Search

Objectives: The primary objective of this study was to compare the effect of therapy with bivalirudin alone versus bivalirudin plus eptifibatide on platelet reactivity measured by turbidometric aggregometry and thrombin-induced platelet-fibrin clot strength (TIP-FCS) measured by thrombelastography in percutaneous coronary intervention (PCI) patients. The secondary aim was to study the relation of platelet aggregation and TIP-FCS to the occurrence of periprocedural infarction.Background: Bivalirudin is commonly administered alone to clopidogrel naïve (CN) patients and to patients on maintenance clopidogrel therapy (MT) undergoing elective stenting. The effect of adding eptifibatide to bivalirudin on platelet reactivity (PR) and TIP-FCS, and their relation to periprocedural infarction in these patients are unknown.Methods: Patients (n = 200) stratified to clopidogrel treatment status were randomly treated with bivalirudin (n = 102) or bivalirudin plus eptifibatide (n = 98). One hundred twenty-eight CN patients were loaded with 600 mg clopidogrel immediately after stenting, and 72 MT patients were not loaded. The PR, TIP-FCS, and myonecrosis markers were serially determined.Results: In CN and MT patients, bivalirudin plus eptifibatide was associated with markedly lower PR at all times (5- and 20-microM adenosine diphosphate-induced, and 15- and 25-microM thrombin receptor activator peptide-induced aggregation; p < 0.001 for all) and reduced mean TIP-FCS (p < 0.05). Patients who had a periprocedural infarction had higher mean 18-h PR (p < 0.0001) and TIP-FCS (p = 0.002).Conclusions: For elective stenting, the addition of eptifibatide to bivalirudin lowered PR to multiple agonists and the tensile strength of the TIP-FCS, 2 measurements strongly associated with periprocedural myonecrosis. Future studies of PR and TIP-FCS for elective stenting may facilitate personalized antiplatelet therapy and enhance the selection of patients for glycoprotein IIb/IIIa blockade. (Peri-Procedural Myocardial Infarction, Platelet Reactivity, Thrombin Generation, and Clot Strength: Differential Effects of Eptifibatide + Bivalirudin Versus Bivalirudin [CLEAR PLATELETS-2]; NCT00370045. [ABSTRACT FROM AUTHOR]

Clopidogrel and aspirin are commonly prescribed anti-platelet medications indicated for patients who have experienced, or are at risk for, ischemic cardiovascular events. The Pharmacogenomics of Anti-Platelet Intervention (PAPI) Study was designed to characterize determinants of clopidogrel and dual anti-platelet therapy (DAPT) response in a healthy cohort of Old Order Amish from Lancaster, PA. Following a loading dose, clopidogrel was taken once a day for 7 days. One hour after the last dose of clopidogrel, 325 mg of aspirin was given. Ex vivo platelet aggregometry was performed at baseline, post-clopidogrel, and post-DAPT. Platelet aggregation measurements were significantly lower after both interventions for all agonists tested (p <0.05), although there was large inter-individual variation in the magnitude of anti-platelet response. Female sex and older age were associated with higher platelet aggregation at all three time-points. Change in aggregation was correlated among the various agonists at each time point. Heritability (h2) of change in platelet aggregation was significant for most traits at all time-points (range h2=0.14-0.57). Utilization of a standardized, short-term intervention provided a powerful approach to investigate sources of variation in platelet aggregation response due to drug therapy. Further, this short-term intervention approach may provide a useful paradigm for pharmacogenomics studies.

Management of severe coronary artery disease (CAD), defined as multivessel disease with or without significant left main artery disease remains a topic for considerable discussion. Although coronary artery bypass graft (CABG) surgery has been the mainstay of treatment, the steady pace of improvement in percutaneous coronary intervention (PCI) continues to beg the question as to whether PCI can perform as well as CABG for these patients. This short review is intended to place the recently published SYNTAX (Synergy between PCI with Taxus and Cardiac Surgery) trial in perspective, considering the previous major clinical trials in this field and to further consider whether PCI can be used appropriately in the management of patients with advanced CAD. The major clinical trials comparing PCI to CABG published prior to SYNTAX are briefly reviewed in chronologic order. The SYNTAX trial is reviewed in more depth and the implications of its results for contemporary clinical management are discussed. PCI has been applied to more advanced forms of CAD as percutaneous technology has evolved from balloon angioplasty to bare metal stents to drug eluting stents. Long-term survival has remained comparable between PCI and CABG patients despite the more advanced nature of disease treated in more recent trials, recognizing that a significant number of patients are excluded from randomization because equivalent revascularization is not achievable percutaneously. Repeat revascularization is more frequently required in PCI patients than in CABG patients. PCI has a role to play, although CABG remains the mainstay of therapy for patients with advanced CAD.

Objectives: We determined the effect of clopidogrel dosing on the incidence of nonresponsiveness (NR) and high post-treatment platelet aggregation (post-PA)., Background: We have reported NR after a 300-mg loading dose. Limited information is available on the comparative effect of a 600-mg loading dose on the incidence of NR and high post-PA., Methods: Clopidogrel responsiveness and post-PA were measured in patients undergoing stenting (n = 190) randomly treated with either a 300-mg or a 600-mg clopidogrel load. Nonresponsiveness was defined as <10% absolute change in platelet aggregation, and high post-PA was defined as >75th percentile aggregation after 300 mg clopidogrel., Results: Nonresponsiveness was lower after 600 mg compared to the 300-mg dose (8% vs. 28% and 8% vs. 32% with 5 and 20 microM ADP, respectively, p < 0.001). Among the patients with high post-PA after 300 mg clopidogrel, 62% to 65% had NR, whereas after the 600-mg dose, all of the patients with high post-PA had NR., Conclusions: A 600-mg clopidogrel loading dose reduces the incidence of NR and high post-PA as compared to a 300-mg dose. Higher dosing strategies and methods to confirm platelet inhibition should be further investigated in order to optimally use clopidogrel in patients undergoing stenting.

Background: If surgeons could "see " through blood and cardiac chamber walls, it would ultimately be unnecessary to open the heart or use cardiopulmonary bypass to perform procedures such as atrial septal defect (ASD) closure or mitral valve repair. Conventional echocardiography generates cross-sectional images that are not satisfactory as the only visualization for surgical procedures, and current 3-Dimensional (3-D) CT and echo systems take several minutes to compose and process a single still frame. Recently, however, the first system for real-time 3-D echocardiography has been developed. This study examines whether real-time 3-D echocardiography can provide images of sufficient anatomic definition, depth perception, and image resolution to substitute for optical visualization in performing ASD closures., Methods: A prototype Volumetrics 3-D echocardiographic system was evaluated in a water bath on a complex-surfaced standard reference model to determine the image resolution and define the ideal imaging parameters. A static image and views of sutures being placed with an endoscopic needle driver and two commercial suture placement devices were evaluated at multiple angles and distances from the target. The resulting images were graded by a blinded reviewer. Once the best imaging parameters were determined, five porcine ASDs were closed with interrupted sutures, running sutures, or a pursestring suture using only echo visualization., Results: The highest quality images were obtained with the probe at a distance of 4-6 cm and at angles perpendicular or 45 degrees forward to the target. Spatial and temporal resolutions were adequate to suture all ASDs closed under only echo guidance., Conclusions: The evaluated real-time 3-D echo system provided adequate spatial and temporal information to act as a guide for surgical procedures.

The key role of endothelin-1 (ET-1) has been recognised in patients with ischaemic heart disease. However, the serial changes of ET-1 during both brief and prolonged ischaemia-reperfusion are poorly known. Serial changes of plasma ET-1 were measured during myocardial stunning (MS) and acute myocardial infarction (AMI). The effects of magnesium (Mg), diltiazem and a MAC-1 inhibitor on the plasma ET-1 were elucidated. Forty-nine swine underwent brief (8 min) or prolonged (50 min) coronary artery occlusion followed by reperfusion. ET-1 plasma concentration was measured by ELISA at prespecified time points. The occlusion was associated with a decline of ET-1 followed by a significant increase during the reperfusion. Mg as well as diltiazem similarly affected the plasma ET-1 by reducing ET-1 release during the first hour of the reperfusion period. MAC-1 inhibition was also associated with decreases of ET-1. Ability of Mg, diltiazem and leumedins to decrease the ET-1 plasma level may have direct clinical implications for the use of these agents in patients with coronary artery disease., (Copyright 1998 The Italian Pharmacological Society)

The important role of fibronectin (Fn) has been recognized in patients with ischemic heart disease. However, serial changes of Fn during both brief and prolonged ischemia-reperfusion are poorly known. Plasma Fn was measured during acute myocardial infarction (AMI) and myocardial stunning (MS), and in the absence of myocardial injury. The effects of magnesium (Mg), diltiazem, and a Mac-1 inhibitor on the level of Fn were elucidated. Forty-nine swine underwent prolonged (50 min) or brief (8 min) coronary artery occlusion followed by reperfusion, while six control animals were free of ischemia. During the AMI experiments, plasma Fn underwent a significant progressive increase. Mg or diltiazem similarly affects the plasma Fn, reducing its release during the entire reperfusion period, and did not influence the plasma Fn in the absence of myocardial injury. Contrarily, Mac-1 inhibition resulted in the Fn elevation in controls, and during the occlusion phase, with no significant effect during reperfusion. There were no changes in the plasma Fn during MS, while inhibition of Mac-1 was associated with the significant increase of Fn during ischemia-reperfusion. Ability of Mg, diltiazem, and leumedins to modulate plasma Fn level may have direct clinical implications for the use of these agents in patients with coronary artery disease.

Alterations in various hemostatic factor have been identified as risk factors for survival after acute myocardial infarction (AMI). However, these clinical data are primarily limited to observations made during the postinfarct stage. We assessed the effects of 50 minutes of left anterior descending artery occlusion on several hemostatic factors and analyzed their changes with outcome in 18 Yorkshire swine. Blood samples were obtained from the systemic circulation at base-line and at 25 and 50 minutes of occlusion. Platelet aggregability and plasma antithrombin-III protein C, protein S, fibronectin, endothelin-1, as well as the metabolites of thromboxane and prostacyclin were measured. Of the 18 swine, 7 survived the infarct and 11 animals developed fatal ventricular fibrillation. Both groups demonstrated significant decreases in platelet aggregation, and a decline in plasma protein S when compared with baseline. Thromboxane, prostacyclin, and ondothelin-1 plasma concentrations were also markedly reduced at the end of occlusion. There were significant changes in antithrombin-III, protein C, and fibronectin levels between surviving animals and those that died of ventricular fibrillation. Acute coronary artery occlusion is associated with substantial changes in the hemostatic factors in swine. Plasma levels of fibronectin, antithrombin-111, and protein C differed between survivors and nonsurvivors and thus might serve as predictors of mortality due to fatal ventricular fibrillation during AMI. The mechanisms of these changes during the acute phase of AMI are unclear. Immediately AMI prognosis may be related to hemostatic changes not only after thrombolysis or spontaneous reperfusion, but also during the occlusion phase as well.

Stent embolization is a rare but acknowledged complication of placement of disarticulated (half) Palmaz-Schatz stents. We report a case in which we diagnosed a previously unrecognized, embolized, undeployed half-stent in the distal LAD, causing slow flow, and then deployed the stent where it lay, resulting in improved flow. The literature on treatment of coronary stent embolization and on cutting and preparing half-stents for deployment is discussed.

Controversy currently exists regarding the use of diltiazem in the treatment of acute myocardial infarction (AMI). due to conflicting results from clinical trials and animal studies. The purpose of this project was to evaluate the changes in the hemostatic profile during AMI following low dose intracoronary diltiazem infusion. Fourteen Yorkshire swine underwent thoracotomy and 50 min LAD occlusion, followed by 3 h of reperfusion. The first group (n = 8) received 2.5 mg of diltiazem intracoronary at a rate of 5.6 micrograms kg min-1 at the onset of reperfusion. The second group (n = 6) received 0.9% saline intracoronary at the onset of reperfusion and served as the control. The dynamics of plasma antithrombin-III (AT-III), Protein C, total Protein S, fibronectin, endothelin-1 (ET-1), and the stable metabolites of thromboxane (TxB2) and prostacyclin (6-keto-PGF1a) were determined at baseline, then twice during occlusion and finally three times during reperfusion. Diltiazem infusion resulted in diminished ET-1 (34.5%), fibronectin (23.2%), and TxB2 (35.6%); and elevated Protein C (29.3%) when compared with controls. We conclude that intracoronary diltiazem favorable influences hemostasis during AMI in swine. The cardioprotective effects of diltiazem during AMI may be related to the improved hemostatic profile and the reduced incidence of thrombotic complications in such patients.

Unlabelled: The purpose of this study was an attempt to extrapolate favorable observations on the effects of magnesium on platelets and haemostasis from animal models to humans. Intravenous magnesium in the treatment of acute myocardial infarction has been tested in several large clinical trials and remains controversial. The mechanism for the cardioprotective properties of magnesium is unknown. Based on experimental studies, several different hypotheses have been advanced to explain the benefits of magnesium including it's effects on platelets and haemostasis. However, few studies have analysed such a relationship in humans. This project was designed to assess the effect of bolus magnesium infusion on ex vivo platelet aggregation and certain haemostatic parameters in healthy volunteers. One gram of magnesium was diluted in 50 ml of D5W and infused over a period of 15 min. The changes of platelet aggregability, plasma antithrombin-III, Protein C, total Protein S, fibronectin, endothelin-1, as well as the metabolites of thromboxane and prostacyclin were determined prior to and immediately after magnesium infusion., Results: Serum magnesium concentration increased from 2.10 +/- 0.08 at baseline to 3.12 +/- 0.13 mg dl-1 (P = 0.0002) post-infusion. Magnesium infusion was associated with a significant elevation in ADP (+19.3%); ristocetin (+13.6%); and collagen-induced platelet aggregation (+14.2%); an increase in plasma antithrombin-III (+10.3%) and thromboxane (+49.4%) when compared to pre-infusion levels. Against this, total Protein S (-20.7%), Protein C (-11.2%) and ET-1 (-26.3%) plasma concentrations were markedly decreased. There were no significant differences in plasma fibronectin and prostacyclin levels. Contrary to expectations, magnesium infusion in human volunteers is associated with platelet activation and mostly small unfavourable changes in certain haemostatic factors. However, the observed changes were small and for the most part remained within the normal physiologic range.

Background: Currently, controversy exists regarding the use of calcium-channel blockers in the treatment of acute myocardial infarction (AMI), due to apparent conflicting results from clinical trials and animal models. One hypothesis to explain such a discrepancy proposes that the timing and duration of drug administration might influence its cardioprotective effect. Pretreatment with calcium-channel blockers or their administration during coronary artery occlusion is associated with the diminished infarct size in animal models. While verapamil failed to reduce infarct size when the drug was given at the onset of reperfusion, similar effects of low dose diltiazem are not known., Methods and Results: This experiment evaluated the effect of intracoronary short term low dose diltiazem administration given immediately with postischemic myocardial reperfusion. Yorkshire swine underwent thoracotomy and 50 min of left anterior descending (LAD) occlusion, followed by 3 h of reperfusion. In the first group, diltiazem (2.5 mg diluted in 60 cc saline) was infused into the LAD over 12 min, beginning with the onset of reperfusion (n=8). In the second group, animals received saline instead of diltiazem and served as controls (n=6). Infarct size was 0.13+/-0.06 g/kg of body weight for diltiazem group, and 0.42+/-0.04 g/kg for controls (P=0.01)., Conclusions: Short-term low dose diltiazem delivered exclusively during early reperfusion can significantly diminish infarct size in swine. Local intracoronary diltiazem may be valuable adjunct in patients subject to myocardial ischemia/reperfusion during coronary artery bypass grafting, primary angioplasty for AMI, or thrombolysis for AMI if given immediately after restoration of coronary blood flow.

The key role of prostanoids has been recognized in patients with ischemic heart disease. However, serial changes of thromboxane and prostacyclin during both brief and prolonged ischemia-reperfusion are poorly known. These plasma prostanoids were measured during myocardial stunning (MS) and acute myocardial infarction (AMI). The effects of magnesium (Mg), diltiazem, and a Mac-1 inhibitor on the level of the stable metabolites of thromboxane (TXB2) and prostacyclin (6-keto-PGF1 alpha) were elucidated. Forty-nine swine underwent brief (8 min) or prolonged (50 min) coronary artery occlusion followed by reperfusion. The occlusion phase was associated with a decline of plasma prostanoids, followed by a significant increase during reperfusion. Mg and diltiazem similarly affected plasma prostanoids by reducing TXB2 release at 1 h of reperfusion. There was, however, no effect on plasma 6-keto-PGF1 alpha. The Mac-1 inhibition was associated with stabilization of both antagonistic prostanoids as well. Ability of Mg, diltiazem, and leumedins to favorably modulate plasma prostanoid levels have direct clinical implications for the use of these agents in patients with coronary artery disease.

Improved cardiovascular morbidity and mortality have been observed in several clinical studies of dietary supplementation with coenzyme Q10 (CoQ10, ubiquinone). Several mechanisms have been proposed to explain the effects of CoQ10, but a comprehensive explanation of its cardioprotective properties is still lacking. One attractive theory links ubiquinone with the inhibition of platelets. The effect of CoQ10 intake on platelet size and surface antigens was examined in human volunteers. Study participants received 100 mg of CoQ10 twice daily in addition to their usual diet for 20 days. Receptor expression was measured by flow cytometry with monoclonal murine anti-human antibodies CD9 (p24), CD42B (Ib), CD41b (IIb), CD61 (IIIa), CD41a (IIb/IIIa), CD49b (VLA-2), CD62p (P selectin), CD31 (PECAM-1), and CD51/CD61 (vitronectin). An increase of total serum CoQ10 level (from 0.6 +/- 0.1 to 1.8 +/- 0.3 micrograms/ml; p < 0.001) was found at protocol termination. Fluorescence intensity was higher for the large platelets when compared with the whole platelet population. Significant inhibition of vitronectin-receptor expression was observed consistently throughout ubiquinone treatment. Reduction of platelet size was observed at the end of CoQ10 supplementation. Inhibition of the platelet vitronectin receptor and a reduction of the platelet size are direct evidence of a link between dietary CoQ10 intake and platelets. These findings may not be fully explained by the known antioxidant and bioenergetic properties of CoQ10. Diminished vitronectin-receptor expression and reduced platelet size resulting from CoQ10 therapy may contribute to the observed clinical benefits in patients with cardiovascular diseases.

Improved cardiovascular morbidity and mortality have been observed in several clinical studies of dietary supplementation with coenzyme Q10 (CoQ10, ubiquinone). Several mechanisms have been proposed to explain the effects of CoQ10. One attractive theory links ubiquinone with the inhibition of platelets. The effect of CoQ10 intake on platelet surface antigens, and certain hemostatic parameters was examined in 15 humans and 10 swine. Study participants received 100 mg of CoQ10 twice daily in addition to their usual diet for 20 days resulting in a three-fold increase of total serum CoQ10 level. We observed a decline in plasma fibronectin (-20.2%), thromboxane B2 (-20.6%), prostacyclin (-23.2%), and endothelin-1 (-17.9%) level. Significant inhibition of vitronectin receptor expression was observed consistently throughout ubiquinone treatment. Inhibition of the platelet vitronectin receptor is a direct evidence of a link between dietary CoQ10 intake, platelets, and hemostasis. These findings may contribute to the observed clinical benefits by a diminished incidence of thrombotic complications in such patients.

There has been some debate regarding the benefit of magnesium (Mg) in the treatment of acute myocardial infarction (AMI) because of conflicting results from recent clinical trials. Several different hypotheses have been advanced to explain the cardioprotective properties of Mg, including the influence of the timing of Mg administration during AMI. This experiment was designed to assess the effect of intracoronary Mg on certain hemostatic parameters that are known to change during an AMI. Yorkshire swine underwent thoracotomy and 50 min left anterior descending artery (LAD) occlusion, followed by 3 h of reperfusion. In the early group, 250 mg of MgSO4 was delivered at the onset of reperfusion (n = 6, Mg-early group). In the second group, MgSO4 was given after 1 h of reperfusion (n = 6, Mg-late group). Six animals received saline instead of Mg and served as controls. The dynamics of plasma antithrombin-III (AT-III), protein C, total protein S, fibronectin, endothelin-1 (ET-1), as well as the stable metabolites of thromboxane (TXB2) and prostacyclin (6-keto-PGFla) were determined at baseline, twice during occlusion, and three times during reperfusion. Mg given at reperfusion onset was associated with a diminished ET-1 (32.9%), decreased fibronectin level (21.7-25.2%), and increased protein C concentrations (31.9-52.3%) when compared with both the control and late Mg group. In summary, intracoronary Mg administered at the onset of reperfusion favorably influenced hemostasis in swine. The beneficial effects of early Mg supplementation in an expanding array of clinical conditions, including AMI, may be directly related to the improved hemostatic profile in such patients.

The use of calcium antagonists and magnesium (Mg) in the treatment of acute myocardial infarction is controversial. We compared changes in hemostasis during acute myocardial infarction after either low-dose intracoronary Mg or diltiazem infusion in 20 Yorkshire swine undergoing thoracotomy and coronary artery occlusion for 50 min, followed by 3 h of reperfusion. The first group received MgSO4 (250 mg), delivered at the onset of reperfusion, the second group received diltiazem (2.5 mg) at the beginning of reperfusion. Six controls received saline. Plasma antithrombin III, protein C, total protein S, fibronectin, endothelin 1, and metabolites of thromboxane and prostacyclin were measured at baseline, twice during occlusion, and three times during reperfusion. Compared to controls, Mg and diltiazem infusion diminished endothelin 1 (32.9 vs. 34.5%) and fibronectin. (21.7 vs. 23.2%), but increased protein C (31.9 vs. 29.3%). Intracoronary Mg, like diltiazem, improved hemostasis in swine.

Epidemiological studies of populations living in areas of low magnesium (Mg) intake have consistently shown a higher cardiovascular morbidity. Several hypotheses have been advanced to explain the cardioprotective properties of magnesium. Few studies, however, have analysed the relation of magnesium to haemostasis. The overall purpose of this project was to assess the association between certain haemostatic variables and magnesium deficiency. This experiment was designed to assess the effect of magnesium deficiency on various haemostatic variables which may relate to cardiovascular morbidity. Twelve female Yorkshire swine were fed for seven weeks on an Mg-sufficient or an Mg-deficient diet. Blood samples were obtained at baseline and after the termination of feeding in order to evaluate platelet aggregability and concentrations of antithrombin-III (AT-III), protein C, total protein S, fibronectin, endothelin-1 (ET-1), as well as the stable metabolites of thromboxane (TxB2) and prostacyclin (6-keto-PGF1 alpha). In animals on an Mg-sufficient diet, there were no significant differences in any of the investigated haemostatic variables. In the Mg-deficient group, a significant decrease in serum magnesium was noted after the feeding period (from 2.0 +/- 0.1 to 1.3 +/- 0.1; P < 0.01). Mg-deficient swine showed significant increases in ADP-induced (33.3 per cent and 59.6 per cent) and collagen-induced (36.6 per cent) platelet aggregation, and decreased plasma antithrombin-III (17.7 per cent) and protein S (14.4 per cent) when compared to baseline. Plasma concentrations of TxB2 (28.7 per cent), protein C (57.2 per cent), and ET-1 (74.9 per cent) were dramatically increased. There were no significant differences in plasma fibronectin and 6-keto-PGF1 alpha levels in the magnesium-depleted animals. We conclude that magnesium deficiency is associated with significant proaggregatory and coagulation alterations. This may contribute to the increased cardiovascular morbidity found in magnesium-deficient populations. The beneficial effects of magnesium supplementation in an expanding array of clinical conditions including cardiovascular disease may, in part, be related to the improved haemostatic profile in such patients.

Plasma antithrombin-III (AT-III), protein S, and protein C were measured during myocardial stunning (MS) and acute myocardial infarction (AMI). The effects of magnesium (Mg), diltiazem, and a Mac-1 inhibitor on their plasma levels were elucidated. Forty-nine open-chest swine underwent brief (8 min) or prolonged (50 min) coronary artery occlusion followed by reperfusion. During MS an increase in the plasma AT-III (from 98.5 +/- 3.38% to 138.1 +/- 3.6%) during the early occlusion phase, without any further changes was observed. The profile of total protein S was not changed during MS. Protein C increased at the end of occlusion (from 45.3 +/- 1.8% to 55.7 +/- 1.4%) reaching a peak (64.5 +/- 1.4%) at the beginning of reperfusion. When compared with controls, no significant differences were found in the antithrombotics profile during MS after pretreatment with Mac-1 inhibitor. For the AMI, the AT-III decreased during occlusion (from 98.5 +/- 3.4% to 61.0 +/- 3.6%). The protein S decreased during occlusion with the lowest level at 1 h of reperfusion (from 71.8 +/- 2.2% to 46.7 +/- 1.0%), followed by an increase during late reperfusion (59.2 +/- 1.5%). Contrarily, protein C increased during occlusion and early reperfusion (from 44.7 +/- 2.6% to 79.4 +/- 2.4%), but declined to 49.6 +/- 2.5% thereafter. In both Mg and diltiazem-treated swine, protein C was higher at the end of occlusion and during the entire reperfusion period compared with controls. Mg and diltiazem therapy was associated with the slight elevation of plasma AT-III. The patterns for protein S level during ischemia-reperfusion were similar with the controls. Protein S was higher at the end of occlusion and through the entire reperfusion in the NPC 15669-treated animals when compared with the controls. Mac-1 inhibition was associated with the elevated protein C during late reperfusion. Ability of Mg, diltiazem, and Mac-1 inhibitor to favorably modulate the plasma level of antithrombotics have direct clinical implications for the use of these agents in patients with acute coronary artery syndromes.

Myocardial stunning (MS) is a transient contractile dysfunction occurring subsequent to an episode of ischemia followed by reperfusion. NPC 15669 is a leumedin, which inhibits leukocyte adhesion to the endothelium by blocking Mac-1 upregulation. The effect of NPC 15669 supplementation on the hemostasis during MS is unknown. We linked the potential changes in the hemostasis with NPC 15669 therapy during mild MS. Twelve Yorkshire swine underwent coronary artery occlusion for 8 min followed by 90 min of reperfusion. NP 15669 (10 mg/kg loading dose followed by constant infusion a 6 mg kg-1 h-1) was administered to 6 of the animals; another swine received saline and served as the controls. Concentrations of antithrombin III (AT-III), protein C, total protein S, fibronectin, endothelin 1 (ET-1) and the stable metabolites of thromboxane (TxB2) and prostacyclin (6-keto-PGF1 alpha) were measured in the systemic circulation. NPC 15669 therapy was associated with diminished ET-1 (37.4%) and 6-keto-PGF1 alpha (47.1%) levels and increased fibronectin (77.6%) concentrations during MS. There were no changes in the plasma concentrations of TxB2, total protein S, protein C and AT-III in the NPC 15669 group when compared with controls. Mild MS in associated with substantial changes in the hemostatic profile. NPC 15669 administration in a swine model of MS affects certain hemostatic parameters. These data provide support for the involvement of cellular mechanisms in the pathogenesis of MS. The ability of leumedins to modulate hemostasis may have implications for their use in cardiovascular disease.

Improved cardiovascular morbidity and mortality have been observed in several clinical studies of dietary supplementation with coenzyme Q10 (CoQ10). We elucidated the effect of CoQ10 on certain hemostatic parameters that may influence the progression of heart disease. Twelve Yorkshire swine were randomized to receive diet supplementation with either CoQ10 or placebo for 20 days. Blood samples were obtained at baseline and at the end of the feeding period. At the end of the protocol, there were no significant differences in hemostatic parameters in the placebo group. A significant increase in total serum CoQ10 level (from 0.39 +/- 0.06 to 0.96 +/- 0.04 microgram/ml, p < 0.001) was noted after the feeding period in the CoQ10-supplemented group. We observed significant inhibition of ADP-induced platelet aggregation (-9.9%) and a decrease in plasma fibronectin (-20.2%), thromboxane B2 (TXB2, -20.6%), prostacyclin (-23.2%), and endothelin-1 (ET-1, -17.9%) level. There were no changes in the plasma concentrations of the natural antithrombotics [antithrombin-III (AT-III), protein S, and protein C] after CoQ10 supplementation. CoQ10 supplementation in a dose of 200 mg daily is associated with mild antiaggregatory changes in the hemostatic profile. Clinical beneficial effects of CoQ10 may be related in part to a diminished incidence of thrombotic complications.

Background: Controversy exists regarding the use of magnesium in the treatment of acute myocardial infarction (AMI) because of apparent conflicting results from clinical trials. One hypothesis to explain the various clinical observations proposes that the timing of magnesium administration significantly influences its therapeutic effect; ie, supraphysiological levels of Mg2+ must be present at the time of reperfusion for magnesium to produce clinical benefit., Methods and Results: These experiments evaluated the effect of varying the timing of magnesium administration during AMI. Female Yorkshire swine (34 to 42 kg) underwent thoracotomy and 50 minutes of left anterior descending coronary artery (LAD) occlusion, followed by 3 hours of reperfusion. In the first group, MgSO4 (250 mg of magnesium diluted in 60 cm3 saline) was infused into the LAD over 12 minutes, beginning immediately with the onset of reperfusion (n = 6, Mg-early group). In the second group, MgSO4 was given after 1 hour of reperfusion (n = 6, Mg-late group). Six pigs received saline instead of magnesium and served as the control group. Lethal arrhythmias were significantly reduced in the Mg-early group. Infarct size was determined by vital staining. Infarct size was 0.16 +/- 0.05 g/kg body wt (Mg-early), 0.35 +/- 0.08 g/kg (Mg-late), and 0.42 +/- 0.04 g/kg for the control group. Compared with the control group, significant (P = .029) reduction in infarct size occurred in the Mg-early group but not in the Mg-late group., Conclusions: We conclude that intracoronary MgSO4 delivered during reperfusion can significantly diminish infarct size in swine, but the timing of administration is critical.

The aim of the present study was to evaluate the relationship between coronary vascular tone and serum magnesium concentration in patients with ischaemic heart disease. Thirty-seven patients were subjected to intracoronary injection of 200 micrograms nitroglycerine after discontinuation of anti-anginal medication for 24 h (group I, n = 23), except if they received calcium channel blockers (group II, n = 14). Coronary angiographic images were acquired before and 2 min after the nitroglycerine injection. Using quantitative coronary arteriography, the luminal diameter of one middle-sized non-stenotic coronary artery segment in each patient was measured before and after nitroglycerine injection, and the percentual increase was computed. In group I, coronary artery diameter changed from 3.0 +/- 0.7 to 3.5 +/- 0.6 mm (mean +/- SD), representing a 16.3 per cent increase. As expected, a more blunted response was observed in group II (from 3.5 +/- 0.8 to 3.7 +/- 1.0 mm, 6.5 per cent increase). However, neither of the groups displayed significant correlation between serum magnesium levels and the measured response to intracoronary nitroglycerine. In conclusion, this study fails to provide any correlation between serum magnesium levels within the normal physiological range and coronary vascular reactivity in patients with ischaemic heart disease.

Background: NPC is a member of the leumedins and is an inhibitor of leukocyte adhesion to endothelium via blockage of integrin binding. NPC 15669 also may have antiplatelet effects. We tested the efficacy of the novel leukocyte recruitment inhibitor NPC 15669 on myocardial stunning (MS) and preconditioned myocardial infarction (MI). Methods and Results: In an open-chested swine model, NPC 15669 (10 rng NPC/kg loading dose followed by constant infusion at 6 mg/kg/hr) was administered in six animals. Myocardial thickening (MT) was determined by epicardial ultrasound. The left anterior descending artery was occluded for 8 minutes followed by 90 minutes of reperfusion, during which myocardial MT was recorded at regular intervals. We have found that treatment with NPC 15669 increases myocardial contractility and significantly decreases MS time compared to controls (26.7 +/- 4.0 minutes vs. 50.0 +/- 4.3 minutes, p =.0026). In NPC 15669-treated animals we observed a reduction of MI size (23.4 +/- 6.7% of tissue at risk became necrotic compared to 53.0 +/- 6.6% in controls, p =.0102). Conclusions: Our data suggest that NPC 15669 significantly reduces myocardial injury in both the stunning and infarction models.

Background: Leumedins inhibit cell adhesion to endothelium via blockage of integrin binding. We tested a hypothesis that the novel leucine derivate NPC 15669 will affect in vitro platelet aggregability (PA) in both human and swine plasma. Methods and Results: Platelet-rich plasma (PRP) was incubated with 200 µ g and 400 µ g of NPC 15669. Then PA was induced by ADP, collagen, thrombin, and ristocetin in the PRP without NPC 15669 and in NPC 15669-treated samples. We have found that PRP incubation with 200 µ g of NPC 15669 significantly decreases PA compared to baseline in all three experimental groups in response to all agonists tested. When PRP was treated with 400 µ g of NPC 15669, dose-dependent reduction of PA was observed only in the human control and swine groups, but not in patients with coronary atherosclerosis. Conclusions: Leumedins, known for their antiinflammatory properties, may have clinical applications related to their effect on platelet function. The mechanism of these effects is unknown, but may be related to the inhibition of platelet-endothelial binding.

Background: Myocardial reperfusion after short durations of ischemia causes prolonged contractile dysfunction (myocardial stunning). Recently it has also been suggested that ischemia-reperfusion results in impaired coronary endothelial function. Since platelet function is, in part, regulated by an intact functioning endothelium, platelet function could be expected to change during ischemia-reperfusion. However, the effect of ischemia and reperfusion on regional and systemic platelet function is unknown. The purpose of this study was to determine the effect of a brief period of myocardial ischemia followed by reperfusion on regional and systemic platelet function. Methods: Fourteen swine in an open-chest model underwent left anterior descending coronary artery (LAD) occlusion for 15 minutes followed by 120 minutes of reperfusion. Platelet aggregability in response to 5 µM ADP was determined simultaneously in the femoral (systemic; N = 14) and great cardiac (regional; N = 9) venous blood at baseline, during occlusion, and at 40 and 90 minutes after reperfusion. LAD blood flow and regional myocardial function were determined by standard methods. Results: Hemodynamics remained stable in all animals. During LAD occlusion platelet aggregability, increased only in the regional coronary circulation (126% of baseline, p =.0001). At 40 minutes of reperfusion systemic platelet aggregahility decreased (86% of baseline, p =.0001) and subsequently increased at 90 minutes at reperfusion in both the systemic (127% of baseline, p =.0001) and regional circula. tions (156% of baseline, p =.0001). Ischemia was evident by the absence ofdistal LAD flow during occlusion that returned during reperfusion and a typical response ofmyocardial stunning in each animal (stunning time = 47.7 +/- 5.2 minutes). Conclusions: This study demonstrates that platelet function is not static during ischemia-reperfusion. Instead, during ischemia regional platelet aggregability is increased. Systemic and regional platelet aggregability also increase during myocardial reperfusion. The mechanism of these responses is unknown but may be related to regional endathelial dysfunction created by ischernia. The response observed could also be explained by the release of proaggregatory mediators in the connary and/or systemic circulation during ischemia-reperfusion. The relative hyeraggregability observed following reperfusion may be relevant for further investigations of coronary artery reocclusion occurring after the relief of myocardial ischemia.

The effect of magnesium deficiency on postischemic myocardial dysfunction (myocardial stunning) in an open-chest swine model was studied. Twelve swine were assigned either to low magnesium diet or control diet. Myocardial stunning was assessed by measuring regional wall thickening by epicardial Doppler before and after brief occlusion (8 min) of the left anterior descending coronary artery. Serum magnesium levels decreased significantly in the experimental group only. Glutathione levels were 42.6% lower in the magnesium deficient swine than in controls. Stunning time was significantly prolonged from 32.8 +/- 3.1 min in the control group to 43.8 +/- 4.6 min in the hypomagnesemic swine. In conclusion, magnesium deficiency is associated with prolonged recovery from myocardial stunning.

Background: Accelerated intravenous dosing is currently the preferred regimen for administering recombinant tissue-type plasminogen activator (rt-PA) in acute myocardial infarction (AMI). This regimen is recommended on the basis of clinical angiographic studies that reported superior 60 and 90 min patency rates. However, continuous infarct-vessel flow characteristics after reperfusion following this regimen are poorly described. The aim of our study was an improved definition of the characteristics of infarct-vessel flow induced by intravenous accelerated rt-PA using an animal model of acute coronary artery thrombosis., Methods: We studied the characteristics of reperfusion induced by an accelerated intravenous rt-PA dosing regimen in a canine coronary artery thrombosis model that simulates AMI. We created a critical stenosis in the left anterior descending coronary artery (LAD) of 24 open-chest dogs. Thrombosis was caused immediately proximal to the stenosis by injection of a blood and thrombin mixture into a segment of the LAD which had intimal damage. Hemodynamics and distal LAD blood flow velocity were recorded continuously. Six animals did not complete the protocol. Animals in the treatment group (n = 10) received an intravenous accelerated rt-PA regimen (1.25 mg/kg total dose) and were observed for 150 min. Eight animals served as controls., Results: None of the control animal arteries reperfused. In the treatment group, reperfusion occurred 31.5 +/- 7.3 min after starting rt-PA dosing in two general patterns. In one pattern, reperfusion onset was gradual and linear; in the other pattern, reperfusion was sudden. LAD flow was cyclical in all animals, with marked variations in magnitude compared with baseline (0-173% of baseline). Reocclusion was common and occurred 30.3 +/- 6.3 min after the initial reperfusion. There were 1.9 +/- 0.4 reocclusions per animal, and two distinct patterns were noted. In one, flow ceased abruptly and, in the second, flow declined gradually in a linear fashion before stopping. Infarct-vessel flow was evident for 103.1 +/- 14.1 min of the 150 min observation period., Conclusions: Reperfusion occurs rapidly after an accelerated dosing regimen of rt-PA. However, the infarct-vessel flow resulting from this therapy is unstable and is associated with early reocclusion and marked variation in flow magnitude. Analysis of continuous infarct-vessel flow velocity patterns suggests at least two different mechanisms for the onset of reperfusion and reocclusion after this therapy. Recognizing several mechanisms of arterial opening and closing may have important implications for methods of stabilizing infarct vessels after reperfusion by accelerated dosing of rt-PA.

We tested the hypothesis that acute, intravenous (i.v.) magnesium (Mg2+) supplementation would protect against myocardial stunning in an in situ swine model of regional ischemia and reperfusion and that a concomitant inhibitory effect on platelet aggregation would be elicited. An open-chest model was used, with transient occlusion of the left anterior descending coronary artery (LAD) for 8 min. Regional contractile function was assessed by measuring wall thickening fraction with epicardial Doppler crystals. One control group (n = 6) and two treatment groups were studied: group I (n = 6) received 750 mg MgSO4 before occlusion; group II (n = 6) received 1 g MgSO4 after the occlusion. Both protocols produced significant hypermagnesemia. In group I, platelet aggregation was measured before and after Mg2+ treatment using platelet-rich plasma (PRP) and various agonists (ADP 5 and 10 mM and collagen 1 mg/ml). As compared with controls, both treatment groups experienced significantly less postischemic dysfunction, with systolic function returning more quickly to baseline. Furthermore, platelet aggregation was significantly decreased immediately after Mg2+ infusion. Inhibition of platelet aggregation induced by Mg2+ treatment occurs concomitantly with significant amelioration of postischemic myocardial dysfunction.

A model of coronary artery thrombosis which: (1) provides a stable thrombus; (2) incorporates intimal injury; (3) has a low mortality rate; (4) responds predictably to thrombolytic therapy, and (5) is technically simple, was developed. Intimal injury was produced proximal to a critical stenosis and followed by the infusion of a blood and thrombin mixture into the injured segment. After thrombus formation flow remained absent in all control animals (n = 7). Microscopy showed intimal injury and coronary thrombosis with platelets adherent to the subendothelium. In animals treated with tissue plasminogen activator (n = 7) flow returned to > 60% of baseline at 20.2 +/- 7.7 min and was cyclical. Mortality and complications were infrequent. This model is useful in investigations of reperfusion therapy.

The effect of magnesium sulphate infusion on ex vivo platelet aggregation in 11 female Yorkshire swine was observed using platelet-rich plasma and different agonists (ADP 5 mM; ADP 10 mM and collagen 1 mg/ml). Infusion of 1 g MgSO4 over 1 h produced a significant decrease in platelet aggregability. A dose-dependent effect of different ADP concentrations on platelet aggregation was noticed. Platelet inhibition was most consistent when using ADP 5 mM. We estimate this concentration of agonist as optimal in swine. The swine model is a good choice for investigation of in vivo platelet activation, especially with regard to cardiovascular research. We conclude that there is an inhibitory effect of supplemental magnesium on ex vivo platelet aggregation in swine with initial normomagnesaemia.

In the picryl chloride contact sensitivity system in mice, i.v. injections of trinitrobenzene sulfonic acid (TNBSA) prevents elicitation of delayed-type hypersensitivity reactions. This suppression is due in part to a non-specific, PG-dependent factor (TNBSA-F) that is induced by i.v. injection of TNBSA and is produced by pooled spleen and lymph node cells in vitro. Inasmuch as a role for lymphokines such as IL-2 has been postulated in delayed-type hypersensitivity, we determined the in vitro effects of TNBSA-F on the responsiveness of HT-2 target cells to IL-2. TNBSA-F induced a dose-dependent unresponsiveness of HT-2 cells to IL-2. The inhibitory activity was not present in supernatants from lymphoid cells of sham-treated mice. In the presence of indomethacin, spleen, and lymph node cells from TNBSA-immunized mice produced a factor whose activity was much reduced compared to TNBSA-F. This suggested that PG were required for TNBSA-F activity. However, PG alone did not induce the unresponsiveness because TNBSA-F but not sham-treated mice had inhibitory activity despite containing similar levels of PGE2. Rather, the combination of i.v. TNBSA injections and PG synthesis during production of TNBSA-F were required to produce a suppressive TNBSA-F. The inhibitory effect of TNBSA-F was not due to the presence of transforming growth factor-beta, soluble immune-response suppressor, INF-gamma, or JE in the factor preparation. Partial characterization showed a single peak of in vitro TNBSA-F activity (molecular mass approximately 35-55 kDa) by Sephadex G-200 gel filtration chromatography and by HPLC. In addition, TNBSA-F retained its activity after multiple cycles of freeze-thaw and heating for 1 h at 56 degrees C. The inhibitory effects of TNBSA-F on IL-2-induced proliferation suggest that suppression of delayed type hypersensitivity after i.v. administration of TNBSA may, in part, be due to a PG-dependent suppressor factor that inhibits the responsiveness of target cells to IL-2.

Myocardial stunning, defined as a reversible decrease in contractility after ischemia and reperfusion, may be a manifestation of reperfusion injury caused by free oxygen radical damage. The aim of this study was to test the hypothesis that pretreatment with coenzyme Q10 (ubiquinone), believed to act as a free radical scavenger, reduces myocardial stunning in a porcine model. Twelve swine were randomized to receive either oral supplementation with coenzyme Q10 or placebo for 20 days. A normothermic open-chest model was used with short occlusion (8 min) of the distal left descending coronary artery followed by reperfusion. Regional contractile function was measured with epicardial Doppler crystals in ischemic and nonischemic segments by measuring thickening fraction of the left ventricular wall during systole. Stunning time was defined as the elapsed time of reduced contractility until return to baseline. Coenzyme Q10 concentrations were measured in blood and homogenized myocardial tissue by high performance liquid chromatography. Plasma levels of reduced coenzyme Q10 (ubiquinol) were higher in swine pretreated with the experimental medication as compared to placebo (mean 0.45 mg/l versus 0.11 mg/l, respectively). Myocardial tissue concentrations, however, did not show any changes (mean 0.79 micrograms/mg dry weight versus 0.74 micrograms/mg). Stunning time was significantly reduced in coenzyme Q10 pretreated animals (13.7 +/- 7.7 min versus 32.8 +/- 3.1 min, P < 0.01). In conclusion, chronic pretreatment with coenzyme Q10 protects ischemic myocardium in an open-chest swine model. The beneficial effect of coenzyme Q10 on myocardial stunning may be due to protection from free radical mediated reperfusion injury. This protective effect seems to be generated by a humoral rather than intracellular mechanism.

The elicitation of delayed-type hypersensitivity (DTH) responses in mice is mediated by the sequential activities of two different Ag-specific, Thy-1+ cells. A required early phase of elicitation is due to DTH-initiating Thy-1+ cells that are CD3- and sIg- and produce Ag-specific factors that act like IgE antibodies in that they sensitize the tissues, so that after local challenge with Ag there is release of the vasoactive amine serotonin. Released serotonin locally recruits and activates CD4+ Th-1 classical DTH effector T cells that secrete lymphokines that attract and activate a nonspecific perivascular infiltrate of circulating, bone marrow-derived leukocytes. The current study used isolated subpopulations of DTH-initiating and DTH-effector T cells to determine whether the two phases of the elicitation of DTH were entirely separate. The contact sensitivity model of DTH was used. Early-acting DTH-initiating cells, and late-acting DTH-effector T cells were either from oxazolone (OX)-immune or picryl chloride (PCl)-immune CBA or BALB/c donors and were transferred to CBA or BALB/c recipients. The results showed that DTH-initiation could be mediated by polyclonal DTH-initiating cells that were Ag mismatched or MHC incompatible with late-acting DTH effector T cells. In fact DTH-initiating cells could be both Ag mismatched and MHC incompatible with late-acting T cells. In addition, potential interactions between different cell populations were ruled out by showing that DTH-initiation could be mediated by a DTH-initiating clone that was Ag or MHC mismatched with the late-acting DTH-effector T cells. Thus, the OX-specific BALB/c clone could initiate DTH for PCl-specific CBA cells in CBA recipients if the recipients were challenged with both OX and PCl, but not when they were challenged with OX or PCl alone. We suggest, at least for the elicitation of DTH reactions in mice, that a more comprehensive description of these responses should accommodate the fact that there are early and late phase responses that each begin with Ag specificity and end with non-specific humoral factors. Inasmuch as the two Thy-1+ cells of DTH can be of different Ag specificity, this suggests that some forms of delayed and chronic inflammation, might be initiated by an immediate hypersensitivity-like immune reactivity to one set of Ag, and could be prolonged and perpetuated by delayed reactivity to another set of Ag.

Elicitation of delayed-type hypersensitivity (DTH) responses is due to the required sequential action of two different antigen (Ag)-specific Thy-1+ cells: early acting, DTH-initiating cells and locally recruited CD4+, alpha beta-TCR+, DTH effector T cells. DTH-initiating cells have an unusual phenotype for Ag-specific cells (Thy-1+, CD5+, CD4-, CD8-, CD3-, sIg-, B220+ (CD45RA+), Mac 1+, IL-2R- and IL-3R+) and act by producing Ag-specific non-IgE factors that sensitize mast cells for release of the vasoactive amine serotonin (5HT) at the local site of elicitation of DTH by challenge. Another mechanism of DTH initiation involves Ag-specific IgE antibodies that also can sensitize mast cells for local serotonin release. Serotonin initiates DTH by activating the local endothelial cells to allow recruitment of DTH effector T cells, and also by activating 5HT-2 receptors on these recruited T cells.

The elicitation of delayed-type hypersensitivity (DTH) reactions in mice is due to the sequential action of two different Ag-specific Thy-1+ cells. An early-acting DTH-initiating cell in the lymphoid organs produces a circulating, Ag-specific factor that is functionally analogous to IgE antibody and initiates DTH by sensitizing the local tissue for release of the vasoactive amine serotonin. In picryl chloride (PC1) or oxazolone (OX) contact sensitivity, this DTH-initiating factor is called PC1-F and OX-F respectively, and is Ag-specific, but MHC-unrestricted. The phenotype of polyclonal DTH-initiating cells was recently shown to be unusual for an Ag-specific cell. The phenotype was: Thy-1+, Lyt-1+ (CD5), triple negative (CD4-, CD8-, and CD3-), B220+ (Ly-5, CD45RA), positive for IL-3 receptors, but not IL-2 receptors, and positive for antibodies that react with a putative constant or framework portion of DTH-initiating factors such as anti-PC1-F antibodies and 14-30 mAb. We report here the generation of an Ag-specific DTH-initiating cell clone from nude mice that were immunized and boosted by contact sensitization with OX. By flow microfluorometry analysis, this clone has a similar unique surface phenotype, and by in vivo assay has the same functional abilities, as polyclonal DTH-initiating cells. The clone produces Ag-specific OX-F that acts in an Ag-specific manner to initiate DTH. Moreover, specific cDNA probes and Northern blot analysis of the clone demonstrated that the Ag-specific DTH-initiating cells are Thy-1+, CD3-, and IL-3R+. Thus, DTH initiation is due to an Ag-specific lymphoid cell, that produces an Ag-specific factor, and that has a unique surface phenotype for Ag-specific cells; namely, Thy-1+, CD5+, sIg-, CD4-, CD8-, CD3-, CD45RA+, IL-2R-, and IL-3R+.

Previous studies demonstrated that the initiation of murine delayed-type hypersensitivity (DTH), as exemplified by contact sensitivity induced by picryl chloride (PCI) or oxazolone (OX), is due to antigen-specific, T cell-derived, DTH-initiating factors called, respectively, PCl-F and OX-F. These factors participate in the extravascular recruitment of CD4+, Th-1, DTH effector T cells in the elicitation of DTH. Related factors also participate, together with nonantigen binding factors derived from CD8+ T cells, to constitute an antigen-specific T cell-derived suppressor factor (TsF) that can down regulate the ability of Th-1 effector T cells to mediate DTH. Since it was shown recently that athymic nude mice can make antigen-specific, DTH-initiating T cell factors, the current study tested whether nude mice also could produce the antigen-specific component of the TsF that suppresses DTH effector T cells. We found that antigen-specific factors from nu/nu mice could complement the nonantigen-binding subfactor produced in normal mice to constitute the whole antigen-specific TsF. Additional studies showed that the successful adoptive cell transfer of DTH-initiating T cell activity from nude mice into normal mice required cyclophosphamide treatment of the recipient. In contrast, transfer of DTH-initiating cell activity from nu/+ mice did not require cyclophosphamide treatment of the recipients. We hypothesized that nude mice lacked contrasuppressor cells. Although nude mice were able to manifest the early, initiating phase of DTH, we found that there was no suppression of early DTH-initiating T cells in nude mice, compared to nu/+. Therefore the production of DTH-initiating T cell factor could be boosted in nude mice. The ability to boost DTH-initiating cells in nude mice should facilitate the development of cell lines and clones with the ability to initiate DTH.

The guanidinium chloride-unfolded state of ribonuclease A was found to be an equilibrium mixture of slow- and fast-refolding forms of the protein chain, as has been suggested. Both forms appear to have the same spectroscopic observables as judged by the relative changes in fluorescence emission and polarization. The equilibrium between them is thermally dependent, with deltaHapp equal to -1.4 kcal/mol. The activation energy Ea is equal to 18 kcal/mol. These findings are consistent with the proposal that cis-trans isomerism of peptide bonds that are NH2-terminal to proline residues is responsible for the slow phase of RNase A refolding. However, the actual dependence of the magnitude of the slow reaction on initial, prefolding temperature cannot be explained by a model in which the proline configurations of the fast refolding form must be identical to those of the native protein, as has been suggested. Instead, the data reveal that, although the native structure of RNase A contains two cis prolines, cis isomers need not be present in the fast-refolding form in order for folding to occur.

An in vitro comparison of corneal endothelial damage caused by polymethylmethacrylate and by silicone intraocular lenses was made. Endothelial damage was assessed by vital staining of corneal endothelium immediately following direct contact with an intraocular lens. Silicone lenses produced less damage than polymethylmethacrylate lenses. The difference could be attributed to a specific type of damage-membrane stripping-seen only after contact with polymethylmethacrylate lenses.

We have recently developed a sensitive limiting dilution (LD) culture system to measure human alloreactive cytotoxic T lymphocyte precursors (CTL-p) in a given lymphoid cell population. We have now used this system to determine frequencies of donor HLA antigen-inducible CTL-p in the peripheral blood of human allograft recipients at various stages after transplantation. All patients (1 pancreas recipient and 9 kidney recipients) were on continuous cyclosporine treatment throughout the study. We report that, in patients with a well-functioning kidney graft (6/9), the number of donor-reactive CTL-p among peripheral blood lymphocytes decreased within 3-8 months after transplantation--in some cases (2/6) more than 10-fold. In contrast, frequencies of CTL-p with specificity for third-part HLA antigens remained largely unaltered in these patients. Furthermore, no decrease of donor-reactive CTL-p frequencies was seen in 3 of 4 patients showing clinical symptoms of graft rejection. These results indicate that functional clonal deletion of antigraft-reactive CTL-p may contribute to the state of graft tolerance in certain patients with a well-functioning kidney allograft.

Cytotoxic T lymphocytes (CTL) recognize antigen in the context of syngeneic MHC class I gene products. The "learning" of MHC restriction is thought to take place during the early intrathymic development of cytotoxic lymphocyte precursors (CLP). This view does not allow for any significant number of "allorestricted" (as opposed to selfrestricted) T cells to occur among mature, peripheral T cells. Recent evidence indicates, however, that large numbers of antigen-specific, allorestricted CLP can be readily detected among splenic T cell populations from several strains of unprimed normal mice. The frequencies of allorestricted CLP as determined under limiting dilution (LD) culture conditions are in fact in the same order of magnitude as frequencies of selfrestricted CLP. These findings were at the origin of the present study, which was designed to investigate whether antigen-specific, allorestricted CTL populations could also be detected among human peripheral blood T lymphocytes. To address this issue we studied the CTL response to virus-infected allogeneic stimulator cells in two different LD systems. In the first system, peripheral T cells from normal donors were cocultured under precisely defined LD conditions with Epstein-Barr virus (EBV)-transformed allogeneic lymphoblastoid cell lines (LCL). Frequencies of CLP that lysed the stimulating LCL ranged from one in 70 to one in 200, while frequencies of CLP that lysed the respective allogeneic ConA blast targets were 3-40-fold lower. The split-well analysis suggested that a large fraction of developing CTL colonies specifically lysed the stimulating LCL targets but neither the respective ConA blasts nor HLA-mismatched third party LCL targets. CTL generated in this culture system thus displayed allorestricted specificity for LCL membrane antigens. Comparable results were obtained in a second LD system where T cells from normal donors were cocultured with mumps virus-infected allogeneic mononuclear cells (MNC) or ConA blasts. One of 600 to one of 2,800 T cells gave rise to a cytotoxic colony that lysed mumps virus-infected stimulator-derived ConA blast target cells. To assess the lytic specificity of the in vitro expanding CTL populations, individual microcultures were split into three aliquots and tested for cytolytic activity against mumps virus-infected and noninfected specific targets as well as mumps virus-infected, HLA-mismatched third party targets. Clonal CTL populations from four of seven donors lysed virus-infected stimulator targets but did not lyse either noninfected stimulator targets or mumps virus-infected third party targets, i.e., they again showed an antigen-specific allorestricted lytic r

A limiting-dilution system was established to measure the frequency of alloreactive cytotoxic T-lymphocyte precursors (CTL-p) in human peripheral blood T cells. Culture medium supplemented with recombinant interleukin-2 enabled clonal expansion of all CTL-p stimulated by allogeneic peripheral blood or spleen cells. The range of CTL-p frequencies in fully HLA-mismatched responder-stimulator combinations was 1:240 to 1:1230. Split-well analysis of individual microwells showed that the cytotoxic T-cell clones generated under limiting-dilution conditions showed exquisite specificity for the stimulating alloantigens. Alloreactive CTL-p were enriched in the OKT4- T-cell subset. This limiting-dilution system was highly reproducible and can thus be applied to investigate human cytotoxic T-cell precursor frequencies in various clinically relevant situations.

The elicitation of delayed-type hypersensitivity (DTH) reactions in mice is caused by the sequential action of two different T cells. An early-acting, DTH-initiating T cell produces an Ag-specific T cell factor, that is analogous to IgE antibody and initiates DTH by sensitizing the local tissues for release of the vasoactive amine serotonin. In picryl chloride or oxazolone contact sensitivity, this T cell factor is Ag-specific, but MHC unrestricted. We, therefore, hypothesized that DTH-initiating T cells are primitive T cells with Ag receptors that can bind Ag without MHC restriction. In order to characterize the origin of this DTH-initiating T cell and the conditions that are necessary for its development, we contact-sensitized various strains of immunodeficient mice. Surprisingly, we found that the early phase of DTH was present in athymic nude mice. In contrast, the early component of DTH was absent in mice with severe combined immunodeficiency. These mice lack T and B cells, but have NK cells. These findings suggested that the early component of DTH was not caused by NK cells, and was caused by cells belonging to a lineage from a rearranging gene family. The early component of DTH in nude mice was Ag specific, was caused by MHC unrestricted Thy-1+ T cells, and was mediated by Ag-binding, Ag-specific T cell factors. We found that DTH-initiating, T cell-derived, Ag-binding molecules from nude mice and normal CBA/J mice had the same functional properties. The early component of DTH was elicited in two different systems (contact sensitivity and SRBC-specific DTH) in two strains of nude mice (BALB/c athymic nudes and CByB6F1/J-nu) from two different suppliers, but not in BALB/c and athymic nudes from a third supplier. From these findings we concluded that DTH-initiating T cells, which produce IgE-like Ag-specific T cell factors, are present in some strains of athymic nude mice and thus are relatively thymic independent T cells.