Light-switching excimer probes for rapid protein monitoring in complex biological fluids

Quantitative protein bioanalysis in complex biological fluids presents considerable challenges in biological studies and disease diagnosis. The major obstacles are the background signals from both the probe and the biological fluids where the proteins reside. We have molecularly engineered light-switching excimer aptamer probes for rapid and sensitive detection of a biomarker protein, platelet-derived growth factor (PDGF). Labeled with one pyrene at each end, the aptamer switches its fluorescence emission from approximately 400 nm (pyrene monomer) to 485 nm (pyrene excimer) upon PDGF binding. This fluorescence wavelength change from monomer to excimer emission is a result of aptamer conformation rearrangement induced by target binding. The excimer probe is able to effectively detect picomolar PDGF in homogeneous solutions. Because the excimer has a much longer fluorescence lifetime (approximately 40 ns) than that of the background (approximately 5 ns), time-resolved measurements were used to eliminate the biological background. We thus were able to detect PDGF in a cell sample quantitatively without any sample pretreatment. This molecular engineering strategy can be used to develop other aptamer probes for protein monitoring. Combined with lifetime-based measurements and molecular engineering, light-switching excimer aptamer probes hold great potential in protein analysis for biomedical studies.     

Canonical pathway of nuclear factor kappa B activation selectively regulates proinflammatory and prothrombotic responses in human atherosclerosis

Nuclear factor kappa B (NF-kappa B) activation has been observed in human atherosclerotic plaques and is enhanced in unstable coronary plaques, but whether such activation has a protective or pathophysiological role remains to be determined. We addressed this question by developing a short-term culture system of cells isolated from human atherosclerotic tissue, allowing efficient gene transfer to directly investigate signaling pathways in human atherosclerosis. We found that NF-kappa B is activated in these cells and that this activity involves p65, p50, and c-Rel but not p52 or RelB. This NF-kappa B activation can be blocked by overexpression of I kappa B alpha or dominant-negative I kappa B kinase (IKK)-2 but not dominant-negative IKK-1 or NF-kappa B-inducing kinase, resulting in selective inhibition of inflammatory cytokines (tumor necrosis factor alpha, IL-6, and IL-8), tissue factor, and matrix metalloproteinases without affecting the antiinflammatory cytokine IL-10 or tissue inhibitor of matrix metalloproteinases. Our results demonstrate that the canonical pathway of NF-kappa B activation that involves p65, p50, c-Rel, and IKK-2 is activated in human atherosclerosis and results in selective up-regulation of major proinflammatory and prothrombotic mediators of the disease.     

Digestive and liver diseases statistics, 2004

BACKGROUND & AIMS: Digestive and liver diseases are associated with substantial morbidity and mortality in the United States. Statistics about the incidence, prevalence, mortality, and resource utilization of digestive and liver diseases in the United States may be cumbersome to obtain because they are scattered in multiple sources. These data may be useful for policy makers, grant applicants, and authors. METHODS: Data on the most common gastrointestinal and liver diseases were collected from large publicly available national databases. Information was collected on inpatient and outpatient gastrointestinal complaints and diagnoses, gastrointestinal cancers, and deaths from common liver diseases. RESULTS: The leading gastrointestinal complaint prompting an outpatient visit is abdominal pain, with 12.2 million annual visits, followed by diarrhea, nausea, and vomiting. Abdominal pain is the leading outpatient gastrointestinal diagnosis, accounting for 5.2 million visits annually, followed by gastroesophageal reflux disease, with 4.5 million visits. Gallstone disease is the most common inpatient diagnosis, with 262,411 hospitalizations and a median inpatient charge of USD$11,584. Colorectal cancer is the most common gastrointestinal cause of death and is the most common gastrointestinal cancer, with an incidence of 54 per 100,000. Among gastrointestinal cancers, primary liver cancer had the highest increase in incidence from 1992 to 2000. CONCLUSIONS: Gastrointestinal and liver diseases are associated with significant outpatient and inpatient healthcare utilization. Following trends in utilization is important for determining allocation of resources for health care and research.     

Quantitation and distribution of beta-tubulin in human cardiac myocytes

Increasing evidence suggests that derangements of cytoskeletal proteins contribute to alterations in intracellular signaling, myocyte function, and the coupling of myocytes to the extracellular matrix during cardiac hypertrophy and failure. Data from animal studies have shown an increased density of beta-tubulin protein in the right or left ventricle subjected to pressure overload, and have demonstrated that interfering with excess polymerization of beta-tubulin improves contractility. We tested the hypothesis that beta-tubulin is increased in human left ventricular hypertrophy and end-stage heart failure. Confocal microscopy of fluorescently labeled beta-tubulin protein revealed an increased density of the beta-tubulin network in cardiomyocytes from both hypertrophied and failing human hearts as compared to cells from nonfailing hearts. Western blot analysis on total heart homogenate showed no change in beta-tubulin when data were normalized to either actin or calsequestrin, although there was a significant increase in failing human hearts when data were normalized only for a constant amount of protein per heart. The mRNA for beta-tubulin was not changed in hypertrophied hearts, but was significantly decreased in failing human hearts. Thus, similar to animal models, we have shown that the density of the microtubular network within the cardiomyocyte is increased in end-stage failing human hearts. We have also shown for the first time that beta-tubulin density is increased in cells from hypertrophied human hearts. Although the functional implications of this finding in the human heart remain to be explored, data from animal studies suggest that increased beta-tubulin protein contributes to cardiac dysfunction.     

Platelet trans fatty acids in relation to angiographically assessed coronary artery disease

Epidemiological and metabolic studies indicate that a higher intake of trans fatty acids (TFA) may be associated with increased risk of coronary heart disease (CHD). In a cross-sectional study of patients who underwent coronary angiography, the relationships between TFAs, measured in platelets, and the degree of coronary artery disease (CAD) were examined in 191 non-diabetic patients (134 men and 57 women). The degree of CAD was quantified by using an angiographic scoring system developed to provide an estimate of the extent of coronary atherosclerosis: an ‘extent score’. The TFA composition of platelets, including palmitelaidic (16:1ω7t), elaidic (18:1ω9t), trans-10-octadecaenoic acid (18:1 ω8t), trans vaccenic (18:1ω7t), trans-12-octadecaenoic acid (18:1ω6t) and linoelaidic (18:2ω6tt) acids, was measured by using gas chromatography and quantified as a percentage of total fatty acids. After adjustment for established CHD risk indicators, including age, gender, cigarette smoking, hypertension and serum total cholesterol concentration, elaidic acid (P = 0.0300) and trans-10-octadecaenoic acid (P = 0.0434) were positively associated with the extent score of CAD. The adjusted associations between other individual TFAs, including palmitelaidic acid (P = 0.1189), vaccenic acid (P = 0.7651), trans-12-octadecaenoic acid (P = 0.0582) and linoelaidic acid (P = 0.8793), and the extent score were not significant. The results of this study, therefore, provide evidence for an association between particular platelet TFAs and the degree of CAD in the patient population studied.     

cagA positive and negative Helicobacter pylori strains are simultaneously present in the stomach of most patients with non-ulcer dyspepsia: relevance to histological damage

^a Institute of Internal Medicine, University of Siena, Siena, Italy, ^b Institute of Pathology, University of Siena, ^c IRIS, Siena, ^d Institute of Surgical Clinics, University of Siena, ^e Institute of Internal Medicine, University of Bologna, Bologna, Italy

Correspondence to: Dr N Figura, Institute of Internal Medicine, University of Siena, Policlinico Le Scotte, viale Bracci, I-53100 Siena, Italy.

Accepted for publication 6 February 1998

Background/Aims—Infection with Helicobacter pylori strains harbouring the cagA gene (cagA+) is associated with an increased risk of developing peptic ulcer and gastric cancer. The aim of this study was to assess whether H pylori isolates with different cagA status were present in patients with non-ulcer dyspepsia, and whether a variable cagA status is relevant to histological gastric mucosal damage and glandular cell proliferation.
Methods—Well separated H pylori colonies (between 2 and 25) from primary plates, per gastric area, for each of 19 patients with non-ulcer dyspepsia were examined for cagA by hybridisation. Western blotting was used to examine both representative colonies for CagA expression and the patients' sera for antibody response to CagA. Glandular gastric cell proliferation was assessed immunohistochemically.
Results—Of the 747 colonies examined, 45.3% were cagA+. All colonies from four patients were cagA+, and all colonies from two patients were cagA−. In 13 patients (68%) both cagA+ and cagA− colonies were found. CagA expression of isolates corresponded to their cagA status. H pylori strains with different CagA molecular masses were present in three patients. Results based on all 19 patients studied showed that the prevalence of cagA+ colonies in areas with mucosal atrophy associated or not with intestinal metaplasia (67.9%) was significantly higher than in normal mucosa (44.7%) and mucosa from patients with chronic gastritis (44.0%) (p< 0.001). High levels of cell proliferation were associated with histological atrophy with or without intestinal metaplasia, but not with the possession of cagA by organisms colonising the same mucosal sites.
Conclusions—Most patients with non-ulcer dyspepsia are infected by both cagA+ and cagA− H pylori colonies. The cagA status of infecting organisms may play a role in the development of atrophy and intestinal metaplasia.
(GUT 1998;:772-778)

Keywords: gastritis; Helicobacter pylori infection;  cagA;  mucosal atrophy;  cell proliferation

     

The association of eating behavior with risk for morbidity in older women

BACKGROUND: Although an influence of eating behavior on dietary intake and physiology has been documented in several studies, the extent to which eating behavior influences long-term health is uncertain. METHODS: Current dietary restraint, disinhibition, and hunger were assessed using the Eating Inventory in 1252 nonsmoking women aged 55 to 65 years. In addition, subjects reported the presence or absence of 22 specific morbidities, along with general demographic information. Logistic regression was used to examine associations between eating behavior scores and morbidity, adjusting for age, prior smoking status, hormone replacement therapy, education level, and body mass index (BMI). RESULTS: In adjusted models excluding BMI, higher disinhibition scores were associated with small increased risks for hypercholesterolemia (odds ratio [OR] 1.04, p =.045), leg cramps (OR 1.05, p =.044), indigestion (OR 1.06, p =.020), and cataract (OR 1.09, p =.036), and a decreased risk of eczema (OR 0.91, p =.008). In addition, higher hunger scores were associated with increased risk of eczema (OR 1.09, p =.026). However, after adjusting for confounding variables plus BMI, higher disinhibition scores were associated with increased risks for low back pain (OR 1.06, p =.031) and constipation (OR 1.10, p =.004), and associations of disinhibition and hunger with eczema were unchanged (OR 0.90, p =.008 and OR 1.09, p =.024, respectively). Dietary restraint was not associated with morbidity in any model. CONCLUSIONS: Higher disinhibition and hunger scores were associated with small alterations in reported morbidity risk in a large population of nonsmoking older women. Although our cross-sectional study design makes the directionality of these relationships unclear, our results suggest at most a relatively minor independent influence of eating behavior constructs on long-term health.     

Arterial compression of the retro-olivary sulcus of the ventrolateral medulla in essential hypertension and diabetes

Pulsatile arterial compression in the retro-olivary sulcus along the surface of the ventrolateral medulla has been postulated as a mechanism in both essential hypertension and diabetes. The objective of this study was to test the independent effect of arterial compression in the retro-olivary sulcus on each of these diseases, using separate logistic regression models to control for other known risk factors. Study design was case-control. The study population consisted of 147 consecutive patients treated for neurological conditions requiring MRI of the posterior cranial fossa. Information on essential hypertension, diabetes, and risk factors for each disease was abstracted from medical records. Presence of arterial compression was determined by blinded review of magnetic resonance images. In the essential hypertension analysis, odds of arterial compression among hypertensive patients were 2.99-times the odds among normotensive subjects (P=0.04), controlling for hypertension risk factors such as age, body mass index, race, diabetes, and family history of hypertension. Of compressed hypertensive subjects, 56% were compressed on the left and 44% were compressed on the right. In the diabetes analysis, odds of arterial compression among diabetic subjects were 1.14-times the odds among nondiabetic subjects (P=0.83). Of compressed diabetic subjects, 60% were compressed on the left, and 40% were compressed on the right. Results suggest that arterial compression of the retro-olivary sulcus may be an independent risk factor for essential hypertension in this population, supporting the postulate for a treatable (with microvascular decompression) neural mechanism for essential hypertension. However, in the diabetic population, the slight increase in the odds of arterial compression was not significant.     

Outcomes of pancreatoduodenectomy in the cirrhotic patient: risk stratification and meta-analysis

Background

Cirrhosis increases the risk of perioperative mortality in gastrointestinal surgery. Though cirrhosis is sometimes considered a contraindication to pancreatoduodenectomy (PD), few data are available in this patient population. The aim of the present study is to identify predictors of outcome in cirrhotic patients undergoing PD.

A randomized trial comparing levo-alpha acetylmethadol with methadone maintenance for patients in primary care settings in Australia

Aims The present study aimed to compare the efficacy of levo‐alpha‐acetylmethadol (LAAM) and methadone, as measured by retention in treatment and heroin use, in a randomized trial conducted under naturalistic conditions. Setting This study is the first randomized trial comparing LAAM with methadone in the primary care setting. Participants were recruited through 29 medical practitioners working in specialist and generalist settings in Australia. Participants Existing methadone maintenance patients, aged 18 years and over and able to give informed consent, were randomized to receive either LAAM or methadone. A total of 93 patients participated. Intervention After being trained in the use of LAAM, existing methadone prescribers were then able to determine an individually tailored treatment regimen for each patient. The trial was an open‐label study. Methadone and LAAM dosing was supervised through local community pharmacies. Participation in ancillary services (e.g. counselling) was optional for all patients. The treatment period for the trial was 12 months. Measurements Baseline, 3‐, 6‐ and 12‐month interviews were conducted. Outcome measures were retention in treatment, self‐reported heroin use and serious adverse events. Findings There were no significant differences between LAAM and methadone on retention in treatment, nor heroin use. There was a trend for LAAM patients to have lower heroin use than methadone patients. Of the seven serious adverse events in the LAAM group, three were not drug‐related. There were two dosing errors. Conclusions This study demonstrates (a) the efficacy of LAAM as a treatment for heroin dependence, and (b) the capacity for LAAM to be effectively delivered in primary care settings by trained general practitioners and pharmacists. The next challenge is to resolve outstanding safety concerns with LAAM.