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1.
Allergy ; 74(12): 2461-2478, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31228873

RESUMO

BACKGROUND: In the house dust mite (HDM) Dermatophagoides pteronyssinus, Der p 1, 2, 5, 7, 21, and 23 have been identified as the most important allergens. The aim of this study was to define hypoallergenic peptides derived from the sequences of the six allergens and to use the peptides and the complete allergens to study antibody, T cell, and cytokine responses in sensitized and nonsensitized subjects. METHODS: IgE reactivity of HDM-allergic and non-HDM-sensitized individuals to 15 HDM allergens was established using ImmunoCAP ISAC technology. Thirty-three peptides covering the sequences of the six HDM allergens were synthesized. Allergens and peptides were tested for IgE and IgG reactivity by ELISA and ImmunoCAP, respectively. Allergenic activity was determined by basophil activation. CD4+ T cell and cytokine responses were determined in PBMC cultures by CFSE dilution and Luminex technology, respectively. RESULTS: House dust mite allergics showed IgE reactivity only to complete allergens, whereas 31 of the 33 peptides lacked relevant IgE reactivity and allergenic activity. IgG antibodies of HDM-allergic and nonsensitized subjects were directed against peptide epitopes and higher allergen-specific IgG levels were found in HDM allergics. PBMC from HDM-allergics produced higher levels of IL-5 whereas non-HDM-sensitized individuals mounted higher levels of IFN-gamma, IL-17, pro-inflammatory cytokines, and IL-10. CONCLUSION: IgG antibodies in HDM-allergic patients recognize peptide epitopes which are different from the epitopes recognized by IgE. This may explain why naturally occurring allergen-specific IgG antibodies do not protect against IgE-mediated allergic inflammation. A mix of hypoallergenic peptides containing T cell epitopes of the most important HDM allergens was identified.


Assuntos
Alérgenos/imunologia , Epitopos de Linfócito T/imunologia , Hipersensibilidade/imunologia , Peptídeos/imunologia , Pyroglyphidae/imunologia , Animais , Antígenos de Dermatophagoides/imunologia , Proteínas de Artrópodes/imunologia , Estudos de Casos e Controles , Cisteína Endopeptidases/imunologia , Citocinas/metabolismo , Ensaio de Imunoadsorção Enzimática , Humanos , Imunoglobulina E/imunologia , Imunoglobulina G/imunologia
2.
Haematologica ; 103(5): 799-809, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29439183

RESUMO

Systemic mastocytosis is a complex disease defined by abnormal growth and accumulation of neoplastic mast cells in various organs. Most patients exhibit a D816V-mutated variant of KIT, which confers resistance against imatinib. Clinical problems in systemic mastocytosis arise from mediator-related symptoms and/or organ destruction caused by malignant expansion of neoplastic mast cells and/or other myeloid cells in various organ systems. DCC-2618 is a spectrum-selective pan KIT and PDGFRA inhibitor which blocks KIT D816V and multiple other kinase targets relevant to systemic mastocytosis. We found that DCC-2618 inhibits the proliferation and survival of various human mast cell lines (HMC-1, ROSA, MCPV-1) as well as primary neoplastic mast cells obtained from patients with advanced systemic mastocytosis (IC50 <1 µM). Moreover, DCC-2618 decreased growth and survival of primary neoplastic eosinophils obtained from patients with systemic mastocytosis or eosinophilic leukemia, leukemic monocytes obtained from patients with chronic myelomonocytic leukemia with or without concomitant systemic mastocytosis, and blast cells obtained from patients with acute myeloid leukemia. Furthermore, DCC-2618 was found to suppress the proliferation of endothelial cells, suggesting additional drug effects on systemic mastocytosis-related angiogenesis. Finally, DCC-2618 was found to downregulate IgE-mediated histamine release from basophils and tryptase release from mast cells. Together, DCC-2618 inhibits growth, survival and activation of multiple cell types relevant to advanced systemic mastocytosis. Whether DCC-2618 is effective in vivo in patients with advanced systemic mastocytosis is currently under investigation in clinical trials.


Assuntos
Proliferação de Células/efeitos dos fármacos , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Mastócitos/patologia , Mastocitose Sistêmica/patologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-kit/antagonistas & inibidores , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/antagonistas & inibidores , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Masculino , Mastócitos/efeitos dos fármacos , Mastócitos/metabolismo , Mastocitose Sistêmica/tratamento farmacológico , Mastocitose Sistêmica/metabolismo , Pessoa de Meia-Idade , Mutação , Células Tumorais Cultivadas
3.
Blood ; 126(26): 2832-41, 2015 Dec 24.
Artigo em Inglês | MEDLINE | ID: mdl-26486787

RESUMO

The Ki-1 antigen (CD30) is an established therapeutic target in patients with Hodgkin lymphoma and anaplastic large-cell lymphoma. We have recently shown that CD30 is expressed abundantly in the cytoplasm of neoplastic mast cells (MCs) in patients with advanced systemic mastocytosis (SM). In the current study, we asked whether CD30 is expressed on the surface of neoplastic MCs in advanced SM, and whether this surface structure may serve as therapeutic target in SM. As assessed by flow cytometry, CD30 was found to be expressed on the surface of neoplastic MCs in 3 of 25 patients (12%) with indolent SM, 4 of 7 patients (57%) with aggressive SM, and 4 of 7 patients (57%) with MC leukemia. The immature RAS-transformed human MC line MCPV-1.1 also expressed cell surface CD30, whereas the KIT-transformed MC line HMC-1.2 expressed no detectable CD30. The CD30-targeting antibody-conjugate brentuximab-vedotin inhibited proliferation in neoplastic MCs, with lower IC50 values obtained in CD30(+) MCPV-1.1 cells (10 µg/mL) compared with CD30(-) HMC-1.2 cells (>50 µg/mL). In addition, brentuximab-vedotin suppressed the engraftment of MCPV-1.1 cells in NSG mice. Moreover, brentuximab-vedotin produced apoptosis in all CD30(+) MC lines tested as well as in primary neoplastic MCs in patients with CD30(+) SM, but did not induce apoptosis in neoplastic MCs in patients with CD30(-) SM. Furthermore, brentuximab-vedotin was found to downregulate anti-IgE-induced histamine release in CD30(+) MCs. Finally, brentuximab-vedotin and the KIT D816V-targeting drug PKC412 produced synergistic growth-inhibitory effects in MCPV-1.1 cells. Together, CD30 is a promising new drug target for patients with CD30(+) advanced SM.


Assuntos
Imunoconjugados/farmacologia , Antígeno Ki-1/biossíntese , Mastócitos/metabolismo , Mastocitose Sistêmica/metabolismo , Animais , Apoptose/efeitos dos fármacos , Brentuximab Vedotin , Proliferação de Células/efeitos dos fármacos , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Xenoenxertos , Humanos , Imuno-Histoquímica , Antígeno Ki-1/antagonistas & inibidores , Mastócitos/patologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Reação em Cadeia da Polimerase
4.
Haematologica ; 102(9): 1519-1529, 2017 09.
Artigo em Inglês | MEDLINE | ID: mdl-28596283

RESUMO

In chronic myeloid leukemia, resistance against BCR-ABL1 tyrosine kinase inhibitors can develop because of BCR-ABL1 mutations, activation of additional pro-oncogenic pathways, and stem cell resistance. Drug combinations covering a broad range of targets may overcome resistance. CDDO-Me (bardoxolone methyl) is a drug that inhibits the survival of leukemic cells by targeting different pro-survival molecules, including STAT3. We found that CDDO-Me inhibits proliferation and survival of tyrosine kinase inhibitor-resistant BCR-ABL1+ cell lines and primary leukemic cells, including cells harboring BCR-ABL1T315I or T315I+ compound mutations. Furthermore, CDDO-Me was found to block growth and survival of CD34+/CD38- leukemic stem cells (LSC). Moreover, CDDO-Me was found to produce synergistic growth-inhibitory effects when combined with BCR-ABL1 tyrosine kinase inhibitors. These drug-combinations were found to block multiple signaling cascades and molecules, including STAT3 and STAT5. Furthermore, combined targeting of STAT3 and STAT5 by shRNA and STAT5-targeting drugs also resulted in synergistic growth-inhibition, pointing to a new efficient concept of combinatorial STAT3 and STAT5 inhibition. However, CDDO-Me was also found to increase the expression of heme-oxygenase-1, a heat-shock-protein that triggers drug resistance and cell survival. We therefore combined CDDO-Me with the heme-oxygenase-1 inhibitor SMA-ZnPP, which also resulted in synergistic growth-inhibitory effects. Moreover, SMA-ZnPP was found to sensitize BCR-ABL1+ cells against the combination 'CDDO-Me+ tyrosine kinase inhibitor'. Together, combined targeting of STAT3, STAT5, and heme-oxygenase-1 overcomes resistance in BCR-ABL1+ cells, including stem cells and highly resistant sub-clones expressing BCR-ABL1T315I or T315I-compound mutations. Whether such drug-combinations are effective in tyrosine kinase inhibitor-resistant patients with chronic myeloid leukemia remains to be elucidated.


Assuntos
Sistemas de Liberação de Medicamentos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Proteínas de Fusão bcr-abl/antagonistas & inibidores , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Células-Tronco Neoplásicas/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Fator de Transcrição STAT3/antagonistas & inibidores , Fator de Transcrição STAT5/antagonistas & inibidores , Adulto , Idoso , Idoso de 80 Anos ou mais , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/genética , Proteínas de Fusão bcr-abl/genética , Proteínas de Fusão bcr-abl/metabolismo , Humanos , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Masculino , Pessoa de Meia-Idade , Células-Tronco Neoplásicas/patologia , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , Fator de Transcrição STAT5/genética , Fator de Transcrição STAT5/metabolismo
5.
J Immunol ; 194(8): 4008-18, 2015 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-25786690

RESUMO

More than 10% of the population in Europe and North America suffer from IgE-associated allergy to grass pollen. In this article, we describe the development of a vaccine for grass pollen allergen-specific immunotherapy based on two recombinant hypoallergenic mosaic molecules, designated P and Q, which were constructed out of elements derived from the four major timothy grass pollen allergens: Phl p 1, Phl p 2, Phl p 5, and Phl p 6. Seventeen recombinant mosaic molecules were expressed and purified in Escherichia coli using synthetic genes, characterized regarding biochemical properties, structural fold, and IgE reactivity. We found that depending on the arrangement of allergen fragments, mosaic molecules with strongly varying IgE reactivity were obtained. Based on an extensive screening with sera and basophils from allergic patients, two hypoallergenic mosaic molecules, P and Q, incorporating the primary sequence elements of the four grass pollen allergens were identified. As shown by lymphoproliferation experiments, they contained allergen-specific T cell epitopes required for tolerance induction, and upon immunization of animals induced higher allergen-specific IgG Abs than the wild-type allergens and a registered monophosphoryl lipid A-adjuvanted vaccine based on natural grass pollen allergen extract. Moreover, IgG Abs induced by immunization with P and Q inhibited the binding of patients' IgE to natural allergens from five grasses better than IgG induced with the wild-type allergens or an extract-based vaccine. Our results suggest that vaccines based on the hypoallergenic grass pollen mosaics can be used for immunotherapy of grass pollen allergy.


Assuntos
Alérgenos , Evolução Molecular Direcionada , Imunização , Phleum , Proteínas de Plantas , Pólen , Rinite Alérgica Sazonal/prevenção & controle , Alérgenos/genética , Alérgenos/imunologia , Alérgenos/farmacologia , Animais , Epitopos de Linfócito T/genética , Epitopos de Linfócito T/imunologia , Epitopos de Linfócito T/farmacologia , Feminino , Humanos , Imunoglobulina E/genética , Imunoglobulina E/imunologia , Imunoglobulina G/genética , Imunoglobulina G/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Phleum/genética , Phleum/imunologia , Proteínas de Plantas/genética , Proteínas de Plantas/imunologia , Proteínas de Plantas/farmacologia , Pólen/genética , Pólen/imunologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/farmacologia , Rinite Alérgica Sazonal/genética , Rinite Alérgica Sazonal/imunologia
6.
Nature ; 478(7370): 524-8, 2011 Aug 03.
Artigo em Inglês | MEDLINE | ID: mdl-21814200

RESUMO

Epigenetic pathways can regulate gene expression by controlling and interpreting chromatin modifications. Cancer cells are characterized by altered epigenetic landscapes, and commonly exploit the chromatin regulatory machinery to enforce oncogenic gene expression programs. Although chromatin alterations are, in principle, reversible and often amenable to drug intervention, the promise of targeting such pathways therapeutically has been limited by an incomplete understanding of cancer-specific dependencies on epigenetic regulators. Here we describe a non-biased approach to probe epigenetic vulnerabilities in acute myeloid leukaemia (AML), an aggressive haematopoietic malignancy that is often associated with aberrant chromatin states. By screening a custom library of small hairpin RNAs (shRNAs) targeting known chromatin regulators in a genetically defined AML mouse model, we identify the protein bromodomain-containing 4 (Brd4) as being critically required for disease maintenance. Suppression of Brd4 using shRNAs or the small-molecule inhibitor JQ1 led to robust antileukaemic effects in vitro and in vivo, accompanied by terminal myeloid differentiation and elimination of leukaemia stem cells. Similar sensitivities were observed in a variety of human AML cell lines and primary patient samples, revealing that JQ1 has broad activity in diverse AML subtypes. The effects of Brd4 suppression are, at least in part, due to its role in sustaining Myc expression to promote aberrant self-renewal, which implicates JQ1 as a pharmacological means to suppress MYC in cancer. Our results establish small-molecule inhibition of Brd4 as a promising therapeutic strategy in AML and, potentially, other cancers, and highlight the utility of RNA interference (RNAi) screening for revealing epigenetic vulnerabilities that can be exploited for direct pharmacological intervention.


Assuntos
Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Proteínas Nucleares/metabolismo , Interferência de RNA , Fatores de Transcrição/metabolismo , Acetilação , Animais , Azepinas/farmacologia , Diferenciação Celular , Linhagem Celular Tumoral , Proliferação de Células , Cromatina/metabolismo , Progressão da Doença , Epigênese Genética/genética , Regulação Neoplásica da Expressão Gênica , Genes myc/genética , Histonas/metabolismo , Humanos , Leucemia Mieloide Aguda/patologia , Camundongos , Transplante de Neoplasias , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/patologia , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/biossíntese , Proteínas Nucleares/genética , RNA Interferente Pequeno/genética , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genética , Triazóis/farmacologia
7.
J Allergy Clin Immunol ; 137(2): 601-609.e8, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26518092

RESUMO

BACKGROUND: Late allergic reactions are common in the course of allergen-specific immunotherapy and even occur with allergy vaccines with reduced IgE reactivity. OBJECTIVE: We sought to study atopy patch test (APT) reactions and T-cell responses to the recombinant birch pollen allergen Bet v 1 and recombinant hypoallergenic T-cell epitope-containing Bet v 1 fragments in patients with birch pollen allergy with and without atopic dermatitis (AD). METHODS: A clinical study was conducted in 15 patients with birch pollen allergy with AD (group 1), 5 patients with birch pollen allergy without AD (group 2), 5 allergic patients without birch pollen allergy (group 3), and 5 nonallergic subjects (group 4) by performing skin prick tests and APTs with rBet v 1 and hypoallergenic rBet v 1 fragments. T-cell, cutaneous lymphocyte antigen (CLA)(+) and CCR4(+) T-cell and cytokine responses were studied by thymidine uptake, carboxyfluorescein diacetate succinimidyl ester staining, and Luminex technology, respectively. RESULTS: rBet v 1 and hypoallergenic rBet v 1 fragments induced APT reactions in not only most of the patients with birch pollen allergy with AD (11/15) but also in most of those without AD (4/5). Patients with birch pollen allergy with AD had higher Bet v 1-specific proliferation of CLA(+) and CCR4(+) T cells compared with patients with birch pollen allergy without AD. There were no differences in Bet v 1-specific CLA(+) and CCR4(+) proliferation and cytokine secretion in patients with and without APT reactions. CONCLUSION: Hypoallergenic rBet v 1 fragments induce T cell-dependent late reactions not only in patients with birch pollen allergy with AD but also in those without AD, which can be determined based on APT results but not based on in vitro parameters.


Assuntos
Alérgenos/imunologia , Antígenos de Plantas/imunologia , Hipersensibilidade Tardia/diagnóstico , Hipersensibilidade Tardia/imunologia , Testes do Emplastro , Linfócitos T/imunologia , Adulto , Betula/efeitos adversos , Citocinas/biossíntese , Dermatite Atópica/diagnóstico , Dermatite Atópica/imunologia , Dermatite Atópica/metabolismo , Feminino , Liberação de Histamina , Humanos , Hipersensibilidade Tardia/metabolismo , Hipersensibilidade Imediata/diagnóstico , Hipersensibilidade Imediata/imunologia , Hipersensibilidade Imediata/metabolismo , Ativação Linfocitária/imunologia , Masculino , Pólen/imunologia , Rinite Alérgica Sazonal/diagnóstico , Rinite Alérgica Sazonal/imunologia , Especificidade do Receptor de Antígeno de Linfócitos T/imunologia , Linfócitos T/metabolismo , Adulto Jovem
8.
Blood ; 123(25): 3951-62, 2014 Jun 19.
Artigo em Inglês | MEDLINE | ID: mdl-24778155

RESUMO

Chronic myeloid leukemia (CML) is a stem cell (SC) neoplasm characterized by the BCR/ABL1 oncogene. Although mechanisms of BCR/ABL1-induced transformation are well-defined, little is known about effector-molecules contributing to malignant expansion and the extramedullary spread of leukemic SC (LSC) in CML. We have identified the cytokine-targeting surface enzyme dipeptidylpeptidase-IV (DPPIV/CD26) as a novel, specific and pathogenetically relevant biomarker of CD34(+)/CD38(─) CML LSC. In functional assays, CD26 was identified as target enzyme disrupting the SDF-1-CXCR4-axis by cleaving SDF-1, a chemotaxin recruiting CXCR4(+) SC. CD26 was not detected on normal SC or LSC in other hematopoietic malignancies. Correspondingly, CD26(+) LSC decreased to low or undetectable levels during successful treatment with imatinib. CD26(+) CML LSC engrafted NOD-SCID-IL-2Rγ(-/-) (NSG) mice with BCR/ABL1(+) cells, whereas CD26(─) SC from the same patients produced multilineage BCR/ABL1(-) engraftment. Finally, targeting of CD26 by gliptins suppressed the expansion of BCR/ABL1(+) cells. Together, CD26 is a new biomarker and target of CML LSC. CD26 expression may explain the abnormal extramedullary spread of CML LSC, and inhibition of CD26 may revert abnormal LSC function and support curative treatment approaches in this malignancy.


Assuntos
Dipeptidil Peptidase 4/metabolismo , Proteínas de Fusão bcr-abl/metabolismo , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Células-Tronco Neoplásicas/metabolismo , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Antineoplásicos/uso terapêutico , Benzamidas/uso terapêutico , Dipeptidil Peptidase 4/genética , Feminino , Proteínas de Fusão bcr-abl/genética , Perfilação da Expressão Gênica , Regulação Leucêmica da Expressão Gênica , Humanos , Mesilato de Imatinib , Subunidade gama Comum de Receptores de Interleucina/deficiência , Subunidade gama Comum de Receptores de Interleucina/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Masculino , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Pessoa de Meia-Idade , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/transplante , Análise de Sequência com Séries de Oligonucleotídeos , Piperazinas/uso terapêutico , Pirimidinas/uso terapêutico , Transplante Heterólogo , Células Tumorais Cultivadas , Adulto Jovem
9.
Blood ; 124(1): 111-20, 2014 Jul 03.
Artigo em Inglês | MEDLINE | ID: mdl-24677542

RESUMO

In systemic mastocytosis (SM), clinical problems arise from factor-independent proliferation of mast cells (MCs) and the increased release of mediators by MCs, but no human cell line model for studying MC activation in the context of SM is available. We have created a stable stem cell factor (SCF) -dependent human MC line, ROSA(KIT WT), expressing a fully functional immunoglobulin E (IgE) receptor. Transfection with KIT D816V converted ROSA(KIT WT) cells into an SCF-independent clone, ROSA(KIT D816V), which produced a mastocytosis-like disease in NSG mice. Although several signaling pathways were activated, ROSA(KIT D816V) did not exhibit an increased, but did exhibit a decreased responsiveness to IgE-dependent stimuli. Moreover, NSG mice bearing ROSA(KIT D816V)-derived tumors did not show mediator-related symptoms, and KIT D816V-positive MCs obtained from patients with SM did not show increased IgE-dependent histamine release or CD63 upregulation. Our data show that KIT D816V is a disease-propagating oncoprotein, but it does not activate MCs to release proinflammatory mediators, which may explain why mediator-related symptoms in SM occur preferentially in the context of a coexisting allergy. ROSA(KIT D816V) may provide a valuable tool for studying the pathogenesis of mastocytosis and should facilitate the development of novel drugs for treating SM patients.


Assuntos
Linhagem Celular , Mastócitos/patologia , Mastocitose Sistêmica/genética , Proteínas Proto-Oncogênicas c-kit/genética , Animais , Western Blotting , Linhagem Celular/citologia , Linhagem Celular/imunologia , Linhagem Celular/metabolismo , Separação Celular , Citometria de Fluxo , Xenoenxertos , Humanos , Imunoglobulina E/imunologia , Imunoglobulina E/metabolismo , Mastócitos/imunologia , Mastócitos/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Transfecção
10.
J Immunol ; 192(10): 4867-75, 2014 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-24733847

RESUMO

Der p 23, a new, major house dust mite (HDM) allergen that is recognized by >70% of HDM-allergic patients, has high allergenic activity and, therefore, must be considered an important component for HDM-specific immunotherapy. We constructed and characterized a hypoallergenic Der p 23 vaccine for HDM immunotherapy. Three nonallergenic peptides from the C-terminal IgE epitope-containing part of Der p 23 (P4, P5) and P6, a mutant peptide containing serines instead of cysteines, were identified. Peptides were fused to the hepatitis B virus-derived PreS domain as recombinant fusion proteins (i.e., PreS-2XP4P5 and PreS-4XP6) that were expressed in Escherichia coli and purified to homogeneity. Compared with Der p 23, PreS-2XP4P5 and PreS-4XP6 showed no relevant IgE reactivity and exhibited considerably reduced allergenic activity in basophil activation tests using blood from HDM-allergic patients. Upon immunization of rabbits, only PreS-2XP4P5 induced high levels of Der p 23-specific IgG Abs that inhibited binding of patients' IgE to Der p 23, comparable to IgG Abs induced with Der p 23, whereas Abs induced with PreS-4XP6 had only low blocking capacity. Additionally, IgG Abs induced with PreS-2XP4P5 inhibited Der p 23-induced basophil activation comparable to IgG Abs induced with Der p 23. Compared with Der p 23, PreS-2XP4P5 induced lower T cell proliferation but higher levels of the tolerogenic cytokine IL-10 and the Th1 cytokine IFN-γ in PBMCs from HDM-allergic patients, indicating an immunomodulatory capacity of the fusion protein. Therefore, PreS-2XP4P5 represents a promising candidate for immunotherapy of HDM-allergic patients.


Assuntos
Alérgenos/farmacologia , Antígenos de Dermatophagoides/farmacologia , Basófilos/imunologia , Pyroglyphidae/imunologia , Vacinas/farmacologia , Alérgenos/genética , Alérgenos/imunologia , Alérgenos/isolamento & purificação , Animais , Antígenos de Dermatophagoides/genética , Antígenos de Dermatophagoides/imunologia , Antígenos de Dermatophagoides/isolamento & purificação , Basófilos/patologia , Proliferação de Células/efeitos dos fármacos , Feminino , Humanos , Imunoglobulina E/imunologia , Interferon gama/imunologia , Interleucina-10/imunologia , Masculino , Coelhos , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/farmacologia , Células Th1/imunologia , Vacinas/genética , Vacinas/imunologia , Vacinas/isolamento & purificação
11.
J Allergy Clin Immunol ; 135(5): 1207-7.e1-11, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25441634

RESUMO

BACKGROUND: Grass pollen is one of the most important sources of respiratory allergies worldwide. OBJECTIVE: This study describes the development of a grass pollen allergy vaccine based on recombinant hypoallergenic derivatives of the major timothy grass pollen allergens Phl p 1, Phl p 2, Phl p 5, and Phl p 6 by using a peptide-carrier approach. METHODS: Fusion proteins consisting of nonallergenic peptides from the 4 major timothy grass pollen allergens and the PreS protein from hepatitis B virus as a carrier were expressed in Escherichia coli and purified by means of chromatography. Recombinant PreS fusion proteins were tested for allergenic activity and T-cell activation by means of IgE serology, basophil activation testing, T-cell proliferation assays, and xMAP Luminex technology in patients with grass pollen allergy. Rabbits were immunized with PreS fusion proteins to characterize their immunogenicity. RESULTS: Ten hypoallergenic PreS fusion proteins were constructed, expressed, and purified. According to immunogenicity and induction of allergen-specific blocking IgG antibodies, 4 hypoallergenic fusion proteins (BM321, BM322, BM325, and BM326) representing Phl p 1, Phl p 2, Phl p 5, and Phl p 6 were included as components in the vaccine termed BM32. BM321, BM322, BM325, and BM326 showed almost completely abolished allergenic activity and induced significantly reduced T-cell proliferation and release of proinflammatory cytokines in patients' PBMCs compared with grass pollen allergens. On immunization, they induced allergen-specific IgG antibodies, which inhibited patients' IgE binding to all 4 major allergens of grass pollen, as well as allergen-induced basophil activation. CONCLUSION: A recombinant hypoallergenic grass pollen allergy vaccine (BM32) consisting of 4 recombinant PreS-fused grass pollen allergen peptides was developed for safe immunotherapy of grass pollen allergy.


Assuntos
Proteínas Recombinantes de Fusão/imunologia , Rinite Alérgica Sazonal/prevenção & controle , Vacinas de Subunidades Antigênicas/imunologia , Vacinas Sintéticas/imunologia , Alérgenos/imunologia , Animais , Basófilos/imunologia , Basófilos/metabolismo , Citocinas/biossíntese , Modelos Animais de Doenças , Feminino , Antígenos de Superfície da Hepatite B/química , Antígenos de Superfície da Hepatite B/genética , Humanos , Imunoglobulina E/sangue , Imunoglobulina E/imunologia , Imunoglobulina G/imunologia , Mediadores da Inflamação/metabolismo , Ativação Linfocitária/imunologia , Linfócitos/imunologia , Linfócitos/metabolismo , Camundongos , Peptídeos/imunologia , Poaceae , Pólen/imunologia , Ligação Proteica , Engenharia de Proteínas , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação
12.
FASEB J ; 28(8): 3540-51, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24760752

RESUMO

Advanced systemic mastocytosis (SM) is an aggressive hematopoietic neoplasm with poor prognosis and short survival times. So far, no curative therapy is available for affected patients. We have identified the cell surface antigen CD52 (CAMPATH-1) as a molecular target expressed abundantly on the surface of primary neoplastic mast cells (MCs) in patients with advanced SM. In contrast, neoplastic MCs of patients with indolent SM and normal MCs expressed only low levels or did not express CD52. To study the mechanisms of CD52 expression and the value of this antigen as a potential therapeutic target, we generated a human MC cell line, designated MCPV-1, by lentiviral immortalization of cord blood-derived MC progenitor cells. Functional studies revealed that activated RAS profoundly promotes surface expression of CD52. The CD52-targeting antibody alemtuzumab induced cell death in CD52(+) primary neoplastic MCs obtained from patients with SM as well as in MCPV-1 cells. NSG mice xenotransplanted with MCPV-1 cells survived significantly longer after treatment with alemtuzumab (median survival: 31 d untreated vs. 46 d treated; P=0.0012). We conclude that CD52 is a novel marker and potential therapeutic target in neoplastic MCs in patients with advanced SM.


Assuntos
Anticorpos Monoclonais Humanizados/uso terapêutico , Antígenos CD/análise , Antígenos de Neoplasias/análise , Antineoplásicos/uso terapêutico , Glicoproteínas/análise , Mastocitose Sistêmica/metabolismo , Terapia de Alvo Molecular , Adulto , Idoso , Alemtuzumab , Animais , Anticorpos Monoclonais Humanizados/farmacologia , Antígenos CD/imunologia , Antígenos de Neoplasias/imunologia , Antineoplásicos/farmacologia , Antígeno CD52 , Linhagem Celular Tumoral , Células Cultivadas , Feminino , Sangue Fetal/citologia , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/fisiologia , Genes ras , Glicoproteínas/imunologia , Humanos , Sistema de Sinalização das MAP Quinases , Masculino , Mastócitos/metabolismo , Mastocitose Sistêmica/tratamento farmacológico , Proteínas de Membrana/genética , Proteínas de Membrana/fisiologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Pessoa de Meia-Idade , Mutação de Sentido Incorreto , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/fisiologia , Proteínas Proto-Oncogênicas c-kit/genética , Proteínas Proto-Oncogênicas p21(ras) , Transdução Genética , Ensaios Antitumorais Modelo de Xenoenxerto , Proteínas ras/genética , Proteínas ras/fisiologia
13.
J Immunol ; 190(7): 3068-78, 2013 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-23440415

RESUMO

Allergen-specific immunotherapy is the only allergen-specific and disease-modifying treatment for allergy. The construction and characterization of a vaccine for birch pollen allergy is reported. Two nonallergenic peptides, PA and PB, derived from the IgE-reactive areas of the major birch pollen allergen Bet v 1 were fused to the hepatitis B surface protein, PreS, in four recombinant fusion proteins containing different numbers and combinations of the peptides. Fusion proteins expressed in Escherichia coli and purified to homogeneity showed a lack of IgE reactivity and allergenic activity when tested with sera and basophils from patients allergic to birch pollen. Compared to Bet v 1 allergen, peptides PA and PB showed reduced T cell activation in PBMCs from allergic patients, whereas PreS fusion proteins induced less IL-5 and more IL-10 and IFN-γ. Immunization of rabbits with the fusion proteins, in particular with a PreS fusion protein 2PAPB-PreS, containing two copies of each peptide, induced high levels of IgG Abs against the major IgE-reactive site on Bet v 1 and related allergens. These IgG Abs inhibited allergic patients' IgE binding to Bet v 1 better than did IgG induced by immunization with complete Bet v 1. Furthermore, 2PAPB-PreS-induced IgG inhibited Bet v 1-induced basophil activation in allergic patients and CD23-facilitated allergen presentation. Our study exemplifies novel beneficial features for a PreS carrier-based peptide vaccine for birch pollen, which, in addition to the established reduction in allergenic activity, include the enhanced focusing of blocking Ab responses toward IgE epitopes, immunomodulatory activity, and reduction of CD23-facilitated allergen presentation.


Assuntos
Antígenos de Plantas/imunologia , Betula/imunologia , Epitopos de Linfócito T/metabolismo , Tolerância Imunológica , Proteínas Recombinantes de Fusão/imunologia , Rinite Alérgica Sazonal/imunologia , Células Th1/imunologia , Vacinas/imunologia , Alérgenos/química , Alérgenos/genética , Alérgenos/imunologia , Animais , Apresentação de Antígeno/imunologia , Antígenos de Plantas/química , Antígenos de Plantas/genética , Reações Cruzadas/imunologia , Epitopos de Linfócito T/biossíntese , Antígenos de Superfície da Hepatite B/química , Antígenos de Superfície da Hepatite B/genética , Humanos , Imunoglobulina E/biossíntese , Imunoglobulina G/biossíntese , Imunofenotipagem , Pólen/imunologia , Coelhos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Rinite Alérgica Sazonal/prevenção & controle , Vacinas Sintéticas
14.
J Allergy Clin Immunol ; 133(3): 836-45.e11, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24182774

RESUMO

BACKGROUND: The major timothy grass pollen allergen Phl p 5 belongs to the most potent allergens involved in hay fever and asthma. OBJECTIVE: This study characterized immune-dominant IgE- and T-cell-recognition sites of Phl p 5. METHODS: Seven peptides, P1 to P7 with a length of 31 to 38 amino acids that spanned the Phl p 5 sequence, were synthesized, characterized by circular dichroism spectroscopy, and tested for IgE reactivity, basophil activation, and T-cell reactivity. Carrier-bound peptides were studied for their ability to induce IgG antibodies in rabbits which recognize Phl p 5 or cross-reactive allergens from different grass species. Peptide-specific antibodies were tested for the capability to inhibit IgE reactivity to Phl p 5 and allergen-induced basophil activation of patients with allergy. RESULTS: The peptides exhibited no secondary structure and showed no IgE reactivity or relevant allergenic activity, indicating that Phl p 5 IgE epitopes are conformational. Except for P3, peptide-specific IgG antibodies blocked IgE binding to Phl p 5 of patients with allergy and cross-reacted with temperate grasses. IgE inhibition experiments and molecular modeling identified several clustered conformational IgE epitopes on the N- as well as C-terminal domain of Phl p 5. P4, which stimulated the strongest T-cell and cytokine responses in patients, was not part of the major IgE-reactive regions. CONCLUSION: Our study shows an interesting dissociation of the major IgE- and T-cell-reactive domains in Phl p 5 which provides a basis for the development of novel forms of immunotherapy that selectively target IgE or T-cell responses.


Assuntos
Imunoglobulina E/imunologia , Proteínas de Plantas/imunologia , Linfócitos T/imunologia , Adolescente , Adulto , Sequência de Aminoácidos , Animais , Basófilos/fisiologia , Criança , Reações Cruzadas , Citocinas/biossíntese , Mapeamento de Epitopos , Feminino , Humanos , Ativação Linfocitária , Masculino , Pessoa de Meia-Idade , Modelos Moleculares , Dados de Sequência Molecular , Proteínas de Plantas/química , Estrutura Secundária de Proteína , Coelhos
15.
Blood ; 119(18): 4242-52, 2012 May 03.
Artigo em Inglês | MEDLINE | ID: mdl-22438247

RESUMO

Aggressive systemic mastocytosis (ASM) and mast cell leukemia (MCL) are advanced hematopoietic neoplasms with poor prognosis. In these patients, neoplastic mast cells (MCs) are resistant against various drugs. We examined the effects of 2 demethylating agents, 5-azacytidine and decitabine on growth and survival of neoplastic MCs and the MC line HMC-1. Two HMC-1 subclones were used, HMC-1.1 lacking KIT D816V and HMC-1.2 exhibiting KIT D816V. Both agents induced apoptosis in HMC-1.1 and HMC-1.2 cells. Decitabine, but not 5-azacytidine, also produced a G(2)/M cell-cycle arrest in HMC-1 cells. Drug-induced apoptosis was accompanied by cleavage of caspase-8 and caspase-3 as well as FAS-demethylation and FAS-re-expression in neoplastic MCs. Furthermore, both demethylating agents were found to synergize with the FAS-ligand in inducing apoptosis in neoplastic MCs. Correspondingly, siRNA against FAS was found to block drug-induced expression of FAS and drug-induced apoptosis in HMC-1 cells. Neither 5-azacytidine nor decitabine induced substantial apoptosis or growth arrest in normal MCs or normal bone marrow cells. Together, 5-azacytidine and decitabine exert growth-inhibitory and proapoptotic effects in neoplastic MCs. These effects are mediated through "FAS-re-expression" and are augmented by the FAS-ligand. Whether epigenetic drugs produce antineoplastic effects in vivo in patients with ASM and MCL remains to be determined.


Assuntos
Antimetabólitos Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Azacitidina/análogos & derivados , Azacitidina/farmacologia , Leucemia de Mastócitos/patologia , Mastócitos/efeitos dos fármacos , Mastocitose Sistêmica/patologia , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Receptor fas/metabolismo , Adulto , Idoso , Sequência de Bases , Linhagem Celular Tumoral/efeitos dos fármacos , Ilhas de CpG , Metilação de DNA/efeitos dos fármacos , DNA de Neoplasias/metabolismo , Decitabina , Sinergismo Farmacológico , Proteína Ligante Fas/fisiologia , Feminino , Humanos , Masculino , Mastócitos/patologia , Metilação/efeitos dos fármacos , Pessoa de Meia-Idade , Dados de Sequência Molecular , Proteínas de Neoplasias/metabolismo , Mutação Puntual , Regiões Promotoras Genéticas/genética , Proteínas Proto-Oncogênicas c-kit/genética , RNA Interferente Pequeno/farmacologia , Receptor fas/antagonistas & inibidores , Receptor fas/genética
16.
Eur J Clin Invest ; 44(12): 1239-45, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25371066

RESUMO

The concept of leukaemic stem cells (LSCs) has been developed to explain the complex cellular hierarchy and biology of leukaemias and to screen for pivotal targets that can be employed to improve drug therapies through LSC eradication in these patients. Some of the newly discovered LSC markers seem to be expressed in a disease-specific manner and may thus serve as major research tools and diagnostic parameters. A useful LSC marker in chronic myeloid leukaemia (CML) appears to be CD26, also known as dipeptidylpeptidase IV. Expression of CD26 is largely restricted to CD34(+) /CD38(-) LSCs in BCR/ABL1(+) CML, but is not found on LSCs in other myeloid or lymphoid neoplasms, with the exception of lymphoid blast crisis of CML, BCR/ABL1p210 + acute lymphoblastic leukaemia, and a very few cases of acute myeloid leukaemia. Moreover, CD26 usually is not expressed on normal bone marrow (BM) stem cells. Functionally, CD26 is a cytokine-targeting surface enzyme that may facilitate the mobilization of LSCs from the BM niche. In this article, we review our current knowledge about the biology and function of CD26 on CML LSCs and discuss the diagnostic potential of this new LSC marker in clinical haematology.


Assuntos
Biomarcadores Tumorais/metabolismo , Dipeptidil Peptidase 4/metabolismo , Leucemia Mielogênica Crônica BCR-ABL Positiva/diagnóstico , Células-Tronco Neoplásicas/metabolismo , Antineoplásicos/uso terapêutico , Benzamidas/uso terapêutico , Biomarcadores Tumorais/fisiologia , Dipeptidil Peptidase 4/fisiologia , Detecção Precoce de Câncer , Previsões , Humanos , Mesilato de Imatinib , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Piperazinas/uso terapêutico , Inibidores de Proteínas Quinases/uso terapêutico , Pirimidinas/uso terapêutico
17.
Haematologica ; 98(9): 1450-7, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23539538

RESUMO

Patients with advanced systemic mastocytosis, including mast cell leukemia, have a poor prognosis. In these patients, neoplastic mast cells usually harbor the KIT mutant D816V that confers resistance against tyrosine kinase inhibitors. We examined the effects of the multi-kinase blocker ponatinib on neoplastic mast cells and investigated whether ponatinib acts synergistically with other antineoplastic drugs. Ponatinib was found to inhibit the kinase activity of KIT G560V and KIT D816V in the human mast cell leukemia cell line HMC-1. In addition, ponatinib was found to block Lyn- and STAT5 activity in neoplastic mast cells. Ponatinib induced growth inhibition and apoptosis in HMC-1.1 cells (KIT G560V(+)) and HMC-1.2 cells (KIT G560V(+)/KIT D816V(+)) as well as in primary neoplastic mast cells. The effects of ponatinib were dose-dependent, but higher IC50-values were obtained in HMC-1 cells harboring KIT D816V than in those lacking KIT D816V. In drug combination experiments, ponatinib was found to synergize with midostaurin in producing growth inhibition and apoptosis in HMC-1 cells and primary neoplastic mast cells. The ponatinib+midostaurin combination induced substantial inhibition of KIT-, Lyn-, and STAT5 activity, but did not suppress Btk. We then applied a Btk short interfering RNA and found that Btk knockdown sensitizes HMC-1 cells against ponatinib. Finally, we were able to show that ponatinib synergizes with the Btk-targeting drug dasatinib to produce growth inhibition in HMC-1 cells. In conclusion, ponatinib exerts major growth-inhibitory effects on neoplastic mast cells in advanced systemic mastocytosis and synergizes with midostaurin and dasatinib in inducing growth arrest in neoplastic mast cells.


Assuntos
Inibidores do Crescimento/administração & dosagem , Imidazóis/administração & dosagem , Mastocitose Sistêmica/tratamento farmacológico , Mastocitose Sistêmica/genética , Proteínas Proto-Oncogênicas c-kit/genética , Piridazinas/administração & dosagem , Estaurosporina/análogos & derivados , Adulto , Idoso , Antineoplásicos/administração & dosagem , Linhagem Celular Tumoral , Sinergismo Farmacológico , Feminino , Humanos , Masculino , Mastocitose Sistêmica/diagnóstico , Pessoa de Meia-Idade , Estaurosporina/administração & dosagem
18.
FASEB J ; 26(2): 894-906, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-22051730

RESUMO

The JAK2 mutation V617F is detectable in a majority of patients with Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs). Enforced expression of JAK2 V617F in mice induces myeloproliferation and bone marrow (BM) fibrosis, suggesting a causal role for the JAK2 mutant in the pathogenesis of MPNs. However, little is known about mechanisms and effector molecules contributing to JAK2 V617F-induced myeloproliferation and fibrosis. We show that JAK2 V617F promotes expression of oncostatin M (OSM) in neoplastic myeloid cells. Correspondingly, OSM mRNA levels were increased in the BM of patients with MPNs (median 287% of ABL, range 22-1450%) compared to control patients (median 59% of ABL, range 12-264%; P < 0.0001). OSM secreted by JAK2 V617F+ cells stimulated growth of fibroblasts and microvascular endothelial cells and induced the production of angiogenic and profibrogenic cytokines (HGF, VEGF, and SDF-1) in BM fibroblasts. All effects of MPN cell-derived OSM were blocked by a neutralizing anti-OSM antibody, whereas the production of OSM in MPN cells was suppressed by a pharmacologic JAK2 inhibitor or RNAi-mediated knockdown of JAK2. In summary, JAK2 V617F-mediated up-regulation of OSM may contribute to fibrosis, neoangiogenesis, and the cytokine storm observed in MPNs, suggesting that OSM might serve as a novel therapeutic target molecule in these neoplasms.


Assuntos
Janus Quinase 2/genética , Janus Quinase 2/metabolismo , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Transtornos Mieloproliferativos/genética , Transtornos Mieloproliferativos/metabolismo , Oncostatina M/metabolismo , Substituição de Aminoácidos , Animais , Sequência de Bases , Medula Óssea/metabolismo , Medula Óssea/patologia , Estudos de Casos e Controles , Linhagem Celular , Citocinas/biossíntese , Técnicas de Silenciamento de Genes , Humanos , Janus Quinase 2/antagonistas & inibidores , Camundongos , Mutação de Sentido Incorreto , Transtornos Mieloproliferativos/patologia , Neovascularização Patológica , Oncostatina M/sangue , Oncostatina M/genética , Fosfatidilinositol 3-Quinases/metabolismo , Policitemia Vera/genética , Policitemia Vera/metabolismo , Policitemia Vera/patologia , Mielofibrose Primária/genética , Mielofibrose Primária/metabolismo , Mielofibrose Primária/patologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fator de Transcrição STAT5/metabolismo , Trombocitemia Essencial/genética , Trombocitemia Essencial/metabolismo , Trombocitemia Essencial/patologia
19.
J Immunol ; 186(9): 5333-44, 2011 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-21451110

RESUMO

Allergic inflammation is based on the cross-linking of mast cell and basophil-bound IgE Abs and requires at least two binding sites for IgE on allergens, which are difficult to characterize because they are often conformational in nature. We studied the IgE recognition of birch pollen allergen Bet v 1, a major allergen for >100 million allergic patients. Monoclonal and polyclonal Abs raised against Bet v 1-derived peptides were used to compete with allergic patients' IgE binding to Bet v 1 to search for sequences involved in IgE recognition. Strong inhibitions of patients' IgE binding to Bet v 1 (52-75%) were obtained with mAbs specific for two peptides comprising aa 29-58 (P2) and aa 73-103 (P6) of Bet v 1. As determined by surface plasmon resonance, mAb2 specific for P2 and mAb12 specific for P6 showed high affinity, but only polyclonal rabbit anti-P2 and anti-P6 Abs or a combination of mAbs inhibited allergen-induced basophil degranulation. Thus, P2 and P6 define a surface patch on the Bet v 1 allergen, which allows simultaneous binding of several different IgE Abs required for efficient basophil and mast cell activation. This finding explains the high allergenic activity of the Bet v 1 allergen. The approach of using peptide-specific Abs for the mapping of conformational IgE epitopes on allergens may be generally applicable. It may allow discriminating highly allergenic from less allergenic allergen molecules and facilitate the rational design of active and passive allergen-specific immunotherapy strategies.


Assuntos
Anticorpos Monoclonais/imunologia , Especificidade de Anticorpos/imunologia , Antígenos de Plantas/imunologia , Mapeamento de Epitopos/métodos , Epitopos de Linfócito B/imunologia , Imunoglobulina E/imunologia , Sequência de Aminoácidos , Animais , Antígenos de Plantas/química , Basófilos/imunologia , Betula/imunologia , Ligação Competitiva , Reações Cruzadas , Ensaio de Imunoadsorção Enzimática , Epitopos de Linfócito B/química , Humanos , Hipersensibilidade/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Peptídeos/química , Peptídeos/imunologia , Pólen/imunologia , Coelhos , Homologia de Sequência de Aminoácidos
20.
J Allergy Clin Immunol ; 130(2): 435-43.e4, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22789398

RESUMO

BACKGROUND: More than 50% of allergic patients have house dust mite (HDM) allergy. Group 1 and 2 allergens are the major HDM allergens. OBJECTIVE: We sought to produce and perform preclinical characterization of a recombinant hypoallergenic combination vaccine for specific immunotherapy of HDM allergy. METHODS: Synthetic genes coding for 2 hybrid proteins consisting of reassembled Der p 1 and Der p 2 fragments with (recombinant Der p 2 [rDer p 2]/1C) and without (rDer p 2/1S) cysteines were expressed in Escherichia coli and purified to homogeneity by means of affinity chromatography. Protein fold was determined by using circular dichroism analysis, allergenic activity was determined by testing IgE reactivity and using basophil activation assays, and the presence of T-cell epitopes was determined based on lymphoproliferation in allergic patients. Mice and rabbits were immunized to study the molecules' ability to induce an allergic response and whether they induce allergen-specific IgG capable of inhibiting allergic patients' IgE binding to the allergens, respectively. RESULTS: rDer p 2/1C and rDer p 2/1S were expressed in large amounts in E coli as soluble and folded proteins. Because of the lack of disulfide bonds, rDer p 2/1S did not form aggregates and was obtained as a monomeric protein, whereas rDer p 2/1C did form aggregates. Both hypoallergens lacked relevant IgE reactivity and had reduced ability to induce allergic inflammation and allergic responses but induced similar T-cell proliferation as the wild-type allergens. Immunization with the hypoallergens (rDer p 2/1S > rDer p 2/1C) induced IgG antibodies in rabbits that inhibited the IgE reactivity of patients with HDM allergy to Der p 1 and Der p 2. CONCLUSION: The preclinical characterization indicates that particularly rDer p 2/1S can be used as a safe hypoallergenic molecule for both tolerance and vaccination approaches to treat HDM allergy.


Assuntos
Antígenos de Dermatophagoides/imunologia , Proteínas de Artrópodes/imunologia , Cisteína Endopeptidases/imunologia , Hipersensibilidade/prevenção & controle , Pyroglyphidae/imunologia , Vacinas/imunologia , Animais , Antígenos de Dermatophagoides/genética , Proteínas de Artrópodes/genética , Clonagem Molecular , Cisteína Endopeptidases/genética , Epitopos de Linfócito T/imunologia , Escherichia coli , Feminino , Humanos , Hipersensibilidade/imunologia , Imunoglobulina E/imunologia , Imunoglobulina E/metabolismo , Imunoglobulina G/imunologia , Imunoglobulina G/metabolismo , Masculino , Camundongos , Ligação Proteica , Pyroglyphidae/genética , Coelhos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Vacinação , Vacinas/genética , Vacinas/uso terapêutico , Vacinas Combinadas , Vacinas Sintéticas
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