Impact of regional climate change on the mosquito vector Aedes albopictus in a tropical island environment: La Réunion.

The recent expansion of Aedes albopictus across continents in both tropical and temperate regions and the exponential growth of dengue cases over the past 50 years represent a significant risk to human health. Although climate change is not the only factor responsible for the increase and spread of dengue cases worldwide, it might increase the risk of disease transmission at global and regional scale. Here we show that regional and local variations in climate can induce differential impacts on the abundance of Ae. albopictus. We use the instructive example of Réunion Island with its varied climatic and environmental conditions and benefiting from the availability of meteorological, climatic, entomological and epidemiological data. Temperature and precipitation data based on regional climate model simulations (3 km × 3 km) are used as inputs to a mosquito population model for three different climate emission scenarios. Our objective is to study the impact of climate change on the life cycle dynamics of Ae. albopictus in the 2070-2100 time horizon. Our results show the joint influence of temperature and precipitation on Ae. albopictus abundance as a function of elevation and geographical subregion. At low-elevations areas, decreasing precipitation is expected to have a negative impact on environmental carrying capacity and, consequently, on Ae. albopictus abundance. At mid- and high-elevations, decreasing precipitation is expected to be counterbalanced by a significant warming, leading to faster development rates at all life stages, and consequently increasing the abundance of this important dengue vector in 2070-2100.

Heat-soaked PCR: an efficient method for DNA amplification with applications to forensic analysis.

Heat-soaked PCR (HS-PCR) is a method for enhancing amplification performed by heating the DNA sample at 94 degrees C in 90 microliters 1.1 x buffer for 30 min. A 10-microliters bolus of concentrated (10x) deoxynucleotides, Taq DNA polymerase and primers prepared without buffer is then added just prior to thermal cycling. We have investigated the application of this method in a variety of forensically important DNA samples and compared it with regular PCR (R-PCR). DNA samples extracted from bone, postmortem tissues, bloodstains and hair contained low concentrations of human DNA or were contaminated with either non- human DNA or hemoglobin degradation products. Optimal conditions for HS-PCR were determined for the 3' ApoB VNTR locus and applied to a centromeric repeat element and to a single-copy locus. HS-PCR consistently and reproducibly enhanced product yield and specificity over R-PCR at all three loci in the entire set of DNA samples. HS-PCR was also effective in overcoming the inhibitory effect of hemoglobin at concentrations that fully impeded R-PCR.

Epinephrine increases facility of outflow and cyclic AMP content in the human eye in vitro.

The physiologic mechanism that underlies the epinephrine-induced increase in facility of outflow (C) in glaucomatous human eyes and normal primate eyes is not completely understood. In this study, a recently developed in vitro human eye perfusion model was used to simultaneously monitor facility and cyclic adenosine monophosphate (AMP) changes in response to epinephrine (EPI). In this system, EPI (2.5 x 10(-5) mol/l) resulted in a maximal 44% increase in C, with an ED50 occurring at approximately 8 x 10(-6) mol/l. The C-increasing effect of 10(-5) mol/l EPI was unaffected by 10(-6) mol/l phentolamine. However, it was completely blocked in the presence of 10(-6) mol/l timolol or 2 x 10(-7) mol/l ICI118,551, suggesting the involvement of beta-2 adrenergic receptors. In biochemical studies, 10(-5) mol/l EPI induced a 12- to 14-fold increase in cyclic AMP in the perfusate of treated eyes. This increase was blocked by ICI118,551. In isolated intact human trabecular tissue, a 10 min incubation with 10(-5) EPI stimulated cyclic AMP by a factor of 2.7 over control levels. After 90 min, cyclic AMP levels were increased 4.2 fold over control levels. Collectively, these results show that the intraocular pressure lowering effect of EPI in the human eye is mediated, at least in part, by an increase in facility of outflow. Furthermore, the facility increase appears to be mediated by beta-2 adrenergic receptors and is correlated in time with increased cyclic AMP production.

Ethacrynic acid induces reversible shape and cytoskeletal changes in cultured cells.

Cell cultures derived from trabecular meshworks of human and bovine eyes and from bovine vascular endothelia were incubated at 37 degrees C for 1 hr with ethacrynic acid (ECA, 0.1-0.5 mmol/l) dissolved in culture medium. At 2 hr after the initial exposure, ECA at concentrations up to 0.4 mmol/l induced a reversible alteration in cell shape in all three cell types that was coincident with a change in the staining pattern of major cytoskeletal components including actin, alpha-actinin, vinculin, and vimentin. Distinct progressive alterations in beta-tubulin also occurred, with initial changes observed 10 min after ECA exposure. The ECA-induced changes in tubulin were blocked in part by preincubation with taxol (which stabilizes the microtubule structure), but they appeared to differ from those occurring with nocodazole (which interferes with tubulin assembly). These results suggest the possibility that ECA-induced increases in outflow facility may be mediated by alterations in the cytoskeletons of outflow pathway cells.

Ethacrynic acid increases facility of outflow in the human eye in vitro.

Anterior segments of human donor eyes were perfused with culture medium at a perfusion pressure of 15 mm Hg in a 5% carbon dioxide environment at 37 degrees C. After determination of a baseline facility of outflow, the perfusion chamber contents were exchanged with either drug vehicle or ethacrynic acid, at concentrations ranging from 0.01 to 0.25 mmol/L, after which postdrug facility was measured in the continuous presence of drug vehicle or ethacrynic acid. Ethacrynic acid increased facility of outflow from 28% to 105% at ethacrynic acid concentrations of 0.01 to 0.25 mmol/L, respectively. No morphologic correlate of the facility increase was observed with 0.01-mmol/L ethacrynic acid, nor were there any signs of cellular toxic effects. At 0.1 mmol/L, separations between trabecular meshwork cells and breaks between inner-wall cells were observed. At 0.25 mmol/L, focal areas of cell swelling and necrosis were noted. This study demonstrated that ethacrynic acid increases outflow facility in the aged human eye at concentrations that produce no apparent toxic effects. Therefore, ethacrynic acid may potentially prove useful in the treatment of glaucoma.

Accommodation and ciliary muscle muscarinic receptors after echothiophate.

Twice daily topical administration of echothiophate for 2 weeks to the eyes of living cynomolgus monkeys produced profound subsensitivity of the accommodative response to pilocarpine and an approximately 50% decrease in the number of specific binding sites for 3H-quinuclidinyl benzilate (3H-QNB) in the ciliary muscle without a change in their affinity. When echothiophate treatment was discontinued, functional cholinergic sensitivity and the number of QNB binding sites both returned to normal over a similar 4-8 week period. Most animals had a modest overshoot of both functional sensitivity and number of binding sites for at least several weeks thereafter. The treated to control eye ratios for the number of binding sites and accommodative response to pilocarpine were correlated and the plot of log treated to control binding site ratio versus treated to control accommodation ratio resembled a dose-response curve. Similarly, the treated versus control eye differences for the two parameters were correlated, with the regression line passing through the 0-0 axis intercept. Collectively, these findings suggest that agonist-induced modulation of functional cholinergic sensitivity in the parasympathetically innervated (as opposed to denervated) ciliary muscle occurs by a muscarinic receptor-mediated mechanism. This system appears to provide a useful model to study the regulation of ciliary muscle cholinergic sensitivity.

Outflow facility studies in the perfused human ocular anterior segment.

We have recently developed a tissue model of the human aqueous outflow pathway involving placement of the eviscerated anterior corneoscleral shell, [with lens and uveal tissue removed but trabecular meshwork (TM) attached] onto a specialized perfusion apparatus. The TM and associated outflow tissues are perfused with culture medium at a physiologically-relevant perfusion pressure in a 5% CO2 environment at 37 degrees C. Under these conditions, the perfused outflow tissues are similar for several days, to the human and/or subhuman primate outflow system in vivo with regard to morphology as well as several functional parameters. Measured facility of outflow (0.271 +/- 0.018 microliters min-1 mmHg-1, n = 79) is similar to facility values obtained by tonography in living human beings. Moreover, outflow facility decreases in a linear fashion with increased perfusion pressure by 1.4% mmHg-1. Finally the removal of the TM results in a 41% decrease in measured outflow resistance. The ability to study viable human outflow tissue for at least several days and the opportunity to establish a model which serves as an alternative to animal testing, point to the potential importance of this technique in investigating the biology of the aqueous outflow system.

Effect of endothelin on outflow facility and accommodation in the monkey eye in vivo.

The effect of the vasoactive peptide, endothelin, on facility of outflow, accommodation, and pupil diameter was measured in the monkey eye in vivo. Endothelin increased the outflow facility 22-71% at approximate anterior chamber concentrations ranging from 10(-10)-10(-7) M. Endothelin-induced accommodation was modest but consistent and statistically significant ranging from 1.61 to a maximum of 2.43 diopters at 10(-9) M and 10(-7) M endothelin, respectively. No change in pupil diameter was noted with any of the administered doses. These data demonstrate an action of endothelin on outflow facility and, together with prior evidence indicating the presence of endothelin receptors on the ciliary muscle, suggest that, like cholinergic agonists, the observed effects of endothelin on outflow facility may be mediated, at least in part, through an action on the ciliary muscle.

Membrane voltage recordings in a cell line derived from human ciliary muscle.

A smooth muscle cell line (H7CM) was established from the ciliary muscle of a 1-day-old human infant. The cultured cells had a normal female karyotype (46 XX) and could be maintained in cell culture for at least 11 generations. A common feature of confluent cultures was the presence of abundant bundles of 6-7 nm microfilaments associated with dense bodies. Both the ultrastructural appearance and the presence of smooth muscle-specific alpha-isoactin (also present in the human ciliary muscle in situ) support the smooth muscle origin of the H7CM cell line. Continuous membrane voltage (Vm) recordings were obtained in confluent monolayers of H7CM cells using glass microelectrodes. Resting Vm in 105 impalements averaged -66.2 +/- 0.7 mV (mean +/- standard error of the mean). In this system, rapid membrane transients induced by changing of the superfusing test solutions were detectable. Relative K+ conductance was characterized, and the contribution of electrogenic sodium/potassium adenosine triphosphatase to Vm was investigated. Under control conditions, H7CM cells were electrically quiescent. However, action potentials could be induced by application of 10 mM barium. Barium-induced action potentials were not abolished by removal of extracellular Na+ nor were they inhibited by the presence of tetrodotoxin. However, they were blocked by verapamil, fulfilling criteria believed to be typical for smooth muscle cells. Acetylcholine, carbachol, and to a lesser extent pilocarpine induced a reversible Vm depolarization. The effect of acetylcholine was blocked by atropine, implying muscarinic receptor involvement in the Vm response. Collectively, these findings show the potential usefulness of cultured ciliary muscle cells in understanding further the cellular mechanisms underlying drug-induced contraction of the human ciliary muscle.

Absence of time-dependent facility increase ("washout") in the perfused enucleated human eye.

During the course of constant-pressure anterior chamber perfusion experiments in eyes from a number of species, measured outflow facility increases progressively with perfusion time. One possible explanation for this phenomenon has been that the technique of perfusion induces a loss of extracellular material from the outflow pathway. Therefore, this general observation has been termed the "wash-out" effect. Data from the authors' laboratories demonstrate that although outflow facility increases in cynomolgus monkey eyes by 26% and 42% from baseline over 90 minutes in vivo and in vitro, respectively, this does not occur in the enucleated human eye. The absence of washout in the human eye probably cannot be explained by postmortem conditions since enucleated monkey eyes undergo a similar magnitude of washout as the monkey eye in vivo. Furthermore, since washout does not occur in the perfused human infant eye, the age of the donor cannot explain the difference in washout properties between human and other primate eyes. The monkey eye in vivo is a valuable model in the study of outflow physiology. However, the difference in washout behavior between human and other primate eyes may point to a potentially important fundamental difference in the biology of the outflow pathways in these two species.

Ciliary muscle choline acetyltransferase and acetylcholinesterase after ciliary ganglionectomy.

Choline acetyltransferase (ChAT; EC 2.3.1.6) and acetylcholinesterase (AChE; EC 3.1.1.7) activities were measured in cynomolgus monkey ciliary muscle 1 month and 6 or more months after ciliary ganglionectomy (CG) or post-ganglionic ciliary neurectomy (PCN). ChAT activity was undetectable and AChE activity was elevated 1 month after CG or PCN, while both averaged about 30% of normal levels 6 or more months after denervation. Four out of six eyes reinnervated by functional criteria 6-12 months after CG or PCN. In one of the two remaining eyes permanently denervated, ChAT was absent from the ciliary muscle. In the other, ChAT activity was about 50% of normal, similar to the reinnervated eyes, but the regenerated cholinergic nerves were not approximated to the ciliary muscle fibers.

Dissociation between the effect of aceclidine on outflow facility and accommodation.

The dose/effect relationships for intracameral doses of aceclidine HCl and pilocarpine HCl on total facility of outflow and accommodative amplitude were compared in the cynomolgus monkey eye in vivo. The largest dose of aceclidine tested (20 micrograms), resulted in an average increase of 270% in outflow facility, whereas accommodation was limited to an average of 5 diopters. In contrast, 20 micrograms of intracameral pilocarpine (which is submaximal in increasing facility) resulted in an average of 19 diopters of accommodation. These results provide further evidence for a dissociation between the accommodative and outflow facility functions of the ciliary muscle.

Ciliary neuromuscular morphology in cynomolgus monkeys after ciliary ganglionectomy.

Cynomolgus monkeys underwent unilateral ciliary ganglionectomy (CG) and/or posterior ciliary neurectomy (PCN). The ciliary muscle was functionally denervated, as evidenced by loss of choline acetyltransferase activity, loss of the accommodative response to topical eserine and electrical stimulation of the Edinger-Westphal nucleus, and supersensitivity of the accommodative response to pilocarpine. Light and electron microscopy carried out 3-28 days after CG/PCN revealed degeneration of myelinated and unmyelinated nerve fibers as evidenced by axonal swelling and shrinkage, mitochondrial degeneration, axoplasmic condensation and vacuolization, and activated, phagocytosing Schwann cells. By 1-3 months after CG/PCN, few non-myelinated axons remained between the muscle bundles, larger nerve bundles were disordered and deteriorated, and Schwann cells filled with lipid and cellular debris were prominent. Beyond 6 months (6-37 months), most eyes were reinnervated by functional criteria. Concurrently, the ciliary neuromuscular morphology appeared virtually normal, exhibiting many non-myelinated axons containing numerous agranular synaptic vesicles and large mitochondria; however, remnants of degenerated axons were still present. In two eyes remaining functionally denervated, many of the unmyelinated axons between the ciliary muscle bundles were swollen or empty, with small, degenerated mitochondria and only rare synaptic vesicles, and were often ensheathed by thickened, condensed Schwann-cell cytoplasm. The muscle fibers were atrophic and separated from each other as well as from the ensheathed nerve fibers. Thus, following CG/PCN, the morphologic and functional evidence of parasympathetic denervation and reinnervation of the ciliary muscle is pathophysiologically and temporally consistent.

Endothelin depolarizes membrane voltage and increases intracellular calcium concentration in human ciliary muscle cells.

The ciliary muscle which is involved in accommodation and regulation of aqueous humour outflow resistance resembles smooth muscle in other parts of the body. In the present investigation we used an established primary cell line (H7CM) to study the effects of endothelin, a novel vasoconstrictor peptide, on membrane voltage (V) and intracellular calcium in cultured human ciliary muscle cells. Membrane voltage was measured in confluent monolayers of H7CM cells using conventional microelectrodes. Intracellular calcium concentration [( Ca]i) was measured in single H7CM cells using the fluorescent calcium indicator fura-2. Under resting conditions V averaged -66.9 +/- 0.7 mV (mean +/- SEM, n = 125). Endothelin (10(-10)-10(-6)M) induced a dose-dependent reversible membrane voltage depolarization and a dose-dependent rise in [Ca]i. The initial calcium peak was followed by a recovery phase during which oscillations of [Ca]i occurred. The initial calcium peak was not dependent on the presence of extracellular calcium and was not abolished in the presence of the calcium antagonist verapamil (10(-4)M). Thus it is probably mediated by a release of calcium from intracellular reservoirs. We conclude that cultured human ciliary muscle cells express a functional endothelin receptor.

Outflow facility studies in the perfused bovine aqueous outflow pathways.

We have recently developed a technique for constant pressure perfusion of the aqueous outflow pathway of the eye. Our preliminary studies, conducted in the calf eye, show surprisingly that the manipulations necessary for preparing the outflow pathways and attached corneoscleral shell for perfusion do not greatly disrupt normal aqueous outflow physiology and anatomy according to the following criteria: 1. facility of outflow is similar before and during outflow pathway perfusion 2. as in the intact eye, facility of outflow decreases with increased IOP 3. removal of outflow resistance tissue greatly increases facility of outflow 4. morphology of outflow tissues remains normal Use of the perfused outflow pathway model may enable the creation of valuable in vitro preparations which may provide much needed information about the pathogenesis of primary open-angle glaucoma.

Time-dependence of perfusion outflow facility in the cynomolgus monkey.

Surgically virgin (N), aniridic (I), and ciliary muscle disinserted (D) cynomolgus monkey eyes had total outflow facility determined in vivo for 88-136 minutes by two-level constant pressure perfusion of the anterior chamber. In all three types of eyes, facility increased during the first hour but stabilized thereafter. Average facility for the second half hour was 15% higher than for the first half hour in N and I eyes, and 10% higher in D eyes. Altered iris muscle tone can therefore not explain the facility increase, while increased ciliary muscle tone might explain part but not all of it. Judicious use of the data may aid interpretation of perfusion experiments conducted under similar conditions.

Dissociation of cholinergic supersensitivity from receptor number in ciliary muscle.

Ciliary ganglionectomy in the cynomolgus monkey produced loss of the accommodative response to electrical stimulation of the Edinger-Westphal nucleus and to topical eserine, concurrent with enhanced responsiveness to topical and systemic pilocarpine. This supersensitivity produced by parasympathetic denervation was not associated with an increase in muscarinic receptor number or affinity measured using specific QNB binding sites in ciliary muscle homogenates. In seven of ten cases, ciliary muscle muscarinic receptors were decreased by 60-84%, while in the remaining three cases, the decrease ranged from 12-37%. In no case was supersensitivity accompanied by an increased number of receptors. Six or more months after ganglionectomy, accommodative responses to central electrical stimulation and topical eserine returned to normal in most animals, indicating parasympathetic reinnervation of the ciliary muscle. Ciliary muscle sensitivity to pilocarpine returned to normal with reinnervation and was associated with recovery of normal receptor number and with normal binding affinity.

Effect of cholinergic drugs on outflow facility after ciliary ganglionectomy.

In cynomolgus monkeys, resting total outflow facility was unaltered 1 and 6 or more months after ciliary ganglionectomy (CG) or postganglionic ciliary neurectomy (PCN). Intraocular pressure (IOP) was decreased in the denervated eye 1 week and 1 month after surgery, but returned to normal after 6 or more months. Although baseline facilities were comparable in CG/PCN and fellow control eyes 6 or more months after surgery, even maximal intracameral doses of pilocarpine did not increase outflow facility in previously denervated eyes, while a normal facility increase occurred in fellow control eyes. However, both previously denervated and fellow control eyes exhibited a large facility increase to both submaximal and greater than maximal intracameral doses of eserine.

Reinnervation of primate ciliary muscle following ciliary ganglionectomy.

Ciliary ganglionectomy and/or postganglionic ciliary neurectomy in the cynomolgus monkey was followed by supersensitivity to intramuscular (i.m.) pilocarpine and lack of response to topical eserine and to electrical stimulation of the Edinger-Westphal nucleus. Normal responsiveness to pilocarpine and eserine returned in most instances after about 6 months. An accommodative response to stimulus of the Edinger-Westphal nucleus was also present and, as in control eyes, could be blocked by hexamethonium. Collectively, these findings demonstrate that parasympathetic innervation to the ciliary muscle mediating accommodation traverses a typical peripheral autonomic synapse, almost certainly located predominantly in the ciliary ganglion; by 6 months after denervation, the ciliary muscles have reinnervated; and the parasympathetic pathway to the eye exhibits plasticity and capacity for regeneration.

Cholinergic drugs alter ciliary muscle response and receptor content.

Topical echothiophate administration to the cynomolgus monkey eye for 5-6.5 months produced marked subsensitivity of the accommodative response to pilocarpine and a 65% decrease in specific high affinity 3H-QNB binding sites (ostensibly indicating muscarinic receptors) in the ciliary muscle. The decrease in 3H-QNB binding sites was quantitatively similar in surgically untouched, totally iridectomized, and ciliary muscle disinserted eyes. Following a 5-month off treatment recovery period, 3H-QNB binding sites increased to more than twice the number in untreated control eyes. In echothiophate-treated eyes whose contralateral eyes had previously received atropine+echothiophate, 3H-QNB binding sites were three to six times more numerous than in other long-term echothiophate-treated eyes, and one to two times more numerous than in untreated controls. Topical pilocarpine administration for 1 day to 7 months reduced ciliary muscle 3H-QNB binding sites by approximately 25%. Alterations in muscarinic receptors during and following cholinergic drug therapy may in part explain the observed subsensitization and recovery of ciliary muscle physiological responses.