Studies on factors which influence cyclic 3', 5'- adenosine monophosphate production in-vivo and in-vitro

Daily variations in plasma (PcAMP), urine (UcAMP), nephrogenous cAMP (NcAMP) and hormones known to affect cAMP metabolism were studied. Circadian variations in PTH (1-84) and NcAMP were observed. Both increased significantly overnight and early morning. PcAMP and glucagon decreased overnight and following meals. PcAMP had greater fluctuations over 24h than glucagon indicating several hormones have transient effects on PcAMP production. A 7h shift of the sleep-wake cycle moved sleep-associated prolactin secretion but caused minimal alteration in PTH (1-84). NcAMP secretion overnight decreased, suggesting PTH (1-84) end organ responses may be modified by other hormones and effectors when sleep is disturbed. PcAMP decreased overnight reaching a nadir corresponding to the lowest glucagon concentration. PcAMP fluctuations were greater when compared to normal 24h profiles. Following a 96h fast serum phosphate, PTH (1-84) and NcAMP circadian rhythms were attenuated. Mean serum calcium and NcAMP production were significantly decreased. Fasting increased variability in PTH (1-84) secretion which may be an important signalling mechanism inducing bone resorption. Serum phosphate fluctuations may play an important role in the genesis of PTH (1-84) and NcAMP circadian rhythms. Mean glucagon concentration was increased with attenuation of the circadian rhythm and mean PcAMP concentration increased. Dissociation of bone and kidney effects of PTH (1-84) in fasting may be due to acute acidosis. In 1°HPT the concerted overnight increase in PTH (1-84) and NcAMP was absent. Parathyroid adenoma resection restored phosphate, PTH (1-84) and NcAMP circadian rhythms. The phosphate increase may stimulate PTH (1-84) secretion which then influences the phosphate rhythm. Mean PcAMP was increased in 1°HPT but 24h PcAMP profiles differed little pre and post surgery. 24h studies indicate control of the circadian rhythms of PTH (1-84), NcAMP and PcAMP is complex. Several hormones act in concert to regulate cAMP generation and end organ responses. Thyrotoxic patients had mean PTH (1-84) and NcAMP lower than normals, hypothyroid and treated hypothyroid patients. Increased serum calcium decreases PTH (1-84) and subsequently NcAMP in thyrotoxicosis. In hypothyroid patients PTH (1-84) and NcAMP are dissociated with mean PTH (1-84) increased compared to thyrotoxic patients but NcAMP remained similar to euthyroid subjects indicating resistance to PTH (1-84) . Thyroxine treated patients have mean PTH (1-84) concentrations lower than euthyroid controls with a relatively increased NcAMP. PcAMP is increased in thyrotoxic and thyroxine treated patients and decreased in hypothyroid patients. Synacthen increased PcAMP with the pattern similar to the cortisol profile. This was either due to simultaneous release of cAMP and cortisol following adrenal stimulation or was a direct effect of cortisol. Dexamethasone decreased NcAMP and UcAMP with a variable effect on PcAMP. Variability in the PcAMP response is probably due to combined effects of dexamethasone and cortisol whereas decreased UcAMP and NcAMP production may represent altered kidney cell sensitivity to PTH (1-84) caused by decreased cortisol. In 1°HPT patients a significant correlation between PTH (1-84) and NcAMP exists (r=0.66, p<0.001). A subgroup of 1°HPT patients have NcAMP inappropriately high for the measured PTH (1-84). Two patients had increased PTHrP but in the majority PTHrP was low and the discrepancy was unexplained. In three patients PcAMP concentration was lower than expected. Twelve 1°HPT patients, divided equally, received atenolol or placebo and were studied prospectively for 6 months. No significant effect of atenolol on any parameter was observed. In patients with Paget's disease treated with bisphosphonate (APD) calcium decreased, PTH (1-84) and NcAMP increased significantly. Patients with HCM prior to therapy with APD had a suppressed or low PTH (1-84) . A high percentage had elevated NcAMP indicating the presence of a circulating humoral factor, probably PTHrP, causing hypercalcaemia. APD therapy in HCM patients resulted in a significant reduction in serum calcium. No patient developed significant hypocalcaemia. PTH (1-84) secretion was stimulated as serum calcium decreased and PTH (1-84) was detectable or elevated whilst some patients remained hypercalcaemic. The absolute concentration rather than rate of decrease of serum calcium was more important in regulating PTH (1-84) secretion. NcAMP correlated poorly with PTH (1-84) in treated HCM patients. An increase then decrease in NcAMP was observed associated with a decrease then increase in TmP04 indicating PTHrP may increase transiently following APD therapy. PTHrP measurement confirmed the hormone's role in the aetiology of HCM and a strong correlation of PTHrP with NcAMP. In HCM patients with breast, lung and kidney malignancy PTHrP secretion commonly results in elevated NcAMP. Patients with HCM and haematological, gastrointestinal and ear-nose-throat malignancies have low circulating PTHrP and normal or low NcAMP. Squamous cell carcinomas commonly secrete PTHrP. 78% of normals had detectable PTHrP which may reflect sample collection into protease inhibitor tubes, age distribution of the normal,population, assay sensitivity and properties of the assay antibodies. Alternatively the assay may be subject to nonspecific binding effects or matrix effects. A subgroup of patients was detected with low PTHrP and low PTH (1-84) with inappropriate or elevated NcAMP. These patients may be producing a factor which stimulates NcAMP production. This factor (s) may be a fragment of PTHrP or PTH not recognised by the IRMAs or may be a new molecule causing hypercalcaemia mediated via cAMP. Superfusion of rat renal tubules was used to study cAMP production in response to PTH stimulation. Optimal conditions producing a consistent, reproducible cAMP response were identified. Tubules were equilibrated on columns for 90 min, prior to stimulation with 2.5 units bovine PTH (1-84) at 10 min intervals. A significant decrease in cAMP production in response to PTH was observed when tubules were perfused with buffer acidic (pH 7.1) relative to normal physiological pH (pH 7.4). Perfusion with buffer relatively alkalotic (pH 7.65) had no significant effect on PTH stimulated cAMP production. 7mM arginine hydrochloride in the perfusate significantly decreased cAMP production. Superfusion of rat renal tubules was a reliable, reproducible method for studying the action of PTH

Interference of Asfotase alfa in immunoassays employing alkaline phosphatase technology

BACKGROUND: Asfotase alfa (STRENSIQ®, Alexion Pharmaceuticals, Inc.) is the only approved treatment for patients with pediatric-onset hypophosphatasia, a disease caused by a mutation in the tissue-nonspecific alkaline phosphatase (TNSALP) gene. ALP is often used as signaling system in routine immunoassays. Because asfotase alfa contains the active site of the full ALP enzyme, it can catalyze the substrate as the antibody-conjugated ALP would within an assay. Therefore, its presence in a treated patient's sample may generate false positive or false negative results. We investigated whether the presence of asfotase alfa within a sample induced interference in immunoassays that utilize ALP or alternative detection systems. METHODS: Asfotase alfa was added to samples at concentrations from 0.08-5 µg/mL and analysed on various immunoassays following manufacturer's instructions. RESULTS: Asfotase alfa was detected in all ALP assays but ALKP1 (RayBiotech). We observed no changes in normetanephrine and noradrenaline (IBL) at any asfotase alfa concentration. However, asfotase alfa notably interfered in an oxytocin (ENZO) assay in nonextracted samples. Extraction using a C18 column eliminated the interference. No interference was observed on automated analyzers using alternative detection system (COBAS fT4 and TSH; Advia Centaur FSH, fT4; Architect LH; FSH). Immulite 2000 fT4, TSH, testosterone and hCG (ALP-based) showed no interference. However, the presence of asfotase alfa resulted in a dose-dependent increase of Troponin I signal. CONCLUSION: The presence of asfotase alfa must be taken into consideration when analyzing blood samples in treated patients to avoid any risk of misinterpretation of false positive/negative results. It is essential that assays be tested for this possible interference

Elevated circulating Sclerostin concentrations in individuals with high bone mass, with and without LRP5 mutations.

CONTEXT: The role and importance of circulating sclerostin is poorly understood. High bone mass (HBM) caused by activating LRP5 mutations has been reported to be associated with increased plasma sclerostin concentrations; whether the same applies to HBM due to other causes is unknown. OBJECTIVE: Our objective was to determine circulating sclerostin concentrations in HBM. DESIGN AND PARTICIPANTS: In this case-control study, 406 HBM index cases were identified by screening dual-energy x-ray absorptiometry (DXA) databases from 4 United Kingdom centers (n = 219 088), excluding significant osteoarthritis/artifact. Controls comprised unaffected relatives and spouses. MAIN MEASURES: Plasma sclerostin; lumbar spine L1, total hip, and total body DXA; and radial and tibial peripheral quantitative computed tomography (subgroup only) were evaluated. RESULTS: Sclerostin concentrations were significantly higher in both LRP5 HBM and non-LRP5 HBM cases compared with controls: mean (SD) 130.1 (61.7) and 88.0 (39.3) vs 66.4 (32.3) pmol/L (both P < .001, which persisted after adjustment for a priori confounders). In combined adjusted analyses of cases and controls, sclerostin concentrations were positively related to all bone parameters found to be increased in HBM cases (ie, L1, total hip, and total body DXA bone mineral density and radial/tibial cortical area, cortical bone mineral density, and trabecular density). Although these relationships were broadly equivalent in HBM cases and controls, there was some evidence that associations between sclerostin and trabecular phenotypes were stronger in HBM cases, particularly for radial trabecular density (interaction P < .01). CONCLUSIONS: Circulating plasma sclerostin concentrations are increased in both LRP5 and non-LRP5 HBM compared with controls. In addition to the general positive relationship between sclerostin and DXA/peripheral quantitative computed tomography parameters, genetic factors predisposing to HBM may contribute to increased sclerostin levels.This study was supported by The Wellcome Trust NIHR Clinical Research Network (portfolio number 5163); and the supporting Comprehensive Local Research Networks included Birmingham and the Black Country, North and East Yorkshire and Northern Lincolnshire, South Yorkshire, West Anglia, and Western. C.L.G. was funded through a Wellcome Trust Clinical Research Training Fellowship (080280/Z/06/Z) and is now funded by Arthritis Research UK (Grant 20000). K.E.S.P. acknowledges the support of Cambridge NIHR Biomedical Research Centre and the Medical Research Council Human Nutrition Research Unit, Cambridge.This is the author accepted manuscript. The final version is available from the Endocrine Society via http://dx.doi.org/10.1210/jc.2013-395

The efficacy and mechanism evaluation of treating idiopathic pulmonary fibrosis with the addition of co-trimoxazole (EME-TIPAC): study protocol for a randomised controlled trial

Background: We hypothesise, based upon the findings from our previous trial, that the addition of co-trimoxazole to standard therapy is beneficial to patients with moderate to severe idiopathic pulmonary fibrosis (IPF). We aim to investigate this by assessing unplanned hospitalisation-free survival (defined as time from randomisation to first non-elective hospitalisation, lung transplant or death) and to determine whether any effect relates to changes in infection and/or markers of disease control and neutrophil activity. Methods/design: The EME-TIPAC trial is a double-blind, placebo-controlled, randomised, multicentre clinical trial. A total of 330 symptomatic patients, aged 40 years old or older, with IPF diagnosed by a multidisciplinary team (MDT) according to international guidelines and a FVC ≤ 75% predicted will be enrolled. Patients are randomised equally to receive either two tablets of co-trimoxazole 480 mg or two placebo tablets twice daily over a median treatment period of 27 (range 12–42) months. All patients receive folic acid 5 mg daily whilst on the trial IMP to reduce the risk of bone marrow depression. The primary outcome for the trial is a composite endpoint consisting of the time to death, transplant or first nonelective hospital admission and will be determined from adverse event reporting, hospital databases and the Office of National Statistics with active tracing of patients missing appointments. Secondary outcomes include the individual components of the primary outcome, (1) King’s Brief Interstitial Lung Disease Questionnaire, (2) MRC Dyspnoea Score, (3) EQ5D, (4) spirometry, (5) total lung-diffusing capacity and (6) routine sputum microbiology. Blood will be taken for cell count, biochemistry and analysis of biomarkers including C-reactive protein and markers of disease. The trial will last for 4 years. Recruitment will take place in a network of approximately 40 sites throughout the UK (see Table 1 for a full list of participating sites). We expect recruitment for 30 months, follow-up for 12 months and trial analysis and reporting to take 4 months. Discussion: The trial is designed to test the hypothesis that treating IPF patients with co-trimoxazole will increase the time to death (all causes), lung transplant or first non-elective hospital admission compared to standard care (https://www.nice.org.uk/guidance/cg163), in patients with moderate to severe disease. The mechanistic aims are to investigate the effect on lung microbiota and other measures of infection, markers of epithelial injury and markers of neutrophil activity. Trial registration: International Standard Randomised Controlled Trials Number (ISRCTN) Registry, ID: 17464641. Registered on 29 January 2015. Keywords: Idiopathic pulmonary fibrosis, Co-trimoxazole, Forced vital capacity, Mortalit

Mutations in Known Monogenic High Bone Mass Loci Only Explain a Small Proportion of High Bone Mass Cases.

High bone mass (HBM) can be an incidental clinical finding; however, monogenic HBM disorders (eg, LRP5 or SOST mutations) are rare. We aimed to determine to what extent HBM is explained by mutations in known HBM genes. A total of 258 unrelated HBM cases were identified from a review of 335,115 DXA scans from 13 UK centers. Cases were assessed clinically and underwent sequencing of known anabolic HBM loci: LRP5 (exons 2, 3, 4), LRP4 (exons 25, 26), SOST (exons 1, 2, and the van Buchem's disease [VBD] 52-kb intronic deletion 3'). Family members were assessed for HBM segregation with identified variants. Three-dimensional protein models were constructed for identified variants. Two novel missense LRP5 HBM mutations ([c.518C>T; p.Thr173Met], [c.796C>T; p.Arg266Cys]) were identified, plus three previously reported missense LRP5 mutations ([c.593A>G; p.Asn198Ser], [c.724G>A; p.Ala242Thr], [c.266A>G; p.Gln89Arg]), associated with HBM in 11 adults from seven families. Individuals with LRP5 HBM (∼prevalence 5/100,000) displayed a variable phenotype of skeletal dysplasia with increased trabecular BMD and cortical thickness on HRpQCT, and gynoid fat mass accumulation on DXA, compared with both non-LRP5 HBM and controls. One mostly asymptomatic woman carried a novel heterozygous nonsense SOST mutation (c.530C>A; p.Ser177X) predicted to prematurely truncate sclerostin. Protein modeling suggests the severity of the LRP5-HBM phenotype corresponds to the degree of protein disruption and the consequent effect on SOST-LRP5 binding. We predict p.Asn198Ser and p.Ala242Thr directly disrupt SOST binding; both correspond to severe HBM phenotypes (BMD Z-scores +3.1 to +12.2, inability to float). Less disruptive structural alterations predicted from p.Arg266Cys, p.Thr173Met, and p.Gln89Arg were associated with less severe phenotypes (Z-scores +2.4 to +6.2, ability to float). In conclusion, although mutations in known HBM loci may be asymptomatic, they only account for a very small proportion (∼3%) of HBM individuals, suggesting the great majority are explained by either unknown monogenic causes or polygenic inheritance.This study was supported by The Wellcome Trust and NIHR CRN (portfolio number 5163). CLG was funded by a Wellcome Trust Clinical Research Training Fellowship (080280/Z/06/Z), the EU 7th Framework Programme under grant agreement number 247642 (GEoCoDE), a British Geriatric Society travel grant, and is now funded by Arthritis Research UK (grant ref 20000). SH acknowledges Arthritis Research UK support (grant ref 19580). KESP acknowledges the support of Cambridge NIHR Biomedical Research Centre. KAW is supported by the core programme of the MRC Nutrition and Bone Health group at MRC Human Nutrition Research, funded by the UK Medical Research Council (Grant code U10590371). EM acknowledges support of the Sheffield Teaching Hospitals Foundation Trust Clinical Research Facility. The SGC is a registered charity (no. 1097737) that receives funds from AbbVie, Bayer, Boehringer Ingelheim, Genome Canada (Ontario Genomics Institute OGI- 055), GlaxoSmithKline, Janssen, Lilly Canada, Novartis Research Foundation, Ontario Ministry of Economic Development & Innovation, Pfizer, Takeda, and Wellcome Trust (092809/Z/10/Z).This is the final version of the article. It first appeared from Wiley via http://dx.doi.org/10.1002/jbmr.270

Ratiometric imaging of minor groove binders in mammalian cells using Raman microscopy

Quantitative drug imaging in live cells is a major challenge in drug discovery and development. Many drug screening techniques are performed in solution, and therefore do not consider the impact of the complex cellular environment in their result. As such, important features of drug-cell interactions may be overlooked. In this study, Raman microscopy is used as a powerful technique for quantitative imaging of Strathclyde-minor groove binders (S-MGBs) in mammalian cells under biocompatible imaging conditions. Raman imaging determined the influence of the tail group of two novel minor groove binders (S-MGB-528 and S-MGB-529) in mammalian cell models. These novel S-MGBs contained alkyne moieties which enabled analysis in the cell-silent region of the Raman spectrum. The intracellular uptake concentration, distribution and mechanism were evaluated as a function of the pKa of the tail group, morpholine and amidine, for S-MGB-528 and S-MGB-529, respectively. Although S-MGB-529 had a higher binding affinity to the minor groove of DNA in solution phase measurements, the Raman imaging data indicated that S-MGB-528 showed a greater degree of intracellular accumulation. Furthermore, using high resolution stimulated Raman scattering (SRS) microscopy the initial localisation of S-MGB-528 was shown to be in the nucleus before accumulation in the lysosome, which was demonstrated using a multimodal imaging approach. This study highlights the potential of Raman spectroscopy for quantitative drug imaging studies and highlights the importance of imaging techniques to investigate drug-cell interactions, to better inform the drug design process

Targeting the MAPK7/MMP9 axis for metastasis in primary bone cancer

From Springer Nature via Jisc Publications RouterHistory: received 2020-03-17, rev-recd 2020-05-24, accepted 2020-06-23, registration 2020-06-24, pub-electronic 2020-07-13, online 2020-07-13, pub-print 2020-08-13Publication status: PublishedFunder: Friends of RosieFunder: THRT, Big C, Paget's AssociationAbstract: Metastasis is the leading cause of cancer-related death. This multistage process involves contribution from both tumour cells and the tumour stroma to release metastatic cells into the circulation. Circulating tumour cells (CTCs) survive circulatory cytotoxicity, extravasate and colonise secondary sites effecting metastatic outcome. Reprogramming the transcriptomic landscape is a metastatic hallmark, but detecting underlying master regulators that drive pathological gene expression is a key challenge, especially in childhood cancer. Here we used whole tumour plus single-cell RNA-sequencing in primary bone cancer and CTCs to perform weighted gene co-expression network analysis to systematically detect coordinated changes in metastatic transcript expression. This approach with comparisons applied to data collected from cell line models, clinical samples and xenograft mouse models revealed mitogen-activated protein kinase 7/matrix metallopeptidase 9 (MAPK7/MMP9) signalling as a driver for primary bone cancer metastasis. RNA interference knockdown of MAPK7 reduces proliferation, colony formation, migration, tumour growth, macrophage residency/polarisation and lung metastasis. Parallel to these observations were reduction of activated interleukins IL1B, IL6, IL8 plus mesenchymal markers VIM and VEGF in response to MAPK7 loss. Our results implicate a newly discovered, multidimensional MAPK7/MMP9 signalling hub in primary bone cancer metastasis that is clinically actionable

Do público e do privado: uma perspectiva de género sobre uma dicotomia moderna

Neste texto propomos uma interpretação crítica da dicotomia histórica entre público e privado como dinâmica fundamental da modernidade. A partir de uma perspectiva de género, discutimos as fronteiras construídas entre espaço coletivo de cidadania e de sociabilidade e espaço individual de intimidade e desigualdade. Argumentamos a favor de uma relação de cumplicidade, ainda que tensa, entre as duas esferas, observando que a vida privada foi, em grande medida, moldada pelas mudanças operadas na vida pública. Recorrendo a diferentes definições de "público", notamos que, à medida que as sociabilidades tradicionais, essencialmente masculinas, estudadas entre outros por Ariès ou Sennett, sofriam uma erosão, crescia o sentimento de intimidade, aumentando igualmente a inclusão do privado no público através do alargamento da cidadania, em consequência das lutas travadas na esfera pública por vários movimentos de emancipação, como o operário ou o feminista. À medida que a pessoa era retirada da comunidade, do clã, do grupo de parentesco, em que eram "naturais" as desigualdades, no sentido aristotélico do termo, ia-se reencontrando progressivamente como indivíduo portador de cidadania. Se o espaço privado se tornou central na definição de uma identidade, ele é também crescentemente atravessado por mecanismos públicos de regulação. Nesse sentido, o movimento de ascensão do privado, que nas últimas décadas tem ocupado espaço de debate, deve ser cuidadosamente reinterpretado