Treatment of aplastic anemia in children with recombinant human granulocyte colony-stimulating factor

Twenty children (aged 1 to 17 years) with severe or moderate aplastic anemia were treated with recombinant human granulocyte colony- stimulating factor (rhG-CSF) at a dose of 400 micrograms/m2 per day administered as a 30-minute intravenous (IV) infusion daily for 2 weeks. This treatment increased the neutrophil counts (2.7- to 28.0- fold) in 12 of the 20 patients. Increasing doses (800 or 1,200 micrograms/m2 per day) were administered to five patients who had not responded to the initial dose, and three showed an increase in neutrophil count. Differential counts of bone marrow (BM) aspirates showed an increase in the myeloid/erythroid ratio. The response was transient, however, and the neutrophil count returned to baseline within 2 to 10 days of discontinuing treatment. No severe toxicity attributable to rhG-CSF was observed. The results suggest that this agent is effective in stimulating granulopoiesis in children with aplastic anemia. Our study also indicates that rhG-CSF will be particularly useful in managing patients with aplastic anemia complicated by bacterial or fungal infection.     

Failure of recombinant human granulocyte-macrophage colony-stimulating factor therapy in aplastic anemia patients with very severe neutropenia

Four patients with very severe aplastic anemia refractory to antilymphocyte globulin were administered recombinant human granulocyte- macrophage--colony stimulating factor (GM-CSF). One patient with minimal residual myelopoiesis responded transiently to two separate courses of GM-CSF at 4 and 8 micrograms/kg/d administered intravenously and another course at 4 micrograms/kg/d administered subcutaneously. Septicemia and bilateral pneumonia that had been resistant to conventional therapy resolved. Three patients with no evidence of residual myelopoiesis did not respond to GM-CSF. In one patient, the dose was increased to 32 micrograms/kg/d with no effect on hematopoiesis. Immediate side effects were minimal at GM-CSF doses up to 16 micrograms/kg/d. GM-CSF may, however, have been involved in the pathophysiology of thrombosis of the inferior vena cava in the patient administered 32 micrograms/kg/d. We conclude that GM-CSF does not induce hematopoiesis in long-standing, severe, treatment-resistant aplastic anemia with complete myelopoietic failure. However, in patients with minimal residual myelopoiesis, GM-CSF could be a promising adjuvant therapy for severe infection.     

Lymphocyte subsets in patients with aplastic anemia

Lymphocyte subsets were enumerated in a group of 31 patients with aplastic anemia. Abnormal numbers of immunoregulatory T-cells were found in some patients: 26% of them showed a reversed helper/suppressor ratio. Seven of 18 patients showed significantly decreased proliferation in response to PWM; this hyporesponsiveness was present in 75% of patients with a reversed helper/suppressor ratio and in 10% of those with a normal helper/suppressor ratio (R = 0.66, P = 0.008). Eight of 18 patients showed suppressor activity over PWM-induced allogeneic cell proliferation. This suppressive activity did not correlate with T-cell phenotype. Of the patients with a low number of T-cells, 73% had responded to treatment, whereas of those patients with a normal number of T-cells, 26% had responded (P = 0.016). The results are consistent with abnormal immune response in selected patients with aplastic anemia, and suggest a possible influence of T-cells on disease process.     

Increased respiratory burst activity of neutrophils in patients with aplastic anemia: effects of granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor.

The superoxide (O2-)-releasing capacity in response to N-formyl-methionyl-leucyl-phenylalanine (FMLP) and the priming effects of recombinant human granulocyte colony-stimulating factor (rhG-CSF) and granulocyte-macrophage colony-stimulating factor (rhGM-CSF) on FMLP-induced O2-release were investigated in neutrophils from 13 patients with aplastic anemia (AA). The O2(-)-releasing capacity of AA neutrophils (0.85 +/- 0.36 nmol/5 min/1 x 10(5) cells, n = 13) was significantly (p < 0.01) increased as compared with that of normal neutrophils (0.24 +/- 0.12 nmol/5 min/1 x 10(5) cells, n = 17). There was no close relationship between the O2(-)-releasing capacity and the peripheral blood neutrophil count or the plasma concentration of C-reactive protein. The plasma concentrations of G-CSF and GM-CSF were not elevated to the detectable levels (< 0.1 ng/ml and < 0.2 ng/ml, respectively) in all patients tested. FMLP-induced O2(-)-release was further enhanced by pretreatment of cells with rhG-CSF or rhGM-CSF for 10 min at 37 degrees C, except that no significant priming by rhG-CSF was observed in five patients. The priming effect of rhGM-CSF was consistently greater than that of rhG-CSF in all patients. The i.v. administration of rhGM-CSF (6 micrograms/kg body weight/day) to one patient resulted in an increase in neutrophil O2(-)-release stimulated by FMLP. These findings indicate that neutrophils from AA patients are already primed in vivo for enhanced release of O2- and that these neutrophil functions are further potentiated by rhG-CSF or rhGM-CSF.     

Long-term follow-up of patients with aplastic anemia and refractory anemia responding to combination therapy with recombinant human granulocyte colony-stimulating factor and erythropoietin

In our previous study, approximately 60% of aplastic anemia (AA) and refractory anemia (RA) patients treated with recombinant human granulocyte colony-stimulating factor (rhG-CSF) and recombinant human erythropoietin (rhEpo) showed a multilineage response. In this study, we analyzed the long-term follow-up of the multilineage responders (multi-R). In the follow-up analysis of 11 multi-R (6 AA and 5 RA), 10 patients (5 AA and 5 RA) were evaluable. The range of time from the start of treatment to the final contact was 50 to 125 months. Analysis of survival times revealed a significant difference between multi-R and non-multi-R among AA patients given this treatment (P = .016). One AA and 1 RA patient among the multi-R developed acute leukemia. Of 7 living multi-R, 3 AA and 2 RA patients did not need transfusion at final contact. Four of them maintained the target hemoglobin concentration of more than 11 g/dL for quality-of-life benefit. The findings suggested that this result is an important advantage of this treatment.     

Asymptomatic cerebral bleeds in patients with aplastic anemia

Several studies suggest that cerebral microbleeds (CMB) seen on gradient echo magnetic resonance imaging (GE-MRI) of brain increase the future risk of intracranial hemorrhage (ICH). We investigated 26 newly diagnosed patients of aplastic anemia (AA) with GE-MRI of brain who were otherwise neurologically asymptomatic. These patients were then further followed up for a period of 6 months for development of overt ICH. The median age of patients was 20.5 years (range 12-40 years). The median values of complete blood counts at the time of diagnosis were hemoglobin 5.7 gm/dl (range 2.5-9.2 gm/dl), white cell count 2,320/μl (800-7,000/μl), and platelet count 11,500/μl (7000-45,000/μl). Three patients were detected to have CMB while two patients had asymptomatic cerebral macrobleeds (>1 cm). During the follow-up period of 6 months, two patients developed spontaneous ICH. None of the two patients had CMB at diagnosis. In conclusion, asymptomatic CMB were found in 11.5 % of patients with AA. The overall prevalence of asymptomatic ICH (CMB & macrobleeds) was 19.2 %. Further studies with larger number of patients are required to clearly delineate the association.     

Defective monocyte-to-macrophage maturation in patients with aplastic anemia

Macrophages (MAC) are important effector cells of the immune system but also play an essential role as regulatory cells in hematopoiesis. They originate from circulating monocytes (MO) as immature precursor cells that undergo terminal differentiation upon migration from the capillary bed into the various tissues. In the presence of serum, MAC maturation from blood MO is observed in vitro and can be followed by the expression of maturation-associated antigens (MAX.1, .3, .11, and .26; transferrin receptor, 13C2, CD16). We have tested blood MO from 22 patients with aplastic anemia (AA) for their capacity to undergo terminal maturation in vitro. After isolation, blood MO in six patients expressed CD14 molecules at low density when compared to normals. On culture for 7 days, in 15 patients various abnormalities could be shown by phenotype analysis using cell-enzyme-linked immunosorbent assay (ELISA) and an immunoperoxidase staining technique of single cells. Abnormalities ranged from the distinctive failure of mature MAC to express single surface antigens (eg, gp64-MAX.1) to complete inhibition of the development of a MAC maturation-associated phenotype. In three patients the maturational defect was found to persist in complete remission after successful therapy with antileukocyte globulin (ALG). Neither in other immunosuppressed or multiple-transfused patients nor in those with bone marrow hypoplasia secondary to cancer chemotherapy and during hematologic reconstitution following autologous bone marrow transplantation (BMT), defective MO maturation in vitro was seen. Our data provide evidence for the existence of serious disorders within the MO-MAC lineage in patients with AA. This observation may either reflect the stem-cell defect or indicate a MAC involvement in the pathogenesis of the disease.     

Measurement of endogenous plasma granulocyte colony-stimulating factor in patients with acquired aplastic anemia by a sensitive chemiluminescent immunoassay

Endogenous plasma granulocyte colony-stimulating factor (G-CSF) concentrations were serially measured in 68 patients with acquired aplastic anemia (AA). A very sensitive chemiluminescent immunoassay (CLEIA) was used to measure the plasma G-CSF concentration. The minimum detection limit of this assay (0.5 pg/mL) was sufficient for the determination of G-CSF concentrations in patients and normal subjects. The plasma G-CSF concentrations were significantly higher in 51 AA patients without signs of infection as compared with healthy control subjects. In AA patients with signs of infection, the G-CSF concentrations appeared to increase during the acute phase. There was a significant negative correlation between plasma G-CSF concentrations and absolute neutrophil counts (ANC) in AA patients without signs of infection. Although a decrease in the plasma G-CSF concentration was observed in all patients who achieved self-sustaining hematopoiesis following bone marrow transplantation (BMT) or immunosuppressive (IS) therapy, it was lower in patients undergoing BMT as compared with those receiving IS therapy for any given degree of neutropenia. Plasma G-CSF concentrations were higher in patients responding to IS therapy than in nonresponders. This study demonstrated the negative feedback regulation of G-CSF that plasma G-CSF concentrations may be useful in predicting the clinical response to IS therapy.     

Autoantibodies frequently detected in patients with aplastic anemia

Although accumulating evidence strongly suggests that aplastic anemia (AA) is a T cell-mediated autoimmune disease, no target antigens have yet been described for AA. In autoimmune diseases, target autoantigens frequently induce not only cellular T-cell responses but also humoral B-cell responses. We hypothesized that the presence of antigen-specific autoantibodies could be used as a "surrogate marker" for the identification of target T-cell autoantigens in AA patients. We screened a human fetal liver library for serologic reactivity against hematopoietic stem/progenitor cell antigens and isolated 32 genes. In 7 of 18 AA patients, an immunoglobulin G (IgG) antibody response was detected to one of the genes, kinectin, which is expressed in all hematopoietic cell lineages tested including CD34+ cells. No response to kinectin was detected in healthy volunteers, multiply transfused non-AA patients, or patients with other autoimmune diseases. Epitope mapping of IgG autoantibodies against kinectin revealed that the responses to several of the epitopes were shared by different AA patients. Moreover, CD8+ cytotoxic T cells raised against kinectin-derived peptides suppressed the colony formation of granulocyte macrophage colony-forming units (CFU-GMs) in an HLA class I-restricted fashion. These results suggest that kinectin may be a candidate autoantigen that is involved in the pathophysiology of AA.     

树突状免疫细胞疗法治疗再生障碍性贫血的临床初步探讨

目的：探讨树突状细胞为主的免疫细胞疗法治疗再生障碍性贫血（AA）的疗效及安全性。方法：选择抗胸腺细胞免疫球蛋白／抗淋巴细胞免疫球蛋白（ATG／ALG）或环孢菌素（Cs-A）治疗无效、HLA配型失败的AA患者共38例,分离其外周血单个核细胞（MNC）,体外培养48h后收集形成的新免疫细胞,洗涤后经静脉回输患者,每疗程后检测患者外周血常规、骨髓象及骨髓活检,流式细胞检测治疗前后外周血T细胞亚群CD4^＋,CD8^＋,CD69^＋水平及CD3^＋、CD8^＋细胞内γ干扰素（IFN-γ）,α-肿瘤坏死因子（TNF-α）表达。结果：38例AA患者外周血MNC经体外细胞因子及钙离子载体混合培养后,均分化成以树突状细胞为主的新型免疫细胞,27例（71％）患者经此种激活的免疫细胞治疗2个疗程后骨髓象、骨髓活检发生显著改变;29例（76．3％）患者在治疗4个疗程后临床症状明显改善,血常规、CD4／CD8比值均较治疗前显著进步;基本治愈6例（15．8％）,缓解8例（21％）,明显进步12例（31．6％）;总有效率68．4％,疗效与Cs—A组接近;治疗后CD3^＋、CD8^＋细胞内IFN-γ明显降低;除部分患者（52．6％）有一过性寒战发热外,未发现明显不良反应;随访6～18个月无复发病例。结论：以树突状细胞为主的免疫细胞对AA患者的骨髓造血及免疫功能具有一定调节作用,临床应用安全、有效。     

Effect of recombinant human granulocyte-macrophage colony-stimulating factor administration on the lymphocyte subsets of patients with refractory aplastic anemia

Human recombinant granulocyte-macrophage colony-stimulating factor (rhGM-CSF) was administered to 14 patients with refractory aplastic anemia (AA). The effect of rhGM-CSF therapy on the lymphocyte phenotype; on the proliferative responses to the mitogen phytohemagglutinin, Candida albicans, and tetanus toxoid antigens; and on the natural killer (NK) activity of the circulating lymphocytes was studied. Samples were collected before (baseline) and twice during the rhGM-CSF administration. The absolute number of circulating lymphocytes remained relatively constant during the first period, but experienced a significant increase (P less than .001) during the second period. The increase was most prominent in the B cells (P less than .001), but the T cells (P less than .016) also increased. Detailed investigation of lymphocyte subsets showed an increase of the markers CD38 (Leu17), HLA-DR, and the transferrin receptor throughout the treatment course giving evidence of lymphoid cell activation. The NK cell activity was suppressed (P less than .008) throughout the treatment. However, proliferative responses to phytohemagglutinin, Candida antigen, and tetanus toxoid were unaffected. Although the mechanism is not yet defined, GM-CSF does induce activation and increase in absolute lymphoid cell number, especially B cells, together with a decrease in NK cytotoxicity. The implication of these immune cell changes in relation to host resistance to microorganisms remains to be established.     

Granulopoietic effects of colony-stimulating factor obtained from urine of patients with aplastic anemia on normal and cyclophosphamide-treated mice

Colony-stimulating factor (CSF) was partially purified from urine of patients with aplastic anemia using DEAE-cellulose and concanavalin A-Sepharose. This partially purified CSF caused significant neutrophilia in the peripheral blood of normal mice by (a) single or continual intraperitoneal injection(s) in vivo, and also revealed a specific activity of 1.4 x 10(3) U/absorbance unit (AU) at 280 nm in vitro, with less than 1 ng/AU endotoxin. In addition, this CSF induced faster recoveries of neutrophils in the peripheral blood and progenitor spleen cells of cyclophosphamide (CY)-treated mice. These findings suggest that the CSF used in this study accelerated the differentiation of the granulocytic cells and the proliferation of granulocyte colony-forming units in the spleen. These effects contributed to a rapid recovery from neutropenia in mice treated with CY.     

Characterization of human megakaryocyte colony-stimulating factor in the urinary extracts from patients with aplastic anemia and idiopathic thrombocytopenic purpura

Using procedures that were effective in the purification of human urinary erythropoietin (Epo), we attempted initial purification of megakaryocyte colony-stimulating factors (CSF) in urinary extracts from patients with aplastic anemia (AA) and idiopathic thrombocytopenic purpura (ITP). Comparison of colony stimulation by purified human Epo and crude urinary extracts revealed: (1) that the pure Epo augments megakaryocyte colony formation in culture and (2) MEG-CSF activity is also present in materials other than Epo in the crude urinary extracts from the two types of patients. Similar to purification of Epo, ethanol precipitation and sulfopropyl-Sephadex chromatography provided twofold and threefold increases in the specific activity of MEG-CSF, respectively. In contrast to Epo, however, significant inactivation of MEG-CSF activity was seen with phenol treatment. The elution profile of MEG-CSF seen on hydroxylapatite chromatography of urinary extracts was different from that of Epo. These data provided a basis for initial steps for purification of MEG-CSF and support the notion that MEG-CSF is distinct from Epo.     

Production of interleukin 3 and granulocyte-macrophage colony-stimulating factor from stimulated blood mononuclear cells in patients with aplastic anemia.

Blood cells from patients with aplastic anemia (AA) were evaluated for the ability to produce interleukin 3 (IL-3) and granulocyte-macrophage colony-stimulating factor (GM-CSF) on stimulation with phytohemagglutinin (PHA) or antilymphocyte globulin (ALG) by the use of an IL-3-dependent cell-line, TF-1, and the GM-CSF-IRMA kit. The IL-3 levels in patients with active AA were significantly lower, both in PHA-stimulated conditioned medium (CM) and in ALG-CM, than those of normal healthy donors (HD; p < 0.01). The degree of reduced IL-3 production in AA patients correlated well with the severity of neutropenia; the level of IL-3 returned to normal after successful treatment with ALG plus methylprednisolone (ALG therapy). On the other hand, GM-CSF production in AA patients varied widely and was only significant in remission patients in PHA-CM; in this case production was higher than that in active AA patients (p < 0.05) or in HD (p < 0.01). Sensitivity to PHA or ALG stimulation was evaluated by the ratio of IL-3 concentrations in ALG-CM versus PHA-CM (ALG/PHA index). The index varied widely from < 0.1 to > 10 in AA patients, contrasting to the clustered values in HD. Seven of the eight patients who had an ALG/PHA index of > 1.0 showed a good clinical response to ALG therapy. However, 12 of the 14 patients who had a lower index (< 1.0) failed to respond. The ALG/PHA index might have an ability to predict the response to ALG therapy.     

Two different types of granulocyte colony-stimulating factor produced by bovine lung tissue

Summary Bovine lung tissue produces two different types of granulocyte colony-stimulating factor (CSF). The high molecular weight (MW) type (CSF-F) of 70,000 d by Sephadex G-100 gel filtration is only found in conditioned medium of homogenized tissue indubated in sealed glass bottles. This species of CSF exclusively stimulates CFU-C of mouse bone marrow, human bone marrow only hardly. The low MW type CSF (CSF-M) of approximately 29,000 d by gel filtration is found mainly in conditioned medium of slightly minced tissue incubated in Petri dishes. It stimulates both human and mouse CFU-C. Methods to prepare both types of CSF are described. By propagating a fibroblast cell line from bovine lung tissue it was found that fibroblasts are the source of the 70,000 d CSF. Indirect evidence suggests that macrophages produce the 29,000 d CSF species.Abbreviations BLCM bovine lung conditioned medium - CFU-C colony forming units in culture - CM conditioned medium - CSF colony stimulating factor - CSF-F colony stimulating factor produced by fibroblasts - CSF-M colony stimulating factor produced by macrophages - MLCM mouse lung conditioned medium - MW molecular weight - RPMI Roswell Park Memorial Institute (medium) - PBS phosphate balanced salt (solution) - SDS sodium dodecyl sulfate     

Clonal hematopoiesis in patients with acquired aplastic anemia.

To determine whether patients with acquired asplastic anemia (AA) exhibit clonal hematopoiesis, we used restriction fragment length polymorphisms of the X-linked genes phosphoglycerate kinase (PGK1) and hypoxanthine phosphoribosyltransferase (HPRT) and the X-linked probe M27 beta. Of the 19 female patients studied, 18 (95%) patients were informative for at least one marker. Of these, eight patients (42%) were heterozygous for PGK1, two (11%) for HPRT, and 16 (84%) for M27 beta. In 13 (72%) patients, a monoclonal pattern was found. Analysis of purified cell suspensions of four of these patients showed that both myeloid and lymphoid cells were of monoclonal origin, indicating the involvement of an early stem cell. The four patients who were studied at presentation all showed a monoclonal pattern. One of these patients showed a spontaneous recovery despite persistent clonal hematopoiesis. The presence of either clonal or polyclonal hematopoiesis did not show a correlation with the response to antithymocyte globulin (ATG) treatment. A relapse after ATG was also seen in a patient exhibiting polyclonal hematopoiesis. Conversely, a monoclonal pattern did not preclude the occurrence of a partial or complete response to ATG. Other potential markers to study clonality, including cytogenetic abnormalities or point mutations of the N-ras protooncogene, were not found in any of the patients. It is concluded that patients with AA may exhibit clonal hematopoiesis. The significance with respect to evolution to disorders with clonal hematopoiesis like paroxysmal nocturnal hemoglobinuria, myelodysplasia, and acute leukemia remains to be determined.