RESUMO
The frequencies, immunophenotype, and function of mucosal-associated invariant T (MAIT) cells were studied in patients with primary Sjögren syndrome (pSS) and healthy controls. MAIT cells were significantly decreased in the peripheral blood (PB) of patients with pSS. Vα7.2+ MAIT cells were detected in the salivary gland tissue from pSS patients, but not in controls, indicating that the reduction of MAIT cells in PB might be due to migration into the target tissue. Furthermore, the residual peripheral blood MAIT cells in pSS patients showed altered immunophenotype and function. While MAIT cells from controls were almost exclusively CD8+ and expressed an effector memory immunophenotype, in pSS patients they were enriched in CD4+ and naïve subpopulations. Consistently, the functional studies demonstrated that MAIT cells from pSS showed a lower level of activation with reduced expression of CD69 and CD154 (CD40L), and a lower production of TNF and IFN-γ. In summary, our findings demonstrate that MAIT cells were reduced and phenotypically and functionally altered in PB of pSS patients. The altered function of MAIT cells in target tissues from pSS patients may result in dysregulation of mucosal immunity leading to microbial damage of mucosal surfaces and subsequent initiation of autoimmune response.
Assuntos
Células Sanguíneas/imunologia , Diferenciação Celular , Células T Matadoras Naturais/imunologia , Síndrome de Sjogren/imunologia , Subpopulações de Linfócitos T/imunologia , Adulto , Proliferação de Células , Estudos de Coortes , Feminino , Humanos , Imunidade nas Mucosas , Memória Imunológica , Interferon gama/metabolismo , Ativação Linfocitária , Masculino , Pessoa de Meia-Idade , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Intra-tumor genetic heterogeneity is a hallmark of cancer. The ability to monitor and analyze these sub-clonal cell populations can be considered key to successful treatment, particularly in the modern era of targeted therapies. Although advances in sequencing technologies have significantly improved our ability to analyze the mutational landscape of tumors, this utility is reduced when considering small, but clinically significant sub-clones, that is, those representing <10% of the tumor burden. We have developed a high-throughput method that utilizes a 17-probe labeled bacterial artificial chromosome contig to quantify sub-clonal populations of cells based on deletion of a single locus. Chronic lymphocytic leukemia (CLL) cells harboring deletion of the short arm of chromosome 17 (del17p), an important prognostic marker for CLL were used to demonstrate the technique. Sub-clones of del17p cells were quantified and isolated from heterogeneous CLL populations using fluorescence in situ hybridization in suspension (FISH-IS) and the locus specific probe set. Using the combination of FISH-IS with the locus-specific probe set enables automated analysis of tens of thousands of cells, accurately quantifying and isolating cells carrying a del17p. Based on the fluorescence intensity of 17p probes, 17p (TP53) deleted cells were identified and sorted using flow cytometric techniques, and enrichment was demonstrated using single nucleotide polymorphism analysis. The ability to separate sub-clones of cells based on genetic heterogeneity, independent of the clone size, highlights the potential application of this method not only in the diagnostic and prognostic setting, but also as an unbiased approach to enable further detailed genetic analysis of the sub-clone with deep sequencing approaches. © 2017 International Society for Advancement of Cytometry.
Assuntos
Citometria de Fluxo/métodos , Ensaios de Triagem em Larga Escala/métodos , Hibridização in Situ Fluorescente/métodos , Leucemia Linfocítica Crônica de Células B/patologia , Deleção Cromossômica , Cromossomos Humanos Par 17/genética , Células Clonais/patologia , Heterogeneidade Genética , Humanos , Leucemia Linfocítica Crônica de Células B/diagnóstico , Leucemia Linfocítica Crônica de Células B/genética , Mutação/genética , Prognóstico , Proteína Supressora de Tumor p53/genéticaRESUMO
The cryopreservation of peripheral blood mononuclear cells (PBMCs) is a routine research laboratory process, enabling long-term storage of primary patient blood samples. Retrospective analysis of these samples has the potential to identify markers that may be associated with prognosis and response to treatment. To draw valid biological conclusions from this type of analysis, it is essential to ensure that any observed changes are directly related to the pathology of the disease rather than the preservation process itself. Therefore, we have investigated 15 cell surface markers that are relevant to chronic lymphocytic leukemia (CLL) on matched fresh and thawed samples to determine the effect of cryopreservation on their detection. We found that the number of CLL cells positive for the markers CD22, CD40, CD49d, CD54, CD69, and CXCR3 was decreased significantly after cryopreservation. In addition, the mean fluorescence intensity of 10 of the 15 markers changed significantly after cryopreservation. These findings demonstrate that care must be taken when interpreting this type of analysis on thawed samples.
Assuntos
Antígenos CD/análise , Antígenos de Neoplasias/análise , Artefatos , Biomarcadores Tumorais/análise , Preservação de Sangue/métodos , Criopreservação , Citometria de Fluxo , Imunofenotipagem , Leucemia Linfocítica Crônica de Células B/sangue , Receptores CXCR/análise , Epitopos/análise , Reações Falso-Negativas , Citometria de Fluxo/métodos , Humanos , Imunofenotipagem/métodos , Leucemia Linfocítica Crônica de Células B/imunologia , Leucemia Linfocítica Crônica de Células B/patologia , Fatores de Tempo , Células Tumorais CultivadasRESUMO
Chronic lymphocytic leukemia (CLL) has an extremely heterogeneous clinical course, and prognostication is based on common genetic abnormalities which are detected by standard cytogenetic methods. However, current methods are restricted by the low number of cells able to be analyzed, resulting in the potential to miss clinically relevant sub-clonal populations of cells. A novel high throughput methodology called fluorescence in situ hybridization in suspension (FISH-IS) incorporates a flow cytometry-based imaging approach with automated analysis of thousands of cells. Here we have demonstrated that the FISH-IS technique is applicable to aneuploidy detection in CLL samples for a range of chromosomes using appropriate centromere probes. This method is able to accurately differentiate between monosomy, disomy and trisomy with a sensitivity of 1% in CLL. An analysis comparing conventional FISH, FISH-IS and laser scanning cytometry (LSC) is presented.
Assuntos
Hibridização in Situ Fluorescente/métodos , Citometria de Varredura a Laser/métodos , Leucemia Linfocítica Crônica de Células B/diagnóstico , Leucemia Linfocítica Crônica de Células B/genética , Trissomia/genética , Cromossomos Humanos/genética , Humanos , Ploidias , Cromossomos Sexuais/genéticaRESUMO
The growing problem of antibiotic-resistant bacteria is a major threat to human health. Paradoxically, new antibiotic discovery is declining, with most of the recently approved antibiotics corresponding to new uses for old antibiotics or structurally similar derivatives of known antibiotics. We used an in silico approach to design a new class of nontoxic antimicrobials for the bacteria-specific mechanosensitive ion channel of large conductance, MscL. One antimicrobial of this class, compound 10, is effective against methicillin-resistant Staphylococcus aureus with no cytotoxicity in human cell lines at the therapeutic concentrations. As predicted from in silico modeling, we show that the mechanism of action of compound 10 is at least partly dependent on interactions with MscL. Moreover we show that compound 10 cured a methicillin-resistant S. aureus infection in the model nematode Caenorhabditis elegans. Our work shows that compound 10, and other drugs that target MscL, are potentially important therapeutics against antibiotic-resistant bacterial infections.