Acta Histochemica Et Cytochemica最新文献

O-linked N-acetylglucosamine (O-GlcNAc) modification, known as O-GlcNAcylation, is a dynamic post-translational modification involving the addition of N-acetylglucosamine to serine or threonine residues. It has emerged as a critical regulator in diabetic pathophysiology. This review summarizes current research on the role of O-GlcNAcylation in hyperglycemia-induced cellular dysfunction, and focuses on vascular smooth muscle cells, renal cytoskeletal proteins, and diabetic complications in animal and human models. Studies reveal that hyperglycemia upregulates O-GlcNAc transferase activity, disrupting the interplay between glycosylation and phosphorylation, thereby impairing signaling pathways and exacerbating vascular proliferation and renal cytoskeletal disorganization. Notable findings include the imbalance of β-actin modifications in diabetic nephropathy, correlated with podocyte damage and glomerular abnormalities. By elucidating these mechanistic pathways, this review underscores the potential of O-GlcNAcylation as a biomarker and a therapeutic target. Future research should focus on tissue-specific effects and pharmacological strategies that mitigate diabetes-induced complications while preserving normal cellular functions.

This study assessed the effectiveness of a combination of platelet-rich plasma (PRP) and β-tricalcium phosphate/polylactic-co-glycolic acid (β-TCP/PLGA) fibers in the treatment of osteoporotic vertebral defects in rats. Seventy-two female Sprague-Dawley rats subjected to ovariectomy to induce osteoporosis were divided into three groups to receive different treatments for critical bone defects created in the lumbar vertebrae. The PRP group received β-TCP/PLGA fibers infused with PRP, the control group received no material, and the other group received the same fibers infused with phosphate-buffered saline (PBS). Over a period of 12 weeks, bone regeneration, macrophage differentiation, and inflammatory responses were evaluated histologically. Compared to the PBS group, the PRP-treated group demonstrated significantly enhanced early stage bone formation, increased expression of osteogenic markers, and a favorable shift in macrophage activity from the M1 inflammatory phenotype to the M2 healing phenotype. These outcomes suggest that the combination of PRP and β-TCP/PLGA fibers not only effectively promotes bone repair under osteoporotic conditions but also modulates the immune response to facilitate better healing, indicating its potential as a beneficial surgical intervention for osteoporotic vertebral fractures.

This study assessed the effectiveness of a combination of platelet-rich plasma (PRP) and β-tricalcium phosphate/polylactic-co-glycolic acid (β-TCP/PLGA) fibers in the treatment of osteoporotic vertebral defects in rats. Seventy-two female Sprague-Dawley rats subjected to ovariectomy to induce osteoporosis were divided into three groups to receive different treatments for critical bone defects created in the lumbar vertebrae. The PRP group received β-TCP/PLGA fibers infused with PRP, the control group received no material, and the other group received the same fibers infused with phosphate-buffered saline (PBS). Over a period of 12 weeks, bone regeneration, macrophage differentiation, and inflammatory responses were evaluated histologically. Compared to the PBS group, the PRP-treated group demonstrated significantly enhanced early stage bone formation, increased expression of osteogenic markers, and a favorable shift in macrophage activity from the M1 inflammatory phenotype to the M2 healing phenotype. These outcomes suggest that the combination of PRP and β-TCP/PLGA fibers not only effectively promotes bone repair under osteoporotic conditions but also modulates the immune response to facilitate better healing, indicating its potential as a beneficial surgical intervention for osteoporotic vertebral fractures.

The long non-coding RNA PVT1 reportedly forms a circular RNA variant (circPVT1). As circPVT1 is expressed in various cancers and has been implicated in promoting cancer cell proliferation and tumor progression, it is considered a potential biomarker and therapeutic target. We previously confirmed that circPVT1 expression varies according to the Gleason pattern, a morphological indicator of malignancy in prostate cancer. In this study, we assessed the expression of circPVT1 using BaseScope^TM assay with prostate cancer tissues and evaluated the correlation with the Grade Group (based on Gleason pattern), an indicator used to morphologically evaluate the degree of malignancy of prostate cancer. The relationship between circPVT1 expression and tumor proliferation was evaluated using cells in which circPVT1 expression was suppressed using the clustered regularly interspaced short palindromic repeats (CRISPR)/RfxCas13d system. BaseScope^TM assay confirmed that circPVT1 expression was significantly higher in Grade Group 2-5 (intermediate- and high-grade groups) than Grade Group 1 (low-grade group). In vitro experiments using the CRISPR/RfxCas13d system showed that specific suppression of circPVT1 expression resulted in a significant reduction in the number of prostate cancer cells. The results of this study suggest that circPVT1 is involved in tumor growth in prostate cancer and may serve as a therapeutic target for moderately and highly malignant prostate cancers that express circPVT1.

Conventional histopathological techniques, such as hematoxylin and eosin staining, are limited to 4-5 μm-thick tissue sections, restricting visualization to two-dimensional planes. Moreover, acquisition of three-dimensional horizontal images from the skin surface remains challenging, hindering precise assessment of tumor margins in skin lesions. This challenge is particularly pronounced in extramammary Paget's disease (EMPD), in which diffuse epidermal tumor cell spread complicates accurate evaluation of lesion extent. We hypothesized that combining horizontal sectioning with identification of individual tumor cells would enhance the determination of surgical margins. In this study, we developed a deep-imaging technique utilizing fluorescent solvatochromic dyes (LipiORDER^® and HistoBright^®) and two-photon microscopy to achieve high-resolution tumor margin visualization in EMPD. This technique enables identification of tumor cells in frozen and paraffin-embedded tissue blocks, as well as in live skin tissue under physiological conditions. Our novel approach holds substantial promise for improving the precision of surgical-margin assessment in EMPD and other cutaneous malignancies.

In the field of histochemistry and cytochemistry (histocytochemistry), fixation is a critical process for preserving biological structures and enabling accurate analysis. Fixation methods, broadly categorized into precipitating and cross-linking techniques, stabilize biomolecules such as proteins, sugars (carbohydrates) and nucleic acids, although lipids often require specific handling due to the loss during a routine procedure. Traditional staining methods have evolved into advanced techniques like immunohistochemistry (IHC) and in situ hybridization (ISH), which allow for precise analysis of the expression of specific molecules. IHC employs antibodies to visualize specific antigens, with fixation playing a vital role in maintaining antigen integrity. However, excessive fixation can mask epitopes, requiring antigen retrieval techniques to restore antigenicity. Microwave-induced retrieval, for instance, enhances staining efficacy while introducing further fixation by promoting molecular interactions. ISH, which targets nucleic acids with specific base sequences, is also sensitive to fixation conditions. Formaldehyde-based fixatives react variably with purines and pyrimidines, affecting hybridization efficiency with a probe. Positive controls like 28S rRNA help to standardize ISH across facilities, ensuring reproducible and reliable results. Variability in fixation protocols among institutions brings fatal defects in achieving consistent results. Shared standards or the use of robust controls can alleviate these issues, enhancing the accuracy and reliability of histocytochemical analyses for both research and clinical applications.

Ibrutinib is an oral irreversible Bruton's tyrosine kinase (BTK) inhibitor that blocks BTK activity by forming covalent bonds with the thiol group of cysteine in the ATP-binding pocket via Michael addition. However, it also reacts with a variety of off-target nonspecific proteins. In this study, we attempted to generate a specific antibody against ibrutinib and develop an immunohistochemical method to detect the ibrutinib-protein conjugates. Ibrutinib has the same amino group as the nucleobase adenine. Paraformaldehyde fixation could not fix it to the tissue via this amino group. However, ibrutinib covalently binds to proteins such as BTKs to exert its action and is therefore immobilized in tissue as ibrutinib-protein conjugates. Thus, immunohistochemistry for ibrutinib detects the location of the ibrutinib-protein conjugates, that is, the sites of covalently bound to the tissue via Michael addition. Using this immunohistochemical method, we visualized the ibrutinib-protein conjugates in the rat gastrointestinal tract (gastric body, duodenum, jejunum, ileum, and colon). This study is the first to elucidate the location of the ibrutinib-protein conjugates in the rat gastrointestinal tract and helps to clarify the mechanism of ibrutinib-induced toxicity.

Endoscopic ultrasound-guided fine-needle aspiration/biopsy (EUS-FNA/B) is critical for determining treatment strategies for patients with pancreatic cancer. However, conventional pathological examination using hematoxylin and eosin (H&E) staining is time-consuming. Microscopy with ultraviolet surface excitation (MUSE) enables rapid pathological diagnosis without requiring slide preparation. This study explores the potential of combining MUSE imaging with a cycle-consistent generative adversarial network (CycleGAN), an image generation algorithm capable of learning translations without paired images, to enhance diagnostic workflows for pancreatic EUS-FNA/B. Thirty-five pancreatic specimens were stained with Terbium/Hoechst 33342, and deep ultraviolet (DUV) fluorescence images were captured by exciting the tissue surface. These fluorescence images, along with H&E-stained formalin-fixed, paraffin-embedded (FFPE) sections from the same specimens, were divided into 256 × 256-pixel segments for CycleGAN training. The algorithm was employed to translate pseudo-H&E images from MUSE test images. The pseudo-H&E images generated by the CycleGAN showed improved inter-pathologist agreement among three pathologists compared with the original MUSE images. We established a technique to perform MUSE imaging on small pancreatic samples obtained through EUS-FNA/B and confirmed that H&E-style translation using CycleGAN simplified interpretation for pathologists. Integrating MUSE imaging with CycleGAN has the potential to offer a rapid, cost-effective, and accurate diagnostic tool.

Squamous cell carcinoma (SCC), a common malignancy affecting the skin, vagina, uterine cervix, anus, larynx, and upper digestive tract, is characterized by significant disruption of cell-cell adhesion in stratified squamous epithelium during tumorigenesis, progression, and metastasis. CALML5, a stratified epithelial-specific protein linked to desmosomal junctions, plays a key role in cell adhesion and is notably downregulated in human papillomavirus (HPV)-associated cervical SCC. Esophageal and pharyngeal cancers, commonly with a squamous cell phenotype, have distinct etiologies: oropharyngeal carcinoma is strongly associated with HPV, whereas esophageal carcinoma is linked to environmental factors such as smoking, alcohol, and diet. To investigate the role of CALML5 in these cancers, we performed immunohistochemical analyses on clinical samples and explored its regulatory mechanisms using in vitro studies with human esophageal SCC cell lines. Our findings revealed that CALML5 expression is suppressed in early-stage esophageal SCC but reactivated at invasive sites in well to moderately differentiated SCC undergoing keratinization. In specialized SCC with sarcomatoid component, CALML5 reactivation occurred alongside aberrant KLF4 expression, highlighting its context-dependent role in tumor progression. Conversely, while HPV-unrelated oropharyngeal SCC exhibited patterns similar to esophageal SCC, HPV-related oropharyngeal SCC consistently showed suppressed CALML5 expression due to impaired KLF4 nuclear translocation. These results suggest that CALML5 functions as a tumor suppressor in HPV-associated cervical SCC but may be reactivated in non-HPV-associated invasive SCC, emphasizing its complex role in SCC pathogenesis and the need for careful interpretation of its expression in clinical contexts.

Gastric cancer (GC), particularly the undifferentiated type, is frequently associated with peritoneal metastasis, which significantly worsens prognosis due to its resistance to conventional treatments. Photodynamic therapy (PDT) is localized treatment using a photosensitizer (PS) activated by light of a specific wavelength to generate cytotoxic reactive oxygen species that induce cell death. Severe adverse events were reported from clinical trials investigating PDT for peritoneal dissemination conducted until the early 2000s, leaving its safety and clinical effectiveness unestablished. The present study explored whether "non-cytotoxic" PDT using talaporfin sodium (TS) could enhance efficacy of chemotherapeutic agents in undifferentiated GC cell line HGC27. Cell viability was evaluated with MTT assay following TS-PDT, and the synergistic effect between non-cytotoxic TS-PDT and anticancer drug SN-38 was assessed. Changes in expression of drug resistance markers were analyzed through qRT-PCR, Western blotting, and immunocytochemistry. We found that non-cytotoxic TS-PDT enhanced the efficacy of chemotherapy in the undifferentiated GC cell line and reduced the expression of C-X-C chemokine receptor type 4, a key marker associated with GC stem-like properties. These findings highlight the potential of non-cytotoxic TS-PDT as a synergistic treatment approach. We conclude that non-cytotoxic TS-PDT could enhance drug sensitivity and offers a promising therapeutic strategy for GC.