Biomedical subjects

Neal S Young

Publications and source records attributed to Neal S Young.

81 records · Page 5Linked to original sources

PubMed2002

The relationship of aplastic anemia and PNH.

Bone marrow failure has been regarded as one of the triad of clinical manifestations of paroxysmal noctumal hemoglobinuria (PNH), and PNH in turn has been described as a late clonal disease evolving in patients recovering from aplastic anemia. Better understanding of the pathophysiology of both diseases and improved tests for cell surface glycosylphosphatidylinositol (GPI)-linked proteins has radically altered this view. Flow cytometry of granulocytes shows evidence of an expanded PNH clone in a large proportion of marrow failure patients at the time of presentation: in our large NIH series, about 1/3 of over 200 aplastic anemia cases and almost 20% of more than 100 myelodysplasia cases. Clonal PNH expansion (rather than bone marrow failure) is strongly linked to the histocompatability antigen HLA.-DR2 in all clinical varieties of the disease, suggesting an immune component to its pathophysiology. An extrinsic mechanism of clonal expansion is also more consistent with knock-out mouse models and culture experiments with primary cells and cell lines, which have failed to demonstrate an intrinsic proliferative advantage for PNH cells. DNA chip analysis of multiple paired normal and PIG-A mutant cell lines and lymphoblastoid cells do not show any consistent differences in levels of gene expression. In aplastic anemia/PNH there is surprisingly limited utilization of the V-beta chain of the T cell receptor, and patients' dominant T cell clones, which are functionally inhibitory of autologous hematopoiesis, use identical CDR3 regions for antigen binding. Phenotypically normal cells from PNH patients proliferate more poorly in culture than do the same patient's PNH cells, and the normal cells are damaged as a result of apoptosis and overexpress Fas. Differences in protein degradation might play a dual role in pathophysiology, as GPI-linked proteins lacking an anchor would be predicted to be processed by the proteasome machinery and displayed in a class I H.A. context, in contrast to the normal pathway of cell surface membrane recycling, lysosomal degradation, and presentation by class II HLA. The strong relationship between a chronic, organ-specific immune destructive process and the expansion of a single mutant stem cell clone remains frustratingly enigmatic but likely to be the result of interesting biologic processes, with mechanisms that potentially can be extended to the role of inflammation in producing premalignant syndromes.

Anemia, Aplastic↗

PubMed2002

Immune pathophysiology of aplastic anemia.

Aplastic anemia (AA) remains an elusive disease. Its pathophysiology is not only fascinating by the seemingly simple findings of cytopenia and marrow hypoplasia, but may also contain key information to the understanding of other fundamental processes such as stem cell regeneration, evolution, and immune control of clonal diseases. Although measurements of blood counts provide an objective tool to assess the disease activity and response to the therapy, immune pathophysiology of AA, as inferred from the successes of immunosuppression, provides only few other clinical clues. Similarly, the current laboratory evidence remains mostly indirect. In spite of the recognition of immune pathways of hematopoietic inhibition and apoptosis in AA, the fundamental question about the nature of the antigen(s) inciting or maintaining the pathologic immune response that ultimately leads to bone marrow failure, remains open. However, recognition of the immune targets may aid in understanding not only the pathogenesis but also many of clinical associations and the late squelae of AA. For example, abnormal cells in AA and myelodysplastic syndrome (MDS) MDS may harbor inciting antigens but the immune response lacks selectivity. Clonal selection pressure may be a result of this process or alternatively, emergence of tolerance could lead to the establishment of abnormal hematopoiesis. Clonal proliferation of large granular lymphocytosis could represent an example of an exaggerated response to an immunodominant hematopoietic antigen. In addition to the traditional functional or phenotypic analysis, pathologic immune response in AA can be studied on molecular level by identifying and quantitating T cell clones based on the presence of unique variable B-chain CDR3 sequences. Detection of clonal expansion is based on the observation that in infections and autoimmune conditions, the presence of antigenic drive will lead to the expansion and overrepresentation of T cell clones recognizing this antigen. However, simple analysis of clonal representation is not sufficient to resolve the complex nature of the immune repertoire in the context of genetic and clinical heterogeneity. Therefore, we analyzed VB and CDR3 repertoire in CD4 and CD8 cells, activated or effector cell subsets. To distinguish truly expanded and likely immunodominant clones, we first studied VB distribution and cloned CDR3 sequences from expanded VB families. Identified clonotypic sequences can be used to design molecular tests to quantitate the strength of pathologic immune response. Clonotype sharing has been confirmed in patients with similar clinical features indicating presence of common antigens. In addition, quantitative analysis showed correlation with the therapy response. Persistence and patterns of clonotypes may be helpful in the classification of immune-mediated marrow failure based on the immune characteristics and will allow inferences into the inciting pathways.

Anemia, Aplastic↗

PubMed2002

Superior growth of glycophosphatidy linositol-anchored protein-deficient progenitor cells in vitro is due to the higher apoptotic rate of progenitors with normal phenotype in vivo.

OBJECTIVE: Recently, phenotypically normal CD34 cells from the marrow of patients with paroxysmal nocturnal hemoglobinuria (PNH) were reported to show impaired growth and elevated Fas receptor expression as compared to glycophosphatidylinositol-anchored protein (GPI-AP)-deficient CD34 cells and CD34 cells from normal individuals. These results are consistent with the theory that PNH cells have an intrinsic growth advantage, but their superior expansion in vitro could also be the outcome of selective extrinsic pressure in vivo. MATERIAL AND METHODS: Growth characteristics, competitive features, and susceptibility to apoptosis of sorted normal or GPI-AP-deficient CD34(+) cells derived from PNH patients were assessed in suspension and methylcellulose cultures. RESULTS: When we directly compared the growth of patients' CD34 cells, separated based on expression of GPI-AP CD55 and CD59, in most of the patients studied, mutant CD34 cells showed higher progeny production and outgrew phenotypically normal CD34 cells derived from PNH patients in mixing experiments. However, their proliferation rate did not exceed that of control CD34 cells. To determine whether deficient growth of phenotypically normal CD34 cells in PNH was secondary to a pre-existing in vivo insult, we determined the fraction of apoptotic cells within fresh normal and PNH CD34 cells. Normal CD34 cells from PNH patients showed a high proportion of apoptotic cells and higher Fas expression, while GPI-AP-deficient and control CD34 cells showed similar, low rates of apoptosis. After correction for pre-existing apoptosis, the proliferation potential of normal and PNH CD34 cells was similar. CONCLUSIONS: These results strongly suggest that clonal expansion of GPI-AP-deficient progenitor cells from PNH patients is due to their selection in the hostile marrow environment of the patient.

Adolescent↗

PubMed2002

A pilot study of the recombinant soluble human tumour necrosis factor receptor (p75)-Fc fusion protein in patients with myelodysplastic syndrome.

Laboratory observations suggest that, in some myelodysplastic syndromes (MDS), immune mechanisms may contribute to the impaired blood cell production. Tumor necrosis factor alpha (TNF-alpha), a potent inhibitor of haematopoiesis, has been hypothesized to mediate suppressive effects in MDS: TNF-alpha levels are elevated and correlated with marrow apoptosis and cytopenia. Inhibition of TNF-alpha production using the soluble TNF receptor (Enbrel) has been successful in rheumatoid arthritis, and we have now applied the same principle to MDS. We determined spontaneous TNF-alpha production by marrow cells in MDS; TNF-alpha production was elevated (> mean + 2 x SD of controls) in > 1/3 of patients, but did not correlate with clinical parameters. Sixteen patients participated in a 3-month pilot study of Enbrel. The drug was well tolerated and 15 patients were evaluable. Of these, one became temporarily (14 weeks) transfusion independent. In another patient, absolute neutrophil count (ANC) rose from 0.5 x 10(9)/l to 0.84 x 10(9)/l. Serious infections were seen in two out of six neutropenic patients. Progression to refractory anaemia with excess blasts in transformation (RAEBt) or leukaemia was observed in three patients. When the effects of Enbrel on haematopoietic colony formation were studied, no significant increase was seen in MDS and there was no correlation with TNF-alpha levels. Although anti-TNF therapy with Enbrel was well tolerated at the dosages used in MDS, its efficacy as a single agent appears low.

Adult↗

PubMed2002

Selective reduction of natural killer T cells in the bone marrow of aplastic anaemia.

T cell-mediated suppression of haematopoiesis is believed to play an important role in the pathophysiology of aplastic anaemia (AA) and in the pancytopenia of some myelodysplastic syndromes (MDS). Natural-killer T (NKT) cells belong to a unique lymphocyte subset that expresses an invariant T-cell receptor (TCR), consisting of Valpha24JalphaQ, and common NK cell surface markers. NKT cells have been hypothesized to play a role in immune regulation, and many human autoimmune conditions are associated with NKT cell deficiency. Here we investigate the role of NKT cells in AA and MDS patients. Flow cytometry demonstrated that NKT cells, unlike other T-lymphocyte subpopulations, were disproportionally decreased in AA and MDS marrow. When we compared variability within the CDR3 region of Valpha24 in CD4-CD8- T cells derived from AA and healthy individuals, the CDR3 size of Valpha24 cells showed a polyclonal distribution in AA patients, while in control subjects a typical oligoclonal or monoclonal pattern was found. Southern blot and sequence analysis of Valpha24 polymerase chain reaction products revealed that the NKT cell-specific JalphaQ region was predominant in control subjects, whereas it was not, or only very weakly, detected in AA and MDS patients. These results show that NKT cells are profoundly decreased in AA and MDS, and their deficiency may, as in other human autoimmune diseases, play a role in the local immune dysregulation in AA and MDS.

Adolescent↗

PubMed2002

Myelodysplastic syndromes.

The myelodysplastic syndromes (MDS) are characterized by hemopoietic insufficiency associated with cytopenias leading to serious morbidity plus the additional risk of leukemic transformation. Therapeutic dilemmas exist in MDS because of the disease's multifactorial pathogenetic features, heterogeneous stages, and the patients' generally elderly ages. Underlying the cytopenias and evolutionary potential in MDS are innate stem cell lesions, cellular/cytokine-mediated stromal defects, and immunologic derangements. This article reviews the developing understanding of biologic and molecular lesions in MDS and recently available biospecific drugs that are potentially capable of abrogating these abnormalities. Dr. Peter Greenberg's discussion centers on decision-making approaches for these therapeutic options, considering the patient's clinical factors and risk-based prognostic category. One mechanism underlying the marrow failure present in a portion of MDS patients is immunologic attack on the hemopoietic stem cells. Considerable overlap exists between aplastic anemia, paroxysmal nocturnal hemoglobinuria, and subsets of MDS. Common or intersecting pathophysiologic mechanisms appear to underlie hemopoietic cell destruction and genetic instability, which are characteristic of these diseases. Treatment results and new therapeutic strategies using immune modulation, as well as the role of the immune system in possible mechanisms responsible for genetic instability in MDS, will be the subject of discussion by Dr. Neal Young. A common morphological change found within MDS marrow cells, most sensitively demonstrated by electron microscopy, is the presence of ringed sideroblasts. Such assessment shows that this abnormal mitochondrial iron accumulation is not confined to the refractory anemia with ring sideroblast (RARS) subtype of MDS and may also contribute to numerous underlying MDS pathophysiological processes. Generation of abnormal sideroblast formation appears to be due to malfunction of the mitochondrial respiratory chain, attributable to mutations of mitochondrial DNA, to which aged individuals are most vulnerable. Such dysfunction leads to accumulation of toxic ferric iron in the mitochondrial matrix. Understanding the broad biologic consequences of these derangements is the focus of the discussion by Dr. Norbert Gattermann.

Anemia, Sideroblastic↗

PubMedDate not supplied

In-vivo dominant immune responses in aplastic anaemia: molecular tracking of putatively pathogenetic T-cell clones by TCR beta-CDR3 sequencing.

BACKGROUND: Aplastic anaemia is a bone-marrow-failure syndrome characterised by low blood-cell counts and fatty bone marrow. In most cases, no obvious aetiological factor can be identified. However, clinical responses to immunosuppression strongly suggest an immune pathophysiology. METHODS: To test the hypothesis that aplastic anaemia results from antigen-specific lymphocyte attack against haemopoietic tissue, we analysed effector immunity, seeking especially dominant specific T-cell responses. Blood samples from 54 patients with aplastic anaemia were subjected to flow cytometry to define T-cell-receptor Vbeta-chain usage and expansion of particular Vbeta subsets. We measured the size distribution of the complementarity-determining region 3 (CDR3) for expanded Vbeta subsets, then cloned and sequenced skewed, oligoclonal, or monoclonal peaks. FINDINGS: Expanded Vbeta subsets were identified in almost all the patients. Over-represented Vbeta subsets from CD8-positive cells showed oligoclonal or monoclonal CDR3 size patterns. The CDR3 sequence repertoire in aplastic anaemia showed much redundancy compared with healthy donors. We identified patient-specific putative pathogenetic clonotypes that were not detectable in controls. In selected patients who were assessed longitudinally, these clonotypes were quantitatively related to disease activity. Selective killing of autologous haemopoietic progenitors by the Vbeta-specific lymphocyte population was shown in one patient. These apparently pathogenetic CDR3 sequences showed homology between individuals, suggesting a role for a "semi-public" immune response in the pathophysiology of aplastic anaemia. INTERPRETATION: In-vivo dominant clonal immune response can be identified in many patients with aplastic anaemia, which is evidence for an underlying antigen-driven immune process. Longitudinal tracking by molecular techniques could inform individual clinical decisions and the development of new treatments in autoimmune diseases. RELEVANCE TO PRACTICE: Although the target of the aberrant immune response is the haemopoietic stem cell, the triggering antigens remain unknown. We combined cell phenotypic, molecular biology, and functional analyses to study the effector arm of immunity in an attempt to establish an immune pathophysiology. Clinical application of such a model could broadly extend to other autoimmune diseases.

Anemia, Aplastic↗

PubMedDate not supplied

Genetic and transcriptional analysis of spindle checkpoint genes in bone marrow failure patients.

The evolution of bone marrow failure syndromes such as aplastic anemia (AA) to clonal hematologic diseases such as myelodysplastic syndrome is well recognized. Cytogenetic abnormalities are commonly seen late events, particularly aneuploidy of chromosomes 7 and 8. A proportion of bone marrow failure patients may also develop aneuploidy that is detectable by fluorescence in situ hybridization but not by standard cytogenetic analysis. The molecular basis for aneuploidy in this setting is currently unknown but may include abnormalities in the mitotic spindle checkpoint. For this reason, we searched for mutations in the mitotic spindle checkpoint genes hBUB1 and hMAD2, and also examined the expression of hBUB1 in cells of bone marrow failure patients. No pathogenic mutations were found in 59 patients. Of 170 bone marrow failure patients, less than one-third expressed hBUB1 transcript. Gene expression profiling confirmed a significant down-regulation of hBUB1 message in patients. We conclude that mutations in mitotic spindle checkpoint genes do not account for aneuploidy in marrow failure states. However, we cannot exclude epigenetic inactivation of hBUB1 as a potential mechanism in some patients.

Aneuploidy↗

PubMedDate not supplied

Recombinant viral-like particles of parvovirus B19 as antigen carriers of anthrax protective antigen.

Viral-like particles (VLPs) of parvovirus B19 were employed as an antigen carrier to present antigenic determinants of Bacillus anthracis. The small-loop peptide and the full-length domain 4 of protective antigen (PA) were chosen as immunogens for presentation on the VLP-capsid surface and subsequent immunization of BALB/c mice. The recombinant VLPs induced anti-PA IgG titers of up to 2.5 x 10(4). Neutralization assays showed that the recombinant VLPs elicited neutralizing anti-PA antibody titers of up to 1:400 and showed potential for the prevention of lethal toxin-induced mortality of mouse-macrophage cells (RAW264.7). In postimmune sera, no anti-PA titers were detected against synthetic small-loop peptide. Recombinant VLPs demonstrated the capacity to retain the immunogenicity of the displayed microbial PA-epitopes and elicited robust levels of anti-PA antibody titers. These findings suggest that the recombinant VLPs of parvovirus B19 have potential as an additional tool in the development of sub-unit vaccines.

Amino Acid Sequence↗