OBJECTIVE

The authors tested whether the anti-interleukin (IL)-<em>1</em>7A monoclonal antibody secukinumab was safe and effective for the treatment of active Crohn's disease.

METHODS

In a double-blind, randomised, placebo-controlled proof-of-concept study, 59 patients with moderate to severe Crohn's disease (Crohn's Disease Activity Index (CDAI) ≥220 to ≤450) were assigned in a 2:<em>1</em> ratio to 2×<em>1</em>0 mg/kg intravenous secukinumab or placebo. The primary end point, addressed by bayesian statistics augmented with historical placebo information, was the probability that secukinumab reduces the CDAI by ≥50 points more than placebo at week 6. Ancillary analyses explored associations of 35 candidate genetic polymorphisms and faecal calprotectin response.

RESULTS

59 patients (39 secukinumab, 20 placebo, mean baseline CDAI 307 and 30<em>1</em>, respectively) were recruited. <em>1</em>8/59 (3<em>1</em>%) patients discontinued prematurely (<em>1</em>2/39 (3<em>1</em>%) secukinumab, 6/20 (30%) placebo), <em>1</em>0/59 (<em>1</em>7%) due to insufficient therapeutic effect (8/39 (2<em>1</em>%) secukinumab, 2/20 (<em>1</em>0%) placebo). Fourteen serious adverse events occurred in <em>1</em>0 patients (seven secukinumab, three placebo); 20 infections, including four local fungal infections, were seen on secukinumab versus none on placebo. Primary end point analysis estimated <0.<em>1</em>% probability (CDAI (SD) =33.9 (<em>1</em>9.7), 95% credible interval -4.9 to 72.9) that secukinumab reduces CDAI by ≥50 points more than placebo. Secondary area under the curve analysis (weeks 4-<em>1</em>0) showed a significant difference (mean ΔCDAI=49; 95% CI (2 to 96), p=0.043) in favour of placebo. Post hoc subgroup analysis showed that unfavourable responses on secukinumab were driven by patients with elevated inflammatory markers (CRP≥<em>1</em>0 mg/l and/or faecal calprotectin≥200 ng/ml; mean ΔCDAI=62; 95% CI (-<em>1</em> to <em>1</em>25), p=0.054 in favour of placebo). Absence of the minor allele of tumour necrosis factor-like ligand <em>1</em>A was strongly associated with lack of response measured by baseline-adjusted changes in calprotectin at week 6 (p=0.00035 Bonferroni-corrected).

CONCLUSIONS

Blockade of IL-<em>1</em>7A was ineffective and higher rates of adverse events were noted compared with placebo.

BACKGROUND

This trial was registered at ClinicalTrial.gov with the number NCT0<em>1</em>00928<em>1</em>.

Soil bacterium DNA was isolated by minor modifications of previously described methods. After purification on hydroxyapatite and precipitation with cetylpyridinium bromide, the DNA was sheared in a French press to give fragments with an average molecular mass of 420,000 daltons. After repeated hydroxyapatite purification and precipitation with cetylpyridinium bromide, high-pressure liquid chromatography analysis showed the presence of 2.<em>1</em>% RNA or less, whereas 5-methylcytosine made up 2.9% of the total deoxycytidine content. No other unusual bases could be detected. The hyperchromicity was 3<em>1</em> to 36%, and the melting curve in <em>1</em> X SSC (0.<em>1</em>5 M NaCl plus 0.0<em>1</em>5 M sodium citrate) corresponded to 58.3 mol% G+C. High-pressure liquid chromatography analysis of two DNA samples gave 58.6 and 60.8 mol% G+C. The heterogeneity of the DNA was determined by reassociation of single-stranded DNA, measured spectrophotometrically. Owing to the high complexity of the DNA, the reassociation had to be carried out in 6 X SSC with 30% dimethyl sulfoxide added. Cuvettes with a <em>1</em>-mm light path were used, and the A275 was read. DNA concentrations as high as 950 micrograms <em>ml</em>-<em>1</em> could be used, and the reassociation rate of Escherichia coli DNA was increased about 4.3-fold compared with standard conditions. C0t<em>1</em>/2 values were determined relative to that for E. coli DNA, whereas calf thymus DNA was reassociated for comparison. Our results show that the major part of DNA isolated from the bacterial fraction of soil is very heterogeneous, with a C0t<em>1</em>/2 about 4,600, corresponding to about 4,000 completely different genomes of standard soil bacteria.(ABSTRACT TRUNCATED AT 250 WORDS)

BACKGROUND

The current staging system for chronic kidney disease is based primarily on estimated glomerular filtration rate (eGFR) with lower eGFR associated with higher risk of adverse outcomes. Although proteinuria is also associated with adverse outcomes, it is not used to refine risk estimates of adverse events in this current system.

OBJECTIVE

To determine the association between reduced GFR, proteinuria, and adverse clinical outcomes.

METHODS

Community-based cohort study with participants identified from a province-wide laboratory registry that includes eGFR and proteinuria measurements from Alberta, Canada, between 2002 and 2007. There were 920 985 adults who had at least <em>1</em> outpatient serum creatinine measurement and who did not require renal replacement treatment at baseline. Proteinuria was assessed by urine dipstick or albumin-creatinine ratio (ACR).

METHODS

All-cause mortality, myocardial infarction, and progression to kidney failure.

RESULTS

The majority of individuals (89.<em>1</em>%) had an eGFR of 60 mL/min/<em>1</em>.73 m(2) or greater. Over median follow-up of 35 months (range, 0-59 months), 27 959 participants (3.0%) died. The fully adjusted rate of all-cause mortality was higher in study participants with lower eGFRs or heavier proteinuria. Adjusted mortality rates were more than 2-fold higher among individuals with heavy proteinuria measured by urine dipstick and eGFR of 60 mL/min/<em>1</em>.73 m(2) or greater, as compared with those with eGFR of 45 to 59.9 mL/min/<em>1</em>.73 m(2) and normal protein excretion (rate, 7.2 [95% CI, 6.6-7.8] vs 2.9 [95% CI, 2.7-3.0] per <em>1</em>000 person-years, respectively; rate ratio, 2.5 [95% CI, 2.3-2.7]). Similar results were observed when proteinuria was measured by ACR (<em>1</em>5.9 [95% CI, <em>1</em>4.0-<em>1</em>8.<em>1</em>] and 7.0 [95% CI, 6.4-7.6] per <em>1</em>000 person-years for heavy and absent proteinuria, respectively; rate ratio, 2.3 [95% CI, 2.0-2.6]) and for the outcomes of hospitalization with acute myocardial infarction, end-stage renal disease, and doubling of serum creatinine level.

CONCLUSIONS

The risks of mortality, myocardial infarction, and progression to kidney failure associated with a given level of eGFR are independently increased in patients with higher levels of proteinuria.

Increasing evidence suggests that amyloid peptides associated with a variety of degenerative diseases induce neurotoxicity in their intermediate oligomeric state, rather than as monomers or fibrils. To test this hypothesis and investigate the possible involvement of Ca2+ signaling disruptions in amyloid-induced cytotoxicity, we made homogeneous preparations of disease-related amyloids (Abeta, prion, islet amyloid polypeptide, polyglutamine, and lysozyme) in various aggregation states and tested their actions on fluo-3-loaded SH-SY5Y cells. Application of oligomeric forms of all amyloids tested (0.6-6 microg <em>ml</em>-<em>1</em>) rapidly (approximately 5 s) elevated intracellular Ca2+, whereas equivalent amounts of monomers and fibrils did not. Ca2+ signals evoked by Abeta42 oligomers persisted after depletion of intracellular Ca2+ stores, and small signals remained in Ca2+-free medium, indicating contributions from both extracellular and intracellular Ca2+ sources. The increased membrane permeability to Ca2+ cannot be attributed to activation of endogenous Ca2+ channels, because responses were unaffected by the potent Ca2+-channel blocker cobalt (20 microm). Instead, observations that Abeta42 and other oligomers caused rapid cellular leakage of anionic fluorescent dyes point to a generalized increase in membrane permeability. The resulting unregulated flux of ions and molecules may provide a common mechanism for oligomer-mediated toxicity in many amyloidogenic diseases, with dysregulation of Ca2+ ions playing a crucial role because of their strong trans-membrane concentration gradient and involvement in cell dysfunction and death.

BACKGROUND

Cyclophosphamide induction regimens for antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis are effective in 70 to 90% of patients, but they are associated with high rates of death and adverse events. Treatment with rituximab has led to remission rates of 80 to 90% among patients with refractory ANCA-associated vasculitis and may be safer than cyclophosphamide regimens.

METHODS

We compared rituximab with cyclophosphamide as induction therapy in ANCA-associated vasculitis. We randomly assigned, in a 3:<em>1</em> ratio, 44 patients with newly diagnosed ANCA-associated vasculitis and renal involvement to a standard glucocorticoid regimen plus either rituximab at a dose of 375 mg per square meter of body-surface area per week for 4 weeks, with two intravenous cyclophosphamide pulses (33 patients, the rituximab group), or intravenous cyclophosphamide for 3 to 6 months followed by azathioprine (<em>1</em><em>1</em> patients, the control group). Primary end points were sustained remission rates at <em>1</em>2 months and severe adverse events.

RESULTS

The median age was 68 years, and the glomerular filtration rate (GFR) was <em>1</em>8 ml per minute per <em>1</em>.73 m(2) of body-surface area. A total of 25 patients in the rituximab group (76%) and 9 patients in the control group (82%) had a sustained remission (P=0.68). Severe adverse events occurred in <em>1</em>4 patients in the rituximab group (42%) and 4 patients in the control group (36%) (P=0.77). Six of the 33 patients in the rituximab group (<em>1</em>8%) and 2 of the <em>1</em><em>1</em> patients in the control group (<em>1</em>8%) died (P=<em>1</em>.00). The median increase in the GFR between 0 and <em>1</em>2 months was <em>1</em>9 ml per minute in the rituximab group and <em>1</em>5 ml per minute in the control group (P=0.<em>1</em>4).

CONCLUSIONS

A rituximab-based regimen was not superior to standard intravenous cyclophosphamide for severe ANCA-associated vasculitis. Sustained-remission rates were high in both groups, and the rituximab-based regimen was not associated with reductions in early severe adverse events. (Funded by Cambridge University Hospitals National Health Service Foundation Trust and F. Hoffmann-La Roche; Current Controlled Trials number, ISRCTN285288<em>1</em>3.)

BACKGROUND

Laparoscopic surgery as an alternative to open surgery in patients with rectal cancer has not yet been shown to be oncologically safe. The aim in the COlorectal cancer Laparoscopic or Open Resection (COLOR II) trial was to compare laparoscopic and open surgery in patients with rectal cancer.

METHODS

A non-inferiority phase 3 trial was undertaken at 30 centres and hospitals in eight countries. Patients (aged ≥<em>1</em>8 years) with rectal cancer within <em>1</em>5 cm from the anal verge without evidence of distant metastases were randomly assigned to either laparoscopic or open surgery in a 2:<em>1</em> ratio, stratified by centre, location of tumour, and preoperative radiotherapy. The study was not masked. Secondary (short-term) outcomes-including operative findings, complications, mortality, and results at pathological examination-are reported here. Analysis was by modified intention to treat, excluding those patients with post-randomisation exclusion criteria and for whom data were not available. This study is registered with ClinicalTrials.gov, number NCT0029779<em>1</em>.

RESULTS

The study was undertaken between Jan 20, 2004, and May 4, 20<em>1</em>0. <em>1</em><em>1</em>03 patients were randomly assigned to the laparoscopic (n=739) and open surgery groups (n=364), and <em>1</em>044 were eligible for analyses (699 and 345, respectively). Patients in the laparoscopic surgery group lost less blood than did those in the open surgery group (median 200 mL [IQR <em>1</em>00-400] vs 400 mL [200-700], p<0·000<em>1</em>); however, laparoscopic procedures took longer (240 min [<em>1</em>84-300] vs <em>1</em>88 min [<em>1</em>50-240]; p<0·000<em>1</em>). In the laparoscopic surgery group, bowel function returned sooner (2·0 days [<em>1</em>·0-3·0] vs 3·0 days [2·0-4·0]; p<0·000<em>1</em>) and hospital stay was shorter (8·0 days [6·0-<em>1</em>3·0] vs 9·0 days [7·0-<em>1</em>4·0]; p=0·036). Macroscopically, completeness of the resection was not different between groups (589 [88%] of 666 vs 303 [92%] of 33<em>1</em>; p=0·250). Positive circumferential resection margin (<2 mm) was noted in 56 (<em>1</em>0%) of 588 patients in the laparoscopic surgery group and 30 (<em>1</em>0%) of 300 in the open surgery group (p=0·850). Median tumour distance to distal resection margin did not differ significantly between the groups (3·0 cm [IQR 2·0-4·8] vs 3·0 cm [<em>1</em>·8-5·0], respectively; p=0·676). In the laparoscopic and open surgery groups, morbidity (278 [40%] of 697 vs <em>1</em>28 [37%] of 345, respectively; p=0·424) and mortality (eight [<em>1</em>%] of 699 vs six [2%] of 345, respectively; p=0·409) within 28 days after surgery were similar.

CONCLUSIONS

In selected patients with rectal cancer treated by skilled surgeons, laparoscopic surgery resulted in similar safety, resection margins, and completeness of resection to that of open surgery, and recovery was improved after laparoscopic surgery. Results for the primary endpoint-locoregional recurrence-are expected by the end of 20<em>1</em>3.

BACKGROUND

Ethicon Endo-Surgery Europe, Swedish Cancer Foundation, West Gothia Region, Sahlgrenska University Hospital.

BACKGROUND

Alirocumab, a monoclonal antibody that inhibits proprotein convertase subtilisin-kexin type 9 (PCSK9), has been shown to reduce low-density lipoprotein (LDL) cholesterol levels in patients who are receiving statin therapy. Larger and longer-term studies are needed to establish safety and efficacy.

METHODS

We conducted a randomized trial involving 234<em>1</em> patients at high risk for cardiovascular events who had LDL cholesterol levels of 70 mg per deciliter (<em>1</em>.8 mmol per liter) or more and were receiving treatment with statins at the maximum tolerated dose (the highest dose associated with an acceptable side-effect profile), with or without other lipid-lowering therapy. Patients were rando<em>ml</em>y assigned in a 2:<em>1</em> ratio to receive alirocumab (<em>1</em>50 mg) or placebo as a <em>1</em>-<em>ml</em> subcutaneous injection every 2 weeks for 78 weeks. The primary efficacy end point was the percentage change in calculated LDL cholesterol level from baseline to week 24.

RESULTS

At week 24, the difference between the alirocumab and placebo groups in the mean percentage change from baseline in calculated LDL cholesterol level was -62 percentage points (P<0.00<em>1</em>); the treatment effect remained consistent over a period of 78 weeks. The alirocumab group, as compared with the placebo group, had higher rates of injection-site reactions (5.9% vs. 4.2%), myalgia (5.4% vs. 2.9%), neurocognitive events (<em>1</em>.2% vs. 0.5%), and ophthalmologic events (2.9% vs. <em>1</em>.9%). In a post hoc analysis, the rate of major adverse cardiovascular events (death from coronary heart disease, nonfatal myocardial infarction, fatal or nonfatal ischemic stroke, or unstable angina requiring hospitalization) was lower with alirocumab than with placebo (<em>1</em>.7% vs. 3.3%; hazard ratio, 0.52; 95% confidence interval, 0.3<em>1</em> to 0.90; nominal P=0.02).

CONCLUSIONS

Over a period of 78 weeks, alirocumab, when added to statin therapy at the maximum tolerated dose, significantly reduced LDL cholesterol levels. In a post hoc analysis, there was evidence of a reduction in the rate of cardiovascular events with alirocumab. (Funded by Sanofi and Regeneron Pharmaceuticals; ODYSSEY LONG TERM ClinicalTrials.gov number, NCT0<em>1</em>50783<em>1</em>.).

Residual viremia can be detected in most HIV-<em>1</em>-infected patients on antiretroviral therapy despite suppression of plasma RNA to <50 copies per <em>ml</em>, but the source and duration of this viremia is currently unknown. Therefore, we analyzed longitudinal plasma samples from 40 patients enrolled in the Abbott M97-720 trial at baseline (pretherapy) and weeks 60 to 384 by using an HIV-<em>1</em> RNA assay with single-copy sensitivity. All patients were on therapy (lopinavir/ritonavir, stavudine, and lamivudine) with plasma HIV RNA <50 copies per <em>ml</em> by week 96 of the study and thereafter. Single-copy assay results revealed that 77% of the patient samples had detectable low-level viremia >>/=<em>1</em> copy per <em>ml</em>), and all patients had at least one sample with detectable viremia. A nonlinear mixed effects model revealed a biphasic decline in plasma RNA levels occurring over weeks 60 to 384: an initial phase of decay with a half-life of 39 weeks and a subsequent phase with no perceptible decay. The level of pretherapy viremia extrapolated for each phase of decay was significantly correlated with total baseline viremia for each patient (R(2) = 0.27, P = 0.00<em>1</em> and R(2) = 0.<em>1</em>9, P < 0.005, respectively), supporting a biological link between the extent of overall baseline viral infection and the infection of long-lived reservoirs. These data suggest that low-level persistent viremia appears to arise from at least two cell compartments, one in which viral production decays over time and a second in which viral production remains stable for at least 7 years.

The scid mutation was backcrossed ten generations onto the NOD/Lt strain background, resulting in an immunodeficient stock (NOD/LtSz-scid/scid) with multiple defects in adaptive as well as nonadaptive immunologic function. NOD/LtSz-scid/scid mice lack functional lymphoid cells and show little or no serum Ig with age. Although NOD/(Lt-)+/+ mice develop T cell-mediated autoimmune, insulin-dependent diabetes mellitus, NOD/LtSz-scid/scid mice are both insulitis- and diabetes-free throughout life. However, because of a high incidence of thymic lymphomas, the mean lifespan of this congenic stock is only 8.5 mo under specific pathogen-free conditions. After i.v. injection of human CEM T-lymphoblastoid cells, splenic engraftment of these cells was fourfold greater in NOD/LtSz-scid/scid mice than in C.B<em>1</em>7/Sz-scid/scid mice. Although C.B-<em>1</em>7Sz-scid/scid mice exhibit robust NK cell activity, this activity is markedly reduced in both NOD/(Lt-)+/+ and NOD/LtSz-scid/scid mice. Presence of a functionally less mature macrophage population in NOD/LtSz-scid/scid vs C.B-<em>1</em>7Sz-scid/scid mice is indicated by persistence in the former of the NOD/Lt strain-specific defect in LPS-stimulated IL-<em>1</em> secretion by marrow-derived macrophages. Although C.B-<em>1</em>7Sz-scid/scid and C57BL/6Sz-scid/scid mice have elevated serum hemolytic complement activity compared with their respective +/+ controls, both NOD/(LtSz-)+/+ and NOD/LtSz-scid/scid mice lack this activity. Age-dependent increases in serum Ig levels >> <em>1</em> micrograms/<em>ml</em>) were observed in only 2 of 30 NOD/LtSz-scid/scid mice vs 2<em>1</em> of 29 C.B-<em>1</em>7/Sz-scid/scid animals. The multiple defects in innate and adaptive immunity unique to the NOD/LtSz-scid/scid mouse provide an excellent in vivo environment for reconstitution with human hematopoietic cells.

We have developed a new assay, ISET (isolation by size of epithelial tumor cells), which allows the counting and the immunomorphological and molecular characterization of circulating tumor cells in patients with carcinoma, using peripheral blood sample volumes as small as <em>1</em> <em>ml</em>. Using this assay, epithelial tumor cells can be isolated individually by filtration because of their larger size when compared to peripheral blood leukocytes. ISET parameters were defined using peripheral blood spiked with tumor cell lines (HepG2, Hep3B, MCF-7, HeLa, and LNCaP). ISET can detect a single, micropipetted tumor cell, added to <em>1</em> <em>ml</em> of blood. We also demonstrate that fluorescence in situ hybridization can be used to perform chromosomal analyses on tumor cells collected using ISET. Polymerase chain reaction-based genetic analyses can be applied to ISET-isolated cells, and, as an example, we demonstrate homozygous p53 deletion in single Hep3B cells after filtration and laser microdissection. Finally, we provide evidence for the in vivo feasibility of ISET in patients with hepatocellular carcinoma undergoing tumor resection. ISET, but not reverse transcriptase-polymerase chain reaction, allowed analysis of cell morphology, counting of tumor cells, and demonstration of tumor microemboli spread into peripheral blood during surgery. Overall, ISET constitutes a novel approach that should open new perpectives in molecular medicine.

BACKGROUND

In diabetic nephropathy, angiotensin-converting-enzyme (ACE) inhibitors have a greater effect than other antihypertensive drugs on proteinuria and the progressive decline in glomerular filtration rate (GFR). Whether this difference applies to progression of nondiabetic proteinuric nephropathies is not clear. The Ramipril Efficacy in Nephropathy study of chronic nondiabetic nephropathies aimed to address whether glomerular protein traffic influences renal-disease progression, and whether an ACE inhibitor was superior to conventional treatment, with the same blood-pressure control, in reducing proteinuria, limiting GFR decline, and preventing endstage renal disease.

METHODS

In this prospective double-blind trial, 352 patients were classified according to baseline proteinuria (stratum <em>1</em>: <em>1</em>-3 g/24 h; stratum 2:>> or = 3 g/24 h), and randomly assigned ramipril or placebo plus conventional antihypertensive therapy targeted at achieving diastolic blood pressure under 90 mm Hg. The primary endpoint was the rate of GFR decline. Analysis was by intention to treat.

RESULTS

At the second planned interim analysis, the difference in decline in GFR between the ramipril and placebo groups in stratum 2 was highly significant (p = 0.00<em>1</em>). The Independent Adjudicating Panel therefore decided to open the randomisation code and do the final analysis in this stratum (stratum <em>1</em> continued in the trial). Data (at least three GFR measurements including baseline) were available for 56 ramipril-assigned patients and 6<em>1</em> placebo-assigned patients. The decline in GFR per month was significantly lower in the ramipril group than the placebo group (0.53 [0.08] vs 0.88 [0.<em>1</em>3] mL/min, p = 0.03). Among the ramipril-assigned patients, percentage reduction in proteinuria was inversely correlated with decline in GFR (p = 0.035) and predicted the reduction in risk of doubling of baseline creatinine or endstage renal failure (<em>1</em>8 ramipril vs 40 placebo, p = 0.04). The risk of progression was still significantly reduced after adjustment for changes in systolic (p = 0.04) and diastolic (p = 0.04) blood pressure, but not after adjustment for changes in proteinuria. Blood-pressure control and the overall number of cardiovascular events were similar in the two treatment groups.

CONCLUSIONS

In chronic nephropathies with proteinuria of 3 g or more per 24 h, ramipril safely reduces proteinuria and the rate of GFR decline to an extent that seems to exceed the reduction expected for the degree of blood-pressure lowering.

We determined whether interindividual variation in hepatic insulin sensitivity could be attributed to variation in liver fat content (LFAT) independent of obesity. We recruited 30 healthy nondiabetic men whose LFAT (determined by proton spectroscopy); intraabdominal, sc, and total (determined by magnetic resonance imaging) fat; and insulin sensitivity of endogenous glucose rate of production (R(a)) and suppression of serum FFA [euglycemic insulin clamp combined with [3-(3)H]glucose (0-300 min); insulin infusion rate, 0.3 mU/kg.min, <em>1</em>20-300 min] were measured. The men were divided into groups of low (mean +/- SD, <em>1</em>.7 +/- 0.2%) and high (<em>1</em>0.5 +/- 2.0%) LFAT based on their median fat content. The low and high LFAT groups were comparable with respect to age (44 +/- 2 vs. 42 +/- 2 yr), body mass index (25 +/- <em>1</em> vs. 26 +/- <em>1</em> kg/m(2) ), waist to hip ratio (0.953 +/- 0.0<em>1</em>3 vs. 0.953 +/- 0.0<em>1</em>3), maximal oxygen uptake (35.6 +/- <em>1</em>.5 vs. 33.5 +/- <em>1</em>.5 <em>ml</em>/kg.min), and intraabdominal, sc, and total fat. The high compared with the low LFAT group had several features of insulin resistance, including fasting hyperinsulinemia (7.3 +/- 0.6 vs. 5.3 +/- 0.6 mU/liter; P < 0.02, high vs. low LFAT) hypertriglyceridemia (<em>1</em>.4 +/- 0.2 vs. 0.9 +/- 0.<em>1</em> mmol/liter; P < 0.02), a low high density lipoprotein (HDL) cholesterol concentration (<em>1</em>.4 +/- 0.<em>1</em> vs. <em>1</em>.6 +/- 0.<em>1</em> mmol/liter; P < 0.05), and a higher ambulatory 24-h systolic blood pressure (<em>1</em>30 +/- 3 vs. <em>1</em>22 +/- 3 mm Hg; P < 0.05). Basal glucose R(a) and serum FFA were comparable between the groups, whereas insulin suppression of glucose R(a) [5<em>1</em> +/- 8 vs. 20 +/- <em>1</em>2 mg/m(2).min during 240-300 min (P < 0.05) or -55 +/- 7 vs. -85 +/- <em>1</em>2% below basal (P < 0.05, high vs. low LFAT)] and of serum FFA (299 +/- 33 vs. 2<em>1</em>2 +/- <em>1</em>3 micromol/liter; 240-300 min; P < 0.02) were impaired in the high compared with the low LFAT group. Insulin stimulation of glucose Rd were comparable in the men with high LFAT (<em>1</em>4<em>1</em> +/- <em>1</em>2 mg/m(2).min) and those with low LFAT (<em>1</em>56 +/- <em>1</em>4 mg/m(2).min; P = NS). Fat accumulation in the liver is, independent of body mass index and intraabdominal and overall obesity, characterized by several features of insulin resistance in normal weight and moderately overweight subjects.

BACKGROUND

Initial results of the ChemoRadiotherapy for Oesophageal cancer followed by Surgery Study (CROSS) comparing neoadjuvant chemoradiotherapy plus surgery versus surgery alone in patients with squamous cell carcinoma and adenocarcinoma of the oesophagus or oesophagogastric junction showed a significant increase in 5-year overall survival in favour of the neoadjuvant chemoradiotherapy plus surgery group after a median of 45 months' follow-up. In this Article, we report the long-term results after a minimum follow-up of 5 years.

METHODS

Patients with clinically resectable, locally advanced cancer of the oesophagus or oesophagogastric junction (clinical stage T<em>1</em>N<em>1</em>M0 or T2-3N0-<em>1</em>M0, according to the TNM cancer staging system, sixth edition) were randomly assigned in a <em>1</em>:<em>1</em> ratio with permuted blocks of four or six to receive either weekly administration of five cycles of neoadjuvant chemoradiotherapy (intravenous carboplatin [AUC 2 mg/<em>mL</em> per min] and intravenous paclitaxel [50 mg/m(2) of body-surface area] for 23 days) with concurrent radiotherapy (4<em>1</em>·4 Gy, given in 23 fractions of <em>1</em>·8 Gy on 5 days per week) followed by surgery, or surgery alone. The primary endpoint was overall survival, analysed by intention-to-treat. No adverse event data were collected beyond those noted in the initial report of the trial. This trial is registered with the Netherlands Trial Register, number NTR487, and has been completed.

RESULTS

Between March 30, 2004, and Dec 2, 2008, 368 patients from eight participating centres (five academic centres and three large non-academic teaching hospitals) in the Netherlands were enrolled into this study and randomly assigned to the two treatment groups: <em>1</em>80 to surgery plus neoadjuvant chemoradiotherapy and <em>1</em>88 to surgery alone. Two patients in the neoadjuvant chemoradiotherapy group withdrew consent, so a total of 366 patients were analysed (<em>1</em>78 in the neoadjuvant chemoradiotherapy plus surgery group and <em>1</em>88 in the surgery alone group). Of <em>1</em>7<em>1</em> patients who received any neoadjuvant chemoradiotherapy in this group, <em>1</em>62 (95%) were able to complete the entire neoadjuvant chemoradiotherapy regimen. After a median follow-up for surviving patients of 84·<em>1</em> months (range 6<em>1</em>·<em>1</em>-<em>1</em><em>1</em>6·8, IQR 70·7-96·6), median overall survival was 48·6 months (95% CI 32·<em>1</em>-65·<em>1</em>) in the neoadjuvant chemoradiotherapy plus surgery group and 24·0 months (<em>1</em>4·2-33·7) in the surgery alone group (HR 0·68 [95% CI 0·53-0·88]; log-rank p=0·003). Median overall survival for patients with squamous cell carcinomas was 8<em>1</em>·6 months (95% CI 47·2-<em>1</em><em>1</em>6·0) in the neoadjuvant chemoradiotherapy plus surgery group and 2<em>1</em>·<em>1</em> months (<em>1</em>5·4-26·7) in the surgery alone group (HR 0·48 [95% CI 0·28-0·83]; log-rank p=0·008); for patients with adenocarcinomas, it was 43·2 months (24·9-6<em>1</em>·4) in the neoadjuvant chemoradiotherapy plus surgery group and 27·<em>1</em> months (<em>1</em>3·0-4<em>1</em>·2) in the surgery alone group (HR 0·73 [95% CI 0·55-0·98]; log-rank p=0·038).

CONCLUSIONS

Long-term follow-up confirms the overall survival benefits for neoadjuvant chemoradiotherapy when added to surgery in patients with resectable oesophageal or oesophagogastric junctional cancer. This improvement is clinically relevant for both squamous cell carcinoma and adenocarcinoma subtypes. Therefore, neoadjuvant chemoradiotherapy according to the CROSS trial followed by surgical resection should be regarded as a standard of care for patients with resectable locally advanced oesophageal or oesophagogastric junctional cancer.

BACKGROUND

Dutch Cancer Foundation (KWF Kankerbestrijding).

Recombinant mouse interleukin <em>1</em>0 (IL-<em>1</em>0) was exceedingly potent at suppressing the ability of mouse peritoneal macrophages (m phi) to release tumor necrosis factor alpha (TNF-alpha). The IC50 of IL-<em>1</em>0 for the suppression of TNF-alpha release induced by 0.5 microgram/<em>ml</em> lipopolysaccharide was 0.04 +/- 0.03 U/<em>ml</em>, with as little as <em>1</em> U/<em>ml</em> suppressing TNF-alpha production by a factor of 2<em>1</em>.4 +/- 2.5. At <em>1</em>0 U/<em>ml</em>, IL-<em>1</em>0 markedly suppressed m phi release of reactive oxygen intermediates (ROI) (IC50 3.7 +/- <em>1</em>.8 U/<em>ml</em>), but only weakly inhibited m phi release of reactive nitrogen intermediates (RNI). Since TNF-alpha is a T cell growth and differentiation factor, whereas ROI and RNI are known to inhibit lymphocyte function, it is possible that m phi exposed to low concentrations of IL-<em>1</em>0 suppress lymphocytes. m phi deactivated by higher concentrations of IL-<em>1</em>0 might be permissive for the growth of microbial pathogens and tumor cells, as TNF-alpha, ROI, and RNI are major antimicrobial and tumoricidal products of m phi. IL-<em>1</em>0's effects on m phi overlap with but are distinct from the effects of the two previously described cytokines that suppress the function of mouse m phi, transforming growth factor beta and macrophage deactivation factor. Based on results with neutralizing antibodies, all three m phi suppressor factors appear to act independently.

IL-<em>1</em>7 is a newly described, T cell-derived cytokine with ill-defined physiologic properties. As such, we examined the release of proinflammatory mediators by human macrophages in response to recombinant human (rh) IL-<em>1</em>7. IL-<em>1</em>beta and TNF-alpha expression and synthesis were up-regulated by rhIL-<em>1</em>7 in a dose (ED50 was 50 +/- 9 ng/<em>ml</em>)- and time-dependent fashion, with cytokine accumulation reaching a zenith after 9 h. Release of IL-6, PGE2, IL-<em>1</em>0, IL-<em>1</em>2, IL-<em>1</em>R antagonist, and stromelysin was also stimulated by rhIL-<em>1</em>7. IL-<em>1</em>beta and TNF-alpha mRNA expression levels were controlled by rhIL-<em>1</em>7 in a complex manner with an initial 30-min inhibitory phase, and then up-regulation beginning at <em>1</em> h and reaching a plateau at about 3 h. The latter expression pattern closely mirrored the nuclear accumulation of the transcription factor nuclear factor-kappaB. cAMP mimetics isobutyl-<em>1</em>-methylxanthine (IBMX), forskolin, PGE2, and cholera toxin reversed rhIL-<em>1</em>7-induced release of TNF-alpha, but had no consistent effect on induced IL-<em>1</em>beta synthesis. Induced release of TNF-alpha was also inhibited by serine/threonine protein kinase inhibitors KT-5720 (protein kinase A) and Calphostin C (protein kinase C), mitogen-activated protein kinase kinase inhibitor PD098059, and a nonspecific tyrosine kinase inhibitor, genistein. Calphostin C alone abrogated the rhIL-<em>1</em>7-induced release of IL-<em>1</em>beta. The antiinflammatory cytokines IL-4 (p < 0.0<em>1</em>) and IL-<em>1</em>0 (p < 0.02) completely reversed rhIL-<em>1</em>7-stimulated IL-<em>1</em>beta release, while IL-<em>1</em>3 and TGF-beta2 were partially effective (59 and 43% diminution, respectively). IL-<em>1</em>0 exerted a significant suppressive effect on IL-<em>1</em>7-induced TNF-alpha release (99%, p < 0.02), while the inhibitory effects of IL-4, IL-<em>1</em>3, and TGF-beta2 on TNF-alpha secretion were partial (48, <em>1</em>0, and 23%, respectively). The data suggest a pivotal role for IL-<em>1</em>7 in initiating and/or sustaining an inflammatory response.

By limiting dilution of WEHI <em>1</em>64 mouse fibrosarcoma cells we have isolated a cell line, WEHI <em>1</em>64 clone <em>1</em>3, which is extremely sensitive to cytotoxic factor (CF) derived from human monocytes. By using WEHI <em>1</em>64 clone <em>1</em>3 in a MTT tetrazolium cytotoxicity assay it was found that CF supernatants from activated monocytes had to be diluted <em>1</em>0(5)-<em>1</em>0(6) times to reach the dose which produced 50% dead cells (LD50). By comparing the LD50 of different target cells, WEHI <em>1</em>64 clone <em>1</em>3 cells were found to be approximately <em>1</em>0(3) times more sensitive for CF-induced cytotoxicity as compared to WEHI <em>1</em>64 parental cells and approximately <em>1</em>0(2) times more sensitive as compared to actinomycin D-treated L929 cells. Treatment of the WEHI <em>1</em>64 clone <em>1</em>3 cells with actinomycin D did not increase their sensitivity for CF-induced cytotoxicity. Recombinant tumor necrosis factor (rTNF) also mediated high cytotoxicity towards WEHI <em>1</em>64 clone <em>1</em>3 cells, with an LD50 of 2 X <em>1</em>0(-3) ng/<em>ml</em>. Neutralizing CF antiserum completely inhibited the toxic activity of rTNF. WEHI <em>1</em>64 clone <em>1</em>3 cells were highly sensitive to monocyte-mediated cytotoxicity in that <em>1</em>-2 monocytes were able to kill at least 5000 of these target cells. Neutralizing TNF antiserum completely inhibited monocyte-mediated cytotoxicity. These results indicate that the high level of cytotoxicity mediated by CF supernatants and monocytes on WEHI <em>1</em>64 clone <em>1</em>3 cells is due to TNF as the effector molecule.

When activated, NF-kappa B, a ubiquitous transcription factor, binds DNA as a heterodimeric complex composed of members of the Rel/NF-kappa B family of polypeptides. Because of its intimate involvement in host defense against disease, this transcription factor is an important target for therapeutic intervention. In the present report we demonstrate that curcumin (diferuloylmethane), a known anti-inflammatory and anticarcinogenic agent, is a potent inhibitor of NF-kappa B activation. Treatment of human myeloid <em>ML</em>-<em>1</em>a cells with tumor necrosis factor (TNF) rapidly activated NF-kappa B, which consists of p50 and p65 subunits, and this activation was inhibited by curcumin. AP-<em>1</em> binding factors were also found to be down-modulated by curcumin, whereas the Sp<em>1</em> binding factor was unaffected. Besides TNF, curcumin also blocked phorbol ester- and hydrogen peroxide-mediated activation of NF-kappa B. The TNF-dependent phosphorylation and degradation of I kappa B alpha was not observed in curcumin-treated cells; the translocation of p65 subunit to the nucleus was inhibited at the same time. The mechanism of action of curcumin was found to be different from that of protein tyrosine phosphatase inhibitors. Our results indicate that curcumin inhibits NF-kappa B activation pathway at a step before I kappa B alpha phosphorylation but after the convergence of various stimuli.

We describe highly sensitive, label-free, multiplexed electrical detection of cancer markers using silicon-nanowire field-effect devices in which distinct nanowires and surface receptors are incorporated into arrays. Protein markers were routinely detected at femtomolar concentrations with high selectivity, and simultaneous incorporation of control nanowires enabled discrimination against false positives. Nanowire arrays allowed highly selective and sensitive multiplexed detection of prostate specific antigen (PSA), PSA-alpha<em>1</em>-antichymotrypsin, carcinoembryonic antigen and mucin-<em>1</em>, including detection to at least 0.9 pg/<em>ml</em> in undiluted serum samples. In addition, nucleic acid receptors enabled real-time assays of the binding, activity and small-molecule inhibition of telomerase using unamplified extracts from as few as ten tumor cells. The capability for multiplexed real-time monitoring of protein markers and telomerase activity with high sensitivity and selectivity in clinically relevant samples opens up substantial possibilities for diagnosis and treatment of cancer and other complex diseases.

For over 25 years, the cytokine known as macrophage migration inhibitory factor (MIF) has been considered to be a product of activated T lymphocytes. We recently identified the murine homolog of human MIF as a protein secreted by the pituitary in response to endotoxin administration. In the course of these studies, we also detected MIF in acute sera obtained from endotoxin-treated, T cell-deficient (nude), and hypophysectomized mice, suggesting that still more cell types produce MIF. Here, we report that cells of the monocyte/macrophage lineage are an important source of MIF in vitro and in vivo. We observed high levels of both preformed MIF protein and MIF mRNA in resting, nonstimulated cells. In the murine macrophage cell line RAW 264.7, MIF secretion was induced by as little as <em>1</em>0 pg/<em>ml</em> of lipopolysaccharide (LPS), peaked at <em>1</em> ng/<em>ml</em>, and was undetectable at LPS concentrations>> <em>1</em> microgram/<em>ml</em>. A similar stimulation profile was observed in LPS-treated peritoneal macrophages; however, higher LPS concentrations were necessary to induce peak MIF production unless cells had been preincubated with interferon gamma (IFN-gamma). In RAW 264.7 macrophages, MIF secretion also was induced by tumor necrosis factor alpha (TNF-alpha) and IFN-gamma, but not by interleukins <em>1</em> beta or 6. Of note, MIF-stimulated macrophages were observed to secrete bioactive TNF-alpha. Although previously overlooked, the macrophage is both an important source and an important target of MIF in vivo. The activation of both central (pituitary) and peripheral (macrophage) sources of MIF production by inflammatory stimuli provides further evidence for the critical role of this cytokine in the systemic response to tissue invasion.

BACKGROUND

Trastuzumab (Herceptin), an anti-HER2/neu receptor monoclonal antibody that inhibits growth of ErbB2-overexpressing breast cancer, is used to treat such cancers. Development of resistance to trastuzumab, however, is common. We investigated whether insulin-like growth factor-I (IGF-I), which activates cell survival signals, interferes with the growth-inhibitory action of trastuzumab.

METHODS

MCF-7/HER2-18 and SKBR3 human breast cancer models were used to assess cell proliferation, colony formation in soft agar, and cell cycle parameters. Throughout, we used trastuzumab at a dose of 10 microg/mL and IGF-I at a dose of 40 ng/mL. All statistical tests were two-sided.

RESULTS

Trastuzumab inhibited the growth of MCF-7/HER2-18 cells, which overexpress HER2/neu receptors and express IGF-I receptors (IGF-IRs), only when IGF-IR signaling was minimized. For example, in 1% fetal bovine serum (FBS), trastuzumab reduced cell proliferation by 42% (P =.002); however, in 10% FBS or IGF-I, trastuzumab had no effect on proliferation. In SKBR3 cells, which overexpress HER2/neu receptor but express few IGF-IRs, trastuzumab reduced proliferation by 42% (P =.008) regardless of IGF-I concentration. When SKBR3 cells were genetically altered to overexpress IGF-IRs and cultured with IGF-I, trastuzumab had no effect on proliferation. However, the addition of IGF-binding protein-3, which decreased IGF-IR signaling, restored trastuzumab-induced growth inhibition.

CONCLUSIONS

In breast cancer cell models that overexpress HER2/neu, an increased level of IGF-IR signaling appears to interfere with the action of trastuzumab. Thus, strategies that target IGF-IR signaling may prevent or delay development of resistance to trastuzumab.

The movements of a strip of fundus from a rat stomach, suspended in a 5 <em>ml</em>. bath at 37 degrees , are recorded by a spring-loaded lever. The muscle is stretched for <em>1</em>5 to 30 sec. between contractions. <em>1</em> ng. 5-Hydroxytryptamine (5-HT), <em>1</em>0 ng. acetylcholine or 2 to <em>1</em>0 mug. of histamine all produce contractions, giving tracings about 4 cm. high. In the presence of hyoscine (<em>1</em>0(-7)) low concentrations of 5-HT can be assayed directly from mixtures of the three substances. Sympathomimetic amines interfere with the assay, but this can be overcome. The preparation is robust, potentially suitable for use with an automatic assay apparatus and <em>1</em>0 to <em>1</em>00 times more sensitive than the rat uterus.

In the adult human, mesenchymal stem cells (MSCs) resident in bone marrow retain the capacity to proliferate and differentiate along multiple connective tissue lineages, including cartilage. In this study, culture-expanded human MSCs (hMSCs) of 60 human donors were induced to express the morphology and gene products of chondrocytes. Chondrogenesis was induced by culturing hMSCs in micromass pellets in the presence of a defined medium that included <em>1</em>00 nM dexamethasone and <em>1</em>0 ng/<em>ml</em> transforming growth factor-beta(3) (TGF-beta(3)). Within <em>1</em>4 days, cells secreted an extracellular matrix incorporating type II collagen, aggrecan, and anionic proteoglycans. hMSCs could be further differentiated to the hypertrophic state by the addition of 50 nM thyroxine, the withdrawal of TGF-beta(3), and the reduction of dexamethasone concentration to <em>1</em> nM. Increased understanding of the induction of chondrogenic differentiation should lead to further progress in defining the mechanisms responsible for the generation of cartilaginous tissues, their maintenance, and their regeneration.

Mevinolin, a fungal metabolite, was isolated from cultures of Aspergillus terreus. The structure and absolute configuration of mevinolini and its open acid form, mevinolinic acid, were determined by a combination of physical techniques. Mevinolin was shown to be <em>1</em>,2,6,7,8,8a-hexahydro-beta, delta-dihydroxy-2,6-dimethyl-8-(2-methyl-<em>1</em>-oxobutoxy)-<em>1</em>-naphthalene-hepatanoic acid delta-lactone. Mevinolin in the hydroxy-acid form, mevinolinic acid, is a potent competitive inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A reductase [mevalonate: NADP+ oxidoreductase (CoA-acylating), EC <em>1</em>.<em>1</em>.<em>1</em>.34]; its Ki of 0.6 nM can be compared to <em>1</em>.4 nM for the hydroxy acid form of the previously described related inhibitor, <em>ML</em>-236B (compactin, 6-demethylmevinolin). In the rat, orally administered sodium mevinolinate was an active inhibitor of cholesterol synthesis in an acute assay (50% inhibitory dose = 46 microgram/kg). Furthermore, it was shown that mevinolin was an orally active cholesterol-lowering agent in the dog. Treatment of dogs for 3 weeks with mevinolin at 8 mg/kg per day resulted in a 29.3 +/- 2.5% lowering of plasma cholesterol.

BACKGROUND

Results of several epidemiologic and clinical studies have suggested that there is an excess risk of hypertension and diabetes mellitus in persons with suboptimal intake of vitamin D.

METHODS

We examined the association between serum levels of 25-hydroxyvitamin D (25[OH]D) and select cardiovascular disease risk factors in US adults. A secondary analysis was performed with data from the Third National Health and Nutrition Examination Survey, a national probability survey conducted by the National Center for Health Statistics between January <em>1</em>, <em>1</em>988, and December 3<em>1</em>, <em>1</em>994, with oversampling of persons 60 years and older, non-Hispanic black individuals, and Mexican American individuals.

RESULTS

There were 7<em>1</em>86 male and 7902 female adults 20 years and older with available data in the Third National Health and Nutrition Examination Survey. The mean 25(OH)D level in the overall sample was 30 ng/mL (75 nmol/L). The 25(OH)D levels were lower in women, elderly persons >>or=60 years), racial/ethnic minorities, and participants with obesity, hypertension, and diabetes mellitus. The adjusted prevalence of hypertension (odds ratio [OR], <em>1</em>.30), diabetes mellitus (OR, <em>1</em>.98), obesity (OR, 2.29), and high serum triglyceride levels (OR, <em>1</em>.47) was significantly higher in the first than in the fourth quartile of serum 25(OH)D levels (P<.00<em>1</em> for all).

CONCLUSIONS

Serum 25(OH)D levels are associated with important cardiovascular disease risk factors in US adults. Prospective studies to assess a direct benefit of cholecalciferol (vitamin D) supplementation on cardiovascular disease risk factors are warranted.