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1.
J Allergy Clin Immunol ; 149(3): 1031-1043, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-34419535

RESUMO

BACKGROUND: House dust mite (HDM) allergens are major elicitors of allergic reactions worldwide. OBJECTIVE: Identification, characterization, and evaluation of diagnostic utility of a new important HDM allergen was performed. METHODS: A cDNA coding for a new Dermatophagoides pteronyssinus (Dp) allergen, Der p 37, was isolated from a Dp expression library with allergic patients' IgE antibodies. Recombinant Der p 37 (rDer p 37) expressed in Escherichia coli was purified, then characterized by mass spectrometry, circular dichroism, and IgE reactivity by ImmunoCAP ISAC technology with sera from 111 clinically defined HDM-allergic patients. The allergenic activity of rDer p 37 was studied by basophil activation and CD4+ T-cell responses by carboxyfluorescein diacetate succinimidyl ester dilution assays. Specific antibodies raised against rDer p 37 were used for the ultrastructural localization of Der p 37 in mites by immunogold transmission electron microscopy. RESULTS: Der p 37, a 26 kDa allergen with homology to chitin-binding proteins, is immunologically distinct from Der p 15, 18, and 23. It is located in the peritrophic membrane of fecal pellets. Der p 37 reacted with IgE antibodies from a third of HDM-allergic patients and induced specific basophil- and CD4+ T-cell activation. Der p 37 IgE-positive patients had significantly higher IgE levels to major HDM allergens, reacted with more HDM allergens, and had a higher risk (odds ratio = 3.1) of asthma compared to Der p 37-negative patients. CONCLUSIONS: Der p 37, a new Dp allergen recognized by a third of HDM-allergic patients, may serve as a surrogate marker for severe HDM sensitization and asthma.


Assuntos
Asma , Hipersensibilidade , Alérgenos , Animais , Antígenos de Dermatophagoides , Proteínas de Artrópodes , Asma/diagnóstico , Poeira , Escherichia coli/genética , Humanos , Imunoglobulina E , Pyroglyphidae
2.
Oncoimmunology ; 5(7): e1171446, 2016 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-27622022

RESUMO

BACKGROUND: Anticancer vaccines could represent a valuable complementary strategy to established therapies, especially in settings of early stage and minimal residual disease. HER-2 is an important target for immunotherapy and addressed by the monoclonal antibody trastuzumab. We have previously generated HER-2 mimotope peptides from phage display libraries. The synthesized peptides were coupled to carriers and applied for epitope-specific induction of trastuzumab-like IgG. For simplification and to avoid methodological limitations of synthesis and coupling chemistry, we herewith present a novel and optimized approach by using adeno-associated viruses (AAV) as effective and high-density mimotope-display system, which can be directly used for vaccination. METHODS: An AAV capsid display library was constructed by genetically incorporating random peptides in a plasmid encoding the wild-type AAV2 capsid protein. AAV clones, expressing peptides specifically reactive to trastuzumab, were employed to immunize BALB/c mice. Antibody titers against human HER-2 were determined, and the isotype composition and functional properties of these were tested. Finally, prophylactically immunized mice were challenged with human HER-2 transfected mouse D2F2/E2 cells. RESULTS: HER-2 mimotope AAV-vaccines induced antibodies specific to human HER-2. Two clones were selected for immunization of mice, which were subsequently grafted D2F2/E2 cells. Both mimotope AAV clones delayed the growth of tumors significantly, as compared to controls. CONCLUSION: In this study, a novel mimotope AAV-based platform was created allowing the isolation of mimotopes, which can be directly used as anticancer vaccines. The example of trastuzumab AAV-mimotopes demonstrates that this vaccine strategy could help to establish active immunotherapy for breast-cancer patients.

3.
Vaccine ; 33(42): 5553-5563, 2015 Oct 13.
Artigo em Inglês | MEDLINE | ID: mdl-26382603

RESUMO

Vaccination with the minor capsid protein L2, notably the 17-36 neutralizing epitope, induces broadly protective antibodies, although the neutralizing titers attained in serum are substantially lower than for the licensed L1 VLP vaccines. Here we examine the impact of other less reactogenic adjuvants upon the induction of durable neutralizing serum antibody responses and protective immunity after vaccination with HPV16 and HPV31 L2 amino acids 17-36 inserted at positions 587 and 453 of VP3, respectively, for surface display on Adeno-Associated Virus 2-like particles [AAVLP (HPV16/31L2)]. Mice were vaccinated three times subcutaneously with AAVLP (HPV16/31L2) at two week intervals at several doses either alone or formulated with alum, alum and MPL, RIBI adjuvant or Cervarix. The use of adjuvant with AAVLP (HPV16/31L2) was necessary in mice for the induction of L2-specific neutralizing antibody and protection against vaginal challenge with HPV16. While use of alum was sufficient to elicit durable protection (>3 months after the final immunization), antibody titers were increased by addition of MPL and RIBI adjuvants. To determine the breadth of immunity, rabbits were immunized three times with AAVLP (HPV16/31L2) either alone, formulated with alum±MPL, or RIBI adjuvants, and after serum collection, the animals were concurrently challenged with HPV16/31/35/39/45/58/59 quasivirions or cottontail rabbit papillomavirus (CRPV) at 6 or 12 months post-immunization. Strong protection against all HPV types was observed at both 6 and 12 months post-immunization, including robust protection in rabbits receiving the vaccine without adjuvant. In summary, vaccination with AAVLP presenting HPV L2 17-36 epitopes at two sites on their surface induced cross-neutralizing serum antibody, immunity against HPV16 in the genital tract, and long-term protection against skin challenge with the 7 most common oncogenic HPV types when using a clinically relevant adjuvant.


Assuntos
Adjuvantes Imunológicos/administração & dosagem , Proteínas do Capsídeo/imunologia , Proteínas Oncogênicas Virais/imunologia , Infecções por Papillomavirus/prevenção & controle , Vacinas contra Papillomavirus/imunologia , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Dependovirus/imunologia , Modelos Animais de Doenças , Epitopos/imunologia , Feminino , Papillomavirus Humano 16 , Camundongos , Camundongos Endogâmicos BALB C , Papillomaviridae/imunologia , Coelhos , Vacinas Sintéticas/imunologia
4.
Viral Immunol ; 27(9): 438-48, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25247267

RESUMO

Adeno-associated viruses (AAVs) are established vectors for gene therapy of different human diseases. AAVs are assembled of 60 capsomers, which can be genetically modified, allowing high-density display of short peptide sequences at their surface. The aim of our study was to evaluate the immunogenicity and safety of an adeno-associated virus-like particle (AAVLP)-displayed B-cell peptide epitope taking ovalbumin (OVA) as a model antigen or allergen from egg, respectively. An OVA-derived B-cell epitope was expressed as fusion protein with the AAV-2 capsid protein of VP3 (AAVLP-OVA) and for control, with the nonrelated peptide TP18 (AAVLP-TP18). Cellular internalization studies revealed an impaired uptake of AAVLP-OVA by mouse BMDC, macrophages, and human HeLa cells. Nevertheless, BALB/c mice immunized subcutaneously with AAVLP-OVA formed similarly high titers of OVA-specific IgG1 compared to mice immunized with the native OVA. The extent of the immune response was independent whether aluminum hydroxide or water in oil emulsion was used as adjuvant. Furthermore, in mice immunized with native OVA, high OVA-specific IgE levels were observed, which permitted OVA-specific mast-cell degranulation in a ß-hexosaminidase release assay, whereas immunizations with AAVLP-OVA rendered background IgE levels only. Accordingly, OVA-immunized mice, but not AAVLP-OVA immunized mice, displayed an anaphylactic reaction with a significant drop of body temperature upon intravenous OVA challenge. From this mouse model, we conclude that AAVLPs that display B-cell epitope peptides on their surface are suitable vaccine candidates, especially in the field of allergy.


Assuntos
Linfócitos B/imunologia , Dependovirus/genética , Portadores de Fármacos , Epitopos de Linfócito B/imunologia , Vetores Genéticos , Ovalbumina/imunologia , Vacinas Virais/imunologia , Adjuvantes Imunológicos/administração & dosagem , Animais , Anticorpos/sangue , Células Cultivadas , Epitopos de Linfócito B/genética , Feminino , Humanos , Imunoglobulina E/sangue , Imunoglobulina G/sangue , Camundongos Endogâmicos BALB C , Ovalbumina/genética , Vacinas Virais/administração & dosagem , Vacinas Virais/genética
5.
J Immunol ; 190(7): 3059-67, 2013 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-23460742

RESUMO

The house dust mite (HDM) Dermatophagoides pteronyssinus is one of most important allergen sources and a major elicitor of allergic asthma. We screened a D. pteronyssinus expression cDNA library with IgE Abs from HDM allergic patients. A cDNA coding for a new major allergen was isolated, which showed sequence homology to peritrophins, which contain chitin-binding domains and are part of the peritrophic matrix lining the gut of arthropods. The mature Der p 23 allergen was expressed in Escherichia coli as an 8-kDa protein without its hydrophobic leader sequence and purified to homogeneity. It reacted with IgE Abs from 74% of D. pteronyssinus allergic patients (n = 347) at levels comparable to the two major HDM allergens, Der p 1 and Der p 2. Thus, Der p 23 represents a new major D. pteronyssinus allergen. Furthermore, rDer p 23 exhibited high allergenic activity as demonstrated by upregulation of CD203c expression on basophils from D. pteronyssinus allergic patients. Immunogold electron microscopy localized the allergen in the peritrophic matrix lining the midgut of D. pteronyssinus as well as on the surface of the fecal pellets. Thus, we identified a new major D. pteronyssinus allergen as peritrophin-like protein. The high allergenic activity of Der p 23 and its frequent recognition as respiratory allergen may be explained by the fact that it becomes airborne and respirable through its association with mite feces. Der p 23 may be an essential component for diagnosis and specific immunotherapy of HDM allergy.


Assuntos
Antígenos de Dermatophagoides/imunologia , Dermatophagoides pteronyssinus/imunologia , Fezes/química , Sequência de Aminoácidos , Animais , Antígenos de Dermatophagoides/química , Antígenos de Dermatophagoides/genética , Antígenos de Dermatophagoides/metabolismo , Sequência de Bases , Basófilos/imunologia , Clonagem Molecular , DNA Complementar/genética , Dermatophagoides pteronyssinus/genética , Humanos , Hipersensibilidade/imunologia , Imunoglobulina E/sangue , Imunoglobulina E/imunologia , Imunoglobulina E/metabolismo , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Dados de Sequência Molecular , Ligação Proteica/imunologia , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
6.
PLoS One ; 7(6): e39741, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22761884

RESUMO

The human papillomavirus (HPV) minor capsid protein L2 is a promising candidate for a broadly protective HPV vaccine yet the titers obtained in most experimental systems are rather low. Here we examine the potential of empty AAV2 particles (AAVLPs), assembled from VP3 alone, for display of L2 epitopes to enhance their immunogenicity. Insertion of a neutralizing epitope (amino acids 17-36) from L2 of HPV16 and HPV31 into VP3 at positions 587 and 453, respectively, permitted assembly into empty AAV particles (AAVLP(HPV16/31L2)). Intramuscularly vaccination of mice and rabbits with AAVLP(HPV16/31L2)s in montanide adjuvant, induced high titers of HPV16 L2 antibodies as measured by ELISA. Sera obtained from animals vaccinated with the AAVLP(HPV16/31L2)s neutralized infections with several HPV types in a pseudovirion infection assay. Lyophilized AAVLP(HPV16/31L2) particles retained their immunogenicity upon reconstitution. Interestingly, vaccination of animals that were pre-immunized with AAV2--simulating the high prevalence of AAV2 antibodies in the population--even increased cross neutralization against HPV31, 45 and 58 types. Finally, passive transfer of rabbit antisera directed against AAVLP(HPV16/31L2)s protected naïve mice from vaginal challenge with HPV16 pseudovirions. In conclusion, AAVLP(HPV16/31L2) particles have the potential as a broadly protective vaccine candidate regardless of prior exposure to AAV.


Assuntos
Papillomaviridae/imunologia , Vacinas Virais/imunologia , Vírion , Adjuvantes Imunológicos/administração & dosagem , Animais , Ensaio de Imunoadsorção Enzimática , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Coelhos , Vacinas Virais/administração & dosagem
7.
Int Arch Allergy Immunol ; 159(3): 253-62, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22722650

RESUMO

BACKGROUND: Diagnosis and immunotherapy of house-dust mite (HDM) allergy is still based on natural allergen extracts. The aim of this study was to analyze commercially available Dermatophagoides pteronyssinus extracts from different manufacturers regarding allergen composition and content and whether variations may affect their allergenic activity. METHODS: Antibodies specific for several D. pteronyssinus allergens (Der p 1, 2, 5, 7, 10 and 21) were used to analyze extracts from 10 different manufacturers by immunoblotting. Sandwich ELISAs were used to quantify Der p 1 and Der p 2 in the extracts. Mite-allergic patients (n = 45) were skin-tested with the extracts and tested for immunoglobulin E (IgE) reactivity to a panel of 10 mite allergens (Der p 1, 2, 4, 5, 7, 8, 10, 14, 20 and 21) by dot blot. RESULTS: Only Der p 1 and Der p 2 were detected in all extracts but their concentrations and ratios showed high variability (Der p 1: 6.0-40.8 µg ml(-1); Der p 2: 1.7-45.0 µg ml(-1)). At least 1 out of 4 allergens (i.e. Der p 5, 7, 10 and 21) was not detected in 8 of the studied extracts. Mite-allergic subjects showed different IgE reactivity profiles to the individual mite allergens, the extracts showed different allergenic activity in skin-prick tests and false-negative results. CONCLUSIONS: Commercially available D. pteronyssinus extracts lack important allergens, show great variability regarding allergen composition and content and some gave false-negative diagnostic test results in certain patients.


Assuntos
Alérgenos/imunologia , Antígenos de Dermatophagoides/imunologia , Proteínas de Artrópodes/imunologia , Cisteína Endopeptidases/imunologia , Dermatite de Contato/imunologia , Dermatophagoides pteronyssinus/imunologia , Adulto , Alérgenos/química , Animais , Anticorpos/sangue , Anticorpos/imunologia , Diversidade de Anticorpos , Antígenos de Dermatophagoides/sangue , Proteínas de Artrópodes/sangue , Misturas Complexas/química , Misturas Complexas/imunologia , Cisteína Endopeptidases/sangue , Dermatite de Contato/sangue , Dermatite de Contato/diagnóstico , Dermatophagoides pteronyssinus/química , Ensaio de Imunoadsorção Enzimática , Reações Falso-Negativas , Humanos , Imunoglobulina E/sangue , Imunoglobulina E/imunologia , Testes Cutâneos
8.
Int Arch Allergy Immunol ; 147(2): 101-9, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-18520154

RESUMO

BACKGROUND: Der p 5 was reported as an important allergen in Dermatophagoides pteronyssinus, which is particularly recognized by patients suffering from asthma. The aim of this study was to produce, by recombinant DNA technology, a folded Der p 5 allergen for diagnostic, therapeutic and preventive purposes. METHODS: Der p 5-encoding cDNA was isolated from a lambda gt11 D. pteronyssinus expression cDNA library and expressed in Escherichia coli. rDer p 5 was purified to homogeneity and characterized by mass spectroscopy and circular dichroism. IgE reactivity was tested with sera from 117 mite-allergic patients and in a basophil histamine release experiment. Der p 5-specific rabbit IgG antibodies were produced for the ultrastructural localization of the allergen in mites by immunogold electron microscopy as well as for cross-reactivity studies. RESULTS: rDer p 5 is a heat-stable protein with predominantly alpha-helical secondary structure which reacted with IgE from 31% of mite-allergic patients' sera and showed no relevant cross-reactivity to group 5 allergens from storage mites and tropical mites. rDer p 5-specific rabbit IgG antibodies inhibited mite-allergic patients' IgE binding to Der p 5 and allowed to localize the allergen in secretory granules of midgut epithelial cells of house dust mites. CONCLUSIONS: The described rDer p 5 molecule may be useful for diagnosis and immunotherapy of house dust mite-allergic patients.


Assuntos
Alérgenos/imunologia , Antígenos de Dermatophagoides/imunologia , Hipersensibilidade/imunologia , Pyroglyphidae/imunologia , Proteínas Recombinantes/imunologia , Alérgenos/química , Alérgenos/genética , Animais , Antígenos de Dermatophagoides/química , Antígenos de Dermatophagoides/genética , Proteínas de Artrópodes , Basófilos/imunologia , Escherichia coli/genética , Liberação de Histamina , Humanos , Hipersensibilidade/diagnóstico , Imunoglobulina E/sangue , Imunoglobulina E/imunologia , Dobramento de Proteína , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética
9.
Blood ; 111(6): 3097-107, 2008 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-18180381

RESUMO

Dasatinib is a multitargeted drug that blocks several tyrosine kinases. Apart from its well-known antileukemic activity, the drug has attracted attention because of potential immunosuppressive and anti-inflammatory effects. We report that dasatinib at 1 microM completely blocks anti-IgE-induced histamine release in blood basophils in healthy donors, and allergen-induced release of histamine in sensitized individuals. In addition, dasatinib inhibited FcepsilonRI-mediated release of IL-4 and IgE-mediated up-regulation of CD13, CD63, CD164, and CD203c in basophils. The effects of dasatinib were dose-dependent (IC(50): 50-500 nM) and specific for FcepsilonRI activation in that the drug failed to inhibit C5a-induced or Ca-ionophore-induced histamine release. Interestingly, at lower concentrations, dasatinib even promoted FcepsilonRI-dependent histamine release in basophils in allergic subjects. In consecutive studies, dasatinib was found to interact with and block several FcepsilonRI downstream targets in basophils, including Btk. Correspondingly, FcepsilonRI-mediated histamine secretion in basophils was markedly reduced in Btk knockout mice and in a patient with Btk deficiency. However, the remaining "low-level" mediator secretion in Btk-deficient cells was fully blocked down again by 1 muM dasatinib. Together, these data suggest that dasatinib inhibits FcepsilonRI-mediated activation of basophils through multiple signaling molecules including Btk. Dasatinib may be an interesting agent for immunologic disorders involving Btk-dependent responses or/and FcepsilonRI activation of basophils.


Assuntos
Basófilos/efeitos dos fármacos , Basófilos/imunologia , Liberação de Histamina/efeitos dos fármacos , Liberação de Histamina/imunologia , Pirimidinas/farmacologia , Receptores de IgE/imunologia , Tiazóis/farmacologia , Adolescente , Adulto , Tirosina Quinase da Agamaglobulinemia , Alérgenos/imunologia , Animais , Basófilos/metabolismo , Células Cultivadas , Dasatinibe , Feminino , Humanos , Interleucina-4/biossíntese , Interleucina-4/metabolismo , Masculino , Camundongos , Pessoa de Meia-Idade , Ligação Proteica , Proteínas Tirosina Quinases/deficiência , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Regulação para Cima
10.
Int Arch Allergy Immunol ; 138(3): 257-66, 2005 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-16215327

RESUMO

BACKGROUND: Dietary intake of wheat can cause two distinct immunologically mediated diseases with severe gastrointestinal manifestations, coeliac disease (CD) and IgE-mediated food allergy. The pathomechanisms underlying these diseases are different, but the profile of the target antigens in wheat has not been compared for the two diseases. METHODS: We compared IgA- and IgE-reactive antigens in wheat using sera from patients with coeliac disease (n = 35) and food allergy to wheat (n = 16) by one- and two-dimensional immunoblotting. Furthermore, the IgG subclass (IgG1-IgG4) reactivity to wheat antigens was studied by enzyme-linked immunosorbent assay. RESULTS: IgA antibodies from CD patients and IgE antibodies from allergic patients recognised distinct profiles of wheat antigens. Furthermore, the IgG subclass responses to wheat antigens were different in CD and wheat-allergic patients. CONCLUSION: This study thus demonstrates that wheat contains antigens/epitopes which are preferentially recognised by CD patients, whereas others elicit IgE-mediated food allergy. This finding suggests that the nature of a food antigen may influence the quality of the pathological immune response in the gut and has implications for the diagnosis and therapy of hypersensitivity to wheat.


Assuntos
Antígenos de Plantas/análise , Doença Celíaca/imunologia , Imunoglobulinas/imunologia , Proteínas de Plantas/análise , Triticum/imunologia , Hipersensibilidade a Trigo/imunologia , Adolescente , Adulto , Idoso , Reações Antígeno-Anticorpo , Antígenos de Plantas/imunologia , Criança , Pré-Escolar , Feminino , Glutens/imunologia , Humanos , Imunoglobulina A/imunologia , Imunoglobulina E/imunologia , Imunoglobulina G/imunologia , Lactente , Masculino , Pessoa de Meia-Idade , Proteínas de Plantas/imunologia
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