[In search of the right diagnosis; the importance of parental values]. / Zoeken naar de juiste diagnose.

A pediatric case study showed that genetic testing to obtain a diagnosis becomes increasingly complex. Expectant parents found out prenatally, with an ultrasound that their child has skeletal abnormalities. The search for a diagnosis that followed after this test result shows how complex this can be. This case study furthermore shows that professionals might have aims for testing which differ from parents' wishes. While professionals in this case wanted to have a diagnosis in an efficient way, the parents involved in this case did not want further testing, supported by their religious believes. As they explained, they will accept their child unconditionally. But when the health problems of their daughter become more severe they accepted testing. This difference in opinions about genetic testing pleas for pre-test counselling which focusses on exploring parental values. Discussing values contributes to a satisfactory diagnostic trajectory and follow-up.

Symposium review: Future of housing for dairy cattle.

The objective of this review was to describe recent changes and expected developments in housing systems for dairy cows. These new developments should create an appropriate production environment for modern high-producing dairy cows and stimulate dairy farming-related developments in management, agro-technology, and equipment. Increased labor efficiency has been an important driver of the change from tie-stall barns to cubicle barns (also known as freestall barns). In future housing systems, the natural behavior of cows, climate control, emissions of ammonia and greenhouse gases, reuse of waste, manure quality, the aesthetics of buildings in the landscape, and capital efficiency are becoming increasingly important elements. To address future requirements, new concepts beyond cubicle barns must be developed. Freewalk housing systems; that is, loose housing systems without cubicles, would meet some of these future demands. These systems operate with composting bedding material or artificial permeable floors as lying and walking areas. However, these barns are still in development. Combinations of cubicle and freewalk housing systems, together with other techniques being developed, might become a major future housing system. Other techniques and systems that are being explored according to sustainability criteria include the multi-climate shed, the CowToilet (Hanskamp AgroTech, Doetinchem, the Netherlands) to separate feces and urine, and multifunctional buildings. These buildings and techniques can be part of land-based or, less commonly, city-based farming systems, such as floating farms.

Invited review: Compost-bedded pack barns for dairy cows.

Compost-bedded pack barns (CBP) are receiving increasing attention as a housing system for dairy cows that has potential to improve animal welfare. This article reviews current scientific knowledge about CBP with the aim of providing a comprehensive tool for producers and researchers using this housing system. In CBP, cows are provided with an open bedded pack area rather than the individual stalls and concrete alleys found in freestall systems. The bedded pack, a mixture of organic bedding and cattle excreta, is cultivated frequently (1-3 times per day) to incorporate fresh manure and air into the pack, thus promoting an aerobic composting process. To function well, CBP generally require a large area per cow. Optimal animal densities over the bedded area range from 7.4 to more than 15 m2/cow depending on several factors, including climate, bedding, pack management, and cow characteristics. Studies have indicated that CBP, compared with conventional systems such as freestall barns, have the potential to improve the welfare of dairy cows. In particular, the main reported benefits include improved comfort during resting, better foot and leg health, and more natural animal behavior. Research has also indicated that adequate udder health can be achieved in CBP. However, because the bedded pack has been shown to contain high bacterial concentrations, proper management is essential to maintain adequate cow cleanliness and reduce the risk of mastitis. Controlling pack moisture is consistently indicated as the most important issue with CBP. Especially under cold and humid weather conditions, large amounts of bedding may be necessary to keep the pack adequately dry and comfortable for the cows. Nevertheless, the improvements in cow health may offset the higher costs of bedding.

Serum Vitamin D and Age-Related Muscle Loss in Afro-Caribbean Men: The Importance of Age and Diabetic Status.

BACKGROUND: Prospective studies examining the potential association of vitamin D with age-related muscle loss have shown inconsistent results. OBJECTIVE: To examine the association between baseline serum 25-hydroxyvitamin D (25(OH)D), 1,25-dihydroxyvitamin D (1,25(OH)2D), and prospective change in lean mass with aging in African ancestry population. We also determined if associations were modulated by age and diabetes mellitus (DM). DESIGN: Prospective observational cohort study. SETTING: Data were collected from a random sub-sample of 574 men, participants of the Tobago Bone Health Study (TBHS). PARTICIPANTS: 574 Afro-Caribbean men, aged 43+ years (mean age: 59.1 ± 10.5), who were randomly selected as the participants in both the baseline and the follow-up visits. MEASUREMENTS: Baseline fasting serum 25(OH)D was measured using liquid chromatography mass spectrometry (LC-MS/MS), and and 1,25(OH)2D was measured using radioimmunosassay (RIA). Changes in dual-energy X-ray absorptiometry (DXA)-measured appendicular lean mass (ALM), and total body lean mass (TBLM) were measured over an average of 6.0 ± 0.5 years. The associations of 25(OH)D and 1,25(OH)2D with ALM and TBLM were assessed by multiple linear regression model after adjusting for potential confounders. RESULTS: When stratifying all men into two groups by age, greater baseline 25(OH)D and 1,25(OH)2D levels were associated with smaller losses of ALM and TBLM in older (age 60+ years) but not in younger (age 43 - 59 years) men. When stratifying by DM status, the associations of 25(OH)D and 1,25(OH)2D with declines in ALM and TBLM were statistically significant only in prediabetic, but not among normal glycemic or diabetic men. CONCLUSION: Higher endogenous vitamin D concentrations are associated with less lean mass loss with aging among older and prediabetic Afro-Caribbean men independent of potential confounders. Our findings raise a possibility that maintaining high serum vitamin D level might be important for musculoskeletal health in elderly and prediabetic African ancestry men.

Limits to the scope of non-invasive prenatal testing (NIPT): an analysis of the international ethical framework for prenatal screening and an interview study with Dutch professionals.

BACKGROUND: The introduction of non-invasive prenatal testing (NIPT) for foetal aneuploidies is currently changing the field of prenatal screening in many countries. As it is non-invasive, safe and accurate, this technique allows for a broad implementation of first-trimester prenatal screening, which raises ethical issues, related, for instance, to informed choice and adverse societal consequences. This article offers an account of a leading international ethical framework for prenatal screening, examines how this framework is used by professionals working in the field of NIPT, and presents ethical guidance for the expansion of the scope of prenatal screening in practice. METHODS: A comparative analysis of authoritative documents is combined with 15 semi-structured interviews with professionals in the field of prenatal screening in the Netherlands. Data were recorded, transcribed verbatim and analysed using thematic analysis. RESULTS: The current ethical framework consists of four pillars: the aim of screening, the proportionality of the test, justice, and societal aspects. Respondents recognised and supported this framework in practice, but expressed some concerns. Professionals felt that pregnant women do not always make informed choices, while this is seen as central to reproductive autonomy (the aim of screening), and that pre-test counselling practices stand in need of improvement. Respondents believed that the benefits of NIPT, and of an expansion of its scope, outweigh the harms (proportionality), which are thought to be acceptable. They felt that the out-of-pocket financial contribution currently required by pregnant women constitutes a barrier to access to NIPT, which disproportionally affects those of a lower socioeconomic status (justice). Finally, professionals recognised but did not share concerns about a rising pressure to test or discrimination of disabled persons (societal aspects). CONCLUSIONS: Four types of limits to the scope of NIPT are proposed: NIPT should generate only test outcomes that are relevant to reproductive decision-making, informed choice should be (made) possible through adequate pre-test counselling, the rights of future children should be respected, and equal access should be guaranteed. Although the focus of the interview study is on the Dutch healthcare setting, insights and conclusions can be applied internationally and to other healthcare systems.

Staphylococcus aureus protects its immune-evasion proteins against degradation by neutrophil serine proteases.

Neutrophils store large quantities of neutrophil serine proteases (NSPs) that contribute, via multiple mechanisms, to antibacterial immune defences. Even though neutrophils are indispensable in fighting Staphylococcus aureus infections, the importance of NSPs in anti-staphylococcal defence is yet unknown. However, the fact that S. aureus produces three highly specific inhibitors for NSPs [the extracellular adherence proteins (EAPs) Eap, EapH1 and EapH2], suggests that these proteases are important for host defences against this bacterium. In this study we demonstrate that NSPs can inactivate secreted virulence factors of S. aureus and that EAP proteins function to prevent this degradation. Specifically, we find that a large group of S. aureus immune-evasion proteins is vulnerable to proteolytic inactivation by NSPs. In most cases, NSP cleavage leads to functional inactivation of virulence proteins. Interestingly, proteins with similar immune-escape functions appeared to have differential cleavage sensitivity towards NSPs. Using targeted mutagenesis and complementation analyses in S. aureus, we demonstrate that all EAP proteins can protect other virulence factors from NSP degradation in complex bacterial supernatants. These findings show that NSPs inactivate S. aureus virulence factors. Moreover, the protection by EAP proteins can explain why this antibacterial function of NSPs was masked in previous studies. Furthermore, our results indicate that therapeutic inactivation of EAP proteins can help to restore the natural host immune defences against S. aureus.

Optimal serum cholesterol concentrations are associated with accelerated bone loss in African ancestry men.

UNLABELLED: We tested if serum lipid and lipoprotein cholesterol levels are associated with longitudinal measures of bone mineral density (BMD) in 1289 African ancestry men. After 6 years of mean follow-up, men with clinically optimal levels of low-density lipoprotein (LDL), high-density lipoprotein (HDL), or triglycerides at baseline experienced the greatest BMD loss, independent of potential confounding factors (all p < 0.05). INTRODUCTION: Studies of lipid and lipoprotein cholesterol associations with bone mineral density (BMD) and bone loss have been inconclusive, and longitudinal data are sparse. Therefore, the aim of this study was to test if fasting serum lipid and lipoprotein cholesterol levels are associated with areal and volumetric BMD and BMD change. METHODS: We determined the association of serum triglycerides, high-density lipoprotein (HDL), and low-density lipoprotein (LDL) cholesterol concentrations with cross-sectional and longitudinal (mean follow-up, 6.1 years) measures of BMD in a cohort of 1289 in African ancestry men (mean age, 56.4 years). Fasting serum triglycerides, HDL, and LDL were measured at baseline concurrent with BMD assessments. Dual-energy X-ray absorptiometry was used to quantify integral hip BMD, and peripheral quantitative computed tomography at the radius and tibia was used to quantify volumetric BMD. Men were categorized as optimal, borderline, or high risk for triglyceride, HDL, and LDL concentrations based on Adult Treatment Panel III guidelines. RESULTS: Lower serum triglyceride or LDL and higher HDL concentrations were associated with lower trabecular BMD at baseline (all p < 0.05). Similarly, men classified as having optimal levels of LDL, HDL, or triglycerides at baseline experienced the greatest integral BMD loss at the hip and trabecular BMD loss at the tibia (all p < 0.05), independent of potential confounding factors. CONCLUSIONS: We found that clinically optimal serum lipid and lipoprotein cholesterol concentrations were associated with accelerated bone loss among Afro-Caribbean men. Further studies are needed to better understand the mechanisms involved and potential clinical significance of these findings.

Antibiotic use in dairy herds in the Netherlands from 2005 to 2012.

The aim of this study was to examine the variation in antibiotic use and the effects of external factors on trends in antibiotic use at the herd level by using the number of daily dosages as an indicator for antibiotic use. For this purpose, antibiotic use was analyzed in 94 dairy herds in the Netherlands from 2005 to 2012. The herds were divided into 3 groups of farmers: one group was guided in their antibiotic use from 2008 to 2010 as part of the project, whereas the other 2 groups were not actively guided. The farms were located in 10 of the 12 provinces and were clients of 32 of the 300 veterinary practices that treat cattle. Sales invoices from the veterinary practices provided the antibiotic and cost data for the participating farmers. The number of animal-defined daily dosages (ADDD) indicates the number of days per year that the average cow in a herd is given antibiotic treatment. The average ADDD for all farms from 2005 to 2012 was 5.86 (standard deviation=2.14); 68% of ADDD were used for udder health, 24% for clinical mastitis and 44% for dry-cow therapy. Variation in ADDD among herds decreased during the study period. The trend in ADDD can be described as having 3 phases: (1) a period of increasing use coinciding with little public concern about antibiotic use (2005-2007), (2) a period of growing awareness and stabilization of use (2007-2010), and (3) a period of decreasing use coinciding with increasing societal concerns (2010-2012). The greatest reduction in use was for drugs other than those used to treat the udder. Drug use for mastitis treatment fell considerably in the final year of the study period, whereas farmers were reluctant to reduce use for dry-cow therapy. Almost 40% of the herds were given less than 2.5 ADDD for dry-cow therapy, which is equivalent to 2.5 tubes per average cow in the herd, and 20% used more than 3 tubes per cow. Use of third- and fourth-generation cephalosporins and fluoroquinolones dropped from 18% of ADDD during 2005 to 2010 to 1% in 2012, with a shift toward penicillins and broad spectrum drugs. The ADDD was 22% lower in 2012 than 2007, the year of the highest usage. The decrease in ADDD over time varied between the 3 groups of farmers. During the second phase of the study, the guided group began to display a reduction in use, whereas the other groups only displayed a significant reduction in the third phase. The reduction in antibiotic use has resulted in lower veterinary costs per cow in recent years.

GRPR-selective PET imaging of prostate cancer using [(18)F]-lanthionine-bombesin analogs.

The gastrin-releasing peptide receptor (GRPR) is overexpressed in a variety of human malignancies, including prostate cancer. Bombesin (BBN) is a 14 amino acids peptide that selectively binds to GRPR. In this study, we developed two novel Al(18)F-labeled lanthionine-stabilized BBN analogs, designated Al(18)F-NOTA-4,7-lanthionine-BBN and Al(18)F-NOTA-2,6-lanthionine-BBN, for positron emission tomography (PET) imaging of GRPR expression using xenograft prostate cancer models. (Methyl)lanthionine-stabilized 4,7-lanthionine-BBN and 2,6-lanthionine-BBN analogs were conjugated with a NOTA chelator and radiolabeled with Al(18)F using the aluminum fluoride strategy. Al(18)F-NOTA-4,7-lanthionine-BBN and Al(18)F-NOTA-2,6-lanthionine-BBN was labeled with Al(18)F with good radiochemical yield and specific activity>30 GBq/µmol for both radiotracers. The logD values measured for Al(18)F-NOTA-4,7-lanthionine-BBN and Al(18)F-NOTA-2,6-lanthionine-BBN were -2.14 ± 0.14 and -2.34 ± 0.15, respectively. In athymic nude PC-3 xenografts, at 120 min post injection (p.i.), the uptake of Al(18)F-NOTA-4,7-lanthionine-BBN and Al(18)F-NOTA-2,6-lanthionine-BBN in prostate cancer (PC-3) mouse models was 0.82 ± 0.23% ID/g and 1.40 ± 0.81% ID/g, respectively. An excess of unlabeled ɛ-aminocaproic acid-BBN(7-14) (300-fold) was co-injected to assess GRPR binding specificity. Tumor uptake of Al(18)F-NOTA-4,7-lanthionine-BBN and Al(18)F-NOTA-2,6-lanthionine-BBN in PC-3 tumors was evaluated by microPET (µPET) imaging at 30, 60 and 120 min p.i. Blocking studies showed decreased uptake in PC-3 bearing mice. Stabilized 4,7-lanthionine-BBN and 2,6-lanthionine-BBN peptides were rapidly and successfully labeled with (18)F. Both tracers may have potential for GRPR-positive tumor imaging.

Patient controlled analgesia with remifentanil versus epidural analgesia in labour: randomised multicentre equivalence trial.

OBJECTIVE: To determine women's satisfaction with pain relief using patient controlled analgesia with remifentanil compared with epidural analgesia during labour. DESIGN: Multicentre randomised controlled equivalence trial. SETTING: 15 hospitals in the Netherlands. PARTICIPANTS: Women with an intermediate to high obstetric risk with an intention to deliver vaginally. To exclude a clinically relevant difference in satisfaction with pain relief of more than 10%, we needed to include 1136 women. Because of missing values for satisfaction this number was increased to 1400 before any analysis. We used multiple imputation to correct for missing data. INTERVENTION: Before the onset of active labour consenting women were randomised to a pain relief strategy with patient controlled remifentanil or epidural analgesia if they requested pain relief during labour. MAIN OUTCOME MEASURES: Primary outcome was satisfaction with pain relief, measured hourly on a visual analogue scale and expressed as area under the curve (AUC), thus providing a time weighted measure of total satisfaction with pain relief. A higher AUC represents higher satisfaction with pain relief. Secondary outcomes were pain intensity scores, mode of delivery, and maternal and neonatal outcomes. Analysis was done by intention to treat. The study was defined as an equivalence study for the primary outcome. RESULTS: 1414 women were randomised, of whom 709 were allocated to patient controlled remifentanil and 705 to epidural analgesia. Baseline characteristics were comparable. Pain relief was ultimately used in 65% (447/687) in the remifentanil group and 52% (347/671) in the epidural analgesia group (relative risk 1.32, 95% confidence interval 1.18 to 1.48). Cross over occurred in 7% (45/687) and 8% (51/671) of women, respectively. Of women primarily treated with remifentanil, 13% (53/402) converted to epidural analgesia, while in women primarily treated with epidural analgesia 1% (3/296) converted to remifentanil. The area under the curve for total satisfaction with pain relief was 30.9 in the remifentanil group versus 33.7 in the epidural analgesia group (mean difference -2.8, 95% confidence interval -6.9 to 1.3). For who actually received pain relief the area under the curve for satisfaction with pain relief after the start of pain relief was 25.6 in the remifentanil group versus 36.1 in the epidural analgesia group (mean difference -10.4, -13.9 to -7.0). The rate of caesarean section was 15% in both groups. Oxygen saturation was significantly lower (SpO2 <92%) in women who used remifentanil (relative risk 1.5, 1.4 to 1.7). Maternal and neonatal outcomes were comparable between both groups. CONCLUSION: In women in labour, patient controlled analgesia with remifentanil is not equivalent to epidural analgesia with respect to scores on satisfaction with pain relief. Satisfaction with pain relief was significantly higher in women who were allocated to and received epidural analgesia. TRIAL REGISTRATION: Netherlands Trial Register NTR2551.

Relative influence of heritability, environment and genetics on serum sclerostin.

SUMMARY: We determined factors associated with serum sclerostin in 446 Afro-Caribbean family members. Age, weight, sex, diabetes and kidney function were associated with sclerostin. Sclerostin was heritable, and nine SNPs in the SOST gene region were associated with sclerostin. Variation in serum sclerostin is a heritable factor that is determined by both genetic and environmental factors. INTRODUCTION: Sclerostin, encoded by the SOST gene, is a Wnt inhibitor that regulates bone mineralization and is a candidate gene locus for osteoporosis. However, little is known about the genetic and non-genetic sources of inter-individual variation in serum sclerostin levels. METHODS: Serum sclerostin was measured in 446 Afro-Caribbean men and women aged 18+ from seven large, multigenerational families (mean family size, 64; 3,840 relative pairs). Thirty-six common single nucleotide polymorphisms (SNP) were genotyped within a 100 kb region encompassing the gene encoding sclerostin (SOST). Genetic and non-genetic factors were tested for association with serum sclerostin. RESULTS: Mean serum sclerostin was 41.3 pmol/l and was greater in men than in women (P < 0.05). Factors associated with higher serum sclerostin were increased age and body weight, male sex, diabetes and decreased glomerular filtration rate, which collectively accounted for 25.4 % of its variation. Residual genetic heritability of serum sclerostin was 0.393 (P < 0.0001). Nine SNPs reached nominal significance with sclerostin. Three of those nine SNPs represented independent association signals (rs851056, rs41455049 and rs9909172), which accounted for 7.8 % of the phenotypic variation in sclerostin, although none of these SNPs surpassed a Bonferroni correction for multiple comparisons. CONCLUSIONS: Serum sclerostin is a heritable trait that is also determined by environmental factors including age, sex, adiposity, diabetes and kidney function. Three independent common SNPs within the SOST region may collectively account for a significant proportion of the variation in serum sclerostin.

Association of volumetric bone mineral density with abdominal aortic calcification in African ancestry men.

SUMMARY: We tested for association between cortical and trabecular volumetric bone mineral density (vBMD) with abdominal aortic calcification (AAC) prevalence in 278 Afro-Caribbean men. AAC was present in 68.3 % of the men. Greater cortical, but not trabecular, vBMD was associated with significantly decreased odds of AAC independent of traditional risk factors. INTRODUCTION: The aim of this study is to assess the prevalence and correlates of AAC in a sample of 278 Afro-Caribbean men (mean age 56) and to test for a largely unexplored association between cortical and trabecular vBMD with AAC prevalence. METHODS: Men were recruited consecutively as part of an ongoing prospective cohort study of body composition in men aged 40+. For this analysis, AAC was assessed by computed tomography of the abdomen from L3 to S1. Aortic calcium was scored using the Agatston method, and prevalence was defined as a score ≥10 to rule out false positives. Men also had BMD assessed using peripheral quantitative computed tomography at 4 % (trabecular vBMD) and 33 % (cortical vBMD) of the radius and tibia. RESULTS: Abdominal aortic calcification was present in 68.3 % of the men. Significant independent predictors of AAC prevalence were increased age, increased BMI, hypertension, and current smoking. Age was the strongest predictor, with each SD (7.8 year) increase in age conferring 2.7 times increased odds of having AAC (P < 0.0001). A one SD greater cortical, but not trabecular, vBMD was associated with a significant decreased odds of AAC prevalence independent of other traditional risk factors (OR 0.65; 95 % CI 0.45-0.92). CONCLUSIONS: Cortical vBMD is inversely associated with AAC presence. This finding suggests that there may be shared physiology between cortical bone compartment remodeling and vascular calcification.

Technical note: A mathematical function to predict daily milk yield of dairy cows in relation to the interval between milkings.

The milk production of a dairy cow is characterized by lactation production, which is calculated from daily milk yields (DMY) during lactation. The DMY is calculated from one or more milkings a day collected at the farm. Various milking systems are in use today, resulting in one or many recorded milk yields a day, from which different calculations are used to determine DMY. The primary objective of this study was to develop a mathematical function that described milk production of a dairy cow in relation to the interval between 2 milkings. The function was partly based on the biology of the milk production process. This function, called the 3K-function, was able to predict milk production over an interval of 12h, so DMY was twice this estimate. No external information is needed to incorporate this function in methods to predict DMY. Application of the function on data from different milking systems showed a good fit. This function could be a universal tool to predict DMY for a variety of milking systems, and it seems especially useful for data from robotic milking systems. Further study is needed to evaluate the function under a wide range of circumstances, and to see how it can be incorporated in existing milk recording systems. A secondary objective of using the 3K-function was to compare how much DMY based on different milking systems differed from that based on a twice-a-day milking. Differences were consistent with findings in the literature.

Skeletal muscle adiposity is associated with serum lipid and lipoprotein levels in Afro-Caribbean men.

OBJECTIVE: When compared with other ethnic groups, African ancestry individuals have lower triglycerides and higher High-density lipoprotein cholesterol (HDL-C) levels, although the mechanisms for these differences remain unclear. A comprehensive array of factors potentially related to fasting serum lipid and lipoprotein levels in African ancestry men was evaluated. DESIGN AND METHODS: Men (1,821) underwent dual-energy X-ray absorptiometry measures of total body fat and quantitative computed tomography assessments of calf skeletal muscle adiposity [subcutaneous and intermuscular adipose tissue (AT), and muscle density as a measure of intra-muscular AT]. RESULTS: Multivariable linear regression analysis identified age (-), total body fat (+), subcutaneous AT (-), fasting glucose (+), fasting insulin (+), diastolic blood pressure (+), and non-African ancestry (+) as independent correlates of triglycerides (all P < 0.05). Total body fat (+), intra-muscular AT (-), and diastolic blood pressure (+) were independent correlates of Low-density lipoprotein cholesterol (LDL-C) (all P < 0.001). Age (+), waist circumference (-), fasting insulin (-), physical activity (+), and alcohol intake (+) were independent correlates of HDL-C (all P < 0.05). CONCLUSIONS: A novel relationship between skeletal muscle adiposity and serum lipid and lipoprotein levels in African ancestry men, independent of total and central adiposity was illuminated. In African ancestry populations, genetic factors are likely a significant determinant of triglycerides levels.

Genetic analysis of serum osteocalcin and bone mineral in multigenerational Afro-Caribbean families.

UNLABELLED: Osteocalcin is a major component of bone matrix. Concentrations of total, carboxylated, and uncarboxylated osteocalcin, are highly heritable and genetically correlated with bone mineral content (BMC) within African ancestry families. INTRODUCTION: Osteocalcin (OC) is a protein constituent of bone matrix and a marker of bone formation. We characterized the heritability of serum OC measures and identified genomic regions potentially involved in the regulation of OC via high-density genome-wide linkage analysis in African ancestry individuals. METHODS: African ancestry individuals (n = 459) were recruited, without regard to health status, from seven probands (mean family size = 66; 4,373 relative pairs). Residual heritability of serum OC measures was estimated and multipoint quantitative trait linkage analysis was performed using pedigree-based maximum likelihood methods. RESULTS: Residual heritabilities of total OC, uncarboxylated OC, carboxylated OC and percent uncarboxylated OC were 0.74 ± 0.10, 0.89 ± 0.08, 0.46 ± 0.10 and 0.41 ± 0.09, respectively. All OC measures were genetically correlated with whole body BMC. We obtained strong evidence of bivariate linkage for percent uncarboxylated OC and whole body BMC on chromosome 17 (logarithm of the odds [LOD] = 3.15, 99 cM). CONCLUSIONS: All forms of OC were highly heritable and genetically correlated with total body BMC in these African ancestry families. The identified linkage region contains several candidate genes for bone and energy metabolism including COL1A1 and TNFRSF11A. Further studies of this genomic region may reveal novel insight into the genetic regulation of OC and bone mineralization.

Genetic analysis of vertebral trabecular bone density and cross-sectional area in older men.

We investigated 383 bone candidate genes for associations between single nucleotide polymorphisms and vertebral trabecular volumetric bone mineral density (vBMD) and cross-sectional area (CSA) in 2,018 Caucasian men aged ≥ 65 years. SNPs in TGFBR3, SOST, KL, CALCR, LEP, CSF1R, PTN, GNRH2, FGFR2, and MEPE were associated with vBMD and SNPs in CYP11B1, DVL2, DLX5, WNT4, and PAX7 were associated with CSA in independent study samples (p < 0.005). INRODUCTION: Vertebral bone mineral density and cross-sectional area are important determinants of vertebral bone strength. Little is known about the specific genetic variants that influence these phenotypes in humans. METHODS: We investigated the potential genetic variants associated with vertebral trabecular volumetric BMD and CSA measured by quantitative computed tomography. We initially tested for association between these phenotypes and 4608 tagging and potentially functional single nucleotide polymorphisms (SNPs) in 383 candidate genes in 862 community-dwelling Caucasian men aged ≥ 65 years in the Osteoporotic Fractures in Men Study. RESULTS: SNP associations were then validated by genotyping an additional 1,156 randomly sampled men from the same cohort. We identified 11 SNPs in 10 genes (TGFBR3, SOST, KL, CALCR, LEP, CSF1R, PTN, GNRH2, FGFR2, and MEPE) that were consistently associated with trabecular vBMD and five SNPs in five genes (CYP11B1, DVL2, DLX5, WNT4, and PAX7) that were consistently associated with CSA in both samples (p < 0.005). CONCLUSION: None of the SNPs associated with trabecular vBMD were associated with CSA. Our findings raise the possibility that at least some of the loci for vertebral trabecular BMD and bone size may be distinct.

Association analysis of 33 lipoprotein candidate genes in multi-generational families of African ancestry.

African ancestry individuals have a more favorable lipoprotein profile than Caucasians, although the mechanisms for these differences remain unclear. We measured fasting serum lipoproteins and genotyped 768 tagging or potentially functional single nucleotide polymorphisms (SNPs) across 33 candidate gene regions in 401 Afro-Caribbeans older than 18 years belonging to 7 multi-generational pedigrees (mean family size 51, range 21-113, 3,426 relative pairs). All lipoproteins were significantly heritable (P<0.05). Gender-specific analysis showed that heritability for triglycerides was much higher (P<0.01) in women than in men (women, 0.62+/-0.18, P<0.01; men, 0.13+/-0.17, P>0.10), but the heritability for LDL cholesterol (LDL-C) was higher (P<0.05) in men than in women (men, 0.79+/-0.21, P<0.01; women, 0.39+/-0.12, P<0.01). The top 14 SNPs that passed the false discovery rate threshold in the families were then tested for replication in an independent population-based sample of 1,750 Afro-Caribbean men aged 40+ years. Our results revealed significant associations for three SNPs in two genes (rs5929 and rs6511720 in LDLR and rs7517090 in PCSK9) and LDL-C in both the family study and in the replication study. Our findings suggest that LDLR and PCSK9 variants may contribute to a variation in LDL-C among African ancestry individuals. Future sequencing and functional studies of these loci may advance our understanding of genetic factors contributing to LDL-C in African ancestry populations.

To protect peptide pharmaceuticals against peptidases.

INTRODUCTION: The major hurdle in the application and delivery of peptide pharmaceuticals is their rapid in vivo breakdown. METHODS: We here combined two approaches to stabilize peptide pharmaceuticals, introduction of D-amino acids and cyclization, by applying an innovative enzymatic method. This method yields peptides with thioether bridges between a D-amino acid and an L-amino acid. On the basis of guidelines concerning the flanking residues of serines/threonines and cysteines, a peptide of interest is designed with serine/threonine and cysteine at appropriate positions to allow their effective participation in cyclization. In Lactococcus lactis the peptide of interest is directly or via a spacer genetically fused to a lantibiotic leader peptide which induces enzyme-catalysed synthesis of a thioether-bridged peptide. The peptide is translocated via a lantibiotic transporter, analysed by mass spectrometry and the leader peptide is removed. Because of its therapeutic relevance and terminal modifications we chose the decapeptide Luteïnizing Hormone Release Hormone (LHRH) as a test case for thioether bridge introduction. The N-terminal pyroglutamate protects against aminopeptidase activity; the amidated C-terminus, which occurs in 50% of all therapeutic peptides, precludes carboxypeptidase action and is essential for optimal receptor interaction. We had Lactococcus posttranslationally introduce a thioether bridge between residues 4 and 7 of the Leu7Cys-LHRH analog QHWSYGCRPG. The N-terminal glutamine of the thioether-bridged peptide could be converted in pyroglutamate. The introduction of the thioether bridge proved to be compatible with subsequent chemical and enzymatic amidation methods. In this way biologically produced thioether LHRH was compared with LHRH isomers obtained by base-assisted sulfur extrusion. RESULTS: Biologically produced thioether LHRH is the most stable thioether LHRH isomer with strongly enhanced proteolytic resistance compared to natural LHRH. DISCUSSION: The data convincingly demonstrate the broad perspective of stereo- and regiospecifically generating cyclized peptide pharmaceuticals with significantly enhanced therapeutic potential.

Withdrawal of fluticasone propionate from combined salmeterol/fluticasone treatment in patients with COPD causes immediate and sustained disease deterioration: a randomised controlled trial.

BACKGROUND: Guidelines recommend inhaled corticosteroids (ICS) as maintenance treatment for patients with chronic obstructive pulmonary disease (COPD) with a post-bronchodilator forced expiratory volume in 1 second (FEV1) <50% predicted and frequent exacerbations, although they have only a small preventive effect on the accelerated decline in lung function. Combined treatment with ICS and long acting beta2 agonists (LABA) may provide benefit to the stability of COPD, but it is unknown if withdrawal of ICS will result in disease deterioration. METHODS: The effects of 1 year withdrawal of the ICS fluticasone propionate (FP) after a 3 month run-in treatment period with FP combined with the LABA salmeterol (S) (500 microg FP + 50 microg S twice daily; SFC) were investigated in patients with COPD in a randomised, double blind study. 497 patients were enrolled from 39 centres throughout the Netherlands; 373 were randomised and 293 completed the study. RESULTS: The drop out rate after randomisation was similar in the two groups. Withdrawal of FP resulted in a sustained decrease in FEV1: mean (SE) change from baseline -4.4 (0.9)% (S) v -0.1 (0.9)% (SFC); adjusted difference 4.1 (95% CI 1.6 to 6.6) percentage points (p<0.001). Corresponding figures for the FEV1/FVC ratio were -3.7 (0.8)% (S) v 0.0 (0.8)% (SFC) (p = 0.002). The annual moderate to severe exacerbation rate was 1.6 and 1.3 in the S and SFC groups, respectively (adjusted rate ratio 1.2; 95% CI 0.9 to 1.5; p = 0.15). The mean annual incidence rate of mild exacerbations was 1.3 (S) v 0.6 (SFC), p = 0.020. An immediate and sustained increase in dyspnoea score (scale 0-4; mean difference between groups 0.17 (0.04), p<0.001) and in the percentage of disturbed nights (6 (2) percentage points, p<0.001) occurred after withdrawal of fluticasone. CONCLUSIONS: Withdrawal of FP in COPD patients using SFC resulted in acute and persistent deterioration in lung function and dyspnoea and in an increase in mild exacerbations and percentage of disturbed nights. This study clearly indicates a key role for ICS in the management of COPD as their discontinuation leads to disease deterioration, even under treatment with a LABA.