免疫毒素用于自体骨髓移植治疗白血病的实验研究及初步临床

目前自体骨髓移植被越来越多地用于白血病治疗,然而在缓解期从病人抽取的骨髓细胞可能含有残留的肿瘤细胞,当大剂量放、化疗后回输给病人,将来有可能引起白血病复发。现在有很多方法可以用来去除这些瘤细胞,免疫毒素是安全有效的方法之一。本文报道了由抗CD2、CD5和CD8单抗与蓖麻毒素组成的抗T免疫毒素,抗CD9、CD10单抗与蓖麻毒素组成的抗C-ALL免疫毒素在体外对靶细胞的特异性细胞毒作用,及用于临床净化骨髓的初步结果。     

A Biological Pacemaker Restored by Autologous Transplantation of Bone Marrow Mesenchymal Stem Cells

Objective：To restore cardiac autonomic pace function by autologous trans- plantation and committed differentiation of hone marrow mesenchymal stern cells, and explore the technique for the treatment of sick sinus syndrome. Methods ： Mesenchymal stern cells isolated from canine bone marrow were culture-expanded and differentiated in vitro by 5-azacytidine. The models of sick sinus syndrome in canines were established by ablating sinus node with radio-frequency technique. Differentiated mesenchymal stem cells labeled by BrdU were autologously transplanted into sinus node area through direct injection. The effects of autologous transplantation of mesenchymal stern cells on cardiac autonomic pace function in sick sinus syndrome models were evaluated by electrocardiography, pathologic and immunohistochemical staining technique. Results：There was distinct improvement on pace function of sick sinus syndrome animal models while differentiated mesenchymal stem cells were auto-transplanted into sinus node area. Mesenchymal stern cells transplanted in sinus node area were differentiated into similar sinus node cells and endothelial cells in vivo, and established gap junction with native cardiomyocytes. Conclusion ： The committed- induced mesenchymal stern cells transplanted into sinus node area can differentiate into a- nalogous sinus node cells and improve pace function in canine sick sinus syndrome models.     

Bone Marrow Transplantation in the Rat

We have analysed the cytological structure of inflammation in the different parenchymal target organs during acute graft-versus-host disease after bone marrow transplantation in the rat. Inflammation (recovery of increased numbers of white cells) was recorded in the liver, skin, and lungs of the allograft recipient compared with the syngeneic graft recipient from day 5 onwards, accompanied by reduced recovery of inflammatory cells from the gut. The major cytological manifestation of the disorder was an increase in the total number of blast cells, large granular lymphocytes (LGL), and lymphocytes in the liver and skin, and of the number of blast cells and LGL in the lung. At the same time, a depletion of LGL and lymphocytes was recorded in the blood, suggesting migration of these cell components to the site of inflammation.     

Monoclonal Antibodies in Conditioning Regimens for Hematopoietic Cell Transplantation

Monoclonal antibodies are increasingly being incorporated in conditioning regimens for autologous or allogeneic hematopoietic cell transplantation (HCT). The benefit of adding rituximab to autologous HCT regimens is purportedly related to in vivo purging of clonal B cells. Randomized trials comparing the addition (or not) of rituximab to high-dose therapy regimens are lacking. No benefit of standard-dose radioimmunotherapy-based regimens for autografting in aggressive lymphomas was seen in a randomized controlled study. The incorporation of rituximab into allogeneic HCT regimens aims to improve responses while reducing nonrelapse mortality resulting from acute graft-versus-host disease. The optimal dose and administration schedule of rituximab in this setting are unknown, and potentially serious complications from increased infections owing to prolonged (and profound) cytopenias or persistent hypogammaglobulinemia are of concern. Radioimmunotherapy-based conditioning for allografting holds promise as a modality to optimize tumor control and synergize adoptive immunotherapy effects, but it remains experimental at this time. The addition of alemtuzumab to allogeneic HCT regimens is associated with prolonged lymphopenia and impaired immune reconstitution, high relapse rates, and serious infections. The optimal dose and schedule of alemtuzumab to avoid prolonged immune paresis remain elusive. It is anticipated that additional monoclonal antibodies will soon become available that can be incorporated into HCT regimens after safety and clinical efficacy are demonstrated.     

Unmanipulated Haploidentical Bone Marrow Transplantation and Posttransplantation Cyclophosphamide for Hematologic Malignancies after Myeloablative Conditioning

Fifty patients with high-risk hematologic malignancies, underwent an unmanipulated haploidentical bone marrow transplantation (BMT), followed by posttransplantation high-dose cyclophosphamide (PT-CY): the myeloablative (MA) conditioning consisted of thiotepa, busulfan, fludarabine (n = 35), or total body irradiation (TBI), fludarabine (n = 15). The median age was 42 years (range, 18-66 years); 23 patients were in remission, 27 had active disease, and 10 patients were receiving a second allograft. Graft-versus-host disease (GVHD) prophylaxis consisted in PT-CY on day +3 and +5, cyclosporine (from day 0), and mycophenolate (from day +1). Three patients died before engraftment, and 2 patients had autologous recovery: 45 patients (90%) had full-donor chimerism on day +30. The median day for neutrophil engraftment was day +18 (range, 13-30 days). The cumulative incidence of grade II-III acute GVHD (aGVHD) was 12%, and of moderate chronic GVHD (cGVHD) 10%. With a median follow-up for surviving patients of 333 days (range, 149-623 days), the cumulative incidence of transplantation-related mortality (TRM) was 18%, and the rate of relapse was 26%. The actuarial 22-month disease-free survival (DFS) rate was 68% for patients in remission and 37% for patients with active disease (P < .001). Causes of death were pneumonia (n = 3), hemorrhage (n = 3), sepsis (n = 3), and relapse (n = 7). In conclusion, an MA conditioning regimen followed by haploidentical BMT with PT-CY results in a low risk of aGVHD and cGVHD and encouraging rates of TRM and DFS.     

HLA-Haploidentical Bone Marrow Transplantation for Hematologic Malignancies Using Nonmyeloablative Conditioning and High-Dose,Posttransplantation Cyclophosphamide

We evaluated the safety and efficacy of high-dose, posttransplantation cyclophosphamide (Cy) to prevent graft rejection and graft-versus-host disease (GVHD) after outpatient nonmyeloablative conditioning and T cell-replete bone marrow transplantation from partially HLA-mismatched (haploidentical) related donors. Patients with advanced hematologic malignancies (n = 67) or paroxysmal nocturnal hemoglobinuria (n = 1) received Cy 50 mg/kg i.v. on day 3 (n = 28) or on days 3 and 4 (n = 40) after transplantation. The median times to neutrophil (>500/μL) and platelet recovery (>20,000/μL) were 15 and 24 days, respectively. Graft failure occurred in 9 of 66 (13%) evaluable patients, and was fatal in 1. The cumulative incidences of grades II-IV and grades III-IV acute (aGVHD) by day 200 were 34% and 6%, respectively. There was a trend toward a lower risk of extensive chronic GVHD (cGVHD) among recipients of 2 versus 1 dose of posttransplantation Cy (P = .05), the only difference between these groups. The cumulative incidences of nonrelapse mortality (NRM) and relapse at 1 year were 15% and 51%, respectively. Actuarial overall survival (OS) and event-free survival (EFS) at 2 years after transplantation were 36% and 26%, respectively. Patients with lymphoid malignancies had an improved EFS compared to those with myelogenous malignancies (P = .02). Nonmyeloablative HLA-haploidentical BMT with posttransplantation Cy is associated with acceptable rates of fatal graft failure and severe aGVHD or cGVHD.     

Lymphocyte Chimerism After Bone Marrow Transplantation

Specific HLA antibodies were used to eliminate donor and recipient cells, respectively, from lymphocyte suspensions prepared from the blood of a child who had been transplanted with bone marrow from an HLA-A- and HLA-B-incompatible, HLA-D-compatible donor. About 70% of the lymphocytes were of donor HLA type, the remaining of recipient type. The phytohemagglutinin-responsive lymphocytes were exclusively limited to the lymphocyte population carrying donor-type HLA antigens. Membrane immunofluorescence investigations of the donor and recipient populations showed a low percentage of IgM-positive lymphocytes in the donor population and an extremely high proportion of IgM-positive lymphocytes in the recipient population. About 90% of the donor lymphocytes were T cells, as judged by their capacity to form rosettes between sheep erythrocytes and T lymphocytes; no cells in the recipient cell population expressed this ability.     

Acceleration of Immune Reconstitution after Bone Marrow Transplantation in Mice by Bone Marrow Stromal

To observe potential effect of the engineered bone marrow stromal cell line QXMSC1 secreting IL-6 (QXMSCIL-6) on accelerating immnune reconstitution in syngeneic bone marrow transplantation in mice, QXMSC1 was transfected with the eukaryocytic expression vector pcDNAIL-6, which contained hIL-6 cDNA by liposome-mediated gene transfecting technique. G418-resistance clone was selected by limiting dilution. The highest secreting clone was selected by ELISA assay and used in animal experiments. The recipient mice (BALB/c) were lethally irradiated and cotransplanted syngeneic bone marrow (10^7/mice) and the QXMSCIIL-6 (5&#215;10^5/mice). Lymphocyte proliferation induced by ConA and LPS, helper T lymphocyte precursor (HTLp), cytotoxic T lymphocyte precursor (CTLp), plaque-forming cell (PFC), delayed type hypersensitivity (DTH) were examined 30, 60 days in post transplantation respectively. The results showed that lymphocytes proliferation to ConA and LPS, HTLp, CTLp increased, DTH and PFC were improved by cografted stromal cells QXMSCIIL-6 on 30, 60 days after BMT. These results demonstrated that the bone marrow stromal cell line QXMSC1 IL-6 transfected with IL-6 (QXMSC11L-6) accelerated immnune reconstitution in syngeneic bone marrow transplantation.     

Bone Marrow Transplantation after Nonmyeloablative Treosulfan Conditioning Is Curative in a Murine Model of Sickle Cell Disease

Allogeneic hematopoietic stem cell transplantation (HSCT) can be curative for patients with sickle cell disease (SCD). However, morbidity associated with myeloablative conditioning and graft-versus-host disease has limited its utility. To this end, autologous HSCT for SCD using lentiviral gene-modified bone marrow (BM) or peripheral blood stem cells has been undertaken, although toxicities of fully ablative conditioning with busulfan and incomplete engraftment have been encountered. Treosulfan, a busulfan analog with a low extramedullary toxicity profile, has been used successfully as part of a myeloablative conditioning regimen in the allogeneic setting in SCD. To further minimize toxicity of conditioning, noncytotoxic monoclonal antibodies that clear stem cells from the marrow niche, such as anti-c-Kit (ACK2), have been considered. Using a murine model of SCD, we sought to determine whether nonmyeloablative conditioning followed by transplantation with syngeneic BM cells could ameliorate the disease phenotype. Treosulfan and ACK2, in a dose-dependent manner, decreased BM cellularity and induced cytopenia in SCD mice. Conditioning with treosulfan alone at nonmyeloablative dosing (3.6?g/kg), followed by transplantation with syngeneic BM donor cells, permitted long-term mixed-donor chimerism. Level of chimerism correlated with improvement in hematologic parameters, normalization of urine osmolality, and improvement in liver and spleen pathology. Addition of ACK2 to treosulfan conditioning did not enhance engraftment. Our data suggests that pretransplant conditioning with treosulfan alone may allow sufficient erythroid engraftment to reverse manifestations of SCD, with clinical application as a preparative regimen in SCD patients undergoing gene-modified autologous HSCT.     

Immunodeficiency after Allogeneic Bone Marrow Transplantation in Man

This study was undertaken to clarify the mechanism behind the severely decreased lymphocyte proliferative response upon stimulation with mitogens and antigens seen after allogeneic bone marrow transplantation (BMT) in man. We investigated eight BMT patients and eight controls and found that the proliferative response of patient cells was reduced both when the cells were stimulated with phytohaemagglutinin (PHA) and when they were stimulated with a combination of phorbol myristate acetate (PMA), which is an activator of protein kinase C (PKC), and the calcium ionophore A23187, which irreversibly opens for calcium transport into the cell (median relative responses were 41 and 37%, respectively). However, the PHA-induced increase in the concentration of intracellular free calcium in post-BMT cells was not significantly different from the values found in control cells and the expression of interleukin 2 (IL-2) receptors (CD25) was only slightly decreased. However, the production of IL-2 was severely decreased in patient cells after stimulation with A23187/PMA (median 3541 units), although it was higher than in PHA-stimulated control cells (median 354 units). These results show that a direct activation of PKC by PMA combined with an increase in intracellular free calcium by A23187 cannot overcome the lymphocyte proliferation deficiency in cells from patients after allogeneic BMT. The data suggests that the defect is affecting the diacylglycerol pathway considerably more than the inositol triphosphate pathway.     

Potential of Gene Therapy in Bone Marrow Transplantation

Gene therapy, initiated as a treatment for inherited disorders such as adenosine deaminase deficiency, is now a promising therapeutic strategy for malignancies and other acquired diseases. In particular, in the field of bone marrow transplantation (BMT) for haematological malignancies, the gene transfer of the suicide gene HSV-TK into donor lymphocytes allows control of the severe complication graft-versus-host disease (GvHD). The transfer of the HSV-TK suicide gene confers selective sensitivity to the drug ganciclovir, allowing in vivo elimination of the donor T-cells if severe GvHD occurs. In Italy, the first pilot study on delayed infusion of genetically engineered donor lymphocytes after T-depleted allogeneic BMT documented efficacy of engineered donor lymphocytes in terms of anti-tumour activity and efficiency of the suicide system. GvHD developed in 3 out of 8 patients and was successfully treated by ganciclovir administration.