Importance of CD71+ Erythrocyte Cell Levels in Prognosis in Patients With ß-Thalassemia.

BACKGROUND/OBJECTIVES: CD71+ erythroid cells (CECs) are immature red blood cells (proerythroblasts, erythroblasts, and reticulocytes). CECs play an important role in the development of sepsis and cancer by causing immunosuppression. We examined the CEC levels in the peripheral blood of beta thalassemia (ßThal) patients and investigated the relationship between CECs and the clinical status of the patients, especially splenectomy. METHODS: Sixty-eight patients with ßThal (46 splenectomized and 22 nonsplenectomized) and 15 healthy controls were included in this study. The hemogram parameters, ferritin, and CECs (flow cytometry method) were measured. RESULTS: It was observed that the CEC level in the patient group was significantly higher than the control group (p < 0.05). CEC levels were found to be significantly higher in patients with splenectomy than in patients with nonsplenectomy (p < 0.05). CEC levels were higher in patients with nontransfusion-dependent ßT (NTD-ßThal) than in patients with transfusion-dependent ßT (TD-ßThal) (p < 0.05). CEC levels were found to be significantly higher in patients with splenectomy than in patients with nonsplenectomy in both TD-ßThal and NTD-ßThal groups (p < 0.05). There was a moderate-negative correlation was detected between CECs and Hb levels (r = -0.467; p < 0.05). CONCLUSIONS: High CEC levels in ßThal patients develop as a result of ineffective erythropoiesis. We think that keeping CEC levels under control is important for prognosis, especially in patients with splenectomy.

Diverse immunological dysregulation, chronic inflammation, and impaired erythropoiesis in long COVID patients with chronic fatigue syndrome.

A substantial number of patients recovering from acute SARS-CoV-2 infection present serious lingering symptoms, often referred to as long COVID (LC). However, a subset of these patients exhibits the most debilitating symptoms characterized by ongoing myalgic encephalomyelitis or chronic fatigue syndrome (ME/CFS). We specifically identified and studied ME/CFS patients from two independent LC cohorts, at least 12 months post the onset of acute disease, and compared them to the recovered group (R). ME/CFS patients had relatively increased neutrophils and monocytes but reduced lymphocytes. Selective T cell exhaustion with reduced naïve but increased terminal effector T cells was observed in these patients. LC was associated with elevated levels of plasma pro-inflammatory cytokines, chemokines, Galectin-9 (Gal-9), and artemin (ARTN). A defined threshold of Gal-9 and ARTN concentrations had a strong association with LC. The expansion of immunosuppressive CD71+ erythroid cells (CECs) was noted. These cells may modulate the immune response and contribute to increased ARTN concentration, which correlated with pain and cognitive impairment. Serology revealed an elevation in a variety of autoantibodies in LC. Intriguingly, we found that the frequency of 2B4+CD160+ and TIM3+CD160+ CD8+ T cells completely separated LC patients from the R group. Our further analyses using a multiple regression model revealed that the elevated frequency/levels of CD4 terminal effector, ARTN, CEC, Gal-9, CD8 terminal effector, and MCP1 but lower frequency/levels of TGF-ß and MAIT cells can distinguish LC from the R group. Our findings provide a new paradigm in the pathogenesis of ME/CFS to identify strategies for its prevention and treatment.

CD71: Role in permafrost immunity.

Iron, an essential constituent of cell metabolism, is transported intra-cellularly bound to the ubiquitous 76 kDa blood glycoprotein transferrin via the transferrin receptor, CD71. Because of its structure, CD71 facilitates the binding and penetration of a large variety of viruses into the host. Among which the hemorrhagic fever-causing New World mammarena viruses (family of single stranded ambisense segmented RNA Arenaviridae), the single stranded positive sense RNA hepatitis C virus, the single stranded negative sense segmented influenza A virus, the single stranded negative sense RNA rabies virus, the single stranded positive sense SARS-CoV2 and possibly many others. In this process, CD71 is associated with the target of the anti-proliferative antibody-1 (CD81) viral co-receptor. In light of the plethora of novel and ancient viruses and microbes emerging from melting eternal glacier ice and permafrost, it is timely and critical to define and characterize interventions, besides the soluble form of CD71 (sCD71), that can abrogate or minimize this novice non-canonical function of CD71.

Cabot rings in a cat with myeloproliferative disease.

A 6-year-old spayed female Scottish Fold cat presented with lethargy and anorexia. A complete blood cell count indicated severe anemia and mild thrombocytopenia. Examination of peripheral blood smears revealed marked changes in the erythroid lineage, including the presence of basophilic stippling and Howell-Jolly bodies as well as an increase in nucleated erythrocytes, polychromatophils, ovalocytes, and schistocytes. Additionally, some erythrocytes contained a ring or figure-eight shaped structure known as a Cabot ring, which were especially observed in polychromatophilic erythrocytes. Hemolytic diseases (Mycoplasma infection and IMHA) were diagnostically excluded, and the cat was treated through prednisolone administration, whole blood transfusion, and administration of vitamins (K2 and B12); however, the anemia progressively worsened. Cabot rings were observed until Day 22 and subsequently disappeared as the number of nucleated RBCs increased, and the erythrocyte lineage shifted to immature population. On Day 42, peripheral blood examination revealed further left shifting and appearance of many rubriblasts. The patient died at home on Day 43. Necropsy revealed neoplastic cells infiltrating the bone marrow and other organs, which were immunopositive to CD71 which is an erythroid lineage marker. In humans, Cabot rings have been observed in megaloblastic anemia, lead poisoning, myelodysplastic syndrome, and myelofibrosis; further, they are thought to be related to stressed bone marrow and dyserythropoiesis. This is the first case report of a cat with Cabot rings, which are suggestive of defects in erythroid lineage production.

The Plasmodium vivax MSP1P-19 is involved in binding of reticulocytes through interactions with the membrane proteins band3 and CD71.

The parasite Plasmodium vivax preferentially invades human reticulocytes. Its merozoite surface protein 1 paralog (PvMSP1P), particularly the 19-kDa C-terminal region (PvMSP1P-19), has been shown to bind to reticulocytes, and this binding can be inhibited by antisera obtained by PvMSP1P-19 immunization. The molecular mechanism of interactions between PvMSP1P-19 and reticulocytes during P. vivax invasion, however, remains unclear. In this study, we analyzed the ability of MSP1P-19 to bind to different concentrations of reticulocytes and confirmed its reticulocyte preference. LC-MS analysis was used to identify two potential reticulocyte receptors, band3 and CD71, that interact with MSP1P-19. Both PvMSP1P-19 and its sister taxon Plasmodium cynomolgi MSP1P-19 were found to bind to the extracellular loop (loop 5) of band3, where the interaction of MSP1P-19 with band3 was chymotrypsin sensitive. Antibodies against band3-P5, CD71, and MSP1P-19 reduced the binding activity of PvMSP1P-19 and Plasmodium cynomolgi MSP1P-19 to reticulocytes, while MSP1P-19 proteins inhibited Plasmodium falciparum invasion in vitro in a concentration-dependent manner. To sum up, identification and characterization of the reticulocyte receptor is important for understanding the binding of reticulocytes by MSP1P-19.

A Novel Homozygous Germline Mutation in Transferrin Receptor 1 (TfR1) Leads to Combined Immunodeficiency and Provides New Insights into Iron-Immunity Axis.

A homozygous missense mutation in the transferrin receptor 1 (TfR1), also known as CD71, leads to a rare inborn error of immunity (IEI) characterized by the impaired lymphocyte activation and proliferation due to defective iron uptake of cells. However, only one causative mutation (c.58T > C, p.Y20H) in the TFRC gene coding for TfR1 has been reported so far. We herein identified a new disease-causing homozygous germline mutation in the TFRC gene (c.64C > T, p.R22W) (referred to as TfR1R22W from now on) in a Turkish patient with combined immunodeficiency (CID). TfR1R22W results in impaired TfR1 internalization similar to previously defined TfR1Y20H mutation. We found that TfR1R22W is associated with severely restricted B and T lymphocyte clonal diversity and impaired T cell activation and cytokine production as well as defective mitochondrial oxidative phosphorylation in helper T cells. In addition, circulating NK, Treg, and MAIT cell populations were significantly decreased in the patient. Using whole transcriptome analysis, we found dysregulated immune homeostasis and novel biological processes associated with TfR1R22W. We also identified a considerable expansion of circulating low-density neutrophils (LDNs) in patient's PBMCs. Overall, TfR1R22W mutation expands the current understanding of the IEI associated with TfR1 dysfunction and provides new insights underlying impaired immune function, lymphocyte diversity, and granulocyte homeostasis.

Transferrin receptor 1 promotes hepatocellular carcinoma progression and metastasis by activating the mTOR signaling pathway.

BACKGROUND: Aberrant iron metabolism is commonly observed in multiple tumor types, including hepatocellular carcinoma (HCC). However, as the key regulator of iron metabolism involved in iron absorption, the role of transferrin receptor (TFRC) in HCC remains elusive. METHODS: The mRNA and protein expression of TFRC were evaluated in paired HCC and adjacent non-tumor specimens. The correlation between TFRC level and clinicopathological features or prognostic significance was also analyzed. The role of TFRC on biological functions was finally studied in vitro and in vivo. RESULTS: The TFRC level was remarkably upregulated in HCC tissues compared to paired peritumor tissues. Overexpressed TFRC positively correlated with serum alpha-fetoprotein, carcinoembryonic antigen, and poor tumor differentiation. Multivariate analysis demonstrated that upregulated TFRC was an independent predictive marker for poorer overall survival and disease-free survival in HCC patients. Loss of TFRC markedly impaired cell proliferation and migration in vitro and notably suppressed HCC growth and metastasis in vivo, while overexpression of TFRC performed an opposite effect. Mechanistically, the mTOR signaling pathway was downregulated with TFRC knockdown, and the mTOR agonist MHY1485 completely reversed the biological inhibition in HCC cells caused by TFRC knockdown. Furthermore, exogenous ferric citrate (FAC) or iron chelator reversed the changed biological functions and signaling pathway expression of HCC cells caused by TFRC knockdown or overexpression, respectively. CONCLUSIONS: Our study indicates that TFRC exerts an oncogenic role in HCC and may become a promising therapeutic target to restrain HCC progression.

Extramedullary hematopoiesis contributes to enhanced erythropoiesis during pregnancy via TGF-ß signaling.

Red blood cells are the predominant cellular component in human body, and their numbers increase significantly during pregnancy due to heightened erythropoiesis. CD71+ erythroid cells (CECs) are immature red blood cells, encompassing erythroblasts and reticulocytes, constitute a rare cell population primarily found in the bone marrow, although they are physiologically enriched in the neonatal mouse spleen and human cord blood. Presently, the mechanisms underlying the CECs expansion during pregnancy remain largely unexplored. Additionally, the mechanisms and roles associated with extramedullary hematopoiesis (EMH) of erythroid cells during pregnancy have yet to be fully elucidated. In this study, our objective was to examine the underlying mechanisms of erythroid-biased hematopoiesis during pregnancy. Our findings revealed heightened erythropoiesis and elevated CECs in both human and mouse pregnancies. The increased presence of transforming growth factor (TGF)-ß during pregnancy facilitated the differentiation of CD34+ hematopoietic stem and progenitor cells (HSPCs) into CECs, without impacting HSPCs proliferation, ultimately leading to enhanced erythropoiesis. The observed increase in CECs during pregnancy was primarily attributed to EMH occurring in the spleen. During mouse pregnancy, splenic stromal cells were found to have a significant impact on splenic erythropoiesis through the activation of TGF-ß signaling. Conversely, splenic macrophages were observed to contribute to extramedullary erythropoiesis in a TGF-ß-independent manner. Our results suggest that splenic stromal cells play a crucial role in promoting extramedullary erythropoiesis and the production of CECs during pregnancy, primarily through TGF-ß-dependent mechanisms.

Murine Bone Marrow Erythroid Cells Have Two Branches of Differentiation Defined by the Presence of CD45 and a Different Immune Transcriptome Than Fetal Liver Erythroid Cells.

Mouse erythropoiesis is a multifaceted process involving the intricate interplay of proliferation, differentiation, and maturation of erythroid cells, leading to significant changes in their transcriptomic and proteomic profiles. While the immunoregulatory role of murine erythroid cells has been recognized historically, modern investigative techniques have been sparingly applied to decipher their functions. To address this gap, our study sought to comprehensively characterize mouse erythroid cells through contemporary transcriptomic and proteomic approaches. By evaluating CD71 and Ter-119 as sorting markers for murine erythroid cells and employing bulk NanoString transcriptomics, we discerned distinctive gene expression profiles between bone marrow and fetal liver-derived erythroid cells. Additionally, leveraging flow cytometry, we assessed the surface expression of CD44, CD45, CD71, and Ter-119 on normal and phenylhydrazine-induced hemolytic anemia mouse bone marrow and splenic erythroid cells. Key findings emerged: firstly, the utilization of CD71 for cell sorting yielded comparatively impure erythroid cell populations compared to Ter-119; secondly, discernible differences in immunoregulatory molecule expression were evident between erythroid cells from mouse bone marrow and fetal liver; thirdly, two discrete branches of mouse erythropoiesis were identified based on CD45 expression: CD45-negative and CD45-positive, which had been altered differently in response to phenylhydrazine. Our deductions underscore (1) Ter-119's superiority over CD71 as a murine erythroid cell sorting marker, (2) the potential of erythroid cells in murine antimicrobial immunity, and (3) the importance of investigating CD45-positive and CD45-negative murine erythroid cells separately and in further detail in future studies.

Anaplastic thyroid cancer cells reduce CD71 levels to increase iron overload tolerance.

BACKGROUND: Follicular thyroid cancer (FTC) is a prevalent form of differentiated thyroid cancer, whereas anaplastic thyroid cancer (ATC) represents a rare, fast-growing, undifferentiated, and highly aggressive tumor, posing significant challenges for eradication. Ferroptosis, an iron-dependent cell death mechanism driven by the excessive production of reactive oxygen species and subsequent lipid peroxidation, emerges as a promising therapeutic strategy for cancer. It has been observed that many cancer cells exhibit sensitivity to ferroptosis, while some other histotypes appear to be resistant, by counteracting the metabolic changes and oxidative stress induced by iron overload. METHODS: Here we used human biopsies and in vitro approaches to analyse the effects of iron-dependent cell death. We assessed cell proliferation and viability through MTT turnover, clonogenic assays, and cytofluorimetric-assisted analysis. Lipid peroxidation assay and western blot were used to analyse molecular mechanisms underlying ferroptosis modulation. Two distinct thyroid cancer cell lines, FTC-133 (follicular) and 8505C (anaplastic), were utilized. These cell lines were exposed to ferroptosis inducers, Erastin and RSL3, while simulating an iron overload condition using ferric ammonium citrate. RESULTS: Our evidence suggests that FTC-133 cell line, exposed to iron overload, reduced their viability and showed increased ferroptosis. In contrast, the 8505C cell line seems to better tolerate ferroptosis, responding by modulating CD71, which is involved in iron internalization and seems to have a role in resistance to iron overload and consequently in maintaining cell viability. CONCLUSIONS: The differential tolerance to ferroptosis observed in our study may hold clinical implications, particularly in addressing the unmet therapeutic needs associated with ATC treatment, where resistance to ferroptosis appears more pronounced compared to FTC.

Diagnosis of erythroid dysplasia by flow cytometry: a review.

INTRODUCTION: The diagnosis of myelodysplastic syndrome (MDS) is complex. Flow cytometric analysis of the myelomonocytic compartment can be helpful, but it is highly subjective and reproducibility by non-specialized groups is unclear. Analysis of the erythroid lineage by flow cytometry is emerging as potentially more reproducible and easier to conduct, while keeping a high diagnostic performance. AREAS COVERED: We review the evidence in this area, including 1) the use of well-established markers - CD71 and CD36 - and other less well-established markers and parameters; 2) the use of flow cytometric scores for the erythroid lineage; and 3) additional aspects, including the emergence of computational tools and the roles of flow cytometry beyond diagnosis. Finally, we discuss the limitations with the current evidence, including 1) the impact of the sample processing protocol and reagents on the results, 2) the lack of a standard gating strategy, and 3) conceptualization and design issues in the available publications. EXPERT OPINION: We end by offering our recommendations for the current use - and our personal take on the value - of the analysis of erythroid lineage by flow cytometry.

Widespread in vivo efficacy of The-0504: A conditionally-activatable nanoferritin for tumor-agnostic targeting of CD71-expressing cancers.

Background: Cancer is still among the leading causes of death all over the world. Improving chemotherapy and minimizing associated toxicities are major unmet medical needs. Recently, we provided a preliminary preclinical evaluation of a human ferritin (HFt)-based drug carrier (The-0504) that selectively delivers the wide-spectrum topoisomerase I inhibitor Genz-644282 to CD71-expressing tumors. The-0504 has so far been evaluated on four different human tumor xenotransplant models (breast, colorectal, pancreatic and liver cancers). Methods: Herein, we extend our studies, by: (a) testing DNA damage in vitro, (b) treating eight additional tumor xenograft models in vivo with The-0504; (c) performing pharmacokinetic (PK) studies in rats; and (d) evaluating The-0504 anti-tumor xenotransplant efficacy by optimizing its administration schedule based on PK considerations. Results: Immunofluorescence demonstrated that The-0504 induces foci expressing the DNA double-strand break marker γH2AX. Expression increases up to 4-fold and is more persistent as compared to free Genz-644282. In vivo studies confirmed a remarkable anti-tumor activity of The-0504, resulting in tumor eradication in most murine xenograft models, regardless of embryological origin (e.g. epithelial, mesenchymal or neuroendocrine), and molecular subtypes. PK studies demonstrated a long persistence of The-0504 in rat serum (half-life of about 40 h as compared to 15 h of the free drug), with a 400-fold increase in peak concentrations as compared to the free drug. On this basis, we reduced The-0504 administration frequency from twice to once per week, with no appreciable loss in therapeutic efficacy in mice. Conclusion: The results presented here confirm that The-0504 is highly active against several human tumor xenotransplants, even when administered less frequently than previously reported. The-0504 may be a good candidate for further clinical development in a tumor histotype-agnostic setting.

Murine Placental Erythroid Cells Are Mainly Represented by CD45+ Immunosuppressive Erythroid Cells and Secrete CXCL1, CCL2, CCL3 and CCL4 Chemokines.

Erythroid cells are emerging players in immunological regulation that have recently been shown to play a crucial role in fetomaternal tolerance in mice. In this work, we set ourselves the goal of discovering additional information about the molecular mechanisms of this process. We used flow cytometry to study placental erythroid cells' composition and BioPlex for the secretome profiling of 23 cytokines at E12.5 and E19.5 in both allogeneic and syngeneic pregnancies. We found that (1) placental erythroid cells are mainly represented by CD45+ erythroid cells; (2) the secretomes of CD71+ placental erythroid cells differ from the ones in syngeneic pregnancy; (3) CCL2, CCL3, CCL4 and CXCL1 chemokines were secreted on each day of embryonic development and in both types of pregnancy studied. We believe that these chemokines lure placental immune cells towards erythroid cells so that erythroid cells can induce anergy in those immune cells via cell-bound ligands such as PD-L1, enzymes such as ARG1, and secreted factors such as TGFß-1.

Immunological impact of the CD71+ RBCs: A potential immune mediator in transfusion.

Donor - recipient sex - mismatched transfusion is associated with increased mortality. The mechanisms for this are not clear, but it may relate to transfusion-related immunomodulation. Recently, CD71+ erythroid cells (CECs), including reticulocytes (CD71+ RBCs) and erythroblasts, have been identified as potent immunoregulatory cells. The proportion of CD71+ RBCs in the peripheral blood is sufficient to play a potential immunomodulatory role. Differences in the quantity of CD71+ RBCs are dependent on blood donor sex. The total number of CD71+ RBCs in red cell concentrates is also affected by blood manufacturing methods, and storage duration. As a component of the total CECs, CD71+ RBCs can affect innate and adaptive immune cells. Phagocytosed CECs directly reduce TNF-α production from macrophages. CECs can also suppress the production of TNF-α production from antigen presenting cells. Moreover, CECs can suppress T cell proliferation thorough immune mediation and / or direct cell-to-cell interactions. Different in their biophysical features compared to mature RBCs, blood donor CD71+ RBCs may be preferential targets for the macrophages. This report summarizes the currently literature supporting an important role for CD71+ RBCs in adverse transfusion reactions including immune mediation and sepsis.

Heme Scavenging and Delivery: The Role of Human Serum Albumin.

Heme is the reactive center of several metal-based proteins that are involved in multiple biological processes. However, free heme, defined as the labile heme pool, has toxic properties that are derived from its hydrophobic nature and the Fe-atom. Therefore, the heme concentration must be tightly controlled to maintain cellular homeostasis and to avoid pathological conditions. Therefore, different systems have been developed to scavenge either Hb (i.e., haptoglobin (Hp)) or the free heme (i.e., high-density lipoproteins (HDL), low-density lipoproteins (LDL), hemopexin (Hx), and human serum albumin (HSA)). In the first seconds after heme appearance in the plasma, more than 80% of the heme binds to HDL and LDL, and only the remaining 20% binds to Hx and HSA. Then, HSA slowly removes most of the heme from HDL and LDL, and finally, heme transits to Hx, which releases it into hepatic parenchymal cells. The Hx:heme or HSA:heme complexes are internalized via endocytosis mediated by the CD91 and CD71 receptors, respectively. As heme constitutes a major iron source for pathogens, bacteria have evolved hemophores that can extract and uptake heme from host proteins, including HSA:heme. Here, the molecular mechanisms underlying heme scavenging and delivery from HSA are reviewed. Moreover, the relevance of HSA in disease states associated with increased heme plasma concentrations are discussed.

Efficacy of Antibodies Targeting TfR1 in Xenograft Mouse Models of AIDS-Related Non-Hodgkin Lymphoma.

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.

Donor sex, pre-donation hemoglobin, and manufacturing affect CD71+ cells in red cell concentrates.

BACKGROUND: Circulating CD71+ red blood cells (RBCs) have been reported to play an immunomodulatory role in vivo, which may contribute to adverse donor-recipient sex-mismatched transfusion outcomes. However, it is not clear how CD71+ RBC quantity in red cell concentrates (RCCs) is affected by manufacturing methods and donor factors such as donor sex, donor age, pre-donation hemoglobin (Hb), venous Hb (Hbv ) levels, and donation frequency. METHODS: We determined CD71+ RBCs and Hb levels in whole blood (WB) from healthy donors (42 male/38 female). Using small-scale red cell filtration (RCF) and whole blood filtration (WBF) methods, leukoreduced RCCs were processed from WB samples (n = 6) and the CD71+ RBCs were determined at days 1, 7, and 28. We examined uni- and multivariate associations among CD71+ RBCs, donor factors, and manufacturing method. RESULTS: Male donors had a higher CD71+ RBC concentration than females (p < .001), especially male donors aged 17-50 years with 1 or 2 WB donations over the previous 12 months. Donors with a Hbv above 155 g/L had a higher CD71+ RBC concentration than an Hbv level below 140 g/L (p < .05). There was a positive correlation between pre-donation Hb and CD71+ RBC concentration (Pearson r = 0.41). WBF RCCs had a higher total number of CD71+ RBCs than RCF-produced RCCs on day 1 (p < .05). DISCUSSION: RCCs have variable numbers of CD71+ RBCs. This makes understanding the impact of donor factors and manufacturing methods on the immunomodulatory effect of CD71+ RBCs critical in exploring donor-recipient sex-mismatched transfusions.

A new approach to the study of Hodgkin lymphoma by flow cytometry.

Hodgkin lymphoma (HL) appears to originate from germinal centre B cells but lacks expression of most B cell markers. In contrast to non-Hodgkin B lymphomas, HL is not routinely diagnosed using flow cytometry techniques, and diagnosis is mainly based on immunohistochemical and cytomorphological pathology studies. Hodgkin and Reed-Sternberg cells are large and fragile, making them difficult to study by flow cytometry. The aim of this study was to characterise the CD71 expression pattern on CD4+ T cells from HL patients and to design a simple flow cytometry algorithm to complement the histopathological diagnosis of HL. The present study suggests the utility of a conventional staining protocol with a simple panel of seven markers (CD15, CD30, CD4, CD8, CD71, CD3, and CD45) and a well-defined analysis strategy. The proposed algorithm uses the CD71 ratio (calculated as the percentage of CD71+ CD4+ T cells divided by the percentage of CD71+ CD45+ CD3- lymphocytes), with a cut-off of 0.5 to establish diagnosis groups as suggestive (≥0.5) or not suggestive (<0.5) of HL. In HL, CD71 expression is higher on CD4+ T lymphocytes than on non-T lymphocytes. In addition, the CD4+ T cell population is increased in HL patients, with no change in amounts of CD8+ T cells. Application of the CD71 ratio algorithm yielded a sensitivity of 82% and specificity of 87%, with 84.61% of patients correctly diagnosed. Although histopathology remains the gold standard for definitive HL diagnosis, the proposed flow cytometry method provides a rapid method to guide the study that would allow a more robust and integrated diagnosis. Moreover, the procedure is easily applicable in most clinical laboratories as it does not require state-of-the-art cytometers and uses standard reagents.

Identification of Transferrin Receptor 1 (TfR1) Overexpressed in Lung Cancer Cells, and Internalization of Magnetic Au-CoFe2O4 Core-Shell Nanoparticles Functionalized with Its Ligand in a Cellular Model of Small Cell Lung Cancer (SCLC).

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.

Immune Transcriptome and Secretome Differ between Human CD71+ Erythroid Cells from Adult Bone Marrow and Fetal Liver Parenchyma.

CD71+ erythroid cells (CECs) were only known as erythrocyte progenitors not so long ago. In present times, however, they have been shown to be active players in immune regulation, especially in immunosuppression by the means of ROS, arginase-1 and arginase-2 production. Here, we uncover organ-of-origin differences in cytokine gene expression using NanoString and protein production using Bio-Plex between CECs from healthy human adult bone marrow and from human fetal liver parenchyma. Namely, healthy human adult bone marrow CECs both expressed and produced IFN-a, IL-1b, IL-8, IL-18 and MIF mRNA and protein, while human fetal liver parenchymaCECs expressed and produced IFN-a, IL15, IL18 and TNF-b mRNA and protein. We also detected TLR2 and TLR9 gene expression in both varieties of CECs and TLR1 and NOD2 gene expression in human fetal liver parenchymaCECs only. These observations suggest that there might be undiscovered roles in immune response for CECs.