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.

Images of oligomeric Kv beta 2, a modulatory subunit of potassium channels.

The Shaker type voltage-gated potassium (K+) channel consists of four pore-forming Kv alpha subunits. The channel expression and kinetic properties can be modulated by auxiliary hydrophilic Kv beta subunits via formation of heteromultimeric Kv alpha-Kv beta complexes. Because each (Kv alpha)4 could recruit more than one Kv beta subunit and different Kv beta subunits could potentially interact, the stoichiometry of alpha-beta and beta-beta complexes is therefore critical for understanding the functional regulation of Shaker type potassium channels. We expressed and purified Kv beta 2 subunit in Sf9 insect cells. The purified Kv beta 2, examined by atomic force and electron microscopy techniques, is found predominately as a square-shaped tetrameric complex with side dimensions of 100 x 100 A2 and height of 51 A. Thus, Kv beta 2 is capable of forming a tetramer in the absence of pore-forming alpha subunits. The center of the Kv beta 2 complex was observed to be the most heavily stained region, suggesting that this region could be part of an extended tubular structure connecting the inner mouth of the ion permeation pathway to the cytoplasmic environment.

Differential stimulation of PKC phosphorylation of potassium channels by ZIP1 and ZIP2.

Targeting of protein modification enzymes is a key biochemical step to achieve specific and effective posttranslational modifications. Two alternatively spliced ZIP1 and ZIP2 proteins are described, which bind to both Kvbeta2 subunits of potassium channel and protein kinase C (PKC) zeta, thereby acting as a physical link in the assembly of PKCzeta-ZIP-potassium channel complexes. ZIP1 and ZIP2 differentially stimulate phosphorylation of Kvbeta2 by PKCzeta. They also interact to form heteromultimers, which allows for a hybrid stimulatory activity to PKCzeta. Finally, ZIP1 and ZIP2 coexist in the same cell type and are elevated differentially by neurotrophic factors. These results provide a mechanism for specificity and regulation of PKCzeta-targeted phosphorylation.

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.

Two distantly positioned PDZ domains mediate multivalent INAD-phospholipase C interactions essential for G protein-coupled signaling.

Drosophila INAD, which contains five tandem protein interaction PDZ domains, plays an important role in the G protein-coupled visual signal transduction. Mutations in InaD alleles display mislocalization of signaling molecules of phototransduction which include the essential effector, phospholipase C-beta (PLC-beta), which is also known as NORPA. The molecular and biochemical details of this functional link are unknown. We report that INAD directly binds to NORPA via two terminally positioned PDZ1 and PDZ5 domains. PDZ1 binds to the C-terminus of NORPA, while PDZ5 binds to an internal region overlapping with the G box-homology region (a putative G protein-interacting site). The NORPA proteins lacking binding sites, which display normal basal PLC activity, can no longer associate with INAD in vivo. These truncations cause significant reduction of NORPA protein expression in rhabdomeres and severe defects in phototransduction. Thus, the two terminal PDZ domains of INAD, through intermolecular and/or intramolecular interactions, are brought into proximity in vivo. Such domain organization allows for the multivalent INAD-NORPA interactions which are essential for G protein-coupled phototransduction.

Seed and Hormonal Regulation of Gibberellin 20-Oxidase Expression in Pea Pericarp.

To understand further how seeds, auxin (4-chloroindole-3-acetic acid [4-Cl-IAA]), and gibberellins (GAs) regulate GA biosynthesis in pea (Pisum sativum L.) pericarp at the molecular level, we studied the expression of GA 20-oxidase in this tissue using northern-blot analysis. Pericarp GA 20-oxidase mRNA levels were highest from prepollination (-2 d after anthesis [DAA]) through anthesis (0 DAA), then decreased 3-fold by 2 DAA, and remained at these levels through 6 DAA. The effects of seeds and hormones (4-Cl-IAA and GA3) on the expression of GA 20-oxidase in pea pericarp were investigated over a 36-h treatment period. GA 20-oxidase mRNA levels in 2 DAA pericarp with seeds remained relatively stable throughout the treatment period; however, when the seeds were removed the pericarp transcript levels declined. When 2 DAA deseeded pericarps were treated with 4-Cl-IAA, a significant increase in GA 20-oxidase mRNA levels was detected within 2 h and transcript levels remained elevated for up to 12 h after 4-Cl-IAA application. GA3 significantly decreased GA 20-oxidase mRNA levels in deseeded pericarp within 2 h of application. These data suggest that the previously reported conversion of GA19 to GA20 in pea pericarp is controlled by seeds, 4-Cl-IAA, and GA3 at least in part by regulating GA 20-oxidase mRNA levels in this tissue.

Influence of Auxin and Gibberellin on in Vivo Protein Synthesis during Early Pea Fruit Growth.

Developing pea fruits (Pisum sativum L.) offer a unique opportunity to study growth and development in a tissue that is responsive to both gibberellins (GAs) and auxin (4-chloroindole-3-acetic acid[4-CI-IAA]). To begin a molecular analysis of the interaction of GAs and auxins in pea fruit development, in vivo labeling with [35S]methionine coupled with two-dimensional gel electrophoresis were used to characterize de novo synthesis of proteins during gibberellic acid (GA3)-, 4-CI-indoleacetic acid-, and seed-induced pea pericarp growth. The most significant and reproducible polypeptide changes were observed between molecular weights of 20 and 60. Comparing about 250 de novo synthesized proteins revealed that seed removal changed the pattern substantially. We identified one class of polypeptides that was uniquely seed induced and five classes that were affected by hormone treatment. The latter included 4-CI-IAA-induced, GA3-induced, GA3- and 4-CI-IAA-induced, 4-CI-IAA-repressed, and GA3- and 4-CI-IAA-repressed polypeptides. Similar patterns of protein expression were associated with both hormone treatments; however, changes unique to GA3 or 4-CI-IAA treatment also indicate that the effects of GA3 and 4-CI-IAA on this process are not equivalent. In general, application of 4-CI-IAA plus GA3 replaced the seed effects on pericarp protein synthesis, supporting our hypothesis that both hormones are involved in pea pericarp development.

Seed and 4-chloroindole-3-acetic acid regulation of gibberellin metabolism in pea pericarp.

In this study, we investigated seed and auxin regulation of gibberellin (GA) biosynthesis in pea (Pisum sativum L.) pericarp tissue in situ, specifically the conversion of [14C]GA19 to [14C]GA20. [14C]GA19 metabolism was monitored in pericarp with seeds, deseeded pericarp, and deseeded pericarp treated with 4-chloroindole-3-acetic acid (4-CI-IAA). Pericarp with seeds and deseeded pericarp treated with 4-CI-IAA continued to convert [14C]GA19 to [14C]GA20 throughout the incubation period (2-24 h). However, seed removal resulted in minimal or no accumulation of [14C]GA20 in pericarp tissue. [14C]GA29 was also identified as a product of [14C]GA19 metabolism in pea pericarp. The ratio of [14C]GA29 to [14C]GA20 was significantly higher in deseeded pericarp (with or without exogenous 4-CI-IAA) than in pericarp with seeds. Therefore, conversion of [14C]GA20 to [14C]GA29 may also be seed regulated in pea fruit. These data support the hypothesis that the conversion of GA19 to GA20 in pea pericarp is seed regulated and that the auxin 4-CI-IAA can substitute for the seeds in the stimulation of pericarp growth and the conversion of GA19 to GA20.

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.