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Biomedical subjects

D Dilloo

Publications and source records attributed to D Dilloo.

At least 19 recordsLinked to original sources
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↗
PubMed1998

Increased transduction efficiency of primary hematopoietic cells by physical colocalization of retrovirus and target cells.

Efficient gene transfer into hematopoietic stem cells offers a number of potential therapeutic applications. However, the relatively low titer of retroviral supernatants and the requirement for cell division to ensure integration have meant that transduction efficiency has been low. We have modified a flowthrough approach to cell transduction and have been able consistently to increase gene transfer efficiency into human hematopoietic progenitor cells. We transduced CD34 cells with retroviral vectors encoding a truncated nerve growth factor receptor (NGFR) or neo. Retroviral supernatant was pulled through 0.2-micron polycarbonated membranes, followed by placement of cells on the filter. In the absence of cytokines, the transduction efficiency of CD34 cells with a NGFR vector was increased 3-11-fold over that obtained at an identical MOI in liquid culture to produce 11%-44% transduction. Furthermore, both Thy1+ and Thy1- subsets in a total CD34 population were transduced with similar efficiency, and transduction with a neo vector, as measured by G418 resistance in clonogenic assays, increased 1.5-5-fold. The mechanism by which gene transfer is improved may reflect colocalization of cells and retrovirus. Costaining of cells transduced on the filter with an NGFR retrovirus with both an NGFR antibody and a gp70 antibody that recognizes viral coat protein revealed high-level coexpression. The levels of in vitro gene transfer we obtain are equivalent to those observed when CD34 cells are cocultured in liquid culture with cytokines. However, culture with cytokines may commit CD34 cells to differentiation and has produced disappointingly low levels of subsequent in vivo gene transfer. Gene marking studies using distinguishable retroviral vectors will provide a means of learning whether the effects of flowthrough transduction genuinely enhance the efficiency of gene transfer to human marrow-repopulating cells.

Antigens, CD↗
PubMed1997

CD40 ligand induces an antileukemia immune response in vivo.

Leukemia cells may express tumor specific antigens in association with Class I and II major histocompatability complex (MHC) molecules. However, lack of expression of conventional costimulator molecules means that these cells tend to induce specific T-cell anergy rather than activation. CD40 ligand (CD40L) is a costimulator molecule that directly activates T cells and may promote antigen presentation by CD40-expressing cells, which include professional antigen presenting cells and B-acute lymphoblastic leukemia (ALL) cells from many patients. We determined whether transgenic expression of CD40L could enhance an antileukemia immune response using a CD40+ murine lymphoblastic (A20) leukemia and a CD40- myeloblastic (WEHI-3) leukemia in a tumor treatment model. Injection of otherwise nonimmunogenic A20 cells in the presence of CD40L induced an immune response active against preexisting A20 tumor at a distant site. Moreover, concomitant local secretion of transgenic interleukin-2 (IL-2) further amplified the antileukemic response induced and increased protection against preexisting tumor. In ex vivo studies, CD40 activation of A20 cells enhances the antigen presenting potential of A20 cells by upregulating expression of B7.1 (CD80), Class I and II MHC molecules, and increases expression of fas antigens. The importance of CD40 activation to the resulting antitumor response is further emphasized by the failure of transgenic CD40L to protect against the CD40- WEHI myeloblastic leukemia. Depletion studies showed the protective effects against A20 cells to be mediated by a combination of CD4+ and CD8+ T lymphocytes and by natural killer (NK) cells. These results suggest a means by which CD40+ leukemia cells may be rendered immunogenic in vivo.

Animals↗
PubMed1997

A novel herpes vector for the high-efficiency transduction of normal and malignant human hematopoietic cells.

Herpes simplex viruses (HSVs) would offer numerous advantages as vectors for gene transfer, but as yet they have not proved capable of transducing hematopoietic cells. Using a genetically inactivated form of HSV that is restricted to a single cycle of replication (disabled single-cycle virus, [DISC-HSV]), we have transduced normal human hematopoietic progenitor cells and primary leukemia blasts with efficiencies ranging from 80% to 100%, in the absence of growth factors or stromal support. Toxicity was low, with 70% to 100% of cells surviving the transduction process. Peak expression of transferred genes occurred at 24 to 48 hours after transduction with the DISC-HSV vector, declining to near background levels by 14 days. Despite this limitation, sufficient protein is produced by the inserted gene to permit consideration of the vector for applications in which transient expression is adequate. One example is the transfer of immunostimulatory genes, to generate leukemia immunogens. Thus, murine A20 leukemia cells transduced with a DISC-HSV vector encoding granulocyte-macrophage colony-stimulating factor were able to stimulate a potent antitumor response in mice, even against pre-existing leukemia. The exceptional transducing ability of the DISC-HSV vector should therefore facilitate genetic manipulation of normal and malignant human hematopoietic cells for biological and clinical investigation.

Animals↗
PubMed1996

High-efficiency transduction of freshly isolated human tumor cells using adenoviral interleukin-2 vectors.

Tumor cells genetically modified to express immunostimulatory molecules can produce high levels of antitumor immunity in rodent models. Although a number of clinical trials are currently in progress to assess the value of the approach in human disease, almost all require ex vivo transduction of cultured tumor cells with retroviral vectors. This process is not feasible for many human malignancies, hampering clinical evaluation of the approach. We have used an E1a,1b/E3 deletion mutant of adenovirus containing either the lacZ or the human interleukin-2 (IL-2) gene to transduce human neuroblastoma cells. This vector transduces fresh neuroblastoma cells and neuroblastoma cell lines with an efficiency of 80-90%, compared to an efficiency of 0-14% obtained with retroviral vectors. Cells transduced with the IL-2 adenovector produce up to 12,000 pg of IL-2/10(6) cells/24 hr. IL-2 adenovector-transduced neuroblasts are immunostimulatory; when they are cultured with patient lymphocytes, they increase the proportion of DR+ T cells and generate major histocompatibility complex (MHC) unrestricted cytotoxic effector cells active against parental (nontransduced) tumor cells. We conclude that IL-2 adenovector can be used to transduce freshly isolated human tumor cells efficiently, which will then produce immunomodulatory quantities of the cytokine. The use of adenoviral rather than retroviral vectors facilitates preparation of human tumor "vaccines" and these vectors are now being used in our clinical study of neuroblastoma patients.

Adenoviruses, Human↗
PubMed1996

Colocalization of retrovirus and target cells on specific fibronectin fragments increases genetic transduction of mammalian cells.

Hematopoietic cells are important targets for genetic modification with retroviral vectors. Attempts at human gene therapy of stem cells have achieved limited success partly because of low gene transfer efficiency. Chymotryptic fragments of the extracellular matrix molecule fibronectin used during infection have been shown to increase transduction of human hematopoietic progenitor cells. Here, we demonstrate that this enhanced gene transfer into mammalian target cells is due to direct binding of retroviral particles to sequences within the fibronectin molecule. Transduction of mammalian cells, including murine long-term repopulating hematopoietic cells, is greatly enhanced when cells are adherent to chimeric fragments containing these retroviral binding sequences. In addition, colocalization of retrovirus and target cells on fibronectin peptides allows targeted transduction of specific cell types by exploiting unique ligand/receptor interactions.

Animals↗
PubMed1996

Combined chemokine and cytokine gene transfer enhances antitumor immunity.

The probability of producing a specific antitumor response should be increased by multiplying the number of T lymphocytes that encounter the malignant cells. We tested this prediction in a murine model, using a recently discovered T-cell chemokine, lymphotactin (Lptn). This chemokine increased tumor cell infiltration with CD4+ lymphocytes but generated little antitumor activity. Coexpression of the T-cell growth factor interleukin-2, however, greatly expanded the T lymphocytes attracted by Lptn, affording protection from the growth of established tumor in a CD4+ and CD8+ T cell-dependent manner. Lesser synergy was seen with GM-CSF. Hence coexpression of a T-cell chemokine and T-cell growth factor potentiates antitumor responses in vivo, suggesting a general strategy to improve cancer immunotherapy.

Animals↗
PubMed1996

Gene marking and gene therapy for transplantation medicine.

The classic application for gene therapy is in the correction of single gene defects, although this has been complicated by the low efficiency of gene transfer into hematopoietic cells. Gene therapy, however, has potential for the modulation of tumor cell growth, drug sensitivity, and antitumor immune responses. In addition, gene marking can be used, in spite of this limited transfer efficiency, to provide information on hematopoiesis, sources of cancer relapse after stem cell transplant, and the relative efficacy of graft manipulation techniques. This article reviews the applications of gene therapy and gene marking in transplantation medicine.

Biomarkers↗
PubMed1996

Differential production of interleukin-3 in human T lymphocytes following either CD3 or CD2 receptor activation.

Interleukin-3 (IL-3) is expressed in T lymphocytes and stimulates the growth of multipotent hematopoietic progenitors. Little is known, however, about the stimuli that lead to IL-3 protein release. We examined IL-3 and granulocyte-macrophage colony-stimulating factor (GM-CSF) mRNA expression and protein secretion in human T lymphocytes following activation via the TCR/CD3 complex, the CD2 receptor, and the IL-2 receptor. GM-CSF mRNA expression and protein release were found in CD3 and CD2 activated T cells with maximum GM-CSF release following stimulation with IL-2. IL-3 protein release is regulated via the CD2 receptor with virtually no IL-3 release after T cell stimulation via CD3. In contrast, IL-3 mRNA accumulation is more pronounced after CD3 activation than after CD2 activation. This suggests that upregulation of IL-3 protein release following T cell stimulation via CD-2 occurs largely at the translational or posttranslational level. These data also indicate that differential control of cytokine production can occur in response to activation of the alternative T cell receptor. Interaction of the T cell CD2-receptor with its natural ligand LFA-3 expressed on stromal cells might represent a regulatory mechanism for rapid release of IL-3, facilitating proliferation of multipotent hematopoietic cells.

CD2 Antigens↗
PubMed1995

Bone marrow fibroblasts from children with aplastic anemia exhibit reduced interleukin-6 production in response to cytokines and viral challenge.

Defects of the bone marrow microenvironment have been implicated in the pathogenesis of aplastic anemia (AA). We examined granulocyte/macrophage colony-stimulating factor (GM-CSF) and IL-6 production in bone marrow fibroblasts from five children with AA in response to cytokines and viral challenge with cytomegalovirus (CMV). Cytokine-stimulated IL-6 production in patients' fibroblasts was significantly reduced, whereas GM-CSF production was found to be comparable to normal controls. Also, CMV-mediated stimulation of IL-6 production was significantly lower in bone marrow fibroblasts from patients with AA. During the late phase of CMV-infection, IL-6 production was suppressed in CMV-infected fibroblasts from patients with AA with 90% inhibition of IL-6 protein production and marked reduction in IL-6 mRNA accumulation. Defective IL-6 production in bone marrow fibroblasts might contribute to hematopoietic suppression in some patients with AA.

Adolescent↗
PubMed1995

IL-2 inhibits proliferation of K562 cells and reduces accumulation of bcr/abl mRNA and oncoprotein.

Cell lines of myeloid origin have been shown to express interleukin-2 receptors (IL-2R). Here, we demonstrate the expression of IL-2R alpha and IL-R beta on the CML blast cell line K562 by FACS analysis and cross-linking assay. Furthermore, we examined the effect of IL-2 on leukemic progenitor growth, employing K562 as a model. Clonogenic growth was assessed after 3 days of culture by colony formation in a serum-free, semi-solid assay system. IL-2 was found to exhibit a dose-dependent suppressive effect on K562 clonogenicity with 48% inhibition of colony formation at 250 U IL-2 and 60% inhibition at 1000 U IL-2. Philadelphia chromosome (Ph)-positive K562 cells possess multiple copies of the bcr/abl fusion gene whose transcript and protein product (p210) is thought to confer growth advantage to CML cells. We therefore investigated IL-2-dependent modulation of bcr/abl mRNA accumulation and p210 protein levels in K562 cells. After 4 h of culture in the presence of IL-2, a 3-15-fold reduction of bcr/abl mRNA accumulation was demonstrated by competitive reverse PCR. Reduction of bcr/abl fusion protein levels was demonstrated at 24 h of IL-2-supplemented cell culture, employing p210 recognizing monoclonal antibodies (mAbs) in FACS analysis. Levels of proliferation marker Ki67 were only marginally affected. We conclude: (1) K562 cells express both IL-2R alpha and IL-R beta; (2) IL-2 inhibits clonogenic growth of K562 in a dose-dependent manner; and (3) IL-2-mediated inhibition of K562 proliferation is preceded by a reduction of bcr/abl mRNA accumulation and p210 protein levels.

Base Sequence↗
PubMed1995

Time course of interferon-gamma production deficiency after autologous and allogeneic stem cell transplantation for malignancies.

While success of autologous bone marrow transplantation (BMT) for malignancies largely depends on the cytotoxicity of the ablative regimen, achievement of relapse-free survival after allogeneic BMT is thought to be enhanced by immunologic effects. We therefore investigated in vivo and in vitro production of interferon-gamma (IFN-gamma), soluble interleukin-2 (IL-2) receptor alpha-chain (sCD25), tumor necrosis factor-alpha (TNF-alpha), and granulocyte-macrophage colony-stimulating factor (GM-CSF) in patients before and during various time periods up to 2 years after autologous and allogeneic BMT. Cytokine levels were assessed in patient plasma and in supernatants of patient-derived peripheral blood mononuclear cells (PBMNC) cultured for 3 days in the presence of T cell-specific stimulation via CD3 plus IL-2. Our studies show that IFN-gamma plasma levels are decreased in autologous graft recipients before and during the first 30 days posttransplant. In allogeneic graft recipients, IFN-gamma plasma levels are also decreased during the first 30 days posttransplant, but otherwise are comparable to normal control (NC) values. In vitro stimulated PBMNC from autologous graft recipients also exhibit an IFN-gamma production defect before and during the first 30 days posttransplant. In contrast, before and up to 30 days after allogeneic BMT, stimulated IFN-gamma production is comparable to NC values but then gradually decreases, reaching its trough levels at between 61 and 180 days post-BMT. The IFN-gamma production defects in both patient groups seem to be specific, as sCD25, TNF-alpha, and GM-CSF production in stimulated PBMNC is normal or even enhanced at any time after autologous or allogeneic BMT. Deficient IFN-gamma production in patient-derived PBMNC does not correlate with variation in monocyte, T cell, or natural killer (NK) cell numbers during the posttransplantation course.

Adolescent↗
PubMed1994

Induction of two distinct natural killer-cell populations, activated T cells and antineoplastic cytokines, by interleukin-2 therapy in children with solid tumors.

Children with poor prognoses regarding solid tumors have benefited from autologous bone marrow transplantation as a treatment modality. Posttransplantation adjuvant interleukin-2 (IL-2) therapy has previously been shown to improve prognosis in adults with neoplastic disease. The improved survival probability has been attributed to IL-2-mediated stimulation of the immune system and its antineoplastic activity. In this study, 10 pediatric patients with solid tumors in complete remission after autologous stem cell transplantation were treated with recombinant IL-2, which was administered in three 5-day cycles of continuous intravenous infusions with a 2-week rest in between cycles. We demonstrated that IL-2 therapy enhanced transplantation-related stimulation of the immune system, on both the cellular and humoral levels. Peripheral blood T and natural killer (NK) cells increased by the factors four and 14, respectively. Activation of the immune system was demonstrated by significantly elevated levels of soluble IL-2 receptor (IL-2R) and increased CD25 surface expression on T lymphocytes. IL-2 also induced significant proliferation of the CD56bright NK-cell subpopulation that appears post-bone marrow transplant (BMT) and is found only in minimal amounts in normal controls. In vivo and in vitro production of tumor cytotoxic cytokines tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) was significantly stimulated following IL-2 therapy, further indicating IL-2-mediated stimulation of the antineoplastic activity of the immune system.

Adolescent↗
PubMed1993

Myeloablative radiochemotherapy and hematopoietic stem-cell rescue in poor-prognosis Ewing's sarcoma.

PURPOSE: The prognosis of patients with multifocal primary and early or multiple relapsed Ewing's sarcoma is poor with conventional chemoradiotherapy and surgery. We evaluated the efficacy and feasibility of a myeloablative regimen administered as consolidation treatment for these patients. PATIENTS AND METHODS: The ablative regimens consisted of simultaneous radiochemotherapy: 12 Gy hyperfractionated total-body irradiation (TBI; two doses of 1.5 Gy for 4 days) plus fractionated high-dose melphalan (30 to 45 mg/m2 for 4 days) followed by high-dose etoposide (40 to 60 mg/kg) with or without carboplatin (900 to 1,500 mg/m2) (hyper-ME +/- C). These regimens were applied in a dose-escalation study that included 17 patients. All patients underwent remission induction chemotherapy and local treatment before myeloablative therapy. Seven patients had multifocal primary Ewing's sarcoma, and 10 had early or multiple relapse. We performed a matched-cohort analysis of the 17 grafted patients with 41 historic controls matched for sex, age, diagnosis, extent of disease, interval from diagnosis to transplant in the transplant group, and interval from diagnosis to relapse in the control group. RESULTS: The probability of relapse in the study patients is 52% at 6 years after the last event before transplantation. In the control group, the probability of relapse at 6 years was 98%. Eight of 17 treated patients are alive in complete remission at a median observation time of 49 months (range, 19 to 76) from the last event before transplantation. Probability of relapse-free survival in the study patients is 45% +/- 12% at 6 years after the last event before transplant, compared with 2% +/- 2% for the historic control group. CONCLUSION: Myeloblative therapy with hyper-ME +/- C radiochemotherapy can improve the prognosis of multifocal primary and early or multiple relapsing Ewing's sarcoma.

Adolescent↗
PubMed1992

Treatment of donor T cell-mediated hematopoietic suppression after haploidentical bone marrow transplantation by T cell modulation in a patient with severe combined immunodeficiency.

An 8-month-old male patient with severe combined immunodeficiency syndrome was transplanted with maternal, haploidentical T cell-depleted bone marrow without prior conditioning therapy. Acute graft-versus-host disease developed 2 weeks post bone marrow transplantation (BMT) and was successfully treated with cortisone. After cortisone withdrawal the patient developed myeloid and B cell depression concomitant with T cell activation. For specific T cell modulation, treatment with the T cell receptor (TCR) alpha beta chain-binding MoAb BMA031 was initiated in combination with cyclosporin A. GM-CSF was given to enhance myeloid reconstitution. About 1 year post BMT, B cell and granulocyte counts were within the normal range with stable chimerism in both lineages. B cell proliferation tests were normal and first signs of in vitro immunoglobulin synthesis occurred.

Agranulocytosis↗
PubMed1991

Differential regulation of lymphokine production by distinct subunits of the T cell interleukin 2 receptor.

Most biologic responses to IL-2 have been attributed to interaction of IL-2 with a high affinity receptor which consists of a heterodimer composed of two distinct IL-2-binding proteins (IL-2R alpha/IL-2R beta). However, both low affinity IL-2R alpha (55 kD) and intermediate affinity IL-2R beta (70-75 kD) also appear to be expressed independently on the cell surface. We investigated the receptor-specific regulatory effects of IL-2 on cytokine production in unstimulated and activated T cells. T cells were activated by stimulation of the antigen receptor complex with anti-CD3 mAb. IL-2 (10(2) U/ml, 1 nM) stimulation of resting cells resulted in a fivefold increase in GM-CSF release but in only minimal IFN-gamma release. IL-2 markedly augmented mRNA expression of GM-CSF but not IFN-gamma in unstimulated T cells. IL-2R beta mAb but not IL-2R alpha mAb decreased IL-2-induced GM-CSF release and mRNA expression from unstimulated T cells. IL-2 concentrations required for GM-CSF release from resting cells suggested ligand binding to an intermediate affinity receptor. GM-CSF and IFN-gamma release from activated T cells increased four- to fivefold in response to 1 nM IL-2 and IL-2 augmented both GM-CSF and IFN-gamma mRNA. IL-2R beta mAb but not IL-2R alpha mAb reduced GM-CSF release and mRNA expression in activated T cells stimulated with 1 nM IL-2. IL-2R alpha blockade markedly decreased IL-2-induced IFN-gamma release and mRNA expression from activated cells, while IL-2R beta blockade had little effect on IFN-gamma production in activated cells. IL-2R alpha blockade altered the affinity of the receptor mediating activated cell GM-CSF release from a high affinity to an intermediate affinity state. These studies indicate an independent role for IL-2R beta in mediating GM-CSF production from T cells. They also suggest that unstimulated and activated T cells, which express distinct IL-2 receptor moieties, mediate release of separate lymphokines and that different subunits of the IL-2 receptor may play an important role in the regulation of cytokine production.

Antibodies, Monoclonal↗