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.

Therapy-induced senescence (TIS) is a component of breast cancer (BC) treatment. Tetraspanins have emerging roles in cancer biology. Tetraspanin 4 (TSPAN4 []) 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 (, and 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 (<0.001). Senescence-associated gene expression analysis revealed consistent upregulation of , and post-NAC (<0.001), while and were significantly downregulated ( and =0.007, respectively). A subset (15/38; 39%) of samples demonstrated upregulation of the TSPAN4 expression post-NAC (<0.01). was upregulated in 54/68 patients post-NAC (<0.00001) and its expression correlated positively with senescence-associated genes. An association between TSPAN4 and TIS post-NAC was identified.

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 and . When combined with osmium tetroxide (OsO) 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 OsO, 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.

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.

The neuroendocrine protein 7B2 plays a crucial role in the maturation and activity regulation of prohormone convertase 2 (PC2). To elucidate the relationship between 7B2 and PC2 expression in endocrine tissues, we generated synthetic peptide antibodies in guinea pigs. The antigenic peptide sequences were selected to correspond to three different positions in the rat amino acid (aa) sequence: The -terminal aa 1-14 is situated immediately following the signal sequence, the middle aa 77-90 contains a part of the proPC2 activation domain, and the -terminal aa 156-168 functions to suppress PC2 activity. These antibodies demonstrated specific reactivity across a diverse array of animal species. The reactivity of these antibodies differed, suggesting that the molecular form of 7B2 differs depending on the endocrine cell, and a different expression pattern was demonstrated in rat and dog pituitary intermediate cells. The colocalization of 7B2 and PC2 in prolactin (PRL) granules in rat pituitary mammotrophs supports the interaction between these proteins. However, the expression intensities of these proteins did not correspond, and epitope-related disparities were detected. These results may be indicative of alterations in the molecular state associated with the dynamics of the interaction between 7B2 and PC2.

The Vibratome, or vibrating microtome allows sectioning of the mouse brain with a reliably established thickness from 50 µm, but they have a strong limitation on the size of the sample suitable for cutting. Herein is described the construction with publicly available element base (from parts for a 3D printer) a so-called 3D-vibrating microtome capable of cutting larger size brain into thin sections, and use it to investigate the limit-attainable values of the minimum slice thickness. Both of these goals have been successfully achieved. Both small mouse slices with a minimum thickness of 30 μm and large whole calf brain slices with a minimum thickness of 150 μm were obtained. Critical features of the effect of blade vibration frequency and sample feed rate on the minimum slice thickness were revealed, In the same way, a clear correlation was established between the minimum achievable thickness and its area.

SummaryOrchiectomy induces atrophy of epithelial cells in the prostate gland, stimulated by androgen deprivation. The objective of the current study was to assess the changes arising from tissue remodeling during short periods of androgen deprivation in the ventral prostate of Mongolian gerbils. Adult male gerbils were divided into groups: control and castrated, euthanized on the 3rd (Ca3d), 7th (Ca7d), and 14th (Ca14d) days post-orchiectomy (n = 7/group). The ventral lobe of the prostate was submitted for histological, stereological, morphometric, serological, and immunohistochemical analyses. Castration promoted a reduction in the weight of the ventral prostate. It altered the relative proportion of prostate tissue compartments, such as a decrease in the epithelium and non-muscular stroma and an increase in the muscular stroma. In addition, we observed that the number of Cd-68 positive cells increased in the Ca3d group, which represents a period of androgen deprivation not previously reported in the literature for Mongolian gerbils. Matrix metalloproteinase-9 quantification revealed a decrease in the Ca7d group compared to the Ca3d. In addition, the frequency of Tumor Necrosis Factor alpha increased in the Ca14d group, influenced by the duration of testosterone deficiency. The findings contribute to the understanding of prostate tissue remodeling after castration, as well as highlighting the rapid alterations in the prostate microenvironment in a short period following castration.

Macrophages have multiple roles in the heart including immune surveillance and extracellular matrix remodeling. Aging increases both collagen deposition and macrophage number in the heart; however, rodent models used to study cardiac macrophages have age-related comorbidities such as atherosclerosis and hypertension. The Fischer 344 rat does not develop these conditions with aging; therefore, the purpose of this study was to evaluate macrophage number and polarization in the hearts of aged (24-month) and young (6-month) Fischer 344 rats. Paraffin-embedded hearts were assessed for collagen deposition and immunolabeled for CD68, CD163, CD206, and galectin-3. Compared with young rats, significantly greater collagen deposition was observed in the old rats. There were no significant differences in CD68 or CD163 cells between age groups, but both CD206 and galectin-3 cells were more numerous in the aged animals. Double-immunofluorescence studies demonstrated that galectin-3 colocalized with both CD68 and CD163, suggesting that galectin-3 is found in cardiac macrophages. Further colocalization studies demonstrated similar proportions of CD68/CD163, CD68/CD163, and CD68/CD163 cells between age groups, suggesting that aging does not affect macrophage polarization. As CD206 and galectin-3 cells promote fibrosis, these results warrant future studies that delineate the specific roles of these cells in the aged heart.

Gold standard for immunohistochemical analyses is the manual assessment by two specialist pathologists. This process is time-consuming, highly dependent on the respective evaluator and often difficult to reproduce. The use of image analysis software, such as ImageJ, QuPath, or CellProfiler, which employ machine learning and/or deep learning mechanisms to perform biomarker analyses, offers a potential solution to these problems. The objective of our study is to evaluate whether digital assessment using the open-source software QuPath is comparable to manual evaluation and to examine the inter-evaluator variability between the two manual evaluators and two software-based evaluations. Six tissue microarrays (TMAs) were constructed for a cohort of 309 patients with primary oral squamous cell carcinoma (OSCC). The tumor tissue and corresponding non-lesional squamous epithelial mucosa specimen were immunohistochemically stained for the biomarkers Ki67, as a nuclear marker; the epidermal growth factor receptor (EGF-R), as a membranous marker; and the major histocompatibility complex class I (MHC-I) heavy chain (HC) expressed on the membrane and in the cytoplasm. The staining pattern was analyzed by two experienced, independent manual evaluators and by QuPath. The percentage of positive cells, for Ki67, and the histoscore (H-score) based on the percentage of positive cells and their staining intensity, for EGF-R and MHC-I, were determined as final values. The results yielded high to excellent spearman correlation coefficients for all three biomarkers (<0.001) in lesional and non-lesional tissues. The Bland-Altman plots demonstrated a high degree of agreement between manual and software-based analysis, as well as inter-evaluator variability demonstrating a high comparability of the evaluation methods. However, a prerequisite for a proper software-based analysis is an accurate, time-consuming annotation of the single specimen, which requires users with a comprehensive understanding of histology and extensive training in QuPath. Once these requirements are met, the software-based analysis offers advantages for large-scale biomarker studies due to objective and reproducible comparability of the stainings leading to a greater accuracy as well as the reuse of established conditions across similar analyses without requiring further operator input.

Immunocytochemistry (IHC) and immunofluorescence (IF) offer crucial diagnostic insights in clinical settings. Most IF assays are performed using fluorophore-conjugated antibodies, but these fluorophores can be subject to issues, such as photobleaching and autofluorescence, that result in lower signal-to-noise ratios (SNR). Gold nanoparticles provide greater signal stability and scatter light well, making them easily separable from the background of biological tissues and improving SNR. In this study, we sought to determine the labeling efficiency, signal quality, and image artifacts of 2.2, 10, and 40 nm diameter gold nanoparticle probes conjugated to antibodies in IF applications on fixed, permeabilized cells in comparison to traditional fluorophores. Overall, micrographs of nanoparticle labels had higher SNR due to lower background signal, and punctate appearances as compared with the continuously distributed signal of the immunofluorescent label. Signal-to-noise ratios varied with nanoparticle diameter, and signal fidelity was worse for keratin versus epithelial growth factor receptor (EGFR). Labeling of EGFR was successful using both extracellular and intracellular epitopes, while poor labeling of keratin 19 with 10 nm diameter nanoparticles was improved by pretreatment with heat and sonication, suggesting hindrance of nanoparticle labels within the fixed, permeabilized cell.

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 HO-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 HO-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.

Prostate cancer (PC) is the second leading cause of cancer-related death in men worldwide. Its progression is marked by significant phenotypic changes in stromal cells and alterations in extracellular matrix (ECM) composition, including variations in collagen fibers, proteoglycans (PGs), and glycosaminoglycans. Tumor cells also modulate ECM secretion through adenosine A3 receptor (A3AR) activity. This study aimed to evaluate stromal cells, ECM components, and A3AR expression in prostate biopsies and explore their association with clinicopathological variables. We analyzed tissue samples from 96 patients diagnosed with PC or benign prostatic hyperplasia (BPH), using immunohistochemistry for alpha-smooth muscle actin and A3AR, and histochemical methods for ECM components. PC samples showed reduced stromal cell content and increased PGs, collagen fibers, and A3AR levels compared with BPH. While we found associations with histological classification, no significant correlations were observed with preoperative prostate-specific antigen levels or clinical risk categories. Our findings suggest that ECM components and A3AR may be involved in PCr progression and hold potential as biomarkers. However, due to the limited number of high-grade cases, further studies are needed to confirm these associations.

Chemokine-like receptor 1 (CMKLR-1) is a G-protein-coupled receptor that functions as the binding site of chemerin. It induces chemotaxis in various immune cells, and it has recently been linked to vascular remodeling in inflammation. In this study, we investigated CMKLR-1 expression in human aorta samples, focusing on its distribution across different cell types and its potential association with clinical and histomorphological data, particularly concerning aortic dissection and aneurysms. We analyzed 62 aorta samples taken from patients undergoing dissection surgery (n = 27) or aneurysm/dilatation (n = 35) using immunohistochemical CMKLR-1 staining. CMKLR-1 expression was primarily observed in the smooth muscle cells (SMCs) of the aortic media layer, and band-like coloring appeared in the unstained center section. CMKLR-1 positivity in the inner and outer parts of the media layer was observed in only a few cases. One-third of the vasa vasorum exhibited staining. Staining in lymphocytes, macrophages, and endothelia was rare. No significant differences in CMKLR-1 expression were found between the dissection and aneurysm cases, and the clinical or histomorphological data. Although CMKLR-1 expression did not distinguish between the studied conditions, its presence in the aortic media, especially in SMCs, is noteworthy.

Glioblastoma is the most frequent and aggressive primary brain cancer in adults, and the prognosis is poor. The neurotrophic factor glial cell-derived neurotrophic factor (GDNF) and its receptors, which are involved in neuronal development, have in experimental studies been suggested to drive tumorigenic processes in glioblastoma, but the role and expression in glioblastoma in patients is under-investigated. The aim of this study was to investigate the expression of GDNF, GDNF family receptor 1-4 (GFRA1-4), and the downstream REarranged during Transfection (RET) receptor in human glioblastoma tissue by RNA in situ hybridization, immunohistochemistry, and immunofluorescence. Expression was quantified by software-based classifiers. The results showed that GDNF was expressed in approximately 10% of tumor cells. The GFRA1 receptor was widely expressed in tumor cells, often colocalizing with the astrocytic tumor cell marker glial fibrillary acidic protein (GFAP), and in a smaller fraction of tumor cells expressing the stem cell markers oligodendrocyte transcription factor 2 (OLIG2) and SRY-Box Transcription Factor 2 (SOX2). The GFRA2 receptor expression was very limited, whereas expression of GFRA3, GFRA4, and RET, respectively, was almost absent. In conclusion, GDNF and its primary receptor GFRA1 were expressed in patient glioblastoma tissue. Potential clinical value needs further investigation.

SummaryThe Wilms' Tumor protein WT1 is a zinc-finger transcription factor with crucial roles in organogenesis, cell differentiation, tissue homeostasis, and oncogenesis. While its expression has been extensively studied in various tissues, its presence in the nervous system, particularly in peripheral glial cells, remains largely unexplored. In this study, we examined WT1 expression in the Schwann cells of mechanosensory corpuscles, nerve bundles, and free nerve endings (FNEs) within human penile tissues. Using single and double immunohistology, we analyzed WT1 coexpression with Schwann cell markers (S100, nestin, SOX10) and its association with axonal (neurofilaments, neuron-specific enolase, tyrosine hydroxylase) and perineurial/endoneurial markers (Glut-1, α-SMA, CD34). We found consistent WT1 cytoplasmic expression in the Schwann cells of Pacinian, Meissner, Krause, genital, Golgi-Mazzoni, and Ruffini-like corpuscles, with variable staining intensity. Confocal microscopy revealed WT1 colocalized with nestin but not S100, suggesting involvement in cytoskeletal organization. In addition, we documented WT1 in myelinating Schwann cells of nerve bundles, with distinct staining patterns in Cajal bands and Schmidt-Lanterman incisures, as well as in non-myelinating Schwann cells of FNEs. This is the first study to describe WT1 expression in sensory corpuscles, implicating it in Schwann cell development, maintenance, or plasticity, with potential relevance for peripheral nerve biology, pathology, and mechanosensation.

Alendronate (ALN), a nitrogen-containing bisphosphonate, is widely used to treat bone disorders. While its inhibitory effect on osteoclast activity is well-established, its impact on the release of extracellular vesicles (EVs) is less understood. This study investigated the effect of ALN on the quantity and size distribution of EVs released by osteoclasts cultured on bovine bone slices pretreated with 10-µM ALN, 100-µM ALN, or vehicle. Raw 264.7 cells were differentiated into osteoclasts using RANK-ligand, and EVs were isolated from conditioned media. Tartrate-resistant acid phosphatase (TRAP) staining, phalloidin staining for actin rings, and nanoparticle tracking analysis (NTA) were performed. TRAP staining showed a significant reduction in the number of TRAP-positive multinucleated cells in the 100-µM ALN group, confirming that high-concentration ALN also impairs osteoclast formation. Phalloidin staining showed a significant decrease in actin ring formation in the 100-µM ALN group, confirming ALN's inhibitory effect on osteoclast activity. NTA revealed a lower total EV concentration in the 100-µM ALN group, with a distinct peak of smaller EVs (<100 nm), suggestive of exosomes. These findings indicate that ALN, especially at higher concentrations, alters the release profile of osteoclast-derived EVs, potentially affecting intercellular communication and bone remodeling beyond its direct inhibition of resorption.

PC12 cells are a cell line originating from rat adrenal medullary chromaffin (AMC) cells. They extend a neurite-like structure in response to nerve growth factor (NGF). Thus, unstimulated and NGF-stimulated PC12 cells are used as models for AMC cells and sympathetic ganglion cells, respectively. However, how closely unstimulated and stimulated PC12 cells resemble AMC cells and sympathetic neurons, respectively, has not been elucidated sufficiently. We explored these issues by using biochemical and immunocytochemical methods. AMC cells and PC12 cells selectively expressed uncoupling protein 3 (UCP3) and uncoupling protein 4 (UCP4), respectively, and glucocorticoid activity inhibited UCP4 expression in PC12 cells. PC12 cells expressed extremely low levels of chromaffin granule-associated proteins, whereas the amount of synaptophysin, a synaptic vesicle-associated protein, was much higher than that in the adrenal medulla. Similar to AMC cells, the muscarinic receptor type 1 was located at the cell periphery in unstimulated PC12 cells, and its expression was markedly enhanced by NGF. Furthermore, NGF stimulation abolished the expression of GABA signaling molecules in PC12 cells. The results suggest that the properties of unstimulated PC12 cells are between those of AMC cells and sympathetic ganglion cells and GABA signaling is intrinsic to AMC cells.

Probably the most sensitive and frequently performed stain for calcium deposits in mammalian tissues is the von Kossa, a stain used for over a century. When originally incorporated into daily use in the histopathology laboratory on paraffin tissue sections, the procedure called for immersion of tissue sections in an aqueous 5% silver nitrate solution and placed in direct sunlight for 20-30 min to make calcium deposits appear black. Although this effective method continues to be used in select locations, the modern laboratory often does not have access to bright sunlight. This study revisited the traditional von Kossa staining method with alternatives in light sources, silver nitrate concentrations, and timing of silver solution exposure and determined that a 1% silver nitrate solution is equally effective as a 5% solution in bright sunlight for 30-60 min. In addition, a 1% silver nitrate solution can be successfully used with exposure to a 3.5W light-emitting diode (LED) daylight lamp for 1 hr, sunlight through a tinted glass window for 1 hr, or overhead room lighting for 2 hr, and the staining reaction is unaffected by temperatures ranging from 1C to 40C.