Uraemic rats made by adenine diet developed severe abnormalities of calcium metabolism in a relatively short period and therefore they may serve as a useful model for the analysis of parathyroid hyperplasia and vascular calcification in chronic renal failure.
A colony‐stimulating factor (CSF) has been purified to homogeneity from the serum‐free medium conditioned by one of the human CSF‐producing tumor cell lines, CHU‐2. The molecule was a hydrophobic glycoprotein (mol. wt 19,000, pI = 6.1 as asialo form) with possible O‐linked glycosides. Amino acid sequence determination of the molecule gave a single NH2‐terminal sequence which had no homology to the corresponding sequence of the other CSFs previously reported. The biological activity was apparently specific for a neutrophilic granulocyte‐lineage of both human and mouse bone marrow cells with a specific activity of 2.7 X 10(8) colonies/10(5) non‐adherent human bone marrow cells/mg protein. The purified CSF can be regarded as a G‐CSF of human origin and will become a useful material for investigation of regulatory mechanisms of human granulopoiesis.
It has been demonstrated that gene transfer by in vivo electroporation of mouse muscle increases the level of gene expression by more than 100-fold over simple plasmid DNA injection. We tested continuous rat erythropoietin (Epo) delivery by this method in normal rats, using plasmid DNA expressing rat Epo (pCAGGS-Epo) as the vector. A pair of electrodes was inserted into the thigh muscles of rat hind limbs and 100 microg of pCAGGS-Epo was injected between the electrodes. Eight 100-V, 50-msec electric pulses were delivered through the electrodes. Each rat was injected with a total of 400 microg of pCAGGS-Epo, which was delivered to the medial and lateral sides of each thigh. The presence of vector-derived Epo mRNA at the DNA injection site was confirmed by RT-PCR. The serum Epo levels peaked at 122.2 +/- 33.0 mU/ml on day 7 and gradually decreased to 35.9 +/- 18.2 mU/ml on day 32. The hematocrit levels increased continuously, from the preinjection level of 49.5 +/- 1.1 to 67.8 +/- 2.2% on day 32 (p < 0.001). In pCAGGS-Epo treated rats, endogenous Epo secretion was downregulated on day 32. In a control experiment, intramuscular injection of pCAGGS-Epo without subsequent electroporation did not significantly enhance the serum Epo levels. These results demonstrate that muscle-targeted pCAGGS-Epo transfer by in vivo electroporation is a useful procedure for the continuous delivery of Epo.
The anemia associated with chronic renal failure is one of the best target diseases for erythropoietin (Epo) gene transfer. We previously reported a short-term (1 month) study of continuous rat Epo delivery by muscle-targeted gene transfer of plasmid DNA expressing rat Epo (pCAGGS-Epo) using in vivo electroporation in normal rats. Here, we performed a long-term pharmacokinetic study of continuous Epo delivery by this method in normal rats and uremic five-sixths nephrectomized rats. In normal rats, Epo gene expression and sufficient erythropoiesis occurred with Epo gene transfer in a dose-dependent manner, and persisted for at least 11
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