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SummaryPeripheral blood from 489 recently diagnosed patients with aplastic anaemia (AA) and 316 with refractory anaemia (RA) of myelodysplastic syndrome was evaluated to characterize CD55

Myleodysplastic syndromes (MDS) are premalignant diseases characterized by cytopenias, myeloid dysplasia, immune dysregulation with association to autoimmunity, and variable risk for acute myeloid leukemia (AML) transformation. Studies of Forkhead-box P3 (FoxP3)+ regulatory T-cells (Tregs) indicate that the number and/or activation state may influence cancer progression in these patients. Focusing on patients with a lower-risk for leukemia transformation, 18 (34.6%) of 52 patients studied displayed an altered Treg compartment compared to age-matched controls. Delineation of unique Treg subsets revealed that an increase in the absolute number of CD4(+)FoxP3(+)CD25(+)CD127(low)CD45RA(−)CD27(−) Tregs (effector memory Tregs; TregEM) was significantly associated with anemia (p=0.046), reduced hemoglobin (p=0.038), and blast counts ≥5% (p=0.006). In healthy donors, this TregEM population constitutes only 2% of all Tregs (1–6 Treg cells/μl) in peripheral blood, but when isolated, exhibit greater suppressive activity in vitro. With a median follow-up of 3.1 years (range-2.7 to 4.9) from sample acquisition, increased numbers of TregEM cells proved to have independent prognostic importance in survival estimates suggesting that enumeration of this Treg subset may be a more reliable indicator of immunological escape than FoxP3+ T-cells as a whole. Based on multivariate analyses, TregEM impacted survival independently from myeloblast characteristics, cytopenias, karyotype and comorbidities. Based on these findings, TregEM cell expansion may be synonymous with human Treg activation and indicate microenvironmental changes conducive to transformation in MDS.

Category Clinical investigations Word countsTotal text word count: 3412 words Abstract word count: 240 words 2 ABSTRACTObjective. Although a number of reports have documented a significantly increased incidence of HLA-DR15 in aplastic anemia (AA), the exact role of HLA-DR15 in the immune mechanisms of AA remains unclear. We herein clarify the difference between DRB1*1501 and DRB1*1502, the 2 DRB1 alleles which determine the presentation of HLA-DR15, in the pathophysiology of AA. Materials and Methods.We investigated the relationships of the patients' HLA-DRB1 allele with both the presence of a small population of CD55 -CD59 -(PNH-type) blood cells and the response to antithymocyte globulin (ATG) plus cyclosporine (CsA) therapy in 140 Japanese AA patients. Results. Of the 30 different DRB1 alleles, only DRB1*1501 (33.6% vs. 12.8%, P c <0.01) and DRB1*1502 (43.6% vs. 24.4%, P c <0.01) displayed significantly higher frequencies among the AA patients than among a control. AA patients possessing HLA-DR15 tended to be old, and especially, the frequency of DRB1*1502 in patients ≥40 years old (52.4%) was markedly higher than that in those <40 years old (16.2%, P c <0.01). Only DRB1*1501 was significantly associated with the presence of a small population of PNH-type cells and it also showed a good response to ATG plus CsA therapy in a univariate analysis. A multivariate analysis showed only the presence of a small population of PNH-type cells to be a significant factor associated with a good response to the immunosuppressive therapy (P<0.01). Conclusion.Although both DRB1*1501 and DRB1*1502 contribute to the development of AA, the methods of contribution differ between the two alleles.3

IntroductionAcquired aplastic anemia (AA), a bone marrow failure syndrome characterized by pancytopenia and bone marrow hypoplasia, has been the subject of study by hematologists for many years, as more than 70% of AA patients improve under immunosuppressive therapies such as antithymocyte globulin (ATG) and cyclosporine (CsA). [1][2][3] The dramatic effects of such T-cell suppressants on in vivo hematopoiesis suggest that immune system attack against hematopoietic stem cells plays an essential role in the development of AA. [4][5][6] However, despite extensive efforts to clarify the immune mechanisms of AA, the key antigens provoking immune response against hematopoietic stem cells remain unknown. This is largely due to a lack of animal models and the heterogeneity of pathogenesis in AA. Lack of good progenitor cell assays in humans has also hindered the elucidation of immune mechanisms in AA.In organ-specific autoimmune diseases, such as insulindependent diabetes mellitus (IDDM) and multiple sclerosis where autoreactive T cells play a primary role in pathogenesis, autoantibodies against target proteins of the pathogenic T cells are often detected. [7][8][9][10] Although such antibodies do not usually contribute to the pathogenesis of T-cell-mediated diseases, detection of the antibodies may prove useful in both identifying autoantigens and diagnosing immune mechanisms underlying the diseases. 11 We recently demonstrated that HLA-DRB1*1501 and increased paroxysmal nocturnal hemoglobinuria (PNH)-type cells represent prognostic markers for the immune mechanisms of AA. 12,13 Extensive investigation of antibodies in the sera of patients possessing HLA-DRB1*1501 and a minor population of PNH-type cells may be useful in identifying novel autoantigens in AA. Using immunofluorescent analysis, we previously found that antibodies to UT-7, a megakaryoblastic cell line, are frequently detectable in sera of AA patients who display increased PNH-type cells (PNH ϩ patients; unpublished observation, T.C. and S.N., May 2001). These antibodies may recognize antigens that elicit T-cell responses against hematopoietic stem cells, allowing expansion of PNH-type stem cells. 14,15 To examine these hypotheses, we screened proteins derived from UT-7 cDNA library using serum from a PNH ϩ patient with HLA-DRB1*1501. Serologic identification of antigens by recombinant expression cloning (SEREX) analysis identified diazepambinding inhibitor-related protein 1 (DRS-1) as an autoantigen that raises both antibody production and T-cell responses to antigenpresenting cells transfected with DRS-1 gene. AA and MDS were diagnosed in patients at Kanazawa University Hospital and other hospitals taking part in the bone marrow failure study group led by the Ministry of Health, Labor, and Welfare of Japan. MDS was diagnosed on the basis of cytopenia in peripheral blood, hypercellularity or normocellularity in the sternal or iliac bone marrow, and presence of dysplasia in at least 2 lineages of bone marrow cells. Cytogenetic abnormalities such as trisomy...

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Although increased blood cell deficiency of glycosyl phosphatidylinositol-anchored membrane proteins has often been detected in patients with aplastic anemia (AA) and myelodysplastic syndrome (MDS), the clinical significance of such paroxysmal nocturnal hemoglobinuria (PNH)-type cells remains to be elucidated. We established a sensitive flow cytometric assay capable of detecting less than 0.01% of CD59-CD55- blood cells in a sample and used the assay to examine a large number of patients with bone marrow failure. An increase in the proportion of PNH-type cells was detectable in approximately 60% of all AA patients and in 20% of all refractory anemia (RA)-MDS patients. The increase was undetectable in patients with RA with an excessive number of blasts, acute myelogenous leukemia, multiple myeloma, or systemic lupus erythematosus. Our study showed that the presence of an increased number of PNH-type cells was predictive of a good response to immunosuppressive therapy and a favorable prognosis among patients with recently diagnosed AA and RA. A sensitive flow cytometric analysis for detection of a small population of PNH-type cells in peripheral blood cells is one of the most important examinations in the management of bone marrow failure.

Reticulated platelets are RNA-rich immature platelets and are thought to reflect the ability of bone marrow to produce platelets (1). Reticulated platelets can be measured by flow cytometry using thiazole orange (2). However , the measurement of the reticulated platelets has not been included in routine examinations because of the complexity of the procedure. Furthermore, the standard method for measurement of reticulated platelets has not been established so far. Some assays use whole blood and others use platelet-rich plasma. Monoclonal antibod-ies specific to platelets used to delineate reticulated plate-lets are varying and normal counts of reticulated platelets are reported to vary from 1% to 20% (2-16). A recent software program using the Sysmex XE2100 fully automated complete blood count analyzer (Sysmex, Kobe, Japan) has facilitated the measurement of RNA-rich immature platelets automatically. Although this instant assay lacks specificity, the measurement of the immature platelet fraction percentage in total platelets (IPF%) may be useful in ascertaining whether thrombo-cytopenia is caused by reduced platelet production or increased metabolic turnover because of platelet destruction. Indeed, IPF% has been successfully utilized to differentiate ITP and aplastic anemia (AA) (17). The measurement of IPF% has also been shown to be useful in predicting platelet recovery following Abstract Objectives: Some patients with myelodysplastic syndrome (MDS) show a marked increase in the percentage of immature platelet fraction (IPF%) despite the absence of severe thrombocytopenia. To determine the significance of such an unbalanced increase in the IPF%, we investigated the IPF% and other laboratory findings of 51 patients recently diagnosed with MDS. Method: Subjects consisted of 80 healthy males, 90 healthy females, and 51 patients with MDS and 20 patients with idiopathic thrombocytopenic purpura (ITP). The IPF and IPF% were determined using a Sysmex XE-2100 system loaded with IPF Master software (XE IPF Master, Sysmex). Platelet counts were measured simultaneously. Results: IPF% and platelet counts of these patients ranged from 1.1% to 25.1% (median, 5.3%) and from 6 to 260 · 10 9 ⁄ L (median, 71 · 10 9 ⁄ L), respectively. Twelve patients showed platelet counts more than 50 · 10 9 ⁄ L with 10% or more IPF%. All of the 12 patients had chromosome abnormalities including monosomy 7 and complex abnormalities involving 7 or 5q. In the other 39 patients who did not show the aberrant IPF% increase, chromosomal abnormalities were seen only in seven patients and none of them had chromosome 7 abnormalities. The IPF% of two patients increased to more than 10% in association with the appearance of monosomy 7. Conclusions: These findings suggest that a high IPF% in MDS patient may be a marker for karyotypic abnormalities with a poor prognosis, including chromosome 7 abnormalities.