RESUMEN
We demonstrate the role of signaling via the glucocorticoid receptor, NR3C1, in differentiation of CD8+ T cell memory. Pharmacological inhibition as well as the short hairpin RNA-mediated knockdown of the receptor hindered memory transition and limited the homeostatic turnover of the activated CD8+ T cells. Dexamethasone exposure of CD8+ T cells expanded during a resolving infection with influenza A virus or a γ-herpesvirus promoted conversion of effector cells into memory cells by modulating cellular metabolism and lowering the accumulation of reactive oxygen species. Reduced reactive oxygen species levels in the responding effector cells upregulated Bcl2 and enhanced survival. The generated virus-specific memory CD8+ T cells were efficiently recalled following challenge of animals with a secondary infection to control it better. The memory-enhancing effect was predominantly evident at low doses of dexamethasone. Therefore, controlled glucocorticoid signaling within the effector CD8+ T cells is crucial for optimal memory differentiation.
RESUMEN
Novel vaccination strategies are crucial to efficiently control tuberculosis, as proposed by the World Health Organization under its flagship program "End TB Strategy." However, the emergence of drug-resistant strains of Mycobacterium tuberculosis (Mtb), particularly in those coinfected with HIV-AIDS, constitutes a major impediment to achieving this goal. We report here a novel vaccination strategy that involves synthesizing a formulation of an immunodominant peptide derived from the Acr1 protein of Mtb. This nanoformulation in addition displayed on the surface a toll-like receptor-2 ligand to offer to target dendritic cells (DCs). Our results showed an efficient uptake of such a concoction by DCs in a predominantly toll-like receptor-2-dependent pathway. These DCs produced elevated levels of nitric oxide, proinflammatory cytokines interleukin-6, interleukin-12, and tumor necrosis factor-α, and upregulated the surface expression of major histocompatibility complex class II molecules as well as costimulatory molecules such as CD80 and CD86. Animals injected with such a vaccine mounted a significantly higher response of effector and memory Th1 cells and Th17 cells. Furthermore, we noticed a reduction in the bacterial load in the lungs of animals challenged with aerosolized live Mtb. Therefore, our findings indicated that the described vaccine triggered protective anti-Mtb immunity to control the tuberculosis infection.
Asunto(s)
Mycobacterium tuberculosis , Tuberculosis , Animales , Células Dendríticas , Epítopos , Ligandos , Mycobacterium tuberculosis/metabolismo , Receptor Toll-Like 2/metabolismo , Tuberculosis/prevención & control , Tuberculosis/microbiología , RatonesRESUMEN
Peste des petits ruminants virus (PPRV) is known to induce transient immunosuppression in infected small ruminants by modulating several cellular pathways involved in the antiviral immune response. Our study shows that the PPRV-coded non-structural proteins C and V can interact with the cellular NF-κB p65 subunit. The PPRV-C protein interacts with the transactivation domain (TAD) while PPRV-V interacts with the Rel homology domain (RHD) of the NF-κB p65 subunit. Both viral proteins can suppress the NF-κB transcriptional activity and NF-κB-mediated transcription of cellular genes. PPRV-V protein expression can significantly inhibit the nuclear translocation of NF-κB p65 upon TNF-α stimulation, whereas PPRV-C does not affect it. The NF-κB-mediated pro-inflammatory cytokine gene expression is significantly downregulated in cells expressing PPRV-C or PPRV-V protein. Our study provides evidence suggesting a role of PPRV non-structural proteins V and C in the modulation of NF-κB signalling through interaction with the NF-κB p65 subunit.
Asunto(s)
Peste de los Pequeños Rumiantes , Virus de la Peste de los Pequeños Rumiantes , Animales , Humanos , Virus de la Peste de los Pequeños Rumiantes/genética , Peste de los Pequeños Rumiantes/metabolismo , Citocinas/genética , Citocinas/metabolismo , FN-kappa B/genética , FN-kappa B/metabolismo , Proteínas Virales/genética , Proteínas Virales/metabolismo , Rumiantes , Expresión Génica , Cabras/genéticaRESUMEN
In this study, we investigated the response of myeloid-derived suppressor cells (MDSCs) during the pathogenesis of an immunoblinding disease of the cornea caused by HSV type 1 infection. We also measured the anti-inflammatory potential of in vitro-differentiated MDSCs in dampening herpetic stromal keratitis resulting from primary ocular HSV1 infection in mice. In the lymphoid organs and inflamed corneal tissues, MDSCs were phenotypically characterized as CD11b+Gr1lo-int cells. Sorted CD11b+Gr1lo-int cells, but not CD11b+Gr1hi cells, suppressed the proliferation and cytokine production by stimulated CD4+ T cells. In vitro-generated MDSCs inhibited the activity of stimulated CD4+ T cells in a predominantly contact-dependent manner. An adoptive transfer of in vitro-generated MDSCs before or after ocular HSV1 infection controlled herpetic stromal keratitis lesions. The transferred MDSCs were primarily recovered from the lymphoid organs of recipients. Surprisingly, MDSCs recipients expanded their endogenous Foxp3+ regulatory T cells (Tregs). We further demonstrated the MDSCs mediated stabilization of Foxp3 expression in already differentiated Tregs and their ability to cause an efficient de novo conversion of Foxp3+ Tregs from stimulated Foxp3-CD4+ T cells. These effects occurred independent of TGF-ß signaling. Therefore, the therapeutic potential of MDSCs could be harnessed as a multipronged strategy to confer an infectious tolerance to the host by activating endogenous regulatory mechanisms.
Asunto(s)
Herpes Simple/inmunología , Herpesvirus Humano 1/inmunología , Tolerancia Inmunológica/inmunología , Inflamación/inmunología , Células Supresoras de Origen Mieloide/inmunología , Traslado Adoptivo/métodos , Animales , Antígeno CD11b/inmunología , Linfocitos T CD4-Positivos/inmunología , Proliferación Celular/fisiología , Femenino , Factores de Transcripción Forkhead/inmunología , Inflamación/virología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Células Supresoras de Origen Mieloide/virología , Transducción de Señal/inmunología , Linfocitos T Reguladores/inmunología , Factor de Crecimiento Transformador beta/inmunologíaRESUMEN
Galectins play diverse roles in pathophysiology of infectious diseases and cancers. Galectin-3 is one of the most studied family member and the only chimeric type lectin. Many aspects of its biogenesis, range of activities, and the disease-modifying potential particularly during microbial infections are yet to be known. We review our current understanding of these issues and also highlight gaps in better defining the immune modulatory potential of galectin-3 during different stages of host responsiveness when an infection sets in. Additionally, we discuss commonly used strategies to disrupt galectin-3 functions both extracellulalry and intracellularly. Existing and improved novel strategies could help fine-tune immune responses to achieve better prognosis of infectious diseases.
Asunto(s)
Antiinfecciosos/inmunología , Enfermedades Transmisibles/inmunología , Galectina 3/inmunología , Animales , HumanosRESUMEN
The host response to viruses includes multiple cell types that have regulatory function. Most information focuses on CD4(+) regulatory T cells that express the transcription factor Foxp3(+) (Tregs), which are the topic of this review. We explain how viruses through specific and non-specific means can trigger the response of thymus-derived natural Tregs as well as induce Tregs. The latter derive under appropriate stimulation conditions either from uncommitted precursors or from differentiated cells that convert to become Tregs. We describe instances where Tregs appear to limit the efficacy of antiviral protective immunity and other, perhaps more common, immune-mediated inflammatory conditions, where the Tregs function to limit the extent of tissue damage that occurs during a virus infection. We discuss the controversial roles that Tregs may play in the pathogenesis of human immunodeficiency and hepatitis C virus infections. The issue of plasticity is discussed, as this may result in Tregs losing their protective function when present in inflammatory environments. Finally, we mention approaches used to manipulate Treg numbers and function and assess their current value and likely future success to manage the outcome of virus infection, especially those that are responsible for chronic tissue damage.
Asunto(s)
Linfocitos T Reguladores/inmunología , Virosis/inmunología , Virus/inmunología , Animales , Humanos , Inmunoterapia Adoptiva , Linfocitos T Reguladores/metabolismo , Virosis/metabolismo , Virosis/terapiaRESUMEN
A monoclonal antibody against the C-type lectin DEC205 (αDEC205) is an effective vehicle for delivery of antigens to dendritic cells through creation of covalent αDEC205-antigen adducts. These adducts can induce antigen-specific T-cell immune responses or tolerance. We exploit the transpeptidase activity of sortase to install modified peptides and protein-sized antigens onto the heavy chain of αDEC205, including linkers that contain nonnatural amino acids. We demonstrate stoichiometric site-specific labeling on a scale not easily achievable by genetic fusions (49 distinct fusions in this report). We conjugated a biotinylated version of a class I MHC-restricted epitope to unlabeled αDEC205 and monitored epitope generation upon binding of the adduct to dendritic cells. Our results show transfer of αDEC205 heavy chain to the cytoplasm, followed by proteasomal degradation. Introduction of a labile dipeptide linker at the N terminus of a T-cell epitope improves proteasome-dependent class I MHC-restricted peptide cross-presentation when delivered by αDEC205 in vitro and in vivo. We also conjugated αDEC205 with a linker-optimized peptide library of known CD8 T-cell epitopes from the mouse γ-herpes virus 68. Animals immunized with such conjugates displayed a 10-fold reduction in viral load.
Asunto(s)
Aminoaciltransferasas/metabolismo , Antígenos CD/inmunología , Antígenos CD/metabolismo , Antígenos Virales/inmunología , Proteínas Bacterianas/metabolismo , Cisteína Endopeptidasas/metabolismo , Epítopos de Linfocito T/inmunología , Lectinas Tipo C/inmunología , Lectinas Tipo C/metabolismo , Receptores de Superficie Celular/inmunología , Receptores de Superficie Celular/metabolismo , Secuencia de Aminoácidos , Animales , Presentación de Antígeno , Antígenos CD/química , Antígenos CD/genética , Antígenos Virales/química , Antígenos Virales/genética , Linfocitos T CD8-positivos/inmunología , Epítopos de Linfocito T/química , Epítopos de Linfocito T/genética , Infecciones por Herpesviridae/inmunología , Infecciones por Herpesviridae/prevención & control , Antígenos de Histocompatibilidad Clase I/metabolismo , Inmunización , Inmunoconjugados/genética , Inmunoconjugados/inmunología , Inmunoconjugados/metabolismo , Lectinas Tipo C/química , Lectinas Tipo C/genética , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Antígenos de Histocompatibilidad Menor , Datos de Secuencia Molecular , Complejo de la Endopetidasa Proteasomal/metabolismo , Ingeniería de Proteínas , Receptores de Superficie Celular/química , Receptores de Superficie Celular/genética , Rhadinovirus/genética , Rhadinovirus/inmunología , Infecciones Tumorales por Virus/inmunología , Infecciones Tumorales por Virus/prevención & control , Vacunas de Subunidad/genética , Vacunas de Subunidad/inmunología , Vacunas Virales/genética , Vacunas Virales/inmunologíaRESUMEN
Antigen presenting cells (APCs) that express a catalytically inactive version of the deubiquitylase YOD1 (YOD1-C160S) present exogenous antigens more efficiently to CD8(+) T cells, both in vitro and in vivo. Compared with controls, immunization of YOD1-C160S mice led to greater expansion of specific CD8(+) T cells and showed improved control of infection with a recombinant -herpes virus, MHV-68, engineered to express SIINFEKL peptide, the ligand for the ovalbumin-specific TCR transgenic OT-I cells. Enhanced expansion of specific CD8(+) T cells was likewise observed on infection of YOD1-C160S mice with a recombinant influenza A virus expressing SIINFEKL. YOD1-C160S APCs retained antigen longer than did control APCs. Enhanced crosspresentation by YOD1-C160S APCs was transporter associated with antigen processing (TAP1)-independent but sensitive to inclusion of inhibitors of acidification and of the proteasome. The activity of deubiquitylating enzymes may thus help control antigenspecific CD8(+) T-cell responses during immunization.
Asunto(s)
Reactividad Cruzada , Mutación Missense , Ubiquitina Tiolesterasa/genética , Ubiquitina Tiolesterasa/inmunología , Transportador de Casetes de Unión a ATP, Subfamilia B, Miembro 2 , Transportadoras de Casetes de Unión a ATP/deficiencia , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/inmunología , Traslado Adoptivo , Animales , Células Presentadoras de Antígenos/enzimología , Células Presentadoras de Antígenos/inmunología , Brefeldino A/farmacología , Linfocitos T CD8-positivos/inmunología , Reactividad Cruzada/efectos de los fármacos , Reactividad Cruzada/genética , Femenino , Concentración de Iones de Hidrógeno , Inmunización , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Ovalbúmina/inmunología , Fragmentos de Péptidos/inmunología , Rhadinovirus/inmunología , Rhadinovirus/patogenicidad , Ubiquitina Tiolesterasa/metabolismoRESUMEN
Stromal keratitis is a chronic immunopathological lesion of the eye caused by HSV-1 infection that can result in blindness. Because the inflammatory lesions are primarily orchestrated by Th1 cells, and to a lesser extent by Th17 cells, inhibiting their activity represents a useful form of therapy. In this study we evaluated the therapeutic potential of galectin-1 (gal-1), an endogenous lectin that in some autoimmune diseases was shown to suppress the functions of Th1 and Th17 cells. Treatment was begun at different times after ocular infection with HSV and the outcome was assessed clinically as well as for effects on various immune parameters. Treatment with recombinant gal-1 significantly diminished stromal keratitis lesion severity and the extent of corneal neovascularization. Treated mice had reduced numbers of IFN-γ- and IL-17-producing CD4(+) T cells, as well as neutrophil infiltration in the cornea. Furthermore, disease severity was greater in gal-1 knockout mice compared with their wild-type counterparts. The many effects of gal-1 treatment include reduction in the production of proinflammatory cytokines and chemokines, increased production of IL-10, and inhibitory effects on molecules involved in neovascularization. To our knowledge, our findings are the first to show that gal-1 treatment represents a useful approach to control lesion severity in a virally induced immunopathological disease.
Asunto(s)
Neovascularización de la Córnea/inmunología , Galectina 1/inmunología , Herpesvirus Humano 1/inmunología , Queratitis Herpética/inmunología , Animales , Ceguera/genética , Ceguera/inmunología , Ceguera/metabolismo , Ceguera/patología , Ceguera/virología , Neovascularización de la Córnea/genética , Neovascularización de la Córnea/metabolismo , Neovascularización de la Córnea/patología , Neovascularización de la Córnea/virología , Galectina 1/genética , Galectina 1/metabolismo , Herpesvirus Humano 1/genética , Herpesvirus Humano 1/metabolismo , Interleucina-10/genética , Interleucina-10/inmunología , Interleucina-10/metabolismo , Queratitis Herpética/genética , Queratitis Herpética/metabolismo , Queratitis Herpética/patología , Ratones , Ratones Noqueados , Células TH1/inmunología , Células TH1/metabolismo , Células TH1/patología , Células Th17/inmunología , Células Th17/metabolismo , Células Th17/patologíaRESUMEN
How therapeutically administered myeloid derived suppressor cells (MDSCs) modulate differentiation of virus-specific CD8+ T cell was investigated. In vitro generated MDSCs from bone marrow precursors inhibited the expansion of stimulated CD8+ T cells but the effector cells in the recipients of MDSCs showed preferential memory transition during Influenza A virus (IAV) or an α- (Herpes Simplex Virus) as well as a γ- (murine herpesvirus 68) herpesvirus infection. Memory CD8+ T cells thus generated constituted a heterogenous population with a large fraction showing effector memory (CD62LloCCR7-) phenotype. Such cells could be efficiently recalled in the rechallenged animals and controlled the secondary infection better. Memory potentiating effects of MDSCs occurred irrespective of the clonality of the responding CD8+ T cells as well as the nature of infecting viruses. Compared to the MDSCs recipients, effector cells of MDSCs recipients showed higher expression of molecules known to drive cellular survival (IL-7R, Bcl2) and memory formation (Tcf7, Id3, eomesodermin). Therapeutically administered MDSCs not only mitigated the tissue damaging response during a resolving IAV infection but also promoted the differentiation of functional memory CD8+ T cells. Therefore, MDSCs therapy could be useful in managing virus-induced immunopathological reactions without compromising immunological memory.
Asunto(s)
Células Supresoras de Origen Mieloide , Ratones , Animales , Linfocitos T CD8-positivos , Memoria Inmunológica , Ratones Endogámicos C57BLRESUMEN
Morphine addiction poses a significant challenge to global healthcare. Current opioid substitution therapies, such as buprenorphine, naloxone and methadone are effective but often lead to dependence. Thus, exploring alternative treatments for opioid addiction is crucial. We have developed a novel vaccine that presents morphine and Pam3Cys (a TLR-2 agonist) on the surface of Acr1 nanoparticles. This vaccine has self-adjuvant properties and targets TLR-2 receptors on antigen-presenting cells, particularly dendritic cells. Our vaccination strategy promotes the proliferation and differentiation of morphine-specific B-cells and Acr1-reactive CD4 T-cells. Additionally, the vaccine elicited the production of high-affinity anti-morphine antibodies, effectively eliminating morphine from the bloodstream and brain in mice. It also reduced the expression of addiction-associated µ-opioid receptor and dopamine genes. The significant increase in memory CD4 T-cells and B-cells indicates the vaccine's ability to induce long-lasting immunity against morphine. This vaccine holds promise as a prophylactic measure against morphine addiction.
Asunto(s)
Células Dendríticas , Morfina , Nanovacunas , Receptor Toll-Like 2 , Animales , Ratones , Linfocitos B/inmunología , Linfocitos B/efectos de los fármacos , Linfocitos B/metabolismo , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD4-Positivos/metabolismo , Células Dendríticas/inmunología , Células Dendríticas/efectos de los fármacos , Células Dendríticas/metabolismo , Morfina/farmacología , Dependencia de Morfina/inmunología , Dependencia de Morfina/tratamiento farmacológico , Nanovacunas/inmunología , Receptores Opioides mu/inmunología , Receptores Opioides mu/metabolismo , Receptor Toll-Like 2/metabolismoRESUMEN
The normal cornea is transparent, which is essential for normal vision, and although the angiogenic factor vascular endothelial growth factor A (VEGF-A) is present in the cornea, its angiogenic activity is impeded by being bound to a soluble form of the VEGF receptor-1 (sVR-1). This report investigates the effect on the balance between VEGF-A and sVR-1 that occurs after ocular infection with HSV, which causes prominent neovascularization, an essential step in the pathogenesis of the vision-impairing lesion, stromal keratitis. We demonstrate that HSV-1 infection causes increased production of VEGF-A but reduces sVR-1 levels, resulting in an imbalance of VEGF-A and sVR-1 levels in ocular tissues. Moreover, the sVR-1 protein made was degraded by the metalloproteinase (MMP) enzymes MMP-2, -7, and -9 produced by infiltrating inflammatory cells that were principally neutrophils. Inhibition of neutrophils, inhibition of sVR-1 breakdown with the MMP inhibitor marimastat, and the provision of exogenous recombinant sVR-1 protein all resulted in reduced angiogenesis. Our results make the novel observation that ocular neovascularization resulting from HSV infection involves a change in the balance between VEGF-A and its soluble inhibitory receptor. Future therapies aimed to increase the production and activity of sVR-1 protein could benefit the management of stromal keratitis, an important cause of human blindness.
Asunto(s)
Neovascularización de la Córnea/inmunología , Neovascularización de la Córnea/virología , Herpes Simple/inmunología , Herpes Simple/virología , Factor A de Crecimiento Endotelial Vascular/metabolismo , Receptor 1 de Factores de Crecimiento Endotelial Vascular/metabolismo , Animales , Línea Celular Transformada , Neovascularización de la Córnea/metabolismo , Femenino , Herpes Simple/metabolismo , Herpesvirus Humano 1/inmunología , Ratones , Ratones Endogámicos C57BL , Unión Proteica/inmunología , Distribución Aleatoria , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Factor A de Crecimiento Endotelial Vascular/biosíntesis , Receptor 1 de Factores de Crecimiento Endotelial Vascular/antagonistas & inhibidoresRESUMEN
After HSV-1 infection, CD8(+) T cells accumulate in the trigeminal ganglion (TG) and participate in the maintenance of latency. However, the mechanisms underlying intermittent virus reactivation are poorly understood. In this study, we demonstrate the role of an inhibitory interaction between T cell Ig and mucin domain-containing molecule 3 (Tim-3)-expressing CD8(+) T cells and galectin 9 (Gal-9) that could influence HSV-1 latency and reactivation. Accordingly, we show that most K(b)-gB tetramer-specific CD8(+) T cells in the TG of HSV-1-infected mice express Tim-3, a molecule that delivers negative signals to CD8(+) T cells upon engagement of its ligand Gal-9. Gal-9 was also upregulated in the TG when replicating virus was present as well during latency. This could set the stage for Gal-9/Tim-3 interaction, and this inhibitory interaction was responsible for reduced CD8(+) T cell effector function in wild-type mice. Additionally, TG cell cultures exposed to recombinant Gal-9 in the latent phase caused apoptosis of most CD8(+) T cells. Furthermore, Gal-9 knockout TG cultures showed delayed and reduced viral reactivation as compared with wild-type cultures, demonstrating the greater efficiency of CD8(+) T cells to inhibit virus reactivation in the absence of Gal-9. Moreover, the addition of recombinant Gal-9 to ex vivo TG cultures induced enhanced viral reactivation compared with untreated controls. Our results demonstrate that the host homeostatic mechanism mediated by Gal-9/Tim-3 interaction on CD8(+) T cells can influence the outcome of HSV-1 latent infection, and manipulating Gal-9 signals might represent therapeutic means to inhibit HSV-1 reactivation from latency.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Galectinas/inmunología , Herpes Simple/inmunología , Receptores Virales/inmunología , Simplexvirus/fisiología , Latencia del Virus/inmunología , Animales , Apoptosis/inmunología , Western Blotting , Linfocitos T CD8-positivos/virología , Separación Celular , Femenino , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Receptor 2 Celular del Virus de la Hepatitis A , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Ganglio del Trigémino/inmunología , Ganglio del Trigémino/virologíaRESUMEN
HSV-1 infection of the cornea leads to a blinding immunoinflammatory lesion of the eye termed stromal keratitis (SK). Recently, IL-17-producing CD4(+) T cells (Th17 cells) were shown to play a prominent role in many autoimmune conditions, but the role of IL-17 and/or of Th17 cells in virus immunopathology is unclear. In this study, we show that, after HSV infection of the cornea, IL-17 is upregulated in a biphasic manner with an initial peak production around day 2 postinfection and a second wave starting from day 7 postinfection with a steady increase until day 21 postinfection, a time point when clinical lesions are fully evident. Further studies demonstrated that innate cells, particularly γδ T cells, were major producers of IL-17 early after HSV infection. However, during the clinical phase of SK, the predominant source of IL-17 was Th17 cells that infiltrated the cornea only after the entry of Th1 cells. By ex vivo stimulation, the half fraction of IFN-γ-producing CD4(+) T cells (Th1 cells) were HSV specific, whereas very few Th17 cells responded to HSV stimulation. The delayed influx of Th17 cells in the cornea was attributed to the local chemokine and cytokine milieu. Finally, HSV infection of IL-17R knockout mice as well as IL-17 neutralization in wild-type mice showed diminished SK severity. In conclusion, our results show that IL-17 and Th17 cells contribute to the pathogenesis of SK, the most common cause of infectious blindness in the Western world.
Asunto(s)
Interleucina-17/inmunología , Queratitis Herpética/inmunología , Simplexvirus/inmunología , Células Th17/inmunología , Animales , Quimiocinas/genética , Quimiocinas/inmunología , Quimiocinas/metabolismo , Interferón gamma/genética , Interferón gamma/inmunología , Interferón gamma/metabolismo , Interleucina-17/genética , Interleucina-17/metabolismo , Queratitis Herpética/genética , Queratitis Herpética/metabolismo , Ratones , Ratones Noqueados , Receptores de Antígenos de Linfocitos T gamma-delta/genética , Receptores de Antígenos de Linfocitos T gamma-delta/inmunología , Receptores de Antígenos de Linfocitos T gamma-delta/metabolismo , Simplexvirus/genética , Simplexvirus/metabolismo , Células TH1/inmunología , Células TH1/metabolismo , Células Th17/metabolismo , Factores de Tiempo , Regulación hacia Arriba/genética , Regulación hacia Arriba/inmunologíaRESUMEN
The protocol is designed to investigate the influence of an anti-cleavage site intrabody in modulating the output of LV(CoV-2 S), a lentivirus-based pseudovirus expressing CoV-2 S protein using HEK293T cells. We clone the single-domain antibody sequence into a lentiviral vector (pLenti-GFP) for intracellular expression and assess not only the viral biogenesis but also the fate of the CoV-2 S protein in such cells. For complete details on the use and execution of this protocol, please refer to Singh et al. (2022).1.
Asunto(s)
COVID-19 , SARS-CoV-2 , Humanos , SARS-CoV-2/genética , Vectores Genéticos/genética , Células HEK293 , Lentivirus/genéticaRESUMEN
In this review, we discuss a variety of immune modulating approaches that could be used to counteract tissue-damaging viral immunoinflammatory lesions which typify many chronic viral infections. We make the point that in several viral infections the lesions can be largely the result of one or more aspects of the host response mediating the cell and tissue damage rather than the virus itself being directly responsible. However, within the reactive inflammatory lesions along with the pro-inflammatory participants there are also other aspects of the host response that may be acting to constrain the activity of the damaging components and are contributing to resolution. This scenario should provide the prospect of rebalancing the contributions of different host responses and hence diminish or even fully control the virus-induced lesions. We identify several aspects of the host reactions that influence the pattern of immune responsiveness and describe approaches that have been used successfully, mainly in model systems, to modulate the activity of damaging participants and which has led to lesion control. We emphasize examples where such therapies are, or could be, translated for practical use in the clinic to control inflammatory lesions caused by viral infections.
Asunto(s)
Modelos Biológicos , Pirimetamina , Humanos , SulfadiazinaRESUMEN
The unprecedented success of mRNA vaccines in managing the COVID-19 pandemic raises the prospect of applying the mRNA platform to other viral diseases of humans and domesticated animals, which may lead to more efficacious vaccines for some agents. We briefly discuss reasons why mRNA vaccines achieved such success against COVID-19 and indicate what other virus infections and disease conditions might also be ripe for control using mRNA vaccines. We also evaluate situations where mRNA could prove valuable to rebalance the status of immune responsiveness and achieve success as a therapeutic vaccine approach against infections that induce immunoinflammatory lesions.
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COVID-19 , Vacunas Virales , Virosis , Animales , Humanos , Vacunas contra la COVID-19/genética , COVID-19/prevención & control , Animales Domésticos , Pandemias/prevención & control , ARN Mensajero/genética , Vacunas de ARNm , Vacunas Virales/genéticaRESUMEN
Morphine is a potent analgesic opiate used to treat chronic pain, mostly in cancer patients. In addition, it is widely used as a drug of abuse. Due to the continuous rise of morphine-associated addiction, there is an urgent need to develop pre-clinical animal models to understand the behavioural pattern of drug dependence and its withdrawal. Recently, the experimental use of zebrafish has attained significance in behavioural neuroscience studies. The literature on zebrafish is conflicting with regard to morphine withdrawal symptoms. Unfortunately, no single model provides comprehensive details to evaluate zebrafish behaviour on opiate exposure. Further, the current models have various limitations, such as short duration, complexity of phenotypes, intricate quantification, and difficulty in studying withdrawal symptoms. Consequently, a firm standardization of the protocol to understand the influence of opiates on physiological and psychological behaviours is required. In this study, we have tried to overcome the shortcomings associated with the existing models and to optimize the protocols involving an array of parameters. We observed that the administration of morphine caused a significant increase in zebrafish behavioural patterns of spiral movements, circular movements, erratic movements, upper transitions, water surface transitions, wall licking, wall licking with upper transitions, wall licking with lower transitions, absolute angle changes, and time spent in the upper compartment. A decline in the freezing bouts and time spent in the lower compartment were noticed. In essence, this study offers a zebrafish model to comprehensively examine changes in behaviour of animals on opiate dependence and its withdrawal. The present study also reported that in zebrafish, the influence of chronic exposure of morphine modulates key gene targets involved in behaviour, neuroinflammation, and autophagy, which directly or indirectly are associated with morphine addiction in a chronic morphine model.
Asunto(s)
Morfina , Alcaloides Opiáceos , Animales , Pez Cebra , Autofagia , Modelos AnimalesRESUMEN
Autophagy is an essential and highly conserved catabolic process in cells, which is important in the battle against intracellular pathogens. Viruses have evolved several ways to alter the host defense mechanisms. PPRV infection is known to modulate the components of a host cell's defense system, resulting in enhanced autophagy. In this study, we demonstrate that the N protein of PPRV interacts with the core components of the class III phosphatidylinositol-3-kinase (PI3K) complex-I and results in the induction of autophagy in the host cell over, thereby expressing this viral protein. Our data shows the interaction between PPRV-N protein and different core components of the autophagy pathway, i.e., VPS34, VPS15, BECN1 and ATG14L. The PPRV-N protein can specifically interact with VPS34 of the PI3K complex-I and colocalize with the proteins of PI3K complex-I in the same sub-cellular compartment, that is, in the cytoplasm. These interactions do not affect the intracellular localization of the different host proteins. The autophagy-related genes were transcriptionally modulated in PPRV-N-expressing cells. The expression of LC3B and SQSTM1/p62 was also modulated in PPRV-N-expressing cells, indicating the induction of autophagic activity. The formation of typical autophagosomes with double membranes was visualized by transmission electron microscopy in PPRV-N-expressing cells. Taken together, our findings provide evidence for the critical role of the N protein of the PPR virus in the induction of autophagy, which is likely to be mediated by PI3K complex-I of the host.
Asunto(s)
Proteínas de la Nucleocápside , Virus de la Peste de los Pequeños Rumiantes , Fosfatidilinositol 3-Quinasas , Autofagia , FosfatidilinositolesRESUMEN
In this communication, we demonstrate that galectin (Gal)-9 acts to constrain CD8(+) T cell immunity to Herpes Simplex Virus (HSV) infection. In support of this, we show that animals unable to produce Gal-9, because of gene knockout, develop acute and memory responses to HSV that are of greater magnitude and better quality than those that occur in normal infected animals. Interestingly, infusion of normal infected mice with alpha-lactose, the sugar that binds to the carbohydrate-binding domain of Gal-9 limiting its engagement of T cell immunoglobulin and mucin (TIM-3) receptors, also caused a more elevated and higher quality CD8(+) T cell response to HSV particularly in the acute phase. Such sugar treated infected mice also had expanded populations of effector as well as memory CD8(+) T cells. The increased effector T cell responses led to significantly more efficient virus control. The mechanisms responsible for the outcome of the Gal-9/TIM-3 interaction in normal infected mice involved direct inhibitory effects on TIM-3(+) CD8(+) T effector cells as well as the promotion of Foxp3(+) regulatory T cell activity. Our results indicate that manipulating galectin signals, as can be achieved using appropriate sugars, may represent a convenient and inexpensive approach to enhance acute and memory responses to a virus infection.