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The article explores a specific type of paroxysmal nocturnal haemoglobinuria (PNH) known as ahemolytic PNH or white cell PNH. Unlike typical PNH, this subtype does not exhibit signs of hemolysis but is characterized by bone marrow failure and thrombophilia. The document suggests that this subtype may be caused by a PIGA mutation in a committed progenitor cell rather than a stem cell. The authors recommend considering screening for ahemolytic PNH in cases of thromboembolic events and bone marrow failure without hemolysis. [Extracted from the article]

COVID-19 did not result in SAA relapse but these results cannot be generalised to patients who may have more severe COVID-19 manifestations due to other underlying comorbidities.13 The timing of relapse post viral infection is not established therefore longer follow-up may lead to better knowledge. The coronavirus disease 2019 (COVID-19) pandemic has caused concern among physicians caring for immunosuppressed patients. No patients required COVID-19 infection-directed therapies or had interruption of ongoing immunosuppression. In patients with concurrent SAA and COVID-19 infection, continuation or initiation of CsA and/or EPAG during SARS-CoV-2 infection does not appear to complicate infectious course. [Extracted from the article]

Summary: Acquired severe aplastic anaemia (SAA) has an immune pathogenesis, and immunosuppressive therapy (IST) with anti‐thymocyte globulin and cyclosporine is effective therapy. Eltrombopag (EPAG) added to standard IST was associated with higher overall and complete response rates in patients with treatment‐naïve SAA compared to a historical IST cohort. We performed a paediatric subgroup analysis of this trial including all patients aged <18 years who received EPAG plus standard IST (n = 40 patients) compared to a historical cohort (n = 87) who received IST alone. Response, relapse, clonal evolution, event‐free survival (EFS), and overall survival were assessed. There was no significant difference in either the overall response rate (ORR) or complete response rate at 6 months (ORR 70% in EPAG group, 72% in historical group, P = 0·78). Adults (≥18 years) had a significantly improved ORR of 82% with EPAG compared to 58% historically (P < 0·001). Younger children had lower response rates than did adolescents. The trend towards relapse was higher and EFS significantly lower in children who received EPAG compared to IST alone. Addition of EPAG added to standard IST did not improve outcomes in children with treatment‐naïve SAA. EPAG in the paediatric population should not automatically be considered standard of care. Registration: clinicaltrials.gov (NCT01623167). [ABSTRACT FROM AUTHOR]

Immune aplastic anaemia (AA) is caused by cytotoxic T lymphocytes (CTLs) that destroy haematopoietic stem and progenitor cells. Enhanced type 1 T helper (Th1) responses and reduced regulatory T cells (Tregs) are involved in the immune pathophysiology. CD24hiCD38hi regulatory B cells (Bregs) suppress CTLs and Th1 responses, and induce Tregs via interleukin 10 (IL‐10). We investigated circulating B‐cell subpopulations, including CD24hiCD38hi Bregs, as well as total B cells, CD4+ T cells, CD8+ T cells and natural killer cells in 104 untreated patients with severe and very severe AA, aged ≥18 years. All patients were treated with standard immunosuppressive therapy (IST) plus eltrombopag. CD24hiCD38hi Bregs were markedly reduced in patients with AA compared to healthy individuals, especially in very severe AA, but residual Bregs remained functional, capable of producing IL‐10; total B‐cell counts and the other B‐cell subpopulations were similar to those of healthy individuals. CD24hiCD38hi Bregs did not correlate with responses to IST, and they recovered to levels present in healthy individuals after therapy. Mature naïve B‐cell counts were unexpectedly associated with IST response. Markedly reduced CD24hiCD38hi Bregs, especially in very severe AA, with recovery after IST suggest Breg deficits may contribute to the pathophysiology of immune AA. [ABSTRACT FROM AUTHOR]

Background & Aims: Short telomeres and genetic telomerase defects are risk factors for some human liver diseases, ranging from non-alcoholic fatty liver disease and non-alcoholic steatohepatitis to cirrhosis. In murine models, telomere dysfunction has been shown to metabolically compromise hematopoietic cells, liver and heart via the activation of the p53-PGC axis. Methods: Tert-and Terc-deficient mice were challenged with liquid high-fat diet. Liver metabolic contents were analysed by CE-TOFMS and liver fat content was confirmed by confocal and electronic microscopy. Results: Tert-deficient but not Terc-deficient mice develop hepatocyte injury and frank steatosis when challenged with liquid high-fat diet. Upon high-fat diet, Tert-/- hepatocytes fail to engage the citric acid cycle (TCA), with an imbalance of NADPH/NADP+ and NADH/NAD+ ratios and depletion of intermediates of TCA cycle, such as cis-aconitic acid. Telomerase deficiency caused an intrinsic metabolic defect unresponsive to environmental challenge. Chemical inhibition of telomerase by zidovudine recapitulated the abnormal Tert-/- metabolic phenotype in Terc-/- hepatocytes. Conclusions: Our findings indicate that in telomeropathies short telomeres are not the only molecular trigger and telomerase enzyme deficiency provokes hepatocyte metabolic dysfunction, abrogates response to environmental challenge, and causes cellular injury and steatosis, providing a mechanism for liver damage in telomere diseases. [ABSTRACT FROM AUTHOR]

Fluorescent cell barcoding (FCB) is a cell-based multiplexing technique for high-throughput flow cytometry. Barcoded samples can be stained and acquired collectively, minimizing staining variability and antibody consumption, and decreasing required sample volumes. Combined with functional measurements, FCB can be used for drug screening, signaling profiling, and cytokine detection, but technical issues are present. We optimized the FCB technique for routine utilization using DyLight 350, DyLight 800, Pacific Orange, and CBD500 for barcoding six, nine, or 36 human peripheral blood specimens. Working concentrations of FCB dyes ranging from 0 to 500 μg/ml were tested, and viability dye staining was optimized to increase robustness of data. A five-color staining with surface markers for Vβ usage analysis in CD4+ and CD8+ T cells was achieved in combination with nine sample barcoding. We provide improvements of the FCB technique that should be useful for multiplex drug screening and for lymphocyte characterization and perturbations in the diagnosis and during the course of disease. Published 2017 by Wiley Periodicals, Inc., on behalf of International Society for Advancement of Cytometry. This article is a US government work and as such, is in the public domain in the United States of America. [ABSTRACT FROM AUTHOR]

The aetiology of paroxysmal nocturnal haemoglobinuria ( PNH) is a somatic mutation in the X-linked phosphatidylinositol glycan class A gene ( PIGA), resulting in global deficiency of glycosyl phosphatidylinositol-anchored proteins ( GPI- APs). This study applied RNA-sequencing to examine functional effects of the PIGA mutation in human granulocytes. CXCR2 expression was increased in GPI- AP- compared to GPI- AP+ granulocytes. Macrophage migration inhibitory factor, a CXCR2 agonist, was significantly higher in plasma of PNH patients. Nuclear factor-κB phosphorylation was upregulated in GPI- AP− compared with GPI- AP+ granulocytes. Our data suggest novel mechanisms in PNH, not obviously predicted by decreased production of the GPI moiety. [ABSTRACT FROM AUTHOR]

Allogeneic haematopoietic stem cell transplantation is curative for severe aplastic anaemia ( SAA) unresponsive to immunosuppressive therapy. To reduce chronic graft- versus-host disease ( GVHD), which occurs more frequently after peripheral blood stem cell ( PBSC) transplantation compared to bone-marrow transplantation ( BMT), and to prevent graft rejection, we developed a novel partial T-cell depleted transplant that infuses high numbers of granulocyte colony-stimulating factor-mobilized CD34+ selected PBSCs combined with a BMT-equivalent dose of non-mobilized donor T-cells. Fifteen patients with refractory SAA received cyclophosphamide, anti-thymocyte globulin and fludarabine conditioning, and were transplanted with a median 8 × 106 CD34+ cells/kg and 2 × 107 non-mobilized CD3+ T-cells/kg from human leucocyte antigen-matched sibling donors. All achieved sustained engraftment with only two developing acute and two developing chronic GVHD. With a 3·5-year median follow-up, 86% of patients survived and were transfusion-independent. When compared to a retrospective cohort of 56 bone-marrow failure patients that received the identical transplant preparative regimen and GVHD prophylaxis with the exception that the allograft contained unmanipulated PBSCs, partial T-cell depleted transplant recipients had delayed donor T-cell chimerism and relative reduction of 75% in the incidence of acute grade II- IV GVHD (13% vs. 52%; P = 0·010) and of 82% in chronic GVHD (13% vs. 72%; P = 0·0004). In multivariate analysis, partial T-cell depleted transplants remained significantly associated with a reduced risk of GVHD. In conclusion, for patients with refractory SAA, this novel transplant strategy achieves excellent engraftment and survival when compared to unmanipulated PBSC transplants and dramatically reduces the incidence of both acute and chronic GVHD. [ABSTRACT FROM AUTHOR]

Although recent observations implicate the importance of telomerase activity in acute myeloid leukaemia ( AML), the roles of epigenetic regulations of the TERT gene in leukaemogenesis, drug resistance and clinical prognosis in AML are not fully understood. We developed a quantitative pyrosequencing-based methylation assay covering the TERT proximal promoter and a partial exon 1 ( TERTpro/Ex1) region and tested both cell lines and primary leukaemia cells derived from AML and AML with preceding myelodysplastic syndrome ( AML/ MDS) patients ( n = 43). Prognostic impact of methylation status of the upstream TERT promoter region was assessed by the Kaplan-Meier method. The activity of the telomerase inhibitor, imetelstat, was measured using leukaemia cell lines. The TERTpro/Ex1 region was highly methylated in all cell lines and primary leukaemia cells showed diverse methylation profiles. Most cases showed hypermethylated regions at the upstream TERTpro/Ex1 region, which were associated with inferior patient survival. TERTpro/Ex1 methylation status was correlated with the cytotoxicity to imetelstat and its combination with hypomethylating agent enhanced the cytotoxicity of imetelstat. AML cell lines and primary blasts harbour distinct TERTpro/Ex1 methylation profiles that could serve as a prognostic biomarker of AML. However, validation in a large cohort of patients is necessary to confirm our findings. [ABSTRACT FROM AUTHOR]

Abnormal telomere lengths have been linked to cancer and other hematologic disorders. Determination of mean telomere content (MTC) is traditionally performed by Southern blotting and densitometry, giving a mean telomere restriction fragment (TRF) value for the total cell population studied. Here, we compared a quantitative Polymerase Chain Reaction approach (qPCR) and a flow cytometric approach, fluorescence in situ hybridization (Flow-FISH), to evaluate telomere content distribution in total patient peripheral blood mononuclear cells or specific cell populations. Flow-FISH is based on in situ hybridization using a fluorescein-labeled peptide nucleic acid (PNA) (CCCTAA)3 probe and DNA staining with propidium iodide. We showed that both qPCR and Flow-FISH provide a robust measurement, with Flow-FISH measuring a relative content longer than qPCR at a single cell approach and that TRF2 fluorescence intensity did not correlate with MTC. Both methods showed comparable telomere content reduction with age, and the rate of relative telomere loss was similar. Published 2016 Wiley Periodicals Inc. This article is a US government work and, as such, is in the public domain in the United States of America. [ABSTRACT FROM AUTHOR]

VEXAS is a heterogenous disease in its clinical manifestations, with multi-organ involvement and variable marrow dysfunction: responses to therapy will need to be carefully categorized across organ systems. While all included patients treated with azacytidine in this cohort met the criteria for MDS based on WHO 2016, the indication for treatment was uncontrolled inflammation despite chronic corticosteroid therapy in 8/11 cases. VEXAS is a new hematologic disease. [Extracted from the article]

Peromyscus leucopus mice share physical similarities with laboratory mice Mus musculus ( MM) but have higher agility and longer lifespan. We compared domesticated P. leucopus linville ( PLL) and M. musculus C57 BL/6 ( MMB6) mice for cellular composition of peripheral blood (PB), bone marrow ( BM) and spleen. PLL mice had significantly fewer platelets and significantly more monocytes in the blood, and notably fewer megakaryocytes in the BM. Spleens of PLL mice were significantly smaller, with 50% fewer cells and reduced 'red pulp'. There was no obvious haematological change in PLL mice between 2-8 and 16-26 months of age, except for a significant increase in blood monocytes. Cellular reactive oxygen species ( ROS) content showed no change with age but differed significantly between different cell types. Treating two to eight month-old PLL mice with antioxidant N-acetylcysteine in drinking water for three months did not affect cellular ROS content, but increased blood leucocytes especially the concentration of monocytes. The low platelets, low megakaryocytes, high monocytes and low splenic erythropoiesis in PLL mice resemble human measurements better than the values seen in MMB6. [ABSTRACT FROM AUTHOR]

Paroxysmal nocturnal hemoglobinuria ( PNH) is an acquired bone marrow disorder caused by expansion of a clone of hematopoietic cells lacking glycosylphosphatidylinositol ( GPI)-anchored membrane proteins. Multiple lines of evidence suggest immune attack on normal hematopoietic stem cells provides a selective growth advantage to PNH clones. Recently, frequent loss of HLA alleles associated with copy number-neutral loss of heterozygosity in chromosome 6p ( CN-6p LOH) in aplastic anemia ( AA) patients was reported, suggesting that AA hematopoiesis 'escaped' from immune attack by loss of HLA alleles. We report here the first case of CN-6p LOH in a Japanese PNH patient only in GPI-anchored protein positive (59%) granulocytes, but not in GPI-anchored protein negative (41%) granulocytes. CN-6p LOH resulted in loss of the alleles A*02:06- DRB1*15:01- DQB1*06:02, which have been reported to be dominant in Japanese PNH patients. Our patient had maintained nearly normal blood count for several years. Our case supports the hypothesis that a hostile immune environment drives selection of resistant hematopoietic cell clones and indicates that clonal evolution may occur also in normal phenotype (non- PNH) cells in some cases. [ABSTRACT FROM AUTHOR]

BACKGROUND: Reports of Monosomy 7 in patients receiving granulocyte-colony-stimulating factor (G-CSF) have raised concerns that this cytokine may promote genomic instability. However, there are no studies addressing whether repeated administration of G-CSF produces Monosomy 7 aneuploidy in healthy donors. STUDY DESIGN AND METHODS: We examined Chromosomes 7 and 8 by fluorescent in situ hybridization (FISH) in CD34+ cells from 35 healthy hematopoietic stem cell transplant (HSCT) donors after G-CSF administration for 5 days and by spectral karyotyping analysis (SKY) in four individuals to assess chromosomal integrity. We also studied 38 granulocyte donors who received up to 42 doses of G-CSF and dexamethasone (Dex) using FISH for Chromosomes 7 and 8. RESULTS: We found no abnormalities in Chromosomes 7 and 8 in G-CSF-mobilized CD34+ cells when assessed by FISH or SKY, nor did we detect aneuploidy in G-CSF- and Dex-treated donors. CONCLUSION: G-CSF does not promote clinically detectable Monosomy 7 or Trisomy 8 aneuploidy in HSCT or granulocyte donors. These findings should be reassuring to healthy HSCT and granulocyte donors. [ABSTRACT FROM AUTHOR]

In this study six versions of recombinant human hoxb4 proteins were produced and their effectiveness evaluated in expanding human haematopoietic stem and progenitor cells in vitro and in vivo. An N-terminal-tat and C-terminal histidine-tagged version of hoxb4 (T-hoxb4-H) showed the highest activity in expanding colony forming cells (CFCs) and long-term culture-initiating cells (LTC-ICs) when used at 50 nmol/l concentration in cell culture. Human cord blood CD34+ cells cultured with 50 nmol/l T-hoxb4-H showed a significant increase in severe-combined immunodeficient mouse-repopulating cells (SRCs). In a mouse model of immune-mediated bone marrow (BM) failure, T-hoxb4-H showed an additive effect with cyclosporine in alleviating pancytopenia. In addition, T-hoxb4-H expanded CFC and LTC-IC on BM samples from patients with refractory severe aplastic anaemia and myelodysplastic syndromes: after culturing with 50 nmol/l T-hoxb4-H for 4 d, BM cells from 10 of the 11 patients showed increases in CFC and LTC-IC, and the increase in LTC-IC was statistically significant in samples from four patients. Recombinant human hoxb4 could be a promising therapeutic agent for BM failure. [ABSTRACT FROM AUTHOR]

Horse anti-thymocyte globulin (h-ATG) and ciclosporin are the initial therapy for most patients with severe aplastic anaemia (SAA), but there is no practical and reliable method to predict response to this treatment. To determine whether pretreatment blood counts discriminate patients with SAA who have a higher likelihood of haematological response at 6 months to immunosuppressive therapy (IST), we conducted a single institution retrospective analysis on 316 SAA patients treated with h-ATG-based IST from 1989 to 2005. In multivariate analysis, younger age, higher baseline absolute reticulocyte count (ARC), and absolute lymphocyte count (ALC) were highly predictive of response at 6 months. Patients with baseline ARC ≥ 25 × 109/l and ALC ≥ 1 × 109/l had a much greater probability of response at 6 months following IST compared to those with lower ARC and ALC (83% vs. 41%, respectively; P < 0·001). This higher likelihood of response translated to greater rate of 5-year survival in patients in the high ARC/ALC group (92%) compared to those with a low ARC/ALC (53%). In the era of IST, the baseline ARC and ALC together serve as a simple predictor of response following IST, which should guide in risk stratification among patients with SAA. [ABSTRACT FROM AUTHOR]

In paroxysmal nocturnal haemoglobinuria (PNH), chronic destruction of PNH red blood cells (RBCs) by complement leads to anaemia and other serious morbidities. Eculizumab inhibits terminal complement-mediated PNH RBC destruction by targeting C5. In the phase III, double-blind, placebo-controlled, TRIUMPH study, eculizumab reduced haemolysis, stabilized haemoglobin levels, reduced transfusion requirements and improved fatigue in patients with PNH. Herein, we explored the effects of eculizumab on measures of anaemia in patients from the TRIUMPH study and the open-label SHEPHERD study, a more heterogeneous population. Eculizumab reduced haemolysis regardless of pretreatment transfusion requirements and regardless of whether or not patients became transfusion-dependent during treatment ( P < 0·001). Reduction in haemolysis was associated with increased PNH RBC counts ( P < 0·001) while reticulocyte counts remained elevated. Eculizumab-treated patients demonstrated significantly higher levels of haemoglobin as compared with placebo in TRIUMPH and relative to baseline levels in SHEPHERD ( P < 0·001 for each study). Eculizumab lowered transfusion requirement across multiple pretreatment transfusion strata and eliminated transfusion support in a majority of both TRIUMPH and SHEPHERD patients ( P < 0·001). Patients who required some transfusion support during treatment with eculizumab showed a reduction in haemolysis and transfusion requirements and an improvement in fatigue. Eculizumab reduces haemolysis and improves anaemia and fatigue, regardless of transfusion requirements. [ABSTRACT FROM AUTHOR]

In aplastic anaemia, T cells have a central role in the pathophysiology of bone marrow destruction. This study showed that T cells from patients with aplastic anaemia expressed decreased T-cell receptor (TCR) ζ-chain protein and mRNA levels compared to healthy controls. Patients with decreased TCR ζ-chain showed an abnormal response in intracellular calcium following stimulation through the TCR. We also observed an altered pattern of the transcription factors CREM α and Elf-1 that are implicated in ζ-chain transcription. We concluded that TCR ζ-chain expression was decreased in the majority of patients with aplastic anaemia, regardless of disease activity or treatment status. [ABSTRACT FROM AUTHOR]

BACKGROUND: Most patients with severe aplastic anemia (sAA) respond to immunosuppression, but a significant number relapse or develop clonal abnormalities such as paroxysmal nocturnal hemoglobinuria, myelodysplasia, or leukemia. In principle, patients without matched sibling donors and older patients might benefit from transplantation of autologous hematopoietic peripheral blood progenitor cells (PBPCs) obtained during remission. Even patients who have clinically recovered from aplastic anemia have diminished hematopoietic progenitor cells, so the practicability of PBPC mobilization in these individuals is unknown. STUDY DESIGN AND METHODS: The feasibility of PBPC mobilization in nine patients with a history of sAA was evaluated. Granulocyte–colony-stimulating factor (10 µg/kg) was administered subcutaneously for 5 days and followed by a 12-L leukapheresis procedure. RESULTS: Only two of the nine patients had sufficient mobilization of CD34 cells to merit collection; in these cases sufficient CD34 cells were obtained for autologous transplantation should the need arise. CONCLUSION: PBPC collection is feasible only in a fraction of recovered AA patients. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The management of patients with severe aplastic anaemia (SAA) who do not have a matched sibling donor and fail a course of horse anti-thymocyte globulin (h-ATG)/ciclosporin (CsA) is uncertain. Repeated courses of ATG-based immunosuppression are often employed; in children and increasingly in adults, alternative donor haematopoietic stem cell transplantation is an option. We analysed the success rate of retreatment with rabbit ATG (r-ATG)/CsA in 43 patients treated at our institution in the last 5 years; 22 were refractory (20 adults; two children) to h-ATG/CsA-based regimens and 21 (17 adults; four children) had relapsed after h-ATG/CsA-based regimens. The overall response rate was 30% in patients who were refractory to h-ATG and 65% in patients who had relapsed following h-ATG. The 1000-d survival in patients who responded to r-ATG was 90% compared with 65% in non-responders. Six patients developed a clonal haematological disorder; two were responders, two were non-responders and in two the evolution occurred before the response could be assessed at 3 months following r-ATG. Thirteen patients died; three were responders, six were non-responders and four patients died prior to 3 months when response was assessed. In our study, the response rate in refractory patients was inferior to what has been previously reported. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

We analysed endothelial cell membrane microparticles (ECMP) in the peripheral blood of patients with paroxysmal nocturnal haemoglobinuria (PNH) ( n = 9), aplastic anaemia (AA) ( n = 10), sickle cell disease (SCD) ( n = 8), and healthy donors (HD) ( n = 11). There was no clinically manifested thrombosis in the PNH or AA group, except one cured thrombophlebitis (PNH), while all SCD patients had a history of vaso-occlusive crises. We used three-colour flow cytometry with blood cell-specific antibodies and antibodies to endothelial antigens CD105 and CD144. Phosphatidylserine-positive microparticles were detected using the annexin V-binding (AVB) assay. The population of CD105+AVB+ ECMP was significantly ( P < 0·05) higher in SCD (median: 0·568 × 109/l; 25–75th percentile range: 0·351–0·976 × 109/l) and PNH (0·401 × 109/l ; 0·19–0·441 × 109/l) patients when compared with AA (0·122 × 109/l; 0·061–0·172 × 109/l) or HD (0·180 × 109/l; 0·137–0·217 × 109/l) group. Even more pronounced differences were observed in ECMP exhibiting a marker of inflammatory stimulation CD54 (CD105+CD54+). Similarly, ECMP that exhibited endothelial specific and proteolysis-sensitive antigen CD144 were increased in SCD and PNH, but not in AA. Elevated CD54+ ECMP may reflect the inflammatory status of endothelial cells in SCD and PNH, while CD144+ ECMP could indicate continuous endothelial stimulation and/or injury. Analysis of circulating ECMP appears promising to provide useful information on the status of the vascular endothelium in PNH and SCD. [ABSTRACT FROM AUTHOR]

Paroxysmal nocturnal haemoglobinuria (PNH) results from acquired mutations in the PIG-A gene of an haematopoietic stem cell, leading to defective biosynthesis of glycosylphosphatidylinositol (GPI) anchors and deficient expression of GPI-anchored proteins on the surface of the cell's progeny. Some laboratory and clinical findings have suggested genomic instability to be intrinsic in PNH; this possibility has been supported by mutation analysis of hypoxanthine-guanine phosphoribosyltransferase ( HPRT) gene abnormalities. However, the HPRT assay examines lymphocytes in peripheral blood (PB), and T cells may be related to the pathophysiology of PNH. We analysed the molecular and functional features of HPRT mutants in PB mononuclear cells from eleven PNH patients. CD8 T cells predominated in these samples; approximately half of the CD8 cells lacked GPI-anchored protein expression, while only a small proportion of CD4 cells appeared to derive from the PNH clone. The HPRT mutant frequency (Mf) in T lymphocytes from PNH patients was significantly higher than in healthy controls. The majority of the mutant T lymphocyte clones were of CD4 phenotype, and they had phenotypically normal GPI-anchored protein expression. In PNH patients, the majority of HPRT mutant clones were contained within the V β2 T cell receptor (TCR) subfamily, which was oligoclonal by complementarity-determining region three (CDR3) size analysis. Our results are more consistent with detection of uniform populations of expanded T cell clones, which presumably acquired HPRT mutations during antigen-driven cell proliferation, and not due to an increased Mf in PNH. HPRT mutant analysis does not support underlying genomic instability in PNH. [ABSTRACT FROM AUTHOR]

T-cell large granular lymphocytic lymphoproliferative disease (T-LGL) is often associated with life-threatening cytopenias. Twenty-five subjects with anaemia and/or neutropenia caused by T-LGL were treated with cyclosporin A (CSA) 5–10 mg/kg/d for at least 3 months. Eighteen patients survived between 35 and 77 months after starting treatment. Fourteen patients [56%; 95% confidence interval (CI) 35–76%] responded to CSA with sustained improvement in the neutrophil count or transfusion independence. Seven had complete normalization of blood counts, and four achieved a durable response only after the addition of erythropoietin. Sustained response required continued low-dose CSA. In a multivariate analysis, HLA-DR4 was highly predictive of CSA responsiveness (odds ratio 18; 95% CI 1·8–184). T-LGL subtype, LGL counts after therapy, lymphocytic marrow infiltration and bone marrow cellularity did not significantly affect the probability of response. We conclude that CSA is effective in inducing haematological responses in HLA-DR4-positive patients and that T-LGL is likely to have an immune pathogenesis. [ABSTRACT FROM AUTHOR]

Summary. Some patients with paroxysmal nocturnal haemoglobinuria (PNH) have bone marrow findings characteristic of myelodysplastic syndrome. We studied nine PNH patients to determine whether these karyotypic abnormalities were more likely to occur in glycosylphosphatidylinositol-anchored protein (GPI-AP)-negative cells. Abnormal chromosome patterns were evident only in the GPI-AP-positive populations of the PNH clone in 8 of 9 cases studied. Purified GPI-AP-negative CD34 cells gave rise only to cells of normal karyotype, whereas the progeny of the GPI-AP-positive CD34 cells showed the karyotypic abnormality. These findings suggest that environmental factors, but not genetic instability of the GPI-AP-deficient clone, foster development or survival of haematopoietic cells with chromosomal abnormalities. [ABSTRACT FROM AUTHOR]

Summary. 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 Vα24JαQ, 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 Vα24 in CD4– CD8– T cells derived from AA and healthy individuals, the CDR3 size of Vα24 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 Vα24 polymerase chain reaction products revealed that the NKT cell-specific JαQ 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. [ABSTRACT FROM AUTHOR]

Summary. Laboratory observations suggest that, in some myelodysplastic syndromes (MDS), immune mechanisms may contribute to the impaired blood cell production. Tumor necrosis factor α (TNF-α), a potent inhibitor of haematopoiesis, has been hypothesized to mediate suppressive effects in MDS: TNF-α levels are elevated and correlated with marrow apoptosis and cytopenia. Inhibition of TNF-α 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-α production by marrow cells in MDS; TNF-α production was elevated (> mean + 2 × 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 × 109 /l to 0·84 × 109 /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-α levels. Although anti-TNF therapy with Enbrel was well tolerated at the dosages used in MDS, its efficacy as a single agent appears low. [ABSTRACT FROM AUTHOR]

Because of the insensitivity of the Ham test, paroxysmal nocturnal haemoglobinuria (PNH) has been inaccurately viewed as a late clonal complication of aplastic anaemia (AA). To clarify the relationship between PNH and marrow failure, we tested for the presence of glycosylphosphatidyl-anchored protein-deficient (GPI-AP) granulocytes in large cohorts of patients with AA, myelodysplasia (MDS), and pure haemolytic PNH. A PNH clone was detected in 32% of new AA patients and 18% of MDS patients. In serial studies, this proportion did not change up to 15 years after diagnosis, suggesting that expansion of aberrant cells is an early event (i.e. prior to initial presentation). For all patients with a PNH clone, on average 14% of PNH granulocytes were found on presentation and 37% at 10 years. Patients with PNH but without cytopenia showed higher percentages of GPI-AP-deficient cells than did those with the AA/PNH syndrome. After immunosuppression, there was no change in the contribution of PNH clone to blood production, arguing against the ‘immune-escape’ theory in PNH. Clinically, a high proportion of GPI-AP-deficient cells correlated with marrow hypercellularity. GPI-AP-deficient cells were similarly present in patients with and without karyotypic abnormalities. Our results indicate that the GPI-AP-deficient clones show quantitative and kinetic differences between classic haemolytic PNH and PNH with marrow failure, in which the evolution rate is low later in the course of the disease. [ABSTRACT FROM AUTHOR]

We sought to optimize and standardize stem cell and lymphocyte doses of T cell-depleted peripheral blood stem cell transplants (T-PBSCT), using delayed add-back of donor T cells (DLI) to prevent relapse and enhance donor immune recovery. Fifty-one patients with haematological malignancies received a T-PBSCT from an HLA-identical sibling, followed by DLI of 1 × 107 and 5 × 107 CD3+ cells/kg on d +45 and +100 respectively. Twenty-four patients were designated as standard risk and twenty-seven patients with more advanced leukaemia were designated as high risk. Median recipient age was 38 years (range 10–56). Median (range) of CD34+ and CD3+ cell transplant doses were 4·6 (2·3–10·9) × 106/kg and 0·83 (0·38–2) × 105/kg respectively. The cumulative probability of acute GVHD was 39%. No patient died from GVHD or its consequences. The probability of developing chronic GVHD was 54% (18% extensive). The probability of relapse was 12% for the standard-risk patients and 66% for high-risk patients. In multivariate analysis, the risk factors for lower disease-free survival and overall survival were high-risk disease, CD34+ dose < 4·6 × 106/kg and CD3+ dose < 0·83 × 105/kg. Predictive factors for chronic GVHD were a T-cell dose at transplant > 0·83 × 105 CD3+ cells/kg. These results further define the impact of CD34 and CD3 cell dose on transplant outcome and show that careful dosing of stem cells and lymphocytes may permit the control and optimization of transplant outcome. [ABSTRACT FROM AUTHOR]

Myelodysplastic syndrome (MDS) and T-cell large granular lymphocytic disease (T-LGL) are bone marrow failure disorders. Successful use of immunosuppressive agents to treat cytopenia in MDS and LGL suggests a common pathophysiology for the two conditions. Of 100 patients with initial diagnoses of either MDS or T-LGL referred to the National Institutes of Health for immunosuppressive treatment of cytopenia, nine had characteristics of both T-LGL and MDS (T-LGL/MDS). Fifteen patients with T-LGL received cyclosporin (CSA) (10 responses). Eight out of nine patients with T-LGL/MDS received CSA (two responses) and one patient received ATG (one response). Of 76 patients with MDS, eight received CSA (one response) and 68 received ATG (21 responses). The response to immunosuppression was significantly lower in patients with T-LGL/MDS and MDS than in patients with T-LGL disease alone (28% vs. 66%, P = 0·01). The proportion of T-helper cells and T-suppressor cells with an activated phenotype (HLA-DR+) was increased in patients with T-LGL, T-LGL/MDS and MDS, but the increase in activated T-suppressor cells in patients with T-LGL/MDS was not statistically significant. Autoreactive T cells may suppress haematopoiesis and contribute to the cytopenia in T-LGL and some patients with MDS, leading to T-LGL/MDS. The lower response rate of MDS or T-LGL/MDS to immunosuppression, compared with T-LGL alone, may reflect the older age and intrinsic stem cell abnormalities in MDS and T-LGL/MDS patients. [ABSTRACT FROM AUTHOR]

Summary Uncertainty remains regarding the safety and tolerability of immunosuppressive therapy (IST) with anti‐thymocyte globulin (ATG) and cyclosporine (CSA) in older patients. We retrospectively analysed two prospective clinical trials of IST in treatment‐naïve severe aplastic anaemia (SAA) to assess safety in older compared to younger patients. Patients ≥18 years of age who had received IST with ATG and CSA +/− eltrombopag (EPAG) were included. Pre‐treatment baseline characteristics and co‐morbidities were assessed as predictors of therapy‐related complications in younger (<60 years) versus older (≥60 years) patients. Out of 245 eligible patients, 54 were older and 191 were younger. Older patients had a similar frequency of SAEs, ICU admissions and hospital length of stay compared to younger patients. Older patients had a higher frequency of cardiac events related to IST, but none resulted in death. Older patients had worse long‐term overall survival, and more relapse and clonal evolution post‐IST. However, older patients who responded to IST had a similar survival at a median follow‐up to younger patients. Disease‐related factors and limited therapeutic options in refractory disease likely contribute to poorer outcomes in older patients, not complications of upfront IST. Therefore, IST should be considered first‐line therapy for most older SAA patients. [ABSTRACT FROM AUTHOR]