Pharmacokinetics of UC781-loaded intravaginal ring segments in rabbits: a comparison of polymer matrices

UC781 is a potent and poorly water-soluble nonnucleoside reverse transcriptase inhibitor being investi- gated as a potential microbicide for preventing sexual transmission of HIV-1. This study was designed to evaluate the in vivo release and pharmacokinetics of UC781 delivered from matrix-type intravaginal ring segments in rabbits. Three polymer matrices (polyurethane, ethylene vinyl acetate copolymer, and silicone elastomer) and two drug loadings (5 and 15 mg/segment) were evaluated in at least one of two independent studies for up to 28 days in vivo. Inter-study comparison of in vivo release, vaginal tissue, and plasma concentrations for similar formulations demonstrated good reproducibility of the animal model. Mean estimates for a 28-day in vivo release ranged from 0.35 to 3.17 mg UC781 per segment. Mean proximal vaginal tissue levels (adjacent to the IVR segment) were 8– 410 ng/g and did not change significantly with time for most formulations. Distal vaginal tissue levels of UC781 were 6- to 49-fold lower than proximal tissue levels. Mean UC781 plasma levels were low for all formulations, at 0.09–0.58 ng/mL. All formulations resulted in similar UC781 concentrations in vaginal tissue and plasma, except the low loading polyurethane group which provided significantly lower levels. Loading dependent release and pharmacokinetics were only clearly observed for the polyurethane matrix. Based on these results, intravaginal ring segments loaded with UC781 led to vaginal tissue concen- trations ranging from below to approximately two orders of magnitude higher than UC781’s EC50 under in vitro conditions (2.8 ng/mL), with little influence by polymer matrix or UC781 loading. Moreover, these findings support the use of rabbit vaginal pharmacokinetic studies in preclinical testing of microbicide intravaginal rings.

The effect of priming on perceptions of dog breed traits

This study examined whether priming cues embedded in mediastyle presentations shaped people's perceptions of specific dog breeds, and in particular, the German shepherd dog (GSD). Two hundred and four adult females were exposed to one of two types of media-style presentation (stories or pictures). Half of the participants in each condition were exposed to versions designed to portray the GSD in a positive light; the remainder to stimuli developed to present the same breed in a negative light. Participants subsequently rated six individual breeds of dog, including the target breed, on a number of traits (e.g., “friendliness,“ “aggression“). Analysis revealed a significant effect of priming on people's perceptions of the GSD. Participants exposed to the negative stimuli perceived this breed as significantly less approachable, and more dangerous and aggressive, than those exposed to the positive stimuli. Priming did not influence the participants' perceptions of other breeds, even those often regarded in a negative light, although there was some evidence of breed-related category-based stereotyping. Overall, results suggest that people's perceptions of dog breeds can be influenced by verbal and visual representations. The results have implications for how dogs are portrayed in the media and other publically available sources of information.

Detection of 3-amino-2-oxazolidinone (AOZ), a tissue-bound metabolite of the nitrofuran furazolidone, in prawn tissue by enzyme immunoassay

Furazolidone, a nitrofuran antibiotic, is banned from use in food animal production within the European Union. Increasingly, compliance with this ban is monitored by use of analytical methods to detect a stable tissue-bound metabolite, 3-amino-2-oxazolidinone (AOZ). Widespread use of furazolidone in poultry and prawns imported into Europe highlighted the urgent need for development of nitrofuran immunoassay screening tests. The first enzyme-linked immunoabsorbant assay for detection of AOZ residues in prawns (shrimps) is now described. Prawn samples were derivatized with o-nitrobenzaldehyde, extracted into ethyl acetate, washed with hexane and applied to a competitive enzyme immunoassay based on a rabbit polyclonal antiserum. Assay limit of detection (LOD) (mean+3 s) calculated from the analysis of 20 known negative cold and warm water prawn samples was 0.1 mug kg(-1). Intra- and interassay relative standard deviations were determined as 18.8 and 38.2%, respectively, using a negative prawn fortified at 0.7 mug kg(-1). The detection capability (CCbeta), defined as the concentration of AOZ at which 20 different fortified samples yielded results above the LOD, was achieved at fortification between 0.4 and 0.7 mug kg(-1). Incurred prawn samples (n=8) confirmed by liquid chromatography coupled with tandem mass spectrometry detection to contain AOZ concentrations between 0.4 and 12.7 mug kg(-1) were all screened positive by this enzyme-linked immunoabsorbant assay. Further data are presented and discussed with regard to calculating assay LOD based on accepting a 5% false-positive rate with representative negative prawn samples. Such an acceptance improves the sensitivity of an ELISA and in this case permitted an LOD of 0.05 mug kg(-1) and a CCbeta of below 0.4 mug kg(-1).

Comparison of two polyclonal antibodies for the detection of 19-nortestosterone in bovine bile by ELISA

19-Nortestosterone (beta-NT) is banned for use as a growth promoter in food animals within the European Union. For regulatory control purposes, urine and bile samples are routinely screened by immunoassay. The aim of the present study was to compare the ability of two immunoassays, using two rabbit polyclonal antibodies raised against two different NT derivatives, to detect NT residues in bovine bile. One antiserum cross-reacted with both alpha-NT and beta-NT (alpha/beta-NT), whereas the other was specific for alpha-NT. Bile samples from 266 slaughtered cattle were deconjugated and analyzed using both antibodies, with all screening positives (>2 ng ml(-1)) confirmed by high resolution gas chromatography mass spectrometry. The alpha/beta-NT and alpha-NT antibody-based ELISAs screened 39 and 44 samples positive, respectively, with NT confirmed in 22 and 39, respectively. The alpha/beta-NT antibody-based ELISA produced a false-negative rate of 44% compared to 0% for the alpha-NT antibody-based ELISA. Supplementary investigations concluded that a matrix effect was a major cause of the marked differences in false-negative rates. This result underlines the necessity to validate immunoassays in the sample matrix.

INTRAVASCULAR ANTI-IGE CHALLENGE IN PERFUSED LUNGS - MEDIATOR RELEASE AND VASCULAR PRESSOR-RESPONSE

Intravascular application of goat anti-rabbit immunoglobulin E (IgE) was used to stimulate parenchymal mast cells in situ in perfused rabbit lungs. Sustained pulmonary arterial pressure rise was evoked in the absence of lung vascular permeability increase and lung edema formation. Early prostaglandin (PG) D2 and histamine release into the perfusate was documented, accompanied by more sustained liberation of cysteinyl leukotrienes (LT), LTB4, and PGI2. The quantities of these inflammatory mediators displayed the following order: histamine > cysteinyl-LT > PGI2 > LTB4 > PGD2. Pressor response and inflammatory mediator release revealed corresponding bell-shaped dose dependencies. Cyclooxygenase inhibition (acetylsalicylic acid) suppressed prostanoid generation, increased LT release, and did not substantially affect pressor response and histamine liberation. BW755 C, a cyclo- and lipoxygenase inhibitor, blocked the release of cysteinyl-LT and markedly reduced the liberation of the other inflammatory mediators as well as the pressor response. The H-1-antagonist clemastine caused a moderate reduction of the anti-IgE-provoked pressure rise. We conclude that intravascular anti-IgE challenge in intact lungs provokes the release of an inflammatory mediator profile compatible with in situ lung parenchymal mast cell activation. Pulmonary hypertension represents the predominant vascular response, presumably mediated by cysteinyl-LT and, to a minor extent, histamine liberation.

Activation of extracellular signal-regulated kinase in proliferative glomerulonephritis in rats

Multiple extracellular mitogens are involved in the pathogenesis of proliferative forms of glomerulonephritis (GN), In vitro studies demonstrate the pivotal role of extracellular signal-regulated kinase (ERK) in the regulation of cellular proliferation in response to extracellular mitogens. In this study, we examined whether this kinase, as a convergence point of mitogenic stimuli, is activated in proliferative GN in vivo, Two different proliferative forms of anti-glomerular basal membrane (GEM) GN in rats were induced and whole cortical tissue as well as isolated glomeruli examined using kinase activity assays and Western blot analysis, Administration of rabbit anti-rat GEM serum to rats, preimmunized with rabbit IgG, induced an accelerated crescentic anti-GEM GN. A significant increase in cortical, and more dramatically glomerular ERK activity was detected at 1, 3, and 7 d after induction of GN, Immunization of Wistar-Kyoto rats with bovine GEM also induced a crescentic anti-GBM GN with an increase of renal cortical ERK activity after 4, 6, and 8 wk, ERK is phosphorylated and activated by the MAP kinase/ERK kinase (MEK), We detected a significant increase in the expression of glomerular MEK in the accelerated form of anti-GEM CN, providing a possible mechanism of long-term activation of ERK in this disease model, In contrast to ERK, activation of stress-activated protein kinase was only detectable at early stages of proliferative GN, indicating these related kinases to serve distinct roles in the pathogenesis of GN, Our observations point to ERK as a putative mediator of the proliferative response to immune injury in GN and suggest that upregulation of MEK is involved in the long-term regulation of ERK in vivo.

Molecular cloning and expression in Escherichia coli K-12 of chromosomal genes determining the O7 lipopolysaccharide antigen of a human invasive strain of E. coli O7:K1

We have cloned and studied the expression in Escherichia coli K-12 of chromosomal rfb genes determining the biosynthesis of the O7 lipopolysaccharide (LPS) antigen from E. coli K1 strain VW187. Two E. coli K-12 strains carrying recombinant cosmids gave positive coagglutination reactions with protein A-rich staphylococcal particles bearing an O7-specific rabbit polyclonal antiserum. Silver-stained polyacrylamide gels of total membranes extracted with hot phenol showed O side chain material which had O7 specificity as determined by immunoblotting experiments. However, the amount of O7 LPS expressed in E. coli K-12 was considerably lower than that produced by the wild-type strain VW187. Deletion and transposition experiments identified a region of about 17 kilobase pairs which is essential for the expression of O7 LPS. The existence of homologies between the O7 LPS genes and other E. coli O side chain genes was investigated by Southern blot hybridization experiments. An O7-specific probe fragment of 15 kilobase pairs did not hybridize to genomic DNA digests of E. coli strains belonging to several different O types, demonstrating that the O7 LPS genes are unique.

Flanking and internal regions of chromosomal genes mediating aerobactin iron uptake systems in enteroinvasive Escherichia coli and Shigella flexneri

We have investigated the presence of the aerobactin system and the location of the aerobactin genes in enteroinvasive strains of Escherichia coli. Also, we cloned the aerobactin region and its flanking sequences from the chromosome of a strain of Shigella flexneri and compared the molecular organization of the aerobactin genes in the two genera. Of the 11 enteroinvasive E. coli strains studied, 5 possessed the aerobactin genes, which were located on the chromosome in each case. These strains produced and utilized aerobactin and also were susceptible to the bacteriocin cloacin-DF13. Restriction endonuclease mapping and hybridization experiments showed that the regions corresponding to the aerobactin-specific sequences were very similar in both enteroinvasive E. coli and S. flexneri. However, differences were found in the region corresponding to the aerobactin receptor gene. The regions flanking the aerobactin system in enteroinvasive E. coli and S. flexneri exhibited some similarities but were different from those in pColV-K30. Under iron-limiting conditions, aerobactin-producing enteroinvasive E. coli and S. flexneri synthesized outer-membrane proteins of 76 and 77 kDa, respectively, which cross-reacted immunologically with rabbit antiserum raised against the 74 kDa pColV-K30-encoded ferric aerobactin receptor.

The personality of “aggressive” and “non-aggressive” dog owners

The relationship between pet-keeping and owner personality has attracted considerable attention. Little focus, however, has been directed towards the personality of pet owners in relation to the type of pet owned. This study therefore explored the personality of pet owners in relation to the type of dog breed owned, focusing specifically on owners of breeds widely considered to be “aggressive” versus those more generally perceived as “non-aggressive”. One hundred and forty seven owners of “aggressive” (German shepherd dogs, Rottweilers) or “non-aggressive” (Labrador retrievers, Golden retrievers) dog breeds completed the Eysenck Personality Questionnaire short scale. Breed of dog owned was significantly related to owners’ psychoticism scores, with people who kept “aggressive” dogs having significantly higher scores on this trait than owners of “non-aggressive” dogs. Dog breed ownership was not significantly related to neuroticism, extraversion or lie scale scores, although male owners of “aggressive” dogs were found to be significantly less neurotic than women who kept “aggressive” or “non-aggressive” dogs. Overall, findings suggest that there is a significant relationship between dog breed ownership and specific personality traits, with owners of breeds widely considered to be “aggressive” harbouring more psychotic tendencies than people who choose to keep dogs with a reputedly less aggressive temperament.

Proper expression of the O-antigen of lipopolysaccharide is essential for the virulence of Yersinia enterocolitica O:8 in experimental oral infection of rabbits

The O-antigen of lipopolysaccharide (LPS) is required for virulence in Yersinia enterocolitica serotype O:8. Here we evaluated the importance of controlling the O-antigen biosynthesis using an in vivo rabbit model of infection. Y. enterocolitica O:8 wild-type strain was compared to three mutants differing in the O-antigen phenotype: (i) the rough strain completely devoid of the O-antigen, (ii) the wzy strain that lacks the O-antigen polymerase (Wzy protein) and expresses LPS with only one repeat unit, and (iii) the wzz strain that lacks the O-antigen chain length determinant (Wzz protein) and expresses LPS without modal distribution of O-antigen chain lengths. The most attenuated strain was the wzz mutant. The wzz bacteria were cleared from the tissues by day 30, the blood parameters were least dramatic and histologically only immunomorphological findings were seen. The level of attenuation of the rough and the wzy strain bacteria was between the wild-type and the wzz strain. Wild-type bacteria were highly resistant to killing by polymorphonuclear leukocytes, the wzz strain bacteria were most sensitive and the rough and wzy strain bacteria were intermediate resistant. These results clearly demonstrated that the presence of O-antigen on the bacterial surface is not alone sufficient for full virulence, but also there is a requirement for its controlled chain length.

Glycation, oxidation, and lipoxidation in the development of the complications of diabetes: a carbonyl stress hypothesis

Modifications of extant plasma proteins, structural proteins,and other macromolecules are enhanced in diabetes because of increased glycation (secondary to increased glucose concentrations) and perhaps because of increased oxidative stress, Increased glycation is present from the time of onset of diabetes, but the relation between diabetes and oxidative stress is less clear: increased oxidative stress may occur later in the course of disease, as vascular damage becomes established, or it may be a feature of uncomplicated diabetes, The combined effects of protein modification by glycation and oxidation may contribute to the development of accelerated atherosclerosis in diabetes and to the development of microvascular complications, Thus, even if not increased by diabetes, variations in oxidative stress may modulate the consequences of hyperglycemia in individual diabetic patients, In this review, the close interaction between glycation and oxidative processes is discussed, and the theme is developed that the most significant modifications of proteins are the result of interactions with reactive carbonyl groups, While glucose itself contains a carbonyl group that is involved in the initial glycation reaction, the most important and reactive carbonyls are formed by free radical-oxidation reactions damaging either carbohydrates (including glucose itself) or lipids, The resulting carbonyl-containing intermediate products then modify proteins, yielding "glycoxidation" and "lipoxidation" products, respectively, This common pathway for glucose and lipid-mediated stress, which may contribute to diabetic complications, is the basis for the carbonyl stress hypothesis for the development of diabetic complications.

Morphometric analysis of corneal endothelial giant cells in normal and traumatized corneas

Corneal endothelial cells from normal and traumatized human, primate, cat and rabbit eyes were studied by specular microscopy. Morphometric analysis was performed on micrographs of corneal endothelium using a semi-automated image analysis system. The results showed that under normal conditions the corneal endothelium of all four species exhibit major morphological similarities (mean cell areas: human 317 +/- 32 microns 2, primate 246 +/- 22 microns2, cat 357 +/- 25 microns 2, rabbit 308 +/- 35 microns 2). The normal corneal endothelium in man was found to be more polymegethous than that of the other species. Trauma to cat, primate and human corneas resulted in a long-term reduction in endothelial cell density and enhanced polymegethism. In contrast, the reparative response of the rabbit ensured the reformation of an essentially normal monolayer following injury. Endothelial giant cells were a normal inclusion in the rabbit corneal endothelium but were only significant in cat, primate and man following trauma. The presence of corneal endothelial giant cells in amitotic corneas may therefore represent a compensatory response in the absence of mitotic potential.

Localised gamma irradiation and experimental intraocular proliferation

A controlled study was undertaken to assess the effect of gamma irradiation on post-traumatic intraocular cellular proliferation. A standard perforating injury in the posterior segment of the rabbit eye was used to induce intraocular cellular proliferation and vitreo-retinal membrane formation. The site of injury was irradiated with an ophthalmic Cobalt60 applicator which provided a continuous source of gamma rays. Non-irradiated eyes developed traction retinal detachments associated with post-traumatic vitreo-retinal membranes. Irradiated eyes developed attenuated membranes or atrophic retinal scars, with the retina remaining attached. The membranes in non-irradiated eyes were highly cellular with abundant collagen, while irradiated membranes had fewer cells within a sparse collagen matrix. The episcleral fibroblasts, on autoradiographic studies appeared to be the main source of the cells that formed the proliferating tissue in both non-irradiated and irradiated eyes. In irradiated eyes both the inflammatory response and division of fibroblasts were delayed and reduced.

α-adrenoceptors do not mediate reflex mydriasis in rabbits

The aim of this study was to identify receptors that mediate reflex mydriasis in pentobarbital-anesthetized rabbits, in which the cervical sympathetic nerve was sectioned unilaterally. Voltage-response curves of pupillary dilation were generated bilaterally by stimulation of the sciatic nerve. Evoked mydriatic responses were mediated mainly by efferent parasympathetic innervation, and, to a lesser extent, by sympathetic innervation. The a-adrenergic antagonist, phenoxybenzamine (0.3 mg/kg, intravenously (i.v.)), antagonized mydriasis of the neurally intact eye, but not that on the sympathectomized side. The a- adrenergic antagonist, RS 79948 (0.3 mg/kg, i.v.), potentiated mydriasis of the normal eye, but was without either a potentiating or inhibitory effect on the mydriasis of the sympathectomized eye. In addition, the dopamine-receptor antagonist, haloperidol (1 mg/kg, i.v.), inhibited evoked mydriasis of the sympathectomized eye. These results suggest that, unlike some other species (cats and rats), a-adrenoceptors do not mediate reflex mydriasis elicited by sciatic-nerve stimulation in the rabbit, and support the previous finding in humans that dopamine receptors may mediate this response.

Studies of alpha-adrenoceptor antagonists on sympathetic mydriasis in rabbits

This study was undertaken to identify the alpha-adrenergic receptor type responsible for sympathetically evoked mydriasis in pentobarbital-anesthetized rabbits. Frequency-response curves of pupillary dilation were generated by stimulation of the preganglionic cervical sympathetic nerve (1-64 Hz). Evoked mydriatic responses were inhibited by systemic administration of nonselective alpha-adrenergic antagonists, phentolamine (0.3-10 mg/kg) and phenoxybenzamine (0.03-0.3 mg/kg), as well as the selective alpha(1)-adrenergic antagonist, prazosin (0.1-1 mg/kg). The alpha(2)-adrenergic antagonist, RS 79948 (0.3 mg/kg, i.v.) was without inhibitory effect, but potentiated the mydriatic response. In addition, the selective alpha(1A)-adrenoceptor antagonist, 5-methylurapidil (0.1-1 mg/kg, i.v.), antagonized the elicited mydriasis in a dose-dependent fashion. Unlike previous observations that prazosin does not block the adrenoceptor in rabbit iris dilator muscle, our results suggest that prazosin is effective in inhibiting neuronally elicited mydriasis in this species, and that alpha(1A)-adrenoceptors appear to mediate the response.