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For the 1319 parvovirus P6 promoter, a 96-nt minimal truncation mutant retained activity in transient reporter gene assays. Deletion of sequences further upstream from this minimal promoter markedly diminished reporter activity in certain cell lines. This upstream region lies within the terminal hairpin from −49 to −157 and contains a 14-nt sequence that is protected by DNase I footprinting. The exact sequence is directly repeated further within the hairpin, suggesting a regulatory role. The hairpin termini of parvoviruses were known to serve as origins of replication and to catalyze virion packaging. We now suggest that, in addition to these functions, they exert cis-acting effects on B19 P6-promoted gene expression.

Perforin is a cytolytic protein expressed mainly in activated cytotoxic T lymphocytes (CTL) and natural killer (NK) cells. In T and NK cells perforin is stored in cytoplasmic granules and is essential for killing via non-Fas-mediated mechanisms. Perforin regulates the translocation of granzyme B from cytotoxic cells into target cells; after entering the target-cell granzyme B migrates to the target cell nucleus to participate in triggering apoptosis. Functional perforin is essential for normal CTL and NK cell function; without perforin CTL and NK cells show reduced or no cytolytic effect. Inherited perforin mutations account for 20–40% of familial hemophagocytic lymphohistiocytosis, an autosomal recessive fatal disease of early childhood characterized by uncontrolled accumulation of activated T cells and macrophages in many organs, increased Th1 cytokines and absent functional perforin. Acquired aplastic anemia (AA), the paradigm of immune mediated bone marrow failure syndromes, is characterized by hematopoietic stem cell destruction by activated T cells and Th1 cytokines. We examined whether mutations in Prf1 occur in AA; peripheral blood DNA samples from 75 patients and 302 controls were analyzed. Three novel nonsynonymous Prf1 mutations among five unrelated patients (ages: 21, 31, 33, 75, and 77 years old), not present in controls, were discovered; two polymorphisms were also identified (H300H, A274A). The mutations were in the coding region of Prf1 gene. In exon 2, arginine was replaced by histidine in one patient (CGT/CAT, R4H) and in 3 patients the same A91V mutation was identified (GCG/GTG, alanine to valine substitution). In exon 3, serine was replaced by isoleucine (S388I; AGC/ATC) in one patient. Germ-line origin of the Prf1 mutations was established by their presence also in DNA from buccal mucosa obtained from affected AA patients. Four of five patients with mutations showed some hemophagocytosis in the bone marrow examination when first diagnosed, but there were no other typical features of hemophagocytic syndrome such as hepatosplenomegaly or altered liver function tests. None of the patients with Prf1 mutations experienced hematologic recovery with immunosuppressive treatment. Perforin protein levels in all patients carrying mutations were very low or absent. By confocal microscopy, CD8 cells from patients with Prf1 mutations had complete absence of perforin granules (perforin and cathepsin D showed the expected pattern of co-localization in controls’ cytotoxic granules). NK cell killing efficiency from patients carrying mutations in a standard Cr51-release cytolytic assay was significantly decreased compared to controls. Prf1 gene mutations may be related to a more severe phenotype of AA associated with marrow hemophagocytosis and failure to respond to immunosuppression. Mutations in the immune regulatory mechanisms identified in young children can manifest in adults without typically associated clinical findings or a suggestive family history. Mechanistically, Prf1 gene mutations help explain the aberrant proliferation and activation of cytotoxic T cells that are destructive of hematopoietic stem cells in AA and may be useful as predictive factors for responses to immunosuppressive treatments and the decision to rapidly undertake stem cell replacement. Prf1 gene mutations are genetic risk factors for bone marrow failure syndromes.

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired hematopoietic stem-cell disorder in which the affected cells are deficient in glycosylphosphatidylinositol (GPI)-anchored proteins. Paroxysmal nocturnal hemoglobinuria is frequently associated with aplastic anemia, although the basis of this relation is unknown.To assess the PNH status of patients with diverse marrow failure syndromes.Correlation of cytofluorometric data with clinical features.Hematology Branch, National Heart, Lung, and Blood Institute, Bethesda, Maryland.115 patients with aplastic anemia, 39 patients with myelodysplasia, 28 patients who had recently undergone bone marrow transplantation, 18 patients with cancer that was treated with chemotherapy, 13 patients with large granular lymphocytosis, 20 controls who had received renal allografts, and 21 healthy participants.Patients with aplastic anemia, myelodysplasia, or renal allografts received antithymocyte globulin.Flow cytometry was used to assess expression of GPI-anchored proteins on granulocytes.Evidence of PNH was found in 25 of 115 (22%) patients with aplastic anemia. No patient with normal GPI-anchored protein expression at presentation developed PNH after therapy (n = 16). Nine of 39 (23%) patients with myelodysplasia had GPI-anchored protein-deficient cells. Abnormal cells were not detected in patients with constitutional or other forms of bone marrow failure or in renal allograft recipients who had received antithymocyte globulin. Aplastic anemia is known to respond to immunosuppressive therapy; in myelodysplasia, the presence of a PNH population was strongly correlated with hematologic improvement after administration of antithymocyte globulin (P = 0.0015).Flow cytometric analysis is superior to the Ham test and permits concomitant diagnosis of PNH in about 20% of patients with myelodysplasia (a rate similar to that seen in patients with aplastic anemia). The presence of GPI-anchored protein-deficient cells in myelodysplasia predicts responsiveness to immunosuppressive therapy. Early emergence of GPI-anchored protein-deficient hematopoiesis in a patient with marrow failure may point to an underlying immune pathogenesis.

Parvovirus B19 (B19) DNA was detected by dot blot hybridization in sera from 5 (17%) of 30 human immunodeficiency virus (HIV)-infected patients with hematocrits (HCT) ofor =24 and 4 (31%) of 13 HRV-infected patients with HCT ofor =20, suggesting that B19 is a reasonably common cause of severe anemia in HIV infection. The anemia promptly remitted after immunoglobulin therapy in 3 of 4 treated patients. The presence of IgM to B19, the clinical circumstance in which anemia developed, and the marrow morphology were poor predictors of chronic B19 infection. DNA hybridization studies of sera from 191 HIV-infected and 117 HIV-seronegative homosexual males attending a clinic in the Seattle area revealed that 1 (0.5%) and 2 (2%) samples, respectively, from the 2 groups contained B19. However, when assayed by polymerase chain reaction (PCR), 5% of the serum samples from HIV-infected persons and 9% from uninfected persons contained B19, although each had an HCT ofor =40. The data argue that anemia results from chronic high-titer B19 infection. Although a negative PCR assay excludes this diagnosis, DNA hybridization may be the more specific serum test.

We recently reported the identification of a novel simian parvovirus in cynomolgus monkeys with severe anemia. We now describe the cloning and sequencing of 4986 nucleotides of the viral DNA. Like the human parvovirus B19, simian parvovirus encapsidates both positive and negative single-stranded DNA. The positive strand contains two large open reading frames, with the left open reading frame encoding the nonstructural protein(s) and the right reading frame encoding the two capsid proteins. Simian parvovirus has little homology with the autonomous parvoviruses or the dependovirus AAV-2 but 50% overall homology with parvovirus B19 DNA. At the amino acid level there was 70% homology with B19 capsid proteins and 50% homology with B19 nonstructural protein. Based on this genetic similarity and with the known tropism of the virus for cynomolgus erythroid precursors, we suggest that this new virus should be classified as a new member of the Erythrovirus genus of the Parvoviridae.

We report three children with congenital anaemia after intrauterine infection with B19 parvovirus. All the fetuses developed hydrops fetalis that was treated by blood transfusion. After delivery the infants had hypogammaglobulinaemia. In all three, sera lacked B19 but viral DNA was found in bone marrow. All were treated with immunoglobulin. One child died and B19 was found in various tissues. In the other two cases, virus could no longer be detected after therapy but the patients remain persistently anaemic. Persistent B19 infection should be suspected in infants with congenital red-cell aplasia.

Fanconi anaemia (FA) is a rare genetic disorder that predisposes to the development of aplastic anaemia and neoplasia. The pathophysiologic hallmark of FA is increased susceptibility to chromosomal breakage. Superoxide metabolism has also been shown to be involved in the cellular pathophysiology of FA. Human SOD (rh-SOD), an enzyme which dismutates superoxide, has recently been cloned and expressed in yeast. We treated four FA patients with a 2-week infusion of rh-SOD (25 mg/kg d daily) to determine whether rh-SOD had any effect on haemopoietic progenitor cell growth or on the abnormal cellular phenotype. We found that lymphocyte chromosomal aberrations induced by diepoxybutane were decreased during rh-SOD treatment in two patients and that bone marrow progenitors were increased in one patient.

B19 parvovirus is absolutely tropic for human erythroid progenitor cells. Among the untested mechanisms underlying this tropism is the possibility of cell-specific positive regulation of the promoter in permissive cells. Using the bacterial chloramphenicol acetyltransferase and firefly luciferase reporter genes, we detected strong activity from the B19 P6 promoter in transfected nonpermissive cells. Very high-level expression was seen in a T lymphoblastoid cell line, CEM. No transcriptional enhancement occurred in an erythropoietin-dependent semipermissive cell line. A putative second B19 promoter at map unit 44 (1344) was nonfunctional and unable to confer tissue specificity. Thus, tropism is unlikely to be regulated at the level of transcriptional initiation from either the P6 or P44 promoter.