Cardiac allograft remodeling after heart transplantation is associated with increased graft vasculopathy and mortality.

The aim of this study was to assess the patterns, predictors and outcomes of left ventricular remodeling after heart transplantation (HTX). Routine echocardiographic studies were performed and analyzed at 1 week, 1 year and 3-5 years after HTX in 134 recipients. At each study point the total cohort was divided into three subgroups based on determination of left ventricle mass and relative wall thickness: (1) NG-normal geometry (2) CR-concentric remodeling and (3) CH-concentric hypertrophy. Abnormal left ventricular geometry was found as early as 1 week after HTX in 85% of patients. Explosive mode of donor brain death was the most significant determinant of CH (OR 2.9, p = 0.01) at 1 week. CH at 1 week (OR 2.72, p = 0.01), increased body mass index (OR 1.1, p = 0.01) and cytomegalovirus viremia (OR - 4.06, p = 0.02) were predictors of CH at 1 year. CH of the cardiac allograft at 1 year was associated with increased mortality as compared to NG (RR 1.87, p = 0.03). CR (RR 1.73, p = 0.027) and CH (RR 2.04, p = 0.008) of the cardiac allograft at 1 year is associated with increased subsequent graft arteriosclerosis as compared to NG.

Low doses of interferon alpha result in more effective clinical natural killer cell activation.

To define critical parameters concerning interferon (IFN) effects upon natural killer (NK) cells in vivo, we gave cancer patients serial weekly intramuscular injections of purified lymphoblastoid IFN in six doses ranging from 10(5) to 3 X 10(7) U. Dose sequences were determined by randomly allocating patients to one of six levels in a latin square ordering scheme. NK cell stimulation, a threefold peak increase above preinjection levels of cytolysis (P = 0.022), occurred in peripheral mononuclear cells (PMC) sampled 24 h postinjection, of 3 X 10(6) U, but was not detectable at any dose in PMC sampled 7 d postinjection. No blunting occurred in NK cell responsiveness to repeated injection of IFN dosages a second time at or several weeks after study completion. At IFN doses of 3 X 10(6), 10(7), and 3 X 10(7) U, a negative correlation existed between the amount of IFN injected and the average extent of NK cell activation (r = -0.423, P less than 0.05). This contrasted with the progressively increasing response of NK cells to in vitro incubation with increasing concentration of up to 3,000 U/ml of IFN. Overnight culturing of PMC sampled before IFN injections resulted in a mean 1.9-fold increase in cytolytic activity (P = 0.0005) and a mean 53% decrease in variance (P = 0.024) between serial preinjection NK cell activity determinations. Cell separation procedures may, therefore, have resulted in NK cell inactivation, from which overnight culturing permitted recovery. We found that maximal NK cell activation at a low IFN dose, decreasing NK cell responsiveness at higher doses, and the need to culture PMC to efficiently detect NK cell boosting may account for disparities in reported effects of IFN on NK cell function.

Role of endogenous atrial natriuretic factor in acute congestive heart failure.

The current studies were designed to investigate the functional significance of elevated endogenous atrial natriuretic factor (ANF) in acute congestive heart failure (CHF). Integrated cardiorenal and endocrine function were measured in three models of acute low-output congestive heart failure with comparably reduced cardiac output (CO) and mean arterial pressure (MAP). Acute CHF was produced by rapid right ventricular pacing (group I, n = 5) which decreases CO and increases atrial pressures and plasma ANF. In group II, n = 5, thoracic inferior vena caval constriction (TIVCC) was produced to decrease venous return and CO but without increases in atrial pressure or plasma ANF. In group III, n = 5, TIVCC was performed and exogenous ANF infused to achieve plasma concentrations observed in acute CHF. In acute CHF with increases in endogenous ANF, sodium excretion (UNaV), renal blood flow (RBF), plasma renin activity (PRA), and plasma aldosterone (PA) were maintained despite decreases in CO and MAP. In contrast, TIVCC with similar reductions in CO and MAP but without increases in ANF resulted in decreases in UNaV and RBF and increases in PRA and PA. Exogenous administration of ANF in TIVCC to mimic levels in acute CHF prevented sodium retention, renal vasoconstriction, and activation of renin and aldosterone. These studies demonstrate that endogenous ANF serves as an important physiologic volume regulator in acute CHF to maintain sodium excretion and possibly participate in the suppression of activation of the renin-angiotensin-aldosterone system despite the stimulus of arterial hypotension.

Identification of atrial natriuretic factor within ventricular tissue in hamsters and humans with congestive heart failure.

In normal mammals, atrial natriuretic factor (ANF) is present within atrial myocardial cells but is absent from ventricular myocardium. In primitive organisms ANF is present within both atria and ventricle, suggesting that the ventricle may participate both in the synthesis and release of the hormone. The current study was designed to test the hypothesis that ventricular ANF develops as a homeostatic response to intravascular volume overload. Studies were performed on cardiac tissue obtained from (i) normal and cardiomyopathic hamsters, (ii) autopsied humans with and without cardiac disease, and (iii) living humans with congestive heart failure (CHF) undergoing diagnostic right ventricular endomyocardial biopsy. The myocardium was examined for the presence of immunoreactive ANF using a two-stage immunohistochemical technique, with nonimmune rabbit sera used as a negative control. There was unequivocal evidence of focal subendocardial deposits of immunoreactive ANF present in both of the ventricles of all six cardiomyopathic hamsters, four of five autopsied human subjects with CHF, and five of seven biopsied humans. No immunoreactive ANF was observed within the ventricular myocardium of control hamsters or normal humans. Utilizing crude tissue homogenates and radioimmunoassay techniques, the quantity of ANF was determined in cardiac atria, ventricles, and noncardiac skeletal muscle. Heart failure is characterized by a reduction in atrial ANF and an increase in ventricular ANF. This study demonstrates immunoreactive ANF is present within the ventricular myocardium in cardiomyopathic hamsters and humans with CHF, and suggests that the ventricle may be capable of responding to chronic volume overload by producing ANF.

Tumor-immunotherapeutic efficacy of Serratia marcescens polyribosomes.

The ability of polyribosomes, obtained from several bacterial species, to suppress the development of cutaneous SaD2 fibrosarcomas in DBA/2 mice were evaluated. Suppression of tumor appearance depended upon the tumor load at the time of treatment, dose of polyribosomes, and species source of polyribosomes, with Serratia marcescens being superior to Escherichia coli, Streptococcus pneumoniae, Mycobacterium bovis (Pasteur), Mycobacterium smegmatis, and Propionibacterium acnes (formerly Corynebacterium parvum). A single injection of 5 or 50 microgram of Serratia polyribosomes at the tumor site 72 hr after the intradermal administration of 1.5 X 10(3) SaD2 cells resulted in 66 to 95% survival. All untreated animals expired within 50 days. Tumor suppression occurred at both flank and footpad sites. Presensitization with polyribosomes and incorporation of polyribosomes into adjuvant were not required for the tumor-suppressive effect. Treatment of Serratia polyribosomes with RNase or pronase reduced the number of survivors. Endotoxin was not detectable with the Limulus amebocyte lysate assay.

Comparative in vivo and in vitro activation of human natural killer cells by two recombinant alpha-interferons differing in antiviral activity.

Natural killer (NK) cell activation by two interferon-alpha subtypes, interferon-alpha A and interferon-alpha D, was examined in vitro and in vivo in eight cancer patients. When assessed in vitro, NK cells in six of eight patients lysed K562 target cells to a greater extent when incubated with interferon-alpha A (1 ng/ml) than with the same concentration of interferon-alpha D. However, when patients were evaluated collectively, no significant difference was detectable in the effectiveness of the two subtypes in enhancing NK cell activity. Patients received the same interferons given as four injections which were randomized with respect to subtype and were separated by intervals of six or more days. NK cell activity was consistently elevated in peripheral mononuclear cells sampled 24 hr but not seven days after injection as compared to peripheral mononuclear cells sampled just prior to each injection (p less than 0.001 for both subtypes). At a given dose level, both interferon subtypes resulted in comparable NK cell activation. However, a negative correlation existed between the amount of interferon administered and the magnitude of enhancement (p less than 0.05). In 16 separate paired determinations, there were eight in which NK cell activity was lower after the second injection of an interferon dose than after the first injection of the same dose (15 or 45 micrograms, irrespective of subtype), and eight in which the alternate pattern occurred. Thus, repeated injection in the same patient of one or the other dose resulted in no consistent changes in the extent of NK cell stimulation. Since the two interferons have a 20-fold difference in specific antiviral activity for human amnion cells and up to an 80-fold difference in human fibroblasts, either different mechanisms are involved in antiviral and NK cell-stimulatory activity, or activities of these two subtypes for cells of different histogenesis vary. Greater NK cell-stimulatory activity therefore occurred at the lowest tested doses, a dose which was less than 10% of previously reported maximally tolerated doses of these interferons.

American cancer society Phase I trial of naturally produced beta-interferon.

Naturally produced beta-interferon was evaluated following i.m. and i.v. administration to 18 patients with advanced cancer. Fever (mean +/- S.E. = 38.1 degrees +/- 1.7 degrees), enhancement of natural killer cell cytotoxicity, and depression of the white blood cell count occurred following a single i.m. injection in the absence of detectable serum antiviral activity. Fever, rigors, and fatigue were dose-limiting toxicities following daily i.v. administration of 10 million units. Tachyphylaxis, as reported following repetitive administration of alpha-interferons, did not occur. Side effects, depression of the white blood cell count, and enhancement of natural killer cell cytotoxicity were similar when beta-interferon was administered daily as a 10-min bolus or as a 6-hr infusion. However, while natural killer cell cytotoxicity increased progressively over 10 days of bolus injections, it was maximal after the initial 6-hr infusion of beta-interferon. Administration of 10 million units of beta-interferon divided equally between a 10-min bolus injection and a 3-hr infusion was well tolerated and resulted in high initial peak and lower sustained serum interferon levels. Based on pharmacokinetic criteria, this schedule of administration can be recommended for further study in Phase II trials. However, in light of the biological activity of beta-interferon following i.m. administration, the level of beta-interferon in the serum may have limited value as a predictor of antitumor response, toxicity, or biological response modification.

Comparison of the biologic effects of two recombinant human interferons alpha (rA and rD) in humans.

IFN-alpha (rD) was investigated to determine the relationship between antiviral activity in vitro and the modulation of biologic effects in vivo. Eight patients with malignancy were given 15 and 45 micrograms weekly injections of IFN-alpha (rA) and IFN-alpha (rD). The frequency of side effects was much lower with IFN-alpha (rD) injections. This was objectively documented both in incidence of side effects (20 versus 45, p less than 0.01) and mean maximum temperature (1 degree C lower with IFN-alpha (rD), p less than 0.002). A bovine cell line, MDBK, was used to measure interferon concentrations in the serum. Geometric mean peak titers and time-to-peak titers were similar with the two recombinant interferon preparations. Although IFN-alpha (rD) has relatively less antiviral activity on human cells, its effect on the total granulocyte count, natural killer (NK) cell cytotoxicity, and 2'5'-A activity was comparable to IFN-alpha (rA). Mean NK cell percent specific 51Cr release was enhanced by both interferons (after 15 micrograms doses, mean percent NK cell cytotoxicity IFN-alpha (rD) preinjection, 10.5% +/- 2.3%; post-injection, 27.2% +/- 4.5%; IFN-alpha (rA), preinjection, 14.4% +/- 3.2%, postinjection, 25.1% +/- 5%). Species-specific antiviral activity of an interferon does not necessarily predict other biologic properties following in vivo administration.

Expression of atrial natriuretic factor in the human ventricle is independent of chamber dilation.

This study investigated the presence of atrial natriuretic factor in ventricular tissue obtained from humans with dilated or restrictive heart disease. In 17 patients with ventricular dilation and impaired systolic function and in 8 patients with restrictive heart disease and preserved systolic function, the presence of ventricular atrial natriuretic factor was investigated in tissue obtained by ventricular endomyocardial biopsy. The objective of the study was to determine if the ventricular presence of atrial natriuretic factor is dependent on ventricular dilation. Left ventricular end-diastolic volume index was greater in the group with dilated cardiomyopathy than in the group with restrictive cardiomyopathy (134 +/- 13 versus 78 +/- 5 ml/m2, p less than 0.05); end-diastolic pressure was elevated in the two groups (20 +/- 2 versus 25 +/- 4 mm Hg, p = NS). With the use of immunohistochemical techniques, ventricular atrial natriuretic factor was clearly detected in 15 of the 17 patients with dilated cardiomyopathy and in 6 of the 8 patients with restrictive cardiomyopathy. This study demonstrates the high prevalence of ventricular atrial natriuretic factor in living patients with either systolic or diastolic dysfunction. Whereas in the atria, stretch or dilation may be an important stimulus, atrial natriuretic factor in the ventricular chamber occurs independent of dilation.

Coexistent pulmonary and portal hypertension: morphologic and clinical features.

Patients with portal hypertension of varying etiology may develop pulmonary artery hypertension. In the present autopsy study, pulmonary and hepatic tissue was studied in 12 patients in whom pulmonary and portal hypertension coexisted. Plexogenic pulmonary arteriopathy was present in 10 patients, 7 of whom had coexistent thromboembolic lesions. One patient had isolated medial hypertrophy, which may be an early stage in the plexogenic category, whereas isolated thromboembolic pulmonary vascular disease was observed in one subject. Hepatic disease was consistent with alcoholic cirrhosis in seven patients, cryptogenic cirrhosis in four and extrahepatic portal hypertension without cirrhosis in one. Thrombocytopenia was present in all 10 patients whose platelet count was determined. This study suggests that pulmonary hypertension associated with portal hypertension commonly has a plexogenic appearance on histologic examination. However, thrombosis (whether embolic or in situ) may also contribute to vascular obstruction.

Enhanced aggregation of human neutrophils by MnCl2 or DTT differentiates the roles of L-selectin and beta 2-integrins.

MnCl2 and dithiothreitol (DTT) enhance the adhesive functions of beta 2 -integrins. We have used these agents and flow cytometry to distinguish the contributions of beta 2-integrins and L-selectin to neutrophil aggregation. Although neither compound induced aggregation, they prolonged N-formyl-methionyl-leucyl-phenylalanine-induced aggregation and produced larger aggregates. Because activated polymorphonuclear granulocytes (PMN) shed L-selectin in the presence of MnCl2, but not DTT, we could evaluate the role of L-selectin in the early and late stages of aggregation. Blocking L-selectin sites with DREG200 Fab and/or beta 2-integrin sites with IB4 Fab indicated that aggregation under all conditions remained beta 2-integrin- and L-selectin-dependent. Disaggregation was integrin-dependent whether L-selectin was present or shed. The disaggregation kinetics suggested that integrin bonds turned over at a slower rate in MnCl2-treated cells. Enhanced aggregation due to DTT and MnCl2 required sustained energy output, suggesting intracellular rather than strictly conformational control. These results provide evidence that PMN aggregation, like leukocyte-endothelial cell adhesion, utilizes L-selectin to form intercellular contacts that are maintained through activated integrins.

Oxidant inhibition of alphaLbeta2 integrin adhesion: evidence for coordinate effects on conformation and cytoskeleton linkage.

Dithiothreitol (DTT) and other dithiol antioxidants with closely spaced thiol pairs strongly activate leukocyte function antigen-1 (LFA-1, alphaLbeta2 integrin) to bind intercellular adhesion molecule-1 (ICAM-1). Because direct biochemical modification of LFA-1 by DTT is not apparently involved, we investigated the possible role of a reduction-oxidation (redox)-sensitive adhesion-regulatory pathway. Phenylarsine oxide (PAO), an oxidant selectively reactive with closely spaced pairs of thiol groups, inhibited LFA-1-dependent adhesion of human natural killer and HSB2 T leukemia cells to murine cells expressing human ICAM-1. PAO also induced disappearance of a conformation-sensitive LFA-1 epitope recognized by KIM127 antibodies and promoted an increase in total apparent cytoskeleton-linked LFA-1 in which a novel cytochalasin D-resistant linkage was involved. Exposure of PAO-pretreated cells to DTT caused a decline in LFA-1/cytoskeleton linkages in conjunction with rapid restoration of KIM127 epitope expression and LFA-1 adhesive function. Implicating an intracellular site of action were findings that (1) an epitope-tagged PAO probe bound predominantly to intracellular proteins but not detectably to immunoprecipitation-purified LFA-1 chains, and (2) membrane permeant but not impermeant dithiol antioxidants reversed PAO adhesion-inhibitory effects. These results support the concept of a reversible redox-sensitive linkage between LFA-1 and cytoskeleton by which oxidants and antioxidants may exert profound opposing effects on LFA-1 conformation and adhesive function.

Beta2-integrins mediate stable adhesion in collisional interactions between neutrophils and ICAM-1-expressing cells.

The aggregation of human neutrophils in suspension has features that are analogous to their attachment to activated endothelium in that both involve selectin and beta2-integrin adhesion receptors. For the collisional interaction that forms neutrophil aggregates in suspension, there is a tethering step in which L-selectin on neutrophils binds PSGL-1. At relatively low shear rates (100-200 s(-1)) firm adhesion is mediated in equal measure by LFA-1 binding to ICAM-3, and Mac-1 binding to an as yet undefined ligand. In this report we used a mouse melanoma cell line expressing an estimated 700,000 ICAM-1 (CD54) to examine the relative roles of LFA-1 and Mac-1 over the kinetics of heterotypic cell adhesion in shear mixed suspensions. Neither heterotypic nor homotypic neutrophil aggregates formed with application of shear alone. However, the rate of aggregation peaked within seconds of chemotactic stimulation. In contrast to homotypic aggregation, neither L-selectin nor its O-glycoprotein ligands on neutrophils contributed to heterotypic adhesion. Adhesion was inhibited in a dose-dependent manner as ICAM-1 was titrated with blocking mAb. A direct interaction between LFA-1 and ICAM-1 was preferred over the first minute of stimulation, whereas at later times adhesion was supported equally by Mac-1. Activation with MnCl2 also favored participation of the constitutively expressed LFA-1. Application of defined shear in a cone and plate viscometer showed that adhesion to the ICAM-1 cells decreased from a maximum level to baseline as shear rate increased up to 400 s(-1) in a manner typical of integrin adhesion alone. In contrast, homotypic aggregation supported by the transition from selectin to integrin binding exhibited an increase in efficiency up to 800 s(-1). The pathophysiological significance of receptor site density and duration of contact in collisional interactions relevant to leukocyte recruitment compared to leukocyte-endothelial cell interactions on surfaces is discussed.

Atrial natriuretic peptide during mineralocorticoid escape in the human.

The relationship between plasma atrial natriuretic peptide (ANP) and mineralocorticoid escape was examined in six normal men (age, 20-32 yr) treated with 0.4 mg/day fludrocortisone acetate for 9-14 days. Urinary sodium excretion decreased from 162 +/- 15 (SEM) meq/24 h before to 97 +/- 10 meq/24 h during fludrocortisone acetate administration (P less than 0.05). Despite continued fludrocortisone acetate administration, sodium excretion subsequently returned to baseline (escape). Plasma ANP increased from 33 +/- 6 pg/ml (control) to 55 +/- 14 pg/ml on the first day of escape (P less than 0.05). Escape was associated with a decrease in PRA from 0.90 +/- 0.22 (control) to 0.26 +/- 0.08 ng/ml X h (escape, P less than 0.05). The escape phenomenon was not associated with a significant change in mean arterial pressure or glomerular filtration rate. This study demonstrates that mineralocorticoid escape is temporally related to a significant increase in circulating ANP.

Lymphocyte subset analysis by flow cytometry. Comparison of three different staining techniques and effects of blood storage.

Flow cytometric analysis enables the researcher and clinician to enumerate lymphocyte subsets in peripheral blood mononuclear cells (PBMC). Often blood samples are collected at one site and then shipped to another site for analysis. Many options in the storage, preparation, and staining of PBMC for flow cytometric analysis exist. Preparation techniques include the conventional Ficoll-Paque (FP) density centrifugation versus the whole blood technique with red blood cells (RBC) lysed using a lysing reagent. In comparing three methods of PBMC preparation, and comparing the staining of fresh blood cells with staining of cells after storage for 24 h at 4 degrees C, analyses show how these different techniques affect the results.

The synergistic effects of cyclosporine and endothelin--demonstration of an important cardiodepressor action.

Cyclosporine represents the foundation for current immunosuppressive therapy following solid organ transplantation. CsA use is associated with renal insufficiency and systemic hypertension. We hypothesized that CsA would enhance the vascular actions of endothelin (ET). Three groups of anesthesized dogs (n = 15) were studied. Group 1 received CsA alone (1 mg/kg), group 2 received ET alone (1 ng/kg/min), and group 3 received combined CsA (1 mg/kg) and ET (1 ng/kg/min). The hemodynamic and renal effects were evaluated after 30 min. Combined treatment resulted in a profound reduction in mean arterial pressure (-62 +/- 14 mmHg (P < .05) and cardiac output (-2.2 +/- 0.4.1/min (P < .05). The reduction in mean arterial pressure and cardiac output were significantly greater than that observed with CsA or ET alone. Systemic vascular resistance was not significantly changed. Combined CsA and ET resulted in a significant reduction in renal blood flow (195 +/- 18 to 101 +/- 11 ml/mm P < .05) but without evidence of active renal vasoconstriction. The decline in GFR (31.8 +/- 5.6 ml/min to being unmeasurable) was of greater magnitude than the change in renal blood flow, suggesting enhanced afferent anteriolar vasoconstriction or an alteration in the ultrafiltration coefficient. These studies demonstrate an important and synergistic cardiodepressor effect when CsA and ET are combined.

Effects of pentoxifylline on renal function and blood pressure in cardiac transplant recipients: a randomized trial.

BACKGROUND: The current success of cardiac transplantation is in part attributable to the development of effective immunosuppressive agents such as cyclosporine. However, concern remains regarding the potential for cyclosporine-induced nephrotoxicity. Animal studies and early reports of renal protective effects of pentoxifylline in bone marrow transplant recipients prompted a randomized trial in cardiac transplant recipients. METHODS: Twenty-nine patients were randomized to receive pentoxifylline 400 mg p.o. t.i.d. or matching placebo for 1 year after cardiac transplantation. Renal function was assessed preoperatively and at 1, 6, and 12 months postoperatively. Glomerular filtration rate and renal plasma flow were measured with iothalamate and para-aminohippurate, respectively. Serum creatinine was also measured. Ambulatory blood pressure monitoring after withdrawal of antihypertensives for 3 days was performed 12 months postoperatively. RESULTS: Twenty-seven patients completed the study. Glomerular filtration rate rose between 1 and 6 months after transplantation, presumably due to the reduction in goal cyclosporine level in that period, and then fell modestly between 6 and 12 months, presumably due to ongoing nephrotoxic effects of cyclosporine. No difference in glomerular filtration rate or creatinine was seen between pentoxifylline and placebo groups at any interval. Renal plasma flow increased modestly between baseline and 6 months in the pentoxifylline group, but not in the placebo group, and then fell between 6 and 12 months. Serum creatinine increased between baseline and 6 months in both groups, apparently due to increased body weight. Results of 18-hr ambulatory blood pressure monitoring obtained 1 year after transplantation was not different between groups. CONCLUSIONS: Renal function declines only modestly in the first year after cardiac transplantation. Pentoxifylline did not attenuate this process and had no effect on blood pressure. The modest decline in renal function may be related to current immunosuppressive strategies.

Performance of in-line microfluidic mixers in laminar flow for high-throughput flow cytometry.

We describe a micromixing approach that is compatible with commercial autosamplers, flow cytometry, and other detection schemes that require the mixing of components that have been introduced into laminarflow. The scheme is based on high-throughput flow cytometry (HyperCyt) where samples from multi-well plates that have been picked up by an autosampler can be separated during delivery by the small air bubbles introduced during the transit of the autosampler probe from well to well. Here, either cell or particle samplesflowing continuously and driven by a syringe are brought together in a Y with reagent samples from wells driven by a peristaltic pump. The mixing is driven by a magnetic microstirrer contained within the sample line. The mixing is assessed using fluorescence of both cell calcium responses and bead-based fluorescence unquenching. In the analysis stream, the particles and reagents are mixed with eithera "wire" or "bar". The bar is more efficient than the wire, and the efficiency of either depends on the spinning action. The high-throughput approach and mixing in HyperCyt integrate autosamplers with submicroliter detection volumes for analysis in flow cytometry or in microfluidic channels.

HTPS flow cytometry: a novel platform for automated high throughput drug discovery and characterization.

The flow cytometer is unique among biomedical analysis instruments because it makes simultaneous and multiple optical measurements on individual cells or particles at high rates. High throughput flow cytometry represents a potentially important multifactorial approach for screening large combinatorial libraries of compounds. Limiting this approach has been the availability of instrumentation and methods in flow cytometry for automated sample handling on the scale required for drug discovery applications. Here, we describe an automated system in which a novel patented fluidics-based pharmacology platform, the HTPS (High Throughput Pharmacological System), is coupled to a flow cytometer using a recently described plug flow-coupling valve technology. Individual samples are aspirated sequentially from microplate wells and delivered to a flow cytometer for rapid multiparametric analysis. For primary screening to detect and quantify cell fluorescence in endpoint assays, a high-speed no-wash protocol enabled processing of 9-10 cell samples/min from 96-well microplates. In an alternate primary screening format, soluble receptor ligands were sampled from microplate wells at rates of 3-4 samples/minute and successfully assessed for the ability to elicit intracellular calcium responses. Experiments with fluorescent beads validated the accurate automated production by the HTPS of exponential and linear gradients of soluble compounds. This feature enabled rapid (2- to 3-min) characterization of the intracellular calcium dose response of myeloid cells to formyl peptide as well as the quantitative relationship between formyl peptide receptor occupancy and cell response. HTPS flow cytometry thus represents a powerful high throughput multifactorial approach to increase the efficiency with which novel bioresponse-modifying drugs may be identified and characterized.

Ventricular septal rupture complicating acute myocardial infarction: identification of simple and complex types in 53 autopsied hearts.

Fifty-three hearts with rupture of the ventricular septum complicating acute myocardial infarction (AMI) were studied. Thirty-three of the hearts were from men (average age 76 years) and 20 were from women (average age 73 years). The study showed 2 types of rupture of the ventricular septum: simple (28 patients) and complex (25 patients). Simple ruptures were direct through-and-through defects. Complex ruptures were associated with serpiginous dissection tracts remote from the primary site of tear of the ventricular septum. Specimens were classified as to the location of the underlying AMI and the level of the septum (apex to base) at which the rupture occurred. Twenty-nine hearts had an inferior AMI and 24 an anterior AMI. Complex ruptures occurred in 20 of the inferior AMIs (69%) and in 5 of the anterior AMIs (21%) (p less than 0.001). Ruptures that involved the inferobasal portion of the septum were much more likely to be complex (94%) than ruptures in all other locations (27%, p less than 0.001). Significant 3-vessel obstructive coronary arterial atherosclerosis was present in 48 hearts. Rupture of a second structure in addition to the ventricular septum was observed in 11 hearts (left ventricular free wall in 9 cases and papillary muscle in 2). The interval from the onset of the AMI to rupture of the septum could be estimated in 22 patients and averaged 4 days (median 2.5 days). Complete heart block reportedly occurred in 6 patients during hospitalization.