Examination of several potential mechanisms for the negative outcome in a clinical stroke trial of enlimomab, a murine anti-human intercellular adhesion molecule-1 antibody: a bedside-to-bench study.

BACKGROUND AND PURPOSE: Enlimomab, a murine monoclonal anti-human intercellular adhesion molecule (ICAM)-1 antibody, had a negative outcome in a multicenter acute-stroke trial. We did a bedside-to-bench study in standardized rat stroke models to explore mechanisms for these untoward results. METHODS: After focal brain ischemia in Wistar rats and spontaneously hypertensive rats (SHR), we administered murine anti-rat ICAM-1 antibody (1A29), subclass-matched murine immunoglobulin (IgG1), or vehicle intravenously. To examine whether rat anti-mouse antibodies were generated against the mouse protein and whether these were deleterious, we sensitized Wistar rats with 1A29 or vehicle 7 days before surgery. Infarct volume, tissue myeloperoxidase activity, neutrophil CD11b expression, and microvascular E-selectin, P-selectin, and ICAM-1 expression were examined 48 hours after surgery. Complement activation was serially assessed for 2 hours after a single injection of either 1A29 or vehicle. RESULTS: 1A29 treatment did not significantly reduce infarct size in either strain. 1A29 sensitization augmented infarct size and generated rat anti-mouse antibodies. Although 1A29 inhibited neutrophil trafficking shown by reduction in brain myeloperoxidase activity, circulating neutrophils were activated and displayed CD11b upregulation. Complement was activated in 1A29-sensitized Wistar rats and 1A29-treated SHR. E-selectin (SHR), endothelial P-selectin (Wistar and SHR), and ICAM-1 (SHR) were upregulated in animals treated with 1A29. CONCLUSIONS: Administration to rats of a murine antibody preparation against ICAM-1, 1A29, elicits the production of host antibodies against the protein, activation of circulating neutrophils, complement activation, and sustained microvascular activation. These observations provide several possible mechanisms for central nervous system-related clinical deterioration that occurred when Enlimomab was given in acute ischemic stroke.

Increased CD8(+) T cells associated with Chlamydia pneumoniae in symptomatic carotid plaque.

BACKGROUND AND PURPOSE: The presence of Chlamydia pneumoniae has been reported in carotid atheroma, but its causative effect in the activation of an atherosclerotic plaque to a prothrombotic state remains unproved. Antigen- mediated activation of T lymphocytes within plaque may represent a mechanism by which infection can result in plaque conversion. The goal of the present study was to characterize the T-cell subtype profile related to the presence of C pneumoniae in patients with symptomatic versus asymptomatic carotid atherosclerosis. METHODS: We studied 14 plaques (5 symptomatic and 9 asymptomatic) positive for C pneumoniae confirmed by polymerase chain reaction and 14 plaques (6 symptomatic and 8 asymptomatic) from age- and stenosis-matched patients negative for C pneumoniae by polymerase chain reaction. T-cell subpopulations of T-helper, T-cytotoxic, and T-memory lymphocytes were identified through indirect enzyme immunohistochemistry with anti-CD3+, anti-CD4+, anti-CD8+, and anti-CD45RO+ monoclonal antibodies, respectively. Results are expressed as the number of positive cells per millimeter squared. RESULTS: In the absence of C pneumoniae, symptomatic plaques had a modest but significant increase of CD3+ (89.6 versus 55.3, P=0.013), CD4+ (57.3 versus 32.7, P=0.01), and CD45RO+ (82.8 versus 43.7, P=0.007), but not CD8+ T cells (28.5 versus 25.5, P=0.245) compared with asymptomatic. However, in the presence of C pneumoniae, there was significant increase of all T-lymphocyte subtypes in symptomatic plaques, including CD8+ (76.8 versus 30.3, P=0.03), CD3+ (192.1 versus 80.4, P=0.004), CD4+ (111.9 versus 37.9, P=0.003), and CD45RO+ (120.2 versus 72.9, P=0.003) cells compared with asymptomatic plaques. With use of 2-way ANOVA, both the presence of chlamydia and symptoms were associated with significantly higher T-cell counts (P<0.005 for all subtypes). CONCLUSIONS: Although all patients with symptomatic disease show a modest elevation in the concentration of intraplaque lymphocytes, a preferential increase in CD8+ class I-restricted T cells is observed in symptomatic carotid plaque positive for C pneumoniae. These data provide incentive to further explore the role of Chlamydia in the modification of immune-mediated mechanisms in active atherosclerotic plaque.

Presence of Chlamydia pneumoniae in human symptomatic and asymptomatic carotid atherosclerotic plaque.

BACKGROUND: Chlamydia pneumoniae has been identified in atherosclerotic plaques of patients with cerebrovascular and cardiovascular disease. However, the direct causative effect of C pneumoniae infection in the activation of atherosclerotic plaque to a prothrombotic state remains to be established. The aim of the present study is to examine the correlation between intraplaque presence of chlamydiae and symptomatic carotid disease in humans. METHODS: Plaques from 37 symptomatic and 57 asymptomatic consenting patients undergoing carotid endarterectomy were snap-frozen, and the tissue was prepared for polymerase chain reaction analysis for Chlamydia pneumoniae per Institutional Review Board-approved protocol. Blood was drawn from each patient at the time of surgery for serological analysis. RESULTS: The overall rate of plaques positive for C pneumoniae was 14.82%, with 5 of 37 (13.5%) plaques from symptomatic patients and 9 of 57 (15.8%) from asymptomatic patients, which revealed a definitive presence of the organism. No association existed between C pneumoniae presence and symptomatic disease (P:=1.0). Also, no association existed between presence of C pneumoniae and severity of stenosis. Finally, seropositivity for anti-chlamydial IgG, IgA, and IgM anti-chlamydial antibodies did not correlate with identification of C pneumoniae in the plaques. However, high-serum anti-chlamydial IgA levels (>/=1:128) were associated with occurrence of symptomatic disease (P=0.03; odds ratio, 2.86; 95% CI, 1.12 to 7.28). CONCLUSIONS: Presence of C pneumoniae as a single factor does not appear to be sufficient to explain the occurrence of cerebrovascular symptoms. Low sensitivity of seropositivity for IgG, IgA, or IgM associated with PCR-identified C pneumoniae presence in the plaque makes it unlikely to be valuable as the single determining factor for actively infected plaque. Association of high-level anti-chlamydial IgA with symptomatic disease suggests that chronic or acute chlamydial infection anywhere in the body could play a role in atherosclerotic plaque activation and be used as a marker to target populations in future stroke prevention trials.

Why do neuroprotective drugs work in animals but not humans?

Many neuroprotective agents that seemed promising in animal studies of ischemic brain injury prove to have no effect when tested in clinical trials, suggesting that fundamental elements of translational research require better definition. A number of modifications have led to improvements in preclinical and human studies since the earliest controlled trials failed to confirm hypotheses suggested by animal data. Continued re-evaluation and sharing of information derived from the laboratory bench or the patient's bedside should eventually lead to effective neuroprotection in acute stroke. Experimental data should be carefully studied to improve the quality of agents coming to clinical trials and to design trial phasing that effectively determines drug safety and efficacy. This article will examine preclinical modeling and its translation to prospective studies of acute stroke therapy and will focus on some potential solutions directed at clinical trial design.

Profile of endothelial and leukocyte activation in Fabry patients.

Fabry disease is an X-linked recessive disorder resulting in the deposition of globotriaosylceramide in numerous cell types including vascular endothelial cells. Because this disease is associated with vascular injury and a high recurrence rate of thrombotic events, measurements of factors regulating endothelium and leukocyte interaction may provide insight into the mechanisms leading to a prothrombotic state. Twenty-five patients with Fabry disease and 25 control subjects participated in the study. Plasma from all 25 Fabry patients and 15 of the 25 controls were studied for multiple endothelial factors. Leukocyte integrins were measured by flow cytometry in 21 Fabry patients and 10 controls. The concentrations of soluble intercellular adhesion molecule-1, vascular cell adhesion molecule-1, P-selectin, and plasminogen activator inhibitor were significantly higher and thrombomodulin was significantly lower in Fabry patients. Expression of the integrin CD11b on monocytes was also significantly higher in the Fabry patients. This study reveals measurable evidence for endothelium and leukocyte activation that is consistent with a prothrombotic state in Fabry patients compared with controls. Further investigations of these findings may help to understand the mechanism of stroke in Fabry disease and provide indicators (or markers) of efficacy of future therapeutic intervention.

Immunological aspects of acute stroke: therapeutic implications.

Recent studies strongly suggest that cerebral ischaemia initiates a focal inflammatory response that results in significant secondary injury to brain tissue,thereby extending the ultimate size of a stroke. Factors involved in this cascade include the release of cytokines that cause a pro-inflammatory and prothrombotic state on cerebral vessel endothelium, the expression of leucocyte adhesion molecules, and the release of chemotactic factors allowing the migration of leucocytes into the area of injured brain tissue causing further damage. Animal studies have clearly demonstrated the detrimental effects of these inflammatory mediators in stroke models and additionally have shown dramatic reduction in infarct size using leucocyte adhesion modification and cytokine receptor blockade. The approach of modifying the effects of inflammatory cytokines and/or limiting leucocyte adhesion and migration into the region of injury holds great promise for identifying agents that will give significant neuronal protection following a stroke.

Progression in acute stroke: value of the initial NIH stroke scale score on patient stratification in future trials.

BACKGROUND AND PURPOSE: The objective was to determine the occurrence of neurological changes during the first 48 hours after acute stroke as it relates to initial stroke severity. METHODS: The National Institutes of Health Stroke Scale (NIHSS) was performed serially for the first 48 hours on 127 consecutive ischemic stroke patients (129 strokes) admitted to the neuroscience intensive care unit. Incidence of stroke progression (a >/=3-point increase on the NIHSS) was recorded and analysis performed to determine its association with initial stroke severity and other demographic and physiological variables. Deficit resolution by 48 hours, defined as an NIHSS score of 0 or 1, measured the frequency of functional recovery predicted by the initial deficit. RESULTS: Overall progression was noted in 31% of events (40/129). Applying Bayes' solution to the observed frequency of worsening, the greatest likelihood of predicting future patient progression occurs with stratification at NIHSS scores of 7. Patients with an initial NIHSS of 7 with a 65.9% (27/41) worsening rate (P<0.000005). Forty-five percent (40/88) of those with an initial score of 7 returned to a normal examination within this period (chi2, P<0.000005). CONCLUSIONS: This study suggests that the early clinical course of the neurological deficit after acute stroke is dependent on the initial stroke severity and that a dichotomy in early outcome exists surrounding an initial NIHSS score of 7. These findings may have significant implications for the design and patient stratification in treatment protocols with respect to primary clinical outcome.

The role of inflammation after acute stroke: utility of pursuing anti-adhesion molecule therapy.

A growing body of evidence, primarily from animal models of cerebral ischemia and preliminary human studies, indicates that inflammatory mechanisms contribute to secondary neuronal injury after acute cerebral ischemia. Ischemia followed by reperfusion rapidly leads to the expression of inflammatory cytokines, particularly tumor necrosis factor-alpha and interleukin-1beta, which stimulate a complex cascade of events involving local endothelial cells, neurons, astrocytes, and perivascular cells. A secondary response includes the release of other cytokines, an increase in components of the coagulation system, an upregulation of cell adhesion molecule expression, and changes in the expression of components of the immune response. The net effect of these events is transformation of the local endothelium to a prothrombotic/proinflammatory state and induction of leukocyte migration to the site of injury. A number of studies have shown that leukocyte migration occurs within hours of reperfusion. Leukocytes accumulate in the injured region, where they cause tissue injury by several mechanisms, including occlusion of microvasculature, generation of oxygen free radicals, release of cytotoxic enzymes, alteration of vasomotor reactivity, and increase in cytokine and chemoattractant release. Monoclonal antibodies against leukocyte adhesion molecules have been shown to reduce infarct volume in animal models of ischemia-reperfusion. However, this treatment failed to show benefit in the Enlimomab Acute Stroke Trial. A number of factors may complicate the use of antibody directed adhesion molecule blockade in acute stroke and will be discussed in this article. Overall, an increased understanding of inflammatory and immunologic mechanisms still offers great potential for reducing acute stroke injury.

Increased endothelial expression of intercellular adhesion molecule-1 in symptomatic versus asymptomatic human carotid atherosclerotic plaque.

BACKGROUND AND PURPOSE: The mechanisms that cause carotid atherosclerotic plaque to become symptomatic remain unclear. Evidence suggests that mediators of inflammation are not only instrumental in the formation of plaque but may also be involved in the rapid progression of atheromatous lesions leading to plaque fissuring, endothelial injury, and intraluminal thrombosis. Our goal is to determine whether intercellular adhesion molecule-1 (ICAM-1), a known component of the inflammatory pathway, is preferentially expressed on symptomatic versus asymptomatic carotid plaques. METHODS: Carotid plaques from symptomatic (n = 25) and asymptomatic (n = 17) patients undergoing carotid endarterectomy with lesions involving >60% stenosis were snap-frozen at the time of surgery. Immunofluorescence studies were performed to measure the percentage of luminal endothelial surface that expressed ICAM-1. The relationships of stroke risk factors, white blood cell count, percent stenosis, and soluble ICAM-1 (sICAM-1) plasma levels to endothelial ICAM-1 expression were investigated. RESULTS: An increased expression of ICAM-1 was found in the high-grade regions of symptomatic (29.5%+/-2.4%, mean+/-SEM) versus asymptomatic (15.7%+/-2.7%, mean+/-SEM) plaques (P=0.002) and in the high-grade versus the low-grade region of symptomatic plaques (29.5+/-2.4, mean+/-SEM, versus 8.9+/-1.6; P<0.001). Plasma sICAM-1 levels were not predictive of symptomatic disease, and no significant correlation between risk factor exposure and endothelial ICAM-1 expression was found. CONCLUSIONS: An elevation in ICAM-1 expression in symptomatic versus asymptomatic plaque suggests that mediators of inflammation are involved in the conversion of carotid plaque to a symptomatic state. The data also suggest a differential expression of ICAM-1, with a greater expression found in the high-grade region than in the low-grade region of the plaque specimen.

Expression of inflammatory mediators and adhesion molecules in human atherosclerotic plaque.

The mechanisms that cause carotid atherosclerotic plaque to become symptomatic remain unclear. Evidence suggests that mediators of inflammation not only are instrumental in the formation of plaque but also may be involved in the rapid progression of atheromatous lesions, leading to plaque fissuring and intraluminal thrombosis. This article reviews the current evidence for the role of inflammatory mediators in atherosclerotic plaque production and maturation. It also includes studies performed in our laboratory to determine if known components of the inflammatory pathway, including cytokines and leukocyte adhesion molecules, are preferentially expressed on symptomatic versus asymptomatic carotid plaques. Carotid plaques from symptomatic and asymptomatic patients undergoing carotid endarterectomy with lesions of greater than 70% stenosis were snap-frozen and stored at -70 degrees C until analysis. Immunofluorescent studies were performed to measure endothelial expression of intercellular adhesion molecule-1 (ICAM-1). ICAM-1 expression was measured in a blinded fashion as percent of luminal endothelial surface of plaque sections. In situ hybridization also was performed to measure expression of message for tumor necrosis factor alpha (TNF-alpha) and ICAM-1 in the plaque by comparing mean optical density between the symptomatic and asymptomatic patients. There was increased expression of ICAM-1 on the endothelial surface in high-grade regions (28%) versus low-grade regions (11%), of plaques from symptomatic patients. There was also a trend toward greater expression of ICAM-1 in the high-grade region of symptomatic plaques versus the high-grade region of asymptomatic plaques. In situ hybridization revealed increased mRNA for TNF-alpha and ICAM-1 in the body of the plaque, preferentially in the high-grade region of plaques from symptomatic patients. The data obtained suggest that a local increase of endothelial inflammatory mediator expression correlates with the clinical setting of thromboembolic ischemia and may play a role in conversion of atheromatous plaque to a prothrombotic state. The data also indicate that this line of investigation deserves further exploration because it may be useful in identifying new mechanisms in patients at risk for stroke and in suggesting possible novel strategies for intervention.

Prospective study of neurological responses to treatment with macrophage-targeted glucocerebrosidase in patients with type 3 Gaucher's disease.

We prospectively evaluated the clinical and biochemical responses to enzyme-replacement therapy (ERT) with macrophage-targeted glucocerebrosidase (Ceredase) infusions in 5 patients (age, 3.5-8.5 years) with type 3 Gaucher's disease. The patients were followed for up to 5 years. Enzyme dosage ranged from 120 to 480 U/kg of body weight/month. Systemic manifestations of the disease regressed in all patients. Neurological deficits remained stable in 3 patients and slightly improved in 1. One patient developed myoclonic encephalopathy. Cognitive deterioration occurred in 1 patient and electroencephalographic deterioration in 2. Sequential cerebrospinal fluid (CSF) samples were obtained during the first 3 years of treatment in 3 patients and were analyzed for biochemical markers of disease burden. Glucocerebroside and psychosine levels were not elevated in these specimens, whereas chitotriosidase and quinolinic acid were elevated in 2 patients. Progressive decrease in the CSF levels of these latter macrophage markers during 3 years of treatment implies a decreased number of Gaucher cells in the cerebral perivascular space. Similar changes were not observed in the patient who had a poor neurological outcome. In conclusion, ERT reverses systemic manifestations of type 3 Gaucher's disease and appears to reduce the burden of Gaucher cells in the brain-CSF compartment in some patients.

Genetics of ischemic stroke.

Advances in the study of cerebrovascular disease suggest that many risk factors for stroke are under genetic influence. Epidemiologic studies show that parental and sibling histories of cerebral ischemic events are associated with an increased risk of stroke. Explanations for familial stroke aggregation include differential phenotypic expression of apolipoprotein (a) and apolipoprotein E, racial variations in the distribution of vascular disease, identification of the autosomal-dominant disorder cerebral autosomal-dominant arteriopathy with subcortical infarcts and leukoencephalopathy, specific point mutations in the mitochondrial-related disorders, and identification of the clinical significance of hereditable coagulopathies. Greater understanding of these factors may lead to early recognition of and intervention in stroke.

Motor performance, histologic damage, and calcium influx in rats treated with NBQX after focal ischemia.

2,3-Dihydroxy-6-nitro-7-sulfamoylbenzo(F)-quinoxaline (NBQX), an alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor antagonist, has been reported to provide neuronal protection after global ischemia. The objectives of this study were to evaluate the neuroprotective effects of NBQX initiated after focal cortical ischemia and to validate a method for measuring functional outcome in this model. Male spontaneously hypertensive rats (SHRs) were exposed to various durations of transient or permanent tandem middle cerebral artery (MCA) occlusion. Studies compared motor performance using balance beam and prehensile-traction tests, calcium-calmodulin (Ca-CaM) binding by immunohistochemistry, and infarct volume between NBQX-treated animals [intravenous (i.v.) 5 mg/kg/h x 6 h or intraperitoneal (i.p.) 30 mg/kg q 30 min x 3 begun postischemia] and controls. All ischemic groups performed less well than sham-operated controls on the motor performance tasks in proportion to the severity of ischemia. No significant improvement in motor performance was noted in the NBQX-treated versus the control animals after 1 h or permanent MCA/CCA occlusion. Treatment with NBQX (i.v. or i.p. dosing) did not reduce Ca-CaM binding after 1 h of occlusion with 1 h of reperfusion or after 2 h of occlusion. Similarly, there was no reduction in infarct size between NBQX-treated and control animals after 24 h of permanent MCA/CCA occlusion (74.6 +/- 7.1 vs. 80.1 +/- 6.0 ml; ns) or after 1 h of occlusion with 23 h of reperfusion (55.1 +/- 4.4 vs. 47.4 +/- 6.2 ml; ns).(ABSTRACT TRUNCATED AT 250 WORDS)

Threshold of calcium disturbances after focal cerebral ischemia in rats. Implications of the window of therapeutic opportunity.

BACKGROUND AND PURPOSE: One explanation for inconclusive results with calcium channel blockers in human acute stroke trials may be incomplete information about the time course of calcium-mediated ischemic neuronal injury. This study explores the temporal relation between duration of focal ischemia and the functional activity of increased intracellular calcium as measured by calcium-calmodulin binding. METHODS: Calcium-calmodulin binding, determined by immunohistochemical assay of free calmodulin, was measured in 60 male spontaneously hypertensive rats after 2 minutes and after 1, 2, 4, and 24 hours of permanent tandem common carotid and middle cerebral artery occlusion, and after 1 and 2 hours of reversible middle cerebral artery occlusion followed by 1 and 22 hours of reperfusion, respectively. Light microscopic histological damage was measured after 1 hour of occlusion with 23 hours of reperfusion and after 24 hours of occlusion. RESULTS: Significant loss of calmodulin staining in the core of the infarction was noted by 1 hour and became maximal after 4 hours of ischemia. No reversal of calmodulin staining loss was noted after reperfusion following 1 and 2 hours of ischemia. Cortical necrosis seen by light microscopy correlated well with the area of maximal calcium-calmodulin binding. The border zone area, represented by a mild loss of calmodulin staining surrounding the central core of maximal binding, gradually decreased in size and became incorporated into the central core after 4 hours of ischemia; it may represent an area of reversible ischemia. CONCLUSIONS: Calcium-calmodulin binding correlates with duration of focal ischemia, and histological neuronal necrosis corresponds to the cortical areas displaying a significant loss of calmodulin staining. Inasmuch as loss of calmodulin staining represents a marker for calcium-mediated activity after ischemia, it suggests a window of opportunity within 4 hours after acute stroke for therapeutic intervention with calcium antagonists.

Temporal relation of calcium-calmodulin binding and neuronal damage after global ischemia in rats.

BACKGROUND AND PURPOSE: This study explores the temporal relation of the severity of ischemia and calcium-calmodulin binding in vulnerable and resistant brain regions in a commonly used model of global ischemia. METHODS: Immunohistochemical assay of free calmodulin unbound to calcium and light microscopic histological damage were measured in rats after 5, 10, or 20 minutes of global ischemia. RESULTS: After 24 hours of reperfusion, decreased calmodulin staining, representing increased calcium influx and calcium-calmodulin binding, correlated with increasing durations of ischemia across all brain regions. Based on a 4-point scale (4, extensive stain; 0, no staining), calmodulin staining after 5 minutes versus 10 minutes of ischemia was 3.2 versus 1.9, respectively (p less than 0.05) and after 10 minutes versus 20 minutes of ischemia was 1.9 versus 1.0, respectively (p less than 0.01). The CA1 region displayed the greatest sensitivity to ischemia. Similar but less dramatic results were seen after 2 hours of reperfusion. After 72 hours of reperfusion, histological damage closely correlated with calcium-calmodulin binding after variable durations of ischemia. A threshold of 10 minutes of ischemia was required to cause calcium-calmodulin binding and irreversible neuronal damage. Surviving neuronal populations showed recovery of calmodulin staining 7 days after ischemia, representing a return of free calmodulin and normal calcium homeostasis. CONCLUSIONS: These correlations between calcium-calmodulin binding, histological damage, and duration of ischemia support the causal role of calcium influx in global ischemic injury and suggest the need for very rapid intervention after ischemia if calcium-mediated damage is to be prevented.

Molecular biology of atherothrombotic brain infarction.

Because reduced high density lipoproteins may contribute to atherothrombotic brain infarction, we performed molecular biologic and metabolic studies to characterize high density lipoprotein metabolism with respect to its role in reverse cholesterol transport, to clone the high density lipoprotein receptor, and to determine gene polymorphism for apolipoprotein A-I, the major protein of high density lipoprotein, because altered structure may impair reverse cholesterol transport. For high density lipoprotein metabolism measurements, high density lipoprotein 3 was isolated, purified, and labeled with iodine-125. The radiolabeled high density lipoprotein 3 was reinjected, and daily blood samples were taken for 10 days. Synthesis rates and fractional catabolic rates were determined from the specific activities and daily decrements. Preliminary data indicate that stroke-prone individuals' fractional catabolic rates for high density lipoprotein 3 are twice those of normal individuals. Also, the conversion of high density lipoprotein 3 to high density lipoprotein 2 is reduced in these individuals, suggesting that high density lipoprotein may be abnormally processed in individuals prone to atherothrombic brain infarctions. We surveyed more than 100 patients with carotid stenosis using a 2.2-kb probe for the apolipoprotein A-I gene. A subset of these patients displays polymorphism with restriction enzymes SacI or PstI. These preliminary findings suggest that gene polymorphism for apolipoprotein A-I may provide a molecular clue of atherothrombic brain infarction.