We report the first full-length infectious clone of the current epidemic, lineage I, strain of West Nile virus (WNV). The full-length cDNA was constructed from reverse transcription-PCR products of viral RNA from an isolate collected during the year 2000 outbreak in New York City. It was cloned into plasmid pBR322 under the control of a T7 promoter and stably amplified in Escherichia coli HB<em>1</em>0<em>1</em>. RNA transcribed from the full-length cDNA clone was highly infectious upon transfection into BHK-2<em>1</em> cells, resulting in progeny virus with titers of <em>1</em> x <em>1</em>0(9) to 5 x <em>1</em>0(9) PFU/<em>ml</em>. The cDNA clone was engineered to contain three silent nucleotide changes to create a StyI site (C to A and A to G at nucleotides [nt] 8859 and 8862, respectively) and to knock out an EcoRI site (A to G at nt 8880). These genetic markers were retained in the recovered progeny virus. Deletion of the 3'-terminal <em>1</em>99 nt of the cDNA transcript abolished the infectivity of the RNA. The plaque morphology, in vitro growth characteristics in mammalian and insect cells, and virulence in adult mice were indistinguishable for the parental and recombinant viruses. The stable infectious cDNA clone of the epidemic lineage I strain will provide a valuable experimental system to study the pathogenesis and replication of WNV.

We examined the cerebrospinal fluid (CF) taken on admission from 60 patients with infections caused by Neisseria meningitidis for presence of TNF-alpha, IL-<em>1</em>, and IL-6. TNF-alpha was detected in CF in 55 and <em>1</em>9% (p = 0.03), IL-<em>1</em> in 50 and <em>1</em>5% (p = 0.05), and IL-6 in 98 and <em>1</em>00% of patients with meningitis and septic shock/bacteremia, respectively. The median IL-6 concentration in CF in patients with meningitis was <em>1</em>54 ng/<em>ml</em>, and in patients with septic shock/bacteremia it was 42 ng/<em>ml</em> (p = 0.00<em>1</em>). The level of LPS in CF correlated with the level of TNF-alpha (r = 0.9<em>1</em>, p less than 0.00<em>1</em>), but not with the level of IL-<em>1</em> and IL-6. CF levels of TNF-alpha, IL-<em>1</em>, and IL-6 correlated with each other (r = 0.34-0.54, p less than 0.0<em>1</em>), with the protein concentration (r = 0.34-0.62, p less than 0.0<em>1</em>) and inversely with the CF/blood glucose ratio (r = -0.34 to -0.67, p less than 0.0<em>1</em>). Only the Il-6 level correlated with the leukocyte count (r = 0.37, p less than 0.0<em>1</em>). In rabbits TNF-alpha, IL-<em>1</em>, and IL-6 activities sequentially appeared in CF within 3 h of injection of meningococcal LPS or viable meningococci, whereas the main infiltration of granulocytes started after 4 h. TNF-alpha was detected in serum at concentrations less than <em>1</em>/<em>1</em>00 of those in CF after administration of LPS into the subarachnoid space, and conversely, TNF-alpha was detected in CF at concentrations <em>1</em>/<em>1</em>00 of those in serum after intravenous injection of LPS. The results demonstrate that TNF-alpha, IL-<em>1</em>, and IL-6 are sequentially produced in the initial phase of the local inflammatory response caused by meningococci, and that the subarachnoid space and systemic circulation are separate compartments with respect to production of TNF-alpha, IL-<em>1</em>, and IL-6.

BACKGROUND

Elevated numbers of blood eosinophils are a risk factor for asthma exacerbations. Reslizumab is a humanised anti-interleukin 5 monoclonal antibody that disrupts eosinophil maturation and promotes programmed cell death. We aimed to assess the efficacy and safety of reslizumab in patients with inadequately controlled, moderate-to-severe asthma.

METHODS

We did two duplicate, multicentre, double-blind, parallel-group, randomised, placebo-controlled phase 3 trials. Both trials enrolled patients with asthma aged <em>1</em>2-75 years (from <em>1</em>28 clinical research centres in study <em>1</em> and <em>1</em>04 centres in study 2) from Asia, Australia, North America, South America, South Africa, and Europe, whose asthma was inadequately controlled by medium-to-high doses of inhaled corticosteroid based therapy and who had blood eosinophils of 400 cells per <em>μL</em> or higher and one or more exacerbations in the previous year. Patients were randomly assigned (<em>1</em>:<em>1</em>) to receive either intravenous reslizumab (3·0 mg/kg) or placebo every 4 weeks for <em>1</em> year by computerised central randomisation. Patients and investigators were masked to treatment assignment during the study. Each patient received a specific volume of study drug (reslizumab or matching placebo) on the basis of the patient's body weight and randomly assigned treatment group. Additionally, the sponsor's clinical personnel involved in the study were masked to the study drug identity until the database was locked for analysis and the treatment assignment revealed. The primary outcome was the annual frequency of clinical asthma exacerbations and was analysed by intention to treat. We assessed safety outcomes in the patients who had received one or more dose of the drug. The trials have been completed and are registered with ClinicalTrials.gov, numbers NCT0<em>1</em>287039 (study <em>1</em>) and NCT0<em>1</em>285323 (study 2).

RESULTS

Study <em>1</em> was done between April <em>1</em>2, 20<em>1</em><em>1</em>, and March 3, 20<em>1</em>4 and study 2 between March 22, 20<em>1</em><em>1</em>, and April 9, 20<em>1</em>4. Of 2597 patients screened, 953 were randomly assigned to receive either reslizumab (n=477 [245 in study <em>1</em> and 232 in study 2]) or placebo (n=476 [244 and 232]). In both studies, patients receiving reslizumab had a significant reduction in the frequency of asthma exacerbations (study <em>1</em>: rate ratio [RR] 0·50 [95% CI 0·37-0·67]; study 2: 0·4<em>1</em> [0·28-0·59]; both p<0·000<em>1</em>) compared with those receiving placebo. Common adverse events on reslizumab were similar to placebo. The most common adverse events were worsening asthma symptoms (<em>1</em>27 [52%] for placebo and 97 [40%] for reslizumab in study <em>1</em>; <em>1</em><em>1</em>9 [5<em>1</em>%] for placebo and 67 [29%] for reslizumab for study 2), upper respiratory tract infections (32 [<em>1</em>3%] and 39 [<em>1</em>6%]; <em>1</em>6 [7%] and eight [3%]), and nasopharyngitis (33 [<em>1</em>4%] and 28 [<em>1</em><em>1</em>%]; 56 [24%] and 45 [<em>1</em>9%]). Two patients in the reslizumab group had anaphylactic reactions; both responded to standard treatment at the study centre and resolved, and the patients were withdrawn from the study.

CONCLUSIONS

These results support the use of reslizumab in patients with asthma and elevated blood eosinophil counts who are inadequately controlled on inhaled corticosteroid-based therapy.

BACKGROUND

Teva Branded Pharmaceutical Products R&D.

OBJECTIVE

The aim of this study was to evaluate the benefits and safety of long-term i.v. iron therapy in iron-deficient patients with heart failure (HF).

RESULTS

CONFIRM-HF was a multi-centre, double-blind, placebo-controlled trial that enrolled 304 ambulatory symptomatic HF patients with left ventricular ejection fraction ≤45%, elevated natriuretic peptides, and iron deficiency (ferritin (<em>1</em>00 ng/<em>mL</em> or <em>1</em>00-300 ng/<em>mL</em> if transferrin saturation <20%). Patients were randomized <em>1</em> : <em>1</em> to treatment with i.v. iron, as ferric carboxymaltose (FCM, n = <em>1</em>52) or placebo (saline, n = <em>1</em>52) for 52 weeks. The primary end-point was the change in 6-min-walk-test (6MWT) distance from baseline to Week 24. Secondary end-points included changes in New York Heart Association (NYHA) class, Patient Global Assessment (PGA), 6MWT distance, health-related quality of life (QoL), Fatigue Score at Weeks 6, <em>1</em>2, 24, 36, and 52 and the effect of FCM on the rate of hospitalization for worsening HF. Treatment with FCM significantly prolonged 6MWT distance at Week 24 (difference FCM vs. placebo: 33 ± <em>1</em><em>1</em> m, P = 0.002). The treatment effect of FCM was consistent in all subgroups and was sustained to Week 52 (difference FCM vs. placebo: 36 ± <em>1</em><em>1</em> m, P < 0.00<em>1</em>). Throughout the study, an improvement in NYHA class, PGA, QoL, and Fatigue Score in patients treated with FCM was detected with statistical significance observed from Week 24 onwards. Treatment with FCM was associated with a significant reduction in the risk of hospitalizations for worsening HF [hazard ratio (95% confidence interval): 0.39 (0.<em>1</em>9-0.82), P = 0.009]. The number of deaths (FCM: <em>1</em>2, placebo: <em>1</em>4 deaths) and the incidence of adverse events were comparable between both groups.

CONCLUSIONS

Treatment of symptomatic, iron-deficient HF patients with FCM over a <em>1</em>-year period resulted in sustainable improvement in functional capacity, symptoms, and QoL and may be associated with risk reduction of hospitalization for worsening HF (ClinicalTrials.gov number NCT0<em>1</em>453608).

Epithelial cell adhesion molecule-positive (EpCAM+) hepatocellular carcinoma (HCC) cells may constitute a tumor-initiating subpopulation in tumorigenic cell lines and HCC specimens. In the present study, EpCAM+ circulating tumor cells (CTCs) were identified prospectively in HCC patients undergoing curative resection, and the prognostic significance and their stem cell-like characteristics were investigated further. Blood samples from <em>1</em>23 HCC patients were tested prior to resection and <em>1</em> month thereafter. CTCs were present in 66.67% of patients, and the cell count measured in 7.5 <em>mL</em> of blood (CTC(7.5) ) ranged between <em>1</em> and 34. Fifty-one patients had CTC(7.5) of ≥2 preoperatively, and these patients developed tumor recurrence earlier than those with CTC(7.5) of <2 CTCs (P < 0.00<em>1</em>). A preoperative CTC(7.5) of ≥2 was an independent prognostic factor for tumor recurrence (P < 0.00<em>1</em>). Its prognostic significance also applied to patients with alpha-fetoprotein (AFP) levels of ≤400 ng/<em>mL</em> or subgroups with low recurrence risk (all P < 0.05). A significant decrease of CTC-positive rates (66.67% to 28.<em>1</em>5%, P < 0.05) and CTC(7.5) values (2.60 ± 0.43 to <em>1</em>.00 ± 0.36, P < 0.05) was observed <em>1</em> month after resection. Patients with consistent CTC(7.5) <2 had lower recurrence rates than those with values consistently ≥2 (<em>1</em>5.5% versus 87.50%, P < 0.00<em>1</em>). EpCAM+ CTCs displayed cancer stem cell biomarkers (CD<em>1</em>33 and ABCG2), epithelial-mesenchymal transition, Wnt pathway activation, high tumorigenic potential, and low apoptotic propensity.

CONCLUSIONS

Stem cell-like phenotypes are observed in EpCAM+ CTCs, and a preoperative CTC(7.5) of ≥2 is a novel predictor for tumor recurrence in HCC patients after surgery, especially in patient subgroups with AFP levels of ≤400 ng/mL or low tumor recurrence risk. EpCAM+ CTCs may serve as a real-time parameter for monitoring treatment response and a therapeutic target in HCC recurrence.

OBJECTIVE

To study the uptake of biodegradable microparticles in Caco-2 cells.

METHODS

Biodegradable microparticles of polylactic polyglycolic acid co-polymer (PLGA 50:50) of mean diameters 0.<em>1</em> micron, <em>1</em> micron, and <em>1</em>0 microns containing bovine serum albumin as a model protein and 6-coumarin as a fluorescent marker were formulated by a multiple emulsion technique. The Caco-2 cell monolayers were incubated with each diameter microparticles (<em>1</em>00 micrograms/<em>ml</em>) for two hours. The microparticle uptake in Caco-2 cells was studied by confocal microscopy and also by quantitating the 6-coumarin content of the microparticles taken up by the cells. The effects of microparticle concentration, and incubation time and temperature on microparticle cell uptake were also studied.

RESULTS

The study demonstrated that the Caco-2 cell microparticle uptake significantly depends upon the microparticle diameter. The 0.<em>1</em> micron diameter microparticles had 2.5 fold greater uptake on the weight basis than the <em>1</em> micron and 6 fold greater than the <em>1</em>0 microns diameter microparticles. Similarly in terms of number the uptake of 0.<em>1</em> micron diameter microparticles was 2.7 x <em>1</em>0(3) fold greater than the <em>1</em> micron and 6.7 x <em>1</em>0(6) greater than the <em>1</em>0 microns diameter microparticles. The efficiency of uptake of 0.<em>1</em> micron diameter microparticles at <em>1</em>00 micrograms/<em>ml</em> concentration was 4<em>1</em>% compared to <em>1</em>5% and 6% for the <em>1</em> micron and the <em>1</em>0 microns diameter microparticles, respectively. The Caco-2 cell microparticle (0.<em>1</em> micron) uptake increased with concentration in the range of <em>1</em>00 micrograms/<em>ml</em> to 500 micrograms/<em>ml</em> which then reached a plateau at higher concentration. The uptake of microparticles increased with incubation time, reaching a steady state at two hours. The uptake was greater at an incubation temperature of 37 degrees C compared to at 4 degrees C.

CONCLUSIONS

The Caco-2 cell microparticle uptake was microparticle diameter, concentration, and incubation time and temperature dependent. The small diameter microparticles (0.<em>1</em> micron) had significantly greater uptake compared to larger diameter microparticles. The results thus suggest that the mechanism of uptake of microparticles in Caco-2 cell is particle diameter dependent. Caco-2 cells are used as an in vitro model for gastrointestinal uptake, and therefore the results obtained in these studies could be of significant importance in optimizing the microparticle-based oral drug delivery systems.

We investigated the ability of cyclosporin A (CsA) and transforming growth factor beta (TGF-beta) to modulate the production of TNF-alpha and TNF-beta and IFN-gamma by unseparated, nonadherent, and adherent PBMC. Treatment of unseparated PBMC with CsA resulted in a significant dose-dependent inhibition of all three cytokines ranging from greater than 90% inhibition for IFN-gamma and TNF-beta, to approximately 70% for TNF-alpha. Pretreatment of unseparated or nonadherent PBMC with TGF-beta inhibited the production of IFN-gamma by 60-70%. However, the inhibition of TNF-alpha and TNF-beta production by these cells was only minimally affected, and at 0.<em>1</em>-<em>1</em> ng/<em>ml</em> TGF-beta could enhance TNF-alpha production by unseparated PBMC. In contrast, pretreatment of adherent PBMC with TGF-beta inhibited the production of TNF-alpha by approximately 60%. TGF-beta also inhibited both TNF-alpha production and tumor cell cytotoxicity mediated by murine peritoneal-derived macrophages. These observations indicate that the biological effects of CsA and TGF-beta on immune functions are of a wider range than previously reported.

OBJECTIVE

To report the short term anatomic and visual acuity response after intravitreal injection of bevacizumab (Avastin, Genentech) in patients with macular edema due to central retinal vein occlusion (CRVO).

METHODS

The authors conducted a retrospective study of patients with macular edema due to CRVO who were treated with at least one intravitreal injection of bevacizumab 1.25 mg in 0.05 mL. Patients underwent Snellen visual acuity testing, optical coherence tomography (OCT) imaging, and ophthalmoscopic examination at baseline and follow-up visits.

RESULTS

There were 16 eyes of 15 consecutive patients with a mean age of 76.1 years (SD 9.8 years). Intravitreal triamcinolone had been previously administered to 9 patients, but all of these patients either had no improvement or had excessive intraocular pressure caused by the triamcinolone. The patients received a mean of 2.8 injections of bevacizumab per eye. No adverse events were observed, including endophthalmitis, clinically evident inflammation, increased intraocular pressure, retinal tears, retinal detachment, or thromboembolic events in any patient. The mean central macular thickness at baseline was 887 microm and decreased to a mean of 372 microm at month 1 (P < 0.001). The mean baseline acuity was 20/600 (logMAR = 1.48) and the mean acuity at month 1 was 20/200 (logMAR = 1.05), a difference that was highly significant (P = 0.001). At last follow-up, a mean of 3 months after the first injection, the mean visual acuity was 20/138 (logMAR = 0.84), which was significantly better than baseline (P < 0.001). Visual acuity improvement, defined as a halving of the visual angle, was seen in 14 of the 16 eyes.

CONCLUSIONS

Initial treatment results of patients with macular edema secondary to CRVO did not reveal any short-term safety concerns. Intravitreal bevacizumab resulted in a significant decrease in macular edema and improvement in visual acuity. The number of patients in this pilot study was limited and the follow-up is too short to make any specific treatment recommendations, but the favorable short-term results suggest further study is needed.

Obesity and weight gain are characterized by increased adipose tissue mass due to an increase in the size of individual adipocytes and the generation of new adipocytes. New adipocytes are believed to arise from resident adipose tissue preadipocytes and mesenchymal progenitor cells. However, it is possible that progenitor cells from other tissues, in particular BM, could also contribute to development of new adipocytes in adipose tissue. We tested this hypothesis by transplanting whole BM cells from GFP-expressing transgenic mice into wild-type C57BL/6 mice and subjecting them to a high-fat diet or treatment with the thiazolidinedione (TZD) rosiglitazone (ROSI) for several weeks. Histological examination of adipose tissue or FACS of adipocytes revealed the presence of GFP(+) multilocular (<em>ML</em>) adipocytes, whose number was significantly increased by ROSI treatment or high-fat feeding. These <em>ML</em> adipocytes expressed adiponectin, perilipin, fatty acid-binding protein (FABP), leptin, C/EBPalpha, and PPARgamma but not uncoupling protein-<em>1</em> (UCP-<em>1</em>), the CD45 hematopoietic lineage marker, or the CDllb monocyte marker. They also exhibited increased mitochondrial content. Appearance of GFP(+) <em>ML</em> adipocytes was contemporaneous with an increase in circulating levels of mesenchymal and hematopoietic progenitor cells in ROSI-treated animals. We conclude that TZDs and high-fat feeding promote the trafficking of BM-derived circulating progenitor cells to adipose tissue and their differentiation into <em>ML</em> adipocytes.

OBJECTIVE

To determine the sensitivity and specificity of elevated serum IgG4 level for the diagnosis of autoimmune pancreatitis (AIP) and its ability to distinguish AIP from pancreatic cancer, its main differential diagnosis.

METHODS

We measured serum IgG4 levels (normal 8-<em>1</em>40 mg/dL) in 5<em>1</em>0 patients including 45 with AIP, <em>1</em>35 with pancreatic cancer, 62 with no pancreatic disease, and 268 with other pancreatic diseases.

RESULTS

Sensitivity, specificity, and positive predictive values for elevated serum IgG4 >><em>1</em>40 mg/dL) for diagnosis of AIP were 76%, 93%, and 36%, respectively, and 53%, 99%, and 75%, respectively, for IgG4 of >280 mg/dL. Among subjects with elevated IgG4, non-AIP subjects (N = 32) differed from AIP subjects (N = 34) in that they were more likely to be female (45%vs 9%, P < 0.00<em>1</em>), less likely to have serum IgG4 >280 mg/dL (<em>1</em>3%vs 7<em>1</em>%, P < 0.00<em>1</em>), or elevation of total IgG (<em>1</em>6%vs 56%, P < 0.00<em>1</em>). Serum IgG4 levels were elevated in <em>1</em>3/<em>1</em>35 (<em>1</em>0%) pancreatic cancer patients; however, only <em>1</em>% had IgG4 levels >280 mg/dL compared with 53% of AIP. Compared with AIP, pancreatic cancer patients were more likely to have CA<em>1</em>9-9 levels of>><em>1</em>00 U/<em>mL</em> (7<em>1</em>%vs 9%, P < 0.00<em>1</em>).

CONCLUSIONS

Elevated serum IgG4 levels are characteristic of AIP. However, mild (<2-fold) elevations in serum IgG4 are seen in up to <em>1</em>0% of subjects without AIP including pancreatic cancer and cannot be used alone to distinguish AIP from pancreatic cancer. Because AIP is uncommon, IgG4 elevations in patients with low pretest probability of having AIP are likely to represent false positives.

OBJECTIVE

This trial tested the hypothesis that combined androgen suppression (CAS) and whole-pelvic (WP) radiotherapy (RT) followed by a boost to the prostate improves progression-free survival (PFS) by <em>1</em>0% compared with CAS and prostate-only (PO) RT. This trial also tested the hypothesis that neoadjuvant and concurrent hormonal therapy (NCHT) improves PFS compared with adjuvant hormonal therapy (AHT) by <em>1</em>0%.

METHODS

Eligibility included localized prostate cancer with an elevated prostate-specific antigen (PSA) < or = <em>1</em>00 ng/<em>mL</em> and an estimated risk of lymph node (LN) involvement of <em>1</em>5%. Between April <em>1</em>, <em>1</em>995, and June <em>1</em>, <em>1</em>999, <em>1</em>,323 patients were accrued. Patients were randomly assigned to WP + NCHT, PO + NCHT, WP + AHT, or PO + AHT. Failure for PFS was defined as the first occurrence of local, regional, or distant disease; PSA failure; or death for any cause.

RESULTS

With a median follow-up of 59.5 months, WP RT was associated with a 4-year PFS of 54% compared with 47% in patients treated with PO RT (P =.022). Patients treated with NCHT experienced a 4-year PFS of 52% versus 49% for AHT (P =.56). When comparing all four arms, there was a progression-free difference among WP RT + NCHT, PO RT + NCHT, WP RT + AHT, and PO RT + AHT (60% v 44% v 49% v 50%, respectively; P =.008). No survival advantage has yet been seen.

CONCLUSIONS

WP RT + NCHT improves PFS compared with PO RT and NCHT or PO RT and AHT, and compared with WP RT + AHT in patients with a risk of LN involvement of <em>1</em>5%.

BACKGROUND

There is little knowledge about clinical variables associated with vitamin D (VitD) insufficiency in asthmatic children.

OBJECTIVE

We sought to investigate disease variables associated with VitD insufficiency in patients with childhood asthma and interaction of VitD with corticosteroid-mediated anti-inflammatory responses.

METHODS

We analyzed 25-hydroxyvitamin D serum levels in <em>1</em>00 asthmatic children to investigate relationships between 25-hydroxyvitamin D levels and patients' characteristics. We determined VitD's effects on dexamethasone (DEX) induction of mitogen-activated protein kinase phosphatase <em>1</em> and IL-<em>1</em>0 in PBMCs.

RESULTS

The median 25-hydroxyvitamin D serum level was 3<em>1</em> ng/mL. Forty-seven percent of subjects had VitD levels in the insufficient range (<30 ng/mL), whereas <em>1</em>7% were VitD deficient (<20 ng/mL). Log(<em>1</em>0) IgE (P = .0<em>1</em>, rho = -0.25) and the number of positive aeroallergen skin prick test responses (P = .02, rho = -0.23) showed a significant inverse correlation with VitD levels, whereas FEV(<em>1</em>) percent predicted (P = .004, rho = 0.34) and FEV(<em>1</em>)/forced vital capacity ratio (P = .0<em>1</em>, rho = 0.30) showed a significant positive correlation with VitD levels. The use of inhaled steroids (P = .0475), use of oral steroids (P = .02), and total steroid dose (P = .00<em>1</em>) all showed significant inverse correlations with VitD levels. The amount of mitogen-activated protein kinase phosphatase <em>1</em> and IL<em>1</em>0 mRNA induced by VitD plus DEX was significantly greater than that induced by DEX alone (P < .0<em>1</em>). In an experimental model of steroid resistance in which DEX alone did not inhibit T-cell proliferation, addition of VitD to DEX resulted in significant dose-dependent suppression of cell proliferation.

CONCLUSIONS

Corticosteroid use and worsening airflow limitation are associated with lower VitD serum levels in asthmatic patients. VitD enhances glucocorticoid action in PBMCs from asthmatic patients and enhances the immunosuppressive function of DEX in vitro.

Mitochondrial proton F0F<em>1</em>-ATPase/ATP synthase synthesizes ATP during oxidative phosphorylation. In this study, we examined the effects of several groups of polyphenolic phytochemicals on the activity of the enzyme. Resveratrol, a stilbene phytoalexin that is present in grapes and red wine, concentration-dependently inhibited the enzymatic activity of both rat brain and liver F0F<em>1</em>-ATPase/ATP synthase (IC(50) of <em>1</em>2 - 28 microM). Screening of other polyphenolic phytochemicals using rat brain F0F<em>1</em>-ATPase activity resulted in the following ranking potency (IC(50) in parenthesis): piceatannol (8 microM>>resveratrol (<em>1</em>9 microM)=(-)epigallocatechin gallate (<em>1</em>7 microM>>(-)epicatechin gallate, curcumin (45 microM>>genistein=biochanin A=quercetin=kaempferol=morin (55 - 65 microM>>phloretin=apigenin=daidzein (approx. <em>1</em>00 microM). Genistin, quercitrin, phloridzin, (+)catechin, (+)epicatechin, (-)epicatechin and (-)epigallocatechin had little effect at similar concentrations. Tannic acid, theaflavins (tea extract) and grape seed proanthocyanidin extract (GSPE) had IC(50) values of 5, 20 and 30 microg <em>ml</em>(-<em>1</em>), respectively. Several monophenolic antioxidants and non-phenolic compounds were ineffective at concentrations of 2<em>1</em>0 microM or higher. The inhibition of F0F<em>1</em>-ATPase by resveratrol and genistein was non-competitive in nature. The effects of polyphenolic phytochemicals were additive. Both resveratrol and genistein had little effect on the Na(+)/K(+)-ATPase activity of porcine cerebral cortex, whereas quercetin had similar inhibitory potency as for F0F<em>1</em>-ATPase. In conclusion, the ATP synthase is a target for dietary phytochemicals. This pharmacological property of these phytochemicals should be included in the examination of their health benefits as well as potential cytotoxicity.

We have studied the effect of fasting on serum leptin levels in normal volunteers. Five normal-weight (BMI < 28, 2 males/3 females) and five obese subjects (BMI>> 28, 2 males/3 females) were fasted (0 Kcal) for 52 h. Mean plasma glucose decreased from 88 +/- 3 to 63 +/- 5 mg/dl, serum insulin from <em>1</em>6 +/- <em>1</em> to <em>1</em>0 +/- <em>1</em> microU/<em>ml</em>, plasma beta-hydroxybutyrate increased from 0.2 +/- 0.<em>1</em> to <em>1</em>.8 +/- 0.4 mumol/<em>ml</em>. Serum leptin levels were higher in the obese than in the normal-weight volunteers (3<em>1</em> +/- <em>1</em>2 vs <em>1</em><em>1</em> +/- 3 ng/<em>ml</em>, p < 0.0<em>1</em>). In the obese, serum leptin decreased from 3<em>1</em> +/- <em>1</em>0 to <em>1</em>2 +/- 5 ng/<em>ml</em> aft552 h (-72%, p < 0.00<em>1</em>); in the normal-weight it decreased from <em>1</em><em>1</em> +/- 3 to 4 +/- 0.5 ng/<em>ml</em> (-64%, p < 0.00<em>1</em>). Serum leptin correlated positively with serum insulin (r = 0.5<em>1</em>, p < 0.00<em>1</em>) and with plasma glucose (r = 0.6<em>1</em>, p < 0.00<em>1</em>). To determine effects of fasting induced decreases in plasma glucose and insulin on serum leptin, four normal subjects (3 males/<em>1</em> female) were fasted for 72 h while their plasma glucose was clamped at basal levels with a variable rate glucose infusion. In these volunteers, serum leptin and insulin concentrations remained unchanged. In summary, the rapid decrease in serum leptin levels during fasting indicated that leptin release was regulated by factors other than changes in body fat mass. The lack of leptin changes during fasting, when basal insulin and glucose levels were maintained at basal levels, suggested that insulin and/or glucose may play a role in the regulation of leptin release.

During a sublethal murine infection with Listeria monocytogenes cells, tumor necrosis factor (TNF) activity was detectable in neither sera nor spleen homogenates at any stage of the infection when a bioassay with L-929 cells (less than 4 U/<em>ml</em>) was used. However, injecting the mice with an immunoglobulin fraction obtained from a rabbit hyperimmunized with recombinant murine TNF-alpha resulted in acceleration of listeriosis. When <em>1</em> mg of anti-TNF antibody was injected per mouse, all the mice died from listeriosis, even though the infectious dose was sublethal for the untreated controls. The antigen-specific elimination of the bacterium from the spleens and livers of anti-TNF antibody-treated mice was delayed, depending on the dose of the antibody injected. Endogenous TNF seemed to be produced early in infection, because suppression of antilisterial resistance was significant when a single injection of anti-TNF antibody was given between day zero and day 2 of infection. The effect of endogenous TNF on antilisterial resistance was due to neither regulation of alpha interferon (IFN-alpha) and IFN-gamma production nor induction of IFN-beta subtype <em>1</em> (IFN-beta <em>1</em>), because anti-TNF antibody treated-mice produced normal levels of IFN-alpha and IFN-gamma in the bloodstream during infection and administration of monoclonal anti-murine IFN-beta <em>1</em> antibody had no effect on the development of listeriosis. Alternatively, the listericidal activity of peritoneal macrophages of L. monocytogenes-infected mice could be abrogated by injection of anti-TNF antibody in vivo. These results suggest that the lower level of TNF is produced endogenously in mice that received L. monocytogenes infection and that it plays an essential role in the host defense against L. monocytogenes infection.

OBJECTIVE

To compare the effectiveness of divalproex sodium with that of lithium and placebo in patients with acute mania.

METHODS

Randomized, double-blind, parallel-group study of treatment outcomes in patients with manic-depressive illness.

METHODS

A total of <em>1</em>79 hospitalized, acutely manic patients meeting the Research Diagnostic Criteria for manic disorder, approximately half of whom had been nonresponsive to lithium previously, were studied at nine university-affiliated hospitals.

METHODS

After a minimum 3-day washout period, random assignment for 2<em>1</em> days to divalproex, lithium, or placebo in a 2:<em>1</em>:2 ratio. Dosage of divalproex and lithium was increased if tolerated to a target concentration of <em>1</em>04<em>1</em> mumol/L (<em>1</em>50 micrograms/<em>mL</em>) or <em>1</em>.5 mmol/L (conventionally expressed as milliequivalents per liter), respectively.

METHODS

Primary outcome measures were changes in the Mania Rating scale derived from the Schedule for Affective Disorders and Schizophrenia.

RESULTS

Intent-to-treat analysis for efficacy was based on data from 68, 35, and 73 patients in the divalproex, lithium, and placebo groups, respectively. Groups were initially comparable except that all eight patients with four or more manic episodes in the previous year were in the divalproex group. In 30%, 33%, and 5<em>1</em>% of the above groups, treatment was prematurely terminated due to lack of efficacy, with fewer premature terminations from divalproex than placebo (P = .0<em>1</em>7). The proportions of patients improving at least 50% were higher for divalproex and lithium groups than for the placebo group: 48% for divalproex (P = .004) and 49% for lithium (P = .025) vs 25% for placebo. Divalproex was as effective in rapid-cycling manic patients as in other patients.

CONCLUSIONS

Both divalproex and lithium were significantly more effective than placebo in reducing the symptoms of acute mania. The efficacy of divalproex appears to be independent of prior responsiveness to lithium.

The aim of this study was to examine the effects of transforming growth factor (TGF) beta<em>1</em> on the phenotype and the biological behavior of pancreatic cancer cell lines with and without mutations in the TGF-beta signaling pathway and to elucidate whether the Ras signaling cascade participates in mediating these effects of TGF-beta<em>1</em>. TGF-beta-responsive (PANC-<em>1</em>, COLO-357, and IMIM-PC<em>1</em>) and nonresponsive (CAPAN<em>1</em> and IMIM-PC2) pancreatic cancer cell lines with activating mutations of the Ki-Ras oncogene were treated with <em>1</em>0 ng/<em>ml</em> TGF-beta<em>1</em> over time. Phenotypic alterations were studied by electron and phase contrast microscopy and by immunohistochemistry and expression analyses of differentiation markers. The influence of TGF-beta on tumor cell scattering, migration, and invasion was determined. The role of the Ras-mitogen-activated protein kinase kinase (MEK)-extracellular signal-regulated kinase (ERK) cascade in mediating TGF-beta-induced morphological and functional effects were studied by pretreatment with the MEK<em>1</em> inhibitor PD 98059 and by measuring ERK2 activation using immune complex kinase assays. TGF-beta<em>1</em> led to a reversible and time-dependent epithelial-mesenchymal transdifferentiation (EMT) in TGF-beta-responsive pancreatic cancer cell lines, characterized by a fibroblastoid morphology and an up-regulation of mesenchymal markers and a down-regulation of epithelial markers. EMT was associated with an increase in tumor cell migration, invasion, and scattering. In the responsive cell lines, TGF-beta<em>1</em> induced a moderate but sustained activation of ERK2. EMT, the concomitant changes in gene expression, and the invasive and migratory potential were reduced or abolished by pretreatment with the selective MEK<em>1</em> inhibitor. Thus, in TGF-beta-responsive pancreatic cancer cells with activating Ki-Ras mutations, TGF-beta<em>1</em> treatment caused an EMT associated with a more invasive phenotype. Cross-talk with the Ras-MEK-ERK-signaling cascade appears to be essential for mediating these effects of TGF-beta<em>1</em>.

OBJECTIVE

Patients with advanced chronic kidney disease (CKD) are in positive phosphorus balance, but phosphorus levels are maintained in the normal range through phosphaturia induced by increases in fibroblast growth factor-23 (FGF23) and parathyroid hormone (PTH). This provides the rationale for recommendations to restrict dietary phosphate intake to 800 mg/d. However, the protein source of the phosphate may also be important.

METHODS

We conducted a crossover trial in nine patients with a mean estimated GFR of 32 ml/min to directly compare vegetarian and meat diets with equivalent nutrients prepared by clinical research staff. During the last 24 hours of each 7-day diet period, subjects were hospitalized in a research center and urine and blood were frequently monitored.

RESULTS

The results indicated that 1 week of a vegetarian diet led to lower serum phosphorus levels and decreased FGF23 levels. The inpatient stay demonstrated similar diurnal variation for blood phosphorus, calcium, PTH, and urine fractional excretion of phosphorus but significant differences between the vegetarian and meat diets. Finally, the 24-hour fractional excretion of phosphorus was highly correlated to a 2-hour fasting urine collection for the vegetarian diet but not the meat diet.

CONCLUSIONS

In summary, this study demonstrates that the source of protein has a significant effect on phosphorus homeostasis in patients with CKD. Therefore, dietary counseling of patients with CKD must include information on not only the amount of phosphate but also the source of protein from which the phosphate derives.

The charges and Lennard-Jones parameters of the TIP3P water potential have been modified to improve its performance under the common condition for molecular dynamics simulations of using Ewald summation in lieu of relatively short nonbonded truncation schemes. These parameters were optimized under the condition that the hydrogen atoms do not have Lennard-Jones parameters, thus making the model independent of the combining rules used for the calculation of nonbonded, heteroatomic interaction energies, and limiting the number of Lennard-Jones calculations required. Under these conditions, this model provides accurate density (rho = 0.997 g/<em>ml</em>) and heat of vaporization (DeltaH(vap) = <em>1</em>0.53 kcal/mol) at 25 degrees C and <em>1</em> atm, but also provides improved structure in the second peak of the O-O radial distribution function and improved values for the dielectric constant (epsilon(0) = 89) and the diffusion coefficient (D = 4.0 x <em>1</em>0(-5) cm(2)/s) relative to the original parametrization. Like the original parameterization, however, this model does not show a temperature density maximum. Several similar models are considered with the additional constraint of trying to match the performance of the optimized potentials for liquid simulation atom force field to that obtained when using the simulation conditions under which it was originally designed, but no model was entirely satisfactory in reproducing the relative difference in free energies of hydration between the model compounds, phenol and benzene. Finally, a model that incorporates a long-range correction for truncated Lennard-Jones interactions is presented, which provides a very accurate dielectric constant (epsilon(0) = 76), however, the improvement in this estimate is on the same order as the uncertainty in the calculation.

We have genetically engineered CD4(+) and CD8(+) T cells with human immunodeficiency virus (HIV) specificity by inserting a gene, CD4zeta, containing the extracellular domain of human CD4 (which binds HIV env) linked to the zeta (zeta) chain of the T-cell receptor (which mediates T-cell activation). Twenty-four HIV-positive subjects received a single infusion of 2 to 3 x <em>1</em>0(<em>1</em>0) autologous CD4zeta-modified CD4(+) and CD8(+) T cells administered with (n = <em>1</em><em>1</em>) or without (n = <em>1</em>3) interleukin-2 (IL-2). Subjects had CD4 counts greater than 50/microL and viral loads of at least <em>1</em>000 copies/<em>mL</em> at entry. T cells were costimulated ex vivo through CD3 and CD28 and expanded for approximately 2 weeks. CD4zeta was detected in <em>1</em>% to 3% of blood mononuclear cells at 8 weeks and 0.<em>1</em>% at <em>1</em> year after infusion, and survival was not enhanced by IL-2. Trafficking of gene-modified T cells to bulk rectal tissue and/or isolated lamina propria lymphocytes was documented in a subset of 5 of 5 patients at <em>1</em>4 days and 2 of 3 at <em>1</em> year. A greater than 0.5 log mean decrease in rectal tissue-associated HIV RNA was observed for at least <em>1</em>4 days, suggesting compartmental antiviral activity of CD4zeta T cells. CD4(+) counts increased by 73/microL at 8 weeks in the group receiving IL-2. There was no significant mean change in plasma HIV RNA or blood proviral DNA in either treatment arm. This sustained, high-level persistence of gene-modified T cells demonstrates the feasibility of ex vivo T-cell gene therapy in HIV-infected adults and suggests the importance of providing HIV-specific T-helper function.

BACKGROUND

The 6-min walk test (6'WT) is a simple measure of functional capacity and predicts survival in patients with moderate heart failure (HF).

METHODS

To assess the role of the 6'WT in the evaluation of patients with advanced HF, 45 patients (age 49 +/- 8 years, mean +/- SD; New York Heart Association class 3.3 +/- 0.6; left ventricular ejection fraction 0.20 +/- 0.06; right ventricular ejection fraction 0.31 +/- 0.11) underwent symptom-limited cardiopulmonary exercise testing and the 6'WT during cardiac transplant evaluation.

RESULTS

Mean 6'WT distance ambulated was 310 +/- 100 m and peak oxygen uptake (peak Vo2) was 12.2 +/- 4.5 mL/kg/min. There was a significant correlation between 6'WT distance ambulated and peak Vo2 (r = 0.64, p < 0.001). Multivariate analysis of patient characteristics, resting hemodynamics, and 6'WT results identified the distance ambulated during the 6'WT as the strongest predictor of peak Vo2 (p < 0.001). 6'WT distance ambulated less than 300 m predicted an increased likelihood of death or pretransplant hospital admission for continuous inotropic or mechanical support within 6 months (p = 0.04), but did not predict long-term overall or event-free survival with a mean follow-up of 62 weeks. Peak Vo2 was the best predictor of long-term overall and event-free survival.

CONCLUSIONS

In patients with advanced HF evaluated for cardiac transplantation, distance ambulated during the 6'WT predicts (1) peak Vo2 and (2) short-term event-free survival.

BACKGROUND

Essential hypertension is associated with impaired endothelium-dependent vasodilation. Inactivation of endothelium-derived nitric oxide by oxygen free radicals participates in endothelial dysfunction in experimental hypertension. To test this hypothesis in humans, we evaluated the effect of antioxidant vitamin C on endothelium-dependent responses in essential hypertensive patients.

RESULTS

In <em>1</em>4 healthy subjects (47.<em>1</em>+/-4.8 years; blood pressure, <em>1</em>20.6+/-4.5/80.9+/-3.5 mm Hg) and <em>1</em>4 essential hypertensive patients (47.3+/-5.<em>1</em> years; blood pressure, <em>1</em>53.9+/-7.<em>1</em>/<em>1</em>02.3+/-4.<em>1</em> mm Hg), we studied forearm blood flow (strain-gauge plethysmography) modifications induced by intrabrachial acetylcholine (0.<em>1</em>5, 0.45, <em>1</em>.5, 4.5, and <em>1</em>5 microg x <em>1</em>00 <em>mL</em>(-<em>1</em>) x min(-<em>1</em>)) or sodium nitroprusside (<em>1</em>, 2, and 4 microg/<em>1</em>00 <em>mL</em> forearm tissue per minute), an endothelium-dependent and -independent vasodilator, respectively, in basal conditions and during infusion of intrabrachial vitamin C (2.4 mg/<em>1</em>00 <em>mL</em> forearm tissue per minute). In hypertensive patients but not in control subjects, vitamin C increased (P<0.0<em>1</em>) the impaired vasodilation to acetylcholine, whereas the response to sodium nitroprusside was unaffected. Moreover, in another <em>1</em>4 hypertensive patients (47.<em>1</em>+/-5.2 years; blood pressure, <em>1</em>55.2+/-6.9/<em>1</em>03.7+/-4.5 mm Hg), the facilitating effect of vitamin C on vasodilation to acetylcholine was reversed by N(G)-monomethyl-L-arginine (<em>1</em>00 microg/<em>1</em>00 <em>mL</em> forearm tissue per minute), a nitric oxide synthase inhibitor, suggesting that in essential hypertension superoxide anions impair endothelium-dependent vasodilation by nitric oxide breakdown. Finally, because in adjunctive 7 hypertensive patients (47.8+/-6.<em>1</em> years; blood pressure, <em>1</em>55.3+/-6.8/<em>1</em>03.5+/-4.3 mm Hg), indomethacin (50 microg/<em>1</em>00 <em>mL</em> forearm tissue per minute), a cyclooxygenase inhibitor, prevented the potentiating effect of vitamin C on vasodilation to acetylcholine, it is possible that in essential hypertension a main source of superoxide anions could be the cyclooxygenase pathway.

CONCLUSIONS

In essential hypertensive patients, impaired endothelial vasodilation can be improved by the antioxidant vitamin C, an effect that can be reversed by the nitric oxide synthase inhibitor N(G)-monomethyl-L-arginine. These findings support the hypothesis that nitric oxide inactivation by oxygen free radicals contributes to endothelial dysfunction in essential hypertension.

Adherence of Plasmodium falciparum-infected erythrocytes to cerebral postcapillary venular endothelium is believed to be a critical step in the development of cerebral malaria. Some of the possible receptors mediating adherence have been identified, but the process of adherence in vivo is poorly understood. We investigated the role of carbohydrate ligands in adherence, and we identified chondroitin sulfate (CS) as a specific receptor for P. falciparum-infected erythrocytes. Parasitized cells bound to Chinese hamster ovary (CHO) cells and C32 melanoma cells in a chondroitin sulfate-dependent manner, whereas glycosylation mutants lacking chondroitin sulfate A (CSA) supported little or no binding. Chondroitinase treatment of wild-type CHO cells reduced binding by up to 90%. Soluble CSA inhibited binding to CHO cells by 99.2 +/- 0.2% at <em>1</em>0 mg/<em>ml</em> and by 72.5 +/- 3.8% at <em>1</em> mg/<em>ml</em>, whereas a range of other glycosaminoglycans such as heparan sulfate had no effect. Parasite lines selected for increased binding to CHO cells and most patient isolates bound specifically to immobilized CSA. We conclude that P. falciparum can express or expose proteins at the surface of the infected erythrocyte that mediate specific binding to CSA. This mechanism of adherence may contribute to the pathogenesis of P. falciparum malaria, but has wider implications as an example of an infectious agent with the capacity to bind specifically to cell-associated or immobilized CS.

We previously found that contrast-induced nephropathy (CIN) complicating percutaneous coronary intervention adversely affects patients with chronic kidney disease (CKD). Therefore, we further investigated whether the predictors and outcome of CIN after percutaneous coronary intervention differ among patients with versus without CKD. Among 7,230 consecutive patients, CIN >>or=25% or>>or=0.5 mg/dl increase in preprocedure serum creatinine 48 hours after the procedure) developed in 38<em>1</em> of <em>1</em>,980 patients (<em>1</em>9.2%) with baseline CKD (estimated glomerular filtration rate [eGFR] <60 <em>ml</em>/min/<em>1</em>.73 m(2)) and in 688 of 5,250 patients (<em>1</em>3.<em>1</em>%) without CKD. Decreased eGFRs, periprocedural hypotension, higher contrast media volumes, lower baseline hematocrit, diabetes, pulmonary edema at presentation, intra-aortic balloon pump use, and ejection fraction <40% were the most significant predictors of CIN in patients with CKD. Apart from intra-aortic balloon pump use, predictors of CIN in patients without CKD were the same as mentioned, plus older age and type of contrast media. Regardless of baseline renal function, CIN correlated with longer in-hospital stay and higher rates of in-hospital complications and <em>1</em>-year mortality compared with patients without CIN. By multivariate analysis, CIN was <em>1</em> of the most powerful predictors of <em>1</em>-year mortality in patients with preexisting CKD (odds ratio 2.37, 95% confidence interval <em>1</em>.63 to 3.44) or preserved eGFR (odds ratio <em>1</em>.78; 95% confidence interval <em>1</em>.22 to 2.60). Thus, regardless of the presence of CKD, baseline characteristics and periprocedural hemodynamic parameters predict CIN, and this complication is associated with worse in-hospital and <em>1</em>-year outcomes.