Biomedical subjects
Publications and source records attributed to A E Warner.
This report describes the case of an appropriately dosed patient who developed maculopathy <8 years after starting hydroxychloroquine (HCQ) therapy for systemic lupus erythematosus. Risk factors and screening for HCQ-associated maculopathy are discussed.
Gap junction communication plays an essential role in follicle growth. Immunocytochemistry and confocal microscopy were used to examine the expression of gap junction connexins of the alpha and beta subfamilies in follicles from primordial to preovulatory stages in the ovaries of prepubertal and adult mice. Connexin-specific antibodies detected alpha(1), alpha(4), alpha(6), beta(1), beta(2) and beta(4) connexins within follicles. In adult ovaries connexin immunolabelling was stronger in larger (more mature) follicles than it was in smaller follicles, with comparatively reduced labelling detected in prepubertal ovaries. In healthy follicles, labelling for alpha subfamily connexins was detected between granulosa cells, whereas labelling for beta subfamily connexins was found in the theca. Labelling for beta subfamily connexins and alpha(4) connexin (preantral stage) was detected on the oocyte surface membrane. In atretic follicles, labelling for beta(4) connexin appeared between the granulosa cells. These results demonstrate that alpha and beta connexin subfamilies are segregated to separate cellular compartments in the mouse follicle. The results are discussed in the light of possible roles for differential gap junctional communication in the regulation of folliculogenesis, oocyte maturation and atresia.
In amphibian embryos, activation of additional sodium pumps in neural plate cell membranes ensures that neural plate-derived neurons differentiate subsequently in the neural tube. When the sodium pump is inhibited during the mid-neural fold stages, neuronal differentiation fails. The effect is irreversible. We find that these events operate through the Na pump beta(3) subunit. When neural plate-specific Na pumps are activated, transcripts for beta(3) decline precipitately during the mid-neural fold stages, first in the neural plate and then in the dorsal mesoderm. As the neural tube closes, beta(3) returns, specifically in motor neurons and interneurons. Inhibition of the Na pump with the cardiac glycoside strophanthidin prevents the normal fall in beta(3) during neurulation: beta(3) is maintained in the neural plate until the neural tube closes, but lost from the dorsal mesoderm. Complete elimination of beta(3) transcripts from dorsal structures then occurs. Inhibiting the Na pump does not induce cell death (assessed by TUNEL staining) in the nervous system. Transcripts for X-Delta, NeuroD, and GSK3beta are not affected by inhibition of the Na pump. Xotch and N-tubulin transcripts fall to very low levels and Xotch disappears permanently from the nervous system. When beta(3) transcript expression is maintained throughout neurulation, by over expression of injected mRNAs, Xotch is eliminated from the neural tube and somites and switches to the ectoderm.
Neurotransmitters regulate neuronal function in the nervous system and modulation of their synthesis, release, and binding by immature neurons and their targets is a major part of nervous system development. We propose that the neurotransmitter noradrenaline regulates neuronal fate during neurulation, before neurons have differentiated. The ability of noradrenaline to induce a neural fate was tested in naive ectoderm caps cut from late blastula stage Xenopus embryos. Noradrenaline (10(-6) M) did not switch on otx-2 or NCAM and did not induce the formation of cement glands. We conclude that noradrenaline cannot induce a neural fate. By contrast, 10(-8) M noradrenaline activated N-tubulin in ectoderm caps expressing the neural inducing molecule noggin by the time intact siblings had become mid-neurulae. Methoxamine, a specific alpha-adrenergic receptor agonist, also activated N-tubulin in noggin-expressing caps. The alpha-adrenergic receptor blocker prazosin inhibited both noradrenaline- and methoxamine-induced activation of N-tubulin. The neurotransmitters dopamine and 5-HT did not activate expression of N-tubulin. XA-1, Otx-2, X-Delta, and Xotch transcripts were not sensitive to noradrenaline. HoxB9, which indicates posteriorization, was not activated by noradrenaline. When intact siblings were at stage 27, many cells in noggin-expressing, noradrenaline-treated caps were stained by the neuron-specific mcAb3A10. We propose that noradrenaline is an important endogenous modulator of neuronal fate, driving noggin-expressing cells to become neurons by binding to alpha-adrenergic receptors and activating a cascade that culminates in the expression of the neuronal markers N-tubulin and 3A10.
The organ uptake of intravenously injected particles was examined in 13 species. All animals were injected intravenously with 198Au colloid and magnetic iron oxide particles. Vascular clearance kinetics of 198Au colloid was similar in all species. Pulmonary uptake of 198Au colloid ranged from 17 to 60% in sheep, calves, pigs, and cats but was <1.1% in monkeys, hyraxes, rabbits, guinea pigs, rats, mice, and chickens. For iron oxide particles, pulmonary uptake ranged from 80 to 99% in sheep, calves, pigs, goats, and cats and 15 to 18% in hamsters, hyraxes, and monkeys and was <10% in rabbits, chicken, mice, rats, and guinea pigs. In all species, the bulk of the remainder of particle uptake was in the liver. Pulmonary intravascular macrophages are the cellular site of lung uptake in calves, cats, pigs, goats, and sheep, whereas monocytes and neutrophils predominate in other species. Kupffer cells were the site of uptake in the liver. Our data show marked species differences in the fate of circulating particles; ruminants, pigs, and cats have extensive pulmonary localization due to phagocytosis by pulmonary intravascular macrophages.
Because mononuclear phagocytes take up perfluorochemical emulsions (PFCE), we examined how prior treatment with PFCE affects the fate of circulating bacteria. Rats were preinjected with three daily intravenous injections of PFCE (2.0 ml/100 g) containing 12.5% (vol/vol) of a 4:1 mixture of F-dimethyl adamantane and F-trimethylbicyclo-nonane, 2.5% (wt/vol) Pluronic F-68 as the emulsifying agent, and 3% (wt/vol) hydroxyethyl starch as the oncotic agent. Pseudomonas aeruginosa or Staphylococcus aureus were injected 4 h after the third PFCE injection. PFCE pretreatment decreased the rate and extent of vascular clearance of P. aeruginosa, with decreased uptake by the liver. Importantly, there were significant decreases in killing of P. aeruginosa in the liver, lungs, spleen, and kidneys of PFCE animals. PFCE did not alter the clearance of S. aureus from the circulation. However, hepatic uptake was reduced, with concomitant increases in lung and kidney uptake. Ultrastructure of Kupffer cells revealed PFCE inclusions and extensive vacuolization. These experiments demonstrate that the clearance kinetics and organ distribution of circulating P. aeruginosa and their subsequent killing are altered by PFCE. Diminished hepatic phagocyte function leads to a decrease in vascular clearance of circulating bacteria, increased uptake in other reticuloendothelial organs, and decreased bactericidal activity versus P. aeruginosa.
Pattern in the developing limb depends on signaling by polarizing region mesenchyme cells, which are located at the posterior margin of the bud tip. Here we address the underlying cellular mechanisms. We show in the intact bud that connexin 43 (Cx43) and Cx32 gap junctions are at higher density between distal posterior mesenchyme cells at the tip of the bud than between either distal anterior or proximal mesenchyme cells. These gradients disappear when the apical ectodermal ridge (AER) is removed. Fibroblast growth factor 4 (FGF4) produced by posterior AER cells controls signaling by polarizing cells. We find that FGF4 doubles gap junction density and substantially improves functional coupling between cultured posterior mesenchyme cells. FGF4 has no effect on cultured anterior mesenchyme, suggesting that any effects of FGF4 on responding anterior mesenchyme cells are not mediated by a change in gap junction density or functional communication through gap junctions. In condensing mesenchyme cells, connexin expression is not affected by FGF4. We show that posterior mesenchyme cells maintained in FGF4 under conditions that increase functional coupling maintain polarizing activity at in vivo levels. Without FGF4, polarizing activity is reduced and the signaling mechanism changes. We conclude that FGF4 regulation of cell-cell communication and polarizing signaling are intimately connected.
Expression of a catalytic alpha subunit of the sodium pump was followed in early Xenopus embryos for correlation with physiological experiments showing that the sodium pump controls cavity expansion and the differentiation of neurones from the neural plate. Two cDNAs (one full length, one partial) for alpha1 subunit isoforms were cloned from a neural plate stage Xenopus library and sequenced. Other isoforms were not detected. Temporal and spatial expression patterns for alpha1 subunit transcripts and protein revealed extensive developmental regulation. At all stages, cells involved in cavity generation (outer ectoderm and cells lining the archenteron) expressed alpha1, transcripts with protein confined to the lateral and basal membranes. Before gastrulation, transcript levels were low and predominantly in animal cells. During gastrulation, alpha1 mRNAs rose significantly. Transcripts and protein were down-regulated in future outer neural plate cells as the mesoderm invaginated. Protein appeared at the blastopore on apical surfaces of lip cells and apposing surfaces of invaginating cells, suggesting that the Na pump opposes entry of fluid. In early neurulae, alpha1 mRNAs rose sharply. Transcript expression remained low in outer neural plate cells and increased in the endoderm, and protein appeared in the notochord. In midneurulae, transcripts returned in outer neural plate cells. Protein expression appeared on basal surfaces of deep neural plate cells and the floor plate, matching physiological observations. After neural tube closure, transcripts were detected in all dorsal structures. Protein was retained in the notochord and floor plate, was eliminated from the outer layer of the neural tube, and appeared on ependymal cells. The results are discussed in relation to previous physiological observations.
PIMs, although present only in selected animal species, may provide a clue to the potential role of mononuclear phagocytes on the vascular side of the air-blood barrier in lung inflammation and injury. We know PIMs localize circulating pathogens to the lungs in animals and concentrate the inflammatory response in the lung parenchyma. In certain disease states-e.g., biliary cirrhosis-pulmonary phagocytes may develop in the pulmonary capillaries, placing the lungs at risk for pathogen localization and subsequent inflammation. The two experimental approaches described last in this article-induction of intravascular macrophages in the lungs of rodents or rabbits and the selective inhibition or destruction of PIMs-offer models to study the role of mononuclear phagocytes in lung injury. The cirrhotic rat, especially, offers a model to investigate the interaction between pulmonary phagocytes and liver disease, including the adhesive molecules necessary for monocytes to adhere and differentiate in pulmonary capillaries. We then can investigate whether such factors arise in human lungs. We now can determine selectively whether macrophages can cause lung injury and what mediators they may contribute to the complex interactions among inflammatory cells, cytokines, proteases, oxygen radicals, and lipid mediators that participate in early sepsis-induced lung injury.
Gadolinium (Gd) suppresses reticuloendothelial functions in vivo by unknown mechanisms. In vitro exposure of rat alveolar macrophages to GdCl3.6H20 caused cell death, as measured by trypan blue permeability, in both dose- and time-dependent fashions. Even a 10-min exposure to Gd caused significant cell death by 24 h. The morphology of Gd-treated cells, pyknosis and karyorrhexis prior to loss of membrane integrity, suggested apoptosis. Upon flow cytometric examination, Gd-treated propidium iodide-excluding cells demonstrated light scatter changes characteristic of apoptotic cells (decreased forward and increased right angle scatter). Gel electrophoresis of DNA from Gd-treated macrophages clearly showed the ladder pattern unique to apoptotic cells. Electron-dense structures containing Gd were observed via electron spectroscopic imaging within phagosomes and also within nuclei (associated with condensed chromatin). Gadolinium, endocytosed by macrophages and distributed to nuclei, causes apoptosis of macrophages in vitro.
What are the ionic requirements for neutrophil (PMN) function and how might altered electrolyte concentrations contribute to airway disease? The in vitro killing of Pseudomonas aeruginosa by human peripheral white blood cells (WBCs) was progressively compromised Na+ concentration was lowered from 124 to 62 mM; at 62 mM Na+, bactericidal activity was 28.8 +/- 7.4% (SE) of normal. In contrast, Cl- concentration affected killing only when lowered to 8 mM. We examined phagocytosis and oxidative metabolism in response to P. aeruginosa or particles opsonized with either immunoglobulin G (IgG) or complement (C'). Phagocytosis of P. aeruginosa and of IgG-coated particles was Na(+)-dependent (31.2 +/- 3.1 and 58.6 +/- 14.2% of normal, respectively, at 62 mM Na+). However, no effect on uptake of C'-coated particles was observed, and the respiratory burst at 70 mM Na+ was normal regardless of stimuli. Thus low Na+ concentration compromises select PMN functions. These results may help explain why airways of cystic fibrosis (CF) patients become colonized with bacteria such as P. aeruginosa. Perhaps the low concentration of Na+ reported for some CF respiratory secretions inhibits bactericidal functions of PMNs, predisposing these patients to airway infections.
Pulmonary intravascular macrophages (PIMs) adhere to the endothelium of lung capillaries and sequester circulating particles and pathogens from the blood. Iron oxide (gamma Fe2O3) 5 mg/kg, administered intravenously, specifically labeled PIMs in situ within the living sheep. Attempts to isolate gamma Fe2O3-labeled PIMs using vascular perfusion (VP) procedures yielded few cells. To improve recovery of PIMs, a proteolytic lung digestion (PLD) procedure was developed. Following PLD, gamma Fe2O3-containing PIMs were recovered by magnets and the amount of gamma Fe2O3 present measured by fluxgate magnetometry. Proteolytic lung digestion recovered 34% of the total gamma Fe2O3 in lung samples and yielded 2 x 10(5) PIMs/g lung with 95% viability. In contrast, VP recovered only 3% of the total gamma Fe2O3 in the lung; furthermore, less than 2% of the recovered gamma Fe2O3 was cell associated. Proteolytic lung digestion followed by magnetic separation is an effective way to recover viable sheep PIMs for in vitro study.
We have characterized a defect in the pulmonary recruitment of neutrophils (PMNs) in rats with experimental endotoxemia. Rats pretreated with intravenous (IV) 0.9% saline (NaCl) showed abundant PMNs in bronchoalveolar lavage (BAL) fluid after intratracheal (IT) lipopolysaccharide (LPS) (5 mg/kg) (54.27 +/- 9.80 x 10(6), n = 7, versus IT saline, 0.73 +/- 0.62 x 10(6), n = 4). In contrast, endotoxemic rats (IV LPS 1.0 mg/kg) failed to show PMN influx after IT LPS (0.40 +/- 0.13 x 10(6) PMNs in BAL fluid, n = 7). Four hours after the IT administration of LPS, the chemotactic activity of BAL fluid from endotoxemic rats (87 +/- 9.92% of maximal chemotaxis toward zymosan-activated serum [ZAS], n = 4) was not significantly different (P > 0.05), from rats pretreated with IV NaCl (61.09 +/- 6.17% of maximal chemotaxis toward ZAS, n = 4). Endotoxemic and control rats showed similar chemotactic gradients in determinations of the BAL/plasma chemotactic activity ratio (BAL/plasma ratio: 2.16 +/- 0.14, n = 4, IV NaCl versus 2.98 +/- 0.14, n = 4, IV LPS, P > 0.05). Serum from untreated rats, rats pretreated with IV NaCl, and endotoxemic rats caused minimal effects on rat PMN chemotaxis in vitro (78.17 +/- 8.16%, 79.29 +/- 7.09%, and 69.28 +/- 9.04% of maximal chemotaxis toward ZAS, respectively, n = 4/group, P > 0.05). Quantitation of PMN adhesion molecules revealed a loss of L-selectin (8 +/- 5% of control group, n = 3), an increase in Mac-1 (776 +/- 82.60% of control group, n = 3), and no change in LFA-1 when normal PMNs were incubated with plasma from rats pretreated with IV LPS (P < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)
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The possibility that monoamines might act as signalling molecules during the early development of the nervous system has been examined in embryos of the amphibian Xenopus laevis. The distributions of 5-hydroxytryptamine, dopamine, noradrenaline and their precursor, dopa, were determined from the fertilized egg up to the late neurula stages using High Performance Liquid Chromatography, formaldehyde-induced fluorescence and antibody staining. 5-hydroxytryptamine was not detected until the tail bud stage. The fertilized egg contained significant concentrations of dopa (10(-6) M) and dopamine (10(-7) M). Both monoamines persisted with little change in concentration up to the late neurula stage. Early neurula stage embryos contained very low levels of noradrenaline. Aldehyde-induced fluorescence showed that monoamines are localized in dorsal regions of the embryo, in ectoderm and mesoderm cells. Monoamines were not present in endoderm cells. Immunocytochemical staining showed dopamine predominantly in the ectoderm, except in future neural regions where it was found also in the mesoderm. Dopamine staining was always most intense in dorsal regions of the embryo. The consequences for subsequent neuronal differentiation of interfering with the biosynthesis and receptor binding of monoamines during neurulation was assayed. Neuronal differentiation was monitored quantitatively in cultures set up as the neural tube closed and qualitatively in intact tadpoles that were left to develop for two days after washout of test reagent. The number of neurons, the number of muscle cells and the total number of differentiated cells were counted after 18-24 hours of culture. Comparison of the number of neurons that differentiated from control and treated embryos showed that inhibition of dopamine beta-hydroxylase, the enzyme catalysing the conversion of dopamine to noradrenaline, during the neural plate stages reduced substantially subsequent neuronal differentiation. The differentiation of myocytes and the total number of differentiated cells were not affected. Exogenous noradrenaline (10(-6) M) or dopamine (10(-6) M) could increase the number of neurons that differentiated subsequently in culture. Interfering with noradrenaline binding to receptors with receptor antagonists during neurulation showed that alpha-adrenergic receptor antagonists reduced substantially the subsequent differentiation of neurons. The differentiation of myocytes and the total number of differentiated cells were not affected. The effect of alpha-adrenergic receptor antagonists was overcome by the simultaneous inclusion of noradrenaline or alpha-receptor agonists, but not agonists at beta-adrenergic receptors. The quantitative reduction in the differentiation of neurons was paralleled by defects in the Central Nervous System of intact tadpoles.(ABSTRACT TRUNCATED AT 400 WORDS)
Removal of circulating particulates (bacteria, cell debris, endotoxin) is accomplished in most species by macrophages resident in the liver and spleen. We have shown that sheep and other species have phagocytic macrophages resident in their pulmonary capillaries. Moreover, these pulmonary intravascular macrophages accomplish the bulk of uptake of injected tracer particles, bacteria, or endotoxin (LPS). Because bacteria or LPS of intestinal origin enter the portal circulation, they would first encounter hepatic mononuclear phagocytes. We sought to determine the extent to which particulates injected into the portal circulation of sheep would be taken up by liver or by lung macrophages. Sheep (four per group) were injected via a mesenteric vein with radiolabeled gold colloid, magnetic iron oxide particles, live Pseudomonas aeruginosa, or 125I E. coli endotoxin. For each, the uptake pattern was determined 1 h after injection. Lung and liver were also fixed to determine the cells responsible for uptake and subsequent inflammatory changes. We found that for circulating gold colloid, iron oxide particles, or bacteria, hepatic uptake predominated, and Kupffer cells were responsible. After hepatic uptake of bacteria, inflammatory changes were confined to the liver. In contrast, nearly 50% of endotoxin escaped hepatic clearance and was subsequently removed by the lungs. We then saw inflammatory changes in both lungs and liver. Thus, hepatic macrophages are active in species with pulmonary intravascular macrophages, partially sparing the lungs from uptake and acute inflammation. Endotoxin, however, may elude hepatic uptake, be sequestered in the lungs, and initiate inflammation there.
Pulmonary intravascular macrophages (PIMs) are an extensive population of mature phagocytic cells adherent to the pulmonary capillary endothelium in selected species. They are not prevalent in lungs of commonly studied laboratory animals, such as rodents, and thus have only been recently appreciated. However, their potential role in host defense and acute lung injury has attracted interest, since a number of studies have demonstrated pulmonary localization of circulating particles, microbes, and endotoxin by PIMs. Those animal species, such as ruminants, that provide useful models of pathogen (or endotoxin)-induced acute lung injury demonstrate rapid pulmonary uptake of bacteria by PIMs. Inflammatory mediators released by activated PIMs may initiate the process and provoke accumulation of neutrophils and platelets. This review summarizes the morphological characteristics of PIMs and their species distribution. The role of these members of the mononuclear phagocyte system, both beneficial and potentially pathogenic, is reviewed. The question of whether PIMs have a role in acute lung injury in humans is also discussed.
Cell-type-specific antibodies have been used to follow the appearance of neurones and glia in the developing nervous system of the amphibian embryo. Differentiated neurones were recognized with antibodies against neurofilament protein while glial cells were identified with antibodies against glial fibrillary acidic protein (GFAP). The appearance of neurones containing the neurotransmitters 5-hydroxytryptamine and dopamine has been charted also. In Xenopus, neurofilament protein in developing neurones was observed occasionally at NF stage 21 and was present reliably in the neural tube and in caudal regions of the brain at stage 23. Antibodies to the low molecular weight fragment of the neurofilament triplet recognized early neurones most reliably. Radial glial cells, identified with GFAP antibody, were identified from stage 23 onwards in the neural tube and caudal regions of the brain. In the developing spinal cord, GFAP staining was apparent throughout the cytoplasm of each radial glial cell. In the brain, the peripheral region only of each glial cell contained GFAP. By stage 36, immunohistochemically recognizable neurones and glia were present throughout the nervous system. In the axolotl, by stage 36 the pattern of neural and glial staining was identical to that observed in Xenopus. GFAP staining of glial cells was obvious at stage 23, although neuronal staining was clearly absent. This implies that glial cells differentiate before neurones. 5-HT-containing cell bodies were first observed in caudal regions of the developing brain on either side of the midline at stage 26. An extensive network of 5-HT neurones appeared gradually, with a substantial subset crossing to the opposite side of the brain through the developing optic chiasma. 5,7-dihydroxytryptamine prevented the appearance of 5-HT. Depletion of 5-HT had little effect on development or swimming behaviour. Dopamine-containing neurones in the brain first differentiated at stage 35-36 and gradually increased in number up to stage 45-47, the latest stage examined. The functional role of 5-HT- or dopamine-containing neurones remains to be elucidated. We conclude that cell-type-specific antibodies can be used to identify neurones and glial cells at early times during neural development and may be useful tools in circumstances where functional identification is difficult.