SEROTONIN AND NEUROPEPTIDE IMMUNOREACTIVITIES IN THE INTRAMOLLUSCAN STAGES OF 3 MARINE TREMATODE PARASITES

Using an indirect immunofluorescence technique interfaced with confocal scanning laser microscopy, whole-mount preparations of three genera of marine trematode larvae, Cryntocotyle lingua, Cercaria emasculans and Himasthla leptosoma, were screened for 5-hydroxytryptamine (5-HT) and selected neuropeptide immunoreactivities (IRs). IRs for pancreatic polypeptide (PP), peptide YY (PYY) and FMRFamide were found in the central nervous systems of the three species of cercariae, immunostaining the paired ganglia and central commissure and the longitudinal nerve cords, with slight differences in both distribution and intensity of IRs being observed for the different antisera used. PP, PYY and FMRFamide IRs were evident in both central and peripheral components of the nervous system in the rediae of C. lingua. 5-HT IR was confined to the peripheral nervous systems of the cercariae of C. emasculans and the rediae of C. lingua, appearing in the form of a network of immunoreactive fibres and associated large cell bodies. A moderate substance P IR was observed in the nervous system of the cercariae of C. lingua. The patterns of immunostaining described were compared with those obtained using antiserum directed to the C-terminal decapeptide amide of neuropeptide F (NPF), a native parasitic peptide from the cestode Moniezia expansa. Results demonstrated that serotoninergic and peptidergic components were present in the nervous systems of all of the trematode larvae studied and that some, if not all, of the IR for PP. PYY and FMRFamide was due to the presence of a trematode NPF homologue.

5-HYDROXYTRYPTAMINE (SEROTONIN)-IMMUNOREACTIVITY IN THE NERVOUS-SYSTEM OF MESOCESTOIDES-CORTI TETRATHYRIDIA (CESTODA, CYCLOPHYLLIDEA)

An indirect immunocytochemical technique has been interfaced with confocal scanning laser microscopy to investigate the occurrence and distribution of serotoninergic (5-HT) nerve elements in Mesocestoides corti tetrathyridia. Cell bodies and nerve fibers immunoreactive to 5-HT were found concentrated in the innervation around the 4 suckers and associated commissures and in the 5 pairs of longitudinal nerve cords and their cross-connectives. Immunoreactivity was evident also in the extensive, peripheral network of fine fibers of the subtegumental region and in the plexus of varicose fibers that innervate the muscle in each of the suckers. In dividing stages of the tetrathyridium, the immunoreactive lateral nerve cords of adjoining progeny were in continuity around the base of the division cleft.

AN IMMUNOCYTOCHEMICAL STUDY OF PUTATIVE NEUROTRANSMITTERS IN THE METACERCARIAE OF 2 STRIGEOID TREMATODES FROM RAINBOW-TROUT (ONCORHYNCHUS-MYKISS)

Whole mounts of the metacercariae of Diplostomum sp. and Cotylurus erraticus from rainbow trout have been treated cytochemically for the demonstration of cholinergic, serotoninergic (5-hydroxytryptamine) and peptidergic elements in the nervous system. Antisera directed against four vertebrate (pancreatic polypeptide, peptide YY, substance P and peptide histidine isoleucine) and two invertebrate peptides (neuropeptide F and FMRFamide) were used in an indirect immunofluorescence procedure in conjunction with confocal scanning laser microscopy (CSLM). Of the seven antisera tested, all except peptide histidine isoleucine showed significant immunoreactivity. Cholinergic and serotoninergic staining was found primarily in the central nervous system (CNS) and in cell bodies associated with the ventral and dorsal nerve cords in both trematodes. Peptidergic immunoreactivity was localised in the CNS and PNS of both genera, revealing an extensive innervation within the holdfast organ and in and around the oral and ventral suckers.

THE CHOLINERGIC, SEROTONINERGIC AND PEPTIDERGIC COMPONENTS OF THE NERVOUS-SYSTEM OF MONIEZIA-EXPANSA (CESTODA, CYCLOPHYLLIDEA)

The central (CNS) and peripheral (PNS) nervous systems of the cyclophyllidean tapeworm, Moniezia expansa, were examined for the presence of cholinergic, serotoninergic and peptidergic elements using enzyme cytochemical and immunocytochemical techniques in conjunction with light and confocal scanning laser microscopy. Cholinesterase activity and 5-hydroxytryptamine- and regulatory peptide-immunoreactivities (IRs) were localized to the nerve fibres and cell bodies of all of the major neuronal components in the CNS of the worm, including the cerebral ganglia and connecting commissure, the 10 longitudinal nerve cords and associated transverse ring commissures. Although each of the 3 systems appeared well developed and comprised a significant portion of the nervous system, the serotoninergic constituent was the most highly developed, consisting of a vast array of nerve fibres and cell bodies distributed throughout the strobila of the worm. A close association of cholinesterase reactivity and peptide-IRs was evident throughout the CNS, indicating the possible co-localization of acetylcholine and neuropeptides. Within the PNS, cholinergic activity and serotoninergic- and peptidergic-IRs occurred in the subtegumental network of nerve fibres and somatic musculature. Although all 3 neurochemical elements were present in the acetabula, they were found in different nerve fibres; only cholinergic and peptidergic cell bodies were found. The common genital opening, vagina and ootype regions of the reproductive system displayed a rich innervation of all 3 types of neuronal populations. Within the peptidergic system, immunostaining with antisera raised to the C-terminus of the neuropeptide Y superfamily of peptides and the invertebrate peptides, neuropeptide F (M. expansa) and FMRFamide was the most prevalent. Limited positive-IR for substance P and neurokinin A were also recorded in the CNS of the worm.

NEUROPEPTIDE F-IMMUNOREACTIVITY IN THE MONOGENEAN PARASITE DICLIDOPHORA-MERLANGI

The localisation and distribution of neuropeptide F (NPF)-immunoreactivity (IR) in the monogenean fish-gill parasite, Diclidophora merlangi, have been investigated by whole-mount immunocytochemistry interfaced with confocal scanning laser microscopy and, at the ultrastructural level, by indirect immunogold labeling. Using antisera directed to intact synthetic NPF (Moniezia expansa, residues 1-39) or to the C-terminal decapeptide (residues 30-39) of synthetic NPF (M. expansa), immunostaining was found throughout the central (CNS) and peripheral nervous systems (PNS), including the innervation of the reproductive system. Immunoreactivity was found to be more intense using the antiserum to the C-terminal decapeptide fragment of NPF. At the subcellular level, gold labeling of NPF-IR was found exclusively over the contents of dense-cored vesicles that occupied nerve axons of both the CNS and the PNS. The distribution pattern of immunostaining for NPF mirrored exactly that previously documented for the vertebrate pancreatic polypeptide (PP) family of peptides and for FMRFamide. This finding and the results of preabsorption experiments strongly suggest that NPF is the predominant native neuropeptide in D. merlangi and that it accounts for most of the immunostaining previously obtained with PP and FMRFamide antisera.

Cholinergic, serotoninergic and peptidergic components of the nervous system of Discocotyle sagittata (Monogenea: Polyopisthocotylea)

Cholinergic, serotoninergic (5-HT) and peptidergic neuronal pathways have been demonstrated in both central and peripheral nervous systems of adult Discocotyle sagittata, using enzyme histochemistry and indirect immunocytochemistry in conjunction with confocal scanning laser microscopy. Antisera to 2 native flatworm neuropeptides, neuropeptide F and the fMRFamide-related peptide (FaRP), GNFFRFamide, were employed to detect peptide immunoreactivity. The CNS is composed of paired cerebral ganglia and connecting dorsal commissure, together with several paired longitudinal nerve cords. The main longitudinal nerve cords (lateral, ventral and dorsal) are interconnected at intervals by a series of annular cross-connectives, producing a ladder-like arrangement typical of the platyhelminth nervous system. At the lever of the haptor, the ventral cords provide nerve roots which innervate each of the 8 clamps. Cholinergic and peptidergic neuronal organisation was similar, but distinct from that of the serotoninergic components. The PNS and reproductive system are predominantly innervated by peptidergic neurones. Copyright (C) 1996 Australian Society for Parasitology. Published by Elsevier Science Ltd.

Immunohistochemical localization of a Shaker-related voltage-gated potassium channel protein in Schistosoma mansoni (Trematoda: Digenea)

We have recently isolated a cDNA (SKV1.1) encoding a Shakei-related K+ channel from the human parasitic trematode Schistosoma mansoni. In order to better understand the functions of SKv1.1 protein, the distribution of SKv1.1 protein in adult S. mansoni was analyzed by immunohistochemistry using a region-specific antibody. SKV1.1 proteins were widely expressed in the nervous and muscular systems. The strongest immunoreactivity (IR) was observed in the nervous system of both male and female. In the nervous system, IR for SKv1.1 proteins was localized in cell bodies and nerve fibers of the anterior ganglia, the central commissure, and the main nerve cords. IR was also observed in the dorsal and the ventral peripheral nerve nets, fine nerve fibers entering into a variety of structures such as the dorsal tubercles, longitudinal and ventral muscle fibers, and oral and ventral suckers. In the muscular system, SKv1.1 proteins were localized to the longitudinal, circular, and ventral muscle fibers of male as well as in isolated muscle fibers where native A-type K+ currents were measured. Moderate IR was also seen in a large number of cell bodies in the parenchyma. These results indicate that SKv1.1 protein may play an important role in the regulation of the excitability of neurons and muscle cells of S. mansoni. (C) 1995 Academic Press, Inc.

A CYTOCHEMICAL STUDY OF THE NERVOUS-SYSTEM OF THE PROTEOCEPHALIDEAN CESTODE, PROTEOCEPHALUS-POLLANICOLA

The localization and distribution of cholinergic, serotoninergic and peptidergic nerve elements in the proteocephalidean tapeworm, Proteocephalus pollanicola, have been investigated by enzyme histochemistry, and by an indirect immunofluorescence technique interfaced with confocal scanning laser microscopy. Cholinesterase (ChE) activity was localized in the major components of the central nervous system (CNS) and the peripheral nervous system (PNS), including the innervation of the reproductive structures of the worm. Serotoninergic (5-HT) nerves were found in the paired cerebral ganglia, transverse commissure and in the 10 longitudinal nerve cords. Antisera to 17 mammalian regulatory peptides and the invertebrate peptide FMRFamide have been used to explore the peptidergic nervous system of the worm. The most extensive immunostaining occurred with antisera raised to members of the neuropeptide Y superfamily, namely neuropeptide Y (NPY), peptide YY (PYY) and pancreatic polypeptide (PP). In all cases, intense immunoreactivity was found in numerous cell bodies and fibres of both the CNS and PNS, including the innervation of the reproductive apparatus. FMRFamide antisera stained the same structures to a comparable degree as those raised to the NPY superfamily. Cholinergic and peptidergic elements were much more prevalent within the CNS, while the serotoninergic nerve fibres tended to dominate in the PNS. The overlap obtained in staining patterns for the peptidergic and cholinergic components suggests that there may be a certain amount of co-localization of peptides with small-molecule transmitter substances in the same neurone. Weak staining for the tachykinin, substance P and for calcitonin gene-related peptide(CGRP) was confined to the major longitudinal nerve cords.

ISOLATION, LOCALIZATION, AND CARDIOVASCULAR ACTIVITY OF TACHYKININS FROM THE STOMACH OF THE BOWFIN AMIA-CALVA

The bowfin is an extant representative of an ancient group of ray-finned fish with evolutionary connections to modern teleosts. A peptide with substance P-like immunoreactivity was isolated from an extract of bowfin stomach and its primary structure was established as Ser-Lys-Ser-His-Gln-Phe-Tyr-Gly-Leu-Met-NH2. This amino acid sequence resembles mammalian substance P only in the COOH-terminal region of the peptide. A second tachykinin with neurokinin A-like immunoreactivity isolated from the extract comprises 23 amino acid residues and shows limited structural similarity to mammalian neuropeptide-gamma. A randomly distributed population of cells in the gastric glands of the bowfin were immunostained with an antiserum raised against substance P, but no immunopositive structures were identified in the surface epithelium, lamina propria, or the nerve plexuses of the submucosa. Bolus injections of synthetic bowfin substance P (0.1-10 nmol/kg) into the bulbus arteriosus of unanesthetized bowfin resulted in a significant and dose-dependent rise in vascular resistance and arterial blood pressure (P < 0.01) and a fall in cardiac output (P < 0.05) without change in heart rate. After 5-10 min, arterial pressure and vascular resistance returned to preinjection levels, but cardiac output significantly (P < 0.05) increased over baseline values. The response to the peptide was unaffected by pretreatment of the animals with phentolamine. The study has shown that the stomach of the bowfin synthesizes tachykinins with novel structural features that display cardiovascular activity in this species.

Functional innervation of Guinea-pig bladder interstitial cells of cajal subtypes: neurogenic stimulation evokes in situ calcium transients

Several populations of interstitial cells of Cajal (ICC) exist in the bladder, associated with intramural nerves. Although ICC respond to exogenous agonists, there is currently no evidence of their functional innervation. The objective was to determine whether bladder ICC are functionally innervated. Guinea-pig bladder tissues, loaded with fluo-4AM were imaged with fluorescent microscopy and challenged with neurogenic electrical field stimulation (EFS). All subtypes of ICC and smooth muscle cells (SMC) displayed spontaneous Ca2+-oscillations. EFS (0.5Hz, 2Hz, 10Hz) evoked tetrodotoxin (1µM)-sensitive Ca2+-transients in lamina propria ICC (ICC-LP), detrusor ICC and perivascular ICC (PICC) associated with mucosal microvessels. EFS responses in ICC-LP were significantly reduced by atropine or suramin. SMC and vascular SMC (VSM) also responded to EFS. Spontaneous Ca2+-oscillations in individual ICC-LP within networks occurred asynchronously whereas EFS evoked coordinated Ca2+-transients in all ICC-LP within a field of view. Non-correlated Ca2+-oscillations in detrusor ICC and adjacent SMC pre-EFS, contrasted with simultaneous neurogenic Ca2+ transients evoked by EFS. Spontaneous Ca2+-oscillations in PICC were little affected by EFS, whereas large Ca2+-transients were evoked in pre-EFS quiescent PICC. EFS also increased the frequency of VSM Ca2+-oscillations. In conclusion, ICC-LP, detrusor ICC and PICC are functionally innervated. Interestingly, Ca2+-activity within ICC-LP networks and between detrusor ICC and their adjacent SMC were synchronous under neural control. VSM and PICC Ca2+-activity was regulated by bladder nerves. These novel findings demonstrate functional neural control of bladder ICC. Similar studies should now be carried out on neurogenic bladder to elucidate the contribution of impaired nerve-ICC communication to bladder pathophysiology.

flp-32 Ligand/Receptor Silencing Phenocopy Faster Plant Pathogenic Nematodes

Restrictions on nematicide usage underscore the need for novel control strategies for plant pathogenic nematodes such as Globodera pallida (potato cyst nematode) that impose a significant economic burden on plant cultivation activities. The nematode neuropeptide signalling system is an attractive resource for novel control targets as it plays a critical role in sensory and motor functions. The FMRFamide-like peptides (FLPs) form the largest and most diverse family of neuropeptides in invertebrates, and are structurally conserved across nematode species, highlighting the utility of the FLPergic system as a broad-spectrum control target. flp-32 is expressed widely across nematode species. This study investigates the role of flp-32 in G. pallida and shows that: (i) Gp-flp-32 encodes the peptide AMRNALVRFamide; (ii) Gp-flp-32 is expressed in the brain and ventral nerve cord of G. pallida; (iii) migration rate increases in Gp-flp-32-silenced worms; (iv) the ability of G. pallida to infect potato plant root systems is enhanced in Gp-flp-32-silenced worms; (v) a novel putative Gp-flp-32 receptor (Gp-flp-32R) is expressed in G. pallida; and, (vi) Gp-flp-32R silenced worms also display an increase in migration rate. This work demonstrates that Gp30 flp-32 plays an intrinsic role in the modulation of locomotory behaviour in G. pallida, and putatively interacts with at least one novel G-protein coupled receptor (Gp-flp-32R). This is the first functional characterisation of a parasitic nematode FLP-GPCR. © 2013 Atkinson et al.

Environmental tobacco smoke exposure and eye disease

Aim: To undertake a systematic review of the literature on the effect of environmental tobacco smoke (ETS) and eye disease. Methods: Medline (1950-January Week 2 2007), EMBASE (1980 to 2007 Week 07), SCOPUS and Science Direct were searched on ETS exposure and eye disease using various combinations of the following terms: passive smoking, environmental tobacco smoke, sidestream smoke, involuntary smoking, secondhand smoke; with eye, conjunctiva, sclera, episclera, cornea, lens, iris, retina, choroid, uvea, optic nerve, uveitis, iritis, blindness, visual loss, cataract, thyroid eye disease, conjunctivitis, age-related macular degeneration, dry eye, tears. The above terms were also used to search abstracts published on The Association for Research in Vision and Ophthalmology Annual Meeting abstracts, from 1995 to 2006, and the grey literature, including PhD and MSc theses/dissertations. A search was further conducted specifically on eye diseases where active smoking has been proposed to be a risk factor, including age-related macular degeneration, Graves ophthalmology, glaucoma, uveitis, refractive errors, strabismus, tobacco-alcohol amblyopia, non-arteritic ischaemic optic neuropathy, Leber optic neuropathy and diabetic retinopathy. Given the scarce number of studies found through the above search, all articles found on ETS and eye disease were included in this review. Results: Seven studies evaluated the possible relationship between ETS and an eye disease. These studies referred to refractive errors in children (n = 2), cataract (n = 1), age-related macular degeneration (n = 3) and Grave ophthalmopathy (n = 1). The data available were insufficient to establish conclusive relationships between ETS and these eye diseases. Conclusion: Very scarce data exist in the literature on the effect of ETS on diseases of the eye. It seems appropriate that ETS should be included in future studies addressing the effect of smoking on eye disease.

Surveillance for ocular hypertension: An evidence synthesis and economic evaluation

OBJECTIVES: To determine effective and efficient monitoring criteria for ocular hypertension [raised intraocular pressure (IOP)] through (i) identification and validation of glaucoma risk prediction models; and (ii) development of models to determine optimal surveillance pathways.

DESIGN: A discrete event simulation economic modelling evaluation. Data from systematic reviews of risk prediction models and agreement between tonometers, secondary analyses of existing datasets (to validate identified risk models and determine optimal monitoring criteria) and public preferences were used to structure and populate the economic model.

SETTING: Primary and secondary care.

PARTICIPANTS: Adults with ocular hypertension (IOP > 21 mmHg) and the public (surveillance preferences).

INTERVENTIONS: We compared five pathways: two based on National Institute for Health and Clinical Excellence (NICE) guidelines with monitoring interval and treatment depending on initial risk stratification, 'NICE intensive' (4-monthly to annual monitoring) and 'NICE conservative' (6-monthly to biennial monitoring); two pathways, differing in location (hospital and community), with monitoring biennially and treatment initiated for a ≥ 6% 5-year glaucoma risk; and a 'treat all' pathway involving treatment with a prostaglandin analogue if IOP > 21 mmHg and IOP measured annually in the community.

MAIN OUTCOME MEASURES: Glaucoma cases detected; tonometer agreement; public preferences; costs; willingness to pay and quality-adjusted life-years (QALYs).

RESULTS: The best available glaucoma risk prediction model estimated the 5-year risk based on age and ocular predictors (IOP, central corneal thickness, optic nerve damage and index of visual field status). Taking the average of two IOP readings, by tonometry, true change was detected at two years. Sizeable measurement variability was noted between tonometers. There was a general public preference for monitoring; good communication and understanding of the process predicted service value. 'Treat all' was the least costly and 'NICE intensive' the most costly pathway. Biennial monitoring reduced the number of cases of glaucoma conversion compared with a 'treat all' pathway and provided more QALYs, but the incremental cost-effectiveness ratio (ICER) was considerably more than £30,000. The 'NICE intensive' pathway also avoided glaucoma conversion, but NICE-based pathways were either dominated (more costly and less effective) by biennial hospital monitoring or had a ICERs > £30,000. Results were not sensitive to the risk threshold for initiating surveillance but were sensitive to the risk threshold for initiating treatment, NHS costs and treatment adherence.

LIMITATIONS: Optimal monitoring intervals were based on IOP data. There were insufficient data to determine the optimal frequency of measurement of the visual field or optic nerve head for identification of glaucoma. The economic modelling took a 20-year time horizon which may be insufficient to capture long-term benefits. Sensitivity analyses may not fully capture the uncertainty surrounding parameter estimates.

CONCLUSIONS: For confirmed ocular hypertension, findings suggest that there is no clear benefit from intensive monitoring. Consideration of the patient experience is important. A cohort study is recommended to provide data to refine the glaucoma risk prediction model, determine the optimum type and frequency of serial glaucoma tests and estimate costs and patient preferences for monitoring and treatment.

FUNDING: The National Institute for Health Research Health Technology Assessment Programme.

A novel three-dimensional culture system for isolation and clonal propagation of neural stem cells using a thermo-reversible gelation polymer

In the present study, we examined the possible utility of a three-dimensional culture system using a thermo-reversible gelation polymer to isolate and expand neural stem cells (NSCs). The polymer is a synthetic biologically inert polymer and gelates at temperatures higher than the gel-sol transition point ( approximately 20 degrees C). When fetal mouse brain cells were inoculated into the gel, spherical colonies were formed ( approximately 1% in primary culture and approximately 9% in passage cultures). The spheroid-forming cells were positive for expression of the NSC markers nestin and Musashi. Under conditions facilitating spontaneous neural differentiation, the spheroid-forming cells expressed genes characteristic to astrocytes, oligodendrocytes, and neurons. The cells could be successively propagated at least to 80 poly-D-lysines over a period of 20 weeks in the gel culture with a growth rate higher than that observed in suspension culture. The spheroids formed by fetal mouse brain cells in the gel were shown to be of clonal origin. These results indicate that the spheroid culture system is a convenient and powerful tool for isolation and clonal expansion of NSCs in vitro.

Developing the specifications of an open angle glaucoma screening intervention in the United Kingdom: a Delphi approach.

Abstract
BACKGROUND:
Glaucoma is a leading cause of blindness. Early detection is advocated but there is insufficient evidence from randomized controlled trials (RCTs) to inform health policy on population screening. Primarily, there is no agreed screening intervention. For a screening programme, agreement is required on the screening tests to be used, either individually or in combination, the person to deliver the test and the location where testing should take place. This study aimed to use ophthalmologists (who were experienced glaucoma subspecialists), optometrists, ophthalmic nurses and patients to develop a reduced set of potential screening tests and testing arrangements that could then be explored in depth in a further study of their feasibility for evaluation in a glaucoma screening RCT.
METHODS:
A two-round Delphi survey involving 38 participants was conducted. Materials were developed from a prior evidence synthesis. For round one, after some initial priming questions in four domains, specialists were asked to nominate three screening interventions, the intervention being a combination of the four domains; target population, (age and higher risk groups), site, screening test and test operator (provider). More than 250 screening interventions were identified. For round two, responses were condensed into 72 interventions and each was rated by participants on a 0-10 scale in terms of feasibility.
RESULTS:
Using a cut-off of a median rating of feasibility of =5.5 as evidence of agreement of intervention feasibility, six interventions were identified from round 2. These were initiating screening at age 50, with a combination of two or three screening tests (varying combinations of tonometry/measures of visual function/optic nerve damage) organized in a community setting with an ophthalmic trained technical assistant delivering the tests. An alternative intervention was a 'glaucoma risk score' ascertained by questionnaire. The advisory panel recommended that further exploration of the feasibility of screening higher risk populations and detailed specification of the screening tests was required.
CONCLUSIONS:
With systematic use of expert opinions, a shortlist of potential screening interventions was identified. Views of users, service providers and cost-effectiveness modeling are now required to identify a feasible intervention to evaluate in a future glaucoma screening trial.