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OBJECTIVE: To evaluate experimental dimethacrylate-based materials containing calcium orthophosphates or calcium silicate particles in terms of their optical, mechanical and Ca2+ release behaviour. METHODS: Dicalcium phosphate dihydrate (DCPD), hydroxyapatite (HAp), beta-tricalcium phosphate (ß-TCP) or calcium silicate (CaSi) particles were added to a photocurable BisGMA/TEGDMA resin (1:1 in mols) at a 30 vol% fraction. Materials containing silanized or non-silanized barium glass particles were used as controls. Degree of conversion (DC) at the top and base of 2-mm thick specimens was determined by ATR-FTIR spectroscopy (n = 5). Translucency parameter (TP) and transmittance (%T) were determined using a spectrophotometer (n = 3). Biaxial flexural strength (BFS) and flexural modulus (FM) were determined by biaxial flexural testing after 24 h storage in water (n = 10). Ca2+ release in water was determined during 28 days by inductively coupled plasma optical emission spectrometry (n = 3). Statistical analysis was performed using ANOVA/Tukey test (DC: two-way; TP, %T; BFS and FM: one-way; Ca2+ release: repeated measures two-way, α = 5 %). RESULTS: CaSi and ß-TCP particles drastically reduced DC at 2 mm, TP and %T (p < 0.001). Compared to both controls, all Ca2+-releasing materials presented lower BFS (p < 0.001) and only the material with DCPD showed significantly lower FM (p < 0.05). The material containing CaSi presented the highest Ca2+ release, while among materials formulated with calcium orthophosphates the use of DCPD resulted in the highest release (p < 0.001). SIGNIFICANCE: CaSi particles allowed the highest Ca2+ release. Notwithstanding, the use of DCPD resulted in a material with the best compromise between optical behaviour, DC, strength and Ca2+ release.
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OBJECTIVE: to evaluate the effect a glass ionomer cement (GIC) containing hydroxyapatite (HAp) or calcium silicate (CaSi) particles on mineral content and mechanical properties of demineralized dentin. Ion release and compressive strength (CS) of the cements were also evaluated. METHODS: GIC (Fuji 9 Gold Label, GC), GIC+ 5%HAp and GIC+ 5%CaSi (by mass) were evaluated. Ion release was determined by induced coupled plasma optical emission spectroscopy (Ca2+/Sr2+) or ion-specific electrode (F-) (n = 3). A composite (Filtek Z250, 3 M ESPE) was used as control in remineralization tests. Demineralized dentin discs were kept in contact with materials in simulated body fluid (SBF) at 37 °C for eight weeks. Mineral:matrix ratio (MMR) was determined by ATR-FTIR spectroscopy (n = 5). Dentin hardness (H) and elastic modulus (E) were determined by nanoindentation (n = 10). CS was tested after 24 h and 7d in deionized water (n = 12). Data were analyzed by ANOVA/Tukey test (α = 0.05). RESULTS: Ca2+ and Sr2+ release was higher for the modified materials (p < 0.05). Only GIC+ 5%HAp showed higher F- release than the control (p < 0.05). All groups showed statistically significant increases in MMR, with no differences among them after 8 weeks (p > 0.05). No differences in dentin H or E were observed among groups (p > 0.05). HAp-modified GIC showed increased initial CS, while adding CaSi had the opposite effect (p < 0.05). After 7 days, GIC+ 5%CaSi presented lower CS in relation to control and GIC+ 5%HAp (p < 0.05). SIGNIFICANCE: GIC modification with HAp or CaSi affected CS and increased ion release; however, none of the groups showed evidence of dentin remineralization in comparison to the negative control.
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Calcio , Cementos de Ionómero Vítreo , Calcio/análisis , Ensayo de Materiales , Cementos de Ionómero Vítreo/farmacología , Cementos de Ionómero Vítreo/química , Durapatita/farmacología , Durapatita/química , Dentina/químicaRESUMEN
OBJECTIVES: To evaluate the effectiveness of spiritual care training on medical students' self-reported competencies. METHODS: This is a quasi-experimental (controlled and non-randomized) study including 115 Brazilian medical students. Participants were enrolled into 2 groups: fourth-year students (n = 64) who received spiritual care training and sixth-year students (n = 51) who did not receive this training - control group (i.e., usual teaching). Participants answered a self-reported Spiritual Care Competence Scale. Comparisons between groups were performed and effect sizes were reported. RESULTS: Providing a spiritual care training resulted in significantly higher self-reported scores for the dimensions of "Assessment" (d = 0.99), "Improvement of care" (d = 0.69), "Counseling (d = 0.88)," "Referral" (d = 0.75), and "Total Spiritual Care" (d = 1.044) as compared to the control group. Likewise, 21 out of 27 items of the Spiritual Care Competence Scale were significantly higher for the intervention group, presenting effect sizes (d) ranging between 0.428 and 1.032. SIGNIFICANCE OF RESULTS: Medical students receiving spiritual care training showed greater self-reported competencies as compared to those in the usual teaching. These results reinforce the importance of promoting spirituality teaching in medical schools.
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Terapias Espirituales , Estudiantes de Medicina , Estudiantes de Enfermería , Humanos , Espiritualidad , Autoinforme , Brasil , Estudiantes de Enfermería/psicologíaRESUMEN
Control of tuberculosis depends on the rapid expression of protective CD4+ T-cell responses in the Mycobacterium tuberculosis (Mtb)-infected lungs. We have recently shown that the immunomodulatory cytokine IL-10 acts intrinsically in CD4+ T cells and impairs their parenchymal migratory capacity, thereby preventing control of Mtb infection. Herein, we show that IL-10 overexpression does not impact the protection conferred by the established memory CD4+ T-cell response, as BCG-vaccinated mice overexpressing IL-10 only during Mtb infection display an accelerated, BCG-induced, Ag85b-specific CD4+ T-cell response and control Mtb infection. However, IL-10 inhibits the migration of recently activated ESAT-6-specific CD4+ T cells into the lung parenchyma and impairs the development of ectopic lymphoid structures associated with reduced expression of the chemokine receptors CXCR5 and CCR7. Together, our data support a role for BCG vaccination in preventing the immunosuppressive effects of IL-10 in the fast progression of Mtb infection and may provide valuable insights on the mechanisms contributing to the variable efficacy of BCG vaccination.
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Mycobacterium tuberculosis , Tuberculosis , Animales , Vacuna BCG , Interleucina-10 , Ratones , Tuberculosis/microbiología , Tuberculosis/prevención & control , VacunaciónRESUMEN
Scavenger receptors are part of a complex surveillance system expressed by host cells to efficiently orchestrate innate immune response against bacterial infections. Stabilin-1 (STAB-1) is a scavenger receptor involved in cell trafficking, inflammation, and cancer; however, its role in infection remains to be elucidated. Listeria monocytogenes (Lm) is a major intracellular human food-borne pathogen causing severe infections in susceptible hosts. Using a mouse model of infection, we demonstrate here that STAB-1 controls Lm-induced cytokine and chemokine production and immune cell accumulation in Lm-infected organs. We show that STAB-1 also regulates the recruitment of myeloid cells in response to Lm infection and contributes to clear circulating bacteria. In addition, whereas STAB-1 appears to promote bacterial uptake by macrophages, infection by pathogenic Listeria induces the down regulation of STAB-1 expression and its delocalization from the host cell membrane.We propose STAB-1 as a new SR involved in the control of Lm infection through the regulation of host defense mechanisms, a process that would be targeted by bacterial virulence factors to promote infection.
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Moléculas de Adhesión Celular Neuronal/inmunología , Quimiocinas/inmunología , Citocinas/inmunología , Listeriosis , Animales , Línea Celular , Humanos , Listeria monocytogenes , Listeriosis/inmunología , Ratones , Ratones Endogámicos C57BL , Receptores Mensajeros de LinfocitosRESUMEN
Leishmania infantum (L. infantum) and Leishmania major (L. major) are phylogenetically related protozoan parasites that cause different pathologies in humans (visceral and cutaneous infections, respectively). Here, we report on how these obligatory intracellular pathogens differentially affect the migration of macrophages. Resorting to gap closure assays of infected murine bone marrow derived macrophages, we observed that L. infantum enhances the mobility of these cells. This is not the case of L. major, whose impact on macrophage migration is null. Resorting to kinase inhibition assays, we witnessed that chemical inhibition of phosphoinositide 3-kinase-γ (PI3Kγ) critically impairs cell mobility in all experimental conditions. Importantly, the blockade of tyrosine kinases with dasatinib also slows down naiÌve and L. major-parasitized cells but not macrophages exposed to L. infantum. The dasatinib-resistant phenotype of L. infantum-infected macrophages aligns with the hypothesis that this parasite invokes a tyrosine kinase-independent pathway to increase the PI3Kγ activity of macrophages and enhance migration.
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Leishmania infantum , Leishmania major , Animales , Humanos , Macrófagos , Ratones , Fosfatidilinositol 3-Quinasa , Fosfatidilinositol 3-Quinasas/genéticaRESUMEN
The transcription factor hypoxia-inducible factor-1 alpha (HIF-1α) is a key regulator of the response and function of myeloid cells in hypoxic and inflammatory microenvironments. To define the role of HIF-1α in tuberculosis, the progression of aerosol Mycobacterium tuberculosis infection was analysed in mice deficient in HIF-1α in the myeloid lineage (mHIF-1α-/- ). We show that myeloid HIF-1α is not required for the containment of the infection, as both wild-type (WT) and mHIF-1α-/- mice mounted normal Th1 responses and maintained control of bacterial growth throughout infection. However, during chronic infection mHIF-1α-/- mice developed extensive lymphocytic inflammatory involvement of the interstitial lung tissue and died earlier than WT mice. These data support the hypothesis that HIF-1α activity coordinates the response of myeloid cells during M. tuberculosis infection to prevent excessive leucocyte recruitment and immunopathological consequences to the host.
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Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Pulmón/metabolismo , Mycobacterium tuberculosis/crecimiento & desarrollo , Células Mieloides/metabolismo , Neumonía/metabolismo , Tuberculosis Pulmonar/metabolismo , Animales , Carga Bacteriana , Células Cultivadas , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Interacciones Huésped-Patógeno , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Pulmón/inmunología , Pulmón/microbiología , Ratones Endogámicos C57BL , Ratones Noqueados , Mycobacterium tuberculosis/inmunología , Células Mieloides/inmunología , Células Mieloides/microbiología , Neumonía/genética , Neumonía/inmunología , Neumonía/microbiología , Transducción de Señal , Tuberculosis Pulmonar/genética , Tuberculosis Pulmonar/inmunología , Tuberculosis Pulmonar/microbiologíaRESUMEN
Granuloma formation is a hallmark of several infectious diseases, including those caused by Mycobacterium sp These structures are composed of accumulations of inflammatory cells, and it has been shown that cytokines such as IFN-γ and TNF-α are required for granuloma assembly during M. avium infections in mice. Macrophages (MΦs) insensitive to IFN-γ (MIIG) mice have MΦs, monocytes, and dendritic cells that are unresponsive to IFN-γ. We observed that although IFN-γ-/- mice present an exacerbated infection, the same is not true for MIIG animals, where the same levels of protection as the wild-type animals were observed in the liver and partial protection in the spleen. Unlike IFN-γ-/- mice, MIIG mice still develop well-defined granulomas, suggesting that IFN-γ-mediated MΦ activation is not required for granuloma assembly. This work also shows that MIIG animals exhibit increased cell recruitment with higher CD4+ T cells numbers as well as increased IFN-γ and TNF-α expression, suggesting that TNF-α may have a role in protection and may compensate the lack of MΦ response to IFN-γ in the MIIG model. TNF-α-deficient MIIG mice (MIIG.TNF-α-/-) exhibited increased bacterial burdens when compared with MIIG mice. These results suggest that in the absence of IFN-γ signaling in MΦs, TNF-α has a protective role against M. avium.
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Interferón gamma/fisiología , Activación de Macrófagos/inmunología , Mycobacterium avium/inmunología , Factor de Necrosis Tumoral alfa/fisiología , Animales , Linfocitos T CD4-Positivos/fisiología , Granuloma/etiología , Ratones , Ratones Endogámicos C57BL , Transducción de SeñalRESUMEN
Anemia is a frequent and challenging complication of mycobacterial infections. We used a model of disseminated Mycobacterium avium infection in mice to investigate the mechanisms of mycobacteria-induced anemia. We found increased formation of RBC in the bone marrow and spleen of infected mice. Infection induced reticulocytosis and the premature egress of immature progenitors to the systemic circulation in an IFN-γ (IFNG)-dependent way. The newly formed RBC had reduced CD47 surface expression and a reduced life span and were phagocytosed in the liver of infected mice, increasing iron recycling in this organ. The increased engulfment and degradation of RBC was independent of IFNG sensing by macrophages. Together, our findings demonstrate that mycobacterial infection alters the formation of erythrocytes, leading to their accelerated removal from circulation and hemolytic anemia. This comprehensive elucidation of the mechanisms underlying mycobacteria-induced anemia has important implications for its efficient clinical management.
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Anemia/etiología , Eritrocitos/fisiología , Interferón gamma/fisiología , Infecciones por Mycobacterium/complicaciones , Animales , Células de la Médula Ósea/citología , Antígeno CD47/análisis , Diferenciación Celular , Eritropoyesis , Hepcidinas/fisiología , Ratones , Ratones Endogámicos C57BL , Infecciones por Mycobacterium/sangre , FagocitosisRESUMEN
O paradesporto no Brasil vem se fomentando devido a um conjunto de motivos, como o fato de termos sediado as Paralimpíadas Mundiais 2016, maior interesse do público/mídia, aumento no número de adeptos das modalidades e construção do centro de treinamento. Além disso, o legado deixado pelo eventose constituiutambém no aumento das expectativas de todosos envolvidos nessemovimento. Sendo assim, buscou-se analisar as expectativas de paratletas em nível escolar. Entrevistou-se 114 atletas brasileiros de ambos os sexos, com idade média de±15,45 anos, participantes das Paralimpíadas Escolares. Identificou-se a relevância da participação nessacompetição na modificação da percepção dos jovensem relação aos seusprojetos futuros e a oportunidade de serem valorizados e vistos com orgulho pelo meio social.
The paradesport in Brazil is being promoted due to a number of reasons, such as the fact that we have hosted the World Paralympics, greater public/media interest, increase in the number of fans of the modalities and construction of the training center. In addition, the legacy left is also an increase in the expectations of all those involved in this movement. Thus, we sought to analyze the expectations of paratletas at school level. We interviewed 114 Brazilian athletes, of both sexes, of age ±15.45 years, participating in School Paralympic Games. It was identified the relevance of participation in this competition in the modification of the perception in relation to future projects and as an opportunity to be seen as an object of valorization and pride in the social environment.
El paradesporto en Brasil se viene fomentando debido diversos motivos, como el hecho de tener ubicadas las Paralimpíadas Mundiales 2016, mayor interés del público/imprenta, mayor número de adeptos de modalidades y construcción del centro de entrenamiento. Además, el legado dejado se constituye también en el aumento de expectativas de todos involucrados en el movimiento. Siendo así, se buscó analizar las expectativas de paratletas a nivel escolar. Entrevistó 114 atletas brasileños, de ambos sexos, de edad ±15,45 años, participantes de las Paralimpíadas Escolares. Se identificó la relevancia de la participación en esta competición en la modificación de la percepción de los proyectos futuros y como oportunidad de ser vistos como objeto de valorización y orgullo por el medio social.
RESUMEN
Immunosuppression is a well-established risk factor for Visceral Leishmaniasis. Post-immunosuppression leishmaniasis is characterized by an increase of parasite burden, hematopoietic disorders and unusual clinical manifestations. Although there are many reports on bone marrow findings in VL, less is known about the relationship between parasite dynamics in this organ and the function of either hematopoietic stem cells and progenitor cells themselves. In the present study, we tackle these issues using a new approach of infecting human stem cells derived from bone marrow with L. infantum. Using this strategy, we show that human hematopoietic stem cells (hHSC) are able to phagocytize L. infantum promastigotes and release modulatory and pro-inflammatory cytokines, mainly TNF-α. Our results demonstrated that L. infantum infection in vitro enhances hematopoiesis, favoring the development of erythrocitic lineage through a mechanism yet unknown. Moreover, we found that L. infantum infection alters the phenotypic profile of the hematopoietic progeny; modifying the surface markers expression of differentiated cells. Thus, our study represents a rare opportunity to monitor the in vitro differentiation of human stem cells experimentally infected by L. infantum to better understand the consequences of the infection on phenotypic and functional profile of the cell progeny.
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Diferenciación Celular/inmunología , Eritropoyesis/inmunología , Células Madre Hematopoyéticas/inmunología , Leishmania infantum/inmunología , Fagocitosis/inmunología , Adulto , Anciano , Células de la Médula Ósea/inmunología , Células de la Médula Ósea/metabolismo , Células de la Médula Ósea/parasitología , Citocinas/inmunología , Citocinas/metabolismo , Femenino , Células Madre Hematopoyéticas/metabolismo , Células Madre Hematopoyéticas/parasitología , Interacciones Huésped-Parásitos/inmunología , Humanos , Leishmania infantum/fisiología , Masculino , Persona de Mediana Edad , Factor de Necrosis Tumoral alfa/inmunología , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
IFN-γ is known to be predominantly produced by lymphoid cells such as certain subsets of T cells, NK cells, and other group 1 innate lymphoid cells. In this study, we used IFN-γ reporter mouse models to search for additional cells capable of secreting this cytokine. We identified a novel and rare population of nonconventional IFN-γ-producing cells of hematopoietic origin that were characterized by the expression of Thy1.2 and the lack of lymphoid, myeloid, and NK lineage markers. The expression of IFN-γ by this population was higher in the liver and lower in the spleen. Furthermore, these cells were present in mice lacking both the Rag2 and the common γ-chain (γc) genes (Rag2-/-γc-/-), indicating their innate nature and their γc cytokine independence. Rag2-/-γc-/- mice are as resistant to Mycobacterium avium as Rag2-/- mice, whereas Rag2-/- mice lacking IFN-γ are more susceptible than either Rag2-/- or Rag2-/-γc-/- These lineage-negative CD45+/Thy1.2+ cells are found within the mycobacterially induced granulomatous structure in the livers of infected Rag2-/-γc-/- animals and are adjacent to macrophages that expressed inducible NO synthase, suggesting a potential protective role for these IFN-γ-producing cells. Accordingly, Thy1.2-specific mAb administration to infected Rag2-/-γc-/- animals increased M. avium growth in the liver. Overall, our results demonstrate that a population of Thy1.2+ non-NK innate-like cells present in the liver expresses IFN-γ and can confer protection against M. avium infection in immunocompromised mice.
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Células Madre Hematopoyéticas/inmunología , Inmunidad Innata , Interferón gamma/biosíntesis , Interferón gamma/genética , Subunidad gamma Común de Receptores de Interleucina/inmunología , Animales , Anticuerpos Monoclonales/administración & dosificación , Proteínas de Unión al ADN/deficiencia , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/inmunología , Granuloma/inmunología , Granuloma/microbiología , Huésped Inmunocomprometido/inmunología , Interferón gamma/inmunología , Subunidad gamma Común de Receptores de Interleucina/deficiencia , Subunidad gamma Común de Receptores de Interleucina/genética , Células Asesinas Naturales/inmunología , Antígenos Comunes de Leucocito/genética , Antígenos Comunes de Leucocito/inmunología , Hígado/citología , Hígado/inmunología , Hígado/microbiología , Macrófagos/inmunología , Ratones , Ratones Endogámicos C57BL , Mycobacterium avium/crecimiento & desarrollo , Mycobacterium avium/inmunología , Óxido Nítrico Sintasa de Tipo II/biosíntesis , Óxido Nítrico Sintasa de Tipo II/genética , Óxido Nítrico Sintasa de Tipo II/inmunología , Bazo/citología , Bazo/inmunología , Antígenos Thy-1/genética , Antígenos Thy-1/inmunologíaRESUMEN
The establishment of mycobacterial infection is characterized by the formation of granulomas, which are well-organized aggregates of immune cells, namely, infected macrophages. The granuloma's main function is to constrain and prevent dissemination of the mycobacteria while focusing the immune response to a limited area. In some cases these lesions can grow progressively into large granulomas which can undergo central necrosis, thereby leading to their caseation. Macrophages are the most abundant cells present in the granuloma and are known to adapt under hypoxic conditions in order to avoid cell death. Our laboratory has developed a granuloma necrosis model that mimics the human pathology of Mycobacterium tuberculosis, using C57BL/6 mice infected intravenously with a low dose of a highly virulent strain of Mycobacterium avium. In this work, a mouse strain deleted of the hypoxia inducible factor 1α (HIF-1α) under the Cre-lox system regulated by the lysozyme M gene promoter was used to determine the relevance of HIF-1α in the caseation of granulomas. The genetic ablation of HIF-1α in the myeloid lineage causes the earlier emergence of granuloma necrosis and clearly induces an impairment of the resistance against M. avium infection coincident with the emergence of necrosis. The data provide evidence that granulomas become hypoxic before undergoing necrosis through the analysis of vascularization and quantification of HIF-1α in a necrotizing mouse model. Our results show that interfering with macrophage adaptation to hypoxia, such as through HIF-1α inactivation, accelerates granuloma necrosis.
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Granuloma/patología , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Macrófagos/metabolismo , Infecciones por Mycobacterium/patología , Necrosis/metabolismo , Animales , Modelos Animales de Enfermedad , Citometría de Flujo , Inmunohistoquímica , Etiquetado Corte-Fin in Situ , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Infecciones por Mycobacterium/metabolismo , Mycobacterium avium , Necrosis/microbiologíaRESUMEN
Granulomas are the hallmark of mycobacterial disease. Here, we demonstrate that both the cell recruitment and the increased glucose consumption in granulomatous infiltrates during Mycobacterium avium infection are highly dependent on interferon-γ (IFN-γ). Mycobacterium avium-infected mice lacking IFN-γ signalling failed to developed significant inflammatory infiltrations and lacked the characteristic uptake of the glucose analogue fluorine-18-fluorodeoxyglucose (FDG). To assess the role of macrophages in glucose uptake we infected mice with a selective impairment of IFN-γ signalling in the macrophage lineage (MIIG mice). Although only a partial reduction of the granulomatous areas was observed in infected MIIG mice, the insensitivity of macrophages to IFN-γ reduced the accumulation of FDG. In vivo, ex vivo and in vitro assays showed that macrophage activated by IFN-γ displayed increased rates of glucose uptake and in vitro studies showed also that they had increased lactate production and increased expression of key glycolytic enzymes. Overall, our results show that the activation of macrophages by IFN-γ is responsible for the Warburg effect observed in organs infected with M. avium.
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Granuloma/inmunología , Interferón gamma/inmunología , Activación de Macrófagos , Macrófagos/inmunología , Mycobacterium avium/inmunología , Tuberculosis/inmunología , Animales , Fluorodesoxiglucosa F18/farmacocinética , Fluorodesoxiglucosa F18/farmacología , Glucólisis/efectos de los fármacos , Glucólisis/genética , Glucólisis/inmunología , Granuloma/genética , Granuloma/microbiología , Granuloma/veterinaria , Interferón gamma/genética , Ácido Láctico/inmunología , Macrófagos/microbiología , Ratones , Ratones Noqueados , Radiofármacos/farmacocinética , Radiofármacos/farmacología , Tuberculosis/genética , Tuberculosis/patología , Tuberculosis/veterinariaRESUMEN
A differential behavior among infected and bystander dendritic cells (DCs) has been explored in different infection models. We have analyzed both populations sorted on contact with visceral Leishmania infantum on a susceptible mice model evaluating the subsequent repercussions on adaptive immune response. Our results demonstrate a clear dichotomy between the immunomodulatory abilities of bystander and infected DCs. The bystander population presents increased levels of IL-12p40 and costimulatory molecules being capable to induce CD4(+) T cell activation with immune protective capabilities. In contrast, infected DCs, which express lower costimulatory molecules and higher levels of IL-10, promote the development of Leishmania Ag-specific, nonprotective T-bet(+)IFN-γ(+)IL-10(+) CD4(+) T cells with an effector phenotype. This specific polarization was found to be dependent on IL-12p70. Splenic infected DCs recovered from chronic infected animals are similarly capable to polarize ex vivo syngeneic naive CD4(+) T cells toward a T-bet(+)IFN-γ(+)IL-10(+) phenotype. Further analysis revealed that only MHC class II(high)-infected DCs were responsible for this polarization. The adoptive transfer of such polarized CD4(+) T cells facilitates visceral leishmaniasis in BALB/c mice in a clear contrast with their counterpart generated with bystander DCs that significantly potentiate protection. Further, we demonstrated that CD4(+) T cells primed by infected DCs in an IL-10 free system, thus deprived of T-bet(+)IFN-γ(+)IL-10(+) population, restore the immune response and reduce parasite load, supporting a deleterious role of IFN-γ(+)IL-10(+) T cells in the maintenance of infection. Overall, our results highlight novel subversion mechanisms by which nonprotective T-bet(+)IFN-γ(+)IL-10(+) T cells are associated with chronicity and prolonged parasite persistence.
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Polaridad Celular/inmunología , Células Dendríticas/inmunología , Antígenos de Histocompatibilidad Clase II/biosíntesis , Interferón gamma/biosíntesis , Interleucina-10/biosíntesis , Leishmaniasis Visceral/inmunología , Proteínas de Dominio T Box/biosíntesis , Linfocitos T/inmunología , Animales , Células Cultivadas , Enfermedad Crónica , Técnicas de Cocultivo , Células Dendríticas/metabolismo , Células Dendríticas/parasitología , Interacciones Huésped-Patógeno/inmunología , Inmunofenotipificación , Interleucina-10/deficiencia , Leishmania infantum/inmunología , Leishmania infantum/patogenicidad , Leishmaniasis Visceral/parasitología , Leishmaniasis Visceral/prevención & control , Ratones , Ratones Endogámicos BALB C , Linfocitos T/metabolismo , Linfocitos T/parasitologíaRESUMEN
OBJECTIVE: To overcome the limitation of bisnaphthalimidopropyldiaaminooctane (BNIPDaoct) low physiological solubility and potentially increase its efficiency against visceral leishmaniasis (VL), a delivery system based on poly(d,l-lactide-co-glycolide) (PLGA) nanoparticles was developed. MATERIALS & METHODS: BNIPDaoct-PLGA nanoparticles were prepared by nanoprecipitation and characterized. Anti-Leishmania activity was evaluated using in vitro and in vivo VL infection models. RESULTS: BNIPDaoct-PLGA nanoparticles were successfully produced and were sized at 156.0 ± 2.8 nm with an encapsulation efficiency of approximately 85%. The PLGA nanoparticles reduced BNIPDaoct cellular toxicity, retained its in vitro anti-leishmanial activity and led to a significant reduction (â¼80%) in the parasite burden in the infected mice spleen when compared with the free drug or amphotericin B. In the liver the effect was less pronounced, with a 30-50% reduction observed between the nanoformulation and the BNIPDaoct per se or the amphotericin B, respectively. CONCLUSION: PLGA nanoparticles provide controlled and effective delivery of BNIPDaoct for treatment of VL.
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Antiprotozoarios/administración & dosificación , Portadores de Fármacos/química , Ácido Láctico/química , Leishmaniasis Visceral/tratamiento farmacológico , Nanopartículas/química , Naftalimidas/administración & dosificación , Ácido Poliglicólico/química , Animales , Antiprotozoarios/farmacocinética , Antiprotozoarios/uso terapéutico , Línea Celular , Humanos , Leishmania donovani/efectos de los fármacos , Ratones , Ratones Endogámicos BALB C , Naftalimidas/farmacocinética , Naftalimidas/uso terapéutico , Copolímero de Ácido Poliláctico-Ácido PoliglicólicoRESUMEN
Invariant NKT (iNKT) cells constitute a versatile T cell subset with important regulatory functions, which are thought to result essentially from their capacity to promptly produce cytokines that influence the Th1/Th2 balance. In this study, we report that these cells can also express Foxp3, an important transcriptional regulator associated with suppressive activity, once they have been exposed to TGF-ß. Foxp3 was expressed by iNKT cells from both peripheral and cord blood. CD4(+) iNKT cells acquired Foxp3 expression preferentially, although a lower proportion of their CD4(-) counterpart also became positive. All Foxp3(+) iNKT cells displayed CD25 but not necessarily CTLA4 or GITR, regardless of the upregulation of these markers in the presence of TGF-ß. Exposure to TGF-ß decreased IL-4 and IFN-γ production while increasing IL-10, independently from Foxp3 expression. IL-17 was not detected. TGF-ß induced high levels of Foxp3, but no suppressor activity, which emerged only in the presence of rapamycin. Peripheral and cord blood Foxp3(+) iNKT cells suppressed the proliferation of conventional autologous and heterologous CD4(+) T cells equally, in a cell contact-dependent and Ag-independent manner. Our findings demonstrate that human iNKT cells become suppressive in the presence of TGF-ß plus rapamycin, thus adding a new facet to their complex functional properties.
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Diferenciación Celular/inmunología , Factores de Transcripción Forkhead/biosíntesis , Inmunosupresores/farmacología , Células T Asesinas Naturales/citología , Células T Asesinas Naturales/inmunología , Sirolimus/farmacología , Linfocitos T Reguladores/inmunología , Factor de Crecimiento Transformador beta/fisiología , Linfocitos T CD4-Positivos/citología , Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD4-Positivos/inmunología , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Sangre Fetal/citología , Sangre Fetal/efectos de los fármacos , Sangre Fetal/inmunología , Humanos , Leucocitos Mononucleares/citología , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/inmunología , Células T Asesinas Naturales/efectos de los fármacos , Linfocitos T Reguladores/citología , Linfocitos T Reguladores/efectos de los fármacosRESUMEN
Parenteral injection of tolerated proteins into orally tolerant mice inhibits the initiation of immunological responses to unrelated proteins and blocks severe chronic inflammatory reactions of immunological origin, such as autoimmune reactions. This inhibitory effect which we have called "indirect effects of oral tolerance" is also known as "bystander suppression." Herein, we show that i.p. injection of OVA + Al(OH)(3) minutes before i.v. injection of Schistosoma mansoni eggs into OVA tolerant mice blocked the increase of pulmonary granulomas. In addition, the expression of ICAM-1 in lung parenchyma in areas outside the granulomas of OVA-orally tolerant mice was significantly reduced. However, at day 18 after granuloma induction there was no difference in immunofluorescency intensity to CD3, CD4, F4/80, andα-SMA per granuloma area of tolerant and control groups. Reduction of granulomas by reexposure to orally tolerated proteins was not correlated with a shift in Th-1/Th-2 cytokines in serum or lung tissue extract.
Asunto(s)
Efecto Espectador , Granuloma/inmunología , Pulmón/metabolismo , Schistosoma mansoni/inmunología , Esquistosomiasis mansoni/inmunología , Administración Oral , Animales , Antígenos CD/metabolismo , Células Cultivadas , Huevos/parasitología , Granuloma/etiología , Granuloma/patología , Granuloma/fisiopatología , Tolerancia Inmunológica , Inmunofenotipificación , Inflamación , Molécula 1 de Adhesión Intercelular/genética , Molécula 1 de Adhesión Intercelular/metabolismo , Pulmón/inmunología , Pulmón/parasitología , Pulmón/patología , Ratones , Ratones Endogámicos C57BL , Ovalbúmina/administración & dosificación , Schistosoma mansoni/patogenicidad , Esquistosomiasis mansoni/complicaciones , Esquistosomiasis mansoni/patología , Esquistosomiasis mansoni/fisiopatologíaRESUMEN
CD1d-reactive invariant NKT (iNKT) cells have been implicated in a number of experimental models of human pathologies. Given the scope of their immunoregulatory activities mediated through distinct cytokine patterns, it has been proposed that this functional diversity originates from distinct iNKT subpopulations. In this study, we report that human CD161(+) iNKT cells are intrinsically endowed with the capacity to generate IL-17, but require TGF-ß, IL-1ß, and IL-23 to carry out this potential. IL-17-producing iNKT cells are already present in cord blood but, in contrast to peripheral blood iNKT cells, they cannot generate IFN-γ. These IL-17 producers respond to aryl hydrocarbon receptor stimulation and express IL-23 receptor and retinoic acid-related orphan receptor C, similar to conventional T helper 17 cells, from which they differ by their restricted ability to coproduce IL-22. In conclusion, IL-17 production by human iNKT cells depends on two critical parameters, namely an intrinsic program and a proinflammatory environment.
Asunto(s)
Inflamación/inmunología , Interleucina-17/biosíntesis , Células T Asesinas Naturales/metabolismo , Ensayo de Inmunoadsorción Enzimática , Humanos , Interferón gamma , Interleucina-1beta/biosíntesis , Interleucina-23/biosíntesis , Interleucinas/biosíntesis , Subfamilia B de Receptores Similares a Lectina de Células NK/inmunología , Células T Asesinas Naturales/inmunología , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/metabolismo , Reacción en Cadena de la Polimerasa , Receptores de Hidrocarburo de Aril/metabolismo , Receptores de Interleucina/metabolismo , Linfocitos T Colaboradores-Inductores/inmunología , Factor de Crecimiento Transformador beta/biosíntesis , Interleucina-22RESUMEN
Understanding the complex interactions between Leishmania and dendritic cells (DCs) is central to the modulation of the outcome of this infection, given that an effective immune response against Leishmania is dependent on the successful activation and maturation of DCs. We report here that Leishmania infantum promastigotes successfully infect mouse bone marrow-derived DCs without triggering maturation, as shown by a failure in the up-regulation of CD40 and CD86 expression, and that parasites strongly counteract the lipopolysaccharide-triggered maturation of DCs. A small increase in interleukin (IL)-12 and IL-10 transcription and secretion and a decrease in IL-6 were observed in infected cells. This arrested DC maturation state is actively promoted by parasites because heat-killed or fixed parasites increased cytokine and costimulatory molecule expression. At a molecular level, L. infantum rapidly induced activation of phosphatidylinositol 3-kinase/Akt and extracellular signal-regulated kinase 1/2, whereas no effect was observed in the c-Jun N-terminal kinase and p38 mitogen-activated protein kinase proinflammatory pathways. Moreover, parasites actively promoted cleavage of the nuclear factor-κB p65(RelA) subunit, causing its impairment. The blockade of phosphatidylinositol 3-kinase/Akt by either treatment of bone marrow-derived DCs with wortmannin or transfection with an Akt dominant-negative mutant resulted in a strong decrease in infection rates, revealing for the first time a crucial role of this pathway on Leishmania engulfment by DCs. Overall, our data indicate that activation of Akt and impairment of nuclear factor-κB are responsible for immunogenicity subversion of L. infantum-infected DCs.