Direct maxacalcitol injection into hyperplastic parathyroids improves skeletal changes in secondary hyperparathyroidism.

Direct maxacalcitol (OCT) injection into a parathyroid gland (PTG) ameliorates several important etiologic factors of resistance to medical treatments for secondary hyperparathyroidism (s-HPT): the upregulations of vitamin D receptor (VDR) and Ca-sensing receptor (CaSR) in PTGs and the regression of PTG hyperplasia by the induction of apoptosis. In this study, we evaluated the bone histomorphology on the basis of maintaining these effects in advanced s-HPT. Five/six nephrectomized Sprague-Dawley rats were fed a high-phosphorus and low-calcium diet for 8 weeks. These rats were divided into four treatment groups: (1) basic uremic (at the baseline), (2) direct OCT single injection into PTGs (DI-OCT) followed by OCT intravenous administration for 4 weeks (IV-OCT), (3) direct vehicle injection and IV-OCT, and (4) no treatment for an additional 4 weeks. The effects of these treatments on serum intact-parathyroid hormone (PTH) level, PTG weight, VDR and CaSR expression levels in PTGs, and bone histomorphometric parameters were investigated. In the DI-OCT+IV-OCT group, the significant decrease in serum intact-PTH level was maintained by the following IV-OCT. A significant decrease in PTG weight and the upregulations of VDR and CaSR expression levels in PTGs were also observed. Bone histomorphometric analysis showed significant improvements in osteitis fibrosa in both cancellous and cortical bones. However, these findings were not observed in the other groups. These results suggest that osteitis fibrosa caused by advanced s-HPT can be successfully reversed by a control of PTH at an appropriate level through the improvement of PTG hyperplasia as induced by DI-OCT+IV-OCT.

Long-term production of erythropoietin after electroporation-mediated transfer of plasmid DNA into the muscles of normal and uremic rats.

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 weeks. Repeated administration of the plasmid DNA effectively produced erythropoiesis. Similar erythropoiesis was observed in the uremic rats, and persisted for more than 15 weeks. Both normal and uremic rats showed a significant decrease in platelet count. Moreover, the uremic rats showed Epo-induced hypertension, which is the major side-effect of recombinant human Epo. These results demonstrate that muscle-targeted pCAGGS-Epo transfer by in vivo electroporation is a useful procedure for the long-term continuous delivery of Epo in both normal and uremic rats.

Continuous erythropoietin delivery by muscle-targeted gene transfer using in vivo electroporation.

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.

[Thoracoabdominal aortic repair without using assisted bypass].

A 41-year-old man, who had undergone descending aortic repair following rupture of the DeBakey type III aortic dissection, underwent thoracoabdominal aneurysm repair 1 year after the first surgery. The operation was performed by partial-clamping and single crossclamping without using assisted bypass or shunt, in order to minimize bleeding ensuing the re-thoracotomy and dissection between lung and the graft.

IL-6 functions in cynomolgus monkeys blocked by a humanized antibody to human IL-6 receptor.

A humanized antibody to the human interleukin-6 receptor (IL-6R), hPM-1, blocked the interleukin-6 (IL-6) functions in normal cynomolgus monkey lymphocytes in vitro. The binding activity of hPM-1 to non-human primate IL-6R was examined in peripheral blood lymphocytes by flow cytometry. PM-1 recognized the IL-6R on T lymphocytes of cynomolgus and rhesus monkeys, but did not on those of marmosets. The homology between human IL-6R and its cynomolgus monkey counterpart was 97.3% in the extracellular domain of the amino acid sequence, as determined by DNA sequencing of the PCR product from peripheral blood mononuclear cells. PM-1 inhibited two functional parameters in vitro in cynomolgus monkeys: (1), T-cell proliferation stimulated by phytohemaglutinin and human IL-6; (2), Immunoglobulin G-production evoked by Staphylococcus aureus Cowan-1- and human IL-6-stimulated B lymphocytes. These data show that hPM-1 binds to and functionally blocks the cynomolgus monkey IL-6 receptors.

In vivo blocking effects of a humanized antibody to human interleukin-6 receptor on interleukin-6 function in primates.

A humanized antibody to human interleukin-6 (IL-6) receptor, MRA, which was constructed by grafting the complementary determining regions, is expected to be useful as a therapeutic agent for IL-6-related diseases, especially multiple myeloma. We examined the ability of MRA to block the in vivo function of IL-6 and its serum concentration profile in primates. Cynomolgus monkeys were intravenously administered with MRA at doses of 0 (vehicle) or 5 mg/kg, then subcutaneously injected with human IL-6 at a dose of 5 micrograms/kg, once a day for 7 days. The injections of IL-6 increased blood platelet counts two-fold and elevated serum C-reactive protein levels to 0.15 to 0.17 mg/ml. These IL-6-induced typical responses were completely inhibited by single pretreatment with MRA. Serum concentrations of MRA were maintained for a long period; some even at one week after administration, were regarded as having sufficient levels to inhibit the myeloma cell growth. These findings suggest that MRA may be effective in the treatment of IL-6-related diseases.

Safety and kinetic properties of a humanized antibody to human interleukin-6 receptor in healthy non-human primates.

A monoclonal antibody, hPM-1, was constructed by grafting the complementarity determining regions to human interleukin-6 (IL-6) receptor, raised in mouse, onto a human antibody backbone (humanized antibody). It is expected to be useful as a therapeutic agent for IL-6-related diseases such as multiple myeloma. To investigate the toxicological and kinetic properties of hPM-1 preliminarily, normal cynomolgus monkeys, which showed cross-reactivity with hPM-1, were intravenously administered with hPM-1 at doses of 0 (vehicle), 4 or 40 mg/kg once a week for 13 weeks. Upon toxicological examination, there were no changes in clinical signs, food consumption, body weights, urinalyses, body temperatures, electrocardiograms, hematological and biochemical parameters including blood platelet counts, serum levels of immunoglobulin G and C-reactive protein, and pathological findings. In a kinetic study, serum concentrations of hPM-1 showed a linearity between doses of 4 and 40 mg/kg. The serum concentrations, even at a dose of 4 mg/kg, were maintained at a high enough level to inhibit the IL-6 functions throughout the period of the study. Concentrations of hPM-1 in bone marrow were almost equal to those in serum. The antibodies against hPM-1 were detected only in one of four monkeys receiving hPM-1. This study suggests that blockage of the IL-6 receptor by hPM-1 does not induce any influence on a healthy living body, and hPM-1 is not toxic under the conditions of this investigation.

Thrombopoietic activity of human interleukin-6.

Thrombopoietin (TPO), a regulatory factor in platelet production, was purified from the conditioned medium of TNK-01 cells cultured in the presence of human interleukin-1. The N-terminal sequence of purified TPO was determined to be VPPGEDSKDVAAPHRQPLT, identical to that of the N-terminal region of human interleukin-6 (IL-6). Two forms of TPO with molecular masses of 24 and 27 kDa were identified as IL-6 by Western analysis using an anti-IL-6 antibody. Commercial recombinant human IL-6 produced in Escherichia coli, stimulated megakaryocyte colony formation in the presence of mouse interleukin-3 and increased the number of peripheral platelets in mice in a dose-dependent manner. From these results, it is concluded that human IL-6 has thrombopoietic activity.

Induction of neutrophilic granulocytosis in mice by administration of purified human native granulocyte colony-stimulating factor (G-CSF).

Mice were subcutaneously (sc) injected once a day for up to 15 days with a purified human native G-CSF sample at a dose of 2.5 micrograms/injection or with control samples with or without added endotoxin. In the G-CSF-treated mice, blood neutrophil counts began to rise as early as 2 hours after the first injection, reached a level 8 times above the preinjection level after 15 days of injections with marked elevation of all progenitor cell levels in spleen, and returned to normal within 48 hours after cessation of the injections. Such neutrophilia was observed even when endotoxin-resistant C3H/HeJ mice were used, but not in control mice. It is possible that repeated G-CSF injections after administration of cyclophosphamide (CY) in mice could accelerate recovery of granulopoiesis with a rather transient rise in blood neutrophil counts.

Purification and characterization of human granulocyte colony-stimulating factor (G-CSF).

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.