N. Zoumbos

Immunologic Abnormalities in Patients Receiving Multiple Blood Transfusions

Concern for the potential risk of the acquired immunodeficiency syndrome among blood cell recipients led us to measure immunologic functions in patients who had received multiple transfusions. Abnormalities of two immunologic tests, natural killer cell function and helper/suppressor (T4/T8) lymphocyte subpopulation ratio, have characterized patients with the acquired immunodeficiency syndrome and are prevalent in populations at risk for this syndrome. Natural killer cell function was severely depressed in multiply transfused patients. However, T4/T8 ratios were normal in this population. The role of chronic antigenic stimulation was studied by measurement of HLA-DR expression on T cells. The expression of HLA-DR antigen is markedly elevated in multiply transfused patients. These results show that chronic exposure to foreign antigens may be associated with abnormalities of immunologic function, but that chronically transfused patients do not have the same immunologic profile as reported in some homosexuals and hemophiliacs.

Prolonged administration of erythropoietin increases erythroid response rate in myelodysplastic syndromes: a phase II trial in 281 patients

Summary. Treatment with recombinant human erythropoietin (rHuEpo) improves anaemia in approximately 20% of patients with myelodysplastic syndromes (MDS). We investigated the potential advantage of a prolonged administration of rHuEpo to achieve higher erythroid response rates (RR) in 281 MDS patients: 118 with refractory anaemia (RA), 77 with refractory anaemia and ringed sideroblasts (RARS), 59 with refractory anaemia with excess of blasts and blast count < 10% (RAEB‐I), and 27 with RAEB and blast count between 11–20% (RAEB‐II). rHuEpo was given subcutaneously at a dose of 150 U/kg thrice weekly, for a minimum of 26 weeks. Response to treatment was evaluated after 12 and 26 weeks of therapy. The overall RR was 45·1%; the RR for RA, RARS, RAEB‐I and RAEB‐II were 48·3%, 58·4%, 33·8% and 13% respectively. A significant increase in RR was observed at week 26 in RA, RARS and RAEB‐I patients, as the response probability increased with treatment duration. The RR was higher in the good cytogenetic prognostic group and serum Epo level of > 150 U/l at baseline predicted for non‐response. The median duration of response was 68 weeks and the overall risk of leukaemic transformation was 21·7%. These results suggest that prolonged administration of rHuEpo produces high and long‐lasting erythroid RR in MDS patients with low blast counts, particularly in those with pretreatment serum Epo levels of < 150 U/l and good cytogenetic prognosis.

Gene transfer into human hematopoietic progenitor cells with an episomal vector carrying an S/MAR element

Episomally maintained self-replicating systems present attractive alternative vehicles for gene therapy applications. Recent insights into the ability of chromosomal scaffold/matrix attachment regions (S/MARs) to mediate episomal maintenance of genetic elements allowed the development of a small circular episomal vector that functions independently of virally encoded proteins. In this study, we investigated the potential of this vector, pEPI-eGFP, to mediate gene transfer in hematopoietic progenitor cell lines and primary human cells. pEPI-eGFP was episomally maintained and conferred sustained eGFP expression even in nonselective conditions in the human cell line, K562, as well as in primary human fibroblast-like cells. In contrast, in the murine erythroleukemia cell line, MEL, transgene expression was silenced through histone deacetylation, despite the vectors episomal persistence. Hematopoietic semisolid cell colonies derived from transfected human cord blood CD34+ cells expressed eGFP, albeit at low levels. After 4 weeks, the vector is retained in approximately 1% of progeny cells. Our results provide the first evidence that S/MAR-based plasmids can function as stable episomes in primary human cells, supporting long-term transgene expression. However, they do not display universal behavior in all cell types.

Genetic modification of hematopoietic stem cells with nonviral systems: past progress and future prospects

Serious unwanted complications provoked by retroviral gene transfer into hematopoietic stem cells (HSCs) have recently raised the need for the development and assessment of alternative gene transfer vectors. Within this context, nonviral gene transfer systems are attracting increasing interest. Their main advantages include low cost, ease of handling and large-scale production, large packaging capacity and, most importantly, biosafety. While nonviral gene transfer into HSCs has been restricted in the past by poor transfection efficiency and transient maintenance, in recent years, biotechnological developments are converting nonviral transfer into a realistic approach for genetic modification of cells of hematopoietic origin. Herein we provide an overview of past accomplishments in the field of nonviral gene transfer into hematopoietic progenitor/stem cells and we point at future challenges. We argue that episomally maintained self-replicating vectors combined with physical methods of delivery show the greatest promise among nonviral gene transfer strategies for the treatment of disorders of the hematopoietic system.

High Alloreactivity of Low-Dose Prophylactic Donor Lymphocyte Infusion in Patients with Acute Leukemia Undergoing Allogeneic Hematopoietic Cell Transplantation with an Alemtuzumab-Containing Conditioning Regimen

The value of prophylactic donor lymphocyte infusion (pDLI) is unclear and differs among diseases and transplantation protocols. Experience with this approach in patients with acute leukemia undergoing hematopoietic cell transplantation (HCT) with an alemtuzumab-incorporating conditioning protocol is lacking. We conducted a single-center prospective study to investigate the applicability and efficacy of prophylactic donor lymphocyte infusion (pDLI) in patients with leukemia undergoing HCT with a low-dose alemtuzumab-containing conditioning regimen. Inclusion criteria were high-risk acute myelogenous leukemia, acute lymphoblastic leukemia, or increasing mixed chimerism. All patients included were tapered off of immunotherapy. Exclusion criteria were a history of ≥ grade II or active graft-versus-host disease (GVHD). Of the 56 consecutive patients who underwent HCT with an alemtuzumab-containing regimen, 15 patients (8 with acute myelogenous leukemia and 7 with acute lymphoblastic leukemia) met the study inclusion criteria and received prophylactic DLI (total of 45 infusions) from 7 sibling donors and 8 unrelated donors. The first infusion was given at a median of 162 days posttransplantation. The median number of DLIs was 3, and the median cumulative CD3(+) cell dose was 2 × 10(6)cells/kg. Six of the 8 patients (75%) who received pDLI while in mixed chimerism converted to stable, complete donor chimerism. Some 47% of DLI recipients developed GVHD (4 acute GVHD and 3 with chronic GVHD) after a median cumulative dose of 2 × 10(6) CD3(+) cells/kg. After a median follow-up of 575 days, 11 (73%) pDLI recipients were alive. All 4 deaths were due to GVHD-related causes. None of the patients who received pDLIs relapsed. Patients with leukemia who received low-dose pDLI after conditioning with alemtuzumab are at low risk for relapse; however, this approach is associated with a relatively high incidence of severe GVHD.

Clinical relevance of balance between type 1 and type 2 immune responses of lymphocyte subpopulations in aplastic anaemia patients.

Immune dysfunction, which leads to the suppression of haemopoiesis by cytokines that are secreted by activated T lymphocytes, is considered to play a key role in the pathogenesis of acquired aplastic anaemia (AAA).

Analysis of lymphocyte subsets in patients with aplastic anaemia

Lymphocyte subsets have been measured in the blood of 28 patients with aplastic anaemia. The mean helper/inducer:suppressor/cytotoxic T lymphocyte ratio (1.24 ± 0.74) was significantly decreased in the total population in comparison to mean ratios in a normal population (1.78±0‐57) and in patients with other haematological diseases (l‐82±0‐92). A reversed ratio (< 1) was present in a large proportion (53%) of aplastic patients, due both to an absolute deficiency of helper/inducer lymphocytes and an increase in suppressor/cytotoxic lymphocytes. Increased HLA‐DR expression, evidence of lymphocyte activation, was present in seven of 12 patients evaluated and was confined to the suppressor/cytotoxic lymphocyte population. In the bone marrow, the percentage of lymphocytes was increased two‐fold compared to normal bone marrow but the ratio of T cell subsets was not abnormal. Two of four patients evaluated after haematopoietic recovery following ATG treatment showed a return to normal of the T4/T8 ratio; in two others a low T4/T8 ratio persisted despite recovery. These results indicate that lymphocyte subset imbalance and lymphocyte activation are present in many patients with aplastic anaemia, either as a contributing factor or as a result of bone marrow failure.

Prognostic significance of immune function parameters in patients with chronic lymphocytic leukaemia

Abstract: We determined immune function parameters in 41 newly diagnosed patients with chronic lymphocytic leukaemia (CLL) and correlated these findings with the clinical data and the subsequent course of the disease. The ratio of helper to suppressor T cells (CD4/CD8), the proportion of circulating natural killer (NK) cells and the NK activity were significantly low in clinical stage B and C patients. Among patients presenting with advanced disease, those who subsequently had a more severe course, characterised mainly by frequent respiratory infections, were found to have at presentation a significantly lower CD4/CD8 ratio (× ± SEM = 0.95 ± 0.09, vs 1.28 ± 0.14), a very low proportion of NK cells (4.78 ± 0.85, vs 11.75 ± 2.1%) and decreased amount of γ‐globulins (0.66 ± 0.08, vs 0.97 ± 0.09 g/dl), in comparison with patients with a much milder later course. These simple parameters of immune function seem to have prognostic value for patients with CLL.

Markers of endothelial and in vivo platelet activation in patients with essential thrombocythemia and polycythemia vera

We investigated endothelial and in vivo platelet activation in a cohort of 52 patients with essential thrombocythemia (ET) and polycythemia vera (PV) before and after cytoreductive treatment, 22 healthy controls, and 17 patients with acute cerebrovascular ischemia (ACVI) and normal platelet counts.We measured platelet expression of CD62P and CD63 antigens and levels of soluble vascular cell adhesion molecule 1 (sVCAM-1).We found increased in vivo platelet activation in all patients with ET and PV, both before and after cytoreductive treatment, compared with controls. In patients with arterial thrombosis, platelet expression of CD62P, and in patients with erythromelalgia, expression of both markers was higher compared with expression in patients without thrombotic complications. In patients with ET and PV before and after treatment, sVCAM-1 expression was increased compared with expression in controls but also compared with expression in patients with ACVI and normal platelet counts. In patients with arterial thrombosis and erythromelalgia, in vivo platelet activation correlated with the level of sVCAM-1. Our findings indicated that in vivo platelet activation reflects intrinsic platelet defects in patients with ET and PV, persists after cytoreductive treatment, and results in endothelial damage, probably through release of angiogenic factors and/or activation of white blood cells.

A novel β‐thalassaemia mutation in the 5’untranslated region of the β‐globin gene

Summary. A thymidine deletion at position +10 of the 5’untranslated region of the β‐globin gene was detected in a β‐thalassaemia intermedia patient carrying a βd́; 39 stop codon mutation on the other chromosome; this new mutation, +10(‐T), was detected by automated fluorescent DNA sequencing and verified by dot‐blot allele‐specific hybridizations.