Journal of Histochemistry & Cytochemistry最新文献

The TIE2 transmembrane receptor protein, known for its role in vascular stability and blood vessel remodeling, has primarily been studied in endothelial cells. This receptor has also been found on several non-endothelial cell types including podocytes, although its presence on podocytes remains a matter of debate. Conventional immunofluorescence approaches applied to membrane proteins are often challenged by the strong tissue autofluorescence spanning green and red parts of the electromagnetic spectrum, sample thickness, and the antibody specificity. Here, we used stimulated emission depletion (STED) microscopy, a super-resolution microscopy method, to detect the TIE2 protein in complex biological tissue with a resolution of less than 50 nm. To further confirm the presence of TIE2 on podocytes and to investigate its localization, we used fluorescence lifetime imaging microscopy (FLIM) to more effectively distinguish between nonspecific autofluorescence and specific emission in cleared and antibody-labeled tissue samples. By correlating these two techniques, we mapped the subcellular localization of TIE2 on mouse podocytes and confirmed its presence on the cell surface facing the Bowman's space, albeit at lower expression levels. This study highlights the potential complementary power of STED and FLIM methods and provides additional evidence that the TIE2 receptor is present on podocytes.

Interstitial cystitis/bladder pain syndrome (IC/BPS) is a heterogeneous condition of uncertain etiology. We assessed bladder collagen characteristics in phenotypically characterized IC/BPS patient subgroups that may influence pathophysiology. Forty-four females (30 IC/BPS; 14 non-IC/BPS) were included. Patients were divided into groups based on Hunner lesions (HL; N=14 or non-HL; N=16) and anesthetic bladder capacity (BC) (BC≤500 cc; low BC; N=17 or BC>500 cc; non-low BC; N=13). Bladder biopsy tissue slides were stained with hematoxylin and eosin or picrosirius red and semi-quantitatively analyzed by a pathologist. CT-FIRE software was used to quantitatively assess lamina propria collagen. All patients with IC/BPS had lower collagen fiber density independent of subgroup (p<0.0001-0.0038) compared to controls. HL had more peri-muscular collagen accumulation (p=0.0061), more acute inflammation (p=0.0364), more severe chronic inflammation (p=0.0088), and narrower collagen fibers than non-HL and controls. Non-low-BC patients had lower collagen density (p=0.0075) and straighter collagen fibers (p=0.0127) than low BC. Low-BC patients had narrower collagen fibers than control (p=0.0096). IC/BPS, regardless of subgroup, is associated with a bladder lamina propria with diminished collagen density. HL, non-low-BC, and low-BC subgroups have unique collagen characteristics. These findings suggest a collagen fiber destruction and redistribution process, which differs by subgroup, and may contribute to pathophysiology of IC/BPS.

Postmortem human brain tissue is an important resource for brain research. Spatial transcriptomics is a novel technology that utilizes formalin-fixed, paraffin-embedded (FFPE) or cryosections, with the former having good cytoarchitecture but poor RNA quality and vice versa for frozen tissue. Sheep brains (n=16) were used to test various protocols that simulate the conditions around the preparation and dissemination of human postmortem FFPE and frozen brain tissue to optimize for future spatial transcriptomic work. FFPE and frozen tissues were investigated for RNA quality, while hematoxylin and eosin-stained cryosections were analyzed by quantifying tissue voids as a proxy for cytoarchitectural integrity. Postmortem interval reduced the RNA integrity number equivalent (RIN^e) of frozen tissue from 7.2 (24 hr) to 4.8 (168 hr). In FFPE tissue, the percentage of RNA fragments greater than 200 nucleotides (DV200) values ranged from 18.9% to 69.01%, with the highest values observed in samples fixed for less than 24 hr. Pretreatment with liquid nitrogen before -80°C storage resulted in the lowest voids (11.6%) in cryosections, but cryoprotectants had little effect. These findings provide researchers with guidelines for tissue preparation in spatial transcriptomics. However, freezing protocols require further refinement to approach the cytoarchitecture of FFPE tissue.

Therapy-induced senescence (TIS) is a component of breast cancer (BC) treatment. Tetraspanins have emerging roles in cancer biology. Tetraspanin 4 (TSPAN4 [NAG2]) has been implicated in tumor progression, however, its association with TIS remains unexplored. We investigated TSPAN4 expression in BC samples from patients who received neoadjuvant chemotherapy (NAC) and its association with TIS markers. Thirty-eight paired pre- and post-NAC BC samples were analyzed using immunohistochemistry (IHC) staining for TSPAN4 and TIS-associated biomarkers (Lamin B1 and Ki67). Pairwise analysis of senescence-related gene expression (LMNB1, MKI67, CDKN1A, ATM, IGFBP7, MMP2, CXCL14, and CCL5) was performed in an independent geneset of 68 paired pre- and post-NAC BC patient samples. NAC reduced the expression of senescence-associated proliferation markers Ki67 and Lamin B1 in BC samples, with 84% and 76% of patients showing decreased expression, respectively (p<0.001). Senescence-associated gene expression analysis revealed consistent upregulation of CDKN1A, ATM, IGFBP7, MMP2, CXCL14, and CCL5 post-NAC (p<0.001), while LMNB1 and MKI67 were significantly downregulated (p<0.0001 and p=0.007, respectively). A subset (15/38; 39%) of samples demonstrated upregulation of the TSPAN4 expression post-NAC (p<0.01). NAG2 was upregulated in 54/68 patients post-NAC (p<0.00001) and its expression correlated positively with senescence-associated genes. An association between TSPAN4 and TIS post-NAC was identified.

Catheter-directed thrombolysis for acute pulmonary thromboembolism (PTE) is associated with a reduction in mortality, although it also carries an increased risk of bleeding. This study aimed to visualize the extent of fibrinolysis within pulmonary artery thrombi using rare specimens obtained via non-obstructive general angioscopy (NOGA). Under direct visualization with NOGA, blood samples were collected from the site of pulmonary artery thrombi and analyzed histologically and immunohistochemically. Fibrinolysis was assessed using the fibrin network index (FNI). The FNI was significantly higher in the Monteplase-treated group than in the untreated group, clearly visualizing the enhanced fibrinolysis induced by Monteplase. In a case where Monteplase was administered in advance for the treatment of deep vein thrombosis (DVT), a marked increase in D-dimer levels was observed; however, the FNI remained low. This suggests that the thrombus may have partially dissolved at the DVT stage, limiting the efficacy of Monteplase by the time the embolus reached the pulmonary artery. The presence of neutrophil extracellular traps (NETs) in association with fibrin was confirmed by immunohistochemical analysis. The influence of NETs in the progression from DVT to PTE was suggested.

This study aimed to develop simple and cytochemical detection methods for identifying macrophages that engulf cholesterol crystals (CCs) and for visualizing the granule enzyme myeloperoxidase (MPO) in putative neutrophil extracellular traps (NETs). Specimens were collected from blood-derived debris using filter paper and non-obstructive general angioscopy (NOGA), and analyzed using various staining techniques. Cytochemical staining methods included supra-vital Sternheimer staining, nonspecific esterase staining, and immunocytochemistry, which effectively detected macrophages actively phagocytosing CCs. MPO was visualized using the diaminobenzidine (DAB) method, which demonstrated MPO-positive NETs adhering to fibrin cores, as well as released and dispersed MPO granules. The diameter of MPO-positive granules ranged from 0.1668 to 0.6502 µm. These findings suggest that simple, rapid, and accessible cytochemical techniques are useful for elucidating the pathophysiology of atherosclerotic lesions, particularly in the context of innate immune responses.

Pancreatic ductal adenocarcinoma (PDAC) is one of the leading causes of cancer-related death worldwide, with a mortality rate almost equal to its incidence, highlighting the urgent need for novel biomarkers and therapeutic targets. This pilot study aims to investigate the potential value of histamine H₃ receptor (H₃R) as a prognostic biomarker for this lethal disease. We analyzed the H₃R expression in PDAC using the RNA sequencing data set from The Cancer Genome Atlas (Pan-Cancer Atlas). In addition, H₃R protein levels were evaluated by immunohistochemistry in 27 PDAC samples and compared with adjacent preneoplastic pancreatic tissue of the same patient, and with 10 non-related healthy pancreatic tissues. This preliminary study shows that the H₃R is barely expressed in healthy tissue. Interestingly, H₃R was detected in 96% of PDAC samples, and its expression in tumoral tissue was significantly higher when compared with its expression in preneoplastic tissue, and it was associated with a better prognosis in terms of overall survival. Present findings suggest that H₃R may serve as a potential prognostic biomarker in PDAC. Future research aimed at elucidating the role of H₃R in PDAC biology and its prognostic value in larger patient cohorts is warranted.

Transmission electron microscopy (TEM) is a key tool for the ultrastructural analysis of biological samples; however, it requires optimized fixation and contrast enhancement methods to achieve accurate results. Here, we evaluated the use of tannic acid as a mordant during primary fixation for TEM processing of Giardia intestinalis and Trichomonas vaginalis. When combined with osmium tetroxide (OsO₄) post-fixation, tannic acid significantly improved in-block contrast in plasma membranes, organelle boundaries, and cytoskeletal elements, while preserving structural integrity. It increased electron density without introducing artifacts and, in some cases, allowed the omission of lead citrate staining, simplifying the protocol and reducing exposure to toxic agents. Even in the absence of OsO₄, samples processed with tannic acid retained sufficient contrast to visualize basal bodies, axonemes, and other cytoskeletal filaments. Moreover, tannic acid enhanced the visualization of poorly characterized structures in the transition zone. We also demonstrate its successful use as a post-staining agent, replacing uranyl acetate for ultrathin sections while maintaining high image quality. These findings support tannic acid as a safe, cost-effective, and efficient alternative to traditional contrast agents, particularly under biosafety constraints, and contribute to the improvement of TEM protocols for studying protozoan morphology and cell biology.

This preliminary study explores the feasibility of identifying novel site-specific biomarkers in keloid disease to enhance understanding of its pathobiology. Keloid scars are clinically and morphologically heterogeneous, showing variable response to therapy. They also differ at the cellular and molecular levels between their actively growing margins and their dormant centers. In addition, keloids behave differently to other fibrous skin tumors, including DSFP and FS. Thus, we performed a high-throughput RNA sequencing and gene/protein analysis on keloid tissue, primary keloid fibroblasts, and keloid-derived immortalized fibroblast cell lines from different sites of the keloid tissue (Extralesional, Peripheral, Middle, and Top). These were compared with normal skin, DFSP, and FS. We identified MTCO1P12 as a common gene transcript exhibiting significantly high expression across all three keloid sites (Peripheral, Middle, and Top), FS, and DFSP compared to the extralesional keloid. Furthermore, three site-specific biomarkers were identified. SLITRK1 was uniquely expressed in the peripheral keloid tissue site and its corresponding fibroblasts. FOXS1 was localized to the middle keloid tissue site and its corresponding fibroblasts. KCNJ6 was exclusively expressed in the top keloid tissue site and its corresponding fibroblasts. It was not found in FS and DFSP. In conclusion, for the first time, we identified and validated three novel site-specific biomarkers within keloid, two of which (SLITRK1 and FOXS1) overlap with more aggressive tumors, while KCNJ6 is unique to keloids. In conclusion, for the first time, we identified and validated three novel site-specific biomarkers in keloids, two of which (SLITRK1 and FOXS1) overlap with more aggressive tumors, while KCNJ6 is unique to keloids. These findings demonstrate the feasibility of identifying spatially distinct molecular signatures in keloids, providing a foundation for future research into targeted therapies.

Type 1 diabetes is an autoimmune disease that leads to beta cell death. To test whether beta cell defects precede diagnosis, the expression of pCREB was surveyed in human islet cells. pCREB is a transcription factor produced by islet cells that regulates the expression of islet cell-specific genes. This analysis indicated that while islet cells of control donors displayed CREB/pCREB in the nucleus of alpha and beta cells, the transcription factor was also found in the cytoplasm of islet cells of normoglycemic GADA donors, donors with two antibodies and of those recently diagnosed. The translocation of CREB/pCREB, which decreases its activity, was correlated with reduced or absent expression of insulin and a protease. These changes suggest an alteration in protein homeostasis. The cytoplasmic localization of CREB/pCREB was transient, since the transcription factor moved to the nuclei of insulin cells of donors with longer standing disease. The fact that altered proteostasis leads to autoinflammation suggests that interventions at an initial stage of the disease, when protein homeostasis could be restored, may prevent the progress of the disease.