Month: February 2020

The PUFAs, once absorbed in the intestines, are then transported, via the bloodstream, to all tissues. Hundreds of small molecules have been identified as metabolites of these few omega-3 and omega-6 precursors in human tissue. Yet, it is the overall balance between omega-3s and omega-6s that seems to modulate many biological processes including the relaxation and contraction of smooth muscle tissue, blood coagulation, and – significantly – inflammation. Although much research demonstrates a potentially important relationship between PUFA intake and the risk of disease, it remains challenging in current dietary intervention studies to accurately evaluate the impact of increased intake of omega-3s by food or supplementation. A frequently confounding factor is the variability inherent in studies of control diets. A different nutritional value may accompany such diets compared with a diet enriched in omega-3– a different composition of fatty acids, for example. Other frequent issues relate to the chemical nature, source, and dose of the omega-3 used in the dietary intervention studies. These issues include the mixed use of the different types of omega-3s, such as EPA and DHA, or the different forms of omega3s, such as triacylglycerols, phospholipids, or ethyl esters. Furthermore, dietary intervention studies in humans are often associated with high individual genetic and environmental variability. All of these factors militate against an accurate evaluation of the biological effects of omega-3s, and no molecular markers of omega-3 intake currently exist. In 2004, the fat-1 transgenic mouse model was developed to eliminate many of the confounders inherent in omega-6/omega-3 research. The mouse was engineered to carry the C. elegans fat1 gene, which can add a double bond into an unsaturated fattyacid hydrocarbon chain, thus converting omega-6 to omega-3 fatty acids. Though the mice are not exposed to an omega-3 diet, this conversion results in an abundance of omega-3 and a reduction in omega-6 fatty acids in their organs. The resulting omega-6/omega-3 fatty acid profile has also been shown to be comparable to those obtained by dietary supplementation. The animals therefore provide a controlled approach for evaluating the effects of a balanced omega-6/omega-3 ratio, one that does not introduce the confounding factors that result from enforcing different test diets. To date, the fat-1 transgenic mouse model has been widely used, and has demonstrated that balancing the omega-6/omega-3 ratio can protect against a wide variety of diseases, including chronic inflammatory diseases and cancer. However, the molecular mechanisms underlying these beneficial effects remain to be fully elucidated. In the present study, we used a multi-platform lipidomic approach to compare the molecular phenotype of fat-1 and WT mice exposed for six months to an identical high-omega-6.

While LT-71 and MEM-75 showed different degrees of transcytosis and recycling. Although these data also show a correlation between affinity and transcytosis, the comparable affinities of antibodies M-A712 and MEM-189, combined to their strikingly different transcytosis behavior, indicate that there may be additional mechanisms governing the intracellular fate of transcytosing antibodies. Finally, we wanted to investigate if transcytosis of pH-dependent TfR antibodies could be blocked by bafilomycin, an inhibitor of endosomal acidification. Figure 6I shows that pre-incubation of hCMEC/D3 cells with bafilomycin strongly reduced basolateral passage of antibody MEM-189, while apical recycling was unaffected. This result confirmed our hypothesis that endosomal acidification is an essential mechanistic step in facilitating the transcytosis of TfR antibodies with reduced affinity at low pH. There is a wealth of literature describing the phenomenon of transcytosis in vivo and in vitro in addition to the classical pathway of receptor-mediated endocytosis and recycling. However, many publications describing in vitro models of protein transcytosis through the blood-brain barrier neglect the magnitude of paracellular flux as opposed to the small amount of transcytosed material. In fact, our results show accumulation of 30 ng of the transferrin ligand or Protein A in the basolateral compartment following incubation with 1 mg/mL of the radiolabel. In order to cope with this limitation, which is especially apparent in the relatively leaky monolayers of hCMEC/D3 cells, we have applied a pulse-chase assay set-up, initially described by Raub and Newton for primary bovine brain endothelial cells. In order to detect the low amounts of transcytosed antibody and at the same time avoid using radiolabeled material, the development of highly sensitive IgG ELISAs has been instrumental for assessing the transcytosis potential of antibodies of different species. Furthermore, the ELISA protocol can be easily adapted for automation which makes it highly attractive in terms of assay throughput. Although several studies addressed the transcytosis of antibodytargeted nanoparticles and immunoliposomes across hCMEC/D3, this is the first study to investigate the transcytosis and the fate of free antibodies targeting receptors capable of mediating transcytosis in immortalized human brain endothelium. Our results support the following conclusions: Apical to basolateral transcytosis of intact transferrin occurs in hCMEC/D3 monolayer cultures. Ligand is also equally recycled to the luminal membrane. Both events are temperature sensitive but not modulated by astrocyte co culture An antibody to the IGF1R is exclusively recycled, while antibodies against the TfR are either degraded in lysosmes or recycled/transcytosed Reduced affinity of antibodies to the transferrin receptor at endosomal pH may enhance antibody transcytosis.