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N S Young

Publications and source records attributed to N S Young.

At least 19 recordsLinked to original sources
PubMed1995

Hematopoietic inhibition by interferon-gamma is partially mediated through interferon regulatory factor-1.

Biologic responses to cytokines are mediated by intracellular pathways involving induction of signaling and metabolic cascades. Interferon (IFN) regulatory factor-1 (IRF-1) is a major transcription factor induced not only by IFN-gamma but also by other cytokines including tumor necrosis factor-alpha (TNF-alpha). Possible IRF-1 binding sequence elements have been located in the promoter regions of several genes, including p53, inducible nitric oxide synthase, and cyclin D1. IFN-gamma and TNF-alpha can inhibit hematopoiesis in vitro and have been implicated in the pathophysiology of bone marrow (BM) failure. We investigated whether the inhibitory effects of these cytokines were intracellularly mediated through the expression of IRF-1 or -2 in target cells. In total BM cells, IRF-1 mRNA expression increased after stimulation with IFN-gamma and TNF-alpha; the stronger effect was observed with IFN-gamma. In contrast, IRF-2 mRNA expression was constitutive and not altered by cytokine stimulation. By gene amplification, low levels of IRF-1 mRNA were present in unstimulated, highly purified CD34+ cells; on exposure to IFN-gamma and TNF-alpha, amplified IRF-1 mRNA showed a much stronger signal than control. When CD34+ cells were treated with IFN-gamma and TNF-alpha, IRF-1 antisense oligodeoxynucleotide (ODN) partially reversed the suppressive effects on CD34+ cell-derived colony formation by IFN-gamma but not those by TNF-alpha. In parallel experiments, IRF-1 antisense ODN decreased both IRF-1 protein and mRNA expression. The effects of ODN were sequence-specific and concentration-dependent. These results suggest that the inhibitory hematopoietic effects of IFN-gamma and TNF-alpha are mediated by different pathways. For IFN-gamma, IRF-1 is involved in the activation of cellular genes responsible for IFN-gamma suppressive effects.

Antigens, CD34↗
PubMed1995

Most of the VP1 unique region of B19 parvovirus is on the capsid surface.

B19 parvovirus is pathogenic in man and a vaccine is desirable. In convalescence after acute infection, the dominant humoral immune response is directed to the minor capsid protein called VP1, which differs from the major capsid protein by an additional NH2-terminal 227 amino acids. We have previously shown that this unique region contains multiple linear neutralizing epitopes. We produced seven recombinant B19 capsids that contained progressively truncated VP1 unique region sequences, each fused to a Flag peptide (AspTyrLysAspAspAspAspLys) at the NH2-terminus. Capsids containing normal VP2 and truncated Flag-VP1 proteins and, in some cases, only truncated Flag-VP1 chimeric proteins, were analyzed by ELISA, affinity chromatography, and electron microscopy using anti-Flag monoclonal antibody. All regions examined showed binding to anti-Flag antibody in multiple assays, indicating that most of the VP1 unique region is external to the capsid and accessible to antibody binding. These results have implications for the design of a B19 parvovirus vaccine and the use of empty capsids for presentation of heterologous protein antigens.

Amino Acid Sequence↗
PubMed1995

Aplastic anaemia.

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Anemia, Aplastic↗
PubMed1995

Goose parvovirus--an autonomous member of the dependovirus genus?

Goose parvovirus is the etiological agent of Derzsy's disease, a fatal hepatitis of young geese. The virus infects geese and Muscovy ducks and can be propagated in the laboratory in primary embryonic goose fibroblasts. To date the virus has only been classified by morphological, biochemical, and culture characteristics as an autonomous parvovirus. We now report the cloning and partial sequencing of 3434 nucleotides of the viral genome. Three overlapping clones were obtained, encoding regions in the nonstructural and capsid coding region. The nucleotide sequence show little homology to other autonomous parvoviruses but 55% homology to the dependovirus AAV2. The homology to AAV2 was also confirmed at the amino acid level (nonstructural protein 55%, capsid coding region 51%). DNA cross hybridization studies indicate an even closer similarity of goose parvovirus to the yet unsequenced human dependoviruses AAV1 and AAV3 than to AAV2. These findings suggest that goose parvovirus may be genetically related to the dependovirus genus rather than to the other autonomous parvoviruses.

Amino Acid Sequence↗
PubMed1995

Cloning and sequencing of the simian parvovirus genome.

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.

Amino Acid Sequence↗
PubMed1995

Fas antigen expression on CD34+ human marrow cells is induced by interferon gamma and tumor necrosis factor alpha and potentiates cytokine-mediated hematopoietic suppression in vitro.

Activation of Fas antigen, a cell surface receptor molecule, by its ligand results in transduction of a signal for cell death. The Fas system has been implicated in target cell recognition, clonal development of immune effector cells, and termination of the cellular immune response. Fas antigen expression on lymphocytes is regulated by interferon gamma (IFN gamma) and tumor necrosis factor alpha (TNF alpha), cytokines that also have inhibitory effects on hematopoiesis. We investigated Fas antigen expression on human marrow cells and the effects of Fas activation on hematopoiesis in vitro. Freshly isolated immature hematopoietic cells, as defined by the CD34 marker, did not express Fas antigen at levels detectable by fluorescent staining. CD34+ cells, which include progenitors and stem cells, showed low levels of Fas expression in culture, even in the presence of growth factors. Stimulation by TNF alpha and IFN gamma markedly increased Fas antigen expression on CD34+ cells. Anti-Fas antibody, which mimics the action of the putative ligand, enhanced IFN gamma- and TNF alpha-mediated suppression of colony formation by bone marrow (BM) in a dose-dependent manner. This effect did not require the presence of accessory cells. Colony formation from mature (CD34+ CD38+) and immature (CD34+ CD38-) progenitor cells and long-term culture initiating cells were susceptible to the inhibitory action of anti-Fas antibody in the presence of IFN gamma and TNF alpha. Apoptosis assays performed on total BM cells and CD34+ cells showed that anti-Fas antibody induced programmed cell death of CD34+ BM cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Antigens, CD↗
PubMed1995

Intensive immunosuppression with antithymocyte globulin and cyclosporine as treatment for severe acquired aplastic anemia.

Immunosuppressive therapy can produce hematologic improvement in a large proportion of patients with severe aplastic anemia. Antithymocyte globulin (ATG) is the current treatment of choice for patients who do not have histocompatible sibling donors or who are otherwise inegligible for allogeneic bone marrow transplantation. About 50% of patients respond to an initial course of ATG, and many nonresponders can be salvaged by subsequent treatment with cyclosporine (CsA). To determine whether simultaneous administration of these agents could further improve response rates, we enrolled 55 patients in a therapeutic trial of 4 days of ATG and 6 months of CsA. Among the 51 patients who had not received previous courses of ATG or CsA, 67% had responded by 3 months, and 78% had responded by 1 year (response was defined as an increase in peripheral blood counts sufficient that a patient no longer met the criteria for severe disease). There was a high incidence of relapse (36% actuarial risk at 2 years), but most relapsed patients responded to additional courses of immunosuppression, and relapse was not associated with a significant survival disadvantage. Evolution to myelodysplastic syndromes and acute leukemia was rare (1 of 51 patients), but the later appearance of paroxysmal nocturnal hemoglobinuria was more common (5 of 51 patients). Actuarial survival was 86% at 1 year and 72% at 2 years. These data support the use of a combination immunosuppressive regimen containing both ATG and CsA as first-line therapy for severe aplastic anemia.

Adolescent↗
PubMed1995

Peptides derived from the unique region of B19 parvovirus minor capsid protein elicit neutralizing antibodies in rabbits.

B19 parvovirus is pathogenic in humans. The virus propagates in the bone marrow, where it is cytotoxic to erythroid progenitor cells. Antibodies appear in blood after infection and neutralize virus in vitro; infection appears to confer lasting immunity. The predominant immune response on immunoblot is to the minor capsid protein (VP1), which differs from the major capsid protein (VP2) by an additional 227 amino acids. We previously demonstrated that antisera directed to a fusion protein containing this unique region or to more limited fusion peptides of 50-100 amino acids each neutralized virus. In the current work, we tested synthetic peptides of about 20 amino acids derived from the VP1 unique region for their ability to elicit a neutralizing antibody response in rabbits. Individual peptides were covalently linked to a lysine core to produce a multivalent antigen. Animals produced antibodies to all 13 synthetic peptides, as determined by ELISA. At 12 weeks, animals injected with one of three peptides--two from the far amino terminus and the third from the center of the unique region--had produced antibodies that completely neutralized virus; by 16 weeks, antisera elicited with another four peptides also were effective. In summary, we identified regions containing neutralizing epitopes within the first 80 amino acids and amino acids 148-205 of the unique region. Our data suggest that synthetic peptides might be useful vaccine reagents for protection against parvovirus infection in humans.

Amino Acid Sequence↗
PubMed1995

Interferon-gamma and tumor necrosis factor-alpha suppress both early and late stages of hematopoiesis and induce programmed cell death.

Increased expression of interferon gamma (IFN-gamma) and tumor necrosis factor alpha (TNF-alpha) in bone marrow failure disorders suggests a possible pathophysiologic role of these cytokines in disease. In this study, we tested the action of TNF-alpha and IFN-gamma on phenotypically and functionally defined stages of hematopoietic development using highly purified progenitor cell populations assayed in standardized culture systems. We hypothesized that the inhibitory effects of IFN-gamma and TNF-alpha might be related to the induction of programmed cell death. In methylcellulose colony assays, IFN-gamma and TNF-alpha inhibited the growth of early hematopoietic cells, including committed CD34+CD38+ progenitor cells and phenotypically less mature CD34+CD38- cells, with 50% decreased colony formation occurring in the range of 750-1,000 U/ml of IFN-gamma and 10-15 ng/ml of TNF-alpha. More potent suppressive effects were observed in cultures supplemented with the combination of both cytokines than in cultures treated with IFN-gamma or TNF-alpha alone. When used at these concentrations, IFN-gamma and TNF-alpha inhibited growth of CD34(+)-enriched long-term culture-initiating cells by 88% and 68%, respectively. IFN-gamma and TNF-alpha triggered apoptosis of total bone marrow and CD34+ cells, recognized by the presence of a characteristic pattern of DNA degradation after low molecular weight DNA extraction, and by detection of apoptotic cells by the in situ terminal deoxynucleotidyl transferase assay. We speculate that chronic exposure of hematopoietic tissue to TNF-alpha and IFN-gamma in vivo may result in broad depletion of the stem and progenitor cell pools. Death of these cells due to apoptosis rather than transient inhibition of proliferation may be responsible for long-lasting hematologic consequences.

ADP-ribosyl Cyclase↗
PubMed1995

Purification of functional recombinant YY1 expressed in Sf9 insect cells.

We expressed a gene for YY1, the multifunctional mammalian transcription factor, with a recombinant baculovirus in insect cells and obtained high levels of protein in cell lysates. A simple and efficient purification method was developed. In the final step, we used DNA affinity chromatography with concatermerized sequence derived from the upstream region of the B19 parvovirus P6 promoter, which has strong affinity for YY1. Approximately 1.8 mg of highly purified YY1 was obtained from 10(9) Sf9 cells. Highly purified YY1 behaved as authentic YY1 on electrophoretic mobility shift assays and in enhancing transcription from the P6 promoter. YY1 produced in a baculovirus system should prove useful for in vitro transcription assays of YY1 and related regulatory proteins.

Animals↗
PubMed1995

Viruses, virulence and pathogenicity.

Pathogenicity is a complex process with stringent requirements of both the host cell and the infecting virion. Among these requirements are a port of entry into host cells, a means of replication for the virus, and a means by which infection damages host cells. Damage to the host can result from multiple mechanisms including transformation, suppression of cellular metabolism, apoptosis, autoimmune responses directed against infected or uninfected tissues, or by molecular mimicry. In the attempt to identify new associations between viral infection and disease, investigators should be mindful that variable host factors as well as viral infection may be required for pathogenesis. Efforts to associate specific viral infections with specific diseases may be obscured by final common pathways through which multiple agents damage host cells in similar ways.

Animals↗
PubMed1995

B19 parvovirus.

B19 parvovirus is pathogenic in man and causes a variety of clinical illnesses, among them several haematological diseases. Acute infection of a host with underlying haemolysis produces transient aplastic crisis; of the midtrimester fetus, hydrops fetalis; and of an immunocompromised patient, pure red cell aplasia. The target of B19 parvovirus infection is the human erythroid progenitor cell. Infection is cytotoxic due to expression of the viral nonstructural protein. The virus can be propagated in cultures of human bone marrow, blood, and fetal liver. Humoral immunity normally terminates infection, and commercially available immunoglobulin can be used to treat persistent infection. Recombinant capsids, produced in a baculovirus system, are suitable as a vaccine reagent.

Adult↗
PubMed1995

Intracellular expression of antibody fragments directed against HIV reverse transcriptase prevents HIV infection in vitro.

We have tested a novel strategy of intracellular immunization to block human immunodeficiency virus (HIV) infection. The expression of a specific antibody within a cell was achieved by transduction of genes that encode for immunoglobulin chains with specificity to viral reverse transcriptase. We demonstrated that inhibition of this enzyme makes cells resistant to HIV infection by blocking an early stage of viral replication. If high efficiency transduction with a stable vector into lymphohaematopoietic stem cells or mature lymphocytes can be achieved, gene transfer-mediated intracellular immunization might be a feasible treatment strategy in AIDS.

Genetic Therapy↗
PubMed1995

Increased expression of Fas antigen on bone marrow CD34+ cells of patients with aplastic anaemia.

Fas antigen, a receptor molecule that mediates signals for programmed cell death, is involved in T-cell-mediated killing of malignant, virus-infected or allogeneic target cells. Interferon-gamma (IFN-gamma) and tumour necrosis factor-alpha (TNF-alpha), potent inhibitors of haemopoiesis, enhance Fas receptor expression on bone marrow (BM) CD34+ cells, and both cytokines render haemopoietic progenitor cells susceptible to Fas-mediated inhibition of colony formation due to the induction of apoptosis. Haemopoietic suppression in aplastic anaemia (AA) has been associated with aberrant IFN-gamma, increased TNF-beta expression, and elevated numbers of activated cytotoxic T-cells in marrow. We have now examined Fas antigen expression in fresh AA BM samples. In normal individuals few CD34+ cells expressed Fas antigen and normal marrow cells had low sensitivity to Fas-mediated inhibition of colony formation. In contrast, in early AA, BM CD34+ cells showed markedly increased percentages of Fas receptor-expressing CD34+ cells, which correlated with increased sensitivity of AA marrow cells to anti-Fas antibody-mediated inhibition of colony formation. The proportion of Fas antigen-bearing cells was lower in recovered patients' BM. Fas antigen was also detected in the marrow of some patients with myelodysplasia, especially the hypocellular variant. These results are consistent with the hypothesis that AA CD34+ cells, probably including haemopoietic progenitor cells, express high levels of Fas receptor due to in vivo exposure to IFN-gamma and/or TNF-alpha and are suitable targets for T-cell-mediated killing. Our results suggest that the Fas receptor/Fas ligand system are involved in the pathophysiology of BM failure.

Adult↗
PubMed1995

Modest truncation of the major capsid protein abrogates B19 parvovirus capsid formation.

In vitro studies have suggested an important role for the minor capsid protein (VP1) unique region and the junction between VP1 and the major capsid protein (VP2) in the neutralizing immune response to B19 parvovirus. We investigated the role of the NH2-terminal region of the major structural protein in capsid structure by expressing progressively more truncated versions of the VP2 gene followed by analysis using immunoblotting and electron microscopy of density gradient-purified particles. Deletion of the first 25 amino acids (aa) of VP2 did not affect capsid assembly. Altered VP2 with truncations to aa 26 to 30, including a single amino acid deletion at position 25, failed to self-assemble but did participate with normal VP2 in the capsid structure. The altered region corresponds to the beginning of the beta A antiparallel strand. Truncations beyond aa 30 were incompatible with either self-assembly or coassembly, probably because of deletion of the beta B strand, which helps to form the core structure of the virus.

Amino Acid Sequence↗
PubMed1995

Human parvovirus B19 can infect cynomolgus monkey marrow cells in tissue culture.

The human pathogenic parvovirus B19 cannot be grown in standard tissue culture but propagates in human bone marrow, where it is cytotoxic to erythroid progenitor cells. We now show that parvovirus B19 can replicate in cynomolgus bone marrow. Cynomolgus monkeys may be a suitable animal model for pathogenesis studies of parvovirus B19.

Animals↗