Human metapneumovirus infection after allogeneic hematopoietic stem cell transplantation.

BACKGROUND: The clinical characteristics of human metapneumovirus (hMPV)-associated lower respiratory tract infection (LRTI) after allogeneic hematopoietic stem cell transplantation (HSCT) is not well described. We describe the clinical course in eight HSCT recipients suffering from hMPV infection. METHODS: We prospectively included all patients with hMPV-associated LRTI after allogeneic HSCT during a period of 1 year. hMPV was diagnosed by multiplex polymerase chain reaction (PCR) from bronchoalveolar lavage (BAL). RESULTS: Eight patients with hMPV-associated LRTI were identified from 93 BAL samples. Three of the eight patients had co-infections with other pathogens. The median age of the patients was 45 years [interquartile range (IQR) 36.8-53.5], the median time posttransplant was 473 days (IQR 251-1,165), 5/8 patients had chronic graft-versus-host disease (cGvHD), and 6/8 patients received immunosuppression. Chest computed tomography (CT) scanning showed a ground-glass pattern in 7/8 patients. Seven of eight patients required hospitalization due to severe symptoms and hypoxemia. All were treated with intravenous immunoglobulin (IVIG), which was combined with oral ribavirin in six patients. The mortality rate was 12.5 % (1/8). CONCLUSIONS: hMPV-associated LRTI in allogeneic HSCT recipients are not uncommon and present with unspecific respiratory symptoms, ground-glass pattern in CT scanning, and co-infection.

Long-term follow-up of metastatic renal cancer patients undergoing reduced-intensity allografting.

SCT from an HLA-compatible sibling donor is an adoptive immunotherapy for cytokine-refractory, metastatic clear-cell renal cell cancer (RCC). However, the recent introduction of targeted therapy compounds has reduced the interest in this therapeutic strategy. We have reanalyzed our series with the aim to assess long-term benefit from allografting. Twenty-five RCC patients received a reduced-intensity allograft from an HLA-identical sibling donor. All patients received a thiotepa, fludarabine and CY conditioning regimen, and a cyclosporine-based GVHD prophylaxis. Best response to allograft was evaluable in 24 patients: 1 CR, 4 PR, 12 minor response/stable disease, 7 progressive disease. One-year survival was 48%, and five-year survival was 20%. At a median observation time of 65 months, five patients are alive, one in CR, one in PR and three with stable disease. By multivariate analysis, C-reactive protein value before transplant, the number of CD34 + infused cells and disease status at day +90 significantly correlated with survival. Survival of patients at favorable/intermediate-risk according to the MSKCC score that underwent allografting was better in comparison to the survival predicted by historical controls. We conclude that 20% of cytokine-refractory RCC patients are alive long-term after allografting. Transplantation is able to induce long-term disease control in a fraction of relapsed RCC patients.

Rapamycin induces a caspase-independent cell death in human monocytes.

The immunosuppressive activity of rapamycin (RAPA) and its efficacy as an anti-rejection agent in organ transplantation have been ascribed principally to its anti-proliferative effects on T cells, while the activity on monocytes is partially unknown. In vitro, RAPA reduced monocyte survival by inducing a caspase-independent cell death. RAPA-induced monocyte cell death (RAPA-CD) was impeded by activation of granulocyte macrophage-colony stimulating factor family receptors or toll-like receptor 4, and by exposure to inflammatory cytokines. In vivo, in patients who received RAPA monotherapy as part of pre-conditioning for islet transplantation, RAPA affected survival of myeloid lineage cells. In the peripheral blood, CD33(+) and CD14(+) cells decreased, whereas lymphocytes appeared unaffected. In the bone marrow, myeloid precursors such as CD15(+) and CD15(+)/CD16(+) were selectively and significantly decreased, but no major cytotoxic effects were observed. The RAPA-CD suggests a dependence of monocytes on mammalian target of RAPA pathways for nutrient usage, and this feature implies that RAPA could be selectively useful as a treatment to reduce monocytes or myeloid cells in conditions where these cells negatively affect patient, suggesting a potential anti-inflammatory action of this drug.

Bone marrow mammaglobin expression as a marker of graft-versus-tumor effect after reduced-intensity allografting for advanced breast cancer.

We assessed mammaglobin (MMG) gene expression in bone marrow (BM) aspirates from patients with advanced breast cancer who had received a reduced-intensity conditioning and stem cell allografting, in order to detect a graft-versus-tumor effect on micrometastatic disease. Nine patients received a reduced-intensity conditioning with fludarabine, cyclophosphamide, and thiotepa, followed by peripheral blood allografting from HLA-identical sibling donors. Nested RT-PCR analysis with sequence-specific primers for MMG was carried out on a monthly basis on BM samples. Three patients had MMG-positive BM, four patients had MMG-negative BM before allografting, and two were undetermined. In two patients, a clinical response after allografting (partial remission) occurred concurrently with the clearance of MMG expression, at a median of 6 months after allografting, following immune manipulation. In two patients, a prolonged stable disease and negative MMG expression occurred after day +360 from allografting. In two patients, progression of the disease was associated with MMG RT-PCR changing from negative to positive. In one case, a disease response occurring after donor lymphocyte infusion and grade II acute GVHD was heralded by negativization of MMG expression. Although preliminary, these data suggest that a graft-versus-breast cancer effect is detectable on micrometastatic BM disease.

Peripheral blood stem cell allograft rejection mediated by CD4(+) T lymphocytes recognizing a single mismatch at HLA-DP beta 1*0901.

Little is known about the molecular characteristics of alloantigens recognized by alloreactive T cells mediating hematologic stem cell graft rejection. In particular, it has never been shown that such alloantigens can be encoded by HLA-DP beta alleles. Indeed, matching for HLA-DP antigens is generally not considered to be of functional importance for the outcome of allogeneic bone marrow or peripheral blood stem cell transplantation. In this study, a case of peripheral blood stem cell allograft rejection was investigated in which the patient and donor differed for a single mismatch at HLA-DP in the rejection direction. Patient-derived T lymphocytes circulating at the time of rejection showed direct ex vivo cytotoxic activity against donor-derived B-lymphoblastoid cells as well as other HLA-DP beta 1*0901--expressing targets. The presence of HLA-DP beta 1*0901--specific effectors in vivo was further confirmed by in vitro stimulation experiments. CD4(+) T-cell lines and clones with specific cytotoxic activity against HLA-DP beta 1*0901--expressing targets including donor B-lymphoblastoid cells were generated both by nonspecific and by donor-specific in vitro stimulation. Taken together, these data demonstrate that HLA-DP can be the target antigen of cytotoxic CD4(+) T lymphocytes involved in peripheral blood stem cell allograft rejection. (Blood. 2001;98:1122-1126)

Effect of HLA matching on outcome of related and unrelated donor transplantation therapy for chronic myelogenous leukemia.

This article examines the diversity and biologic role of human lymphocyte antigen (HLA) genes as related to marrow transplantation for chronic myelogenous leukemia (CML). A better understanding of the nature and function of HLA variation is necessary as unrelated marrow transplantation evolves into a safe and effective treatment for CML. HLA matching is an important aspect of donor selection criteria and has a role in engraftment as well as the development of graft-versus-host disease and tolerance after transplant.

HSV-TK gene transfer into donor lymphocytes for control of allogeneic graft-versus-leukemia.

In allogeneic bone marrow transplantation (allo-BMT), donor lymphocytes play a central therapeutic role in both graft-versus-leukemia (GvL) and immune reconstitution. However, their use is limited by the risk of severe graft-versus-host disease (GvHD). Eight patients who relapsed or developed Epstein-Barr virus-induced lymphoma after T cell-depleted BMT were then treated with donor lymphocytes transduced with the herpes simplex virus thymidine kinase (HSV-TK) suicide gene. The transduced lymphocytes survived for up to 12 months, resulting in antitumor activity in five patients. Three patients developed GvHD, which could be effectively controlled by ganciclovir-induced elimination of the transduced cells. These data show that genetic manipulation of donor lymphocytes may increase the efficacy and safety of allo-BMT and expand its application to a larger number of patients.

Evaluation of recombinant human erythropoietin to facilitate autologous blood donation before surgery in anaemic patients with cancer of the gastrointestinal tract.

The aim of this study was to determine whether the preoperative administration of recombinant human erythropoietin (rHuEPO) could increase the rate of autologous blood donation and reduce the perioperative need for homologous blood in anaemic patients with cancer. Twenty-two anaemic (haematocrit less than 34 per cent), iron-deficient (iron less than 700 micrograms/l) patients, with gastric or colorectal cancer scheduled for elective surgery, were allocated randomly to two groups. The first (n = 11) received iron saccharate 200 mg/day intravenously for 12 consecutive days. The second (n = 11) received rHuEPO subcutaneously (300 units/kg as first administration, and 100 units/kg 4, 8 and 12 days later) with supplemental iron. On days 4, 8 and 12, if the haematocrit was greater than 34 per cent, patients donated one unit (350 ml) of autologous blood. In the iron group the mean haematocrit did not change from admission (31 per cent) to day 12 of treatment (31 per cent), and no patient could donate autologous blood. In the rHuEPO group, eight patients donated two units of autologous blood and three donated one unit. Four patients in the iron group received perioperative transfusion of homologous blood compared with none in the rHuEPO group. Administration of rHuEPO facilitated the donation of autologous blood and reduced perioperative homologous blood transfusion in anaemic patients with cancer.

Gene therapy in peripheral blood lymphocytes and bone marrow for ADA- immunodeficient patients.

Adenosine deaminase (ADA) deficiency results in severe combined immunodeficiency, the first genetic disorder treated by gene therapy. Two different retroviral vectors were used to transfer ex vivo the human ADA minigene into bone marrow cells and peripheral blood lymphocytes from two patients undergoing exogenous enzyme replacement therapy. After 2 years of treatment, long-term survival of T and B lymphocytes, marrow cells, and granulocytes expressing the transferred ADA gene was demonstrated and resulted in normalization of the immune repertoire and restoration of cellular and humoral immunity. After discontinuation of treatment, T lymphocytes, derived from transduced peripheral blood lymphocytes, were progressively replaced by marrow-derived T cells in both patients. These results indicate successful gene transfer into long-lasting progenitor cells, producing a functional multilineage progeny.

Transfer of the HSV-tk gene into donor peripheral blood lymphocytes for in vivo modulation of donor anti-tumor immunity after allogeneic bone marrow transplantation.

The infusion of donor lymphocytes after allogeneic bone marrow transplantation is a promising therapeutic tool for achieving a graft versus leukemia (GvL) effect in case of leukemic relapse (1-7), and for the treatment of other complications related to the severe immunosuppressive status of transplanted patients, such as Epstein Barr virus-induced lymphoproliferative disorders (EBV-BLPD) (8) or reactivation of CMV infection (9). Although the delay in the administration of T lymphocytes is expected to reduce the risk of severe GvHD, this risk is still present at higher doses of donor T-cells. The transfer of a suicide gene into donor lymphocytes could allow the in vivo selective elimination of cells responsible for severe GvHD. Additionally, under appropriate conditions, it may allow in vivo modulation of donor anti-tumor responses, and to separate GvL from GvHD. Finally, crucial questions concerning survival and function of donor lymphocytes could be answered by their gene marking. Previous studies documented that T lymphocytes are suitable targets for gene transfer through retroviral vectors (10, 11). This protocol has been designed to evaluate in the contest of allogeneic BMT: 1--the safety of increasing doses of donor lymphocytes transduced with a suicide retroviral vector; 2--the efficacy in terms of survival and immunologic potential of donor lymphocytes after in vitro activation, gene transduction, and immunoselection; 3--the possibility of in vivo down regulation of GvHD by the administration of ganciclovir to patients treated by tk-transduced donor lymphocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

Gene therapy and apheresis.

Gene therapy is becoming a real therapeutic chance in some genetic disorders. The first candidates to this approach are genetic diseases which involve hematopoiesis, because of the facility for the collection and the manipulation of hematopoietic progenitors. Apheresis techniques, which are able to collect a great number of mononuclear cells from peripheral blood, are ideal for obtaining a large number of cells which can be transfected. Future uses of gene therapy techniques could be: the treatment of hematopoietic genetic disorders, procedures of gene marking, and the manipulation of normal hematopoietic cells with the objective of increasing their resistance to myelotoxic drugs.

Transfer of the ADA gene into bone marrow cells and peripheral blood lymphocytes for the treatment of patients affected by ADA-deficient SCID.

Severe combined immunodeficiency (SCID) caused by deficiency of the enzyme adenosine deaminase (ADA) is the first genetic disorder considered for human somatic cell gene therapy. ADA-SCID patients can be cured by HLA-matched sibling donor bone marrow transplantation. Alternative transplantation strategies as well as enzyme replacement are being tested in those patients who do not have a suitable matched sibling donor. Some ADA-SCID patients may not be candidates for cytoablation due to infectious damage to the lung or liver, or may have a milder phenotype that does not justify the risks associated with haploidentical bone marrow transplantation. Replacement therapy with PEG-ADA has resulted in improvement in growth, a variable increase in the number of peripheral blood lymphocytes, and a decrease in the incidence of severe infections. Another approach to the treatment of severe genetic diseases is now represented by somatic cell gene therapy. We and others have conducted experiments in vitro and in vivo that have documented that T-lymphocytes are suitable vehicles for gene transfer. Although the pluripotent stem cell remains the ideal target cell for somatic cell gene therapy of disorders of the hematopoietic system, the use of T-lymphocytes as gene therapy vehicles is specifically indicated for ADA-deficient patients where they represent the affected cells. Furthermore, the selective engraftment of T-cells only, following bone marrow transplantation, has resulted in reconstitution of cellular and humoral immunity. A model for the functional analysis in vivo of the human immune system has been utilized for the preclinical evaluation of this approach.(ABSTRACT TRUNCATED AT 250 WORDS)