Recombinant canine distemper virus strain Snyder Hill expressing green or red fluorescent proteins causes meningoencephalitis in the ferret.

The propensity of canine distemper virus (CDV) to spread to the central nervous system is one of the primary features of distemper. Therefore, we developed a reverse genetics system based on the neurovirulent Snyder Hill (SH) strain of CDV (CDV(SH)) and show that this virus rapidly circumvents the blood-brain and blood-cerebrospinal fluid (CSF) barriers to spread into the subarachnoid space to induce dramatic viral meningoencephalitis. The use of recombinant CDV(SH) (rCDV(SH)) expressing enhanced green fluorescent protein (EGFP) or red fluorescent protein (dTomato) facilitated the sensitive pathological assessment of routes of virus spread in vivo. Infection of ferrets with these viruses led to the full spectrum of clinical signs typically associated with distemper in dogs during a rapid, fatal disease course of approximately 2 weeks. Comparison with the ferret-adapted CDV(5804P) and the prototypic wild-type CDV(R252) showed that hematogenous infection of the choroid plexus is not a significant route of virus spread into the CSF. Instead, viral spread into the subarachnoid space in rCDV(SH)-infected animals was triggered by infection of vascular endothelial cells and the hematogenous spread of virus-infected leukocytes from meningeal blood vessels into the subarachnoid space. This resulted in widespread infection of cells of the pia and arachnoid mater of the leptomeninges over large areas of the cerebral hemispheres. The ability to sensitively assess the in vivo spread of a neurovirulent strain of CDV provides a novel model system to study the mechanisms of virus spread into the CSF and the pathogenesis of acute viral meningitis.

The influence of obesity and consequent insulin resistance on coronary risk factors in medically treated patients with coronary disease.

OBJECTIVE: Obesity promotes the development and progression of coronary heart disease (CHD), in part, through its association with hyperlipidemia, hypertension, clotting abnormalities and insulin resistance. We assessed whether these relationships persist in patients with established CHD treated with evidence-based preventive pharmacologic therapies. DESIGN AND SUBJECTS: We performed a cross-sectional study of 74 adults with CHD and a body mass index (BMI) of >27 kg m(-2) (mean 32+/-4). The mean age of subjects was 64+/-9 years (range 44-84 years). MEASUREMENTS: Obesity measures included weight, BMI, waist, fat mass, intra-abdominal fat and subcutaneous fat. Risk factor measures included insulin sensitivity, fasting insulin level, lipid profiles, blood pressure, C-reactive protein (hs-CRP), plasminogen activator inhibitor (PAI-1) and platelet reactivity. Medication use included aspirin (99%), statin (84%), beta-blocker (71%), ACE inhibitor or blocker (37%) and clopidogrel (28%). RESULTS: There was no direct relationship between obesity parameters and risk factor measures of lipid concentrations, blood pressure, clotting abnormalities or platelet reactivity except for a modest relationship between visceral fat and hs-CRP (r=0.30, P=0.02). However, increased BMI, waist circumference, fat mass, total abdominal fat and abdominal subcutaneous fat all correlated with insulin sensitivity (r-values -0.30 to -0.45, P-values 0.01 to <0.001) and insulin concentrations. Insulin sensitivity, in turn, was the best predictor of PAI-1, triglycerides, high-density lipoprotein (HDL) levels, cholesterol/HDL levels (all P<0.01) and platelet reactivity (R=0.34, P=0.02). CONCLUSIONS: Use of preventive pharmacologic therapies obviated the expected relationship between adiposity and CHD risk factors. However, a residual effect of insulin resistance is left untreated. Total adiposity and central adiposity were strong predictors of insulin sensitivity, which in turn predicted cardiac risk factors such as lipid concentrations, PAI-1 and platelet reactivity. Thus, while evidence-based pharmacologic treatments may diminish the statistical relationship between obesity and many cardiac risk factors, adiposity negatively impacts CHD risk by reducing tissue insulin sensitivity.

Development of a challenge-protective vaccine concept by modification of the viral RNA-dependent RNA polymerase of canine distemper virus.

We demonstrate that insertion of the open reading frame of enhanced green fluorescent protein (EGFP) into the coding sequence for the second hinge region of the viral L (large) protein (RNA-dependent RNA polymerase) attenuates a wild-type canine distemper virus. Moreover, we show that single intranasal immunization with this recombinant virus provides significant protection against challenge with the virulent parental virus. Protection against wild-type challenge was gained either after recovery of cellular immunity postimmunization or after development of neutralizing antibodies. Insertion of EGFP seems to result in overattenuation of the virus, while our previous experiments demonstrated that the insertion of an epitope tag into a similar position did not affect L protein function. Thus, a desirable level of attenuation could be reached by manipulating the length of the insert (in the second hinge region of the L protein), providing additional tools for optimization of controlled attenuation. This strategy for controlled attenuation may be useful for a "quick response" in vaccine development against well-known and "new" viral infections and could be combined efficiently with other strategies of vaccine development and delivery systems.

Advantages of using recombinant measles viruses expressing a fluorescent reporter gene with vibratome slice technology in experimental measles neuropathogenesis.

AIMS: In this study of experimental measles neuropathogenesis, the utility of enhanced green fluorescent protein (EGFP) as a sensitive indicator of measles virus (MV) cell-to-cell spread in the central nervous system (CNS) has been assessed in vibratome-cut brain slices to demonstrate the degree and mechanism of viral spread in the rodent CNS. METHODS: Recombinant MVs expressing EGFP were visualized at different levels in 200-microm vibratome-cut brain sections from infected animals by confocal scanning laser microscopy (CSLM). Comparison was made with 7-microm microtome sections, stained for the N protein of measles by immunocytochemistry (ICC). RESULTS: The recombinant viruses were readily visualized in infected brain tissue, with no loss of neuropathogenicity. No difference was found in the sites of infection when MV infection was detected through EGFP fluorescence or by ICC. MV-infected cells were detected in the cerebral cortex, olfactory bulb and tract, hippocampus, thalamus, hypothalamus, ependyma and subventricular zone. However, the 200-microm vibratome-cut sections and confocal microscopy proved excellent for demonstrating virus distribution in neurites and for in-depth analysis of the extent of tract infection in the white matter of the cerebral hemispheres such as selective infection of the internal capsule and anterior commissure. CONCLUSIONS: The use of self-tracing recombinant MVs, viewed in thick vibratome-cut sections by CSLM, demonstrated that in experimental MV neuropathogenesis the infection is selective and spreads predominately by neurites using defined anatomical pathways.

Size-exclusion chromatography with on-line ultraviolet, proton nuclear magnetic resonance and mass spectrometric detection and on-line collection for off-line Fourier transform infrared spectroscopy.

The coupling of HPLC with UV detection and on-line NMR spectroscopy and mass spectrometry combined with a dedicated interface for the collection of the chromatographic eluent for subsequent Fourier transform (FT) IR has been investigated using a number of polymer additives as model compounds. Size-exclusion chromatography was performed using deuterated chloroform as eluent with the separation monitored on-line by UV detection at 254 nm and on-flow 1H-NMR and MS. The effluent from the NMR probe was directed to a dedicated HPLC interface where it was deposited on a germanium plate for subsequent FT-IR. NMR and MS spectra were successfully obtained for 2,6-di-tert.-butyl-4-methylphenol, octadecyl-3-(3,5-di-tert.-butyl-4-hydroxyphenyl) propionate (Irganox 1076) and diisooctyl phthalate on-line and FT-IR spectra for all three compounds were obtained off-line. Practical problems encountered with this multiple hyphenation are described.

Photosynthetic responses of native and introduced C4 grasses from Venezuelan savannas.

Introduced African grasses are invading the grasslands of the Venezuelan savannas and displacing the native grasses. This work, which is part of a program to understand the reasons for the success of the African grasses, specifically investigates whether introduced and native grasses differ in some photosynthetic characteristics.The responses to photon flux density, leaf temperature, leaf-air vapour pressure difference and leaf water potential of leaf photosynthetic rate of two introduced African C4 grasses (Hyparrhenia rufa and Melinis minutiflora) and of a lowland and a highland population of a native Venezuelan grass (Trachypogon plumosus) grown under controlled conditions were compared. These responses in all three species were typical of tropical C4 pasture grasses. The introduced grasses had higher maximum leaf conductance, net photosynthetic rates, and optimum temperature (H. rufa only) for photosynthesis than T. plumosus. However, T. plumosus was able to continue photosynthesis to lower leaf water potentials than the two introduced grasses, and the efficiency which it utilized water, light and mineral nutrients to fix carbon were similar to those of the introduced grasses.The higher rates of leaf photosynthesis of the introduced grasses contributed to, but only partially explained, the higher growth rates compared to T. plumosus. The higher growth rates and nutrient concentration of the introduced grasses are consistent with their ability to establish rapidly, compete successfully for resources, and displace T. plumosus from moist, fertile sites. Conversely, the slower growth rate, lower nutrient concentrations, and superior water relations characteristics are consistent with the capacity of T. plumosus to resist invasion by introduced grasses in poorer sites.

Comparative responses of the Savanna grasses Cenchrus ciliaris and Themeda triandra to defoliation.

Two perennial tussock grasses of savannas were compared in a glasshouse study to determine why they differed in their ability to withstand frequent, heavy grazing; Cenchrus ciliaris is tolerant and Themeda triandra is intolerant of heavy grazing. Frequent defoliation at weekly intervals for six weeks reduced shoot biomass production over a subsequent 42 day regrowth period compared with previously undefoliated plants (infrequent) in T. triandra, but not in C. ciliaris. Leaf area of T. triandra expanded rapidly following defoliation but high initial relative growth rates of shoots were not sustained after 14 days of regrowth because of reducing light utilising efficiency of leaves. Frequently defoliated plants were slower in rate of leaf area expansion and this was associated with reduced photosynthetic capacity of newly formed leaves, lower allocation of photosynthate to leaves but not lower tiller numbers. T. triandra appears well adapted to a regime where defoliation is sufficiently infrequent to allow carbon to be fixed to replace that used in initial leaf area expansion. In contrast, C. ciliaris is better adapted to frequent defoliation than is T. triandra, because horizontally orientated nodal tillers are produced below the defoliation level. This morphological adaptation resulted in a 10-fold higher leaf area remaining after defoliation compared with similarly defoliated T. triandra, which together with the maintenance of moderate levels of light utilising efficiency, contributed to the higher leaf area and shoot weight throughout the regrowth period.

Stomatal responses to humidity in Opuntia inermis in relation to control of CO2 and H2O exchange patterns.

At constant cladode temperature the stomatal resistance of O. inermis increased when the cladode-air vapor pressure difference was increased and stomatal resistance decreased when the cladode-air vapor pressure difference was lowered. Net CO2 fixation in the dark was very responsive to these humidity dependent changes in stomatal resistance. Net CO2 fixation and stomatal resistance in the light did not respond to changes in cladode-air vapor pressure differences in the light under the conditions tested. When temperature response functions for dark CO2 fixation were examined at constant ambient humidity, the reduction in dark CO2 fixation at higher temperatures was largely due to stomatal closure in response to the increased vapor pressure difference. The water requirement for net CO2 fixation in the dark at typical nocturnal vapor pressure differences was about 10 times lower than that of net CO2 fixation in the light at vapor pressure differences typical of the late afternoon. The role of the stomatal responses to humidity in determining the patterns and rates of net CO2 exchange in the light or dark, and its possible ecological significance is discussed.

Comparison between pressure-volume and dewpoint-hygrometry techniques for determining the water relations characteristics of grass and legume leaves.

The water relations characteristics of three grass species (Panicum maximum var. trichoglume, Cenchrus ciliaris, Heteropogon contortus), and a legume (Macroptilium atropurpureum) grown in the field were measured using both a modified pressure/volume technique with pressure bomb measurements on single leaves and a dewpoint hygrometry technique applied to fresh and to frozen and thawed leaf discs.The two techniques agreed well in the estimates of osmotic potential at full turgor and the water potential at zero turgor. However, for parameters such as the relative water content at zero turgor, bound water and bulk modulus of elasticity there was a poor correlation between the estimates from the two methods. The pressure/volume technique gave less variable results and is more convenient for field use than the hygrometry technique. The determination of the modulus of elasticity from various functions relating pressure potential to relative water content is discussed.

Hazards of laser smoke during endodontic therapy.

The purpose of this study was to evaluate the potential for spreading bacterial contamination from the root canal to the patient and the dental team via the smoke produced by the laser. Five extracted teeth were deliberately inoculated with a specific strain of Escherichia coli. The canals were subjected to an agron laser. The smoke plume was captured and cultured. All of the cultures were positive for growth of the E. coli used. It was concluded that the laser smoke does present a hazard of bacterial dissemination and that precautions must be taken to protect against spreading infections when using lasers in the root canal.

Chronic pain associated with a foreign body left under the soft tissue flap during periapical surgery.

A case is reported of a patient who requested treatment for chronic pain of 7 yr duration. After several unsuccessful endodontic procedures, a cotton pellet was discovered under the soft tissue near the apex of the left maxillary cuspid. It was theorized that the cotton was left under the tissue flap during a previous apicoectomy procedure. After the cotton was removed the patient subsequentially reported that the pain was relieved.

Retrievability of Thermafil plastic cores using organic solvents.

Twenty distal roots of extracted mandibular first and second molars were instrumented, then obturated using Thermafil obturating material with solid plastic core carriers. To simulate a retreatment process, the gutta-percha was softened using one of four solvents, chloroform, xylene, eucalyptol, or halothane. A K file was used to advance the solvent into the gutta-percha and to engage the plastic carrier. In all but one case, the plastic carriers were easily removed from the root canal. It was concluded that the plastic carriers used with the Thermafil obturation media do not present a difficult obstacle for removal should the root require retreatment.

Long-term sealing ability of a calcium hydroxide sealer.

A calcium hydroxide sealer (Sealapex) was compared with a zinc oxide and eugenol sealer (Tubli-Seal) over a 32-wk interval to examine solubility in an in vitro simulation. After obturation with gutta-percha and the appropriate sealer, specimens were immediately immersed in a saline solution to challenge the sealers' solubility. The solutions were changed weekly to allow for a continued dissolution of the sealers and to prevent establishment of an equilibrium between the solution and the sealers. The 2- and 32-wk specimens were removed from the solutions, immersed in India ink for 3 days, and then made transparent by a clearing process. Microscopic examination was used to determine the linear penetration of the ink for each tooth. Results revealed that Sealapex statistically had no greater dissolution (based upon linear penetration) than Tubli-Seal at both 2 and 32 wk. It is suggested that Sealapex has a sealing ability comparable to Tubli-Seal and can withstand long-term exposure to tissue fluids without significant leakage. This may allow time for the biochemical action of the calcium hydroxide to stimulate physiological calcification of the apical foramen.

Factors affecting the negotiability of second mesiobuccal canals in maxillary molars.

Factors affecting the negotiability of MB2 canals were evaluated by studying 87 extracted maxillary molars that had undergone previous endodontic treatment in the endodontic technique laboratory. The mesiobuccal roots were resected and radiographed, after which a #08 file was used in an attempt to negotiate those roots with the potential for a second mesiobuccal canal. The file was then intentionally separated in the MB2 canals that could be negotiated. The roots were decalcified, cleared, and observed under a stereomicroscope. Several factors that could interfere with the total or partial negotiation of MB2 canals were identified and included accumulation of debris and sealer that blocked access to these canals, dentinal debris produced with the pathfinding instrument, the presence of anatomical variations, diffuse calcifications, and pulp stones.

Rubber latex allergy and the endodontic patient.

Increasingly, patients with rubber latex allergy are being seen in dental offices. Health care workers, persons allergic to certain foods and those with certain medical histories are potentially latex-sensitive. This article presents the case of a patient with a history of severe rubber latex allergy and the associated management of an endodontically involved tooth. The possibility of the latex allergic patient being sensitive to gutta-percha obturation material is also raised.

Effect of preflaring on Root ZX apex locators.

The Root ZX apex locator is an example of a generation of apex locators that identify the terminus of the canal by measuring a ratio between two electrical impedances. Studies have shown this device to have a high degree of accuracy. However, the manufacturer warns that the performance of these devices is limited by the presence of calcifications and dentinal shaving obstructions. An in vitro study was designed to determine if preflaring of canals would facilitate the passage of files to the apical foramen by eliminating cervical interferences and to see what effect this would have on the performance of the Root ZX apex locator. Thirty-two canals were divided into two groups. Group 1 was not manipulated before use of the Root ZX apex locator and served as control. In group 2, the canals were preflared before the use of the Root Zx apex locator. The working length files were secured in place and measured with the linear measurement tool used by the Visilog 5 imaging program. Results of this study suggest that preflaring of canals will allow working length files to more consistently reach the apical foramen (p = 0.015), which in turn increases the efficacy of the Root ZX apex locator.

Streptococcus pneumoniae exposure is associated with human metapneumovirus seroconversion and increased susceptibility to in vitro HMPV infection.

It remains largely unknown which factors determine the clinical outcome of human metapneumovirus (HMPV) infections. The aim of the present study was to analyse whether exposure to bacterial pathogens can influence HMPV infections. From 57 children, serum samples and colonization data for Haemophilus influenzae, Moraxella catarrhalis, Staphylococcus aureus and Streptococcus pneumoniae were collected at 1.5, 6, 14 and 24 months of age. Seroconversion rates to HMPV were determined and related to bacterial carriage. Frequent nasopharyngeal carriage (≥2 times in the first 2 years of life) of S. pneumoniae, but not of the other three pathogens, was associated with increased seroconversion rates of infants to HMPV at the age of 2 years (frequently vs. less exposed, 93% vs. 59%; p <0.05). Subsequently, the susceptibility of well-differentiated normal human bronchial epithelial cells (wd-NHBE) pre-incubated with bacterial pathogens to in vitro HMPV infection was evaluated. Pre-incubation of wd-NHBE with S. pneumoniae resulted in increased susceptibility to infection with HMPV-enhanced green fluorescent protein (EGFP), as determined by enumeration of EGFP-positive cells. This was not the case for cells pre-incubated with H. influenzae, M. catarrhalis on S. aureus. We conclude that exposure to S. pneumoniae can modulate HMPV infection.