[This corrects the article DOI: 10.1016/j.jpi.2023.100298.].

Macrophages play a crucial role in the homeostasis of lymph nodes. Their phenotypes and functions are dictated by the molecular composition of the microenvironment. The presence of tumor cells can influence the microenvironment, leading to changes in the cellular functions of tumor-associated macrophages (TAMs). Cellular alterations often correlate with histomorphometric and distributional changes. This study aims to characterize these pathomic modifications resulting from tumor cell infiltration by comparing them to reactive conditions. We assessed CD68+ and CD163+ TAMs in 160 three-dimensional (3D) images of human lymphoid tissue sections derived from 82 cases comprising six different diagnoses. To investigate TAM profiles, we employed a hybrid approach involving computer vision and graph theoretical algorithms. For calculating the histomorphometric features of TAMs, we utilized an image analysis pipeline with IMARIS Advanced Tracking Software. The distributions of TAMs were characterized using cell graphs. The incorporation of 3D tissue analysis in targeted areas of thick tissue sections revealed specific patterns of histomorphometric and distributional changes in TAMs. In chronic lymphocytic leukemia and diffuse large B-cell lymphoma, these distinctive alteration patterns demonstrated entity specificity. Furthermore, pathomic alterations displayed possible correlations with established functional aberrations in lymphomas. These findings imply that the pathomic properties of TAMs mirror their functional aberrations. Through validation and generalization via molecular pathological examinations, this approach has the potential to unveil and understand functional aberrations in histopathological assessments.

The A20 binding inhibitor of nuclear factor-kappa B (NF-κB)-1 (ABIN-1) serves as a ubiquitin sensor and autophagy receptor, crucial for modulating inflammation and cell death. Our previous in vitro investigation identified the microtubule-associated protein 1A/1B-light chain 3 (LC3)-interacting region (LIR) motifs 1 and 2 of ABIN-1 as key mitophagy regulators. This study aimed to explore the in vivo biological significance of ABIN1-LIR domains using a novel CRISPR-engineered ABIN1-ΔLIR1/2 mouse model, which lacks both the LIR motifs. Comprehensive morphological, serum, and tissue histochemical analyses revealed increased body fat and liver weights, altered serum and hepatic lipid profiles, and substantial hepatic lipid droplet accumulation, indicative of altered hepatic lipid metabolism, dyslipidemia, and hepatic steatosis in ABIN1-ΔLIR1/2 mice. Transcriptomic, metabolomic, and lipidomic analyses indicated dysregulated hepatic mitochondrial metabolism, favoring lipogenesis. Mechanistically, LIR1/2 deletion inhibited the expression and activity of transcription factor EB (TFEB) and AMP-activated protein kinase β1 (AMPKβ1), resulting in compromised autophagy and lipophagy. ABIN1 interacted with TFEB and colocalization was observed in both the cytoplasmic and nuclear compartments of hepatocytes. Impaired mitophagy was evidenced by the decreased expression of parkin and optineurin, along with increased levels of mitochondrial cytochrome c oxidase subunit II. These findings were corroborated by liver biopsies of patients with metabolic dysfunction-associated steatotic liver disease. Thus, this study underscores the functional role of ABIN1-LIR motifs in modulating the ABIN1-AMPK-TFEB axis, which is critical for mitochondria-associated lipid metabolism and mitophagy, offering insights into the mechanistic pathways contributing to the pathogenesis of steatosis-associated liver diseases with potential therapeutic implications.NEW & NOTEWORTHY Having identified LC3-interacting region (LIR) motifs 1/2 of A20 binding inhibitor of NF-κB-1 (ABIN-1) as mitophagy regulators in vitro, this study generated CRISPR-engineered ABIN1-ΔLIR1/2 mice lacking both LIR motifs to elucidate its in vivo significance. These mice exhibit enhanced hepatic lipid droplet accumulation, dysregulated mitochondrial metabolism, and impaired mitophagy, through modulation of the AMPK-TFEB axis. Liver biopsies from patients with metabolic dysfunction-associated steatotic liver disease corroborate these findings, suggesting therapeutic implications for liver diseases.

The cellular compartments in the lymph node form dynamic networks, enabling coordinated innate and adaptive immunological responses. This compartmentalization of the lymph node into subcompartments, such as the T and B zones, has been proven to be beneficial. The study of lymph node microarchitecture has yielded new insights into a range of fields, including anatomy, pathology and biological processes. This review focuses on three-dimensional (3D) and four-dimensional (4D) investigations of human lymph nodes, with a particular emphasis on comparisons with data obtained from mice. It will discuss the findings of 3D/4D investigations of human lymph nodes. The investigation of the immune system in 3D space and time offers numerous advantages over the analysis of thin tissue sections. It provides data that is not visible in two-dimensional (2D) representations. A comparison of volumes, surfaces, cell speeds, cell contact numbers, contact duration times, morphologies and other variables can be made in the context of immune responses and lymphomas. The evaluation of data, the application of statistics and the use of machine learning have all been demonstrated to be valuable. In conditions of reactivity and neoplasia, T cells are the fastest-moving cells. In contrast, B cells show slower movement and higher turning angles in reactive lymphoid tissue and lymphomas. Even slower than B cells are reticulum cells, like follicular dendritic reticulum cells (FDC) of the B zones and macrophages. Fast T cells are especially found in Hodgkin lymphomas and mantle cell lymphomas. Contact times between T and B cells differ between different lymphoma types and may prove useful in defining lymphomas. 4D technologies, which evaluate living tissue slices, are suitable for use in testing checkpoint blockers (such as nivolumab) and other therapeutic drugs or cells. Following incubation with nivolumab, the duration of contacts between CD4-positive T cells and CD30-positive Hodgkin-Reed-Sternberg cells was documented. The preliminary data indicate that 3D and 4D experiments in hematopathology may facilitate new insights into diagnostics, biology, and clinical applications, including the development of new lymphoma classifications.

This study deals with a 4D investigation of lymphocytes in human tissue under reactive and neoplastic conditions. The immune system's response to pathogens highly depends on cell interaction and movement, which makes it essential to analyze these dynamics. To achieve this, we observed cells and their movement in 4D. Human lymphoid tissue was examined, including 8 cases with 23 movies of hyperplastic tissue from the pharyngeal tonsil and 12 cases with 35 movies of lymphadenitis. Additionally, there were 4 cases involving 16 movies of marginal zone lymphoma (MZL), 3 cases with 19 movies of nodular lymphocyte predominant Hodgkin lymphoma (NLPHL), 3 cases with 6 movies of follicular lymphoma grade 1/2 (FL), and 2 cases with 10 movies of diffuse large B-cell lymphoma (DLBCL). We tracked the movement, analyzed the activity of B cells and PD1-positive T cells under reactive conditions, and compared the results to different types of lymphomas. In this study, CD20-positive B cells were examined in the context of CD35 staining. CD35 stains follicular dendritic cells (FDC), an indication for a germinal center, so we primarily analyzed B cells in the germinal center areas and partially in the immediate periphery. We categorized cells by defining track types (Low motion cells, Moving and turning in place, Long distance movement) to describe their movement pattern and action types (Passive cells, Interactive cells, Active cells) to describe their behavior and interactions. In neoplastic tissue, slower cellular dynamics and fewer interactions were observed compared to reactive tissue. This indicates differences in cellular behavior between reactive and neoplastic tissues, with a more dynamic cellular environment observed in reactive tissue.

Follicular lymphoma (FL) represents the most prevalent subtype of non-Hodgkin's-lymphoma in Western Europe and the United States. While the examination of two-dimensional histological slides remains the gold standard method for diagnosing FL stages, three-dimensional analysis provides additional insights, particularly regarding cellular morphology, spatial relationships and network connectivity. This investigation assessed the tumor-related morphological destruction of fibroreticular cell (FRC) networks bordering germinal centres in FL. A confocal laser scanning technology and a digital three-dimensional analysis system were used. Quantitive measurements included the length of fibroblastic reticular walls surrounding the germinal centres as well as the size of the gaps and holes within these structures. Three-dimensional analysis revealed progressive structural degradation and a reduction in mechanical barrier integrity, with differences observed between low- and high-grade FL. High-grade FL exhibited greater network destruction. Fibroblastic reticular cell networks' wall length demonstrated a consistent decline across all grades. The lengths of these walls and wall-like structures in FL grades 1 or 2 were similar to reactive germinal centres seen in lymphadenitis, as well as the gap size. The gaps are thought to be responsible for B- and T-cell exchange. This work demonstrated the massive destruction of neoplastic germinal centres in grades 3a and 3b FL. In grade 3b, this was accompanied by a likely dysfunctional mechanical border of the germinal centre and the near-complete loss of structural integrity. Under physiological conditions, gaps and holes regulate lymphoid traffic. Under reactive conditions, only a few specific T-cells can access the germinal centre. Under neoplastic conditions, the diameter of these gaps increases as grades increase, culminating in complete structural disruption in grade 3b. The mechanical destruction was found to begin at one pole of the germinal centre, as evidenced by localized decay and fragmentation of FRC walls on one side. Fibroblastic reticular cell networks are critical for maintaining chemokine gradients to ensure compartmentalisation of lymphoid structures. Their ongoing degradation in FL of the networks leads to a morphological loss of function. This is due to the blurring of various lymph node zones.

Lymph nodes function according to cellular and structural regulations. When these rules deviate from the benign equilibrium, dysregulations occur leading to the onset of diseases. In the development of malignant lymph node diseases, processes can be observed that consistently follow the same pattern. This study aims to define the structural and cellular parameters of the reactive lymph node and to demonstrate how lymph nodes change during tumorigenesis. We analysed benign cases diagnosed as Lymphadenitis (8 patients). Malignant cases with the diagnosis of Nodular Sclerosis classical Hodgkin Lymphoma (5 patients), Mixed Cellularity classical Hodgkin Lymphoma (5 patients), Follicular Lymphoma (7 patients), and diffuse large B-cell Lymphoma (2 patients) were selected. Confocal microscopy was used to visualise immune cells and their compartments in 3D. Based on the fibroblastic reticular cell network we defined five compartments in reactive lymph nodes: Subcapsular sinus, marginal sinus, follicle, T-zone, and medulla. We analysed the cellular composition based on dendritic cells, macrophages, T cells and B cells and extended this analysis to include the presence of extracellular vesicles. During tumorigenesis, the compartmentalisation of the lymph node is progressively destroyed. Despite this ongoing destruction and loss of strict compartmental delineations, at least two distinct structural regions are still visible, defined as follicle-like and T-zone-like compartments. A comparison between reactive and neoplastic cases reveals a progressive cellular and structural homogenisation. Higher masses of CD8+ T cells were found under neoplastic conditions and higher masses of CD30+ were found in Hodgkin Lymphoma. The volume of CD20+ cells in follicles was consistently lower in malignant tissues compared to benign, but higher in T-zones in malignant cases. An increase in vesicles was detected in most neoplasms. These findings offer new insights into cellular and structural remodelling, deepening our understanding of tumorigenesis and paving the way for more precise therapeutic interventions.

The human immune system is determined by the functionality of the human lymph node. With the use of high-throughput techniques in clinical diagnostics, a large number of data is currently collected. The new data on the spatiotemporal organization of cells offers new possibilities to build a mathematical model of the human lymph node - a virtual lymph node. The virtual lymph node can be applied to simulate drug responses and may be used in clinical diagnosis. Here, we review mathematical models of the human lymph node from the viewpoint of cellular processes. Starting with classical methods, such as systems of differential equations, we discuss the values of different levels of abstraction and methods in the range from artificial intelligence techniques formalism.

Germinal centers (GCs) are some of the most important structures in the human immune system. As such, their cell types and functions have been thoroughly investigated. B cells, T cells, follicular dendritic cells (FDCs), and macrophages have widely been found to typically be aggregated in GCs. However, the amount of space occupied by each of these cell types has yet to be investigated. In this study, we conducted confocal laser-based 3D cell-volume quantification of typical GC cells under reactive conditions in lymphadenitis and investigated how volume proportions change during GC development. For this investigation, we used anti-CD3 (T cells), anti-CD20 and anti-Pax5 (B cells), anti-CD23 (FDCs), anti-CD68 (macrophages), and DAPI (nuclear staining). We detected average proportions of about 11% CD3, 9% CD20, 6% CD23, and 2% CD68 in the largest possible regions of interest within GCs. Interestingly, these values remained steady relatively independent of GC size. The remarkably low B cell proportion can be attributed to technical constraints given the use of the CD20 antibody in 3D. Applying the B cell marker Pax5, we found that about 44% of the volume was occupied by B cells after extrapolating the volume of B cell nuclei to that of whole B cells. We concluded that Pax5 is more suitable than anti-CD20 for 3D B cell quantification in GCs. The substantial unstained volume in GCs raises the question of whether other cell types fill these open spaces. Our 3D investigation enabled a unique morphological and volumetric evaluation of GC cells that balance their overall volumes in GCs.

Therapy resistance is still a major reason for treatment failure in colorectal cancer (CRC). Previously, we identified the E3 ubiquitin ligase TRIM25 as a novel suppressor of caspase-2 translation which contributes to the apoptosis resistance of CRC cells towards chemotherapeutic drugs. Here, we report the executioner caspase-7 as being a further target of TRIM25. The results from the gain- and loss-of-function approaches and the actinomycin D experiments indicate that TRIM25 attenuates caspase-7 expression mainly through a decrease in mRNA stability. The data from the RNA pulldown assays with immunoprecipitated TRIM25 truncations indicate a direct TRIM25 binding to caspase-7 mRNA, which is mediated by the PRY/SPRY domain, which is also known to be highly relevant for protein–protein interactions. By employing TRIM25 immunoprecipitation, we identified the heterogeneous nuclear ribonucleoprotein H1 (hnRNPH1) as a novel TRIM25 binding protein with a functional impact on caspase-7 mRNA stability. Notably, the interaction of both proteins was highly sensitive to RNase A treatment and again depended on the PRY/SPRY domain, thus indicating an indirect interaction of both proteins which is achieved through a common RNA binding. Ubiquitin affinity chromatography showed that both proteins are targets of ubiquitin modification. Functionally, the ectopic expression of caspase-7 in CRC cells caused an increase in poly ADP-ribose polymerase (PARP) cleavage concomitant with a significant increase in apoptosis. Collectively, the negative regulation of caspase-7 by TRIM25, which is possibly executed by hnRNPH1, implies a novel survival mechanism underlying the chemotherapeutic drug resistance of CRC cells. The targeting of TRIM25 could therefore offer a promising strategy for the reduction in therapy resistance in CRC patients.

The simulation of immune response is a challenging task because quantitative data are scarce. Quantitative theoretical models either focus on specific cell–cell interactions or have to make assumptions about parameters. The broad variation of, e.g., the dimensions and abundance between lymph nodes as well as between individual patients hampers conclusive quantitative modeling. No theoretical model has been established representing a consensus on the set of major cellular processes involved in the immune response. In this paper, we apply the Petri net formalism to construct a semi-quantitative mathematical model of the lymph nodes. The model covers the major cellular processes of immune response and fulfills the formal requirements of Petri net models. The intention is to develop a model taking into account the viewpoints of experienced pathologists and computer scientists in the field of systems biology. In order to verify formal requirements, we discuss invariant properties and apply the asynchronous firing rule of a place/transition net. Twenty-five transition invariants cover the model, and each is assigned to a functional mode of the immune response. In simulations, the Petri net model describes the dynamic modes of the immune response, its adaption to antigens, and its loss of memory.

In recent years, medical disciplines have moved closer together and rigid borders have been increasingly dissolved. The synergetic advantage of combining multiple disciplines is particularly important for radiology, nuclear medicine, and pathology to perform integrative diagnostics. In this review, we discuss how medical subdisciplines can be reintegrated in the future using state-of-the-art methods of digitization, data science, and machine learning. Integration of methods is made possible by the digitalization of radiological and nuclear medical images, as well as pathological images. 3D histology can become a valuable tool, not only for integration into radiological images but also for the visualization of cellular interactions, the so-called connectomes. In human pathology, it has recently become possible to image and calculate the movements and contacts of immunostained cells in fresh tissue explants. Recording the movement of a living cell is proving to be informative and makes it possible to study dynamic connectomes in the diagnosis of lymphoid tissue. By applying computational methods including data science and machine learning, new perspectives for analyzing and understanding diseases become possible.

Histological sections of the lymphatic system are usually the basis of static (2D) morphological investigations. Here, we performed a dynamic (4D) analysis of human reactive lymphoid tissue using confocal fluorescent laser microscopy in combination with machine learning. Based on tracks for T-cells (CD3), B-cells (CD20), follicular T-helper cells (PD1) and optical flow of follicular dendritic cells (CD35), we put forward the first quantitative analysis of movement-related and morphological parameters within human lymphoid tissue. We identified correlations of follicular dendritic cell movement and the behavior of lymphocytes in the microenvironment. In addition, we investigated the value of movement and/or morphological parameters for a precise definition of cell types (CD clusters). CD-clusters could be determined based on movement and/or morphology. Differentiating between CD3- and CD20 positive cells is most challenging and long term-movement characteristics are indispensable. We propose morphological and movement-related prototypes of cell entities applying machine learning models. Finally, we define beyond CD clusters new subgroups within lymphocyte entities based on long term movement characteristics. In conclusion, we showed that the combination of 4D imaging and machine learning is able to define characteristics of lymphocytes not visible in 2D histology.

Classical Hodgkin lymphoma (cHL) is one of the most common malignant lymphomas in Western Europe. It is diagnosed on the basis of histological sections by pathologists using a light microscope. The tumor cells, the Hodgkin- and Reed Sternberg cells (HRS), are visualized by morphology and positive response for the CD30-antigen. The same antigen can also be detected by immunohistochemistry on a reactive counterpart, showing CD30+ cells in special immunoreactions, such as inflammations of lymph nodes (lymphadenitis). CD30+ cells in reactive and neoplastic conditions are surrounded by lymphocytes and histiocytes, forming a micromilieu that enables the survival of the tumor cells, as well as their reactive counterparts. This study deals with an investigation of CD30+-surrounding cells using a confocal laser technology, visualizing the contacts of reactive and neoplastic CD30+ cells with CD68+ macrophages and CD163+ macrophages as well as to PD1+ lymphocytes and B cells (CD20+). CD4 immunostains were not included, because CD4+ cells were too numerous for clear dissection of single cells. 3D images visualized the, so-called, connectomes. Clear differences in the number of contacts between CD30-reactive and neoplastic cells (HRS) with macrophages and B lymphocytes were visible. Lymphadenitis and Mixed Cellularity type of classical Hodgkin Lymphoma (cHL) differed in that Mixed Cellularity (MC) cHL had more connections to macrophages (CD163+) and lower number of connections to B cells (CD20+). The connectomes of both Hodgkin variants MCcHL and Nodular Sclerosis cHL (NScHL) mainly differed in the number of contacts to CD163+ macrophages, which was higher in MCcHL. Investigating the volumes of CD30+ -reactive and neoplastic cells, we found out that reactive cells showed lesser volumes, which correlated with the number of contacts. The comparison between 2D and 3D images, including 3D prints, demonstrated clear advantages of the 3D method. 3D images visualized significantly more and clearly defined intercellular contacts. Complicated cellular networks and their contacts became especially evident in volume and surface evaluations, as well as in 3D prints.

This study deals with 3D laser investigation on the border between the human lymph node T-zone and germinal centre. Only a few T-cells specific for antigen selected B-cells are allowed to enter germinal centres. This selection process is guided by sinus structures, chemokine gradients and inherent motility of the lymphoid cells. We measured gaps and wall-like structures manually, using IMARIS, a 3D image software for analysis and interpretation of microscopy datasets. In this paper, we describe alpha-actin positive and semipermeable walls and wall-like structures that may hinder T-cells and other cell types from entering germinal centres. Some clearly defined holes or gaps probably regulate lymphoid traffic between T- and B-cell areas. In lymphadenitis, the morphology of this border structure is clearly defined. However, in case of malignant lymphoma, the wall-like structure is disrupted. This has been demonstrated exemplarily in case of angioimmunoblastic T-cell lymphoma. We revealed significant differences of lengths of the wall-like structures in angioimmunoblastic T-cell lymphoma in comparison with wall-like structures in reactive tissue slices. The alterations of morphological structures lead to abnormal and less controlled T- and B-cell distributions probably preventing the immune defence against tumour cells and infectious agents by dysregulating immune homeostasis.

Human lymph nodes play a central part of immune defense against infection agents and tumor cells. Lymphoid follicles are compartments of the lymph node which are spherical, mainly filled with B cells. B cells are cellular components of the adaptive immune systems. In the course of a specific immune response, lymphoid follicles pass different morphological differentiation stages. The morphology and the spatial distribution of lymphoid follicles can be sometimes associated to a particular causative agent and development stage of a disease. We report our new approach for the automatic detection of follicular regions in histological whole slide images of tissue sections immuno-stained with actin. The method is divided in two phases: (1) shock filter-based detection of transition points and (2) segmentation of follicular regions. Follicular regions in 10 whole slide images were manually annotated by visual inspection, and sample surveys were conducted by an expert pathologist. The results of our method were validated by comparing with the manual annotation. On average, we could achieve a Zijbendos similarity index of 0.71, with a standard deviation of 0.07.

AIMS:The examination of histological sections is still the gold standard in diagnostic pathology. Important histopathological diagnostic criteria are nuclear shapes and chromatin distribution as well as nucleus-cytoplasm relation and immunohistochemical properties of surface and intracellular proteins. The aim of this investigation was to evaluate the benefits and drawbacks of three-dimensional imaging of CD30+ cells in classical Hodgkin Lymphoma (cHL) in comparison to CD30+ lymphoid cells in reactive lymphoid tissues.MATERIALS AND RESULTS:Using immunoflourescence confocal microscopy and computer-based analysis, we compared CD30+ neoplastic cells in Nodular Sclerosis cHL (NScCHL), Mixed Cellularity cHL (MCcHL), with reactive CD30+ cells in Adenoids (AD) and Lymphadenitis (LAD). We confirmed that the percentage of CD30+ cell volume can be calculated. The amount in lymphadenitis was approx. 1.5%, in adenoids around 2%, in MCcHL up to 4,5% whereas the values for NScHL rose to more than 8% of the total cell cytoplasm. In addition, CD30+ tumour cells (HRS-cells) in cHL had larger volumes, and more protrusions compared to CD30+ reactive cells. Furthermore, the formation of large cell networks turned out to be a typical characteristic of NScHL.CONCLUSION:In contrast to 2D histology, 3D laser scanning offers a visualisation of complete cells, their network interaction and spatial distribution in the tissue. The possibility to differentiate cells in regards to volume, surface, shape, and cluster formation enables a new view on further diagnostic and biological questions. 3D includes an increased amount of information as a basis of bioinformatical calculations.

Histopathological methods based on 2 μm thin sections are routinely used in pathological anatomical diagnosis. Many medical disciplines already rely on a 3D representation, regarding visualization and imaging techniques. Pathology in particular uses different tissue visualizations to make the final diagnosis. Thereby, a standard 2D histological section only represents a flat snapshot of a three-dimensional complex cell system. Despite that, 3D cell analysis is not yet standardly used in clinical routine. This work used 3D analysis systems to investigate the morphological alterations of the fibroblastic reticular cell network inside human lymph nodes during neoplastic transformation and evaluates the added value of 3D visualizations in tissue interpretation. We investigated the surface and volume quotient, cell cross-linking and percentage cell volume of the fibroblastic reticular cell (FRC) network inside Lymphadenopathy (follicular hyperplasia) (LAD), Follicular Lymphoma Grade 1 (FL1), Nodular Sclerosis classical Hodgkin Lymphoma (NScHL) and Angioimmunoblastic T-Cell Lymphoma (AITL). We found that the average quotient of LAD and FL1 differed from those of NScHL and AITL, indicating that the surface and volume quotient changes in the course of neoplastic transformation. This is probably due to an increased network convolution, while the total cell volume remains the same at about 2%. In conclusion, this paper describes the tumor-related morphological changes of the FRC network, which would have been difficult to achieve without the use of 3D analysis systems.