PubMed Health⌕ Search

Biomedical subjects

M W Lowdell

Publications and source records attributed to M W Lowdell.

At least 19 recordsLinked to original sources
PubMed2006

The sesquiterpene lactone parthenolide induces selective apoptosis of B-chronic lymphocytic leukemia cells in vitro.

We have studied the in vitro actions of the sesquiterpene lactone parthenolide (PTL) on cells isolated from patients with chronic lymphocytic leukemia (CLL). Dye reduction viability assays showed that the median LD(50) for PTL was 6.2 muM (n=78). Fifteen of these isolates were relatively resistant to the conventional agent chlorambucil but retained sensitivity to PTL. Brief exposures to PTL (1-3 h) were sufficient to induce caspase activation and commitment to cell death. Chronic lymphocytic leukemia cells were more sensitive towards PTL than were normal T lymphocytes or CD34(+) haematopoietic progenitor cells. The mechanism of cell killing was via PTL-induced generation of reactive oxygen species, resulting in turn in a proapoptotic Bax conformational change, release of mitochondrial cytochrome c and caspase activation. Parthenolide also decreased nuclear levels of the antiapoptotic transcription factor nuclear factor-kappa B and diminished phosphorylation of its negative regulator IkappaB. Killing of CLL cells by PTL was apparently independent of p53 induction. This is the first report showing the relative selectivity of PTL towards CLL cells. The data here warrant further investigation of this class of natural product as potential therapeutic agents for CLL.

Apoptosis↗
PubMed2003

In vitro co-stimulation with anti-CD28 synergizes with IL-12 in the generation of T cell immune responses to leukaemic cells; a strategy for ex-vivo generation of CTL for immunotherapy.

The existence of an immune based graft-versus-leukaemia (GvL) effect highlighted the prospect of managing relapsed leukaemias with T cell-based adoptive immunotherapy. Thus, various strategies have been explored for the in vitro expansion of acute myeloid leukaemia (AML)-specific T cells. In a popular approach, AML blasts have been genetically modified to express co-stimulatory molecules essential for effective T cell priming. One such tactic has been the modification of AML cells to express the B7/CD80 co-stimulatory molecule that binds to CD28 on T cells initiating events that culminate in enhanced cytokine production, proliferation and development of effector functions by T cells. The success of these strategies has been limited by difficulties in attaining sufficient transduction efficiencies and associated high levels of CD80 expression. We demonstrate that these problems can be circumvented by using anti-CD28 monoclonal antibody. Furthermore, we show that the synergistic relationship between CD80/CD28 pathway and interleukin 12 cytokine (IL-12), documented in the generation of cytotoxic T lymphocytes (CTL) for solid tumours, also applies to AML. CD28/IL-12 synergy facilitated the proliferation of allogeneic T cells in response to stimulation with primary AML blasts. The synergy also favoured generation of a Th1-type immune response, evidenced by gamma interferon (IFN-gamma) secretion and facilitated naive and memory T cell proliferation. Unlike some methods of in vitro T cell expansion, use of CD28/IL-12 synergy left T cells in the physiologically appropriate CD45RA-/CCR7- subsets known to be associated with immediate cytotoxic functions.

Acute Disease↗
PubMed2003

New advances in acute graft-versus-host disease prophylaxis.

Immunocompetent donor T cells in Allogeneic Haematopoietic Stem Cell grafts mediate acute Graft versus Host Disease (GvHD), still a major cause of recipient morbidity and mortality post transplant. Despite the advent of high resolution HLA-typing and matching at HLA loci, acute GvHD remains a significant problem, even in HLA matched siblings, due primarily to minor histocompatability antigen mismatches. Treatment of GvHD remains ineffective and highly immunosuppressive and the challenge to find effective methods of prevention continues. Non selective removal of donor T cells from the graft has been proven to be effective in preventing GvHD but the beneficial effects of donor T cells, namely effective immune reconstitution and anti tumour activity, are lost. This review considers mechanisms by which acute GvHD may be prevented in the context of the current model of GvHD immunopathogenesis, with a special emphasis on the recent techniques of selective removal or destruction of donor allogeneic T cells that have been described.

Acute Disease↗
PubMed2003

Natural killer cells in haematopoietic stem cell transplantation.

Natural killer cells represent the predominant lymphoid cell in the peripheral blood for many months after allogeneic or autologous stem cell transplant and their role in immunity to pathogens during this period is established. However, following the largely unsuccessful trials of NK and IL-2 activated NK cells for the treatment of haematological malignancies in the 1980's and 90's, their role in tumour immunology was discredited. Over the past ten years we have come to understand some of the complex regulatory pathways involved in NK cell activation and we are now in a position to capitalise upon this knowledge. This review presents our current state of understanding of NK cell regulation and highlights the role of these cells in engraftment, graft-versus-host disease, anti-leukaemia activity and post-transplant infection.

Animals↗
PubMed2003

Quality issues in stem cell and immunotherapy laboratories.

The advent of the Code of Practice for Tissue Banks has led to the requirement for quality systems to be established in all laboratories involved in the production or processing of all cellular tissues to be used therapeutically. The quality system is all-encompassing from process validations and quality assurance to the standard of facilities and staff training. This seems self-evident to those working within the transfusion field but is a relatively novel concept to many hospital laboratories preparing transplant products such as bone marrow or peripheral blood derived haematopoietic stem cells. This review places the current guidelines in an historical context and explains many of the central tenets and requirements of the Code of Practice while outlining a process to facilitate preparation for accreditation.

Accreditation↗
PubMed2002

Functional impairment of human T-lymphocytes following PHA-induced expansion and retroviral transduction: implications for gene therapy.

The immune function of retrovirus-mediated gene modified (GM) T cells is critical for a beneficial effect to follow their adoptive transfer into patients. Recent clinical data show that GM T cells expanded with PHA have reduced function in vivo. However, little functional analysis of PHA stimulation is available. Our results show that expansion of T cells with PHA impairs their ability to respond (proliferation, cytotoxicity and IFN gamma and perforin expression) to allogeneic stimulation or viral antigens in vitro. Conversely, CD3/CD28-based protocols can preserve this immune function. Retroviral transduction did not alter the functional profile induced by polyclonal stimulation. We investigated the mechanisms leading to this functional effect, and identified differential effects of PHA and CD3/CD28 on the distribution of CCR7/CD45RA T cell functional subsets, which may explain the functional differences observed. While CD3/CD28 stimulation parallels the lineage differentiation pattern induced by antigens in physiological conditions, PHA induces a skewed distribution of the CCR7/CD45RA functional T cell subsets, with near disappearance of the subpopulations that display the effector phenotype. Overall, this study demonstrates a functional disadvantage for transduction protocols based on PHA, uncovers mechanisms that may explain this functional effect, and provides us with information to design and select transduction protocols with an improved functional outcome.

CD28 Antigens↗
PubMed2001

Caspase 8 activation independent of Fas (CD95/APO-1) signaling may mediate killing of B-chronic lymphocytic leukemia cells by cytotoxic drugs or gamma radiation.

Ligation of the cell-surface Fas molecule by its ligand (Fas-L) or agonistic anti-Fas monoclonal antibodies results in the cleavage and activation of the cysteine protease procaspase 8 followed by the activation of procaspase 3 and by apoptosis. In some leukemia cell lines, cytotoxic drugs induce expression of Fas-L, which may contribute to cell killing through the ligation of Fas. The involvement of Fas, Fas-L, and caspase 8 was studied in the killing of B-cell chronic lymphocytic leukemia (B-CLL) cells by chlorambucil, fludarabine, or gamma radiation. Spontaneous apoptosis was observed at 24-hour incubation, with additional apoptosis induced by each of the cytotoxic treatments. Although Fas mRNA expression was elevated after exposure to chlorambucil, fludarabine, or gamma radiation, Fas protein levels only increased after irradiation. Therefore, Fas expression may be regulated by multiple mechanisms that allow the translation of Fas mRNA only in response to restricted cytotoxic stimuli. None of the cytotoxic stimuli studied here induced Fas-L expression. An agonistic anti-Fas monoclonal antibody (CH-11) did not significantly augment apoptosis induction by any of the death stimuli. A Fas-blocking antibody (ZB4) did not inhibit spontaneous, chlorambucil-, fludarabine-, or radiation-induced apoptosis. However, procaspase 8 processing was induced by all cytotoxic stimuli. These data suggest that the Fas/Fas-L signaling system does not play a major role in the induction of apoptosis in B-CLL cells treated with cytotoxic drugs or radiation. However, Fas-independent activation of caspase 8 may play a crucial role in the regulation of apoptosis in these cells.

Antibodies, Monoclonal↗
PubMed2001

Use of Leu3a/3b for the accurate determination of CD4 subsets for measurement of intracellular cytokines.

BACKGROUND: Identification of human T-helper cell subsets is possible by measurement of intracellular cytokines after coincubation of lymphocytes with phorbol myristate acetate (PMA), calcium ionophore, and brefeldin A for up to 20 h. However, exposure to PMA leads to internalization of membrane CD4 and to loss of resolution of the CD4+ cells. Detection of CD3+CD8- cells or preselection of CD4+ cells prior to stimulation is more cumbersome than direct measurement of CD4+ cells. We report the use of the Leu3a/Leu3b multiclone for the accurate determination of CD4 cells after PMA stimulation. METHODS: Peripheral blood lymphocytes were isolated from healthy normal donors and the proportion of CD3+ / CD4+ T cells was determined by flow cytometry before and after incubation with PMA, calcium ionophore, and brefeldin A for 20 h using a variety of anti-CD4 monoclonal antibodies. RESULTS: The Leu3a/3b multiclone reagent was the only anti-CD4 monoclonal antibody capable of resolving more than 98% of the initial CD4+ events after incubation with PMA. CONCLUSIONS: The higher signal-to-noise ratio associated with Leu3a3b reagent, compared with other CD4-specific antibodies available, allows the direct and accurate identification of the CD4 subset even after PMA treatment of cells.

CD4 Antigens↗
PubMed2001

Identification of both myeloid CD11c+ and lymphoid CD11c- dendritic cell subsets in cord blood.

Dendritic cells (DCs) are the most potent antigen-presenting cells described to date. In human peripheral blood, both myeloid and lymphoid subsets of DCs have been identified. In contrast, cord blood (CB) DCs have recently been described as being exclusively of the immature CD11c- lymphoid DC subset. Using an alternative method of enrichment, based on a negative selection system, both lymphoid (HLA-DR+ CD123+++ CD11c- CD33-) and myeloid (HLA-DR++ CD123+ CD11c+ CD33+) DCs were identified in CB. Although the majority of CB DCs showed a lymphoid phenotype, a significant number of CD11c+ myeloid DCs (25.6% +/- 14.5%, n = 13) were also present. Other markers, such as CD80 and CD83, were negative in both subsets. Analyses of the allostimulatory capacity of both subsets showed that freshly isolated CB lymphoid DCs failed to induce a potent allostimulation of naive CB T cells. These features are therefore consistent with previous work reporting an immature phenotype for lymphoid DCs in adult blood. The significance of the inverted CD11c+/CD11c- ratio observed in CB DCs (1:3) with respect to adult blood DCs (3:1) remains to be explained.

Antigens, CD↗
PubMed2001

Acute myeloid leukaemia cells secrete a soluble factor that inhibits T and NK cell proliferation but not cytolytic function--implications for the adoptive immunotherapy of leukaemia.

Evidence of an immune mediated graft-versus-leukaemia effect has led to the belief that T and NK cell based adoptive immunotherapy can constitute effective treatment for relapsed leukaemias. However, work on solid tumours has shown this strategy may be hampered, by an immune escape mechanism in which tumour secreted immunosuppressive factors compromise T and NK cell function. Indeed, acute myeloid leukaemia (AML) cells secrete immunosuppressive factors that block the synthesis of Th1 type cytokines in T cells. We demonstrate here that this immunosuppression, mediated by both HL60 AML cell line and primary AML blasts, inhibits T and NK cell proliferation but not cytolytic activity. Supernatants from HL60 cell line and primary AML blasts inhibited T cell proliferation to mitogenic and alloantigen stimulation but had no effect on cytolytic function. Similarly, the proliferation of NK cells to IL-2 and IL-15 stimulation was inhibited whilst their cytolytic function, shown by lysis of AML blasts, K562 and Daudi cells remained unaffected. The failure of T and NK cells to proliferate was not due to effector cell apoptosis. Indeed, removal of lymphocytes from the immunosuppressive environment partially restored their capacity to respond to mitogenic stimulation. T cells exposed to immunosuppressive supernatants did not increase expression of mitotic inhibitory proteins that arrest cell division, thereby ruling this out as a mechanism of operation for this immunosuppression. T cell expansion requires antigen stimulation, usually provided in the form of AML blasts, therefore our data suggest that NK cells may be more practical for the immunotherapy of AML.

Cell Division↗
PubMed2000

Immunotherapy of AML: future directions.

Immunotherapy in the form of allogeneic GvL has been curing AML patients for nearly 30 years but our understanding of the mechanisms has been poor. Our rapidly evolving understanding of the human immune system and the concomitant technical developments in ex vivo cell manipulation, the vision of funding bodies, the dedication of clinical and research staff, and above all the commitment of our patients, promise substantial progress in the treatment of this disease in the year 2000 and beyond.

Acute Disease↗
PubMed1999

Selective removal of alloreactive cells from haematopoietic stem cell grafts: graft engineering for GVHD prophylaxis.

One of the main goals in allogeneic bone marrow transplantation is the abrogation of graft-versus-host disease with the preservation of antileukaemia and antiviral activity. We have established a novel system for the selective removal of alloreactive lymphocytes from donor grafts while retaining an effective allogeneic response to third-party stimulator cells. Initial feasibility studies were done with unrelated HLA-mismatched pairs and then extended into the matched setting. Mononuclear cells from HLA-matched donors were cocultured with irradiated recipient cells prestimulated with cytokines (gamma-IFN and TNF-alpha) in a modified mixed lymphocyte culture (MLC). Alloreactive donor lymphocytes were identified by expression of CD69, an early activation marker and selectively removed by paramagnetic bead sorting. The remaining 'non-alloreactive' lymphocytes were tested in proliferative assays against the original matched recipient and to a third-party donor. A mean depletion of proliferative capacity to 11.5 +/- 9.9% of the original matched recipient response was achieved while the residual third-party response was largely preserved at 77.8 +/- 20.9% which should translate into improved immune reconstitution and preservation of antiviral activity. The non-alloreactive lymphocytes could also possess functional antileukaemia activity. Moreover, the alloreactive cells are easily recoverable in this selective T cell depletion strategy for cryopreservation and ready for immediate access as therapeutic donor lymphocyte infusions in cases of frank relapse post transplant.

Antigens, CD↗
PubMed1999

Generation of autologous immunity to acute myeloid leukaemia and maintenance of complete remission following interferon-alpha treatment.

Interferon-alpha (IFN-alpha) is established as part of the treatment for chronic myeloid leukaemia, although its precise mode of action remains largely unknown. Its use in acute myeloid leukaemia (AML) has been limited. We have previously documented autologous cytolytic activity against AML blasts in patients after autologous bone marrow transplantation. Here we present a patient with poor-risk AML who relapsed from first complete remission (CR) and was unwilling to undergo high-dose chemotherapy with stem cell rescue. In second chemotherapy-induced CR, the patient had no evidence of antileukaemia cytolytic activity in an in vitro assay, and she commenced IFN-alpha (Roferon). She subsequently developed high levels of leukaemia-specific cytotoxicity, and has remained in second CR for two years. These findings support the use of IFN-alpha in patients with poor-risk AML, and suggest that one mechanism of action may be immunological.

Adult↗
PubMed1998

The effect of T cell depletion with Campath-1M on immune reconstitution after chemotherapy and allogeneic bone marrow transplant as treatment for leukaemia.

The prophylactic use of T cell depletion (TCD) strategies for the prevention of graft-versus-host disease (GVHD) following allogeneic stem cell transplantation remains widespread. Initial reports of high incidence of graft rejection after TCD BMT led to a move away from this approach but improved conditioning regimens have reduced this risk substantially. The use of TCD has also been associated with higher relapse risk post-BMT although the success of donor leukocyte infusion (DLI) as treatment for relapse has reduced this problem, especially in chronic myeloid leukaemia (CML). Currently the use of TCD BMT is increasing particularly due to the relative increase in BMT from non-related donors for whom TCD is the optimal GVHD prophylaxis. However, doubts remain over the long-term effect on the reconstituted immune system of recipients of TCD BMT, particularly in adult recipients. In this study we have undertaken a detailed sequential analysis in 23 patients who received allo-grafts from HLA-identical sibling donors after high-dose chemo/radiotherapy for acute or chronic leukaemia. Of these patients, 11 received non-manipulated grafts, five received 'partially TCD' (PTCD) and a further seven received 'fully TCD' (FTCD) bone marrow. T cell depletion was performed ex vivo by Campath-1M plus autologous serum as a source of complement. Partial TCD describes grafts with a T cell reduction of 1-2 log. Full TCD refers to grafts with a reduction of >2.5 log. The decision regarding the optimal degree of TCD was clinical and was based upon the perceived relative risk of relapse based upon the disease and remission status. All patients were monitored for up to 12 months post-BMT with regard to reconstitution of T and NK cell subsets. T cell depletion at either level was associated with a slower recovery of CD4 cells. This was most marked in the FTCD recipients and lasted throughout the period of study. CD8 cell recovery was also slower in the TCD recipients but this normalised throughout the 12 months post-BMT. The ratio of CD45RA+:CD45RO+ increased in all recipients after month 3. This suggests that a degree of extra-thymic T cell maturation can occur in recipients of allogeneic BMT. NK cell recovery was more rapid in the TCD recipients and these differences were maintained throughout the first year.

Adult↗
PubMed1997

Temporal dynamics of CD69 expression on lymphoid cells.

Lymphocyte activation remains an area of intense interest to immunologists and cell biologists and the dynamics of expression of surface molecules during the process are widely studied. The CD69 C-type lectin is reportedly the earliest activation antigen on lymphocytes and can be detected within hours of mitogenic stimulation. Recently reports have described differential activation dynamics with respect to different antigenic or mitogenic stimuli. This study has investigated the dynamics of CD69 expression over time after mitogenic, allogeneic, cytokine and target cell mediated activation of T-cell and NK cell subsets. It is apparent that the dynamics of CD69 expression differ with respect to the cell type and the method of stimulation. Mitogenic stimulation resulted in the most rapid expression of CD69 on both T- and NK cells while alloantigen stimulation induced a far slower response. Target cell stimulation of NK cells gave paradoxical results in that the CD69 + ve subset increased as a proportion of the total NK cells but did not increase in number. This was due to the selective binding of CD69 - ve NK cells to the target cells and their subsequent loss from the lymphoid gate. We confirmed this by showing that CD69 + ve NK cells do not lyse K562 target cells. This observation demonstrates the caution needed in the analysis of flow cytometric data based solely upon relative proportions of cells within discrete subsets.

Antigens, CD↗
PubMed1997

Can repeated plasma donation by asymptomatic HIV-infected individuals delay the onset of AIDS?

Healthy HIV-positive regular donors of plasma in a programme of passive immunotherapy for AIDS patients were studied over a period of about two years. None developed symptoms of clinical progression; most seemed to make substantial gains of CD4 cells by comparison with asymptomatic individuals who were not donating. The effects of donation did not seem to diminish with repetition, and donor CD4 counts tended towards stabilizing within normal limits. Asymptomatic HIV-positive individuals were compared immunologically with 'normals' and people with AIDS, using a battery of 25 measurements on peripheral blood. The immunological profiles of donor and non-donor asymptomatics, indistinguishable at the start, became dissimilar: donors' profiles resembled AIDS less, non-donors became less like 'normal' and a few non-donor results could not be distinguished from AIDS. Improvement in the CD4 counts and amelioration of the immunological profile in donors provide prima facie evidence that plasmapheresis may be therapeutic for asymptomatic HIV-positive people. Further studies are justified.

Acquired Immunodeficiency Syndrome↗