Neural immunoregulation: emerging roles for nerves in immune homeostasis and disease.

In this review, James Downing and Jaleel Miyan outline emerging evidence for neural mechanisms that contribute to specific categories of host defence. Involvement of direct innervation in the adaptive control of immunological responses complements an established view of neuroendocrine-immune modulation. The challenge remains to understand the integrative and homeostatic functions of 'hardwiring' of peripheral immune effector sites, its bearing on disorder and potential for therapeutic modification.

Neuronal and glial cell types revealed by NADPH-diaphorase histochemistry in the retina of a teleost fish, the grass goby (Zosterisessor ophiocephalus, Perciformes, Gobiidae).

The grass goby is a mud-burrowing fish with a rich retinal vasculature appropriate to its hypoxic habitat. NADPH-diaphorase histochemistry was performed on retinal sections and wholemounts to reveal cells that contain nitric oxide synthase and so may be presumed to synthesise nitric oxide, a gaseous intercellular messenger with many roles including vasodilation. Structures that were consistently stained by this method included cone ellipsoids, horizontal cells, Müller cells and their processes, large displaced ganglion cells in the inner nuclear layer (identified by their axons), large interstitial ganglion cells in the inner plexiform layer, and capillary endothelial cells. In wholemounts, horizontal cells were seen to form a regular pattern, contacting each other at their dendritic terminals. Some cells in the ganglion cell layer were weakly stained, but stained bipolar and amacrine cells were not seen. The diaphorase-positive large ganglion cells all formed large, sparsely branched dendritic trees, arborizing near the scleral border of the inner plexiform layer. The displaced and interstitial cells seemed to belong to distinct morphological types, the interstitial cells having smaller somata and trees. Analysis of their spatial distributions in one representative retina confirmed this: the displaced cells formed a highly regular mosaic with a mean spacing (nearest-neighbour distance) of 303 microm, whereas the interstitial cells formed a separate mosaic, almost as regular but with a smaller mean spacing of 193 microm, rising to 217 microm in a sample that excluded the area retinae temporalis. Spatial correlogram analysis showed that these two mosaics were spatially independent. Nitric oxide probably has many roles in the retina. The presence of its synthetic enzyme in Müller cells, which communicate with retinal blood vessels, is consistent with a role in the control of retinal blood flow. Its function in large, mosaic-forming retinal ganglion cells is unknown.

Rapid progesterone actions on thymulin-secreting epithelial cells cultured from rat thymus.

Many soluble factors of neural, endocrine, paracrine and autocrine origin are present in the thymus and modulate its function. Long-term effects of sex steroids have been documented for thymocytes and cells of the thymic microenvironment. In this report we examine rapid actions of progesterone upon aspects of epithelial cell physiology. Progesterone (0.1-10 microM) was applied to cultured thymulin-secreting thymic epithelial cells (TS-TEC) and changes in transmembrane potential, transmembrane current, intracellular calcium levels and thymulin secretion were assessed. Rapid changes in electrophysiology and intracellular calcium provide evidence for a membrane-bound progesterone receptor in these cells, in addition to classical cytoplasmic receptors. Application of progesterone to TS-TEC caused electrophysiological changes in 56% of cells (n = 40), activating an inward current (-24 +/- 9 pA at 1 microM, n = 7, p < 0.02) and dose-dependent depolarization (7.1 +/- 1.8 mV at 1 microM, n = 19, p < 0.01). Intracellular calcium levels, monitored by the ratiometric fluorescent calcium indicator fura-2, increased within seconds of progesterone (1 microM) application. Progesterone (1 microM) increased thymulin levels in supernatant, as measured by ELISA, above the levels in the preapplication period (142 +/- 16% of the preapplication period, n = 3, p < 0.02). This effect was reduced in the presence of cobalt chloride which blocks voltage-dependent calcium channels. In addition, TS-TEC in culture were immunoreactive to antibody AG7. This antibody was raised to a membrane-bound antigen involved in calcium influx subsequent to progesterone binding in sperm. Thus we suggest that progesterone acts upon many aspects of TS-TEC physiology through both cytoplasmic and membrane-bound receptors.

NADPH diaphorase-positive dendritic profiles in rat thymus are discrete from autofluorescent cells, immunoreactive for inducible nitric oxide synthase, and show strain-specific abundance differences.

Predisposition to autoimmune disorder in Lewis rats has been associated with abnormal hypothalamic regulation of circulating steroids, leading to inadequate suppression of T helper 1 (Th1) cell-mediated inflammatory reactions. In addition, autoimmune syndromes can be triggered within formerly resistant animals, following damage to the negative selection process of the thymus. A contribution to the autoimmune-susceptible phenotype may therefore derive from the status of thymic tolerance. One mechanism of intrathymic negative selection may involve nitric oxide. Because inducible nitric oxide synthase (iNOS) is known to be inhibitable by steroids, its expression might be different within strains having neuroendocrine disturbance. We report on a study to compare intrathymic iNOS expression in autoimmune-prone Lewis rats with other resistant strains. Interdigitating cells (IDC), darkly stained for diaphorase, were confirmed as immunoreactive for iNOS. They were located towards the medullary side of an accumulation of unstained, but autofluorescent cells (presumed to be macrophages) that circumscribes the corticomedullary zone. The role of iNOS+ IDC in the apoptotic deletion of T cells has been suggested by other studies. Despite the blunted steroidal condition reported for Lewis, nitrergic cell abundance was shown, by quantitative analysis of histochemical stain, to be on average approximately twofold lower compared with resistant strains (Fischer and Sprague-Dawley). This trend was evident in males and females, and confirmed by independent observers. We hypothesize that an intrathymic, iNOS-dependent mechanism may be important for the suppression of potentially autoreactive T-cell clones.

Neuropeptides exert direct effects on rat thymic epithelial cells in culture.

To determine if major thymic neuropeptides and neurotransmitters can directly influence the functional activity of cultured rat thymic epithelium, neuropeptides and neurotransmitters were applied, and intercellular communication, proliferation, and thymulin secretion assessed. After injections of a mixture of lucifer yellow dextran (too large to pass gap junctions) and cascade blue (which does) into single cells, some neuropeptides decrease dye coupling: 0.1 mM GABA (P < 0.0001), 100 nM NPY (P < 0.0001), 100 nM VIP (P < 0.001), 100 nM CGRP (P < 0.001), 100 nM SP (P < 0.01), and 0.1 mM histamine (P < 0.01), whereas 0.1 mM 5-HT, 1 mM acetylcholine, and 1 microM isoproterenol (beta-adrenergic agonist) had no effect. Proliferation (incorporation of tritiated thymidine) was increased by CGRP (P = 0.004) and histamine (P < 0.02), but decreased by isoproterenol (P = 0.002), 5-HT (P = 0.003), and acetylcholine (P < 0.05). The percentage of multinucleate cells was decreased after isoproterenol (2.5%), and increased after 5-HT (21.3%), GABA (15%), and histamine (15.1%). Compared to controls, thymulin in the supernatant was decreased after challenge with acetylcholine (52%), isoproterenol (71%), 5-HT (73%), and histamine (84%). This study demonstrates direct effects of neuropeptides and neurotransmitters on functional aspects of cultured thymic epithelial cells.

Modulation of dye-coupling and proliferation in cultured rat thymic epithelium by factors involved in thymulin secretion.

Cultures of rat thymic epithelium were used to measure the effect of thymulin secretagogues on dye-coupling and proliferation. Dye-coupling was assessed after the injection of lucifer yellow dextran which cannot permeate the connexin pore of gap junctions and the smaller, permeant cascade blue. In addition to gap junctional communication, larger intercellular bridges were demonstrated by the transfer of lucifer yellow dextran between cells. The extent of intercellular communication was found to be influenced by both cell density and the number of passages. In control cultures, intercellular communication was reduced in cell groups of low (< 20 cells/group) or high cell densities (> 100 cells/group) compared with groups of 20-60 cells. The highest coupling indices were found in subcultures 20-30. Taking these factors into account, significant decreases in coupling index were observed after pretreatment of test cultures with factors known to influence the secretion of thymulin (5 U/ml interleukin 1 (alpha and beta), 1 microM progesterone, 1 microM oestrogen, 1 microM testosterone, 1 ng/ml adrenocorticotropic hormone, 100 nM rat growth hormone) but 7.5 ng/ml thymulin had no effect on dye-coupling. The nonspecific gap junction uncoupler, octanol, abolished dye-coupling. Cellular proliferation, as measured by the uptake of tritiated thymidine, showed that the same factors that reduced coupling also increased proliferation. None of these factors affected the number of multinucleate cells present, except interleukin-1beta which caused a significant reduction in the average number of nuclei per cell. Thus rat thymic epithelium in vitro provides a model for the study of the direct action of factors on cells of the thymic microenvironment.

Transient block of respiratory chain by cyanide triggers NADPH-diaphorase activity (a marker for nitric oxide synthase) in Dunning rat prostatic epithelium.

Synthesis of nitric oxide (NO) has been shown in the glandular epithelium of human prostate, with highest levels in the peripheral zone. This location is believed to be the main source of prostatic cancer. The ability of stromal cells to produce NO may contribute to the malignant process. Since solid tumours are prone to hypoxia and malignant progression, experiments were undertaken to test the effect of respiratory block on the induction of nitric oxide synthase (NOS) by a Dunning rat prostatic epithelial line. A metastatic phenotype (Mat-LyLu) was treated in vitro with brief exposure to cyanide in order to mimic transient hypoxic stress. NADPH-diaphorase activities in paraformaldehyde-fixed cells was used to follow the expression of NOS. NADPH-diaphorase activity was found to be inducible by a range of factors, including mechanical damage and infection of cultures. Cyanide induced a dose-dependent staining that was statistically greater than in untreated cells. Consistent with diaphorase staining being a marker for the inducible isoform of NOS (iNOS), induction and enhancement of staining, respectively, was observed in response to treatment with lipopolysaccharide or withdrawal of dexamethasone supplement. Results demonstrate that prostatic epithelia can be triggered in culture to express iNOS by transient oxidative stress in the form of respiratory poisoning by NaCN. Paradoxically, nitric oxide production by epithelia within hypoxic zones of solid tumours may contribute to the promotion and/or inhibition of tumorigenesis.

The application of in vitro isolation, cryopreservation and patch-clamp microelectrode recording methods to adult rat thymic nurse cells.

Characterisation of the ionic mechanisms possessed by immune cells has begun to reveal a range of transmembrane ion channel properties which may have immunological significance. Since thymic epithelial cells appear to influence selection of the T cell repertoire, understanding their membrane physiology may be of importance. A method is therefore outlined for the targeting of patch-clamp electrophysiological measurements to acutely isolated, fresh and cryopreserved adult rat thymic nurse cells. These cells represent a discrete and predominantly cortical population of epithelium. Cells were separated by enzymatic and mechanical dispersal of thymus, enriched by sedimentation and identified on the basis of their characteristic lympho-epithelial, multi-cellular morphologies. Phase-bright cells retaining this anatomical form survived freezing and the voltage-gated conductances in such cells are described. The merits of this approach include the preparation and storage of mature differentiated phenotypes of immune cells for in vitro studies. Their preservation permits temporal separation of experiments, correlative experimentation on identical samples, optimises cell preparation in terms of cost and labour, and reduces animal and tissue requirements.

Differential expression of voltage-activated Na+ currents in two prostatic tumour cell lines: contribution to invasiveness in vitro.

The voltage-gated ionic currents of two rodent prostatic cancer cell lines were investigated using the whole-cell patch clamp technique. The highly metastatic Mat-Ly-Lu cells expressed a transient, inward Na+ current (blocked by 600 nM tetrodotoxin), which was not found in any of the weakly metastatic AT-2 cells. Although both cell lines expressed a sustained, outward K+ current, this occurred at a significantly higher density in the AT-2 than in the Mat-Ly-Lu cells. Incubation of the Mat-Ly-Lu cell line with 600 nM tetrodotoxin significantly reduced the invasive capacity of the cells in vitro. Under identical conditions, tetrodotoxin had no effect on the invasiveness of the AT-2 cells.

Multiple nitric oxide synthase systems in adult rat thymus revealed using NADPH diaphorase histochemistry.

Nitric oxide (NO) has become recognized as a multifunctional mediator, with roles in vascular physiology, neurotransmission and non-specific immune defense. The histochemical marker associated with the neural and endothelial form of NO synthase (NOS), reduced nicotinamide adenine dinucleotide diaphorase (NADPHd), has enabled the indirect localization of potential sites of NO production. Innervation of the thymus and its immunological functions made this tissue a candidate for utilization of various NO systems. In the present study on adult rat thymus, multiple cellular sites expressing NADPHd activity, thereby implicated as sites of NOS activity, have been identified using morphological criteria alone: blood vessel endothelium, dendritic cells, deep cortical or medullary stromal cells, intrinsic neuron-like profiles, granulocytes (possibly neutrophils) and fat cells. In addition, the availability to the thymic microenvironment of another form of NOS in macrophages, which is not stained by the diaphorase technique, was supported by the observation of these cells at corticomedullary and cortical locations. These results indicate that a wide variety of possible immunomodulatory roles can be expected for NO in the thymus including the induction of tolerance, major histocompatibility complex (MHC) restriction, lymphocyte trafficking and regulation of thymic endocrine output.

Peptide modulation of ACh receptor desensitization controls neurotransmitter release from chicken sympathetic neurons.

1. The distribution and release of substance P (SP) in embryonic chicken lumbar sympathetic ganglia was examined with the use of immunohistochemistry and radioimmunoassay, respectively. SP immunoreactivity was detected in nerve fibers surrounding individual sympathetic neurons and was released by ganglionic depolarization. 2. Effects of SP on nicotinic acetylcholine receptor (AChR) function was assayed in embryonic sympathetic neurons in vitro by whole-cell patch clamp. SP (0.1-20 microM) accelerated the rate of decay (desensitization) of ACh-induced currents. The AChR desensitization time course is biphasic and described by the sum of two exponential functions dependent on agonist concentration (time constant of the faster component, tau f = 1-2 s, and the slower time constant, tau s = 10-25 s). SP selectively decreased tau s and the contribution of the slow component to the overall rate of current decay. The effects of SP on desensitization were concentration dependent and reversible. SP slowed recovery from desensitization by 2.5-fold. 3. SP shifted the dose-response curve for ACh-induced desensitization, reducing the concentration of ACh required to produce half-maximal desensitization by approximately twofold. 4. Preapplication of SP was equivalent to SP applied together with ACh in accelerating AChR desensitization. SP did not alter the time course of currents elicited by nondesensitizing concentrations of ACh, carbamylcholine (CARB), or dimethylphenylpiperazinium (DMPP). These data suggest that AChR activation is neither necessary nor sufficient for the peptide to modulate receptor function. A kinetic model of the effects of SP on specific steps in AChR desensitization is presented. 5. SP enhanced the rate of decay of synaptic currents in sympathetic neurons innervated in vitro, decreasing the synaptic current duration by up to 80%. 6. Effects of SP on neurotransmitter release from sympathetic neurons were evaluated by measuring the release of [3H]-norepinephrine (NE). ACh and CARB stimulated NE release in a concentration- and calcium-dependent manner. SP alone had no effect on NE secretion, but the peptide inhibited NE release induced by ACh or CARB by 40-50%. 7. Although agonists specific for either nicotinic or muscarinic receptors stimulated release of NE, SP selectively inhibited the nicotinic component of transmitter secretion. Thus SP suppressed NE release induced by DMPP by up to 80% but had no effect on muscarine or depolarization-induced NE secretion. 8. Parallel studies of the modulatory effects of SP on whole-cell currents and NE secretion revealed that SP inhibition of transmitter release from sympathetic neurons is directly proportional to the extent of potentiation of AChR desensitization.(ABSTRACT TRUNCATED AT 400 WORDS)

Electrophysiological effects of tachykinin analogues on ganglion cell activity in cyprinid fish retina.

Electrical spike activity of ganglion cells has been recorded extracellularly in the teleost (roach) retina, and effects of a variety of tachykinins studied at a working concentration of 1 microM. Application of substance P mostly caused a slow and prolonged increase in background activity. In contrast, the response to carbachol was very brisk and short-lasting. Substance P and physalaemin predominantly induced an enhancement of 'On' and 'Off' components of light-evoked responses, whilst eledoisin and neurokinin A were mostly inhibitory. All effects were independent of chromatic and spatial aspects of the responses. Interestingly, in the presence of a tachykinin antagonist, 'Spantide' [D-Arg1,D-Pro2, D-Trp7.9, Leu11]SP, the profile of the effect of substance P reversed, inhibitory actions becoming much more common. Taken together, the results suggest that a tachykinin system utilising two subtypes of the receptor may be active in the roach retina and these may be involved in differential control of visual sensitivity.

Communication intervention for individuals with dual sensory and intellectual impairments.

For individuals with dual sensory and intellectual impairments, the lack of effective communication skills places severe limitations on their learning potential and sense of belonging. Educational intervention for this population has recognized the critical need to develop communication skills, yet the complexity of the individual's needs continues to plague progress. Unique needs and situations of individuals in this category, coupled with extremely limited numbers of trained and experienced professionals, challenge the field to develop creative means of addressing this vital issue. Documented case studies provide the field with examples of potential options to employ when addressing the communicative needs of individuals with dual sensory and intellectual impairments. However, these case studies provide partial communication systems that address partial communicative needs. Providing individuals having severe sensory, intellectual, and other disabilities with a true language that can meet all communication needs, both receptive and expressive, is still beyond our grasp. Future efforts with this focus will need to combine early intervention, creative technology, and principles of normalization in order to be successful.

Ten-year follow up comparing anterior and posterior chamber intraocular lens implants.

Results achieved in eyes following a single surgeon's first 200 procedures consisting of intracapsular cataract extraction and implantation of a Choyce-Tennant anterior chamber lens (AC-IOL) (1977 to 1980) are compared with those achieved following the same surgeon's first 200 procedures consisting of extracapsular cataract extraction and implantation of a posterior chamber intraocular lens (PC-IOL) (1980 to 1982). For the AC-IOL eyes, follow up ranged from 11.1 to 14.2 years (mean, 12.0 years); for the PC-IOL eyes, from 9.5 to 10.8 years (mean, 10.0 years). The AC-IOL eyes had many early problems: pupillary block (7%), iritis (15%), and secondary glaucoma (8%). Four percent developed corneal edema, 1.5% vitritis, and 2% localized iris holes under the lens. Ectropion uveae appeared in 8.5%, indicating some ongoing inflammation. One and one-half percent of these lenses were removed or exchanged. Seventy-nine and one-half percent of these eyes had 20/40 or better vision at 10 years; 4% had visual loss along with corneal edema or vitritis, apparently related to the AC-IOLs. The PC-IOL eyes had comparatively few lens-related complications: 1% corneal edema, 1.5% iritis, 1% vitritis, and 2% secondary glaucoma. Some localized trapping of the pupil occurred in 8%. One percent of the PC-IOLs were removed, and 1% decentered, requiring McCannell sutures. Final visual acuity at 10 years was 20/40 or better in 77.5%. None of the PC-IOL eyes had decreased vision related to the lens implant.

Cat retinal ganglion cells show transient responses to acetylcholine and sustained responses to L-glutamate.

The neuropharmacological basis for the different receptive field properties of cat retinal ganglion cells was investigated using whole cell voltage-clamp recordings from acutely dissociated adult tissue. Subclasses of physiologically characterised ganglion cells were determined on the basis of (i) their soma diameters and (ii) their projection to central visual nuclei (identified by microinjection of fluorescent dyes into the lateral geniculate and/or superior colliculus). The sensitivities of all categories of ganglion cells, prepared from peripheral retina were found to be similar for gamma-amino butyric acid, glycine, acetylcholine and glutamate. The kinetics of desensitisation differed among receptor subtypes, revealing possible physiologically significant molecular specialisations that could be involved in shaping synaptic transmission.

Biconvex intraocular lenses and Nd:YAG capsulotomy: experimental comparison of surface damage with different poly(methyl methacrylate) formulations.

Biconvex posterior chamber lenses have optical advantages and decrease the risk of capsular opacification, but they are more likely to be pitted during ND:YAG capsulotomy because of apposition of the lens to the capsule. This study reports the likelihood of surface damage to different formulations of poly(methyl methacrylate) at the energy levels required to open posterior capsules. Molded lenses are more easily damaged than higher molecular weight lathe-cut materials (P less than .01), as expected. However, by keeping energy output low, even injection-molded lenses showed minimal damage, with mean pit size 39 +/- 39 microns at 1 mJ. By using a converging contact lens, low power, and keeping the focus behind the capsule, damage to all materials tested should be clinically insignificant.

Quantitative analysis of cone photoreceptor-horizontal cell connectivity patterns in the retina of a cyprinid fish: electron microscopy of functionally identified and HRP-labelled horizontal cells.

Horizontal cells generating photopic luminosity and biphasic/chromaticity-type S-potentials were identified and intracellularly labelled with horseradish peroxidase in the retina of the roach. The synaptic connectivity patterns of the horizontal cell dendrites within pedicles of different spectral types of cone were then quantitatively studied by electron microscopy. Luminosity-type responses were generated by H1-like horizontal cells contacting similar numbers of red- and green-sensitive cones and very few blue-sensitive cones. Most dendritic contacts were lateral to synaptic ribbons. Central contacts with ribbons were made almost exclusively within red-sensitive cone pedicles. Biphasic/chromaticity-type S-potentials were generated by H2-like horizontal cells. The dendrites of the latter contacted green- and blue-sensitive cones, both at central and lateral sites at synaptic ribbons. An attempt was made to correlate cone ribbon connectivity patterns and spectral characteristics of the horizontal cells according to several hypotheses, some proposed in earlier studies.

Amacrine cells in the retina of a cyprinid fish: functional characterization and intracellular labelling with horseradish peroxidase.

Forty amacrine cells in retinae of a cyprinid fish, the roach, were intracellularly labelled with horseradish peroxidase following electrophysiological identification as sustained depolarizing, sustained hyperpolarizing or transient units. Labelled cells were analysed by light microscopy and compared with a catalogue of amacrine cells established in a previous Golgi study on the same species. About 30% of the cell types characterized by the Golgi method were encountered in the present study. When intracellularly labelled cells were differentiated on the basis of their dendritic organization in the plane of the retina, a given electrophysiological response pattern was found to be generated by different morphological types, and vice versa. However, examination of the ramification patterns of the dendrites within the inner plexiform layer (i.e. in the radial dimension of the retina), showed that this morphological parameter of a given amacrine cell could be correlated with its light-evoked response. Several amacrine cell types were found to possess special distal dendrites which arose from the main dendritic branches and extended well over a mm in the retina. Distal dendrites were oriented tangentially with respect to the optic nerve papilla, but did not appear to be involved in any synaptic connectivity. It is concluded that the Golgi-based classification is a valuable tool for identifying intracellularly labelled amacrine cells. However, although the correlation between layering of dendrites in the inner plexiform layer and electrophysiology was generally good, additional physiological parameters would be required to determine whether more extensive parallels exist between structural and functional characteristics of amacrine cells. Alternatively, the considerable morphological diversity of amacrine cells may be of limited physiological significance.

A horizontal cell selectively contacts blue-sensitive cones in cyprinid fish retina: intracellular staining with horseradish peroxidase.

A horizontal cell selectively contacting blue-sensitive cones has been intracellularly stained with horseradish peroxidase in the retina of a cyprinid fish, the roach. The light microscopical morphology of the cell belonged to the H3 category of horizontal cells found in cyprinid fish retinae. In response to spectral stimuli, the cell generated chromaticity-type S-potentials that were hyperpolarizing to blue and depolarizing to yellow-orange. A red-sensitive hyperpolarizing component was absent possibly because of suppression of the negative feedback pathway between luminosity-type (H1) horizontal cells and green-sensitive cones.