C anine a -L-iduronidase (iduronidase) deficiency is a model of the hum an lysosom al storage disorder m ucopolysaccharidosis type I (M PS I). W e used this canine m odel to evaluate the therapeutic potential of hem atopoietic stem cell (H SC ) gene therapy for enzyme deficiencies. In previous studies, iduronidase-deficient dogs infused with autologous marrow cells genetically m odified to express iduronidase had long-term engraftment with provirally marked cells, but there was no evidence of proviral iduronidase expression or clinical im provem ent. The presence of hum oral and cellular imm une responses against iduronidase apparently abrogated the therapeutic potential of H SC gene therapy in these experim ents. To evaluate HSC gene therapy for canine M PS I in the absence of a confounding im m une response, we have now performed in utero adoptive transfer of iduronidase-transduced M PS I m arrow cells into preim m une fetal pups. In three separate experiments, 17 m idgestation fetal pups were injected with 0.5± 1.5 3 10 7 norm al or M PS I allogeneic long-term m arrow culture (LTM C ) cells transduced with neo r -or iduronidase-containing retroviral vectors. Nine norm al and three M PS I pup s survived the neonatal period and demonstrated engraftment of provirally m arked progenitors at levels of up to 12% for up to 12 months. H ow ever, the proportion of provirally m arked circulating leukocytes was , 1% . Neither iduronidase enzyme nor proviral-specific transcripts were detected in blood or m arrow leukocytes of any M PS I dog. Hum oral imm une responses to iduronidase were not detected in neonates, even after ª boostingº with autologous iduronidase-transduced LTM C cells. All M PS I dogs died at 8± 11 m onths of age from com plications of M PS I disease with no evidence of am elioration of M PS I disease. O ur results suggest that iduronidase-transduced prim itive hem atopoietic progenitors can engraft in fetal recipients, contribute to hem atopoiesis, and induce im m unologic nonresponsiveness to iduronidase in M PS I dogs. However, the therapeutic potential of HSC gene transfer in this m odel of iduronidase deficiency appears to be limited by poor m aintenance of proviral iduronidase gene expression and relatively low levels of genetically corrected circulating leukocytes. 1521 OVERVIEW SU M M A RY G enetically m odified H SC s have been proposed as vehicles for gene therapy of single-gene inherited disorders, such as enzym e deficien cies. W e evaluated the ability of H SC s to am eliorate disease sym ptom s in a canine m odel of iduronidase deficien cy. In previous studies adoptive transfer of iduronidase-transduced hem atopoietic progenitors to iduronidase-deficient dogs resu lted in engraftm ent of provirus-positive cells; how ever, no clinical benefit was evident. Im m une responses against the therapeutic iduronidase protein and cells expressing it were detected. The im m une response probably abrogated any potential therapeutic impact of H SC gene therapy. In the work reported here, adoptive transf...

Levels of serum granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) in patients with various leukocyte disorders were estimated by enzyme-linked immunosorbent assay (ELISA). Some cases of acute myelogenous leukemia and aplastic anemia showed elevated serum levels of G-CSF and/or GM-CSF, whereas almost all of 23 healthy controls showed G-CSF and GM-CSF levels lower than 100 pg/ml. High levels of both types of CSF were noted in patients with granulocytosis due to infection. These levels became lower after resolution of the infection. Daily changes in serum CSF levels were also examined in a patient with autoimmune neutropenia, and it was found that the peripheral neutrophilic granulocyte count changed almost in parallel with the serum G-CSF level but not with GM-CSF, following the pattern with a delay of about 4-5 h, suggesting the possibility that G-CSF mainly regulates peripheral neutrophil circulation.

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The level of granulocyte-macrophage colony-stimulating factor (GM-CSF) in the cerebrospinal fluid from 14 infants and children with meningitis and 6 patients who suffered other diseases besides meningitis was measured by our sensitive enzyme linked immunosorbent assay for GM-CSF. The minimal detection level of GM-CSF was 40 pg/ml. Six of 9 patients (67%) with aseptic meningitis had detectable GM-CSF in cerebrospinal fluid and the concentrations of GM-CSF ranged from 49 to 114 pg/ml (mean 72 pg/ml), whereas none of 5 patients with bacterial meningitis or 6 patients with other diseases besides meningitis had detectable GM-CSF levels. There was no clear correlation between the GM-CSF levels in cerebrospinal fluid and the leukocyte count in either peripheral blood or cerebrospinal fluid, or the concentration of protein or glucose in cerebrospinal fluid.

Granulocyte colony-stimulating factor (G-CSF) in the cerebrospinal fluid from patients with meningitis was measured by our modified enzyme- linked immunosorbent assay for G-CSF. The minimal detection level was 20 pg/mL G-CSF. In patients with bacterial meningitis, the G-CSF levels in the cerebrospinal fluid were extremely elevated, showing a mean value of approximately 1,500 pg/mL. On the other hand, G-CSF levels in the cerebrospinal fluid from 67% patients with aseptic meningitis were moderately increased, showing a mean value of about 80 pg/mL, whereas G- CSF levels in 33% samples remained undetectable. The G-CSF levels and neutrophil counts in the cerebrospinal fluid were proven to be related by Spearman's rank correlation coefficient analysis (r = .724). These elevations of G-CSF levels at inflammation sites associated with bacterial meningitis may indicate that G-CSF plays an important role in the combat of bacterial infections.