Characteristics of neuropathic pain and its relationship with quality of life in 72 patients with spinal cord injury.

STUDY DESIGN: A cross-sectional study. OBJECTIVES: Neuropathic pain (NP) after spinal cord injury (SCI) tends to be hard to treat, and its heterogeneous properties make it difficult to identify and characterize. This study was conducted to assess the characteristics of SCI-related NP in detail. SETTING: A single hospital for SCI rehabilitation. METHODS: This study included 72 patients who were seen at our hospital in 2012 and 2013 and who had sustained SCI at least 3 months before enrollment. The patients completed the Neuropathic Pain Symptom Inventory (NPSI) and the Short Form (SF)-36 Health Inventory. The NPSI score was analyzed for correlations with clinical presentations of SCI and SF-36 subitems. RESULTS: Paresthesia/dysesthesia was the most common subtype of NP after SCI. With regard to location, below-level superficial NP was significantly more intense than at-level pain. Patients who underwent surgery showed significantly less evoked pain compared with patients with non-surgery. Patients reported significantly more severe pain if >1 year had elapsed after the SCI. Patients with an American Spinal Injury Association Impairment Scale grade of B for completeness of injury reported more intense NP than those with other grades. Among the SF-36 subitems, NP correlated significantly with bodily pain, general health and mental health. CONCLUSION: NP in SCI patients was significantly associated with the location of pain, the time period since the injury, surgery and quality-of-life factors. A more detailed understanding of the characteristics of NP may contribute to better strategies for relieving the pain associated with SCI.

Oral administration of recombinant human acid α-glucosidase reduces specific antibody formation against enzyme in mouse.

Animal and human studies of enzyme replacement therapy for Pompe disease have indicated that antibodies generated against the infused recombinant human acid α-glucosidase (rhGAA) can negatively impact therapeutic outcome. In this study, we show that oral administration of rhGAA into mice can reduce the titer of anti-rhGAA antibody following immunization with rhGAA. Oral administration of rhGAA is safe and antigen specific, it offers advantages over other immunosuppressive drugs.

Neonatal gene transfer using lentiviral vector for murine Pompe disease: long-term expression and glycogen reduction.

Pompe disease results from the deficiency of the lysosomal enzyme acid alpha-glucosidase (GAA), leading to accumulated glycogen in the heart and the skeletal muscles, which causes cardiomyopathy and muscle weakness. In this study, we tested the feasibility of gene therapy for Pompe disease using a lentivirus vector (LV). Newborn GAA knockout mice were treated with intravenous injection of LV encoding human GAA (hGAA) through the facial superficial temporal vein. The transgene expression in the tissues was analyzed up to 24 weeks after treatment. Our results showed that the recombinant LV was efficient not only in increasing the GAA activity in tissues but also in decreasing their glycogen content. The examination of histological sections showed clearence of the glycogen storage in skeletal and cardiac muscles 16 and 24 weeks after a single vector injection. Levels of expressed hGAA could be detected in serum of treated animals until 24 weeks. No significant immune reaction to transgene was detected in most treated animals. Therefore, we show that LV-mediated delivery system was effective in correcting the biochemical abnormalities and that this gene transfer system might be suitable for further studies on delivering GAA to Pompe disease mouse models.

Effects of endogenous and exogenous prostaglandin E2 on the proliferation and differentiation of a mouse cementoblast cell line (OCCM-30).

BACKGROUND: Cementum formation is considered to be a critical event for successful regeneration of periodontal tissues. Cementoblasts share many characteristics with osteoblasts. Prostaglandin E(2) (PGE(2)) is an important local factor in bone metabolism. Although the effects of PGE(2) on osteoblasts are well known, its effects on cementoblasts have not yet been established. We examined the effects of PGE(2) on proliferation and differentiation in a mouse cementoblast cell line, OCCM-30 cells. METHODS: OCCM-30 cells were treated with three concentrations of PGE(2) (10, 100, and 1,000 ng/ml). Cell number, alkaline phosphatase (ALP) activity, and expression for mineralization-related genes were determined. Osteoprotegerin (OPG) and receptor activator of nuclear factor-kappa B (NF-kappaB) ligand (RANKL) expression were also examined by real-time polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA). RESULTS: The addition of PGE(2) at the highest dose used in this study suppressed cell proliferation of OCCM-30 cells. The expression of mineralization-related marker mRNA, such as type 1 collagen, ALP, bone sialoprotein (BSP), and osteocalcin (OCN), was constitutively detected in OCCM-30 cells. PGE(2) dose dependently stimulated ALP activity and BSP-mRNA expression in OCCM-30 cells at day 3. Transcripts for OPG and RANKL and the protein level of OPG in culture media were upregulated with PGE(2) stimulation. CONCLUSION: These results demonstrate that PGE(2) suppressed cementoblast proliferation but stimulated ALP activity and the BSP-mRNA level, suggesting a role of PGE(2) in controlling cementoblast differentiation, and further indicate that PGE(2) modulates RANKL and OPG expression in cementoblasts; the increase of OPG secreted from cementoblasts with PGE(2) stimulation may be essential to protect the root surface from resorption.

Enzyme-linked immunosorbent assay for human manganese-containing superoxide dismutase and its content in lung cancer.

A manganese-containing superoxide dismutase (Mn-SOD) was purified from human liver. Polyclonal antibody for the Mn-SOD was prepared in goat, and a simple and specific enzyme-linked immunosorbent assay (ELISA) for the Mn-SOD was developed. This assay was found to be sensitive to nanogram amounts of the enzyme. With respect to Mn-SOD levels of normal lung tissues, a positive correlation (r = 0.92, P less than .001) was observed between the results of enzymatic assay and those of immunochemical assay by ELISA. In lung carcinoma tissue the enzyme activity was in the same order of magnitude as in uninvolved tissues. However, the enzyme content determined by ELISA was significantly higher in adenocarcinoma than in the uninvolved lung tissue, whereas no significant difference from the control was observed in other histologic types. The discrepancy between the enzyme activity and immunoreactive content suggested that in the adenocarcinoma of the lung an immunoreactive but enzymatically inactive Mn-SOD protein existed and that a high content of this enzyme was characteristic of lung adenocarcinoma.

A monoclonal antibody against gamma-glutamyltransferase from human primary hepatoma: its use in enzyme-linked immunosorbent assay of sera of cancer patients.

A monoclonal antibody, gamma-120, was raised against a highly purified gamma-glutamyltransferase (gamma GT) from human primary hepatoma. The antibody preferentially bound to the small subunit of gamma GT from human hepatoma and kidney as evidenced by immunoblot analysis. Weak binding to the normal liver enzyme could be detected by solid-phase enzyme-linked immunosorbent assay (ELISA). With the use of this antibody, an ELISA was developed for the quantitation of immunoreactive gamma GT in human serum. Sera of 188 normal control subjects displayed a low level (9.4 micrograms/ml) of immunoreactive gamma GT. Highly elevated levels of immunoreactive gamma GT were detected in the sera of patients with primary hepatoma, metastatic liver cancer, pancreatic cancer, and certain types of lung cancer. Slightly elevated levels of immunoreactive gamma GT were seen in the sera of patients with liver cirrhosis. The levels of gamma GT were within a normal range in the sera of patients with gastrointestinal cancers and patients with nonmalignant diseases such as hepatitis and gallstones. The antibody has been shown to be useful for the diagnosis of some of the neoplastic diseases.

Measurement of human serum immunoreactive gamma-glutamyl transpeptidase in patients with malignant tumors using enzyme-linked immunosorbent assay.

An enzyme-linked immunosorbent assay for human immunoreactive gamma-glutamyl transpeptidase (gamma-GTP) was developed. This assay was found to be simple, reproducible, and sensitive to 10 ng of the enzyme. Serum immunoreactive gamma-GTP content was significantly elevated in patients with various malignant tumors including liver cell cancer, lung cancer, gastric cancer, esophageal cancer, and colorectal cancer. On the other hand, in sera of patients with nonneoplastic diseases, the immunoreactive gamma-GTP content was not significantly elevated. No correlation was found between the serum levels of gamma-GTP determined by enzymatic assay and enzyme-linked immunosorbent assay, which indicates that, due to the presence of endogenous inhibitors and/or activators in sera, the enzyme activity may not reflect the true amount of enzyme protein. The measurement of immunoreactive gamma-GTP protein in sera appears to be useful for the detection and monitoring of certain malignant tumors.

Increase in the glucosylated form of erythrocyte Cu-Zn-superoxide dismutase in diabetes and close association of the nonenzymatic glucosylation with the enzyme activity.

Human erythrocytes contain glucosylated and nonglucosylated Cu-Zn-superoxide dismutases which can be separated by boronate affinity chromatography. The percentage of the glucosylated form is significantly increased in the erythrocytes of patients with diabetes as compared to normal erythrocytes. The nonglucosylated form of Cu-Zn-superoxide dismutase, which was washed through the boronate column, was glucosylated in vitro upon exposure to radioactive or non-radioactive D-glucose. Incorporation of D-glucose into the protein was observed, and with the increase in glucosylation, the enzymatic activity decreased, indicating that the glucosylation of the enzyme led to a low active form. This is the first demonstration that superoxide dismutase is glucosylated in erythrocytes and that the glucosylation leads to the inactivation of the enzyme.

High levels of Mn-superoxide dismutase in serum of patients with neuroblastoma and in human neuroblastoma cell lines.

Levels of serum manganese superoxide dismutase (Mn-SOD) in normal children aged from 1 to 14 years and children with various hematological and malignant diseases were determined by enzyme-linked immunosorbent assay (ELISA). In the normal children, the serum Mn-SOD levels gradually increased in proportion to age. By 8 years of age, the Mn-SOD level was nearly at the adult level. The normal values of serum Mn-SOD (mean +/- SD) of children below 4 and above 8 years old were 48 +/- 10.2 ng/ml and 84 +/- 22.5 ng/ml, respectively. Assuming the upper limit of normal Mn-SOD level in serum to be the mean value +/- 2 SD of children at each age, high serum levels of Mn-SOD were found for 8 of 12 patients with neuroblastoma, three of four patients with Wilms tumor, and four of five patients with acute myeloid leukemia. The patients with neuroblastoma exhibited a transient increase in Mn-SOD following chemotherapy, but after 1 week the levels decreased markedly to the control levels. The changes in serum Mn-SOD levels in the patients with neuroblastoma correlated well with the levels of neuron-specific enolase. Mn-SOD was intensely stained in bone marrow cells of patients whose cancer cells had moved into the bone marrow. High levels of Mn-SOD were also found in cultured human neuroblastoma cells. These data indicate that Mn-SOD is expressed in neuroblastoma cells, may serve as one of the diagnostic and prognostic markers for the neuroblastoma, and may be useful to predict the effectiveness of chemotherapy for neuroblastoma and the recurrence of this disease.

Purification of Cu-Zn-superoxide dismutase from human erythrocytes by immunoaffinity chromatography. Evidence for the presence of isoelectric heterogeneity.

Cu-Zn-superoxide dismutase (SOD) was prepared in highly purified form from human erythrocytes by immunoaffinity chromatography using anti-human Cu-Zn-SOD goat IgG. The purified SOD had a high specific activity and gave a single band on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. On isoelectric focusing and immunoblotting several bands with enzyme activity and antigenicity were discerned.

Animal model of closed angle glaucoma in albino mutant quails.

The intraocular pressure of imperfect albino mutant quails with a sex-linked recessive gene was measured by using a cannulating method, and morphological changes of the iridocorneal angle were observed with a gonioprism. Albino mutant quails were characterized by an elevated intraocular pressure and lens enlargement at 6 mo after hatching. Initially the iridocorneal angle opened and eventually closed. The usefulness of the closed angle glaucoma as an animal model system for human diseases was discussed.

Measurement of retinal permeability to sodium fluorescein in vitro.

Fluorescein permeabilities of isolated dog retinal pigment epithelium (RPE)-choroid and sensory retina were measured individually. Retina to choroid (outward) permeability of sodium fluorescein was significantly larger than choroid to retina (inward) permeability at RPE-choroid under both open- and short-circuit conditions. Outward permeability was decreased by the addition of 10(-3) M KCN and of 10(-4) M probeneside but was not affected by 10(-6) M ouabain. These drugs decreased transepithelial potential and short-circuit current. No directional difference of fluorescein permeability for sensory retina was found. Active transport of fluorescein across RPE from retina to choroid is suggested. Fluorescein permeability of sensory retina was larger than inward but less than outward permeability of RPE-choroid. The result indicates that the sensory retina acts as a diffusion barrier for sodium fluorescein.

Possible mechanisms underlying mammary carcinogenesis in female Wistar rats by nitrofurazone.

Mechanisms underlying mammary carcinogenesis in female rat given nitrofurazone (NF) were examined. Experiment I: female Wistar rats were divided into three groups, and given diets containing 0, 500 or 1000 ppm NF for 5 weeks. At terminal sacrifice, body and uterus weights were the same in all groups, although ovary weights in NF-treated animals were significantly higher than in control animals, the increase being dose-dependent. Serum prolactin (PRL) concentrations in NF-treated groups at 17:00 h on the day of proestrus were also dose-dependently higher than that in control group. Experiment II: a two-stage rat mammary carcinogenesis protocol was performed. Rats were divided into four groups, Groups 2 and 4 being treated by 9,10-dimethyl-1,2-benzanthracene (DMBA) at 7-weeks-old. Groups 3 and 4 were given diets containing 1000 ppm of NF between 8 and 27 weeks of age, when all surviving rats were autopsied. DMBA-treated animals demonstrated mammary tumors at high incidences, 91.1 and 90.5%, respectively, in Groups 2 and 4, no tumor development being observed without the initial carcinogen exposure (Groups 1 and 3). The mean tumor weights and the mean numbers of tumors per tumor-bearing rats in Group 4 were increased as compared with Group 2, albeit not significantly. Serum PRL (proestrus day at 17:00 h) and progesterone (PG) (diestrus day at 10:00 h) concentrations in NF-treated animals (Groups 3 and 4) were significantly higher than those in untreated rats (Groups 1 and 2). These results suggest that increases of serum PRL and PG concentrations by NF may be the most important factors regarding its promotion of mammary tumor growth and/or enhancement of mammary carcinogenesis in female rats.

Elevated serum manganese superoxide dismutase in acute leukemias.

We measured the serum levels of manganese-superoxide dismutase (Mn-SOD) in acute myeloid leukemia (AML) and acute lymphoid leukemia (ALL). Serum Mn-SOD level for normal subjects was 94.1 +/- 23.5 ng/ml (mean +/- S.D.), the levels for AML and ALL patients were 159.6 +/- 77.1 ng/ml and 154.4 +/- 77.0 ng/ml, respectively. The serum Mn-SOD levels were unrelated to individual intracellular Mn-SOD levels, but correlated well with serum lactate dehydrogenase values. Regression of the leukemia was accompanied by decrease in the serum level of Mn-SOD. Serum Mn-SOD may thus serve as a measure of the activity of the disease.

Effects of humid heat exposure on human sleep stages and body temperature.

The objective of this study was to confirm the effect of humid heat exposure on sleep stages and body temperature. Seven healthy male volunteers with a mean age of 22.7+/-1.63, served as the subjects. The experiments were carried out under four different conditions of room temperature and relative humidity: 29 degrees C RH 50% (29/50), 29 degrees C RH 75% (29/75), 35 degrees C RH 50% (35/50), and 35 degrees C RH 75% (35/75). The subjects wearing only shorts slept from 23:00 to 7:00 on a bed, which was covered with a 100% cotton sheet. EEG, EOG, and mental EMG were recorded through the night. Rectal temperature (Tr) and skin temperature were measured continuously. The 35/75 condition caused more wake and a lower sleep efficiency index (SEI) and stage S3+S4 than 29/50 and 29/75. Stage REM and stage 3 were significantly decreased at 35/75 than at 29/50 and 35/50. Tr was maintained at a higher level at 35/75 than under the other conditions. Mean skin temperature was higher at 35/50 and 35/75 than at 29/50 and 29/75. These results suggest that humid heat exposure during night sleep increases the thermal load to supress the sleep-evoked Tr decrease, stage 3, SWS, and REM, and increase wakefulness.

Topical tacalcitol (1,24-(R)-dihydroxyvitamin D3) induces a transient increase in thymidine incorporation and calmodulin content in pig epidermis following tape stripping in vivo.

Tape stripping induces transient increase in keratinocyte proliferation in vivo. The effects of tacalcitol (1,24-(R)-dihydroxyvitamin D3) ointment on the cell kinetics of pig epidermis after the tape stripping were investigated. The tacalcitol ointment (2 micrograms/g) was applied once to the back of pigs immediately after the tape stripping. The pig epidermal cell kinetics were analyzed at various times following the treatment. Tape stripping transiently increased thymidine incorporation of keratinocytes; the maximal effect was observed at 24 h. Tape stripping-induced increase in thymidine incorporation was markedly augmented by tacalcitol treatment. At 24 h following the tape stripping DNA-flow cytometry revealed an accelerated transition from G0/1 to S phase of cell cycle in tacalcitol treated epidermis. There was no significant difference, however, in mitotic counts and G2/M phase fractions between tape stripping-treated and tape stripping plus tacalcitol ointment-treated epidermis. We also measured calmodulin content of pig epidermis following the treatments. Although tape stripping slightly increased calmodulin content of pig epidermis, this was statistically not significant. Tape stripping plus tacalcitol ointment treatment resulted in a significant increase in calmodulin content at 24 h following the treatment. There was no significant difference in calmodulin content between tape stripping treated- and tape stripping plus tacalcitol-treated epidermis.

gamma-Glutamyl transpeptidase of rat liver and hepatoma tissues: an enzyme immunoassay and immunostaining studies.

A sensitive enzyme immunoassay method for rat gamma-glutamyl transpeptidase was developed that was able to quantitate between 5 to 500 ng of the enzyme. The enzyme contents of fetal liver, adult liver, hyperplastic nodules, and various tissues including hepatoma were determined. Normal rat liver contained very little enzyme, while fetal liver and hepatoma tissue contained high levels. The gamma-glutamyl transpeptidase content of the liver was assayed during the process of 3'-methyl 4-dimethyl-aminoazobenzene-induced hepatocarcinogenesis. The content was increased at an early stage, then decreased, and again increased at the stage when hepatoma developed. The change of the enzyme content determined by an enzyme immunoassay was highly consistent with that determined by enzymatic assay. This indicates that the increased activity of the enzyme as previously reported was accompanied by an increased immunoreactive enzyme protein. Immunohistochemical studies on the hyperplastic nodule revealed the presence of gamma-glutamyl transpeptidase in the hepatocyte membrane.

Damage of hippocampal neurons caused by cobalt focus in the cerebral cortex of rats.

The relationship between the focus of cobalt seizures in the cerebral cortex and neuron loss in the hippocampus, as well as the CD50 of pentylenetetrazol was examined in rats. Spike activity in EEG frequently appeared 4 days after cobalt application and reached a peak 8-16 days after cobalt application, which was sometimes accompanied by jerks in the limbs. Changes in the CD50 value showed a two-step pattern, i.e., the first decrease in CD50 appeared one day after application of cobalt and continued at the same value until the fourth day. Then a second gradual decrease of CD50 was observed from the fourth day to eighth day and continued at the same value until 20 days after cobalt application. Neuron loss in the CA1 area of the hippocampus was observed as early as two days after cobalt application and the degree of neuron loss progressively increased until the 20th day. These findings suggest that neuron loss in the hippocampus following cobalt-induced seizures is not a result of generalized convulsions.

Release of zinc from the brain of El (epilepsy) mice during seizure induction.

Brain distribution after i.v. injection of 65ZnCl2 into El mice, an animal model of genetically determined epilepsy, was studied by autoradiography to study the utilization of zinc in the brain. The distribution of 65Zn in the brain of El mice 6 days after injection was almost the same as that of ddY (normal) mice, suggesting that the uptake of zinc by the brain of El mice is normal. To study the movement of zinc in the brain in the course of seizure induction, the concentrations of 65Zn in the brain of seizure-afflicted and untreated control El mice were compared 20 days after 65Zn injection. The concentration of 65Zn in the brain of seized El mice was overall lower than that of control El mice; the concentration of 65Zn was decreased notably in the piriform cortex and amygdaloid nuclei complex during convulsion. These results suggest that the release of zinc from the El mouse brain is enhanced during convulsion. The decrease in actively functioning zinc in the brain may be associated with the increase in susceptibility to seizure in the El mouse.