The prevalence and economic burden of chronic airway disorders such as asthma is on the rise annually. Allergic asthma is characterized by chronic airway inflammation, airway hyper-responsiveness (AHR), and structural airway remodeling due to increased smooth muscle mass. Most allergic asthma occurs due to the overproduction of immunoglobulin E (IgE) antibodies against common allergens. Classically, IgE has been shown to modulate airway smooth muscle (ASM) contraction/relaxation which is believed to be the underlying cause of airway hyperreactivity. However, the molecular mechanisms underlying IgE effects on ASM cell are not established. Recently, the high-affinity Fc receptor for IgE (FcεRI) has been identified in human ASM cells in vitro and in vivo within bronchial biopsies of allergic asthma patients. However, it is unknown whether FcεRI activation on ASM can modulate the immune response within the airways. We hypothesized that the IgE-FcεRI interaction plays a key role in inducing phenotypic and functional changes in ASM cells that eventually contributes to the establishment of airway inflammation, AHR, and remodeling. We sought to investigate the regulation, effector functions, and underlying mechanisms of FcεRI activation in ASM cells. Our work shows that the proinflammatory tumor necrosis factor (TNF) and T helper type 2 (Th2) cytokine interleukin (IL)-4 enhanced the FcεRI abundance and amplified the IgE-induced chemokine (eotaxin-1/CCL11, RANTES/CCL5, IL-8/CXCL8, and IP-10/CXCL10) release in ASM cells via transcriptional mechanisms. Both TNF and IgE induced a novel, Th2-favoring cytokine thymic stromal lymphopoietin (TSLP) through the activation of spleen tyrosine kinase (Syk), and nuclear factor kappa B (NF-κB) and activator protein-1 (AP-1). In addition, IgE induced de novo DNA synthesis and ASM cell proliferation via mitogen-activated protein kinases (MAPKs) and signal-transducer and activator of transcription 3 (STAT3) activation. Collectively, our data suggest that the IgE-induced FcεRI activation leads to the expression of multiple chemokines in ASM which may indirectly recruit inflammatory cells and promote allergic airway inflammation; IgE induces TSLP which can promote the Th2 immune responses within the airways; and IgE may potentially induce airway remodeling by directly inducing ASM cell proliferation. Therefore, targeting the IgE-FcεRI network on ASM may offer a novel therapeutic strategy in allergic asthma.

Polymorphonuclear neutrophils (PMNs) are important effector cells in host defense and the inflammatory response to antigen. The involvement of PMNs in inflammation is mainly mediated by the Fc receptor family, including IgE receptors. Recently, we have shown that human PMNs from allergic asthmatic subjects express the high affinity receptor, FceRI. In this study, we have examined the regulation of FceRI by human PMNs in vitro and in vivo during the allergic pollen season. First we studied the pattern of expression of FceRI in PMNs during the pollen allergic and outside the pollen season. Peripheral blood neutrophils were isolated from adult atopic asthmatics (AA) (n=17), allergic non asthmatics (ANA) (n=15) and healthy donors (n=16) by dextran, ficoll gradient centrifugation and magnetic cell sorting (MACS). Surface, total protein and mRNA expression of FceRI were investigated in the three groups by FACS, immunocytochemistry (ICC) and fluorescent in situ hybridization (FISH) respectively. Secondly, we investigated the effect of Th-2 cytokines which are known to regulate IgE receptor expression. PMNs from atopic asthmatic subjects were stimulated in vitro with Th-2 cytokines (IL-4, IL-9, GM-CSF) and Th-1 cytokine IFN-gamma. Finally we determined whether the expression of FceRIbeta chain correlated with the surface expression of FceRIalpha chain in PMNs. Irrespective of the season, PMNs from atopic asthmatic subjects showed increased expression of FceRIalpha chain in surface, total protein and mRNA compared to atopic non asthmatics and healthy donors (n=20). Interestingly, FceRIalpha chain surface and mRNA expression increased significantly during pollen season compared to non pollen season (P=0.001) in PMNs isolated from AA (n=9) in contrast to healthy donors and ANA (n=8). Furthermore similar pattern of FceRI expression were observed in vitro when PMNs were stimulated with Th2 cytokines. IL-4, IL-9 and GM-CSF showed increased protein and mRNA expression of FceRIalpha chain at 6 and 18hrs (n=6) whereas IFN-gamma down regulated the mRNA expression of FceRIalpha chain at 6hrs. Also, irrespective of season AA (n=11) subjects showed increased expression of FceRI beta chain when compared to ANA (n=10) and healthy donors (n=9). Western blot analysis showed increased FceRI beta protein in atopic asthmatic subjects (n=4). Interestingly irrespective of the groups, there was a positive correlation r = 0.8054 between total protein expression of beta chain with surface expression of alpha chain of FceRI in neutrophils. Our data suggest that the expression of FceRI in neutrophils of atopic asthmatic patients is highly regulated. Our in vitro studies provide evidence that Th-2 cytokines such as IL-9, IL-4 and GM-CSF up-regulate the expression of FceRI. Furthermore we show evidence of increased expression of FceRIbeta chain in neutrophils of atopic asthmatic subjects. Collectively these results suggest that FceRI mediated neutrophil dependent activation may play a key role in allergic diseases.

The prevalence and economic burden of chronic airway disorders such as asthma is on the rise annually. Allergic asthma is characterized by chronic airway inflammation, airway hyper-responsiveness (AHR), and structural airway remodeling due to increased smooth muscle mass. Most allergic asthma occurs due to the overproduction of immunoglobulin E (IgE) antibodies against common allergens. Classically, IgE has been shown to modulate airway smooth muscle (ASM) contraction/relaxation which is believed to be the underlying cause of airway hyperreactivity. However, the molecular mechanisms underlying IgE effects on ASM cell are not established. Recently, the high-affinity Fc receptor for IgE (FcεRI) has been identified in human ASM cells in vitro and in vivo within bronchial biopsies of allergic asthma patients. However, it is unknown whether FcεRI activation on ASM can modulate the immune response within the airways. We hypothesized that the IgE-FcεRI interaction plays a key role in inducing phenotypic and functional changes in ASM cells that eventually contributes to the establishment of airway inflammation, AHR, and remodeling. We sought to investigate the regulation, effector functions, and underlying mechanisms of FcεRI activation in ASM cells. Our work shows that the proinflammatory tumor necrosis factor (TNF) and T helper type 2 (Th2) cytokine interleukin (IL)-4 enhanced the FcεRI abundance and amplified the IgE-induced chemokine (eotaxin-1/CCL11, RANTES/CCL5, IL-8/CXCL8, and IP-10/CXCL10) release in ASM cells via transcriptional mechanisms. Both TNF and IgE induced a novel, Th2-favoring cytokine thymic stromal lymphopoietin (TSLP) through the activation of spleen tyrosine kinase (Syk), and nuclear factor kappa B (NF-κB) and activator protein-1 (AP-1). In addition, IgE induced de novo DNA synthesis and ASM cell proliferation via mitogen-activated protein kinases (MAPKs) and signal-transducer and activator of transcription 3 (STAT3) activation. Collectively, our data suggest that the IgE-induced FcεRI activation leads to the expression of multiple chemokines in ASM which may indirectly recruit inflammatory cells and promote allergic airway inflammation; IgE induces TSLP which can promote the Th2 immune responses within the airways; and IgE may potentially induce airway remodeling by directly inducing ASM cell proliferation. Therefore, targeting the IgE-FcεRI network on ASM may offer a novel therapeutic strategy in allergic asthma.

Polymorphonuclear neutrophils (PMNs) are important effector cells in host defense and the inflammatory response to antigen. The involvement of PMNs in inflammation is mainly mediated by the Fc receptor family, including IgE receptors. Recently, we have shown that human PMNs from allergic asthmatic subjects express the high affinity receptor, FceRI. In this study, we have examined the regulation of FceRI by human PMNs in vitro and in vivo during the allergic pollen season. First we studied the pattern of expression of FceRI in PMNs during the pollen allergic and outside the pollen season. Peripheral blood neutrophils were isolated from adult atopic asthmatics (AA) (n=17), allergic non asthmatics (ANA) (n=15) and healthy donors (n=16) by dextran, ficoll gradient centrifugation and magnetic cell sorting (MACS). Surface, total protein and mRNA expression of FceRI were investigated in the three groups by FACS, immunocytochemistry (ICC) and fluorescent in situ hybridization (FISH) respectively. Secondly, we investigated the effect of Th-2 cytokines which are known to regulate IgE receptor expression. PMNs from atopic asthmatic subjects were stimulated in vitro with Th-2 cytokines (IL-4, IL-9, GM-CSF) and Th-1 cytokine IFN-gamma. Finally we determined whether the expression of FceRIbeta chain correlated with the surface expression of FceRIalpha chain in PMNs. Irrespective of the season, PMNs from atopic asthmatic subjects showed increased expression of FceRIalpha chain in surface, total protein and mRNA compared to atopic non asthmatics and healthy donors (n=20). Interestingly, FceRIalpha chain surface and mRNA expression increased significantly during pollen season compared to non pollen season (P=0.001) in PMNs isolated from AA (n=9) in contrast to healthy donors and ANA (n=8). Furthermore similar pattern of FceRI expression were observed in vitro when PMNs were stimulated with Th2 cytokines. IL-4, IL-9 and GM-CSF showed increased protein and mRNA expression of FceRIalpha chain at 6 and 18hrs (n=6) whereas IFN-gamma down regulated the mRNA expression of FceRIalpha chain at 6hrs. Also, irrespective of season AA (n=11) subjects showed increased expression of FceRI beta chain when compared to ANA (n=10) and healthy donors (n=9). Western blot analysis showed increased FceRI beta protein in atopic asthmatic subjects (n=4). Interestingly irrespective of the groups, there was a positive correlation r = 0.8054 between total protein expression of beta chain with surface expression of alpha chain of FceRI in neutrophils. Our data suggest that the expression of FceRI in neutrophils of atopic asthmatic patients is highly regulated. Our in vitro studies provide evidence that Th-2 cytokines such as IL-9, IL-4 and GM-CSF up-regulate the expression of FceRI. Furthermore we show evidence of increased expression of FceRIbeta chain in neutrophils of atopic asthmatic subjects. Collectively these results suggest that FceRI mediated neutrophil dependent activation may play a key role in allergic diseases.

Egg is one of the major food allergens that affects 1.6~3.2% of the infants and young children population. The objective of this study is to reduce the egg white IgE binding ability by lactobacilli or Aspergillus oryzae fermentation. Modifications of egg white proteins during fermentation were analyzed by Ninhydrin method, Ellman method, SDS-PAGE, ELISA, and MALDI-TOF-MS. Tryptone supplementation and acidification are necessary to grow lactobacilli in egg white. Egg whites were fermented by L sanfranciscensis, L. sakei, and L. delbrueckii subsp. delbrueckii individually for 96 h; and Aspergillus oryzae for 120 h. The IgE binding ability of egg white was significantly reduced (~50%) by L. delbrueckii subsp. delbrueckii after 48 h of incubation and almost eliminated by Aspergillus oryzae after 24 h of inoculation. In addition to slight modification of ovomucoid (the dominant egg allergen), no substantial protein degradation was observed during fermentation. / Food Science and Technology

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2002. / Härtill 5 uppsatser.

In the past decades, there has been a global raise in the prevalence of allergic diseases and, in parallel, suggestive manifestations of such diseases have been observed in patients with Human Immunodeficiency Virus/Acquired Immune Deficiency Syndrome (HIV/Aids), however, there are few studies concerning the pediatric population. Objective: estimating the prevalence of allergic diseases on subjects under 18 years old who have HIV/Aids in the Association of Municipalities of Laguna Region (AMUREL) region and relating clinical-immunological characteristics of infection and atopy. Methods: a cross sectional study has been carried out with 29 outpatient aged under 18 years old followed in the HIV/Aids specialized clinic of the region, from February to October 2012. A questionnaire of the International Study for Asthma and Allergies in Childhood (ISAAC) protocol has been applied, data collection from the medical records investigating socio-demographic and laboratory data. Blood has been collected for total IgE serum dosage and Radioallergosorbent Test (RAST) for mites, cockroaches, and epithelium of animals, feathers, and fungi. The data were compiled on the computerized base of Excel 2007 and the analysis was carried out on the Prism 6 program. The following tests were applied: t-student test, chi-squared test, Fisher’s exact test and Mann-Whitney U test, when indicated, where values of p<0,05 were considered significant. Results: among the 29 evaluated individuals, the prevalence of symptoms of allergic diseases was 65.5% (IC95% 56.1-74.8), rhinitis being the most frequent 44.8% (IC95% 35.0-54.5), followed by asthma 37.9% (IC95% 28.3-47.4) and eczema 27.6% (IC95% 18.8-36.3). The RAST test was positive in 20.7% of the individuals with no significant difference among the groups. There was no significant difference concerning total IgE serum levels among individuals with and without records of allergic disease symptoms. Nevertheless, a high frequency of elevated total IgE measures 40.7% and a relation between elevated IgE and clinical stage of the HIV/Aids disease have been observed. A relation between CD8+ cell count and the prevalence of allergic diseases symptoms has also been obtained (p=0.014). Conclusion: there has been a high prevalence of allergic disease as well as high frequency of elevated total IgE serum. The relation elevated IgE with clinical stage deserves future inquiry, with serial measurements of total IgE serum for the identification of aggravation of HIV/Aids. The association between CD8+ count and prevalence of allergic diseases symptoms corroborates studies that have proved the role of these cells in the development of allergic diseases. / Submitted by Rogele Pinheiro (rogele.pinheiro@unisul.br) on 2017-10-23T17:20:01Z No. of bitstreams: 1 106415_Leandro.pdf: 467294 bytes, checksum: cd848f6be942abd36683496da1a38cca (MD5) / Approved for entry into archive by Caroline Correa da Cruz (caroline.cruz@unisul.br) on 2017-10-23T20:16:33Z (GMT) No. of bitstreams: 1 106415_Leandro.pdf: 467294 bytes, checksum: cd848f6be942abd36683496da1a38cca (MD5) / Made available in DSpace on 2017-10-23T20:16:33Z (GMT). No. of bitstreams: 1 106415_Leandro.pdf: 467294 bytes, checksum: cd848f6be942abd36683496da1a38cca (MD5) Previous issue date: 2013-03-05 / Nas últimas décadas houve um aumento global na prevalência de doenças alérgicas e, em paralelo, sugestivas manifestações dessas doenças têm sido observadas em pacientes com Vírus da Imunodeficiência Humana/Síndrome da Imunodeficiência Humana Adquirida (HIV/Aids), no entanto, poucos são os estudos dirigidos à população pediátrica. Objetivo: estimar a prevalência de doenças alérgicas em menores de 18 anos com HIV/Aids da região da Associação dos Municípios da Região de Laguna (AMUREL) e relacionar características clínico-imunológicas de infecção e atopia. Métodos: realizou-se estudo epidemiológico transversal com 29 pacientes menores de 18 anos, em acompanhamento nos serviços especializados em HIV/Aids da região, de fevereiro a outubro de 2012. Aplicou-se o questionário do protocolo Estudo Internacional de Asma e Alergia na Infância e Adolescência (ISAAC), levantamento de dados nos prontuários médicos investigando dados sociodemográficos e laboratoriais. Foi coletado sangue para dosagem de Imunoglobulina E (IgE) sérica total e Radioallergosorbent Test (RAST) para ácaros, barata, epitélios de animais, penas e fungos. Os dados foram compilados em base informatizada do Excel 2007 e a análise realizada no programa Prism 6. Aplicou-se os testes: tstudent, qui-quadrado, prova exata de Fischer e teste Mann-Whitney, quando indicados, sendo considerados significativos valores de p<0,05 Resultados: entre os 29 indivíduos avaliados, a prevalência de sintomas de doenças alérgicas foi de 65,5% (IC95% 56,1-74,8), sendo a mais frequente a rinite 44,8% (IC95% 35,0-54,5), seguida de asma 37,9% (IC95% 28,3-47,4) e eczema 27,6% (IC95% 18,8-36,3). O RAST foi positivo em 20,7% dos indivíduos, sem diferença significativa entre os grupos. Não houve diferença significativa quanto aos níveis de IgE sérica total, entre indivíduos com e sem relato de sintomas de doença alérgica. Contudo, observou-se alta frequência de medidas de IgE sérica total elevada 40,7% e relação entre IgE elevada e estádio clínico da doença HIV/Aids. Obteve-se ainda associação entre contagem de células CD8+ e a prevalência de sintomas de doenças alérgicas (p=0,014). Conclusão: houve alta prevalência de doença alérgica, assim como alta frequência de níveis elevados de IgE sérica total. A relação IgE elevada com estádio clínico, sugere-se investigações futuras, com medidas seriadas da IgE total para verificar se há agravamento do HIV/Aids. A associação entre contagem de CD8+ e prevalência de sintomas de doenças alérgicas corrobora estudos que provaram o papel dessas células no desenvolvimento de doenças alérgicas.

Submitted by Marcelo Andrade Silva (marcelo.andradesilva@ufpe.br) on 2015-03-09T14:18:41Z No. of bitstreams: 2 DISSERTAÇÃO CASSIA GISELLE DE OLIVEIRA NÓBREGA.pdf: 1673463 bytes, checksum: 460066c3a78447d401976dede5d04300 (MD5) license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) / Made available in DSpace on 2015-03-09T14:18:41Z (GMT). No. of bitstreams: 2 DISSERTAÇÃO CASSIA GISELLE DE OLIVEIRA NÓBREGA.pdf: 1673463 bytes, checksum: 460066c3a78447d401976dede5d04300 (MD5) license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Previous issue date: 2014 / A intensidade da resposta alérgica pode ser alterada em indivíduos sensibilizados por Ascaris lumbricoides, ou seja, com presença de anticorpos específicos no soro. Quanto à IgE anti-Asc, este anticorpo tem sido relatado como um fator de risco para asma, mas sobre a IgG1anti-Asc pouco é sabido. Este trabalho teve como objetivo verificar a associação entre IgE, IgG1 e IgG4 anti-Asc no soro e a presença de asma, bem como, entre asma e níveis de citocinas, valores absolutos de neutrófilos e eosinófilos, em pacientes com IgE ou IgG1 anti-Asc no soro. Para isto, crianças de 2 a 14 anos de idade, residentes na Região Metropolitana do Recife (n=104), asmáticas ou não asmáticos, sem infecção, tiveram as amostras de sangue coletadas. Foi realizado o leucograma e a cultura celular do sangue periférico. As células foram cultivadas, estimulados ou não com PHA, e os sobrenadantes submetidos à dosagem de citocinas por CBA. Os isótipos IgE, IgG1 e IgG4 anti-Asc, no soro, foram mensurados por ELISA. Foram formados 8 grupos de estudo: asma IgE anti-Asc positivo e negativo; controle (paciente não asmáticos) IgE anti-Asc positivo e negativo; asma IgG1 anti-Asc positivo e negativo; controle IgG1 anti-Asc positivo e negativo. Foi observado que não houve diferença na quantidade de indivíduos com IgE positivo e negativo entre os grupos asma e controle, bem como nos níveis deste anticorpo. O mesmo foi notado para a presença de IgG1 anti-Asc, porém com níveis mais elevados de anticorpo nos indivíduos controles. Não foram detectados níveis de IgG4 anti-Asc em pacientes asmáticos ou controles. Foram encontrados maiores níveis de IL-6, TNF-α e número de eosinófilos nos pacientes asmáticos em comparação aos controles. Este perfil se manteve nos pacientes asma IgE anti-Asc negativo, quando comparado aos controles negativo. Nos pacientes do grupo asma IgG1 anti-Asc positivo, foram observados maiores níveis de IL-6 e eosinófilos, em comparação aos do grupo controle positivo. No grupo asma IgG1 negativo houve maiores números de neutrófilos e eosinófilos, em comparação aos pacientes controle negativo. Os grupos asmáticos, independente da presença de IgE anti-Asc, apresentaram maior frequência de indivíduos com IL-10 e IFN-. Apenas os indivíduos do grupo asma IgG1 anti-Asc negativo apresentaram maior frequência de pacientes IL-10 e IFN-γ. Diante disso, o fato de ter asma favoreceu a produção de IL-6, TNF-α e eosinófilos, bem como IL-10 e IFN-γ, independente da presença da IgE anti-Asc. Contudo, em pacientes asmáticos a presença da IgG1 anti-Asc parece interferir melhorando a produção de IL-6, eosinófilos, mas não de IL-10 e IFN-γ.

While food hypersensitivity can be a life-threatening problem, its scope is yet to be fully developed. More work is needed to further define its parameters but basic food hypersensitivity has been significantly clarified in the decade of the 80's to become standard practice for most updated allergists. Studies related to inhalation of food antigens remains within the purview of research centers as does other immunologic processes. The diagnosis of food hypersensitivity remains dependent on the medical history with test like elimination diets, skin testing, and RAST. Double-blind, placebo-controlled, food challenges (DBPCFC) provide the most definitive support for the association between certain symptoms and a specific food.

Indiana University-Purdue University Indianapolis (IUPUI) / Follicular T helper (TFH) and regulatory (TFR) cells are two key classes of CD4+ T cells found in germinal centers (GCs). The primary role of TFH cells is to help B cells form GCs to produce high-affinity antibodies during an infection while the role of TFR cells remains controversial. The transcriptional repressor Bcl6 is essential for the differentiation of TFH, TFR and GCB cells and understanding signaling pathways that induce Bcl6 and TFH cell differentiation are important. We observed that Bcl6 is highly up-regulated in activated CD4 T cells following glucose deprivation by a pathway involving the metabolic sensor AMP kinase. The transcription factor Blimp1 represses both TFH cell differentiation and Bcl6 expression, and we show the major role of Blimp1 on TFH cell differentiation is to repress Bcl6 expression and not other genes in the TFH differentiation pathway. We also found Bcl6 positively regulates expression of the key TFH cell receptor PD-1 by inhibiting the repression of PD-1 by the transcription factor Tbet. The roles of TFH and TFR cells in controlling allergen-specific IgE were investigated using a peanut allergy model and strains of mice with alterations in the TFH and TFR pathways. We found TFR cells unexpectedly play an essential role in promoting and maintaining IgE production and anaphylaxis, as well as the GC reaction. Compared to control mice, TFR-deficient mice lacked circulating peanut-specific IgE and anaphylactic responses were significantly weakened. Mechanistically, TFR cells require Blimp1 controlled IL-10 to promote GCB cell survival and IgE production. Blocking IL-10 signals mimicked the loss of IgE levels in TFR-deficient mice and rescued mice from anaphylaxis. Overall, these studies have defined novel roles of Bcl6, TFH and TFR cells in regulating antibody production by the GC reaction, and provide greater understanding of how allergic immune responses are controlled. / 2019-11-21