苯中毒致再生障碍性贫血的细胞免疫功能研究

目的:探讨细胞免疫功能与苯中毒性再生障碍性贫血(再障)发生、发展及转归的关系。方法:根据16例苯中毒再障患者T淋巴细胞亚群有无异常,分为A组和B组。T细胞亚群正常的患者为A组,CD4 /CD8 比值降低者为B组。A组应用雄激素、粒细胞集落刺激因子(G-CSF)和促红细胞生成素(Epo)联合治疗,B组应用以上方案联合环孢素A(CsA)治疗。外周血细胞水平恢复正常后G-CSF、Epo及CsA逐渐减量。结果:所有病例治疗后外周血细胞计数、骨髓涂片正常,治疗前CD4 /CD8 降低者,治疗后恢复正常。结论:部分苯中毒性再障患者,即使无免疫抑制剂的应用,造血干细胞的损伤也能逐渐恢复。因此,对苯中毒性再障患者,常规检查外周血T淋巴细胞及其亚群的变化,以预测应用免疫抑制剂的可行性及其有效性。     

Hodgkin lymphoma accompanied by aplastic anemia and polyclonal expansion of large granular lymphocytes

Immunologic abnormalities have been described in patients with Hodgkin lymphoma, including autoimmune hemolytic anemia and immune thrombocytopenic purpura. The concurrent diagnoses of Hodgkin lymphoma and acquired aplastic anemia, however, is extremely rare. We report a 56-year-old Japanese female patient with severe aplastic anemia and increased large granular lymphocytes prior to the recurrence of Hodgkin lymphoma. After being in remission for 10 years from Hodgkin lymphoma, she developed progressive pancytopenia. The large granular lymphocytes (expressed CD3+ CD8+ TCRalphabeta+) had a polyclonal distribution, the serum-soluble FasL concentration was significantly elevated, and bone marrow biopsy showed severely hypocellular bone marrow without infiltration of abnormal lymphocytes. No lymphadenopathy was observed that would suggest a relapse of Hodgkin lymphoma. A diagnosis of aplastic anemia was made, and treatment with corticosteroids and cyclosporine was initiated. Two months later, she suddenly developed celiac and mediastinal lymphadenopathy. She underwent one cycle of chemotherapy before she died of progressive pancytopenia. Autopsy revealed the recurrence of Hodgkin lymphoma, nodular sclerosis in the lymph nodes and markedly hypocellular bone marrow. Although autoimmune disorders are described in Hodgkin lymphoma, our case shows a rare instance of a patient who had aplastic anemia as the first manifestation of a relapse of Hodgkin lymphoma.     

Lymphoproliferative disease of 'lak cell' precursor large granular lymphocytes in association with celiac disease

We have investigated a case of lymphoproliferative disease of large granular lymphocytes (LDGL) occurring in association with celiac disease, anemia, neutropenia, and carcinomas of the endometrium, breast, and skin. The large granular lymphocyte (LGL) proliferation was monoclonal, T cell in origin, with T cell receptor β-chain gene rearrangement, and a CD3⁺, CD8⁺, CD16^± phenotype. In spite of the high frequency of LGL, natural killer (NK) cell activity was absent. Stimulation with interleukin-2 in vitro, however, resulted in high lymphokine-activated killer (LAK) cell activity against NK-resistant targets. The T-cell nature of the LAK precursor cells is in contrast to the majority seen in normal peripheral blood. Therapeutic trials of cyclosporin A, low-dose cyclophosphamide, and levamisole were unsuccessful in reducing transfusion requirements. This case is unique in the association of LDGL with celiac disease. It is also unique in that the patient had been followed for several years prior to the onset of the LDGL. The case extends the list of lymphoproliferative disorders documented to be associated with celiac disease and, conversely, adds to our knowledge of lymphoproliferative disorder of LGL and its 'dysimmune' manifestations.     

Damaged DNA in lymphocytes of aplastic anemia.

The size of single-stranded DNA in lymphocytes in GO stage from 22 patients with acquired primary and secondary aplastic anemia was estimated by alkaline sucrose gradient centrifugation. The average size was 9.3 (+/-0.3) times 10(7) daltons. The lymphocytes of patients contained significantly more single-strand breaks in DNA, compared to those of normal persons. The difference in size of single-stranded DNA which had been present in nontransformed lymphocytes could also be observed in transformed lymphocytes. Some characteristic differences could be observed in the sedimentation patterns of single-stranded DNA in the lymphocytes of patients with aplastic anemia and those of normal persons. The single-strand breaks in DNA suggested that the repair processes were disturbed in the DNA molecules of circulating lymphocytes from patients with acquired primary and secondary aplastic anemia.     

Natural history of paroxysmal nocturnal hemoglobinuria clones in patients presenting as aplastic anemia

Objective: To investigate the natural history of paroxysmal nocturnal hemoglobinuria (PNH) clones in patients with acquired aplastic anemia (AA). Patients and Methods: Twenty‐seven patients with AA and a detectable PNH clone were monitored for a median of 5.7 years (range1.5–11.5 years). Twenty‐two patients received high‐dose cyclophosphamide (HiCy) therapy. The erythrocyte and granulocyte PNH clone sizes were measured using flow cytometry and analyzed via CellQuest software. PE‐conjugated anti‐glycophorin A, anti‐CD15, FITC‐conjugated anti‐CD59, and FLAER staining were used to define glycosylphosphatidylinositol‐AP‐deficient cells. Results: We found a linear relationship between PNH clone size and the development of intravascular hemolysis, assessed by lactate dehydrogenase (LDH) values (Pearson correlation coefficient = 0.80, P < 0.001 for erythrocyte PNH clones; and Pearson correlation coefficient = 0.73, P < 0.0001 for granulocyte PNH clones). An erythrocyte PNH size of 3–5% and granulocyte PNH size of 23% were the thresholds to predict hemolysis as measured by an elevated LDH (receiver operating characteristic analyses with AUC = 0.96 for erythrocyte PNH clone sizes and AUC = 0.88 for granulocyte PNH clone sizes). Patients with small (≤15%) initial PNH clone sizes were less likely to develop an elevated LDH (mean ± SD: 236.9 ± 109.9 vs. 423.1 ± 248.8; P = 0.02). Over time, the PNH clone sizes remained stable in 25.9% of patients; 48.1% experienced a rise in the PNH clone size; and 25.9% experienced a decrease. Conclusion: The risk of developing clinically significant PNH after HiCy therapy appears to be low in AA patients with PNH clones, especially for those with small initial PNH clones and for those who respond to HiCy therapy.     

Chronic lymphocytic leukemia presenting in association with aplastic anemia

An unusual case of chronic lymphocytic leukemia presenting in association with aplastic anemia is discussed, along with a review of the few previously reported cases. Possible humoral and cellular autoimmune mechanisms are examined as possible causes of marrow aplasia in this setting.     

DNA microarray analysis of T cell-type lymphoproliferative disease of granular lymphocytes

Lymphoproliferative disease of granular lymphocytes (LDGL) is characterized by the clonal proliferation of large granular lymphocytes of either T- or natural killer cell origin. To better understand the nature of T cell-type LDGL, we purified the CD4-CD8+ proliferative fractions from LDGL patients (n=4) and the surface marker-matched T cells isolated from healthy volunteers (n=4), and compared the expression profiles of 3456 genes using DNA microarray. Through this analysis, we identified a total of six genes whose expression was active in the LDGL T cells, but silent in the normal ones. Interestingly, expression of the gene for interleukin (IL) 1beta was specific to LDGL T cells, which was further confirmed by the examination of the serum level of IL-1beta protein. Given its important role in inflammatory reactions, the disease-specific expression of IL-1beta may have a causative relationship with the LDGL-associated rheumatoid arthritis. Spectratyping analysis of the T-cell receptor repertoire also proved the monoclonal or oligoclonal nature of LDGL cells. These data have shown that microarray analysis with a purified T-cell subset is an efficient approach to investigate the pathological condition of Tcell-type LDGL.     

Celiac disease presenting as iron-deficiency anemia in northern India.

BACKGROUND: Adult celiac disease is infrequent in India. Iron-deficiency anemia as its presenting manifestation is still rarer. METHODS: We investigated patients with refractory iron-deficiency anemia attending the hematology clinic of a tertiary-care hospital for celiac disease. The diagnosis of celiac disease was based on histology, serology and response to treatment. RESULTS: Of 19 patients with refractory iron-deficiency anemia seen from April 1998 to March 2000, 11 were diagnosed to have celiac disease. Four of these had abnormal D-xylose test and 3 had fat malabsorption. All 11 patients responded to gluten-free diet with improvement in hematological parameters. CONCLUSION: Patients with refractory iron-deficiency anemia of unknown cause should be investigated for subclinical celiac disease.     

Danazol as first-line therapy for aplastic anemia

Immunosuppressive therapy (IST) with anti-thymocyte globulin (ATG) plus cyclosporine A (CsA) is the standard treatment for aplastic anemia (AA) patients not eligible for allogeneic hematopoietic stem cell transplantation (HSCT). In the absence of ATG + CsA, androgens continue to be a treatment option. We documented the clinical evolution of AA patients treated with danazol instead of ATG + CsA. AA patients lacking both, human leukocyte antigen-matched donor and access to IST, were treated with danazol and modern support therapy and compared with those receiving a HSCT. Overall survival (OS), response rates, and death risk odds were calculated. Fifty AA patients were studied. Thirteen received a HSCT and 37 danazol and support therapy. Median daily dose of danazol was 400 mg (300 to 600 mg), administered during a median of 12 months. Five-year OS was higher for patients receiving HSCT (92%) compared to the danazol group (41%) (P = 0.001). Overall response rate was 46% (17/37) in the danazol-treated group and the median time to initial response was 3 months (1–27). Tendency to achieve remission was similar among severity groups (P = 0.094). The only adverse side effect recorded on the danazol group was an episode of gastrointestinal bleeding. No patient treated with danazol suffered clonal evolution of his/her disease. Although ATG plus CsA is the therapy of choice for AA patients without a donor when neither HSCT nor IST is available, danazol remains an acceptable therapeutic option for AA patients.     

Chronic lymphoproliferative disease of large granular lymphocytes

An 80-year-old patient has been followed for hepato- and splenomegaly, hemolytic anemia, neutropenia with lymphocytosis with large granular lymphocyte predominance in his peripheral blood, with infiltration of bone marrow, liver and probably also spleen. Determination of surface markers of proliferating lymphocytes in peripheral blood showed a mixed phenotype of T suppressor/cytotoxic and natural killer cells (SIg-, E+, T3+, T8+, EAC+, Leu7-, N901+, NK9+, VIB C5 and VIB E3-, Ia-). An in vitro cytotoxic test showed the functional inactivity of the cells tested also after human leukocyte interferon stimulation. Chromosomal analysis neither of peripheral blood lymphocytes nor of bone marrow cells proved the monoclonality marker. Following long-term prednisone therapy, the improvement of anemia, later also neutropenia accompanied by the decrease of lymphocytes has been achieved. As the disease present in our patient was distinguished only in recent years and in our country has not been reported yet, the details on its clinical, morphologic, hematologic, cytogenetic and mainly immunophenotypic characteristics are given in this paper. The problems concerning classification of the disease and determination of its biological nature are discussed.     

Circulating activated suppressor T lymphocytes in hepatitis-associated aplastic anaemia

Lymphocyte subpopulations were measured in the blood of 21 children with severe aplastic anaemia (SAA). Five children with hepatitis-associated AA appeared to constitute a unique group. Compared with children with idiopathic AA, four of five children had a striking increase in percentage of activated suppressor T lymphocytes, which were defined by binding of both anti-Leu 2a and anti-HLA DR monoclonal antibodies (children with hepatitis-associated AA, 21.8 +/- 19.9% [mean +/- SD], children with idiopathic AA, 2.9 +/- 1.2%). However, there was no difference in the mean absolute number of the activated suppressor T lymphocytes between the two groups, because the mean number of lymphocytes was markedly less in children with hepatitis-associated AA (0.28 +/- 0.06 x 10(9)/l) than in those with idiopathic AA (1.64 +/- 0.74 x 10(9)/l). The mean helper: suppressor T lymphocyte ratio was significantly lower in children with hepatitis-associated AA (0.58 +/- 0.74) than in those with idiopathic AA (1.22 +/- 0.44). On the other hand, we did not find such a remarkable alteration of lymphocyte subpopulations in children with non-A non-B hepatitis who did not develop AA. These findings suggested that an imbalance of lymphocyte subpopulations and T lymphocyte activation may have pathogenetic relevance in some of the children with hepatitis-associated AA.     

Acquired pure red cell aplasia associated with lymphoproliferative disease of granular T lymphocytes.

Acquired pure red cell aplasia (PRCA) can be associated with lymphoproliferative disease of granular T lymphocytes (T-LDGL), also known as T-cell large granular lymphocyte leukemia. Fifteen adult patients with PRCA associated with T-LDGL comprise this study. Neutropenia and rheumatoid arthritis were uncommon. All patients responded to immunosuppressive therapy. The 2 most commonly used treatments were prednisone and cyclophosphamide +/- corticosteroids, producing overall response rates of 50% and 60%, respectively. Treatment with cyclophosphamide was associated with a more durable remission (median, 60 versus 7.5 months). After a median follow-up of 67 months, 2 patients died of treatment-related complications, one from myelodysplasia and another from cyclosporine-induced renal failure. The clinical course and treatment responses of PRCA associated with T-LDGL in this series were similar to the general group of PRCA. Because T-LDGL is frequently underdiagnosed, it is likely that a significant proportion of idiopathic or primary PRCA is in fact secondary to T-LDGL.     

Danazol increases T regulatory cells in patients with aplastic anemia

Objectives: Danazol is an attenuated androgen and is used in the treatment of aplastic anemia (AA) in resource constraint settings. We chose to study the role of CD4+ CD25^high CD127^low FoxP3+ T regulatory cells (T-regs) in the pathophysiology of AA and their response to treatment with Danazol alone or in combination with immunosuppressive treatment (IST).

Methods: T-regs’ percentages of 25 acquired idiopathic AA patients and 25 healthy controls who completed study protocol were analyzed by performing multicolor flowcytometry on peripheral blood samples.

Results: More than one-third (36%) of AA patients in our study received Danazol as monotherapy, whereas less than a third (32%) each received standard doses of IST with equine Anti Thymocyte Globulin (ATG) and Cyclosporine combination, or Cyclosporine and Danazol combination, respectively. Results showed that all AA patients had a significantly lower percentage of T-regs at the time of diagnosis when compared to healthy controls (p?p?Discussion: The rise in T-regs’ percentage was higher in patients treated with Danazol alone when compared to standard IST (ATG with Cyclosporine), or Cyclosporine with Danazol combinations (p?=?0.585).

Conclusion: We conclude that Danazol also leads to increase in T-regs in acquired idiopathic AA.     

Lymphoproliferative disease of granular lymphocytes with T-cell receptor gamma delta-positive phenotype: restricted usage of T-cell receptor gamma and delta subunit genes

Lymphoproliferative disease of granular lymphocytes (LDGL) is characterized by more than 0.5 x 109/L of proliferating granular lymphocytes in the peripheral blood. Because of its rarity, the characteristics of LDGL with T-cell receptor (TCR) gammadelta phenotype (gammadeltaT-LDGL) have not yet been identified. This report describes the clinical, hematological, and immunological findings of four patients with this disease. In two cases, the clinical course was indolent and the other two patients required various therapies. The cells had a common immunophenotype: CD3+, CD4-, CD16+, CD56-, CD57-, CD122-, TCR-gammadelta+, and three were CD8-positive. The immunopurified TCR-gammadelta cells from the patients expressed only Vgamma9 and Vdelta1. Spectratyping and sequencing showed mono- or oligoclonality for TCRgamma and TCRdelta subunit genes. Soluble Fas ligand in sera was significantly elevated in all patients. These findings suggest that gammadeltaT-LDGL qualifies as a distinct disease entity.