Sonicated unilamellar liposomes containing phosphatidylethanolamine and palmitoythomocysteine fuse rapidly when the medium pH is lowered from 7 to 5. Liposome fusion was demonstrated by (i) mixing of the liposomal lipids as shown by resonance energy transfer, (it) gel filtration, and (iii) electron microscopy. The pH-sensitive fusion of liposomes was observed only when palmitoylhomocysteine (-20 MATERIALS AND METHODS Materials. Palmitoylhomocysteine (PamHcy) was synthesized and purified as described (8). Dioleoyl phosphatidylethanolamine (PtdEtn), dioleoyl phosphatidylcholine (PtdCho), and bovine brain phosphatidylserine (PtdSer) were used in this study. All phospholipids, including N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-PtdEtn (N-NBD-PtdEtn) and N-(lissamine rhodamine B-sulfonyl)-PtdEtn (N-RhPtdEtn), were purchased from Avanti Biochemicals. Cholesterol and calcein were obtained from Sigma.Liposome Preparation. Solvent-free lipid films were suspended in phosphate-buffered saline (pH 7.4) at 10 ,mol/ml and sonicated at room temperature for 15 min with a bath sonicator (Laboratory Supplies, Hicksville, NY). Various lipid compositions were used as indicated in the text. Fluorescence-labeled liposomes containing 1 mol % each of N-NBD-PtdEtn and N-Rh-PtdEtn were prepared identically as the unlabeled liposomes.Liposome Fusion. Ten microliters of labeled liposomes and various amounts of unlabeled liposomes were added to 2 ml of phosphate buffered saline. After the relative fluorescence was measured, 5-20 ,pl of HCl at various concentrations was added to achieve the desired pH while the sample was vigorously mixed. After about 2 min at room temperature, appropriate amounts of NaOH were added to return the pH to 7.4. The relative fluorescence of the sample was again measured.Fluorescence Measurements. A Perkin-Elmer LS 5 fluorescence spectrophotometer was used. The emission spectrum was taken for each sample and samples were excited at 468 nm. The excitation and emission slit widths were 5 nm and 3 nm, respectively. Light scattering was about 5-6% of the total fluorescence signal. The ratio, R, of N-NBDPtdEtn emission at 530 nm to the N-Rh-PtdEtn emission at 580 nm is a sensitive measure of the efficiency of the resonance energy transfer between N-NBD-PtdEtn and N-RhPtdEtn (9). The value of R was 0.20 for the unfused liposomes, due to the high efficiency of energy transfer. As the labeled liposomes fused with the unlabeled liposomes, dilution of the fluorescent lipids occurred, resulting in a decrease in the efficiency of energy transfer and an increase in R values (7,9). Hence the increase in R value is a quantitative measure for the degree of liposome fusion. A total mixing of lipids upon complete fusion would result in a maximal R value that is determined by the ratio of unlabeled to labeled liposomes in the fusion mixture. The percent of liposome fusion used in this communication is defined as % fusion RfR -1 100, n [1] in which Ri and Rf are the R values before and after the fusion reaction, respectively, a...

Phosphatidylserine (PS) is asymmetrically distributed in mammalian cell membranes, being preferentially localized in the inner leaflet. Some studies have suggested that a disturbance in the normal asymmetric distribution of PS-e.g., PS exposure in the outer leaflet ofthe cell membrane, which can occur upon platelet activation as well as in certain pathologic red cells-serves as a potent procoagulant surface and as a signal for triggering their recognition by macrophages. These studies suggest that the regulation of PS distribution in cell membranes may be critical in controlling coagulation and in determining the survival ofpathologic cells in the circulation. In this paper we describe a sensitive technique, based on PS-dependent prothrombinase complex activity, for assessing the amount of PS on the external leaflet of intact viable cells. Our results indicate that tumorigenic, undifferentiated murine erythroleukemic cells express 7-to 8-fold more PS in their outer leaflet than do their differentiated, nontumorigenic counterparts. Increased expression of PS in the tumorigenic cells directly correlated with their ability to be recognized and bound by macrophages.Macrophages play an important role as effector cells in host defense against cancer metastasis (1) and viral diseases (2). When appropriately activated, macrophages are able to recognize and destroy a variety of tumorigenic and virusinfected cells, including cells resistant to other host defenses such as T cells and natural killer cells (1), while leaving normal cells unharmed. Macrophage-mediated tumor cell killing has been shown to be independent of such cell characteristics as surface receptors, drug resistance, cell cycle, and metastatic potential (1,3).The mechanism responsible for the ability of mononuclear phagocytes to discriminate between normal and pathologic cells is not known. The broad spectrum of target cells susceptible to macrophage-mediated cytolysis might suggest, however, that a uniform surface moiety could be involved in target cell recognition.An interesting feature of some cell membranes is the asymmetric distribution of membrane phospholipids between the two leaflets of the bilayer (4). In red blood cells (RBC), for example, most membrane phospholipids show some preference for either leaflet, whereas phosphatidylserine (PS) is the only phospholipid that adopts a totally asymmetric distribution, being localized exclusively in the cell's inner leaflet (5-7). Although the mechanisms responsible for maintaining an asymmetric distribution of PS are still unclear, recent evidence suggests that the preservation of PS in the cell's inner leaflet is of central importance in cellular physiology. For example, the exposure of PS that occurs in activated platelets (8) and in sickled RBC (9, 10) regulates hemostasis by serving as a potent procoagulant surface (11, 12) and as a signal for triggering the recognition of these cells by macrophages (13). Related experiments have shown that artificially generated phospholipid vesicles (14,15) a...

Three commercially available porcine-derived biologic meshes were implanted in an Old World primate abdominal wall resection repair model to compare biological outcome as a predictor of clinical efficacy. Tissues were explanted over a 6-month period and evaluated for gross pathology, wound healing strength, mesenchymal cellular repopulation, vascularity, and immune response. In vivo functional outcomes were correlated with in vitro profile for each material. Small intestinal submucosa-based implants demonstrated scar tissue formation and contraction, coincident with mesh pleating, and were characterized by immediate and significant cellular and humoral inflammatory responses. Porcine dermal-based grafts demonstrated significant graft pleating, minimal integration, and an absence of cellular repopulation and vascularization. However, a significant cellular immune response surrounded the grafts, coincident with poor initial wound healing strengths. In vivo observations for the three porcine-derived mesh products correlated with individual in vitro profiles, indicating an absence of characteristic biochemical markers and structural integrity. This correlation suggests that in vivo results observed for these mesh products are a direct consequence of specific manufacturing processes that yield modified collagen matrices. The resulting loss of biological and structural integrity elicits a foreign body response while hindering normal healing and tissue integration.

The use of AlloDerm to partially enclose implants effectively prevented formation of a capsule in areas where AlloDerm contacted the implant at 10 weeks. Long-term studies will be required to determine whether this is a durable result that can be reproduced in humans.

The combination of a nondamaging process, successful removal of cells, and reduction of the xenogeneic alpha-Gal antigens from the porcine dermal matrix, while maintaining an intact extracellular matrix, is critical to its ability to remodel and integrate into host tissue, leading to its overall acceptance.