Large scale high-throughput DNA sequencing studies have identified clonal hematopoiesis (CH) as a clinical phenomenon characterized by a disproportionately large clonal population in the ...hematopoietic system with a shared mutational background. CH originates through mutations in hematopoietic stem and progenitor cells (HSPCs) which provide a proliferative advantage over unmutated HSPCs and has been characterized as a risk factor for myeloid neoplasm (MN) development. Large population studies found that CH is an age-related event which is commonly found in association with milder phenotypes such as cytopenia, mild monocytosis, intravascular hemolysis or chronic inflammation. More importantly, the vast majority of individuals with CH are asymptomatic and healthy people of advanced age, where the impact of CH is thus considered to be of indeterminate potential (CHIP). These conditions are sometimes referred to as benign to facilitate distinction from overt MN but, despite this definition, may still result in severe illness, reduced overall survival, and increased risk of hematologic neoplasms development and all-cause mortality. The purpose of this review is to describe clinical conditions associated with CH, the clinical significance of CH-related clinical phenotypes, and the determinants of progression from CH to overt MN following the paradigmatic example of SF3B1-driven CH.
Investigating the role that host erythrocyte proteins play in malaria infection is hampered by the genetic intractability of this anucleate cell. Here we report that reticulocytes derived through in ...vitro differentiation of an enucleation-competent immortalized erythroblast cell line (BEL-A) support both successful invasion and intracellular development of the malaria parasite Plasmodium falciparum. Using CRISPR-mediated gene knockout and subsequent complementation, we validate an essential role for the erythrocyte receptor basigin in P. falciparum invasion and demonstrate rescue of invasive susceptibility by receptor re-expression. Successful invasion of reticulocytes complemented with a truncated mutant excludes a functional role for the basigin cytoplasmic domain during invasion. Contrastingly, knockout of cyclophilin B, reported to participate in invasion and interact with basigin, did not impact invasive susceptibility of reticulocytes. These data establish the use of reticulocytes derived from immortalized erythroblasts as a powerful model system to explore hypotheses regarding host receptor requirements for P. falciparum invasion.
Neutrophils are granulocytes with essential antimicrobial effector functions and short lifespans. During infection or sterile inflammation, emergency granulopoiesis leads to release of immature ...neutrophils from the bone marrow, serving to boost circulating neutrophil counts. Steady state and emergency granulopoiesis are incompletely understood, partly due to a lack of genetically amenable models of neutrophil development.
We optimised a method for ex vivo production of human neutrophils from CD34
haematopoietic progenitors. Using flow cytometry, we phenotypically compared cultured neutrophils with native neutrophils from donors experiencing emergency granulopoiesis, and steady state neutrophils from non-challenged donors. We carry out functional and proteomic characterisation of cultured neutrophils and establish genome editing of progenitors.
We obtain high yields of ex vivo cultured neutrophils, which phenotypically resemble immature neutrophils released into the circulation during emergency granulopoiesis. Cultured neutrophils have similar rates of ROS production and bacterial killing but altered degranulation, cytokine release and antifungal activity compared to mature neutrophils isolated from peripheral blood. These differences are likely due to incomplete synthesis of granule proteins, as demonstrated by proteomic analysis.
Ex vivo cultured neutrophils are genetically tractable via genome editing of precursors and provide a powerful model system for investigating the properties and behaviour of immature neutrophils.
The process of reticulocyte maturation into fully mature erythrocytes that occurs in circulation is known to be characterised by a complex interplay between loss of cell surface area and volume, ...removal of remnant cell organelles and redundant proteins, and highly selective membrane and cytoskeletal remodelling. However, the mechanisms that underlie and drive these maturational processes in vivo are currently poorly understood and, at present, reticulocytes derived through in vitro culture fail to undergo the final transition to erythrocytes. Here, we use high-throughput proteomic methods to highlight differences between erythrocytes, cultured and endogenous reticulocytes. We identify a cytoskeletal protein, non-muscle myosin IIA (NMIIA) as exhibiting differential abundance and phosphorylation status between reticulocytes and erythrocytes and localize it in the proximity of autophagosomal vesicles. An ex vivo circulation system was developed to simulate the mechanical shear component of circulation and demonstrated that mechanical stimulus is necessary, but insufficient for reticulocyte maturation. Using this system in concurrence with NMII inhibition, we demonstrate involvement of NMIIA in reticulocyte remodelling and propose a previously undescribed mechanism of shear stress-responsive vesicle clearance that is crucial for reticulocyte maturation.
Macrophages have previously been characterized based on phenotypical and functional differences into suggested simplified subtypes of MØ, M1, M2a and M2c. These macrophage subtypes can be generated ...in a well-established primary monocyte culture model that produces cells expressing accepted subtype surface markers. To determine how these subtypes retain functional similarities and better understand their formation, we generated all four subtypes from the same donors. Comparative whole-cell proteomics confirmed that four distinct macrophage subtypes could be induced from the same donor material, with > 50% of 5435 identified proteins being significantly altered in abundance between subtypes. Functional assessment highlighted that these distinct protein expression profiles are primed to enable specific cell functions, indicating that this shifting proteome is predictive of meaningful changes in cell characteristics. Importantly, the 2552 proteins remained consistent in abundance across all macrophage subtypes examined, demonstrating maintenance of a stable core proteome that likely enables swift polarity changes. We next explored the cross-polarization capabilities of preactivated M1 macrophages treated with dexamethasone. Importantly, these treated cells undergo a partial repolarization toward the M2c surface markers but still retain the M1 functional phenotype. Our investigation of polarized macrophage subtypes therefore provides evidence of a sliding scale of macrophage functionality, with these data sets providing a valuable benchmark resource for further studies of macrophage polarity, with relevance for cell therapy development and drug discovery.
Myelodysplastic syndromes with ring sideroblasts (MDS-RS) commonly develop from hematopoietic stem cells (HSC) bearing mutations in the splicing factor SF3B1 (SF3B1mt). Direct studies into MDS-RS ...pathobiology have been limited by a lack of model systems that fully recapitulate erythroid biology and RS development and the inability to isolate viable human RS. Here, we combined successful direct RS isolation from patient samples, high-throughput multiomics analysis of cells encompassing the SF3B1mt stem-erythroid continuum, and functional assays to investigate the impact of SF3B1mt on erythropoiesis and RS accumulation. The isolated RS differentiated, egressed into the blood, escaped traditional nonsense-mediated decay (NMD) mechanisms, and leveraged stress-survival pathways that hinder wild-type hematopoiesis through pathogenic GDF15 overexpression. Importantly, RS constituted a contaminant of magnetically enriched CD34+ cells, skewing bulk transcriptomic data. Mis-splicing in SF3B1mt cells was intensified by erythroid differentiation through accelerated RNA splicing and decreased NMD activity, and SF3B1mt led to truncations in several MDS-implicated genes. Finally, RNA mis-splicing induced an uncoupling of RNA and protein expression, leading to critical abnormalities in proapoptotic p53 pathway genes. Overall, this characterization of erythropoiesis in SF3B1mt RS provides a resource for studying MDS-RS and uncovers insights into the unexpectedly active biology of the "dead-end" RS.
Ring sideroblast isolation combined with state-of-the-art multiomics identifies survival mechanisms underlying SF3B1-mutant erythropoiesis and establishes an active role for erythroid differentiation and ring sideroblasts themselves in SF3B1-mutant myelodysplastic syndrome pathogenesis.
Nasal dermoids are uncommon midline congenital lesions in the nose, usually diagnosed in the first years of life. Imaging is mandatory to evaluate local and intracranial extension and treatment ...consists in surgical excision. This study aims to review the experience of the department in managing pediatric nasal dermoids using a dorsal rhinotomy surgical approach.
Retrospective case series of pediatric nasal dermoids treated at a tertiary university teaching hospital over a period of seven years.
Nine children were treated during this period. Clinical presentation was a dermoid sinus-cyst in seven cases and a cystic lesion in two. Pre-operative imaging revealed extension of the lesion to the foramen cecum in three cases. Surgery was performed via vertical dorsal rhinotomy in all patients, and associated endoscopic surgery was used in three patients. Reconstruction with autologous material was performed in three cases. No complications or recurrences were registered during the follow-up.
In the presented series, a vertical dorsal rhinotomy incision has provided good functional and aesthetic results. The possibility of nasal dermoid intracranial extension should be accessed with imaging but remains uncommon. In its absence, this approach may be useful and can be paired with other techniques, such as nasal endoscopy, to achieve the best outcomes.
Los dermoides nasales son lesiones congénitas poco frecuentes de la línea media de la nariz, que suelen diagnosticarse en los primeros años de vida. Las imágenes son obligatorias para evaluar la extensión local e intracraneal y el tratamiento consiste en la escisión quirúrgica. Este estudio tiene como objetivo revisar la experiencia del departamento en el manejo de los dermoides nasales pediátricos mediante un abordaje quirúrgico de rinotomía dorsal.
Serie de casos retrospectivos de dermoides nasales pediátricos tratados en un hospital universitario de tercer nivel durante un período de siete años.
Durante este período se trató a nueve niños. La presentación clínica fue un quiste con seno en siete casos y una lesión quística en dos. Las imágenes preoperatorias revelaron extensión de la lesión al agujero ciego en tres casos. La cirugía se realizó mediante rinotomía dorsal vertical en todos los pacientes y en tres pacientes se utilizó cirugía endoscópica asociada. En tres casos se realizó reconstrucción con material autólogo. No se registraron complicaciones ni recurrencias durante el seguimiento.
En la serie presentada, una incisión de rinotomía dorsal vertical ha proporcionado buenos resultados funcionales y estéticos. Se debe acceder a la posibilidad de extensión intracraneal del dermoide nasal mediante imágenes, pero sigue siendo poco común. En su ausencia, este enfoque puede resultar útil y puede combinarse con otras técnicas, como la endoscopia nasal, para lograr los mejores resultados.
Introduction Rigid esophagoscopy (RE) has long been a part of otolaryngology practice. In the past decades, the procedure was less commonly performed due to the advances and availability of flexible ...endoscopic techniques. This study aims to describe the outcomes of RE performed to treat foreign body ingestion and to evaluate risk factors associated with postoperative complications. Methods Patients who underwent RE to treat foreign body ingestion in an otolaryngology emergency department of a Portuguese tertiary university hospital, between 2010 and 2020, were included. A total of 162 cases were analyzed, and data was collected retrospectively. Results The most common foreign bodies were meat bone (31.5%, n = 47), food impaction (28.8%, n = 43), and fish bone (19.5%, n = 29). The proximal esophagus was by far the most frequent location (80%, n = 118). Esophageal perforation occurred in 8% (13 patients), and there was a 2.5% (n = 4) mortality rate. The odds ratio of an esophageal perforation if the foreign body was completely or partially located outside the proximal esophagus was 4.67 times that of a foreign body exclusively in the proximal esophagus (OR = 4.67 95% CI: 1.39-15.72; p = 0.016; Fisher's exact test). Conclusion RE remains an effective and important technique in the management of ingested foreign bodies, particularly if endoscopic removal is unsuccessful. Foreign body location outside the proximal esophagus was associated with esophageal perforation.
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