Pathophysiology of Hemolysis in Infections with Hemophilus influenzae Type b

Abstract The capsular polysaccharide of Hemophilus influenzae type b, polyribosyl ribitol phosphate (PRP), is released from growing organisms during human infection and can be found in body fluids. It binds to untreated erythrocytes. Many patients with invasive infections with this organism develop significant hemolysis, but the mechanism has been unclear. We have found that PRP binds to human erythrocytes in vivo. PRP-coated erythrocytes have a shortened circulation time in mice, but do not lyse spontaneously or fix complement. PRP-coated erythrocytes exposed to antiserum to H. influenzae type b are undamaged in the absence of complement, but are rapidly and effectively lysed in the presence of an intact complement system both in vitro and in vivo in mice. PRP-coated red cells are taken up by liver and spleen. Antiserum to PRP increases hepatic uptake of PRP-coated red cells more than splenic, and appears to induce intravascular, complementmediated hemolysis, as well as extravascular hemolysis. Patients with invasive infection develop hemolysis when circulating PRP and antibody to PRP are present simultaneously. PRP can sometimes be detected on patient erythrocytes when free PRP is present in serum, but this is an inconsistent finding. The hemolytic anemia that occurs during human infection with H. influenzae type b may be due to absorption of PRP to red cells and immune destruction of sensitized erythrocytes. The process requires an intact complement system; both complement-mediated cell lysis and extravascular hemolysis contribute to red cell destruction

Natural gene therapy in monozygotic twins with Fanconi anemia

Monozygotic twin sisters, with nonhematologic symptoms of Fanconi anemia (FA), were discovered to be somatic mosaics for mutations in the FANCA gene. Skin fibroblasts, but not lymphocytes or committed hematopoietic progenitors, were sensitive to DNA cross-linking agents. Molecular analysis revealed, in skin cells of both twins, a frameshift causing deletion in exon 27 (2555ΔT) and an exon 28 missense mutation (2670G>A/R880Q). The latter resulted in primarily cytoplasmic expression and reduced function of the mutant FANCA (R880Q) protein. Surprisingly, the same acquired exon 30 missense change (2927G>A/E966K) was detected in the hematopoietic cells of both sisters, but not in their fibroblasts, nor in either parent. This compensatory mutation existed in cis with the maternal exon 28 mutation, and it restored function and nuclear localization of the resulting protein. Both sisters have been free of hematologic symptoms for more than 2 decades, suggesting that this de novo mutation occurred prenatally in a single hematopoietic stem cell (HSC) in one twin and that descendants of this functionally corrected HSC, via intra-uterine circulation, repopulated the blood lineages of both sisters. This finding suggests that treating FA patients with gene therapy might require transduction of only a few hematopoietic stem cells

Oxymetholone Therapy of Fanconi Anemia Suppresses Osteopontin Transcription and Induces Hematopoietic Stem Cell Cycling

Summary: Androgens are widely used for treating Fanconi anemia (FA) and other human bone marrow failure syndromes, but their mode of action remains incompletely understood. Aged Fancd2−/− mice were used to assess the therapeutic efficacy of oxymetholone (OXM) and its mechanism of action. Eighteen-month-old Fancd2−/− mice recapitulated key human FA phenotypes, including reduced bone marrow cellularity, red cell macrocytosis, and peripheral pancytopenia. As in humans, chronic OXM treatment significantly improved these hematological parameters and stimulated the proliferation of hematopoietic stem and progenitor cells. RNA-Seq analysis implicated downregulation of osteopontin as an important potential mechanism for the drug’s action. Consistent with the increased stem cell proliferation, competitive repopulation assays demonstrated that chronic OXM therapy eventually resulted in stem cell exhaustion. These results expand our knowledge of the regulation of hematopoietic stem cell proliferation and have direct clinical implications for the treatment of bone marrow failure. : In this article, Zhang and colleagues show that 18-month-old Fancd2−/− mice recapitulated key human Fanconi anemia phenotypes, including peripheral pancytopenia and macrocytosis. Chronic oxymetholone treatment improved these hematological parameters and stimulated the proliferation of hematopoietic stem and progenitor cells, but eventually resulted in stem cell exhaustion. RNaseq analysis implicated downregulation of osteopontin as an important mechanism for the drug’s action

Germ cell defects and hematopoietic hypersensitivity to gamma-interferon in mice with a targeted disruption of the Fanconi anemia C gene

Fanconi anemia (FA) is an autosomal recessive chromosome instability syndrome characterized by progressive bone marrow (BM) failure, skeletal defects, and increased susceptibility to malignancy. FA cells are hypersensitive to DNA cross-linking agents, oxygen and have cell cycle abnormalities. To develop an animal model of the disease we generated mice homozygous for a targeted deletion of exon 9 of the murine FA complementation group C gene (fac). Mutant mice had normal neonatal viability and gross morphology, but their cells had the expected chromosome breakage and DNA cross-linker sensitivity. Surprisingly, male and female mutant mice had reduced numbers of germ cells and females had markedly impaired fertility. No anemia was detectable in the peripheral blood during the first year of life, but the colony forming capacity of marrow progenitor cells was abnormal in vitro in mutant mice. Progenitor cells from fac knock-out mice were hypersensitive to interferon gamma. This previously unrecognized phenotype may form the basis for BM failure in human FA

TNF-α induces leukemic clonal evolution ex vivo in Fanconi anemia group C murine stem cells

The molecular pathogenesis of the myeloid leukemias that frequently occur in patients with Fanconi anemia (FA) is not well defined. Hematopoietic stem cells bearing inactivating mutations of FA complementation group C (FANCC) are genetically unstable and hypersensitive to apoptotic cytokine cues including IFN-γ and TNF-α, but neoplastic stem cell clones that arise frequently in vivo are resistant to these cytokines. Reasoning that the combination of genetic instability and cytokine hypersensitivity might create an environment supporting the emergence of leukemic stem cells, we tested the leukemia-promoting effects of TNF-α in murine stem cells. TNF-α exposure initially profoundly inhibited the growth of Fancc–/– stem cells. However, longer-term exposure of these cells promoted the outgrowth of cytogenetically abnormal clones that, upon transplantation into congenic WT mice, led to acute myelogenous leukemia. TNF-α induced ROS-dependent genetic instability in Fancc–/– but not in WT cells. The leukemic clones were TNF-α resistant but retained their characteristic hypersensitivity to mitomycin C and exhibited high levels of chromosomal instability. Expression of FANCC cDNA in Fancc–/– stem cells protected them from TNF-α–induced clonal evolution. We conclude that TNF-α exposure creates an environment in which somatically mutated preleukemic stem cell clones are selected and from which unaltered TNF-α–hypersensitive Fancc–/– stem cells are purged