UNLABELLED: The development of functional B lymphocytes during chicken embryogenesis relies on a series of tightly regulated processes. Precursor B cells migrate from the spleen via the blood to the bursa of Fabricius, where they colonize the bursal follicles to undergo further maturation and differentiation. To better understand the molecular mechanisms underlying early B cell migration in the chicken embryo, transcriptome analysis of B cells isolated from the spleen, blood, and bursa at embryonic days (ED) 12, ED14, and ED16 was performed. These findings suggest that sphingosine-1-phosphate (S1P) and its receptors regulate B cell presence in the bloodstream, while CCR7 and CXCR4 guide B cells to the bursa. Additionally, integrins and cell adhesion molecules, such as PECAM1, appear to facilitate transendothelial migration into the bursal mesenchyme. This study highlights a coordinated interplay between chemokines, integrins and cell adhesion molecules involved in B cell recruitment and colonization of the bursa microenvironment. These findings enhance our understanding of early B cell migration and shed light on the mechanisms governing B cell trafficking during chicken embryonic development. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12864-025-11749-w.

• Klíčová slova
• Adaptive immunity, B cell development, B cell migration, Bursa of Fabricius, Cell adhesion molecules, Chicken embryonic development, Transcriptomics,
• Publikační typ
• časopisecké články MeSH

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field.

• Klíčová slova
• Autophagosome, LC3, cancer, flux, lysosome, macroautophagy, neurodegeneration, phagophore, stress, vacuole,
• MeSH
• autofagie * fyziologie   MeSH
• autofagozomy MeSH
• biologické markery MeSH
• biotest normy   MeSH
• lidé MeSH
• lyzozomy MeSH
• proteiny spojené s autofagií metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH
• směrnice MeSH
• Názvy látek
• biologické markery MeSH
• proteiny spojené s autofagií MeSH

Secondary diversification of the Ig repertoire occurs through somatic hypermutation (SHM), gene conversion (GCV), and class switch recombination (CSR)-three processes that are initiated by activation-induced cytidine deaminase (AID). AID targets Ig genes at orders of magnitude higher than the rest of the genome, but the basis for this specificity is poorly understood. We have previously demonstrated that enhancers and enhancer-like sequences from Ig genes are capable of stimulating SHM of neighboring genes in a capacity distinct from their roles in increasing transcription. Here, we use an in vitro proteomics approach to identify E-box, MEF2, Ets, and Ikaros transcription factor family members as potential binders of these enhancers. ChIP assays in the hypermutating Ramos B cell line confirmed that many of these factors bound the endogenous Igλ enhancer and/or the IgH intronic enhancer (Eμ) in vivo. Further investigation using SHM reporter assays identified binding sites for E2A and MEF2B in Eμ and demonstrated an association between loss of factor binding and decreases in the SHM stimulating activity of Eμ mutants. Our results provide novel insights into trans-acting factors that dictate SHM targeting and link their activity to specific DNA binding sites within Ig enhancers.

• Klíčová slova
• AID, E2A, MEF2B, Ramos B cell line, Somatic hypermutation,
• MeSH
• geny pro imunoglobuliny MeSH
• kur domácí MeSH
• lidé MeSH
• somatická hypermutace imunoglobulinových genů fyziologie   MeSH
• transkripční faktory genetika   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• transkripční faktory MeSH

In mice, the ability of naive T (TN) cells to mount an effector response correlates with TCR sensitivity for self-derived Ags, which can be quantified indirectly by measuring surface expression levels of CD5. Equivalent findings have not been reported previously in humans. We identified two discrete subsets of human CD8+ TN cells, defined by the absence or presence of the chemokine receptor CXCR3. The more abundant CXCR3+ TN cell subset displayed an effector-like transcriptional profile and expressed TCRs with physicochemical characteristics indicative of enhanced interactions with peptide-HLA class I Ags. Moreover, CXCR3+ TN cells frequently produced IL-2 and TNF in response to nonspecific activation directly ex vivo and differentiated readily into Ag-specific effector cells in vitro. Comparative analyses further revealed that human CXCR3+ TN cells were transcriptionally equivalent to murine CXCR3+ TN cells, which expressed high levels of CD5. These findings provide support for the notion that effector differentiation is shaped by heterogeneity in the preimmune repertoire of human CD8+ T cells.

• MeSH
• aktivace lymfocytů imunologie   MeSH
• biologické markery MeSH
• buněčná diferenciace imunologie   MeSH
• CD8-pozitivní T-lymfocyty imunologie   metabolismus   MeSH
• dospělí MeSH
• imunofenotypizace MeSH
• imunologická paměť MeSH
• kultivované buňky MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladý dospělý MeSH
• myši MeSH
• receptory CXCR3 metabolismus   MeSH
• senioři MeSH
• T-lymfocyty - podskupiny imunologie   metabolismus   MeSH
• věkové faktory MeSH
• zvířata MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladý dospělý MeSH
• mužské pohlaví MeSH
• myši MeSH
• senioři MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Intramural MeSH
• Názvy látek
• biologické markery MeSH
• CXCR3 protein, human MeSH Prohlížeč
• receptory CXCR3 MeSH

Despite the development of several cultivation methods, the rate of discovery of microorganisms that are yet-to-be cultivated outpaces the rate of isolating and cultivating novel species in the laboratory. Furthermore, no current cultivation technique is capable of selectively isolating and cultivating specific bacterial taxa or phylogenetic groups independently of morphological or physiological properties. Here, we developed a new method to isolate living bacteria solely based on their 16S rRNA gene sequence. We showed that bacteria can survive a modified version of the standard fluorescence in situ hybridization (FISH) procedure, in which fixation is omitted and other factors, such as centrifugation and buffers, are optimized. We also demonstrated that labelled DNA probes can be introduced into living bacterial cells by means of chemical transformation and that specific hybridization occurs. This new method, which we call live-FISH, was then combined with fluorescence-activated cell sorting (FACS) to sort specific taxonomic groups of bacteria from a mock and natural bacterial communities and subsequently culture them. Live-FISH represents the first attempt to systematically optimize conditions known to affect cell viability during FISH and then to sort bacterial cells surviving the procedure. No sophisticated probe design is required, making live-FISH a straightforward method to be potentially used in combination with other single-cell techniques and for the isolation and cultivation of new microorganisms.

• MeSH
• Bacillus genetika   MeSH
• Bacteria genetika   MeSH
• bakteriální RNA genetika   MeSH
• DNA sondy MeSH
• fylogeneze MeSH
• hybridizace in situ fluorescenční * MeSH
• mikrobiologické techniky * MeSH
• průtoková cytometrie MeSH
• RNA ribozomální 16S genetika   MeSH
• separace buněk * MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• bakteriální RNA MeSH
• DNA sondy MeSH
• RNA ribozomální 16S MeSH

CD molecules are surface molecules expressed on cells of the immune system that play key roles in immune cell-cell communication and sensing the microenvironment. These molecules are essential markers for the identification and isolation of leukocytes and lymphocyte subsets. Here, we present the results of the first phase of the CD Maps study, mapping the expression of CD1-CD100 (n = 110) on 47 immune cell subsets from blood, thymus, and tonsil using an eight-color standardized EuroFlow approach and quantification of expression. The resulting dataset included median antibody binding capacities (ABCs) and percentage of positivity for all markers on all subsets and was developed into an interactive CD Maps web resource. Using the resource, we examined differentially expressed proteins between granulocyte, monocyte, and dendritic cell subsets, and profiled dynamic expression of markers during thymocyte differentiation, T-cell maturation, and between functionally distinct B-cell subset clusters. The CD Maps resource will serve as a benchmark of antibody reactivities ensuring improved reproducibility of flow cytometry-based research. Moreover, it will provide a full picture of the surfaceome of human immune cells and serves as a useful platform to increase our understanding of leukocyte biology, as well as to facilitate the identification of new biomarkers and therapeutic targets of immunological and hematological diseases.

• Klíčová slova
• B-cell, CD marker, T-cell, expression profiling, flow cytometry, lymphocyte, monocyte, surfaceome,
• MeSH
• B-lymfocyty imunologie   metabolismus   MeSH
• CD antigeny biosyntéza   MeSH
• datové soubory jako téma MeSH
• dendritické buňky imunologie   metabolismus   MeSH
• dítě MeSH
• dospělí MeSH
• granulocyty imunologie   metabolismus   MeSH
• imunofenotypizace MeSH
• internet MeSH
• leukocyty imunologie   metabolismus   MeSH
• lidé MeSH
• lymfopoéza MeSH
• monocyty imunologie   metabolismus   MeSH
• peptidové mapování MeSH
• podskupiny lymfocytů imunologie   metabolismus   MeSH
• předškolní dítě MeSH
• průtoková cytometrie MeSH
• reprodukovatelnost výsledků MeSH
• separace buněk MeSH
• T-lymfocyty imunologie   metabolismus   MeSH
• Check Tag
• dítě MeSH
• dospělí MeSH
• lidé MeSH
• předškolní dítě MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• CD antigeny MeSH

These guidelines are a consensus work of a considerable number of members of the immunology and flow cytometry community. They provide the theory and key practical aspects of flow cytometry enabling immunologists to avoid the common errors that often undermine immunological data. Notably, there are comprehensive sections of all major immune cell types with helpful Tables detailing phenotypes in murine and human cells. The latest flow cytometry techniques and applications are also described, featuring examples of the data that can be generated and, importantly, how the data can be analysed. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid, all written and peer-reviewed by leading experts in the field, making this an essential research companion.

• MeSH
• alergologie a imunologie normy   MeSH
• fenotyp MeSH
• konsensus MeSH
• lidé MeSH
• průtoková cytometrie metody   normy   MeSH
• separace buněk metody   normy   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

The plasminogen system is harnessed in a wide variety of physiological processes, such as fibrinolysis, cell migration, or efferocytosis; and accordingly, it is essential upon inflammation, tissue remodeling, wound healing, and for homeostatic maintenance in general. Previously, we identified a plasminogen receptor in the mannose 6-phosphate/insulin-like growth factor 2 receptor (M6P/IGF2R, CD222). Here, we demonstrate by means of genetic knockdown, knockout, and rescue approaches combined with functional studies that M6P/IGF2R is up-regulated on the surface of macrophages, recognizes plasminogen exposed on the surface of apoptotic cells, and mediates plasminogen-induced efferocytosis. The level of uptake of plasminogen-coated apoptotic cells inversely correlates with the TNF-α production by phagocytes indicating tissue clearance without inflammation by this mechanism. Our results reveal an up-to-now undetermined function of M6P/IGF2R in clearance of apoptotic cells, which is crucial for tissue homeostasis.

• Klíčová slova
• M6P/IGF2R, efferocytosis, macrophages, plasminogen, tissue homeostasis,
• MeSH
• buněčná diferenciace účinky léků   MeSH
• fagocytóza účinky léků   MeSH
• fibroblasty účinky léků   metabolismus   MeSH
• genový knockout MeSH
• Jurkat buňky MeSH
• lidé MeSH
• makrofágy cytologie   účinky léků   metabolismus   MeSH
• myši MeSH
• plazminogen farmakologie   MeSH
• receptor IGF typ 2 metabolismus   MeSH
• THP-1 buňky MeSH
• TNF-alfa metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• plazminogen MeSH
• receptor IGF typ 2 MeSH
• TNF-alfa MeSH