Antibodies against saline-soluble components of skeletal muscle in myasthenia gravis

Summary Antibodies against phosphate-buffered-saline extracts (SE) of non-acetylcholine receptor (AChR) skeletal muscle antigens were found in patients with myasthenia gravis (MG). The antigenicity of SE was distributed in three fractions with molecular masses of over 200 kDa, 90–150 kDa and 7–14 kDa on gel filtration. These fractions shared common antigenicities. Further analysis of 90–150 kDa fractions on sodium dodecyl sulphate polyacrylamide gel electrophoresis showed five major bands, ranging from 105 kDa to 275 kDa. The antibodies against SE were detected in 52% (58/112) of the MG patients; incidence and titres were higher in the thymoma group (n=21; 90% and 0.872 respectively) than in the non-thymoma group (n=91; 43% and 0.200, P<0.001). In patients without a thymoma, these antibodies were frequently observed in late-onset disease and the severe generalized form (P<0.01). In 4 of 7 ocular MG patients without anti-AChR antibodies, low but appreciable levels of anti-SE antibodies were found. In 73% (11/15) of generalized MG patients treated with prednisolone and thymectomy, anti-SE antibody titres changed in association with those of anti-AChR antibodies and with the clinical course. Both antibody titres increased synchronously in patients who developed crises.     

Effects of ketanserin on the pacemaker activity and contractility in the isolated, blood-perfused dog atrium

Effects of ketanserin on the pacemaker activity and atrial contractility and on 5-hydroxytryptamine (5-HT)-induced cardiac responses were investigated in the isolated, blood-perfused dog atrium. Ketanserin (1-300 micrograms) injected into the sinus node artery evoked a transient positive followed by a negative chronotropic effect, and a dose above 30 micrograms of ketanserin produced a dose-dependent negative inotropic effect with a little positive one. Ketanserin-induced negative chronotropic and inotropic effects were not affected by atropine in a dose which blocked ACh-induced responses. Propranolol inhibited positive inotropic responses to ketanserin and norepinephrine and significantly augmented the negative chronotropic and inotropic responses to ketanserin. Imipramine did not affect the transient positive followed by negative chronotropic and the negative inotropic responses to ketanserin but it induced the positive cardiac responses following the negative ones. Tetrodotoxin, phentolamine, and diphenhydramine did not modify the effects of ketanserin. From these results, it is concluded that ketanserin might induce the direct negative chronotropic and inotropic effects and the indirect effects by catecholamine release. Ketanserin-induced catecholamine release might not be due to tyramine-like or nicotine-like action. Ketanserin significantly inhibited a low dose (3 micrograms) of 5-HT-induced negative chronotropic and inotropic effects, suggesting the possibility of 5-HT2 receptors in the isolated dog atrium.     

Neonate blood IgE levels on filter paper as indicators of atopic disease

Measurements of IgE levels in the blood of neonates were investigated using filter paper for blood collection in mass screening of congenital metabolic disorders. Time-resolved fluoroimmunometric assay system for the measurement of filter paper blood IgE levels was also studied. In an analysis of the present results, IgE values of at least 0.015U/ml, the measurement limit, were considered as high. High IgE levels in filter paper blood were seen in 28 (7.2%) of the 389 cases. When the relation with serum IgE levels at 18 months of age was investigated in 134 of 389 subjects, high serum IgE levels were also found in about 86.7% of the subjects with high IgE levels in filter paper blood. In addition, when the relation between family history of atopic disease and presence of atopic disease in the first 18 months of age was investigated in 203 of the 389 subjects, about 90% of the subjects with a family history of atopic disease and high IgE levels in filter paper blood developed atopic disease. Since filter paper blood is routinely collected in Japan, IgE levels in this blood should be widely checked for the prediction of onset of atopic disease in infants.     

Functional linkage of the cardiac ATP-sensitive K+ channel to the actin cytoskeleton

The role of the cytoskeleton in the rundown and reactivation of adenosine triphosphate (ATP) sensitive K⁺ channels (KATP channels) was examined by perturbing selectively the intracellular surface of inside-out membrane patches excised from guinea-pig ventricular myocytes. Actin filament-depolymerizing agents (cytochalasins and desoxyribonuclease I) accelerated channel rundown, while actin filament stabilizer (phalloidin) or phosphatidylinositol biphosphate (PIP₂; inhibitor of F-actin-severing proteins) inhibited spontaneous and/or Ca²⁺-induced rundown. When rundown was induced by cytochalasin D or by long exposure to high Ca²⁺, channel activity could not be restored by exposure to MgATP, but application of F-actin with MgATP could reinstitute channel activity. The processes of rundown and reactivation of cardiac K_ATP channels may thus be influenced by the assembly and disassembly of the actin cytoskeletal network, which provides a novel regulatory mechanism of this channel.     

Accumulation of prion protein in muscle fibers of experimental chloroquine myopathy: in vivo model for deposition of prion protein in non-neuronal tissues

Prion protein (PrP) is known to accumulate in some non-neuronal tissues under conditions unrelated to prion diseases. The biochemical and biological nature of such accumulated PrP molecules, however, has not been fully evaluated. In this study, we established experimental myopathy in hamsters by long-term administration of chloroquine, and we examined the nature of the PrP molecules that accumulated. PrP accumulation was immunohistochemically demonstrated in autophagic vacuoles in degenerated muscle fibers, and this was accompanied by the accumulation of other molecules related to the neuropathogenesis of prion diseases such as clathrin, cathepsin B, heparan sulfate, and apolipoprotein J. Accumulated PrP molecules were partially insoluble in detergent solution and were slightly less sensitive to proteinase K digestion than normal cellular PrP. Muscle homogenates containing these PrP molecules did not cause disease in inoculated hamsters. The findings indicate that the PrP molecules that accumulated in muscle fibers have distinct biochemical and biological properties. Therefore, experimental chloroquine myopathy is a novel and useful model to investigate the mechanism of deposition of PrP in non-neuronal tissues and might provide new insights in the pathogenesis of prion diseases.     

Relationship between glucose transporter and changes in the absorptive system in small intestinal absorptive cells during the weaning process

In the present study, we investigated the changes in the localization of the glucose transporter GLUT2 and the fructose transporter GLUT5 in small intestinal absorptive cells during postnatal development, especially during the weaning period, using immunohistochemistry and confocal laser scanning microscopy. In the jejunum, GLUT2 was observed within the apical and basolateral membrane domain of absorptive cells, especially in the middle part of the villi. In the suckling rat ileum, GLUT2 was found within the apical and basolateral membrane domain of absorptive cells, but after 18 or 19 days after birth, GLUT2 was found mainly within the apical membrane domain. GLUT5 was observed within the apical membrane domain of absorptive cells in the suckling rat jejunum. In the 18- or 19-day-old rat jejunum, GLUT5 was localized within the apical and basolateral membrane domain of absorptive cells in the lower part of the villi, but after weaning, GLUT5 was found within the apical and basolateral membrane domain of absorptive cells throughout the entire villi. In the suckling rat ileum, there was little GLUT5 in the absorptive cells. In the 18- or 19-day-old rat ileum, GLUT5 was localized within the apical membrane domain of absorptive cells in the lower part of the villi, but after weaning, GLUT5 was observed mainly within the apical membrane domain of absorptive cells throughout the entire villi. These results suggest that the localization of glucose transporters corresponds with a shift from neonatal-suckling to weaned absorptive cells during postnatal development.     

Generation of functional and mature dendritic cells from cord blood and bone marrow CD34+ cells by two-step culture combined with calcium ionophore treatment

The object of this study is to explore a culture method to generate a large number of functional and mature dendritic cells (DC) from human CD34+ hematopoietic progenitor cells. In the present study, we used a two-step method combined with calcium ionophore to induce DC from cord blood (CB) or normal human bone marrow (BM) CD34+ progenitor cells. The two-step method consists of 10 days of first step culture for the expansion and proliferation of CD34+ hematopoietic progenitor cells in the presence of SCF, IL-3, IL-6, G-CSF, and 7--11 days of second step culture for the induction of DC in the presence of GM-CSF, IL-4 and TNF-alpha. By the two-step culture, total nucleated cells were increased 208+/-66 (+/-SD, n=13), or 94+/-29 (n=5)-fold in the culture of CB or BM cells, respectively, compared with the number of CD34+ cells at the time of starting culture. Out of the total nucleated cells, 23 +/-10.4% of cells in CB cell culture and 25 +/-5% of cells in the BM cell culture acquired DC characteristic phenotypes, which were marked expressions of CD1a, HLA-DR, co-stimulatory molecules such as CD80, CD40, and adhesion molecule such as CD58. In allogeneic mixed leukocyte reaction (MLR), two-step cultured cells showed potent allo-stimulatory capacity. With this two-step culture, the absolute number of CD1a+ cells that co-expressed HLA-DR, CD80, CD40 and CD58 was enhanced approximately 3 times in CB cell culture and 1.9 times in BM cell culture, compared with the commonly used one-step culture method for the generation of DC from CD34+ cells using SCF, GM-CSF and TNF-alpha. However, on these DC generated in the two-step culture, the expressions of co-stimulatory molecule CD86 and mature DC marker CD83 were not sufficient. By the treatment of two-step cultured cells with calcium ionophore agent (A23187), the expression of co-stimulatory molecules such as CD86 and CD80 (especially CD86) was up-regulated. Besides, the expression of mature DC marker CD83 was remarkably induced by treatment with A23187 for a short duration (24 h). Consistent with the up-regulation of surface molecules CD86, CD80 and CD83, the two-step cultured cells treated with A23187 also showed a stronger allo-stimulatory capacity compared with the cells without A23187 treatment. In conclusion, the present study demonstrated that the two-step culture method effectively improved the yield of CD1a+ DC generated from CD34+ cells, and the phenotypes and functions of these CD1a+ DC could be enhanced efficiently by treatment with a calcium ionophore agent.     

Split tolerance between spleen and lymph node cells in severe combined immunodeficiency mice grafted with AKR fetal liver cells

Severe combined Immunodeficient (SCID) mice defective in stemcells for T and B cells appear to be an ideal host for constructionof chimeric mice. When bone marrow cells are used as a sourceof stem cells, however, host SCID mice do not always show sufficientreconstitutlon. In this study, fetal liver cells from AKR embryoswere transplanted into SCID mice without prior irradiation.This treatment induced full reconstltution of lymphopoiesisas evaluated by flow cytometry analysis and serum Ig production2 months after transplantation. Thus, fetal liver cells seemto be a better source for reconstitutlon of SCID mice than bonemarrow cells. Lymph node (LN) cells of these mice (FLT mice)had no proliferatlve or cytotoxlc activities against eitherhost-type (C.B-17) or donor-type (AKR) spleen cells. However,spleen cells from FLT mice exhibited marked proliferatlve andcytotoxlc activities against C.B-17 cells, with no activitiesagainst AKR cells. Spirt tolerance against C.B-17 cells In spleenand LN cells was not a transient phenomenon, since similar resultswere obtained from a cytotoxic T lymphocyte assay 4 months later.In spite of the strong host reactivity in vitro, aberrationof clonal deletion or development of a graft-versus-host diseasewas not seen in FLT mice. As IL-2 induced the host reactivityof LN cells in a mixed lymphocyte reaction, potentially host-reactiveT cells were present in LN but were rendered anerglc. Tolerancein FLT mice seems to be regulated by a peripheral mechanism.We supposed that the split tolerance in FLT mice was inducedby the different antigenicity between the spleen and LN.     

Effect of alkyl groups on the rate constants of propagation and termination in the radical polymerization of dialkyl itaconates

Polymerization of dialkyl itaconates with dimethyl azoisobutyrate ( 5 ) was studied in benzene at 50°C by means of electron spin resonance (ESR). The monomers used are dimethyl ( 1 ), diethyl ( 2 ), dibutyl ( 3 ) and di-2-ethylhexyl ( 4 ) itaconates. All the polymerization systems involve ESR-observable propagating polymer radicals under the actual polymerization conditions. The polymerization rate (R_p) and degree of polymerization of the resulting polymer increase in going from shorter to longer alkyl groups. The ESR-determined rate constants of propagation (k_p) and termination (k_t) decrease as the alkyl chain becomes longer. k_p of 1 is 3,3 times higher than that of 4 , while k_t of 1 is 590 times higher than that of 4 . Thus, the steric effect due to the alkyl group suppresses much more termination than propagation, leading to the fact that R_p increases as the alkyl group becomes larger.