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Background: Persistent mixed chimerism represents a state in which recipient and donor cells stably co-exist after hematopoietic stem cell transplantation. However, since in most of the studies reported in literature the engraftment state was observed in the nucleated cells, in this study we determined the donor origin of the mature erythrocytes of patients with persistent mixed chimerism after transplantation for hemoglobinopathies. Results were compared with the engraftment state observed in singly picked out burst-forming unit - erythroid colonies and in the nucleated cells collected from the peripheral blood and from the bone marrow., Design and Methods: The donor origin of the erythrocytes was determined analyzing differences on the surface antigens of the erythrocyte suspension after incubation with anti-ABO and/or anti-C, -c, -D, -E and -e monoclonal antibodies by a flow cytometer. Analysis of short tandem repeats was used to determine the donor origin of nucleated cells and burst-forming unit - erythroid colonies singly picked out after 14 days of incubation., Results: The proportions of donor-derived nucleated cells in four transplanted patients affected by hemoglobinopathies were 71%, 46%, 15% and 25% at day 1364, 1385, 1314 and 932, respectively. Similar results were obtained for the erythroid precursors, analyzing the donor/recipient origin of the burst-forming unit - erythroid colonies. In contrast, on the same days of observation, the proportions of donor-derived erythrocytes in the four patients with persistent mixed chimerism were 100%, 100%, 73% and 90%. Conclusions Our results showed that most of the erythrocytes present in four long-term transplanted patients affected by hemoglobinopathies and characterized by the presence of few donor engrafted nucleated cells were of donor origin. The indication that small proportions of donor engrafted cells might be sufficient for clinical control of the disease in patients affected by hemoglobinopathies is relevant, although the biological mechanisms underlying these observations need further investigation.

Background and Objectives: A combination of 8-methoxypsoralen and ultraviolet-A radiation (PUVA) is used for the treatment of T cell-mediated disorders, including chronic graft-versus-host disease. The mechanisms of action of this therapy, referred to as extracorporeal phototherapy, have not been fully elucidated. PUVA is known to induce apoptosis in T lymphocytes collected by apheresis, however scarce information is available concerning the apoptotic pathways activated by PUVA., Design and Methods: We used Jurkat human T leukemia cells and normal T lymphocytes to analyze the PUVA-triggered caspase activation pattern by means of immunoblot analysis, in vitro caspase activity assays, and selective caspase inhibitors coupled to flow cytometric analysis., Results: PUVA treatment induced activation of apical caspases-9 and -8, and of effector caspases-3 and -7 in Jurkat cells and human T lymphocytes. While activation of caspase-9 occurred as early as 1 h after PUVA treatment of Jurkat cells, procaspase-8 cleavage was delayed and was detected 6 h after the exposure. Also in normal T lymphocytes, cleavage of caspase-8 was subsequent to activation of caspase-9. PUVA-dependent proteolytic cleavage of procaspase-8 was blocked by inhibitors selective for either caspase-9 or -3. Moreover, procaspase-8 was cleaved in vitro by activated caspase-3, which gave rise to proteolytic fragments equivalent to those generated in vivo., Interpretation and Conclusions: Activation of caspase-8 in PUVA-treated Jurkat cells and normal T lymphocytes is secondary to up-regulation of caspase-9. Overall, our results identify caspase-9 as the critical upstream caspase initiating apoptosis by PUVA in Jurkat T-cells and human T lymphocytes.

Background and Objectives: In chronic lymphocytic leukemia (CLL) B-cells are refractory to activation signals and to apoptosis. CD40 triggering, however, rescues CLL B-cells from their anergic state and upregulates the FAS receptor. We therefore studied whether CD40 triggering enhances CLL B-cell sensitivity to fludarabine, and receptors or cytokines potentially involved in apoptosis., Design and Methods: CD40-activation of CLL B-cells was carried out by co-culture with CD40L-transfected cells. After fludarabine treatment, apoptosis was evaluated by propidium iodide (PI), annexin-V/PI or DiOC6 staining and flow cytometry analysis. Modulation of Bcl-2, of tumor necrosis factor receptor (TNFRI/II) and release of tumor necrosis factor (TNF)alpha/interferon (IFN)gamma was also analyzed. Furthermore, addition of caspase-inhibitors or anti-TNFalpha/-IFNgamma monoclonal antibodies to fludarabine-treated cells allowed us to determine the mediators of apoptosis. Student's t tests or ANOVA variance statistical analysis were performed to evaluate whether any differences observed might be considered significant., Results: CD40 triggering enhanced fludarabine sensitivity of CLL B-cells, downmodulated Bcl-2 and upregulated TNFRI/II. Caspases 1 and 6 were the major caspases involved in fludarabine apoptosis induction in resting B cells, while only anti-TNFalpha/-IFNgamma monoclonal antibodies reduced apoptosis in activated cells. In agreement with this observation, autocrine production of TNFalpha and IFNgamma by CD40-activated CLL B cells was found., Interpretation and Conclusions: B-cells from a considerable proportion of CLL cases studied (11/20) are more prone to fludarabine-induced apoptosis after CD40 triggering; accordingly Bcl-2 expression was lower in activated cells. Moreover, upregulation of TNFRI/II, release of TNFalpha and IFNgamma, and inhibition of apoptosis by anti-TNFalpha/-IFNgamma monoclonal antibodies in CD40-activated cells strongly suggest that these cytokines may play a role in sensitizing B-cells to fludarabine treatment.

Background and Objective: In this study we describe a newly established CD30+ Epstein Barr virus (EBV)-infected B cell line derived from an EBV-infected B cell culture (utilized, once irradiated, as a feeder) which showed a B clonal rearrangement and strong CD30 antigen expression., Design and Methods: The cells injected into SCID mice were able to grow giving rise to CD30+ solid tumors with the morphologic features of an anaplastic large cell lymphoma (ALCL). Thus we tried to establish a model to investigate the potency of immunoconjugates containing a CD30 monoclonal antibody (Ber-H2) and ribosome-inactivating proteins (saporin, momordin and ricin A-chain) as toxic moieties., Results: We observed a strong cytotoxic activity of the anti-CD30 immunotoxins on the in vitro growth of D430B cells. High levels of anti-tumor activity were also observed in vivo, in the SCID mouse model., Interpretation and Conclusions: The antitumor immunotoxin therapy was sccessful in our chosen animal model, the effecacy seeming to be associated with strength of CD30 expression. Our data suggest that immunotoxins should be tested (before use) on the tumor cells of the subject to be treated and that immunotoxins should be directed to different tumor-associated antigens to avoid selection of cell populations with different antigenic mosaics.

Patients affected by Glanzmann's thrombasthenia often require blood and platelet transfusions due to bleeding. They may develop antibodies against platelet antigens and become refractory to platelet transfusions. In this study we present our approach to platelet transfusion in a thrombasthenic patient with platelet antibodies directed against gpIIb/IIIa. We found that platelets from two HPA1b/b donors were weakly positive when matched with the patient's serum. The patient was submitted to dental surgery and platelets were transfused before and after surgery to prevent bleeding. The patient did not experience transfusion reaction and good platelet recovery was observed.

On the basis of the experience derived from the study of 2,000 biopsies, the authors critically review the problem of the newly generated monoclonal antibodies (mAbs) working in paraffin sections. The reliability and drawbacks of the most commonly used reagents are examined, with special reference to their employment in the diagnosis of malignant lymphomas. The authors underline that only a few mAbs are really specific, since most reagents give rise to a certain number of cross-reactivities. In the light of this, a wide panel of antibodies should always be applied in the diagnosis of lymphoid tumours in order to avoid possible misinterpretations, which can actually lead to wrong therapeutical decisions.

A column immunoadsorption method, based on the high affinity between the protein Avidin and the vitamin Biotin has been used to obtain positive CD34+ or S313+ marrow cells selection. Cell suspensions from marrow samples of six healthy subjects were incubated either the monoclonal antibody S313 or HPCA 1 (My 10-like) and a biotinilated goat anti-mouse Immunoglobulins antiserum, passed over a column containing an Avidin coated Polyacrylamide matrix, at the flow rate of 1 ml/min. The phenotype and the clonogenic efficiency of the recovered adherent cell fraction were studied by cytofluorimetric analysis and haemopoietic progenitors short term cultures. The results obtained show a mean CD34+ and S313+ cells recovery greater than 50% with a lower stem cells enrichment. Although these data could not considered optimal for clinical application in haematologic neoplasias, these preliminary studies demonstrate the possible use of the method for autologous bone marrow transplantation.

An assay based on the inhibition of the cloning capacity in a plasma clot semisolid medium assay has been used to test the sensitivity of the Raji cell line to lymphokine-activated killer (LAK) cells. This method overcomes some limitations intrinsic to the widely employed 51Cr release assay and always shows a higher degree of sensitivity. No inhibition of colony growth was found when the effector cells were plated without prior pre-incubation with interleukin 2 or with the addition of the medium derived from the LAK cells. Though more time-consuming than the classic 51Cr release assay, this technique does not require radioactive material. This test may be suitable for a more precise evaluation of LAK activity and for the study of the mechanisms involved in cell killing.

The mixed lymphocyte reactions are usually performed by the uptake of 3H thymidine (3H TdR) in the study of histocompatibility for allogeneic bone marrow transplantation. Bromodeoxyuridine (BrdUrd), an analogue of thymidine, can be used as an alternative marker of proliferation. In this study we compared the evaluation of the proliferative activity of alloreactive lymphocytes in MLR by 3H TdR and BrdUrd incorporation in flow cytometry. The results show that BrdUrd is able to recognize proliferative activity at least 24-48 hours before 3H TdR, and allows discrimination of proliferation of a few cells despite its proximity to the autologous controls. However, the large quantity of cells needed for every combination is presently a disadvantage of the method.