Journal of Histochemistry & Cytochemistry最新文献

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.

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 N-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 C-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.

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.

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.

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.

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.

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 (p<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.

Vascular pericytes are widely present across the human body and crucial in regulating vascular flow, permeability, and homeostasis. In the human retina, pericytes are important for forming and maintaining the blood-retinal barrier, as well as for autoregulation of blood flow. Pericyte loss has been implicated in various pathological conditions. Visualization of pericytes by immunofluorescence (IF) staining provides valuable information on pericyte number, morphology, location, and on expression of anatomic and functional markers. However, species-specific differences in pericyte marker expression exist. In this study, we aimed to develop a novel IF co-staining protocol to detect the pericyte markers NG2, PDGFRβ, αSMA, CD13, and RFC1 in human retinal flatmounts. Unlike retinal sections, retinal flatmounts enable 3D visualization of pericyte distribution across the entire vascular network. Key optimizations included tailoring the fixation method, blocking buffer composition and antibody solvent, as well as using jasplakinolide to enhance αSMA detection. Our protocol successfully enabled double staining of NG2 and PDGFRβ, as well as αSMA and PDGFRβ, whereas CD13 and RFC1 expression was not detectable in human retinal flatmounts. This novel 3D IF protocol enhances in situ visualization of human retinal pericytes, enabling accurate studies of their role in vascular health and disease to aid targeted therapy development.

Pulmonary arterial hypertension (PAH) is a lethal condition where expansion of the vascular extracellular matrix contributes to increased pulmonary vascular resistance. Versican, a chondroitin sulfate proteoglycan, is known to accumulate in vascular lesions of PAH and hyaluronan and tenascin-C, binding partners of versican, are elevated in PAH. The specific distribution and localization of versican isoforms, their cleavage products, and binding partners in vascular lesions of PAH had not been studied previously. Versican has five distinct isoforms, V0-V4, identified by the arrangement of its chondroitin-sulfate attachment regions, GAGα and GAGβ. Here, tissue from idiopathic PAH was imaged with synchrotron-based phase-contrast micro-CT and analyzed by histology, immunohistochemistry, and in situ hybridization. Plasma concentration of versican in PAH patients and controls was measured using ELISA. GAGα- and GAGβ-containing isoforms were identified in pulmonary arteriopathy of all patients. However, immunohistochemical staining of N-terminal G1 domain (versican G1) and C-terminal G3 domain (versican G3) using specific antibodies did not consistently co-localize. Tenascin-C was occasionally found in neointima, but also in thin-walled collateral vessels. Hyaluronan accumulated in the neointima, co-localizing with both versican G3 and the neoepitope DPEAAE. DPEAAE did not co-localize with the corresponding neoepitope of the C-terminal fragment generated by cleavage, possibly indicating motility of fragments. Patient plasma had a higher concentration of versican G3-containing fragments, compared to controls. The distribution of versican isoforms, cleavage products, and binding partners demonstrated here warrants further investigation of their functional roles in PAH, versican G3 was reinforced as a potential biomarker for PAH.

The understanding of cardiomyopathies is hindered by a lack of quantitative histologic data. To address this methodical gap, we wrote a MATLAB-based image analysis platform to quantify nuclear and cellular disarray. We validated its utility in an animal model of tachycardiomyopathy (T-CM), whose ultrastructural remodeling processes have only partially been characterized and differ substantially from more prevalent cardiomyopathies. Six rabbits received right ventricular pacemaker implants. Three animals were paced incrementally up to 380 bpm for 30 days to induce T-CM. In three control rabbits, the pacemaker remained inactive (SHAM). Left ventricular tissue was collected, fixed in formalin, embedded in paraffin, stained, and digitized for nuclear morphometry, texture analysis, orientation analysis, and vascular architecture evaluation. Nuclear segmentation performed by the software was highly accurate, closely matching manual counts (mean manual nuclear count per slide = 81.3 ± 3.8, mean automated nuclear count per slide = 81.9 ± 4.3, r = 0.981, p<0.001). In T-CM, nuclei were enlarged [SHAM (a.u.) = 2362, T-CM (a.u.) = 2660, p=0.0042]. Texture patterns differed between the groups with higher nuclear contrast in T-CM [SHAM (a.u.) = 0.0169, T-CM (a.u.) = 0.0247, p=0.0149], highlighting structural remodeling at the nuclear level. Median vessel size increased in T-CM [SHAM (a.u.) = 1532, T-CM (a.u.) = 2421, p<0.0001]. In conclusion, our MATLAB-based image analysis platform allows high-throughput quantification of nuclear and extracellular disarray. It identified enlargement of nuclei and increased nuclear contrast as part of ultrastructural remodeling in tachycardiomyopathy.