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M W Lowdell

Publications and source records attributed to M W Lowdell.

32 records · Page 2Linked to original sources
PubMed1997

Construction and biological characterization of an interleukin-12 fusion protein (Flexi-12): delivery to acute myeloid leukemic blasts using adeno-associated virus.

Interleukin-12 (IL-12) is a cytokine that exhibits pleiotropic effects on lymphocytes and natural killer cells and has been shown to have promise for the immunotherapy of cancer. The combination of the immune costimulatory molecule B7.1 and IL-12 has been shown to be synergistic for T cell activation. By transfecting tumor cells with both IL-12 and B7.1 cDNAs, it may be possible to use these modified targets as vaccines. A major obstacle in designing a vector to deliver these genes results from the structure of IL-12. Functional IL-12 is a heterodimer composed of two distinct subunits that are encoded by separate genes on different chromosomes. Production of functional IL-12 requires the coordinated expression of both genes. This presents several problems in vectors, particularly those in which additional genes, either a co-stimulatory gene or a selectable marker, are inserted. Therefore, we have constructed a single cDNA that encodes a single-chain protein, called Flexi-12, which retains all of the biological characteristics of recombinant IL-12 (rIL-12). The monomeric polypeptide Flexi-12 is able to induce the proliferation of phytohemagglutinin (PHA) blasts, induce PHA blasts to secrete interferon-gamma (IFN-gamma) and additionally, by preincubation, enhance the killing of K562 targets by PBLs. These phenomena are in a dose-dependent manner comparable to that seen with rIL-12. We have also shown that tyrosine phosphorylation of the STAT 4 transcription factor, which has been shown to be unique to the IL-12 signaling pathway, occurs with Flexi-12 at levels similar to those seen with rIL-12. We have packaged Flexi-12 into a recombinant adeno-associated virus (AAV) and used this vector to infect acute myeloid leukemic (AML) blasts. Infected AML blasts produced between 2 and 6 ng of IL-12/10(6) cells per ml per 48 hr. These studies also confirm that AAV is an efficient delivery vehicle for cytokines to leukemic cells. Direct analysis of these modified cells acting as tumor vaccines is underway.

Amino Acid Sequence↗
PubMed1997

Analysis of apoptosis in relation to tissue destruction associated with Hashimoto's autoimmune thyroiditis.

The level of apoptosis has been investigated in thyroid tissue from eight patients with Graves's disease, one with Hashitoxicosis, three with Hashimoto's thyroiditis, and five patients with multinodular goitre, using flow cytometry and an in situ immunofluorescence technique. Cryostat sections have also been studied for Bcl-2 and APO-1/Fas expression in the thyrocytes and infiltrating lymphocytes, to determine their susceptibility to apoptosis. An increased level of apoptosis was detected in Hashimoto's glands. This was associated with decreased Bcl-2 staining and a patchy APO-1/Fas reactivity on thyrocytes. In addition, APO-1/Fas expression was noted within the germinal centres of lymphoid follicles. It is suggested that the dysregulation of apoptosis-related genes could be an important factor in the progression of destructive thyroid autoimmune disease.

Apoptosis↗
PubMed1997

The in vitro detection of anti-leukaemia-specific cytotoxicity after autologous bone marrow transplantation for acute leukaemia.

Anti-leukaemia activity after allogeneic bone marrow transplantation has been studied extensively but its antigen specificity and effector cell phenotype remain unknown. Here we report a study in three recipients of autologous bone marrow transplantation done as part of the treatment for acute leukaemia, in whom we were able to detect innate specific anti-leukaemia activity post-transplant. One patient maintained selective cell-mediated cytolytic activity against her autologous leukaemic cells in the absence of cytolysis of her normal bone marrow mononuclear cells (BMMC). She remains in complete remission 3 years after ABMT for acute myeloid leukaemia (M5). A second patient was transplanted for acute lymphoblastic leukaemia and had detectable anti-leukaemia activity up to 20 weeks post-ABMT. At this point anti-leukaemia activity could no longer be demonstrated and the patient suffered a relapse 2 weeks later. A third patient was transplanted for AML (M4 Eo) and lacked detectable leukaemia-specific immune reactivity at 1, 3 and 6 months post-ABMT. She relapsed 6 months after her ABMT and returned to complete remission after further chemotherapy. She commenced treatment with alpha interferon and regained NK function. Furthermore, she developed high level cytolytic activity against her autologous leukaemic cells in the absence of activity against her remission bone marrow samples. She remains in complete remission 17 months after her initial relapse. This is the first report of an apparent association between in vitro leukaemia-specific cytolytic activity in individual patients after ABMT and clinical outcome. It encourages the theory that autologous immunomodulation may be useful in the future treatment of leukaemia.

Acute Disease↗
PubMed1997

Immunocytochemical detection of breast cancer cells: a comparison of three attachment factors.

The evaluation of contaminating breast cancer cells in hematopoietic grafts is of considerable importance for monitoring the efficiency of purging procedures. We report a comparison of three systems for the in vitro detection and enumeration of metastatic breast cancer cells. Breast cancer cells from established cell lines were mixed with Daudi cells at dilutions ranging from 1:10 to 1:1,000,000, and a predetermined number were fixed in defined areas on microscope slides coated with one of the following attachment factors: (i) Cell-Tak Cell and Tissue Adhesive, (ii) 0.1% solution of Poly-L-Lysine, or (iii) Cel-Line HTC Super Cured slides. We employed a specificity-proven pancytokeratin antibody (A45-B/B3) and the alkaline phosphatase-antialkaline phosphatase (APAAP) staining technique. In multiple experiments, one breast cancer cell in 1,000,000 Daudi cells could reliably be detected in the Cell-Tak and Cel-Line systems and 1 in 100,000, with the Poly-L-Lysine system. The observed number of seeded cells showed a highly significant correlation with the number of cells seeded (p < 0.0001 in all cases). Finally, we used the Cell-Tak method to evaluate clinical material from various sources: from patients with primary carcinomas of the breast, prechemotherapy, and during various chemotherapeutic regimens, as well as from patients with metastatic disease. The system consistently detected tumor cells in bone marrow samples from these patients. All peripheral blood samples from patients with metastatic disease tested positive at incidences ranging from 5 to 19/10(6) peripheral blood mononuclear cells. This is a simple and reliable technique that allows rapid screening of large cell numbers with high resolution of positive cells.

Alkaline Phosphatase↗
PubMed1997

Detection of adeno-associated virus type 2 in sorted human bone marrow progenitor cells.

Wild-type adeno-associated virus (wtAAV) is a helper-dependent human parvovirus which has the ability to integrate into the genome of a wide variety of human cells, including those of the hemopoietic lineages. Recombinant adeno-associated virus (rAAV) is becoming a good candidate for virally mediated gene therapy. rAAV is likely to be a safe vector in clinical gene transfer, as it has never been associated with any disease despite previous studies showing that up to 70% of adults are seropositive for wtAAV. Seroconversion appears to occur early in life. wtAAV is an upper respiratory tract virus that is gut secreted, but little is known about the integration of latent wtAAV in hemopoietic lineages. Unlike retroviruses, which have been the most common vehicles for gene transfer to date, wtAAV appears to have a preferred integration site in the target cell which has been termed AAVS1. Several studies have shown that wtAAV can only integrate into only one of the pair of chromosome 19 in a cell. This may have implications for the use of rAAV in gene transfer because patients with latent virus would be refractory to further infection with rAAV. We used a polymerase chain reaction (PCR) assay to detect the presence of wtAAV in the bone marrow samples from 106 patients who presented at our institution. We were able to detect the presence of integrated virus in 18 whole marrow samples. Subsequently CD34+ and CD3+ cell subsets were sorted from the cryopreserved marrow of three PCR-positive individuals to assess integration of virus in these cell lineages. In all three samples tested, we were unable to detect wtAAV virus in the CD34+ hematological precursor cells, but a detectable level of integrated viral DNA was demonstrated in the CD3+ cell fraction. Our findings therefore suggest that CD34+ cells might remain a good target for rAAV-mediated gene transfer despite previous wtAAV infection.

Adolescent↗
PubMed1996

External quality assurance for CD34 cell enumeration--results of a preliminary national trial. Royal Microscopical Society Clinical Flow Cytometry Group QA Schemes.

With the development of haematopoietic stem cell (HSC) mobilisation strategies and the associated technical expertise in leukapheresis has come the need for accurate and reproducible enumeration of HSC in the peripheral blood. Enumeration of HSC is not only required for timing of the harvest but is valuable in determining that the minimum number of HSC required for successful engraftment has been collected. In order to establish a minimum number of HSC required, results from multiple institutions performing such transplants need to be assessed. Clearly, to compare results from multiple centres requires confidence in the reproducibility of the assay. We have evaluated an established EQAS method which has already been proven in the external quality assurance of CD4 measurement in clinical samples to assess the inter- and intra-laboratory reproducibility of CD34 measurement. Fifteen laboratories participated in two distributions in which 28 samples were analysed. Standardised methods were not employed, laboratories using their routine methods. Participants reported their results in terms of '% positive of total leukocytes' and 'absolute number of CD34+/microliters'. A wide range of clinical samples was despatched and analysed with CD34 cell content ranging from 0.08-19.31% positive. The coefficients of variance (CV) associated with the estimations of relative proportions and absolute numbers were maximally 100.1 and 136.6%, respectively. This study highlights the need for external quality assurance and standardisation of the methodology of this assay.

Antigens, CD34↗
PubMed1995

VLA-6 (CDw49f) is an important adhesion molecule in NK cell-mediated cytotoxicity following autologous or allogeneic bone marrow transplantation.

Graft-vs.-leukemia (GVL) is postulated to be the principal mechanism responsible for continued remission after allogeneic bone marrow transplantation (BMT). The specific cytotoxic effectors mediating this effect are as yet undefined, but the major histocompatibility complex (MHC)-nonrestricted lysis of tumor cell lines by natural killer (NK) and lymphokine-activated killer (LAK) cells from recipients of allogeneic BMTs has been proposed as an in vitro correlate of GVL. In vitro culture or treatment in vivo with interleukin-2 (IL-2) is associated with enhanced NK cytotoxicity and lysis of NK-resistant targets (LAK cytotoxicity). NK, LAK, and cytotoxic T lymphocytes (CTL) have cytotoxic properties against autologous and allogeneic leukemic targets. These immune effector cells require receptor-ligand interaction for target recognition and adhesion via specific molecules such as integrins, a group of heterodimeric transmembrane glycoproteins. The integrins include the very late activation (VLA) subfamily, which all share the same beta 1 subunit but have distinct chains. VLA-6 (CDw49f) has been identified on NK cells and binds to laminin, a basement membrane protein found on malignant tumor cells but not normal cells. Monoclonal antibodies (mAbs) to laminin have been found to inhibit in vitro cytotoxicity of the tumor cell line K562, suggesting an important role for VLA-6 in this interaction. The specific aim of this study was to investigate the role of VLA-6 in the interactions of the tumor cell lines K562 and Daudi with peripheral blood lymphocytes (PBL) acting as effectors in cell-mediated cytotoxicity from normal volunteers, patients recovering from chemotherapy, and patients recovering from autologous or allogeneic BMT. In over 96% of assays, incubation of effector cells with anti-CDw49f mAbs led to detectable inhibition of NK and LAK cell-mediated cytotoxicity. More notably, the degree of anti-VLA6-induced suppression of LAK activity was significantly greater in the normal donors than in any of the patient groups, despite a significantly lower incidence of expression of VLA-6 on NK cells from controls than from patients. This implies a reduced role for this adhesion molecule in LAK activity following some form of in vivo stimulation. This hypothesis is supported by the observation that addition of exogenous IL-2 to the cultures ameliorated the effect of VLA-6 blockade, although the incidence and level of VLA-6 expression was unchanged by IL-2. In contrast, VLA-6 blocking led to a greater reduction in NK activity of BMT recipients than of normal donors, demonstrating that the VLA-6 adhesion pathway is important in this group of patients. These results indicate that the VLA-6-laminin interaction is important in normal NK-target interaction but may play a less significant role in the innate cytotoxic response post-BMT, perhaps reflecting subtle differences in the subsets of NK cells present in BMT recipients compared with normal donors.

Antibodies, Monoclonal↗
PubMed1992

Early T-cell defects in pre-type 1 diabetes.

Alterations of lymphocyte subsets have been recently reported in pre-type 1 diabetes but the relation with other immunological markers, in particular islet cell antibodies (ICA), is still unknown. In the present study, we have investigated prospectively changes of lymphocyte subsets in 86 first-degree relatives of patients affected by type 1 diabetes and correlated such modifications with ICA titres. Among individuals with persistent ICA, 8 had ICA titres of more than 20 JDF units, 14 had ICA titres between 5 and 20 JDF units and 64 had ICA titres between 0 and 5 JDF units. First-degree relatives with ICA titres of more than 20 JDF units had significantly decreased proportions of CD3 cells. This reduction was predominantly in the CD4 subset, giving rise to a decreased CD4/CD8 lymphocyte ratio. Those with ICA titres between 5 and 20 JDF units showed abnormalities in both CD3 and CD4 lymphocytes, but not in CD4/CD8 lymphocyte ratio. Further characterization of the CD4 cell subset was performed using three other monoclonal antibodies, CD45RO (UCHL1), CD45RA and CD29, phenotyping memory T-cells, the inducer cells of suppressor function and helper-inducer cells, respectively. The proportions of total CD45RO and CD45RA were not significantly different among first-degree relative with distinct ICA titres in a cross-sectional study, whereas a trend towards a reduced proportion of CD4/CD45RA cells was observed. The longitudinal study demonstrated that individuals potentially susceptible to the development of type 1 diabetes and who possess high titres of ICA have impairment of CD4/CD8 lymphocyte ratio, mainly due to a reduction in the CD4 subset.(ABSTRACT TRUNCATED AT 250 WORDS)

Antibodies, Monoclonal↗
PubMed1992

Decline in CTL and antibody responses to HIV-1 p17 and p24 antigens in HIV-1-infected hemophiliacs irrespective of disease progression. A 5-year follow-up study.

CTL and antibody responses to HIV-1 p17 and p24 antigens were monitored from 1986-1991, in 4 hemophiliacs. The patients had been infected with HIV-1 between 1980 and 1984. Two patients have remained asymptomatic while two progressed to AIDS in 1990. CTL were boosted by culturing with peptides from p17 aa 86-115, or p24 aa 265-279; and aa 270-373 or PHA. Lysis was measured on autologous or allogeneic targets pulsed with peptides or infected with recombinant vaccinia virus carrying HIV-1 gag or influenza A matrix genes. Antibodies to p17 and p24 were tested by ELISA using peptides and by Western blotting. High levels of CTL activity to p17 and p24 antigens could be generated only with lymphocytes from the two asymptomatic patients between 1986 and 1989, but these responses were absent in 1990 and 1991. Antibodies to p17 peptides disappeared in parallel with CTL activity. Antibodies to some p24 peptides also declined but most patients retained activity to others. In all patients a > or = 3-fold increase in CD8+ cell numbers occurred over time and accompanied the decline of CTL and antibody responses. The loss of CTL and p17 antibodies occurred irrespective of whether patients remained asymptomatic or progressed to AIDS in the intervening two years.

Cell Line, Transformed↗
PubMed1992

Absence of a specific effect of free radicals on HLA-B27.

The spondylitis associated HLA-B27 epitope includes a characteristic unpaired cysteine at amino acid position 67. On some B27 molecules the thiol (-SH) side chain of this residue seems to be available for chemical interactions. The possibility that free radicals produced during inflammation might specifically affect this group was investigated in this work. Cells bearing HLA-B27 were exposed to free radicals generated by ultraviolet irradiation or hydrogen peroxide, and HLA antigens were then measured by flow cytometry. Binding of monoclonal antibodies to B27 was not affected. These results do not support a specific susceptibility of HLA-B27 to damage by free radicals, despite its apparently vulnerable structure.

4-Chloromercuribenzenesulfonate↗
PubMed1991

Cellular mechanisms of graft-versus-host disease in a mouse model.

Female CBA/H (H-2k, Mlsb) mice alloimmunized prior to and during syngeneic pregnancy with DBA/2 (H-2d, Mlsa) splenocytes gave rise to offspring which resisted graft-versus-host disease (GVHD) following neonatal intraperitoneal inoculation of high doses of DBA/2 spleen cells. Lymphocytes from GVHD-resistant mice tested after 6 weeks of age were unresponsive to DBA/2 stimulator cells in 72 h mixed lymphocyte cultures. Isotope uptake measured 24 h after culture, however, indicated that a considerable early response was made to DBA/2 which later declined. Proliferative responses to BALB/c were also depressed but no early response to this strain was detected. FACS analysis of T-lymphocyte profiles of the GVHD-resistant CBA/H mice revealed a 100% increase in the Lyt-2+ subpopulation compared to normal CBA/H mice. Significant increases in Lyt-2+ cells were also noted in in vitro cultures of CBA/H lymphocytes responding to GVHD-resistant CBA/H stimulators. Lymphocytes from GVHD-resistant mice suppressed the proliferative responses of normal CBA/H lymphocytes to alloantigenic but not mitogenic stimulation. Suppression of alloantigenic responses were shown to be specific to DBA/2 and did not affect the response to BALB/c stimulator cells, indicating that both anergy and specific suppressor cells were operative in inducing unresponsiveness.

Animals↗
PubMed1990

IgG subclass levels and immune reconstitution after T cell-depleted allogeneic bone marrow transplantation.

Defects of humoral immunity are well documented after bone marrow transplantation (BMT). Immunoglobulin recovery can be impaired and selective deficiencies of IgG subclasses have been reported. The nature of these deficiencies may reflect patterns of infection in the post-BMT period. We studied immunoglobulin and IgG subclass recovery in 20 long term (greater than 100 days) survivors of T depleted allogeneic BMT. Although there was no fall in mean levels of IgG, IgM or IgA for the patient group, 14 patients (70%) developed a deficiency of one or more immunoglobulin isotype at some stage post-BMT. Eight patients (40%) had deficiency of IgG, IgA and IgM and six had selective deficiencies. When IgG subclasses were measured it was seen that mean levels of IgG2 and IgG4 fell post-BMT with trough levels occurring at around 120 days post-transplant. Sixty per cent of patients developed IgG2 subclass deficiency and of these patients 78% had an associated IgG4 deficiency. Deficiencies of IgG1 and IgG3 were less common and less prolonged than those of IgG2 and IgG4; in addition, mean levels of IgG1 and IgG3 showed a rise early post-BMT. In conclusion, a majority of our patients developed immunoparesis following BMT, usually at around 120 days after transplantation. IgG2 subclass deficiency, often in association with IgG4 deficiency, is common and may occur despite normal total IgG levels. Deficiencies of immunoglobulin and IgG subclasses may persist for longer than 1 year post-BMT. Differing profiles of immunoglobulin and IgG subclass recovery may help dictate patterns of infection in long-term survivors of BMT.

Adult↗