Osteoblast CFTR Inactivation Reduces Differentiation and Osteoprotegerin Expression in a Mouse Model of Cystic Fibrosis-Related Bone Disease

Low bone mass and increased fracture risk are recognized complications of cystic fibrosis (CF). CF-related bone disease (CFBD) is characterized by uncoupled bone turnover—impaired osteoblastic bone formation and enhanced osteoclastic bone resorption. Intestinal malabsorption, vitamin D deficiency and inflammatory cytokines contribute to CFBD. However, epidemiological investigations and animal models also support a direct causal link between inactivation of skeletal cystic fibrosis transmembrane regulator (CFTR), the gene that when mutated causes CF, and CFBD. The objective of this study was to examine the direct actions of CFTR on bone. Expression analyses revealed that CFTR mRNA and protein were expressed in murine osteoblasts, but not in osteoclasts. Functional studies were then performed to investigate the direct actions of CFTR on osteoblasts using a CFTR knockout (Cftr−/−) mouse model. In the murine calvarial organ culture assay, Cftr−/− calvariae displayed significantly less bone formation and osteoblast numbers than calvariae harvested from wildtype (Cftr+/+) littermates. CFTR inactivation also reduced alkaline phosphatase expression in cultured murine calvarial osteoblasts. Although CFTR was not expressed in murine osteoclasts, significantly more osteoclasts formed in Cftr−/− compared to Cftr+/+ bone marrow cultures. Indirect regulation of osteoclastogenesis by the osteoblast through RANK/RANKL/OPG signaling was next examined. Although no difference in receptor activator of NF-κB ligand (Rankl) mRNA was detected, significantly less osteoprotegerin (Opg) was expressed in Cftr−/− compared to Cftr+/+ osteoblasts. Together, the Rankl:Opg ratio was significantly higher in Cftr−/− murine calvarial osteoblasts contributing to a higher osteoclastogenesis potential. The combined findings of reduced osteoblast differentiation and lower Opg expression suggested a possible defect in canonical Wnt signaling. In fact, Wnt3a and PTH-stimulated canonical Wnt signaling was defective in Cftr−/− murine calvarial osteoblasts. These results support that genetic inactivation of CFTR in osteoblasts contributes to low bone mass and that targeting osteoblasts may represent an effective strategy to treat CFBD.     

Survey of Innate Immune Responses to Burkholderia pseudomallei in Human Blood Identifies a Central Role for Lipopolysaccharide

B. pseudomallei is a gram-negative bacterium that causes the tropical infection melioidosis. In northeast Thailand, mortality from melioidosis approaches 40%. As exemplified by the lipopolysaccharide-Toll-like receptor 4 interaction, innate immune responses to invading bacteria are precipitated by activation of host pathogen recognition receptors by pathogen associated molecular patterns. Human melioidosis is characterized by up-regulation of pathogen recognition receptors and pro-inflammatory cytokine release. In contrast to many gram-negative pathogens, however, the lipopolysaccharide of B. pseudomallei is considered only weakly inflammatory. We conducted a study in 300 healthy Thai subjects to investigate the ex vivo human blood response to various bacterial pathogen associated molecular patterns, including lipopolysaccharide from several bacteria, and to two heat-killed B. pseudomallei isolates. We measured cytokine levels after stimulation of fresh whole blood with a panel of stimuli. We found that age, sex, and white blood cell count modulate the innate immune response to B. pseudomallei. We further observed that, in comparison to other stimuli, the innate immune response to B. pseudomallei is most highly correlated with the response to lipopolysaccharide. The magnitude of cytokine responses induced by B. pseudomallei lipopolysaccharide was significantly greater than those induced by lipopolysaccharide from Escherichia coli and comparable to many responses induced by lipopolysaccharide from Salmonella minnesota despite lower amounts of lipid A in the B. pseudomallei lipopolysaccharide preparation. In human monocytes stimulated with B. pseudomallei, addition of polymyxin B or a TLR4/MD-2 neutralizing antibody inhibited the majority of TNF-α production. Challenging existing views, our data indicate that the innate immune response to B. pseudomallei in human blood is largely driven by lipopolysaccharide, and that the response to B. pseudomallei lipopolysaccharide in blood is greater than the response to other lipopolysaccharide expressing isolates. Our findings suggest that B. pseudomallei lipopolysaccharide may play a central role in stimulating the host response in melioidosis.     

Critical period for estrogen‐dependent motoneuron dendrite growth is coincident with ERα expression in target musculature

The spinal cord of rats contains the sexually dimorphic, steroid‐sensitive motoneurons of the spinal nucleus of the bulbocavernosus (SNB). In males, SNB dendrite growth is dependent on gonadal steroids: dendrite growth is inhibited after castration, but supported in androgen‐ or estrogen‐treated castrated males. Furthermore, estrogenic support of SNB dendrite growth is mediated by estrogen action at the target musculature, inhibited by estrogen receptor (ER) blockade at the muscle and supported by local estradiol treatment. However, this estrogenic support is restricted to the early postnatal period, after which the morphology of SNB dendrites is insensitive to estrogens. To test if the developmentally restricted effects of estrogens on SNB dendrite growth coincide with the transient expression of ER in the target musculature, ERα expression was assessed during development and in adulthood. ERα expression in extra‐Muscle fiber cells was greatest from postnatal day 7 (P7) to P14 and declined after P21. Because this pattern of ERα expression coincided with the period of estrogen‐dependent dendrite growth, we tested if limiting hormone exposure to the period of maximal ERα expression in extra‐muscle fiber cells could fully support estrogen‐dependent SNB dendrite growth. We restricted estradiol treatment in castrated males from P7 to P21 and assessed SNB dendritic morphology at P28. Treating castrates with estradiol implants at the muscle from P7 to P21 supported dendrite growth to normal levels through P28. These data suggest that the transient ERα expression in target muscle could potentially define the critical period for estrogen‐dependent dendrite growth in SNB motoneurons. © 2011 Wiley Periodicals, Inc. Develop Neurobiol, 2013     

Application of Multiplexed Kinase Inhibitor Beads to Study Kinome Adaptations in Drug-Resistant Leukemia

Protein kinases play key roles in oncogenic signaling and are a major focus in the development of targeted cancer therapies. Imatinib, a BCR-Abl tyrosine kinase inhibitor, is a successful front-line treatment for chronic myelogenous leukemia (CML). However, resistance to imatinib may be acquired by BCR-Abl mutations or hyperactivation of Src family kinases such as Lyn. We have used multiplexed kinase inhibitor beads (MIBs) and quantitative mass spectrometry (MS) to compare kinase expression and activity in an imatinib-resistant (MYL-R) and -sensitive (MYL) cell model of CML. Using MIB/MS, expression and activity changes of over 150 kinases were quantitatively measured from various protein kinase families. Statistical analysis of experimental replicates assigned significance to 35 of these kinases, referred to as the MYL-R kinome profile. MIB/MS and immunoblotting confirmed the over-expression and activation of Lyn in MYL-R cells and identified additional kinases with increased (MEK, ERK, IKKα, PKCβ, NEK9) or decreased (Abl, Kit, JNK, ATM, Yes) abundance or activity. Inhibiting Lyn with dasatinib or by shRNA-mediated knockdown reduced the phosphorylation of MEK and IKKα. Because MYL-R cells showed elevated NF-κB signaling relative to MYL cells, as demonstrated by increased IκBα and IL-6 mRNA expression, we tested the effects of an IKK inhibitor (BAY 65-1942). MIB/MS and immunoblotting revealed that BAY 65-1942 increased MEK/ERK signaling and that this increase was prevented by co-treatment with a MEK inhibitor (AZD6244). Furthermore, the combined inhibition of MEK and IKKα resulted in reduced IL-6 mRNA expression, synergistic loss of cell viability and increased apoptosis. Thus, MIB/MS analysis identified MEK and IKKα as important downstream targets of Lyn, suggesting that co-targeting these kinases may provide a unique strategy to inhibit Lyn-dependent imatinib-resistant CML. These results demonstrate the utility of MIB/MS as a tool to identify dysregulated kinases and to interrogate kinome dynamics as cells respond to targeted kinase inhibition.     

Factors Affecting the Delivery,Access, and Use of Interventions to Prevent Malaria in Pregnancy in Sub-Saharan Africa: A Systematic Review and Meta-Analysis

Background

Malaria in pregnancy has important consequences for mother and baby. Coverage with the World Health Organization–recommended prevention strategy for pregnant women in sub-Saharan Africa of intermittent preventive treatment in pregnancy (IPTp) and insecticide-treated nets (ITNs) is low. We conducted a systematic review to explore factors affecting delivery, access, and use of IPTp and ITNs among healthcare providers and women.

Methods and Results

We searched the Malaria in Pregnancy Library and Global Health Database from 1 January 1990 to 23 April 2013, without language restriction. Data extraction was performed by two investigators independently, and data was appraised for quality and content. Data on barriers and facilitators, and the effect of interventions, were explored using content analysis and narrative synthesis. We conducted a meta-analysis of determinants of IPTp and ITN uptake using random effects models, and performed subgroup analysis to evaluate consistency across interventions and study populations, countries, and enrolment sites. We did not perform a meta-ethnography of qualitative data.Ninety-eight articles were included, of which 20 were intervention studies. Key barriers to the provision of IPTp and ITNs were unclear policy and guidance on IPTp; general healthcare system issues, such as stockouts and user fees; health facility issues stemming from poor organisation, leading to poor quality of care; poor healthcare provider performance, including confusion over the timing of each IPTp dose; and women''s poor antenatal attendance, affecting IPTp uptake. Key determinants of IPTp coverage were education, knowledge about malaria/IPTp, socio-economic status, parity, and number and timing of antenatal clinic visits. Key determinants of ITN coverage were employment status, education, knowledge about malaria/ITNs, age, and marital status. Predictors showed regional variations.

Conclusions

Delivery of ITNs through antenatal clinics presents fewer problems than delivery of IPTp. Many obstacles to IPTp delivery are relatively simple barriers that could be resolved in the short term. Other barriers are more entrenched within the overall healthcare system or socio-economic/cultural contexts, and will require medium- to long-term strategies. Please see later in the article for the Editors'' Summary     

Separate and overlapping relationships of inattention and hyperactivity/impulsivity in children and adolescents with attention-deficit/hyperactivity disorder

There is debate regarding the dimensional versus categorical nature of attention-deficit/hyperactivity disorder (ADHD). This study utilized confirmatory factor analysis to examine this issue. ADHD symptoms rated on interviews and rating scales from a large sample of individuals (ages 3–17, 74 % male, 75 % Caucasian) with ADHD were examined (n = 242). Four potential factor structures were tested to replicate prior findings in a sample with a wide age range and included only participants who met DSM-IV-TR diagnostic criteria for ADHD. Correlations with executive function measures were performed to further assess the separability and validity of the derived factors. The data support a bifactor model with a general ADHD factor and two specific factors, inattention and hyperactivity/impulsivity. Importantly, the individual factors were also differentially correlated with executive functioning measures. This study adds to a growing literature suggesting both a general component to ADHD, as well as dimensional traits of inattention and hyperactivity/impulsivity, associated with distinct executive functioning profiles. The presence of a general underlying factor contraindicates separating the inattentive and combined subtypes of ADHD into distinct disorders.     

Hospital Differences in Cesarean Deliveries in Massachusetts (US) 2004–2006: The Case against Case-Mix Artifact

Objective

We examined the extent to which differences in hospital-level cesarean delivery rates in Massachusetts were attributable to hospital-level, rather than maternal, characteristics.

Methods

Birth certificate and maternal in-patient hospital discharge records for 2004–06 in Massachusetts were linked. The study population was nulliparous, term, singleton, and vertex births (NTSV) (n = 80,371) in 49 hospitals. Covariates included mother''s age, race/ethnicity, education, infant birth weight, gestational age, labor induction (yes/no), hospital shift at time of birth, and preexisting health conditions. We estimated multilevel logistic regression models to assess the likelihood of a cesarean delivery

Results

Overall, among women with NTSV births, 26.5% births were cesarean, with a range of 14% to 38.3% across hospitals. In unadjusted models, the between-hospital variance was 0.103 (SE 0.022); adjusting for demographic, socioeconomic and preexisting medical conditions did not reduce any hospital-level variation 0.108 (SE 0.023).

Conclusion

Even after adjusting for both socio-demographic and clinical factors, the chance of a cesarean delivery for NTSV pregnancies varied according to hospital, suggesting the importance of hospital practices and culture in determining a hospital''s cesarean rate.     

The FTO A/T Polymorphism and Elite Athletic Performance: A Study Involving Three Groups of European Athletes

Objective

The FTO A/T polymorphism (rs9939609) is a strong candidate to influence obesity-related traits. Elite athletes from many different sporting disciplines are characterized by low body fat. Therefore, the aim of this study was to assess whether athletic status is associated with the FTO A/T polymorphism.

Subjects and Methods

A large cohort of European Caucasians from Poland, Russia and Spain were tested to examine the association between FTO A/T polymorphism (rs9939609) and athletic status. A total of 551 athletes were divided by type of sport (endurance athletes, n = 266 vs. sprint/power athletes, n = 285) as well as by level of competition (elite-level vs. national-level). The control group consisted of 1,416 ethnically-matched, non-athletic participants, all Europeans. Multinomial logistic regression analyses were conducted to assess the association between FTO A/T genotypes and athletic status/competition level.

Results

There were no significantly greater/lesser odds of harbouring any type of genotype when comparing across athletic status (endurance athletes, sprint/power athletes or control participants). These effects were observed after controlling for sex and nationality. Furthermore, no significantly greater/lesser odds ratios were observed for any of the genotypes in respect to the level of competition (elite-level vs. national-level).

Conclusion

The FTO A/T polymorphism is not associated with elite athletic status in the largest group of elite athletes studied to date. Large collaborations and data sharing between researchers, as presented here, are strongly recommended to enhance the research in the field of exercise genomics.     

In Vitro Formation of β Cell Pseudoislets Using Islet-Derived Endothelial Cells

β cell pseudoislets (PIs) are used for the in vitro study of β-cells in a three-dimensional (3-D) configuration. Current methods of PI induction require unique culture conditions and extensive mechanical manipulations. Here we report a novel co-culture system consisting of high passage β-cells and islet-derived endothelial cells (iECs) that results in a rapid and spontaneous formation of free-floating PIs. PI structures were formed as early as 72 h following co-culture setup and were preserved for more than 14 d. These PIs, composed solely of β-cells, were similar in size to that of native islets and showed an increased percentage of proinsulin-positive cells, increased insulin gene expression in response to glucose stimulation, and restored glucose-stimulated insulin secretion when compared to β-cells cultured as monolayers. Key extracellular matrix proteins that were absent in β-cells cultured alone were deposited by iECs on PIs and were found in and around the PIs. iEC-induced PIs are a readily available tool for examining β cell function in a native 3-D configuration and can be used for examining β-cell/iEC interactions in vitro.