Abstract Background Autologous hematopoietic stem cell transplantation (aHSCT) using hematopoietic progenitor cells (HPCs) has become an important therapeutic modality for patients with high‐risk malignancies. Current literature on standardized method for HPC apheresis in children is sparse and failure rate reported as high as 30%. Patients/Methods A retrospective study of 125 pediatric patients with high‐risk malignancies undergoing aHSCT in Western Australia between 1997 and 2016 was conducted. Results Mobilization was achieved by means of chemotherapy and granulocyte colony‐stimulating factor (G‐CSF). Patients underwent apheresis the day after CD34 + counts reached ≥20/µL and an additional dose of G‐CSF. Peripheral arterial and intravenous lines were inserted in pediatric intensive care unit under local anesthetic and/or sedation, omitting the need for general anesthesia as well as facilitating an uninterrupted apheresis flow. Larger apheresis total blood volumes were processed in patients weighing ≤20 kg. The minimal dose of ≥2 × 10 6 CD34 + cells/kg was successfully collected in 98.4% of all patients. The optimal dose of 3‐5 × 10 6 CD34 + cells/kg was collected in 96% of patients scheduled for a single aHSCT, 87.5% for tandem, and 100% for triple aHSCT. All HPC collections were completed in one apheresis session. Mobilization after ≤3 chemotherapy cycles and cycles including cyclophosphamide resulted in a significantly higher yield of CD34 + cells. Conclusion Our approach to HPC mobilization by means of chemotherapy and single myeloid growth factor combined with optimal collection timing facilitated by continuous apheresis flow resulted in highly effective HPC harvest in children and adolescents with high‐risk cancers.
The master transcription factor nuclear factor (erythroid-derived 2)-like 2 (Nrf2) regulates the expression of antioxidant and phase II-metabolizing enzymes by activating the antioxidant response element (ARE) and thereby protects cells and tissues from oxidative stress. Pulmonary complications remain the leading cause of death in human immunodeficiency virus (HIV)-1-infected individuals, who display systemic oxidative stress and glutathione deficiency that can be modeled in transgenic rats where HIV-1-related viral proteins decrease glutathione levels and cause epithelial barrier dysfunction within the alveolar space by as yet unknown mechanisms. We hypothesized that HIV-1-related proteins inhibit Nrf2-mediated antioxidant defenses and thereby disrupt the normally tight alveolar epithelial barrier. Nrf2 RNA silencing dampened Nrf2/ARE activity, decreased the expression of the tight junction proteins zonula occludens-1, occludin, and claudin-18, increased paracellular permeability of alveolar epithelial monolayers derived from wild-type rats, and therefore reproduced the effects of HIV-1 transgene expression on the epithelial barrier that we had previously described. In contrast, upregulating Nrf2 activity, either by plasmid-mediated overexpression or treatment with the Nrf2 activator sulforaphane, increased the expression of ARE-dependent antioxidants, including NAD(P)H dehydrogenase, quinone 1 and glutathione, improved the expression of tight junction proteins, and restored the ability to form tight barriers in alveolar epithelial cells from HIV-1 transgenic rats. Taken together, these new findings argue that HIV-1-related proteins downregulate Nrf2 expression and/or activity within the alveolar epithelium, which in turn impairs antioxidant defenses and barrier function, thereby rendering the lung susceptible to oxidative stress and injury. Furthermore, this study suggests that activating the Nrf2/ARE pathway with the dietary supplement sulforaphane could augment antioxidant defenses and lung health in HIV-1-infected individuals.
Excessive alcohol use increases the risk of acute lung injury and pneumonia. Chronic alcohol ingestion causes oxidative stress within the alveolar space, including near depletion of glutathione (GSH), which impairs alveolar epithelial and macrophage function, in experimental animals and human subjects. However, the fundamental mechanism(s) by which alcohol induces such profound lung oxidative stress is unknown. Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) is a redox-sensitive master transcription factor that regulates activation of the antioxidant response element (ARE). As the alveolar epithelium controls GSH levels within the alveolar space, we hypothesized that alcohol also decreases Nrf2 expression and/or activation within the alveolar epithelium. In this study, we determined that alcohol ingestion in vivo or direct alcohol exposure in vitro down-regulated the Nrf2-ARE pathway in lung epithelial cells, decreased the expression of antioxidant genes, and lowered intracellular GSH levels. RNA silencing of Nrf2 gene expression in alveolar epithelial cells in vitro decreased expression of these same antioxidant genes, and likewise lowered intracellular GSH levels, findings that mirrored the effects of alcohol. In contrast, treating alcohol-exposed alveolar epithelial cells in vitro with the Nrf2 activator, sulforaphane, preserved Nrf2 expression, ARE activation, intracellular GSH levels, and epithelial barrier function. These new experimental findings implicate down-regulation of the Nrf2-ARE signaling pathway as a fundamental mechanism by which alcohol causes profound oxidative stress and alveolar epithelial dysfunction, and suggest that treatments, such as sulforaphane, that activate this pathway could mitigate the pathophysiological consequences of alcohol on the lung and other organs.
Background: Using an experimental model of airway fibrosis following lung transplantation, we recently showed that chronic alcohol ingestion by donor rats amplifies airway fibrosis in the recipient. Associated with alcohol‐mediated amplification of airway fibrosis is increased transforming growth factor β‐1(TGFβ 1 ) and α‐smooth muscle actin expression. Other studies have shown that interleukin‐13 (IL‐13) modulates TGFβ 1 signaling during experimentally‐induced airway fibrosis. Therefore, we hypothesized that IL‐13 is a component of alcohol‐mediated amplification of pro‐fibrotic mediators in the alcoholic lung. Methods: To test this hypothesis, we analyzed tracheal epithelial cells and type II alveolar cells from control‐ or alcohol‐fed rats, alcohol‐treated mouse lung fibroblasts, and human bronchial epithelial cells in vitro for expression of various components of the IL‐13 signaling pathway. Signaling via the IL‐13 pathway was assessed by measuring levels of phosphorylated signal transducers and activators of transcription‐6 (STAT6). In addition, we performed heterotopic tracheal transplantation using control‐fed and alcohol‐fed donor rats and analyzed tracheal allografts for expression of components of the IL‐13 signaling pathway by RT‐PCR and immunocytochemical analyses. Results: Interleukin‐13 expression was detected in type II alveolar epithelial cells and human bronchial epithelial cells, but not in lung fibroblasts. IL‐13 expression was decreased in whole lung and type II cells in response to alcohol exposure. In all cell types analyzed, expression of IL‐13 signaling receptor (IL‐13Rα 1 ) mRNA was markedly increased. In contrast, mRNA and protein expression of the IL‐13 decoy receptor (IL‐13Rα 2 ) were decreased in all cells analyzed. Exposure to alcohol also increased STAT6 phosphorylation in response to IL‐13 and lipopolysaccharide. Conclusions: Data from multiple cell types in the pulmonary system suggest that IL‐13 and its receptors play a role in alcohol‐mediated activation of pro‐fibrotic pathways. Taken together, these data suggest that alcohol primes the airway for increased IL‐13 signaling and subsequent tissue remodeling upon injury such as transplantation.
Chronic alcohol exposure in rats induces oxidative stress in the lung via the angiotensin II pathway and elicits an increase in expression of the profibrotic mediator, transforming growth factor β1 (TGF-β1). In addition, fibronectin expression is increased in the lungs of alcohol-fed rats. These data strongly suggest that profibrotic mechanisms are activated in response to chronic alcohol exposure, and if true, this would suggest that alcohol abuse might increase the risk of fibrotic lung diseases. Consistent with this hypothesis, we have recently determined that alcohol ingestion increases TGF-β1 expression and collagen deposition in an experimental model of obliterative bronchiolitis (OB) following tracheal transplantation in rats. Another profibrotic cytokine, interleukin-13 (IL-13), has been shown to be an important mediator of tissue remodeling/fibrosis in the lung and may act upstream of the TGF-β1 pathway. We hypothesized that alterations in the expression of IL-13 or its receptors could contribute to alcohol-mediated amplification of profibrotic mediators in the alcoholic lung. To test this hypothesis, we analyzed whole lungs and type II alveolar epithelial cells isolated from alcohol-fed rats, as well as mouse lung fibroblasts exposed to alcohol in vitro, for various components of the IL-13 signaling pathway. Gene expression of IL-13, along with the α1 and α2 subunits of its receptor (IL-13Rα1 and IL-13Rα2), was examined by assessing mRNA levels by RT-PCR. In parallel, IL-13Rα2 protein expression was determined by immunostaining. We found that IL-13Rα2 protein was expressed in the whole lung, lung fibroblasts, and type II epithelial cells. RT-PCR analyses indicated that whole lung expresses all three components of the IL-13 signaling pathway. Although lungs from alcohol-fed rats displayed decreased IL-13 mRNA expression, IL-13Rα1 expression was increased, and a marked elevation in IL-13Rα1mRNA expression was observed in alcohol-treated primary lung fibroblasts. Associated with the increase in IL-13Rα1 mRNA in lung fibroblasts was an increase in α-smooth muscle actin protein expression, suggesting fibroblast transdifferentiation into myofibroblasts, which is a cardinal feature of fibrotic diseases such as idiopathic pulmonary fibrosis and bronchiolitis obliterans. These data suggest that IL-13 and its receptors play a role in alcohol-mediated activation of profibrotic mechanisms. In particular, we speculate that alcohol abuse increases IL-13Rα1 expression, which could heighten the response of cells in the lung to IL-13, thereby amplifying TGF-β1-induced fibroblast activation and subsequent tissue remodeling.
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