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OBJECTIVES: Immune monitoring is an important aspect in diagnostics and clinical trials for patients with compromised immune systems. Flow cytometry is the standard method for immune cell counting but faces limitations. Best practice guidelines are available, but lack of standardization complicates compliance with e.g., in vitro diagnostic regulations. Limited sample availability forces immune monitoring to predominantly use population-based reference intervals. Epigenetic qPCR has evolved as alternative with broad applicability and low logistical demands. Analytical performance specifications (APS) have been defined for qPCR in several regulated fields including testing of genetically modified organisms or vector-shedding. METHODS: APS were characterized using five epigenetic qPCR-based assays quantifying CD3+, CD4+, CD8+ T, B and NK cells in light of regulatory requirements. RESULTS: Epigenetic qPCR meets all specifications including bias, variability, linearity, ruggedness and sample stability as suggested by pertinent guidelines and regulations. The assays were subsequently applied to capillary blood from 25 normal donors over a 28-day period. Index of individuality (IoI) and reference change values were determined to evaluate potential diagnostic gains of individual reference intervals. Analysis of the IoI suggests benefits for individual over population-based references. Reference change values (RCVs) show that changes of approx. Fifty percent from prior measurement are suggestive for clinically relevant changes in any of the 5 cell types. CONCLUSIONS: The demonstrated precision, long-term stability and obtained RCVs render epigenetic cell counting a promising tool for immune monitoring in clinical trials and diagnosis.
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Epigénesis Genética , Células Asesinas Naturales , Humanos , Citometría de FlujoRESUMEN
OBJECTIVE: The diagnosis of RA patients remains a challenge, especially in ACPA-negative disease. Novel T-cell subsets, particularly Th17 may be useful, although data on Th17 frequency using flow cytometry in RA are conflicting. We investigated whether a novel epigenetic qPCR assay for the quantification of Th17 could differentiate patients with RA from those with symptoms evolving towards an alternative diagnosis. METHODS: We used a qPCR assay measuring the extent of the methylation at a key position in the IL-17 and CD4 genes. Assays were performed on whole blood from 49 healthy controls (HC) and 165 early arthritis clinic patients. Flow cytometry was further used to detect the expression of CXCR4 on Th17 cells. RESULTS: In 75 inflammatory arthritis patients who progressed to RA, the qPCR assays showed significantly fewer Th17 cells compared with 90 patients who did not (P<0.0001). Regression models demonstrated a high predictive value for RA development (75.8% correct prediction), and particularly for the ACPA-negative group (n = 125) where Th17 and swollen joint count (SJC) were the only predictors (73% correct prediction). The chemokine receptor CXCR4 had significantly higher expression on Th17 from early RA patients (n = 11) compared with HC (n = 15). CONCLUSION: The results of the epigenetic qPCR assay showed that low levels of Th17 cells were predictive of developing RA, particularly in the ACPA-negative patients. This could have value for insights into pathogenesis and management. The results suggest the recruitment of Th17 to the inflammatory disease site, consistent with high CXCR4 expression.
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Artritis Reumatoide/diagnóstico , Reacción en Cadena en Tiempo Real de la Polimerasa/estadística & datos numéricos , Células Th17/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores/sangre , Femenino , Citometría de Flujo , Humanos , Masculino , Persona de Mediana Edad , Valor Predictivo de las Pruebas , Receptores CXCR4/sangre , Adulto JovenRESUMEN
BACKGROUND: Laboratory-based, mechanistic, and prognosis studies suggest that a shift from antitumor immunity towards tumor-immune tolerance plays a major role in carcinogenesis. However, prospective epidemiological studies on the consequences of differing immune tolerance levels prior to clinical manifestation are missing. METHODS: A case-cohort study embedded in EPIC-Heidelberg was conducted comprising incident cases of breast (n = 399), colorectal (n = 185), lung (n = 149), and prostate (n = 378) cancer, which occurred during 6.6 years of follow-up, and a subcohort (n = 807). Foxp3+ regulatory T-lymphocytes and CD3+ T-lymphocytes were measured by quantitative polymerase chain reaction-based DNA methylation analysis in prediagnostic leukocyte samples. Hazard ratios (HRs) for associations of cancer risk with the ratio of both parameters, the "cellular ratio of immune tolerance" (ImmunoCRIT), were estimated using Cox regression models. All statistical tests were two-sided. RESULTS: ImmunoCRIT values were positively associated with the risk of lung (highest vs lowest tertile, HR = 1.98, 95% confidence interval = 1.06 to 3.69, P trend = .0263) and colorectal cancer (HR = 1.59, 95% CI = 0.99 to 2.54, P trend = .0069) after multivariable adjustment, but not with prostate cancer risk. Regarding breast cancer significant heterogeneity by estrogen receptor (ER) status was observed (P heterogeneity = .02), and the ImmunoCRIT was associated with the risk of ER-negative breast cancer (HR = 3.34, 95% CI = 1.52 to 7.35, P trend ≤ .001), but not ER-positive breast cancer. CONCLUSION: The present study indicates that increased peripheral immune tolerance may be an independent risk factor for lung, colorectal, and ER-negative breast cancer, whereas its role on the development of prostate and ER-positive breast tumors remains uncertain.
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Neoplasias de la Mama/inmunología , Neoplasias Colorrectales/inmunología , Tolerancia Inmunológica , Neoplasias Pulmonares/inmunología , Neoplasias de la Próstata/inmunología , Linfocitos T Reguladores/inmunología , Anciano , Neoplasias de la Mama/química , Complejo CD3/análisis , Estudios de Casos y Controles , Metilación de ADN , Europa (Continente) , Femenino , Estudios de Seguimiento , Factores de Transcripción Forkhead/análisis , Humanos , Masculino , Persona de Mediana Edad , Oportunidad Relativa , Modelos de Riesgos Proporcionales , Estudios Prospectivos , Receptores de Estrógenos/análisis , Medición de Riesgo , Factores de RiesgoRESUMEN
TGF-beta induces apoptosis and inhibits the proliferation of EBV-negative B-lymphoma cell lines. In contrast, EBV-immortalized B cells are resistant to both the proapoptotic and the antiproliferative activities of TGF-beta. We have generated a lymphoblastoid cell line, in which we can switch on and off the EBV-specific transcriptional program driven by EBNA2. When these cells express the EBNA2-driven phenotype, they are resistant to TGF-beta-mediated growth arrest. We used this cell line to readdress the question of how EBV can overcome the antiproliferative TGF-beta activity. We show here that EBV-driven cells remain TGF-beta-responsive since TGF-beta target genes are readily induced. Thus, EBV can overcome TGF-beta-mediated growth arrest without interfering with the core machinery of the TGF-beta signaling pathway, which links ligand binding to the induction of TGF-beta target genes.