RESUMO
INTRODUCTION: Regular reviews of medications, including prescription reviews and adherence reviews, are vital to support pharmacological effectiveness and optimize health outcomes for patients. Despite being more likely to report a long-term illness that requires medication when compared to their white counterparts, individuals from ethnic minority communities are less likely to engage with regular medication reviews, with inequalities negatively affecting their access. It is important to understand what barriers may exist that impact the access of those from ethnic minority communities and to identify measures that may act to facilitate improved service accessibility for these groups. METHODS: Semi-structured interviews were conducted between June and August 2021 using the following formats as permitted by governmental COVID-19 restrictions: in person, over the telephone or via video call. Perspectives on service accessibility and any associated barriers and facilitators were discussed. Interviews were audio-recorded and transcribed verbatim. Reflexive thematic analysis enabled the development of themes. QSR NVivo (Version 12) facilitated data management. Ethical approval was obtained from the Health Research Authority (ref: 21/HRA/1426). RESULTS: In total 20 participants from ethnic minority communities were interviewed; these participants included 16 UK citizens, 2 refugees and 2 asylum seekers, and represented a total of 5 different ethnic groups. Three themes were developed from the data regarding the perceived barriers and facilitators affecting access to medication reviews and identified approaches to improve the accessibility of such services for ethnic minority patients. These centred on (1) building knowledge and understanding about medication reviews; (2) delivering medication review services; and (3) appreciating the lived experience of patients. CONCLUSION: The results of this study have important implications for addressing inequalities that affect ethnic minority communities. Involving patients and practitioners to work collaboratively in coproduction approaches could enable better design, implementation and delivery of accessible medication review services that are culturally competent. PATIENT OR PUBLIC CONTRIBUTION: The National Institute for Health Research Applied Research Collaboration and Patient and Public Involvement and Engagement group at Newcastle University supported the study design and conceptualization. Seven patient champions inputted to ensure that the research was conducted, and the findings were reported, with cultural sensitivity.
Assuntos
Tratamento Farmacológico da COVID-19 , Etnicidade , Minorias Étnicas e Raciais , Feminino , Acessibilidade aos Serviços de Saúde , Humanos , Revisão de Medicamentos , Grupos Minoritários , Pesquisa QualitativaRESUMO
INTRODUCTION: Medicines-centred consultations are vital to support medicine effectiveness and optimize health outcomes for patients. However, inequalities negatively impact ethnic minority populations when accessing medicines advice. It is important to identify opportunities to improve access for these communities however, knowledge of how best to achieve this is lacking; this study will generate recommendations to improve access to medicines advice from community pharmacies for people from ethnic minority communities. METHODS: A series of codesign workshops, with four groups of patient-stakeholders, were conducted between September-November 2021; they took place in-person or via video call (adhering to COVID-19 restrictions). Existing evidence-based perceptions affecting access to medicines advice were critiqued and recommendations were generated, by use of reflexive thematic analysis, to improve access for ethnic minority patients. The workshops were audio-recorded and transcribed verbatim. QSR NVivo (Version 12) facilitated data analysis. RESULTS: Twelve participants were recruited using purposive sampling; including eight UK citizens, two asylum seekers and two participants in receipt of residency visas. In total, four different ethnic minority groups were represented. Each participant took part in a first and second workshop to share and cocreate recommendations to improve access to medicines advice in community pharmacies. Three recommendations were developed and centred on: (i) delivering and providing culturally competent medicines advice; (ii) building awareness of accessing medicines advice from community pharmacies; and (iii) enabling better discussions with patients from ethnic minority communities. CONCLUSIONS: These recommendations have the potential to support community pharmacy services to overcome ethnic inequalities affecting medicines advice; service commissioners should consider these findings to best meet the needs of ethnic minority patients. Cultural competence training for community pharmacy staff could support the creation of pharmacies as inclusive healthcare settings. Collaborative working with ethnic minority communities could enable specific tailoring of medicines-centred services to best meet their needs. PATIENT OR PUBLIC CONTRIBUTION: The National Institute for Health Research (NIHR) and Newcastle University Patient and Public Involvement and Engagement group had extensive input in the study design and conceptualization. Seven patient champions were appointed to the steering group to ensure that the research was conducted, and findings were reported, with cultural competence. TRIAL REGISTRATION: Not applicable.
Assuntos
Tratamento Farmacológico da COVID-19 , Farmácias , Humanos , Etnicidade , Grupos Minoritários , Minorias Étnicas e Raciais , Acessibilidade aos Serviços de SaúdeRESUMO
Human interleukin-12 (hIL-12) is a heparin-binding cytokine whose activity was previously shown to be enhanced by heparin and other sulfated glycosaminoglycans. The current study investigated the mechanisms by which heparin increases hIL-12 activity. Using multiple human cell types, including natural killer cells, an IL-12 indicator cell line, and primary peripheral blood mononuclear and T cells, along with bioactivity, flow cytometry, and isothermal titration calorimetry assays, we found that heparin-dependent modulation of hIL-12 function correlates with several of heparin's biophysical characteristics, including chain length, sulfation level, and concentration. Specifically, only heparin molecules longer than eight saccharide units enhanced hIL-12 activity. Furthermore, heparin molecules with three sulfate groups per disaccharide unit outperformed heparin molecules with one or two sulfate groups per disaccharide unit in terms of enhanced hIL-12 binding and activity. Heparin also significantly reduced the EC50 value of hIL-12 by up to 11.8-fold, depending on the responding cell type. Cytokine-profiling analyses revealed that heparin affected the level, but not the type, of cytokines produced by lymphocytes in response to hIL-12. Interestingly, although murine IL-12 also binds heparin, heparin did not enhance its activity. Using the gathered data, we propose a model of hIL-12 stabilization in which heparin serves as a co-receptor enhancing the interaction between heterodimeric hIL-12 and its receptor subunits. The results of this study provide a foundation for further investigation of heparin's interactions with IL-12 family cytokines and for the use of heparin as an immunomodulatory agent.
Assuntos
Heparina/farmacologia , Interleucina-12/farmacologia , Animais , Fenômenos Biofísicos , Calorimetria , Citocinas/biossíntese , Relação Dose-Resposta a Droga , Feminino , Citometria de Fluxo , Células HEK293 , Heparina/química , Heparitina Sulfato/metabolismo , Humanos , Interleucina-12/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Ligação Proteica , Receptores de Interleucina-2/metabolismo , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Transdução de Sinais/efeitos dos fármacos , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismoRESUMO
Post-transcriptional regulatory mechanisms of several complex and simple retroviruses and retroelements have been elucidated, with the exception of the gammaretrovirus family. We found that, similar to the other retroviruses, gag gene expression of MuLV and XMRV depends on post-transcriptional regulation mediated via an RNA sequence overlapping the pro-pol open reading frame, termed the Post-Transcriptional Element (PTE). PTE function can be replaced by heterologous RNA export elements, e.g. CTE of simian type D retroviruses. Alternatively, Gag particle production is achieved using an RNA/codon optimized gag gene. PTE function is transferable and can replace HIV Rev-RRE-regulated expression of HIV gag. Analysis of PTE by SHAPE revealed a highly structured RNA comprising seven stem-loop structures, with the 5' and 3' stem-loops forming an essential bipartite signal. MuLV and XMRV PTE share 98% identity and have highly similar RNA structures, with changes mostly located to single-stranded regions. PTE identification strongly suggests that all retroviruses and retroelements share common strategies of post-transcriptional gene regulation to produce Gag. Expression depends on complex RNA structures embedded within retroviral mRNA, in coding regions or the 3' untranslated region. These specific structures serve as recognition signals for either cellular or viral proteins.
Assuntos
Regulação Viral da Expressão Gênica , Vírus da Leucemia Murina de Moloney/genética , RNA Mensageiro/química , RNA Viral/química , Sequências Reguladoras de Ácido Ribonucleico , Vírus Relacionado ao Vírus Xenotrópico da Leucemia Murina/genética , Produtos do Gene gag/genética , Produtos do Gene gag/metabolismo , Células HEK293 , Células HeLa , Humanos , Vírus da Leucemia Murina de Moloney/metabolismo , Conformação de Ácido Nucleico , RNA Mensageiro/metabolismo , Vírus Relacionado ao Vírus Xenotrópico da Leucemia Murina/metabolismo , Produtos do Gene gag do Vírus da Imunodeficiência Humana/genética , Produtos do Gene gag do Vírus da Imunodeficiência Humana/metabolismoRESUMO
BACKGROUND: None of the HIV T-cell vaccine candidates that have reached advanced clinical testing have been able to induce protective T cell immunity. A major reason for these failures may have been suboptimal T cell immunogen designs. METHODS: To overcome this problem, we used a novel immunogen design approach that is based on functional T cell response data from more than 1,000 HIV-1 clade B and C infected individuals and which aims to direct the T cell response to the most vulnerable sites of HIV-1. RESULTS: Our approach identified 16 regions in Gag, Pol, Vif and Nef that were relatively conserved and predominantly targeted by individuals with reduced viral loads. These regions formed the basis of the HIVACAT T-cell Immunogen (HTI) sequence which is 529 amino acids in length, includes more than 50 optimally defined CD4(+) and CD8(+) T-cell epitopes restricted by a wide range of HLA class I and II molecules and covers viral sites where mutations led to a dramatic reduction in viral replicative fitness. In both, C57BL/6 mice and Indian rhesus macaques immunized with an HTI-expressing DNA plasmid (DNA.HTI) induced broad and balanced T-cell responses to several segments within Gag, Pol, and Vif. DNA.HTI induced robust CD4(+) and CD8(+) T cell responses that were increased by a booster vaccination using modified virus Ankara (MVA.HTI), expanding the DNA.HTI induced response to up to 3.2% IFN-γ T-cells in macaques. HTI-specific T cells showed a central and effector memory phenotype with a significant fraction of the IFN-γ(+) CD8(+) T cells being Granzyme B(+) and able to degranulate (CD107a(+)). CONCLUSIONS: These data demonstrate the immunogenicity of a novel HIV-1 T cell vaccine concept that induced broadly balanced responses to vulnerable sites of HIV-1 while avoiding the induction of responses to potential decoy targets that may divert effective T-cell responses towards variable and less protective viral determinants.
Assuntos
Vacinas contra a AIDS/imunologia , Infecções por HIV/imunologia , Infecções por HIV/virologia , HIV-1/imunologia , Linfócitos T/imunologia , Alelos , Sequência de Aminoácidos , Animais , Antivirais/imunologia , Linfócitos T CD4-Positivos/imunologia , Estudos de Coortes , Epitopos/imunologia , Epitopos de Linfócito T/imunologia , Feminino , Células HEK293 , Haplótipos , Antígenos de Histocompatibilidade Classe I/genética , Humanos , Imunidade Celular , Imunidade Humoral , Memória Imunológica , Macaca mulatta , Masculino , Camundongos Endogâmicos C57BL , Peptídeos/química , Peptídeos/imunologia , Linfócitos T Citotóxicos/imunologia , VacinaçãoRESUMO
IL-12 is a 70-kDa heterodimeric cytokine composed of the p35 and p40 subunits. To maximize cytokine production from plasmid DNA, molecular steps controlling IL-12p70 biosynthesis at the posttranscriptional and posttranslational levels were investigated. We show that the combination of RNA/codon-optimized gene sequences and fine-tuning of the relative expression levels of the two subunits within a cell resulted in increased production of the IL-12p70 heterodimer. We found that the p40 subunit plays a critical role in enhancing the stability, intracellular trafficking, and export of the p35 subunit. This posttranslational regulation mediated by the p40 subunit is conserved in mammals. Based on these findings, dual gene expression vectors were generated, producing an optimal ratio of the two subunits, resulting in a ~1 log increase in human, rhesus, and murine IL-12p70 production compared with vectors expressing the wild type sequences. Such optimized DNA plasmids also produced significantly higher levels of systemic bioactive IL-12 upon in vivo DNA delivery in mice compared with plasmids expressing the wild type sequences. A single therapeutic injection of an optimized murine IL-12 DNA plasmid showed significantly more potent control of tumor development in the B16 melanoma cancer model in mice. Therefore, the improved IL-12p70 DNA vectors have promising potential for in vivo use as molecular vaccine adjuvants and in cancer immunotherapy.
Assuntos
Subunidade p35 da Interleucina-12/biossíntese , Subunidade p40 da Interleucina-12/biossíntese , Multimerização Proteica , Animais , Humanos , Imunoterapia , Subunidade p35 da Interleucina-12/genética , Subunidade p35 da Interleucina-12/imunologia , Subunidade p40 da Interleucina-12/genética , Subunidade p40 da Interleucina-12/imunologia , Macaca mulatta , Melanoma/genética , Melanoma/imunologia , Melanoma/metabolismo , Melanoma/terapia , Camundongos , Neoplasias Experimentais/genética , Neoplasias Experimentais/imunologia , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/terapia , Estabilidade Proteica , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologiaRESUMO
To identify the most promising vaccine candidates for combinatorial strategies, we compared five SIV vaccine platforms including recombinant canary pox virus ALVAC, replication-competent adenovirus type 5 host range mutant RepAd, DNA, modified vaccinia Ankara (MVA), peptides and protein in distinct combinations. Three regimens used viral vectors (prime or boost) and two regimens used plasmid DNA. Analysis at necropsy showed that the DNA-based vaccine regimens elicited significantly higher cellular responses against Gag and Env than any of the other vaccine platforms. The T cell responses induced by most vaccine regimens disseminated systemically into secondary lymphoid tissues (lymph nodes, spleen) and effector anatomical sites (including liver, vaginal tissue), indicative of their role in viral containment at the portal of entry. The cellular and reported humoral immune response data suggest that combination of DNA and viral vectors elicits a balanced immunity with strong and durable responses able to disseminate into relevant mucosal sites.
Assuntos
Imunidade Celular , Vírus da Imunodeficiência Símia/imunologia , Vacinas Virais/imunologia , Animais , Antígenos Virais/imunologia , Linfócitos T CD8-Positivos , Células Cultivadas , Feminino , Macaca mulattaRESUMO
Combinatorial HIV/SIV vaccine approaches targeting multiple arms of the immune system might improve protective efficacy. We compared SIV-specific humoral immunity induced in rhesus macaques by five vaccine regimens. Systemic regimens included ALVAC-SIVenv priming and Env boosting (ALVAC/Env); DNA immunization; and DNA plus Env co-immunization (DNA&Env). RepAd/Env combined mucosal replication-competent Ad-env priming with systemic Env boosting. A Peptide/Env regimen, given solely intrarectally, included HIV/SIV peptides followed by MVA-env and Env boosts. Serum antibodies mediating neutralizing, phagocytic and ADCC activities were induced by ALVAC/Env, RepAd/Env and DNA&Env vaccines. Memory B cells and plasma cells were maintained in the bone marrow. RepAd/Env vaccination induced early SIV-specific IgA in rectal secretions before Env boosting, although mucosal IgA and IgG responses were readily detected at necropsy in ALVAC/Env, RepAd/Env, DNA&Env and DNA vaccinated animals. Our results suggest that combined RepAd priming with ALVAC/Env or DNA&Env regimen boosting might induce potent, functional, long-lasting systemic and mucosal SIV-specific antibodies.
Assuntos
Imunidade nas Mucosas/imunologia , Vacinas contra a SAIDS/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Vírus da Imunodeficiência Símia/imunologia , Vacinação/métodos , Vacinas contra a AIDS/administração & dosagem , Vacinas contra a AIDS/imunologia , Animais , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Citotoxicidade Celular Dependente de Anticorpos/imunologia , Quimioterapia Combinada , ELISPOT , Produtos do Gene env/imunologia , Humanos , Imunoglobulina A/sangue , Imunoglobulina A/imunologia , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Macaca mulatta , Vacinas contra a SAIDS/administração & dosagem , Síndrome de Imunodeficiência Adquirida dos Símios/prevenção & controle , Síndrome de Imunodeficiência Adquirida dos Símios/virologia , Vírus da Imunodeficiência Símia/fisiologia , Fatores de Tempo , Vacinas de DNA/administração & dosagem , Vacinas de DNA/imunologia , Vacinas de Subunidades Antigênicas/administração & dosagem , Vacinas de Subunidades Antigênicas/imunologiaRESUMO
Kaposi's sarcoma-associated herpesvirus (KSHV [human herpesvirus 8; HHV-8]) open reading frame 57 (ORF57) is a viral early protein participating in posttranscriptional regulatory events, such as splicing, RNA stabilization, and protein expression. Recent data suggest that ORF57 recruits the transcription and export (TREX) complex to viral RNA and exports these transcripts to the cytoplasm. In this study, we show that although ORF57 promotes expression of a selection of KSHV viral intronless RNAs, it is not a bona fide export factor.
Assuntos
Núcleo Celular/metabolismo , Herpesvirus Humano 8/genética , Processamento Pós-Transcricional do RNA/genética , RNA Viral/metabolismo , Proteínas Virais/genética , Transporte Ativo do Núcleo Celular/genética , Transporte Ativo do Núcleo Celular/fisiologia , Northern Blotting , Oligonucleotídeos/genética , Fases de Leitura Aberta/genética , Proteínas Virais/metabolismoRESUMO
Kaposi's sarcoma-associated herpesvirus (KSHV) encodes ORF57, which promotes the accumulation of specific KSHV mRNA targets, including ORF59 mRNA. We report that the cellular export NXF1 cofactors RBM15 and OTT3 participate in ORF57-enhanced expression of KSHV ORF59. We also found that ectopic expression of RBM15 or OTT3 augments ORF59 production in the absence of ORF57. While RBM15 promotes the accumulation of ORF59 RNA predominantly in the nucleus compared to the levels in the cytoplasm, we found that ORF57 shifted the nucleocytoplasmic balance by increasing ORF59 RNA accumulation in the cytoplasm more than in the nucleus. By promoting the accumulation of cytoplasmic ORF59 RNA, ORF57 offsets the nuclear RNA accumulation mediated by RBM15 by preventing nuclear ORF59 RNA from hyperpolyadenylation. ORF57 interacts directly with the RBM15 C-terminal portion containing the SPOC domain to reduce RBM15 binding to ORF59 RNA. Although ORF57 homologs Epstein-Barr virus (EBV) EB2, herpes simplex virus (HSV) ICP27, varicella-zoster virus (VZV) IE4/ORF4, and cytomegalovirus (CMV) UL69 also interact with RBM15 and OTT3, EBV EB2, which also promotes ORF59 expression, does not function like KSHV ORF57 to efficiently prevent RBM15-mediated nuclear accumulation of ORF59 RNA and RBM15's association with polyadenylated RNAs. Collectively, our data provide novel insight elucidating a molecular mechanism by which ORF57 promotes the expression of viral intronless genes.
Assuntos
Regulação Viral da Expressão Gênica , Herpesvirus Humano 8/metabolismo , Fases de Leitura Aberta/fisiologia , Proteínas de Ligação a RNA/metabolismo , Proteínas Virais/metabolismo , Núcleo Celular/genética , Núcleo Celular/metabolismo , Citoplasma/genética , Citoplasma/metabolismo , Células HEK293 , Células HeLa , Herpesvirus Humano 8/genética , Humanos , Proteínas de Transporte Nucleocitoplasmático/genética , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Fases de Leitura Aberta/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Viral/genética , RNA Viral/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas Virais/genéticaRESUMO
Retrovirus replication requires specialized transport mechanisms to export genomic mRNA from the nucleus to the cytoplasm of the infected cell. This regulation is mediated by a combination of viral and/or cellular factors that interact with cis-acting RNA export elements linking the viral RNA to the cellular CRM1 or NXF1 nuclear export pathways. Endogenous type D murine LTR retrotransposons (musD) were reported to contain an RNA export element located upstream of the 3'-LTR. Although functionally equivalent, the musD export element, termed the musD transport element, is distinct from the other retroviral RNA export elements, such as the constitutive transport element of simian/Mason-Pfizer monkey retroviruses and the RNA transport element found in rodent intracisternal A-particle LTR retrotransposons. We demonstrate here that the minimal RNA transport element (musD transport element) of musD comprises multiple secondary structure elements that presumably serve as recognition signals for the cellular export machinery. We identified two classes of tertiary interactions, namely kissing loops and a pseudoknot. This work constitutes the first example of an RNA transport element requiring such structural motifs to mediate nuclear export.
Assuntos
RNA/metabolismo , Regiões 3' não Traduzidas , Animais , Transporte Biológico , Células HeLa , Humanos , Camundongos , Modelos Genéticos , Mutagênese Sítio-Dirigida , Conformação de Ácido Nucleico , Oligonucleotídeos Antissenso/genética , Regiões Promotoras Genéticas , Retroelementos/genética , Retroviridae/genética , Sequências Repetidas TerminaisRESUMO
BACKGROUND AND AIMS: We aimed to validate a nurse-led process using electronic health records to identify those at risk of familial hypercholesterolaemia (FH) for genetic diagnosis in primary care. METHODS: Those at risk of FH were identified using searches developed and refined locally and implemented in primary care by a trained nurse; they were invited for further assessment and genetic testing if indicated. Family members at risk of FH were identified and invited for cascade testing. RESULTS: In total 94,444 patient records were screened (expected prevalence of FH (1 in 250); 377). Of 176 records which already had a diagnostic for FH, 15 had been genetically confirmed and one was undergoing DNA testing. A further 572 (0.61%) were identified as high risk of FH. After desktop screening, 113 (15%) were invited for further assessment. Of these, 73 individuals attended the primary care clinic (64%) of whom 61 (54%) underwent proband genetic testing. Pathogenic variants were detected in 22 cases (36%) and variants of unknown significance in a further 4 cases; a total of 26 probands (43%) were therefore referred for family cascade testing. CONCLUSIONS: An optimised FH identification pathway, based on the NICE CG71 recommendations for systematic searching of primary care electronic health records, can be deployed successfully in primary care settings.
Assuntos
Hiperlipoproteinemia Tipo II , Medicina Estatal , Testes Genéticos , Humanos , Hiperlipoproteinemia Tipo II/diagnóstico , Hiperlipoproteinemia Tipo II/epidemiologia , Hiperlipoproteinemia Tipo II/genética , Programas de Rastreamento , Atenção Primária à SaúdeRESUMO
BACKGROUND: Cardiac allograft vasculopathy (CAV) limits the long-term success of cardiac transplantation. The incidence of CAV is increased in patients with elevated plasma levels of oxidized lipids or fibrin deposition within right heart biopsy (RHB) specimens. The present study investigated whether tissue factor (TF), the expression of which is regulated by oxidized lipids, is upregulated in patients with CAV. METHODS AND RESULTS: A TF score was developed to quantify TF expression in RHB specimens from 63 consecutive patients undergoing routine annual posttransplantation RHB and coronary angiography. In patients >2 years (3.0+/-0.8 years) posttransplantation (n=35), a high TF score was observed with greater frequency (75% versus 26%, P<0.004) in patients with CAV than those without CAV. In patients <2 years (0.87+/-0.48 years) posttransplantation (n=28) without evidence of CAV, the TF score was determined and patients were followed up prospectively. A high TF score had a positive predictive value of 78.6% for the development of CAV, and a low TF score had a negative predictive value of 100%. CONCLUSIONS: These data demonstrate that early TF expression predicts subsequent development of CAV. Increased TF expression could link the elevated levels of oxidized LDL and fibrin deposition known to precede CAV. These findings suggest that TF may play a role in the pathophysiology of CAV and could offer a potential prognostic tool and a novel target for the prevention of CAV, possibly with antioxidants or inhibitors of the TF pathway.
Assuntos
Doença da Artéria Coronariana/diagnóstico , Doença da Artéria Coronariana/metabolismo , Transplante de Coração , Tromboplastina/biossíntese , Adulto , Biomarcadores/análise , Angiografia Coronária , Doença da Artéria Coronariana/etiologia , Doença da Artéria Coronariana/patologia , Seguimentos , Humanos , PrognósticoRESUMO
Replication of retroviruses and transposition of endogenous retroelements exploits a unique mechanism of post-transcriptional regulation as a means of exporting their incompletely-spliced mRNAs (which serve as both the genomic RNA and the template for protein synthesis). Following discovery of the Rev response element (RRE) that mediates nucleocytoplasmic export of the full-length and singly-spliced human immunodeficiency virus type 1 (HIV-1) genome, equivalent cis-acting regulatory elements have been characterized for both complex and simple retroviruses and retroelements, together with the obligate viral and host proteins with which they interact. The exception to this is the gammaretrovirus family of simple retroviruses, exemplified by reticuloendotheliosis virus (REV), murine leukemia virus (MLV) and xenotropic MLV-related retrovirus (XMRV). In this commentary, we discuss our recent data that reported structural and functional data on the MLV/XMRV post-transcriptional regulatory element (designated the PTE). The PTE was characterized by a highly-structured region of multiple stem-loops (SL1 - SL7) overlapping the pro and 5' portion of the pol open reading frames, comprising a bipartite export signal whose structures are separated by â¼1400 nt. In addition, structural probing suggested that SL3 nucleotides were involved in pseudoknot formation. These data, when compared with RNA transport elements of complex retroviruses (HIV) and simple murine retrotransposons (musD), collectively present an emerging picture that long-range tertiary interactions are critical mediators of their biological function.
RESUMO
A panel of SIVmac251 transmitted Env sequences were tested for expression, function and immunogenicity in mice and macaques. The immunogenicity of a DNA vaccine cocktail expressing SIVmac239 and three transmitted SIVmac251 Env sequences was evaluated upon intradermal or intramuscular injection followed by in vivo electroporation in macaques using sequential vaccination of gp160, gp120 and gp140 expressing DNAs. Both intradermal and intramuscular vaccination regimens using the gp160 expression plasmids induced robust humoral immune responses, which further improved using the gp120 expressing DNAs. The responses showed durability of binding and neutralizing antibody titers and high avidity for>1 y. The intradermal DNA delivery regimen induced higher cross-reactive responses able to neutralize the heterologous tier 1B-like SIVsmE660_CG7V. Analysis of cellular immune responses showed induction of Env-specific memory responses and cytotoxic granzyme B(+) T cells in both vaccine groups, although the magnitude of the responses were ~10x higher in the intramuscular/electroporation group. The cellular responses induced by both regimens were long lasting and could be detected ~1 y after the last vaccination. These data show that both DNA delivery methods are able to induce robust and durable immune responses in macaques.
Assuntos
Eletroporação/métodos , Vacinas contra a SAIDS/imunologia , Vacinação/métodos , Vacinas de DNA/imunologia , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Reações Cruzadas , Feminino , Produtos do Gene env/genética , Produtos do Gene env/imunologia , Injeções Intradérmicas , Injeções Intramusculares , Macaca mulatta , Camundongos , Camundongos Endogâmicos BALB C , Vacinas contra a SAIDS/administração & dosagem , Vírus da Imunodeficiência Símia/genética , Vírus da Imunodeficiência Símia/imunologia , Linfócitos T Citotóxicos/imunologia , Fatores de Tempo , Vacinas de DNA/administração & dosagemRESUMO
The control of mRNA degradation and translation are important aspects of gene regulation. Recent results suggest that translation repression and mRNA decapping can be intertwined and involve the formation of a quiescent mRNP, which can accumulate in cytoplasmic foci referred to as P bodies. The Pat1 protein is a key component of this complex and an important activator of decapping, yet little is known about its function. In this work, we analyze Pat1 in Saccharomyces cerevisiae function by deletion and functional analyses. Our results identify two primary functional domains in Pat1: one promoting translation repression and P-body assembly and a second domain promoting mRNA decapping after assembly of the mRNA into a P-body mRNP. In addition, we provide evidence that Pat1 binds RNA and has numerous domain-specific interactions with mRNA decapping factors. These results indicate that Pat1 is an RNA binding protein and a multidomain protein that functions at multiple stages in the process of translation repression and mRNA decapping.