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Background The recombinant protein-based vaccine, NVX-CoV2373, demonstrated 89.7% efficacy against coronavirus disease 2019 (COVID-19) in a phase 3, randomized, observer-blinded, placebo-controlled trial in the United Kingdom. The protocol was amended to include a blinded crossover. Data to the end of the placebo-controlled phase are reported. Methods Adults aged 18–84 years received 2 doses of NVX-CoV2373 or placebo (1:1) and were monitored for virologically confirmed mild, moderate, or severe COVID-19 (onset from 7 days after second vaccination). Participants who developed immunoglobulin G (IgG) against nucleocapsid protein but did not show symptomatic COVID-19 were considered asymptomatic. Secondary outcomes included anti-spike (S) IgG responses, wild-type virus neutralization, and T-cell responses. Results Of 15 185 participants, 13 989 remained in the per-protocol efficacy population (6989 NVX-CoV2373, 7000 placebo). At a maximum of 7.5 months (median, 4.5) postvaccination, there were 24 cases of COVID-19 among NVX-CoV2373 recipients and 134 cases among placebo recipients, a vaccine efficacy of 82.7% (95% confidence interval [CI], 73.3%–88.8%). Vaccine efficacy was 100% (95% CI, 17.9%–100.0%) against severe disease and 76.3% (95% CI, 57.4%–86.8%) against asymptomatic disease. High anti-S and neutralization responses to vaccination were evident, together with S-protein–specific induction of interferon-γ secretion in peripheral blood T cells. Incidence of serious adverse events and adverse events of special interest were similar between groups. Conclusions A 2-dose regimen of NVX-CoV2373 conferred a high level of ongoing protection against asymptomatic, symptomatic, and severe COVID-19 through >6 months postvaccination. A gradual decrease of protection suggests that a booster may be indicated. Clinical Trials Registration EudraCT, 2020-004123-16.

Background The safety and immunogenicity profile of COVID-19 vaccines when administered concomitantly with seasonal influenza vaccines have not yet been reported. We therefore aimed to report the results of a substudy within a phase 3 UK trial, by evaluating the safety, immunogenicity, and efficacy of NVX-CoV2373 when co-administered with licensed seasonal influenza vaccines. Methods We did a planned exploratory substudy as part of the randomised, observer-blinded, placebo-controlled, phase 3 trial of the safety and efficacy of the COVID-19 vaccine (NVX-CoV2373) by co-administrating the influenza vaccine at four study hospitals in the UK. Approximately, the first 400 participants meeting the main study entry criteria—with no contraindications to influenza vaccination—were invited to join the substudy. Participants of the main study were randomly assigned (1:1) to receive two intramuscular injections of either NVX-CoV2373 (5 μg) or placebo (normal saline) 21 days apart; participants enrolled into the substudy were co-vaccinated with a single (0·5 mL) intramuscular, age-appropriate (quadrivalent influenza cell-based vaccine [Flucelvax Quadrivalent; Seqirus UK, Maidenhead] for those aged 18–64 years and adjuvanted trivalent influenza vaccine [Fluad; Seqirus UK, Maidenhead] for those ≥65 years), licensed, influenza vaccine on the opposite deltoid to that of the first study vaccine dose or placebo. The influenza vaccine was administered in an open-label manner and at the same time as the first study injection. Reactogenicity was evaluated via an electronic diary for 7 days after vaccination in addition to monitoring for unsolicited adverse events, medically attended adverse events, and serious adverse events. Immunogenicity was assessed with influenza haemagglutination inhibition and SARS-CoV-2 anti-spike protein IgG assays. Vaccine efficacy against PCR-confirmed, symptomatic COVID-19 was assessed in participants who were seronegative at baseline, received both doses of study vaccine or placebo, had no major protocol deviations affecting the primary endpoint, and had no confirmed cases of symptomatic COVID-19 from the first dose until 6 days after the second dose (per-protocol efficacy population). Immunogenicity was assessed in participants who received scheduled two doses of study vaccine, had a baseline sample and at least one post-vaccination sample, and had no major protocol violations before unmasking (per-protocol immunogenicity population). Reactogenicity was analysed in all participants who received at least one dose of NVX-CoV2373 or placebo and had data collected for reactogenicity events. Safety was analysed in all participants who received at least one dose of NVX-CoV2373 or placebo. Comparisons were made between participants of the substudy and the main study (who were not co-vaccinated for influenza). This study is registered with ClinicalTrials.gov, number NCT04583995. Findings Between Sept 28, 2020, and Nov 28, 2020, a total of 15 187 participants were randomised into the main phase 3 trial, of whom 15 139 received treatment (7569 received dose one of NVX-CoV2373 and 7570 received dose one of placebo). 431 participants were co-vaccinated with a seasonal influenza vaccine in the substudy (217 received NVX-CoV2373 plus the influenza vaccine and 214 received placebo plus the influenza vaccine). In general, the substudy participants were younger, more racially diverse, and had fewer comorbid conditions than those in the main study. Reactogenicity events were more common in the co-administration group than in the NVX-CoV2373 alone group: tenderness (113 [64·9%] of 174 vs 592 [53·3%] of 1111) or pain (69 [39·7%] vs 325 [29·3%]) at injection site, fatigue (48 [27·7%] vs 215 [19·4%]), and muscle pain (49 [28·3%] vs 237 [21·4%]). Incidences of unsolicited adverse events, treatment-related medically attended adverse events, and serious adverse events were low and balanced between the co-administration group and the NVX-CoV2373 alone group. No episodes of anaphylaxis or deaths were reported within the substudy. Co-administration resulted in no change to influenza vaccine immune response although a reduction in antibody responses to the NVX-CoV2373 vaccine was noted. NVX-CoV2373 vaccine efficacy in the substudy (ie, participants aged 18 to

BackgroundThe recombinant protein-based vaccine, NVX-CoV2373, demonstrated 89.7% efficacy against coronavirus disease 2019 (COVID-19) in a phase 3, randomized, observer-blinded, placebo-controlled trial in the United Kingdom. The protocol was amended to include a blinded crossover. Data to the end of the placebo-controlled phase are reported.MethodsAdults aged 18–84 years received 2 doses of NVX-CoV2373 or placebo (1:1) and were monitored for virologically confirmed mild, moderate, or severe COVID-19 (onset from 7 days after second vaccination). Participants who developed immunoglobulin G (IgG) against nucleocapsid protein but did not show symptomatic COVID-19 were considered asymptomatic. Secondary outcomes included anti-spike (S) IgG responses, wild-type virus neutralization, and T-cell responses.ResultsOf 15 185 participants, 13 989 remained in the per-protocol efficacy population (6989 NVX-CoV2373, 7000 placebo). At a maximum of 7.5 months (median, 4.5) postvaccination, there were 24 cases of COVID-19 among NVX-CoV2373 recipients and 134 cases among placebo recipients, a vaccine efficacy of 82.7% (95% confidence interval [CI], 73.3%–88.8%). Vaccine efficacy was 100% (95% CI, 17.9%–100.0%) against severe disease and 76.3% (95% CI, 57.4%–86.8%) against asymptomatic disease. High anti-S and neutralization responses to vaccination were evident, together with S-protein–specific induction of interferon-γ secretion in peripheral blood T cells. Incidence of serious adverse events and adverse events of special interest were similar between groups.ConclusionsA 2-dose regimen of NVX-CoV2373 conferred a high level of ongoing protection against asymptomatic, symptomatic, and severe COVID-19 through >6 months postvaccination. A gradual decrease of protection suggests that a booster may be indicated.

Hypocalcaemia is a frequent, and potentially dangerous complication of total thyroidectomy occurring secondary to devascularisation of the parathyroid glands. This quality improvement (QI) project was undertaken in a large Ear, Nose and Throat (ENT) department in the East of England over a one year period. The project aimed to improve postoperative guideline compliance by optimising the recognition and management of patients at risk of hypocalcaemia. This process focussed on improving parathyroid hormone (PTH) and calcium blood testing, prophylactic calcium prescribing and the subsequent monitoring and management of hypocalcaemia. A baseline audit was conducted to determine the initial guideline compliance. The QI process subsequently involved the introduction of a new intraoperative PTH pathway and the amendment of trust guidelines. In addition, there was a focus on improving clinician awareness of guidelines, junior doctor education, communication between operating surgeons and junior doctors and the optimisation of patient handover. The project saw a significant improvement in the monitoring of hypocalcaemia (from 22.2% to 83.3% for patients with an intermediate risk of hypocalcaemia) and in the prescribing of prophylactic calcium supplements from 7.5% to 43.5%. The measurement of PTH at four hours improved from 42.5% to 52.2%. By optimising postoperative care, this QI project improved patient safety as well as impacting on the duration, and overall cost, of inpatient stay.

BackgroundCovid-19 vaccines are urgently needed, especially against emerging variants. NVX-CoV2373 is a recombinant severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2 rS) nanoparticle vaccine containing trimeric full-length SARS-CoV-2 spike glycoprotein and Matrix-M adjuvant.MethodsA phase 3, randomized, observer-blinded, placebo-controlled trial was conducted in adults 18-84 years old who received two intramuscular 5-µg doses, 21 days apart, of NVX-CoV2373 or placebo (1:1) across 33 sites in the United Kingdom. The primary efficacy endpoint was virologically confirmed symptomatic Covid-19 with onset 7 days after second vaccination in serologically negative participants.ResultsA total of 15,187 participants were randomized, of whom 7569 received NVX-CoV2373 and 7570 received placebo; 27.2% were 65 years or older, 44.7% had comorbidities and 4.2% had baseline serological evidence of SARS-CoV-2. There were 10 cases of Covid-19 among NVX-CoV2373 recipients and 96 cases among placebo recipients, with symptom onset at least 7 days after second vaccination; NVX-CoV2373 was 89.7% (95% confidence interval, 80.2 to 94.6) effective in preventing Covid-19, with no hospitalizations or deaths reported. There were five cases of severe Covid-19, all in the placebo group. Post hoc analysis revealed efficacies of 96.4% (73.8 to 99.5) and 86.3% (71.3 to 93.5) against the prototype strain and B.1.1.7 variant, respectively. Vaccine efficacy was similar across subgroups, including participants with comorbidities and those ≥65 years old. Reactogenicity was generally mild and transient. The incidence of serious adverse events was low and similar in the two groups.ConclusionA two-dose regimen of NVX-CoV2373 conferred 89.7% protection against a blend of prototype and variant Covid-19, demonstrated high efficacy against the B.1.1.7 variant, and had a reassuring safety profile.(Funded by Novavax, Inc. EudraCT number, 2020-004123-16).

Introduction Hypocalcaemia is a frequent, and potentially dangerous, complication of total thyroidectomy [1, 2]. This quality improvement (QI) project was undertaken in a large ENT department in the East of England over a year. The project improved postoperative guideline compliance by optimising the recognition and management of patients at risk of hypocalcaemia. This process focussed on improving parathyroid hormone (PTH) and calcium blood testing, appropriate prescribing and the monitoring and management of hypocalcaemia. Method Following a baseline audit the QI process subsequently involved the introduction of a new intraoperative PTH pathway and the amendment of trust guidelines. In addition, there was a focus on improving clinician awareness of guidelines, junior doctor education, communication between operating surgeons and junior doctors and the optimisation of patient handover. Results The measurement of PTH at four hours improved from 42.5% to 52.2%. The project saw a significant improvement in the monitoring of hypocalcaemia (from 22.2% to 83.3% for patients with an intermediate risk of hypocalcaemia) and in the prescribing of prophylactic calcium supplements from 7.5% to 43.5%. Conclusions By optimising postoperative care this QI project improved patient safety as well as impacting on the duration, and overall cost, of inpatient stay.

Background The purpose of this trial was to test if the Norfolk Diabetes Prevention Study (NDPS) lifestyle intervention, recently shown to reduce the incidence of type 2 diabetes in high-risk groups, also improved glycaemic control in people with newly diagnosed screen-detected type 2 diabetes. Methods We screened 12,778 participants at high risk of type 2 diabetes using a fasting plasma glucose and glycosylated haemoglobin (HbA1c). People with screen-detected type 2 diabetes were randomised in a parallel, three-arm, controlled trial with up to 46 months of follow-up, with a control arm (CON), a group-based lifestyle intervention of 6 core and up to 15 maintenance sessions (INT), or the same intervention with additional support from volunteers with type 2 diabetes trained to co-deliver the lifestyle intervention (INT-DPM). The pre-specified primary end point was mean HbA1c compared between groups at 12 months. Results We randomised 432 participants (CON 149; INT 142; INT-DPM 141) with a mean (SD) age of 63.5 (10.0) years, body mass index (BMI) of 32.4 (6.4) kg/m2, and HbA1c of 52.5 (10.2) mmol/mol. The primary outcome of mean HbA1c at 12 months (CON 48.5 (9.1) mmol/mol, INT 46.5 (8.1) mmol/mol, and INT-DPM 45.6 (6.0) mmol/mol) was significantly lower in the INT-DPM arm compared to CON (adjusted difference −2.57 mmol/mol; 95% CI −4.5, −0.6; p = 0.007) but not significantly different between the INT-DPM and INT arms (−0.55 mmol/mol; 95% CI −2.46, 1.35; p = 0.57), or INT vs CON arms (−2.14 mmol/mol; 95% CI −4.33, 0.05; p = 0.07). Subgroup analyses showed the intervention had greater effect in participants p = 0.02). This effect was most significant in the INT-DPM arm (−6.01 mmol/mol; 95% CI −9.56, −2.46 age 65 years old; p = 0.007). The use of oral hypoglycaemic medication was associated with a significantly lower mean HbA1c but only within the INT-DPM arm compared to CON (−7.0 mmol/mol; 95% CI −11.5, −2.5; p = 0.003). Conclusion The NDPS lifestyle intervention significantly improved glycaemic control after 12 months in people with screen-detected type 2 diabetes when supported by trained peer mentors with type 2 diabetes, particularly those receiving oral hypoglycaemics and those under 65 years old. The effect size was modest, however, and not sustained at 24 months. Trial registration ISRCTN34805606. Retrospectively registered 14.4.16

Adipose tissue contains self-renewing multipotent cells termed mesenchymal stromal cells. In situ, these cells serve to expand adipose tissue by adipogenesis, but their multipotency has gained interest for use in tissue regeneration. Little is known regarding the repertoire of receptors expressed by adipose-derived mesenchymal stromal cells (AD-MSCs). The purpose of this study was to undertake a comprehensive analysis of purinergic receptor expression. Mesenchymal stromal cells were isolated from human subcutaneous adipose tissue and confirmed by flow cytometry. The expression profile of purinergic receptors was determined by quantitative real-time PCR and immunocytochemistry. The molecular basis for adenine and uracil nucleotide-evoked intracellular calcium responses was determined using Fura-2 measurements. All the known subtypes of P2X and P2Y receptors, excluding P2X2, P2X3 and P2Y12 receptors, were detected at the mRNA and protein level. ATP, ADP and UTP elicited concentration-dependent calcium responses in mesenchymal cells (N = 7–9 donors), with a potency ranking ADP (EC50 1.3 ± 1.0 μM) > ATP (EC50 2.2 ± 1.1 μM) = UTP (3.2 ± 2.8 μM). Cells were unresponsive to UDP (

Leukocytes sense extracellular ATP, a danger-associated molecular pattern, released during cellular stress and death, via activation of cell surface P2X and P2Y receptors. Here, we investigate P2 receptor expression in primary human monocyte-derived macrophages and receptors that mediate ATP-evoked intracellular [Ca2+]i signals and cytokine production in response to ATP concentrations that exclude P2X7 receptor activation. Expression of P2X1, P2X4, P2X5, P2X7, P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, and P2Y13 was confirmed by quantitative RT-PCR and immunocytochemistry. ATP elicited intracellular Ca2+ responses in a concentration-dependent fashion (EC50 = 11.4 ± 2.9 μM, n = 3). P2Y11 and P2Y13 activations mediated the amplitude of [Ca2+]i response, whereas P2X4 activation, but not P2X1 or P2X7, determined the duration of Ca2+ response during a sustained phase. ATP mediated gene induction of CXCL5, a proinflammatory chemokine. P2X4 antagonism (PSB-12062 or BX430) inhibited ATP-mediated induction of CXCL5 gene expression and secretion of CXCL5 by primary macrophage. Inhibition of CXCL5 secretion by P2X4 antagonists was lost in the absence of extracellular Ca2+. Reciprocally, positive allosteric modulation of P2X4 (ivermectin) augmented ATP-mediated CXCL5 secretion. P2X7, P2Y11, or P2Y13 receptor did not contribute to CXCL5 secretion. Together, the data reveals a role for P2X4 in determining the duration of ATP-evoked Ca2+ responses and CXCL5 secretion in human primary macrophage.

Phosphaturic mesenchymal tumours are a heterogeneous set of bone and soft tissue neoplasms that can cause a number of paraneoplastic syndromes such as tumour induced osteomalacia. The term phosphaturic comes from the common finding that these tumours secrete high levels of fibroblast growth factor 23 which causes renal phosphate wasting leading to hypophosphatemia. Phosphaturic mesenchymal tumours are rare and diagnosis is difficult. A very active 68 year old male presented with bone pain and muscle weakness. He was hypophosphataemic and total alkaline phosphatase was markedly elevated. The patient was placed on vitamin D supplementation but his condition progressed. In the fifth year of presentation the patient required the use of a wheelchair and described “explosive” bone pain on physical contact. Serum 1,25 dihydroxyvitamin D was low and serum fibroblast growth factor 23 was significantly elevated, raising suspicion of a phosphaturic mesenchymal tumour. A lesion was detected in his left femoral head and the patient underwent a total hip replacement. The patient displayed a rapid improvement to his condition and during a three year follow up period he returned to an active lifestyle. As molecular testing may help provide a robust diagnosis and is particularly useful in rare diseases we took a next generation sequencing approach to identify a differential expression of small RNAs in the resected tumour. Small RNAs are non-coding RNA molecules that play a key role in regulation of gene expression and can be used as specific biomarkers. We found an upregulation of miR-197. We also found a downregulation of miR-20b, miR-144 and miR-335 which is a small RNA profile typical of osteosarcoma. MiR-21, the most frequently upregulated microRNA in cancer, was downregulated. We conclude that the specific small RNA profile is typical of osteosarcoma except for the downregulation of oncogenic miR-21. Transcriptional plasticity of miR-197, which is computationally predicted to target fibroblast growth factor 23 messenger RNA, may be upregulated in a cellular effort to correct the ectopic expression of the protein.

Aim: To investigate factors influencing diagnostic discordance for non-diabetic hyperglycaemia and Type 2 diabetes. Methods: Some 10 000 adults at increased risk of diabetes were screened with HbA1c and fasting plasma glucose (FPG). The 2208 participants with initial HbA1c ≥ 42 mmol/mol (≥ 6.0%) or FPG ≥ 6.1 mmol/l were retested after a median 40 days. We compared the first and second HbA1c results, and consequent diagnoses of non-diabetic hyperglycaemia and Type 2 diabetes, and investigated predictors of discordant diagnoses. Results: Of 1463 participants with non-diabetic hyperglycaemia and 394 with Type 2 diabetes on first testing, 28.4% and 21.1% respectively had discordant diagnoses on repeated testing. Initial diagnosis of non-diabetic hyperglycaemia and/or impaired fasting glucose according to both HbA1c and FPG criteria, or to FPG only, made reclassification as Type 2 diabetes more likely than initial classification according to HbA1c alone. Initial diagnosis of Type 2 diabetes according to both HbA1c and FPG criteria made reclassification much less likely than initial classification according to HbA1c alone. Age, and anthropometric and biological measurements independently but inconsistently predicted discordant diagnoses and changes in HbA1c. Conclusions: Diagnosis of non-diabetic hyperglycaemia or Type 2 diabetes with a single measurement of HbA1c in a screening programme for entry to diabetes prevention trials is unreliable. Diagnosis of non-diabetic hyperglycaemia and Type 2 diabetes should be confirmed by repeat testing. FPG results could help prioritise retesting. These findings do not apply to people classified as normal on a single test, who were not retested.

Primary hyperparathyroidism is the third most common endocrine disorder in the western world with an estimated prevalence of 1-4 per 1000.The only curative treatment is surgical resection. Minimally invasive surgical techniques are preferred over bilateral neck exploration due to reduced risk of surgical complications (1.2% vs 3%) and seven-fold shorter hospital stay with cost reduction of up to 50% (1); however this requires accurate preoperative localisation. Various imaging modalities evolved over time to identify pathological parathyroid glands pre-operatively. Imaging modality which is non-invasive, accurate and has minimal radiation exposure should be preferred as a first line option. Planar Technetium-99m Sestamibi scintigraphy coupled with Ultrasonography (MIBI/USS) has conventionally been used as the localisation study of choice. Single Photon Emission Computed Tomography (SPECT)/CT has been introduced recently and adopted by our centre as a first line investigation. The aim of the current study was to compare the accuracy of MIBI/USS versus SPECT/CT in localising pathological parathyroid glands preoperatively and assess the influence of imaging modality on surgical outcomes. A total of 135 patients with confirmed primary hyperparathyroidism were studied retrospectively in a tertiary centre over 24 months period. 61 patients underwent MIBI/USS and 74 had SPECT/CT as first line imaging. 82% of patients were female with mean age of 67 years. Preoperatively Sestamibi -SPECT/CT identified the laterality of the adenoma in 78.4% of cases, compared to 49.2% in MIBI/USS (p

The world diabetes population quadrupled between 1980 and 2014 to 422 million and the enormous impact of Type 2 diabetes is recognised by the recent creation of national Type 2 diabetes prevention programmes. There is uncertainty about how to correctly risk stratify people for entry into prevention programmes, how combinations of multiple ‘at high risk’ glycemic categories predict outcome, and how the large recently defined ‘at risk’ population based on an elevated glycosylated haemoglobin (HbA1c) should be managed. We identified all 141,973 people at highest risk of diabetes in our population, and screened 10,000 of these with paired fasting plasma glucose and HbA1c for randomisation into a very large Type 2 diabetes prevention trial. Baseline discordance rate between highest risk categories was 45.6%, and 21.3–37.0% of highest risk glycaemic categories regressed to normality between paired baseline measurements (median 40 days apart). Accurate risk stratification using both fasting plasma glucose and HbA1c data, the use of paired baseline data, and awareness of diagnostic imprecision at diagnostic thresholds would avoid substantial overestimation of the true risk of Type 2 diabetes and the potential benefits (or otherwise) of intervention, in high risk subjects entering prevention trials and programmes.

White adipocytes are key regulators of metabolic homeostasis, which release stored energy as free fatty acids via lipolysis. Adipocytes possess both basal and stimulated lipolytic capacity, but limited information exists regarding the molecular mechanisms that regulate basal lipolysis. Here, we describe a mechanism whereby autocrine purinergic signaling and constitutive P2Y2 receptor activation suppresses basal lipolysis in primary human in vitro differentiated adipocytes. We found that human adipocytes possess cytoplasmic calcium tone due to ATP secretion and constitutive P2Y2 receptor activation. Pharmacological antagonism or knockdown of P2Y2 receptors increases intracellular cAMP levels and enhances basal lipolysis. P2Y2 receptor antagonism works synergistically with phosphodiesterase inhibitors in elevating basal lipolysis, but is dependent upon adenylate cyclase activity. Mechanistically, we suggest that the increased calcium tone exerts an anti-lipolytic effect by suppression of calcium-sensitive adenylate cyclase isoforms. We also observed that acute enhancement of basal lipolysis following P2Y2 receptor antagonism alters the profile of secreted adipokines leading to longer term adaptive decreases in basal lipolysis. Our findings reveal that basal lipolysis and adipokine secretion are controlled by autocrine purinergic signaling in human adipocytes.

Background: Pregnant women with type 1 diabetes are a high-risk population who are recommended to strive for optimal glucose control, but neonatal outcomes attributed to maternal hyperglycaemia remain suboptimal. Our aim was to examine the effectiveness of continuous glucose monitoring (CGM) on maternal glucose control and obstetric and neonatal health outcomes. Methods: In this multicentre, open-label, randomised controlled trial, we recruited women aged 18-40 years with type 1 diabetes for a minimum of 12 months who were receiving intensive insulin therapy. Participants were pregnant (≤13 weeks and 6 days' gestation) or planning pregnancy from 31 hospitals in Canada, England, Scotland, Spain, Italy, Ireland, and the USA. We ran two trials in parallel for pregnant participants and for participants planning pregnancy. In both trials, participants were randomly assigned to either CGM in addition to capillary glucose monitoring or capillary glucose monitoring alone. Randomisation was stratified by insulin delivery (pump or injections) and baseline glycated haemoglobin (HbA1c). The primary outcome was change in HbA1c from randomisation to 34 weeks' gestation in pregnant women and to 24 weeks or conception in women planning pregnancy, and was assessed in all randomised participants with baseline assessments. Secondary outcomes included obstetric and neonatal health outcomes, assessed with all available data without imputation. This trial is registered with ClinicalTrials.gov, number NCT01788527. Findings: Between March 25, 2013, and March 22, 2016, we randomly assigned 325 women (215 pregnant, 110 planning pregnancy) to capillary glucose monitoring with CGM (108 pregnant and 53 planning pregnancy) or without (107 pregnant and 57 planning pregnancy). We found a small difference in HbA1c in pregnant women using CGM (mean difference -0·19%; 95% CI -0·34 to -0·03; p=0·0207). Pregnant CGM users spent more time in target (68% vs 61%; p=0·0034) and less time hyperglycaemic (27% vs 32%; p=0·0279) than did pregnant control participants, with comparable severe hypoglycaemia episodes (18 CGM and 21 control) and time spent hypoglycaemic (3% vs 4%; p=0·10). Neonatal health outcomes were significantly improved, with lower incidence of large for gestational age (odds ratio 0·51, 95% CI 0·28 to 0·90; p=0·0210), fewer neonatal intensive care admissions lasting more than 24 h (0·48; 0·26 to 0·86; p=0·0157), fewer incidences of neonatal hypoglycaemia (0·45; 0·22 to 0·89; p=0·0250), and 1-day shorter length of hospital stay (p=0·0091). We found no apparent benefit of CGM in women planning pregnancy. Adverse events occurred in 51 (48%) of CGM participants and 43 (40%) of control participants in the pregnancy trial, and in 12 (27%) of CGM participants and 21 (37%) of control participants in the planning pregnancy trial. Serious adverse events occurred in 13 (6%) participants in the pregnancy trial (eight [7%] CGM, five [5%] control) and in three (3%) participants in the planning pregnancy trial (two [4%] CGM and one [2%] control). The most common adverse events were skin reactions occurring in 49 (48%) of 103 CGM participants and eight (8%) of 104 control participants during pregnancy and in 23 (44%) of 52 CGM participants and five (9%) of 57 control participants in the planning pregnancy trial. The most common serious adverse events were gastrointestinal (nausea and vomiting in four participants during pregnancy and three participants planning pregnancy). Interpretation: Use of CGM during pregnancy in patients with type 1 diabetes is associated with improved neonatal outcomes, which are likely to be attributed to reduced exposure to maternal hyperglycaemia. CGM should be offered to all pregnant women with type 1 diabetes using intensive insulin therapy. This study is the first to indicate potential for improvements in non-glycaemic health outcomes from CGM use. Funding: Juvenile Diabetes Research Foundation, Canadian Clinical Trials Network, and National Institute for Health Research.

Leukocytes sense extracellular ATP, a danger-associated molecular pattern, released during cellular stress and death, via activation of cell surface P2X and P2Y receptors. Here, we investigate P2 receptor expression in primary human monocyte-derived macrophages and receptors that mediate ATP-evoked intracellular [Ca2+]i signals and cytokine production in response to ATP concentrations that exclude P2X7 receptor activation. Expression of P2X1, P2X4, P2X5, P2X7, P2Y1, P2Y2, P2Y4, P2Y6, P2Y11 and P2Y13 was confirmed by qRT-PCR and immunocytochemistry. ATP elicited intracellular Ca2+ responses in a concentration-dependent fashion (EC50 = 11.4 ± 2.9 μM, N=3). P2Y11 and P2Y13 activations mediated the amplitude of [Ca2+]i response, whilst P2X4 activation, but not P2X1 or P2X7, determined the duration of Ca2+ response during a sustained phase. ATP-mediated gene induction of CXCL5, a pro-inflammatory chemokine. P2X4 antagonism (PSB-12062 or BX-430) inhibited ATP-mediated induction of CXCL5 gene expression and secretion of CXCL5 by primary macrophage. Inhibition of CXCL5 secretion by P2X4 antagonists was lost in the absence of extracellular Ca2+. Reciprocally, positive allosteric modulation of P2X4 (ivermectin) augmented ATP-mediated CXCL5 secretion. P2X7, P2Y11 or P2Y13 receptor did not contribute to CXCL5 secretion. Together, the data reveals a role for P2X4 in determining the duration of ATP-evoked Ca2+ responses and CXCL5 secretion in human primary macrophage.

In this addendum to the above paper, the Society for Endocrinology Clinical Committee and the original authors provide additional advice on the dose equivalence of calcium gluconate and calcium chloride. The ‘Severe hypocalcaemia’ section, which appeared in the September 2016 issue of Endocrine Connections (volume 5, page G8, https://doi.org/10.1530/EC-16-0056 reads as follows: Severe hypocalcaemia Severe hypocalcaemia: serum calcium1.9 mmol/L and/or symptomatic at any level below reference range. This is a medical emergency Administer i.v. calcium gluconate Initially, give 10–20 mL 10% calcium gluconate in 50–100 mL of 5% dextrose i.v. over 10 min with ECG monitoring. This can be repeated until the patient is asymptomatic. It should be followed up with a calcium gluconate infusion as follows: Dilute 100 mL of 10% calcium gluconate (10 vials) in 1 L of Normal saline or 5% dextrose and infuse at 50–100 mL/h. (Calcium chloride can be used as an alternative to calcium gluconate, but it is more irritant to veins and should only be given via a central line) Titrate the rate of infusion to achieve normocalcaemia and continue until treatment of the underlying cause has taken effect The Society for Endocrinology Clinical Committee and the original authors now provide this additional advice on the dose equivalence of calcium gluconate and calcium chloride, as follows: Each 10 mL vial of 10% calcium gluconate contains 2.2 mmol of calcium. Calcium chloride for i.v. administration is available in a number of preparations including, commonly, 10 mL of 7.35% calcium chloride (which contains 5 mmol of calcium) and 5 mL of 14.7% calcium chloride (which also contains 5 mmol of calcium). Great care should be taken since other preparations are also available. If using calcium chloride in place of calcium gluconate, 4.4 mL of 7.35% calcium chloride or 2.2 mL of 14.7% calcium chloride for i.v. administration should therefore be used as equivalent to 10 mL of 10% calcium gluconate. If other calcium chloride preparations are used, the required volume should be calculated, aiming to achieve 2.2–4.4 mmol i.v. loading bolus followed by a 1.1–2.2 mmol/h maintenance infusion.

A 71-year-old woman who had been taking ibandronate for 10 years presented to an Endocrinology Department with persistent mid-thigh pain. Pelvic X-ray showed bilateral femoral cortical expansion, indicating impending atypical femoral fractures (AFFs). AFFs have been linked to long-term bisphosphonate therapy and have morbidity and mortality similar to that of hip fractures. Such fractures can be averted by regular reviews of bisphosphonate therapy and vigilance for prodromal symptoms. This patient’s bisphosphonate therapy was stopped, and fractures were avoided by treatment with vitamin D and parathyroid hormone.

Despite currently available therapies, most patients diagnosed with acute myeloid leukemia (AML) die of their disease. Tumor-host interactions are critical for the survival and proliferation of cancer cells; accordingly, we hypothesize that specific targeting of the tumor microenvironment may constitute an alternative or additional strategy to conventional tumor-directed chemotherapy. Because adipocytes have been shown to promote breast and prostate cancer proliferation, and because the bone marrow adipose tissue accounts for up to 70% of bone marrow volume in adult humans, we examined the adipocyte-leukemia cell interactions to determine if they are essential for the growth and survival of AML. Using in vivo and in vitro models of AML, we show that bone marrow adipocytes from the tumor microenvironment support the survival and proliferation of malignant cells from patients with AML. We show that AML blasts alter metabolic processes in adipocytes to induce phosphorylation of hormone-sensitive lipase and consequently activate lipolysis, which then enables the transfer of fatty acids from adipocytes to AML blasts. In addition, we report that fatty acid binding protein-4 (FABP4) messenger RNA is upregulated in adipocytes and AML when in coculture. FABP4 inhibition using FABP4 short hairpin RNA knockdown or a small molecule inhibitor prevents AML proliferation on adipocytes. Moreover, knockdown of FABP4 increases survival in Hoxa9/Meis1-driven AML model. Finally, knockdown of carnitine palmitoyltransferase IA in an AML patient-derived xenograft model improves survival. Here, we report the first description of AML programming bone marrow adipocytes to generate a protumoral microenvironment.

We describe the case of a patient who presented with a right-sided glomus jugulare tumor and bilateral glomus vagale tumors. These proved to be nonmalignant paragangliomas on histopathological analysis. Genetic analysis revealed a germline heterozygous missense mutation (Pro81Leu) in the succinate dehydrogenase subunit D ( SDHD) gene. We discuss the clinical presentations of the familial paraganglioma syndrome type 1, which is caused by mutations in SDHD, and the implications for the clinical diagnosis and care of such patients.

Familial juvenile hyperuricemic nephropathy (FJHN), which is inherited as an autosomal dominant disorder, is characterized by hyperuricemia, a low fractional renal excretion of urate, and chronic renal failure that is associated with interstitial fibrosis. Studies in 4 families (3 European and 1 Japanese) have mapped the gene causing autosomal dominant FJHN to chromosome 16p11-p13. To refine this location we have pursued linkage studies in 7 European families with autosomal dominant FJHN and used 11 chromosome 16p11-p13 polymorphic loci whose order has been established as 16pter-D16S3069-D16S3060-D16S3041-D16S3036-D16S3046-[D16S403,D16S417]-D16S420-D16S3113-D16S401-D16S3133-16cen. Cosegregation between these polymorphic loci and FJHN was observed in 5 of the families, and linkage was established between FJHN and 6 loci (peak LOD score, 5.32 with D16S417, at 0% recombination), with the most likely location of FJHN being within a 22-centimorgan interval flanked centromerically by D16S401 and telomerically by D16S3069. Furthermore, FJHN in 2 families was found not to be linked to chromosome 16p11-p13, thereby demonstrating genetic heterogeneity. Thus, 5 additional families with FJHN showing linkage to chromosome 16p11-p13 loci have been identified, and genetic heterogeneity has been demonstrated in more than 25% of FJHN families. These results will facilitate the characterization of this gene regulating urate metabolism.

Toll-like receptors (TLRs) are central to innate immunity and yet their expression is widespread and not restricted to professional inflammatory cells. TLRs have been reported on adipocytes and have been implicated in obesity-associated pathologies such as diabetes. Why TLRs are found on adipocytes is not clear although one hypothesis is that they may coordinate energy utilization for the energy intensive process of an immune response. We have explored TLR signalling in primary human in vitro differentiated adipocytes and investigated the specific adapter molecules that are involved. Only lipopolysaccharide (LPS), poly(I:C), Pam3CSK4 and MALP-2 could induce the production of IL-6, IL-8 and MCP-1 by adipocytes. Poly(I:C) alone caused a strong induction of type I interferons, as assessed by IP-10 production. Using siRNA, it was confirmed that LPS-dependent signalling in adipocytes occurs via TLR4 utilizing the adapter molecules MyD88, Mal and TRIF and caused rapid degradation of IκBα. Pam3CSK4 signalling utilized TLR2, MyD88 and Mal (but not TRIF). However, the response to poly(I:C) observed in these cells appeared not to require TRIF, but MyD88 was required for induction of NFκB-dependent cytokines by Poly(I:C). Despite this, IκBα degradation could not be detected in poly(I:C) stimulated adipocytes at any time-point up to 4 h. Indeed, IL-6 transcription was not induced until 8-16 h after exposure. These data suggest that Pam3CSK4 and LPS signal via the expected routes in human adipocytes, whereas poly(I:C)/TLR3 signalling may act via a TRIF-independent, MyD88-dependent route.

The calcium-sensing receptor (CaSR) is a G-protein-coupled receptor that has an extracellular bilobed venus flytrap domain (VFTD) predicted to contain five calcium (Ca(2+))-binding sites. To elucidate the structure-function relationships of the VFTD, we investigated 294 unrelated probands with familial hypocalciuric hypercalcaemia (FHH), neonatal severe primary hyperparathyroidism (NSHPT) or autosomal dominant hypocalcaemic hypercalciuria (ADHH) for CaSR mutations and performed in vitro functional expression studies and three-dimensional modelling of mutations involving the VFTD. A total of 70 different CaSR mutations were identified: 35 in FHH, 10 in NSHPT and 25 in ADHH patients. Furthermore, a CaSR variant (Glu250Lys) was identified in FHH and ADHH probands and demonstrated to represent a functionally neutral polymorphism. NSHPT was associated with a large proportion of truncating CaSR mutations that occurred in the homozygous or compound heterozygous state. Thirty-four VFTD missense mutations were identified, and 18 mutations were located within 10 Å of one or more of the predicted Ca(2+)-binding sites, particularly at the VFTD cleft, which is the principal site of Ca(2+) binding. Mutations of residues 173 and 221, which are located at the entrance to the VFTD cleft binding site, were associated with both receptor activation (Leu173Phe and Pro221Leu) and inactivation (Leu173Pro and Pro221Gln), thereby highlighting the importance of these residues for entry and binding of Ca(2+) by the CaSR. Thus, these studies of disease-associated CaSR mutations have further elucidated the role of the VFTD cleft region in Ca(2+) binding and the function of the CaSR.

One of the most common ways to demonstrate a direct protein-protein interaction in vitro is the glutathione-S-transferse (GST)-pulldown. Here we report the detailed characterization of a putative interaction between two transcription factor proteins, GATA-1 and Kruppel-like factor 3 (KLF3/BKLF) that show robust interactions in GST-pulldown experiments. Attempts to map the interaction interface of GATA-1 on KLF3 using a mutagenic screening approach did not yield a contiguous binding face on KLF3, suggesting that the interaction might be non-specific. NMR experiments showed that the proteins do not interact at protein concentrations of 50-100 μM. Rather, the GST tag can cause part of KLF3 to misfold. In addition to misfolding, the fact that both proteins are DNA-binding domains appears to introduce binding artifacts (possibly nucleic acid bridging) that cannot be resolved by the addition of nucleases or ethidium bromide (EtBr). This study emphasizes the need for caution in relying on GST-pulldown results and related methods, without convincing confirmation from different approaches.

Familial benign hypocalciuric hypercalcaemia (FBHH) is a genetically heterogeneous disorder that consists of three designated types, FBHH1, FBHH2 and FBHH3, whose chromosomal locations are 3q21.1, 19p and 19q13, respectively. FBHH1 is caused by mutations of a calcium-sensing receptor (CaSR), but the abnormalities underlying FBHH2 and FBHH3 are unknown. FBHH3, also referred to as the Oklahoma variant (FBHH(Ok)), has been mapped to a 12cM interval, flanked by D19S908 and D19S866. To refine the location of FBHH3, we pursued linkage studies using 24 polymorphic loci. Our results establish a linkage between FBHH3 and 17 of these loci, and indicate that FBHH3 is located in a 4.1 Mb region flanked centromerically by D19S112 and telomerically by rs245111, which in the syntenic region on mouse chromosome 7 contains four Casr-related sequences (Gprc2a-rss). However, human homologues of these Gprc2a-rss were not found and a comparative analysis of the 22.0 Mb human and 39.3 Mb mouse syntenic regions showed evolutionary conservation of two segments that were inverted with loss from the human genome of 11.6 Mb that contained the four Gprc2a-rss. Thus, FBHH3 cannot be attributed to Gprc2a-rss abnormalities. DNA sequence analysis of 12 other genes from the interval that were expressed in the parathyroids and/or kidneys did not detect any abnormalities, thereby indicating that these genes are unlikely to be the cause of FBHH3. The results of this study have refined the map location of FBHH3, which will facilitate the identification of another CaSR or a mediator of calcium homeostasis.

Obesity is regarded as a pro-inflammatory state. It is associated with low circulating levels of the adipokine, adiponectin, which is considered to be an anti-inflammatory. However, adiponectin knockout mice do not consistently demonstrate pro-inflammatory phenotypes, suggesting more complexity in the in vivo immunomodulatory effects of adiponectin than originally anticipated. Moreover, adiponectin exerts pro-inflammatory effects in some experimental systems. This contradiction has been resolved by hypothesizing that adiponectin induces tolerance to inflammatory stimuli, notably Toll-like receptor (TLR) ligands. We noticed that this effect resembled lipopolysaccharide (LPS) tolerance and therefore tested adiponectin from a variety of sources for LPS contamination. All adiponectin tested carried low levels of LPS in the range of 1-30 pg/microg of adiponectin, sufficient to produce final LPS concentrations in the pg/ml range under experimental conditions. We found that induction of tolerance to TLR ligands by adiponectin in human monocyte-derived macrophages could be reproduced by such LPS concentrations. Moreover, the LPS antagonist, polymixin B, substantially inhibited induction of tolerance by adiponectin. Furthermore, polymixin B and a naturally occurring antagonist LPS were able to partially attenuate induction of tumour necrosis factor-alpha and interleukin-6 in human monocyte-derived macrophages by adiponectin. Polymixin B also inhibited nuclear factor-kappaB and mitogen-activated protein kinase signalling elicited by adiponectin. We therefore propose that some of adiponectin's immunomodulatory effects, in particular, its TLR-tolerising actions in human monocyte-derived macrophages, may be confounded by induction of tolerance by contaminating LPS.

This study describes a novel approach to generate conditionally immortalized preadipocyte cell lines from white adipose tissue (IMWAT) that can be induced to differentiate into white adipocytes even after expansion in culture. Such adipocytes express markers of white fat such as peroxisome proliferator-activated receptor gamma and aP2 but not brown fat markers, have an intact insulin signaling pathway, and express proinflammatory cytokines. They can be readily transduced with adenoviral vectors, allowing them to be used to investigate the consequences of the depletion of specific adipocyte factors using short hairpin RNA. This approach has been used to study the effect of reduced expression of the nuclear receptor corepressor receptor interacting protein 140 (RIP140), a regulator of adipocyte function. The depletion of RIP140 results in changes in metabolic gene expression that resemble those in adipose tissue of the RIP140 null mouse. Thus, IMWAT cells provide a novel model for adipocytes that are derived from preadipocytes rather than fibroblasts and provide an alternative system to primary preadipocytes for the investigation of adipocyte function.

The covalent attachment of ubiquitin-like proteins to their substrates represents an unusual posttranslational modification in that the modifier itself is a small polypeptide of around 100 amino acids (48). Ubiquitin, the founding member of the family, is well known as a modifier that directs proteins to the proteasome. Ubiquitin is also involved in other cellular processes, including the regulation of intracellular transport and gene activation (33, 67). Small ubiquitin-like modifier (SUMO) has been extensively studied recently. The enzymatic reactions involved in SUMO modification are analogous to those seen in ubiquitin modification and entail an E1-activating enzyme, consisting of an Aos1/Uba2 (SAE1/SAE2) heterodimer, the E2-conjugating enzyme Ubc9, and an E3 ligase that promotes the transfer of SUMO from the E2 enzyme to substrate proteins (29, 32). Although E1 and E2 enzymes are typically sufficient to support sumoylation in vitro, it appears than in vivo E3 ligases also play a part in the process. Thus far, the protein inhibitors of activated STATs (PIAS), the PIAS-like protein Zimp10, the polycomb protein Pc2, and the nuclear pore component RanBP2 have been identified as E3 ligases (16, 18, 19, 24, 38, 43, 52). Sumoylation is a reversible and dynamic process, and several SUMO proteases have also been described previously (30). The functional consequences of SUMO attachment differ from substrate to substrate and in many cases are not understood at the molecular level. To date, sumoylation has been reported to affect diverse cellular processes such as nuclear transport, maintenance of genome integrity, DNA repair, enzymatic activity, mitochondrial fission, signal transduction, and transcriptional regulation (11, 12, 39, 49, 50, 65, 66). Remarkably, over half of the presently identified SUMO substrates are transcription factors or coregulators of transcription, and in most cases, modification with SUMO leads to the attenuation of transcriptional activation (49, 66). Thus, mutation of the sumoylation sites and thereby elimination of sumoylation of Sp3, p300, Elk-1, c-Jun, c-Myb, C/EBP, AP2, and diverse nuclear receptors enables them to become more potent activators (1, 2, 8, 10, 20, 31, 34, 40, 41, 46, 58, 61, 66, 68). Interestingly, the so-called synergy control motif that limits the transcriptional synergy of many transcription factors is essentially identical to the SUMO consensus sequence, further suggesting that SUMO conjugation is mechanistically involved in transcriptional attenuation (14, 15). Precisely how sumoylation causes the attenuation of activation is not yet understood, but SUMO modification has been shown to target transcription factors into repressive subnuclear structures and PML bodies and also to promote the recruitment of histone deacetylases (10, 43, 69). It is also likely that SUMO itself could act as a repressor when directed to certain promoters (14, 41, 68). Furthermore, a recent study indicated that sumoylation of histone H4 also correlates with transcriptional repression and facilitates recruitment of histone deacetylase 1 (HDAC1) and HP1 (54). In addition to its role in limiting the activity of transactivation domains, the sumoylation of transcriptional repressors might also be required for their silencing activity (66). A number of transcriptional corepressors, such as the histone deacetylases HDAC1, HDAC4, HDAC6, and HDAC9 and the corepressor C-terminal binding protein (CtBP), have been shown to be subject to sumoylation (5, 22, 26, 36). We have now examined the transcriptional repressor basic Kruppel-like factor/Kruppel-like factor 3 (BKLF/KLF3) and tested the effect of sumoylation on its ability to repress target promoters. BKLF belongs to the mammalian Sp/Kruppel-like factor family, of which there are presently 24 members (Sp1 to Sp8 and KLF1 to KLF16). KLF proteins are characterized by a distinctive DNA-binding domain at the C terminus of the protein that consists of three Kruppel-like C2H2 zinc fingers. Outside this domain there is little homology among the known KLF proteins (17, 37). BKLF is abundant in erythroid cells, and it has been shown to function as a strong transcriptional repressor on several target promoters (4, 62). The repression domain of BKLF has been mapped to the N-terminal region and was found to associate with the transcriptional corepressor CtBP through the short CtBP interaction motif PXDLT (62, 63). Disruption of the BKLF-CtBP interaction leads to a significant reduction of the repression potential of BKLF in cellular assays. Importantly, the elimination of the BKLF-CtBP interaction does not completely abolish the repressive properties of BKLF, suggesting that BKLF may recruit additional cofactors to regulate transcription (62). Here we report that BKLF also interacts with the E2-conjugating enzyme Ubc9 and is consequently a target for SUMO modification. We provide evidence that PIAS family members, but not the Polycomb protein Pc2, function as E3 SUMO ligases for BKLF in vitro and in vivo. Site-directed mutagenesis and deletion analysis identified two lysines, K10 and K197, as the sumoylation sites. Mutation of these residues compromises the repression activity of BKLF. More importantly, mutations of both the sumoylation sites and the CtBP binding motif in BKLF switch it from a strong repressor to an activator of transcription. These results show that SUMO modification and CtBP recruitment act in synergy to repress transcription and link SUMO modification to transcriptional repression by BKLF.

In this addendum to the above paper, the Society for Endocrinology Clinical Committee and the original authors provide additional advice on the dose equivalence of calcium gluconate and calcium chloride. The ‘Severe hypocalcaemia’ section, which appeared in the September 2016 issue of Endocrine Connections (volume 5, page G8, https://doi.org/10.1530/EC-16-0056 reads as follows: Severe hypocalcaemia Severe hypocalcaemia: serum calcium

Summary background Familial isolated hyperparathyroidism (FIHP) is an autosomal dominant disorder characterized by uniglandular or multiglandular parathyroid tumours that occur in the absence of other endocrine tumours. The disorder may represent either an early stage of multiple endocrine neoplasia type 1 (MEN1), or an allelic variant of MEN1, or a distinct entity involving another locus. We have explored these possibilities in seven families in whom primary hyperparathyroidism occurred as the sole endocrinopathy. methods Seven FIHP families were ascertained and venous blood samples obtained from 35 members (17 affected and 18 unaffected) for DNA sequence analysis of the MEN1 gene. The mean (± SD) follow-up period in the 17 affected members was 15·06 (± 8·83) years. results Four heterozygous germline mutations of the MEN1 gene were identified. These consisted of two 4-bp intragenic deletions that would result in prematurely truncated proteins, and two missense (Asp153Val and Ala411Pro) mutations. Furthermore, analysis of parathyroid tumour DNA from one individual revealed a loss of the wild-type allele and retention of the mutant allele, consistent with Knudson's ‘two-hit’ model of hereditary cancer and a tumour suppressor role for MEN1 in FIHP. conclusions Our results provide further support for FIHP being a distinct allelic variant of MEN1, and an analysis of the 16 mutations reported to date indicate that FIHP is associated with a higher frequency of missense MEN1 mutations.

Gout, which is commonly associated with hyperuricemia, affects 0.2% of the population. Hyperuricemia has a heterogeneous etiology that may be due to either over production and/or reduced renal clearance, of urate. In order to identify the mechanisms underlying reduced excretion of urate, we undertook positional cloning studies of familial juvenile hyperuricaemic nephropathy (FJHN), which is an autosomal dominant disorder characterized by hyperuricaemia, a low fractional renal excretion of urate, and chronic renal failure that is associated with interstitial fibrosis. The FJHN locus has been previously localized to a 22 centiMorgan interval flanked centromerically by D16S401 and telomerically by D16S3069, on chromosome 16p11-p13. This interval contains over 120 genes and we selected 13 renal expressed sequences to search for mutations in 5 unrelated FJHN families that contained 21 affected and 24 unaffected members. This revealed 5 heterozygous missense mutations (Cys77Tyr, Cys126Arg, Asn128Ser, Cys255Tyr and Cys300Gly) that altered evolutionary conserved residues in the gene encoding UROMODULIN. UROMODULIN, which is an 85 Kda glycoprotein, has roles in renal stone formation, the modulation of immune responses, and urothelial cytoprotection. The results of our studies, which have identified the gene causing FJHN, now indicate a further, novel role for UROMODULIN in urate metabolism.