Safety and Efficacy of NVX-CoV2373 Covid-19 Vaccine.

BACKGROUND: Early clinical data from studies of the NVX-CoV2373 vaccine (Novavax), a recombinant nanoparticle vaccine against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that contains the full-length spike glycoprotein of the prototype strain plus Matrix-M adjuvant, showed that the vaccine was safe and associated with a robust immune response in healthy adult participants. Additional data were needed regarding the efficacy, immunogenicity, and safety of this vaccine in a larger population. METHODS: In this phase 3, randomized, observer-blinded, placebo-controlled trial conducted at 33 sites in the United Kingdom, we assigned adults between the ages of 18 and 84 years in a 1:1 ratio to receive two intramuscular 5-µg doses of NVX-CoV2373 or placebo administered 21 days apart. The primary efficacy end point was virologically confirmed mild, moderate, or severe SARS-CoV-2 infection with an onset at least 7 days after the second injection in participants who were serologically negative at baseline. RESULTS: A total of 15,187 participants underwent randomization, and 14,039 were included in the per-protocol efficacy population. Of the participants, 27.9% were 65 years of age or older, and 44.6% had coexisting illnesses. Infections were reported in 10 participants in the vaccine group and in 96 in the placebo group, with a symptom onset of at least 7 days after the second injection, for a vaccine efficacy of 89.7% (95% confidence interval [CI], 80.2 to 94.6). No hospitalizations or deaths were reported among the 10 cases in the vaccine group. Five cases of severe infection were reported, all of which were in the placebo group. A post hoc analysis showed an efficacy of 86.3% (95% CI, 71.3 to 93.5) against the B.1.1.7 (or alpha) variant and 96.4% (95% CI, 73.8 to 99.5) against non-B.1.1.7 variants. Reactogenicity was generally mild and transient. The incidence of serious adverse events was low and similar in the two groups. CONCLUSIONS: A two-dose regimen of the NVX-CoV2373 vaccine administered to adult participants conferred 89.7% protection against SARS-CoV-2 infection and showed high efficacy against the B.1.1.7 variant. (Funded by Novavax; EudraCT number, 2020-004123-16.).

The predictive role of symptoms in COVID-19 diagnostic models: A longitudinal insight.

To investigate the symptoms of SARS-CoV-2 infection, their dynamics and their discriminatory power for the disease using longitudinally, prospectively collected information reported at the time of their occurrence. We have analysed data from a large phase 3 clinical UK COVID-19 vaccine trial. The alpha variant was the predominant strain. Participants were assessed for SARS-CoV-2 infection via nasal/throat PCR at recruitment, vaccination appointments, and when symptomatic. Statistical techniques were implemented to infer estimates representative of the UK population, accounting for multiple symptomatic episodes associated with one individual. An optimal diagnostic model for SARS-CoV-2 infection was derived. The 4-month prevalence of SARS-CoV-2 was 2.1%; increasing to 19.4% (16.0%-22.7%) in participants reporting loss of appetite and 31.9% (27.1%-36.8%) in those with anosmia/ageusia. The model identified anosmia and/or ageusia, fever, congestion, and cough to be significantly associated with SARS-CoV-2 infection. Symptoms' dynamics were vastly different in the two groups; after a slow start peaking later and lasting longer in PCR+ participants, whilst exhibiting a consistent decline in PCR- participants, with, on average, fewer than 3 days of symptoms reported. Anosmia/ageusia peaked late in confirmed SARS-CoV-2 infection (day 12), indicating a low discrimination power for early disease diagnosis.

Safety and Efficacy of the NVX-CoV2373 Coronavirus Disease 2019 Vaccine at Completion of the Placebo-Controlled Phase of a Randomized Controlled Trial.

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.

UK national chronic hypoparathyroidism audit.

OBJECTIVES: Individuals with chronic hypoparathyroidism may experience suboptimal medical care with high frequency of unplanned hospitalisation and iatrogenic harm. In 2015 the European Society for Endocrinology published consensus guidelines on the management of chronic hypoparathyroidism. We set out to audit compliance with these guidelines. METHODS: Using these recommendations as audit standards we worked with the Society for Endocrinology and Parathyroid UK to conduct a national audit of management of chronic hypoparathyroidism in the United Kingdom. Endocrine leads in 117 endocrine departments were invited to participate in the survey by completing a data collection tool on up to 5 sequential cases of chronic hypoparathyroidism seen in their outpatient clinics in the preceding 12 months. Data were collected on 4 treatment standards and 9 monitoring standards. Data on hospitalisations and Quality of Life monitoring were also collected. RESULTS: Responses were received from 22 departments giving a response rate of 19%, concerning 80 individual cases. The mean age of subjects was 48.4 years. The main findings were that the commonest cause of hypoparathyroidism was post surgical (66.3%). Treatments taken by the group included activated vitamin D analogues (96.3%), oral calcium salts (66.3%), vitamin D supplements (17.5%), thiazide diuretics (5%) and rhPTH1-34 (1.3%). Compliance with the audit standards varied between 98.8% and 60% for the treatment standards and between 91.3% and 20% for the monitoring standards. Some of the areas of weakness revealed include low rates of 24 h urinary calcium excretion monitoring, serum magnesium monitoring and low rates of renal imaging where indicated. In addition and importantly, 16.3% of subjects had experienced at least one hospital admission in the preceding 12 months. CONCLUSION: We conclude that further improvements in the UK national standard of management of chronic hypoparathyroidism should be made and that this will benefit both quality of life, morbidity and potentially mortality in this group of patients.

Effects of the Norfolk diabetes prevention lifestyle intervention (NDPS) on glycaemic control in screen-detected type 2 diabetes: a randomised controlled trial.

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 < 65 years old (difference in mean HbA1c compared to CON -4.76 mmol/mol; 95% CI -7.75, -1.78 mmol/mol) than in older participants (-0.46 mmol/mol; 95% CI -2.67, 1.75; interaction 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 and -0.22 mmol/mol; 95% CI -2.7, 2.25; aged > 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.

Constitutive P2Y2 receptor activity regulates basal lipolysis in human adipocytes.

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 signalling and constitutive P2Y2 receptor activation suppresses basal lipolysis in primary human in vitro-differentiated adipocytes. We found that human adipocytes possess cytoplasmic Ca2+ 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 Ca2+ tone exerts an anti-lipolytic effect by suppression of Ca2+-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 demonstrate that basal lipolysis and adipokine secretion are controlled by autocrine purinergic signalling in human adipocytes.

ATP Evokes Ca2+ Responses and CXCL5 Secretion via P2X4 Receptor Activation in Human Monocyte-Derived Macrophages.

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.

Leukemic blasts program bone marrow adipocytes to generate a protumoral microenvironment.

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.

P2Y2 and P2Y6 receptor activation elicits intracellular calcium responses in human adipose-derived mesenchymal stromal cells.

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 (< 30 µM) and UDP-glucose (< 30 µM). ATP responses were attenuated by selective P2Y2 receptor antagonism (AR-C118925XX; IC50 1.1 ± 0.8 µM, 73.0 ± 8.5% max inhibition; N = 7 donors), and UTP responses were abolished. ADP responses were attenuated by the selective P2Y6 receptor antagonist, MRS2587 (IC50 437 ± 133nM, 81.0 ± 8.4% max inhibition; N = 6 donors). These data demonstrate that adenine and uracil nucleotides elicit intracellular calcium responses in human AD-MSCs with a predominant role for P2Y2 and P2Y6 receptor activation. This study furthers understanding about how human adipose-derived mesenchymal stromal cells can respond to external signalling cues.

Enhanced GABAA-Mediated Tonic Inhibition in Auditory Thalamus of Rats with Behavioral Evidence of Tinnitus.

Accumulating evidence suggests a role for inhibitory neurotransmitter dysfunction in the pathology of tinnitus. Opposing hypotheses proposed either a pathologic decrease or increase of GABAergic inhibition in medial geniculate body (MGB). In thalamus, GABA mediates fast synaptic inhibition via synaptic GABAA receptors (GABAARs) and persistent tonic inhibition via high-affinity extrasynaptic GABAARs. Given that extrasynaptic GABAARs control the firing mode of thalamocortical neurons, we examined tonic GABAAR currents in MGB neurons in vitro, using the following three groups of adult rats: unexposed control (Ctrl); sound exposed with behavioral evidence of tinnitus (Tin); and sound exposed with no behavioral evidence of tinnitus (Non-T). Tonic GABAAR currents were evoked using the selective agonist gaboxadol. Months after a tinnitus-inducing sound exposure, gaboxadol-evoked tonic GABAAR currents showed significant tinnitus-related increases contralateral to the sound exposure. In situ hybridization studies found increased mRNA levels for GABAAR δ-subunits contralateral to the sound exposure. Tin rats showed significant increases in the number of spikes per burst evoked using suprathreshold-injected current steps. In summary, we found little evidence of tinnitus-related decreases in GABAergic neurotransmission. Tinnitus and chronic pain may reflect thalamocortical dysrhythmia, which results from abnormal theta-range resonant interactions between thalamus and cortex, due to neuronal hyperpolarization and the initiation of low-threshold calcium spike bursts (Walton and Llinás, 2010). In agreement with this hypothesis, we found tinnitus-related increases in tonic extrasynaptic GABAAR currents, in action potentials/evoked bursts, and in GABAAR δ-subunit gene expression. These tinnitus-related changes in GABAergic function may be markers for tinnitus pathology in the MGB.

Effect of tissue inhibitor of metalloproteinases 3 on DLK1 shedding in cultured human pre-adipocytes and implications for adipose tissue remodelling.

BACKGROUND: Metabolically unhealthy obesity is associated with adipose tissue inflammation and increased risk of type 2 diabetes. Dysfunctional adipose tissue remodelling has been implicated in development of metabolically unhealthy obesity, but the pathogenesis remains poorly characterised. We hypothesised that in health, tissue inhibitor of metalloproteinases 3 (TIMP3) modulates adipose tissue remodelling by regulating extracellular matrix turnover and shedding of the adipogenic regulator DLK1, but that in adipose tissue inflammation it might drive development of metabolically unhealthy obesity. METHODS: Primary pre-adipocyte and in-vitro-differentiated adipocyte cultures were established from abdominal subcutaneous adipose tissue donated by healthy women undergoing breast reconstruction. Cells were seeded onto collagen I, and subsequently treated with differentiation medium or tumour necrosis factor (TNF) alpha (50 ng/mL). Adenoviral transduction allowed TIMP3 overexpression. Media and lysates were collected for quantitative RT-PCR, and immunoblot and hydroxyproline release assays. Statistical analysis was performed with t testing or ANOVA. FINDINGS: Induction of differentiation in human pre-adipocytes reduced TIMP3 mRNA levels by 75% (n=3, p<0·0001). Hydroxyproline release by differentiating pre-adipocytes was 2·3 times greater than that by control-treated cells (mean 5·66 µg/mL [SD 0·77] vs 2·45 [0·36], p<0·0001) indicating greater collagen I degradation. TNF alpha reduced TIMP3 mRNA levels by 66% in in-vitro-differentiated adipocytes (n=3, p<0·0001); reduced TIMP3 expression was confirmed by western blot. Shedding of soluble DLK1 (sDLK1) by pre-adipocytes was increased by TNF alpha and by overexpression of adenovirally delivered TIMP3 compared with control conditions, as confirmed by immunoblot (n=3). Addition of recombinant human sDLK1 (500 pM) to pre-adipocyte cultures reduced adipogenesis, as assessed by oil red O staining (n=2). INTERPRETATION: We have shown that TIMP3 is downregulated in adipogenesis, and by inflammatory signals in adipocytes. Furthermore, TIMP3 modulates sDLK1 shedding and collagen I degradation. TIMP3 is known to inhibit ADAM17 (DLK1 sheddase) and MMP14 (implicated in extracellular matrix turnover). TIMP3 might therefore integrate inflammatory signals with adipose remodelling. Subversion of remodelling pathways by chronic adipose inflammation might lead to maladaptive adipose expansion and metabolically unhealthy obesity. FUNDING: British Heart Foundation, Diabetes Research and Wellness Foundation Open Funding 2011.

The role of metalloproteinases and their tissue inhibitors in adipose tissue remodelling and whole-body lipid distribution: a cross-sectional clinical study.

BACKGROUND: Metabolically unhealthy obesity is associated with insulin resistance. Dysfunctional adipose tissue remodelling might explain features of this disorder, such as chronic white adipose tissue inflammation, adipocyte hypertrophy, and ectopic lipid deposition. Metalloproteinases and their tissue inhibitors (TIMPs) have been implicated in human adipose tissue remodelling. In a cross-sectional study, we investigated the association of adipose metalloproteinase and TIMP expression with whole-body lipid distribution and insulin resistance. METHODS: Healthy women undergoing elective surgery donated fasting blood samples (for calculation of homoeostasis model assessment of insulin resistance [HOMA2-IR], the primary outcome). At operation 2 cm(3) biopsy samples of subcutaneous and visceral adipose tissue were obtained. 1 cm(3) was fixed, paraffin-embedded, and stained for adipocyte size quantification, and RNA was extracted from the remaining tissue for quantitative RT-PCR analysis. The women also underwent whole-body MRI for analysis of fat distribution. FINDINGS: 26 women were recruited (mean age 50·3 years, SD 13·1) into five body-mass index categories (18·5-24·9 kg/m(2) [n=12, 46·1%], 25-29·9 [n=6, 23·1%], 30-34·9 [n=3, 11·5%], 35-39·9 [n=3, 11·5%], >40 [n=2, 7·8%]). Mean fasting glucose was 5·29 mmol/L (SD 0·66), mean fasting insulin 71·29 pmol/L (47·72), and mean HOMA2-IR 1·35 (0·91). HOMA2-IR correlated with body-mass index (r=0·73, p<0·0001), subcutaneous and visceral adipose tissue volumes (r=0·94 and r=0·87, respectively; both p<0·0001), and hepatic fat fraction (r=0·57, p=0·013). Visceral adipose tissue MMP14 expression correlated strongly with hepatic fat fraction (r=0·944, p<0·0001), HOMA2-IR (r=0·74, p=0·01), and visceral adipose tissue volume (r=0·74, p=0·036). Subcutaneous adipose tissue TIMP3 expression correlated with subcutaneous adipocyte area (r=0·72, p=0·029), but not with HOMA2-IR (r=-0·53, p=0·062). INTERPRETATION: The results suggest that metalloproteinases and TIMPs regulate adipose tissue remodelling and distribution. MMP14 has been implicated in collagen turnover in pre-adipocyte differentiation, whereas TIMP3 may modulate the shedding of DLK1, a regulator of adipogenesis. In our concurrent in-vitro study, we have shown that human adipocytes express metalloproteinases and TIMPs, and that their expression varies with inflammatory stimulation. These proteins might therefore integrate inflammatory signals with dysregulated adipose remodelling in metabolically unhealthy obesity. FUNDING: British Heart Foundation, Diabetes Research & Wellness Foundation Open Funding 2011.

Single unit hyperactivity and bursting in the auditory thalamus of awake rats directly correlates with behavioural evidence of tinnitus.

Tinnitus is an auditory percept without an environmental acoustic correlate. Contemporary tinnitus models hypothesize tinnitus to be a consequence of maladaptive plasticity-induced disturbance of excitation-inhibition homeostasis, possibly convergent on medial geniculate body (MGB, auditory thalamus) and related neuronal networks. The MGB is an obligate acoustic relay in a unique position to gate auditory signals to higher-order auditory and limbic centres. Tinnitus-related maladaptive plastic changes of MGB-related neuronal networks may affect the gating function of MGB and enhance gain in central auditory and non-auditory neuronal networks, resulting in tinnitus. The present study examined the discharge properties of MGB neurons in the sound-exposure gap inhibition animal model of tinnitus. MGB single unit responses were obtained from awake unexposed controls and sound-exposed adult rats with behavioural evidence of tinnitus. MGB units in animals with tinnitus exhibited enhanced spontaneous firing, altered burst properties and increased rate-level function slope when driven by broadband noise and tones at the unit's characteristic frequency. Elevated patterns of neuronal activity and altered bursting showed a significant positive correlation with animals' tinnitus scores. Altered activity of MGB neurons revealed additional features of auditory system plasticity associated with tinnitus, which may provide a testable assay for future therapeutic and diagnostic development.

Brown and white adipose tissues: intrinsic differences in gene expression and response to cold exposure in mice.

Brown adipocytes dissipate energy, whereas white adipocytes are an energy storage site. We explored the plasticity of different white adipose tissue depots in acquiring a brown phenotype by cold exposure. By comparing cold-induced genes in white fat to those enriched in brown compared with white fat, at thermoneutrality we defined a "brite" transcription signature. We identified the genes, pathways, and promoter regulatory motifs associated with "browning," as these represent novel targets for understanding this process. For example, neuregulin 4 was more highly expressed in brown adipose tissue and upregulated in white fat upon cold exposure, and cell studies showed that it is a neurite outgrowth-promoting adipokine, indicative of a role in increasing adipose tissue innervation in response to cold. A cell culture system that allows us to reproduce the differential properties of the discrete adipose depots was developed to study depot-specific differences at an in vitro level. The key transcriptional events underpinning white adipose tissue to brown transition are important, as they represent an attractive proposition to overcome the detrimental effects associated with metabolic disorders, including obesity and type 2 diabetes.

Identification of 70 calcium-sensing receptor mutations in hyper- and hypo-calcaemic patients: evidence for clustering of extracellular domain mutations at calcium-binding sites.

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.

Mice with behavioral evidence of tinnitus exhibit dorsal cochlear nucleus hyperactivity because of decreased GABAergic inhibition.

Tinnitus has been associated with increased spontaneous and evoked activity, increased neural synchrony, and reorganization of tonotopic maps of auditory nuclei. However, the neurotransmitter systems mediating these changes are poorly understood. Here, we developed an in vitro assay that allows us to evaluate the roles of excitation and inhibition in determining the neural correlates of tinnitus. To measure the magnitude and spatial spread of evoked circuit activity, we used flavoprotein autofluorescence (FA) imaging, a metabolic indicator of neuronal activity. We measured FA responses after electrical stimulation of glutamatergic axons in slices containing the dorsal cochlear nucleus, an auditory brainstem nucleus hypothesized to be crucial in the triggering and modulation of tinnitus. FA imaging in dorsal cochlear nucleus brain slices from mice with behavioral evidence of tinnitus (tinnitus mice) revealed enhanced evoked FA response at the site of stimulation and enhanced spatial propagation of FA response to surrounding sites. Blockers of GABAergic inhibition enhanced FA response to a greater extent in control mice than in tinnitus mice. Blockers of excitation decreased FA response to a similar extent in tinnitus and control mice. These findings indicate that auditory circuits in mice with behavioral evidence of tinnitus respond to stimuli in a more robust and spatially distributed manner because of a decrease in GABAergic inhibition.

Noise as an Extrinsic Variable in the Animal Research Facility.

Animal research facilities are noisy environments. The high air change rates required in animal housing spaces tend to create higher noise levels from the heating and cooling systems. Housing rooms are typically constructed of hard wall material that is easily cleaned but simultaneously highly reverberant, meaning that the sound cannot be controlled/attenuated so the sounds that are generated bounce around the room uncontrolled. (Soft, sound-absorbing surfaces generally cannot be used in animal facilities because they collect microbes; various wall surface features and sound control panel options are available, although rarely used.) In addition, many of our husbandry tasks such as cage changing, animal health checks, cleaning, and transporting animals produce high levels of noise. Finally, much of the equipment we have increasingly employed in animal housing spaces, such as ventilated caging motors, biosafety, or procedure cabinets, can generate high levels of background noise, including ultrasound. These and many additional factors conspire to create an acoustic environment that is neither naturalistic nor conducive to a stress-free environment. The acoustic variability both within and between institutions can serve as an enormous confounder for research studies and a threat to our ability to reproduce studies over time and between research laboratories. By gaining a better appreciation for the acoustic variables, paired with transparency in reporting the levels, we might be able to gain a better understanding of their impacts and thereby gain some level of control over such acoustic variables in the animal housing space. The result of this could improve both animal welfare and study reproducibility, helping to address our 3Rs goals of Replacement, Reduction, and Refinement in the animal biomedical research enterprise.

Diminished cortical inhibition in an aging mouse model of chronic tinnitus.

Flavoprotein autofluorescence imaging was used to examine auditory cortical synaptic responses in aged animals with behavioral evidence of tinnitus and hearing loss. Mice were exposed to noise trauma at 1-3 months of age and were assessed for behavioral evidence of tinnitus and hearing loss immediately after the noise trauma and again at ~24-30 months of age. Within 2 months of the final behavioral assessment, auditory cortical synaptic transmission was examined in brain slices using electrical stimulation of putative thalamocortical afferents, and flavoprotein autofluorescence imaging was used to measure cortical activation. Noise-exposed animals showed a 68% increase in amplitude of cortical activation compared with controls (p = 0.008), and these animals showed a diminished sensitivity to GABA(A)ergic blockade (p = 0.008, using bath-applied 200 nm SR 95531 [6-Imino-3-(4-methoxyphenyl)-1(6H)-p yridazinebutanoic acid hydrobromide]). The strength of cortical activation was significantly correlated to the degree of tinnitus behavior, assessed via a loss of gap detection in a startle paradigm. The decrease in GABA(A) sensitivity was greater in the regions of the cortex farther away from the stimulation site, potentially reflecting a greater sensitivity of corticocortical versus thalamocortical projections to the effects of noise trauma. Finally, there was no relationship between auditory cortical activation and activation of the somatosensory cortex in the same slices, suggesting that the increases in auditory cortical activation were not attributable to a generalized hyperexcitable state in noise-exposed animals. These data suggest that noise trauma can cause long-lasting changes in the auditory cortical physiology and may provide specific targets to ameliorate the effects of chronic tinnitus.

Acoustic experience alters the aged auditory system.

OBJECTIVES: Presbyacusis, one of the most common ailments of the elderly, is often treated with hearing aids, which serve to reintroduce some or all of those sounds lost to peripheral hearing loss. However, little is known about the underlying changes to the ear and brain as a result of such experience with sound late in life. The present study attempts to model this process by rearing aged CBA mice in an augmented acoustic environment (AAE). DESIGN: Aged (22-23 months) male (n = 12) and female (n = 9) CBA/CaJ mice were reared in either 6 weeks of low-level (70 dB SPL) broadband noise stimulation (AAE) or normal vivarium conditions. Changes as a function of the treatment were measured for behavior, auditory brainstem response thresholds, hair cell cochleograms, and gamma aminobutyric acid neurochemistry in the key central auditory structures of the inferior colliculus and primary auditory cortex. RESULTS: The AAE-exposed group was associated with sex-specific changes in cochlear pathology, auditory brainstem response thresholds, and gamma aminobutyric acid neurochemistry. Males exhibited significantly better thresholds and reduced hair cell loss (relative to controls) whereas females exhibited the opposite effect. AAE was associated with increased glutamic acid decarboxylase (GAD67) levels in the inferior colliculus of both male and female mice. However, in primary auditory cortex AAE exposure was associated with increased GAD67 labeling in females and decreased GAD67 in males. CONCLUSIONS: These findings suggest that exposing aged mice to a low-level AAE alters both peripheral and central properties of the auditory system and these changes partially interact with sex or the degree of hearing loss before AAE. Although direct application of these findings to hearing aid use or auditory training in aged humans would be premature, the results do begin to provide direct evidence for the underlying changes that might be occurring as a result of hearing aid use late in life. These results suggest the aged brain retains significantly anatomical, electrophysiological, and neurochemical plasticity.

Time course of tinnitus development following noise exposure in mice.

Gap-induced prepulse inhibition of acoustic startle (GPIAS) has been used in rats and mice to study the problem of tinnitus. The current study demonstrates that similar methods can be used to study the temporal development of tinnitus over time in middle-aged mice. Six-month-old mice on a mixed C57Bl6 × 129 background were anesthetized with isoflurane and exposed to unilateral noise (n = 15), or sham exposure for controls (n = 8), for 1 hr (16-kHz octave band signal, 116-dB SPL). Tinnitus was tested in eight different sound frequency bands before and at postexposure time points of 1, 3-4, 7, 14, 21, and 30 days and monthly thereafter until 7 months postexposure. Noise-exposed mice displayed a number of changes in GPIAS consistent with the presence of hyperacusis and tinnitus. Noise exposure was associated with acute tinnitus measured 1 day later at several frequencies at and above the exposure frequency center. Consistent, chronic tinnitus then emerged in the 24-kHz range. Several time points following noise exposure suggested evidence of hyperacusis, often followed temporally by the development of deficits in GPIAS (reflecting tinnitus). Temporal development of these changes following noise exposure are discussed in the context of the interactions among aging, noise exposure, and the associated neurochemical changes that occur at early stages of auditory processing.