Journal of Histochemistry & Cytochemistry最新文献

Formalin-fixed paraffin-embedded tissue (FFPET), which is the most widely used pathology archive, usually has low-quality DNA and RNA due to extensive nucleic acid crosslinking. RNA fluorescence in situ hybridization (RNA-FISH) has been increasingly utilized in research and clinical settings to diagnose disease pathology. In this study, the effect of RNA degradation over archival time on RNA-FISH signals in FFPET and fresh frozen tissue (FFT) was systematically assessed. RNAscope multiplex fluorescent assay with the four house-keeping-gene (HKG) probes UBC, PPIB, POLR2A, and HPRT1 was performed on 62 archived breast cancer samples (30 FFPETs and 32 FFTs). As expected, the number of RNAscope signals in FFPETs is lower than in FFTs in an archival duration-dependent fashion. The RNA degradation in FFPETs is most pronounced in high-expressor HKGs, UBC and PPIB, than in low-to-moderate expressors POLR2A and HPRT1 (p<0.0001). Analysis of RNA expression over time showed that PPIB, which has the highest signal, was the most degraded in both adjusted transcript and H-score quantification methods (R² = 0.35 and R² = 0.33, respectively). This proves that although the RNAscope probes are designed to detect fragmented RNA, performing a sample quality check using HKGs is strongly recommended to ensure accurate results.

SummaryPrevious studies have suggested that chromogranin A (CgA) is a partner molecule of secretogranin III (SgIII). In mouse pituitary corticotroph-derived AtT-20 cells, SgIII plays a role in sorting CgA/hormone aggregates into secretory granules (SGs). Although CgA expression is equivocal, CgB is clearly detectable in the rat pituitary corticotrophs. Therefore, we hypothesized that CgB shares a function with CgA in pituitary corticotrophs. In the binding assays, CgB, similar to CgA, showed binding activity to SgIII under weakly acidic conditions and in the presence of Ca²⁺. Considering the differences in animal species, the different abilities of antibodies, and the conditions of tissue fixation and thin sectioning in immunofluorescence histochemistry, we found that CgA was expressed in a small population (approximately 10%), and its expression intensity was weaker than that of CgB (>98%) in rodent pituitary corticotrophs. In addition, similar to CgA, CgB and SgIII were colocalized in adrenocorticotropic hormone (ACTH) granules. The labeling of CgA and CgB was not completely consistent, and CgB colocalized with SgIII in many granules. These results suggest that there are multiple sorting systems for ACTH granules in pituitary corticotrophs and that the SgIII/CgB complex behaves more dominantly than the SgIII/CgA complex, which has somewhat different properties.

The pancreatic islet vasculature comprises microvascular endothelial cells surrounded by mural cells (pericytes). Both cell types support the islet by providing (1) a conduit for delivery and exchange of nutrients and hormones; (2) paracrine signals and extracellular matrix (ECM) components that support islet development, architecture, and endocrine function; and (3) a barrier against inflammation and immune cell infiltration. In type 2 diabetes, the islet vasculature becomes inflamed, showing loss of endothelial cells, detachment, and/or trans-differentiation of pericytes, vessel dilation, and excessive ECM deposition. While most work to date has focused either on endothelial cells or pericytes in isolation, it is very likely that the interaction between these cell types and disruption of that interaction in diabetes are critically important. In fact, dissociation of pericytes from endothelial cells is an early, key feature of microvascular disease in multiple tissues/disease states. Moreover, in beta-cell replacement therapy, co-transplantation with microvessels versus endothelial cells alone is substantially more effective in improving survival and function of the transplanted cells. Ongoing studies, including characterization of islet vascular cell signatures, will aid in the identification of new therapeutic targets aimed at improving islet function and benefiting people living with all forms of diabetes.

Over a period of almost 30 years, Gomori was one of the most prolific and productive investigators in the emerging field of enzyme histochemistry and was recognized by his peers as a pioneer in developing methods for the histochemical demonstration of hydrolytic enzyme activity, most notably phosphatases, esterases, and lipases. Gomori also made important contributions to diabetes research by developing histological techniques that reliably stained the insulin-secreting B cell of the pancreatic islets of Langerhans. Gomori's aldehyde fuchsin staining method was standard for pathological and physiological studies on islet B cells in relation to diabetes and obesity until insulin antibodies became widely available for immunohistochemical identification of B cells. Gomori was a founding member of The Histochemical Society in 1950. When the HCS established the Journal of Histochemistry and Cytochemistry in 1953, Gomori served as one of the first Associate Editors. He also served as President of The Histochemical Society.

Allergic fungal rhinosinusitis (AFRS) shares similarities with eosinophilic chronic rhinosinusitis (ECRS), both characterized by intractable nasal polyps. The key distinction lies in the presence of fungal infection within the nasal cavity. While ECRS nasal polyps are known for significant infiltration of M2 macrophages and eosinophils, as well as an increase in high endothelial venule (HEV)-like vessels, these features are less commonly reported in AFRS. This study compared clinicopathological findings between AFRS (n=10), ECRS (n=12), and non-ECRS (n=10) patients' nasal polyps using immunohistochemical analysis for CD163 and CD68 to assess the M2/M1 macrophage ratio, and peripheral lymph node addressin (PNAd) and CD34 to evaluate the proportion of HEV-like vessels. AFRS showed a significantly higher number of CD163-positive M2 macrophages and an increased M2/M1 ratio compared with ECRS. However, the percentage of HEV-like vessels and the number of eosinophils infiltrating the nasal polyps were similar in both AFRS and ECRS. The observed increase in M2 macrophages in AFRS nasal polyps is presumed to be induced by fungal infection in the nasal cavity, in comparison with ECRS. These results highlight the distinctive immunological profiles of AFRS and ECRS, emphasizing the role of macrophage polarization in their pathogenesis.

Hematoxylin and eosin (H&E)-stained slides inevitably deteriorate over time, frequently becoming unreadable. Reutilizing these slides can reduce the need for additional serial sections, particularly when the target region is no longer available in the tissue block. This study aims to develop efficient protocols for recycling faded H&E-stained slides, providing benefits for future research on stored samples. Seventy-one faded slides, representing a variety of tissue types and pathologies, were randomly divided into two groups. Slides were de-stained and re-stained using the conventional procedure and a modified Tris and HCl procedure. Three observers independently assessed all slides based on predefined parameters. The stability of the re-stained slides was re-assessed in 6 months. The modified Tris and HCl method yielded significantly higher scores compared to the conventional method for crispness of staining, nuclear staining, cytoplasmic staining, and vibrancy of staining (p < 0.05), as well as greater durability, as evidenced by minimal score reduction 6 months after staining. Thus, incorporating a Tris and HCl step into the process effectively enhances and restores faded H&E slides, offering a valuable technique for revitalizing histology slides for future research and educational purposes.

Trefoil factor family (TFF) peptides have been examined primarily in the gastrointestinal tract, where they play an important role in the epithelial regeneration. The therapeutic effects of TFFs, particularly the TFF3 protein, have been well studied in humans and in animal models of gastrointestinal injury, whereas little is known about their occurrence and function in the urinary bladder. In this study, we investigated the presence, location, and function of Tff3 in the urinary bladders of wild-type mice (Tff3^WT) and compared them with Tff3 knockout mice (Tff3^KO) using molecular and microscopic methods at the light and electron microscopic level. Our results show that Tff3 is expressed in the superficial cells of the urothelium, where it colocalizes with the uroplakin UP1b as one of the fundamental structural components of the apical plasma membrane, which is an important component of the blood-urine permeability barrier. Analysis of the urothelium with experimentally induced injury revealed that injury is more severe in Tff3^KO mice and urothelial regeneration is attenuated compared with Tff3^WT mice, suggesting that Tff3 plays a fine-tuned role in homeostasis and protection of the urothelium. This study provides the first data on the precise location and function of Tff3 in the bladder epithelium. (J Histochem Cytochem XX. XXX-XXX, XXXX).

Intestinal tuft cells are rare cells that regulate diverse functions. They harbor chemosensory receptors and signal to the mucosal immune system in response to external stimuli, though their full function and structure remain unclear. Named for their apical "tuft" of long actin-rich microvilli, tuft cells facilitate chemoreception and other physiological responses. In enterocytes, microvilli are stabilized by intermicrovillar adhesion complexes (IMACs) composed of several proteins, including cadherin-related family member-2 (CDHR2) and cadherin-related family member-5 (CDHR5), Myosin 7b, and Usher syndrome type 1 C (USH1C). We hypothesized that IMACs would be enriched in tuft cells to regulate microvillar organization. Immunostaining of murine intestinal tissue revealed that CDHR2 and CDHR5 colocalize with the tuft cell markers, DCLK1, phospho-EGFR, advillin, and cytokeratin 18. CDHR2 was dispersed throughout murine tuft cells, while CDHR5 was concentrated on the apical surface. USH1C and Myosin 7b were present in tuft cells, but at lower levels. Human single-cell RNA sequencing revealed robust CDHR2 and CDHR5 expression in tuft cells in the small intestine and colon. Immunostaining of human intestinal tissue confirmed CDHR2 and CDHR5 localization to the apical surface of tuft cells. Our findings demonstrate that protocadherins are key components of murine and human intestinal tuft cells.

Melatonin plays a major role in regulating the sleep-wake cycle and enhancing testosterone production. We investigated the short-term effects of melatonin treatment for 14 consecutive days in the cryptorchidism model. We categorized experimental mice into Sham (S), Orchiopexy (O), Melatonin (Mel), and Orchiopexy + Melatonin (OMel) groups. Surgery involved inducing cryptorchidism in the left testis for seven days, followed by orchiopexy. The Mel group's testes did not descend, but they received melatonin injections after seven days of cryptorchidism. The OMel group underwent both orchiopexy and melatonin treatment. Both O and Mel groups exhibited decreased sperm and round-headed sperm in the epididymis. Significant increases were observed in the numbers of giant cells and negative Nectin-3 cells at p-value<0.05. The pattern of Cadm1 expression changed, and Nectin-2 and Nectin-3 co-expression was lacking in abnormal spermatids. Sertoli cell cytoplasm in both O and Mel groups exhibited autophagosomes and multivesicular bodies, which correlated with increased cyclooxygenase-2 expression. However, Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive cell numbers increased significantly in all treatment groups compared to the S group. Our study found that the combination of orchiopexy and melatonin positively influenced the expression of cell adhesion molecules (Cadm1, Nectin-2, and Nectin-3) involved in spermatogenesis, while reducing giant cells, autophagosomes, and apoptosis.

Heterotrimeric extracellular matrix proteins laminins are mostly deposited at basal membranes and are important in repair and neoplasia. Here, we localize laminin beta 2 (LAMB2) at the sites of blood-brain barrier (BBB). Microvasculature (MV) of normal brain is endowed with complete LAMB2 coverage. In contrast, its cognate protein laminin beta 1 (LAMB1) is absent in MV of normal brain but emerges at the sprouting tip of a growing vessels. Similarly, vascular proliferation in high-grade gliomas (HGG) is accompanied by marked overexpression of LAMB1, whereas LAMB2 shows deficient deposition. We find that many brain pathologies with presence of post-gadolinium enhancement (PGE) on magnetic resonance imaging (MRI) show disruption of LAMB2 vascular ensheathment. Inhibition of vascular endothelial growth factor signaling in HGG blocks angiogenesis, suppresses PGE in HGG, prevents expression of LAMB1, and restores LAMB2 vascular coverage. Analysis of single-cell RNA sequencing (scRNA-seq) databases shows that in quiescent brain LAMB2 is predominantly expressed by BBB-associated pericytes (PCs) and endothelial cells (ECs), whereas neither cell types produce LAMB1. In contrast, in HGG, both LAMB1 and 2 are overexpressed by endothelial precursor cells, a phenotypically unique immature group, specific to proliferating hyperplastic MV.