Lymphocytes and antibody in retrovirus-induced feline pure red cell aplasia

The possible role of antibody and T-lymphocytes was investigated in the pure red cell aplasia (PRCA) associated with feline leukemia virus, subgroup C (FeLV-C), infection. In previous studies, erythroid colony-forming cells were undetectable in marrow culture of cats with PRCA. Yet erythroid burst-forming cells (BFU-E) remained, suggesting that BFU-E were able to differentiate in vitro but not in vivo. It was inferred that immunologic suppression may contribute to the pathogenesis of feline PRCA, and the interactions of antibody and T-lymphocytes with erythroid and granulocyte-macrophage progenitors were studied. Incubation of normal or PRCA marrow cells with PRCA serum or IgG concentrated from this serum and then complement (C') failed to decrease hematopoietic colony growth when compared to the results obtained with cultures of marrow cells incubated with C' alone. In crossover coculture studies, T-cells from Safari cats with PRCA had no inhibitory effect on colony growth from normal or autologous PRCA marrow cells. For the determination of whether feline PRCAs were associated with a clonal T-cell process, lymphocytes were obtained periodically from glucose-6-phosphate dehydrogenase (Glc-6-PD) heterozygous cats following FeLV-C infection and were expanded with a crude preparation of interleukin-2. The ratios of Glc-6-PD enzyme types in these samples did not change as cats developed anemia, suggesting that the inhibition of erythropoiesis was not associated with the clonal expansion of T-cells. These studies, therefore, do not support the premise that feline PRCA results from the interaction of antibody or T-cells with erythroid progenitors.     

Epstein-Barr virus associated diffuse large B-cell lymphoma complicated by autoimmune hemolytic anemia and pure red cell aplasia.

A 53-year old man with systemic lymphadenopathy and hepatosplenomegaly was diagnosed with diffuse large B cell-lymphoma after inguinal lymph node biopsy. Anemia was noted, direct and indirect Coombs tests were positive, and the haptoglobin level was low. However, the bone marrow aspirate revealed erythroid aplasia. Co-existing autoimmune haemolytic anemia (AIHA) and pure red cell aplasia (PRCA) were diagnosed. In situ hybridization with Epstein-Barr virus (EBV) encoded small RNA (EBER) showed positive findings in lymphoma cells. Southern blot hybridization revealed immunoglobulin heavy chain gene rearrangement and a clonal EBV terminal repeat, indicating monoclonal proliferation of EBV in infected B cells. The patient was treated with CHOP, resulting in a complete remission (CR). AIHA and PRCA subsided after 3 courses of chemotherapy. In conclusion, this case demonstrates not only the association of B-cell lymphoma with autoimmune disorders but also the involvement of EBV in these conditions.     

Antineoplastic dolastatins: potent inhibitors of hematopoietic progenitor cells.

Dolastatins 10 and 15, isolated from the shell-less marine mollusk Dolabella auricularia, are potent antineoplastic agents with unknown myelotoxic effects in vivo. The goal of this study was to determine whether the dolastatins inhibit the proliferation of normal hematopoietic progenitor cells. Assays to test inhibition of colony formation and of cell proliferation were performed in vitro with bone marrow cell preparations enriched for progenitor cells and with progenitor cell lines, respectively, using varying drug concentrations and exposure times. Dolastatins 10 and 15 both inhibited human and murine bone marrow cell colony formation in a concentration-dependent manner, with the concentration required for half maximal inhibition ranging from 0.1 to 1 pg/mL for dolastatin 10 and from 10 to 100 pg/mL for dolastatin 15. These concentrations are 25-fold to 100-fold lower than the concentration required for antineoplastic activity. Complete inhibition of human bone marrow cell colony formation was observed at concentrations of 10-100 pg/mL for dolastatin 10 and 1000-10,000 pg/mL for dolastatin 15. Committed progenitor cells and multipotential progenitor cells were similarly inhibited. The magnitude of inhibition of human hematopoietic cell colony formation was dependent on pre-exposure time to dolastatins 10 and 15, with a reversible effect up to 8 hours and with a 24-hour preincubation resulting in maximal (100%) and irreversible inhibition. Dolastatin 10 at a concentration of 10-100 pg/mL limited the proliferation of six human and four murine hematopoietic progenitor cell lines, as measured by tritiated thymidine incorporation, to between 34% and 83% of that occurring in the absence of the drug. These results indicate that the dolastatins are potent inhibitors of normal hematopoietic progenitor cell proliferation.     

重组人集落刺激因子对脐带血和骨髓造血祖细胞体外培养的影响

分别应用单层琼脂培养和甲基纤维素培养方法，同期观察了重组人粒细胞集落刺激因子（ｒｈＧ－ＣＳＦ）、重组人粒单细胞集落刺激因子（ｒｈＧ－ＣＳＦ）、重组人白细胞介素－３（ｒｈＩＬ－３）和红细胞生成素（ＥＰＯ），对脐带血和骨髓造血祖细胞的影响。结果显示：脐带血中含有丰富的造血祖细胞，其早期的造血祖细胞数量与骨髓相似，脐带血的造血祖细胞的增殖能力更强。     

Pure red cell aplasia in association with malignant histiocytosis

The authors report a case of malignant histiocytosis complicated with pure red cell aplasia. Erythrophagocytic histiocytes were found only rarely and no histiocyte ingested erythroblasts. Neither serum immunoglobulin G nor peripheral blood T-cells have any inhibitory activity to erythroid colony growth from allogeneic bone marrow. Pure red cell aplasia is an unusual complication in malignant histiocytosis.     

Lineage alteration in precursor B cell acute lymphoblastic leukemia following T cell lymphoblastic lymphoma.

A 10-year-old girl was diagnosed with lymphoblastic lymphoma; staging evaluation revealed a large mediastinal mass and normal peripheral blood and bone marrow morphology. Tumor cell immunologic marker analysis and Southern blot gene rearrangement studies demonstrated a T-cell lineage. She achieved a complete remission following multi-agent chemotherapy; however, 19 months following initial diagnosis while on maintenance therapy, she presented with typical acute lymphoblastic leukemia (ALL). The bone marrow was replaced by lymphoblasts, though the mediastinum was normal and there was no peripheral lymphadenopathy. Repeat immunophenotypic and genotypic studies demonstrated a precursor B-cell ALL lineage without expression of the T-cell surface antigens present on the original neoplasm. Repeat genotypic analysis showed immunoglobulin heavy and light chain gene rearrangements without the T-cell receptor gamma and beta gene rearrangements noted in the original lymphoblastic lymphoma. The complete alteration of lineage in these lymphoblastic processes suggests the de novo occurrence of a second neoplasm or, alternatively, an ALL relapse from a lineage-uncommitted neoplastic lymphoid progenitor cell.     

Stimulation of B-lymphocyte colony formation in vitro by sera from patients with leukaemia or lymphoma.

Studies were made on the effects of 665 sera, from normal donors or patients with various diseases, on B-lymphocyte colony formation in agar by mouse spleen cells. Undiluted serum from most normal donors inhibited colony formation, but 43-53% of sera from patients with histiocytic lymphoma, lymphocytic lymphoma or Hodgkin's disease stimulated colony formation, serum activity correlating with the stage of the disease. Moderate colony-stimulating activity was observed with serum taken from patients with acute lymphoid or myeloid leukaemia following, but not prior to, chemotherapy. Colony stimulating activity was not correlated with the blood group of serum donors and could not be ascribed to the presence of endotoxin, red cells or mouse red cell haemagglutinins in the active sera. Elevated colony stimulating activity was not observed in sera from patients with non-neoplastic disorders ot haemopoiesis or with diseases of other organ systems.     

Effect of lymphokine-activated killer cell fraction on the development of human hematopoietic progenitor cells

Lymphokine-activated killer (LAK) cells from cultures of human peripheral blood mononuclear cells with recombinant interleukin-2 (rIL-2) have been clinically used in adoptive immunotherapy for cancer patients. To study their influence on human hematopoiesis, the LAK cell fraction was cocultured with marrow nonphagocytic cells from normal subjects in an assay system of hematopoietic progenitors. The fraction suppressed colony growth from relatively mature erythroid progenitors in a dose-dependent manner. Although unactivated cells, which were produced without IL-2, augmented the growth of early erythroid progenitors, the LAK cell fraction did not. This fraction suppressed colony growth from mature granulocyte-macrophage progenitors (day 7 CFU-GM) especially with an 18-h preincubation prior to coculture. It also suppressed both immature granulocyte-macrophage progenitors (day 14 CFU-GM) and multipotential hematopoietic progenitors. The suppressive effects were observed on colony growth from autologous marrow cells as well as allogeneic marrow cells. The suppression of day 7 CFU-GM colony growth by supernatants due to preincubation with marrow cells and the LAK cell fraction suggested that the humoral factor contributes to the suppression by the LAK cell fraction. These data suggest that the LAK cell fraction suppresses the development of human hematopoietic progenitor cells.     

侵袭型非霍奇金淋巴瘤合并纯红细胞再生障碍性贫血

目的:研究侵袭性非霍奇金淋巴瘤并发纯红细胞再生障碍性贫血(PRCA)的临床特点和治疗结果。方法:报告两例分别并发于非特指型外周T细胞淋巴瘤(PTCL-NOS)和弥漫大B细胞淋巴瘤(DLBCL)的PRCA,并复习相关文献。结果:在联合化疗治疗后,DLBCL及其相关的PRCA均获完全缓解,而PTCL-NOS虽获缓解,但其相关的PRCA未好转,加用泼尼松治疗后PRCA缓解。结论:NHL相关的PRCA在联合化疗或免疫抑制治疗后可获完全缓解,且可不需维持治疗。     

Growth kinetics and blast-colony forming cell binding capacity of aplastic anaemic stromal cells

The kinetics of bone marrow cell growth and a special function of stromal cells (the capability of binding blast colony forming cells) were studied in patients with aplastic anaemia (AA). All 10 patients studied showed faster growth of bone marrow stromal cells. The time for a confluent stromal layer formation was 24.5 days for AA bone marrow as opposed to 33.0 days for normal bone marrow. This faster growth rate could also be observed if normal bone marrow cells, depleted of plastic non-adherent fraction, were plated, suggesting that at least one of the reasons for altered stromal cell growth kinetics in AA is the changes in the ratio of plastic adherent/non-adherent cells. Functionally, i.e. in supporting the growth of normal bone marrow blast colonies, AA stromal layers did not differ from that of normal stromal layers, independently of the clinical state of the disease (AA or SAA; in one patient before or after ATG treatment; in two patients after successful allogenic bone marrow transplantation). Moreover, in some AA patients this blast colony forming cell binding function of AA stromal layers could also be detected in samples cultured without hydrocortisone (i.e. in the absence of fat cells), suggesting that AA stroma also differs qualitatively from normal stroma without inducing a defective microenvironment for stem cell homing.     

Avoidance of bone marrow suppression using A-5021 as a nucleoside analog for retrovirus-mediated herpes simplex virus type I thymidine kinase gene therapy

Gene therapy using the herpes simplex virus thymidine kinase (HSV-TK) gene combined with an anti-herpes drug, ganciclovir (GCV), has been applied for human diseases, especially for cancer treatment. However, bone marrow toxicity has been the most consistent adverse effect of GCV treatment in clinical settings. We evaluated the cytotoxic activity of a novel guanosine analog, (1'S,2'R)-9[[1',2'-bis(hydroxymethyl)cycloprop-1'-yl]methyl]guanin e (A-5021), against retrovirus-mediated HSV-TK gene-transduced human lung cancer cells. The bone marrow toxicity of A-5021 and GCV was studied by colony formation assay in both rodent and human bone marrow specimens. We demonstrated that A-5021 had potent cytotoxic activity equal to that of GCV against the retroviral vector-mediated HSV-TK gene-transduced lung cancer cell lines. Further, phosphorylated A-5021 could be transferred to neighboring cells, and this analog killed HSV-TK- neighboring cells, as was the case for GCV. In contrast, A-5021 did not exhibit an inhibitory effect on bone marrow progenitor cells and colony formation (the 50% inhibitory concentration of the colony-forming units in culture = >100 microg/mL in human bone marrow specimens and >66 microg/mL in rodent bone marrow specimens). These results indicate that A-5021 has potent cytotoxic activity as a nucleoside analog for gene therapy using HSV-TK gene, and can be used much more safely than GCV.     

Growth kinetics and blast-colony forming cell binding capacity of aplastic anaemic stromal cells.

The kinetics of bone marrow cell growth and a special function of stromal cells (the capability of binding blast colony forming cells) were studied in patients with aplastic anaemia (AA). All 10 patients studied showed faster growth of bone marrow stromal cells. The time for a confluent stromal layer formation was 24.5 days for AA bone marrow as opposed to 33.0 days for normal bone marrow. This faster growth rate could also be observed if normal bone marrow cells, depleted of plastic non-adherent fraction, were plated, suggesting that at least one of the reasons for altered stromal cell growth kinetics in AA is the changes in the ratio of plastic adherent/non-adherent cells. Functionally, i.e. in supporting the growth of normal bone marrow blast colonies, AA stromal layers did not differ from that of normal stromal layers, independently of the clinical state of the disease (AA or SAA; in one patient before or after ATG treatment; in two patients after successful allogenic bone marrow transplantation). Moreover, in some AA patients this blast colony forming cell binding function of AA stromal layers could also be detected in samples cultured without hydrocortisone (i.e. in the absence of fat cells), suggesting that AA stroma also differs qualitatively from normal stroma without inducing a defective microenvironment for stem cell homing.     

Primary bone marrow T-cell anaplastic large cell lymphoma with triple m gradient

We present a case of a 60-year-old male patient with primary bone marrow anaplastic large cell lymphoma. He was admitted to the hospital with the symptoms of anemia and fever. There was no evidence of lymphadenopathy or splenomegaly. Immunoelectrophoresis showed the presence of a triple M gradient (double IgM and an IgG), with the IgG and one of the IgM paraproteins functioning as a cryoglobulin. The patient had no hepatitis C virus infection. Bone marrow biopsy showed massive CD30-positive, ALK-negative large lymphoid cell infiltration of T-cell origin with anaplastic morphology. PCR analysis of lymphoid cells separated from the bone marrow demonstrated the presence of a B/T hybrid genotype disorder with no evidence of the t(2;5), nor t(1;2) translocations. The patient entered a period of remission following CHOP chemotherapy. The patient subsequently died of sepsis as a consequence of serious humoral immunodeficiency.     

侵袭型非霍奇金淋巴瘤合并纯红细胞再生障碍性贫血

目的：研究侵袭性非霍奇金淋巴瘤并发纯红细胞再生障碍性贫血（PRCA）的临床特点和治疗结果。方法：报告两例分别并发于非特指型外周T细胞淋巴瘤（PTCL-NOS）和弥漫大B细胞淋巴瘤（DLBCL）的PRCA,并复习相关文献。结果：在联合化疗治疗后,DLBCL及其相关的PRCA均获完全缓解,而PTCL-NOS虽获缓解,但其相关的PRCA未好转,加用泼尼松治疗后PRCA缓解。结论：NHL相关的PRCA在联合化疗或免疫抑制治疗后可获完全缓解,且可不需维持治疗。     

Effects of sera from patients with malignant lymphomas on granulocytic progenitor cells (CFU-C): Changes after treatment

Serum from normal subjects, under certain methodologic conditions, increases the colony-forming ability of normal bone marrow. Sera from eight patients with malignant lymphoma reduced the colony-forming ability of normal marrow. The results suggest the presence in these patients of a toxic or inhibitory serum factor rather than the absence of a stimulating factor.Longitudinal studies done in four patients show that this activity is difficult to attribute only to the feedback control of granulopoiesis and that other activities associated with the disease probably intervene.When the effect of sera from three patients on their own marrow and on a normal receptor marrow was compared, the reactivity of their granulocytic progenitor cells (CFU-C) was similar to the reactivity of CFU-C in the normal receptor marrow.Finally, the bone-marrow colony-forming ability of the patients studied and the size of their CFU-C compartment were found to be subnormal or reduced. The significance of these findings together with a serum factor that reduces the colony-forming ability of normal receptor marrow is discussed.     

Effects of recombinant human tumor necrosis factor on highly enriched hematopoietic progenitor cell populations from normal human bone marrow and peripheral blood and bone marrow from patients with chronic myeloid leukemia

Previous studies using unseparated normal human bone marrow cells have indicated that recombinant tumor necrosis factor alpha (rTNF-alpha) can inhibit the in vitro colony growth by normal granulocyte/macrophage (CFU-GM) and erythroid (BFU-E) progenitor cells in a dose-dependent manner. In the present studies, by using very low numbers of highly enriched normal bone marrow progenitor cell populations as target cells, we have extended these previous findings to provide convincing evidence that erythroid and myeloid colony growth suppression by rTNF-alpha is manifested by a direct interaction between rTNF-alpha and CFU-GM and BFU-E progenitor cells. In addition, the sensitivity of normal peripheral blood and chronic myeloid leukemia bone marrow CFU-GM and BFU-E colony growth to inhibition by rTNF-alpha was examined and found to be comparable with that of normal bone marrow CFU-GM and BFU-E. Although the continuous presence of high doses of rTNF-alpha (5000 units/ml) was required in methylcellulose cultures for maximal CFU-GM (90%) and BFU-E (70%) colony suppression, short-term exposure (24 to 72 hr) of normal bone marrow-enriched progenitor cells to rTNF-alpha, in the absence of hematopoietic growth factors, was sufficient to irreversibly suppress up to 50 to 65% of CFU-GM colony growth. In contrast, the number of BFU-E colonies was increased under these conditions. If, however, hematopoietic growth factors (Mo-T-cell-conditioned medium and erythropoietin) were present during preincubation of the cells with rTNF-alpha, BFU-E were then slightly suppressed while the extent of CFU-GM inhibition remained essentially the same. The suppressive effect of rTNF-alpha on erythroid and myeloid progenitor cell growth appears to be most pronounced on the more primative stages of committed progenitor cell development, since inhibition of CFU-GM- and BFU-E-derived colony growth progressively decreased with the delayed addition of rTNF-alpha to methylcellulose cultures. [3H]Thymidine incorporation was also inhibited by rTNF-alpha in normal bone marrow-enriched progenitor cell populations stimulated to proliferate in liquid culture by colony-stimulating factors. This effect was transient, however, since the activity of rTNF-alpha declined after the first 24 h of culture at 37 degrees C, particularly at low doses of rTNF-alpha where the activity was completely lost after 48 h of culture. This loss of activity appeared to be due to a decreased sensitivity of progenitor cells to the antiproliferative effects of tumor necrosis factor (TNF) after an initial exposure rather than a lack of available TNF.(ABSTRACT TRUNCATED AT 400 WORDS)     

Differential cytotoxicity of deoxyguanosine and 8-aminoguanosine for human leukemic cell lines and normal bone marrow progenitor cells

The inhibitory effect of deoxyguanosine (GdR) alone or in combination with the purine nucleoside phosphorylase (PNP) inhibitor, 8-aminoguanosine (AG) was tested on human T, B, cALL and myeloid leukaemia cell lines and on normal human bone marrow haemopoietic progenitor cells. GdR was found to be toxic to T-leukaemia cells. AG (100μm ) alone did not have any inhibitory effect, but when used with GdR (2·5 × 10^?5m ) a synergistic effect was seen towards T cells. Incubation with GdR and AG resulted in a marked decrease in cell viability (> 90 per cent in three and > 75 per cent in four of 5 T leukaemic cell lines tested at 72 h). This drug combination did not inhibit the growth of non-T leukaemic cells and was also non-toxic to normal bone marrow multipotent progenitor cells (CFU-GEMM) in vitro. Adenosine deaminase (ADA) acts consecutively with PNP in purine degradation. The addition of an ADA inhibitor, deoxycoformycin and deoxyadenosine, however, did not enhance the toxicity of GdR and AG for T cell leukaemia. The possibility of using GdR and AG for in vitro removal of residual T leukaemic blasts with the sparing of normal bone marrow cells, prior to autologous bone marrow transplantation should be further explored.     

Pure red cell aplasia associated with parvovirus B19 infection in T-large granular lymphocyte leukemia

There have been few reports of large granular lymphocyte (LGL) leukemia with neutropenia complicated with pure red cell aplasia (PRCA) that developed after human parvovirus (HPV) B19 infection. We report here the case of a 35-year-old female who developed HPV B19-associated PRCA with T-LGL leukemia. LGL count of peripheral blood was lower than 2 x 10(9) l(-1), although phenotypic analysis of LGL showed CD3+, CD16-, CD56-, CD57+ with double positive for CD3 and CD57, and genetic study showed the clonal rearrangement of T-cell receptor gene. Microscopically, the patient's bone marrow showed characteristic giant proerythroblasts. A serologic study of HPV B19 was positive for IgM, but negative for IgG, with a positive result on Dot-blot hybridization assay for HPV B19 DNA. Severe anemia and reticulocytopenia ameliorated without treatment 10 days after the initial examination, but slight anemia, neutropenia, a moderate increase of LGL counts with rearrangement of TCR gene, and positive result of HPV B19 DNA has persisted 7 months after the initial examination. We suggest that this viral infection may play an etiologic role in some patients with LGL leukemia who develop PRCA.     

Establishment of a follicular lymphoma cell line (FLK-1) dependent on follicular dendritic cell-like cell line HK.

A novel cell line, FLK-1, was established from bone marrow cells of a patient with follicular lymphoma by means of co-culture with follicular dendritic cell (FDC)-like cell line HK. Immunophenotypic analysis showed that FLK-1 expressed CD10, CD19, CD20, CD38, IgG and HLA-DR, which is a typical feature of germinal center B cells. Cytogenetic analysis of FLK-1 demonstrated t(14;18)(q32;q21) translocation involving BCL2 and immunoglobulin heavy chain genes. Especially noteworthy is that the growth of FLK-1 was found to be dependent on a FDC line, HK. When HK cells were removed from the culture, FLK-1 cells stopped growing and eventually died. An apoptotic mechanism appeared to be involved as indicated by the presence of chromosome condensation and DNA ladder formation. The culture experiment using micropore membranes showed that soluble factor(s) of HK cells supported the growth, while direct cell-to-cell contact appeared to be necessary for longterm cell proliferation. These findings suggest the importance of the micro-environment for follicular lymphoma cells to grow. The FLK-1 cell line may thus prove to be useful for studying the growth mechanism of follicular lymphoma and provide new insights into the pathogenesis of follicular lymphoma.     

Effects of recombinant human tumor necrosis factor alpha, recombinant human gamma-interferon, and prostaglandin E on colony formation of human hematopoietic progenitor cells stimulated by natural human pluripotent colony-stimulating factor, pluripoietin alpha, and recombinant erythropoietin in serum-free cultures

The influences of pure human pluripotent colony-stimulating factor, highly purified pluripoietin alpha, pure recombinant human tumor necrosis factor alpha, pure recombinant human gamma-interferon, and natural prostaglandin E1 (PGE1) were evaluated on colony formation of multipotential and erythroid progenitor cells in the presence of recombinant erythropoietin and hemin and on colony formation of granulocyte-macrophage progenitors in normal human marrow cultured in the presence or absence of serum. Serum was replaced by bovine serum albumin, iron-saturated transferrin, cholesterol, and calcium chloride. Increasing concentrations of pluripotent colony-stimulating factor and pluripoietin alpha stimulated increasing numbers of colonies from nonadherent low-density T-lymphocyte-depleted cells in the absence and presence of serum. Growth was usually greater in the presence of serum and on a unit basis pluripoietin alpha was more active than pluripotent colony-stimulating factor. Recombinant human tumor necrosis factor alpha and recombinant human gamma-interferon suppressed colony formation colony forming unit-granulocyte-macrophage, burst forming unit-erythroid, and colony forming unit-granulocyte-erythroid-macrophage-megakaryocyte; PGE1 suppressed colony formation by colony-forming unit-granulocyte-macrophage, stimulated colony formation by burst forming unit-erythroid, and had no effects on colony formation by colony forming unit-granulocyte-erythroid-macrophage-megakaryocyte in both serum-containing and serum-free medium. The PGE1 enhancing effects on erythroid colony formation required T-lymphocytes. Thus, results are similar using serum-containing and serum-free cultures of human bone marrow cells and serum-free defined culture medium can be used to study the mechanism of action of purified natural and recombinant growth and suppressor molecules in vitro.