Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 10 de 10
Filtrar
1.
BMC Microbiol ; 19(1): 43, 2019 02 18.
Artigo em Inglês | MEDLINE | ID: mdl-30777007

RESUMO

BACKGROUND: Rodentibacter (R.) pneumotropicus colonizes the respiratory and urogenital tracts of laboratory mice with a reported moderate serological prevalence from 4 to 13%. Thus, regular tests to identify this pathogen in mice are recommended for animal facilities. However, a recent study indicated that current serological assays are partly insensitive, as C57BL/6 and BALB/c mice infected with R. pneumotropicus were incorrectly screened as seronegative. RESULTS: Here, we report a systematic analysis of protein and lipopolysaccharides antigens by immunoblot and ELISA that allowed establishing a sensitive test system able to differentiate between R. pneumotropicus and the closely related species R. heylii. Furthermore, the main immunogen, designated as 'characteristic antigen for Rodentibacter of laboratory origin 1' (CARLO-1), was identified by two-dimensional gel electrophoresis followed by immunoblot and tandem mass spectrometry in a preparation of outer membrane proteins. An indirect ELISA relying on the recombinantly expressed protein provided high sensitivity, specificity, and selectivity. The corresponding carlo1 gene was highly conserved (> 97%) among 21 isolates of R. pneumotropicus and R. heylii. CONCLUSION: The newly identified protein CARLO-1 is well suited for the sensitive and specific serological detection of Rodentibacter infections in mice. Indirect differentiation of R. pneumotropicus and R. heylii infections may be possible using an ELISA based on a whole-cell antigen preparation. All four established ELISA systems using a whole-cell preparation, lipopolysaccharides, outer-membrane proteins and protein CARLO-1 as antigen, respectively, outperformed a commercial ELISA in terms of sensitivity.


Assuntos
Antígenos de Bactérias/imunologia , Proteínas de Bactérias/imunologia , Ensaio de Imunoadsorção Enzimática , Infecções por Pasteurellaceae/diagnóstico , Infecções por Pasteurellaceae/veterinária , Pasteurellaceae/imunologia , Animais , Feminino , Immunoblotting , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Pasteurellaceae/patogenicidade , Infecções por Pasteurellaceae/imunologia , Sensibilidade e Especificidade
2.
J Virol Methods ; 262: 72-78, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30144945

RESUMO

Precise health monitoring of laboratory animals is a critical factor for surveillance and accuracy of animal experiments. Rotavirus epizootic diarrhea of infant mice (EDIM) leads to infections in mice that can influence animal studies, e.g., by altering the intestinal physiology. Thus, the aim of this study was establishing a highly sensitive and specific ELISA for the serological detection of EDIM infections in rodents. First, virus proteins were separated by SDS-PAGE and immunogenic proteins were visualized by immunoblotting and identified after in-gel digestion by tandem mass spectrometry. Subsequently, the major immunogen VP6 (virus protein 6) was expressed in Escherichia coli in high yields, purified by affinity chromatography, and used to establish an indirect ELISA. The diagnostic sensitivity and specificity were both above 99 % and the selectivity better than 98.7 % for animals infected by other pathogens listed by the Federation of Laboratory Animal Science Associations. Importantly, the Strep-rVP6-His-ELISA was more sensitive than a commercial virus-based ELISA and is a time- and cost-efficient complement to EDIM-specific immune-fluorescence assays. In conclusion, the assay can improve health monitoring by reducing the risk of missed EDIM infections in animal housing facilities, thereby improving animal welfare, reliability of animal studies, and protection of precious mice breeds.


Assuntos
Anticorpos Antivirais/sangue , Antígenos Virais/imunologia , Proteínas do Capsídeo/imunologia , Ensaio de Imunoadsorção Enzimática/métodos , Infecções por Rotavirus/diagnóstico , Rotavirus/imunologia , Adjuvantes Imunológicos , Administração Intranasal , Animais , Antígenos Virais/genética , Proteínas do Capsídeo/genética , Escherichia coli/genética , Imunização , Ciência dos Animais de Laboratório , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Reprodutibilidade dos Testes , Rotavirus/isolamento & purificação , Infecções por Rotavirus/imunologia , Sensibilidade e Especificidade
3.
Virol J ; 15(1): 114, 2018 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-30049287

RESUMO

BACKGROUND: Reovirus type-3 infections cause severe pathologies in young mice and thus influence animal experiments in many ways. Therefore, the Federation of Laboratory Animal Science Associations (FELASA) recommends an annual screening in laboratory mice as part of a thorough health monitoring program. Based on the high protein sequence homology among the different reovirus serotypes, immunofluorescence antibody assay and other indirect methods relying on the whole virus are presumably cross-reactive to antibodies triggered by mammalian orthoreovirus infections independent of the serotype. METHODS: The serotype-specific protein σ-1 was expressed in Escherichia coli with an N-terminal Strep-tag and a C-terminal His-tag. The purified Strep-rσ-1-His-construct was used to develop an indirect ELISA by testing defined positive and negative sera obtained by experimental infection of mice as well as field sera. RESULTS: The Strep-rσ-1-His-ELISA provided high sensitivity and specificity during validation. Notably, a high selectivity was also observed for sera positively tested for other relevant FELASA-listed pathogens. Screening of field samples indicated that a commercial reovirus type-3-based ELISA might be cross-reactive to other murine reovirus serotypes and thus produces false-positive results. CONCLUSIONS: The prevalence of reovirus type-3 might be overestimated in German animal facilities and most likely in other countries as well. The occurrence of other reovirus serotypes, however, raises the question if murine health monitoring programs should be extended to these pathogens.


Assuntos
Orthoreovirus Mamífero 3/classificação , Infecções por Reoviridae/imunologia , Infecções por Reoviridae/virologia , Proteínas do Core Viral/imunologia , Animais , Cromatografia Líquida de Alta Pressão , Ensaio de Imunoadsorção Enzimática , Hemaglutinação , Testes de Hemaglutinação , Camundongos , Infecções por Reoviridae/diagnóstico , Sorogrupo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Espectrometria de Massas em Tandem , Proteínas do Core Viral/genética
4.
BMC Microbiol ; 18(1): 45, 2018 05 30.
Artigo em Inglês | MEDLINE | ID: mdl-29848308

RESUMO

BACKGROUND: Mice are a natural host for Rodentibacter (R.) pneumotropicus. Despite specific monitoring, it is still one of the most important infectious agents in laboratory animals. The objective of this study was to determine the virulence of a prevalent pathotype of R. pneumotropicus and characterize the host response in a new animal model. RESULTS: Intranasal infection of C57BL/6 and BALB/c mice with a R. pneumotropicus strain (JF4Ni) bearing the genes of the three known repeats in toxin (RTX) toxins resulted in an unprecedented high mortality and morbidity above 50 and 80%, respectively. Morbidity was associated with severe weight loss as well as conjunctivitis and dyspnea. A main pathology was a catarrhal purulent to necrotic bronchopneumonia. Specific immune globuline (Ig) A was detected in tracheonasal lavages of most surviving mice which were still colonized by R. pneumotropicus. Furthermore, all surviving animals showed a distinct production of IgG antibodies. To differentiate T-helper cell (Th) 1 and Th2 immune responses we used subclasses of IgGs as indicators. Mean ratios of IgG2b to IgG1 were below 0.8 in sera drawn from both mice strains prior infection and from BALB/c mice post infection. In contrast, C57BL/6 mice had a mean IgG2b/IgG1 ratio of 1.6 post infection indicating a Th1 immune response in C57BL/6 versus a Th2 response in BALB/c mice associated with a tenfold higher bacterial load in the lung. In accordance with a Th1 response high antigen-specific IgG2c titers were detected in the majority of surviving C57BL/6 mice. CONCLUSIONS: R. pneumotropicus JF4Ni is a highly virulent strain causing severe pneumonia and septicemia after intranasal infection of C57BL/6 and BALB/c mice. Persisting infections in the two mice strains are associated with Th1 and Th2 immune responses, respectively, and differences in the bacterial burden of the lung. The described model is ideally suited for future vaccination studies using the natural host.


Assuntos
Imunidade Humoral , Imunoglobulina G/metabolismo , Infecções por Pasteurella/imunologia , Pasteurella pneumotropica/patogenicidade , Animais , Pulmão/microbiologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Infecções por Pasteurella/mortalidade , Pasteurella pneumotropica/imunologia , Pneumonia Bacteriana/imunologia , Pneumonia Bacteriana/microbiologia , Sepse/imunologia , Sepse/microbiologia , Células Th1/imunologia , Células Th2/imunologia
5.
Microbes Infect ; 20(2): 101-110, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29024796

RESUMO

Streptobacillus (S.) moniliformis is a rat-associated zoonotic pathogen that occasionally causes disease in other species. We investigated the working hypothesis that intranasal infection might lead to different immune responses in C57BL/6 and BALB/c mice associated with distinct pathologies. This study confirmed with 75% mortality the known high susceptibility of C57BL/6 mice to Streptobacillus moniliformis infection in comparison to BALB/c mice which did not develop signs of disease. Main pathologies in C57BL/6 mice were purulent to necrotizing lymphadenitis and pneumonia. Significant seroconversion was recorded in surviving mice of both strains. Differentiation of IgG-subclasses revealed mean ratios of IgG2b to IgG1 below 0.5 in sera of all mice prior to infection and of BALB/c mice post infection. In contrast, C57BL/6 mice had a mean IgG2b/IgG1 ratio of 2.5 post infection indicating a Th1 immune response in C57BL/6 versus a Th2 response in BALB/c mice. Evaluation of different sentinel systems revealed that cultural and serological investigations of these animals might not be sufficient to detect infection. In summary, an intranasal S. moniliformis infection model in C57BL/6 mice leading to purulent to necrotizing inflammations in the lung, the lymph nodes and other organs associated with a Th1 immune response is described.


Assuntos
Infecções por Fusobacterium/imunologia , Infecções por Fusobacterium/patologia , Streptobacillus , Animais , Modelos Animais de Doenças , Feminino , Infecções por Fusobacterium/microbiologia , Especificidade de Hospedeiro/imunologia , Imunoglobulina G/sangue , Inflamação/imunologia , Pulmão/microbiologia , Pulmão/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Streptobacillus/fisiologia , Células Th1/imunologia
6.
Angew Chem Int Ed Engl ; 55(15): 4826-30, 2016 Apr 04.
Artigo em Inglês | MEDLINE | ID: mdl-26938787

RESUMO

Promotion of cell adhesion on biomaterials is crucial for the long-term success of a titanium implant. Herein a novel concept is highlighted combining very stable and affine titanium surface adhesive properties with specific cell binding moieties in one molecule. A peptide containing L-3,4-dihydroxyphenylalanine was synthesized and affinity to titanium was investigated. Modification with a cyclic RGD peptide and a heparin binding peptide (HBP) was realized by an efficient on-resin combination of Diels-Alder reaction with inverse electron demand and Cu(I) catalyzed azide-alkyne cycloaddition. The peptide was fluorescently labeled by thiol Michael addition. Conjugating the cyclic RGD and HBP in one peptide gave improved spreading, proliferation, viability, and the formation of well-developed actin cytoskeleton and focal contacts of osteoblast-like cells.


Assuntos
Adesão Celular , Peptídeos/química , Titânio/química , Cromatografia Líquida de Alta Pressão , Espectrometria de Massas por Ionização por Electrospray
7.
Eur J Pharm Biopharm ; 85(1): 69-77, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23454053

RESUMO

The control of the interaction between biological systems and surfaces plays a major role in the development of bioactive implants. Random absorbance of different compounds of the body liquids attach at the implant site after surgery. This protein layer triggers the activation of immune cells and is a breeding ground for pathogens, which may induce inflammation processes. Many efforts have been made to block these fouling processes such as PEGylation and unspecific coatings. These systems lead to bioinert implant surfaces that lower the inflammation potential of implanted materials. In contrast, the biomimetic approach attempts the functionalization of implant surfaces with compounds such as peptides, proteins, or sugars that form an artificial layer on the implant that corresponds to the naturally occurring extracellular matrix. This enables the controlled recruitment of cells that improve the healing processes or enhance the osseointegration into the implanted material. An improved connection of implants with cells that enhances the healing processes or tightens the connection of implants with the surrounding tissue is obtained by this approach. However, for bioactive functionalization of implant materials, efficient and robust immobilization techniques are required. Peptides owing to their low-toxicity and their multifunctionality are interesting agents that can act as molecular glue to surfaces. Here, an overview is provided of the development of surface binding peptides, the molecular mechanisms of peptide-surface interactions, and the application of surface binding peptides in the development of multifunctional biomaterials that facilitate beneficial characteristics in vitro and in vivo.


Assuntos
Materiais Biocompatíveis/química , Bioprótese/efeitos adversos , Peptídeos/química , Adesividade , Adsorção , Animais , Materiais Biocompatíveis/efeitos adversos , Biomimética/tendências , Técnicas de Visualização da Superfície Celular , Humanos , Proteínas Imobilizadas/efeitos adversos , Proteínas Imobilizadas/química , Cinética , Teste de Materiais/métodos , Oligopeptídeos/efeitos adversos , Oligopeptídeos/biossíntese , Oligopeptídeos/química , Peptídeos/efeitos adversos , Peptídeos/metabolismo , Propriedades de Superfície
8.
Chem Biodivers ; 9(11): 2648-58, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-23161641

RESUMO

Cyclic Arg-Gly-Asp (RGD) peptides show remarkable affinity and specificity to integrin receptors and mediate important physiological effects in tumor angiogenesis. Additionally, they are one of the keyplayers in improving the biocompatibility of biomaterials. The fully biodegradable polymer poly(lactic-co-glycolic acid) (PLGA) is frequently used for biomedical implants and can be applied as nanoparticles for drug delivery. The aim of this work was the generation of a lipidated c[RGDfK] peptide including a second functionality for coating of hydrophobic PLGA. Therefore, we established a general and straightforward strategy for the introduction of two different modifications into the same c[RGDfK] peptide. This allowed the generation of a palmitoylated integrin-binding lipopeptide that shows high affinity to PLGA. Additionally, we coupled 5(6)-carboxyfluorescein to the second site for modification to enable sensitive quantification of the immobilized lipopeptide on PLGA. In conclusion, we present a synthesis protocol that enables the preparation of c[RGDfK] lipopeptides with a strong affinity to PLGA and an additional site for modifications. This will provide the opportunity to introduce a variety of effector molecules site-specifically to the c[RGDfK] lipopeptide, which will enable the introduction of multifunctionality into c[RGDfK]-coated PLGA devices or nanoparticles.


Assuntos
Fluoresceínas/química , Corantes Fluorescentes/química , Ácido Láctico/química , Lipídeos/química , Oligopeptídeos/química , Ácido Poliglicólico/química , Acilação , Ciclização , Fluoresceínas/síntese química , Corantes Fluorescentes/síntese química , Integrinas/metabolismo , Ácido Láctico/síntese química , Ligantes , Lipídeos/síntese química , Oligopeptídeos/síntese química , Ácido Poliglicólico/síntese química , Copolímero de Ácido Poliláctico e Ácido Poliglicólico
9.
Bioconjug Chem ; 23(10): 2129-37, 2012 Oct 17.
Artigo em Inglês | MEDLINE | ID: mdl-22989005

RESUMO

Multifunctionality is gaining more and more importance in the field of improved biomaterials. Especially peptides feature a broad chemical variability and are versatile mediators between inorganic surfaces and living cells. Here, we synthesized a unique peptide that binds to SiO(2) with nM affinity. We equipped the peptide with the bioactive integrin binding c[RGDfK]-ligand and a fluorescent probe by stepwise Diels-Alder reaction with inverse electron demand and copper(I) catalyzed azide-alkyne cycloaddition. For the first time, we report the generation of a multifunctional peptide by combining these innovative coupling reactions. The resulting peptide displayed an outstanding binding to silicon oxide and induced a significant increase in cell spreading and cell viability of osteoblasts on the oxidized silicon surface.


Assuntos
Materiais Biocompatíveis/química , Química Click , Oligopeptídeos/química , Peptídeos Cíclicos/química , Dióxido de Silício/química , Silício/química , Alcinos/química , Azidas/química , Materiais Biocompatíveis/síntese química , Materiais Biocompatíveis/farmacologia , Biotina/metabolismo , Catálise , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Cobre/química , Reação de Cicloadição , Desenho de Fármacos , Ensaio de Imunoadsorção Enzimática , Corantes Fluorescentes/química , Humanos , Integrina alfaVbeta3/metabolismo , Oligopeptídeos/síntese química , Oligopeptídeos/farmacologia , Osteoblastos/citologia , Osteoblastos/efeitos dos fármacos , Peptídeos Cíclicos/síntese química , Peptídeos Cíclicos/farmacologia , Propriedades de Superfície
10.
Biochemistry ; 50(45): 9817-25, 2011 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-21999704

RESUMO

We provide a protocol for the preparation of fully active Y2 G protein-coupled receptors (GPCRs). Although a valuable target for pharmaceutical research, information about the structure and dynamics of these molecules remains limited due to the difficulty in obtaining sufficient amounts of homogeneous and fully active receptors for in vitro studies. Recombinant expression of GPCRs as inclusion bodies provides the highest protein yields at lowest costs. But this strategy can only successfully be applied if the subsequent in vitro folding results in a high yield of active receptors and if this fraction can be isolated from the nonactive receptors in a homogeneous form. Here, we followed that strategy to provide large quantities of the human neuropeptide Y receptor type 2 and determined the folding yield before and after ligand affinity chromatography using a radioligand binding assay. Directly after folding, we achieved a proportion of ~25% active receptor. This value could be increased to ~96% using ligand affinity chromatography. Thus, a very homogeneous sample of the Y2 receptor could be prepared that exhibited a K(D) value of 0.1 ± 0.05 nM for the binding of polypeptide Y, which represents one of the natural ligands of the Y2 receptor.


Assuntos
Receptores de Neuropeptídeo Y/química , Receptores de Neuropeptídeo Y/metabolismo , Cromatografia de Afinidade , Humanos , Técnicas In Vitro , Ligantes , Modelos Moleculares , Neuropeptídeo Y/metabolismo , Peptídeo YY/metabolismo , Dobramento de Proteína , Ensaio Radioligante , Receptores de Neuropeptídeo Y/genética , Receptores de Neuropeptídeo Y/isolamento & purificação , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA