Estimation of initial phenobarbital dosing in term neonates with moderate-to-severe hypoxic ischaemic encephalopathy following perinatal asphyxia.

WHAT IS KNOWN AND OBJECTIVE: Phenobarbital is the first-line treatment of seizures in asphyxiated neonates; however, due to the high pharmacokinetic variability in this population, there is no consensus on the optimal dosage regimen. This study was conducted to identify variables that affect phenobarbital fate during routine clinical care and then to evaluate the dosage schedule that could be applied in term asphyxiated neonates with respect to achieving the target therapeutic range. METHODS: Phenobarbital pharmacokinetics was calculated based on serum concentrations measurements using one-compartmental model. Body weight, body surface area, gestational age, creatinine clearance, total bilirubin, alanine aminotransferase, aspartate aminotransferase, international normalized ratio, Apgar scores, umbilical cord arterial pH and base excess were explored as covariates in linear regression models. Based on this analysis, phenobarbital loading and maintenance dose regimen were projected. RESULTS AND DISCUSSION: In the whole study population (N = 36), phenobarbital volume of distribution, clearance and half-life median (interquartile range) values were 0.49 (0.38-0.59) L/kg, 0.0045 (0.0034-0.0055) L/h/kg and 75.1 (60.2-103.3) hours, respectively. The drug volume of distribution was associated with body weight, length and body surface area, whereas clearance was not in relationship with any explored features. Weight-normalized loading dose of 15 mg/kg and weight-normalized daily maintenance dose of 3 mg/kg proved to be optimal in our study population to reach phenobarbital therapeutic range. WHAT IS NEW AND CONCLUSIONS: This study presents basis for phenobarbital initial dosing in term asphyxiated neonates during first week of life. Phenobarbital weight-normalized loading dose of 15 mg/kg lead to simulated target peak concentrations in 72% of neonates, weight-normalized maintenance dose of 3 mg/kg lead to steady state within therapeutic window in the same proportion of patients.

[Big surprise in the lesser pelvis]. / Große Überraschung im kleinen Becken.

A 17-year-old young man presented at our clinic with asymptomatic microhematuria. Ultrasonography and computer tomography found an intraperitoneal lesion of unknown dignity located on top of the bladder. Surgical exploration and histological examination confirmed the diagnosis of a secondary pelvic spleen, a lien bipartitus.

[Predictors for presence of residual tumor in follow-up transurethral resection of bladder tumors: single center results]. / Vorhersageparameter für das Vorliegen eines Residualtumors bei der TUR-B-Nachresektion: single-center-Ergebnisse.

BACKGROUND: European Association of Urology (EAU) guidelines recommend a follow-up transurethral resection of bladder tumors (reTUR-B) for intermediate and high-risk non-muscle invasive bladder cancer (NMIBC) 2-6 weeks after the initial resection. The purpose of this study was to find parameters which indicate the presence of residual tumor in reTUR-B and to evaluate the prognostic value. PATIENTS AND METHODS: The data from all patients treated with TUR-B between January 2005 and December 2008 were retrospectively evaluated. The residual tumor rate was correlated with age, sex, staging, grading, risk group, multifocality and surgeon's level of training. RESULTS: A total number of 555 TUR-B operations were carried out and 179 patients received reTUR-B according to the EAU guidelines. Age (p=0.8), sex (p=0.7), initial staging (p=0.2), initial grading (p=0.3) and surgeon's level of training (p=0.7) did not have an impact on the rate of residual tumor in reTUR-B. Tumors categorized as high risk according to the EAU risk score in initial TUR-B (p<0.01) and multifocality (p=0.01) were associated with significantly higher rates of residual tumor. CONCLUSIONS: A reTUR-B is strongly indicated in high risk bladder tumors as well as multifocal tumors showing a significantly increased residual tumor rate. Other clinical parameters showed no prognostic value for the existence of residual tumor in reTUR-B.

Local treatment of hand-foot syndrome with uridine/thymidine: in vitro appraisal on a human keratinocyte cell line HaCaT.

5-fluorouracil (5-FU) is one of the most commonly used antineoplastic drugs in the anticancer therapy. The hand-foot (HF) syndrome (palmar-plantar erythrodysesthesia) is an adverse effect frequently related to long-term i.v. administration of 5-FU or its orally applicable prodrug capecitabine. Its severity can even lead to interruption of the otherwise effective anticancer therapy. Tentative practice in some clinics has shown that topical application of 10% uridine ointment is beneficial for calming down the HF syndrome. This study is focused on verifying the alleged protective activity of uridine in the in vitro model of cultured human keratinocyte cell line HaCaT. We also tested the protective effects of thymidine alone or uridine-thymidine combination. The cellular viability time progression was measured in order to evaluate the effect of protective agents by three different types of cytopathogenicity tests-NTCA test (non-destructive test of cellular activity), modified MTT test and RTCA (real-time cell analyser, Roche). All three methods proved the ability of uridine and uridine-thymidine combination to protect keratinocytes against 5-FU damage in vitro. While thymidine alone did not show any remarkable effect, the thymidine-uridine combination demonstrated enhanced protective activity compared to uridine alone. Our findings provided the supporting rationale for using uridine or uridine-thymidine ointments in the HF syndrome local therapy.

[Clinical endpoints during rabies vaccine control tests]. / Klinische Endpunkte bei der Tollwutimpfstoffprüfung.

All warm-blooded animals, including humans, are susceptible to rabies. The infection with the virus leads inevitably to the death of the recipient. Vaccines and anti-serums are at present the only possibility to prevent rabies infections in the human and veterinary medicine. In order to be able to guarantee the production of a reliably safe and effective vaccine, each batch has to be tested. These tests contain animal experiments. Alone for efficacy tests of rabies vaccines done for each batch far over one hundred mice are used. In a defined time period in challenge experiments the number of deaths rates of immunised animals are compared with non vaccinated control animals. At present several replacement methods for this test are in development. However, so far none of them has been validated in an international multicenter study. The modifications necessary for example in the WHO (World Health Organization) or O.I.E. (Office International d"Epizooties) guidelines will need several more years. Therefore, this test method will be internationally used in the foreseeable future. In order to avoid unnecessary suffering of the animals, we looked for signs, which can be used to replace lethality as criterion. For this purpose score-sheets were developed, on which the observed clinical signs were recorded. The decrease of body-temperature, which was measured with transplanted transponders, occurred too late to be usable. A clear reduction of the body weight is the earliest sign of an illness. Slow and circular movements, followed by cramps and paralyses, are the first neurological symptoms of rabies. The combination of these parameters can serve as a reliable indicator for humane endpoints. This saves the mice on average between four and five days of the most stressful phase of the experiment and is a clear "refinement" of the test conditions in the sense of the 3R. A video which shows the clinical signs is available.

Identifying women with cervical neoplasia: using human papillomavirus DNA testing for equivocal Papanicolaou results.

CONTEXT: A Papanicolaou (Pap) test result of atypical squamous cells of undetermined significance (ASCUS) presents a clinical challenge. Only 5% to 10% of women with ASCUS harbor serious cervical disease, but more than one third of the high-grade squamous intraepithelial lesions (HSILs) in screening populations are identified from ASCUS Pap test results. OBJECTIVE: To determine whether human papillomavirus (HPV) DNA testing of residual material from liquid-based Pap tests and referral of cases found to be HPV-positive directly to colposcopy could provide sensitive detection of underlying HSILs in women with ASCUS Pap results, compared with repeat Pap testing. DESIGN AND SETTING: Natural history of women with ASCUS Pap smear results, all of whom had liquid-based cytology, HPV testing, and subsequent repeat Pap tests and colposcopy with histologic evaluation, conducted at 12 gynecology clinics in a large managed care organization between October 1995 and June 1996. PARTICIPANTS: From a cohort of 46009 women who had routine cervical examinations, 995 women with Pap test results of ASCUS who consented to participate were identified. MAIN OUTCOME MEASURES: Cervical histology, HPV test results, and repeat Pap smear results, and sensitivity of HPV testing to identify patients found to have HSIL+ histology. RESULTS: Of 995 participants with ASCUS Pap test results, 973 had both a definitive histologic diagnosis and HPV result. Sixty-five (6.7%) had histologic HSIL or cancer. For women with histologic HSIL+, the HPV test was positive in 89.2% (95% confidence interval [CI], 78.4%-95.2%), and the specificity was 64.1 % (95% CI, 60.9%-67.2%). The repeat Pap smear result was abnormal in 76.2% (95% CI, 63.5%-85.7%). Triage based on HPV testing only or on repeat Pap testing only would refer similar proportions (approximately 39%) to colposcopy. The sensitivity of HPV DNA testing for HSIL was equivalent to, if not greater than, that of the repeat Pap test. We further estimated that an HPV-based algorithm including the immediate colposcopy of HPV-positive women, and then repeat Pap testing of all others, would provide an overall sensitivity of 96.9% (95% CI, 88.3%-99.5%). CONCLUSIONS: For women with ASCUS Pap tests, HPV DNA testing of residual specimens collected for routine cervical cytology can help identify those who have underlying HSIL. By testing the specimen collected at initial screening, the majority of high-risk cases can be identified and referred for colposcopy based on a single screening.

Determining the utility and effectiveness of the NeoPath AutoPap 300 QC System used routinely.

OBJECTIVE: To evaluate routine use of the NeoPath AutoPap 300 QC System (AP 300) as it influences diagnostic quality and operations in a large cytology laboratory. STUDY DESIGN: During a three-month period, 35,143 conventionally prepared consecutive cervical cytologic smears taken from non-high-risk women and evaluated as negative by our staff of 25 cytotechnologists were selected for processing by four AP 300 instruments. Slides flagged for review by the AP 300 were reevaluated by our five quality control (QC) cytotechnologists. False negative (FN) results were compared with results of our current practice (CP), random-selection QC method, used during the preceding six months. RESULTS: A 240% increase was seen in the FN detection rate for atypical squamous cells of undetermined significance (ASCUS) and squamous intraepithelial lesions (SIL) (n AP = 65 FN in 5,034 QC, n CP = 77 FN in 22,052 QC) and a 744% increase in the FN detection rate for low and high grade SIL (n AP = 24 FN/5,034 QC, n CP = 12 FN/22,052 QC). The rate of overcall by cytotechnologists did not increase. The QC ASCUS/SIL ratio improved. FN biopsy correlation increased from 45% to 85% (n CP = 17/38 agreement, n AP = 23/27 agreement). Turnaround time increased by one or more days for negative and 1.5 days for QC result reporting. Sensitivity varied among instruments. CONCLUSION: More FN results and greater specificity were seen using the AP 300 than using CP. As with other instrumentation, each laboratory should establish acceptable ranges of performance and baseline values for sensitivity and specificity.

The Production of Polyclonal Antibodies in Laboratory Animals. The Report and Recommendations of ECVAM Workshop 35.

This is the report of the thirty-fifth of a series of workshops organised by the European Centre for the Validation of Alternative Methods (ECVAM). ECVAM's main goal, as defined in 1993 by its Scientific Advisory Committee, is to promote the scientific and regulatory acceptance of alternative methods which are of importance to the biosciences and which reduce, refine or replace the use of laboratory animals. One of the first priorities set by ECVAM was the implementation of procedures which would enable it to become well informed about the state-of-the-art of non-animal test development and validation, and the potential for the possible incorporation of alternative tests into regulatory procedures. It was decided that this would be best achieved by the organisation of ECVAM workshops on specific topics, at which small groups of invited experts would review the current status of various types of in vitro tests and their potential uses, and make recommendations about the best ways forward (1). This joint ECVAM/FELASA (Federation of European Laboratory Animal Science Associations) workshop on The Immunisation of Laboratory Animals for the Production of Polyclonal Antibodies was held in Utrecht (The Netherlands), on 20-22 March 1998, under the co-chairmanship of Coenraad Hendriksen (RIVM, Bilthoven, The Netherlands) and Wim de Leeuw (Inspectorate for Health Protection, The Netherlands). The participants, all experts in the fields of immunology, laboratory animal science, or regulation, came from universities, industry and regulatory bodies. The aims of the workshop were: a) to discuss and evaluate current immunisation procedures for the production of polyclonal antibodies (including route of injection, animal species and adjuvant ); and b) to draft recommendations and guidelines to improve the immunisation procedures, with regard both to animal welfare and to the optimisation of immunisation protocols. This report summarises the outcome of the discussions and includes a number of recommendations and a set of draft guidelines (included in Appendix 1).

Time interval effect on repeat cervical smear results.

OBJECTIVE: Second cervical smears obtained at short time intervals often exhibit a lesser degree of abnormality than the first smear. We studied the effect of time interval between smears on diagnoses in two large, distinctive cohorts. STUDY DESIGN: Patients with two or more satisfactory smears with at least one smear or a cervical biopsy showing atypical squamous cells of undetermined significance or greater were selected. Patients were divided into four subsets by test intervals (days) (< or = 45, 46-90, 91-120, > 120) and compared statistically. RESULTS: The distribution of differences between results for the short-interval subsets (< 120) was significantly different (P < .01) from the interval subset > 120 days. At short intervals the results revealed loss of sensitivity in the second smear as compared to the initial smear and concurrent biopsies. CONCLUSION: Rapidly repeated cervical smears show poor agreement with the biopsy and may be misleading. This effect is most pronounced when the interval is < 45 days. Colposcopists should consider whether concurrent smears shortly after an abnormal smear are worth performing, given the loss of sensitivity.

16-BAC/SDS-PAGE: a two-dimensional gel electrophoresis system suitable for the separation of integral membrane proteins.

Integral membrane proteins, particularly those with more than one transmembrane domain, have traditionally been difficult to separate by two-dimensional electrophoretic methods. Here we report the adaptation of a previously published procedure [D. E. Macfarlane (1989) Anal. Biochem. 176, 457-463] for the analytical and semipreparative separation of membrane proteins. The first dimension involves discontinuous gel electrophoresis in an acidic buffer system using the cationic detergent benzyldimethyl-n-hexadecylammonium chloride (16-BAC). The second dimension consists of discontinuous SDS-PAGE. Using carbonate-washed membranes of synaptic vesicles and clathrin-coated vesicles as examples, we demonstrate that complex membrane protein mixtures can be resolved with a resolution at least fivefold higher than that of one-dimensional SDS-PAGE. Protein patterns are highly reproducible and proteins with single or multiple transmembrane domains are resolved as clearly distinct spots. Smearing of bands or losses of protein are minimal. Several spots were identified by immunoblotting and internal sequencing. Thus, this method is suitable for the analytical and semipreparative characterization of membrane proteins derived from complex biological samples.

An anion binding site that regulates the glutamate transporter of synaptic vesicles.

Glutamate, the major excitatory neurotransmitter of the mammalian central nervous system, is stored in synaptic vesicles and released by exocytosis upon depolarization of the presynaptic nerve terminal. Synaptic vesicles possess an active glutamate-specific transporter that is driven by an electrochemical proton gradient across the vesicle membrane and requires chloride for maximal activity. In this study, we have characterized the role of chloride in vesicular glutamate transport using 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), a potent inhibitor of anion translocators. DIDS inhibited glutamate uptake with an IC50 of 0.7 microM or less. In contrast, all energy gradient parameters (membrane potential, pH gradient, and ATPase activity) required at least 5-fold higher concentration of DIDS for inhibition. Furthermore, high concentrations of chloride but not of glutamate or other anions prevented DIDS inhibition of glutamate uptake. In contrast to uptake, glutamate efflux from glutamate-loaded vesicles was independent of chloride over a wide concentration range. However, efflux was still susceptible to DIDS inhibition. DIDS inhibition was prevented by excess chloride. We conclude that the vesicular glutamate transporter possesses a DIDS-sensitive chloride binding site on the cytoplasmic side, distinct from the substrate binding site, which regulates transport activity.

Functional reconstitution of the gamma-aminobutyric acid transporter from synaptic vesicles using artificial ion gradients.

The gamma-aminobutyric acid transporter of rat brain synaptic vesicles was reconstituted in proteoliposomes, and its activity was studied in response to artificially created membrane potentials or proton gradients. Changes of the membrane potential were monitored using the dyes oxonol VI and 3,3'-diisopropylthiodicarbocyanine iodide, and changes of the H+ gradient were followed using acridine orange. An inside positive membrane potential was generated by the creation of an inwardly directed K+ gradient and the subsequent addition of valinomycin. Under these conditions, valinomycin evoked uptake of [3H]GABA which was saturable. Similarly, [3H]glutamate uptake was stimulated by valinomycin, indicating that both transporters can be driven by the membrane potential. Proton gradients were generated by the incubation of K(+)-loaded proteoliposomes in a buffer free of K+ or Na+ ions and the subsequent addition of nigericin. Proton gradients were also generated via the endogenous H+ ATPase by incubation of K(+)-loaded proteoliposomes in equimolar K+ buffer in the presence of valinomycin. These proton gradients evoked nonspecific, nonsaturable uptake of GABA and beta-alanine but not of glycine in proteoliposomes as well as protein-free liposomes. Therefore, transporter activity was monitored using glycine as an alternative substrate. Proton gradients generated by both methods elicited saturable glycine uptake in proteoliposomes. Together, our data confirm that the vesicular GABA transporter can be energized by both the membrane potential and the pH gradient and show that transport can be achieved by artificial gradients independently of the endogenous proton ATPase.

P29: a novel tyrosine-phosphorylated membrane protein present in small clear vesicles of neurons and endocrine cells.

A novel membrane protein from rat brain synaptic vesicles with an apparent 29,000 Mr (p29) was characterized. Using monospecific polyclonal antibodies, the distribution of p29 was studied in a variety of tissues by light and electron microscopy and immunoblot analysis. Within the nervous system, p29 was present in virtually all nerve terminals. It was selectively associated with small synaptic vesicles and a perinuclear region corresponding to the area of the Golgi complex. P29 was not detected in any other subcellular organelles including large dense-core vesicles. The distribution of p29 in various subcellular fractions from rat brain was very similar to that of synaptophysin and synaptobrevin. The highest enrichment occurred in purified small synaptic vesicles. Outside the nervous system, p29 was found only in endocrine cell types specialized for peptide hormone secretion. In these cells, p29 had a distribution very similar to that of synaptophysin. It was associated with microvesicles of heterogeneous size and shape that are primarily concentrated in the centrosomal-Golgi complex area. Secretory granules were mostly unlabeled, but their membrane occasionally contained small labeled evaginations. Immunoisolation of subcellular organelles from undifferentiated PC12 cells with antisynaptophysin antibodies led to a concomitant enrichment of p29, synaptobrevin, and synaptophysin, further supporting a colocalization of all three proteins. P29 has an isoelectric point of approximately 5.0 and is not N-glycosylated. It is an integral membrane protein and all antibody binding sites are exposed on the cytoplasmic side of the vesicles. Two monoclonal antibodies raised against p29 cross reacted with synaptophysin, indicating the presence of related epitopes. P29, like synaptophysin, was phosphorylated on tyrosine residues by endogenous tyrosine kinase activity in intact vesicles.