Background Tumor necrosis factor alpha (TNF-α) increases in patients with severe congestive heart failure (CHF) and cachexia. Two naturally occurring modulators of TNF-α activity have been identified in human serum. These two soluble proteins are the extracellular domains of the TNF receptors (sTNF-RI and sTNF-RII, respectively). The determination of circulating sTNF-Rs could provide us with some additional information about the activation of this cytokine in CHF. Methods and Results This study was undertaken to examine the concentration of sTNF-Rs and of bioactive and antigenic TNF-α in 37 consecutive patients with various degrees of CHF compared with that of 26 age-matched healthy subjects. Antigenic TNF-α increased (from 14.3±7.08 to 33.5±13.1 pg/mL, P <.001) in preterminal patients with severe CHF (New York Heart Association [NYHA] class IV). In these patients, sTNF-Rs were also increased (sTNF-RI from 1.17±0.43 to 4.43±2.14 ng/mL and sTNF-RII from 2.2±0.44 to 7.55±2.28 ng/mL, P <.001). When measured by cytolytic bioassay, TNF-α was undetectable (<100 pg/mL). Addition of 625 pg/mL recombinant human TNF-α (rhTNF-α), corresponding in the bioassay to 60% of the lethal dose, to the serum of healthy subjects resulted in a significant increase of the expected cytotoxicity (from 625 to 1290±411 pg/mL, P <.001). Addition of the same dose of rhTNF-α to the serum of patients with mild to moderate CHF (NYHA classes II and III) increased the cytotoxicity from 625 to 877±132 pg/mL, P <.001. In 4 patients with severe CHF (class IV), the expected cytotoxicity was completely inhibited, whereas it was reduced from 625 to 263±198 pg/mL, P <.001, in the remaining 8 patients. Ten patients died within 1 month of entry into the study. They had the highest level of sTNF-RII (8.18±1.92 ng/mL). sTNF-RII was a more powerful independent indicator of mortality than TNF-α, sTNF-RI, NYHA class, norepinephrine, and atrial natriuretic peptide. Conclusions Measurement of sTNF-Rs, in addition to antigenic and bioactive TNF-α, is essential for evaluation of the activation of this cytokine in CHF. Both sTNF-Rs increase in preterminal patients with severe CHF and might inhibit the in vitro cytotoxicity of TNF-α. Antigenic TNF-α also increases in severe CHF. The increased levels of sTNF-RII independently correlate with poor short-term prognosis.

The purified amino-terminal fragment (ATF) of human urokinase plasminogen activator (residues 1-135), which is not required for activation of plasminogen, binds with high affinity to specific plasma membrane receptors on U937 monocytes. Intact urokinase efficiently competes for 125I-labeled ATF binding; 50% competition occurs with 1 nM urokinase. A large part of receptor-bound urokinase remains on the cell surface for at least 2 hr at 37 degrees C. Differentiation of U937 monocytes into macrophage-like cells specifically increases ATF binding 10- to 20-fold. These results suggest an important role for urokinase in monocyte/macrophage biology: the native enzyme binds to the cells with the amino-terminal domain; the catalytic, carboxyl-terminal domain remains exposed on the cell surface to stimulate localized proteolysis and facilitate cell migration.

Isoaspartate formation in extracellular matrix proteins, by aspartate isomerization or asparagine deamidation, is generally viewed as a degradation reaction occurring in vivo during tissue aging. For instance, non-enzymatic isoaspartate formation at RGD-integrin binding sites causes loss of cell adhesion sites, which in turn can be enzymatically "repaired" to RGD by protein-l-isoAsp-O-methyltransferase. We show here that isoaspartate formation is also a mechanism for extracellular matrix activation. In particular, we show that deamidation of Asn263 at the Asn-Gly-Arg (NGR) site in fibronectin N-terminal region generates an alpha(v)beta3-integrin binding site containing the L-isoDGR sequence, which is enzymatically "deactivated" to DGR by protein-L-isoAsp-O-methyltransferase. Furthermore, rapid NGR-to-isoDGR sequence transition in fibronectin fragments generates alpha(v)beta3 antagonists (named "isonectins") that competitively bind RGD binding sites and inhibit endothelial cell adhesion, proliferation, and tumor growth. Time-dependent generation of isoDGR may represent a sort of molecular clock for activating latent integrin binding sites in proteins.

Neuroblastoma, the most common solid tumor of infancy derived from the sympathetic nervous system, continues to present a formidable clinical challenge. Sterically stabilized immunoliposomes (SIL) have been shown to enhance the selective localization of entrapped drugs to solid tumors, with improvements in therapeutic indices. We showed that SIL loaded with doxorubicin (DXR) and targeted to the disialoganglioside receptor GD₂ [aGD₂-SIL(DXR)] led to a selective inhibition of the metastatic growth of experimental models of human neuroblastoma. By coupling NGR peptides that target the angiogenic endothelial cell marker aminopeptidase N to the surface of DXR-loaded liposomes [NGR-SL(DXR)], we obtained tumor regression, pronounced destruction of the tumor vasculature, and prolonged survival of orthotopic neuroblastoma xenografts. Here, we showed good liposome stability, long circulation times, and enhanced time-dependent tumor accumulation of both the carrier and the drug. Antivascular effects against animal models of lung and ovarian cancer were shown for formulations of NGR-SL(DXR). In the chick embryo chorioallantoic assay, NGR-SL(DXR) substantially reduced the angiogenic potential of various neuroblastoma xenografts, with synergistic inhibition observed for the combination of NGR-SL(DXR) with aGD₂-SIL(DXR). A significant improvement in antitumor effects was seen in neuroblastoma-bearing animal models when treated with the combined formulations compared with control mice or mice treated with either tumor- or vascular-targeted liposomal formulations, administered separately. The combined treatment resulted in a dramatic inhibition of tumor endothelial cell density. Long-term survivors were obtained only in animals treated with the combined tumor- and vascular-targeted formulations, confirming the pivotal role of combination therapies in treating aggressive metastatic neuroblastoma. (Cancer Res 2006; 66(20): 10073-82)