Balance of Treg vs. T-helper cells in the transition from symptomless to lesional psoriatic skin.

BACKGROUND: In the pathogenesis of psoriasis, proinflammatory T cells are strongly involved in the inflammatory process, where regulatory T-cell (Treg) function is impaired. OBJECTIVES: As effective Treg function is associated with a numerical balance between Treg and effector T cells, we wondered whether Treg/T-helper cell ratios may be associated with certain stages of the inflammatory process. We opted for the margin zone model as a dynamic approach. METHODS: From nine patients with chronic plaque psoriasis, 3-mm punch biopsies were obtained from the centre and margin of the lesion, perilesional skin and distant uninvolved skin. Skin biopsies of 10 healthy volunteers were included as a control. Samples were analysed using immunohistochemistry and immunofluorescence. RESULTS: In the transition from symptomless to lesional skin, a significant increase of CD3+, CD4+ and Foxp3+ cells was found. In seven of nine patients the ratio of Treg (Foxp3+) vs. CD4+ T cells was higher in the distant uninvolved skin than in the perilesional and lesional skin. Interestingly, the Foxp3/CD4 ratio in the distant uninvolved skin was even higher than in the skin of healthy controls. Notably, we found that most of the interleukin (IL)-17 expression was not related to CD4+ cells, but to mast cells. CONCLUSIONS: The relatively high Foxp3/CD4 ratio in symptomless skin of patients with psoriasis suggests an active immune controlling mechanism distant from the psoriatic plaque. In the margin and centre of the plaque the ratio appears skewed towards effector cells associated with inflammation. IL-17, an important driver of the psoriatic process, is mostly related to mast cells, and only sporadically to T cells.

Reflectance confocal microscopy: an effective tool for monitoring ultraviolet B phototherapy in psoriasis.

BACKGROUND: In vivo reflectance confocal microscopy (RCM) is a novel, noninvasive imaging technique which enables imaging of skin at a cellular resolution comparable to conventional microscopy. OBJECTIVES: We performed a pilot study to evaluate RCM as a noninvasive tool for monitoring ultraviolet (UV) B phototherapy in psoriasis. METHODS: In six patients with psoriasis, lesional and nonlesional skin was selected for RCM imaging using a standardized protocol. Well-known histological features of psoriasis were visualized: parakeratosis, acanthosis, agranulosis, papillomatosis, presence of epidermal inflammatory cells, increased number of papillary capillaries and increased capillary blood flow. RCM imaging was performed before the first irradiation with UVB phototherapy, after nine irradiations, at clearance and 12 weeks after clearance. In four patients, 4-mm punch biopsies were obtained and stained with haematoxylin-eosin. Additionally, immunohistochemical staining was performed with monoclonal antibodies specific for CD31, CD3, filaggrin, K16, Ki67 and CD1a for correlation to RCM images. RESULTS: There was a high correlation between clinical, RCM and histological features. Normalization of RCM and histological features corresponded highly to clinical improvement of psoriasis. CONCLUSIONS: This study is the first to establish the use of RCM as an effective tool for noninvasive monitoring of UVB phototherapy in patients with psoriasis. Potentially, RCM could be used in many other skin diseases for monitoring therapeutic response on a cellular level in a clinical or research setting.

Early development of descending supraspinal pathways: a tracing study in fixed and isolated rat embryos.

Early brainstem-spinal cord projections were studied in the rat using the carbocyanine dye DiI in fixed embryos and biotinylated dextran amine (BDA) in an isolated embryonic brain-spinal cord preparation. A system of staging embryos was used that allows direct comparison with data in other mammals. With both techniques it was shown that in embryos of at least 12 days of age (E12), i.e., at the time of closure of the posterior neuropore, already a variety of brainstem centers innervate the spinal cord. In the interstitial nucleus of the fasciculus longitudinalis medialis and various parts of the reticular formation - mesencephalic, pontine as well as medullary - DiI or BDA labelled neurons were observed. Mainly large immature, bipolar neurons were labeled. In later stages (E13, E14) the number of labeled neurons increased and more mature, multipolar cells were found. Labeled neurons were also observed in the vestibular nuclear complex and in the medullary raphe. Just below the cerebellum a conspicuous small group of neurons was found labeled in a position reminiscent of the locus coeruleus. Comparison with available data on the time of neuron origin of brainstem neurons suggests that interstitiospinal and reticulospinal neurons start projecting spinalwards shortly after they are generated. The earliest brainstem projections to the spinal cord all pass via the fasciculus longitudinalis medialis.

The use of tracers in explants of the developing reticular formation and spinal cord of Xenopus laevis.

The development of reticulospinal projections to the lumbar spinal cord is studied by using a collagen co-culture system. Outgrowing reticulospinal fibers seem to grow out in a straightforward direction, without a specific preference for the lumbar or tail spinal cord. Carbocyanine tracers such as DiI, DiO and DiA are used to label the outgrowing fibers or their parent cell bodies. In double labeling studies contacts of outgrowing reticulospinal fibers with lumbar motoneurons are analyzed. The advances of a confocal laser scanning microscope for such studies are illustrated.

The dorsal column-medial lemniscal projection of anuran amphibians.

The efferent connections of the dorsal column nucleus (DCN) of the anuran amphibians Rana ridibunda and Xenopus laevis have been studied by means of bidirectionally transported tracers. Efferent projections from the DCN innervate the spinal cord, tegmentum of the brain stem, cerebellum, torus semicircularis and thalamus. The pattern of connectivity of the anuran DCN is largely comparable to that of amniotic vertebrates although some peculiarities are found.

Early development of dorsal column-medial lemniscal projections in the clawed toad, Xenopus laevis.

In Xenopus laevis fluorescent dextran amines were applied to study the development of the dorsal column-medial lemniscal projection: rhodamine dextran amine was applied at the mesodiencephalic border to retrogradely label the cells of origin of the medial lemniscus in the dorsal column nucleus (DCN); fluorescein dextran amine to the spinal cord to anterogradely label the primary afferent projections to the DCN. The first mesodiencephalic projections were found at stage 51, i.e. almost immediately after spinal afferent fibers had reached the DCN.

Early development of rubrospinal and cerebellorubral projections in Xenopus laevis.

In Xenopus laevis HRP was applied at the spinomedullary border at various stages of development. In these experiments labeled rubrospinal neurons were observed from stage 48 on. HRP applications to the mesencephalic tegmentum showed, from stage 49 on, retrogradely labeled neurons in the cerebellar nucleus, particularly contralaterally. These data suggest that anuran cerebellorubral projections arise early, well before the rubrospinal innervation of the spinal cord is complete.

Observations on the development of ascending spinal pathways in the clawed toad, Xenopus laevis.

The development of ascending spinal pathways has been studied in the clawed toad, Xenopus laevis. From stage 35 (hatching) on, HRP was applied at the spinomedullary border or to the area of the developing dorsal column nucleus, to analyze the development of ascending spinal pathways to the brain stem, and the onset and development of spinal projections to the dorsal column nucleus, respectively. Several populations of spinal neurons with ascending projections at least as far as the spinomedullary border were successively labeled. In early stages ascending spinal projections arise from Rohon-Beard cells and ascending interneuron populations located at the margin of the gray and white matter, i.e., marginal neurons. The ascending interneuron populations could be characterized as dorsolateral commissural and commissural interneurons projecting contralaterally, and as ipsilaterally projecting ascending interneurons and distinguished by Roberts and co-workers. Such a subdivision could be made until about stage 57. Then these ascending and commissural interneuron populations become intermingled with other populations of ascending tract neurons. Rohon-Beard cells could be labeled, more or less shrunken, until stage 55. Around stage 48 (at the time of the appearance of the limb buds) spinal ganglion cells could be labeled from the spinomedullary border and the developing dorsal column nucleus. At stage 48 such ascending primary spinal afferents were found to arise only from non-limb-bud-innervating dorsal root ganglia. Gradually also the limb-bud-innervating ganglia give rise to ascending collaterals, so that by stage 53 all spinal ganglia send ascending collaterals to the brain stem. The number of cells of origin of secondary spinal afferents to the brain stem increases during development, and their distribution becomes more extensive. Particularly impressive is a large population of neurons in the dorsal horn projecting ipsilaterally to the dorsal column nucleus. Part of the latter population represents non-primary spinal afferents to the dorsal column nucleus.

Development of olivocerebellar fibers in the clawed toad, Xenopus laevis: a light and electron microscopical HRP study.

An anterograde tracer study was undertaken to provide a light- and electron microscopical description of climbing fiber development in the clawed toad, Xenopus laevis, ranging from premetamorphic stages to the adult state. The inferior olive was unilaterally labeled with horseradish peroxidase and the contralateral climbing fiber morphology investigated. At early stages of development, only undifferentiated fibers were observed in the rostral alar plate. At later stages, these fibers form large varicosities, which contact presumed cerebellar Purkinje cells. Finger-like protrusions arising from the Purkinje cell somata penetrate the climbing fiber varicosities and form synaptic specializations at these contact sites. In older tadpoles, a large variety of climbing fiber morphologies was found displaying a mediolateral gradient. At dorsolateral cerebellar areas long and straight climbing fibers follow the Purkinje cell primary dendrites. However, in ventromedial areas pericellular baskets or nests were found on presumed Purkinje cell somata. These pericellular nests were found throughout development but were not observed in adult animals. Both pericellular nests and real climbing fibers make synaptic contacts on spiny protrusions of the Purkinje cell's somatic or dendritic surface. In several cases, labeled as well as unlabeled climbing fiber profiles were observed on the same Purkinje cell, indicating multiple, convergent innervation. Also, divergent Purkinje cell innervation was found. In conclusion, this study shows that anuran climbing fiber development encompasses stages and processes similar to those observed in mammals. The only principal difference with climbing fiber development in mammals is the low degree of synchrony observed in anurans.

Development of spinocerebellar afferents in the clawed toad, Xenopus laevis.

The development of spinocerebellar projections in the clawed toad, Xenopus laevis, was studied with horseradish peroxidase as an anterograde and retrograde tracer. Early in development cells of origin of spinocerebellar projections were found, contralaterally, in or close to the medial motor column. In older tadpoles ipsilaterally projecting spinal neurons were also labeled from the cerebellum. These are virtually indistinguishable from the large primary motoneurons that occupy a very similar position in the spinal cord. Most of the labeled spinal cells were found in the thoracic spinal cord; they lie halfway between the brachial and lumbar secondary motor columns. Surprisingly, no primary spinocerebellar projection arising from dorsal root spinal ganglion cells could be demonstrated in X. laevis tadpoles and adult toads. Therefore, fibers in the cerebellum that were labeled anterogradely from the spinal cord can be expected to originate exclusively from the secondary spinocerebellar tract cells. These fibers appear to cross the cerebellum in or at the border of the granular layer. The present data suggest that in X. laevis early in the development of the cerebellum a distinct secondary spinocerebellar projection is already present, originating in neurons that can be compared with the "spinal border cells" in mammals. The relative sparseness of this secondary spinocerebellar projection and the apparent absence of primary spinocerebellar afferents probably indicate that spinocerebellar pathways are only of minor importance in X. laevis. The possibility remains, however, that the expansion of the secondary spinocerebellar pathway only starts when metamorphosis has been completed.

Brain stem afferents to the anterior dorsal ventricular ridge in a lizard (Varanus exanthematicus).

The anterior dorsal ventricular ridge (ADVR), a large intraventricular protrusion in the reptilian forebrain, receives information from many different sensory modalities and in turn, projects massively onto the striatum. The ADVR possesses functional similarities to the mammalian isocortex and may perform complex sensory integrations. The ADVR in lizards is composed of three longitudinal zones which receive visual, somatosensory and acustic information, respectively. These projections are relayed via thalamic nuclei. Previous retrograde tracer studies also suggested brain stem projections to the ADVR arising in the midbrain reticular formation and in certain monoaminergic brain stem nuclei (substantia nigra, locus coeruleus and nucleus raphes superior). In the present study the powerful retrograde fluorescent tracer 'Fast Blue' was applied as a slow-release gel to the ADVR of the savanna monitor lizard, Varanus exanthematicus. Thalamic projections were confirmed and various direct brain stem projections to the ADVR were demonstrated. Brain stem afferents to the ADVR were found from the laminar nucleus of the torus semicircularis (possibly comparable to the mammalian periaqueductal gray), from the midbrain reticular formation, from the substantia nigra (pars compacta and reticulata) and the adjacent ventral tegmental area, from the nucleus raphes superior, from the locus coeruleus, from the parabrachial region, from the nucleus of the lateral lemniscus and even from the most caudal part of the brain stem (a few neurons in the nucleus of the solitary tract and lateral reticular formation, possibly comparable to the mammalian A2 and A1 groups, respectively). These data strongly suggest direct ADVR projections from the parabrachial region (related to visceral and taste information) as well as distinct catecholaminergic (presumably dopaminergic: substantia nigra, ventral tegmental area and, noradrenergic: locus coeruleus, respectively) and serotonergic projections (nucleus raphes superior).

A possible pain control system in a non-mammalian vertebrate (a lizard, Gekko gecko).

In a lizard (Gekko gecko) the anterograde tracer PHA-L was microiontophoretically applied to the predominantly serotonergic nucleus raphes inferior. Extensive spinal projections from the rostral magnocellular part of this nucleus were demonstrated to the superficial layers of the dorsal horn and to the intermediate zone, more sparsely to the ventral horn. But, in addition, retrogradely labeled neurons were found in and just below a periventricular cell group in tegmentum mesencephali, i.e. the laminar nucleus of the torus semicircularis, a cell group which receives spinal afferents and projects to the spinal cord as the mammalian periaqueductal gray. These data suggest the presence of a three-tiered pain control system in a lizard composed of projections from the laminar nucleus of the torus semicircularis to the rostral part of the inferior raphe nucleus which in its turn projects to the superficial layers of the dorsal horn of the spinal cord.

The distribution of motoneurons supplying hind limb muscles in the clawed toad, Xenopus laevis.

The distribution of motoneurons in the lumbar spinal cord (spinal segments 8-10) of the clawed toad, Xenopus laevis, was studied with the horseradish peroxidase technique. In a total of 13 different hind limb muscles this tracer was applied in a slow-release gel. Motoneurons innervating a particular hind limb muscle were clustered in longitudinally arranged motor pools. Motor pools of different muscles did show considerable overlap both in the rostrocaudal and transverse plane. But, the various motor pools clearly show a somatotopic organization of motoneurons even in such a condensed lumbar spinal cord as in Xenopus laevis. Motoneurons innervating more distally positioned muscles are generally found in more caudal segments, while proximal muscles (with the exception of the m. adductor magnus) are supplied by motoneurons more or less throughout the lumbar enlargement. Flexor muscles usually are innervated by motoneurons situated ventrolaterally in the ventral horn, extensor muscles by dorsomedially found motoneurons. This pattern is particularly apparent for proximal (thigh) muscles, less so for more distal (shank and foot) muscles. The present data are in keeping with those obtained with the retrograde cell degeneration technique in ranid frogs and are consistent with observations in other tetrapods, although a more clear separation of motor pools is evident in "higher" vertebrates such as birds and mammals.

Collateralization of descending pathways from the brainstem to the spinal cord in a lizard, Varanus exanthematicus.

With the multiple fluorescent retrograde tracer technique, the collateralization in the spinal cord of descending supraspinal pathways was studied in a lizard, Varanus exanthematicus. Fast Blue (FB) gels were implanted unilaterally in the spinal gray matter of the cervical enlargement and Nuclear Yellow (NY) gels were implanted ipsilaterally in two series of experiments in all spinal funiculi of the lumbar enlargement or in midthoracic spinal segments, respectively. All brainstem nuclei known to project to the spinal cord in reptiles were found to give rise to branching axons that may influence widely separate levels of the spinal cord. The number of double-labeled FB-NY cells varied in these brainstem nuclei from none to half the number of neurons projecting to the cervical enlargement. Highly collateralizing projections (expressed as the percentage of double-labeled neurons, DL) were found to arise from the nucleus raphes inferior, the contralateral nucleus reticularis superior pars lateralis, the contralateral nuclei vestibulares ventromedialis and descendens, and the ipsilateral nucleus reticularis inferior pars ventralis. A lower percentage of DL neurons was noted for the contralateral nucleus ruber and bilaterally for the nucleus reticularis medius and nucleus reticularis inferior. Extensive brainstem projections directed to cervical and high thoracic spinal levels originate from the area lateralis hypothalami, the nucleus of the fasciculus longitudinalis medialis, the contralateral nucleus cerebellaris medialis, and from the nucleus tractus solitarii. Projections preferentially directed to midthoracic or lower levels of the spinal cord were found to arise from the ipsilateral locus coeruleus, the contralateral nucleus reticularis superior pars lateralis, the nucleus reticularis inferior pars ventralis, the nucleus reticularis inferior, and the nucleus raphes inferior. In contrast to findings in mammals, in Varanus exanthematicus the red nucleus, the nucleus vestibularis ventrolateralis, and certain parts of the reticular formation did not display a clear-cut somatotopic organization. In general two different patterns of collateralization can grossly be discerned: a gradual decrease of spinal collaterals caudalward, which can be interpreted as a certain specificity of such projections; and a constant number of collateral nerve fibers throughout the spinal cord that can be interpreted as either a nonspecific or, in contrast, a highly specific system, focussed exclusively on the cervical and lumbar enlargements.

The fasciculus longitudinalis medialis in the lizard Varanus exanthematicus. 2. Vestibular and internuclear components.

In the present study the vestibular components of the fasciculus longitudinalis medialis (flm) were investigated in the lizard Varanus exanthematicus with various tracing techniques: anterograde transport of horseradish peroxidase to study vestibulo-oculomotor and vestibulospinal projections, the multiple retrograde fluorescent tracer technique for the cells of origin of such projections. Internuclear projections between the oculomotor and abducens nuclei could also be studied in this way. Rather extensive vestibulo-ocular projections passing via the flm were demonstrated. Mainly ipsilateral ascending projections arise in the dorsolateral vestibular nucleus, mainly contralateral ascending projections in the ventromedial vestibular nucleus and adjacent parts of the ventrolateral and descending vestibular nuclei. Furthermore, distinct bilateral ascending projections of the nucleus prepositus hypoglossi were demonstrated. Extensive vestibulospinal projections pass via the flm and form the medial vestibulospinal tract. This largely contralateral descending pathway arises predominantly in the ventromedial and descending vestibular nuclei. Terminal structures presumably arising in the ventromedial and descending vestibular nuclei were found on contralateral neurons, probably motoneurons innervating neck muscles. Vestibular neurons with both ascending (presumably to extra-ocular motoneurons) and descending projections to the spinal cord are present in all vestibular nuclei, although preferentially in the ventromedial vestibular nucleus and adjacent parts of the ventrolateral and descending vestibular nuclei. However, also in the dorsolateral vestibular nucleus a substantial number of double labeled neurons were found. These vestibular neurons with both vestibulomesencephalic and vestibulospinal projections are probably involved in combined movements of eyes and head. Evidence for reciprocal internuclear connections between the oculomotor and abducens nuclei was found. Neurons in the dorsal part of the oculomotor nucleus probably project to the ipsilateral abducens nucleus, while neurons in the abducens nucleus most likely project to the contralateral oculomotor nucleus. These reciprocal internuclear connections between the oculomotor and abducens nuclei probably play an important role in conjugate horizontal eye movements.

Cerebellar efferents in the lizard Varanus exanthematicus. II. Projections of the cerebellar nuclei.

The projections of the cerebellar nuclei have been studied in the lizard Varanus exanthematicus with various experimental anatomical techniques. In anterograde degeneration experiments (lesions of the cerebellar peduncle) both ascending and decending contralateral projections were found. Ascending fibers which could be traced from the cerebellar commissure ventralward decussated at the level of the trochlear and oculomotor nuclei. These fibers coursed rostralward to the mesodiencephalic junction. With anterograde tracing techniques (3H-leucine and HRP) this tract was found to terminate in the nucleus ruber and the interstitial nucleus of the fasciculus longitudinalis medialis. Moreover, retrograde tracer studies (HRP, "Fast Blue") showed that this tract appeared to arise mainly in the lateral cerebellar nucleus. With both anterograde degeneration and tracing techniques (3H-leucine and HRP) a bundle of fibers could be followed, which decussates in the basal part of the cerebellum and passes dorsally around the contralateral medial cerebellar nucleus to the lateral side of the brainstem. This contralaterally descending projection system was found, lateral to the vestibular nuclear complex, and as far caudally as the descending vestibular nucleus, to terminate on various vestibular nuclei. Horseradish peroxidase studies showed that this contralaterally descending projection system originates mainly in the medial cerebellar nucleus, but ipsilaterally descending projections were also found. With the fluorescent double labeling technique ("Fast Blue" and "Nuclear Yellow") the projections of the cerebellar nuclei described above were confirmed. Furthermore, double labeling revealed neurons in both cerebellar nuclei (especially the medial nucleus) that project to both the mesencephalon and the cervical spinal cord. The present results indicate that the efferent connections of the cerebellar nuclei in the lizard Varanus exanthematicus are organized as two main projections, an ascending projection comparable to the mammalian brachium conjunctivum arising in the lateral cerebellar nucleus, and a descending projection comparable to the mammalian hook bundle (fasciculus uncinatus), originating mainly in the medial cerebellar nucleus. Such projections are common for terrestrial vertebrates.

Ascending and descending axon collaterals efferent from the brainstem reticular formation. A retrograde fluorescent tracer study in the lizard, Varanus exanthematicus.

The existence of divergent axon collaterals of neurons in the reticular formation has been studied with fluorescent tracers in a lizard. It appeared that ascending and descending projections arise in at least partially overlapping fields. However, only few reticular or raphe neurons were found with both ascending and descending projections.

The fasciculus longitudinalis medialis in the lizard Varanus exanthematicus. 1. Interstitiospinal, reticulospinal and vestibulospinal components.

With the horseradish peroxidase (HRP) technique the various descending components of the medial longitudinal fasciculus (flm) have been studied in the lizard Varanus exanthematicus. After wheat germ agglutinin conjugated HRP injections at the spinomedullary border, retrogradely labeled fibers passing via the flm could be traced to various parts of the magnocellular rhombencephalic reticular formation, the descending and ventromedial vestibular nuclei and the interstitial nucleus of the flm. By implanting HRP slow-release gels into the flm the trajectory and site of termination of various components of the flm have been analysed. The interstitiospinal tract passes via the dorsal part of the flm. Reticulospinal fibers arising in the nucleus reticularis superior and nucleus reticularis medius take a position ventral to the interstitiospinal fibers. Vestibulospinal projections via the flm are found in its ventral part and arise mainly in the contralateral ventromedial and descending vestibular nuclei. A strong vestibulocollic projection to cervical motoneurons should be noted. The positional relations of the various fiber components within the flm found in a lower vertebrate such as the lizard Varanus exanthematicus are comparable to those in mammals.

Cerebellar connections in Xenopus laevis. An HRP study.

In the present study the cerebellar afferents in the clawed toad Xenopus laevis have been analysed with the horseradish peroxidase (HRP) technique. In addition, data on the efferent connections of the cerebellum could be gathered, based on the phenomenon of anterograde transport of HRP. Cerebellar afferents in Xenopus laevis appear to arise mainly in the vestibular nuclear complex, in a primordial inferior olive and in the spinal cord. Both primary (arising in the ipsilateral vestibular ganglion) and secondary vestibulocerebellar projections were found. A distinct crossed olivocerebellar projection to the molecular layer of the cerebellum was found. Two spinocerebellar pathways are present in Xenopus laevis, as in other anurans, viz. an ipsilateral dorsal spinocerebellar tract, presumably arising in dorsal root ganglion cells, and a larger ventral pathway, bilaterally arising in the spinal gray matter. The latter tract mainly originates in the ventrolateral and ventromedial spinal fields. Furthermore, a secondary trigeminocerebellar projection arising in the descending trigeminal nucleus, a cerebellar projection arising in the dorsal column nucleus, a small projection arising in a possible primordium of the mammalian nucleus prepositus hypoglossi, a raphecerebellar projection, and a small cerebellar projection originating in the ipsilateral mesencephalic tegmentum were demonstrated. Cerebellar efferents in Xenopus laevis are mainly aimed at the vestibular nuclear complex. A distinct ipsilateral cerebellovestibular projection present throughout the vestibular nuclear complex presumably arises in Purkyn e cells, a smaller contralateral projection in the cerebellar nucleus. In addition, a small primordial brachium conjunctivum, projecting to the red nucleus, was noted. The basic pattern of cerebellar connections as suggested for terrestrial vertebrates (ten Donkelaar and Bangma 1984) is also found in the permanently aquatic anuran Xenopus laevis.