Rapid polyclonal desensitization with antibodies to IgE and FcεRIα.

BACKGROUND: Rapid desensitization, a procedure in which persons allergic to an antigen are treated at short intervals with increasing doses of that antigen until they tolerate a large dose, is an effective, but risky, way to induce temporary tolerance. OBJECTIVE: We wanted to determine whether this approach can be adapted to suppress all IgE-mediated allergies in mice by injecting serially increasing doses of monoclonal antibodies (mAbs) to IgE or FcεRIα. METHODS: Active and passive models of antigen- and anti-IgE mAb-induced IgE-mediated anaphylaxis were used. Mice were desensitized with serially increasing doses of anti-IgE mAb, anti-FcεRIα mAb, or antigen. Development of shock (hypothermia), histamine and mast cell protease release, cytokine secretion, calcium flux, and changes in cell number and FcεRI and IgE expression were evaluated. RESULTS: Rapid desensitization with anti-IgE mAb suppressed IgE-mediated immediate hypersensitivity; however, some mice developed mild anaphylaxis during desensitization. Rapid desensitization with anti-FcεRIα mAb that only binds FcεRI that is not occupied by IgE suppressed both active and passive IgE-mediated anaphylaxis without inducing disease. It quickly, but temporarily, suppressed IgE-mediated anaphylaxis by decreasing mast cell signaling through FcεRI, then slowly induced longer lasting mast cell unresponsiveness by removing membrane FcεRI. Rapid desensitization with anti-FcεRIα mAb was safer and longer lasting than rapid desensitization with antigen. CONCLUSION: A rapid desensitization approach with anti-FcεRIα mAb safely desensitizes mice to IgE-mediated anaphylaxis by inducing mast cell anergy and later removing all mast cell IgE. Rapid desensitization with an anti-human FcεRIα mAb may be able to prevent human IgE-mediated anaphylaxis.

Correlation of screening and confirmatory results in tiered immunogenicity testing by solution-phase bridging assays.

Biotherapeutic proteins induce undesired immune responses that can affect drug efficacy and safety. For this reason, immunogenicity assessment is an integral part of drug development and is mandated by the regulatory authorities. Immunogenicity is typically evaluated by a tiered approach consisting of a screening assay followed by a competitive inhibition with unlabeled drug serving as confirmatory assay and additional characterization of the immune response. The confirmatory assay is intended to reduce the number of false positive responses generated in the screening tier and ensure that all samples are correctly classified as positive or negative. The positive-negative sample decisions are based on screening and confirmatory assay cut points that are statistically derived through evaluation of drug-naive samples. In this paper, we describe the analysis of cut point data for the presence of statistical correlation between the screening and confirmatory results. Data were obtained from validations of solution-phase bridging assays for detection of anti-drug antibodies against monoclonal antibody therapeutics. All data sets showed moderate to strong positive correlation, indicating that the screening and confirmatory assays were not independent and were likely to generate similar information. We present theoretical evidence that correlated results may be a general feature of the tiered approach when the same test platform is used for both screening and confirmatory assays. The competitive inhibition test, therefore, may be of limited value beyond reduction of the overall false positive rate. Our results indicate that similar sample results could be obtained by using just the screening assay with the false positive rate set to 1%.

Pollen and plant food profilin allergens show equivalent IgE reactivity.

BACKGROUND: Profilins are commonly involved in polysensitization of allergic patients; therefore, appropriate markers should be used in component-resolved diagnosis. OBJECTIVE: To evaluate the immunological equivalence between profilins from pollens and plant-derived foods, to be used in component-resolved diagnosis. METHODS: Specific immunoglobulin (Ig) G antibodies against pollen and fruit profilins, as well as sera from patients allergic to mustard, melon, or olive pollen, were used. Purified profilins from mustard seeds, fruit melon, and chenopod and birch pollen were assayed in immunoblotting, enzyme-linked immunosorbent assay (ELISA), and ELISA inhibition assays. RESULTS: Significant correlation was found in the response of purified profilins by ELISA and immunoblotting for both specific IgG and IgE. The highest levels of IgE binding were obtained for olive pollen-allergic patients, which could be related to the route of sensitization. The responses of individual patients to profilins were also similar and independent of the sensitizing source. The inhibition between pairs of allergens was generally higher than 70%, indicating that profilins share most of the IgE epitopes. Modeling of mimotopes in the conformational structure of the implicated profilins supports their strong cross-reactivity obtained experimentally. CONCLUSIONS: No correlation exists between the level of IgE response of individual patients to specific profilins and the corresponding theoretical sensitizing source, suggesting that the sensitization could be attributable to any profilin present in the environment of the patients. This would bear out the use of most profilins as a common marker for polysensitization in component-resolved diagnosis and for therapeutic approaches.

Glycoengineered Pichia produced anti-HER2 is comparable to trastuzumab in preclinical study.

Mammalian cell culture systems are used predominantly for the production of therapeutic monoclonal antibody (mAb) products. A number of alternative platforms, such as Pichia engineered with a humanized N-linked glycosylation pathway, have recently been developed for the production of mAbs. The glycosylation profiles of mAbs produced in glycoengineered Pichia are similar to those of mAbs produced in mammalian systems. This report presents for the first time the comprehensive characterization of an anti-human epidermal growth factor receptor 2 (HER2) mAb produced in a glycoengineered Pichia, and a study comparing the anti-HER2 from Pichia, which had an amino acid sequence identical to trastuzumab, with trastuzumab. The comparative study covered a full spectrum of preclinical evaluation, including bioanalytical characterization, in vitro biological functions, in vivo anti-tumor efficacy and pharmacokinetics in both mice and non-human primates. Cell signaling and proliferation assays showed that anti-HER2 from Pichia had antagonist activities comparable to trastuzumab. However, Pichia-produced material showed a 5-fold increase in binding affinity to FcγIIIA and significantly enhanced antibody dependant cell-mediated cytotoxicity (ADCC) activity, presumably due to the lack of fucose on N-glycans. In a breast cancer xenograft mouse model, anti-HER2 was comparable to trastuzumab in tumor growth inhibition. Furthermore, comparable pharmacokinetic profiles were observed for anti-HER2 and trastuzumab in both mice and cynomolgus monkeys. We conclude that glycoengineered Pichia provides an alternative production platform for therapeutic mAbs and may be of particular interest for production of antibodies for which ADCC is part of the clinical mechanism of action.

Pectin methylesterases of pollen tissue, a major allergen in olive tree.

Olive tree (Olea europaea) pollen is a main cause of allergy in Mediterranean areas and North America. A novel allergen, Ole e 11, has been detected by proteomic techniques. Protein bands binding IgE from allergic sera were excised from a 2D electrophoresis gel and analysed by Edman degradation and MALDI-TOF MS. Four peptides were sequenced and used for designing primers to clone the cDNA codifying the protein. Ole e 11 consists of a 342 amino acid length polypeptide with a molecular mass of 37.4 kDa and a pI of 7.8. The allergen was identified as a pectin methylesterase and showed low identity with other members of this family from foods such as those from carrot (23%), orange (25%) and tomato (24%), and higher identity with those from Arabidopsis thaliana (57%) and Salsola kali (54%) pollen. The protein was overproduced in Pichia pastoris, purified, and characterized as an active enzyme. CD analysis rendered 3%alpha-helix, 50%beta-sheet and 27%beta-turns for its secondary structure, which is in agreement with other pectin methylesterase structures. The recombinant protein was demonstrated to be immunologically equivalent to the natural form by immunoblotting, indirect ELISA and inhibition experiments, using polyclonal antiserum and sera from olive pollen allergic patients. The prevalence fluctuated between 55.9% and 75.6% in three different allergic populations. The availability of this new olive pollen allergen could improve the component-resolved diagnosis. Its allergenic relevance is stepped up by the biotechnological use of these enzymes to improve organoleptic properties in processing foods and further confirms the need to include it in an accurate diagnosis.

Allergy to Salsola Kali in a Salsola incanescens-rich area: role of extensive cross allergenicity.

BACKGROUND: Pollens from the Salsola spp. are an important source of respiratory allergy in tropical countries. Our aim was to characterize the IgE binding proteins of S. incanescens pollen extract and study its cross-reactivity with S. kali pollen allergens. METHODS: Prick tests with S. kali and S. incanescens pollen extracts were performed on eight respiratory allergy patients from Mashhad, Northeast Iran. The antigenic profiles and IgE-binding patterns of S. kali and S. incanescens pollen extracts were compared by SDS-PAGE and Western blotting, using individual sera from the salsola pollen-sensitive patients. Cross-reactivity of proteins in the two weeds was assessed by IgE- immunoblotting inhibition. RESULTS: S. kali and S. incanescens pollen extracts showed similar IgE-binding profiles in Western blotting. The IgE binding components of 39, 45, 66 and 85 kDa were detected in both pollen extracts. Furthermore, inhibition of the immunoblots revealed extensive inhibition of IgE binding to proteins and a close relationship between these two weeds allergens. CONCLUSIONS: S. incanescens pollen is a potent allergen source with several IgE binding components that shows a close allergenic relationship with S. kali. Our results suggest that in S. incanescens-rich areas, S. kali pollen extracts could be used as a diagnostic reagent for allergic patients to S. incanescens pollen.

Clinical features of four cases with cashew nut allergy and cross-reactivity between cashew nut and pistachio.

BACKGROUND: Few cases of cashew nut (CN) allergy have been reported in Japan. We evaluated the clinical features of 4 cases with CN allergy and investigated the allergens involved. METHODS: In order to investigate the cross-reactivity between CN and pistachios, we performed ImmunoCAP inhibition tests using sera of 4 cases with positive histories of CN allergy and positive results of specific IgE measurement (ImmunoCAP) and skin prick tests. Furthermore, we analyzed the molecular weights of allergens of CN and pistachios by IgE-immunoblotting. RESULTS: Of the 4 cases (male : female = 1:3), there were 3 cases (patient #2-4) and 1 case (patient #1) of anaphylaxis and oral allergy syndrome, respectively. The initial symptom was an oropharyngeal symptom in 3 of the 4 cases, of which 2 cases developed anaphylaxis within 10 minutes after eating only a few pieces of CN. All 4 cases reacted positively to the skin prick test with CN, although 1 case of anaphylaxis tested negatively for CN by ImmunoCAP. Additionally, in 2 cases, IgE-binding to CN and pistachio were inhibited with both pistachios and CN, indicating cross-reactivity between CN and pistachios. IgE-immunoblotting of CN using sera from the 4 cases revealed 2 bands at molecular weights of approximately 33 kd and 42 kd, whereas that of pistachios showed a single band at 36 kd. However, IgE in all 4 sera did not bind to rAna o 2. CONCLUSIONS: In CN allergy, a small amount of CN could induce a severe anaphylactic reaction. Moreover, in cases of suspected CN allergy, reactions to not only CN but also pistachio, which could be cross-reactive to CN, should be examined.

Allergenicity and immunogenicity of the major mugwort pollen allergen Art v 1 chemically modified by acetylation.

BACKGROUND: Treating allergies with modified allergens is an approach to make the treatment safer and more efficient. Art v 1 is the most prominent allergen of mugwort pollen and a significant cause of hayfever around Europe. The aim of this study was to reduce the allergenicity of Art v 1 by acetylation, and to investigate the capacity of the modified protein to generate blocking antibodies. METHODS: The reduction of allergenicity of Art v 1 following acetylation was monitored by immunoblot, ELISA inhibition using a pool of sera from mugwort pollen allergic patients, basophil activation assay and by skin prick testing of mugwort-allergic patients. Rabbits were immunized against Art v 1 and acetylated Art v 1 (acArt v 1) and the rabbit antisera were tested for their capacity to block human IgE binding in ELISA. Human T cell proliferation against Art v 1 and acArt v 1 was examined in peripheral blood mononuclear cells (PBMCs) of mugwort pollen allergic patients and cytokine release in PBMC cultures was monitored. RESULTS: Acetylation of Art v 1 gave a derivative of reduced allergenicity in the in vitro and ex vivo tests applied. The skin test reactivity to acArt v 1 was significantly reduced in 19 patients when compared with the reactivity to Art v 1. Rabbit antibodies to acArt v 1 and Art v 1 showed similar capacity to block human IgE binding to Art v 1 in inhibition ELISA. Both proteins were able to induce proliferation of PBMCs and CD3/CD4(+) cells of mugwort-allergic patients. Release of IL-5 was significantly reduced in cultures stimulated with acArt v 1. CONCLUSIONS: Art v 1 modified by acetylation had a significantly reduced allergenicity in vitro and in vivo, while its immunogenicity was retained. Modification of allergens by acetylation could be a new strategy for allergen-specific immunotherapy.

Clinical and immunobiochemical characterization of airborne Delonix regia (Gulmohar tree) pollen and cross-reactivity studies with Peltophorum pterocarpum pollen: 2 dominant avenue trees from eastern India.

BACKGROUND: Delonix regia and Peltophorum pterocarpum pollen are important aeroallergens for type 1 hypersensitivity in the tropics. The IgE-binding proteins of D regia and their cross-allergenity with P pterocarpum pollen have not been evaluated. OBJECTIVES: To isolate and characterize the IgE-binding proteins of D regia pollen for the first time and to investigate the cross-allergenity with P pterocarpum pollen belonging to the same family (Leguminosae). METHODS: Allergenic activities were determined by in vivo and in vitro analyses. Pollen extract was fractionated by a combination of 2 columns (diethyl amino ethyl Sephadex and Sephacryl S-200). Protein components were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, periodic acid-Schiff staining, and immunoblotting. In vitro inhibition tests were performed to evaluate the cross-reactivity. RESULTS: The skin prick test results of the patients with respiratory allergies in Calcutta, India, showed 31.1% positivity with D regia pollen. Nine IgE-reactive protein components were found in the crude extract. An optimum IgE-reactive fraction was resolved into 4 subfractions. Subfraction A, which showed maximum IgE reactivity, contained 2 (96- and 66-kDa) IgE-reactive protein components. The 66-kDa component was found to be glycoprotein. Remarkable cross-reactivity between D regia and P pterocarpum pollen was found on IgE enzyme-linked immunosorbent assay inhibition and dot blotting. Shared IgE-binding components (66, 56, 32, 28, 25, and 23 kDa) were observed between D regia and P pterocarpum pollen extracts, whereas the 96- and 43-kDa components were specific to D regia. CONCLUSION: The purification of the IgE-binding proteins and the identification of the shared/cross-reactive proteins in these taxonomically related pollen members should be helpful for the diagnosis and therapy of patients susceptible to these pollens.

Llama single-chain antibody that blocks lipopolysaccharide binding and signaling: prospects for therapeutic applications.

Sepsis is a considerable health problem and a burden on the health care system. Endotoxin, or lipopolysaccharide (LPS), present in the outer membrane of gram-negative bacteria, is responsible for more than 50% of the sepsis cases and is, therefore, a legitimate target for therapeutic approaches against sepsis. In this study, we selected and characterized a llama single-chain antibody fragment (VHH) directed to Neisseria meningitidis LPS. The VHH, designated VHH 5G, showed affinity to purified LPS as well as to LPS on the surfaces of the bacteria. Epitope mapping using a panel of N. meningitidis mutants revealed that VHH 5G recognizes an epitope in the inner core of LPS, and as expected, the VHH proved to have broad specificity for LPS from different bacteria. Furthermore, this VHH blocked binding of LPS to target cells of the immune system, resulting in the inhibition of LPS signaling in whole blood. Moreover, it was found to remove LPS efficiently from aqueous solutions, including serum. The selected anti-LPS VHH is a leading candidate for therapies against LPS-mediated sepsis.

Arginine-rich anti-vascular endothelial growth factor (anti-VEGF) hexapeptide inhibits collagen-induced arthritis and VEGF-stimulated productions of TNF-alpha and IL-6 by human monocytes.

Vascular endothelial growth factor (VEGF) has been suggested to play a critical role in the pathogenesis of rheumatoid arthritis (RA). We previously identified a novel RRKRRR hexapeptide that blocked the interaction between VEGF and its receptor through the screening of peptide libraries. In this study, we investigated whether anti-VEGF peptide RRKRRR (dRK6) could suppress collagen-induced arthritis (CIA) and regulate the activation of mononuclear cells of RA patients. A s.c. injection of dRK6 resulted in a dose-dependent decrease in the severity and incidence of CIA and suppressed synovial infiltration of inflammatory cells in DBA/1 mice. In these mice, the T cell responses to type II collagen (CII) in lymph node cells and circulating IgG Abs to CII were also dose-dependently inhibited by the peptides. In addition, VEGF directly increased the production of TNF-alpha and IL-6 from human PBMC. Synovial fluid mononuclear cells of RA patients showed a greater response to VEGF stimulation than the PBMC of healthy controls. The major cell types responding to VEGF were monocytes. Moreover, anti-VEGF dRK6 inhibited the VEGF-induced production of TNF-alpha and IL-6 from synovial fluid mononuclear cells of RA patients and decreased serum IL-6 levels in CIA mice. In summary, we observed first that dRK6 suppressed the ongoing paw inflammation in mice and blocked the VEGF-induced production of proinflammatory cytokines. These data suggest that dRK6 may be an effective strategy in the treatment of RA, and could be applied to modulate various chronic VEGF-dependent inflammatory diseases.

The staphylococcal superantigen-like protein 7 binds IgA and complement C5 and inhibits IgA-Fc alpha RI binding and serum killing of bacteria.

The staphylococcal superantigen-like proteins (SSLs) are close relatives of the superantigens but are coded for by a separate gene cluster within a 19-kb region of the pathogenicity island SaPIn2. rSSL7 (formally known as SET1) bound with high affinity (K(D), 1.1 nM) to the monomeric form of human IgA1 and IgA2 plus serum IgA from primate, pig, rat, and horse. SSL7 also bound the secretory form of IgA found in milk from human, cow, and sheep, and inhibited IgA binding to cell surface FcalphaRI (CD89) and to a soluble form of the FcalphaRI protein. In addition to IgA, SSL7 bound complement factor C5 from human (K(D), 18 nM), primate, sheep, pig, and rabbit serum, and inhibited complement-mediated hemolysis and serum killing of a Gram-negative organism Escherichia coli. SSL7 is a superantigen-like protein secreted from Staphylococcus aureus that blocks IgA-FcR interactions and inhibits complement, leading to increased survival of a sensitive bacterium in blood.

Association of diamine oxidase and S-adenosylhomocysteine hydrolase in Nicotiana tabacum extracts.

The oxidative deamination of methylated putrescine by a diamine oxidase activity (DAO) is an important step in the biosynthesis of nicotine in tobacco and tropane alkaloids in several Solanaceous plants. A polyclonal rabbit antiserum was previously developed to a purported purified DAO enzyme from Nicotiana tabacum. The antiserum bound to a single 53 kDa protein and immunoprecipitated 80% of DAO activity from tobacco root extracts. In an effort to obtain DAO cDNAs, this antiserum was used to screen a tobacco cDNA expression library and three distinct immunoreactive cDNA clones were isolated. These cDNAs encoded predicted proteins that were either identical or nearly identical to predicted S-adenosylhomocysteine hydrolase (SAHH) from two Nicotiana species. Thus, the rabbit antiserum was not specific to DAO, even though it immunodepleted the majority of DAO activity from root extracts. Alternative hypotheses to explain the DAO immunodepletion results (such as poisoning of DAO activity or that SAHH is a bifunctional enzyme) were tested and ruled out. Therefore, we hypothesize that SAHH associates with DAO as part of a larger multienzyme complex that may function in planta as a nicotine metabolic channel.

Molecular characterization of polygalacturonases as grass pollen-specific marker allergens: expulsion from pollen via submicronic respirable particles.

Grass pollen belong to the most important allergen sources involved in the elicitation of allergic asthma. We have isolated cDNAs coding for Bermuda grass (Cynodon dactylon) and timothy grass (Phleum pratense) pollen allergens, belonging to a family of pectin-degrading enzymes (i.e., polygalacturonases). The corresponding allergens, termed Cyn d 13 and Phl p 13, represent glycoproteins of approximately 42 kDa and isoelectric points of 7.5. rPhl p 13 was expressed in Escherichia coli and purified to homogeneity. Immunogold electron microscopy using rabbit anti-rPhl p 13 Abs demonstrated that in dry pollen group 13, allergens represent primarily intracellular proteins, whereas exposure of pollen to rainwater caused a massive release of cytoplasmic material containing submicronic particles of respirable size, which were coated with group 13 allergens. The latter may explain respiratory sensitization to group 13 allergens and represents a possible pathomechanism in the induction of asthma attacks after heavy rainfalls. rPhl p 13 was recognized by 36% of grass pollen allergic patients, showed IgE binding capacity comparable to natural Phl p 13, and induced specific and dose-dependent basophil histamine release. Epitope mapping studies localized major IgE epitopes to the C terminus of the molecule outside the highly conserved functional polygalacturonase domains. The latter result explains why rPhl p 13 contains grass pollen-specific IgE epitopes and may be used to diagnose genuine sensitization to grass pollen. Our finding that rabbit anti-rPhl p 13 Abs blocked patients' IgE binding to the allergen suggests that rPhl p 13 may be used for immunotherapy of sensitized patients.

Generation of an allergy vaccine by disruption of the three-dimensional structure of the cross-reactive calcium-binding allergen, Phl p 7.

The grass pollen allergen, Phl p 7, belongs to a family of highly cross-reactive calcium-binding pollen allergens. Because Phl p 7 contains most of the disease-eliciting epitopes of pollen-derived calcium-binding allergens, hypoallergenic variants were engineered according to the x-ray crystal structure of Phl p 7 for allergy vaccination. In three recombinant variants, amino acids essential for calcium binding were mutated, and two peptides comprising the N- and C-terminal half were obtained by synthetic peptide chemistry. As determined by circular dichroism analysis and size exclusion chromatography coupled to mass spectrometry, recombinant mutants showed altered structural fold and lacked calcium-binding capacity, whereas the two synthetic peptides had completely lost their structural fold. Allergic patients' IgE Ab binding was strongest reduced to the variant containing two mutations in each of the two calcium-binding sites and to the peptides. Basophil histamine release and skin test experiments in allergic patients identified the peptides as the vaccine candidates with lowest allergenic activity. Immunization of rabbits with the peptides induced IgG Abs that blocked allergic patients' IgE binding to Phl p 7 and inhibited allergen-induced basophil degranulation. Our results indicate that disruption of an allergen's three-dimensional structure represents a general strategy for the generation of hypoallergenic allergy vaccines, and demonstrate the importance of allergen-specific IgG Abs for the inhibition of immediate allergic symptoms.

Autoantibodies against cerebral muscarinic cholinoceptors in Sjögren syndrome: functional and pathological implications.

Previous studies have demonstrated that antibodies against muscarinic acetylcholine receptors (mAChRs) from exocrine glands, correlates with Sjögren syndrome (SS) in the majority of patients. The aim of the present investigation was to establish if serum IgG antibodies present in SS interacts with cerebral mAChRs. Results show that anti-cerebral IgG are present in the sera of 40% SS patients studied. Autoantibodies were able to interact with mAChRs of cerebral frontal cortex membranes inhibiting the [(3)H]QNB binding to its specific receptor. Moreover, tested by ELISA and dot blot they recognized the synthetic peptides corresponding to the second extracellular loop of human M(1) and M(3) mAChR. In addition, the corresponding affinity-purified anti-M(1) and anti-M(3) peptide IgGs displayed an agonistic activity, stimulating phosphoinositide hydrolysis. The results support the notion that serum IgG autoantibodies in SS patients target cerebral mAChRs may have some role in the pathogenesis of higher cognitive dysfunction present in SS patients.

A major allergen from pollen defines a novel family of plant proteins and shows intra- and interspecies [correction of interspecie] cross-reactivity.

Olive tree (Olea europaea) pollen is a main cause of allergy associated with extensive areas of Europe and North America. Ole e 10, a small (10.8 kDa) and acidic (pI 5.8) protein, has been identified as a major allergen from the olive pollen, isolated, and characterized. Circular dichroism analysis gave 17% alpha helix, 33% beta sheet, and 21% beta turn for its secondary structure. Based on amino acid sequences of tryptic peptides, the protein was cloned and sequenced. The allergen consists of a single polypeptide chain of 102 aa, with a signal peptide of 21 residues. Ole e 10 showed homology with the C-terminal domain of another olive allergen, Ole e 9 (1,3-beta-glucanase, 53% identity), with deduced sequences from Arabidopsis thaliana genes (42-46% identity) and with polypeptide segments (Cys boxes) of proteins involved in yeast development (Epd1/Gas-1p/Phr2 families; 42-43% similarity). Ole e 10 showed 55% prevalence for olive-allergic patients and exhibited an IgE response dependent on its conformation. Remarkable IgE cross-reactivity was detected with Ole e 9, but no correlation was observed between the individual IgE responses to both allergens. Ole e 10 shares IgE B cell epitopes with proteins from Oleaceae, Gramineae, Betulaceae, Chenopodiaceae, Cupressaceae, Ambrosia, and Parietaria pollens, latex, and vegetable foods, such as tomato, kiwi, potato, and peach. These data indicate that Ole e 10 is a new pan-allergenic plant protein that shows notable intra- and interspecie IgE cross-reactivity and is a powerful candidate to be involved in pollen-latex-fruit syndrome.

Grass pollen immunotherapy induces mucosal and peripheral IL-10 responses and blocking IgG activity.

T regulatory cells and IL-10 have been implicated in the mechanism of immunotherapy in patients with systemic anaphylaxis following bee stings. We studied the role of IL-10 in the induction of clinical, cellular, and humoral tolerance during immunotherapy for local mucosal allergy in subjects with seasonal pollinosis. Local and systemic IL-10 responses and serum Ab concentrations were measured before/after a double-blind trial of grass pollen (Phleum pratense, Phl P) immunotherapy. We observed local increases in IL-10 mRNA-positive cells in the nasal mucosa after 2 years of immunotherapy, but only during the pollen season. IL-10 protein-positive cells were also increased and correlated with IL-10 mRNA(+) cells. These changes were not observed in placebo-treated subjects or in healthy controls. Fifteen and 35% of IL-10 mRNA signals were colocalized to CD3(+) T cells and CD68(+) macrophages, respectively, whereas only 1-2% of total CD3(+) cells and 4% of macrophages expressed IL-10. Following immunotherapy, peripheral T cells cultured in the presence of grass pollen extract also produced IL-10. Immunotherapy resulted in blunting of seasonal increases in serum allergen Phl p 5-specific IgE, 60- to 80-fold increases in Phl p 5-specific IgG, and 100-fold increases in Phl p 5-specific IgG4. Post-immunotherapy serum exhibited inhibitory activity, which coeluted with IgG4, and blocked IgE-facilitated binding of allergen-IgE complexes to B cells. Both the increases in IgG and the IgG "blocking" activity correlated with the patients' overall assessment of improvement. Thus, grass pollen immunotherapy may induce allergen-specific, IL-10-dependent "protective" IgG4 responses.

Dominating IgE-binding epitope of Bet v 1, the major allergen of birch pollen, characterized by X-ray crystallography and site-directed mutagenesis.

Specific allergy vaccination is an efficient treatment for allergic disease; however, the development of safer vaccines would enable a more general use of the treatment. Determination of molecular structures of allergens and allergen-Ab complexes facilitates epitope mapping and enables a rational approach to the engineering of allergen molecules with reduced IgE binding. In this study, we describe the identification and modification of a human IgE-binding epitope based on the crystal structure of Bet v 1 in complex with the BV16 Fab' fragment. The epitope occupies approximately 10% of the molecular surface area of Bet v 1 and is clearly conformational. A synthetic peptide representing a sequential motif in the epitope (11 of 16 residues) did not inhibit the binding of mAb BV16 to Bet v 1, illustrating limitations in the use of peptides for B cell epitope characterization. The single amino acid substitution, Glu(45)-Ser, was introduced in the epitope and completely abolished the binding of mAb BV16 to the Bet v 1 mutant within a concentration range 1000-fold higher than wild type. The mutant also showed up to 50% reduction in the binding of human polyclonal IgE, demonstrating that glutamic acid 45 is a critical amino acid also in a major human IgE-binding epitope. By solving the three-dimensional crystal structure of the Bet v 1 Glu(45)-Ser mutant, it was shown that the change in immunochemical activity is directly related to the Glu(45)-Ser substitution and not to long-range structural alterations or collapse of the Bet v 1 mutant tertiary structure.

Mechanisms of endotoxin tolerance in human intestinal microvascular endothelial cells.

Lipopolysaccharide (endotoxin) tolerance is well described in monocytes and macrophages, but is less well characterized in endothelial cells. Because intestinal microvascular endothelial cells exhibit a strong immune response to LPS challenge and play a critical regulatory role in gut inflammation, we sought to characterize the activation response of these cells to repeated LPS exposure. Primary cultures of human intestinal microvascular endothelial cells (HIMEC) were stimulated with LPS over 6-60 h and activation was assessed using U937 leukocyte adhesion, expression of E-selectin, ICAM-1, VCAM-1, IL-6, IL-8, manganese superoxide dismutase, HLA-DR, and CD86. Effect of repeat LPS stimulation on HIMEC NF-kappaB and mitogen-activated protein kinase (MAPK) activation, generation of superoxide anion, and Toll-like receptor 4 expression was characterized. LPS pretreatment of HIMEC for 24-48 h significantly decreased leukocyte adhesion after subsequent LPS stimulation. LPS pretreatment inhibited expression of E-selectin, VCAM-1, IL-6, and CD86, while ICAM-1, IL-8, and HLA-DR were not altered. Manganese superoxide dismutase expression increased with repeated LPS stimulation, with a reduction in intracellular superoxide. NF-kappaB activation was transiently inhibited by LPS pretreatment for 6 h, but not at later time points. In contrast, p44/42 MAPK, p38 MAPK, and c-Jun N-terminal kinase activation demonstrated inhibition by LPS pretreatment 24 or 48 h prior. Toll-like receptor 4 expression on HIMEC was not altered by LPS. HIMEC exhibit endotoxin tolerance after repeat LPS exposure in vitro, characterized by diminished activation and intracellular superoxide anion concentration, and reduced leukocyte adhesion. HIMEC possess specific mechanisms of immunoregulatory hyporesponsiveness to repeated LPS exposure.