We have recently developed a new technique to objectively identify erythrocyte cohorts of defined age in mouse blood. The technique (termed double in vivo biotinylation, DIB) involves an initial biotinylation of all erythrocytes in circulation, followed after a few days by a second biotinylation, at a lower density, that labels the biotin-negative erythrocytes that have entered since the first biotinylation. The proportions of biotin^high, biotin^low, and biotin^negative erythrocytes are enumerated by flow cytometry. The DIB technique allows us to track age-related changes on erythrocyte cohorts (Protocol A), and to simultaneously identify very young and older erythrocyte populations in the blood (Protocol B). Using this technique, we have reexamined: i) the relationship between age and buoyant density of erythrocytes, ii) erythrocyte destruction through a random removal mechanism, and iii) the expression of phosphatidylserine on aging erythrocytes. We have also used the DIB technique to study age-related changes in the expression of various markers like CD47 and CD147 and green autofluorescence in aging erythrocyte populations.

Single wall Carbon Nanotubes (SWCNTs) are hydrophobic and do not disperse in aqueous solvents. Acid functionalization of SWCNTs results in attachment of carboxy and sulfonate groups to carbon atoms and the resulting acid functionalized product (AF-SWCNTs) is negatively charged and disperses easily in water and buffers. In the present study, effect of AF-SWCNTs on blood erythrocytes was examined. Incubation of mouse erythrocytes with AF-SWCNTs and not with control SWCNTs, resulted in a dose and time dependent lysis of erythrocyte. Using fluorescence tagged AF-SWCNTs, binding of AF-SWCNTs with erythrocytes could be demonstrated. Confocal microscopy results indicated that AF-SWCNTs could enter the erythrocytes. Treatment with AF-SWCNTs resulted in exposure of hydrophobic patches on erythrocyte membrane that is indicative of membrane damage. A time and dose dependent increase in externalization of phosphatidylserine on erythrocyte membrane bilayer was also found. Administration of AF-SWCNTs through intravenous route resulted in a transient anemia as seen by a sharp decline in blood erythrocyte count accompanied with a significant drop in blood haemoglobin level. Administration of AF-SWCNTs through intratracheal administration also showed significant decline in RBC count while administration through other routes (gavage and intra-peritoneal) was not effective. By using a recently developed technique of a two step in vivo biotinylation of erythrocytes that enables simultaneous enumeration of young (age <10 days) and old (age>40 days) erythrocytes in mouse blood, it was found that the in vivo toxic effect of AF-SWCNTs was more pronounced on older subpopulation of erythrocytes. Subpopulation of old erythrocytes fell after treatment with AF-SWCNTs but recovered by third day after the intravenous administration of AF-SWCNTs. Taken together our results indicate that treatment with AF-SWCNTs results in acute membrane damage and eventual lysis of erythrocytes. Intravenous administration of AF-SWCNTs resulted in a transient anemia in which older erythrocytes are preferably lysed.

Addition of polydispersed acid functionalised single-walled carbon nanotubes (AF-SWCNTs) significantly suppressed alloimmune cytotoxic T cell (CTL) response generated in a mixed lymphocyte reaction (MLR) between spleen cells from C57BL/6 (H-2(b)) and BALB/c (H-2(d)) mice. AF-SWCNTs treatment also decreased CD69 expression, enhanced apoptotic response in T cells and reduced significantly the recovery of live CD4⁺ and CD8⁺ T cells from MLR cultures. A two to threefold increase was noticed in the binding/uptake of AF-SWCNTs by T cells in MLR cultures as compared with control cultured T cells. Confocal microscopy confirmed the internalization of AF-SWCNTs by live CD8⁺ T cells in MLR cultures. Administration of AF-SWCNTs suppressed the generation of anti-P815 CTL response in C57BL/6 mice and the recovery of T-cell populations from the spleens. The results demonstrate a suppressive effect of AF-SWCNTs on CTL response and provide an insight into the mechanism of this suppression.

Interactions between poly-dispersed acid functionalized single walled carbon nanotubes (AF-SWCNTs) and primary lung epithelial (PLE) cells were studied. Peritoneal macrophages (PMs, known phagocytic cells) were used as positive controls in this study. Recovery of live cells from cultures of PLE cells and PMs was significantly reduced in the presence of AF-SWCNTs, in a time and dose dependent manner. Both PLE cells as well as PMs could take up fluorescence tagged AF-SWCNTs in a time dependent manner and this uptake was significantly blocked by cytochalasin D, an agent that blocks the activity of acto-myosin fibers and therefore the phagocytic activity of cells. Confocal microscopic studies confirmed that AF-SWCNTs were internalized by both PLE cells and PMs. Intra-trachially instilled AF-SWCNTs could also be taken up by lung epithelial cells as well as alveolar macrophages. Freshly isolated PLE cells had significant cell division activity and cell cycling studies indicated that treatment with AF-SWCNTs resulted in a marked reduction in S-phase of the cell cycle. In a previously standardized system to study BCG antigen presentation by PLE cells and PMs to sensitized T helper cells, AF-SWCNTs could significantly lower the antigen presentation ability of both cell types. These results show that mouse primary lung epithelial cells can efficiently internalize AF-SWCNTs and the uptake of nanotubes interfered with biological functions of PLE cells including their ability to present BCG antigens to sensitized T helper cells.

For more than 20 years, Dr. Florence Bourgeois, MD, MPH has held multiple positions at the intersection of pediatrics, therapeutics, regulatory science, health informatics and research integrity. A graduate of Yale University, Harvard School of Public Health and Washington University School of Medicine in St. Louis, Dr. Bourgeois’ record transcends academia and medicine. In addition to being a faculty member at Harvard Medical School and the Division of Emergency Medicine and the Computational Health Informatics Program (CHIP) at Boston Children’s Hospital, she is the Director of PedRx, which aims to advance the development and evidence-based use of novel therapeutics for children globally. As the Scientific Director of the Boston Children’s Hospital Biobank, Dr. Bourgeois’ efforts focus on expanding physicians’ capacity to conduct research in pediatric populations. As the Co-Director of the Harvard-MIT Center for Regulatory Science, Dr. Bourgeois also spearheads cross-disciplinary collaborations between academia, biotechnology and pharmaceutical companies, and regulatory agencies. She is the recipient of an Innovation in Regulatory Science Award from the Burroughs Wellcome Fund and has served as an Expert Visitor to the European Medicines Agency to analyze the EU’s pediatric drug legislation. As part of this interview, we examine the relationship between clinical trials and drug development in pediatrics as well as their ethical and social impacts.