BACKGROUND

Measurement of (<em>1</em>-->3)-beta-D-Glucan (BG) has emerged as an adjunct diagnostic strategy for invasive fungal infections (IFI).

METHODS

Subjects at 6 clinical sites in the United States were enrolled as either fungal infection-negative subjects (n = <em>1</em>70) or subjects with proven or probable IFI according to European Organization for the Research and Treatment of Cancer/Mycoses Study Group criteria (n = <em>1</em>63). A central laboratory and 4 sites performed assays. A single sample was obtained per patient and was evaluated using an assay to detect serum BG derived from fungal cell walls (range, 0 to>> 7000 pg/mL).

RESULTS

At a cutoff of 60 pg/mL, the sensitivity and specificity of the assay were 69.9% and 87.<em>1</em>%, respectively, with a positive predictive value (PPV) of 83.8% and a negative predictive value (NPV) of 75.<em>1</em>%. At a cutoff value of 80 pg/mL, the sensitivity and specificity were 64.4% and 92.4%, respectively, with a PPV of 89% and an NPV of 73%. Of the <em>1</em>07 patients with proven candidiasis, 8<em>1</em>.3% had positive results at a cutoff value of 60 pg/mL, and 77.6% had positive results at a cutoff value of 80 pg/mL. Of the <em>1</em>0 patients with aspergillosis, 80% had positive results at cutoff values of 60 and 80 pg/mL. The 3 subjects diagnosed with Fusarium species had positive results at a cutoff value of 60 pg/mL. Patients infected with Mucor or Rhizopus species (both of which lack BG) had negative results at both cutoff values, and of the <em>1</em>2 patients with Cryptococcus infection, 3 had positive results at a cutoff value of 60 pg/mL, and 2 had positive results at a cutoff value of 80 pg/mL. Of the subjects with proven positive results who were receiving antifungal therapy (n = <em>1</em><em>1</em>8), 72.9% had results positive for BG at a cutoff value of 60 pg/mL, and 69.5% had results positive for BG at a cutoff value of 80 pg/mL. The interlaboratory sample test r2 was 0.93.

CONCLUSIONS

Reproducible assay results with high specificity and high PPV in a multicenter setting demonstrate that use of an assay to detect serum BG derived from fungal cell walls is a useful diagnostic adjunct for IFI.

BACKGROUND

The prevalence, prognostic import, and impact of renal insufficiency on the benefits of ACE inhibitors and beta-blockers in community-dwelling patients with heart failure are uncertain.

RESULTS

We analyzed data from a prospective cohort of 754 patients with heart failure who had ejection fraction, serum creatinine, and weight measured at baseline. Median age was 69 years, and 43% had an ejection fraction>> or =35%. By the Cockcroft-Gault equation, <em>1</em><em>1</em>8 patients (<em>1</em>6%) had creatinine clearances < or =30 <em>mL</em>/min and 30<em>1</em> (40%) had creatinine clearances between 30 and 59 <em>mL</em>/min. During follow-up (median 926 days), 385 patients (37%) died. Even after adjustment for all other prognostic factors, survival was significantly associated with renal function (P=0.002) in patients with either systolic or diastolic dysfunction; patients exhibited a <em>1</em>% increase in mortality for each <em>1</em>-<em>mL</em>/min decrease in creatinine clearance. The associations with <em>1</em>-year mortality reductions were similar for ACE inhibitors (OR 0.46 [95% CI 0.26 to 0.82] versus OR 0.28 [95% CI 0.<em>1</em><em>1</em> to 0.70]) and beta-blockers (OR 0.40 [95% CI 0.23 to 0.70] versus OR 0.4<em>1</em> [95% CI 0.<em>1</em>9 to 0.85]) in patients with creatinine clearances <60 <em>mL</em>/min versus>> or =60 <em>mL</em>/min, although these drugs were used less frequently in patients with renal insufficiency.

CONCLUSIONS

Renal insufficiency is more prevalent in patients with heart failure than previously reported and is an independent prognostic factor in diastolic and systolic dysfunction. ACE inhibitors and beta-blockers were associated with similar reductions in mortality in patients with and without renal insufficiency.

BACKGROUND

Nearly half of patients with heart failure have a preserved ejection fraction (HFpEF). Symptoms of exercise intolerance and dyspnea are most often attributed to diastolic dysfunction; however, impaired systolic and/or arterial vasodilator reserve under stress could also play an important role.

RESULTS

Patients with HFpEF (n=<em>1</em>7) and control subjects without heart failure (n=<em>1</em>9) generally matched for age, gender, hypertension, diabetes mellitus, obesity, and the presence of left ventricular hypertrophy underwent maximal-effort upright cycle ergometry with radionuclide ventriculography to determine rest and exercise cardiovascular function. Resting cardiovascular function was similar between the 2 groups. Both had limited exercise capacity, but this was more profoundly reduced in HFpEF patients (exercise duration <em>1</em>80+/-7<em>1</em> versus 455+/-<em>1</em>84 seconds; peak oxygen consumption 9.0+/-3.4 versus <em>1</em>4.4+/-3.4 <em>mL</em> x kg(-<em>1</em>) x min(-<em>1</em>); both P<0.00<em>1</em>). At matched low-level workload, HFpEF subjects displayed approximately 40% less of an increase in heart rate and cardiac output and less systemic vasodilation (all P<0.05) despite a similar rise in end-diastolic volume, stroke volume, and contractility. Heart rate recovery after exercise was also significantly delayed in HFpEF patients. Exercise capacity correlated with the change in cardiac output, heart rate, and vascular resistance but not end-diastolic volume or stroke volume. Lung blood volume and plasma norepinephrine levels rose similarly with exercise in both groups.

CONCLUSIONS

HFpEF patients have reduced chronotropic, vasodilator, and cardiac output reserve during exercise compared with matched subjects with hypertensive cardiac hypertrophy. These limitations cannot be ascribed to diastolic abnormalities per se and may provide novel therapeutic targets for interventions to improve exercise capacity in this disorder.

A seroepidemiologic study of the prevalence of Helicobacter pylori infection in Japan was performed, and the relationship between serum pepsinogen I and II levels (markers of gastritis and gastric atrophy) and H. pylori infection was investigated. Four hundred and eighteen asymptomatic children and adults were studied. The prevalence of anti-H. pylori immunoglobulin G antibody increased with age. For persons born after <em>1</em>950, the frequency of H. pylori infection increased at approximately <em>1</em>% per year; for those born before <em>1</em>950 the prevalence was high (70%-80%) and relatively constant. Serum pepsinogen I and II levels were significantly higher in H. pylori-infected volunteers than in H. pylori-uninfected volunteers [5<em>1</em>.6 +/- 3 vs. 42.9 +/- 2 ng/<em>mL</em> (P less than 0.05) for pepsinogen I; <em>1</em>6.0 +/- <em>1</em> vs. 7.5 +/- 0.8 ng/<em>mL</em> (P less than 0.00<em>1</em>) for pepsinogen II]. The ratio of pepsinogen I to pepsinogen II was significantly lower in H. pylori-infected volunteers (3.5 +/- 0.2) than in uninfected volunteers (6.3 +/- 0.3; P less than 0.00<em>1</em>). The apparent decrease in prevalence of H. pylori accompanying the Westernization of Japan may eventually be accompanied by a reduction in the frequency of atrophic gastritis, the precursor lesion of the epidemic form of gastric carcinoma, and ultimately result in a decrease in the incidence of gastric carcinoma in Japan.

BACKGROUND

By using a novel experimental system for the study of reactive fibroblast differentiation at the molecular level, we describe the induction of the alpha-isoform of smooth muscle actin by transforming growth factor-beta (TGF-beta <em>1</em>) in human normal breast gland fibroblasts.

METHODS

The experimental system allowed fibroblasts to plate and remain quiescent and nonreactive but at the same time stay sensitive to environmental cues for more than 2 weeks after explantation. Most important, the biological activity of growth factors and cytokines could be studied without cells entering the cell cycle, thus serving as a model for stromal reactions with little cell turnover.

RESULTS

By use of double-labeling immunocytochemistry, isoelectric focusing, two-dimensional gel electrophoresis, and fluorography, evidence is presented that the effect of TGF-beta <em>1</em> is dose-dependent with a maximal response above 80 pg/ml and a course of 6 days. No other growth factor/cytokine tested (platelet-derived growth factor, interleukin-<em>1</em>, insulin-like growth factor-<em>1</em>, acidic fibroblast growth factor, basic fibroblast growth factor, epidermal growth factor, and interferon-gamma) could induce alpha-smooth muscle actin on their own or potentiate the effect of TGF-beta <em>1</em>. In an inhibitory assay, only basic fibroblast growth factor was found to prevent the action of TGF-beta <em>1</em>. The relative contribution of TGF-beta <em>1</em>-like activity to carcinoma cell induced alpha-smooth muscle actin in fibroblasts was deciphered using TGF-beta neutralizing antibodies and medium conditioned by two different breast carcinoma cell lines. Conditioned medium elicited a fibroblast response indistinguishable from that obtained with exogenously added TGF-beta <em>1</em>, and indeed neutralization attributed the entire response to the sole effect of secreted TGF-beta <em>1</em>-like activity.

CONCLUSIONS

These results provide a strategy for the molecular characterization of epithelial-stromal interaction in breast neoplasia.

Metabolic properties of the four subclasses of human IgG were investigated by performing 47 turnover studies in individuals with normal IgG serum concentrations, as well as in patients with an increased level of one of the subclasses. Studies in <em>1</em>2 subjects with normal IgG serum concentration showed that the average biologic half-life of G(<em>1</em>), G(2), and G(4) was 2<em>1</em> days, while that of G(3) was only 7.<em>1</em> days. Fractional catabolic rates of G(<em>1</em>), G(2), and G(4) were 6.9 to 8% of the intravascular pool per day. G(3), however, had a higher fractional catabolic rate, amounting to <em>1</em>6.8% of the intravascular pool per day. Distribution of the subclasses was such that the intravascular compartment contained 5<em>1</em>-54% of the total body pools of G(<em>1</em>), G(2), and G(4), but 64% of the total body pool of G(3).The short survival and high fractional catabolic rate of G(3) is an inherent property of these molecules, and is not due to denaturation during isolation and radiolabeling. This was demonstrated by studies of a patient with a serum G(3)-myeloma protein. The survival of her own protein, separately labeled either in vivo with guanidoarginine-(<em>1</em>4)C or in vitro with (<em>1</em>25)I, was determined in the patient. Survivals of the in vivo and in vitro labeled proteins were identical.G(<em>1</em>) and G(3) serum concentrations and synthetic rates were determined. The mean serum concentration of G(<em>1</em>) was 6.8 mg/<em>ml</em> and that of G(3) was 0.7 mg/<em>ml</em>, while their synthetic rates were 25.4 and 3.4 mg/kg per day respectively. The low serum concentration of IgG(2) thus results from a combination of high catabolic and low synthetic rates. Studies in <em>1</em>0 patients with multiple myeloma showed that an elevated serum concentration of any IgG subclass was associated with shortened biologic half-life and increased fractional catabolic rate of all subclasses. The implications of this concentration-catabolism relationship are discussed. The serum concentration of nonmyeloma IgG was usually low in myeloma patients and the synthesis of nonmyeloma IgG was somewhat decreased, suggesting that low serum concentrations of nonmyeloma IgG result from decreased synthesis, as well as from an increased fractional catabolic rate.

Relatively little is known about the exact mechanisms used by Bacillus subtilis in its behavior as a biocontrol agent on plants. Here, we report the development of a sensitive plant infection model demonstrating that the bacterial pathogen Pseudomonas syringae pv tomato DC3000 is capable of infecting Arabidopsis roots both in vitro and in soil. Using this infection model, we demonstrated the biocontrol ability of a wild-type B. subtilis strain 605<em>1</em> against P. syringae. Arabidopsis root surfaces treated with B. subtilis were analyzed with confocal scanning laser microscopy to reveal a three-dimensional B. subtilis biofilm. It is known that formation of biofilms by B. subtilis is a complex process that includes secretion of surfactin, a lipopeptide antimicrobial agent. To determine the role of surfactin in biocontrol by B. subtilis, we tested a mutant strain, M<em>1</em>, with a deletion in a surfactin synthase gene and, thus, deficient in surfactin production. B. subtilis M<em>1</em> was ineffective as a biocontrol agent against P. syringae infectivity in Arabidopsis and also failed to form robust biofilms on either roots or inert surfaces. The antibacterial activity of surfactin against P. syringae was determined in both broth and agar cultures and also by live-dead staining methods. Although the minimum inhibitory concentrations determined were relatively high (25 microg <em>mL</em>(-<em>1</em>)), the levels of the lipopeptide in roots colonized by B. subtilis are likely to be sufficient to kill P. syringae. Our results collectively indicate that upon root colonization, B. subtilis 605<em>1</em> forms a stable, extensive biofilm and secretes surfactin, which act together to protect plants against attack by pathogenic bacteria.

Using real-time quantitative PCR, cell-free EBV DNA was detectable in the plasma of 96% (55 of 57) of nasopharyngeal carcinoma (NPC) patients (median concentration, 2<em>1</em>058 copies/<em>ml</em>) and 7% (3 of 43) of controls (median concentration, 0 copies/<em>ml</em>). Advanced-stage NPC patients had higher plasma EBV DNA levels than those with early-stage disease. At <em>1</em> month after completion of radiotherapy, plasma EBV DNA was undetectable in 7 of <em>1</em>5 subjects (47%) but remained high in the remaining 8 subjects (53%). Clinical examination revealed that all of the former seven subjects had complete tumor regression, whereas six of the eight latter subjects exhibited evidence of disease persistence or had developed distant metastases. These results suggest that quantitative analysis of plasma EBV DNA may be a useful clinical and research tool in the screening and monitoring of NPC patients.

A PCR assay was developed for the detection and identification of Candida and Aspergillus species. The design of the oligonucleotide primer pair as well as the species-specific probes used for species identification was derived from a comparison of the sequences of the <em>1</em>8S rRNA genes of various fungal pathogens. The primers targeted a consensus sequence for a variety of fungal pathogens. The assay was tested for sensitivity and specificity with <em>1</em>34 fungal and 85 nonfungal isolates. To assess clinical applicability, 60<em>1</em> blood samples from four defined groups were tested: group A (n = 35), controls; groups B to D (n = 86), patients with febrile neutropenia, without fungal colonization (group B; n = 29) and with fungal colonization (group C; n = 36); and patients with documented invasive fungal infection (IFI) (group D; n = 2<em>1</em>). The assay detected and, by species-specific hybridization, identified most of the clinically relevant Candida and Aspergillus species at <em>1</em> CFU/<em>ml</em> of blood. Amplification was <em>1</em>00% sensitive for all molds and yeasts tested, with Histoplasma capsulatum being the only non-Aspergillus species hybridizing with the Aspergillus spp. probe. None of 35 group A patients and only 3 of 65 group B and C patients were PCR positive. The sensitivity of the assay for specimens from patients with IFI (2<em>1</em> patients in group D) was <em>1</em>00% if two specimens were tested. For specificity, 3 of <em>1</em>89 specimens from patients at risk but with negative cultures were positive by the assay, for a specificity of 98%. PCR preceded radiological signs by a median of 4 days (range, 4 to 7 days) for <em>1</em>2 of <em>1</em>7 patients with hepatosplenic candidiasis or pulmonary aspergillosis. For the <em>1</em>0 patients with IFI responding to antifungal therapy, PCR assays became persistently negative after <em>1</em>4 days of treatment, in contrast to the case for <em>1</em><em>1</em> patients, who remained PCR positive while not responding to antifungal therapy. Thus, the described PCR assay allows for the highly sensitive and specific detection and identification of fungal pathogens in vitro and in vivo. Preliminary data from the screening of a selected group of patients revealed some value in the early diagnosis and monitoring of antifungal therapy.

Elevated levels of ambient particulate matter (PM(<em>1</em>0)) have been associated with increased cardiopulmonary morbidity and mortality. We previously showed that the deposition of particles in the lung induces a systemic inflammatory response that includes stimulation of the bone marrow. This marrow response is related to mediators released by alveolar macrophages (AM) and in this study we measured cytokines produced by human AM exposed to ambient particles of different composition and size. Identified cytokines were also measured in the circulation of healthy young subjects exposed to air pollutants during the <em>1</em>997 Southeast Asian forest fires. Human AM were incubated with particle suspensions of residual oil fly ash (ROFA), ambient urban particles (EHC 93), inert carbon particles, and latex particles of different sizes (0.<em>1</em>, <em>1</em>, and <em>1</em>0 microm) and concentrations for 24 h. Tumor necrosis factor-alpha (TNF-alpha) increases in a dose-dependent manner when AM were exposed to EHC 93 particles (p < 0.02). The TNF response of AM exposed to different sizes of latex particles was similar. The latex (<em>1</em>58 +/- 3<em>1</em>%), inert carbon (<em>1</em>79 +/- 32%), and ROFA (2<em>1</em>6 +/- 34%) particles all show a similar maximum TNF response (percent change from baseline) whereas EHC 93 (<em>1</em>,020 +/- 2<em>1</em>2%, p < 0.05) showed a greater maximum response that was similar to lipopolysaccharide (LPS) <em>1</em> microg/<em>ml</em> (8<em>1</em>2 +/- 320%). Macrophages incubated with an optimal dose of EHC 93 particles (0.<em>1</em> mg/<em>ml</em>) also produce a broad spectrum of other proinflammatory cytokines, particularly interleukin (IL)-6 (p < 0.0<em>1</em>), IL-<em>1</em> beta (p < 0.05), macrophage inflammatory protein-<em>1</em> alpha (MIP-<em>1</em> alpha) (p < 0.05), and granulocyte macrophage colony-stimulating factor (GM-CSF) (p < 0.0<em>1</em>) with no difference in concentrations of the anti-inflammatory cytokine IL-<em>1</em>0 (p = NS). Circulating levels of IL-<em>1</em> beta, IL-6, and GM-CSF were elevated in subjects exposed to high levels of PM(<em>1</em>0) during an episode of acute air pollution. These results show that a range of different particles stimulate AM to produce proinflammatory cytokines and these cytokines are also present in the blood of subjects during an episode of acute atmospheric air pollution. We postulate that these cytokines induced a systemic response that has an important role in the pathogenesis of the cardiopulmonary adverse health effects associated with atmospheric pollution.

AMD-3<em>1</em>00, a bicyclam, is a novel agent that uniquely inhibits the entry of human immunodeficiency virus type <em>1</em> (HIV-<em>1</em>) into CD4(+) T cells via selective blockade of the chemokine CXCR-4 receptor. Twelve healthy volunteers were given AMD-3<em>1</em>00 as a single <em>1</em>5-min intravenous infusion at <em>1</em>0, 20, 40, or 80 microg/kg. Five subjects also received a single subcutaneous injection of AMD-3<em>1</em>00 (40 or 80 microg/kg). Three subjects received two escalating oral doses each (80 and <em>1</em>60 microg/kg). All subjects tolerated their dose(s) well without any grade 2 toxicity or dose adjustment. Six subjects experienced mild, transient symptoms, primarily gastrointestinal in nature and not dose related. All subjects experienced a dose-related elevation of the white blood cell count, from <em>1</em>.5 to 3.<em>1</em> times the baseline, which returned to the baseline 24 h after dosing. AMD-3<em>1</em>00 demonstrated dose proportionality for the maximum drug concentration in serum (C(max)) and the area under the concentration-time curve from 0 h to infinity (AUC(0-infinity)) over the entire dose range. At the highest intravenous dose (80 microg/kg), the median C(max) was 5<em>1</em>5 (range, 470 to 52<em>1</em>) ng/<em>ml</em> and the AUC(0-infinity) was <em>1</em>,044 (range, 980 to <em>1</em>,403) ng-h/<em>ml</em>. The median systemic absorption after subcutaneous dosing was 87% (range, 67 to <em>1</em>06%). No drug was detectable in the blood following oral dosing. Using a two-compartment model, the median pharmacokinetic parameter estimates (ranges) were as follows: volume of distribution, 0.34 (0. 27 to 0.36) liter/kg; clearance, <em>1</em>.30 (0.97 to <em>1</em>.34) liters/h; elimination half-life, 3.6 (3.5 to 4.9) h. After a single, well-tolerated intravenous dose of AMD-3<em>1</em>00, concentrations were sustained for <em>1</em>2 h above the in vitro antiretroviral 90% inhibitory concentrations and for 8 h above antiviral concentrations identified in the SCID-hu Thy/Liv mouse model of HIV infection.

OBJECTIVE

The predictive value of glomerular structure on progression of renal disease was examined in patients with Type II (non-insulin-dependent) diabetes and microalbuminuria (urinary albumin-to-creatinine ratio = 30-299 mg/g).

METHODS

Kidney biopsy specimens were obtained from <em>1</em>6 diabetic Pima Indians (6 men, <em>1</em>0 women). Progression of renal disease was assessed by measuring urinary albumin excretion 4 years after the biopsy (UAE(4 years)) and by computing the change in urinary albumin excretion during the study (Delta UAE).

RESULTS

At baseline, the duration of diabetes averaged <em>1</em>3.3 years (range = 4.0-23.8 years) and the mean glomerular filtration rate was <em>1</em>59 <em>ml</em> x min(-<em>1</em>) x <em>1</em>.73 m(-2) (range = 98 - 239 <em>ml</em> x min(-<em>1</em>) x <em>1</em>.73 m(-2)). Median urinary albumin excretion was 67 mg/g (range = 25-<em>1</em>36 mg/g) and it increased to 625 mg/g (range = 9-<em>1</em>347<em>1</em> mg/g) after 4 years; <em>1</em>0 subjects (63 %; 4 men, 6 women) developed macroalbuminuria (urinary albumin-to-creatinine ratio>>/= 300 mg/g). Neither mean arterial pressure nor HbA(<em>1</em> c) changed substantially during follow-up. Among the glomerular morphologic characteristics, the number of visceral epithelial cells, or podocytes, per glomerulus was the strongest predictor of renal disease progression (UAE(4 years), r = -0.49, p = 0.05; DeltaUAE, r = -0.57, p = 0.02), with fewer cells predicting more rapid progression. Glomerular basement membrane thickness did not predict progression (UAE(4 years), r = 0.<em>1</em><em>1</em>, p = 0.67; DeltaUAE, r = 0.09, p = 0.73) and mesangial volume fraction had only a modest effect (UAE(4 years,) r = 0.42, p = 0.<em>1</em><em>1</em>; DeltaUAE, r = 0.48, p = 0.06).

CONCLUSIONS

Whether lower epithelial cell number per glomerulus among those that progressed was due to cellular destruction, a reduced complement of epithelial cells, or both is uncertain. Nevertheless, these findings suggest that podocytes play an important part in the development and progression of diabetic renal disease. [Diabetologia (<em>1</em>999) 42: <em>1</em>34<em>1</em>-<em>1</em>344]

Folate derivatives are important in experimental colorectal carcinogenesis; low folate intake, particularly with substantial alcohol intake, is associated with increased risk. The enzyme 5,<em>1</em>0-methylenetetrahydrofolate reductase (MTHFR) catalyzes the conversion of 5,<em>1</em>0-methylenetetrahydrofolate, required for purine and thymidine syntheses, to 5-methyltetrahydrofolate, the primary circulatory form of folate necessary for methionine synthesis. A common mutation (677C->>T) in MTHFR reduces enzyme activity, leading to lower levels of 5-methyltetrahydrofolate. To evaluate the role of folate metabolism in human carcinogenesis, we examined the associations of MTHFR mutation, plasma folate levels, and their interaction with risk of colon cancer. We also examined the interaction between genotype and alcohol intake. We used a nested case-control design within the Physicians' Health Study. Participants were ages 40-84 at baseline when alcohol intake was ascertained and blood samples were drawn. During <em>1</em>2 years of follow-up, we identified 202 colorectal cancer cases and matched them to 326 cancer-free controls by age and smoking status. We genotyped for the MTHFR polymorphism and measured plasma folate levels. Men with the homozygous mutation (<em>1</em>5% in controls) had half the risk of colorectal cancer [odds ratio (OR), 0.49; 95% confidence interval (CI), 0.27-0.87] compared with the homozygous normal or heterozygous genotypes. Overall, we observed a marginal significant increased risk of colorectal cancer (OR, <em>1</em>.78; 95% CI, 0.93-3.42) among those whose plasma folate levels indicated deficiency (<3 ng/<em>ml</em>) compared with men with adequate folate levels. Among men with adequate folate levels, we observed a 3-fold decrease in risk (OR, 0.32; 95% CI, 0.<em>1</em>5-0.68) among men with the homozygous mutation compared with those with the homozygous normal or heterozygous genotypes. However, the protection due to the mutation was absent in men with folate deficiency. In men with the homozygous normal genotype who drank little or no alcohol as reference, those with the homozygous mutation who drank little or no alcohol had an 8-fold decrease in risk (OR, 0.<em>1</em>2; 95% CI, 0.03-0.57), and for moderate drinkers, a 2-fold decrease in risk (OR, 0.42; 95% CI, 0.<em>1</em>5-<em>1</em>.20); no decrease in risk was seen in those drinking <em>1</em> or more drinks/day. Our findings provide support for an important role of folate metabolism in colon carcinogenesis. In particular, these results suggest that the 677C->>IT mutation in MTHFR reduces colon cancer risk, perhaps by increasing 5,<em>1</em>0-methylenetetrahydrofolate levels for DNA synthesis, but that low folate intake or high alcohol consumption may negate some of the protective effect.

OBJECTIVE

Docosahexaenoic acid (DHA; 22:6 n-3) and arachidonic acid (AA; 20:4 n-6) are important for development of the central nervous system in mammals. There is a growth spurt in the human brain during the last trimester of pregnancy and the first postnatal months, with a large increase in the cerebral content of AA and DHA. The fetus and the newborn infant depend on maternal supply of DHA and AA. Our hypothesis was that maternal intake of DHA during pregnancy and lactation is marginal and that high intake of this fatty acid would benefit the child. We examined the effect of supplementing pregnant and lactating women with very-long-chain n-3 polyunsaturated fatty acids (PUFAs; cod liver oil) on mental development of the children, compared with maternal supplementation with long-chain n-6 PUFAs (corn oil).

METHODS

The study was randomized and double-blinded. Pregnant women were recruited in week <em>1</em>8 of pregnancy to take <em>1</em>0 <em>mL</em> of cod liver oil or corn oil until 3 months after delivery. The cod liver oil contained <em>1</em><em>1</em>83 mg/<em>1</em>0 <em>mL</em> DHA, 803 mg/<em>1</em>0 <em>mL</em> eicosapentaenoic acid (20:5 n-3), and a total of 2494 mg/<em>1</em>0 <em>mL</em> summation operator n-3 PUFAs. The corn oil contained 4747 mg/<em>1</em>0 <em>mL</em> linoleic acid (<em>1</em>8:2 n-6) and 92 mg/<em>1</em>0 <em>mL</em> alpha-linolenic acid (<em>1</em>8:3 n-3). The amount of fat-soluble vitamins was identical in the 2 oils (<em>1</em><em>1</em>7 micro g/<em>mL</em> vitamin A, <em>1</em> micro g/<em>mL</em> vitamin D, and <em>1</em>.4 mg/<em>mL</em> dl-alpha-tocopherol). A total of 590 pregnant women were recruited to the study, and 34<em>1</em> mothers took part in the study until giving birth. All infants of these women were scheduled for assessment of cognitive function at 6 and 9 months of age, and 262 complied with the request. As part of the protocol, <em>1</em>35 subjects from this population were invited for intelligence testing with the Kaufman Assessment Battery for Children (K-ABC) at 4 years of age. Of the <em>1</em>35 invited children, 90 came for assessment. Six children did not complete the examination. The K-ABC is a measure of intelligence and achievement designed for children aged 2.5 years through <em>1</em>2.5 years. This multisubtest battery comprises 4 scales: Sequential Processing, Simultaneous Processing, Achievement (not used in the present study), and Nonverbal Abilities. The Sequential Processing and Simultaneous Processing scales are hypothesized to reflect the child's style of problem solving and information processing. Scores from these 2 scales are combined to form a Mental Processing Composite, which serves as the measure of intelligence in the K-ABC.

RESULTS

We received dietary information from 76 infants (4<em>1</em> in the cod liver oil group and 35 in the corn oil group), documenting that all of them were breastfed at 3 months of age. Children who were born to mothers who had taken cod liver oil (n = 48) during pregnancy and lactation scored higher on the Mental Processing Composite of the K-ABC at 4 years of age as compared with children whose mothers had taken corn oil (n = 36; <em>1</em>06.4 [7.4] vs <em>1</em>02.3 [<em>1</em><em>1</em>.3]). The Mental Processing Composite score correlated significantly with head circumference at birth (r = 0.23), but no relation was found with birth weight or gestational length. The children's mental processing scores at 4 years of age correlated significantly with maternal intake of DHA and eicosapentaenoic acid during pregnancy. In a multiple regression model, maternal intake of DHA during pregnancy was the only variable of statistical significance for the children's mental processing scores at 4 years of age.

CONCLUSIONS

Maternal intake of very-long-chain n-3 PUFAs during pregnancy and lactation may be favorable for later mental development of children.

The emergence of multidrug-resistant strains of Mycobacterium tuberculosis has resulted in increased interest in the fluoroquinolones (FQs) as antituberculosis agents. To investigate the frequency and mechanisms of FQ resistance in M. tuberculosis, we cloned and sequenced the wild-type gyrA and gyrB genes, which encode the A and B subunits of the DNA gyrase, respectively; DNA gyrase is the main target of the FQs. On the basis of the sequence information, we performed DNA amplification for sequencing and single-strand conformation polymorphism analysis to examine the presumed quinolone resistance regions of gyrA and gyrB from reference strains (n = 4) and clinical isolates (n = 55). Mutations in codons of gyrA analogous to those described in other FQ-resistant bacteria were identified in all isolates (n = <em>1</em>4) for which the ciprofloxacin MIC was>> 2 micrograms/<em>ml</em>. In addition, we selected ciprofloxacin-resistant mutants of Mycobacterium bovis BCG and M. tuberculosis Erdman and H37ra. Spontaneously resistant mutants developed at a frequency of <em>1</em> in <em>1</em>0(7) to <em>1</em>0(8) at ciprofloxacin concentrations of 2 micrograms/<em>ml</em>, but no primary resistant colonies were selected at higher ciprofloxacin concentrations. Replating of those first-step mutants selected for mutants with high levels of resistance which harbored gyrA mutations similar to those found among clinical FQ-resistant isolates. The gyrA and gyrB sequence information will facilitate analysis of the mechanisms of resistance to drugs which target the gyrase and the implementation of rapid strategies for the estimation of FQ susceptibility in clinical M. tuberculosis isolates.

BACKGROUND

Erythropoietin (EPO) is a critical regulator for the proliferation of immature erythroid precursors, but its role as a potential cytoprotectant in the cerebrovasculature system has not been defined.

RESULTS

We examined the ability of EPO to regulate a cascade of apoptotic death-related cellular pathways during anoxia-induced vascular injury in endothelial cells (ECs). EC injury was evaluated by trypan blue, DNA fragmentation, membrane phosphatidylserine (PS) exposure, protein kinase B activity, mitochondrial membrane potential, and cysteine protease induction. Exposure to anoxia alone rapidly increased genomic DNA fragmentation from 2+/-<em>1</em>% to 40+/-5% and membrane PS exposure from 3+/-2% to 56+/-5% over 24 hours. Administration of a cytoprotective concentration of EPO (<em>1</em>0 ng/<em>mL</em>) prevented DNA destruction and PS exposure. Cytoprotection by EPO was completely abolished by cotreatment with anti-EPO neutralizing antibody, which suggests that EPO was necessary and sufficient for the prevention of apoptosis. Protection by EPO was intimately dependent on the activation of protein kinase B (Akt<em>1</em>) and the maintenance of mitochondrial membrane potential. Subsequently, EPO inhibited caspase 8-, caspase <em>1</em>-, and caspase 3-like activities that were linked to mitochondrial cytochrome c release.

CONCLUSIONS

The present work serves to illustrate that EPO can offer novel cytoprotection during ischemic vascular injury through direct modulation of Akt<em>1</em> phosphorylation, mitochondrial membrane potential, and cysteine protease activity.

BACKGROUND

Diabetes is regarded as a coronary heart disease risk equivalent-ie, people with the disorder have a risk of coronary events similar to those with previous myocardial infarction. We assessed whether chronic kidney disease should be regarded as a coronary heart disease risk equivalent.

METHODS

We studied a population-based cohort with measures of estimated glomerular filtration rate (eGFR) and proteinuria from Alberta, Canada. We used validated algorithms based on hospital admission and medical-claim data to classify participants with baseline history of myocardial infarction or diabetes and to ascertain which patients were admitted to hospital for myocardial infarction during follow-up (the primary outcome). For our primary analysis, we defined baseline chronic kidney disease as eGFR <em>1</em>5-59·9 <em>mL</em>/min per <em>1</em>·73 m(2) (stage 3 or 4 disease). We used Poisson regression to calculate unadjusted rates and relative rates of myocardial infarction during follow-up for five risk groups: people with previous myocardial infarction (with or without diabetes or chronic kidney disease), and (of those without previous myocardial infarction), four mutually exclusive groups defined by the presence or absence of diabetes and chronic kidney disease.

RESULTS

During a median follow-up of 48 months (IQR 25-65), <em>1</em><em>1</em>,340 of <em>1</em>,268,029 participants (<em>1</em>%) were admitted to hospital with myocardial infarction. The unadjusted rate of myocardial infarction was highest in people with previous myocardial infarction (<em>1</em>8·5 per <em>1</em>000 person-years, 95% CI <em>1</em>7·4-<em>1</em>9·8). In people without previous myocardial infarction, the rate of myocardial infarction was lower in those with diabetes (without chronic kidney disease) than in those with chronic kidney disease (without diabetes; 5·4 per <em>1</em>000 person-years, 5·2-5·7, vs 6·9 per <em>1</em>000 person-years, 6·6-7·2; p<0·000<em>1</em>). The rate of incident myocardial infarction in people with diabetes was substantially lower than for those with chronic kidney disease when defined by eGFR of less than 45 <em>mL</em>/min per <em>1</em>·73 m(2) and severely increased proteinuria (6·6 per <em>1</em>000 person-years, 6·4-6·9 vs <em>1</em>2·4 per <em>1</em>000 person-years, 9·7-<em>1</em>5·9).

CONCLUSIONS

Our findings suggest that chronic kidney disease could be added to the list of criteria defining people at highest risk of future coronary events.

BACKGROUND

Alberta Heritage Foundation for Medical Research.

The mosquito-borne Zika virus (ZIKV) is responsible for an explosive ongoing outbreak of febrile illness across the Americas. ZIKV was previously thought to cause only a mild, flu-like illness, but during the current outbreak, an association with Guillain-Barré syndrome and microcephaly in neonates has been detected. A previous study showed that ZIKV requires murine adaptation to generate reproducible murine disease. In our study, a low-passage Cambodian isolate caused disease and mortality in mice lacking the interferon (IFN) alpha receptor (A<em>1</em>29 mice) in an age-dependent manner, but not in similarly aged immunocompetent mice. In A<em>1</em>29 mice, viremia peaked at ∼<em>1</em>0(7) plaque-forming units/<em>mL</em> by day 2 postinfection (PI) and reached high titers in the spleen by day <em>1</em>. ZIKV was detected in the brain on day 3 PI and caused signs of neurologic disease, including tremors, by day 6. Robust replication was also noted in the testis. In this model, all mice infected at the youngest age (3 weeks) succumbed to illness by day 7 PI. Older mice (<em>1</em><em>1</em> weeks) showed signs of illness, viremia, and weight loss but recovered starting on day 8. In addition, AG<em>1</em>29 mice, which lack both type I and II IFN responses, supported similar infection kinetics to A<em>1</em>29 mice, but with exaggerated disease signs. This characterization of an Asian lineage ZIKV strain in a murine model, and one of the few studies reporting a model of Zika disease and demonstrating age-dependent morbidity and mortality, could provide a platform for testing the efficacy of antivirals and vaccines.

BACKGROUND

Renal denervation (RDN) with radiofrequency ablation substantially reduces blood pressure in patients with treatment-resistant hypertension. We assessed the long-term antihypertensive effects and safety.

METHODS

Symplicity HTN-<em>1</em> is an open-label study that enrolled <em>1</em>53 patients, of whom <em>1</em><em>1</em><em>1</em> consented to follow-up for 36 months. Eligible patients had a systolic blood pressure of at least <em>1</em>60 mm Hg and were taking at least three antihypertensive drugs, including a diuretic, at the optimum doses. Changes in office systolic blood pressure and safety were assessed every 6 months and reported every <em>1</em>2 months. This study is registered with ClinicalTrials.gov, numbers NCT00483808, NCT00664638, and NCT00753285.

RESULTS

88 patients had complete data at 36 months. At baseline the mean age was 57 (SD <em>1</em><em>1</em>) years, 37 (42%) patients were women, 25 (28%) had type 2 diabetes mellitus, the mean estimated glomerular filtration rate was 85 (SD <em>1</em>9) mL/min per <em>1</em>·73 m(2), and mean blood pressure was <em>1</em>75/98 (SD <em>1</em>6/<em>1</em>4) mm Hg. At 36 months significant changes were seen in systolic (-32·0 mm Hg, 95% CI -35·7 to -28·2) and diastolic blood pressure (-<em>1</em>4·4 mm Hg, -<em>1</em>6·9 to -<em>1</em><em>1</em>·9). Drops of <em>1</em>0 mm Hg or more in systolic blood pressure were seen in 69% of patients at <em>1</em> month, 8<em>1</em>% at 6 months, 85% at <em>1</em>2 months, 83% at 24 months, and 93% at 36 months. One new renal artery stenosis requiring stenting and three deaths unrelated to RDN occurred during follow-up.

CONCLUSIONS

Changes in blood pressure after RDN persist long term in patients with treatment-resistant hypertension, with good safety.

BACKGROUND

Ardian LLC/Medtronic Inc.

Green tea and tea polyphenols have been studied extensively as cancer chemopreventive agents in recent years. The bioavailability and metabolic fate of tea polyphenols in humans, however, are not clearly understood. In this report, the pharmacokinetic parameters of (-)-epigallocatechin-3-gallate (EGCG), (-)-epigallocatechin (EGC), and (-)-epicatechin (EC) were analyzed after administration of a single oral dose of green tea or decaffeinated green tea (20 mg tea solids/kg) or EGCG (2 mg/kg) to eight subjects. The plasma and urine levels of total EGCG, EGC, and EC (free plus conjugated forms) were quantified by HPLC coupled to an electrochemical detector. The plasma concentration time curves of the catechins were fitted in a one-compartment model. The maximum plasma concentrations of EGCG, EGC, and EC in the three repeated experiments with green tea were 77.9 +/- 22.2, 223.4 +/- 35.2, and <em>1</em>24.03 +/- 7.86 ng/<em>ml</em>, respectively, and the corresponding AUC values were 508.2 +/- 227, 945.4 +/- 438.4, and 529.5 +/- 244.4 ng x h x <em>ml</em>(-<em>1</em>), respectively. The time needed to reach the peak concentrations was in the range of <em>1</em>.3-<em>1</em>.6 h. The elimination half-lives were 3.4 +/- 0.3, <em>1</em>.7 +/- 0.4, and 2.0 +/- 0.4 h, respectively. Considerable interindividual differences and variations between repeated experiments in the pharmacokinetic parameters were noted. Significant differences in these pharmacokinetic parameters were not observed when EGCG was given in decaffeinated green tea or in pure form. In the plasma, EGCG was mostly present in the free form, whereas EGC and EC were mostly in the conjugated form. Over 90% of the total urinary EGC and EC, almost all in the conjugated forms, were excreted between 0 and 8 h. Substantial amounts of 4'-O-methyl EGC, at levels higher than EGC, were detected in the urine and plasma. The plasma level of 4'-O-methyl EGC peaked at <em>1</em>.7 +/- 0.5 h with a half life of 4.4 +/- <em>1</em>.<em>1</em> h. Two ring-fission metabolites, (-)-5-(3',4',5'-trihydroxyphenyl)-gamma-valerolactone (M4) and (-)-5-(3',4'-dihydroxyphenyl)-valerolactone (M6), appeared in significant amounts after 3 h and peaked at 8-<em>1</em>5 h in the urine as well as in the plasma. These results may be useful for designing the dose and dose frequency in intervention studies with tea and for development of biomarkers of tea consumption.

Macrophages exposed to IFN-gamma and infected with amastigotes of Leishmania major develop the capacity to eliminate the intracellular pathogen. This antimicrobial activity of activated macrophages correlates with the initiation of nitrogen oxidation of L-arginine, yet other reports suggest that two signals are required for induction of this biochemical pathway for effector activity. In the present studies, macrophages treated with up to <em>1</em>00 U/<em>ml</em> IFN-gamma, or <em>1</em>00 ng LPS, or <em>1</em>0(7) amastigotes produced minimal quantities (less than 9 microM) of NO2- and failed to develop cytotoxic effector activities. In contrast, the combination of IFN-gamma and either LPS (greater than 0.<em>1</em> ng) or amastigotes (<em>1</em>0(6) induced high concentrations (much greater than 30 microM) of NO2- and macrophage cytotoxicity against intra- and extracellular targets. The induction of nitrogen oxidation by amastigotes could be dissociated from LPS-induced events by <em>1</em>) performing the assays in the presence of polymyxin B (which blocked LPS effects, but not amastigote effects), 2) determining the threshold of IFN-gamma required to prime cells for subsequent trigger (<em>1</em> U/<em>ml</em> for LPS trigger effects; <em>1</em>0-fold higher for amastigotes), and 3) determining the heat sensitivity of the two trigger agents (amastigote effects abolished at <em>1</em>00 degrees C; LPS effects unaffected at this temperature). Further, culture fluids from amastigote-infected macrophages did not contain detectable LPS (less than 6 pg/<em>ml</em>). Possible parasite and cell-associated factors that could contribute to the induction of nitrogen oxidation and cytotoxic activity of IFN-gamma treated macrophages were examined: only certain intact microorganisms, LPS from a variety of bacteria, and the cytokine TNF alpha were effective. Both NO2- production and intracellular killing were abolished by the addition of anti-TNF-alpha mAb in the assay. TNF-alpha was produced by amastigote-infected macrophages and IFN-gamma dramatically enhanced secretion of this cytokine; IFN-gamma alone had no effect. Endogenous TNF-alpha produced during infection of macrophages with L. major acted in an autocrine fashion to trigger the production of L-arginine-derived toxic nitrogen intermediates that killed the intracellular parasites.

BACKGROUND

Although the addition of the HCV NS3/4A protease inhibitors boceprevir and telaprevir to pegylated interferon (peginterferon) alfa plus ribavirin has improved sustained virological response (SVR) in treatment-naive and treatment-experienced patients infected with hepatitis C virus (HCV) genotype <em>1</em>, the regimens have a high pill burden and are associated with increased rates and severity of adverse events, such as anaemia and rash. The efficacy and safety of the combination of simeprevir, a one pill, once-daily, oral HCV NS3/4A protease inhibitor, plus peginterferon alfa 2a plus ribavirin were assessed in treatment-naive patients with HCV genotype <em>1</em> infection.

METHODS

In QUEST-<em>1</em>, a phase 3, randomised, double-blind multicentre trial undertaken in <em>1</em>3 countries (Australia, Europe, North America, Puerto Rico, and New Zealand), 394 patients (aged ≥<em>1</em>8 years) with chronic HCV genotype <em>1</em> infection and no history of HCV treatment, stratified by HCV subtype and host IL28B genotype, were randomly assigned in a 2:<em>1</em> ratio with a computer-generated allocation sequence to receive simeprevir (<em>1</em>50 mg once daily, orally) plus peginterferon alfa 2a plus ribavirin for <em>1</em>2 weeks, followed by peginterferon alfa 2a plus ribavirin (simeprevir group), or placebo orally plus peginterferon alfa 2a plus ribavirin for <em>1</em>2 weeks, followed by peginterferon alfa 2a plus ribavirin (placebo group). Treatment duration was 24 weeks or 48 weeks in the simeprevir group according to criteria for response-guided therapy (ie, HCV RNA <25 IU/mL [undetectable or detectable] at week 4 and <25 IU/mL undetectable at week <em>1</em>2) and 48 weeks in the placebo group. Patients, study personnel, and the sponsor were masked to the treatment group assignment. The primary efficacy endpoint was sustained virological response <em>1</em>2 weeks after the planned end of treatment (SVR<em>1</em>2) and was assessed with an intention-to-treat analysis. The results of the primary analysis (week 60) are presented for safety and SVR<em>1</em>2. This trial is registered with ClinicalTrials.gov, number NCT0<em>1</em>289782.

RESULTS

Treatment with simeprevir, peginterferon alfa 2a, and ribavirin was superior to placebo, peginterferon alfa 2a, and ribavirin (SVR<em>1</em>2 in 2<em>1</em>0 [80%] patients of 264 vs 65 [50%] of <em>1</em>30, respectively, adjusted difference 29·3% [95% CI 20·<em>1</em>-38·6; p<0·000<em>1</em>). Adverse events in the first <em>1</em>2 weeks of treatment led to discontinuation of simeprevir in two ((<em>1</em>%) patients and discontinuation of placebo in one patient ((<em>1</em>%); fatigue (<em>1</em>06 [40%] vs 49 [38%] patients, respectively) and headache (8<em>1</em> [3<em>1</em>%] vs 48 [37%], respectively) were the most common adverse events. The prevalences of anaemia (42 [<em>1</em>6%] vs <em>1</em>4 [<em>1</em><em>1</em>%], respectively) and rash (72 [27%] vs 33 [25%]) were similar in the simeprevir and placebo groups. Addition of simeprevir did not increase severity of patient-reported fatigue and functioning limitations, but shortened their duration.

CONCLUSIONS

Simeprevir once daily with peginterferon alfa 2a and ribavirin shortens therapy in treatment-naive patients with HCV genotype <em>1</em> infection without worsening the adverse event profiles associated with peginterferon alfa 2a plus ribavirin.

BACKGROUND

Janssen Infectious Diseases-Diagnostics.

The effect of early aggressive antihypertensive treatment on kidney function in diabetic nephropathy was studied prospectively in ten insulin-dependent diabetics (mean age 29 years). During the mean pretreatment period of 29 (range 23-38) months the glomerular filtration rate (GFR) decreased significantly and the urinary albumin excretion rate and arterial blood pressure rose significantly. During the 39 month (range 28-48) period of antihypertensive treatment with metoprolol, hydralazine, and frusemide (furosemide) or thiazide, arterial blood pressure fell from <em>1</em>44/97 mm Hg (mean of all pretreatment values) to <em>1</em>28/84 mm Hg (mean of all post-treatment values), urinary albumin excretion from 977 micrograms/min to 433 micrograms/min, and GFR from 80 to 62 <em>ml</em>/min/<em>1</em> . 73 m2. The rate of decline in GFR decreased from 0.9<em>1</em> <em>ml</em>/min/month before treatment to 0.39 <em>ml</em>/min/month (range 0.08 to 0.68 <em>ml</em>/min/month) during treatment.

Within <em>1</em>5 min of endotracheal intubation, the resolution of pulmonary edema was studied over the next <em>1</em>2 h in 34 mechanically ventilated patients by (<em>1</em>) serial measurements of the alveolar-arterial oxygen difference, (2) the extent of edema on the initial and follow-up chest radiograph, and (3) by an initial and final measurement of total protein and albumin concentration in sequential samples of pulmonary edema fluid. Based on the oxygenation and chest radiographic data, 24 patients clinically improved and <em>1</em>0 patients did not improve. In the <em>1</em>0 patients who did not clinically improve (3, hydrostatic edema; 7, permeability edema), there was no change in the final edema fluid protein concentration (4.<em>1</em> +/- <em>1</em>.<em>1</em> g/<em>1</em>00 <em>ml</em>) compared with the initial edema fluid protein concentration (4.2 +/- <em>1</em>.0 g/<em>1</em>00 <em>ml</em>) (p = ns). However, in the 24 patients who clinically improved (<em>1</em>5, hydrostatic edema; 9, permeability edema), there was an increase in every patient's final edema protein concentration (5.6 +/- 2.3 g/<em>1</em>00 <em>ml</em>) compared with their initial edema protein concentration (3.8 +/- <em>1</em>.2 g/<em>1</em>00 <em>ml</em>) (p less than 0.0<em>1</em>). In <em>1</em>3 of these 24 patients, the final edema fluid concentration (7.3 +/- <em>1</em>.6 g/<em>1</em>00 <em>ml</em>) exceeded the final plasma protein concentration (5.6 +/- 0.8 g/<em>1</em>00 <em>ml</em>) by a mean value of <em>1</em>.7 g/<em>1</em>00 <em>ml</em> protein. The data provide the first evidence in humans to support the hypothesis that active ion transport across the alveolar epithelial barrier is the primary mechanism for clearance of edema fluid from the air spaces of the lung.(ABSTRACT TRUNCATED AT 250 WORDS)