Effects of extraction buffer on the solubility and immunoreactivity of the pacific oyster allergens

Despite recent technological advances, novel allergenic protein discovery is limited by their low abundance, often due to specific physical characteristics restricting their recovery during the extraction process from various allergen sources. In this study, eight different extraction buffers were compared for their ability to recover proteins from Pacific oyster (Crassostrea gigas). The protein composition was investigated using high resolution mass spectrometry. The antibody IgE-reactivity of each extract was determined using a pool of serum from five shellfish-allergic patients. Most of the investigated buffers showed good capacity to extract proteins from the Pacific oyster. In general, a higher concentration of proteins was recovered using high salt buffers or high pH buffers, subsequently revealing more IgE-reactive bands on immunoblotting. In contrast, low pH buffers resulted in a poor protein recovery and reduced IgE-reactivity. Discovery of additional IgE-reactive proteins in high salt buffers or high pH buffers was associated with an increase in allergen abundance in the extracts. In conclusion, increasing the ionic strength and pH of the buffer improves the solubility of allergenic proteins during the extraction process for oyster tissue. This strategy could also be applied for other difficult-to-extract allergen sources, thereby yielding an improved allergen panel for increased diagnostic efficiency

Identification of novel oyster allergens using a combined transcriptomic and proteomic approach for improved component resolved diagnosis

Background: Increasing production and consumption of mollusc is associated with the rise in prevalence of mollusc allergy worldwide, currently ranging from 0.15% to 1.3% of the general population. However, the elucidation of mollusc allergens for better diagnostics still lags behind other seafood groups such as fish and crustacean. Genomic data have been utilized previously for improved identification of non-food allergens by performing similarity searching using the BLAST program. Based on the published genome of the Pacific oyster (Crassostrea gigas) we aimed to identify the complete potential oyster allergen repertoire using ioinformatics analysis, and to investigate identified protein allergenicity using a combination of immuno-chemical methods and proteomic analysis. Results: Ninety-five potential allergenic proteins of the Pacific oyster were discovered using in silico analyses. These proteins were of same protein family and had more than 50% amino acid identity with their homologous allergens. The allergenicity of these proteins was characterized using a combination of immunoassay and transcriptome-derived proteomics analyses. However The 2D-immunoblotting results showed only twenty two IgE-reactive spots in the raw extract of the Pacific oyster, and six spots in the heated extract. The identity of these IgE-reactive proteins was investigated by mass spectrometry. Sixteen allergens were identified, some with two or more isoforms. Conclusions: The combination of genomics coupled to proteomics and IgE-reactivity profiling is a powerful method for the identification of novel allergens from food sources. Using this combination approach we were able to expand the current knowledge on IgE-reactivity to various proteins of the Pacific oyster. These newly identified allergens and knowledge of their gene sequences will facilitate the development of improved component resolved diagnosis and future immunotherapy approach for oyster allergy

Collagen-an important fish allergen for improved diagnosis

Background Fish collagen is widely used in medicine, cosmetics, and the food industry. However, its clinical relevance as an allergen is not fully appreciated. This is likely due to collagen insolubility in neutral aqueous solutions, leading to low abundance in commercially available in vitro and skin prick tests for fish allergy. Objective To investigate the relevance of fish collagen as an allergen in a large patient population (n = 101). Methods Acid-soluble collagen type I was extracted from muscle and skin of Atlantic salmon, barramundi, and yellowfin tuna. IgE binding to collagen was analyzed by ELISA for 101 fish-allergic patients. Collagen-sensitized patients' sera were tested for IgE binding to parvalbumin from the same fish species. IgE cross-linking was analyzed by rat basophil leukemia assay and basophil activation test. Protein identities were confirmed by mass spectrometry. Results Purified fish collagen contained type I α1 and α2 chains and their multimers. Twenty-one of 101 patients (21%) were sensitized to collagen. Eight collagen-sensitized patients demonstrated absence of parvalbumin-specific IgE to some fish species. Collagen induced functional IgE cross-linking, as shown by rat basophil leukemia assay performed using 6 patients' sera, and basophil activation test using fresh blood from 1 patient. Collagen type I α chains from barramundi and Atlantic salmon were registered at www.allergen.org as Lat c 6 and Sal s 6, respectively. Conclusions IgE sensitization and IgE cross-linking capacity of fish collagen were demonstrated in fish-allergic patients. Inclusion of relevant collagen allergens in routine diagnosis is indicated to improve the capacity to accurately diagnose fish allergy

Optimal foreign borrowing: The impact of the planning horizon on the half and full debt cycle

Shrimp is one of the predominant causes of food allergy among adults, often presenting with severe reactions. Current in vitro diagnostics are based on quantification of patient specific-IgE (sIgE) to shrimp extract. Tropomyosin is the known major shrimp allergen, but IgE sensitisation to other allergens is poorly characterised. In this study, the binding of IgE to various shrimp allergens, additional to tropomyosin, was investigated using sera from 21 subjects who had clinical reactions to one or more shellfish species. Total shrimp-sIgE was quantified using ImmunoCAP, while allergen-sIgEs were quantified using immunoblotting and mass spectrometry, and immuno-PCR to recombinant shrimp tropomyosin. Sixty-two percent of subjects (13/21) were positive to shrimp by ImmunoCAP. IgE from 43% of subjects (9/21) bound tropomyosin, while an additional 29% of subjects (6/21) demonstrated IgE-binding solely to other shrimp allergens, including sarcoplasmic calcium-binding protein, arginine kinase and hemocyanin. Furthermore, IgE sensitisation to other shrimp allergens was demonstrated in 50% of subjects (4/8) who were ImmunoCAP negative. The lack of standardised shrimp allergens and inadequacy of current extracts for shrimp allergy diagnosis is highlighted by this study. Comprehensive knowledge of less studied allergens and their inclusion in component-resolved diagnostics will improve diagnostic accuracy, benefitting the wider population suffering from shellfish allergy

Induction of IgG2 and IgG4 B-cell memory following sublingual immunotherapy for ryegrass pollen allergy

Background: While treatment for atopic rhinitis is aimed mostly to relieve symptoms, only allergen-specific immunotherapy (AIT) is targeted to modify the natural history of allergic diseases. This results in sustained clinical tolerance, even when treatment has stopped. The immunomodulatory effects of AIT are attributed mainly to increased regulatory T-cell function and increased allergen-specific IgG4, yet little is known about the effect on the memory B-cell compartment. Objective: We aimed to examine the effects of AIT on the IgE- and IgG subclass-expressing memory B cells. Methods: We recruited 29 patients with atopic seasonal rhinoconjunctivitis and performed a longitudinal analysis of the peripheral immune compartment before, during, and after sublingual immunotherapy (SLIT) for allergy to temperate grass pollen, predominantly to ryegrass pollen (RGP; Lolium perenne). Using flow cytometry on peripheral blood mononuclear cells and serum immunoassays, we analyzed the effects of a 4 months preseasonal treatment regimen comprising two or three courses in consecutive years on circulating IgE+ and IgG+ memory B cells and allergen-specific Ig levels. Results: SLIT increased RGP-specific serum IgG2 and IgG4, as well as the frequencies of IgG2 + and IgG4 + memory B cells, whereas no effect was observed on the IgE+ memory B-cell compartment. Furthermore, SLIT enhanced proportions of regulatory T cells specific to RGP. These changes were associated with clinical improvement. Conclusion: Our data provide evidence for immunological effects of SLIT on B-cell memory. Skewing responses toward IgG2 and IgG4 subclasses might be a mechanism to suppress IgE-mediated allergic responses

Definition of the viral targets of protective HIV-1-specific T cell responses

<p>Abstract</p> <p>Background</p> <p>The efficacy of the CTL component of a future HIV-1 vaccine will depend on the induction of responses with the most potent antiviral activity and broad HLA class I restriction. However, current HIV vaccine designs are largely based on viral sequence alignments only, not incorporating experimental data on T cell function and specificity.</p> <p>Methods</p> <p>Here, 950 untreated HIV-1 clade B or -C infected individuals were tested for responses to sets of 410 overlapping peptides (OLP) spanning the entire HIV-1 proteome. For each OLP, a "protective ratio" (PR) was calculated as the ratio of median viral loads (VL) between OLP non-responders and responders.</p> <p>Results</p> <p>For both clades, there was a negative relationship between the PR and the entropy of the OLP sequence. There was also a significant additive effect of multiple responses to beneficial OLP. Responses to beneficial OLP were of significantly higher functional avidity than responses to non-beneficial OLP. They also had superior in-vitro antiviral activities and, importantly, were at least as predictive of individuals' viral loads than their HLA class I genotypes.</p> <p>Conclusions</p> <p>The data thus identify immunogen sequence candidates for HIV and provide an approach for T cell immunogen design applicable to other viral infections.</p

CTL Responses of High Functional Avidity and Broad Variant Cross-Reactivity Are Associated with HIV Control

Cytotoxic T lymphocyte (CTL) responses targeting specific HIV proteins, in particular Gag, have been associated with relative control of viral replication in vivo. However, Gag-specific CTL can also be detected in individuals who do not control the virus and it remains thus unclear how Gag-specific CTL may mediate the beneficial effects in some individuals but not in others. Here, we used a 10mer peptide set spanning HIV Gag-p24 to determine immunogen-specific T-cell responses and to assess functional properties including functional avidity and cross-reactivity in 25 HIV-1 controllers and 25 non-controllers without protective HLA class I alleles. Our data challenge the common belief that Gag-specific T cell responses dominate the virus-specific immunity exclusively in HIV-1 controllers as both groups mounted responses of comparable breadths and magnitudes against the p24 sequence. However, responses in controllers reacted to lower antigen concentrations and recognized more epitope variants than responses in non-controllers. These cross-sectional data, largely independent of particular HLA genetics and generated using direct ex-vivo samples thus identify T cell responses of high functional avidity and with broad variant reactivity as potential functional immune correlates of relative HIV control

The structure of the caspase recruitment domain of BinCARD reveals that all three cysteines can be oxidized

The caspase recruitment domain (CARD) is present in death-domain superfamily proteins involved in inflammation and apoptosis. BinCARD is named for its ability to interact with Bcl10 and inhibit downstream signalling. Human BinCARD is expressed as two isoforms that encode the same N-terminal CARD region but which differ considerably in their C-termini. Both isoforms are expressed in immune cells, although BinCARD-2 is much more highly expressed. Crystals of the CARD fold common to both had low symmetry (space group P1). Molecular replacement was unsuccessful in this low-symmetry space group and, as the construct contains no methionines, first one and then two residues were engineered to methionine for MAD phasing. The double-methionine variant was produced as a selenomethionine derivative, which was crystallized and the structure was solved using data measured at two wavelengths. The crystal structures of the native and selenomethionine double mutant were refined to high resolution (1.58 and 1.40 Å resolution, respectively), revealing the presence of a cis-peptide bond between Tyr39 and Pro40. Unexpectedly, the native crystal structure revealed that all three cysteines were oxidized. The mitochondrial localization of BinCARD-2 and the susceptibility of its CARD region to redox modification points to the intriguing possibility of a redox-regulatory role

PIF Genes Mediate the Effect of Sucrose on Seedling Growth Dynamics

As photoautotrophs, plants can use both the form and amount of fixed carbon as a measure of the light environment. In this study, we used a variety of approaches to elucidate the role of exogenous sucrose in modifying seedling growth dynamics. In addition to its known effects on germination, high-resolution temporal analysis revealed that sucrose could extend the number of days plants exhibited rapid hypocotyl elongation, leading to dramatic increases in ultimate seedling height. In addition, sucrose changed the timing of daily growth maxima, demonstrating that diel growth dynamics are more plastic than previously suspected. Sucrose-dependent growth promotion required function of multiple phytochrome-interacting factors (PIFs), and overexpression of PIF5 led to growth dynamics similar to plants exposed to sucrose. Consistent with this result, sucrose was found to increase levels of PIF5 protein. PIFs have well-established roles as integrators of response to light levels, time of day and phytohormone signaling. Our findings strongly suggest that carbon availability can modify the known photomorphogenetic signaling network