Expression Levels Of Molecules Research Articles

Adhesion of pancreatic tumor cell clusters by desmosomal molecules enhances early liver metastases formation

Desmosomes are intercellular adhesion complexes providing mechanical coupling and tissue integrity. Previously, a correlation of desmosomal molecule expression with invasion and metastasis formation in several tumor entities was described together with a relevance for circulating tumor cell cluster formation. Here, we investigated the contribution of the desmosomal core adhesion molecule desmoglein-2 (DSG2) to the initial steps of liver metastasis formation by pancreatic cancer cells using a novel ex vivo liver perfusion mouse model. We applied the pancreatic ductal adenocarcinoma cell line AsPC-1 with and without a knockout (KO) of DSG2 and generated mouse lines with a hepatocyte-specific KO of the known interacting partners of DSG2 (DSG2 and desmocollin-2). Liver perfusion with DSG2 KO AsPC-1 cells led to smaller circulating cell clusters and a reduced number of cells adhering to murine livers compared to control cells. While this was independent of the expression levels of desmosomal adhesion molecules in hepatocytes, we show that increased cluster size of cancer cells, which correlates with stronger cell–cell adhesion and expression of desmosomal molecules, is a major factor contributing to the early phase of metastatic spreading. In conclusion, impaired desmosomal adhesion results in reduced circulating cell cluster size, which is relevant for seeding and attachment of metastatic cells to the liver.

Modulation of MHC expression by interferon-gamma and its influence on PBMC-mediated cytotoxicity in canine mast cell tumour cells

Immunotherapy is a promising alternative treatment for canine mast cell tumour (MCT). However, evasion of immune recognition by downregulating major histocompatibility complex (MHC) molecules might decline treatment efficiency. Enhancing MHC expression through interferon-gamma (IFN-γ) is crucial for effective immunotherapy. In-house and reference canine MCT cell lines derived from different tissue origins were used. The impacts of IFN-γ treatment on cell viability, expression levels of MHC molecules, as well as cell apoptosis were evaluated through the MTT assay, RT-qPCR and flow cytometry. The results revealed that IFN-γ treatment significantly influenced the viability of canine MCT cell lines, with varying responses observed among different cell lines. Notably, IFN-γ treatment increased the expression of MHC I and MHC II, potentially enhancing immune recognition and MCT cell clearance. Flow cytometry analysis in PBMCs-mediated cytotoxicity assays showed no significant differences in overall apoptosis between IFN-γ treated and untreated canine MCT cell lines across various target-to-effector ratios. However, a trend towards higher percentages of late and total apoptotic cells was observed in the IFN-γ treated C18 and CMMC cell lines, but not in the VIMC and CoMS cell lines. These results indicate a variable response to IFN-γ treatment among different canine MCT cell lines. In summary, our study suggests IFN-γ's potential therapeutic role in enhancing immune recognition and clearance of MCT cells by upregulating MHC expression and possibly promoting apoptosis, despite variable responses across different cell lines. Further investigations are necessary to elucidate the underlying mechanisms and evaluate IFN-γ's efficacy in in vivo models.

Lung antimicrobial proteins and peptides: From host defense to therapeutic strategies.

Representing severe morbidity and mortality globally, respiratory infections associated with chronic respiratory diseases, including complicated pneumonia, asthma, interstitial lung disease, and chronic obstructive pulmonary disease, are a major public health concern. Lung health and the prevention of pulmonary disease rely on the mechanisms of airway surface fluid secretion, mucociliary clearance, and adequate immune response to eradicate inhaled pathogens and particulate matter from the environment. The antimicrobial proteins and peptides contribute to maintaining an antimicrobial milieu in human lungs to eliminate pathogens and prevent them from causing pulmonary diseases. The predominant antimicrobial molecules of the lung environment include human α- and β-defensins and cathelicidins, among numerous other host defense molecules with antimicrobial and antibiofilm activity such as PLUNC (palate, lung, and nasal epithelium clone) family proteins, elafin, collectins, lactoferrin, lysozymes, mucins, secretory leukocyte proteinase inhibitor, surfactant proteins SP-A, and SP-D, and RNases. It has been demonstrated that changes in antimicrobial molecule expression levels are associated with regulating inflammation, potentiating exacerbations, pathological changes, and modifications in chronic lung disease severity. Antimicrobial molecules also display roles in both anticancer and tumorigenic effects. Lung antimicrobial proteins and peptides are promising alternative therapeutics for treating and preventing multidrug-resistant bacterial infections and anticancer therapies.

Effects of Aeromonas infection on the immune system, physical barriers and microflora structure in the intestine of juvenile grass carp (Ctenopharyngodon idella)

Grass carp (Ctenopharyngodon idella) is an intensively cultured and economically important herbivorous fish species in China, but its culture is often impacted by Aeromonas pathogens such as Aeromonas hydrophila and Aeromonas veronii. In this study, healthy grass carp were separately infected with A. hydrophila or A. veronii for 12, 24, 48 or 72 h. The results showed that the mRNA expression levels of intestinal inflammatory factors (tnf-α, il-1β and il-8), complement factors (c3 and c4), antimicrobial peptides (hepcidin, nk-lysin and β-defensin-1), immunoglobulins (igm and igt), and immune pathway-related signaling molecules (tlr1, tlr2, tlr4, myd88, irak4, irak1, traf6, nf-κb p65 and ap-1) were differentially upregulated in response to A. hydrophila and A. veronii challenge. Additionally, the expression levels of the intestinal pro-apoptotic genes tnfr1, tnfr2, tradd, caspase-8, caspase-3 and bax were significantly increased, whereas the expression of the inhibitory factor bcl-2 was significantly downregulated, indicating that Aeromonas infection significantly induced apoptosis in the intestine of grass carp. Moreover, the expression of intestinal tight junction proteins (occludin, zo-1, claudin b and claudin c) was significantly decreased after infection with Aeromonas. Histopathological analysis indicated the Aeromonas challenge caused severe damage to the intestinal villi with adhesions and detachment of intestinal villi accompanied by severe inflammatory cell infiltration at 12 h and 72 h. The 16S rRNA sequencing results showed that Aeromonas infection significantly altered the structure of the intestinal microflora of the grass carp at the phylum (Proteobacteria, Fusobacteria, Bacteroidetes and Firmicutes) and genus (Proteus, Cetobacterium, Bacteroides, and Aeromonas) levels. Take together, the findings of this study revealed that Aeromonas infection induces an intestinal immune response, triggers cell apoptosis, destroys physical barriers and alters microflora structure in the intestine of juvenile grass carp; the results will help to reveal the pathogenesis of intestinal bacterial diseases in grass carp.

High-fat diet-induced increase in glucocorticoids contributes to adipogenesis in obese mice

BackgroundThis study was designed to examine how glucocorticoids (GCs) induced by a long-term ingestion of high-fat diet (HFD) mediate the HFD-induced adipose expansion and obesity. Material and methodsTo address this goal, we used a unique L/L mouse model that fails to induce its corticosterone (CORT) level, a major type of GCs in rodents, after prolonged exposure to an HFD. ResultsWe found that, after receiving a 12-week HFD feeding, the L/L mice show less weight gain, milder adipose expansion, and higher plasma levels of triglycerides than the wild-type mice. These changes were reversed by replenishing CORT to L/L mice. When examining the expression levels of various molecules linked to lipid uptake and de novo lipogenesis in CORT-induced adipose expansion, we observed a reduction in the expression of adipose preadipocyte factor 1 (Pref-1), a key regulator in adipogenesis. In 3T3-L1 preadipocyte-like cells, dexamethasone, an agonist of the glucocorticoid receptor, also reduced expressions of Pref-1 and facilitated intracellular accumulation of lipids. ConclusionsOur results suggest that fat ingestion-induced release of CORT contributes to adipose expansion and development of obesity and highlight the pathogenic role of CORT-mediated downregulation of adipose Pref-1 in diet-induced obesity.

The association between endometrial polyps and insulin resistance from the expression of PI3K and AKT proteins perspective

BackgroundInsulin resistance (IR) induces hyperinsulinemia, which activates downstream signaling pathways such as the phosphatidylinositol-3-kinase/protein kinase B (PI3K/AKT) pathway, ultimately leading to abnormal proliferation and apoptosis of endometrial cells. This is thought to be a key pathogenic mechanism underlying the development of endometrial polyps (EP). This study aims to investigate the relationship between IR and the development of EP, the expression levels of downstream signaling molecules, including PI3K and AKT, and related laboratory parameters were examined.MethodsA total of 100 patients who visited the gynecology outpatient clinic of Zhongda Hospital affiliated with Southeast University from May 2021 to March 2023 and were diagnosed with abnormal endometrial echoes by vaginal ultrasound and underwent hysteroscopic diagnostic curettage were enrolled in this study. General data and relevant hematological indicators were compared, and intraoperative specimens were obtained for pathological examination. Possible factors influencing the development of endometrial polyps were analyzed using Pearson correlation analysis and logistic regression analysis.ResultsIn terms of body mass index, waist circumference, fasting insulin, insulin resistance index, serum total testosterone, and free testosterone index, women of childbearing age in the endometrial polyp group had higher values than those in the non-polyp group, while sex hormone-binding globulin in the endometrial polyp group was lower than that in the non-polyp group, and the differences were statistically significant (P < 0.05). The expression scores and mRNA expression levels of PI3K and AKT proteins were higher in the EP group than in the non-EP group (p < 0.05). Pearson correlation analysis showed a positive correlation between HOMA-IR and the expression scores of PI3K and AKT proteins (p < 0.01).ConclusionsInsulin resistance and abnormal activation of the phosphatidylinositol 3-kinase/protein kinase B signaling pathway may be potential pathogenic mechanisms for the development of endometrial polyps.

Mechanism of Dabugan Decoction in treatment of generalized anxiety disorder based on network pharmacology and experimental verification

This study aims to explore the mechanism of Dabugan Decoction in the treatment of generalized anxiety disorder(GAD) based on network pharmacology, molecular docking, and animal experiments. Network pharmacology and molecular docking technology were used to obtain the possible targets and related signaling pathways of Dabugan Decoction in the treatment of GAD. The GAD rat model was established, and the corresponding drugs were given by gavage after randomization. After 28 days of continuous intervention, the anxiety state of rats was detected, and the pathological changes of the hippocampus were detected in each group. ELISA and Western blot were used to detect the protein expression levels of related molecules. A total of 65 drug compounds in Dabugan Decoction were obtained, involving 403 targets of action, 7 398 disease targets of GAD, and 279 common targets of &quot;drug-disease&quot;. The key nodes in the protein-protein interaction(PPI) network were Akt1, TNF, IL-6, TP53, IL-1β, etc. Function analysis of Gene Ontology(GO) and enrichment analysis of Kyoto Encyclopedia of Genes and Genomes(KEGG) showed that the PI3K-Akt signaling pathway was the most important pathway. The results of molecular docking showed that the core components of the drug had good binding activity with the corresponding key targets. Animal experiments showed that Dabugan Decoction could effectively improve the anxiety behavior of rats and increase the open arm end movement distance and total distance of rats in the elevated cross labyrinth, the number and stay time of entering the open box, and the time(%) and the number of entering the center of the open field. At the same time, HE staining and Nicil staining showed that the number of hippocampal nerve cells in rats increased, and they were closely arranged. The damage to the cell body was improved, and there was an increase in Nissl substances in the cells. The expression of TNF-α, IL-6, and IL-1β in rat hippocampus decreased, and the expression of TP53, p-Akt1, and p-PI3K increased. The mechanism may be related to the activation of the PI3K-Akt signaling pathway and the inhibition of inflammatory response. Dabugan Decoction can play a good therapeutic and regulatory role in GAD, reflecting the overall effect of traditional Chinese medicine(TCM) compound and the characteristics of multiple targets and multiple pathways. At the same time, it is preliminarily discussed that the state of GAD may be improved by Dabugan Decoction via-activating PI3K-Akt signaling pathway and inhibiting inflammatory response and anti-apoptosis, thus providing experimental data support for the clinical application of Dabugan Decoction.

Identification of Circular RNAs as Biomarker Candidates in Lung Cancer Treatment.

Lung cancer is the most common malignancy and among the leading cause of cancer death worldwide. Therefore, there is an important need for biomarkers that can be used in the early diagnosis of the disease and in the follow-up of treatment. Circular RNAs (circRNAs) have a covalently closed circular structure that lacks 3' and 5' polar ends and is resistant to RNAase enzymes. Due to these properties, they can be stably found in body fluids. Therefore, they can serve as potential biomarkers in the diagnosis, monitoring of therapeutic response and prognosis of cancer. In our study, we aimed to investigate the expression levels of circRNA molecules in the treatment of lung cancer and to determine those that have the potential to be biomarkers. In this in vitro study, expression levels of 163 circRNAs were investigated in A549 cells, a non-small cell lung cancer cell line, before and after treatment with carboplatin and pemetrexed. Total RNA isolation and cDNA synthesis were performed after treatments. Expression levels of circRNA genes were determined by RT-qPCR method with the designed divergent primer sequences. The study revealed the characterisation of differentially expressed circRNAs by treatment in lung cancer cells. Of them, hsa_circ_0001320 is not expressed in cancer cells, is expressed only after treatment, and increased the level of its expression in response to combination therapy. As a result, while carboplatin, pemetrexed, and combined drug applications changed the expression levels of some circRNAs in lung cancer cells, some circRNAs were expressed only after treatment. In treatment follow-up and management, hsa_circ_0001320 has been identified as potential biomarker candidate.

The Role of Notch Signaling Pathway in Adult Patients with Epstein-Barr Virus-induced Infectious Mononucleosis

To investigate the changes of Notch signaling molecules and Th22 cells in adult patients with infectious mononucleosis (IM), and assess the regulatory function of Notch signaling inhibition to Th22 cells. Forty-two IM patients and twenty-one healthy controls were enrolled in this study. Their peripheral blood was collected, from which plasma and peripheral blood mononuclear cells (PBMCs) were isolated. Plasma interleukin (IL)-17 and IL-22 were measured by enzyme-linked immunosorbent assay. The percentages of CD3+ CD4+ IL-17+ Th17 cells and CD3+ CD4+ IL-22+ Th22 cells were investigated by flow cytometry. The mRNA relative levels corresponding to Th17 transcription factor retinoic acid related orphan receptor γt (RORγt), Th22 transcription factor aryl hydrocarbon receptor (AhR), and Notch signaling pathway molecules (including Notch receptors, Notch ligands, Notch downstream molecules) were semi-quantified by real-time PCR. CD4+ T cells were purified and stimulated with γ-secretase inhibitor (GSI). Cellular proliferation, Th17 and Th22 percentage, IL-17 and IL-22 secretion, transcription factor mRNA were measured in response to GSI stimulation. The relative expression levels of Notch1 and Notch2 mRNA in PBMCs of IM group were 13.58±3.18 and 4.73±1.16, respectively, which were significantly higher than 1.09±0.12 and 1.07±0.15 in PBMCs of control group (both P < 0.001). However, there were no significant differences in relative expression levels of Notch3 and Notch4 mRNA between IM group and control group (P >0.05). The relative expression levels of Notch ligands (including DLL1 and Jagged1 ) mRNA and Notch downstream molecules (including Hes1, Hes5, and Hey1 ) were increased in IM group compared with control group (all P < 0.001). In IM group, the Th17 and Th22 percentage were 5.03%±1.15% and 4.48%±1.29%, respectively, which were both higher than 4.36%±0.82% and 3.83%±0.55% in control group (both P < 0.05). In IM group, the IL-17 and IL-22 level were (301.1±53.82) and (101.2±16.45) pg/ml, respectively, which were both higher than (237.2±72.18) and (84.75±11.83) pg/ml in control group (both P < 0.001). In IM group, the relative expression levels of RORγt and AhR mRNA were 1.25±0.22 and 1.21±0.12, respectively, which were both higher than 0.99±0.15 and 1.04±0.11 in control group (both P < 0.001). There were no remarkable differences in CD4+ T cell proliferation, Th17 percentage, IL-17 secretion, and relative expression level of RORγt mRNA between cells with GSI stimulation and without GSI stimulation (P >0.05). GSI stimulation reduced Th22 percentage, IL-22 secretion, and relative expression level of AhR mRNA compared with non-stimulation (all P < 0.05). Notch signaling pathway regulates IL-22 secretion by CD4+ T cells via AhR in IM patients. Notch-AhR-Th22 pathway may take part in the pathogenesis of IM.

Effect of SLAMF8 on the phagocytosis of tumor-associated macrophages in colorectal cancer.

e14571 Background: Phagocytosis plays an essential role in innate immunity and is key in bridging innate immunity and adaptive immunity. Phagocytosis checkpoints promote tumor immune escape by inhibiting phagocytosis by tumor-associated macrophages (TAMs). SLAMF8, which is mainly expressed on the surface of TAMs, may be involved in regulating the tumor immune microenvironment. However, its regulation of the phagocytosis of TAMs remains unclear. The present study aims to investigate the effect of SLAMF8 on the phagocytosis of TAMs and to provide a new target for tumor immunotherapy. Methods: The dataset including 458 colorectal cancer (CRC) tissues from the TCGA database was used to analyze the correlation between the expression of SLAMF8 and several classical phagocytosis checkpoint molecules, and the above correlation was further verified by the RNA-sequencing data of 20 CRC tissues in our center. In vitro, the effects of SLAMF8 expression level on tumor cell proliferation and macrophage phagocytosis were also evaluated. RNA sequencing was used to analyze the effect of SLAMF8 on the expression of genes related to promoting macrophage phagocytosis. The effect of SLAMF8 on the expression levels of proteins related to FcR-mediated phagocytosis was detected by flow cytometry. Results: The results from the TCGA dataset and our validation dataset both showed that the SLAMF8 expression was significantly positively correlated with the expression levels of classical phagocytosis checkpoint molecules such as SIGLEC11, SIGLEC10, SIGLEC9, CD274and LILRB1. In vitro, functional tests showed that the phagocytosis of CT26 CRC cells by macrophages was significantly enhanced after SLAMF8 knockdown. RNA sequencing results suggested that the expression levels of molecules related to FcR-mediated phagocytosis were significantly upregulated after the decreased expression of SLAMF8.Flow cytometry analysis confirmed that SLAMF8 inhibited the expression levels of FcR-mediated phagocytosis related proteins such as CD16/32, CD64, and CD11b. Conclusions: SLAMF8 may release the “don’t-eat me” signal to inhibit FcR-mediated phagocytosis of tumor cells by macrophage. Further studies are needed to investigate the underlying mechanism of SLAMF8 on the regulation of macrophage phagocytosis and to test its possibility as a new immunotherapy target.

Association of N-adhesin and focal adhesion kinase with frequency of tumor spread through the airspace (STAS) in stage I lung carcinoma and correlation with recurrence.

e20020 Background: Spread through air spaces (STAS) is one of the many modes of lung cancer dissemination. Aberrant activation of epithelial-mesenchymal transition (EMT) has been associated with enhanced tumor cell migration and invasiveness, a process that is accompanied by a decrease in adhesion molecules that maintain contact between epithelial cells, leading to cell separation and increased cell motility. Our objectives were to investigate the effect of adhesion molecule expression levels on STAS occurrence and postoperative recurrence in patients undergoing radical resection for stage I lung carcinoma. Methods: We examined the association of adhesion molecules with STAS as the primary endpoint and the Disease-Free Survival (DFS) as a secondary endpoint. E-cadherin, P-cadherin, N-cadherin, focal adhesion kinase (FAK), epithelial cell adhesion molecule (EpCAM) neural cell adhesion molecule 1 (NCAM1), vascular cell adhesion molecule 1 (VCAM1), intercellular cell adhesion molecule-1 (ICAM-1) were analyzed by immunohistochemistry in patients undergoing radical resection for stage I lung carcinoma. Associations between those adhesion molecules expression levels and STAS were determined by using the chi-square test and logistic regression model. DFS was analyzed by using the log-rank test and Cox proportional risk model. Results: As of January 1, 2024, 12 of 60 patients undergoing radical resection for stage I lung carcinoma had a disease recurrence. All of 60 patients’ tissue specimens were retrospectively analyzed, and there were no significant differences between patients with STAS-positive(n=30) and STAS-negative(n=30) in baseline clinicopathologic features, except for histological growth patterns, and lymphovascular invasion.Higher incidence of STAS was significantly associated with low expression of E-cadherin (P=0.002), high expression of N-cadherin (P=0.018) and FAK (P=0.027), and males (P=0.026), independent of tumor size, age, and clinical stage. Multivariate analysis showed that STAS (P=0.025), low E-cadherin/high N-cadherin (P=0.038), low E-cadherin/high FAK (P=0.025), and high N-cadherin/FAK (P=0.003) were significantly correlated with a higher risk of recurrence. In addition, females (P=0.034), and high N-cadherin/FAK (P=0.032) were associated with a high risk of recurrence in patients with STAS. Conclusions: Low expression of e-cadherin, high expression of N-cadherin and FAK were independent predictors of higher incidence of STAS. Tumors with low E-cadherin/high N-cadherin, low E-cadherin/high FAK, and high N-cadherin/FAK expression were important predictors of recurrence in patients with stage I lung carcinoma. Additionally, high N-cadherin/FAK expression in tumors provides information about a higher risk of recurrence in STAS-positive stage I lung carcinoma.

Synergistic effect of chrysin and kaempferol in ameliorating Cerebral Ischemic Reperfusion injury in rat by controlling expression of proinflammatory mediators NF-κB and STAT3

ABSTRACT Objectives The two flavonoids kaempferol and chrysin are known to possess anti-inflammatory and antioxidant activities. In addition, these two flavonoids were reported to display synergistic effects against inflammation. The present study aims to provide an analysis of the combined effects of kaempferol and chrysin on ischemic rat brain induced by endothelin-1. Methods The neurological deficit score and infarct area of the brain were determined post drug treatment. Histopathological sections displayed the morphological changes in the brain tissue. The brain tissues were processed for assessing the antioxidant and anti-inflammatory activity by measuring superoxide dismutase activity, catalase activity, level of reduced glutathione, brain malondialdehyde, and amount of calcium. The expression level of inflammatory molecules was analyzed by western blotting and immunohistochemistry. Results The infarct area, neurological score and NF-κB and STAT3 expression levels were significantly reduced. Discussion The analysis of neuroprotective synergistic activity of kaempferol and chrysin indicated the therapeutic potential of the combination in alleviating cerebral ischemia by controlling expression of proinflammatory mediators.

Brunonianines D-F, three new C19-diterpenoid alkaloids from the Delphinium brunonianum, with therapeutic effect on ovarian cancer in vitro and in vivo

The current standard treatment for ovarian cancer consists of surgery to reduce the size of the tumor, followed by treatment with chemotherapeutic drugs, which have major side effects. Therefore, finding a new natural product drug with fewer side effects is a strategy. Delphinium brunonianum (D. brunonianum) is a traditional Tibetan medicine, mainly from southern Tibet, China, whereas the chemical constituents in this plant remain elusive. The major metabolites in the dichloromethane fraction of D. brunonianum were analyzed and purified by HPLC and various column chromatography techniques. Nine diterpenoid alkaloids (1–9) and one amide alkaloid (10) were isolated from D. brunonianum, including three novel C19-type diterpenoid alkaloids (Brunonianines D-F) (1–3). Their structures were elucidated by 1D/2D NMR, HR-ESI-MS and single-crystal X-ray diffraction analyses. All compounds were evaluated for toxicity in four tumor cell lines. Most of the compounds exhibited potent inhibitory effects on Skov-3 cell lines, with IC50 values ranging from 2.57 to 8.05 μM. The western blotting experiment was used to further analyze the expression levels of molecules in the Bax/Bcl-2/Caspase-3 signaling pathway for compound 1. Molecular docking was performed to predict the binding modes of Brunonianine D with target proteins. In vivo experiments were also performed and evaluated in real time by monitoring the size of the Skov-3 tumor. Additionally, tumor H&E staining and the TUNEL assay used to evaluate anti-tumor effects.

Prediction of TNFRSF9 expression and molecular pathological features in thyroid cancer using machine learning to construct Pathomics models.

The TNFRSF9 molecule is pivotal in thyroid carcinoma (THCA) development. This study utilizes Pathomics techniques to predict TNFRSF9 expression in THCA tissue and explore its molecular mechanisms. Transcriptome data, pathology images, and clinical information from the cancer genome atlas (TCGA) were analyzed. Image segmentation and feature extraction were performed using the OTSU's algorithm and pyradiomics package. The dataset was split for training and validation. Features were selected using maximum relevance minimum redundancy recursive feature elimination (mRMR_RFE) and modeling conducted with the gradient boosting machine (GBM) algorithm. Model evaluation included receiver operating characteristic curve (ROC) analysis. The Pathomics model output a probabilistic pathomics score (PS) for gene expression prediction, with its prognostic value assessed in TNFRSF9 expression groups. Subsequent analysis involved gene set variation analysis (GSVA), immune gene expression, cell abundance, immunotherapy susceptibility, and gene mutation analysis. High TNFRSF9 expression correlated with worsened progression-free interval (PFI) and acted as an independent risk factor [hazard ratio (HR) = 2.178, 95% confidence interval (CI) 1.045-4.538, P = 0.038]. Nine pathohistological features were identified. The GBM Pathomics model demonstrated good prediction efficacy [area under the curve (AUC) 0.819 and 0.769] and clinical benefits. High PS was a PFI risk factor (HR = 2.156, 95% CI 1.047-4.440, P = 0.037). Patients with high PS potentially exhibited enriched pathways, increased TIGIT gene expression, Tregs infiltration (P < 0.0001), and higher rates of gene mutations (BRAF, TTN, TG). The GBM Pathomics model constructed based on the pathohistological features of H&E-stained sections well predicted the expression level of TNFRSF9 molecules in THCA.

Rapamycin Induces Phenotypic Alterations in Oral Cancer Cells That May Facilitate Antitumor T Cell Responses.

In this study, we investigated the antitumor immunomodulatory effects of rapamycin in oral cancer. We examined the proliferation, apoptosis, and migration of cancer cells and investigated the cell surface expression levels of immune accessory molecules and T cell immune responses in vitro. We investigated the effect of in vivo administration of rapamycin on immune cell distribution and T cell immune responses in oral tumor-bearing mice. Rapamycin treatment significantly inhibited OSCC cell proliferation and migration, increased apoptotic cell death, and upregulated cell surface expression of several immune accessory and adhesion molecules, including CD40, CD83, PD-L1, PD-L2, MHC class I, P-selectin, and VCAM-1. These cancer cells augmented T cell proliferation. In vivo rapamycin administration significantly attenuated mouse tumor growth with an increased proportion of immune cells, including CD4+ T cells, CD8+ T cells, and dendritic cells (DCs); decreased the proportion of immune suppressive cells, such as myeloid-derived suppressor cells and regulatory T cells; enhanced DC maturation and upregulated the surface expression of CD40, CD86, and ICAM-1. Our results suggest that the therapeutic effect of mTOR inhibition in oral cancer can cause direct antitumor and immunomodulatory effects.

Inhibition of the rapamycin-insensitive mTORC1 /4E-BP1 axis attenuates TGF-β1-induced fibrotic response in human Tenon's fibroblasts

Subconjunctival fibrosis is the major cause of failure in both conventional and modern minimally invasive glaucoma surgeries (MIGSs) with subconjunctival filtration. The search for safe and effective anti-fibrotic agents is critical for improving long-term surgical outcomes. In this study, we investigated the effect of inhibiting the rapamycin-insensitive mTORC1/4E-BP1 axis on the transforming growth factor-beta 1(TGF-β1)-induced fibrotic responses in human Tenon's fibroblasts (HTFs), as well as in a rat model of glaucoma filtration surgery (GFS). Primary cultured HTFs were treated with 3 ng/mL TGF-β1 for 24 h, followed by treatment with 10 μM CZ415 for additional 24 h. Rapamycin (10 μM) was utilized as a control for mTORC1/4E-BP1 signaling insensitivity. The expression levels of fibrosis-associated molecules were measured using quantitative real-time PCR, Western blotting, and immunofluorescence analysis. Cell migration was assessed through the scratch wound assay. Additionally, a rat model of GFS was employed to evaluate the anti-fibrotic effect of CZ415 in vivo. Our findings indicated that both rapamycin and CZ415 treatment significantly reduced the TGF-β1-induced cell proliferation, migration, and the expression of pro-fibrotic factors in HTFs. CZ415 also more effectively inhibited TGF-β1-mediated collagen synthesis in HTFs compared to rapamycin. Activation of mTORC1/4E-BP signaling following TGF-β1 exposure was highly suppressed by CZ415 but was only modestly inhibited by rapamycin. Furthermore, CZ415 was found to decrease subconjunctival collagen deposition in rats post GFS. Our results suggest that rapamycin-insensitive mTORC1/4E-BP1 signaling plays a critical role in TGF-β1-driven collagen synthesis in HTFs. This study demonstrated that inhibition of the mTORC1/4E-BP1 axis offers superior anti-fibrotic efficacy compared to rapamycin and represents a promising target for improving the success rate of both traditional and modern GFSs.

Widespread alterations in systemic immune profile are linked to lung function heterogeneity and airway microbes in cystic fibrosis

BackgroundExcessive inflammation and recurrent airway infections characterize people with cystic fibrosis (pwCF), a disease with highly heterogeneous clinical outcomes. How the overall immune response is affected in pwCF, its relationships with the lung microbiome, and the source of clinical heterogeneity have not been fully elucidated. MethodsPeripheral blood and sputum samples were collected from 28 pwCF and an age-matched control group. Systemic immune cell subsets and surface markers were quantified using multiparameter flow cytometry. Lung microbiome composition was reconstructed using metatranscriptomics on sputum samples, and microbial taxa were correlated to circulating immune cells and surface markers expression. ResultsIn pwCF, we found a specific systemic immune profile characterized by widespread hyperactivation and altered frequencies of several subsets. These included substantial changes in B-cell subsets, enrichment of CD35+/CD49d+ neutrophils, and reduction in dendritic cells. Activation markers and checkpoint molecule expression levels differed from healthy subjects. CTLA-4 expression was increased in Tregs and, together with impaired B-cell subsets, correlated with patients' lung function. Concentrations and frequencies of key immune cells and marker expression correlated with the relative abundance of commensal and pathogenic bacteria in the lungs. ConclusionThe CF-specific immune signature, involving hyperactivation, immune dysregulation with alteration in Treg homeostasis, and impaired B-cell function, is a potential source of lung function heterogeneity. The activity of specific microbes contributes to disrupting the balance of the immune response. Our data provide a unique foundation for identifying novel markers and immunomodulatory targets to develop the future of cystic fibrosis treatment and management.

Prognostic Value of Histone Acetyl Transferase 1 (HAT-1) and Inflammatory Signatures in Pancreatic Cancer.

Pancreatic cancer is a type of gastrointestinal tumor with a growing incidence and mortality worldwide. Pancreatic ductal adenocarcinoma (PDAC) constitutes 90% of cases, and late-stage diagnosis is common, leading to a 5-year survival rate of less than 10% in high-income countries. The use of biomarkers has different proven translational applications, facilitating early diagnosis, accurate prognosis and identification of potential therapeutic targets. Several studies have shown a correlation between the tissue expression levels of various molecules, measured through immunohistochemistry (IHC), and survival rates in PDAC. Following the hallmarks of cancer, epigenetic and metabolic reprogramming, together with immune evasion and tumor-promoted inflammation, plays a critical role in cancer initiation and development. In this study, we aim to explore via IHC and Kaplan-Meier analyses the prognostic value of various epigenetic-related markers (histones 3 and 4 (H3/H4), histone acetyl transferase 1 (HAT-1), Anti-Silencing Function 1 protein (ASF1), Nuclear Autoantigenic Sperm Protein (NASP), Retinol Binding Protein 7 (RBBP7), importin 4 (IPO4) and IPO5), metabolic regulators (Phosphoglycerate mutase (PGAM)) and inflammatory mediators (allograft inflammatory factor 1 (AIF-1), interleukin 10 (IL-10), IL-12A and IL-18) in patients with PDAC. Also, through a correlation analysis, we have explored the possible interconnections in the expression levels of these molecules. Our results show that higher expression levels of these molecules are directly associated with poorer survival rates in PDAC patients, except in the case of IL-10, which shows an inverse association with mortality. HAT1 was the molecule more clearly associated with mortality, with a hazard risk of 21.74. The correlogram demonstrates an important correlation between almost all molecules studied (except in the case of IL-18), highlighting potential interactions between these molecules. Overall, our study demonstrates the relevance of including different markers from IHC techniques in order to identify unexplored molecules to develop more accurate prognosis methods and possible targeted therapies. Additionally, our correlation analysis reveals potential interactions among these markers, offering insights into PDAC's pathogenesis and paving the way for targeted therapies tailored to individual patient profiles. Future studies should be conducted to confirm the prognostic value of these components in PDAC in a broader sample size, as well as to evaluate the possible biological networks connecting them.

Differential Gene Expression in Porcine Lung Compartments after Experimental Infection with Mycoplasma hyopneumoniae.

Mycoplasma hyopneumoniae (Mhyo) is the causative agent of porcine enzootic pneumonia (EP), as well as one of the main pathogens involved in the porcine respiratory disease complex. The host-pathogen interaction between Mhyo and infected pigs is complex and not completely understood; however, improving the understanding of these intricacies is essential for the development of effective control strategies of EP. In order to improve our knowledge about this interaction, laser-capture microdissection was used to collect bronchi, bronchi-associated lymphoid tissue, and lung parenchyma from animals infected with different strains of Mhyo, and mRNA expression levels of different molecules involved in Mhyo infection (ICAM1, IL-8, IL-10, IL-23, IFN-α, IFN-γ, TGF-β, and TNF-α) were analyzed by qPCR. In addition, the quantification of Mhyo load in the different lung compartments and the scoring of macroscopic and microscopic lung lesions were also performed. Strain-associated differences in virulence were observed, as well as the presence of significant differences in expression levels of cytokines among lung compartments. IL-8 and IL-10 presented the highest upregulation, with limited differences between strains and lung compartments. IFN-α was strongly downregulated in BALT, implying a relevant role for this cytokine in the immunomodulation associated with Mhyo infections. IL-23 was also upregulated in all lung compartments, suggesting the potential involvement of a Th17-mediated immune response in Mhyo infections. Our findings highlight the relevance of Th1 and Th2 immune response in cases of EP, shedding light on the gene expression levels of key cytokines in the lung of pigs at a microscopic level.

AKT, p-AKT, ERK1/2 and p-ERK1/2 in Mural Granulosa Cells Are Not Correlated to Different Ovarian Stimulation Protocols in Patients Undergoing Assisted Reproductive Treatment.

(1) Background: In this paper we aim to study the relationship between the expression levels of molecules involved in apoptotic/survival pathways, considered as molecular markers of oocyte competence (i.e., AKT, p-AKT, ERK1/2, and p-ERK1/2) in mural granulosa cells (MGCs) and the administration of r-FSH alone or combined with exogenous r-LH, in ovarian stimulation protocol. Moreover, we aim to evaluate oocyte competence by comparing normally cleaved embryos that were transferred in the uterus, with embryos that were arrested during in vitro culture. (2) Methods: The study included 34 normo-responder women undergoing ICSI procedures. All subjects were divided into two groups. Group A consisted of 18 women stimulated with r-FSH and used as a control group; Group B consisted of 14 women stimulated with r-FSH combined with r-LH. The MGCs were obtained from individual follicles. Immunoblot analyses were carried out to analyze the AKT, p-AKT, ERK1/2, and p-ERK1/2 levels in MGCs and to correlate them with the ovarian stimulation protocol. Furthermore, the oocyte competence was evaluated, for each follicle, according to the development of the embryo during in vitro culture and the pregnancy outcome. (3) Results: We found no significant difference in the levels of molecules in isolated MGCs between groups A and B. These results, in light of our previous research, suggest for the first time, to our knowledge, that cumulus cells and mural granulosa cells in the same follicle show different expression levels of molecules involved in the apoptotic mechanism. (4) Conclusions: Our results could clarify some controversial data in the literature where cumulative cell pools of cumulus and granulosa were analyzed, described as ovarian follicle cells, and used as markers of oocyte competence. In this paper, we found evidence that cumulus and granulosa cells need to be analyzed separately.