Genotypes and infection sites in an outbreak of multidrug-resistant Pseudomonas aeruginosa.

An outbreak of multidrug-resistant (MDR) Pseudomonas aeruginosa occurred in an acute care hospital in Japan, which lasted for more than three years. During January 2006 to June 2009, 59 hospitalised patients with MDR P. aeruginosa were mainly detected by urine culture in the first half, whereas isolation from respiratory tract samples became dominant in the latter half of the outbreak. Non-duplicate MDR P. aeruginosa isolates were available from 51 patients and all isolates were positive for bla(VIM-2). Pulsed-field gel electrophoresis (PFGE) analysis categorised the isolates into three major clusters; types A, B and C with eight, 19 and 21 isolates, respectively. The outbreak started with patients harbouring PFGE type A strains, followed by type B, and type C strains. Multivariate analysis demonstrated that patients with PFGE type C strains were more likely to be detected by respiratory tract samples (odds ratio: 11.87; 95% confidence interval: 1.21-116.86). Improved aseptic urethral catheter care controlled PFGE type A and type B strains and improvement in respiratory care procedures finally contained the transmission of PFGE type C strains.

Quality control of diphtheria tetanus acellular pertussis combined (DTaP) vaccines in Japan.

Diphtheria tetanus acellular pertussis combined (DTaP) vaccines have been successfully used in Japan by controlling their potencies and toxicities with animal models. In accordance with the recent practical introduction of DTaP vaccines of various formulations, a question has been raised in other nations as to the efficacy of a quality control system based on animal tests and standard preparations. The World Health Organization issued its guidelines on the production and quality control of acellular pertussis vaccines in 1998 along with the concept of quality control by ensuring that production lots were consistent with clinical trial lots, rather than by comparing them with standard preparations in traditional laboratory tests. However, because it is not feasible to evaluate the combined use of vaccines from different manufacturers in a clinical study, the alternative trend of quality control may give rise to a difficulty in rationalizing the practical immunizations to use vaccines of different brands in a mixed consequence. A standardized national regulation system to ensure the equivalence of approved products would be essential for such an immunization practice. The success of the Japanese DTaP vaccination suggests the possibility of an effective quality control of DTaP vaccines by means of standardized test systems.

Identification of cardiac abnormal structures with harmonic power Doppler contrast echocardiography.

We describe two cases in which echocardiographic image enhancement with an intravenous contrast agent using harmonic power Doppler (HPD) imaging established the diagnosis of abnormal structures in the left ventricle (LV).

Analysis of Bordetella pertussis isolates collected in Japan before and after introduction of acellular pertussis vaccines.

Because of recent concern that whole-cell pertussis vaccination can drive antigenic divergence of circulating isolates of Bordetella pertussis, we compared 12 clinical isolates of B. pertussis collected in Japan, the first country to introduce acellular pertussis vaccines, with the vaccine strain. We used pulsed-field gel electrophoresis, sequencing of ptx and prn genes and expression of fimbriae. Most of the isolates collected before or after introduction of acellular vaccine possess similar restriction patterns. They contain ptx genes and prn alleles similar to the vaccine strain and to European isolates collected before the introduction of vaccination. Two recently collected isolates exhibiting a different pulsed-field gel electrophoresis pattern possess ptxS1 and prn alleles similar to the alleles harbored by European isolates circulating currently. Our preliminary results suggest that, if acellular pertussis vaccine-induced antigenic divergence exists, it is likely to be a slow or rare process.

Use of antiserum-coated latex particles for serotyping Streptococcus pneumoniae.

The Quellung reaction provides a standard means for serotyping Streptococcus pneumoniae, but it requires microscopic examination with skillful technique. We have developed an improved agglutination method with anti-rabbit IgG-coated latex particles, which are sensitized with pooled antisera for serotyping/serogrouping S. pneumoniae. Our method is as specific and sensitive as the Quellung test, and much easier to perform.

Effects of a dual L/N-type Ca(2+) channel blocker cilnidipine on neurally mediated chronotropic response in anesthetized dogs.

We investigated the effects of an L-type and N-type Ca(2+) channel blocker, cilnidipine, on neurally mediated chronotropic responses to clarify the anti-autonomic profile of cilnidipine in anesthetized dogs. Pretreatment with cilnidipine (0.3, 1.0 and 3.0 microg/kg, i.v.), which decreased mean blood pressure by 5 to 31 mm Hg, inhibited the changes in heart rate and plasma norepinephrine concentration induced by bilateral carotid artery occlusion, whereas it had no effect on vagal nerve stimulation-induced bradycardia. These results suggest that antihypertensive and antisympathetic doses of cilnidipine fail to influence chronotropic responses mediated by parasympathetic nerve activation in the in vivo canine heart.

Stimulation of Bordetella pertussis adenylate cyclase toxin intoxication by its hemolysin domain.

The internalization of the N-terminal catalytic domain of Bordetella pertussis adenylate cyclase toxin (ACT) across the cytoplasmic membrane has been considered to occur independently from protein-protein interactions which can lead to oligomerization required for hemolytic activity by its C-terminal hemolysin domain. Here we report that when added in excess, this hemolysin domain stimulates the internalization, suggesting the involvement of protein-protein interactions in cell-invasive activity of ACT, as well as its hemolytic activity.

Selectivity of dihydropyridines for cardiac L-type and sympathetic N-type Ca2+ channels.

The blocking effects of cilnidipine and other dihydropyridines on L-type cardiac Ca2+ channels (I(Ca,L)) and N-type sympathetic Ca2+ channel currents (I(Ca,N)) were studied using a whole-cell patch-clamp technique. At -80 mV, cilnidipine had little inhibitory effect below concentrations of 1 microM on I(Ca,L) (IC50 value; 17 microM). However, 1 microM cilnidipine strongly shifted the steady-state inactivation curve of I(Ca,L) toward negative potentials without changing the current-voltage relationship. Each action of cilnidipine was characterized by a high affinity for the inactivated channel in preference to the resting channel. The IC50 values of dihydropyridines for I(Ca,L) were in the range between 0.01 and 10 microM, and those for I(Ca,N) were between 3 and 30 microM. Cilnidipine had the strongest affinity for I(Ca,N) among the dihydropyridines tested. These results suggest that cilnidipine did not cause hypotension-evoked tachycardia deficiency by depression of cardiac L-type channels but by sympathetic N-type channels blockade.

Suppression of platelet aggregation by Bordetella pertussis adenylate cyclase toxin.

The effect of Bordetella pertussis adenylate cyclase toxin (ACT) on platelet aggregation was investigated. This cell-invasive adenylate cyclase completely suppressed ADP (10 microM)-induced aggregation of rabbit platelets at 3 micrograms/ml and strongly suppressed thrombin (0. 2 U/ml)-induced aggregation at 10 micrograms/ml. The suppression was accompanied by marked increase in platelet intracellular cyclic AMP (cAMP) content and was diminished by the anti-ACT monoclonal antibody B7E11. A catalytically inactive point mutant of ACT did not show the suppressive effect. Since an increase of cAMP content is a known cause of platelet dysfunction, these results indicate that the observed platelet inactivation was due to the catalytic activity of ACT through increase of intracellular cAMP.

Serotyping of Streptococcus pyogenes isolated from common and severe invasive infections in Japan, 1990-5: implication of the T3 serotype strain-expansion in TSLS. The Working Group for Group A Streptococci in Japan.

To clarify the relationship between the epidemics of severe invasive group A streptococcal infections (streptococcal Toxic Shock-Like Syndrome: TSLS) and common group A streptococcal infections in Japan, we examined the T serotypes of S. pyogenes strains (group A streptococci) isolated from clinical specimens of the streptococcal infections (17999 cases) in the period 1990-5, including the severe infections (TSLS) (29 cases) in the period 1992-5. Characteristic points of the analyses were: (1) dominant serotypes of the infections in these periods were T12, T4, T1, T28 and TB3264, which were consistently isolated; (2) isolates of T3 rapidly increased through 1990 to 1994 while T6 decreased in the period 1990-3; (3) when Japanese area was divided into three parts, T3 serotype tended to spread out from the north-eastern to the south-western area; (4) strains of T3 and T1 serotypes were dominant in the TSLS. Dominant-serotype strains of streptococcal infections did not always induce severe infections and dominance of T3 serotype in the TSLS seemed to be correlated with the increase of T3 in streptococcal infections. These results may indicate that certain clones of S. pyogenes are involved in the pathogenesis of the TSLS.

IFN-gamma-mediated protection against intracerebral challenge with Bordetella pertussis in mice.

Mice inoculated with whole cell pertussis vaccine (WCV) acquired protection against intracerebral challenge with Bordetella pertussis without any appreciable antibody production against pertussis toxin or filamentous hemagglutinin. Spleen cells from mice immunized with WCV produced a significant amount of IFN-gamma upon stimulation in vitro with WCV. Furthermore, mice inoculated with recombinant IFN-gamma along with a suboptimal dose of WCV survived longer than those that received WCV alone. These results suggest that, in WCV-immune mice, IFN-gamma plays an important role in protection against intracerebral challenge with B. pertussis.

Hemodialysis effect on serum boron level in the patients with long term hemodialysis.

Serum and dialysate boron levels in 17 patients with long term hemodialysis (HD) were determined by inductively coupled plasma emission spectrometry (ICPES). Serum boron level was compared with the value of age matched 467 healthy controls and the relationship between serum and dialysate boron level was analyzed. The results showed that serum boron level was significantly higher at the beginning of HD, and lower at the completion of HD in comparison with controls. Although the dialysate was contaminated with trace boron, HD resulted in an excessive decrease of serum boron, rather than boron exposure from the dialysate. Boron hemodialyzability was almost proportional to the gradient of the boron level at the beginning of HD and it could be controlled by the adjustment of the gradient. In conclusion, the serum boron level was very much disturbed in long term HD patients. If boron excess in serum at the beginning of HD, or deficiency at the completion of HD may contribute to the complications of HD patients, fine adjustment and close surveillance of the gradient should be taken into account.

Nitric oxide induction by pertussis toxin in mouse spleen cells via gamma interferon.

We examined the major pathogenic substances of Bordetella pertussis for the ability to induce nitric oxide, and important biological function of macrophages, via gamma interferon in spleen cells. B. pertussis, which produces a variety of pathogenic substances, including pertussis toxin and filamentous hemagglutinin, causes a severe respiratory disease. Nitric oxide was detected in the culture fluid of spleen cells stimulated with pertussis toxin or its B oligomer but not in the culture fluid of spleen cells stimulated with the A protomer of pertussis toxin or with filamentous hemagglutinin. Incubation of the peritoneal exudate macrophages with pertussis toxin, B oligomer, A protomer, or filamentous hemagglutinin induced little nitric oxide, whereas incubation with gamma interferon induced a significant amount of nitric oxide. The induction of nitric oxide in spleen cells stimulated with pertussis toxin was completely inhibited by anti-gamma interferon antibody. The treatment of spleen cells with anti-Thy-1.2 antibody plus complement followed by stimulation with pertussis toxin decreased the secretion of gamma interferon and nitric oxide. These results suggest that gamma interferon from T lymphocytes stimulated with pertussis toxin induces nitric oxide.

[Antihypertensive effects of repeated oral administration of cilnidipine, a novel calcium antagonist, in 2K1C renal hypertensive dogs].

Antihypertensive effects of repeated oral administration of cilnidipine in 2K1C renal hypertensive dogs were compared with those of nicardipine. On the first day, oral administration of cilnidipine (3 mg/kg) or nicardipine (3 mg/kg) markedly lowered both systolic and diastolic blood pressure 1 hr after administration. The hypotensive effects of cilnidipine were longer compared with those of nicardipine. Both drugs elevated the heart rate and plasma renin activity. On the 8th and 15th days, similar responses were obtained by repeated administrations of cilnidipine and nicardipine. After withdrawal of these drugs, no rebound phenomena in blood pressure were observed. The changes in mean blood pressure were correlated with plasma cilnidipine or nicardipine concentrations that were obtained at each time of blood pressure measurement (r = -0.598; P < 0.001 and r = -0.594; P < 0.001, respectively). These results suggest that stable and long-acting antihypertensive effects of cilnidipine for 15 consecutive days in renal hypertensive dogs are related to the change in plasma drug concentrations.

Development and usefulness of the gelatin-particle-agglutination test for titration of antibodies against diphtheria, pertussis and tetanus toxins.

The gelatin-particle-agglutination (PA) test for titrating antibodies against diphtheria, pertussis and tetanus toxins was developed and used for assaying 65 sera from healthy children to assess the antitoxin acquisition in relation to the administration of adsorbed diphtheria-purified pertussis-tetanus (DPT) combined vaccine. The antitoxin titers obtained by the PA test and the conventional methods were correlated well; the correlation coefficient of the diphtheria antitoxin titers between the PA test and the cell culture method was 0.908, that of the tetanus antitoxin titers between the PA test and the passive hemagglutination test 0.968, and that of anti-pertussis toxin titers between the PA test and polystyrene-ball ELISA 0.885. The PA test was shown to be useful in both developed and developing countries, since it is simple to perform, sensitive and specific, and the three antitoxins can be titrated by the same procedure.

A clinical study on SSP (silver spike point) electro-therapy combined with splint therapy for temporo-mandibular joint dysfunction.

When the functional limits of the muscles related to the temporo-mandibular joint and adjacent tissue exceed their anatomical capability, pain, crepitation, and functional abnormality appear as the main complaints. Although the precise nature of the condition is unknown, pain at the temporo-mandibular joint sometimes in combination with muscular tension is assumed to be due to compression of the myoneural mechanism. It is reported that occlusal lifting using a splint enables the alleviation of this muscular tension. On the other hand, there are only a few reports on the usefulness of SSP therapy for Temporo-Mandibular Joint Dysfunction. We studied the efficacy of SSP therapy combined with splint therapy in 33 patients diagnosed as having Temporo-Mandibular Joint Dysfunction who consulted our department primarily due to pain, and report our findings below. Evaluation of the results was conducted 2 weeks later. Very beneficial results were seen in 6 cases. Beneficial results were seen in 7 cases. Slightly beneficial results were seen in 18 cases, while there were no changes found in 2 cases. When combined SSP and splint therapies were conducted for Temporo-Mandibular Joint Dysfunction, favorable results were seen in about 90% of the cases.

Post-transfusion hepatitis type B: long incubation period and poor prognosis in compromised hosts.

Seven cases of post-transfusion hepatitis type B [PTH(B)] were investigated. PTH(B) developed in 4 patients more than 65 years old and in 4 patients after treatment of a malignant disease (2 cases of gastric cancer and one each of ovarian cancer and chronic myelogenous leukemia, respectively). The mean incubation period was 78 days (70-90) in patients with non-malignant diseases and 147 days (105-200) in patients with malignant diseases. The symptoms of acute hepatic failure developed in 6 patients and 5 of them expired. One fatal case revealed 4 units of blood and an investigation of 4 donors revealed that one of them was an HBsAg carrier with negative serum HBsAg by the reverse passive hemagglutination (RPHA) method. From these results, it was concluded that compromised hosts such as aged patients or patients with malignant diseases are apt to contract severe PTH(B) with a long incubation period when the transfused blood contains small amounts of HBV.

Mechanisms whereby insulin increases diacylglycerol in BC3H-1 myocytes.

We previously suggested that insulin increases diacylglycerol (DAG) in BC3H-1 myocytes, both by increases in synthesis de novo of phosphatidic acid (PA) and by hydrolysis of non-inositol-containing phospholipids, such as phosphatidylcholine (PC) and phosphatidylethanolamine (PE). We have now evaluated these insulin effects more thoroughly, and several potential mechanisms for their induction. In studies of the effect on PA synthesis de novo, insulin stimulated [2-3H]glycerol incorporation into PA, DAG, PC/PE and total glycerolipids of BC3H-1 myocytes, regardless of whether insulin was added simultaneously with, or after 2 h or 3 or 10 days of prelabelling with, [2-3H]glycerol. In prelabelled cells, time-related changes in [2-3H]glycerol labelling of DAG correlated well with increases in DAG content: both were maximal in 30-60 s and persisted for 20-30 min. [2-3H]Glycerol labelling of glycerol 3-phosphate, on the other hand, was decreased by insulin, presumably reflecting increased utilization for PA synthesis. Glycerol 3-phosphate concentrations were 0.36 and 0.38 mM before and 1 min after insulin treatment, and insulin effects could not be explained by increases in glycerol 3-phosphate specific radioactivity. In addition to that of [2-3H]glycerol, insulin increased [U-14C]glucose and [1,2,3-3H]glycerol incorporation into DAG and other glycerolipids. Effects of insulin on [2-3H]glycerol incorporation into DAG and other glycerolipids were half-maximal and maximal at 2 nM- and 20 nM-insulin respectively, and were not dependent on glucose concentration in the medium, extracellular Ca2+ or protein synthesis. Despite good correlation between [3H]DAG and DAG content, calculated increases in DAG content from glycerol 3-phosphate specific radioactivity (i.e. via the pathway of PA synthesis de novo) could account for only 15-30% of the observed increases in DAG content. In addition to increases in [3H]glycerol labelling of PC/PE, insulin rapidly (within 30 s) increased PC/PE labelling by [3H]arachidonic acid, [3H]myristic acid, and [14C]choline. Phenylephrine, ionophore A23187 and phorbol esters did not increase [2-3H]glycerol incorporation into DAG or other glycerolipids in 2-h-prelabelling experiments; thus activation of the phospholipase C which hydrolyses phosphatidylinositol, its mono- and bis-phosphate, Ca2+ mobilization, and protein kinase C activation, appear to be ruled out as mechanisms to explain the insulin effect on synthesis de novo of PA, DAG and PC.(ABSTRACT TRUNCATED AT 400 WORDS)

Insulin provokes co-ordinated increases in the synthesis of phosphatidylinositol, phosphatidylinositol phosphates and the phosphatidylinositol-glycan in BC3H-1 myocytes.

BC3H-1 myocytes were cultured in the presence of [3H]inositol or [3H]glucosamine during their entire growth cycle to ensure that all lipids containing inositol and glucosamine were labelled to isotopic equilibrium or maximal specific radioactivity. After such labelling, a lipid (or group of lipids), which was labelled with both inositol and glucosamine, was observed to migrate between phosphatidylinositol 4-phosphate and phosphatidylinositol (PI) in two different t.l.c. systems. Insulin provoked rapid, sizeable, increases in the inositol-labelling of this lipid (presumably a PI-glycan), and these increases were similar to those observed in PI and PI phosphates. Our results indicate that insulin provokes co-ordinated increases in the net synthesis de novo of PI and its derivatives, PI phosphates and the PI-glycan, in BC3H-1 myocytes. This increase in synthesis of PI may serve as the mechanism for replenishing the PI-glycan during stimulation of its hydrolysis by insulin. Moreover, increases in the content of the PI-glycan may contribute to increases in the generation of head-group 'mediators' during insulin action.