Novel in vitro diagnosis of equine allergies using a protein array and mathematical modelling approach: a proof of concept using insect bite hypersensitivity.

Insect bite hypersensitivity (IBH) is a seasonal recurrent skin allergy of horses caused by IgE-mediated reactions to allergens present in the saliva of biting insects of the genus Culicoides, and possibly also Simulium and Stomoxys species. In this work we show that protein microarrays containing complex extracts and pure proteins, including recombinant Culicoides allergens, can be used as a powerful technique for the diagnosis of IBH. Besides the obvious advantages such as general profiling and use of few microliters of samples, this microarray technique permits automation and allows the generation of mathematical models with the calculation of individual risk profiles that can support the clinical diagnosis of allergic diseases. After selection of variables on influence on the projection (VIP), the observed values of sensitivity and specificity were 1.0 and 0.967, respectively. This confirms the highly discriminatory power of this approach for IBH and made it possible to attain a robust predictive mathematical model for this disease. It also further demonstrates the specificity of the protein array method on identifying a particular IgE-mediated disease when the sensitising allergen group is known.

Prediction of tolerance in children with IgE mediated cow's milk allergy by microarray profiling and chemometric approach.

The sera of a retrospective cohort (n=41) composed of children with well characterized cow's milk allergy collected from multiple visits were analyzed using a protein microarray system measuring four classes of immunoglobulins. The frequency of the visits, age and gender distribution reflected real situation faced by the clinicians at a pediatric reference center for food allergy in São Paulo, Brazil. The profiling array results have shown that total IgG and IgA share similar specificity whilst IgM and in particular IgE are distantly related. The correlation of specificity of IgE and IgA is variable amongst the patients and this relationship cannot be used to predict atopy or the onset of tolerance to milk. The array profiling technique has corroborated the clinical selection criteria for this cohort albeit it clearly suggested that 4 out of the 41 patients might have allergies other than milk origin. There was also a good correlation between the array data and ImmunoCAP results, casein in particular. By using qualitative and quantitative multivariate analysis routines it was possible to produce validated statistical models to predict with reasonable accuracy the onset of tolerance to milk proteins. If expanded to larger study groups, the array profiling in combination with the multivariate techniques show potential to improve the prognostic of milk allergic patients.

Multiple protein extract microarray for profiling human food-specific immunoglobulins A, M, G and E.

Existing food immunoglobulin (Ig) tests require large volumes of serum, are limited to one immunoglobulin class, are not amenable to high throughput analysis and only give a limited picture of the immunological response to food antigens. Conversely a new generation of Component Resolved Diagnostic systems using pure proteins is highly specific and totally dependent on the availability of the protein in its recombinant or natural origin form. Here we demonstrate a proof-of-concept of a microarray test based on protein extracts of food components. Our approach relies on innovations on three different fronts: the novelty of using arrayed food samples sequentially extracted with detergent and chaotropic agents, the ability to measure four different Ig classes simultaneously and the ability to analyse the generated data via a suitable bioinformatics/statistical analysis interface. This approach combines high numerical power of microarrays with automation, high throughput analysis and enables detailed investigation of the Ig profiles to food antigens. The prototype shown contains extracts of approximately 350 food ingredients that cover most of the food products found in the UK. Here we showed that the use of a sequential extraction technique to solubilise and then denature food samples has its benefits in the assessment of variations in antigenicity when tested with human sera. A patient dependent degree of class specificity was observed with human sera (IgG specificity correlates well with IgA>IgM>>>>>IgE). Besides generating a simultaneous profile for IgA, IgM, IgG and IgE the array system has shown good discrimination between challenge responders in atopic and non-atopic individuals. Poly- and mono-specific IgE responders were easily identified. The mathematical modelling of specific IgE content showed good correlations when compared with established IgE antibody testing assay (UniCAP). Although in its proof-of-principle stages, the immune profiling technique described here has the potential to provide unique insights into exposure/sensitization and establish relationships between specific immunoglobulin classes and subclasses against food protein antigens. In further developments, the immune profiling technique could also be extended to other related areas such as parasite and bacterial gut infection. Full analyses of large longitudinal and retrospective clinical trials are on going to determine the positive and negative predictive values of the technique.

A novel tool for the detection of allergic sensitization combining protein microarrays with human basophils.

BACKGROUND: Protein microarray (PM) is a powerful alternative to costly or labour-intensive diagnostic for the large-scale detection of allergen-specific IgE. In this study, we established a proof-of-concept that coupling the diversity of protein array with the biological output of basophilic cells is a feasible proposition. METHOD: Human basophils purified from the peripheral blood of healthy donors were stripped, re-sensitized with the serum or IgE preparation to be tested, and incubated with manually spotted protein array chips (FAST slides). The basophilic cell lines KU-812 and RBL-703/21 likewise sensitized were compared with peripheral blood basophils by the same approach. Purified basophils or other basophilic cells were incubated with FAST slides for various periods of time, washed, and cell binding was visualized by light microscopy. Basophil activation, indicating the effective cross-linking of IgE by allergens, was monitored via up-regulation of basophil activation surface marker (CD 63). RESULTS: Purified stripped peripheral basophils, re-sensitized with the serum of a grass pollen-allergic patient, displayed strong binding to anti-IgE antibody and grass pollen extract with relatively low unspecific binding. Similar results were obtained with RBL-703/21, which may be a good replacement for peripheral basophils to avoid the costly, cumbersome and time-consuming basophil purification. CONCLUSION: Our data suggest that coupling the diversity of a PM approach with the potential functionality and biological activity of a cell-based test is feasible and may result in a new system to detect allergic sensitization.

Influence of plant lipids on immune responses in mice to the major Brazil nut allergen Ber e 1.

BACKGROUND: Lipids, particularly bacterial lipopolysaccharide, can impact on immune responses to proteins, with low doses enhancing type 2 responses. OBJECTIVE: We have examined the influence of natural plant lipid extracts on antibody responses provoked in mice by recombinant Ber e 1, the major allergen in Brazil nuts. METHODS: BALB/c strain mice were immunized (by intraperitoneal injection) with natural or recombinant Ber e l produced in Pichia pastoris and admixed with various lipid fractions isolated from Brazil nuts. Serum samples were analysed for specific IgE antibody by homologous passive cutaneous anaphylaxis assay and for IgG by enzyme-linked immunosorbant assay. RESULTS: Exposure to recombinant (lipid-free) Ber e 1 alone failed to induce detectable IgG or IgE antibody. Co-administration of the total lipid fraction (with reduced triglyceride levels), sterol-rich, or polar lipid fractions, resulted in marked adjuvant effects on IgG and IgE. However, the beta-sitosterol and glycolipid-rich fractions were associated with only low-level IgG antibody, and had little impact on IgE antibody production. Natural Ber e 1 containing endogenous lipids also provoked IgG and IgE antibody responses. Identical IgE and IgG antibody responses were detected regardless of whether natural or recombinant Ber e 1 was used as substrates for analyses. CONCLUSION: Endogenous Brazil nut lipids are required for the induction of optimal antibody responses to Ber e 1 in the BALB/c strain mouse. Appropriate antibody binding sites are present on both natural and recombinant forms of Ber e 1, suggesting that the impact of lipid is at the induction phase, rather than antibody recognition, and is possibly required for efficient antigen presentation.

Phosphorylcholine mimics the effects of ES-62 on macrophages and dendritic cells.

Modulation of macrophage/dendritic cell (DC) cytokine production by the filarial nematode phosphorylcholine (PC)-containing product, ES-62, is mediated by Toll-like receptor (TLR) 4 and signal transduction depends on the TLR adaptor MyD88. Intriguingly, comparison of TLR4 knock-out (ko) mice with TLR4 mutant C3H/HeJ mice indicates that ES-62 cytokine responses are not dependent on the Pro712 residue of TLR4, which is crucial for the response to bacterial lipopolysaccharide (LPS). Because other immunomodulatory effects of ES-62 have been attributed to PC we have now investigated, using PC conjugated to ovalbumin (PC-Ova), whether PC is responsible for the interaction of ES-62 with TLR4. PC-Ova mimicked the modulation of interleukin (IL)-12 production by ES-62 in a TLR4- and MyD88-dependent manner and as with native ES-62, PC-Ova effects were not dependent on Pro712. Furthermore, both native ES-62 and PC-Ova suppressed Akt phosphorylation, whereas neither altered the activation of p38 or Erk MAP kinases. To rule out any role for the ES-62 protein component, we tested a PC-free recombinant ES-62 (rES-62) generated in the yeast Pichia pastoris. Surprisingly, rES-62 also modulated IL-12 production, but in a TLR4/MyD88-independent manner. Furthermore, rES-62 strongly activated both the p38 and Erk MAP kinases and Akt. However, recent biophysical analysis suggests there are differences in folding/shape between native and rES-62 and hence data obtained with the latter should be treated with caution. Nevertheless, although our study indicates that PC is likely to be primarily responsible for the modulation of cytokine production observed with native ES-62, an immunomodulatory role for the protein component cannot be ruled out.

Lack of immunological cross-reactivity between parasite-derived and recombinant forms of ES-62, a secreted protein of Acanthocheilonema viteae.

The longevity of filarial nematodes is dependent on secreted immunomodulatory products. Previous investigation of one such product, ES-62, has suggested a critical role for post-translationally attached phosphorylcholine (PC) moieties. In order to further investigate this, ES-62 lacking PC was produced, using the Pichia pastoris recombinant gene expression system. Unlike parasite-derived ES-62, which is tetrameric the recombinant material was found to consist of a mixture of apparently stable tetramers, dimers and monomers. Nevertheless, the recombinant protein was considered to be an adequate PC-free ES-62 as it was recognized by existing antisera against the parasite-derived protein. However, subsequent to this, recognition of parasite-derived ES-62 by antibodies produced against the recombinant protein was found to be absent. In an attempt to explain this, recombinant ES-62 was subjected to structural analysis and was found to (i) contain 3 changes in amino acid composition; (ii) demonstrate significant alterations in glycosylation; (iii) show major differences in protein secondary structure. The effects of these alterations in relation to the observed change in immunogenicity were investigated and are discussed. The data presented clearly show that recognition by existing antibodies is insufficient proof that recombinant proteins can be used to mimic parasite-derived material in studies on nematode immunology and vaccination.

In vitro stability and immunoreactivity of the native and recombinant plant food 2S albumins Ber e 1 and SFA-8.

BACKGROUND: The ability of an intact protein to reach the circulatory system may be a prerequisite to allergenicity and many allergens, particularly those from plant foods, have been found to be consistently more resistant to digestion by pepsin than other proteins. OBJECTIVE: This study assessed the pepsinolytic stability of native 2S albumins from Brazil nut and sunflower seed and their recombinant versions produced in Pichia pastoris. The physicochemical stability of native and recombinant Brazil nut 2S albumins and recombinant sunflower seed 2S albumin was also assessed. The immunoreactivity of native Brazil nut 2S albumin and recombinant 2S albumins was compared using serum from patients allergic to Brazil nuts and animals immunized with native 2S albumins. METHODS: Digestibility was measured in simulated gastric fluid followed by SDS-PAGE. Circular dichroism spectra were used to analyse unfolding, as proteins were denatured by temperature, pH and guanidinium chloride. Immunoreactivity was assessed by immunoblot, RAST and ELISA. RESULTS: Brazil nut 2S albumin was significantly more resistant to proteolytic digestion than other Brazil nut proteins. It was also resistant to thermally and chemically induced denaturation. Equally high resistance to proteolytic digestion was observed with sunflower seed 2S albumin. The recombinant albumins mirrored their native counterparts in stability and immunoreactivity. CONCLUSION: The important food allergen Brazil nut 2S albumin is as stable to digestion as is sunflower seed 2S albumin, whose allergenicity has yet to be determined. The 2S albumins and their recombinant counterparts could not be easily denatured by physicochemical treatments. The results suggest that 2S albumin is the only Brazil nut protein to reach the gut immune system intact. The production of properly folded recombinant proteins will facilitate mechanistic studies as well as diagnostic testing and antigen-based therapies.

Comparison of modular and non-modular xylanases as carrier proteins for the efficient secretion of heterologous proteins from Penicillium funiculosum.

Genes encoding three enzymes with xylanase activity from the filamentous fungus Penicillium funiculosum are described. Two of the encoded xylanases are predicted to be modular in structure with catalytic and substrate-binding domains separated by a serine and threonine-rich linker region; the other had none of these properties and was non-modular. In order to develop P. funiculosum as a host for the secreted production of heterologous proteins, each of the xylanases was assessed for use as a carrier protein in a fusion strategy. We show that one of the modular xylanases (encoded by xynA) was an effective carrier protein but the other (encoded by xynB) and the non-modular xylanase (encoded by xynC) were not effective as secretion carriers. We show that the beta-glucuronidase (GUS) protein from Escherichia coli is secreted by P. funiculosum when expressed as an XYNA fusion but that the secreted GUS protein, cleaved in vivo from XYNA, is glycosylated and enzymatically inactive.

Use of a histone H4 promoter to drive the expression of homologous and heterologous proteins by Penicillium funiculosum.

Two genes encoding histone H4 (H4.1 and H4.2) from Penicillium funiculosum have been cloned and characterised. Structurally, the histone H4.1 gene is divergently linked to the histone H3 gene and the two genes are separated by approximately 800 bp. The transcription of the histone H4.1 and H4.2 genes in P. funiculosum appears to be distinctively regulated. Histone H4.1 mRNA showed a high steady-state level during the early stages of batch culture that decreased as growth reached the stationary phase. In contrast, the expression of the histone H4.2 gene was lower than that of H4.1 throughout batch growth and increased gradually with time. In order to expand the industrial application of P. funiculosum as a host for the production of heterologous proteins, the promoter of the histone H4.1 gene was successfully used to drive the expression of an intracellular bacterial enzyme, beta-glucuronidase, and a secreted homologous enzyme, xylanase C. The constitutive secretion of xylanase C was achieved in the absence of other xylanases by batch fermentation in the presence of glucose.