Electrochemical Biosensor for Markers of Neurological Esterase Inhibition.

A novel, integrated experimental and modeling framework was applied to an inhibition-based bi-enzyme (IBE) electrochemical biosensor to detect acetylcholinesterase (AChE) inhibitors that may trigger neurological diseases. The biosensor was fabricated by co-immobilizing AChE and tyrosinase (Tyr) on the gold working electrode of a screen-printed electrode (SPE) array. The reaction chemistry included a redox-recycle amplification mechanism to improve the biosensor's current output and sensitivity.

Integrated Experimental and Theoretical Studies on an Electrochemical Immunosensor.

Electrochemical immunosensors (EIs) integrate biorecognition molecules (e.g., antibodies) with redox enzymes (e.

Cyclophosphamide is neuroprotective in a gerbil model of transient severe focal cerebral ischemia: correlation with effects of tirilazad mesylate (U-74006F).

Using a gerbil model of severe, temporary focal ischemia (3 h unilateral carotid occlusion), preliminary experiments identified an involvement of neutrophils in the reperfusion injury to the ischemic hemisphere. The present experiments were designed to (1) quantitate the temporal accumulation of neutrophils in the gerbil model, (2) determine if cyclophosphamide-induced neutropenia provided cytoprotection to the ischemic hemisphere, and (3) attempt to correlate the cytoprotective efficacy of tirilazad mesylate with possible effects on postischemic neutrophil accumulation. Following 3 h of unilateral carotid occlusion, animals were collected at increasing times of reperfusion and the CA1 region of the hippocampus and the lateral cortex were assessed for postischemic neuronal damage using a semiquantitative index (N.

A non-radioactive iothalamate and p-aminohippuric acid high-performance liquid chromatographic method for simultaneously measuring glomerular filtration rate and renal blood flow in the rat.

A high-performance liquid chromatographic (HPLC) assay method has been developed for the quantitative determination of iothalamate and p-aminohippuric acid (PAH) concentrations in serum and urine samples in the male rat. Glomerular filtration rate (GFR) was measured as clearance of iothalamate, while effective renal blood flow (ERBF) was measured as clearance of PAH. The method is simple, rapid and sensitive and detects iothalamate and PAH in rat serum and urine following administration of bolus doses and continuous infusions of iothalamate and PAH.

Sulfhydryl compounds, captopril, and MPG inhibit complement-mediated myocardial injury.

Factors including complement activation, neutrophil infiltration, and oxygen-derived free radicals have been implicated in the pathogenesis of myocardial tissue injury during ischemia and reperfusion. Certain sulfhydryl-containing compounds have been shown to inhibit complement activation. The sulfhydryl compounds captopril and N-(2-mercaptopropionyl)-glycine (MPG) are antioxidant compounds that previously have been shown to protect the myocardium from ischemia and reperfusion-induced damage.

Soluble complement receptor type 1 prevents human complement-mediated damage of the rabbit isolated heart.

The purpose of this study was to determine if recombinant human soluble CR1 (sCR1) could prevent tissue damage associated with the activation of human complement. Directly mediated human complement-dependent myocardial injury was induced in the rabbit isolated heart perfused with a Krebs-Henseleit buffer containing 6% human plasma. There were three study groups: 1) 6% heat-inactivated human plasma (control); 2) 6% normal human plasma (NHP); or 3) 6% normal human plasma + 20 nM sCR1 (NHP + sCR1).

Effects of complement activation in the isolated heart. Role of the terminal complement components.

The mechanisms of the complement-mediated myocardial injury associated with ischemia and reperfusion have not been elucidated fully. Complement activation may directly mediate injury through actions of the anaphylatoxins C3a and C5a or generation of the membrane attack complex C5b-9. A model was developed to examine the direct effects of complement activation on heart function, assess myocardial tissue damage, and determine which complement components mediate tissue injury.

The use of sodium dodecyl sulfate-polyacrylamide gel electrophoresis to detect renal damage in Sprague-Dawley rats treated with gentamicin sulfate.

Renal toxicity is a common manifestation to the exposure of laboratory animals and humans to a wide range of xenobiotics. Traditional methods for evaluating renal damage by clinical chemistry such as blood urea nitrogen (BUN) and serum creatinine are not sensitive to early, mild changes. The use of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to measure the molecular weight spectrum of urinary proteins allows for an evaluation of the functional changes associated with renal damage.

Complement levels in septic primates treated with anti-C5a antibodies.

During gram-negative sepsis it is known that endotoxin activates complement by the alternate pathway. The complement anaphylatoxin C5a, a result of this activation, is thought to play a key role in attracting and activating neutrophils in the lungs, leading to the adult respiratory distress syndrome. Complement levels were measured in primates made septic by Escherichia coli infusions.

Differential prostacyclin production by human umbilical vasculature.

Using the avidin-biotin immunohistochemical technique with rabbit antihuman 6-keto-prostaglandin (PG)-F1 alpha (6KPGF), we studied the distribution of the stable prostacyclin metabolite, 6KPGF, in 14 formaldehyde-fixed human umbilical cords. All umbilical veins demonstrated intense endothelial cell staining. None of the arteries stained.

Inhibition of C1q functions by RHP, a protein elevated in sera from patients with rheumatoid arthritis.

We have previously shown that serum levels of C1q, unbound to C1r X C1s, are elevated in rheumatoid arthritis. We have also shown that RHP, a newly described serum protein which affects the C1q-anti C1q precipitin reaction, is also present at elevated levels in rheumatoid arthritis. We now show that RHP inhibits the hemolytic activity of C1q, disaggregates C1, and inhibits the ability of C1q bound to latex beads or to aggregated IgG to enhance the oxidative metabolism of neutrophils.

Plasma catecholamines in acute magnesium deficiency in weanling rats.

A controlled study was conducted to quantitate plasma catecholamines in magnesium-deficient weanling rats experiencing the seizure-shock episode. Eighty-four male Sprague-Dawley rats each weighing 35.6 +/- 0.

Effects of anti-C5a antibodies on the adult respiratory distress syndrome in septic primates.

In vitro and in vivo studies have suggested that human complement component C5a plays a key role in neutrophil injury in the adult respiratory distress syndrome (ARDS). First, using leukocyte aggregometry, we demonstrated that the addition of a recently developed rabbit anti-human polyclonal antibody to C5a des arg to endotoxin-activated plasma prevented leukocyte aggregation in vitro. We then administered the anti-C5a des arg antibody to septic primates (Macaca fascicularis).

Bioluminescent enzyme immunoassay for estriol. Use of reversibly inactivated bacterial luciferase as label.

A bioluminescent enzyme immunoassay using estriol labeled with reversibly inactivated bacterial luciferase is described. An estriol derivative bearing an alkylthiolsulfonate is linked to the cysteinyl thiols of luciferase by formation of mixed disulfide linkages; thus, luciferase becomes inactive. After immunoassay, the inactive luciferase of the label bound to the immunoprecipitate is reactivated by incubation with dithiothreitol and the luciferase activity then is quantitated by a 20-s reaction performed with an automated luminometer (LKB 1251).

Complement activation and clearance in acute illness and injury: evidence for C5a as a cell-directed mediator of the adult respiratory distress syndrome in man.

The appearance of the adult respiratory distress syndrome (ARDS) during the course of acute illness is believed to result, in part, from intrapulmonary neutrophil sequestration and degranulation induced by circulating inflammatory mediators. To evaluate the role of complement-neutrophil interactions in the pathogenesis of ARDS in man, 34 patients suffering from intra-abdominal sepsis (seven), multisystem trauma (15), or acute pancreatitis (12) were serially studied with regard to neutrophil migratory responses to C5a and F-Met-Leu-Phe, lysosomal content of beta-glucuronidase and lysozyme, and simultaneously obtained plasma levels of immunoreactive C3adesArg and C5adesArg. Nineteen patients developed ARDS.

Immunohistochemical localization of 6-keto-PGF1-alpha in canine coronary vasculature.

The localization of the prostacyclin metabolite, 6-keto-PGF1-alpha, in canine coronary vasculature was accomplished using immunohistochemical techniques (avidin-biotin method of immunoperoxidase staining). Six-keto-PGF1-alpha was localized to the intimal endothelial cell layer of epicardial and intramyocardial arteries and veins. No specific staining was seen in the the media or adventitia of canine coronary vasculature, or in capillaries, or myocardial fibers.

Complement-induced expression of cryptic receptors on the neutrophil surface: a mechanism for regulation of acute inflammation in trauma.

We explored the hypothesis that identified changes in neutrophil function in patients with acute injury result from in vivo exposure to C5a. To evaluate this hypothesis, we performed a battery of tests on 26 trauma patients (14 with blunt injury, 12 with penetrating injury). Measured were plasma levels of the complement activation products C3a and C5a; neutrophil chemotaxis to C5a and N-formyl-methionyl-leucyl-phenylalanine (FMLP); neutrophil receptors for FMLP and C3b; and superoxide response to FMLP and serum-opsonized zymosan.

Heterophile antibodies in the serum of children with nephrotic syndrome.

Serum of children with the nephrotic syndrome contained high titers of a (19S) IgM antibody against sheep, horse, guinea pig, rat, and rabbit red blood cells but not against cow red blood cells. There was high correlation between high titers of antisheep antibodies and active nephrotic syndrome in the children with minimal change nephrotic syndrome. The antibody differed from the Paul-Bunnell antibody found in patients with infectious mononucleosis and from the anti-Forssman, Hangautziu-Deicher antibody found in patients with horse serum sickness.

Manufacture of antithymocyte globulin (ATGAM) for clinical trials.

Methods are described for preparing large amounts of horse anti-human thymocyte globulin (ATG, ATGAM; The Upjohn Company) for clinical use. These methods have been used since 1968 to provide material for clinical trials. Characteristics of 40 lots of ATG are summarized.