Biomedical subjects

Neal S Young

Publications and source records attributed to Neal S Young.

At least 19 recordsLinked to original sources

PubMed2021

Somatic Mutations in UBA1 Define a Distinct Subset of Relapsing Polychondritis Patients With VEXAS.

OBJECTIVE: Somatic mutations in UBA1 cause a newly defined syndrome known as VEXAS (vacuoles, E1 enzyme, X-linked, autoinflammatory, somatic syndrome). More than 50% of patients currently identified as having VEXAS met diagnostic criteria for relapsing polychondritis (RP), but clinical features that characterize VEXAS within a cohort of patients with RP have not been defined. We undertook this study to define the prevalence of somatic mutations in UBA1 in patients with RP and to create an algorithm to identify patients with genetically confirmed VEXAS among those with RP. METHODS: Exome and targeted sequencing of UBA1 was performed in a prospective observational cohort of patients with RP. Clinical and immunologic characteristics of patients with RP were compared based on the presence or absence of UBA1 mutations. The random forest method was used to derive a clinical algorithm to identify patients with UBA1 mutations. RESULTS: Seven of 92 patients with RP (7.6%) had UBA1 mutations (referred to here as VEXAS-RP). Patients with VEXAS-RP were all male, were on average ≥45 years of age at disease onset, and commonly had fever, ear chondritis, skin involvement, deep vein thrombosis, and pulmonary infiltrates. No patient with VEXAS-RP had chondritis of the airways or costochondritis. Mortality was greater in VEXAS-RP than in RP (23% versus 4%; P = 0.029). Elevated acute-phase reactants and hematologic abnormalities (e.g., macrocytic anemia, thrombocytopenia, lymphopenia, multiple myeloma, myelodysplastic syndrome) were prevalent in VEXAS-RP. A decision tree algorithm based on male sex, a mean corpuscular volume >100 fl, and a platelet count <200 ×103 /μl differentiated VEXAS-RP from RP with 100% sensitivity and 96% specificity. CONCLUSION: Mutations in UBA1 were causal for disease in a subset of patients with RP. This subset of patients was defined by disease onset in the fifth decade of life or later, male sex, ear/nose chondritis, and hematologic abnormalities. Early identification is important in VEXAS given the associated high mortality rate.

Aged↗

PubMed2007

PTPN22 620W allele is not associated with aplastic anemia.

The 1858C/T variant in PTPN22 imparts a gain of function mutation dysregulating T-cell stimulation and is associated with an array of autoimmune diseases. Using a case-control design, we compared the frequency of this polymorphism in 91 patients with acquired aplastic anemia to 132 ethnically matched controls. Representation of the PTPN22 variant was not significantly different between the two populations, suggesting that this gene polymorphism does not contribute to the etiology of aplastic anemia. Aplastic anemia thus joins a list of autoimmune diseases that commonly lack a major humoral disease component and do not associate with the PTPN22 variant.

Adolescent↗

PubMed2006

Age-dependent accumulation of mtDNA mutations in murine hematopoietic stem cells is modulated by the nuclear genetic background.

Alterations in mitochondrial DNA (mtDNA) and consequent loss of mitochondrial function underlie the mitochondrial theory of aging. In this study, we systematically analyzed the mtDNA control region somatic mutation pattern in 2864 single hematopoietic stem cells (HSCs) and progenitors, isolated by flow cytometry sorting on Lin(-)Kit(+)CD34(-) parameters from young and old C57BL/6 (B6) and BALB/cBy (BALB) mice, to test the hypothesis that the accumulated mtDNA mutations in HSCs were strain-correlated and associated with HSC functional senescence during aging. An increased level of mtDNA mutations in single HSCs was observed in old B6 when compared with young B6 mice (P=0.003); in contrast, no significant age-dependent accumulation of mutations was observed in BALB mice (old versus young, P=0.202) and the level of mutations in both young and old BALB mice was close to that of old B6 mice (P>0.280). Cellular reactive oxygen species (ROS) in mouse HSCs could not be correlated with the level of mtDNA mutations in these cells, although B6 mice had a higher proportion of ROS(-) cells when compared with the BALB mice. Propagation assays of single HSCs showed B6 cells form larger colonies compared with cells from BALB mice, irrespective of age and mtDNA mutation load. We infer from our data that age-related mtDNA somatic mutation accumulation in mouse HSCs is influenced by the nuclear genetic background and that these mutations may not obviously correlate to either cellular ROS content or HSC senescence.

Aging↗

PubMed2006

Distinct EBV and CMV reactivation patterns following antibody-based immunosuppressive regimens in patients with severe aplastic anemia.

The natural history of EBV and CMV reactivation and the potential for serious complications following antibody-based immunosuppressive treatment for bone marrow failure syndromes in the absence of transplantation is not known. We monitored blood for EBV and CMV reactivation by polymerase chain reaction (PCR) weekly in 78 consecutive patients (total of 99 immunosuppressive courses) with aplastic anemia. Four regimens were studied: (1) HC, horse ATG/cyclosporine; (2) HCS, horse ATG/CsA/sirolimus; (3) RC, rabbit ATG/CsA; and (4) CP, alemtuzumab. There were no cases of EBV or CMV disease, but EBV reactivation occurred in 82 (87%) of 94 and CMV reactivation in 19 (33%) of 57 seropositive patients after starting immunosuppression. The median peak EBV copies were higher in the RC group when compared with HC, HCS, and alemtuzumab (P < .001). The median duration of PCR positivity for EBV was higher in the RC group compared with HC, HCS, and alemtuzumab (P = .001). Subclinical reactivation of both EBV and CMV is common and nearly always self-limited in patients with bone marrow failure receiving immunosuppression; different regimens are associated with different intensity of immunosuppression as measured by viral load and lymphocyte count; and viral reactivation patterns differ according to immunosuppressive regimens.

Adolescent↗

PubMed2006

CD34 cells from patients with trisomy 8 myelodysplastic syndrome (MDS) express early apoptotic markers but avoid programmed cell death by up-regulation of antiapoptotic proteins.

CD34 cells from patients with trisomy 8 myelodysplastic syndrome (MDS) are distinguished from other MDS cells and from normal hematopoietic cells by their pronounced expression of apoptotic markers. Paradoxically, trisomy 8 clones can persist in patients with bone marrow failure and expand following immunosuppression. We previously demonstrated up-regulation of c-myc and CD1 by microarray analysis. Here, we confirmed these findings by real-time polymerase chain reaction (PCR), demonstrated up-regulation of survivin, c-myc, and CD1 protein expression, and documented comparable colony formation by annexin(+) trisomy 8(-) CD34(+) and annexin(-) CD34 cells. There were low levels of DNA degradation in annexin(+) trisomy 8 CD34 cells, which were comparable with annexin(-) CD34 cells. Trisomy 8 cells were resistant to apoptosis induced by gamma irradiation. Knock-down of survivin by siRNA resulted in preferential loss of trisomy 8 cells. These results suggest that trisomy 8 cells undergo incomplete apoptosis and are nonetheless capable of colony formation and growth.

Annexins↗

PubMed2006

The complement inhibitor eculizumab in paroxysmal nocturnal hemoglobinuria.

BACKGROUND: We tested the safety and efficacy of eculizumab, a humanized monoclonal antibody against terminal complement protein C5 that inhibits terminal complement activation, in patients with paroxysmal nocturnal hemoglobinuria (PNH). METHODS: We conducted a double-blind, randomized, placebo-controlled, multicenter, phase 3 trial. Patients received either placebo or eculizumab intravenously; eculizumab was given at a dose of 600 mg weekly for 4 weeks, followed 1 week later by a 900-mg dose and then 900 mg every other week through week 26. The two primary end points were the stabilization of hemoglobin levels and the number of units of packed red cells transfused. Biochemical indicators of intravascular hemolysis and the patients' quality of life were also assessed. RESULTS: Eighty-seven patients underwent randomization. Stabilization of hemoglobin levels in the absence of transfusions was achieved in 49% (21 of 43) of the patients assigned to eculizumab and none (0 of 44) of those assigned to placebo (P<0.001). During the study, a median of 0 units of packed red cells was administered in the eculizumab group, as compared with 10 units in the placebo group (P<0.001). Eculizumab reduced intravascular hemolysis, as shown by the 85.8% lower median area under the curve for lactate dehydrogenase plotted against time (in days) in the eculizumab group, as compared with the placebo group (58,587 vs. 411,822 U per liter; P<0.001). Clinically significant improvements were also found in the quality of life, as measured by scores on the Functional Assessment of Chronic Illness Therapy-Fatigue instrument (P<0.001) and the European Organization for Research and Treatment of Cancer Quality of Life Questionnaire. Of the 87 patients, 4 in the eculizumab group and 9 in the placebo group had serious adverse events, none of which were considered to be treatment-related; all these patients recovered without sequelae. CONCLUSIONS: Eculizumab is an effective therapy for PNH.

Adolescent↗

PubMed2006

Functional characterization of natural telomerase mutations found in patients with hematologic disorders.

Human telomerase hTERC RNA serves as a template for the catalytic hTERT protein to synthesize telomere repeats at chromosome ends. We have recently shown that some patients with bone marrow failure syndromes are heterozygous carriers for hTERC or hTERT mutations. These sequence variations usually lead to a compromised telomerase function by haploinsufficiency. Here, we provide functional characterization of an additional 8 distinct hTERT sequence variants and 5 hTERC variants that have recently been identified in patients with dyskeratosis congenita (DC) or aplastic anemia (AA). Among the mutations, 2 are novel telomerase variants that were identified in our cohort of patients. Whereas most of the sequence variants modulate telomerase function by haploinsufficiency, 2 hTERC variants with sequence changes located within the template region appear to act in a dominant-negative fashion. Inherited telomerase gene mutations, therefore, operate by various mechanisms to shorten telomere lengths, leading to limited marrow stem cell reserve and renewal capacity in patients with hematologic disorders.

Adult↗

PubMed2006

Granulocyte colony-stimulating factor preferentially stimulates proliferation of monosomy 7 cells bearing the isoform IV receptor.

Granulocyte colony-stimulating factor (GCSF) administration has been linked to the development of monosomy 7 in severe congenital neutropenia and aplastic anemia. We assessed the effect of pharmacologic doses of GCSF on monosomy 7 cells to determine whether this chromosomal abnormality developed de novo or arose as a result of favored expansion of a preexisting clone. Fluorescence in situ hybridization (FISH) of chromosome 7 was used to identify small populations of aneuploid cells. When bone marrow mononuclear cells from patients with monosomy 7 were cultured with 400 ng/ml GCSF, all samples showed significant increases in the proportion of monosomy 7 cells. In contrast, bone marrow from karyotypically normal aplastic anemia, myelodysplastic syndrome, or healthy individuals did not show an increase in monosomy 7 cells in culture. In bone marrow CD34 cells of patients with myelodysplastic syndrome and monosomy 7, GCSF receptor (GCSFR) protein was increased. Although no mutation was found in genomic GCSFR DNA, CD34 cells showed increased expression of the GCSFR class IV mRNA isoform, which is defective in signaling cellular differentiation. GCSFR signal transduction via the Jak/Stat system was abnormal in monosomy 7 CD34 cells, with increased phosphorylated signal transducer and activation of transcription protein, STAT1-P, and increased STAT5-P relative to STAT3-P. Our results suggest that pharmacologic doses of GCSF increase the proportion of preexisting monosomy 7 cells. The abnormal response of monosomy 7 cells to GCSF would be explained by the expansion of undifferentiated monosomy 7 clones expressing the class IV GCSFR, which is defective in signaling cell maturation.

Antigens, CD34↗

PubMed2006

Mitochondrial DNA sequence variation in single cells from leukemia patients.

A high frequency of mtDNA somatic mutation has been observed in many tumors as well as in aging tissues. In this study, we analyzed the mtDNA control region sequence variation in 3534 single normal cells and individual blasts from 18 patients with leukemia and 10 healthy donors, to address the mutation process in leukemic cells. We found significant differences in mtDNA sequence, as represented by the number of haplotypes and the mean number of cells with each nonaggregate haplotype in a population of cells, in patients compared to controls. Patients with similar clinical leukemia types, particularly acute myeloid leukemia (AML), did not show a uniform pattern of sequence variation in single blasts. Some patients at relapse presented a complex shift of major haplotypes in single cells. Four patients showed high frequencies of cells containing mutations 189, 260, 16150, and 16488, respectively, as a result of clonal expansion and could be considered as potential markers for their respective disease progression. To our knowledge, this is the first large-scale study of mtDNA variation in single malignant cells. Our results suggest that the somatic mutation process in leukemia is complex, leading to diverse levels of genetic alterations due to either intrinsic aspects of leukemia pathophysiology or chemotherapy effects.

Adolescent↗

PubMed2006

Parvovirus B19 empty capsids as antigen carriers for presentation of antigenic determinants of dengue 2 virus.

For the production of dengue-vaccine candidates, empty capsids, or virus-like particles (VLPs), of parvovirus B19 that carry dengue 2-specific epitopes were employed as antigen carriers. Two epitopes (comprising amino acids 352-368 and 386-397) of domain BIII of the envelope glycoprotein were chosen to produce recombinant B19 VLPs for immunization of BALB/c mice. Serum samples from immunized mice revealed that recombinant B19 VLPs elicited strong humoral immune responses. In summary, this B19 VLP-vaccine platform produced high (> or =2.0 x 10(5)) anti-dengue 2 titers and robust (< or =1 120) 50%-plaque-reduction neutralization test (PRNT(50)) titers, which effectively neutralized live dengue 2 virus in PRNT(50) assays.

Animals↗

PubMed2006

Current concepts in the pathophysiology and treatment of aplastic anemia.

Aplastic anemia, an unusual hematologic disease, is the paradigm of the human bone marrow failure syndromes. Almost universally fatal just a few decades ago, aplastic anemia can now be cured or ameliorated by stem-cell transplantation or immunosuppressive drug therapy. The pathophysiology is immune mediated in most cases, with activated type 1 cytotoxic T cells implicated. The molecular basis of the aberrant immune response and deficiencies in hematopoietic cells is now being defined genetically; examples are telomere repair gene mutations in the target cells and dysregulated T-cell activation pathways. Immunosuppression with antithymocyte globulins and cyclosporine is effective at restoring blood-cell production in the majority of patients, but relapse and especially evolution of clonal hematologic diseases remain problematic. Allogeneic stem-cell transplant from histocompatible sibling donors is curative in the great majority of young patients with severe aplastic anemia; the major challenges are extending the benefits of transplantation to patients who are older or who lack family donors. Recent results with alternative sources of stem cells and a variety of conditioning regimens to achieve their engraftment have been promising, with survival in small pediatric case series rivaling conventional transplantation results.

Anemia, Aplastic↗

PubMed2006

Brief communication: Successful treatment of pure red-cell aplasia with an anti-interleukin-2 receptor antibody (daclizumab).

BACKGROUND: Pure red-cell aplasia (PRCA) is a rare hematologic disease characterized by anemia, reticulocytopenia, and absence of bone marrow erythroid precursors. Most patients respond to some form of immunosuppressive treatment, but few prospective clinical trials have been performed. OBJECTIVE: To examine the efficacy of a humanized monoclonal antibody to the interleukin-2 receptor (daclizumab) for treating PRCA. DESIGN: Pilot study. SETTING: Federal government research hospital. PATIENTS: 15 patients with idiopathic PRCA. INTERVENTION: Daclizumab, 1 mg/kg of body weight, every 2 weeks for a total of 5 infusions. Pure red-cell aplasia was defined as transfusion-dependent anemia with a reticulocyte count of 60 x 10(9) cells/L or less and bone marrow showing absent or diminished erythroid precursors. MEASUREMENTS: Response to therapy was assessed by transfusion independence, increments in reticulocyte count, and nontransfused hemoglobin. RESULTS: Daclizumab had little toxicity. Of the 15 patients, 6 patients (40%) responded to treatment. All responders became transfusion-independent and achieved normal or near-normal hemoglobin values and normal reticulocyte counts. LIMITATIONS: The study was unblinded, and the number of patients was too small to assess drug safety reliably. CONCLUSIONS: Daclizumab seems safe. Its efficacy in this pilot protocol suggests that expanded study in PRCA and other bone marrow failure syndromes is warranted.

Adolescent↗

PubMed2006

T-bet, a Th1 transcription factor, is up-regulated in T cells from patients with aplastic anemia.

In aplastic anemia, immune destruction of hematopoietic cells results in bone marrow failure. Type 1 cytokines, especially IFN-gamma, have been implicated in the pathophysiology of T-cell-mediated, Fas-mediated stem cell apoptosis of hematopoietic cells. Here, we show that the transcription factor T-bet (T-box expressed in T cells) is increased in T cells from patients with aplastic anemia. Patients' T-bet bound directly to the proximal site of the IFN-gamma promoter without any prior stimulation, in contrast to healthy controls. Increased levels of Itk kinase participated in T-bet up-regulation and active transcription of the IFN-gamma gene observed in these patients. Blocking PKC-, a kinase that lies downstream of Itk kinase, decreased T-bet protein and IFN-gamma intracellular levels. These data suggest that the increased IFN-gamma levels observed in aplastic anemia patients are the result of active transcription of the IFN-gamma gene by T-bet. Blocking the transcription of the IFN-gamma gene with kinase inhibitors might lead to the development of novel therapeutic agents for patients with aplastic anemia and other autoimmune diseases.

Adolescent↗

PubMed2006

Relative incidence of agranulocytosis and aplastic anemia.

Agranulocytosis and aplastic anemia are both rare, life-threatening blood dyscrasias. Agranulocytosis is mainly caused by medicines, whereas the etiology of aplastic anemia is largely unexplained. In two epidemiologic studies using the same methods, we observed a striking inverse relationship between the incidence of the two diseases in different regions, including five countries in Europe, and Israel and Thailand. The annual incidence of agranulocytosis ranged from 1.1 to 4.9 cases per million, and that of aplastic anemia, from 0.7 to 4.1 per million; the inverse correlation was consistent among the regions (R2 = 0.74). There is no clear explanation for this previously unreported pattern, but it seems unlikely to be due to methodology.

Agranulocytosis↗

PubMed2006

Genetic variation in telomeric repeat binding factors 1 and 2 in aplastic anemia.

OBJECTIVE: Abnormal telomere shortening has been observed in a subset of individuals with aplastic anemia (AA). We hypothesized that genetic variation in two genes critical in telomere biology, TERF1 and TERF2, could be a risk factor for AA. METHODS: The proximal promoter and all coding regions of TERF1 and TERF2 were sequenced in 47 individuals with acquired AA. Regions with genetic variation were sequenced in an additional 95 AA patients and 289 healthy controls. Single nucleotide polymorphism (SNP) frequencies were analyzed using co-dominant and dominant models and haplotypes determined. Functional studies evaluated telomerase activity, telomere and telomeric overhang lengths, and TRF2 protein expression in select patients. RESULTS: Two nonsynonymous amino acid changes were detected, one in exon 9 of TERF1 and another in exon 6 of TERF2. These sequence variants resulted in conservative amino acid changes and were not predicted to alter TRF1 or TRF2 protein expression or function. SNP and haplotype analyses in acquired AA patients suggested that one variant allele, in intron 9 of TERF1, and haplotype could be associated with increased risk for aplastic anemia (OR 1.59, 95% confidence interval 1.06-2.39, p = 0.033). TERF2 SNPs and haplotypes were not significantly associated with aplastic anemia. CONCLUSIONS: It is possible that a common genetic variant in TERF1 is associated with risk for AA but additional studies are required. Highly penetrant, non-synonymous, or insertion-deletion mutations in TERF1 and TERF2 were not identified and therefore are not likely to be major genetic risk factors for the development of AA.

Anemia, Aplastic↗

PubMed2006

Nonisotopic detection of microRNA using digoxigenin labeled RNA probes.

MicroRNAs (miRNAs) are an important class of endogenously derived, small approximately 22 nucleotide noncoding regulatory RNAs that have recently become implicated in development, cell regulation and cancers of various tissues. Here we report a nonisotopic Northern analysis method for miRNA detection using 3'-digoxigenin (DIG)-labeled RNA oligo probes. Northern blot analysis was performed using miRNA or total RNA fractions extracted from human leukemic cell lines, and blots were hybridized with either 32P- or DIG-labeled RNA probe for miR-181, miR-155 or miR-16. A labeled probe for U6 small nuclear RNA served as an internal control. The use of DIG-labeled RNA probes was equally sensitive compared to 32P-labeled probes in detecting miRNA quantities as low as 50 ng. The ability to use nonisotopic methods and yet obtain sensitive and reliable results offers an advantage to investigators who prefer to avoid isotopes.

Blotting, Northern↗

PubMed2006

Overcoming graft rejection in heavily transfused and allo-immunised patients with bone marrow failure syndromes using fludarabine-based haematopoietic cell transplantation.

Allogeneic haematopoietic cell transplantation (HCT) can cure a variety of non-malignant haematological disorders. Although transplant outcomes for selected patients with severe aplastic anaemia (SAA) and paroxysmal nocturnal haemoglobinuria (PNH) have improved, older age, allo-immunisation from transfusions, prior immunosuppressive therapy and a prolonged time from diagnosis to transplantation are associated with worse outcome. Because of its potent immunosuppressive effects, we investigated a fludarabine-based non-myeloablative conditioning regimen in patients with transfusion-dependent non-malignant haematological disorders at increased risk for graft rejection with conventional transplant conditioning. Twenty-six patients with transfusion dependent/anti-thymocyte globulin (ATG)-refractory SAA, PNH or pure red cell aplasia underwent HCT from a human leucocyte antigen (HLA)-compatible relative. Transplant conditioning consisted of cyclophosphamide (120 mg/kg) and fludarabine (125 mg/m2) with or without ATG. Ciclosporine, alone or combined with mycophenolate mofetil or methotrexate, was used as graft-versus-host disease (GVHD) prophylaxis. All patients achieved durable engraftment and transfusion-independence. Twenty-four of 26 patients are alive at a median of 21 months following transplantation. Although a high cumulative incidence of acute (65% grades II-IV, 54% grades III-IV) and chronic GVHD (56%) was observed, only one patient died from transplant-related causes (cumulative incidence 7%). These data show that HCT following fludarabine-based non-myeloablative conditioning results in durable engraftment and excellent survival in SAA and PNH patients at high risk for graft rejection.

Adolescent↗

PubMed2006

Treatment of severe aplastic anaemia with combined immunosuppression: anti-thymocyte globulin, ciclosporin and mycophenolate mofetil.

Severe aplastic anaemia (SAA) can be successfully treated with immunosuppressive therapies or haematopoietic stem cell transplantation (HSCT). Response rates with horse anti-thymocyte globulin (h-ATG) plus ciclosporin (CsA) are about 60-70%, and robust responders have an excellent long-term survival. We introduced a third immunosuppressive agent to standard h-ATG/CsA, mycophenolate mofetil (MMF), in an attempt to improve the response rate and survival, and to decrease the relapse rate and clonal evolution to myelodysplasia. A total of 104 consecutive patients with SAA were treated with h-ATG/CsA/MMF between May 1999 and June 2003 at the National Institutes of Health Clinical Center. The overall response rate at 6 months was 62%, with 24 (37% of responders) patients relapsing at a median of 389 d from ATG. Nine patients showed evidence of clonal evolution following ATG. After a median follow up of 42 months, the median survival among responders was not reached and among non-responders was 58 months. Over half of the relapses occurred during MMF administration. Despite a strong theoretical rationale for its use, MMF did not result in the improvement of response or relapse rates when compared with historical standard h-ATG/CsA.

Adolescent↗