Intravascular Cell Research Articles

In Vivo Visualization of Intravascular Patrolling Immune Cells in the Primate Eye.

Insight into the immune status of the living eye is essential as we seek to understand ocular disease and develop new treatments. The nonhuman primate (NHP) is the gold standard preclinical model for therapeutic development in ophthalmology, owing to the similar visual system and immune landscape in the NHP relative to the human. Here, we demonstrate the utility of phase-contrast adaptive optics scanning light ophthalmoscope (AOSLO) to visualize immune cell dynamics on the cellular scale, label-free in the NHP. Phase-contrast AOSLO was used to image preselected areas of retinal vasculature in five NHP eyes. Images were registered to correct for eye motion, temporally averaged, and analyzed for immune cell activity. Cell counts, dimensions, velocities, and frequency per vessel were determined manually and compared between retinal arterioles and venules. Based on cell appearance and circularity index, cells were divided into three morphologies: ovoid, semicircular, and flattened. Immune cells were observed migrating along vascular endothelium with and against blood flow. Cell velocity did not significantly differ between morphology or vessel type and was independent of blow flood. Venules had a significantly higher cell frequency than arterioles. A higher proportion of cells resembled "flattened" morphology in arterioles. Based on cell speeds, morphologies, and behaviors, we identified these cells as nonclassical patrolling monocytes (NCPMs). Phase-contrast AOSLO has the potential to reveal the once hidden behaviors of single immune cells in retinal circulation and can do so without the requirement of added contrast agents that may disrupt immune cell behavior.

Development of a peptide-generated antibody to rabbit hemorrhagic disease virus 2 VP60 and its immunohistochemical application in natural cases.

Rabbit hemorrhagic disease virus 2 (RHDV2) has spread across the United States infecting and causing death in domestic and wild rabbits. Immunohistochemistry (IHC) would be a useful tool for the detection of RHDV2 antigen in tissues as it is inexpensive and readily achievable in most diagnostic laboratories. However, there is no readily available antibody for this purpose. To fill this void, we generated an RHDV2 capsid protein VP60-specific antibody in chicken eggs and validated the antibody using formalin-fixed tissues from 5 domestic rabbits naturally infected with RHDV2. Viral antigen was detected immunohistochemically in various tissues, most prominently in hepatocytes and macrophages in liver, and in macrophages in spleen and cecal lymphoid tissue. Intravascular mononuclear cells in lung and renal tubular and biliary epithelium also were immunolabeled. Both nuclear and cytoplasmic immunolabeling were observed. This peptide-generated antibody is a potentially useful tool as an adjunct to reverse-transcription PCR or in situ hybridization for detection of RHDV2 in tissues.

Bioengineering of vascularized porcine flaps using perfusion-recellularization

Large volume soft tissue defects greatly impact patient quality of life and function while suitable repair options remain a challenge in reconstructive surgery. Engineered flaps could represent a clinically translatable option that may circumvent issues related to donor site morbidity and tissue availability. Herein, we describe the regeneration of vascularized porcine flaps, specifically of the omentum and tensor fascia lata (TFL) flaps, using a tissue engineering perfusion-decellularization and recellularization approach. Flaps were decellularized using a low concentration sodium dodecyl sulfate (SDS) detergent perfusion to generate an acellular scaffold with retained extracellular matrix (ECM) components while removing underlying cellular and nuclear contents. A perfusion-recellularization strategy allowed for seeding of acellular flaps with a co-culture of human umbilical vein endothelial cell (HUVEC) and mesenchymal stromal cells (MSC) onto the decellularized omentum and TFL flaps. Our recellularization technique demonstrated evidence of intravascular cell attachment, as well as markers of endothelial and mesenchymal phenotype. Altogether, our findings support the potential of using bioengineered porcine flaps as a novel, clinically-translatable strategy for future application in reconstructive surgery.

Differential in vivo labeling with barcoded antibodies allows for simultaneous transcriptomic profiling of airway, lung tissue and intravascular immune cells.

Single-cell RNA sequencing (scRNA-seq) is the state-of-the-art approach to study transcriptomic signatures in individual cells in respiratory health and disease. However, classical scRNA-seq approaches provide no spatial information and are performed using either bronchoalveolar lavage fluid (BAL) or lung single cell suspensions to assess transcript levels in airway and tissue immune cells, respectively. Herein we describe a simple method to simultaneously characterize transcriptomic features of airway, lung parenchymal and intravascular immune cells based on differential in vivo labeling with barcoded antibodies. In addition to gaining basic spatial information, this approach allows for direct comparison of cells within different anatomical compartments. Furthermore, this method provides a time- and cost-effective alternative to classical scRNA-seq where lung and BAL samples are processed individually, reducing animal and reagent use. We demonstrate the feasibility of this approach in a preclinical mouse model of bacterial lung infection comparing airway, parenchymal and vasculature neutrophils early after infection.

Abstract 17854: Imaging Intravascular Macrophages Using Micro-Sized Superparamagnetic Decoys

Introduction: During inflammatory processes, intravascular macrophages are capable of phagocytizing circulating debris in the circulation. Hypothesis: Intravenous injection of contrast-loaded decoys could be used to label intravascular macrophages and map their distribution by non-invasive imaging. Methods: We synthetized superparamagnetic decoys with hydrodynamic diameters from 20 nm to 4.5 μm. Experimental models of neuroinflammation (intrastriatal injection of LPS or TNF) and ischemic stroke (thrombin model and ferric chloride model) were performed in mice. High resolution 3D T2*-weighted MRI sequences were performed after intravenous injection of the superparamagnetic decoys. Results: In experimental models of neuroinflammation, intravenous injection of superparamagnetic decoys with a diameter of 1 μm (but neither smaller nor larger particles) reveals intravascular phagocytic cells in the cerebral circulation, in a dose dependent manner. In ischemic stroke, we found that intravenous injection of the decoys leads to numerous signal voids in the vessels in and around the infarct core at 48 hours (Figure 1a-b). Interestingly, the intravascular phagocytic activity appears much higher in permanent rather than transient models of ischemic stroke. Pre-treatment with anti-CD11a blocking antibodies significantly reduced the number of intravascular macrophages in stroke. Histological studies confirmed that injected decoys are phagocytized by intravascular cells which express typical markers of the monocyte/macrophage lineage including CX3CR1 dependent GFP expression and CD68 (Figure 1c). Conclusions: Intravenous injection of superparamagnetic decoys in the micrometer size range allows to image intravascular macrophages. This new method allows to study the pathophysiological roles of intravascular macrophages in experimental models and could be easily translated to humans.

Platelet Angiopoietin-1 Protects Against Experimental Lung Metastasis By Limiting Tumor Cell Extravasation

In addition to their fundamental roles in hemostasis, platelets can contribute to both tumor growth and metastasis. However, despite being a reservoir for pro-angiogenic and metastatic cytokines, platelets also harbor negative regulators of tumor progression. One such regulator is angiopoietin-1 (Angpt1), an angiogenic cytokine that preserves vascular integrity by promoting cell-cell endothelial interactions downstream of its receptor, the receptor tyrosine kinase Tie-2. In addition, Angpt1 also protects against tumor cell metastasis through an unidentified mechanism. Although platelets are believed to be the primary source of Angpt1 in circulation, the contributions of platelet-derived Angpt1 to tumor growth and metastasis have not been investigated. In this study, we demonstrate that platelet Angpt1 is selectively upregulated in the spontaneous PyMT breast cancer mouse model, but not pre-clinical models of melanoma or non-malignant inflammation (colitis), suggesting a distinct contribution of platelet Angpt1 in breast cancer. To test whether deletion of platelet Angpt1 affects tumor growth or metastasis, we generated mice selectively lacking Angpt1 in megakaryocytes and their platelet progeny (Angpt1 fl/flPF4 Cre, referred to as Angpt1 Plt KO). Plasma Angpt1 was undetectable in Angpt1 Plt KO mice, comparable to mice deficient in the megakaryocyte differentiation factor thrombopoietin, which display approximately 10% of platelet counts. Taken together, these findings establish that platelets are the principal source of circulating Angpt1. In tumor implantation studies, orthotopic tumor growth and vascularization were unaffected in Angpt1 Plt KO mice in pre-clinical models of breast cancer and melanoma. However, Angpt1 Plt KO mice had increased experimental metastasis during both early (3-24 hours) and late (10 days, Figure 1) stages of tumor colonization in both breast cancer and melanoma models, revealing an important role for platelet Angpt1 in restraining the latter stages of tumor metastasis following tumor engraftment. Next, we sought to mechanistically determine how platelet Angpt1 limits tumor cell metastasis. Static adhesion assays demonstrated that platelets lacking Angpt1 did not affect tumor cell attachment to endothelial cells when compared to platelets containing Angpt1. However, Angpt1 Plt KO mice had increased tumor cell retention and extravasation in their lungs following experimental metastasis (3-24 hours), suggesting that platelet Angpt1 limits endothelial permeabilization and the trans-endothelial migration of tumor cells out of blood vessels. Most importantly, all intravascular tumor cells were found within platelet-rich microthrombi immediately after sequestration in the lungs. We therefore conclude that localized Angpt1 released by activated platelets at the site of tumor cell arrest likely reprograms the surrounding endothelium to limit successful tumor cell extravasation. These findings reveal an unexpected role for platelets in restricting tumor cell metastasis.

Advances in the design, generation, and application of tissue-engineered myocardial equivalents.

Due to the limited regenerative ability of cardiomyocytes, the disabling irreversible condition of myocardial failure can only be treated with conservative and temporary therapeutic approaches, not able to repair the damage directly, or with organ transplantation. Among the regenerative strategies, intramyocardial cell injection or intravascular cell infusion should attenuate damage to the myocardium and reduce the risk of heart failure. However, these cell delivery-based therapies suffer from significant drawbacks and have a low success rate. Indeed, cardiac tissue engineering efforts are directed to repair, replace, and regenerate native myocardial tissue function. In a regenerative strategy, biomaterials and biomimetic stimuli play a key role in promoting cell adhesion, proliferation, differentiation, and neo-tissue formation. Thus, appropriate biochemical and biophysical cues should be combined with scaffolds emulating extracellular matrix in order to support cell growth and prompt favorable cardiac microenvironment and tissue regeneration. In this review, we provide an overview of recent developments that occurred in the biomimetic design and fabrication of cardiac scaffolds and patches. Furthermore, we sift in vitro and in situ strategies in several preclinical and clinical applications. Finally, we evaluate the possible use of bioengineered cardiac tissue equivalents as in vitro models for disease studies and drug tests.

The Anti-Inflammatory Action of Pulsed Radiofrequency-A Hypothesis and Potential Applications.

In 2013, it was reported that pulsed radiofrequency (PRF) could be applied to obtain a systemic anti-inflammatory effect. Patients with chronic pain and patients with an inflammatory condition from other disciplines could potentially profit from this finding. At that time, intravenous application was used, but since then, it became clear that it could be applied transcutaneously as well. This procedure was named RedoxPRF. This can be used both for regional and for systemic application. Recently, the basic element of the mode of action has been clarified from the analysis of the effects of PRF on a standard model of muscle injury in rats. The objective of this paper is to present a hypothesis on the mode of action of RedoxPRF now that the basic mechanism has become known. Cell stress causes an increased production of free radicals, disturbing the redox equilibrium, causing oxidative stress (OS) either directly or secondarily by other types of stress. Eventually, OS causes inflammation and an increased sympathetic (nervous) system activity. In the acute form, this leads to immune paralysis; in the chronic form, to immune tolerance and chronic inflammation. It is hypothesized that RedoxPRF causes a reduction of free radicals by a recombination of radical pairs. For systemic application, the target cells are the intravascular immune cells that pass through an activated area as on an assembly line. Hypothesis conclusions: 1. RedoxPRF treatment works selectively on OS. It has the unique position of having a point of engagement at the most upstream level of the train of events. 2. RedoxPRF has the potential of being a useful tool in the treatment of inflammatory diseases and possibly of stage 4 cancer. 3. In the treatment of chronic pain, RedoxPRF is an entirely new method because it is different from ablation as well as from stimulation. We propose the term "functional restoration". 4. Controlled studies must be conducted to develop this promising new field in medicine further.

Loose Tumor Cells in Pulmonary Arteries of Lung Adenocarcinoma Resection Specimens: No Correlation With Survival, Despite High Prevalence.

Loose tumor cells and tumor cell clusters can be recognized in the lumen of intratumoral pulmonary arteries of resected non-small cell lung cancer specimens. It is unclear whether these should be considered tumor-emboli, and as such could predict a worsened prognosis. To investigate the nature and prognostic impact of pulmonary artery intraluminal tumor cells. This multicenter study involved an exploratory pilot study and a validation study from 3 institutions. For the exploratory pilot study, a retrospective pulmonary resection cohort of primary adenocarcinomas, diagnosed between November 2007 and November 2010, were scored for the presence of tumor cells, as well as potentially other cells in the intravascular spaces, using hematoxylin-eosin and cytokeratin 7 (CK7) stains. In the validation part, 2 retrospective cohorts of resected pulmonary adenocarcinomas, between January 2011 and December 2016, were included. Recurrence-free survival (RFS) and overall survival (OS) data were collected. In the pilot study, CK7+ intravascular cells, mainly tumor cells, were present in 23 of 33 patients (69.7%). The 5-year OS for patients with intravascular tumor cells was 61%, compared with 40% for patients without intravascular tumor cells (P = .19). In the validation study, CK7+ intravascular tumor cells were present in 41 of 70 patients (58.6%). The 5-year RFS for patients with intravascular tumor cells was 80.0%, compared with 80.6% in patients without intravascular tumor cells (P = .52). The 5-year OS rates were, respectively, 82.8% and 71.6% (P = .16). Loose tumor cells in pulmonary arterial lumina were found in most non-small cell lung cancer resection specimens and were not associated with a worse RFS or OS. Therefore, most probably they represent an artifact.

Alpha4 beta7 integrin controls Th17 cell trafficking in the spinal cord leptomeninges during experimental autoimmune encephalomyelitis.

Th1 and Th17 cell migration into the central nervous system (CNS) is a fundamental process in the pathogenesis of experimental autoimmune encephalomyelitis (EAE), the animal model of multiple sclerosis (MS). Particularly, leptomeningeal vessels of the subarachnoid space (SAS) constitute a central route for T cell entry into the CNS during EAE. Once migrated into the SAS, T cells show an active motility behavior, which is a prerequisite for cell-cell communication, in situ reactivation and neuroinflammation. However, the molecular mechanisms selectively controlling Th1 and Th17 cell trafficking in the inflamed leptomeninges are not well understood. By using epifluorescence intravital microscopy, we obtained results showing that myelin-specific Th1 and Th17 cells have different intravascular adhesion capacity depending on the disease phase, with Th17 cells being more adhesive at disease peak. Inhibition of αLβ2 integrin selectively blocked Th1 cell adhesion, but had no effect on Th17 rolling and arrest capacity during all disease phases, suggesting that distinct adhesion mechanisms control the migration of key T cell populations involved in EAE induction. Blockade of α4 integrins affected myelin-specific Th1 cell rolling and arrest, but only selectively altered intravascular arrest of Th17 cells. Notably, selective α4β7 integrin blockade inhibited Th17 cell arrest without interfering with intravascular Th1 cell adhesion, suggesting that α4β7 integrin is predominantly involved in Th17 cell migration into the inflamed leptomeninges in EAE mice. Two-photon microscopy experiments showed that blockade of α4 integrin chain or α4β7 integrin selectively inhibited the locomotion of extravasated antigen-specific Th17 cells in the SAS, but had no effect on Th1 cell intratissue dynamics, further pointing to α4β7 integrin as key molecule in Th17 cell trafficking during EAE development. Finally, therapeutic inhibition of α4β7 integrin at disease onset by intrathecal injection of a blocking antibody attenuated clinical severity and reduced neuroinflammation, further demonstrating a crucial role for α4β7 integrin in driving Th17 cell-mediated disease pathogenesis. Altogether, our data suggest that a better knowledge of the molecular mechanisms controlling myelin-specific Th1 and Th17 cell trafficking during EAE delevopment may help to identify new therapeutic strategies for CNS inflammatory and demyelinating diseases.

Update on C-Cell Neuroendocrine Neoplasm: Prognostic and Predictive Histopathologic and Molecular Features of Medullary Thyroid Carcinoma.

Medullary thyroid carcinoma (MTC) is a C-cell-derived epithelial neuroendocrine neoplasm. With the exception of rare examples, most are well-differentiated epithelial neuroendocrine neoplasms (also known as neuroendocrine tumors in the taxonomy of the International Agency for Research on Cancer [IARC] of the World Health Organization [WHO]). This review provides an overview and recent evidence-based data on the molecular genetics, disease risk stratification based on clinicopathologic variables including molecular profiling and histopathologic variables, and targeted molecular therapies in patients with advanced MTC. While MTC is not the only neuroendocrine neoplasm in the thyroid gland, other neuroendocrine neoplasms in the thyroid include intrathyroidal thymic neuroendocrine neoplasms, intrathyroidal parathyroid neoplasms, and primary thyroid paragangliomas as well as metastatic neuroendocrine neoplasms. Therefore, the first responsibility of a pathologist is to distinguish MTC from other mimics using appropriate biomarkers. The second responsibility includes meticulous assessment of the status of angioinvasion (defined as tumor cells invading through a vessel wall and forming tumor-fibrin complexes, or intravascular tumor cells admixed with fibrin/thrombus), tumor necrosis, proliferative rate (mitotic count and Ki67 labeling index), and tumorgrade (low- or high-grade) along with the tumor stage and the resection margins. Given the morphologic and proliferative heterogeneity in these neoplasms, an exhaustive sampling is strongly recommended. Routine molecular testing for pathogenic germline RET variants is typically performed in all patients with a diagnosis of MTC; however, multifocal C-cell hyperplasia in association with at least a single focus of MTC and/or multifocal C-cell neoplasia are morphological harbingers of germline RET alterations. It is of interest to assess the status of pathogenic molecular alterations involving genes other than RET like theMET variants in MTC families with no pathogenic germline RET variants. Furthermore, the status of somatic RET alterations should be determined in all advanced/progressive or metastatic diseases, especially when selective RET inhibitor therapy (e.g., selpercatinib or pralsetinib) is considered. While the role of routine SSTR2/5 immunohistochemistry remains to be further clarified, evidence suggests that patients with somatostatin receptor (SSTR)-avid metastatic disease may also benefit from the option of 177Lu-DOTATATE peptide radionuclide receptor therapy. Finally, the authors of this review make a call to support the nomenclature change of MTC to C-cell neuroendocrine neoplasm to align this entity with the IARC/WHO taxonomy since MTCs represent epithelial neuroendocrine neoplasms of endoderm-derived C-cells.

Optimized workflow to modify microRNA expression in primary human intravascular cells

BackgroundA comprehensive dissection of the role of microRNAs (miRNAs) in gene regulation and subsequent cell functions requires a specific and efficient knockdown or overexpression of the miRNA of interest; these are achieved by transfecting the cell of interest with a miRNA inhibitor or a miRNA mimic, respectively. Inhibitors and mimics of miRNAs with a unique chemistry and/or structural modifications are available commercially and require different transfection conditions. Here, we aimed to investigate how various conditions affect the transfection efficacy of two miRNAs with high and low endogenous expression, miR-15a-5p and miR-20b-5p respectively, in human primary cells.ResultsMiRNA inhibitors and mimics from two commonly used commercial vendors were employed, i.e., mirVana (Thermo Fisher Scientific) and locked nucleic acid (LNA) miRNA (Qiagen). We systematically examined and optimized the transfection conditions of such miRNA inhibitors and mimics to primary endothelial cells and monocytes using either a lipid-based carrier (lipofectamine) for delivery or an unassisted uptake. Transfection of LNA inhibitors with either phosphodiester (PE)- or phosphorothioate (PS)-modified nucleotide bonds, delivered using a lipid-based carrier, efficiently downregulated the expression levels of miR-15a-5p already 24 h following transfection. MirVana miR-15a-5p inhibitor displayed a less efficient inhibitory effect, which was not improved 48 h following a single transfection or two consecutive transfections. Interestingly, LNA-PS miR-15a-5p inhibitor efficiently reduced the levels of miR-15a-5p when delivered without a lipid-based carrier in both ECs and monocytes. When using a carrier, mirVana and LNA miR-15a-5p and miR-20b-5p mimics showed similar efficiency 48 h following transfection to ECs and monocytes. None of the miRNA mimics effectively induced overexpression of the respective miRNA when given to primary cells without a carrier.ConclusionLNA miRNA inhibitors efficiently downregulated the cellular expression of miRNA, such as miR-15a-5p. Furthermore, our findings suggest that LNA-PS miRNA inhibitors can be delivered in the absence of a lipid-based carrier, whereas miRNA mimics need the aid of a lipid-based carrier to achieve sufficient cellular uptake.

Single-cell RNA sequencing of human lung innate lymphoid cells in the vascular and tissue niche reveals molecular features of tissue adaptation.

Innate lymphoid cells (ILCs) are sentinels of healthy organ function, yet it is unknown how ILCs adapt to distinct anatomical niches within tissues. Here, we used a unique humanized mouse model, MISTRG mice transplanted with human hematopoietic stem and progenitor cells (HSPCs), to define the gene signatures of human ILCs in the vascular versus the tissue (extravascular) compartment of the lung. Single-cell RNA sequencing in combination with intravascular cell labeling demonstrated that heterogeneous populations of human ILCs and natural killer (NK) cells occupied the vascular and tissue niches in the lung of HSPC-engrafted MISTRG mice. Moreover, we discovered that niche-specific cues shape the molecular programs of human ILCs in the distinct sub-anatomical compartments of the lung. Specifically, extravasation of ILCs into the lung tissue was associated with the upregulation of genes involved in the acquisition of tissue residency, cell positioning within the lung, sensing of tissue-derived signals, cellular stress responses, nutrient uptake, and interaction with other tissue-resident immune cells. We also defined a core tissue signature shared between human ILCs and NK cells in the extravascular space of the lung, consistent with imprinting by signals from the local microenvironment. The molecular characterization of human ILCs and NK cells in the vascular and tissue niches of the lung provides new knowledge on the mechanisms of ILC tissue adaptation and represents a resource for further studies.

Serum Sialyl Fibronectin Is an Indicator of Good Prognosis in Thyroid Cancer.

Sialyl-fibronectin (S-FN), a type of glycoprotein like Sialyl Lewisa and MAC1 thyroid cancer biomarkers, has been found to be expressed in thyroid cancer. In this study, we examined the usefulness of serum S-FN as a biomarker, as well as prognostic factor in various tumors including thyroid cancer. Using the MoAb JT-95, an ELISA kit was created and S-FN levels in sera (blood S-FN) of a total of 182 cases were investigated. Analysis included 63 cases of thyroid cancer, 33 cases of thyroid benign tumors, 7 cases of parathyroid benign tumors, and 79 cases of breast cancer. The incidence of blood S-FN-positive cases was 24 (38.0%) in 63 examined patients with thyroid cancer. Out of 40 examined benign neck tumor cases, 16/40 (40%) were S-FN-positive. Out of 79 examined breast cancer cases, 20/79 (25.3%) were S-FN-positive, with significant differences between thyroid and breast cancer cases (p=0.007). Regarding the association of blood S-FN expression and prognosis in thyroid cancer, there was a significant difference between 39 blood S-FN-negative cases and 15 recurrent or metastatic cases in terms of progression and pathological factors: tumor size, lymph node metastasis, extra-membrane infiltration, and clinical stage. All 63 assessed thyroid cancer cases also had a significant difference in these factors. There were no significant differences between these factors in the 24 blood S-FN-positive thyroid cancer cases. In cases of recurrent metastasis, intravascular cells were observed in all recurrent metastasis patients of both groups. Regarding staining of intravascular infiltrating cells, recurrent metastasis appeared at a higher rate in cases with S-FN-negative infiltrating cells. S-FN presence in blood can be used as an indicator of good prognosis in thyroid cancer patients.

Intravascular Large Cell non-Hodgkin Lymphoma in Subacute-Chronic Subdural Hematoma: a case report

Introduction: Intravascular lymphoma (IVL) is a rare subtype of non-Hodgkin extranodal lymphoma with characterized by a positive CD20 B cell proliferation in small lumens and medium-sized blood vessels, except veins and large arteries. Blood vessels in the central nervous system (CNS) and the skin is most often affected. These tumors usually occur in older adults and aggressive. Methodology: Descriptive study of the case report type, whose data were obtained from the patient’s medical record and has received the patient's consent to be published. Case presentation: This paper reports a 63-year-old man, who suffered Parkinson disease and neurological symptoms for 6 months. From imaging, there is a chronic subacute subdural hematoma in the right frontotemporoparietal area. Operation performed, histopathological examination of the tissue with Hematoxylin-Eosin staining and immunohistochemical examination confirmed diagnosis Intravascular Large Cell non-Hodgkin Lymphoma (IVLCL) type B-cell CD20 (+) in subacute-chronic subdural hematoma (SDH). Discussion: This tumor is characterized by selective growth of lymphoma cells within the lumen of blood vessels, particularly capillaries, and with the exception of the larger arteries and veins, occurred in adults, with a median age of 67 years. Sixty five percent show neurological symptoms with rapidly progressive cognitive dysfunction or subacute dementia. The cause of chronic SDH in this patient is unknown, but it can occur due to increasing age, brain atrophy, and enlargement of the subdural space, relatively minor trauma. Conclusion: This lymphoma is aggressive with a poor prognosis but a chemotherapy regimen with rituximab significantly improves the clinical outcome of this patient, with an overall survival of 3 years 60-81%.

A single-cell map of vascular and tissue lymphocytes identifies proliferative TCF-1+ human innate lymphoid cells.

Innate lymphoid cells (ILCs) play important roles in tissue homeostasis and host defense, but the proliferative properties and migratory behavior of especially human ILCs remain poorly understood. Here we mapped at single-cell resolution the spatial distribution of quiescent and proliferative human ILCs within the vascular versus tissue compartment. For this purpose, we employed MISTRG humanized mice as an in-vivo model to study human ILCs. We uncovered subset-specific differences in the proliferative status between vascular and tissue ILCs within lymphoid and non-lymphoid organs. We also identified CD117-CRTH2-CD45RA+ ILCs in the spleen that were highly proliferative and expressed the transcription factor TCF-1. These proliferative ILCs were present during the neonatal period in human blood and emerged early during population of the human ILC compartment in MISTRG mice transplanted with human hematopoietic stem and progenitor cells (HSPCs). Single-cell RNA-sequencing combined with intravascular cell labeling suggested that proliferative ILCs actively migrated from the local vasculature into the spleen tissue. Collectively, our comprehensive map reveals the proliferative topography of human ILCs, linking cell migration and spatial compartmentalization with cell division.

Time-domain ultrasound as prior information for frequency-domain compressive ultrasound for intravascular cell detection: A 2-cell numerical model

This study proposes a new method for the detection of a weak scatterer among strong scatterers using prior-information ultrasound (US) imaging. A perfect application of this approach is in vivo cell detection in the bloodstream, where red blood cells (RBCs) serve as identifiable strong scatterers. In vivo cell detection can help diagnose cancer at its earliest stages, increasing the chances of survival for patients. This work combines time-domain US with frequency-domain compressive US imaging to detect a 20-μ MCF-7 circulating tumor cell (CTC) among a number of RBCs within a simulated venule inside the mouth. The 2D image reconstructed from the time-domain US is employed to simulate the reflected and scattered pressure field from the RBCs, which is then measured at the location of the receivers. The RBCs are tagged one time by a human operator and another time, automatically, by template-based computer vision. Next, the resulting signal from the RBCs is subtracted from the measured total signal in frequency domain to generate the scattered-field data, coming from the CTC alone. Feeding that signal and the background pressure field into a norm-one-based compressive sensing code enables detecting the CTC at various locations. As errors could arise in determining the location of the RBCs and their acoustic properties in the real world, small errors (up to 10% in the former and 5% in the latter) are purposefully introduced to the model, to which the proposed method is shown to be resilient. Localization errors are smaller than 12 μ when a human tags the RBCs and smaller than 25 μ when computer vision is applied. Despite its limitations, this study, for the first time, reports the results of combining two US modalities aimed at cell detection and introduces a unique and useful application for ultrahigh-frequency US imaging. It should be noted that this method can be used in detecting weak scatterers with ultrasound waves in other applications as well.

WHOLE EXOME SEQUENCING DETERMINES PLAUSIBLE VARIANTS IN AN ORAL COMPOSITE HEMANGIOENDOTHELIOMA WITH APPARENT YAP1-MAML2 FUSION

<h3>Introduction</h3> Composite hemangioendothelioma (CHE) is considered a borderline malignant vascular tumor. Materials and Methods: 21-year-old female presented with painless left mandibular vestibular 1 cm mass of less than a year duration. H&E evaluation necessitated immunohistochemical studies that included CD31, CD34, D2-40, ERG, CAMTA1, and type IV collagen (COLIV). FISH was performed to investigate the presence of <i>YAP1</i> and <i>MAML2</i> aberrations and WES was performed to possibly identify causative gene variants. <h3>Results</h3> Non-encapsulated infiltrating tumor featured dilated vascular channels lined by cells exhibiting occasionally hobnail/matchstick-like arrangement. Intravascular cell proliferations and hyaline globules were also present. Lobular cell aggregates of spindle and epithelioid cells and slit-like spaces exhibiting a retiform or angiosarcomatous morphology were noted. Signet-ring and adipocyte-like cells were identified. Mitotic activity was exceptionally rare. Vascular spaces and the stroma featured lymphocytes and plasma cells. Neoplastic cells were positive for CD31, CD34, D2-40 and ERG, negative for CAMTA1 while COLIV highlighted the plasmalemma of most neoplastic cells, vessels and hyaline globules. FISH revealed inversion gaps supporting <i>YAP1MAML2</i> fusion. WES identified three missense mutations (a) <i>FLT1</i> [c.3046A>G; p.R1016G], (b) <i>PIK3CA</i> [c.3140A>T; p.H1047L], (c) <i>C11orf42</i> [c.910G>C; p.A304P], and one mitochondrial DNA frameshift insertion in <i>MT-ND4</i> [c.1107_1108insC; p.P370fs]. <h3>Conclusions</h3> 1. Histopathology and FISH support the diagnosis of CHE 2. FLT1 (VGFR1 precursor) and PIK3CA variants may be driver mutations. 3. Alternatively, PIK3CA may relate to tumor growth/enlargement 4. C11orf42 variant may be also tumorigenic 5. MT-ND4(NADH: Ubiquinone Oxidoreductase Core Subunit 4) variant possibly leads to altered reactive oxygen species hindering apoptosis.

Overview of the 2022 WHO Classification of Adrenal Cortical Tumors

The new WHO classification of adrenal cortical proliferations reflects translational advances in the fields of endocrine pathology, oncology and molecular biology. By adopting a question–answer framework, this review highlights advances in knowledge of histological features, ancillary studies, and associated genetic findings that increase the understanding of the adrenal cortex pathologies that are now reflected in the 2022 WHO classification. The pathological correlates of adrenal cortical proliferations include diffuse adrenal cortical hyperplasia, adrenal cortical nodular disease, adrenal cortical adenomas and adrenal cortical carcinomas. Understanding germline susceptibility and the clonal-neoplastic nature of individual adrenal cortical nodules in primary bilateral macronodular adrenal cortical disease, and recognition of the clonal-neoplastic nature of incidentally discovered non-functional subcentimeter benign adrenal cortical nodules has led to redefining the spectrum of adrenal cortical nodular disease. As a consequence, the most significant nomenclature change in the field of adrenal cortical pathology involves the refined classification of adrenal cortical nodular disease which now includes (a) sporadic nodular adrenocortical disease, (b) bilateral micronodular adrenal cortical disease, and (c) bilateral macronodular adrenal cortical disease (formerly known primary bilateral macronodular adrenal cortical hyperplasia). This group of clinicopathological entities are reflected in functional adrenal cortical pathologies. Aldosterone producing cortical lesions can be unifocal or multifocal, and may be bilateral with no imaging-detected nodule(s). Furthermore, not all grossly or radiologically identified adrenal cortical lesions may be the source of aldosterone excess. For this reason, the new WHO classification endorses the nomenclature of the HISTALDO classification which uses CYP11B2 immunohistochemistry to identify functional sites of aldosterone production to help predict the risk of bilateral disease in primary aldosteronism. Adrenal cortical carcinomas are subtyped based on their morphological features to include conventional, oncocytic, myxoid, and sarcomatoid subtypes. Although the classic histopathologic criteria for diagnosing adrenal cortical carcinomas have not changed, the 2022 WHO classification underscores the diagnostic and prognostic impact of angioinvasion (vascular invasion) in these tumors. Microscopic angioinvasion is defined as tumor cells invading through a vessel wall and forming a thrombus/fibrin-tumor complex or intravascular tumor cells admixed with platelet thrombus/fibrin. In addition to well-established Weiss and modified Weiss scoring systems, the new WHO classification also expands on the use of other multiparameter diagnostic algorithms (reticulin algorithm, Lin–Weiss–Bisceglia system, and Helsinki scoring system) to assist the workup of adrenal cortical neoplasms in adults. Accordingly, conventional carcinomas can be assessed using all multiparameter diagnostic schemes, whereas oncocytic neoplasms can be assessed using the Lin–Weiss–Bisceglia system, reticulin algorithm and Helsinki scoring system. Pediatric adrenal cortical neoplasms are assessed using the Wieneke system. Most adult adrenal cortical carcinomas show > 5 mitoses per 10 mm2 and > 5% Ki67. The 2022 WHO classification places an emphasis on an accurate assessment of tumor proliferation rate using both the mitotic count (mitoses per 10 mm2) and Ki67 labeling index which play an essential role in the dynamic risk stratification of affected patients. Low grade carcinomas have mitotic rate of ≤ 20 mitoses per 10 mm2, whereas high-grade carcinomas show > 20 mitoses per 10 mm2. Ki67-based tumor grading has not been endorsed in the new WHO classification, since the proliferation indices are continuous variables rather than being static thresholds in tumor biology. This new WHO classification emphasizes the role of diagnostic and predictive biomarkers in the workup of adrenal cortical neoplasms. Confirmation of the adrenal cortical origin of a tumor remains a critical requirement when dealing with non-functional lesions in the adrenal gland which may be mistaken for a primary adrenal cortical neoplasm. While SF1 is the most reliable biomarker in the confirmation of adrenal cortical origin, paranuclear IGF2 expression is a useful biomarker in the distinction of malignancy in adrenal cortical neoplasms. In addition to adrenal myelolipoma, the new classification of adrenal cortical tumors has introduced new sections including adrenal ectopia, based on the potential role of such ectopic tissue as a possible source of neoplastic proliferations as well as a potential mimicker of metastatic disease. Adrenal cysts are also discussed in the new classification as they may simulate primary cystic adrenal neoplasms or even adrenal cortical carcinomas in the setting of an adrenal pseudocyst.

Overview of the 2022 WHO Classification of Parathyroid Tumors.

The 2022 WHO classification reflects increases in the knowledge of the underlying pathogenesis of parathyroid disease. In addition to the classic characteristic features of parathyroid neoplasms, subtleties in histologic features which may indicate an underlying genetic abnormality reflect increased understanding of the clinical manifestations, histologic, and genetic correlation in parathyroid disease. The importance of underlying genetic aberrancies is emphasized due to their significance to the care of the patient. Traditionally, the term "parathyroid hyperplasia" has been applied to multiglandular parathyroid disease; however, the concept of hyperplasia is generally no longer supported in the context of primary hyperparathyroidism since affected glands are usually composed of multiple "clonal" neoplastic proliferations. In light of these findings and management implications for patient care, the 2022 WHO classification endorses primary hyperparathyroidism-related multiglandular parathyroid disease (multiglandular multiple parathyroid adenomas) as a germline susceptibility-driven multiglandular parathyroid neoplasia. From such a perspective, pathologists can provide additional value to genetic triaging by recognizing morphological and immunohistochemical harbingers of MEN1, CDKN1B, MAX, and CDC73-related manifestations. In the current WHO classification, the term "parathyroid hyperplasia" is now used primarily in the setting of secondary hyperplasia which is most often caused by chronic renal failure. In addition to expansion in the histological features, including those that may be suggestive of an underlying genetic abnormality, there are additional nomenclature changes in the 2022 WHO classification reflecting increased understanding of the underlying pathogenesis of parathyroid disease. The new classification no longer endorses the use of "atypical parathyroid adenoma". This entity is now being replaced with the term of "atypical parathyroid tumor" to reflect a parathyroid neoplasm of uncertain malignant potential. The differential diagnoses of atypical parathyroid tumor are discussed along with the details of worrisome clinical and laboratory findings, and also features that define atypical histological and immunohistochemical findings to qualify for this diagnosis. The histological definition of parathyroid carcinoma still requires one of the following findings: (i) angioinvasion (vascular invasion) characterized by tumor invading through a vessel wall and associated thrombus, or intravascular tumor cells admixed with thrombus, (ii) lymphatic invasion, (iii) perineural (intraneural) invasion, (iv) local malignant invasion into adjacent anatomic structures, or (v) histologically/cytologically documented metastatic disease. In parathyroid carcinomas, the documentation of mitotic activity (e.g., mitoses per 10mm2) and Ki67 labeling index is recommended. Furthermore, the importance of complete submission of parathyroidectomy specimens for microscopic examination, and the crucial role of multiple levels along with ancillary biomarkers have expanded the diagnostic workup of atypical parathyroid tumors and parathyroid carcinoma to ensure accurate characterization of parathyroid neoplasms. The concept of parafibromin deficiency has been expanded upon and term "parafibromin deficient parathyroid neoplasm" is applied to a parathyroid neoplasm showing complete absence of nuclear parafibromin immunoreactivity. Nucleolar loss is considered as abnormal finding that requires further molecular testing to confirm its biological significance. The 2022 WHO classification emphasizes the role of molecular immunohistochemistry in parathyroid disease. By adopting a question-answer framework, this review highlights advances in knowledge of histological features, ancillary studies, and associated genetic findings that increase the understanding of the underlying pathogenesis of parathyroid disease that are now reflected in the updated classification and new entities in the 2022 WHO classification.