Search

SORL1 is strongly associated with both sporadic and familial forms of Alzheimer’s disease (AD), but a lack of information about alternatively spliced transcripts currently limits our understanding of the role of SORL1 in AD. Here, we describe a SORL1 transcript (SORL1-38b) characterized by inclusion of a novel exon (E38b) that encodes a truncated protein. We identified E38b-containing transcripts in several brain regions, with the highest expression in the cerebellum and showed that SORL1-38b is largely located in neuronal dendrites, which is in contrast to the somatic distribution of transcripts encoding the full-length SORLA protein (SORL1-fl). SORL1-38b transcript levels were significantly reduced in AD cerebellum in three independent cohorts of postmortem brains, whereas no changes were observed for SORL1-fl. A trend of lower 38b transcript level in cerebellum was found for individuals carrying the risk variant at rs2282649 (known as SNP24), although not reaching statistical significance. These findings suggest synaptic functions for SORL1-38b in the brain, uncovering novel aspects of SORL1 that can be further explored in AD research.

Introduction Depression is expected to be the leading cause of disability worldwide by 2030. The prevalence is increasing and is two-fold higher in women than in men, women being particular at high risk during hormonal transition phases such as pregnancy and the postpartum period. The objective for this trial was to assess the effect of supervised group exercise on psychological well-being and symptoms of depression among pregnant women with or at high risk of depression. Material and methods This study was undertaken at Department of Obstetrics, Copenhagen University Hospital, Rigshospitalet, Denmark, from August 2016-September 2018. Pregnant women with a current or previous history of depression or/and anxiety requiring treatment within the last ten years, or use of antidepressants three months prior to or during pregnancy, were randomly assigned to 12 weeks supervised group exercise from 17-22 weeks of gestation twice weekly, or to a control group. The primary outcome was self-reported psychological well-being at 29-34 weeks of gestation, measured by the five-item World Health Organization Well-being Index (WHO-5). Secondary outcomes included delivery outcomes and psychological well-being (WHO-5) eight weeks postpartum. Results The intention-to-treat analysis showed no significant effect on psychological well-being on the primary outcome. Mean WHO-5 score in the intervention group was 2.0 (95% CI: -1.3 to 5.2, P=0.2) higher than in the control group. Per protocol analysis of women who attended ≥75% of the exercise sessions showed a statistically significant higher WHO-5 mean relative to controls at gestational weeks 29-34. Eight weeks postpartum the intervention group reported higher psychological well-being than the control group, mean difference in WHO-5 of 5.5 (95% CI: 1.0 to 10.1, P=0.04). Conclusions Supervised group exercise did not improve psychological well-being for women with or at high risk of depression at 29-34 weeks of gestation. Eight weeks postpartum the intervention group reported significantly higher psychological well-being than the control group. Based on our results, supervised exercise in groups is a safe complementary course of treatment alongside the existing antenatal care.

The few established causal genes in Alzheimer’s disease (AD), mutations in APP and PSENs, have been functionally characterized using biomarkers, capturing an in vivo profile reflecting the disease’s initial preclinical phase. SORL1, a gene encoding the endosome recycling receptor SORLA, epidemiologically behaves as a causal gene when truncating mutations lead to partial loss of protein function. Here, in an effort to test whether SORL1 can indeed function as an AD causal gene, we used CRISPR-Cas9-based gene editing to develop a novel model of SORL1 haploinsufficiency in Göttingen Minipigs taking advantage of porcine models for biomarker investigations. SORL1 haploinsufficiency in young minipigs was found to phenocopy the preclinical in vivo profile of AD observed with other causal genes, resulting in spinal fluid abnormalities in Aβ and tau, with no evident neurodegeneration or amyloid plaque formation. These studies provide functional support that SORL1 is a bona fide causal gene in AD, and when taken together with recent insight on other AD-causal genes, support the idea that dysfunctional endosomal recycling is a dominant pathogenic pathway in the disease.

Background and objective During pregnancy, absence from work increases significantly. Job adjustments have been shown to decrease absences; however, studies show only half of pregnant women who need job adjustments receive them. Little is known about the viewpoints of managers and possible challenges in the management of pregnant employees. The aim of this study was to investigate the experiences and considerations of managers in relation to managing pregnant hospital staff members and to describe the experiences of an active management policy for pregnant individuals. Methods A qualitative study based on five focus group interviews was conducted at five public hospitals in Zealand, Denmark with participation of 19 hospital managers, from 17 different wards, representing six different medical specialties. The interviews took place from February to May 2019. Thematic analysis was used to analyze the data. Results Four themes were identified: (1) The everyday management, (2) Managerial dilemmas, (3) Acknowledging the workplace culture, and (4) Dialogue as a means for the working relationship. The managers’ experiences revolved around investing a lot of effort into the working relationship with pregnant staff members by adjusting job tasks and work schedules while balancing work tasks between all staff members. The dialogue was considered central in order to identify the needs of the individual staff member. Conclusions Overall, management dialogue constituted a central tool in order to identify the needs of the individual staff member. A proactive and open approach increased the chances of a fruitful dialogue. The individual staff member, the influence of the workplace culture, and the everyday management of the workplace all shaped the experiences of the managers. The concept of an active management policy for pregnant individuals was perceived to entail useful elements, but also as replicating what managers already did.

The few established causal genes in Alzheimer’s disease (AD), mutations in APP and PSENs, have been functionally characterized using biomarkers, capturing an in vivo profile reflecting the disease’s initial preclinical phase. SORL1, a gene encoding the endosome recycling receptor SORLA, epidemiologically behaves as a causal gene when truncating mutations lead to partial loss of protein function. Here, in an effort to test whether SORL1 can indeed function as an AD causal gene, we used CRISPR-Cas9-based gene editing to develop a novel model of SORL1 haploinsufficiency in Gottingen Minipigs taking advantage of porcine models for biomarker investigations. SORL1 haploinsufficiency in young minipigs was found to phenocopy the preclinical in vivo profile of AD observed with other causal genes, resulting in spinal fluid abnormalities in Ab and tau, with no evident neurodegeneration or amyloid plaque formation. These studies provide functional support that SORL1 is a bona fide causal gene in AD, and when taken together with recent insight on other AD-causal genes, support the idea that dysfunctional endosomal recycling is a dominant pathogenic pathway in the disease.

Urosepsis is a potentially life-threatening, systemic reaction to uropathogenic bacteria entering the bloodstream of the host. One of the hallmarks of sepsis is early thrombocyte activation with a following fall in circulating thrombocytes as a result of intravascular aggregation and sequestering of thrombocytes in the major organs. Development of a thrombocytopenic state is associated with a poorer outcome of sepsis. Uropathogenic Escherichia coli frequently produce the pore-forming, virulence factor &alpha, haemolysin (HlyA), of which the biological effects are mediated by ATP release and subsequent activation of P2 receptors. Thus, we speculated that inhibition of thrombocyte P2Y1 and P2Y12 receptors might ameliorate the septic response to HlyA-producing E. coli. The study combined in vitro measurements of toxin-induced thrombocyte activation assessed as increased membrane abundance of P-selectin, fibronectin and CD63 and data from in vivo murine model of sepsis-induced by HlyA-producing E. coli under infusion of P2Y1 and P2Y12 antagonists. Our data show that the P2Y1 receptor antagonist almost abolishes thrombocyte activation by pore-forming bacterial toxins. Inhibition of P2Y1, by constant infusion of MRS2500, markedly increased the survival in mice with induced sepsis. Moreover, MRS2500 partially prevented the sepsis-induced depletion of circulating thrombocytes and dampened the sepsis-associated increase in proinflammatory cytokines. In contrast, P2Y12 receptor inhibition had only a marginal effect in vivo and in vitro. Taken together, inhibition of the P2Y1 receptor gives a subtle dampening of the thrombocyte activation and the cytokine response to bacteraemia, which may explain the improved survival observed by P2Y1 receptor antagonists.

Background In Scandinavia, women of childbearing age represent nearly 50% of the overall workforce. Two-thirds of these women spend a considerable time on sick leave during their pregnancies. Low back pain accounts for a significant portion of all pregnancy-related sick leave. However, pregnant women's experiences with pain-induced sick leave remains unexplored. Objective The study aimed to investigate women's experiences with sick leave in relation to pregnancy-induced low back pain. Methods An inductive, qualitative study based on semi-structured, in-depth, face-to-face interviews with 19 purposefully selected Danish women. Interviews were analysed by means of thematic content analysis. Results The analysis revealed 4 categories: (1) Stuck in a diagnosis, (2) Inflexibility of the labour market, (3) Adapting to reduced capacity for work, and (4) Being socially excluded. The women's experiences revolved around disruption of their physical functioning and expected capacity for work, a loss of professional identity, and a sense of inflexibility and exclusion from important relationships at work. Conclusions Our findings illuminate the possibilities for workplace adjustments with the intention of reducing time spent on sick leave, maintaining pregnant women's affiliation with their workplace, and a need to explore the role of healthcare professionals in addressing women's supportive needs in relation to sick leave.

Urosepsis is a severe condition often caused by Escherichia coli that spontaneously have ascended the urinary tract to the kidneys causing pyelonephritis and potentially bacteraemia. The number of sepsis cases has been steadily increasing over the last decades, and there are still no specific, molecular supportive therapies for sepsis to supplement antibiotic treatment. P2X1 receptors are expressed by a number of immune cells including thrombocytes, which presently have been established as an important player in the acute immune response to bacterial infections. P2X1 receptor-deficient mice have been shown to be relatively protected against urosepsis, with markedly reduced levels of circulating proinflammatory cytokines and intravascular coagulation. However, here we show that continuous intravenous infusion with P2X1 receptor antagonist markedly accelerates development of a septic response to induced bacteraemia with uropathogenic E. coli. Mice exposed to the P2X1 receptor antagonists die very early with haematuria, substantially elevated plasma levels of proinflammatory cytokines, massive intravascular coagulation and a concomitant reduction in circulating thrombocytes. Interestingly, infusion of P2X1 receptor antagonists causes a marked acute reduction in circulating thrombocytes and a higher number of bacteria in the blood. These data support the notion that the number of functional thrombocytes is important for the acute defence against bacteria in the circulation and that the P2X1 receptor potentially could be essential for this response.

Severe urinary tract infections are commonly caused by sub-strains of Escherichia coli secreting the pore-forming virulence factor α-hemolysin (HlyA). Repeated or severe cases of pyelonephritis can cause renal scarring that subsequently can lead to progressive failure. We have previously demonstrated that HlyA releases cellular ATP directly through its membrane pore and that acute HlyA-induced cell damage is completely prevented by blocking ATP signaling. Local ATP signaling and P2X 7 receptor activation play a key role in the development of tissue fibrosis. This study investigated the effect of P2X 7 receptors on infection-induced renal scarring in a murine model of pyelonephritis. Pyelonephritis was induced by injecting 100 million HlyA-producing, uropathogenic E. coli into the urinary bladder of BALB/cJ mice. A similar degree of pyelonephritis and mortality was confirmed at day 5 after infection in P2X 7 +/+ and P2X 7 −/− mice. Fibrosis was first observed 2 weeks after infection, and the data clearly demonstrated that P2X 7 −/− mice and mice exposed to the P2X 7 antagonist, brillian blue G, show markedly less renal fibrosis 14 days after infection compared with controls (P < 0.001). Immunohistochemistry revealed comparable early neutrophil infiltration in the renal cortex from P2X 7 +/+ and P2X 7 −/− mice. Interestingly, lack of P2X 7 receptors resulted in diminished macrophage infiltration and reduced neutrophil clearance in the cortex of P2X 7 −/− mice. Hence, this study suggests the P2X 7 receptor to be an appealing antifibrotic target after renal infections.

α-Haemolysin (HlyA) from uropathogenic Escherichia coli has been demonstrated to be a significant virulence factor for ascending urinary tract infections. Once the E. coli reach the well-vascularised kidneys, there is a high risk of bacteraemia and a subsequent septic host response. Despite this, HlyA has the potential to accelerate the host response both directly and via its ability to facilitate adenosine triphosphate release from cells. It has not been settled whether HlyA aggravates bacteraemia into a septic state. To address this, we used an E. coli strain in a model of acute urosepsis that was either transfected with a plasmid containing the full HlyA operon or one with deletion in the HlyA gene. Here, we show that HlyA accelerates the host response to E. coli in the circulation. Mice exposed to HlyA-producing E. coli showed massively increased proinflammatory cytokines, a substantial fall in circulating thrombocytes, extensive haematuria, and intravascular haemolysis. This was not seen in mice exposed to either E. coli that do not secrete HlyA or vehicle controls. Consistent with the massive host response to the bacteria, the mice exposed to HlyA-producing E. coli died exceedingly early, whereas mice exposed to E. coli without HlyA production and vehicle controls survived the entire observation period. These data allow us to conclude that HlyA is a virulence factor that accelerates a state of bacteraemia into fulminant sepsis in a mouse model.

Background: Focal congenital hyperinsulinism (CHI) may be cured by resection of the focal, but often non-palpable, pancreatic lesion. The surgical challenge is to minimize removal of normal pancreatic tissue. Aim: To evaluate the results of intraoperative ultrasound-guided, tissue-sparing pancreatic resection in CHI patients at an international expert center. Methods: Retrospective study of CHI patients treated at Odense University Hospital, Denmark, between January 2010 and March 2017. Results: Of 62 consecutive patients with persistent CHI, 24 (39%) had focal CHI by histology after surgery. All patients had a paternal ABCC8 or KCNJ11 mutation and a focal lesion by 18F-DOPA-PET/CT. Intraoperative ultrasound localized the focal lesion in 16/20 patients (sensitivity 0.80), including one ectopic lesion in the duodenal wall. Intraoperative ultrasound showed no focal lesion in 11/11 patients with diffuse CH (specificity 1.0). The positive predictive value for focal histology was 1.0, negative predictive value 0.73. Tissue-sparing pancreatic resection (focal lesion enucleation, local resection of tail or uncinate process) was performed in 67% (n = 16). In 11/12 having tissue-sparing resection and intraoperative ultrasound, the location of the focal lesion was exactly identified. Eight patients had resection of the pancreatic head or head/body, four with Roux-en-Y, three with pancreatico-gastrostomy and one without reconstruction. None had severe complications to surgery. Cure of hypoglycaemia was seen in all patients after one (n = 21) or two (n = 3) pancreatic resections. Conclusion: In focal CHI, tissue-sparing pancreatic resection was possible in 67%. Intraoperative ultrasound was a helpful supplement to the mandatory use of genetics, preoperative 18F-DOPA-PET/CT and intraoperative frozen sections.

Background: Maternal smoking is still a major public health problem posing the risk of several negative health outcomes for both the pregnant woman and her offspring. The prevalence of maternal smoking in Denmark and other high-income countries has decreased continuously since the 1980s, and a prevalence below 10% of women who continue to smoke during pregnancy has been reported in studies after 2010. Previous studies have shown that low socioeconomic status is associated with maternal smoking. Information from the Danish Birth Register about maternal smoking shows that the prevalence of women who report to smoke in pregnancy has decreased continuously with 23.3% who reported ever smoking in pregnancy in 2000, 12.9% in 2010 and 9.0% in 2017. The aim of this study was to estimate the prevalence of maternal smoking at the time of conception and at 20 weeks of gestation in a regional Danish population, to describe differences in maternal characteristics among smokers, quitters and never-smokers, and to estimate predictors of smoking at the time of conception. Methods: A cross-sectional study was conducted among pregnant women receiving antenatal care at the Department of Obstetrics, Zealand University Hospital, Denmark from August 2015 to March 2016 (n = 566). The main outcome was smoking at the time of conception and at 20 weeks of gestation. The questionnaire also collected information about maternal, health-related and sociodemographic characteristics. Descriptive analysis was conducted, and multivariate logistic regression analysis was used to assess the potential associated predictors (adjusted odds ratio). Results: The prevalence of self-reported smoking at the time of conception was 16% (n = 90) and 6% smoked at 20 weeks of gestation (n = 35), as 61% of smokers quit smoking during early pregnancy. Multiple logistic regression analysis showed that significant predictors for smoking at conception were the socioeconomic factors; ≤12 years of education, shift work and being unemployed. Conclusion: The prevalence of self-reported maternal smoking in this regional Danish population of pregnant women is lower than seen in previous studies. However, predictors for smoking at the time of conception remain to be factors of low socioeconomic status confirming a social inequality in maternal smoking. Women at risk of smoking during pregnancy must be identified in early pregnancy or even before pregnancy and be offered interventions to help them quit smoking.

BACKGROUND: Low back pain is a common condition among childbearing women, causing physical disability and an increased risk of sick leave and obstetric complications.AIMS: To assess the prevalence and intensity of low back pain during pregnancy, to describe the physical disability and sick leave in relation to the severity of low back pain and to identify predictors of moderate to severe low back pain in socio-demographic, health and obstetric characteristics among childbearing women.METHODS: A cohort study was undertaken (n=566) during August 2015 to March 2016. Questionnaires were used to obtain information about low back pain intensity, physical disability due to low back pain and sick leave at 20 and 32weeks of gestation. Of the 654 eligible women, 87% completed the first questionnaire.FINDINGS: Three out of four reported any low back pain at 20weeks of gestation, and nine out of ten women at 32weeks. Of these women, one in three reported moderate to severe pain at 20weeks, increasing to half of the women at 32weeks. Both sick leave and physical disability increased with increasing low back pain scores. Pre-pregnancy low back pain, multiparity and lower level of education were all identified as predictors of moderate to severe low back pain.CONCLUSIONS: Women with pre-pregnancy low back pain, multiparity and lower level of education, represent the group of women with the highest risk of moderate to severe low back pain during pregnancy and should be payed special attention.

Background Evidence has shown that there are several physical and mental advantages of exercise during pregnancy. Despite this, the recommendations for exercise during pregnancy are poorly fulfilled. The aim of this study was to illuminate non-exercising pregnant women’s views and experiences concerning exercise before and during pregnancy. Method The study had a qualitative design with an inductive approach and was analysed by content analysis. A total of 16 individual and face-to-face interviews were conducted with healthy pregnant women, mainly in the third trimester and living in Sweden. The participating women had not been exercising 3 months before pregnancy or during pregnancy. Results The main category “Insurmountable now, but possible in the future” was based on the four categories: “Lost and lack of routines”, “Feelings of inadequacy”, “Having a different focus” and “Need for support”. The women experienced that their lack of routines was a major barrier that prevented them from exercising. Other factors that contributed were, for example, pregnancy-related problems, long working days and prioritizing family life. The women described it as difficult to combine exercise with their focus on the pregnancy and they missed continuous support from the antenatal care provider. The women expressed a need for suggestions concerning exercise during pregnancy and follow-up on previous counselling, especially when pregnancy-related issues arose. Information about easily accessible alternatives or simple home exercises was requested. They felt immobile and were not satisfied with their inactivity and tried to partly compensate with everyday activities. The women identified the postpartum period as an important possibility for becoming more active, for their own sake, but also because they wanted to become role models for their children. Conclusion Continuous support during pregnancy is needed concerning exercise. Pregnancy is mostly a barrier that prevents exercise for this group of women but, at the same time, may be a motivator and a possibility for better health. As the result showed that these women were highly motivated to a life-style change post-pregnancy, it may be crucial to support previously non-exercising women postpartum. Electronic supplementary material The online version of this article (10.1186/s12884-018-2035-3) contains supplementary material, which is available to authorized users.

Objective We aimed to investigate the prevalence of sick leave and self-reported reasons given for sick leave during pregnancy. Furthermore, we aimed to estimate the frequency of long-term sick leave during pregnancy in relation to pre-baseline maternal characteristics and to identify predictors of long-term sick leave. Method Data from 508 employed pregnant women seeking antenatal care was collected by questionnaires from August 2015 to March 2016. The questionnaires, which were filled in at 20 and 32 weeks of gestation, provided information on maternal characteristics, the number of days spent on sick leave and the associated reasons. Descriptive statistics and logistic regression analysis were applied. Results The prevalence of sick leave was 56% of employed pregnant women in the first 32 weeks of gestation and more than one in four reported long-term sick leave (>20 days, continuous or intermittent). Low back pain was the reason most frequently stated. Fewer than one in ten stated that their sick leave was due to work-related conditions. Positive predictors of long-term sick leave were multiparity, pre-pregnancy low back pain and mental disease, while an advanced degree education was a negative predictor. Conclusions The prevalence of sick leave was 56% in the first 32 weeks of gestation and more than one in four women reported long-term sick leave. The majority of reasons for sick leave were pregnancy-related and low back pain was the most frequently given reason.

BACKGROUND: Low back pain is highly prevalent among pregnant women, but evidence of an effective treatment are still lacking. Supervised exercise-either land or water based-has shown benefits for low back pain, but no trial has investigated the evidence of an unsupervised water exercise program on low back pain. We aimed to assess the effect of an unsupervised water exercise program on low back pain intensity and days spent on sick leave among healthy pregnant women.METHODS: In this randomised, controlled, parallel-group trial, 516 healthy pregnant women were randomly assigned to either unsupervised water exercise twice a week for a period of 12 weeks or standard prenatal care. Healthy pregnant women aged 18 years or older, with a single fetus and between 16-17 gestational weeks were eligible. The primary outcome was low back pain intensity measured by the Low Back Pain Rating scale at 32 weeks. The secondary outcomes were self-reported days spent on sick leave, disability due to low back pain (Roland Morris Disability Questionnaire) and self-rated general health (EQ-5D and EQ-VAS).RESULTS: Low back pain intensity was significantly lower in the water exercise group, with a score of 2.01 (95% CI 1.75-2.26) vs. 2.38 in the control group (95% CI 2.12-2.64) (mean difference = 0.38, 95% CI 0.02-0.74 p = 0.04). No difference was found in the number of days spent on sick leave (median 4 vs. 4, p = 0.83), disability due to low back pain nor self-rated general health. There was a trend towards more women in the water exercise group reporting no low back pain at 32 weeks (21% vs. 14%, p = 0.07).CONCLUSIONS: Unsupervised water exercise results in a statistically significant lower intensity of low back pain in healthy pregnant women, but the result was most likely not clinically significant. It did not affect the number of days on sick leave, disability due to low back pain nor self-rated health.TRIAL REGISTRATION: ClinicalTrials.gov NCT02354430.

Objective To investigate the extent to which Danish women attending antenatal care plan their pregnancies and to determine the association between pregnancy planning and the intake of folic acid, alcohol consumption and smoking habits prior to conception and before the 16th week of gestation. Methods A cross-sectional survey of 258 women. Main outcome measures: intake of folic acid, alcohol consumption and smoking. Pregnancy planning was assessed by the London Measure of Unplanned Pregnancy (LMUP) and the five graded Swedish Pregnancy Planning Scale. Results Most (77%) of the participants reported that their pregnancies were very or fairly well planned. Higher median LMUP scores were observed in women taking folic acid (p < 0.001), in those consuming less alcohol, and in women who stopped smoking prior to pregnancy (p = 0.043). However, 43% of the respondents with a high degree of pregnancy planning and 98% of those with a low degree of planning had not taken folic acid prior to pregnancy. Binge drinking during early pregnancy was reported by 20% of women with a high degree of planned pregnancy and 31% of those with a low degree (p = 0.1). Conclusion Pregnancy planning was associated with a healthier lifestyle but still many women could improve their lifestyle in connection to pregnancy. Their level of alcohol consumption is higher than that recommended for best pregnancy outcome. © 2014 The European Society of Contraception and Reproductive Health.

Non-influenzae commensal Haemophilus species of low pathogenicity may be difficult to discriminate from Haemophilus influenzae. We investigated the level of misidentifications in respiratory specimens from cystic fibrosis patients and evaluated the colonisation dynamics of genuine H. influenzae isolates. One hundred and ninety-two presumptive H. influenzae isolates were re-examined by assessment of marker genes sodC and fucK, and isolates with aberrant genotypes were subjected to multilocus sequence typing. Misidentifications (3%) were mainly caused by failure to identify porphyrin-synthesising strains, and only a single strain (0.5%) could be classified as ‘non-haemolytic Haemophilus haemolyticus’. Sequential isolates of confirmed H. influenzae isolates from individual patients were typed by pulsed-field gel electrophoresis. Despite the routine prescription of antimicrobial therapy, the majority of H. influenzae isolates were identical with at least one of the strains cultured from the two preceding positive samples from the same patient.

Background Pregnant women with depression and/or anxiety prior to pregnancy are at higher risk of preterm birth, breastfeeding problems, postpartum depression, and disruption of the mother-infant attachment. It is well documented that exercise improves psychological well-being in nonpregnant subjects with symptoms of depression. However, in only a few small studies have researchers examined the effect of exercise on symptoms of depression among pregnant women. We hypothesize that physiotherapist-supervised group exercise for pregnant women at risk of antenatal depression increases their psychological well-being. This paper describes the study protocol of a randomized controlled trial (RCT) on a supervised group exercise intervention for pregnant women with a current or previous history of depression and/or anxiety. Methods/design The RCT is being carried out at the Department of Obstetrics, Rigshospitalet, Copenhagen University Hospital, in the period 2016–2019. The inclusion criteria are pregnant women ≥18 years of age with depression and/or anxiety requiring treatment by a psychiatrist or a psychologist within the last 10 years and/or intake of antidepressants in the 3 months prior to conception and/or during pregnancy. The women must have appropriate Danish language skills, be pregnant with a single fetus, give written informed consent, and be at 17–22 gestational weeks when the intervention begins. The primary outcome is psychological well-being (the five-item World Health Organization Well-being Index). Secondary outcomes are symptoms of depression (Edinburgh Postnatal Depression Scale), functional ability (General Health Questionnaire), clinical symptoms of anxiety (State-Trait Anxiety Inventory), sleep quality and sleep disturbances (Pittsburgh Sleep Quality Index), and pregnancy and delivery outcomes. The intervention is supervised group exercise twice weekly for 12 weeks. The control group will receive standard antenatal care. On the basis of sample size calculation, a total of 300 women will be randomly assigned to either the intervention or the control group in a ratio of 1:1. Discussion The trial is expected to contribute to the body of knowledge used in planning antenatal care for pregnant women at risk of depression. Trial registration ClinicalTrials.gov, NCT02833519. Registered on 19 May 2016. Electronic supplementary material The online version of this article (doi:10.1186/s13063-017-1938-z) contains supplementary material, which is available to authorized users.

Background: Postponing parenthood has steadily increased during the past decades in Western countries. This trend has affected the size of families in the direction of fewer children born per couple. In addition, higher maternal age is associated with an increased risk of pregnancy-related complications such as prematurity and foetal death, while higher paternal age increases the risk of miscarriage and affects time-to-pregnancy. Hence, understanding the circumstances and reflections that influence the decision is greatly needed and little is known about potential gender difference influencing the choice. The aim was to investigate attitudes towards parenthood, intentions for childbirth and knowledge about fertility issues among men and women. Methods: We conducted a cross-sectional study based on a validated 49-item questionnaire among students, who attended selected mandatory lectures at a Danish university college in February to April 2016. The participation rate was 99%, and 517 completed the questionnaire. Results: Though the majority of all participants wished to have children in the future (> 86%), there was significant difference between the genders (p = 0.002). Women rated having children to be more important than men did (p < 0.001), while men rated higher the likelihood of abstaining from having children if faced with infertility (p = 0.003). Knowledge about fertility issues was similar between genders including poor knowledge about the age-related decline in female fertility. While women found it more important to have children before being 'too old' (p = 0.04), still more than 40% of all respondents intended to have their last child after the age of 35 years. For both genders the most important prerequisite for parenthood was having a partner to share responsibility with. Perceived or experienced life changes related to parenthood were generally positive such as personal development. Conclusion: The majority of respondents wished to have children, but many desired to have these after the biological decline in female fertility. The moderate knowledge level among both genders uncovered in this study is of concern. Future research should address the potential link between fertility knowledge and planning of parenthood. We may benefit from intervention studies examining the effect of routine preconception care.

The osteoclast initiates resorption by creating a resorption lacuna. The ruffled border surrounding the lacunae arises from exocytosis of lysosomes. To dissolve the inorganic phase of the bone, the vacuolar adenosine triphosphatase, located in the ruffled border, pumps protons into the resorption lacunae. The electroneutrality of the lacunae is maintained by chloride transport through the chloride-proton antiporter chloride channel 7. Inhibition of either proton or chloride transport prevents bone resorption. The aims of this study were to validate the human osteoclastic microsome- based influx assay with respect to lysosomal acidification and assess whether it is a reliable test of a compound's ability to inhibit acidification. Investigated were the expression levels of the lysosomal acidification machinery, the activation of the assay by adenosine triphosphate, H(+) and Cl(-) dependency, the effect of valinomycin, inhibitor sensitivity, and the ion profile of the human osteoclast microsomes. The expression level of chloride channel 7 was increased in the human osteoclastic microsomes compared with whole osteoclasts. Acid influx was induced by 1.25 mM adenosine triphosphate. Further 1.1 μM valinomycin increased the acid influx by 129%. Total abrogation of acid influx was observed using both H(+) and Cl(-) ionophores. Finally, investigation of the anion profile demonstrated that Cl(-) and Br(-) are the preferred anions for the transporter. In conclusion, the acid influx assay based on microsomes from human osteoclasts is a useful tool for detection of inhibitors of the osteoclastic acidification machinery, and thus may aid the identification of effective drugs for osteoporosis that target the acid secretion by osteoclasts.

Osteopetrosis is the result of mutations affecting osteoclast function. Careful analyses of osteopetrosis have provided instrumental information on bone remodeling, including the coupling of bone formation to bone resorption. Based on a range of novel genetic mutations and the resulting osteoclast phenotypes, we discuss how osteopetrosis models have clarified the function of the coupling of bone formation to bone resorption, and the pivotal role of the osteoclast and their function in this phenomenon. We highlight the distinct possibility that osteoclast activities can be divided into two separate avenues: bone resorption and control of bone formation.

Osteoclasts possess a large amount of ion transporters, which participate in bone resorption; of these, the vacuolar-adenosine trisphosphatase (V-ATPase) and the chloride-proton antiporter ClC-7 acidify the resorption lacuna. However, whether other ion transporters participate in this process is currently not well understood. We used a battery of ion channel inhibitors, human osteoclasts, and their subcellular compartments to perform an unbiased analysis of the importance of the different ion transporters for acidification of the resorption lacuna in osteoclasts. CD14(+) monocytes from human peripheral blood were isolated, and mature osteoclasts were generated using RANKL and M-CSF. The human osteoclasts were (1) used for acridine orange assays for evaluation of lysosomal acidification, (2) used for bone resorption assays, (3) used for generation of osteoclasts membranes for acid influx experiments, or (4) lysed in trizol for mRNA isolation for Affymetrix array analysis. Inhibitors targeted toward most of the ion transporters showed low potency in the acidification-based assays, although some inhibitors, such as carbonic anhydrase II and the sodium-hydrogen exchanger (NHE) inhibitors, reduced resorption potently. In contrast, inhibitors targeted at V-ATPase and ClC-7 potently inhibited both acidification and resorption, as expected. We here show evidence that acidification of the resorption lacuna is mainly mediated by V-ATPase and ClC-7. Furthermore, a group of other ion transporters, including carbonic anhydrase II, the NHEs, and potassium-chloride cotransporters, are all involved in resorption but do not seem to directly be involved in acidification of the lysosomes.

Collecting duct-derived endothelin (ET)-1 is an autocrine inhibitor of Na+ and water reabsorption; its deficiency causes hypertension and water retention. Extracellular fluid volume expansion increases collecting duct ET-1, thereby promoting natriuresis and diuresis; however, how this coupling between volume expansion and collecting duct ET-1 occurs is incompletely understood. One possibility is that volume expansion increases tubular fluid flow. To investigate this, cultured IMCD3 cells were subjected to static or flow conditions. Exposure to a shear stress of 2 dyn/cm2 for 2 h increased ET-1 mRNA content by ∼2.3-fold. Absence of perfusate Ca2+, chelation of intracellular Ca2+, or inhibition of Ca2+ signaling (calmodulin, Ca2+/calmodulin-dependent kinase, calcineurin, PKC, or phospholipase C) prevented the flow response. Evaluation of possible flow-activated Ca2+ entry pathways revealed no role for transient receptor potential (TRP)C3, TRPC6, and TRPV4; however, cells with TRPP2 (polycystin-2) knockdown had no ET-1 flow response. Flow increased intracellular Ca2+ was blunted in TRPP2 knockdown cells. Nonspecific blockade of P2 receptors, as well as specific inhibition of P2X7 and P2Y2 receptors, prevented the ET-1 flow response. The ET-1 flow response was not affected by inhibition of either epithelial Na+ channels or the mitochondrial Na+/Ca2+ exchanger. Taken together, these findings provide evidence that in IMCD3 cells, flow, via polycystin-2 and P2 receptors, engages Ca2+-dependent signaling pathways that stimulate ET-1 synthesis.

Background Exercise during pregnancy is associated with health benefits for both the mother and the fetus, and is therefore recommended in several national guidelines. Only few studies investigate whether these guideline recommendations are met. The aims of this study were 1. To assess the prevalence of pregnant women meeting the Danish recommendations for exercise during early pregnancy, 2. To identify pre-pregnancy factors associated with a lower probability for meeting the recommendations, and 3. To describe which types of exercise pregnant women prefer before and during pregnancy. Methods We conducted a cross-sectional study based on a questionnaire during the first trimester among 7,915 women participating in the prospective Copenhagen Pregnancy Cohort. Associations were estimated by multivariate regression analyses. Results In early pregnancy, 38 % of the study population met the recommendation for exercise from the Danish Health and Medicines Authority (≥3.5 hours a week). Multiparity, previous miscarriage use of assisted reproductive technology, no engagement in exercise before pregnancy, smoking, pregnancy following assisted reproductive technology, overweight, not understanding Danish language and a low educational level were all factors associated a lower probability for meeting the recommendations. The preferred types of exercise before and during pregnancy were bicycling, brisk walking, running and strength training. The proportion of women engaged in any type of exercise decreased in early pregnancy with the exception of swimming and aquatic exercise. Conclusions In this cohort, more than one-third met the Danish recommendation for exercise during early pregnancy. Exercise in pregnancy is still an issue to address because the most vulnerable groups of pregnant women do not exercise. This is a cause of concern because it may reflect social inequalities in health and highlights the need for a structural and systematic approach to preconception care and early antenatal counselling.

Urinary tract infections are commonly caused by α-hemolysin (HlyA)-producing Escherichia coli. In erythrocytes, the cytotoxic effect of HlyA is strongly amplified by P2X receptors, which are activated by extracellular ATP released from the cytosol of the erythrocytes. In renal epithelia, HlyA causes reversible [Ca2+]i oscillations, which trigger interleukin-6 (IL-6) and IL-8 release. We speculate that this effect is caused by HlyA-induced ATP release from the epithelial cells and successive P2 receptor activation. Here, we demonstrate that HlyA-induced [Ca2+]i oscillations in renal epithelia were completely prevented by scavenging extracellular ATP. In accordance, HlyA was unable to inflict any [Ca2+]i oscillations in 132-1N1 cells, which lack P2R completely. After transfecting these cells with the hP2Y2 receptor, HlyA readily triggered [Ca2+]i oscillations, which were abolished by P2 receptor antagonists. Moreover, HlyA-induced [Ca2+]i oscillations were markedly reduced in medullary thick ascending limbs isolated from P2Y2 receptor-deficient mice compared with wild type. Interestingly, the following HlyA-induced IL-6 release was absent in P2Y2 receptor-deficient mice. This suggests that HlyA induces ATP release from renal epithelia, which via P2Y2 receptors is the main mediator of HlyA-induced [Ca2+]i oscillations and IL-6 release. This supports the notion that ATP signaling occurs early during bacterial infection and is a key player in the further inflammatory response. Background: The virulence factor HlyA elicits [Ca2+]i oscillations in renal epithelial cells. Results: These oscillations and following IL-6 release are reduced by inhibition or lack of P2Y2 receptors. Conclusion: The effects of HlyA in renal epithelial cells are mediated by P2Y2 receptors. Significance: ATP release and P2Y2 receptor activation are essential parts of the early interaction between Escherichia coli and the renal epithelium.

Background. In early postpartum haemorrhage (PPH), a low concentration of fibrinogen is associated with excessive subsequent bleeding and blood transfusion. We hypothesized that pre-emptive treatment with fibrinogen concentrate reduces the need for red blood cell (RBC) transfusion in patients with PPH. Methods. In this investigator-initiated, multicentre, double-blinded, parallel randomized controlled trial, we assigned subjects with severe PPH to a single dose of fibrinogen concentrate or placebo (saline). A dose of 2 g or equivalent was given to all subjects independent of body weight and the fibrinogen concentration at inclusion. The primary outcome was RBC transfusion up to 6 weeks postpartum. Secondary outcomes were total blood loss, total amount of blood transfused, occurrence of rebleeding, haemoglobin ,58 g litre 21 , RBC transfusion within 4 h, 24 h, and 7 days, and as a composite outcome of ‘severe PPH’, defined as a decrease in haemoglobin of .40 g litre 21 , transfusion of at least 4 units of RBCs, haemostatic intervention (angiographic embolization, surgical arterial ligation, or hysterectomy), or maternal death. Results. Of the 249 randomized subjects, 123 of 124 in the fibrinogen group and 121 of 125 in the placebo group were included in the intention-to-treat analysis. At inclusion the subjects had severe PPH, with a mean blood loss of 1459 (SD 476) ml and a mean fibrinogen concentration of 4.5 (SD 1.2) g litre 21 . The intervention group received a mean dose of 26 mg kg 21 fibrinogen concentrate, thereby significantly increasing fibrinogen concentration compared with placebo by 0.40 g litre 21 (95% confidence interval, 0.15 –0.65; P¼0.002). Postpartum blood transfusion occurred in 25 (20%) of the fibrinogen group and 26 (22%) of the placebo group (relative risk, 0.95; 95% confidence interval, 0.58 –1.54; P¼0.88). We found no difference in any predefined secondary outcomes, per-protocol analyses, or adjusted analyses. No thromboembolic events were detected. Conclusions. We found no evidence for the use of 2 g fibrinogen concentrate as pre-emptive treatment for severe PPH in patients with normofibrinogenaemia. Clinical trial registration. ClinicalTrials.gov: http://clinicaltrials.gov/show/NCT01359878. Published protocol: http://www.trialsjournal.com/content/pdf/1745-6215-13-110.pdf.

Bone resorption is solely mediated by osteoclasts. Therefore, a pure osteoclast population is of high interest for the investigation of biological aspects of the osteoclasts, such as the direct effect of growth factors and hormones, as well as for testing and characterizing inhibitors of bone resorption. We have established a pure, stable, and reproducible system for purification of human osteoclasts from peripheral blood. We isolated CD14-positive (CD14+) monocytes using anti-CD14-coated beads. After isolation, the monocytes are differentiated into mature osteoclasts by stimulation with macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor kappaB ligand (RANKL). Osteoclast formation was only observed in the CD14+ population, not in the CD14- population, and only in the presence of both M-CSF and RANKL, confirming that the CD14+ system is a pure population of osteoclast precursors. No expression of osteoclast markers was observed in the absence of RANKL, whereas RANKL dose-dependently induced the expression of cathepsin K, tartrate-resistant acid phosphatase (TRACP), and matrix metallo proteinase (MMP)-9. Furthermore, morphological characterization of the cells demonstrated that actin rings were only formed in the presence of RANKL. Moreover, the osteoclasts were capable of forming acidic resorption lacunae, and inhibitors of lysosomal acidification attenuated this process. Finally, we measured the response to known bone resorption inhibitors, and found that the osteoclasts were sensitive to these and thereby provided a robust and valid method for interpretation of the effect of antiresorptive compounds. In conclusion, we have established a robust assay for developing osteoclasts that can be used to study several biological aspects of the osteoclasts and which in combination with the resorption marker CTX-I provides a useful tool for evaluating osteoclast function in vitro.

Osteoclasts degrade bone matrix by secretion of hydrochloric acid and proteases. We studied the processes involved in the degradation of the organic matrix of bone in detail and found that lysosomal acidification is involved in this process and that MMPs are capable of degrading the organic matrix in the absence of cathepsin K. Introduction: Osteoclasts resorb bone by secretion of acid by the vacuolar H+-adenosine triphosphatase (V-ATPase) and the chloride channel ClC-7, followed by degradation of the matrix, mainly collagen type I, by cathepsin K and possibly by matrix metalloproteinases (MMPs). However, the switch from acidification to proteolysis and the exact roles of both the ion transporters and the proteinases still remain to be studied. Materials and Methods: We isolated CD14+ monocytes from human peripheral blood from either controls or patients with autosomal dominant osteopetrosis type II (ADOII) caused by defective ClC-7 function and cultured them in the presence of RANKL and macrophage-colony stimulating factor (M-CSF) to generate osteoclasts. We decalcified cortical bovine bone slices and studied the osteoclasts with respect to morphology, markers, and degradation of the decalcified matrix in the presence of various inhibitors of osteoclast acidification and proteolysis, using normal calcified bone as a reference. Results: We found that ADOII osteoclasts not only have reduced resorption of the calcified matrix, but also 40% reduced degradation of the organic phase of bone. We found that both acidification inhibitors and cathepsin K inhibitors reduced degradation of the organic matrix by 40% in normal osteoclasts, but had no effect in the ADOII osteoclasts. Furthermore, we showed that inhibition of MMPs leads to a 70% reduction in the degradation of the organic bone matrix and that MMPs and cathepsin K have additive effects. Finally, we show that osteoclastic MMPs mediate release of the carboxyterminal telopeptide of type I collagen (ICTP) fragment in the absence of cathepsin K activity, and therefore, to some extent, are able to compensate for the loss of cathepsin K activity. Conclusions: These data clearly show that osteoclastic acidification of the lysosomes plays a hitherto nonrecognized role in degradation of the organic matrix. Furthermore, these data shed light on the complicated interplay between acidification dependent and independent proteolytic processes, mediated by cathepsin K and the MMPs, respectively.

Patients with defective osteoclastic acidification have increased numbers of osteoclasts, with decreased resorption, but bone formation that remains unchanged. We demonstrate that osteoclast survival is increased when acidification is impaired, and that impairment of acidification results in inhibition of bone resorption without inhibition of bone formation. We investigated the role of acidification in human osteoclastic resorption and life span in vitro using inhibitors of chloride channels (NS5818/NS3696), the proton pump (bafilomycin) and cathepsin K. We found that bafilomycin and NS5818 dose dependently inhibited acidification of the osteoclastic resorption compartment and bone resorption. Inhibition of bone resorption by inhibition of acidification, but not cathepsin K inhibition, augmented osteoclast survival, which resulted in a 150 to 300% increase in osteoclasts compared to controls. We investigated the effect of inhibition of osteoclastic acidification in vivo by using the rat ovariectomy model with twice daily oral dosing of NS3696 at 50 mg/kg for 6 weeks. We observed a 60% decrease in resorption (DPYR), increased tartrate-resistant acid phosphatase levels, and no effect on bone formation evaluated by osteocalcin. We speculate that attenuated acidification inhibits dissolution of the inorganic phase of bone and results in an increased number of nonresorbing osteoclasts that are responsible for the coupling to normal bone formation. Thus, we suggest that acidification is essential for normal bone remodeling and that attenuated acidification leads to uncoupling with decreased bone resorption and unaffected bone formation.

Several studies have documented that length gain often lags behind weight gain during infancy and early childhood, suggesting that linear growth is partly regulated by initial body mass or fatness. To investigate this hypothesis, we analysed data from four longitudinal studies on growth of infants in the first 12 months: (1) U.S. breast‐fed and formula‐fed infants (n = 89); (2) breast‐fed infants in Ghana (n = 190); (3) normal birthweight, breast‐fed infants in Honduras (n = 108); and (4) term, low‐birthweight breast‐fed infants in Honduras (n = 119). The dependent variable was length gain during each 3‐month interval (1– 4, 2–5, 3–6, 4–7, 5–8, 6–9, 7–10, 8–11 and 9–12 months). Three main independent variables were examined: initial weight‐for‐length z‐score (W/L), weight change during the prior 3 months, and initial skinfold thickness. Controlling for maternal height, infant sex, and initial length‐for‐age z‐score, length gain was positively correlated with initial W/L and prior weight change during all age intervals and with initial skinfold thickness at 3 and 4 months (r = 0.15–0.36; P

Fuel utilization in two adult patients with the myopathic form of very long-chain acyl-CoA dehydrogenase (VLCAD) deficiency and five healthy subjects was investigated with stable isotopes during exercise at 50% of VO2max. The findings indicate that residual VLCAD activity in the patients is sufficient to maintain normal oxidation of fat at rest, but that fat oxidation rate cannot increase above basal levels during exercise. This can cause an energy deficit and intramuscular accumulation of fat intermediates that may induce the exercise-induced symptoms.

This study was designed to determine the effect of the in vivo administration of epigallocatechin gallate (EGCG) and epicatechin gallate (ECG) on enzymes involved in the synthesis and metabolism of estradiol. EGCG (12.5, 25, or 50 mg/kg/day, i.p.) or ECG (12.5 or 25 mg/kg/day, i.p.) was administered to female Swiss-Webster mice for 7 days. The chemicals were well tolerated by the mice with the exception of EGCG given at 50 mg/kg, which resulted in severe hepatic necrosis and a 67% mortality rate. Following the administration of nontoxic doses of EGCG and ECG, aromatase (CYP19), CYP3A, CYP1A, and catechol O-methyltransferase (COMT) were measured. Additionally, the activity of CYP2E1 was determined, since this CYP450 isoform is important in the bioactivation of numerous carcinogens. The results demonstrated that ovarian aromatase activity was inhibited 56% by EGCG (25 and 12.5 mg/kg), but not ECG, while hepatic CYP3A catalytic activity and polypeptide levels were increased 31 +/- 4 and 47 +/- 2%, respectively, by 25 mg/kg of EGCG. However, ECG (but not EGCG) inhibited CYP1A catalytic activity and polypeptide levels (31 +/- 5 and 47 +/- 5%, respectively). Hepatic and renal COMT, as well as renal CYP3A remained unchanged following catechin dosing. Hepatic CYP2E1 catalytic activity and polypeptide levels were significantly increased (37 +/- 3 and 22 +/- 3%) following administration of EGCG (25 mg/kg). These results indicate that EGCG modulates enzymes responsible for both the synthesis and metabolism of estradiol, which may provide a potential mechanism for the reported action of EGCG, reported action as an inhibitor of breast tumor growth.

Pelvic girdle pain is a frequent cause of sick leave among pregnant women in Denmark. Studies regarding prevention of pelvic girdle pain are sparse. The aim of this study was to examine the association between physical exercise and pelvic girdle pain in pregnancy.A nested case-control study within the Danish National Birth Cohort (n = 5304).This study used self-reported data on pelvic girdle pain obtained from an interview six months after childbirth. Information on physical exercise was obtained from the pregnancy interview around gestational week 16. The association was estimated using logistic regression analysis.Physical exercise in pregnancy was associated with decreased risk of overall pelvic girdle pain (OR = 0.87; 95% CI: 0.77-0.99, p = 0.028). Tests for trend indicated decreasing odds for pelvic girdle pain with increasing number of hours per week spent on exercise (p 0.001). Compared to no exercise, swimming was associated with a decreased risk of pelvic girdle pain (OR = 0.73; 95% CI: 0.58-0.91, p = 0.005).The findings suggest a possible protective effect of physical exercise on pelvic girdle pain during pregnancy.

Introduction Low back pain during pregnancy is common and associated with sick leave. Studies suggest that exercise may reduce low back pain during pregnancy. Before carrying out a randomised controlled trail with individual water exercise as intervention a qualitative feasibility study was done. Objective To explore women's views and experiences of the acceptability and benefits of and possible barriers to the standardised individual unsupervised water exercise intervention. Materials and Methods Eleven women were interviewed after participating in a water exercise intervention. Content analysis was used. Results Four main categories emerged: motivation to participate, attitudes towards the exercise programme, perception of benefits, and acceptability of supportive components. The women had a desire to stay physically active during pregnancy and found water exercise a suitable, type of exercise to perform during pregnancy. The intervention was experienced to have benefits on both their physical health and their mental well-being. Crowded swimming pools were perceived as the greatest barrier. Conclusion It is feasible to perform a RCT using the described intervention. The intervention was accepted by the participants because it supported their desire to be physically active during pregnancy. The main barrier was crowded swimming pools and this issue must be addressed in a future RCT.

Forty-four of 48 Burkholderia cepacia complex strains cultured from Danish cystic fibrosis patients were Burkholderia multivorans , a distribution of species that has not been reported before. Although cases of cross infections were demonstrated, no major epidemic clone was found. The species distribution may represent the sporadic acquisition of bacteria from the environment.

Background Normal osteoclasts resorb bone by secretion of acid and proteases. Recent studies of patients with loss of function mutations affecting either of these processes have indicated a divergence in osteoclastic phenotypes. These difference in osteoclast phenotypes may directly or indirectly have secondary effects on bone remodeling, a process which is of importance for the pathogenesis of both osteoporosis and osteoarthritis. We treated human osteoclasts with different inhibitors and characterized their resulting function. Methods Human CD14 + monocytes were differentiated into mature osteoclasts using RANKL and M-CSF. The osteoclasts were cultured on bone in the presence or absence of various inhibitors: Inhibitors of acidification (bafilomycin A1, diphyllin, ethoxyzolamide), inhibitors of proteolysis (E64, GM6001), or a bisphosphonate (ibandronate). Osteoclast numbers and bone resorption were monitored by measurements of TRACP activity, the release of calcium, CTX-I and ICTP, as well as by counting resorption pits. Results All inhibitors of acidification were equally potent with respect to inhibition of both organic and inorganic resorption. In contrast, inhibition of proteolysis by E64 potently reduced organic resorption, but only modestly suppressed inorganic resorption. GM6001 alone did not greatly affect bone resorption. However, when GM6001 and E64 were combined, a complete abrogation of organic bone resorption was observed, without a great effect on inorganic resorption. Ibandronate abrogated both organic and inorganic resorption at all concentrations tested [0.3-100 μM], however, this treatment dramatically reduced TRACP activity. Conclusions We present evidence highlighting important differences with respect to osteoclast function, when comparing the different types of osteoclast inhibitors. Each class of osteoclast inhibitors will lead to different alterations in osteoclast quality, which secondarily may lead to different bone qualities.

Background Breast and prostate cancer patients often develop lesions of locally high bone turnover, when the primary tumor metastasizes to the bone causing an abnormal high bone resorption at this site. The objective of the present study was to determine whether local increased bone turnover in breast and prostate cancer patients is associated with an increase in cartilage degradation and to test in vitro whether osteoclasts or cathepsin K alone generate CTXII from human bone. Methods The study included 132 breast and prostate cancer patient, where presence of bone metastases was graded according to the Soloway score. Total bone resorption (CTXItotal) and cartilage degradation (CTXII) were determined. Results Breast and prostate cancer patients with bone metastases revealed significant increased levels of CTXItotal at Soloway scores 1 and higher compared to patients without bone metastases (p < 0.001). CTXII was statistically elevated at score 3 and 4 (p < 0.01). CTXII/CTXItotal significantly decreased at score 3 and 4 (p < 0.001). Levels of CTXItotal, CTXII and CTXII/CTXItotal changed +900%, +130%, and -90%, respectively at Soloway score 4 compared to score 0. The in vitro experiments revealed that osteoclasts released CTXI fragments but not CTXII from bone specimens. The same was observed for cathepsin K. Conclusion Data suggest that an uncoupling between bone resorption and cartilage degradation occurs in breast and lung cancer patient.

Objective—To characterize physiologic responses of ball pythons (Python regius) following a minor surgical procedure and investigate the effects of 2 commonly used analgesics on this response. Animals—15 healthy ball pythons. Procedures—Snakes were randomly assigned to receive 1 of 3 treatments: meloxicam (0.3 mg/kg [0.14 mg/lb]; n = 5), butorphanol (5 mg/kg [2.3 mg/lb]; 5), or saline (0.9% NaCl) solution (5) before catheterization of the vertebral artery. Plasma concentrations of catecholamines and cortisol, blood pressure, heart rate, and blood gas values were measured at various times for 72.5 hours after catheterization. The 72.5-hour point was defined as baseline. Results—Heart rate of ball pythons increased significantly during the first hour following surgery. Mean plasma epinephrine concentration increased slightly at 2.5 hours after surgery, whereas mean plasma cortisol concentration increased beginning at 1.5 hours, reaching a maximum at 6.5 hours. Mean blood pressure increased within the first hour but returned to the baseline value at 2.5 hours after surgery. After 24.5 hours, blood pressure, heart rate, and plasma hormone concentrations remained stable at baseline values. There were no significant differences in values for physiologic variables between snakes that received saline solution and those that received meloxicam or butorphanol. Conclusions and Clinical Relevance—Measurement of physiologic variables provides a means of assessing postoperative pain in snakes. Meloxicam and butorphanol at the dosages used did not decrease the physiologic stress response and did not appear to provide analgesic effects in ball pythons.

Osteoclasts are the sole cells possessing the ability to resorb calcified bone matrix. This occurs via secretion of hydrochloric acid mediated by the V-ATPase and the chloride channel ClC-7. Loss of acidification leads to osteopetrosis characterized by ablation of bone resorption and increased osteoclast numbers, indicating increased life span of the osteoclasts. To investigate the role of the inorganic phase of bone with respect to osteoclast life span, we used the V-ATPase inhibitor bafilomycin and the calcium uptake antagonist ryanodine on human osteoclasts cultured on calcified and decalcified bone slices. Bafilomycin inhibited bone resorption and increased osteoclast survival on calcified but not decalcified bones. Ryanodine attenuated calcium uptake and thereby augmented osteoclast survival on calcified bones. In summary, we found that acidification leading to calcium release from bone during resorption controls osteoclast survival, potentially explaining the increased numbers of osteoclasts in patients with osteopetrosis.

Dissolution of the inorganic phase of bone by the osteoclasts mediated by V-ATPase and ClC-7 is a prerequisite for bone resorption. Inhibitors of osteoclastic V-ATPase or ClC-7 are novel approaches for inhibition of osteoclastic bone resorption. By testing natural compounds in acidification assays, diphyllin was identified. We characterized diphyllin with respect to the pharmacological effects on osteoclasts. Introduction: Osteoclastic acidification of the resorption lacuna and bone resorption requires activity of both V-ATPase and the chloride channel ClC-7. Inhibition of these processes represents a novel approach for treatment of bone metabolic disorders. We identified diphyllin, a novel inhibitor of V-ATPase, and characterized this natural compound with respect to activity in human osteoclasts. Materials and Methods: Diphyllin was tested in the acid influx assay and V-ATPase assay using bovine chromaffin granules. Human osteoclasts were generated from CD14+ monocytes cultured with macrophage-colony stimulating factor (M-CSF ) and RANKL. The effect of diphyllin on lysosomal acidification in human osteoclasts was studied using acridine orange. The effect of diphyllin on bone resorption by osteoclasts was measured as release of C-terminal cross-linked telopeptide of type I collagen (CTX-I ) and calcium into the supernatants and by scoring pit area. Osteoclast number, TRACP activity, and cell viability were measured. Furthermore, the effect of diphyllin on bone nodule formation was tested using the mouse osteoblast cell line MC3T3-E 1. Results: In the acid influx assay, diphyllin potently inhibited the acid influx (IC50 = 0.6 nM). We found that diphyllin inhibited V-ATPase with an IC50 value of 17 nM, compared with 4 nM for bafilomycin A1. Moreover, diphyllin dose-dependently inhibited lysosomal acidification in human osteoclasts. Furthermore, we found that diphyllin inhibited human osteoclastic bone resorption measured by CTX-I (IC50 = 14 nM), calcium release, and pit area, despite increasing TRACP activity, numbers of osteoclasts, and cell viability. Finally, diphyllin showed no effect on bone formation in vitro, whereas bafilomycin A1 was toxic. Conclusions: We identified a natural compound that potently inhibits V-ATPase and thereby lysosomal acidification in osteoclasts, which leads to abrogation of bone resorption. Because recent studies indicate that inhibition of the osteoclastic acidification leads to inhibition of resorption without inhibiting formation, we speculate that diphyllin is a potential novel treatment for bone disorders involving excessive resorption.

Estrogen deficiency arising with the menopause promotes marked acceleration of bone resorption, which can be restored by hormone replacement therapy. The inhibitory effects of estrogen seem to involve indirect cytokine- mediated effects via supporting bone marrow cells, but direct estrogen-receptor mediated effects on the bone-resorbing osteoclasts have also been proposed. Little information is available on whether estrogens modulate human osteoclastogenesis or merely inhibit the functional activity of osteoclasts. To clarify whether estrogens directly modulate osteoclastic activities human CD14+ monocytes were cultured in the presence of M-CSF and RANKL to induce osteoclast differentiation. Addition of 0.1-10 nM 17beta-estradiol to differentiating osteoclasts resulted in a dose-dependent reduction in tartrate resistant acid phosphatase (TRACP) activity reaching 60% at 0.1 nM. In addition, 17beta-estradiol inhibited bone resorption, as measured by the release of the C-terminal crosslinked telopeptide (CTX), by 60% at 0.1 nM, but had no effect on the overall cell viability. In contrast to the results obtained with differentiating osteoclasts, addition of 17beta-estradiol (0.001-10 nM) to mature osteoclasts did not affect bone resorption or TRACP activity. We investigated expression of the estrogen receptors, using immunocytochemistry and Western blotting. We found that ER-alpha is expressed in osteoclast precursors, whereas ER- beta is expressed at all stages, indicating that the inhibitory effect of estrogen on osteoclastogenesis is mediated by ER-alpha for the major part. In conclusion, these results suggest that the in vivo effects of estrogen are mediated by reduction of osteoclastogenesis rather than direct inhibition of the resorptive activity of mature osteoclasts.

Background Bone resorption is initiated by osteoclastic acidification of the resorption lacunae. This process is mediated by secretion of protons through the V-ATPase and chloride through the chloride antiporter ClC-7. To shed light on the intracellular signalling controlling extracellular acidification, we screened a protein kinase inhibitor library in human osteoclasts. Methods Human osteoclasts were generated from CD14+ monocytes. The effect of different kinase inhibitors on lysosomal acidification in human osteoclasts was investigated using acridine orange for different incubation times (45 minutes, 4 and 24 hours). The inhibitors were tested in an acid influx assay using microsomes isolated from human osteoclasts. Bone resorption by human osteoclasts on bone slices was measured by calcium release. Cell viability was measured using AlamarBlue. Results Of the 51 compounds investigated only few inhibitors were positive in both acidification and resorption assays. Rottlerin, GF109203X, Hypericin and Ro31-8220 inhibited acid influx in microsomes and bone resorption, while Sphingosine and Palmitoyl-DL-carnitine-Cl showed low levels of inhibition. Rottlerin inhibited lysosomal acidification in human osteoclasts potently. Conclusions In conclusion, a group of inhibitors all indicated to inhibit PKC reduced acidification in human osteoclasts, and thereby bone resorption, indicating that acid secretion by osteoclasts may be specifically regulated by PKC in osteoclasts.