RESUMEN
PURPOSE: To test whether analyzing DEPArray (Menarini Silicon Biosystems) isolated single B cells from the vitreous fluid can reveal crucial genomic and clinicopathological features to distinguish patients with vitreoretinal lymphoma (VRL) from those with chronic inflammation using immunoglobulin heavy chain (IGH), disease biomarker myeloid differentiation primary response 88 (MYD88)L265P mutation, and copy number profiling. DESIGN: A single-center, retrospective study. PARTICIPANTS: Remnant vitreous biopsies from 7 patients with VRL and 4 patients with chronic inflammation were acquired for molecular analysis. METHODS: Vitreous fluid samples were prefixed in PreservCyt (Hologic) and underwent cytologic analysis and immunohistochemistry examination. Single cells were isolated using the DEPArray NxT system, followed by downstream genomic analysis. MAIN OUTCOME MEASURES: The frequencies of the dominant IGH and MYD88L265P mutation and the genome-wide copy number aberration (CNA) profiles of individual vitreous-isolated B cells were characterized. RESULTS: An average of 10 to 13 vitreous B cells were used in the single-cell IGH and MYD88 analyses. Higher frequencies of dominant IGH (88.8% ± 13.2%) and MYD88L265P mutations (35.0% ± 31.3%) were detected in patients with VRL than in patients with chronic inflammation (65.9% ± 13.4% and 1.5% ± 2.6% for IGH and MYD88L265P, respectively). In a cytology-proven VRL case, all 15 vitreous isolated B cells were derived from the same clone with 100% paired IGH: immunoglobulin light chain (IGK) sequences. Genome-wide copy number profiling revealed a high degree of similarity between B cells from the same patient with VRL, with extensive gains and losses at the same areas across the whole genome. In addition, 14 of 15 B cells showed a BCL2/JH t(14;18) translocation, confirming cellular malignancy with a clonal origin. Clustering analysis of the copy number profiles revealed that malignant B cells derived from different patients with VRL had no common genome-wide signatures. CONCLUSIONS: Single B-cell genomic characterization of the IGH, MYD88L265P mutation, and copy number profile enables VRL diagnosis. Because our study involved only a small cohort, these meaningful proof-of-concept data now warrant further investigation in a larger patient cohort.
Asunto(s)
Linfocitos B/metabolismo , Inflamación/genética , Linfoma de Células B Grandes Difuso/diagnóstico , Mutación , Factor 88 de Diferenciación Mieloide/genética , Retina/patología , Neoplasias de la Retina/diagnóstico , Linfocitos B/patología , Biopsia , Línea Celular , Enfermedad Crónica , Análisis Mutacional de ADN , ADN de Neoplasias/análisis , Estudios de Factibilidad , Genómica , Inflamación/diagnóstico , Inflamación/etiología , Linfoma de Células B Grandes Difuso/complicaciones , Linfoma de Células B Grandes Difuso/genética , Factor 88 de Diferenciación Mieloide/metabolismo , Retina/metabolismo , Neoplasias de la Retina/complicaciones , Neoplasias de la Retina/genética , Cuerpo Vítreo/metabolismo , Cuerpo Vítreo/patologíaRESUMEN
Mycobacterium tuberculosis thrives within macrophages by residing in phagosomes and preventing them from maturing and fusing with lysosomes. A parallel transcriptional survey of intracellular mycobacteria and their host macrophages revealed signatures of heavy metal poisoning. In particular, mycobacterial genes encoding heavy metal efflux P-type ATPases CtpC, CtpG, and CtpV, and host cell metallothioneins and zinc exporter ZnT1, were induced during infection. Consistent with this pattern of gene modulation, we observed a burst of free zinc inside macrophages, and intraphagosomal zinc accumulation within a few hours postinfection. Zinc exposure led to rapid CtpC induction, and ctpC deficiency caused zinc retention within the mycobacterial cytoplasm, leading to impaired intracellular growth of the bacilli. Thus, the use of P(1)-type ATPases represents a M. tuberculosis strategy to neutralize the toxic effects of zinc in macrophages. We propose that heavy metal toxicity and its counteraction might represent yet another chapter in the host-microbe arms race.
Asunto(s)
ATPasas de Translocación de Protón Bacterianas/metabolismo , Macrófagos/metabolismo , Mycobacterium tuberculosis/enzimología , Tuberculosis/metabolismo , Zinc/metabolismo , Animales , ATPasas de Translocación de Protón Bacterianas/genética , Células Cultivadas , Femenino , Humanos , Macrófagos/efectos de los fármacos , Macrófagos/microbiología , Ratones , Ratones Endogámicos BALB C , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , Tuberculosis/microbiología , Zinc/toxicidadRESUMEN
BACKGROUND: Transcriptional profiling using microarrays provides a unique opportunity to decipher host pathogen cross-talk on the global level. Here, for the first time, we have been able to investigate gene expression changes in both Mycobacterium tuberculosis, a major human pathogen, and its human host cells, macrophages and dendritic cells. METHODOLOGY/PRINCIPAL FINDINGS: In addition to common responses, we could identify eukaryotic and microbial transcriptional signatures that are specific to the cell type involved in the infection process. In particular M. tuberculosis shows a marked stress response when inside dendritic cells, which is in accordance with the low permissivity of these specialized phagocytes to the tubercle bacillus and to other pathogens. In contrast, the mycobacterial transcriptome inside macrophages reflects that of replicating bacteria. On the host cell side, differential responses to infection in macrophages and dendritic cells were identified in genes involved in oxidative stress, intracellular vesicle trafficking and phagosome acidification. CONCLUSIONS/SIGNIFICANCE: This study provides the proof of principle that probing the host and the microbe transcriptomes simultaneously is a valuable means to accessing unique information on host pathogen interactions. Our results also underline the extraordinary plasticity of host cell and pathogen responses to infection, and provide a solid framework to further understand the complex mechanisms involved in immunity to M. tuberculosis and in mycobacterial adaptation to different intracellular environments.