RESUMO
Stem cell therapy in cat patients needs a high quantity of mesenchymal stromal/stem cells (MSCs) requiring in vitro propagation under culture conditions which may potentially impact cellular characteristics and genetic stability. This study aimed to assess the in vitro characteristics and cytogenetic stability of cat adipose tissue-derived MSCs (cAT-MSCs). For this purpose, morphological features, clonogenic potential, and proliferative capacity of cAT-MSCs were assessed at passages 2 (P2), P4, and P6. Multipotency and immunophenotype were evaluated. Cytogenetic analyses were conducted up to P6. The cAT-MSCs exhibited a spindle-shaped morphology in early passages. The doubling time increased from 2.5 days at P2 to 9.4 at P4 and 10.5 at P6, accompanied by the observation of nuclear abnormalities such as cluster formation, karyorrhexis, karyolysis, and a decline in the mitotic index at P4. Cells demonstrated multipotency capacity and were CD45-, CD90+, and CD44+. Metaphase analysis at P2 and P4 revealed some indications of structural instability such as gaps, breaks, deletions, duplications, and early chromatid segregation, but these alterations did not show an increase across passages. In conclusion, cAT-MSCs decreased their proliferative capacity after P4, accompanied by morphological alterations and signs of structural instability.
RESUMO
Dogs are the main source of animal and human cystic echinococcosis caused by the Cestode parasite Echinococcus granulosus. Dog vaccination seems to be a good strategy to control this parasitic disease. Here we present the development of a polymeric nanoparticle-based oral vaccine for dogs against Echinococcus granulosus delivered in enteric-coated capsules. To achieve our target, we encapsulated two recombinant antigens into biodegradable polymeric nanoparticles in the presence of Monophosphoryl lipid A as an adjuvant to ensure efficient delivery and activation of a protective mucosal immune response. The formulated delivery system showed a nanoparticle size less than 200 nm with more than 80 % antigen encapsulation efficiency and conserved integrity and immunogenicity. The nanoparticle surface was coated with chitosan to enhance adhesion to the gut mucosa and a subsequent antigen delivery. Chitosan-coated nanoparticles showed a higher cell internalization in murine macrophages and dendritic cells as well as a higher penetration into Caco-2 cells in vitro. Antigen-loaded nanoparticles were freeze-dried and enteric-coated capsules were filled with the obtained powder. The obtained results show a promising nanoparticles delivery system for oral vaccination.
Assuntos
Quitosana , Equinococose , Echinococcus granulosus , Vacinas , Cães , Humanos , Animais , Camundongos , Echinococcus granulosus/fisiologia , Células CACO-2 , Equinococose/prevenção & controle , Equinococose/parasitologia , AntígenosRESUMO
OBJECTIVES: The aim of this study was to isolate feline dental pulp stem cells (fDPSCs) and characterize their clonogenic and proliferative abilities, as well as their multipotency, immunophenotype and cytogenetic stability. METHODS: Dental pulp was isolated by explant culture from two cats <1 year old at post mortem. Their clonogenicity was characterized using a colony-forming unit fibroblast assay, and their proliferative ability was quantified with a doubling time assay in passages 2, 4 and 6 (P2, P4 and P6, respectively). Multipotency was characterized with an in vitro trilineage differentiation assay in P2, and cells were immunophenotyped in P4 by flow cytometry. Chromosomic stability was evaluated by cytogenetic analysis in P2, P4 and P6. RESULTS: The fDPSCs displayed spindle and epithelial-like morphologies. Isolated cells showed a marked clonogenic capacity and doubling time was maintained from P2 to P6. Trilineage differentiation was obtained in one sample, while the other showed osteogenic and chondrogenic differentiation. Immunophenotypic analysis showed fDPSCs were CD45-, CD90+ and CD44+. Structural and numerical cytogenetic aberrations were observed in P2-P4. CONCLUSIONS AND RELEVANCE: In this study, fDPSCs from two cats were isolated by explant culture and immunophenotyped. Cells displayed clonogenic and proliferative ability, and multipotency in vitro, and signs of chromosomic instability were observed. Although a larger study is needed to confirm these results, this is the first report of fDPSC isolation and in vitro characterization.
Assuntos
Polpa Dentária , Células-Tronco , Gatos , Animais , Diferenciação Celular , Citometria de Fluxo/veterinária , Células Cultivadas , Proliferação de CélulasRESUMO
Since dogs play a central role in the contamination of humans and livestock with Echinococcus granulosus, the development of an effective vaccine for dogs is essential to control the disease caused by this parasite. For this purpose, a formulation based on biodegradable polymeric nanoparticles (NPs) as delivery system of recombinant Echinococcus granulosus antigen (tropomyosin EgTrp) adjuved with monophosphoryl lipid A (MPLA) has been developed. The obtained nanoparticles had a size of approximately 200 nm in diameter into which the antigen was correctly preserved and encapsulated. The efficiency of this system to deliver the antigen was evaluated in vitro on canine monocyte-derived dendritic cells (cMoDCs) generated from peripheral blood monocytes. After 48 h of contact between the formulations and cMoDCs, we observed no toxic effect on the cells but a strong internalization of the NPs, probably through different pathways depending on the presence or not of MPLA. An evaluation of cMoDCs activation by flow cytometry showed a stronger expression of CD80, CD86, CD40 and MHCII by cells treated with any of the tested formulations or with LPS (positive control) in comparison to cells treated with PBS (negative control). A higher activation was observed for cells challenged with EgTrp-NPs-MPLA compared to EgTrp alone. Formulations with MPLA, even at low ratio of MPLA, give better results than formulations without MPLA, proving the importance of the adjuvant in the nanoparticles structure. Moreover, autologous T CD4+ cell proliferation observed in presence of cMoDCs challenged with EgTrp-NPs-MPLA was higher than those observed after challenged with EgTrp alone (p<0.05). These first results suggest that our formulation could be used as an antigen delivery system to targeting canine dendritic cells in the course of Echinococcus granulosus vaccine development.
Assuntos
Antígenos de Protozoários/administração & dosagem , Células Dendríticas/imunologia , Cães/parasitologia , Equinococose/prevenção & controle , Echinococcus granulosus/imunologia , Vacinas Protozoárias/administração & dosagem , Adjuvantes Imunológicos/administração & dosagem , Animais , Antígenos de Protozoários/genética , Antígenos de Protozoários/imunologia , Linfócitos T CD4-Positivos/imunologia , Diferenciação Celular , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Células Dendríticas/efeitos dos fármacos , Cães/sangue , Cães/imunologia , Portadores de Fármacos/química , Portadores de Fármacos/toxicidade , Equinococose/imunologia , Equinococose/parasitologia , Equinococose/veterinária , Echinococcus granulosus/genética , Imunogenicidade da Vacina , Lipídeo A/análogos & derivados , Lipídeo A/química , Lipídeo A/toxicidade , Ativação Linfocitária/imunologia , Monócitos/fisiologia , Nanopartículas/química , Nanopartículas/toxicidade , Poliésteres/química , Poliésteres/toxicidade , Cultura Primária de Células , Vacinas Protozoárias/genética , Vacinas Protozoárias/imunologia , Testes de Toxicidade Aguda , Tropomiosina/administração & dosagem , Tropomiosina/genética , Tropomiosina/imunologia , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologiaRESUMO
Rhipicephalus microplus is a cattle-specific tick, causing considerable losses in the livestock industry. The identification of molecules responsible for modulation of host defenses during different parasite stages can help in the development of alternative methods, such as vaccination, to control tick infestations. Hq05, a protein of unknown function identified in the tick Haemaphysalis qinghaiensis, induced a significant protective immune response when used as a vaccine in sheep. In the present study, we investigated Bm05br, the Hq05 homologous gene from R. microplus. Besides H. qinghaiensis, Bm05br homologous found in other tick species such as Rhipicephalus annulatus, Rhipicephalus sanguineus sensu lato, Haemaphysalis longicornis and Ixodes scapularis were comparatively analyzed. Bm05br expression profile in different R. microplus tissues and life-stages was determined by qRT-PCR and Western blot. Bm05br was detected in ovaries, salivary glands and the fat body of both partially and fully engorged females. The highest transcription levels were observed in partially engorged females fat body and salivary glands. Gene knockdown by RNAi reduced egg hatching rate and the weight of tick larvae obtained from treated group, when compared to controls. These results indicate that Bm05br may be involved in R. microplus reproduction. Together with its distribution and high sequence conservation across different tick species, our data suggest Bm05br as a potential antigen for development of a multispecies anti-tick vaccine.
Assuntos
Antígenos/genética , Antígenos/imunologia , Rhipicephalus/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Artrópodes/química , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/metabolismo , Clonagem Molecular , Regulação da Expressão Gênica , Estágios do Ciclo de Vida , Interferência de RNA , Coelhos , Proteínas Recombinantes , Especificidade da EspécieRESUMO
Fasciola hepatica is the agent of fasciolosis, a foodborne zoonosis that affects livestock production and human health. Although flukicidal drugs are available, re-infection and expanding resistance to triclabendazole demand new control strategies. Understanding the molecular mechanisms underlying the complex interaction with the mammalian host could provide relevant clues, aiding the search for novel targets in diagnosis and control of fasciolosis. Parasite survival in the mammalian host is mediated by parasite compounds released during infection, known as excretory/secretory (E/S) products. E/S products are thought to protect parasites from host responses, allowing them to survive for a long period in the vertebrate host. This work provides in-depth proteomic analysis of F. hepatica intra-mammalian stages, and represents the largest number of proteins identified to date for this species. Functional classification revealed the presence of proteins involved in different biological processes, many of which represent original findings for this organism and are important for parasite survival within the host. These results could lead to a better comprehension of host-parasite relationships, and contribute to the development of drugs or vaccines against this parasite.
Assuntos
Fasciola hepatica/crescimento & desenvolvimento , Proteínas de Helminto/metabolismo , Fígado/parasitologia , Proteômica/métodos , Animais , Fasciola hepatica/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Redes Reguladoras de Genes , Interações Hospedeiro-ParasitaRESUMO
Various classes of endopeptidases and their inhibitors facilitate blood feeding and digestion in ticks. Cystatins, a family of tight-binding and reversible inhibitors of cysteine endopeptidases, have recently been found in several tick tissues. Moreover, vaccine trials using tick cystatins have been found to induce protective immune responses against tick infestation. However, the mode of action of tick cystatins is still poorly understood, limiting the elucidation of their physiological role. Against this background, we have investigated sequence characteristics and immunogenic properties of 5 putative cystatins from Rhipicephalus (Boophilus) microplus from Brazil and Uruguay. The similarity of the deduced amino acid sequences among cystatins from the Brazilian tick strain was 27-42%, all of which had a secretory signal peptide. The cystatin motif (QxVxG), a glycine in the N-terminal region, and the PW motif in the second hairpin loop in the C-terminal region are highly conserved in all 5 cystatins identified in this study. Four cysteine residues in the C terminus characteristic of type 2 cystatins are also present. qRT-PCR revealed differential expression patterns among the 5 cystatins identified, as well as variation in mRNA transcripts present in egg, larva, gut, salivary glands, ovary, and fat body tissues. One R. microplus cystatin showed 97-100% amino acid similarity between Brazilian and Uruguayan isolates. Furthermore, by in silico analysis, antigenic amino acid regions from R. microplus cystatins showed high degrees of homology (54-92%) among Rhipicephalus spp. cystatins. Three Brazilian R. microplus cystatins were expressed in Escherichia coli, and immunogenicity of the recombinant proteins were determined by vaccinating mice. Western blotting using mice sera indicated cross-reactivity between the cystatins, suggesting shared epitopes. The present characterization of Rhipicephalus spp. cystatins represents an empirical approach in an effort to evaluate the physiological role of cystatins in a larger context of targeting them for use in future tick control strategies.
Assuntos
Doenças dos Bovinos/parasitologia , Cistatinas/química , Cistatinas/imunologia , Rhipicephalus/imunologia , Infestações por Carrapato/veterinária , Sequência de Aminoácidos , Animais , Brasil , Bovinos , Doenças dos Bovinos/imunologia , Biologia Computacional , Cistatinas/genética , DNA Complementar/química , DNA Complementar/genética , Feminino , Expressão Gênica , Camundongos , Dados de Sequência Molecular , Filogenia , RNA Mensageiro/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Rhipicephalus/química , Rhipicephalus/genética , Alinhamento de Sequência , Análise de Sequência de DNA , Infestações por Carrapato/imunologia , Infestações por Carrapato/parasitologiaRESUMO
BACKGROUND: One form of sepsis, or endotoxic shock, is a hyperactivated systemic response caused by excessive expression of proinflammatory mediators, which results from Gram-negative bacterial lipopolysaccharide-stimulated Toll-like receptor-4 signaling. This lipopolysaccharide signaling is known to consist of a MyD88-dependent nuclear factor-κB-mediated pathway that results in production of proinflammatory mediators (tumor necrosis factor-α, interleukin-6, intercellular adhesion molecule-1, vascular cell adhesion molecule-1, inducible nitric oxide synthase, cyclooxygenase-2) and a MyD88-independent interferon regulatory factor-mediated pathway that regulates production of Type 1 interferon-inducible proteins (interferon γ-induced protein-10, monocyte chemotactic protein-1). In prior studies, phenylmethimazole markedly decreased virally induced Toll-like receptor-3 expression and signaling and significantly suppressed murine colitis in an experimental model wherein lipopolysaccharide is known to play an important role. OBJECTIVE: In this study, we probed the hypothesis that phenylmethimazole inhibits lipopolysaccharide-mediated Toll-like receptor-4 signaling and is efficacious in attenuating inflammatory changes and improving survival in an in vivo murine model of endotoxic shock. DESIGN: Experimental animal model. SETTING: University laboratory. SUBJECTS: Male C57BL/6J mice weighing 18-22 g. INTERVENTIONS: Phenylmethimazole (1 mg/kg) was administered intraperitoneally to mice before a lethal lipopolysaccharide challenge (25 mg/kg). RAW264.7 mouse macrophage cells were pretreated with phenylmethimazole followed by lipopolysaccharide stimulation. MEASUREMENTS AND MAIN RESULTS: : Macroscopic observations revealed that phenylmethimazole was significantly protective in controlling clinical manifestations of endotoxic shock and death under conditions wherein flunixin of meglumine and prednisolone were marginally effective. A combination of enzyme-linked immunosorbent assay, Northern blot, reverse transcriptase-polymerase chain reaction, immunohistochemistry, and Western blot analyses showed that phenylmethimazole attenuated lipopolysaccharide-induced increases in production of proinflammatory cytokines (tumor necrosis factor-α, interleukin-6, interferon-γ), endothelial cell adhesion molecules (intercellular adhesion molecule-1, vascular cell adhesion molecule-1), inducible nitric oxide synthase and cyclooxygenase-2, interferon regulatory factor-1, interferon-inducible proteins (interferon γ-induced protein-10, monocyte chemotactic protein-1), and signal transducer and activator of transcription-1 phosphorylation in multiple tissues in mice. Consistent with these observations, electrophoretic mobility shift assay demonstrated that phenylmethimazole inhibited in vitro lipopolysaccharide-induced nuclear factor-κB and interferon regulatory factor-1 activation in RAW 264.7 mouse macrophages. CONCLUSIONS: Collectively, these results provide direct evidence that phenylmethimazole diminishes lipopolysaccharide-induced MyD88-dependent as well as MyD88-independent signaling pathways and is protective in an experimental model of endotoxic shock.
Assuntos
Citocinas/biossíntese , Citocinas/efeitos dos fármacos , Metimazol/análogos & derivados , Choque Séptico/imunologia , Choque Séptico/prevenção & controle , Tionas/uso terapêutico , Animais , Modelos Animais de Doenças , Inflamação/imunologia , Masculino , Metimazol/uso terapêutico , Camundongos , Camundongos Endogâmicos C57BL , Choque Séptico/metabolismoRESUMO
Ulcerative colitis is an autoimmune-inflammatory disease characterized by abnormally increased expression of Toll-like receptor-4 (TLR4) in colonic epithelial cells, increased production of pro-inflammatory cytokines (e.g., TNF-alpha, IL-1beta, IL-6, IL-12), chemokines (e.g., IP-10), and endothelial cell adhesion molecules (e.g., VCAM-1), plus enhanced leukocyte infiltration into colonic interstitium. Previously, we have shown that phenyl methimazole (C10) markedly decreases virally-induced TLR-3 expression and signaling and potently inhibits both TNF-alpha-induced VCAM-1 expression and the resultant leukocyte-endothelial cell adhesion. In this study we probed the hypothesis that C10 is efficacious in a TLR-4- and VCAM-1-associated murine model [the dextran sulfate sodium (DSS) model] of human colitis. C10 was administered intraperitoneally coincident with or after DSS treatment was initiated. Macroscopic colon observations revealed that C10 significantly reversed DSS-induced shortening of the colon (P<0.05) and reduced the presence of blood in the colon. Histological analyses of colonic tissues revealed that C10 distinctly attenuated both DSS-induced edema as well as leukocyte infiltration in the colonic mucosa and resulted in pronounced protection against DSS-induced crypt damage (P<0.001). Northern blot analyses and immunohistochemistry of colonic tissue revealed that C10 markedly diminished DSS-induced expression of pertinent inflammatory mediators: TNF-alpha, IL-1beta, IL-6, IL-12, IP-10, TLR-4 and VCAM-1. Most importantly, C10 significantly improved survival and protected mice against DSS-induced colitic-death: 75% by comparison to 12.5% with identical treatment with DMSO-control (log rank test: P=0.005). These results provide direct evidence that C10 suppresses DSS-induced colitis by inhibiting expression of key inflammatory mediators and leukocyte infiltration, and is a potentially attractive therapeutic for colitis.
Assuntos
Colite Ulcerativa/prevenção & controle , Metimazol/análogos & derivados , Tionas/uso terapêutico , Receptor 4 Toll-Like/antagonistas & inibidores , Molécula 1 de Adesão de Célula Vascular/imunologia , Animais , Northern Blotting , Colite Ulcerativa/imunologia , Colite Ulcerativa/metabolismo , Colite Ulcerativa/patologia , Citocinas/biossíntese , Citocinas/imunologia , Sulfato de Dextrana , Modelos Animais de Doenças , Imuno-Histoquímica , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/imunologia , Mucosa Intestinal/patologia , Masculino , Metimazol/farmacologia , Metimazol/uso terapêutico , Camundongos , Camundongos Endogâmicos C57BL , Tionas/farmacologia , Receptor 4 Toll-Like/biossíntese , Molécula 1 de Adesão de Célula Vascular/biossínteseRESUMO
The expression of certain endothelial cell adhesion molecules (ECAMs) is increased in the vasculature of the inflamed bowel (e.g., colitis), thereby providing an opportunity for targeted drug delivery. We recently demonstrated that biodegradable particles conjugated with ligands to ECAMs exhibit significant selective adhesion to ECAM expressing endothelium. In the present study, we used a murine model of colitis to determine whether poly(lactic acid)-poly(ethylene glycol) particles conjugated with a VCAM-1 ligand (alpha-V) exhibit enhanced adhesion to colitic vasculature. In post-capillary venules of the colon, significantly more alpha-V particles accumulate in colitic mice relative to (i) control mice (i.e., selectivity) and (ii) particles bearing a control ligand (i.e., ligand efficiency). The selectivity and ligand efficiency of alpha-V particles were a function of the total number of particles infused. The highest selectivity observed within our test regime was 3, while ligand efficiency increased linearly with the number of particles injected to a value of 24. This work represents a significant step towards achieving a targeted drug delivery scheme for the treatment of inflammatory bowel disease and indicates that the efficiency of targeting is dependent on the dose regime.