Report of the 3rd Havemeyer workshop on allergic diseases of the Horse, Hólar, Iceland, June 2007.

Allergic diseases occur in most mammals, although some species such as humans, dogs and horses seem to be more prone to develop allergies than others. In horses, insect bite hypersensitivity (IBH), an allergic dermatitis caused by bites of midges, and recurrent airway obstruction (RAO), a hyperreactivity to stable born dust and allergens, are the two most prevalent allergic diseases. Allergic diseases involve the interaction of three major factors: (i) genetic constitution, (ii) exposure to allergens, and (iii) a dysregulation of the immune response determined by (i) and (ii). However, other environmental factors such as infectious diseases, contact with endotoxin and degree of infestation with endoparasites have been shown to influence the prevalence of allergic diseases in humans. How these factors may impact upon allergic disease in the horse is unknown at this time. The 3rd workshop on Allergic Diseases of the Horse, with major sponsorship from the Havemeyer Foundation, was held in Hólar, Iceland, in June 2007 and focussed on immunological and genetic aspects of IBH and RAO. This particular venue was chosen because of the prevalence of IBH in exported Icelandic horses. The incidence of IBH is significantly different between Icelandic horses born in Europe or North America and those born in Iceland and exported as adults. Although the genetic factors and allergens are the same, exported adult horses show a greater incidence of IBH. This suggests that environmental or epigenetic factors may contribute to this response. This report summarizes the present state of knowledge and summarizes important issues discussed at the workshop.

Infection of cultured rat myotubes and neurons from the spinal cord by rabies virus.

Rabies virus multiplication was investigated in cultured primary rat myotubes and neurons. The susceptibility of these two cell types to fixed rabies challenge virus strain (CVS) was monitored by fluorescence and virus titration. Differentiated rat myotubes were susceptible to rabies virus infection, and showed an increasing accumulation of viral material from day one to day four. However, these cells did not release infective viral particles, nor did they accumulate infectious virions in the cytoplasm. In contrast, infected neurons released large amounts of infectious particles. Electron microscopy observation of infected myotubes showed minor alterations and the presence of typical viral inclusions in the cytoplasm without mature virions assembling viral membranes. Competition binding experiments show that alpha-bungarotoxin inhibits rabies virus infection from 10(-5) to 10(-7) M, whereas lower toxin concentrations failed to have any effect. These data do not confirm the hypothesis of a fixed rabies virus amplification step at the site of the viral entry. On the other hand, the high susceptibility of peripheral neurons to rabies virus infection is an argument for the direct uptake of virions by these cells. The restrictive viral multiplication in the myotubes is an alternative explanation for the local persistence of rabies virus at the site of inoculation.

Axonal secretion of Reelin by Cajal-Retzius cells: evidence from comparison of normal and Reln(Orl) mutant mice.

A novel secretory pathway has been identified in the study of mice homozygous for the Reln(Orl) mutation, a line characterised by the defective secretion of the large extracellular matrix glycoprotein Reelin. By using both light and electron microscopy, immunohistochemical studies for Reelin in these mutants identified morphological changes in their Cajal-Retzius cells (CR cells). The CR cells of the mutant displayed the characteristic features of bipolar, tangentially elongated neurons with a dendritic proximal pole and an axonal cone at the opposite end of the soma. At either pole, cisterns of prominent rough endoplasmic reticulum (RER) were found to be rich in Reelin. However, the Reelin-positive RER cisterns of the axonal cones were hugely dilated in homozygous Reln(Orl) mice as compared with their wild type counterparts. CR cell axons displayed beads throughout their length, each contained a smooth spheroidal cistern filled with Reelin-immunoreactive fibrillar material, and were increased in number and size in Reln(Orl) mice. RER phenotype was rescued in the Reln(Alb2) mice, a mutation in which no Reelin protein is produced. We propose that the RER dilations viewed in the Reln(Orl) mutation are due to the accumulation of the defective Reelin protein, and the large axonal beads in Reln(Orl) mice reflect the accumulation of truncated Reelin as the result of defects in its secretion. These observations point to an original, hitherto unrecognised, mechanism of secretion by bulk transport in smooth cisterns from the axonal cone into the axon, followed by secretion in the cortical marginal zone from the axonal cisterns that we have named axonal reelin reservoirs.

A simple and rapid flow cytometric method for routine assessment of baker's yeast uptake by human polymorphonuclear leukocytes.

A new method for measuring uptake of baker's yeast (BY) by human polymorphonuclear leukocytes (PMN) using flow cytometry is described. The method correlates excellently with the visual method, is reproducible and provides a means for investigating the early phases of the phagocytic process as well as the phagocytic capacity of PMN. This quick and accurate method allows the counting of large numbers of cells, and monitoring of the process of particle uptake and has a considerable potential in the routine assessment of polymorph function in various clinical situations.

Cajal-Retzius cell ontogenesis and death in mouse brain visualized with horseradish peroxidase and electron microscopy.

The ontogenetic development of Cajal-Retzius cells was studied in mouse by local application of horseradish peroxidase over the developing neocortex, revelation with 3,3'-diaminobenzidene and examination from horizontal thick sections. Cajal-Retzius cells were completely stained in Golgi-like fashion. The Cajal-Retzius cells were seen to be elongated spindle-shaped bipolar neurons with their main processes horizontally oriented. They were exclusively located in the first cortical layer and were connected to the cortex surface by the numerous vertical appendages. Except for these appendages, the Cajal-Retzius cells were two-dimensional, with an immature structure at their tips resembling a growth cone. Cajal-Retzius cell dendrites were up to 400-microns-long and reached their maximal length prenatally. Their axon and its collaterals were very fine and sometimes measured several millimetres. It followed a random but planar trajectory confined to the first layer. Healthy Cajal-Retzius bearing growth cones were seen until one week after birth when signs of Cajal-Retzius cell degeneration began to occur and intensified in the days that followed. Rough endoplasmic reticulum and Golgi complex swelling along with a progressive darkening of the Cajal-Retzius cells were revealed by electron microscopy, strongly suggesting that most Cajal-Retzius cells disappear from the first cortical layer. Usually neuronal death is the result of cell deafferentation following synapse retraction; however, this effect does not seem to apply to Cajal-Retzius cells engaged in the process of death since normal synaptic junctions were seen on them. No signs of the morphological transformation of Cajal-Retzius cells into persisting horizontal first layer cells were observed. The concept of dual origin of neocortex is discussed in light of the similar fate of Cajal-Retzius cells and subplate neurons which both are transient neurons.

Immunocytochemical localization of the intermediate filament protein peripherin in adult mouse adrenal chromaffin cells in culture.

Peripherin is the main intermediate filament protein in sympathetic neurons. Immunoreactivity to peripherin was studied in mouse adrenal chromaffin cells after 6 days in culture, and compared to immunoreactivity to tyrosine hydroxylase used as a general marker of chromaffin cells in culture. Most of the cells immunoreactive to tyrosine hydroxylase were rounded, with a glandular phenotype and a few of them had processes. The cells reactive to peripherin only constituted a small proportion of the chromaffin cells (2%), and most of them sent out processes. However, not all the cells with processes were reactive for peripherin. These results did not change in the presence of nerve growth factor. The discussion focuses on the significance of the sub-population of cells reactive to peripherin. We suggest that these cells resemble the small granule chromaffin cells, regarded as an intermediate cell type between glandular cells and neurons. The cells that expressed peripherin here are compared to those selected to form the PC12 clone. The presence of peripherin in only a few of the cells sending out neurite-like processes is discussed in relation to the expression of other neurofilament proteins in developing cells and to the influence of non-chromaffin cells.

Neurobehavioral evaluation of Reln-rl-orl mutant mice and correlations with cytochrome oxidase activity.

The Reln-rl-orl mutation is characterized by a marked deficit in cerebellar granule cell and Purkinje cell number as well as ectopias in cerebellum, hippocampus, and neocortex. By comparison to Balb/c controls, Reln-rl-orl mutants did not alternate spontaneously in a T-maze and were deficient for visuomotor guidance in a water maze. Despite cerebellar ataxia and motor coordination impairments on stationary beam, coat-hanger, and rotorod tests, the horizontal motor activity of Reln-rl-orl mutants was not reduced in an open-field. The elevated cytochrome oxidase (CO) activity in Purkinje cells and the reduced CO activity in the roof nuclei (interpositus and dentate) of the mutants were associated with poor performance on the small stationary beam. In addition, deficient CO activity of the granular layer of the motor cortex was associated with shorter latencies before falling from the larger stationary beam and a lower number of rears in the open-field. Conversely, elevated CO activity in the polymorphic layer of primary somatosensory cortex was congruent with higher latencies before falling from the same apparatus, indicating functional compensation.

Ontogenesis of NADPH-diaphorase neurons in the mouse forebrain.

Demonstration of NADPH diaphorase (NADPH-d) activity in the nervous system has recently gained considerable interest since it has been shown that this enzyme is a nitric oxide synthase (NOS). Therefore, histochemical staining of NADPH-d activity provides a specific labelling of neurons that use nitric oxide (NO). In this work, spatiotemporal distribution of NADPH-d neurons has been determined during forebrain ontogenesis. NADPH-d neurons first appeared between embryonic days 15 and 16 and were confined to the fronto-lateral aspect of the incipient corpus striatum and cortical subplate but were not present in the cortical plate. Until birth, NADPH-d neurons differentiated progressively in cortical subplate and striatum in rostro-caudal and latero-medial directions. For both regions, the adult cortical pattern was established during the first postnatal week. The pattern of genesis of NADPH-d neurons might be related to the spatiotemporal ontogenesis of catecholaminergic afferents to the forebrain described previously in the literature.

Decrease of tailed, asymmetric 16S acetylcholinesterase in rat superior cervical ganglion neurons in vitro after potassium depolarization: partial antagonist action of a calcium-channel blocker.

In primary cell cultures of rat superior cervical ganglia the tailed, asymmetric 16S molecular form of acetylcholinesterase (AChE) is preferentially decreased after potassium depolarization. This effect is not related to noradrenergic differentiation elicited by high potassium concentration. Moreover there is a partial antagonist action of a calcium-channel blocker, methoxyverapamil (D-600). These results suggest that membrane electrical activity exerts a regulatory control on AChE, and Ca2+ fluxes play an important role in regulatory events of AChE biosynthesis.

Regional brain variations of cytochrome oxidase activity in Relnrl-orl mutant mice.

Cell malpositioning has been described in laminated structures of the spontaneous mutation, reeler, including the cerebellum, the hippocampus, and the neocortex. Despite the ectopic positions of different neuronal populations, the specificity of synaptic connections is maintained. The metabolic consequences of this form of neuropathology were examined in Reln(rl) mutant mice by quantitative measures of cytochrome oxidase (CO) activity, a mitochondrial enzyme essential for oxidative metabolism in neurons. Despite severe tissue disorganization but in line with the intact synaptic organization, the reeler mutation did not affect global metabolic activity of the laminated structures of the brain. CO activity, however, was altered in specific subregions of the cerebellum, hippocampus, and neocortex, as well as in septum and various brainstem (medial pontine, paramedial reticular, paragigantocellular reticular) regions anatomically related to these structures, attesting to large functional alterations in Reln(rl-orl) brain. Metabolic activity variations were also detected in the ventral tegmental area and ventral neostriatum of the mesolimbic dopaminergic pathway. The results are discussed and compared to the regional CO variations found in other ataxic mice, in regard to the structural defects, the integrity of the connections, and the mutation-specific effects.

[The perforation organ of Thais lapillus L. (Gasteropodes, Prosobranches). Optical and electron microscopic study]. / L'organe de perforation de Thais lapillus L. (Gastéropodes, Prosobranche). Etude en microscopie optique et électronique

The histologic and cytologic structure of the boring organ of Thais lapillus has been studied with both light and electron microscopy. The organ evaginates from the foot and has a dome-like form. It has a spongy center which contain nerves, muscles and blood vessels, these originate in the foot and continue into the boring organ via a short stalk. The organ is invested externally by a continuous simple epithelium constituted of extremely elongated cells (150 to 300 mu). There are three radial zones from the basal to the apical aspect of the cells : basally the cells are overlapping, here are located the nuclei, the Golgi apparatus, the rough endoplasmic reticulum and secretory granules; organelles which in their character suggest a secretory function for these cells. In the middle zone cells are separated into "groups" by large spaces occupied by blood sinuses; at this level are located numerous elongated mitochondria with closely apposed, angulated cristae. In addition there are microtubules arranged parallel to the main cellular axis, whose role may be to facilitate rapid transit of secretory granules from the basal to the apical zone of the epithelium. In the apical zone the cells expand in tightly interdigitating folds, having a height of 2 mu, which are joined together by junctions of various types. This zone is the only junctional area between the epithelial cells of this expanding and contracting boring organ. A network of microfilaments which penetrates the cellular folds at this level, probably plays an important role in the invagination of the organ. Finally at the apex, the epithelial cells have a high "brush border" of long microvilli whose plasmalemma is coated internally by particulate subunits of 30 A. This peculiarity may be an other fact to support the hypothesis that hydrogen ions are emited by these cells, by the action of carbonic anhydrase located perhaps in these subunits.

[Identification of Cajal-Retzius cells during neocortical ontogenesis in mice with fluorescent carbocyanine]. / Identification des cellules de Cajal-Retzius durant l'ontogenèse du néocortex de la souris à l'aide d'une carbocyanine fluorescente.

Cajal-Retzius cells, which are present transiently in the first layer of the mammalian neocortex, have been revealed in the mouse by DiI. This lipophilic fluorescent dye, locally applied over the cortex after formaldehyde fixation, allowed the global view of cortical cells. During ontogenesis, Cajal-Retzius cells retained their initial characteristic bipolar shape and orientation parallel to the meningeal surface. The bright fluorescent light emitted by this dye allowed visualization of the labelled cells by "microtomoscopy" using a confocal scanning laser microscope and analysis of the detailed aspect of these neurons and of their connections.

An in vivo and in vitro study of rabies virus infection of the rat superior cervical ganglia.

In the attempt to develop a homogeneous neuronal model to study rabies pathogenesis in vivo and in vitro, the superior cervical ganglia (SCG) were chosen because of their unique features. In vivo infection of the SCG was attempted by inoculation of fixed rabies virus into the anterior eye chamber. However, viral by this route as well as intracerebrally failed to infect this neuronal organ in adult rats whereas the infection was poorly efficient in 24 hours newborn rats. Dissociated cell cultures from the rat embryo SCG were infected in vitro and examined for the presence of rabies specific antigen and release of virus particles in the supernatant. Despite the presence of rabies nucleoprotein in the cytoplasm and the presence of typical Negri bodies, neurons from the rat SCG produced few particles as observed by electron microscopy and no increase in virus yields could be detected by titration of viral infectivity during the infectious cycle. Our observations indicate that although rabies virus is neurotropic as shown in previous studies, all neuronal tissues are not equally susceptible to this viral infection. The resistance of the SCG to rabies virus infection in vivo does not seems to be a lack of accessibility of this organ to infection since other authors had shown that it could be infected by herpes virus. Both in vitro and in vivo experiments show that although neurons from the SCG are susceptible to rabies virus infection, infected cells do not produce rabies infectious virions efficiently.

Mechanism of rabies virus entry into CER cells.

The early steps of rabies virus (CVS) infection in vitro were studied in chicken embryo-related (CER) cells. The infection was monitored by looking for specific intracytoplasmic viral inclusions using anti-rabies fluorescein isothiocyanate at 24 h after the addition of virus. The attachment of rabies virus to CER cells was shown to be inhibited by pretreatment of the cells with neuraminidase. These cells recovered their susceptibility to rabies virus infection 6 h after removal of the enzyme. Treatment of CER cells with neuraminidase after the viral attachment step did not inhibit infection. The subsequent delivery of infectious virions into acid prelysosomal vacuoles or lysosomes was studied using lysosomotropic agents. Ammonium chloride and chloroquine were used to prevent the virus fusion step thus preventing infection. Both drugs were shown to inhibit the early steps of infection, NH4Cl having a much earlier effect than chloroquine. The two drugs had no effect on the attachment step nor did NH4Cl inhibit virus multiplication. The use of metabolic inhibitors (2-deoxy-D-glucose and sodium azide) shows that the entry of rabies virus into CER cells does not require the involvement of cellular energy processes. In electron microscopy studies, the presence of rabies virus particles was detected in coated pits and coated vesicles as well as in uncoated vesicles, and later in lysosomes. These data indicate that the mechanism by which rabies virus enters CER cells is probably through adsorptive endocytosis and does not require the participation of cellular metabolic active processes.