Indium-111 radiolabelling of a brain-penetrant Aß antibody for SPECT imaging.

Background: The development of bispecific antibodies that can traverse the blood-brain barrier has paved the way for brain-directed immunotherapy and when radiolabelled, immunoPET imaging. The objective of this study was to investigate how indium-111 (111In) radiolabelling with compatible chelators affects the brain delivery and peripheral biodistribution of the bispecific antibody RmAb158-scFv8D3, which binds to amyloid-beta (Aß) and the transferrin receptor (TfR), in Aß pathology-expressing tg-ArcSwe mice and aged-matched wild-type control mice. Methods: Bispecific RmAb158-scFv8D3 (biAb) was radiolabelled with 111In using CHX-A"-DTPA, DOTA, or DOTA-tetrazine (DOTA-Tz). Affinity toward TfR and Aß, as well as stability, was investigated in vitro. Mice were then intravenously administered with the three different radiolabelled biAb variants, and blood samples were collected for monitoring pharmacokinetics. Brain concentration was quantified after 2 and 72 h, and organ-specific retention was measured at 72 h by gamma counting. A subset of mice also underwent whole-body Single-photon emission computed tomography (SPECT) scanning at 72 h after injection. Following post-mortem isolation, the brains of tg-ArcSwe and WT mice were sectioned, and the spatial distribution of biAb was further investigated with autoradiography. Results: All three [111In]biAb variants displayed similar blood pharmacokinetics and brain uptake at 2 h after administration. Radiolabelling did not compromise affinity, and all variants showed good stability, especially the DOTA-Tz variant. Whole-body SPECT scanning indicated high liver, spleen, and bone accumulation of all [111In]biAb variants. Subsequent ex vivo measurement of organ retention confirmed SPECT data, with retention in the spleen, liver, and bone - with very high bone marrow retention. Ex vivo gamma measurement of brain tissue, isolated at 72 h post-injection, and ex vivo autoradiography showed that WT mice, despite the absence of Aß, exhibited comparable brain concentrations of [111In]biAb as those found in the tg-ArcSwe brain. Conclusions: The successful 111In-labelling of biAb with retained binding to TfR and Aß, and retained ability to enter the brain, demonstrated that 111In can be used to generate radioligands for brain imaging. A high degree of [111In]biAb in bone marrow and intracellular accumulation in brain tissue indicated some off-target interactions or potential interaction with intrabrain TfR resulting in a relatively high non-specific background signal.

Iron Is Critical for Mucosal-Associated Invariant T Cell Metabolism and Effector Functions.

Mucosal-Associated Invariant T (MAIT) cells are a population of innate T cells that play a critical role in host protection against bacterial and viral pathogens. Upon activation, MAIT cells can rapidly respond via both TCR-dependent and -independent mechanisms, resulting in robust cytokine production. The metabolic and nutritional requirements for optimal MAIT cell effector responses are still emerging. Iron is an important micronutrient and is essential for cellular fitness, in particular cellular metabolism. Iron is also critical for many pathogenic microbes, including those that activate MAIT cells. However, iron has not been investigated with respect to MAIT cell metabolic or functional responses. In this study, we show that human MAIT cells require exogenous iron, transported via CD71 for optimal metabolic activity in MAIT cells, including their production of ATP. We demonstrate that restricting iron availability by either chelating environmental iron or blocking CD71 on MAIT cells results in impaired cytokine production and proliferation. These data collectively highlight the importance of a CD71-iron axis for human MAIT cell metabolism and functionality, an axis that may have implications in conditions where iron availability is limited.

Reducing neonatal Fc receptor binding enhances clearance and brain-to-blood ratio of TfR-delivered bispecific amyloid-ß antibody.

Recent development of amyloid-ß (Aß)-targeted immunotherapies for Alzheimer's disease (AD) have highlighted the need for accurate diagnostic methods. Antibody-based positron emission tomography (PET) ligands are well suited for this purpose as they can be directed toward the same target as the therapeutic antibody. Bispecific, brain-penetrating antibodies can achieve sufficient brain concentrations, but their slow blood clearance remains a challenge, since it prolongs the time required to achieve a target-specific PET signal. Here, two antibodies were designed based on the Aß antibody bapineuzumab (Bapi) - one monospecific IgG (Bapi) and one bispecific antibody with an antigen binding fragment (Fab) of the transferrin receptor (TfR) antibody 8D3 fused to one of the heavy chains (Bapi-Fab8D3) for active, TfR-mediated transport into the brain. A variant of each antibody was designed to harbor a mutation to the neonatal Fc receptor (FcRn) binding domain, to increase clearance. Blood and brain pharmacokinetics of radiolabeled antibodies were studied in wildtype (WT) and AD mice (AppNL-G-F). The FcRn mutation substantially reduced blood half-life of both Bapi and Bapi-Fab8D3. Bapi-Fab8D3 showed high brain uptake and the brain-to-blood ratio of its FcRn mutated form was significantly higher in AppNL-G-F mice than in WT mice 12 h after injection and increased further up to 168 h. Ex vivo autoradiography showed specific antibody retention in areas with abundant Aß pathology. Taken together, these results suggest that reducing FcRn binding of a full-sized bispecific antibody increases the systemic elimination and could thereby drastically reduce the time from injection to in vivo imaging.

Anti-tumor immunotherapy using engineered bacterial outer membrane vesicles fused to lysosome-targeting chimeras mediated by transferrin receptor.

The lysosome-targeting chimera (LYTAC) approach has shown promise for the targeted degradation of secreted and membrane proteins via lysosomes. However, there have been challenges in design, development, and targeting. Here, we have designed a genetically engineered transferrin receptor (TfR)-mediated lysosome-targeting chimera (TfR-LYTAC) that is efficiently internalized via TfR-mediate endocytosis and targets PD-L1 for lysosomal degradation in cultured cells but not in vivo due to short half-life and poor tumor targeting. A delivery platform was developed by fusing TfR-LYTAC to the surface of bacterial outer membrane vesicles (OMVs). The engineered OMV-LYTAC combines PD-1/PD-L1 pathway inhibition with LYTAC and immune activation by bacterial OMVs. OMV-LYTAC significantly reduced tumor growth in vivo. We have provided a modular and simple genetic strategy for lysosomal degradation as well as a delivery platform for in vivo tumor targeting. The study paves the way for the targeting and degradation of extracellular proteins using the TfR-LYTAC system.

Host receptor-targeted therapeutic approach to counter pathogenic New World mammarenavirus infections.

Five New World mammarenaviruses (NWMs) cause life-threatening hemorrhagic fever (HF). Cellular entry by these viruses is mediated by human transferrin receptor 1 (hTfR1). Here, we demonstrate that an antibody (ch128.1/IgG1) which binds the apical domain of hTfR1, potently inhibits infection of attenuated and pathogenic NWMs in vitro. Computational docking of the antibody Fab crystal structure onto the known structure of hTfR1 shows an overlapping receptor-binding region shared by the Fab and the viral envelope glycoprotein GP1 subunit that binds hTfR1, and we demonstrate competitive inhibition of NWM GP1 binding by ch128.1/IgG1 as the principal mechanism of action. Importantly, ch128.1/IgG1 protects hTfR1-expressing transgenic mice against lethal NWM challenge. Additionally, the antibody is well-tolerated and only partially reduces ferritin uptake. Our findings provide the basis for the development of a novel, host receptor-targeted antibody therapeutic broadly applicable to the treatment of HF of NWM etiology.

Insights from targeting transferrin receptors to develop vaccines for pathogens of humans and food production animals.

While developing vaccines targeting surface transferrin receptor proteins in Gram-negative pathogens of humans and food production animals, the common features derived from their evolutionary origins has provided us with insights on how improvements could be implemented in the various stages of research and vaccine development. These pathogens are adapted to live exclusively on the mucosal surfaces of the upper respiratory or genitourinary tract of their host and rely on their receptors to acquire iron from transferrin for survival, indicating that there likely are common mechanisms for delivering transferrin to the mucosal surfaces that should be explored. The modern-day receptors are derived from those present in bacteria that lived over 320 million years ago. The pathogens represent the most host adapted members of their bacterial lineages and may possess factors that enable them to have strong association with the mucosal epithelial cells, thus likely reside in a different niche than the commensal members of the bacterial lineage. The bacterial pathogens normally lead a commensal lifestyle which presents challenges for development of relevant infection models as most infection models either exclude the early stages of colonization or subsequent disease development, and the immune mechanisms at the mucosal surface that would prevent disease are not evident. Development of infection models emulating natural horizontal disease transmission are also lacking. Our aim is to share our insights from the study of pathogens of humans and food production animals with individuals involved in vaccine development, maintaining health or regulation of products in the human and animal health sectors.

Iron deprivation restrains the differentiation and pathogenicity of T helper 17 cell.

Iron plays a critical role in immune responses. However, its role in T helper cell differentiation and function remains poorly understood. In this study, it is shown that the restraint of iron availability through blocking CD71-mediated iron endocytosis impaired the differentiation and pathogenicity of TH 17 cells. Administrations of anti-CD71 mAb could relieve the development of experimental autoimmune encephalomyelitis (EAE). Mechanistically, the iron deficiency due to the blocking of CD71 enhanced IL-2 expression, which further restrained the differentiation of TH 17 cells. Meanwhile, CD71 blockade impaired histone modifications of Il17 gene and reduced the recruitment of RORγt to Il17a locus. In sum, the findings reveal that iron plays a pivotal role in regulating TH 17 cell differentiation and function in autoimmune diseases.

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.

Targeting TfR1 with the ch128.1/IgG1 Antibody Inhibits EBV-driven Lymphomagenesis in Immunosuppressed Mice Bearing EBV+ Human Primary B-cells.

Epstein-Barr virus (EBV) is a human gammaherpesvirus associated with the development of hematopoietic cancers of B-lymphocyte origin, including AIDS-related non-Hodgkin lymphoma (AIDS-NHL). Primary infection of B-cells with EBV results in their polyclonal activation and immortalization. The transferrin receptor 1 (TfR1), also known as CD71, is important for iron uptake and regulation of cellular proliferation. TfR1 is highly expressed in proliferating cells, including activated lymphocytes and malignant cells. We developed a mouse/human chimeric antibody targeting TfR1 (ch128.1/IgG1) that has previously shown significant antitumor activity in immunosuppressed mouse models bearing human malignant B-cells, including multiple myeloma and AIDS-NHL cells. In this article, we examined the effect of targeting TfR1 to inhibit EBV-driven activation and growth of human B-cells in vivo using an immunodeficient NOD.Cg-Prkdcscid Il2rgtm1Wjl /SzJ [NOD/SCID gamma (NSG)] mouse model. Mice were implanted with T-cell-depleted, human peripheral blood mononuclear cells (PBMCs), either without EBV (EBV-), or exposed to EBV in vitro (EBV+), intravenously via the tail vein. Mice implanted with EBV+ cells and treated with an IgG1 control antibody (400 µg/mouse) developed lymphoma-like growths of human B-cell origin that were EBV+, whereas mice implanted with EBV+ cells and treated with ch128.1/IgG1 (400 µg/mouse) showed increased survival and significantly reduced inflammation and B-cell activation. These results indicate that ch128.1/IgG1 is effective at preventing the growth of EBV+ human B-cell tumors in vivo, thus, indicating that there is significant potential for agents targeting TfR1 as therapeutic strategies to prevent the development of EBV-associated B-cell malignancies. SIGNIFICANCE: An anti-TfR1 antibody, ch128.1/IgG1, effectively inhibits the activation, growth, and immortalization of EBV+ human B-cells in vivo, as well as the development of these cells into lymphoma-like tumors in immunodeficient mice.

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.

Novel method for screening functional antibody with comprehensive analysis of its immunoliposome.

Development of monoclonal antibody is critical for targeted drug delivery because its characteristics determine improved therapeutic efficacy and reduced side-effect. Antibody therapeutics target surface molecules; hence, internalization is desired for drug delivery. As an antibody-drug conjugate, a critical parameter is drug-to-antibody ratio wherein the quantity of drugs attached to the antibody influences the antibody structure, stability, and efficacy. Here, we established a cell-based immunotoxin screening system to facilitate the isolation of functional antibodies with internalization capacities, and discovered an anti-human CD71 monoclonal antibody. To overcome the limitation of drug-to-antibody ratio, we employed the encapsulation capacity of liposome, and developed anti-CD71 antibody-conjugated liposome that demonstrated antigen-antibody dependent cellular uptake when its synthesis was optimized. Furthermore, anti-CD71 antibody-conjugated liposome encapsulating doxorubicin demonstrated antigen-antibody dependent cytotoxicity. In summary, this study demonstrates the powerful pipeline to discover novel functional antibodies, and the optimal method to synthesize immunoliposomes. This versatile technology offers a rapid and direct approach to generate antibodies suitable for drug delivery modalities.

Brain pharmacokinetics of anti-transferrin receptor antibody affinity variants in rats determined using microdialysis.

Receptor-mediated transcytosis (RMT) is used to enhance the delivery of monoclonal antibodies (mAb) into the central nervous system (CNS). While the binding to endogenous receptors on the brain capillary endothelial cells (BCECs) may facilitate the uptake of mAbs in the brain, a strong affinity for the receptor may hinder the efficiency of transcytosis. To quantitatively investigate the effect of binding affinity on the pharmacokinetics (PK) of anti-transferrin receptor (TfR) mAbs in different regions of the rat brain, we conducted a microdialysis study to directly measure the concentration of free mAbs at different sites of interest. Our results confirmed that bivalent anti-TfR mAb with an optimal dissociation constant (KD) value (76 nM) among four affinity variants can have up to 10-fold higher transcytosed free mAb exposure in the brain interstitial fluid (bISF) compared to lower and higher affinity mAbs (5 and 174 nM). This bell-shaped relationship between KD values and the increased brain exposure of mAbs was also visible when using whole-brain PK data. However, we found that mAb concentrations in postvascular brain supernatant (obtained after capillary depletion) were almost always higher than the concentrations measured in bISF using microdialysis. We also observed that the increase in mAb area under the concentration curve in CSF compartments was less significant, which highlights the challenge in using CSF measurement as a surrogate for estimating the efficiency of RMT delivery. Our results also suggest that the determination of mAb concentrations in the brain using microdialysis may be necessary to accurately measure the PK of CNS-targeted antibodies at the site-of-actions in the brain.

Blood-brain barrier transport using a high affinity, brain-selective VNAR antibody targeting transferrin receptor 1.

Transfer across the blood-brain barrier (BBB) remains a significant hurdle for the development of biopharmaceuticals with therapeutic effects within the central nervous system. We established a functional selection method to identify high affinity single domain antibodies to the transferrin receptor 1 (TfR1) with efficient biotherapeutic delivery across the BBB. A synthetic phage display library based on the variable domain of new antigen receptor (VNAR) was used for in vitro selection against recombinant human TfR1 ectodomain (rh-TfR1-ECD) followed by in vivo selection in mouse for brain parenchyma penetrating antibodies. TXB2 VNAR was identified as a high affinity, species cross-reactive VNAR antibody against TfR1-ECD that does not compete with transferrin or ferritin for receptor binding. IV dosing of TXB2 when fused to human Fc domain (TXB2-hFc) at 25 nmol/kg (1.875 mg/kg) in mice resulted in rapid binding to brain capillaries with subsequent transport into the brain parenchyma and specific uptake into TfR1-positive neurons. Likewise, IV dosing of TXB2-hFc fused with neurotensin (TXB2-hFc-NT) at 25 nmol/kg resulted in a rapid and reversible pharmacological response as measured by body temperature reduction. TXB2-hFc did not elicit any acute adverse reactions, bind, or deplete circulating reticulocytes or reduce BBB-expressed endogenous TfR1 in mice. There was no evidence of target-mediated clearance or accumulation in peripheral organs except lung. In conclusion, TXB2 is a high affinity, species cross-reactive, and brain-selective VNAR antibody to TfR1 that rapidly crosses the BBB and exhibits a favorable pharmacokinetic and safety profile and can be readily adapted to carry a wide variety of biotherapeutics from blood to brain.

CD71+ Erythroid Cells in Human Neonates Exhibit Immunosuppressive Properties and Compromise Immune Response Against Systemic Infection in Neonatal Mice.

Newborns are highly susceptible to infectious diseases. The underlying mechanism of neonatal infection susceptibility has generally been related to their under-developed immune system. Nevertheless, this notion has recently been challenged by the discovery of the physiological abundance of immunosuppressive erythroid precursors CD71+erythroid cells (CECs) in newborn mice and human cord blood. Here, as proof of concept, we show that these cells are also abundant in the peripheral blood of human newborns. Although their frequency appears to be more variable compared to their counterparts in mice, they rapidly decline by 4 weeks of age. However, their proportion remains significantly higher in infants up to six months of age compared to older infants. We found CD45 expressing CECs, as erythroid progenitors, were the prominent source of reactive oxygen species (ROS) production in both humans and mice. Interestingly, a higher proportion of CD45+CECs was observed in the spleen versus bone marrow of neonatal mice, which was associated with a higher ROS production by splenic CECs compared to their siblings in the bone marrow. CECs from human newborns suppressed cytokine production by CD14 monocytes and T cells, which was partially abrogated by apocynin in vitro. Moreover, the depletion of CECs in neonatal mice increased the number of activated effector immune cells in their spleen and liver, which rendered them more resistant to Listeria monocytogenes infection. This was evident by a significant reduction in the bacteria load in the spleen, liver and brain of treated-mice compared to the control group, which enhanced their survival rate. Our finding highlights the immunoregulatory processes mediated by CECs in newborns. Thus, such tightly regulated immune system in newborns/infants may explain one potential mechanism for the asymptomatic or mild COVID-19 infection in this population.

Identification and in vivo characterization of a brain-penetrating nanobody.

BACKGROUND: Preclinical models to determine blood to brain transport ability of therapeutics are often ambiguous. In this study a method is developed that relies on CNS target-engagement and is able to rank brain-penetrating capacities. This method led to the discovery of an anti-transferrin receptor nanobody that is able to deliver a biologically active peptide to the brain via receptor-mediated transcytosis. METHODS: Various nanobodies against the mouse transferrin receptor were fused to neurotensin and injected peripherally in mice. Neurotensin is a neuropeptide that causes hypothermia when present in the brain but is unable to reach the brain from the periphery. Continuous body temperature measurements were used as a readout for brain penetration of nanobody-neurotensin fusions after its peripheral administration. Full temperature curves were analyzed using two-way ANOVA with Dunnett multiple comparisons tests. RESULTS: One anti-transferrin receptor nanobody coupled to neurotensin elicited a drop in body temperature following intravenous injection. Epitope binning indicated that this nanobody bound a distinct transferrin receptor epitope compared to the non-crossing nanobodies. This brain-penetrating nanobody was used to characterize the in vivo hypothermia model. The hypothermic effect caused by neurotensin is dose-dependent and could be used to directly compare peripheral administration routes and various nanobodies in terms of brain exposure. CONCLUSION: This method led to the discovery of an anti-transferrin receptor nanobody that can reach the brain via receptor-mediated transcytosis after peripheral administration. This method could be used to assess novel proteins for brain-penetrating capabilities using a target-engaging readout.

Purification of pancreatic endocrine subsets reveals increased iron metabolism in beta cells.

OBJECTIVES: The main endocrine cell types in pancreatic islets are alpha, beta, and delta cells. Although these cell types have distinct roles in the regulation of glucose homeostasis, inadequate purification methods preclude the study of cell type-specific effects. We developed a reliable approach that enables simultaneous sorting of live alpha, beta, and delta cells from mouse islets for downstream analyses. METHODS: We developed an antibody panel against cell surface antigens to enable isolation of highly purified endocrine subsets from mouse islets based on the specific differential expression of CD71 on beta cells and CD24 on delta cells. We rigorously demonstrated the reliability and validity of our approach using bulk and single cell qPCR, immunocytochemistry, reporter mice, and transcriptomics. RESULTS: Pancreatic alpha, beta, and delta cells can be separated based on beta cell-specific CD71 surface expression and high expression of CD24 on delta cells. We applied our new sorting strategy to demonstrate that CD71, which is the transferrin receptor mediating the uptake of transferrin-bound iron, is upregulated in beta cells during early postnatal weeks. We found that beta cells express higher levels of several other genes implicated in iron metabolism and iron deprivation significantly impaired beta cell function. In human beta cells, CD71 is similarly required for iron uptake and CD71 surface expression is regulated in a glucose-dependent manner. CONCLUSIONS: This study provides a novel and efficient purification method for murine alpha, beta, and delta cells, identifies for the first time CD71 as a postnatal beta cell-specific marker, and demonstrates a central role of iron metabolism in beta cell function.

TfR1 Extensively Regulates the Expression of Genes Associated with Ion Transport and Immunity.

Transferrin receptor 1 (TfR1), encoded by the TFRC gene, is the gatekeeper of cellular iron uptake for cells. A variety of molecular mechanisms are at work to tightly regulate TfR1 expression, and abnormal TfR1 expression has been associated with various diseases. In the current study, to determine the regulation pattern of TfR1, we cloned and overexpressed the human TFRC gene in HeLa cells. RNA-sequencing (RNA-seq) was used to analyze the global transcript levels in overexpressed (OE) and normal control (NC) samples. A total of 1669 differentially expressed genes (DEGs) were identified between OE and NC. Gene ontology (GO) analysis was carried out to explore the functions of the DEGs. It was found that multiple DEGs were associated with ion transport and immunity. Moreover, the regulatory network was constructed on basis of DEGs associated with ion transport and immunity, highlighting that TFRC was the node gene of the network. These results together suggested that precisely controlled TfR1 expression might be not only essential for iron homeostasis, but also globally important for cell physiology, including ion transport and immunity.

Physiological blood-brain transport is impaired with age by a shift in transcytosis.

The vascular interface of the brain, known as the blood-brain barrier (BBB), is understood to maintain brain function in part via its low transcellular permeability1-3. Yet, recent studies have demonstrated that brain ageing is sensitive to circulatory proteins4,5. Thus, it is unclear whether permeability to individually injected exogenous tracers-as is standard in BBB studies-fully represents blood-to-brain transport. Here we label hundreds of proteins constituting the mouse blood plasma proteome, and upon their systemic administration, study the BBB with its physiological ligand. We find that plasma proteins readily permeate the healthy brain parenchyma, with transport maintained by BBB-specific transcriptional programmes. Unlike IgG antibody, plasma protein uptake diminishes in the aged brain, driven by an age-related shift in transport from ligand-specific receptor-mediated to non-specific caveolar transcytosis. This age-related shift occurs alongside a specific loss of pericyte coverage. Pharmacological inhibition of the age-upregulated phosphatase ALPL, a predicted negative regulator of transport, enhances brain uptake of therapeutically relevant transferrin, transferrin receptor antibody and plasma. These findings reveal the extent of physiological protein transcytosis to the healthy brain, a mechanism of widespread BBB dysfunction with age and a strategy for enhanced drug delivery.

Human Fallopian Tube Epithelial Cell Culture Model To Study Host Responses to Chlamydia trachomatis Infection.

Chlamydia trachomatis infection of the human fallopian tubes can lead to damaging inflammation and scarring, ultimately resulting in infertility. To study the human cellular responses to chlamydial infection, researchers have frequently used transformed cell lines that can have limited translational relevance. We developed a primary human fallopian tube epithelial cell model based on a method previously established for culture of primary human bronchial epithelial cells. After protease digestion and physical dissociation of excised fallopian tubes, epithelial cell precursors were expanded in growth factor-containing medium. Expanded cells were cryopreserved to generate a biobank of cells from multiple donors and cultured at an air-liquid interface. Culture conditions stimulated cellular differentiation into polarized mucin-secreting and multiciliated cells, recapitulating the architecture of human fallopian tube epithelium. The polarized and differentiated cells were infected with a clinical isolate of C. trachomatis, and inclusions containing chlamydial developmental forms were visualized by fluorescence and electron microscopy. Apical secretions from infected cells contained increased amounts of proteins associated with chlamydial growth and replication, including transferrin receptor protein 1, the amino acid transporters SLC3A2 and SLC1A5, and the T-cell chemoattractants CXCL10, CXCL11, and RANTES. Flow cytometry revealed that chlamydial infection induced cell surface expression of T-cell homing and activation proteins, including ICAM-1, VCAM-1, HLA class I and II, and interferon gamma receptor. This human fallopian tube epithelial cell culture model is an important tool with translational potential for studying cellular responses to Chlamydia and other sexually transmitted pathogens.

Therapeutic TVs for Crossing Barriers in the Brain.

Brain disorders are at the leading edge of global disease burden worldwide. Effective therapies are lagging behind because most drugs cannot reach their targets in the brain because of the blood-brain barrier (BBB). The new development of a BBB transport vehicle may bring us a step closer to solve this problem.