Background: An antibody specific to small-molecule inhibitor-bound TNF has enabled the development of target occupancy biomarker assays to support the development of novel treatments for autoimmune disorders. Materials & methods: ELISAs were developed for inhibitor-bound and total TNF to determine the percentage of TNF occupancy in samples from stimulated blood. Inhibitor-saturated samples allowed measurement of total and inhibitor-bound TNF in a single electrochemiluminescence immunoassay. Results: TNF occupancy was proportional to inhibitor concentration in plasma samples. An electrochemiluminescence method for inhibitor-bound TNF was validated for use as a potential clinical occupancy biomarker assay. Conclusion: Development of these assays has allowed measurement of a target occupancy biomarker, which has supported progression of the first small-molecule inhibitors of TNF.

OBJECTIVES This phase 2 proof-of-concept study (NCT02610543) assessed efficacy, safety and effects on salivary gland inflammation of seletalisib, a potent and selective PI3Kδ inhibitor, in patients with moderate-to-severe primary Sjögren's syndrome (PSS). METHODS Adults with PSS were randomized 1:1 to seletalisib 45 mg/day or placebo, in addition to current PSS therapy. Primary end points were safety and tolerability and change from baseline in EULAR Sjögren's Syndrome Disease Activity Index (ESSDAI) score at week 12. Secondary end points included change from baseline at week 12 in EULAR Sjögren's Syndrome Patient Reported Index (ESSPRI) score and histological features in salivary gland biopsies. RESULTS Twenty-seven patients were randomized (seletalisib n = 13, placebo n = 14); 20 completed the study. Enrolment challenges led to early study termination with loss of statistical power (36% vs 80% planned). Nonetheless, a trend for improvement in ESSDAI and ESSPRI [difference vs placebo: -2.59 (95% CI: -7.30, 2.11; P=0.266) and -1.55 (95% CI: -3.39, 0.28), respectively] was observed at week 12. No significant changes were seen in saliva and tear flow. Serious adverse events (AEs) were reported in 3/13 of patients receiving seletalisib vs 1/14 for placebo and 5/13 vs 1/14 discontinued due to AEs, respectively. Serum IgM and IgG concentrations decreased in the seletalisib group vs placebo. Seletalisib demonstrated efficacy in reducing size and organisation of salivary gland inflammatory foci and in target engagement, thus reducing PI3K-mTOR signalling compared with placebo. CONCLUSION Despite enrolment challenges, seletalisib demonstrated a trend towards clinical improvement in patients with PSS. Histological analyses demonstrated encouraging effects of seletalisib on salivary gland inflammation and organisation. TRIAL REGISTRATION https://clinicaltrials.gov, NCT02610543.

Mutations in two genes can result in activated PI3Kδ syndrome (APDS), a rare immunodeficiency disease with limited therapeutic options. Seletalisib, a potent, selective PI3Kδ inhibitor, was evaluated in patients with APDS1 and APDS2. In the phase 1b study (European Clinical Trials Database 2015-002900-10) patients with genetic and clinical confirmation of APDS1 or APDS2 received 15-25 mg/d seletalisib for 12 wk. Patients could enter an extension study (European Clinical Trials Database 2015-005541). Primary endpoints were safety and tolerability, with exploratory efficacy and immunology endpoints. Seven patients (median age 15 years; APDS1 n = 3; APDS2 n = 4) received seletalisib; five completed the phase 1b study. For the extension study, four patients entered, one withdrew consent (week 24), three completed ≥84 wk of treatment. In the phase 1b study, patients had improved peripheral lymphadenopathy (n = 2), lung function (n = 1), thrombocyte counts (n = 1), and chronic enteropathy (n = 1). Overall, effects were maintained in the extension. In the phase 1b study, percentages of transitional B cells decreased, naive B cells increased, and senescent CD8 T cells decreased (human cells); effects were generally maintained in the extension. Seletalisib-related adverse events occurred in four of seven patients (phase 1b study: hepatic enzyme increased, dizziness, aphthous ulcer, arthralgia, arthritis, increased appetite, increased weight, restlessness, tendon disorder, and potential drug-induced liver injury) and one of four patients had adverse events in the extension (aphthous ulcer). Serious adverse events occurred in three of seven patients (phase 1b study: hospitalization, colitis, and potential drug-induced liver injury) and one of four patients had adverse events in the extension (stomatitis). Patients with APDS receiving seletalisib had improvements in variable clinical and immunological features, and a favorable risk-benefit profile was maintained for ≤96 wk.

Mutations in two genes can result in activated PI3K delta syndrome (APDS), a rare immunodeficiency disease with limited therapeutic options. Seletalisib, a potent, selective PI3K delta inhibitor, was evaluated in patients with APDS1 and APDS2. In the phase lb study (European Clinical Trials Database 2015-002900-10) patients with genetic and clinical confirmation of APDS1 or APDS2 received 15-25 mg/d seletalisib for 12 wk. Patients could enter an extension study (European Clinical Trials Database 2015-005541). Primary endpoints were safety and tolerability, with exploratory efficacy and immunology endpoints. Seven patients (median age 15 years; APDS1 n = 3; APDS2 n = 4) received seletalisib; five completed the phase lb study. For the extension study, four patients entered, one withdrew consent (week 24), three completed >= 84 wk of treatment. In the phase lb study, patients had improved peripheral lymphadenopathy (n = 2), lung function (n = 1), thrombocyte counts (n = 1), and chronic enteropathy (n = 1). Overall, effects were maintained in the extension. In the phase lb study, percentages of transitional B cells decreased, naive B cells increased, and senescent CD8 T cells decreased (human cells); effects were generally maintained in the extension. Seletalisib-related adverse events occurred in four of seven patients (phase lb study: hepatic enzyme increased, dizziness, aphthous ulcer, arthralgia, arthritis, increased appetite, increased weight, restlessness, tendon disorder, and potential drug-induced liver injury) and one of four patients had adverse events in the extension (aphthous ulcer). Serious adverse events occurred in three of seven patients (phase lb study: hospitalization, colitis, and potential drug-induced liver injury) and one of four patients had adverse events in the extension (stomatitis). Patients with APDS receiving seletalisib had improvements in variable clinical and immunological features, and a favorable risk-benefit profile was maintained for <= 96 wk.

Background: Seletalisib is a potent, selective oral inhibitor of phosphoinositide-3 kinase delta (PI3Kδ). Preclinical data have shown that the PI3Kδ pathway is upregulated within salivary glands of patients with PSS and contributes to disease pathogenesis.1 Objectives: To assess the efficacy and safety of seletalisib in patients with PSS. Methods: In this Phase II, double-blind, proof of concept study (NCT02610543), patients with PSS having an EULAR Sjögren's Syndrome Disease Activity Index (ESSDAI) score ≥5 were randomised 1:1 to seletalisib once daily or placebo (PBO) in addition to current PSS therapy for 12 weeks. The primary endpoint was change from baseline in ESSDAI at Week 12. The study was designed to have 80% power to detect a difference of 3.8 points in change from baseline in ESSDAI between seletalisib and PBO at Week 12 and required 58 patients to complete treatment. Other endpoints included EULAR Sjögren's Syndrome Patient Reported Index (ESSPRI), salivary gland biopsy changes, Schirmer's I test, immunoglobulin concentrations and incidence of treatment-emergent adverse events (TEAEs). Results: The study was terminated early due to slow recruitment, which led to study power decreasing to 36%. Twenty of 27 patients randomised (seletalisib n=13, PBO n=14) completed treatment. Demographic characteristics were generally similar between groups. Mean (SE) change from baseline in ESSDAI at Week 12 was seletalisib –5.4 (1.7) vs PBO –2.8 (1.5); treatment difference vs PBO (95% CI) was –2.59 (–7.30, 2.11; p=0.266). The percentages of patients achieving a ≥3 point reduction in ESSDAI were seletalisib 66.7% vs PBO 54.5%. Post-hoc Bayesian analyses of treatment difference showed an 86.5% probability of being superior to PBO and a 48.8% probability of a >3 point difference from PBO. Clinically notable improvements in some secondary endpoints were also observed in the seletalisib group (Table 1). Minor salivary gland biopsies had broadly similar histological features across groups at baseline. At Week 12, seletalisib treatment led to a reduction in the size and cellular organisation of mononuclear inflammatory cell foci vs PBO (Table 2). TEAEs were reported by 13/13 (100.0%) seletalisib and 13/14 (92.9%) PBO patients; most frequently reported: diarrhoea (5/13 [38.5%] vs 0/14 [0%]) and headache (3/13 [23.1%] vs 2/14 [14.3%]). Serious TEAEs were reported by 3/13 (23.1%) vs 1/14 (7.1%), and discontinuations due to TEAEs by 5/13 (38.5%) vs 1/14 (7.1%) seletalisib and PBO patients, respectively. Conclusion: Although this Phase II PSS study was terminated early due to slow recruitment, seletalisib demonstrated a trend to clinical improvement in patients with PSS and acceptable safety and tolerability. Histological analyses demonstrated encouraging effects of seletalisib on the organisation and extent of salivary gland lymphocytic infiltration in patients with PSS. References [1] Nayar S, et al. Ann Rheum Dis. 2019;78:249–260 Acknowledgement: Funded by UCB Pharma Disclosure of Interests: Maria Juarez Shareholder of: UCB Pharma, Employee of: UCB Pharma, Nieves Diaz Shareholder of: UCB Pharma, Employee of: UCB Pharma, Geoffrey I. Johnston Shareholder of: UCB Pharma, Pfizer, AstraZeneca, Employee of: UCB Pharma (and AstraZeneca + Pfizer previously), Saba Nayar: None declared, Andrew Payne Employee of: UCB Pharma, Eric Helmer Employee of: Galapagos, UCB Pharma, Takeda, Johnson & Johnson, Dionne Cain Employee of: UCB Pharma, Paulette Williams Shareholder of: UCB Pharma, Employee of: UCB Pharma, Wan Fai Ng: None declared, Benjamin Fisher Consultant for: Novartis, Roche, MedImmune, Bristol-Myers Squibb, Jacques-Eric Gottenberg Grant/research support from: Bristol-Myers Squibb, Grant/research support from: Bristol-Myers Squibb, Consultant for: Bristol-Myers Squibb, Lilly, Pfizer, Sanofi-Genzyme, UCB Pharma, Consultant for: Bristol-Myers Squibb, Eli Lilly, UCB, Sanofi-Genzyme, Pfizer, Giuliana Guggino Grant/research support from: Laborest, Pfizer, Consultant for: Novartis, Abbvie, Speakers bureau: Sandoz, Xavier Mariette Grant/research support from: Servier, Consultant for: AstraZeneca, Bristol-Myers Squibb, GlaxoSmithKline, Janssen, Pfizer, UCB Pharma, Marika Kvarnström: None declared, Valerie Devauchelle-Pensec Grant/research support from: Roche-Chugai, Speakers bureau: MSD, BMS, UCB, Roche, Jose Rosas Consultant for: Abbvie, Amgen, Bristol, Janssen, Lilly, Merck Sharp & Dohme, Pfizer, UCB Pharma, Speakers bureau: Abbvie, Amgen, Bristol, Janssen, Lilly, Merck Sharp & Dohme, Pfizer, UCB Pharma, Juan Sanchez-Burson Speakers bureau: Lilly, Janssen, Pfizer, Roberto Giacomelli Grant/research support from: Pfizer, Actelion, Speakers bureau: Actelion, Bristol-Myers Squibb, Merck Sharp & Dohme, Abbvie, Pfizer, Sobi, Roche, Francesca Barone Grant/research support from: GlaxoSmithKline, Roche, UCB Pharma, Actelion, ONO Pharmaceutical, Consultant for: GlaxoSmithKline, Roche, Actelion, ONO Pharmaceutical, Simon J. Bowman Grant/research support from: Previously UCB Pharma (to University of Birmingham) and Roche, Consultant for: 2016-7: Novartis, Mitsubishi Tanabe Pharma 2017-8: AstraZeneca, MedImmune, GFK, Xtlbio, ONO Pharmaceutical 2018-9: Novartis, AstraZeneca, UCB Pharma

ABSTRACTBackground: Although primarily a neurodegenerative process, there is increasing awareness of peripheral disease mechanisms in Parkinson's disease. To investigate disease processes in accessible patient cells, we studied peripheral blood mononuclear cells in recently diagnosed PD patients and rapid eye movement‐sleep behavior disorder patients who have a greatly increased risk of developing PD. We hypothesized that peripheral blood mononuclear cells may recapitulate cellular pathology found in the PD brain and investigated these cells for mitochondrial dysfunction and oxidative stress.Methods: Peripheral blood mononuclear cells were isolated and studied from PD patients, rapid eye movement‐sleep behavior disorder patients and age‐ and sex‐matched control individuals from the well‐characterized Oxford Discovery cohort. All participants underwent thorough clinical assessment.Results: Initial characterization showed that PD patients had elevated levels of CD14 + monocytes and monocytes expressing C‐C motif chemokine receptor 2. Mitochondrial dysfunction and oxidative stress were increased in PD patient peripheral blood mononuclear cells, with elevated levels of mitochondrial reactive oxygen species specifically in patient monocytes. This was combined with reduced levels of the antioxidant superoxide dismutase in blood cells from PD patients and, importantly, also in rapid eye movement‐sleep behavior disorder patients. This mitochondrial dysfunction was associated with a concomitant increase in glycolysis in both PD and rapid eye movement‐sleep behavior disorder patient blood cells independent of glucose uptake or monocyte activation.Conclusions: This work demonstrates functional bioenergetic deficits in PD and rapid eye movement‐sleep behavior disorder patient blood cells during the early stages of human disease. © 2018 The Authors. Movement Disorders published by Wiley Periodicals, Inc. on behalf of International Parkinson and Movement Disorder Society.

Objectives Systemic lupus erythematosus (SLE) is a heterogeneous autoimmune disease associated with diffuse immune cell dysfunction. CD40–CD40 ligand (CD40L) interaction activates B cells, antigen-presenting cells and platelets. CD40L blockade might provide an innovative treatment for systemic autoimmune disorders. We investigated the safety and clinical activity of dapirolizumab pegol, a polyethylene glycol conjugated anti-CD40L Fab' fragment, in patients with SLE. Methods This 32-week randomised, double-blind, multicentre study (NCT01764594) evaluated repeated intravenous administration of dapirolizumab pegol in patients with SLE who were positive for/had history of antidouble stranded DNA/antinuclear antibodies and were on stable doses of immunomodulatory therapies (if applicable). Sixteen patients were randomised to 30 mg/kg dapirolizumab pegol followed by 15 mg/kg every 2 weeks for 10 weeks; eight patients received a matched placebo regimen. Randomisation was stratified by evidence of antiphospholipid antibodies. Patients were followed for 18 weeks after the final dose. Results No serious treatment-emergent adverse events, thromboembolic events or deaths occurred. Adverse events were mild or moderate, transient and resolved without intervention. One patient withdrew due to infection. Efficacy assessments were conducted only in patients with high disease activity at baseline. Five of 11 (46%) dapirolizumab pegol-treated patients achieved British Isles Lupus Assessment Group-based Composite Lupus Assessment response (vs 1/7; 14% placebo) and 5/12 (42%) evaluable for SLE Responder Index-4 responded by week 12 (vs 1/7; 14% placebo). Mechanism-related gene expression changes were observed in blood RNA samples. Conclusions Dapirolizumab pegol could be an effective biological treatment for SLE. Further studies are required to address efficacy and safety. Trial registration number NCT01764594.

Background CD40-CD40L is a critical costimulatory pathway necessary for normal immune function (including B cell differentiation, germinal center formation and antibody isotype switching) and strongly implicated in the pathogenesis of autoimmune disorders such as SLE. In a randomised, placebo-controlled, parallel group Phase 1 study, dapirolizumab pegol (DZP), a pegylated anti-CD40L Fab9, was administered to patients with active SLE on stable background therapy (NCT01764594). Objectives To examine the effect of DZP on peripheral blood RNA transcripts associated with immune cell subsets and SLE disease activity as biomarkers for proof of biological activity. Methods Twenty-four subjects were randomised (2:1) to receive DZP or placebo and 22 subjects completed the study. Subjects randomised to DZP received a loading dose of 30 mg/kg followed by 15 mg/kg every 2 weeks for a total of 6 doses and were subsequently followed for 18 weeks. For transcriptional analyses, blood was collected in PAXgene tubes at Weeks 0, 2, 4, 8, 12, 20 and 28. A panel of 96 genes was selected for analysis by qPCR including genes expressed in plasma cells, B cells and other immune cells. Also included were transcripts associated with SLE disease activity, such as the Type I Interferon (IFN) signature. For the analysis, genes were grouped into functional domains (such as B cells, T cells, IFN) and decision criteria were designed to identify consistent expression changes within a domain. At a gene level, a positive signal required a >2 fold up- or down-regulated change from baseline, and occurring on at least 2 successive visits. Differences in changes from baseline between DZP and placebo groups were considered significant at <0.0894 (to achieve a 5% false positive rate at a functional domain level). Results Changes in genes within the plasma cell and B cell domains had the greatest degree of statistical significance. Among the plasma cell genes, the DZP group exhibited rapid decreases in the expression of several immunoglobulin-associated genes (secretory IgA, IgG, Igk, Igl, J chain) starting at Week 2 and maintained over the treatment period. Among the B cell genes, the DZP group showed a transient increase in CD19 and CD20 RNA transcripts at Week 2 that was mirrored by flow cytometry data. Additionally, there were transient increases in membrane IgD, membrane IgM and TCL1A genes which are associated with naïve B cells. Several of the patients in the DZP group also exhibited a ≥2 fold reduction in the expression of IFN-responsive genes (MX1, OAS1, IFITM3, G1P2) for at least 2 time points. No other consistent changes in other functional group expression patterns were observed. Conclusions In summary, DZP treatment resulted in changes in peripheral blood RNA transcripts found in plasma cells and B cells consistent with the inhibition of CD40L:CD40 signaling. A trend in the reduction of the IFN signature was also observed with DZP. The utility of this approach to monitor biological activity, early clinical response and patient stratification will be further explored. Disclosure of Interest A. Ranger: None declared, N. Allaire Employee of: Biogen, P. Colman Employee of: Biogen, C. Wager Employee of: Biogen, H. Li Employee of: Biogen, A. Thai Employee of: Biogen, P. Cullen Employee of: Biogen, C. Otoul Employee of: UCB Pharma, J. Czerkowicz Employee of: Biogen, C. Roberts Employee of: Biogen, C. Chamberlain Shareholder of: UCB Pharma, Employee of: UCB Pharma, L. Burkly Employee of: Biogen, G. Johnston Shareholder of: UCB Pharma, Employee of: UCB Pharma

Background Dipeptidyl-peptidase-4 (DPP4/CD26) has been recently identified as a marker for a special fibroblast lineage responsible for the tissue remodeling during physiological wound healing. Fibrotic disease may be considered as a consequence of persistent, exaggerated and uncontrolled tissue repair processes. Systemic sclerosis (SSc) is associated with the highest mortality among the connective tissue disorders and effective antifibrotic therapies are still lacking. DPP4 inhibitors are already used in treatment of diabetes. Objectives The aim of the study was to characterize the DPP4 positive cells, investigate the expression of DPP4 in SSc skin and to evaluate the antifibrotic effect of DPP4 inhibitors in preclinical models of systemic sclerosis. Methods Mouse fibroblasts were isolated and DPP4 positive cells characterized by fluorescence activating cell sorting. Expression of DPP4/CD26 in human and murine skin was analyzed by immunofluorescence. DPP4 inhibitors were tested in two different concentrations administered orally (Sitagliptin 3mg/kg/d and 10mg/kg/d, Vildagliptin 1,5mg/kg/d and 15mg/kg/d) in bleomycin induced skin fibrosis and in sclerodermatous chronic graft-versus-host disease mouse model (cGvHD). The antifibrotic effect on skin was assessed by hydroxyproline assay, alpha smooth muscle cells quantification and measuring the dermal thickness. Inflammatory infiltrate was assessed by CD45 immunofluorescence staining. Results We have demonstrated that DPP4/CD26 positive cells are a unique population of cells implicated in fibrosis. DPP4/CD26 positive cells were increased not only in experimental fibrosis, but also in skin biopsies from SSc patients as compared to healthy volunteers. Treatment with DPP4 inhibitor reduced dermal thickness in both mouse models (p<0.05). The differentiation of resting fibroblasts into myofibroblasts was also significantly decreased (p<0.05) in all treatment groups. Hydroxyproline content of the skin diminished by 40% in comparison with NaCl injected mice or syngeneic transplanted mice. Moreover, DDP4 inhibitors reduced the inflammatory infiltrate in a dose dependent manner in the bleomycin injected skin. Conclusions DPP4/CD26 identifies a subpopulation of fibrosis-promoting fibroblasts that plays a key role in the pathogenesis of fibrosis in SSc. Moreover, inhibitors of DPP4 show a significant antifibrotic effect in several mouse models of SSc in well tolerated doses. These results may have direct clinical implications as DPP4 inhibitors are already in clinical use for diabetes. Acknowledgement AS received an EULAR Scientific Training Bursary (2014) and was an Articulum Fellow (2015). Disclosure of Interest None declared

Several SNPs located in or around the IL28B gene are associated with response of patients infected with Hepatitis C virus to treatment with pegylated interferon-α ⁺/⁻ ribavirin or with spontaneous clearance of the virus. The results of such studies are so compelling that future treatment approaches are likely to involve clinical decisions being made on the basis of a patient's genotype. Since IL28B is a paralogue of IL28A with greater than 95% sequence identity, it is possible that without genotyping assay specificity, sequences in IL28A may contribute to genotype identification, and potentially confound treatment decisions. This study aimed to 1) examine DNA sequences in IL28B surrounding each of the reported associated SNPs and the corresponding regions in IL28A; and 2) develop a robust assay for rs12979860, the most 'cosmopolitan' SNP most strongly associated with treatment response across all global populations studied to date. Bioinformatic analysis of genomic regions surrounding IL28A and IL28B demonstrated that 3 SNPs were unique to IL28B, whereas the remaining 6 SNP regions shared >93% identity between IL28A and IL28B. Using a panel of DNA samples, PCR amplification followed by Sanger sequencing was used to examine IL28B SNPs and the corresponding regions in IL28A. For the overlapping SNPs, all 6 in IL28B were confirmed to be polymorphic whereas the corresponding positions in IL28A were monomorphic. Based upon IL28A and IL28B sequence data, a specific TaqMan® assay was developed for SNP rs12979860 that was 100% concordant to the sequence-derived genotypes. Analysis using a commercial assay identified one discordant result which led to a change in their genotype-calling algorithm. Where future treatment decisions are made upon the results of genotyping assays, it is very important that results are concordant with data from a sequence-based format. This is especially so in situations where designing specific PCR primers is a challenge.

BACKGROUND:Type 2 diabetes is a risk factor for progression of non-alcoholic fatty liver disease (NAFLD) to fibrosis and cirrhosis. We examined the prevalence of advanced liver disease in people with type 2 diabetes and analysed the effectiveness of liver function tests (LFTs) as a screening tool. METHODS:Participants (n = 939, aged 61-76 years) from the Edinburgh Type 2 Diabetes Study, a randomly selected population of people with type 2 diabetes, underwent abdominal ultrasonography. Hyaluronic acid (HA) and platelet count/spleen diameter ratio (PSR) were used as non-invasive markers of hepatic fibrosis and portal hypertension. Subjects were screened for secondary causes of liver disease that excluded them from a diagnosis of NAFLD. The efficacy of LFTs [alanine aminotransferase (ALT) and gamma-glutamyltransferase (GGT)] in screening for liver disease was determined. RESULTS:Cirrhosis was identified by ultrasound in four participants (0.4%). Ten (1.1%) had evidence of portal hypertension (PSR < 909), and two (0.2%) had hepatocellular carcinoma. Fifty-three participants (5.7%) had evidence of hepatic fibrosis (HA > 100 ng/ml in the absence of joint disease); a further 169 had HA > 50 ng/ml. In participants with NAFLD-related fibrosis (HA > 100 ng/ml), 12.5% had an elevated ALT level and 17.5% had an elevated GGT level. CONCLUSION:The prevalence of hepatic fibrosis and cirrhosis were lower than expected. The use of LFTs to screen for liver disease missed most cases of fibrosis predicted by raised HA levels.

AIM: To compare ultrasound gradings of steatosis with fat fraction (FF) on magnetic resonance spectroscopy (MRS; the non-invasive reference standard for quantification of hepatic steatosis), and evaluate inter- and intraobserver variability in the ultrasound gradings.MATERIALS AND METHODS: Triple grading of hepatic ultrasound examination was performed by three independent graders on 131 people with type 2 diabetes. The stored images of 60 of these individuals were assessed twice by each grader on separate occasions. Fifty-eight patients were pre-selected on the basis of ultrasound grading (normal, indeterminate/mild steatosis, or severe steatosis) to undergo H-1-MRS. The sensitivity and specificity of the ultrasound gradings were determined with reference to MRS data, using two cut-offs of FF to define steatosis, >= 9% and >= 6.1%.RESULTS: Median (intraquartile range) MRS FF (%) in the participants graded on ultrasound as normal, indeterminate/mild steatosis, and severe steatosis were 4.2 (1.2-5.7), 4.1 (3.1-8.5) and 19.4(12.9-27.5), respectively. Using a liver FF of >= 6.1% on MRS to denote hepatic steatosis, the unadjusted sensitivity and specificity of ultrasound gradings (severe versus other grades of steatosis) were 71 and 100%, respectively. Interobserver agreement within one grade was observed in 79% of cases. Exact intraobserver agreement ranged from 62 to 87%.CONCLUSION: Hepatic ultrasound provided a good measure of the presence of significant hepatic steatosis with good intra- and interobserver agreement. The grading of a mildly steatotic liver was less secure and, in particular, there was considerable overlap in hepatic FF with those who had a normal liver on ultrasound. (C) 2011 The Royal College of Radiologists. Published by Elsevier Ltd. All rights reserved.

OBJECTIVE Type 2 diabetes is an established risk factor for development of hepatic steatosis and nonalcoholic fatty liver disease (NAFLD). We aimed to determine the prevalence and clinical correlates of these conditions in a large cohort of people with type 2 diabetes. RESEARCH DESIGN AND METHODS A total of 939 participants, aged 61–76 years, from the Edinburgh Type 2 Diabetes Study (ET2DS)—a large, randomly selected population of people with type 2 diabetes—underwent liver ultrasonography. Ultrasound gradings of steatosis were compared with magnetic resonance spectroscopy in a subgroup. NAFLD was defined as hepatic steatosis in the absence of a secondary cause (screened by questionnaire assessing alcohol and hepatotoxic medication use, plasma hepatitis serology, autoantibodies and ferritin, and record linkage to determine prior diagnoses of liver disease). Binary logistic regression was used to analyze independent associations of characteristics with NAFLD. RESULTS Hepatic steatosis was present in 56.9% of participants. After excluding those with a secondary cause for steatosis, the prevalence of NAFLD in the study population was 42.6%. Independent predictors of NAFLD were BMI, lesser duration of diabetes, HbA1c, triglycerides, and metformin use. These remained unchanged after exclusion of participants with evidence of hepatic fibrosis from the group with no hepatic steatosis. CONCLUSIONS Prevalences of hepatic steatosis and NAFLD were high in this unselected population of older people with type 2 diabetes, but lower than in studies in which ultrasound gradings were not compared with a gold standard. Associations with features of the metabolic syndrome could be used to target screening for this condition.

Adverse drug reactions are a major clinical problem. Drug-induced hepatotoxicity constitutes a large percentage of these reactions. A thorough understanding of the genetic events, specifically, the early "decision-making" processes underlying biological changes caused by drugs and metabolites, is required. To assist in the understanding of these events, we have employed the model hepatotoxin, paracetamol (APAP), and GeneChip technology to investigate global genetic events seen after nontoxic and toxic doses in the mouse. Mice were dosed [vehicle, nontoxic APAP (1 mmol/ kg), and toxic APAP (3.5 mmol/kg)], and individual hepatic RNA samples were hybridized to separate chips to determine interanimal variation. Statistical analysis detected 175 CD-1 mouse genes that were significantly regulated (P < 4.1 x 10(-6)), and nonsignificant genes were discarded. For clarity, the significantly regulated genes were then binned into categories according to their major function-antioxidant, glutathione, metabolism, transcription, immune, and apoptosis. There was no hepatic stress observed after dosing 1 mmol/kg APAP, when measured by serum alanine aminotransferase levels. Hepatic toxicity was observed at both 4 and 24 h after a 3.5 mmol/kg dose of APAP. Time course expression profiles for selected genes have been created. These results demonstrate that most active gene expression occurs around 4 h after a toxic dose of APAP. Down-regulation of these genes is observed over 24 h, coinciding with the development of overt toxicity. These data provide a deeper understanding of the in vivo time course of physiological responses of the liver to chemical stress and provide a logical step forward for the investigation of new chemical entities demonstrated positive in chemically reactive metabolite screens. The complete data set can be viewed at http:// www.ebi.ac.uk/arrayexpress/. The accession number is E-MEXP-82.

We have cloned cDNAs encoding three human alpha-1 adrenergic receptor (AR) subtypes and characterized pharmacological properties of the expressed receptor protein. A number of significant sequence corrections have been identified and compared with previously published data, at both nucleotide and amino acid levels; the most major differences occur for the human alpha-1a/dAR. Pharmacological characterization was performed simultaneously using six cloned alpha-1AR subtypes (human and rat alpha-1a/d, human and hamster alpha-1b, human and bovine alpha-1c) stably expressed in rat-1 fibroblasts at approximately equal receptor concentrations (1-2 pmol/mg of total protein). In general, human alpha-1AR subtypes have similar pharmacology compared to their rat, hamster and bovine homologs, although a few minor species differences important for alpha-1AR classification are noted. In addition, much lower inactivation (approximately 20%) by the alkylating agent chloroethylclonidine is noted in this study compared to previous reports for both human and bovine alpha-1cAR membrane preparations. All six alpha-1AR subtypes couple to phosphoinositide hydrolysis in a pertussis toxin-insensitive manner, including the cloned human alpha-1a/dAR which had not been expressed previously. In spite of significant sequence differences between human alpha-1ARs and their other species counterparts, previously established ligand selectivity remains fairly comparable. In summary, these data represent the first side-by-side comparison of pharmacological properties between species homologs of alpha-1AR subtypes and should facilitate the development of alpha-1AR subtype selective drugs for clinical use.

It has been suggested by a number of investigators that glycoproteins may play a role in the development and/or maintenance of synapses in the mammalian CNS. For many synaptic glycoproteins, however, little precise structural or functional information is available. In an effort to isolate probes specific to individual glycoproteins, we have screened a rat brain cDNA expression library with a mixed polyclonal antibody directed against concanavalin A-binding synaptic junctional glycoproteins. Using this approach, we have previously reported the cloning of SC1, a putative extracellular matrix glycoprotein found in adult brain (Johnston et al., Neuron 2:165-176, 1990). We now report the cloning and characterization of a second novel cDNA, which has been designated SC2. Northern blots show that this cDNA recognizes a 1.2-kb mRNA that is present throughout postnatal development in the rat. It is expressed at high levels in brain and is also found at lower levels in several other tissues. In situ hybridization suggests that the SC2 mRNA is strongly expressed by many types of neurons. Sequence data reveals a single open reading frame in the cDNA, encoding a putative hydrophobic protein with a calculated molecular weight of 36.1 kDa. Sequence analysis reveals some similarity between SC2 and 5 alpha-reductase, a microsomal membrane protein important in testosterone metabolism.

Endothelial-leukocyte adhesion molecule 1 is a cell surface glycoprotein expressed by cytokine-activated endothelium that mediates the adhesion of blood neutrophils. Endothelial-leukocyte adhesion molecule 1 is a member of the selectin family of cell adhesion molecules each of which contain an amino-terminal lectin-like domain, followed by an epidermal growth factor-like domain and a variable number of short consensus repeats similar to those found in complement binding proteins. Genomic clones encoding the ELAM gene were isolated and the organization of the ELAM gene was determined. The gene, which is present in a single copy in the human genome, contains 14 exons spanning about 13 kilobases of DNA. The positions of exon-intron boundaries correlate with the putative functional subdivisions of the protein. Introns are found at similar positions in all of the six complement regulatory repeats, suggesting that these elements arose by internal gene duplication. A consensus TATAA element is located upstream of the transcriptional start site. The ELAM promoter contains an inverted CCAAT box and consensus NF-kappa B- and AP-1-binding sites. The ELAM gene was assigned to the q12 greater than qter region of human chromosome 1 by analysis of human-mouse hybrid cell lines. Two other members of the selectin gene family, the leukocyte adhesion molecule 1 (LAM-1, TQ1, LEC-CAM 1, or Leu-8) and the granule membrane protein 140 (GMP-140, PADGEM, or CD62) have been localized to the long arm of chromosome 1, as have the structurally related complement binding proteins, suggesting that these genes may share a common evolutionary history.