RESUMO
Viral infection activates the innate immune system, which recognizes viral components by a variety of pattern recognition receptors and initiates signalling cascades leading to the production of pro-inflammatory cytokines. To date, signalling cascades triggered after virus recognition are not fully characterized and are investigated by many research groups. The critical role of the E3 ubiquitin ligase Pellino3 in antibacterial and antiviral response is now widely accepted, but the precise mechanism remains elusive. In this study, we sought to explore Pellino3 role in the retinoic acid-inducible gene I (RIG-I)-dependent signalling pathway. In this work, the molecular mechanisms of the innate immune response, regulated by Pellino3, were investigated in lung epithelial cells during influenza B virus infection. We used wild-type and Pellino3-deficient A549 cells as model cell lines to examine the role of Pellino3 ligase in the type I interferon (IFN) signalling pathway. Our results indicate that Pellino3 is involved in direct ubiquitination and degradation of the TRAF3, suppressing interferon regulatory factor 3 (IRF3) activation and interferon beta (IFNß) production.
Assuntos
Influenza Humana , Fator 3 Associado a Receptor de TNF , Humanos , Fator 3 Associado a Receptor de TNF/genética , Fator 3 Associado a Receptor de TNF/metabolismo , Fator Regulador 3 de Interferon/genética , Fator Regulador 3 de Interferon/metabolismo , Regulação para Baixo , Imunidade Inata , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
IFN-I is the key regulatory component activating and modulating the response of innate and adaptive immune system to bacterial as well as viral pathogens. IFN-I promotes the expression of IFN-induced genes (ISG) and, consequently, the production of chemokines, e.g., CXCL10. Those chemokines control migration and localization of immune cells in tissues, and, thus, are critical to the function of the innate immune system during infection. Consequently, the regulation of IFN-I signaling is essential for the proper induction of an immune response. Our previous study has shown that E3 ubiquitin ligase Pellino3 positively regulates IFNß expression and secretion. Herein, we examined the role of Pellino3 ligase in regulating CXCL10 expression in response to IFNß stimulation. Our experiments were carried out on murine macrophage cell line (BMDM) and human monocytes cell line (THP-1) using IFNß as a IFNAR ligand. We demonstrate that Pellino3 is important for IFNß-induced phosphorylation and nuclear translocation of STAT1/STAT2/IRF9 complex which interacts with CXCL10 promoter and enhances its expression. In this study, we characterize a novel molecular mechanism allowing Pellino3-dependent modulation of the IFNß-induced response in BMDM and THP-1 cell lines.
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Quimiocina CXCL10 , Interferon Tipo I , Animais , Humanos , Camundongos , Quimiocina CXCL10/genética , Quimiocina CXCL10/metabolismo , Interferon Tipo I/metabolismo , Ligases/metabolismo , Macrófagos/metabolismo , Monócitos/metabolismo , Transdução de Sinais , Interferon beta/farmacologiaRESUMO
Activation of TLR7 by small imidazoquinoline molecules such as R848 or R837 initiates signaling cascades leading to the activation of transcription factors, such as AP-1, NF-κB, and interferon regulatory factors (IRFs) and afterward to the induction of cytokines and anti-viral Type I IFNs. In general, TLRs mediate these effects by utilizing different intracellular signaling molecules, one of them is Mal. Mal is a protein closely related to the antibacterial response, and its role in the TLR7 pathways remains poorly understood. In this study, we show that Mal determines the expression and secretion of IFNß following activation of TLR7, a receptor that recognizes ssRNA and imidazoquinolines. Moreover, we observed that R848 induces Mal-dependent IFNß production via ERK1/2 activation as well as the transcription factor IRF7 activation. Although activation of TLR7 leads to NF-κB-dependent expression of IRF7, this process is independent of Mal. We also demonstrate that secretion of IFNß regulated by TLR7 and Mal in macrophages and dendritic cells leads to the IP-10 chemokine expression. In conclusion, our data demonstrate that Mal is a critical regulator of the imidazoquinolinones-dependent IFNß production via ERK1/2/IRF7 signaling cascade which brings us closer to understanding the molecular mechanism's regulation of innate immune response.
Assuntos
Fator Regulador 7 de Interferon/genética , Interferon beta/genética , Glicoproteínas de Membrana/genética , Proteínas Proteolipídicas Associadas a Linfócitos e Mielina/genética , Receptor 7 Toll-Like/genética , Animais , Citocinas/genética , Humanos , Imunidade Inata/genética , Interferon Tipo I/genética , Sistema de Sinalização das MAP Quinases/genética , Camundongos , Camundongos Knockout , NF-kappa B/genética , Quinolonas/toxicidade , Fator de Transcrição AP-1/genéticaRESUMO
Lanthanide doped nanoparticles (Ln:NPs) hold promise as novel luminescent probes for numerous applications in nanobiophotonics. Despite excellent photostability, narrowband photoluminescence, efficient anti-Stokes emission and long luminescence lifetimes, which are needed to meet the requirements of multiplexed and background free detection at prolonged observation times, concern about their toxicity is still an issue for both in vivo and in vitro applications. Similar to other chemicals or pharmaceuticals, the very same properties that are desirable and potentially useful from a biomedical perspective can also give rise to unexpected and hazardous toxicities. In engineered bionanomaterials, the potentially harmful effects may originate not only from their chemical composition but also from their small size. The latter property enables the nanoparticles to bypass the biological barriers, thus allowing deep tissue penetration and the accumulation of the nanoparticles in a number of organs. In addition, nanoparticles are known to possess high surface chemical reactivity as well as a large surface-to-volume ratio, which may seriously affect their biocompatibility. Herein we survey the underlying mechanisms of nanotoxicity and provide an overview on the nanotoxicity of lanthanides and of upconverting nanoparticles.
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Nanopartículas/química , Barreira Hematoencefálica/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Regulamentação Governamental , Humanos , Elementos da Série dos Lantanídeos/química , Nanopartículas/metabolismo , Nanopartículas/toxicidade , Espécies Reativas de Oxigênio/metabolismo , Propriedades de SuperfícieRESUMO
Monoclonal antibodies (mAbs) are biomolecules of great scientific and practical significance. In contrast to polyclonal antibodies from immune sera, they are homogeneous and monospecific, since they are produced by hybridoma cells representing a clone arising from a single cell. The successful technology was described for the first time in 1975; the inventors were later awarded the Nobel Prize. Currently, mAbs are broadly used as a research tool, in diagnostics and medicine in particular for the treatment of cancer or in transplantology. About 47 therapeutics based on monoclonal antibodies are now available in the US and Europe, and the number is still growing. Production of monoclonal antibodies is a multistage, time-consuming and costly process. Growing demand for these molecules creates space for research focused on improvements in hybridoma technology. Lower costs, human labor, and time are important goals of these attempts. In this article, a brief review of current methods and their advances is given.
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Anticorpos Monoclonais/imunologia , Formação de Anticorpos/imunologia , Células Produtoras de Anticorpos/metabolismo , Hibridomas , Anticorpos Monoclonais/metabolismo , Anticorpos Monoclonais/uso terapêutico , Europa (Continente) , Humanos , Hibridomas/citologia , Hibridomas/imunologia , Hibridomas/metabolismo , Neoplasias/tratamento farmacológicoRESUMO
In a previous attempt to generate a protective vaccine against Candida albicans, a ß-mannan tetanus toxoid conjugate showed poor immunogenicity in mice. To improve the specific activation toward the fungal pathogen, we aimed to target Dectin-1, a pattern-recognition receptor expressed on monocytes, macrophages, and dendritic cells. Laminarin, a ß-glucan ligand of Dectin-1, was incorporated into the original ß-mannan tetanus toxoid conjugate providing a tricomponent conjugate vaccine. A macrophage cell line expressing Dectin-1 was employed to show binding and activation of Dectin-1 signal transduction pathway by the ß-glucan-containing vaccine. Ligand binding to Dectin-1 resulted in the following: 1) activation of Src family kinases and Syk revealed by their recruitment and phosphorylation in the vicinity of bound conjugate and 2) translocation of NF-κB to the nucleus. Treatment of immature bone marrow-derived dendritic cells (BMDCs) with tricomponent or control vaccine confirmed that the ß-glucan-containing vaccine exerted its enhanced activity by virtue of dendritic cell targeting and uptake. Immature primary cells stimulated by the tricomponent vaccine, but not the ß-mannan tetanus toxoid vaccine, showed activation of BMDCs. Moreover, treated BMDCs secreted increased levels of several cytokines, including TGF-ß and IL-6, which are known activators of Th17 cells. Immunization of mice with the novel type of vaccine resulted in improved immune response manifested by high titers of Ab recognizing C. albicans ß-mannan Ag. Vaccine containing laminarin also affected distribution of IgG subclasses, showing that vaccine targeting to Dectin-1 receptor can benefit from augmentation and immunomodulation of the immune response.
Assuntos
Células Dendríticas/metabolismo , Sistemas de Liberação de Medicamentos , Lectinas Tipo C/administração & dosagem , Toxoide Tetânico/administração & dosagem , Toxoide Tetânico/imunologia , beta-Glucanas/metabolismo , Animais , Sítios de Ligação/imunologia , Linhagem Celular , Células Dendríticas/imunologia , Sistemas de Liberação de Medicamentos/métodos , Epitopos/imunologia , Epitopos/metabolismo , Glucanos , Lectinas Tipo C/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Polissacarídeos/imunologia , Polissacarídeos/metabolismo , Toxoide Tetânico/metabolismo , Trissacarídeos/administração & dosagem , Trissacarídeos/imunologia , Trissacarídeos/metabolismo , Vacinas Conjugadas/administração & dosagem , Vacinas Conjugadas/imunologia , Vacinas Conjugadas/metabolismo , beta-Glucanas/imunologiaRESUMO
The expression of the HER2 (human epidermal growth factor receptor 2) protein in cancer cells is a well-established cancer marker used for diagnostic and therapeutic purposes in modern treatment protocols, especially in breast cancer. The gold-standard immunohistochemical diagnostic methods with the specific anti-HER2 antibodies are utilized in the clinic to measure expression level of the membrane-bound receptor. However, a soluble extracellular domain (ECD) of HER2 is released to the extracellular matrix, thus the blood assays for HER2 measurements present an attractive way for HER2 level determination. There is a need for accurate and validated assays that can be used to correlate the concentration of the circulating HER2 protein with disease clinical manifestations. Here we describe two monoclonal antibodies binding HER2 with a unique sequence of the complementarity-determining regions that recognize HER2 ECD. Development and validation of the sandwich enzyme-linked immunosorbent assay (ELISA) for quantification of the soluble HER2 in a variety of biological samples is also presented. The assay provides HER2 quantitation within a concentrations range from 1.56 to 100 ng/ml with sensitivity at the level of 0.5 ng/ml that meets the expectations for measurements of HER2 in the blood and tumor tissue samples. The method presents satisfactory intra- and inter-assay precision and accuracy for immunochemical quantification of biomarkers in biological samples. The utility of the generated monoclonal anti-HER2 antibodies has been confirmed for use in the precise measurement of HER2 (both cell-bound and soluble) in several types of biological material, including serum, solid tumor tissue, and cell culture medium. Additionally, the developed immunochemical tools have a potential for HER2 detection, not only in a wide range of sample types but also independently of the sample storage/pre-processing, allowing for comprehensive HER2 analysis in tissue (IHC), cultured cells (immunofluorescence) and blood (ELISA).
Assuntos
Anticorpos Monoclonais , Neoplasias da Mama , Humanos , Feminino , Anticorpos Monoclonais/uso terapêutico , Receptor ErbB-2 , Neoplasias da Mama/metabolismo , Ensaio de Imunoadsorção Enzimática/métodos , Células Cultivadas , Biomarcadores TumoraisRESUMO
One of the main parameters characterizing steel is tensile strength. Conducting actual research is time consuming and expensive. For this reason, the technique uses simplified methods that allow one to quickly estimate the resistance of the material to fatigue. They are conducted mainly by computer methods. For the proper development of programs to determine the fatigue parameters of steel, solid data preparation is necessary. Unfortunately, some studies are performed on materials produced in laboratory conditions, which is only an approximation of the actual production conditions. Real alloys contain natural impurities which can affect their properties. Therefore, it is important to use real results obtained on an industrial scale for analysis including computer simulations. One of the important parameters that can be used to describe the properties of steel is the scatter index. It is the quotient of the average distance between the pollution and the average size of the pollution. This parameter makes it possible to take into account the fatigue strength of steel, taking into account the size of impurities and the distance between these impurities. The paper attempted to determine the scatter index and its impact on the fatigue resistance coefficient for steel melted in an industrial 140 ton electric furnace. The tests were carried out on structural steel with an average carbon content of 0.26%. The steel was hardened and tempered in all temperature tempering ranges (low, medium, and high). The fatigue resistance coefficient in the scatter index function was determined and discussed for each of the applied heat treatment parameters.
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In the course of evolution, humankind has used many construction materials [...].
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Aluminum-silicon alloys require modification due to their coarse-grained microstructures and resulting low strength properties. So far, research into the modification process has focused on the use of various chemical components and technological processes, the tasks of which are to refine the microstructure and, thus, increase the mechanical properties of the alloy. In this paper, the answer to the question of whether the form of the modifier influences the modification effect of the hypoeutectic silumin will be found. The tests were carried out using the popular silumin AlSi7Mg. To answer our research question, the alloy was modified under comparable conditions using the following elements: Ti, B, and master alloys AlTi1.5 and AlB1.5. Modifiers in the form of Sr and master alloy AlSr1.5 were also used. All mentioned modifiers were produced and introduced into the liquid alloy in the form of a powder and a rod. Master alloys AlSr1.5 were also produced via cooling from the liquid state through cooling in air and the second variant at a speed of 200 °C/s (in the form of powder and a thin strip). The microstructure and mechanical properties were analyzed based on the following measures: tensile strength, elongation, and hardness of silumin. Based on the conducted research, it was found that the form of the modifier also affects the modification effect visible in the form of changes in the microstructure and mechanical properties. For the powder-modified alloy, greater fineness in the eutectic phase (α and B phases) and an increase in all analyzed mechanical properties were obtained.
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Bacteriophages (phages) are viruses infecting bacteria. They are widely present in the environment, food, and normal microflora. The human microbiome is a mutually interdependent network of bacteria, bacteriophages, and human cells. The stability of these tri-kingdom interactions may be essential for maintaining immunologic and metabolic health. Phages, as with each other's antigens, may evoke an immune response during a human's lifetime and induce specific antibody generation. In this manuscript, we labeled these antibodies as naturally generated. Naturally generated antibodies may be one of the most important factors limiting the efficacy of phage therapy. Herein, we attempted to determine the physiological level of these antibodies specific to a population bacteriophage named I11mO19 in human sera, using an ELISA-based assay. First, we purified the phage particles and assessed the immunoreactivity of phage proteins. Then, affinity chromatography was performed on columns with immobilized phage proteins to obtain a fraction of human polyclonal anti-phage antibodies. These antibodies were used as a reference to elaborate an immunoenzymatic test that was used to determine the level of natural anti-phage antibodies. We estimated the average level of anti-I11mO19 phage antibodies at 190 µg per one milliliter of human serum. However, immunoblotting revealed that cross-reactivity occurs between some proteins of I11mO19 and two other coliphages: T4 and ΦK1E. The antigens probably share common epitopes, suggesting that the determined level of anti-I11mO19 phage might be overestimated and reflects a group of antibodies reactive to a broad range of other E. coli phages. Anti-I11mO19 antibodies did not react with Pseudomonas bacteriophage F8, confirming specificity to the coliphage group. In this work, we wanted to show whether it is possible to determine the presence and level of anti-phage antibodies in nontargeted-immunized sera, using an immunoenzymatic assay. The conclusion is that it is possible, and specific antibodies can be determined. However, the specificity refers to a broader coliphage group of phages, not only the single phage strain.
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The biological importance of lipopolysaccharides (LPS), components of bacterial cell wall has not been explained sufficiently. The glycine present in these structures could play an important role in the immunological response after bacterial infections and during sepsis. In our studies we obtained synthetic and stable substituted glycinated 1-thioglycosides derivatives of monosaccharides, e.g., D-glucose or D-galactose as well as disaccharides, e.g., melibiose and lactose. The conditions of acylation reactions were validated and specific products were separated by using chromatography methods. Their structures were confirmed by NMR. These compounds were conjugated with carrier proteins e.g., bovine serum albumin and horse myoglobin. Prior to conjugation proteins were modified with glycidol to create the protein-diol intermediates and subsequent periodate oxidation of the glycol moieties to generate the reactive aldehyde functionalities. Modified and formylated carrier proteins were conjugated with acylated thioglycosides in the presence of sodium cyanoborohydride. Subsequently, the products obtained were analyzed in SDS-PAGE and separated by using HW-55S gel-filtration chromatography. The immunoreactivity of selected glycinated glycoconjugates were studied in ELISA assays with specific anti-aminoacylated glyconjugate antibodies obtained after rabbit immunization with Escherichia coli K12 C600 core oligosaccharide glycine-containing glycoconjugate. The differences in the immunoreactivity of different glycinated 1-thioglycosides were observed. The received glycine-acylated glycoconjugates could mimic the non-sugar substituents localized in various bacterial LPS. These synthetic compounds could be candidates for their use as glycoconjugate vaccines in protection against serious bacterial infections, e.g.. sepsis.
Assuntos
Glicoconjugados/síntese química , Fatores Imunológicos/síntese química , Tioglicosídeos/síntese química , Animais , Glicoconjugados/química , Glicoconjugados/imunologia , Fatores Imunológicos/química , Fatores Imunológicos/imunologia , Espectroscopia de Ressonância Magnética , Coelhos , Tioglicosídeos/química , Tioglicosídeos/imunologiaRESUMO
Non-metallic inclusions are one of the many factors influencing the strength of materials operating under variable loads. Their influence on the strength of the material depends not only on the morphology of the impurities themselves, but it is also closely related to the microstructure of the material. This microstructure is the matrix for non-metallic inclusions. This article discusses the results of a study investigating the effect of non-metallic inclusions on the fatigue strength of structural steel during rotary bending. The study was performed at 12 heats produced in an industrial plant's 140-ton electric furnaces. Six heats were desulphurised, and six were refined with argon. This paper presents the bending fatigue strength of steel hardened and tempered at different temperatures, subject to the relative volume of inclusions. This paper also presents the dimensional structure of non-metallic inclusions divided by different two technologies. The research shows that the main fraction of non-metallic inclusions is Al2O3; the most numerous were impurities with a diameter of less than 2 µm; argon refining does not affect the proportion of non-metallic inclusions of large dimensions (with a diameter of over 15 µm); the influence of non-metallic inclusions on the strength of the steel is also related to the microstructure of the steel constituting the matrix of inclusions.
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A protective ß-mannan trisaccharide epitope from the Candida albicans cell wall phosphomannan has been synthesized and activated for copolymerization with acrylamide. The resulting glycopolymer displayed 33 trisaccharide haptens and was derivatized for conjugation to the immunogenic carrier protein, chicken serum albumin. The resulting conjugate achieves a high degree of oligosaccharide substitution while limiting the sites of substitution on the protein. The murine immune response against this conjugate was compared with the response to a trisaccharide-tetanus toxoid conjugate vaccine. The glycopolymer was shown to induce a more robust immune response with higher trisaccharide-specific antibody titers and with a significantly larger proportion of responding mice developing antibodies that bound the target, native cell wall antigen of C. albicans.
Assuntos
Candida albicans/química , Epitopos/química , Glicoconjugados/síntese química , Mananas/síntese química , Trissacarídeos/síntese química , Acrilamida/química , Animais , Candida albicans/citologia , Galinhas , Epitopos/imunologia , Feminino , Glicoconjugados/química , Glicoconjugados/imunologia , Mananas/química , Mananas/imunologia , Camundongos , Conformação Molecular , Estrutura Molecular , Albumina Sérica/química , Toxoide Tetânico/síntese química , Toxoide Tetânico/química , Toxoide Tetânico/imunologia , Trissacarídeos/química , Trissacarídeos/imunologia , Vacinação , Vacinas Conjugadas/química , Vacinas Conjugadas/imunologiaRESUMO
The tether employed to covalently attach ß-mannan disaccharide glycoconjugates influences the specificity of rabbit antibodies that protect against Candida albicans. Two glycoconjugates containing (1 â 2)-ß-mannan disaccharides linked to chicken serum albumin (CSA) either via a structurally uniform or via a stereodiversified spacer were prepared and evaluated in immunization trials in mice and rabbits. Immunization with conjugate vaccine possessing a structurally diversified linker induced higher IgG titers against Candida albicans cell wall phosphomannan than a conjugate with a structurally uniform linker. These results suggest that affinity maturation and the specific antibody response can be shifted towards recognition of the desired hapten by employing a linker with diversified configuration.
Assuntos
Glicoconjugados/química , Glicoconjugados/imunologia , Haptenos/química , Haptenos/imunologia , Animais , Especificidade de Anticorpos , Candida albicans/imunologia , Sequência de Carboidratos , Mananas/química , Mananas/imunologia , Camundongos , Coelhos , Albumina Sérica/imunologia , Vacinas Conjugadas/química , Vacinas Conjugadas/imunologiaRESUMO
We demonstrate that interactions between multimeric receptors and multivalent ligands are dramatically enhanced by recruiting a complementary templating receptor such as an endogenous multimeric protein but only when individual ligands are attached to a polymer as preorganized, covalent, heterobifunctional pairs. This effect cannot be replicated by a multivalent ligand if the same recognition elements are independently arrayed on the scaffold. Application of this principle offers an approach to create high-avidity inhibitors for multimeric receptors. Judicious selection of the ligand that engages the templating protein allows appropriate effector function to be incorporated in the polymeric construct, thereby providing an opportunity for therapeutic applications. The power of this approach is exemplified by the design of exceptionally potent Escherichia coli Shiga toxin antagonists that protect transgenic mice that constitutively express a human pentraxin, serum amyloid P component.
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Antibacterianos/química , Escherichia coli O157/efeitos dos fármacos , Toxina Shiga I/antagonistas & inibidores , Animais , Antibacterianos/síntese química , Antibacterianos/farmacologia , Escherichia coli O157/metabolismo , Humanos , Ligantes , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Polímeros/síntese química , Polímeros/química , Polímeros/farmacologia , Componente Amiloide P Sérico/metabolismo , Toxina Shiga I/químicaRESUMO
Sensing of viral particles and elements that initiate mechanisms of immune response is an intrinsic ability of mammalian cells. Regulatory cytokines and antiviral mediators are released after triggering of complex signaling cascades in response to interaction of pathogen particles with pattern recognition receptors (PRRs) leading to the production of interferons (IFN) and proinflammatory cytokines. Viral RNA in the cytoplasm constitute a potent danger molecule that recognition is performed by RIG-I-like receptors, the most common group of receptors in mammalian cells, capable to recognize a foreign RNA. It is known that the E3 ubiquitin ligase Pellino3 plays an important role in antibacterial and antiviral response, but its involvement in the RLR pathways remains poorly understood. In this study, we investigate the molecular mechanisms of the innate immune response in BMDMs (immortalized macrophages from mouse bone marrow) during VSV infection. Here, we present evidence that the activation of the RIG-I/Pellino3/ERK1/2 pathway in BMDMs is crucial for the protection against VSV. We demonstrate that during infection, viral particles replicate in Pellino3 knockout BMDMs more effectively than in wild-type cells. Increased viral replication resulting in cell lysis and death is aid by impaired synthesis of IFN-I and inflammatory cytokines as a consequence of disturbances in the ERK1/2 pathway regulation.
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Imunidade Inata/imunologia , Ativação de Macrófagos/imunologia , Macrófagos/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Citocinas/metabolismo , Imunidade Inata/genética , Interferons/metabolismo , Ativação de Macrófagos/fisiologia , Camundongos , RNA Viral/metabolismo , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Replicação Viral/imunologiaRESUMO
This study reports a novel impedimetric immunosensor for protein D detection in purified and bacterial (Haemophilus influenzae, Hi) samples. The detection was based on antigen recognition by anti-protein D antibodies (apD) immobilised at the maze-like boron-doped carbon nanowall electrodes (B:CNW). The B:CNW electrodes were synthesised, and their surface was characterised by scanning electron microscopy (SEM) and X-ray photoelectron spectroscopy (XPS) methods. The sensor was prepared in a two-step procedure: apD were covalently linked on the previously modified B:CNW electrodes using diazonium salt. Modification steps were controlled by electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) measurements. The immunosensor exhibited excellent electrochemical performance, stability, satisfactory sensitivities, and linear ranges for antigen detection. Protein D was detected down to 2.39 × 102fg/mL with a linear range extending from 3.37 × 10-11to 3.37 × 10-3µg/mL (in purified sample). Next, Hi's LOD was 5.20 × 102CFU/mL with a linear range of 8.39 × 101-8.39 × 103CFU/mL. Selectivity studies showed no reaction with negative samples as Streptococcus pyogenes, Streptococcus pneumoniae or Bordetella parapertussis bacteria. Therefore, the new approach is suitable for rapid and quantitative detection of Hi, and is a good candidate for further tests on clinical samples.
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Técnicas Biossensoriais , Boro , Carbono , Espectroscopia Dielétrica , Técnicas Eletroquímicas , Eletrodos , Haemophilus influenzae , Imunoensaio , Limite de DetecçãoRESUMO
Zwitterionic polysaccharides (ZPSs) from Bacteroides fragilis and Streptococcus pneumoniae display unique T-cell activities. The first synthesis of a hexasaccharide representing two repeating units of the zwitterionic capsular polysaccharide from S. pneumoniae type 1 (Sp1) is reported. Key elements of the approach are stereoselective construction of 1,4-cis-alpha-galactose linkages based on a reactive trichloroacetimidate donor that incorporates a 6-O-acetyl group, which may contribute to the high alpha selectivity in glycosylation. After assembly of the fully protected hexasaccharide from five monosaccharide synthons 2-4, 24 and 25, selective deprotection of the primary hydroxyl groups of the four galactose residues followed by oxidation to the corresponding uronic acids provides hexasaccharide 19. The trisaccharide counterpart 1 was synthesized in similar fashion from three synthons, 2-4. This approach employed both conventional and dehydrative glycosylation methodologies and avoids the use of poorly reactive uronic acid derived glycosyl donors and acceptors.