Conversion of the severe to the moderate disease phenotype with donor leukocyte microchimerism in canine leukocyte adhesion deficiency.

Leukocyte adhesion deficiency-1 (LAD-1), a genetic immunodeficiency disease characterized by life-threatening bacterial infections, results from the defective adherence and migration of leukocytes due to mutations in the leukocyte integrin CD18 molecule. Canine LAD (CLAD) represents the canine homologue of the severe phenotype of LAD-1 in children. In previous studies we demonstrated that non-myeloablative stem cell transplantation from matched littermates resulted in mixed donor-host chimerism and reversal of the disease phenotype in CLAD. In this study, we describe two CLAD dogs with less than 2% donor leukocyte chimerism following non-myeloablative transplant. Both dogs are alive more than 24 months after transplant with an attenuated CLAD phenotype resembling the moderate deficiency phenotype of LAD. The improvement in the CLAD phenotype with very low levels of donor CD18(+) leukocytes correlated with the preferential egress of the CD18(+) neutrophils into extravascular sites. The clinical response with very low levels of donor CD18(+) leukocytes in CLAD supports using this model for testing gene therapy strategies since the low levels of gene-corrected hematopoietic cells expected with hematopoietic gene therapy would likely have a therapeutic effect in CLAD.

Molecular biology of leukemia.

Identification and characterization of leukemia-related chromosomal translocations have had significant impact on all aspects of the management of acute leukemia, including its diagnosis, assignment of prognosis, and development of an appropriate treatment plan. Several genes are recurrent targets of chromosomal abnormalities, suggesting that they play a key role in leukemogenesis. Significant progress has been made to define potentially unifying molecular mechanisms of leukemic transformation. Hopefully, these findings will provide the basis for molecularly targeted therapies for leukemia.

Discordant detection of monosomy 7 by GTG-banding and FISH in a patient with Shwachman-Diamond syndrome without evidence of myelodysplastic syndrome or acute myelogenous leukemia.

The myelodysplastic syndromes (MDS) are a group of hematologic disorders commonly affecting elderly persons and often leading to acute myelogenous leukemia (AML). Although rare in children, when MDS does occur, it is frequently part of a congenital disorder such as Shwachman-Diamond syndrome (SDS). Monosomy 7 and/or deletion of part or all of 7q are poor prognostic signs in MDS and AML, although the pathophysiologic relationship between this finding and MDS or AML is unclear. Shwachman-Diamond syndrome is an inherited illness characterized by exocrine pancreatic insufficiency and by congenital neutropenia. Patients with SDS are at increased risk of developing myelodysplastic syndrome (MDS) and acute myelogenous leukemia (AML). Because monosomy 7 is a poor prognostic sign in MDS and AML, establishing its presence is important. However, different methods of detection of monosomy 7 may lead to different results in some patients. We present the case of a 10-year-old girl known to have SDS, who had a bone marrow aspiration and biopsy done to rule out MDS and AML. By light microscopy, the patient's bone marrow was unremarkable. GTG-banding showed the following karyotype: 45,XX,-C[3]/47,XX,+C[1]/46,XX[45]. Fluorescence in situ hybridization (FISH) was performed with a chromosome 7-specific alpha-satellite probe (D7Z1). Almost all (373 of 376) cells exhibited only one chromosome 7 signal. A second marrow aspiration done 6 months later showed an essentially normal karyotype by GTG-banding. Fluorescence in situ hybridization with the same chromosome 7 probe showed 230 of 250 cells to be monosomic for chromosome 7. A whole chromosome 7 painting probe demonstrated disomy for chromosome 7 in 90 of 90 cells; however, subtle heteromorphism in the centromeric regions of the 2 copies of chromosome 7 was noted in some cells. This case demonstrates that FISH and GTG-banding can give discordant results, that the two should be viewed as complementary technologies, and that both have a place in a full karyotypic analysis. Furthermore, this case demonstrates for the first time that heteromorphism and/or subtle structural abnormalities of chromosome 7, previously associated with MDS and AML, can exist without clinical or morphologic signs of these illnesses. It will be of interest to further study the relationship, if any, between SDS and various structural abnormalities of chromosome 7 in MDS and AML, and to elucidate the molecular mechanisms of pathogenesis, physiology, and treatment of these disorders.

Multiple SH3 domain interactions regulate NADPH oxidase assembly in whole cells.

Src homology 3 (SH3) domains mediate specific protein-protein interactions crucial for signal transduction and protein subcellular localization. Upon phagocyte stimulation, two SH3 domain-containing cytosolic components of the NADPH oxidase, p47phox and p67phox, are recruited to the membrane where they interact with flavocytochrome b558 to form an activated microbicidal oxidase. Deletion analysis of p47phox and p67phox in transfected K562 cells demonstrated multiple SH3-mediated interactions between p47phox and the transmembrane flavocytochrome b558 and also between the cytosolic components themselves. The core region of p47phox (residues 151-284), spanning both SH3 domains, was required for flavocytochrome-dependent translocation and oxidase activity in whole cells. Furthermore, translocation of p67phox occurred through interactions of its N-terminal domain (residues 1-246) with p47phox SH3 domains. Both of these interactions were promoted by PMA activation of cells and were influenced by the presence of other domains in both cytosolic factors. Deletion analysis also revealed a third SH3 domain-mediated interaction involving the C-termini of both cytosolic factors, which also promoted p67phox membrane translocation. These data provide evidence for a central role for p47phox in regulation of oxidase assembly through several SH3 domain interactions.

A bicistronic retrovirus vector containing a picornavirus internal ribosome entry site allows for correction of X-linked CGD by selection for MDR1 expression.

Chronic granulomatous disease (CGD) is an inherited hematologic disorder involving failure of phagocytic cell oxidase to produce superoxide (O2-.), resulting in recurrent infections. The success of retrovirus gene therapy for hematopoietic diseases will be limited both by the efficiency of ex vivo transduction of target cells and by the ability of corrected cells to replace uncorrected cells in vivo. Using MFG-based retrovirus vectors containing oxidase genes, we have previously demonstrated in vitro correction of CGD, but transduction rates were low. In the present study we explore a strategy for providing a selective growth advantage to transduced cells, while retaining the single promoter feature of MFG responsible for high virus titer and enhanced protein production. We constructed a bicistronic retrovirus producing a single mRNA encoding both the therapeutic gene for the X-linked form of CGD (X-CGD), gp91phox, and the selectable human multidrug resistance gene, MDR1 linked together by the encephalomyocarditis virus internal ribosome entry site (IRES). As a control we constructed a bicistronic vector with the polio virus IRES element and using the bacterial neomycin resistance gene (neor) as the selective element. In Epstein-Barr virus transformed B (EBV-B) cells from an X-CGD patient, a tissue culture model of CGD, we show correction of the CGD defect and complete normalization of the cell population using either of these vectors and appropriate selection (vincristine for MDR1 and G418 for neor). Using a chemiluminescence assay of O2-. production, populations of cells transduced with either vector demonstrated initial correction levels of from less than 0.1% up to 2.7% of normal EBV-B cell oxidase activity. With either construct, cell growth under appropriate selection enriched the population of transduced cells, resulting in correction of X-CGD EBV-B cells to a level of O2-. production equalling or exceeding that of normal EBV-B cells. These studies show that a therapeutic gene can be linked to a resistance gene by an IRES element, allowing for selective enrichment of cells expressing the therapeutic gene. Furthermore, the use of MDR1 as a selective element in our studies validates an important approach to gene therapy that could allow in vivo selection and is generalizable to a number of therapeutic settings.

ELF magnetic fields, breast cancer, and melatonin: 60 Hz fields block melatonin's oncostatic action on ER+ breast cancer cell proliferation.

In this study we investigated whether a 60 Hz magnetic field can act at the cellular level to influence the growth of human estrogen-dependent breast cancer cells. Our experimental design assessed cell proliferation of a human breast cancer cell line, MCF-7, in the absence or the presence of melatonin which inhibits growth at a physiological concentration of 10(-9) M. In three experiments, continuous exposure to average sinusoidal 60 Hz magnetic fields of 1.90 +/- 0.01, 2.40 +/- 0.70, and 2.53 +/- 0.50 mG, or simultaneous exposure in matched incubators to average 60 Hz magnetic fields of 10.4 +/- 2.12, 11.95 +/- 2.73, and 11.95 +/- 3.28 mG, respectively, had no effect on cell proliferation in the absence of melatonin. When MCF-7 cells were cultured in the presence of 10(-9) M melatonin, an 18% inhibition of growth was observed for cells in a 2.40 +/- 0.70 mG field. This effect was blocked by a 60 Hz magnetic field of 11.95 +/- 2.75 mG. In a second experiment, a 27% inhibition of MCF-7 cell growth was observed for cells in a 2.53 +/- 0.50 mG magnetic field, and this was blocked by a 60 Hz magnetic field of 11.95 +/- 3.28 mG. These results provide the first evidence that ELF frequency magnetic fields can act at the cellular levels to enhance breast cancer cell proliferation by blocking melatonin's natural oncostatic action. In addition, there appears to be a dose threshold between 2 and 12 mG. The mechanism(s) of action is unknown and may involve modulation of signal transduction events associated with melatonin's regulation of cell growth.