Allergen-Specific Immunotherapy Alters the Frequency,as well as the FcR and CLR Expression Profiles of Human Dendritic Cell Subsets

Allergen-specific immunotherapy (AIT) induces tolerance and shifts the Th2 response towards a regulatory T-cell profile. The underlying mechanisms are not fully understood, but dendritic cells (DC) play a vital role as key regulators of T-cell responses. DCs interact with allergens via Fc receptors (FcRs) and via certain C-type lectin receptors (CLRs), including CD209/DC-SIGN, CD206/MR and Dectin-2/CLEC6A. In this study, the effect of AIT on the frequencies as well as the FcR and CLR expression profiles of human DC subsets was assessed. PBMC was isolated from peripheral blood from seven allergic donors before and after 8 weeks and 1 year of subcutaneous AIT, as well as from six non-allergic individuals. Cells were stained with antibodies against DC subset-specific markers and a panel of FcRs and CLRs and analyzed by flow cytometry. After 1 year of AIT, the frequency of CD123⁺ DCs was increased and a larger proportion expressed FcεRI. Furthermore, the expression of CD206 and Dectin-2 was reduced on CD141⁺ DCs after 1 year of treatment and CD206 as well as Dectin-1 was additionally down regulated in CD1c⁺ DCs. Interestingly, levels of DNGR1/CLEC9A on CD141⁺ DCs were increased by AIT, reaching levels similar to cells isolated from non-allergic controls. The modifications in phenotype and occurrence of specific DC subsets observed during AIT suggest an altered capacity of DC subsets to interact with allergens, which can be part of the mechanisms by which AIT induces allergen tolerance.     

Evaluation of ITS rDNA as a complement to mitochondrial gene sequences for phylogenetic studies in freshwater mussels: an example using Unionidae from north-western Europe

Mitochondrial inheritance in the Unionidae is complex since mitochondria can be inherited from both parents. An increased rate of recombination could lead to erroneous homology assessments, which could cause problems for phylogenetic reconstruction. For this reason we investigated the possibility of using a nuclear marker, the ribosomal internal transcribed spacer region, for phylogenetic studies in the Unionidae, as a complement and comparison to two of the most widely used mitochondrial genes today. The nuclear ribosomal internal transcribed spacer region ( ITS1 , 5.8S , ITS2 ) was sequenced from 72 specimens representing six of the seven species of Unionidae mussels occurring in NW Europe: Unio pictorum , U. tumidus , U. crassus , Anodonta anatina , A. cygnea and Pseudanodonta complanata. Sequences from Margaritifera margaritifera were used as an outgroup . The ITS sequences of all species were found to have low intragenomic and infraspecific variation. Compared to mitochondrial genes ( 16S and COI ) they show an intermediate genetic diversity. Phylogenetic analyses produce tree topologies that are congruent with those resulting from analyses of the mitochondrial sequences. Likewise, an incongruence length difference (ILD) test showed no significant incongruence between data sets, indicating that if recombination has occurred it has not produced any conflicting patterns. Best-resolved and supported trees are obtained when gaps are treated as a fifth character state. A combined analysis of the three gene regions shows that Unio crassus and U. pictorum are more closely related than either to U. tumidus . Pseudanodonta is nested within Anodonta as sister taxon to A. cygnea . Advice on how to best preserve mussel material for DNA studies is provided.     

Neuropeptide K: isolation, structure and biological activities of a novel brain tachykinin

A 36 amino acid residue peptide, which contains a substance K sequence at its C-terminus has been isolated from porcine brain extracts. The primary structure of the peptide, designated neuropeptide K (NPK), was found to be: (sequence; see text) This N-terminally extended form of substance K is present in a high concentration in the brain. The peptide is highly biologically active with regard to gallbladder contraction, protein extravasation, hypotension and bronchial smooth muscle spasm and may act as an additional tachykinin neuromessenger.     

Multiple tachykinins (neurokinin A, neuropeptide K and substance P) in capsaicin-sensitive sensory neurons in the guinea-pig

The occurrence of tachykinins in sensory neurons of the guinea-pig was studied by means of radioimmunoassay combined with ion-exchange and high-performance liquid chromatography as well as by immunohistochemistry. Antisera raised against kassinin (antiserum K12), neurokinin A (NKA) (antiserum NKA2) and substance P (SP) (antisera SP25 and SP2) were used. Antiserum K12 detected NKA, neuropeptide K (NPK) and a component eluting in the position of eledoisin (ELE) in extracts of the lung and ureter. Neurokinin B (NKB) was, however, not found. Neutral water extraction favored recovery of NKA and of the ELE-like component, while NPK was found only in acid extracts. The SP antisera detected two immunoreactive components of which the major form coeluted with synthetic SP. Capsaicin pretreatment depleted all these various forms of immunoreactivity in several peripheral organs including the ureter and lung. The immunoreactivity detected by antisera K12 or SP25 in radioimmunoassay had a similar regional distribution pattern in peripheral tissues. Immunohistochemical examination revealed that antiserum NKA2 stained the same spinal ganglion cells as the SP2 antiserum. The distribution of capsaicin-sensitive nerve fibers stained by these two antisera was also identical in peripheral organs such as the ureter, inferior mesenteric ganglion, heart and lung. It is concluded that multiple tachykinins, including SP, NKA, NPK and an ELE-like peptide, are present in capsaicin-sensitive sensory nerves in the guinea-pig. This finding can most likely be related to the origin of SP, NKA and NPK from the same precursor molecule, subsequent posttranslational tissue processing and axonal transport to terminal regions.     

VIP and PHI in cat neurons: co-localization but variable tissue content possible due to differential processing

The concentrations of vasoactive intestinal polypeptide (VIP) and the peptide with NH2- terminal histidine and COOH-terminal isoleucine (PHI) in various peripheral tissues and some areas in the CNS of the cat were compared with their immunohistochemical localization. The VIP levels in the gastrointestinal tract were 3 to 6 times higher than PHI levels. Much (up to 10-fold) higher VIP than PHI levels were also observed in the genitourinary tract as well as in the lung and heart. In the neurohypophysis, however, the VIP/PHI ratio was close to 1. Gel-permeation chromatography revealed that VIP- and PHI-immunoreactivity (IR) in the intestine, pancreas and brain consisted of three larger molecular forms in addition to the 'standard' peptides. These larger forms which had overlapping elution positions may represent prepro-VIP/PHI forms. The immunohistochemical analysis revealed that VIP- and PHI-IR was present in the same ganglion cells in the intestine, pancreas, uterus and sympathetic ganglia. Furthermore, the terminal networks for these two peptides were very similar in the periphery. In the median eminence of the hypothalamus and in the posterior lobe of the pituitary, considerably more nerves were PHI- than VIP-IR. This observation was in parallel to a low VIP/PHI ratio. In conclusion, VIP and PHI seem to co-exist in most neuronal systems. Although the ratio of VIP and PHI on the precursor gene is 1:1, differences in posttranslational processing may create a considerably higher content of VIP than PHI in most terminal areas.     

Evolution of black grouse leks: female preferences benefit males in larger leks

Leks, communal display grounds in which males display and femalesonly attend to mate, represent one of the most remarkable outcomesof sexual selection. There have been no detailed studies thatcompare the behavior of males and females between leks of differentsizes to test if there is any benefit for male clumping andto test the many hypotheses suggested to explain lekking. Inthe black grouse, Tetrao tetrix, larger leks have many morefemale visits and copulations, leading to a higher average malemating success. Females visiting larger leks are also more likelyto mate, indicating that female preferences of males are importantfor the evolution of leks. Yearling males seldom copulate, buttheir presence on the lek increases the mating success of adultmales, suggesting that lek size per se and not only male qualityaffects female preferences for larger leks. The distributionof males over lek sizes agrees with a game theory model of idealfree distribution with unequal competitors, in which less competitivemales go to smaller leks.     

Vascular control of the pig nasal mucosa: distribution and effect of somatostatin in relation to noradrenaline and neuropeptide Y

By means of immunohistochemistry and radioimmunoassay (RIA), we have investigated the possible occurrence of somatostatin (SOM)-like immunoreactivity (-LI) in the autonomic innervation of the pig nasal mucosa. SOM-immunoreactive (-IR) fibres were present around nasal arteries, arterioles and venous sinusoids. Double-labelling experiments revealed that SOM-LI was co-localized with the noradrenaline (NA) markers tyrosine hydroxylase and dopamine-β-hydroxylase as well as with neuropeptide Y (NPY) in a subpopulation of neurons in the superior cervical sympathetic ganglion and in perivascular nerve terminals. Furthermore, SOM-LI was also present in perivascular fibres containing vasoactive intestinal polypeptide (VIP) and NPY of presumably parasympathetic origin. The parasympathetic fibres that were associated with glands contained peptide histidine isoleucine (PHI), VIP and NPY but not SOM, suggesting that in the nasal mucosa SOM-IR is restricted to perivascular nerves. As revealed by RIA, the content of SOM-LI in biopsies of both nasal mucosa and superior cervical sympathetic ganglion was about 12 pmol/g and the reverse phase HPLC characterisation of SOM-LI shown two separate peaks for SOM-28 and SOM-14.     

Detection of neuropeptide Y-like immunoreactivity and messenger RNA in rat platelets: the effects of vinblastine, reserpine, and dexamethasone on NPY expression in blood cells.

Rat plasma contains high basal levels (220 pmol/liter) of neuropeptide Y (NPY)-like immunoreactivity (LI) compared to pig (30 pmol/liter) and man (25 pmol/liter). The platelet-enriched fraction (PEF), obtained from rat blood contained 10,061 pmol/g NPY-LI. However, in human and pig blood, the PEF contained very low levels of NPY-LI. Gradient centrifugation of rat blood showed the highest concentration of NPY-LI (10.8 +/- 0.4 pmol/g) in the platelet fraction. The mononuclear cell fraction contained 1.64 +/- 0.16 pmol/g, whereas only 0.56 +/- 0.06 pmol/g of NPY-LI was found in the red blood cell/polymorphonuclear cell fraction. Characterization of NPY-LI in rat plasma and platelets by high-pressure liquid chromatography showed one predominating peak which coeluted with synthetic NPY (1-36) as well as three minor peaks, one of which coeluted with oxidized NPY. Analysis of NPY messenger RNA (mRNA) in bone marrow of the rat revealed a 0.79-kb-long NPY mRNA. This size is intermediate to the 0.82-kb NPY mRNA in brain and the 0.76-kb NPY mRNA in spleen. The highest level of NPY mRNA in rat blood was found in the mononuclear cell fraction but NPY mRNA was also detected in the platelet fraction. No NPY mRNA was detected in bone marrow or blood from pig and rabbit or from human blood or bone marrow. Forty-eight hours after treatment of rats with vinblastine the content of NPY mRNA and NPY-LI in rat blood was decreased, while the level of NPY-LI in bone marrow was markedly enhanced. Reserpine treatment caused an increase in NPY mRNA content in bone marrow and spleen. After administration of dexamethasone the level of NPY mRNA increased in both spleen and peripheral blood cells with increased NPY-LI content in the spleen. It is concluded that in addition to megakaryocytes in spleen and bone marrow, platelets and possibly also lymphocytes/monocytes in peripheral blood of the rat contain NPY mRNA and peptide. The expression of NPY mRNA in bone marrow, spleen, and blood is influenced by vinblastine, reserpine, and dexamethasone.     

Coupling of the polyamine and iron metabolism pathways in the regulation of proliferation: Mechanistic links to alterations in key polyamine biosynthetic and catabolic enzymes

Many biological processes result from the coupling of metabolic pathways. Considering this, proliferation depends on adequate iron and polyamines, and although iron-depletion impairs proliferation, the metabolic link between iron and polyamine metabolism has never been thoroughly investigated. This is important to decipher, as many disease states demonstrate co-dysregulation of iron and polyamine metabolism. Herein, for the first time, we demonstrate that cellular iron levels robustly regulate 13 polyamine pathway proteins. Seven of these were regulated in a conserved manner by iron-depletion across different cell-types, with four proteins being down-regulated (i.e., acireductone dioxygenase 1 [ADI1], methionine adenosyltransferase 2α [MAT2α], Antizyme and polyamine oxidase [PAOX]) and three proteins being up-regulated (i.e., S-adenosyl methionine decarboxylase [AMD1], Antizyme inhibitor 1 [AZIN1] and spermidine/spermine-N¹-acetyltransferase 1 [SAT1]). Depletion of iron also markedly decreased polyamine pools (i.e., spermidine and/or spermine, but not putrescine). Accordingly, iron-depletion also decreased S-adenosylmethionine that is essential for spermidine/spermine biosynthesis. Iron-depletion additionally reduced ³H-spermidine uptake in direct agreement with the lowered levels of the polyamine importer, SLC22A16. Regarding mechanism, the “reprogramming” of polyamine metabolism by iron-depletion is consistent with the down-regulation of ADI1 and MAT2α, and the up-regulation of SAT1. Moreover, changes in ADI1 (biosynthetic) and SAT1 (catabolic) partially depended on the iron-regulated changes in c-Myc and/or p53. The ability of iron chelators to inhibit proliferation was rescuable by putrescine and spermidine, and under some conditions by spermine. Collectively, iron and polyamine metabolism are intimately coupled, which has significant ramifications for understanding the integrated role of iron and polyamine metabolism in proliferation.