CD23, the low affinity receptor for IgE, is expressed mainly on B cells and has been shown to regulate IgE production. Previously, recombinant mouse and human CD23 were constructed with a trimerizing isoleucine zipper motif attached in frame to the N-terminus of the entire extracellular CD23 (lz-ECCD23). The goal was to examine the role of the necessity of the CD23 stalk for binding IgE. Using PCR-based mutagenesis to delete the majority of the stalk, binding to IgE was lost. Further studies examined the effect of lz-ECCD23 in preventing IgE from binding FcεRI and therefore acting as a therapeutic agent. It was determined that the lz-ECCD23 construct was capable of doing this, albeit most effectively at 4°C rather than at physiological temperature. In addition, antibodies to the stalk region of CD23 were developed and assessed for their capacity to modulate IgE. Rabbit anti-CD23stalk (RAS) antibodies were found to inhibit IgE production in IL-4/antiCD40 stimulated B cells. The inhibition observed was dependent on the Fc portion of the antibody, implicating a role for FcγRIIb, an inhibitory receptor, in the IgE reduction. It was also shown that the addition of anti-stalk antibodies caused significant release of soluble CD23 (sCD23). Finally, I show that optimal IgE production was cell density dependent and was achieved through the addition of IL-21 and/or IL-10 to IL-4/antiCD40 stimulated B cells. While IgE production is inversely proportional to plated cell densities, it is directly correlated to increased cellular division, as determined by CFSE staining, and to increased cellular differentiation, as determined by FACS analysis for differentiation markers. This work is the first demonstration that IgE production in humans is dependent on cell density, that IL-21 affects all isotypes tested, and that maximal Ig production is found at lower cell densities, correlating with increased cell division. I also show for the first time that the increase in IgE observed after IL-10 addition to IL-4 and anti-CD40 stimulated cells correlates with increased cellular division. When IL-10 and IL-21 were added together, there was a synergistic increase in IgE, but interestingly, no further cell division was seen, suggesting an increase in differentiation.

Les allergies alimentaires à l'arachide et au lait posent un problème majeur de santé publique, en particulier chez les enfants. Nous avons développé un modèle cellulaire de dégranulation, afin d'analyser si la liaison d'un allergène à ses IgE spécifiques, mesurée in vitro, a bien la capacité de déclencher la phase effectrice de la réaction allergique. Ce modèle repose sur l'utilisation de la lignée cellulaire RBL SX-38 développée par JP Kinet, mastocytes de rat immortalisés et modifiés pour exprimer le récepteur de haute affinité aux IgE humaines. Le développement de ce modèle pour son utilisation en microplaques de 96 puits a consisté notamment en l'optimisation de différents paramètres critiques pour la réalisation des 2 phases du test : la phase de sensibilisation des cellules par les IgE de patients allergiques et la phase de déclenchement par les allergènes auxquels le patient est sensibilisé. Ce modèle a permis de montrer l'importance des caséines et des albumines 2S (Ara h 2 et Ara h 6) dans cette étape clé de la réaction allergique au lait et à l'arachide, confirmant les observations sérologiques et cliniques disponibles. L'effet de traitements thermiques sur l'allergénicité d'un allergène majeur de l'arachide (Ara h 1), a été évalué dans le cadre du programme européen EuroPrevall. Les résultats obtenus montrent la diminution de la réactivité d'Ara h 1 après chauffage de la protéine isolée en solution. Par contre, la même protéine préparée et purifiée à partir d'arachide grillée présente une forte réactivité. Ces résultats suggèrent qu'un traitement à température élevée de la graine entraine une augmentation de l'allergénicité de Ara h 1 du fait de ses interactions avec les autres constituants de la graine. Le modèle cellulaire de dégranulation développé apparaît être un outil pertinent pour l'étude de la fonctionnalité biologique de l'interaction allergène-IgE, permettant une meilleure compréhension de la relation entre la structure d'une protéine alimentaire et son allergénicité, voire une évaluation du risque allergique des aliments ne nécessitant pas d'essais cliniques systématiques.

<p>Immunoglobulin E (IgE) occurs exclusively in mammals and is one of five immunoglobulin (Ig) classes found in man. Unlike other isotypes, IgE is best known for its pathological effects, whereas its physiological role remains somewhat elusive. </p><p>To trace the emergence of IgE and other post-switch isotypes we have studied Ig expression in two monotreme species, the duck-billed platypus (<i>Ornithorhynchus anatinus</i>) and the short-beaked echidna (<i>Tachyglossus aculeatus</i>), leading to the cloning of IgE, two IgG isotypes in platypus and echidna IgE. The presence of IgE and the conservation of the overall structure in all extant mammalian lineages indicates an early appearance in mammalian evolution and a selective advantage of structural maintenance. Furthermore, both of the two highly divergent platypus γ-chains have three constant domains. Hence, the major evolutionary changes that gave rise to the IgE and IgG isotypes of present day mammals occurred before the separation of monotremes from the marsupial and placental lineages, estimated to have occurred 150-170 million years ago.</p><p>As the central mediator in atopic allergy, IgE is a prime target in the development of preventive treatments. This thesis describes an active immunization strategy that has the potential to reduce IgE to a clinically significant extent. The active vaccine component is a chimeric IgE molecule, Cε2-Cε3-Cε4. The receptor-binding target domain, Cε3, is derived from the recipient species, whereas the flanking domains, acting both as structural support and to break T-cell tolerance, are derived from an evolutionarily distant mammal. Vaccination of ovalbumin-sensitized rats resulted in a substantial reduction in total IgE in three out of four strains, accompanied by a significant reduction in skin-reactivity upon allergen challenge. No cross-linking activity was observed and the response to vaccination was reversible with time. The apparent safety and efficacy of the vaccine suggest that active immunization against IgE has the potential to become a therapeutic method for humans. </p><p>Furthermore, the cloning and expression of the pig (<i>Sus scrufa</i>) ε-chain will facilitate the development of sensitive and specific assays for pig IgE, thus increasing the possibilities of using the pig model in future studies of IgE-mediated reactions.</p>

Immunoglobulin E (IgE) occurs exclusively in mammals and is one of five immunoglobulin (Ig) classes found in man. Unlike other isotypes, IgE is best known for its pathological effects, whereas its physiological role remains somewhat elusive. To trace the emergence of IgE and other post-switch isotypes we have studied Ig expression in two monotreme species, the duck-billed platypus (Ornithorhynchus anatinus) and the short-beaked echidna (Tachyglossus aculeatus), leading to the cloning of IgE, two IgG isotypes in platypus and echidna IgE. The presence of IgE and the conservation of the overall structure in all extant mammalian lineages indicates an early appearance in mammalian evolution and a selective advantage of structural maintenance. Furthermore, both of the two highly divergent platypus γ-chains have three constant domains. Hence, the major evolutionary changes that gave rise to the IgE and IgG isotypes of present day mammals occurred before the separation of monotremes from the marsupial and placental lineages, estimated to have occurred 150-170 million years ago. As the central mediator in atopic allergy, IgE is a prime target in the development of preventive treatments. This thesis describes an active immunization strategy that has the potential to reduce IgE to a clinically significant extent. The active vaccine component is a chimeric IgE molecule, Cε2-Cε3-Cε4. The receptor-binding target domain, Cε3, is derived from the recipient species, whereas the flanking domains, acting both as structural support and to break T-cell tolerance, are derived from an evolutionarily distant mammal. Vaccination of ovalbumin-sensitized rats resulted in a substantial reduction in total IgE in three out of four strains, accompanied by a significant reduction in skin-reactivity upon allergen challenge. No cross-linking activity was observed and the response to vaccination was reversible with time. The apparent safety and efficacy of the vaccine suggest that active immunization against IgE has the potential to become a therapeutic method for humans. Furthermore, the cloning and expression of the pig (Sus scrufa) ε-chain will facilitate the development of sensitive and specific assays for pig IgE, thus increasing the possibilities of using the pig model in future studies of IgE-mediated reactions.

Background: The reasons why some individuals become IgE-sensitised and allergic are largely unknown, though genetic- and early life environmental factors seem to be of importance. Objective: The overall aim of this thesis was to investigate the relationship between IgE-sensitisation and allergic disease, viral infections, genetic markers and early life cytokines. Results: IgE-sensitised children were found to have reduced numbers of IL-12 producing cord blood mononuclear cells (CBMC), whereas children diagnosed with eczema were found to have reduced numbers of IFN-γ producing CBMC. When dividing the children into early onset of IgE-sensitisation and late onset of IgE-sensitisation we found that the children with an early onset had low numbers of PHA-induced IL-4, IL-12 and IFN-γ secreting CBMC. At the age of two there was a general exacerbation of cytokine responses in the IgE-sensitised children, and the results were similar for the children with early onset IgE-sensitisation. Children with a late onset IgE-sensitisation were more similar to the non-sensitised children, but with a specific increase in the response to cat allergen (IL-4 and IFN-γ). The mothers of IgE-sensitised children, were just as their children, found to have an exaggerated cytokine response as compared to mothers of non-sensitised children. Maternal responses correlated well to the responses seen in the child, though the samples were taken two years after delivery. Cytomegalovirus (CMV) infection in early life was associated to reduced numbers of IL-4, and increased numbers of IFN-γ producing cells at the age of two. No association between CMV seropositivity and IgE-sensitisation was seen. Epstein-Barr virus (EBV) infection, on the other hand, was inversely correlated with IgE –sensitisation, whereas no statistically significant association to cytokine production could be seen. We also showed that the IL12B 1188 C-allele was associated to having a positive skin prick test at the age of two. The rare alleles of the three SNPs investigated (IL12B 1188C, IL12RB1132C and IRF1 1688A) were all associated to low IL-12 production at birth. Conclusions: Our results indicate that allergic diseases are complex traits, and that both the genetic and the cytokine background differ between the different allergic diseases. We can also conclude that the time of onset seem to play a role when investigating IgE-sensitisation, and that perhaps early and late onset IgE-sensitisation have partly different causes. CMV and EBV infection early in life are associated to a protective cytokine profile and to protection from IgE-sensitisation, respectively, again indicating the heterogeneity and the complexity of allergic diseases.

The main subject of this thesis is to design a voltage peak detector for FPW-based IgE measurement systems. Therefore, two different peak detectors are proposed. The first voltage peak detector basically samples the input signal twice (double sampling) to reduce the ripples appearing during the sample and hold modes. This voltage peak detector also resolves the detection error of conventional voltage peak detectors when they are used to detect the output signal of FPW-based biosensors.The fastest signal which this voltage peak detector can detect is 10 MHz. The second voltage peak detector is composed of a coupling capacitor, an unity gain buffer, an 8th order voltage control voltage source(VCVS) low pass filter, and a non-inverting amplifier. The major difference of this design from the previous one is to filter and amplify the input signal. The specification requirements of the operational transconductance amplifier in this voltage peak detector can be relaxed thereafter. The resolution and performance of the sensing system are also improved. By replacing the conventional power MOS by a non-inverting amplifier, the charging time is reduced and over charge hazard is avoided. Besides, the speed of the entire system is enhanced. The fastest signal which this voltage peak detector can detect is 50 MHz and the precision is 0.357 %.

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Thesis (Ph.D. in Immunology) -- University of Colorado Denver, 2008. / Typescript. Includes bibliographical references (leaves 122-129). Free to UCD Anschutz Medical Campus. Online version available via ProQuest Digital Dissertations;

Submitted by Hiolanda Rêgo (hiolandarego@gmail.com) on 2017-02-15T15:30:00Z No. of bitstreams: 1 Dissertação_ICS_ Fernanda Garcia Guerra.pdf: 320427 bytes, checksum: f3c495155faf56748788adbb54a6f36c (MD5) / Approved for entry into archive by Delba Rosa (delba@ufba.br) on 2017-03-23T13:41:01Z (GMT) No. of bitstreams: 1 Dissertação_ICS_ Fernanda Garcia Guerra.pdf: 320427 bytes, checksum: f3c495155faf56748788adbb54a6f36c (MD5) / Made available in DSpace on 2017-03-23T13:41:01Z (GMT). No. of bitstreams: 1 Dissertação_ICS_ Fernanda Garcia Guerra.pdf: 320427 bytes, checksum: f3c495155faf56748788adbb54a6f36c (MD5) / CNPq; PPGIM – UFBA / O Lúpus Eritematoso Sistêmico (LES) é uma doença reumática autoimune, classificada como uma reação de hipersensibilidade tipo III, que cursa com exuberante produção de auto-anticorpos de diferentes especificidades, e reação inflamatória crônica. O LES acomete predominantemente pessoas do sexo feminino, com prevalência de 5-50 casos/100.000. Contudo, estudos epidemiológicos têm mostrado diferenças raciais na prevalência e aspectos clínicos e laboratoriais do LES. Diferenças têm sido demonstradas principalmente na freqüência dos auto-anticorpos contra antígenos nucleares extraíveis (ENA, extractable nuclear antigens) e de anticorpos IgG anti-DNA fita dupla. Existem poucos dados sobre a prevalência destes auto-anticorpos em pacientes brasileiros, principalmente usando imunoensaios sensíveis. Assim, neste estudo foram investigadas as freqüências de anticorpos antinúcleo dos isotipos IgG e IgE com especificidade antigênica para as proteínas nucleares SSA, SSB, Sm e U1-RNP, além de anticorpos IgG anti-DNA fita dupla. Soros de 21 pacientes do sexo feminino e de 26 doadoras sadias, idade entre 15-65 anos foram inicialmente triados para a presença de anticorpos antinucleares IgG e IgE através de reação de imunofluorescência indireta (IFI, FAN-IgG e FAN-IgE) com células HEp-2. Anticorpos IgG anti-DNA fita dupla, e IgG e IgE anti-ENA foram investigados por técnica de ELISA (Enzyme-Linked Immunosorbent Assay) indireto, usando fase sólida coberta com os autoantígenos purificados. A concentração sérica de IgE foi determinada por ELISA de captura. Todos os soros dos pacientes com LES (100%) foram reativos no teste de FAN-IgG (mediana do título = 640), enquanto 15/21 (71%) amostras reagiram no teste de FAN-IgE. Anticorpos IgG anti-DNA fita dupla foram detectados em 12/21 (52%) soros (mediana do título = 777 UI/ml, sensibilidade de 35,5%). Quatorze soros reagiram em ELISA-IgG para anticorpos anti-ENA, apresentando as seguintes sensibilidades: anti-RNP = 40%; anti-SSA e anti-Sm = 20%, e anti SSB = 10,5%. Anticorpos IgE anti-ENA foram detectados em sete soros, apresentando o teste de ELISA-IgE uma sensibilidade de 2,5% para 13 anticorpos anti-SSA e SSB, e de 5,3% e 17,6% para anti-Sm e anti-RNP, respectivamente. Os testes de ELISA-IgE para anticorpos anti-Sm e anti-SSA mostraram 100% de especificidade, enquanto uma especificidade de 95% foi encontrada para os testes com anticorpos anti-SSB e anti-RNP. Um aumento na concentração de IgE sérica for observado em 6 (29%) das amostras (mediana = 426 UI/ml), existindo uma correlação positiva entre os títulos de FAN-IgG e IgG anti-RNP (r = 0.796, P = 0.002), entre os títulos de IgG e IgE anti-RNP (r = 0.594, P = 0.0045) e também entre IgE anti-RNP e IgE total (r = 0.680, P = 0.0007). Concluindo, a freqüência de FAN-IgG e anticorpos IgG anti- SSA, SSB e anti-Sm nos pacientes brasileiros com LES concordou com os resultados de outros estudos internacionais, existindo contudo uma forte predominância de anticorpos anti-RNP nestes indivíduos.

Milk is one of the eight major food allergens. Cow's milk allergy is the most common allergy in children under 2 years of age. About 1.6 to 2.8 percent of children under this age are reported to have cow's milk allergy. Casein, β-lactoglobulin and α-lactalbumin are major milk protein allergens. Nonthermal treatments like high intensity ultrasound, ultraviolet (UV) light and nonthermal plasma treatments have been reported in the literature to be effective in reducing the allergenicity of different food proteins. Hence it was expected for these treatments to reduce cow milk allergenicity. The objective of this study was to investigate the effect of high intensity ultrasound, nonthermal atmospheric plasma and UV-C light treatments in reducing the allergenicity of isolated major milk proteins. Sonics Vibracell VC 505 ultrasonic liquid processor was used to perform high intensity ultrasound treatments. UV light treatments were performed using a DDK Scientific Corporation UV tunnel. A nonthermal atmospheric plasma setup assembled in Department of Microbiology lab was used to perform plasma treatments. Samples were analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to estimate the change in protein concentration and enzyme linked immuno sorbent assays (ELISA) to observe the change in IgE binding. A one-way analysis of variance was conducted to evaluate the relationship between treatment time and percent IgE binding at 95% confidence level. Further investigation was conducted with nuclear magnetic resonance (NMR) spectroscopy on treated casein to assess any change in the structure of protein. SDS-PAGE results for ultrasound and plasma treatments didn't show any change in gel band intensities for casein, β-lactoglobulin and α-lactalbumin indicating no significant change in protein concentration. Ci-ELISA analysis showed that there was no significant difference (p>0.05) in IgE binding values for control and treated samples in ultrasound and plasma treatment conditions tested in this study. The intensities of all the three protein bands in SDS-PAGE gel were reduced by UV-C light treatment at 15 min treatment time. In Ci-ELISA, there was a significant difference (p< 0.05) in IgE binding values for control and treated samples and a reduction in allergenicity of proteins (25% reduction for casein and 28% reduction for whey protein fractions) was observed. Further investigations using in vivo clinical trials need to be conducted to confirm this result. NMR results didn't show any noticeable changes in the structures of casein with all three different treatments. In conclusion, UV-C light treatment can reduce the allergenicity of isolated major milk proteins to some extent. High intensity ultrasound and nonthermal atmospheric plasma treatments failed to generate effective results for reducing allergenicity at the conditions tested in this study. Higher intensity and longer treatment conditions might yield better results with ultrasound treatment. Different power and gas flow rates used to generate plasma with direct exposure of proteins might yield better results towards reducing the allergenicity of major milk allergens.