RESUMEN
PURPOSE: Transferrin receptor (TFR), a membrane protein that has a critical role in the transport of iron into cells, is known to be a ferroptosis-related marker. Although TFR is reported to be abundantly expressed in tumor cells, its relationship with ferroptosis inducers in hepatocellular carcinoma (HCC) remains unclear. METHODS: The authors performed immunohistochemical staining of TFR and divided 350 HCC patients into two groups according to its expression. They analyzed the association between TFR expression and prognosis or clinicopathologic factors. In addition, the regulation of malignant activity and its effect on the efficacy of ferroptosis inducers were investigated in vitro. RESULTS: For this study, 350 patients were divided into TFR-positive (n =180, 51.4%) and TFR-negative (n = 170, 48.6%) groups. The TFR-positive group had more hepatitis B surface antigen (HBs-Ag) (p = 0.0230), higher α-fetoprotein (AFP) levels (p = 0.0023), higher des-gamma-carboxyprothrombin (DCP) levels (p = 0.0327), a larger tumor size (p = 0.0090), greater proportions of Barcelona Clinic Liver Cancer (BCLC) stage B or C (p = 0.0005), poor differentiation (p < 0.0001), and microscopic intrahepatic metastasis (p = 0.0066). In the multivariate analyses, TFR expression was an independent prognostic factor in disease-free survival (p = 0.0315). In vitro, TFRC knockdown decreased cell motility. In addition, TFRC knockdown abolished artesunate (AS)-, lenvatinib-, and sorafenib-induced ferroptosis in HCC cell lines. The study demonstrated that simultaneous treatment of AS with multi-kinase inhibitor augmented the ferroptosis-inducing effects of AS in HCC cell lines. CONCLUSION: TFR expression is a poor prognostic factor in HCC, but its expression increases sensitivity to ferroptosis-inducing agents.
Asunto(s)
Carcinoma Hepatocelular , Ferroptosis , Neoplasias Hepáticas , Humanos , Carcinoma Hepatocelular/patología , Neoplasias Hepáticas/patología , Pronóstico , Receptores de Transferrina/análisisRESUMEN
BACKGROUND: The pathways that control protein transport across the blood-brain barrier (BBB) remain poorly characterized. Despite great advances in recapitulating the human BBB in vitro, current models are not suitable for systematic analysis of the molecular mechanisms of antibody transport. The gaps in our mechanistic understanding of antibody transcytosis hinder new therapeutic delivery strategy development. METHODS: We applied a novel bioengineering approach to generate human BBB organoids by the self-assembly of astrocytes, pericytes and brain endothelial cells with unprecedented throughput and reproducibility using micro patterned hydrogels. We designed a semi-automated and scalable imaging assay to measure receptor-mediated transcytosis of antibodies. Finally, we developed a workflow to use CRISPR/Cas9 gene editing in BBB organoid arrays to knock out regulators of endocytosis specifically in brain endothelial cells in order to dissect the molecular mechanisms of receptor-mediated transcytosis. RESULTS: BBB organoid arrays allowed the simultaneous growth of more than 3000 homogenous organoids per individual experiment in a highly reproducible manner. BBB organoid arrays showed low permeability to macromolecules and prevented transport of human non-targeting antibodies. In contrast, a monovalent antibody targeting the human transferrin receptor underwent dose- and time-dependent transcytosis in organoids. Using CRISPR/Cas9 gene editing in BBB organoid arrays, we showed that clathrin, but not caveolin, is required for transferrin receptor-dependent transcytosis. CONCLUSIONS: Human BBB organoid arrays are a robust high-throughput platform that can be used to discover new mechanisms of receptor-mediated antibody transcytosis. The implementation of this platform during early stages of drug discovery can accelerate the development of new brain delivery technologies.
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Anticuerpos/metabolismo , Bioingeniería/métodos , Barrera Hematoencefálica/metabolismo , Organoides/metabolismo , Receptores de Transferrina/metabolismo , Transcitosis/fisiología , Animales , Anticuerpos/análisis , Astrocitos/química , Astrocitos/metabolismo , Barrera Hematoencefálica/química , Barrera Hematoencefálica/citología , Células Cultivadas , Técnicas de Cocultivo , Células Endoteliales/química , Células Endoteliales/metabolismo , Humanos , Organoides/química , Organoides/citología , Pericitos/química , Pericitos/metabolismo , Receptores de Transferrina/análisisRESUMEN
Mature erythrocytes are the major metabolic regulators by transporting oxygen throughout the body. However, their precursors and progenitors defined as CD71+ Erythroid Cells (CECs) exhibit a wide range of immunomodulatory properties. Here, we uncover pronounced sexual dimorphism in CECs. We found female but not male mice, both BALB/c and C57BL/6, and human females were enriched with CECs. CECs, mainly their progenitors defined as CD45+CECs expressed higher levels of reactive oxygen species (ROS), PDL-1, VISTA, Arginase II and Arginase I compared to their CD45- counterparts. Consequently, CECs by the depletion of L-arginine suppress T cell activation and proliferation. Expansion of CECs in anemic mice and also post-menstrual cycle in women can result in L-arginine depletion in different microenvironments in vivo (e.g. spleen) resulting in T cell suppression. As proof of concept, we found that anemic female mice and mice adoptively transferred with CECs from anemic mice became more susceptible to Bordetella pertussis infection. These observations highlight the role of sex and anemia-mediated immune suppression in females. Notably, enriched CD45+CECs may explain their higher immunosuppressive properties in female BALB/c mice. Finally, we observed significantly more splenic central macrophages in female mice, which can explain greater extramedullary erythropoiesis and subsequently abundance of CECs in the periphery. Thus, sex-specific differences frequency in the frequency of CECs might be imprinted by differential erythropoiesis niches and hormone-dependent manner.
Asunto(s)
Antígenos CD/análisis , Células Eritroides/inmunología , Terapia de Inmunosupresión , Receptores de Transferrina/análisis , Caracteres Sexuales , Traslado Adoptivo , Anemia/inmunología , Animales , Arginasa/análisis , Arginina/metabolismo , Antígeno B7-H1/análisis , Bordetella pertussis , Recuento de Células , Técnicas de Cocultivo , Citocinas/metabolismo , Células Eritroides/química , Eritropoyesis , Ciclo Estral/inmunología , Femenino , Hormonas Esteroides Gonadales/fisiología , Hematopoyesis Extramedular , Humanos , Activación de Linfocitos , Macrófagos/fisiología , Masculino , Proteínas de la Membrana/análisis , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Especies Reactivas de Oxígeno/análisis , Bazo/patología , Linfocitos T/inmunologíaRESUMEN
Flow cytometry is a useful ancillary tool for the diagnosis of nodal B cell lymphomas. Well-established antigens have diagnostic limitations. This study aimed to assess the expression of CD71, CD81, CD44 and CD39 by flow cytometry in B cell lymphomas. Expression of these 4 antigens was queried in 185 samples with a diagnosis of a B cell lymphoma according to a histological examination of the lymph node and the World Health Organization (WHO) classification (follicular lymphoma [FL, n = 96], diffuse large B cell lymphoma/High grade B cell lymphoma [DLBCL/HGBH, n = 48], marginal zone lymphoma/lymphoplasmacytic lymphoma [MZL/LPL, n = 14], chronic lymphocytic leukemia/small lymphocytic lymphoma [CLL, n = 10], mantle cell lymphoma [MCL, n = 11], Burkitt lymphoma [BL, n = 4] and other [n = 2]). CD81 was bright and CD44 was dim in germinal center-derived malignancies, particularly aggressive lymphomas (BL and CD10-positive DLBCL/HGBL). CD81 was very dim in CLL. CD71 was bright in aggressive lymphomas (DLBCL/HGBL and BL). CD39 was bright in CD10-negative DLBCL. CD71 appeared valuable in the differential diagnosis between indolent and aggressive lymphomas, CD39 between CD10-negative DLBCL and MZL/LPL and CD81 between MCL and CLL. To conclude, we report the expression of CD71, CD81, CD44 and CD39 by FC in B cell lymphomas. Further studies will have to determine the value they add to specific FC panels.
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Antígenos CD/análisis , Apirasa/análisis , Citometría de Flujo/métodos , Receptores de Hialuranos/análisis , Linfoma de Células B/inmunología , Receptores de Transferrina/análisis , Tetraspanina 28/análisis , Femenino , Humanos , MasculinoRESUMEN
Ineffective erythropoiesis and iron overload are common in myelodysplastic syndromes (MDS). Erythroferrone (ERFE) and growth/differentiation factor 15 (GDF15) are two regulators of iron homeostasis produced by erythroid progenitors. Elevated systemic levels of ERFE and GDF15 in MDS are associated with dysregulated iron metabolism and iron overload, which is especially pronounced in MDS with SF3B1 gene mutations. However, the role of ERFE and GDF15 in MDS pathogenesis and their influence on disease progression are largely unknown. Here, we analyzed the expression of ERFE and GDF15 in CD71+ erythroid progenitors of n = 111 MDS patients and assessed their effects on patient survival. The expression of ERFE and GDF15 in MDS was highly aberrant. Unexpectedly, ERFE expression in erythroprogenitors was highly relevant for MDS prognosis and independent of International Prognostic Scoring System (IPSS) stratification. Although ERFE expression was increased in patients with SF3B1 mutations, it predicted overall survival (OS) in both the SF3B1wt and SF3B1mut subgroups. Of note, ERFE overexpression predicted superior OS in the IPSS low/Int-1 subgroup and in patients with normal karyotype. Similar observations were made for GDF15, albeit not reaching statistical significance. In summary, our results revealed a strong association between ERFE expression and MDS outcome, suggesting a possible involvement of ERFE in molecular MDS pathogenesis.
Asunto(s)
Antígenos CD/análisis , Células Precursoras Eritroides/metabolismo , Síndromes Mielodisplásicos/metabolismo , Hormonas Peptídicas/biosíntesis , Receptores de Transferrina/análisis , Adulto , Anciano , Anciano de 80 o más Años , Células Precursoras Eritroides/química , Femenino , Factor 15 de Diferenciación de Crecimiento/biosíntesis , Factor 15 de Diferenciación de Crecimiento/genética , Humanos , Estimación de Kaplan-Meier , Masculino , Persona de Mediana Edad , Síndromes Mielodisplásicos/mortalidad , Síndromes Mielodisplásicos/terapia , Hormonas Peptídicas/genética , Fosfoproteínas/genética , Modelos de Riesgos Proporcionales , Factores de Empalme de ARN/genética , Resultado del Tratamiento , Adulto JovenRESUMEN
BACKGROUND: iron deficiency in pulmonary tuberculosis (TB) patients may weaken their immune system, causing difficulty in overcoming the infection. Accurate diagnosis of iron deficiency anemia (IDA) in pulmonary TB patients is essential. In order to prove the iron deficient state, diagnosis should focus on inflammatory factors, which could highly affect the outcome of iron status, such as measurement of serum ferritin (SF). Soluble Transferrin Receptor (sTfR) is the best parameter to diagnose iron deficiency in the inflammatory condition. This study aimed to understand the role of sTfR to identify IDA in TB patients. METHODS: cross-sectional study were applied to 3 study groups: anemic pulmonary TB (68 subjects), IDA (7 subjects), and non-anemic pulmonary TB (15 subjects). The test averages and correlations between sTfR, SF, and other hematological parameters were measured and analyzed. RESULTS: significant differences of sTfR were found in the anemic TB group compared to the IDA group and also between the IDA and non-anemic TB groups (p<0.0001). However, there was no significant difference (p>0.05) between TB anemic and non-anemic groups. We also found no significant difference between the TB anemic sub-group with normal levels of sTfR compared with the non-anemic group. There was no significant difference of sTfR levels between sub-group of increasing sTfR and group IDA (p>0.05). However, there was strong correlation between sTfR and SF in the IDA group (r=-0.89; p=0.007). CONCLUSION: the findings suggest verifying the sTfR amount in anemic patients with pulmonary TB suffering from inflammation, so that the iron deficiency could be more accurately identified and properly treated.
Asunto(s)
Anemia Ferropénica/diagnóstico , Anemia/diagnóstico , Ferritinas/análisis , Deficiencias de Hierro , Receptores de Transferrina/análisis , Tuberculosis Pulmonar/complicaciones , Adulto , Anemia/complicaciones , Anemia/patología , Anemia Ferropénica/complicaciones , Anemia Ferropénica/patología , Estudios Transversales , Femenino , Humanos , Inflamación/sangre , Inflamación/complicaciones , Masculino , Persona de Mediana Edad , Solubilidad , Tuberculosis Pulmonar/sangreRESUMEN
Although the basic process of receptor-mediated endocytosis (RME) is well established, certain specific aspects, like the endosomal redox state, remain less characterized. Previous studies used chemically labeled ligands or antibodies with a FRET (fluorescence resonance energy transfer) probe to gauge the redox activity of the endocytic pathway with a limitation being their inability to track the apo receptor. New tools that allow direct labeling of a cell surface receptor with synthetic probes would aid in the study of its endocytic pathway and function. Herein, we use a peptide ligase, butelase 1, to label the human transferrin receptor 1 (TfR1) in established human cell lines with a designer disulfide FRET probe. This strategy enables us to obtain real-time live cell imaging of redox states in TfR1-mediated endocytosis, attesting a reducing environment in the endosomal compartments and the dynamics of TfR1 trafficking. A better understanding of endocytosis of different cell surface receptors has implications in designing strategies that hijack this natural process for intracellular drug delivery.
Asunto(s)
Antígenos CD/análisis , Disulfuros/química , Endosomas/química , Transferencia Resonante de Energía de Fluorescencia , Colorantes Fluorescentes/química , Receptores de Transferrina/análisis , Antígenos CD/metabolismo , Endosomas/metabolismo , Humanos , Oxidación-Reducción , Receptores de Transferrina/metabolismoRESUMEN
The aim of our study was to isolate populations of keratinocyte stem cells based on the expression of cell surface markers and to investigate whether the culture could affect their phenotype. keratinocytes from human oral mucosa were sorted based on the expression of the epithelial stem cell markers p75NTR and CD71. We also examined the co-expression of other epithelial stem markers such as integrins ß1 and α6 and their stem cell-like proprieties in in vitro assays. Three passages after being sorted by MACS, more than 93% of the p75NTR+ve cells lost the expression of p75NTR, while 5.46% of the p75NTR-ve gained it. Within the small population of the p75NTR+ve cells, 88% co-expressed other epithelial stem cell markers such as integrins ß1 and α6, while only 28% of p75NTR-ve cells co-expressed these markers. These results were confirmed by sorting cells by FACS. Additionally, when double staining was used for sorting cells, 99% of the p75NTR+veCD71-ve and 33% of the p75NTR-veCD71+ve cells expressed both integrins, but just one week after culture, only 1.74% of the p75NTR+veCD71-ve cells still expressed p75NTR and only 0.32% still expressed CD71. Similar results were obtained when co-culturing p75NTR+ve and p75NTR-ve populations before analysis. Our results suggest that phenotype changes may be part of an intrinsic cellular mechanism to conserve levels of protein expression as they may found in the human body. In addition, in vitro culture may not offer ideal conditions for epithelial stem cell maintenance due to phenotype changes under standard culture conditions.
Asunto(s)
Técnicas de Cultivo de Célula/métodos , Células Epiteliales/citología , Queratinocitos/citología , Mucosa Bucal/citología , Fenotipo , Células Madre/citología , Antígenos CD/análisis , Biomarcadores/análisis , Separación Celular/métodos , Citometría de Flujo/métodos , Humanos , Proteínas del Tejido Nervioso/análisis , Receptores de Factor de Crecimiento Nervioso/análisis , Receptores de Transferrina/análisis , Reproducibilidad de los ResultadosRESUMEN
BACKGROUND: Extracorporeal photopheresis (ECP) is a leukapheresis-based cellular therapy that is used with increasing frequency worldwide to treat various T-cell-mediated diseases. Currently, the inhibition of T-cell proliferation after photopheresis is analysed frequently using time-consuming assays including radioactive thymidine assays or carboxyfluorescein succinimidyl ester (CFSE) staining. We investigated whether simple surface T-cell staining using surrogate markers of T-cell proliferation can replace time-consuming measurement of T-cell proliferation in ECP quality control. STUDY DESIGN AND METHODS: T-cell activation markers were investigated by flow cytometry after ECP. Candidates were validated by direct comparison with the classical CFSE T-cell proliferation inhibition test and apoptosis staining. Finally, surface T-cell staining was performed in patient samples in comparison with classical methods. RESULTS: CD71 expression exhibited the fastest and most robust upregulation, which was detectable as early as 6-8 h after T-cell stimulation and almost completely abrogated by ECP. In a direct comparison with the CFSE T-cell proliferation assay, suppression of CD71 expression after ECP was almost identical and detectable as early as 16 h after stimulation in peripheral blood mononuclear cells of healthy donors. Furthermore, in direct comparison with classical apoptosis staining, the inhibition delta of CD71 after ECP was significantly higher. Moreover, in patients under T-cell suppressive therapy, T-cell-dependent CFSE and CD71 assays exhibited decreased sensitivity to detect ECP treatment and were inferior in comparison to apoptosis staining. CONCLUSION: Surface CD71 analysis represents a very simple quality control alternative to detect ECP-mediated T-cell proliferation inhibition in normal PBMC samples devoid of T-cell suppressive drugs.
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Antígenos CD/análisis , Citometría de Flujo/métodos , Fotoféresis/normas , Control de Calidad , Receptores de Transferrina/análisis , Linfocitos T/metabolismo , Adulto , Apoptosis , Proliferación Celular , Femenino , Humanos , Leucocitos Mononucleares , Activación de Linfocitos , Fotoféresis/métodos , Linfocitos T/fisiologíaRESUMEN
Abstract The aim of our study was to isolate populations of keratinocyte stem cells based on the expression of cell surface markers and to investigate whether the culture could affect their phenotype. keratinocytes from human oral mucosa were sorted based on the expression of the epithelial stem cell markers p75NTR and CD71. We also examined the co-expression of other epithelial stem markers such as integrins β1 and α6 and their stem cell-like proprieties in in vitro assays. Three passages after being sorted by MACS, more than 93% of the p75NTR+ve cells lost the expression of p75NTR, while 5.46% of the p75NTR-ve gained it. Within the small population of the p75NTR+ve cells, 88% co-expressed other epithelial stem cell markers such as integrins β1 and α6, while only 28% of p75NTR-ve cells co-expressed these markers. These results were confirmed by sorting cells by FACS. Additionally, when double staining was used for sorting cells, 99% of the p75NTR+veCD71-ve and 33% of the p75NTR-veCD71+ve cells expressed both integrins, but just one week after culture, only 1.74% of the p75NTR+veCD71-ve cells still expressed p75NTR and only 0.32% still expressed CD71. Similar results were obtained when co-culturing p75NTR+ve and p75NTR-ve populations before analysis. Our results suggest that phenotype changes may be part of an intrinsic cellular mechanism to conserve levels of protein expression as they may found in the human body. In addition, in vitro culture may not offer ideal conditions for epithelial stem cell maintenance due to phenotype changes under standard culture conditions.
Asunto(s)
Humanos , Fenotipo , Células Madre/citología , Queratinocitos/citología , Técnicas de Cultivo de Célula/métodos , Células Epiteliales/citología , Mucosa Bucal/citología , Receptores de Transferrina/análisis , Biomarcadores/análisis , Antígenos CD/análisis , Separación Celular/métodos , Reproducibilidad de los Resultados , Receptores de Factor de Crecimiento Nervioso/análisis , Citometría de Flujo/métodos , Proteínas del Tejido Nervioso/análisisRESUMEN
KCNQ1 (Kv7.1 or KvLQT1) plays important physiological roles in various tissues forming potassium channels with KCNE subunits. Among the channels formed by KCNQ1 and KCNE subunits, the best studied is the slow delayed rectifier potassium channel in the heart, the IKs (KCNQ1/KCNE1) channel, which is critical for repolarization of cardiac action potential. The KCNQ1 channel is internalized by Nedd4/Nedd4-like ligase-dependent ubiquitination. It is also reported that phosphorylation of KCNE1 by PKC results in internalization of the KCNQ1/KCNE1 channel. Because we have observed down-regulation of KCNQ1/KCNE1 currents by activation of the α1-adrenergic receptor (α1AR) that activates PKC, this study investigated whether α1AR causes internalization of the KCNQ1 protein. We fused HaloTag to the extracellular region of KCNQ1 (Halo-KCNQ1) and co-expressed it with α1ARs in HEK293 cells. The KCNQ1 protein on the cell surface was selectively labeled with membrane-impermeable HaloTag ligands, and changes in its localization were monitored by confocal fluorescence microscopy. Activation of α1AAR and α1BAR caused marked internalization of KCNQ1, which was not KCNE1-dependent. Internalization of KCNQ1 by α1AR activation was inhibited by disruption of the PY motif or the YXXΦ motif in the C-terminus. Double staining for the receptor and the channel revealed that KCNQ1 internalization was independent of α1AR internalization. Our results suggest that α1AR-mediated direct internalization of KCNQ1 is AP2/clathrin-dependent and may be triggered by ubiquitination of KCNQ1 via the AMP dependent kinase (AMPK)/Nedd4-2 pathway. When phenylephrine was applied to rat neonatal cardiomyocytes transfected with KCNQ1 and α1AR, the KCNQ1 protein was internalized. The internalization of KCNQ1 by α1AR would affect pathophysiology in a variety of tissues expressing KCNQ1, which merits further in vivo study.
Asunto(s)
Canal de Potasio KCNQ1/metabolismo , Receptores Adrenérgicos alfa 1/fisiología , Proteínas Quinasas Activadas por AMP/fisiología , Animales , Células HEK293 , Humanos , Miocitos Cardíacos/metabolismo , Proteína Quinasa C/fisiología , Ratas , Ratas Sprague-Dawley , Receptores de Transferrina/análisisRESUMEN
Pancreatic cancer is a highly lethal malignancy associated with tissues of the pancreas. Early diagnosis and effective treatment are crucial to improving the survival rate of patients with pancreatic cancer. In a previous study, we employed the cell-SELEX strategy to obtain an ssDNA aptamer termed XQ-2d with high binding affinity for pancreatic cancer. Here, we first identify CD71 as the XQ-2d-binding target. We found that knockdown of CD71 abolished the binding of XQ-2d and that the binding affinity of XQ-2d is associated with membrane-bound CD71, rather than total CD71 levels. Competitive analysis revealed that XQ-2d shares the same binding site on CD71 with transferrin (Tf), but not anti-CD71 antibody. We then used a surface energy transfer (SET) nanoruler to measure the distance between the binding sites of XQ-2d and anti-CD71 antibody, and it was about 15 nm. Furthermore, we did molecular dynamics simulation to clarify that the spatial structure of XQ-2d and Tf competitively binding to CD71. We also engineered XQ-2d-mediated targeted therapy for pancreatic cancer, using an XQ-2d-based complex for loading doxorubicin (Dox). Because CD71 is overexpressed not only in pancreatic cancer but also in a variety of tumors, our work provides a systematic novel way of studying a potential biomarker and also promising tools for cancer diagnosis and therapy, opening new doors for effective cancer theranostics.
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Antígenos CD/análisis , Aptámeros de Nucleótidos/química , Receptores de Transferrina/análisis , Sitios de Unión , Biomarcadores de Tumor/análisis , Línea Celular Tumoral , Humanos , Modelos Moleculares , Neoplasias Pancreáticas/diagnóstico , Transferrina/análisisRESUMEN
Chronic mountain sickness (CMS) has a higher incidence in the plateau region and is characterized by excessive erythrocytosis and hypoxemia. Bcl-2 family plays an important role in the process of erythropoiesis and the regulation of apoptosis. This study aimed to examine the change in apoptosis of erythroblasts in CMS patients and explore the involvement of Bcl-2 family. Bone marrow mononuclear cells (BMMNCs) were isolated by density gradient centrifugation from 18 CMS patients and 17 control participants. The apoptotic rate, mitochondrial membrane potential (MMP), the protein expression of caspase-3, TNFR, Fas, Bcl-2, Bax and Cyt-C were examined by flow cytometry, and mRNA expression was determined by real-time PCR. The results showed that apoptotic rate of erythroblasts was lower and MMP was higher in CMS group than in control group. The mRNA and protein expression levels of Bcl-2 were higher while Bax level was lower in CMS group than in control group. In CMS group, the apoptosis rate of CD71+ erythroblasts was negatively correlated with the ratio of CD71+ cells in BMMNCs and positively correlated with hemoglobin level. In conclusion, erythroblasts apoptosis is decreased due to the regulation of the expression of Bcl-2 family members in the erythroblasts of CMS patients.
Asunto(s)
Mal de Altura/sangre , Apoptosis , Eritroblastos/metabolismo , Policitemia/etiología , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Antígenos CD/análisis , Células de la Médula Ósea/patología , Estudios de Casos y Controles , Enfermedad Crónica , Regulación hacia Abajo , Eritroblastos/patología , Hemoglobinas/análisis , Humanos , Potencial de la Membrana Mitocondrial , Cultivo Primario de Células , Receptores de Transferrina/análisis , Proteína X Asociada a bcl-2/metabolismoRESUMEN
BACKGROUND: Maternal anemia has adverse consequences for the mother-infant dyad. To evaluate whether and how milk nutrient content may change in ways that could "buffer" infants against the conditions underlying maternal anemia, this study assessed associations between milk macronutrients and maternal iron-deficiency anemia (IDA), non-iron-deficiency anemia (NIDA), and inflammation. METHODS: A secondary analysis of cross-sectional data and milk from northern Kenya was conducted (n = 204). The combination of hemoglobin and transferrin receptor defined IDA/NIDA. Elevated serum C-reactive protein defined acute inflammation. The effects of IDA, NIDA, and inflammation on milk macronutrients were evaluated in regression models. RESULTS: IDA (ß = 0.077, p = .022) and NIDA (ß = 0.083, p = .100) predicted higher total protein (ln). IDA (ß = -0.293, p = .002), NIDA (ß = -0.313, p = .047), and inflammation (ß = -0.269, p = .007) each predicted lower fat (ln); however, anemia accompanying inflammation predicted higher fat (ß = 0.655, p = .007 for IDA and ß = 0.468, p = .092 for NIDA). NIDA predicted higher lactose (ß = 1.020, p = .003). CONCLUSIONS: Milk macronutrient content both increases and decreases in the presence of maternal anemia and inflammation, suggesting a more complicated and dynamic change than simple impairment of nutrient delivery during maternal stress. Maternal fat delivery to milk may be impaired under anemia. Mothers may buffer infant nutrition against adverse conditions or poor maternal health by elevating milk protein (mothers with IDA/NIDA), lactose (mothers with NIDA), or fat (mothers with anemia and inflammation). This study demonstrates the foundational importance of maternal micronutrient health and inflammation or infection for advancing the ecological understanding of human milk nutrient variation.
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Anemia Ferropénica , Inflamación , Leche Humana/química , Adulto , Anemia , Anemia Ferropénica/sangre , Anemia Ferropénica/epidemiología , Proteína C-Reactiva/análisis , Estudios Transversales , Femenino , Humanos , Inflamación/sangre , Inflamación/epidemiología , Kenia , Fenómenos Fisiologicos Nutricionales Maternos/fisiología , Valor Nutritivo/fisiología , Receptores de Transferrina/análisis , Adulto JovenRESUMEN
Low effectiveness and resistance to treatments are commonplace in disorders of the central nervous system (CNS). These issues concern mainly the blood-brain barrier (BBB), which preserves homeostasis in the brain and protects this organ from toxic molecules and biohazards by regulating transport through it. BBB shuttles-short peptides able to cross the BBB-are being developed to help therapeutics to cross this barrier. BBB shuttles can be discovered by massive exploration of chemical diversity (e.g. computational means, phage display) or rational design (e.g. derivatives from a known peptide/protein able to cross). Here we present the selection of a peptide shuttle (HAI) from several candidates and the subsequent in-depth in vitro and in vivo study of this molecule. In order to explore the chemical diversity of HAI and enhance its biostability, and thereby its bioactivity, we explored two new protease-resistant versions of HAI (i.e. the retro-D-version, and a version that was N-methylated at the most sensitive sites to enzymatic cleavage). Our results show that, while both versions of HAI are resistant to proteases, the retro-D-approach preserved better transport properties.
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Barrera Hematoencefálica/metabolismo , Péptidos de Penetración Celular/síntesis química , Péptidos de Penetración Celular/farmacocinética , Receptores de Transferrina/análisis , Animales , Péptidos de Penetración Celular/química , Sistemas de Liberación de Medicamentos , Diseño de Fármacos , Estabilidad de Medicamentos , Humanos , Péptido Hidrolasas/metabolismo , Permeabilidad , RatasRESUMEN
Progress towards an in-depth understanding of the final steps of the erythroid lineage development is paramount for many hematological diseases. We have characterized the final stages of reticulocyte maturation from bone marrow to peripheral blood using for the first time single-cell Mass Cytometry (CyTOF). We were able to measure the expression of 31 surface markers within a single red blood cell (RBC). We demonstrate the validity of CyTOF for RBC phenotyping by confirming the progressive reduction of transferrin receptor 1 (CD71) during reticulocyte maturation to mature RBC. We highlight the high-dimensional nature of mass cytometry data by correlating the expression of multiple proteins on individual RBCs. We further describe a more drastic reduction pattern for a component of the alpha4/beta1 integrin CD49d at the very early steps of reticulocyte maturation in bone marrow and directly linked with the mitochondria remnants clearance pattern. The enhanced and accurate RBC phenotyping potential of CyTOF described herein could be beneficial to decipher RBC preferences, as well as still not well understood receptor-ligand interaction of some hemotropic parasites such as the malaria causing agent Plasmodium vivax.
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Técnicas Citológicas/instrumentación , Eritrocitos/metabolismo , Análisis de la Célula Individual/métodos , Animales , Antígenos CD/análisis , Biomarcadores/análisis , Diferenciación Celular , Linaje de la Célula , Técnicas Citológicas/métodos , Eritrocitos/fisiología , Humanos , Inmunofenotipificación , Integrina alfa4/análisis , Receptores de Transferrina/análisis , Reticulocitos/fisiologíaRESUMEN
Maintaining an intact tumor environment is critical for quantitation of receptor-ligand engagement in a targeted drug development pipeline. However, measuring receptor-ligand engagement in vivo and non-invasively in preclinical settings is extremely challenging. We found that quantitation of intracellular receptor-ligand binding can be achieved using whole-body macroscopic lifetime-based Förster Resonance Energy Transfer (FRET) imaging in intact, live animals bearing tumor xenografts. We determined that FRET levels report on ligand binding to transferrin receptors conversely to raw fluorescence intensity. FRET levels in heterogeneous tumors correlate with intracellular ligand binding but strikingly, not with ubiquitously used ex vivo receptor expression assessment. Hence, MFLI-FRET provides a direct measurement of systemic delivery, target availability and intracellular drug delivery in preclinical studies. Here, we have used MFLI to measure FRET longitudinally in intact and live animals. MFLI-FRET is well-suited for guiding the development of targeted drug therapy in heterogeneous tumors in intact, live small animals.
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Sistemas de Liberación de Medicamentos , Transferencia Resonante de Energía de Fluorescencia/instrumentación , Neoplasias/metabolismo , Imagen Óptica/instrumentación , Receptores de Transferrina/metabolismo , Transferrina/metabolismo , Animales , Antineoplásicos/administración & dosificación , Antineoplásicos/farmacocinética , Línea Celular Tumoral , Diseño de Equipo , Femenino , Colorantes Fluorescentes/análisis , Colorantes Fluorescentes/metabolismo , Humanos , Ratones Desnudos , Neoplasias/tratamiento farmacológico , Receptores de Transferrina/análisis , Transferrina/análisis , Imagen de Cuerpo Entero/instrumentaciónRESUMEN
Little is known about the renal responses to acute iron overloading. This study measured the renal tubular expression of transferrin receptor-1 (TfR1), cubilin/megalin receptors, hepcidin, ferroportin, and ferritin chains following subacute intoxication of 40 male Wistar rats with a single oral dose of ferrous iron (300 mg/kg). The animals were randomly subdivided into 4 equal subgroups at the time of necropsy (1, 2, 4, and 8 hr). The results were compared with the controls ( n=15) and with the chronic group ( n=15), which received iron for 4 weeks (75 mg/kg/day; 5 days/week). Although both toxicity models inhibited TfR1, they upregulated the cubilin/megalin receptors and hepcidin, and triggered iron deposition in tubular cells. The ferritin heavy-chain and ferroportin were downregulated in the 2-hr and 4-hr acute subgroups, whereas chronic toxicity promoted their expression, compared with controls. Moreover, the 4-hr and 8-hr subgroups had higher intracellular Fe+2 and marked cell apoptosis compared with the chronic group. In conclusion, the kidney appears to sustain iron reabsorption in both intoxication models. However, the cellular iron storage and exporter proteins were differentially expressed in both models, and their inhibition post-acute toxicity might contribute toward the intracellular accumulation of Fe+2, oxidative stress, and ferroptosis.
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Sobrecarga de Hierro/patología , Hierro/análisis , Riñón/patología , Enfermedad Aguda , Animales , Apoptosis , Caspasas/análisis , Enfermedad Crónica , Ensayo de Inmunoadsorción Enzimática/métodos , Ferritinas/análisis , Ferritinas/sangre , Técnica del Anticuerpo Fluorescente/métodos , Hepcidinas/análisis , Hepcidinas/sangre , Hierro/sangre , Sobrecarga de Hierro/sangre , Riñón/metabolismo , Proteína 2 Relacionada con Receptor de Lipoproteína de Baja Densidad/análisis , Masculino , Estrés Oxidativo , Ratas Wistar , Receptores de Superficie Celular/análisis , Receptores de Transferrina/análisisRESUMEN
BACKGROUND: Acute lymphoblastic leukemia (ALL) is the commonest childhood cancer. Transferrin receptor 1 (CD71) is a trans-membrane glycoprotein which has important role in iron homeostasis by acting as a gatekeeper regulating iron uptake from transferrin and is an attractive target for anti-cancer agents, particularly those that aim to induce lethal iron deprivation in malignant hematopoietic cells. AIM OF THE WORK: To assess the prognostic value of Transferrin receptor -1 (CD71) in children with newly diagnosed ALL. PATIENTS AND METHODS: This study was carried out on 75 patients with newly diagnosed ALL. Transferrin receptor-1 expression was analyzed on the bone marrow blasts by flow cytometry at time of diagnosis with positive CD71 expression is considered when ≥20% of malignant cells express this marker while negative expression is considered when <20% of malignant cells express this marker. RESULTS: Transferrin receptor-1 positive expression was detected in 45 patients (60%) while negative expression was found in the remaining 30 patients (40%). CD71 expression was significantly higher on T- ALL patients compared with B-ALL patients. Positive CD71 expression at diagnosis was significantly associated with bad clinical and laboratory prognostic factors as lymphadenopathy, higher white blood cell count, higher hemoglobin level, lower platelets count, and higher blast cells in peripheral blood and bone marrow and higher lactate dehydrogenase levels'. There were significant differences in disease free survival (DFS) and overall survival (OS) between positive and negative CD71 expression groups with significantly shorter DFS and OS in positive CD71 expression group compared to negative group. CONCLUSION AND RECOMMENDATIONS: 'Positive Transferrin receptor -1 (CD71) expression in patients with ALL is adverse prognostic factor and should be taken in consideration in designing future therapeutic strategies based on patient- specific risk factors'.
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Antígenos CD/análisis , Biomarcadores de Tumor/análisis , Leucemia-Linfoma Linfoblástico de Células Precursoras/inmunología , Receptores de Transferrina/análisis , Adolescente , Niño , Preescolar , Supervivencia sin Enfermedad , Femenino , Humanos , Masculino , Leucemia-Linfoma Linfoblástico de Células Precursoras/mortalidad , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Leucemia-Linfoma Linfoblástico de Células Precursoras/terapia , Valor Predictivo de las Pruebas , Pronóstico , Factores de Riesgo , Factores de TiempoRESUMEN
Transferrin receptor (TfR) is overexpressed on the surface of many cancer cells due to its vital roles in iron circulation and cellular respiration. Soluble transferrin receptor (sTfR), a truncated extracellular form of TfR in serum, is an important marker of iron deficiency anemia (IDA) and bone marrow failure in cancer patients. More recently, sTfR level in urine has been related to a specific kidney disease of Henoch-Schönlein purpura nephritis (HSPN). Despite the universal significance of sTfR, there is still a lack of a simple and sensitive method for the quantification of sTfR. Furthermore, it is desirable to have a probe that can detect both TfR and sTfR for further comparison study. In this work, we developed a water-soluble AIE-peptide conjugate with aggregation-induced emission (AIE) characteristics. Taking advantage of the negligible emission from molecularly dissolved tetraphenylethene (TPE), probe TPE-2T7 was used for the light-up detection of sTfR. The probe itself is nonemissive in aqueous solution, but it turns on its fluorescence upon interaction with sTfR to yield a detection limit of 0.27 µg/mL, which is much lower than the sTfR level in IDA patients. Furthermore, a proof-of-concept experiment validates the potential of the probe for diagnosis of HSPN by urine test.