[Retrospective analysis of the relationship between HUS incidence and antibiotics among patients with Escherichia coli O157 enterocolitis in the Sakai outbreak].

An outbreak of Escherichia coli O157:H7 infection occurred in July 1996 in Sakai City. About 5000 children were infected, 122 of whom developed hemolytic uremic syndrome (HUS). In this outbreak, almost all patients were administrated some type of antibiotics. The effects of antibiotics on E. coli O157 associated hemorrhagic colitis (HC) have been controversial. In this study, we focused on the effects of antibiotics on development of HUS in the HUS in the Sakai outbreak. We retrospectively determined the antibiotics administrated within three days after the onset of HC, clinical courses, and laboratory data of 301 patients who were hospitalized and identified as Escherichia coli O157 infection by stool culture, from results of questionnaires sent by the Osaka Prefecture Medical Association to hospitals in Osaka Prefecture. The antibiotics used could be identified for 216 patients. The incidence of HUS among these patients was 11.6%. They were divided into 19 groups based on the type of antibiotics administrated. The incidence of HUS in the new quinolone (3.7%) group was low, but was high in the intravenous cephalosporin (18.2%) group. The differences in the incidence of HUS among the 19 antibiotic groups was significant (p < 0.05) on analysis of covariance which eliminated the contributions of variables including age, sex and laboratory data. These findings indicate that the suitable antibiotics can prevent the development of E. coli O157-associated HUS.

Structure and expression of a cyst specific protein of Acanthamoeba castellanii.

The life cycle of Acanthamoeba is divided into a growth-division phase and two distinctive processes of cellular differentiation, termed encystment and excystment. Polyacrylamide gel electrophoresis revealed that a specific protein of 21 kDa in molecular weight occurs in the cyst, but not in the trophozoite stages of A. castellanii Neff strain. This cyst-specific protein, designated as CSP21, was purified from guanidine-HCl extract of cyst wall and anti-CSP21 antibody was produced. Immunoblotting of proteins extracted from a variety of species of Acanthamoeba genus suggested that the antibody is specific for group II amoebae, therefore, providing a useful tool for Acanthamoebae taxonomy. A cDNA clone for A. castellanii CSP21 was isolated by immunoscreening of a cDNA expression library constructed from mRNA of amoebae at encysting stage. The deduced primary structure indicated that CSP21 is a hydrophilic protein showing no significant homology with peptides thus far published. RNA blot analysis showed that the expression of CSP21 mRNA was restricted within early stages of encystment, suggesting that the biosynthesis of CSP21 is regulated at mRNA level.

Chromosome size polymorphisms in the genus acanthamoeba electrokaryotype by pulsed-field gel electrophoresis.

Twenty-eight strains from 12 species from the genus Acanthamoeba, including five isolates from amoebic keratitis patients, were subjected to molecular karyotyping by pulsed-field gel electrophoresis. 9 to 21 chromosome-sized DNA bands ranging from 200 kb to 3 Mb in size were detected. Molecular karyotypes also showed a wide multifariousness, i.e. there existed inter- and intraspecific heterogeneity. The five isolates from amoebic keratitis patients did not exhibit characteristic molecular karyotypes distinguishable from environmental isolates. Although karyotypic heterogeneity was observed within group I amoeba, they are distinguishable from those of group II and III. Strains having identical restriction fragment length polymorphism profiles of mtDNA did not have an identical molecular karyotype, i.e. weak correlation was found between molecular karyotypes and mtDNA restriction fragment length polymorphism profiles.

Suppression of delayed-type hypersensitivity and IgE antibody responses to ovalbumin by intranasal administration of Escherichia coli heat-labile enterotoxin B subunit-conjugated ovalbumin.

Oral administration of a small amount of antigen conjugated cholera toxin B subunit is known to induce tolerance to the antigen. In the present experiments, whether nasal administration of allergen conjugated to Escherichia coli heat-labile toxin B subunit (LTB) induced tolerance was examined in BALB/c mice. A single administration of a small amount of LTB-coupled ovalbumin (OVA) suppressed the induction of delayed-type hypersensitivity and IgE antibody responses to OVA which was administered parenterally after nasal administration of LTB-coupled OVA. The antigen-specific suppression was abrogated by the addition of the holotoxin to LTB-coupled OVA. The suppression, induced by nasal administration with a small amount of allergen conjugated to a mucosa-binding molecule, may be applicable for preventing the development of allergy.

Synchronized endocytosis studied in the oocyte of a temperature-sensitive mutant of Drosophila melanogaster.

This study demonstrates that endocytosis in the oocyte of Drosophila melanogaster is reversibly blocked at the stage of pit formation by the temperature-sensitive, single-gene mutant, shibirts1. Uptake of horeradish peroxidase conjugated with wheat-germ agglutinin was observed to be normal in mutant oocytes at 19 degrees C, but was blocked at 29 degrees C. After 10 min at 29 degrees C, there was a build-up of coated pits along invaginations of the plasma membrane. Also, the endosomal compartment consisting of tubules, bulbs, and small yolk spheres, disappeared. Lowering the temperature to 19 degrees C after 10 min at 29 degrees C released a synchronized wave of endocytosis into a cytoplasm cleared of uptake-related organelles. By observing this synchronized wave after exposure to 19 degrees C for varying durations, we determined that endocytosis proceeds as follows: coated pits/vesicles----tubules----small yolk spheres----mature yolk spheres. The observations suggest that these organelles transform one into another within this sequence.

Membrane pinch-off and reinsertion observed in living cells of Drosophila.

The garland cell of Drosophila is a nephrocyte which takes up waste products from the haemolymph. Endocytosis is thought to occur by the pinch-off of coated vesicles from deep invaginations of the plasma membrane called labyrinthine channels. Electron microscopic studies show that the length of these channels is variable, depending on the relative rates of membrane pinch-off and reinsertion (recycling). Thus, in wild-type garland cells, if the temperature is raised from 19 degrees C to 30 degrees C, the channels shorten, because at high temperature the pinch-off rate exceeds the reinsertion rate. On the other hand, in garland cells of the temperature-sensitive, single-gene mutant shibirets1 (shi), in which endocytosis is reversibly blocked at the pinch-off stage at 30 degrees C, the labyrinthine channels elongate considerably, as membrane insertion proceeds while pinch-off is blocked. The rates of membrane pinch-off and insertion were quantitated in living garland cells by observing the changes in the capacitance of the whole cell membrane which occur as a result of changes in the total area of the plasma membrane. In wild-type cells, the capacitance gradually decreased as the temperature was raised to 30 degrees C, reflecting the shortening of the channels. In shi cells, the capacitance decreased between 19 degrees C and 26 degrees C but then began to increase at higher temperatures as the blockage of endocytosis caused by the shi gene took effect, causing the channels to elongate. The observations suggest that in shi cells the surface area of the cell more than doubles in 12 min by channel elongation. Estimates of the amount of membrane which is pinched off and reinserted were made.

[Enzyme immunoassay of 17-hydroxyprogesterone in dried blood spot on filter paper using specific antibody for 17-hydroxyprogesterone].

Specific antiserum for 17-hydroxyprogesterone (17-OH-P) was prepared by immunizing 7 alpha-(2-carboxyethylthio)-17-OH-P conjugated bovine serum albumin (BSA) in rabbits. Using this antiserum, 17-OH-P enzyme immunoassay for dried blood spots on filter paper was established. As a label, alkaline phosphatase was coupled covalently with 7 alpha-carboxy-methylthio-17-OH-P by carbodiimide method. B/F separation was carried out by the addition of anti-rabbit IgG goat antiserum. All specimens used were punched out with a paper puncher of 3mm diameter. The assay sensitivity was 2pg/tube, which was estimated by two standard deviation at zero concentrations of the calibration curve. Cross reactivities of this antibody were as follows: 11-deoxycortisol (8.21%), 17-OH-pregnenolone (3.33%), progesterone (1.67%), 11-deoxycorticosterone (0.31%), cortisol (0.16%), pregnenolone-3-sulfate Na salt (0.03%), dehydroepiandrosterone (DHEA) (less than 0.03%), 16 alpha-OH-DHEA (less than 0.03%), DHEA-3-glucuronide (less than 0.03%), DHEA-3-sulfate Na salt (less than 0.03%), pregnenolone (less than 0.02%). Intra- and inter-assay coefficient of variations were 4-14% and 9-18%, respectively. In normal babies, 17-OH-P concentrations measured directly (without sample extraction) were below 23pg/disk (n=204). The histogram of 17-OH-P level in normal babies obtained by the direct method was distributed lower than that obtained by the enzyme immunoassay system (range: 4-79pg/disk, n=268) which used antibody raised against 17-OH-P-3-O-carboxymethyloxime conjugated BSA (Enzaplate, Sapporo Diagnostic Laboratory).(ABSTRACT TRUNCATED AT 250 WORDS)

Serodiagnosis of scrub typhus with antigens immobilized on nitrocellulose sheet.

This study was designed to develop a simple method for serodiagnosis of scrub typhus. The basis of the method is detection of anti-Rickettsia tsutsugamushi antibody in patient serum by reaction with antigens dot blotted on a nitrocellulose sheet (NCS). The final evaluation of the reaction is performed by observing the color intensity which develops as a result of sequential treatments of the NCS with peroxidase-conjugated anti-human immunoglobulin G or immunoglobulin M antibody and with the substrate of the enzyme. After various trials, we found that the best results were obtained by using a purified antigen which adhered to an NCS at 0.2 to 2 micrograms of protein per dot and a test serum diluted 1,000- to 4,000-fold. Under these conditions, almost all antibody-positive sera showed a distinct color at the dot on the NCS, so that a positive reaction could be distinguished by the naked eye from a negative reaction with antibody-negative sera, which developed only a faint color. A comparison of the results of screening of antibody-positive and -negative sera by this method and the immunofluorescence test showed that both methods produced similar results. From these results, it is concluded that this dot immunoassay can be useful for the serodiagnosis of scrub typhus.

Characterization of polypeptides in Rickettsia tsutsugamushi: effect of preparative conditions on migration of polypeptides in polyacrylamide gel electrophoresis.

The polypeptide compositions and antigenic components of Rickettsia tsutsugamushi were analyzed by modifying the solubilization conditions prior to polyacrylamide gel electrophoresis and by using monoclonal antibodies in immunoblotting experiments. Several polypeptides were converted to larger or smaller molecules by using various conditions for rickettsial sample preparation. Solubilization of a sample in 2-mercaptoethanol-containing buffer resulted in conversion of high-molecular-weight polypeptides to smaller polypeptides and conversion of some of the 43-kilodalton (43K) polypeptide to a 46K polypeptide. The heat modifiability of selected polypeptides was shown by heating samples at 100 degrees C. A major polypeptide on the rickettsial surface which showed strain-specific antigenicity appeared at the 43K position in samples solubilized at 37 degrees C but moved to the 56K position after samples were heated at 100 degrees C. Immunoblotting with an anti-56K polypeptide monoclonal antibody demonstrated that the reactive antigens existed predominantly as the higher-molecular-weight polypeptides. These polypeptides were converted to 43K polypeptides at 37 degrees C or the 56K polypeptides at 100 degrees C by cleavage of disulfide linkages with 2-mercaptoethanol treatment.

N tau-Ribosylhistidine, a novel histidine derivative in urine of histidinemic patients. Isolation, structure, and tissue level.

On amino acid analysis of urine of histidinemic patients, an unidentified compound was eluted in a position between beta-aminoisobutyric acid and gamma-aminobutyric acid. This compound was purified to homogeneity from the urine by a combination of extraction with 80% ethanol, repeated column chromatography on Bio-Rad AG-50, and high performance liquid chromatography on a strongly cationic ion exchanger. The compound yielded free histidine on hydrolysis in an evacuated sealed tube with 0.1-6.0 M HCl at 145 degrees C for 5 h, but not at 100 degrees C for 24 h. This compound was determined to be N tau-ribosylhistidine by 1H and 13C NMR spectroscopies. The urinary content of this material in normal and histidinemic children was 17.8 +/- 13.4 (n = 10) and 126 +/- 51 (n = 14) mumol/g creatinine (mean +/- S.D.), respectively, and were closely correlated with those of urinary histidine. The renal clearance value of N tau-ribosylhistidine in humans was 96% of that of creatinine. When rats were fed on diets rich in histidine, the urinary excretion of N tau-ribosylhistidine increased greatly and was well correlated with the intake of histidine.

Semiautomated enzyme immunoassay of thyrotropin as a mass screening test for neonatal hypothyroidism.

A sensitive, simple, and rapid semiautomated sandwich enzyme immunoassay (EIA) was developed for measuring thyrotropin in dried blood samples on filter paper for use in screening for neonatal hypothyroidism. Good correlation was found between values for thyrotropin determined by this method and those determined by radioimmunoassay (RIA) (r=0.94). In pilot tests on 17,160 newborn infants in the general population, five cases of primary hypothyroidism were detected by both EIA and RIA. The recall rate was slightly highter in EIA than in RIA.

Histamine metabolism in patients with histidinemia: determination of urinary levels of histamine, N tau-methylhistamine, imidazole acetic acid, and its conjugate(s).

Histamine metabolism in histidinemic patients was studied by measuring the urinary levels of histamine and its metabolites. The urinary excretions of histamine, N tau-methylhistamine, imidazole acetic acid, and its conjugate(s) were higher in patients with histidinemia than in controls, and these levels of excretion were correlated with the plasma histidine level. The urinary histamine levels of patients with eczema-like dermatitis were twice that of those without dermatitis. The urinary excretion of 3-methylhistidine showed a close correlation with the urinary histidine excretion. Thus, it was concluded that histamine metabolism is higher in histidinemic patients than in normal controls.

Location of DNA-binding proteins and disulfide-linked proteins in vaccinia virus structural elements.

Treatment with sodium dodecyl sulfate (SDS) converted the vaccinia virus strain IHD-J into particles of two types: (i) ghosts which possessed a thin-membrane vesicle derived from basement part of the virus membrane with attached lateral bodies and a membranous structure derived from the core wall and (ii) aggregates of a DNA-nucleoprotein eluted from the core. These particles lacked lipids, and all the viral phospholipids were detected in the SDS-soluble fraction. The viral membrane was composed of an SDS-soluble coat layer and the basement membrane, and the basement membrane was maintained by a mechanism other than the lipid bilayer. By comparisons of protein species in morphologically distinct subviral particles prepared by several solubilizing methods, protein compositions of viral structural elements were suggested as follows: 25,000-molecular-weight viral protein-17,000-molecular-weight viral protein ( VP25K - VP17K ), viral basement membrane; VP13 . 8K , major component of the lateral body; VP70K , VP69K , VP66K , and VP64K , minor components of the lateral body; VP61K , outer layer of core wall; VP57K - VP22K , inner layer of core wall; and VP27K - VP13K , nucleoprotein. These structural elements found in the SDS-insoluble particles dissolved in the same SDS solution under reducing conditions, indicating that the disulfide linkages seem to have a principal role in maintaining their morphological integrity. VP57K , VP27K , VP13 . 8K , and VP13K were revealed to possess affinity for DNA. Denatured calf thymus DNA and viral DNA in double- or single-stranded form associated equally well with these proteins, but RNA did not bind. Therefore, it was strongly suggested that disulfide-linked VP27K - VP13K represented the nucleoproteins of vaccinia virus. A structural model of vaccinia virus is proposed and discussed.

HLA-A and -B antigens in Japanese patients with congenital hypothyroidism and their parents.

The frequency of HLA-A and B antigens were studied in 32 Japanese patients with congenital hypothyroidism due to thyroid dysgenesis, and in their parents. The incidence of the Aw24 antigen was significantly higher in 27 mothers of patients with ectopic thyroid (91.3%, corrected P less than 0.037) and it seemed to be slightly higher in patients (77.7%) than in controls (56.8%). The Aw24 antigen was also found in 4 patients with thyroid hypoplasia and their mothers. No difference was found in the incidences of the antigen in the fathers of patients and in controls. The haplotype frequencies were not significantly different in controls, patients and their parents. These findings suggest that the gene for susceptibility to congenital hypothyroidism due to thyroid dysgenesis is closely linked to the gene for the HLA-A locus of the patients' mothers.

Isolation of Rickettsia tsutsugamushi antigenically different from Kato, Karp, and Gilliam strains from patients.

Rickettsia tsutsugamushi strains from three recent patients of Tsutsugamushi disease in Niigata Prefecture were isolated primarily in mice and then in L cell cultures. By this procedure, low virulent strains to mice, as well as high virulent ones, could be isolated and cultivated serially in L cell cultures, suggesting the usefulness of L cells for isolation of this species of rickettsia. Each newly isolated strain was identified as a member of R. tsutsugamushi from the results of cross immunological tests and morphological observation. On the other hand, it was recognized that one of these rickettsiae showed immunological properties distinguishable from the prototype strains of Kato, Karp, and Gilliam by the cross complement fixation test, and also had low virulence in mice.

Electron microscopic studies on intracellular multiplication of Rickettsia tsutsugamushi in L cells.

The mechanism and kinetics of intracellular growth of Rickettsia tsutsugamushi were investigated by electron microscopic observations, parallel with quantitative analysis by counting the rickettsiae seen in electron micrographs and by plaque assay for infectivity of the culture. The observations demonstrated the existence of electron-less dense and -dense types of rickettsiae in the early stage of infection, binary fission and the process of release of the microorganisms in the host cell cytoplasm and from the cell surface, formation of abnormally long rickettsiae, and the process of lysis of the host cell in the later stage of infection with vacuole formation between the inner and outer leaflets of the host cell nuclear membrane. Separate titrations of infectivity of the cells and the culture fluid showed a very slow increase in infectivity in the culture fluid compared with the intracellular titer, suggesting that the progeny rickettsiae stay in the cell or at the cell surface for a relatively long period. Doubling time of the rickettsia was found to be about 9 hr.

Influence of pimozide on hypothalamo-pituitary function in children with behavioral disorders.

Hypothalamo-pituitary functions were examined in thirteen children with behavioral disorders (six with hyperkinesia, four with autism, two with tic and one with schizophrenia) before and during treatment with pimozide, an antidopaminergic drug. The mean (+/- S.E.M.) basal serum PRL level (24.5 +/- 4.2 ng/ml) during pimozide treatment was significantly higher than that (12.4 +/- 3.2 ng/ml) before treatment. Hyperresponse of PRL to TSH releasing hormone (TRH) was observed in five (three with hyperkinesia, one with tic and one with autism) of the thirteen patients before treatment and in seven (four with hyperkinesia, two with autism and one with tic) during treatment. Mean TSH response during treatment was not significantly different from that before treatment. However, three of the four autistic children showed hyperresponse of TSH to TRH before treatment, whereas only one also showed a hyperresponse during treatment. The pimozide treatment had no demonstrable influence on GH or cortisol secretion in response to insulin-induced hypoglycemia, or on serum T4 and T3 levels.