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.

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.

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.

Quantitative comparison of planar and SPECT normal data files of thallium-201, technetium-99m-sestamibi, technetium-99m-tetrofosmin and technetium-99m-furifosmin.

UNLABELLED: In recent years, several of 99mTc-labeled myocardial perfusion imaging agents have been developed, such as 99mTc-sestamibi, 99mTc-tetrofosmin and 99mTc-furifosmin. Although images obtained with these new tracers have a general similar appearance, there are differences in the myocardial kinetics, body distribution, general quality of images and imaging protocols. The aim of this study was to quantitatively compare normal exercise planar and SPECT data files obtained with 201TI and 99mTc-labeled agents. METHODS: Lower-limit-of-normal curves were generated for each specific radiopharmaceutical from normal subjects with low (< 3%) pretest likelihood of coronary artery disease using circumferential count distribution profiles from planar and SPECT exercise images. Lower-limit-of-normal curves were statistically compared using the nonparametric Kruskall-Wallis and Wilcoxon tests. RESULTS: Planar and SPECT lower-limit-of-normal curves generated for each radiopharmaceutical showed general similarities. Statistically significant differences among the lower-limit-of-normal curves were found in the planar left anterior oblique view and in the planar left lateral view (p < 0.05 for each). No statistically significant differences existed between lower-limit-of-normal curves of various radiopharmaceuticals on the planar anterior view and on SPECT imaging. CONCLUSION: For quantitative analysis of planar images, radiopharmaceutical-specific normal data files are mandatory. Although SPECT normal data files of various radiopharmaceuticals are not statistically different, they are not identical. It appears, nevertheless, prudent to use radiopharmaceutical-specific normal data files for quantitative analysis of SPECT images.

Temperature-sensitive mutants of fowl plague virus defective in the intracellular transport of the hemagglutinin.

Nine mutants of fowl plague virus with temperature-sensitive defects in the biosynthesis of the hemagglutinin have been characterized by analyzing the processing and the intracellular location of this glycoprotein in MDCK and chick embryo cells. It was found that with all of these mutants the transport of the hemagglutinin to the cell surface was impeded at the non-permissive temperature. There were differences, however, in the site of the block. With mutants tsl, ts227, ts478 and ts658 the precursor HA was not cleaved and the oligosaccharide side chains remained sensitive to endoglucosaminidase H. When the hemagglutinin was analyzed in permeabilized cells by immunofluorescence, usually only cytoplasmic labeling was seen. Immunofluorescence of non-permeabilized cells and hemadsorption revealed that the hemagglutinin did not reach the cell surface. In contrast, the hemagglutinin of mutants ts79, ts482, ts532, ts546 and ts651 was cleaved and oligosaccharides were processed to the endoglucosaminidase H-resistant form at non-permissive temperature. In permeabilized cells, the cytoplasm and juxtanuclear regions typical for the Golgi apparatus were labeled by immunofluorescence. Except for ts482, ts532 and ts546 which were leaky, hemagglutinin could not be detected at the cell surface. These observations indicate that, with the first group of mutants, hemagglutinin transport is usually arrested already in the rough endoplasmic reticulum, whereas with the second group it is inhibited at a late stage between the Golgi apparatus and the plasma membrane.

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.

Immunoreactive thymosin alpha 1 in human thymus and thymoma.

Thymosin alpha 1-like immunoreactivity was assessed in human thymus and thymoma tissue extracts by means of a new radioimmunoassay that included an anti-thymosin alpha 1 mouse monoclonal antibody. Thymosin alpha 1-like immunoreactivity levels decreased with age in normal thymuses but not in thymomas. The average thymosin alpha 1-like immunoreactivity level was 45.0 +/- 52.1 ng/mg protein in normal thymuses and 273.9 +/- 205.0 ng/mg protein in thymomas. The average thymosin alpha 1-immunoreactivity level in thymomas was higher than that in normal thymuses. Thymosin alpha 1-like immunoreactivity levels in thymomas appeared to have no relationship to the clinical stage of the thymoma or associated diseases. When viewed according to histologic characteristics, the average thymosin alpha 1-like immunoreactivity level in polygonal cell thymomas (382.5 +/- 192.6 ng/mg protein) was significantly higher than that in the spindle cell thymoma (101.8 +/- 81.2 ng/mg protein). When viewed according to the degree of lymphocyte infiltration, thymomas could be classified according to four grades: absent, scant, moderate, and predominant. In predominant or moderate thymomas, the average thymosin alpha 1-like immunoreactivity level was higher than that in scant or absent thymomas. Also, thymosin alpha 1-like immunoreactivity levels in thymuses of patients with myasthenia gravis were relatively higher than those in patients with normal thymuses.

Inhibitory factor of microtubule assembly from sea urchin egg cortex. I. Preparation and characterization.

A new inhibitory factor of the microtubule (MT) assembly system was isolated from unfertilized sea urchin egg cortex. This factor not only suppressed spontaneous brain MT assembly, but also induced depolymerization of the reconstituted MTs. The factor did not suppress initial MT growth initiated by ciliary outer fiber fragments but the assembled MTs were soon depolymerized with time. The inhibitory activity was heat-stable but sensitive to trypsin or urea. The mode of the inhibition was distinct from the inhibitory effects of RNA on the MT assembly. The inhibitory factor partially purified on DEAE-Sephadex A-50 completely inhibited tubulin polymerization in a factor: tubulin ratio of 0.013.

Evidence that a polysaccharide from the cortex of sea urchin egg inhibits microtubule assembly through its binding to microtubule-associated proteins.

A new factor that not only inhibits brain microtubule assembly but also causes depolymerization of pre-assembled microtubules was purified from unfertilized sea urchin egg cortices. The purified fraction contained neither proteins nor nucleic acids. The inhibitory activity of the fraction was heat-stable and insensitive to various proteases, but decrease on treatment with glycosidases or with periodic acid. The purified factor was identified as a polysaccharide by analysis of the sugar composition. The sedimentation constant of the factor was estimated to be 9S by sucrose density gradient centrifugation. Polyacrylamide gel electrophoresis of the factor revealed a single broad band stained with periodic acid-Schiff reagent. The factor completely inhibited microtubule assembly at a factor: microtubule protein ratio of 0.03 (w/w). The polysaccharide was found to bind to microtubule-associated proteins, thereby inhibiting microtubule assembly.

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.

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.

A microstructural study of spinal cord edema.

The experimental spinal cord edema was produced in a cat by the infusion method of Marmarou. Horseradish peroxidase (HRP) dissolved in autoserum of a cat was used as a tracer. After laminectomy, a 30-gauge needle was inserted into the intumescentia cervicalis. A total amount of 20 microliters of a tracer was infused at a rate of 10 microliters/hr. The structural features were studied immediately and 3 days after infusion. Immediately after infusion, HRP was noted in the infused white and gray matters. Though the perivascular space in the white matter at the infused site was widely distended and filled with HRP, the space in the gray matter was not distended but filled with HRP. HRP which was observed along vessels led to the surface of the spinal cord. Swelling of astrocyte was not observed. Three days after infusion, the extracellular space and the perivascular space in the infused white matter were still expanded but were not filled with HRP. The fine structural features were similar to the findings as seen in Marmarou's infusion type of brain edema. Using this model, it seems to be feasible to study the resolution process of spinal cord edema.