Abnormal T-cell repertoire is consistent with immune process underlying the pathogenesis of paroxysmal nocturnal hemoglobinuria.
(17/413)
Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal disorder of the hematopoietic stem cell (HSC). Somatic mutations in the PIG-A gene result in the deficiency of several glycosylphosphatidylinositol-linked proteins from the surface of blood cells. This explains intravascular hemolysis but does not explain the mechanism of bone marrow failure that is almost invariably seen in PNH. In view of the close relationship between PNH and idiopathic aplastic anemia (IAA), it has been suggested that the 2 disorders might have a similar cellular pathogenesis, namely, that autoreactive T-cell clones are targeting HSCs. In this paper, we searched for abnormally expanded T-cell clones by size analysis of the complementarity-determining region 3 (CDR3) in the beta variable chain (BV) messenger RNA (mRNA) of the T-cell receptor (TCR) in 19 patients with PNH, in 7 multitransfused patients with hemoglobinopathy. and in 11 age-matched healthy individuals. We found a significantly higher degree of skewness in the TCR BV repertoire of patients with PNH, compared with controls (R(2) values 0.82 vs 0.91, P <.001). The mean frequency of skewed families per individual was increased by more than 2-fold in patients with PNH, compared with controls (28% +/- 19.6% vs 11.4% +/- 6%, P =.002). In addition, several TCR BV families were significantly more frequently skewed in patients with PNH than in controls. These findings provide experimental support for the concept that PNH, like IAA, has an immune pathogenesis. In addition, the identification of expanded T-cell clones by CDR3 size analysis will help to investigate the effect of HSC-specific T cells on normal and PNH HSCs. (+info)
Hematopoietic progenitor cells in the blood and bone marrow in various hematologic disorders.
(18/413)
Hematopoietic progenitor cells are present in the blood and the bone marrow. Changes in the numbers of hematopoietic progenitor cells reflect alteration of pluripotent stem cells. We discuss such changes in common hematologic diseases including aplastic anemia, paroxysmal nocturnal hemoglobinuria (PNH) and thalassemia. In aplastic anemia, the numbers of burst forming units-erythroid (BFU-E) and colony-forming units-granulocyte-macrophage (CFU-GM) are much decreased; the decrease still exists after recovery from therapy. In PNH, the numbers of progenitor cells are low, even in the presence of marrow hypercellularity. In thalassemia, the numbers of progenitor cells are much increased; more pronounced in splenectomized patients. (+info)
Paroxysmal nocturnal hemoglobinuria testing by flow cytometry. Evaluation of the REDQUANT and CELLQUANT kits.
(19/413)
The commercially available REDQUANT and CELLQUANT kits were evaluated in a clinical flow cytometry laboratory using healthy volunteers and a small series of 5 patients with paroxysmal nocturnal hemoglobinuria (PNH). Most samples also were tested with a "home brew" antibody panel for CD66b expression on granulocytes and CD14 expression on monocytes. The REDQUANT and CELLQUANT kits in tandem correctly identified all 5 PNH samples with no false-positive results. The 1 PNH sample from a patient with a negative Ham test result also was identified correctly. One marker, CD59 on granulocytes, when considered alone, had a high false-positive rate, and I suggest lowering the fluorescence intensity criteria for identifying decreased antigen expression. The home-brew tube also performed well, identifying all 5 patients, and may serve as a supplement in difficult specimens. The evaluated kits performed well. However, the validation series was small, which is likely to be a problem at any single institution. The provision of a reproducible manufacturer-defined expression level for normal cells adds another degree of confidence that the test correctly identifies patients with this potentially life-threatening disorder while avoiding false-positive results. (+info)
Immunophenotypic analysis of B cells in PNH: insights into the generation of circulating naive and memory B cells.
(20/413)
Peripheral blood B cells in patients with paroxysmal nocturnal hemoglobinuria (PNH) comprise variable mixtures of normal B cells produced before the onset of disease and glycosylphosphatidylinositol (GPI)-deficient B cells derived from the PNH hematopoietic stem cell. In a detailed phenotypic analysis of 29 patients with PNH, this study shows consistent phenotypic differences between PNH B cells and residual normal B cells. In the majority of patients with active disease, PNH B cells comprised mainly naive cells with a CD27(-)IgM(+)IgD(strong+)IgG(-) phenotype. The proportion of CD27(+) memory cells within this compartment was related to disease duration (Spearman [r(s)] 0.403; P =.030). In PNH patients with predominantly GPI-deficient hematopoiesis, that is, a large granulocyte PNH clone, the residual normal B cells had a predominantly memory (CD27(+)) phenotype. Furthermore, the majority of these memory B cells were not immunoglobulin (Ig) class switched and had an IgM(+)IgD(+)IgG(-) phenotype. Using PNH as a novel model with which to study B lymphopoiesis, this study provides direct evidence that production of new naive B cells occurs throughout life and that the major population of long-lived memory B cells are IgM(+)IgD(+). Moreover, studies of GPI(-) B cells in 2 patients in remission from PNH suggest that the life span of a B-cell clone can be more than 24 years. (+info)
In vitro proliferation and differentiation of megakaryocytic progenitors in patients with aplastic anemia, paroxysmal nocturnal hemoglobinuria, and the myelodysplastic syndromes.
(21/413)
It has previously been shown that patients with aplastic anemia (AA) have a stem cell defect both of proliferation and differentiation. This has been shown by long-term bone marrow (BM) culture, long-term initiating cell assays, and committed progenitor assays. We present, for the first time, data on megakaryocyte (Mk) colony formation from purified BM CD34(+) cells from patients with AA. The results are compared with those from normal controls and from patients with paroxysmal nocturnal hemoglobinuria (PNH) and the myelodysplastic syndromes (MDSs). Those treated for AA had previously received immunosuppression (antithymocyte globulin and/or cyclosporin). No patients had received bone marrow transplantation. A total of 13 AA patients (five untreated, eight treated), six PNH, six MDS, and 13 normal donors were studied. BM CD34(+) cells were purified by indirect labeling and then cultured in a collagen-based Mk assay kit (MegaCult-C, StemCell Technologies). The cultures were fixed on day 12, and the Mk colonies were identified by the alkaline phosphatase anti-alkaline phosphatase technique using the monoclonal antibody CD41 (GP IIb/IIIa). The slides were scored for Mk colony-forming units (CFU-Mks) (3-20 and >20 cells), Mk burst-forming units (BFU-Mks) (>50 cells), and mixed colonies. The results show that total Mk colony formation in AA was significantly lower than in normal donors (p<0.0001), both in untreated patients/nonresponders to treatment (p = 0.0001) and in complete/partial responders (p<0.002). There was no significant difference in Mk colony formation in treated and untreated patients (p = 0.05). Patients with AA had a lower total colony formation than PNH patients (p = 0.0002). PNH patients exhibited lower colony formation than normal controls (p = 0.03), as shown by MDS patients, although the considerable number of variables resulted in a lack of statistically significant difference from normal controls (p = 0.2). We have now shown that Mk colony formation from purified BM CD34(+) cells is significantly reduced, supporting previous evidence that AA results from a stem cell defect. (+info)
Homozygous human C3 deficiency. The role of C3 in antibody production, C-1s-induced vasopermeability, and cobra venom-induced passive hemolysis.
(22/413)
Studies of the family of a patient with marked deficiency of the third component of complement (C3) demonstrated that the patient was homozygous for a blank allele at the C3 locus, C3-. Metabolic studies with purified radiolabeled C3 in the patient revealed a mildly elevated fractional catabolic rate and a markedly reduced synthesis rate, consistent with a lack of C3 synthesis as the patient's primary defect. There was also a mild increase in the rate of conversion of purified C3 added to her serum and incubated at 37 degrees C in vitro. Major blood group-compatible erythrocytes from a patient with paroxysmal nocturnal hemoglobinuria had the same shortened survival in the C3-deficient patient as in a normal control. Although no leukocytosis developed in the patient in spontaneous infection by pyogenic organisms, there was a normal leukocytosis in response to the injection of thyphoid vaccine. The intradermal injection of C-1s, which produces a marked increase in vasopermeability in the skin of normal subjects, produced no definite change in the patient, possibly implicating C3 or a protein in the alternative pathway as the normal mediator of this response. The patient's serum exhibited near-normal immune adherence activity, confirming the lack of requirement of C3 for this function. C5 inactivation and passive hemolysis of unsensitized guinea pig erythrocytes occurred normally in C3-deficient serum on incubation with cobra venom factor, indicating that C3 is not required for these reactions. The patient's humoral antibody response to both protein and carbohydrate antigens was entirely normal, making it unlikely that C3 is required for antigen processing. (+info)
BACKGROUND: Heme oxygenase (HO) is the rate-limiting enzyme in the degradation of heme; its inducible isozyme, HO-1, protects against acute heme protein-induced nephrotoxicity and other forms of acute tissue injury. This study examines the induction of HO-1 in the kidney chronically inflamed by heme proteins and the functional significance of such an induction of HO-1. METHODS: Studies were undertaken in a patient with chronic tubulointerstitial disease in the setting of paroxysmal nocturnal hemoglobinuria (PNH), in a rat model of chronic tubulointerstitial nephropathy caused by repetitive exposure to heme proteins, and in genetically engineered mice deficient in HO-1 (HO-1 -/-) in which hemoglobin was repetitively administered. RESULTS: The kidney in PNH evinces robust induction of HO-1 in renal tubules in the setting of chronic inflammation. The heme protein-enriched urine from this patient, but not urine from a healthy control subject, induced expression of HO-1 in renal tubular epithelial cells (LLC-PK1 cells). A similar induction of HO-1 and related findings are recapitulated in a rat model of chronic inflammation induced by repetitive exposure to heme proteins. Additionally, in the rat, the administration of heme proteins induces monocyte chemoattractant protein (MCP-1). The functional significance of HO-1 so induced was uncovered in the HO-1 knockout mouse: Repeated administration of hemoglobin to HO-1 +/+ and HO-1 -/- mice led to intense interstitial cellular inflammation in HO-1 -/- mice accompanied by striking up-regulation of MCP-1 and activation of one of its stimulators, nuclear factor-kappaB (NF-kappaB). These findings were not observed in similarly treated HO-1 +/+ mice or in vehicle-treated HO-1 -/- and HO-1 +/+ mice. CONCLUSION: We conclude that up-regulation of HO-1 occurs in the kidney in humans and rats repetitively exposed to heme proteins. Such up-regulation represents an anti-inflammatory response since the genetic deficiency of HO-1 markedly increases activation of NF-kappaB, MCP-1 expression, and tubulointerstitial cellular inflammation. (+info)
Post-transfusion purpura: a heterogeneous syndrome.
(24/413)
Three new patients with post-transfusion purpura (PTP) are described. As the manifestations in two differ significantly from those of previously reported cases, they serve to expand the definition of this syndrome. Although all 14 previously reported cases have occurred in Pl-A1-negative females, one of our patients was a Pl-A-negative male. Moreover, a female whose postrecovery platelets possessed the Pl-A1 antigen is described. Antiplatelet antibody activity was detected in all three patients by the 51Cr release test; in contrast, only one reacted in the complement (C) fixation assay. Serum obtained during the acute episode from the PlA1-positive patient reacted against platelets from four of 11 normals by C fixation and against platelets from 48 of 53 normals by 51Cr release, including five of nine Pl-A1-negative platelet samples. This case represents the first instance of PTP in which the platelet isoantibody was not specifically directed against the Pl-A1 antigen. These observations suggest that PTP may be a more heterogeneous disorder than previously realized. (+info)