To define the mechanism(s) responsible for the negative inotropic effects of tumor necrosis factor-a (TNFa) in the adult heart, we examined the functional effects of TNFa in the intact left ventricle and the isolated adult cardiac myocyte. Studies in both the ventricle and the isolated adult cardiac myocyte showed that TNFa exerted a concentration-and time-dependent negative inotropic effect that was fully reversible upon removal of this cytokine. Further, treatment with a neutralizing anti-TNFa antibody prevented the negative inotropic effects of TNFa in isolated myocytes. A cellular basis for the above findings was provided by studies which showed that treatment with TNFa resulted in decreased levels of peak intracellular calcium during the systolic contraction sequence; moreover, these findings did not appear to be secondary to alterations in the electrophysiological properties of the cardiac myocyte. Further studies showed that increased levels of nitric oxide, de novo protein synthesis, and metabolites of the arachidonic acid pathway were unlikely to be responsible for the TNFa-induced abnormalities in contractile function. Thus, these studies constitute the initial demonstration that the negative inotropic effects of TNFa are the direct result of alterations in intracellular calcium homeostasis in the adult cardiac myocyte. (J. Clin. Invest.

Context Cocaine dependence, which affects 2.5 million Americans annually, has no FDA approved pharmacotherapy. Objective To evaluate the immunogenicity, safety, and efficacy of a novel cocaine vaccine to treat cocaine dependence. Design 24 week Phase IIb randomized double-blind placebo-controlled trial with efficacy assessed during weeks 8 to 20 and follow-up to week 24. Setting Cocaine and opioid dependent persons recruited from 2003–2005 from greater New Haven, CT. Participants 115 methadone maintained subjects (67% male, 87% Caucasian, aged 18–46) were randomized to vaccine or placebo and 82% completed the trial. Most smoked crack cocaine along with using marijuana (18%), alcohol (10%), and non-prescription opioids (44%). Intervention Over 12 weeks 109/115 subjects received five vaccinations of placebo or succinylnorcocaine linked to cholera B protein. Main Outcome Measure Semi-quantitative urinary cocaine metabolite levels measured thrice weekly with positive cutoff of 300 ng/ml. Results The 38% of vaccinated subjects who attained serum IgG anti-cocaine levels ≥ 43 µg/mL (high IgG) had significantly more cocaine-free urines than those with < 43 µg/mL (low IgG) and the placebo subjects during weeks 9 to 16 (45% vs 35%). The proportion of subjects having a 50% reduction in cocaine use was significantly greater in the high IgG than low IgG subjects (0.53 vs. 0.23) (P<0.04). The most common side effects were injection site induration and tenderness. There were no treatment related serious adverse events, withdrawals, or deaths. Conclusions Attaining high (≥ 43 µg/mL) IgG anti-cocaine antibody levels was associated with significantly reduced cocaine use, but only 38% of the vaccinated attained these IgG levels and they had only 2 months of adequate cocaine blockade. Thus, we need improved vaccines and boosters.

Experimental models of acute ischemic myocardial injury indicate that the inflammatory response after the ischemic event contributes to tissue damage. This is especially apparent with reperfusion of the ischemic tissue. In such models some therapeutic strategies designed to reduce neutrophil accumulation or function have resulted in apparently beneficial effects. Although such findings are encouraging, interventions into these pathological processes using specific molecular targets will require greater understanding of specific mechanisms. Current evidence indicates that potential sites of therapeutic intervention will be found in pathways leading to complement activation, generation of lipid-derived mediators, adhesion of neutrophils to endothelial cells and cardiac myocytes, and activation of neutrophil secretory processes releasing, for example, proteolytic enzymes and reactive oxygen. Understanding the dynamic interplay between the mediators, adhesion pathways, and secretory processes that results in myocardial damage will allow a rational approach to controlling the detrimental inflammatory consequences of ischemia and reperfusion.

Beginning in the first 60 minutes after reperfusion, C5a, TGF-beta 1, and MCP-1, acting sequentially, promote infiltration of monocytes into formerly ischemic myocardium. These events may promote the healing of myocardial injury facilitated by reperfusion.

Background-A key component of reperfusion of myocardial infarction is an immediate inflammatory response, which enhances tissue repair. Matrix turnover is crucial to tissue repair, and matrix metalloproteinases (MMPs) are key enzymes involved in matrix degradation. The hypothesis tested is that one inflammation-based effector of tissue repair is the secretion and activation of MMP-9 by infiltrating neutrophils. Methods and Results-Cardiac lymph and tissue were assayed for latent and active MMP-2 and MMP-9 by zymography and immunochemistry. Dual-labeling immunofluorescence determined the cellular source of MMP-9 protein. Isolated canine neutrophils were incubated with preischemic and postischemic cardiac lymph in the presence and absence of collagen-fibronectin pads, and the supernatants were assayed for latent and active MMP-9. MMP-9 increased during the first hours of reperfusion in both lymph supernatants and myocardial extracts, and this increase was of neutrophil origin. MMP-9 in the cardiac lymph remained latent but was activatable. In contrast, MMP-9 in the myocardium was in both latent and active forms. In situ zymography demonstrated that activated MMP-9 surrounded the infiltrated neutrophils. When postischemic cardiac lymph was incubated with neutrophils in vitro, MMP-9 secretion and activation occurred only in the presence of a collagen-fibronectin substrate; preischemic cardiac lymph did not induce significant secretion or activation. Conclusions-Infiltrating neutrophils are an early source of MMP-9 after reperfusion, and a portion of MMP-9 in the myocardium is active. Infiltrating neutrophils may localize MMP-9 activation by secreting MMP-9 and as a source of activating proteases.

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MCP-1 mRNA is induced in the endothelium of a specific class of small veins immediately after reperfusion. MCP-1 induction is confined to the previously ischemic area that has been reperfused. We suggest a significant role for MCP-1 in monocyte trafficking in the reperfused myocardium.

Background. We have previously demonstrated that chemotactic factors released from the ischemic canine myocardium peak early during reperfusion and that they elicit neutrophil adherence reactions in vitro that are dependent on the CD18 glycoprotein family. In this study we investigated the hypothesis that neutrophil localization in ischemic canine myocardium in vivo occurs over a similar time course during early reperfusion and involves a CD18-dependent mechanism.Methods and Results. We occluded the circumflex coronary artery for 1 hour in acute, open-chest dogs, followed by reperfusion for 1, 2, 3, or 4 hours. Regional myocardial blood flow was determined using radiolabeled microspheres, and localization was traced using technetium-99m-labeled autologous neutrophils. In the first hour of reperfusion, neutrophil localization occurred preferentially within the subendocardial region and was inversely related to flow. Neutrophil localization diminished across the ischemic myocardium from endocardium to epicardium but remained negatively related to flow in the midmyocardial region. Regardless of flow, little neutrophil localization occurred in the subepicardial region. Neutrophil localization was greatest in the first hour of reperfusion and diminished thereafter. By 4 hours of reperfusion, the rate of localization was markedly attenuated relative to 1 hour. Dogs given anti-CD18 monoclonal antibody R15.7 (1 mg/kg i.v.) before occlusion underwent 1 hour of occlusion followed by 1 hour of reperfusion. When compared with 1-hour reperfusion controls, the R15.7-treated dogs demonstrated significant attenuation of neutrophil localization in the subendocardial region.Conclusions. These data support the concepts that rapid neutrophil localization during reperfusion occurs within regions of previous myocardial ischemia and that neutrophils preferentially localize within the subendocardial region. The rate of neutrophil localization is greatest within the first hour after the initiation of reperfusion, and localization is, at least in