Neurodevelopmental outcome in full-term newborns with refractory neonatal seizures.

AIM: This retrospective study describes the prognosis of full-term newborns with refractory neonatal seizures, comparing the need for treatment with two versus three or more antiepileptic drugs. METHODS: We reviewed our database (January 2002-December 2007) to include newborns with refractory neonatal seizures and abnormal electroencephalogram. Group A consisted of 17 newborns with two antiepileptic drugs. Group B consisted of 29 newborns with three or more antiepileptic drugs. Outcome was determined at 2 years of age using the Dutch Bayley Scales of Infant Development or a neurodevelopmental classification scheme. RESULTS: Group A and group B were comparable regarding to a variety of demographic and aetiologic factors. Thirteen newborns died before 2 years of age and one was lost to follow-up. Normal development at 2 years of age was found in 50% and 5% for group A and B, respectively. Severe neurodevelopmental delay at 2 years of age was found in 30% and 68% for group A and B, respectively. CONCLUSION: The number of antiepileptic drugs probably reflects increased seizure burden and is--in that way--related to poor outcome. This may be useful information for early prediction of adverse neurological outcome in the first days of life.

Prognostic models for stillbirth and neonatal death in very preterm birth: a validation study.

OBJECTIVES: To validate externally 2 prognostic models for stillbirth and neonatal death in very preterm infants who are either known to be alive at the onset of labor or admitted for neonatal intensive care. PATIENTS AND METHODS: All infants, with gestational age 22 to 32 weeks, of European ethnicity, known to be alive at the onset of labor (n = 17 582) and admitted for neonatal intensive care (n = 11 578), who were born in the Netherlands between January 1, 2000, and December 31, 2007. The main outcome measures were stillbirth or death within 28 days for infants known to be alive at the onset of labor and death before discharge from the NICU for infants admitted for intensive care. Model performance was studied with calibration plots and c statistic. RESULTS: Of the infants known to be alive at the onset of labor, 16.7% (n = 2939) died during labor or within 28 days of birth, and 7.8% (n = 908) of the infants admitted for neonatal intensive care died before discharge from intensive care. The prognostic model for infants known to be alive at the onset of labor showed good calibration and excellent discrimination (c statistic 0.92). The prognostic model for infants admitted for neonatal intensive care showed good calibration and good discrimination (c statistic 0.82). CONCLUSIONS: The 2 prognostic models for stillbirth and neonatal death in very preterm Dutch infants showed good performance, suggesting their use in clinical practice in the Netherlands and possibly other Western countries.

Long-term neurodevelopmental outcome of monochorionic and matched dichorionic twins.

BACKGROUND: Monochorionic (MC) twins are at increased risk for perinatal mortality and serious morbidity due to the presence of placental vascular anastomoses. Cerebral injury can be secondary to haemodynamic and hematological disorders during pregnancy (especially twin-to-twin transfusion syndrome (TTTS) or intrauterine co-twin death) or from postnatal injury associated with prematurity and low birth weight, common complications in twin pregnancies. We investigated neurodevelopmental outcome in MC and dichorionic (DC) twins at the age of two years. METHODS: This was a prospective cohort study. Cerebral palsy (CP) was studied in 182 MC infants and 189 DC infants matched for weight and age at delivery, gender, ethnicity of the mother and study center. After losses to follow-up, 282 of the 366 infants without CP were available to be tested with the Griffiths Mental Developmental Scales at 22 months corrected age, all born between January 2005 and January 2006 in nine perinatal centers in The Netherlands. Due to phenotypic (un)alikeness in mono-or dizygosity, the principal investigator was not blinded to chorionic status; perinatal outcome, with exception of co-twin death, was not known to the examiner. FINDINGS: Four out of 182 MC infants had CP (2.2%) - two of the four CP-cases were due to complications specific to MC twin pregnancies (TTTS and co-twin death) and the other two cases of CP were the result of cystic PVL after preterm birth - compared to one sibling of a DC twin (0.5%; OR 4.2, 95% CI 0.5-38.2) of unknown origin. Follow-up rate of neurodevelopmental outcome by Griffith's test was 76%. The majority of 2-year-old twins had normal developmental status. There were no significant differences between MC and DC twins. One MC infant (0.7%) had a developmental delay compared to 6 DC infants (4.2%; OR 0.2, 95% 0.0-1.4). Birth weight discordancy did not influence long-term outcome, though the smaller twin had slightly lower developmental scores than its larger co-twin. CONCLUSIONS: There were no significant differences in occurrence of cerebral palsy as well as neurodevelopmental outcome between MC and DC twins. Outcome of MC twins seems favourable in the absence of TTTS or co-twin death.

Functioning at school age of moderately preterm children born at 32 to 36 weeks' gestational age.

OBJECTIVE: To study outcome of low-risk moderately preterm birth between 32 and 36/7 weeks' gestation. METHODS: 377 Moderately preterm children (M: 34.7, SD: 1.2 complete weeks), without need for neonatal intensive care and without dysmaturity or congenital malformations, were compared with 182 term children and assessed at eight years (M: 8.9, SD: 0.54). School situation, IQ, sustained attention, behavior problems, and attention-deficit/hyperactivity characteristics were studied. RESULTS: Special education was attended by 7.7% of the moderately preterm children, more than twice the rate of 2.8% in the general Dutch population of this age. Additional exploration for two preterm subgroups of 32 to 33 versus 34 to 36 weeks' gestation showed a need for special education in 9.7% versus 7.3% and a significant difference in grade retention for 30% versus 17%, respectively. Of the children attending mainstream primary schools, grade retention was found in 19% of the preterm versus 8% of the comparison children. Adjusting for maternal education, a group difference of 3 points was found in IQ. The preterm children needed more time for the sustained attention task. The preterm children had more behavior problems (specifically internalizing problems with 27% scoring above the borderline cut-off), as well as more attention-deficit/hyperactivity disorder characteristics (specifically attention deficits). CONCLUSIONS: Cognitive and emotional regulation difficulties affect functioning of moderately preterm children, as school problems, a slightly lower IQ, attention and behavioral problems are found when they are compared with term-born children. Identification and monitoring of precursors of these problems at younger age is needed in view of prevention purposes.

Functional outcomes and participation in young adulthood for very preterm and very low birth weight infants: the Dutch Project on Preterm and Small for Gestational Age Infants at 19 years of age.

OBJECTIVE: Young adults who were born very preterm or with a very low birth weight remain at risk for physical and neurodevelopmental problems and lower academic achievement scores. Data, however, are scarce, hospital based, mostly done in small populations, and need additional confirmation. METHODS: Infants who were born at < 32 weeks of gestation and/or with a birth weight of < 1500 g in The Netherlands in 1983 (Project on Preterm and Small for Gestational Age Infants) were reexamined at age 19. Outcomes were adjusted for nonrespondents using multiple imputation and categorized into none, mild, moderate, or severe problems. RESULTS: Of 959 surviving young adults, 74% were assessed and/or completed the questionnaires. Moderate or severe problems were present in 4.3% for cognition, 1.8% for hearing, 1.9% for vision, and 8.1% for neuromotor functioning. Using the Health Utility Index and the London Handicap Scale, we found 2.0% and 4.5%, respectively, of the young adults to have > or = 3 affected areas in activities and participation. Special education or lesser level was completed by 24%, and 7.6% neither had a paid job nor followed any education. Overall, 31.7% had > or = 1 moderate or severe problems in the assessed areas. CONCLUSIONS: A total of 12.6% of young adults who were born very preterm and/or with a very low birth weight had moderate or severe problems in cognitive or neurosensory functioning. Compared with the general Dutch population, twice as many young adults who were born very preterm and/or with a very low birth weight were poorly educated, and 3 times as many were neither employed nor in school at age 19.

Lower mortality but higher neonatal morbidity over a decade in very preterm infants.

Better perinatal care has led to better survival of very preterm children, but may or may not have increased the number of children with cerebral and pulmonary morbidity. We therefore investigated the relationship between changes in perinatal care during one decade, and short-term outcome in very preterm infants. Perinatal risk factors and their effects on 28-day and in-hospital mortality, and on intraventricular haemorrhage and bronchopulmonary dysplasia (BPD) in survivors, were compared in two surveys of very preterm singleton infants in the Netherlands. Between 1983 and 1993, 28-day mortality decreased from 52.1% to 31.8% in infants of 25-27 weeks' gestation and from 15.2% to 11.3% in infants of 28-31 weeks' gestation. The incidence of intraventricular haemorrhage in survivors did not change (44.4% and 43.3% in infants of 25-27 weeks' gestation, and 29.0% and 24.0% in infants of 28-31 weeks' gestation). The incidence of BPD in survivors increased from 40.3% to 60.0% in infants of 25-27 weeks' gestation and remained similar in infants of 28-31 weeks' gestation (8.5% and 9.8% respectively). In multivariable analysis, higher mortality was associated with congenital malformation, low gestational age, low birthweight, no administration of steroids before birth, low Apgar scores and intraventricular haemorrhage, in 1983 as well in 1993, and with male gender in 1993. The effect of maternal age on mortality diminished significantly between 1983 and 1993. Intraventricular haemorrhage in surviving children was associated with low gestational age and artificial ventilation, both in 1983 and in 1993. The effect of artificial ventilation on the incidence of intraventricular haemorrhage diminished significantly between 1983 and 1993. BPD was associated with low gestational age and artificial ventilation, both in 1983 and in 1993, and with low birthweight and caesarean section in 1993. We conclude that the better survival of very preterm infants, especially of those of 25-27 weeks' gestation, has been accompanied by a similar incidence (and thus with an increased absolute number) of children with intraventricular haemorrhage and by an increased incidence of children with BPD.

Outcome of perinatal care for very preterm infants at 5 years of age: a comparison between 1983 and 1993.

Perinatal mortality in very preterm infants has decreased by up to 50% during the last decades. Studies of changes of long-term outcome are inconclusive. We studied the visual, auditory, neuromotor, cognitive and behavioural development of two geographically defined populations of very preterm, singleton infants, born in 1983 and in 1993, and analysed the relationship between perinatal risk factors and outcomes. The incidence of disabling cerebral palsy increased from 6.0% to 11.1% (OR 2.45 [95% CI 1.11, 5.38]). Impaired vision and strabismus decreased significantly, presumably by continuous monitoring of pO(2). Hearing problems, the need for special education and the incidence of behavioural problems did not change over time. The proportion of children who showed optimal performance in every developmental domain increased from 29.5% in 1983 to 43.2% in 1993. Cerebral palsy was associated with male gender in 1983, with low Apgar score and intraventricular haemorrhage in 1993, and with seizures both in 1983 and in 1993. The intensiveness of neonatal treatment has increased, leading to the survival of many more healthy infants, but at the cost of more infants with cerebral damage. Modern perinatal care is no longer limited by the devastating effects of pulmonary problems as it was in the past, but fails to safeguard cerebral integrity in very preterm infants.

Is paediatric assessment of motor development of very preterm and low-birthweight children appropriate?

AIM: To determine whether paediatricians that examine, in regular clinical practice, very preterm and very-low-birthweight children at 5 y of age detect neurological impairments and functional motor problems in these children. METHODS: We compared a paediatric judgement, a standardized neurological examination (Touwen examination) and a screening of motor development (Denver Developmental Screening Test; DDST) with the Movement ABC in 396 5-y-old very preterm and low-birthweight children. RESULTS: The Movement ABC detected clinically important motor disorders in 20.5% and borderline disturbances in 22.5% of the children. Compared to the Movement ABC, the sensitivity of the paediatric judgement was 0.19, Touwen examination 0.62 and DDST 0.52; the negative predictive values were 0.61, 0.74 and 0.69, respectively. CONCLUSION: Paediatric assessment of motor development in 5-y-old very preterm and low-birthweight children generally is not sensitive enough to detect functional motor problems. The Movement ABC should be added to the assessment of the motor development of very preterm and low-birthweight children at 5 y of age.

Transfection effect of microbubbles on cells in superposed ultrasound waves and behavior of cavitation bubble.

The combination of ultrasound and ultrasound contrast agents (UCAs) is able to induce transient membrane permeability leading to direct delivery of exogenous molecules into cells. Cavitation bubbles are believed to be involved in the membrane permeability; however, the detailed mechanism is still unknown. In the present study, the effects of ultrasound and the UCAs, Optison on transfection in vitro for different medium heights and the related dynamic behaviors of cavitation bubbles were investigated. Cultured CHO-E cells mixed with reporter genes (luciferase or beta-gal plasmid DNA) and UCAs were exposed to 1 MHz ultrasound in 24-well plates. Ultrasound was applied from the bottom of the well and reflected at the free surface of the medium, resulting in the superposition of ultrasound waves within the well. Cells cultured on the bottom of 24-well plates were located near the first node (displacement node) of the incident ultrasound downstream. Transfection activity was a function determined with the height of the medium (wave traveling distance), as well as the concentration of UCAs and the exposure time was also determined with the concentration of UCAs and the exposure duration. Survival fraction was determined by MTT assay, also changes with these values in the reverse pattern compared with luciferase activity. With shallow medium height, high transfection efficacy and high survival fraction were obtained at a low concentration of UCAs. In addition, capillary waves and subsequent atomized particles became significant as the medium height decreased. These phenomena suggested cavitation bubbles were being generated in the medium. To determine the effect of UCAs on bubble generation, we repeated the experiments using crushed heat-treated Optison solution instead of the standard microbubble preparation. The transfection ratio and survival fraction showed no additional benefit when ultrasound was used. These results suggested that cavitation bubbles created by the collapse of UCAs were a key factor for transfection, and their intensities were enhanced by the interaction of the superpose ultrasound with the decreasing the height of the medium. Hypothesizing that free cavitation bubbles were generated from cavitation nuclei created by fragmented UCA shells, we carried out numerical analysis of a free spherical bubble motion in the field of ultrasound. Analyzing the interaction of the shock wave generated by a cavitation bubble and a cell membrane, we estimated the shock wave propagation distance that would induce cell membrane damage from the center of the cavitation bubble.

Hepatic vein transit time of SonoVue: a comparative study with Levovist.

PURPOSE: To prospectively compare transit times of Levovist and SonoVue in healthy volunteers and patients with biopsy-proved hepatitis C-related liver disease. MATERIALS AND METHODS: Institutional review board approval and informed consent were obtained. Forty patients and 25 healthy volunteers were examined. Subjects fasted, a bolus of SonoVue (0.6 mL) was injected into a cubital fossa vein, and hepatic venous time-intensity profiles were measured with spectral Doppler tracing. This was repeated with two injections of Levovist (2 g) and another injection of SonoVue. Time-intensity curves of spectral Doppler signals of right and middle hepatic veins were analyzed. A sustained signal intensity increase of 10% above baseline levels indicated hepatic vein transit time (HVTT). Carotid artery audio intensity was measured in volunteers. Analysis of variance and t tests were used for statistical analysis. RESULTS: Twelve patients had mild hepatitis; 18, moderate or severe hepatitis; and 10, cirrhosis. Mean HVTTs in control, mild hepatitis, moderate or severe hepatitis, and cirrhosis groups were 38.3 seconds +/- 2.4 (standard error), 47.5 seconds +/- 6.5, 29.5 seconds +/- 10.8, and 17.6 seconds +/- 5.0, respectively, with Levovist (P < .001) and 29.4 seconds +/- 6.9, 27.4 seconds +/- 9.3, 22.9 seconds +/- 4.7, and 16.4 seconds +/- 4.9, respectively, with SonoVue (P < .001). HVTT decreased as severity increased at imaging with both contrast agents. There was no significant difference in HVTT between mild and moderate hepatitis groups with SonoVue; however, there were significant differences in HVTT between all patient groups with Levovist. HVTT of SonoVue was shorter than that of Levovist in all groups (P < .001) except the cirrhosis group; in this group, HVTT of the two contrast agents was similar (P = .05). No difference was observed in mean cardiopulmonary transit time for SonoVue or Levovist (9.1 seconds +/- 2.4 [standard error] and 8.4 seconds +/- 2.5, respectively, P = .18). CONCLUSION: HVTT was significantly shorter with SonoVue than with Levovist; there was no significant difference in cardiopulmonary transit time.

Pluronic block copolymers: novel functions in ultrasound-mediated gene transfer and against cell damage.

Pluronics have been investigated as vectors for drug and gene delivery in vitro and in vivo and were demonstrated to have high efficiency for gene transfer in vivo. However, they alone do not enhance gene transfer in vitro. We examined three pluronics, F127, L61 and P85, for their effects on ultrasound (US)-mediated gene transfer in three cell lines, 3T3-MDEI, C2C12 and CHO. The polymers showed differential effects on cell viability and transfection efficiency in a dose-dependent manner. All the polymers were unable to facilitate gene transfer when used alone, but enhanced US-mediated gene transfer significantly at concentrations around the critical micelle concentration in the three cell lines. F127 showed no significant toxicity at any concentration and protected the cells against US-mediated damage at a high concentration. L61 decreased cell viability significantly in a dose-dependent manner, whereas P85 showed mild toxicity when its concentration was at or above 0.05%.

Delivery of oligodeoxynucleotides into human saphenous veins and the adjunct effect of ultrasound and microbubbles.

Therapy with naked oligodeoxynucleotides (ODNs, molecular weight: 3000 to 7500) provides an elegant means of modulating gene expression without the problems associated with conventional gene therapy, but the relatively low transfer efficiency on intravascular administration is a limitation to clinical application. Ultrasound, which can be potentiated by microbubbles, shows promise as a method of delivering macromolecules such as plasmid DNA and other transgenes into cells. Since uptake of molecules into cells depends on their molecular weight, it might be expected that the delivery of ODNs, which are relatively small, will be facilitated by ultrasound and microbubbles. In the present study, we delivered ODNs into veins using ultrasound and microbubbles. First, we quantified the uptake of fluorescent-labeled ODNs into intact ex vivo human saphenous veins and isolated smooth muscle cells from the veins, evaluating the effect of ultrasound and microbubbles on uptake. Ultrasound potentiated the delivery of ODN in cells, except at high concentrations. When intact veins were studied, we achieved nuclear localization of fluorescent-labeled ODNs in cells. This increased with increasing concentration and incubation time and was not potentiated by ultrasound, even when microbubbles were used. We then applied a therapeutic ODN (antisense to intercellular adhesion molecule 1, ICAM-1) to vein samples and documented a functional inhibition of gene expression in a sequence-specific manner at the protein level with immunohistochemistry and western blot analysis. Again, no significant difference was seen with adjunct ultrasound. These observations suggest high diffusion of ODNs into human saphenous veins in this ex vivo model, indicating potential applications to inhibition of vascular bypass graft occlusion and other vasculopathies. Although microbubble-ultrasound was of value with cells in culture, it was not beneficial with intact veins.

Gene transfer with microbubble ultrasound and plasmid DNA into skeletal muscle of mice: comparison between commercially available microbubble contrast agents.

PURPOSE: To compare three commercial microbubble contrast agents (Optison, SonoVue, and Levovist) for their effect on gene delivery in skeletal muscle in conjunction with the use of therapeutic ultrasound. MATERIALS AND METHODS: The study was approved by the Animal Care and Use Committee. Plasmid DNA (10 microg) encoding green fluorescent protein (GFP) was mixed with microbubbles (or saline control) and injected into the tibialis anterior muscle of mice with and without adjunct ultrasound (1 MHz, 2 W/cm2, 30 seconds, 20% duty cycle). The efficiencies of GFP transgene expression were determined with four experimental conditions: (a) plasmid and saline as control (six mice), (b) plasmid and Optison (six mice), (c) plasmid and SonoVue (four mice), and (d) plasmid and Levovist (air based, four mice). The right legs were exposed to ultrasound, while the left legs were unexposed. Transfection efficiency was assessed by counting the number of GFP-positive fibers. Tissue damage was assessed by measuring the maximal-damage area on serial sections. RESULTS: When ultrasound was applied, both SonoVue and Optison significantly improved (P < .05) gene transfection efficiency. Optison was also effective (P < .05) even when no ultrasound was applied, which is consistent with previous studies. Levovist without ultrasound decreased the level of transfection (P < .05), with increased tissue damage. CONCLUSION: Both non-air-based agents show promise in gene delivery in skeletal muscle with undetectable tissue damage. Enhanced gene transfer with additional ultrasound was achieved only with SonoVue.

Can Doppler sonography grade the severity of hepatitis C-related liver disease?

OBJECTIVE: Many authors have claimed that Doppler sonography indexes are of value in grading and assessing diffuse liver disease. However, there is much controversy regarding the reliability and reproducibility of these techniques. We performed a prospective study to evaluate whether these methods can grade disease in a well-stratified cohort of patients with hepatitis C virus (HCV)-related liver disease. SUBJECTS AND METHODS: Sixty-five patients with biopsy-proven HCV-related liver disease were recruited, and Doppler sonography was performed by one operator. The patients were classified into one of the following three groups on the basis of the Ishak-modified histologic activity index (HAI) fibrosis (F) and necroinflammatory (NI) scores: mild hepatitis (F < or = 2 and NI < or = 3), moderate or severe hepatitis (3 < or = F < 6 or NI > or = 4), or cirrhosis (F = 6/6). We measured the following Doppler indexes: main hepatic artery peak velocity (Vmax) and resistive index, main portal vein peak velocity (Vmax), and maximal portal vein diameter and circumference that allowed calculation of the portal vein congestive index (portal vein area and portal vein velocity). The ratio of the hepatic artery velocity (Vmax) to the portal vein velocity (Vmax) was also calculated, and the phasicity (triphasic, biphasic, or monophasic) of the hepatic veins of each patient was recorded. We also measured the maximal spleen length longitudinally. RESULTS: A total of 65 patients with liver disease (mild hepatitis, n = 20; moderate or severe hepatitis, n = 25; cirrhosis, n = 20) with biopsy-proven HCV-related liver disease were studied. Optimal hepatic arterial traces were obtained in only 30 patients and portal vein circumference in 18 patients. No significant differences were observed in the Doppler indexes with increasing severity of liver disease. Five (29%) of 17 patients with mild hepatitis had an abnormal hepatic vein trace (i.e., biphasic or monophasic) compared with 11 (55%) of 20 patients with moderate or severe hepatitis and 12 (60%) of 20 patients with cirrhosis. The only index to show a significant intergroup difference was splenic length (analysis of variance, p < 0.001), but there was still overlap between the groups. CONCLUSION: Doppler-derived indexes, which have previously been recommended for the assessment of severity in chronic liver disease, are difficult to reproduce reliably and therefore have a limited clinical role in the noninvasive assessment of hepatic fibrosis or inflammation.

A novel technique to measure splanchnic transit time using microbubble ultrasound.

OBJECTIVE: The objective of this study was to measure splanchnic transit time by intravenous injection of a microbubble. MATERIALS AND METHODS: Ten volunteers were examined before and after eating. After Doppler indices of splanchnic circulation were obtained, the superior mesenteric artery (SMA) and vein (SMV) were simultaneously interrogated using power Doppler ultrasound after intravenous injection of a microbubble. Contrast arrival in the SMA and subsequently the SMV was recorded and splanchnic transit time calculated from differences in the time-intensity curves. RESULTS: Splanchnic transit time decreased significantly after eating (mean 11 vs. 6.9 seconds; P = 0.007), reflecting splanchnic hemodynamics. Between-subject variability attributable to repeated measurements was least for the SMA resistive index (17%) but 56% for the new index, suggesting poor reproducibility. CONCLUSION: Splanchnic transit time may be measured by microbubble injection but is subject to considerable measurement error. Newer microbubbles and imaging methods may allow more reproducible measurements.

Optimisation of ultrasound-mediated gene transfer (sonoporation) in skeletal muscle cells.

Ultrasound (US) is a promising tool for facilitating direct gene transfer to skeletal muscle, but no systematic optimisation study has been performed. We exposed H2K myoblast cells to US with varying intensity of exposure and duration to evaluate its effect on cell viability and transfection efficiency using as endpoints transfection rate, average fluorescence intensity (fluorescence normalised by the number of transfected cells) and overall expression (the product of transfection rate and average fluorescence intensity) as indices. Cell viability decreased with exposure time and intensity, consistent with previous findings. Optimal setting of US was observed at the range of 0.5 to 1 W cm(-2) with duration of 20 s, producing maximum efficiency (transfection = 4.5%) in gene transfection with minimum cell toxicity (cell viability = 83%). Higher intensity alone or in combination with low intensity and long duration did not improve cell viability and transfection. The increase of eGFP (enhanced green fluorescence protein) plasmid concentration up to 200 microg per mL was related to an increase in average fluorescence intensity and overall expression. However, transfection rate saturated when DNA concentration reached 50 microg per mL despite initial increase with DNA concentration. The average fluorescence intensity was linearly proportional to the logarithm of DNA concentration, suggesting a diffusion-based model for DNA uptake under sonoporation. We conclude that low-intensity US irradiation provides a safe and effective alternative for gene delivery.

Evidence for spleen-specific uptake of a microbubble contrast agent: a quantitative study in healthy volunteers.

PURPOSE: To evaluate the pharmacokinetics of the microbubble contrast agent BR1. MATERIALS AND METHODS: Twenty healthy volunteers were injected via arm vein with a 1.2-mL bolus of BR1. Ultrasonographic images of liver and right kidney and of spleen and left kidney were obtained intermittently for 5 minutes with low-mechanical-index software (to minimize microbubble destruction) that shows stationary microbubbles in green. Percentage total uptake was calculated as the number of green pixels in the region of interest for each organ over time, divided by the total pixels. Relative uptake, the ratio of total uptake in liver to that in right kidney and of total uptake in spleen to that in left kidney, and differential uptake, the difference in total uptake between liver and right kidney and between spleen and left kidney, were calculated. Total uptake for each organ was plotted against time, and the gradient of a best-fit straight line was calculated. Wilcoxon signed rank test was used to compare mean uptake values in each subject. Mann-Whitney U test was used for comparisons in sex and age. RESULTS: Total uptake declined over 5 minutes in left and right kidney and in liver (from 88% +/- 10% [1 minute] to 67% +/- 14% [5 minutes]), but not in spleen (range, 90%-99%). Mean relative uptake +/- 1 SD for spleen increased from 2.3 +/- 0.7 (1 minute) to 3.7 +/- 2.3 (5 minutes) (P =.005) but for liver was constant: 2.1 +/- 0.9 (1 minute) and 2.3 +/- 0.4 (5 minutes) (P =.06). Mean differential uptake +/- 1 SD for spleen increased from 51.3% +/- 14.9% (1 minute) to 65.0% +/- 9.1% (5 minutes) (P =.002). Significant difference was seen over time in total uptake gradients between spleen and left kidney (P =.014) but not between liver and right kidney or right and left kidney. No difference was seen between men and women or with age. CONCLUSION: BR1 produces spleen-specific enhancement that is longer (5 minutes) than the blood pool phase.

Improved detection of hepatic metastases with pulse-inversion US during the liver-specific phase of SHU 508A: multicenter study.

PURPOSE: To compare conventional B-mode ultrasonography (US) alone with the combination of conventional B-mode US and contrast material-enhanced (SHU 508A) late-phase pulse-inversion US for the detection of hepatic metastases by using dual-phase spiral computed tomography (CT) as the standard of reference. MATERIALS AND METHODS: One hundred twenty-three patients underwent conventional US, US in the liver-specific phase of SHU 508A, and single-section spiral CT. US and CT images were assessed by blinded readers. Differences in sensitivity, specificity, and the number and smallest size of metastases at conventional and contrast-enhanced US were compared by using CT as the standard of reference. Lesion conspicuity was assessed objectively (quantitatively) and subjectively by one reader before and after contrast material administration. RESULTS: In 45 of 80 (56%) patients with metastases, more metastases were seen at contrast-enhanced US than at conventional US. In three of these patients, conventional US images appeared normal. The addition of contrast-enhanced US improved sensitivity for the detection of individual metastases from 71% to 87% (P <.001). On a patient basis, sensitivity improved from 94% to 98% (P =.44), and specificity improved from 60% to 88% (P <.01). Contrast enhancement improved the subjective conspicuity of metastases in 66 of 75 (88%) patients and the objective contrast by a mean of 10.8 dB (P <.001). Contrast-enhanced US showed more metastases than did CT in seven patients, and CT showed more than did contrast-enhanced US in one of 22 patients in whom an independent reference (magnetic resonance imaging, intraoperative US, or pathologic findings) was available. CONCLUSION: Contrast-enhanced US improved sensitivity and specificity in the detection of hepatic metastases.

Inhibition of beta-lactamases by monocyclic acyl phosph(on)ates.

The cyclic acyl phosph(on)ates, 1-hydroxy-5-phenyl-2,6-dioxaphosphorinone(3)-1-oxide, its 4-phenyl isomer, and the phosphonate (2-oxo) analogue of the latter inhibited typical class A (TEM-2) and class C (Enterobacter cloacae P99) beta-lactamases in a time-dependent fashion. No enzyme-catalyzed turnover was detected in any case. The interactions occurring were interpreted in terms of the reaction scheme E + I left arrow over right arrow EI left arrow over right arrow EI', where EI is a reversibly formed noncovalent complex, and EI' is a covalent complex. Reactions of the cyclic phosphates with the P99 beta-lactamase were effectively irreversible, while that of the 4-phenyl cyclic phosphate with the TEM beta-lactamase was slowly reversible. The 4-phenyl cyclic phosphate was generally the most effective inhibitor, both kinetically and thermodynamically, with second-order rate constants of inactivation of both enzymes around 10(4) s(-1) M(-1). This compound also bound noncovalently to both enzymes, with dissociation constants of 25 microM from the P99 enzyme and 100 microM from the TEM. It is unusual to find an inhibitor equally effective against the TEM and P99 enzymes; the beta-lactamase inhibitors currently employed in medical practice (e.g., clavulanic acid) are significantly more effective against class A enzymes. The results of lysinoalanine analysis after hydroxide treatment of the inhibited enzymes and of a (31)P nuclear magnetic resonance spectrum of one such complex were interpreted as favoring a mechanism of inactivation by enzyme acylation rather than phosphylation. Molecular modeling of the enzyme complexes of the 4-phenyl phosphate revealed bound conformations where recyclization and thus reactivation of the enzyme would be difficult. The compounds studied were turned over slowly or not at all by acetylcholinesterase and phosphodiesterase I.