Intracellular Expression Levels Research Articles

An integrative lipidomics and transcriptomics study revealing Bavachin and Icariin synergistically induce idiosyncratic liver injury

Objectives: Reports of traditional Chinese medicine (TCM)-related liver injury have increased over recent years; however, identifying susceptibility-related components and biomarkers remains challenging due to the heterogeneous nature of TCM and idiosyncratic drug-induced liver injury (IDILI). Psoraleae Fructus (PF) and Epimedii Folium (EF), commonly found in TCM prescriptions, have been implicated in IDILI, but their constituents and underlying mechanisms are poorly understood. Methods: In this study, we identified bavachin (Bav) and icariin (Ica) as susceptibility components for IDILI in PF and EF using a TNF-α-mediated mouse model. Lipidomics and transcriptomics were used to investigate their related mechanism. Results: Liver biochemistry and histopathology analyses revealed that co-exposure to Bav, Ica, and a non-toxic dose of TNF-α prestimulation induced significant liver injury, while Bav and Ica alone did not. Lipidomics identified seven differentially abundant metabolites in the Bav/Ica/TNF-α group compared to the Ica/TNF-α or Bav/TNF-α groups, mainly enriched in alpha-linolenic acid (ALA), arachidonic acid (AA), and linoleic acid (LA) metabolic pathways. Additionally, transcriptomics revealed 49 differentially expressed genes (DEGs) in the Bav/TNF-α vs Bav/Ica/TNF-α and Ica/TNF-α vs Bav/Ica/TNF-α groups, primarily associated with the PI3K/AKT/mTOR signaling pathway and sphingolipid metabolism. Integrative lipidomics and transcriptomics analyses identified significant positive correlations between five differential metabolites (DMs) – PC (O-16:0_14:1), PG (22:1_20:3), PI (16:0_14:1), PS (18:0_19:2), and TG (17:0_18:2_22:5) – and ten DEGs – Nr0b2, Btbd19, Btg2, Fam222a, Fam83f, Gtse1, Anln, Gja4, Srrm4, and Zfp13. Conslusions: Collectively, these results suggest that alterations in intracellular metabolism and gene expression levels may contribute to the synergistic induction of IDILI by the incompatible pair Bav and Ica in the presence of TNF-α.

TRAF4 promotes lung cancer development by activating tyrosine kinase of EGFR

Objective: To explore the role of tumor necrosis factor receptor-associated factor 4 (TRAF4) in promoting the abnormal activation of epidermal growth factor receptor (EGFR) and its effect on lung cancer cell proliferation, migration and invasion. Methods: Tumor tissues from patients who underwent lung adenocarcinoma resection at The First Affiliated Hospital of Second Military Medical University, from January 2015 to May 2017 were collected, and the expressions of TRAF4 and Ki-67 in lung cancer tissues were detected by immunohistochemistry, the mRNA levels of Cyclin D and Vimentin were detected by real-time fluorescence quantitative PCR (qRT-PCR). The effect of TRAF4 on the tumor growth ability of lung cancer A549 cells was investigated by the xenograft model, the effect of TRAF4 or EGFR on the tumor proliferation ability was detected by using cell counting kit 8 (CCK8) and BrdU assay, and the migration and invasion abilities of tumor cells were detected by Transwell assay. Different structural domain deletion expression vectors of TRAF4 and EGFR were constructed to transfect cells, and the interaction mode of TRAF4 and EGFR was investigated by immunoprecipitation assay. Results: The expression of TRAF4 in non-small cell lung cancer (NSCLC) tissues was positively correlated with the expressions of Ki-67, cyclin D, and vimentin (r2: 0.438, 0.695, and 0.736, respectively, all P＜0.01). Immunohistochemical assay of tumor tissues from NSCLC patients showed that tissues with high expression of TRAF4 were also high in Ki-67. Patients with high TRAF4 expression (TRAF4 positivity >30%) had a shorter progression-free survival (PFS) time than that of patients with low TRAF4 expression (TRAF4 positivity ≤30%) (median PFS of 12 and 19 months, respectively; P=0.034). Traf4-/- cells had a weakened proliferative capacity than traf4+/+ cells and formed tumors with smaller size (P＜0.05). The expression level of Ki-67 in the tumor tissues formed by traf4-/- cells [(45.6±8.7)%] was lower than that in the tumor tissues formed by traf4+/+ cells [(62.3±10.3)%, P=0.015], the mRNA levels of cyclin D (1.01±0.15) and vimentin (1.01±0.12) in the traf4-/- cells were lower than those of the traf4+/+ cells (3.41±0.32 and 3.12±0.18, respectively, both P＜0.05).The western blot results showed that, with the elevated intracellular expression level of TRAF4, phosphorylation level of EGFR was significantly increased in both wild-type EGFR and activation mutant EGFR-expression cells. The capacities of proliferation, migration and invasion of A549 cells was weakened after EGFR knockdown (all P＜0.01). Immunoprecipitation experiments showed that TRAF4 binds to the peptide segment of the near-membrane region of EGFR through the TRAF structural domain, and the mutual binding between EGFR molecules was enhanced under TRAF4 overexpression conditions. Increasing TRAF4 expression promoted EGFR molecular phosphorylation and activation of downstream signaling. Conclusions: TRAF4 expression is elevated in NSCLC tissues and tumor cells, which promotes tumor proliferation, migration and invasion. TRAF4 directly binds to EGFR molecules, enhances its own phosphorylation and activates the downstream signaling pathway by promoting the interaction between EGFR molecules.

Pterostilbene suppresses head and neck cancer cell proliferation via induction of apoptosis.

Head and neck cancer (HNC) is one of the most prevalent causes of death worldwide, and so discovering anticancer agents for its treatment is very important. Pterostilbene (PS) is a trans-stilbene reported to be beneficial in managing various cancers. The objective of the study was to evaluate the cytotoxic, antiproliferative, proapoptotic, and antimigrative effect of PS on HEp-2, SCC-90, SCC-9, FaDu, and Detroit-551 cell lines. MTT and live/dead assays were employed to assess cell viability, while a cell migration test was performed to evaluate wound healing capacity. The mRNA, protein, and intracellular expression levels of CASP-3, BAX, and BCL-2 genes were evaluated by real-time PCR, western blotting, and immunofluorescence staining. Annexin V-PI staining was conducted to assess the amounts of viable, apoptotic, and necrotic cells. The results revealed that PS displayed cytotoxic, antiproliferative activity in a dose-dependent manner in HNC cells by upregulating CASP-3 and BCL-2 while downregulating BCL-2 in the apoptotic pathway. The proapoptotic properties were confirmed by the annexin-V-IP results. Moreover, PS displayed a significant suppressive efficacy on the migration capacity of HNC cells. The present study provides proof that PS has the prospective to be improved as an attractive anticancer agent against HNC following advanced studies.

Architectural engineering of Cyborg Bacteria with intracellular hydrogel

Synthetic biology primarily uses genetic engineering to control living cells. In contrast, recent work has ushered in the architectural engineering of living cells through intracellular materials. Specifically, Cyborg Bacteria are created by incorporating synthetic PEG-based hydrogel inside cells. Cyborg Bacteria do not replicate but maintain essential cellular functions, including metabolism and protein synthesis. Thus far, Cyborg Bacteria have been engineered using one primary composition of intracellular hydrogel components. Here, we demonstrate the versatility of controlling the physical and biochemical aspects of Cyborg Bacteria using different structures of hydrogels. The intracellular cell-gel architecture is modulated using a different photoinitiator, PEG-diacrylate (PEG-DA) of different molecular weights, 4arm PEG-DA, and dsDNA-PEG. We show that the molecular weight of the PEG-DA affects the generation and metabolism of Cyborg Bacteria. In addition, we show that the hybrid dsDNA-PEG intracellular hydrogel controls protein expression levels of the Cyborg Bacteria through post-transcriptional regulation and polymerase sequestration. Our work creates a new frontier of modulating intracellular gel components to control Cyborg Bacteria function and architecture.

The Effect of ZnO Nanoparticles Functionalized with Glutamine and Conjugated with Thiosemicarbazide on Triggering of Apoptosis in the Adenocarcinoma Gastric Cell Line.

Gastric carcinoma is the fourth most common malignancy worldwide. Conjugation of metal nanoparticles with thiosemicarbazones has shown considerable anti-cancer potential. Zinc oxide nanoparticles (ZnO NPs) were synthesized, functionalized by glutamine, and conjugated with thiosemicarbazide (ZnO@Gln-TSC). Fourier transform infrared spectroscopy, X-ray diffraction, scanning electron microscopy and transmission electron microscopy imaging, energy-dispersive X-ray, DLS, and zeta potential were used to characterize the NPs. The toxicity of ZnO NPs, TSC, ZnO@Gln-TSC NPs, and oxaliplatin in AGS cells and ZnO NPs and ZnO@Gln-TSC NPs in HEK293 cells was investigated by MTT assay. Cell apoptosis was evaluated by flow cytometry, caspase-3 activity, and Hoechst staining assays. The intra-cellular reactive oxygen species level and expression level of the CASP3 gene in AGS cells treated with ZnO@Gln-TSC NPs were evaluated. The NPs were in the size range of 20 to 70 nm. The DLS and zeta potential were 374 nm and -31.7 mV, respectively. In MTT, the IC50 of ZnO, TSC, oxaliplatin, and ZnO@Gln-TSC NPs for AGS cells were 130, 80.5, 67.7, and 9.8 μg/mL, respectively, and the IC50 of ZnO and ZnO@Gln-TSC NPs for HEK293 cells were 215 and 150.5 μg/mL, respectively. Flow cytometry showed higher apoptosis in the cell treated with the NPs and TSC. Apoptotic features, including cell shrinkage, were recognized. A significant increase of 5.9 folds in the level of ROS was noticed. The activity of caspase-3 and the expression level of the CASP3 gene were increased by1.83 and 1.6 folds after exposure to ZnO@Gln-TSC NPs, respectively. This study revealed the anti-cancer potential of ZnO@Gln-TSC NPs to be used for gastric cancer treatment after further in vitro and in vivo assays.

IGFBP2/ITGA5 promotes gefitinib resistance via activating STAT3/CXCL1 axis in non-small cell lung cancer

There is a paucity of comprehensive knowledge pertaining to the underlying mechanisms leading to gefitinib resistance in individuals diagnosed NSCLC harboring EGFR-sensitive mutations who inevitably develop resistance to gefitinib treatment within six months to one year. In our preceding investigations, we have noted a marked upregulation of IGFBP2 in the neoplastic tissues of NSCLC, predominantly in the periphery of the tissue, implying its plausible significance in NSCLC. Consequently, in the current research, we delved into the matter and ascertained the molecular mechanisms that underlie the participation of IGFBP2 in the emergence of gefitinib resistance in NSCLC cells. Firstly, the expression of IGFBP2 in the bronchoalveolar lavage fluid and lung cancer tissues of 20 NSCLC patients with gefitinib tolerance was found to be significantly higher than that of non-tolerant patients. Furthermore, in vitro and in vivo experiments demonstrated that IGFBP2 plays a significant role in the acquisition of gefitinib resistance. Mechanistically, IGFBP2 can activate STAT3 to enhance the transcriptional activity of CXCL1, thereby increasing the intracellular expression level of CXCL1, which contributes to the survival of lung cancer cells in the gefitinib environment. Additionally, we identified ITGA5 as a key player in IGFBP2-mediated gefitinib resistance, but it does not function as a membrane receptor in the process of linking IGFBP2 to intracellular signaling transduction. In conclusion, this study demonstrates the promoting role and mechanism of IGFBP2 in acquired gefitinib resistance caused by non-EGFR secondary mutations, suggesting the potential of IGFBP2 as a biomarker for gefitinib resistance and a potential intervention target.

Simple Fluorescence Labeling Method Enables Detection of Intracellular Distribution and Expression Level of Retinoid X Receptors.

There is no straightforward method to visualize the intracellular distribution of nuclear receptors, such as retinoid X receptors (RXRs), which are trafficked between the cytosol and nucleus. Here, in order to develop a simple fluorescence labeling method for RXRs, we designed and synthesized compound 4, consisting of an RXR-selective antagonist, CBTF-EE (2), linked via an ether bond to the fluorophore nitrobenzoxadiazole (NBD). Compound 4 is nonfluorescent, but the ether bond (-O-NBD) reacts with biothiols such as cysteine and homocysteine to generate a thioether (-S-NBD), followed by intramolecular Smiles rearrangement with an amino group such as that of lysine to form a fluorescent secondary amine (-NH-NBD) adjacent to the binding site. Fluorescence microscopy of intact or RXR-overexpressing MCF-7 cells after incubation with 4 enabled us to visualize RXR expression as well as nuclear transfer of RXR induced by the agonist bexarotene (1).

Cell-type specific molecular expression levels by restricted-dimensional cytometry.

Cytometric analysis has been commonly used to delineate distinct cell subpopulations among peripheral blood mononuclear cells by the differential expression of surface receptors. This capability has reached its apogee with high-dimensional approaches such as mass cytometry and spectral cytometry that include simultaneous assessment of 20-50 analytes. Unfortunately, this approach also engenders significant complexity with analytical and interpretational pitfalls. Here, we demonstrate a complementary approach with restricted-dimensionality to assess cell-type specific intracellular molecular expression levels at exceptional levels of precision. The expression of five analytes was individually assessed in four mononuclear cell-types from peripheral blood. Distinctions in expression levels were seen between cell-types and between samples from different donor groups. Mononuclear cell-type specific molecular expression levels distinguished pregnant from nonpregnant women and G-CSF-treated from untreated persons. Additionally, the precision of our analysis was sufficient to quantify a novel relationship between two molecules-Rel A and translocator protein-by correlational analysis. Restricted-dimensional cytometry can provide a complementary approach to define characteristics of cell-type specific intracellular protein and phosphoantigen expression in mononuclear cells.

Inflammatory factors secreted from endothelial cells induced by high glucose impair human retinal pigment epithelial cells

Abstract Objectives Diabetic retinopathy (DR) is a retinal disease that arises from impaired glucose tolerance and leads to retinal microvascular leakages. Recent studies have indicated that DR pathogenesis is linked to dysfunctional retinal pigment epithelial (RPE) cells. Methods Investigating the potential interplay between endothelial cells (ECs) and RPE cells by treating ECs with high glucose (HG) and evaluating the function of cytokines released from ECs on the growth of RPE cells. Results The results revealed that high glucose-stimulated Human Umbilical Vein Endothelial Cells (HUVECs) activated the NF-κB signaling pathway, increased intracellular levels of reactive oxygen species (ROS) and expression of caspase 3 while also elevating HUVECs delivery of cytokines such as VEGF, TNF-α, IL-6, and IL-1β. Conclusions As a result of our study, cytokines released from HG-treated HUVECs impede the growth of ARPE-19 in vitro, highlighting the importance of functional ECs for exploring the underlying mechanisms of vascular-associated retinal dysfunction. Inflammatory factors secreted from endothelial cells induced by high glucose impair human retinal pigment epithelial cells.

Pyruvate Kinase M2 Nuclear Translocation Regulate Ferroptosis-Associated Acute Lung Injury in Cytokine Storm.

Cytokine storm (CS) is linked with macrophage dysfunction and acute lung injury (ALI), which can lead to patient mortality. Glycolysis is preferentially exploited by the pro-inflammatory macrophages, in which pyruvate kinase M2 (PKM2) is a critical enzyme. The mechanism underlying the link between CS and ALI involves cell death, with the recently discovered programmed cell death known as ferroptosis being involved. However, the relationship between the glycolysis and ferroptosis in the context of CS-related ALI remains unclear. CS-associated ALI induced by poly I:C (10mg/kg, i.v) and LPS (5mg/kg, i.p) (IC: LPS) exhibit significant ferroptosis. Ferrostatin-1 (ferroptosis inhibitor) treatment attenuated IC:LPS‑induced mortality and lung injury. Moreover, Alveolar macrophage (AM) from IC:LPS model exhibited enhanced glycolysis and PKM2 translocation. The administration of ML-265(PKM2 monomer/dimer inhibitor) resulted in the formation of a highly active tetrameric PKM2, leading to improved survival and attenuation of ALI. Furthermore, ML-265 treatment decreased ferroptosis and restored the balance between anaerobic glycolysis and oxidative phosphorylation. Notably, in patients with lung infection, intracellular expression level of PKM2 were correlated with circulating inflammation. Enhanced ferroptosis and PKM2 nuclear translocation was noticed in CD14+ blood monocytes of lung infection patients with CS. In conclusion, PKM2 is a key regulatory node integrating metabolic reprograming with intra-nuclear function for the regulation of ferroptosis. Targeting PKM2 could be explored as a potential means in the future to prevent or alleviate hyper-inflammatory state or cytokines storm syndrome with aberrant ferroptotic cell death.

Deciphering the origin of developmental stability: The role of intracellular expression variability in evolutionary conservation.

Progress in evolutionary developmental biology (evo-devo) has deepened our understanding of how intrinsic properties of embryogenesis, along with natural selection and population genetics, shape phenotypic diversity. A focal point of recent empirical and theoretical research is the idea that highly developmentally stable phenotypes are more conserved in evolution. Previously, we demonstrated that in Japanese medaka (Oryzias latipes), embryonic stages and genes with high stability, estimated through whole-embryo RNA-seq, are highly conserved in subsequent generations. However, the precise origin of the stability of gene expression levels evaluated at the whole-embryo level remained unclear. Such stability could be attributed to two distinct sources: stable intracellular expression levels or spatially stable expression patterns. Here we demonstrate that stability observed in whole-embryo RNA-seq can be attributed to stability at the cellular level (low variability in gene expression at the cellular levels). We quantified the intercellular variations in expression levels and spatial gene expression patterns for seven key genes involved in patterning dorsoventral and rostrocaudal regions during early development in medaka. We evaluated intracellular variability by counting transcripts and found its significant correlation with variation observed in whole-embryo RNA-seq data. Conversely, variation in spatial gene expression patterns, assessed through intraindividual left-right asymmetry, showed no correlation. Given the previously reported correlation between stability and conservation of expression levels throughout embryogenesis, our findings suggest a potential general trend: the stability or instability of developmental systems-and the consequent evolutionary diversity-may be primarily anchored in intrinsic fundamental elements such as the variability of intracellular states.

Ilex paraguariensis A.St.-Hil. improves lipid metabolism in high-fat diet-fed obese rats and suppresses intracellular lipid accumulation in 3T3-L1 adipocytes via the AMPK-dependent and insulin signaling pathways.

Obesity is closely associated with several chronic diseases, and adipose tissue plays a major role in modulating energy metabolism. This study aimed to determine whether Mate, derived from I. paraguariensis A.St.-Hil., ameliorates lipid metabolism in 3T3-L1 adipocytes and high-fat diet (HFD)-fed obese Sprague-Dawley (SD) rats. 3T3-L1 adipocytes were cultured for 7 days, following which intracellular lipid accumulation and expression levels of lipid metabolism-related factors were examined. Dorsomorphin was used to investigate the potential pathways involved, particularly the adenosine monophosphate-activated protein kinase (AMPK)- dependent pathway. Mate was administered to rat HFD-fed obese SD models for 8 consecutive weeks. The expression of lipid metabolism-related factors in the organs and tissues collected from dissected SD rats was evaluated. Mate suppressed intracellular lipid accumulation in 3T3-L1 adipocytes, increased the protein and gene expression levels of AMPK, hormone sensitive lipase (HSL), calmodulin kinase kinase (CaMKK), liver kinase B1 (LKB1), protein kinase A (PKA), CCAAT/enhancer binding protein β (C/EBPβ), insulin receptor b (IRβ), and insulin receptor substrate 1 (IRS1) (Tyr465), and decreased those of sterol regulatory element binding protein 1C (Srebp1c), fatty acid synthase (FAS), peroxisome-activated receptor γ (PPARγ), and IRS1 (Ser1101). Furthermore, an AMPK inhibitor abolished the effects exerted by Mate on intracellular lipid accumulation and HSL and FAS expression levels. Mate treatment suppressed body weight gain and improved serum cholesterol levels in HFD-fed obese SD rats. Treatment with Mate increased the protein and gene expression levels of AMPK, PKA, Erk1/Erk2 (p44/p42), and uncoupling protein 1 and reduced those of mammalian target of rapamycin, S6 kinase, Srebp1c, ap2, FAS, Il6, Adiponectin, Leptin, and Fabp4 in rat HFD-fed obese SD models. Mate suppressed intracellular lipid accumulation in 3T3-L1 adipocytes and improved lipid metabolism in the epididymal adipose tissue of HFD-fed obese SD rats via the activation of AMPK-dependent and insulin signaling pathways.

Antibacterial activity and mechanism of chelerythrine against Streptococcus agalactiae.

Streptococcus agalactiae (S.agalactiae), also known as group B Streptococcus (GBS), is a highly infectious pathogen. Prolonged antibiotic usage leads to significant issues of antibiotic residue and resistance. Chelerythrine (CHE) is a naturally occurring benzophenidine alkaloid and chelerythrine chloride (CHEC) is its hydrochloride form with diverse biological and pharmacological activities. However, the antibacterial mechanism of CHEC against GBS remains unclear. Thus, this study aims to investigate the in vitro antibacterial activity of CHEC on GBS and elucidate its underlying mechanism. The antibacterial effect of CHEC on GBS was assessed using inhibitory zone, minimum inhibitory concentration (MIC), and minimum bactericidal concentration (MBC) assays, as well as by constructing a time-kill curve. The antibacterial mechanism of CHEC was investigated through techniques such as scanning electron microscopy (SEM) and transmission electron microscopy (TEM), measurement of alkaline phosphatase (AKP) activity, determination of Na+ K+, Ca2+ Mg2+-adenosine triphosphate (ATP) activity, observation of membrane permeability, and analysis of intracellular reactive oxygen species (ROS) and mRNA expression levels of key virulence genes. The results demonstrated that the inhibition zone diameters of CHEC against GBS were 14.32 mm, 12.67 mm, and 10.76 mm at concentrations of 2 mg/mL, 1 mg/mL, and 0.5 mg/mL, respectively. The MIC and MBC values were determined as 256 μg/mL and 512 μg/mL correspondingly. In the time-kill curve, 8 × MIC, 4 × MIC and 2 × MIC CHEC could completely kill GBS within 24 h. SEM and TEM analyses revealed significant morphological alterations in GBS cells treated with CHEC including shrinkage, collapse, and leakage of cellular fluids. Furthermore, the antibacterial mechanism underlying CHEC's efficacy against GBS was attributed to its disruption of cell wall integrity as well as membrane permeability resulting in extracellular release of intracellular ATP, AKP, Na+ K+, Ca2+ Mg2+. Additionally CHEC could increase the ROS production leading to oxidative damage and downregulating mRNA expression levels of key virulence genes in GBS cells. In conclusion, CHEC holds potential as an antimicrobial agent against GBS and further investigations are necessary to elucidate additional molecular mechanisms.

Association between lactate metabolism‑related molecules and venous thromboembolism: A study based on bioinformatics and an invitro model.

Venous thromboembolism (VTE) is characterized by a high recurrence rate and adverse consequences, including high mortality. Damage to vascular endothelial cells (VECs) serves a key role in VTE and lactate (LA) metabolism is associated with VEC damage. However, the pathogenesis of VTE and the role of lactate metabolism-related molecules (LMRMs) remain unclear. Based on the GSE48000 dataset, the present study identified differentially expressed (DE-)LMRMs between healthy individuals and those with VTE. Thereafter, LMRMs were used to establish four machine learning models, namely, the random forest, support vector machine and generalized linear model (GLM) and eXtreme gradient boosting. To verify disease prediction efficiency of the models, nomograms, calibration curves, decision curve analyses and external datasets were used. The optimal machine learning model was used to predict genes involved in disease and an in vitro oxygen-glucose deprivation (OGD) model was used to detect the survival rate, LA levels and LMRM expression levels of VECs. A total of four DE-LMRMs, solute carrier family 16 member 1 (SLC16A1), SLC16A7, SLC16A8 and SLC5A12 were obtained and GLM was identified as the best performing model based on its ability to predict differential expression of the embigin, lactate dehydrogenase B, SLC16A1, SLC5A12 and SLC16A8 genes. Additionally, SLC16A1, SLC16A7 and SLC16A8 served key roles in VTE and the OGD model demonstrated a significant decrease in VEC survival rate as well as a significant increase and decrease in intracellular LA and SLC16A1 expression levels in VECs, respectively. Thus, LMRMs may be involved in VTE pathogenesis and be used to build accurate VTE prediction models. Further, it was hypothesized that the observed increase in intracellular LA levels in VECS was associated with the decrease in SLC16A1 expression. Therefore, SLC16A1 expression may be an essential target for VTE treatment.

A pH-Responsive and MRI-Functional nanomedicine enabling targeted delivery of simvastatin in atherosclerosis

Abstract Background Simvastatin is commonly used for the treatment of atherosclerosis. However, it causes side effects in some patients and not always effective. Moreover, although its treatment effects are often attributed to its cholesterol-lowering activity, some in vitro studies suggest that simvastatin also exerts antioxidant and anti-inflammatory effects. Purpose To develop an atherosclerotic plaque microenvironment-responsive nanoparticular system to deliver and release Gadolinium (Gd3+) for magnetic resonance imaging (MRI) and simvastatin for treatment within the plaque. Methods A nanoparticle was synthesized with nanoscale coordination polymers (NCPs), a pH-responsive linker, Gd3+, and simvastatin (ST), i.e. a ST/NCP-PEG nanoparticle. The biological effects of ST/NCP-PEG nanoparticle were tested on RAW264.7 macrophages. Four weeks old ApoE-/- mice were fed on high fat diet for 4 weeks and then randomly divided into three groups for a 8-week treatment regime via weekly intravenous administration of: 1) NCP-PEG nanoparticle without ST (control); 2) ST/NCP-PEG nanoparticles; or 3) free ST. Results We have developed a pH-responsive ST/NCP-PEG nanoparticle carrying an MRI contrast agent Gd3+ and a drug molecule ST, with an 80 nm diameter in spherical shape. This nanoparticle collapsed into biodegradable components while releasing both Gd3+ and ST under the atherosclerotic plaque microenvironment with a pH 5.6. ST/NCP-PEG nanoparticles exerted strong anti-oxidant and anti-inflammatory effects in vitro, as determined by an intracellular ROS indicator DCF-DA and expression levels of cytokines (e.g. IL-6, TNF-α and MCP-1), respectively. Upon intravenous administration, ST/NCP-PEG nanoparticles, as demonstrated by IVIS imaging system, accumulated specifically in the plaques in a time-dependent manner, reaching the peak concentration at 24 hours post administration. The plaques were readily visualized by MR imaging (T1-weighted signal) due to the enrichment of Gd3+. After 8 weeks treatment, compared to the empty NCP-PEG nanoparticle, treatment with ST/NCP-PEG and free ST reduced plaque lesion size by 55% (p<0.001) and 29% (p<0.05), respectively. Moreover, ST/NCP-PEG nanoparticles showed a larger reduction in plaque lesions than free ST (p<0.05). Oil red O staining of both the whole aorta and the aortic roots generated similar results in terms of plaque lesion changes. Mechanistically, both ST/NCP-PEG and free ST reduced ROS production and increased the ratios of M2/M1 macrophages within the plaque, with ST/NCP-PEG nanoparticle treatment exerted larger therapeutic effects. Moreover, long-term treatment with ST/NCP-PEG nanoparticles did not show obvious toxicity in all major organs. Conclusions An atherosclerotic plaque microenvironment-responsive MR imaging functional nanoparticle for targeted delivery of ST to the plaque could diagnose atherosclerotic plaque and improve treatment efficacy of ST.

Melatonin as a Potential Therapeutic Tool in Allergic Rhinitis Induced by House-Dust Mite

Melatonin is known as an important regulator of circadian rhythm in humans. In the literature, there are no studies evaluating the efficacy of melatonin in the management of allergic rhinitis (AR) or nasal polyps (Np). Np tissue was taken from nasal cavity and mucosal tissue (Mu) was taken from the nasal septal area. Melatonin (25-200nM) and Mite Allergen (2.5-12.5%) were prepared in complete media. Cell viability, apoptosis, intracellular reactive oxygen species production and gene expression levels were determined. Our results showed that there is no toxic effect of Melatonin, Mite and their combination which was given to Np-MSCs and Mu-MSCs. Melatonin significantly reduced reactive oxygen species levels in both mite-treated Np-MSCs and Mu-MSCs. Indoleamine 2,3-dioxygenase level was significantly decreased in melatonin-treated cells. Cyclooxygenase-1 level was significantly decreased in melatonin-treated healthy and allergic Np-MSCs while there was no significant difference in 100 and 150nM Melatonin-treated Mu-MSCs. Interestingly, 50nM Melatonin significantly increased Cyclooxygenase-1 level in Mu-MSCs. 50, 100 and 150nm Melatonin significantly decreased Interleukin-6 level in Mite-treated Np-MSCs. In addition, 100 and 150nM Melatonin significantly decreased Interleukin-6 level in Mite-treated Mu-MSCs. Melatonin has well-established anti-oxidant and anti-neoplastic activity, could be a promising therapeutic agent in the treatment of AR and nasal polyposis.

The SP/NK1R system promotes the proliferation of breast cancer cells through NF-κB-mediated inflammatory responses.

Numerous molecules have been introduced to participate in the formation of breast cancer, the most common malignancy in women. Among them, neuropeptide substance P (SP) and its related receptor neurokinin-1 receptor (NK1R) have attracted unprecedented attention in tumorigenesis processes. In this study, we investigated the effect of the SP/NK1R pathway on the induction of oxidative stress in breast cancer and examine the therapeutic potential of NK1R inhibition in this malignancy. MCF-7 cells were treated with varying concentrations of SP and aprepitant, an FDA-approved NK1R antagonist, either as a single drug or in a combined modality. Resazurin assay was used to evaluate the anti-cancer ability of aprepitant. The alteration in the intracellular levels of reactive oxygen species (ROS) and gene expression were determined using ROS assay and the qRT-PCR analysis, respectively. The stimulation of the SP/NK1R axis in the MCF-7 cells was coupled with the accumulation of ROS as well as upregulation of NF-κB and its related pro-inflammatory cytokines, including tumor necrosis factor (TNF)-α and IL-6. In contrast, the suppression of NK1R by aprepitant halted the viability of MCF-7 cells, at least partly due to p53-mediated upregulation of p21. Moreover, aprepitant attenuated the oncogenic properties of SP by preventing the oxidative property of this neuropeptide. Overall, our results suggest that the SP/NK1R pathway might play a critical role in breast cancer pathogenesis, probably through inducing ROS/NF-κB-mediated inflammatory responses. Moreover, it seems that blockage of the axis has promising therapeutic value against breast cancer cells. Schematic representation proposed for the plausible mechanism by which the stimulation of the SP/NK1R might induce oxidative stress in breast cancer-derived MCF-7 cells. Once SP interacts with NK1R, this signaling axis could disturb the balance between the expression of p53 and NF-κB, an event that leads to the accumulation of ROS within MCF-7 cells. The produced ROS, in turn, elevates the expression of pro-inflammatory cytokines (TNF-α and IL-6) and downregulates the expression of p21. On the other hand, aprepitant, an antagonist of NK1R, could reduce the survival of proliferative capacity of MCF-7 cells by decreasing the intracellular levels of ROS and p53-mediated up-regulation of p21. Along with the effect on p53, aprepitant could also reduce the expression of NF-κB and its related pro-inflammatory cytokines.

Alcohol use disorder (AUD) is associated with enhanced sensitivity to cellular lipopolysaccharide challenge.

Inflammation has been associated with alcohol use disorder (AUD). A novel method to characterize AUD-related immune signaling involves probing Toll-like receptor (TLR)-4 stimulated monocyte production of intracellular cytokines (ICCs) via lipopolysaccharide (LPS). We evaluated relationships between AUD and ICC production at rest and after LPS stimulation. We analyzed blood samples from 36 participants (AUD N = 14; Controls N = 22), collected across time, with ICC expression assessed at rest (i.e., unstimulated) and following stimulation with LPS (i.e., a total of 5 repeated unstimulated or stimulated measures/participant). Markers assessed included tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), TNF-α and IL-6 co-expression, and interferon (IFN). For each marker, we constructed linear mixed models with AUD, LPS, and timepoint as fixed effects (BMI as covariate), allowing for random slope and intercept. AUD × LPS was included as an interaction. For TLR4-stimulated monocyte production of TNF-α, there were effects of AUD (p < 0.01), LPS (p < 0.001), and AUD × LPS interaction (p < 0.05), indicating that individuals with AUD showed greater unstimulated- and stimulated monocyte expression of TNF-α. Similarly, for TLR4-stimulated monocyte co-expression of TNF-α and IL-6, there were effects of AUD (p < 0.01), LPS (p < 0.001), and AUD × LPS interaction (p < 0.05). No AUD or LPS effects were found for IL-6. Timepoint effects were observed on IL-6 and TNF-α/IL-6 co-expression (p < 0.001). Finally, for IFN there were also effects of AUD (p < 0.05), LPS (p < 0.001), and AUD × LPS (p < 0.001). Individuals with AUD showed greater resting or unstimulated levels of intracellular monocyte expression of TNF-α and IL-6/TNF-α co-expression than controls. AUD was associated with increases in TLR4-stimulated monocyte production of TNF-α and co-production of IL-6 and TNF-α. This is, to our knowledge, the first study to investigate relationships between AUD and monocyte production of proinflammatory cytokines, at rest and in response to TLR4 stimulation with LPS. The study extends previous findings on the roles of proinflammatory cytokines in AUD and serves as a critical proof of concept for the use of this method to probe neuroimmune mechanisms underlying AUD.

Discovery of a novel marker for human granulocytes and tissue macrophages: RTL1 revisited.

Here, retrotransposon-like 1 (RTL1) is introduced as a marker for circulating and tissue neutrophils, tissue macrophages, and tumor-associated macrophages (TAM) and neutrophils (TAN). Anti-RTL1 polyclonal and monoclonal antibodies were produced, and their reactivity was examined by Western blotting (WB), ELISA, and immunostaining of human normal and cancer tissues. The reactivity of the anti-RTL1 antibodies with peripheral blood leukocytes and a panel of hematopoietic cell lines was examined. The generated antibodies specifically detected RTL1 in the WB of the placenta and U937 cells. The polyclonal antibody showed excellent reactivity with tissue-resident macrophages, Hofbauer cells, alveolar and splenic macrophages, Kupffer cells, and inflammatory cells in the tonsil, appendix, and gallbladder. In vitro GM-CSF-differentiated macrophages also showed a high level of intracellular RTL1 expression. TAM and TAN also showed excellent reactivity with this antibody. Almost all circulating granulocytes but not lymphocytes or monocytes expressed RTL1 at their surface. Serial sections of the appendix stained with CD15 and RTL1 and placenta stained with CD68 and RTL1 showed a considerable overlap in RTL1 expression in CD15+ granulocytes and CD68+ macrophages. A small percentage of myelomonocytic cell lines was positive for surface RTL1, while promyelocytic, monocytic, megaloblastic, and lymphoblastic cell lines were negative. Endothelial cells of normal and cancer tissues highly expressed RTL1. RTL1 could be considered a new marker for different normal tissue macrophages, TAM, circulating and tissue neutrophils, and TAN.

CD26lowPD-1+ CD8 T cells are terminally exhausted and associated with leukemia progression in acute myeloid leukemia.

Acute myeloid leukemia (AML) is a devastating blood cancer with poor prognosis. Novel effective treatment is an urgent unmet need. Immunotherapy targeting T cell exhaustion by blocking inhibitory pathways, such as PD-1, is promising in cancer treatment. However, results from clinical studies applying PD-1 blockade to AML patients are largely disappointing. AML is highly heterogeneous. Identification of additional immune regulatory pathways and defining predictive biomarkers for treatment response are crucial to optimize the strategy. CD26 is a marker of T cell activation and involved in multiple immune processes. Here, we performed comprehensive phenotypic and functional analyses on the blood samples collected from AML patients and discovered that CD26lowPD-1+ CD8 T cells were associated with AML progression. Specifically, the percentage of this cell fraction was significantly higher in patients with newly diagnosed AML compared to that in patients achieved completed remission or healthy controls. Our subsequent studies on CD26lowPD-1+ CD8 T cells from AML patients at initial diagnosis demonstrated that this cell population highly expressed inhibitory receptors and displayed impaired cytokine production, indicating an exhaustion status. Importantly, CD26lowPD-1+ CD8 T cells carried features of terminal exhaustion, manifested by higher frequency of TEMRA differentiation, increased expression of transcription factors that are observed in terminally exhausted T cells, and high level of intracellular expression of granzyme B and perforin. Our findings suggest a prognostic and predictive value of CD26 in AML, providing pivotal information to optimize the immunotherapy for this devastating cancer.