RESUMEN
Background and Aims: Mental health institutions and community organizations have had difficulty recruiting patients and caregivers onto their Patient, Family, and Community Advisory Committees (PFACs). Previous research has focused on barriers and enablers of engaging patients and caregivers who have advisory experience. This study acknowledges the experiential difference between patients and caregivers by focusing only on caregivers; further, we compare the barriers and enablers between advising versus non-advising caregivers of loved ones with mental illness. Methods: Data from a cross-sectional survey codesigned by researchers, staff, clients, and caregiver affiliated with a tertiary mental health center were completed by n = 84 caregivers (n = 40 past/current PFAC advising caregivers; n = 44 non-advising caregivers). Results: Caregivers were disproportionately female and late middle-aged. Advising and non-advising caregivers differed on employment status. There were no differences of the demographics of their care-recipients. More non-advising caregivers reported being hindered from PFAC engagement by family-related duties and interpersonal demands. Finally, more advising caregivers considered being publicly acknowledged as important. Conclusions: Advising and non-advising caregivers of loved ones with mental illness were similar in demographics and in reporting the enablers and hindrances that impact PFAC engagement. Nevertheless, our data highlights specific considerations that institutions/organizations should consider when recruiting and retaining caregivers on PFACs. Patient or Public Contribution: This project was led by a caregiver advisor to address a need she saw in the community. The surveys were codesigned by a team of two caregivers, one patient, and one researcher. The surveys were reviewed by a group of five caregivers external to the project. The results of the surveys were discussed with two caregivers involved directly with the project.
RESUMEN
Toxoplasma gondii is an obligate intracellular protozoan parasite. This apicomplexan is the causative agent of toxoplasmosis, a leading cause of central nervous system disease in AIDS. It has long been known that T. gondii interferes with major histocompatibility complex class II (MHC-II) antigen presentation to attenuate CD4(+) T cell responses and establish persisting infections. Transcriptional downregulation of MHC-II genes by T. gondii was previously established, but the precise mechanisms inhibiting MHC-II function are currently unknown. Here, we show that, in addition to transcriptional regulation of MHC-II, the parasite modulates the expression of key components of the MHC-II antigen presentation pathway, namely, the MHC-II-associated invariant chain (Ii or CD74) and the peptide editor H2-DM, in professional antigen-presenting cells (pAPCs). Genetic deletion of CD74 restored the ability of infected dendritic cells to present a parasite antigen in the context of MHC-II in vitro. CD74 mRNA and protein levels were, surprisingly, elevated in infected cells, whereas MHC-II and H2-DM expression was inhibited. CD74 accumulated mainly in the endoplasmic reticulum (ER), and this phenotype required live parasites, but not active replication. Finally, we compared the impacts of genetic deletion of CD74 and H2-DM genes on parasite dissemination toward lymphoid organs in mice, as well as activation of CD4(+) T cells and interferon gamma (IFN-γ) levels during acute infection. Cyst burdens and survival during the chronic phase of infection were also evaluated in wild-type and knockout mice. These results highlight the fact that the infection is influenced by multiple levels of parasite manipulation of the MHC-II antigen presentation pathway.
Asunto(s)
Antígenos de Diferenciación de Linfocitos B/inmunología , Antígenos de Histocompatibilidad Clase II/inmunología , Toxoplasma/inmunología , Toxoplasmosis/inmunología , Animales , Presentación de Antígeno , Células Presentadoras de Antígenos/inmunología , Antígenos de Diferenciación de Linfocitos B/genética , Linfocitos T CD4-Positivos , Células Dendríticas/inmunología , Células Dendríticas/parasitología , Femenino , Antígenos de Histocompatibilidad Clase II/genética , Humanos , Interferón gamma/genética , Interferón gamma/inmunología , Macrófagos/inmunología , Macrófagos/parasitología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Toxoplasma/genética , Toxoplasma/fisiología , Toxoplasmosis/genética , Toxoplasmosis/parasitologíaRESUMEN
The obligate intracellular protozoan parasite Toxoplasma gondii interferes with major histocompatibility complex class II antigen presentation to dampen host CD4(+) T cell responses. While it is known that T. gondii inhibits major histocompatibility complex class II gene transcription and expression in infected host cells, the mechanism of this host manipulation is unknown. Here, we show that soluble parasite proteins inhibit IFNγ-induced expression of major histocompatibility complex class II on the surface of the infected cell in a dose-dependent response that was abolished by protease treatment. Subcellular fractionation of T. gondii tachyzoites revealed that the major histocompatibility complex class II inhibitory activity co-partitioned with rhoptries and/or dense granules. However, parasite mutants deleted for single rhoptries or dense granules genes (ROP1, 4/7, 14, 16 and 18 or GRA 2-9 and 12 knock-out strains) retained the ability to inhibit expression of major histocompatibility complex class II. In addition, excreted/secreted antigens released by extracellular tachyzoites displayed immunomodulatory activity characterized by an inhibition of major histocompatibility complex class II expression, and reduced expression and release of TNFα by macrophages. Tandem MS analysis of parasite excreted/secreted antigens generated a list of T. gondii secreted proteins that may participate in major histocompatibility complex class II inhibition and the modulation of host immune functions.
Asunto(s)
Antígenos de Histocompatibilidad Clase II/genética , Interferón gamma/inmunología , Macrófagos/inmunología , Proteínas Protozoarias/inmunología , Toxoplasma/inmunología , Toxoplasmosis/inmunología , Animales , Presentación de Antígeno , Femenino , Antígenos de Histocompatibilidad Clase II/inmunología , Interacciones Huésped-Parásitos , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Proteínas Protozoarias/genética , Toxoplasma/genética , Toxoplasma/fisiología , Toxoplasmosis/genética , Toxoplasmosis/parasitologíaRESUMEN
Neonatal immune development begins in pregnancy and continues into lactation and may be affected by maternal diet. We investigated the possibility that maternal protein deficiency (PD) during a chronic gastrointestinal (GI) nematode infection could impair neonatal immune development. Beginning on d 14 of pregnancy, mice were fed protein-sufficient (PS; 24%) or protein-deficient (PD; 6%) isoenergetic diets and were infected weekly with either 0 (sham) or 100 Heligmosomoides bakeri larvae. Pups were killed on d 2, 7, 14, and d 21 and dams on d 20 of lactation. Lymphoid organs were weighed. Cytokine concentration in maternal and pup serum and in milk from pup stomachs and lymphoid cell populations in pup spleen and thymus were determined using luminex and flow cytometry, respectively. GI nematode infection increased Th2 cytokines (IL-4, IL-5, IL-13), IL-2, IL-10, and eotaxin in serum of dams whereas PD reduced IL-4 and IL-13. The lower IL-13 in PD dams was associated with increased fecal egg output and worm burdens. Maternal PD increased vascular endothelial growth factor in pup milk and eotaxin in pup serum. Maternal infection increased eotaxin in pup serum. Evidence of impaired neonatal immune development included reduced lymphoid organ mass in pups associated with both maternal infection and PD and increased percentage of T cells and T:B cell ratio in the spleen associated with maternal PD. Findings suggest that increases in specific proinflammatory cytokines as a result of the combination of infection and dietary PD in dams can impair splenic immune development in offspring.
Asunto(s)
Citocinas/metabolismo , Enfermedades Gastrointestinales/inmunología , Enfermedades del Sistema Inmune/etiología , Fenómenos Fisiologicos Nutricionales Maternos , Infecciones por Nematodos/inmunología , Complicaciones Parasitarias del Embarazo/inmunología , Deficiencia de Proteína/fisiopatología , Animales , Animales Recién Nacidos , Animales no Consanguíneos , Citocinas/sangre , Heces/parasitología , Femenino , Enfermedades Gastrointestinales/complicaciones , Enfermedades Gastrointestinales/metabolismo , Enfermedades Gastrointestinales/parasitología , Heligmosomatoidea/inmunología , Heligmosomatoidea/aislamiento & purificación , Heligmosomatoidea/fisiología , Enfermedades del Sistema Inmune/congénito , Enfermedades del Sistema Inmune/inmunología , Enfermedades del Sistema Inmune/metabolismo , Lactancia/sangre , Lactancia/inmunología , Lactancia/metabolismo , Subgrupos Linfocitarios/inmunología , Subgrupos Linfocitarios/metabolismo , Tejido Linfoide/inmunología , Tejido Linfoide/metabolismo , Masculino , Ratones , Leche/metabolismo , Infecciones por Nematodos/complicaciones , Infecciones por Nematodos/metabolismo , Infecciones por Nematodos/parasitología , Carga de Parásitos , Embarazo , Complicaciones Parasitarias del Embarazo/sangre , Complicaciones Parasitarias del Embarazo/metabolismo , Deficiencia de Proteína/complicaciones , Distribución AleatoriaRESUMEN
Effective control of the intracellular protozoan parasite Toxoplasma gondii depends on the activation of antigen-specific CD8(+) T-cells that manage acute disease and prevent recrudescence during chronic infection. T-cell activation in turn, requires presentation of parasite antigens by MHC-I molecules on the surface of antigen presenting cells. CD8(+) T-cell epitopes have been defined for several T. gondii proteins, but it is unclear how these antigens enter into the presentation pathway. We have exploited the well-characterized model antigen ovalbumin (OVA) to investigate the ability of parasite proteins to enter the MHC-I presentation pathway, by engineering recombinant expression in various organelles. CD8(+) T-cell activation was assayed using 'B3Z' reporter cells in vitro, or adoptively-transferred OVA-specific 'OT-I' CD8(+) T-cells in vivo. As expected, OVA secreted into the parasitophorous vacuole strongly stimulated antigen-presenting cells. Lower levels of activation were observed using glycophosphatidyl inositol (GPI) anchored OVA associated with (or shed from) the parasite surface. Little CD8(+) T-cell activation was detected using parasites expressing intracellular OVA in the cytosol, mitochondrion, or inner membrane complex (IMC). These results indicate that effective presentation of parasite proteins to CD8(+) T-cells is a consequence of active protein secretion by T. gondii and escape from the parasitophorous vacuole, rather than degradation of phagocytosed parasites or parasite products.
Asunto(s)
Antígenos de Protozoos/inmunología , Linfocitos T CD8-positivos/inmunología , Activación de Linfocitos/inmunología , Toxoplasmosis/inmunología , Toxoplasmosis/parasitología , Vacuolas/inmunología , Traslado Adoptivo , Animales , Células Presentadoras de Antígenos/inmunología , Western Blotting , Células Cultivadas , Femenino , Técnica del Anticuerpo Fluorescente , Humanos , Complejo Mayor de Histocompatibilidad/inmunología , Ratones , Ratones Endogámicos C57BL , Ovalbúmina/fisiología , Fracciones Subcelulares , Toxoplasma/genética , Toxoplasma/inmunología , Toxoplasmosis/metabolismo , Vacuolas/parasitologíaRESUMEN
BACKGROUND: Microarrays are invaluable tools for genome interrogation, SNP detection, and expression analysis, among other applications. Such broad capabilities would be of value to many pathogen research communities, although the development and use of genome-scale microarrays is often a costly undertaking. Therefore, effective methods for reducing unnecessary probes while maintaining or expanding functionality would be relevant to many investigators. RESULTS: Taking advantage of available genome sequences and annotation for Toxoplasma gondii (a pathogenic parasite responsible for illness in immunocompromised individuals) and Plasmodium falciparum (a related parasite responsible for severe human malaria), we designed a single oligonucleotide microarray capable of supporting a wide range of applications at relatively low cost, including genome-wide expression profiling for Toxoplasma, and single-nucleotide polymorphism (SNP)-based genotyping of both T. gondii and P. falciparum. Expression profiling of the three clonotypic lineages dominating T. gondii populations in North America and Europe provides a first comprehensive view of the parasite transcriptome, revealing that ~49% of all annotated genes are expressed in parasite tachyzoites (the acutely lytic stage responsible for pathogenesis) and 26% of genes are differentially expressed among strains. A novel design utilizing few probes provided high confidence genotyping, used here to resolve recombination points in the clonal progeny of sexual crosses. Recent sequencing of additional T. gondii isolates identifies >620 K new SNPs, including ~11 K that intersect with expression profiling probes, yielding additional markers for genotyping studies, and further validating the utility of a combined expression profiling/genotyping array design. Additional applications facilitating SNP and transcript discovery, alternative statistical methods for quantifying gene expression, etc. are also pursued at pilot scale to inform future array designs. CONCLUSIONS: In addition to providing an initial global view of the T. gondii transcriptome across major lineages and permitting detailed resolution of recombination points in a historical sexual cross, the multifunctional nature of this array also allowed opportunities to exploit probes for purposes beyond their intended use, enhancing analyses. This array is in widespread use by the T. gondii research community, and several aspects of the design strategy are likely to be useful for other pathogens.
Asunto(s)
Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Toxoplasma/genética , Animales , Exones/genética , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Genotipo , Interacciones Huésped-Parásitos/genética , Humanos , Ratones , Modelos Genéticos , Parásitos/genética , Filogenia , Polimorfismo de Nucleótido Simple/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reproducibilidad de los Resultados , Especificidad de la EspecieRESUMEN
The Toxoplasma gondii population consists of multiple strains, defined by genotype and virulence. Previous studies have established that protective immunity to this organism is mediated by IL-12, which drives T cells to produce IFN-gamma. Paradoxically, although type I and type II strains of T. gondii both induce IL-12 and IFN-gamma in the mouse, type I parasites are lethal, whereas type II strains establish chronic infection. The cellular basis for these strain-dependent differences remains unclear. To better understand these events, the CD8(+) T cell and dendritic cell (DC) responses to transgenic, OVA-expressing type I RH (RH OVA) and type II Prugniuad (Pru OVA) parasites were examined. Pru OVA-infected mice developed a robust DC response at the site of infection and the draining lymph node and generated a population of endogenous OVA-specific CD8(+) T cells. In contrast, RH OVA-infected mice had fewer DCs and OVA-specific CD8(+) T cells. RH OVA-infected mice given preactivated OVA-specific CD8(+) T cells were protected, suggesting that reduced DC-derived signals contributed to the low OVA-specific CD8(+) T cell numbers observed during type I infection. Indeed, DC depletion prior to Pru OVA infection resulted in a failure to generate activated OVA-specific CD8(+) T cells, and IL-12p70 treatment during RH OVA infection modestly increased the number of Ag-specific cells. Together, these data are consistent with a model of immunity to T. gondii in which strain-dependent DC responses shape the generation of Ag-specific CD8(+) T cells and determine the outcome of infection.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Células Dendríticas/inmunología , Células Dendríticas/parasitología , Activación de Linfocitos/inmunología , Toxoplasma/inmunología , Toxoplasma/patogenicidad , Toxoplasmosis Animal/inmunología , Animales , Linfocitos T CD8-positivos/parasitología , Linfocitos T CD8-positivos/patología , Proliferación Celular , Células Cultivadas , Células Dendríticas/patología , Epítopos de Linfocito T/administración & dosificación , Epítopos de Linfocito T/genética , Epítopos de Linfocito T/inmunología , Femenino , Activación de Linfocitos/genética , Recuento de Linfocitos , Linfopenia/genética , Linfopenia/inmunología , Linfopenia/parasitología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Ovalbúmina/administración & dosificación , Ovalbúmina/biosíntesis , Ovalbúmina/genética , Toxoplasma/genética , Toxoplasmosis Animal/genética , Toxoplasmosis Animal/patología , Virulencia/inmunologíaRESUMEN
Toxoplasma gondii is a leading cause of congenital birth defects, as well as a cause for ocular and neurological diseases in humans. Its cytoskeleton is essential for parasite replication and invasion and contains many unique structures that are potential drug targets. Therefore, the biogenesis of the cytoskeletal structure of T. gondii is not only important for its pathogenesis, but also of interest to cell biology in general. Previously, we and others identified a new T. gondii cytoskeletal protein, TgMORN1, which is recruited to the basal complex at the very beginning of daughter formation. However, its function remained largely unknown. In this study, we generated a knock-out mutant of TgMORN1 (DeltaTgMORN1) using a Cre-LoxP based approach. We found that the structure of the basal complex was grossly affected in DeltaTgMORN1 parasites, which also displayed defects in cytokinesis. Moreover, DeltaTgMORN1 parasites showed significant growth impairment in vitro, and this translated into greatly attenuated virulence in mice. Therefore, our results demonstrate that TgMORN1 is required for maintaining the structural integrity of the parasite posterior end, and provide direct evidence that cytoskeleton integrity is essential for parasite virulence and pathogenesis.
Asunto(s)
Citoesqueleto/genética , Genes Protozoarios , Proteínas Protozoarias/metabolismo , Toxoplasma/genética , Virulencia/genética , Animales , Western Blotting , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/metabolismo , Citoesqueleto/metabolismo , Técnica del Anticuerpo Fluorescente , Técnicas de Inactivación de Genes , Ratones , Proteínas Protozoarias/genética , Toxoplasma/metabolismo , Toxoplasma/patogenicidadRESUMEN
To better understand the initiation of CD8(+) T cell responses during infection, the primary response to the intracellular parasite Toxoplasma gondii was characterized using 2-photon microscopy combined with an experimental system that allowed visualization of dendritic cells (DCs) and parasite specific CD8(+) T cells. Infection with T. gondii induced localization of both these populations to the sub-capsular/interfollicular region of the draining lymph node and DCs were required for the expansion of the T cells. Consistent with current models, in the presence of cognate antigen, the average velocity of CD8(+) T cells decreased. Unexpectedly, infection also resulted in modulation of the behavior of non-parasite specific T cells. This TCR-independent process correlated with the re-modeling of the lymph node micro-architecture and changes in expression of CCL21 and CCL3. Infection also resulted in sustained interactions between the DCs and CD8(+) T cells that were visualized only in the presence of cognate antigen and were limited to an early phase in the response. Infected DCs were rare within the lymph node during this time frame; however, DCs presenting the cognate antigen were detected. Together, these data provide novel insights into the earliest interaction between DCs and CD8(+) T cells and suggest that cross presentation by bystander DCs rather than infected DCs is an important route of antigen presentation during toxoplasmosis.
Asunto(s)
Linfocitos T CD8-positivos/patología , Células Dendríticas/patología , Microscopía de Fluorescencia por Excitación Multifotónica/métodos , Toxoplasma/fisiología , Toxoplasmosis/patología , Análisis de Varianza , Animales , Linfocitos T CD8-positivos/metabolismo , Linfocitos T CD8-positivos/parasitología , Movimiento Celular , Células Dendríticas/metabolismo , Células Dendríticas/parasitología , Citometría de Flujo , Cinética , Ganglios Linfáticos/metabolismo , Ratones , Ratones Transgénicos , Toxoplasmosis/metabolismoRESUMEN
Multiple studies have established that the ability of CD8(+) T cells to act as cytolytic effectors and produce gamma interferon is important in mediating resistance to the intracellular parasite Toxoplasma gondii. To better understand the generation of the antigen-specific CD8(+) T-cell responses induced by T. gondii, mice were immunized with replication-deficient parasites that express the model antigen ovalbumin (OVA). Class I tetramers specific for SIINFEKL were used to track the OVA-specific endogenous CD8(+) T cells. The peak CD8(+) T-cell response was found at day 10 postimmunization, after which the frequency and numbers of antigen-specific cells declined. Unexpectedly, replication-deficient parasites were found to induce antigen-specific cells with faster kinetics than replicating parasites. The generation of optimal numbers of antigen-specific CD8(+) effector T cells was found to require CD4(+) T-cell help. At 7 days following immunization, antigen-specific cells were found to be CD62L(low), KLRG1(+), and CD127(low), and they maintained this phenotype for more than 70 days. Antigen-specific CD8(+) effector T cells in immunized mice exhibited potent perforin-dependent OVA-specific cytolytic activity in vivo. Perforin-dependent cytolysis appeared to be the major cytolytic mechanism; however, a perforin-independent pathway that was not mediated via Fas-FasL was also detected. This study provides further insight into vaccine-induced cytotoxic T-lymphocyte responses that correlate with protective immunity to T. gondii and identifies a critical role for CD4(+) T cells in the generation of protective CD8(+) T-cell responses.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Vacunas Antiprotozoos/inmunología , Toxoplasma/inmunología , Animales , Linfocitos T CD4-Positivos/fisiología , Citotoxicidad Inmunológica , Inmunización , Inmunofenotipificación , Cinética , Ratones , Ratones Endogámicos C57BL , Ovalbúmina/inmunologíaRESUMEN
Infection with the parasite Toxoplasma gondii leads to the induction of a Th1-type response dominated by IFN-gamma production and control of this pathogen. Cells of the innate immune system are essential in initiating this response both through the production of IL-12 as well as the presentation of parasite-derived Ags to MHC-restricted T cells. Although dendritic cells (DCs) have been implicated in these events, the contribution of individual DC populations remains unclear. Therefore, multiparameter flow cytometry was used to identify and characterize subsets of murine DCs during acute toxoplasmosis. This approach confirmed that infection leads to the expansion and activation of conventional DC (cDC) subsets. Unexpectedly, however, this analysis further revealed that plasmacytoid DCs are also expanded and that these cells up-regulate MHC class II and costimulatory molecules associated with their acquired ability to prime naive CD4(+) T cells. Furthermore, T. gondii-activated plasmacytoid DCs produce high levels of IL-12 and both plasmacytoid DC maturation and cytokine production are dependent on TLR11. Together these studies suggest that pDCs are a prominent DC subset involved in the initial stages of T. gondii infection, presenting parasite Ags and producing cytokines that are important for controlling infection.
Asunto(s)
Células Dendríticas/inmunología , Interferón gamma/inmunología , Interleucina-12/inmunología , Células Plasmáticas/inmunología , Células TH1/inmunología , Toxoplasma/inmunología , Toxoplasmosis/inmunología , Animales , Presentación de Antígeno/genética , Presentación de Antígeno/inmunología , Antígenos de Protozoos/inmunología , Antígenos de Histocompatibilidad Clase II/genética , Antígenos de Histocompatibilidad Clase II/inmunología , Interferón gamma/genética , Interleucina-12/genética , Ratones , Ratones Noqueados , Células TH1/parasitología , Receptores Toll-Like/inmunología , Toxoplasmosis/genética , Regulación hacia Arriba/genética , Regulación hacia Arriba/inmunologíaRESUMEN
Challenge with the intracellular protozoan parasite Toxoplasma gondii induces a potent CD8+ T-cell response that is required for resistance to infection, but many questions remain about the factors that regulate the presentation of major histocompatibility complex class I (MHC-I)-restricted parasite antigens and about the role of professional and nonprofessional accessory cells. In order to address these issues, transgenic parasites expressing ovalbumin (OVA), reagents that track OVA/MHC-I presentation, and OVA-specific CD8+ T cells were exploited to compare the abilities of different infected cell types to stimulate CD8+ T cells and to define the factors that contribute to antigen processing. These studies reveal that a variety of infected cell types, including hematopoietic and nonhematopoietic cells, are capable of activating an OVA-specific CD8+ T-cell hybridoma, and that this phenomenon is dependent on the transporter associated with antigen processing and requires live T. gondii. Several experimental approaches indicate that T-cell activation is a consequence of direct presentation by infected host cells rather than cross-presentation. Surprisingly, nonprofessional antigen-presenting cells (APCs) were at least as efficient as dendritic cells at activating this MHC-I-restricted response. Studies to assess whether these cells are involved in initiation of the CD8+ T-cell response to T. gondii in vivo show that chimeric mice expressing MHC-I only in nonhematopoietic compartments are able to activate OVA-specific CD8+ T cells upon challenge. These findings associate nonprofessional APCs with the initial activation of CD8+ T cells during toxoplasmosis.
Asunto(s)
Células Presentadoras de Antígenos/inmunología , Antígenos de Protozoos/metabolismo , Linfocitos T CD8-positivos/inmunología , Antígenos de Histocompatibilidad Clase I/inmunología , Toxoplasma/inmunología , Toxoplasmosis/inmunología , Animales , Antígenos de Protozoos/inmunología , Femenino , Genes Reporteros , Activación de Linfocitos/fisiología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Transgénicos , Ovalbúmina/genética , Ovalbúmina/inmunología , Ovalbúmina/metabolismoRESUMEN
Aside from being the precursors of the Ab-secreting cells, B cells are engaged in other immune functions such as Ag presentation to T cells or cytokine production. These functions may contribute to the pathogenic role of B cells in a wide range of autoimmune diseases. We demonstrate that B cells acquire the capacity to amplify IFN-gamma production by CD4 and CD8 T cells during the course of the Th1 inflammatory response to Toxoplasma gondii infection. Using the two following different strategies, we observed that B cells from T. gondii-infected mice, but not from naive mice, induce higher IFN-gamma expression by splenic host T cells: 1) reconstitution of B cell-deficient mice with B cells expressing an alloantigen different from the recipients, and 2) adoptive transfer of B and T cells into RAG-/- mice. In vitro assays allowing the physical separation of T and B cells demonstrate that Ag-primed B cells enhance IFN-gamma production by T cells in a contact-dependent fashion. Using an OVA-transgenic strain of T. gondii and OVA-specific CD4 T cells, we observed that the proinflammatory effect of B cells is neither Ag specific nor requires MHCII expression. However, TNF-alpha expressed on the surface of B cells appears to mediate in part the up-regulation of IFN-gamma by the effector T cells.
Asunto(s)
Linfocitos B/metabolismo , Interferón gamma/biosíntesis , Linfocitos T/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Presentación de Antígeno/inmunología , Linfocitos B/inmunología , Quimiocina CCL3 , Quimiocina CCL4 , Quimiocinas CC/metabolismo , Femenino , Antígenos de Histocompatibilidad Clase II/inmunología , Inflamación/inmunología , Activación de Linfocitos/inmunología , Proteínas Inflamatorias de Macrófagos/metabolismo , Ratones , Ratones Noqueados , Bazo/inmunología , Bazo/metabolismo , Linfocitos T/inmunología , Factor de Necrosis Tumoral alfa/deficiencia , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
Despite its large size and the numerous processes in which it is implicated, neither the identity nor the functions of the proteins targeted to the yeast vacuole have been defined comprehensively. In order to establish a methodological platform and protein inventory to address this shortfall, we refined techniques for the purification of 'proteomics-grade' intact vacuoles. As confirmed by retention of the preloaded fluorescent conjugate glutathione-bimane throughout the fractionation procedure, the resistance of soluble proteins that copurify with this fraction to digestion by exogenous extravacuolar proteinase K, and the results of flow cytometric, western and marker enzyme activity analyses, vacuoles prepared in this way retain most of their protein content and are of high purity and integrity. Using this material, 360 polypeptides species associated with the soluble fraction of the vacuolar isolates were resolved reproducibly by 2D gel electrophoresis. Of these, 260 were identified by peptide mass fingerprinting and peptide sequencing by MALDI-MS and liquid chromatography coupled to ion trap or quadrupole TOF tandem MS, respectively. The polypeptides identified in this way, many of which correspond to alternate size and charge states of the same parent translation product, can be assigned to 117 unique ORFs. Most of the proteins identified are canonical vacuolar proteases, glycosidases, phosphohydrolases, lipid-binding proteins or established vacuolar proteins of unknown function, or other proteases, glycosidases, lipid-binding proteins, regulatory proteins or proteins involved in intermediary metabolism, protein synthesis, folding or targeting, or the alleviation of oxidative stress. On the basis of the high purity of the vacuolar preparations, the electrophoretic properties of the proteins identified and the results of quantitative proteinase K protection measurements, many of the noncanonical vacuolar proteins identified are concluded to have entered this compartment for breakdown, processing and/or salvage purposes.
Asunto(s)
Proteoma/análisis , Proteínas de Saccharomyces cerevisiae/análisis , Saccharomyces cerevisiae/metabolismo , Vacuolas/metabolismo , Electroforesis en Gel Bidimensional , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Microscopía Fluorescente , Proteoma/genética , Proteoma/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
IL-10 plays a vital role in controlling the inflammatory response during acute Toxoplasma gondii infection, however the production of IL-10 during the chronic phase of toxoplasmosis has been associated with parasite persistence. To address this paradox, the production and effect of IL-10 in the brain during toxoplasmic encephalitis (TE) was investigated. Analysis of brain mononuclear cells (BMNC) from chronically infected mice revealed that infiltrating macrophages and CD4(+) T cells were the major sources of IL-10. Endogenous levels of IL-10 inhibited the production of IL-12, IFN-gamma, TNF-alpha, and IL-6 from both hematopoetic and non-hematopoetic cells in the brain, as well as anti-microbial activity of astrocytes. Furthermore, IL-10-/- mice that progressed to the chronic phase of infection had equivalent parasite burden to WT mice but developed a lethal inflammatory response within the brain characterized by increased numbers of CD4(+) T cells and macrophages, and elevated production of inflammatory cytokines. Finally, partial depletion of CD4(+) T cells decreased the severity of the inflammation in the brain and allowed IL-10-/- mice to survive infection. Together these results point to a vital role for IL-10 in the control of CD4(+) T cell mediated inflammation in the brain during TE.
Asunto(s)
Encefalitis/inmunología , Encefalitis/prevención & control , Mediadores de Inflamación/fisiología , Interleucina-10/fisiología , Toxoplasmosis Cerebral/inmunología , Toxoplasmosis Cerebral/prevención & control , Animales , Astrocitos/inmunología , Astrocitos/metabolismo , Astrocitos/patología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/parasitología , Linfocitos T CD4-Positivos/patología , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Células Cultivadas , Enfermedad Crónica , Regulación hacia Abajo/inmunología , Encefalitis/parasitología , Encefalitis/patología , Femenino , Mediadores de Inflamación/metabolismo , Interleucina-10/biosíntesis , Interleucina-10/deficiencia , Interleucina-10/genética , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Ratones Noqueados , Toxoplasma/crecimiento & desarrollo , Toxoplasma/inmunología , Toxoplasmosis Cerebral/patologíaRESUMEN
The study of the immune response to Toxoplasma gondii has provided numerous insights into the role of T cells in resistance to intracellular infections. However, the complexity of this eukaryote pathogen has made it difficult to characterize immunodominant epitopes that would allow the identification of T cells with a known specificity for parasite antigens. As a consequence, analysis of T-cell responses to T. gondii has been based on characterization of the percentage of T cells that express an activated phenotype during infection and on the ability of these cells to produce cytokines in response to complex mixtures of parasite antigens. In order to study specific CD4(+) T cells responses to T. gondii, recombinant parasites that express a truncated ovalbumin (OVA) protein, in either a cytosolic or a secreted form, were engineered. In vitro and in vivo studies reveal that transgenic parasites expressing secreted OVA are able to stimulate T-cell receptor-transgenic OVA-specific CD4(+) T cells to proliferate, express an activated phenotype, and produce gamma interferon (IFN-gamma). Furthermore, the adoptive transfer of OVA-specific T cells into IFN-gamma(-/-) mice provided enhanced protection against infection with the OVA-transgenic (but not parental) parasites. Together, these studies establish the utility of this transgenic system to study CD4(+)-T-cell responses during toxoplasmosis.
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Linfocitos T CD4-Positivos/inmunología , Ovalbúmina/inmunología , Toxoplasma/inmunología , Toxoplasmosis Animal/inmunología , Traslado Adoptivo , Animales , Animales Modificados Genéticamente , Femenino , Ingeniería Genética , Interferón gamma/biosíntesis , Ratones , Ratones Endogámicos BALB C , Ovalbúmina/genética , Proteínas Recombinantes/inmunologíaRESUMEN
Parasite differentiation is commonly associated with transitions between complex life cycle stages and with long-term persistence in the host, and it is therefore critical for pathogenesis. In the protozoan parasite Toxoplasma gondii, interconversion between rapidly growing tachyzoites and latent encysted bradyzoites is accompanied by numerous morphological and metabolic adaptations. In order to explore early cell biological events associated with this differentiation process, we have exploited fluorescent reporter proteins targeted to various subcellular locations. Combining these markers with efficient in vitro differentiation and time-lapse video microscopy provides a dynamic view of bradyzoite development in living cultures, demonstrating subcellular reorganization, maintenance of the mitochondrion, and missegregation of the apicoplast. Bradyzoites divide asynchronously, using both endodyogeny and endopolygeny, and are highly motile both within and between host cells. Cysts are able to proliferate without passing through an intermediate tachyzoite stage, via both the migration of free bradyzoites and the fission of bradyzoite cysts, suggesting a mechanism for dissemination during chronic infection.
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Diferenciación Celular/fisiología , Proteínas Protozoarias/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Toxoplasma/citología , Toxoplasma/fisiología , Animales , Forma de la Célula , Fibroblastos/citología , Fibroblastos/parasitología , Fibroblastos/fisiología , Humanos , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Mitocondrias/metabolismo , Proteínas Protozoarias/genética , Proteínas Recombinantes de Fusión/genética , Toxoplasma/genética , Vacuolas/parasitologíaRESUMEN
Despite its noted ability to induce strong cellular immunity, and its known susceptibility to IFN-gamma-dependent immune effector mechanisms, the protozoan Toxoplasma gondii is a highly successful parasite, able to replicate, disseminate, and either kill the host or, more commonly, establish resistant encysted life forms before the emergence of protective immune responses. We sought to understand how the parasite gains the advantage. Using transgenic clonal parasite lines engineered to express fluorescent markers in combination with dendritic cells (DC) grown from the bone marrow of wild-type mice or transgenic mice expressing fluorescent protein-tagged MHC class II molecules, we used flow cytometry and fluorescence microscopy to analyze the responses of infected DC to both invasion by the parasite and subsequent DC maturation signals. We found that T. gondii preferentially invades immature dendritic cells but fails to activate them in the process, and renders them resistant to subsequent activation by TLR ligands or the immune-system-intrinsic maturation signal CD40L. The functional consequences of T. gondii-mediated suppression of DC activation are manifested in a relative inability of infected immature DC to activate naive CD4(+) Th lymphocytes, or to secrete cytokines, such IL-12 and TNF-alpha, that play important roles in innate and/or adaptive immunity. The findings reveal that T. gondii suppresses the ability of immature DC to participate in innate immunity and to induce adaptive immune responses. The ability of T. gondii to temporarily evade recognition could provide a selective advantage that permits dissemination and establishment before adaptive immune response initiation.
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Células Dendríticas/inmunología , Células Dendríticas/parasitología , Toxoplasma/inmunología , Toxoplasmosis/inmunología , Animales , Diferenciación Celular/inmunología , Células Cultivadas , Células Dendríticas/citología , Femenino , Citometría de Flujo , Activación de Linfocitos/inmunología , Masculino , Ratones , Ratones Transgénicos , Microscopía Fluorescente , Microscopía por VideoRESUMEN
We report the functional characterization of a new UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase (ppGalNAc-T) (EC 2.4.1.41) from the human disease-causing parasite, Toxoplasma gondii. This glycosyltransferase is denoted as T. gondii ppGalNAc-T3. These enzymes are responsible for the initial step of mucin-type O-glycosylation: the transfer of GalNAc from the UDP-GalNAc nucleotide sugar donor onto a peptide acceptor. Following an in silico analysis of the publicly available T. gondii DNA database, we used molecular biology approaches to identify and isolate the cDNA encoding this enzyme. The resulting type II membrane protein contains N-terminal cytoplasmic, transmembrane, and C-terminal lumenal domains. Conceptual translation of the cDNA sequence also reveals a stem region and the presence of several important sequence motifs. When the recombinant construct was expressed in stably transfected Drosophila melanogaster S2 cells, the purified protein exhibited glycosyltransferase activity in vitro against glycopeptide, but not "naked" peptide, acceptors. In addition, using reverse transcriptase-PCR, T. gondii ppGalNAc-T3 mRNA was equivalently expressed during the tachyzoite and bradyzoite developmental stages. The identification of T. gondii ppGalNAc-T3 as a functional "follow-up" glycopeptide glycosyltransferase further confirms that this human parasite has its own enzymatic O-glycosylation machinery.