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Biomedical subjects

C M Rooney

Publications and source records attributed to C M Rooney.

At least 19 recordsLinked to original sources
PubMed2001

An Epstein-Barr virus deletion mutant associated with fatal lymphoproliferative disease unresponsive to therapy with virus-specific CTLs.

There is a growing interest in using antigen-specific T cells for the treatment of human malignancy. For example, adoptive transfer of Epstein-Barr virus (EBV)-specific cytotoxic T lymphocytes (CTLs) has been effective prophylaxis and treatment of EBV-associated lymphoproliferative disease in immunocompromised patients. For all immunotherapies, however, there has been a hypothetical concern that mutations in tumor-specific antigens may lead to tumor escape. We now demonstrate that such events may indeed occur, with lethal outcome. A patient who developed lymphoma after marrow transplantation received donor-derived, EBV-specific CTLs but died with progressive disease. The tumor cells proved substantially less sensitive to cytolysis than the EBV-transformed B-cell line used for CTL generation. The major cytolytic activity of the donor CTL was directed against 2 HLA-A11-restricted epitopes in the viral EBNA-3B antigen. Sequence analysis of this gene in the tumor virus revealed a 245-base pair deletion, which removed these 2 CTL epitopes. Hence, the viral antigen in the tumor had mutated in a way that allowed escape from CTLs. Analysis of EBV polymorphisms demonstrated that before CTL infusion, more than one virus was present, including a virus with wild-type EBNA-3B. After CTL infusion, only the virus with the EBNA-3B deletion could be detected, suggesting that the infused CTLs had selected a resistant strain in vivo. Such an occurrence, even when polyclonal CTL lines are used against genetically stable virus antigens, suggests that escape mutants may be a serious problem when CTL therapy is directed against more unstable tumor cell-derived targets.

Adolescent↗
PubMed2000

CD20 monoclonal antibody (rituximab) for therapy of Epstein-Barr virus lymphoma after hemopoietic stem-cell transplantation.

After bone marrow transplantation (BMT) using T-cell-depleted marrow from an unrelated donor or HLA-mismatched related donor, the risk of developing lymphoproliferative disease associated with the Epstein-Barr virus (EBV) ranges from 1% to 25%. We have shown that administration of donor-derived EBV-specific cytotoxic T lymphocytes (CTL) is effective prophylaxis and treatment for this complication, and we routinely generate CTL for high-risk patients. However, EBV lymphoma can occur in recipients of matched-sibling transplants for whom CTL are unavailable or in patients for whom CTL administration is contraindicated. We report on 3 such patients, who were successfully and safely treated with rituximab, a CD20 monoclonal antibody. The patients remain disease free 7, 8, and 9 months, respectively, after therapy. We conclude that CD20 antibody may be a useful alternative treatment strategy in patients with EBV lymphoma after BMT. (Blood. 2000;95:1502-1505)

Adolescent↗
PubMed2000

Transduction of human PBMC-derived dendritic cells and macrophages by an HIV-1-based lentiviral vector system.

Professional antigen-presenting cells, such as dendritic cells (DCs) and macrophages, are target cells for gene therapy of infectious disease and cancer. However, transduction of DCs and macrophages has proved difficult by most currently available gene transfer methods. Several recent studies have shown that lentiviral vector systems can efficiently transduce many nondividing and differentiated cell types. In this study, we examined the gene transfer to DCs and macrophages using a lentiviral vector system. Human DCs were propagated from the adherent fraction of peripheral blood mononuclear cells (PBMCs) by culture in medium containing GM-CSF, IL-4, and TNF-alpha. Human macrophages were propagated from adherent PBMCs in medium containing GM-CSF. High titers of a replication-defective vesicular stomatitis virus glycoprotein G pseudotyped HIV-1-based vector encoding the enhanced yellow fluorescent protein were produced. In immature DCs (culture days 3 and 5), transduction efficiencies of 25 to 35% were achieved at a multiplicity of infection of 100. However, the transduction efficiency was decreased in more mature DCs (culture day 8 or later). Furthermore, monocyte-derived macrophages were also transduced by the lentiviral vector system. In addition, Alu-LTR PCR demonstrated the integration of the HIV-1 provirus into the cellular genome of the transduced DCs and macrophages. Allogeneic mixed lymphocyte reactions revealed similar antigen-presenting functions of untransduced and lentivirally transduced DCs. Thus, the results of this study demonstrate that both PBMC-derived DCs and macrophages can be transduced by lentiviral vectors.

Alu Elements↗
PubMed2000

The use of cytotoxic t cells for the prevention and treatment of epstein-barr virus induced lymphoma in transplant recipients.

Epstein-Barr Virus Lymphoproliferative Diseases (EBV-LPD) can affect patients receiving allogeneic hemopoietic stem cell or solid organ transplant. They are caused by impairment of EBV specific CD8+ cytotoxic T-lymphocyte (CTL) response due to the immunosuppression that follows these procedures. Despite different therapies LPD can still have a rapid and lethal course. A promising solution is the application of adoptive immunotherapy approaches such as ex vivo generated EBV-specific CTLs for prevention and therapy. Their infusion has been demonstrated to be safe and effective in allogeneic hemopoietic stem cell transplant recipients and their use after allogeneic solid organ transplant is also under evaluation.

Adoptive Transfer↗
PubMed1999

Transfer of EBV-specific CTL to prevent EBV lymphoma post bone marrow transplant.

EBV-associated lymphoproliferative disorders (EBV-LPD) are a significant problem after hemopoietic stem cell transplantation from unrelated donors or mismatched family members. Risk factors include T-cell depletion, MHC mismatch, and intensity of immunosuppression. New therapeutic strategies involve cellular immunotherapy approaches and both donor T-cells and EBV-specific cytotoxic T lymphocytes (CTLs) have proven to be effective therapies. EBV-specific CTL has also proved to have a major impact on the incidence of this complication when used prophylactically.

Antibody Specificity↗
PubMed1999

Characterization of mutations in the gene doublecortin in patients with double cortex syndrome.

Mutations in the X-linked gene doublecortin, which encodes a protein with no dear structural homologues, are found in pedigrees in which affected females show "double cortex" syndrome (DC; also known as subcortical band heterotopia or laminar heterotopia) and affected males show X-linked lissencephaly. Mutations in doublecortin also cause sporadic DC in females. To determine the incidence of doublecortin mutations in DC, we investigated a cohort of eight pedigrees and 47 sporadic patients with DC for mutations in the doublecortin open reading frame as assessed by single-stranded conformational polymorphism analysis. Mutations were identified in each of the eight DC pedigrees (100%), and in 18 of the 47 sporadic DC patients (38%). Identified mutations were of two types, protein truncation mutations and single amino acid substitution mutations. However, pedigrees with DC displayed almost exclusively single amino acid substitution mutations, suggesting that patients with these mutations may have less of a reproductive disadvantage versus those patients with protein truncation mutations. Single amino acid substitution mutations were tightly clustered in two regions of the open reading frame, suggesting that these two regions are critical for the function of the Doublecortin protein.

Brain Diseases↗
PubMed1999

Autoimmune disease induced by dendritic cell immunization against leukemia.

Induction of an optimal immune response will likely be a prerequisite for successful immunotherapy of human leukemias and other malignancies. Dendritic cells are highly effective at inducing an immune response to antigens to which the host is unresponsive, while transgenic expression of the costimulator molecule CD40 ligand (gp39/CD154) and the T cell growth factor interleukin 2 (IL2) are also able to augment immune responsiveness. We therefore investigated whether a combination of these two distinctive approaches to immunostimulation could safely increase the anti-tumor immune response compared to each stimulus alone. We injected BALB/CBYJ mice with syngeneic dendritic cells (DC) exposed to A20 lymphoblastic leukemia cell-derived peptides and proteins which had been acid-eluted from the cell surface. In additional mice, the pulsed DC were mixed with genetically modified syngeneic fibroblasts that were expressing CD40 ligand or secreting interleukin 2 (IL2). Three days after their third, weekly, vaccination, they were challenged with parental A20 cells. Tumor growth was suppressed by responses to pulsed DC alone (P < 0.02). This suppression was further enhanced when pulsed DC were coinjected with fibroblasts expressing CD40 ligand and IL2 (P < 0.0005 compared to DC alone) even though CD40 ligand and IL2-expressing fibroblasts alone offered no significant protection in this model. Mice receiving the full complement of immunostimulants either failed to develop visible tumors or developed small tumors which quickly necrosed and regressed, allowing the mice to become long term tumor-free survivors. Antibody mediated depletion of either CD4+ or CD8+ T-cell subset significantly reduced the level of protection afforded by the vaccination. However, it became evident that this intensive stimulation of the immune system lead not only to tumor eradication but also to destruction of cells bearing normal self antigens. Hence, 60 days after challenge with A20 cells all mice in the DC/IL2/CD40 ligand group developed a severe, systemic autoimmune disorder that resembled graft versus host disease and manifest itself by significant peripheral blood cytotoxicity against autologous fibroblasts, blood dyscrasias, gross hepatosplenomegaly, cachexia and fur loss. This phenomenon depended on CD8+ cytotoxic T lymphocytes. Our results therefore suggest that the most effective strategies of immunotherapy against leukemia may also exceed the threshold of anergic cells, leading to a loss of self tolerance to normal self-antigens and the induction of an CD8+ anti-self effector response.

Animals↗
PubMed1999

Lymphoproliferative disorders involving Epstein-Barr virus after hemopoietic stem cell transplantation.

Lymphoproliferative disorders involving uncontrolled expansion of donor-derived B cells infected with Epstein-Barr virus (EBV) are a significant problem after hemopoietic stem cell transplantation. Risk factors, which include T cell depletion, major histocompatibility complex mismatch, and intensity of immunosuppression illustrate the importance of T cell immune surveillance. Recent studies have identified viral and host factors that affect the T-cell response to EBV. Monitoring EBV load in the blood by polymerase chain reaction allows early identification of high-risk patients and early institution of therapy. Adoptive immunotherapy approaches using donor T cells have proven effective and EBV-specific cytotoxic T lymphocytes have also been used successfully for prophylaxis. The simplest way of preventing EBV lymphoproliferation, however, may be to deplete B cells from the donor marrow prior to infusion to prevent the transmission of EBV-infected B cells.

B-Lymphocytes↗
PubMed1998

Infusion of cytotoxic T cells for the prevention and treatment of Epstein-Barr virus-induced lymphoma in allogeneic transplant recipients.

Epstein-Barr virus (EBV) causes potentially lethal immunoblastic lymphoma in up to 25% of children receiving bone marrow transplants from unrelated or HLA-mismatched donors. Because this complication appears to stem from a deficiency of EBV-specific cytotoxic T cells, we assessed the safety and efficacy of donor-derived polyclonal (CD4(+) and CD8(+)) T-cell lines as immunoprophylaxis and treatment for EBV-related lymphoma. Thirty-nine patients considered to be at high risk for EBV-induced lymphoma each received 2 to 4 intravenous infusions of donor-derived EBV-specific T lymphocytes, after they had received T-cell-depleted bone marrow from HLA-matched unrelated donors (n = 33) or mismatched family members (n = 6). The immunologic effects of this therapy were monitored during and after the infusions. Infused cells were identified by detection of the neo marker gene. EBV-specific T cells bearing the neo marker were identified in all but 1 of the patients. Serial analysis of DNA detected the marker gene for as long as 18 weeks in unmanipulated peripheral blood mononuclear cells and for as long as 38 months in regenerated lines of EBV-specific cytotoxic T cells. Six patients (15.5%) had greatly increased amounts of EBV-DNA on study entry (>2, 000 genome copies/10(6) mononuclear cells), indicating uncontrolled EBV replication, a complication that has had a high correlation with subsequent development of overt lymphoma. All of these patients showed 2 to 4 log decreases in viral DNA levels within 2 to 3 weeks after infusion and none developed lymphoma, confirming the antiviral activity of the donor-derived cells. There were no toxic effects that could be attributed to prophylactic T-cell therapy. Two additional patients who did not receive prophylaxis and developed overt immunoblastic lymphoma responded fully to T-cell infusion. Polyclonal donor-derived T-cell lines specific for EBV proteins can thus be used safely to prevent EBV-related immunoblastic lymphoma after allogeneic marrow transplantation and may also be effective in the treatment of established disease.

Adolescent↗
PubMed1998

Epstein-Barr virus (EBV)-specific cytotoxic T lymphocytes for the treatment of patients with EBV-positive relapsed Hodgkin's disease.

Adoptive transfer of Epstein-Barr virus (EBV)-specific cytotoxic T lymphocytes (CTLs) is effective prophylaxis and treatment of EBV-positive immunoblastic lymphoma in immunocompromised patients. In 50% of patients with Hodgkin's disease, the tumor cells are EBV antigen-positive and may therefore also be suitable targets for treatment with virus-specific CTLs. However, Hodgkin's disease may produce several inhibitory effects on immune induction and effector function in vivo, which may preclude the generation or effector function of CTLs reactive against EBV viral proteins, including those expressed by the tumor cells. We have investigated whether EBV-specific CTLs could be generated ex vivo from 13 patients with Hodgkin's disease: nine with active relapsed disease and four who were in clinical remission after a first or subsequent relapse. CTL lines were successfully generated from nine of 13 patients (five active disease, four remission). Although these lines had an abnormal pattern of expansion comparable to EBV-specific CTLs generated from normal donors, their phenotype was normal except for reduced expression of the zeta chain of the T-cell receptor (TCR). Their cytotoxicity was also compared to EBV-specific lines generated from normal donors and included activity against LMP2a, one of the three weakly immunogenic viral antigens expressed by Hodgkin's tumor cells. To assess the activity of the CTLs in vivo, they were gene-marked and infused into three patients with multiply relapsed disease. The CTLs persisted for more than 13 weeks postinfusion and retained their potent antiviral effects in vivo, thereby enhancing the patient immune response to EBV. This approach may therefore have value in the treatment of EBV-positive Hodgkin's disease.

Adolescent↗
PubMed1998

Treatment of relapsed Hodgkin's disease using EBV-specific cytotoxic T cells.

Donor-derived Epstein-Barr virus (EBV)-specific cytotoxic T lymphocytes (CTL) are successful in the prevention and treatment of Epstein-Barr virus (EBV)-associated lymphoproliferative disease (LPD) in allogeneic bone marrow transplant (BMT) recipients [1, 2]. This finding prompted us to use a similar approach to the treatment of relapsed EBV-positive Hodgkin's disease [3]. Autologous EBV-specific CTL lines could be generated on the first or second attempt from 11 of 15 patients with Hodgkin's disease. Peripheral blood TCR zeta-chain levels were low, but increased in the activated CTL lines. Three patients have received gene-marked autologous CTL. The first two patients experienced alleviation of stage B symptoms and a drop in peripheral blood EBV load. However, this situation reversed between 6 and 12 weeks after infusion, when chemotherapy and radiation were reinstated. Both patients eventually progressed and died. The third patient had a pleural effusion, which increased after CTL infusion. Analysis of the pleural effusion revealed both tumor cells and levels of marker gene over 100 fold greater than in peripheral blood. The infused CTL line showed activity against LMP2. The patient initially improved and then remained stable for over eight months after CTL infusion, but now has progressive disease. We currently are evaluating methods for introducing the LMP2 gene into dendritic cells and using these to present LMP2 to autologous T cells. Using both retrovirus and herpesvirus vectors to express LMP2 in dendritic cells, LMP2-specific CTL were successfully generated from individuals who were EBV-seronegative or who were non-responsive to LMP2 when presented on autologous LCL. In future protocols, LMP2-specific CTL will be used for treatment.

Dendritic Cells↗
PubMed1998

Immunotherapy for Epstein-Barr virus-associated cancers.

Epstein-Barr virus (EBV)-associated lymphoproliferative disease (EBV-LPD) is a frequently fatal complication of organ transplantation and human immunodeficiency virus (HIV) infection. We have studied the safety and efficacy of adoptively transferred, gene-marked virus-specific cytotoxic T lymphocytes (CTLs) as prophylaxis and treatment of EBV-LPD in recipients of T-cell-depleted allogeneic bone marrow. In 42 patients treated prophylactically, no toxicity was experienced. None of these patients developed EBV-LPD, in contrast with eight of 53 (15%) patients who did not receive prophylactic CTL. Three patients who had not received CTL developed aggressive disease and received CTL as treatment. Gene-marked CTL homed to tumor sites and selective accumulation of marker gene was detected in tumor tissues. Tumors regressed completely in two patients, but the third died of respiratory failure. Infused CTLs persisted for up to 3 years in vivo, they rapidly reconstituted EBV-specific immune responses to levels seen in normal individuals, and they reduced high viral titers by two to three logs. We are now using autologous EBV-specific CTL to treat patients with relapsed EBV-positive Hodgkin's disease and we are developing methods for the generation of antigen-specific lines. This approach could be applied to patients with HIV who develop EBV-LPD, using CTL derived early in the course of HIV infection.

B-Lymphocytes↗
PubMed1998

Gene and cell transfer for specific immunotherapy.

The realisation that human tumor cells may express and process tumor specific and tumor associated antigens has increased interest in immunotherapeutic approaches to cancer treatment. This interest has been coupled with a burgeoning ability to genetically modify tumor cells and components of the immune system, in an effort to maximize the anti-neoplastic response. In a number of settings, gene modified tumor vaccines, cytotoxic T cells and dendritic cells are producing both immunomodulation and clinically evident benefits. Continued exploration of this approach seems well justified.

Adenoviridae↗
PubMed1998

EBV specific CTL: a model for immune therapy.

We have been generating Epstein-Barr virus specific cytotoxic T cells for patients at high risk of developing EBV driven lymphoma. To discover the fate of the cells in vivo, we first marked them genetically, using a retroviral vector. Our results in 51 patients show that the approach is safe, that the CTL persist for several years and that they are able to mediate anti-viral and anti-tumor effects. We are now studying other virally-linked malignancies to discover whether a similar approach will be of therapeutic value.

Bone Marrow Transplantation↗
PubMed1997

Control of virus-induced lymphoproliferation: Epstein-Barr virus-induced lymphoproliferation and host immunity.

Epstein-Barr virus (EBV) is a latent herpesvirus that is associated with a number of tumors. EBV-infected cells show three patterns of latency ranging from type 1, where only one EBV-encoded antigen is expressed, to type 3, where all nine latent cycle proteins encoded by EBV are expressed. Malignancies exhibiting the type 3 latency pattern are highly immunogenic and occur only in immunocompromised patients. It has recently been shown that adoptive immunotherapy with EBV-specific cytotoxic T lymphocytes is an effective therapy for such tumors. Immunotherapy strategies and approaches to increase tumor immunogenicity are now being evaluated in tumors expressing type 2 latency.

Animals↗