Cytotoxic T cell polyepitope vaccines delivered by ISCOMs.

CD8 alphabeta cytotoxic T lymphocyte (CTL) polyepitope or polytope vaccines have traditionally been delivered using recombinant vector or DNA based delivery modalities. Here we show the delivery of polytope vaccines in the form of either synthetic polypeptides or recombinant polytope proteins by ImmunoStimulatory COMplexes (ISCOMs(R)). Induction of multiple protective CTL responses by these polytope-ISCOM formulations were comparable to viral vector or DNA based delivery modalities as assessed by IFNgamma ELISpot, chromium release and viral challenge assays. Measurement of CTL responses specific for the different epitopes revealed immunodominance patterns, which were largely independent of the vaccine vector or the order of the epitopes in the polytope. ISCOMs thus emerge as a viable human delivery modality for protein-based polytope vaccines.

Inhibition of NF-kappaB activation by a dominant-negative mutant of IkappaBalpha.

The activity of the transcription factor NF-kappaB is tightly regulated by the inhibitory molecule IkappaBalpha. Upon stimulation, IkappaBalpha is rapidly degraded and NF-kappaB translocates to the nucleus to induce gene expression. The IkappaBalpha degradation is preceded by phosphorylation, suggesting that this event plays a role in the activation of NF-kappaB. In this study, we have mutated three potential phosphorylation sites in porcine IkappaBalpha and found that expression of the Ser32 mutant of IkappaBalpha (IS32A), but not Tyr42 or Ser262 mutants or wild-type IkappaBalpha, blocked the activation of NF-kappaB by TNF-alpha. These results suggest that the Ser32 residue, a potential casein kinase II phosphorylation site, is critical for NF-kappaB activation.

Glomerular fibrinolytic activity in anti-GBM glomerulonephritis in rabbits.

Fibrin is an important mediator of injury in severe proliferative forms of glomerulonephritis (GN). Normal glomeruli express fibrinolytic activity, which may protect against the injurious effects of fibrin deposition. Changes in glomerular fibrinolytic activity (GFA) may play an important role in modulating fibrin accumulation in GN. To study the changes in GFA associated with fibrin deposition in GN, autologous phase anti-glomerular basement antibody initiated GN (anti-GBM GN) was studied in rabbits. Net GFA was significantly reduced in association with glomerular fibrin deposition (1.3 +/- 0.8 ng fibrin lysed/10(3) glomeruli/2 hr, normal 57.1 +/- 25.4 ng fibrin lysed/10(3) glomeruli/2 hr, P < 0.02). Reduced GFA in fibrin associated GN was associated with decreased expression of tissue type plasminogen activator (tPA) and increased expression of plasminogen activator inhibitor type-1 (PAI-1) and glomerular macrophage infiltration. In a fibrin independent model of anti-GBM induced GN (heterologous phase), with equivalent injury (proteinuria), net GFA was increased (174 +/- 64 ng fibrin lysed/10(3) glomeruli/2 hr). This was associated with increased tPA and uPA, and decreased PAI-1 in the absence of significant macrophage infiltration. These studies demonstrate that fibrin deposition in GN is associated with a net reduction of GFA, attributable to reduced expression of plasminogen activators and augmentation of PAI-1. Reduction of GFA may potentiate glomerular fibrin deposition and consequent glomerular injury. The association between glomerular macrophage influx and reduction in GFA suggests that this change may be directed by macrophages.

Procoagulant activity expression by macrophages from atheromatous vascular plaques.

Embolization of thrombi from ulcerated plaques is an important cause of morbidity from atherosclerotic carotid artery disease. Factors controlling thrombus formation on these lesions are not well understood. Macrophages were isolated from atherosclerotic plaques to assess their potential to promote local fibrin deposition. Plaques were collected from 11 patients undergoing carotid endarterectomy and 9 patients undergoing reconstructive procedures for atherosclerotic disease of their distal aorta or femoral arteries. Blood was also collected concurrently to isolate monocytes. Procoagulant activity (PCA) of carotid macrophages (8.6 +/- 4.1 mU/10(6) cells) was significantly higher than that of macrophages from non-carotid lesions (0.35 +/- 0.20 mU/10(6) cells; P less than 0.05) or blood monocytes from either group of patients. The PCA of carotid plaque macrophages from patients with recent emboli was 16.1 +/- 8.4 mU/10(6) cells (n = 5) compared to 2.4 +/- 0.8 mU/10(6) cells (n = 6) for plaque macrophages from assymptomatic carotid endarterectomy patients. Carotid macrophage PCA was factor V and factor VII dependent. Its functional activity was inhibited by an anti-tissue factor antibody, and immunohistochemical studies on tissue sections from carotid plaques showed tissue factor in areas where macrophages were abundant. These studies demonstrate that macrophages within carotid artery plaques have augmented procoagulant activity compared with blood monocytes and macrophages from other atherosclerotic lesions and indicate that carotid plaque macrophages are activated. Augmented macrophage PCA may contribute to thrombus formation on ulcerated plaques.