Because animal studies have demonstrated that mechanical ventilation at high volume and pressure can be deleterious to the lungs, limitation of airway pressure, allowing hypercapnia if necessary, is already used for ventilation of acute respiratory distress syndrome (ARDS). Whether a systematic and more drastic reduction is necessary is debatable. A multicenter randomized study was undertaken to compare a strategy aimed at limiting the end-inspiratory plateau pressure to 25 cm H2O, using tidal volume (VT) below <em>1</em>0 <em>ml</em>/kg of body weight, versus a more conventional ventilatory approach (with regard to current practice) using VT at <em>1</em>0 <em>ml</em>/kg or above and close to normal PaCO2. Both arms used a similar level of positive end-expiratory pressure. A total of <em>1</em><em>1</em>6 patients with ARDS and no organ failure other than the lung were enrolled over 32 mo in 25 centers. The two groups were similar at inclusion. Patients in the two arms were ventilated with different VT (7.<em>1</em> +/- <em>1</em>.3 versus <em>1</em>0.3 +/- <em>1</em>.7 <em>ml</em>/kg at Day <em>1</em>, p < 0.00<em>1</em>) and plateau pressures (25.7 +/- 5. 0 versus 3<em>1</em>.7 +/- 6.6 cm H2O at Day <em>1</em>, p < 0.00<em>1</em>), resulting in different PaCO2 (59.5 +/- <em>1</em>5.0 versus 4<em>1</em>.3 +/- 7.6 mm Hg, p < 0.00<em>1</em>) and pH (7.28 +/- 0.09 versus 7.4 +/- 0.09, p < 0.00<em>1</em>), but a similar level of oxygenation. The new approach did not reduce mortality at Day 60 (46.6% versus 37.9% in control subjects, p = 0.38), the duration of mechanical ventilation (23.<em>1</em> +/- 20.2 versus 2<em>1</em>.4 +/- <em>1</em>6. 3 d, p = 0.85), the incidence of pneumothorax (<em>1</em>4% versus <em>1</em>2%, p = 0. 78), or the secondary occurrence of multiple organ failure (4<em>1</em>% versus 4<em>1</em>%, p = <em>1</em>). We conclude that no benefit could be observed with reduced VT titrated to reach plateau pressures around 25 cm H2O compared with a more conventional approach in which normocapnia was achieved with plateau pressures already below 35 cm H2O.

We recently demonstrated that muscle protein synthesis was stimulated to a similar extent in young and elderly subjects during a 3-h amino acid infusion. We sought to determine if a more practical bolus oral ingestion would also produce a similar response in young (34 +/- 4 yr) and elderly (67 +/- 2 yr) individuals. Arteriovenous blood samples and muscle biopsies were obtained during a primed (2.0 micromol/kg) constant infusion (0.05 micromol.kg(-<em>1</em>).min(-<em>1</em>)) of L-[ring-2H5]phenylalanine. Muscle protein kinetics and mixed muscle fractional synthetic rate (FSR) were calculated before and after the bolus ingestion of <em>1</em>5 g of essential amino acids (EAA) in young (n = 6) and elderly (n = 7) subjects. After EAA ingestion, the rate of increase in femoral artery phenylalanine concentration was slower in elderly subjects but remained elevated for a longer period. EAA ingestion increased FSR in both age groups by approximately 0.04%/h (P < 0.05). However, muscle intracellular (IC) phenylalanine concentration remained significantly higher in elderly subjects at the completion of the study (young: <em>1</em><em>1</em>5.6 +/- 5.4 nmol/<em>ml</em>; elderly: <em>1</em>50.2 +/- <em>1</em>9.4 nmol/<em>ml</em>). Correction for the free phenylalanine retained in the muscle IC pool resulted in similar net phenylalanine uptake values in the young and elderly. EAA ingestion increased plasma insulin levels in young (6.<em>1</em> +/- <em>1</em>.2 to 2<em>1</em>.3 +/- 3.<em>1</em> microIU/<em>ml</em>) but not in elderly subjects (3.0 +/- 0.6 to 4.3 +/- 0.4 microIU/<em>ml</em>). Despite differences in the time course of plasma phenylalanine kinetics and a greater residual IC phenylalanine concentration, amino acid supplementation acutely stimulated muscle protein synthesis in both young and elderly individuals.

Corticosteroids are the preeminent antiinflammatory agents although the molecular mechanisms that impart their efficacy have not been defined. The endothelium plays a critical role in inflammation by directing circulating leukocytes into extravascular tissues by expressing adhesive molecules for leukocytes [e.g., endothelial-leukocyte adhesion molecule <em>1</em> (ELAM-<em>1</em>) and intercellular adhesion molecule <em>1</em> (ICAM-<em>1</em>)]. We therefore determined whether corticosteroids suppress inflammation by inhibiting endothelial expression of adhesion molecules for neutrophils (polymorphonuclear leukocytes). Preincubation of endothelial cells with endotoxin [lipopolysaccharide (LPS), <em>1</em> microgram/<em>ml</em>] led to a 4-fold increase in subsequent adherence of polymorphonuclear leukocytes (P < 0.000<em>1</em>, n = <em>1</em>0) to endothelial cells, an increase that was markedly attenuated when endothelial cells were treated with dexamethasone (IC50 < <em>1</em> nM, P < 0.000<em>1</em>, n = 6 or 7) during preincubation with LPS. Moreover, the steroid receptor agonist cortisol (<em>1</em>0 microM), but not its inactive metabolite tetrahydrocortisol (<em>1</em>0 microM), diminished LPS-induced endothelial cell adhesiveness. Further evidence that the action of dexamethasone was mediated through ligation of corticosteroid receptors [human glucocorticoid receptors (hGRs)] was provided by experiments utilizing the steroid antagonist RU-486. RU-486 (<em>1</em>0 microM), which prevents translocation of ligated hGR to the nucleus by inhibiting dissociation of hGR from heat shock protein 90, completely aborted the effect of dexamethasone on adhesiveness of endothelial cells (P < 0.0005, n = 3). Treatment of endothelial cells with LPS (<em>1</em> microgram/<em>ml</em>) stimulated transcription of ELAM-<em>1</em>, as shown by Northern blot analysis, and expression of membrane-associated ELAM-<em>1</em> and ICAM-<em>1</em>, as shown by quantitative immunofluorescence (both P < 0.00<em>1</em>, n = 9). Dexamethasone markedly inhibited LPS-stimulated accumulation of mRNA for ELAM-<em>1</em> and expression of ELAM-<em>1</em> and ICAM-<em>1</em> (IC50 < <em>1</em>0 nM, both P < 0.00<em>1</em>, n = 4-9); inhibition of expression by dexamethasone was reversed by RU-486 (both P < 0.005, n = 4-6). As in the adhesion studies, cortisol but not tetrahydrocortisol inhibited expression of ELAM-<em>1</em> and ICAM-<em>1</em> (both P < 0.005, n = 3 or 4). In contrast, sodium salicylate (<em>1</em> mM) inhibited neither adhesion nor expression of these adhesion molecules. These studies suggest that antagonism by dexamethasone of endotoxin-induced inflammation is a specific instance of the general biological principle that the glucocorticoid receptor is a hormone-dependent regulator of transcription.

Responses to highly active antiretroviral therapy (HAART) in correctional settings and their sustained benefit in prisoners after release are currently not known. To examine the human immunodeficiency virus type <em>1</em> (HIV-<em>1</em>) RNA level (VL) and CD4 lymphocyte response to HAART during incarceration and upon reentry to the correctional system, we conducted a retrospective cohort study of longitudinally linked demographic, pharmacy, and laboratory data from the Connecticut prison system. During incarceration, the mean CD4 lymphocyte count increased by 74 lymphocytes/ mu L, and the mean VL decreased by 0.93 log<em>1</em>0 copies/<em>mL</em> (P<.000<em>1</em>). Fifty-nine percent of the subjects achieved a VL of <400 copies/<em>mL</em> at the end of each incarceration period. For the 27% of subjects who were reincarcerated, the mean CD4 lymphocyte count decreased by 80 lymphocytes/ mu L, and the mean VL increased by <em>1</em>.<em>1</em>4 log<em>1</em>0 (P<.000<em>1</em>). Although HAART use resulted in impressive VL and CD4 lymphocyte outcomes during the period of incarceration, recidivism to prison was high and was associated with a poor outcome. More effective community-release programs are needed for incarcerated patients with HIV disease.

BACKGROUND

The continuing, randomised, multinational, phase IIB POWER <em>1</em> and 2 studies aim to evaluate efficacy and safety of darunavir in combination with low-dose ritonavir in treatment-experienced HIV-<em>1</em>-infected patients. We did a pooled subgroup analysis to update results at week 48 for patients receiving the recommended dose of darunavir-ritonavir compared with those receiving other protease inhibitors (PIs).

METHODS

After 24-week dose-finding phases and primary efficacy analyses, patients randomised to receive darunavir-ritonavir were given 600/<em>1</em>00 mg twice daily, and patients receiving control PIs continued on assigned treatment into the longer-term, open-label phase; all patients continued on optimised background regimen. We assessed patients who had reached week 48 or discontinued earlier at the time of analysis; for the darunavir-ritonavir group, only patients who received 600/<em>1</em>00 mg twice daily from baseline were included. Analyses were intention-to-treat. The POWER 2 study (TMC<em>1</em><em>1</em>4-C202) is registered with ClinicalTrials.gov (NCT0007<em>1</em>097).

RESULTS

At week 48, 67 of <em>1</em><em>1</em>0 (6<em>1</em>%) darunavir-ritonavir patients compared with <em>1</em>8 of <em>1</em>20 (<em>1</em>5%) of control PI patients had viral load reductions of <em>1</em> log<em>1</em>0 copies per mL or greater from baseline (primary endpoint; difference in response rates 46%, 95% CI 35%-57%, p<0.000<em>1</em>). Based on a logistic regression model including stratification factors (baseline number of primary PI mutations, use of enfuvirtide, baseline viral load) and study as covariates, the difference in response was 50% (odds ratio <em>1</em><em>1</em>.72, 95% CI 5.75-23.89). In the darunavir-ritonavir group, rates of adverse events were mostly lower than or similar to those in the control group when corrected for treatment exposure. No unexpected safety concerns were identified.

CONCLUSIONS

Efficacy responses with darunavir-ritonavir 600/<em>1</em>00 mg twice daily plus optimised background regimen were greater than those with control PI and were sustained to at least week 48, with favourable safety and tolerability in treatment-experienced patients. This regimen could expand the treatment options available for such patients.

At least 24 different serotypes were detected in populations of Borrelia hermsii that originated from a single organism. These serotypes were identified by staining with specific fluoresceinated antisera prepared against cloned populations of living organisms of each type. In the order of decreasing frequency, the <em>1</em>0 types more often encountered were 7, which was clearly dominant, and 2, <em>1</em>7, 24, <em>1</em>3, 2, <em>1</em>, 2<em>1</em>, <em>1</em><em>1</em>, and <em>1</em>2. Each of the 24 types were shown to change to 7 or more other serotypes. Spirochetemia in mice was persistent, and relapses occurred when the concentration of organisms was sufficient for detection by visual means. After mice were inoculated with a single organism, peak spirochetemia usually occurred on day 4, after which clearance of organisms occurred, and an apparently pure population was replaced by a mixed population consisting of as many as seven variants. These types persisted for 2-3 d before being replaced by other types. Conversions occurred constantly and were independent of relapses. The rate of conversion in mice treated with cyclophosphamide to delay antibody production was comparable to that of controls. Spontaneous conversion was clearly demonstrated in tubes of fortified Kelly's medium inoculated with a single organism of type 7 or 2<em>1</em>. <em>1</em><em>1</em> different variants appeared in eight cultures of type 2<em>1</em> by the time growth had reached 4 X <em>1</em>0(6)-<em>1</em>0(7) organisms/<em>ml</em>. The rate of spontaneous change was estimated to be or approximately <em>1</em>0(-4)-<em>1</em>0(-3) per cell per generation.

BACKGROUND

Patients with systolic heart failure (HF) who develop secondary pulmonary hypertension (PH) have reduced exercise capacity and increased mortality compared with HF patients without PH. We tested the hypothesis that sildenafil, an effective therapy for pulmonary arterial hypertension, would lower pulmonary vascular resistance and improve exercise capacity in patients with HF complicated by PH.

RESULTS

Thirty-four patients with symptomatic HF and PH were randomized to <em>1</em>2 weeks of treatment with sildenafil (25 to 75 mg orally 3 times daily) or placebo. Patients underwent cardiopulmonary exercise testing before and after treatment. The change in peak VO2 from baseline, the primary end point, was greater in the sildenafil group (<em>1</em>.8+/-0.7 <em>mL</em> x kg(-<em>1</em>) x min(-<em>1</em>)) than in the placebo group (-0.27 <em>mL</em> x kg(-<em>1</em>) x min(-<em>1</em>); P=0.02). Sildenafil reduced pulmonary vascular resistance and increased cardiac output with exercise (P<0.05 versus placebo for both) without altering pulmonary capillary wedge or mean arterial pressure, heart rate, or systemic vascular resistance. The ability of sildenafil treatment to augment peak VO2 correlated directly with baseline resting pulmonary vascular resistance (r=0.74, P=0.002) and indirectly with baseline resting right ventricular ejection fraction (r=-0.64, P=0.0<em>1</em>). Sildenafil treatment also was associated with improvement in 6-minute walk distance (29 m versus placebo; P=0.047) and Minnesota Living With Heart Failure score (-<em>1</em>4 versus placebo; P=0.0<em>1</em>). Subjects in the sildenafil group experienced fewer hospitalizations for HF and a higher incidence of headache than those in the placebo group without incurring excess serious adverse events.

CONCLUSIONS

Phosphodiesterase 5 inhibition with sildenafil improves exercise capacity and quality of life in patients with systolic HF with secondary PH.

BACKGROUND

The goal of this study was to characterize viral loads and factors affecting viral clearance in persons with severe influenza.

METHODS

This was a <em>1</em>-year prospective, observational study involving consecutive adults hospitalized with influenza. Nasal and throat swabs were collected at presentation, then daily until <em>1</em> week after symptom onset. Real-time reverse-transcriptase polymerase chain reaction to determine viral RNA concentration and virus isolation were performed. Viral RNA concentration was analyzed using multiple linear or logistic regressions or mixed-effect models.

RESULTS

One hundred forty-seven inpatients with influenza A (H3N2) infection were studied (mean age+/-standard deviation, 72+/-<em>1</em>6 years). Viral RNA concentration at presentation positively correlated with symptom scores and was significantly higher than that among time-matched outpatients (control subjects). Patients with major comorbidities had high viral RNA concentration even when presenting>2 days after symptom onset (mean+/-standard deviation, 5.06+/-<em>1</em>.85 vs 3.62+/-2.<em>1</em>3 log<em>1</em>0 copies/mL; P=.005; beta, +0.86 [95% confidence interval, +0.03 to +<em>1</em>.68]). Viral RNA concentration demonstrated a nonlinear decrease with time; 26% of oseltamivir-treated and 57% of untreated patients had RNA detected at <em>1</em> week after symptom onset. Oseltamivir started on or before symptom day 4 was independently associated with an accelerated decrease in viral RNA concentration (mean beta [standard error], -<em>1</em>.<em>1</em>9 [0.43] and -0.68 [0.33] log<em>1</em>0 copies/mL for patients treated on day <em>1</em> and days 2-3, respectively; P<.05) and viral RNA clearance at <em>1</em> week (odds ratio, 0.<em>1</em>0 [95% confidence interval, 0.03-0.35] and 0.30 [0.<em>1</em>0-0.90] for patients treated on day <em>1</em>-2 and day 3-4, respectively). Conversely, major comorbidities and systemic corticosteroid use for asthma or chronic obstructive pulmonary disease exacerbations were associated with slower viral clearance. Viral RNA clearance was associated with a shorter hospital stay (7.0 vs <em>1</em>3.5 days; P=.00<em>1</em>).

CONCLUSIONS

Patients hospitalized with severe influenza have more active and prolonged viral replication. Weakened host defenses slow viral clearance, whereas antivirals started within the first 4 days of illness enhance viral clearance.

OBJECTIVE

The primary objectives of this trial were to define the maximum tolerated dose (MTD) and to characterize the toxicities and pharmacokinetics of depsipeptide (FR90<em>1</em>228) given on a day-<em>1</em> and day-5 schedule every 2<em>1</em> days. A secondary objective of the trial was to seek evidence of antineoplastic activity.

METHODS

Patients with advanced or refractory neoplasms received depsipeptide by a 4-h i.v. infusion on days <em>1</em> and 5 of a 2<em>1</em>-day cycle. On the basis of preclinical data suggesting that depsipeptide may have significant cardiac toxicity, patients were treated while receiving continuous cardiac monitoring and were followed with serial cardiac enzyme determinations, electrocardiograms (ECGs), and nuclear ventriculograms (MUGA scans). The starting dose of the trial was <em>1</em> mg/m(2), and dose escalations proceeded through a total of eight dose levels to a maximum of 24.9 mg/m(2). Toxicities were graded using the National Cancer Institute common toxicity criteria, and pharmacokinetics were determined using a liquid chromatography/tandem mass spectrometry method.

RESULTS

Patients (37) received a total of 88 cycles of treatment on study (range: one to eight cycles). Dose-limiting toxicity (DLT) was observed, and the MTD exceeded at a dose of 24.9 mg/m(2). The DLTs included grade-3 fatigue (3 patients), grade-3 nausea and vomiting (<em>1</em> patient), grade-4 thrombocytopenia (2 patients), and grade-4 cardiac arrhythmia (<em>1</em> patient, atrial fibrillation). The MTD was defined at the seventh dose level (<em>1</em>7.8 mg/m(2)). Reversible ST/T changes and mild reversible dysrhythmias were observed on the post-treatment ECG. There were no clinically significant changes in left ventricular ejection fraction. One patient achieved a partial response. The plasma disposition of depsipeptide was well described by a first-order, two-compartment model. The mean volume of distribution, clearance, t(<em>1</em>/2alpha) and t(<em>1</em>/2beta) at a dose of <em>1</em>7.8 mg/m(2) was: 8.6 liters/m(2), <em>1</em><em>1</em>.6 liters/h/m(2), 0.42 h, and 8.<em>1</em> h, respectively. The mean maximum plasma concentration at the MTD was 472.6 ng/ml (range: 249-577.8 ng/ml). Biological assays showed that the serum levels achieved could cause the characteristic cell cycle effects of this agent when serum was added to PC3 cells in culture, as well as increased histone acetylation in patient-derived peripheral blood mononuclear cells.

CONCLUSIONS

The MTD of depsipeptide given on a day-<em>1</em> and -5 schedule every 2<em>1</em> days is <em>1</em>7.8 mg/m(2). The DLTs are fatigue, nausea, vomiting, and transient thrombocytopenia and neutropenia. Whereas cardiac toxicity was anticipated based on preclinical data, there was no evidence of myocardial damage. However, reversible ECG changes with ST/T wave flattening were regularly observed. Biologically active serum concentrations were achieved, and <em>1</em> patient obtained a partial response. The recommended Phase II dose is <em>1</em>7.8 mg/m(2) administered on day <em>1</em> and 5 of a 2<em>1</em>-day cycle.

OBJECTIVE

The efficacy and safety of hepatic resection (HR) to treat patients with Barcelona Clinic Liver Cancer (BCLC) stage B and C hepatocellular carcinoma (HCC) was retrospectively assessed.

BACKGROUND

Although guidelines from the European Association for the Study of Liver Disease and the American Association for the Study of Liver Disease do not recommend HR for treating BCLC stage B/C HCC, several Asian and European studies have come to the opposite conclusions.

METHODS

A consecutive sample of <em>1</em>259 patients with BCLC stage B/C HCC who underwent HR (n = 908) or transarterial chemoembolization (TACE, n = 35<em>1</em>) were included. Moreover, propensity score-matched patients were analyzed to adjust for any baseline differences. In parallel with this retrospective clinical study, the MEDLINE database was searched for studies evaluating the efficacy and safety of HR for BCLC stage B/C HCC.

RESULTS

Among our patient sample, the 90-day mortality rate in the HR group was 3.<em>1</em>%. HR provided a survival benefit over TACE at <em>1</em>, 3, and 5 years (88% vs 8<em>1</em>%, 62% vs 33%, and 39% vs <em>1</em>6%, respectively; all P < 0.00<em>1</em>). Propensity scoring and subgroup analyses based on tumor size, tumor number, presence or absence of macrovascular invasion, and portal hypertension (PHT) also showed that HR was associated with better long-term survival than TACE. All 36 studies identified in our literature search reported that HR is associated with good long-term survival and low morbidity. Multivariate analyses revealed that alpha-fetoprotein more than or equal to 400 ng/<em>mL</em>, diabetes mellitus, macrovascular invasion, and PHT are independent predictors of poor prognosis in patients with BCLC stage B/C HCC.

CONCLUSIONS

Our clinical and literature analyses suggest that in patients with HCC with preserved liver function, the presence of large, solitary tumors, multinodular tumors, macrovascular invasion, or PHT are not contraindications for HR.

A significant need exists for long-term degradable biomaterials which can slowly and predictably transfer a load-bearing burden to developing biological tissue. In this study Bombyx mori silk fibroin yarns were incubated in <em>1</em>mg/<em>ml</em> Protease XIV at 37 degrees C to create an in vitro model system of proteolytic degradation. Samples were harvested at designated time points up to <em>1</em>2 weeks and (<em>1</em>) prepared for scanning electron microscopy (SEM), (2) lyophilized and weighed, (3) mechanical properties determined using a servohydraulic Instron 85<em>1</em><em>1</em>, (4) dissolved and run on a SDS-PAGE gel, and (5) characterized with Fourier transform infrared spectroscopy. Control samples were incubated in phosphate-buffered saline. Fibroin was shown to proteolytically degrade with predictable rates of change in fibroin diameter, failure strength, cycles to failure, and mass. SEM indicated increasing fragmentation of individual fibroin filaments from protease-digested samples with time of exposure to the enzyme; particulate debris was present within 7 days of incubation. Gel electrophoresis indicated a decreasing amount of the silk 25 kDa light chain and a shift in the molecular weight of the heavy chain with increasing incubation time in protease. Results support that silk is a mechanically robust biomaterial with predictable long-term degradation characteristics.

OBJECTIVE

The antigen, falciparum malaria protein <em>1</em> (FMP<em>1</em>), represents the 42-kDa C-terminal fragment of merozoite surface protein-<em>1</em> (MSP-<em>1</em>) of the 3D7 clone of P. falciparum. Formulated with AS02 (a proprietary Adjuvant System), it constitutes the FMP<em>1</em>/AS02 candidate malaria vaccine. We evaluated this vaccine's safety, immunogenicity, and efficacy in African children.

METHODS

A randomised, double-blind, Phase IIb, comparator-controlled trial.The trial was conducted in <em>1</em>3 field stations of one mile radii within Kombewa Division, Nyanza Province, Western Kenya, an area of holoendemic transmission of P. falciparum. We enrolled 400 children aged <em>1</em>2-47 months in general good health.Children were randomised in a <em>1</em>ratio<em>1</em> fashion to receive either FMP<em>1</em>/AS02 (50 microg) or Rabipur(R) rabies vaccine. Vaccinations were administered on a 0, <em>1</em>, and 2 month schedule. The primary study endpoint was time to first clinical episode of P. falciparum malaria (temperature>>/=37.5 degrees C with asexual parasitaemia of>>/=50,000 parasites/microL of blood) occurring between <em>1</em>4 days and six months after a third dose. Case detection was both active and passive. Safety and immunogenicity were evaluated for eight months after first immunisations; vaccine efficacy (VE) was measured over a six-month period following third vaccinations.

RESULTS

374 of 400 children received all three doses and completed six months of follow-up. FMP<em>1</em>/AS02 had a good safety profile and was well-tolerated but more reactogenic than the comparator. Geometric mean anti-MSP-<em>1</em>(42) antibody concentrations increased from<em>1</em>.3 microg/mL to 27.3 microg/mL in the FMP<em>1</em>/AS02 recipients, but were unchanged in controls. 97 children in the FMP<em>1</em>/AS02 group and 98 controls had a primary endpoint episode. Overall VE was 5.<em>1</em>% (95% CI: -26% to +28%; p-value = 0.7).

CONCLUSIONS

FMP<em>1</em>/AS02 is not a promising candidate for further development as a monovalent malaria vaccine. Future MSP-<em>1</em>(42) vaccine development should focus on other formulations and antigen constructs.

BACKGROUND

Clinicaltrials.gov NCT00223990.

Increased angiogenesis has recently been recognized in active multiple myeloma (MM). Since vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) are two key mediators of angiogenesis, we characterized the production of VEGF, b-FGF and interleukin-6 (IL-6) (a MM growth and survival factor) in MM cell lines and Epstein-Barr virus (EBV) transformed B cell lines from MM patients, patient MM cells, as well as bone marrow stromal cells (BMSCs) from normal healthy donors and MM patients. We detected secretion of VEGF, but no bFGF and IL-6, in MM cell lines (MM.<em>1</em>S, RPMI 8226 and U266); EBV transformed B cell lines from MM patients (IM-9, HS-Sultan and ARH77); MM cell lines resistant to doxorubicin (RPMI-DOX40), mitoxantrone (RPMI-MR20), melphalan (RPMI-LR5) and dexamethasone (MM.<em>1</em>R); and patient MM cells (MM<em>1</em> and MM2). BMSCs from MM patients and normal donors secreted VEGF, b-FGF and IL-6. Importantly, when MM cells were adhered to BMSCs, there was a significant increase in VEGF (<em>1</em>.5- to 3.<em>1</em>-fold) and IL-6 (<em>1</em>.9- to 56-fold) secretion. In contrast, the bFGF decreased in co-cultures of BMSCs and MM cells. Paraformaldehyde fixation of BMSCs or MM cells prior to adhesion revealed that VEGF was produced both from BMSCs and MM cells, though it may come primarily from BMSCs in some cultures. IL-6 was produced exclusively in BMSCs, rather than MM cells. Moreover, when MM cells were placed in Transwell insert chambers to allow their juxtaposition to BMSCs without cell to cell contact, induction of VEGF and IL-6 secretion persisted, suggesting the importance of humoral factors. Addition of exogenous IL-6 (<em>1</em>0 ng/<em>ml</em>) increased VEGF secretion by BMSCs. Conversely, VEGF (<em>1</em>00 ng/<em>ml</em>) significantly increased IL-6 secretion by BMSCs. Moreover, anti-human VEGF (<em>1</em> microg/<em>ml</em>) and anti-human IL-6 (<em>1</em>0 microg/<em>ml</em>) neutralizing antibodies reduced IL-6 and VEGF secretion, respectively, in cultures of BMSCs alone and co-cultures of BMSCs and MM cells. Finally, thalidomide (<em>1</em>00 microM) and its immunomodulatory analog IMiD<em>1</em>-CC4047 (<em>1</em> microM) decreased the upregulation of IL-6 and VEGF secretion in cultures of BMSCs, MM cells and co-cultures of BMSCs with MM cells. These data demonstrate the importance of stromal-MM cell interactions in regulating VEGF and IL-6 secretion, and suggest additional mechanisms whereby thalidomide and IMiD<em>1</em>-CC4047 act against MM cells in the BM millieu.

High-intensity interval training (HIT) induces skeletal muscle metabolic and performance adaptations that resemble traditional endurance training despite a low total exercise volume. Most HIT studies have employed 'all out', variable-load exercise interventions (e.g. repeated Wingate tests) that may not be safe, practical and/or well tolerated by certain individuals. Our purpose was to determine the performance, metabolic and molecular adaptations to a more practical model of low-volume HIT. Seven men (2<em>1</em> + or - 0.4 years, V(O2peak) = 46 + or - 2 <em>ml</em> kg(-<em>1</em>) min(-<em>1</em>)) performed six training sessions over 2 weeks. Each session consisted of 8-<em>1</em>2 x 60 s intervals at approximately <em>1</em>00% of peak power output elicited during a ramp V(O2) peak test (355 + or - <em>1</em>0 W) separated by 75 s of recovery. Training increased exercise capacity, as assessed by significant improvements on both 50 kJ and 750 kJ cycling time trials (P < 0.05 for both). Skeletal muscle (vastus lateralis) biopsy samples obtained before and after training revealed increased maximal activity of citrate synthase (CS) and cytochrome c oxidase (COX) as well as total protein content of CS, COX subunits II and IV, and the mitochondrial transcription factor A (Tfam) (P < 0.05 for all). Nuclear abundance of peroxisome proliferator-activated receptor gamma co-activator <em>1</em>alpha (PGC-<em>1</em>alpha) was approximately 25% higher after training (P < 0.05), but total PGC-<em>1</em>alpha protein content remained unchanged. Total SIRT<em>1</em> content, a proposed activator of PGC-<em>1</em>alpha and mitochondrial biogenesis, was increased by approximately 56% following training (P < 0.05). Training also increased resting muscle glycogen and total GLUT4 protein content (both P < 0.05). This study demonstrates that a practical model of low volume HIT is a potent stimulus for increasing skeletal muscle mitochondrial capacity and improving exercise performance. The results also suggest that increases in SIRT<em>1</em>, nuclear PGC-<em>1</em>alpha, and Tfam may be involved in coordinating mitochondrial adaptations in response to HIT in human skeletal muscle.

Tumor necrosis factor-alpha (TNF-alpha) has a key role in skeletal disease in which it promotes reduced bone formation by mature osteoblasts and increased osteoclastic resorption. Here we show that TNF inhibits differentiation of osteoblasts from precursor cells. TNF-alpha treatment of fetal calvaria precursor cells, which spontaneously differentiate to the osteoblast phenotype over 2<em>1</em> days, inhibited differentiation as shown by reduced formation of multilayered, mineralizing nodules and decreased secretion of the skeletal-specific matrix protein osteocalcin. The effect of TNF was dose dependent with an IC50 of 0.6 ng/<em>ml</em>, indicating a high sensitivity of these precursor cells. Addition of TNF-alpha from days 2-2<em>1</em>, 2-<em>1</em>4, 7-<em>1</em>4, and 7-<em>1</em>0 inhibited nodule formation but addition of TNF after day <em>1</em>4 had no effect. Partial inhibition of differentiation was observed with addition of TNF on only days 7-8, suggesting that TNF could act during a critical period of phenotype selection. Growth of cells on collagen-coated plates did not prevent TNF inhibition of differentiation, suggesting that inhibition of collagen deposition into matrix by proliferating cells could not, alone, explain the effect of TNF. Northern analysis revealed that TNF inhibited the expression of insulin-like growth factor I (IGF-I). TNF had no effect on expression of the osteogenic bone morphogenic proteins (BMPs-2, -4, and -6), or skeletal LIM protein (LMP-<em>1</em>), as determined by semiquantitative RT-PCR. Addition of IGF-I or BMP-6 to fetal calvaria precursor cell cultures enhanced differentiation but could not overcome TNF inhibition, suggesting that TNF acted downstream of these proteins in the differentiation pathway. The clonal osteoblastic cell line, MC3T3-E<em>1</em>-<em>1</em>4, which acquires the osteoblast phenotype spontaneously in postconfluent culture, was also studied. TNF inhibited differentiation of MC3T3-E<em>1</em>-<em>1</em>4 cells as shown by failure of mineralized matrix formation in the presence of calcium and phosphate. TNF was not cytotoxic to either cell type as shown by continued attachment and metabolism in culture, trypan blue exclusion, and Alamar Blue cytotoxicity assay. These results demonstrate that TNF-alpha is a potent inhibitor of osteoblast differentiation and suggest that TNF acts distal to IGF-I, BMPs, and LMP-<em>1</em> in the progression toward the osteoblast phenotype.

BACKGROUND

Hepatocellular carcinoma (HCC) is prevalent worldwide and improvements in timely and effective diagnosis are needed. We assessed whether measurement of Dickkopf-<em>1</em> (DKK<em>1</em>) in serum could improve diagnostic accuracy for HCC.

METHODS

We analysed data for patients with HCC, chronic hepatitis B virus (HBV) infection, liver cirrhosis, and healthy controls, recruited from two Chinese centres between December, 2008, and July, 2009. A validation cohort matched for age and sex was recruited from another centre in China between July, 20<em>1</em>0, and June, 20<em>1</em><em>1</em>. DKK<em>1</em> was measured in serum by ELISA by independent researchers who had no access to patients' clinical information. We used receiver operating characteristics (ROC) to calculate diagnostic accuracy.

RESULTS

We assessed serum DKK<em>1</em> in 83<em>1</em> participants: 424 with HCC, 98 with chronic HBV infection, 96 with cirrhosis, and 2<em>1</em>3 healthy controls. The validation cohort comprised 453 participants: 209 with HCC, 73 with chronic HBV infection, 72 with cirrhosis, and 99 healthy controls. Levels of DKK<em>1</em> in serum were significantly higher in patients with HCC than in all controls. ROC curves showed the optimum diagnostic cutoff was 2·<em>1</em>53 ng/mL (area under curve [AUC] 0·848 [95% CI 0·820-0·875], sensitivity 69·<em>1</em>%, and specificity 90·6% in the test cohort; 0·862 [0·825-0·899], 7<em>1</em>·3%, and 87·2% in the validation cohort). Similar results were noted for early-stage HCC (0·865 [0·835-0·895], 70·9%, and 90·5% in the test cohort; 0·896 [0·846-0·947], 73·8%, and 87·2% in the validation cohort). Furthermore, DKK<em>1</em> maintained diagnostic accuracy for patients with HCC who were α-fetoprotein (AFP) negative (0·84<em>1</em> [0·80<em>1</em>-0·882], 70·4%, and 90·0% in the test cohort; 0·869 [0·8<em>1</em>5-0·923], 66·7%, and 87·2% in the validation cohort), including for patients with early-stage HCC (0·870 [0·829-0·9<em>1</em><em>1</em>], 73·<em>1</em>%, and 90·0% in the test cohort; 0·893 [0·804-0·983], 72·2%, and 87·2% in the validation cohort), compared with all controls. Raised concentrations of DKK<em>1</em> in serum could differentiate HCC from chronic HBV infection and cirrhosis (0·834 [0·798-0·87<em>1</em>], 69·<em>1</em>%, and 84·7% in the test cohort; 0·873 [0·832-0·9<em>1</em>3], 7<em>1</em>·3%, and 90·6% in the validation cohort). Moreover, measurement of DKK<em>1</em> and AFP together improved diagnostic accuracy for HCC versus all controls compared with either test alone (0·889 [0·866-0·9<em>1</em>3], 73·3%, and 93·4% in the test cohort; 0·888 [0·856-0·920], 78·5%, and 87·2% in the validation cohort).

CONCLUSIONS

DKK<em>1</em> could complement measurement of AFP in the diagnosis of HCC and improve identification of patients with AFP-negative HCC and distinguish HCC from non-malignant chronic liver diseases.

BACKGROUND

National Key Basic Research Programme of China, National Key Sci-Tech Special Projects of Infectious Diseases, National Natural Science Foundation of China, Research Fund for the Doctoral Programme of Higher Education of China.

Endothelial dysfunction associated with atherosclerosis has been attributed to alterations in the L-arginine-nitric oxide (NO)-cGMP pathway or to an excess of endothelin-<em>1</em> (ET-<em>1</em>). The 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) have been shown to ameliorate endothelial function. However, the physiological basis of this observation is largely unknown. We investigated the effects of Atorvastatin and Simvastatin on the pre-proET-<em>1</em> mRNA expression and ET-<em>1</em> synthesis and on the endothelial NO synthase (eNOS) transcript and protein levels in bovine aortic endothelial cells. These agents inhibited pre-proET-<em>1</em> mRNA expression in a concentration- and time-dependent fashion (60-70% maximum inhibition) and reduced immunoreactive ET-<em>1</em> levels (25-50%). This inhibitory effect was maintained in the presence of oxidized LDL (<em>1</em>-50 microg/<em>ml</em>). No significant modification of pre-proET-<em>1</em> mRNA half-life was observed. In addition, mevalonate, but not cholesterol, reversed the statin-mediated decrease of pre-proET-<em>1</em> mRNA levels. eNOS mRNA expression was reduced by oxidized LDL in a dose-dependent fashion (up to 57% inhibition), whereas native LDL had no effect. Statins were able to prevent the inhibitory action exerted by oxidized LDL on eNOS mRNA and protein levels. Hence, these drugs might influence vascular tone by modulating the expression of endothelial vasoactive factors.

BACKGROUND

Lithium is a widely used and effective treatment for mood disorders. There has been concern about its safety but no adequate synthesis of the evidence for adverse effects. We aimed to undertake a clinically informative, systematic toxicity profile of lithium.

METHODS

We undertook a systematic review and meta-analysis of randomised controlled trials and observational studies. We searched electronic databases, specialist journals, reference lists, textbooks, and conference abstracts. We used a hierarchy of evidence which considered randomised controlled trials, cohort studies, case-control studies, and case reports that included patients with mood disorders given lithium. Outcome measures were renal, thyroid, and parathyroid function; weight change; skin disorders; hair disorders; and teratogenicity.

RESULTS

We screened 5988 abstracts for eligibility and included 385 studies in the analysis. On average, glomerular filtration rate was reduced by -6·22 <em>mL</em>/min (95% CI -<em>1</em>4·65 to 2·20, p=0·<em>1</em>48) and urinary concentrating ability by <em>1</em>5% of normal maximum (weighted mean difference -<em>1</em>58·43 mOsm/kg, 95% CI -229·78 to -87·07, p<0·000<em>1</em>). Lithium might increase risk of renal failure, but the absolute risk was small (<em>1</em>8 of 3369 [0·5%] patients received renal replacement therapy). The prevalence of clinical hypothyroidism was increased in patients taking lithium compared with those given placebo (odds ratio [OR] 5·78, 95% CI 2·00-<em>1</em>6·67; p=0·00<em>1</em>), and thyroid stimulating hormone was increased on average by 4·00 iU/<em>mL</em> (95% CI 3·90-4·<em>1</em>0, p<0·000<em>1</em>). Lithium treatment was associated with increased blood calcium (+0·09 mmol/L, 95% CI 0·02-0·<em>1</em>7, p=0·009), and parathyroid hormone (+7·32 pg/<em>mL</em>, 3·42-<em>1</em><em>1</em>·23, p<0·000<em>1</em>). Patients receiving lithium gained more weight than did those receiving placebo (OR <em>1</em>·89, <em>1</em>·27-2·82, p=0·002), but not those receiving olanzapine (0·32, 0·2<em>1</em>-0·49, p<0·000<em>1</em>). We recorded no significant increased risk of congenital malformations, alopecia, or skin disorders.

CONCLUSIONS

Lithium is associated with increased risk of reduced urinary concentrating ability, hypothyroidism, hyperparathyroidism, and weight gain. There is little evidence for a clinically significant reduction in renal function in most patients, and the risk of end-stage renal failure is low. The risk of congenital malformations is uncertain; the balance of risks should be considered before lithium is withdrawn during pregnancy. Because of the consistent finding of a high prevalence of hyperparathyroidism, calcium concentrations should be checked before and during treatment.

BACKGROUND

National Institute for Health Research Programme Grant for Applied Research.

Although there is consensus that instrumental conditioning depends on the encoding of action-outcome associations, it is not known where this learning process is localized in the brain. Recent research suggests that the posterior dorsomedial striatum (pDMS) may be the critical locus of these associations. We tested this hypothesis by examining the contribution of N-methyl-D-aspartate receptors (NMDARs) in the pDMS to action-outcome learning. Rats with bilateral cannulae in the pDMS were first trained to perform two actions (left and right lever presses), for sucrose solution. After the pre-training phase, they were given an infusion of the NMDA antagonist 2-amino-5-phosphonopentanoic acid (APV, <em>1</em> mg/<em>mL</em>) or artificial cerebral spinal fluid (ACSF) before a 30-min session in which pressing one lever delivered food pellets and pressing the other delivered fruit punch. Learning during this session was tested the next day by sating the animals on either the pellets or fruit punch before assessing their performance on the two levers in extinction. The ACSF group selectively reduced responding on the lever that, in training, had earned the now devalued outcome, whereas the APV group did not. Experiment 2 replicated the effect of APV during the critical training session but found no effect of APV given after acquisition and before test. Furthermore, Experiment 3 showed that the effect of APV on instrumental learning was restricted to the pDMS; infusion into the dorsolateral striatum did not prevent learning. These experiments provide the first direct evidence that, in instrumental conditioning, NMDARs in the dorsomedial striatum are involved in encoding action-outcome associations.

Neural stem/progenitor cells (NSCs) are capable of self-renewal and differentiation into all types of neural lineage under different biochemical and topographical cues. In this study, we cultured rat hippocampus-derived adult NSCs (rNSCs) on laminin-coated electrospun Polyethersulfone (PES) fiber meshes with average fiber diameters of 283+/-45 nm, 749+/-<em>1</em>53 nm and <em>1</em>452+/-3<em>1</em>2 nm; and demonstrated that fiber diameter of PES mesh significantly influences rNSC differentiation and proliferation. Under the differentiation condition (in the presence of <em>1</em> microM retinoic acid and <em>1</em>% fetal bovine serum), rNSCs showed a 40% increase in oligodendrocyte differentiation on 283-nm fibers and 20% increase in neuronal differentiation on 749-nm fibers, in comparison to tissue culture polystyrene surface. SEM imaging revealed that cells stretched multi-directionally to follow underlying 283-nm fibers, but extended along a single fiber axis on larger fibers. When cultured on fiber meshes in serum free medium in the presence of 20 ng/<em>mL</em> of FGF-2, rNSCs showed lower proliferation and more rounded morphology compared to that cultured on laminin-coated 2D surface. As the fiber diameter decreased, higher degree of proliferation and cell spreading and lower degree of cell aggregation were observed. This collective evidence indicates fiber topography can play a vital role in regulating differentiation and proliferation of rNSCs in culture.

BACKGROUND

Mutations in the type II receptor for bone morphogenetic protein (BMPR-II), a receptor member of the transforming growth factor-beta (TGF-beta) superfamily, underlie many cases of familial and sporadic primary pulmonary hypertension (PPH). We postulated that pulmonary artery smooth muscle cells (PASMCs) from patients with PPH might demonstrate abnormal growth responses to TGF-beta superfamily members.

RESULTS

For studies of (3)H-thymidine incorporation or cell proliferation, PASMCs (passages 4 to 8) were derived from main pulmonary arteries. In control cells, 24-hour incubation with TGF-beta(<em>1</em>) (<em>1</em>0 ng/<em>mL</em>) or bone morphogenetic protein (BMP)-2, -4, and -7 (<em>1</em>00 ng/<em>mL</em>) inhibited basal and serum-stimulated (3)H-thymidine incorporation, and TGF-beta(<em>1</em>) and BMPs inhibited the proliferation of serum-stimulated PASMCs. In contrast, TGF-beta(<em>1</em>) stimulated (3)H-thymidine incorporation (200%; P<0.00<em>1</em>) and cell proliferation in PASMCs from PPH but not from patients with secondary pulmonary hypertension. In addition, BMPs failed to suppress DNA synthesis and proliferation in PASMCs from PPH patients. Reverse transcription-polymerase chain reaction of PASMC mRNA detected transcripts for type I (TGF-betaRI, Alk-<em>1</em>, ActRI, and BMPRIB) and type II (TGF-betaRII, BMPR-II, ActRII, ActRIIB) receptors. Receptor binding and cross-linking studies with (<em>1</em>25)I-TGF-beta(<em>1</em>) confirmed that the abnormal responses in PPH cells were not due to differences in TGF-beta receptor binding. Mutation analysis of PASMC DNA failed to detect mutations in TGF-betaRII and Alk-<em>1</em> but confirmed the presence of a mutation in BMPR-II in <em>1</em> of 5 PPH isolates.

CONCLUSIONS

We conclude that PASMCs from patients with PPH exhibit abnormal growth responses to TGF-beta(<em>1</em>) and BMPs and that altered integration of TGF-beta superfamily growth signals may contribute to the pathogenesis of PPH.

OBJECTIVE

We hypothesized that preconditioning (PC) with stromal-derived factor <em>1</em> alpha (SDF-<em>1</em>) significantly enhances cell survival, proliferation, and engraftment of bone marrow-derived mesenchymal stem cells (MSCs) via SDF-<em>1</em>/CXCR4 signaling.

RESULTS

MSCs were cultured and then incubated in medium for 60 min without SDF-<em>1</em> (control group) or with SDF-<em>1</em> 0.05 microg/mL (SDF-<em>1</em> group) or CXCR4-selective antagonist, AMD 3<em>1</em>00 (AMD) (5 microg/mL, AMD group) or SDF-<em>1</em> and AMD (0.05 microg/mL, 5 microg/mL, respectively, SDF-<em>1</em>+AMD group). MSCs were treated for 60 min, washed in normal medium, and then exposed to H2O2 (<em>1</em>00 micromol/L) for 60 min to determine the effects of various treatments on cell injury, viability, and proliferation. For in vivo studies, rats were grouped (n = 6) after left anterior descending coronary artery ligation to receive 20 microL Dulbecco's modified Eagle's medium without cells or with 5 x <em>1</em>0(5) non-preconditioned MSCs (control group), SDF-<em>1</em> preconditioned MSCs (SDF-<em>1</em> group), AMD (AMD group), or MSCs treated with SDF-<em>1</em> plus AMD (SDF-<em>1</em>+AMD group). Heart function, infarct size, fibrosis, and MSC proliferation and differentiation in infarcted myocardium were determined after 4 weeks. In vitro data showed a marked increase in cell viability and proliferation following SDF-<em>1</em> PC. In vivo data in preconditioned group showed a robust cell proliferation, reduction in infarct size and fibrosis, and significant improvement in cardiac function. Effects of SDF-<em>1</em> PC were abrogated by CXCR4 antagonist.

CONCLUSIONS

We conclude that PC with the chemokine SDF-<em>1</em> suppresses MSCs apoptosis, enhances their survival, engraftment, and vascular density, and improves myocardial function via SDF/CXCR4 signaling. Chemokine PC is a novel approach for enhancing stem cell survival and regeneration of infarcted myocardium.

BACKGROUND

Experimental data suggest that manipulation of alveolar fluid clearance with beta-agonists can accelerate the resolution of alveolar edema and improve survival.

OBJECTIVE

To determine if a sustained infusion of intravenous salbutamol (albuterol) would accelerate the resolution of alveolar edema in adult patients with acute lung injury (ALI) or acute respiratory distress syndrome (ARDS).

METHODS

This was a single-center, double-blind, randomized controlled trial. Patients with ALI/ARDS were randomized to treatment with intravenous salbutamol (<em>1</em>5 microg kg(-<em>1</em>) h(-<em>1</em>)) or placebo for 7 d. The primary endpoint was extravascular lung water measured by thermodilution (PiCCO) at Day 7.

RESULTS

Sixty-six patients were screened; of these, 40 met the inclusion criteria and were enrolled during 200<em>1</em>-2003. Patients in the salbutamol group had significantly lower lung water at Day 7 than the placebo group (9.2 +/- 6 vs. <em>1</em>3.2 +/- 3 ml kg(-<em>1</em>); 95% confidence interval difference, 0.2-8.3 ml kg(-<em>1</em>); p = 0.038). Plateau airway pressure was lower at Day 7 in the salbutamol group (23.9 +/- 3.8 cm H2O) versus placebo (29.5 +/- 7.2 cm H2O; p = 0.049). There was a trend toward lower Murray lung injury score at Day 7 in the salbutamol group (<em>1</em>.7 +/- 0.9) versus placebo (2.0 +/- 0.6; p = 0.2). Patients in the salbutamol group had a higher incidence of supraventricular arrhythmias (26 vs. <em>1</em>0%; p = 0.2).

CONCLUSIONS

Although further research is required to confirm the efficacy and safety of intravenous salbutamol in ALI/ARDS, this trial provides the first proof of principle that, in humans with ALI/ARDS, sustained treatment with intravenous beta-agonists reduces extravascular lung water.

A factor which inhibits osteoclast-like cell formation was found in the conditioned medium of human embryonic lung fibroblasts, IMR-90. The factor, termed osteoclastogenesis inhibitory factor, OCIF, was purified to homogeneity. OCIF is a heparin-binding basic glycoprotein and has been isolated as a monomer with an apparent molecular weight (Mr) of 60,000 and a homodimer with a Mr of <em>1</em>20,000. The N-terminus of OCIF is blocked and the determination of internal amino acid sequences revealed that OCIF has no homology to known proteins. OCIF inhibited in a dose-dependent manner osteoclastogenesis elicited through three distinct signaling pathways stimulated by <em>1</em> alpha,25-dihydroxy vitamin D3, parathyroid hormone, and interleukin-<em>1</em><em>1</em>, respectively, in a dose range of <em>1</em> to 40 ng/<em>ml</em> (IC50 = 4 to 6 ng/<em>ml</em>). OCIF neither inhibits bone resorption by mature osteoclasts nor exerts any other biological activities. These data strongly suggest that OCIF is a novel cytokine which specifically inhibits osteoclastogenesis.