Hajime Nishiya

Effect of particle size on alumina-induced production of reactive oxygen metabolites by human leukocytes

To assess the role of crystal size in the biologic response, we quantitated the generation of reactive oxygen metabolites in human polymorphonuclear leukocytes (PMNs) or mononuclear leukocytes (MNs) with a chemiluminescence assay using 6 alpha-alumina preparations with average diameters of 0.6, 0.8, 3.2, 7.5, 28 and 68 microns on the basis of equal weights (1 mg/ml), and also on the basis of surface areas (100 cm2/ml). On an equal weight alumina particles induced PMN chemiluminescence in the following order of magnitude: 3.2 microns > 7.5 microns, 0.8 micron > 0.6 micron > 28 microns, 68 microns. The particulates induced MN chemiluminescence: 3.2 microns > 0.8 micron > 0.6 micron > 7.5 microns > 28 microns, 68 microns. On the basis of identical surface areas, alumina elicited PMN chemiluminescence: 3.2 microns > 7.5 microns, 0.8 micron > 0.6 micron, 28 microns, 68 microns. The particulates induced MN chemiluminescence: 3.2 microns > 0.8 micron > 0.6 micron, 7.5 microns > 28 microns, 68 microns. The maximal effect of particle size on the alumina-induced production of reactive oxygen metabolites by human leukocytes was seen at around 3 microns.

Polymerase chain reaction assay for specific identification of Candida guilliermondii (Pichia guilliermondii)

The incidence rates of fungemia caused by Candida guilliermondii have been increasing over the past several years. Although still relatively rare (1.3% of all cases of fungemia in Japan), most cases of C. guilliermondii fungemia occur in patients with cancer or hematological malignancy and their mortality rate is high. As C. guilliermondii tends to be resistant to various antifungal agents, early identification of this pathogen and treatment with an appropriate antifungal agent are required to improve survival rates in these patients. However, it is extremely difficult to differentiate C. guilliermondii (Pichia guilliermondii) from members of the C. famata complex. To date, identification based on DNA sequencing has been the only reliable method for the identification of fungal groups. Here, we used a polymerase chain reaction (PCR)-based method that we developed for the simple and reliable identification of C. guilliermondii (P. guilliermondii). A pair of specific primers was designed corresponding to the 18S rDNA sequence. The PCR system was applied to isolates from fungemia patients. These yeasts could not be identified with CHROMagar Candida, but were successfully identified using this PCR-based system.

Exotic myiasis caused by 19 larvae of Cordylobia anthropophaga in Namibia and identified using molecular methods in Japan

A case of exotic myiasis caused by tumbu fly (Cordylobia anthropophaga) parasitism acquired while travelling in the Republic of Namibia is reported. This is the fifth case reported in Japan, and is very unusual in that the patient was infected with 19 larvae. This is also the first case diagnosed using molecular methods in Japan. We cultured the extracted larvae in vitro and successfully obtained pupae.

真菌 (ムーコル) 性脳動脈瘤の1剖検例

An autopsy of rupture of Mucor cerebral aneurysm, not diagnosed during the patients life, was experienced. A 63-year-old female was admitted to our hospital with the chief complaint of disturbance of consciousness and high fever. Her past histories were diabetes mellitus, liver cirrhosis and nasal sinusitis. The remarkable findings on admission were moderate inflammatory data, high blood sugar level in serum and ascites. Brain CT film revealed a non-enhanced low-density area in the frontal region. The cerebrospinal fluid showed bloody color and white blood cell counts were 3300/microliter (mostly neutrophils). Under our suspected of bacterial encephalomeningitis, intravenous cefotaxime and ampicillin therapy was started immediately. Cultures of cerebrospinal fluid for bacteria were negative. The disturbance of her consciousness gradually improved under general treatment. However, her conscious level suddenly became a coma on the 6th hospital day and she died on the 9th hospital day. An autopsy revealed Mucor at the site of the rupture of the cerebral aneurysm.

Priming effect of hydroxyapatite on the chemiluminescence response in human polymorphonuclear leukocytes

In order to determine whether hydroxyapatite modulates the response of polymorphonuclear leukocyte (PMN) to oxidative stimuli, human PMNs were incubated with a non‐activating concentration (1 or 10 μg/ml) of hydroxyapatite prior to stimulation with N‐formyl‐methionyl‐leucyl‐phenylalanine (FMLP; 0.1 or 1 μM), phorbol 12‐myristate 13‐acetate (PMA; 100 pg/ml), sodium fluoride (50 μM), zymosan (1 μg/ml), or the calcium ionophore A23187 (0.1 μM). Chemiluminescence was measured with an automatic microcomputer‐controlled luminescence analyzer at 37°C. Hydroxyapatite alone did not stimulate chemiluminescence at concentrations below 10 μg/ml. Levels 300–400% higher than ‘stimulus only’ controls without preincubation with hydroxyapatite have been recorded. This synergism between hydroxyapatite and subsequent stimuli reveals a new activity of hydroxyapatite and suggests that particulate material may prepare PMNs for an exaggerated inflammatory response to other phlogistic mediators. This is the first report demonstrating PMNs primed with particulate material.

Effects of 2(3)-Tert-Butyl-4-Hydroxyanisole Pretreatment on Cefpiramide Binding to Mouse Glutathione S-Transferases

Binding of cefpiramide (CPM) and other beta-lactam antimicrobial agents to 2(3)-tert-butyl-4-hydroxyanisole (BHA)-induced liver glutathione (GSH) S-transferases (EC 2.5.1.18) from CD-1 mice was studied. A marked induction of hepatic GSH S-transferase from mice fed BHA was observed. Gel chromatography of liver cytosol from mice fed BHA showed an increased binding of CPM, cefotetan and cefazolin to BHA-induced GSH S-transferases. The extent of their binding to GSH S-transferase seemed to be correlated with the extent of their excretion into the bile. Binding of CPM to the GSH S-transferase fraction was inhibited by both indocyanine green, which is known to bind liver GSH S-transferases intensively, and by cefoperazon, which is mainly excreted into the bile. This study suggests that GSH S-transferases are the main binding proteins of CPM in the liver cytosol fraction and play an important role as carrier proteins of CPM and some antimicrobial agents in mouse liver.

A case study of measles vaccination for university students during the measles outbreak in Tokyo, Japan, 2007

In April 2007, seven students belonging to the same class at Teikyo University developed measles. To prevent the spread of infection, 27 of 106 students in the same class who had low anti-measles antibody titers as measured by hemagglutination inhibition (HI) assay were vaccinated. After the outbreak had subsided, the HI values were investigated in 103 students, and they answered questionnaires about their health condition during the period of the outbreak and their previous clinical histories of measles, including vaccination records. There was no new case of measles after introduction of the vaccination program. However, the HI titers of 42% of the students who were not vaccinated in this program were significantly elevated. Fever and catarrhal signs occurred in 7 of these students with pre-exposure titers of 8 or less. The post-exposure HI titers of 71% of students who were unaffected by measles and had high HI titers (>8) before the epidemic did not increase. These results suggested that people with low HI titers may become potential carriers of measles and that measurement of pre-exposure HI anti-measles antibody titer is a useful method for selection of candidates to undergo vaccination.

The usefulness of changing focus during examination using Gram staining as initial diagnostic clue for infective tuberculosis

Gram staining is a useful technique for detecting bacteria but is highly questionable in detecting Mycobacterium tuberculosis. Its detection generally requires special staining, such as Ziehl–Neelsen staining. We experienced three cases in which tuberculosis was first suggested by Gram staining of sputum or pus, confirmed by Ziehl–Neelsen staining, and diagnosed by polymerase chain reaction or culture. To find colorless tubercle bacilli in clinical samples with various organisms, varying the focus to slightly longer and shorter during study of the slides is indispensable. We present criteria for detecting infective pulmonary tuberculosis in Gram staining. First, in the ordinary focus, weakly stained, thin, gram-positive bacilli are found; second, with a slightly longer focus distance, the thin, cord-like, conspicuous gram-positive bacilli can be observed; and third, with a shorter focus distance, the gram-positive bacilli have changed into the brightened, colorless, or ghost ones. Four laboratory technologists each evaluated 20 Gram-stained samples after being lectured on the criteria, with no prior information about the sample. They accurately evaluated the presence of the bacilli in Gram-stained preparations in more than 90% of samples containing 3+ bacilli on Ziehl–Neelsen staining. Gram staining is available as an easy and rapid initial clue to recognize highly infective tuberculosis.

Comparative study of the effects of cefodizime and HBW 538 in potentiating the production of reactive oxygen species by human polymorphonuclear leukocytes.

The immunomodulatory activity of cefodizime (CDZM), an aminothiazolylcephalosporin, was compared to that of HBW 538, a derivative of the CDZM side chain at position 3 (the mercaptothiazolyl group) in respect to the production of reactive oxygen species (ROS) by human whole blood and polymorphonuclear leukocytes (PMN) in vitro. Ten-fold diluted whole blood and PMN from healthy individuals were incubated with CDZM or HBW 538 alone at the concentrations of 1, 10, or 100 micrograms/ml, or CDZM or HBW 538 at 100 micrograms/ml in combination with tumor necrosis factor-alpha (TNF-alpha) at 100 U/ml or lipopolysaccharide (LPS) at 1 microgram/ml. The production of ROS was measured by a chemiluminescence (CL) assay in which luminol was added to a mixture and after which the PMN or whole blood were stimulated with nonopsonized zymosan or phorbol myristate acetate. The following results were obtained: (1) The CL responses of whole blood and PMN were slightly but not significantly enhanced by CDZM at 100 micrograms/ml, whereas both CL responses were significantly enhanced by exposure to HBW 538 at 10 and 100 micrograms/ml. (2) The enhanced PMN CL response which followed priming with TNF-alpha or LPS was not augmented by CDZM but was significantly augmented by HBW 538. These results indicate that the ability of the HBW 538 molecule to enhance the production of ROS by stimulated PMN and to act agonistically with TNF-alpha or LPS is abrogated when HBW 538 is part of the CDZM molecule.(ABSTRACT TRUNCATED AT 250 WORDS)

Three Cases of Mycobacterium tuberculosis Infection Initially Recognized by Focus Changing Examination in Gram Staining

If tubercle bacilli could be easily detected in clinical samples with gram staining, it would be possible to detect tuberculosis more promptly and easily, which may contribute to tuberculosis control. In this paper, we present three infective tuberculosis cases in which gram staining easily detected tubercle bacilli before Ziehl-Neelsen (Z-N) staining and diagnosed by polymerase chain reaction (PCR) or culture.