Pharmacokinetics and distribution of voriconazole in body fluids of dogs after repeated oral dosing.

The goal of this project was to determine the pharmacokinetics of voriconazole and its concentration in cerebrospinal fluid (CSF), aqueous humor, and synovial fluid in five healthy dogs following once daily oral dose of 6 mg/kg for 2 weeks. Body fluid and plasma drug concentrations were determined by high-performance liquid chromatography (HPLC). Mild to moderate gastrointestinal adverse effects were seen. The mean AUC0-24 : minimum inhibitory concentration (MIC) ratio was 15.23 for a chosen MIC of 1 µg/mL, which is lower than the recommended target of 20-25 and also lower than previously reported in dogs, perhaps reflecting induction of metabolizing enzymes by multiple dosing. Voriconazole concentrations in the CSF, aqueous humor, and synovial fluid were only 13-30% the concurrent plasma concentration, which is lower than previously reported in other species. Results of this study suggest that twice daily, administration may be necessary to maintain therapeutic plasma concentrations in dogs but further studies are warranted.

Efficacy of gallium maltolate against Lawsonia intracellularis infection in a rabbit model.

Antimicrobial efficacy against Lawsonia intracellularis is difficult to evaluate in vitro, thus, the effects of gallium maltolate's (GaM) were investigated in a rabbit model for equine proliferative enteropathy (EPE). Juvenile (5-6-week-old) does were infected with 3.0 × 10(8) L. intracellularis/rabbit and allocated into three groups (n = 8). One week postinfection, one group was treated with GaM, 50 mg/kg; one, with doxycycline, 5 mg/kg; and one with a sham-treatment (control). Feces and blood were collected daily and weekly, respectively, to verify presence of L. intracellularis fecal shedding using qPCR, and seroconversion using immunoperoxidase monolayer assay. Rabbits were sacrificed after 1 week of treatment to collect intestinal tissues focusing on EPE-affected sections. Intestinal lesions were confirmed via immunohistochemistry. No difference was noted between treatments regarding EPE-lesions in jejunum (P = 0.51), ileum (P = 0.74), and cecum (P = 0.35), or in L. intracellularis fecal shedding (P = 0.64). GaM and doxycycline appear to have similar efficacy against EPE in infected rabbits.

Pharmacokinetics of gallium maltolate in Lawsonia intracellularis-infected and uninfected rabbits.

Oral gallium maltolate (GaM) pharmacokinetics (PK) and intestinal tissue (IT) concentrations of elemental gallium ([Ga]) and iron ([Fe]) were investigated in a rabbit model of equine proliferative enteropathy (EPE). New Zealand white does (uninfected controls and EPE-infected, n = 6/group) were given a single oral GaM dose (50 mg/kg). Serial blood samples were collected from 0 to 216 h post-treatment (PT) and IT samples after euthanasia. Serology, qPCR, and immunohistochemistry confirmed, or excluded, EPE. Blood and IT [Ga] and [Fe] were determined using inductively coupled plasma-mass spectrometry. PK parameters were estimated through noncompartmental approaches. For all statistical comparisons on [Ga] and [Fe] α = 5%. The Ga log-linear terminal phase rate constant was lower in EPE rabbits vs. uninfected controls [0.0116 ± 0.004 (SD) vs. 0.0171 ± 0.0028 per hour; P = 0.03]; but half-life (59.4 ± 24.0 vs. 39.4 ± 10.8 h; P = 0.12); Cmax (0.50 ± 0.21 vs. 0.59 ± 0.42 µg/mL; P = 0.45); tmax (1.75 ± 0.41 vs. 0.9 ± 0.37 h; P = 0.20); and oral clearance (6.743 ± 1.887 vs. 7.208 ± 2.565 L/h; P = 0.74) were not. IT's [Ga] and [Fe] were higher (P < 0.0001) in controls. In conclusion, although infection reduces IT [Ga] and [Fe], a 48 h GaM dosing interval is appropriate for multidose studies in EPE rabbits.

Involvement of the CD95 (APO-1/Fas) receptor/ligand system in multiple sclerosis brain.

Immunohistochemical methods were used to search for Fas receptor/Fas ligand system involvement in multiple sclerosis (MS) white matter brain lesions. We found large numbers of Fas ligand (Fas-L)-bearing cells present in two acute lesions and 12 of 16 chronic MS lesions, and very few positive cells in non-inflammatory controls. Four of six brains from non-MS neuropathologic conditions associated with inflammation and white matter disease were, however, also positive for Fas-L. Double staining with cell-specific markers revealed that the pattern of ligand-positive cells in chronic MS lesions was complex and composed of several different cell types which were primarily resident glial cells with a small overlay of macrophages. Fas/APO 1 (CD95) receptor expression in MS tissue was also evaluated and marked upregulation of the receptor was found. In addition, Fas receptor was induced, but to a lesser extent, in numerous control brains. The observations that TUNEL-positive dying cells were present in MS lesions and showed excellent co-localization with Fas-L, indicate that the Fas death system may contribute to plaque pathogenesis and could lead to the development of a new category of therapeutic agents for MS.

Effect of titanium oxide nanoparticle incorporation into nm thick coatings deposited using an atmospheric pressure plasma.

This study reports on the use of an atmospheric plasma technique to incorporate metal oxide nanoparticles into nm thick siloxane coatings. Titanium dioxide (TiO2) particles with diameters of 30-80 nm, were mixed with a number of different siloxanes-polydimethylsiloxane, hexamethyldisiloxane and tetraethylorthosilicate (TEOS). The TiO2/TEOS mixture was found to give the most stable suspension, possibly due to the higher surface tension of TEOS compared with the other siloxanes. TiO2/TEOS mixtures with 2 to 10% by weight of the metal oxide were prepared and were then nebulised into a helium/oxygen atmospheric plasma. Polyethylene terepthalate (PET) and silicon wafer substrates were passed through this plasma using a reel-to-reel substrate manipulation system. SEM combined with EDX was used to examine the distribution of the metal oxide particles in the resultant coatings. The TEOS coating thickness without TiO2 addition was 9 nm. The composite coating consisted of a relatively homogeneous distribution of small agglomerates of the TiO2 nanoparticles in TEOS. A linear increase in the titanium surface concentration was observed with increase in the quantity of TiO2 added into the siloxane precursor. The chemical functionality of the siloxane coating was examined using FTIR spectroscopy and no significant spectrum differences was observed with the incorporation of the different concentrations of TiO2 into the polymer. There were also no changes observed in coating surface energy with TiO2 incorporation. Coating morphology was examined using optical profilometry and surface roughness (Ra) values increased from typical values of 0.8 nm for the TEOS coating to 4.1 nm for the TiO2/TEOS coating. The adhesion of the deposited coatings was compared using fragmentation tests. These were carried out through uniaxial tensile loading. The coating cracking pattern after applied strain of 20% was not observed to change significantly with the addition of TiO2 into the siloxane.

Rapid inhibition of pinocytosis in baby hamster kidney (BHK-21) cells following infection with vesicular stomatitis virus.

Infection of baby hamster kidney cells with vesicular stomatitis virus (VSV) caused a reduced rate of pinocytosis (as judged by the uptake of horseradish peroxidase) after 1 h, and maximum inhibition (60-80%) was observed at 4-6 h. This inhibition occurred 2-3 h before release of virus or changes in cell morphology. Analytical cell fractionation of homogenates of VSV-infected cells indicated that the horseradish peroxidase taken up by pinocytosis was transferred to lysosomes. The inhibition of pinocytosis required viral gene expression: little or no inhibition was detected in cells infected with UV-irradiated virus, wild-type virus in the presence of cycloheximide, or a temperature-sensitive mutant which failed to synthesize viral proteins. When cells were infected with temperature-sensitive viruses with mutations in the five VSV genes, an inhibition of pinocytosis was observed only when the viral transmembrane glycoprotein was present on the surface of the cells.

Kinetics and residues after intraperitoneal procaine penicillin G administration in lactating dairy cows.

This paper describes the pharmacokinetic profile of procaine penicillin G after intraperitoneal (IP) administration in eight lactating dairy cows. Procaine pencillin G (PPG, 21 000 IU/kg) was deposited into the abdominal cavity of each cow following an incision in the right paralumbar fossa. Blood and milk samples were taken over the following 10 days, at which point the cows were euthanized. Plasma, milk, muscle, liver, and kidney penicillin concentrations were determined by HPLC, with a limit of quantification of 5 ng/mL for plasma and milk and 40 ng/g for tissue samples. A noncompartmental method was used to analyze plasma kinetics. The mean pharmacokinetic parameters (+/-SD) were: C(max), 5.5 +/- 2.6 microg/mL; T(max), 0.75 +/- 0.27 h; AUC(0-infinity), 10.8 +/- 4.9 microg x h/mL; MRT, 2.2 +/- 0.9 h. All milk from treated cows contained detectable penicillin residues for a minimum of three milkings (31 h) and maximum of five milkings (52 h) after administration. Concentrations of penicillin in all muscle, liver, and kidney samples taken 10 days postadministration were below the limit of quantification. Necropsy examinations revealed foci of hemorrhage on the rumenal omentum of most cows but peritonitis was not observed. Systemic inflammation as determined by change in leukogram or plasma fibrinogen was noted in one cow. The results of this study demonstrate that IP PPG is absorbed and eliminated rapidly in lactating dairy cows.

Characterization and applications of auto-locked vacuum-sealed diode lasers for precision metrology.

We demonstrate the performance characteristics of a new class of vacuum-sealed, autolocking diode laser systems and their applications to precision metrology. The laser is based on adaptations of a design that uses optical feedback from an interference filter and it includes a vacuum-sealed cavity, an interchangeable base-plate, and an autolocking digital controller. A change of the base-plate allows operation at desired wavelengths in the visible and near infrared spectral range, whereas the autolocking ability allows the laser to be tuned and frequency stabilized with respect to atomic, molecular, and solid-state resonances without human intervention using a variety of control algorithms programmed into the same controller. We characterize the frequency stability of this laser system based on the Allan deviation (ADEV) of the beat note and of the lock signal. We find that the ADEV floor of 2 × 10-12 and short-term linewidth of ∼200 kHz are strongly influenced by current noise and vacuum sealing. Reducing the current noise and cavity pressure decreases the ADEV floor and increases the averaging time at which the floor occurs, which is a signature of long-term stability. We also show that evacuating the cavity to ∼1 Torr reduces the range of the correction signal of the feedback loop by approximately one order of magnitude, thereby increasing the lock range of the controller. The long-term stability allows the laser to be incorporated into a commercial gravimeter for accurate measurements of gravitational acceleration at the level of a few parts-per-billion, which are comparable to values obtained with an iodine-stabilized He-Ne laser. The autolocking and pattern-matching features of the controller allow the laser to be tuned and stabilized with respect to a temperature tunable transmission spectrum of a fiber-Bragg grating. This capability may be suitable for the development of a differential absorption LIDAR transmitter that can generate data at both on-line and off-line lock points using a single laser.

The effects of ageing on the distribution of vesicular acetylcholine transporter immunoreactive inputs to pelvic motoneurons of male Wistar rats.

Age-related changes in the number and size of large cholinergic terminals immunoreactive for vesicular acetylcholine transporter (VAChT), were documented for the dorsolateral nucleus (DLN), retrodorsolateral nucleus (RDLN) and spinal nucleus of the bulbospongiosus (SNB) of the lumbosacral spinal cord of male rats. The most significant changes were a large increase in the number and size of cholinergic terminals within the DLN of aged animals, together with a small decrease in terminal number within the RDLN. No significant age-associated differences in VAChT labeling were seen within the SNB. In both age groups, SNB motoneurons projecting to the levator ani muscle received about 9 to 10 contacts from large cholinergic terminals. Ultrastructural examination of the terminals revealed structures likely to be postsynaptic subsurface cisterns that are characteristic of type C terminal boutons. Since both the DLN and SNB contain motoneurons innervating pelvic muscles and sphincters, these findings provide further evidence for a central cholinergic influence on micturition and sexual reflexes and suggest that this may remain robust in the face of ageing.

Interferon treatment inhibits pinocytosis.

Treating mouse L cells with crude or purified mouse interferon inhibited fluid-phase pinocytosis. Inhibition was maximum at 24 h after treatment with 1,000 U of interferon per ml and was dose dependent and reversible with time. Pinocytosis was inhibited when human and chicken embryo cells were treated with homologous, but not heterologous, interferons.

Purification and identification of a 7.6-kDa protein in media conditioned by superinvasive cancer cells.

BACKGROUND: Selection of the human drug sensitive and invasive cell line (MDA-MB-435S-F) with the chemotherapeutic agent paclitaxel, resulted in the development of drug resistant cell lines displaying enhanced invasion-related characteristics. MATERIALS AND METHODS: Serum-free conditioned media from the human cancer drug-sensitive and invasive cell line (MDA-MB-435S-F) and its paclitaxel-resistant superinvasive variant (MDA-MB-435S-F/Taxol10p4pSI) were analyzed using Surface enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS). RESULTS: A differentially expressed protein was observed at 7.6 kDa, which was 4-fold up-regulated in MDA-MB-435S-F/Taxol10p4pSI. The differentially expressed protein was identified using matrix-assisted laser desorption ionization tandem time-of-flight mass spectrometry (MALDI-TOF/TOF MS), as a fragment of bovine transferrin. The transferrin receptor was also found to be overexpressed in the superinvasive cell line. CONCLUSION: Cleavage of serum proteins such as transferrin could provide a valuable source of markers for malignant tumours and could also play a role in aspects of cancer pathogenesis, such as tumour cachexia.

Protein and chemotherapy profiling of extracellular vesicles harvested from therapeutic induced senescent triple negative breast cancer cells.

Triple negative breast cancer (TNBC) is an aggressive subtype with relatively poor clinical outcomes and limited treatment options. Chemotherapy, while killing cancer cells, can result in the generation of highly chemoresistant therapeutic induced senescent (TIS) cells that potentially form stem cell niches resulting in metastases. Intriguingly, senescent cells release significantly more extracellular vesicles (EVs) than non-senescent cells. Our aim was to profile EVs harvested from TIS TNBC cells compared with control cells to identify a potential mechanism by which TIS TNBC cells maintain survival in the face of chemotherapy. TIS was induced and confirmed in Cal51 TNBC cells using the chemotherapeutic paclitaxel (PTX) (Taxol). Mass spectrometry (MS) analysis of EVs harvested from TIS compared with control Cal51 cells was performed using Ingenuity Pathway Analysis and InnateDB programs. We demonstrate that TIS Cal51 cells treated with 75 nM PTX for 7 days became senescent (senescence-associated ß-galactosidase (SA-ß-Gal) positive, Ki67-negative, increased p21 and p16, G2/M cell cycle arrest) and released significantly more EVs (P=0.0002) and exosomes (P=0.0007) than non-senescent control cells. Moreover, TIS cells displayed an increased expression of the multidrug resistance protein 1/p-glycoprotein. MS analysis demonstrated that EVs derived from senescent Cal51 cells contained 142 proteins with a significant increased fold change compared with control EVs. Key proteins included ATPases, annexins, tubulins, integrins, Rabs and insoluble senescence-associated secretory phenotype (SASP) factors. A fluorescent analogue of PTX (Flutax-2) allowed appreciation of the removal of chemotherapy in EVs from senescent cells. Treatment of TIS cells with the exosome biogenesis inhibitor GW4869 resulted in reduced SA-ß-Gal staining (P=0.04). In summary, this study demonstrates that TIS cells release significantly more EVs compared with control cells, containing chemotherapy and key proteins involved in cell proliferation, ATP depletion, apoptosis and the SASP. These findings may partially explain why cancer senescent cells remain viable despite chemotherapeutic challenge.

Interleukin 8: an autocrine growth factor for malignant mesothelioma.

Interleukin 8 (IL-8) is a potent chemokine that also has a direct growth-potentiating effect on certain tumors. In the present study, we determined IL-8 levels in human malignant mesothelioma (MM) effusions and congestive heart failure pleural fluids. We also investigated antigenic IL-8 production by different MM cell lines, and we describe the role of IL-8 in the autocrine growth regulation of MMs. Mesothelial (CRL-9444 = MC) and MM (CRL-2081 = MM-1, CRL-5915 = MM-2, and CRL-5820 = MM-3) cell lines were grown using standard culture methods. The bioactive IL-8 levels were measured in supernatants of cultured cells by ELISA, and the expression of cell-associated immunoreactive IL-8 was observed by immunohistochemistry. The proliferative activity was determined by thymidine ([3H]thymidine) incorporation and also by direct cell counts after incubation with varying concentrations of IL-8 in the presence/absence of specific polyclonal IL-8 antibody. We found significantly higher levels of IL-8 in mesothelioma pleural fluids than congestive heart failure and a time-dependent increase in IL-8 levels in MM-1 and MM-2 cell supernatants during 96 h of incubation. IL-8 levels were nearly undetectable in MM-3 and MC cell line supernatants. In MM-1 and MM-2 cells, IL-8 caused a dose-dependent increase of [3H]thymidine incorporation to maximal levels of 46.3 +/- 3.6% and 12.3 +/- 1.6% (P < 0.001), respectively, when compared with serum-free medium as control. Neutralization of IL-8 significantly decreased proliferative activity of MM-1 and MM-2. IL-8 did not induce proliferative activity in MM-3 and MC cells. We conclude that IL-8 had a direct growth-potentiating activity in MMs.

A survey of undergraduate orthodontic training and orthodontic practices by general dental practitioners.

PURPOSE OF THE STUDY: To assess satisfaction with undergraduate orthodontic training, the variety of treatments undertaken in general practice, practitioners' perceived competence in orthodontics and the level of interest in continuing education in orthodontics. DESIGN: Cross-sectional questionnaire-based study. MATERIALS AND METHODS: A questionnaire was mailed to 520 general practitioners in Dublin, Kildare and Wicklow as listed in Irish Dental Council Register of Dentists 2003. RESULTS: Forty-six percent of dentists responded. More than half (54 per cent) of the respondents were satisfied with both academic and clinical aspects of undergraduate training. Twenty-nine percent regularly perform orthodontic treatment. Only 60 percent feel comfortable treating orthodontic emergencies. Over 70 percent have either already attended or aspire to attend further training in orthodontic diagnosis and interceptive orthodontics. CONCLUSIONS: Our study indicates that in the greater Dublin area, graduates (those qualified less than 10 years) are increasingly satisfied with undergraduate teaching. Orthodontic treatment is performed regularly in general dental practice with interceptive procedures most often carried out. While ability to deal with orthodontic emergencies is not universal, practitioners do appear confident to perform a variety of orthodontic procedures. Interest in continuing education in orthodontics is very high. Our study indicates that participation in continuing education in orthodontics appears to translate into greater provision of orthodontic care in general practice.

Brain dystrophin-glycoprotein complex: persistent expression of beta-dystroglycan, impaired oligomerization of Dp71 and up-regulation of utrophins in animal models of muscular dystrophy.

BACKGROUND: Aside from muscle, brain is also a major expression site for dystrophin, the protein whose abnormal expression is responsible for Duchenne muscular dystrophy. Cognitive impairments are frequently associated with this genetic disease, we therefore studied the fate of brain and skeletal muscle dystrophins and dystroglycans in dystrophic animal models. RESULTS: All dystrophin-associated glycoproteins investigated were reduced in dystrophic muscle fibres. In Dp427-deficient mdx brain and Dp71-deficient mdx-3cv brain, the expression of alpha-dystroglycan and laminin was reduced, utrophin isoforms were up-regulated and beta-dystroglycan was not affected. Immunofluorescence localization of beta-dystroglycan in comparison with glial, endothelial and neuronal cell markers revealed co-localization of von Willebrand factor with beta-dystroglycan. Its expression at the endothelial-glial interface was preserved in dystrophin isoform-deficient brain from mdx and mdx-3cv mice. In addition, chemical crosslinking revealed that the Dp71 isoform exists in mdx brain predominantly as a monomer. CONCLUSIONS: This suggests an association of beta-dystroglycan with membranes at the vascular-glial interface in the forebrain. In contrast to dystrophic skeletal muscle fibres, dystrophin deficiency does not trigger a reduction of all dystroglycans in the brain, and utrophins may partially compensate for the lack of brain dystrophins. Abnormal oligomerization of the dystrophin isoform Dp71 might be involved in the pathophysiological mechanisms underlying abnormal brain functions.

Evidence for multiple sclerosis as an infectious disease.

Evidence for a viral cause of multiple sclerosis (MS) is indirect since no infectious agent has been reproducibly isolated from MS tissues nor has viral genome or antigen been consistently identified. The occurrence of spontaneous human and animal models of demyelination, serologic studies, and epidemiologic data provide persuasive circumstantial evidence for an infectious trigger in this disease. Potential mechanisms for viral induced demyelination include persistent infection of host tissues or immune mediated organ damage either in the presence or absence of the infectious agent. Any proposed viral candidate should cause demyelination in animals or man and the pattern of infection should be consistent with the unique geographic features of MS epidemiology. In addition, serologic studies should support an infection by the agent and/or viral genome should be detected in MS tissues. At this time no virus can be unequivocally linked to MS but cumulative evidence is more supportive of canine distemper virus than other viruses.

Disease markers in acute multiple sclerosis.

Three serum components that are known to become elevated during inflammation and tissue destruction, C-reactive protein, C3 proactivator, and orosomucoid, which are acute phase reactants, and serum lgM were measured in patients with multiple sclerosis (MS) hospitalized with acute exacerbations. Significantly elevated levels of one or more of these serum components were found in 12 of 13 patients with clinically active MS. Serial studies in two patients revealed that clinical improvement was accompanied by a decline in the serum levels of these factors. These findings suggest that measurement of these serum proteins may be of value in assessing progress of disease activity in MS patients.

Steroid therapy in multiple sclerosis. Point of view.

A number of studies have demonstrated that high-dose corticosteroid treatment improves the rate of recovery from acute exacerbations of multiple sclerosis. The beneficial effect is more rapidly and consistently produced by high-dose corticosteroid administration than with adrenocorticotropic hormone. The most rapid improvement in clinical condition and cerebrospinal fluid parameters of patients has been observed with short courses of very high-dose intravenous therapy. This form of treatment would be most advantageous for patients with severe and rapidly progressive exacerbations. Ultimately, the duration of treatment will depend on the patient's response to treatment, tolerance of corticosteroid withdrawal, and the occurrence of significant complications of therapy. However, in our experience, a three- to four-month slowly tapered course of oral therapy is often needed, and is usually well tolerated, with few serious side effects when given once daily in the morning.

Household pets among veterans with multiple sclerosis and age-matched controls. Pilot survey.

A pilot study was carried out among 22 Vietnam-era male US veterans with multiple sclerosis (MS) and 55 age- and sex-matched controls for prior exposure to dogs, cats, and animals with a distemperlike illness. No difference in dog ownership, or sick animals, and subsequent human illness was found in the group with MS or the control group. However, the distribution of dogs by indoor-outdoor status, as reported by patients with MS or controls, showed significant variation by tier of residence. Indoor dogs were more common in northern than southern latitudes, and this may be an important finding in light of the variation in the risk of MS with latitude.