Relation of soluble and platelet P-selectin to early outcome in patients with acute myocardial infarction after thrombolytic therapy.

Pre- and post-treatment platelet and soluble P-selectin were measured in a group of patients enrolled in the GUSTO-III study and were correlated with clinical outcomes. A peak in soluble P-selectin levels at 3 hours after thrombolytic therapy and lower baseline platelet P-selectin were associated with successful thrombolysis.

Role of soluble and platelet-bound P-selectin in discriminating cardiac from noncardiac chest pain at presentation in the emergency department.

BACKGROUND: It has been reported that selectins participate in the pathogenesis of acute coronary syndromes by modulating platelet-leukocyte-endothelium interactions. Elevated P-selectin level also has been observed in the clinical setting of myocardial ischemia and reperfusion; however, its utility in differentiating cardiac from noncardiac origins of chest pain is unknown. METHODS AND RESULTS: Soluble and platelet fractions of P-selectin were measured for 122 patients with chest pain and 14 healthy persons acting as controls. Patients with a cardiac problem (unstable angina, congestive heart failure, acute myocardial infarction) had significantly elevated levels of soluble P-selectin (156.0 +/- 58.8 ng/mL, P =.002) and platelet-bound P-selectin (11.7% +/- 6.4% positive cells, P =.013) compared with the P-selectin profile among controls (102.6 +/- 29.0 ng/mL, 4.1% +/- 1.2% positivity) and among patients with noncardiac chest pain (114.7 +/- 36.6 ng/mL, 5.7% +/- 2.9% positivity). With a cutpoint of 10% positivity for membrane and 120 ng/mL for soluble P-selectin, the sensitivities were 0.442 and 0. 558, and the specificities were 0.915 and 0.553. CONCLUSIONS: When a patient arrives in the emergency department, measurement of membrane P-selectin may serve as an additional diagnostic tool to detect heightened platelet activity, which is most prevalent among patients with a cardiac origin of chest pain. However, low sensitivity limits the utility of the P-selectin profile alone in suitably differentiating acute coronary syndromes within the overall population of patients with chest pain.

The flow cytometer model markedly affects measurement of ex vivo whole blood platelet-bound P-selectin expression in patients with chest pain: are we comparing apples with oranges.

Surface expression of P-selectin is known to be a marker of platelet activation in patients with acute coronary syndromes. However, direct comparisons of flow cytometer data may be obscured by differences in methodology, artifactual platelet activation during washing procedures, choice of antibodies, absence of control measurements, and possible observer bias due to unblinded data collection. We sought to test the hypothesis that the model of flow cytometer represents another variable affecting P-selectin measurements. Platelet P-selectin in whole blood was measured by FACScan (Becton Dickinson, Inc., San Diego, CA, USA) or EPICS XL (Coulter Corporation, Hialeah, FL, USA) flow cytometry in 338 patients presenting with chest pain to the emergency departments of three community hospitals as part of a multicenter diagnostic trial. Platelet expression of P-selectin (% of cell positivity) was consistently higher for each discharge diagnosis when measured with FACScan flow cytometer (13.2+/-4.1 for myocardial infarction, 10.0+/-3.6 for unstable angina, 9.9+/-3.5 for heart failure, 4.7+/-0.1 for gastrointestinal illness, and 6.3+/-0.7 for patients with noncardiac chest pain) when compared with results obtained from the EPICS XL instrument (2.4+/-0.2, 2.5+/-0.2, 2.5+/-0.1, 1.8+/-0.1, and 2.3+/-0.1 respectively, p=0.0001 for all groups). This study reveals marked discrepancies in the level of platelet P-selectin measurement based exclusively on the model of flow cytometer used. If P-selectin is to become a diagnostic tool for differentiating an etiology of chest pain, standardized measurements must be defined for each model of flow cytometer.

Risk stratification of emergency department patients with acute coronary syndromes using P-selectin.

OBJECTIVES: We compared the predictive properties of P-selectin to creatine kinase, MB fraction (CK-MB) for detecting acute myocardial infarction (AMI), acute coronary syndromes (ACS) and serious cardiac events upon emergency department (ED) arrival. BACKGROUND: Practioners detecting early diagnosis of ACS have focused on cardiac markers of myocardial injury. Plaque rupture/platelet aggregation precedes myocardial ischemia. Therefore, markers of platelet aggregation may detect ACS earlier than cardiac markers. METHODS: Consecutive patients with potential ACS presenting to an urban university ED were identified by research assistants who screened all ED patients between November 12, 1997 and January 31, 1998. Whole blood was drawn at presentation and 1 h later and rapidly stained and fixed for membrane P-selectin assay and plasma was separated for soluble P-selectin assay. Creatine kinase, MB fraction values were determined using standard immunoassay techniques. Clinical history and hospital course were followed daily. Outcomes were AMI, ACS (AMI and unstable angina) and serious cardiac events. Receiver operator characteristic curves were derived for CK-MB, and soluble and membrane-bound P-selectin to determine the optimal cutoff values. Predictive properties were calculated with 95% confidence intervals. RESULTS: A total of 263 patients were enrolled. They had a mean age of 56.5+/-14 years; 52% were male. There were 22 patients with AMI; 87 patients with ACS and 54 patients with serious cardiac events. Creatine kinase, MB fraction had a higher specificity for detection of AMI, ACS and serious cardiac events than both soluble and membrane-bound P-selectin. At the time of ED presentation, the specificity of CK-MB, and soluble and membrane-bound P-selectin for AMI was 91% versus 76% versus 71%; for ACS, 95% versus 79% versus 71%, and for serious cardiac events, 91% versus 76% versus 72% (p < 0.05). The sensitivities for AMI were 50% versus 45% versus 32%; for ACS, 26% versus 35% versus 30%, and for serious cardiac events, 29% versus 35% versus 36%. CONCLUSIONS: Although theoretically attractive, the use of soluble and membrane-bound P-selectin for risk stratification of chest pain patients at the time of ED presentation does not appear to have any advantages over the use of CK-MB.

A nonhuman primate model for the selective elimination of CD8+ lymphocytes using a mouse-human chimeric monoclonal antibody.

Nonhuman primates provide valuable animal models for human diseases. However, studies assessing the role of cell-mediated immune responses have been difficult to perform in nonhuman primates. We have shown that CD8+ lymphocyte-mediated immunity in rhesus monkeys can be selectively eliminated using the mouse-human chimeric anti-CD8 monoclonal antibody cM-T807. In vitro, this antibody completely blocked antigen-specific expansion of cytotoxic T cells and decreased major histocompatibility complex class I-restricted, antigen-specific lysis of target cells but did not mediate complement-dependent cell lysis. In vivo administration of cM-T807 in rhesus monkeys resulted in near total depletion of CD8+ T cells from the blood and lymph nodes for up to 6 weeks. This depletion was not solely complement-dependent and persisted longer in adults than in juveniles. Preservation of B cell and CD4+ T cell function in monkeys depleted of CD8+ lymphocytes was demonstrated by their ability to develop humoral immune responses to the administered chimeric monoclonal antibody. Furthermore, during CD8+ lymphocyte depletion, monkeys developed delayed-type hypersensitivity reactions comprised only of CD4+ T cells but not CD8+ T cells. This CD8+ lymphocyte depletion model should prove useful in defining the role of cell-mediated immune responses in controlling infectious diseases in nonhuman primates.

Control of viremia in simian immunodeficiency virus infection by CD8+ lymphocytes.

Clinical evidence suggests that cellular immunity is involved in controlling human immunodeficiency virus-1 (HIV-1) replication. An animal model of acquired immune deficiency syndrome (AIDS), the simian immunodeficiency virus (SIV)-infected rhesus monkey, was used to show that virus replication is not controlled in monkeys depleted of CD8+ lymphocytes during primary SIV infection. Eliminating CD8+ lymphocytes from monkeys during chronic SIV infection resulted in a rapid and marked increase in viremia that was again suppressed coincident with the reappearance of SIV-specific CD8+ T cells. These results confirm the importance of cell-mediated immunity in controlling HIV-1 infection and support the exploration of vaccination approaches for preventing infection that will elicit these immune responses.

Increased baseline levels of platelet P-selectin, and platelet-endothelial cell adhesion molecule-1 in patients with acute myocardial infarction as predictors of unsuccessful thrombolysis.

BACKGROUND: Platelets play a pivotal role in the pathogenesis of acute myocardial infarction and their activation can cause thrombolysis to fail. METHODS: Baseline aggregation of platelets and expression of major surface receptors measured by flow cytometry were compared with clinical outcome after thrombolysis for 23 patients enrolled in the GUSTO-III trial. RESULTS: Failure to reperfuse (n = 3) and recurrent ischemia (n = 2) were observed in five patients who subsequently underwent emergency angioplasty. These patients were treated later in their course of disease than were the 18 patients who were successfully reperfused and remained free of recurrent ischemia. Greater than normal expression of platelet P-selectin (33.7 +/- 1.1 versus 28.1 +/- 1.9, P = 0.01) and expression of platelet--endothelial cell adhesion molecule-1 (PECAM-1; (57.1 +/- 2.3 versus 50.2 +/- 2.8, P = 0.02) were observed in members of the group with recurrent ischemia or failed reperfusion. CONCLUSION: Greater than normal baseline expression of P-selectin and PECAM-1 platelet receptors was correlated to delayed and unsuccessful coronary thrombolysis among patients presenting with acute myocardial infarction.

Changes in hemostasis after parenteral magnesium in myocardial ischemia-reperfusion: from animal studies to clinical trials.

There has been some debate regarding the benefit of parenteral magnesium (Mg) in the treatment of acute myocardial infarction (AMI), due to conflicting results from animal studies and recent clinical trials. Several different hypotheses, proposing antiplatelet, and antithrombotic properties have been advanced to explain the cardioprotective properties of Mg during AMI. Although early clinical results were promising, there were serious flaws in the design and the logistics of small trials including out of date methodology, absence of control groups, and possible observer bias due to unblinded data collection. Moreover, the latest large-scale megatrial Fourth International Study of Infarct Survival (ISIS-4) provided conflicting data and caused major controversy, which will be difficult to resolve. Proponents of Mg therapy have suggested that the potential benefit was not seen in ISIS-4 because Mg was administered too late. Further clinical trials, well-designed, and carefully conducted, should elucidate possible benefits of parenteral Mg during myocardial injury, especially in conjunction with new and aggressive reperfusion techniques. The benefits of parenteral Mg in an expanding array of clinical conditions, including AMI, may be directly related to an improved hemostatic profile. This review summarizes the latest, and often confusing data on the effects of Mg on certain hemostatic characteristics which may be directly relevant to the existing controversy.

Depressed platelet status in an elderly patient with hemorrhagic stroke after thrombolysis for acute myocardial infarction. GUSTO-III Investigators.

BACKGROUND: Impaired platelet function has been reported in acute myocardial infarction (AMI) and stroke. However, prospective data on the changes of platelet status in patients before the occurrence of hemorrhagic stroke after thrombolytic therapy are unavailable. CASE DESCRIPTION: An 86-year-old male patient was among the 23 AMI patients enrolled in the platelet study for the GUSTO-III trial. He received 325 mg of aspirin daily for at least 6 years, suffered an AMI, and was successfully reperfused with alteplase, but after 44 hours developed a large hemorrhagic stroke resulting in paraplegia. Platelet aggregation and receptor expression were measured by flow cytometry and ELISA before thrombolysis and at 3, 6, 12, and 24 hours thereafter. The percentage of platelet aggregation was lower in the stroke patient at every time point when induced by 5 micromol/L of ADP, by 10 micromol/L of ADP, and by thrombin than in the rest of the AMI group. Ristocetin and collagen-induced aggregability were within the group range. Decreased platelet glycoprotein Ib, IIb, IIIa, and IIb/IIIa and vitronectin receptor expression were observed in the stroke patient. No other differences in p24 (CD9), very late antigen-2, P-selectin, and platelet/endothelial cell adhesion molecule-1 expression were determined. CONCLUSIONS: Profound depression of platelet status preceded the occurrence of hemorrhagic stroke in an elderly long-term aspirin user treated with thrombolytic therapy. Initial "exhausted" platelets may be responsible for the increased risk for hemorrhagic stroke after coronary thrombolysis.

Gene family use and somatic mutation in primary and secondary fluorescein-specific IgM antibody responses.

A comparative analysis of the DNA sequences of primary and secondary IgM, fluorescein-specific antibodies was performed. These antibodies were secreted by hybridomas generated following fusion of immunized BALB/c mouse lymphocytes and SP2/0 myeloma cells. Our results show that primary and secondary fluorescein-specific IgM antibodies use a variety of segments from the variable region of the immunoglobulin heavy chain locus (VH), with members of the J558 and 7183 VH gene families predominating in both populations. D regions from the DF116 and DSP2 families were used exclusively in our primary antibody sample and predominated in the secondary response. In the primary antibodies, 15 out of 18 definable D regions were transcribed in reading frame one, but in the secondary antibodies the three reading frames were used stochastically. Secondary IgM antibodies showed a higher frequency of somatic mutation than their primary counterparts, but we could detect no evidence of selection for mutations in the complementarity determining regions as compared with the framework regions. It appears that fusion of secondary cells, 3-6 days after immunization, is able to 'capture' the IgM-producing population of B cells at a stage in their development following mutation but prior to antigenic selection.

Antilipid A monoclonal antibody HA-1A decreases the capacity of bacterial lipopolysaccharide to activate human vascular endothelial cells by an immune adherence mechanism.

Human monoclonal IgM antibody HA-1A, which recognizes the lipid A component of bacterial lipopolysaccharide (LPS), has been shown to reduce mortality in Gram negative septicemia. The vascular endothelial lining of blood vessels, which controls leucocyte traffic and activation, as well as haemostatic balance, may be one of the primary targets of LPS action during sepsis. In earlier studies we have described HA-1A-induced immune adherence of LPS to complement receptors on erythrocytes, and showed that pre-incubation with HA-1A, in the presence of complement and red blood cells, markedly reduced LPS-induced cytokine production from peripheral blood mononuclear cells. In the present study, we measured the effect of immune adherence of LPS in the presence of HA-1A on the responses of cultured endothelial cells, and found that subsequent expression of adhesion molecules such as E-selectin, ICAM-1 and VCAM-1, and secretion of the cytokines interleukin-6 and granulocyte-macrophage colony stimulating factor were markedly reduced. Moreover, the ability of LPS to increase levels of tissue factor procoagulant activity on endothelial cells was markedly diminished by LPS immune adherence to HA-1A. This decrease in endothelial activation in response to LPS following immune adherence to HA-1A may play a significant role in the protective effect of HA-1A in vivo during the course of Gram negative sepsis.

Antilipid a monoclonal antibody HA-1A: immune complex clearance of endotoxin reduces TNF-alpha, IL-1 beta and IL-6 production.

HA-1A is a human monoclonal IgM antibody which recognizes the lipid A component of lipopolysaccharide (LPS). This antibody has reduced mortality in the septic shock syndrome resulting from Gram negative bacteria, in which many of the manifestations are considered to be due to cellular activation and secretion of cytokines, most notably TNF-alpha. However HA-1A does not directly neutralize LPS effectively in vitro, and studies reported to date have not defined its mechanism of action. Here we demonstrate that HA-1A, which in the presence of complement promotes immune adherence, may inhibit LPS action by facilitating its sequestration on red blood cells and clearance to an extent that cytokine production is reduced. Incubation of LPS at clinically significant (pg/ml) does with HA-1A at therapeutic levels (e.g. 10 micrograms/ml) and complement resulted in LPS association with erythrocyte CR1 receptors. This reduced the ability of the residual, free LPS by 50-70% to induce the secretion of TNF-alpha, IL-1 beta and IL-6 from normal blood mononuclear cells. This mechanism is likely to be operative in vivo, and could account for the protective effect of HA-1A, and its reduction of TNF-alpha production in vivo.

Effects of isotype and Fc region on in vitro function of a mouse/human chimeric CD4 antibody.

Murine CD4 mAbs have shown potential for the treatment of allograft rejection and autoimmune disorders including rheumatoid arthritis. Clinical usefulness of the murine mAbs has been limited by immunogenicity and a short circulating half-life. Mouse/human chimeric antibodies have been constructed, composed of the variable region of M-T412 (a murine G2a mAb specific for the human CD4 molecule) and human G1 (cM-T412 G1) or G4 (cM-T412 G4) Fc regions. F(ab')2 and F(ab) fragments of the murine G2a and chimeric G1 mAbs were generated by enzymatic digestion. The chimeric mAbs and all fragments retained the avidity and specificity of the murine M-T412 and were evaluated in in vitro assays measuring Ig production by pokeweed mitogen (PWM)-stimulated peripheral blood mononuclear cells (PBMC), sIL-2R produced by phytohemagglutinin-stimulated PBMC, and proliferation in response to tetanus toxoid, CD3 mAb plus IL-2, and mixed lymphocyte response (MLR). When PBMC were stimulated with tetanus toxoid, 10 ng/ml of cM-T412 G1 inhibited proliferation by 90%, while neither the cM-T412 G4, M-T412 G2a, nor any mAb fragment produced > 65% inhibition, even at 1000-fold higher concentrations. A similar pattern of inhibition was observed in MLR assays. In contrast, the F(ab')2 fragment of the cM-T412 G1 was as effective as the whole antibody in inhibiting PWM-stimulated IgM synthesis and PBMC proliferation in response to stimulation by a CD3 mAb plus IL-2.(ABSTRACT TRUNCATED AT 250 WORDS)

High-level expression and characterization of a mouse-human chimeric CD4 antibody with therapeutic potential.

The use of murine anti-CD4 monoclonal antibodies (MAbs) has shown considerable promise for the treatment of allograft rejection and rheumatoid arthritis. We have constructed mouse-human anti-CD4 antibodies with the goal of increasing their clinical potential by decreasing immunogenicity and improving effector functions. The chimeric antibodies were constructed by cloning the heavy and light chain variable regions of M-T412, a murine antibody raised against the human CD4 antigen, and joining them to the human G1, G4, or kappa constant regions in mammalian expression vectors. After transfection into mouse myeloma cells, stable cell lines were isolated that secrete up to 140 micrograms/ml chimeric antibody in static culture. The chimeric antibodies were equivalent to the murine antibody in their binding characteristics and relative affinities. However, the chimeric M-T412 MAbs have enhanced activity when compared to the murine G2a MAb in mediating antibody-dependent cell-mediated cytotoxicity using human CD4+ target and effector cells.

Temporal variations in the fine specificity of IgM anti-fluorescyl antibodies.

This study compares the fine specificities of the primary and secondary fluorescein (FITC)-specific immunoglobulin M (IgM) repertoires in BALB/c mouse serum and monoclonal antibodies (MoAb) and has found reproducible, immunization-dependent differences. FITC and four of its homologues; iodoacetamido fluorescein (IAF), dichlorotriazinyl aminofluorescein (DTAF), substituted rhodamine isothiocyanate (XRITC) and tetramethyl rhodamine isothiocyanate (TRITC), each conjugated to bovine serum albumin (BSA), were used to determine reactivity patterns of serum IgM from mice immunized once or twice with FITC-haemocyanin (FITC-Hy). Reactivity patterns were also obtained for 20 IgM MoAb, eight of which were produced by fusions of SP2/0 myeloma cells with splenocytes from mice immunized once (primary) and 12 from mice immunized twice (secondary) with FITC-Hy. Each MoAb exhibited a unique fine specificity pattern, evidence of extensive heterogeneity in the FITC-specific repertoire. Reactivities of IgM MoAb with certain homologues were found to be more characteristic of either the primary or secondary response. Polyclonal serum IgM also showed reproducible immunization-dependent variations in fine specificity. Such a pattern could result from idiotypic suppression of primary antibodies, from the expansion of subsets of IgM memory cells utilizing novel genes and/or from somatic mutation absent in primary IgM antibodies.

Fluorescein-specific hybridomas derived from primary mice exhibit more stringent growth requirements than do hybrids from pre-immune animals.

Although the generation of antigen-specific hybridoma cell lines from animals which have been multiply immunized is now a routine procedure, the derivation of hybridomas following a single in vivo antigen injection has proven to be much more difficult to accomplish. We show that the addition of an interleukin-containing supernatant derived from rat spleen cells which have been stimulated with concanavalin A (ConA SN) to hybrids after the initial cloning step results in the consistently successful isolation of IgM anti-fluorescein specific hybridomas. However, addition of the same supernatant to fused cultures simultaneously with the addition of the HAT selection medium results in the loss growing cells. In contrast to the situation with primary hybridomas, the growth of secondary hybridomas is inhibited by the addition of ConA SN at the cloning step. Following successful cloning, there is a time-dependent variation in the sensitivity of all cell lines to ConA SN.