Chronic myeloid leukemia arising in a progenitor common to T cells and myeloid cells.

Until recently, T cells were believed not to be involved in chronic myeloid leukemia. We describe an example of CML in T lymphoblastic crisis with massive generalized lymphadenopathy in which the blasts were CD2(+), CD5(+), and CD7(+), variably CD1(+) and CD3(+), and both responded to and could be induced to produce the T cell growth factor, interleukin-2. Additionally, the blasts were shown to contain the CML-related tyrosine kinase P210bcr-abl rather than the smaller kinase associated with Ph1(+) ALL. Finally, the participation of the T lymphoid lineage in the CML clone was proven by the presence of the same BCR rearrangement in blasts as in granulocytes, suggesting the existence of a bone marrow progenitor common to the T cell and myeloid lineages.

Effect of different promoters on expression of genes introduced into hematopoietic and marrow stromal cells by electroporation.

Electroporation is a simple and relatively efficient means of introducing genes into hematopoietic cells. However, achieving and maintaining high levels of gene expression in transfected hematopoietic progenitor cells remains problematic. In order to address this problem we examined the effect of different viral and cellular promoters on the transient expression of reporter genes transferred into K562, KG1a, and human marrow stromal cells. We found that although the Rous sarcoma virus long terminal repeat was most active in K562 cells in a transient expression assay, the murine cytomegalovirus immediate early promoter was strikingly more active than the other promoters in KG1a cells as well as in the marrow stromal cell population. Long-term stable gene expression was also demonstrated in stromal cells. We infer that the murine cytomegalovirus immediate early promoter may be highly active in human hematopoietic progenitor cells and that human marrow stromal cells may be an attractive vehicle for gene delivery.

Bead transfection: rapid and efficient gene transfer into marrow stromal and other adherent mammalian cells.

We report a simple, rapid, efficient and cost-effective method of gene transfer into bone marrow stromal and other adherent mammalian cells. Our approach involves brief incubation of cells with glass beads in a solution containing the DNA to be transferred. We optimized the technique using COS cells (SV40 transformed kidney cell line from African green monkey) and a transient expression assay for chloramphenicol acetyl transferase (CAT). Factors affecting gene transfer include size and condition of the beads and DNA concentration, but not DNA conformation. Gene transfer efficiency, assessed in a transient expression assay for beta-galactosidase activity, was 5 and 3% in nontransformed human bone marrow stromal cells and COS cells, respectively. Long-term stable expression with the selectable marker, neomycin phosphotransferase, was demonstrated in clonogenic COS cells at a frequency of 27%. Southern analysis of resistant clones revealed the transferred DNA to be integrated in low copy number at one or two sites in the host cell genome. Comparison with electroporation and DEAE-dextran indicates that bead transfection is more efficient than the latter and less costly than either of these methods. In view of its simplicity and because the use of retroviral sequences can be avoided, bead transfection may be an attractive means of gene insertion for gene therapy.

Early embryonic expression of murine coagulation system components.

The development of the embryonic coagulation system, and its contribution to the maintenance of vascular integrity during the formation of embryonic blood vessels, remain poorly understood. We have characterized the temporal expression patterns of 27 hemostasis-related genes during murine development. We show that, although most coagulation and fibrinolysis-related factors are expressed coordinately by 7.5 dpc, several, including FIX, FXII and PAI2, are not detectable until later developmental timepoints. The expression of hemostasis-specific genes prior to the formation of a functional circulatory system supports the view that some coagulation factors have additional non-hemostatic functions during development. In addition, the discordant expression of some factors suggests that the embryonic hemostatic system may be distinct from that of the adult. These analyses will help to elucidate the regulation of hemostasis during embryonic/vascular development, and will provide a framework to facilitate the interpretation of gene inactivation studies.

Diazepam improves aspects of social behaviour and neuron activation in NMDA receptor-deficient mice.

NR1 knockdown (NR1KD) mice are genetically modified to express low levels of the NR1 subunit of N-methyl-D-aspartate (NMDA) receptors, and show deficits in affiliative social behaviour. In this study, we determined which brain regions were selectively activated in response to social stimulation and asked whether differences in neuronal activation could be observed in mice with reduced sociability. Furthermore, we aimed to determine whether brain activation patterns correlated with the amelioration of social deficits through pharmacological intervention. The cingulate cortex, lateral septal nuclei, hypothalamus, thalamus and amygdala showed an increase in c-Fos immunoreactivity that was selective for exposure to social stimuli. NR1KD mice displayed a reduction in social behaviour and a reduction in c-Fos immunoreactivity in the cingulate cortex and septal nuclei. Acute clozapine did not significantly alter sociability; however, diazepam treatment did increase sociability and neuronal activation in the lateral septal region. This study has identified the lateral septal region as a neural substrate of social behaviour and the GABA system as a potential therapeutic target for social dysfunction.

Splenic infarction and tissue accumulation of crystals associated with the use of clofazimine (Lamprene; B663) in the treatment of pyoderma gangrenosum.

A male patient 68 years, suffering from pyoderma gangrenosum which was resistant to conventional treatment, received clofazimine 400 mg daily for 5 months, then reducing to 300 mg daily for the next 6 months. Eleven months after starting the drug, he was admitted to hospital with severe abdominal pain, laparotomy revealing infarction of the spleen, with violaceous congestion of the small bowel. The spleen was removed and post-operative recovery was satisfactory. Histopathological examination of the spleen (removed at operation) and of tissue from a duodenal biopsy (taken postoperatively) showed large numbers of striations and outlines suggestive of crystal deposition. Mesenteric lymph node revealed a massive accumulation of crystals in cortical and medullary sinuses. The findings emphasize that clofazamine should not be used in high dosage over prolonged periods of time, except under close clinical and laboratory supervision, and for conditions not amenable to other drugs.