Key Points The best survival benefit of HSCT is observed in patients with FA who are transplanted before 10 years with bone marrow after a fludarabine-based regimen. Long-term outcome of patients with FA after transplantation is mainly affected by secondary malignancies and chronic graft-versus-host disease.
Tumor necrosis factor alpha (TNF-␣) production is abnormally high in IntroductionThe Fanconi anemia (FA) proteins play an important role in regulating genome stability, 1 but there is little evidence that the loss of the genoprotection per se in FA cells accounts for the molecular pathogenesis of the bone-marrow failure characteristic of this disease. In fact there is evidence that at least some of these proteins are multifunctional 2 and participate in canonical signaling pathways in hematopoietic cells. [2][3][4][5][6][7][8] Fanconi anemia, complementation group C (FANCC)-deficient cells, for example, are hypersensitive to the apoptotic effects of tumor necrosis factor-␣ (TNF-␣). [4][5][6][7][8][9] In addition, FA cells overproduce TNF-␣ for reasons that have not yet been fully explained. [10][11][12] Most importantly, there is clear evidence that overproduction of and hypersensitivity to TNF-␣ in hematopoietic cells of Fancc Ϫ/Ϫ mice results in bone marrow hypoplasia 13,14 and that long-term ex vivo exposure of murine Fancc Ϫ/Ϫ hematopoietic cells to both growth factors and TNF-␣ results in the evolution of cytogenetically marked preleukemic clones. 9 Therefore, the hematopoietic phenotype of FA may evolve from the overproduction of precisely the cytokine to which FA stem cells are hypersensitive. We designed gene expression microarray experiments by using marrow cells from both patients with FA and normal volunteers in part to seek potential clues to the mechanisms by which FA cells overproduce TNF-␣.Recognizing that transcriptomal analysis would not reveal aspects of the FA phenotype that were controlled translationally or posttranslationally, we also conducted a proteomics analysis. We based our experimental design on an accepted function of the FA "nuclear core complex," that is, its capacity to facilitate monoubiquitinylation of both Fanconi anemia, complementation group I and Fanconi anemia, complementation group D2 (FANCD2). 15,16 Although it is clear that monoubiquitinylation, at least of FANCD2, is required for the avoidance of genotoxicity, 17 it seemed to us unlikely that 8 individual FA genes encoding the "core complex proteins" should have evolved to control the monoubiquitinylation of merely 1 or 2 nuclear proteins. Therefore, reasoning that ubiquitinylation of a variety of other proteins might also be influenced by the core FA proteins, we designed a proteomics survey of ubiquitinylated proteins in FA-C cells and isogenic controls. We reasoned that this approach might lead to the identification of other proteins underubiquitinylated in mutant cells. As reported herein, the gene expression microarray analysis revealed a significant overrepresentation of overexpressed ubiquitin pathway genes in the mutant cells. We therefore took into account the alternative possibility that some ubiquitinylated proteins might be found uniquely in the mutant cells.Indeed, one such protein, Toll-like receptor 8 (TLR8), did appear in the ubiquitin-positive fractions only in FANCC-mutant cells. Given that TLR8 activ...
discontinuation: 60% compared to 7AE6% in the slow tapering group (P = 0AE001). Cumulative incidence of MDS/AML was 8% at 10 years, with a significant correlation with both G-CSF cumulative dose and second IST. This long-term follow-up of children with AAA shows that IST with a slow CyA tapering course is an effective treatment with a low-relapse rate in these cases.
Fanconi anemia, complementation group C (FANCC)-deficient hematopoietic stem and progenitor cells are hypersensitive to a variety of inhibitory cytokines, one of which, TNF␣, can induce BM failure and clonal evolution in Fancc-deficient mice. FANCC-deficient macrophages are also hypersensitive to TLR activation and produce TNF␣ in an unrestrained fashion. Reasoning that suppression of inhibitory cytokine production might enhance hematopoiesis, we screened small molecules using TLR agonist-stimulated FANCCand Fanconi anemia, complementation IntroductionBM failure is a nearly universal complication of Fanconi anemia (FA), an inherited disease caused by biallelic inactivating mutations of any one of 15 genes. [1][2][3][4] FA gene products collectively facilitate responses to DNA damage, 1 and therefore it is often presumed (notwithstanding a lack of direct evidence supporting the idea) that hematopoietic defects simply reflect attrition of hematopoietic stem cells (HSCs) that have specifically suffered excessive DNA damage. An alternative explanation is that the FA proteins are multifunctional and play a direct role in stem cell maintenance, and therefore, DNA damage in FA HSCs is not necessarily required to suppress their function. [5][6][7][8][9] In normal cells, for example, Fanconi anemia, complementation group C (FANCC) modulates the hematopoietic inhibitory effects of TNF␣, IFN␥, and MIP-1␣, each of which normally function to suppress hematopoiesis. 6,10-12 FANCC influences TNF␣ responsiveness at least in part by modulating the activation state of the IFN-inducible double-stranded RNA activated protein kinase. 13 FANCC also suppresses the activation potential of certain TLR pathways in normal mononuclear phagocytes. 14 Therefore, in hematopoietic tissues, FANCC deficiency results in a TLR-dependent overproduction of TNF␣, one of the cytokines to which the stem-cell pool is uniquely intolerant. 10,[15][16][17][18][19] These abnormalities are important elements in the pathogenesis of BM failure. 6,20 There is also experimental evidence that this TNF␣-inhibitory loop is a selective pressure that enhances the ultimate emergence of TNF-resistant leukemic and preleukemic clones. [21][22][23] Therefore, interdiction of TNF␣-induced BM failure, particularly in ways that might have an additional favorable influence on IFN␥-and MIP-1␣-activated signaling pathways, may improve BM function and reduce the likelihood of clonal evolution by improving the fitness landscape and altering the coefficient of selection. 21 Seeking small molecules with these attributes, in the present study we exploited the TLR-hypersensitive phenotype as a screening tool to identify therapeutic agents that might suppress that pathway in FA cells. Using a TLR8-hypersensitive, FANCCdeficient mononuclear phagocyte cell line that we described previously, 14 we screened 75 small molecules, approximately 50 of which were kinase inhibitors. We identified 2 inhibitors, BIRB 796 and dasatinib, that functioned to suppress the TLR-dependent overproduction...
Twelve paediatric patients with aplastic anaemia and two groups of normal control subjects underwent flow cytometric analysis for intracytoplasmic expression of γ interferon (γ‐IFN) and tumour necrosis factor α (TNF‐α) in bone marrow and peripheral blood CD4+ and CD8+ cells. The same cytokines were tested, by immunoassay, in culture supernatants from unstimulated bone marrow mononuclear cells (MNCs). Marrow CD4+ and CD8+ cells expressing γ‐IFN and TNF‐α were significantly increased in the patients in comparison with normal control subjects (P from < 0·05 to < 0·0001 in the different cellular subsets). Patients' marrow CD4+ and CD8+ cells containing γ‐IFN and TNF‐α were significantly increased when compared with the same cell fractions from paired peripheral blood samples (P from < 0·05 to < 0·001 in the various cellular subsets). In the supernatant of marrow MNCs, γ‐IFN and TNF‐α were detected in four out of eight and five out of eight cases, respectively, whereas neither cytokine was traceable in the control subjects. Patients' peripheral blood CD4+ and CD8+ cells containing γ‐IFN and TNF‐α were not significantly increased in comparison with those from normal control subjects. Whereas patients with favourable and unfavourable outcomes had no significantly different proportions of marrow γ‐IFN+/CD4+ and γ‐IFN+/CD8+ cells, the percentages of marrow CD4+ and CD8+ cells containing TNF‐α were significantly lower in subjects with favourable than in those with unfavourable outcome. Overall, these findings show that, in aplastic patients, T cells overexpressing γ‐IFN and TNF‐α concentrate in the bone marrow and that intracytoplasmic expression of TNF‐α in marrow CD4+ and CD8+ cells is associated with an unfavourable clinical course.
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