Knowledge and satisfaction of pharmacists attending an educational workshop on evidence-based management of low back pain.

Pharmacists are well positioned to provide quality care to people with low back pain (LBP). Education and training can equip pharmacists with the knowledge to optimally manage LBP in primary care. The aim of this study was to investigate the knowledge and satisfaction of pharmacists who attended a 2-h educational workshop on the evidence-based management of LBP. Case-based learning, underpinned by key adult learning principles, was one teaching method used to deliver important educational messages. Knowledge was assessed using a questionnaire consisting of multiple-choice, true/false questions and a written vignette based on a real-life clinical case scenario. Written feedback from pharmacists was used to gauge the success and limitations of the intervention. One hundred and ninety-three pharmacists completed the in-house assessment. Pharmacists demonstrated an accurate understanding of evidence-based pharmacological management of LBP, with all identifying paracetamol as the first-line drug choice for non-specific LBP. Ninety-nine per cent of pharmacists identified the symptoms presented in the vignette as a syndrome representing a significant clinical red flag requiring urgent referral. This educational intervention has delivered key messages on LBP management to pharmacists. There is a continued need for educational interventions addressing common conditions.

Tryptophan metabolism to kynurenine is a potential novel contributor to hypotension in human sepsis.

OBJECTIVES: To determine whether tryptophan metabolism to kynurenine contributes to the direct regulation of vascular tone in human septic shock. BACKGROUND: Indoleamine 2,3-dioxygenase 1 is an inducible enzyme that converts tryptophan to kynurenine and shares functional similarities with inducible nitric oxide synthase. Recently, kynurenine has been identified as an endothelium-derived relaxing factor produced during inflammation, raising the possibility that this novel pathway may contribute to hypotension in human sepsis. DESIGN: Prospective, matched, single-center, cohort study. SETTINGS: Intensive care unit of a tertiary teaching hospital matched to control subjects from the general medical ward and healthy volunteers. SUBJECTS: Patients (n = 16) with septic shock had indoleamine 2,3-dioxygenase activity assessed as the kynurenine-to-tryptophan ratio, and the severity of hypotension was determined by their inotrope requirements. Healthy and blood pressure-matched nonseptic control subjects were also studied. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Tissues from septic and control patients were stained for the presence of indoleamine 2,3-dioxygenase 1. Indoleamine 2,3-dioxygenase activity increased up to ninefold in patients with septic shock and was significantly higher than in the two control groups (p < .01). Indoleamine 2,3-dioxygenase activity was strongly correlated with inotrope requirements (p < .001). Indoleamine 2,3-dioxygenase protein was expressed in inflamed cardiac tissue as well as in endothelial cells of resistance vessels in hearts and kidneys from subjects who died from sepsis. CONCLUSIONS: : Indoleamine 2,3-dioxygenase 1 is expressed in resistance vessels in human sepsis and Indoleamine 2,3-dioxygenase activity correlates with hypotension in human septic shock. Indoleamine 2,3-dioxygenase 1 is thus a potential novel contributor to hypotension in sepsis.

A comparison of multiple shRNA expression methods for combinatorial RNAi.

RNAi gene therapies for HIV-1 will likely need to employ multiple shRNAs to counter resistant strains. We evaluated 3 shRNA co-expression methods to determine their suitability for present use; multiple expression vectors, multiple expression cassettes and single transcripts comprised of several dsRNA units (aka domains) with each being designed to a different target. Though the multiple vector strategy was effective with 2 shRNAs, the increasing number of vectors required is a major shortcoming. With single transcript configurations we only saw adequate activity from 1 of 10 variants tested, the variants being comprised of 2 - 3 different target domains. Whilst single transcript configurations have the most advantages on paper, these configurations can not yet be rapidly and reliably re-configured for new targets. However, our multiple cassette combinations of 2, 3 and 4 (29 bp) shRNAs were all successful, with suitable activity maintained in all positions and net activities comparable to that of the corresponding single shRNAs. We conclude that the multiple cassette strategy is the most suitably developed for present use as it is easy to design, assemble, is directly compatible with pre-existing shRNA and can be easily expanded.

The use of a human papillomavirus 18 promoter for tissue-specific expression in cervical carcinoma cells.

The use of tissue-specific promoter elements in the treatment of cervical cancer has been explored in this paper. The P(105) promoter of human papillomavirus 18 (HPV18) was utilised to direct tissue-specific expression in a number of cell types. Expression was examined in three cervical carcinoma cell lines: HeLa (HPV18 positive), SiHa (HPV16 positive), and C33A cells (HPV negative); the epithelial cell line, H1299; and the foetal fibroblast cell line, MRC5, utilising a luciferase expression vector. Expression was highest in the cervical cell lines by a factor of at least 80. The effect of a number of mutations in the P(105) promoter on expression levels was examined. Three deletion constructs of the long control region (LCR) were investigated: an 800 bp fragment (LCR800), a 400 bp fragment (LCR400), and a 200 bp fragment (LCR200), as well as the full length product LCR of HPV18 (LCR1000). The LCR800 construct of the HPV18 P(105) promoter had the highest level of expression in the cervical cell lines and was also highest in the HPV18-positive HeLa cell line. Site-directed mutagenesis was then employed on the LCR800 construct to create four further constructs that each had inactivating mutations in one of the four E2 binding sites (E2BSs). Overall, this study indicated that the LCR800 construct of the HPV18 P(105) promoter could be utilised as a tissuerestricted promoter in cervical cancer cells.

Biphasic glucocorticoid-dependent regulation of Wnt expression and its inhibitors in mature osteoblastic cells.

Glucocorticoids exert both anabolic and catabolic effects on bone. Previously, we reported that endogenous glucocorticoids control mesenchymal lineage commitment and osteoblastogenesis through regulation of Wnt signaling in osteoblasts. Here, we investigated the effects of glucocorticoids on Wnt expression in mature osteoblasts. Mature osteoblasts and their immature progenitors were separately isolated from Col2.3-GFP transgenic mice in which mature osteoblasts are identifiable through GFP expression. mRNA levels of Wnt2, Wnt2b, Wnt4, Wnt5a, Wnt10b, and Wnt11 were 4- to 12-fold higher in osteoblasts compared to their progenitors (P < 0.05). Expression of Wnt7b and Wnt10b in osteoblasts was modulated by corticosterone (CS), in a biphasic fashion with 3- to 3.5-fold upregulation at 10 nM CS (P < 0.01) and 50% downregulation at 100 nM CS (P < 0.05). CS 100 nM also increased expression of the Wnt inhibitors sFRP-1 and DKK-1 two- to threefold (P < 0.05). We conclude that the contrasting anabolic and catabolic effects of glucocorticoids on bone are, at least in part, mediated through the regulation of Wnt expression and its inhibitors in mature osteoblasts.

The effects of educational interventions on pharmacists' knowledge, attitudes and beliefs towards low back pain.

BACKGROUND: Practitioner beliefs and attitudes towards low back pain (LBP) influence treatment decisions. Little is known about pharmacists' knowledge, attitudes and beliefs towards LBP. OBJECTIVES: To investigate the effect of educational interventions on pharmacists' knowledge, attitudes and beliefs towards LBP. Setting Sydney Metropolitan Area. METHODS: Knowledge, attitudes and beliefs was measured using the "Pharmacists' Back Beliefs Questionnaire", with items from two previously reported questionnaires on back beliefs. Responses from pharmacists attending a 2-h educational workshop on LBP (n = 204) and pharmacists recruiting participants for a LBP clinical trial (n = 66) were compared to responses from a control group of pharmacists (n = 65) to allow an evaluation of the two interventions. Responses from workshop participants were also evaluated before and after the session. Participants indicated their agreement with statements about LBP on a 5-point Likert scale. Preferred responses were based on guidelines for the evidence-based management of LBP. The primary analysis evaluated total score on the nine-inevitability items of the Back Beliefs Questionnaire ("inevitability score"). MAIN OUTCOME MEASURE: Inevitability score. RESULTS: There was no significant difference in inevitability score between LBP clinical trial pharmacists and the control group [mean difference (MD) 0.47 (95 % CI -1.35 to 2.29; p = 0.61)]. The educational workshop led to a significant and favourable change in inevitability score (MD 7.23 p < 0.001) and notable changes in responses to misconceptions regarding bed rest and the need for imaging (p < 0.001) among participating pharmacists. CONCLUSIONS: Pharmacists attending the educational workshop provided the most compelling evidence that education specifically aimed at delivering evidence-based information can be successful in changing practitioner knowledge, beliefs and attitudes towards LBP.

Lessons learned during implementation of therapeutic hypothermia for neonatal hypoxic ischemic encephalopathy in a regional transport program in Ontario.

BACKGROUND: Therapeutic hypothermia (TH) is the first intervention to consistently show improved neurological outcomes in neonates with hypoxic ischemic encephalopathy (HIE). Since the recent introduction of TH for HIE in many centres, reviews of practices during the implementation of TH in Canada have not been published. OBJECTIVE: To determine if eligible neonates are being offered TH and to identify any barriers to the effective implementation of TH. METHODS: A retrospective review of neonates referred to a regional tertiary centre at a gestational age of 35 weeks or more with HIE was conducted. RESULTS: Among 41 neonates referred, 29 (71%) were eligible for TH; among eligible patients, five were moribund and excluded, and TH was initiated in 16 (67%) of the remaining 24. Reasons for not cooling in eight eligible patients included a delay in referral (n=5, median age at referral was 14 h) and a failure to recognize the severity of HIE (n=3). Among cooled patients, median times were the following: 116 min for age at referral; 80 min for time from referral to transport team arrival; and 358 min for age at initiation of cooling. Seven (44%) patients had cooling initiated after 6 h of age. CONCLUSION: A significant proportion of eligible patients were not offered TH, and in many cooled patients, initiation of cooling was delayed beyond the recommended 6 h. For eligible patients to benefit from TH, it is imperative that all birthing centres be made aware that TH is now widely available as an important treatment option, but also that TH is a time-sensitive therapy requiring rapid identification and referral. In the region studied, for eligible patients, referring hospitals should initiate passive cooling before arrival of the transport team. Referring hospitals should be prepared to provide early, yet safe initiation of passive cooling by having the appropriate equipment, and having staff trained in the use and monitoring of rectal temperatures.

Haemodynamic changes after delivery room surfactant administration to very low birth weight infants.

INTRODUCTION: Surfactant replacement therapy (SRT) reduces respiratory morbidity and mortality in premature infants. The goal of this study was to characterise the effects of delivery room SRT on the ductus arteriosus and early neonatal haemodynamics. METHODS: A prospective observational study was conducted in preterm infants of less than 32 weeks' gestation who received SRT within 30 min of birth. Serial echocardiography was performed before and after SRT. Characteristics of the ductus arteriosus, myocardial performance, right ventricular output (RVO) and left ventricular output (LVO) and the ratio of RVO:LVO were measured. RESULTS: Sixteen babies, born at 28.3+/-1.3 weeks' gestation and weighing 1289+/-224 g, were studied. SRT was associated with an improvement in the arterial oxygen tension:fractional inspired oxygen ratio (p<0.001), increased systolic and decreased diastolic arterial pressure (p<0.05). The ductus arteriosus was patent in all and transductal flow was unrestrictive and exclusively left-to-right after SRT. An increase in transductal diameter (p<0.001), left atrium:aortic ratio (p=0.006) but a decrease in left ventricular end-diastolic dimension (p=0.02) was identified. CONCLUSION: SRT administration was followed by increased RVO but decreased LVO, resulting in an increased RVO:LVO ratio and an increase in ductal size. Delivery room administration of SRT is associated with major haemodynamic changes. The impact of these changes needs prospective evaluation.

Glucocorticoid-dependent Wnt signaling by mature osteoblasts is a key regulator of cranial skeletal development in mice.

Glucocorticoids are important regulators of bone cell differentiation and mesenchymal lineage commitment. Using a cell-specific approach of osteoblast-targeted transgenic disruption of intracellular glucocorticoid signaling, we discovered a novel molecular pathway by which glucocorticoids, mainly through the mature osteoblast, regulate the cellular mechanisms that govern cranial skeleton development. Embryonic and neonatal transgenic mice revealed a distinct phenotype characterized by hypoplasia and osteopenia of the cranial skeleton; disorganized frontal, parietal and interparietal bones; increased suture patency; ectopic differentiation of cartilage in the sagittal suture; and disturbed postnatal removal of parietal cartilage. Concurrently, expression of Mmp14, an enzyme essential for calvarial cartilage removal, was markedly reduced in parietal bone and cartilage of transgenic animals. Expression of Wnt9a and Wnt10b was significantly reduced in osteoblasts with disrupted glucocorticoid signaling, and accumulation of beta-catenin, the upstream regulator of Mmp14 expression, was decreased in osteoblasts, chondrocytes and mesenchymal progenitors of transgenic mice. Supracalvarial injection of Wnt3a protein rescued the transgenic cranial phenotype. These results define novel roles for glucocorticoids in skeletal development and delineate how osteoblasts--under steroid hormone control--orchestrate the intricate process of intramembranous bone formation by directing mesenchymal cell commitment towards osteoblastic differentiation while simultaneously initiating and controlling cartilage dissolution in the postnatal mouse.

Osteoblasts directly control lineage commitment of mesenchymal progenitor cells through Wnt signaling.

Lineage commitment of mesenchymal progenitor cells is still poorly understood. Here we demonstrate that Wnt signaling by osteoblasts is essential for mesenchymal progenitor cells to differentiate away from a default adipogenic into an osteoblastic lineage. Dominant adipogenesis and reduced osteoblastogenesis were observed in calvarial cell cultures from transgenic mice characterized by osteoblast-targeted disruption of glucocorticoid signaling. This phenotypic shift in mesenchymal progenitor cell commitment was associated with reciprocal regulation of early adipogenic and osteoblastogenic transcription factors and with a reduction in Wnt7b and Wnt10b mRNA and beta-catenin protein levels in transgenic versus non-transgenic cultures. Transwell co-culture of transgenic mesenchymal progenitor cells with wild type osteoblasts restored commitment to the osteoblast lineage. This effect was blocked by adding sFRP1, a Wnt inhibitor, to the co-culture. Treatment of transgenic cultures with Wnt3a resulted in stimulation of osteoblastogenesis and suppression of adipogenesis. Our findings suggest a novel cellular mechanism in bone cell biology in which osteoblasts exert direct control over the lineage commitment of their mesenchymal progenitor through Wnt signaling. This glucocorticoid-dependent forward control function indicates a central role for osteoblasts in the regulation of early osteoblastogenesis.