Complement blockade with a C1 esterase inhibitor in paroxysmal nocturnal hemoglobinuria.

Paroxysmal nocturnal hemoglobinuria (PNH) is a rare, clonal, hematopoietic stem cell disorder that manifests with a complement-mediated hemolytic anemia, bone marrow failure, and a propensity for thrombosis. These patients experience both intra- and extravascular hemolysis in the context of underlying complement activation. Currently eculizumab effectively blocks the intravascular hemolysis PNH. There remains an unmet clinical need for a complement inhibitor with activity early in the complement cascade to block complement at the classical and alternative pathways. C1 esterase inhibitor (C1INH) is an endogenous human plasma protein that has broad inhibitory activity in the complement pathway through inhibition of the classical pathway by binding C1r and C1s and inhibits the mannose-binding lectin-associated serine proteases in the lectin pathway. In this study, we show that commercially available plasma derived C1INH prevents lysis induced by the alternative complement pathway of PNH erythrocytes in human serum. Importantly, C1INH was able to block the accumulation of C3 degradation products on CD55 deficient erythrocytes from PNH patient on eculizumab therapy. This could suggest a role for inhibition of earlier phases of the complement cascade than that currently inhibited by eculizumab for incomplete or nonresponders to that therapy.

Assessing dietary intake among infants and toddlers 0-24 months of age in Baltimore, Maryland, USA.

OBJECTIVE: To characterize food and nutrient intake and develop a population-specific food list to be used as a comprehensive dietary assessment tool for Baltimore infants and toddlers aged 0-24 months. The data were used to inform the Growing Leaps and Bounds (GLB) program, which promotes early obesity prevention among Baltimore infants and toddlers. RESEARCH METHODS & PROCEDURES: A cross-sectional dietary survey using 24-hour recalls among randomly selected primary caregivers of infants and toddlers was conducted. RESULTS: Data were collected from 84 children, (response rate 61%) 45 boys; 39 girls. Mean daily energy intakes were 677 kcal, 988 kcal, and 1,123 kcal for children 0-6 months, 7-12 months and 13-24 months, respectively. Infants 0-6 months had higher percentage of energy from fat (48%) than infants 7-12 months (34%) and 13-24 months (31%). Mean daily intakes for all nutrients among 0-12 months old were ≥ Dietary Reference Intakes (DRI), while toddlers 13-24 months had inadequate vitamins A, D, and E intake. Breastfeeding occurred in 33% of infants and toddlers 0 to 6 months, while less than 3% of those aged 7 to 24 months were breastfed. A 104-item food list with eight food and drink categories was developed. CONCLUSIONS: Infants were formula fed with a higher frequency than they were breastfed. The consumption of high-sugar and high-fat foods (e.g. sweetened drinks, French fries) increased with each age group, which can increase the risk of childhood obesity.

The small population of PIG-A mutant cells in myelodysplastic syndromes do not arise from multipotent hematopoietic stem cells.

BACKGROUND: Patients with paroxysmal nocturnal hemoglobinuria harbor clonal glycosylphosphatidylinositol-anchor deficient cells arising from a multipotent hematopoietic stem cell acquiring a PIG-A mutation. Many patients with aplastic anemia and myelodysplastic syndromes also harbor small populations of glycosylphosphatidylinositol-anchor deficient cells. Patients with aplastic anemia often evolve into paroxysmal nocturnal hemoglobinuria; however, myelodysplastic syndromes seldom evolve into paroxysmal nocturnal hemoglobinuria. Here, we investigate the origin and clonality of small glycosylphosphatidylinositol-anchor deficient cell populations in aplastic anemia and myelodysplastic syndromes. DESIGN AND METHODS: We used peripheral blood flow cytometry to identify glycosylphosphatidylinositol-anchor deficient blood cells, a proaerolysin-resistant colony forming cell assay to select glycosylphosphatidylinositol-anchor deficient progenitor cells, a novel T-lymphocyte enrichment culture assay with proaerolysin selection to expand glycosylphosphatidylinositol-anchor deficient T lymphocytes, and PIG-A gene sequencing assays to identify and analyze PIG-A mutations in patients with aplastic anemia and myelodysplastic syndromes. RESULTS: Twelve of 15 aplastic anemia patients were found to harbor a small population of glycosylphosphatidylinositol-anchor deficient granulocytes; 11 of them were found to harbor a small population of glycosylphosphatidylinositol-anchor deficient erythrocytes, 10 patients were detected to harbor glycosylphosphatidylinositol-anchor deficient T lymphocytes, and 3 of them were detected only after T-lymphocyte enrichment in proaerolysin selection. PIG-A mutation analyses on 3 patients showed that all of them harbored a matching PIG-A mutation between CFU-GM and enriched T lymphocytes. Two of 26 myelodysplastic syndromes were found to harbor small populations of glycosylphosphatidylinositol-anchor deficient granulocytes and erythrocytes transiently. Bone marrow derived CD34(+) cells from 4 patients grew proaerolysin-resistant colony forming cells bearing PIG-A mutations. No glycosylphosphatidylinositol-anchor deficient T lymphocytes were detected in myelodysplastic syndrome patients. CONCLUSIONS: In contrast to aplastic anemia and paroxysmal nocturnal hemoglobinuria, where PIG-A mutations arise from multipotent hematopoietic stem cells, glycosylphosphatidylinositol-anchor deficient cells in myelodysplastic syndromes appear to arise from more committed progenitors.

Silencing of genes required for glycosylphosphatidylinositol anchor biosynthesis in Burkitt lymphoma.

OBJECTIVE: To investigate the mechanism of glycosylphosphatidylinositol (GPI) anchor deficiency in Burkitt lymphoma cell lines. METHODS: We identified a large GPI anchor protein deficient population in three different Burkitt lymphoma cell lines through proaerolysin treatment of the cells and flow cytometry analysis using a proaerolysin variant (FLAER). The mechanism of GPI anchor protein deficiency was studied by DNA gene sequencing, a cell-free assay to investigate the GPI anchor biosynthetic pathway, microarray analysis, and quantitative real-time polymerase chain reaction. RESULTS: Burkitt lymphoma cell lines harbor large populations of FLAER(neg) cells, which are resistant to proaerolysin. In all three cell lines, silencing of a gene involved in an early step in GPI-anchor biosynthesis was responsible for the lack of GPI-anchored proteins on the cell surface. Quantitative polymerase chain reaction and microarray analysis demonstrate that the level of mRNA for PIGL and PIGY is lower in the FLAER(neg) Ramos cells and that mRNA levels of PIGY are reduced in the Akata and Daudi cells. Hypermethylation of these genes was associated with the low levels of mRNA and treatment of the cells with 5-aza-2' deoxycytidine restored cell surface GPI-anchored proteins to the FLAER(neg) cells. CONCLUSION: GPI-anchored protein deficiency in Burkitt lymphoma cells is not due to a genetic mutation (e.g., PIGA); rather, the lack of GPI-anchored proteins results from transcriptional silencing of PIGL and PIGY.

Glycosylphosphatidylinositol-anchored protein deficiency confers resistance to apoptosis in PNH.

OBJECTIVE: Investigate the contribution of PIG-A mutations to clonal expansion in paroxysmal nocturnal hemoglobinuria (PNH). MATERIALS AND METHODS: Primary CD34+ hematopoietic progenitors from PNH patients were assayed for annexin-V positivity by flow cytometry in a cell-mediated killing assay using autologous effectors from PNH patients or allogeneic effectors from healthy controls. To specifically assess the role of the PIG-A mutation in the development of clonal dominance and address confounders of secondary mutation and differential immune attack that can confound experiments using primary cells, we established an inducible PIG-A CD34+ myeloid cell line, TF-1. Apoptosis resistance was assessed after exposure to allogeneic effectors, NK92 cells (an interleukin-2-dependent cell line with the phenotype and function of activated natural killer [NK] cells), tumor necrosis factor (TNF)-alpha, and gamma-irradiation. Apoptosis was measured by annexin-V staining and caspase 3/7 activity. RESULTS: In PNH patients, CD34+ hematopoietic progenitors lacking glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-AP(-)) were less susceptible than GPI-AP+ CD34+ precursors to autologous (8% vs 49%; p < 0.05) and allogeneic (28% vs 58%; p < 0.05) cell-mediated killing from the same patients. In the inducible PIG-A model, GPI-AP(-) TF-1 cells exhibited less apoptosis than induced, GPI-AP+ TF-1 cells in response to allogeneic cell-mediated killing, NK92-mediated killing, TNF-alpha, and gamma-irradiation. GPI-AP(-) TF-1 cells maintained resistance to apoptosis when effectors were raised against GPI-AP(-) cells, arguing against a GPI-AP being the target of immune attack in PNH. NK92-mediated killing was partially inhibited with blockade by specific antibodies to the stress-inducible GPI-AP ULBP1 and ULBP2 that activate immune effectors. Clonal competition experiments demonstrate that the mutant clone expands over time under proapoptotic conditions with TNF-alpha. CONCLUSION: PIG-A mutations contribute to clonal expansion in PNH by conferring a survival advantage to hematopoietic progenitors under proapoptotic stresses.

PIG-A mutations in normal hematopoiesis.

Paroxysmal nocturnal hemoglobinuria (PNH) is caused by phosphatidylinositol glycan-class A (PIG-A) mutations in hematopoietic stem cells (HSCs). PIG-A mutations have been found in granulocytes from most healthy individuals, suggesting that these spontaneous PIG-A mutations are important in the pathogenesis of PNH. It remains unclear if these PIG-A mutations have relevance to those found in PNH. We isolated CD34+ progenitors from 4 patients with PNH and 27 controls. The frequency of PIG-A mutant progenitors was determined by assaying for colony-forming cells (CFCs) in methylcellulose containing toxic doses of aerolysin (1 x 10(-9) M). Glycosylphosphatidylinositol (GPI)-anchored proteins serve as receptors for aerolysin; thus, PNH cells are resistant to aerolysin. The frequency of aerolysin resistant CFC was 14.7 +/- 4.0 x 10(-6) in the bone marrow of healthy donors and was 57.0 +/- 6.7 x 10(-6) from mobilized peripheral blood. DNA was extracted from individual day-14 aerolysin-resistant CFCs and the PIG-A gene was sequenced to determine clonality. Aerolysin-resistant CFCs from patients with PNH exhibited clonal PIG-A mutations. In contrast, PIG-A mutations in the CFCs from controls were polyclonal, and did not involve T cells. Our data confirm the finding that PIG-A mutations are relatively common in normal hematopoiesis; however, the finding suggests that these mutations occur in differentiated progenitors rather than HSCs.

Natural history of paroxysmal nocturnal haemoglobinuria using modern diagnostic assays.

Paroxysmal nocturnal haemoglobinuria (PNH) is an uncommon, acquired disorder of blood cells caused by mutation of the phosphatidylinositol glycan class A (PIG-A) gene. The disease often manifests with haemoglobinuria, peripheral blood cytopenias, and venous thrombosis. The natural history of PNH has been documented in retrospective series; but there has only been one study that correlated the more sensitive and specific flow cytometric assays that have become available in the last decade with severe symptoms associated with PNH. In a retrospective analysis of 49 consecutive patients with PNH evaluated at Johns Hopkins, large PNH clones were associated with an increased risk for thrombosis as well as haemoglobinuria, abdominal pain, oesophageal spasm, and impotence. Of the 14 (29%) patients that developed thrombosis, nine died; six of these from complications related to thromboses. According to logistic regression modelling, for a 10% change in PNH clone size, the odds ratio for risk of thrombosis was estimated to be 1.64. No patient with <61% PNH granulocytes developed a thrombosis, whereas 12 of 22 patients (54.5%) with > or =61% PNH granulocytes manifested with thrombosis. These data not only confirm that the size of the PNH clone correlates with the risk for thrombosis, but they also suggest a correlation of PNH clone size to more symptomatic PNH.

Enhanced cytotoxicity of rituximab following genetic and biochemical disruption of glycosylphosphatidylinositol anchored proteins.

Rituximab, an anti-CD20 monoclonal antibody used to treat B cell lymphoproliferative disorders and autoimmune diseases, kills cells through complement dependent cytotoxicity, antibody-dependent cellular toxicity and apoptosis. A mechanism of resistance to rituximab is upregulation of the complement regulatory proteins, CD59 and CD55. Paroxysmal nocturnal hemoglobinuria (PNH) is a hematopoietic disorder caused by PIGA mutations that lead to a loss of all glycosylphospatidylinositol (GPI)-anchored proteins including, CD55 and CD59. We compared the cytotoxic activity of rituximab against a PNH B cell line, LD -, and the isogenic cell line LD - PIGA + in which GPI-anchor expression was restored by stable transfection of PIGA. The PNH cell line was more sensitive to rituximab-mediated killing than the LD - PIGA + cells. Biochemical disruption of GPI anchors with phosphatidylinositol specific phospholipase C (PIPLC), a phospholipase that cleaves GPI-anchored proteins, also increased rituximab-mediated killing. Thus, genetic and biochemical interruption of GPI anchor proteins augments sensitivity to rituximab.