Selective cloning, characterization, and production of the Culicoides nubeculosus salivary gland allergen repertoire associated with equine insect bite hypersensitivity

Salivary gland proteins of Culicoides spp. have been suggested to be among the main allergens inducing IgE-mediated insect bite hypersensitivity (IBH), an allergic dermatitis of the horse. The aim of our study was to identify, produce and characterize IgE-binding salivary gland proteins of Culicoides nubeculosus relevant for IBH by phage surface display technology. A cDNA library constructed with mRNA derived from C. nubeculosus salivary glands was displayed on the surface of filamentous phage M13 and enriched for clones binding serum IgE of IBH-affected horses. Ten cDNA inserts encoding putative salivary gland allergens were isolated and termed Cul n 2 to Cul n 11. However, nine cDNA sequences coded for truncated proteins as determined by database searches. The cDNA sequences were amplified by PCR, subcloned into high level expression vectors and expressed as hexahistidine-tagged fusion proteins in Escherichia coli. Preliminary ELISA results obtained with these fusions confirmed the specific binding to serum IgE of affected horses. Therefore, the putative complete open reading frames derived from BLAST analyses were isolated by RACE-PCR and subcloned into expression vectors. The full length proteins expressed in Escherichia coli showed molecular masses in the range of 15.5-68.7 kDa in SDS-PAGE in good agreement with the masses calculated from the predicted protein sequences. Western blot analyses of all recombinant allergens with a serum pool of IBH-affected horses showed their ability to specifically bind serum IgE of sensitized horses, and ELISA determinations yielded individual horse recognition patterns with a frequency of sensitization ranging from 13 to 57%, depending on the allergen tested. The in vivo relevance of eight of the recombinant allergens was demonstrated in intradermal skin testing. For the two characterized allergens Cul n 6 and Cul n 11, sensitized horses were not available for intradermal tests. Control horses without clinical signs of IBH did not develop any relevant immediate hypersensitivity reactions to the recombinant allergens. The major contribution of this study was to provide a repertoire of recombinant salivary gland allergens repertoire from C. nubeculosus potentially involved in the pathogenesis of IBH as a starting basis for the development of a component-resolved serologic diagnosis of IBH and, perhaps, for the development of single horse tailored specific immunotherapy depending on their component-resolved sensitization patterns.

Cloning of IgE-binding proteins from Simulium vittatum and their potential significance as allergens for equine insect bite hypersensitivity

Insect bite hypersensitivity (IBH) is an allergic dermatitis of horses caused by bites of Culicoides and sometimes Simulium spp. The aim of this investigation was to identify Simulium allergens associated with IBH. A phage surface display cDNA library expressing recombinant Simulium vittatum salivary gland proteins was screened using sera of IBH-affected horses sensitized to S. vittatum salivary gland proteins as shown in immunoblot, resulting in the identification of seven cDNAs encoding IgE-binding proteins. The deduced amino acid sequences of these proteins showed sequence similarities to antigen 5 like protein (Sim v 1), to a serine protease inhibitor (Sim v 2), to two alpha-amylases (Sim v 3 and Sim v 4), and to three S. vittatum erythema proteins (SVEPs). The cDNA inserts were subcloned and expressed as [His](6)-tagged protein in Escherichia coli and purified using Ni(2+)-chelate affinity chromatography. Mice were immunised with the seven recombinant proteins and the antibodies tested against the recombinant proteins and salivary gland extract (SGE) of S. vittatum and Culicoides nubeculosus in immunoblot analyses. r-Sim v 1 specific mouse Abs recognized a band of about 32 kDa in immunoblots of both S. vittatum and C. nubeculosus SGE, detectable also by serum IgE of IBH-affected horses. Preincubation of horse serum with r-Sim v 1 completely inhibited IgE binding to the 32 kDa band demonstrating the presence of cross-reactive antigen 5 like proteins in both SGE. Determination of IgE levels against the r-Sim v proteins and crude S. vittatum extract by ELISA in sera from 25 IBH-affected and 20 control horses showed that IBH-affected horses had significantly higher IgE levels than controls against r-Sim v 1, 2, 3, 4 and S. vittatum extract, whereas the r-SVEP showed only marginal IgE binding. Further analyses showed that 60% of IBH-affected horses reacted to r-Sim v 1, suggesting that this could be a major allergen for IBH. Forty to twenty percent of the IBH-affected horses reacted with r-Sim v 2, 3 or 4. Combination of the results obtained with the 4 r-Sim v proteins showed that 92% of the IBH-affected but only 15% of the healthy horses had IgE levels against one or more of the 4 r-Sim v proteins. Seventy percent of the healthy horses had detectable IgE against S. vittatum extract, indicating a low specificity of the detection system used. Optimization of the ELISA system will be required to determine reliable cut-off values for the IBH-related allergens. Their in vivo relevance needs to be carefully assessed.

Response surface modeling of the interaction between propofol and sevoflurane

BACKGROUND: Propofol and sevoflurane display additivity for gamma-aminobutyric acid receptor activation, loss of consciousness, and tolerance of skin incision. Information about their interaction regarding electroencephalographic suppression is unavailable. This study examined this interaction as well as the interaction on the probability of tolerance of shake and shout and three noxious stimulations by using a response surface methodology. METHODS: Sixty patients preoperatively received different combined concentrations of propofol (0-12 microg/ml) and sevoflurane (0-3.5 vol.%) according to a crisscross design (274 concentration pairs, 3 to 6 per patient). After having reached pseudo-steady state, the authors recorded bispectral index, state and response entropy and the response to shake and shout, tetanic stimulation, laryngeal mask airway insertion, and laryngoscopy. For the analysis of the probability of tolerance by logistic regression, a Greco interaction model was used. For the separate analysis of bispectral index, state and response entropy suppression, a fractional Emax Greco model was used. All calculations were performed with NONMEM V (GloboMax LLC, Hanover, MD). RESULTS: Additivity was found for all endpoints, the Ce(50, PROP)/Ce(50, SEVO) for bispectral index suppression was 3.68 microg. ml(-1)/ 1.53 vol.%, for tolerance of shake and shout 2.34 microg . ml(-1)/ 1.03 vol.%, tetanic stimulation 5.34 microg . ml(-1)/ 2.11 vol.%, laryngeal mask airway insertion 5.92 microg. ml(-1) / 2.55 vol.%, and laryngoscopy 6.55 microg. ml(-1)/2.83 vol.%. CONCLUSION: For both electroencephalographic suppression and tolerance to stimulation, the interaction of propofol and sevoflurane was identified as additive. The response surface data can be used for more rational dose finding in case of sequential and coadministration of propofol and sevoflurane.

Class-switzched B cells display response to therapeutic B-cell depletion in rheumatoid arthritis

Introduction Reconstitution of peripheral blood (PB) B cells after therapeutic depletion with the chimeric anti-CD20 antibody rituximab (RTX) mimics lymphatic ontogeny. In this situation, the repletion kinetics and migratory properties of distinct developmental B-cell stages and their correlation to disease activity might facilitate our understanding of innate and adaptive B-cell functions in rheumatoid arthritis (RA). Methods Thirty-five 'RTX-naïve' RA patients with active arthritis were treated after failure of tumour necrosis factor blockade in an open-label study with two infusions of 1,000 mg RTX. Prednisone dose was tapered according to clinical improvement from a median of 10 mg at baseline to 5 mg at 9 and 12 months. Conventional disease-modifying antirheumatic drugs were kept stable. Subsets of CD19+ B cells were assessed by flow cytometry according to their IgD and CD27 surface expression. Their absolute number and relative frequency in PB were followed every 3 months and were determined in parallel in synovial tissue (n = 3) or synovial fluid (n = 3) in the case of florid arthritis. Results Six of 35 patients fulfilled the European League Against Rheumatism criteria for moderate clinical response, and 19 others for good clinical response. All PB B-cell fractions decreased significantly in number (P < 0.001) after the first infusion. Disease activity developed independently of the total B-cell number. B-cell repopulation was dominated in quantity by CD27-IgD+ 'naïve' B cells. The low number of CD27+IgD- class-switched memory B cells (MemB) in the blood, together with sustained reduction of rheumatoid factor serum concentrations, correlated with good clinical response. Class-switched MemB were found accumulated in flaring joints. Conclusions The present data support the hypothesis that control of adaptive immune processes involving germinal centre-derived, antigen, and T-cell-dependently matured B cells is essential for successful RTX treatment.

First thoughts on surface tonal patterns in Amawaka, a Panoan language of Peru and Brazil

Amawaka ([ɑmɨ̃ˈwɐkɑ]) is a highly endangered and underdocumented tonal language of the Headwaters (Fleck 2011) subgroup of the Panoan family in the Southwest Amazon Basin, spoken by approximately 200 people. Undocumented phonetic and phonological phenomena of Amawaka include its tonal structure, both in terms of surface realizations and the patterns underlying these realizations. Original audiovisual data from the author’s fieldwork in various Amawaka communities at the Peru-Brazil border will illuminate the as-yet obscure tonal systematicity of the language. Unlike other elements, monosyllabic bimoraic phonological nominal words with long vowels display variation in their surface realization. All the words with the open back unrounded /ɑ/, like /ˈkɑ̀:/ (patarashca, a traditional Amazonian dish), /ˈnɑ̀:/ “mestizo” etc. [with the exception of /ˈtɑ:/ “reed”, which surfaces with either a H or L tone] bear a low tone in isolation. This realization contrasts with all the encountered nominal monosyllables with vowels from the close and close-mid front and central spectrum /i, ɘ, ɨ, ɨ̃/, which clearly surface as high tone words in isolation, for example /ˈmɨ̃́:/ (a clay-lick for animals), /ˈwí:/ “Anopheles, spp. mosquito”. Monosyllables with close-mid back rounded /o/ have a less restrictive pitch that varies among speakers from low to high realizations, and sometimes even across the speech tokens from an individual speaker, e.g. /wó:/ or /wō:/ “hair”, /ɧō:/ or /ɧò:/ (a type of tarantula). Phrasal tonal phonology is more complex, when these three kinds of monosyllables appear in larger noun phrases. Some retain the same surface tones as their isolation form, while others seem to vary freely in their surface realization, e.g. /ˈtɘ́:.nɑ̀:/ or /ˈtɘ́:.nɑ́:/ ‘one mestizo’. Yet other monosyllables, e.g. /mɑ̀:/, exhibit a falling tone when preceded by a H syllable, suggesting probably latent tone sandhi phenomena, e.g /ˈtɘ́:.mɑ̂:/ (one clay-lick for parrots). In disyllabic, trisyllabic and quadrisyllabic nouns, tonal and stress patterns generally seem to be more consistent and tend to be retained both in isolation and in larger intonational phrases. Disyllabic nouns, for instance, surface as L-H or L-L when a glottal stop is in coda position. The association of L with a glottal stop is a feature that occurs in other Panoan languages as well, like Capanahua (Loos 1969), and more generally it is an areal feature, found in other parts of Amazonia (Hyman 2010). So, tone has significant interactions with the glottal stop and glottalization, which generally co-occurs with L. The data above suggest that the underlying tonal system of Amawaka is much more complex than the privative one-tone analysis (/H/ vs. Ø, i.e. lack of tone) that was proposed by Russell and Russell (1959). Evidence from field data suggests either an equipollent (Hyman 2010) two-tone opposition between /H/ and /L/, or a hybrid system, with both equipollent and privative features; that is, /H/ vs. /L/ vs. either Ø or /M/. This first systematic description of Amawaka tone, in conjunction with ongoing research, is poised to address broader questions concerning interrelationships between surface/underlying tone and other suprasegmental features, such as nasality, metrical stress, and intonation. References Fleck, David W. 2011. Panoan languages and linguistics. In Javier Ruedas and David W. Fleck (Eds.), Panoan Histories and Interethnic Identities, To appear. Hyman, Larry. 2010. Amazonia and the typology of tone systems. Presented at the conference Amazonicas III: The structure of the Amazonian languages. Bogotá. Loos, Eugene E. 1969. The phonology of Capanahua and its grammatical basis. Norman: SIL and U. Oklahoma. Russell, Robert & Dolores. 1959. Syntactotonemics in Amahuaca (Pano). Série Lingüistica Especial, 128-167. Publicaçoes do Museu Nacional, Rio de Janeiro, Brasil.

A Noachian source region for the "Black Beauty" meteorite, and a source lithology for Mars surface hydrated dust?

The Martian surface is covered by a fine-layer of oxidized dust responsible for its red color in the visible spectral range (Bibring et al., 2006; Morris et al., 2006). In the near infrared, the strongest spectral feature is located between 2.6 and 3.6 mu m and is ubiquitously observed on the planet (Jouglet et al., 2007; Milliken et al., 2007). Although this absorption has been studied for many decades, its exact attribution and its geological and climatic implications remain debated. We present new lines of evidence from laboratory experiments, orbital and landed missions data, and characterization of the unique Martian meteorite NWA 7533, all converging toward the prominent role of hydroxylated ferric minerals. Martian breccias (so-called "Black Beauty" meteorite NWA7034 and its paired stones NWA7533 and NWA 7455) are unique pieces of the Martian surface that display abundant evidence of aqueous alteration that occurred on their parent planet (Agee et al., 2013). These dark stones are also unique in the fact that they arose from a near surface level in the Noachian southern hemisphere (Humayun et al., 2013). We used IR spectroscopy, Fe-XANES and petrography to identify the mineral hosts of hydrogen in NWA 7533 and compare them with observations of the Martian surface and results of laboratory experiments. The spectrum of NWA 7533 does not show mafic mineral absorptions, making its definite identification difficult through NIR remote sensing mapping. However, its spectra are virtually consistent with a large fraction of the Martian highlands. Abundant NWA 7034/7533 (and paired samples) lithologies might abound on Mars and might play a role in the dust production mechanism. (C) 2015 Elsevier B.V. All rights reserved.

OSIRIS observations of meter-sized exposures of H2O ice at the surface of 67P/Churyumov-Gerasimenko and interpretation using laboratory experiments

Since OSIRIS started acquiring high-resolution observations of the surface of the nucleus of comet 67P/Churyumov-Gerasimenko, over one hundred meter-sized bright spots have been identified in numerous types of geomorphologic regions, but mostly located in areas receiving low insolation. The bright spots are either clustered, in debris fields close to decameter-high cliffs, or isolated without structural relation to the surrounding terrain. They can be up to ten times brighter than the average surface of the comet at visible wavelengths and display a significantly bluer spectrum. They do not exhibit significant changes over a period of a few weeks. All these observations are consistent with exposure of water ice at the surface of boulders produced by dislocation of the weakly consolidated layers that cover large areas of the nucleus. Laboratory experiments show that under simulated comet surface conditions, analog samples acquire a vertical stratification with an uppermost porous mantle of refractory dust overlaying a layer of hard ice formed by recondensation or sintering under the insulating dust mantle. The evolution of the visible spectrophotometric properties of samples during sublimation is consistent with the contrasts of brightness and color seen at the surface of the nucleus. Clustered bright spots are formed by the collapse of overhangs that is triggered by mass wasting of deeper layers. Isolated spots might be the result of the emission of boulders at low velocity that are redepositioned in other regions.