Search Results (27)

The basal cell maintains the airway’s respiratory epithelium as the putative resident stem cell. Basal cells are known to self-renew and differentiate into airway ciliated and secretory cells. However, it is not clear if every basal cell functions as a stem cell. To address functional heterogeneity amongst the basal cell population, we developed a novel monoclonal antibody, HLO1-6H5, that identifies a subset of KRT5+ (cytokeratin 5) basal cells. We used HLO1-6H5 and other known basal cell-reactive reagents to isolate viable airway subsets from primary human airway epithelium by Fluorescence Activated Cell Sorting. Isolated primary cell subsets were assessed for the stem cell capabilities of self-renewal and differentiation in the bronchosphere assay, which revealed that bipotent stem cells were, at minimum 3-fold enriched in the HLO1-6H5+ cell subset. Crosslinking-mass spectrometry identified the HLO1-6H5 target as a glycosylated TFRC/CD71 (transferrin receptor) proteoform. The HLO1-6H5 antibody provides a valuable new tool for identifying and isolating a subset of primary human airway basal cells that are substantially enriched for bipotent stem/progenitor cells and reveals TFRC as a defining surface marker for this novel cell subset. Full article

Angiotensin-converting enzyme 2 (ACE2) is considered a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) receptor of high importance, but due to its non-ubiquitous expression, studies of other proteins that may participate in virus internalisation have been undertaken. To date, many alternative receptors have been discovered. Their functioning may provide an explanation for some of the events observed in severe COVID-19 that cannot be directly explained by the model in which ACE2 constitutes the central point of infection. Diabetes mellitus type 2 (T2D) can induce severe COVID-19 development. Although many mechanisms associated with ACE2 can lead to increased SARS-CoV-2 virulence in diabetes, proteins such as basigin (CD147), glucose-regulated protein 78 kDa (GRP78), cluster of differentiation 4 (CD4), transferrin receptor (TfR), integrins α₅β₁/α_vβ₃, or ACE2 co-receptors neuropilin 2 (NRP2), vimentin, and even syalilated gangliosides may also be responsible for worsening the COVID-19 course. On the other hand, some others may play protective roles. Understanding how diabetes-associated mechanisms can induce severe COVID-19 via modification of virus receptor functioning needs further extensive studies. Full article

Heat stress (HS) is a significant concern in broiler chickens, which is vital for global meat supply in the dynamic field of poultry farming. The impact of heat stress on the ileum and its influence on the redox homeostatic genes in chickens remains unclear. We hypothesized that adding zinc to the feed of heat-stressed broilers would improve their resilience to heat stress. However, this study aimed to explore the effects of organic zinc supplementation under HS conditions on broiler chickens’ intestinal histology and regulation of HS index genes. In this study, 512 Xueshan chickens were divided into four groups: vehicle, HS, 60 mg/kg zinc, and HS + 60 mg/kg zinc groups. Findings revealed that zinc supply positively increased the VH and VH: CD in the ileum of the broilers compared to the HS group, while CD and VW decreased in Zn and HS+Zn supplemented broilers. Zn administration significantly increased superoxide dismutase (SOD), catalase (CAT), glutathione (GSH), and decreased the enzymatic activities of reactive oxygen species (ROS) and malondialdehyde (MDA) compared to the HS group. In addition, Zn administration significantly increased relative ATP, complex I, III, and V enzyme activity compared to the HS group. Furthermore, the expression of acyl-CoA synthetase long-chain family member 4 (ACSL4), lactate transporter 3 (LPCAT3), peroxiredoxin (PRX), and transferrin receptor (TFRC) in the protein levels was extremely downregulated in HS+Zn compared to the HS group. Zn supply significantly decreased the enrichment of RORγ, P300, and SRC1 at target loci of ACSL4, LPCAT3, and PRX compared to the HS group. The occupancies of histone active marks H3K9ac, H3K18ac, H3K27ac, H3K4me1, and H3K18bhb at the locus of ACSL4 and LPCAT3 were significantly decreased in HS+Zn compared to the HS group. Moreover, H3K9la and H3K18la at the locus of ACSL4 and LPCAT3 were significantly decreased in HS+Zn compared to the HS group. This study emphasizes that organic Zn is a potential strategy for modulating the oxidative genes ACSL4, LPCAT3, PRX, and TFRC in the ileum of chickens via nuclear receptor RORγ regulation and histone modifications. Full article

Background: Transferrin receptor 1 (TfR1), glucose transporter 1 (GLUT1), and CD98hc are candidates for targeted therapy at the blood–brain barrier (BBB). Our objective was to challenge the expression of TfR1, GLUT1, and CD98hc in brain capillaries using the histone deacetylase inhibitor (HDACi) valproic acid (VPA). Methods: Primary mouse brain capillary endothelial cells (BCECs) and brain capillaries isolated from mice injected intraperitoneally with VPA were examined using RT-qPCR and ELISA. Targeting to the BBB was performed by injecting monoclonal anti-TfR1 (Ri7217)-conjugated gold nanoparticles measured using ICP-MS. Results: In BCECs co-cultured with glial cells, Tfrc mRNA expression was significantly higher after 6 h VPA, returning to baseline after 24 h. In vivo Glut1 mRNA expression was significantly higher in males, but not females, receiving VPA, whereas Cd98hc mRNA expression was unaffected by VPA. TfR1 increased significantly in vivo after VPA, whereas GLUT1 and CD98hc were unchanged. The uptake of anti-TfR1-conjugated nanoparticles was unaltered by VPA despite upregulated TfR expression. Conclusions: VPA upregulates TfR1 in brain endothelium in vivo and in vitro. VPA does not increase GLUT1 and CD98hc proteins. The increase in TfR1 does not result in higher anti-TfR1 antibody targetability, suggesting targeting sufficiently occurs with available transferrin receptors without further contribution from accessory VPA-induced TfR1. Full article

Hepcidin is upregulated by increased body iron stores and inflammatory cytokines. It is associated with cardiovascular events, arterial stiffness, and increased iron accumulation in human atheroma with hemorrhage. However, it is unknown whether the expression of hepcidin in human carotid plaques is related to plaque severity and whether hepcidin expression differs between men and women. Carotid samples from 58 patients (38 males and 20 females) were immunostained with hepcidin, macrophages, ferritin, and transferrin receptor. Immunocytochemistry of hepcidin was performed on THP-1 macrophages exposed to iron or 7betahydroxycholesterol. Hepcidin expression significantly increases with the progression of human atherosclerotic plaques. Plaques of male patients have significantly higher levels of hepcidin. Expressions of hepcidin are significantly correlated with the accumulation of CD68-positive macrophages and transferrin receptor 1 (TfR1) and apoptosis. In vitro, hepcidin is significantly increased in macrophages exposed to iron and moderately increased following 7-oxysterol treatment. In the cultured cells, suppression of hepcidin protected against macrophage cell death, lysosomal membrane permeabilization, and oxidative stress. Hepcidin may play a crucial role in the development and progression of atherosclerosis. The differential expression of hepcidin in male and female patients and its significant correlations with plaque severity, highlight the potential of hepcidin as a biomarker for risk stratification and therapeutic targeting in atherosclerosis. Full article

This study aimed to investigate whether cyclophosphamide (C) and adriamycin (A) induction therapy (IT) prior to nivolumab could enhance the efficacy of nivolumab in previously treated patients with non-squamous (NSQ) non-small-cell lung cancer (NSCLC) with less than 10% programmed death-ligand 1 (PD-L1) expression. Twenty-two enrolled patients received four cycles of CA-IT every 3 weeks. Nivolumab was given 360 mg every 3 weeks from the second cycle and 480 mg every 4 weeks after four cycles of CA-IT. The median progression-free survival (PFS) and overall survival (OS) were 2.4 months and 11.6 months, respectively. Fluorescence-activated cell sorting revealed the lowest ratio of myeloid-derived suppressor cells (MDSCs) to CD8+T-cells in the responders. Proteomic analysis identified a consistent upregulation of extracellular matrix-receptor interactions and phagosome pathways in the responders. Among the differentially expressed proteins, the transferrin receptor protein (TFRC) was higher in the responders before treatment (fold change > 1.2). TFRC validation with an independent cohort showed the prognostic significance of either OS or PFS in patients with low PD-L1 expression. In summary, CA-IT did not improve nivolumab efficacy in NSQ-NSCLCs with low PD-L1 expression; however, it induced decreasing MDSC, resulting in a durable response. Higher baseline TFRC levels predicted a favorable response to nivolumab in NSCLC with low PD-L1 expression. Full article

Hyperforin (HPF) is an acylphloroglucinol compound found abundantly in Hypericum perforatum extract which exhibits antidepressant, anti-inflammatory, antimicrobial, and antitumor activities. Our recent study revealed a potent antimelanoma effect of HPF, which hinders melanoma cell proliferation, motility, colony formation, and induces apoptosis. Furthermore, we have identified glutathione peroxidase-4 (GPX-4), a key enzyme involved in cellular protection against iron-induced lipid peroxidation, as one of the molecular targets of HPF. Thus, in three BRAF-mutated melanoma cell lines, we investigated whether iron unbalance and lipid peroxidation may be a part of the molecular mechanisms underlying the antimelanoma activity of HPF. Initially, we focused on heme oxygenase-1 (HO-1), which catalyzes the heme group into CO, biliverdin, and free iron, and observed that HPF treatment triggered the expression of this inducible enzyme. In order to investigate the mechanism involved in HO-1 induction, we verified that HPF downregulates the BTB and CNC homology 1 (BACH-1) transcription factor, an inhibitor of the heme oxygenase 1 (HMOX-1) gene transcription. Remarkably, we observed a partial recovery of cell viability and an increase in the expression of the phosphorylated and active form of retinoblastoma protein when we suppressed the HMOX-1 gene using HMOX-1 siRNA while HPF was present. This suggests that the HO-1 pathway is involved in the cytostatic effect of HPF in melanoma cells. To explore whether lipid peroxidation is induced, we conducted cytofluorimetric analysis and observed a significant increase in the fluorescence of the BODIPY C-11 probe 48 h after HPF administration in all tested melanoma cell lines. To discover the mechanism by which HPF triggers lipid peroxidation, along with the induction of HO-1, we examined the expression of additional proteins associated with iron homeostasis and lipid peroxidation. After HPF administration, we confirmed the downregulation of GPX-4 and observed low expression levels of SLC7A11, a cystine transporter crucial for the glutathione production, and ferritin, able to sequester free iron. A decreased expression level of these proteins can sensitize cells to lipid peroxidation. On the other hand, HPF treatment resulted in increased expression levels of transferrin, which facilitates iron uptake, and LC3B proteins, a molecular marker of autophagy induction. Indeed, ferritin and GPX-4 have been reported to be digested during autophagy. Altogether, these findings suggest that HPF induced lipid peroxidation likely through iron overloading and decreasing the expression of proteins that protect cells from lipid peroxidation. Finally, we examined the expression levels of proteins associated with melanoma cell invasion and metastatic potential. We observed the decreased expression of CD133, octamer-4, tyrosine-kinase receptor AXL, urokinase plasminogen activator receptor, and metalloproteinase-2 following HPF treatment. These findings provide further support for our previous observations, demonstrating the inhibitory effects of HPF on cell motility and colony formation in soft agar, which are both metastasis-related processes in tumor cells. Full article

Transferrin receptor 1 (TfR1), also known as CD71, is a transmembrane protein involved in the cellular uptake of iron and the regulation of cell growth. This receptor is expressed at low levels on a variety of normal cells, but is upregulated on cells with a high rate of proliferation, including malignant cells and activated immune cells. Infection with the human immunodeficiency virus (HIV) leads to the chronic activation of B cells, resulting in high expression of TfR1, B-cell dysfunction, and ultimately the development of acquired immunodeficiency syndrome-related B-cell non-Hodgkin lymphoma (AIDS-NHL). Importantly, TfR1 expression is correlated with the stage and prognosis of NHL. Thus, it is a meaningful target for antibody-based NHL therapy. We previously developed a mouse/human chimeric IgG3 specific for TfR1 (ch128.1/IgG3) and showed that this antibody exhibits antitumor activity in an in vivo model of AIDS-NHL using NOD-SCID mice challenged intraperitoneally with 2F7 human Burkitt lymphoma (BL) cells that harbor the Epstein-Barr virus (EBV). We have also developed an IgG1 version of ch128.1 that shows significant antitumor activity in SCID-Beige mouse models of disseminated multiple myeloma, another B-cell malignancy. Here, we aim to explore the utility of ch128.1/IgG1 and its humanized version (hu128.1) in mouse models of AIDS-NHL. To accomplish this goal, we used the 2F7 cell line variant 2F7-BR44, which is more aggressive than the parental cell line and forms metastases in the brain of mice after systemic (intravenous) administration. We also used the human BL cell line JB, which in contrast to 2F7, is EBV-negative, allowing us to study both EBV-infected and non-infected NHL tumors. Treatment with ch128.1/IgG1 or hu128.1 of SCID-Beige mice challenged locally (subcutaneously) with 2F7-BR44 or JB cells results in significant antitumor activity against different stages of disease. Treatment of mice challenged systemically (intravenously) with either 2F7-BR44 or JB cells also showed significant antitumor activity, including long-term survival. Taken together, our results suggest that targeting TfR1 with antibodies, such as ch128.1/IgG1 or hu128.1, has potential as an effective therapy for AIDS-NHL. Full article

Superparamagnetic iron oxide nanoparticles (SPIONs) may act as an excellent theragnostic tool if properly coated and stabilized in a biological environment, even more, if they have targeting properties towards a specific cellular target. Humanized Archaeoglobus fulgidus Ferritin (HumAfFt) is an engineered ferritin characterized by the peculiar salt-triggered assembly-disassembly of the hyperthermophile Archaeoglobus fulgidus ferritin and is successfully endowed with the human H homopolymer recognition sequence by the transferrin receptor (TfR1 or CD71), overexpressed in many cancer cells in response to the increased demand of iron. For this reason, HumAfFt was successfully used in this study as a coating material for 10 nm SPIONs, in order to produce a new magnetic nanocarrier able to discriminate cancer cells from normal cells and maintain the potential theragnostic properties of SPIONs. HumAfFt-SPIONs were exhaustively characterized in terms of size, morphology, composition, and cytotoxicity. The preferential uptake capacity of cancer cells toward HumAfFt-SPIONs was demonstrated in vitro on human breast adenocarcinoma (MCF7) versus normal human dermal fibroblast (NHDF) cell lines. Full article

Lung cancer is, currently, one of the main malignancies causing deaths worldwide. To date, early prognostic and diagnostic markers for small cell lung cancer (SCLC) have not been systematically and clearly identified, so most patients receive standard treatment. In the present study, we combine quantitative proteomics studies and the use of magnetic core-shell nanoparticles (mCSNP’s), first to identify a marker for lung cancer, and second to functionalize the nanoparticles and their possible application for early and timely diagnosis of this and other types of cancer. In the present study, we used label-free mass spectrometry in combination with an ion-mobility approach to identify 220 proteins with increased abundance in small cell lung cancer (SCLC) cell lines. Our attention was focused on cell receptors for their potential application as mCSNP’s targets; in this work, we report the overexpression of Transferrin Receptor (TfR1) protein, also known as Cluster of Differentiation 71 (CD71) up to a 30-fold increase with respect to the control cell. The kinetics of endocytosis, evaluated by a flow cytometry methodology based on fluorescence quantification, demonstrated that receptors were properly activated with the transferrin supported on the magnetic core-shell nanoparticles. Our results are important in obtaining essential information for monitoring the disease and/or choosing better treatments, and this finding will pave the way for future synthesis of nanoparticles including chemotherapeutic drugs for lung cancer treatments. Full article

(1) Background: Protease-activated receptor 1 (PAR1) has regulatory functions in inflammation, atherogenesis, and atherothrombosis. Chronic iron administration accelerates arterial thrombosis. Intraplaque hemorrhage and hemoglobin catabolism by macrophages are associated with dysregulated iron metabolism and atherosclerotic lesion instability. However, it remains unknown whether expressions of PAR1 in human atherosclerotic lesions are related to plaque severity, accumulation of macrophages, and iron-related proteins. We investigated the expression of PAR1 and its relation to the expression of ferritin and transferrin receptors in human carotid atherosclerotic plaques and then explored potential connections between their expressions, plaque development, and classical risk factors. (2) Methods: Carotid samples from 39 patients (25 males and 14 females) were immunostained with PAR1, macrophages, ferritin, and transferrin receptor. Double immunocytochemistry of PAR1 and ferritin was performed on THP-1 macrophages exposed to iron. (3) Results: PAR1 expression significantly increases with the patient’s age and the progression of human atherosclerotic plaques. Expressions of PAR1 are significantly correlated with the accumulation of CD68-positive macrophages, ferritin, and transferrin receptor 1 (TfR1), and inversely correlated with levels of high-density lipoprotein. In vitro, PAR1 is significantly increased in macrophages exposed to iron, and the expression of PAR1 is colocalized with ferritin expression. (4) Conclusions: PAR1 is significantly related to the progression of human atherosclerotic lesions and the patient’s age. PAR1 is also associated with macrophage infiltration and accumulation of iron metabolic proteins in human atherosclerotic lesions. Cellular iron-mediated induction of PAR1 and its colocalization with ferritin in macrophages may further indicate an important role of cellular iron in atherothrombosis. Full article

Up to 4 million patients with signs of myocardial ischemia have no obstructive coronary artery disease (CAD). The absence of precise guidelines for diagnosis and treatment in non-obstructive CAD encourages the scientific community to fill the gap knowledge, to provide non-invasive and less expensive diagnostic tools. The aim of our study was to explore the biological profile of Ischemia with Non-Obstructive Coronary Arteries (INOCA) patients with microvascular dysfunction compared to patients presenting with obstructive chronic coronary syndrome (ObCCS) in order to find specific hallmarks of each clinical condition. We performed a gene expression array from peripheral blood mononuclear cells (PBMCs) isolated from INOCA (n = 18) and ObCCS (n = 20) patients. Our results showed a significantly reduced gene expression of molecules involved in cell adhesion, signaling, vascular motion, and inflammation in INOCA as compared to the ObCCS group. In detail, we found lower expression of Platelet and Endothelial Cell Adhesion Molecule 1 (CD31, p < 0.0001), Intercellular Adhesion Molecule-1 (ICAM1, p = 0.0004), Tumor Necrosis Factor (TNF p = 0.0003), Transferrin Receptor (TFRC, p = 0.002), and Vascular Endothelial Growth Factor A (VEGFA, p = 0.0006) in the INOCA group compared with ObCCS. Meanwhile, we observed an increased expression of Hyaluronidase (HYAL2, p < 0.0001) in INOCA patients in comparison to ObCCS. The distinct expression of molecular biomarkers might allow an early and non-invasive differential diagnosis between ObCCS and INOCA, improving clinical management and treatment options, in the era of personalized medicine. Full article

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to have a significant impact on global public health. Multiple mechanisms for SARS-CoV-2 cell entry have been described; however, the role of transferrin receptor 1 (TfR1) in SARS-CoV-2 infection has received little attention. We used ferristatin II to induce the degradation of TfR1 on the surface of Vero cells and to study the consequences of such treatment on the viability of the cells and the replication of SARS-CoV-2. We demonstrated that ferristatin II is non-toxic for Vero cells in concentrations up to 400 µM. According to confocal microscopy data, the distribution of the labeled transferrin and receptor-binding domain (RBD) of Spike protein is significantly affected by the 18h pretreatment with 100 µM ferristatin II in culture medium. The uptake of RBD protein is nearly fully inhibited by ferristatin II treatment, although this protein remains bound on the cell surface. The findings were well confirmed by the significant inhibition of the SARS-CoV-2 infection of Vero cells by ferristatin II with IC₅₀ values of 27 µM (for Wuhan D614G virus) and 40 µM (for Delta virus). A significant reduction in the infectious titer of the Omicron SARS-CoV-2 variant was noted at a ferristatin II concentration as low as 6.25 µM. We hypothesize that ferristatin II blocks the TfR1-mediated SARS-CoV-2 host cell entry; however, further studies are needed to elucidate the full mechanisms of this virus inhibition, including the effect of ferristatin II on other SARS-CoV-2 receptors, such as ACE2, Neuropilin-1 and CD147. The inhibition of viral entry by targeting the receptor on the host cells, rather than the viral mutation-prone protein, is a promising COVID-19 therapeutic strategy. Full article

In patients treated for prostate cancer (PCa) with radical prostatectomy (RP), determining the risk of extraprostatic extension (EPE) and nodal involvement (NI) remains crucial for planning nerve-sparing and extended lymphadenectomy. The study aimed to determine proteins that could serve as immunohistochemical markers of locally advanced PCa. To select candidate proteins associated with adverse pathologic features (APF) reverse-phase protein array data of 498 patients was retrieved from The Cancer Genome Atlas. The analysis yielded 6 proteins which were then validated as predictors of APF utilizing immunohistochemistry in a randomly selected retrospective cohort of 53 patients. For univariate and multivariate analysis, logistic regression was used. Positive expression of TfR1 (OR 13.74; p = 0.015), reduced expression of CD49b (OR 10.15; p = 0.013), and PSA (OR 1.29; p = 0.013) constituted independent predictors of EPE, whereas reduced expression of e-cadherin (OR 10.22; p = 0.005), reduced expression of CD49b (OR 24.44; p = 0.017), and PSA (OR 1.18; p = 0.002) were independently associated with NI. Both models achieved high discrimination (AUROC 0.879 and 0.888, respectively). Immunohistochemistry constitutes a straightforward tool that might be easily utilized before RP. Expression of TfR1 and CD49b is associated with EPE, whereas expression of e-cadherin and CD49b is associated with NI. Since following immunohistochemical markers predicts respective APFs independently from PSA, in the future they might supplement existing preoperative nomograms or be implemented in novel tools. Full article

Anaplastic thyroid carcinoma (ATC) is the most fatal and rapidly evolving endocrine malignancy invading the head and neck region and accounts for up to 50% of thyroid cancer-associated deaths. Deregulation of the microRNA (miRNA) expression promotes thyroid carcinoma progression by modulating the reorganization of the ATC transcriptome. Here, we applied comparative miRNA–mRNA sequencing on a cohort of 28 thyroid carcinomas to unravel the association of deregulated miRNA and mRNA expression. This identified 85 miRNAs significantly deregulated in ATC. By establishing a new analysis pipeline, we unraveled 85 prime miRNA–mRNA interactions supporting the downregulation of candidate tumor suppressors and the upregulation of bona fide oncogenes such as survivin (BIRC5) in ATC. This miRNA-dependent reprogramming of the ATC transcriptome provided an mRNA signature comprising 65 genes sharply distinguishing ATC from other thyroid carcinomas. The validation of the deregulated protein expression in an independent thyroid carcinoma cohort demonstrates that miRNA-dependent oncogenes comprised in this signature, the transferrin receptor TFRC (CD71) and the E3-ubiquitin ligase DTL, are sharply upregulated in ATC. This upregulation is sufficient to distinguish ATC even from poorly differentiated thyroid carcinomas (PDTC). In sum, these findings provide new diagnostic tools and a robust resource to explore the key miRNA–mRNA regulation underlying the progression of thyroid carcinoma. Full article