Little is known about fungal biofilms, which may cause infection and antibiotic resistance. In this study, biofilm formation by different Candida species, particularly Candida albicans and C. parapsilosis, was evaluated by using a clinically relevant model of Candida biofilm on medical devices. Candida biofilms were allowed to form on silicone elastomer and were quantified by tetrazolium (XTT) and dry weight (DW) assays. Formed biofilm was visualized by using fluorescence microscopy and confocal scanning laser microscopy with Calcofluor White (Sigma Chemical Co., St. Louis, Mo.), concanavalin A-Alexafluor 488 (Molecular Probes, Eugene, Oreg.), and FUN-<em>1</em> (Molecular Probes) dyes. Although minimal variations in biofilm production among invasive C. albicans isolates were seen, significant differences between invasive and noninvasive isolates (P < 0.00<em>1</em>) were noted. C. albicans isolates produced more biofilm than C. parapsilosis, C. glabrata, and C. tropicalis isolates, as determined by DW assays (P was <0.00<em>1</em> for all comparisons) and microscopy. Interestingly, noninvasive isolates demonstrated a higher level of XTT activity than invasive isolates. On microscopy, C. albicans biofilms had a morphology different from that of other species, consisting of a basal blastospore layer with a dense overlying matrix composed of exopolysaccharides and hyphae. In contrast, C. parapsilosis biofilms had less volume than C. albicans biofilms and were comprised exclusively of clumped blastospores. Unlike planktonically grown cells, Candida biofilms rapidly (within 6 h) developed fluconazole resistance (MIC,>><em>1</em>28 microg/<em>ml</em>). Importantly, XTT and FUN-<em>1</em> activity showed biofilm cells to be metabolically active. In conclusion, our data show that C. albicans produces quantitatively larger and qualitatively more complex biofilms than other species, in particular, C. parapsilosis.

Vascular endothelial growth factor (VEGF) induces the proliferation of endothelial cells and is a potent angiogenic factor that binds to heparin. We have therefore studied the effect of heparin upon the interaction of VEGF with its receptors. Heparin, at concentrations ranging from 0.<em>1</em> to <em>1</em>0 micrograms/<em>ml</em>, strongly potentiated the binding of <em>1</em>25I-VEGF to its receptors on endothelial cells. Scatchard analysis of <em>1</em>25I-VEGF binding indicates that <em>1</em> microgram/<em>ml</em> heparin induces an 8-fold increase in the apparent density of high affinity binding sites for VEGF, but does not significantly affect the dissociation constant of VEGF. Cross-linking experiments showed that heparin strongly potentiates the formation of the <em>1</em>70-, <em>1</em>95- and 225-kDa <em>1</em>25I-VEGF-receptor complexes on endothelial cells. At high <em>1</em>25I-VEGF concentrations (4 ng/<em>ml</em>), heparin preferentially enhanced the formation of the <em>1</em>70- and <em>1</em>95-kDa complexes. Preincubation of the cells with heparin, followed by extensive washes, produced a similar enhancement of subsequent <em>1</em>25I-VEGF binding. The binding of <em>1</em>25I-VEGF was completely inhibited following digestion of endothelial cells with heparinase and could be restored by the addition of exogenous heparin to the digested cells. The enhancing effect of heparin facilitated the detection of VEGF receptors on cell types that were not known previously to express such receptors. Our results suggest that cell surface-associated heparin-like molecules are required for the interaction of VEGF with its cell surface receptors.

To assess the differences in proteolytic activity of acute and chronic wound environments, wound fluids were collected from acute surgical wounds (22 samples) and chronic wounds (25 samples) of various etiologies, including mixed vessel disease ulcers, decubiti and diabetic foot ulcers. Matrix metalloproteinase (MMP) activity measured using the Azocoll assay was significantly elevated by 30 fold in chronic wounds (median 22.8 microg MMP Eq/<em>ml</em>) compared to acute wounds (median 0.76 microg MMP Eq/<em>ml</em>) (p < 0.00<em>1</em>). The addition of the matrix metalloproteinase inhibitor Illomostat decreased the matrix metalloproteinase activity by approximately 90% in all samples, confirming that the majority of the activity measured was due to matrix metalloproteinases. Gelatin zymograms indicated predominantly elevated matrix metalloproteinase-9 with smaller elevations of matrix metalloproteinase-2. In addition tissue inhibitor of metalloproteinase-<em>1</em> levels were analyzed in a small subset of acute and chronic wounds. When tissue inhibitor of metalloproteinase-<em>1</em> levels were compared to protease levels there was an inverse correlation (p = 0.02, r = - 0.78). In vitro degradation of epidermal growth factor was measured by addition of <em>1</em>25I labelled epidermal growth factor to acute and chronic wound fluid samples. There was significantly higher degradation of epidermal growth factor in chronic wound fluid samples (mean 28.<em>1</em>%) compared to acute samples (mean 0.6%). This also correlated to the epidermal growth factor activity of these wound fluid samples (p < 0. 00<em>1</em>, r = 0.64). Additionally, the levels of proteases were assayed in wound fluid collected from <em>1</em>5 venous leg ulcers during a nonhealing and healing phase using a unique model of chronic wound healing in humans. Patients with nonhealing venous leg ulcers were admitted to the hospital for bed rest and wound fluid samples were collected on admission (nonhealing phase) and after 2 weeks (healing phase) when the ulcers had begun to heal as evidenced by a reduction in size (median <em>1</em>2%). These data showed that the elevated levels of matrix metalloproteinase activity decreased significantly as healing occurs in chronic leg ulcers (p < 0.0<em>1</em>). This parallels the processes observed in normally healing acute wounds. This data also supports the case for the addition of protease inhibitors in chronic wounds in conjunction with any treatments using growth factors.

The purpose of this investigation was to compare the results from four commonly used maximal treadmill stress tests: Balke, Bruce, Ellestad, and a continuous multistage running protocol. The results compared serial and maximal heart rate, metabolic demands, and ECG determinations. Fifty-one healthy men, 35 to 55 years of age, volunteered for this study and were dichotomized into trained and untrained subjects. Regression analyses showed all the tests to correlate highly. No significant differences were found between tests at maximum for V02, heart rate, and blood pressure, except for V02 for the Balke as compared to the running protocol (39 vs. 4<em>1</em> <em>ml</em>./Kg-min). The Balke protocol showed lower values at maximum in VE and RP than the other three tests as well as the most gradual rate of progression in MET cost (0.5 METS per minute). The increase for the Bruce and Ellestad tests was from <em>1</em> to <em>1</em>.5 METS per minute, and a rapid initial increase (9 METS in the first 3 minutes) made the running test undesirable as a screening method. Although serial plots of heart rate and MET costs were similar to those previously reported for different population samples, the present data further refined these values. Finally, a nomograph comparing treadmill time and V02, max. for the Balke, Bruce, and Ellestad tests was developed from these data.

BACKGROUND

Immune activation in patients with chronic heart failure may be secondary to endotoxin (lipopolysaccharide) action. We investigated the hypothesis that altered gut permeability with bacterial translocation and endotoxaemia would be increased in patients with oedema secondary to congestive heart failure.

METHODS

We compared 20 patients who had chronic heart failure with recent-onset peripheral oedema (mean age 64 years [SD <em>1</em>0], New York Heart Association [NYHA] class 3.3 [0.7]), 20 stable non-oedematous patients with chronic heart failure (mean age 63 years [<em>1</em>9], NYHA class 2.6 [0.7]), and <em>1</em>4 healthy volunteers (mean age 55 years [<em>1</em>6]). Biochemical markers of endotoxaemia, inflammation, and immune activation were measured. Ten patients were studied within <em>1</em> week of complete resolution of oedema. Five patients survived longer than 6 months and were restudied again after remaining free of oedema for more than 3 months.

RESULTS

Mean endotoxin concentrations were higher in oedematous patients with chronic heart failure than in stable patients with chronic heart failure (0.74 [SD 0.45] vs 0.37 EU/mL [0.23], p=0.0009) and controls (0.46 EU/mL [0.2<em>1</em>], p=0.02). Oedematous patients had the highest concentrations of several cytokines. After short-term diuretic treatment, endotoxin concentrations decreased from 0.84 EU/mL [0.49] to 0.45 EU/mL [0.2<em>1</em>], p<0.05) but cytokines remained raised. After freedom of oedema for more than 3 months after oedema resolved, endotoxin concentrations remained unchanged from the previous visit (0.49 EU/mL [0.06], p=0.45).

CONCLUSIONS

Raised concentrations of endotoxin and cytokines are found in patients with chronic heart failure during acute oedematous exacerbation. Intensified diuretic treatment can normalise endotoxin concentrations. Our preliminary findings suggest that endotoxin may trigger immune activation in patients with chronic heart failure during oedematous episodes.

Samples of the expressed fixed oil from different sources of Nigella sativa seeds were examined by thin-layer and gas chromatography for content of fixed oils and thymoquinone, and these substances were tested as possible inhibitors of eicosanoid generation and membrane lipid peroxidation. The crude fixed oil and pure thymoquinone both inhibited the cyclooxygenase and 5-lipoxygenase pathways of arachidonate metabolism in rat peritoneal leukocytes stimulated with calcium ionophore A23<em>1</em>87, as shown by dose-dependent inhibition of thromboxane B2 and leukotriene B4, respectively. Thymoquinone was very potent, with approximate IC50 values against 5-lipoxygenase and cyclo-oxygenase of < <em>1</em> microgram/<em>ml</em> and 3.5 micrograms/<em>ml</em>, respectively. Both substances also inhibited non-enzymatic peroxidation in ox brain phospholipid liposomes, but thymoquinone was about ten times more potent. However, the inhibition of eicosanoid generation and lipid peroxidation by the fixed oil of N. sativa is greater than is expected from its content of thymoquinone (ca. 0.2% w/v), and it is possible that other components such as the unusual C20:2 unsaturated fatty acids may contribute also to its anti-eicosanoid and antioxidant activity. These pharmacological properties of the oil support the traditional use of N. sativa and its derived products as a treatment for rheumatism and related inflammatory diseases.

BACKGROUND

Antiretroviral agents active against drug-resistant HIV-<em>1</em> are needed for treatment-experienced patients. The aim of this trial was to assess the efficacy, safety, and tolerability of TMC<em>1</em>25 (etravirine), a non-nucleoside reverse transcriptase inhibitor (NNRTI).

METHODS

DUET-<em>1</em> is a continuing, multinational randomised, double-blind, placebo-controlled, phase III trial. Treatment-experienced adult patients with virological failure on stable antiretroviral therapy, documented genotypic evidence of NNRTI resistance, viral load over 5000 copies per mL, and three or more primary protease inhibitor mutations were randomly assigned to receive 200 mg TMC<em>1</em>25 or placebo twice daily. All patients also received darunavir with low-dose ritonavir and investigator-selected nucleoside reverse transcriptase inhibitors. Enfuvirtide use was optional. The primary endpoint was a confirmed viral load below 50 copies per mL at week 24 (FDA time-to-loss of virological response algorithm). Analyses were done by intention to treat. This trial is registered with ClinicalTrials.gov, with the number NCT00254046.

RESULTS

6<em>1</em>2 patients were randomised and treated (304 in the TMC<em>1</em>25 group, 308 in the placebo group). By week 24, 42 (<em>1</em>4%) patients in the TMC<em>1</em>25 group and 56 (<em>1</em>8%) in the placebo group had discontinued, mainly due to virological failure. At week 24, <em>1</em>70 (56%) patients in the TMC<em>1</em>25 group and <em>1</em><em>1</em>9 (39%) patients in the placebo group achieved a confirmed viral load of less than 50 copies per mL (difference in response rates <em>1</em>7%; 95% CI 9-25; p=0.005). Most adverse events were mild or moderate in severity. The type and incidence of adverse events, including neuropsychiatric events, seen with TMC<em>1</em>25 were generally comparable with placebo, with the exception of rash (6<em>1</em> [20%] patients on TMC<em>1</em>25 vs 30 [<em>1</em>0%] on placebo) and diarrhoea (36 [<em>1</em>2%] patients on TMC<em>1</em>25 vs 63 [20%] on placebo).

CONCLUSIONS

In treatment-experienced patients with NNRTI resistance, treatment with TMC<em>1</em>25 achieved better virological suppression at week 24 than did placebo. The safety and tolerability profile of TMC<em>1</em>25 was generally comparable with placebo.

BACKGROUND

Uric acid levels are increased in patients with kidney dysfunction. We tested the hypothesis that uric acid may be associated with kidney disease progression.

METHODS

Cohort study.

METHODS

5,808 participants of the Cardiovascular Health Study.

METHODS

Uric acid levels.

METHODS

Kidney disease progression was defined as a decrease in estimated glomerular filtration rate (GFR) of 3 mL/min/1.73 m(2) per year or greater >>or=0.05 mL/s) and as incident chronic kidney disease (CKD). Measures of kidney function were estimated GFR using the Modification of Diet in Renal Disease Study equation.

RESULTS

Higher quintiles of uric acid levels were associated with greater prevalences of estimated GFR less than 60 <em>mL</em>/min/1.73 m(2) (<1.00 <em>mL</em>/s) of 7%, 14%, 12%, 25%, and 42% for quintiles 1 (<or=4.41 mg/dL [<or=262 micromol/L]), 2 (4.41 to 5.20 mg/dL [262 to 309 micromol/L]), 3 (5.21 to 5.90 mg/dL [310 to 351 micromol/L]), 4 (5.91 to 6.90 mg/dL [352 to 410 micromol/L]), and 5 (>6.90 mg/dL [>410 micromol/L]), respectively. In comparison, there was only a modest, but significant, association between quintiles of uric acid levels and progression of kidney function decrease, with adjusted odds ratios of 1.0, 0.88 (95% confidence interval [CI], 0.64 to 1.21), 1.23 (95% CI, 0.87 to 1.75), 1.47 (95% CI, 1.04 to 2.07), and 1.49 (95% CI, 1.00 to 2.22) for quintiles 1 through 5, respectively. No significant association was found between uric acid level and incident CKD (adjusted odds ratio, 1.00; 95% CI, 0.89 to 1.14).

CONCLUSIONS

Measurements of albuminuria were not available.

CONCLUSIONS

Uric acid levels are associated strongly with prevalent CKD. In comparison, greater uric acid levels had a significant, but much weaker, association with progression of kidney disease.

Imatinib is a potent and selective inhibitor of the protein tyrosine kinase Bcr-Abl, platelet-derived growth factor receptors (PDGFRalpha and PDGFRbeta) and KIT. Imatinib is approved for the treatment of chronic myeloid leukaemia (CML) and gastrointestinal stromal tumour (GIST), which have dysregulated activity of an imatinib-sensitive kinase as the underlying pathogenetic feature. Pharmacokinetic studies of imatinib in healthy volunteers and patients with CML, GIST and other cancers show that orally administered imatinib is well absorbed, and has an absolute bioavailability of 98% irrespective of oral dosage form (solution, capsule, tablet) or dosage strength (<em>1</em>00 mg, 400 mg). Food has no relevant impact on the rate or extent of bioavailability. The terminal elimination half-life is approximately <em>1</em>8 hours. Imatinib plasma concentrations predictably increase by 2- to 3-fold when reaching steady state with 400mg once-daily administration, to 2.6 +/- 0.8 microg/<em>mL</em> at peak and <em>1</em>.2 +/- 0.8 microg/<em>mL</em> at trough, exceeding the 0.5 microg/<em>mL</em> (<em>1</em> micromol/L) concentrations needed for tyrosine kinase inhibition in vitro and leading to normalisation of haematological parameters in the large majority of patients with CML irrespective of baseline white blood cell count. Imatinib is approximately 95% bound to human plasma proteins, mainly albumin and alpha<em>1</em>-acid glycoprotein. The drug is eliminated predominantly via the bile in the form of metabolites, one of which (CGP 74588) shows comparable pharmacological activity to the parent drug. The faecal to urinary excretion ratio is approximately 5:<em>1</em>. Imatinib is metabolised mainly by the cytochrome P450 (CYP) 3A4 or CYP3A5 and can competitively inhibit the metabolism of drugs that are CYP3A4 or CYP3A5 substrates. Interactions may occur between imatinib and inhibitors or inducers of these enzymes, leading to changes in the plasma concentration of imatinib as well as coadministered drugs. Hepatic and renal dysfunction, and the presence of liver metastases, may result in more variable and increased exposure to the drug, although typically not necessitating dosage adjustment. Age (range <em>1</em>8-70 years), race, sex and bodyweight do not appreciably impact the pharmacokinetics of imatinib.

Elevated plasma concentrations of TNF receptors <em>1</em> and 2 (TNFR<em>1</em> and TNFR2) predict development of ESRD in patients with type 2 diabetes without proteinuria, suggesting these markers may contribute to the pathogenesis of renal decline. We investigated whether circulating markers of the TNF pathway determine GFR loss among patients with type <em>1</em> diabetes. We followed two cohorts comprising 628 patients with type <em>1</em> diabetes, normal renal function, and no proteinuria. Over <em>1</em>2 years, 69 patients developed estimated GFR less than 60 <em>mL</em>/min per <em>1</em>.73 m(2) (<em>1</em>6 per <em>1</em>000 person-years). Concentrations of TNFR<em>1</em> and TNFR2 were strongly associated with risk for early renal decline. Renal decline was associated only modestly with total TNFα concentration and appeared unrelated to free TNFα. The cumulative incidence of estimated GFR less than 60 <em>mL</em>/min per <em>1</em>.73 m(2) for patients in the highest TNFR2 quartile was 60% after <em>1</em>2 years compared with 5%-<em>1</em>9% in the remaining quartiles. In Cox proportional hazards analysis, patients with TNFR2 values in the highest quartile were threefold more likely to experience renal decline than patients in the other quartiles (hazard ratio, 3.0; 95% confidence interval, <em>1</em>.7-5.5). The risk associated with high TNFR<em>1</em> values was slightly less than that associated with high TNFR2 values. TNFR levels were unrelated to baseline free TNFα level and remained stable over long periods within an individual. In conclusion, early GFR loss in patients with type <em>1</em> diabetes without proteinuria is strongly associated with circulating TNF receptor levels but not TNFα levels (free or total).

Toxin A of Clostridium difficile causes severe inflammatory enterocolitis in man and animals that appears to be mediated in part by acute inflammatory cells that migrate into the toxin A-exposed mucosa. To determine the direct effects of toxin A on intestinal epithelial permeability and structure in the absence of other modulating factors, we used cultured monolayers of a human intestinal epithelial cell line (T84). A toxin A concentration of 7 x <em>1</em>0(-<em>1</em>) micrograms/<em>ml</em> (3 x <em>1</em>0(-9) M) nearly abolished monolayer transepithelial resistance within 6-8 h. This marked permeability defect occurred while the monolayers were still confluent. Dual sodium-mannitol flux studies localized the permeability defect to the intercellular tight junction. Cytotoxicity assays and morphological evaluation using Nomarski optics and electron microscopy failed to demonstrate any evidence of cell damage at the time the maximum resistance response was observed. Fluorescent staining for F actin, however, revealed a marked decrease in fluorescent intensity in toxin-treated monolayers versus controls. These data show that toxin A can directly affect the barrier function of this model intestinal epithelium and initially does so by selectively enhancing tight junction permeability. Furthermore, cytoskeletal structure is markedly altered over the same time course, although the integrity of individual cells is maintained. Because the cytoskeleton of intestinal epithelial cells is known to be capable of regulating tight junction permeability, we speculate that the above effects of toxin A on epithelial barrier function result from alterations of the cytoskeleton.

Inositol trisphosphate (IP3) was previously shown to release Ca2+ from a nonmitochondrial store in sea urchin eggs. In this study, egg homogenates and purified microsomes were monitored with either fura 2 or Ca2+-sensitive minielectrodes to determine whether other stimuli would induce Ca2+ release. Pyridine nucleotides (whose concentrations are known to change at fertilization) were found to release nearly as much Ca2+ as did IP3. Average releases/<em>ml</em> of homogenate were: 0.6 microM IP3, <em>1</em>0.9 nmol of Ca2+; 50 microM NADP, 7.3 nmol of Ca2+; and <em>1</em>00 microM NAD, 6.5 nmol of Ca2+ (n = 6). Specificity was demonstrated by screening a series of other phosphorylated metabolites, and none was found to reproducibly release Ca2+. Calcium release induced by IP3 or NADP was immediate, whereas a lag of <em>1</em>-4 min occurred with NAD. This lag before NAD-induced Ca2+ release led to the discovery that a soluble egg factor (Mr greater than <em>1</em>00,000) converts NAD into a highly active metabolite that releases Ca2+ without a lag. The NAD metabolite (E-NAD) was purified to homogeneity by high pressure liquid chromatography and produced half-maximal Ca2+ release at about 40 nM. Injection of E-NAD into intact eggs produced both an increase in intracellular Ca2+ (as assayed with indo-<em>1</em>) and a cortical reaction. Following Ca2+ release by each of the active agents (IP3, NAD, and NADP), the homogenates resequestered the released Ca2+ but were desensitized to further addition of the same agent. A series of desensitization experiments showed that homogenates desensitized to any two of these agents still responded to the third, indicating the presence of three independent Ca2+ release mechanisms. This is further supported by experiments using Percoll density gradient centrifugation in which NADP-sensitive microsomes were partially separated from those sensitive to IP3 and NAD.

An estimate of glomerular filtration rate has been derived for children from body length (L, in centimeters) and plasma creatinine (Pcr, in milligrams per deciliter): GFR = 0.55 L/Pcr. The near universality of this estimate in children led us to seek a similar formula for estimating GFR in full-term infants during the first year of life. We measured Pcr in <em>1</em>37 healthy infants and performed creatinine clearance (Ccr) studies in 63 of them aged greater than or equal to 5 days. Beyond the first week, Pcr averaged 0.39 +/- 0.0<em>1</em> (0.<em>1</em>0 SD) mg/dl. The estimate of GFR from 0.55 L/Pcr overestimated Ccr by 24% (P less than 0.00<em>1</em>). Based on the calculation of a new constant from Ccr X Pcr/L, GFR was more accurately estimated from 0.45 L/Pcr (mean difference of Ccr - 0.45 L/Pcr = -0.4 +/- 3.7 (SE) <em>ml</em>/min X <em>1</em>.73 m2) in full-term infants between <em>1</em> and 52 weeks of age. Because the constant 0.45 and Pcr do not change significantly during this period, GFR can be approximated at the bedside from body length of the healthy full-term infant (GFR = 0.45 L/0.39 = <em>1</em>.<em>1</em> L).

OBJECTIVE

The aim of this evidence-based report was to review pertinent randomized controlled studies that describe hemodynamic goals in acute, critically ill patients and to evaluate outcome of resuscitation therapy in association with physiologic, clinical, and therapeutic influences.

METHODS

MEDLINE was the source of randomized controlled studies written in English. The inclusion criteria were acutely ill, high-risk elective surgery, trauma, and septic patients. The goals of therapy were to resuscitate to either normal or supranormal values; the latter were described as a cardiac index of >4.5 L x min(-<em>1</em>) x m(-2), pulmonary artery occlusion pressure of (<em>1</em>8 mm Hg, oxygen delivery of >600 <em>mL</em> x min(-<em>1</em>) x m(-2), and oxygen consumption of>><em>1</em>70 <em>mL</em> x min(-<em>1</em>) x m(-2). The outcome criterion was survival or death. We found 2<em>1</em> randomized clinical trials described in 20 articles. The studies were divided into groups based on the time that goals were implemented (i.e., "early," 8 to <em>1</em>2 hrs postoperatively or before organ failure, vs. "late," or after onset of organ failure) and the severity of illness, determined by the control group mortality as >20% (<em>1</em>2 studies) or (<em>1</em>5% (nine studies).

RESULTS

In severely ill patients (control mortalities group >20%), six studies had a 23% mortality difference (p <.05) between the control and protocol groups with early optimization, but seven studies optimized after the development of organ failure did not have significantly improved mortality. Moreover, outcome was not significantly improved in less severely ill patients (control mortalities group (<em>1</em>5%) and normal values as goals or when therapy did not improve oxygen delivery.

CONCLUSIONS

Review of 2<em>1</em> randomized controlled trials with various approaches to treatment revealed statistically significant mortality reductions, with hemodynamic optimization, when patients with acute critical illness were treated early to achieve optimal goals before the development of organ failure, when there were control group mortalities of >20% and when therapy produced differences in oxygen delivery between the control and protocol groups.

BACKGROUND

Pegylated interferon (peginterferon) alfa 2a or 2b plus ribavirin regimens were the standard of care in patients with hepatitis C virus (HCV) infection, but the sustained virological response can be suboptimum in patients with HCV genotype <em>1</em> infection. The efficacy, safety, and tolerability of the combination of simeprevir, a one-pill, once-daily, oral HCV NS3/4A protease inhibitor versus placebo, plus peginterferon alfa 2a or 2b plus ribavirin was assessed in treatment-naive patients with HCV genotype <em>1</em> infection.

METHODS

In the QUEST-2, phase 3 study, done at 76 sites in <em>1</em>4 countries (Europe, and North and South Americas), patients with confirmed chronic HCV genotype <em>1</em> infection and no history of HCV treatment were randomly assigned with a computer-generated allocation sequence in a ratio of 2:<em>1</em> and stratified by HCV genotype <em>1</em> subtype and host IL28B genotype to receive simeprevir (<em>1</em>50 mg once daily, orally), peginterferon alfa 2a (<em>1</em>80 μg once weekly, subcutaneous injection) or 2b (according to bodyweight; 50 μg, 80 μg, <em>1</em>00 μg, <em>1</em>20 μg, or <em>1</em>50 μg once weekly, subcutaneous injection), plus ribavirin (<em>1</em>000-<em>1</em>200 mg/day or 800-<em>1</em>400 mg/day, orally; simeprevir group) or placebo (once daily, orally), peginterferon alfa 2a or 2b, plus ribavirin (placebo group) for <em>1</em>2 weeks, followed by just peginterferon alfa 2a or 2b plus ribavirin. Total treatment duration was 24 weeks or 48 weeks (simeprevir group) based on criteria for response-guided therapy (ie, HCV RNA <25 IU/mL undetectable or detectable at week 4 and undetectable week <em>1</em>2) or 48 weeks (placebo). Patients, study personnel, and the sponsor were masked to treatment assignment. The primary efficacy endpoint was sustained virological response at <em>1</em>2 weeks after the planned end of treatment (SVR<em>1</em>2). Analyses were by intention to treat. The trial is registered with ClinicalTrials.gov, number NCT0<em>1</em>290679. Results from the primary (SVR<em>1</em>2, week 60) analysis are presented.

RESULTS

209 (8<em>1</em>%) of 257 patients in the simeprevir group and 67 (50%) of <em>1</em>34 in the placebo group had SVR<em>1</em>2 (adjusted difference 32·2%, 95% CI 23·3-4<em>1</em>·2; p<0·000<em>1</em>). The incidences of adverse events were similar in the simeprevir and placebo groups at <em>1</em>2 weeks (246 [96%] vs <em>1</em>30 [97%]) and for the entire treatment (249 [97%] vs <em>1</em>32 [99%]), irrespective of the peginterferon alfa used. The most common adverse events were headache, fatigue, pyrexia, and influenza-like illness at <em>1</em>2 weeks (95 [37%) vs 45 [34%], 89 [35%] vs 52 [39%], 78 [30%] vs 48 [36%], and 66 [26%] vs 34 [25%], respectively) and for the entire treatment (<em>1</em>00 [39%] vs 49 [37%], 94 [37%] vs 56 [42%], 79 [3<em>1</em>%] vs 53 [40%], and 66 [26%] vs 35 [26%], respectively). Rash and photosensitivity frequencies were higher in the simeprevir group than in the placebo group (6<em>1</em> [24%] vs <em>1</em>5 [<em>1</em><em>1</em>%] and ten [4%] vs one [(<em>1</em>%], respectively). There was no difference in the prevalence of anaemia between the simeprevir and placebo groups (35 [<em>1</em>4%] vs 2<em>1</em> [<em>1</em>6%], respectively, at <em>1</em>2 weeks, and 53 [2<em>1</em>%] vs 37 [28%], respectively, during the entire treatment).

CONCLUSIONS

Addition of simeprevir to either peginterferon alfa 2a or peginterferon alfa 2b plus ribavirin improved SVR in treatment-naive patients with HCV genotype <em>1</em> infection, without worsening the known adverse events associated with peginterferon alfa plus ribavirin.

BACKGROUND

Janssen Infectious Diseases-Diagnostics.

The phase IIb, double-blind, placebo-controlled PILLAR trial investigated the efficacy and safety of two different simeprevir (SMV) doses administered once-daily (QD) with pegylated interferon (Peg-IFN)-α-2a and ribavirin (RBV) in treatment-naïve patients with HCV genotype <em>1</em> infection. Patients were randomized to one of five treatments: SMV (75 or <em>1</em>50 mg QD) for <em>1</em>2 or 24 weeks or placebo, plus Peg-IFN and RBV. Patients in the SMV arms stopped all treatment at week 24 if response-guided therapy (RGT) criteria were met; patients not meeting RGT continued with Peg-IFN and RBV until week 48, as did patients in the placebo control group. Sustained virologic response (SVR) rates measured 24 weeks after the planned end of treatment (SVR24) were 74.7%-86.<em>1</em>% in the SMV groups versus 64.9% in the control group (P < 0.05 for all comparisons [SMV versus placebo], except SMV 75 mg for 24 weeks). Rapid virologic response (HCV RNA <25 IU/<em>mL</em> undetectable at week 4) was achieved by 68.0%-75.6% of SMV-treated and 5.2% of placebo control patients. According to RGT criteria, 79.2%-86.<em>1</em>% of SMV-treated patients completed treatment by week 24; 85.2%-95.6% of these subsequently achieved SVR24. The adverse event profile was generally similar across the SMV and placebo control groups, with the exception of mild reversible hyperbilirubinemia, without serum aminotransferase abnormalities, associated with higher doses of SMV.

CONCLUSIONS

SMV QD in combination with Peg-IFN and RBV significantly improves SVR rates, compared with Peg-IFN and RBV alone, and allows the majority of patients to shorten their therapy duration to 24 weeks.

We studied the effects of exercise training in patients with chronic heart failure attributed to left ventricular dysfunction (ejection fraction, 24 +/- <em>1</em>0%). Twelve ambulatory patients with stable symptoms underwent 4-6 months of conditioning by exercising 4.<em>1</em> +/- 0.6 hr/wk at a heart rate corresponding to 75% of peak oxygen consumption. Before and after training, patients underwent maximal bicycle exercise testing with direct measurement of central hemodynamic, leg blood flow, and metabolic responses. Exercise training resulted in a decrease in heart rate at rest and submaximal exercise and a 23% increase in peak oxygen consumption from <em>1</em>6.8 +/- 3.8 to 20.6 +/- 4.7 <em>ml</em>/kg/min (p less than 0.0<em>1</em>). Heart rate, arterial lactate, and respiratory exchange ratio were unchanged at peak exercise after training. Maximal cardiac output tended to increase from 8.9 +/- 2.7 to 9.9 +/- 3.2 <em>1</em>/min and contributed to improved peak oxygen consumption in some patients, although this change did not reach statistical significance (p = 0.<em>1</em>3). Rest and exercise measurements of left ventricular ejection fraction, left ventricular end-diastolic volume, and left ventricular end-systolic volume were unchanged. Right atrial, pulmonary arterial, pulmonary capillary wedge, and systemic arterial pressures were not different after training. Training induced several important peripheral adaptations that contributed to improved exercise performance. At peak exercise, systemic arteriovenous oxygen difference increased from <em>1</em>3.<em>1</em> +/- <em>1</em>.4 to <em>1</em>4.6 +/- 2.3 <em>ml</em>/dl (p less than 0.05). This increase was associated with an increase in peak-exercise leg blood flow from 2.5 +/- 0.7 to 3.0 +/- 0.8 l/min (p less than 0.0<em>1</em>) and an increase in leg arteriovenous oxygen difference from <em>1</em>4.5 +/- <em>1</em>.3 to <em>1</em>6.<em>1</em> +/- <em>1</em>.9 <em>ml</em>/dl (p = 0.07). Arterial and femoral venous lactate levels were markedly reduced during submaximal exercise after training, even though cardiac output and leg blood flow were unchanged at these workloads. Thus, ambulatory patients with chronic heart failure can achieve a significant training effect from long-term exercise. Peripheral adaptations, including an increase in peak blood flow to the exercising leg, played an important role in improving exercise tolerance.(ABSTRACT TRUNCATED AT 400 WORDS)

OBJECTIVE

An emerging model of metabolic syndrome and type 2 diabetes is of adipose dysfunction with leukocyte recruitment into adipose leading to chronic inflammation and insulin resistance (IR). This study sought to explore potential mechanisms of inflammatory-induced IR in humans with a focus on adipose tissue.

METHODS

We performed a 60-h endotoxemia protocol (3 ng/kg intravenous bolus) in healthy adults (n = 20, 50% male, 80% Caucasian, aged 27.3 +/- 4.8 years). Before and after endotoxin, whole-blood sampling, subcutaneous adipose biopsies, and frequently sampled intravenous glucose tolerance (FSIGT) testing were performed. The primary outcome was the FSIGT insulin sensitivity index (S(i)). Secondary measures included inflammatory and metabolic markers and whole-blood and adipose mRNA and protein expression.

RESULTS

Endotoxemia induced systemic IR as demonstrated by a 35% decrease in S(i) (3.<em>1</em>7 +/- <em>1</em>.66 to 2.06 +/- 0.73 x <em>1</em>0(-4) [microU * <em>ml</em>(-<em>1</em>) * min(-<em>1</em>)], P < 0.005), while there was no effect on pancreatic beta-cell function. In adipose, endotoxemia suppressed insulin receptor substrate-<em>1</em> and markedly induced suppressor of cytokine signaling proteins (<em>1</em> and 3) coincident with local activation of innate (interleukin-6, tumor necrosis factor) and adaptive (monocyte chemoattractant protein-<em>1</em> and CXCL<em>1</em>0 chemokines) inflammation. These changes are known to attenuate insulin receptor signaling in model systems.

CONCLUSIONS

We demonstrate, for the first time in humans, that acute inflammation induces systemic IR following modulation of specific adipose inflammatory and insulin signaling pathways. It also provides a rationale for focused mechanistic studies and a model for human proof-of-concept trials of novel therapeutics targeting adipose inflammation in IR and related consequences in humans.

BACKGROUND

Overweight and obesity are well-established risk factors for cardiovascular disease and decline in kidney function in individuals with existing chronic kidney disease (CKD). Conversely, their association with the development of CKD is less clear.

METHODS

We evaluated the association between body mass index (BMI) and risk for CKD in a cohort of <em>1</em><em>1</em>,<em>1</em>04 initially healthy men who participated in the Physicians' Health Study and provided a blood sample after <em>1</em>4 years. BMI was calculated from self-reported weight and height. We estimated glomerular filtration rate (GFR) by using the abbreviated equation from the Modification of Diet in Renal Disease Study and defined CKD as GFR less than 60 <em>mL</em>/min/<em>1</em>.73 m2 ((<em>1</em> <em>mL</em>/s/<em>1</em>.73 m2).

RESULTS

After an average <em>1</em>4-year follow-up, <em>1</em>,377 participants (<em>1</em>2.4%) had a GFR less than 60 <em>mL</em>/min/<em>1</em>.73 m2 ((<em>1</em> <em>mL</em>/s/<em>1</em>.73 m2). Higher baseline BMI was associated consistently with increased risk for CKD. Compared with participants in the lowest BMI quintile (<22.7 kg/m2), those in the highest quintile (>26.6 kg/m2) had an odds ratio (OR) of <em>1</em>.45 (95% confidence interval [CI], <em>1</em>.<em>1</em>9 to <em>1</em>.76; P trend <0.00<em>1</em>) after adjusting for potential confounders. We found similar associations by using different categories of BMI. Compared with men who remained within a +/-5% range of their baseline BMI, those who reported a BMI increase greater than <em>1</em>0% had a significant increase in risk for CKD (OR, <em>1</em>.27; 95% CI, <em>1</em>.06 to <em>1</em>.53).

CONCLUSIONS

In this large cohort of initially healthy men, BMI was associated significantly with increased risk for CKD after <em>1</em>4 years. Strategies to decrease CKD risk might include prevention of overweight and obesity.

There exists a unique group of persons who are able to durably control HIV in the absence of therapy. The mechanisms of control in these persons remain poorly defined. In this study, we examined CD8(+) T-cell responses in blood and rectal mucosa from <em>1</em>7 "elite controllers" (viral load < 75 copies/<em>mL</em>), <em>1</em><em>1</em> "viremic controllers" (75-2000 copies/<em>mL</em>), <em>1</em>4 noncontrollers >> <em>1</em>0,000 copies/<em>mL</em>), and <em>1</em>0 antiretroviral-treated persons (< 75 copies/<em>mL</em>). Production of interferon-gamma, interleukin-2, tumor necrosis factor-alpha, macrophage inflammatory protein-<em>1</em> beta, and CD<em>1</em>07a by CD8(+) T cells in response to HIV-<em>1</em> Gag stimulation was measured using flow cytometry. Our hypothesis was that "polyfunctional" T cells producing multiple antiviral factors would be most abundant in mucosal tissues of HIV controllers. Mucosal CD8(+) T-cell responses were significantly stronger and more complex in controllers than in antiretroviral-suppressed persons (P = .0004). The frequency of 4-function responses in rectal mucosa was higher in controllers than in noncontrollers and patients on therapy (P < .000<em>1</em>). Mucosal responses in controllers were frequently stronger and more complex than blood responses. These findings demonstrate that many controllers mount strong, complex HIV-specific T-cell responses in rectal mucosa. These responses may play an important role in mucosal immune surveillance, as suggested by their relative enrichment among persons who control HIV in the absence of therapy.

Twenty-eight human isolates of Escherichia coli from Argentina and Spain and eight veterinary isolates received from the Ministry of Agriculture Fisheries and Foods in the United Kingdom required 2 to>> <em>1</em>28 micrograms of ciprofloxacin per <em>ml</em> for inhibition. Fragments of gyrA and parC encompassing the quinolone resistance-determining region were amplified by PCR, and the DNA sequences of the fragments were determined. All isolates contained a mutation in gyrA of a serine at position 83 (Ser83) to an Leu, and 26 isolates also contained a mutation of Asp87 to one of four amino acids: Asn (n = <em>1</em>4), Tyr (n = 6), Gly (n = 5), or His (n = <em>1</em>). Twenty-four isolates contained a single mutation in parC, either a Ser80 to Ile (n = <em>1</em>7) or Arg (n = 2) or a Glu84 to Lys (n = 3). The role of a mutation in gyrB was investigated by introducing wild-type gyrB (pBP548) into all isolates; for three transformants MICs of ciprofloxacin were reduced; however, sequencing of PCR-derived fragments containing the gyrB quinolone resistance-determining region revealed no changes. The analogous region of parE was analyzed in 34 of 36 isolates by single-strand conformational polymorphism analysis and sequencing; however, no amino acid substitutions were discovered. The outer membrane protein and lipopolysaccharide profiles of all isolates were compared with those of reference strains, and the concentration of ciprofloxacin accumulated (with or without <em>1</em>00 microM carbony cyanide m-chlorophenylhydrazone [CCCP] was determined. Twenty-two isolates accumulated significantly lower concentrations of ciprofloxacin than the wild-type E. coli isolate; nine isolates accumulated less then half the concentration. The addition of CCCP increased the concentration of ciprofloxacin accumulated, and in all but one isolate the percent increase was greater than that in the control strains. The data indicate that high-level fluoroquinolone resistance in E. coli involves the acquisition of mutations at multiple loci.

Muscle glycogen stores are depleted during exercise and are rapidly repleted during the recovery period. To investigate the mechanism for this phenomenon, untrained male rats were run for 45 min on a motor-driven treadmill and the ability of their muscles to utilize glucose was then assessed during perfusion of their isolated hindquarters. Glucose utilization by the hindquarter was the same in exercised and control rats perfused in the absence of added insulin; however, when insulin (30-40,000 muU/<em>ml</em>) was added to the perfusate, glucose utilization was greater after exercise. Prior exercise lowered both, the concentration of insulin that half-maximally stimulated glucose utilization (exercise, <em>1</em>50 muU/<em>ml</em>; control, 480 muU/<em>ml</em>) and modestly increased its maximum effect. The increase in insulin sensitivity persisted for 4 h following exercise, but was not present after 24 h. The rate-limiting step in glucose utilization enhanced by prior exercise appeared to be glucose transport across the cell membrane, as in neither control nor exercised rats did free glucose accumulate in the muscle cell. Following exercise, the ability of insulin to stimulate the release of lactate into the perfusate was unaltered; however its ability to stimulate the incorporation of [(<em>1</em>4)C]glucose into glycogen in certain muscles was enhanced. Thus at a concentration of 75 muU/<em>ml</em> insulin stimulated glycogen synthesis eightfold more in the fast-twitch red fibers of the red gastrocnemius than it did in the same muscle of nonexercised rats. In contrast, insulin only minimally increased glycogen synthesis in the fast-twitch white fibers of the gastrocnemius, which were not glycogen-depleted. The uptake of 2-deoxyglucose by these muscles followed a similar pattern suggesting that glucose transport was also differentially enhanced. Prior exercise did not enhance the ability of insulin to convert glycogen synthase from its glucose-6-phosphate-dependent (D) to its glucose-6-phosphate-independent (<em>1</em>) form. On the other hand, following exercise, insulin prevented a marked decrease in muscle glucose-6-phosphate, which could have diminished synthase activity in situ. The possibility that exercise enhanced the ability of insulin to convert glycogen synthase D to an intermediate form of the enzyme, more sensitive to glucose-6-phosphate, remains to be explored. These results suggest that following exercise, glucose transport and glycogen synthesis in skeletal muscle are enhanced due at least in part to an increase in insulin sensitivity. They also suggest that this increase in insulin sensitivity occurs predominantly in muscle fibers that are deglycogenated during exercise.

The metabolism of low density lipoprotein (LDL, beta lipoprotein) was studied in <em>1</em>0 normal individuals and <em>1</em>0 patients with familial type II hyperlipoproteinemia using purified radioiodinated LDL. Over 97% of the label was bound to the protein moiety of LDL and therefore the turnover data reflect the fate and distribution of LDL-apoprotein. Comparison of the metabolic behavior of biologically screened and unscreened labeled LDL preparations in dogs as well as the analysis of the urinary excretion of radioiodide derived from labeled LDL degradation in humans indicated that no significant denaturation resulted from the isolation, purification, and labeling techniques. The plasma concentration of LDL-cholesterol in normals was <em>1</em>05+/-2<em>1</em> mg/<em>1</em>00 <em>ml</em> (mean +/-<em>1</em> SD) in contrast to 254+/-47 mg/<em>1</em>00 mg in patients with type II hyperlipoproteinemia; these values corresponded to LDL-apoprotein concentrations of 63+/-<em>1</em>3 mg/<em>1</em>00 <em>ml</em> and <em>1</em>53+/-30 mg/<em>1</em>00 <em>ml</em>, respectively. Despite these differences in concentration, the synthetic rate of LDL-apoprotein in both groups was not significantly different (<em>1</em>4.43+/-<em>1</em>.75 mg/kg per day in normals vs. <em>1</em>5.0<em>1</em>+/-<em>1</em>.7<em>1</em> mg/kg per day in type II) nor was there any difference in the fraction of the total exchangeable LDL which was in the intravascular space (68.4+/-4.3% vs. 73.3+/-5.2%). However, the fractional catabolic rate of LDL in normal individuals differed significantly from that of patients with type II hyperlipoproteinemia (0.462+/-0.077/day in normals vs. 0.237+/-0.044/day in type II) and correspondingly the biological half-life of LDL was significantly prolonged (3.08+/-0.35 days normals vs. 4.68+/-0.44 days in type II). These data indicate that the pathologic elevation of plasma LDL concentration in the individuals with type II hyperlipoproteinemia studied here is due to a decreased fractional rate of LDL degradation rather than to an abnormality of LDL synthesis. This defect of catabolism may be the primary defect in type II hyperlipoproteinemia or, alternatively, may be secondary to an underlying abnormality in lipid metabolism.

BACKGROUND

It is commonly stated in the literature on human exposure to bisphenol A (BPA) that food is the predominant BPA exposure source, and that BPA is rapidly and completely cleared from the body. If this is correct, BPA levels in fasting individuals should decrease with increased fasting time.

OBJECTIVE

We set out to investigate the relationship between urine BPA concentration and fasting time in a population-based sample.

METHODS

We modeled log BPA urine concentration as a function of fasting time, adjusted for urine creatinine and other confounders, in <em>1</em>,469 adult participants in the 2003-2004 National Health and Nutrition Examination Survey. We estimated the BPA "population-based half-life" (pop(<em>1</em>/2)) for a fasting time of 0-24 hr, < 4.5 hr, 4.5-8.5 hr, and>> 8.5 hr.

RESULTS

The overall pop(<em>1</em>/2) for the 0- to 24-hr interval was 43 hr [95% confidence interval (CI), 26-<em>1</em><em>1</em>9 hr]. Among those reporting fasting times of 4.5-8.5 hr (n = 44<em>1</em>), BPA declined significantly with fasting time, with a pop(<em>1</em>/2) of 4.<em>1</em> hr (95% CI, 2.6-<em>1</em>0.6 hr). However, within the fasting time intervals of 0-4.5 hr (n = <em>1</em>29) and 8.5-24 hr (n = 899), we saw no appreciable decline. Fasting time did not significantly predict highest >> <em>1</em>2 ng/mL) or lowest (below limit of detection) BPA levels.

CONCLUSIONS

Overall, BPA levels did not decline rapidly with fasting time in this sample. This suggests substantial nonfood exposure, accumulation in body tissues such as fat, or both. Explaining these findings may require experimental pharmacokinetic studies of chronic BPA exposure, further examination of BPA levels and effects in fat, and a search for important nonfood sources.