Efficient gene delivery to the adult and fetal CNS using pseudotyped non-integrating lentiviral vectors.

Non-integrating lentiviral vectors show considerable promise for gene therapy applications as they persist as long-term episomes in non-dividing cells and diminish risks of insertional mutagenesis. In this study, non-integrating lentiviral vectors were evaluated for their use in the adult and fetal central nervous system of rodents. Vectors differentially pseudotyped with vesicular stomatitis virus, rabies and baculoviral envelope proteins allowed targeting of varied cell populations. Efficient gene delivery to discrete areas of the brain and spinal cord was observed following stereotactic administration. Furthermore, after direct in utero administration (E14), sustained and strong expression was observed 4 months into adulthood. Quantification of transduction and viral copy number was comparable when using non-integrating lentivirus and conventional integrating vector. These data support the use of non-integrating lentiviral vectors as an effective alternative to their integrating counterparts in gene therapy applications, and highlight their potential for treatment of inherited and acquired neurological disorders.

Abnormal cytogenetic clones in patients with aplastic anaemia: response to immunosuppressive therapy.

We report the response to immunosuppressive therapy with antithymocyte globulin (ATG) and cyclosporin or oxymetholone in 13 cases of aplastic anaemia (AA) with an abnormal cytogenetic clone detected at or sometime after diagnosis. Blood and bone marrow examination showed no distinctive morphological features of myelodysplasia (MDS) at diagnosis. Haematological response occurred promptly in eight cases; the remainder responded after additional immunosuppression with or without oxymetholone. Three patients had a late relapse of AA, treated successfully by allogeneic bone marrow transplantation in one; the others responded to oxymetholone. Transformation to MDS or acute leukaemia was not observed after a median follow-up of 4.1 years (range 1.2-11.2). In four patients the cytogenetic clone disappeared after treatment.

G-CSF-mobilized CD34 peripheral blood stem cells are significantly less apoptotic than unstimulated peripheral blood CD34 cells: role of G-CSF as survival factor.

The mechanism of release of CD34+ cells into the peripheral blood (PB) after mobilization treatment with chemotherapy and/or growth factors is not clearly understood. Growth factors may induce increased proliferation and self renewal within the stem cell compartment. It is possible that they alter adhesion molecule profiles or other progenitor:stroma interactions, to allow release of these cells into the periphery. However, CD34+ cells are present in the PB under steady-state conditions, albeit in low number. Growth factors such as granulocyte colony-stimulating factor (G-CSF) may promote the survival of CD34+ cells in the PB by suppressing apoptosis. In order to test this hypothesis, we have quantitated apoptotic cells in the CD34+ fraction of peripheral blood stem cell (PBSC) collections, using two-colour flow cytometry, after staining with anti-CD34 antibody and the fluorescent DNA binding agent, 7-amino actinomycin D (7AAD). 7AAD differentially stains live, apoptotic and dead cells, due to the altered accessibility of DNA in each subpopulation. We have shown a significant reduction in the proportion of apoptotic cells in the CD34+ population mobilized by G-CSF compared to CD34+ cells in unstimulated PB, consistent with the theory that G-CSF is acting, at least in part, by suppressing apoptosis. In addition, we found that G-CSF mobilized CD34+ cells are less apoptotic than CD34+ cells of unstimulated normal bone marrow, indicating that, at the doses used, G-CSF is significantly altering the survival capacity of the mobilized cells.

The sensitivity of Fanconi anaemia group C cells to apoptosis induced by mitomycin C is due to oxygen radical generation, not DNA crosslinking.

Fanconi's anaemia (FA) is characterized by increased spontaneous and induced chromosome fragility. This has been widely regarded to be due to a defect in DNA crosslink repair, because of the sensitivity of cells to known DNA crosslinking agents such as mitomycin C (MMC) and diepoxybutane (DEB). Although Fanconi cells are also sensitive to molecular oxygen, and may be protected by antioxidants, this has generally been considered to be a secondary phenomenon. However, it has recently been demonstrated that the FAC protein, coded for by the Fanconi anaemia gene for complementation group C, is strictly cytoplasmic and does not enter the nucleus even after DNA damage, which seems inconsistent with a role in DNA repair. We have studied the effects of MMC and oxygen on apoptotic cell death in FA group C (FA-C) and normal lymphoblastoid cell lines. Hyperoxia alone failed to induce apoptosis in either FA-C or normal cells. At ambient oxygen, MMC is known to generate oxygen free radicals, whereas decreased oxygen tension facilitates the metabolic activation of MMC for DNA crosslinking. We therefore studied the effects of MMC at 20% and 5% oxygen to favour oxygen radical generation or DNA crosslinking respectively. FA-C cells showed increased sensitivity compared to normal cells for the induction of apoptosis by MMC at 20% oxygen. When cells were treated with MMC at 5% oxygen we found no increased sensitivity of Fanconi cells to MMC when compared to normal cells. These results imply a role for oxygen free radicals, but not for DNA crosslinking, in the sensitivity of FA cells to MMC.

Platinum agents and secondary myeloid leukaemia: two cases treated only with platinum-based drugs.

With the increasing use of chemotherapy for many different primary malignancies, secondary or therapy-related acute myeloid leukaemias (AML) and myelodysplastic syndromes (MDS) are becoming more common. The risk of developing sAML has been estimated to be between 2% and 10%, depending upon the type, duration and dosage of previous therapy (Michels et al, 1985; Shulman, 1993; Robinson & Mertens, 1993; Ballen & Antin, 1993). It is therefore one of the most serious long-term complications of current cancer treatment and is likely to increase as longer survival rates for the primary tumour are achieved. An increasing range of drugs have been reported to cause sAML, including the alkylating agents, the epipodophyllotoxins and the anthracyclines, both as single agents and in combination (Pedersen-Bjergaard & Philip, 1991; Pedersen-Bjergaard & Rowley, 1994). We report two cases of secondary AML in which platinum compounds were the sole prior chemotherapy.

The use of 7-amino actinomycin D in identifying apoptosis: simplicity of use and broad spectrum of application compared with other techniques.

The detection and quantitation of apoptotic cells is becoming increasingly important in the investigation of the role of apoptosis in cellular proliferation and differentiation. The pathogenesis of hematologic disorders such as aplastic anemia and the development of neoplasia are believed to involve dysregulation of apoptosis. To quantitate accurately the proportion of apoptosis cells within different cell types of a heterogeneous cell population such as blood or bone marrow, a method is required that combines the analysis of large numbers of cells with concurrent immunophenotyping of cell surface antigens. In this study, we have evaluated such a method using the fluorescent DNA binding agent, 7-amino actinomycin D (7AAD), to stain three diverse human cell lines, induced to undergo apoptosis by three different stimuli. Flow cytometric analysis defines three populations on the basis of 7AAD fluorescence and forward light scatter. We have shown by cell sorting and subsequent morphological assessment and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling that the populations defined by 7AAD represent live, apoptotic, and late-apoptotic/dead cells. This method is quick, simple, reproducible, and cheap and will be a valuable tool in the investigation of the role of apoptosis in normal physiology and in disease states.

Increased apoptosis in aplastic anemia bone marrow progenitor cells: possible pathophysiologic significance.

We have quantitated apoptotic cells by flow cytometry in human bone marrow (BM) and peripheral blood (PB) from normal donors and aplastic anemia (AA) patients, using the fluorescent DNA-binding dye 7-amino actinomycin D (7AAD). No significant difference was found in baseline percent apoptosis between normal and AA samples. Serum deprivation induced cell death to a greater degree in AA samples than in normal samples, but this was not significant. Using dual staining with anti CD34 antibody and 7AAD, we have shown that CD34+ progenitors in normal PB are significantly more apoptotic than those in normal BM. AA BM CD34+ cells contain a significantly greater proportion of apoptotic cells than normal BM CD34+ cells. Those AA patients with the lowest absolute number of CD34+ cells showed the highest proportion of apoptotic CD34+ cells. This appears to be related to clinical severity (transfusion dependence) at the time of study. We conclude that apoptosis is accelerated in AA BM progenitors and that this may contribute to the stem cell deficiency characteristic of this disorder.

Successful bone marrow transplant for Fanconi anaemia in transformation.

We present a patient who was diagnosed as suffering from Fanconi anaemia at the age of 36 years. At the time of diagnosis his bone marrow showed features of pre-leukaemic transformation. He received an allogeneic bone marrow transplant (BMT) from his HLA-identical sibling. The post-transplant course was unremarkable with evidence of trilineage engraftment at day +32 and no acute or chronic GVHD. He is well with sustained engraftment and no haematological evidence of Fanconi anaemia 18 months post-transplant.

The radiological features of adult T-cell leukaemia/lymphoma.

Adult T-cell leukaemia/lymphoma (ATLL) results from a monoclonal expansion of T-cells infected with the human T-cell lymphotropic virus type 1 (HTLV-1). Six cases of ATLL, three males and three females all from the Caribbean and with a mean age of 55 years, have been reviewed retrospectively, illustrating the wide spectrum of radiological changes. One patient had extensive mediastinal adenopathy, hitherto an unrecorded finding in ATLL.

Lung abscess: a neglected cause of life threatening haemoptysis.

Three cases who presented with life threatening haemoptysis are reported, all of whom required surgery to control the bleeding. In all three patients chronic lung abscess was responsible for the haemoptysis. Even in the absence of typical clinical or radiographic features of an abscess this diagnosis should be considered in any patient presenting with life threatening haemoptysis.