Comparison of Glucose Oxidase and Peroxidase as Labels for Antibody in Enzyme-linked Immunosorbent Assay

Horse-radish peroxidase and glucose oxidase were each separately conjugated to identical aliquots of goat IgG containing anti-human IgG antibodies. We used a minor modification of the periodate method of Nakane and Kawaoi to covalently bind each enzyme to IgG. Glucose oxidase conjugates proved superior to peroxidase conjugates based on the following qualities. The glucose oxidase conjugates had 1) usable dilutions 2 to 10 times greater than peroxidase conjugates, 2) much lower background or control non-specific activities, and 3) nearly twice the sensitivity as expressed by absorbance change vs. change in antigen. Comparison of the antibody titers showed the glucose oxidase conjugate with 80% and the peroxidase conjugate with 20% of the original goat antibody.     

Advances in Imaging of Cardiac Allograft Rejection

Endomyocardial biopsy with its inherent invasiveness and morbidity calls for the development of noninvasive imaging methods to evaluate heart transplant recipients. While conventional imaging technologies report on anatomical and metabolic changes in heart grafts, macrophage-targeted imaging could allow disease detection before gross anatomical and functional changes have occurred. One important approach in magnetic resonance–based molecular imaging exploits an increased T2/T2* relaxation effect, occurring when phagocytic cells localized in the heart graft take up iron-oxide nanoparticles. This methodology of nanoparticle reporting on immune cell accumulation in the graft combined with precise functional and morphological information of cardiac MRI has potential to supplant endomyocardial biopsy. The use of multifunctional nanoparticles fit for multiple imaging modalities (magnetic, optical, and nuclear) will help improve methods of ex vivo and in vitro imaging of allograft rejection and also further our knowledge of allograft rejection by providing a tool for nondestructive serial in vivo assessment.     

A preliminary investigation into the psychometric properties of the Dublin Extrapersonal Neglect Assessment (DENA): A novel screening tool for extrapersonal neglect

Extrapersonal neglect is one clinical manifestation that can occur following stroke. Existing neglect assessment procedures have been criticised for lengthy administration and do not assess how extrapersonal space is affected. This study investigated the psychometric properties of a new, time-efficient screening tool for extrapersonal neglect. Full ethical approval was granted and consent obtained from 50 participants with first-time stroke. Participants were screened for extrapersonal neglect on two consecutive days by two raters using the Dublin Extrapersonal Neglect Assessment (DENA) to test inter-rater reliability. Construct validity of the DENA was investigated by comparing the DENA to the Catherine Bergego Scale (CBS). Additional analyses were calculated between the DENA and the extrapersonal items of the CBS (CBS-E). The kappa statistic, intraclass correlation coefficients (ICCs) and Bland Altman analyses were calculated to determine excellent inter-rater reliability (ICC 0.971, κ?=?.876) and significant correlation between the DENA and the CBS and CBS-E (ICC 0.870, 0.934, κ?=?.793, .833, respectively). Bland Altman analyses demonstrated acceptable levels of agreement between the DENA raters, and the DENA and CBS, with no systematic differences evident. The DENA provides clinicians with a quick and psychometrically sound screening tool for extrapersonal neglect to ensure this impairment is addressed in stroke rehabilitation.     

Cystatin C deficiency increases elastic lamina degradation and aortic dilatation in apolipoprotein E-null mice

The pathogenesis of atherosclerosis and abdominal aortic aneurysm involves substantial proteolysis of the arterial extracellular matrix. The lysosomal cysteine proteases can exert potent elastolytic and collagenolytic activity. Human atherosclerotic plaques have increased cysteine protease content and decreased levels of the endogenous inhibitor cystatin C, suggesting an imbalance that would favor matrix degradation in the arterial wall. This study tested directly the hypothesis that impaired expression of cystatin C alters arterial structure. Cystatin C-deficient mice (Cyst C-/-) were crossbred with apolipoprotein E-deficient mice (ApoE-/-) to generate cystatin C and apolipoprotein E-double deficient mice (Cyst C-/-ApoE-/-). After 12 weeks on an atherogenic diet, cystatin C deficiency yielded significantly increased tunica media elastic lamina fragmentation, decreased medial size, and increased smooth muscle cell and collagen content in aortic lesions of ApoE-/- mice. Cyst C-/-ApoE-/- mice also showed dilated thoracic and abdominal aortae compared with control ApoE-/- mice, although atheroma lesion size, intimal macrophage accumulation, and lipid core size did not differ between these mice. These findings demonstrate directly the importance of cysteine protease/protease inhibitor balance in dysregulated arterial integrity and remodeling during experimental atherogenesis.     

The yield and the predictors of esophageal pathology when upper endoscopy is used for the initial evaluation of dysphagia

BACKGROUND: The utility of EGD when used as an initial test for the evaluation of dysphagia is unclear. The objective was to determine the yield and the predictive factors of significant pathology when EGD is performed as the initial test to evaluate dysphagia. METHODS: This is a retrospective analysis of a computerized database. Data on patients who underwent EGD for dysphagia were retrieved from the endoscopy database of 6 endoscopy units. Patients who had undergone prior esophageal evaluation, failed EGD, or who had a history of prior upper-GI pathology were excluded. Univariate and multivariable logistic regression analyses were performed to evaluate any relation between endoscopic findings and presenting clinical features. RESULTS: A total of 1649 patients with dysphagia (mean age 56.7 years, standard deviation 16.4; M:F 3:2) were analyzed. Abnormal findings at EGD were found in 70% (1150) of the patients, and a major pathology was seen in 54% (898). Male gender (p=0.0001), heartburn (p=0.0007), and odynophagia (p=0.0001) predicted the presence of major pathology. Cancer was found in 4% (70) of patients and was predicted by male gender (p=0.0002), age (p=0.01), and weight loss (p=0.04). The esophagus was normal in 29% (483) of patients and was predicted by female gender (p=0.0001) and the absence of heartburn (p=0.0004) but not age. There was a lack of details on patients' presentation and clinical history and an absence of long-term clinical follow-up. CONCLUSIONS: EGD is an effective and an appropriate tool for the initial evaluation of patients presenting with dysphagia. Early EGD should be considered, particularly, in male patients aged more than 40 years old who concomitantly report heartburn, odynophagia, or weight loss.     

In vitro evaluation of antibiotic prophylaxis in the prevention of biliary stent blockage

BACKGROUND: Bacterial adherence and biofilm formation are important factors in the blockage of biliary stents. Clinical studies with oral antibiotic prophylaxis to prevent stent blockage have produced conflicting results. The aim of this study was to evaluate the in vitro effect of single antibiotic (ciprofloxacin, ceftazidime, or ampicillin) treatment on adherence of Escherichia coli and Enterococcus to plastic stents. METHODS: Selected clinical isolates of E coli and Enterococcus were perfused through a modified Robbins device containing segments of polyethylene stents. The stents were removed daily and the number of bacteria attached was measured. The effect of antibiotic treatment on bacterial adherence was tested by the perfusion of individual antibiotics into separate modified Robbins devices using a side-arm adaptor and the results were compared with saline controls. RESULTS: Compared with the saline controls, ciprofloxacin and ceftazidime caused a 10- to 100-fold reduction in the number of E coli attached to the stents, whereas ampicillin had no effect on adherence of E coli. Ampicillin caused a 5- to 10-fold reduction in Enterococcus adherence but there was no change with ceftazidime. Sustained reduction in E coli adherence was observed with prolonged ciprofloxacin perfusion. CONCLUSION: Timely treatment with appropriate antibiotics reduced bacterial adherence in vitro and may be potentially beneficial in the prevention of stent blockage.     

From vulnerable plaque to vulnerable patient: a call for new definitions and risk assessment strategies: Part II

Atherosclerotic cardiovascular disease results in >19 million deaths annually, and coronary heart disease accounts for the majority of this toll. Despite major advances in treatment of coronary heart disease patients, a large number of victims of the disease who are apparently healthy die suddenly without prior symptoms. Available screening and diagnostic methods are insufficient to identify the victims before the event occurs. The recognition of the role of the vulnerable plaque has opened new avenues of opportunity in the field of cardiovascular medicine. This consensus document concludes the following. (1) Rupture-prone plaques are not the only vulnerable plaques. All types of atherosclerotic plaques with high likelihood of thrombotic complications and rapid progression should be considered as vulnerable plaques. We propose a classification for clinical as well as pathological evaluation of vulnerable plaques. (2) Vulnerable plaques are not the only culprit factors for the development of acute coronary syndromes, myocardial infarction, and sudden cardiac death. Vulnerable blood (prone to thrombosis) and vulnerable myocardium (prone to fatal arrhythmia) play an important role in the outcome. Therefore, the term "vulnerable patient" may be more appropriate and is proposed now for the identification of subjects with high likelihood of developing cardiac events in the near future. (3) A quantitative method for cumulative risk assessment of vulnerable patients needs to be developed that may include variables based on plaque, blood, and myocardial vulnerability. In Part I of this consensus document, we cover the new definition of vulnerable plaque and its relationship with vulnerable patients. Part II of this consensus document will focus on vulnerable blood and vulnerable myocardium and provide an outline of overall risk assessment of vulnerable patients. Parts I and II are meant to provide a general consensus and overviews the new field of vulnerable patient. Recently developed assays (eg, C-reactive protein), imaging techniques (eg, CT and MRI), noninvasive electrophysiological tests (for vulnerable myocardium), and emerging catheters (to localize and characterize vulnerable plaque) in combination with future genomic and proteomic techniques will guide us in the search for vulnerable patients. It will also lead to the development and deployment of new therapies and ultimately to reduce the incidence of acute coronary syndromes and sudden cardiac death. We encourage healthcare policy makers to promote translational research for screening and treatment of vulnerable patients.     

Changing concepts of atherogenesis

This review discusses three stages in the life history of an atheroma: initiation, progression and complication. Recruitment of mononuclear leucocytes to the intima characterizes initiation of the atherosclerotic lesion. Specific adhesion molecules expressed on the surface of vascular endothelial cells mediate leucocyte adhesion: the selectins and members of the immunoglobulin superfamily such as vascular cell adhesion molecule-1 (VCAM-1). Once adherent, the leucocytes enter the artery wall directed by chemoattractant chemokines such as macrophage chemoattractant protein-1 (MCP-1). Modified lipoproteins contain oxidized phospholipids which can elicit expression of adhesion molecule and cytokines implicated in early atherogenesis. Progression of atheroma involves accumulation of smooth muscle cells which elaborate extracellular matrix macromolecules. These processes appear to result from an eventual net positive balance of growth stimulatory versus growth inhibitory stimuli, including proteins (cytokines and growth factors) and small molecules (e.g. prostanoids and nitric oxide). The clinically important complications of atheroma usually involve thrombosis. Arterial stenoses by themselves seldom cause acute unstable angina or acute myocardial infarction. Indeed, sizeable atheroma may remain silent for decades or produce only stable symptoms such as angina pectoris precipitated by increased demand. Recent research has furnished new insight into the molecular mechanisms that cause transition from the chronic to the acute phase of atherosclerosis. Thrombus formation usually occurs because of a physical disruption of atherosclerotic plaque. The majority of coronary thromboses result from a rupture of the plaque's protective fibrous cap, which permits contact between blood and the highly thrombogenic material located in the lesion's lipid core, e.g. tissue factor. Interstitial collagen accounts for most of the tensile strength of the plaque's fibrous cap. The amount of collagen in the lesion's fibrous cap depends upon its rate of biosynthesis stimulated by factors released from platelets (e.g. transforming growth factor beta or platelet-derived growth factor), but inhibited by gamma interferon, a product of activated T cells found in plaques. Degradation by specialized enzymes (matrix metalloproteinases) also influences the level of collagen in the plaque's fibrous cap. Such studies illustrate how the application of cellular and molecular approaches has fostered a deeper understanding of the pathogenesis of atherosclerosis. This increased knowledge of the basic mechanisms enables us to understand how current therapies for atherosclerosis may act. Moreover, the insights derived from recent scientific advances should aid the discovery of new therapeutic targets that would stimulate development of novel treatments. Such new treatments could further reduce the considerable burden of morbidity and mortality due to this modern scourge, and reduce reliance on costly technologies that address the symptoms rather than the cause of atherosclerosis.