Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 284
Filtrar
1.
J Photochem Photobiol B ; 198: 111560, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31336216

RESUMO

Previous studies revealed significant impact on cancer cell by mid-infrared (MIR) radiation. However, the effects of narrow band MIR on immune reaction and infectious disease are still unknown. In this study, an enhanced innate immune response was observed through the interaction between Leptospiral outer membrane protein (LipL32) and toll-like receptor 2 (TLR2). Thereafter, human kidney proximal tubular cells (HK-2 cells) initiated a serial reaction of enhanced MCP-1 production. The 6 µm narrow bandwidth light source emitted by waveguide thermal emitter (WTE) was applied to induce carbonyl group (CO bond) stretching vibration during the stage of antigen-receptor complex formation. The amount of MCP-1 gene expression had 2.5 folds increase after narrow band MIR illumination comparing to non-MIR illumination at low dose LipL32 condition. Besides, both ELISA and confocal microscopy results also revealed that the chemokine concentration increased significantly after narrow band MIR illumination either at low or high concentration of LipL32. Furthermore, a specific phenomenon that narrow band MIR can amplify the signal of weak immune response by enhancing sensitivity of the interaction between antigen and receptor was observed. This study exhibits clear evidence that the narrow band MIR exposure can modulate the early immune response of infectious disease and play a potential role to develop host-directed therapy in the future.


Assuntos
Proteínas da Membrana Bacteriana Externa/farmacologia , Raios Infravermelhos , Lipoproteínas/farmacologia , Proteínas da Membrana Bacteriana Externa/imunologia , Linhagem Celular , Sobrevivência Celular/efeitos da radiação , Quimiocina CCL2/genética , Quimiocina CCL2/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos da radiação , Humanos , Túbulos Renais Proximais/citologia , Túbulos Renais Proximais/efeitos dos fármacos , Túbulos Renais Proximais/metabolismo , Leptospira/metabolismo , Lipoproteínas/imunologia
2.
Microbiol Immunol ; 63(7): 261-268, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31209918

RESUMO

Pasteurella multocida is one of the most important bacteria responsible for diseases of animals. Crude extracts from sonicated P. multocida strain Dainai-1, which is serotype A isolated from bovine pneumonia, were found to inhibit proliferation of mouse spleen cells stimulated with Con A. The crude extract was purified by cation and anion exchange chromatography and hydroxyapatite chromatography. Its molecular weight was 27 kDa by SDS-PAGE and it was named PM27. PM27 was found to inhibit proliferation of mouse spleen cells stimulated with Con A as effectively as did the crude extract; however, its activity was lost after heating to 100°C for 20 min. PM27 did not directly inhibit proliferation of HT-2 cells, which are an IL-2-dependent T cell line, nor did it modify IL-2 production by Con A-stimulated mouse spleen cells. The N-terminal amino acid sequence of PM27 was determined and BLAST analysis revealed its identity to uridine phosphorylase (UPase) from P. multocida. UPase gene from P. multocida Dainai-1 was cloned into expression vector pQE-60 in Escherichia coli XL-1 Blue. Recombinant UPase (rUPase) tagged with His at the C-terminal amino acid was purified with Ni affinity chromatography. rUPase was found to inhibit proliferation of mouse spleen cells stimulated with Con A; however, as was true for PM27, its activity was lost after heating to 100°C for 20 min. Thus, PM27/UPase purified from P. multocida has significant antiproliferative activity against Con A-stimulated mouse spleen cells and may be a virulence factor.


Assuntos
Proteínas da Membrana Bacteriana Externa/farmacologia , Proliferação de Células/efeitos dos fármacos , Pasteurella multocida/metabolismo , Uridina Fosforilase/isolamento & purificação , Uridina Fosforilase/farmacologia , Sequência de Aminoácidos , Animais , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Bovinos , Linhagem Celular/efeitos dos fármacos , Escherichia coli/genética , Humanos , Interleucina-2/metabolismo , Camundongos , Peso Molecular , Pasteurella multocida/genética , Fosforilases , Proteínas Recombinantes , Baço , Linfócitos T/efeitos dos fármacos , Uridina Fosforilase/genética , Uridina Fosforilase/metabolismo
3.
Mol Biol Rep ; 46(2): 2493-2504, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30919211

RESUMO

Osteoarthritis (OA) is a degenerative joint disease, in which low-grade inflammation plays an important role at the initiating step. Low-doses of LPS-induced inflammation in the plasma activate chondrocytes and promote the secretion proinflammatory cytokines, leading to secondary inflammation. Blocking OA-associated TLR activation is a promising strategy for the development of suitable therapies. Here, we want to find some bacteria-derived peptides that can block TLR signaling in chondrocytes more efficiently. Based on previous studies, we screened 12 TIR domain-derived peptides for their effects on NF-кB activation induced by LPS, IL-1ß or TNF-α in murine ATDC-5 cells. We evaluated their effects on LPS-induced cytokine expression and secretion. Among them, two bacteria-derived peptides, TcpC-DD and TcpB-DD, showed the most potent inhibitory activities. In comparison with TcpB-DD, TcpC-DD exhibited broader TLR-inhibitory specificity during inflammation in chondrocytes. Furthermore, both TcpC-DD and TcpB-DD displayed strong inhibition of LPS- and IL-1ß-induced catabolic reactions in chondrocytes. However, only TcpC-DD exhibited obvious suppression of TNF-α-induced catabolism. In conclusion, we identified two novel inhibitory peptides that modulate catabolism in chondrocytes and innate immune responses, and these peptides could be used to develop novel therapeutic strategies for OA.


Assuntos
Proteínas da Membrana Bacteriana Externa/farmacologia , Condrócitos/imunologia , Imunidade Inata/efeitos dos fármacos , Animais , Bactérias/metabolismo , Proteínas da Membrana Bacteriana Externa/metabolismo , Linhagem Celular , Células Cultivadas , Citocinas/metabolismo , Humanos , Imunidade Inata/imunologia , Inflamação/metabolismo , Interleucina-1beta/metabolismo , Camundongos , NF-kappa B/metabolismo , Osteoartrite/imunologia , Osteoartrite/fisiopatologia , Peptídeos/metabolismo , Domínios Proteicos , Receptores de Interleucina-1/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fator de Necrose Tumoral alfa/metabolismo
4.
Protoplasma ; 256(4): 951-969, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30793222

RESUMO

Metacaspase, as hypersensitive response (HR) executors, has been identified in many plant species. Previously, the entire gene family of metacaspase has been uncovered, but there are still questions that remain unclear regarding HR-regulating gene members. In this study, based on metacaspase expression during different grapevine genotypes interacting with Plasmopara viticola, we identified MC2 and MC5 as candidates involved in HR. We overexpressed both metacaspases as GFP fusions in tobacco BY-2 cells to address subcellular localization and cellular functions. We found MC2 located at the ER, while MC5 was nucleocytoplasmic. In these overexpressor lines, cell death elicited by the bacterial protein harpin, is significantly enhanced, indicating MC2 and MC5 mediated defence-related programmed cell death (PCD). This effect was mitigated, when the membrane-located NADPH oxidase was inhibited by the specific inhibitor diphenylene iodonium, or when cells were complemented with methyl jasmonate, a crucial signal of basal immunity. Both findings are consistent with a role of MC2 and MC5 in cell death-related immunity. Using a dual-luciferase reporter system in grapevine cells we demonstrated both MC2 and MC5 promoter alleles from V. rupestris were more responsive to harpin than those from V. vinifera cv 'Müller-Thurgau', while they were not induced by MeJA as signal linked with basal immunity. These findings support a model, where MC2 and MC5 act specifically as executors of the HR.


Assuntos
Caspases/genética , Doenças das Plantas/microbiologia , Proteínas de Plantas/genética , Vitis/microbiologia , Acetatos/metabolismo , Proteínas da Membrana Bacteriana Externa/farmacologia , Caspases/metabolismo , Morte Celular/genética , Ciclopentanos/metabolismo , Resistência à Doença/genética , Resistência à Doença/imunologia , Regulação da Expressão Gênica de Plantas , Interações Hospedeiro-Patógeno/genética , Oomicetos/patogenicidade , Oxilipinas/metabolismo , Filogenia , Células Vegetais/microbiologia , Imunidade Vegetal , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Regiões Promotoras Genéticas , Explosão Respiratória , Tabaco/genética , Vitis/genética
5.
Mol Immunol ; 108: 1-7, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30739075

RESUMO

Our previous data demonstrated that Friend leukemia virus integration 1 (Fli-1), an ETS transcription factor, governs pericyte loss and vascular dysfunction in cecal ligation and puncture-induced murine sepsis by regulating essential pyroptosis markers including caspase-1. However, whether Fli-1 regulates caspase-1 expression levels in vitro and how Fli-1 regulates caspase-1 remain unknown. Our present work further demonstrated that overexpressed Fli-1 significantly increased caspase-1 and IL-18 expression levels in cultured mouse lung pericytes. Bacterial outer membrane vesicles (OMVs) have been found to induce cell pyroptosis through transferring LPS intracellularly. Using OMVs to induce an in vitro model of pyroptosis, we observed that OMVs significantly increased protein levels of Fli-1 in mouse lung pericytes. Furthermore, knockdown of Fli-1 by siRNA blocked OMVs-induced caspase-1, caspase-11 and IL-18 expression levels. As caspase-1 was predicted as a potential target of Fli-1, we cloned murine caspase-1 promoter into a luciferase construct. Our data demonstrate for the first time that Fli-1 regulates caspase-1 expression by directly binding to its promoter regions measured by chromatin immunoprecipitation (ChIP) assay and luciferase reporter system. In summary, our findings demonstrated a novel role and mechanism of Fli-1 in regulating caspase-1 expression in lung pericytes.


Assuntos
Caspase 1/imunologia , Regulação Enzimológica da Expressão Gênica/imunologia , Proteína Proto-Oncogênica c-fli-1/imunologia , Animais , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/farmacologia , Caspase 1/genética , Escherichia coli K12/química , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Interleucina-18/genética , Interleucina-18/imunologia , Pulmão , Camundongos , Pericitos , Proteína Proto-Oncogênica c-fli-1/genética
6.
Scand J Trauma Resusc Emerg Med ; 26(1): 61, 2018 Jul 24.
Artigo em Inglês | MEDLINE | ID: mdl-30041663

RESUMO

BACKGROUND: Streptococcus pyogenes is a Gram positive bacterial species commonly involved in sepsis. Invasive strains express virulence factors such as the M1 protein. M1 protein forms complexes with fibrinogen leading to a cytokine storm in plasma contributing to the development of septic shock and organ failure. In experimental animals M1 protein causes vascular nitric oxide production and hyporesponsiveness to pressors, but it is not known whether it affects the human vascular wall. METHODS: Human omental arteries obtained during surgery were incubated in vitro with M1 protein or lipopolysaccharide (LPS) as positive control, with or without plasma. After 48 h, contractile response to noradrenaline was measured, and levels of nitrite/nitrate and the cytokines interleukin (IL)-1ß, IL-6, IL-8, IL-10, and tumor necrosis factor (TNF)-α in the incubation medium were measured. A second set of arteries were incubated with or without main components of plasma (immunoglobulin G, albumin or fibrinogen), in the presence of M1 protein followed by cytokine measurement. RESULTS: Artery segments incubated with M1 protein and plasma contracted weaker in response to noradrenaline, and levels of IL-6 and IL-8 were significantly higher compared to after incubation with M1 protein alone. Incubation with M1 protein and fibrinogen resulted in elevated levels of IL-6 and IL-8, while incubation with M1 protein and albumin or immunoglobulin G did not affect the levels. Neither any of the other cytokines nor nitrite/nitrate was detected in the medium in any of the incubation conditions. CONCLUSIONS: The study shows that M1 protein of Streptococcus pyogenes has a direct effect on the human vascular wall in the presence of plasma, demonstrated both as a diminished contractile response to noradrenaline and increased cytokine production. The effect of plasma was attributed to fibrinogen. The findings suggest that M1 protein contributes to the development of septic shock through impairment of the contractility of the vascular wall.


Assuntos
Antígenos de Bactérias/farmacologia , Artérias/metabolismo , Proteínas da Membrana Bacteriana Externa/farmacologia , Proteínas de Transporte/farmacologia , Citocinas/metabolismo , Fibrinogênio/metabolismo , Choque Séptico/metabolismo , Infecções Estreptocócicas/metabolismo , Streptococcus pyogenes/isolamento & purificação , Idoso , Idoso de 80 Anos ou mais , Artérias/patologia , Feminino , Humanos , Pessoa de Meia-Idade , Omento/irrigação sanguínea , Choque Séptico/microbiologia , Choque Séptico/patologia , Infecções Estreptocócicas/patologia
7.
PLoS One ; 13(6): e0200112, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29953523

RESUMO

Chlamydia pecorum is a mucosal infection, which causes debilitating disease of the urinary tract, reproductive tract and ocular sites of koalas (Phascolarctos cinereus). While antibiotics are available for treatment, they are detrimental to the koalas' gastrointestinal tract microflora leaving the implementation of a vaccine as an ideal option for the long-term management of koala populations. We have previously reported on the successes of an anti-chlamydial recombinant major outer membrane protein (rMOMP) vaccine however, recombinant protein based vaccines are not ideal candidates for scale up from the research level to small-medium production level for wider usage. Peptide based vaccines are a promising area for vaccine development, because peptides are stable, cost effective and easily produced. In this current study, we assessed, for the first time, the immune responses to a synthetic peptide based anti-chlamydial vaccine in koalas. Five healthy male koalas were vaccinated with two synthetic peptides derived from C. pecorum MOMP and another five healthy male koalas were vaccinated with full length recombinant C. pecorum MOMP (genotype G). Systemic (IgG) and mucosal (IgA) antibodies were quantified and pre-vaccination levels compared to post-vaccination levels (12 and 26 weeks). MOMP-peptide vaccinated koalas produced Chlamydia-specific IgG and IgA antibodies, which were able to recognise not only the genotype used in the vaccination, but also MOMPs from several other koala C. pecorum genotypes. In addition, IgA antibodies induced at the ocular site not only recognised recombinant MOMP protein but also, whole native chlamydial elementary bodies. Interestingly, some MOMP-peptide vaccinated koalas showed a stronger and more sustained vaccine-induced mucosal IgA antibody response than observed in MOMP-protein vaccinated koalas. These results demonstrate that a synthetic MOMP peptide based vaccine is capable of inducing a Chlamydia-specific antibody response in koalas and is a promising candidate for future vaccine development.


Assuntos
Proteínas da Membrana Bacteriana Externa/farmacologia , Vacinas Bacterianas/farmacologia , Infecções por Chlamydia/prevenção & controle , Infecções por Chlamydia/veterinária , Chlamydia , Peptídeos/farmacologia , Phascolarctidae , Vacinação , Animais , Proteínas da Membrana Bacteriana Externa/imunologia , Vacinas Bacterianas/imunologia , Infecções por Chlamydia/imunologia , Peptídeos/imunologia
8.
Theriogenology ; 104: 173-178, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-28863350

RESUMO

Embryo slow freezing protocols include a nucleation induction step called manual seeding. This step is time consuming, manipulator dependent and hard to standardize. It requires access to samples, which is not always possible within the configuration of systems, such as differential scanning calorimeters or cryomicroscopes. Ice nucleation can be induced by other methods, e.g., by the use of ice nucleating agents. Snomax is a commercial preparation of inactivated proteins extracted from Pseudomonas syringae. The aim of our study was to investigate if Snomax can be an alternative to manual seeding in the slow freezing of mouse embryos. The influence of Snomax on the pH and osmolality of the freezing medium was evaluated. In vitro development (blastocyst formation and hatching rates) of fresh embryos exposed to Snomax and embryo cryopreserved with and without Snomax was assessed. The mitochondrial activity of frozen-thawed blastocysts was assessed by JC-1 fluorescent staining. Snomax didn't alter the physicochemical properties of the freezing medium, and did not affect embryo development of fresh embryos. After cryopreservation, the substitution of manual seeding by the ice nucleating agent (INA) Snomax did not affect embryo development or embryo mitochondrial activity. In conclusion, Snomax seems to be an effective ice nucleating agent for the slow freezing of mouse embryos. Snomax can also be a valuable alternative to manual seeding in research protocols in which manual seeding cannot be performed (i.e., differential scanning calorimetry and cryomicroscopy).


Assuntos
Proteínas da Membrana Bacteriana Externa/farmacologia , Criopreservação/métodos , Crioprotetores/farmacologia , Embrião de Mamíferos/efeitos dos fármacos , Animais , Técnicas de Cultura , Desenvolvimento Embrionário/efeitos dos fármacos , Feminino , Congelamento , Concentração de Íons de Hidrogênio , Masculino , Camundongos , Concentração Osmolar
9.
Nat Commun ; 8(1): 626, 2017 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-28931823

RESUMO

Gram-negative bacteria actively secrete outer membrane vesicles, spherical nano-meter-sized proteolipids enriched with outer membrane proteins, to the surroundings. Outer membrane vesicles have gained wide interests as non-living complex vaccines or delivery vehicles. However, no study has used outer membrane vesicles in treating cancer thus far. Here we investigate the potential of bacterial outer membrane vesicles as therapeutic agents to treat cancer via immunotherapy. Our results show remarkable capability of bacterial outer membrane vesicles to effectively induce long-term antitumor immune responses that can fully eradicate established tumors without notable adverse effects. Moreover, systematically administered bacterial outer membrane vesicles specifically target and accumulate in the tumor tissue, and subsequently induce the production of antitumor cytokines CXCL10 and interferon-γ. This antitumor effect is interferon-γ dependent, as interferon-γ-deficient mice could not induce such outer membrane vesicle-mediated immune response. Together, our results herein demonstrate the potential of bacterial outer membrane vesicles as effective immunotherapeutic agent that can treat various cancers without apparent adverse effects.Bacterial outer membrane vesicles (OMVs) contain immunogens but no study has yet examined their potential in treating cancer. Here, the authors demonstrate that OMVs can suppress established tumours and prevent tumour metastasis by an interferon-γ mediated antitumor response.


Assuntos
Adenocarcinoma/imunologia , Proteínas da Membrana Bacteriana Externa/farmacologia , Quimiocina CXCL10/efeitos dos fármacos , Neoplasias do Colo/imunologia , Interferon gama/efeitos dos fármacos , Interleucina-8/efeitos dos fármacos , Vesículas Transportadoras , Aciltransferases/genética , Animais , Proteínas da Membrana Bacteriana Externa/metabolismo , Quimiocina CXCL10/imunologia , Escherichia coli , Proteínas de Escherichia coli/genética , Células HEK293 , Humanos , Imunoterapia , Interferon gama/imunologia , Interleucina-8/imunologia , Camundongos , Transplante de Neoplasias , Organismos Geneticamente Modificados
10.
J Med Chem ; 60(18): 7745-7763, 2017 09 28.
Artigo em Inglês | MEDLINE | ID: mdl-28829599

RESUMO

Fatty acyl analogues of muramyldipeptide (MDP) (abbreviated N-L18 norAbuGMDP, N-B30 norAbuGMDP, norAbuMDP-Lys(L18), norAbuMDP-Lys(B30), norAbuGMDP-Lys(L18), norAbuGMDP-Lys(B30), B30 norAbuMDP, L18 norAbuMDP) are designed and synthesized comprising the normuramyl-l-α-aminobutanoyl (norAbu) structural moiety. All new analogues show depressed pyrogenicity in both free (micellar) state and in liposomal formulations when tested in rabbits in vivo (sc and iv application). New analogues are also shown to be selective activators of NOD2 and NLRP3 (inflammasome) in vitro but not NOD1. Potencies of NOD2 and NLRP3 stimulation are found comparable with free MDP and other positive controls. Analogues are also demonstrated to be effective in stimulating cellular proliferation when the sera from mice are injected sc with individual liposome-loaded analogues, causing proliferation of bone marrow-derived GM-progenitors cells. Importantly, vaccination nanoparticles prepared from metallochelation liposomes, His-tagged antigen rOspA from Borrelia burgdorferi, and lipophilic analogue norAbuMDP-Lys(B30) as adjuvant, are shown to provoke OspA-specific antibody responses with a strong Th1-bias (dominance of IgG2a response). In contrast, the adjuvant effects of Alum or parent MDP show a strong Th2-bias (dominance of IgG1 response).


Assuntos
Acetilmuramil-Alanil-Isoglutamina/análogos & derivados , Acetilmuramil-Alanil-Isoglutamina/farmacologia , Adjuvantes Imunológicos/farmacologia , Antígenos de Superfície/farmacologia , Proteínas da Membrana Bacteriana Externa/farmacologia , Vacinas Bacterianas/farmacologia , Borrelia burgdorferi/imunologia , Lipoproteínas/farmacologia , Acetilmuramil-Alanil-Isoglutamina/química , Acetilmuramil-Alanil-Isoglutamina/imunologia , Adjuvantes Imunológicos/química , Animais , Formação de Anticorpos , Antígenos de Superfície/química , Antígenos de Superfície/imunologia , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/imunologia , Vacinas Bacterianas/química , Vacinas Bacterianas/imunologia , Feminino , Células HEK293 , Humanos , Imunização , Lipoproteínas/química , Lipoproteínas/imunologia , Doença de Lyme/imunologia , Doença de Lyme/microbiologia , Camundongos , Camundongos Endogâmicos BALB C , Proteína 3 que Contém Domínio de Pirina da Família NLR/agonistas , Proteína 3 que Contém Domínio de Pirina da Família NLR/imunologia , Células RAW 264.7
11.
Zhejiang Da Xue Xue Bao Yi Xue Ban ; 46(2): 144-150, 2017 03 25.
Artigo em Chinês | MEDLINE | ID: mdl-28752705

RESUMO

Objective: To prepare a nano-carrier based on combining bacterial outer membrane vesicles (OMV) with three block polymer pluronic F127 (PEO100-PPO65-PEO100) (OMV-F127) and to investigate its immunological activity. Methods: Attenuated salmonella (sal) was cultivated. OMV were separated by centrifugal ultrafiltration or ultrasonication, and OMV-F127 was prepared by mechanical extrudation method. The protein contents and compositions were tested with BCA and SDS-PAGE; the morphology of OMV, F127 and OMV-F127 were observed with FM and TEM; the particle sizes and their zeta potential were determined with DLS. Mouse macrophage RAW246.7 cells were treated with OMV-F127 (50 µg/mL, 100 µg/mL) in vitro, and the concentrations of IL-12, TNF-α and IFN-γ in culture supernatant were measured with ELISA kits. Results: The contents of protein in separated OMV by centrifugal ultrafiltration and ultrasonication were 2.8 mg/mL and 2.7 mg/mL, respectively. SDS-PAGE showed the marker protein OmpF/C in OMV. Under the FM and TEM, ball-like structure of F127 and OMV-F127 was observed. Size analysis revealed that the diameters of OMV, F127 and OMV-F127 were 72±2 nm, 90±3 nm and 92±2 nm, respectively. ELISA tests revealed that OMV-F127 significantly stimulated the secretion of IL-12, TNF-α and IFN-γ in RAW246.7 cells. Conclusion: A nano-carrier based on bacterial outer membrane vesicles has been prepared, which can stimulate the secretion of cytokines and may have immunomodulatory effects.


Assuntos
Proteínas da Membrana Bacteriana Externa , Citocinas , Polietilenos , Polipropilenos , Animais , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/farmacologia , Secreções Corporais/efeitos dos fármacos , Citocinas/análise , Eletroforese em Gel de Poliacrilamida , Fatores Imunológicos/química , Fatores Imunológicos/farmacologia , Camundongos , Polietilenos/química , Polietilenos/farmacologia , Polipropilenos/química , Polipropilenos/farmacologia , Células RAW 264.7 , Salmonella/imunologia
12.
Anal Chem ; 89(11): 5998-6005, 2017 06 06.
Artigo em Inglês | MEDLINE | ID: mdl-28467855

RESUMO

Currently, reliable valving on integrated microfluidic devices fabricated from rigid materials is confined to expensive and complex methods. Freeze-thaw valves (FTVs) can provide a low cost, low complexity valving mechanism, but reliable implementation of them has been greatly hindered by the lack of ice nucleation sites within the valve body's small volume. Work to date has required very low temperatures (on the order of -40 °C or colder) to induce freezing without nucleation sites, making FTVs impractical due to instrument engineering challenges. Here, we report the use of ice-nucleating proteins (INPs) to induce ice formation at relatively warm temperatures in microfluidic devices. Microfluidic channels were filled with buffers containing femtomolar INP concentrations from Pseudomonas syringae. The channels were cooled externally with simple, small-footprint Peltier thermoelectric coolers (TECs), and the times required for channel freezing (valve closure) and thawing (valve opening) were measured. Under optimized conditions in plastic chips, INPs made sub-10 s actuations possible at TEC temperatures as warm as -13 °C. Additionally, INPs were found to have no discernible inhibitory effects in model enzyme-linked immunosorbent assays or polymerase chain reactions, indicating their compatibility with microfluidic systems that incorporate these widely used bioassays. FTVs with INPs provide a much needed reliable valving scheme for rigid plastic devices with low complexity, low cost, and no moving parts on the device or instrument. The reduction in freeze time, accessible actuation temperatures, chemical compatibility, and low complexity make the implementation of compact INP-based FTV arrays practical and attractive for the control of integrated biochemical assays.


Assuntos
Proteínas da Membrana Bacteriana Externa/farmacologia , Microfluídica/instrumentação , Ensaio de Imunoadsorção Enzimática/economia , Ensaio de Imunoadsorção Enzimática/instrumentação , Congelamento , Microfluídica/economia , Microfluídica/normas , Reação em Cadeia da Polimerase/economia , Reação em Cadeia da Polimerase/instrumentação , Temperatura Ambiente
13.
J Insect Sci ; 17(2)2017 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-28365771

RESUMO

Defense elicitors are products that activate acquired defense responses in plants, thus rendering the plants less susceptible to attack by a broad range of pests. We demonstrated previously under laboratory conditions that foliar applications of the defense elicitors Actigard (acibenzolar-S-methyl), Employ (harpin protein), or ODC (chitosan) to potted pear trees (Pyrus communis L.) each caused an increase in mortality of Cacopsylla pyricola (Förster) (Hemiptera: Psyllidae) nymphs and altered the settling and oviposition behavior of the adults. In this study, we monitored C. pyricola populations over a 3-yr period on orchard-grown trees treated with water (untreated control), Actigard, Employ, or ODC. Fewer nymphs were observed on trees treated with elicitors compared with untreated trees in both 2014 and 2016. A similar but statistically nonsignificant pattern was observed in 2015 when nearly 30% fewer nymphs were observed on trees treated with elicitors versus untreated controls. Observed reductions in psyllid numbers by defense elicitors were modest and do not warrant the use of these products alone for managing C. pyricola. However, these products are often used for management of fire blight, and our observations that elicitors also reduce C. pyricola populations may be useful for system-wide integrated pest management approaches.


Assuntos
Proteínas da Membrana Bacteriana Externa/farmacologia , Quitosana/farmacologia , Hemípteros/fisiologia , Pyrus/efeitos dos fármacos , Tiadiazóis/farmacologia , Animais , Ninfa/fisiologia , Dinâmica Populacional
14.
J Vector Borne Dis ; 54(4): 317-327, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29460861

RESUMO

Background & objectives: The nature of the rickettsial antigens and the immune response generated by them, have been the subject of exhaustive research so that a suitable vaccine can be developed. Till date evaluations of Rickettsia rickettsii antigens that induce both humoral and cellular responses in animal models have only shown partial protection and short-term immunological memory. This study was aimed to evaluate the immune response induced by DNA plasmids generated from the OmpA and OmpB genes of R. rickettsii in peripheral blood mononuclear cells of rickettsial (sensitized) patients compared to healthy subjects. Methods: Plasmids OmpA-49, OmpB-15 and OmpB-24 were generated in the pVAX vector. Macrophages derived from the THP-1 cell line were transfected in vitro with the plasmids and were co-cultured with T-lymphocytes from sensitized subjects and healthy subjects to evaluate cell proliferation and cytokine production. Results: The OmpB-24 plasmid induced proliferative response in human lymphocytes, with production of IL-2, IFN-γ, IL-12p70, IL-6 and TNF-α, likely due to the presence of conserved epitopes among R. rickettsii, R. typhi and R. felis (differing from 1 to 3 amino acids) during the construction of the plasmids. Interpretation & conclusion: DNA sequences of rickettsial epitopes can be cloned into the pVAX vector. Constructed plasmids can generate a proliferative response and produce cytokines in vitro, in co-culture of transfected macrophages with sensitized human lymphocytes. Plasmid OmpB-24 proved to be the most immunogenic with respect to plasmids OmpA-49 and OmpB-15.


Assuntos
Antígenos de Bactérias/imunologia , Proteínas da Membrana Bacteriana Externa/imunologia , Leucócitos Mononucleares/efeitos dos fármacos , Rickettsia rickettsii/imunologia , Adolescente , Adulto , Idoso , Anticorpos Antibacterianos/sangue , Antígenos de Bactérias/farmacologia , Proteínas da Membrana Bacteriana Externa/farmacologia , Citocinas/biossíntese , Citocinas/efeitos dos fármacos , Feminino , Humanos , Leucócitos Mononucleares/imunologia , Masculino , Pessoa de Meia-Idade , Plasmídeos/imunologia , Rickettsia rickettsii/química , Adulto Jovem
15.
Artigo em Inglês | MEDLINE | ID: mdl-28018863

RESUMO

Achieving cross-protective efficacy against multiple bacterial strains or serotypes is an important goal of vaccine design. Enterotoxigenic Escherichia coli (ETEC) is an important cause of diarrheal disease in underdeveloped nations. We have been interested in identifying and characterizing ETEC antigens that generate protective immune responses independent of ETEC colonization factor (CF) expression. Our previous studies used proteomics to identify the ETEC MipA, Skp, and ETEC_2479 proteins as effective in protecting mice from homologous challenge with ETEC H10407 using a pulmonary inoculation model. This model permits analysis of mouse survival, bacterial clearance, and the production of secretory IgA (sIgA) and has been employed previously for studies of enteric pathogens for which robust oral challenge models do not exist. MipA belongs to a family of proteins involved in remodeling peptidoglycan. Skp rescues misdirected outer membrane proteins. ETEC_2479 is predicted to function as an outer membrane porin. These proteins are conserved in pathogenic ETEC strains as well as in commensal Proteobacteria. Antibodies produced against the ETEC MipA, Skp, and ETEC_2479 proteins also reduced the adherence of multiple ETEC strains differing in CF type to intestinal epithelial cells. Here we characterized the ability of 10 heterologous ETEC strains that differ in CF type to cause clinical signs of illness in mice after pulmonary challenge. ETEC strains C350C1A, E24377A, E7476A, WS2173A, and PE360 caused variable degrees of lethality in this mouse model, while ETEC strains B7A, WS6866B, 2230, ARG-2, and 8786 did not. Subsequent challenge experiments in which mice were first vaccinated intranasally with MipA, Skp, or ETEC_2479, when combined with cholera toxin, showed both that each antigen was protective and that protection was strongly correlated with fecal IgA concentrations. We conclude that the MipA, Skp, or ETEC_2479 antigens generate protection in the mouse pulmonary challenge model against ETEC strains that express different CFs.


Assuntos
Antígenos de Bactérias/imunologia , Proteínas da Membrana Bacteriana Externa/imunologia , Proteínas de Ligação a DNA/imunologia , Escherichia coli Enterotoxigênica/imunologia , Infecções por Escherichia coli/prevenção & controle , Proteínas de Escherichia coli/imunologia , Vacinas contra Escherichia coli/imunologia , Proteínas de Fímbrias/imunologia , Chaperonas Moleculares/imunologia , Animais , Anticorpos Antibacterianos/imunologia , Antígenos de Bactérias/farmacologia , Proteínas da Membrana Bacteriana Externa/farmacologia , Toxina da Cólera/farmacologia , Proteínas de Ligação a DNA/farmacologia , Modelos Animais de Doenças , Infecções por Escherichia coli/imunologia , Infecções por Escherichia coli/microbiologia , Proteínas de Escherichia coli/farmacologia , Vacinas contra Escherichia coli/administração & dosagem , Feminino , Proteínas de Fímbrias/farmacologia , Imunoglobulina A Secretora/imunologia , Pulmão/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Chaperonas Moleculares/farmacologia , Peptidoglicano/imunologia , Porinas/imunologia , Porinas/farmacologia
16.
Sci Rep ; 6: 38240, 2016 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-27905535

RESUMO

The demand for improved pertussis vaccines is urgent due to the resurgence of whooping cough. A deeper understanding of the mode of action of pertussis vaccines is required to achieve this improvement. The vaccine-induced effects of a candidate outer membrane vesicle vaccine (omvPV) and a classical protective but reactogenic whole cell vaccine (wPV) were comprehensively compared in mice. The comparison revealed essential qualitative and quantitative differences with respect to immunogenicity and adverse effects for these vaccines. Both vaccines stimulated a mixed systemic Th1/Th2/Th17 response. Remarkably, omvPV evoked higher IgG levels, lower systemic pro-inflammatory cytokine responses and enhanced splenic gene expression than wPV. The omvPV-induced transcriptome revealed gene signatures of the IFN-signaling pathway, anti-inflammatory signatures that attenuate LPS responses, anti-inflammatory metabolic signatures, and IgG responses. Upon intranasal challenge, both immunized groups were equally efficient in clearing Bordetella pertussis from the lungs. This study importantly shows that immunization with omvPV provides a milder inflammatory responses but with equal protection to bacterial colonization and induction of protective antibody and Th1/Th17 type immune responses compared to wPV. These results emphasize the potential of omvPV as a safe and effective next-generation pertussis vaccine.


Assuntos
Anticorpos Antibacterianos/imunologia , Proteínas da Membrana Bacteriana Externa/imunologia , Vacinas Bacterianas/imunologia , Bordetella pertussis/imunologia , Regulação da Expressão Gênica/imunologia , Imunoglobulina G/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Animais , Proteínas da Membrana Bacteriana Externa/farmacologia , Vacinas Bacterianas/farmacologia , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C
17.
Antimicrob Agents Chemother ; 60(8): 4757-63, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27216069

RESUMO

Gram-negative bacteria are notoriously resistant to a variety of high-molecular-weight antibiotics due to the limited permeability of their outer membrane (OM). The basis of OM barrier function and the genetic factors required for its maintenance remain incompletely understood. Here, we employed transposon insertion sequencing to identify genes required for Vibrio cholerae resistance to vancomycin and bacitracin, antibiotics that are thought to be too large to efficiently penetrate the OM. The screen yielded several genes whose protein products are predicted to participate in processes important for OM barrier functions and for biofilm formation. In addition, we identified a novel factor, designated vigA (for vancomycin inhibits growth), that has not previously been characterized or linked to outer membrane function. The vigA open reading frame (ORF) codes for an inner membrane protein, and in its absence, cells became highly sensitive to glycopeptide antibiotics (vancomycin and ramoplanin) and bacitracin but not to other large antibiotics or detergents. In contrast to wild-type (WT) cells, the vigA mutant was stained with fluorescent vancomycin. These observations suggest that VigA specifically prevents the periplasmic accumulation of certain large antibiotics without exerting a general role in the maintenance of OM integrity. We also observed marked interspecies variability in the susceptibilities of Gram-negative pathogens to glycopeptides and bacitracin. Collectively, our findings suggest that the OM barrier is not absolute but rather depends on specific OM-antibiotic interactions.


Assuntos
Antibacterianos/farmacologia , Elementos de DNA Transponíveis/genética , Farmacorresistência Bacteriana/genética , Vibrio cholerae/efeitos dos fármacos , Vibrio cholerae/genética , Bacitracina/farmacologia , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/farmacologia , Glicopeptídeos/genética , Peso Molecular , Mutagênese Insercional/genética , Vancomicina/farmacologia
18.
Cell Microbiol ; 18(12): 1723-1738, 2016 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-27121139

RESUMO

Gingival junctional epithelial cell apoptosis caused by periodontopathic bacteria exacerbates periodontitis. This pathological apoptosis is involved in the activation of transforming growth factor ß (TGF-ß). However, the molecular mechanisms by which microbes induce the activation of TGF-ß remain unclear. We previously reported that Aggregatibacter actinomycetemcomitans (Aa) activated TGF-ß receptor (TGF-ßR)/smad2 signalling to induce epithelial cell apoptosis, even though Aa cannot bind to TGF-ßR. Additionally, outer membrane protein 29 kDa (Omp29), a member of the Aa Omps family, can induce actin rearrangements via focal adhesion kinase (FAK) signalling, which also plays a role in the activation of TGF-ß by cooperating with integrin. Accordingly, we hypothesized that Omp29-induced actin rearrangements via FAK activity would enhance the activation of TGF-ß, leading to gingival epithelial cell apoptosis in vitro. By using human gingival epithelial cell line OBA9, we found that Omp29 activated TGF-ßR/smad2 signalling and decreased active TGF-ß protein levels in the extracellular matrix (ECM) of cell culture, suggesting the transactivation of TGF-ßR. Inhibition of actin rearrangements by cytochalasin D or blebbistatin and knockdown of FAK or integrinß1 expression by siRNA transfection attenuated TGF-ßR/smad2 signalling activity and reduction of TGF-ß levels in the ECM caused by Omp29. Furthermore, Omp29 bound to fibronectin (Fn) to induce its aggregation on integrinß1, which is associated with TGF-ß signalling activity. All the chemical inhibitors and siRNAs tested blocked Omp29-induced OBA9 cells apoptosis. These results suggest that Omp29 binds to Fn in order to facilitate Fn/integrinß1/FAK signalling-dependent TGF-ß release from the ECM, thereby inducing gingival epithelial cell apoptosis via TGF-ßR/smad2 pathway.


Assuntos
Aggregatibacter actinomycetemcomitans/genética , Proteínas da Membrana Bacteriana Externa/genética , Células Epiteliais/microbiologia , Fibronectinas/genética , Quinase 1 de Adesão Focal/genética , Integrina beta1/genética , Fator de Crescimento Transformador beta/genética , Aggregatibacter actinomycetemcomitans/metabolismo , Apoptose/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas da Membrana Bacteriana Externa/farmacologia , Linhagem Celular Transformada , Citocalasina D/farmacologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Fibronectinas/metabolismo , Quinase 1 de Adesão Focal/metabolismo , Regulação da Expressão Gênica , Gengiva/metabolismo , Gengiva/microbiologia , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Interações Hospedeiro-Patógeno , Humanos , Integrina beta1/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Receptor do Fator de Crescimento Transformador beta Tipo I , Receptores de Fatores de Crescimento Transformadores beta/genética , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Transdução de Sinais , Proteína Smad2/antagonistas & inibidores , Proteína Smad2/genética , Proteína Smad2/metabolismo , Fator de Crescimento Transformador beta/antagonistas & inibidores , Fator de Crescimento Transformador beta/metabolismo
19.
PLoS One ; 11(4): e0154153, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27111850

RESUMO

Opa proteins are major surface-expressed proteins located in the Neisseria meningitidis outer membrane, and are potential meningococcal vaccine candidates. Although Opa proteins elicit high levels of bactericidal antibodies following immunisation in mice, progress towards human clinical trials has been delayed due to previous findings that Opa inhibits T cell proliferation in some in vitro assays. However, results from previous studies are conflicting, with different Opa preparations and culture conditions being used. We investigated the effects of various Opa+ and Opa- antigens from N. meningitidis strain H44/76 in a range of in vitro conditions using peripheral blood mononuclear cells (PBMCs) and purified CD4+ T cells, measuring T cell proliferation by CFSE dilution using flow cytometry. Wild type recombinant and liposomal Opa proteins inhibited CD4+ T cell proliferation after stimulation with IL-2, anti-CD3 and anti-CD28, and these effects were reduced by mutation of the CEACAM1-binding region of Opa. These effects were not observed in culture with ex vivo PBMCs. Opa+ and Opa- OMVs did not consistently exert a stimulatory or inhibitory effect across different culture conditions. These data do not support a hypothesis that Opa proteins would be inhibitory to T cells if given as a vaccine component, and T cell immune responses to OMV vaccines are unlikely to be significantly affected by the presence of Opa proteins.


Assuntos
Antígenos de Bactérias/farmacologia , Proteínas da Membrana Bacteriana Externa/farmacologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Leucócitos Mononucleares/efeitos dos fármacos , Meningite Meningocócica/prevenção & controle , Neisseria meningitidis/imunologia , Antígenos de Bactérias/genética , Antígenos de Bactérias/imunologia , Antígenos CD/genética , Antígenos CD/imunologia , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/imunologia , Sítios de Ligação , Antígenos CD28/antagonistas & inibidores , Antígenos CD28/genética , Antígenos CD28/imunologia , Complexo CD3/genética , Complexo CD3/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/microbiologia , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/imunologia , Proliferação de Células/efeitos dos fármacos , Separação Celular , Expressão Gênica , Humanos , Imunomodulação/efeitos dos fármacos , Interleucina-2/farmacologia , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/microbiologia , Ativação Linfocitária/efeitos dos fármacos , Meningite Meningocócica/imunologia , Meningite Meningocócica/microbiologia , Vacinas Meningocócicas/biossíntese , Neisseria meningitidis/genética , Cultura Primária de Células , Ligação Proteica , Isoformas de Proteínas/genética , Isoformas de Proteínas/imunologia , Isoformas de Proteínas/farmacologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/farmacologia , Vacinação
20.
Appl Microbiol Biotechnol ; 100(11): 4791-801, 2016 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-27098257

RESUMO

Outer membrane vesicles (OMVs) produced by Gram-negative bacteria have been intensively investigated in recent times. Vesicle formation models have been proposed, some factors affecting the process were established, and important roles vesicles play in vital activities of their producing cells were determined. Studies of pathogenic bacterial vesicles contribute to understanding the causes of acute infection and developing drugs on their basis. Despite intensive research, issues associated with the understanding of vesicle biogenesis, the mechanisms of bacterium-bacterium and pathogen-host interactions with participation of vesicles, still remain unresolved. This review discusses some results obtained in the research into OMVs of Lysobacter sp. XL1 VKM B-1576. This bacterium secretes into the environment a spectrum of bacteriolytic enzymes that hydrolyze peptidoglycan of competing bacteria, thus leading to their lysis. One of these enzymes, lytic endopeptidase L5, has been shown not only to be secreted by means of vesicles but also to be involved in their formation. As part of vesicles, the antimicrobial potential of L5 enzyme has been found to be considerably expanded. Vesicles have been shown to have a therapeutic effect in respect of anthrax infection and staphylococcal sepsis modelled in mice. The scientific basis for constructing liposomal antimicrobial preparations from vesicle phospholipids and recombinant bacteriolytic enzyme L5 has been formed.


Assuntos
Anti-Infecciosos/farmacologia , Proteínas da Membrana Bacteriana Externa/farmacologia , Lysobacter/fisiologia , Biogênese de Organelas , Vesículas Transportadoras/fisiologia , Animais , Antraz/tratamento farmacológico , Bacteriólise , Modelos Animais de Doenças , Endopeptidases/farmacologia , Bactérias Gram-Positivas/efeitos dos fármacos , Interações Hospedeiro-Patógeno , Lysobacter/enzimologia , Camundongos , Peptidoglicano/metabolismo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA