Randomized, double-blind, placebo-controled clinical trial of sublingual immunotherapy in natural rubber latex allergic patients

<p>Abstract</p> <p>Background</p> <p>Natural rubber latex allergy is a common and unsolved health problem. Since the avoidance of exposure is very difficult, immunotherapy is strongly recommended, but before its use in patients, it is essential to prove the efficacy and safety of extracts.</p> <p>The aim of the present randomised, double-blind, placebo-controlled clinical trial was to assess the efficacy and tolerability of latex sublingual immunotherapy in adult patients undergoing permanent latex avoidance.</p> <p>Methods</p> <p>Twenty-eight adult latex-allergic patients (5 males and 23 females), with mean age of 39 years (range 24-57) were randomized to receive a commercial latex-sublingual immunotherapy or placebo during one year, followed by another year of open, active therapy. The following outcomes were measured at baseline and at the end of first and second year of follow-up: skin prick test, gloves-use score, conjunctival challenge test, total and specific IgE, basophil activation test, and adverse reactions monitoring.</p> <p>Results</p> <p>No significant difference in any of the efficacy <it>in vivo </it>variables was observed between active and placebo groups at the end of the placebo-controlled phase, nor when each group was compared with their baseline values at the end of the two year-study. An improvement in the average percentage of basophils activated was observed. During the induction phase, 4 reactions in the active group and 5 in the placebo group were recorded. During the maintenance phase, two patients dropped out due to pruritus and to acute dermatitis respectively.</p> <p>Conclusion</p> <p>Further studies are needed to evaluate latex-sublingual immunotherapy, since efficacy could not be demonstrated in adult patients with avoidance of the allergen.</p> <p>Trial registration number</p> <p><a href="http://www.anzctr.org.au/ACTRN12611000543987.aspx">ACTRN12611000543987</a></p

Purinergic modulation of microglial cell activation

Microglial cells are resident macrophages in the brain and their activation is an important part of the brain immune response and the pathology of the major CNS diseases. Microglial activation is triggered by pathological signals and is characterized by morphological changes, proliferation, phagocytosis and the secretion of various cytokines and inflammatory mediators, which could be both destructive and protective for the nervous tissue. Purines are one of the most important mediators which regulate different aspects of microglial function. They could be released to the extracellular space from neurons, astrocytes and from the microglia itself, upon physiological neuronal activity and in response to pathological stimuli and cellular damage. Microglial activation is regulated by various subtypes of nucleotide (P2X, P2Y) and adenosine (A1, A2A and A3) receptors, which control ionic conductances, membrane potential, gene transcription, the production of inflammatory mediators and cell survival. Among them, the role of P2X7 receptors is especially well delineated, but P2X4, various P2Y, A1, A2A and A3 receptors also powerfully participate in the microglial response. The pathological role of microglial purine receptors has also been demonstrated in disease models; e.g., in ischemia, sclerosis multiplex and neuropathic pain. Due to their upregulation and selective activation under pathological conditions, they provide new avenues in the treatment of neurodegenerative and neuroinflammatory illnesses

The non-specific lipid transfer protein, Ara h 9, is an important allergen in peanut

Background Plant food allergy in the Mediterranean area is mainly caused by non-specific lipid transfer proteins (nsLTP). The aim of this study was to characterize peanut nsLTP in comparison with peach nsLTP, Pru p 3, and assess its importance in peanut allergy. Methods Peanut-allergic patients from Spain (n = 32) were included on the basis of a positive case history and either a positive skin prick test or specific IgE to peanut. For comparison, sera of 41 peanut-allergic subjects from outside the Mediterranean area were used. Natural Ara h 9 and two isoforms of recombinant Ara h 9, expressed in Pichia pastoris, were purified using a two-step chromatographic procedure. Allergen characterization was carried out by N-terminal sequencing, circular dichroism (CD) spectroscopy, immunoblotting, IgE inhibition tests and basophil histamine release assays. Results Compared with natural peanut nsLTP, the recombinant proteins could be purified in high amounts from yeast supernatant (X45 mg/L). The identity of the proteins was verified by N-terminal amino acid sequencing and with rabbit nsLTP-specific antibodies. CD spectroscopy revealed similar secondary structures for all preparations and Pru p 3. The Ara h 9 isoforms showed 62\u201368% amino acid sequence identity with Pru p 3. IgE antibody reactivity to rAra h 9 was present in 29/32 Spanish and 6/41 non-Mediterranean subjects. Recombinant Ara h 9 showed strong cross-reactivity to nPru p 3 and similar IgE-binding capacity as nAra h 9. The two Ara h 9 isoforms displayed similar IgE reactivity. In peanutallergic patients with concomitant peach allergy, Ara h 9 showed a weaker allergenic potency than Pru p 3 in histamine release assays. Conclusions Ara h 9 is a major allergen in peanut-allergic patients from the Mediterranean area. Ara h 9 is capable of inducing histamine release from basophils, but to a lesser extent than Pru p 3

Dominant-negative H-Ras (H-Ras_S17NDN) prevents SNX27b-dependent down-regulation of GIRK channels.

<p><b>A</b>, Schematic shows GIRK2c and GIRK3 with PDZ-binding motif and GIRK2a and GIRK3-RR, which lack motifs that interact with SNX27-PDZ <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059800#pone.0059800-Balana1" target="_blank">[7]</a>. <b>B</b>, Examples of baclofen-induced (100 µM) and Ba²⁺-sensitive (1 mM Ba²⁺) currents in HEK293 cells transfected with cDNA for GABA_B1a/B2, GIRK2c/GIRK3 and either empty vector (Control), H-Ras_S17NDN or H-Ras_S17NDN plus SNX27b. <b>C</b>, Bar graph shows average I_Baclofen for GIRK2c/GIRK3 alone (–47.7±8.5 pA⋅pF⁻¹, n = 6), GIRK2c/GIRK3 and H-Ras_S17NDN (–22.0±3.8 pA⋅pF⁻¹, n = 19) or GIRK2c/GIRK3, SNX27b and H-Ras_S17NDN (–23.7±4.7 pA⋅pF⁻¹, n = 16). <b>D</b>, Bar graph shows I_Baclofen for control (GIRK2a/GIRK3-RR alone) (–18.8±3.0 pA⋅pF⁻¹, n = 10) and GIRK2a/GIRK3-RR plus H-Ras_S17NDN (–18.6±2.0 pA⋅pF⁻¹, n = 11). <b>E</b>, Bar graph shows average I_Barium for GIRK2c/GIRK3 alone (–27.6±9.8 pA⋅pF⁻¹, n = 6), GIRK2c/GIRK3 and H-Ras_S17NDN (–7.3±2.2 pA⋅pF⁻¹, n = 18) or GIRK2c/GIRK3, SNX27b and H-Ras_S17NDN (–7.1±1.5 pA⋅pF⁻¹, n = 14). <b>F</b>, Bar graph shows average I_Barium for GIRK2a/GIRK3-RR alone (−2.49±0.9 pA⋅pF⁻¹, n = 10), GIRK2c/GIRK3 and H-Ras_S17NDN (−1.84±0.4 pA⋅pF⁻¹, n = 11). **P<0.05, one way ANOVA followed by Bonferroni post hoc test; n.s. – not significant.</p

Ras-association domain of sorting nexin 27 is critical for regulating expression of GIRK potassium channels

G protein-gated inwardly rectifying potassium (GIRK) channels play an important role in regulating neuronal excitability. Sorting nexin 27b (SNX27b), which reduces surface expression of GIRK channels through a PDZ domain interaction, contains a putative Ras-association (RA) domain with unknown function. Deleting the RA domain in SNX27b (SNX27b-DRA) prevents the down-regulation of GIRK2c/GIRK3 channels. Similarly, a point mutation (K305A) in the RA domain disrupts regulation of GIRK2c/GIRK3 channels and reduces H-Ras binding in vitro. Finally, the dominant-negative H-Ras (S17N) occludes the SNX27b-dependent decrease in surface expression of GIRK2c/GIRK3 channels. Thus, the presence of a functional RA domain and the interaction with Ras-like G proteins comprise a novel mechanism for modulating SNX27b control of GIRK channel surface expression and cellular excitability

Deletion of RA domain in SNX27b impairs functional regulation of GIRK channels.

<p><b>A</b>, Cartoon shows model of GIRK channels regulation by SNX27b. GIRK channels recycle through endosomal compartments. SNX27b associating with GIRK2c/3 channels in the early endosome (EE) reduces plasma membrane expression (PM) by targeting some channels to the late endosome (LE). <b>B</b>, SNX27 contains three functional domains; PDZ, PX and RA. GIRK2c and GIRK3 contain a C-terminal PDZ binding motif (-E(S/N)ESKV). <b>C,</b> Examples of baclofen-induced (100 µM) and Ba²⁺-sensitive (1 mM Ba²⁺) currents in HEK293T cells transfected with cDNA for GABA_B1a/B2 receptors, GIRK2c/GIRK3 and either control vector, SNX27b or SNX27b-ΔRA. Agonist-independent basal currents are revealed by inhibition with 1 mM Ba²⁺. <b>D</b>, Average baclofen-induced current densities (I_Baclofen) for control (–41.3±5.2 pA⋅pF⁻¹, n = 8), SNX27b (–11.0±3.6 pA⋅pF⁻¹, n = 8) and SNX27b- ΔRA (–55.9±8.2 pA⋅pF⁻¹, n = 13) with GIRK2c/GIRK3. <b>E</b>, Average I_Baclofen for control (–15.7±3.6 pA⋅pF⁻¹, n = 6) and SNX27b-ΔRA (–18.6±4.9 pA⋅pF⁻¹, n = 5) with GIRK1/GIRK3 (**P<0.05, one way ANOVA followed by Bonferroni <i>post hoc</i> test; n.s. – not significant).</p

Deletion of RA domain in SNX27b affects localization of GIRK2c/3 channels monitored with BiFC.

<p><b>A</b>, <i>Left</i>, Schematic shows placement of split YFP on GIRK2c and GIRK3. Note the C-terminal domains are free to interact with other proteins. <i>Right</i>, BiFC-tagged GIRK2c/3 channels are functional. Current-voltage plot is shown for ^CYGIRK2c/^NYGIRK3 channels. Baclofen (100 µM) activates and Ba²⁺ (1 mM) inhibits inwardly rectifying current. HEK293T cells were transfected with cDNA encoding GABA_B1a, GABA_B2 and ^CYGIRK2c/^NYGIRK3. Average baclofen-induced current densities were –13.2±6.0 pA⋅pF⁻¹ (n = 3) for ^CYGIRK2c/^NYGIRK3. <b>B</b>, HEK293 cells were co-transfected with ^CYGIRK2c, ^NYGIRK3 and either control cDNA (<i>i</i>), wild-type SNX27b (<i>ii</i>), SNX27b-ΔRA (deletion of Asp272-Trp358) (<i>iii</i>) or SNX27b-Y51L (a PDZ mutation) (<i>iv</i>). Green fluorescence in images represents molecular recombination of ^CYGIRK2c/^NYGIRK3 heterotetramers. Coexpression of wild-type SNX27b induced formation of puncta. By contrast, ^CYGIRK2c/^NYGIRK3 fluorescence was diffuse in the cytoplasm for SNX27b-ΔRA and for SNX27b-Y51L, similar to control. Inset shows zoom of boxed area. <b>C,</b> SNX27b-ΔRA exhibited a pattern of punctate expression similar that of wild-type SNX27b. YFP was fused to the C-terminus of SNX27b or SNX27b-ΔRA to directly visualize expression. HEK293T cells were transfected with cDNA for SNX27b-YFP and SNX27bΔRA-YFP. Scale bar: 10 µm.</p

K305A point mutation in SNX27b RA domain disrupts functional regulation of GIRK2c/GIRK3 channels.

<p><b>A</b>, Alignment of two different RA domains: a RID (Ras interacting domain) from RalGDS and a RA domain of RGL, with RA domain of SNX27. Residues implicated in Ras binding in Raf and RalGDS domains <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059800#pone.0059800-Block1" target="_blank">[28]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059800#pone.0059800-Vetter1" target="_blank">[30]</a> are highlighted in red. <b>B</b>, High-resolution structure shows R20, K32 and K52 at the binding interface of RID and Ras <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059800#pone.0059800-Huang1" target="_blank">[26]</a>. <b>C,</b> RA domain mutations impair the ability of SNX27b to induce formation of GIRK2c/3 puncta using BiFC. HEK293T cells were co-transfected with cDNA for ^CYGIRK2c/^NYGIRK3 and either wild-type SNX27b (<b>i</b>), SNX27b-R276A (<b>ii</b>), SNX27b-R288A/K291A (<b>iii</b>) or SNX27b-K305A (<b>iv</b>). Green fluorescence indicates molecular complementation of ^CYGIRK2c/^NYGIRK3. Scale bar: 10 µm. <b>D</b>, Colocalization of wild-type SNX27b (SNX27b-YFP; green) and SNX27b-K305A (anti-SNX27; green) with an early endosomal marker (anti-EEA1, green). Scale bar: 5 µm. <b>E</b>, Average I_Baclofen currents for control (–53.4±9.4 pA⋅pF⁻¹, n = 5), SNX27b (–8.05±3.44 pA⋅pF⁻¹, n = 6) and SNX27b-K305A (–52.9±8.8 pA⋅pF⁻¹, n = 10) with GIRK2c/GIRK3 channels.</p