Monoclonal antibody-defined correlations in melanoma between levels of GD2 and GD3 antigens and antibody-mediated cytotoxicity.

A monoclonal antibody is described that specifically detects the ganglioside antigens GD2 and GD3, binding preferentially to GD2, in melanoma. Antibody specificity was demonstrated with solid-phase radioimmunoassay and enzyme-linked immunosorbent assay as well as by immunostaining on thin-layer chromatography plates using structurally characterized gangliosides. Binding of both the IgG3 antibody and its IgG2a switch variant were assayed on live cells by cytofluorography and by immunoperoxidase staining on frozen tissue sections. The binding patterns correlated with antitumor activity in antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity assays with human effector cells and complement in an 111In-release assay using cell lines derived from the same individual. The significant level of killing in all tumor cells tested that express GD2, GD3, or both, suggests the importance of multiple specificity towards tumor antigens, i.e., binding of a monoclonal antibody to two or more tumor-associated antigens.

Efficacy of antioxidant therapies in transient focal ischemia in mice.

BACKGROUND AND PURPOSE: Ginkgo biloba extract (EGb) and alpha-lipoic acid (LA) are commercially available "antioxidant supplements" with a variety of actions that may be beneficial during acute stroke. These actions include inhibiting platelet and leukocyte activation and adhesion, reducing free radical generation, and increasing cerebral blood flow. Both EGb and LA have been shown to be neuroprotective in cell culture and global central nervous system ischemia models. In this study we investigated the neuroprotective efficacy of EGb and LA in a clinically relevant, transient focal central nervous system ischemic model. METHODS: In the EGb study, 60 adult C57blk mice were randomized to treatment with EGb given orally (via gavage) for 7 days: low dose, 50 EGb mg/kg daily; high dose, 100 mg/kg daily; matched placebo. On day 7, reversible middle cerebral artery occlusion was produced by advancing a silicone-coated 8-0 filament into the internal carotid artery for 45 minutes followed by reperfusion. At 24 hours, the animals were evaluated on a 28-point clinical scale, and infarct volume was determined with the use of triphenyltetrazolium chloride. In the LA study, 24 C57blk mice were treated with 100 mg/kg SC of LA or placebo 1.5 hours before transient MCAO, as in the EGb study. RESULTS: In the EGb study, values for infarct volume at 24 hours were as follows (mean+/-SD): low dose (n=18), 13+/-5 mm(3); high dose (n=22), 22+/-12 mm(3); placebo (n=20), 20+/-10 mm(3) (P:=0.03 overall; P=0.02, low dose versus placebo). Infarct percentage of hemisphere values were as follows: low dose, 14+/-5%; high dose, 21+/-11%; placebo, 20+/-9% (P=0.03 overall; P=0.02, low dose versus placebo). Ten percent of the high-dose group showed significant intracerebral hemorrhage (ICH) within the infarct, while no ICH was seen in the other groups. Neurological function scores were as follows: low dose, 11.8+/-1.5; high dose, 11.4+/-1.7; placebo, 11.3+/-1.8 (P=NS). In the LA study, infarct volume was as follows: 100 mg/kg LA (n=12), 16.8+/-8.3 mm(3); placebo (n=12), 27.2+/-14.6 mm(3) (P<0.05). LA also produced a significant improvement in neurological function at 24 hours: LA, 9.5+/-1.2; placebo, 11.2+/-1.8 (P=0.02). There was no evidence of ICH in any of the animals. CONCLUSIONS: Both oral EGb and LA therapies produced significant reductions in stroke infarct volume. However, for EGb this beneficial effect appears to be dose related, with higher doses potentially increasing the risk of ICH.

Fiber intakes, stool frequency, and stool weights of subjects consuming self-selected diets.

Neutral detergent fiber (NDF) contents of duplicate collections of food consumed by 29 men and women were determined for 7 days four times during the year. The ratio of analyzed NDF to calculated crude fiber intakes was approximately 2. Although mean NDF intake was 7.7 g/day for females and 9.5 g/day for males, the majority of the subjects had NDF intakes of from 4 to 9 g/day. Fiber-containing foods consumed with the greatest frequency were vegetables. NDF intakes were greater in the winter than in the spring and summer. Caloric and total carbohydrate intakes, number of stools, and fecal weights were greater for the males than for the females. Fecal weights were significantly correlated with caloric intake, NDF intake, and number of stools. It is suggested that the insoluble fiber measured by the NDF method is likely to be better correlated with fecal weight than the total dietary fiber which includes soluble fibers.

Impact of variation in carbohydrate intake on mineral utilization by vegetarians.

Dietary intakes of two groups of vegetarians (of Asian Indian and of American origin) residing in the United States were compared with intakes of a group of nonvegetarians. Mineral intakes and balances were determined for all three groups for a 7-d period. The vegetarians had a significantly higher percent of energy intake from carbohydrate and significantly higher intakes of crude fiber (CF) and neutral detergent fiber (NDF) than did the nonvegetarians. The American vegetarians had significantly higher intakes of magnesium, iron, and copper than did the other groups. Manganese intakes were significantly higher for the two vegetarian groups than for the nonvegetarians. Calcium, iron, zinc, and copper balances were not significantly different among the groups. Magnesium and manganese balances were significantly more negative for the American vegetarians than for the other two groups. In general, the higher percent of carbohydrate intake or the higher level of fiber intake did not appear to affect mineral utilization by the vegetarians.

Microalbuminuria in ischemic stroke.

OBJECTIVES: To determine (1) the incidence of microalbuminuria in patients with recent ischemic stroke, (2) its relationship to risk factors for stroke, (3) its prevalence in the major subtypes of ischemic stroke, and (4) its potential for identifying patients at increased risk for recurrent stroke, myocardial infarction, or vascular death. DESIGN: Prospective case-control study. SETTING: Outpatient clinics at the medical centers affiliated with the Department of Veterans Affairs and Oregon Health Sciences University in Portland, Ore. PATIENTS: A total of 186 older men and women (median age, 65 years) who were enrolled in a prospective study of risk factors for recurrent stroke, including 97 patients with recent (6-8 weeks) ischemic stroke, 51 with similar clinical risk factors for stroke, including 24 with a history of remote stroke or transient ischemic attack, and 38 community-dwelling volunteers. RESULTS: Microalbuminuria was 3 times more prevalent in patients with recent stroke (29%) than in those with clinical risk factors for stroke (10%), and was undetectable in healthy elderly controls (P<.001). The presence of microalbuminuria in recent stroke as well as in the combined recent and remote stroke or transient ischemic attack group (n = 121) was predicted by diabetes (odds ratio [OR], 8.4; 95% confidence interval [CI], 2.6-27.0; P<.001; serum albumin levels (OR, 0.12; 95% CI, 0.03-0.50; P<.005); age (OR, 1.1; 95% CI, 1.0-1.2; P<.01), and ischemic heart disease (OR, 3.0; 95% CI, 1.0-9.1; P<.05). Among patients with recent stroke the prevalence of microalbuminuria did not differ among major ischemic stroke subtypes, ie, atheroembolic, 23%; cardioembolic, 30%; and lacunar, 33%. During a mean +/- SD of 1.5 +/- 0.9 years of follow-up, 20% of patients with recent stroke, 14% with risk factors for stroke, and 0% of healthy elderly volunteers had vascular end points (P<.004), with events being as frequent in patients with microalbuminuria (32%) as in patients with macroalbuminuria (33%). After controlling for major clinical risk factors, microalbuminuria remained an independently significant predictor of future stroke in the combined recent stroke and remote stroke or transient ischemic attack group (Cox proportional hazard ratio, 4.9; 95% CI, 1.4-17.6; P<.01). CONCLUSIONS: Microalbuminuria is a common finding in patients with cerebrovascular disease and is associated with increased risk for stroke even after correction for the presence of confounding clinical risk factors. These data suggest that microalbuminuria merits further examination as a potentially inexpensive and easily measured marker of increased risk for stroke.

Association of intracranial stenosis with cortical symptoms or signs.

The association of the presence of cortical symptoms or signs and anterior circulation intracranial stenosis in patients with anterior circulation ischemia and no known extracranial carotid or cardiac etiology was studied. Fifteen percent (5/33) of patients with cortical symptoms or signs had symptomatic intracranial stenosis of 50% or more by MR angiography or angiography, compared with 0% (0/15) of those without such findings (p < 0.005).

A phase III randomized efficacy trial of 2000 mg citicoline in acute ischemic stroke patients.

BACKGROUND: Citicoline may reduce CNS ischemic injury by stabilizing cell membranes and reducing free radical generation. Previous safety and efficacy trials in patients who have had acute strokes suggested that citicoline may improve neurologic outcome with minimal side effects. OBJECTIVE: To determine the safety and efficacy of citicoline treatment in acute stroke patients. METHOD: An 118-center, randomized, double-blind, efficacy trial in 899 patients compared placebo (n = 446) with citicoline (n = 453) (1000 mg PO twice a day) for 6 weeks, with a 6-week post-treatment follow-up period. Patients with acute (< or =24 hours) ischemic strokes clinically thought to be in the middle cerebral artery territory with NIH Stroke Scale (NIHSS) scores > or =8 were enrolled. RESULTS: Mean time to treatment was 13 hours for both groups and mean age was 67 years for those receiving placebo and 68 years for those receiving citicoline. Mean baseline NIHSS scores were 14.5 for placebo and 13.9 for citicoline (p = 0.06); medians were 14 for placebo and 13 for citicoline (p = 0.04). The incidence and type of side effects were similar between the groups. There were no between-group differences on the planned primary analysis, percent of patients with a > or =7-point NIHSS score change at 90 days (placebo 51%, citicoline 52%). There were no between-group differences on the other planned secondary analyses at 90 days, including mortality. However, post hoc analyses using standard "excellent recovery" measures suggested a possible treatment effect on the modified Rankin 0 or 1 (last observation carried forward: placebo 20%, citicoline 26%; p = 0.025) as well as a global outcome statistic. CONCLUSIONS: Citicoline was safe but ineffective in improving the outcome of patients with acute ischemic stroke as measured by the planned analyses. Post hoc analyses suggest that a modest treatment effect may have been seen if more traditional analyses had been used.

Persistent inflammatory response in stroke survivors.

OBJECTIVE: Goals were to determine how long acute-phase markers remain elevated after ischemic stroke and how marker levels relate to stroke risk factors, stroke mechanism, and subsequent vascular events. METHODS: Fibrinogen (FIB), C-reactive protein (CRP), leukocytes (WBC), neutrophils (PMN), interleukin-6, and interleukin-1 receptor antagonist were measured at stroke onset and at 6 weeks, 6 months, and 1 year after enrollment, or until a vascular event occurred in 136 acute ischemic stroke patients, 76 patients with comparable risk factors for stroke, and 48 age-balanced healthy subjects. RESULTS: Multivariate logistic analysis showed that prior stroke and FIB level predicted new events in stroke patients (p < 0.04 for both), whereas congestive heart failure (p < 0.02) and creatinine level (p < 0.006) were predictive in at-risk patients. After controlling for infection, FIB, CRP, and PMN levels at baseline were higher in at-risk but not in stroke patients with recurrent events (p < 0.05 for all). At 1 year, FIB levels remained elevated in event-free stroke survivors compared with levels in the risk and control groups (p < 0.001 for both). FIB also remained higher in stroke survivors who had atheroembolism (AE) compared with non-AE stroke survivors (381+/-72 versus 342+/-78 mg/dL, p < 0.02). Peripheral vascular disease was an independent predictor (p < 0.0001) of longitudinal FIB in stroke survivors. Of note, both WBC and PMN levels were chronically elevated in patients with stroke risk factors and in stroke survivors (p < 0.0001 for both) compared with healthy elderly subjects. CONCLUSIONS: Most acute-phase markers decline gradually after stroke, but FIB remains significantly elevated and is associated with increased risk for recurrent vascular events.

A randomized dose-response trial of citicoline in acute ischemic stroke patients. Citicoline Stroke Study Group.

Citicoline (CDP-choline) is a key intermediary in the biosynthesis of phosphatidylcholine, an important component of the neural cell membrane. It has been shown to produce beneficial effects in both animal models and non-US clinical stroke trials. This study comprised a randomized (3 doses of citicoline to 1 placebo), vehicle-controlled, double-blind trial at 21 US centers. Treatment was to be started within 24 hours of stroke onset and was continued orally for 6 weeks. Final outcome assessments were at 12 weeks. Two hundred fifty-nine patients were enrolled, with approximately 65 in each of the four groups. Mean time from stroke onset to treatment was 14.5 hours, and there were no significant differences in baseline characteristics between the four groups except for patient weight. A significant difference between the groups, favoring citicoline treatment, was seen in terms of functional outcome as measured by the Barthel Index and Rankin scale, neurologic evaluation as measured by the National Institutes of Health (NIH) stroke scale, and cognitive function as measured by the Mini Mental Status Examination. When the baseline NIH stroke scale was used as a covariate, both the 500-mg citicoline group and the 2,000-mg citicoline group had a significant improvement in terms of the percent of patients who had a favorable outcome on the Barthel Index at 90 days. There were no drug-related serious adverse events or deaths in this study. This study suggests that oral citicoline can be used safely with minimal side effects in acute stroke treatment. Citicoline appears to improve functional outcome and reduce neurologic deficit with 500 mg of citicoline appearing to be the optimal dose.

Synthetic approaches to endothelin receptor antagonists in clinical development.

The increasing structural and stereochemical complexity of new drug candidates continues to pose numerous synthetic challenges for pharmaceutical process development. Often the implementation of new methodologies, and/or the novel utilization of existing methodologies becomes an essential aspect of developing cost-effective and practical syntheses for new chemotherapeutics. An excellent case in point, and highlighted in this review, are the novel synthetic processes developed for some of the leading endothelin receptor antagonists currently in clinical development.

Circulating intercellular adhesion molecule-1 levels and neutrophil adhesion in stroke.

To examine whether changes in leukocyte adhesion properties occur during stroke, we measured circulating serum intercellular adhesion molecule 1 (cICAM-1) levels and neutrophil adhesion in acute stroke, patients at high risk of stroke, and in matched controls. Levels of cICAM-1 were significantly lower in the stroke group (186.2 +/- 15.6 ng ml-1) compared to controls (257.7 +/- 24.8) and risks (257.7 +/- 16.5). Neutrophil adhesion was significantly higher in the stroke group (23.6 +/- 4.3%; n = 14) compared to controls (9.7 +/- 2.3%; n = 12) and risks (12.7 +/- 2.5%; n = 13). These data suggest that changes in leukocyte adhesion dynamics are occurring in acute stroke.

Lazaroids. CNS pharmacology and current research.

The 21-aminosteroids (lazaroids) are inhibitors of lipid membrane peroxidation and appear to function as oxygen free radical scavengers. The therapeutic potential of the lazaroid tirilazad mesylate has been extensively studied in several CNS disorders. Tirilazad and related compounds have been found to be highly beneficial in spinal cord trauma. Spinal cord injury studies utilising tirilazad are currently underway to determine the optimal combination of medications. Tirilazad has also been found to be beneficial in experimental head injury models, however current clinical studies have failed to confirm this efficacy, due in part to difficulties in obtaining therapeutic drug concentrations. Clinical studies using tirilazad in subarachnoid haemorrhage have been more promising. It has been shown to be beneficial in terms of reducing vasospasm and cerebral infarction associated with subarachnoid haemorrhage, and has now been approved in several European countries in this indication. Results from US studies are expected shortly. Finally, tirilazad has also been extensively tested in a variety of stroke models. Although it appears to be highly beneficial in experimental models, the clinical studies to date have failed to confirm this efficacy. Again, this failure appears to be due largely to inadequate drug concentrations having so far been tested.

Doxycycline treatment reduces ischemic brain damage in transient middle cerebral artery occlusion in the rat.

Agents that inhibit leukocyte adhesion including intercellular adhesion molecule-1 antibodies (anti-ICAM-1) have shown beneficial effects in experimental central nervous system (CNS) ischemia. Doxycycline inhibits leukocyte function in vitro by binding divalent cations and reduces spinal cord reperfusion injury. The authors used a clinically relevant model of focal CNS reperfusion injury to test whether treatment with doxycycline would reduce cerebral ischemic damage and improve functional outcome. Reversible middle cerebral artery occlusion was produced in adult Sprague-Dawley rats by advancing a filament into the internal carotid artery for 2 h. Animals received either i.p. doxycycline (10 mg/kg) (N = 13) or saline (N = 11) 30 min before ischemia, followed by 10 mg/kg every 8 h x 6. Both functional assessment (5 point neurologic scale) and infarct volume was evaluated at 48 h. Functional efficacy: doxycycline 0.5 +/- 0.2 (mean +/- SE) vs control 1.3 +/- 0.3 (p = 0.03). Infarct volume: doxycycline 56 +/- 18 mm3 vs control 158 +/- 44 mm3 (p = 0.03); This protective effect supports the role of doxycycline in reducing CNS reperfusion injury.

The influence of antiadhesion therapies on leukocyte subset accumulation in central nervous system ischemia in rats.

Although treatment with agents that block leukocyte function, including anti-ICAM-1 and doxycycline, reduces experimental central nervous system (CNS) ischemic injury, it is not known how leukocyte subset accumulation is affected by these agents. Using the rat two-vessel occlusion model and immunohistochemistry, we investigated granulocyte (PMN) and monocyte/macrophage (M phi) accumulation at 1 and 4 d postischemia. A total of 24 animals were randomized to sham surgery, or to ischemia with saline, anti-ICAM-1, or doxycycline treatments. No leukocytes were observed in sham animals. At 24 h postischemia, there was a moderate infiltration of PMN and M phi in untreated animals that was significantly decreased with either treatment. At 4 d after ischemia no PMN were identified, with extensive M phi accumulation occurring in untreated animals that was only partially reduced with doxycycline treatment. These results confirm that both anti-ICAM-1 and doxycycline treatments reduce PMN and M phi infiltration at 24 h. Delayed M phi accumulation occurs despite treatment, suggesting that some of these cells represent transformed resident microglia.

Potential of anticytokine therapies in central nervous system ischaemia.

Central nervous system (CNS) ischaemia is associated with an acute inflammatory response which appears to potentiate CNS injury, especially following reperfusion. This response includes the release of inflammatory mediators called cytokines including IL-1 and TNF-alpha, which triggers the production of additional cytokines including IL-6 and activates leukocytes which infiltrate into the CNS. Increased expression of cytokines has been demonstrated to occur in the first few hours after CNS ischaemia. Preliminary clinical studies suggest that plasma levels of IL-6 are correlated with functional recovery while brain levels of cytokines have been demonstrated to increase following experimental ischaemia. Although there are no current clinical 'anti-cytokine' treatment studies for stroke, experimental studies modulating IL-1 and TNF-alpha have shown neuroprotection.

A highly enantioselective conjugate reduction of 3-arylinden-1-ones using bakers' yeast for the preparation of (S)-3-arylindan-1-ones.

[formula: see text] The bakers' yeast reduction of 3-(1,3-benzodioxol-5-yl)-6-propoxy-1H-inden-1-one 4 has been shown to give (S)-3-(1,3-benzodioxol-5-yl)-2,3-dihydro-6-propoxy-1H-indan-1-one 6 in 65% yield with high enantioselectivity (> 99.0% ee), a key intermediate for the synthesis of the endothelin receptor antagonist SB 217242. In addition, the substituted 3-arylinden-1-ones 10a-e gave equally high enantioselectivity for the 3-arylindan-1-one products 13a-e. Mechanistic studies of the reaction indicate the operative pathway to be an asymmetric conjugate reduction, wherein the hydride transfer from NAD(P)H occurs from the Re-face of the indenone substrate.

Effect of protein kinase C modulation on outcome of experimental CNS ischemia.

Protein kinase C (PKC) is an important intracellular regulator, and its activity may play a central role in the modulation of neuronal ischemic damage. Staurosporine and the compound H-7 are potent in vitro inhibitors of PKC, and 1,2-oleoylacetylglycerol (OAG) is an effective activator. We administered these compounds through a spinal subarachnoid catheter and demonstrated in vivo alteration of spinal cord PKC activity. We then tested the effects of altering PKC activity in a well-established rabbit model of reversible spinal cord ischemia. Animals within each experimental group were subjected to a range of spinal cord ischemic durations by temporary occlusion of the infrarenal abdominal aorta. Compared to control, both staurosporine and H-7 significantly shortened the duration of ischemia that the animals could tolerate, without developing permanent paraplegia. OAG resulted in an insignificant lengthening of the ischemic duration that the animals could withstand. The worsening of ischemic outcome by PKC inhibitors suggests that the enzyme is important for maintaining neurologic function under ischemic conditions, possibly secondary to modulation of intracellular calcium levels.

Treatment with conotoxin, an 'N-type' calcium channel blocker, in neuronal hypoxic-ischemic injury.

Therapeutic efficacy of calcium channel blockers in stroke remains controversial, but previously used agents bind almost exclusively to L-type calcium channels. The newly-discovered N-type calcium channel is specific to neurons, and therapy involving blockade of this site has not been previously attempted. We assessed the neuroprotective effect of omega-conotoxin GVIA (CgTx), a blocker of N-type calcium channels, using both in vitro hypoxic injury to rat cortical neurons and an in vivo model of reversible spinal cord ischemia in the rabbit. In cell cultures, CgTx inhibited hypoxia-induced 45Ca accumulation and neuronal injury minimally, compared to the NMDA antagonist ketamine. In vivo, the duration of spinal cord ischemia which produced permanent paraplegia in 50% of control animals (ET50) was 24.0 +/- 2.6 min. Animals treated 2 h prior to ischemia with 0.5 nmol CgTx in the subarachnoid space had an ET50 of 26.9 +/- 1.8 min (P = 0.36). Animals treated 24 h prior to ischemia (all had persistent systemic tremor) had a ET50 of 28.9 +/- 1.8 min (P = 0.13). We conclude that pharmacologic modulation of the N-type calcium channel does not provide a significant protective effect against neuronal hypoxic-ischemic injury.

Temporal modulation of cytokine expression following focal cerebral ischemia in mice.

There is increasing evidence that the inflammatory response plays an important role in CNS ischemia. The murine model of focal ischemia, however, remains incompletely characterized. In this study we examined expression of several cytokines and the vascular adhesion molecule E-selectin, in order to characterize the molecular events following stroke in the C57BL/6J mouse. Using a multi-probe RNAse protection assay (RPA), mRNA for 19 cytokines was analyzed following permanent and transient occlusion of the middle cerebral artery in mice. In addition, samples from the same mice were analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR) to evaluate E-selectin mRNA expression levels. Several cytokine mRNAs showed a similar expression pattern in both permanent and transient CNS ischemia while others showed a temporal expression pattern that was dependent on the type of stroke. For both models, mRNA levels of TNFalpha rose early (4 h) followed by IL-6 (10-18 h) and a comparatively late increase (96 h) in TGFbeta1. IL-1alpha, IL-1beta and IL-1ra levels showed a model dependent shift in temporal expression. Reperfusion appeared to delay the induction of these cytokines. Temporal changes in cytokine mRNA expression in the mouse CNS occur following ischemic damage. Our findings demonstrate the utility and power of multi-probe RPA for evaluation of changes in cytokine mRNA levels. Moreover, this study is, to our knowledge the first to show temporal changes in cytokine mRNA in mouse cerebral ischemia, forming a basis for further exploration of the roles of these cytokines in modulating ischemic neuronal damage in this model.

Noninvasive screening of extracranial carotid disease: duplex sonography with angiographic correlation.

Initial experience with a pulsed Doppler device coupled to a high resolution real-time sonographic scanner for noninvasive evaluation of extracranial carotid disease is reported. The primary objective was to evaluate patients with asymptomatic cervical bruits and/or equivocal histories of transient cerebral ischemia to determine which of them required further study with angiography. An assessment of the degree of extracranial carotid stenosis as well as the severity of atheromatous plaque formation was made and subsequently compared with the findings at angiography. During a 455 day period, a total of 501 patients were examined. In 150 arteries with complete angiography, correlation disclosed the noninvasive duplex technique to be 92% accurate in predicting which arteries would be significantly diseased. It is proposed that careful sonographic investigation of the extracranial carotid system is a safe and useful screening test for certain patients at risk for cerebrovascular accident.