Post-secretion neutralization of transgene-derived effect: soluble erythropoietin receptor/IgG1Fc expressed in liver neutralizes erythropoietin produced in muscle.

BACKGROUND: The regulation of transgene expression is a key issue for the development of safe gene therapy. Various strategies have been used to regulate protein production at the levels of transgene expression, transcription, translation, and secretion. Neutralization following secretion is another important backup system to prevent super-therapeutic levels of a protein from being expressed by gene transfer. METHODS: We tested whether the soluble human erythropoietin receptor (EpoR)/IgG(1)Fc could neutralize the rat Epo at the post-secretory level and suppress erythrocytosis. RESULTS: To assess whether soluble human EpoR could bind rat Epo in vitro, we used the Epo-dependent human leukemic cell line, AS-E2. EpoR/IgG(1)Fc significantly inhibited the growth of AS-E2 cells in Epo-containing medium. To test this neutralization effect of EpoR/IgG(1)Fc in vivo, we first transferred pCAGGS-Epo into rat muscle by in vivo electroporation, confirmed erythropoiesis for 3 weeks, and then delivered EpoR/IgG(1)Fc by liver-targeted gene transfer via tail-vein injection with hydrodynamics-based transfection. Reticulocyte counts and hematocrit levels in rats that received pCAGGS-EpoR/IgG(1)Fc injections were significantly lower than in rats that received pCAGGS-EpoR, pCAGGS-IgG(1)Fc, or no injection. CONCLUSIONS: These results demonstrate that liver-targeted pCAGGS-EpoR/IgG(1)Fc transfer by tail-vein injection with hydrodynamics-based transfection is useful for neutralizing Epo delivered by in vivo electroporation. This backup strategy at the level of post-secretion could facilitate the clinical application of gene therapy in the future.

High-level expression of naked DNA delivered to rat liver via tail vein injection.

BACKGROUND: High levels of foreign gene expression in mouse hepatocytes can be achieved by rapid tail vein injection of a large volume of a naked DNA solution, the 'hydrodynamics-based procedure'. Rats are more tolerant of the frequent phlebotomies required for monitoring blood parameters than mice, and thus are better for some biomedical research. METHODS: We tested this technique for the delivery of a therapeutic protein in normal rats, using a rat erythropoietin (Epo) expression plasmid vector, pCAGGS-Epo. RESULTS: We obtained maximal Epo expression when the DNA solution was injected in a volume of 25 ml (approximately 100 ml/kg body weight) within 15 s. We observed a dose-response relationship between serum Epo levels and the amount of injected DNA up to 800 microg. Using quantitative real-time PCR, the vector-derived Epo mRNA expression was mainly detected in the liver. When a lacZ expression plasmid was injected similarly, beta-galactosidase was exclusively detected in the liver, mainly in hepatocytes. Toxicity attributable to the technique was mild and transient, as assessed by histochemical analysis. Epo gene expression and erythropoiesis occurred with Epo gene transfer in a dose-dependent manner, and persisted for at least 12 weeks, the last time point examined. Repeated administration of the plasmid DNA also effectively led to erythropoiesis. CONCLUSIONS: These results demonstrate that gene transfer into the liver via rapid tail vein injection can easily be achieved in the rat, which is more than 10 times larger than the mouse, and has significant value for gene function analysis in rats.

Carcinosarcoma of the uterus: immunohistochemical and genetic analysis of clonality of one case.

BACKGROUND: Carcinosarcomas of the uterus are characterized by admixtures of malignant epithelial and stromal cells, and their histogenesis remains controversial. CASE: An operated case of carcinosarcoma of the uterus in a 49-year-old woman is reported with clonal analysis. The tumor was composed of carcinomatous, sarcomatous, and transitional elements in the frontal wall of the uterine body and therefore was diagnosed as a carcinosarcoma. On immunohistochemical analysis, the sarcomatous component proved negative for epithelial membrane antigen and keratin while both components were positive for vimentin. Analysis of X-chromosome inactivation showed the same pattern throughout and additionally, the same K-ras and p53 mutations were homogeneously detected. Microsatellite instability analysis showed loss of heterozygosity at D5S346 in the sarcomatous but not the carcinomatous component. CONCLUSIONS: This tumor appears monoclonal in line with the combination tumor theory, with late divergence in genetic alteration in the sarcomatous elements.

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.

Five cases of Diphyllobothrium nihonkaiense infection with discovery of plerocercoids from an infective source, Oncorhynchus masou ishikawae.

Five persons from 2 families residing at Miyama Town, Mie Prefecture, Japan, ingested fresh raw fish Oncorhynchus sp. on 9 May 1999 that was caught at Owase district in Mie. They all expelled diphyllobothriid cestodes 11-37 days after ingesting the fish. The parasites were morphologically identical to Diphyllobothrium nihonkaiense Yamane et al., 1986. Five plerocercoids were detected from a portion of the fish. Nucleotide sequence of a region of the cytochrome c oxidase subunit I gene of mitochondrial DNA from an adult worm was identical with that from the plerocercoid. The fish was identified as Oncorhynchus masou ishikawae according to the nucleotide sequence of the nuclear ribosomal second internal transcribed spacer region II gene. This is the first record of D. nihonkaiense plerocercoids from O. m. ishikawae.

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.

Association of remnant-like particle cholesterol with coronary artery disease in patients with normal total cholesterol levels.

BACKGROUND: Limited information is available as to whether there is a difference in the association of lipid and fibrinolytic variables with coronary artery disease according to the presence or absence of elevated serum total cholesterol. We examined the levels of various lipid and fibrinolytic variables including remnant-like particle cholesterol (RLP-C). RLP-C is a recently established simple assay method for the estimation of triglyceride-rich lipoprotein remnants. METHODS AND RESULTS: Levels of total cholesterol, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol (HDL-C), triglyceride, lipoprotein(a), RLP-C, uric acid, blood glucose, tissue plasminogen activator, tissue plasminogen activator inhibitor type 1, antithrombin III, and protein C were measured in 208 patients who underwent diagnostic coronary angiograms. Of these 208 patients, 57 were hypercholesterolemic (> or =220 mg/dL) and 151 were normocholesterolemic. HDL-C showed significant differences between patients with and those without angiographically determined coronary artery stenosis in both hypercholesterolemic and normocholesterolemic patients (P =.0025 and P =.0003, respectively). Both RLP-C and uric acid showed significant differences only in the normocholesterolemic subgroup (P =.0006 and P =.0060, respectively). This difference in uric acid was not significant by multivariable analysis. The ratio of RLP-C/HDL-C was demonstrated to be highly significantly (P <.0001) associated with coronary artery stenosis in patients with normal total cholesterol, whereas there was no statistically significant association in the hypercholesterolemic patient subgroup. CONCLUSIONS: Our current study disclosed that RLP-C levels are strongly associated with coronary artery disease, especially in patients with normal total cholesterol levels. Moreover, RLP-C/HDL-C ratio may be even more significantly associated with the presence of coronary artery stenosis in normocholesterolemic patients.

Predictive value of remnant-like particles cholesterol/high-density lipoprotein cholesterol ratio as a new indicator of coronary artery disease.

BACKGROUND: There is as yet no definite consensus on the predictive value of the various lipid profiles and fibrinolytic parameters that became available in clinical use recently for coronary artery disease. METHODS: Levels of lipoprotein(a), high-density lipoprotein cholesterol (HDL-C), remnant-like particles cholesterol (RLP-C), tissue plasminogen activator (TPA), TPA inhibitor, antithrombin III, and protein C were measured in 124 patients who underwent diagnostic coronary angiograms. RESULTS: Of these patients, 37 had no significant stenoses (group N) and 87 had significant stenoses (group S). There were no significant differences in patient characteristics between the two groups. HDL-C was significantly lower (p = 0.0071 ) and RLP-C was significantly higher (p = 0.0022) in group S. When a product and a ratio of each of two factors were calculated, RLP-C/HDL-C was demonstrated to be a highly significant predictor for coronary artery stenoses (p < 0.0001). There were also significant increases in RLP-C/HDL-C levels with increasing number of vessels involved (r = 0.359, p < 0.0001 ). CONCLUSION: Our present study disclosed the predictive value of RLP-C/HDL-C ratio as a new indicator of coronary artery disease.

Rhabdomyolysis and myocardial damage induced by palytoxin, a toxin of blue humphead parrotfish.

A 55-year-old man had rhabdomyolysis and myocardial damage induced by palytoxin. Weakness and myalgia of four extremities occurred five hours after eating a fish. Rhabdomyolysis developed and the serum creatine phosphokinase (CK) was elevated to 40,000 IU/l on the 3rd day. Gastric lavage with activated charcoal and forced mannitol-alkaline diuresis therapy were performed. The patient recovered with no complication such as renal failure. In this case, palytoxin was suggested to induce myocardial damage which was demonstrated by an elevation of the myosin light chain level and a change in electrocardiogram.

Enhancement of neurite outgrowth by the soluble form of human L1 (neural cell adhesion molecule).

L1, a neural cell adhesion molecule, is involved in neurite outgrowth, migration and fasciculation. Although L1 is a membrane glycoprotein expressed on neural cells, the soluble form of L1 is generated in vivo by proteolysis. In the present study, a stable transfectant of Chinese hamster ovary (CHO) cells secreting human L1 without cytoplasmic and membrane spanning domains was generated, and the function of the secreted L1 was examined. Explants from embryonic chick brain stem were cultured on a substrate coated with polyethylenimine (PEI) alone, on substrate-bound L1 or in medium containing soluble L1. The neurites induced by L1, both the substrate-bound form and the soluble form, were 2-3 times longer than those cultured on PEI. The ability of the soluble L1 to induce neurite formation was slightly greater than that of the substrate L1. The present results demonstrated that neurite outgrowth was induced not only by substrate-bound L1 but also by soluble L1. Soluble L1 could be a pharmaceutical candidate for the promotion of nerve regeneration.

Oxidized low density lipoprotein caused CNS neuron cell death.

Death induced by oxidized low density lipoproteins (oxLDL) to embryonic CNS neuronal and neuroblastoma cells was investigated. Cell damage and viability were evaluated by LDH leakage and the MTT method, respectively. Dose- and time-dependent degeneration of neurons occurred after oxLDL (1-100 microg/ml) treatment but was absent after native low density lipoproteins (LDL). This degeneration was mediated, in part, by apoptosis because increased TUNEL and Hoechst dye-positive staining was observed. These effects occurred in the absence of microglia. However, DNA degradation was not detected. The cytotoxicity was attenuated by pre-treatment with antioxidants. These results suggest that oxidation by oxLDL may be important in neurocytotoxicity in the brain.

Axonal growth-related cell surface molecule, neurin-1, involved in neuron-glia interaction.

We purified and characterized a novel axonal growth-related molecule, neurin-1, which is anchored to the surface membrane via a phosphatidylinositol (PI) linkage. This molecule was detected by a combination of phosphatidylinositol-specific phospholipase C (PI-PLC) treatment from detergent-soluble mouse brain membranes and subsequent Western blot analysis with monoclonal antibody (MAb 2A). Neurin-1 is immunologically distinct from other known axonal growth associated surface glycoproteins. In immunoblots of embryonic mouse brain membrane, the MAb 2A recognized a single band at approximately 68 kDa, and showed that neurin-1 is mainly associated with fiber-containing regions of developing embryonic mouse brain. Expression is immunohistochemically similar to that of cell adhesion molecule L1, but in comparison, neurin-1 appears somewhat later. Late in embryonic development, neurin-1 appeared to be more stage- and region-specific. Its precise localization at the neural cell surface membranes was confirmed by immuno-electron microscopy using labeled and cultured live nerve cells. Neurin-1 was found only on the surface of the axon and growth cone. Neurin-1, otherwise termed PI anchor protein, corresponds closely in function to the other PI-anchored cell adhesion molecules. Anti-neurin-1 antibody (MAb 2A), however, perturbs the axonal growth and neural cell migration from the astrocyte feeder layer cultures. These results suggest that neurin-1 is one of the important cell surface molecules mediated in the neuron and glial cell interaction.

Impaired plasticity of neurons in aging. Biochemical, biophysical, and behavioral studies.

Age-related correlation of impaired plasticity of neurons (biochemical and biophysical aspects) and behavioral alterations were investigated in young (3.5 months) and extremely aged (approximately 40 months) female Wistar rats. Age-dependent significant differences in second messenger (cAMP and Ins (1,4,5)P3) concentration and signal transduction via muscarinic and dopaminergic receptors were found. The results point to the specifically impaired coupling between dopamine D1 receptor and GS protein, which underlies normal brain aging. However, cholinergic neurotransmission may be modulated at another level in extremely aged rats. Thus, it appears that the site of affection in coupling of receptor and G protein and/or G protein-dependent signal transduction in aging cannot be generalized. This indicates that alterations in the coupling of signal transduction depend on diverse neurotransmitter receptors with advanced age. The age-dependent alterations in the cAMP and PI signal pathways could be due to changes in the physical properties of the membranes. To support this hypothesis, age-dependent changes in the physical state and the biochemical composition of synaptosomal membranes from the cortex, cerebellum, and striatum were examined by measuring the steady-state fluorescence amisotropy of the membrane probes 1,6-diphenyl-1,3,5-hexatriene (DPH), trimethylammonium-DPH (TMA-DPH), and trimethylammoniumpropyl-DPH (TMAP-DPH). Significant differences in the physical properties of the synaptosomal membranes existed between young and very aged rats, expressed by a higher anisotropy in the 40-month-old rat brain tissue. The changes in the physical properties of the membranes were in line with the determined age-dependent alterations in the chemical composition, e.g., the increase in cholesterol content of the aged membranes.

Signal transduction in aging.

The possible age-related involvement of two different signal transduction pathways in the rat CNS was investigated. In the phosphytidyl inostiol (PI) response, higher phospholipase-C (PL-C) activity and drastically higher (almost 2.5-fold) inositol (1,4,5)trisphosphate (Ins(1,4,5) P(3)) concentration in the corpus striatum (caudate-putamen) of extremely old (approximately 40 months) female Wistar rats in comparison to young adult (approximately 3.5 months) rats were observed. In the adenosine 3':5'-cyclic monophosphate (cAMP) cascade, a significantly higher endogenus cAMP level and a significant decline of the adenylate cyclase (AC, ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1.) activity were observed in striatal tissue from young rats in comparison with aged rats. Binding saturation experiments with [(3)H]SCH 23390 at the dopamine (DA) D(1) receptor (D(1)) and [(3)H]spiperone at the DA D(2) receptor (D(2)) revealed no change in the affinity (K(d)) but a significant decrease in the density (B(max)) of D(1) (-31%, p<0.005) and of D(2) (-22%, p<0.05), respectively, in the aged versus young striata. DA seems to slightly inhibit total inositol phosphate formation and this effect was antagonized by (-)-sulpiride. A significant decrease (p<0.05) in the AC activity stimulated by 10 muM DA in the senescent compared to the young animals was monitored. Apparently, the age-related decline of the AC activity was independent of changes of G(S) and G(i) activity.

Alterations of inositol phosphate turnover in striatum of aged rats.

Age-related inositol phosphate turnover in the rat central nervous system was investigated. Higher phospholipase-C activity and drastically higher (almost 2.5-fold) inositol 1,4,5-trisphosphate concentration in the corpus striatum (caudate-putamen) of extremely old (approximately 40 months) female Wistar rats in comparison to the young adult (approximately 3.5 months) rats were observed. Dopamine seems to slightly inhibit total inositol phosphate formation and this effect was antagonized by (-)-sulpiride.

Age-related alteration in signal transduction: involvement of the cAMP cascade.

In the present study, we have investigated the involvement of the cAMP signal transduction pathways in young and aged rats. A significantly higher endogenous adenosine 3':5'-cyclic monophosphate (cAMP) level and a significant decline of the adenylate cyclase [AC, ATP pyrophosphate-lyase (cyclizing), EC.4.6.1.1.] activity were observed in striatal tissue from young rats (3 months) in comparison to aged rats (approximately 40 months). In the nucleus accumbens (NA), no age-dependent changes in the cAMP concentration and in the AC basal activity were found. To address the question, whether the interactions of guanine nucleotide-binding protein (G-protein) subunits (G alpha s and Gi) with AC have changed in the aging process, various pharmacological agents that modulate the AC activity (e.g., beta, tau-imidoguanine 5'-triphosphate (GppNHp), sodium fluoride (NaF), forskolin (FSK), and the combinations of GppNHp plus FSK, NaF plus FSK, and NaF plus ethanol (ETOH)) were applied. In addition, a [3H]FSK binding test was carried out. In striatal and NA tissue, the stimulation of the AC activity by FSK was inhibited by GppNHp (via Gi-protein) and was superadditive by the combination of FSK and NaF (via Gs-protein). The absolute AC activity upon stimulation by all agents used was significantly lower in the aged striatum compared to young striatum. In the NA, however, the AC activity showed an age-dependent reduction only upon FSK and upon FSK plus GppNHp stimulation. There was no difference in the specific [3H]FSK binding to the G alpha s protein-coupled catalytic subunit of the AC between young and aged animals both in the striatum and NA.(ABSTRACT TRUNCATED AT 250 WORDS)

Functional roles of cyclic guanosine 3',5'-monophosphate analogue in cerebral vasodilation.

1. Vasodilating effects of cyclic nucleotides in cerebral vasculature were examined using membrane permeable cyclic nucleotide analogues, 8-bromoguanosine 3',5'-cyclic monophosphate (8-Br-cGMP) and 8-bromoadenosine 3',5'-cyclic monophosphate (8-Br-cAMP). 2. In isolated canine basilar artery (CBA), 8-Br-cGMP but not 8-Br-cAMP, significantly inhibited Ca(2+)-induced and agonist [serotonin(5-HT), prostaglandin(PG)F2 alpha or endothelin]-induced contraction, in a concentration-dependent manner. 3. When Ca2+ was depleted from intracellular store sites by pretreatment with A23187, 8-Br-cGMP but not 8-Br-cAMP strongly attenuated contractions induced by Ca(2+)-influx. 4. Neither 8-Br-cGMP nor 8-Br-cAMP modified contraction induced by caffeine which elicits Ca2+ release from intracellular Ca2+ store. 5. 8-Br-cGMP lowered the high K(+)-induced sustained [Ca2+]i elevation. 6. These results suggest that, at least in CBA, cGMP exerts its inhibitory effect on the contraction induced by influx of Ca2+, by reducing the level of [Ca2+]i and reducing [Ca2+]i sensitivity of the contractile machinery.

Altered dopamine receptor mediated signal transmission in the striatum of aged rats.

Striatal membranes of very old (40 months) as against young (3 months) female Wistar rats were used. Binding saturation experiments with [3H]SCH 23390 at the dopamine (DA) D1 receptor (D1) and [3H]spiperone at the DA D2 receptor (D2) revealed no change in the affinity (Kd) but a significant decrease in the density (Bmax) of D1 (-31%, P < 0.005) and of D2 (-22%, P < 0.05), respectively, in the aged vs. young striata. Displacement of either [3H]SCH 23390 or [3H]spiperone binding by DA displayed biphasic curves. The Hill coefficient (nH) was significantly increased in the senescent compared with the young of D1 (0.72 +/- 0.04 vs. 0.61 +/- 0.03, P < 0.025) but unchanged of D2 (0.49 +/- 0.04 vs. 0.51 +/- 0.02). The proportion of the high-affinity agonist binding state (Rhigh) was significantly decreased (P < 0.025) in the older (20.9 +/- 3.2%) in comparison with the young (30.6 +/- 2.0%) in D1 but increased non-significantly in D2 (47.9 +/- 2.6 vs. 40.5 +/- 5.1%). Calculating the resulting Bmax from Scatchard and displacement analyses of each single aged and young animal revealed a highly significant reduction (P < 0.001) of the high-affinity agonist binding state of D1 (-53%) as well as a non-significant reduction of D2 (-8%) in the older. Simultaneously, a significant 57% decrease (P < 0.01) in the adenylate cyclase (AC) activity stimulated by 10 microM DA in the senescent compared with the young animals was monitored. The DA stimulation of AC was reversed in both cases by the addition of 200 nM of the D1 antagonist SCH 23390.

BY-1949 elicits vasodilation via preferential elevation of cyclic GMP levels within the cerebral artery: possible involvement of endothelium-mediated mechanisms.

The pharmacological mechanisms by which BY-1949, a novel dibenzoxazepine derivative, increases in regional cerebral blood flow, were investigated using the canine basilar artery in vitro. BY-1949 inhibited contractions elicited by serotonin (5-HT), prostaglandin (PG) F2 alpha, endothelin and phorbol-12,13-diacetate (PDA), respectively, to the same extent. In addition, pretreatment of the artery with methylene blue significantly suppressed the vasodilating effect of BY-1949. BY-1949 also dose dependently suppressed contractions of the basilar artery induced by CaCl2 (Ca2+) in a non-competitive manner. Biochemical studies disclosed that BY-1949 significantly increased cyclic GMP without causing any apparent change in cyclic AMP. These increases in cyclic GMP were virtually abolished after the endothelial cells were removed. These results strongly suggest that the increased regional cerebral blood flow induced by BY-1949 is explicable, at least partly, in terms of a preferential elevation of cyclic GMP within the cerebral vasculature, where the endothelium plays a pivotal role.