A Holstein cow-calf model for the transfer of ciprofloxacin through milk after a long-term intravenous infusion.

This study is part of an ongoing effort to develop animal models that provide milk and sufficient infant (offspring) plasma samples to fully describe a drug's pharmacokinetics to quantitate the risk to the nursing infant. Ciprofloxacin was administered to six healthy Holstein cows as a constant rate intravenous infusion (flow rate was weight adjusted) to achieve a steady-state concentration of approximately 300 ng/mL for 7 days. Plasma and milk samples were collected from the cow at regular intervals over the course of the 7 days. The plasma and milk samples were analyzed for ciprofloxacin by high-performance liquid chromatography. The milk was fed to calves, and calf plasma samples were analyzed to study the lactational transfer of ciprofloxacin from dam to nursing neonate. Remarkably, concentrations of ciprofloxacin in milk were 45 times higher than plasma drug concentrations in the dam. Approximately 6% of the administered dose was transferred to the milk, resulting in an average oral dose of 0.5 mg/kg to the calves with every feeding. The drug did not accumulate in the calves, and plasma concentrations were between one-tenth and one-fifth the plasma concentrations of the dam.

The evolution of prenatal screening and diagnosis and its impact on an unselected population over an 18-year period.

OBJECTIVE: To review changes in and impact of prenatal screening and diagnosis. DESIGN: Population-based congenital anomaly register study. SETTING: Oxfordshire. POPULATION: Congenital anomalies confirmed and those suspected prenatally, delivered 1991-2008. METHODS: Analysis of proportions of congenital anomalies confirmed and those suspected prenatally. MAIN OUTCOME MEASURES: Birth prevalence, prenatal detection rates, pregnancy outcomes. RESULTS: A total of 2651 (2.3%) infants/fetuses had a congenital anomaly diagnosed. There were 3839 suspected or confirmed cases, 2847 due to a prenatal suspicion, of which 1659 had an anomaly confirmed at delivery, and 1188 false-positive diagnoses, 91% due to reporting ultrasound normal variants. The percentage of prenatal notifications rose from 48% in 1991-93 to 83-88% from 1996 to 2003 and dropped to 61% in 2006-08, partly reflecting changes in the reporting of normal variants. Reporting these increased the prenatal diagnosis rate from 53 to 63% with an increase in false-positive rate from 0.09 to 1.04%. A total of 722 (44% of prenatally detected affected fetuses) resulted in termination; 48% of these had chromosome anomalies, 34% had isolated structural anomalies, 7% had multiple anomalies, 10% had familial disorders; 42% had lethal anomalies and 58% would probably have survived the neonatal period giving an estimated 20% reduction in birth prevalence of congenital anomalies compatible with survival because of terminations. CONCLUSION: There has been an improvement in prenatal detection of congenital anomalies over the two decades studied. The recognition that reporting normal variants, although increasing prenatal detection rates, leads to an increase in false-positive diagnoses has had an impact on practice that has redressed the balance between these two effects.

Intra- and interobserver variability in fetal ultrasound measurements.

OBJECTIVE: To assess intra- and interobserver variability of fetal biometry measurements throughout pregnancy. METHODS: A total of 175 scans (of 140 fetuses) were prospectively performed at 14-41 weeks of gestation ensuring an even distribution throughout gestation. From among three experienced sonographers, a pair of observers independently acquired a duplicate set of seven standard measurements for each fetus. Differences between and within observers were expressed in measurement units (mm), as a percentage of fetal dimensions and as gestational age-specific Z-scores. For all comparisons, Bland-Altman plots were used to quantify limits of agreement. RESULTS: When using measurement units (mm) to express differences, both intra- and interobserver variability increased with gestational age. However, when measurement of variability took into account the increasing fetal size and was expressed as a percentage or Z-score, it remained constant throughout gestation. When expressed as a percentage or Z-score, the 95% limits of agreement for intraobserver difference for head circumference (HC) were ± 3.0% or 0.67; they were ± 5.3% or 0.90 and ± 6.6% or 0.94 for abdominal circumference (AC) and femur length (FL), respectively. The corresponding values for interobserver differences were ± 4.9% or 0.99 for HC, ± 8.8% or 1.35 for AC and ± 11.1% or 1.43 for FL. CONCLUSIONS: Although intra- and interobserver variability increases with advancing gestation when expressed in millimeters, both are constant as a percentage of the fetal dimensions or when reported as a Z-score. Thus, measurement variability should be considered when interpreting fetal growth rates.

Taking immunogenicity assessment of therapeutic proteins to the next level.

Therapeutic proteins provide innovative and effective therapies for numerous diseases. However, some of these products are associated with unwanted immunogenicity that may lead to clinical consequences such as reduced or loss of efficacy, altered pharmacokinetics (PK), general immune and hypersensitivity reactions, and neutralisation of the natural counterpart (e.g. the physiological hormone). Regulatory guidance on immunogenicity assessment needs to take into consideration a great diversity of products, indications and patient populations as well as constantly advancing manufacturing technologies. Such guidance needs to be sufficiently specific while, at the same time, allowing interactive discussion and adjusted benefit-risk weighing of each product on a case-by-case basis, e.g. for a unique treatment of a life threatening disease acceptable treatment risks may differ considerably from the ones in case of less serious disease. This theme was the focus of the international conference "Taking immunogenicity assessment of therapeutic proteins to the next level", held at the Paul-Ehrlich-Institut in Langen, Germany, on the 10-11. June 2010. The objectives of the conference were to highlight how the field could move from that of a mere description of risk factors to a system of risk assessment and mitigation, as well as an understanding of the impact of unwanted immunogenicity on the overall benefit/risk consideration for a medicinal product. More than 150 experts from industry, academia and regulatory authorities worldwide discussed the phenomenon of undesired immunogenicity from different perspectives. The conference focussed on issues relevant to three areas: (1) new European guidelines that are currently the subject of discussion; (2) testing strategies for immunogenicity assessment; and (3) scientific progress on the product-related factors that may contribute to the development of pathogenesis of immunogenicity, in particular in the field of protein aggregation and post-translational modifications. This report provides an overview of issues, insights, and conclusions that were discussed and achieved during the meeting.

Intermittent mydriasis associated with carotid vascular occlusion.

PurposeTo describe two cases of stereotyped, intermittent, neurologically isolated, unilateral mydriasis in patients with a history of acquired internal carotid artery (ICA) occlusive disease on the ipsilateral side.PatientsTwo patients with intermittent mydriasis.MethodsCase Series.ResultsCase one: A 78-year-old man experienced 10 episodes of intermittent, unilateral, and painless mydriasis in the left eye and had 100% occlusion of the left ICA artery due to atherosclerotic disease. Case two: A 26-year-old woman with history of migraine developed new painless, intermittent episodes of unilateral mydriasis after sustaining chest trauma and was diagnosed with subsequent dissection and 65% occlusion of the ipsilateral ICA. Neither patient developed permanent anisocoria.ConclusionBenign episodic unilateral mydriasis (BEUM) typically presents in young women with a history of migraine. To our knowledge, these are the first cases of episodic, unilateral, neurologically isolated mydriasis associated with occlusive disease of the ICA in the English language ophthalmic literature. We hypothesize that transient dysfunction of the autonomic nervous system related to the ICA disease may account for the intermittent mydriatic episodes in these patients and we recommend consideration for imaging of the ICA in patients with atypical features for BEUM (for example, old age or males, non-isolated mydriasis, or recent trauma).

A whole system approach to improving mortality associated with acute kidney injury.

BACKGROUND: Acute kidney injury (AKI) is in the main managed by non-nephrologists, many who feel challenged by or lack awareness of the complexity that the renal element adds to their patients' care. National reports have raised major concerns about the quality of care and have predicted that mortality reductions of 30% are achievable with good medical practice. AIM: This quality improvement project evaluated whether a whole system approach could improve outcomes for patients with AKI. DESIGN AND METHODS: Quality improvement methodology was used to understand hospital patterns, processes and professional knowledge. Change concepts were developed which included management of patients at risk, staff education and awareness program, development of a patient specific electronic alert to prompt diagnosis, easy to remember care bundle (ABCDE-IT), dedicated outreach team and patient and family empowerment leaflet. RESULTS: Statistical process control analysis was used to verify outcomes over time. A shift in the in-hospital mortality rate corresponded to a relative 23.2% reduction in mortality and was sustained over the next 33 months (P < 0.0001). The favourable shift in mortality was temporally distinct from the improved AKI detection rate. This timeframe corresponded to lying below the 99.8% lower confidence limit in comparison with all English acute trusts for comparative AKI specific SHMI/HSMR mortality rates. Length of stay also reduced shortly after onset of the project by 14.1% or 2.6 day reduction (P < 0.0001). CONCLUSION: This project demonstrated that an integrated, whole-system approach is necessary to ensure sustained improvements in AKI mortality and length of stay.

Outcome of antenatally suspected congenital cystic adenomatoid malformation of the lung: 10 years' experience 1991-2001.

OBJECTIVE: To determine the outcome of antenatally suspected congenital cystic adenomatoid malformation of the lung (CCAM) over a 10 year period. METHODS: This is a retrospective study of all babies diagnosed antenatally in the Prenatal Diagnosis Unit and delivered in Oxford between 1991 and 2001. Data were obtained from the Oxford Congenital Anomaly Register, theatre records, and histopathology reports. RESULTS: Twenty eight cases of CCAM were diagnosed antenatally. Five pregnancies were terminated. Data are available on all 23 of the pregnancies that continued and resulted in two neonatal deaths and 21 surviving babies. Eleven of the 23 cases (48%) showed some regression of the lesion antenatally, and four of these cases appeared to resolve completely on prenatal ultrasound. Three of the 23 babies (13%) were symptomatic in the early neonatal period, and three developed symptoms shortly afterwards. Seventeen of the 23 babies (74%) were asymptomatic, of whom 12 had abnormalities on chest radiograph or computed tomography scan and had elective surgery. Two babies (8%) had completely normal postnatal imaging, and three had abnormalities which resolved in the first year of life. Seventeen of the 23 babies (74%) had surgery. Histology at surgery was heterogeneous. Of the 23 live births, all 21 survivors (91%) are well at follow up or have been discharged. CONCLUSIONS: All babies diagnosed antenatally with CCAM require postnatal imaging with computed tomography irrespective of signs of antenatal resolution. In asymptomatic infants, the recommendations are close follow up and elective surgery for persistent lesions within the first year of life. Histology at surgery was heterogeneous, and this should be considered when counselling parents.

Crystal structures of HIV-1 reverse transcriptases mutated at codons 100, 106 and 108 and mechanisms of resistance to non-nucleoside inhibitors.

Leu100Ile, Val106Ala and Val108Ile are mutations in HIV-1 reverse transcriptase (RT) that are observed in the clinic and give rise to resistance to certain non-nucleoside inhibitors (NNRTIs) including the first-generation drug nevirapine. In order to investigate structural mechanisms of resistance for different NNRTI classes we have determined six crystal structures of mutant RT-inhibitor complexes. Val108 does not have direct contact with nevirapine in wild-type RT and in the RT(Val108Ile) complex the biggest change observed is at the distally positioned Tyr181 which is > 8 A from the mutation site. Thus in contrast to most NNRTI resistance mutations RT(Val108Ile) appears to act via an indirect mechanism which in this case is through alterations of the ring stacking interactions of the drug particularly with Tyr181. Shifts in side-chain and inhibitor positions compared to wild-type RT are observed in complexes of nevirapine and the second-generation NNRTI UC-781 with RT(Leu100Ile) and RT(Val106Ala), leading to perturbations in inhibitor contacts with Tyr181 and Tyr188. Such perturbations are likely to be a factor contributing to the greater loss of binding for nevirapine compared to UC-781 as, in the former case, a larger proportion of binding energy is derived from aromatic ring stacking of the inhibitor with the tyrosine side-chains. The differing resistance profiles of first and second generation NNRTIs for other drug resistance mutations in RT may also be in part due to this indirect mechanism.

Structural mechanisms of drug resistance for mutations at codons 181 and 188 in HIV-1 reverse transcriptase and the improved resilience of second generation non-nucleoside inhibitors.

Mutations at either Tyr181 or Tyr188 within HIV-1 reverse transcriptase (RT) give high level resistance to many first generation non-nucleoside inhibitors (NNRTIs) such as the anti-AIDS drug nevirapine. By comparison second generation inhibitors, for instance the drug efavirenz, show much greater resilience to these mutations. In order to understand the structural basis for these differences we have determined a series of seven crystal structures of mutant RTs in complexes with first and second generation NNRTIs as well as one example of an unliganded mutant RT. These are Tyr181Cys RT (TNK-651) to 2.4 A, Tyr181Cys RT (efavirenz) to 2.6 A, Tyr181Cys RT (nevirapine) to 3.0 A, Tyr181Cys RT (PETT-2) to 3.0 A, Tyr188Cys RT (nevirapine) to 2.6 A, Tyr188Cys RT (UC-781) to 2.6 A and Tyr188Cys RT (unliganded) to 2.8 A resolution. In the two previously published structures of HIV-1 reverse transcriptase with mutations at 181 or 188 no side-chain electron density was observed within the p66 subunit (which contains the inhibitor binding pocket) for the mutated residues. In contrast the mutated side-chains can be seen in the NNRTI pocket for all seven structures reported here, eliminating the possibility that disordering contributes to the mechanism of resistance. In the case of the second generation compounds efavirenz with Tyr181Cys RT and UC-781 with Tyr188Cys RT there are only small rearrangements of either inhibitor within the binding site compared to wild-type RT and also for the first generation compounds TNK-651, PETT-2 and nevirapine with Tyr181Cys RT. For nevirapine with the Tyr188Cys RT there is however a more substantial movement of the drug molecule. We conclude that protein conformational changes and rearrangements of drug molecules within the mutated sites are not general features of these particular inhibitor/mutant combinations. The main contribution to drug resistance for Tyr181Cys and Tyr188Cys RT mutations is the loss of aromatic ring stacking interactions for first generation compounds, providing a simple explanation for the resilience of second generation NNRTIs, as such interactions make much less significant contribution to their binding.

Lipoprotein-associated phospholipase A(2), platelet-activating factor acetylhydrolase: a potential new risk factor for coronary artery disease.

A specific and robust immunoassay for the lipoprotein-associated phospholipase A(2) (Lp-PLA(2)), platelet-activating factor acetylhydrolase, is described for the first time. The immunoassay was used to evaluate possible links between plasma Lp-PLA(2) levels and atherosclerosis risk amongst susceptible individuals. Such an investigation was important because Lp-PLA(2) participates in the oxidative modification of low density lipoprotein by cleaving oxidised phosphatidylcholines, generating lysophosphatidylcholine and oxidised free fatty acids. The majority of Lp-PLA(2) was found associated with LDL (approximately 80%) and, as expected, enzyme levels were significantly positively correlated to LDL cholesterol. Plasma Lp-PLA(2) levels were significantly elevated in patients with angiographically proven coronary artery disease (CAD) when compared with age-matched controls, even though LDL cholesterol levels did not differ significantly. Indeed, when included in a general linear model with LDL cholesterol and other risk factors, Lp-PLA(2) appeared to be an independent predictor of disease status. We propose, therefore, that plasma Lp-PLA(2) mass should be viewed as a potential novel risk factor for CAD that provides information related to but additional to traditional lipoprotein measurements.

Interaction of a plasmin A-chain/t-PA B-chain hybrid enzyme with plasma inhibitors in vivo and in vitro.

A hybrid plasminogen activator consisting of the "A" chain of plasmin linked to the "B" chain of rt-PA was inhibited in vitro in human and guinea pig plasmas 4 to 5-fold more rapidly than its parent activator, two-chain t-PA. Using zymographic and autoradiographic techniques together with the use of immunodepleted plasma the major inhibitor was identified as alpha-2-antiplasmin. The pharmacokinetic profile of the hybrid in guinea pigs was determined by two different methods: disappearance of fibrinolytic activity and removal of radiolabelled hybrid from the circulation. Fibrinolytic activity was cleared rapidly via inhibitory mechanisms, whilst radiolabelled material was cleared considerably more slowly due to the formation of hybrid-inhibitor complexes. When the active site of the hybrid was reversibly acylated inhibitory mechanisms were evaded and a prolonged pharmacokinetic profile of activity was observed.

Amniotic fluid concentrations of dimeric inhibins, activin A and follistatin in pregnancy.

OBJECTIVE: The feto-placental unit is the major source of circulating concentrations of inhibin A and activin A in human pregnancy. The aim of this study was to measure the amniotic fluid concentrations of inhibin A, inhibin B, activin A and follistatin in pregnancies bearing male and female fetuses. DESIGN AND METHOD: Amniotic fluid samples collected by amniocentesis were stored at -20 degrees C. Dimeric inhibins, 'total' activin A and 'total' follistatin were measured using specific two-site enzyme immunoassays. Samples were assayed blindly and the information on fetal sex was obtained from the cytogenetics laboratory. RESULTS: Data show that amniotic fluid concentrations of inhibin A, inhibin B and activin A gradually increase with gestation whilst concentrations of follistatin are similar between weeks 15 and 20 of pregnancy. Mean amniotic fluid levels of inhibin A and inhibin B at 16 and 17 weeks gestation and mean activin A levels at 15 and 16 weeks gestation are considerably lower in pregnancies with male (n=24) compared with female (n=28) fetuses. Levels of follistatin are not different in the male and female fetal pregnancies at any studied gestation. CONCLUSIONS: The results indicate that amniotic fluid contains high concentrations of inhibins (A and B), activin A and follistatin in early pregnancy suggesting that these hormones are produced by the fetal membranes and may be involved in the development of the fetus.

Prenatal determination of fetal rhesus D status by DNA amplification of peripheral blood of rhesus-negative mothers.

We have developed a sensitive PCR-based assay for the RhD gene and used it to detect circulating fetal cells from RhD-positive fetuses from peripheral blood of RhD-negative mothers. With further improvement in diagnostic accuracy, this assay may have implications in the management of RhD-sensitized pregnancies in women whose partners are heterozygous for the RhD gene. Further studies are required to determine the relationship between maternal anti-D levels and circulating fetal cell numbers.

Retained cocaine conditioned place preference in D1 receptor deficient mice.

The role of the D1 dopamine receptor subtype in mediating cocaine effects was examined in mice in which the D1 receptor gene had been ablated by homologous recombination. Cocaine reward was assessed by conditioned place preference experiments using mice which had either one allele (+/-) or both alleles (-/-) of the D1 dopamine receptor gene disrupted and in their wild type (+/+) littermates. Cocaine conditioning resulted in similar increases in preference for drug-paired environments in mice of each of the three genotypes. Cocaine did not alter locomotor activity levels in homozygous, D1 knockout mice -/-, whereas increased activity was noted in both +/+ and +/- animals. These results are consistent with the idea that the D1 receptor is involved in the locomotor stimulant effects of cocaine, but has little role in a major test of the rewarding and reinforcing effects of the drug.

Omphalocele and pericardial effusion: possible sonographic markers for the pentalogy of Cantrell or its variants.

BACKGROUND: The pentalogy of Cantrell consists of defects involving the diaphragm, abdominal wall, pericardium heart, and lower sternum. CASES: We report three cases of the pentalogy of Cantrell (variant form), involving an omphalocele complicated by an anterior diaphragmatic hernia. In two cases, a pericardial effusion was noted at antenatal scanning; the case without a pericardial effusion had an intact diaphragmatic pericardium at surgical repair. CONCLUSION: The presence of a pericardial effusion in association with an omphalocele should prompt a detailed search for other features of the pentalogy of Cantrell or its variants.

Circadian rhythm in bladder volumes in the term human fetus.

Circadian rhythms have been identified in a variety of maternal and fetal biophysical and endocrinologic parameters. The authors have undertaken a 24-hour study to identify the normal variation in fetal bladder volumes in the healthy, term human fetus. A significant decrease in fetal bladder volumes occurred between 2400 hours and 0600 hours when compared with other times of the day. It is suggested that this fall in fetal bladder volumes may be related to fetal cardiovascular or adrenal gland function.

Uterine-peritoneal amniotic fluid leakage: an unusual complication of intrauterine shunting.

Bilateral pleuroamniotic shunting was performed at 33 weeks' gestation in a fetus with bilateral hydrothorax, hydrops, and gross polyhydramnios. The procedure was successful, but acute amniotic fluid leakage into the maternal peritoneal cavity occurred soon after. This produced marked maternal discomfort and transient oligohydramnios, with consequent fetal distress. Expectant management was adopted in view of fetal lung immaturity. Resolution of maternal ascites occurred within 24 hours and the fetal heart rate normalized as amniotic fluid reaccumulated. The pregnancy progressed uneventfully thereafter.

Intrauterine transfusion in an Rh-immunized twin pregnancy: a case report of successful outcome and a review of the literature.

Fifteen sets of twins have been reported among 2331 pregnancies complicated by Rh alloimmunization of sufficient severity to warrant intrauterine transfusions. Four of the 15 sets were managed in Winnipeg, Canada. One of the four is described in detail in the present report. Serial amniocenteses (N = 15) and intrauterine transfusions (N = 8) were used in the management of the dizygous affected twin fetuses with a favorable outcome. Factors contributing to the survival of the twins are described.

Preventive intervention for maltreated preschool children: impact on children's behavior, neuroendocrine activity, and foster parent functioning.

OBJECTIVE: This article describes the results of a pilot study that evaluated the effectiveness of the Early Intervention Foster Care (EIFC) program in the period immediately following a child's placement in a new foster home. METHOD: Data were collected from an EIFC group, a regular foster care group, and a community comparison group-each with 10 participants-via questionnaires for children and their caretakers and salivary cortisol sampling. RESULTS: EIFC foster parents adopted and maintained positive parenting strategies, EIFC children's behavioral adjustment improved, and changes occurred in several salivary cortisol measures. Moreover, regular foster care children exhibited decrements in functioning in several areas over the same time period. CONCLUSIONS: Results are discussed with regard to how such research fits into a larger program of prevention research for high-risk preschool children.

Contrast sonography for uterine cavity assessment: a comparison of conventional two-dimensional with three-dimensional transvaginal ultrasound; a pilot study.

OBJECTIVE: To compare conventional two- (2-D) and three-dimensional (3-D) scanning of the uterine cavity with and without saline contrast medium. DESIGN: Observational pilot study. SETTING: University-based fertility service. PATIENT(S): Ten IVF patients requiring uterine cavity assessment. INTERVENTION(S): Two-dimensional and 3-D transvaginal scans before and after injection of saline into the uterine cavity. MAIN OUTCOME MEASURE(S): Number and type of uterine cavity abnormalities detected by each technique. RESULT(S): The 2-D scanning suggested cavity abnormalities in 4 of 10 women (fibroids, 3; hyperechoeic thick endometrium, 1). The 3-D scanning confirmed these and revealed one additional abnormality suggestive of a uterine septum. The 2-D scanning with saline injection diagnosed abnormalities in 5 of 10 (uterine septum, 1; fibroids, 3; endometrial polyp, 1). The 3-D contrast scanning with saline did not add any further information to 2-D contrast scanning with saline. CONCLUSION(S): In this pilot study, 3-D scanning to assess the uterine cavity appeared to offer no advantages over conventional 2-D contrast sonography.