Volume 133, Number 3 (Supplement) • PSRC Abstract Supplement
producing two dividing cells and 10% one differentiated and one proliferating cell. Here too, division outcomes were independent of neighbours or close relations. These stem-like cells generated colonies containing hundreds of cells, mostly proliferating. Combinatorial statistics using the timelapse data predicted the distribution of clone sizes in a large sample of cells (n=1462) cultured for 7days, indicating the probabilities of each type of division were balanced as in vivo. Hypersupplementation (20ng/mL) or withdrawal (0ng/mL) of EGF, or addition of R-Spondin to the culture imbalanced the generation of proliferating or differentiating cells indicating that the fate of the progenitor-like cells was subject to external regulation. Transcriptional analysis revealed a distinct pattern of gene expression in 8-cell colonies derived from stem and progenitor-like cells. Validation of these results by immunostaining revealed the proportion of cells expressing differentiation associated genes in progenitor-derived colonies in accordance with the timelapse data.
Nicholas J Albano, BS; Daniel A Cuzzone, MD; Swapna Ghanta, MD; Seth Z Aschen, BS; Gina T Farias-Eisner, BA; Ira L Savetsky, MD; Jason C Gardenier, MD; Walter J Joseph, BS; Jeremy S Torrisi, BA; Babak J Mehrara, MD Memorial Sloan-Kettering Cancer Center, New York, NY Introduction: Obesity induced inflammation is a major contributor to morbidity from a variety of disorders including atherosclerosis, cancer, and metabolic syndrome. Although many studies have focused on the cardiovascular effects of obesity, the negative effects of dietary changes on the lymphatic system remain largely unknown. This gap in our knowledge is important since the lymphatic system is a critical physiologic regulator of inflammatory responses, a key mechanism by which the negative effects of obesity are exerted. We have previously shown that obesity results in significant architectural changes in peripheral lymph nodes. The purpose of these experiments was to determine the functional effects of these changes on immune responses. Methods: We used a well-described diet induced obesity (DIO) model in which adult male C57BL/6J mice were fed a high fat (60%) and compared with control mice fed a normal chow diet for 10 weeks. To test humoral immunity, we vaccinated DIO or control mice with ovalbumin (OVA) and analyzed serum anti-OVA IgG1 titers 3 weeks after vaccination. Additionally, B and T cells were isolated from peripheral lymph nodes, cultured, and re-stimulated in vitro to analyze cytokine responses using ELISA. To assess cellular immunity, we induced contact hypersensitivity responses in the ear using 1-fluoro-2,4-dinitrobenzene (DNFB). Responses were measured using caliper measurements, immunohistochemistry and flow cytometry to analyzed gross and cellular inflammatory responses, respectively. Finally, we analyzed inflammatory lymph node lymphangiogenesis in obese and control animals to determine how draining lymph nodes respond to inflammatory stimuli. Results: Consistent with our previous studies, we found that DIO mice had significantly smaller lymph nodes as compared with controls; however, despite these changes, DIO mice demonstrated comparable anti-OVA IgG1 titers and B cell cytokine production as compared with controls. In contrast, T cells isolated from DIO mice demonstrated a significant decrease in both interferon gamma (IFN-g) and IL4 production after stimulation in vitro (P
producing two dividing cells and 10% one differentiated and one proliferating cell. Here too, division outcomes were independent of neighbours or close relations. These stem-like cells generated colonies containing hundreds of cells, mostly proliferating. Combinatorial statistics using the timelapse data predicted the distribution of clone sizes in a large sample of cells (n=1462) cultured for 7days, indicating the probabilities of each type of division were balanced as in vivo. Hypersupplementation (20ng/mL) or withdrawal (0ng/mL) of EGF, or addition of R-Spondin to the culture imbalanced the generation of proliferating or differentiating cells indicating that the fate of the progenitor-like cells was subject to external regulation. Transcriptional analysis revealed a distinct pattern of gene expression in 8-cell colonies derived from stem and progenitor-like cells. Validation of these results by immunostaining revealed the proportion of cells expressing differentiation associated genes in progenitor-derived colonies in accordance with the timelapse data.
Nicholas J Albano, BS; Daniel A Cuzzone, MD; Swapna Ghanta, MD; Seth Z Aschen, BS; Gina T Farias-Eisner, BA; Ira L Savetsky, MD; Jason C Gardenier, MD; Walter J Joseph, BS; Jeremy S Torrisi, BA; Babak J Mehrara, MD Memorial Sloan-Kettering Cancer Center, New York, NY Introduction: Obesity induced inflammation is a major contributor to morbidity from a variety of disorders including atherosclerosis, cancer, and metabolic syndrome. Although many studies have focused on the cardiovascular effects of obesity, the negative effects of dietary changes on the lymphatic system remain largely unknown. This gap in our knowledge is important since the lymphatic system is a critical physiologic regulator of inflammatory responses, a key mechanism by which the negative effects of obesity are exerted. We have previously shown that obesity results in significant architectural changes in peripheral lymph nodes. The purpose of these experiments was to determine the functional effects of these changes on immune responses. Methods: We used a well-described diet induced obesity (DIO) model in which adult male C57BL/6J mice were fed a high fat (60%) and compared with control mice fed a normal chow diet for 10 weeks. To test humoral immunity, we vaccinated DIO or control mice with ovalbumin (OVA) and analyzed serum anti-OVA IgG1 titers 3 weeks after vaccination. Additionally, B and T cells were isolated from peripheral lymph nodes, cultured, and re-stimulated in vitro to analyze cytokine responses using ELISA. To assess cellular immunity, we induced contact hypersensitivity responses in the ear using 1-fluoro-2,4-dinitrobenzene (DNFB). Responses were measured using caliper measurements, immunohistochemistry and flow cytometry to analyzed gross and cellular inflammatory responses, respectively. Finally, we analyzed inflammatory lymph node lymphangiogenesis in obese and control animals to determine how draining lymph nodes respond to inflammatory stimuli. Results: Consistent with our previous studies, we found that DIO mice had significantly smaller lymph nodes as compared with controls; however, despite these changes, DIO mice demonstrated comparable anti-OVA IgG1 titers and B cell cytokine production as compared with controls. In contrast, T cells isolated from DIO mice demonstrated a significant decrease in both interferon gamma (IFN-g) and IL4 production after stimulation in vitro (P
