RESUMO
Influenza viruses constitute a major threat to human health globally. The viral surface glycoprotein hemagglutinin (HA) is the immunodominant antigen, contains the site for binding to the cellular receptor (RBS), and it is the major target of neutralizing antibody responses post-infection. We developed llama-derived single chain antibody fragments (VHHs) specific for type A influenza virus. Four VHHs were identified and further characterized. VHH D81 bound residues in the proximity of the C-terminal region of HA1 of H1 and H5 subtypes, and showed weak neutralizing activity, whereas VHH B33 bound residues in the proximity of the N-terminal region of the HA's stem domain (HA2) of H1, H5, and H9 subtypes, and showed no neutralizing activity. Of most relevance, VHHs E13 and G41 recognized highly conserved conformational epitopes on the H1 HA's globular domain (HA1) and showed high virus neutralizing activity (ranging between 0.94 to 0.01µM), when tested against several human H1N1 isolates. Additionally, E13 displayed abrogated virus replication of a panel of H1N1 strains spanning over 80 years of antigenic drift and isolated from human, avian, and swine origin. Interestingly, E13 conferred protection in vivo at a dose as low as 0.05 mg/kg. Mice treated with E13 intranasally resulted in undetectable virus challenge loads in the lungs at day 4 post-challenge. The transfer of sterilizing pan-H1 immunity, by a dose in the range of micrograms given intranasally, is of major significance for a monomeric VHH and supports the further development of E13 as an immunotherapeutic agent for the mitigation of influenza infections.
Assuntos
Anticorpos Neutralizantes , Camelídeos Americanos , Glicoproteínas de Hemaglutininação de Vírus da Influenza , Vírus da Influenza A Subtipo H1N1 , Infecções por Orthomyxoviridae , Anticorpos de Domínio Único , Animais , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Humanos , Vírus da Influenza A Subtipo H1N1/imunologia , Anticorpos de Domínio Único/imunologia , Anticorpos Neutralizantes/imunologia , Camundongos , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/prevenção & controle , Infecções por Orthomyxoviridae/virologia , Camelídeos Americanos/imunologia , Anticorpos Antivirais/imunologia , Feminino , Influenza Humana/imunologia , Influenza Humana/prevenção & controle , Influenza Humana/virologia , Epitopos/imunologia , Cães , Camundongos Endogâmicos BALB CRESUMO
The specific roles of Notch in progressive adulthood neurodegenerative disorders have begun to be unraveled in recent years. A number of independent studies have shown significant increases of Notch expression in brains from patients at later stages of sporadic Alzheimer's disease (AD). However, the impact of Notch canonical signaling activation in the pathophysiology of AD is still elusive. To further investigate this issue, 2-month-old wild-type (WT) and hemizygous McGill-R-Thy1-APP rats (Tg(+/-)) were injected in CA1 with lentiviral particles (LVP) expressing the transcriptionally active fragment of Notch, known as Notch Intracellular Domain (NICD), (LVP-NICD), or control lentivirus particles (LVP-C). The Tg(+/-) rat model captures presymptomatic aspects of the AD pathology, including intraneuronal amyloid beta (Aß) accumulation and early cognitive deficits. Seven months after LVP administration, Morris water maze test was performed, and brains isolated for biochemical and histological analysis. Our results showed a learning impairment and a worsening of spatial memory in LVP-NICD- as compared to LVP-C-injected Tg(+/-) rats. In addition, immuno histochemistry, ELISA multiplex, Western blot, RT-qPCR, and 1H-NMR spectrometry of cerebrospinal fluid (CSF) indicated that chronic expression of NICD promoted hippocampal vessel thickening with accumulation of Aß in brain microvasculature, alteration of blood-brain barrier (BBB) permeability, and a decrease of CSF glucose levels. These findings suggest that, in the presence of early Aß pathology, expression of NICD may contribute to the development of microvascular abnormalities, altering glucose transport at the BBB with impact on early decline of spatial learning and memory.
Assuntos
Doença de Alzheimer/patologia , Vasos Sanguíneos/patologia , Glucose/metabolismo , Hipocampo/metabolismo , Transtornos da Memória/patologia , Receptores Notch/química , Receptores Notch/metabolismo , Memória Espacial , Doença de Alzheimer/líquido cefalorraquidiano , Doença de Alzheimer/complicações , Doença de Alzheimer/fisiopatologia , Animais , Transporte Biológico , Barreira Hematoencefálica/metabolismo , Barreira Hematoencefálica/patologia , Região CA1 Hipocampal/metabolismo , Região CA1 Hipocampal/patologia , Modelos Animais de Doenças , Vetores Genéticos/metabolismo , Células HEK293 , Hipocampo/patologia , Hipocampo/fisiopatologia , Humanos , Inflamação/patologia , Lentivirus/genética , Transtornos da Memória/complicações , Transtornos da Memória/fisiopatologia , Microvasos/patologia , Domínios Proteicos , Espectroscopia de Prótons por Ressonância Magnética , Ratos Transgênicos , Ratos WistarRESUMO
When disrupted, iron homeostasis negatively impacts oligodendrocyte (OLG) differentiation and impairs myelination. To better understand myelin formation and OLG maturation, in vivo and in vitro studies were conducted to evaluate the effect of iron deficiency (ID) not only on OLG maturation but also on astrocytes (AST) and microglial cells (MG). In vivo experiments in an ID model were carried out to describe maturational events during OLG and AST development and the reactive profile of MG during myelination when iron availability is lower than normal. In turn, in vitro assays were conducted to explore proliferating and maturational states of each glial cell type derived from control or ID conditions. Studies targeted NG2, PDGFRα, CNPAse, CC1, and MBP expression in OLG, GFAP and S100 expression in AST, and CD11b, ED1, and cytokine expression in MG, as well as BrDU incorporation in the three cell types. Our results show that ID affected OLG development at early stages, not only reducing their maturation capacity but also increasing their proliferation and affecting their morphological complexity. AST ID proliferated more than control ones and were more immature, much like OLG. Cytokine expression in ID animals reflected an anti-inflammatory state which probably influenced OLG maturation. These results show that ID conditions alter all glial cells and may impact myelin formation, which could be regulated by a mechanism involving a cross talk between AST, MG, and oligodendrocyte progenitors (OPC).
Assuntos
Anemia Ferropriva/metabolismo , Astrócitos/metabolismo , Ferro/metabolismo , Microglia/metabolismo , Oligodendroglia/metabolismo , Animais , Encéfalo/metabolismo , Diferenciação Celular/fisiologia , Homeostase/fisiologia , Bainha de Mielina/metabolismo , Ratos , Ratos WistarRESUMO
Transforming growth factor beta 1 (TGF-beta1), an anti-inflammatory cytokine, has been shown to have pro-neurogenic effects on adult Neural Stem Cells (aNSC) from the dentate gyrus and in vivo models. Here, we expanded the observation of the pro-neurogenic effect of TGF-beta1 on aNSC from the subventricular zone (SVZ) of adult rats and performed a functional genomic analysis to identify candidate genes to mediate its effect. 10 candidate genes were identified by microarray analysis and further validated by qRT-PCR. Of these, Fibulin-2 was increased 477-fold and its inhibition by siRNA blocks TGF-beta1 pro-neurogenic effect. Curiously, Fibulin-2 was not expressed by aNSC but by a GFAP-positive population in the culture, suggesting an indirect mechanism of action. TGF-beta1 also induced Fibulin-2 in the SVZ in vivo. Interestingly, 5 out of the 10 candidate genes identified are known to interact with integrins, paving the way for exploring their functional role in adult neurogenesis. In conclusion, we have identified 10 genes with putative pro-neurogenic effects, 5 of them related to integrins and provided proof that Fibulin-2 is a major mediator of the pro-neurogenic effects of TGF-beta1. These data should contribute to further exploring the molecular mechanism of adult neurogenesis of the genes identified and the involvement of the integrin pathway on adult neurogenesis.
Assuntos
Células-Tronco Adultas/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Células-Tronco Neurais/metabolismo , Neurogênese , Fator de Crescimento Transformador beta1/farmacologia , Células-Tronco Adultas/citologia , Células-Tronco Adultas/efeitos dos fármacos , Animais , Astrócitos/metabolismo , Proteínas de Ligação ao Cálcio/genética , Células Cultivadas , Proteínas da Matriz Extracelular/genética , Integrinas/metabolismo , Ventrículos Laterais/citologia , Ventrículos Laterais/crescimento & desenvolvimento , Ventrículos Laterais/metabolismo , Células-Tronco Neurais/citologia , Células-Tronco Neurais/efeitos dos fármacos , Ratos , Ratos Wistar , Fator de Crescimento Transformador beta1/metabolismoRESUMO
BACKGROUND: Bovine herpesvirus type 1 (BoHV-1) is the causative agent of respiratory and genital tract infections; causing a high economic loss in all continents. Use of marker vaccines in IBR eradication programs is widely accepted since it allows for protection of the animals against the disease while adding the possibility of differentiating vaccinated from infected animals.The aim of the present study was the development and evaluation of safety and efficacy of a glycoprotein E-deleted (gE-) BoHV-1 marker vaccine strain (BoHV-1ΔgEßgal) generated by homologous recombination, replacing the viral gE gene with the ß-galactosidase (ßgal) gene. RESULTS: In vitro growth kinetics of the BoHV-1ΔgEßgal virus was similar to BoHV-1 LA. The immune response triggered by the new recombinant strain in cattle was characterized both as live attenuated vaccine (LAV) and as an inactivated vaccine. BoHV-1ΔgEßgal was highly immunogenic in both formulations, inducing specific humoral and cellular immune responses. Antibody titers found in animals vaccinated with the inactivated vaccine based on BoHV-1ΔgEßgal was similar to the titers found for the control vaccine (BoHV-1 LA). In the same way, titers of inactivated vaccine groups were significantly higher than any of the LAV immunized groups, independently of the inoculation route (p < 0.001). Levels of IFN-γ were significantly higher (p < 0.001) in those animals that received the LAV compared to those that received the inactivated vaccine. BoHV-1ΔgEßgal exhibited an evident attenuation when administered as a LAV; no virus was detected in nasal secretions of vaccinated or sentinel animals during the post-vaccination period. BoHV-1ΔgEßgal, when used in either formulation, elicited an efficient immune response that protected animals against challenge with virulent wild-type BoHV-1. Also, the deletion of the gE gene served as an immunological marker to differentiate vaccinated animals from infected animals. All animals vaccinated with the BoHV-1ΔgE ßgal strain were protected against disease after challenge and shed significantly less virus than control calves, regardless of the route and formulation they were inoculated. CONCLUSIONS: Based on its attenuation, immunogenicity and protective effect after challenge, BoHV-1ΔgEßgal virus is an efficient and safe vaccine candidate when used either as inactivated or as live attenuated forms.
Assuntos
Infecções por Herpesviridae/veterinária , Herpesvirus Bovino 1/metabolismo , Proteínas Virais/metabolismo , Vacinas Virais/imunologia , Animais , Bovinos , Linhagem Celular , Cães , Feminino , Deleção de Genes , Regulação Viral da Expressão Gênica/fisiologia , Infecções por Herpesviridae/prevenção & controle , Infecções por Herpesviridae/virologia , Herpesvirus Bovino 1/genética , Herpesvirus Bovino 1/imunologia , Gravidez , Complicações Infecciosas na Gravidez/prevenção & controle , Complicações Infecciosas na Gravidez/veterinária , Complicações Infecciosas na Gravidez/virologia , Vacinas Atenuadas , Vacinas de Produtos Inativados , Proteínas Virais/genética , Vacinas Virais/efeitos adversosRESUMO
Neuroinflammation has received increased attention as a target for putative neuroprotective therapies in Parkinson's Disease (PD). Two prototypic pro-inflammatory cytokines interleukin-1ß (IL-1) and tumor necrosis factor-α (TNF) have been implicated as main effectors of the functional consequences of neuroinflammation on neurodegeneration in PD models. In this review, we describe that the functional interaction between these cytokines in the brain differs from the periphery (e.g., their expression is not induced by each other) and present data showing predominantly a toxic effect of these cytokines when expressed at high doses and for a sustained period of time in the substantia nigra pars compacta (SN). In addition, we highlight opposite evidence showing protective effects of these two main cytokines when conditions of duration, amount of expression or state of activation of the target or neighboring cells are changed. Furthermore, we discuss these results in the frame of previous disappointing results from anti-TNF-α clinical trials against Multiple Sclerosis, another neurodegenerative disease with a clear neuroinflammatory component. In conclusion, we hypothesize that the available evidence suggests that the duration and dose of IL-1ß or TNF-α expression is crucial to predict their functional effect on the SN. Since these parameters are not amenable for measurement in the SN of PD patients, we call for an in-depth analysis to identify downstream mediators that could be common to the toxic (and not the protective) effects of these cytokines in the SN. This strategy could spare the possible neuroprotective effect of these cytokines operative in the patient at the time of treatment, increasing the probability of efficacy in a clinical setting. Alternatively, receptor-specific agonists or antagonists could also provide a way to circumvent undesired effects of general anti-inflammatory or specific anti-IL-1ß or TNF-α therapies against PD.
RESUMO
Pituitary tumors are the most common primary intracranial neoplasms. Although most pituitary tumors are considered typically benign, others can cause severe and progressive disease. The principal aims of pituitary tumor treatment are the elimination or reduction of the tumor mass, normalization of hormone secretion and preservation of remaining pituitary function. In spite of major advances in the therapy of pituitary tumors, for some of the most difficult tumors, current therapies that include medical, surgical and radiotherapeutic methods are often unsatisfactory and there is a need to develop new treatment strategies. Gene therapy, which uses nucleic acids as drugs, has emerged as an attractive therapeutic option for the treatment of pituitary tumors that do not respond to classical treatment strategies if the patients become intolerant to the therapy. The development of animal models for pituitary tumors and hormone hypersecretion has proven to be critical for the implementation of novel treatment strategies and gene therapy approaches. Preclinical trials using several gene therapy approaches for the treatment of anterior pituitary diseases have been successfully implemented. Several issues need to be addressed before clinical implementation becomes a reality, including the development of more effective and safer viral vectors, uncovering novel therapeutic targets and development of targeted expression of therapeutic transgenes. With the development of efficient gene delivery vectors allowing long-term transgene expression with minimal toxicity, gene therapy will become one of the most promising approaches for treating pituitary adenomas.