Evaluation of Fluorescence Microsphere Immunoassay for Detection of Antibodies to Rift Valley Fever Virus Nucleocapsid Protein and Glycoproteins.

Rift Valley fever virus (RVFV) is a mosquito-borne, zoonotic virus that infects ruminants, including cattle, sheep, goats, camels, and buffalo. Multiplexing diagnostic assays that can simultaneously detect antibodies against multiple RVFV antigens offer a high-throughput test for disease surveillance and vaccine evaluations. We describe the improvement and evaluation of a previously developed fluorescence microsphere immunoassay (FMIA) for the detection of IgG and IgM antibodies against the RVFV glycoprotein (Gn) and the immunogenic nucleocapsid protein (Np). Well-characterized vaccinated and experimentally infected ruminant sera were used for the evaluation of the assay. Recombinant viral proteins were produced and then coupled to polystyrene magnetic beads for analysis using the Luminex MAGPIX system with xMAP technology. The FMIA was performed in parallel with virus neutralization tests. Our results revealed the highest median fluorescence intensity (MFI) values for the detection of IgG antibodies against RVFV Np, indicating that this antigen would be a good candidate for a screening assay. The Np and Gn targets could differentiate infected animals from animals vaccinated with a candidate subunit vaccine formulation based on the RVFV Gn and Gc proteins. The results presented in this report demonstrate that FMIA provides a rapid and robust serological diagnostic tool for the detection of antibodies against RVFV. The targets developed in this assay provide the basis for the development of a companion diagnostic test for an RVFV Gn/Gc subunit vaccine that is capable of differentiating infected from vaccinated animals (DIVA), as well as a multiplex serodiagnostic assay that can simultaneously screen for several ruminant diseases.

Utility of third trimester sonographic measurements for predicting SGA in cases of fetal gastroschisis.

OBJECTIVE: To assess the accuracy of different sonographic estimated fetal weight (EFW) cutoffs, and combinations of EFW and biometric measurements for predicting small for gestational age (SGA) in fetal gastroschisis. STUDY DESIGN: Gastroschisis cases from two centers were included. The sensitivity, specificity, positive and negative predictive values (PPV and NPV) were calculated for different EFW cutoffs, as well as EFW and biometric measurement combinations. RESULTS: Seventy gastroschisis cases were analyzed. An EFW<10% had 94% sensitivity, 43% specificity, 33% PPV and 96% NPV for SGA at delivery. Using an EFW cutoff of <5% improved the specificity to 63% and PPV to 41%, but decreased the sensitivity to 88%. Combining an abdominal circumference (AC) or femur length (FL) z-score less than -2 with the total EFW improved the specificity and PPV but decreased the sensitivity. CONCLUSION: A combination of a small AC or FL along with EFW increases the specificity and PPV, but decreases the sensitivity of predicting SGA.

Abnormal profile of human nucleolytic activity as a test for cancer.

Serum samples from patients with various malignancies including acute nonlymphocytic leukemia (ANLL), brain tumor (BT), Hodgkin's disease (HD), and non Hodgkin's lymphoma (NHL) were evaluated for nucleolytic activity against six synthetic polynucleotides: polyadenylic acid, polyuridylic acid, polycytidylic acid, polyguanylic acid, polyadenylic-polyuridylic acid, and polyguanylic-polycytidylic acid; The enzyme activity was determined spectrophotometrically by following the degradation of substrate to acid-soluble nucleotides. Most patients had elevated serum RNase activity at the 95% confidence level when compared to 30 controls. Included in this group were 67% of patients with ANLL, 46% of patients with BT, 73% of patients with HD, and 67% of patients with NHL. These data confirmed the earlier suggestion that elevated serum nuclease activity is found in patients with neoplastic disease. However, whether or not a serum was identified as abnormal depended on the substrate used in the assay; this underscored the need to test samples against a variety of polynucleotides. Alterations in serum nucleolytic activity represent an important marker of neoplastic disease and can serve as the basis for a useful clinical screening device.

3-(Iodoacetamido)-benzoylurea: a novel cancericidal tubulin ligand that inhibits microtubule polymerization, phosphorylates bcl-2, and induces apoptosis in tumor cells.

3-(Iodoacetamido)-benzoylurea (3-IAABU) is a newly synthesized antitubulin compound with a molecular weight of 347. 3-IAABU exhibited anticancer activity in a variety of tumor cell lines with ID90 in the range of 0.015-0.29 microM for leukemic cells and 0.06-0.92 microM for solid tumors. Higher selectivity against malignant cells was observed with 3-IAABU than that with vinblastine and paclitaxel. It inhibits microtubule assembly in tubulin systems either with or without microtubule-associated proteins (ID50 was 0.1 microM and 1.2 microM, respectively) and microtubule depolymerization was not affected, indicating an inhibition of polymerization by binding of 3-IAABU to the heterodimeric subunit of tubulin. 3-IAABU was shown to inhibit the binding of colchicine, a subunit binding compound, but did not inhibit binding of vinblastine and guanosine 5'-triphosphate/guanosine 5'-diphosphate, indicating that colchicine site corresponds to the site that 3-IAABU locates. Tumor cells treated with 3-IAABU showed scattered chromosomes in metaphase. Normal microtubule architecture or spindle apparatus was absent in these cells; instead, punctuated aggregates of tubulin were found by an immunofluorescent staining. Cell cycle analyses showed an accumulation of tumor cells at M phase after a 4-h treatment with 3-IAABU. The phosphorylated bcl-2 representative of an inactivated form of the oncoprotein was found in the cells 12 h after treatment with 3-IAABU. These cells progressed to apoptosis within 16 h. As a new tubulin ligand, 3-IAABU could be a promising agent in cancer chemotherapy.

Methods of collecting and evaluating non-clinical cardiac electrophysiology data in the pharmaceutical industry: results of an international survey.

OBJECTIVE: To assess current practice in the pharmaceutical industry for assessing the potential for QT interval prolongation by non-cardiovascular medicinal products. METHODS: The survey was based on responses from the Toxicology and (Safety) Pharmacology laboratories (a total of 74 laboratories) of 54 companies based in Europe, Japan/Asia and the USA, received between January and March 1999. RESULTS: All 54 companies conducted preclinical in vivo electrocardiography (EGG) evaluation of new active substances (NASs). Thirty of these companies also conducted in vitro cardiac electrophysiology studies on their compounds. The majority of in vivo work was done in conscious beagle dogs. There was no consistency within the industry in defining the magnitude of change in QT interval that is considered biologically important. Most companies considered a change greater than 10% to be important, although the design of the studies suggested that group sizes used may not give sufficient statistical power to detect this size of change. Bazett's formula was used by 41% of laboratories to correct QT for changes in heart rate, despite the fact that this formula is generally deemed to be unsuitable for use in dogs. For studies in anaesthetised dogs, the majority of laboratories used barbiturate anaesthesia, but researchers should be aware of the effects of this and some other anaesthetic agents on QT interval. As for in vitro cardiac electrophysiology, there was wide diversity in the testing methodologies, particularly with regard to the test species and tissue type. As with QT prolongation, there was no consensus on the degree of action potential prolongation to cause concern. For both in vitro and in vivo testing, the majority of companies tested a minimum of three dose (or concentration) levels in order to ascertain any dose-response relationship. CONCLUSIONS: The survey provides a snapshot of the practice in the industry prior to any internationally-agreed consensus on the most effective and efficient approaches to minimising the risk of QT prolongation by new drugs in man. It must be stated that for any given methodology, the 'majority view' in the industry is not necessarily best practice.

Relationships between preclinical cardiac electrophysiology, clinical QT interval prolongation and torsade de pointes for a broad range of drugs: evidence for a provisional safety margin in drug development.

OBJECTIVE: To attempt to determine the relative value of preclinical cardiac electrophysiology data (in vitro and in vivo) for predicting risk of torsade de pointes (TdP) in clinical use. METHODS: Published data on hERG (or I(Kr)) activity, cardiac action potential duration (at 90% repolarisation; APD(90)), and QT prolongation in dogs were compared against QT effects and reports of TdP in humans for 100 drugs. These data were set against the free plasma concentrations attained during clinical use (effective therapeutic plasma concentrations; ETPC(unbound)). The drugs were divided into five categories: (1) Class Ia and III antiarrhythmics; (2) Withdrawn from market due to TdP; (3) Measurable incidence/numerous reports of TdP in humans; (4) Isolated reports of TdP in humans; (5) No reports of TdP in humans. RESULTS: Data from hERG (or I(Kr)) assays in addition to ETPC(unbound) data were available for 52 drugs. For Category 1 drugs, data for hERG/I(Kr) IC(50), APD(90), QTc in animals and QTc in humans were generally close to or superimposed on the ETPC(unbound) values. This relationship was uncoupled in the other categories, with more complex relationships between the data. In Category 1 (except amiodarone), the ratios between hERG/I(Kr) IC(50) and ETPC(unbound) (max) ranged from 0.1- to 31-fold. Similar ranges were obtained for drugs in Category 2 (0.31- to 13-fold) and Category 3 (0.03- to 35-fold). A large spread was found for Category 4 drugs (0.13- to 35700-fold); this category embraced an assortment of mechanisms ranging from drugs which may well be affecting I(Kr) currents in clinical use (e.g. sparfloxacin) to others such as nifedipine (35700-fold) where channel block is not involved. Finally, for the majority of Category 5 drugs there was a >30-fold separation between hERG/I(Kr) activity and ETPC(unbound) values, with the notable exception of verapamil (1.7-fold), which is free from QT prolongation in man; this is probably explained by its multiple interactions with cardiac ion channels. CONCLUSIONS: The dataset confirms the widely-held belief that most drugs associated with TdP in humans are also associated with hERG K(+) channel block at concentrations close to or superimposed upon the free plasma concentrations found in clinical use. A 30-fold margin between C(max) and hERG IC(50) may suffice for drugs currently undergoing clinical evaluation, but for future drug discovery programmes, pharmaceutical companies should consider increasing this margin, particularly for drugs aimed at non-debilitating diseases. However, interactions with multiple cardiac ion channels can either mitigate or exacerbate the prolongation of APD and QT that would ensue from block of I(Kr) currents alone, and delay of repolarisation per se is not necessarily torsadogenic. Clearly, an integrated assessment of in vitro and in vivo data is required in order to predict the torsadogenic risk of a new candidate drug in humans.

Serial aEEG recordings in a cohort of extremely preterm infants: feasibility and safety.

OBJECTIVE: Amplitude-integrated electroencephalography (aEEG) monitoring is increasing in the neonatal population, but the safety and feasibility of performing aEEG in extremely preterm infants have not been systematically evaluated. STUDY DESIGN: Inborn infants 23(0/7) to 28(6/7) weeks gestation or birth weight 401 to 1000 g were eligible. Serial, 6-h aEEG recordings were obtained from first week of life until 36 weeks postmenstrual age. Adverse events were documented, and surveys evaluated the impact of the aEEGs on routine care. Success of performing aEEGs according to protocol and aEEG quality were assessed. RESULT: A total of 102 infants were enrolled, with 755 recordings performed. 83% of recordings were performed according to schedule, and 96% were without adverse event. Bedside nurses reported no interference with routine care for 89% of recordings. 92% of recordings had acceptable signal quality. CONCLUSION: Serial aEEG monitoring is safe in preterm infants, with few adverse events and general acceptance by nursing staff.

A comparison of motor behaviours in groups of rats distinguished by their climbing response to apomorphine.

Administration of apomorphine hydrochloride (0.5 mg kg-1 s.c.) to adult male or female Wistar rats previously acclimatized to the test environment induced climbing behaviour in approximately 50% of animals examined. The proportion of animals climbing was related to age, being maximal at 8-9 weeks. Those animals showing an initial climbing response to apomorphine (0.5 mg kg-1 s.c.), climbed when challenged with this dose of apomorphine on subsequent occasions. In 'climbing' animals the intensity of response was related to the dose of apomorphine administered; no dose-response relationship was observed in 'non-climbing' animals. No overall differences in the spontaneous motor behaviour of 'climbing' and 'non-climbing' animals were apparent as assessed by measurement of spontaneous climbing behaviour, by holeboard activity, and by locomotor activity measured in either photocell cages or in a treadwheel. There was no overall difference in the ability of apomorphine to induce locomotor activity or stereotyped behaviour in 'climbing' and 'non-climbing' animals. However, the administration of apomorphine induced rearing and treadwheel activity only in those animals classified as 'climbers'. There was no difference between the number (Bmax) of specific [3H]-spiperone binding sites or the dissociation constant (KD) in striatal or mesolimbic tissue preparations for 'climbing' and 'non-climbing' rats. The ability of an animal to climb in response to apomorphine appears to be dependent on an ability to orient vertically, since this is a component of behaviour common to climbing, rearing, and treadwheel activity. The ability to climb does not appear to be related to differences in dopamine receptor numbers in brain or to the penetration of apomorphine into brain.

FILM (5-fluorouracil, ifosfamide, leucovorin and mitomycin C), an alternative chemotherapy regimen suitable for the treatment of advanced breast cancer in the 'out-patient' setting.

FILM, a combination of 5-fluorouracil (5-FU) 750 mg/m(2), ifosfamide 1 g/m(2), leucovorin 200 mg/m(2) and mitomycin C 6 mg/m(2) (alternate cycles), was administered to 24 chemo-naive patients with inoperable disease, locally advanced or metastatic. Up to 6 x 3-weekly cycles of FILM were administered on an out-patient basis. Responses included 8 patients in complete remission (CR) and 12 showing a partial response (PR) (83%). Following analysis of these results, the FILM regimen was introduced as a standard out-patient protocol at the North Middlesex Hospital, United Kingdom. A further 66 patients have been treated in this setting. Retrospective analysis of these data confirm the trial results and allow conclusions regarding tolerability, toxicity, duration of response and survival to be drawn from a total cohort of 90 patients. A total of 524 cycles have been administered. Nineteen cycles (4%) were delayed owing to slow recovery of white blood cells (WBC), but no dose reductions were necessary. Five blood transfusions were required for anaemia. The most frequent non-haematological toxicities included nausea, vomiting and fatigue. Of 80 patients treated for inoperable or locally advanced disease, 56 (70%) remain in remission, and 69 (86%) remain alive after 5 years.

Influence of aerobic fitness on cardiovascular responses during slow head-down tilting.

This study examined the influence of cardiorespiratory fitness (CRF) on mean heart rate (HR), systolic blood pressure (SBP), and diastolic blood pressure (DBP) responses to slow head-down tilting. Twenty-four young, healthy volunteers were tilted randomly from the horizontal plane to -30, -60, and -90 degrees with lower limbs extended or flexed. A multivariate analysis of variance between three independent variables (level of CRF, tilt angle, and lower limb position) and three dependent variables (mean HR, SBP, and DBP) was performed on the cardiovascular changes from an initial horizontal baseline value (absolute change) and from a horizontal value that immediately preceded tilting angles (relative change). The results for significant absolute cardiovascular changes indicated a CRF influence on HR increase (F = 6.42, p less than .05), a tilt-angle influence on SBP increase (F = 9.56, p less than .001), and DBP increase (F = 6.49, p less than .01) and an interaction influence between CRF level and limb position for DBP (F = 5.83, p less than .05). Significant relative cardiovascular change was noted for tilt-angle influence on HR (F = 9.04, p less than or equal to .001). We conclude, therefore, that physical therapists should consider the CRF of individuals and the tilt angle to be used before they conduct slow head-down tilting for therapy.

The pre-clinical assessment of QT interval prolongation: a comparison of in vitro and in vivo methods.

The literature was searched for in vivo dog studies reporting QT prolongation and in vitro studies reporting increased myocardial action potential duration, which indicates the potential to prolong QT interval, for nine non-cardiac drugs that have been reported to produce QT prolongation in man. The drugs were: astemizole; terfenadine; erythromycin; sparfloxacin; cisapride; probucol; terodiline; risperidone and sertindole. 1. There were reports of the appropriate finding with in vitro methods for six of the drugs and with in vivo methods for seven of the drugs. No reports were found concerning the remaining drugs with each method. This indicates that both methods are effective and each method would have correctly identified the drugs in question as having the potential to prolong the QT interval in man in all cases for which studies were reported. 2. This suggests that, if properly conducted, either method alone is sufficient for the pre-clinical assessment of QT interval prolongation. This does not support the routine use of both methods before the administration of new drugs to man.

Relationship between QT interval and heart rate in Alderley Park beagles.

The QT interval and heart rate were measured from the electrocardiogram of 1608 Alderley Park beagles, and the mean QT intervals for 28 values of heart rates between 84 and 192 beats per minute were calculated. These were analysed by linear regression and ranked by Pearson's correlation coefficient for each of eight equations, all of which have been proposed to describe the relationship between QT interval and heart rate. The equation QT=b(square root RR), upon which the calculation of QTc using Bazett's formula is based, did not adequately describe the relationship. QT=b(cube root RR) was a more adequate one-parameter equation. The first-ranked equation was 1/QT=a+b(HR), but its use presents practical and interpretational difficulties; consequently, the second-ranked equation, QT=a+b(log(e)HR), is recommended for routine use.

Outcomes of extremely preterm infants following severe intracranial hemorrhage.

OBJECTIVE: Severe intracranial hemorrhage (ICH) is an important prognostic variable in extremely preterm (EPT) infants. We examined imaging and clinical variables that predict outcomes in EPT infants with severe ICH. STUDY DESIGN: Retrospective analysis of 353 EPT infants with severe ICH. Outcomes were compared by examining: (i) unilateral vs bilateral ICH; and (ii) presence vs absence of hemorrhagic parenchymal infarction (HPI). Regression analyses identified variables associated with death or neurodevelopmental impairment (NDI). RESULT: Bilateral ICH and HPI had higher rates of adverse outcomes and were independently associated with death/NDI. HPI was the most important variable for infants of lower birth weight, and bilateral ICH for larger infants. For infants surviving to 36 weeks, shunt placement was most associated with death/NDI. CONCLUSION: Bilateral ICH and the presence of HPI in EPT infants with severe ICH are associated with death/NDI, though the importance depends on birth weight and survival to 36 weeks.

Therapeutic hypothermia during neonatal transport: data from the California Perinatal Quality Care Collaborative (CPQCC) and California Perinatal Transport System (CPeTS) for 2010.

OBJECTIVE: To evaluate cooling practices and neonatal outcomes in the state of California during 2010 using the California Perinatal Quality Care Collaborative and California Perinatal Transport System databases. STUDY DESIGN: Database analysis to determine the perinatal and neonatal demographics and outcomes of neonates cooled in transport or after admission to a cooling center. RESULT: Of the 223 infants receiving therapeutic hypothermia for hypoxic ischemic encephalopathy (HIE) in California during 2010, 69% were cooled during transport. Despite the frequent use of cooling in transport, cooling center admission temperature was in the target range (33-34 °C) in only 62 (44%). Among cooled infants, gestational age was <35 weeks in 10 (4.5%). For outborn and transported infants, chronologic age at the time of cooling initiation was >6 h in 20 (11%). When initiated at the birth hospital, cooling was initiated at <6 h of age in 131 (92.9%). CONCLUSION: More than half of the infants cooled in transport do not achieve target temperature by the time of arrival at the cooling center. The use of cooling devices may improve temperature regulation on transport.

A leak-free technique for open insertion of peritoneal dialysis catheters.

A technique for open insertion of acute peritoneal dialysis catheters without excessive postoperative leakage of dialysate fluid is described. By using the laparotomy incision only for exploration to guide catheter placement by counter puncture, one can obtain a much more accurate and water-tight closure of the lineal alba than is possible when the catheter exists through the incision.

Immediate reconstruction after mastectomy: team approach.

A review of 198 cases of breast reconstruction done at the time of mastectomy for carcinoma of the breast shows an extremely low morbidity. The technique of using a tissue-expander prosthesis enables immediate breast reconstruction without any fear of impairing prognosis, of spreading disease, or of impeding adjuvant therapy.

The risk assessment of faecal contamination by MAR indexing of Escherichia coli.

Multiple Antibiotic Resistance (MAR) index of faecal indicator bacterium Escherichia coli, isolated from water and sediment in the Bhavani river was employed to assess the quality of the river, and the risk of contamination. The MAR index of all the stations exceeded the high risk level (0.250) of contamination. The study of MAR index for individual isolates also showed high resistance against the tested antibiotics and it was found that these antibiotic resistant E. coli were distributed throughout the length of river sampled. It is concluded that the river is contaminated with faecal enteric bacteria, originating from high risk sources such as night soil, commercial poultry farms etc. It is implied that the quality of river water is not safe for drinking purpose and that adequate measures may be taken before use.

beta-Tubulin mutation suppresses microtubule dynamics in vitro and slows mitosis in vivo.

Microtubule (MT) dynamics vary both spatially and temporally within cells and are thought to be important for proper MT cellular function. Because MT dynamics appear to be closely tied to the guanosine triphosphatase (GTPase) activity of beta-tubulin subunits, we examined the importance of MT dynamics in the budding yeast S. cerevisiae by introducing a T107K point mutation into a region of the single beta-tubulin gene, TUB2, known to affect the assembly-dependent GTPase activity of MTs in vitro. Analysis of MT dynamic behavior by video-enhanced differential interference contrast microscopy, revealed that T107K subunits slowed both the growth rates and catastrophic disassembly rates of individual MTs in vitro. In haploid cells tub2-T107K is lethal; but in tub2-T107K/tub2-590 heterozygotes the mutation is viable, dominant, and slows cell-cycle progression through mitosis, without causing wholesale disruption of cellular MTs. The correlation between the slower growing and shortening rates of MTs in vitro, and the slower mitosis in vivo suggests that MT dynamics are important in budding yeast and may regulate the rate of nuclear movement and segregation. The slower mitosis in mutant cells did not result in premature cytokinesis and cell death, further suggesting that cell-cycle control mechanisms "sense" the mitotic slowdown, possibly by monitoring MT dynamics directly.