Abnormal expression of b10 cell frequencies: possible relation to pathogenesis and disease severity of aplastic anemia

SUMMARY OBJECTIVE: Aplastic anemia (AA) is an immune-mediated disease that destroys hematopoietic cells through activated T lymphocytes. B lymphocyte-mediated humoral immunity also plays an important role in the pathogenesis of AA. Regulatory B cell (Breg) subpopulation, which is defined as "B10", secretes interleukin 10 (IL-10). The objective of our experiment was to investigate whether the scale-down proportion of B10 cells in AA patients may play a key role in the pathogenesis. METHODS: A total of 38 AA patients (14 SAA patients and 24 NSAA patients) and 20 healthy control subjects were included. All subjects did not suffer from autoimmune diseases or any other diseases affecting the immune system, such as infectious diseases. Bone marrow mononuclear cells (PBMCs) were isolated and analyzed by Flow cytometry (FCM) and Immunofluorescence double-labeling assay. The relationship between the relative proportions of B10 and ProB10 and their associations to AA, as well as disease severity, were assessed by common clinical indicators and then examined. RESULTS: Our analyses revealed AA patients had significantly lower proportions of peripheral B10 and B10pro compared to healthy controls. SAA patients had a substantially lower percentage of B10 cells and B10pro cells compared to NSAA patients. In addition, B10 cells and B10pro cells were negatively correlated with absolute neutrophil counts, hemoglobin levels and platelet, and absolute reticulocyte counts in AA patients. CONCLUSIONS: The present study attempted to elucidate the potential role of the scale-down proportion of B10 cells in the pathogenesis of AA.

RESUMO OBJETIVO: A anemia aplástica (AA) é uma doença imunomediada que destrói células hematopoiéticas por meio dos linfócitos T ativados. A imunidade humoral mediada por linfócitos B também desempenha um papel importante na patogênese da AA. A subpopulação de células B reguladoras (Breg), que é definida como "B10", secreta interleucina 10 (IL-10). No experimento, investigou-se se a proporção reduzida de células B10 nos pacientes de AA pode desempenhar um papel-chave na patogênese. MÉTODOS: Um total de 38 pacientes de AA (14 pacientes de anemia aplástica grave e 24 pacientes de anemia aplástica não grave) e 20 indivíduos de controle saudáveis foram incluídos. Todos os indivíduos não sofriam de doenças autoimunes ou de quaisquer outras doenças que afetam o sistema imunológico, tais como doenças contagiosas. As células mononucleares da medula óssea (PBMCs) eram isoladas e analisadas por citometria de fluxo (FCM) e ensaio de dupla marcação por imunofluorescência. A relação entre as proporções relativas de células B10 e as células ProB10 e as suas associações à AA, assim como a gravidade da doença avaliada por indicadores clínicos comuns, foram examinadas. RESULTADOS: Nossas análises revelaram que os pacientes de AA têm proporções significativamente menores de células B10 e células ProB10 periféricas em comparação com indivíduos de controle saudáveis. Os pacientes de anemia aplástica grave tiveram uma percentagem substancialmente menor de células B10 e células B10pro em comparação com pacientes de anemia aplástica não grave. Além disso, as células B10 e B10pro foram negativamente correlacionadas com contagens absolutas de neutrófilos, níveis de hemoglobina e plaquetas e contagem de reticulócitos absolutos nos pacientes de AA. CONCLUSÕES: Além disso, o estudo presente tentou elucidar o papel imunorregulatório potencial das células B10 na patogênese da AA e fornecer uma nova estratégia para a aplicação de imunoterapia baseada na célula B para tratar a AA no futuro.

Recent advances in treatment of aplastic anemia

Recent advances in the treatment of aplastic anemia (AA) made most of patients to expect to achieve a long-term survival. Allogeneic stem cell transplantation (SCT) from HLA-matched sibling donor (MSD-SCT) is a preferred first-line treatment option for younger patients with severe or very severe AA, whereas immunosuppressive treatment (IST) is an alternative option for others. Horse anti-thymocyte globuline (ATG) with cyclosporin A (CsA) had been a standard IST regimen with acceptable response rate. Recently, horse ATG had been not available and replaced with rabbit ATG in most countries. Subsequently, recent comparative studies showed that the outcomes of patients who received rabbit ATG/CsA were similar or inferior compared to those who received horse ATG/CsA. Therefore, further studies to improve the outcomes of IST, including additional eltrombopag, are necessary. On the other hand, the upper age limit of patients who are able to receive MSD-SCT as first-line treatment is a current issue because of favorable outcomes of MSD-SCT of older patients using fludarabine-based conditioning. In addition, further studies to improve the outcomes of patients who receive allogeneic SCT from alternative donors are needed. In this review, current issues and the newly emerging trends that may improve their outcomes in near futures will be discussed focusing the management of patients with AA.

Paroxysmal nocturnal hemoglobinuria in childhood and adolescence--a retrospective analysis of 18 cases.

OBJECTIVE: To assess the clinical and hematological profile of PNH in children. METHODS: Clinical and laboratory features of children with PNH diagnosed in the past six years at our centre were reviewed. Various investigations done included a complete blood count and peripheral smear examination, plasma hemoglobin, urine hemosiderin, acid ham test, sucrose lysis test, immunophenotyping of erythrocytes by sephadex column gel card and of granulocytes by flow cytometry. There were 18 children with a marked male predominance (M 14: F 4). RESULTS: Pallor, jaundice, dark urine and bleeding manifestations were the major presenting complaints. One girl suffered an arterial stroke. All children had cytopenia in at least one cell line. Children were treated with danazol, stanazolol, prednisolone and cyclosporin A variously. Overall response rate was 61%. Children with classical PNH performed slightly better with response rates of 66% (6/9) as compared to aplastic anemia-PNH group which has a response rate of 55% (5/9). Amongst various variables only danazol correlated with better response (p=0.029). CONCLUSION: PNH is an uncommon disease in children and should be included in the differential diagnosis of children presenting with cytopenia.

XmnI polymorphism is associated with fetal hemoglobin levels in hypoplastic syndromes

CONTEXTO E OBJETIVO: O aumento adquirido da hemoglobina fetal (HbF) já foi implicado como fator prognóstico em distúrbios diseritropoiéticos. Nossos objetivos foram de examinar elevações adquiridas na HbF em pacientes com anemia aplástica (AA) e hemoglobinúria paroxística noturna (PNH), e de avaliar se há associação entre a presença de polimorfismos XmnI e de região de controle de locus gênico 5' (LCR-HS2) e os níveis de HbF. TIPO DE ESTUDO E LOCAL: Estudo longitudinal no Serviço de Hematologia e Transfusão de Sangue da Universidade Federal de São Paulo – Escola Paulista de Medicina.MÉTODOS: Estudamos um grupo de 37 pacientes com AA e/ou PNH. Reação de polimerase em cadeia (PCR) e digestão enzimática foram usadas para analisar polimorfismos XmnI; e PCR para clonagem e sequenciamento automático dos polimorfismos HS2. RESULTADOS: O nível médio de HbF foi de 2,32%, mas não houve diferença significativa entre o nível de HbF dos pacientes AA e PNH (p = 0.46). Os níveis de HbF menores que 1,0% mostraram correlação estatisticamente significativa com ausência do polimorfismo XmnI (+) (p = 0.007). CONCLUSÕES: Ausência de polimorfismo XmnI está associado com diminuição de HbF. Mais estudos são necessários para confirmar estas observações e fazer comparações sobre tratamento, prognóstico e sobrevida.

Effects of serum from aplastic anemia patients on the expression of cyclin D3 isoform in umbilical cord blood CD34+ cells / 华中科技大学学报（医学）（英德文版）

The pathogenesis of aplastic anemia (AA) was explored and the effects of AA serum on the expression of crucial cyclin D isoform (cyclin D3) in umbilical cord blood hematopoietic stem/progenitor cells were observed. The CD34+ cells were isolated from the cord blood with MIDI-MACS Semi-solid methylcellulose culture technique was used to measure the formation of CFU-GM; The expression level of cyclin D3 was assayed by semi-quantitative RT-PCR and Western-blot after the hematopoietic stem/progenitor cells were incubated in AA serum. The results showed that the AA serum could inhibit the formation of CFU-GM and down regulate the expression level of the cyclin D3 at the mRNA and protein level respectively. In conclusion, the AA serum could inhibit the proliferation of hematopoietic stem cells and down regulate level of cyclin D3, which might be one mechanism of hematopoiesis inhibition in AA.

Red cell indices for distinguishing macrocytosis of aplastic anaemia and megaloblastic anaemia.

Megaloblastic anaemia and aplastic anaemia are important causes of pancytopenia in India. Since both may have presence of macrocytes, peripheral smear examination alone may pose a difficulty in distinction between the two in the absence of macro-ovalocytes and hypersegmented neutrophils. The present study was conducted to evaluate the role of red cell indices in differentiation between macrocytosis of aplastic anaemia and megaloblastic anaemia. Haemogram from 25 cases each of biopsy proven megaloblastic anaemia and aplastic anaemia were reviewed. It was observed that MCV was greater than 97 fl in 15 cases of aplastic anemia (mean MCV 109.7 fl), and 25 cases of megaloblastic anaemia (mean MCV 113.2 fl). Hb, MCV & MCHC were comparable in the two groups. However, mean RDW in megaloblastic anaemia (mean 87.7 fl) was significantly higher than those in aplastic anaemia (mean 71.4 fl). The difference in RDW of patients with megaloblastic anaemia and aplastic anaemia was statistically significant. We conclude that RDW can be of help to differentiate between the two conditions.

Effects of Adjuvant Androgen on Anemia and Nutritional Parameters in Chronic Hemodialysis Patients Using Low-dose Recombinant Human Erythropoietin

BACKGROUND: Recombiant human erythropoietin (epoetin) has greatly contributed to improvement of the anemia of chronic renal failure patients on hemodialysis. However, the reduced erythropoietic effect to epoetin and its high cost have induced lots of supplementary treatments. Therefore, we performed a prospective study to evaluate the effects of adjuvant low-dose androgen therapy in patients using a lower-dose of epoetin than the commonly recommended dose on anemia and the nutritional parameters. METHODS: 17 patients of hemoglobin (Hgb) less than 9 g/dL even after being treated with 1,000 U epoetin subcutaneously (s.c.) 3 times per week on a stable status for more than 6 months, who were on hemodialysis at our institution were examined. They were injected with the same dose of epoetin s.c. and nandrolone decanoate 100 mg intramuscularly (i.m) weekly for another 6 months. Blood test was performed every month before therapy for 6 months and after therapy for 6 months and the mean values were reviewed for comparison. RESULTS: Hgb (7.75+/-0.9 vs 8.99+/-1.39 g/dL, p < 0.01) and hematocrit (Hct) (23.68+/-2.85 vs 26.66+/-3.91%, p < 0.01) were apparently changed before and after adjuvant therapy. Hgb and Hct, weekly dose of epoetin were not statistically different in 9 male patients before and after adjuvant therapy. The weekly dose of epoetin was not statistically different in 8 female patients, but Hgb and Hct (8.02+/-0.6 vs 9.72+/-1.31 g/dL, 24.54+/-1.7 vs 28.74+/-3.06%, p < 0.01) were statistically different before and after adjuvant therapy. In comparison between male and female groups, weekly doses of epoetin and nandrolone decanoate were significantly greater in the female group than the male group (epoetin; 50.66+/-6.23 vs 61.18+/-8.76 U/kg/week, nandrolone decanoate; 1.69+/-0.2 vs 2.04+/-0.29 mg/kg/week, p < 0.05). CONCLUSION: Our data show that the adjuvant androgen therapy is effective for the anemia of hemodialysis patients who did not recover from anemia even after being continuously treated with low-dose epoetin.

Deficiencia de proteínas de superficie ancladas por glucosilfosfatidilinositol en células de pacientes mestizos mexicanos con hipoplasia medular / Clycosilphosphatidylinositol anchored cell surface proteins deficiency in mexican mestizo patients with aplastic anemia

Se estudiaron de manera prospectiva 10 sujetos con diagnóstico histológico de hipoplasia medular para identificar por medio de citometría de flujo las características de las moléculas de superficie CD55 y CD59, ancladas a la superficie celular a través de glucosilfosfatidilinositol (GPI). En cinco se identificó una distribución anormal de estas moléculas; las pruebas de hemólisis ácida, por insulina y sacarosa, así como la investigación de hemosiderinuria fueron anormales en dos de los cinco pacientes. Cinco de ellos fueron tratados con globulina antitimocito y ciclosporina-A y tres se encuentran en remisión completa, en tanto que cinco enfermos fueron tratados con andrógenos y ninguno logró la remisión. De los pacientes en remisión completa, uno tuvo trastornos en las moléculas ancladas por GPI ocurren con frecuencia en pacientes con hipoplasia medular, que la identificación de estas alteraciones es más sensible que las pruebas tradicionales para investigar hemoglobinuria paroxística nocturna (HPN), que las formas aplásticas de HPN son frecuentes en nuestro país y que el tratamiento con inmunosupresión intensa puede ser efectivos en algunas formas hipoplásicas de la HPN. La identificación citofluorográfica de las alteraciones en las moléculas ancladas por GPI debieran reemplazar a las pruebas ®tradicionales¼ para identificar a la HPN

A case of chronic graft-versus-host-disease following allogeneic peripheral blood stem cell rescue for poor graft function after bone marrow transplantation

To overcome poor graft function after allogeneic bone marrow transplantation (BMT), the use of peripheral blood stem cells (PBSC) instead of bone marrow is gaining more popularity because of its advantages. There may, however, be an increased risk of graft-versus-host-disease (GVHD) because of the large number of lymphocytes present in a leukapheresis product. An 18-year-old man with severe aplastic anemia underwent an allogeneic BMT using his HLA-identical sister. After initial excellent graft take for 8 months, his blood counts gradually decreased to 2.8 x 10(9)/L of white cells and 28 x 10(9)/L of platelets with marrow cellularity of < 10%. After allogeneic granulocyte-colony stimulating factor mobilized PBSC rescue, the patient's blood counts recovered satisfactorily. Around 1 year after the boost, he developed chronic GVHD that responded to prednisolone and cyclosporin A. He is now well on low-dose steroids at day +1055 after PBSC rescue. The present case is the first experience of a long-term follow-up who underwent allogeneic PBSC rescue in Korea.

Inhibition of hematopoiesis by a plasma factor in a case of aplastic anemia associated with systemic lupus erythematosus

Systemic Lupus Erythematosus (SLE) may be associated with inhibition of hematopoiesis mediated by antibodies, T-cells or both. A 41-year-old woman with a five-year history of SLE treated with prednisone was admitted to Cabrini Medical Center in New York. The patient complained of fever, chills, arthralgias, general malaise, weakness and dyspnea on exertion, and showed malar rash, pallor, and a systolic ejection murmur along the left sternal border. Admission work up included a CBC with evidence of moderate pancytopenia, a normal EKG, and a normal chest X-ray. The patient's anemia was symptomatic and required a transfusion of packed red blood cells (PRBC's). Bone marrow biopsy and aspiration revealed an aplastic marrow with few hypoplastic islands of hematopoietic elements. The patient was treated with plasmapheresis, achieving immediate progress towards recovery. Bone marrow culture studies (erythroid BFU-E, and myeloid CFU-GM) were done by incubating various titers of the patient's acute phase plasma with normal bone marrow cells. This was done to determine if the patient's plasma contained any hematopoietic inhibitory activity, as has been reported in other cases. Our experiments demonstrated marked inhibition of erymathropoiesis and myelopoiesis in vitro, when various titers of the patient's plasma were included in the culture media. Control plasma produced no inhibition. These studies support the hypothesis that a circulating antibody which inhibits hematopoiesis may be produced in SLE patients with aplastic anemia, and be responsible for it

Globulina antitimocito en el tratamiento de la anemia aplástica adquirida: informe de cinco casos / Anithymocytic globulin in the treatment of aplastic anemia. Five clinic cases

Se informa de la experiencia obtenida en cinco niños con diagnóstico de anemia aplástica adquirida, a quienes se les administró globulina antitimocito. Uno de los niños no había recibido ningún tratamiento previo. Los otros cuatro habían recibido medicación con andrógenos (mesterolona) con pobre respuesta; dos de los cuales tenían menos de un mes de recibir este medicamento. Las reacciones secundarias observadas fueron: eritema máculopapular, prurito, artralgias y fiebre. En ningún caso hubo necesidad de suspender el tratamiento por intolerancia. A todos los niños se les administró mesterolona después de la aplicación de la globulina antitimocito. Se practicó un estudio de médula ósea antes del tratamiento y 12 semanas después de iniciado éste. No se observaron cambios en las diferentes series celulares. Dos de los niños fallecieron meses después por hemorragias e infección. Tres continúan con aplicación de mesterolona. Dos de ellos han tenido menor frecuencia de internamientos para transfusión de sangre y presentan evidente mejoría de la celularidad de médula ósea

Alteración de la distribución de subpoblaciones T en la sangre periférica de pacientes con anemia severa idiopática / Changes in distribution of T subpopulations in the peripheral blood of patients with severe idiopathic anemia

Se efectuaron estudios inmunológicos en células mononucleares de sangre periférica de 17 pacientes con diagnóstico de aplasia severa de médula ósea (AMO): a) fenotipo linfocitario; b) respuesta proliferativa a la PHA; c) valoración de la producción de interleukina 2 (IL2) y expresión del antígeno CD25 (Tac). El rango de edad fue de 4 a 25 años. De los diecisiete enfermos 15 presentaron una disminución significativa de la relación CD4/CD8 (0,72 + ou- 0,19 VN: 1,8 + ou - 0,6). La respuesta proliferativa a la PHA fue normal en el 80% y sólo 2 pacientes mostraron una respuesta disminuida. La producción de IL2 por células mononucleares estimuladas con PHA se encontró significativamente aumentada con deficiencia en la expresión del antígeno CD25. En los 2 pacientes restantes observamos una relación CD4/CD8 normal, no respuesta proliferativa a la PHA e hipoproducción de IL2. Estos datos sugieren la existencia de diferentes grupos de AMO que presentan grados de compromiso inmunológico vaiable y que podrían delinearse mediante perfiles de laboratorio. Por otra parte, la alteración en la distribución de poblaciones reguladoras, fundamentalmente a expensas de una disminución absoluta de la subpoblación CD4+ en la mayoría de nuestros pacientes, asociada a la hiperproducción de IL2 y alteración diferente de expresión del antígeno Tac sugiere la existencia de alteraciones funcionales de esta subpoblación en pacientes con AMO

Therapy for pediatric aplastic anemia: 7 years experience at the Puerto Rico Pediatric Oncology Program

Nine pediatric patients with aplastic or hypoplastic anemia were seen at the Pediatric Hematology-Oncology service of the University of Puerto Rico, from 1980 to first trimester of 1987. Included were eitht males and one female ranging from twenty months to sixteen 8/12 years (mean 13 years). Hematomas and fever were the most common complaints. The interval since the first symptoms and the diagnosis varied from one day to one week. The peripheral cell count was abnormal in all patients; pancytopenia being the most common finding. Five of seven who received antithymocyte globulin (ATG-AM), showed improvement in their peripheral count. The median time interval from the end of ATG-AM and response was two months (range 2 weeks to 15 months)

Abnormalities of hematopoietic progenitor cells in patients with aplastic anemia after hematologic recovery.

Hematopoietic progenitor cells were studied in 11 patients with aplastic anemia who had hematologic recovery after androgen therapy. The mean numbers of colonies derived from erythroid and granulocyte-macrophage progenitor cells (BFU-E and CFU-GM) were markedly decreased compared to normal controls. Cell-mediated suppression of colony growth as detected by coculture studies was observed in 5 patients; 4 patients for CFU-GM and one for both CFU-GM and BFU-E. It is thus concluded that the pool of hematopoietic stem cells in patients after hematologic recovery is still not fully reconstituted. In addition, this impaired reconstitution appears due in some cases to cell mediated suppression of progenitor colony growth.

Anemia hipoplásica / Aplastic anemia

Se presenta un paciente de seis años de edad, afectado de anemia hipoplástica congénita. Se efectúa un análisis de la etiopatogenia, diagnóstico, pronóstico y de las posibilidades terapéuticas.