Transport of IRW, an ovotransferrin-derived antihypertensive peptide, in human intestinal epithelial Caco-2 cells.

IRW is an egg ovotransferrin-derived ACE inhibitory peptide. The purpose of this study was to evaluate the stability and transcellular transport of IRW in Caco-2 cell monolayers. The stability of IRW was monitored on the apical (AP) surface while its transport was studied from AP to basal (BL) and from BL to AP surfaces. The results revealed that IRW is resistant against intestinal peptidase up to 60 min. Transport of IRW was not affected by addition of wortamanin, a transcytosis inhibitor. However, in the presence of cytochalasin D, a gap junction disruptor, transport of IRW was significantly increased, suggesting a possible passive transport from AP to BL surface. A higher transport of IRW from AP to BL surface than that from BL to AP surface suggests a passive-mediated transport. Moreover, in the presence of glycyl-sarcosine, a substrate for peptide transporter PepT 1, transport of IRW was reduced from AP to BL surface. The above observations showed atypical transport of IRW in Caco-2 cell monolayers. Thus, IRW may possibly be absorbed intact into the site of action for controlling hypertension.

The intrasplenic circulation of three formulations of the same protein antigen.

This paper presents a kinetic study of the intrasplenic circulation of three formulations of the protein antigen conalbumin including the soluble form and two liposomal formulations, one encapsulated in the internal aqueous milieu and one surface-linked to the liposomal vehicle. These formulations differ not only in their physical status but also in their immunostimulating properties and were chosen in an attempt to correlate the movements of antigen in lymphoid tissues with the immune response elicited. The presence of conalbumin was followed over a period of 21 days using, as a detection system, an antibody that we developed and which allows for the visualization of antigenic peptides such as those presented at the surface of antigen-presenting cells (APC). The results demonstrate that the amount of antigen accessing to the spleen, its time of residency and the pathway it follows are all profoundly influenced by the form under which it penetrates the immune system. The results also indicate that the marked initial preferences of an antigen for either B cells, marginal zone macrophages (MZM) or metallophilic macrophages (MM) are of fundamental importance in determining the fate of this antigen in the spleen. It is concluded that the exact formulation of an antigen is as crucial to the regulation of the immune response as is the nature of this antigen. It is further concluded that liposomes can be used efficiently to modify the formulation of an antigen and can contribute as such to the induction of specific immune functions by driving the antigen towards some privileged immune cell populations.

Trafficking of liposomal antigen to the trans-Golgi of murine macrophages requires both liposomal lipid and liposomal protein.

Major histocompatibility complex (MHC) class I molecules found on antigen-presenting cells present peptides derived from cytoplasmic proteins to T cells. In contrast, peptides from exogenous proteins are mostly presented by class II molecules. It has been well established that liposomes can serve as an efficient delivery system for entry of exogenous protein antigens into the MHC class I pathway. Our previous studies utilizing fluorophore-labeled proteins encapsulated in liposomes demonstrated that after phagocytosis of the liposomes by bone marrow-derived macrophages (BMs), the processed peptides were subsequently visualized in the trans-Golgi, while free conalbumin was excluded from the trans-Golgi area. In the present study, we investigated whether liposomal lipids follow the same intracellular route as the liposomal proteins after phagocytosis by BMs. Multilamellar liposomes with different lipid compositions that also contained fluorescent phospholipids (empty liposomes) were incubated with murine BMs. Our results indicate that although empty liposomes were avidly phagocytosed by macrophages, the fluorescent liposomal lipids did not localize to any particular area of the cell but were distributed throughout the cell. In contrast, when a protein was encapsulated in the liposomes, the liposomal lipids were no longer dispersed throughout the cell, but were concentrated and localized in the trans-Golgi area. Furthermore, when the liposomes contained a fluorescent-labeled protein, the fluorescent peptides also localized to the trans-Golgi. These results demonstrate that the combination of both liposomal lipids and liposomal protein is required for Golgi-specific targeting of liposomal antigens. Transport of both liposomal lipids and liposomal proteins to the Golgi complex, a major subcellular organelle in the passage of MHC class I molecules, might explain why antigens encapsulated in liposomes readily induce cytotoxic T lymphocytes.

Differential biodistribution of encapsulated and surface-linked liposomal antigens.

The biodistribution of liposomal antigens either encapsulated in or surface-linked to liposomes of similar composition was studied over time following intravenous injection and the results analyzed in relation to adjuvanticity. The two formulations were shown to behave very differently in vivo. While encapsulated antigen was rapidly focused to liver and spleen as expected, surface-linked antigen exhibited a more disseminated distribution which parallels that of the free protein. In dual-labelling experiments, it was also shown that encapsulated antigen remains associated with its liposomal vehicle in contrast to surface-linked antigen which is rapidly dissociated. This dissociation was apparently neither due to an exchange with plasma lipoproteins nor to a direct action of blood constituents. Besides, it was found that surface-linked antigen was rapidly accumulated in the carcass. We propose that the retention of the surface-linked antigen in the carcass results from a pre-processing of the protein involving more probably mononuclear phagocytes. This pre-processing might in turn favor the dissociation of the protein from the liposomes in a form that allows its dissemination in the whole organism and its interaction with more efficient antigen presenting cells such as for example Langerhans or dendritic cells.