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Considering polygenic risk scores (PRSs) in individual risk prediction is increasingly implemented in genetic testing for hereditary breast cancer (BC) based on next-generation sequencing (NGS). To calculate individual BC risks, the Breast and Ovarian Analysis of Disease Incidence and Carrier Estimation Algorithm (BOADICEA) with the inclusion of the BCAC 313 or the BRIDGES 306 BC PRS is commonly used. The PRS calculation depends on accurately reproducing the variant allele frequencies (AFs) and, consequently, the distribution of PRS values anticipated by the algorithm. Here, the 324 loci of the BCAC 313 and the BRIDGES 306 BC PRS were examined in population-specific database gnomAD and in real-world data sets of five centers of the German Consortium for Hereditary Breast and Ovarian Cancer (GC-HBOC), to determine whether these expected AFs can be reproduced by NGS-based genotyping. Four PRS loci were non-existent in gnomAD v3.1.2 non-Finnish Europeans, further 24 loci showed noticeably deviating AFs. In real-world data, between 11 and 23 loci were reported with noticeably deviating AFs, and were shown to have effects on final risk prediction. Deviations depended on the sequencing approach, variant caller and calling mode (forced versus unforced) employed. Therefore, this study demonstrates the necessity to apply quality assurance not only in terms of sequencing coverage but also observed AFs in a sufficiently large cohort, when implementing PRSs in a routine diagnostic setting. Furthermore, future PRS design should be guided by the technical reproducibility of expected AFs across commonly used genotyping methods, especially NGS, in addition to the observed effect sizes.
Assuntos
Neoplasias da Mama , Herança Multifatorial , Humanos , Feminino , Neoplasias da Mama/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Sequenciamento de Nucleotídeos em Larga Escala/normas , Técnicas de Genotipagem/métodos , Técnicas de Genotipagem/normas , Predisposição Genética para Doença , Testes Genéticos/métodos , Testes Genéticos/normas , Frequência do Gene , Algoritmos , Estratificação de Risco GenéticoRESUMO
BACKGROUND: Forkhead-Box-Protein P3 (FoxP3) is a transcription factor and marker of regulatory T cells, converting naive T cells into Tregs that can downregulate the effector function of other T cells. We previously detected the expression of FoxP3 in retinal pigment epithelial (RPE) cells, forming the outer blood-retina barrier of the immune privileged eye. METHODS: We investigated the expression, subcellular localization, and phosphorylation of FoxP3 in RPE cells in vivo and in vitro after treatment with various stressors including age, retinal laser burn, autoimmune inflammation, exposure to cigarette smoke, in addition of IL-1ß and mechanical cell monolayer destruction. Eye tissue from humans, mouse models of retinal degeneration and rats, and ARPE-19, a human RPE cell line for in vitro experiments, underwent immunohistochemical, immunofluorescence staining, and PCR or immunoblot analysis to determine the intracellular localization and phosphorylation of FoxP3. Cytokine expression of stressed cultured RPE cells was investigated by multiplex bead analysis. Depletion of the FoxP3 gene was performed with CRISPR/Cas9 editing. RESULTS: RPE in vivo displayed increased nuclear FoxP3-expression with increases in age and inflammation, long-term exposure of mice to cigarette smoke, or after laser burn injury. The human RPE cell line ARPE-19 constitutively expressed nuclear FoxP3 under non-confluent culture conditions, representing a regulatory phenotype under chronic stress. Confluently grown cells expressed cytosolic FoxP3 that was translocated to the nucleus after treatment with IL-1ß to imitate activated macrophages or after mechanical destruction of the monolayer. Moreover, with depletion of FoxP3, but not of a control gene, by CRISPR/Cas9 gene editing decreased stress resistance of RPE cells. CONCLUSION: Our data suggest that FoxP3 is upregulated by age and under cellular stress and might be important for RPE function.
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Degeneração Macular , Epitélio Pigmentado da Retina , Animais , Humanos , Camundongos , Ratos , Células Cultivadas , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Inflamação/genética , Inflamação/metabolismo , Degeneração Macular/genética , Degeneração Macular/metabolismo , Degeneração Macular/patologia , Epitélio Pigmentado da Retina/metabolismo , Epitélio Pigmentado da Retina/patologia , Pigmentos da Retina/genética , Pigmentos da Retina/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Male breast cancer (mBC) is associated with a high prevalence of pathogenic variants (PVs) in the BRCA2 gene; however, data regarding other BC predisposition genes are limited. In this retrospective multicenter study, we investigated the prevalence of PVs in BRCA1/2 and 23 non-BRCA1/2 genes using a sample of 614 patients with mBC, recruited through the centers of the German Consortium for Hereditary Breast and Ovarian Cancer. A high proportion of patients with mBC carried PVs in BRCA2 (23.0%, 142/614) and BRCA1 (4.6%, 28/614). The prevalence of BRCA1/2 PVs was 11.0% in patients with mBC without a family history of breast and/or ovarian cancer. Patients with BRCA1/2 PVs did not show an earlier disease onset than those without. The predominant clinical presentation of tumor phenotypes was estrogen receptor (ER)-positive, progesterone receptor (PR)-positive, and HER2-negative (77.7%); further, 10.2% of the tumors were triple-positive, and 1.2% were triple-negative. No association was found between ER/PR/HER2 status and BRCA1/2 PV occurrence. Comparing the prevalence of protein-truncating variants (PTVs) between patients with mBC and control data (ExAC, n = 27,173) revealed significant associations of PTVs in both BRCA1 and BRCA2 with mBC (BRCA1: OR = 17.04, 95% CI = 10.54−26.82, p < 10−5; BRCA2: OR = 77.71, 95% CI = 58.71−102.33, p < 10−5). A case-control investigation of 23 non-BRCA1/2 genes in 340 BRCA1/2-negative patients and ExAC controls revealed significant associations of PTVs in CHEK2, PALB2, and ATM with mBC (CHEK2: OR = 3.78, 95% CI = 1.59−7.71, p = 0.002; PALB2: OR = 14.77, 95% CI = 5.02−36.02, p < 10−5; ATM: OR = 3.36, 95% CI = 0.89−8.96, p = 0.04). Overall, our findings support the benefit of multi-gene panel testing in patients with mBC irrespective of their family history, age at disease onset, and tumor phenotype.
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Reconstitution of the T cell repertoire after allogeneic stem cell transplantation is a long and often incomplete process. As a result, reactivation of Epstein-Barr virus (EBV) is a frequent complication that may be treated by adoptive transfer of donor-derived EBV-specific T cells. We generated donor-derived EBV-specific T cells by stimulation with peptides representing defined epitopes covering multiple HLA restrictions. T cells were adoptively transferred to a patient who had developed persisting high titers of EBV after allogeneic stem cell transplantation for angioimmunoblastic T-cell lymphoma (AITL). T cell receptor beta (TCRß) deep sequencing showed that the T cell repertoire of the patient early after transplantation (day 60) was strongly reduced and only very low numbers of EBV-specific T cells were detectable. Manufacturing and in vitro expansion of donor-derived EBV-specific T cells resulted in enrichment of EBV epitope-specific, HLA-restricted T cells. Monitoring of T cell clonotypes at a molecular level after adoptive transfer revealed that the dominant TCR sequences from peptide-stimulated T cells persisted long-term and established an EBV-specific TCR clonotype repertoire in the host, with many of the EBV-specific TCRs present in the donor. This reconstituted repertoire was associated with immunological control of EBV and with lack of further AITL relapse.
Assuntos
Infecções por Vírus Epstein-Barr , Transplante de Células-Tronco Hematopoéticas , Transferência Adotiva , Epitopos , Herpesvirus Humano 4/fisiologia , Humanos , Peptídeo T , Peptídeos , Linfócitos TRESUMO
One of the hallmarks of B lymphoid malignancies is a B cell clone characterized by a unique footprint of clonal immunoglobulin (IG) gene rearrangements that serves as a diagnostic marker for clonality assessment. The EuroClonality/BIOMED-2 assay is currently the gold standard for analyzing IG heavy chain (IGH) and κ light chain (IGK) gene rearrangements of suspected B cell lymphomas. Here, the EuroClonality-NGS Working Group presents a multicentre technical feasibility study of a novel approach involving next-generation sequencing (NGS) of IGH and IGK loci rearrangements that is highly suitable for detecting IG gene rearrangements in frozen and formalin-fixed paraffin-embedded tissue specimens. By employing gene-specific primers for IGH and IGK amplifying smaller amplicon sizes in combination with deep sequencing technology, this NGS-based IG clonality analysis showed robust performance, even in DNA samples of suboptimal DNA integrity, and a high clinical sensitivity for the detection of clonal rearrangements. Bioinformatics analyses of the high-throughput sequencing data with ARResT/Interrogate, a platform developed within the EuroClonality-NGS Working Group, allowed accurate identification of clonotypes in both polyclonal cell populations and monoclonal lymphoproliferative disorders. This multicentre feasibility study is an important step towards implementation of NGS-based clonality assessment in clinical practice, which will eventually improve lymphoma diagnostics.
Assuntos
Rearranjo Gênico/genética , Genes de Imunoglobulinas/genética , Estudos de Viabilidade , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias kappa de Imunoglobulina/genética , Linfoma de Células B/genética , Transtornos Linfoproliferativos/genéticaRESUMO
The degree and type of T cell infiltration influence rectal cancer prognosis regardless of classical tumor staging. We asked whether clonal expansion and tumor infiltration are restricted to selected-phenotype T cells; which clones are accessible in peripheral blood; and what the spatial distribution of their target antigens is. From five rectal cancer patients, we isolated paired tumor-infiltrating T cells (TILs) and T cells from unaffected rectum mucosa (TUM) using 13-parameter FACS single cell index sorting. TCRαß sequences, cytokine, and transcription factor expression were determined with single cell sequencing. TILs and TUM occupied distinct phenotype compartments and clonal expansion predominantly occurred within CD8+ T cells. Expanded TIL clones identified by paired TCRαß sequencing and exclusively detectable in the tumor showed characteristic PD-1 and TIM-3 expression. TCRß repertoire sequencing identified 49 out of 149 expanded TIL clones circulating in peripheral blood and 41 (84%) of these were PD-1- TIM-3-. To determine whether clonal expansion of predominantly tumor-infiltrating T cell clones was driven by antigens uniquely presented in tumor tissue, selected TCRs were reconstructed and incubated with cells isolated from corresponding tumor or unaffected mucosa. The majority of clones exclusively detected in the tumor recognized antigen at both sites. In summary, rectal cancer is infiltrated with expanded distinct-phenotype T cell clones that either i) predominantly infiltrate the tumor, ii) predominantly infiltrate the unaffected mucosa, or iii) overlap between tumor, unaffected mucosa, and peripheral blood. However, the target antigens of predominantly tumor-infiltrating TIL clones do not appear to be restricted to tumor tissue.
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OBJECTIVE: Refractory coeliac disease (RCD) is a potentially hazardous complication of coeliac disease (CD). In contrast to RCD type I, RCD type II is a precursor entity of enteropathy-associated T-cell lymphoma (EATL), which is associated with clonally expanding T-cells that are also found in the sequentially developing EATL. Using high-throughput sequencing (HTS), we aimed to establish the small-intestinal T-cell repertoire (TCR) in CD and RCD to unravel the role of distinct T-cell clonotypes in RCD pathogenesis. DESIGN: DNA extracted from duodenal mucosa specimens of controls (n=9), active coeliacs (n=10), coeliacs on a gluten-free diet (n=9), RCD type I (n=8), RCD type II (n=8) and unclassified Marsh I cases (n=3) collected from 2002 to 2013 was examined by TCRß-complementarity-determining regions 3 (CDR3) multiplex PCR followed by HTS of the amplicons. RESULTS: On average, 106 sequence reads per sample were generated consisting of up to 900 individual TCRß rearrangements. In RCD type II, the most frequent clonotypes (ie, sequence reads with identical CDR3) represent in average 42.6% of all TCRß rearrangements, which was significantly higher than in controls (6.8%; p<0.01) or RCD type I (6.7%; p<0.01). Repeat endoscopies in individual patients revealed stability of clonotypes for up to several years without clinical symptoms of EATL. Dominant clonotypes identified in individual patients with RCD type II were unique and not related between patients. CD-associated, gliadin-dependent CDR3 motifs were only detectable at low frequencies. CONCLUSIONS: TCRß-HTS analysis unravels the TCR in CD and allows detailed analysis of individual TCRß rearrangements. Dominant TCRß sequences identified in patients with RCD type II are unique and not homologous to known gliadin-specific TCR sequences, supporting the assumption that these clonal T-cells expand independent of gluten stimulation.
Assuntos
Doença Celíaca/diagnóstico , Doença Celíaca/metabolismo , Genes Codificadores da Cadeia beta de Receptores de Linfócitos T/genética , Linfócitos T/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores/metabolismo , Estudos de Casos e Controles , Doença Celíaca/classificação , Doença Celíaca/genética , Diagnóstico Diferencial , Dieta Livre de Glúten/métodos , Duodeno/patologia , Feminino , Genes Codificadores da Cadeia beta de Receptores de Linfócitos T/imunologia , Humanos , Imunossupressores/uso terapêutico , Mucosa Intestinal/patologia , Intestino Delgado/patologia , Masculino , Pessoa de Meia-Idade , Valor Preditivo dos Testes , Estudos Retrospectivos , Sensibilidade e EspecificidadeRESUMO
BACKGROUND: The aim was to determine the patients' characteristics, comorbidity, and inpatient treatment features of very old otorhinolaryngology patients (80+ years) compared to old patients (75-79 years). METHODS: A single-center cohort study in a tertiary and university care center was performed with 144 old and 143 very old patients who were hospitalized in 2012. Predictors for differences between old and very old patients were analyzed univariately and multivariately using regression models. RESULTS: Ear (30 %) and nose/paranasal sinus (23 %) diseases were the most frequent reasons for hospitalization. Baseline and disease characteristics were not different between the two groups of elderly patients. Duration of hospitalization was no longer in very old patients (p = 0.827). Mobility (p = 0.017), dietary intake (p = 0.017), and having hearing aid (p < 0.0001) were independent comorbidity predictors in very old patients compared to old patients. Polymedication was found less frequently in very old patients (p = 0.017). To take cardiovascular drugs (p = 0.009) or psychotherapeutic drugs (p = 0.045) were independent permanent medication predictors in very old patients compared to old patients. About half of the patients received a surgical treatment (52 %) and the other half a conservative treatment (48 %). The very old patients received significantly more often an antibiotic treatment (p < 0.0001). Complication rates for surgical cases and non-surgical cases were not different (p = 0.686 and p = 0.524, respectively). CONCLUSIONS: Although comorbidity continues to increase in hospitalized very old compared to old otorhinolaryngology patients, most of the disease, treatment and treatment related complication characteristics seem not to change significantly from old to very old patients.
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Avaliação Geriátrica , Pacientes Internados/estatística & dados numéricos , Otorrinolaringopatias/epidemiologia , Fatores Etários , Idoso , Idoso de 80 Anos ou mais , Feminino , Seguimentos , Alemanha/epidemiologia , Hospitalização , Humanos , Masculino , Morbidade/tendências , Otorrinolaringopatias/terapia , Fatores de RiscoAssuntos
Cateterismo Periférico/métodos , Eletrocardiografia/métodos , Neoplasias Otorrinolaringológicas/tratamento farmacológico , Procedimentos Cirúrgicos Otorrinolaringológicos , Equipe de Assistência ao Paciente , Ultrassonografia de Intervenção/métodos , Dispositivos de Acesso Vascular , Quimioterapia Adjuvante , Contraindicações , Desenho de Equipamento , Humanos , VeiasRESUMO
Multinucleated giant cells (MGCs) form by fusion of macrophages and are presumed to contribute to the removal of debris from tissues. In a systematic in vitro analysis, we show that IL-4-induced MGCs phagocytosed large and complement-opsonized materials more effectively than their unfused M2 macrophage precursors. MGC expression of complement receptor 4 (CR4) was increased, but it functioned primarily as an adhesion integrin. In contrast, although expression of CR3 was not increased, it became functionally activated during fusion and was located on the extensive membrane ruffles created by excess plasma membrane arising from macrophage fusion. The combination of increased membrane area and activated CR3 specifically equips MGCs to engulf large complement-coated targets. Moreover, we demonstrate these features in vivo in the recently described complement-dependent therapeutic elimination of systemic amyloid deposits by MGCs. MGCs are evidently more than the sum of their macrophage parts.
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Células Gigantes/metabolismo , Interleucina-4/farmacologia , Fagocitose/efeitos dos fármacos , Amiloide/metabolismo , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Antígeno CD11c/metabolismo , Antígenos CD18/metabolismo , Complemento C3/deficiência , Complemento C3/genética , Complemento C3/metabolismo , Cricetinae , Células Gigantes/imunologia , Humanos , Integrina alfaXbeta2/metabolismo , Fator Estimulador de Colônias de Macrófagos/farmacologia , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Ratos , Receptores de IgG/metabolismo , Acetato de Tetradecanoilforbol/farmacologia , Regulação para Cima/efeitos dos fármacosRESUMO
Accumulation of CD3(+) T-cell receptor (TCR)αß(+)CD4(-)CD8(-) double-negative T cells (DNT) is a hallmark of autoimmune lymphoproliferative syndrome (ALPS). DNT origin and differentiation pathways remain controversial. Here we show that human ALPS DNT have features of terminally differentiated effector memory T cells reexpressing CD45RA(+) (TEMRA), but are CD27(+)CD28(+)KLRG1(-) and do not express the transcription factor T-bet. This unique phenotype was also detected among CD4(+) or CD8(+) ALPS TEMRA cells. T-cell receptor ß deep sequencing revealed a significant fraction of shared CDR3 sequences between ALPS DNT and both CD4(+) and CD8(+)TEMRA cells. Moreover, in ALPS patients with a germ line FAS mutation and somatic loss of heterozygosity, in whom biallelic mutant cells can be tracked by absent Fas expression, Fas-negative T cells accumulated not only among DNT, but also among CD4(+) and CD8(+)TEMRA cells. These data indicate that in human Fas deficiency DNT cannot only derive from CD8(+), but also from CD4(+) T cells. Furthermore, defective Fas signaling leads to aberrant transcriptional programs and differentiation of subsets of CD4(+) and CD8(+) T cells. Accumulation of these cells before their double-negative state appears to be an important early event in the pathogenesis of lymphoproliferation in ALPS patients.
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Síndrome Linfoproliferativa Autoimune/imunologia , Síndrome Linfoproliferativa Autoimune/patologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/patologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/patologia , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/patologia , Receptor fas/deficiência , Receptor fas/genética , Adolescente , Adulto , Síndrome Linfoproliferativa Autoimune/genética , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Criança , Pré-Escolar , Estudos de Associação Genética , Mutação em Linhagem Germinativa , Humanos , Memória Imunológica , Antígenos Comuns de Leucócito/metabolismo , Perda de Heterozigosidade , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Proteínas com Domínio T/metabolismo , Subpopulações de Linfócitos T/metabolismo , Adulto JovemRESUMO
Adoptive immunotherapy against viral infections is a promising treatment option for patients after hematopoietic stem cell transplantation. However, the generation of virus-specific T cells is either cost-intensive or time-consuming. We developed the first GMP-compliant protocol to generate donor-derived adenovirus (HAdV), cytomegalovirus, and Epstein-Barr virus-specific T-cell lines (TCLs) within 12 days by the use of overlapping polypeptides derived from different viruses in combination with IL-15. Two patients after undergoing haploidentical hematopoietic stem cell transplantation with HAdV viremia displaying rising viral loads despite treatment with cidofovir received 1×10 donor-derived short-term expanded HAdV-specific TCLs per kg body weight. In both patients, HAdV-specific T cells could be detected by IFN-γ-ELISpot 30 and 22 days postinfusion, and resulted in complete clearance or >1.5 log reduction of viral load within 15 and 18 days, respectively. This protocol facilitates rapid and cost-effective generation of virus-specific TCLs, which appear to provide an effective treatment option.
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Infecções por Adenovirus Humanos/imunologia , Infecções por Adenovirus Humanos/terapia , Adenovírus Humanos/imunologia , Imunoterapia Adotiva , Especificidade do Receptor de Antígeno de Linfócitos T/imunologia , Linfócitos T/imunologia , Linfócitos T/transplante , Infecções por Adenovirus Humanos/virologia , Adenovírus Humanos/classificação , Pré-Escolar , Evolução Fatal , Feminino , Doença Enxerto-Hospedeiro/etiologia , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Humanos , Imunoterapia Adotiva/métodos , Imunoterapia Adotiva/normas , Lactente , Masculino , Peptídeos/farmacologia , Linfócitos T/efeitos dos fármacos , Linfócitos T Citotóxicos/efeitos dos fármacos , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/transplante , Transplante Homólogo , Resultado do Tratamento , Carga ViralRESUMO
Previous studies have proposed varying causes for idiopathic sudden sensorineural hearing loss (SSNHL), including vascular occlusion, ruptured inner ear membrane, acoustic tumours and circulatory disturbances in the inner ear. The objective of this study was to characterise the autonomic regulation in 19 SSNHL patients in comparison to 19 healthy age-gender matched normal-hearing control subjects (CON) in order to improve the diagnostics of vascular caused hearing loss in SSNHL patients. A high-resolution short-term electrocardiogram (ECG) and the continuous noninvasive blood pressure signal were simultaneously recorded under resting conditions (30min). Linear and nonlinear indices of heart rate- and blood pressure variability (HRV, BPV) were calculated to characterise autonomic regulation. The results showed that HRV analysis did not produce significantly different results between SSNHL and CON, whereas linear and nonlinear BPV indices showed significant differences between both groups (p<0.01). This study was the first to show an altered cardiovascular regulation in SSNHL patients when compared to CON subjects, based on continuous blood pressure analysis. This was characterised by reduced variability, complexity and dynamics of blood pressure time series in SSNHL. These findings may contribute to an improved classification of the controversially discussed causes of SSNHL and, in addition, may lead to improved diagnostic strategies for a subgroup of SSNHL patients whose hearing loss is caused by cardiovascular factors.
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Sistema Nervoso Autônomo/fisiopatologia , Perda Auditiva Súbita/diagnóstico , Perda Auditiva Súbita/fisiopatologia , Idoso , Pressão Sanguínea/fisiologia , Eletrocardiografia , Entropia , Feminino , Frequência Cardíaca/fisiologia , Humanos , Masculino , Pessoa de Meia-Idade , Análise de Componente Principal , Análise Espectral , Fatores de TempoRESUMO
OBJECTIVE: We propose that the fetal heart is highly resilient to hypoxic stress. Our objective was to elucidate the human fetal gene expression profile in response to simulated ischemia and reperfusion to identify molecular targets that account for the innate cardioprotection exhibited by the fetal phenotype. METHODS: Primary cultures of human fetal cardiac myocytes (gestational age, 15-20 weeks) were exposed to simulated ischemia and reperfusion in vitro by using a simulated ischemic buffer under anoxic conditions. Total RNA from treated and baseline cells were isolated, reverse transcribed, and labeled with Cy3 or Cy5 and hybridized to a human cDNA microarray for expression analysis. This analysis revealed a highly significant (false discovery rate, <3%) suppression of interleukin 6 transcript levels during the reperfusion phase confirmed by means of quantitative polymerase chain reaction (0.25 +/- 0.11-fold). Interleukin 6 signaling during ischemia and reperfusion was assessed at the protein expression level by means of Western measurements of interleukin 6 receptor, the signaling subunit of the interleukin 6 receptor complex (gp130), and signal transducer of activated transcription 3. Posttranslational changes in the protein kinase B signaling pathway were determined on the basis of the phosphorylation status of protein kinase B, mitogen-activated protein kinase, and glycogen synthase kinase 3beta. The effect of suppression of a prohypertrophic kinase, integrin-linked kinase, with short-interfering RNA was determined in an ischemia and reperfusion-stressed neonatal rat cardiac myocyte model. Endogenous secretion of interleukin 6 protein in culture supernatants was measured by enzyme-linked immunosorbent assay. RESULTS: Human fetal cardiac myocytes exhibited a significantly lower rate of apoptosis induction during ischemia and reperfusion and after exposure to staurosporine and recombinant interleukin 6 compared with that observed in neonatal rat cardiac myocytes ( P < .05 for all comparisons, analysis of variance). Exposure to exogenously added recombinant interleukin 6 increased the apoptotic rate in both rat and human fetal cardiac myocytes ( P < .05). Short-interfering RNA-mediated suppression of integrin-linked kinase, a prohypertrophy upstream kinase regulating protein kinase B and glycogen synthase kinase 3beta phosphorylation, was cytoprotective against ischemia and reperfusion-induced apoptosis in neonatal rat cardiac myocytes ( P < .05). CONCLUSIONS: Human fetal cardiac myocytes exhibit a uniquely adaptive transcriptional response to ischemia and reperfusion that is associated with an apoptosis-resistant phenotype. The stress-inducible fetal cardiac myocyte gene repertoire is a useful platform for identification of targets relevant to the mitigation of cardiac ischemic injury and highlights a novel avenue involving interleukin 6 modulation for preventing the cardiac myocyte injury associated with ischemia and reperfusion.
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Modelos Animais de Doenças , Doenças Fetais/metabolismo , Traumatismo por Reperfusão Miocárdica/metabolismo , Miócitos Cardíacos/metabolismo , Adaptação Fisiológica , Fatores Etários , Animais , Apoptose/genética , Western Blotting , Células Cultivadas , Ensaio de Imunoadsorção Enzimática , Doenças Fetais/embriologia , Doenças Fetais/genética , Doenças Fetais/prevenção & controle , Regulação da Expressão Gênica no Desenvolvimento/genética , Quinase 3 da Glicogênio Sintase/fisiologia , Glicogênio Sintase Quinase 3 beta , Humanos , Interleucina-6/análise , Interleucina-6/fisiologia , MAP Quinase Quinase 1/fisiologia , Traumatismo por Reperfusão Miocárdica/embriologia , Traumatismo por Reperfusão Miocárdica/genética , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Análise de Sequência com Séries de Oligonucleotídeos , Oxirredução , Fenótipo , Fosforilação , Reação em Cadeia da Polimerase , Processamento de Proteína Pós-Traducional/fisiologia , Proteínas Serina-Treonina Quinases/fisiologia , Proteínas Proto-Oncogênicas/fisiologia , Proteínas Proto-Oncogênicas c-akt , Ratos , Transdução de Sinais/fisiologia , Ativação Transcricional/fisiologiaRESUMO
BACKGROUND: The global myocardial stress response during cardiac surgery has not been systematically studied, nor is it known whether the response of the neonatal myocardium is intrinsically different from that of older children. To determine the age-related molecular basis of this response, we conducted microarray-based differential gene expression profiling on right ventricular tissue samples acquired in patients of varying ages with right ventricular outflow tract obstruction. METHODS: We studied gene expression profiles in 24 patients during operations for lesions involving right ventricular outflow tract obstruction age stratified into group I (7 patients, aged 5 to 66 days; mean, 30 days) and group II (17 patients, aged 4 months to 12.5 years; mean, 2.8 years). Myocardial samples were taken from the right ventricular outflow tract after aortic occlusion and archived in liquid nitrogen. RNA isolation, fluorescence labeling of complementary DNA, hybridization to spotted arrays containing 19,008 characterized or unknown human complementary DNAs, and quantitative fluorescence scanning of gene-expression intensity were performed at the University of Toronto Health Network Microarray Centre. Data were analyzed with the Significance Analysis for Microarrays program. Minimum Information About Microarray Experiments-compliant, log2-normalized data sets were compared to ascertain potential statistical differences in gene expression between patient groups. RESULTS: There were no hospital deaths or major postoperative morbid events. We identified 50 transcripts differentially expressed in the neonatal group (the predicted false discovery rate was <0.8 transcripts). The neonatal pattern of gene expression (group I) was dominated by genes with literature-validated cardioprotective, antihypertrophic, and antiproliferative properties, including increases in atrial natriuretic peptide, protein phosphatase 2A, small GTPase rap1, and protein inhibitor of activated STAT protein, PIASy. Several transcripts have not been previously reported in heart. CONCLUSIONS: Neonatal myocardium has a unique pattern of gene expression, which may result from developmental (age-related) differences or reflect a more severe disease phenotype independent of age effects per se. The neonatal transcript profile seems to reflect a stress-induced protective program composed of genes with functions diametrically opposed to those expected to be related to the pathogenesis of critical right ventricular outflow tract obstruction, thus revealing a novel and compensatory antidisease transcriptional response in the neonatal heart.
Assuntos
Perfilação da Expressão Gênica , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina , Obstrução do Fluxo Ventricular Externo/genética , Obstrução do Fluxo Ventricular Externo/cirurgia , Fator Natriurético Atrial/genética , Procedimentos Cirúrgicos Cardíacos , Proteínas de Transporte/genética , Criança , Pré-Escolar , DNA Complementar/análise , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Miocárdio/química , Análise de Sequência com Séries de Oligonucleotídeos , Fosfoproteínas Fosfatases/genética , Reação em Cadeia da Polimerase , Proteínas Inibidoras de STAT Ativados , Proteína Fosfatase 2 , RNA Mensageiro/análiseRESUMO
PURPOSE: This study was undertaken to evaluate the antiadhesive properties of a polymeric agent in infants undergoing staged surgical correction of congenital heart abnormalities. DESCRIPTION: Sixteen infants having staged surgical repair were treated with a polymeric matrix at the completion of the initial surgery. There were 5 untreated controls. The tenacity and extent of adhesions at five separate regions of the heart were evaluated at the follow-up surgery. EVALUATION: For all sites combined, there was a threefold difference in median tenacity scores in favor of the experimental treatment (1.0 vs 3.0, p < 0.01). Significant differences were achieved separately at the right ventricle and the anterior surface of the great vessels (p = 0.02 for both comparisons). Analysis of adhesion scores reflecting the extent of adhesions similarly favored the experimental treatment for all sites (80 vs 270, p < 0.01), with significant differences persisting at the right atrium (p < 0.01) and the anterior surface of the great vessels (p = 0.04). There were no treatment-related adverse events. CONCLUSIONS: Use of this polymeric agent at the completion of open cardiac surgery may prevent the occurrence or reduce the severity of pericardial adhesions.