Expression of membrane-bound IL-15 by bone marrow fibroblast-like stromal cells in aplastic anemia

In order to explore the relationship between IL-15 and aplastic anemia (AA), bone marrow (BM) fibroblast-like stromal cells (BMFSCs) were obtained from BM samples of 23 AA patients by density centrifugation and primary culturing in vitro. Indirect immunofluorescence labeling as well as flow cytometry and confocal laser scanning microscopy analysis were used to determine the expression of membrane-bound IL-15 (mIL-15) on the surface of BMFSCs derived from AA patients (AA-BMFSCs). The effects of IFN-gamma and cyclosporin A (CsA) on the expression of mIL-15 were also investigated. [(3)H]thymidine incorporation test as well as specific antibody inhibition and Transwell separation experiment was adopted to functionally evaluate the expression of mIL-15 on the surface of AA-BMFSCs. mIL-15 was found to be over-expressed on the surface of AA-BMFSCs. IFN-gamma further significantly up-regulated its expression, which, however, was inhibited by CsA. Interestingly, a tight correlation was found between the expression of mIL-15 and IL-15Ralpha on the surface of AA-BMFSCs. AA-BMFSCs had the capability to stimulate the proliferation of T lymphocytes, which was partly or completely inhibited by using neutralizing anti-IL-15Ralpha antibody, neutralizing anti-IL-15 antibody, blocking anti-IL-2/15Rgamma(c) mAb or Transwell chambers with a 0.3-mum pore size membrane to block the direct cell-to-cell contact between AA-BMFSCs and T cells. Apparently, BMFSCs as the most important component of BM hematopoietic microenvironment usually over-express mIL-15 in AA patients. Therefore, AA-BMFSCs may indirectly participate in the T cell-mediated destruction of hematopoietic progenitors in AA by recruiting T cells to BM and stimulating them in situ.     

The complex pathophysiology of acquired aplastic anaemia

Immune‐mediated destruction of haematopoietic stem/progenitor cells (HSPCs) plays a central role in the pathophysiology of acquired aplastic anaemia (aAA). Dysregulated CD8⁺ cytotoxic T cells, CD4⁺ T cells including T helper type 1 (Th1), Th2, regulatory T cells and Th17 cells, natural killer (NK) cells and NK T cells, along with the abnormal production of cytokines including interferon (IFN)‐γ, tumour necrosis factor (TNF)‐α and transforming growth factor (TGF)‐β, induce apoptosis of HSPCs, constituting a consistent and defining feature of severe aAA. Alterations in the polymorphisms of TGF‐β, IFN‐γ and TNF‐α genes, as well as certain human leucocyte antigen (HLA) alleles, may account for the propensity to immune‐mediated killing of HSPCs and/or ineffective haematopoiesis. Although the inciting autoantigens remain elusive, autoantibodies are often detected in the serum. In addition, recent studies provide genetic and molecular evidence that intrinsic and/or secondary deficits in HSPCs and bone marrow mesenchymal stem cells may underlie the development of bone marrow failure.     

Analysis of 65 Turkish patients with congenital aplastic anemia (Fanconi anemia and non-Fanconi anemia): Hacettepe experience

During the last 14 years, 65 unrelated patients were diagnosed as having constitutional aplastic anemia (CAA). In 52 of 65 patients the diepoxybutane (DEB) test was positive. Comparison of several hematological and clinical parameters in Fanconi anemia (FA) (DEB⁺) and non-Fanconi anemia (non-FA) (DEB^-) patients disclosed no statistically significant differences. The study indicated that in Turkey there were no peculiarities in associated congenital abnormalities in FA and non-FA. The rate of consanguinity was 78% in FA and 46% in non-FA, suggesting that also among the non-FA group recessively inherited disorders are hidden. The mean age at diagnosis in FA was 7.7 ± 4.4 (1.8–12) and in non-FA 7.8 ± 3.8 (2–15) years. Nine out of 52 FA and five out of 13 non-FA patients died during the follow-up period. Five of the 52 FA patients developed malignancies, three of them had acute myeloblasts leukemia (AML), one a squamous cell carcinoma of the gingiva, and another a hepatocellular carcinoma. Peliosis hepatica occurred in three of the FA and one of the non-FA patients. A total of seven patients stayed in remission without any medication. The remaining 58 patients were given 2–5 mg/kg of oxymetholone and 5 mg prednisolone treatment. Because of sustained remission, oxymetholone therapy was terminated in four of the 45 FA and two of the 13 non-FA patients. Detailed examination of the pedigrees of all of patients indicated the presence of multiple congenital anomalies. In seven of 52 FA and one of 13 non-FA patients there was increased risk for AML and/or other cancers among family members.     

Defective T-cell differentiation in acquired immune deficiency syndrome (AIDS)

A decline in T-cell lymphocyte number is the central characteristic of acquired immune deficiency syndrome (AIDS). The reason for the loss of these cells is not well understood. We investigated the hypothesis that defects in T-cell differentiation contributed to T-cell loss using anin vitro colony assay that measures T-cell precursor (CFU-T) frequency. The results indicate a substantial generalized decrease in CFU-T in people with AIDS (P<0.01), most of whom have Kaposi's sarcoma, and an occasionally severe decrease in CFU-T in people with ARC. Some of the cells from low colony formers suppressed colony formation by control cells. In addition, plasma from people with AIDS was less supportive of colony growth than control plasma. Decreased Ia expression on adherent mononuclear cells did not correlate with colony formation. A defect in T-cell repopulation can help explain the loss of T cells associated with AIDS.     

慢性再生障碍性贫血患者免疫功能改变及意义

目的　观察慢性再生障碍性贫血 (CAA)患者红细胞和淋巴细胞免疫功能的改变 ,探讨红细胞和T淋巴细胞免疫功能之间及机体免疫功能和CAA发病之间的关系。方法　采用免疫酶标比色法、免疫比浊法、单克隆抗体免疫萤光法、流式细胞仪检测分析等 ,观察比较CAA患者和对照组的各项免疫指标。结果　与对照组相比较 ,CAA患者IgG、IgA、IgM、CD5 8、RBC C3bRR等显著降低 (P <0 .0 1) ,CD3、CD4、CD8、CD4 /CD8比值等明显异常。结论　CAA患者表现多种免疫功能改变 ;主要在骨髓内分化、增生的红细胞、B淋巴细胞的免疫功能明显低下 ,在骨髓外分化、增生的T淋巴细胞亚群的表达多异常升高。     

流式细胞术对骨髓增生异常综合征和再生障碍性贫血患者骨髓细胞免疫表型的分析

检测骨髓增生异常综合征(MDS)和再生障碍性贫血(AA)患者骨髓细胞免疫表型特征,探讨其对二者发病机制、分型诊断及鉴别诊断的临床意义。选用多种单克隆抗体,应用直接免疫荧光法采用流式细胞术CD45/SSC设门,对23例MDS、14例AA、9例正常对照的骨髓细胞各免疫标志的表达率进行检测。结果:MDS组与正常对照组相比其造血干/祖细胞抗原CD34、HLA-DR、髓系早期抗原CD13、CD33、单核系抗原CD14、T淋巴细胞抗原CD7的表达率增高,髓系成熟抗原CD15、B淋巴系抗原CD19、CD20的表达率降低,DI值增高;由RA进展至RAEB,CD34、HLA-DR、CD13、CD33、CD14、CD7的表达率逐渐增高,CD15、CD3、CD19、CD20的表达率逐渐减低,DI值逐渐增高。AA组与正常对照组相比其CD34、CD13、CD33、CD15的表达率降低,CD3、CD7、CD25、CD22的表达率增高。MDS组与AA组相比其CD34、HLA-DR、CD13、CD33、CD14的表达率增高,CD3、CD5、CD7、CD15、CD19、CD20、CD22、CD25的表达率显著降低。MDS和AA患者骨髓细胞免疫表型分析,有助于揭示二者的发病机制,为临床提供新的诊断、分型及鉴别诊断方法。     

Invasive bronchopulmonary aspergillosis leading to rapidly progessive respiratory failure in a patient with severe aplastic anemia

Summary A 22-year-old man with severe aplastic anemia was treated with antilymphocyte globulin, prednisone, and oxymetholone. Fourteen days after initiation of treatment he developed a fulminant mediastinal and subcutaneous emphysema leading to respiratory failure refractory to mechanical ventilation. Fiberoptic bronchoscopy showed nodular lesions typical of aspergillus. Cultures of bronchial mucus revealedAspergillus fumigatus as the responsible pathogen.     

The significance of antilymphocyte antibodies in patients with acquired immune deficiency syndrome (AIDS) and their sexual partners

Antilymphocyte antibodies have been demonstrated in autoimmune diseases, acute viral infections, and acquired immune deficiency syndrome (AIDS) by using either the conventional microlymphocytotoxicity or the double fluorescence technique. In the present study, we used both methods to detect the antilymphocyte antibodies and to characterize further their immunologic significance in patients with AIDS and their sexual partners. The results using the conventional microlymphocytotoxicity method demonstrated that 8 of 10 patients with AIDS and 6 of 10 partners had significant levels of antilymphocyte antibodies which were reactive with B and T cells at cold and warm temperatures. A significant loss in antibody activity following absorption with B, T, and Daudi cells andStaphylococcus aureus, but not platelets or red cells, indicated that these antibodies are not directed to HLA class I antigens but, rather, to antigens that are common to both groups of lymphocytes. There is a close association between antilymphocyte antibodies and lymphopenia in patients but not in partners. Antibodies against lymphocyte subclasses [helper (T4) and suppressor (T8)] were detected by the double fluorescence staining technique, which employs C6-deficient serum as a nonlytic source of complement, and demonstrated the binding of antibodies to target cells, in contrast to lysing of the target cells as in the microlymphocytotoxicity method. The results of this assay showed that antibodies were directed to both populations, and there was no correlation or association between the absolute numbers of peripheral T4 and T8 cells and the percentage of antibody binding. Taken together, there appear to be at least two kinds of antilymphocyte antibodies: lymphocytotoxic antibodies detected by the conventional microlymphocytotoxicity assay and noncytotoxic antibodies detected by the double fluorescence staining technique. The former may be responsible in part for the lymphopenia. The latter may alter lymphocyte function. The patients and partners who had antilymphocyte antibodies also had anti-HTLV-III antibodies, although there was not any close correlation between titers. These findings support the possibility that both types of antibodies occur as part of a generalized immune response, possibly stimulated by the same viral agent.     

Kaposi's sarcoma in acquired immune deficiency syndrome (AIDS)

Of 22 patients with Kaposi's sarcoma, 16 had the acquired immune deficiency syndrome (AIDS). The histological pattern in AIDS differs from the more familiar classical Kaposi's sarcoma. The features most useful in making the diagnosis are: dissection of collagen; lymphatic vessel like spaces; angiomatoid lesions; premonitory sign; and spindle cell proliferation. It is important to examine multiple levels of small biopsy specimens and to be cautious in making the diagnosis of patch Kaposi's sarcoma in the presence of recent or healed ulceration and at sites of previous trauma. Only four of 16 patients with AIDS had evidence of systemic Kaposi's sarcoma, supporting the view that Kaposi's sarcoma in AIDS does not necessarily have an aggressive clinical course.     

The role of RIP1 and RIP3 in the development of aplastic anemia induced by cyclophosphamide and busulphan in mice

This study aimed to investigate the role of RIP1 and RIP3 in the pathogenesis of aplastic anemia (AA) induced by cyclophosphamide and busulphan in mice. Animals were randomly divided into three groups: the control group, the AA group, and the Nec-1 group. Mouse AA model was established by intraperitoneal injection of cyclophosphamide (40 mg/kg/d) and busulfan (20 mg/kg/d) for 12 days. The Nec-1 group mice received intraperitoneal injection of Nec-1 (2 mg/kg/d) for 12 days prior to intraperitoneal injection of cyclophosphamide (40 mg/kg/d) and busulfan (20 mg/kg/d) for 12 days. The control mice received intraperitoneal injection of equal volume of saline. At 12 h after the last intraperitoneal injection, blood and bone marrow tissues were collected from mice. Peripheral blood cells were analyzed using hematology analyzer and the histological changes of bone marrow tissues were examined using scanning electron microscopy (SEM). The levels of RIP3 and RIP3 in bone marrow were measured using Western blot analysis and the interaction of RIP1 and RIP3 proteins was investigated on the basis of immunoprecipitation analysis. ELISA was used to measure the levels of IL-6, TNF-α, and FLT-3L in bone marrow tissue supernatant. Apoptosis and necrosis of bone marrow cells were analyzed using flow cytometry. Western blot showed that the expression of RIP1 and RIP3 was significantly increases in AA mice compared to the normal controls. Immunoprecipitation detected the pro-necrotic RIP1-RIP3 complex, suggesting that RIP1 and RIP3 mediated necroptosis may involved in the damage of bone marrow cells. Compared to the AA mice, Nec-1 group mice exhibited significantly increase of peripheral blood cells and mononuclear cells in bone marrow tissues and decrease of the apoptosis/necrosis of bone marrow cells. In addition, we observed significant decrease of IL-6, TNF-α, and FLT-3L in bone marrow tissue supernatant in the Nec-1 group mice compared to AA mice. Our results suggest that Nec-1 can prevent the development of AA by inhibiting bone marrow cells necrosis and the production of inflammatory mediators. RIP1 and RIP3-mediated necroptosis may involve in the pathogenesis of AA induced by cyclophosphamide and busulfan in mice.     

Lymphocytotoxic antibodies in the acquired immune deficiency syndrome (AIDS)

The acquired immune deficiency syndrome (AIDS) manifested by opportunistic infections or Kaposi's sarcoma is a newly recognized and often fatal disease. Three patients seen in Syracuse, New York, were noted to have lymphopenia and persistent serum lymphocytotoxic antibodies (LCTAs). In a double blinded study, 25 serum samples were coded and sent to us by the Centers for Disease Control Task Force on Kaposi's Sarcoma and Opportunistic Infections. Samples from 5 patients with Pneumocystis carinii pneumonia, 5 patients with Kaposi's sarcoma, 5 presumably healthy homosexual males, 5 presumably healthy heterosexual males, and 5 presumably healthy heterosexual females were included. Of the ten AIDS patients, nine had "positive" or "suspicious" results on testing for lymphocytotoxic antibodies. The five heterosexual male and five heterosexual female controls had "negative" results. Of the five homosexual male "controls," three had "positive" or "suspicious" LCTA results. Two of these three "controls" were available for follow-up. Both showed deficiencies in studies of their cell-mediated immunity. Lymphocytotoxic antibodies may participate in the ongoing immunodeficiency seen in AIDS.     

Cytogenetic differentiation of Fanconi anemia, “idiopathic” aplastic anemia,and Fanconi anemia heterozygotes

We have analyzed chromosome breaks in 8 patients with Fanconi anemia (FA), 42 with “idiopathic” aplastic anema (AA), 15 first-degree relatives of FA patients, and 13 controls. Their lymphocytes were treated in culture with three concentrations of mitomycin-C (MMC). A 60-fold increase in breaks was observed in FA patients as compared to AA patients, regardless of severity of clinical signs. The MMC-stress test was standardized to clearly differentiate FA from other pancytopenias in doubtful cases. Also, the effect of storage of MMC in solution was investigated. The data on SCEs of 12 subjects tested, 10 mo apart, showed an inverse relationship between length of storage of MMC and chromosome damage. The 10-month-old solution induced only one half as many SCEs as it induced at 4 months. Further, the usefulness and power of diepoxybutane (DEB) in detection of FA heterozygotes was investigated in 12 first-degree relatives of patients with Fanconi anemia and 12 healthy controls. The mean number of chromosome breaks per mitosis by DEB stress in obligate heterozygotes was 0.08 in comparison to 0.06 in controls. Four of twelve control subjects showed proportions of breaks almost identical to or higher than those of FA heterozygotes, ie, 0.12, 0.10, 0.10, and 0.11 breaks per mitosis. The responses of healthy controls to DEB could be separated into two groups: one with mean chromosome breaks of 0.11 per mitosis, and a second with mean breaks of 0.04 per mitosis. Thus, it appears that heterozygote detection by DEB stress of cultured lymphocytes is not unequivocal.     

Transient bone marrow hypoplasia preceding T-Cell acute lymphoblastic leukemia: a case report

BackgroundAcute lymphoblastic leukemia (ALL) is the most common childhood malignancy and is characterised by hyperproliferation of malignant lymphocytes in the bone marrow. Rarely, ALL may be preceded by a period of pancytopenia and bone marrow hypoplasia which spontaneously recovers. This phenomenon, which has not before been described in T-cell ALL, is referred to as transient bone marrow hypoplasia.Case presentationA 5-year-old boy who presented with high-grade fever and generalised lymphadenopathy, was found to have pancytopenia on peripheral blood count and bone marrow hypoplasia. He was observed over a one-month period during which his bone marrow and peripheral blood counts recovered spontaneously. Symptoms recurred after 4 months and he was found to have blast infiltration of the bone marrow and diagnosed with T-cell ALL.ConclusionCases of transient bone marrow hypoplasia or overt aplastic anemia with spontaneous recovery and then followed by B-cell ALL or Acute Myeloid Leukemia have been described previously in the medical literature. This is the first case of transient bone marrow hypoplasia resulting into ALL of T-cell immunophenotype. While marrow hypoplasia preceding ALL remains poorly understood, it suggests an antecedent environmental insult to lymphoid progenitors or a germline abnormality that predisposes to lymphoid dysplasia. This may provide clues to the hitherto unknown pathophysiological process and etiological factors that precede the majority of childhood ALL cases. This case enlightens pediatricians about the existence of such rare cases so as to periodically follow up children with pancytopenia and/or bone marrow hypoplasia for prolonged periods even after apparent recovery.     

干细胞白血病基因在再障和正常骨髓基质细胞中的表达

目的：了解白血病干细胞(SCL)基因在再生障碍性贫血(AA)骨髓基质细胞 (BMSC)及骨髓细胞中的表达情况。方法：收集9例AA和33例正常骨髓标本。体外长期培养扩增BMSC，并收集悬浮细胞。运用反转录聚合酶链反应酶联免疫吸附测定(RT-PCR-ELISA)检测SCL基因在BMSC和悬浮细胞中的表达，分析表达率，并以管家基因β2微球蛋白(β2M)为内参照进行半定量分析。结果：SCL基因在AA的BMSC中的表达率(22.2％)低于正常对照组(69.7％，P<0.05)。SCL基因在AA的悬浮细胞中的表达率(87.5％)高于其对应的BMSC(P<0.05)。PCR-ELISA半定量分析结果显示，SCL基因在正常对照组悬浮细胞中的表达水平高于其对应的BMSC(P<0.05)。结论：SCL基因在AA的BMSC中的相对低表达状态提示可能与AA造血调控异常有关。     

An atypical case of Fanconi anemia in elderly sibs

We describe a 56-year-old woman suspected of Fanconi anemia on the basis of the following clinical findings: microcephaly, short stature, congenital deafness, and the clinical findings in her deceased brother. Hematologic or other signs of malignancy were absent. The diagnosis was confirmed by demonstrating hypersensitivity of her lymphocytes to mitomycin C (MMC). Cell fusion experiments indicated that the patient belongs to complementation group A. The patient's brother died at the age of 50 of heart and renal failure, and anemia. He had clinical findings similar to those of his sister, and a horseshoe kidney. From 31 years on he had thrombocytopenia and leucopenia. Both patients had insulin-dependent diabetes mellitus. A chromosomal breakage test carried out elsewhere before his death failed to demonstrate MMC hypersensitivity of his lymphocytes, which led to the investigation of his sister. To our knowledge these two cases are the oldest Fanconi anemia patients reported thus far. Am. J. Med. Genet. 68:362–366, 1997. © 1997 Wiley-Liss, Inc.     

环孢素A治疗再障和骨髓增生异常综合征T淋巴细胞异常激活的变化

研究再生障碍性贫血(AA)用环孢素A(CsA)治疗前后和骨髓增生异常综合征(MDS)患者外周血CD4+、CD8+T淋巴细胞培养前后早期激活标志CD69的表达及其意义。将外周血在PHA20μg/ml条件下进行全血细胞培养,于0h和4h分别用双色免疫荧光标记流式细胞仪对CD4+、CD8+T淋巴细胞CD69的表达进行分析。发现PHA刺激前初治SAA和MDS-RA+MDS-RAS患者CD4+、CD8+细胞CD69的表达率增高,CAA与RAEB+RAEB-T患者CD8+细胞CD69的表达率增高;PHA刺激后AA与MDS患者CD4+、CD8+细胞表达CD69明显增强,AA患者CD4+细胞CD69的表达率高于CD8+细胞。CsA治疗后SAA患者PHA刺激前CD4+、CD8+细胞CD69的表达率较治疗前明显减低,CAA患者CD8+细胞CD69的表达率较治疗前明显减低。治疗后AA患者PHA刺激后CD4+、CD8+细胞CD69的表达率较治疗前明显减低。CsA治疗有效的AA患者治疗前PHA刺激前后CD4+、CD8+细胞CD69的表达率明显增高,治疗后明显减低。初治AA患者PHA刺激前后CD4+细胞CD69的表达率均明显高于MDS患者。说明T细胞早期活化及其活化潜能增强,以及产生针对自身造血干/祖细胞的细胞毒效应在AA和MDS发病中起重要作用,CsA能抑制AA患者T细胞的早期激活。     

Biopsy pathology of acquired immune deficiency syndrome (AIDS).

Between January 1982 and May 1986 279 biopsy specimens from 82 patients with acquired immune deficiency syndrome (AIDS) were examined. A wide variety of infectious conditions were diagnosed, the commonest being Pneumocystis pneumonia (n = 36), cytomegalovirus (n = 21), a variety of fungi (n = 8), mycobacteria (n = 7). Kaposi's sarcoma was the commonest tumour (n = 40), and there were two cases of extranodal lymphoma. Striking features were the unusual sites of disease and the occasional paucity of organisms.     

Lymphocyte-reactive antibodies in acquired immune deficiency syndrome

Antilymphocyte antibodies were studied using the Terasaki microcytotoxicity technique in 21 gay patients including 7 with Kaposi's sarcoma, 5 with opportunistic infection, and 9 with lymphadenopathy syndrome. A significant increase in lymphocyte-reactive antibody was noted in 61% of this group. Similar studies using serum from 25 apparently healthy gay males showed lymphocytotoxic antibody in only one instance. When isolated T-cell subsets (OKT4+ or OKT8+) were utilized, a few sera from acquired immune deficiency syndrome (AIDS) patients apparently showed preferential killing for helper-inducer or suppressor-cytotoxic T-cell subsets, however, this showed no correlation with T-cell phenotypic profiles of OKT4 or OKT8+ cells in concurrent peripheral blood studies. When isolated normal OKT4 or OKT8 T-cell subsets were treated with complement and AIDS serum showing apparent T-cell subset specificity and treated T cells added to B cells, macrophages, and OKT4 or OKT8 cells cultured with pokeweed mitogen, no discernible effect on either help or suppression of IgG or IgM synthesis was recorded.     

Treatment of the acquired immune deficiency syndrome

The acquired immune deficiency syndrome (AIDS) and AIDS-related complex (ARC) are sequelae of immune system injury initiated by a novel human retrovirus [human T-lymphotrophic virus strain III/lymphadenopathy-associated virus (HTLV III/LAV)]. The resulting spectrum of immune deficiency sets the stage for opportunistic infection and malignancy. In this review, we consider progress made in the treatment and prevention of AIDS and HTLV III/LAV infection. Immunomodulator and antiviral approaches are discussed.