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Biomedical subjects

A D Romaschin

Publications and source records attributed to A D Romaschin.

At least 19 recordsLinked to original sources
PubMed1998

A rapid assay of endotoxin in whole blood using autologous neutrophil dependent chemiluminescence.

A rapid (30 min) whole blood assay for the detection of lipopolysaccharide (LPS) is described. This chemiluminescent (CL) assay utilizes the CR1 and CR3 receptor-induced oxidant production of polymorphonuclear leucocytes as a detection platform. The differential priming of neutrophils in whole blood by LPS-antibody complexes allows the specificity of the assay to be achieved. Oxidant released in response to complement opsonized zymosan results in luminol oxidation and subsequent light emission. This is dependent on heat labile putative complement proteins in the plasma. The assay consists of a control which measures baseline whole blood neutrophil oxidant production. The test assay contains murine monoclonal IgM antibody against the Lipid A epitope of LPS and measures the enhanced chemiluminescent response of the neutrophils in the presence of LPS-antibody complexes. Maximal sensitivity of the CL assay is dependent upon optimal antigen-antibody equivalence and duration of pre-incubation with the whole blood sample. The quantification of LPS is possible by inclusion of a positive control containing a maximally reactive LPS dose (800 pg/ml Escherichia coli 055:B5 LPS at an antibody concentration of 0.8 microg/assay). The CL assay is insensitive to variations in patient neutrophil concentration over a minimum range of 0.5 to 20 x 10(9) cells/l. The CL assay is widely reactive with the LPS of many strains of gram negative bacteria but not with the cell wall products of gram positive bacteria or Candida and Aspergillus. In comparison to acid extraction chromogenic LAL, the CL assay demonstrates superior recovery precision and accuracy in in vitro studies. This was reproducible over a wide range of LPS concentrations (0.017-1.6 EU/ml or 20-2000 pg/ml). This assay may be a clinically useful tool for the diagnosis of infection or endotoxin in patients.

Antibodies, Bacterial↗
PubMed1996

Sequential ischemia/reperfusion results in contralateral skeletal muscle salvage.

Sequential ischemia/reperfusion in a paired canine gracilis muscle model resulted in significant muscle salvage. In this model, one randomly chosen gracilis muscle was subjected to 5 h of ischemia followed by 48 h of in vivo reperfusion. The contralateral (second) muscle was then made ischemic and reperfused using the same protocol. Muscle necrosis was determined at the end of 48 h of reperfusion. A mean 60% reduction in muscle necrosis was observed in the second group of muscles. Analysis of tissue adenine nucleotides indicated that significant sparing of ATP utilization occurred in the second muscle group during ischemia. Preliminary analysis of tissue heat shock proteins (HSP) showed that the second group of muscles had a different pattern of HSP expression before the onset of ischemia. The results suggest that reduced ATP utilization and altered HSP expression in the second muscle play a role in the tissue salvage observed in this sequential muscle ischemia model.

Adenosine Triphosphate↗
PubMed1996

Plasma activation of neutrophil CD18 after skeletal muscle ischemia: a potential mechanism for late systemic injury.

Reperfusion of acutely ischemic skeletal muscle is associated with neutrophil activation, which may augment local injury or cause damage to distant organs. Polymorphonuclear neutrophil glycoprotein CD18 plays a role in this injury, since its blockade substantially reduces damage; however, its mechanisms of control during reperfusion are poorly understood. The purpose of this study was to investigate the importance of circulating plasma factors to CD18-dependent neutrophil function during reperfusion and to relate these to quantitative expression of CD18. Eight rabbits were subjected to hindlimb ischemia for 5 h, followed by 48 h of reperfusion. Plasma collected at seven intervals was incubated with unstimulated neutrophils from uninjured rabbits. CD18-specific neutrophil activation was evaluated by quantifying adherence to protein-coated polystyrene and by measuring oxidant production, detected by chemiluminescence after exposure to complement-opsonized zymosan. CD18 was quantified cytofluorometrically. Plasma collected at end ischemia and during early reperfusion affected no significant alterations of adhesion, oxidant production, or CD18. Late reperfusion plasma (between 8 and 48 h) significantly increased adherence and oxidant production (to 4.11 +/- 0.61 and 2.60 +/- 0.32 times the values of preischemic plasma, P < 0.006). Peak adherence, oxidant production, and CD18 expression were evoked synchronously by 24 h plasma. CD18 expression increased only at 24 h and did not increase proportional to increases in adherence and oxidant production. Control plasma (nonischemic, n = 5) elicited no significant differences of any inflammatory measure during sham ischemia or reperfusion. These results indicate that endogenous mediators may evoke a progressive systemic inflammatory response after ischemia by stimulating CD18-dependent neutrophil function in a delayed but prolonged manner.

Animals↗
PubMed1994

Salvage of postischemic skeletal muscle by monoclonal antibody blockade of neutrophil adhesion molecule CD18.

Reperfusion of ischemic skeletal muscle is associated with neutrophil (PMN) adherence to damaged endothelium and PMN-mediated tissue destruction. Neutrophils may attach to endothelium through surface adhesive molecules, such as CD18. The purpose of this study was to determine whether monoclonal antibody blockade of CD18 would reduce skeletal muscle necrosis associated with ischemia and reperfusion. In rabbits, an entire hindlimb was rendered ischemic for 4 hr, followed by 48 hr of in vivo reperfusion. Animals were allocated to one of five treatment groups: ischemia/reperfusion without treatment (I/R controls), I/R plus treatment with the anti-CD18 antibody IB4 (end-ischemic 2 mg/kg dose), I/R plus treatment with an identical dose of isotype-matched control Ig, I/R plus anterior compartment fasciotomy, or I/R plus both IB4 and fasciotomy. After 48 hr of reperfusion anterior tibial muscle necrosis was assessed (by tetrazolium staining and computerized planimetry), wet:dry muscle weights (W:D) were determined, and muscle PMN sequestration was measured by myeloperoxidase (MPO) activity. IB4-treated animals exhibited markedly reduced muscle MPO activity, compared to untreated animals. Although all interventions reduced edema formation (W:D ratios), none did so significantly. IB4 treatment reduced muscle necrosis when used alone (to 28 +/- 7%, vs. 48% +/- 6% in untreated controls), however this was not statistically significant (P = 0.06).2+ Fasciotomy significantly reduced necrosis (to 22 +/- 2%, P < 0.05); however, the addition of IB4 to fasciotomy resulted in necrosis that was significantly lower than that after fasciotomy alone (12 +/- 4%, P < 0.05 vs fasciotomy group) and the least necrosis of any group.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Surface modification of the biomedical polymer poly(ethylene terephthalate).

X-ray photoelectron spectroscopy was used to characterize modified surfaces of a biomedically important polymer, poly(ethylene terephthalate). Several modification schemes were investigated and direct silanization with 3-aminopropyltriethoxysilane was found to be the optimum procedure, resulting in an aminated surface. Surface coverage of up to 100% was achieved with retention of the polymeric structural integrity. Further activation of the silanized surface was accomplished with two cross-linkers, glutaraldehyde and sebacoyl chloride. A simple biomolecule, L-cysteine, was successfully immobilized onto a surface pre-treated with 3-aminopropyltriethoxysilane and glutaraldehyde, with a coverage of 42%.

Biocompatible Materials↗
PubMed1993

Differential stimulation of macrophage procoagulant activity by vascular grafts.

PURPOSE: The mechanism by which some graft materials are more thrombogenic than others is poorly understood. We hypothesized that differential induction of macrophage procoagulant activity (PCA) by various materials may contribute to variable thrombogenicity. METHODS: Thioglycollate-elicited murine peritoneal macrophages were added to disks of Dacron and expanded polytetrafluoroethylene (ePTFE). After adherence, macrophages were incubated with and without endotoxin (lipopolysaccharide) and then recovered by sonication for determination of PCA with a one-step clotting bioassay. RESULTS: PCA was significantly higher in cells after incubation on Dacron compared with ePTFE both in the absence of lipopolysaccharide (243 +/- 76 vs 68 +/- 39 mU, n = 4) and after stimulation with lipopolysaccharide (491 +/- 137 vs 139 +/- 41 mU, n = 4) (p < 0.01, analysis of variance). Using factor-deficient plasmas, we found that this PCA was consistent with tissue factor. This differential induction of PCA was related to increased macrophage adherence to Dacron compared to that to ePTFE (9374 +/- 1158 vs 2111 +/- 330 cells/mm2; n = 4; p < 0.01, analysis of variance). CONCLUSIONS: The thrombogenic nature of Dacron correlates with its ability to adhere macrophages and induce PCA. Strategies aimed at modulating these effects may reduce the thrombogenicity of vascular grafts and therefore potentially the incidence of graft thrombosis.

Analysis of Variance↗
PubMed1992

Systemic phospholipase A2 and cachectin levels in adult respiratory distress syndrome and multiple-organ failure.

In this clinical study we have prospectively measured plasma phospholipase A2 (PLA2) activity and tumor necrosis factor (TNF) levels in ventilated intensive care unit (ICU) patients with (n = 9) and without (n = 12) evidence of respiratory distress syndrome (ARDS) and multiple-organ failure (MOF). The median peak TNF concentration in control patients was 40 ng/L (range less than 40-100 ng/L) and in ARDS patients 231 ng/L (range 100-2550 ng/L; p less than 0.001). All of the control patients were discharged alive from the ICU, whereas 6 of 9 ARDS patients died in the ICU. In 6 ARDS patients, it was possible to measure more than 4 consecutive plasma TNF levels. Of these 6 patients, the 3 with persistent elevations in systemic TNF above 230 ng/L succumbed (p less than 0.05, one-tailed). Patients with ARDS also had parallel elevations in plasma PLA2 activity above controls. These elevations were significant for arterial PLA2 activity but not for venous PLA2 activity. Our study suggests that serial measurement of plasma (arterial or venous) TNF levels may have (1) prognostic and (2) etiologic significance in ICU patients with ARDS and MOF.

Critical Care↗
PubMed1992

Prolonged adenine nucleotide resynthesis and reperfusion injury in postischemic skeletal muscle.

Skeletal muscle ischemia results in energy depletion and intracellular acidosis. Reperfusion is associated with impaired adenine nucleotide resynthesis, edema formation, and myocyte necrosis. The purpose of these studies was to define the time course of cellular injury and adenine nucleotide depletion and resynthesis in postischemic skeletal muscle during prolonged reperfusion in vivo. The isolated canine gracilis muscle model was used. After 5 h of ischemia, muscles were reperfused for either 1 or 48 h. Lactate and creatine phosphokinase (CPK) release during reperfusion was calculated from arteriovenous differences and blood flow. Adenine nucleotides, nucleosides, bases, and creatine phosphate were quantified by high-performance liquid chromatography, and muscle necrosis was assessed by nitroblue tetrazolium staining. Reperfusion resulted in a rapid release of lactate, which paralleled the increase in blood flow, and a delayed but prolonged release of CPK. Edema formation and muscle necrosis increased between 1 and 48 h of reperfusion (P less than 0.05). Recovery of energy stores during reperfusion was related to the extent of postischemic necrosis, which correlated with the extent of nucleotide dephosphorylation during ischemia (r = 0.88, P less than 0.001). These results suggest that both adenine nucleotide resynthesis and myocyte necrosis, which are protracted processes in reperfusing skeletal muscle, are related to the extent of nucleotide dephosphorylation during ischemia.

Adenine Nucleotides↗
PubMed1991

Improved biocompatibility of silicone rubber by removal of surface entrapped air nuclei.

Biomaterials activate the complement system which is important since C3a promotes platelet aggregation and release, and C5a activates neutrophils that may augment coagulation. Tiny air nuclei (microbubbles) are found in the surface roughness of biomaterials on exposure to a liquid, therefore two interfaces exist: (a) a blood/biomaterial, and (b) a blood/air interface. Experiments were carried out that documented that air bubbles activate complement and augment in vitro platelet aggregation in human plasma. The air nuclei were removed from the surface of silicone rubber by a technique termed denucleation to determine if complement activation and platelet aggregation could be reduced. We observed a significant reduction in C3a and C5a in the plasma samples incubated with denucleated silicone rubber as compared to the control samples (p less than 0.001, ANOVA). The plasma incubated with the denucleated silicone caused reduced platelet aggregation as compared to the plasma incubated with the control silicone when added to a platelet suspension (p less than 0.001, ANOVA). Surface chemical analysis by x-ray photo-electron spectroscopy (XPS) showed no change in the silicone rubber surface after the denucleation procedure.

Air↗
PubMed1990

European versus North American cardioplegia: comparison of Bretschneider's and Roe's cardioplegic solutions in a canine model of cardiopulmonary bypass.

Roe's and Bretschneider's crystalloid cardioplegic solutions were compared in a canine model of total cardiopulmonary bypass with 4.5 hours of hypothermic (27 degrees C) ischemic arrest and 60 minutes of reperfusion. Bretschneider's solution (Group I, six dogs) preserved tissue adenosine triphosphate (ATP) near control levels and maintained coronary effluent pH near 7.0 throughout the ischemic interval, while Roe's solution (Group II, six dogs) allowed progressive acidosis and depletion of ATP (P less than 0.005 versus control). Group I had supranormal left ventricular function during reperfusion (greater than 100% of pre-arrest function) but Group II regained only 40-75% of pre-arrest function. Group I had 2.82% +/- 3.61% necrosis of heart mass and Group II 9.33% +/- 8.26 (P less than 0.10). We conclude that Bretschneider's solution provided better myocardial protection than Roe's solution. The development of acidosis in the Roe group suggests that the more effective buffering of Bretschneider's solution with histidine is the probable basis for its superiority.

Animals↗
PubMed1990

Exogenous magnesium chloride-adenosine triphosphate administration during reperfusion reduces the extent of necrosis in previously ischemic skeletal muscle.

The lower extremity may be exposed to prolonged periods of ischemia, resulting in depletion of intracellular energy stores in the affected skeletal muscle. The role of adenine nucleotide reduction and failure of resynthesis on reperfusion in determining the extent of muscle necrosis was investigated in this study, in addition to the possible beneficial effects of the addition of exogenous adenosine triphosphate-magnesium chloride during early reperfusion. The isolated paired canine gracilis muscle model was used. After 4 hours of normothermic ischemia in group I, a perfusate Krebs-Henseleit solution plus the gradual reintroduction of oxygenated blood flow was compared to standard reperfusion. In group II, a similar infusion protocol was used, with the addition of 2 mmol/L adenosine triphosphate-magnesium chloride and compared to normal reperfusion. Adenosine triphosphate-magnesium chloride resulted in the salvage of skeletal muscle, 57% +/- 12% versus 44% +/- 14% (p less than 0.05, n = 6 pairs). Reperfusion with the solution alone increased the resulting necrosis (42% +/- 13% vs 60% +/- 20%, n = 6 pairs). Adenine nucleotide stores were not increased, but oxygen consumption was increased by magnesium chloride-adenosine triphosphate (p less than 0.05, analysis of variance [ANOVA]). A clear relationship was demonstrated between the fall in energy stores, as measured by a change in energy charge potential from preischemia to end ischemia levels, and the extent of resulting necrosis (p less than 0.01). In summary, the addition of 2 mmol/L to an infusion of Krebs-Henseleit solution during reperfusion results in significant salvage of skeletal muscle.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenine Nucleotides↗
PubMed1990

The effect of ischemia/reperfusion on adenine nucleotide metabolism and xanthine oxidase production in skeletal muscle.

Prolonged ischemia to skeletal muscle as occurs after an acute arterial occlusion results in alterations in adenine nucleotide metabolism. Adenosine triphosphate continues to be used for cellular functions, and an ischemia-induced degradation of phosphorylated adenine nucleotides is initiated. In this experiment we demonstrated the time-dependent aspect of adenine nucleotide depletion during ischemia and the production of large quantities of soluble precursors. In addition, we studied the rate of conversion of xanthine dehydrogenase to xanthine oxidase, a potential source of oxygen-free radicals, after controlled periods of total normothermic ischemia (4 hours and 5 hours) and during the reperfusion phase. During ischemia complete depletion of creatine phosphate occurred in both groups, and adenosine triphosphate fell from 22.1 +/- 1.3 to 10.3 +/- 1.4 mumol/gm dry weight after 4 hours and from 21.6 +/- 0.7 to 3.9 +/- 0.8 mumol/gm dry weight after 5 hours (p less than 0.05). During reperfusion, creatine phosphokinase resynthesis occurred in both groups, but adenosine triphosphate levels were not significantly increased (p greater than 0.05). A washout of lipid soluble products of adenine nucleotide metabolism occurred equally in both groups. The relationship between phosphorylated adenine nucleotides as measured by the energy charge potential fell significantly in both groups (p less than 0.05), but after the shorter period of ischemia (4 hours it returned to normal during early reperfusion but did not after 5 hours of ischemia. There was 21% +/- 4% necrosis after 4 hours and 51% +/- 8% after 5 hours of ischemic stress when assessed at 48 hours. In conclusion, the degree of adenine nucleotide degeneration as determined primarily by the length of the ischemic period, may be the most important determinant of the ultimate extent of skeletal muscle ischemic necrosis that results from an acute interruption of circulation.

Adenine Nucleotides↗
PubMed1990

Subcellular distribution of peroxidized lipids in myocardial reperfusion injury.

Previous studies in our laboratory have demonstrated the peroxidation of myocardial phospholipid in a canine model of reversible global normothermic ischemia and reperfusion while on cardiopulmonary bypass. The present study examines the distribution of phospholipid peroxidation products in three major cellular organelle fractions of myocardium prepared by established centrifugal fractionation procedures (sarcolemma, sarcoplasmic reticulum, and mitochondria). These organelles were isolated from control (nonischemic) and ischemic-reperfused myocardium harvested during early reperfusion (5 min), when previous studies indicated maximal peroxidative injury in whole myocardial biopsies. Utilizing a more rapid analytic procedure for measuring phospholipid containing the conjugated diene chromophore in the polyunsaturated fatty acyl substituents, we were able to establish the fidelity of this procedure by comparing the results obtained with it to the previous more laborious analytic procedure (involving phospholipid hydrolysis with phospholipase A2 and subsequent derivatization for high-pressure liquid chromatography followed by gas chromatographic-mass spectrometric analysis). Analysis of phospholipid extracts from organelle fractions for evidence of peroxidative conjugated diene formation revealed that sarcolemmal membranes had the highest content of oxidized phospholipid containing the conjugated diene chromophore (mean 2.2 +/- 1.2 nmol phospholipid-conjugated diene/mumol phospholipid phosphorus, P less than 0.02 compared with control). Both sarcoplasmic reticulum and mitochondrial membranes were also peroxidized but to a much smaller extent (mean 0.4 +/- 0.2 and 0.3 +/- 0.25 nmol phospholipid conjugated diene/mumol phospholipid phosphorus).

Animals↗
PubMed1990

Complement activation and white cell sequestration in postischemic skeletal muscle.

After skeletal muscle ischemia, tissue damage is augmented during reperfusion. White blood cells (WBCs) and complement proteins may participate in the reperfusion injury. The purpose of this study was to define the kinetics of classical and alternative pathway complement activation and WBC sequestration by postischemic skeletal muscle during the first 48 h of reperfusion in vivo. The isolated canine gracilis muscle model was used. Systemic levels of the complement proteins factor B (alternative pathway) and C4 (classical pathway) were quantitated by hemolytic assay. WBC sequestration was measured by gracilis arterial-venous WBC differences and tissue myeloperoxidase activity. Reperfusion was associated with an 18% decrease in systemic factor B levels but no consistent change in systemic C4 levels. WBCs were sequestered during the first 4 h of reperfusion, and tissue myeloperoxidase activity was elevated 97-fold after 48 h of reperfusion. These results suggest that skeletal muscle ischemia-reperfusion stimulates 1) activation of the alternative but not the classical complement pathway and 2) an immediate and prolonged sequestration of WBCs.

Animals↗
PubMed1990

Calcium paradox in an in vivo model of multidose cardioplegia and moderate hypothermia. Prevention with diltiazem or trace calcium levels.

The production and prevention of calcium paradox injury in myocardium was studied in a canine model of cardiopulmonary bypass with multidose, moderately hypothermic, crystalloid cardioplegic solution. During 4 1/2 hours of global ischemia, three groups of six dogs each received one of three histidine-buffered cardioplegic solutions (500 ml initially and 250 ml every 30 minutes) at 27 degrees C. Group 1 cardioplegic solution was calcium free, group 2 solution contained a trace amount of calcium chloride (70 mumols /L), and group 3 cardioplegic solution was calcium free but contained diltiazem (150 micrograms/kg body weight). Left ventricular function measured as percent control of developed pressure revealed significantly greater (p less than 0.05) recovery in groups 2 and 3. Triphenyltetrazolium chloride staining showed 35% +/- 9% (mean +/- standard error) of heart mass necrosis in group 1 versus 0% and 0.5% +/- 0.4% in groups 2 and 3, respectively (p less than 0.001). Electron microscopy revealed ultrastructural changes characteristic of calcium paradox injury in group 1 myocardium. Calcium paradox injury was produced in an in vivo model of global myocardial ischemia and multidose cardioplegia despite moderate hypothermia and non-coronary collateral flow. The addition of either trace levels of calcium or diltiazem to the cardioplegic solution was effective in preventing this injury.

Animals↗
PubMed1989

Asanguineous reperfusion in a canine model of cardiopulmonary bypass and controlled postischemic work.

This study was designed to test the hypothesis that asanguineous reperfusion with a standard crystalloid cardioplegic solution results in improved myocardial salvage after a period of global ischemia. Four groups of 6 dogs each were placed on cardiopulmonary bypass. Control group A (work only) performed two hours of controlled work by contracting against a saline-filled left intraventricular balloon. Control group B (ischemia only) underwent 45 minutes of global normothermic ischemia before simple blood reperfusion while supported on bypass. Groups C and D were subjected to ischemia and reperfusion as in group B, followed by controlled work stress as in group A. Group D, however, received 500 mL of St. Thomas' Hospital solution immediately before blood reperfusion. Morphological analysis showed no significant injury in groups A and B, whereas group C had 11.4% +/- 2.4% necrosis of heart mass versus 2.5% +/- 1.1% in group D (p less than 0.001). Biochemical data from left ventricular biopsies showed no significant differences between groups B, C, and D. Functional analyses showed deterioration of diastolic compliance in group C (p less than 0.05), although a significant difference in systolic functional indexes could not be detected. Myocardial protection and salvage was improved by initial reperfusion with an asanguineous cardioplegic solution versus reperfusion with blood alone.

Animals↗
PubMed1989

An enzymatic method for oxalate automated with the Cobas Fara centrifugal analyzer.

Measurement of oxalate in urine has been automated for use with the Cobas Fara centrifugal analyzer. No sample pretreatment other than (a critical) pH adjustment is required. Between-run CVs were less than 4%. Results were linearly related to oxalate concentration to 1000 mumol/L. Ascorbic acid ingestion, up to 5 g of ascorbate daily, caused no demonstrable interference with the assay. This practical, automated method for assaying urinary oxalate is substantially faster than other chemical or enzymatic methods.

Aldehyde Oxidoreductases↗