High prevalence of vertebral fractures in seizure patients with normal bone density receiving chronic anti-epileptic drugs.

People with epilepsy who take certain medications are at risk for developing osteoporosis and fractures of the vertebrae that commonly go undiagnosed. By using technology available in a bone density scan, we observed at least one fracture in many subjects with bone density in the normal and osteopenic range. PURPOSE/INTRODUCTION: Chronic use of antiepileptic drugs (AEDs), both enzyme-inducing (phenytoin, phenobarbital, carbamazepine, and primidone) and non-enzyme-inducing (i.e., valproate), is recognized as a cause of secondary osteoporosis. Vertebral compression fractures (VF) are the most common type of osteoporotic fractures and may confer an increased risk of future hip, wrist, and vertebral fractures. Vertebral compression fractures in the general population are frequently asymptomatic, and under-diagnosed. The purpose of this study is to describe the prevalence of VF in a cohort of male veterans with epilepsy on chronic AEDs. METHODS: The cohort for this study consisted of 146 male veterans who carried a diagnosis of epilepsy and were chronic users of AEDs known to cause osteoporosis (phenobarbital, phenytoin, carbamazepine, primidone, and valproate). Chronic AED use was defined as receiving an AED for at least 2 years. Subjects were previously seen in the osteoporosis clinic and had been evaluated by a dual-energy X-Ray absormetry (DXA) instrument including morphometric studies following a standard vertebral fracture assessment (VFA) protocol during the same DXA imaging acquisition session. RESULTS: The mean age was 63 years. Low bone mineral density defined as osteoporosis or osteopenia was observed in 29% and 43% respectively. We observed at least one VF in 41 % of the subjects who had normal BMD, 54% in the osteopenic range, and 75% in the osteoporotic range. CONCLUSIONS: By performing a VFA in addition to standard bone densitometric studies, we disclosed a large prevalence of compression fractures in individuals with epilepsy chronically treated with AEDs who had BMDs in the normal and osteopenic ranges. The addition of VFA or other imaging methods to evaluate VF should be included in the evaluation of bone health in individuals with epilepsy receiving AEDs since it may modify treatment recommendations to prevent future osteoporotic fractures.

Geography but not alternative host species explain the spread of raccoon rabies virus in Vermont.

In North America, the raccoon-associated variant of rabies virus (RRV) is of special concern, given its relatively rapid spread throughout the eastern USA and its potential public health impact due to high raccoon host densities in urban areas. Northward expansion of this epizootic included an outbreak in the Canadian province of Quebec in 2006-2009 due to trans-border spread from the State of Vermont. To inform a more proactive approach to future control efforts, this study uses phylogenetic analyses to explore the role of geography and alternative carnivore hosts in the dynamics of RRV spread within Vermont. Specifically, we sought to examine whether striped skunks, a species frequently infected by RRV, could be part of the maintenance host community. Whole genome sequencing of 160 RRV samples from Vermont and neighbouring US states were used for fine-scale phylogeographic analyses. Results, together with the complete surveillance record of raccoon rabies since its entry into Vermont in 1994, document incursions by two distinct viral lineages and identify topographical features of the landscape which have significantly influenced viral spread, resulting in a complex distribution pattern of viral variants throughout the state. Results of phylogenetic cluster analysis and discrete state reconstruction contained some evidence of skunk-to-skunk and skunk-to-raccoon transmission but overall failed to support a role for skunks as alternative maintenance hosts.

Mapping the distribution of the main host for plague in a complex landscape in Kazakhstan: An object-based approach using SPOT-5 XS, Landsat 7 ETM+, SRTM and multiple Random Forests.

Plague is a zoonotic infectious disease present in great gerbil populations in Kazakhstan. Infectious disease dynamics are influenced by the spatial distribution of the carriers (hosts) of the disease. The great gerbil, the main host in our study area, lives in burrows, which can be recognized on high resolution satellite imagery. In this study, using earth observation data at various spatial scales, we map the spatial distribution of burrows in a semi-desert landscape. The study area consists of various landscape types. To evaluate whether identification of burrows by classification is possible in these landscape types, the study area was subdivided into eight landscape units, on the basis of Landsat 7 ETM+ derived Tasselled Cap Greenness and Brightness, and SRTM derived standard deviation in elevation. In the field, 904 burrows were mapped. Using two segmented 2.5 m resolution SPOT-5 XS satellite scenes, reference object sets were created. Random Forests were built for both SPOT scenes and used to classify the images. Additionally, a stratified classification was carried out, by building separate Random Forests per landscape unit. Burrows were successfully classified in all landscape units. In the 'steppe on floodplain' areas, classification worked best: producer's and user's accuracy in those areas reached 88% and 100%, respectively. In the 'floodplain' areas with a more heterogeneous vegetation cover, classification worked least well; there, accuracies were 86 and 58% respectively. Stratified classification improved the results in all landscape units where comparison was possible (four), increasing kappa coefficients by 13, 10, 9 and 1%, respectively. In this study, an innovative stratification method using high- and medium resolution imagery was applied in order to map host distribution on a large spatial scale. The burrow maps we developed will help to detect changes in the distribution of great gerbil populations and, moreover, serve as a unique empirical data set which can be used as input for epidemiological plague models. This is an important step in understanding the dynamics of plague.

Rapid identification of the raccoon rabies virus variant using a real-time reverse-transcriptase polymerase chain reaction.

The raccoon-associated variant of rabies virus (RRV) is enzootic throughout the eastern seaboard of the United States with frequent incursions into Canada. Many wildlife management agencies are actively engaged in control programmes targeting elimination of this disease and rapid identification of raccoon rabies cases is crucial to the success of these operations. This report documents the development of a reverse transcriptase real-time PCR (RT-qPCR) that specifically identifies this rabies virus variant (RRV RT-qPCR) and which can be readily multiplexed with a generic rabies virus RT-qPCR for use as a typing tool. Using a large collection of rabies virus samples representative of the variants circulating around the world, but with a focus on those occurring in the Americas, the RRV RT-qPCR was 100% sensitive and 99.31% specific. To further apply these assays for diagnostic purposes, addition of an RT-qPCR targeting the host ß-actin mRNA, which serves as an internal amplification control, in a triplex format was shown to yield highly comparable results using a subset of our viral collection. Use of these assays for early and accurate identification of this viral variant will help to optimize the utilization of resources required for control of this disease.

The basic reproduction number for complex disease systems: defining R(0) for tick-borne infections.

Characterizing the basic reproduction number, R(0), for many wildlife disease systems can seem a complex problem because several species are involved, because there are different epidemiological reactions to the infectious agent at different life-history stages, or because there are multiple transmission routes. Tick-borne diseases are an important example where all these complexities are brought together as a result of the peculiarities of the tick life cycle and the multiple transmission routes that occur. We show here that one can overcome these complexities by separating the host population into epidemiologically different types of individuals and constructing a matrix of reproduction numbers, the so-called next-generation matrix. Each matrix element is an expected number of infectious individuals of one type produced by a single infectious individual of a second type. The largest eigenvalue of the matrix characterizes the initial exponential growth or decline in numbers of infected individuals. Values below 1 therefore imply that the infection cannot establish. The biological interpretation closely matches that of R(0) for disease systems with only one type of individual and where infection is directly transmitted. The parameters defining each matrix element have a clear biological meaning. We illustrate the usefulness and power of the approach with a detailed examination of tick-borne diseases, and we use field and experimental data to parameterize the next-generation matrix for Lyme disease and tick-borne encephalitis. Sensitivity and elasticity analyses of the matrices, at the element and individual parameter levels, allow direct comparison of the two etiological agents. This provides further support that transmission between cofeeding ticks is critically important for the establishment of tick-borne encephalitis.

Schizosaccharomyces pombe ras1 and byr1 are functionally related genes of the ste family that affect starvation-induced transcription of mating-type genes.

We have further investigated the function of the ras1 and byr1 genes, which were previously shown to be critical for sexual differentiation in fission yeast cells. Several physiological similarities between strains containing null alleles of these genes supports the idea that ras1 and byr1 are functionally closely related. Furthermore, we have found that byr1 is allelic to ste1, one of at least 10 genes which when mutated can cause sterility. Since ras1 had previously been found to be allelic to ste5, both ras and byr genes are now clearly shown to be a part of the ste gene family, thus confirming their close functional relationship. The observation that the mating-type loci could overcome the sporulation block of ras1 and byr1 mutant strains prompted investigation of the role of the ras-byr pathway in the induction of the mating-type gene transcripts upon nitrogen starvation. By Northern analysis of RNA preparations from strains carrying wild-type or mutant ras1 alleles and grown to different stages of the growth cycle, we have shown that ras1 plays an important role in inducing the Pi transcript of the mating-type loci and the mei3 gene transcript. These observations provide a molecular basis for the role of the ste gene family, including ras1 and byr1, in meiosis and indicate that further characterization of other ste genes would be very useful for elucidating the mechanism of ras1 function in fission yeast cells.

Importance of bird-to-bird transmission for the establishment of West Nile virus.

West Nile virus (WNV) is principally considered to be maintained in a mosquito-bird transmission cycle. Under experimental conditions, several other transmission routes have been observed, but the significance of these additional routes in nature is unknown. Here, we derive an expression for the basic reproduction number (R0) for WNV including all putative routes of transmission between birds and mosquitoes to gauge the relative importance of these routes for the establishment of WNV. Parameters were estimated from published experimental results. Sensitivity analysis reveals that R0 is sensitive to transmission between birds via close contact, but not to mosquito-to-mosquito transmission. In seasons or in areas where the mosquito-to-bird ratio is low, bird-to-bird transmission may be crucial in determining whether WNV can establish or not. We explain the use of R0 as a flexible tool to measure the risk of establishment of vector-borne diseases.

Effects of (+/-)3,4-methylenedioxymethamphetamine, (+/-)3,4-methylenedioxyamphetamine and methamphetamine on temperature and activity in rhesus macaques.

Severe and malignant hyperthermia is a frequently reported factor in emergency department (ED) visits and fatalities in which use of amphetamine drugs, such as (+/-)3,4-methylenedioxymethamphetamine (MDMA), (+/-)3,4-methylenedioxyamphetamine (MDA) and (+)methamphetamine (METH), is confirmed. Individuals who use "ecstasy" are also often exposed, intentionally or otherwise, to several of these structurally-related compounds alone or in combination. In animal studies the degree of (subcritical) hyperthermia is often related to the severity of amphetamine-induced neurotoxicity, suggesting health risks to the human user even when emergency medical services are not invoked. A clear distinction of thermoregulatory risks posed by different amphetamines is therefore critical to understand factors that may produce medical emergency related to hyperthermia. The objective of this study was therefore to determine the relative thermoregulatory disruption produced by recreational doses of MDMA, MDA and METH in nonhuman primates. Body temperature and spontaneous home cage activity were monitored continuously in six male rhesus monkeys via radiotelemetric devices. The subjects were challenged intramuscularly with 0.56-2.4 mg/kg MDMA, 0.56-2.4 mg/kg MDA and 0.1-1.0 mg/kg METH. All three amphetamines significantly elevated temperature; however the time course of effects differed. The acute effect of METH lasted hours longer than MDA or MDMA and a disruption of nighttime circadian cooling was observed as long as 18 h after 1.0 mg/kg METH and 1.78-2.4 mg/kg MDA, but not after MDMA. Activity levels were only reliably increased by 0.32 mg/kg METH. It is concluded that while all three substituted amphetamines produce hyperthermia in rhesus monkeys, the effects do not depend on elevated locomotor activity and exhibit differences between compounds. The results highlight physiological risks posed both by recreational use of the amphetamines and by current trials for clinical MDMA use.

Comparison between magnetic force microscopy and electron back-scatter diffraction for ferrite quantification in type 321 stainless steel.

Several analytical techniques that are currently available can be used to determine the spatial distribution and amount of austenite, ferrite and precipitate phases in steels. The application of magnetic force microscopy, in particular, to study the local microstructure of stainless steels is beneficial due to the selectivity of this technique for detection of ferromagnetic phases. In the comparison of Magnetic Force Microscopy and Electron Back-Scatter Diffraction for the morphological mapping and quantification of ferrite, the degree of sub-surface measurement has been found to be critical. Through the use of surface shielding, it has been possible to show that Magnetic Force Microscopy has a measurement depth of 105-140 nm. A comparison of the two techniques together with the depth of measurement capabilities are discussed.

Site-directed mutagenesis of large plasmids.

A protocol combining recombination PCR and long-distance PCR is demonstrated to be highly accurate and rapid for site-directed mutagenesis of large (> 10 kb) plasmids. Application of this protocol to the generation of mutant rabies virus glycoproteins expressed by the baculovirus/insect cell system illustrates the usefulness of this approach in facilitating structure-function relationships in this important eukaryotic expression system.

Antigenic and genetic divergence of rabies viruses from bat species indigenous to Canada.

Antigenic characterisation of over 350 chiropteran rabies viruses of the Americas, especially from species reported rabid in Canada, distinguished 13 viral types. In close accord with this classification, nucleotide sequencing of representative isolates, at both the N and G loci, identified four principal phylogenetic groups (I-IV), sub-groups of which circulated in particular bat species. Amongst the North American bat viruses, there was a notable division between group I specimens associated with colonial, non-migratory bats (Myotis sp. and Eptesicus fuscus) and those of group II harbored by solitary, migratory species (Lasiurus sp. and Lasionycteris noctivagans). Certain species of Myotis were clearly identified as rabies reservoirs, an observation often obscured previously by their frequent infection by viral variants of other chiroptera. An additional group (III) apparently circulates in E. fuscus, whilst viruses harbored by both insectivorous and haematophagus bats of Latin America clustered to a separate clade (group IV). Comparison of the predicted N and G proteins of these viruses with those of strains of terrestrial mammals indicated a similarity in structural organisation regardless of host species lifestyle. Finally, these sequences permitted examination of the evolutionary relationship of American bat rabies viruses within the Lyssavirus genus.

Uterine and fetal Doppler flow changes from a single dose of a long-acting intranasal decongestant.

Oxymetazoline, an alpha-adrenergic agent, is the active vasoconstrictor in long-acting intranasal decongestants. This investigation studied the effects of oxymetazoline on the maternal and fetal circulations. Twelve healthy gravid patients in the third trimester (27-39 weeks) underwent flow velocity measurements by the same sonographer using a pulsed Doppler system (ATL Ultramark 4 machine). Maternal and fetal indices were recorded 15 minutes before dosing, immediately thereafter, and at 15-minute intervals during the first 2 hours after the drug was given. No significant changes were found in either the maternal blood pressures or pulse rates. Blood flow velocities did not change significantly from baseline for the uterine arcuate artery, fetal aorta, or umbilical artery circulations. In no case did absolute blood flow velocity decrease significantly or systolic/diastolic ratios elevate to worrisome values. For uncomplicated pregnancies, we conclude there are no significant acute changes in the maternal and fetal circulations after a single dose of intranasal oxymetazoline.

Influence of suspension on the oxidative burst by rat neutrophils.

The influence of spaceflight on the oxidative burst of neutrophils is not known. The present study was designed to evaluate the influence of antiorthostatic suspension, a ground-based modeling system designed to simulate certain aspects of weightlessness that occur after spaceflight, on the capacity of rat neutrophils to express the oxidative burst, an important host defense mechanism against microbial pathogens. Rats were suspended in whole body harnesses in the antiorthostatic orientation for a 3- or 7-day period. Control rats were suspended orthostatically or allowed to remain in vivarium cages without the attachment of any suspension materials. After suspension, peripheral blood was harvested and neutrophils were isolated by density gradient centrifugation. The enriched neutrophil preparations were stimulated with N-formyl-methionyl-leucine-phenylalanine and phorbol myristic acid to induce the oxidative burst. It was found that neutrophils isolated from suspended animals released the same levels of superoxide anion as did vivarium control animals that were not suspended, indicating that whole body suspension did not alter this aspect of rat neutrophil function.

The AB-180 circulatory support system: summary of development and plans for phase I clinical trial.

BACKGROUND: The AB-180 circulatory support system is a small, durable, efficient centrifugal pump with low thrombogenic potential. The device was designed to provide a fully implantable, left ventricular assist system for short-term support to address the issues of systemic anticoagulation, thrombus formation, infection, and cost. METHODS: Extensive bench and animal studies were performed to validate the mechanical integrity of the device and its functionality as an implant. RESULTS: These studies demonstrated anticoagulation requirements, established operating guidelines, incorporated safety systems, and demonstrated safety and efficacy. CONCLUSIONS: The AB-180 fulfills the stated goals on initial evaluation. A phase I human trial is underway.

Polymerase chain reaction protocols for rabies virus discrimination.

The development of RT PCR methodology has facilitated greatly the genetic characterisation of many rabies viruses (RVs), distinct strains of which persist in certain host species reservoirs within geographically defined regions. The relative temporally conserved nature of certain regions of the RV genome, particularly the N gene, permits development of rapid molecular methods for RV typing. Two main strategies have been applied to viral discrimination: (1) restriction fragment length polymorphism (RFLP) of PCR products and (2) strain-specific PCR (SS-PCR), in which sequences of specific viral strains are amplified differentially using strain-specific primers. Both these approaches have yielded methods of value to rabies epidemiological studies and control programs in Ontario. These procedures have facilitated the identification of intra-strain variants of the arctic fox strain, the only terrestrial RV strain persisting in the area, and they allow rapid discrimination of this strain from those circulating in insectivorous bat reservoirs and from the foreign raccoon strain, which continues to spread throughout the northeastern US and threatens to enter Ontario. Such methods can be adapted readily for use in other regions harbouring multiple overlapping RV reservoirs.

The design of strain-specific polymerase chain reactions for discrimination of the racoon rabies virus strain from indigenous rabies viruses of Ontario.

Since its recognition as a discrete epizootic in Florida in the early 1950s, the raccoon strain of rabies virus (RV) has spread over almost the entire eastern seaboard of the US and now threatens to enter the southernmost regions of Canada. To characterise this RV strain in more detail, nucleotide sequencing of the N and G genes, encoding the nucleoprotein and glycoprotein, respectively, of representative isolates has been undertaken. This sequence information generated a conserved restriction map of the N gene, thereby permitting unequivocal identification of this strain by molecular techniques. Comparisons of the predicted nucleoprotein and glycoprotein products with those of other RV strains identified a number of amino acid sequence variations conserved only in the raccoon strain. This information was used to design strain-specific primers targeted to the N gene sequences encoding these residues. The incorporation of these primers into a multiplex polymerase chain reaction (PCR) protocol permitted easy and rapid discrimination between the raccoon RV strain and indigenous Ontario RVs.

Detection of bovine immunodeficiency-like virus by the polymerase chain reaction.

This report describes a test for the bovine lentivirus, bovine immunodeficiency-like virus, in which polymerase chain reaction (PCR) technology is employed. Two pairs of oligonucleotide primers directed to sequences within the coding regions of the gag and pol genes generated the expected PCR products from molecular BIV clones and from DNA of BIV-infected cell cultures but not from DNA of uninfected cultures. Data indicating the specificity and sensitivity of these PCRs for BIV detection and the potential utility of this technology for diagnostic applications are presented.

Neurofibromatosis and interstitial lung disease.

The literature suggests that 10% to 20% of adult patients with neurofibromatosis have associated interstitial lung disease. Characteristics of such involvement, as present in the case reported herein, include bilateral lower lobe fibrosis and may include bullous and cystic changes in advanced cases. In addition to pulmonary fibrosis, neurofibromatosis may have other intrathoracic associations; including "dumbbell" neurofibromas, intercostal neurofibromas, and intrathoracic meningoceles.

Effects of mitomycin C and carboplatin pretreatment on multidrug resistance-associated P-glycoprotein expression and on subsequent suppression of tumor growth by doxorubicin and paclitaxel in human metastatic breast cancer xenografted nude mice.

Overexpression of P-glycoprotein (Pgp), multidrug resistance-associated protein (MRP), and several other proteins has been associated with development of multidrug resistance by cancer cells, which represents a significant obstacle to successful treatment by chemotherapy. We had previously demonstrated that a single noncytotoxic dose of mitomycin C (MMC), carboplatin, or one of several other DNA cross-linking agents suppressed mRNA expression of the mdr1 gene coding for Pgp, leading to a subsequent suppression of Pgp protein levels and a concomitant decrease in drug efflux. Pretreatment with MMC led to a 5- to 10-fold decrease in the ED50 for cell killing by a subsequent agent such as the Pgp substrate, doxorubicin, but did not affect killing by the non-Pgp substrate, cisplatin. In this study, we report that MMC and carboplatin each significantly suppressed Pgp protein levels in human MDA-MB-435 cells xenografted as solid tumors into the lateral mammary fat pads of female nude mice, with a similar time course as had previously been observed in cell culture. Pretreatment of mice with MMC or carboplatin 48-72 h prior to receiving either doxorubicin or paclitaxel caused a significantly greater reduction in tumor growth rate compared to either agent alone or the combination given simultaneously. These data suggest that a combination chemotherapy regimen consisting of a DNA cross-linking agent given to modulate the MDR phenotype, followed by a second cytotoxic agent, may be an effective treatment for human patients with de novo or late stage acquired multidrug-resistant malignancies.