas acute preferential activation of raphe-located 5-HT1A autoreceptors impairs performance. CONCLUSIONS AND IMPLICATIONS: We successfully established and validated a novel, simple and robust OPS task and observed a diverging profile of response with 'biased' 5-HT1A receptor agonists based on their targeting of receptors in distinct brain regions. Our data suggest that the post-synaptic 5-HT1A receptor consists of a potential novel molecular target to improve pattern separation performance.

Topological switching and orbiting dynamics of colloidal spheres dressed with chiral nematic solitons.

Metastable configurations formed by defects, inclusions, elastic deformations and topological solitons in liquid crystals are a promising choice for building photonic crystals and metamaterials with a potential for new optical applications. Local optical modification of the director or introduction of colloidal inclusions into a moderately chiral nematic liquid crystal confined to a homeotropic cell creates localized multistable chiral solitons. Here we induce solitons that "dress" the dispersed spherical particles treated for tangential degenerate boundary conditions, and perform controlled switching of their state using focused optical beams. Two optically switchable distinct metastable states, toron and hopfion, bound to colloidal spheres into structures with different topological charges are investigated. Their structures are examined using Q-tensor based numerical simulations and compared to the profiles reconstructed from the experiments. A topological explanation of observed multistability is constructed.

Higher body mass index in adults at diagnosis of the slowly progressive form of type 1 diabetes mellitus is associated with lower risk HLA genes.

We hypothesised that higher body weight, a proposed risk factor for type 1 diabetes mellitus, would be associated with increased penetrance of lower risk genes. In adults at diagnosis of the slowly progressive form of type 1 diabetes mellitus we found that higher body mass index was associated with the absence of the highest risk HLA genes.

Common and well-documented HLA alleles: 2012 update to the CWD catalogue.

We have updated the catalogue of common and well-documented (CWD) human leukocyte antigen (HLA) alleles to reflect current understanding of the prevalence of specific allele sequences. The original CWD catalogue designated 721 alleles at the HLA-A, -B, -C, -DRB1, -DRB3/4/5, -DQA1, -DQB1, and -DPB1 loci in IMGT (IMmunoGeneTics)/HLA Database release 2.15.0 as being CWD. The updated CWD catalogue designates 1122 alleles at the HLA-A, -B, -C, -DRB1, -DRB3/4/5, -DQA1, -DQB1, -DPA1 and -DPB1 loci as being CWD, and represents 14.3% of the HLA alleles in IMGT/HLA Database release 3.9.0. In particular, we identified 415 of these alleles as being 'common' (having known frequencies) and 707 as being 'well-documented' on the basis of ~140,000 sequence-based typing observations and available HLA haplotype data. Using these allele prevalence data, we have also assigned CWD status to specific G and P designations. We identified 147/151 G groups and 290/415 P groups as being CWD. The CWD catalogue will be updated on a regular basis moving forward, and will incorporate changes to the IMGT/HLA Database as well as empirical data from the histocompatibility and immunogenetics community. This version 2.0.0 of the CWD catalogue is available online at cwd.immunogenomics.org, and will be integrated into the Allele Frequencies Net Database, the IMGT/HLA Database and National Marrow Donor Program's bioinformatics web pages.

Secreted semaphorin 5A suppressed pancreatic tumour burden but increased metastasis and endothelial cell proliferation.

BACKGROUND: Our earlier reports demonstrated that membrane-bound semaphorin 5A (SEMA5A) is expressed in aggressive pancreatic cancer cells and tumours, and promotes tumour growth and metastasis. In this study, we examine whether (1) pancreatic cancer cells secrete SEMA5A and (2) that secreted SEMA5A modulates certain phenotypes associated with tumour progression, angiogenesis and metastasis through various other molecular factors and signalling proteins. METHODS AND RESULTS: In this study, we show that human pancreatic cancer cell lines secrete the extracellular domain (ECD) of SEMA5A (SEMA5A-ECD) and overexpression of mouse Sema5A-ECD in Panc1 cells (not expressing SEMA5A; Panc1-Sema5A-ECD; control cells - Panc1-control) significantly increases their invasion in vitro via enhanced ERK phosphorylation. Interestingly, orthotopic injection of Panc1-Sema5A-ECD cells into athymic nude mice results in a lower primary tumour burden, but enhances the micrometastases to the liver as compared with Panc1-control cells. Furthermore, there is a significant increase in proliferation of endothelial cells treated with conditioned media (CM) from Panc1-Sema5A-ECD cells and a significant increase in microvessel density in Panc1-Sema5A-ECD orthotopic tumours compared with those from Panc1-control cells, suggesting that the increase in liver micrometastases is probably due to increased tumour angiogenesis. In addition, our data demonstrate that this increase in endothelial cell proliferation by Sema5A-ECD is mediated through the angiogenic molecules - interleukin-8 and vascular endothelial growth factor. CONCLUSION: Taken together, these results suggest that a bioactive, secreted form of Sema5A-ECD has an intriguing and potentially important role in its ability to enhance pancreatic tumour invasiveness, angiogenesis and micrometastases.

Use of class I and class II HLA loci for predicting age at onset of type 1 diabetes in multiple populations.

AIMS/HYPOTHESIS: The study aimed to assess, in multiple populations, the role of HLA alleles on early and late age at onset of type 1 diabetes. METHODS: Stepwise linear regression models were used to determine which HLA class I and class II risk alleles to include. High-resolution genotyping data for patients from the Type 1 Diabetes Genetics Consortium (T1DGC) collection (n = 2,278) and four independent cohorts from Denmark, Sardinia and the USA (Human Biological Data Interchange [HBDI] and Joslin Diabetes Center) (n = 1,324) (total n = 3,602) were used to assess the role of HLA variation on age of onset and predict early onset (age ≤ 5 years) and late onset (age ≥ 15 years) of type 1 diabetes. RESULTS: In addition to carriage of HLA class I alleles A*24:02, B*39:06, B*44:03 and B*18:01, HLA class II DRB1-DQB1 loci significantly contributed to age at onset, explaining 3.4% of its variance in the combined data. HLA genotypes, together with sex, were able to predict late onset in all cohorts studied, with AUC values ranging from 0.58 to 0.63. Similar AUC values (0.59-0.70) were obtained for early onset for most cohorts, except in the Sardinian study, in which none of the models tested had significant predictive power. CONCLUSIONS/INTERPRETATION: HLA associations with age of onset are consistent across most white populations and HLA information can predict some of the risk of early and late onset of type 1 diabetes. Considerable heterogeneity was observed between Sardinian and other populations, particularly with regard to early age of onset.