Lack of clinical efficacy of rituximab in the treatment of autoimmune neutropenia and pure red cell aplasia: implications for their pathophysiology.

We describe 11 patients with severe refractory autoimmune cytopenias treated with the anti-CD20 monoclonal antibody rituximab. Six patients had autoimmune neutropenia (AIN), two had pure red cell aplasia (PRCA), one had AIN and autoimmune haemolytic anaemia, one had AIN and immune thrombocytopaenia purpura (ITP) and one had PRCA and ITP. Rituximab was administered at a dose of 375 mg/m(2) as an intravenous infusion weekly for 4 weeks. Six of eight patients with AIN and all three patients with PRCA did not respond. Two patients died: one with resistant AIN and autoimmune haemolytic anaemia died of pneumocytis pneumonia infection, and one with PRCA and ITP died of an acute exacerbation of bronchiectasis. Rituximab in AIN and PRCA appears to be less effective than Campath-1H when compared to historical data from our group. This supports the hypothesis that T cells may be important in the pathophysiology of AIN and PRCA.

Marrow transplants from matched unrelated donors for aplastic anaemia using alemtuzumab, fludarabine and cyclophosphamide based conditioning.

Graft failure, regimen-related toxicity and graft-versus-host disease (GVHD) are the critical barriers to unrelated donor transplants for aplastic anaemia (AA). We investigated the use of a novel conditioning regimen consisting of alemtuzumab (humanized CD52 antibody), fludarabine and cyclophosphamide in seven patients with AA, who underwent bone marrow transplant procedure using matched unrelated donors. The aetiology of AA was acquired (n=3), Fanconi's (n=3) and congenital (n=1). Median age was 13 years (range 8-35). All the donors were fully matched for HLA class I and II antigens using high-resolution typing. All the patients engrafted at a median of 18 days (range 13-35). Two patients died of transplant-related complications: one of adenovirus disease and the other developed extensive chronic GVHD of skin followed by cytomegalovirus (CMV) disease. Three patients developed Grade II acute GVHD disease (GVHD); none had Grade III-IV acute GVHD. Of the six evaluable patients, only one developed chronic GVHD. We conclude that this conditioning regimen for unrelated donor transplants for AA is sufficiently immunosuppressive to allow stable engraftment and appears to have a favourable impact on the incidence and severity of GVHD, warranting further investigation.

c-kit protein (stem cell factor receptor) expression on cells with erythroid characteristics and on normal human bone marrow without the use of monoclonal antibodies.

Stem cell factor (SCF) is a determining and crucial element in the development of early hematopoietic cells. The SCF receptor protein has been identified as the product of the protooncogene c-kit and has been detected using monoclonal antibodies (MAbs) on a broad selection of erythroid, myeloid, and lymphoid cell lines as well as on bone marrow mononuclear cells (BMMNC). SCF is known to increase both the number and size of burst-forming unit-erythroid (BFU-E) colonies in normal human BM culture in a dose-dependent fashion. A detailed study of the involvement of SCF and its receptor c-kit in normal erythropoiesis will help elucidate intrinsic irregularities of anemias such as Diamond Blackfan Anemia, an aregenerative congenital anemia. Abnormalities of this heterogeneous disorder are confined to the red cell lineage and are thought to arise through a defect at the stem/progenitor cell level. Our in vitro studies suggest that SCF therapy will influence BFU-E production in at least a portion of these patients, although in another group, SCF response is limited or absent. Additionally, further investigations have shown a possible c-kit signaling defect that clearly necessitates further c-kit characterization. To parallel this, we, therefore, attempted to study the relationship of c-kit with its ligand. This report describes a nonradioactive method for detecting SCF receptors that varies from conventional assays in that the fluorescent label conjugated to the SCF/c-kit complex is connected via an extended-ester linkage that reduces steric influence and promotes full normal structural ligand binding of the SCF to its receptor.(ABSTRACT TRUNCATED AT 250 WORDS)

Interaction of granulocyte-macrophage colony-stimulating factor and interleukin 3 in human long-term bone marrow culture.

Granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin 3 (IL-3), or a combination of both growth factors were added weekly to normal human long-term bone marrow cultures (LTBMC). GM-CSF had a greater effect on the total nonadherent cell population than the committed progenitor cells (granulocyte-macrophage colony-forming units, CFUgm), whereas IL-3 had the opposite effect and stimulated the expansion of greater numbers of CFUgm than GM-CSF. The combination of both factors had an additive effect on CFUgm. The longevity of the growth factor-treated cultures was not reduced. These data indicate that IL-3 stimulates an earlier progenitor cell population than GM-CSF and that a combination of the two factors should be more effective in vivo and could be applied to the expansion of bone marrow progenitor cells in culture before bone marrow transplantation.

Mechanism of suppression of normal hemopoietic activity by lymphokine-activated killer cells and their products.

Interleukin 2 (IL-2)-activated lymphocytes (lymphokine-activated killer [LAK] cells) have been shown to inhibit the formation of autologous human granulocyte-macrophage hemopoietic progenitors (granulocyte-macrophage colony-forming units, CFU-GM) in vitro. Effects of LAK cells on these progenitors may include a number of different mechanisms. LAK cells are potent cytotoxic lymphocytes capable of lysing certain normal autologous cells. They also produce cytokines known to inhibit hemopoiesis (interferon gamma [IFN-gamma] and tumor necrosis factor alpha [TNF-alpha]) or enhance it (granulocyte-macrophage colony-stimulating factor, GM-CSF). In our current study we analyzed the mechanism of suppression of autologous CFU-GM by LAK cells. Our results suggest that LAK cells are not directly cytotoxic to normal CFU-GM. We show that it is possible to abolish the hemopoiesis-inhibiting activity of LAK cells without abrogating their cytotoxicity against tumor cell lines using inhibitors of DNA synthesis, namely hydroxyurea or irradiation.

The novel monoclonal antibody By114 helps detect the early emergence of a paroxysmal nocturnal hemoglobinuria clone in aplastic anemia.

The newly described monoclonal antibody By114 has been used with flow cytometry to investigate the status of the 90-kD glycosylphosphatidyl-inositol (GPI)-anchored component of CD66 (CD66c) on neutrophils from nine patients with paroxysmal nocturnal hemoglobinuria (PNH), seven with aplastic anemia/PNH, and 63 with aplastic anemia (AA) and a negative Ham's test. We have found that By114 is a sensitive indicator for recognizing patients with PNH and has helped delineate a group of nine patients with aplastic anemia and a negative Ham's test who have evidence of a larger PNH clone than indicated by other monoclonal antibodies (mAbs). By114 is a valuable marker for detecting the emergence of a PNH clone before the Ham's test becomes positive and is a more sensitive detector of deficient GPI-anchored proteins than other mAbs.

Increased apoptosis in aplastic anemia bone marrow progenitor cells: possible pathophysiologic significance.

We have quantitated apoptotic cells by flow cytometry in human bone marrow (BM) and peripheral blood (PB) from normal donors and aplastic anemia (AA) patients, using the fluorescent DNA-binding dye 7-amino actinomycin D (7AAD). No significant difference was found in baseline percent apoptosis between normal and AA samples. Serum deprivation induced cell death to a greater degree in AA samples than in normal samples, but this was not significant. Using dual staining with anti CD34 antibody and 7AAD, we have shown that CD34+ progenitors in normal PB are significantly more apoptotic than those in normal BM. AA BM CD34+ cells contain a significantly greater proportion of apoptotic cells than normal BM CD34+ cells. Those AA patients with the lowest absolute number of CD34+ cells showed the highest proportion of apoptotic CD34+ cells. This appears to be related to clinical severity (transfusion dependence) at the time of study. We conclude that apoptosis is accelerated in AA BM progenitors and that this may contribute to the stem cell deficiency characteristic of this disorder.

In vitro response of normal and aplastic anemia bone marrow to mast cell growth factor and in combination with granulocyte-macrophage colony-stimulating factor and interleukin-3.

We have examined the effect of mast cell growth factor (MGF), granulocyte-macrophage colony-stimulating factor (GM-CSF), and interleukin-3 (IL-3), singly or in combination, on the growth of normal and aplastic anemia (AA) bone marrow in clonogenic assay and long-term bone marrow culture (LTBMC). MGF stimulated colony-forming unit-granulocyte/macrophage (CFU-GM), burst-forming unit-erythroid (BFU-E), and mixed colony-forming unit (consisting of granulocyte-macrophage and erythroid elements) (CFU-GEM) colony formation from both normal and AA marrow. The three-factor combination stimulated the greatest number of colonies. Marrow from less severely affected AA patients was stimulated to produce the highest number of colonies, and a normal response was possible if progenitors were present. When added to LTBMC, MGF alone had little effect. GM-CSF and IL-3 stimulated increased numbers of progenitor cells harvested each week from normal and AA LTBMC. This resulted in normal colony numbers in some patients, the majority of whom were less severely affected than the patients who did not respond in LTBMC. The three-factor combination was additive for normal CFU-GM production. However, no further increases in AA LTBMC resulted from the addition of MGF to GM-CSF and IL-3. The partial correction in clonogenic assay with MGF in some AA patients raises the possibility of therapeutic benefit. We failed to demonstrate increased progenitor cell numbers in AA LTBMC, however. Further studies may overcome possible limitations to progenitor cell proliferation.

Separation of human blast progenitors from granulocytic, erythroid, megakaryocytic, and mixed colony-forming cells by "panning" on cultured marrow-derived stromal layers.

Primitive myeloid progenitor cells will adhere to stromal feeder layers of human bone-marrow-derived adherent cells grown in the presence of methylprednisolone (MP+ layers). These progenitors form colonies of blast cells on the MP+ stromal layers, but not on stromal layers grown in the absence of MP (MP- layers). The present study was designed to determine whether this failure of colony formation was caused by inability of the progenitors to adhere to the MP- layers or inability to proliferate in their presence. We also compared the capacities of the blast progenitors to adhere to MP+ and MP- stromal cells with those of mixed (GEMM-CFC), erythroid (BFU-E), megakaryocytic (Mk-CFC), and granulocyte-macrophage (GM-CFC) colony-forming cells. Incubation of bone marrow mononuclear cells with MP+ stromal layers removed 90% of the blast progenitors, but did not remove the majority of the GEMM-CFC, BFU-E, Mk-CFC, and GM-CFC; incubation of bone marrow mononuclear cells with MP- stromal layers did not remove the blast progenitors or the GEMM-CFC, BFU-E, Mk-CFC, and GM-CFC. Thus, the blast progenitors adhere to MP+ stromal feeder layers, but not to MP- stromal layers. In this respect they differ from the other more mature colony-forming cells that do not show any marked tendency to adhere to either type of stromal layer.

Comparisons of the composition of fat cells obtained from the marrow of normal individuals or of subjects with aplastic anemia and from bone marrow cultures.

The lipid composition of fat cells in the bone marrow of normal individuals and patients with aplastic anemia was investigated, using thin-layer and gas-liquid chromatography. The results were compared to those obtained from fat cells grown in vitro. Thin-layer chromatography (t.l.c) revealed that triacylglycerols were the major component of normal and aplastic marrow fat cells and of fat cells in culture. Analysis by gas-liquid chromatography (g.l.c) of the triacylglycerols, diacylglycerols, and nonesterified fatty acids detected minor differences between fat cells from normal and aplastic marrow. Although there were no statistically significant differences in the proportions of saturated fatty acids and their unsaturated analogues, the relative amounts of gamma-linolenic acid were increased in aplastic anemia and those of linoleic and myristoleic acids were reduced. In contrast, fat cells obtained from in vitro cultures contained a much higher proportion of unsaturated fatty acids. These findings indicate that there is no marked abnormality in the composition of bone marrow fat cells in aplastic anemia, but that minor differences can be detected; the fatty acid composition of bone marrow fat cells is not reproduced in vitro.

The role of antilymphocyte globulin in the treatment of chronic acquired bone marrow failure.

Antilymphocyte globulin is an immunoglobulin preparation prepared from heterologous serum after the animal (horse or rabbit) has been immunised with human lymphocytes, obtained from the thymus (antithymocyte globulin, ATG) or thoracic duct (antilymphocyte globulin, ALG). The rationale for the use of ALG in the treatment of chronic acquired marrow failure is based on its immunosuppressive activity and the fact that a proportion of cases of bone marrow failure, whether affecting single or multiple haemopoietic cell lines are due to immune-mediated suppression of haemopoiesis. In addition, in vitro studies have shown that ALG also has an immunostimulatory effect on lymphokine and haemopoietic growth factor production, and may therefore directly stimulate haemopoietic progenitor cells. ALG has been used for the treatment of aplastic anaemia and acquired chronic marrow failure affecting single cell lines namely pure red cell aplasia (PRCA), amegakaryocytic thrombocytopenia and chronic neutropenia due to immune inhibition of granulopoiesis ('acquired white cell aplasia'). ALG is used for treatment of non-severe aplastic anaemia (NSAA) and in those cases of severe aplastic anaemia (SAA) where allogeneic transplantation is not possible or is not indicated. Treatment with ALG results in 75% long term survival for NSAA and 40-50% for SAA although there is a very severe subgroup of SAA defined by peripheral blood neutrophils of less than 0.2 x 10(9)/l who rarely benefit from ALG therapy. For those patients who do not respond a second course of ALG can be given later using ALG from a different animal source.(ABSTRACT TRUNCATED AT 250 WORDS)

Use of cyclosporin A in allogeneic bone marrow transplantation for severe aplastic anemia.

We report on 18 months of experience with cyclosporin A (Cy A) in allogeneic bone marrow transplantation for severe aplastic anemia (SAA). Twenty-three patients treated with Cy A for postgraft immunosuppression are described and compared with 14 similar patients with SAA in whom methotrexate (MTX) was used. The early results are encouraging with 73% survival in the Cy A group compared with 43% in the MTX group. The improvement is partly attributable to the low incidence of graft failure. Graft-versus-host disease (GVHD) remains a problem with an overall incidence of 70% in Cy A-treated aplastic patients, although mortality has been ony 14%. Toxicity attributable to Cy A has so far been acceptable and nephrotoxicity is usually mild and reversible. However, three aplastic patients have developed clinically significant renal impairment while receiving both Cy A and aminoglycoside antibiotics.

Epstein-Barr virus (EBV) associated B-cell lymphoproliferative disease following HLA identical sibling marrow transplantation for aplastic anaemia in a patient with an EBV seronegative donor.

BACKGROUND: B-cell lymphoproliferative disorders (BLPD*) caused by Epstein-Barr virus (EBV) occurring after allogeneic bone marrow transplantation (BMT) are usually of donor origin. Treatment such as discontinuation of immunosuppression may be successful in some cases, but infusion of donor T cells results in successful eradication of EBV BLPD in most cases. METHODS AND RESULTS: We report a case of EBV positive aggressive BLPD after HLA matched sibling BMT for aplastic anaemia. The tumour completely regressed after withdrawal of cyclosporin and donor lymphocyte infusion. However, although the tumor was of donor origin, the donor serum was negative for antibodies to EBV antigens and no EBV-specific cytotoxicity was detected in donor peripheral blood mononuclear cells. The recipient was seropositive for EBV before BMT. CONCLUSIONS: We speculate that a 'second primary' EBV infection occurred involving donor cells in the recipient during BMT immunosuppression, with subsequent outgrowth of donor-derived BLPD. EBV infection may have been by an endogenous EBV isolate, from external sources, or from third party transfusions.

Unrelated donor marrow transplantation between 1977 and 1987 at four centers in the United Kingdom.

Retrospectively we analyzed the histocompatibility data and clinical results of bone marrow transplantation in 51 patients who received marrow from unrelated donors (UD) from 1977 to 1987 at one of four UK BMT centers. We compared the results with those obtained in 51 transplants carried out at the same centers using HLA-identical (ID) sibling donors. Of the UD/recipient pairs 32 (63%) were serologically identical for HLA A, B, and DR antigens, and 37% showed varying degrees of mismatch. UD-BMT primary diagnoses were: severe aplastic anemia or Fanconi's anemia (n = 17), acute leukemia (n = 11), chronic myeloid leukemia (n = 21), and other conditions (n = 2). T cell depletion of the graft was associated with a significant improvement in survival in both UD and ID-BMT. Graft failure was more common in recipients of UD than of ID transplants (13 [25%] vs. 5 [10%] P = 0.05) but there was no significant difference in the frequency of acute or chronic graft-versus-host disease. Actuarial survival was superior for recipients of ID transplants (UD vs. ID: 49% vs. 78%, respectively, at 3 months; 32% vs. 63% at one year). Reduced survival for recipients of UD-BMT was confirmed in case control regression analysis (relative risk 3.0, P = 0.01). Nevertheless in patients whose only alternative is a partially mismatched family donor we think that UD-BMT is justified.

A comparison of 111In with 52Fe and 99mTc-sulfur colloid for bone marrow scanning.

Under most circumstances 52Fe, 111In, and colloid show a similar distribution of marrow. The lesser uptake of 111In by liver and spleen may occasionally be of value in permitting visualization of that portion of the spinal marrow obscured by these organs in the colloid scan. However, in red cell aplasia, when there is dissociation between phagocytic and erythropoietic functions, scanning with 111In gives no information about erythropoietic tissue distribution. Therefore, indium cannot be used as an analog for iron in the study of the hematopoietic system.

Regulation of granulocyte and macrophage colony-stimulating factor production by phytohaemagglutinin-treated blood mononuclear cells.

The colony-stimulating activity (CSA) of medium conditioned by phytohaemagglutinin (PHA)-stimulated blood mononuclear cells was tested using granulocyte-macrophage colony-forming cells (GM-CFC) from normal bone marrow. Low concentrations of the conditioned medium stimulated granulocytic colony-forming cells (CFC) which formed colonies by the seventh day of incubation; higher concentrations stimulated the formation of macrophage colonies which were not seen until the end of the second week in culture. The colony-stimulating activities could not be demonstrated in adherent cell-depleted bone marrow cultures. This dependence of activity on adherent cells was confirmed by incubating different concentrations of conditioned medium with isolated adherent cells and then testing for colony-stimulating activity in cultures of non-adherent bone marrow cells. The activities of conditioned media following exposure to adherent cells corresponded to the results seen when the conditioned medium from PHA-stimulated mononuclear cell cultures was used to stimulate agar cultures of unseparated marrow. The results suggest that PHA-responsive mononuclear cells (probably T lymphocytes) may modulate the regulation of colony-stimulating factor (CSF) production by adherent colony-stimulating cells (CSC).

4-Hydroperoxycyclophosphamide inhibits proliferation by human granulocyte-macrophage colony-forming cells (GM-CFC) but spares more primitive progenitor cells.

Despite its considerable toxicity to haemopoietic colony-forming cells, 4-hydroperoxycyclophosphamide (4-HC) has successfully been used to purge marrow of leukaemic cells before it is used to rescue patients from high-dose chemoradiotherapy. These conflicting observations indicate that haemopoietic progenitor cells that are not detected by the established colony-forming assays survive exposure to 4-HC and repopulate the marrow. The recent finding that murine spleen colony-forming cells (CFU-S) are resistant to 4-HC [Porcellini A, et al. (1983) Expl Hemat. 11 (suppl 14) 331 (abstract)] [14] also indicates that sensitivity to 4-HC can be used to distinguish primitive progenitor cells from committed progenitor cells. As part of a study on the nature of a population of blast colony-forming cells in human bone marrow, we tested their sensitivity to 4-HC to see whether they also are spared by the drug. We found that 4-HC had much less effect on the blast colony-forming cells than on the granulocyte-macrophage colony-forming cells (GM-CFC). This result suggests that the blast-colony-forming cells may be early human haemopoietic progenitor cells.

Choice of bone marrow transplantation as treatment for severe aplastic anaemia.

Bone marrow transplantation is the first line of treatment for all patients with aplastic anaemia who have an identical twin. Age is not an important consideration in these circumstances. For patients with an HLA identical sibling donor bone marrow transplantation should be offered as a matter of urgency to those patients with SAA under the age of 50. The most controversial issue is the use of unrelated volunteers, or non-sibling family members who are phenotypically matched or who have a minor degree of mismatch of the HLA system. A strong case could be made for offering this form of transplant to all patients under the age of 20 who have VSAA and using it as the second line of treatment in patients who fail to respond to ALG after 4 months if they have VSAA.

Fatal autoimmune pancytopenia following bone marrow transplantation for aplastic anaemia.

We report a case of autoimmune pancytopenia 10 months after allogeneic bone marrow transplantation (BMT) for severe aplastic anaemia (SAA). The autoimmune haemolytic anaemia (AIHA) and immune thrombocytopenic purpura (ITP) were refractory to conventional immunosuppressive therapy which included steroids, azathioprine, vincristine and intravenous immunoglobulin. Splenectomy led to a recovery of the thrombocytopenia but the haemolysis continued despite further immunosuppressive therapy. Four months after the onset of haemolysis granulocyte-specific antibodies were detected. The patient subsequently received total lymph node irradiation (TLI) with a peripheral blood stem cell transplant (PBSCT) from his original donor, but died 9 days later from cerebral aspergillosis. The severe nature of autoimmune cytopenias and their lack of response to conventional treatment following allogeneic BMT is discussed further.

Liver function abnormality following treatment with antithymocyte globulin for aplastic anaemia.

We have observed transient elevations of serum alanine transaminase (ALT) levels in patients with aplastic anaemia who have been treated with antithymocyte globulin (ATG). Out of 18 patient episodes analysed retrospectively over a 12 month period, 15 experienced increases in ALT levels with values ranging from 1.2 to 18.5 times the upper limit of normal. In 11 of 15 episodes this was transient with ALT values returning to normal by 30 days, but in two patients this persisted for 6 months, and in a further two, until death at 34 and 145 days from unrelated causes. There was no evidence of acute viral infection or reactivation and no other drug toxicity could be implicated. We conclude that this may represent either a non-specific binding effect of ATG to hepatocytes or infection with an unidentified agent.