Comparison of listeriolysin O and phospholipases PlcA and PlcB activities, and initial intracellular growth capability among food and clinical strains of Listeria monocytogenes.

AIMS: We investigated whether Listeria monocytogenes strains differ in their ability to escape from the primary phagosome after internalization into human intestinal epithelial cells. METHODS AND RESULTS: Food and clinical strains were used to study specific alleles; the activities of listeriolysin O (LLO) and phospholipases PlcA and PlcB, which promote rupture of the phagocytic vacuole; and initial intracellular bacterial growth in Caco-2 cells. Results showed no difference in LLO activities between food and clinical strains or among serotypes. In contrast, the LLO truncation mutant lacked detectable haemolytic activity and intracellular growth. PlcA and PlcB produced by the strains of serotypes 4b/4e and 1/2b exhibited significantly lower activities than those of serotypes 1/2a and 1/2c. In contrast, the strains of serotype 1/2b grew significantly faster than those of serotypes 4b/4e and 1/2a. Moreover, the PrfA truncation mutants lacked LLO and phospholipases activities and did not show intracellular growth. CONCLUSIONS: We determined that LLO and PrfA mutants exert a significant effect on intracellular growth, although it was unclear from this study whether PlcA and PlcB alleles affect escape from vacuoles. SIGNIFICANCE AND IMPACT OF THE STUDY: This study estimates that low-virulence L. monocytogenes strains associated with escape ability from the primary vacuoles are not widely distributed among food strains.

Haplotype-based, case-control study of the receptor (calcitonin) activity-modifying protein (RAMP) 1 gene in essential hypertension.

The adrenomedullin receptor is a complex molecule that comprises the calcitonin-receptor-like receptor (CRLR) and the receptor-activity-modifying protein (RAMP). RAMP1 is a vasodilation factor, and RAMP1-deficient mice (RAMP1(-/-)) exhibit inflammatory responses with a significant transient increase in serum calcitonin-gene-related peptide levels and proinflammatory cytokines when compared with RAMP1(+/+) mice. The purpose of the present study was to investigate the relationships between essential hypertension (EH) and RAMP1 gene single-nucleotide polymorphisms (SNPs) or haplotypes in a Japanese population via a case-control study. Based on a database search of the National Center of Biotechnology Information website and the HapMap project, we chose six RAMP1 gene SNPs and performed an association study involving 263 patients with EH and 267 age-matched normotensive (NT) subjects. There was no significant difference between the EH and NT groups with regard to overall distribution of genotypes or SNP alleles. However, the haplotype-based case-control analysis revealed that there was a significant difference between the EH and NT groups with regard to overall distribution of the allele combinations at three SNPs-rs3754701-rs3769048-rs10199956-(P=0.002). The T-A-T haplotype was significantly more common in the EH group (10.3%) than in the NT control group (6.1%) (P=0.047). These results suggested that this T-A-T RAMP1 gene haplotype might have utility as a genetic marker for EH and that the RAMP1 gene or a neighbouring gene may be associated with increased susceptibility to EH.

Intracellular bacteria recognition contributes to maximal interleukin (IL)-12 production by IL-10-deficient macrophages.

Interleukin (IL)-12 is a key factor that induces T helper cell type 1-mediated immunity and inflammatory diseases. In some colitis models, such as IL-10 knock-out (KO) mice, IL-12 triggers intestinal inflammation. An abundant amount of IL-12 is produced by intestinal macrophages in response to stimulation by commensal bacteria in IL-10 KO mice. Intact bacteria are more potent inducers of macrophage IL-12 production than cell surface components in this model. This suggested that cell surface receptor signalling and intracellular pathogen recognition mechanisms are important for the induction of IL-12. We addressed the importance of intracellular recognition mechanisms and demonstrated that signal transducers and activator of transcription 1 (STAT1) signalling activated bacterial phagocytosis and was involved in the induction of abnormal IL-12 production. In IL-10 KO mouse bone marrow-derived (BM) macrophages, Escherichia coli stimulation induced increased IL-12p70 production compared to lipopolysaccharide combined with interferon (IFN)-γ treatment. Significant repression of IL-12 production was achieved by inhibition of phagocytosis with cytochalasin D, and inhibition of de novo protein synthesis with cycloheximide. Induction of IFN regulatory factors-1 and -8, downstream molecules of STAT1 and the key transcription factors for IK-12 transcription, following E. coli stimulation, were mediated by phagocytosis. Interestingly, STAT1 was activated after stimulation with E. coli in IL-10 KO BM macrophages, although IFN-γ could not be detected. These data suggest that molecules other than IFN-γ are involved in hyper-production mechanisms of IL-12 induced by E. coli stimulation. In conclusion, enteric bacteria stimulate excessive IL-12p70 production in IL-10 KO BM macrophages via phagocytosis-dependent signalling.

Ultrasound predictors of mortality in monochorionic twins with selective intrauterine growth restriction.

OBJECTIVES: The aim of this study was to evaluate the use of ultrasound assessment to predict risk of mortality in expectantly managed monochorionic twin fetuses with selective intrauterine growth restriction (sIUGR). METHODS: This was a retrospective study of 101 monochorionic twin pregnancies diagnosed with sIUGR before 26 weeks of gestation. All patients were under expectant management during the observation period. At the initial evaluation, the presence or absence of each of the following abnormalities was documented: oligohydramnios; stuck twin phenomenon; severe IUGR < 3(rd) centile of estimated fetal weight; abnormal Doppler in the umbilical artery; and polyhydramnios in the larger twin. The relationships between these ultrasound findings and mortality of sIUGR fetuses were evaluated using multiple logistic regression analysis. RESULTS: Of 101 sIUGR twins, 22 (21.8%) fetuses suffered intrauterine demise and nine (8.9%) suffered neonatal death; 70 (69.3%) survived the neonatal period. Multiple logistic regression analysis revealed that the stuck twin phenomenon (odds ratio (OR): 14.5; 95% CI: 2.2-93.2; P = 0.006) and constantly absent diastolic flow in the umbilical artery (OR: 29.4; 95% CI: 3.3-264.0; P = 0.003) were significant risk factors for mortality. CONCLUSIONS: Not only abnormal Doppler flow in the umbilical artery but also severe oligohydramnios should be recognized as important indicators for mortality in monochorionic twins with sIUGR.

Monochorionic monoamniotic twin pregnancies with two yolk sacs may not be a rare finding: a report of two cases.

The exact determination of amnionicity is a major issue for the clinical management of monochorionic twin pregnancies, due to the high risk of perinatal mortality and morbidity in monochorionic monoamniotic (MCMA) twins. Counting the number of yolk sacs is believed to be a good indicator of amnionicity in the early first trimester, and it has previously been suggested that the number of yolk sacs is equal to amnionicity in both MCMA and monochorionic diamniotic twin pregnancies. However, the accuracy of the relationship between number of yolk sacs and amnionicity has recently been called into question. To the best of our knowledge, no previous reports have shown two yolk sacs in MCMA twin pregnancies. We report two cases of MCMA twins with two yolk sacs on first-trimester ultrasonography, and confirmed monoamnionicity in the second trimester showing umbilical cord entanglement. Postnatal examination showed an MCMA placenta in both cases, and entangled umbilical cords confirmed monoamnionicity. The possibility of monoamnionicity must still be suspected when two yolk sacs are detected early in the first trimester on ultrasound examination in monochorionic twin pregnancies.

Validation of Quintero stage III sub-classification for twin-twin transfusion syndrome based on visibility of donor bladder: characteristic differences in pathophysiology and prognosis.

OBJECTIVE: To validate the Quintero stage III subclassification for twin-twin transfusion syndrome (TTTS) based on visibility of the bladder of the donor twin. METHODS: Between July 2002 and August 2006, there were 131 pregnant Japanese women affected by severe TTTS before 26 weeks' gestation, treated with fetoscopic laser surgery at five centers in Japan, whose pregnancies continued beyond 22 weeks. Outcome data were available in all cases and surviving infants were followed up for at least 6 years. This study focused on the Stage III TTTS patients. These were subclassified into Stage III atypical (abnormal Doppler flow with visible donor bladder) and Stage III classical (abnormal Doppler flow with non-visible donor bladder) groups. Perioperative data and postnatal outcomes were compared between the groups. RESULTS: Seven Stage I, 22 Stage II, 82 Stage III and 20 Stage IV pregnancies continued beyond 22 weeks. There was a significantly higher incidence of absent or reversed end-diastolic velocity in the umbilical artery (UA-AREDV) of the donor in Stage III atypical than in Stage III classical patients (83.8% vs. 53.3%, P = 0.004). Stage III atypical cases also had a significantly higher incidence of arterioarterial (AA) anastomoses (72.9% vs. 17.8%, P < 0.001) and intrauterine fetal demise (IUFD) of the donor (43.2% vs. 13.3%, P = 0.002). However, there were no differences in overall survival or in abnormal brain scans of surviving infants. Donors with both UA-AREDV and AA anastomoses had a significantly higher incidence of IUFD compared with the others (53.3%, P < 0.001). CONCLUSIONS: Quintero stage III atypical was characterized by a high incidence of AA anastomoses and UA-AREDV of the donor, resulting in IUFD. Subclassification of Stage III based on visibility of the bladder of the donor twin was adequate for and compatible with differentiating prognosis and pathophysiology.

Morphologic evaluation of the caudal end of the inferior petrosal sinus using 3D rotational venography.

BACKGROUND AND PURPOSE: The inferior petrosal sinus (IPS) is the main transvenous access route used to examine or treat lesions involving the cavernous sinus. To carry out these procedures successfully, one must have a detailed knowledge of the anatomy of the venous system around the junction of the IPS and the internal jugular vein (IJV). MATERIALS AND METHODS: Eighty-three sides in 63 patients (26 men, 37 women; mean, 56.5 years of age) were examined by using 3D rotational venography (3DRV). RESULT: The drainage patterns of the IPS could be classified into the following 6 types, with emphasis on the level of IPS-IJV junction: type A, the IPS drains into the jugular bulb in 1/83 sides (1.2%); type B, the IPS drains into the IJV at the level of the extracranial opening of the hypoglossal canal in 29/83 sides (34.9%); type C, the IPS drains into the lower extracranial IJV in 31/83 sides (37.3%); type D, the IPS forms a plexus and has multiple junctions to the IJV near the jugular foramen in 5/83 sides (6.0%); type E, the IPS drains directly into the vertebral venous plexus (VVP) with no connection to the IJV in 3/83 sides (3.6%); and type F, the IPS is absent in 14/83 sides (16.9%). Each type is also characterized by the way of anastomosis with the VVP. CONCLUSION: This classification seemed to be rational from the embryologic viewpoint, and it may be useful in establishing treatment strategies that involve endovascular manipulation via the IPS.

Quality control system for mass screening in Japan.

Japan was the first country to establish a nationwide quality control system. When the Japanese Federal Government initiated Nationwide Neonatal Screening in 1977, the system officially included a Quality Control (QC) System that should cover all screening laboratories in Japan. This QC system is quite different from that for usual clinical chemistry. The aim of the National QC System for Neonatal Screening is evaluation of the accuracy of the tests and evaluation of the ability to detect suspicious samples with very mild abnormalities. For accomplishing the aim, the QC center established an inter-laboratory QC survey Screening laboratories having weak points can be identified through the inter-laboratory QC survey, and the Center must find a way to improve the ability of these screening laboratories. This requires a nationwide consensus regarding the cut-off levels of tested materials. Based on the cooperation of the Societies For Mass-screening, of Inborn Errors of Metabolism and of Pediatric Endocrinology, we set low cutoff levels for each compound to minimize the number of false negative cases. The system also included the evaluation of the quality of essential screening reagents and the special filter paper for blood collection (in partnership with the production companies). For this purpose, we developed some new methods for evaluating the standard-compounds for the various screening tests exactly, except in the case of TSH screening.

Risk stratification using serum concentrations of cardiac troponin T in patients with end-stage renal disease on chronic maintenance dialysis.

BACKGROUND: It has been recently suggested that cardiac troponin T (cTnT) may be more sensitive than troponin I (cTnI) for subclinical myocardial cell injury in patients on chronic dialysis. METHODS: We prospectively compared the predictive value of cTnT with cTnI, atrial (ANP) and brain natriuretic peptide (BNP) in 100 consecutive outpatients on chronic dialysis without acute coronary syndromes over a period of 3 months, and assessed whether the combination of cTnT with clinical information including age, duration of dialysis, and medical histories was useful for risk stratification of these patients. During the 2-year follow-up period, 19 patients died, mostly due to cardiac causes (53%). RESULTS: The area under the receiver operator characteristic (ROC) curve for the cTnT as predictor of both overall and cardiac death was significantly greater than the area under the cTnI curve (p < 0.0001 and p = 0.01), the BNP curve (p < 0.001 and p < 0.01) or the ANP curve (p < 0.0001 and p < 0.005). In a stepwise multivariate Cox regression analysis, only cTnT (p < 0.05 and p < 0.01) and a history of heart failure requiring hospitalization (p < 0.05 and p < 0.005) were independent predictors of both all cause and cardiac mortality. Using parameters of cTnT > or =0.1 microg/l and/or history of heart failure, the overall and cardiac mortality rate for the low risk group (n=66) were 4.5% and 1.5%, respectively, 40% and 16% for the intermediate risk group (n=25), and 67% and 56% for the high risk group (n=9). CONCLUSION: cTnT concentrations offer a higher prognostic accuracy than cTnI, ANP and BNP in patients on chronic dialysis. The combination of elevated cTnT and a history of heart failure may be a highly effective means of risk stratification of these patients.

[Endoscopic transthoracic sympathectomy (ETS) with a fine 2-mm thoracoscope in palmar hyperhidrosis].

Endoscopic transthoracic sympathectomy (ETS) is an efficient, safe, minimally invasive procedure, and requires only a short period of hospitalization. We performed bilateral ETS using a thoracoscope 2 mm in diameter. We performed 120 ETS for the treatment of palmar hyperhidrosis from August 1997 to April 2000. The patient was placed in the semi-sitting position under general anesthesia, one-lung ventilation being used; the operation was performed with 2-mm two-puncture method. The sympathetic chain could be observed through parietal pleura riding on the costovertebral junctions. A 2-mm Kirschner wire was employed as an electrode, and the second and third thoracic sympathetic chains were electro cauterized. A thoracic drain was not used. The needling sites were only 2 mm in size and could be fixed without suture. For the patient, a small scar of this size means virtually no scar. The operative times were from 11 min to 81 min in bilateral ETS. Immediately and dramatic decrease in the sweat excretion in the palms was noted in all patients. The 95% patients were highly satisfied with the results. The commonest side effects were compensatory sweating. This procedure is recommended as the method of choice for the surgical treatments of palmar hyperhidrosis.

Detection of a coronary arterial thrombus by indium-111-oxine-labeled platelet scintigraphy.

Coronary arteriography revealed significant left anterior descending coronary artery stenosis in a 72-year-old man with a history of myocardial infarction. Stenting of the stenotic vessel was performed. Twelve hours after stenting the patient complained of chest pain but emergent coronary arteriography did not show sign of any coronary arterial stenosis. Under suspicion of coronary thrombus formation, indium-111-oxine-labeled platelet scintigraphy was performed 5 days after stenting, and revealed accumulation of indium-111-oxine in the area corresponding to the stent implantation site.

Detection of inflammation in aortic aneurysms with indium 111-oxine--labeled leukocyte imaging.

BACKGROUND: The exact cause of aortic aneurysms is not completely understood. Histologically, the atherosclerotic lesions present in an aneurysm contain numerous inflammatory cells. This finding represents active atherosclerosis, which can cause lesion expansion. In this study we investigated the role of scintigraphy in the evaluation of inflammation in aortic aneurysms. METHODS AND RESULTS: We performed imaging using indium 111-oxine--labeled leukocytes in 14 patients with aortic aneurysms (10 thoracic and 4 abdominal) diagnosed by computed tomography. Peripheral blood evidence of inflammation was assessed on the same day. In 8 patients who subsequently underwent graft replacement of the aneurysm, the excised specimen was examined for evidence of inflammatory infiltration and correlated with the scintigraphic findings. Scintigraphic accumulation of labeled leukocytes was present in 10 of the 14 patients. Although all patients had a small increase in the erythrocyte sedimentation rate, there was no significant difference in the erythrocyte sedimentation rate between patients with positive and negative scintigram results. In 5 of the 8 surgical patients with positive scintigram results, the resected specimens demonstrated numerous inflammatory cells in the adventitia of the aortic wall and atherosclerotic changes in the media. There was no correlation between the presence of periaortic inflammatory adhesions at the time of surgery and the scintigraphic results. CONCLUSIONS: The accumulation of In-111-oxine--labeled leukocytes is a potentially useful scintigraphic marker of inflammatory infiltration in aortic aneurysms.

Usefulness of indium-111-oxine-labeled leukocyte scintigraphy in diagnosis of inflammation associated with chronic aortic dissection.

BACKGROUND: Patients with chronic aortic dissection require monitoring for indications of disease progression. In present study, inflammation adjacent to associated aortic wall was evaluated by indium-111-oxine-labeled leukocyte scintigraphy, scince inflammation of the blood vessel wall often associates with progression of chronic aortic dissection. METHODS AND RESULTS: Fifteen patients with aortic dissection underwent indium-111-oxine-labeled leukocyte scintigraphy. Seven showed positive images at sites corresponding to the actual sites of the dissociated aorta. Four patients with positive images underwent surgery. Histologic examination revealed inflammatory and necrotic changes of the aortic wall. During a mean follow-up period of 2.3 years, progression of aortic dissection was observed in two of the seven patients with positive intimal imaging. CONCLUSION: Indium-111-oxine-labeled leukocyte scintigraphy may be a useful noninvasive technique to assess the persistent inflammation in patients with chronic aortic dissection.

[Problems for analysis of regional wall motion using ECG gated myocardial SPECT imaging].

Several issues arose during analysis of cardiac function using ECG-gated myocardial SPECT imaging. First, the analysis program did not depending on a direct radioactivity counts as in equilibrium radionuclide angiography, but on the extraction of the myocardial margin. Consequently, spatial resolution was an important consideration in the analysis. The auto-analyzing program could be used in cases with poor quality images. Poor time resolution was also a weak point of the gated SPECT program. In the present study, we compared observation of regional wall motion obtained by Quantitative gated SPECT program (QGS) and echocardiography in patients with subacute phase myocardial infarction. The QGS indices of regional wall motion were correlated with the echocardiography indices, although mild asynergy could not be detected by any of the QGS indices. Further basic research on the analysis of regional wall motion using gated SPECT is needed.

Plasma concentration of brain natriuretic peptide as a biochemical marker for the evaluation of right ventricular overload and mortality in chronic respiratory disease.

The purpose of this study was to evaluate whether the plasma brain natriuretic peptide (BNP) concentration is a useful marker of right ventricular (RV) overload and whether it has prognostic value as a predictor of death in patients with chronic respiratory disease (CRD). We measured the plasma BNP and atrial natriuretic peptide (ANP) concentrations in 31 consecutive patients with CRD who underwent right-heart catheterization to evaluate pulmonary hypertension. All patients were followed for >12 months. The plasma BNP concentration closely correlated with the mean pulmonary artery pressure and pulmonary vascular resistance (r=0.62, P<0.0005 and r=0. 85, P<0.0001), and showed a weak linear correlation with cardiac output (r=-0.36, P<0.05). During the follow-up period, 5 (16%) end-stage CRD deaths (4 RV heart failure and 1 respiratory infection) and 2 non-end-stage CRD deaths occurred. In a stepwise multivariate Cox proportional-hazards regression analysis including age, sex, BNP, ANP, hemodynamic variables and the ratio of PaO(2) to fraction of inspired oxygen, only BNP (P<0.05) was an independent predictor of end-stage CRD death. The upward and leftward shift in the receiver operating characteristic curve between patients with end-stage CRD death and those without was greater for BNP than for ANP. Our findings suggest that the plasma BNP concentration may be an inexpensive, simple and useful marker of RV overload and end-stage CRD death in CRD patients. These preliminary results need to be confirmed in a large series of CRD patients.

Pulmonary stenosis in recipient twins in twin-to-twin transfusion syndrome: report on 3 cases and review of literature.

This report describes 3 cases of pulmonary stenosis in the recipient twin in twin-twin transfusion syndrome. Fetal echocardiography showed cardiomegaly, tricuspid valve regurgitation, and increased reverse flow in the inferior vena cava, as signs of congestive heart failure in all 3 cases. We diagnosed 2 cases of pulmonary stenosis by fetal echocardiography prenatally and confirmed our findings in all 3 cases postnatally. Two cases underwent postnatal balloon valvuloplasty to release the pulmonary valvular stenosis in neonatal period. The third one died soon after delivery and autopsy showed a slightly thickened pulmonary valve. One of the cases was diagnosed in the early second trimester (20 weeks of pregnancy), the earliest detection of fetal pulmonary stenosis reported in literature. The presence of high peak velocity of the pulmonary artery at 20 weeks of pregnancy preceded the development of pulmonary stenosis in this case. This supports the hypothesis that alterations in fetal hemodynamics may result in structural cardiac abnormality.