Gastrointestinal (GI) tract damage by chemotherapy or radiation limits their efficacy in cancer treatment. Radiation has been postulated to target epithelial stem cells within the crypts of Lieberkühn to initiate the lethal GI syndrome. Here, we show in mouse models that microvascular endothelial apoptosis is the primary lesion leading to stem cell dysfunction. Radiation-induced crypt damage, organ failure, and death from the GI syndrome were prevented when endothelial apoptosis was inhibited pharmacologically by intravenous basic fibroblast growth factor (bFGF) or genetically by deletion of the acid sphingomyelinase gene. Endothelial, but not crypt, cells express FGF receptor transcripts, suggesting that the endothelial lesion occurs before crypt stem cell damage in the evolution of the GI syndrome. This study provides a basis for new approaches to prevent radiation damage to the bowel.

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activated species.3 It is well known that fixed, postmitotic Mitochondrial damage may be a major cause of cellucells accumulate different age pigments, especially lipofuscin. lar aging. So far, this hypothesis had only been tested A considerable amount of this pigment may derive from inusing isolated mitochondria. The aim of this study was jured mitochondria. 4 This led us to propose a hypothesis 5 of to investigate the involvement of mitochondria in aging cell aging, according to which senescence is a by-product of using whole liver cells and not isolated mitochondria oxy-radical attack to the mitochondrial genome. only. Using flow cytometry, we found that age is associRecently, several studies have shown changes in mitochonated with a decrease in mitochondrial membrane potendria on aging. Age-related changes in mitochondrial respiratial (30%), an increase in mitochondrial size, and an intory function and in mitochondrial transport systems have crease in mitochondrial peroxide generation (23%).been reported. [6][7][8] All these changes were found in experiments Intracellular peroxide levels were also increased. The using isolated mitochondria. However, these effects could be number of mitochondria per cell and inner mitochoncaused by altered susceptibility of old mitochondria to the drial membrane mass did not change. Gluconeogenesis stress caused by the isolation procedure. Moreover, because from glycerol or fructose (mitochondrial-independent) intact cells were not used, the mitochondrial-cytosolic interdid not change with age, whereas it did from lactate actions were also ignored. This may lead to errors. The iso-(mitochondrial-dependent). The change in the rate of lated hepatocyte is an excellent model for aging studies. 9 This gluconeogenesis was not accompanied by changes in is especially useful when attempting to study aging at the any of the following parameters: phosphoenolpyruvate cellular level, using whole cells. carboxykinase or pyruvate carboxylase activities or miTo our knowledge, no attempts have been made to correlate tochondrial ATP/ADP or cytosolic NADH/NAD / ratios. age-associated changes in physiological functions of intact This was caused by a decreased rate of malate export cells with specific biochemical or molecular changes in mito-(to 20% of the controls) from mitochondria. The impairchondria. The aim of this study was to test whether mitochonment of the mitochondrial malate transporter is postdrial performance is impaired with aging, using intact liver transcriptional because its expression in Xenopus oocells. To this end, we have measured the rate of biochemical cytes using polyadenylated RNA from livers of young or pathways, gluconeogenesis, and ketogenesis, which critically old animals did not change. Ketogenesis from oleate also depend on mitochondrial function. In addition, we have used fell in hepatocytes from old rats. Our results show, for flow cytometry, which allows a noninvasive analysis of indithe first time in intact cells, a correlation between agevidual cells, to study ...

Rhodamine 123 (Rh123) is widely used as a flow cytometric probe for mitochondrial membrane potential (MMP) in metabolic, pharmacologic, and toxicological studies. However, the use of relatively high concentrations of Rh123 (up to 10 pg/ml) and prolonged incubation times (up to 1 h), including washing steps, may be inconvenient for certain applications in which labile cells are used or which demand rapid or repeated analysis. In this paper we describe a rapid kinetic assay of MMP in isolated rat hepatocytes, based upon the quantitation of the initial rate of Rh123 uptake by living cells, selected by their scattering properties. The results indicate that at an appropriate dye-to-cell ratio (in our experiments, 50 ng Rh123/ml for 250,000300,000 cells/ml), the initial rate of Rh123 uptake is a highly reproducible and sensitive parameter for estimation of MMP, as demonstrated by the effects of substrates and inhibitors of the glycolytic pathway and mitochondrial respiration. Because of its simplicity, rapidity (about 5 min) and metabolic implications, this assay would be also suitable for the routine evaluation of metabolic state of cell suspensions, as a complementary test to the standard dualstaining tests of viability. Other possible applications in screening pharmacologic and toxicological analysis are discussed. o 1994 Wiley-Liss, Ine.Key terms: Mitochondria, mitochondrial membrane potential, rhodamine 123, flow cytometry, hepatocyte, glucose metabolism Mitochondrial membrane potential (MMP) is a key parameter to assess cellular energy metabolism under physiological and pathological conditions (5). However, the conventional approach of studying isolated mitochondria in vitro does not allow integration of the mitochondrial function into the whole cell.Flow cytometry provides convenient methods for the analysis of MMP in situ. Recently, most flow cytometric studies on mitochondrial function have been performed with rhodamine 123 (Rh123), a lipophilic cationic fluorochrome which is incorporated by mitochondria according to transmembrane potential (6,9,11,14,20,22). Rh123 has been applied extensively in flow cytometric studies of mitochondrial metabolism (2,30,33) and metabolic heterogeneity (341, cell activation (81, differentiation (71, oncogenic transformation (32), stem cell characterization and purification (37,381, aging (16), and apoptotic death (lo), as well as for assessment of cytotoxicity (20,22,23,24) and drug resistance (15,17). Some of the multiple applications of Rh123 have been reviewed by Ronot et al. (26,271 and Chen (5,6).The most usual techniques to study MMP by flow cytometry in whole cells follow the one described by Johnson et al. (13,14), with slight modifications. Following a 30-60 min equilibration of cells with Rh123

BackgroundThis open-label, first-in-human, phase 1 study evaluated the safety, pharmacokinetics, pharmacodynamics, and maximum tolerated dose (MTD) of AMG 228, an agonistic human IgG1 monoclonal antibody targeting glucocorticoid-induced tumor necrosis factor receptor−related protein (GITR), in patients with refractory advanced solid tumors.MethodsAMG 228 was administered intravenously every 3 weeks (Q3W). Dose escalation was in two stages: single-patient cohorts (3, 9, 30, and 90 mg), followed by “rolling six” design (n = 2–6; 180, 360, 600, 900, and 1200 mg). Primary endpoints included incidence of dose-limiting toxicities (DLTs), AEs, and pharmacokinetics. Additional endpoints were objective response and pharmacodynamic response.ResultsThirty patients received AMG 228, which was well tolerated up to the maximum planned dose (1200 mg). No DLTs occurred; the MTD was not reached. The most common treatment-related AEs were fatigue (13%), infusion-related reaction (7%), pyrexia (7%), decreased appetite (7%), and hypophosphatemia (7%). Two patients had binding anti−AMG 228 antibodies (one at baseline); no neutralizing antibodies were detected. AMG 228 exhibited target-mediated drug disposition, and serum exposure was approximately dose proportional at 180–1200 mg and greater than dose proportional at 3–1200 mg. Doses > 360 mg Q3W achieved serum trough coverage for 95% in vitro GITR occupancy. Despite GITR coverage in peripheral blood and tumor biopsies, there was no evidence of T-cell activation or anti-tumor activity.ConclusionsIn patients with advanced solid tumors, AMG 228 Q3W was tolerable up to the highest tested dose (1200 mg), exhibited favorable pharmacokinetics, and provided target coverage indicating a pharmacokinetic profile appropriate for longer intervals. However, there was no evidence of T-cell activation or anti-tumor activity with AMG 228 monotherapy.Trial registrationClinicalTrials.gov, NCT02437916.Electronic supplementary materialThe online version of this article (10.1186/s40425-018-0407-x) contains supplementary material, which is available to authorized users.