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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ABSTRACT1α,25-dihydroxyvitamin D3 [Calcitriol or 1,25(OH) 2 D 3 ] is an important active metabolite involved in multiple functions but its calcemic effect in vivo limits its therapeutic applications. On the other hand, 22-oxa-1α,25-dihydroxyvitamin D 3 (22-oxacalcitriol or 22-Oxa-1α,25-D 3 ), a low calcemic analog of vitamin D3 (VitD3), has been widely used as a drug for the secondary hyperparathyroidism. Here, we investigated immunomodulating effect of these two VitD3 derivatives on the differentiation of type-1 immunoregulatory cells such as dendritic cells (DC1), cytotoxic T cells (Tc1) and helper T cells (Th1). BALB/c mouse bone marrow-derived DC (BMDC1) induced by culture with Th1 condition (GM-CSF, IL-3, IL-12 and IFN-γ) expressed higher levels of MHC Class I and Class II molecules and co-stimulatory molecules compared with BMDC0 induced by neutral condition (GM-CSF + IL-3). In addition, BMDC1 showed stronger immunostimulating activity to induce alloantigen (H-2 d )-specific cytotoxic T lymphocytes (CTL) compared with BMDC0. However, if VitD3 derivatives were added into the culture for BMDC1 induction, the expression of functional molecules and type-1 IFNs were greatly inhibited. Moreover, VitD3 derivative-treated BMDC1 lost their immunostimulating activity to induce alloantigen-specific IFN-γ-producing Tc1. In addition, it was demonstrated that the addition of VitD3 derivatives inhibited the differentiation of IFN-γ-producing Th1 cells from ovalbumin (OVA)-specific naive Th cells, while it rather augmented the differentiation of IL-4-or IL-10-producing Th2 cells. There was no significant difference in immunomodulating activity between 1,25(OH) 2 D 3 and 22-Oxa-1α,25-D 3 . Thus, VitD3 derivatives are demonstrated to inhibit the functional differentiation of DC1, Tc1 and Th1 cells, which play a critical role in type-1 cellular immune responses.

The most important etiological factors of resistance to medical treatments for secondary hyperparathyroidism are the decreased contents of the vitamin D receptor (VDR) and Ca-sensing receptor (CaSR) in parathyroid cells and a severely swollen parathyroid gland (PTG) as a result of hyperplasia. The effects of direct maxacalcitol (OCT) injection into PTG in terms of these factors were investigated in this study. The PTG of Sprague-Dawley rats that were 5/6 nephrectomized and fed a high-phosphate diet were treated by a direct injection of OCT (DI-OCT) or vehicle (DI-vehicle). The changes in serum intact parathyroid hormone (PTH), Ca 2؉, and phosphorus levels, in VDR and CaSR expression levels in parathyroid cells, and in Ca 2؉ -PTH curves were examined. Apoptosis was analyzed by the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling method and DNA electrophoresis for PTG. DI-OCT markedly decreased serum intact PTH level, and a significant difference in this level between DI-OCT and DI-vehicle was observed. However, serum Ca 2؉ and phosphorus levels did not changed markedly in both groups. The upregulations of both VDR and CaSR, the clear shift to the left downward in the Ca 2؉ -PTH curve, and the induction of apoptosis after DI-OCT were observed. These findings were not observed in the DI-vehicle-treated rats. Moreover, these effects of DI-OCT were confirmed by the DI-OCT into one PTG and DI-vehicle alone into another PTG in the same rat. DI-OCT may introduce simultaneous VDR and CaSR upregulations and the regression of hyperplastic PTG, and these effects may provide a strategy for strongly suppressing PTH levels in very severe secondary hyperparathyroidism. S econdary hyperparathyroidism (SHPT) resulting from ESRD causes not only renal osteodystrophy but also cardiovascular disorders because of ectopic calcification in vascular tissues. A low level of parathyroid hormone (PTH) causes adynamic bone disease; easily increases serum calcium (Ca), phosphorus (P), and CaϫP levels; and may contribute to a poor prognosis in patients with ESRD. Thus, the maintenance of appropriate levels of PTH, Ca, and P is required for the improvement of the prognosis and quality of life of these patients (1-3).SHPT develops in conditions of P retention, low Ca level, and the inactivation of vitamin D (VD) (4 -6); therefore, the control of P level and supplementations of Ca and VD are necessary in patients with SHPT as preventive or medical treatment. However, advanced SHPT with severely swollen parathyroid glands (PTG) as a result of hyperplasia is resistant to these medical treatments because of the low contents of VD receptor (VDR) and Ca-sensing receptor (CaSR) in parathyroid cells (PTC) (7,8). When SHPT progresses into such status, it is considered irreversible. Thus, patients with very severe SHPT require parathyroidectomy-autotransplantation (PTx-AT), which may be complicated by some problems, such as the necessity of general anesthesia, the hyperor hypofunction of autotransplanted PTG, and mental suffering.The ...

Immune balance controlled by Th1 and Th2 cells is critical for the protection of host from pathogenic invasion while its imbalance becomes the cause of various immune disorders including autoimmune diseases. Cytokines, such as IL-12 and IL-4, are critical factor to drive the differentiation of naïve CD4(+) T cells to Th1 or Th2 cells. In addition to cytokines, steroid hormones have been demonstrated to affect on the control of Th1/Th2 balance and the onset of autoimmune diseases. Here, we will propose a new concept that immunosteroid, which is designated as a steroid produced by immunoregulatory cells, also play a critical role for regulation of Th1/Th2 balance. First example of immunosteroid is Th2-dependently produced progesterone. Th2 cells, but not Th1 cells expressed P450scc and 20alpha-HSD and produced progesterone from 22R-hydroxycholesterol in cooperation with 3beta-HSD-expressing mouse fibroblasts. Th2-dependently produced progesterone induced apoptotic cell death of Th1 cells and inhibited the differentiation of Th1 cells. While Th2 cells were escaped from toxic effect of progesterone by metabolizing it to non-toxic 20alpha-hydroxyprogesterone with 20alpha-HSD. Second example of immunosteroid is dendritic cell (DC)-dependently produced 1alpha,25-dihydroxyvitamin D3 [1,25(OH)(2)D] secosteroid hormone, which has been demonstrated to inhibit autoimmune diseases. We found that 25-hydroxyvitamin D3 1alpha-hydroxylase, which metabolize 25-hydroxyvitamin D3 (inactive form) to 1,25(OH)(2)D was expressed in Th2-cytokine induced bone marrow-derived DC2 but not Th1-cytokine induced DC1. Moreover, 1,25(OH)(2)D was significantly inhibited DC1-induced type1 immunity. Thus, we initially demonstrated the critical role of immunosteroids in the control of Th1/Th2 balance influencing on the onset of autoimmune diseases. Therefore, it will be an important issue to investigate the possible role of immunosteroids for the regulation of autoimmune diseases.