Cobalt ions stimulate a fibrotic response through matrix remodelling, fibroblast contraction and release of pro-fibrotic signals from macrophages.

Many studies report the adverse responses to metal-on-metal (MoM) hip prostheses, with tissues surrounding failed MoM hip prostheses revealing abundant tissue necrosis and fibrosis. These local effects appear to be initiated by metal ions released from the prosthesis causing the secretion of inflammatory mediators. However, little is known about the effect of the metal ions on tissue remodelling and pseudotumor formation, which are also associated with the failure of MoM hip prostheses. The peri-prosthetic soft tissue masses can lead to pain, swelling, limited range of joint movement and extensive tissue lesion. To elucidate this cellular response, a multidisciplinary approach using both two- and three-dimensional (2D and 3D) in vitro culture systems was employed to study the effects of Co2+ and Cr3+ on human fibroblast activation and mechanobiology. Co2+ induced a fibrotic response, characterised by cytoskeletal remodelling and enhanced collagen matrix contraction. This was associated with increased cell stiffness and contractile forces as measured by atomic force microscopy and traction force microscopy, respectively. These effects were triggered by the generation of reactive oxygen species (ROS). Moreover, this fibrotic response was enhanced in the presence of macrophages, which increased the prevalence of a-smooth muscle actin (a-SMA)-positive fibroblasts and collagen synthesis. Cr3+ did not show any significant effect on fibroblast activation. Co2+ promoted matrix remodelling by fibroblasts that was further enhanced by macrophage signalling. Use of alternative implant materials or manipulation of this fibrotic response could provide an opportunity for enhancing the success of prostheses utilising CoCr alloys.

Developing a set of consensus indicators to support maternity service quality improvement: using Core Outcome Set methodology including a Delphi process.

OBJECTIVE: To develop a core metric set to monitor the quality of maternity care. DESIGN: Delphi process followed by a face-to-face consensus meeting. SETTING: English maternity units. POPULATION: Three representative expert panels: service designers, providers and users. MAIN OUTCOME MEASURES: Maternity care metrics judged important by participants. METHODS: Participants were asked to complete a two-phase Delphi process, scoring metrics from existing local maternity dashboards. A consensus meeting discussed the results and re-scored the metrics. RESULTS: In all, 125 distinct metrics across six domains were identified from existing dashboards. Following the consensus meeting, 14 metrics met the inclusion criteria for the final core set: smoking rate at booking; rate of birth without intervention; caesarean section delivery rate in Robson group 1 women; caesarean section delivery rate in Robson group 2 women; caesarean section delivery rate in Robson group 5 women; third- and fourth-degree tear rate among women delivering vaginally; rate of postpartum haemorrhage of ≥1500 ml; rate of successful vaginal birth after a single previous caesarean section; smoking rate at delivery; proportion of babies born at term with an Apgar score <7 at 5 minutes; proportion of babies born at term admitted to the neonatal intensive care unit; proportion of babies readmitted to hospital at <30 days of age; breastfeeding initiation rate; and breastfeeding rate at 6-8 weeks. CONCLUSIONS: Core outcome set methodology can be used to incorporate the views of key stakeholders in developing a core metric set to monitor the quality of care in maternity units, thus enabling improvement. TWEETABLE ABSTRACT: Achieving consensus on core metrics for monitoring the quality of maternity care.

Caesarean scar pregnancy in the UK: a national cohort study.

OBJECTIVE: To estimate the incidence of caesarean scar pregnancy (CSP) and to describe the management outcomes associated with this condition. DESIGN: A national cohort study using the UK Early Pregnancy Surveillance Service (UKEPSS). SETTING: 86 participating Early Pregnancy Units. POPULATION: All women diagnosed in the participating units with CSP between November 2013 and January 2015. METHODS: Cohort study of women identified through the UKEPSS monthly mailing system. MAIN OUTCOME MEASURES: Incidence, clinical outcomes and complications. RESULTS: 102 cases of CSP were reported, with an estimated incidence of 1.5 per 10 000 (95% CI 1.1-1.9) maternities. Full outcome data were available for 92 women. Management was expectant in 21/92 (23%), medical in 15/92 (16%) and surgical in 56/92 (61%). The success rates of expectant, medical and surgical management were 43% (9/21), 46% (7/15) and 96% (54/56), respectively. The complication rates were 15/21 (71%) with expectant, 9/15 (60%) with medical and 20/56 (36%) with surgical management. Discharge from care (median number of days) was 82 (range 37-174) with expectant, 21 (range 10-31) with medical and 11 (range 4-49) with surgical management. CONCLUSIONS: Surgical management appears to be associated with a high success rate, low complication rate and short post-treatment follow up. TWEETABLE ABSTRACT: Surgery for CSP appears to be successful, with low complication rates and short post-treatment follow up.

Hormone-secreting adrenal tumours cause severe hypertension and high rates of poor pregnancy outcome; a UK Obstetric Surveillance System study with case control comparisons.

OBJECTIVE: To examine the management and outcomes of adrenal tumours in pregnancy. DESIGN: A national observational, cohort study over 4 years using the UK Obstetric Surveillance System (UKOSS). SETTING: Consultant-led obstetric units. PATIENTS: Women with phaeochromocytoma, primary aldosteronism or Cushing's syndrome diagnosed before or during pregnancy. METHODS: Clinical features of UKOSS cases were compared with those of women with adrenal tumours reported from 1985-2015. Nested case-control comparisons involving the UKOSS cases as well as those identified in the literature were performed for pregnancy outcome data using UKOSS controls with uncomplicated singleton (n = 2250) pregnancy and data from the Office of National Statistics (ONS). MAIN OUTCOME MEASURES: Incidence, management and frequency of adverse maternal and offspring outcomes of adrenal tumours in pregnancy. RESULTS: Fifteen pregnant women met the inclusion criteria: ten phaeochromocytoma, three primary aldosteronism and two Cushing's syndrome. All of the tumours had an incidence rate <2 per 100 000 pregnancies. Clinical symptoms were similar to those in non-pregnant women due to the hormones released. All women had severe hypertension, and in those diagnosed in pregnancy prior to conception. There was a significantly increased risk of adverse pregnancy outcomes in affected women, with increased rates of stillbirth, preterm labour and operative delivery. CONCLUSIONS: Adrenal tumours are associated with increased risks for pregnant women and their babies. Data on these tumours to inform practice are limited and international collaborative efforts are likely to be needed. TWEETABLE ABSTRACT: Study of hormone-secreting adrenal tumours in pregnancy linked with high BP and high rates of fetal morbidity.

The use of stomas in the early management of Hirschsprung disease: Findings of a national, prospective cohort study.

BACKGROUND/PURPOSE: Primary pull-through without a stoma has become preferred practice in managing Hirschsprung disease (HD). The aims of this study were to establish stoma rate and identify factors associated with stoma formation in a population-based cohort in the UK and Ireland. METHODS: Live-born infants with HD were prospectively identified in all 28 specialist pediatric surgical units in the UK and Ireland between October 2010 to September 2012. Method of colonic decompression was recorded and multivariable logistic regression was used to identify factors associated with stoma formation. RESULTS: 305 infants with HD were identified. Rectal washouts were initially used in 86% (263) with a defunctioning stoma formed as the primary management in 13% (39). Ultimately, 36% (111) required a stoma prior to definitive surgery. Compared to infants managed with rectal washouts alone; infants managed with a stoma were more likely to have a transition zone proximal to the splenic flexure, Down (or another) syndrome, and HD diagnosis established more than 28days after presentation. CONCLUSIONS: Although rectal washouts are commonly employed, a stoma prior to definitive surgery was required in 36% of infants in a national cohort. Delayed diagnosis, aganglionosis proximal to the splenic flexure and presence of other anomalies are associated with stoma formation. TYPE OF STUDY AND LEVEL OF EVIDENCE: Prognosis study (high-quality prospective cohort study with 80% follow-up and all patients enrolled at same time point in disease).

Factors associated with maternal mortality at advanced maternal age: a population-based case-control study.

OBJECTIVE: This study aimed to examine the factors associated with maternal mortality among women aged ≥35 years. DESIGN: Unmatched population based case-control study. SETTING: United Kingdom. POPULATION: Between 2009 and 2012, 105 cases of maternal deaths aged ≥35 years were extracted from the surveillance database of the MBRRACE-UK confidential enquiries into maternal deaths in the UK. In addition, 766 controls aged ≥35 years were identified from the UK Obstetric Surveillance System (2005-2012). METHODS: Risk factors known to be associated with maternal mortality and morbidity and for which data were available were examined for their association with maternal mortality among women ≥35 years using logistic regression analysis. MAIN OUTCOME MEASURES: Odds ratios and 95% confidence intervals associated with maternal death. RESULTS: Five factors were found to be significantly associated with increased odds of death among women aged ≥35 years: smoking during pregnancy (adjusted odds ratio (aOR) 2.06, 95% CI 1.13-3.75), inadequate use of antenatal care (aOR 23.62, 95% CI 8.79-63.45), medical co-morbidities (aOR 5.92, 95% CI 3.56-9.86) and previous pregnancy problems (aOR 2.06, 95% CI 1.23-3.45). The odds associated with death increased by 12% per year increase in age (aOR 1.12, 95% CI 1.02-1.22). CONCLUSION: Age was associated with maternal mortality even after adjusting for other known risk factors. Importantly, this study showed an association between maternal mortality and smoking among women aged 35 years or older. It emphasises the importance of public health action to reduce smoking levels and address trends in rising maternal age. TWEETABLE ABSTRACT: Smoking is a risk factor for maternal death for those aged over 35 years.

IFT88 influences chondrocyte actin organization and biomechanics.

OBJECTIVES: Primary cilia are microtubule based organelles which control a variety of signalling pathways important in cartilage development, health and disease. This study examines the role of the intraflagellar transport (IFT) protein, IFT88, in regulating fundamental actin organisation and mechanics in articular chondrocytes. METHODS: The study used an established chondrocyte cell line with and without hypomorphic mutation of IFT88 (IFT88(orpk)). Confocal microscopy was used to quantify F-actin and myosin IIB organisation. Viscoelastic cell and actin cortex mechanics were determined using micropipette aspiration with actin dynamics visualised in live cells transfected with LifeACT-GFP. RESULTS: IFT88(orpk) cells exhibited a significant increase in acto-myosin stress fibre organisation relative to wild-type (WT) cells in monolayer and an altered response to cytochalasin D. Rounded IFT88(orpk) cells cultured in suspension exhibited reduced cortical actin expression with reduced cellular equilibrium modulus. Micropipette aspiration resulted in reduced membrane bleb formation in IFT88(orpk) cells. Following membrane blebbing, IFT88(orpk) cells exhibited slower reformation of the actin cortex. IFT88(orpk) cells showed increased actin deformability and reduced cortical tension confirming that IFT regulates actin cortex mechanics. The reduced cortical tension is also consistent with the reduced bleb formation. CONCLUSIONS: This study demonstrates for the first time that the ciliary protein IFT88 regulates fundamental actin organisation and the stiffness of the actin cortex leading to alterations in cell deformation, mechanical properties and blebbing in an IFT88 chondrocyte cell line. This adds to the growing understanding of the role of primary cilia and IFT in regulating cartilage biology.

R-spondins: novel matricellular regulators of the skeleton.

R-spondins are a family of four matricellular proteins produced by a variety of cell-types. Structurally, R-spondins contain a TSR1 domain that retains the tryptophan structure and a modified cysteine-rich CSVCTG region. In addition, the R-spondins contain two furin repeats implicated in canonical Wnt signaling. R-spondins positively regulate canonical Wnt signaling by reducing Wnt receptor turnover and thereby increasing beta-catenin stabilization. R-spondins are prominently expressed in the developing skeleton and contribute to limb formation, particularly of the distal digit. Additionally, results suggest that R-spondins may contribute to the maintenance of adult bone mass by regulating osteoblastogenesis and bone formation.

Surface topography regulates wnt signaling through control of primary cilia structure in mesenchymal stem cells.

The primary cilium regulates cellular signalling including influencing wnt sensitivity by sequestering ß-catenin within the ciliary compartment. Topographic regulation of intracellular actin-myosin tension can control stem cell fate of which wnt is an important mediator. We hypothesized that topography influences mesenchymal stem cell (MSC) wnt signaling through the regulation of primary cilia structure and function. MSCs cultured on grooves expressed elongated primary cilia, through reduced actin organization. siRNA inhibition of anterograde intraflagellar transport (IFT88) reduced cilia length and increased active nuclear ß-catenin. Conversely, increased primary cilia assembly in MSCs cultured on the grooves was associated with decreased levels of nuclear active ß-catenin, axin-2 induction and proliferation, in response to wnt3a. This negative regulation, on grooved topography, was reversed by siRNA to IFT88. This indicates that subtle regulation of IFT and associated cilia structure, tunes the wnt response controlling stem cell differentiation.

Contemporary management and outcomes for infants born with oesophageal atresia.

BACKGROUND: Reports on the management and outcome of rare conditions, such as oesophageal atresia, are frequently limited to case series reporting single-centre experience over many years. The aim of this study was to identify all infants born with oesophageal atresia in the UK and Ireland to describe current clinical practice and outcomes. METHODS: This was a prospective multicentre cohort study of all infants born with oesophageal atresia and/or tracheo-oesophageal fistula in 2008-2009 in the UK and Ireland to record current clinical management and early outcomes. RESULTS: A total of 151 infants admitted to 28 paediatric surgical units were identified. Some aspects of perioperative management were universal, including oesophageal decompression, operative technique and the use of transanastomotic tubes. However, there were a number of areas where clinical practice varied considerably, including the routine use of perioperative chest drains, postoperative contrast studies and antireflux medication, with each of these being employed in 30-50 per cent of patients. There was a trend towards routine postoperative ventilation. CONCLUSION: The prospective methodology used in this study can help identify practices that all surgeons employ and also those that few surgeons use. Areas of clinical equipoise can be recognized and avenues for further research identified.

Myocardial infarction in pregnancy and postpartum in the UK.

AIM: Cardiac disease is a leading cause of maternal death in the developed world, responsible for one-fifth of all maternal deaths in the UK. The aim of this study was to estimate the incidence of myocardial infarction (MI) in pregnancy and up to one week postpartum in the UK and describe risk factors, management and outcomes. METHODS: A prospective population-based study with nested case control analysis used the UK Obstetric Surveillance System to identify all women in the UK with MI in pregnancy (in the years 2005-2010). A control group of 1360 women was used for comparison. Multivariable unconditional logistic regression was conducted to identify potential risk factors for MI in pregnancy and calculate adjusted odds ratios with 95% confidence intervals. RESULTS: Twenty-five cases of MI in pregnancy were reported, giving an estimated incidence of 0.7 per 100,000 maternities (95%CI 0.5-1.1). Maternal age, smoking, hypertension, twin pregnancy and pre-eclampsia were independently associated with MI in pregnancy. Fifteen (60%) women underwent coronary angiography; nine (60%) had coronary atherosclerosis, three (21%) had coronary artery dissection, one (7%) had a coronary thrombus and two (13%) had normal coronary arteries. Nine women had angioplasty +/- stenting and two were thrombolysed. No women died. CONCLUSIONS: Many risk factors are both recognisable and modifiable. Management of MI in pregnancy was highly variable indicating a clear need for further information regarding the safety and outcomes of different interventions. The addition of pregnancy status as a compulsory field in cardiac audit databases would enable routine collection of this information.

Operational plasticity enables hsp104 to disaggregate diverse amyloid and nonamyloid clients.

It is not understood how Hsp104, a hexameric AAA+ ATPase from yeast, disaggregates diverse structures, including stress-induced aggregates, prions, and α-synuclein conformers connected to Parkinson disease. Here, we establish that Hsp104 hexamers adapt different mechanisms of intersubunit collaboration to disaggregate stress-induced aggregates versus amyloid. To resolve disordered aggregates, Hsp104 subunits collaborate noncooperatively via probabilistic substrate binding and ATP hydrolysis. To disaggregate amyloid, several subunits cooperatively engage substrate and hydrolyze ATP. Importantly, Hsp104 variants with impaired intersubunit communication dissolve disordered aggregates, but not amyloid. Unexpectedly, prokaryotic ClpB subunits collaborate differently than Hsp104 and couple probabilistic substrate binding to cooperative ATP hydrolysis, which enhances disordered aggregate dissolution but sensitizes ClpB to inhibition and diminishes amyloid disaggregation. Finally, we establish that Hsp104 hexamers deploy more subunits to disaggregate Sup35 prion strains with more stable "cross-ß" cores. Thus, operational plasticity enables Hsp104 to robustly dissolve amyloid and nonamyloid clients, which impose distinct mechanical demands.

Primary cilia elongation in response to interleukin-1 mediates the inflammatory response.

Primary cilia are singular, cytoskeletal organelles present in the majority of mammalian cell types where they function as coordinating centres for mechanotransduction, Wnt and hedgehog signalling. The length of the primary cilium is proposed to modulate cilia function, governed in part by the activity of intraflagellar transport (IFT). In articular cartilage, primary cilia length is increased and hedgehog signaling activated in osteoarthritis (OA). Here, we examine primary cilia length with exposure to the quintessential inflammatory cytokine interleukin-1 (IL-1), which is up-regulated in OA. We then test the hypothesis that the cilium is involved in mediating the downstream inflammatory response. Primary chondrocytes treated with IL-1 exhibited a 50% increase in cilia length after 3 h exposure. IL-1-induced cilia elongation was also observed in human fibroblasts. In chondrocytes, this elongation occurred via a protein kinase A (PKA)-dependent mechanism. G-protein coupled adenylate cyclase also regulated the length of chondrocyte primary cilia but not downstream of IL-1. Chondrocytes treated with IL-1 exhibit a characteristic increase in the release of the inflammatory chemokines, nitric oxide and prostaglandin E2. However, in cells with a mutation in IFT88 whereby the cilia structure is lost, this response to IL-1 was significantly attenuated and, in the case of nitric oxide, completely abolished. Inhibition of IL-1-induced cilia elongation by PKA inhibition also attenuated the chemokine response. These results suggest that cilia assembly regulates the response to inflammatory cytokines. Therefore, the cilia proteome may provide a novel therapeutic target for the treatment of inflammatory pathologies, including OA.

Influenza A/H1N1v in pregnancy: an investigation of the characteristics and management of affected women and the relationship to pregnancy outcomes for mother and infant.

BACKGROUND: In April 2009 a novel influenza A virus (AH1N1v) of swine origin (swine flu) emerged, spreading rapidly and achieving pandemic status in June 2009. Pregnant women were identified as being at high risk of severe influenza-related complications and as a priority group for vaccination against AH1N1v. Limited information was available about the maternal and fetal risks of AH1N1v infection or of antiviral drug or AH1N1v vaccine use in pregnancy. OBJECTIVES: To assess rates of and risk factors for adverse outcomes following AH1N1v infection in pregnancy and to assess the adverse effects of the antiviral drugs and vaccines used in prevention and management. METHODS: Prospective national cohort studies were conducted to identify pregnant women who were (1) suspected to be infected with AH1N1v or being treated with antiviral medication in primary care; (2) vaccinated against AH1N1v; and (3) admitted to hospital with confirmed AH1N1v. Characteristics of women with influenza-like illness (ILI) in primary care were compared with those of women without symptoms accepting or declining immunisation. Characteristics of women admitted to hospital with confirmed AH1N1v infection in pregnancy were compared with a historical cohort of over 1200 women giving birth in the UK who were uninfected with AH1N1v. Outcomes examined in hospitalised women included maternal death, admission to an intensive care unit, perinatal mortality and preterm birth. Risk factors for hospital and intensive care unit admission were examined in a full regression model. RESULTS: The weekly incidence of ILI among pregnant women averaged 51/100,000 over the study period. Antiviral drugs were offered to 4.8% [95% confidence interval (CI) 4.0% to 5.9%] and vaccination to 64.8% (95% CI 64.7% to 68.9%) of registered pregnant women. Ninety pregnant women with ILI presenting in primary care were reported to the research team, 55 of whom were prescribed antiviral drugs and in 42 (76%) cases this was within 2 days of symptom onset. After comparison with 1329 uninfected pregnant women offered vaccination, pre-existing asthma was the only maternal factor identified as increasing risk of ILI presentation [adjusted odds ratio (OR) 2.0, 95% CI 1.0 to 3.9]. Maternal obesity and smoking during pregnancy were also associated with hospital admission with AH1N1v infection. Overall, 241 pregnant women were admitted to hospital with laboratory-confirmed AH1N1v infection. Eighty-three per cent of these women were treated with antiviral agents, but only 6% received antiviral treatment before hospital admission. Treatment within 2 days of symptom onset was associated with an 84% reduction in the odds of admission to an intensive therapy unit (OR 0.16, 95% CI 0.08 to 0.34). Women admitted to hospital with AH1N1v infection were more likely to deliver preterm; a three times increased risk was suggested compared with an uninfected population cohort (OR 3.1, 95% CI 2.1 to 4.5). CONCLUSIONS: Earlier treatment with antiviral agents is associated with improved outcomes for pregnant women and further actions are needed in future pandemics to ensure that antiviral agents and vaccines are provided promptly to pregnant women, particularly in the primary care setting. Further research is needed on longer-term outcomes for infants exposed to AH1N1v influenza, antiviral drugs or vaccines during pregnancy.

Peripartum hysterectomy in the UK: management and outcomes of the associated haemorrhage.

OBJECTIVES: To identify women undergoing peripartum hysterectomy in the UK and to describe the causes, management and outcome of the associated haemorrhage. DESIGN: A population-based descriptive study using the UK Obstetric Surveillance System (UKOSS). SETTING: All 229 hospitals with consultant-led maternity units in the UK. POPULATION: All women in the UK delivering between February 2005 and February 2006. METHODS: Prospective case identification through the UKOSS monthly mailing. MAIN OUTCOME MEASURES: Rates with 95% CIs. Odds ratio estimates. RESULTS: Three hundred and eighteen women underwent peripartum hysterectomy. The most commonly reported causes of haemorrhage were uterine atony (53%) and morbidly adherent placenta (39%). Women were not universally managed with uterotonic therapies. Fifty women were unsuccessfully managed with B-Lynch or other brace suture prior to hysterectomy, 28 with activated factor VII and 9 with arterial embolisation. Twenty-one percent of women suffered damage to other structures, 20% required a further operation and 19% were reported to have additional severe morbidity. Bladder damage was more likely in women with placenta accreta (OR 3.41, 95% CI 1.55-7.48) than in women with uterine atony. There were no significant differences in outcomes between women undergoing total or subtotal hysterectomy. Two women died; case fatality 0.6% (95% CI 0-1.5%). CONCLUSIONS: For each woman who dies in the UK following peripartum hysterectomy, more than 150 survive. The associated haemorrhage is managed in a variety of ways and not universally according to existing guidelines. Further investigation of the outcomes following some of the more innovative therapies for control of haemorrhage is needed.

Randomized, double-blind comparison of patient-controlled epidural infusion vs nurse-administered epidural infusion for postoperative analgesia in patients undergoing colonic resection.

BACKGROUND: There is little published evidence of the analgesic efficacy of patient-controlled epidural analgesia (PCEA) for postoperative pain relief. The aim of this study was to compare the analgesic efficacy of epidural infusion of bupivacaine 0.125% and fentanyl 4 microg ml(-1) administered by either PCEA with a background infusion or nurse-administered continuous epidural infusion (CEI) after major intra-abdominal surgery. METHODS: In a double-blind, randomized clinical trial, 205 adult patients undergoing colonic resection by laparotomy received either PCEA or CEI. Pain scores were recorded via a four-point verbal rating scale at 1, 2, 3, 4, 8, 12, 24, 48, and 72 h after surgery. The administration of epidural top-ups and systemic analgesia over the same period was also recorded, and patient satisfaction questionnaires completed. RESULTS: The median area under the curve of pain against time was significantly lower in the PCEA group (2 vs 24, P<0.001) as were median summary pain scores on movement (0.67 vs 1.33, P<0.001). Significantly fewer patients in the PCEA group received one or more epidural top-ups (13 vs 36%, P = 0.0002) or any systemic analgesics (41 vs 63%, P = 0.0021). Patients in the PCEA group were significantly more likely to be very satisfied than in the CEI group (76 vs 43%, P<0.0001). CONCLUSIONS: PCEA provides greater analgesic efficacy than CEI for postoperative analgesia after major intra-abdominal surgery, and a decreased requirement for physician or nurse intervention.

Purinergic pathway suppresses the release of .NO and stimulates proteoglycan synthesis in chondrocyte/agarose constructs subjected to dynamic compression.

Mechanical loading plays a fundamental role in the physiological and pathological processes of articular cartilage. The application of dynamic compression to chondrocytes cultured in agarose, downregulates the release of nitric oxide (NO) and enhances cell proliferation and proteoglycan synthesis. We hypothesize that the observed metabolic changes in response to dynamic compression involve a purinergic signaling pathway. Chondrocyte/agarose constructs were subjected to dynamic compression (15%, 1 Hz, 48 h) in the presence of antagonists for the purinergic pathway. Gadolinium was used as a putative inhibitor of stretch-activated calcium ion channels including adenosine 5'-triphosphate (ATP) release channels; suramin was employed as a P2 receptor antagonist and apyrase was used to catalyze the hydrolysis of extracellular ATP. The data presented demonstrate that in the absence of the inhibitor, dynamic compression suppressed .NO release. Treatment with gadolinium and suramin caused a compression-induced upregulation of .NO release, a response abolished with apyrase. Compression-induced stimulation of cell proliferation was reversed with gadolinium, suramin, or apyrase. By contrast, compression-induced stimulation of proteoglycan synthesis was abolished under all treatment conditions. Thus, the purinergic pathway is important in suppressing the release of .NO and stimulation of proteoglycan synthesis. Indeed, high levels of .NO could trigger a downstream catabolic response and mediate the compression-induced inhibition of cell proliferation. The current study demonstrates for the first time the importance of a purinergic pathway in mediating the metabolic response to dynamic compression and suppressing an inflammatory effect.

Role of polymorphic human cytochrome P450 enzymes in estrone oxidation.

Estrogen and its metabolites are believed to play important roles in breast cancer. The influence of genetic polymorphisms in the enzymes responsible for formation and disposition of estrogen on breast cancer risk may shed light on the importance of estrogen metabolites in this disease. However, for such studies to be valid, it is important to correctly identify the enzymes involved in estrogen bioactivation. Therefore, we assessed the human cytochrome P450-dependent oxidation of estrone using substrate concentrations that more closely approximate the maximum expected concentrations in breast tissue. The in vitro metabolism of estrone by recombinant human cytochrome P450 enzymes and human liver microsomes was studied. The formation of estrone metabolites (2-hydroxyestrone, 4-hydroxyestrone, and 16alpha-hydroxyestrone) was monitored by high-performance liquid chromatography. 2-Hydroxyestrone formation was catalyzed predominantly by CYP1A2, CYP1A1, and CYP1B1 enzymes; 4-hydroxyestrone formation was catalyzed predominantly by CYP1B1, CYP1A2, and CYP1A1 enzymes; and 16alpha-hydroxyestrone formation was catalyzed predominantly by CYP2C19, CYP1A1, and CYP3A5. This study confirms the important role of members of the CYP1 family in the 2-hydroxylation and 4-hydroxylation of estrone, but the enzymes identified as responsible for the 16alpha-hydroxylation of estrone are different from those previously identified. The relative importance of these enzymes in vivo would depend on the specific tissue expression of the enzymes. These enzymes are all known to be genetically variant in the human population, and additional studies to assess the role CYP1A2, CYP2C19, and CYP3A5 in breast cancer risk are indicated.

Spinocerebellar ataxia type 14: study of a family with an exon 5 mutation in the PRKCG gene.

We report our observations in an Australian family with spinocerebellar ataxia type 14 (SCA 14). We describe a novel mutation in exon 5 of the PRKCG gene, altering a highly conserved cysteine to a phenylalanine at codon 150, and record the detailed clinical observations in six affected family members.