Immunohistochemical Staining Method Research Articles

Arginase Pathway Markers of Immune-Microenvironment in Thymic Epithelial Tumors and Small Cell Lung Cancer

BackgroundKey regulators of antitumor immunity such as arginase-1 and the adenosine pathway may have an important role in modulating the effect of immunotherapy. Here, we investigated the expression profile of these immune-related biomarkers in thymic epithelial tumors (TETs) and small cell lung cancer (SCLC), 2 solid tumors where immune checkpoint inhibitors have activity. Materials and MethodsImmunohistochemical staining was performed using tissue microarrays of 123 TET (110 thymoma and 13 thymic carcinoma) and 125 SCLC cases. The expression profile of the following immune-related biomarkers was assessed: arginase-1, CD39, CD73, A2AR, PD-L2, and CD15. The expression profile was also correlated with clinical data. ResultsNo sample was positive for arginase-1. In the adenosine pathway, the prevalence of positive staining for CD39, CD73, and A2AR was 4.9%, 2.5%, and 69.2%, in TETs and 0%, 1.7%, and 50.8%, in SCLC. The multivariate analysis showed that CD39 expression was significantly associated with worse disease related survival (hazard ratio [HR], 10.36; 95% confidence interval [CI]: 2.01-53.47; P= .005) and a shorter time-to progression (HR, 11.35; 95% CI, 2.11-61.23; P = .005) in TETs. Other biomarkers were not associated with disease related survival or time to progression in TETs. No biomarker was associated with survival in SCLC. ConclusionArginase-1 was not detectable in TETs and SCLC. Expression of markers in the adenosine pathway were present in both TETs and SCLC. CD39 expression in tumor cells may identify subsets of patients with TETs with an unfavorable prognosis.

МОРФОЛОГИЧЕСКИЕ ИЗМЕНЕНИЯ СОСУДИСТОЙ СИСТЕМЫ ПЕЧЕНИ КРЫС ПОД ВОЗДЕЙСТВИЕМ ТИОАЦЕТАМИДА

Background. Angiogenesis plays a key role in the progression of liver fibrosis. However, the available data on morphological changes in the liver vascular system are insufficient and contradictory. Objective. The aim of the work is to study the morphological changes in the liver vascular system of rats under the influence of thioacetamide. Material and methods. Fibrosis and cirrhosis of the liver in Wistar rats were induced with thioacetamide given at a dose of 200 mg/kg of animal weight for 17 weeks. To study morphological changes, we used classical and immunohistochemical staining methods. Microscopic analysis was performed using OLYMPUS BX51 microscope and image analysis software ImageScope Color and cellSens Standard. Results. The introduction of a solution of thioacetamide through the stomach leads to a gradual increase in the progression of pathological changes. In addition, it permits to track all stages of cirrhosis development and morphological restructuring of the liver vascular system. Throughout the experiment there was intensive capillarization of the parenchyma sinusoids and neoangiogenesis in the portal tracts and connective tissue septa manifested by the formation of many venules and small veins. We also observed an increase in the area of interlobular veins, which in some places had reached gigantic proportions. Three morphological phenotypes of CD34-positive cells were revealed. In the interlobular arteries as well as interlobular, central and sublobular veins, these cells had an elongated shape and a rod-shaped dark-colored nucleus. During the transformation of liver fibrosis into cirrhosis CD34-positive cells of an elongated shape with light roundedelongated nuclei were observed in the sinusoids closer to the periphery of individual false nodules. Rounded cells with dark-colored nuclei were present in the connective tissue near the hepatic triads, in the connective tissue septa among the cells of the infiltrate and between the sharply increased number of bile ducts. Conclusions. The established complex phenotypic changes in sinusoidal endothelial cells prove a close connection between fibrogenesis and neoangiogenesis. They probably play a leading role in the development of fibrosis and restructuring of the venous system of the portal vein.

Expression of B7-H3 Correlates with PD-L1 and Poor Prognosis in Patients with Cervical Cancer.

PurposeThe purpose of this study was to investigate B7 homolog 3 (B7-H3) expression patterns and define its associations with programmed cell-death ligand 1 (PD-L1), pathological features, and survival in patients with cervical cancer.Patients and MethodsImmunohistochemical staining was used to investigate B7-H3 and PD-L1 expression in tissue microarrays from 552 patients with stage IB1 and IIA1 cervical cancer, including 406 with squamous cell carcinoma and 146 with endocervical adenocarcinoma.ResultsB7-H3 was expressed in the tumor cells (TCs) of 32.1% of the samples as well as in the stromal cells of 92.9% of the specimens. B7-H3 was co-expressed with PD-L1 in 21.0% of the samples, while only one or the other was expressed in 41.7% of the samples. B7-H3 expression in TCs was more frequent in squamous cell carcinoma, PD-L1-positive samples, and tissues from patients with lymph node metastasis; moreover, its expression was an independent predictor of shorter survival.ConclusionB7-H3 positivity in TCs is a promising prognostic biomarker, and targeting B7-H3 alone or in combination with PD-1/PD-L1 may be a potential immunotherapeutic strategy for patients with cervical cancer.

Ferula elaeochytrisin sıçan kalbinde irisin düzeylerine etkisi

Purpose: The aim of this study was to determine the levels of irisin in the heart, which is a vital organ, in rats of different ages and to reveal whether Ferula elaeochytris (FE) has an effect on the expression of the irisin. 
 Materials and Methods: Our study was designed in six groups, young (4 months), middle age (12 months), old (24 months) rats which were given FE extract for 8 weeks and their control groups. After that their heart tissues were taken and irisin levels were measured by immunohistochemical staining method. 
 Results: The obtained results were analyzed by performing histoscoring. It was observed that the level of irisin in old rats was statistically significantly lower in young and middle age rats. In addition, irisin level of in all groups administered with FE extract was found significantly increased. 
 Conclusion: FE extract increases the level of irisin through other pathways besides its protective effect against tissue damage.

RIPK4 Suppresses the Invasion and Metastasis of Hepatocellular Carcinoma by Inhibiting the Phosphorylation of STAT3.

Receptor interacting serine/threonine kinase 4 (RIPK4) is a member of the threonine/serine protein kinase family; it plays related functions in a variety of tumours, but its biological function has not been fully revealed. It has been reported that it is differentially expressed in hepatocellular carcinoma (HCC). Our research aimed to reveal the role of RIPK4 in the progression of HCC and to reveal the biological behaviour of RIPK4 in HCC. We analysed the differences in RIPK4 expression in HCC by using a publicly available data set. By using PCR, Western blotting and immunohistochemical staining methods, we detected the expression level of RIPK4 in HCC patient specimens and studied the relationship between the expression of RIPK4 and the clinicopathological features of HCC patients. The prognostic data were combined to analyse the relationship between RIPK4 and HCC patient survival and tumour recurrence. We found that the expression level of RIPK4 in nontumour tissues was significantly higher than that in tumour tissues, and the level of RIPK4 was significantly positively correlated with postoperative survival and recurrence in HCC patients. Further, our study found that RIPK4 inhibits the progression of HCC by influencing the invasion and metastasis of HCC and that overexpression of RIPK4 reduces the invasion and metastasis of HCC by inhibiting epithelial-mesenchymal transition (EMT) and the STAT3 pathway. In in vivo experiments, overexpression of RIPK4 stably inhibited HCC metastasis. To summarize, our research revealed the relationship between RIPK4 and the prognosis of patients with HCC. We discovered that RIPK4 affects the invasion and metastasis of HCC through the EMT and STAT3 pathways. Targeted inhibition of the RIPK4 gene and the STAT3 pathway may be potential therapeutic strategies for inhibiting the postoperative recurrence and metastasis of HCC.

CT Image Examination Based on Virtual Reality Analysis in Clinical Diagnosis of Gastrointestinal Stromal Tumors.

With the advancement and development of medical equipment, CT images have become a common lung examination tool. This article mainly studies the application of CT imaging examination based on virtual reality analysis in the clinical diagnosis of gastrointestinal stromal tumors. Before extracting suspected lymph nodes from a CT image of the stomach, the CT image sequence is preprocessed first, which can reduce the cumbersomeness of subsequent extraction of suspected lymph nodes and speed up the subsequent processing. According to medical knowledge, CT images of the stomach show that lymph nodes mainly exist in the adipose tissue around the gastric wall, but there are no lymph nodes in the subcutaneous fat outside the chest. The most basic gray value in the image and the neighborhood average difference feature related to gray level are used as the primary features of visual attention detection. When extracting the neighborhood average difference feature, we use a 3∗3 sliding window method to traverse each point of the pixel matrix in the image, thereby calculating the feature value of each pixel in the image. After the feature extraction is completed, it is necessary to calibrate the data and make a training data set. The SP immunohistochemical staining method was used. The specimens were fixed with 10% formaldehyde, routinely embedded in paraffin, sectioned, and stained with HE. The tumor tissue was determined by immunohistochemistry, and the reagents were products of Maixin Company. All patients were followed up by regular outpatient review, letters, and visits or phone calls. The data showed that immunohistochemical tumor cells showed positive staining for CD117 (14/15, 93.3%) and CD34 (10/15, 66.7%). The results show that the application of virtual reality technology to CT imaging examination can significantly improve the diagnostic accuracy of gastrointestinal stromal tumors.

Diagnostic value of glycophorin-A in comparison with P57 immunohistochemical staining method in differentiating complete and partial molar pregnancies

IntroductionCurrent histomorphological criteria in distinguishing two subtypes of hydatidiform moles has considerable inter-observer variability and limitations. In this regard, ancillary studies can aid pathologist to obtain an accurate diagnosis. Herein, we evaluated the utility of Glycophorin-A (GLA) in differentiating complete and partial moles. Materials and methodsIn this case-control study, formalin-fixed paraffin-embedded blocks of 47 patients with pathologic diagnosis of complete and 42 partial hydatidiform moles were included and the diagnoses were confirmed by immunohistochemistry (IHC) for P57. Sections from all samples were stained for GLA using IHC method. Using 2 × 2 tables, the sensitivity, specifity, Positive and Negative Predictive Values (PPV and NPV) as well as accuracy of GLA were determined. ResultsPrimary pathologic diagnosis was changed in 7.1% and types of hydatidiform mole were specified in 11.9% of the cases after review of the slides and IHC study for P57. NRBCs were found in 52.7% of the PM cases and none of CMs by pathologist in H&E sections. IHC study for GLA revealed positive result in one case of complete moles (2%) and 31 case of partial mole samples (73.8%). It was negative in 98% of the complete mole and 11 (26.2%) of partial mole cases. DiscussionThe results of this study showed a significant association between GLA immunoreactivity and type of molar pregnancy. Diagnostic sensitivity, specificity and accuracy of this marker for discrimination of molar pregnancy were 73.8%, 98% and 86.5%, respectively. Therefore, this marker can be utilized in differentiating partial and complete hydatidiform mole.

MO435MULTIMODAL IMAGING FOR MOLECULAR TISSUE ANALYSIS

Abstract Background and Aims MALDI mass spectrometric imaging (MALDI MSI) is a powerful histologic tool for the analysis of biomolecules in tissue samples. MALDI MSI measurements result in a high sensitivity and accuracy of spatial distribution of biomolecules in tissue samples. For more detailed analysis of MALDI MSI data and correlation between the molecular and microscopic levels, a combination of MALDI MSI data and histological staining is essential. By combining MALDI MSI data and histological data, much more information are obtained than by analyzing both methods individually. Therefore, MALDI MSI datasets and histological staining were fused to a 3D model presenting a biomolecule distribution of the whole organ and provides more information than a single tissue section. We have developed, established and validated an algorithm for an automatic registration of MALDI data with different histological image data for cross-process evaluation of multimodal datasets to create 3D models. This multimodal imaging approach simplifies and improves molecular analyses of tissue samples in clinical research and diagnosis. Method The datasets for fusion and creation of a 3D model consist of mass spectrometric data, histological and immunohistochemical staining methods. Histological tissue sections of a whole mouse kidney were prepared. For MALDI MSI data, organ sections were analyzed by using a Rapiflex mass-spectrometer. Results A mathematical registration was used to achieve a perfect superposition of the individual histological sections of mass spectrometric data. It is feasible to combine mass spectrometric data, histological and immunohistochemical datasets in high numbers and reconstruct the measured mouse kidney. By using different imaging methods, a variety of information about tissue structure as well as tissue changes and protein distributions can be obtained. The fusion of the data also offers a virtual incision of the organ from arbitrary angle and level. The algorithms are adapted to take the data fusion automatically offering a high-throughput approach for clinical diagnostics and the possibility to involved artificial intelligence in its interpretation in research. Conclusion A successful fusion of MALDI MSI data and different histological and immunohistochemical staining datasets of a whole organ is performed.

Clinicopathological significance of SOX4 and epithelial-mesenchymal transition markers in patients with laryngeal squamous cell carcinoma

ObjectiveSex-determining region-Y-related high-mobility-group box 4 (SOX4) is associated with the metastasis and prognosis of many cancer types. However, studies on the role of SOX4 in laryngeal squamous cell carcinoma (LSCC) are few, and hence the mechanism is unclear. Epithelial–mesenchymal transition (EMT) allows neoplastic cells to gain the plasticity and motility required for tumor progression and metastasis. This study aimed to analyze the relationship between SOX4 and EMT, and their relationship with clinicopathological factors and related prognosis. MethodsImmunohistochemical staining was used to detect the positive expression of SOX4 protein, EMT-related transcription factor protein, and related marker protein in 127 LSCC tissue samples. At the same time, data on various parameters of clinical pathology and postoperative survival were collected. ResultsThe positive expression rate of SOX4 and Slug in LSCC was related to pathological differentiation, Lymph node metastasis (LNM), and pathological TNM of a tumor. The expression rates of ZEB1, E-cadherin, N-cadherin, and β-catenin in LSCC correlated with LNM and pTNM. The expression of SOX4, combined expression of SOX4 and ZEB1, and LNM were independent prognostic factors for the total survival time of patients with LSCC. ConclusionsIn summary, SOX4 was vital in the LSCC EMT process, which might be mediated by transcription factor ZEB1. SOX4 and ZEB1 might serve as potential biomarkers of metastasis and prognosis, as well as promising therapeutic targets of LSCC.

Prospects for predicting the course of nodular sclerosis Hodgkin lymphoma by morphometric analysis of CD163-positive macrophages

Background. In present days, much attention is paid to the study of the interrelation between the macrophage/hystiocytic microenvironment and the tumor substrate in lymphoproliferative disorders.Objective. The article is devoted to the morphometric and morphological assessment of CD163-positive macrophages in nodular sclerosis Hodgkin lymphoma.Materials and methods. Formalin fixed, paraffin-embedded (FFPE) lymph node samples of 45 patients were used for the study. To identify and visualize CD163-positive cells in the test material, an immunohistochemical staining method was used.Results. The study shows that the morphometric and morphological analysis of CD163-positive cells can be an effective and promising criterion for representing them as potential predictors of the disease course. Immunohistochemical study of 45 cases using the CD163 marker revealed a difference in the nature of macrophages localization in the lymph nodes nodules. The dependence of CD163-expressing cells number on the disease course was determined.Conclusion. The data obtained can be used to stratify patients with nodular sclerosis of classical Hodgkin lymphoma into risk groups and to determine personalized approaches to treatment. Immunohistochemical determination of the CD163 marker can be used in the complex diagnosis of the causes of refractoriness to the first and subsequent lines of therapy.

Effect of catechin on dietary AGEs absorption and cytotoxicity in Caco-2 cells

Maillard reaction produces advanced glycation end products (AGEs) that endanger human health. This study investigated the protective effect of (+)-catechin (CC) on different types of dietary AGEs absorption and cytotoxicity in Caco-2 cells. Our results showed that CC had higher inhibitory rate on peptide bound-AGEs absorption than free Nɛ-carboxymethyl lysine (CML), which dropped to 36.24% and 32.21% when treated with 20 and 50 μM CC. The reasons might be that CC could repair the loose tight junction (ZO-1) and down-regulation of protein-coupling peptide carrier 1 (PEPT-1) expression in Caco-2 cells which were in accordance with molecular docking results. Additionally, CC could remarkably decreased the protein levels of receptor of AGEs (RAGE), mitogen-activated protein kinases (MAPK) and nuclear factor-kappa B (NF-κB) that detected by western blotting and immunohistochemical staining method. Taken together, these findings demonstrated that CC may inhibit AGEs absorption and protected Caco-2 cells against RAGE-MAPK-NF-κB signaling suppression.

Prognostic significance of PD-L1-positive cancer-associated fibroblasts in patients with triple-negative breast cancer

BackgroundCancer-associated fibroblasts (CAFs) are some of the most abundant components of the tumour microenvironment. A recent study suggested that in some cancers, CAFs express programmed death ligand 1 (PD-L1), which can act as a prognostic marker. The aim of this study was to investigate the clinicopathological significance of CAF PD-L1 expression in patients with triple-negative breast cancer (TNBC) and to identify the most suitable primary antibody for immunostaining for CAF PD-L1.MethodsImmunohistochemical staining (primary antibodies of 73–10, SP142, and E1L3N) and tissue microarrays were used to analyse the expression profiles of PD-L1 in CAF in 61 patients with TNBC who underwent surgery. Overall survival (OS) was compared based on CAF PD-L1 expression, and the risk factors for OS were analysed. The relationship between clinicopathological parameters and survival was also examined.ResultsThirty-four (55.7%) patients were positive for CAF PD-L1 (73–10) expression. Compared with CAF PD-L1 negativity, there was a significant correlation between CAF PD-L1 positivity and better OS (p = 0.029). CAF PD-L1 expression, evaluated using SP-142 or E1L3N, did not correlate with OS. CAF PD-L1-positivity (73–10) correlated significantly with better prognosis in multivariate analyses (hazard ratio: 0.198; 95% confidence interval: 0.044–0.891; p = 0.035).ConclusionsCAF PD-L1 expression is a novel marker for a better prognosis of patients with TNBC, and the 73–10 assay may be suitable for immunostaining CAF PD-L1.

An immunohistochemical panel of three small ubiquitin-like modifier genes predicts outcomes of patients with triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is a highly heterogeneous and aggressive disease. Developing new candidate biomarkers for chemotherapy response and possible therapeutic targets has become an urgent clinical need. Small ubiquitin-like modifiers (SUMOs) mediate post-translational modifications (SUMOylation) has been shown to be involved in numerous biological processes. However, the role of SUMOylation in TNBC has yet to be elucidated. The mRNA expression of SUMO1/2/3 was analyzed by the Cancer Genome Atlas (TCGA) and Gene Expression Omnibus database (GEO) databases (N=412). We also evaluated the SUMO1/2/3 protein expression in 212 TNBC patients using immunohistochemical (IHC) staining method. A classifier with Least absolute shrinkage and selection operator (LASSO) Cox regression model was then built based on the associations between the expression of SUMO1/2/3 proteins and the disease-free survival (DFS) of TNBC patients. Elevated SUMO1/2/3 levels were indicated to be associated with a poorer overall survival (OS) and DFS for TNBC patients. With the LASSO model, we built a classifier based on the IHC scores of SUMO1/2/3 proteins and named it the 'SB classifier'. Patients with SB classifier-defined high score were found to have an unfavorable response to chemotherapy [hazard ratio (HR) 4.04, 95% confidence interval (CI): 2.14-7.63; P<0.0001]. A nomogram was then developed to identify which patients might benefit from chemotherapy. Finally, our results also suggested that the activation of SUMOylation pathway in TNBC might be induced by MYC signaling. We constructed a reliable prognostic and predictive tool for TNBC patients treated with chemotherapy, which could facilitate individualized counseling and management.

SCG3 Protein Expression in Glioma Associates With less Malignancy and Favorable Clinical Outcomes.

Introduction: Secretogranin III (SCG3) physiologically participates in neurotransmitter storage/transport and is widely expressed in neuroendocrine tumors. However, there is no report on SCG3 protein expression in gliomas. Methods: The method of immunohistochemical staining on a glioma tissue microarray was utilized to detect SCG3 protein expression and investigate the correlations of its expression with clinicopathological and genetic features in gliomas. The RNA-seq data of SCG3 in The Cancer Genome Atlas database was exploited to explore these correlations at the transcriptional level. Results: There were 57.5% (130/226) glioma cases having SCG3 cytoplasmic staining in the tissue microarray. SCG3 expression inversely correlated with malignancy grade at both transcriptional and protein levels. The highest level was observed in oligodendroglial tumors, especially in oligodendrogliomas (ODs) with IDH-mutation/1p19q-codeletion. The lowest SCG3 expression was observed in glioblastomas (GBMs), especially in the mesenchymal subtype. Nearly a half of GBM cases (44.4%, 64/144) had any discernible SCG3 staining, and were defined as SCG3-positive by the microarray study. SCG3-positive GBM cases exhibited improved overall survival as compared with the SCG3-negative cases (29.3 vs. 14.5 months; Hazard ratio, 0.364; 95% CI, 0.216–0.612; p < 0.001). A multivariate Cox regression analysis also revealed SCG3 positivity as an independent favorable prognosticator in GBM patients. Conclusion: SCG3 protein expression inversely correlates with glioma malignancy and predicts favorable outcomes in GBM patients.

Abstract PS4-11: Identification and validation of stromal immunotype predict survival and benefit from neoadjuvant chemotherapy in patients of breast cancer

Abstract Background Immune infiltration of breast cancer is associated with clinical outcome. A growing number of research suggests the immune response were composed by variously functionally distinct immune cell. But it’s not clear which kinds of cell play the important role. The aim of this study was to determine whether differences in the cellular composition of the immune infiltrate in breast cancer influence survival and treatment response, and construct a stromal immunotype which could predict the response of neoadjuvant therapy and survival. Patients and Methods A total of 1502 ER negative breast cancers from TCGA and METABRIC cohort were used to infer the proportions of 22 subsets of immune cells, Another 320 ER negative breast cancer patients from Guangdong Provincial People’s Hospital in the validation cohort were also included in the study. Immune cell infiltration was evaluated by immunohistochemical staining or CIBERSORT method, Five immune features were selected out of 22 immune features to construct immunotype based on LASSO Cox regression model. ResultOf the cell subsets investigated, CD8+ T cells (hazard ratio [HR] = 0.064, 95% CI 0.018-0.234; p &amp;lt; 0.001), CD4+ T cells (HR 0.072, 95% CI 0.013-0.382; p = 0.002), B cells (HR 0.04, 95% CI 0.005-0.340; p = 0.003) , M1 macrophages(HR 0.09, 95% CI 0.016-0.502; p = 0.006) were associated with favourable outcome. T regulatory cells(HR 10.791, 95% CI 2.059-58.444; p = 0.005) emerged as the most strongly associated with poor outcome. Using the LASSO model, we classified ER negative breast cancer patients into stromal immunotype A subgroup (CD8+T cellshigh CD4+T cellshigh B cellhigh M1 macrophageshigh Treglow) and stromal immunotype B subgroup (CD8+T cellslow CD4+T cellslow B celllow M1 macrophageslow Treg high). Significant differences were found between immunotype A and immunotype B in the combined cohort with 10-year overall survival (66.2% vs. 49.8%; P&amp;lt;0.001) and 10-year disease-free survival (63.8% vs. 44.4%; P&amp;lt;0.001). Stromal immunotype was revealed to be an independent prognostic indicator in multivariate analysis in all cohorts separately, and also showed to be related to pCR in neoadjuvant chemotherapy. Finally, stromal immunotype A showed higher immune checkpoint molecules (PD-L1, PD-1, CTLA-4) expression and three important cytokines expression profiles (IL-2, INF-γ and TGF-β). Conclusion The stromal immunotypes could predict survival and recurrence of ER negative breast cancer patients effectively. Furthermore, the immunotypes might be a practical predictive tool for immunotherapy and neoadjuvant chemotherapy. Citation Format: Fei Ji, Ciqiu Yang, Liulu Zhang, Mei Yang, Jieqing Li, Hongfei Gao, Teng Zhu, Minyi Cheng, Kun Wang. Identification and validation of stromal immunotype predict survival and benefit from neoadjuvant chemotherapy in patients of breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS4-11.

The S100 calcium-binding protein A11 promotes hepatic steatosis through RAGE-mediated AKT-mTOR signaling

RationaleNonalcoholic fatty liver disease (NAFLD), the most common cause of chronic liver disease, has become an increasingly severe public health problem. However, the underlying mechanism for the occurrence and development of NAFLD remains largely unknown. S100 calcium-binding protein A11 (S100A11) is a multifunctional protein previously reported to be a poor prognostic indicator of hepatocellular carcinoma, while the role of S100A11 affects NAFLD is still not clear. MethodsImmunohistochemical staining was performed using human NAFLD and control biopsy specimens. Serum level of S100A11 were analyzed by Elisa assays. The S100A11 over-expressed/ knocked-down model was established in vitro or in vivo. The expression levels of genes related to lipid metabolism in liver tissue were performed by quantitative PCR and western blotting. Hepatic lipid accumulation was determined by biochemical measurements and histochemistry. ResultsWe showed that the concentration of serum S100A11 was significantly elevated in NAFLD patients, and expression of S100A11 was remarkedly increased in the livers of NAFLD patients and mouse models. Overexpression of S100A11 in vivo markedly increased liver steatosis, body weight, and serum aspartate aminotransaminase (AST) levels. Mechanistically, our results demonstrated that S100A11 acted as a positive regulator of AKT/mTOR signaling to induce lipid synthesis and aggravate lipid deposition. ConclusionsThese results provide evidence for a novel role of S100A11 that contributes to hepatic steatosis, suggesting that targeting S100A11 may be an alternative approach for the treatment of NAFLD.

RGFP966, a selective HDAC3 inhibitor, ameliorates allergic and inflammatory responses in an OVA-induced allergic rhinitis mouse model

RGFP966 is a selective inhibitor of histone deacetylase 3 (HDAC3) playing crucial roles in triggering allergic and inflammatory responses. Whereas, its role in allergic rhinitis (AR) remains uncertain. This study sought to illustrate the role and mechanism of HDAC3 inhibitor RGFP966 on allergic and inflammatory responses in murine AR. RGFP966 administration was applied on murine AR. HE staining, PAS staining, toluidine blue staining, immunohistochemistry staining and real-time PCR methods were used to assess eosinophils, goblet cells, mast cells, HDAC3 positive cells and mRNA levels in nasal tissues of mice. HDAC3 activities in nasal tissues were quantified with HDAC3 Activity Assay Kit. We collected blood and nasal lavage fluid (NLF) of mice for assaying IgE, inflammatory cytokines and inflammatory cells. Results indicated that RGFP966 intervention attenuated sneezing, nose rubbing, IgE, inflammatory cytokines, eosinophils, goblet cells, mast cells, inflammatory cells, HDAC3 levles and activities in RGFP966 treated mice. In conclusion, RGFP966 might reduce HDAC3 expression and HDAC3 activities, and then eosinophils and mast cells recruitment, goblet cells proliferation and inflammatory cytokines levels are decreased, resulting in the alleviation of allergic and inflammatory responses in AR mice.

In-Depth Immune-Oncology Studies of the Tumor Microenvironment in a Humanized Melanoma Mouse Model.

The presence and interaction of immune cells in the tumor microenvironment is of significant importance and has a great impact on disease progression and response to therapy. Hence, their identification is of high interest for prognosis and treatment decisions. Besides detailed phenotypic analyses of immune, as well as tumor cells, spatial analyses is an important parameter in the complex interplay of neoplastic and immune cells—especially when moving into focus efforts to develop and validate new therapeutic strategies. Ex vivo analysis of tumor samples by immunohistochemistry staining methods conserves spatial information is restricted to single markers, while flow cytometry (disrupting tissue into single cell suspensions) provides access to markers in larger numbers. Nevertheless, this comes at the cost of scarifying morphological information regarding tissue localization and cell–cell contacts. Further detrimental effects incurred by, for example, tissue digestion include staining artifacts. Consequently, ongoing efforts are directed towards methods that preserve, completely or in part, spatial information, while increasing the number of markers that can potentially be interrogated to the level of conventional flow cytometric methods. Progression in multiplex immunohistochemistry in the last ten years overcame the limitation to 1–2 markers in classical staining methods using DAB with counter stains or even pure chemical staining methods. In this study, we compared the multiplex method Chipcytometry to flow cytometry and classical IHC-P using DAB and hematoxylin. Chipcytometry uses frozen or paraffin-embedded tissue sections stained with readily available commercial fluorophore-labeled antibodies in repetitive cycles of staining and bleaching. The iterative staining approach enables sequential analysis of a virtually unlimited number of markers on the same sample, thereby identifying immune cell subpopulations in the tumor microenvironment in the present study in a humanized mouse melanoma model.

The effect of miR-471-3p on macrophage polarization in the development of diabetic cardiomyopathy

AimsThe imbalance of M1/M2 macrophage ratio promotes the occurrence of diabetic cardiomyopathy (DCM), but the precise mechanisms are not fully understood. The aim of this study was to investigate whether miR-471-3p/silent information regulator 1 (SIRT1) pathway is involved in the macrophage polarization during the development of DCM. MethodsImmunohistochemical staining was used to detect M1 and M2 macrophages infiltration in the heart tissue. Flow cytometry was used to detect the proportion of M1 and M2 macrophages. Expression of miR-471-3p was quantified by real time quantitative-PCR. Transfection of miRNA inhibitor into RAW264.7 cells was performed to investigate the underlying mechanisms. Bioinformatics methods and western blotting were used to explore the target gene of miR-471-3p and further confirmed by dual luciferase reporter assay. Key findingsWe observed that M1 macrophages infiltration in the heart of tissue in DCM while M2 type was decreased. M1/M2 ratio was increased significantly in bone marrow-derived macrophages (BMDMs) from db/db mice and in RAW264.7 cells treated with advanced glycation end products (AGEs). Meanwhile, miR-471-3p was significantly upregulated in RAW264.7 cells induced by AGEs and inhibition of miR-471-3p could reduce the inflammatory polarization of macrophages. Bioinformatics analysis identified SIRT1 as a target of miR-471-3p. Both dual luciferase reporter assay and western blotting verified that miR-471-3p negatively regulated SIRT1 expression. SIRT1 agonist resveratrol could downregulate the increased proportion of M1 macrophages induced by AGEs. ConclusionOur results indicated that the development of DCM was related to AGEs-induced macrophage polarized to M1 type through a mechanism involving the miR-471-3p/SIRT1 pathway.

Secretome improves testosterone and androgen-binding protein production in testicular dysfunction rats induced by cisplatin.

Objective:This study was determined to see the effects of secretome on cellular production of testosterone and androgen-binding protein (ABP) using immunohistochemistry in testicle dysfunction due to cisplatin.Materials and Methods:Forty-eight rats were divided into four groups: the healthy group, the group with testicular dysfunction, the secretome-treated group with 0.2 ml/kg body weight (BW), and the secretome-treated group with 0.3 ml/kg BW. The immunohistochemistry staining method is used to find out testosterone and ABP reactivity in tissue organs.Results:Very strong testosterone and ABP immunoreactivity were found in Leydig cells of normal testes. While in the Leydig cells of cisplatin-induced testicles, testosterone and ABP immunoreactivity were not observed. Testosterone and ABP were observed 1 week after the second secretome injection. The number of testosterone-immunoreactive cells in the low dose group from week 1 to 3 was 0, 19, and 32, respectively. From week 1 until week 4, the high dose group was 0, 29, 33, and 65, respectively. The number of ABP-immunoreactive cells from the first week until the third week in the low dose group was 0, 28, and 34, respectively. The high dose group from the first week until the fourth week was 0, 26, 58, and 83, respectively. The number of cells that produce testosterone and ABP increased from week 2 to 4.Conclusion:The administration of secretome could increase the number of immunoreactive cells toward testosterone and ABP in testicular dysfunction.