Journal of Histochemistry & Cytochemistry最新文献

Vesicular monoamine transporter 2 (VMAT2) is an integral membrane protein that packages monoamines, including dopamine, norepinephrine, serotonin, and histamine, into synaptic vesicles. VMAT2 is normally expressed in sympathetic, enteric, and central nervous system neurons and in neuroendocrine cells. Although VMAT2 immunoreactivity was originally reported in a wide range of neuroendocrine neoplasms (NENs), its diagnostic value has been largely overlooked in recent practice due to reliance on established markers such as chromogranin A (CgA), synaptophysin (SYP), and the transcription factors ISLET1 and INSM1. To reassess the relevance of VMAT2, we evaluated its expression relative to CgA, SYP, ISLET1, and INSM1 in 60 consecutively diagnosed NENs from diverse anatomic sites and tumor grades. VMAT2 was positive in 37 of 39 (95%) well-differentiated neuroendocrine tumors (NETs; grades 1-3), in all adrenal paragangliomas (7 of 7) and in one medullary thyroid carcinoma, showing sensitivity comparable to CgA and INSM1 and slightly lower than SYP. VMAT2 was detected in 7 of 12 (58%) neuroendocrine carcinomas. Sensitivity was highest in small-intestinal and gastric NETs and more variable in pancreatic NETs. These results support VMAT2 as a robust, differentiation-linked neuroendocrine marker whose inclusion in modern immunohistochemical panels may improve diagnostic accuracy.

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.

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.

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.

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.

SummaryThis study examines the involvement of T cell autocrine hyaluronan (HA) with the immunological synapse (IS) that mediates T cell activation by antigen-presenting cells (APCs). Three-dimensional (3D) confocal images of mouse CD4⁺ T cells interacting with B cell lymphoma (A20) APCs in vitro showed HA to be primarily on the T cell side of the IS, appearing as a compact mass indenting the T cell nucleus. Similar 3D imaging of CD4⁺ T cells forming a pseudo-IS on anti-CD3 antibody-coated glass showed HA in the vicinity of the IS in dense masses or complex, arched, columnar structures. Affinity/immunofluorescence labeling studies confirmed the HA masses and columns were cytoplasmic, located beneath the cortical actin layer but outside the nucleus. In T cells forming a pseudo-IS, the HA-binding protein RHAMM was localized to cortical cytoplasm and had limited spatial overlap with cytoplasmic HA. Pre-exposure of T cells to the HA synthesis inhibitor 4-methylumbelliferone (4-MU) or to the HA-binding peptide Pep-1 inhibited IS formation with A20 APCs. Moreover, actin ring development in T cell pseudo-IS was inhibited by pre-exposure to 4-MU, but not by pre-exposure to Pep-1. Collectively, our previous and present studies suggest a complex role for cell surface and cytoplasmic T cell autocrine HA in IS formation and T cell receptor signaling.

SummaryThis study aimed to verify the effect of peroxiredoxin 1 (PRDX1) in wound healing. PRDX1 and tumor necrosis factor receptor-associated factor 4 (TRAF4) expressions in human umbilical vein endothelial cells (HUVECs) were assessed by real-time quantitative PCR and Western blot. Cell proliferation, migration, and angiogenesis were assessed by cell counting kit-8, wound healing, and tube formation assay. The reactive oxygen species level was measured using 2'-7'-dichlorodihydrofluorescein diacetate. Senescence-associated β-galactosidase staining assay was used to detect cells senescence. The relationship among PRDX1, TRAF4, and UBE3A was determined by co-immunoprecipitation. Upregulation of PRDX1 promoted the proliferation, migration, and angiogenesis of HUVECs. Meanwhile, PRDX1 overexpression inhibited oxidative stress and senescence of HUVECs by H₂O₂-induced. Furthermore, overexpression of PRDX1 inhibited the degradation of TRAF4 to activate PI3K/AKT/VEGF axis via binding to UBE3A. The effect of PRDX1 on H₂O₂-induced oxidative stress and senescence was reversed by TRAF4 silence. The promotion of PRDX1 on proliferation, migration, and angiogenesis was canceled by knockdown of TRAF4. PRDX1 inhibited oxidative stress and senescence via restraining the degradation of TRAF4 by binding to UBE3A, eventually accelerating wound healing.