ADAMTS13 autoantibodies cloned from patients with acquired thrombotic thrombocytopenic purpura: 2. Pathogenicity in an animal model.

BACKGROUND: Acquired thrombotic thrombocytopenic purpura (TTP) is a potentially fatal disease in which ultralarge von Willebrand factor (UL-VWF) multimers accumulate as a result of autoantibody inhibition of the VWF protease, ADAMTS13. Current treatment is not specifically directed at the responsible autoantibodies and in some cases is ineffective or of transient benefit. More rational, reliable, and durable therapies are needed, and a human autoantibody-mediated animal model would be useful for their development. Previously, TTP patient anti-ADAMTS13 single-chain variable-region fragments (scFv's) were cloned that inhibited ADAMTS13 proteolytic activity in vitro and expressed features in common with inhibitory immunoglobulin G in patient plasma. Here, pathogenicity of these scFv's is explored in vivo by transfecting mice with inhibitory antibody cDNA. STUDY DESIGN AND METHODS: Hydrodynamic tail vein injection of naked DNA encoding human anti-ADAMTS13 scFv was used to create sustained ADAMTS13 inhibition in mice. Accumulation of UL-VWF multimers was measured and formation of platelet (PLT) thrombi after focal or systemic vascular injury was examined. RESULTS: Transfected mice expressed physiological plasma levels of human scFv and developed sustained ADAMTS13 inhibition and accumulation of unprocessed UL-VWF multimers. Induced focal endothelial injury generated PLT thrombi extending well beyond the site of initial injury, and systemic endothelial injury induced thrombocytopenia, schistocyte formation, PLT thrombi, and death. CONCLUSIONS: These results demonstrate for the first time the ability of human recombinant monovalent anti-ADAMTS13 antibody fragments to recapitulate key pathologic features of untreated acquired TTP in vivo, validating their clinical significance and providing an animal model for testing novel targeted therapeutic approaches.

Hydrogen peroxide promotes aging-related platelet hyperactivation and thrombosis.

BACKGROUND: The incidence of thrombotic events increases during aging, but the mechanisms are not well understood. To investigate the prothrombotic role of oxidative stress during aging, we tested the hypothesis that aged mice overexpressing the antioxidant enzyme glutathione peroxidase-1 (Gpx1) are protected from experimental thrombosis. METHODS AND RESULTS: Susceptibility to carotid artery thrombosis was first examined in wild-type C57BL/6J mice. After photochemical injury of the carotid artery, the time to stable occlusion was significantly shorter in 12- and 18-month-old mice compared with 4-month-old mice (P<0.01). Unlike wild-type mice, transgenic mice overexpressing Gpx1 (Gpx1 Tg) did not exhibit shortened times to occlusion of the carotid artery at 12 or 18 months of age. Wild-type mice also exhibited increased susceptibility to venous thrombosis after inferior vena cava ligation at 12 or 18 months of age (P<0.05 versus 4 months of age). Gpx1 Tg mice were protected from this aging-related enhanced susceptibility to venous thrombosis. Age-dependent platelet hyperactivation, evidenced by increased hydrogen peroxide, fibrinogen binding, and activation of fibrinogen receptor αIIbß3, was observed in thrombin-activated platelets from wild-type but not Gpx1 Tg mice (P<0.05). Enhanced platelet activation responses in aged mice were also prevented by polyethylene glycol-catalase or apocynin, an inhibitor of NADPH oxidase. Aged mice displayed increased intraplatelet expression of p47(phox) and superoxide dismutase-1, suggesting a mechanistic pathway for increased hydrogen peroxide generation. CONCLUSIONS: Our findings demonstrate that hydrogen peroxide is a key mediator of platelet hyperactivity and enhanced thrombotic susceptibility in aged mice.

Red blood cells mediate the onset of thrombosis in the ferric chloride murine model.

Application of ferric chloride (FeCl(3)) to exposed blood vessels is widely used to initiate thrombosis in laboratory mice. Because the mechanisms by which FeCl(3) induces endothelial injury and subsequent thrombus formation are little understood, we used scanning electron and brightfield intravital microscopy to visualize endothelial damage and thrombus formation occurring in situ. Contrary to generally accepted belief, FeCl(3) does not result in appreciable subendothelial exposure within the time frame of thrombosis. Furthermore, the first cells to adhere to FeCl(3)-treated endothelial surfaces are red blood cells (RBCs) rather than platelets. Energy dispersive x-ray spectroscopy demonstrated that ferric ions predominantly localize to endothelial-associated RBCs and RBC-derived structures rather than to the endothelium. With continuing time points, RBC-derived structures rapidly recruit platelets, resulting in large complexes that subsequently enlarge and coalesce, quickly covering the endothelial surface. Further studies demonstrated that neither von Willebrand factor nor platelet glycoprotein Ib-α receptor (GPIb-α) is required for RBCs to adhere to the endothelium, and that deficiency of GPIb-α greatly abrogated the recruitment of platelets to the endothelial-associated RBC material. These findings illuminate the mechanisms of FeCl(3)-mediated thrombosis and reveal a previously unrecognized ability of RBCs to participate in thrombosis by mediating platelet adhesion to the intact endothelial surface.

Manganese superoxide dismutase depletion in murine hematopoietic stem cells perturbs iron homeostasis, globin switching, and epigenetic control in erythrocyte precursor cells.

Heme synthesis partially occurs in the mitochondrial matrix; thus there is a high probability that enzymes and intermediates important in the production of heme will be exposed to metabolic by-products including reactive oxygen species. In addition, the need for ferrous iron for heme production, Fe/S coordination, and other processes occurring in the mitochondrial matrix suggests that aberrant fluxes of reactive oxygen species in this compartment might perturb normal iron homeostasis. Manganese superoxide dismutase (Sod2) is an antioxidant enzyme that governs steady-state levels of the superoxide in the mitochondrial matrix. Using hematopoietic stem cell-specific conditional Sod2 knockout mice we observed increased superoxide concentrations in red cell progeny, which caused significant pathologies including impaired erythrocytes and decreased ferrochelatase activity. Animals lacking Sod2 expression in erythroid precursors also displayed extramedullary hematopoiesis and systemic iron redistribution. Additionally, the increase in superoxide flux in erythroid precursors caused abnormal gene regulation of hematopoietic transcription factors, globins, and iron-response genes. Moreover, the erythroid precursors also displayed evidence of global changes in histone posttranslational modifications, a likely cause of at least some of the aberrant gene expression noted. From a therapeutic translational perspective, mitochondrially targeted superoxide-scavenging antioxidants partially rescued the observed phenotype. Taken together, our findings illuminate the superoxide sensitivity of normal iron homeostasis in erythrocyte precursors and suggest a probable link between mitochondrial redox metabolism and epigenetic control of nuclear gene regulation during mammalian erythropoiesis.

ADAMTS13 deficiency exacerbates VWF-dependent acute myocardial ischemia/reperfusion injury in mice.

Epidemiologic studies suggest that elevated VWF levels and reduced ADAMTS13 activity in the plasma are risk factors for myocardial infarction. However, it remains unknown whether the ADAMTS13-VWF axis plays a causal role in the pathophysiology of myocardial infarction. In the present study, we tested the hypothesis that ADAMTS13 reduces VWF-mediated acute myocardial ischemia/reperfusion (I/R) injury in mice. Infarct size, neutrophil infiltration, and myocyte apoptosis in the left ventricular area were quantified after 30 minutes of ischemia and 23.5 hours of reperfusion injury. Adamts13(-/-) mice exhibited significantly larger infarcts concordant with increased neutrophil infiltration and myocyte apoptosis compared with wild-type (WT) mice. In contrast, Vwf(-/-) mice exhibited significantly reduced infarct size, neutrophil infiltration, and myocyte apoptosis compared with WT mice, suggesting a detrimental role for VWF in myocardial I/R injury. Treating WT or Adamts13(-/-) mice with neutralizing Abs to VWF significantly reduced infarct size compared with control Ig-treated mice. Finally, myocardial I/R injury in Adamts13(-/-)/Vwf(-/-) mice was similar to that in Vwf(-/-) mice, suggesting that the exacerbated myocardial I/R injury observed in the setting of ADAMTS13 deficiency is VWF dependent. These findings reveal that ADAMTS13 and VWF are causally involved in myocardial I/R injury.

Alternatively-spliced extra domain A of fibronectin promotes acute inflammation and brain injury after cerebral ischemia in mice.

BACKGROUND AND PURPOSE: The fibronectin isoform containing the alternatively spliced extra domain A (EDA(+)-FN) is normally absent from the circulation, but plasma levels of EDA(+)-FN can become markedly elevated in several human pathological conditions associated with inflammation including ischemic stroke. It remains unknown whether EDA(+)-FN contributes to stroke pathogenesis or is simply an associative marker. Several in vitro studies suggest that EDA(+)-FN can activate Toll-like receptor 4, an innate immune receptor that triggers proinflammatory responses. We undertook a genetic approach in mice to investigate the ability of EDA(+)-FN to mediate inflammatory brain damage in a focal cerebral ischemia/reperfusion injury model. METHODS: We used genetically modified EDA(+/+) mice, which constitutively express EDA(+)-FN. Extent of injury, neurological outcome, and inflammatory mechanisms were assessed after 1-hour cerebral ischemia/23-hour reperfusion injury and compared with wild-type mice. RESULTS: We found that EDA(+/+) mice developed significantly larger infarcts and severe neurological deficits that were associated with significant increased neutrophil and macrophage infiltration as quantitated by immunohistochemistry. Additionally, we found upregulation of nuclear factor-κB, cyclo-oxygenase-2, and inflammatory cytokines tumor necrosis factor-α, interleukin-1ß, and interleukin-6 in the EDA(+/+) mice compared with wild-type mice. Interestingly, increased brain injury and neurological deficits were largely abrogated in EDA(+/+) mice by treatment with a specific Toll-like receptor 4 inhibitor. CONCLUSIONS: These findings provide the first evidence that EDA(+)-FN promotes inflammatory brain injury after ischemic stroke and suggest that the elevated levels of plasma EDA(+)-FN observed in chronic inflammatory conditions could worsen injury and outcome in patients after acute stroke.

The CXCR4/CXCR7/SDF-1 pathway contributes to the pathogenesis of Shiga toxin-associated hemolytic uremic syndrome in humans and mice.

Hemolytic uremic syndrome (HUS) is a potentially life-threatening condition. It often occurs after gastrointestinal infection with E. coli O157:H7, which produces Shiga toxins (Stx) that cause hemolytic anemia, thrombocytopenia, and renal injury. Stx-mediated changes in endothelial phenotype have been linked to the pathogenesis of HUS. Here we report our studies investigating Stx-induced changes in gene expression and their contribution to the pathogenesis of HUS. Stx function by inactivating host ribosomes but can also alter gene expression at concentrations that minimally affect global protein synthesis. Gene expression profiling of human microvascular endothelium treated with Stx implicated a role for activation of CXCR4 and CXCR7 by their shared cognate chemokine ligand (stromal cell-derived factor-1 [SDF-1]) in Stx-mediated pathophysiology. The changes in gene expression required a catalytically active Stx A subunit and were mediated by enhanced transcription and mRNA stability. Stx also enhanced the association of CXCR4, CXCR7, and SDF1 mRNAs with ribosomes. In a mouse model of Stx-mediated pathology, we noted changes in plasma and tissue content of CXCR4, CXCR7, and SDF-1 after Stx exposure. Furthermore, inhibition of the CXCR4/SDF-1 interaction decreased endothelial activation and organ injury and improved animal survival. Finally, in children infected with E. coli O157:H7, plasma SDF-1 levels were elevated in individuals who progressed to HUS. Collectively, these data implicate the CXCR4/CXCR7/SDF-1 pathway in Stx-mediated pathogenesis and suggest novel therapeutic strategies for prevention and/or treatment of complications associated with E. coli O157:H7 infection.

Blood group O and black race are independent risk factors for thrombotic thrombocytopenic purpura associated with severe ADAMTS13 deficiency.

BACKGROUND: It has been postulated that blood group O subjects may be partially protected against thrombotic thrombocytopenic purpura (TTP) because they have lower plasma levels of von Willebrand factor. STUDY DESIGN AND METHODS: The Oklahoma TTP Registry enrolled 301 consecutive patients from November 13, 1995 (when systematic ADAMTS13 measurements began), through 2009; 281 (93%) patients had ADAMTS13 measurements. Patients were designated as having severe ADAMTS13 deficiency when the activity measurement by either method was less than 10%. ABO blood group was determined in all 281 patients. The observed frequency of blood group O was compared to the expected frequency. The association between severe ADAMTS13 deficiency and blood group, race, sex, and age were analyzed by logistic regression. RESULTS: The frequency of blood group O was unexpectedly and significantly greater than the race-ethnicity-adjusted expected frequency in 65 patients with severe ADAMTS13 deficiency (60.0% vs. 47.4%, p = 0.042) but not in 216 patients without severe ADAMTS13 deficiency (44.9% vs. 46.5%, p = 0.639). Blood group O and race-ethnicity were independently associated with severe ADAMTS13 deficiency among patients with TTP. The probability for severe ADAMTS13 deficiency was 45.8% with O and 32.1% with non-O blood groups for black patients and 24.1% with O and 15.1% with non-O blood groups for white patients. CONCLUSION: Among patients with TTP and severe ADAMTS13 deficiency the relative frequency of patients with blood group O was greater than expected, suggesting that blood group O may be a risk factor for TTP associated with severe ADAMTS13 deficiency.

Shiga toxin B subunits induce VWF secretion by human endothelial cells and thrombotic microangiopathy in ADAMTS13-deficient mice.

Diarrhea-associated hemolytic uremic syndrome (D+HUS) is the most common cause of acute renal failure among children. Renal damage in D+HUS is caused by Shiga toxin (Stx), which is elaborated by Shigella dysenteriae and certain strains of Escherichia coli, in North America principally E coli O157:H7. Recent studies demonstrate that Stx also induces von Willebrand factor (VWF) secretion by human endothelial cells and causes thrombotic thrombocytopenic purpura, a disease with similarities to D+HUS, in Adamts13(-/-) mice. Stx occurs in 2 variants, Stx1 and Stx2, each of which is composed of 1 catalytically active A subunit that is responsible for cytotoxicity, and 5 identical B subunits that mediate binding to cell-surface globo-triaosylceramide. We now report that B subunits from Stx1 or Stx2 can stimulate the acute secretion of VWF in the absence of the cytotoxic A subunit. This rapid effect requires binding and clustering of globotriaosylceramide, and depends on plasma membrane cholesterol and caveolin-1 but not clathrin. Furthermore, similar to Stx2 holotoxin, the isolated Stx2B subunits induce thrombotic microangiopathy in Adamts13(-/-) mice. These results demonstrate the existence of a novel Stx B-induced lipid raft-dependent signaling pathway in endothelial cells that may be responsible for some of the biological effects attributed previously to the cytotoxic Stx A subunit.

Correction of ADAMTS13 deficiency by in utero gene transfer of lentiviral vector encoding ADAMTS13 genes.

Deficiency of A Disintegrin And Metalloprotease with ThromboSpondin (ADAMTS13) results in thrombotic thrombocytopenic purpura (TTP). Plasma infusion or exchange is the only effective treatment to date. We show in this study that an administration of a self-inactivating lentiviral vector encoding human full-length ADAMTS13 and a variant truncated after the spacer domain (MDTCS) in mice by in utero injection at embryonic days 8 and 14 resulted in detectable plasma proteolytic activity (approximately 5-70%), which persisted for the length of the study (up to 24 weeks). Intravascular injection via a vitelline vein at E14 was associated with significantly lower rate of fetal loss than intra-amniotic injection, suggesting that the administration of vector at E14 may be a preferred gestational age for vector delivery. The mice expressing ADAMTS13 and MDTCS exhibited reduced sizes of von Willebrand factor (vWF) compared to the Adamts13(-/-) mice expressing enhanced green fluorescent protein (eGFP). Moreover, the mice expressing both ADAMTS13 and MDTCS showed a significant prolongation of ferric chloride-induced carotid arterial occlusion time as compared to the Adamts13(-/-) expressing eGFP. The data demonstrate the successful correction of the prothrombotic phenotypes in Adamts13(-/-) mice by a single in utero injection of lentiviral vectors encoding human ADAMTS13 genes, providing the basis for developing a gene therapy for hereditary TTP in humans.

The combined roles of ADAMTS13 and VWF in murine models of TTP, endotoxemia, and thrombosis.

Ultralarge von Willebrand factor (UL-VWF) multimers are thought to play a central role in pathogenesis of the disease thrombotic thrombocytopenic purpura (TTP); however, experimental evidence in support of this hypothesis has been difficult to establish. Therefore, to examine directly the requirement for VWF in TTP pathogenesis, we generated ADAMTS13-deficient mice on a TTP-susceptible genetic background that were also either haploinsufficient (Vwf+/-) or completely deficient (Vwf-/-) in VWF. Absence of VWF resulted in complete protection from shigatoxin (Stx)-induced thrombocytopenia, demonstrating an absolute requirement for VWF in this model (Stx has been shown previously to trigger TTP in ADAMTS13-deficient mice). We next investigated the requirements for ADAMTS13 and VWF in a murine model of endotoxemia. Unlike Stx-induced TTP findings, LPS-induced thrombocytopenia and mortality were not affected by either VWF or ADAMTS13 deficiency, suggesting divergent mechanisms of thrombocytopenia between these 2 disorders. Finally, we show that VWF deficiency abrogates the ADAMTS13-deficient prothrombotic state, suggesting VWF as the only relevant ADAMTS13 substrate under these conditions. Together, these findings shed new light on the potential roles played by ADAMTS13 and VWF in TTP, endotoxemia, and normal hemostasis.

Thrombotic thrombocytopenic purpura in humans and mice.

Thrombotic thrombocytopenic purpura (TTP) is a disorder of blood coagulation that presents classically with the pentad of fever, thrombocytopenia, microangiopathic hemolytic anemia, renal dysfunction and mental status changes. However, the clinical presentation can be quite variable making the diagnosis difficult in many cases. "Hyaline" microthrombi composed primarily of platelets and Von Willebrand Factor (VWF) are found in the small vessels of affected organs and represent the pathological hallmark of the disease. The accompanying tissue ischemia is thought to explain the clinical TTP signs and symptoms. Pathogenesis of TTP has been linked to dysfunction of ADAMTS13, a metalloprotease whose only known substrate is VWF. Interestingly, further investigation into the natural history of TTP has demonstrated that ADAMTS13 deficiency likely is necessary, but not sufficient for the development of this disease, suggesting that additional genetic and/or environmental factors are required for TTP pathogenesis. Recently, a mouse model of TTP was established that recapitulates many of the key clinical features of this disease, including the requirement for further genetic and environmental factors in addition to ADAMTS13 deficiency. Therefore, in addition to being useful for the direct study of disease pathophysiology in vivo, this mouse model may also play a key role in elucidating some of the important environmental and genetic contributors to disease pathogenesis. Here we will review TTP in humans, and then discuss recent information gained from the analysis of ADAMTS13-deficient mice.

Systemic antithrombotic effects of ADAMTS13.

The metalloprotease ADAMTS13 (a disintegrin-like and metalloprotease with thrombospondin type I repeats 13) cleaves highly adhesive large von Willebrand factor (VWF) multimers after their release from the endothelium. ADAMTS13 deficiency is linked to a life-threatening disorder, thrombotic thrombocytopenic purpura (TTP), characterized by platelet-rich thrombi in the microvasculature. Here, we show spontaneous thrombus formation in activated microvenules of Adamts13-/- mice by intravital microscopy. Strikingly, we found that ADAMTS13 down-regulates both platelet adhesion to exposed subendothelium and thrombus formation in injured arterioles. An inhibitory antibody to ADAMTS13 infused in wild-type mice prolonged adhesion of platelets to endothelium and induced thrombi formation with embolization in the activated microvenules. Absence of ADAMTS13 did not promote thrombi formation in alphaIIbbeta3 integrin-inhibited blood. Recombinant ADAMTS13 reduced platelet adhesion and aggregation in histamine-activated venules and promoted thrombus dissolution in injured arterioles. Our findings reveal that ADAMTS13 has a powerful natural antithrombotic activity and recombinant ADAMTS13 could be used as an antithrombotic agent.

Shigatoxin triggers thrombotic thrombocytopenic purpura in genetically susceptible ADAMTS13-deficient mice.

Thrombotic thrombocytopenic purpura (TTP) is a life-threatening illness caused by deficiency of the vWF-cleaving protease ADAMTS13. Here we show that ADAMTS13-deficient mice are viable and exhibit normal survival, although vWF-mediated platelet-endothelial interactions are significantly prolonged. Introduction of the genetic background CASA/Rk (a mouse strain with elevated plasma vWF) resulted in the appearance of spontaneous thrombocytopenia in a subset of ADAMTS13-deficient mice and significantly decreased survival. Challenge of these mice with shigatoxin (derived from bacterial pathogens associated with the related human disease hemolytic uremic syndrome) resulted in a striking syndrome closely resembling human TTP. Surprisingly, no correlation was observed between plasma vWF level and severity of TTP, implying the existence of TTP-modifying genes distinct from vWF. These data suggest that microbe-derived toxins (or possibly other sources of endothelial injury), together with additional genetic susceptibility factors, are required to trigger TTP in the setting of ADAMTS13 deficiency.

ADAMTS13 turns 3.

It has now been 3 years since the von Willebrand factor (VWF)-cleaving protease implicated in thrombocytopenic purpura (TTP) pathogenesis was identified as ADAMTS13 (a disintegrin-like and metalloprotease with thrombospondin type 1 motif 13). More than 50 ADAMTS13 mutations resulting in familial TTP have been reported. Considerable progress has also been realized toward understanding the role of ADAMTS13 in normal hemostasis, as well as the mechanisms by which ADAMTS13 deficiency contributes to TTP pathogenesis. Measurement of ADAMTS13 activity in TTP and other pathologic conditions also remains a focus of a substantial clinical research effort. Building on these studies, continued investigation of ADAMTS13 and VWF holds considerable promise for advancing the understanding of TTP pathogenesis and should lead to improved diagnosis and treatment for this important hematologic disease.

Contribution of Drosophila DEG/ENaC genes to salt taste.

The ability to detect salt is critical for the survival of terrestrial animals. Based on amiloride-dependent inhibition, the receptors that detect salt have been postulated to be DEG/ENaC channels. We found the Drosophila DEG/ENaC genes Pickpocket11 (ppk11) and Pickpocket19 (ppk19) expressed in the larval taste-sensing terminal organ and in adults on the taste bristles of the labelum, the legs, and the wing margins. When we disrupted PPK11 or PPK19 function, larvae lost their ability to discriminate low concentrations of Na(+) or K(+) from water, and the electrophysiologic responses to low salt concentrations were attenuated. In both larvae and adults, disrupting PPK11 or PPK19 affected the behavioral response to high salt concentrations. In contrast, the response of larvae to sucrose, pH 3, and several odors remained intact. These results indicate that the DEG/ENaC channels PPK11 and PPK19 play a key role in detecting Na(+) and K(+) salts.

A novel chromosomal inversion at 11q23 in infant acute myeloid leukemia fuses MLL to CALM, a gene that encodes a clathrin assembly protein.

Rearrangements involving the MLL gene at chromosome band 11q23 are common in infant acute myeloid leukemias (AMLs). We recently encountered an infant patient with rapidly progressive AML whose leukemic cells harbored a previously undescribed MLL rearrangement involving an inversion of 11q [inv(11)(q14q23)]. We used panhandle PCR to determine that this rearrangement juxtaposed the MLL (Mixed-Lineage Leukemia) gene to the CALM (Clathrin Assembly Lymphoid Myeloid leukemia) gene at 11q14-q21. The CALM protein participates in recruitment of clathrin to internal membrane surfaces, thereby regulating vesicle formation in both endocytosis and intracellular protein transport. Intriguingly, CALM has been identified in other cases of AML as a translocation partner for the AF10 gene, which has independently been found to be an MLL partner in AML. We identified the MLL-CALM fusion transcript (but not the reciprocal CALM-MLL transcript) in leukemia cell RNA by RT-PCR. The predicted 1803 amino acid MLL-CALM fusion protein includes amino-terminal MLL domains involved in transcriptional repression, and carboxy-terminal CALM-derived clathrin-binding domains. The genomic breakpoint in MLL is in the 7th intron (within the breakpoint cluster region); the corresponding CALM breakpoint is in the 7th CALM intron. In contrast, breakpoints in CALM-AF10 translocations lie in the 17th-19th CALM introns (30 kb downstream); also, in these translocations, CALM provides the 5' end of the fusion transcript. Together with its previously recognized association with AF10 in AML, the identification of CALM as an MLL fusion partner suggests that interference with clathrin-mediated trafficking pathways may be an underappreciated mechanism in leukemogenesis.

Rituximab for refractory childhood autoimmune hemolytic anemia.

BACKGROUND: Chronic childhood autoimmune hemolytic anemia is an uncommon disorder that is associated with significant morbidity. Treatment with high dose steroids, splenectomy and frequent blood transfusions results in a myriad of complications including growth failure, bone demineralization, Cushing's syndrome, immunosuppression, and transfusional hemosiderosis. OBJECTIVES: To investigate the efficacy of the monoclonal anti-CD20 antibody, rituximab, in treating children with AIHA. METHODS: Four children with chronic AIHA, including two with prior splenectomy, who were dependent on high dose steroids and refractory to other immunosuppressive regimens were treated with four to six weekly doses of rituximab at a dose of 375 mg/m2. RESULTS: All four patients became transfusion-independent and were taken off prednisone completely. Adverse effects included infusion-related reactions that were mild, and infectious complications of Pneumocystis carinii pneumonia and varicella pneumonia. CONCLUSIONS: Treatment with rituximab appears promising for refractory AIHA; if may obviate the need for prednisone and may result in sustained disease remissions in some patients.