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Busulfan liver metabolism depends on glutathione, a crucial mediator of cellular and systemic stress. Here we investigated 40 polymorphisms at 27 loci involved in hepatic glutathione homeostasis, with the aim of testing their impact on the clinical outcome of 185 busulfan-conditioned allogeneic transplants. GSTA2 S112T serine allele homozygosity is an independent prognostic factor for poorer survival (RR=2.388), for increased any time- and 100-day transplant-related mortality (RR=4.912 and RR=5.185, respectively). The genotype also predicts a wider busulfan area under the concentration-time curve (1214.36±570.06 vs. 838.10±282.40 mMol*min) and higher post-transplant bilirubin serum levels (3.280±0.422 vs. 1.874+0.197 mg/dL). In vitro, busulfan elicits pro-inflammatory activation (increased NF-KappaB activity and interleukin-8 expression) in human hepatoma cells. At the same time, the drug down-regulates a variety of genes involved in bilirubin liver clearance: constitutive androstane receptor, multidrug resistance-associated protein, solute carrier organic anion transporters, and even GSTA2. It is worthy of note that GSTA2 also acts as an intra-hepatic bilirubin binding protein. These data underline the prognostic value of GSTA2 genetic variability in busulfan-conditioned allotransplants and suggest a patho-physiological model in which busulfan-induced inflammation leads to the impairment of post-transplant bilirubin metabolism.

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 Thalassemia major can be cured with allogeneic hematopoietic stem cell transplantation. Persistent mixed chimerism develops in around 10% of transplanted thalassemic patients, but the biological mechanisms underlying this phenomenon are poorly understood.Design and Methods The presence of interleukin-10-producing T cells in the peripheral blood of eight patients with persistent mixed chimerism and five with full donor chimerism was investigated. A detailed characterization was then performed, by T-cell cloning, of the effector and regulatory T-cell repertoire of one patient with persistent mixed chimerism, who developed stable split erythroid/lymphoid chimerism after a hematopoietic stem cell transplant from an HLA-matched unrelated donor.Results Higher levels of interleukin-10 were produced by peripheral blood mononuclear cells from patients with persistent mixed chimerism than by the same cells from patients with complete donor chimerism or normal donors. T-cell clones of both host and donor origin could be isolated from the peripheral blood of one, selected patient with persistent mixed chimerism. Together with effector T-cell clones reactive against host or donor alloantigens, regulatory T-cell clones with a cytokine secretion profile typical of type 1 regulatory cells were identified at high frequencies. Type 1 regulatory cell clones, of both donor and host origin, were able to inhibit the function of effector T cells of either donor or host origin in vitro.Conclusions Overall these results suggest that interleukin-10 and type 1 regulatory cells are associated with persistent mixed chimerism and may play an important role in sustaining long-term tolerance in vivo. These data provide new insights into the mechanisms of peripheral tolerance in chimeric patients and support the use of cellular therapy with regulatory T cells following hematopoietic stem cell transplantation.

Busulfan liver metabolism depends on glutathione, a crucial mediator of cellular and systemic stress. Here we investigated 40 polymorphisms at 27 loci involved in hepatic glutathione homeostasis, with the aim of testing their impact on the clinical outcome of 185 busulfan-conditioned allogeneic transplants. GSTA2 S112T serine allele homozygosity is an independent prognostic factor for poorer survival (RR=2.388), for increased any time- and 100-day transplant-related mortality (RR=4.912 and RR=5.185, respectively). The genotype also predicts a wider busulfan area under the concentration-time curve (1214.36 ± 570.06 vs. 838.10 ± 282.40 mMol*min) and higher post-transplant bilirubin serum levels (3.280 ± 0.422 vs. 1.874+0.197 mg/dL). In vitro, busulfan elicits pro-inflammatory activation (increased NF-KappaB activity and interleukin-8 expression) in human hepatoma cells. At the same time, the drug down-regulates a variety of genes involved in bilirubin liver clearance: constitutive androstane receptor, multidrug resistance-associated protein, solute carrier organic anion transporters, and even GSTA2. It is worthy of note that GSTA2 also acts as an intra-hepatic bilirubin binding protein. These data underline the prognostic value of GSTA2 genetic variability in busulfan-conditioned allotransplants and suggest a patho-physiological model in which busulfan-induced inflammation leads to the impairment of post-transplant bilirubin metabolism.

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: Thalassemia major can be cured with allogeneic hematopoietic stem cell transplantation. Persistent mixed chimerism develops in around 10% of transplanted thalassemic patients, but the biological mechanisms underlying this phenomenon are poorly understood., Design and Methods: The presence of interleukin-10-producing T cells in the peripheral blood of eight patients with persistent mixed chimerism and five with full donor chimerism was investigated. A detailed characterization was then performed, by T-cell cloning, of the effector and regulatory T-cell repertoire of one patient with persistent mixed chimerism, who developed stable split erythroid/lymphoid chimerism after a hematopoietic stem cell transplant from an HLA-matched unrelated donor., Results: Higher levels of interleukin-10 were produced by peripheral blood mononuclear cells from patients with persistent mixed chimerism than by the same cells from patients with complete donor chimerism or normal donors. T-cell clones of both host and donor origin could be isolated from the peripheral blood of one, selected patient with persistent mixed chimerism. Together with effector T-cell clones reactive against host or donor alloantigens, regulatory T-cell clones with a cytokine secretion profile typical of type 1 regulatory cells were identified at high frequencies. Type 1 regulatory cell clones, of both donor and host origin, were able to inhibit the function of effector T cells of either donor or host origin in vitro., Conclusions: Overall these results suggest that interleukin-10 and type 1 regulatory cells are associated with persistent mixed chimerism and may play an important role in sustaining long-term tolerance in vivo. These data provide new insights into the mechanisms of peripheral tolerance in chimeric patients and support the use of cellular therapy with regulatory T cells following hematopoietic stem cell transplantation.

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.