RESUMEN
OBJECTIVE: Salmonella enterica infections can lead to Reactive Arthritis (ReA), which can exhibit an association with human leucocyte antigen (HLA)-B*27:05, a molecule prone to misfolding and initiation of the unfolded protein response (UPR). This study examined how HLA-B*27:05 expression and the UPR affect the Salmonella life-cycle within epithelial cells. METHODS: Isogenic epithelial cell lines expressing two copies of either HLA-B*27:05 and a control HLA-B*35:01 heavy chain (HC) were generated to determine the effect on the Salmonella infection life-cycle. A cell line expressing HLA-B*27:05.HC physically linked to the light chain beta-2-microglobulin and a specific peptide (referred to as a single chain trimer, SCT) was also generated to determine the effects of HLA-B27 folding status on S.enterica life-cycle. XBP-1 venus and AMP dependent Transcription Factor (ATF6)-FLAG reporters were used to monitor UPR activation in infected cells. Triacin C was used to inhibit de novo lipid synthesis during UPR, and confocal imaging of ER tracker stained membrane allowed quantification of glibenclamide-associated membrane. RESULTS: S.enterica demonstrated enhanced replication with an altered cellular localisation in the presence of HLA-B*27:05.HC but not in the presence of HLA-B*27:05.SCT or HLA-B*35:01. HLA-B*27:05.HC altered the threshold for UPR induction. Salmonella activated the UPR and required XBP-1 for replication, which was associated with endoreticular membrane expansion and lipid metabolism. CONCLUSIONS: HLA-B27 misfolding and a UPR cellular environment are associated with enhanced Salmonella replication, while Salmonella itself can activate XBP-1 and ATF6. These data provide a potential mechanism linking the life-cycle of Salmonella with the physicochemical properties of HLA-B27 and cellular events that may contribute to ReA pathogenesis. Our observations suggest that the UPR pathway maybe targeted for future therapeutic intervention.
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Células Epiteliales/citología , Antígeno HLA-B27/metabolismo , Infecciones por Salmonella/microbiología , Salmonella enterica/metabolismo , Respuesta de Proteína Desplegada/fisiología , Factor de Transcripción Activador 6/metabolismo , Artritis Reactiva/microbiología , Ciclo Celular , Línea Celular , Antígeno HLA-B35/metabolismo , Humanos , Prohibitinas , Infecciones por Salmonella/complicaciones , Proteína 1 de Unión a la X-Box/metabolismoRESUMEN
With 296 million cases estimated worldwide, chronic hepatitis B virus (HBV) infection is the most common risk factor for hepatocellular carcinoma (HCC). HBV-encoded oncogene X protein (HBx), a key multifunctional regulatory protein, drives viral replication and interferes with several cellular signalling pathways that drive virus-associated hepatocarcinogenesis. This review article provides a comprehensive overview of the role of HBx in modulating the various hallmarks of HCC by supporting tumour initiation, progression, invasion and metastasis. Understanding HBx-mediated dimensions of complexity in driving liver malignancies could provide the key to unlocking novel and repurposed combinatorial therapies to combat HCC.
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Carcinoma Hepatocelular , Hepatitis B Crónica , Neoplasias Hepáticas , Transactivadores , Proteínas Reguladoras y Accesorias Virales , Carcinoma Hepatocelular/patología , Carcinoma Hepatocelular/virología , Virus de la Hepatitis B/metabolismo , Hepatitis B Crónica/complicaciones , Humanos , Neoplasias Hepáticas/patología , Neoplasias Hepáticas/virología , Transactivadores/metabolismo , Proteínas Reguladoras y Accesorias Virales/metabolismoRESUMEN
Peptide-loaded Major Histocompatibility Complex (pMHC) class I molecules can be expressed in a single chain trimeric (SCT) format, composed of a specific peptide fused to the light chain beta-2 microglobulin (ß2m) and MHC class I heavy chain (HC) by flexible linker peptides. pMHC SCTs have been used as effective molecular tools to investigate cellular immunity and represent a promising vaccine platform technology, due to their intracellular folding and assembly which is apparently independent of host cell folding pathways and chaperones. However, certain MHC class I HC molecules, such as the Human Leukocyte Antigen B27 (HLA-B27) allele, present a challenge due to their tendency to form HC aggregates. We constructed a series of single chain trimeric molecules to determine the behaviour of the HLA-B27 HC in a scenario that usually allows for efficient MHC class I molecule folding. When stably expressed, a pMHC SCT incorporating HLA-B27 HC formed chaperone-bound homodimers within the endoplasmic reticulum (ER). A series of HLA-B27 SCT substitution mutations revealed that the F pocket and antigen binding groove regions of the HLA-B27 HC defined the folding and dimerisation of the single chain complex, independently of the peptide sequence. Furthermore, pMHC SCTs can demonstrate variability in their association with the intracellular antigen processing machinery.
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Antígeno HLA-B27 , Antígenos de Histocompatibilidad Clase I , Presentación de Antígeno , Genes MHC Clase I , Antígeno HLA-B27/genética , Antígenos de Histocompatibilidad Clase I/genética , Humanos , Chaperonas Moleculares/genética , Péptidos/genéticaRESUMEN
Epidemiological data consistently rank hepatocellular carcinoma (HCC) as one of the leading causes of cancer-related deaths worldwide, often posing severe economic burden on health care. While the molecular etiopathogenesis associated with genetic and epigenetic modifications has been extensively explored, the biological influence of the emerging field of epitranscriptomics and its associated aberrant RNA modifications on tumorigenesis is a largely unexplored territory with immense potential for discovering new therapeutic approaches. In particular, the underlying cellular mechanisms of different hallmarks of hepatocarcinogenesis that are governed by the complex dynamics of m6A RNA methylation demand further investigation. In this review, we reveal the up-to-date knowledge on the mechanistic and functional link between m6A RNA methylation and pathogenesis of HCC.
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Type I interferons (IFNs) are produced by most cells in response to virus infection and stimulate a program of anti-viral gene expression in neighboring cells to suppress virus replication. Type III IFNs have similar properties, however their effects are limited to epithelial cells at mucosal surfaces due to restricted expression of the type III IFN receptor. Rotavirus (RV) replicates in intestinal epithelial cells that respond predominantly to type III IFNs, and it has been shown that type III rather than type I IFNs are important for controlling RV infections in vivo. The RV NSP1 protein antagonizes the host type I IFN response by targeting IRF-3, IRF-5, IRF-7, or ß-TrCP for proteasome-mediated degradation in a strain-specific manner. Here we provide the first demonstration that NSP1 proteins from several human and animal RV strains antagonize type III as well as type I IFN induction. We also show that NSP1 is a potent inhibitor of IRF-1, a previously undescribed property of NSP1 which is conserved among human and animal RVs. Interestingly, all NSP1 proteins were substantially more effective inhibitors of IRF-1 than either IRF-3 or IRF-7 which has significance for evasion of basal anti-viral immunity and type III IFN induction in the intestinal epithelium.
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Células Epiteliales/virología , Interferón Tipo I/antagonistas & inhibidores , Interferones/antagonistas & inhibidores , Rotavirus/inmunología , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/inmunología , Animales , Células Epiteliales/inmunología , Células HEK293 , Humanos , Factor 1 Regulador del Interferón/antagonistas & inhibidores , Factor 1 Regulador del Interferón/inmunología , Interferón Tipo I/inmunología , Interferones/inmunología , Intestinos/citología , Rotavirus/química , Rotavirus/aislamiento & purificaciónRESUMEN
The Epstein-Barr virus (EBV) nuclear antigen (EBNA)1 contains a glycine-alanine repeat (GAr) domain that appears to protect the antigen from proteasomal breakdown and, as measured in cytotoxicity assays, from major histocompatibility complex (MHC) class I-restricted presentation to CD8+ T cells. This led to the concept of EBNA1 as an immunologically silent protein that although unique in being expressed in all EBV malignancies, could not be exploited as a CD8 target. Here, using CD8+ T cell clones to native EBNA1 epitopes upstream and downstream of the GAr domain and assaying recognition by interferon gamma release, we show that the EBNA1 naturally expressed in EBV-transformed lymphoblastoid cell lines (LCLs) is in fact presented to CD8+ T cells via a proteasome/peptide transporter-dependent pathway. Furthermore, LCL recognition by such CD8+ T cells, although slightly lower than seen with paired lines expressing a GAr-deleted EBNA1 protein, leads to strong and specific inhibition of LCL outgrowth in vitro. Endogenously expressed EBNA1 is therefore accessible to the MHC class I pathway despite GAr-mediated stabilization of the mature protein. We infer that EBNA1-specific CD8+ T cells do play a role in control of EBV infection in vivo and might be exploitable in the control of EBV+ malignancies.
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Linfocitos T CD8-positivos/inmunología , Antígenos Nucleares del Virus de Epstein-Barr/inmunología , Alanina , Secuencia de Aminoácidos , Linfoma de Burkitt , Línea Celular Tumoral , Infecciones por Virus de Epstein-Barr/genética , Infecciones por Virus de Epstein-Barr/inmunología , Antígenos Nucleares del Virus de Epstein-Barr/genética , Glicina , Herpesvirus Humano 4/inmunología , Humanos , Complejo Mayor de Histocompatibilidad , Fragmentos de Péptidos/inmunología , Secuencias Repetitivas de AminoácidoRESUMEN
Viruses that establish lifelong latent infections must ensure that the viral genome is maintained within the latently infected cell throughout the life of the host, yet at the same time must also be capable of avoiding elimination by the immune surveillance system. Gammaherpesviruses, which include the human viruses Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus, establish latent infections in lymphocytes. Infection of this dynamic host-cell population requires that the viruses have appropriate strategies for enabling the viral genome to persist while these cells go through rounds of mitosis, but at the same time must avoid detection by host CD8(+) cytotoxic T lymphocytes (CTLs). The majority of gammaherpesviruses studied have been found to encode a specific protein that is critical for maintenance of the viral genome within latently infected cells. This protein is termed the genome maintenance protein (GMP). Due to its vital role in long-term latency, this offers the immune system a crucial target for detection and elimination of virus-infected cells. GMPs from different gammaherpesviruses have evolved related strategies that allow the protein to be present within latently infected cells, but to remain effectively hidden from circulating CD8(+) CTLs. In this review, I will summarize the role of the GMPs and highlight the available data describing the immune-evasion properties of these proteins.
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Gammaherpesvirinae/inmunología , Genoma Viral , Evasión Inmune , Proteínas Virales/fisiología , Animales , Antígenos Virales/fisiología , Antígenos Nucleares del Virus de Epstein-Barr/inmunología , Antígenos Nucleares del Virus de Epstein-Barr/fisiología , Gammaherpesvirinae/clasificación , Humanos , Proteínas Nucleares/fisiología , Linfocitos T Citotóxicos/inmunología , Latencia del VirusRESUMEN
Herpesvirus saimiri (HVS) establishes a persistent infection in squirrel monkeys by maintaining its episome within T lymphocytes. The product of open reading frame 73 (ORF73) plays a key role in episomal maintenance and is the functional homologue of Epstein-Barr virus EBNA1 and Kaposi's sarcoma-associated herpesvirus LANA1 proteins. There is little sequence homology among these proteins, although all contain a central domain of repeating amino acids. The repeat domains of EBNA1 and LANA1 enhance the stability of these proteins and cause a retardation in self-protein synthesis, leading to poor recognition by CD8(+) cytotoxic T lymphocytes (CTL). The HVS ORF73 repeat domain is composed of a glutamic acid and glycine repeat linked to a glutamic acid and alanine repeat (EG-EA repeat). Here we show that the EG-EA repeat similarly causes a reduction in the recognition of ORF73 by CD8(+) CTL. However, deletion of the EG-EA repeat from HVS ORF73 had no affect on the stability of the protein or its rate of translation. In contrast, the presence of the EG-EA repeat was found to decrease the steady-state levels of ORF73 mRNA. The inhibitory properties of the EG-EA repeat were maintained when transferred to a heterologous protein, and manipulation of the repeat revealed that the motif EEAEEAEEE was sufficient to cause a reduction in recognition of ORF73 by CD8(+) CTL. Thus, the EG-EA repeat of HVS ORF73 plays a role in immune evasion but utilizes a mechanism distinct from that of the EBNA1 and LANA1 repeats.
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Linfocitos T CD8-positivos/inmunología , Herpesvirus Saimiriino 2/inmunología , Antígenos de Histocompatibilidad Clase I/inmunología , ARN Mensajero/biosíntesis , ARN Viral/biosíntesis , Proteínas Virales/biosíntesis , Proteínas Virales/inmunología , Línea Celular , Humanos , Secuencias Repetitivas de Aminoácido/genética , Secuencias Repetitivas de Aminoácido/inmunología , Proteínas Virales/genéticaRESUMEN
PURPOSE: Specific markers of circulating tumor cells may be informative in managing lung cancer. Because the RE-1 silencing transcription factor (REST/NRSF) is a transcriptional repressor that is inactivated in neuroendocrine lung cancer, we identified REST-regulated transcripts (CHGA, CHGB, SCG3, VGF, and PCSK1) for evaluation as biomarkers in peripheral blood. EXPERIMENTAL DESIGN: Transcripts were screened across lung cancer and normal cell lines. Candidates were assessed by reverse transcription-PCR and hybridization of RNA extracted from the peripheral blood of 111 lung cancer patients obtained at clinical presentation and from 27 cancer-free individuals. RESULTS: Expression profiling revealed multiple chromogranin transcripts were readily induced on REST depletion, most notably SCG3 was induced >500-fold. The SCG3 transcript was also overexpressed by 12,000-fold in neuroendocrine compared with nonneuroendocrine lung cancer cells. In peripheral blood of lung cancer patients and cancer-free individuals, we found that SCG3 was more tumor-specific and more sensitive than other chromogranin transcripts as a biomarker of circulating tumor cells. Overall, 36% of small cell lung cancer (SCLC) and 16% of non-SCLC patients scored positively for normalized SCG3 transcript. This correlated with worse survival among SCLC patients with limited disease (n = 33; P = 0.022) but not extensive disease (n = 29; P = 0.459). Interestingly, the subcohort of 6 SCLC patients with resistance to platinum/etoposide chemotherapy all scored positively for peripheral blood SCG3 transcript (P = 0.022). CONCLUSIONS: SCG3 mRNA, a component of the REST-dependent neurosecretory transcriptional profile, provides a sensitive prognostic biomarker for noninvasive monitoring of neuroendocrine lung cancer.
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Biomarcadores de Tumor/sangre , Cromograninas/sangre , Neoplasias Pulmonares/sangre , Tumores Neuroendocrinos/sangre , Carcinoma Pulmonar de Células Pequeñas/sangre , Adulto , Anciano , Anciano de 80 o más Años , Línea Celular Tumoral , Dermatoglifia del ADN , Femenino , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Masculino , Persona de Mediana Edad , Pronóstico , ARN Mensajero/sangre , Carcinoma Pulmonar de Células Pequeñas/tratamiento farmacológicoRESUMEN
Human herpesvirus 6B (HHV6B) infects over 90% of the population, and normally establishes a latent infection, where episodes of reactivation are asymptomatic. However, in immunocompromised patients HHV6B reactivation is associated with high morbidity and mortality. Cellular immunotherapy has been utilised against other herpesvirus in immunocompromised settings. However, limited information on the immune response against HHV6B has hampered the development of immunotherapy for HHV6B-driven disease. In this study, we have analysed the cellular immune response against four HHV6B antigens in a panel of 30 healthy donors. We show that the base-line level of T cell reactivity in peripheral blood is very low to undetectable. A short-term reactivation step enabled expansion of T cell responses, and all donors responded to at least 1 antigen, but more commonly 3 or 4. A hierarchy of immunogenicity was determined with antigens U90 and U54 being co-dominant, followed by U11 and U39. Putative CD8+ T cell epitopes were mapped to U90 and U11, predicted to be presented in the context of HLA-A1, A29, B39 and C6. T cells reactive against these novel epitopes were able to recognise virus-infected cells. Our data is supportive of the application and on-going development of T cell immunotherapy against HHVB-driven disease in the immunocompromised host.
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Cytomegalovirus (CMV), Epstein-Barr virus (EBV), and adenovirus (Ad) cause significant morbidity and mortality in immunocompromised patients undergoing allogeneic stem cell transplantation. We have established a procedure to generate polyclonal cytotoxic T lymphocyte (CTL) populations with specificity against Ad and CMV or against Ad and EBV. Healthy donor-derived dendritic cells (DCs) were transduced with recombinant adenovirus encoding either CMV pp65 or EBV EBNA3C and used to stimulate autologous T cells. Stimulated T lymphocytes displayed specific simultaneous cytotoxicity against CMV and adenovirus and to a lesser extent against adenovirus and EBV. Recombinant vaccinia virus encoding individual adenovirus proteins showed that the T cell response to the adenovirus was directed mainly against the capsid protein hexon. The frequency of IFN-gamma-secreting T cells was 0.02% for adenovirus alone, and 0.05 and 0.14% for adenoviruses encoding EBNA3C and pp65, respectively. pp65-specific CTLs killed autologous fibroblasts infected with the laboratory strain CMV AD169. The culture conditions were specific as alloreactive T cells were not expanded. Therefore, this approach could be considered in order to generate efficient virus cytolytic T cells to be used as adoptive immunotherapy in transplanted patients.
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Adenoviridae/genética , Células Dendríticas/metabolismo , Inmunoterapia/métodos , Fosfoproteínas/metabolismo , Linfocitos T Citotóxicos/metabolismo , Linfocitos T Citotóxicos/virología , Proteínas de la Matriz Viral/metabolismo , Separación Celular , Fibroblastos/metabolismo , Citometría de Flujo , Técnicas de Transferencia de Gen , Proteínas Fluorescentes Verdes , Humanos , Interferón gamma/metabolismo , Proteínas Luminiscentes/metabolismo , Microscopía Fluorescente , Fosfoproteínas/genética , Plásmidos/metabolismo , Factores de Tiempo , Transducción Genética , Proteínas de la Matriz Viral/genéticaRESUMEN
For recognition by CD8(+) lymphocytes, peptides derived from cytosolically processed antigen need to access MHC class I molecules en route to the target cell surface. This normally requires peptide transport into the endoplasmic reticulum via the transporter associated with antigen presentation (TAP) complex. However, as recent work with Epstein-Barr virus illustrates, TAP-independent presentation pathways also exist and are growing in number.