Advancing glioblastoma treatment through iron metabolism: A focus on TfR1 and Ferroptosis innovations.

Glioblastoma (GBM) represents a formidable challenge in oncology, characterized by aggressive proliferation and poor prognosis. Iron metabolism plays a critical player in GBM progression, with dysregulated iron uptake and utilization contributing to tumor growth and therapeutic resistance. Iron's pivotal role in DNA synthesis, oxidative stress, and angiogenesis underscores its significance in GBM pathogenesis. Elevated expression of iron transporters, such as transferrin receptor 1 (TfR1), highlights the tumor's reliance on iron for survival. Innovative treatment strategies targeting iron dysregulation hold promise for overcoming therapeutic challenges in GBM management. Approaches such as iron chelation therapies, induction of ferroptosis to nanoparticle-based drug delivery systems exploit iron-dependent vulnerabilities, offering avenues for enhance treatment efficacy and improve patient outcomes. As research advances, understanding the complexities of iron-mediated carcinogenesis provides a foundation for developing precision medicine approaches tailored to combat GBM effectively. This review explores the intricate relationship between iron metabolism and GBM, elucidating its multifaceted implications and therapeutic opportunities. By consolidating the latest insights into iron metabolism in GBM, this review underscores its potential as a therapeutic target for improving patient care in combination with the standard of care approach.

Anaemia and iron deficiency associate with polymorphism TMPRSS6 rs855791 in Brazilian children attending day care centres.

Fe-deficiency anaemia is a major public health concern in children under 5 years of age. TMPRSS6 gene, encoding matriptase-2 protein, is implicated in Fe homoeostasis and has been associated with anaemia and Fe status in various populations. The aim of this cross-sectional study was to investigate the associations between the single nucleotide polymorphism (SNP) TMPRSS6 rs855791 and biomarkers of anaemia and Fe deficiency in Brazilian children attending day care centres. A total of 163 children aged 6-42 months were evaluated. Socio-economic, demographic, biochemical, haematological, immunological and genotype data were collected. Multiple logistic and linear regressions with hierarchical selection were used to assess the effects of independent variables on categorised outcomes and blood marker concentrations. Minor allele (T) frequency of rs855791 was 0·399. Each copy of the T allele was associated with a 4·49-fold increased risk of developing anaemia (P = 0·005) and a 4·23-fold increased risk of Fe deficiency assessed by serum soluble transferrin receptor (sTfR) (P < 0·001). The dose of the T allele was associated with an increase of 0·18 mg/l in sTfR concentrations and reductions of 1·41 fl and 0·52 pg in mean corpuscular volume (MCV) and mean corpuscular haemoglobin (MCH), respectively. In conclusion, the T allele of SNP TMPRSS6 rs855791 was significantly associated with anaemia and Fe deficiency assessed by sTfR in Brazilian children attending day care centres. The effect was dose dependent, with each copy of the T allele being associated with lower MCV and MCH and higher concentrations of sTfR.

Evaluating the Diagnostic Performance of Hemoglobin in the Diagnosis of Iron Deficiency Anemia in High-Altitude Populations: A Scoping Review.

We evaluated the available literature on the diagnostic performance of hemoglobin (Hb) in the diagnosis of iron deficiency anemia (IDA) in high-altitude populations. We searched PubMed, Web of Science, Scopus, Embase, Medline by Ovid, the Cochrane Library, and LILCAS until 3 May 2022. We included studies that evaluated the diagnostic performance (sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), receiver operating characteristic (ROC) curves, and accuracy) of Hb (with and without an altitude correction factor) compared to any iron deficiency (ID) marker (e.g., ferritin, soluble transferrin receptor (sTFR), transferrin saturation, or total body iron (TBI)) in populations residing at altitudes (≥1000 m above sea level). We identified a total of 14 studies (with 4522 participants). We found disagreement in diagnostic performance test values between the studies, both in those comparing hemoglobin with and in those comparing hemoglobin without a correction factor for altitude. Sensitivity ranged from 7% to 100%, whereas specificity ranged from 30% to 100%. Three studies reported higher accuracy of uncorrected versus altitude-corrected hemoglobin. Similarly, two studies found that not correcting hemoglobin for altitude improved the receiver operating characteristic (ROC) curves for the diagnosis of iron deficiency anemia. Available studies on high-altitude populations suggest that the diagnostic accuracy of Hb is higher when altitude correction is not used. In addition, the high prevalence of anemia in altitude regions could be due to diagnostic misclassification.

Iron metabolism disorders of patients with chronic paracoccidioidomycosis.

Paracoccidioidomycosis (PCM) is caused by Paracoccidioides spp.; during infection, some host mechanisms limit the availability of iron, thereby reducing its reproduction. However, Paracoccidioides spp. can evade the immune defense and, even under limited iron conditions, use this mineral for growth and dissemination. This study evaluated the iron metabolism of 39 patients who were diagnosed with chronic PCM from 2013 to 2021. The forms of iron before treatment and at the time of clinical cure were evaluated based on the following: serum ferritin levels (storage iron); total iron-binding capacity (TIBC) and transferrin saturation (TSAT) level (transport iron); red blood cell (RBC), hemoglobin (Hb), hematocrit (HCT), and soluble transferrin receptor (sTfR) levels; and sTfR/log ferritin ratio (functional iron). The mean age of the patients was 54.5 years (±6.7 years). Most patients were men (97.4%), rural workers (92.1%), and smokers (84.6%); furthermore, most had moderate disease severity (66.7%). After achieving clinical cure, we observed that serum ferritin levels decreased, and parameters of functional iron increased. The extent of alteration in these parameters were more pronounced in severe cases than in to mild or moderate cases. Furthermore, moderate correlations were observed between C-reactive protein and the Hb (r = -0.500; p = 0.002), RBC (r = -0.461; p = 0.005), HCT (r = -0.514; p = 0.001), and iron levels (r = -0.491; p = 0.002). However, it is possible to infer that PCM interferes with functional and storage iron because improvements in these parameters after treatment as well as associations with disease severity were observed. PCM can lead to anemia of inflammation, which can be differentiated from iron deficiency anemia by a careful investigation of the iron form parameters.

Prevalence of Anemia and Iron Deficiency in Women of Reproductive Age in Cuba and Associated Factors.

This study aims to evaluate the prevalence of anemia and iron deficiency in women of reproductive age and the association with inflammation, global overweight, adiposity, and menorrhagia. A sample design of women of reproductive age from the Eastern, Central, and Havana Regions was carried out. Biochemical determinations of hemoglobin, serum ferritin, soluble transferrin receptors, leukocytes, C-reactive protein, alpha-1 acid glycoprotein, and homocysteine were performed. Serum ferritin was also adjusted by inflammation. Nutritional status was assessed, and menstrual characteristics were collected by survey. A total of 742 women were studied. The prevalence of anemia was 21.4%, iron storage deficiency at 16.0%, and erythropoietic dysfunction at 5.4%, with inflammation at 47.0% and elevated homocysteine at 18.6%. Global overweight was 46.2% and increased adiposity at 58.4%. Anemia is associated with iron deposition deficiency (OR = 3.023 (1.816-5.033)) and with erythropoietic deficiency (OR = 5.62 (3.03-10.39)), but not with inflammation, global overweight, and adiposity. Global overweight was found to be associated with inflammation (OR = 2.23 (1.41-3.53)). Anemia was associated with heavy menstrual bleeding (OR = 1.92 (1.34-2.76)). Homocysteine was associated with inflammation (OR = 2.05 (1.08-3.90)), but not with anemia. In conclusion, anemia in Cuba is classified as a moderate public health problem, but not iron deficiency. A high prevalence of overweight and obesity was found, associated with inflammation, but not with anemia or iron deficiency. Heavy menstrual bleeding is a factor associated with anemia.

COVID-19 compromises iron homeostasis: Transferrin as a target of investigation.

IMPORTANCE: Since the beginning of the COVID-19 pandemic, numerous metabolic alterations have been observed in individuals with this disease. It is known that SARS-CoV-2 can mimic the action of hepcidin, altering intracellular iron metabolism, but gaps remain in the understanding of possible outcomes in other pathways involved in the iron cycle. OBJECTIVE: To profile iron, ferritin and hepcidin levels and transferrin receptor gene expression in patients diagnosed with COVID-19 between June 2020 and September 2020. DESIGN, SETTING AND PARTICIPANTS: Cross-sectional study that evaluated iron metabolism markers in 427 participants, 218 with COVID-19 and 209 without the disease. EXPOSURES: The primary exposure was positive diagnose to COVID-19 in general population of Santo André and São Bernardo cities. The positive and negative diagnose were determinate through RT-qPCR. MAIN OUTCOMES AND MEASURES: Devido a evidências de alterações do ciclo do ferro em pacientes diagnosticados com COVID-19 e devido a corregulação entre hepcidina e receptor de transferrina, uma análise da expressão gênica deste último, poderia trazer insights sobre o estado de ferro celular. A hipótese foi confirmada, mostrando aumento da expressão de receptor de transferrina concomitante com redução do nível de hepcidina circulante. RESULTS: Serum iron presented lower values in individuals diagnosed with COVID-19, whereas serum ferritin presented much higher values in infected patients. Elderly subjects had lower serum iron levels and higher ferritin levels, and men with COVID-19 had higher ferritin values than women. Serum hepcidin was lower in the COVID-19 patient group and transferrin receptor gene expression was higher in the infected patient group compared to controls. CONCLUSIONS AND RELEVANCE: COVID-19 causes changes in several iron cycle pathways, with iron and ferritin levels being markers that reflect the state and evolution of infection, as well as the prognosis of the disease. The increased expression of the transferrin receptor gene suggests increased iron internalization and the mimicry of hepcidin action by SARS-CoV-2, reduces iron export via ferroportin, which would explain the low circulating levels of iron by intracellular trapping.

Antibody-Based Inhibition of Pathogenic New World Hemorrhagic Fever Mammarenaviruses by Steric Occlusion of the Human Transferrin Receptor 1 Apical Domain.

Pathogenic clade B New World mammarenaviruses (NWM) can cause Argentine, Venezuelan, Brazilian, and Bolivian hemorrhagic fevers. Sequence variability among NWM glycoproteins (GP) poses a challenge to the development of broadly neutralizing therapeutics against the entire clade of viruses. However, blockade of their shared binding site on the apical domain of human transferrin receptor 1 (hTfR1/CD71) presents an opportunity for the development of effective and broadly neutralizing therapeutics. Here, we demonstrate that the murine monoclonal antibody OKT9, which targets the apical domain of hTfR1, can sterically block cellular entry by viral particles presenting clade B NWM glycoproteins (GP1-GP2). OKT9 blockade is also effective against viral particles pseudotyped with glycoproteins of a recently identified pathogenic Sabia-like virus. With nanomolar affinity for hTfR1, the OKT9 antigen binding fragment (OKT9-Fab) sterically blocks clade B NWM-GP1s and reduces infectivity of an attenuated strain of Junin virus. Binding of OKT9 to the hTfR1 ectodomain in its soluble, dimeric state produces stable assemblies that are observable by negative-stain electron microscopy. A model of the OKT9-sTfR1 complex, informed by the known crystallographic structure of sTfR1 and a newly determined structure of the OKT9 antigen binding fragment (Fab), suggests that OKT9 and the Machupo virus GP1 share a binding site on the hTfR1 apical domain. The structural basis for this interaction presents a framework for the design and development of high-affinity, broadly acting agents targeting clade B NWMs. IMPORTANCE Pathogenic clade B NWMs cause grave infectious diseases, the South American hemorrhagic fevers. Their etiological agents are Junin (JUNV), Guanarito (GTOV), Sabiá (SABV), Machupo (MACV), Chapare (CHAV), and a new Sabiá-like (SABV-L) virus recently identified in Brazil. These are priority A pathogens due to their high infectivity and mortality, their potential for person-to-person transmission, and the limited availability of effective therapeutics and vaccines to curb their effects. While low homology between surface glycoproteins of NWMs foils efforts to develop broadly neutralizing therapies targeting NWMs, this work provides structural evidence that OKT9, a monoclonal antibody targeting a single NWM glycoprotein binding site on hTfR1, can efficiently prevent their entry into cells.

Iduronate-2-sulfatase fused with anti-hTfR antibody, pabinafusp alfa, for MPS-II: A phase 2 trial in Brazil.

In Hunter syndrome (mucopolysaccharidosis II [MPS-II]), systemic accumulation of glycosaminoglycans (GAGs) due to a deficiency of iduronate-2-sulfatase (IDS), caused by mutations in the IDS gene, leads to multiple somatic manifestations and in patients with the severe (neuronopathic) phenotype, also to central nervous system (CNS) involvement. These symptoms cannot be effectively treated with current enzyme-replacement therapies, as they are unable to cross the blood-brain barrier (BBB). Pabinafusp alfa, a novel IDS fused with an anti-human transferrin receptor antibody, was shown to penetrate the BBB and to address neurodegeneration in preclinical studies. Subsequent phase 1/2 and 2/3 clinical studies in Japan have shown marked reduction of GAG accumulation in the cerebrospinal fluid (CSF), along with favorable clinical responses. A 26-week, open-label, randomized, parallel-group phase 2 study was conducted in Brazil to further evaluate the safety and efficacy of intravenously administered pabinafusp alfa at 1.0, 2.0, and 4.0 mg/kg/week in MPS-II patients. The safety profiles in the three dosage groups were similar. Neurodevelopmental evaluation suggested positive neurocognitive signals despite a relatively short study period. The 2.0-mg/kg group, which demonstrated marked reductions in substrate concentrations in the CSF, serum, and urine, was considered to provide the best combination regarding safety and efficacy signals.

Hepcidin and other indicators of iron status, by alpha-1 acid glycoprotein levels, in a cohort of Mexican infants.

The purpose of this study was to describe the changes in iron status indicators at 6 and 12 months of age, controlling by inflammation by measuring alpha-1 acid glycoprotein (AGP). This longitudinal study included 48 healthy-term singleton infants with birth weight ≥ 2500 g, born in hospitals of the Mexican Institute for Social Security. Complete blood count, ferritin, soluble transferrin receptor (sTfR), hepcidin, and AGP were measured in blood at 6 and 12 months of age. sTfR/ferritin ratio and total body iron (TBI) stores were calculated. Hemoglobin and sTfR/ferritin ratio increased with age, while ferritin and TBI decreased. In infants without inflammation, hepcidin, sTfR, and MVC did not show significant changes from 6 to 12 months of age, while ferritin and TBI decreased. In infants with inflammation, hepcidin, TBI, and ferritin levels increased, while hemoglobin and sTfR/ferritin ratio decreased. MVC and sTfR did not change significantly in the presence or absence of inflammation. Hepcidin concentration correlated positively and significantly with ferritin and TBI stores and showed significant negative correlation with sTfR/ferritin ratio. Our study showed that, in absence of inflammation and ID, during the first year of life, physiological changes occur in hemoglobin and ferritin levels as well as in indicators derived from ferritin and sTfR; in contrast, hepcidin and sTfR did not show significant change. However, hepcidin concentration was lower in infants with ID and was higher when inflammation was present, supporting that infants have a functional hepcidin response to changes in iron stores.

Classic and emergent indicators for the assessment of human iron status.

Iron deficiency is the leading cause of anemia, a significant global public health problem. Different methods exist for assessing iron nutritional status, including laboratory tests that focus on storage, transportation, and iron functional compartment parameters. Classical markers such as bone marrow, serum iron, ferritin, hemoglobin, erythrocyte parameters, transferrin, transferrin receptors, and zinc protoporphyrin are discussed in this review. Additional parameters calculated from these indicators, including transferrin saturation, ferritin index and Thomas plot, and some emergent parameters such as hepcidin, erythroferrone, and low hemoglobin density are also discussed. There is no a single indicator for assessing iron nutritional status. Therefore, the use of more than one indicator may be the best practice to obtain the correct diagnosis, also considering the influence of inflammation/infection on many of these indicators. The constant validation of the current parameters, the improvement of assessment methods, and the identification of new indicators will be the key to refine the assessment of iron nutritional status and the right choice of treatment for its improvement.

Relationship between soluble transferrin receptor and type 2 diabetes mellitus: A meta-analysis.

Obesity may play a role in the association between sTfR and T2DM. Analysis of the review carried out by Liu et al. titled "Iron metabolism and type 2 diabetes mellitus: A meta-analysis and systematic review".

Vitamin A deficiency regulates the expression of ferritin in young male Wistar rats / A deficiência de vitamina A regula a expressão de ferritina em ratos Wistar machos jovens

ABSTRACT Objective Iron deficiency and vitamin A deficiency are two of the main micronutrient deficiencies. Both micronutrients are essential for human life and children's development. This study aimed to investigate the effects of vitamin A deficiency on ferritin and transferrin receptors' expression and its relationship with iron deficiency. Methods Five diets with different vitamin A-to-iron ratios were given to thirty five 21-day-old male Wistar rats (separated in groups of seven animals each). The animals received the diet for six weeks before being euthanized. Serum iron and retinol levels were measured as biochemical parameters. Their duodenums, spleens, and livers were analyzed for the expression of ferritin and transferrin receptors by Western Blotting. Results Regarding biochemical parameters, the results show that when both vitamin A and iron are insufficient, the serum iron content (74.74µg/dL) is significantly lower than the control group (255.86µg/dL). The results also show that vitamin A deficiency does not influence the expression of the transferrin receptor, but only of the ferritin one. Conclusion Vitamin A deficiency regulates the expression of ferritin in young male Wistar rats.

RESUMO Objetivo A deficiência de ferro e de vitamina A são duas das principais deficiências de micronutrientes, sendo que ambos são essenciais para a vida humana e o desenvolvimento das crianças. O objetivo deste estudo foi investigar o efeito da deficiência de vitamina A na expressão de ferritina e o receptor de transferrina e sua relação com a deficiência de ferro. Métodos Cinco dietas com diferentes proporções de vitamina A para ferro foram administradas a 35 ratos Wistar machos de 21 dias de vida (sete animais por grupo). Os animais receberam a dieta por seis semanas antes de serem eutanasiados. Os níveis séricos de ferro e retinol foram medidos como parâmetros bioquímicos. Duodeno, baço e fígado foram analisados quanto à expressão de ferritina e o receptor de transferrina por Western Blotting. Resultados Em relação aos parâmetros bioquímicos, os resultados mostram que quando a vitamina A e o ferro são insuficientes, o teor de ferro sérico (74.74µg/dL) é significativamente menor do que no grupo controle (255.86µg/dL). Os resultados também mostram que a deficiência de vitamina A não influencia a expressão do receptor da transferrina, mas da ferritina. Conclusão A deficiência de vitamina A regula a expressão de ferritina em ratos Wistar machos jovens.

Correcting ß-thalassemia by combined therapies that restrict iron and modulate erythropoietin activity.

ß-Thalassemia intermedia is a disorder characterized by ineffective erythropoiesis (IE), anemia, splenomegaly, and systemic iron overload. Novel approaches are being explored based on the modulation of pathways that reduce iron absorption (ie, using hepcidin activators like Tmprss6-antisense oligonucleotides [ASOs]) or increase erythropoiesis (by erythropoietin [EPO] administration or modulating the ability of transferrin receptor 2 [Tfr2] to control red blood cell [RBC] synthesis). Targeting Tmprss6 messenger RNA by Tmprss6-ASO was proven to be effective in improving IE and splenomegaly by inducing iron restriction. However, we postulated that combinatorial strategies might be superior to single therapies. Here, we combined Tmprss6-ASO with EPO administration or removal of a single Tfr2 allele in the bone marrow of animals affected by ß-thalassemia intermedia (Hbbth3/+). EPO administration alone or removal of a single Tfr2 allele increased hemoglobin levels and RBCs. However, EPO or Tfr2 single-allele deletion alone, respectively, exacerbated or did not improve splenomegaly in ß-thalassemic mice. To overcome this issue, we postulated that some level of iron restriction (by targeting Tmprss6) would improve splenomegaly while preserving the beneficial effects on RBC production mediated by EPO or Tfr2 deletion. While administration of Tmprss6-ASO alone improved the anemia, the combination of Tmprss6-ASO + EPO or Tmprss6-ASO + Tfr2 single-allele deletion produced significantly higher hemoglobin levels and reduced splenomegaly. In conclusion, our results clearly indicate that these combinatorial approaches are superior to single treatments in ameliorating IE and anemia in ß-thalassemia and could provide guidance to translate some of these approaches into viable therapies.

The Action of JAK/STAT3 and BMP/HJV/SMAD Signaling Pathways on Hepcidin Suppression by Tucum-do-Cerrado in a Normal and Iron-Enriched Diets.

The Brazilian savanna fruit, tucum-do-cerrado (Bactris setosa Mart.) reduces hepatic hepcidin levels. Therefore, we investigated the effect of tucum-do-cerrado on the TfR/HFE and/or BMP/HJV/SMAD and JAK/STAT pathways, in normal and excess iron conditions. Rats were treated with: control diet (CT); control diet +15% tucum-do-cerrado (Tuc); iron-enriched diet (+Fe); or iron-enriched diet +15% tucum-do-cerrado (Tuc+Fe). Tucum-do-cerrado (Tuc) decreased hepatic Hamp and Hjv mRNA levels but did not alter Bmp6, Smad7, Tfr1, and Hfe mRNA levels; pSMAD1/5/8 and pSTAT3 protein levels; labile iron pool (LIP); and inflammatory biomarkers, compared to the CT group. The iron-enriched diet increased Hamp mRNA levels, as well as pSMAD1/5/8 and pSTAT3 protein levels, while no difference was observed in Hjv, Bmp6, Smad7, Tfr1, and Hfe mRNA levels and LIP compared to the CT group. The association of tucum-do-cerrado with the iron-enriched diet (Tuc+Fe) decreased Hamp, Hjv, Bmp6, and Hfe mRNA levels and pSTAT3 protein content compared to the +Fe group, while increased Hamp and decreased Hfe mRNA levels compared to the Tuc group. Therefore, the inhibition of hepatic hepcidin by tucum-do-cerrado consumption may involve the downregulation of intestinal Dmt1 and hepatic Hjv expression and deacetylation mediated by SIRT1 by a mechanism that is independent of tissue iron content. However, in excess iron conditions, the modulation of hepatic hepcidin expression by tucum-do-cerrado seems to be partially mediated by the inflammatory signaling pathway, as well as involves the chelating activity of tucum-do-cerrado.

Variability in the cleavage of exosome-associated transferrin receptor questions the utility of clinically useful soluble transferrin receptor assays for dogs, cats, and horses.

Whole transferrin receptor (TfR) is present in reticulocyte exosomes. Soluble transferrin receptor (sTfR) is cleaved from whole TfR in human plasma, with the remnant cytoplasmic domain (cTfR) remaining membrane associated. In humans, sTfR is a biomarker that can detect iron deficiency in the presence of inflammatory disease. This condition is still a diagnostic dilemma in veterinary species. We aimed to (1) confirm the presence of exosomes and exosome-associated TfR in the serum of dogs, cats, and horses; and (2) to assess and compare the proportion of cTfR to total (cTfR + whole) in exosomal membranes of healthy and diseased dogs and cats and in healthy horses to indirectly predict their anticipated levels of circulating sTfR. We used discarded serum and whole blood samples from canine and feline patients, separated into healthy and diseased groups based on the health status of each patient, and healthy equine participants from a previous study. Ultracentrifugation, followed in some experiments by OptiPrep discontinuous density gradient fractionation, was used to isolate exosomes. Exosomes and associated TfR were identified using TEM and Western blot for TfR, respectively. Densitometry tracings of Western blots of serum exosomes were used to measure the proportion of cTfR to total TfR. Extracellular vesicles compatible with exosomes were successfully isolated and expressed TfR. The proportion of cTfR in dogs was greater than 50%, indicating that a majority of the whole TfR was cleaved to produce sTfR (and remnant cTfR). There was significant interindividual variation and no significant difference between healthy and diseased animals. The proportion of cTfR in cats was very low at 11%, indicating that very little sTfR was likely produced. There was a small yet significant difference between healthy and diseased cats. Healthy horses do not appear to cleave exosome-associated TfR. Diagnosis of iron deficiency in the presence of inflammatory disease remains a challenge in veterinary medicine. Our results indicate that TfR is poorly or unpredictably cleaved in veterinary species, revealing that there are species differences in exosomal TfR handling. These data suggest that development of an assay for the detection and quantification of sTfR in the species investigated may not be warranted.

Extracellular vesicles containing the transferrin receptor as nanocarriers of apotransferrin.

Previous work by our group has shown the pro-differentiating effects of apotransferrin (aTf) on oligodendroglial cells in vivo and in vitro. Further studies showed the remyelinating effect of aTf in animal demyelination models such as hypoxia/ischemia, where the intranasal administration of human aTf provided brain neuroprotection and reduced white matter damage, neuronal loss, and astrogliosis in different brain regions. These data led us to search for a less invasive and controlled technique to deliver aTf to the CNS. To such end, we isolated extracellular vesicles (EVs) from human and mouse plasma and different neuron and glia conditioned media and characterized them based on their quality, quantity, identity, and structural integrity by western blot, dynamic light scattering, and scanning electron microscopy. All sources yielded highly pure vesicles whose size and structures were in keeping with previous literary evidence. Given that, remarkably, EVs from all sources analyzed contained Tf receptor 1 (TfR1) in their composition, we employed two passive cargo-loading strategies which rendered successful EV loading with aTf, specifically through binding to TfR1. These results unveil EVs as potential nanovehicles of aTf to be delivered into the CNS parenchyma, and pave the way for further studies into their possible clinical application in the treatment of demyelinating diseases.

Phenotype changes of oral epithelial stem cells after in vitro culture.

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.

Influence of heat stress on reference genes stability in heart and liver of two chickens genotypes.

INTRODUCTION: Real-time polymerase chain reaction (RT-qPCR) is an important tool for analyzing gene expression. However, before analyzing the expression of target genes, it is crucial to normalize the reference genes, in order to find the most stable gene to be used as an endogenous control. A gene that remains stable in all samples under different treatments is considered a suitable normalizer. In this sense, we aimed to identify stable reference genes for normalization of target genes in the heart and liver tissues from two genetically divergent groups of chickens (Cobb 500® commercial line and Peloco backyard chickens) under comfort and acute heat stress environmental conditions. Eight reference genes (ACTB, HPRT1, RPL5, EEF1, MRPS27, MRPS30, TFRC and LDHA) were analyzed for expression stability. The samples were obtained from 24 chickens, 12 from the backyard Peloco and 12 from the Cobb 500® line, exposed to two environmental conditions (comfort and heat stress). Comfort temperature was 23°C and heat stress temperature was 39.5°C for one hour. Subsequently, the animals were euthanized, and heart and liver tissue fragments were collected for RNA extraction and amplification. To determine the stability rate of gene expression, three different statistical algorithms were applied: BestKeeper, geNorm and NormFinder, and to obtain an aggregated stability list, the RankAgregg package of R software was used. RESULTS: The most stable genes using BestKeeper tool, including the two factors (genetic group and environmental condition), were LDHA, RPL5 and MRPS27 for heart tissue, and TFRC, RPL5 and EEF1 for liver tissue. Applying geNorm algorithm, the best reference genes were RPL5, EEF1 and MRPS30 for heart tissue and LDHA, EEF1 and RPL5 for liver. Using the NormFinder algorithm, the best normalizer genes were EEF1, RPL5 and LDHA in heart, and EEF1, RPL5 and ACTB in liver tissue. In the overall ranking obtained by RankAggreg package, considering the three algorithms, the RPL5, EEF1 and LDHA genes were the most stable for heart tissue, whereas RPL5, EEF1 and ACTB were the most stable for liver tissue. CONCLUSION: According to the RankAggreg tool classification based on the three different algorithms (BestKeeper, geNorm and NormFinder), the most stable genes were RPL5, EEF1 and LDHA for heart tissue and RPL5, EEF1 and ACTB for liver tissue of chickens subjected to comfort and acute heat stress environmental conditions. However, the best reference genes may vary depending on the experimental conditions of each study, such as different breeds, environmental stressors, and tissues analyzed. Therefore, the need to perform priori studies to assay the best reference genes at the outset of each study is emphasized.

Phenotype changes of oral epithelial stem cells after in vitro culture

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.