RESUMO
Adjuvants are the helper substances that increase vaccine efficacy by enhancing the potency and longevity of specific immune responses to antigens. Most existing fish vaccines are presented in the form of oil-based emulsions delivered by intraperitoneal injection. The characterization of their mode of action is a valuable aid to future vaccine development, particularly for the potential identification and stimulation of specific immunological pathways related to the desired protective response. This study characterized the expression of selected immune-related genes in the peritoneal cavity, head kidney and spleen following the administration of two adjuvanted-bacterial vaccines thought to induce humoral (Montanide™ ISA 763A VG) or humoral and cell mediated (Montanide™ ISA 761 VG) immune responses, to determine if differences in responsiveness are readily apparent. The most informative site was the spleen, where Montanide™ ISA 763A VG + bacterin gave rise to upregulation of genes driving T-cell/lymphoid responses, namely IL-2, IL-15 and IL-21. This combined with upregulation of IFNγ1 and IFNγ2, IL-4/13B2, p35A1 and p40 (B1 and C) indicated that the induction of Th1 and possibly Th2 immunity was occurring in fish vaccinated with this adjuvant. Perhaps the most intriguing finding was the lack of a detectable Th1 response in fish given Montanide™ ISA 761 VG + bacterin, suggesting some other arm of the immune system is activated to give protection. Whatever the reason for the different responses detected, it is clear from the present study that the adjuvant used has a major impact on the responses elicited. Since these differences are readily detectable it allows, in principle, their use to help select the most appropriate adjuvants for inclusion into fish vaccines, where the type of response elicited may need to be tailored to a particular pathogen to confer protection.
Assuntos
Adjuvantes Imunológicos/farmacologia , Aeromonas salmonicida , Vacinas Bacterianas/imunologia , Doenças dos Peixes/prevenção & controle , Infecções por Bactérias Gram-Negativas/veterinária , Manitol/análogos & derivados , Oncorhynchus mykiss/imunologia , Animais , Citocinas/genética , Citocinas/metabolismo , Regulação da Expressão Gênica/imunologia , Infecções por Bactérias Gram-Negativas/prevenção & controle , Rim Cefálico/metabolismo , Macrófagos Peritoneais , Manitol/farmacologia , Oncorhynchus mykiss/microbiologiaRESUMO
Mammalian perivisceral adipose has been shown to play an important role in the regulation of the peritoneal immune responses. Recently it has been demonstrated that peritoneal antigens are collected by leukocytes within the visceral adipose mass, and a broad range of immunomodulatory genes are differentially expressed in adipose tissue after intraperitoneal vaccination in rainbow trout. To assess the immune cell component in adipose, immunohistochemical analysis was used to examine B-cell, T-cell and antigen presenting cell (APC) numbers and distribution in rainbow trout adipose tissue 24 and 72â¯h post vaccination in comparison to control fish. The results of this study support previous work on mammals with omental milky spots in naïve fish found to contain APCs and T-cells which then increased in size, number and complexity following vaccination. It suggests that following peritoneal stimulation the visceral adipose mass in fish likely plays an important role in vaccine antigen uptake and presentation by APCs, as well as subsequent T-cell activation and differentiation.
Assuntos
Tecido Adiposo/imunologia , Células Apresentadoras de Antígenos/imunologia , Linfócitos B/imunologia , Doenças dos Peixes/imunologia , Oncorhynchus mykiss/imunologia , Linfócitos T/imunologia , Aeromonas salmonicida/fisiologia , Animais , Diferenciação Celular/imunologia , Infecções por Bactérias Gram-Negativas/imunologia , Infecções por Bactérias Gram-Negativas/veterinária , Imuno-Histoquímica/veterinária , Ativação Linfocitária/imunologia , Fatores de Tempo , Vacinação/veterináriaRESUMO
OBJECTIVE: The effects of vaccinating 18-day-old chicken embryos with the combination of recombinant Eimeria profilin plus Clostridium perfringens (C. perfringens) NetB proteins mixed in the Montanide IMS adjuvant on the chicken immune response to necrotic enteritis (NE) were investigated using an Eimeria maxima (E. maxima)/C. perfringens co-infection NE disease model that we previously developed. METHODS: Eighteen-day-old broiler embryos were injected with 100 µL of phosphate-buffered saline, profilin, profilin plus necrotic enteritis B-like (NetB), profilin plus NetB/Montanide adjuvant (IMS 106), and profilin plus Net-B/Montanide adjuvant (IMS 101). After post-hatch birds were challenged with our NE experimental disease model, body weights, intestinal lesions, serum antibody levels to NetB, and proinflammatory cytokine and chemokine mRNA levels in intestinal intraepithelial lymphocytes were measured. RESULTS: Chickens in ovo vaccinated with recombinant profilin plus NetB proteins/IMS106 and recombinant profilin plus NetB proteins/IMS101 showed significantly increased body weight gains and reduced gut damages compared with the profilin-only group, respectively. Greater antibody response to NetB toxin were observed in the profilin plus NetB/IMS 106, and profilin plus NetB/IMS 101 groups compared with the other three vaccine/adjuvant groups. Finally, diminished levels of transcripts encoding for proinflammatory cytokines such as lipopolysaccharide-induced tumor necrosis factor-α factor, tumor necrosis factor superfamily 15, and interleukin-8 were observed in the intestinal lymphocytes of chickens in ovo injected with profilin plus NetB toxin in combination with IMS 106, and profilin plus NetB toxin in combination with IMS 101 compared with profilin protein alone bird. CONCLUSION: These results suggest that the Montanide IMS adjuvants potentiate host immunity to experimentally-induced avian NE when administered in ovo in conjunction with the profilin and NetB proteins, and may reduce disease pathology by attenuating the expression of proinflammatory cytokines and chemokines implicated in disease pathogenesis.
RESUMO
Route of vaccine delivery can greatly impact the immunogenicity, efficacy and safety of the vaccine. Four groups of piglets were immunised transdermally (t.d.), intradermally (i.d.) or intramuscularly (i.m.) with the same doses of antigen in combination with a water-in-oil-in-water emulsion adjuvant Montanide™ ISA 201 VG or with a microemulsion adjuvant Montanide™ IMS 1313 VG N ST (Seppic, France). The last group was left without vaccination as a control group. All animals were subsequently exposed to the infection induced by Actinobacillus pleuropneumoniae (App). The immune response was evaluated with respect to the intensity of systemic and mucosal antibody formation, their isotype characterisation and rate of cell-mediated immunity. These findings were compared with the intensity of adverse local reactions and level of protection in experimental challenge. Monitoring of the local reaction at the injection site after each administration showed that microemulsion adjuvant IMS 1313 was less reactogenic than the water-in-oil-in-water emulsion ISA 201. In terms of efficacy, both dermal administrations were less immunogenic than the i.m route. The i.m. injection induced higher anti-App9 IgG and IgM titres. Nevertheless, IgG1 and IgG2 isotypes analysis revealed a close immunological profile between i.m. and i.d. routes. The concentration of IFN-γ from peripheral blood after in vitro restimulation with the specific antigen was only increased in the i.m. group at the day of challenge (D35) and two weeks after (D49). Interestingly, the smallest gross pulmonary lesions were observed in the i.d. vaccinated group (3.4%) compared to the control group (39.4%) and to groups with other routes of administration. Taken together, these results suggest that i.d. administration of vaccines is a promising approach. Even the i.d. vaccine was more reactogenic and slightly less immunogenic than the i.m. vaccine, its protection effectiveness seemed to be superior.
Assuntos
Infecções por Actinobacillus , Actinobacillus pleuropneumoniae , Doenças dos Suínos , Suínos , Animais , Administração Cutânea , Emulsões , Imunização/veterinária , Imunização/métodos , Vacinação/métodos , Vacinação/veterinária , Adjuvantes Imunológicos , Imunoglobulina G , Imunidade , Infecções por Actinobacillus/prevenção & controle , Infecções por Actinobacillus/veterinária , Vacinas Bacterianas , Anticorpos Antibacterianos , Doenças dos Suínos/prevenção & controleRESUMO
Two-photon laser scanning microscopy (2PLSM) is an important tool for in vivo tissue imaging with sub-cellular resolution, but the penetration depth of current systems is potentially limited by sample-induced optical aberrations. To quantify these, we measured the refractive index n' in the somatosensory cortex of 7 rats in vivo using defocus optimization in full-field optical coherence tomography (ff-OCT). We found n' to be independent of imaging depth or rat age. From these measurements, we calculated that two-photon imaging beyond 200 µm into the cortex is limited by spherical aberration, indicating that adaptive optics will improve imaging depth.
Assuntos
Encéfalo/fisiologia , Microscopia Confocal/métodos , Fótons , Refratometria , Tomografia de Coerência Óptica/métodos , Envelhecimento/fisiologia , Animais , Vasos Sanguíneos/anatomia & histologia , Masculino , Ratos , Ratos Wistar , Gravação em VídeoRESUMO
In mammals, visceral adipose is increasingly seen as playing an important role in immune function with numerous pro-inflammatory, anti-inflammatory and immune-modulating proteins and peptides being identified in adipocytes. Adipose is also now known as a tissue that has an important role in the regulation of peritoneal immune responses. Despite this, only lately has consideration been given to visceral adipose as an important immune tissue in fish, especially in the context of intraperitoneal vaccination. The present study demonstrates that fish visceral adipose is capable of expressing a large range of immune molecules in response to stimulation with a live bacterium (A. salmonicida), a bacterial PAMP (Y. ruckeri flagellin), and the pro-inflammatory cytokines IL-1ß, TNF-α3 and IFN-γ. Following infection and stimulation with flagellin and IL-1ß a large upregulation of pro-inflammatory and antimicrobial molecules was seen, with a high degree of overlap. TNF-α treatment affected relatively few genes and the effects were more modest. IFN-γ had the smallest impact on adipose but IFN-γ inducible genes showed some of the largest effects. Overall, it is clear that adipose tissue should be considered an active immune site in fish, capable of participating in and influencing immune responses.
Assuntos
Tecido Adiposo/imunologia , Aeromonas salmonicida/imunologia , Infecções por Bactérias Gram-Negativas/imunologia , Oncorhynchus mykiss/imunologia , Peritônio/imunologia , Animais , Proteínas de Peixes/genética , Proteínas de Peixes/metabolismo , Flagelina/imunologia , Regulação da Expressão Gênica , Injeções Intraperitoneais , Interferon gama/genética , Interferon gama/metabolismo , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Moléculas com Motivos Associados a Patógenos/imunologia , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo , VacinaçãoRESUMO
To define the bottlenecks that restrict antigen expression after oral administration of viral-vectored vaccines, we tracked vectors derived from the human adenovirus type 5 at whole body, tissue, and cellular scales throughout the digestive tract in a murine model of oral delivery. After intragastric administration of vectors encoding firefly luciferase or a model antigen, detectable levels of transgene-encoded protein or mRNA were confined to the intestine, and restricted to delimited anatomical zones. Expression of luciferase in the form of multiple small bioluminescent foci in the distal ileum, cecum, and proximal colon suggested multiple crossing points. Many foci were unassociated with visible Peyer's patches, implying that transduced cells lay in proximity to villous rather than follicle-associated epithelium, as supported by detection of transgene-encoded antigen in villous epithelial cells. Transgene-encoded mRNA but not protein was readily detected in Peyer's patches, suggesting that post-transcriptional regulation of viral gene expression might limit expression of transgene-encoded antigen in this tissue. To characterize the pathways by which the vector crossed the intestinal epithelium and encountered sentinel cells, a fluorescent-labeled vector was administered to mice by the intragastric route or inoculated into ligated intestinal loops comprising a Peyer's patch. The vector adhered selectively to microfold cells in the follicle-associated epithelium, and, after translocation to the subepithelial dome region, was captured by phagocytes that expressed CD11c and lysozyme. In conclusion, although a large number of crossing events took place throughout the intestine within and without Peyer's patches, multiple firewalls prevented systemic dissemination of vector and suppressed production of transgene-encoded protein in Peyer's patches.
Assuntos
Adenovírus Humanos/genética , Adenovírus Humanos/imunologia , Vetores Genéticos/genética , Vetores Genéticos/imunologia , Transgenes/genética , Transgenes/imunologia , Administração Oral , Animais , Feminino , Expressão Gênica , Genes Reporter , Vetores Genéticos/administração & dosagem , Humanos , Imunização , Mucosa Intestinal/imunologia , Mucosa Intestinal/metabolismo , Camundongos , Especificidade de Órgãos , Nódulos Linfáticos Agregados/imunologia , Nódulos Linfáticos Agregados/metabolismo , Fagócitos/metabolismo , Transporte Proteico , VacinaçãoRESUMO
Vaccination is the most effective way to control swine influenza virus (SIV) in the field. Classical vaccines are based on inactivated antigens formulated with an oil emulsion or a polymeric adjuvant. Standard adjuvants enhance the humoral response and orient the immune response toward a Th2 response. An important issue is that current vaccines do not protect against new strains. One approach to improve cross-protection is to enhance Th1 and cytotoxic responses. The development of adjuvants orienting the immune response of inactivated vaccines toward Th1/Cytotoxic responses would be highly beneficial. This study shows that the water in oil in water emulsion adjuvant Montanide™ ISA 201 VG allows the induction of anti-influenza CD8 T cell in mice and induces homologous protection against an H1N1 challenge in swine. Such adjuvants that induce both humoral and cell-mediated immunity could improve the protection conferred by SIV vaccines in the field.
Assuntos
Adjuvantes Imunológicos/administração & dosagem , Linfócitos T CD8-Positivos/imunologia , Vacinas contra Influenza/administração & dosagem , Vacinas contra Influenza/imunologia , Infecções por Orthomyxoviridae/patologia , Infecções por Orthomyxoviridae/prevenção & controle , Animais , Modelos Animais de Doenças , Feminino , Vírus da Influenza A Subtipo H1N1/imunologia , Camundongos Endogâmicos C57BL , Suínos , Vacinas de Produtos Inativados/administração & dosagem , Vacinas de Produtos Inativados/imunologiaRESUMO
Chickens were immunized subcutaneously with an Eimeria recombinant profilin protein plus Montanide™ ISA 70 VG (ISA 70) or Montanide™ ISA 71 VG (ISA 71) water-in-oil adjuvants, or with profilin alone, and comparative RNA microarray hybridizations were performed to ascertain global transcriptome changes induced by profilin/ISA 70 vs. profilin alone and by profilin/ISA 71 vs. profilin alone. While immunization with profilin/ISA 70 vs. profilin alone altered the levels of more total transcripts compared with profilin/ISA 71 vs. profilin alone (509 vs. 296), the latter was associated with a greater number of unique biological functions, and a larger number of genes within these functions, compared with the former. Further, canonical pathway analysis identified 10 pathways that were associated with genes encoding the altered transcripts in animals immunized with profilin/ISA 71 vs. profilin alone, compared with only 2 pathways in profilin/ISA 70 vs. profilin alone. Therefore, ISA 71 was selected as a candidate adjuvant in conjunction with profilin vaccination for in vivo disease protection studies. Vaccination with profilin/ISA 71 was associated with greater body weight gain following E. acervulina infection, and decreased parasite fecal shedding after E. maxima infection, compared with profilin alone. Anti-profilin antibody levels were higher in sera of E. maxima- and E. tenella-infected chickens vaccinated with profilin/ISA 71 compared with profilin alone. Finally, the levels of transcripts encoding interferon-γ, interleukin (IL)-2, IL-10, and IL-17A were increased in intestinal lymphocytes from E. acervulina-, E. maxima-, and/or E. tenella-infected chickens vaccinated with profilin/ISA 71 compared with profilin alone. None of these effects were seen in chickens injected with ISA 71 alone indicating that the adjuvant was not conferring non-specific immune stimulation. These results suggest that profilin plus ISA 71 augments protective immunity against selective Eimeria species in chickens.
Assuntos
Coccidiose/prevenção & controle , Manitol/análogos & derivados , Ácidos Oleicos/farmacologia , Profilinas/imunologia , Vacinação/métodos , Animais , Peso Corporal/efeitos dos fármacos , Peso Corporal/imunologia , Galinhas , Coccidiose/genética , Coccidiose/imunologia , Citocinas/genética , Intestinos/imunologia , Linfócitos/efeitos dos fármacos , Linfócitos/imunologia , Linfócitos/metabolismo , Manitol/farmacologia , Oocistos/efeitos dos fármacos , Oocistos/imunologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Baço/imunologia , Transcriptoma/efeitos dos fármacos , Transcriptoma/imunologiaRESUMO
Myelin sheath disruption is responsible for multiple neuropathies in the central and peripheral nervous system. Myelin imaging has thus become an important diagnosis tool. However, in vivo imaging has been limited to either low-resolution techniques unable to resolve individual fibers or to low-penetration imaging of single fibers, which cannot provide quantitative information about large volumes of tissue, as required for diagnostic purposes. Here, we perform myelin imaging without labeling and at micron-scale resolution with >300-µm penetration depth on living rodents. This was achieved with a prototype [termed deep optical coherence microscopy (deep-OCM)] of a high-numerical aperture infrared full-field optical coherence microscope, which includes aberration correction for the compensation of refractive index mismatch and high-frame-rate interferometric measurements. We were able to measure the density of individual myelinated fibers in the rat cortex over a large volume of gray matter. In the peripheral nervous system, deep-OCM allows, after minor surgery, in situ imaging of single myelinated fibers over a large fraction of the sciatic nerve. This allows quantitative comparison of normal and Krox20 mutant mice, in which myelination in the peripheral nervous system is impaired. This opens promising perspectives for myelin chronic imaging in demyelinating diseases and for minimally invasive medical diagnosis.
Assuntos
Imagem Molecular/métodos , Bainha de Mielina/química , Tomografia de Coerência Óptica/métodos , Animais , Córtex Cerebral/química , Córtex Cerebral/ultraestrutura , Proteína 2 de Resposta de Crescimento Precoce/genética , Feminino , Masculino , Camundongos , Camundongos Knockout , Microscopia , Bainha de Mielina/ultraestrutura , Ratos , Ratos Wistar , Nervo Isquiático/química , Nervo Isquiático/ultraestruturaRESUMO
In order to understand how neuronal circuits control locomotory patterns it is necessary to record neuronal activity of freely behaving animals. Here, using a new automated system for simultaneous recording of behavior and neuronal activity in freely moving Caenorhabditis elegans on standard agar plates, we show that spontaneous reversals from forward to backward locomotion reflect precisely the activity of the AVA command interneurons. We also witness spontaneous activity transients in the PLM sensory neurons during free behavior of the worm in standard conditions. We show that these activity transients are coupled to short spontaneous forward accelerations of the worm.
Assuntos
Caenorhabditis elegans/fisiologia , Locomoção/fisiologia , Neurônios/fisiologia , Animais , Caenorhabditis elegans/anatomia & histologia , Cálcio/metabolismo , Sinalização do Cálcio/fisiologia , Processamento de Imagem Assistida por Computador , Interneurônios/fisiologia , Técnicas Analíticas Microfluídicas , Microscopia de Fluorescência , Mutação/fisiologia , Rede Nervosa/fisiologia , Células Receptoras Sensoriais/fisiologiaRESUMO
Most animals display multiple behavioral states and control the time allocation to each of their activity phases depending on their environment. Here we develop a new quantitative method to analyze Caenorhabditis elegans behavioral states. We show that the dwelling and roaming two-state behavior of C. elegans is tightly controlled by the concentration of food in the environment of the animal. Sensory perception through the amphid neurons is necessary to extend roaming phases while internal metabolic perception of food nutritional value is needed to induce dwelling. Our analysis also shows that the proportion of time spent in each state is modulated by past nutritional experiences of the animal. This two-state behavior is regulated through serotonin as well as insulin and TGF-beta signaling pathways. We propose a model where food nutritional value is assessed through internal metabolic signaling. Biogenic amines signaling could allow the worm to adapt to fast changes in the environment when peptide transcriptional pathways may mediate slower adaptive changes.