Two cases of aniridia in Llanwenog sheep.
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Animals↗
Biomedical subjects
D J Gould
Publications and source records attributed to D J Gould.
Avoiding the postdoctoral glut: an alternative route to a career in academia.
Dramatic increases in the number of PhDs granted during the last several decades have not been balanced by comparable increases in the number of academic positions, producing a glut of postdoctoral fellows. One major reason for the burgeoning numbers of postdoctoral fellows is a widespread perception that there is no alternative route to a career in academia other than through gaining expertise in bench research. This paper suggests that considerable numbers of postdoctoral fellows who are interested in securing a tenure-track position at a major medical or research university should consider directing their postgraduate training toward teaching. Teaching at universities is becoming a specialized field partly because researchers are unable to keep up with the explosion of information, new instructional theories, methodologies, and technology. Thus, some individuals who are currently in a postdoctoral holding pattern could use those years to increase their teaching competence. Such an "educational" postdoctoral position may offer a faster route to a career in academia as universities continue to recognize the need for teaching specialists.
Biological Science Disciplines↗
A novel doxycycline inducible autoregulatory plasmid which displays "on"/"off" regulation suited to gene therapy applications.
The development of transcriptionally controlled systems which function in eukaryotic cells are important for achieving regulated gene expression in gene therapy. In this study we combined the components of the tetracycline-inducible system in self-contained retroviral and plasmid vectors. Regulated reporter gene expression from the autoregulatory plasmid pGTRTL in response to doxycycline (Dox) induction surpasses the expression observed from other self-contained retroviral and plasmid vectors. Induction kinetics and expression levels of luciferase and the therapeutic molecule, truncated soluble complement receptor 1 (sCR1) were characterised in a mouse fibroblast and a human neuroblastoma cell line. The regulatory characteristics of the plasmids were shown to be optimal for gene therapy applications, as there was a rapid reduction in expression levels following removal of Dox. Co-transfection of cells with an autoregulatory plasmid and a Dox inducible enhanced green fluorescent protein (EGFP) plasmid demonstrated the feasibility of using this plasmid combination to achieve parallel regulation of two genes of interest in a single cell under the control of Dox. These novel autoregulatory plasmids display the requirements for gene therapy applications in chronic conditions which are remitting/relapsing such as rheumatoid arthritis or multiple sclerosis, where novel protein therapeutics and combination therapies are needed. Gene Therapy (2000) 7, 2061-2070.
Animals↗
Squamous cell carcinoma in an area of necrobiosis lipoidica diabeticorum: a case report.
Ulceration in an area of necrobiosis lipoidica diabeticorum is a frequent complication but malignant transformation is rare. One such case is reported in a diabetic patient and the literature is reviewed.
Carcinoma, Squamous Cell↗
Canine monocytic ehrlichiosis presenting as acute blindness 36 months after importation into the UK.
A four-year-old Labrador retriever developed sudden-onset blindness, associated with bilateral uveitis, intraocular haemorrhage and retinal detachment. It had been imported into the UK from Sardinia 36 months before presentation. Haematological abnormalities included non-regenerative anaemia, thrombocytopenia and neutropenia. Serum and urine protein electrophoresis demonstrated a monoclonal gammopathy. An immunofluorescent antibody test for Ehrlichia canis was positive, with a titre of 1:320, confirming a diagnosis of chronic monocytic ehrlichiosis. This case highlights how the prolonged subclinical phase of monocytic ehrlichiosis could enable infected dogs to enter the UK without signs of disease. Chronic monocytic ehrlichiosis should be considered in dogs which have been imported from E canis-endemic countries and present with bleeding disorders and gammopathy, even if signs develop many years after importation.
Acute Disease↗
Pemphigus foliaceus in association with systemic lupus erythematosus, and subsequent lymphoma in a cocker spaniel.
A seven-year-old neutered male cocker spaniel was presented with an 11-month history of generalised bacterial dermatitis. There were skin lesions over the entire body, which were round, slightly raised and encrusted. Skin biopsies were collected and the histological findings were consistent with pemphigus foliaceus. Immunohistochemical staining by the indirect immunoperoxidase method was positive, with desmosomal deposition of immunoglobulin (Ig) G. Haematological analysis revealed a regenerative anaemia and profound thrombocytopenia, while a Coombs' test was positive for polyvalent canine Coombs' reagent and anti-dog IgG. An antinuclear antibody test was positive, with a titre of 10,240. An ophthalmic examination demonstrated low tear production (keratoconjunctivitis sicca). Seven months after initial referral, the dog was re-presented with severe generalised peripheral lymphadenopathy. Radiographic evaluation of the thorax and abdomen revealed enlarged cranial mediastinal and sublumbar lymph nodes. Tru-Cut biopsy from an enlarged lymph node confirmed the diagnosis of lymphoma, which was phenotyped as a B-cell tumour. The diagnosis in this case was systemic lupus erythematosus, with the unusual feature of pemphigus foliaceus, and subsequent development of B-cell lymphoma. The case adds further to knowledge of the protean clinical presentations of canine autoimmune diseases and provides additional evidence for the potential association between autoimmunity and immune-system neoplasia in this species.
Animals↗
The effects of mitotic inhibition on the spinal cord response to the superimposed injuries of spinal cord hemisection and peripheral axotomy.
The present study was carried out to test the hypothesis that dividing microglia are responsible for the depression of crossed phrenic nerve activity documented at 2 weeks postphrenicotomy in an injury model which superimposes the effects of spinal cord injury on peripheral axotomy. Crossed phenic nerve activity is defined as the respiratory activity recorded from the phrenic nerve during the crossed phrenic phenomenon (CPP) which is a respiratory reflex induced by respiratory stress following an ispsilateral spinal cord hemisection. Young adult female Sprague-Dawley rats were subjected to left intrathoracic phrenicotomies. Cytarabine (Cyt-A, a powerful antimitotic drug) or saline-filled miniosmotic pumps were then implanted into the cisterna magna and 2 weeks were allowed to pass at which time the CPP was induced by a left C2 spinal cord hemisection and transection of the contralateral phrenic nerve. Control studies including bromodeoxyuridine labeling of mitotic cells and a triple immunofluorescent protocol were carried out to verify that microglial cells were the primary cell type undergoing mitosis in the current injury model and that Cyt-A completely inhibited cellular proliferation. Quantitative electrophysiological analysis of crossed phrenic nerve activity showed that there is a statistically significant depression of activity at 2 weeks postphrenicotomy when animals were infused with saline compared to controls. Crossed phrenic nerve activity levels were not significantly different, however, from control levels when 2-week postphrenicotomized rats were infused with Cyt-A. Immunofluorescent studies showed that the majority of cells dividing in response to phrenicotomy were microglia. Furthermore, there were no astrocytes seen dividing at any time. From the results, we conclude that activated microglial cells may be responsible for the depression in crossed phrenic activity normally seen 2 weeks postphrenicotomy. Further, the activation of microglia may be related to the astrocytic response to injury. The activated microglial cell may be acting as a coordinator of various aspects of the injury response. Alternatively, the activation of microglia may be a necessary step in the cascade of multiple events that take place in the spinal cord after injury.
Animals↗
Potentiation of TNF-alpha toxicity by conjugation with ricin A-chain.
Hybrid toxins containing a cytokine moiety have been used effectively to selectively kill cells expressing the complementary cytokine receptor both in vivo and in vitro. To date all cytokines incorporated into hybrid toxins, e.g. interleukin 2 are biologically active as monomers, so attachment of a toxin group causes minimal interference with the cytokine structure. By contrast, the pro-inflammatory and anti-cancer cytokine tumour necrosis factor alpha (TNF-alpha) is biologically active as a homotrimer in which the grooves created between the hydrophobically associated monomers form the receptor binding region, so maintenance of this structure is crucial for activity. In this report the authors show that TNF-alpha can be modified by reaction with a crosslinking agent and by subsequent attachment of the toxin ricin A-chain without loss of TNF-alpha cytotoxic activity in the WEHI assay. Structural association of the hybrid toxin composed of TNF-alpha and ricin A-chain was confirmed by Western blot analysis. The hybrid toxin was toxic to HeLa cells (IC50=4 pM) not sensitive to native TNF-alpha, and sensitive WEHI cells with substantially increased lethality (LD50=0.01 fM). This increased TNF-alpha cytotoxic activity suggests that hybrid toxins containing TNF-alpha may have therapeutic applications in the treatment of cancer.
Cell Line↗
A complex photodermatosis: solar urticaria progressing to polymorphic light eruption.
We report an unusual case of localized solar urticaria which progressed on each occasion to polymorphic light eruption (PLE); this was initially noted following provocation by narrow band ultraviolet B (311-313 nm) phototherapy.
Adult↗
Pathway-specific effects of calcitonin gene-related peptide on irideal arterioles of the rat.
1. Arteriolar diameter and membrane voltage have been measured to investigate the actions of calcitonin gene-related peptide (CGRP) in rat irideal arterioles. 2. Activation of sensory nerves inhibited sympathetic vasoconstriction, reduced the accompanying 40-50 mV depolarization by 90% and caused a 4 mV hyperpolarization. 3. The inhibition of vasoconstriction was prevented by either preincubation in L-NAME (10 microM), to inhibit nitric oxide production, by preincubation in the cell-permeant adenylate cyclase inhibitor dideoxyadenosine (1 mM) or by preincubation in the ATP-sensitive potassium channel blocker glibenclamide (10 microM). The subsequent addition of a nitric oxide donor to the glibenclamide solution inhibited nerve-mediated vasoconstriction, suggesting that the potassium channel involvement preceded the production of nitric oxide. The small hyperpolarization was not affected by L-NAME. 4. Nerve-mediated vasodilatation persisted in the presence of L-NAME (10 microM) but was abolished with the CGRP1 receptor antagonist CGRPS-37. 5. In arterioles preconstricted with the alpha 2-adrenoceptor agonist UK-14304 (100 nM), exogenous CGRP caused a hyperpolarization and a dose-dependent vasodilatation, neither of which was affected by L-NAME (10 microM). 6. In arterioles preconstricted with 30 mM KCl, CGRP (10 nM) caused vasodilatation but not hyperpolarization, suggesting that the hyperpolarization was not causal to the vasodilatation. 7. Forskolin (30 nM), in the presence of L-NAME to present effects due to nitric oxide, caused vasodilatation. 8. These results suggest that CGRP inhibits sympathetic nerve-mediated vasoconstriction through sequential increases in cyclic AMP and nitric oxide, while vasodilatation results from increases in cyclic AMP alone. The production of nitric oxide, but not its mechanism of action, appears to be dependent on the activation of ATP-sensitive potassium channels. The possible sites of action of these two pathways are discussed.
Animals↗
Glial changes in the phrenic nucleus following superimposed cervical spinal cord hemisection and peripheral chronic phrenicotomy injuries in adult rats.
The objective of the present study was to characterize the microglial and astroglial reaction in the phrenic nucleus following either an ipsilateral C2 spinal cord hemisection, a peripheral phrenicotomy, or a combination of the two injuries in the same adult rat. The present study used three different fluorescent markers and a confocal laser image analysis system to study glial cells and phrenic motoneurons at the light microscopic level. Young adult female rats were divided into one combined injury group (left phrenicotomy and left C2 spinal hemisection with periods of 1 to 4 weeks between injuries, N = 12) and three other groups consisting of noninjured animals (N = 3), animals that received C2 hemisection only (N = 3), and animals with phrenicotomy only (survival periods of 2 (N = 3) and 4 (N = 3) weeks after phrenicotomy). Fluorogold was injected into the diaphragm to label phrenic motoneurons in all animals. Microglia and astrocytes were labeled with Texas red and fluorescein, respectively, and were visualized simultaneously along with phrenic motoneurons. The results suggest that the microglial and astrocytic response in the superimposed injury model are similar to the glial reactions characteristically seen in a peripheral axotomy alone model. These reactions include proliferation and migration of microglial cells along the perineuronal surface (peaking at 2 weeks) and the hypertrophy of astrocytes (peaking at 4 weeks). In addition, the increase in astrocytic tissue, which is characteristically seen in response to axotomy alone, is significantly enhanced in the superimposed injury model. Also, there is a large and rapid increase in GFAP-positive astrocytes within 24 hours after hemisection alone. The information gained from the present study will aid in determining, predicting, and eventually manipulating central nervous system responses to multiple injuries with the objective of reestablishing function in the damaged CNS.
Animals↗
Distribution of bacteria on hands and the effectiveness of brief and thorough decontamination procedures using non-medicated soap.
Our perception of the role of hand washing in the clinical situation is based on experimental studies in which test-bacteria are usually inoculated onto the skin surface and removed using hand washing preparations containing antiseptics. In this study, we have investigated the distribution of bacteria on the hands of volunteers and the effectiveness of long (3 minute) and brief (10 second) washes in removing both naturally-occurring and artificially-inoculated bacteria (Micrococcus sp.), using only soap and water. There was a tenfold reduction in median counts of artificially inoculated bacteria following both long and brief washes. However, less than 50% of naturally-occurring bacteria were removed and, for hands previously disinfected by immersion in 70% ethanol, the washing procedure increased bacterial counts. In both unwashed hands, and hands washed following a strict protocol, the mean variation in counts of naturally-occurring bacteria at different sites (wrists, dorsal surface, palmar surface, fingertips and interdigital spaces) was only two-fold. The efficiency of recovery of naturally-occurring organisms was estimated by repeated swabbing, to be more than 60%. The data question the value of typical hand wash procedures recommended by many authorities for use in clinical situations and of the perfunctory hand washes frequently adopted by nursing staff in busy wards. Experimental evidence is required to justify procedures and to identify the precise circumstances in which they are of value.
Disinfectants↗
Cloning of canine rom-1 and its investigation as a candidate gene for generalized progressive retinal atrophies in dogs.
Generalized progressive retinal atrophy (gPRA) represents a genetically heterogenous group of retinal degenerations affecting pedigree dogs. Currently, we are using a candidate gene approach in an attempt to identify mutations causing gPRA in dogs. Here we report the cloning, sequencing and analysis of canine rom-1, a structural gene of the rod photoreceptor. Single-stranded conformation polymorphism (SSCP) analysis was used to look for polymorphisms segregating with gPRA in the English cocker spaniel, Labrador retriever, miniature poodle, miniature long-haired dachshund, Tibetan terrier, miniature schnauzer, Cardigan Welsh corgi and Irish wolfhound. Further investigation involved DNA sequencing and restriction fragment length polymorphism (RFLP) analysis. Our studies revealed the presence of three polymorphisms, none of which segregated with disease phenotype. Haplotype analysis identified four rom-1 alleles. Our results indicate that rom-1 is unlikely to be a cause of gPRA in the breeds of dog examined.
Animals↗
Electrical properties of iridial arterioles of the rat.
When intracellular recordings were made from iridial arterioles, the cells had membrane potentials of about -65 mV and perivascular nerve stimulation evoked a membrane depolarization. When these cells were labeled with lucifer yellow, all cells that responded to perivascular nerve stimulation had the morphological characteristics of smooth muscle cells. Cells with the morphological characteristics of endothelial cells were never stained. When impaled with two separate recording electrodes, the smooth muscle layer was shown to form an electrical syncytium with a membrane time constant of approximately 80 ms and an electrical length constant of approximately 900 microns. At the ultrastructural level, areas of close apposition were frequently observed between adjacent smooth muscle cells and between adjacent endothelial cells. On the other hand, at contacts between smooth muscle and endothelial cells, the membranes characteristically had much larger separations. The observations show that individual smooth muscle cells are electrically coupled to their neighbors, but the morphological studies raise the possibility that in these arterioles the endothelial and muscle layers are electrically separate.
Animals↗
Sensory nerves play an efferent role in the function of the arterioles, but not the dilator muscle, of the rat iris.
We have studied the expression, distribution and function of receptors for the sensory neurotransmitters, substance P (SP) and calcitonin gene-related peptide (CGRP) in the dilator muscle and arterioles of the rat iris. Immunohistochemical studies showed that the sensory fibres containing these peptides are distributed throughout the connective tissue stroma of the iris and in association with the larger arterioles, but do not come into close association with the dilator muscle cells. Using reverse-transcription polymerase chain reaction, we have shown that both NK1 and NK3 receptor message is expressed by iris tissue, comprising both dilator muscle and stromal tissue. Binding sites for the NK1 agonist, [Sar9, Met(O2)11]-substance P (SarSP), and for CGRP are confined to the stromal layer and to the larger arterioles within that layer and do not appear to be associated with the dilator muscle itself. Application of either SarSP or CGRP produced both a vasodilatation and an inhibition of sympathetic nerve-induced vasoconstriction of the larger arterioles. Neither SarSP nor CGRP altered the resting tone of the dilator nor were they capable of modulating the contractions due to sympathetic nervous activity. These results suggest that the sensory fibres perform an efferent role in the larger irideal arterioles while their presence in the irideal stroma appears not to modulate the activity of the dilator muscle.
Adrenergic Fibers↗
Recent advances in understanding the spectrum of canine generalised progressive retinal atrophy.
Canine generalised progressive retinal atrophy (gPRA) is a large and ever-increasing collection of naturally occurring, heterogeneous, progressive disorders. Most are inherited in an autosomal recessive manner and new, breed-specific forms continue to be described. The gPRAs cause photoreceptor cell death and subsequent retinal degeneration, culminating in blindness. In humans, similar inherited retinal dystrophies are recognised as retinitis pigmentosa and macular dystrophy. Molecular biological studies have revealed disease-causing mutations in several genes in humans and also in mice with retinal dystrophies. Recently, molecular genetic techniques have identified the cause of one form of gPRA in Irish setters while important candidate genes have been investigated in other breeds. Identification of mutations responsible for different forms of gPRA allows carrier and predegenerate animals to be detected using DNA-based tests. Such genetic tests will greatly facilitate the eradication of these diseases in different breeds.
Animals↗
Alpha-adrenoceptor activation of a chloride conductance in rat iris arterioles.
Changes in membrane potential associated with alpha-adrenoceptor-mediated contraction of rat iris arterioles after nerve stimulation (10 Hz, 1 s) have been measured with conventional intracellular recording techniques. Two different types of intracellular responses were recorded. Cells that show a depolarization are proposed to represent the arteriolar smooth muscle cells because the characteristics of the depolarization are correlated with those of the contraction. Cells that show no response or a small hyperpolarization in response to nerve stimulation are proposed to represent the endothelial cells of the arteriole. Both the depolarization and the contraction were abolished by tetrodotoxin (1 microM), benextramine (10 microM), and prazosin (0.1 mM), indicating that they result from nerve-mediated activation of alpha-adrenoceptors. A small but significant part of the contraction (30%) and the depolarization (11%) was nifedipine sensitive (10 microM). Caffeine (1 mM) abolished the contraction and reduced the depolarization by one-half. Reducing the external chloride concentration also abolished the contraction and reduced the depolarization by 90%. Flufenamic acid (250 mM) abolished both the contraction and the depolarization. It is suggested that, in iris arterioles, the activation of synaptic alpha-adrenoceptors leads to the release of intracellular calcium that activates both the chloride channels in the cell membrane leading to depolarization and the intracellular contractile apparatus leading to vasoconstriction.
Animals↗