Immunohistochemical Staining Techniques Research Articles

Abstract PO1-27-11: Survival Benefit of Adjuvant Endocrine Therapy in Patients with Estrogen Receptor Low-Positive Breast Cancer

Abstract Background: Estrogen receptor (ER) low-positive (1–10%) breast cancer is a very rare subtype that accounts for only 2–5% of all breast cancer cases and was introduced as a new category in the 2020 American Society of Clinical Oncology/College of American Pathologist Guidelines. There are limited data on the overall benefit of endocrine therapy for patients with ER low-positive cancers. The objective of this study was to analyze the clinicopathological characteristics and treatment patterns in patients with ER low-positive breast cancer and to compare disease-free survival (DFS) between patients who received and did not receive endocrine therapy. Methods: A retrospective search of the prospectively maintained database of our institution identified consecutive women with ER low-positive (1–10%) primary invasive breast cancer who underwent curative surgery at Seoul National University Hospital (Seoul, Korea) between January 2010 and April 2021. Standard immunohistochemical staining techniques were used to assess the level of ER, progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) expression, and semiquantitative scores (0–100%) were reported for ER and PR. DFS was defined as the time from surgery to the development of any recurrence and was compared between women who received and did not receive adjuvant endocrine therapy using univariable and multivariable Cox proportional hazards regression. Results: A total of 721 women (median age, 51 years; range, 24–83 years) with ER low-positive cancer were included in the analysis. Overall, 28% (203/721) had AJCC stage I, 42% (304/721) had stage II, and 30% (214/721) had stage III. PR expression was negative (< 1%) in 79% (571/721), low-positive (1–10%) in 12% (90/721), and high-positive ( >10%) in 8% (60/721). HER2 expression was negative in 54% (390/721) and positive in 46% (331/721). Most women (88% [634/721]) received adjuvant endocrine therapy, including all women with PR expression >10%. Of the 661 women with PR expression ≤10%, 87 (13%) did not receive adjuvant endocrine therapy. HER2-targeted therapy was performed in 81% (268/331) of women with HER2-positive cancer. During a median follow-up of 5.7 years, 98 (14%) had recurrence (25 locoregional/contralateral and 73 distant metastases). AJCC stage III (HR, 3.6; 95% CI: 2.4, 5.4; P < .001) and receiving total mastectomy (HR, 1.8; 95% CI: 1.2, 2.8; P = .004) were associated with worse DFS. Receiving adjuvant endocrine therapy was independently associated with better DFS in the entire cohort (HR, 0.46; 95% CI: 0.28, 0.76; P = .002) and in the HER2-negative subgroup (HR, 0.40; 95% CI: 0.22, 0.75; P = .004). In the HER2-positive subgroup, receiving adjuvant endocrine therapy was not significantly associated with better DFS (HR, 0.55; 95% CI: 0.23, 1.32; P = .18). Conclusion: In this large cohort of women with ER low-positive breast cancers, adjuvant endocrine therapy was administered in the majority of cases, including those with PR >10% expression. Receiving adjuvant endocrine therapy was associated with better DFS, especially when HER2 expression was negative. Citation Format: Su Hyun Lee, Han Suk Ryu, Myoung-jin Jang, Kyung-Hun Lee, Han-Byoel Lee. Survival Benefit of Adjuvant Endocrine Therapy in Patients with Estrogen Receptor Low-Positive Breast Cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-27-11.

Angiopoietin 1 Relieves Osteolysis by Promoting Macrophage Mitophagy Through the TBK1-SQSTM1 Pathway to Inhibit AIM2 Inflammasome-Mediated Pyroptosis.

Osteolysis resulting from wear particles and subsequent aseptic loosening is a leading cause of revision surgery of artificial joints. The underlying pathogenesis of particle-induced osteolysis (PPO) has remained largely uncertain. Addressing how to mitigate osteolysis caused by wear particles presents a significant challenge for orthopedic surgeons. This study aimed to explore the molecular mechanism by which Angiopoietin (Ang-1) inhibits osteoclast activation to alleviate osteolysis. RAW264.7 mouse macrophages were stimulated with LPS or RANKL to induce osteoclast formation. Additionally, titanium (Ti) particles (50mg) were subperiosteally implanted around the cranial suture of mice to establish a calvarial osteolysis model. Ang-1, a member of the pro-angiogenic factor protein family and an important inflammatory regulator molecule, was utilized in this model. TRAP staining was utilized to detect osteoclast activation, while a western blot was conducted to identify key proteins associated with mitophagy and pyroptosis. Scanning electron microscopy was employed to observe the morphology and dimensions of Ti particles. Additionally, a combination of micro-CT, H&E, Masson's trichrome, and immunohistochemical staining techniques were applied to analyze the calvarial samples. Results indicated that Ang-1 could inhibit LPS- or RANKL-induced osteoclastogenesis and alleviate Ti particle-induced calvarial osteolysis in mice. TBK-1, a key signaling molecule involved in initiating mitophagy, was found to be mechanistically enhanced by Ang-1 through promoting TBK-1 phosphorylation in macrophages. This process inhibited AIM2 inflammasome-mediated pyroptosis and impeded osteoclastogenesis. Overall, this research uncovers a novel mechanism by which Ang-1 can attenuate inflammatory osteolysis, potentially offering a new therapeutic approach for PPO.

Overexpression of cyclin F/CCNF as an independent prognostic factor for poor survival in clear cell renal cell carcinoma

Cyclin F (encoded by CCNF gene) has been reported to be implicated in the pathobiology of several human cancers. However, its potential clinical significance in clear cell renal cell carcinoma (ccRCC) remains unknown. The present study aimed to evaluate the potential significance of cyclin F, assessed by immunohistochemical (IHC) staining and molecular (bioinformatics) techniques, as a prognostic marker in ccRCC in relation to clinicopathological features and outcomes. IHC staining was performed using two independent ccRCC tissue array cohorts, herein called tissue macroarray (TMA)_1 and tissue microarray (TMA)_2, composed of 108 ccRCCs and 37 histologically normal tissues adjacent to the tumor (NAT) and 192 ccRCCs and 16 normal kidney samples, respectively. The mRNA expression data were obtained from The Cancer Genome Atlas (TCGA) and the Gene Expression Omnibus (GEO) public datasets, followed by bioinformatics analysis of biological mechanisms underlying prognosis. The relationship between immune cell infiltration level and CCNF expression in ccRCC was investigated using the Tumor Immune Estimation Resource 2.0 (TIMER2) and Gene Expression Profiling Interactive Analysis 2 (GEPIA2). Cyclin F expression was significantly elevated in ccRCC lesions compared to both NAT and normal renal tissues. Likewise, CCNF mRNA was markedly increased in ccRCCs relative to non-cancerous tissues. In all analyzed cohorts, tumors with features of more aggressive behavior were more likely to display cyclin F/CCNF-high expression than low. Furthermore, patients with high cyclin F/CCNF expression had shorter overall survival (OS) times than those with low expression. In addition, multivariable analysis revealed that cyclin F/CCNF-high expression was an independent prognostic factor for poor OS in ccRCC. Enrichment analysis for mechanistically relevant processes showed that CCNF and its highly correlated genes initiate the signaling pathways that eventually result in uncontrolled cell proliferation. CCNF expression was also correlated with immune cell infiltration and caused poor outcomes depending on the abundance of tumor-infiltrating immune cells in ccRCC. Our findings suggest that cyclin F/CCNF expression is likely to have an essential role in ccRCC pathobiology through regulating multiple oncogenic signaling pathways and affecting the tumor immune microenvironment and may serve as prognostic biomarker and promising therapeutic target in ccRCC.

125 Contribution of Sensory Neuron-associated Macrophages to Neuropathic Pain via Up-regulation of Calcium Channel in C Fiber Sensory Neurons in Dorsal Root Ganglion: An Animal Model Study

INTRODUCTION: Animal models have demonstrated that sensory neuron-associated macrophages (sNAMs) play a key role in the initiation and persistence of neuropathic pain. The Cav3.2 calcium channel (Cav3.2) is also known to be involved in modulating neuropathic pain in peripheral sensory neurons. METHODS: Male Sprague-Dawley rats underwent unilateral tight ligation of the lumbar 5 spinal nerve and were divided into three groups: a control group (SHAM), a group that underwent ligation without, and with minocycline (SNL and SNL+Mino). The mechanical pain threshold of the hind paw was assessed with von-Frey filament application. Immunohistochemical staining techniques were used to evaluate the levels of sNAM, Cav3.2, and C-fiber sensory neurons with anti-Iba1, anti-Cav3.2, and IB4 antibodies, respectively. Cytokine levels were analyzed using cytokine arrays and western blotting. RESULTS: The SNL group had a significantly lower pain threshold compared to the control group (p < 0.001). In the DRG, neuropathic pain led to an accumulation of macrophages and increased their ability to attach to Cav 3.2. The expression of Cav 3.2 then triggered the activation of IB4 cells in the DRG. Additionally, cytokine array and western blot analyses revealed a higher expression rate of TNF-α in the SNL group compared to the control group (p = 0.048). Treatment with minocycline resulted in increased pain threshold, decreased number of macrophages in the DRG, and interruption of the attachment between macrophages and Cav 3.2, leading to lower expression of TNF-α. CONCLUSIONS: In conclusion, the results of this study suggest that sNAMs may play a role in the development of neuropathic pain by increasing the expression of Cav3.2 through the upregulation of TNF-α release in peripheral sensory nociceptive neurons.

Microscopic Focus on the Thyroid Follicles of the One-Humped Camel (Camelus dromedarius).

The microstructure of the thyroid gland of the one-humped camel (Camelus dromedarius) was described using morphometric, histological, immunohistochemical staining, and ultrastructural standard techniques. The follicular secretory units of the thyroid glands displayed comparable and variable sizes where the large follicles were situated at the peripheral margins; however, the small ones were located in the central region. Semisquamous epithelium (low cuboidal) lined the large thyroid follicles, while high cuboidal (columnar) epithelium lined the small ones. Our electron microscopic findings revealed that the low-sized cuboidal follicular cells lack organelles and are hypoactive. The high cuboidal follicular cells are active cells and rich in cellular organelles such as cisternae of rough endoplasmic reticulum, mitochondria, colloid droplets, scrolled Golgi apparatus, and secretory vesicles. A few degenerate follicular cells appeared on rare occasions. The parafollicular cells appeared with a more prominent and conspicuous nucleus than the follicular cells. The follicular cells were classified as active, inactive, or degenerated using transmission electron microscopy. The follicular and parafollicular cells showed calcitonin-positive immunoreactivity. Overall, the presented results showed particular convergences of the morphostructural aspects of the thyroid gland of C. dromedarius to that of other mammals with some distinctive features to cope with their physiology and harsh niche.

Transcriptome-Wide N6-Methyladenosine Alternations in Pulmonary Arteries of Monocrotaline-Induced Pulmonary Arterial Hypertension in Rats and Novel Therapeutic Targets.

N6-methyladenosine (m6A) is a post-transcriptional epigenetic change with transcriptional stability and functionality regulated by specific m6A-modifying enzymes. However, the significance of genes modified by m6A and enzymes specific to m6A regulation in the context of pulmonary arterial hypertension (PAH) remains largely unexplored. MeRIP-seq and RNA-seq were applied to explore variances in m6A and RNA expression within the pulmonary artery tissues of control and monocrotaline-induced PAH rats. Functional enrichments were analyzed using the Gene Ontology and Kyoto Encyclopedia of Genes and Genomes. To screen candidate m6A-related genes, the STRING and Metascape databases were used to construct a protein-protein interaction network followed by a real-time PCR validation of their expression. The expression level of an m6A regulator was further investigated using immunohistochemical staining, immunofluorescence, and Western blot techniques. Additionally, proliferation assays were conducted on primary rat pulmonary artery smooth muscle cells (PASMCs). We identified forty-two differentially expressed genes that exhibited either hypermethylated or hypomethylated m6A. These genes are predominantly related to the extracellular matrix structure, MAPK, and PI3K/AKT pathways. A candidate gene, centromere protein F (CENPF), was detected with increased expression in the PAH group. Additionally, we first identified an m6A reader, leucine rich pentatricopeptide repeat containing (LRPPRC), which was downregulated in the PAH rat model. The in vitro downregulation of Lrpprc mediated by siRNA resulted in the enhanced proliferation and elevated expression of Cenpf mRNA in primary rat PASMCs. Our study revealed a modified transcriptome-wide m6A landscape and associated regulatory mechanisms in the pulmonary arteries of PAH rats, potentially offering a novel target for therapeutic strategies in the future.

HSPB6 Deficiency Promotes the Development of Aortic Dissection and Rupture

To better understand the pathogenesis of acute type A aortic dissection, high-sensitivity liquid chromatography–tandem mass spectrometry/mass spectrometry (LC-MS/MS)-based proteomics and phosphoproteomics approaches were used to identify differential proteins. Heat shock protein family B (small) member 6 (HSPB6) in aortic dissection was significantly reduced in human and mouse aortic dissection samples by real-time PCR, western blotting, and immunohistochemical staining techniques. Using an HSPB6-knockout mouse, we investigated the potential role of HSPB6 in β-aminopropionitrile monofumarate-induced aortic dissection. We found increased mortality and increased probability of ascending aortic dissection after HSPB6 knockout compared with wild-type mice. Mechanistically, our data suggest that HSPB6 deletion promoted vascular smooth muscle cell apoptosis. More importantly, HSPB6 deletion attenuated cofilin activity, leading to excessive smooth muscle cell stiffness and eventually resulting in the development of aortic dissection and rupture. Our data suggest that excessive stiffness of vascular smooth muscle cells caused by HSPB6 deficiency is a new pathogenetic mechanism leading to aortic dissection.

Effects of VX765 on osteoarthritis and chondrocyte inflammation in rats

To investigate the effects and underlying mechanisms of VX765 on osteoarthritis (OA) and chondrocytes inflammation in rats. Chondrocytes were isolated from the knee joints of 4-week-old Sprague Dawley (SD) rats. The third-generation cells were subjected to cell counting kit 8 (CCK-8) analysis to assess the impact of various concentrations (0, 1, 5, 10, 20, 50, 100 μmol/L) of VX765 on rat chondrocyte activity. An in vitro lipopolysaccharide (LPS) induced cell inflammation model was employed, dividing cells into control group, LPS group, VX765 concentration 1 group and VX765 concentration 2 group without obvious cytotoxicity. Western blot, real-time fluorescence quantitative PCR, and ELISA were conducted to measure the expression levels of inflammatory factors-transforming growth factor β 1 (TGF-β 1), interleukin 6 (IL-6), and tumor necrosis factor α (TNF-α). Additionally, Western blot and immunofluorescence staining were employed to assess the expressions of nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase 1 (HO-1). Thirty-two SD rats were randomly assigned to sham surgery group (group A), OA group (group B), OA+VX765 (50 mg/kg) group (group C), and OA+VX765 (100 mg/kg) group (group D), with 8 rats in each group. Group A underwent a sham operation with a medial incision, while groups B to D underwent additional transverse incisions to the medial collateral ligament and anterior cruciate ligament, with removal of the medial meniscus. One week post-surgery, groups C and D were orally administered 50 mg/kg and 100 mg/kg VX765, respectively, while groups A and B received an equivalent volume of saline. Histopathological examination using HE and safranin-fast green staining was performed, and Mankin scoring was utilized for evaluation. Immunohistochemical staining technique was employed to analyze the expressions of matrix metalloproteinase 13 (MMP-13) and collagen type Ⅱ. The CCK-8 assay indicated a significant decrease in cell viability at VX765 concentrations exceeding 10 μmol/L ( P<0.05), so 4 μmol/L and 8 μmol/L VX765 without obvious cytotoxicity were selected for subsequent experiments. Following LPS induction, the expressions of TGF-β 1, IL-6, and TNF-α in cells significantly increased when compared with the control group ( P<0.05). However, intervention with 4 μmol/L and 8 μmol/L VX765 led to a significant decrease in expression compared to the LPS group ( P<0.05). Western blot and immunofluorescence staining demonstrated a significant upregulation of Nrf2 pathway-related molecules Nrf2 and HO-1 protein expressions by VX765 ( P<0.05), indicating Nrf2 pathway activation. Histopathological examination of rat knee joint tissues and immunohistochemical staining revealed that, compared to group B, treatment with VX765 in groups C and D improved joint structural damage in rat OA, alleviated inflammatory reactions, downregulated MMP-13 expression, and increased collagen type Ⅱ expression. VX765 can improve rat OA and reduce chondrocyte inflammation, possibly through the activation of the Nrf2 pathway.

TMEM64 aggravates the malignant phenotype of glioma by activating the Wnt/β-catenin signaling pathway

Transmembrane protein 64 (TMEM64), a member of the family of transmembrane protein, is an α-helical membrane protein. Its precise role in various types of tumors, including glioma, is unclear. This study used immunohistochemical (IHC) staining, western blotting, and quantitative reverse transcription polymerase chain reaction (qRT-PCR) techniques to show that TMEM64 expression was significantly higher in glioma cells and tissues compared to normal cells and tissues, respectively. Additionally, a correlation between high TMEM64 expression and higher grade as well as a worse prognosis was found. TMEM64 enhanced cell proliferation and tumorigenicity while inhibiting glioma cell apoptosis in vitro and in vivo, according to loss- and gain-of-function studies. Mechanistically, it was discovered that TMEM64 increased the malignant phenotype of gliomas by accelerating the translocation of β-catenin from the cytoplasm to the nucleus, thereby activating the Wnt/β-catenin signaling pathway. Stimulation with the Wnt/β-catenin signaling pathway activator CHIR-99021 successfully reversed the malignant phenotype of glioma; however, these effects were inhibited upon TMEM64 silencing. Stimulation with the Wnt/β-catenin signaling pathway inhibitor XAV-939 successfully rescued the malignant phenotype of glioma, which was promoted upon TMEM64 overexpression. Our results provide that TMEM64 as a novel prognostic biomarker and a potential treatment target for glioma.

Automated Whole Slide Imaging for Label-Free Histology using Photon Absorption Remote Sensing Microscopy.

Peak SNR and in-focus acquisitions are achieved across entire tissue sections using the scattering signal from the PARS detection beam to measure the optimal focal plane. Whole slide images (WSI) are seamlessly stitched together using a custom contrast leveling algorithm. Identical tissue sections are subsequently H&E stained and brightfield imaged. The one-to-one WSIs from both modalities are visually and quantitatively compared. PARS WSIs are presented at standard 40x magnification in malignant human breast and skin samples. We show correspondence of subcellular diagnostic details in both PARS and H&E WSIs and demonstrate virtual H&E staining of an entire PARS WSI. The one-to-one WSI from both modalities show quantitative similarity in nuclear features and structural information. PARS WSIs are compatible with existing digital pathology tools, and samples remain suitable for histochemical, immunohistochemical, and other staining techniques. This work is a critical advance for integrating label-free optical methods into standard histopathology workflows.

Diagnostic Accuracy of Cd1a Immuno Histochemical Evaluation in Comparison with H &amp; E and Giemsa for Cutaneous Leishmaniasis

Background: Cutaneous leishmaniasis remains a global health challenge, with diagnosis largely dependent on the visualization of Leishmania parasites in infected tissue. Traditional staining methods, such as Hematoxylin and Eosin (H&amp;E) and Giemsa, have been the cornerstone of histopathological diagnosis, but they come with limitations in sensitivity and specificity. Objective: This study aimed to evaluate the diagnostic accuracy of CD1a immunohistochemical staining in the detection of leishmania amastigotes in skin biopsies and compare its efficacy with H&amp;E and Giemsa staining methods. Methods: A total of 110 skin biopsy samples with a strong clinical suspicion of cutaneous leishmaniasis were examined using H&amp;E, Giemsa, and CD1a immunohistochemical staining techniques. The sensitivity and specificity of these methods were analyzed and compared to ascertain the most effective diagnostic approach. Results: The study identified leishmania amastigotes in 78% of the cases using H&amp;E staining, while Giemsa staining resulted in amastigote detection in 82% of cases. Notably, CD1a immunohistochemical staining showed a remarkable sensitivity, identifying amastigotes in 95% of the samples. The increase in detection rate with CD1a staining was statistically significant when compared to H&amp;E (p&lt;0.05), with CD1a revealing amastigotes in an additional 23% of cases where H&amp;E staining was negative. Giemsa staining was able to detect amastigotes in three cases that were not visible with H&amp;E, suggesting its potential role as a complementary technique in certain diagnostic scenarios. Conclusion: The study concludes that CD1a immunohistochemical staining significantly enhances the detection of leishmania amastigotes and should be considered as an adjunct to conventional staining methods in the histopathological diagnosis of cutaneous leishmaniasis. Future diagnostic protocols could benefit from the incorporation of CD1a staining to improve accuracy.

The role of ARL4C in predicting prognosis and immunotherapy drug susceptibility in pan-cancer analysis.

Background: ARLs, which are a class of small GTP-binding proteins, play a crucial role in facilitating tumor tumorigenesis and development. ARL4C, a vital member of the ARLs family, has been implicated in the progression of tumors, metastatic dissemination, and development of resistance to therapeutic drugs. Nevertheless, the precise functional mechanisms of ARL4C concerning tumor prognosis and immunotherapy drug susceptibility remain elusive. Methods: By combining the GTEx and TCGA databases, the presence of ARL4C was examined in 33 various types of cancer. Immunohistochemistry and immunofluorescence staining techniques were utilized to confirm the expression of ARL4C in particular tumor tissues. Furthermore, the ESTIMATE algorithm and TIMER2.0 database were utilized to analyze the tumor microenvironment and immune infiltration associated with ARL4C. The TISCH platform facilitated the utilization of single-cell RNA-seq datasets for further analysis. ARL4C-related immune escape was investigated using the TISMO tool. Lastly, drug sensitivity analysis was conducted to assess the sensitivity of different types of tumors to compounds based on the varying levels of ARL4C expression. Results: The study found that ARL4C was highly expressed in 23 different types of cancer. Moreover, the presence of high ARL4C expression was found to be associated with a poor prognosis in BLCA, COAD, KIRP, LGG, and UCEC. Notably, ARL4C was also expressed in immune cells, and its high expression was found to be correlated with cancer immune activation. Most importantly, the drug sensitivity analysis revealed a positive correlation between ARL4C expression and the heightened sensitivity of tumors to Staurosporine, Midostaurin, and Nelarabine. Conclusion: The findings from our study indicate that the expression level of ARL4C may exert an influence on cancer development, prognosis, and susceptibility to immunotherapy drugs. In addition, the involvement of ARL4C in the tumor immune microenvironment has expanded the concept of ARL4C-targeted immunotherapy.

Transcranial burst electrical stimulation contributes to neuromodulatory effects in the rat motor cortex.

Transcranial Burst Electrical Stimulation (tBES) is an innovative non-invasive brain stimulation technique that combines direct current (DC) and theta burst stimulation (TBS) for brain neuromodulation. It has been suggested that the tBES protocol may efficiently induce neuroplasticity. However, few studies have systematically tested neuromodulatory effects and underlying neurophysiological mechanisms by manipulating the polarity of DC and TBS patterns. This study aimed to develop the platform and assess neuromodulatory effects and neuronal activity changes following tBES. Five groups of rats were exposed to anodal DC combined with intermittent TBS (tBES+), cathodal DC combined with continuous TBS (tBES-), anodal and cathodal transcranial direct current stimulation (tDCS+ and tDCS-), and sham groups. The neuromodulatory effects of each stimulation on motor cortical excitability were analyzed by motor-evoked potentials (MEPs) changes. We also investigated the effects of tBES on both excitatory and inhibitory neural biomarkers. We specifically examined c-Fos and glutamic acid decarboxylase (GAD-65) using immunohistochemistry staining techniques. Additionally, we evaluated the safety of tBES by analyzing glial fibrillary acidic protein (GFAP) expression. Our findings demonstrated significant impacts of tBES on motor cortical excitability up to 30 min post-stimulation. Specifically, MEPs significantly increased after tBES (+) compared to pre-stimulation (p = 0.026) and sham condition (p = 0.025). Conversely, tBES (-) led to a notable decrease in MEPs relative to baseline (p = 0.04) and sham condition (p = 0.048). Although tBES showed a more favorable neuromodulatory effect than tDCS, statistical analysis revealed no significant differences between these two groups (p > 0.05). Additionally, tBES (+) exhibited a significant activation of excitatory neurons, indicated by increased c-Fos expression (p < 0.05), and a reduction in GAD-65 density (p < 0.05). tBES (-) promoted GAD-65 expression (p < 0.05) while inhibiting c-Fos activation (p < 0.05), suggesting the involvement of cortical inhibition with tBES (-). The expression of GFAP showed no significant difference between tBES and sham conditions (p > 0.05), indicating that tBES did not induce neural injury in the stimulated regions. Our study indicates that tBES effectively modulates motor cortical excitability. This research significantly contributes to a better understanding of the neuromodulatory effects of tBES, and could provide valuable evidence for its potential clinical applications in treating neurological disorders.

The association of claudin-18 and androgen receptor expression in prostatic carcinoma: an immunohistochemical study

BackgroundClaudin-18 (CLDN18) is a recently identified anticancer therapeutic target with promising results for various gastrointestinal malignancies. The role of CLDN18 in prostatic carcinoma has not been investigated. The aim of this study was to investigate CLDN18 and androgen receptor (AR) expression in prostatic carcinoma and to link these findings with other clinicopathological characteristics. This retrospective study was carried out on 120 cases of prostatic lesions, including 100 cases of prostatic carcinoma and 20 cases of benign prostatic hyperplasia. The immunohistochemical staining technique was used to evaluate the expression of both CLDN18 and AR in prostatic carcinoma in relation to clinicopathological parameters.ResultsCLDN18 expression was completely absent in benign prostatic tissue, while it was detected in the membrane of 30 (30%) of studied carcinoma cases, with a statistically significant difference (p = 0.046). In contrast to other variables, a statistically significant relationship was identified between CLDN18 expression and Gleason Grade group (p = 0.000), stage (p = 0.03), and nodal metastasis (p = 0.000). The expression of the androgen receptor was detected in the nucleus of 96 (96%) of the cancer cases under study, with no statistically significant difference between the studied groups (p = 0.427). A statistically significant relation was found between AR expression and Gleason Grade group (p = 0.03) and stage (p = 0.01), while no relation with other variables was detected. AR expression and CLDN18 expression were shown to be statistically significantly correlated (p = 0.002).ConclusionsCLDN18 was expressed in prostatic carcinoma and correlated with an adverse tumor outcome. CLDN18 may be regulated by AR. CLDN18 could be a candidate therapeutic marker for the treatment of prostatic carcinoma.

Dopaminergic Projections from the Hypothalamic A11 Nucleus to the Spinal Trigeminal Nucleus Are Involved in Bidirectional Migraine Modulation

Clinical imaging studies have revealed that the hypothalamus is activated in migraine patients prior to the onset of and during headache and have also shown that the hypothalamus has increased functional connectivity with the spinal trigeminal nucleus. The dopaminergic system of the hypothalamus plays an important role, and the dopamine-rich A11 nucleus may play an important role in migraine pathogenesis. We used intraperitoneal injections of glyceryl trinitrate to establish a model of acute migraine attack and chronicity in mice, which was verified by photophobia experiments and von Frey experiments. We explored the A11 nucleus and its downstream pathway using immunohistochemical staining and neuronal tracing techniques. During acute migraine attack and chronification, c-fos expression in GABAergic neurons in the A11 nucleus was significantly increased, and inhibition of DA neurons was achieved by binding to GABA A-type receptors on the surface of dopaminergic neurons in the A11 nucleus. However, the expression of tyrosine hydroxylase and glutamic acid decarboxylase proteins in the A11 nucleus of the hypothalamus did not change significantly. Specific destruction of dopaminergic neurons in the A11 nucleus of mice resulted in severe nociceptive sensitization and photophobic behavior. The expression levels of the D1 dopamine receptor and D2 dopamine receptor in the caudal part of the spinal trigeminal nucleus candalis of the chronic migraine model were increased. Skin nociceptive sensitization of mice was slowed by activation of the D2 dopamine receptor in SP5C, and activation of the D1 dopamine receptor reversed this behavioral change. GABAergic neurons in the A11 nucleus were activated and exerted postsynaptic inhibitory effects, which led to a decrease in the amount of DA secreted by the A11 nucleus in the spinal trigeminal nucleus candalis. The reduced DA bound preferentially to the D2 dopamine receptor, thus exerting a defensive effect against headache.

Comparing the effect of tissue adhesive and suturing material on collagen I/III ratio in abdominal skin wounds: an experimental study

Background: The skin closure procedure should be technically simple, acceptable, quick, and cost-effective. Sutures remain the technique’s mainstay, however tissue adhesive is becoming more used in clinical practice. Collagen ratios of types I and III play a significant role as postoperative wound healing parameters. Here, the authors aim to examine the collagen I/III ratio of tissue adhesive vs. non-absorbable sutures for abdominal skin closure in Wistar albino rats. Material and methods: The authors allocated 20 rats into four experimental groups. Wounds in groups 1 and 3 were sealed with tissue adhesive (cyanoacrylate), while those in groups 2 and 4 were closed using suture material (monofilament non-absorbable nylon). Groups 1 and 2 were sacrificed on postoperative day (POD) 4, while those in groups 3 and 4 were euthanized on POD 7. Skin samples (1×0.5 cm) were collected for analysis, and the collagen I/III ratios were determined using immunohistochemistry staining techniques. Results: The levels of collagen I and III expression did not exhibit statistically significant differences between tissue adhesive and nylon suture groups at either POD 4 (P=0.052, P=0.513) or POD 7 (P=0.125, P=0.80). Similarly, the collagen I/III ratio did not significantly differ between the two groups at POD 4 (1.23±2.26 vs. 0.70±0.24; P=0.47) or POD 7 (0.68±0.96 vs. 0.77±1.22; P=0.857). Conclusions: There were no statistical significance difference in collagen I/III ratio between the tissue adhesive and suture material groups, suggesting that the choice of wound closure material may not influence the abdominal skin closure.

Deep learning approach for accurate prostate cancer identification and stratification using combined immunostaining of cytokeratin, p63, and racemase.

Prostate cancer (PCa) is the most frequently diagnosed cancer in men worldwide, affecting around 1.4 million individuals. Current PCa diagnosis relies on histological analysis of prostate biopsy samples, an activity that is both time-consuming and prone to observer bias. Previous studies have demonstrated that immunostaining of cytokeratin, p63, and racemase can significantly improve the sensitivity and the specificity of PCa detection compared to traditional H&E staining. This study introduces a novel approach that combines diagnosis-specific immunohistochemical (IHC) staining and deep learning techniques to provide reliable stratification of prostate glands. Our approach leverages a customized segmentation network, called K-PPM, that incorporates adaptive kernels and multiscale feature integration to enhance the functional information of IHC. To address the high class-imbalance problem in the dataset, we propose a weighted adaptive patch-extraction and specific-class kernel update. Our system achieved noteworthy results, with a mean Dice Score Coefficient of 90.36% and a mean absolute error of 1.64 % in specific-class gland quantification on whole slides. These findings demonstrate the potential of our system as a valuable support tool for pathologists, reducing workload and decreasing diagnostic inter-observer variability. Our study presents innovative approaches that have broad applicability to other digital pathology areas beyond PCa diagnosis. As a fully automated system, this model can serve as a framework for improving the histological and IHC diagnosis of other types of cancer.

Simvastatin improves learning and memory impairment via gut-brain axis regulation in an ovariectomized/D-galactose Alzheimer's rat model

AimAlzheimer’s disease (AD) is the most prevalent form of dementia with multiple etiology and no effective remedy. Statins are a group of medicines that are basically used to lower cholesterol. However, several studies have recently done to assess the potential relationship between statins use and dementia but presented controversial results. MethodsIn this study, using ovariectomy and D‐galactose injection, a model of AD was induced in female rats, and then the protective effects of oral administration of simvastatin were investigated. shuttle box and Y-maze tests were done to assess the animals’ learning and memory performance. Using GC-MC, ELISA, Immunohistochemistry and tissue staining techniques, changes in the amount of short-chain fatty acids (SCFAs), plasma and hippocampus neuroinflammatory markers and histological changes in the intestine and hippocampus were assessed in sham, disease and treatment groups. Key findingsOral administration of simvastatin improved the gut microbiome activity (increased the amount of SCFAs in fecal samples) and strengthened the tight junctions of intestinal cells. Moreover, simvastatin reduced the amount of TNF-α and IL-1β in plasma and hippocampus. Also, cell death and Amyloid plaques notably decreased in the simvastatin-treated hippocampal tissue. All these physiological changes led to better performance in behavioral tasks in the treatment group in comparison to the disease group. SignificanceThese findings provide evidence that simvastatin may improve gut-brain axis followed by improvement in learning and memory via an anti-inflammatory effect.

Identification and validation of immune-associated NETosis subtypes and biomarkers in anti-neutrophil cytoplasmic antibody associated glomerulonephritis.

NETosis is a new form of cell death, marked by DNA chromatin release from dead neutrophils. While it aids in microbe defense, it may worsen inflammation in autoimmune diseases, causing tissue harm. The impact of NETosis on Anti-neutrophil Cytoplasmic Antibody-associated Glomerulonephritis (ANCA-GN) remains unexplored and requires investigation. First, a weighted gene co-expression network analysis (WGCNA) was conducted to uncover differential expression of neutrophil extranuclear trap-associated genes (DE-NETs) in ANCA-GN. The NETosisScore model was established through the single sample gene set enrichment analysis (ssGSEA), which categorized all patients into high-risk and low-risk groups. The accuracy of model was assessed by ROC curve. The biological function of various subgroups was explored through Gene Set Variation Analysis (GSVA), while the abundance of immune cell infiltration was measured with CIBERSORT. Furthermore, the key NETosis-related genes (NRGs) were identified using threemachine learning algorithms, and their relationship with renal function was analyzed through the NephroseqV5 database. Through the application of qPCR and immunohistochemical staining techniques, the mRNA and protein expression levels of NRGs were determined in patients with ANCA-GN andcontrol. A NETosisScore model was developed from 18 DE-NETs using the ssGSEA algorithm. The model's ability to predict ANCA-GN patients with a ROC AUC of 0.921. The high-risk group in ANCA-GN showed enrichment of immune-related pathways and greater infiltration of immune cells, as revealed by KEGG enrichment analysis and CIBERSORT. Using three machine learning algorithms, we identified six NRGs. Significant positive correlations were found between NRGs and CCR, macrophages, T-cell co-inhibition, and TIL. Further KEGG analysis revealed that the functions of NRGs may be closely related to the toll-like receptor signaling pathway. The levels of NRGs increased as kidney function declined and were positively correlated with Scr (serum creatinine) and negatively correlated with GFR (glomerular filtration rate), qPCR analysis showed increased expression of most NRGs in ANCA-GN patients. Furthermore, immunohistochemical staining confirmed higher expression of all NRGs in ANCA-GN patients. NETosisScore model accurately predicts high-risk patients in ANCA-GN with enriched immune pathways, 6 NRGs identified as potential biomarkers.

High-risk human papillomavirus prevalence among patients with cervical cancer at Moewardi General Hospital in Surakarta, Indonesia

Link of Video Abstract: https://www.youtube.com/watch?v=Ryf_-hvseVc Introduction: In Indonesia, cervical cancer is the second most prevalent type of cancer among the general population. Cervical cancer has the second-highest mortality rate of all cancers. Infection with the Human Papilloma Virus (HPV) is one of the causes that starts the growing phase of cervical cancer. The purpose of this study, therefore, is to determine the prevalence of HPV infection in patients with cervical cancer who were examined at Dr. Moewardi Hospital and lived in the ex-Surakarta Residency. Methods: Cervical tissue biopsy at the Anatomical Pathology Installation of Dr. Moewardi General Hospital Surakarta which had been diagnosed as cervical cancer was examined by the detection of E6 protein from HPV 16 and HPV 18 using immunohistochemical staining techniques. The data obtained would be analyzed based on the characteristics of the patient's age and the degree of histopathological malignancy with a p-value &lt; 0.05 deemed significant. Results: Overall, the prevalence of high-risk HPV infection was 77.8% (35/45), with women aged between 41-55 years having the highest prevalence of HPV infection (45.7, 16/35), followed by women aged 56-70 (42.9%), women over 70 (8.6%) and women under 40 (2.9%). The results of statistical tests showed that no significant relationship was found between HPV infection and age (p = 0.428). High-risk HPV infection was most prevalent in the poorly differentiated group (54.3%), followed by the well-differentiated group (34.3%) and the moderately differentiated group (11.4%). The results of statistical tests revealed a significant association between HPV infection and tumor grade (p = 0.027). Conclusion: To lower patient morbidity and mortality, it is crucial to receive a prophylactic HPV vaccine due to the incidence of HPV 16 and 18 infections in patients with cervical cancer at the Regional Hospital Dr. Moewardi Surakarta, with a prevalence of 77.8%.