Impact of glucose fluctuation and monocyte subsets on coronary plaque rupture.

BACKGROUND AND AIMS: It remains unclear whether glycemic fluctuation can affect plaque rupture in acute myocardial infarction (AMI). Here we investigate the impact of glucose fluctuation on plaque rupture, as observed by optical coherence tomography (OCT), and monocyte subsets in patients with AMI. METHODS AND RESULTS: We studied 37 consecutive patients with AMI. All patients underwent OCT examination, which revealed 24 patients with plaque rupture and 13 patients without plaque rupture at the culprit site. Peripheral blood sampling was performed on admission. Three monocyte subsets (CD14(+)CD16(-), CD14(bright)CD16(+), and CD14(dim)CD16(+)) were assessed by flow cytometry. Glycemic variability, expressed as the mean amplitude of glycemic excursion (MAGE), was determined by a continuous glucose monitoring system 7 days after the onset of AMI. MAGE was significantly higher in the rupture patients than in the non-rupture patients (P=0.036). Levels of CD14(bright)CD16(+) monocytes from the rupture patients were significantly higher than those from the non-rupture patients (P=0.042). Of interest, levels of CD14(bright)CD16(+) monocytes correlated positively and significantly with MAGE (r=0.39, P=0.02). CONCLUSION: Dynamic glucose fluctuation may be associated with coronary plaque rupture, possibly through the preferential increase in CD14(bright)CD16(+) monocyte levels.

Increased expression of TDP-43 in the skin of amyotrophic lateral sclerosis.

OBJECTIVES: Transactivation-responsive DNA-binding protein-43 (TDP-43) was identified as a major component of the ubiquitin-positive inclusions in sporadic amyotrophic lateral sclerosis (ALS). However, there has been no study of TDP-43 in ALS skin. The present study investigates TDP-43 in ALS skin. MATERIALS AND METHODS: We made a quantitative immunohistochemical study of the expression of TDP-43 in the skin from 15 patients with ALS and 15 control subjects. RESULTS: The proportion of TDP-43-positive (TDP-43+) cells in the epidermis in ALS patients was significantly higher (P < 0.001) than in controls. There was a significant positive relationship (r = 0.62, P < 0.02) between the proportion and duration of illness in ALS patients. The optical density of TDP-43+ cells in the epidermis in ALS patients is markedly stronger (P < 0.001) than in controls. There was a significant positive relation (r = 0.72, P < 0.01) between the immunoreactivity and duration of illness in ALS patients. CONCLUSIONS: These data suggest that changes of TDP-43 in ALS skin are likely to be related to the disease process and that metabolic alterations of TDP-43 may take place in the skin of patients with ALS.

Immunity against breast cancer by TERT DNA vaccine primed with chemokine CCL21.

Human telomerase reverse transcriptase (TERT) has been considered a potential tumor-associated antigen for active-specific immunotherapy. However, effective specific tumor antigen-specific immunity has been difficult to induce consistently by various TERT vaccine formulations. New adjuvant strategies have been employed, such as utilizing chemokines to attract T cells and antigen-presenting cells. Chemokine adjuvant strategies may enhance tumor antigen-specific immunity induced by vaccines. Therefore, we utilized chemokine ligand 21 (CCL21) as an adjuvant with a xenogeneic TERT DNA vaccine to induce tumor antigen-specific immunity against TERT-expressing breast cancer. The TERT DNA vaccine consisted of a plasmid containing the COOH terminal end of the TERT (cTERT) gene, encapsulated in multilayered liposomes with hemagglutinating virus of Japan coating. We demonstrated that CCL21 treatment before cTERT DNA vaccine, given intramuscularly, induced significantly higher anti-TERT specific cell-mediated immunity compared to cTERT DNA vaccine alone. Effective tumor antigen-specific immunity was shown both in prophylactic and therapeutic regimens against TS/A murine breast cancer. The study demonstrated that CCL21 administration before cTERT DNA vaccination significantly augmented tumor antigen-specific immunity against breast cancer.

Safety of ofloxacin (OFLX) and fosfomycin sodium (FOM) ear drops.

OBJECTIVE: The objective of this study is to evaluate the safety of two ear drops, Ofloxacin (OFLX: Taribid Otic Solution, Daiichi Seiyaku) and Fosfomycin sodium (FOM: Fosmicin S, Meiji Seiyaku). METHODS: Albino guinea pigs were used as experimental animals, and the ototoxicity was evaluated by means of threshold changes in the compound action potentials (CAP), when topically applied to the middle ear cavity of the guinea pig. The sound stimuli applied were; click sound, with tone bursts of 8 kHz, 4 kHz, and 2 kHz. In one group of animals, after one application of the ear drops in the right middle ear cavity, the change in CAP was compared with a contralateral saline control at 24h, one week, and four weeks. In other group of animals, the ear drops were applied into the middle ear cavity for seven consecutive days and the CAP was measured at 24h. RESULTS: At 24h the CAP threshold for click, 8 and 4 kHz elevated significantly for both the saline and ear drop treatment, but the threshold returned to normal when measured at 7 days and 28 days. Seven consecutive days of ear drops administration resulted in no reduction in the CAP for either ear drops. CONCLUSIONS: Based on the lack of changes in the CAP, these two ear drops studied did not show any significant ototoxicities.

Cutaneous infiltration with Waldenström macroglobulinemia.

A 59-year-old Japanese man was diagnosed as Waldenström macroglobulinema. With impaired general performance and a 2-year history of pruritic eruptions that were initially confined to the forearms, but later involved the face, limbs, and trunk. A skin biopsy that was performed on the forehead showed infiltration with abnormal cells of the dermis around the central vessels. PCR analysis of the skin biopsy showed immunoglobulin heavy chain rearrangement. As the rearrangement band occurred at the same site as that of the bone marrow aspiration, skin infiltration with abnormal cells was proven.

Usefulness of repeated direct intratumoral gene transfer using hemagglutinating virus of Japan-liposome method for cytosine deaminase suicide gene therapy.

To investigate the feasibility of repeated gene transfection in suicide gene therapy against human solid tumors by a combination of 5- fluorocytosine (5-FC) and its converting enzyme, cytosine deaminase (CD), we repeatedly transfected the yeast CD gene into the human pancreatic cancer cell line BXPC3 using the hemagglutinating virus of Japan-liposome in a new gene transfer method. The in vivo growth of the s.c. transplanted BXPC3 tumor in nude mice given CD-gene transfection was significantly suppressed by i.p. injection of 5-FC when compared with tumors treated with the control vector. Furthermore, the tumor transfected with the CD gene during a 7-day interval was suppressed much more than that of a single transfection. These results suggest that repeated transfection of the suicide gene together with the combination of 5-FC and the yeast CD gene using the hemagglutinating virus of Japan-liposome gene transfer method may be useful for the treatment of human solid tumors, including pancreatic cancer.

Detection of peritoneal micrometastases of gastric carcinoma with green fluorescent protein and carcinoembryonic antigen promoter.

The aim of this study was to specifically visualize micrometastases in the peritoneal cavity, which cannot be detected by conventional methods, by using enhanced Green Fluorescent Protein (EGFP) containing carcinoembryonic antigen (CEA) promoter in an upstream position. In in vitro experiments, two cell lines from human gastric cancer, MKN45 and MKN1, and a cell line from human fibrosarcoma, HT1080, were transduced with pCEA-EGFP, which contains the CEA promoter region. MKN45 and MKN1, which expressed CEA mRNA, showed positive fluorescence after transduction of pCEA-EGFP, whereas HT1080 did not. In in vivo experiments, 7 days after 10(7) MKN45 had been injected into the peritoneal cavity of BALB/c nude mice, pCEA-EGFP was transduced in the peritoneal cavity using a fusogenic liposome with the envelope protein of Hemagglutinating Virus of Japan on the surface. On the peritoneum of the abdominal wall, fluorescent nodules were detected by fluorescence stereomicroscopy. These nodules had a minimal size of approximately 0.15 mm and could not be detected by conventional stereomicroscopy or macroscopy. They were histologically confirmed to be cancer cells by H&E staining. The results suggest that visualization of peritoneal micrometastasis of gastric cancer using CEA promoter and EGFP can offer a new strategy for diagnosis of micrometastasis.

Effects of cyanide on stearyl-coenzyme A desaturase activities in microsomes from various mammalian tissues.

The effect of potassium cyanide on the desaturase activity for stearyl-CoA in microsomes of various mammalian tissues has been investigated. Potassium cyanide inhibited the desaturase activities in microsomes from livers of rat, hen, guinea pig and rat lung, but not the activities in microsomes from rabbit liver, pig thyroid and bovine adrenocortex, It is concluded that the so-called "cyanide-sensitive factor" does not seem to be common to all the desaturase activities of stearyl-CoA in the microsomes of mammalian tissues.

A prominent feature of the conversion of P-450 to P-420 of cytochrome P-450B1 among the cytochrome P-450 isozymes.

Conversion of cytochrome P-450 to P-420 was observed with the use of three isozymes of P-450, P-450PB, P-450MC and P-450B1. The last one, which was isolated and characterized in our laboratories, is the cytochrome P-450 with high affinity for cytochrome b5. Of these isozymes, cytochrome P-450B1 is predominantly fast in the rate of conversion from P-450 to P-420 in the reduced state under carbon monoxide. p-Nitroanisole, which is the substrate of P-450B1 for demethylation, accelerated the conversion, whereas the effects of the compound on the rate of conversion of the other P-450S were small. The effect of cholate on the conversion was distinct and rapid but not very selective among the isozymes. Stabilization with glycerol for prevention of the conversion was found to be effective, for any of these isozymes. No remarkable difference was observed in the stability of the oxidized state among these isozymes when detected in the CO-reduced form. The rates of the reaction from the oxidized to the CO-reduced form were measured with these isozymes. The rate of P-450B1 was the highest in both the medium with glycerol and that without glycerol. Circular dichroism was measured with respect to conversion of P-450 to P-420. The absorption at 450 nm was related to the significant circular dichroism, while the increased absorption at 420 nm due to the conversion was not accompanied by distinct circular dichroism. These data support the concept that the heme vicinity of cytochrome P-450B1 is more labile in the structure of the reduced form under carbon monoxide than those of the other isozymes.

Radiation protection system at the RIKEN RI beam factory.

The RIKEN RI (radioactive isotope) Beam Factory is scheduled to commence operations in 2006, and its maximum energy will be 400 MeV u(-1) for ions lighter than Ar and 350 MeV u(-1) for uranium. The beam intensity will be 1 pmicroA (6 x 10(12) particles s(-1)) for any element at the goal. For the hands-on-maintenance and the rational shield thickness of the building, the beam loss must be controlled with several kinds of monitors. Three types of radiation monitors will be installed. The first one consists of a neutron dose equivalent monitor and an ionisation chamber, which are commercially available area monitors. The second one is a conventional hand-held dose equivalent monitor wherein the logarithmic signal is read by a programmable logic controller based on the radiation safety interlock system (HIS). The third one is a simple plastic scintillator called a beam loss monitor. All the monitors have threshold levels for alarm and beam stop, and HIS reads all these signals.

Abnormalities of Purkinje cell arborization in brindled mouse cerebellum. A Golgi study.

The cerebellum of the hemizygous brindled mouse (MObr/y), a murine model of Kinky hair disease (KHD) in human beings, was investigated chronologically using the Golgi technique. In 15-day-old MObr/y, Purkinje cells showed considerable changes in their arborization such as perisomatic dendrite-like processes, numerous spine-like protrusions from somata and stem dendrites, focal swellings of stem and distal dendrites and generally poor development of dendritic trees. These changes closely resembled those of KHD. Similar changes except for the focal swellings of dendrites, could be found in control mice at day eight but never after day 12. In the MObr/y receiving intraperitoneal injections of cupric chloride (CuCl2) on postnatal (PN) days seven and ten, Purkinje cells appeared similar, if not identical, to those of controls at PN day 15. Focal swellings of dendrites transiently reappeared in treated animals after PN day 23 but spontaneously subsided by day 110. These results suggested that normal arborization of Purkinje cells in MObr/y is at least in part due to delayed maturation, which is correctable by cupric chloride (CuCl2) treatment. The "weeping willow" deformity, which characterizes Purkinje cells in KHD in humans were not observed in MObr/y. Because other neuronal populations, which are known to be deficient in KHD, appeared well preserved in the murine mutant, these dendritic deformities may be secondary to the loss of other neurons.

Ultrastructural and morphometric studies of Purkinje cells of brindled mouse after administration of cupric chloride.

The mitochondrial and dendritic changes in Purkinje cells, which developed transiently in cupric chloride treated brindled mice, were investigated chronologically with light and electron microscopy. Both changes occurred predominantly in the anterior lobe of the cerebellum. The maximal mitochondrial changes coincided with dendritic changes, suggesting that these alterations were causally related. In the focally swollen dendrites there were disruption of neurotubules, abnormal mitochondria with electron-lucent or electron-dense matrix and large lamellar bodies. Quantitative analysis of the dendritic spine revealed significant differences in the spine area and synaptic length between the brindled mice and normal littermates.

Molecular characterisation of glutamate dehydrogenase gene defects in Japanese patients with congenital hyperinsulinism/hyperammonaemia.

Congenital hyperinsulinism and hyperammonaemia (CHH) is caused by dysregulation of glutamate dehydrogenase (GDH). We characterised the GDH gene in two Japanese patients with CHH. Patient 1 showed late-onset and mild hypoglycaemic episodes and mild hyperammonaemia, compared with patient 2. In GDH activity of lymphoblasts, patient 1 showed twofold higher basal GDH activity than control subjects and mild insensitivity for GTP inhibition. Patient 2 showed severe insensitivity for GTP inhibition, and similar allosteric stimulation by ADP in the controls. Genetic studies identified heterozygous and de novo L413V and G446D mutations in patients 1 and 2, respectively. COS cell expression study confirmed that both mutations were disease-causing gene. The insensitivity for GTP inhibition in L413V and G446D was emphasised in COS cell expression system as a result of the dosage effect of mutant GDH gene. L413V showed less impairment of GDH than G446D based on biochemical and genetic results, which was consistent with the clinical phenotype. Based on the structure of bovine GDH, G446D was located in GTP binding site of pivot helix and its surroundings, while L413V was located in alpha-helix of antenna-like structure. These different locations of mutations gave different effects on GDH enzyme. The antenna-like structure plays an important role in GDH activity.

Rate enhancement of the electron transfer of the adrenodoxin-adrenodoxin reductase system by inorganic and nucleotide phosphates.

Phosphate and pyrophosphate increased the rate of reduction of adrenodoxin by NADPH-adrenodoxin reductase and NADPH, pyrophosphate being one order more effective than the former. However, the cytochrome c reduction by the electron transport system was inhibited in the presence of inorganic (pyro)phosphate. On the other hand, ADP and ATP enhanced the rates of reduction of both adrenodoxin and cytochrome c through adrenodoxin by the electron transport system. GTP also enhanced the rate of reduction of cytochrome c by this system, whereas AMP showed no appreciable enhancement. These inorganic and nucleotide phosphates did not affect the rate of ferricyanide reduction by the reductase.

Effect of age and carbon tetrachloride on cytokine concentrations in rat liver.

Interleukin-1beta (IL-1beta), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-alpha) concentrations were measured in livers of young-adult and old rats administered carbon tetrachloride or vehicle. IL-1beta levels were higher and IL-6 levels were lower in old rats than in young-adult rats. Carbon tetrachloride treatment increased IL-1beta and decreased TNF-alpha and IL-6. The elevation in IL-1beta was diminished by aging. These results indicate that the increase in carbon tetrachloride hepatotoxicity that occurs in old age could be related to a dysregulation of inflammatory cytokines.

Electronmicroscopic study on biopsied rectal mucosa in adrenoleukodystrophy.

Rectal mucosal biopsies were performed for seven members of five families with adrenoleukodystrophy (ALD). Many histiocytes contained characteristic cytoplasmic lamellar inclusions that were identical to those seen in adrenocortical cells, brain macrophages, and Schwann cells of affected patients. The ultrastructural features were seen in all patients and also in two asymptomatic younger brothers who had presymptomatic ALD according to assay of very-long-chain fatty acids.

Electronmicroscopy of conjunctival biopsy in mannosidosis.

Electronmicroscopic examination was performed on conjunctival biopsies from two adolescent siblings with mannosidosis. Fibroblasts and endothelial cells contained membrane-bound vacuoles and vesicles that contained homogeneous osmiophilic globules. These vesicles seem to be pathognomonic of mannosidosis. Plasma cells also contained membrane-bound vacuoles, suggesting inhibition of the immunoglobulin production.

Decreased type IV collagen of skin and serum in patients with amyotrophic lateral sclerosis.

OBJECTIVE: To study type IV collagen of skin and serum in patients with ALS. BACKGROUND: Collagen abnormalities of skin have been reported in ALS patients. However, little is known concerning type IV collagen in ALS. METHODS: We studied type IV collagen immunoreactivity of skin and measured serum levels of the 7S fragment of the N-terminal domain of type IV collagen (7S collagen) in patients with ALS and control subjects. RESULTS: The basement membrane as well as blood vessels of skin in ALS patients was weakly positive for type IV collagen as compared with those of diseased control subjects. This weak immunostaining became more pronounced as ALS progressed. The optical density for type IV collagen immunoreactivity in ALS patients was significantly lower (p < 0.001) than in diseased control subjects and was significantly decreased with duration of illness (r = -0.85, p < 0.01). Serum 7S collagen levels in patients with ALS were significantly decreased (p < 0.01) as compared with those in diseased and healthy control subjects and were negatively and significantly associated with duration of illness (r = -0.81, p < 0.001). There was an appreciable positive correlation between concentrations of serum 7S collagen and the density for type IV collagen immunoreactivity in ALS patients (r = 0.81, p < 0.02). CONCLUSIONS: These data suggest that a metabolic alteration of type IV collagen may take place in the skin of ALS patients and that the decreased levels of serum 7S collagen may reflect a decreased type IV collagen immunoreactivity of skin in patients with ALS.

Alterations of skin glycosaminoglycans in patients with ALS.

BACKGROUND AND OBJECTIVE: Collagen abnormalities of skin have been reported among patients with ALS. However, little is known concerning glycosaminoglycans of the skin in ALS. Our objective was to clarify morphologic and biochemical findings of skin glycosaminoglycans among patients with ALS. METHODS: We performed morphologic studies and biochemical analysis of glycosaminoglycans of skin from 8 patients with ALS, 6 patients with other neurologic or muscular diseases (control group A), and 7 patients without neurologic disorders (control group B). RESULTS: The wide spaces that separate collagen bundles reacted strongly with Alcian blue stain in skin from patients with ALS and stained more markedly as ALS progressed. Staining with Alcian blue was virtually eliminated by Streptomyces hyaluronidase. The content of hyaluronic acid was significantly higher (p < 0.001) among patients with ALS than in control groups A and B. There was a significant positive correlation between content of hyaluronic acid and duration of illness among patients with ALS (r = 0.88, p < 0.01). However, there was no significant difference in content of dermatan sulfate, chondroitin sulfate-4S, or chondroitin sulfate-6S between patients with ALS and control groups A and B. There was also an appreciable positive correlation between optical density of Alcian blue and content of hyaluronic acid among patients with ALS (r = 0.92, p < 0.01). CONCLUSIONS: The data suggest that a metabolic alteration of glycosaminoglycans related to the increased amount of hyaluronic acid may take place in the skin of patients with ALS.

Loss of serotonin-containing neurons in the raphe of patients with myotonic dystrophy: a quantitative immunohistochemical study and relation to hypersomnia.

Hypersomnia occurs frequently in patients with myotonic dystrophy (MyD). We performed a quantitative immunohistochemical study of serotonin (5-HT)-containing neurons linked to hypersomnia in the dorsal raphe nucleus (DRN) and the superior central nucleus (SCN) in 8 patients with MyD, 5 of whom showed hypersomnia, and in 12 age-matched controls. The densities of 5-HT neurons in the DRN and the SCN were significantly lower in MyD patients with hypersomnia than in MyD patients without hypersomnia and controls. These data suggest that the loss of 5-HT neurons of the DRN and the SCN is associated with the presence of hypersomnia in MyD.