Desiccation tolerance in the resurrection plant Barbacenia graminifolia involves changes in redox metabolism and carotenoid oxidation.

Desiccation tolerance in vegetative tissues enables resurrection plants to remain quiescent under severe drought and rapidly recover full metabolism once water becomes available. Barbacenia graminifolia is a resurrection plant that occurs at high altitudes, typically growing on rock slits, exposed to high irradiance and limited water availability. We analyzed the levels of reactive oxygen species (ROS) and antioxidants, carotenoids and its cleavage products, and stress-related phytohormones in fully hydrated, dehydrated, and rehydrated leaves of B. graminifolia. This species exhibited a precise adjustment of its antioxidant metabolism to desiccation. Our results indicate that this adjustment is associated with enhanced carotenoid and apocarotenoids, α-tocopherol and compounds of ascorbate-glutathione cycle. While α-carotene and lutein increased in dried-leaves suggesting effective protection of the light-harvesting complexes, the decrease in ß-carotene was accompanied of 10.2-fold increase in the content of ß-cyclocitral, an apocarotenoid implicated in the regulation of abiotic stresses, compared to hydrated plants. The principal component analysis showed that dehydrated plants at 30 days formed a separate cluster from both hydrated and dehydrated plants for up to 15 days. This regulation might be part of the protective metabolic strategies employed by this resurrection plant to survive water scarcity in its inhospitable habitat.

Recombinant antigen 5 from Polybia paulista wasp venom (Hymenoptera, Vespidae): Antigen-specific antibody production and functional profile of CD4+ T cells in the immune response.

Polybia paulista is a neotropical social wasp related to severe accidents and allergic reactions cases, including anaphylaxis, in southeastern Brazil. Antigen 5 (Poly p 5) is a major allergenic protein from its venom with potential use for component-resolved diagnostic. Therefore, the previous characterization of the immune response profile triggered by Poly p 5 should be evaluated. Recombinant Poly p 5 (rPoly p 5) was used to sensitize BALB/c mice with six weekly intradermal doses, and the specific antibody production and the functional profile of CD4+ T cells were assessed. rPoly p 5 induced the production of specific immunoglobulins (sIg) sIgE, sIgG1 and sIgG2a, which could recognize natural Poly p 5 presented in the venom of four different wasp species. rPoly p 5 stimulated in vitro the CD4+ T cells from immunized mice, which showed a significant proliferative response. These antigen-specific CD4+T cells produced IFN-γ and IL-17A cytokines and increased ROR-γT transcription factor expression. No differences between the control group and sensitized mice were found in IL-4 production and GATA-3 and T-bet expression. Interestingly, increased CD25+FoxP3+ regulatory T cells (Tregs) frequency was observed in the splenocyte cell cultures from rPoly p 5 immunized mice after the in vitro stimulation with both P. paulista venom extract and rPoly p 5. Here we showed that rPoly p 5 induces antigen-specific antibodies capable of recognizing Antigen 5 in the venom of four wasp species and modulates antigen-specific CD4+ T cells to IFN-γ production response associated with a Th17 profile in sensitized mice. These findings emphasize the potential use of rPoly p 5 as an essential source of a major wasp allergen with significant immunological properties.

Noncoding RNAs responsive to nitric oxide and their protein-coding gene targets shed light on root hair formation in Arabidopsis thaliana.

An overview of the total Arabidopsis thaliana transcriptome, described previously by our research group, pointed some noncoding RNA (ncRNA) as participants in the restoration of hair-root phenotype in A. thaliana rhd6 mutants, leading us to a deeper investigation. A transcriptional gene expression profiling of seedling roots was performed aiming to identify ncRNA responsive to nitric oxide (GSNO) and auxin (IAA), and their involvement in root hair formation in the rhd6 null mutant. We identified 3,631 ncRNAs, including new ones, in A. thaliana and differential expression (DE) analysis between the following: 1) GSNO-treated rhd6 vs. untreated rhd6, 2) IAA-treated rhd6 vs. untreated rhd6, 3) GSNO-treated rhd6 vs. IAA-treated rhd6, and 4) WS-2 vs. untreated rhd6 detected the greatest number of DE genes in GSNO-treated rhd6. We detected hundreds of in silico interactions among ncRNA and protein-coding genes (PCGs), highlighting MIR5658 and MIR171 precursors highly upregulated in GSNO-treated rhd6 and wild type, respectively. Those ncRNA interact with many DE PCGs involved in hormone signaling, cell wall development, transcription factors, and root hair formation, becoming candidate genes in cell wall modulation and restoration of root hair phenotype by GSNO treatment. Our data shed light on how GSNO modulates ncRNA and their PCG targets in A. thaliana root hair formation.

Water retention and metabolic changes improve desiccation tolerance in Barbacenia graminifolia (Velloziaceae).

Barbacenia graminifolia is a Velloziaceae species endemic to the campos rupestres in Serra do Cipó, Minas Gerais state (Brazil). This biome is characterised by high irradiance and limited water conditions. Unlike other resurrection plants, B. graminifolia can maintain a high hydric status (>80%) after 28 days of water suppression before desiccation. We investigated the physiological and metabolic mechanisms associated with structural changes that allow B. graminifolia to maintain hydration under a prolonged water deficit and to recover after desiccation. After 30 days of water deficit, desiccated plants exhibited chlorophyll degradation, a 178.4% and 193.7% increase in total carotenoids and MDA, respectively, and twice the CAT and APX activity compared to hydrated plants. The metabolite profile showed increased amino acids, carbohydrates, saturated fatty acids and benzoic acids during dehydration, while trichloroacetic acid cycle acids were higher in hydrated and rehydrated plants. Anatomical and ultrastructural data corroborated the physiological and metabolic changes and revealed the presence of mucilaginous cells with high water retention capacity. Our data indicated that combined strategies of assimilatory metabolism shutdown, accumulation of compatible solutes and antioxidant compounds, increase in hydrophilic molecules, changes in the composition of membrane lipids and remodelling of cell organelles conditioned the efficiency of B. graminifolia in delaying water loss, tolerating further desiccation and quickly recovering after rehydration. These attributes evidence that this species is well adapted to cope with adverse environmental conditions, mainly directing the metabolism to an efficient antioxidant response and improving its capacity to retain water during the dry season.

A comparative study between Fusarium solani and Neocosmospora vasinfecta revealed differential profile of fructooligosaccharide production.

Fructooligosaccharides (FOS) are fructose-based oligosaccharides employed as additives to improve the food's nutritional and technological properties. The rhizosphere of plants that accumulate fructopolysaccharides as inulin has been revealed as a source of filamentous fungi. These fungi can produce FOS either by inulin hydrolysis or by biosynthesis from sucrose, including unusual FOS with enhanced prebiotic properties. Here, we investigated the ability of Fusarium solani and Neocosmospora vasinfecta to produce FOS from different carbon sources. Fusarium solani and N. vasinfecta grew preferentially in inulin instead of sucrose, resulting in the FOS production as the result of endo-inulinase activities. N. vasinfecta was also able to produce the FOS 1-kestose and 6-kestose from sucrose, indicating transfructosylating activity, absent in F. solani. Moreover, the results showed how these carbon sources affected fungal cell wall composition and the expression of genes encoding for ß-1,3-glucan synthase and chitin synthase. Inulin and fructose promoted changes in fungal macroscopic characteristics partially explained by alterations in cell wall composition. However, these alterations were not directly correlated with the expression of genes related to cell wall synthesis. Altogether, the results pointed to the potential of both F. solani and N. vasinfecta to produce FOS at specific profiles.

Changes in cell wall composition and ultrastructure related to desiccation during the seed maturation of Paubrasilia echinata (brazilwood).

Paubrasilia echinata (brazilwood) is an endangered native tree from the Brazilian Atlantic Forest whose seeds tolerate maturation drying, but, unlike classic orthodox seeds, they quickly lose viability after shedding. This work analyzed the biochemical and ultrastructural changes during the maturation of brazilwood seeds, with particular attention to the cell walls and organization of the cellular components. The physiological seed maturity was accompanied by increased starch content and decreased soluble sugars. Arabinose increased considerably and was the predominant cell-wall sugar during maturation, suggesting a rise in arabinans that contribute to greater cell wall flexibility. This increase was consistent with the cell wall infolding observed in the hypocotyl axis and cotyledons during the maturation of brazilwood seeds. Ultrastructural analyses showed changes in the number and distribution of protein bodies and amyloplasts and the reorganization of lipid droplets into large drops or masses during seed desiccation. Our findings demonstrate that brazilwood seeds behave like other orthodox seeds during maturation, performing the cell wall and metabolic changes before the major decline in the seed water content. However, the high vacuolization and reorganization of lipid bodies observed at 65 DAA suggest that cell deterioration occurs to some extent at the end of the maturation period and could be responsible for reducing the longevity of the brazilwood dried seeds.

Biological and Inflammatory Effects of Antigen 5 from Polybia paulista (Hymenoptera, Vespidae) Venom in Mouse Intraperitoneal Macrophages.

The social wasp Polybia paulista (Hymenoptera, Vespidae) is highly aggressive, being responsible for many medical occurrences. One of the most allergenic components of this venom is Antigen 5 (Poly p 5). The possible modulation of the in vitro immune response induced by antigen 5 from P. paulista venom, expressed recombinantly (rPoly p 5), on BALB/c mice peritoneal macrophages, activated or not with LPS, was assessed. Here, we analyzed cell viability changes, expression of the phosphorylated form of p65 NF-κB subunit, nitric oxide (NO), proinflammatory cytokines production, and co-stimulatory molecules (CD80, CD86). The results suggest that rPoly p 5 does not affect NO production nor the expression of co-stimulatory molecules in mouse peritoneal macrophages. On the other hand, rPoly p 5 induced an increase in IL-1ß production in non-activated macrophages and a reduction in the production of TNF-α and MCP-1 cytokines in activated macrophages. rPoly p 5 decreased the in vitro production of the phosphorylated p65 NF-κB subunit in non-activated macrophages. These findings suggest an essential role of this allergen in the polarization of functional M2 macrophage phenotypes, when analyzed in previously activated macrophages. Further investigations, mainly in in vivo studies, should be conducted to elucidate Polybia paulista Ag5 biological role in the macrophage functional profile modulation.

Cross-Reactive Carbohydrate Determinant in Apis mellifera, Solenopsis invicta and Polybia paulista Venoms: Identification of Allergic Sensitization and Cross-Reactivity.

Allergic reactions to Hymenoptera venom, which could lead to systemic and even fatal symptoms, is characterized by hypersensitivity reactions mediated by specific IgE (sIgE) driven to venom allergens. Patients multisensitized to sIgE usually recognize more than one allergen in different Hymenoptera species. However, the presence of sIgE directed against Cross-Reactive Carbohydrate Determinant (CCD), which occurs in some allergens from Hymenoptera venom, hampers the identification of the culprit insects. CCD is also present in plants, pollen, fruits, but not in mammals. Bromelain (Brl) extracted from pineapples is a glycoprotein commonly used for reference to sIgE-CCD detection and analysis. In sera of fifty-one Hymenoptera allergic patients with specific IgE ≥ 1.0 KU/L, we assessed by immunoblotting the reactivity of sIgE to the major allergens of Apis mellifera, Polybia paulista and Solenopsis invicta venoms. We also distinguished, using sera adsorption procedures, the cases of CCD cross-reaction using Brl as a marker and inhibitor of CCD epitopes. The presence of reactivity for bromelain (24-28 kDa) was obtained in 43% of the patients, in which 64% presented reactivity for more than one Hymenoptera venom in radioallergosorbent (RAST) tests, and 90% showed reactivity in immunoblot analysis to the major allergens of Apis mellifera, Polybia paulista and Solenopsis invicta venoms. Sera adsorption procedures with Brl lead to a significant reduction in patients' sera reactivity to the Hymenoptera allergens. Immunoblotting assay using pre- and post-Brl adsorption sera from wasp-allergic patients blotted with non-glycosylated recombinant antigens (rPoly p1, rPoly p5) from Polybia paulista wasp venom showed no change in reactivity pattern of sIgE that recognize allergen peptide epitopes. Our results, using Brl as a marker and CCD inhibitor to test sIgE reactivity, suggest that it could complement diagnostic methods and help to differentiate specific reactivity to allergens' peptide epitopes from cross-reactivity caused by CCD, which is extremely useful in clinical practice.

Functional Profile of Antigen Specific CD4+ T Cells in the Immune Response to Phospholipase A1 Allergen from Polybia paulista Venom.

Insect venom can cause systemic allergic reactions, including anaphylaxis. Improvements in diagnosis and venom immunotherapy (VIT) are based on a better understanding of an immunological response triggered by venom allergens. Previously, we demonstrated that the recombinant phospholipase A1 (rPoly p 1) from Polybia paulista wasp venom induces specific IgE and IgG antibodies in sensitized mice, which recognized the native allergen. Here, we addressed the T cell immune response of rPoly p 1-sensitized BALB/c mice. Cultures of splenocytes were stimulated with Polybia paulista venom extract and the proliferation of CD8+ and CD4+ T cells and the frequency of T regulatory cells (Tregs) populations were assessed by flow cytometry. Cytokines were quantified in cell culture supernatants in ELISA assays. The in vitro stimulation of T cells from sensitized mice induces a significant proliferation of CD4+ T cells, but not of CD8+ T cells. The cytokine pattern showed a high concentration of IFN-γ and IL-6, and no significant differences to IL-4, IL-1ß and TGF-ß1 production. In addition, the rPoly p 1 group showed a pronounced expansion of CD4+CD25+FoxP3+ and CD4+CD25-FoxP3+ Tregs. rPoly p 1 sensitization induces a Th1/Treg profile in CD4+ T cell subset, suggesting its potential use in wasp venom immunotherapy.

Improved production of the recombinant phospholipase A1 from Polybia paulista wasp venom expressed in bacterial cells for use in routine diagnostics.

Phospholipase A1 (PLA1) is one of the three major allergens identified in the venom of P. paulista (Hymenoptera: Vespidae), a clinically relevant wasp from southeastern Brazil. The recombinant form of this allergen (rPoly p 1) could be used for the development of molecular diagnostic of venom allergy. Early attempts to produce rPoly p 1 using Escherichia coli BL21 (DE3) cells rendered high yields of the insoluble rPoly p 1 but with low levels of solubilized protein recovery (12%). Here, we aimed to improve the production of rPoly p 1 in E. coli by testing different conditions of expression, solubilization of the inclusion bodies and protein purification. The results showed that the expression at 16 °C and 0.1 mM of IPTG increased the production of rPoly p 1, still in the insoluble form, but with high solubilized protein yields after incubation with citrate-phosphate buffer with 0.15 M NaCl, 6 M urea, pH 2.6 at 25 ºC for 2 h. The venom allergen was also cloned in pPICZαA vector for soluble expression as a secreted protein in Pichia pastoris X-33 cells, rendering almost undetectable levels (nanograms) in the culture supernatant. In contrast, a sevenfold increase of the solubilized and purified rPoly p 1 yields (1.5 g/L of fermentation broth) was obtained after improved production in E. coli. The identity of the protein was confirmed with an anti-His antibody and MS spectra. Allergen-specific IgE (sIgE)-mediated recognition was evaluated in immunoblotting with sera of allergic patients (n = 40). Moreover, rPoly p 1 showed high levels of diagnostic sensitivity (95%). The optimized strategy for rPoly p 1 production described here, will provide the amounts of allergen necessary for the subsequent protein refolding, immunological characterization steps, and ultimately, to the development of molecular diagnostic for P. paulista venom allergy.

Metabolic Changes on the Acquisition of Desiccation Tolerance in Seeds of the Brazilian Native Tree Erythrina speciosa.

Erythrina speciosa Andrews (Fabaceae) is a native tree of Atlantic forest from Southern and Southeastern Brazil. Although this species is found in flooded areas, it produces highly desiccation tolerant seeds. Here, we investigated the physiological and metabolic events occurring during seed maturation of E. speciosa aiming to better understand of its desiccation tolerance acquisition. Seeds were separated into six stages of maturation by the pigmentation of the seed coat. Water potential (WP) and water content (WC) decreased gradually from the first stage to the last stage of maturation (VI), in which seeds reached the highest accumulation of dry mass and seed coat acquired water impermeability. At stage III (71% WC), although seeds were intolerant to desiccation, they were able to germinate (about 15%). Desiccation tolerance was first observed at stage IV (67% WC), in which 40% of seeds were tolerant. At stage V (24% WC), all seeds were tolerant to desiccation and at stage VI all seeds germinated. Increased deposition of the arabinose-containing polysaccharides, which are known as cell wall plasticizers polymers, was observed up to stage IV of seed maturation. Raffinose and stachyose gradually increased in axes and cotyledons with greater increment in the fourth stage. Metabolic profile analysis showed that levels of sugars, organic, and amino acids decrease drastically in embryonic axes, in agreement with lower respiratory rates during maturation. Moreover, a non-aqueous fractionation revealed a change on the proportions of sugar accumulation among cytosol, plastid, and vacuoles between the active metabolism (stage I) and the dormant seeds (stage VI). The results indicate that the physiological maturity of the seeds of E. speciosa is reached at stage V and that the accumulation of raffinose can be a result of the change in the use of carbon, reducing metabolic activity during maturation. This work confirms that raffinose is involved in desiccation tolerance in seeds of E. speciosa, especially considering the different subcellular compartments and suggests even that the acquisition of desiccation tolerance in this species occurs in stages prior to the major changes in WC.

Increasing atmospheric CO2 and canopy temperature induces anatomical and physiological changes in leaves of the C4 forage species Panicum maximum.

Changes in leaf anatomy and ultrastructure are associated with physiological performance in the context of plant adaptations to climate change. In this study, we investigated the isolated and combined effects of elevated atmospheric CO2 concentration ([CO2]) up to 600 µmol mol-1 (eC) and elevated temperature (eT) to 2°C more than the ambient canopy temperature on the ultrastructure, leaf anatomy, and physiology of Panicum maximum Jacq. grown under field conditions using combined free-air carbon dioxide enrichment (FACE) and temperature free-air controlled enhancement (T-FACE) systems. Plants grown under eC showed reduced stomatal density, stomatal index, stomatal conductance (gs), and leaf transpiration rate (E), increased soil-water content (SWC) conservation and adaxial epidermis thickness were also observed. The net photosynthesis rate (A) and intrinsic water-use efficiency (iWUE) were enhanced by 25% and 71%, respectively, with a concomitant increase in the size of starch grains in bundle sheath cells. Under air warming, we observed an increase in the thickness of the adaxial cuticle and a decrease in the leaf thickness, size of vascular bundles and bulliform cells, and starch content. Under eCeT, air warming offset the eC effects on SWC and E, and no interactions between [CO2] and temperature for leaf anatomy were observed. Elevated [CO2] exerted more effects on external characteristics, such as the epidermis anatomy and leaf gas exchange, while air warming affected mainly the leaf structure. We conclude that differential anatomical and physiological adjustments contributed to the acclimation of P. maximum growing under elevated [CO2] and air warming, improving the leaf biomass production under these conditions.

Production of inulin- and neolevan-type fructooligosaccharides by Penicillium janczewskii Zaleski CCIBt 3352.

Fructooligosaccharides (FOS) are fructose-based oligosaccharides employed as additives to improve the nutritional and technological properties of foods. The rhizosphere of inulin-accumulating plants from the Cerrado (Brazilian savanna) harbor fungi capable of synthesizing FOS from sucrose through the transfructosylating activity of ß-fructosyltransferases and/or ß-fructofuranosidases. Here, we investigated the ability of Penicillium janczewskii Zaleski CCIBt 3352, a fungus isolated from the rhizosphere of Chrysolaena obovata (Asteraceae), to produce FOS in a medium supplemented with sucrose concentrations of 30, 100, or 150 g L-1 . Hydrolytic activity on sucrose was observed in culture filtrates; however, at 150 g L-1 sucrose, the accumulation of 8 g L-1 1-kestose (inulin-type FOS) and 7.3 g L-1 neokestose (neolevan-type FOS) was observed, the latter being a type of FOS not commonly produced by filamentous fungi. In addition, minor amounts of four unidentified oligosaccharides, with a high degree of polymerization, were detected. The production of FOS was also observed in enzymatic assays, indicating the presence of extracellular enzymes with transfructosylating activity in the culture filtrates. Our findings demonstrate the feasibility of isolating promising microorganisms, for the production of FOS-synthesizing enzymes, from the rhizosphere of fructan-producing plants of the Brazilian Cerrado.

Warming and water deficit impact leaf photosynthesis and decrease forage quality and digestibility of a C4 tropical grass.

Global warming is predicted to cause more intense extreme events such as heat waves, flooding and severe droughts, producing significant effects on agriculture. In tropics, climate change will severely impact livestock production affecting water availability, forage quality and food for cattle. We investigated the isolated and combined effects of soil water deficit (wS) and + 2°C increase in canopy temperature (eT) on leaf gas exchange, chlorophyll fluorescence, carbohydrate content, forage quality and in vitro dry matter digestibility (IVDMD) of a field-grown C4 tropical forage grass Panicum maximum Jacq. using a temperature-free air-controlled enhancement (T-FACE) system. The wS and eT treatments showed no effects on photosystem II photochemistry. However, wS under ambient temperature decreased net photosynthesis rate (A), stomatal conductance (gs ) and maximum rate of carboxylation of Rubisco (Vcmax ), leading to a reduced starch content in leaves. A 16% reduction in leaf dry mass (LDM) and reduction in forage quality by increasing fibers, reducing crude protein (CP) and decreasing the IVDMD was also observed by effect of wS. Warming under adequate soil moisture (eT) significantly increased LDM by 25% but reduced the forage quality, increasing the lignin content and reducing starch, CP and digestibility. The combined wSeT treatment reduced A, gs , Vcmax and the forage quality. When compared to control, the lignin content in leaves increased by 43, 28 and 17% in wS, eT and wSeT, respectively, causing a significant reduction in IVDMD. We concluded that despite physiological mechanisms to acclimate to warming, both warming and water deficit will impair the quality and digestibility of C4 tropical pastures.

Current Advances in Immunological Studies on the Vespidae Venom Antigen 5: Therapeutic and Prophylaxis to Hypersensitivity Responses.

Although systemic reactions caused by allergenic proteins present in venoms affect a small part of the world population, Hymenoptera stings are among the main causes of immediate hypersensitivity responses, with risk of anaphylactic shock. In the attempt to obtain therapeutic treatments and prophylaxis to hypersensitivity responses, interest in the molecular characterization of these allergens has grown in the scientific community due to the promising results obtained in immunological and clinical studies. The present review provides an update on the knowledge regarding the immune response and the therapeutic potential of Antigen 5 derived from Hymenoptera venom. The results confirm that the identification and topology of epitopes, associated with molecular regions that interact with antibodies, are crucial to the improvement of hypersensitivity diagnostic methods.

Suppression of a single BAHD gene in Setaria viridis causes large, stable decreases in cell wall feruloylation and increases biomass digestibility.

Feruloylation of arabinoxylan (AX) in grass cell walls is a key determinant of recalcitrance to enzyme attack, making it a target for improvement of grass crops, and of interest in grass evolution. Definitive evidence on the genes responsible is lacking so we studied a candidate gene that we identified within the BAHD acyl-CoA transferase family. We used RNA interference (RNAi) silencing of orthologs in the model grasses Setaria viridis (SvBAHD01) and Brachypodium distachyon (BdBAHD01) and determined effects on AX feruloylation. Silencing of SvBAHD01 in Setaria resulted in a c. 60% decrease in AX feruloylation in stems consistently across four generations. Silencing of BdBAHD01 in Brachypodium stems decreased feruloylation much less, possibly due to higher expression of functionally redundant genes. Setaria SvBAHD01 RNAi plants showed: no decrease in total lignin, approximately doubled arabinose acylated by p-coumarate, changes in two-dimensional NMR spectra of unfractionated cell walls consistent with biochemical estimates, no effect on total biomass production and an increase in biomass saccharification efficiency of 40-60%. We provide the first strong evidence for a key role of the BAHD01 gene in AX feruloylation and demonstrate that it is a promising target for improvement of grass crops for biofuel, biorefining and animal nutrition applications.

Phospholipase A1-based cross-reactivity among venoms of clinically relevant Hymenoptera from Neotropical and temperate regions.

Molecular cross-reactivity caused by allergen homology or cross-reactive carbohydrate determinants (CCDs) is a major challenge for diagnosis and immunotherapy of insect venom allergy. Venom phospholipases A1 (PLA1s) are classical, mostly non-glycosylated wasp and ant allergens that provide diagnostic benefit for differentiation of genuine sensitizations from cross-reactivity. As CCD-free molecules, venom PLA1s are not causative for CCD-based cross-reactivity. Little is known however about the protein-based cross-reactivity of PLA1 within vespid species. Here, we address PLA1-based cross-reactivity among ten clinically relevant Hymenoptera venoms from Neotropical and temperate regions including Polybia paulista (paulistinha) venom and Vespula vulgaris (yellow jacket) venom. In order to evaluate cross-reactivity, sera of mice sensitized with recombinant PLA1 (rPoly p 1) from P. paulista wasp venom were used. Pronounced IgE and IgG based cross-reactivity was detected for wasp venoms regardless the geographical region of origin. The cross-reactivity correlated well with the identity of the primary sequence and 3-D models of PLA1 proteins. In contrast, these mice sera showed no reaction with honeybee (HBV) and fire ant venom. Furthermore, sera from patients monosensitized to HBV and fire ants did not recognize the rPoly p 1 in immunoblotting. Our findings reveal the presence of conserved epitopes in the PLA1s from several clinically relevant wasps as major cause of PLA1-based in vitro cross-reactivity. These findings emphasize the limitations but also the potential of PLA1-based HVA diagnostics.

Heterologous Expression, Purification and Immunoreactivity of the Antigen 5 from Polybia paulista Wasp Venom.

Polybia paulista (Hymenoptera: Vespidae) is responsible for a high number of sting accidents and anaphylaxis events in Southeast Brazil, Argentina and Paraguay. The specific detection of allergy to the venom of this wasp is often hampered by the lack of recombinant allergens currently available for molecular diagnosis. Antigen 5 (~23 kDa) from P. paulista venom (Poly p 5) is a highly abundant and glycosylated allergenic protein that could be used for development of component-resolved diagnosis (CRD). Here, we describe the cloning and heterologous expression of the antigen 5 (rPoly p 5) from P. paulista venom using the eukaryotic system Pichia pastoris. The expression as a secreted protein yielded high levels of soluble rPoly p 5. The recombinant allergen was further purified to homogeneity (99%) using a two-step chromatographic procedure. Simultaneously, the native form of the allergen (nPoly p 5) was purified from the wasp venom by Ion exchange chromatography. The rPoly p 5 and nPoly p 5 were then submitted to a comparative analysis of IgE-mediated immunodetection using sera from patients previously diagnosed with sensitization to wasp venoms. Both rPoly p 5 and nPoly p 5 were recognized by specific IgE (sIgE) in the sera of the allergic individuals. The high levels of identity found between nPoly p 5 and rPoly p 5 by the alignment of its primary sequences as well as by 3-D models support the results obtained in the immunoblot. Overall, we showed that P. pastoris is a suitable system for production of soluble rPoly p 5 and that the recombinant allergen represents a potential candidate for molecular diagnosis of P.paulista venom allergy.

Oral Tolerance Induced by OVA Intake Ameliorates TNBS-Induced Colitis in Mice.

INTRODUCTION: Literature data have shown that the consumption of dietary proteins may cause modulatory effects on the host immune system, process denominated oral tolerance by bystander suppression. It has been shown that the bystander suppression induced by dietary proteins can improve inflammatory diseases such as experimental arthritis. Here, we evaluated the effects of oral tolerance induced by ingestion of ovalbumin (OVA) on TNBS-induced colitis in mice, an experimental model for human Crohn's disease. METHODS AND RESULTS: Colitis was induced in BALB/c mice by instilling a single dose of TNBS (100 mg/kg) in ethanol into the colon. Tolerized mice received OVA (4mg/mL) dissolved in the drinking water for seven consecutive days, prior to or concomitantly with the intrarectal instillation. Control groups received protein-free water and ethanol by intrarectal route. We observed that either the prior or concomitant induction of oral tolerance were able to reduce the severity of colitis as noted by recovery of body weight gain, improvement of clinical signs and reduction of histological abnormalities. The in vitro proliferation of spleen cells from tolerant colitic mice was lower than that of control mice, the same as the frequencies of CD4+ T cells secreting IL-17 and IFN-γ. The frequencies of regulatory T cells and T cells secreting IL-10 have increased significantly in mice orally treated with OVA. The levels of inflammatory cytokines (IL-17A, TNF-α, IL-6 and IFN-γ) were lower in supernatants of cells from tolerant colitic mice, whereas IL-10 levels were higher. CONCLUSION: Our data show that the modulation of immune response induced by oral tolerance reduces the severity of experimental colitis. Such modulation may be partially attributed to the increase of Treg cells and reduction of pro-inflammatory cytokines in peripheral lymphoid organs of tolerant mice by bystander suppression.