RESUMEN
Multiparameter flow cytometry (MFC)-based clonality assessment is a powerful method of diagnosis and follow-up in monoclonal gammopathy of undetermined significance (MGUS) and multiple myeloma (MM). However, the relevance of intraclonal heterogeneity in immunophenotypic studies remains poorly understood. The main objective of this work was to characterize the different immunophenotypic subclones in MGUS and MM patients and to investigate their correlation with disease stages. An 8-color MFC protocol with 17 markers was used to identify the subclones within the neoplastic compartment of 56 MGUS subjects, 151 newly diagnosed MM patients, 30 MM subjects in complete remission with detectable minimal residual disease, and 36 relapsed/refractory MM patients. Two or more clusters were observed in > 85% of MGUS subjects, 75% of stage I MM patients, and < 15% in stage III. Likewise, a significant correlation between the dominant subclone size, secondary cytogenetic features, and changes in the expression of CD27, CD44, and CD81 was detected. The loss of intraclonal equilibrium may be an important factor related with kinetics and risk of progression not well considered to date in MFC studies. The MFC strategy used in this work can provide useful biomarkers in MGUS and MM.
Asunto(s)
Biomarcadores/metabolismo , Citometría de Flujo/métodos , Mieloma Múltiple/diagnóstico , Paraproteinemias/diagnóstico , Aberraciones Cromosómicas , Humanos , Receptores de Hialuranos/metabolismo , Inmunofenotipificación , Mieloma Múltiple/metabolismo , Mieloma Múltiple/patología , Estadificación de Neoplasias , Paraproteinemias/metabolismo , Paraproteinemias/patología , Tetraspanina 28/metabolismo , Miembro 7 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/metabolismoRESUMEN
BACKGROUND: Acute pancreatitis is an inflammatory disorder of the pancreas that is responsible for significant morbidity and mortality. The inflammasome pathway has acquired significant relevance in the pathogenesis of many inflammatory disorders, but its role in patients with acute pancreatitis still awaits clarification. METHODS: We performed a prospective study in which 27 patients with acute pancreatitis and 16 healthy controls were included. We isolated peripheral blood mononuclear cells (PBMCs) and we assessed the expression and activation of different inflammasomes as well as their association with the clinical course of the disease. RESULTS: Our results show that PBMCs from patients with acute pancreatitis have elevated expression of several components of the inflammasome complex, including the inflammasome-forming receptor absent in melanoma 2 (AIM2), early during the onset of the disease. Activation of the AIM2 or NLRP3 inflammasomes in PBMCs from patients with acute pancreatitis results in exacerbated IL-1ß and IL-18 production compared with PBMCs from healthy controls. Furthermore, both AIM2 mRNA expression and AIM2-mediated production of IL-1ß by PBMCs correlated with increased systemic inflammation in these patients. Last, AIM2 expression was further increased in those patients that developed transient or persistent organ failure (moderate or severe acute pancreatitis). CONCLUSIONS: Our data demonstrates that AIM2 inflammasome expression and activation is increased early during the course of acute pancreatitis, and suggests that AIM2 activation may affect systemic inflammation and organ failure in these patients.
Asunto(s)
Proteínas de Unión al ADN/biosíntesis , Proteínas de Unión al ADN/metabolismo , Inflamasomas , Pancreatitis/patología , Enfermedad Aguda , Adulto , Anciano , Femenino , Humanos , Inmunidad Celular , Inflamación/patología , Interleucina-18/biosíntesis , Interleucina-18/genética , Interleucina-1beta/biosíntesis , Interleucina-1beta/genética , Masculino , Persona de Mediana Edad , Monocitos , Estudios Prospectivos , ARN Mensajero/biosíntesis , ARN Mensajero/genéticaRESUMEN
BACKGROUND: Treatment with non-selective beta-blockers (NSBB) has been associated with anti-inflammatory and anti-cancer effects in patients with cirrhosis. This study aims to analyze the impact of chronic NSBB treatment on immune activation and disease progression in stable outpatients with cirrhosis. METHODS: In this prospective follow-up of 150 patients with cirrhosis, 39 received treatment with NSBB. Blood samples were taken every 6-9 months, and immune and adrenergic variables were measured. Mixed linear models were used to assess the effect of NSBB on these variables over time. Multivariate Cox regression was used to study associations with adverse clinical events (hepatocellular carcinoma, death, or liver transplant). RESULTS: Median follow-up was 1635 days. NSBB treatment was associated with significantly lower levels of IL-6 (ß - 4.7; 95% confidence interval [CI] -6.9, -2.6) throughout the study. During follow-up, 11 patients developed hepatocellular carcinoma, 32 died, and 4 underwent liver transplant. Patients with higher concentrations of IL-10, IL-6 and IFN-γ developed more clinical events. Event-free survival was significantly better in patients treated with NSBB (hazard ratio 0.36, 95% CI 0.18, 0.71) in a multivariate Cox regression adjusted for Child-Pugh-Score, esophageal varices, and platelets. CONCLUSION: Chronic treatment with NSBB in patients with stable cirrhosis gives rise to a different state of immune activation, characterized by lower concentrations of IL-6 over time, and it is associated with a reduced risk of adverse event (death, hepatocellular carcinoma, or transplant), after controlling for disease severity.
Asunto(s)
Carcinoma Hepatocelular , Neoplasias Hepáticas , Humanos , Estudios Prospectivos , Estudios Longitudinales , Carcinoma Hepatocelular/tratamiento farmacológico , Carcinoma Hepatocelular/inducido químicamente , Interleucina-6 , Antagonistas Adrenérgicos beta/uso terapéutico , Cirrosis Hepática/patología , Neoplasias Hepáticas/tratamiento farmacológico , Neoplasias Hepáticas/inducido químicamenteRESUMEN
An unprecedented global social and economic impact as well as a significant number of fatalities have been brought on by the coronavirus disease 2019 (COVID-19), produced by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Acute SARS-CoV-2 infection can, in certain situations, cause immunological abnormalities, leading to an anomalous innate and adaptive immune response. While most patients only experience mild symptoms and recover without the need for mechanical ventilation, a substantial percentage of those who are affected develop severe respiratory illness, which can be fatal. The absence of effective therapies when disease progresses to a very severe condition coupled with the incomplete understanding of COVID-19's pathogenesis triggers the need to develop innovative therapeutic approaches for patients at high risk of mortality. As a result, we investigate the potential contribution of promising combinatorial cell therapy to prevent death in critical patients.
RESUMEN
The performance of a laboratory-developed IgG/IgA flow cytometry-based immunoassay (FCI) using Jurkat T cells stably expressing full-length native S protein was compared against Elecsys electrochemiluminiscent (ECLIA) Anti-SARS-CoV-2 S (Roche Diagnostics, Pleasanton, CA, USA), and Liaison SARS-CoV-2 TrimericS IgG chemiluminiscent assay (CLIA) (Diasorin S.p.a, Saluggia, IT) for detection of SARS-CoV-2-specific antibodies. A total of 225 serum/plasma specimens from 120 acute or convalescent COVID-19 individuals were included. Overall, IgG/IgA-FCI yielded the highest number of positives (n = 179), followed by IgA-FCI (n = 177), Roche ECLIA (n = 175), IgG-FCI (n = 172) and Diasorin CLIA (n = 154). For sera collected early after the onset of symptoms (within 15 days) IgG/IgA-FCI also returned the highest number of positive results (52/72; 72.2%). Positive percent agreement between FCI and compared immunoassays was highest for Roche ECLIA, ranging from 96.1 (IgG/IgA-FCI) to 97.7% (IgG-FCI), whereas negative percent agreement was higher between FCI and Diasosin CLIA, regardless of antibody isotype. The data suggest that FCI may outperform Roche ECLIA and Diasorin CLIA in terms of clinical sensitivity for serological diagnosis of SARS-CoV-2 infection.
Asunto(s)
Anticuerpos Antivirales/sangre , Citometría de Flujo/métodos , Inmunoensayo/métodos , SARS-CoV-2/inmunología , Glicoproteína de la Espiga del Coronavirus/inmunología , Anticuerpos Antivirales/inmunología , Prueba Serológica para COVID-19 , Humanos , Células Jurkat , Estudios Retrospectivos , Sensibilidad y EspecificidadRESUMEN
Background: Multiparametric Flow Cytometry (MFC) is an essential tool to study the involved cell lineages, the aberrant differentiation/maturation patterns and the expression of aberrant antigens in acute myeloid leukemia (AML). The characterization of leukemia-associated immunophenotypes (LAIPs) at the moment of diagnosis is critical to establish reproducible strategies for the study of measurable residual disease using MFC (MFC-MRD). Methods: In this study, we identify and characterize LAIPs by comparing the leukemic populations of 145 AML patients, using the EuroFlow AML/ MDS MFC panel, with six databases of normal myeloid progenitors (MPCs). Principal component analysis was used to identify and characterize the LAIPs, which were then used to generate individual profiles for MFC-MRD monitoring. Furthermore, we investigated the relationship between the expression patterns of LAIPs and the different subtypes of AML. The MFC-MRD study was performed by identifying residual AML populations that matched with the LAIPs at diagnosis. To further validate this approach, the presence of MRD was also assessed by qPCR (qPCR-MRD). Finally, we studied the association between MFC-MRD and progression-free survival (PFS). Results: The strategy used in this study allowed us to describe more than 300 different LAIPs and facilitated the association of specific phenotypes with certain subtypes of AML. The MFC-MRD monitoring based on LAIPs with good/strong specificity was applicable to virtually all patients and showed a good correlation with qPCR-MRD and PFS. Conclusions: The described methodology provides an objective method to identify and characterize LAIPs. Furthermore, it provides a theoretical basis to develop highly sensitive MFC-MRD strategies.
RESUMEN
SARS-CoV-2 is the virus that causes the disease called COVID-19, which has caused the worst pandemic of the century. Both, to know the immunological status of general population and to evaluate the efficacy of the vaccination process that is taking place around the world, serological tests represent a key tool. Classic serological tests, based on colorimetric techniques, such as ELISA or CLIA, continue to be the most widely used option. However, a real improvement in results is still needed. We developed a highly sensitive and specific FCM assay that allows the detection of IgG and IgA antibodies, directed against the native and functional S-protein of SARS-CoV-2 exposed on the membrane of a transfected cell line, up to 8 months after infection.
Asunto(s)
Anticuerpos Antivirales/inmunología , COVID-19/inmunología , Citometría de Flujo , Inmunoglobulina A/inmunología , Inmunoglobulina G/inmunología , SARS-CoV-2/inmunología , Glicoproteína de la Espiga del Coronavirus/inmunología , Adulto , Anciano , Femenino , Humanos , Células Jurkat , Masculino , Persona de Mediana EdadRESUMEN
The role of decentralized assessment of measurable residual disease (MRD) for risk stratification in acute myeloid leukemia (AML) remains largely unknown, and so it does which methodological aspects are critical to empower the evaluation of MRD with prognostic significance, particularly if using multiparameter flow cytometry (MFC). We analyzed 1076 AML patients in first remission after induction chemotherapy, in whom MRD was evaluated by MFC in local laboratories of 60 Hospitals participating in the PETHEMA registry. We also conducted a survey on technical aspects of MRD testing to determine the impact of methodological heterogeneity in the prognostic value of MFC. Our results confirmed the recommended cutoff of 0.1% to discriminate patients with significantly different cumulative-incidence of relapse (-CIR- HR:0.71, P < 0.001) and overall survival (HR: 0.73, P = 0.001), but uncovered the limited prognostic value of MFC based MRD in multivariate and recursive partitioning models including other clinical, genetic and treatment related factors. Virtually all aspects related with methodological, interpretation, and reporting of MFC based MRD testing impacted in its ability to discriminate patients with different CIR. Thus, this study demonstrated that "real-world" assessment of MRD using MFC is prognostic in patients at first remission, and urges greater standardization for improved risk-stratification toward clinical decisions in AML.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Citometría de Flujo/métodos , Trasplante de Células Madre Hematopoyéticas/mortalidad , Quimioterapia de Inducción/mortalidad , Leucemia Mieloide Aguda/patología , Recurrencia Local de Neoplasia/patología , Neoplasia Residual/patología , Anciano , Terapia Combinada , Progresión de la Enfermedad , Femenino , Estudios de Seguimiento , Humanos , Leucemia Mieloide Aguda/terapia , Masculino , Persona de Mediana Edad , Recurrencia Local de Neoplasia/terapia , Neoplasia Residual/terapia , Pronóstico , Sistema de Registros , Tasa de Supervivencia , Trasplante HomólogoRESUMEN
Hepatic immune function is compromised during cirrhosis. This study investigated the immune features of liver sinusoidal endothelial cells (LSECs) in two experimental models of cirrhosis. Dendritic cells, hepatic macrophages, and LSECs were isolated from carbon tetrachloride and bile duct-ligated rats. Gene expression of innate receptors, bacterial internalization, co-stimulatory molecules induction, and CD4+ T cell activation and differentiation were evaluated. Induced bacterial peritonitis and norfloxacin protocols on cirrhotic rats were also carried out. LSECs demonstrated an active immunosurveillance profile, as shown by transcriptional modulation of different scavenger and cell-adhesion genes, and their contribution to bacterial internalization. LSECs significantly increased their expression of CD40 and CD80 and stimulated CD4+ T cell activation marker CD71 in both models. The pro-inflammatory Th17 subset was expanded in CCl4-derived LSECs co-cultures. In the bile duct ligation (BDL) model, CD4+ T cell differentiation only occurred under induced bacterial peritonitis conditions. Differentiated pro-inflammatory Th cells by LSECs in both experimental models were significantly reduced with norfloxacin treatment, whereas Foxp3 tolerogenic Th CD4+ cells were expanded. Conclusion: LSECs' participation in the innate-adaptive immune progression, their ability to stimulate pro-inflammatory CD4+ T cells expansion during liver damage, and their target role in norfloxacin-induced immunomodulation granted a specific competence to this cell population in cirrhosis.
Asunto(s)
Células Presentadoras de Antígenos/patología , Células Endoteliales/patología , Cirrosis Hepática/inmunología , Cirrosis Hepática/patología , Hígado/patología , Células Th17/inmunología , Inmunidad Adaptativa/efectos de los fármacos , Animales , Células Presentadoras de Antígenos/efectos de los fármacos , Biomarcadores/metabolismo , Diferenciación Celular/efectos de los fármacos , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Proliferación Celular/efectos de los fármacos , Endocitosis/efectos de los fármacos , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Escherichia coli/metabolismo , Inmunidad Innata/efectos de los fármacos , Activación de Linfocitos/efectos de los fármacos , Masculino , Viabilidad Microbiana/efectos de los fármacos , Monitorización Inmunológica , Norfloxacino/farmacología , Ratas Sprague-Dawley , Receptores Inmunológicos/metabolismo , Linfocitos T Reguladores/efectos de los fármacos , Linfocitos T Reguladores/inmunología , Transcripción Genética/efectos de los fármacosRESUMEN
Intestinal permeability to translocation of bacterial products is increased in cirrhosis. Regulatory T cells (Tregs) remain central to the interplay between the host and microbial milieu. We propose that Tregs are involved in promoting gut barrier integrity and a balanced interaction with gut microbiota-derived short-chain fatty acids (SCFAs). Carbon tetrachloride cirrhosis was induced in wild-type and recombination activating gene 1 (Rag1)-/- mice. Naive T cells and Treg cells were transferred into Rag1 -/- mice. Intestinal permeability was assessed in vivo after lipopolysaccharide (LPS) oral administration, and bacterial DNA presence was evaluated in mesenteric lymph nodes. Transcript and protein levels of tight-junction (TJ) proteins were measured in colonic tissue. Intestinal T helper profile in response to Escherichia coli (E. coli) was determined by flow cytometry. SCFAs were measured by gas chromatography-mass spectrometry in colonic content before and after E. coli challenge. Rag1 -/- mice showed significantly increased permeability to LPS and bacterial DNA translocation rate compared with control mice. Naive T and Treg cotransfer significantly reduced gut permeability to bacterial antigen translocation and restored TJ protein expression in Rag1 -/- mice. Naive T and Treg replenishment in Rag1 -/- mice restrained proinflammatory differentiation of intestinal lymphocytes in response to E. coli. The main SCFA concentration resulted in significant reduction in Rag1 -/- mice after E. coli administration but remained unaltered after naive T and Tregs cotransfer. The reduced expression of SCFA receptors induced by E. coli was reestablished following naive T and Treg reconstitution in Rag1 -/- mice. Conclusion: The restriction of gut permeability, local inflammatory differentiation, and loss of bacteria-derived SCFAs foster the value of Tregs in preventing bacterial translocation in cirrhosis.
RESUMEN
We describe an exceptional case of erythroid sarcoma in a pediatric patient as a growing orbital mass with no evidence of morphologic bone marrow involvement, who was finally diagnosed of pure erythroid sarcoma based on histopathology and flow cytometry criteria. We discuss the contribution of standardized eight-color flow cytometry as a rapid and reliable diagnostic method. The use of normal bone marrow databases allowed us to identify small aberrant populations in bone marrow and later confirm the diagnosis in the neoplastic tissue.
Asunto(s)
Sistema Nervioso Central/patología , Citometría de Flujo , Leucemia/patología , Infiltración Leucémica/diagnóstico , Linfoma/patología , Adolescente , Adulto , Anciano , Biomarcadores/líquido cefalorraquídeo , Niño , Preescolar , Femenino , Humanos , Lactante , Leucemia/líquido cefalorraquídeo , Infiltración Leucémica/líquido cefalorraquídeo , Linfoma/líquido cefalorraquídeo , Masculino , Persona de Mediana Edad , Adulto JovenRESUMEN
No disponible