RESUMEN
BACKGROUND: Virtual patients (VPs) are widely used in health professions education. When they are well integrated into curricula, they are considered to be more effective than loosely coupled add-ons. However, it is unclear what constitutes their successful integration. The aim of this study was to identify and synthesise the themes found in the literature that stakeholders perceive as important for successful implementation of VPs in curricula. METHODS: We searched five databases from 2000 to September 25, 2023. We included qualitative, quantitative, mixed-methods and descriptive case studies that defined, identified, explored, or evaluated a set of factors that, in the perception of students, teachers, course directors and researchers, were crucial for VP implementation. We excluded effectiveness studies that did not consider implementation characteristics, and studies that focused on VP design factors. We included English-language full-text reports and excluded conference abstracts, short opinion papers and editorials. Synthesis of results was performed using the framework synthesis method with Kern's six-step model as the initial framework. We appraised the quality of the studies using the QuADS tool. RESULTS: Our search yielded a total of 4808 items, from which 21 studies met the inclusion criteria. We identified 14 themes that formed an integration framework. The themes were: goal in the curriculum; phase of the curriculum when to implement VPs; effective use of resources; VP alignment with curricular learning objectives; prioritisation of use; relation to other learning modalities; learning activities around VPs; time allocation; group setting; presence mode; VPs orientation for students and faculty; technical infrastructure; quality assurance, maintenance, and sustainability; assessment of VP learning outcomes and learning analytics. We investigated the occurrence of themes across studies to demonstrate the relevance of the framework. The quality of the studies did not influence the coverage of the themes. CONCLUSIONS: The resulting framework can be used to structure plans and discussions around implementation of VPs in curricula. It has already been used to organise the curriculum implementation guidelines of a European project. We expect it will direct further research to deepen our knowledge on individual integration themes.
Asunto(s)
Curriculum , Humanos , Educación de Pregrado en Medicina , Simulación de Paciente , Participación de los Interesados , Empleos en Salud/educaciónRESUMEN
BACKGROUND: As Ukraine struggles with the education of healthcare professionals due to the war, we aimed to identify the specific effects of the war on medical education, the resulting needs, and the expected consequences for schools, faculty, staff, students, and the healthcare system. METHODS: In October and November 2022, we performed a survey of students, faculty, and staff of medical schools in Ukraine and conducted semi-structured interviews with faculty leaders (i.e., rectors, vice-rectors). We conducted a descriptive analysis of the survey's closed-ended questions. The survey and the interviews included open-ended questions about war-related restrictions to teaching and learning, resulting needs, and expected consequences, for which we applied a thematic analysis. RESULTS: We received 239 survey responses (N = 49 faculty and staff, N = 190 students) and conducted nine interviews with faculty leaders across Ukraine. Most survey participants indicated that they had experienced restrictions or changes to their work or study due to the war (86% of faculty and staff, 69% of students). From the thematic analysis of the survey and interviews, we identified eight themes: disruption of teaching, increased workload, mental stress, financial restrictions, non-war related needs, international cooperation, quality of education, and prospects of future professionals. The quality of healthcare education in Ukraine was threatened, and schools, faculty, staff, and students were under great strain. While already established international cooperation has been supportive, some needs have still not been addressed. CONCLUSIONS: We hope that our findings will help researchers and educators from abroad contribute to meeting Ukraine's needs in medical education.
Asunto(s)
Curriculum , Educación Médica , Humanos , Facultades de Medicina , Estudiantes , DocentesRESUMEN
BACKGROUND: Virtual patients (VPs) are a suitable method for students to train their clinical reasoning abilities. We describe a process of developing a blueprint for a diverse and realistic VP collection (prior to VP creation) that facilitates deliberate practice of clinical reasoning and meets educational requirements of medical schools. METHODS: An international and interdisciplinary partnership of five European countries developed a blueprint for a collection of 200 VPs in four steps: (1) Defining the criteria (e.g., key symptoms, age, sex) and categorizing them into disease-, patient-, encounter- and learner-related, (2) Identifying data sources for assessing the representativeness of the collection, (3) Populating the blueprint, and (4) Refining and reaching consensus. RESULTS: The blueprint is publicly available and covers 29 key symptoms and 176 final diagnoses including the most prevalent medical conditions in Europe. Moreover, our analyses showed that the blueprint appears to be representative of the European population. CONCLUSIONS: The development of the blueprint required a stepwise approach, which can be replicated for the creation of other VP or case collections. We consider the blueprint an appropriate starting point for the actual creation of the VPs, but constant updating and refining is needed.
Asunto(s)
Competencia Clínica , Razonamiento Clínico , Europa (Continente) , HumanosRESUMEN
Achromatopsia (ACHM) is a rare autosomal recessively inherited retinal disease characterized by congenital photophobia, nystagmus, low visual acuity, and absence of color vision. ACHM is genetically heterogeneous and can be caused by biallelic mutations in the genes CNGA3, CNGB3, GNAT2, PDE6C, PDE6H, or ATF6. We undertook molecular genetic analysis in a single female patient with a clinical diagnosis of ACHM and identified the homozygous variant c.778G>C;p.(D260H) in the CNGA3 gene. While segregation analysis in the father, as expected, identified the CNGA3 variant in a heterozygous state, it could not be displayed in the mother. Microsatellite marker analysis provided evidence that the homozygosity of the CNGA3 variant is due to partial or complete paternal uniparental isodisomy (UPD) of chromosome 2 in the patient. Apart from the ACHM phenotype, the patient was clinically unsuspicious and healthy. This is one of few examples proving UPD as the underlying mechanism for the clinical manifestation of a recessive mutation in a patient with inherited retinal disease. It also highlights the importance of segregation analysis in both parents of a given patient or especially in cases of homozygous recessive mutations, as UPD has significant implications for genetic counseling with a very low recurrence risk assessment in such families.
Asunto(s)
Cromosomas Humanos Par 2/genética , Defectos de la Visión Cromática/patología , Canales Catiónicos Regulados por Nucleótidos Cíclicos/genética , Padre , Mutación , Disomía Uniparental , Adolescente , Defectos de la Visión Cromática/genética , Femenino , Genes Recesivos , Humanos , Masculino , Linaje , FenotipoRESUMEN
Visual impairment due to inherited ophthalmic disorders is amongst the most common disabilities observed in populations practicing consanguineous marriages. Here we investigated the molecular genetic basis of an unselected broad range of ophthalmic disorders in 20 consanguineous families from Arab villages of Israel and the Palestinian Authority. Most patients had little or very poor prior clinical workup and were recruited in a field study. Homozygosity mapping followed by candidate gene sequencing applying conventional Sanger sequencing or targeted next generation sequencing was performed in six families. In the remaining 14 families, one affected subject per family was chosen for whole exome sequencing. We discovered likely disease-causing variants, all homozygous, in 19 of 20 independent families (95%) including a previously reported novel disease gene for congenital nystagmus associated with foveal hypoplasia. Moreover, we found a family in which disease-causing variants for two collagenopathies - Stickler and Knobloch syndrome - segregate within a large sibship. Nine of the 19 distinct variants observed in this study were novel. Our study demonstrated a very high molecular diagnostic yield for a highly diverse spectrum of rare ophthalmic disorders in Arab patients from Israel and the Palestinian Authority, even with very limited prior clinical investigation. We conclude that 'genetic testing first' may be an economic way to direct clinical care and to support proper genetic counseling and risk assessment in these families.
Asunto(s)
Árabes/genética , Enfermedades Hereditarias del Ojo/genética , Mutación , Enfermedades Hereditarias del Ojo/epidemiología , Enfermedades Hereditarias del Ojo/etnología , Femenino , Sitios Genéticos , Humanos , Israel , Masculino , LinajeRESUMEN
Herein we present a consanguineous family with three children affected by foveal hypoplasia with infantile nystagmus, following an autosomal recessive mode of inheritance. The patients showed normal electroretinography responses, no signs of albinism, and no anterior segment or brain abnormalities. Upon whole exome sequencing, we identified a homozygous mutation (c.1861C>T;p.Q621*) in the aryl hydrocarbon receptor (AHR) gene that perfectly co-segregated with the disease in the larger family. AHR is a ligand-activated transcription factor that has been intensively studied in xenobiotic-induced toxicity. Further, it has been shown to play a physiological role under normal cellular conditions, such as in immunity, inflammatory response and neurogenesis. Notably, knockout of the Ahr gene in mouse impairs optic nerve myelin sheath formation and results in oculomotor deficits sharing many features with our patients: the eye movement disorder in Ahr-/- mice appears early in development and presents as conjugate horizontal pendular nystagmus. We therefore propose AHR to be a novel disease gene for a new, recessively inherited disorder in humans, characterized by infantile nystagmus and foveal hypoplasia.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Homocigoto , Nistagmo Congénito/genética , Hipoplasia del Nervio Óptico/genética , Receptores de Hidrocarburo de Aril/genética , Animales , Niño , Electrorretinografía/métodos , Femenino , Humanos , Masculino , Ratones , Mutación/genética , Malformaciones del Sistema Nervioso/genética , Malformaciones del Sistema Nervioso/patología , Nistagmo Congénito/diagnóstico , Hipoplasia del Nervio Óptico/patología , LinajeRESUMEN
Spinocerebellar ataxia type 3 (SCA3) is caused by the expansion of CAG repeats in the ATXN3 gene leading to an elongated polyglutamine tract in the ataxin-3 protein. Previously, we demonstrated that symptoms of SCA3 are reversible in the first conditional mouse model for SCA3 directing ataxin-3 predominantly to the hindbrain. Here, we report on the effects of transgenic ataxin-3 expression in forebrain regions. Employing the Tet-off CamKII-promoter mouse line and our previously published SCA3 responder line, we generated double transgenic mice (CamKII/MJD77), which develop a neurological phenotype characterized by impairment in rotarod performance, and deficits in learning new motor tasks as well as hyperactivity. Ataxin-3 and ubiquitin-positive inclusions are detected in brains of double transgenic CamKII/MJD77 mice. After turning off the expression of pathologically expanded ataxin-3, these inclusions disappear. However, the observed phenotype could not be reversed, very likely due to pronounced apoptotic cell death in the frontal brain. Our data demonstrate that cerebellar expression is not required to induce a neurological phenotype using expanded ATXN3 as well as the pronounced sensibility of forebrain neurons for toxic ataxin-3.
Asunto(s)
Ataxina-3/genética , Lóbulo Frontal/metabolismo , Enfermedad de Machado-Joseph/genética , Enfermedad de Machado-Joseph/metabolismo , Neuronas/metabolismo , Expansión de Repetición de Trinucleótido , Animales , Ataxina-3/metabolismo , Conducta Animal , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/genética , Modelos Animales de Enfermedad , Lóbulo Frontal/patología , Expresión Génica , Estudios de Asociación Genética , Predisposición Genética a la Enfermedad , Inmunohistoquímica , Enfermedad de Machado-Joseph/patología , Ratones , Ratones Transgénicos , Degeneración Nerviosa/genética , Degeneración Nerviosa/metabolismo , Degeneración Nerviosa/patología , Especificidad de Órganos/genética , Agregado de Proteínas , Agregación Patológica de Proteínas , Desempeño PsicomotorRESUMEN
The original version of this Article contained an error in the spelling of the author Anja K. Mayer, which was incorrectly given as Anja Kathrin Mayer. This has now been corrected in both the PDF and HTML versions of the Article.
RESUMEN
Mutations in CNGA3 and CNGB3, the genes encoding the subunits of the tetrameric cone photoreceptor cyclic nucleotide-gated ion channel, cause achromatopsia, a congenital retinal disorder characterized by loss of cone function. However, a small number of patients carrying the CNGB3/c.1208G>A;p.R403Q mutation present with a variable retinal phenotype ranging from complete and incomplete achromatopsia to moderate cone dysfunction or progressive cone dystrophy. By exploring a large patient cohort and published cases, we identified 16 unrelated individuals who were homozygous or (compound-)heterozygous for the CNGB3/c.1208G>A;p.R403Q mutation. In-depth genetic and clinical analysis revealed a co-occurrence of a mutant CNGA3 allele in a high proportion of these patients (10 of 16), likely contributing to the disease phenotype. To verify these findings, we generated a Cngb3R403Q/R403Q mouse model, which was crossbred with Cnga3-deficient (Cnga3-/-) mice to obtain triallelic Cnga3+/- Cngb3R403Q/R403Q mutants. As in human subjects, there was a striking genotype-phenotype correlation, since the presence of 1 Cnga3-null allele exacerbated the cone dystrophy phenotype in Cngb3R403Q/R403Q mice. These findings strongly suggest a digenic and triallelic inheritance pattern in a subset of patients with achromatopsia/severe cone dystrophy linked to the CNGB3/p.R403Q mutation, with important implications for diagnosis, prognosis, and genetic counseling.
Asunto(s)
Defectos de la Visión Cromática , Canales Catiónicos Regulados por Nucleótidos Cíclicos , Heterocigoto , Activación del Canal Iónico , Mutación Missense , Células Fotorreceptoras Retinianas Conos , Enfermedades de la Retina , Sustitución de Aminoácidos , Animales , Defectos de la Visión Cromática/genética , Defectos de la Visión Cromática/metabolismo , Defectos de la Visión Cromática/patología , Canales Catiónicos Regulados por Nucleótidos Cíclicos/genética , Canales Catiónicos Regulados por Nucleótidos Cíclicos/metabolismo , Modelos Animales de Enfermedad , Células HEK293 , Humanos , Ratones , Ratones Transgénicos , Mutación , Células Fotorreceptoras Retinianas Conos/metabolismo , Células Fotorreceptoras Retinianas Conos/patología , Enfermedades de la Retina/genética , Enfermedades de la Retina/metabolismo , Enfermedades de la Retina/patologíaRESUMEN
PurposePart of the hidden genetic variation in heterogeneous genetic conditions such as inherited retinal diseases (IRDs) can be explained by copy-number variations (CNVs). Here, we explored the genomic landscape of IRD genes listed in RetNet to identify and prioritize those genes susceptible to CNV formation.MethodsRetNet genes underwent an assessment of genomic features and of CNV occurrence in the Database of Genomic Variants and literature. CNVs identified in an IRD cohort were characterized using targeted locus amplification (TLA) on extracted genomic DNA.ResultsExhaustive literature mining revealed 1,345 reported CNVs in 81 different IRD genes. Correlation analysis between rankings of genomic features and CNV occurrence demonstrated the strongest correlation between gene size and CNV occurrence of IRD genes. Moreover, we identified and delineated 30 new CNVs in IRD cases, 13 of which are novel and three of which affect noncoding, putative cis-regulatory regions. Finally, the breakpoints of six complex CNVs were determined using TLA in a hypothesis-neutral manner.ConclusionWe propose a ranking of CNV-prone IRD genes and demonstrate the efficacy of TLA for the characterization of CNVs on extracted DNA. Finally, this IRD-oriented CNV study can serve as a paradigm for other genetically heterogeneous Mendelian diseases with hidden genetic variation.
Asunto(s)
Mapeo Cromosómico , Variaciones en el Número de Copia de ADN , Genoma Humano , Genómica , Sistemas de Lectura Abierta , ARN no Traducido , Enfermedades de la Retina/genética , Alelos , Proteínas Relacionadas con las Cadherinas , Cadherinas/genética , Bases de Datos Genéticas , Proteínas del Ojo/genética , Predisposición Genética a la Enfermedad , Estudio de Asociación del Genoma Completo , Genómica/métodos , Humanos , Secuencias Reguladoras de Ácidos Nucleicos , Enfermedades de la Retina/diagnóstico , Análisis de Secuencia de ADN , Eliminación de SecuenciaRESUMEN
We report ophthalmic and genetic findings in patients with autosomal recessive retinitis pigmentosa (RP), cone-rod dystrophy (CRD) or cone dystrophy (CD) harboring potential pathogenic variants in the CDHR1 gene. Detailed ophthalmic examination was performed in seven sporadic and six familial subjects. Mutation screening was done using a customized next generation sequencing panel targeting 105 genes implicated in inherited retinal disorders. In one family, homozygosity mapping with subsequent candidate gene analysis was performed. Stringent filtering for rare and potentially disease causing variants following a model of autosomal recessive inheritance led to the identification of eleven different CDHR1 variants in nine index cases. All variants were novel at the time of their identification. In silico analyses confirmed their pathogenic potential. Minigene assays were performed for two non-canonical splice site variants and revealed missplicing for the mutant alleles. Mutations in CDHR1 are a rare cause of retinal dystrophy. Our study further expands the mutational spectrum of this gene and the associated clinical presentation.
Asunto(s)
Cadherinas/genética , Distrofias de Conos y Bastones/genética , Mutación , Proteínas del Tejido Nervioso/genética , Retinitis Pigmentosa/genética , Proteínas Relacionadas con las Cadherinas , Distrofias de Conos y Bastones/patología , Estudios de Asociación Genética , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Retinitis Pigmentosa/patologíaRESUMEN
Achromatopsia is a rare autosomal recessive cone disorder characterized by color vision defects, photophobia, nystagmus, and severely reduced visual acuity. The disease is caused by mutations in genes encoding crucial components of the cone phototransduction cascade (CNGA3, CNGB3, GNAT2, PDE6C, and PDE6H) or in ATF6, involved in the unfolded protein response. CNGB3 encoding the beta subunit of the cyclic nucleotide-gated ion channel in cone photoreceptors is the major achromatopsia gene. Here, we present a comprehensive spectrum of CNGB3 mutations and their prevalence in a cohort of 1074 independent families clinically diagnosed with achromatopsia. Of these, 485 (45.2%) carried mutations in CNGB3. We identified a total of 98 different potentially disease-causing CNGB3 variants, 58 of which are novel. About 10% of patients with CNGB3 mutations only harbored a single heterozygous variant. Therefore, we performed quantitative real-time PCR in 43 of such single heterozygotes in search of the missing allele, followed by microarray-based comparative genomic hybridization and breakpoint mapping. We discovered nine different heterozygous copy number variations encompassing one to 10 consecutive exons in 16 unrelated patients. Moreover, one additional patient with a homozygous CNGB3 deletion encompassing exons 4-18 was identified, highlighting the importance of CNV analysis for this gene.
Asunto(s)
Defectos de la Visión Cromática/diagnóstico , Defectos de la Visión Cromática/genética , Canales Catiónicos Regulados por Nucleótidos Cíclicos/genética , Variaciones en el Número de Copia de ADN , Mutación , Alelos , Mapeo Cromosómico , Segregación Cromosómica , Hibridación Genómica Comparativa , Análisis Mutacional de ADN , Exones , Efecto Fundador , Genotipo , Humanos , Tasa de MutaciónAsunto(s)
Síndrome de Bardet-Biedl/genética , Predisposición Genética a la Enfermedad , Niño , Femenino , Genotipo , Humanos , Masculino , FenotipoRESUMEN
Retinal dystrophies (RD) constitute a group of blinding diseases that are characterized by clinical variability and pronounced genetic heterogeneity. The different nonsyndromic and syndromic forms of RD can be attributed to mutations in more than 200 genes. Consequently, next generation sequencing (NGS) technologies are among the most promising approaches to identify mutations in RD. We screened a large cohort of patients comprising 89 independent cases and families with various subforms of RD applying different NGS platforms. While mutation screening in 50 cases was performed using a RD gene capture panel, 47 cases were analyzed using whole exome sequencing. One family was analyzed using whole genome sequencing. A detection rate of 61% was achieved including mutations in 34 known and two novel RD genes. A total of 69 distinct mutations were identified, including 39 novel mutations. Notably, genetic findings in several families were not consistent with the initial clinical diagnosis. Clinical reassessment resulted in refinement of the clinical diagnosis in some of these families and confirmed the broad clinical spectrum associated with mutations in RD genes.
Asunto(s)
Mutación , Distrofias Retinianas/genética , Variaciones en el Número de Copia de ADN , Exoma , Proteínas del Ojo/genética , Femenino , Estudios de Asociación Genética , Heterogeneidad Genética , Predisposición Genética a la Enfermedad , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Masculino , Tasa de Mutación , Linaje , Fenotipo , Distrofias Retinianas/diagnósticoRESUMEN
Several genes have been implicated in the autosomal recessive form of cone-rod dystrophy (CRD), but the majority of cases remain unsolved. We identified a homozygous interval comprising two known genes associated with the autosomal recessive form of CRD, namely RAB28 and PROM1, in a consanguineous family with clinical evidence of CRD. Both genes proved to be mutation negative upon sequencing of exons and canonical splice sites but whole-genome sequencing revealed a private variant located deep in intron 18 of PROM1. In silico and functional analyses of this variant using minigenes as splicing reporters revealed the integration of a pseudoexon in the mutant transcript, thereby leading to a premature termination codon and presumably resulting in a functional null allele. This is the first report of a deep intronic variant that acts as a splicing mutation in PROM1. The detection of such variants escapes the exon-focused techniques typically used in genetic analyses. Sequencing the entire genomic regions of known disease genes might identify more causal mutations in the autosomal recessive form of CRD.
Asunto(s)
Antígenos CD/genética , Exones/genética , Predisposición Genética a la Enfermedad , Genoma Humano , Glicoproteínas/genética , Intrones/genética , Mutación/genética , Péptidos/genética , Retinitis Pigmentosa/genética , Antígeno AC133 , Secuencia de Aminoácidos , Antígenos CD/química , Secuencia de Bases , Análisis Mutacional de ADN , Femenino , Glicoproteínas/química , Células HEK293 , Homocigoto , Humanos , Masculino , Datos de Secuencia Molecular , Linaje , Péptidos/química , Empalme del ARN/genéticaRESUMEN
PURPOSE: To identify the genetic defect in a consanguineous Israeli Muslim Arab family with juvenile retinitis pigmentosa (RP). METHODS: DNA samples were collected from the index patient, her parents, her affected sister, and two non-affected siblings. Genome-wide linkage analysis with 250 K single nucleotide polymorphism (SNP) arrays was performed using DNA from the two affected patients. Owing to consanguinity in the family, we applied homozygosity mapping to identify the disease-causing gene. The candidate gene SPATA7 was screened for mutations with PCR amplifications and direct Sanger sequencing. RESULTS: Following high-density SNP arrays, we identified several homozygous genomic regions one of which included the SPATA7 gene. Several mutations in SPATA7 have been reported for various forms of retinal dystrophy, including Leber congenital amaurosis (LCA) and juvenile RP. PCR-based sequence content mapping, long-distance PCR amplifications, and subsequent sequencing analysis revealed a homozygous 63.4 kb large deletion that encompasses the 5' part of the SPATA7 gene including exons 1-5. The mutation showed concordant segregation with the phenotype in the family as expected for autosomal recessive mode of inheritance and is consistent with a diagnosis of juvenile RP. CONCLUSIONS: We report a novel homozygous large deletion in SPATA7 associated with juvenile RP in a consanguineous Israeli Muslim Arab family. This is the first larger deletion mutation reported for SPATA7.
Asunto(s)
Secuencia de Bases , Consanguinidad , Proteínas de Unión al ADN/genética , Homocigoto , Retinitis Pigmentosa/genética , Eliminación de Secuencia , Adolescente , Adulto , Árabes , Preescolar , Exones , Femenino , Genes Recesivos , Humanos , Islamismo , Israel , Masculino , Datos de Secuencia Molecular , Linaje , Retinitis Pigmentosa/patologíaRESUMEN
Hepatitis D virus (HDV) superinfection of hepatitis B virus (HBV) carriers causes severe liver disease and a high rate of chronicity. Therefore, a vaccine protecting HBV carriers from HDV superinfection is needed. To protect from HDV infection an induction of virus-specific T cells is required, as antibodies to the two proteins of HDV, p24 and p27, do not neutralize the HBV-derived envelope of HDV. In mice, HDV-specific CD8(+) and CD4(+) T cell responses were induced by a DNA vaccine expressing HDV p27. In subsequent experiments, seven naive woodchucks were immunized with a DNA prime and adenoviral boost regimen prior to simultaneous woodchuck hepatitis virus (WHV) and HDV infection. Five of seven HDV-immunized woodchucks were protected against HDV infection, while acute self-limiting WHV infection occurred as expected. The two animals with the breakthrough had a shorter HDV viremia than the unvaccinated controls. The DNA prime and adenoviral vector boost vaccination protected woodchucks against HDV infection in the setting of simultaneous infection with WHV and HDV. In future experiments, the efficacy of this protocol to protect from HDV infection in the setting of HDV superinfection will need to be proven.
Asunto(s)
Virus de la Hepatitis B de la Marmota/inmunología , Hepatitis B/complicaciones , Hepatitis D/prevención & control , Virus de la Hepatitis Delta/inmunología , Sobreinfección/prevención & control , Linfocitos T/inmunología , Adenoviridae , Animales , Anticuerpos Antivirales/sangre , Aspartato Aminotransferasas/metabolismo , Western Blotting , Cartilla de ADN/genética , Ensayo de Inmunoadsorción Enzimática , Femenino , Vectores Genéticos/inmunología , Hepatitis D/complicaciones , Inmunización Secundaria , Marmota , Ratones , Plásmidos/genética , Sobreinfección/virología , Vacunas Virales/genética , Vacunas Virales/inmunologíaRESUMEN
Induction of hepatitis B virus (HBV)-specific cytotoxic T cells by therapeutic immunization may be a strategy to treat chronic hepatitis B. In the HBV animal model, woodchucks, the application of DNA vaccine expressing woodchuck hepatitis virus (WHV) core antigen (WHcAg) in combination with antivirals led to the prolonged control of viral replication. However, it became clear that the use of more potent vaccines is required to overcome WHV persistence. Therefore, we asked whether stronger and more functional T-cell responses could be achieved using the modified vaccines and an optimized prime-boost vaccination regimen. We developed a new DNA plasmid (pCGWHc) and recombinant adenoviruses (AdVs) showing high expression levels of WHcAg. Mice vaccinated with the improved plasmid pCGWHc elicited a stronger WHcAg-specific CD8(+) T-cell response than with the previously used vaccines. Using multicolor flow cytometry and an in vivo cytotoxicity assay, we showed that immunization in a DNA prime-AdV boost regimen resulted in an even more vigorous and functional T-cell response than immunization with the new plasmid alone. Immunization of naïve woodchucks with pCGWHc plasmid or AdVs induced a significant WHcAg-specific degranulation response prior to the challenge, this response had not been previously detected. Consistently, this response led to a rapid control of infection after the challenge. Our results demonstrate that high antigen expression levels and the DNA prime-AdV boost immunization improved the T-cell response in mice and induced significant T-cell responses in woodchucks. Therefore, this new vaccination strategy may be a candidate for a therapeutic vaccine against chronic HBV infection.
Asunto(s)
Adenoviridae/inmunología , Antígenos del Núcleo de la Hepatitis B/inmunología , Vacunas contra Hepatitis B/inmunología , Virus de la Hepatitis B/inmunología , Hepatitis B/inmunología , Linfocitos T/inmunología , Vacunas de ADN/inmunología , Adenoviridae/genética , Animales , Células Cultivadas , Modelos Animales de Enfermedad , Femenino , Hepatitis B/virología , Antígenos del Núcleo de la Hepatitis B/administración & dosificación , Antígenos del Núcleo de la Hepatitis B/genética , Vacunas contra Hepatitis B/administración & dosificación , Vacunas contra Hepatitis B/genética , Virus de la Hepatitis B/genética , Humanos , Inmunización Secundaria , Marmota , Ratones , Ratones Endogámicos C57BL , Vacunas de ADN/administración & dosificación , Vacunas de ADN/genéticaRESUMEN
BACKGROUND, AIM, AND SCOPE: Lungs are permanently and simultaneously challenged by airborne microorganisms and airborne pollutants. Temporal increase of airborne particulate matter (APM), a potential carrier for extractable organic matter (EOM), degrades the situation of pulmonary patients. The Ah receptor (AhR) has been described as an important factor influencing the immunological challenge by viral infections. Molecular mechanisms underlying epidemiological observations are not well understood. Cytokine secretion (IL-6, IL-8, and TGF-beta) from human bronchial epithelial cells (Beas2B) was determined as an indicator for immune responses upon co-stimulation with an artificial analog of viral dsRNA [polyinosinic/polycytidylic acid, (PIC)] and EOM of Standard Reference Material 1649a (SRM). Since polycyclic aromatic hydrocarbons are major components of APM usually acting via the AhR, particular focus was on AhR involvement. MATERIALS AND METHODS: Cytokine secretion was demonstrated by enzyme-linked immunosorbent assay. To mimic the activation of organic matter during contact of particles with the human lung, Soxhlet extraction of SRM was performed. In some experiments, the AhR was blocked by alpha-naphthoflavone. RESULTS: Microbial stimulation (PIC) induced Beas2B cytokine release, whereas isolated exposure to EOM of APM did not. Co-stimulation with EOM and PIC increased IL-8 secretion, whereas neither IL-6 nor TGF-beta was affected. Blocking of the AhR suppressed the release of IL-8. DISCUSSION: Organic compounds adsorbed on airborne particulate matter influence the cytokine secretion of lung epithelial cells induced by pathogen-associated molecular patterns. RECOMMENDATIONS AND PERSPECTIVES: Further investigation of these observations is required to understand the molecular mechanisms underlying adverse health effects of APM reported in epidemiological studies.
Asunto(s)
Contaminantes Atmosféricos/toxicidad , Bronquios/efectos de los fármacos , Citocinas/metabolismo , Material Particulado/toxicidad , Mucosa Respiratoria/efectos de los fármacos , Contaminantes Atmosféricos/normas , Benzo(a)pireno , Benzoflavonas/toxicidad , Bronquios/inmunología , Línea Celular , Humanos , Interleucina-6/metabolismo , Interleucina-8/antagonistas & inhibidores , Interleucina-8/metabolismo , Material Particulado/normas , Poli C/toxicidad , Hidrocarburos Policíclicos Aromáticos/toxicidad , Receptores de Hidrocarburo de Aril/antagonistas & inhibidores , Receptores de Hidrocarburo de Aril/metabolismo , Estándares de Referencia , Mucosa Respiratoria/inmunología , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
The upper airways are prone to contact with pathogenic as well as non-pathogenic microbes, therefore immune recognition principles have to be tightly controlled. Here we show that human BEAS-2B bronchial epithelial cells inhibited secretion of the pro-inflammatory cytokines TNF-alpha and IL-12 by monocytes, macrophages and dendritic cells. This inhibitory effect could be transferred by supernatant of resting BEAS-2B cells and was also observed when primary murine tracheal epithelial cells were prepared. In contrast to inhibition of pro-inflammatory cytokine secretion epithelial cell-conditioned dendritic cells showed increased expression of IL-10 and arginase-1, thus displaying properties of alternative activation. Accordingly, Toll-like receptor-mediated up-regulation of CD40, CD86 and PD-L2 (CD273) on murine dendritic cells was reduced in the presence of bronchial epithelial cell supernatant. However, expression of negative regulatory PD-L1 (CD274) was increased and dendritic cell induced proliferation of T lymphocytes was diminished. Epithelial cells also showed a direct inhibitory effect on T lymphocyte proliferation and this was due to the constitutive secretion of TGF-beta by bronchial epithelial cells. Moreover, epithelial cell-conditioned T lymphocytes showed increased differentiation towards IL-10-producing Tr1 cells. The results indicate that bronchial epithelial cells induce a non-inflammatory microenvironment that regulates local immune homeostasis.